Structural and chemical organization of teeth 2.

628 36 184MB

English Pages [510] Year 1967

Report DMCA / Copyright

DOWNLOAD FILE

Polecaj historie

Structural and chemical organization of teeth 2.

Table of contents :
Front Cover
Structural and Chemical Organization of Teeth
Copyright Page
Table of Contents
List of Contributors
Preface
Contents of Volume I
Section 3. DEFINITIVE STRUCTURAL ORGANIZATION OF THE TOOTH
Chapter 12. Microanatomy and Histochemistry of Dentine
I. Introduction. Classification of Dentines
II. Osteodentine, Distribution and Structure
III. Vasodentine, Distribution and Structure
IV. Plicidentine, Distribution and Structure
V. Orthodentine
VI. Nonmammalian Orthodentine
VII. Mammalian Orthodentine
VIII. Summary
References
Chapter 13. Ultrastructure of Dentine
I. Introduction
II. Structural Characteristics
III. The Relation of Structure to Some Aspects of Physiology
IV. Pathologic Alterations
V. Concluding Remarks
References
Chapter 14. Microanatomy and Histochemistry of Enamel
I. Introduction
II. Methods of Investigation
III. The Prisms
IV. Retzius Lines
V. Hunter-Schreger Lines
VI. Enamel Tufts and Lamellae
VII. Enamel Spindles
VIII. Enamel Cuticle
IX. Surface Layer of Enamel
X. Developmental Hypomineralizations
XI. Innermost Part of the Enamel
XII. Similarity of Structures in Contralateral Teeth
XIII. Histochemistry of the Adult Enamel
XIV. Enamel Structures in Some Mammals
XV. Concluding Remarks
References
Chapter 15. Ultrastructure of Enamel
I. Methods for the Investigation of the Ultrastructure of Mature Tooth Enamel
II. Units of Construction of Enamel
III. Ultrastructure of Human Enamel and That of Some Other Primates
IV. Conclusion
References
Section 4. PHYSICAL AND CHEMICAL ORGANIZATION OF THE TOOTH
Chapter 16. Crystalline Organization of Dental Mineral
I. Introduction
II. Crystallographic Investigations and Methods
III. The Mineral Phase in Dental Hard Tissues. Results of Investigations
IV. Conclusion
References
Chapter 17. Chemistry of the Mineral Phase of Dentine
I. Introduction
II. Preparation of Dentine for Analysis
III. Analytical Data on the Inorganic Composition of Dentine
IV. Nutrition and the Inorganic Composition of Dentine
V. Inorganic Composition of Dentine in Diseased and Other Abnormal States
VI. Nature of the Mineral Phase in Dentine
VII. Summary
References
Chapter 18. Chemistry of the Mineral Phase of Enamel
I. Introduction
II. Concentrations of Inorganic and Organic Material and Water
III. The Mineral Phase
IV. Surface Chemistry—Distribution in Depth of Various Constituents
V. Topical Treatment of the Enamel Surface
VI. Enamel Caries
VII. Concluding Remarks
References
Chapter 19. Chemical Organization of the Organic Matrix of Dentine
I. Introduction
II. The Organic Components of Dentine
III. Chemical Balance Sheet for Dentine
IV. Changes in the Organic Components of Dentine
V. Mineralization of the Organic Matrix
VI. Concluding Remarks
Addendum
References
Note Added in Proof
Chapter 20. Chemical Organization of the Organic Matrix of Enamel
I. Introduction
II. Immature Matrix
III. Maturation of the Organic Matrix
IV. Mature Matrix
V. Altered Enamel
VI. Concluding Remarks
References
Section 5. THE ORGANIZATION OF THE DENTAL SUPPORTING TISSUES
Chapter 21. Connective Tissue Components of the Periodontium
I. General Introduction
II. Development
III. Organization of the Definitive Supporting Tissues
IV. Pathology of the Supporting Tissues
V. Concluding Remarks
References
Chapter 22. The Structure and Physiology of the Dento-gingival Junction
I. The Histogenesis of the Dento-gingival Junction
II. The Nature of the Connection between Epithelium and Enamel
III. The Apical Shift of the Dento-gingival Junction
IV. The Structure and Chemistry of the Crevicular Epithelium
V. The Physiology of the Gingival Crevice
VI. Conclusion
References
Author Index
Subject Index

Citation preview

STRUCTURAL AN D CHEMICAL ORGANIZATION OF TEETH Volume II

CONTRIBUTORS Å. W . F.

B R U D E V O LD

J. E. H.

M.

A.-G. G.

B R A D F O RD

E A S T OE FULLMER G U S T A F S ON

G U S T A F S ON

J . - G.

HELMCKE

ERLING HARAL D S. L . R.

J O H A N S EN LÔE

R O W L ES

S Ô R E M A RK

M . V.

S T A CK

OTTO

R.

TRAUTZ

Structural and Chemical Organization of Teeth Edited by A. E. W. MILES LONDON HOSPITAL MEDICAL COLLEGE LONDON, ENGLAND

VOLUM E

ð

1967

ACADEMIC

PRESS

New York and London

C O P Y R I G HT

©

1 9 6 7,

BY A C A D E M I C PRESS I N C .

AL L RIGHTS RESERVED N O PART OF THI S BOOK MA Y BE REPRODUCED I N AN Y FORM BY

PHOTOSTAT,

MICROFILM ,

OR

BY

OTHER

MEANS,

W I T H O U T W R I T T EN PERMISSION FROM THE PUBLISHERS.

ACADEMI C PRESS INC. Il l N EW

F I F TH

AVENUE

Y O R K, Í . Y .

1 0 0 03

United Kingdom Edition Published by ACADEMI C PRESS INC. ( L O N D O N)

LTD.

BERKELEY SQUARE H O U S E, L O N D ON W .

1

Library of Congress Catalog Card Number: 65-26405

PRINTED BY THE ST CATHERINE PRESS LTD., BRUGES, BELGIUM

Science is organized knowledge HERBERT SPENCER

This page intentionally left blank

LIST OF CONTRIBUTORS Numbers in parenthese s indicate the pages on which the authors' contributions begin.

E. W. Bradford, Department of Dental Surgery, The Dental School, University of Bristol, Bristol, England (3) F. Brudevold, Forsyth Dental Center, Harvard School of Dental Medicine, Boston, Massachusetts (247) J. E. Eastoe, Department of Dental Science, Royal College of Surgeons, London, England (279) H.

M . Fullmer, National Institute of Dental Research, National Institutes of Health, Public Health Service, United States Department of Health, Education, and Welfare, Bethesda, Maryland (349)

A.-G. Gustafson, Department of Oral Histopathology, Faculty of Odontology, University of Lund (Tandlàkarhogskolan), Malmo, Sweden (75) G. Gustafson, Department of Oral Histopathology, Faculty of Odontology, University of Lund (Tandlàkarhogskolan), Malmo, Sweden (75)

J.-G. Helmcke, Forschungsgruppe fur Mikromorphologie im Fritz-Haber-Institut, Berlin, West Germany (135) Erling Johansen , The University of Rochester School of Medicine and Dentistry and Strong Memorial Hospital, Rochester, New York (35) Harald Loe, Department of Periodontology, The Royal Dental College, Aarhus, Denmark (415) S. L. Rowles, Department of Dental Pathology, Birmingham University, Birmingham, England (201) R. Soremark, Harvard School of Dental Boston, Massachusetts (247) M.

Medicine,

V. Stack, Dental Research Unit (Medical Research Council), The Dental School, University of Bristol, Bristol, England (317)

Otto R. Trautz, Department of Biochemistry, New York University College of Dentistry, and The Murry and Leonie Guggenheim Institute for Dental Research, New York, New York (165)

This page intentionally left blank

PREFACE which tend to thwart this predisposition are equally important for the total understandin g of the biology of organisms. This is especially true of processe s such as bacterial disease , which represen t the interplay with other biological systems. However, it is necessar y to set some limit s to the scope of a work of this kind and this book is concerned with " n o r m a l" processes . Nevertheless , consideration of disease has been admitted wherever disease processe s can be said to throw light upon normal structure. I n a work such as this, in which a single theme is broken down to a number of components , some overlap between chapters is essentia l for comprehensivenes s and for the expression of divergencies of view. Disagreemen t is a far more stimulating state than harmonious agreemen t and these divergencies should serve to provoke thought. The first section of this work, consisting of the first six chapters, is general and introductory. This applies particularly to Chapter 1 which incorporates a general account of dentitions. Apart from providing the background for the remainder of the book this general account of a very large subject wil l be useful to readers whose training has not been primarily in zoology. The remaining four sections deal systematically wit h organization of teeth in terms of definitive states and their development , and with organization at various levels of resolution: the microscopic or microanatomica l level, as revealed by the light microscope; the submicroscopi c structure; and the chemical and physical organization of the teeth and supporting structures. I t has been said that science is measurement . This is surely too narrow a definition, rather science is accurate observation, and this includes measure -

I n the past 20 years or so there has been a rapid, almost explosive increase in fundamenta l knowledge about teeth, their environments and development. This book arises out of the evident need for this knowledge, at present widely scattered , to be drawn together and correlated in a form that would render it readily accessible to advanced students and research workers in diverse fields. Quite apart from the sheer magnitude of the task, the collation of this knowledge and especially its proper evaluation, involving as it does a variety of disciplines, is an undertaking which required the integrated efforts of a team of authors chosen from workers actively engaged in investigation in the fields covered by the various chapters. The treatment has been as comprehensiv e as possible in both breadth and depth. The aim has been to adopt an overall view of teeth in vertebrates as a whole. Nevertheless , often because wider information is not available, in some areas the emphasis lies on the mammalian, or even more narrowly on the human state. The intention has been to emphasize the dynamic aspects of each structure, to trace wherever possible its history— phylogenetic and ontogenetic. I n depth, the aim has been to embrace all aspects of structure and chemistry, at all levels of organization, ranging from that resolved by the light microscope down to the molecular state. The relatively new disciplines of histochemistry and molecular biology furnish bridges between chemistry and what is conventionally understood by the term structure, and these have received the emphasis warranted by advances on these fronts. The biological forces whereby an organism has a powerful predisposition to pass its lif e cycle in a " n o r m a l" state and the biological forces or circumstance s ix

χ

PREFACE

ment, but to this might be added, as quite inseparable from the matter of accurate observation, accurate communication to others of the observation and the ideas that stem from it. Without clear and unambiguous communication, however important and accurate the observation, there is no advance in the sum total of knowledge: the authors who have collaborated in this effort have performed a valuable service to their colleagues . I n performing the editorial functions I have been aided in many ways by a number of people. In particular I wish to mention, Dr. D. F. G. Poole, Dr. A. Boyde and Dr. J. P. Waterhouse . To

Dr. Waterhouse I am in addition grateful for his preparation of the index—an important feature of a work which is largely a work of reference. In the early stages of planning the book I had the benefit of the collaboration of Dr. R. C. Greulich for which I am very grateful. For much sustained and patient assistance , quite apart from typing of manuscripts and other secretarial functions, I am very much indebted to my secretary, Miss Y. M . Davis. November 1966

A . E. W. MILE S

CONTENTS vii

List of Contributors Preface

ix

Contents of Volume I

xv

Section 3.

DEFINITIVE STRUCTURAL ORGANIZATION OF T H E T O O T H

Chapter 12.

Microanatomy and Histochemistry of Dentine

E. W . BRADFORD

I. Introduction. Classification of Dentines II . Osteodentine , Distribution and Structure III . Vasodentine , Distribution and Structure IV . Plicidentine, Distribution and Structure V. Orthodentine VI . Nonmammalian Orthodentine VII . Mammalian Orthodentine VIII . Summary Reference s

Chapter 13.

3 5 7 8 9 10 12 31 32

Ultrastructure of Dentine

E R L I N G JOHANSEN

I. II . III . IV . V.

Introduction Structural Characteristic s The Relation of Structure to Some Aspects of Physiology Pathologic Alterations Concluding Remarks Reference s

Chapter 14.

35 37 63 65 69 72

Microanatomy and Histochemistry of Enamel

G . GUSTAFSON AN D A . - G . GUSTAFSON

I. II . III .

Introduction Methods of Investigation The Prisms

^ ^6 2

xi

xii

CONTENTS

IV . V. VI . VII . VIII . IX . X. XL XII . XIII . XIV . XV .

Retzius Lines Hunter-Schrege r Lines Enamel Tufts and Lamellae Enamel Spindles Enamel Cuticle Surface Layer of Enamel Developmenta l Hypomineralizations Innermost Part of the Enamel Similarity of Structures in Contralatera l Teeth Histochemistry of the Adult Enamel Enamel Structures in Some Mammals Concluding Remarks Reference s

Chapter 15.

89 100 104 110 Il l 112 116 123 124 124 126 128 129

Ultrastructure of Enamel

J . - G. HELMCK E

I. II . III . IV .

\.

Methods for the Investigation of the Ultrastructure of Mature Tooth Enamel Units of Construction of Enamel Ultrastructure of Human Enamel and That of Some Other Primates Conclusion Reference s

Section 4.

Chapter 16. O T TO R.

I. II . III . IV .

S. L .

I. II . III . IV . V. VI . VII .

PHYSICAL A N D CHEMICAL ORGANIZATION OF THE TOOTH

Crystalline Organization of Dental Mineral T R A U TZ

Introduction Crystallographic Investigations and Methods The Mineral Phase in Dental Hard Tissues. Results of Investigations Conclusion Reference s

Chapter 17.

135 140 145 161 162

165 166 188 197 197

Chemistry of the Mineral Phase of Dentine

ROWLES

Introduction Preparation of Dentine for Analysis Analytical Data on the Inorganic Composition of Dentine Nutrition and the Inorganic Composition of Dentine Inorganic Composition of Dentine in Disease d and Other Abnormal States Nature of the Mineral Phase in Dentine Summary Reference s

201 202 206 222 230 233 237 238

CONTENTS Chapter 18.

xiii

Chemistry of the Mineral Phase of Enamel

F . BRUDEVOLD AN D R . SÔREMARK

I. II . III . IV . V. VI . VII .

Introduction Concentration s of Inorganic and Organic Material and Water The Mineral Phase Surface Chemistry—Distribution in Depth of Various Constituents Topical Treatment of the Enamel Surface Enamel Caries Concluding Remarks Reference s

Chapter 19.

247 248 249 251 265 269 271 272

Chemical Organization of the Organic Matrix of Dentine

J. E. EASTOE

I. II . III . IV . V. VI .

Introduction The Organic Components of Dentine Chemical Balance Sheet for Dentine Changes in the Organic Components of Dentine Mineralization of the Organic Matrix Concluding Remarks Addendum Reference s Note Added in Proof

Chapter 20. M.

I. II . III . IV . V. VI .

279 283 300 301 303 309 310 312 315

Chemical Organization of the Organic Matrix of Enamel

V . STACK

Introduction Immature Matrix Maturation of the Organic Matrix Mature Matrix Altered Enamel Concluding Remarks Reference s

Section 5. Chapter 2 1.

317 318 324 327 339 341 342

THE ORGANIZATION OF T H E DENTAL SUPPORTING T I S S U E S

Connective Tissue Components of the Periodontium

H. M . FULLME R

I. II . III . IV . V.

General Introduction Development Organization of the Definitive Supporting Tissues Pathology of the Supporting Tissues Concluding Remarks Reference s

349 349 358 401 402 404

CONTENTS

xiv Chapter 22.

The Structure and Physiology of the Dento-gingival Junction

H A R A L D L ÔE

I. II . III . IV . V. VI .

The Histogenesi s of the Dento-gingival Junction The Nature of the Connection between Epithelium and Enamel The Apical Shift of the Dento-gingival Junction The Structure and Chemistry of the Crevicular Epithelium The Physiology of the Gingival Crevice Conclusion Reference s

5

417 2

437 6 1

^2

Author Index

^

Subject Index

3

CONTENTS OF VOLUM E I Section 1.

Chapter 6.

General Organization

R. W .

Chapter 1. The History and General Organization of Dentitions A . E. W.

MILE S AN D D. F. G.

Section 2. Structural Organization of the Tooth During Development

POOLE

Chapter 2. Phylogeny of Tooth Tissues: Evolution of Some Calcified Tissues in Early Vertebrates T.

Chapter 7. The Microanatomy mistry of Dentinogenesi s

and

Histoche-

Í . Â . B . SYMONS

0RVIG

Chapter 3. Phylogeny of Tooth Tissues: Enameloi d and Enamel in Recent Vertebrates , with a Note on the History of Cementum D.

Innervation of Dental Tissues

FEARNHEAD

F. G.

Chapter 8.

Ultrastructure of Dentinogenesi s

SHOSABURO

TAKUM A

Chapter 9. Microanatomy and Histochemistry of Amelogenesis

POOLE

E D W A RD J. R E I TH AN D EARL O.

Chapter 4. Fundamenta l Aspects of Tooth Morphogenesis W. A. G A U NT AND A. E. W.

Chapter 10. ROBERT M .

MILE S

Chapter 11.

Ultrastructure of Amelogenesis F R A NK AN D J O HN

J. H.

R.

Author Index—Subject Index

ROCKERT

xv

NALBANDPA N

Maturation of Enamel

Chapter 5. Vascular Supply of Dental Tissues, Including Lymphatics L. DE C. H. SAUNDERS AN D H. Ô. E.

BUTCHER

ALLA N

This page intentionally left blank

STRUCTURAL AN D CHEMICAL ORGANIZATION OF TEETH Volume II

2

This page intentionally left blank

SECTION

3

Definitive Structural Organization of the Tooth

This page intentionally left blank

C H A P T E R

12

MICROANATOM Y AN D HISTOCHEMISTRY E. W .

OF DENTINE

BRADFORD

I. Introduction. Classification of Dentines

3

II . Osteodentine , Distribution and Structure

5

III . Vasodentine , Distribution and Structure

7

IV . Plicidentine, Distribution and Structure

8

V. Orthodentine

9

VI . Nonmammalian Orthodentine

10

VII . Mammalian Orthodentine A . Primary Curvatures B. Secondar y Curvatures C. Controversy on Structure of the Dentine D. Present Concept E. The Submicroscopi c Structure of Dentine F. Changes in Dentine G. Species Differences

12 13 14 14 17 24 26 30

VIII . Summary

31

Reference s

32

I. INTRODUCTION. CLASSIFICATION OF DENTINES A study of published work would suggest a general definition of dentine as a tissue which is situated superficially in the body and forms the basis of the teeth of vertebrates and of the exoskeletons of the elasmobranch s and some primitive agnathans . It is of mesoderma l origin, develops in a centripetal direction from a dermal papilla or dental pulp and in the mature state is usually mineralized (see Volume I, Chapters 2 and 3). Such a definition, though perfectly satisfactory

if teeth only are considered , is found to be unsatisfactory in other contexts: when considering for instance the range of tissues in the exoskeleton s of primitive vertebrates , some of which closely resemble dentine as found in teeth. It is known that these dermal plates form the basis of the membrane bones of the skull of the more specialized vertebrates, so at least some of the tissues of these dermal plates should be considered to be primitive bone. I t has become fashionable therefore to conceive 3

4

Ε.

W. B R A D F O R D

of a series of tissues which are intermediate between bone and dentine, and to classify dentine according to whether it does or does not resemble bone. A t different times during the last 30 years, various workers (Tretjakoff, 1926; Thomasett, 1928; and 0rvig, 1951) have shown that some of the borderline tissues previously considered to be dentine are so similar, both structurally and developmentally , to bone that they should be classified as bone. Thus Tretjakoff found that the basal layers of the exoskeleton of certain teleosts contain canals which house, not processe s of the odontoblast cells, but fibres of Sharpey and the tissue is therefore not dentine. A similar argument can be put forward when considering the basal layers of the teeth and scales of the elasmobranch s where fibres of a connective tissue nature are inserted into a mineralized tissue to form an attachment for the tooth (Fig. 1) (Bradford, 1954). Tretjakoff (1926) did not define explicitly what he meant by dentine but implied that it is characterize d by tubules which house cell processes . 0rvig (1951) found that in teeth which consist of several dentinal osteons (such as the eagle rays,

Myliobatis, cementing

and guitar fishes, Rhinobatis) the substanc e between the osteons is Table 1 DENTINE

A. Mantle type: i.e., the tissue forming an outer layer to the tooth. Vitrodentine (Rose, 1898), mantle dentine (Weidenreich, 1925), durodentine (Schmidt and Keil, 1958), fibrodentine (Thomasett, 1928), palliai dentine (Lison, 1941), mesoderma l enamel (Kvam, 1946). These tissues have been discusse d at some length in Chapters 2 and 3 (Volume I). B. Circumpulpal type: Tomes Osteodentin e

Thomasett Osteodentine s a. osteodentin e b. pseudodentin e c. syndentine

0rvig Osteodentine s a. normal b. special

Orthodentine

Dentines with an open pulp chamber a. orthodentine b. vasodentine

Orthodentines a. orthodentine b. vasodentine

Vasodentine Plicidentine

M

VV

IN Fig. 1. Longitudinal section of the base of a tooth of Raia bat is, demineralized , stained with Masson's trichrome to show the insertion of the fibres of the fibrous ligament into the mineralized tooth tissue. E, Epithelial reflection; F, attaching fibres; M , mineralized tooth tissue, χ320.

12. M I C R O A N A T O M Y

AND

HISTOCHEMISTRY

Ô

OF

DENTINE

5

on the one hand and orthodentine on the other wil l be retained in this discussion as it wil l be seen to be a valid and useful division.

II. OSTEODENTINE, DISTRIBUTION A N D STRUCTURE

Ñ

T

*

Fig. 2, Ground transverse section of a dentinal osteon of Rhinobatis sp. showing the general arrangemen t of the system. P, Central pulp canal; T, highly branched radiating tubules; //, incremental lines of deposition of the matrix; /, intersystem tissue, χ 220.

structurally different from dentine in that it contains lacunae which he presumed to be occupied in vivo by osteocytes (Fig. 2). He therefore categorized it as bone. Thomasett (1928) recognized the validity of dividing dentines into three groups, the surface layer of fibrodentine, osteoid resembling bone and nonosteoid. The first type might be called dentine of the mantle type (Weidenreich, 1925) and has been adequately discusse d in Chapters 2 and 3 (Volume I), the second type might be termed osteodentin e since it bears more resemblanc e to bone than to mammalian dentine and the third group may be termed orthodentine and includes those tissues which resemble mammalian dentine more closely than bone. The division of the circumpulpal dentines (Weidenreich, 1925) into osteodentin e

These tissues can be defined only in terms of their histological appearance . Each tooth is composed of one or more denticles or dentinal osteons. Individual denticles develop on the surface of a dental papilla which remains as a central pulp chamber. Initiall y there is laid down a regular dense surface layer which could be the tooth surface in a tooth consisting of a single dentinal osteon, or interosteon tissue in a tooth composed of many dentinal osteons. A hyaline mineralized matrix is deposited on this framework in an incremental manner so that the completed matrix exhibits incremental lines of growth. This matrix is penetrate d by radiating canals which are wide at their points of origin, taper rapidly and divide dichotomously (Fig. 2). A typical example of osteodentin e is the simple monoconodont tooth of the pike (Esox lucius). A n examination of ground longitudinal sections of the teeth of Esox lucius shows that each consists of a single denticle composed of three elements (Fig. 3), a central pulp cavity, a layer containing a network of canals (osteodentine ) and a dense outer layer. The dense outer layer is thickest over the tip of the tooth becoming gradually reduced in thickness towards the base of the tooth. This outer layer is penetrated to some distance by canaliculi which arise from the surface canals of the canalicular layer. There is no sharp line of demarcation between this dense outer layer and the rest of the tooth in ground sections but in demineralized sections there are obvious differences in structure (Fig. 4). The differentiation of a dense outer layer from the remainder of the tooth in ground sections is obvious only in fairly thick sections. In thin longitudinal ground sections both portions of the tooth

6

Ε.

W.

Fig. 3. Ground longitudinal section close to the base of a tooth of Esox lucius unstained. P, Central pulp cavity ; CD, circumpulpal dentine (osteodentine) ; MD, dense outer layer of mantle type, χ 142.

BRADFORD

Fig. 4. Demineralized transverse section of Esox lucius showing variations in the structure of P9 Central pulp cavity; CD, circumpulpal dentine tine); MD, outer layer of mantle type. Masson's

tooth of the tissue. (osteoden trichrome.

X 1 4 2.

are completely permeate d by the mounting medium so that in transmitted light the tooth presents a uniform appearanc e due to the similar refractive index of the parts. In polarized light also such sections are uniform in appearance . The outer layer is composed of dentine of the mantle type and is the concern of Chapter 3 (Volume I). The canals in osteodentin e are very irregular both in cross section and in the direction they pursue. They anastomos e frequently with adjacent canals and there are marked dilatations at points of intersection. The incremental pattern of growth shows as regular incremental lines concentric wit h boundaries of the central pulp cavity and not of the individual canals. The canals and canaliculi are occupied by cellular tissue, not merely prolongations of cells as wil l be met with in orthodentine but whole cell bodies. The nuclei of the cells in the canaliculi are long and slender,

are contained within the lumen of the canal and usually coincide with junction points of two or more branches . Each branch is occupied by a long slender cell process. The canaliculi, canals and soft tissue spaces of the pulp are occupied by a tissue which is indistinguishable from the tissue found in the marrow spaces of the bone. This description agrees with the general conceptions of osteodentine as set out by Thomasett and 0rvig. A glance at Table 1 shows the general measure of agreemen t which exists on the classification of various types of dentine. There are, however, one or two points of disagreement , a discussion of which wil l help to emphasize the differences between osteodentine and orthodentine. The first is the assignmen t of vasodentine to one group or the other, and the second the position of the group called plicidentine by Tomes (1898).

12. M I C R O A N A T O M Y

AND

HISTOCHEMISTRY

III. VASODENTINE, DISTRIBUTION A N D STRUCTURE Vasodentine is so called because it is penetrate d by blood vessels. It is found in a few members of the Gadidae such as the haddock {Gadus aeglifinius) and cod {Gadus callarias) and in the related Merluccidae such as the hake (Merluccius merluccius). Ground longitudinal sections of these teeth show them to be composed of four elements (Fig. 5): a central pulp cavity, a hyaline matrix penetrated by a regular system of canals, a compact outer layer covering the whole tooth and a highly mineralized tooth tip peripheral to the compact layer. The two outer layers are of the mantle type whereas the hyaline matrix is of circumpulpal type and has been called vasodentine . Demineralized sections show that the tissue is permeate d by canals

Fig. 5. Ground longitudinal section of tooth of Merluccius merluccius, embedde d in methyl methacrylate , unstained. P, Central pulp cavity; CD, circumpulpal dentine (vasodentine); MD, outer layer; He, hypermineralize d tip. χ 90.

OF

DENTINE

7

which contain blood vessels and other soft tissue cells. The lateral branches of the main canals are not large enough to accommodat e blood vessels and contain only mesenchym e cells and their processes . Closer examination shows that mesenchyme cells are associate d with the blood vessels in the large canals, especially at points where the canals branch. Though vasodentine of this type is a constant feature of the teeth of the Gadidae there is a marked variation in its degree of vascularization . Discussion of this tissue has tended to centre around Merluccius merluccius, which shows the greatest and most regular degree of vascularity. Where vascularity is not marked, as in Gadus callarius, there is a marked incremental pattern in the hyaline matrix. A comparison of Figs. 3 and 5 shows that in ground sections the only obvious difference between the osteodentin e of Esox lucius and the vasodentine of Merluccius merluccius is the difference in the pattern formed by the canals within the hyaline matrix. The pulpal surface of this hyaline matrix is lined by what appear to be large columnar cells similar to mammalian odontoblasts , and this has been adduced as evidence for including this type of dentine with mammalian dentine. Mummery (1924, p. 283), however, considered these columnar shaped bodies to be fibres and not cells and thought that the formative cells of this tissue were small round osteoblas t type cells situated between the columnar bodies. Whether or not this theory is correct, columnar bodies also line the tissue spaces of the bone of attachment and the tissue spaces of the surrounding jaw bone. Fischer (1938) and Kohlenberger (1940) maintain that the absence of odontoblast processe s is secondar y because processe s of these cells are present during the formative period but disappea r at a later stage. Kerr (1958), however, states that odontoblast processe s are never present even during development. From the histological point of view therefore there is reason for considering vasodentine to be one more modification of osteodentine .

Ε.

8

W.

IV. PLICIDENTINE, DISTRIBUTION AND STRUCTURE Tomes (1898) defined plicidentine as dentine in which the tubules radiate perpendicula r to the boundary of a highly convoluted pulp: "I t is merely dentine with its pulp folded up and wrinkled into a greater or lesser degree of complexity". He noted that the degree of complexity varies from simple lateral folding, as in the teeth of Varanidae (Fig. 6), to complicated lateral folding, as in the tooth bases of Labyrinthodontia, and that the convolutions could have an axis parallel to the long axis of the tooth, in which case "such a tooth might be said to be built up of a series of small parallel fused denticles" (Fig. 7.). 0rvig and Thomasett (Table 1) do not agree that it is possible to distinguish on histological grounds

BRADFORD

a tissue as defined by Tomes. In any case, the peculiar distribution of this tissue in vertebrates suggests that a careful consideration of each particular example is necessary . Tomes listed vertebrates in which plicidentine is found (see accompanyin g tabulation). Class Elasmobranch s Pisces Amphibia Reptilia Mammalia

Vertically folded Myliobatis Pristis

Laterally folded Zygobatis Lepidosteus Labyrinthodon Varanus

Orycteropus

The type of dentine found in Myliobatis, Zygobatis and Lepidosteus correspond s in every detail with the description already given for osteodentine .

Lc

Ñ

Fig. 6. Ground transverse section of the crown of the tooth of Varanus sp. showing lateral convolution of the dentine, unstained. Lc, Laterally convoluted orthodentine; P, pulp cavity with lateral convolutions, χ 85.

Fig. 7. Ground transverse section of the base of a tooth of Varanus sp. showing vertical convolution of the dentine unstained. Ve, Vertically convoluted orthodentine; P, pulp cavities ; /, intersystem tissue ; L, lacunae, χ 180.

12. M I C R O A N A T O M Y

AND

HISTOCHEMISTRY

OF

DENTINE

9

Ñ

τ

Fig. 8. Ground transverse section of a rostral tooth of Pristis microdon showing the appearanc e of fused dentinal osteons, unstained. P, Pulp cavity; T, highly branched radiating tubules; /, intersystem tissue, χ 198. (From Bradford, 1957.)

I n these fishes the teeth are composed of many dentinal osteons. The rostral teeth of the sawfish Pristis microdon consist of a tissue unlike any other so far described (Bradford, 1957). Ground sections suggest superficially that they are composed of a series of fused dentinal osteons with canaliculi radiating into a hyaline matrix (Fig. 8.). Closer examination of both ground and decalcified sections shows that the matrix, far from being hyaline and almost structureless , is composed of rods of tissue running the whole length of the tooth (Fig. 9.). Further work needs to be carried out on fresh specimens to compare the oral teeth with the rostral ones and to study the mode of formation of the rostral teeth. For the present, this tissue should not be considered to be a form of circumpulpal dentine but to have more in common with the mantle type of dentine or the enamels of higher vertebrates .

Fig. 9. A portion of the same section shown in Fig. 8 at higher magnification. Note transverse sections of the rods of which the tissue is composed , χ 1500. (From Bradford, 1957.)

Until more information is available on the structure of the teeth of Orycteropus (aardvark) than can be obtained from the occasiona l ground transverse sections upon which the descriptions in the literature are based (Adloff , 1930; Anthony, 1934), it is not possible to classify the dentine of this mammal with any confidence. The remaining examples of plicidentine wil l be discusse d as modifications of orthodentine.

V. ORTHODENTINE Structurally orthodentine is a unique tissue and, providing that well-fixed specimens of tissue are available, there is no difficult y in its identification and differentiation from other dentines. It may be defined as a mineralized collagenous tissue

Ε.

10

W.

surrounding a mesoderma l papilla or dental pulp. It s formative cells remain within the dental pulp but, at some stage in the developmen t of the tissue, these cells posses s long dentinal processe s which penetrate almost the whole thickness of the dentine. Orthodentine which is in structure fundamentally the same as mammalian orthodentine occurs in some members of all extant classes of vertebrates . If, in accord with the view of 0rvig, it occurs in placoderms, it is probable that this tissue is of equal antiquity to bone and osteodentin e and did not evolve from one of the types of osteodentine , as has been frequently suggested . What is more probable is that orthodentine, osteodentin e and bone are all modifications of a common ancestra l mineralized tissue. Orthodentine as defined above has been found in representative s of the following classes :

BRADFORD

1. Primitive, with irregularly sized tubules and much branching. 2. Intermediate, with regular parallel tubules running the full thickness of the tissue. 3. Advanced, in which each tubule is surrounded by a hypercalcified layer of peritubular dentine.

VI. NONMAMMALIAN ORTHODENTINE The primitive type has been found in members of class Chondrichthyes , most easily studied in the teeth of the thornback ray, Raia clavata, but

Chondrichthyes Osteichthyes Amphibia Reptilia Mammalia Raia bat is, Raia clavata Labrus maculatus Rana temporaria Lacertilia, Crocodilia, Colubridae Al l dentate mammals with the exception of Orycteropus

I t is certain that orthodentine occurs in many other members of Chondrichthyes and Osteichthyes but the distribution has not yet been comprehensivel y charted. During the evolution of the higher mammals, orthodentine has undergone very few changes in structure. Nevertheles s it can be classified into three types:

Fig. 10. Ground longitudinal section of a tooth of Raia clavata, unstained. P, Central pulp cavity; CD, circumpulpal orthodentine; MD, outer layer; S, structureless tissue, χ 80.

12. M I C R O A N A T O M Y

AND

H I S T O C H E M I S T RY

is apparently present in the teeth, scales and spines of other members of the elasmobranchs . The dentine of Raia clavata consists of a hyaline matrix penetrate d by a system of parallel tubules which run from the pulpal surface to the under surface of the enameloid with which the tooth is capped (Fig. 10). Some of the tubules cross the junction between these two tissues to pass for a short distance into the enameloid. In the mature tooth, however, the pulpal ends of the tubules are sealed off by a thick layer of structureles s tissue. In longitudinal sections the tubules appear to be very regular, but closer investigation, especially of transverse sections, shows that they branch repeatedly, have an irregular outline and vary considerably in size (Fig. 11). The rapidity with which sections of this tissue can be ground suggests that it is not very highly mineralized. The individual tubules are not surrounded by a hypermineralize d layer of tissue.

Fig. 11. Ground transverse section of the orthodentine of Raia clavata, unstained. T, Tubules of irregular cross section; Br, branches . X1500.

OF

DENTINE

11

The intermediate type of orthodentine has been found in Osteichthyes (the wrasse, Labrus maculatus), Amphibia (Rana temporaria), Reptilia (Crocodilus niloticus) and some of the more primitiv e mammals. In the teeth of Labrus maculatus the orthodentine forms the major portion of the tooth and consists of a hyaline matrix which is penetrated by a series of parallel tubules (Fig. 12). These tubules run from the pulpal surface to the enameloid and some cross the boundary and run for some distance into the enameloid. A t the boundary between the two tissues the tubules form a system of fine branches , but there appears to be no anastomosi s between the branches . The tubules are about 3 μ in diameter, are regular in size and cross section and branch infrequently, except at the enameloid-dentin e border (Fig. 13). I n the mature tooth the central ends of the tubules may be sealed off by the deposition of osteodentin e withi n the central pulp cavity. I n decalcified sections of the teeth of the frog {Rana temporaria) the lining of the dentinal tubules stains differently from the dentine matrix. The formative cells of the tissue are columnar or spindle-shape d cells each of which sends a long dentinal process running into a dentinal tubule (Fig. 14). The intermediate type of orthodentine exhibits no change in tubule size as between ground and decalcified sections (Figs. 15 and 16). This is due to the absence of any hypermineralize d area around the tubules. The dentine, whilst being more highly mineralized than the primitive type, is still not so highly mineralized as the advanced type. In demineralized sections the collagen fibre bundles are easily differentiated from the cementing substance between the bundles. In reptiles in which the teeth are gomphose d the orthodentine is covered by a layer of cementum into which are attached the fibres of the periodontal membrane. A more complicated arrangemen t occurs in, for example, snakes and lizards where the teeth are rigidly attached to the jaws by ankylosis. In the varanid lizards the single shell of orthodentine covering the dental pulp is convoi-

12

Ε.

W.

BRADFORD

Fig. 12. Ground longitudinal section of a tooth of Labrus maculatus, unstained. OrD, Orthodentine; MD, outer layer; T, dentinal tubules, χ 160.

Fig. 13. Ground transverse section of the orthodentine of Labrus maculât us, unstained, χ 1000.

uted in a plane transverse to the long axis of the tooth and, nearer the base of attachment , is convoluted in a plane parallel to the long axis (Figs. 6 and 7). The external morphology is associate d with the labyrinthine structure of the plicidentine in the varanids which is similar to that of certain extinct amphibians and crossopterygians . I t cannot in any way be said to resemble the regular osteodentin e discusse d earlier.

VII. MAMMALIAN ORTHODENTINE The orthodentines found in Mammalia appear when examined in longitudinal section to be essentially similar in structure. A study of the tissue in sections ground transverse to the path of the tubules, however, shows that there may be considerable variations in structure from one species to another. These differences take two forms: (1) the division into those vertebrates which

Fig. 14. Longitudinal section of a tooth of Rana temporaria, demineralized . T, Dentinal tubule. O, odontoblast of the pulp. Masson's trichrome. x 560.

12. M I C R O A N A T O M Y

AND

HISTOCHEMISTRY

OF

DENTINE

13

Fig. 16. Transverse section of orthodentine of Crocodilus niloticus. Masson's trichrome. χ 1530.

Fig. 15. Ground transverse section of the orthodentine of Crocodilus niloticus, unstained. Any hypermineralize d layer which may be present around the individual dentinal tubule is difficult to differentiate from optical artifacts. A comparison with Fig. 16 shows that demineralization does not produce an increase in the size of the tubule, χ 1530.

appear to have the intermediate type of orthodentine and those which have the more advanced type; (2) the variation in the relative proportions of the two types of dentine matrix, intertubular and peritubular (see below). A s an introduction to a discussion of mammalian dentine, human dentine wil l be described as the typical example about which most knowledge is available. H u m an dentine is typified by a hard mineralized matrix surrounding a central pulp cavity. The matrix is permeate d by tubules which run from this central pulp cavity to the enameldentine junction in the tooth crown, and almost to the dentine-cementu m junction in the root.

The tubules are usually described as running parallel to each other but, though this is approximately true, the distance between the centres of adjacent tubules may be as great as 15 ì at the periphery and as littl e as 6 μ at the pulpal surface. A.

PRIMARY

CURVATURES

The tubules of orthodentine describe two curvatures; the larger or primary curvature may best be observed in the cervical region of ground longitudinal sections of the tooth where each tubule describes a shallow open double curve so that the outermost part of the tubule presents a convexity towards the crown of the tooth whereas the innermost part near the pulp presents a concavity towards the crown (Fig. 17). This, however, is only the course of the majority of the tubules. The tubules which rise from the tips of the pulp cornua to the tips of the dentine cusps are straight or almost straight, as also are the short tubules in the apical one-third of the root.

14

Ε.

W.

BRADFORD

begins only when the root of the tooth begins to be formed.^A more likely explanation is that the pulp must necessaril y retreat from the surface on which dentine is being deposited; since deposition occurs on the roof of the pulp cavity in addition to the walls, the pulp and odontoblasts must retreat apically. The distance which an odontoblast on the lateral wall moves apically is in fact less than the thickness of dentine deposited on the roof of the pulp cavity. The dentine of the apical third of the root, in which there are no primary curvatures, is produced when there is littl e increment of dentine occurring on the roof of the coronal pulp cavity. Where abrupt changes occur in the direction of tubules, all tubules are affected and the resultant line which appears in the dentine is known as a Schreger line of dentine (see section VII , C, 6, a, p. 2 1 ). B. SECONDARY CURVATURES

Fig. 17. Ground longitudinal section of a human tooth t of the primary curvatures of the showing the arrangemen dentinal tubules. E, enamel; P, pulp. The incisai tip of the crown is to the right, χ 50.

From the cervix towards the apex the tubules in the root become less and less curved (Bradford, 1 9 5 1 ). This suggests that during the developmen t of the tissue there is an overall movement of the odontoblast cells in an apical direction. The movement of cells becomes smaller towards the apex with littl e or no movement in the apical third of the root. Two possible explanations might be advanced for this movement. If the base of the tooth follicl e is considered as a fixed point then the whole tooth moves away from this point during the deposition of the calcified tissues. If the rate of dentine formation exceeds that of new pulp tissue then the dentine would move away from the pulp. Out of accord with this explanation, however, is the fact that primary curvatures are most marked in the dentine of the crown. A t the time the crown is being formed the follicl e appears to enlarge approximately equally in all directions ; elongation

The so-called secondar y curvatures, usually described as of corkscrew form, are not really curvatures of the dentinal tubules. The spiral curvature is pursued by the odontoblast process withi n the original dentinal tubule. This wil l be dealt with in greater detail later (p. 2 0 ). This spiral course of the odontoblast process is an extremely close one with a wavelength of approximately 12 μ so that within the thickness of a section ground at right angles to the tubules more than one complete phase of the spiral occurs, as can be detected by focussing up and down through the thickness of the section. C . CONTROVERSY ON STRUCTURE OF THE DENTINE

The details of dentine structure differ according to whether one is examining dentine near the external surface of crown or root or close to the pulp. There is, of course, a basic pattern and most of the differences are merely variations of one or other aspects of this pattern or are due to progressive changes that occur in the mature tissue (Figs. 18 and 1 9) Microscopic examination of dentine is rendered difficul t because the tissue is highly mineralized

12. M I C R O A N A T O M Y

AND

HISTOCHEMISTRY

OF

DENTINE

15

Fig. 18. Ground transverse section of human dentine. Note the eccentric positions of the odontoblast processe s at a single stage of their spiral path. OPr, Odontoblast process ; PtD, peritubular dentine ; ItD, intertubular dentine. Masson's light green, χ 1500. (From Bradford, 1950b.)

Fig. 19. Transverse section of human dentine demineralized in 5% nitric acid and stained in haematoxylin and eosin. Tomes fibre (TF) is seen to be separate d from the intertubular dentine (ItD) by a space, χ 1200. (From Bradford, 1955.)

and must therefore be observed in ground or demineralized sections. In ground sections the dentine is translucent and difficult to stain so that the production of optical artifacts or misinterpretation of optical appearance s is easy. The unmineralized structures, namely odontoblast processes , present in the tissue are close in size to the resolving power of the optical microscopes that are in general use, i.e. about 0.5 μ. Observation of the tubules or odontoblast processe s in true longitudinal and transverse section is extremely difficult ; indeed the actual path and configuration of a single dentinal tubule from the pulp to the enameldentine junction has not yet been observed and recorded photographically. Demineralization of the dentine and serial sectioning might be expected to overcome many of these difficulties but, as wil l be seen later, this technique introduces as many problems as it solves because of the occurrence of

considerable shrinkage and the destruction of various components of the matrix. Many papers have been written on the subject of the structure of mammalian and especially human dentine. Some are concerned with observations and many with theories. The reader who wishes to trace the history of this in detail is referred to the bibliographies in Fleischman (1905), Hanazawa (1917) and von Saal (1930). Many of these papers add littl e to the sum total of knowledge; so for the more general reader and for those merely concerned with fact, the research into the study of human dentine can be divided usefully into three phases . The first phase lasted from Owen to John Tomes and includes the names of such famous microscopists as Kôlliker (1889), Neumann (1863) and von Ebner (1902). During this period the main microscopic features of the tissue were noted and

3

16

Ε. W.

named; the contour lines of Owen, the incremental lines of von Ebner, Tomes fibres, etc. The dentine was observed by these early workers almost exclusively in the calcified state, in small pieces or in ground sections. It was observed that in sections in which the tubules were cut longitudinally the dark lines of the tubules had highly translucent peripheral zones which it was thought could be an optical artifact. In sections in which the tubules Neumann (1863)

R o m e r (1905) Sheath of Neumann • T o m e s fibre A r e a of low mineralization-

F l e i s c h m a n (1905) Hanazawa (1917) (decalcified sections)

F l e i s c h m a n (1905) Hanazawa (1917) (ground s e c t i o n s ) Sheath of Neumann T o m e s fibre Contraction s p a c e

Walkhoff (1921)

m von Saal (1930)

Sheath of Neumann Radial f i b r e s T o m e s fibre Perifibrillar space Intermediate zone

P r e s e n t concept Outer h y p o m i n e r a l i z e d l a y e r (line of junction) P e r i t u b u l a r dentine Inner h y p o m i n e r a l i z e d l a y e r Dentinal p r o c e s s of t h e odontoblast Radial f i l a m e n t s Intertubular dentine

Fig. 20. Diagram to illustrate the differences in concept of the structure of human orthodentine. The term Tomes fibre here refers to the structure remaining in the tubule after demineralization .

BRADFORD

were cut transversely , however, each tubule appeared to be surrounded by a frequently eccentric cuff or halo. These cuffs were investigated by Neumann (1863) and thus came to be known as the sheaths of Neumann (Fig. 20). A t this time therewere two dominant problems in respect of dentine; firstly, the structural basis for the sensitivity of the tissue, and secondly, the nature of the tissue of which the sheath of Neumann is composed (Figs. 18 and 20). Among those who worked on these problems in the subsequen t years are Fleischman (1905), Walkhoff (1921), Hanazawa (1917) and Churchill (1932) but no decisive answers emerged. Improvements in the technique of demineralization and embedding tissues for sectioning seemed very likely to increase the chance of successfu l study of these problems. However, this was not the case because new types of artifact were substituted for those associate d with the study of the relatively thick ground section. Fleischman (1905) published the results of a series of observations whereby he showed conclusively that in demineralized sections a Tomes fibre 1-2 μ in diameter was contained withi n a dentinal tubule 5 μ in diameter (Fig. 19). He found no evidence of any tissue between the fibre and the dentine matrix. Therefore he concluded that the sheaths of N e u m a nn seen in ground sections are an artifact probably produced optically due to the observation of the opaque tubule in a thick section (50-100 μ) of a translucent material. He suggeste d that in vivo the fibres of Tomes are approximately 5 μ in diameter and completely fil l the tubule and that demineralization and embedding caused the fibre to shrink to 1-2 μ. He found that the tubule wall in demineralized sections stained more heavily than the rest of the dentine and regarded this stained layer as the sheath of Neumann. This became the generally accepted view although it was challenged by a number of workers. Walkhoff (1921) showed that in some ground sections of dentine it was possible to stain differentially the cuff as seen by Neumann. However, this was not considered significant by other investigators.

12. M I C R O A N A T O M Y

AND

HISTOCHEMISTRY

Hanazawa (1917) repeated the work of Fleischman and extended it but, despite the fact that there was a certain amount of contradictory evidence in his results, he expresse d confirmation of the generally accepted opinion. Churchill (1932) tried to find a more satisfactory explanation for the appearanc e of the sheaths of Neumann, but without success , and came to the conclusion that even the heavily stained tubule boundary seen by Fleischman and Hanazawa in demineralized sections was an artifact. Noyes (1930), Fish (1933) and Macgregor (1936) produced supporting evidence that solid particles of dye would in certain circumstances penetrate into these large dentinal tubules seen in demineralized sections. Careful work by von Saal (1930) on ground sections never reached the textbooks though in some respects he approached very close to the present day concept of the structure of the tissue. The basic reason for the controversy is therefore seen to be that the two views of the structure of the dentine derived from ground sections on the one hand and decalcified sections on the other are irreconcilable (Fig. 20). This was recognized by Hanazawa although he did not pursue the idea far enough to bring him within reach of the solution of the problem. The third phase started about 1949 with the emergence of new concepts of the structure of the tissue. New techniques , such as the embedding of tissue in methyl methacrylate , allowed the study of ground sections of dentine of the order of 7-10 μ in thickness; that is comparable in thickness to demineralized sections. Using such techniques , it was shown (Bradford, 1949, 1950a,b, 1951) that: 1. The thinner the section the more definite becomes the separation between the dentinal process of the odontoblast and the dentine and the more closely does the section resemble a demineralized section. For the greater part of its length the odontoblast process is no more than 2 μ in diameter. 2. The dentine matrix and the odontoblast process in the mature tissue can be stained differentially from whatever separate s them and this undoubtedly is not a space (Fig. 18.)

OF

DENTINE

17

3. Scratch marks and cracks can be seen running in the dentine matrix across the intervening tissue to terminate in the soft tissue of the odontoblast process. 4. The tissue between the odontoblast process and the dentine matrix in mature dentine gives a reaction for calcium with the appropriate stains. 5. In developing dentine this intervening tissue can be stained differentially from the dentine matrix and the odontoblast process (see Volume I, Chapter 7). 6. In the orthodentine of some mammals this intervening tissue appears to be absent and in others to be present in large quantity (Fig. 33). A s a result of these findings, Bradford (1950b, 1951) postulated that this intervening tissue, now termed the peritubular dentine, is highly mineralized and, lik e enamel, disappear s almost completely during normal methods of demineralization and, as a corollary to this, that the Tomes fibre as seen in demineralized sections is a summation of the dentinal process of the odontoblast, the inner hypomineralized layer of the peritubular dentine and the remnants of the matrix of the peritubular dentine. D . PRESENT CONCEPT

The simplest way to establish a concept of the microstructure of mature human dentine is to trace the developmen t of the tissue from the dental papilla. To this extent some overlap with the account given in Chapter 7 of Volume I is unavoidable. The odontoblasts are embedde d in the gelatinous ground substanc e of the pulp which forms the basis of the matrix of the dentine. 1. The Fibres of von Korff The first sign of dentine formation is the accumulation of argyrophilic fibres between the odontoblasts. These fibres were first noted by von Korff (1930) as running in bundles towards the forming dentine surface parallel to the long axes of the odontoblasts . There is still controversy about the

18

Ε. W.

BRADFORD

direction of these fibres in this region as there is no doubt that at the dentinal end of the odontoblasts these fibres are already aligned parallel to the dentine surface and at right angles to the long axes of the odontoblasts . The structures known as von Korff fibres are fibrillar in nature, are argyrophilic and appear to be of spiral form. They pursue a course from the area central to the odontoblasts between which they pass in order to reach the dentine surface. It is equally true, however, that they can be seen best in the young odontoblast layer, and that their appearanc e is dependen t upon the type of fixative used, showing up best in tissue fixed in the harsher fixatives such as Zenker. The problem of the nature of these structures and their relationship to the formation of dentine requires much more investigation.

Fig. 21. Longitudinal section of human dentine and pulp, undemineralized . P, Pulp; PM, pulpodentinal membrane; 0, odontoblast layer; PrD, predentine; ItD, mature intertubular dentine; Cs, forming calcospherites ; MF, scalloped mineralizing front of mature intertubular dentine. Masson's trichrome. χ 500. (From Bradford, 1950a.)

2. The Pulpodentinal

Membrane

I n the region of the dentinal end of the odontoblasts the von Korff fibres may well form the pulpodentinal membrane (Bradford, 1950a) (Fig. 21) where they form a delicate lacework around the dentinal processes . They may be stained with Weigert's haematoxylin as well as with silver at this stage in their development , and may therefore be classed as precollagenou s as far as their staining is concerned . The fibres when first formed are embedde d in the soft gelatinous ground substanc e of the dental pulp. They run at right angles to the dentinal tubules and tangentially to the lumina of the tubules (Kramer, 1951). They appear to run in one plane only, the fibres of adjacent planes not being

Fig. 22. Undemineralize d section of the pulpodentinal membrane parallel to the pulpodentinal junction. The fibres lie in a plane at right angles to the tubules and run tangentially to them. Wilder's silver stain, χ 1200. (From Bradford, 1958.)

12. M I C R O A N A T O M Y

AND

HISTOCHEMISTRY

OF

DENTINE

19

coincident in direction (Fig. 22). These tangential fibres thus outline a polygonal lumen for the original dentinal tubule which is about 5 μ in diameter. In normal dentine this polygonal shape is masked by the degree of mineralization but slow partial demineralization , including such as occurs in dental caries, brings out this basic polygonal shape. The portion of the dentine containing these fibres has been termed the intertubular dentine (Bradford, 1951) and this term wil l be retained. 3. The Intertubular

Dentine

The intertubular dentine undergoes some as yet not understood chemical change which allows calcification to occur. The argyrophilic precollagenous fibres of the pulpodentinal membrane become collagenous in the predentine where typical 640 Â banding can be seen (Frank and Nalbandian, 1963). Judging from its staining reactions, the ground substanc e changes continually through the pulp ground substance , pulpodentinal membrane, early predentine, predentine and intermediate dentine as part of the process of providing a medium in which mineralization can occur. However, the nature of these changes is not understood . The final stage, characterize d by the intermediate dentine, can be considered as the mineralization point associate d with an appearanc e of acid mucopolysaccharides . These substance s bind all basic dyes such as haematoxylin, and dyes based on metals such as silver, gold, mercury, and also basic lead acetate, to give metachromasi a with toluidine blue (Sylvén, 1947). Wit h increasing calcification the tissue loses most of its characteristic staining reactions. The degree of mineralization of the intertubular dentine does not reach such a point at which it cannot be cut whilst undemineralize d wit h razor blade attachment s (Bradford, 1951) or glass knives. The reason for the difficult y in cutting the mature whole tissue wil l be considered later. 4. The Peritubular

Dentine

A t some point within the predentine an additional tissue starts to be laid down between the intertubular dentine and the odontoblast process. This

Fig. 23. Undemineralize d transverse section of late predentine stained with silver nitrate (Cajal). A thin layer of peritubular dentine is present within the dentinal tubules. OPr, Odontoblast process; PtD, peritubular dentine; ItDy intertubular dentine, x 1200. (From Bradford, 1951.)

has come to be known as the peritubular dentine. Presumably this tissue is laid down by the odontoblast itself. With the apposition of new tissue to the walls of the tubule there is a correspondin g reduction in the diameter of the odontoblast process (Fig. 23). Whereas the process is approximately 5 μ in diameter at the pulpodentinal junction it becomes restricted to 1-2 μ at the level of the intermediate dentine. A t this level also, metachromatic mucopolysaccharides appear in this tissue indicating the occurrence of changes associate d with mineralization. Gradual mineralization occurs through the next 100 jut or so of dentine (Symons, 1962). Using dentine from the horse, Equus cabalus, in which the peritubular dentine may reach a thickness of 10 μ, it is possible to separate this tissue in impure form by grinding and flotation (Bradford, 1963). The heavier fractions containing full y mineralized peritubular dentine had a specific gravity of 2.4-2.5

20

Ε.

W.

whereas the main bulk of the dentine had a specific gravity of 2.1-2.2. This high degree of mineralization makes sectioning of the undemineralize d tissue difficult. a. The inner hypomineralized layer. The deposition of material on the walls of the tubules is a continuous process until the lumen of the tubule is obliterated. Until complete closure occurs, the full y mineralized peritubular dentine is always separate d from the remains of the dentinal process of the odontoblast by an unmineralized or hypomineralized layer containing metachromati c mucopolysaccharides . This may be termed the internal hypomineralized layer (Shroff et al, 1954). b. The outer hypomineralized layer. Owing to the time lapse between the formation of the intertubular dentine and the peritubular dentine, the two tissues are separate d by a narrow zone of unmineralized or hypomineralized tissue, in the

Fig. 24. Ground transverse section of human dentine stained with silver nitrate and reduced in sunlight. OPr, Odontoblast process; Ho, outer hypomineralized layer; PtD, peritubular dentine ; ItD, intertubular dentine, χ 1500. (From Bradford, 1951.)

BRADFORD

same way as adjacent bone lamellae are separate d by resting lines. This zone may be termed the line of junction or outer hypomineralized layer (Fig. 24). I n suitable stained sections of mature dentine it is therefore possible to identify the following structures: (1) intertubular dentine; (2) outer hypomineralized layer; (3) peritubular dentine; (4) inner hypomineralized layer; (5) dentinal process of the odontoblast. It is unlikely, however, that all these structures wil l be seen in one and the same section produced by a single technique. 5. The Dentinal Process of the Odontoblast The odontoblast at an early stage of its development is a cuboidal cell with a number of processes , the largest of which are directed towards the periphery of the dental papilla (Kramer, 1951). When the formation of dentine commence s one or more of these processe s of each cell becomes entrapped. As the deposition of dentine proceeds the number of cell processe s is thus reduced until each cell possesse s a single elongated process. This mode of formation results in each dentinal process of the mature tissue having a number of terminal branches immediately subjacent to the enamel and cementum. It has always been assumed , probably correctly, that in lif e the dentinal tubule is occupied throughout its entire length by the dentinal process of the odontoblast. There is no direct evidence that this is so as the minute quantity of cytoplasm which would be present in the terminal branches is beyond the limi t of resolution of the light microscope. At an early stage of developmen t whilst it is still possible to produce good undemineralize d sections of pulp, predentine and dentine and whilst the dentinal process is 5 μ in diameter, continuity of the cytoplasm of the process of the cell and cell body and similarity of staining reactions are easily observed. Where a considerable thickness of mineralized dentine is present, continuity of cytoplasm can be observed with certainty only in the predentine ; more deeply in the thickness of dentine, in older dentine, the tubule contents become shrivelled (Nalbandian and Frank, 1962). This could be due

12. M I C R O A N A T O M Y

AND

HISTOCHEMISTRY

to the slowness with which fixatives penetrate highly mineralized tissue. Warwick James (1957) suggeste d that in fact the odontoblast process does not occupy the dentinal tubule except in the early stages, though the evidence he produced for this theory was not convincing. 6. Structural Markings in the Dentine There has been considerable confusion and argument concerning the structural markings in the tissue ever since 1845 when Owen classed two different phenomena as "contour lines". One series of markings is caused by the congruence of the primary curvatures of the dentinal tubules. These are best termed Schreger lines or markings, and the others, which are due to discontinuities in the formation or mineralization of the dentine, are known as incremental lines. a. Schreger lines or markings. These may be seen in their simplest form on transversel y ground surfaces of the crowns of human teeth. Two concentric rings may be seen, each ring corresponding to one primary curvature of the dentinal tubules. In the tusk of the walrus, Rosmarus rosmarus, where the dentine is much thicker and where the curvature on the tubules is more abrupt, the Schreger markings are much more obvious. Since, however, the curvatures of only groups of tubules are congruent the markings appear as discontinuous lines. In the elephant tusk (Miles and White, 1960), where also curvatures of tubules are congruent in regular alternating groups, the curvatures in adjacent groups are exactly opposite in direction. This produces a lozenge, diamond or basket weave pattern, according to the plane of section. The deposition of dentine is sometimes stopped or slowed down during systemic metabolic disturbances. The dentine formed after this period wil l contain tubules with a slightly different direction and curvature from that found in the pre-existing dentine so that a marked Schreger line may be found in this area. The second series of markings are those caused by variations in structure and/or composition of the

OF

DENTINE

21

dentine during the processe s of deposition and mineralization. b. Incremental lines. Three different incremental markings may be seen in the dentine, which are perhaps easiest to differentiate from each other by their size. The most marked are the contour lines which can sometimes be seen with a hand lens on the polished dentine surface of longitudinal sections of teeth. These lines are records of disturbances of rhythm of deposition of the tissue. When viewed in transverse sections of the tooth these lines are concentric with the pulp cavity but in longitudinal sections they are not parallel to the outer surface of the dentine nor to the final dentine pulp junction of the completed teeth nor to each other. They correspond to the position of the dentine-pulp junction, or more probably the position of the intermediate dentine at the time at which the disturbance occurred. Such a marked disturbance frequently occurs at birth leaving a line in the dentine in the affected teeth correspondin g in time to the birth (Rushton, 1939) or neonatal line (Schour, 1939) in the enamel. Each of these contour lines is merely an exaggera tion of the normal incremental lines of growth in the dentine, marking the rhythm of deposition of the tissue. Though on average these incremental lines are 25 μ apart, there are considerable variations depending upon the portion of the tooth (Fig. 25). The third type of line is best seen in dentine which has been demineralized , either during preparation of sections or by dental caries, and stained in bulk with a silver stain such as one of the many modification of Bielschowsky's silver stain. These lines when present in the tissue are extremely regular and about 3-4 μ apart. By their presence or absence and their contour, the dentine may be divided into three portions (Fig. 26). There is an outer portion close to the enamel or cementum in which these lines are absent but in which can be noted the arcade formation (see p. 25). This formation can also be seen when polarized light is used (Schmidt and Keil, 1958). There are many small tightly packed arcades towards the

22

Ε.

W.

Fig. 25. Demineralized longitudinal section of human dentine stained haematoxylin and eosin showing contour lines in the crown of the tooth, χ 60.

periphery but fewer and larger arcades are present towards the pulp. On the pulpal aspect of this arcade layer is a portion of dentine in which the lines are present arranged in concentric rings rather lik e innumerable small finger prints. The more peripheral lines of these systems are continuous with those of adjacent systems. On the pulpal aspect of this portion is a layer in which the lines are present but run though the tissue concentric with the dentine-pulp junction. The significance of this staining of the dentine is far from clear. If the staining were due to incremental deposition it would be expected that this would show in routine sections of the tissue. This is not so, and generally is only to be seen in

BRADFORD

Fig. 26. Demineralized transverse section of the root of a human tooth stained in bulk with Bielschowsky silver stain. OZ, Outer zone with arcades ; MZ , middle zone with a globular arrangement ; / Z, inner zone with concentric layers, χ 480.

sections of tissue which have been stained in bulk after demineralization . Schmidt and Keil found that the arcade appearanc e was accentuate d when viewed under polarized light if the tissue was extracted with potassium hydroxide and glycerol to remove the organic material. They therefore came to the conclusion that the arcade appearanc e was a function of the crystallite arrangemen t and that the centre of the tip of each arcade corresponde d to one centre of crystallization ; this centre then dictated the crystallite orientation in successiv e deposits of dentine. This concept appears to run counter to the generally accepted view that mineralization of mineralized collagenous tissue is inotropic, the orientation of the crystallites being dictated by the orientation of the collagen fibres. Keil (1934), however, assume d that if the arcade formation is a function of the crystallites

12. M I C R O A N A T O M Y

AND

HISTOCHEMISTRY

OF

DENTINE

23

then the appearanc e of arcades in silver and gold preparations may be explained by the deposition of metallic particles on the walls of the spaces previously occupied by crystals. Even an explanation such as this for the arcade pattern does not make any easier an explanation of the circular and linear markings in the other two layers of the dentine. The only rational explanation so far advanced for these markings is that they are evidence of a diffusion phenomeno n occurring at the time of mineralization of the tissue; that is, they have connections with the Liesegang phenomeno n (Mummery, 1924, pp. 137 and 281). 7. Interglobular

Dentine

The mineralization of the dentine does not always follow a linear pattern. A t times when there is a high rate of deposition of matrix resulting in a broad predentine layer, centres of mineralization arise spontaneousl y in this broad layer producing globules of mature mineralized tissue (Fig. 21). These globules, or calcospherites , normally fuse together and leave no trace of their union in the completed tissue; but at times of extremely rapid deposition, or disturbed general metabolism, fusion may be incomplete and so-called interglobular spaces occur and the dentine may be termed interglobular dentine (Figs. 27 and 17). A consideration of this tissue is useful as it gives an insight into the sequence s of formation of the matuie tissue. I n ground sections of teeth, interglobular dentine is seen to be composed of translucent mineralized globules partially separate d from each other by opaque unmineralized or hypomineralized material. A t one time this separating material was considered to be an empty space, but it is now known to contain hypomineralized matrix. If such a section is demineralized the distinction between the globules and intervening tissue is lost and the demineralized tissue is uniform and stains uniformly. This suggests that the difference between the two areas is merely one of mineralization. If, however, we consider the formation of these globules, they form within the predentine so that any tissue be-

Fig. 27. Ground longitudinal section of human dentine unstained. Ig, Interglobular area in human dentine showing the scalloped edges of adjacent calcospherites . x 145.

tween the globules might be expected to be predentine. Predentine , however, has its own characteristic staining properties which are quite distinct from the tissue found between the globules. This in turn suggests that changes have occurred in the matrix between the globules to transform it into hypomineralized or unmineralized mature dentine. I t may therefore be necessar y to add yet another stage to the pattern of formation of mature intertubular dentine, i.e., pulpodentinal membrane, early predentine and late predentine, intermediate dentine (mature intertubular dentine matrix and mineralizing intertubular dentine). Indeed in stained sections of undemineralize d developing dentine it is frequently possible to discern two advancing edges of mature dentine, one staining more deeply than the other. 8. Granular Layer of Tomes In nearly all teeth, a further zone of irregular dentine formation is to be seen adjacent to the

24

Ε.

W.

cementum and is known as the "granular layer of T o m e s" (Fig. 28). This irregular layer differs from the interglobular layer both in appearanc e and in position within the tissue, yet littl e or nothing is known of the reason for its existence nor in what way the so-called granules differ from the intervening tissue. It is said to be confined to the root but may be seen frequently under the poorly calcified cervical

BRADFORD

9. Hyaline Layer of Hopewell-Smith

(1919)

The granular layer of Tomes seems to develop by the coalescenc e of globules of mature calcified dentine within a matrix similar to that of the predentine. The hyaline layer is interposed between tissue which is undoubtedly dentine and tissue which is definitely cementum but there is some question as to which cells are responsible for its formation (Blackwood, 1957), odontoblasts or cementoblasts . I t appears , however, to be the first tissue deposited at the dentine-cementu m boundary and therefore is probably dentine. The granular layer and the hyaline layer can be seen on the outside of the root dentine for the first half of the root, but more apically these tissues become so similar to the cementum in appearanc e that the three tissues appear inextricably mixed. Al l these features of irregular dentine matrix formation are exaggerate d in teeth having hypoplastic dentine. E. T HE SUBMICROSCOPIC STRUCTURE OF DENTINE

Fig. 28. Ground longitudinal section of human dentine adjacent to the cementum . T, Dentinal tubule with branches ; G, granular layer of Tomes; Hy, hyaline layer of HopewellSmith; PC, primary cementum . Stained Cajal silver in bulk and toned with gold chloride, χ 700.

enamel. If the coronal dentine be divided into two portions, as suggeste d by Weidenreich, mantle and circumpulpal, then the interglobular dentine lies within the circumpulpal dentine of the crown, and sometimes the first portion of the root, whereas the mantle dentine of the crown seems to correspond to the granular layer of Tomes and the hyaline layer of Hopewell-Smith in the root.

The submicroscopi c structure of the dentine still requires considerable investigation. Though the basis of the matrix is undoubtedly collagen fibres, the exact arrangemen t of these fibres is still in doubt. Weidenreich (1925) maintained that the collagen fibres of the mantle dentine run parallel to the dentinal tubules from the periphery towards the pulp though the evidence for this is suspect. The structural framework of dentine is a network of collagen fibres impregnated with crystallites of hydroxyapatite. Since, in certain directions, these structural elements are parallel to each other, dentine lends itself to analysis with the polarizing microscope. To date, it is with this instrument that much of the information on the finer structure of dentine has been obtained (Schmidt and Keil, 1958). Collagen fibres show strongly positive birefringence relative to length, and hydroxyapatite is weakly negative. Collagen may be studied by removing the mineral by demineralization and the hydroxyapatite may be studied by

12. M I C R O A N A T O M Y

AND

HISTOCHEMISTRY

removing the collagen by boiling in glycerinepotash solution. However, the birefringence of the collagen is so much stronger than that of the mineral that even though most of the crystallites are arranged parallel to collagen fibres, in intact sections of dentine any birefringence shown is due to the organic material. The distribution of the collagen fibres wil l be considered first. A s already indicated, dentine is laid down incrementally, the increments being seen in the adult tissue as the incremental lines. The collagen fibres are arranged in a network in planes parallel wit h these incremental lines. In intact, or demineralized tangential sections parallel with the incremental planes, the section as a whole shows very littl e birefringence. The dentinal tubules penetrate this plane perpendicularly or obliquely and, around each tubule, there is a more regular arrangemen t of fibres over short distances causing weak polarization crosses to appear around the sections of the tubules. I n longitudinal sections of the tooth, which cut almost perpendicularly through the incremental layers, many collagen fibres now li e in the section plane, parallel with each other and to the direction of the incremental lines (foliate texture); therefore both intact and demineralized dentine in longitudinal sections show strong positive birefringence. During mineralization, the collagen fibres appear to act as seeding sites for the hydroxyapatite so that vast numbers of mineral crystallites become aligned parallel to the collagen fibres. If the collagen fibres are removed from sections of adult dentine, and provided that the deproteinized mineral is mounted in a liquid of refractive index 1.55-1.6 in order to eliminate " f o r m" birefringence, then the birefringence of the section wil l be found to be negative with respect to the direction of the incremental lines. The process whereby hydroxyapatite becomes aligned parallel with existing collagen fibres has been termed "inotropic" mineralization (Schmidt and Keil, 1958) and this type of crystallite orientation is perhaps the commones t found in both dentine and bone (see also Volume I, Chapters 2

OF

DENTINE

25

and 3). But it is not the only type of crystallite orientation to be found. In many mammals (Schmidt and Keil , 1958) and certain mammal-like reptiles (Poole, 1956) a second, well marked orientation, in which crystallites are spheritically arranged, occurs as a result of a "globular" type of mineralization (Schmidt and Keil, 1958). Where this occurs, the dentine, in intact or deproteinized sections, shows bright globules, or more usually, distorted arcades transecte d by polarization crosses . The negative sign of these polarization crosses indicates that the crystallites of the negatively biréfringent hydroxyapatite are radially arranged. It has been suggeste d that the size and shape of these globules depends upon the rate of secretion of calcific material by the odontoblasts . Schmidt and Keil (1958) have described a third type of crystallite orientation in human dentine and the dentine of other mammals. In sections containing longitudinal sections of dentinal tubules, the tubules often seem somewhat brighter than their immediate surroundings , the extra brightness being lost when the tubule is arranged parallel wit h the vibration direction of either polarizer or analyzer. This indicates that even though there is a background of crystallites either parallel to the collagen fibres or spheritically arranged, yet other crystallites are parallel to the dentinal tubules. I t is highly likely that these crystallites are in the peritubular zone. Transverse sections of tubules of horse dentine show distorted polarization crosses in the peritubular area. This could be consistent with crystallites orientated rather obliquely to the axis of the tubule. X-ray diffraction studies (Thewlis, 1940) first indicated that the mineral in dentine was hydroxyapatite and that the crystallite size was very small, as has more recently been confirmed wit h the electron microscope. " F i b r e" X-ray diffraction studies have indicated no preferential orientation of crystallites within the dentine, and this would be expected since the X-ray collimator sizes used (0.5 mm) would produce diffraction patterns from wide areas of dentine and fail to resolve the details of crystallite orientation indi-

Ε.

26

W.

BRADFORD

cated by the polarizing microscope. The electron microscope has confirmed that crystallites are aligned parallel to the collagen fibres but, as yet, none of the other crystallite arrangement s has been identified. O Pr F.

CHANGES IN DENTINE

1. In Peritubular

>

Dentine

The peritubular dentine is laid down initiall y in the late predentine by the deposition of unmineralized material on the inner aspect of the walls of the dentinal tubules by the odontoblasts . Deposition is very rapid at first, most of the matrix being completed within 10-20/* of dentine thickness, but slows down so that complete obliteration of the tubule may take many years. Mineralization of the matrix occurs slowly and is usually complete at a distance of 100-150 μ from the dentine-predentin e junction. There is considerable variation, however, from tubule to tubule in the speed of mineralisation. In the region where the intertubular dentine is apparently completely mineralized but the peritubular dentine is still mineralizing it is frequently possible to obtain undemineralize d sections using a razor blade attachment to the microtome. This supports the view that the greater part of the mineral salts withi n the tissue are in the peritubular dentine. Because of the highly mineralized nature of the mature peritubular dentine, and of the small quantities present, investigation of the arrangements of crystallites and chemical analysis of the matrix have not been achieved. It is also difficult to obtain routine demineralized sections of the tissue since both calcium salts and matrix tend to disappear (Fig. 29). So far it has been possible to trace the developmen t of the matrix by electron microscopy (Frank and Nalbandian, 1963) and by histochemical staining (Symons, 1961) and to obtain a figure for the degree of mineralization of this tissue in dentine of the horse (Bradford, 1963) as measure d by the specific gravity of the tissue (see section VII , D, 4, p. 19-20).

*

α

f

*>.{ P tD

ry At

A

*

\.

Ύ

^ It D

Fig. 29. Transvers e section of human dentine demineralized in ethylenediamin e tetracetic acid and sectioned using polyethylene glycol as embedding agent stained with gentian violet. OPr, Odontoblast process; PtD, peritubular dentine; ItD, intertubular dentine, χ 1500. (From Bradford, 1955.)

The tissue never seems to mineralize completely so that strands of hypomineralized tissue remain connecting the inner and outer hypomineralized layers. These are essentially strands or sheets of hypomineralized tissue and must not be confused wit h the true branches of the dentinal process of the odontoblast. Similar strands which may or may not be continuations of the former, pass out into the intertubular dentine and are continuous with similar strands associate d with adjacent tubules. These are well seen in the dentine of the koala bear Phascolarctos cinereus (Fig. 30). The fine mesfrwork so formed can be observed by removing all collagenous protein from the dentine and then decalcifying in a sufficiently viscous fluid for support. The degree of mineralization of the peritubular dentine appears to be dependen t upon the degree of mineralization of the surrounding intertubular

12. M I C R O A N A T O M Y

AND

HISTOCHEMISTRY

OF

DENTINE

27

Fig. 30. Ground transverse section of dentine of Phascolarctos cinereus unstained. OPr, Odontoblast process ; PtD, peritubular dentine; ItD, intertubular dentine; Ho, strands of hypomineralized tissue, χ 1200.

Fig. 31. Ground transverse section of dentine of Dugong dugong unstained. PtD, Peritubular dentine present in mineralized form adjacent to mineralized intertubular dentine; OPr, dentinal tubule passing through an interglobular area, x 1500.

dentine. The relationship seems similar to that between enamel and dentine formation (see Volume I, Chapter 4, p. 186) in that deposition of enamel does not commence until a layer of dentine is present covering the dentine pulp. So formation and mineralization of peritubular dentine occurs after the formation of intertubular dentine. Deposition of crystalline hydroxyapatite occurs after crystallization in the intertubular dentine and probably occurs centripetally to give a radial arrangemen t of crystallites, though this is not yet established . If the peritubular dentine is adjacent to hypomineralized intertubular dentine or unmineralized intertubular dentine, as in interglobular dentine, it does not calcify to the same extent. Blake (1958) has shown that where a dentinal tubule runs through an interglobular area the peritubular dentine is hypomineralized . He also showed that

peritubular dentine in this position could be induced to mineralize by placing sections in a mineralizing solution. This relationship between hypomineralized peritubular dentine and interglobular dentine is well shown in hypoplastic dentine and in degenerat e dentine as found in the teeth of Dugong dugong (Fig. 31). The interchange and deposition of mineral salts in the forming peritubular dentine after apparent completion of the tissue is well illustrated by the uptake of tetracyclines in the area. Atkinson and Harcourt (1962) have shown that the fluorescence of the dentine in cases of neonatal tetracycline administration is due to a very large extent to the deposition of tetracycline in the peritubular dentine. I n exceptionally large tubules such as sometimes arise from the tip of the dentine cusp, the Reisendentin-kanalchen of Keil, or in the occlusal dentine

28

Ε.

W.

of the mandibular incisor teeth, the peritubular dentine does not mineralize completely. It is also possible that the tubules of dead tracts of Fish may contain unmineralized or hypomineralized peritubular dentine (Bradford, 1960). It is also not known what occupies the spindles, the extension of the dentinal tubules into the enamel, but this also is probably hypomineralized peritubular dentine. a. Sclerosis. The account so far of the structure of dentine is strictly accurate for dentine only in some phases of its formation from growth to maturity. Further age changes occur in the tissue itself and modifications in the type of dentine deposited by the cells of the pulp occur. These latter modifications known as secondar y dentine are adequatel y dealt with in Chapter 7 (Volume I). The changes which occur within the tissue are physiological and occur even in unerupted teeth. The physiological changes may, however, be speeded up by external stimuli such as the penetration of solutions to the enamel-dentin e junction through the enamel cap, or by attrition. The same changes also seem to occur even more rapidly in some individuals in response to pathological conditions such as tooth fracture, erosion and caries. The dentine gradually changes in structure and physical properties with the age of the tooth and depending to some extent on the stimuli to which the tooth is subjected. There is a slow but steady deposition of peritubular dentine on the tubule walls which leads ultimately to obliteration of the lumen (Fig. 32). The closure of the tubules or sclerosis of the dentine occurs first in the narrowest tubules. Thus the terminal branches of the tubules in the crown of the tooth and the narrow tubules of the root apex are the first to close (Bradford, 1960). The progressive sclerosis of the tissue produces an increased brittleness in the affected areas and also makes the tissue more translucent. This latter property has been used by Gustafson (1950) as one of the criteria by which it is possible to estimate the age of an individual for forensic purposes . The loss of elasticity of the dentine could be due solely to the rigidity imparted by the full y mineral-

BRADFORD

Fig. 32. Ground transverse section of human dentine unstained. Section shows various stages in the closure of the dentinal tubule with mineralized material, χ 1500. (From Bradford, 1960.)

ized tubules or there may be an accompanyin g increase in the degree of mineralization of the intertubular dentine. Loss of elasticity also occurs, however, in teeth from which the pulp has been extirpated, which suggests that changes , such as alteration in the degree of hydration, occur in the collagen of the intertubular dentine. These changes may be similar to senile changes which occur in other collagenous tissues of the body. b. Dead tracts (Fish, 1930), opaque dentine (von Beust, 1931), metamorphosed dentine (Bodecker, 1944). When relatively thick longitudinal ground sections of teeth are cleared and mounted in balsam it can be seen that most dentinal tubules have been penetrate d by the clearing agent with conse-

12. M I C R O A N A T O M Y

AND

HISTOCHEMISTRY

quent raising of the refractive index. Groups of tubules, however, particularly those associate d wit h small carious lesions, areas of attrition and permeable cervical enamel appear black as if no clearing agent had penetrate d them. The black groups of tubules have been termed "dead tracts". This appearanc e of the dentine may be pathological as a reaction to caries or due to physiological changes occurring primarily in the pulp. The essentia l feature appears to be a loss of continuity of the contents of the dentinal tubule with the odontoblast cell and with consequen t degeneratio n or necrosis of the tubule contents (von Beust, 1931 ; Rushton, 1940). The tubules tend to be sealed at the enamel end and with a translucent substanc e (eburnoid: Fish, 1933) at the pulpal end. The tubules are penetrate d with difficult y by clearing and mounting media so that dead-tract tubules appear black in ground sections compared with those tubules which have been penetrate d by the mounting medium. No difference has yet been demonstrate d between the appearanc e in demineralized sections of dead tracts and that of normal dentine. There is therefore only speculation as to what occurs within the tubules. There appears to be a physiological relationship between sclerosis and dead-tract formation. Both may be classed as reactions of the dentine to stimulation under certain conditions. Dead tracts, however, are more likely to occur with greater stimulation. They occur most frequently in the crown of the tooth where the tubules are largest and where the pulp has least opportunity to produce a complete mineralized closure (Lefkowitz, 1942). Given a constant stimulus they occur more frequently in teeth in which senescen t changes can be presumed to be occurring in the pulp (Bodecker, 1944). 2. In Physical

Properties

The physical properties of dentine have been extensively discusse d by Leicester (1949). Since this time additional information has become available concerning the mechanica l properties of

OF

DENTINE

29

the tissue. Peyton, Mahler and Herskenov (1952) and Tyldesley (1959) studied the elasticity of dentine and obtained a value of 1.7 for Young's modulus of elasticity. The breaking stress was found to be 38,000 psi. Density measurement s quoted by Leicester and other authors since may be satisfactory for comparative studies providing that test samples are treated identically but these measurement s should not be considered to be correct in absolute terms. There are two principal reasons for this which stem from the fact that dentine is a mixture containing organic collagen fibres, inorganic crystals of apatite and microscopic spaces . These spaces allow the ingress of the liquids used for flotation and once penetration has occurred much of the liqui d wil l remain even after the normal techniques of drying have been carried out (Stack, personal communication, 1965). The collagen fibres of the organic matrix are readily affected physically by some of the liquids used for flotation such as acetone and bromoform and these physical changes may well affect the results. It is essentia l that inert liquids which do not produce swelling should be used (Mohanarakrishna n and Ramathan, 1964). Changes have been shown to occur in the structure of the dentine under the influence of attrition and dental caries but these histological changes do not seem to be matched by changes in the physical properties of the tissue. F or instance, closure of the dentinal tubules in sclerosis might be expected to change the degree of overall mineralization of the tissue and also its hardness but Bhussry and Hess (1963) have shown that in pooled samples of dentine from various age groups, 10-70 years of age, the differences were not statistically significant. Conversely, Emslie and Stack (1958) showed that root dentine in its outer third where sclerosis is occurring has a much higher microhardnes s number than the inner root dentine. Hardness has also been shown to increase in areas of sclerosis associate d with attrition and dental caries. One possible explanation of this lack of agreemen t may be that hardness may not necessaril y be associate d wit h an increase in density.

30

Ε.

W.

BRADFORD

Clinical observation of teeth from human subjects suggests that teeth from the old are more brittl e than teeth from the young. This could be due to an increasing rigidity imparted to the tissue by the fully sclerotic tubules. The observed loss of elasticity might also be due to a change in the collagen fibres of the intertubular dentine, such changes being similar to those which occur in collagen in other parts of the body. The latter explanation is supported to some extent by the fact than in dead teeth in which sclerosis cannot proceed, there is nevertheles s an increased tendency to fracture. Against these clinical impressions , however, must be set the work of Tyldesley (1959), who found that the modulus of elasticity of the dentine did not vary significantly with age. G . SPECIES DIFFERENCES

A s noted earlier, the variations in the structure of mammalian dentine take several forms: (1) a variation in the degree of complexity of the pattern formed by the dentinal tubules; (2) a variation in the degree of hypoplasia of the tissue; (3) the presence or absence of a hypermineralize d layer of peritubular dentine; (4) a variation in the relative amounts of intertubular and peritubular dentine. A survey of Eutheria (Bradford, 1954) looking at ground sections of the dentine of the teeth of one or two members of each order shows that wellformed dentine of the highly specialized type with marked, well-mineralized peritubular dentine is to be found in the orders Primate, Carnivora, Hyracoidea, Perissodactyla , Artiodactyla, Cetacea , Sirenia, Proboscidea . The less specialized dentine with no differentiation of peritubular dentine occurs in the orders Insectivora, Chiroptera, Rodentia, Lagomorpha. In these latter animals it is not possible to state categorically that hypermineralize d peritubular dentine does not exist. Microscopy of ground sections of the teeth of these animals is rendered difficul t by smallness of the teeth and small quantities of peritubular dentine would be very difficult to differentiate from optical artifacts under these

circumstances . The dentine of these latter groups does resemble in all respects that found in Reptilia. I t would be convenient if the presence of hypermineralized peritubular dentine could be looked upon as a specialized characteristic occurring only in the more advanced members of Mammalia. This, however, is not wholly true as this same division into highly specialized and less specialized can be seen within a single order, Marsupialia. Thus, though the dentine of the kangaroo Macropus giganteus appears to be of a less specialized type, there is no doubt about the differentiation of peritubular dentine in the koala Phascolarctos cinereus (Fig. 30). This peculiar distribution of a characteristic suggests that an accurate charting of its distribution is essentia l and also that perhaps valuable information on the evolution of the Metatheria and Eutheria might be obtained by a study of the presence or absence of this character in the cynodont and dicynodont reptiles. Withi n the group of animals showing good differentiation of peritubular dentine there are great variations in the relative proportions of peritubular dentine to intertubular dentine. It has been noted already that in any individual tooth there is a gradual diminution in the quantity of intertubular dentine from the periphery to the pulp. There are also variations in size of original dentinal tubules from one area of tooth to another. In addition, the proportion of the two types of dentine may vary in different teeth in a single dentition. These variations, however, are never as great as those which occur from animal to animal. The two extremes so far found are certain zones of dentine in the molar of the horse (Equus cabalus) (Fig. 33) where the proportion of peritubular dentine is greatest and the dentine of the tusk of Elephas maximus where the proportion of peritubular dentine is smallest (Fig. 34). It is of interest to note that in these two animals the average distance of tubule centre to tubule centre is approximately the same. A s peritubular dentine is more highly mineralized than intertubular dentine, the relative proportions of the two wil l affect the overall hardness and the

12. M I C R O A N A T O M Y

AND

HISTOCHEMISTRY

OF

DENTINE

31

It D

Fig. 33. Ground transverse section of dentine of Equus cabalus unstained. OPr, Odontoblast process; PtD, peritubular dentine; ItD, intertubular dentine, χ 1500. (From Bradford, 1963.)

resistance to abrasion of the dentine. The greatest development of the peritubular dentine is to be found in the Perissodactyl a and Artiodactyla, in which teeth of limited growth withstand considerable attrition. Conversely in those animals in which wear caused by attrition is made good by continued tooth growth, the proportion of peritubular dentine is at a minimum. I n mammals in which there has been a steady evolutionary tendency to reduction of the dentition as a whole, there appear to have been concurrent changes in the histological structure of the tooth and of the dentine (Bradford, 1954). Thus in the dugong, in which the dentition is reduced to two upper incisors and two molars in each jaw in the adult, the dentine is wholly of the interglobular type, similar in all respects to what in other mammals would be termed hypoplastic. In 4

Fig. 34. Ground transverse section of dentine of Elephas maximus unstained. OPr, Odontoblast process; PtD, peritubular dentine; ItD, intertubular dentine, χ 1200.

the edentate s the amount of orthodentine in any teeth that are present is much reduced though it may be normal in structure and the central pulp canal fill s with another mineralized tissue. In the aardvark, the process has progresse d even further so that orthodentine is missing from the tooth, the tooth being composed of another mineralized tissue which has variously been termed plicidentine or osteodentine .

VIII. SUMMARY Dentine is a term that has been shown to embrace a number of tissues which differ greatly in structure. I t is now generally accepted that these tissues may be divided into three groups. Those of the first group, which form the outer coverings of the teeth of many fishes, have been discusse d in Chapters

32

Ε.

W.

BRADFORD

2 and 3 (Volume I). The second and third groups comprise the tissues which form the main bulk of the teeth of all animals. Tissues of these latter types may be differentiated into those which resemble the dentine of mammals, termed orthodentine, and those which bear some resemblance s to bone, termed osteodentines . Vasodentine , which has in the past been classed as a form of orthodentine, has been shown to be more akin to osteodentine and should be grouped accordingly. Both osteodentin e and orthodentine are to be found in members of the more primitive orders of vertebrates and there is no evidence in extant animals that the one developed from the other. There are still dental tissues about which insufficient is known to classify them ; the tissue forming the rostral teeth of Pristidae and the tissue forming the teeth of Orycteropus are two examples. There may well be other examples that would come to light in a systematic survey of the histological structure of the teeth and dermal appendage s of vertebrates . Since the teeth and dermal appendage s are the only palaeontologica l remains of many extinct species, it is essentia l that at some time in the near future an accurate description and classification of the tissues forming the teeth and scales of known livin g and extinct species should be carried out. Though there is a broad consensu s of opinion as to what constitutes osteodentine , nevertheles s the tissue is very pleomorphic, varying not only from species to species but even within the individual tooth. The one constant feature of the tissue is that, where it occurs in a tooth, it forms the medium of attachmen t of the tooth to the underlying bone in truly ankylosed teeth, to the fibrous membrane in the elasmobranch s and to the hinge in the hinged attachment s of bony fishes. In view of this tissue relationship perhaps osteodentin e should be considered as a relation of cementum rather than of orthodentine. Orthodentine, as present in extant animals, exhibits a number of variations in structure and a classification has been suggested . This classification should not be accepted without confirmatory

evidence. For instance the use of higher magnifications might well show that peritubular dentine is present in many instances where it cannot be differentiated with the optical microscope. Though it is now accepted that there are structural and chemical differences between peritubular dentine and intertubular dentine, littl e is known about the chemical differences—whethe r for instance the matrix of peritubular dentine is collagenous. Indeed despite the effort which has been put into the study of dentine both human and comparative over the past 120 years since Owen there is a great deal still to be learned.

References Adloff , P. (1930). Tomessch e Kornerschicht, Interglobulardentin und Vasodentin — in einigen Sâugetierzâhnen , zugleich ein Beitrag zur Kenntnis des Gebisses von Orycteropus und zur Stammesgeschicht e dieser Tierform. Vjschr. Zahnheilk. 46, 207-258. Anthony, M . R. (1934). La dentition de l'Oryctérope. Morphologic Développement . Structure. Interprétation. Ann. Sci. nat. (Zool.) 17, 290-322. Atkinson, H. F. and Harcourt, J. K. (1962). Tetracyclines in human dentine. Nature, Lond. 195, 508-509. Bhussry, B. R. and Hess, W. C. (1963). Ageing in enamel and dentin. J. Geront. 18, 343-344. Blackwood, H. J. J. (1957). Intermediate cementum. Brit, dent. J. 102, 345-350. Blake, G. C. (1958). The peritubular translucent zones in human dentine. Brit. dent. J. 104, 57-64. Bodecker, C. F. (1944). "Fundamental s of Dental Histology and Embryology Including Clinical Applications", 4th ed. Columbia Univ. Press, New York. Bradford, E. W. (1949). An histological study of human dentine with special reference to the innervation and permeability of the tissue. M.D.S. Thesis, University of Sheffield. Bradford, E. W. (1950a). An investigation of the structure of the pulpo-dentinal junction. Brit. dent. J. 88, 55-58. Bradford, E. W. (1950b). The identity of Tomes' fibre. Brit. dent. J. 89, 203-209. Bradford, E. W. (1951). The interpretation of ground sections of dentine. Brit. dent. J. 90, 303-308. Bradford, E. W. (1954). An histological investigation of the dentinal tissues of vertebrates with special reference to the phylogeny of dentine and its bearing upon the structure

12. M I C R O A N A T O M Y

AND

HISTOCHEMISTRY

of the tissue in man. D.D.Sc. Thesis, University of St. Andrews. Bradford, E. W. (1955). The interpretation of decalcified sections of human dentine. Brit. dent. J. 98, 153-158. Bradford, E. W. (1957). The structure of the rostral teeth and rostrum of Pristis microdon. J. dent. Res. 36, 663-668. Bradford, E. W. (1958). The maturation of the dentine. Brit. dent. J. 105, 212-216. Bradford, E. W. (1960). The dentine, a barrier to caries. Brit. dent. J. 109, 387-398. Bradford, E. W. (1963). An histochemica l and biochemical study of the peritubular dentine. Ann. Histochem. 8,21-24. Churchill, H. R. (1932). "Human Odontograph y and Histology". Lea & Febiger, Philadelphia, Pennsylvania . Emslie, R. D. and Stack, M. V. (1958). The microhardnes s of roots of teeth with periodontal disease . Dent. Practit. dent Rec. 9, 101-103. Fischer, H. (1938). Uber Bau und Entwicklung des Gadidaezahnes . Z. Zellforsch. 27, 726-744. Fish, E. W. (1930). Lesions of the dentin and their significance in the production of dental caries. J. Amer. dent. Ass. 17, 992-1008. Fish, E. W. (1933). "A n Experimental Investigation of Enamel, Dentine and Dental Pulp". J. Bale Sons & Danielsson, London. Fleischman, L. (1905). Uber Bau und Inhalt des Dentinkanâlchens. Arch. mikr. Anat. 66, 297-310. Frank, R. M. and Nalbandian, J. (1963). Comparative aspects of developmen t of dental hard structures. J. dent. Res. 42, 422-^-37. Gustafson, G. (1950). Age determinations on teeth. / . Amer. dent. Ass. 41, 45-54. Hanazawa, K. (1917). A study of the minute structure of dentin, especially of the relation between the dentinal tubules and fibrils. Dent. Cosmos 59, 125-148 and 271-300. Hopewell-Smith, A. (1919). "The Normal and Pathological Histology of the Mouth", 2nd ed., Vol. 1. Churchill, London. James, W. W. (1957). A further study of dentine. Trans. Zool. Soc. Lond. 29, 1-66. Keil, A. (1934). Uber Doppelbrechun g und Feinbau des menschliche n Zahnbeins. Z. Zellforsch. 21, 635-652. Kerr, T. (1958). Development and structure of some actinopterygian and urodele teeth. Proc. Zool. Soc. Lond. 133, 401-422. Kôlliker, Á., ed. (1889). "Handbuch der Gewebelehr e des Menschen." Engelmann, Leipzig. Kohlenberger, H. (1940). Zur Kenntnis des Vasodentins . Z. mikr.-anat. Forsch. 48, 416-477. Kramer, I. R. H. (1951). Distribution of collagen fibres in the dentine matrix. Brit. dent. J. 91, 1-7. Kvam, T. (1946). Comparative study of the ontogentic and phylogenetic developmen t of dental enamel. Norske Tandlaegeforen. Tid. 56, Suppl., 1-198.

OF

DENTINE

33

Lefkowitz, W. (1942). The "vitality " of the calcified dental tissues. V. Protective metaporphosi s of the dentin. / . dent. Res. 21, 423-428. Leicester, H. M. (1949). "Biochemistry of the Teeth". Kimpton, London. Lison, L. (1941). Recherche s sur la structure et l'histogenès e des dents des poissons dipneustes . Arch. Biol., Paris 52, 279-320. Macgregor, A. (1936). An experimenta l investigation of the lymphatic system of the teeth and jaws. Proc. Roy. Soc. Med. 29, 1237-1272. Miles, A . E . W. and White, J. W.(1960). Ivory.Proc. Roy.Soc. Med. 53, 775-780. Mohanarakrishnan , V. and Ramathan , M. (1964). Studies in collagen fibres. I. Density. Leather Sci., Madras 11, 260-266. Mummery, J. H. (1924). "The Microscopic and General Anatomy of the Teeth", 2nd ed. Oxford Univ. Press, London and New York. Nalbandian, J. and Frank, R. M. (1962). Microscopie électronique de l'odontoblaste humain et de son prolongement au cours de la dentinogenèse . C. R. Soc. BioL, Paris 156, 1498-1499. Neumann, E. (1863). "Beitrag zur Kenntnis des normalen Zahnbein- und Knochengewebes" . Vogel, Leipzig. Noyés, F. B. (1930). " A Textbook on Dental Histology and Embryology", 4th ed. Kimpton, London. 0rvig, T. (1951). Histologie studies of Placoderms and fossil Elasmobranchs . I. The endoskeleton , with remarks on the hard tissues of lower vertebrates in general. ArkivZool. [2] 2, 321-454. Owen, R. (1845). "Odontography; or, a Treatise on the Comparative Anatomy of the Teeth; their Physiological Relations, Mode of Development, and Microscopic Structure, in the Vertebrate Animals", 2 vols. Hippolyte, Baillière, London. Peyton, F. Á., Mahler, D. B. and Herskenov, B. (1952). Physical properties of dentine. / . dent. Res. 31, 366-370. Poole, D. F. G. (1956). The structure of the teeth of some mammel-like reptiles. Quart. J. micr. Sci. 97, 303-312. Romer, O. (1905). Erbringung des Beweises , dass die Tomessche n Dentinfasern identisch sind mit den von Kolliker zuerst beschriebene n Dentinkanâlchen . Dtsch. Mschr. Zahnkeilk. 23, 695-704. Rose, C. R. (1898). Uber die verschiedene n Abânderunge n der Hartgewebe bei niederen Wirbeltieren. Anat. Anz. 14, 21-23 and 33-69. Rushton, M. A. (1939). The birefringence of deciduous tooth enamel formed before and after birth. Brit. dent. J. 67, 1-10. Rushton, M. A. (1940). Observations on Fish's "dead tracts" in dentine. Brit. dent. J. 68, 11-13. Schmidt, W. J. and Keil, A. (1958). "Di e gesunde n und die erkrankten Zahngeweb e des Menschen und der

34

Ε.

W.

Wirbeltiere im Polarisationmikroskop" . Carl Hanser, Miinchen. Schour, I. (1939). Studies in tooth development . / . dent. Res. 18, 91-102. Shroff, F. R., Williamson, Ê. I., Bertaud, W. S. and Hall, D. M. (1954). The nature of the odontoblast process. Oral Surg. 9, 423-443. Sylvén, Â. (1947). Cartilage and chondroitin sulphate. II . Chondroitin sulphate and the physiological ossification of cartilage. / . Bone Jt. Surg. 29, 973-976. Symons, Í . Â. B. (1961). A histochemica l study of the intertubular and peritubular matrices in normal human dentine. Arch, oral Biol. 5, 239-248. Symons, Í . Â. B. (1962). A histochemica l study of the odontoblast process. Arch, oral Biol. 7, 455-462. Thewlis, J. (1940). The structure of the teeth as shown by X-ray examination. M.R.C. Spec. Rep. Ser. 238. Thomasett, J. J. (1928). Essaie de classification des variétés de dentine chez les poissons. C.R. Acad. Sci., Paris 187, 1075-1176. Tomes, C. S. (1898). Upon the structure and developmen t

BRADFORD of the enamel of the elasmobranc h fishes. Phil. Trans. B190, 443-464. Tretjakoff, D. K. (1926). Die Z hn e der Plectognathen . Z. wiss. Zool. 127, 619-644. Tyldesley, W. R. (1959). The mechanica l properties of human enamel and dentine. Brit. dent. J. 106, 269-278. von Beust, T. B. (1931). Reactions of the dentinal fibril to external irritation. / . Amer. dent. Ass. 18, 1060-1071. von Ebner, V. (1902). Die Z hne . In "Handbuch der Gewebelehr e des Menschen" (A. Kôlliker, éd.). Engelmann, Leipzig. von Korff, K. (1930). (Jber das Wachstum der Dentingrundsubstan z verschiedene r Wirbeltiere. Z. mikr.-anat. Forsch. 22, 445-467. von Saal, R. (1930). Beobachtunge n uber den feineren Bau des menschliche n Zahnbeins. Z. Zellforsch. 11, 638-657. Walkhoff, O. (1921). "Lehrbuch der Zahnheilkunde". Barth, Leipzig. Weidenreich, F. (1925). Uber den Schmelz der Wirbeltiere und seine Beziehunge n zum Zahnbein. Z. Anat. Entwiekl.Gesch. 79, 292-351.

CHAPTER

13

ULTRASTRUCTURE O F DENTINE ERLING

JOHANSEN

I. Introduction

35

II . Structural Characteristic s A. Cellular Components B. Extracellular Components

37 37 45

III . The Relation of Structure to Some Aspects of Physiology IV . Pathologic Alterations

63 · ·

V. Concluding Remarks

65 69

Reference s

72

I. INTRODUCTION Our knowledge of dentine has been considerably expanded within the last two decades through ultrastructural research . Wit h successfu l application of the methods of polarization microscopy, X-ray diffraction and electron microscopy it has been possible to observe, record and describe in considerable detail the morphologic characteristics of dentine from the cellular down to the molecular and, indeed, atomic levels. Electron microscopic observations on the organic portion of the tissue have revealed elaborate fine structures in the cellular component and an intricate feltwork of fibrils in the extracellular collagenous matrix. Studies of the mineral phase by means of electron microscopy, electron diffraction and X-ray diffraction have yielded data on the size and shape of apatite crystallites and on the spatial arrangement of atoms within their structure. Some information on the structural relationship of the

apatite crystallites to elements of the organic matrix has also been obtained through ultrastructural research . The resulting picture of the molecular organization of dentine is providing e a structural basis for a more comprehensiv understandin g of the physiology and pathology of the tissue. Although several methods of study have contributed to our present knowledge of the ultrad structure of dentine, the information to be presente in this chapter wil l be based primarily upon electron microscopic observations . The early contributions through studies of replicas and shadowed preparad but the emphasis tions wil l be cited and discusse wil l be on findings from recent studies of thin sec. Since the tions and of homogenate preparations latter experimenta l procedures afford direct visual, the illustrative ization of structural components material has been selected from these studies. 35

13. U L T R A S T R U C T U R E

II. STRUCTURAL CHARACTERISTICS Dentine, lik e other connective tissues, consists of a relatively large amount of extracellular substance and a rather small amount of cellular material. The extracellular components occur mainly in the form of a densely mineralized collagenous matrix organized around canalicular structures. This mineralized matrix forms the body of the tooth, encloses the dental pulp and provides attachmen t and support for enamel and cementum. Thus, dentine may be characterize d as the supporting framework of the tooth. However, the vital processe s of the tissue are invested in the cellular component of dentine, the odontoblasts. Other cellular elements, such as osteoclast s during resorption and nerves, may also be found withi n dentine. The role of these latter elements wil l be considered briefly in the discussion of some aspects of the physiology of dentine. A.

CELLULA R COMPONENTS

OF

DENTINE

37

extensions. When the apparently predetermine d full thickness of dentine has been achieved, matrix formation ceases or slows down to an almost imperceptible rate. However, during the quiescent stages that follow, the odontoblasts appear to retain their ability to participate in further dentine development, as is shown by secondar y dentine formation. Based on circumstantial evidence, it has been thought that odontoblasts play an important role in the synthesis of constituents of the organic matrix of dentine, particularly the collagen. Furthermore, the cells of dentine have been implicated in the initiation and transmission of sensory stimuli in dentine. A s light microscopic studies have not been conclusive in providing evidence in support of these contentions, electron microscopic observations on the fine structure of the odontoblast and of its protoplasmic extension assume special significance. 1.

Odontoblasts

The structural relation of the odontoblast to the matrix of dentine is unique in that the nucleuscontaining portion of the cell is situated at the internal border of the tissue with a cytoplasmic process of the cell extending into the system of dentinal canals. This structural relationship of cellular and extracellular components of full y formed dentine represent s an anatomical arrangement establishe d in dentinogenesis . A t the beginning of dentine formation the odontoblasts are located close to the enamel-dentin e and dentinecementum junctions. As matrix formation proceeds the cells recede, leaving behind protoplasmic

The ultrastructure of the odontoblast has been studied most extensively in developing dentine of mouse, rat and human teeth (Nylen and Scott, 1958; Noble, Carmichael and Rankine, 1962; Bruns, personal communication, 1962). I n the developing tissue, irrespective of species, the odontoblasts appear as elongated cells with the nucleus placed close to the basal end (Fig. 1). During active matrix formation, the cells display highly developed endoplasmic reticulum, distended Golgi apparatus and numerous mitochondria (Figs. 2-4). The odontoblast of full y formed h u m an and rat dentine shares several characteristic s with

Figs. 1-4. Odontoblasts from rat incisor shown in longitudinal section to demonstrat e the arrangemen t and distribution of cytoplasmic organelles; tissues stained with l communication, lead hydroxide. (From Bruns, persona 1962.)

Fig. 2. Higher magnification showing the thin limitin g or plasma membrane (PM) which envelops the cell, the endoplasmic reticulum (ER) with associate d ribosomes (/?), Golgi apparatus (G), vacuoles (V) and mitochondria (MI). (Approx. X 10,800.)

Fig. 1. The nucleus (Ë0 is placed at the basal end of the cell; the endoplasmic reticulum (ER) occupies most of the cytoplasm; the Golgi apparatus (G) is located approximately in the centre of the cell. (Approx. x 5,000.)

Fig. 3. A double membrane (DM) surrounds the nucleus (N); endoplasmic reticulum (ER) with ribosome particles (R). (Approx. x 23,700.) Fig. 4. Mitochondrium showing outer and typical inner membrane s and cristae. (Approx. x 15,800.)

ERLING

JOHANSEN

13. U L T R A S T R U C T U R E

OF

DENTINE

39

the odontoblast of the developing tissue (Bruns, personal communication, 1962; Johanse n and Parks, 1962 and unpublished data, 1962; Frank, 1966). After dentinogenesi s the cell retains its columnar shape with the nucleus placed close to its basal end. Two membranes separate d by a narrow space surround the nucleus (Fig. 3), enclosing its chromatin material as well as one or two nucleoli. Openings or nuclear pores, which in some locations appear to be continuous with the endoplasmic reticulum, have been observed in the outer membrane. The cytoplasmic organelles of the cell, the endoplasmic reticulum, the Golgi apparatus and the mitochondria are, however, fewer in number and of smaller size (Fig. 5) than in developing dentine. A s in the forming tissue, they are located primarily in the distal portion of the cell but show a decreasin g concentration towards the predentine. These organelles have their own structural characteristic s making them readily identifiable within the cell. The endoplasmic reticulum appears as tubular vesicles or cisternae arranged wit h their long axes approximately parallel to the long axis of the cell. The walls of the cisternae are studded with ribonucleoprotein particles. The odontoblast also contains a number of mitochondria distributed throughout its cytoplasm. These bodies show double outer membranes as well as typical inner membrane s and cristae (Fig. 4). The Golgi complex located in the central part of the cell consists of numerous small vesicles and some paired membrane s which, when extended, display a vacuolar appearance . The distal end of the cell shows membrane-boun d vacuoles of varying

sizes that contain amorphous or finely granular material. A feltwork of fine fibrils with a diameter of approximately 50-80 Â has been noted within the cytoplasm (Fig. 6). Furthermore, glycogen deposits in a rosette-like arrangemen t have been observed in some cells (Frank, 1966). The entire odontoblast is enveloped by a limitin g or plasma membrane and, where adjacent cells occur in close proximity, desmosome-lik e intercellular connections have been noted (Frank, 1966). In this region of closely packed cells, bundles of collagenous fibrils (von Korff fibres) have been found in intercellular spaces (Fig. 7) (Johanse n and Parks, 1962). I n the vicinity of the predentine a structure resembling the terminal bar apparatus has been described; its function is believed to be that of binding the cells together and providing support (Frank, 1966). The odontoblasts , whose diameter commonly measure s 4-5 μ in their widest portion, gradually taper to 3 μ as they enter the canals of predentine (Fig. 8). A comparison of the extent, distribution and number of cytoplasmic organelles of odontoblasts in developing and full y formed dentine reveals smaller, less extended and fewer structures in the full y formed tissue (Noble et al., 1962; Frank, 1966). This finding might reflect a quiescent state followin g the formation of the full thickness of dentine. Odontoblasts are, however, capable of further participation in dentine developmen t as is seen in secondar y dentine formation. Whether such participation is preceded by renewed proliferation and extension of cytoplasmic organelles has not been determined.

Figs. 5-7. Odontoblasts from fully formed human permanen t tooth in cross section to demonstrat e the relation of cells, the distribution and arrangemen t of cytoplasmic organelles and the occurrence of bundles of collagenous fibrils between the cells in the region of the nuclei. Stained with lead hydroxide. (Figs. 5 and 6 from Johanse n and Parks, unpublished work, 1962; Fig. 7 from Johanse n and Parks, 1962.)

fibril s (CF) appear in spaces between individual cells; nucleus (N). (Approx. χ 9,100.) The polystyrene latex spherules (courtesy of Dow Chemical Co.) are approximately 0.26 μ in diameter. Fig. 6. Higher magnification of single odontoblast displaying fine filaments (FF) within the cytoplasm and some cytoplasmic organelles. (Approx. χ 14,500)

Fig. 5. Some cells show endoplasmic reticulum (ER), mitochondria (MI) and vesicles ( VE) ; bundles of collagenous

Fig. 7. Bundles of collagenous fibril s between odontoblasts (O). (Approx. x 27,400.)

13. U L T R A S T R U C T U R E

The function of the odontoblast in matrix formation has not been settled through electron microscopic studies, but the abundanc e of its cytoplasmic organelles points to an active role. During dentinogenesi s the degree of developmen t of the endoplasmic reticulum is in accord with a high degree of protein synthesis, and the distended Golgi apparatus indicates production of substance s for extracellular destination. These materials, which possibly are contained in the vacuoles observed in the cytoplasm of the odontoblasts , may represent precursors for either the collagenous or polysaccharid e constituent of the matrix. The diminution of cytoplasmic organelles following completion of dentinogenesi s also supports the idea that the odontoblast plays an active role in the synthesis of organic matrix, provided that this reduction in size and number is not considered incompatible with further synthesizing activity. Ultrastructural studies of odontoblasts have so far yielded no additional information on the lif e cycle or lif e span of these highly specialized cells. The process by which undifferentiated cells of the pulp are transformed into odontoblasts in response to tissue injury has not yet been studied. Neither have observations been made on the possible replacemen t of individual cells, nor on the relation of newly differentiated odontoblasts to establishe d dentinal canals and their contents. 2. Odontoblast

Process

The odontoblast and its cytoplasmic extension have generally been regarded as one structural unit and electron microscopic studies on full y formed permanen t human dentine have substantiated this concept (Johanse n and Parks, 1962; Frank, 1966). Observations on longitudinal and cross sections of odontoblasts in the vicinity of the predentine-pul p border have revealed continuity of the plasma membrane as the cell enters the Fig. 8. The predentine-pul p border of fully formed human permanen t tooth showing distal ends of odontoblasts (O) and their extensions into the dentinal canals (OP). The matrix of predentine (PD) presents a feltwork

OF

DENTINE

41

dentinal canal (Figs. 8-10). Likewise, the protoplasm extends without interruption into the predentine, where it displays structural characteristics quite similar to those observed in the distal end of the cell. A t this level, the odontoblast process contains some cytoplasmic organelles including endoplasmic reticulum and mitochondria. These are, however, fewer in number, smaller and less distinct than those observed closer to the nucleus. Also, in the predentine area a number of vacuolar inclusions have been observed as well as fine intracellular fibrils (Frank, 1966). The limitin g membrane of the odontoblast process appears thin and resembles the plasma membrane of the cell body (Figs. 8 and 9). Closely associate d wit h some odontoblast processes , but separate d from them by a narrow space of 50-200 Â , are structures recently described by Frank (1966) as unmyelinated nerve fibres. These structures, also noted by Johanse n and Parks (unpublished observations , 1962) in the dentinal canals of predentine (Fig. 11) and at the predentine-dentin e border (Fig. 12), occupy or form concavities or depression s on the surface of the odontoblast process. The functional relation of these nerve-like structures to the odontoblast process is, however, not clear from present observations . The fine structure of the odontoblast process peripheral to the predentine-dentin e border has proved rather difficult to study because of the high mineral content of dentine proper. The hardness of the tissue has made it difficult to obtain sufficiently thin sections for examination after staining and inadequate penetration of fixatives has been another problem. Attempts were made to overcome these obstacles by demineralizing the tissue and by utilizing various fixatives such as formalin, alcohol or buffered osmic acid solution (Johanse n and Parks, 1962). In studies where these procedures were employed, some dentinal canals displayed of relatively narrow collagenous fibrils. Stained with phosphotungstic acid. (Approx. χ 14,100.) (From Johanse n and Parks, 1962.)

ERLING

JOHANSEN

13.

ULTRASTRUCTURE

OF

DENTINE

43

collagenous fibrils, as shown in Figs. 13 and 22. Others contained aggregate s of granular material partially filling the lumen (Figs. 13 and 16), and some appeare d empty (Figs. 13 and 15). The latter observations most likely reflect insufficient penetration of fixatives, or structural changes referable to the demineralization procedure. In a recent study of undemineralize d tissues fixed in glutaraldehyde , Frank (1966) appears to have overcome these technical difficulties to a considerable degree. His studies of the area adjacent to the predentine-dentin e border showed that the odontoblast process continues into this part of dentine without marked alteration. Unmyelinated nerve fibres were associate d with some processe s but these were smaller in diameter and contained fewer mitochondria than the corresponding structures in predentine. Within the odontoblast processe s a few cytoplasmic organelles were observed, and the fine intracellular fibrils noted in predentine were found to extend into this portion of the cell. Also present were vacuoles of various sizes containing fine granular material. Further peripherally these vacuoles seemed to rupture, discharging their contents between the membrane of the odontoblast process and the calcified wall of the dentinal canal. Noncalcified collagenous fibrils with typical cross-bandin g and surrounded by an amorphous ground substanc e could also be discerned in this location. In the outermost region the dentinal canals contained large vacuoles of lipi d material which appeare d to compress the surrounding odontoblast process, giving it a hyaline appearance . It is also of interest that in this study of undemineralize d tissues some peripheral canals were found to contain amorphous material.

I n some of the early ultrastructural studies on replicas and shadowed preparations of dentine, the odontoblast processe s were seen as either hollow tubes (Scott and Wyckoff, 1947; Syrrist and Gustafson, 1951) or solid structures (Scott, 1955). Comparison of these findings with the observations just presente d suggests that the hollow tubes most likely represente d empty dentinal canals. If this assumption is correct, the membranous structures seen in replicas and in shadowed preparations might be a modification of the matrix rather than the limitin g membrane of the odontoblast process as considered at the time. The solid structures filling the lumen of the canals could either represen t the odontoblast process or substances contained within the dentinal canals, depending upon their location. The structural relation of the odontoblast process to the wall of the dentinal canal is of interest in relation to the circulation of tissue flui d within dentine. F or continued mineralization of the tissue and for sclerosis of dentinal canals to take place, movement of electrolytes to the peripheral areas of dentine is necessary . This could be achieved without difficult y if a space existed between the plasma membrane of the odontoblast process and the matrix as is suggeste d by some electron micrographs (Figs. 8 and 10). However, if the odontoblast process completely fills the lumen as suggeste d in other studies (Frank, 1966), extracellular circulation would be minimal and an intracellular transport mechanism for the elements of the mineral component must be considered. It is conceivable that both circumstance s exist in dentine at the same time. This would be possible if the odontoblast as a livin g cell were capable of alrering its morphology in response to

Figs. 9 and 10. Odontoblasts and odontoblast processe s from fully formed human permanen t tooth cut in cross section in the region of the predentine-pul p border and within the predentine. (From Johanse n and Parks, 1962.)

cells show some endoplasmic reticulum and a few vesicles. Stained with lead hydroxide. (Approx. χ 10,800.)

Fig. 9. In the pulp adjacent to the predentine the odontoblasts are separate d by a matrix containing a few collagenous fibril s and bundles of collagenous fibrils (CF). Individual

Fig. 10. Within the predentine the odontoblast processe s do not appear to fil l the lumina of the dentinal canals. No membranou s structure is seen surrounding the lumen of the dentinal canal. Cf. Figs. 13 and 16. Stained with phosphotungsti c acid. (Approx. χ 19,500.)

ERLING

JOHANSEN

13. U L T R A S T R U C T U R E

stimuli and changing local conditions. The observed differences in structural relationship between the odontoblast and the wall of the dentinal canal might, however, be due to fixation artifacts, a possibility that cannot be ruled out. B. EXTRACELLULA R COMPONENTS

The extracellular substanc e of dentine, which represents the greater part of the tooth, occurs mainly in the form of a densely mineralized collagenous matrix. The principal constituents of this system are the collagen fibril s and the apatite crystallites which jointly impart the qualities of hardness , resilience, rigidity and structural stability, making dentine well adapted to its mechanical functions. The absence of resorptive mechanisms further ensures a tissue of enduring form. This does not mean, however, that the extracellular components of dentine remain constant throughout the lif e span of the tooth. Wit h advancing age there is an increase in both organic and mineral constituents which is brought about by appositional growth. This occurs through secondary dentine formation at the pulpal surfaces resulting in added thickness of the tissue and also through progressive sclerosis of dentinal canals with gradual obliteration of their lumina. I n mature dentine one may therefore distinguish between the original matrix formed during tooth development and that formed subsequentl y through appositional growth. Structural irregularities such as incremental lines may be encountere d in both types of matrices while interglobular dentine in the coronal portion of the tooth and the granular layer of Tomes in the root portion are usually associate d with the original matrix.

Figs. 11 and 12. Odontoblast processe s from fully formed human permanen t tooth in cross section and apparent unmyelinated nerve fibres in close associatio n with the processes . Stained with lead hydroxide. (Fig. 11 from Johanse n and Parks, unpublished observations , 1962; n and Parks, 1962.) Fig. 12 from Johanse

OF

DENTINE

45

Ultrastructural studies on the extracellular components of mature dentine have mainly been concerned with the identification and characteriza tion of the collagenous fibrils, the apatite crystallites and their arrangemen t within the tissue. The nature and characteristic s of the junctions and borders of dentine have also been explored, but very littl e work has been carried out on structural irregularities and developmenta l defects in dentine. 1. Organic Phase The organic matrix of dentine consists primarily of collagen with small amounts of mucopolysaccharides , and possibly some lipids. These various substance s have been identified and quantitated by chemical methods (Chapter 19), and their distribution has been determined by light microscopic and histochemica l procedures (Chapter 12). Through electron microscopic studies additional information on the structural organization of the matrix has been obtained, especially on the collagenous fibrils and their distribution. a. Collagenous framework. The collagen fibres observed in demineralized dentine by light microscopy have been found to consist of aggregate s of fibrils when studied with the electron microscope. These fibrils were first recognized in replicas and shadowed preparations of demineralized dentine and identified as collagen on the basis of their 640-700 Â cross-bandin g (Rouiller, Huber and Rutishausen , 1952; Bernick et al., 1952; Scott, 1955; Frank, 1959). Other fibrils in similarly prepared specimens failed to display this banding typical of collagen (Scott and Wyckoff, 1950). Although the cause of this difference in appearanc e was ascribed to the presence of ground substance masking the structural details of some Fig. 11. The structure resembling an unmyelinated nerve fibre (NF) contains mitochondria-like bodies with small, clear, circular spaces of varying diameter. (Approx. χ 19,500.) Fig. 12. At the predentine-dentin e border the fibrelik e structure (NF) in this section appears smaller and contains only one dark-staining body. (Approx. x 33,600.)

13. U L T R A S T R U C T U R E

OF

DENTINE

47

fibril s (Rouiller et al, 1952; Bernick et al, 1952), questions remained regarding their identity. Application of staining procedures with heavy-meta l preparations such as phosphotungsti c acid, lead solutions and uranyl acetate have subsequentl y eliminated this interference from ground substanc e (Johanse n and Parks, 1962), and sections stained wit h these preparations have clearly shown that the fibrous elements of dentine are collagenous in nature (Figs. 13-19). I n addition to the already mentioned cross-band s at approximately 640-700 Â intervals, dentinal collagen displays bands and interbands (Figs. 14a, b, c) resembling those noted in other naturally occurring mammalian collagens and in certain reconstituted collagens. These similarities indicate structural kinship between collagens from dentine and those from other tissues and permit application of findings on reconstituted collagen to the understandin g of dentinal collagen. Work on soluble collagens has led to the conclusion that collagenous fibrils represen t aggregate s of monomeric macromolecule s of rather constant size and molecular weight (Schmitt, Gross and Highberger, 1955; Schmitt, 1959). These structural units, referred to as tropocollagen molecules, have been characterize d as long, thin rods approximately 2800 ÷ 15 Â with a molecular weight of approximately 360,000. The tropocollagen molecule consists of a three stranded helix of amino acids with axial repeats occurring at 28.6 Â intervals (Crick and Rich, 1955; Rich and Crick, 1955). Polymerization of these macromolecule s takes place by end-to-end interaction resulting in long protofibrils which in turn constitute

the collagenous fibrils observed in electron microscopy. The conspicuous cross-banding s occurring at approximately 700 Â intervals along the fibril represent the junctions between these macromolecules. The occurrence of five such junctions along a 2800 Â distance of the fibrils is the result of an orderly staggering of tropocollagen molecules. The affinity of these regions for phosphotungstic acid stain is thought to indicate concentrations of arginine and lysine in the terminal regions of tropocollagen molecules (Kixhn, Grassman n and Hofmann, 1957). The less conspicuous bands found within the 700 Â period are thought to represen t areas with side chains of relatively large size, while the interbands contain side chains of smaller size. I n dentine these tropocollagen molecules appear to be polymerized into collagenous fibrils of varying widths. This has become apparent through measurement s of fibrils from different regions of demineralized dentine (Johanse n and Parks, 1962). The widest fibrils occurred in dentine proper and were located in intercanalicular areas. The majority of these fibrils showed widths of 600-700 Â. In contrast, those measuied in pericanalicular zones were considerably narrower with diameters in the range 250-500 Â . Collagenous fibrils of intermediate width, ranging from 400 to 500 Â , were found in the unmineralized predentine surrounding the pulp chamber. These regional differences in width suggest dimensional changes concomitant wit h the mineralization process. The fact that intercanalicular fibrils generally appear wider than those of predentine is indicative of appositional growth in intercanalicular areas. If this

Figs. 13-14. Matrix from demineralized dentine of fully formed human permanen t tooth illustrating feltwork of collagenous fibril s and cross-bandin g on individual fibrils. (From Johanse n and Parks, 1962.)

Fig. 14a. Two collagenous fibril s from predentine with cross-band s in register. Stained with lead hydroxide. (Approx. X 75,500.)

Fig. 13. The collagenous fibril s appear in longitudinal, oblique and cross-sectiona l views in matrix obtained from the vicinity of the predentine-dentin e border. A membranou s structure (MS) surrounds the lumen (L) of the canal. Stained with phosphotungsti c acid. (Approx. 17,800.) 5

Fig. 14b. Collagenous fibrils from intercanalicular area with major bands at approximately 700 intervals and asymmetrically placed interbands. (Approx. χ 49,000.) Fig. 14c. Collagenous fibril s displaying slit-lik e spaces (SS) possibly occupied by crystallites prior to demineralization. Stained with phosphotungsti c acid. (Approx. x 49,800.)

13. U L T R A S T R U C T U R E

OF

DENTINE

49

does take place, the necessar y precursors might have been located within the area at the time of mineralization, but it is also possible that they arrived with the mineral elements. However, the possibility cannot be discounted that the difference in width is an effect of mineralization. This hypothesis is based on the observation that demineralized collagenous fibril s often display slit-lik e spaces which in the intact tissue are believed to be occupied by crystallites (Fig. 14c). The apparent diminution of fibril s in pericanalicular areas is also difficul t to explain, but it might indicate separation of groups of protofibrils into small fibrils during mineralization although this does not seem very likely. More plausible is the possibilit y that the small fibrils of pericanalicular areas were formed subsequen t to mineralization of the erstwhile predentinal matrix in connection with the developmen t of the hypermineralize d zone. The arrangemen t and distribution of collagenous matrix has been observed in replicas (Rouiller et al, 1952; Scott, 1955) as well as in unstained and stained sections of demineralized dentine (Johanse n and Parks, 1962). The results of these studies have shown that the collagenous fibrils of the matrix are arranged in a trellis-like feltwork (Figs. 13, 15, and 16). This conclusion is based on the fact that collagenous fibrils in intercanalicular, pericanalicular and predentine zones may follow random courses in relation to the long axes of dentinal canals. I n sections stained with phosphotungsti c acid this is well illustrated by the occurrence of oblique, longitudinal and crosssectional views of fibrils in every field selected. Departures from this arrangemen t are, however, noted in some specimens . F or instance, in inter-

canalicular matrix a fan-like arrangemen t of fibrils reminiscent of von Korff fibres has been observed (Fig. 17). Around dentinal canals, groups of fibrils have appeare d to follow a circular or oblique course (Rouiller et al, 1952; Scott, 1955), whil e other groups of fibrils have displayed an orientation essentially parallel to the long axis of the dentinal canal (Figs. 18 and 19) (Johansen , unpublished data, 1965). The fibrils in all these various arrangement s appear to form a continuous structure throughout dentine with individual fibrils extending across zone borders without interruption even though the widths of fibrils vary between zones. Furthermore, the narrow fibrils of pericanalicular zones appear to be continuous with a membranous structure surrounding the lumen of dentinal canals (Fig. 16). This observation, together with a possible preferred orientation of fibrils adjacent to the dentinal canal, suggests that the membranous structure surrounding the lumen is at least in part collagenous in nature and that it belongs to the extracellular matrix (Rouiller et al, 1952; Johanse n and Parks, 1962). The membranous structure surrounding dentinal canals has been seen in replicas (Gerould, 1944; Syrrist, 1949; Syrrist and Gustafson, 1951 ; Helmke and Jahn, 1952), in homogenate preparations (Johansen , unpublished studies, 1963), in unstained sections (Bernick et al, 1952; Arwil l and Bloom, 1954) and in stained sections (Johanse n and Parks, 1962). The fullest view of this membrane has been obtained in replicas and in homogenate preparations after isolation from the rest of the tissue (Syrrist and Gustafson, 1951; Scott, 1953; Johansen , unpublished work, 1963). It has been found to consist of one, but sometimes two, main

Figs. 15 and 16. Matrix from demineralized dentine of full y formed human permanen t tooth showing pericanalicular zones, intercanalicular areas and a membranou s structure surrounding the lumina of dentinal canals. Stained with phosphotungsti c acid. (From Johanse n and Parks, 1962.)

canalicular areas exhibit wider fibril s more densely arranged. (Approx. x 15,800.) Fig. 16. Higher magnification showing continuity between the fibril s of the pericanalicular zone and both the membranous structure surrounding the lumen and the denser intercanalicular matrix. Granular material within dentinal canal may represen t remnants of cytoplasm from the odontoblast process. (Approx. x 38,600.)

Fig. 15. The pericanalicular zone (PZ) displays feltwork of sparsely distributed delicate fibril s while inter-

13. U L T R A S T R U C T U R E

stems from which secondar y and tertiary branches extend laterally (Fig. 20). In cross-sectiona l views the membrane may show a double lumen (Fig. 21) or a single lumen (Fig. 22) and bulbous expansions of the lumina occur at the origins of lateral branches (Fig. 16). Some investigators have described banding on the fibril s associate d with this structure (Helwig and Menke, 1949; Menke, 1950) but such fibril s are not apparent in the accompanyin g illustration (Fig. 20). This could, however, be a result of the procedures employed, since collagen at times wil l become denatured during microscopy if the specimen has not been sufficiently dehydrated prior to study. Observations on stained sections from various parts of dentine have shown that this membrane surrounds the lumen of the dentinal canal throughout the tissue except in the predentine (Figs. 8 and 10). Cross-sectiona l views of dentinal canals furthermore show that the membrane is thinnest in the vicinity of the predentine (Fig. 13) and considerably wider towards the peripheral part of the dentine (Figs. 16, 22). The interpretation that the membrane around dentinal canals represent s a specific arrangemen t of the matrix has been a matter of controversy. On the basis of replica studies it has been suggeste d that this structure represent s the limitin g membrane of the odontoblast process (Syrrist and Gustafson, 1951; Helmcke and Jahn, 1952; Scott, 1955). However, this suggestion does not seem compatible wit h the finding that the plasma membrane in areas of the predentine, where it can be clearly

Figs. 17-19. Matrix from demineralized dentine of fully formed human permanen t tooth illustrating particular arrangement s of collagenous fibril s in intercanalicular areas and adjacent to the lumen of the dentinal canal. Stained with phosphotungsti c acid. (Fig. 17 from Johanse n and Parks, 1962; Figs. 18-19 from Johansen , unpublished work, 1963.) Fig. 17. Collagenous fibril s from intercanalicular matrix diverging from a single fasciculus reminiscent of von Korff fibres. (Approx χ 49,000.) Fig. 18. Oblique section of membranou s structure surrounding dentinal canal displaying collagenous fibril s

OF

DENTINE

51

visualized (Figs. 11 and 12), is a delicate structure less than 100 Â in thickness. In peripheral areas the membranous structure is much wider, being more analogous to an extracellular capsule. The possibility that the pericanalicular membrane contains a high concentration of ground substanc e cannot be overlooked as banding is often difficult to discern. Also possible, but less likely, is the explanation that this membranous sheath contains a variety of organic molecules derived from the tissue flui d as well as from the protoplasm of the odontoblast and its processes . The relation of this membrane to the hyalinized odontoblast process described by Frank (1966) also deserves further attention. b. Other organic constituents. Fully formed dentine contains small amounts of mucopolysaccha rides distributed throughout the mineralized portion of the tissue. This constituent of dentine occurs in the form of polymers based on a repeat unit of modified hexose molecules such as glucosamine and galactosamine . These polymers have, however, not been resolved with the electron microscope to the extent of revealing structural characteristics . Their presence is merely noted by the masking of fibrils due to their close association with the collagenous matrix in intercanalicular areas (Fig. 23) and their amorphous appearanc e in pericanalicular zones (Plackovâ and Stëpânek , 1960). The mucopolysaccharide s may play a role in the nucleation phenomena in mineralization and

arranged with their long axes parallel to the long axis of the lumen (L). (Approx. x 16,200.) Fig. 19. Higher magnification of membranou s structure exhibiting cross-bandin g typical of collagen; lumen (L). (Approx. x 33,600.) Fig. 20. A branched tubular structure isolated from triturated dentine most likely representin g the membranou s structure seen surrounding dentinal canals in sections. The tubular structure consists of two main parallel trunks (T) to which secondar y (SB) and tertiary branches (TB) are attached. Specimen not stained. (Approx. x 13,700.)

ERLING

J O H A N S EN

13. U L T R A S T R U C T U R E

also have a function in the linkage of mineral and organic components of dentine. These possibilities were explored in a correlated electron microscopic and histochemica l study of hyperand hypomineralized rat dentine produced by injected fluoride and strontium (Yaeger, 1963a,b). I n this study it was found that a decrease d mucopolysaccharid e content of hypomineralized areas was associate d with fewer but larger crystallites while hypermineralize d areas contained an increased number of crystallites of normal diameter and larger amounts of mucopolysaccharides . However, additional studies along these lines are needed for clarification of the function of mucopolysaccharide s in dentine and for the characterizatio n of structural defects. Dentine also contains small amounts of lipids but their distribution within the tissue has not been clearly established . It is known from histochemical and electron microscopic studies that lipid-containing vacuoles are located within dentinal canals. The occurrence of lipids within the dentinal matrix has not been reported even though lipi d aggregate s might be visualized in osmiumfixed tissues. On the basis of removal of organic material through lipi d extraction procedures , apparent lipi d deposits were noted in pericanalicular regions (Shroff et al, 1956). However, this observation remains to be confirmed in stained preparations . 2. Mineral Phase The mineral phase of dentine has been identified as an apatite in the form of small crystallites. This conclusion is based on extensive X-ray Figs. 21-22. Dentinal canals from fully formed human permanen t tooth illustrating double lumen (Fig. 21) and single lumen containing collagenous fibril s (Fig. 22). Stained with phosphotungsti c acid. (From Johansen , unpublished work, 1962.) Fig. 21. Two membranou s structures shown in oblique section within demineralized pericanalicular zone (PZ) probably representin g two parallel trunks as shown in Fig. 20 (F). (Approx. χ 49,000.)

OF

DENTINE

53

diffraction data and an increasing volume of electron microscopic evidence. These methods of study have also yielded information on the size and shape of the crystallites as well as on the relative concentration of mineral within the tissue. a. Distribution of mineral. Electron microscopic studies of full y formed dentine have revealed a distinct pattern of mineral concentration within the tissue. This was first recognized in studies of replicas which showed a difference in degree of mineralization between the matrix immediately surrounding dentinal canals (pericanalicular zone) and that of intervening areas (intercanalicular). Based on the appearanc e of the dentinal matrix after polishing, and following acid treatment, it was concluded by some investigators that the pericanalicular zone was hypomineralized (Menke, 1950) and by others that it was hypermineralize d (Rouiller et al, 1952; Takuma et al, 1956; Shroff et al, 1954; Takuma, 1960a; Awazawa, 1962). Studies on ultrathin sections of the mineralized tissue have subsequentl y confirmed that the pericanalicular zone is hypermineralize d (Fig. 24) (Frank, 1959; Johanse n and Parks, 1959, 1962; T a k u ma 1960b). Such studies have also establishe d that a hypermineralize d zone exists around the lateral branches of dentinal canals (Fig. 25) and that the electron density of this zone (Fig. 26) corresponds to that of main dentinal canals (Fig. 24) (Johansen , unpublished data, 1965). The difference in mineral content between the pericanalicular zone and intervening matrix is illustrated in Fig. 24 where a sharp line of demarcation is seen separating the pericanalicular zone from the intercanalicular area. The illustration Fig. 22. Oblique and cross sectional views of collagenous fibril s within the lumen of dentinal canal. (Approx. x 28,200.) Fig. 23. Low power view of section of demineralized human dentine stained with lead hydroxide. Individual collagenous fibril s are discerned with difficulty, possibly due to the presence of ground substance . With this technique the border of the canals always stained darker than the intercanalicular matrix. (Approx. x 12,500.) (From Johansen and Parks, 1962.)

ERLING

J O H A N S EN

13. U L T R A S T R U C T U RE

OF

DENTINE

55

further shows randomly distributed light and dark fields within the intercanalicular areas, suggesting regional variation in mineral concentration within these areas. Whether this represent s a true variation or an apparent one due to the orientation of crystallites and/or uneven thickness of sections has not been established . Some of the dark fields could possibly represen t obliquely or longitudinally cut pericanalicular zones of branches of dentinal canals. The hypermineralize d pericanalicular zone of primary and secondar y dentinal canals is not of uniform width along the course of its canal. The zone appears widest in the middle of dentine proper wit h a gradual narrowing towards the enameldentine and dentine-cementu m junctions. In the vicinit y of the predentine-dentin e border it is inconspicuous or not perceptible (Takuma, 1960a). The growth of this zone after dentinogenesi s appears to occur by gradual apposition of mineral on the walls of the canals resulting in a narrowing of the lumina (Fig. 27) (Nalbandian, Gonzales and Sognnaes , 1960). Diminution of the lumen may also occur by mineral deposition within canals in response to injury or irritation such as dental caries (Fig. 28). Both of these processe s frequently lead to partial or complete obliteration of canals as shown in Figs. 29 and 30. b. The apatite crystallites. A major contribution of ultrastructural research to the study of dentine was the identification of the mineral phase as an apatite. This was first achieved by Gross (1926) with X-ray diffraction methods and has subsequentl y been substantiate d by numerous investigators employing similar procedures (Roseberry, Hastings and Morse, 1931; Thewlis, 1932; Trautz et al, 1953). It has also been shown that normal, transparen t and opaque dentine from the same tooth have identical X-ray diffraction patterns (Bale, Hodge and Warren, 1934). Selected

area diffraction studies with the electron microscope have also confirmed the apatite nature of dentine mineral (Johanse n and Parks, 1960). The designation of the mineral phase of dentine as an apatite is based on the characteristic s of its X-ray diffractograms. These diffractograms, as well as those obtained on bone and enamel, have revealed atomic arrangement s similar to those of naturally occurring mineral apatites (deJong, 1926; Thewlis, 1932). While X-ray diffractograms of dentine can only be obtained on aggregate s of crystallites (powder patterns), the naturally occurring apatites can be studied in single crystals. For this reason, calculations of the spatial arrangement of constituent atoms were first accomplishe d on naturally occurring apatites (Mehmel, 1930; Naray-Szabo , 1930). In subsequen t studies on biological and laboratory-produce d apatites, comparable data have been developed and refined for synthetic hydroxyapatites and for the mineral of dentine, bone and enamel (Carlstrom and Engstrom, 1956). Investigations of this nature have establishe d that apatites consist of a repeating unit termed the unit cell. The spatial arrangemen t of atoms making up this unit cell has been defined by Trautz (1955) as follows: ' T he hexagona l unit cell which is the smallest space unit of the structure containing all the crystallographic symmetry elements of the whole crystal, is a parallelepipedo n whose edges are formed by the two horizontal " a" axes, enclosing an angle of 120°, and by the vertical " c " axis at right angles to the " a" axes. The unit cell contains 10Ca++, 6 P 0 4 — , and 2 OH~ ions. The phosphate oxygens are arranged in tetrahedra l groups enclosing the phosphorus and are tied more strongly to it than to the C a+ + ions, which are intersperse d between the P 04 groups holding them in the structure together. When the crystal grows from the solution, the P 04 groups as

Fig. 24. Mineralized matrix from dentine of fully developed human permanen t tooth illustrating dentinal canals (C) surrounded by a hypermineralize d pericanalicular zone (PZ) approximately 0.5 μ wide. The intercanalicular

matrix (IC) shows an irregular but generally less dense distribution of mineral matter, occasionally displaying a banded appearance . (Approx. X 17,800.) (From Johanse n and Parks, 1962.)

13. U L T R A S T R U C T U R E

such are built into the crystal. The two OH~ ions sit on the hexagona l " c " axis, each surrounded by three C a+ + ions at the same level. The other four C a+ + ions occupy positions on the two vertical trigonal axes which pass through the cell at one third and two thirds along the long cell diagonal." However, the unit cell is not of constant composition; an isomorphous substitution of C a + + by S r+ +, M g + + and perhaps N a + and of O H" by F~ has been observed. The exact location of the carbonate in apatites has not been agreed upon but in the well-crystallized mineral carbonate apatites a considerable substitution of P 0 4 by C 0 3 has been detected by the shortening of the a axis (Trautz, 1960). A surface location of the carbonate as generally considered for biological apatites would, however, not affect the length of the a axis. A s an example of the dimensions of the unit cell, the values reported by Trautz (1960) for human enamel containing 2 - 3% of C 0 3 by weight and 0.01% F are given: a axis = 9.441 Â ; c axis = 6.884 Â. X-ray diffraction data have also formed the basis for calculations of the shape and size of the crystallites of dentine. The results arrived at by this procedure could support the concepts of both needle-shape d and plate-like structures (Trautz et al., 1953). The computations further indicated that average lengths of crystallites are between 200-300 Â (Bale et al, 1934; Jensen and Moeller, 1948; Trautz et al, 1953). However, fir m conclusions on these points could not be reached because of the small size of the crystallites and their random distribution within the tissue. Figs. 25-30. Mineralized matrix of dentine of fully formed human permanen t tooth illustrating secondar y canals and various degrees of occlusion of primary dentinal canals. Fig. 25. Low power micrograph of intercanalicular matrix with secondar y canals (SC) surrounded by hypermineralized pericanalicular zones. (Approx. χ 12,500.) Fig. 26. Higher magnification of a secondar y canal demonstratin g the difference in degree of mineralization between hypermineralize d pericanalicular zone and the surrounding intercanalicular matrix. (Approx. χ 33,600.)

OF

DENTINE

57

The earliest electron microscopic observations on the crystallites of dentine resulted in conflicting conclusions as to their size and shape. Some investigators referred to the crystallites as plate-like (Watson and Avery, 1954; Little, 1955) while others considered them to be needle-like structures (Takuma, 1960a,b; Pautard, 1960). The basis for this controversy is readily apparent upon examination of electron micrographs of thin sections of dentine as seen in Fig. 31. These illustrations show that some crystallites appear as narrow, dense images resembling needles while others are definitely plate-like in form. Some punctate images are also apparent. A t the edge of sections where crystallites can be seen without superimposition , plate-like crystallites always predominate (Fig. 32). These findings raised the possibility of dentine containing both plate-like and needle-shape d crystallites. To resolve this problem, a study was carried out with stereoscopi c techniques which made it possible to observe individual crystallites at different angles (Johanse n and Parks, 1960). These studies showed that crystallites optimally orientated with regard to the axis of til t would appear as narrow dense profiles in one view and as broad, less dense structures in another view (Fig. 34a,b). It would be expected that if some or all of the narrow dense profiles were needle-like structures they would display approximately the same outline and density irrespective of the tilt . However, since the crystallites did change in the manner indicated, it was concluded that the crystallites of dentine are plate-like structures which, when viewed on edge, present a narrow dense Fig. 27. Increased thickness of the pericanalicular zone resulting in narrowing of the lumen. (Approx. x 15,400.) Fig. 28. Higher magnification of single canal showing partial occlusion due to mineral deposits (MD). (Approx. x 33,600.) Fig. 29. Two narrow lumina of dentinal canals surrounded by heavy mineral deposits. (Approx. x 15,400.) Fig. 30. Nearly complete occlusion of canalicular lumen by mineral deposits. (Approx. χ 15,400.)

58

ERLING

J O H A N S EN

13. U L T R A S T R U C T U R E

profile. The punctate images which always appeare d in areas of possible superimposition of structures were interpreted as representin g views of crystallite fragments in one or more layers. These observations made on sections have subsequentl y been confirmed in the study of collagenous fibrils isolated from homogenat e preparations (Figs. 35 and 36) (Johanse n and Parks, 1962). I n order to establish definite sizes of individual crystallites, measurement s were made on structures that on the basis of our stereoscopi c techniques could be identified as single crystallites (Johanse n and Parks, 1960). The average thickness was found to be 20-35 , and lengths up to 1000 Â were measured . A t the predentine-dentin e border the crystallites were found to be generally smaller and less dense than those of dentine proper (Fig. 33). These dimensions indicate that 100-200 unit cells extend along the c axis to establish the length of the crystallites. The values for thickness indicate that the crystallites are made up of 2-5 unit cells in this dimension. The impression has further been gained that the broad surfaces of individual crystallites appear pitted and irregular, indicating considerable variation in actual thickness (Johansen, unpublished observations , 1965). 3. Collagen-Crystallite

Relationship

Thin sections of fully formed dentine studied wit h the electron microscope often reveal a banded pattern in intercanalicular areas suggesting a

Figs. 31-34. Mineralized matrix of dentine of fully formed human permanen t tooth illustrating the morphology of apatite crystallites. (From Johanse n and Parks, 1960, 1962.) Fig. 31. Thin section of intercanalicular matrix of undemineralize d sound dentine embedde d in methacrylate displaying a lack of uniformity in density of mineral deposition. Narrow dense profiles, punctate images and platelik e structures can be seen. (Approx. χ 66,000.) Fig. 32. The crumbled edge of a thin section of unembedde d sound dentine showing the crystallites in broad surface view in the thinnest parts and in edge view (dense narrow profiles) in the thicker parts. (Approx. χ 85,500.)

OF

DENTINE

59

special relationship between crystallite distribution and the periodicity of collagenous fibrils (Fig. 25). This relationship could not, however, be conveniently and reliably investigated in sections due to the difficult y in obtaining long segments of single fibrils free of superimposition . For this reason individual fibrils and groups of fibrils were isolated from homogenat e preparations and subjected to study (Johanse n and Parks, 1962). The results of this investigation showed that the crystallites are located within, as well as on the surface, of the fibrils and that their arrangemen t along the fibril is orderly. Where the crystallite-collagen complex appeare d intact, the crystallites were orientated with their long axes parallel to, or approximately parallel to, the long axis of the fibril (Figs. 35 and 36). The orderly arrangemen t was also apparent in the banding or structural periodicity manifested by these fibrils (Figs. 35a and 36a). The periodic unit, which was approximately 700 Â in length and corresponde d to the main bands noted in demineralized and unmineralized collagen (Figs. 14a,b,c), had a light and dark component (Figs. 35a and 36b). Most frequently the dark band was broader than the light one, but sometimes they were of nearly equal width. The structural basis for these bands could not be determined, but they appeare d to reflect the orientation rather than the length of the crystallites. Measurement s on crystallite dimensions frequently revealed lengths two thirds to one and a half times

Fig. 33. Thin section of embedde d unstained dentine showing the mineralization front of the predentine-dentin e border. The crystallites in this region are generally smaller, less dense and more sparsely distributed than in fully mineralized dentine. (Approx. χ 85,500.) Figs. 34a and 34b. Two aspects of the same field were obtained by taking a micrograph before and after tiltin g the specimen through 30° ( ± 1 5° from the horizontal). Profiles, of crystallites lying more or less parallel to the axis of til t (arrow), that appear thin in one view are appreciably broader in the other view, thus indicating the plate-like shape of the crystallites. (Approx. χ 75,500.)

13. U L T R A S T R U C T U R E

that of the repeating period. It was also of interest that where two or more fibril s were tightly bound together in the matrix, the alternating light and dark bands were in register across the fibrils. A similar arrangemen t was observed in stained preparations of unmineralized and demineralized dentine where adjacent fibril s displayed cross bandings in register (Figs. 14a,c). Individual fibrils obtained from the vicinity of the predentine-dentin e border also displayed banding, but only in the thicker parts (Figs. 35c and 36c). The crystallites associate d wit h these fibrils were considerably smaller than those encountere d in peripheral dentine as already noted in sections. Of special interest was the observation that some fibrils were calcified through part of their length and not in the remainder (Figs. 12 and 35c). Other fibrils from such preparations displayed unmineralized segments between calcified parts (Fig. 36c). It is assume d that such fibrils were orientated approximately parallel to the calcification front with a small loop projecting into the predentine. The nature of the relationship between the collagenous fibrils and the apatite crystallites can at present only be speculate d upon. The fact that the crystallites in intercanalicular areas are closely associate d with the collagenous fibrils is suggestive of the fibril playing a role in the nucleation phenomenon. Several mechanism s have been considered by which this might occur in the mineralization of bone, including specific electric charge distribution along the fibril, spatial configuration of certain amino acids for binding of calcium or phosphate ions, and specific amino acid groups serving as templates for apatite lattice formation (Neuman and Neuman, 1958; Glimcher, 1960). Whil e such mechanism s may be operational in Figs. 35a-35c. Relationship between apatite crystallites and collagen in isolated collagenous fibrils from homogenize d dentine preparations . The crystallites are orientated with their long axes parallel to the long axis of the fibrils. Alternating light and dark transverse bands are seen clearly in (a) and (b), and faintly on the left side of (c). (From Johanse n and Parks, 1962.)

OF

DENTINE

61

intercanalicular areas, their contribution in the mineralization of pericanalicular zones is more doubtful. This view is based on the finding that pericanalicular zones contain a high concentration of crystallites and only few and small collagenous fibrils. The observation that individual fibrils at the predentine-dentin e border display both mineralized and unmineralized segments , further complicates the picture and suggests the participation of other substances , lik e mucopolysaccha rides, in the nucleation process. 4.

Junctions and Surrounding Borders

Dentine is surrounded by tissues which differ to an extreme degree in physical characteristic s and composition. In the coronal portion the external surface of dentine is covered by the inert enamel and in the root portion by the densely mineralized and avascular cementum. The borders between these tissues and dentine are the socalled enamel-dentin e and dentine-cementu m junctions which in light microscopy appear as distinct structures. The internal surface of dentine abuts on the highly vascularized dental pulp with the interphase of the tissues being the predentine-pul p border. The nature of these various boundaries has been studied with the electron microscope and the findings wil l be presente d and related to the general phenomeno n of junctions between tissues. a. Enamel-dentine junction. Epithelial and adjoining connective tissues are normally separate d by membranous structures. Electron microscopic studies of epidermal tissues of mammals have revealed an apparently homogeneou s 350 thick membrane which follows the basal contours of the epithelial cells. This membrane is separate d Fig. 35a. Three interconnecte d fibrils, two of which are joined to form a larger unit at the left. (Approx. x 90,900.) Fig. 35b. Single fibril exhibiting large crystallites in both broad surface and edge views. (Approx. χ 138,200.) Fig. 35c. Fibrils isolated from the predentine-dentin e border area, each displaying a mineralized (T) and a nonmineralized (U) segment. Note small size of crystallites. (Approx. X 73,800.)

13. U L T R A S T R U C T U RE

from the cells by a space of about 300 Â (Montagna, 1962). Prior to matrix formation, a similar membrane and space have been observed between the ameloblasts and the odontoblasts in the continuously growing incisors of mice (Nylen and Scott, 1958) and rats (Fig. 37) (Bruns, personal communication, 1962). Wit h subsequen t scalloping of the enamel-dentin e junction during matrix formation and mineral deposition, this membrane probably disappears (Fig. 38) as the large crystallites of enamel and the small crystallites of dentine appear in juxtaposition in the fully mineralized tissue (Figs. 39a,b) (Johansen , unpublished work, 1965; Hohling, 1961). The new junction between enamel and dentine thus consists of interdigitating crystallites, a structural arrangemen t that probably forms the basis for the strong union between the two tissues.

OF

DENTINE

63

(Fig. 40) (Johansen , unpublished data, 1965; Herting, 1963). c. Predentine-pulp border. The predentine-pul p border represent s the interphase between two rather dissimilar connective tissues, the dentine and the pulp. The fact that the matrix adjacent to the pulp, the predentine, is not mineralized establishe s a transitional zone between the highly vascularized pulp and the heavily mineralized dentine. The inner border of the predentine is readily recognized by its rather densely arranged collagenous fibrils as compared to the adjacent pulp (Figs. 8 and 9).

III. THE RELATION OF STRUCTURE TO SOME ASPECTS OF PHYSIOLOGY

b. Dentine-cementum junction. Adjoining connective tissues are usually integrated and show no demonstrabl e separating membrane. In view of this structural organization, the nature of the dentine-cementu m junction, which is readily visualized in light microscopy, is of special interest. Electron microscopic studies of developing mouse teeth have shown that during root formation a thin membrane occurs between the developing dentine and Hertwig's epithelial root sheath. Wit h the disintegration of the root sheath and formation of cementum this membrane apparently disappears as no distinct border has been discerned between the calcified matrices of dentine and cementum (Selvig, 1963). Likewise, in full y formed rat and human teeth, the collagenous fibrils of dentine and cementum are in direct apposition

One of the features of teeth which are of importance in the maintenanc e of the dentition as a characteristic of the vertebrates is the fact that the extracellular components of mature dentine are excluded from active participation in the general metabolism of the body. Ordinarily the tissue appears insensitive and irresponsive to hormonal and other factors governing metabolic processes . This condition is in marked contrast to the high sensitivity of the tissue in its formative phase (Kreshover, 1960). Disturbances in metabolism during matrix formation and calcification often result in structural aberrations which in extent and distribution are proportional to the disturbance. These defects, in the form of incremental lines and interglobular dentine, remain apparently unaltered throughout the lif e span of the tooth.

Figs. 36a-36c. Relationship between apatite crystallites and collagen in isolated collagenous fibrils from homogenize d dentine preparations . (From Johanse n and Parks, 1962.)

Fig. 36b. Here the difference in length of the dark and light components of the periodic fibrilla r unit appears less marked than in (a). The lengths of some of the more obvious segments are marked on the picture. (Approx. x 84,700.)

Fig. 36a. Two fibril s are joined together to form a single unit in the lower half of the picture; the periodicity of both fibrils is seen to be in register. Here the dark component of the periodic unit seems to be longer than the light component. (Approx. χ 110,400.) 6

Fig. 36c. This fibril , taken from the dentine-predentin e border region, shows an unmineralized segment between two mineralized segments . The crystallites are considerably smaller than in mature dentine. (Approx. χ 98,400.)

ERLING

J O H A N S EN

13. U L T R A S T R U C T U RE

OF

DENTINE

65

Isotope studies have, however, shown that there is a passive ionic exchange between dentine and the surrounding tissue fluid (Sognnae s and Shaw, 1952). This phenomeno n may be related to subtle changes in the composition of the mineral phase noted with increasing age of the tooth (Brudevold, 1957). The structural stability of dentine is readily related to qualities inherent in the extracellular and cellular components of the tissue. The collagenous fibril s of the organic matrix are inert metabolically and essentially insoluble in the tissue fluid. The apatite crystallites of the mineral phase are also stable structures and effectively protected against dissolution by the tissue flui d which is supersaturate d with regard to common ions (Neuman and Neuman, 1958). Breakdown of these structural elements by forces normally operating within the body can therefore be achieved only through cellular resorption. Such a mechanism plays no role in the normal physiology of dentine except in the resorption of roots in preparation for the shedding of deciduous teeth. However, under pathological conditions cellular resorption of the dentine of permanen t teeth is occasionally encountere d in periodontal disease , hyperparathyroidis m and radiation injury (Sognnaes , 1963). The cells responsible for dentine resorption resemble osteoclast s and they cannot be considered a normal cellular component of dentine. The odontoblast, which is the only cell constantly associate d with the tissue, does not posses s resorptive capabilities. Its primary function appears to be

appositional through the formation of constituents of the organic matrix. The presence of apparent nerve fibres in close association with some odontoblast processe s provides one explanation for the sensitivity of dentine. This finding does not, however, rule out the participation of the odontoblast and its processe s in the initiation and transmission of sensory stimuli in dentine (Avery and Rapp, 1959). Neither does it exclude the possibility that a hydrodynamic mechanism exists for the stimulation of nerve fibres in the pulp (Brannstrom and Astrôm, 1964). More detailed observations on the relation of the odontoblasts and their processe s to the pulpal nerve fibres are necessar y before the strutural basis for the sensitivity of dentine can be settled.

Figs. 37-40. Enamel-dentin e and dentine-cementu m junctions from rat incisor and rat molar. (Figs. 37 and 38 from Bruns, personal communication, 1962.)

Fig. 39a. In fully developed dentine (D) from the rat incisor the large crystallites of the enamel (E) are in juxtaposition with the small crystallites of dentine (see arrows). (Approx. χ 46,900.)

Fig. 37. At the developing end of the rat incisor a space (S) occurs between the limitin g membrane of the ameloblast (A) and a membranou s structure marking the border of the developing dentinal matrix (DDM). Note the straight course of this membrane . (Approx. χ 15,400.) Fig. 38. With mineralization of both the enamel (E) and the dentinal matrix (D) scalloping of the border occurs with interdigitation of the two tissues. (Approx. x 12,900.)

IV. PATHOLOGIC ALTERATIONS The structural stability inherent in dentine is often not sufficient to uphold the integrity of the tissue when it is exposed to deleterious substance s present in the oral cavity. Changes in structure and composition frequently result when such substance s in the form of acids and enzymes accumulate in bacterial plaques and remain in contact wit h tooth surfaces for prolonged periods of time. Under these circumstance s demineralization, partial remineralization and proteolysis of underlying tissues take place with formation of carious lesions (Johansen , 1963, 1965). Alterations

Fig. 39b. Higher magnification of crystallites in juxtaposition. (Approx. χ 67,200.) Fig. 40. Dentine-cementu m junction of rat molar illustrating continuity of the mineralized collagenous matrices of the two tissues. Dentine (D); cementum (CE). (Approx. χ 13,700.)

13. U L T R A S T R U C T U R E

in the ultrastructure of dentine brought about by these processe s wil l be considered briefly. Soft carious dentine from advanced lesions of human teeth studied in sections has revealed the typical morphologic pattern of sound dentine, namely that of dentinal canals, intercanalicular matrix and frequently hypermineralize d pericanalicular zones (Figs. 41 and 46) (Johanse n and Parks, 1961). The relative proportions of these various structures are, however, different as the extent of intercanalicular matrix is reduced while dentinal canals are distended. The lumina of most canals are filled with bacteria that have replaced the cellular components normally occupying dentinal canals (Figs. 41 and 46) (Scott and Albright, 1954; Bernick, Warren and Baker, 1954). Other canals appear empty, containing neither bacteria nor cellular substance . Between the lesion and the pulp, these canals usually contain heavy mineral deposits which occlude their lumina (transparen t dentine), but also a few patent canals are encountered in this zone (Takuma and Kurahashi, 1962). Studies on the organic phase of carious dentine after demineralization and staining with phosphotungstic acid revealed a number of changes in the matrix (Johanse n and Parks, 1961; Johansen , 1965). The membrane which in the sound tissue surrounds the canalicular lumen is frequently absent or partially missing in the canals containing bacteria (Fig. 41), while the empty canals retain the membrane . In the adjacent pericanalicular

Figs. 41-45. Carious dentinal matrix from demineralized fully formed human permanen t teeth. Stained with phosphotungstic acid. (Figs. 41, 42 and 45 from Johansen , 1965; Figs. 43 and 44 from Johanse n and Parks, 1962.) Fig. 41. Longitudinal section of dentinal canal filled with bacteria (B). A membranou s structure similar to that lining the canal in the sound tissue is evident at M but missing at the arrow to the left. The intercanalicular matrix displays distinct collagenous fibril s in cross section (CF). The pericanalicular zone (PZ) contains fine filaments. (Approx. χ 12,000.)

OF

DENTINE

67

areas the matrix consists of fine filaments (Fig. 42) or of a fine granular substanc e quite different in appearanc e from the narrow collagenous fibrils seen in the correspondin g location of sound dentine. It has not been determined whether this change in structure represent s a replacemen t by extraneous material, an alteration in the collagen or possibly a combination of both. Some changes were also detected in the matrix of intercanalicular areas, but only in the superficial parts of lesions. In this location groups of fibrils appeare d to have been destroyed and replaced by microorganisms while other groups of fibrils were in a state of partial breakdown (Fig. 43). However, many fibrils appear unaffected by the carious process, as can be seen in Fig. 44. In deeper parts the collagenous matrix of intercanalicular areas may be remarkably well-preserved with individual fibrils displaying the cross-bandin g typical of collagen even after total loss of associate d crystallites (Fig. 45). Observations on the mineral phase of carious dentine show a marked decreas e in density of crystallite distribution indicating a generalized demineralization of the tissue (Johanse n and Parks, 1961). The decreas e is most noticeable in intercanalicular areas where sparsely distributed crystallites are scattered more or less randomly (Figs. 46-48). Pericanalicula r zones also show a decreas e in mineral content, but remain relatively densely mineralized when compared to intercanalicular areas (Fig. 47). Individual crystallites observed

Fig. 42. Pericanalicula r zone (PZ) displaying fine filaments; adjacent intercanalicular matrix shows collagenous fibril s (CF). (Approx. x 28,200.) Fig. 43. A region of intercanalicular matrix where apparent breakdown of collagenous fibril s (CF) is taking place; some fibril s appear granular and are surrounded by a fine-textured material. (Approx. χ 28,200.) Fig. 44. Seemingly (Approx. x 31,100.)

unaltered

collagenous

fibrils.

Fig. 45. This isolated fibril from homogenat e preparation, devoid of crystallites, displays cross banding. Unstained. (Approx. χ ^24,900.)

13. U L T R A S T R U C T U R E

in pericanalicular as well as in intercanalicular areas are plate-like in shape and in general resemble those of the sound tissue (Figs. 48 and 49). They are also found to be arranged wit h their long axes parallel to the long axes of collagenous fibrils when studied in homogenat e preparations (Fig. 50). Some carious lesions of human dentine studied wit h soft X-ray techniques have been found to contain densely mineralized bodies scattered throughout the tissue (Banez and Johansen , 1964, 1965). Their sizes vary from a few microns up to more than one hundred microns and their borders are usually well defined. A more detailed examination of these formations with the electron microscope have revealed that some consist of large elongated crystallites (Figs. 51a, b), never observed in the sound tissue. A t the border, these large crystallites are intermingled with the smaller plate-like crystallites usually found in carious dentine (Fig. 51b). In the centre, the large crystallites occupy intercanalicular areas, pericanalicular zones and the lumina of dentinal canals (Fig. 51c). U p on demineralization such formations reveal a matrix different from that commonly observed in carious dentine. The collagenous fibrils of intercanalicular areas vary considerably in size and frequently display a granular appearanc e (Fig. 5Id). They are separate d by clear spaces that presumably contained crystallites prior to demineralization . I n the pericanalicular zone the matrix consists Figs. 46-50. Distribution of mineral and morphology of crystallites observed in carious dentine from fully formed human permanen t teeth. (Figs. 46-50 from Johanse n and Parks, 1962.) Fig. 46. Undemineralize d soft carious dentine near superficial edge of lesion, showing three dentinal canals (C). The canals are distended and filled with micro-organisms (B). The intercanalicular matrix is largely, but incompletely, demineralized by the carious process and contains some scattered micro-organisms (B). A heavily mineralized discontinuous lining of the canals appears in some places as a black border at the edge of the intercanalicular matrix. (Approx. x 11,200.) Fig. 47. Undemineralize d section of soft carious dentine displaying a thick densely mineralized layer interposed

OF

DENTINE

69

of fine filaments and granular material (Fig. 5Id). The most probable explanation for these formations is that they have resulted from a remineralization process of the carious tissue, and that they do not represent the original dentinal matrix. This point of view is supported by the fact that some of these densely mineralized formations are definitely the products of remineralization because they occupy spaces created through the destruction of the tissue by the carious process (Fig. 52). It should be noted, however, that the crystallites of these particular formations differ from those previously discusse d as they are more irregular in shape and more variable in size. The frequency of relatively large crystallites is also higher in the latter formations which resemble those observed by Lenz (1955) within the lumina of dentinal canals.

V. CONCLUDING REMARKS This chapter presents a consideration of the ultrastructure of full y formed dentine based on electron microscopic and X-ray diffraction studies. The main cellular component of dentine, the odontoblast, has been characterize d in considerable detail as to structure and organization. The finding of fewer and smaller cytoplasmic organelles in the odontoblast of full y formed dentine as compared to the developing tissue is interpreted as suggesting between the lumen of the canal (C) and the extensively demineralized intercanalicular matrix (IC). (Approx. X 31,100.) Fig. 48. Undemineralize d soft carious dentine exhibiting sparsely distributed crystallites in broad surface view and in narrow profile view. (Approx. x 63,900.) Fig. 49. Undemineralize d soft carious dentine showing the somewhat crumbled edge of a section taken from unembedde d material. The crystallites appear thin and dark in edge view and broad and pale in broad surface view. (Approx. x 83,000.) Fig. 50. Isolated collagenous fibril from homogenat e preparation displaying faint banding in some areas. A few crystallites in both broad surface view and narrow profile view are seen associate d with the fibril. (Approx. x 90,500.)

ERLING

J O H A N S EN

13. U L T R A S T R U C T U RE

active participation of the cell in matrix formation. Further studies are, however, needed to substantiat e this contention. The answer might be found in ultrastructural studies on odontoblasts participating in secondar y dentine formation following a quiescent period upon completion of dentinogenesis . Combined autoradiographi c and electron microscopic studies would further help in elucidating the role of the odontoblast in matrix formation by the identification and localization of labelled constituents within cytoplasmic organelles or in the matrix or in both. Further studies are also needed to identify the pulpal cells possessin g the potential of differentiating into odontoblasts . I t would furthermore be of interest to observe the structural changes accompanyin g this process of cell differentiation in response to irritation and injury. Such studies might also clarify the structural relation of newly formed odontoblasts to existing dentinal canals in conjunction with limited cell destruction in the odontoblast layer.

OF

DENTINE

71

The odontoblast process within predentine and withi n the adjacent dentine proper displays structures similar to those observed in the distal portion of the odontoblast. In peripheral parts of dentine technical difficulties associate d with fixation and sectioning have so far prevented detailed observation on this part of the process. However, lipi d vacuoles and granular material have been observed withi n dentinal canals in this location but these may be manifestations of poor fixation.

The recent identification of nerve-like structures in close association with odontoblast processe s is of great interest having regard to the high sensitivit y of dentine, but further studies are needed to clarify the relation of these nerve fibres to the odontoblast processes . Also, the peripheral extension of these nerve-like structures needs elucidation as the enamel-dentin e and dentinecementum junctions are particularly sensitive. Another cellular component associate d with dentine is the osteoclas t during dentinal resorption, but no ultrastructural studies have been conducted on dentine being broken down by this process. The collagenous matrix of the extracellular component of dentine occurs as a feltwork of fine fibrils. The width of these fibril s varies in different parts of dentine. Fibrils of relatively large diameter characterize the intercanalicular matrix while pericanalicular zones contain fibril s of smaller diameter. Predentine contains collagenous fibrils of intermediate width. Several possible explanations have been set forth for these regional differences, but additional studies are required for definitive conclusions. I n close association with the collagenous matrix a membranous structure is observed surrounding dentinal canals. The nature of this membrane has not been conclusively established , but on the basis of replica studies it has been regarded by some workers as the plasma membrane of the odontoblast process and by others as a constituent

Figs. 51-52. Densely mineralized body in dentine of carious lesion of fully formed human permanen t tooth studied in sections. (From Banez and Johansen , 1964, 1965.)

Fig. 51c. Central portion of the densely mineralized body with large crystallites occupying intercanalicular areas (IC) and also completely obliterating the lumen of dentinal canal (ODC). (Approx. X 10,400.)

Fig. 51a. Low power view of the border between the densely mineralized body (DMB) and the extensively demineralized surrounding matrix (PM). (Approx. x 14,500.) Fig. 51b. Higher magnification of border shown in Fig. 51a. Note the large size and elongated form of crystallites within the densely mineralized body. The smaller crystallites normally found in carious dentine are also intersperse d between the large crystallites. (Approx. χ 40,300.)

Fig. 51d. Central portion of the densely mineralized body, demineralized and stained with phosphotungsti c acid. A few collagenous fibril s show banding but the majorit y appear finely granular and without bands. The pericanalicular zone (PZ) displays fine fibril s and a granular substance . (Approx. χ 27,400.) Fig. 52. Two dentinal canals (C) with remaining hypermineralized pericanalicular zones (PZ) and a densely mineralized body probably representin g a secondar y mineralization with minerals derived from the saliva. (Approx. x 12,000.)

72

ERLING

of the extracellular matrix. Observations on sections of dentine stained with heavy metals support the latter hypothesis. Additional investigations are, however, needed to settle this controversy. The mucopolysaccharid e constituent of the extracellular component has been noted primarily because of its masking effect on the collagenous fibrils. Its possible role in nucleation was discussed . The mineral phase of dentine has with X-ray diffraction methods been identified as an apatite on the basis of the spatial arrangemen t of atoms. Wit h electron microscopic techniques the crystallites have been visualized as plate-like and needlelik e structures. Through the application of stereoscopic techniques it has been demonstrate d that this difference in crystallite appearanc e can be explained on the basis of plate-like structures being viewed from different angles. The distribution of mineral within dentine occurs in a distinct pattern in relation to dentinal canals. I t has been found that intercanalicular matrix is relatively densely mineralized while a zone of hypermineralization exists around dentinal canals except in the vicinity of the predentine. Similar hypermineralized pericanalicular zones are observed around lateral branches of canals. These formations are thought to represen t inward appositional growth of pericanalicular matrix. The relation of apatite crystallites to collagenous fibrils can be observed in fibrils isolated from homogenized preparations of dentine and in these the apatite crystallites appear to be closely associated with the collagenous fibrils with the long axes of both structures arranged parallel. The calcified fibrils also show a banded appearanc e wit h a light and dark segment making up a periodic unit. These observations indicate that the collagenous matrix plays a role in the nucleation phenomenon , but the lack of mineralization of predentinal collagen remains unexplained. The various junctions and borders of dentine are best considered in relation to the general phenomeno n of junctions between tissues. The boundary between enamel and dentine (enameldentine junction) is delineated by the large crystal-

J O H A N S EN

lites of enamel and the small crystallites of dentine which occur in juxtaposition. However, the dentinecementum junction cannot be discerned as a border because the collagenous fibrils of dentine and cementum appear to be in direct apposition. The relation of the structure of dentine to some aspects of its physiology and pathology has been briefly considered especially with regard to the fact that the extracellular components of dentine are stable structures which do not participate actively in the general body metabolism. Other factors which contribute to the inherent stability of dentine are the super-saturatio n of the tissue flui d with regard to the ions of the apatite lattice and the slow rate of turnover of collagen in the internal environment of the body. However, when dentine is exposed to deleterious substance s originating in the oral cavity it is frequently altered in structure and composition. The ultrastructural changes observed in resultant carious lesions have been referred to. References Arwill , T. and Bloom, G. (1954). Some remarks on the structure of dentine as revealed by the electron microscope. Acta odont. scand. 12, 185-192. Avery, J. K. and Rapp, R. (1959). Investigation of the mechanism of neural impulse transmission in human teeth. Oral Surg. 12, 190-198. Awazawa, Y. (1962). Electron microscope investigation of the dentin with particular regard to the nature of the area surrounding the odontoblast process. / . Nihon Univ. Sch. Dent. 6, 31-54. Bale, W. F., Hodge, H. C. and Warren, S. L. (1934). Roentgen-ray diffraction studies of enamel and dentin. Amer. J. Roentgenol 32, 369-376. Banez, L. Ï . N. and Johansen , E. (1964). Correlated soft x-ray and electron microscopic studies on selected areas of carious dentin. / . dent. Res. 43, Suppl., 850-851 (Abstract). Banez, L. Ï . N. and Johansen , E. (1965). Further observations on selected areas of carious dentin utilizing correlated soft x-ray and electron microscopic techniques . Preprint. Abstr. Int. Ass. dent. Res., 43rd gen. Meet., Toronto, 1965 No. 126. Bernick, S., Baker, R. F., Rutherford, R. L. and Warren, O. (1952). Electron microscopy of enamel and dentin. /. Amer. dent. Ass. 46, 68-696.

13. U L T R A S T R U C T U R E Bernick, S., Warren, Ï . and Baker, R. F. (1954). Electron microscopy of carious dentin. / . dent. Res. 33, 20-26. Br nnstrom , M. and Astrôm, A. (1964). A study on the mechanism s of pain elicited from the dentin. / . dent. Res. 43, 619-625. Brudevold, F. (1957). Changes in enamel with age. Proc. 25th Year Celebration Univ. Rochester dent. Res. Fellowship Program pp. 185-192. Carlstrôm, D. and Engstrôm, A. (1956). Ultrastructure and distribution of mineral salts in bone tissue. In "The Biochemistry and Physiology of Bone" (G. H. Bourne, ed.), pp. 149-178. Academic Press, New York. Crick, F. H. C. and Rich, A. (1955). Structure of polyglycine. II . Nature 175, 780-781. deJong, W. F. (1926). Le substanc e minéral dans les os. Rec. Trav. chim. Pays-Bas 45, 445-448. Frank, R. M. (1959). Electron microscopy of undecalcified sections of human adult dentine. Arch, oral Biol. 1, 29-32. Frank, R. M. (1966). Étude au microscope électronique de l'odontoblaste et du canalicule dentinaire humain. Arch, oral Biol. 11. 179-199. Gerould, C. H. (1944). Ultramicrostructure s of human tooth as revealed by the electron microscope. / . dent. Res. 23, 239-245. Glimcher, M. J. (1960). Specificity of the molecular structure of organic matrices in mineralization. In "Calcification in Biological Systems", Publ. No. 64, pp. 421^187. Amer. Ass. Advanc. Sci., Washington, D. C . Gross, R. (1926). "Di e kristalline Strucktur von Dentin und Zahnschmelz" , Festschr. , p. 59. Zahnnârztl. Inst. Univ. Greifswald, Berlin. Helmcke, J.-G. and Jahn, B. (1952). Elektronenmikrosko pische Untersuchunge n uber das Dentin im menschliche n Zahn. Naturwissenschaften 39, 492-493. Helwig, G. and Menke, E. (1949). Elektronenmikroskopi e an Zellfortstze n im menschliche n Zahnbein. Naturwissenschaften 36, 281-283. Herting, H. C. (1963). Elektronenmikroskopisch e Untersuchunge n uber das Zahnwurzelzemen t des Menschen. Proc. 9th ORCA Congr. dent. Caries, Paris, 1962 pp. 303312. Pergamon Press, Oxford. Hôhling, H. J. (1961). Elektronenmikroskopisch e Untersuchunge n am gesunde n und kariosen Dentin mit Hilf e der Abdruckmethode und der Schnittmethod e an kompakter, nichtentmineralisierte r Substanz . Z. Zellforsch. 53, 192-200. Jensen , A. T. and Moeller, A. (1948). Determination of size and shape of the apatite particles in different dental enamels and in dentin by the x-ray method. / . dent. Res. 27, 524-531. Johansen , E. (1963). Ultrastructural and chemical observations on dental caries. In "Mechanisms of Hard Tissue Destruction", Publ. No. 75, pp. 187-211. Amer. Ass. Advanc. Sci., Washington, D. C.

OF

DENTINE

73

Johansen , E. (1965). Electron microscopic and chemical studies of carious lesions with reference to the organic phase of affected tissue. Ann. N.Y. Acad. Sci. 131, 776-685. Johansen , E. and Parks, H. F. (1959). Preliminary electronmicroscopic observations on carious dentin. / . dent. Res. 38, 693 (Abstract). Johansen , E. and Parks, H. F. (1960). Electron microscopic observations on the three-dimensiona l morphology of apatite crystallites of human dentin and bone. / . Biophys. Biochem. Cytol. 1, Ί4Ζ-Ί46. Johansen , E. and Parks, H. F. (1961). Electron-microscopi c observations on soft carious human dentin. / . dent. Res. 40, 235-248. Johansen , E. and Parks, H. F. (1962). Electron-microscopi c observations on sound human dentine. Arch, oral Biol. 7, 185-193. Kreshover, S. J. (1960). Metabolic disturbance s in tooth formation. Ann. N.Y. Acad. Sci. 86, 161-167. Kuhn, K., Grassmann , W. and Hofmann, U. (1957). (jber die Bindung der Phosphowalframs ure im Kollagen. Naturwissenschaften 44, 538-539. Lenz, H. (1955). Elektronenmikroskopische r Nachweis der Dentinverânderunge n durch Karies. Deut. Zahn- Mundu. Kieferheilk. 22, 24-33. Little, K. (1955). Electron microscope studies on teeth. /. dent. Res. 34, 778 (Abstract). Mehmel, M. (1930). The structure of apatite. Z. Krist. 75, 323-331. Menke, E. (1950). Elektronenmikroskopi e an der menschlichen Zahnhartsubstanz . Z. Anat. Entw.Gesch. 115, 1-18. Montagna, W. (1962). The epidermis. "The Structure and Function of Skin", 2nd ed., pp. 14-121. Academic Press, New York. Nalbandian, Á., Gonzales, F. and Sognnaes , R. F. (1960). Sclerotic changes in root dentin of human teeth as observed by optical, electron and x-ray microscopy. / . dent. Res. 39, 598-607. Nâray-Szabo , S. (1930). Structure of fluorapatite. Z. Krist. 75, 387-398. Neuman, W. F. and Neuman, M. W. (1958). "The Chemical Dynamics of Bone Mineral". Univ. of Chicago Press, Chicago, Illinois. Noble, H. W., Carmichael, A. F. and Rankine, D. M. (1962). Electron microscopy of human developing dentine. Arch, oral Biol. 7, 395-399. Nylen, M. U. and Scott, D. B. (1958). An electron microscopic study of the early stages of dentinogenesis . Publ. Hlth. Serv. Publ, Wash. 613. Pautard, F. G. E. (1960). Calcification in unicellular organism. In "Calcification of Biological Tissues", Publ. No. 64, pp. 1-14. Amer. Ass. Advanc. Sci., Washington, D. C.

74

ERLING

Plackovâ, A. and Stëpânek , J. (1960). Zur Kenntnis der peritubulâren Zone des Dentins. Z. Zellforsch. 52, 730738. Rich, A. and Crick, F. H. C. (1955). The structure of collagen. Nature 176, 915-916. Roseberry, H. H., Hastings, A. B. and Morse, J. K. (1931). X-ray analysis of bone and teeth. J. biol. Chem. 90, 395-407. Rouiller, C , Huber, L. and Rutishauser , E. (1952). La structure de la dentine. Étude comparée de l'os et l'ivoir e au microscope électronique. Acta anat. 16, 16-28. Schmitt, F. O. (1959). Interaction properties of elongate protein macromolecule s with particular reference to collagen (Tropocollagen). Rev. mod. Phys. 31, 349-358. Schmitt, F. O., Gross, J. and Highberger, J. H. (1955). Fibrous proteins and their biological significance. Symp. Soc. exp. Biol. 9, 148-162. Scott, D. B. (1953). Recent contributions in dental histology by use of the electron microscope. Int. dent. J. 4, 64-95. Scott, D. B. (1955). The electron microscopy of enamel and dentin. Ann. N.Y. Acad. Sci. 50, 575-584. Scott, D. B. and Albright, J. T. (1954). Electron microscopy of carious enamel and dentin. Oral Surg. 7, 64-78. Scott, D. B. and Wyckoff, R. W. G. (1947). Electron microscopy of tooth structure by the shadowed collodion replica method. Publ. Hlth. Rep. Wash. 62, 1513-1516. Scott, D. G. and Wyckoff, R. W. G. (1950). Electron microscopy of human dentin. / . dent. Res. 29, 556-560. Selvig, K. A. (1963). Electron microscopy of Hertwig's epithelial sheath and of early dentin and cementum formation in the mouse incisor. Acta odont. scand. 21, 175-186. Shroff, F. R., Williamson, Ê. I. and Bertaud, W. S. (1954). Electron microscope studies of dentin. Oral Surg. 7, 662-670. Shroff, F. R., Williamson, Ê. I., Bertaud, W. S. and Hall D. M. (1956). Further electron microscope studies of dentin. Oral Surg. 9, 432-443. Sognnaes , R. F. (1963). Dental hard tissue destruction with special reference to idiopathic erosions. In "Mecha-

J O H A N S EN nisms of Hard Tissue Destruction", Publ. No. 75, pp. 9 1152. Amer. Ass. Advanc. Sci., Washington, D. C. Sognnaes , R. F. and Shaw, J. H. (1952). Salivary and pulpal contribution to the radioactive phosphorus uptake in enamel and dentin. J. Amer. dent. Ass. 44, 489-505. Syrrist, A. (1949). An introduction in electron microscopy with some results from histological investigations of enamel and dentine. Odont. Tidskr. 67, 79-105. Syrrist, A. and Gustafson, G. (1951). A contribution to the techniques of the electron microscopy of dentine. Odont. Tidskr. 59, 500-513. Takuma, S. (1960a). Preliminary report on the mineralization of human dentin. / . dent. Res. 39, 964-972. Takuma, S. (1960b). Electron microscopy of the structure around the dentinal tubule. / . dent. Res. 39, 973-981. Takuma, S. and Kurahashi, Y. (1962). Electron microscopy of various zones in a carious lesion in human dentine. Arch, oral Biol. 7, 439-444. Takuma, S., Kurahashi, Y., Yoshioka, N. and Yamaguchi, A. (1956). Some consideration s of the microstructure of dental tissues revealed by the electron microscope. Oral Surg. 9, 328-343. Thewlis, J. (1932). X-ray analysis of teeth. Brit. J. Radiol. [N.S.] 5, 353-359. Trautz, O. R. (1955). X-ray diffraction of biological and synthetic apatites. Ann. N.Y. Acad. Sci. 60, 696-712. Trautz, O. R. (1960). Crystallographic studies of calcium carbonate phosphate . Ann. N.Y. Acad. Sci. 85, 145-160. Trautz, O. R., Klein, E., Fessenden , E. and Addelston, H. K. (1953). The interpretation of the x-ray diffractograms obtained from human dental enamel. / . dent. Res. 32, 420-431. Watson, M. L. and Avery, J. K. (1954). The developmen t of the hamster lower incisor as observed by electron microscopy. Amer. J. Anat. 95, 109-162. Yaeger, J. A. (1963a). Microscopy of the response of rodent dentin to injected fluoride. Anat. Rec. 145, 139-147. Yaeger, J. A. (1963b). Fine structure of the matrix of the response in rat incisor dentin to injected strontium. /. dent. Res. 42, 1178-1182.

C H A P T E R 14

MICROANATOM Y AN D HISTOCHEMISTRY OF ENAMEL G. G U S T A F S O N

AND A . - G .

GUSTAFSON

I. Introduction

76

II . Methods of Investigation

76

III . The Prisms A . Prism Sheath B. Interprismatic Substanc e C. Cross-Striation or Segmentatio n of the Prisms D. Discussion

82 82 83 83 84

IV . Retzius Lines A . Rhythmic Incremental Lines B. Pathologic Retzius Lines C. Discussion

89 89 90 97

V. Hunter-Schrege r Lines A . Discussion

100 101

VI . Enamel Tufts and Lamellae A. Discussion

104 107

VII . Enamel Spindles A. Discussion

Ð0 110

VIII . Enamel Cuticle A. Discussion

111 HI

IX . Surface Layer of Enamel A. Discussion

112 114

X . Developmenta l Hypomineralizations A . Discussion

116 120

XI . Innermost Part of the Enamel

123

A . Discussion

123

XII . Similarity of Structures in Contralatera l Teeth

124

XIII . Histochemistry of the Adult Enamel

124

XIV . Enamel Structures in Some Mammals A . The Dog B. Rodents and Lagomorphs C. Comparative Studies XV . Concluding Remarks

126 127 127 128 128

Reference s

129 75

76

G.

G U S T A F S ON

AND

I. INTRODUCTION Mammalian enamel, particularly human enamel, has been the subject of innumerable investigations, using a variety of histological methods. Different characteristics of the enamel, such as its hardness , its chemical composition and the submicroscopi c arrangemen t of its constituents , have been studied. Current ideas on enamel structure are greatly influenced by the role attributed to the enamelforming cells, the ameloblasts . In recent years there have been many workers in this field who consider amelogenesi s to be a process of secretion. This concept involves certain ideas concerning the nature of the secreted material and its transformation from an unorganized state to fibril s and crystals. Certain views on the interrelation between the organic and the inorganic constituents are dependen t upon the same theory. The material secreted by the ameloblasts appears homogeneou s under the light microscope but, at a submicroscopic level, it has been shown to consist of organic matrix in the form of submicroscopi c fibrils. These fibrils have preferred orientations and later have an orientating influence on the crystallites. This observation has an important bearing on the interpretation of certain features seen under the light microscope. I t is the purpose of this chapter to show which structural details, seen under the light microscope, may be of importance when other methods are used. Microanatomy is thus not only a subject in itself but also a necessar y introduction to submicroscopi c studies. About two thousand papers concerning different aspects of enamel have been published. As it wil l not be possible to consider all of these, literature earlier than 1945 wil l not be referred to. Welldocumented reviews of the earlier literature are available in G. Gustafson (1945), Berggren (1947), Leicester (1949), Jansen and Visser (1950), Erausquin (1953, 1961), Hals (1953), Sullivan (1953), Watson and Avery (1954), Kérébel (1955), Darling and Crabb (1956), Bouyssou, Bouyssou and Teulie (1957), Hammarlund-Essle r (1958),

A . - G.

G U S T A F S ON

Schmidt and Keil (1958), Turner (1958), A.-G. Gustafson (1959), Schule (1962) and Sicher (1962). I n spite of the great number of investigations, there is no agreemen t about the real nature of enamel. It wil l be the purpose of this chapter to describe the structure of enamel and to explain how differences in opinion have arisen.

II. METHODS OF INVESTIGATION Because of its extreme hardness it is very difficult to investigate enamel with ordinary histological methods. Although it is impossible to preserve enamel using routine methods of decalcification, there are some decalcifying methods whereby the organic matrix is retained fairly well. Such a method has been devised by Manley, Brain and Marsland (1955) and improved by Brain in 1962. However, results from this decalcification procedure are dependen t more on the skill of the operator than on the method used. According to these workers, fixation is very important and careful handling of the nearly decalcified specimens in the final stages is essential. They have shown that with their method it is possible to obtain decalcified specimens of enamel in which the whole organic part appears to have been preserved . Alternative methods involve supporting the specimen externally by an agar gel which is permeable to decalcifying and embedding substances (Hurst, Nuckolls and Conlon, 1953). The use of a decalcifying fluid of high viscosity helps to support the fragile organic matrix but in any case handling the specimen with extreme care is essentia l at all stages. Sundstrom (1966a,c) has reported promising results in the decalcification of thin ground sections with chromium sulphate solutions which are presumed to exert a simultaneous tanning effect. Decalcification in vacuo was tried by Frank and Deluzarche (1950) and by Frank (1952) using a pressure of about 100 mm of mercury obtained by means of a water-pump. Sognnae s (1948), who analyzed as far as possible the various factors

14. M I C R O A N A T O M Y

AND

H I S T O C H E M I S T RY

concerned in the disruption and loss of the organic matrix on decalcification, adopted opposite measures and decalcified under increased pressure with the idea of preventing the formation of carbon dioxide bubbles within the substanc e of the enamel. Even if it were easily possible to retain the whole organic matrix of enamel in decalcified sections, it is still necessar y to investigate the enamel without previous decalcification because of the important role played by the inorganic material which constitutes the greater part of the enamel. The chief advantage of undecalcified enamel is the undisturbed relationship between the different constituents. Attempts have therefore been made to obtain ground sections thin enough for microscopic examination with transmitted light. The thickness of ground sections used before 1945 was so great that it interfered with the transmission of light. Thus, in investigations of ground sections of enamel, optical disturbance s gave misleading appearances , and consequentl y false interpretations , of enamel structures. Atkinson (1950) was able to cut serial sections of both hard and soft tissues down to 25 μ without previous decalcification. This thickness was a littl e less than that previously used by most investigators (Jansen , 1950), but it was still too great to allow free transmission of the light. Embedding in plastic and serial grinding of thin sections of both hard and soft tissues were also used by Hammarlund-Essle r (1955). Her sections were used for microradiographic and autoradiogra phic procedures but were not thin enough for histologic study. A s she used her sections for microradiographic work, she was more concerned that they be piano-parallel (Hammarlund-Essler , 1958). Piano-parallel sections of about 17 μ thickness were obtained by Hallén and Rockert (1958) after embedding the mineralized tissues in methyl methacrylate. The pieces were sawn into sections 3-4 mm thick and thereafter attached to the flat end of a steel cylinder with a layer of glue less than 0.5 μ thick. The final control of the thickness was made with a "microkator" and an "unevenness " of 10.3 % was found.

OF

ENAMEL

77

I n a later publication, Hallén and Rockert (1960) gave more information about the technique. They were now able to obtain sections thinner than 10 μ by using a certain adhesive allowing parallel lapping. The sections were measure d with the "microkator" giving an indication of thickness wit h an accuracy of ± 0 .1 μ. Sections as thin as 30 μ could be prepared routinely, the much thinner sections being obtained with special care. The first really thin good ground sections were made by Fremlin, Mathieson and Hardwick (1961) using a method in which the sections were fixed to glass during the grinding. Fremlin et al. considered two criteria in determining the desired optical thickness of the section. According to them, there is no need to reduce the thickness below the depth of field of the objective to be used, and it is important not to reduce the thickness so far that there is insufficient difference in optical density to permit a distinction to be made between different parts of the specimen. " T he section should be thin enough to eliminate unwanted structures but sometimes it may be desirable to ensure that it is thick enough for the observer to determine the relation between the layer of the specimen observed and the layers above and below." "The optimal thickness for enamel should be 6-7 μ or less for sections parallel to the prisms and perhaps 3-4 μ or less for transverse sections." The most disturbing artifacts present in very thin ground sections are splits due to drying out of the section, shrinkage of the adhesive and embedde d diamond dust (Hardwick, Martin and Davies, 1965). Wit h the method described by Fremlin et al, it is possible to obtain ground sections in which at least part of the enamel fulfil s the optical requirements . Unfortunately, it is rarely possible to obtain sections of enamel of even thickness throughout. The present authors therefore have tried to improve this technique with the aid of a special device for holding the section during grinding (Fig. 1). If, as proposed by Fremlin et al., the glass to which the section is attached is held by hand during grinding, the edges of the section become much thinner than its middle. The mechanica l device prevents the

78

G. G U S T A F S ON

AND

Fig. 1. Device for making thin ground sections of teeth and bone, a, section of tooth; b, cylinder on which the section is glued; c, outer cylinder with flange; d, glass slab.

slight tiltin g of the glass that occurs in handgrinding. Even with this mechanica l device, Sundstrom (1966a) found that it is relatively difficult , if not impossible, to get absolutely pianoparallel sections with the same thickness over the whole area. The method of glueing was of particular importance. The specimen had to be glued to the carrier in a cold state—otherwis e there was more glue in the middle of the section than at the sides and then the section became wedge-shaped . If the sections were ground on paper there was a tendency for them to become thicker in the middle than at the sides. If fine powder was used for the final grinding and polishing, the tendency to produce a wedge-shap e was diminished, and the amount of taper was of the order of 1 μ. The physical properties of the specimen also affected its piano-parallelism . This was most obvious in the inner part of the enamel, i.e. the area comprising the Hunter-Schrege r bands, especially near the enamel-dentin e junction. Variations in thickness also develop from subtle irregularities produced either by scratching or by preferential removal of material due to different resistance between enamel subunits, e.g. prism sheaths and interprismatic substance . Surface structure is determined by the internal structure of enamel. It is likely that prisms lying parallel to the surface are more easily ground away than prisms perpendicula r to the surface. Investigations on dog enamel showed that the thicker parts correspond to areas of enamel where the prisms run perpendicula r to the plane of the section.

A . - G.

G U S T A F S ON

But it is also clear that the most important limitin g factor in making piano-parallel sections is the glueing procedure. I t is possible to get ground sections less than 10 μ thick which, within limited areas, are piano-parallel to a degree allowing even microradiography . These thin ground sections are also suitable for investigation with phase-contras t illumination, which hitherto has been of littl e value because of the excessive thickness of the sections. Mathieson and Fremlin (1963) point out, however, that the sensitivity of phase-contras t illumination to small surface irregularities constitutes a disadvantage . Serious faults in the surface may arise from imperfections present in the underlying structure. There may be a difference in hardness and brittleness between the prism core and the outer parts of the prisms or the interprismatic substance . A selective loss may thus occur in grinding or polishing, due to preferential ripping out of fragments rather than of a gradual grinding away of the softer parts. Fully calcified enamel exhibits negative intrinsic birefringence whereas partially calcified enamel may, under certain conditions, show predominantly positive form birefringence (Schmidt and Keil, 1958; Allan, 1960a; Crabb and Darling, 1962; Myers, 1955). Normal enamel exhibits very littl e form birefringence, but if it is present and can be altered or eliminated by imbibition, then a deficiency of mineral salts is indicated. The submicroscopic , inorganic crystallites of enamel are negatively biréfringent relative to their elongated " c " axes and, as enamel is usually negatively biréfringent with respect to prism direction, for many years it was concluded that the crystallites within the prism and interprismatic substance were parallel with the long axes of the prisms. It was therefore suppose d that the degree of birefringence could be used for the estimation of the degree of mineralization of enamel. However, several factors are now known to limi t the use of birefringence measurement s for this purpose. I n the first place, electron microscope studies, X-ray diffraction studies and even the polarizing microscope itself have shown that not all the

14. M I C R O A N A T O M Y

AND

H I S T O C H E M I S T RY

crystallites in enamel are parallel with the axes of the prisms. I n sections parallel with the long axis of the tooth, there is almost always a deviation from the long axis of the prisms averaging, according to Frank (1959), 18° ( ± 7 ° ). However, he found that at the periphery of the prisms, and in the interprismatic substance , deviation as great as 45° existed. These findings were in accord with the results obtained with the polarizing microscope by Lyon and Darling (1957). Poole and Brooks (1961), using X-ray diffraction studies as well as the polarizing microscope, believed there to be a gradual change in the orientation of crystallites from one side of the prism to the other. Sognnaes , Frank and Kern (1960) concluded that crystallites are preferentially orientated roughly along the long axes of the prisms and Nalbandian and Frank (1962) also concluded that the crystallites within the prism, that is inside the prism sheath, are approximately parallel to the long axis of the prism or diverge from its centre in a feather-like configuration. F r om these and other investigations it can be deduced that deviations in crystallite arrangement , particularly in the outer part of the prism and interprismatic substance , do exist so that, as stated by Helmcke (1955, 1959a,b,c, 1960a,b, 1964), the effects produced in polarized light may depend not only upon quantitative differences but also on the orientation of crystallites. Nevertheless , as a relatively gross estimation of the degree of mineralization, the polarizing microscope may be applied to the main part of the prism where the crystallites are largely parallel with the prism axis. C r a bb (1959) is of the opinion that the intrinsic birefringence of enamel gives an indication of its degree of mineralization. Enamel possesse s a very small organic component of keratin-like nature which shows positive intrinsic birefringence. Investigations into the strength of this positive birefringence (Allan, 1960a; Schmidt, 1963a) indicate that it is so weak as to play no detectable part in the total birefringence of the enamel in contrast to the organic matrix of dentine (Chapter 12). 7

OF

ENAMEL

79

The investigations in polarized light of human enamel have been based partly on the assumption that in deficient enamel there are submicroscopi c spaces which, when filled with a substanc e having a refractive index different from that of the enamel, are positively doubly refractive. The presence of these submicroscopi c spaces has been confirmed mainly by imbibition experiments . Sognnae s et al. (1960) in their electron microscope investigations also have found evidence of such spaces . They write: "The mineralization in the enamel of adult human teeth may be quite variable, as suggeste d by the differences in birefringence observed in the polarizing microscope investigations by G. Gustafson (1945) and G. Gustafson and Payen (1957) and Schmidt and Keil (1958). Our electron microscopic observations of nondecalcified sections indicate that these variations are morphologically related to demonstrable differences in the inorganic crystallization of enamel within small localized submicroscopical areas of an enamel rod. Such differences have been detected from one rod to another and even in limited zones within the same rod". A s investigations with polarized light are by themselves relatively unreliable, a number of investigations have been carried out to check the results. A.-G. Gustafson (1959) and G. Gustafson and Gustafson in 1961 compared results obtained wit h polarized light and microradiography . They found fairly good conformity between results from the two methods. Such comparisons had, however, already been carried out by Hodson in 1955. He examined ground sections placed in water and could easily detect the areas of poor calcification. He also compared the photographs taken in this way with prints of X-ray negatives. Alla n (1963) also compared results obtained by microradiography with those obtained by polarization microscopy. He showed that, when he immersed the ground sections in cedar wood oil, there were lines within the enamel which were isotropic or weakly negatively biréfringent. He could show that strongly negative lines correlated well with the radiopaque lines shown in microradio-

80

G. G U S T A F S ON

AND

graphs and that isotropic lines corresponde d with radiolucent lines. He also compared ground sections wit h decalcified enamel where the retained insoluble matrix showed many chromophobic and chromophili c lines which, on the whole, corresponde d well wit h the striae shown by microradiograph y and polarization microscopy. Thus there was a clear relationship between the results obtained with the three methods. Hals (1953) compared the appearance s seen in polarized light with those obtained with fluorescent microscopy. He found that negatively biréfringent sections were nonfluorescen t whereas areas which were not biréfringent exhibited fluorescence. Unfortunately it is very difficult to show in a single photograph the correspondenc e between appearance s seen in polarized light and microradiographs, because biréfringent areas do not show up i n the photomicrograph s taken in polarized light if the long axes of the biréfringent particles are parallel to one of the two directions of vibration of the polarizing filters of the microscope. One of the present authors (A.-G. Gustafson, unpublished work, 1965) has overcome this by taking the photographs in polarized light with polarizers which rotate during the whole time of exposure (compare Figs. 28a and 28b). I n such photographs the effect of the direction of the crystallites within the prism is more or less eliminated if they are parallel to the plane of the section. Usually, however, the extinction position deviates from the direction of the prisms (Lyon and Darling, 1957). If the crystallites are perpendicula r to that plane they will , of course, show no birefringence. Crystallites may deviate not only in relation to the long axis of the prisms but also in relation to the plane of section. The prisms do not run parallel to each other, except in small areas particularly in the inner third of the enamel (Hunter-Schrege r bands) ; there is thus an apparent decrease in the intrinsic birefringence. The imbibition medium may penetrate the section or it may not; in cases of penetration there may be areas which show negative birefringence in spite of having a low mineral content. This applies, however,

A . - G.

G U S T A F S ON

particularly to carious areas (G. Gustafson, 1957). The estimation of mineralization with the aid of the compensato r Red 1 is naturally only a gross one and by no means quantitative. It must be borne in mind that air-imbibed sections placed in Canada balsam exaggerat e the differences but can be used to show clearly morphological details. The interpretation of the polarized light image of mature enamel therefore offers many difficulties and polarization microscopy is not very suitable for the study of the presumed or real variations in mineralization (see Carlstrom, 1964). Baud and Held (1956) made microradiograph s of ground sections of enamel and then stained the same sections with a 1 % solution of silver nitrate. Those parts of the sections which stained most heavily with the silver were those parts which had the lowest absorption of X-rays, i.e. were least mineralized. Microradiography has also been used by Engfeldt, Bergman and Hammarlund-Essle r (1954), Hammarlund-Essle r (1958) and many others. According to Hutchinson, Rowland and Fosdick (1963), radiographic examination of the enamel is not feasible unless sections are one rod thick. Furthermore, the section must contain the entire rod rather than two half-rods if the radiographs are to be meaningful. They therefore recommend the use of a modified Fremlin technique to produce sections of enamel 4-6 μ thick. Another control of microradiograph s was carried out by Avery (1963), who was able to confirm the microradiographic findings with comparative investigations of microhardness . Comparative investigations were also carried out wit h microhardnes s and polarized light in investigations by G. Gustafson and Klin g (1948). Mi crohardnes s investigations on the enamel are extremely difficult . Very shortly after the tooth is removed from the body the enamel becomes brittle and the indentations are thus not very distinct. The thinness of some of the structural details such as Retzius lines makes it necessar y to use indentations so small that their width is less than that of the striae measured . This could be done when a load

14. M I C R O A N A T O M Y

AND

H I S T O C H E M I S T RY

as light as 9 gm was used. It is extremely difficul t to measure such small indentations. Furthermore the hardness is not directly related to the real amount of mineral present; the binding substance , i.e. the organic matter, also contributes to the degree of hardness . Usually only ground surfaces are investigated for microhardnes s but Caldwell et al. (1957) found that practically all intact surfaces can be tested satisfactorily although with somewhat greater variations than with polished surfaces. The difficulties in determining the so-called microhardnes s have also been discusse d by Ryge, Foley and Fairhurst (1961). They prefer the term micro-indentation-hardnes s instead of simply microhardness . On the enamel it had to be carried out with low loads, and considerable variations were found in relation to the loads, the rates of load application, the length of time the load was borne, method of specimen preparation and also in the interpretation of the data. Knapp, Avery and Costich (1958) tried to overcome the difficulties of using ground sections by converting the original organic and inorganic materials to coloured plastic and gelatine, respectively. The organic material was extracted from samples of teeth with ethylenediamin e and the enamel was then infiltrated with coloured plastic. Following decalcification the specimen was impregnated with gelatine and sections were cut. The proportion of coloured plastic was relatively greater between the prisms, suggesting that the interprismatic substanc e contains less mineral than the prisms themselves . In these specimens the rod boundaries and the spaces occupied by interprismatic substance s were more distinct than can be observed using thicker conventional ground sections of enamel itself. Quite new methods of investigation have been introduced by Boyde, Switsur and Stewart (1963) using X-ray emission microanalysis and ion-beam erosion etching. The X-ray emission microanalysis gives information about the elemental composition of the surface layer of the specimen. Both methods reveal differences in composition (e.g. the degree of

OF

ENAMEL

81

mineralization) and ion-beam etching may also reveal differences in structure (e.g. the orientation of hydroxyapatite crystallites). Both qualitative and quantitative data can be obtained, which can be related to the topography of the surface of the specimen. The authors consider, however, that accurate measurement s in elemental microanalysis and crystallite orientation in relation to histological details cannot be expected before the developmen t of a method for the production of thin (about 5 μ) parallel-sided sections with undamage d surfaces. Grinding by conventional methods damages a surface layer of enamel to a thickness which has yet to be established . Investigations in ultraviolet light were carried out by Jansen and Visser (1950). The in vivo staining techniques used by Berggren (1947) have been succeede d by a special technique for demonstrating the passag e of tissue flui d through the enamel (Bergman, 1963a,b). The drawback inherent in thick ground sections and the investigation of natural and ground surfaces have been partly overcome by the development of replica methods. Wit h these methods the cleaned and dried surface is covered by a thin layer of collodion. The hardened fil m is removed, placed on a slide and covered with a cover glass. The method was improved by D. B. Scott and Wyckoff (1946a,b, 1947), who shadowed the surface of the replica with metal. This allowed the different details of such surfaces to be seen more clearly. This method was somewhat unreliable as the manipulation was very critical because the results depended on the angle of the shadowing and the quality of the metal. Grimbert and Pigeat (1961) studied transparen t replicas directly with phasecontrast microscopy. Replica methods have greatest value in the study of teeth in situ (Mannerberg, 1960; Heuser, 1956, 1961) but replicas from ground and etched surfaces are also very useful. The electron microscopy of enamel is the subject of other chapters of this book but it is appropriate here to lay emphasis on the importance of approaching the problem of the structure of enamel by as many methods as possible—for example, by

82

G.

G U S T A F S ON

AND

comparing observations made with light microscopy with those obtained by electron microscopy (Helmcke, 1959a,b,c, 1960a, 1964; Helmcke, Schulz and Scott 1961, 1963).

III. THE PRISMS Mammalian enamel appears to be composed of individual units, the so-called prisms. These run from the enamel-dentin e junction to the outer surface of the enamel. Many authors object to the term " p r i s m" and the most frequently used alternative, especially in American literature, is " r o d ". Held (1947) preferred the term " c o r d o n" as the true prismatic form is relatively uncommon. Similarly Wustrov (1951) pointed out that the term " p r i s m" implies a shape of geometric precision which is rarely, if ever, present. In our view, since the exact true nature and shape of the so-called prisms are not yet full y understood , there is littl e point in changing the name. In any case it may be suppose d that they are, strictly speaking, neither rods nor cordons because they are composed of segments placed one upon the other. Each prism is surrounded by a prism sheath, a concentration of organic material at the periphery of the prism. The interior of the prism is composed of very small crystals of hydroxyapatite. The prisms do not li e immediately adjacent to each other but are separate d by the interprismatic substance , which can be abundant but sometimes is not evident. The prisms do not run perpendicularly to the outer surface of the enamel but pursue an undulating path so that layers of enamel are formed i n which all prisms run in the same direction. This gives rise to the Hunter-Schrege r bands which are found in the inner half of the enamel. I n the long axis of the teeth there are bands near the dentine in which there is less inorganic material, or more organic, than in the remainder of the enamel. These bands, the enamel tufts, reach about

A . - G.

G U S T A F S ON

a third of the way through the enamel. Similar bands also run in the long axis of the tooth and reach the enamel surface. These are the lamellae. The tooth is covered by different layers one or two of which are formed by the ameloblasts and another is acquired after the tooth has erupted. This latter is not strictly part of the enamel proper. The enamel is not uniformly mineralized but shows, besides complex structures mentioned, variations due to faults, either in the organic matrix or in mineralization. These defects may be in the course of the incremental lines or they may be more diffuse. Al l these variations, be they normal or pathological, have been the subject of intensive research into their origin and structure. We wil l now review the different structures, first according to our own interpretation and then according to the views of other workers. A . PRISM SHEATH

The prism sheath can be demonstrate d by practically any histological method, in both decalcified sections and undecalcified ground sections or ground surfaces. It can be impregnated by various dyes, be they "ordinary" dyes or fluorescent ones. Various fluids may penetrate the enamel along the prism sheath, both in vivo and in vitro. Because of this, the prism sheath was long considered to be a wholly organic membrane but this view was abandone d some years ago. Electron microscopy has shown that enamel is composed of submicroscopic fibres and submicroscopi c crystallites. Thus, the prism sheath is a region particularly rich in fibril s which, at the low magnification of the light microscope, appear as a continuous layer of organic matter. Only in some areas do the prisms in cross-sectio n appear to be surrounded on all sides by a prism sheath. In other areas the sheaths may assume a horseshoe form and in others they may look more lik e a keyhole. They can assume many forms which are dependen t on their relationship to their neighbours (Fig. 2).

14. M I C R O A N A T O M Y

ι ι

AND

ι

-

H I S T O C H E M I S T RY

OF

ENAMEL

83

to be more mineralized than the prism sheath. The amount of interprismatic substanc e varies immensely and sometimes is missing entirely, leaving the prism sheaths in contact with one another. In normal well-mineralized enamel the interprismatic substance is highly mineralized, as can be seen when a surface of cross-cut prisms is etched with acid (G. Gustafson, 1945). The relatively small amount of the interprismatic substanc e renders it difficul t to identify in the light microscope, particularly in ground sections which are not extremely thin. C . CROSS-STRIATION OR SEGMENTATION OF THE PRISMS

Fig. 2. Diagrammatic representatio n of prisms, prism sheaths and interprismatic substance . (A) Cross-cut prisms: interprismatic substance ; b, prism sheaths ; and c, prism core. (B) Longitudinally cut prisms. Between the cross-striation s (d) are the prism segments . (C) Cross-cut prisms with no interprismatic substance . The prism sheaths are close to each other. (D) Cross-cut prisms with prism sheaths open on one side. Thus the interprismatic substanc e is in immediate contact with the prism core. The form of the prisms varies according to the relation of the axes of adjacent prisms or groups of prisms. The horsesho e form can therefore vary a great deal in details. B . INTERPRISMATIC SUBSTANCE

The prisms and their sheaths are not usually in contact with each other. They are separate d by the interprismatic substanc e which is generally thought

Early investigators referred to segmentatio n of the prisms, which was later called cross-striation because the prisms appear to be divided by regular transverse lines into segments about 4-6 μ in length and, as this is about the same as the width of the prisms, the segments are practically square in section. In polarized light, the prisms appear clearly segmente d (Fig. 3) especially in certain areas of the enamel. The cross-striation can also be seen in decalcified sections and it was therefore thought that the striations are richer in organic material than the rest of the prism. Microradiography (Fig. 4) indeed sometimes shows that the narrow bands of the cross-striation s are less mineralized than the main part of the prism. Variations in mineralization within different segments produce variations in character of the enamel. Where it is well mineralized the segments and cross-striation s are not easily seen and in some places may be invisible. Such areas are most common near the dentine and sometimes near the enamel surface in so-called nonprismatic enamel. I n areas of enamel in which the prisms are not uniformly mineralized it is possible to find isolated, well-mineralized segments . These are perfectly square in section and sometimes there is a row of single segments indicating a layer of enamel formed at one time. This gives rise to a Retzius line (Fig. 5).

84

G.

G U S T A F S ON

AND

Fig. 3. Ground section of human enamel in polarized light (crossed polars). The prisms are divided into negatively biréfringent square segments . Arrow indicates the direction of the prisms, χ 420.

A . - G.

G U S T A F S ON

Fig. 4. Micro radiograph of ground section of human enamel showing segments of some of the prisms. Arrow indicates a prism with cross-striation . χ 700. (From A.-G. Gustafson, 1959.)

The disturbance giving rise to Retzius lines may involve one or more segments , and thus the lines may vary in width, as wil l be shown later. I n some areas, the prism segments have a different appearanc e in polarized light. Even in the 45° position, the centres of the segments show no negative birefringence while the outer part of the prisms and the intersegmenta l bands show positive birefringence (Figs. 6 and 7). The intersegmenta l bands are usually single but, in some cases and in some areas, there is a double band effect. It might be suppose d that this is due to an optical phenomenon but careful examination shows that such is not the case. D.

DISCUSSION

The enamel prisms are visible as separate elements from the very beginning of enamel formation and they appear in the same position and the same relations to each other throughout formation and maturation.

Fig. 5. A ground section in which most of the enamel is isotropic with single rows of negatively biréfringent (light) segments , some of which are perfectly square. The prism sheaths show faint positive birefringence. Arrows indicate rows of negative segments . Human enamel, x 546. (From A.-G. Gustafson, 1959.)

14. M I C R O A N A T O M Y

AND

H I S T O C H E M I S T RY

OF

ENAMEL

85

Fig. 6. An area of hypomineralized human enamel showing cross-striation s as double lines. The arrow indicates direction of the prisms, x 1080. (From A.-G. Gustafson, 1959.)

The thickness or width of the prisms has been the subject of only a few investigations. Maj (1947) found that the prisms in the inner zone have a diameter that is less than that of those in the outer zone (4.867 and 5.285 ì , respectively). Heuser (1961) in a replica study found that the difference between the areas of the inner and the outer surfaces of the enamel is compensate d for not only by increase in the width of the prisms but also in the amount of the interprismatic substance . The interpretation of the structural detail of enamel, seen with the light microscope, has been very much discusse d i n the literature. Wit h new instruments, particularly the electron microscope, new concepts have been put forward and old interpretations of appearance s in the light microscope have been questioned . The structures which have given rise to most controversy are the prism sheath and the interprismatic substance . These were first observed with the light microscope but have also been distinguished by some investigators with the electron microscope. A m o ng the early electron microscopists, Syrrist (1949) could, in replicas, demon-

Fig. 7. Incremental Retzius lines with bending of the prisms towards the neck of the tooth (below) in polarized light. This is best seen in the area indicated by the arrow. Human enamel, x 325. (From G. Gustafson, 1945.)

strate the prism sheaths and also the fact that they contained calcific matter. The pseudo-replica s described in his paper were among the first to demonstrate the enamel crystallites directly. D . B. Scott et al. (1952), in electron micrographs, were able to distinguish all the principal features recognized with the light microscope, namely enamel prisms, interprismatic substanc e and prism sheaths, and attributed them to differences in the density and direction of the network of submicroscopic fibril s which are visible with the electron microscope but not with the light microscope. These workers found that the prisms in electron micrographs had about the same dimensions as those observed with the light microscope. This was confirmed with shadowed replicas and with thin sections of demineralized enamel. Well-defined sheaths made up of closely packed submicroscopi c fibril s could be seen. Sognnaes (1955) also stated that the histomorphology of the enamel could be attributed to the

86

G. G U S T A F S ON

AND

organic framework. Jansen and Visser (1950) found that the prism sheaths could be well stained with fluorescent dyes. Wislocki and Sognnae s (1950) showed that the prism sheaths as well as crossstriations exhibited bright red metachromasi a when stained with toluidine blue, that they stained intensely with methylene blue (showing basophilia) and gave a positive reaction for phosphatas e (Gomori method). The prism sheaths also stained brown with Prussian blue, thus indicating the presence of sulphydryl groups. Helmcke (1960b) has been unable to find anything lik e prism sheaths and interprismatic substance with electron microscopy and attributes their appearanc e under the light microscope to a summation effect due to the thickness of the sections. Where sheath-like structures are present he attributes them to the effect of demineralization by acids (1964). According to Helmcke, prism sheaths are not found in non-demineralize d developing enamel. He found that in places where the so-called prism sheaths are thin there is no membrane but only a pattern of points produced by cross-section s of closely packed fibrils. It should be noted that Helmcke used a method by which replicas are taken from fractured surfaces without any other sort of preparation (Syrrist and Gustafson, 1951). Helmcke's opinion is supported by Fearnhea d (1963), who found no definite organic fibres greater than 20 A in diameter in denaturate d developing enamel, although definitive fibres could be produced by decalcification or fixation procedures . Darling (1964), on the other hand, is unable to accept Helmcke's idea that the prism sheath is simply a concentration of insoluble matrix produced by the process of demineralization . I t is true that most ground sections used for optical studies give an "average" or composite picture of the material of which the enamel is composed. A s it is most likely that there are no qualitative but only quantitative differences between prism core, prism sheath and interprismatic substance, however, such a "summation effect" may be of great value in disclosing differences which

A . - G.

G U S T A F S ON

are not apparent with methods such as replicas which do not give this effect. In investigations of quantitative differences it is clear that summation is of the greatest importance. Before 1945 the prism sheath was thought of as being something lik e a membrane. This view has now been refuted and it is believed that the prism sheath is composed of the same materials as the rest of the enamel, the difference merely being one of concentration and of different orientation from the interprismatic fibrilla r network (Frank, 1957). According to Kérébel (1961a,b), it is not possible to explain the prism sheath as an optical phenomenon only. In his view the prism is the anatomic unit of the enamel even if it is not the biologic unit. According to Frank (1959), the various types of prism sheath are seen in electron micrographs of decalcified sections, just as with the light microscope; some prisms lack both sheaths and interprismatic substance . In any case the concentration of crystallites is such in undecalcified sections that interprismatic substanc e is seen only at high magnifications. It is of interest in this connection that Crabb (1959) found that during the formation of enamel the centre of the prism is mineralized first. According to Carlstrom (1964), "the prism boundaries are not resolved even when using an oil-immersion objective; they are seen only because they represen t sudden jumps in optical density". The prism boundaries or so-called prism sheaths are certainly thinner than 0.2 μ. According to Hardwick, Martin and Davies (1965), in its most typical form the body of the prism is enclosed by a horseshoe-shape d sheath which appears to be less than 0.5 μ in thickness. Practically all investigations from the earliest times have shown that the prism sheaths have a different shape in different parts of the enamel. Fujita (1953) found that only in the deeper parts of the enamel close to the dentine, where the prisms are round or oval, were they surrounded completely by prism sheaths . In other places about half the prism sheaths have an arcade form although many other types are to be found. Fujita refuted

14. M I C R O A N A T O M Y

AND

H I S T O C H E M I S T RY

the old idea that the arcade form is dependen t upon the angle at which the prism is sectioned (see also Hals, 1953). D. B. Scott (1955), who found the same configuration of prisms both with light microscopy and with electron microscopy, noted round scalelik e (arcade-shaped , three-sided) , hexagona l and polygonal prisms as well as ones of very irregular outline. Only 2 % appeare d hexagona l or round, wit h complete sheaths , the most frequent form being arcade-shaped . A single band of sheath material could be shared by two adjacent prisms. Frank (1957) confirmed that the same forms seen wit h the light microscope could be seen even more clearly with the electron microscope. If the prisms are a product of secretion they must show a variable configuration depending on the direction and the relationship to surrounding prisms. The prisms never run straight from the enamel-dentin e junction outwards and thus one may find arcade-like sheaths and sheaths with long lateral "wing processes " as described long ago and also more recently (Fujita, 1953; Helmcke, 1964). Even "keyhole" forms have been described. The form is obviously dependen t on the direction in which the prisms are cut, so that there is almost no limi t to the variations. Every type can be related to different areas of the enamel, as reported by Fujita. The form of the prism seems to have some relationship to the amount of interprismatic substance present (J. H. Scott and Symons, 1958), for when the circular form is found the interprismatic substanc e is usually abundant, whereas when the hexagona l form is present it is scanty or absent. "Interprismatic substance " has been found by so many investigators using so many different optical methods that the existence of a region between the prism sheaths seems beyond dispute It s existence has, however, been denied by Helmcke (1955), who could find no indication of it, even in abnormal enamel, when studying replicas in the electron microscope. This was in accord with earlier investigations by Bernick et al. (1952), who could find no interprismatic substanc e when using

OF

ENAMEL

87

the electron microscope, in spite of finding at the periphery of each rod a definite organic sheath having a different density from the prism interior. The sheaths appeare d to be contiguous with each other. Bernick et al. incidentally described processe s of rods which projected between other adjoining rods. These would correspond with the "win g processes " of light microscopy. D. B. Scott and Wyckoff (1946b) used replicas which were shadowed by metal and investigated under the light microscope. Whenever prism ends were visible they were separate d by 1-3 μ of interprismatic material. This thickness varied considerably even within a small area and on different sides of a single prism. In ground sections etched wit h various acids they were also able to demonstrate that interprismatic substanc e etched to a greater degree than the prisms themselves . Jansen and Visser (1950) found, with fluorescence microscopy, an unstained zone between two stained lines which they interpreted as prism sheaths . The unstained zone should thus represen t the interprismatic substance . Fremlin and Mathieson (1962), using phasecontrast microscopy, concluded that the horsesho e shape of prisms in transverse section represent s their true shape. They found that there is between the prisms some substanc e which could not be attributed simply to a sudden change in refractive index. In several sections the bodies of the prisms occupied only half or less of the total area of enamel. In high magnification microradiograph s the prisms are delineated by a layer of low X-ray absorption, and Engfeldt et al. (1954) considered this to be due to low mineralization of the interprismatic substance . According to Held (1947), the interprismatic substanc e is rich in organic elements. Frank (1957) found interprismatic substanc e with the electron microscope both in replicas and in decalcified sections. But in the majority of cases prisms were close to each other sharing a common prism sheath with no intervening interprismatic substance . Nalbandian and Frank (1962), with the electron

88

G. G U S T A F S ON

AND

microscope, found that the prism sheaths do not encircle the prisms completely and, unlike Helmcke, they found "interprismatic substance " which they concluded was slightly more calcified than the prism sheath itself. As mentioned earlier, according to these workers, the apatite crystallites in the "interprismatic substance " run in a different direction from those within the prisms. This view is in accord with that held by most electron microscopists. This deviation in direction of the crystallites in the main part of the prism makes investigation of the interprismatic substanc e with polarized light difficult , if not impossible. The existence of interprismatic substanc e can be demonstrate d but its nature cannot be evaluated with the aid of polarized light alone. The cross-striation of prisms has been described by Manley et al. (1955) on decalcified sections. They state that the interior of each prism is divided into a series of units produced by the activity of the enamel-forming cells, the junctions between successiv e units being represente d by fine striations. According to Sicher (1962) also, the prisms are built up of segments separate d by dark lines which produce a striated appearanc e and are due to a rhythmic formation of the organic matrix. These transverse striations demarcate rod segments and become more visible with the action of mild acids. They also are well-marked in insufficiently calcified enamel. According to Hanazawa (1957), transverse striations are in all probability of the same nature as prism sheaths . In the view of Wustrov (1951), the segmentatio n of prisms is primarily due to rhythmic differences in the secretion of the organic matrix by the ameloblasts , and the difference in the degree of mineralization between the crossstriations and the prism segments is secondary . Darling (1964) has estimated that the prism segments are approximately 6 μ wide and 6 μ long and, according to Hardwick, Martin and Davies (1965), the length of the segments varies between 3 and 10 μ. I n decalcified normal enamel, Schmidt and Keil (1958) found cross-striation in the form of mem-

A . - G.

G U S T A F S ON

branes perpendicula r to the long axes of the prisms. Thus, according to them, the cross-striation was preformed in the organic matrix. Generally such a concentration of organic matter coincided wit h low mineralization. Various widths of crossstriation were seen but these differences were irregular and without periodicity. Alla n (1963), using decalcified sections from contralateral teeth, showed staining characteristic s of the striations indicative of variations in both the organic and the mineral content. I n all probability the transverse striations which appear dark in ground sections have the same characteristics as sheaths . This interpretation, which has been accepted without question by dental histologists for many decades , has been disputed by Helmcke (1963), who claimed that the cross-striation is not due to a greater amount of organic substance . He considered it very unlikely that cross-striation s could be produced by preformed organic matrix. Helmcke, Schulz and Scott (1963) have produced evidence, based upon shadowed positive carbon replicas for electron microscopy, pseudoreplica s and true replicas, that cross-striation s are due to periodic differences in the width of the prisms. They could find no quantitative differences at the regions of crossstriation and concluded that the variations in organization are due to optical effects. The idea that the cross-striation is produced from a pattern which is establishe d in the organic matrix was recently supported by Schmidt (1963a), who concluded on the basis of a polarized light study that such details as the cross-striation could not be due to alternating crystallization of hydroxyapatite. Cross-striation s could be found in practically every ground section in ordinary transmitted light. However, they could not be found quite as easily in some areas and, close to the enameldentine junction, they were absent. It is thus likely that the cross-striation s are real configurations. In the area of visible cross-striations , the incident light perpendicula r to the axis is reflected and scattered differently from that in other areas. In ordinary light, Schmidt could demonstrat e the

14. M I C R O A N A T O M Y

AND

H I S T O C H E M I S T RY

OF

ENAMEL

89

cross-striations as alternating light and dark lines perpendicular to the long axes of the prisms. He found a greater take-up of dyes by the dark lines compared with the light bands and concluded that this indicated greater porosity in the dark lines. Leimgruber (1949) considered that cross-striations were due to an optical phenomeno n produced by the crystallographic pattern and that they did not necessaril y indicate any differences in degree of mineralization. It should be noted that crossstriations as observed with the light microscope have not been demonstrate d in electron micrographs. A n old argument is used by Boyde (1965) who says: "I t should be noted that the concept of the development of the cross-striation s of the prisms given by Gustafson (1959) and by Scott and Nylen (1960) results in my opinion, from the erroneous identification of rows of cross-striation s of prisms as the cross-striation s of longitudinally-sectione d prisms".

IV. RETZIUS LINES A . RHYTHMI C INCREMENTAL

LINES

There are two factors responsible for the various structures seen in enamel; firstly, differences in degree of mineralization, and secondly, differences in direction of the prisms. I n longitudinal sections of the tooth it can be seen that the enamel is formed by relatively broad layers separate d from each other by narrower lines. The broad layers are of the same width, whereas the narrow lines may show differences in width and intensity (Figs. 7-11). The Swedish scientist Anders Retzius described the lines named after him in the middle of the 18th century. He described two configurations, one of which he called bands and the other lines. By bands he meant a zone of enamel one, two or more prism segments wide, and by lines he meant the narrow boundaries between the zones, boundaries or lines which are actually produced by the joining up of a series of ac-

Fig. 8. A much broader and more prominent band than is usual in human enamel and produced by abrupt changes in prism direction. Polarized light, χ 450.

centuated cross-striation s of adjacent prisms. In the later literature, in general no clear distinction has been made between the bands and the lines, the two terms being used indiscriminately. However, usually the terms Retzius lines and Retzius bands both refer to what Retzius described originally as lines. On the sides of teeth the Retzius lines reach the surface at an angle (Fig. 10) which varies in different places. A t the tops of cusps the lines do not reach the surface at all (Fig. 12). I n crosssections of teeth the lines run as concentric circles parallel to the enamel-dentin e junction and parallel to the outer surface. I t is obvious that the broader bands represen t layers of enamel formed continuously whereas the narrow bands represen t layers in which there is a change in the prisms usually seen as bending

90

G.

G U S T A F S ON

AND

(Fig. 8) but sometimes disturbance s in the formation of the prism segments can be seen. The Retzius lines are approximately equidistant from each other and it is clear that this represent s rhythmic formation, probably connected with the mechanism of production of crown morphology which is not, however, accomplishe d by variations in the direction of prisms only but probably also by differences in prism width or by inclusion of supplementar y prisms. B . PATHOLOGIC RETZIUS LINES

1. Variations in

Mineralization

Any disturbance in cell activity can influence the composition of the cell secretions and therefore the organic matrix, the inorganic material or both. It is

A . - G.

G U S T A F S ON

not clear if a lack of minerals is primarily responsible for disturbance s in mineralization or if such a disturbance is due to some changes already present in the organic matrix. Variations in mineral content visible in polarized light, microradiograph s and dark-field illumination wil l be considered first. Pathologic Retzius lines vary in width and it can be difficult to distinguish between a broad pathologic line and adjacent areas of hyper- or hypomineralization of a kind that will be described later. A s discusse d under methods of investigation, SectionII,p. 76, it is believed that negatively biréfringent areas and bands in the enamel relative to the long axis of the prism are more mineralized than positively biréfringent or isotropic areas. This can, however, not be proved from photomicrograph s in polarized light alone as the direction of the crystals

Fig. 9. Incrementa l Retzius lines (dark) and between them zones of negatively biréfringent enamel. The Retzius lines end on the enamel surface. Human enamel, x 345. (From A.-G. Gustafson, 1959.)

14. M I C R O A N A T O M Y

AND

H I S T O C H E M I S T RY

OF

ENAMEL

91

Fig. 10. Incremental Retzius lines ending on the surface of human enamel. The lines show a step-like appearance . The inner (right-hand) side of each line is demarcate d sharply by lines transverse to the prism axes but on the other side towards the outer enamel surface the demarcation is much less sharp and less segmented . The lines are first isotropic and then slightly negatively biréfringent, χ 1100. (From A.-G. Gustafson, 1959.)

92

G. G U S T A F S ON

AND

A . - G.

G U S T A F S ON

Fig. 12. Ground section through the tip of a human tooth showing that the Retzius lines do not reach the enamel surface but run in a curve from one side to the other. Polarized light, χ 40.

14. M I C R O A N A T O M Y

AND

H I S T O C H E M I S T RY

OF

ENAMEL

93

Fig. 13. (a) Ground section of human enamel in polarized light with crossed polars. Some Retzius lines are highly negatively biréfringent (arrows) and some are not. x 65.

(b) Microradiograph of the same area showing that the negatively biréfringent Retzius lines are radiopaque and highly mineralized, χ 72. (From G. Gustafson and Gustafson, 1961).

cannot be deduced from such photomicrographs . Confirmatory evidence can be obtained, however, from microradiograph s or by X-ray diffraction of small restricted areas. If ground sections are placed in thick Canada balsam after drying in air, which actually means imbibition with air as thick balsam does not penetrate noncarious enamel, and investigated in polarized light (Fig. 13a) and then by microradiography (Fig. 13b), there is usually a close correspondenc e between areas with high negative birefringence and those of high radiopacity. A s seen previously (Fig. 11), direct comparison between the polarized light appearance s and microradiography can be made in the comparison microscope. It is, however, clear from the nature

of the two methods that polarized light gives more detail than microradiograph y because the latter shows only the inorganic material. Controls can also be made using dark-field microscopy with which homogeneou s Retzius lines appear dark and are more transparen t than their surroundings . A highly mineralized band or area is more homogene ous and therefore more transparent . I n Figs. 13a and b the two prominent Retzius lines are on this basis more mineralized than the surrounding enamel and may be called "hypermineralized". I t is possible (G. Gustafson and Kling, 1948) to demonstrate Retzius lines of greater hardness than the surrounding enamel. Strictly speaking, the expression "hyper"-mineralization is incorrect since it is impossible to

Fig. 14. High magnification of an area with broad incremental Retzius lines. The dark bands are isotropic and the light bands negatively biréfringent. Note the square prism segments . Human enamel, χ 1050. (From A.-G. Gustafson, 1961.)

Fig. 15. A broad Retzius line showing sharply delineated prism segments on the dentine side (below) whereas on the side towards the enamel surface (above) they are less sharply delineated, less well-mineralized and are isotropic and even positively biréfringent. Human enamel, χ 1100. (From A.-G. Gustafson, 1959.)

14. M I C R O A N A T O M Y

AND

H I S T O C H E M I S T RY

OF

ENAMEL

95

determine whether or not the degree of mineralization in the well-mineralized lines is greater than " n o r m a l ". This expression is used here for practical reasons as a term of comparison, i.e. with the apparently less mineralized surroundings , and is thus relative. Retzius lines may also be less mineralized than their surroundings and are said to be " h y p o "mineralized (Fig. 14). I n fact these are more common and show a great variation in appearance . Usually they are isotropic or even positively biréfringent (in Canada balsam), and they too can be made up of one single row of prism segments , or as is often the case, a number of segments . There is one important feature of Retzius lines which may affect several adjacent prism segments (Fig. 15). The side of the dark band towards the dentine is sharply delineated, with well-developed square segments , whereas the other side, towards the enamel surface, is less sharply delineated with indistinct segments showing a gradual increase in negative birefringence. This phenomeno n has been

described by Darling (1963) in connection with dental caries which, as he points out, tends to spread mainly along the striae of Retzius from which the lesion "tends to spread into the overlying enamel". There are great variations in Retzius lines, some of which are shown in Figs. 14-17. The " n o r m a l" enamel may end with a single row of highly negatively biréfringent segments followed by isotropic or even positively biréfringent segments (Fig. 16). Even under these circumstance s the external side of the line is less distinct than the internal one. Instances are to be met where the line is ill-defined on both sides (Fig. 17). One and the same Retzius line can very rarely be hypermineralize d for a certain distance while the rest is hypomineralized (Fig. 18). This variation i n the mineralization of simultaneousl y formed prism sections suggests that a local factor must play a role. Even the isotropic (or positive) segments are sometimes perfectly square (Fig. 19) and can be

Fig. 16. In this Retzius line, crossing the prisms, there is, towards the dentine (below), a row of highly negatively biréfringent segments . The Retzius line itself shows isotropy and the border line towards the surface of the enamel (above) is not sharp. Human enamel, χ 170.

Fig. 17. A rare example of a Retzius line in which both borders are unsharp. Photographe d with rotating polars. Human enamel, χ 300.

8

96

G. G U S T A F S ON

AND

A . - G.

G U S T A F S ON

Fig. 19. Retzius lines composed of rows of isotropic (dark) prism segments some of which are strikingly rectangular, χ 893. Human enamel. (From A.-G. Gustafson, 1959.)

Fig. 18. An unusual example of a Retzius line composed of negatively biréfringent segments (below) and of isotropic segments (above). Human enamel, χ 600. (From A.-G. Gustafson, 1959.)

compared with the well-mineralized segments in Fig. 5.

(negative)

2. Retzius Lines with Variation in Width of the Prism Core and the Interprismatic Substance A rare type of line disturbance is one in which the interprismatic substanc e is wider than normal (Fig. 20). These lines are not sharply delineated as the interprismatic substanc e has the form of spools extending over the area of many prism segments . The prism itself can continue over this line undisturbed but less broad. I n some places the prisms show a positive birefringence or isotropy in the area of the line. This type of line can also be seen on microradiograph s (Fig. 21).

Fig. 20. A Retzius line which appears to be produced by widening of the interprismatic substanc e and narrowing of the prisms. The limit s of the band are not sharply defined. Polarized light. Human enamel, x 333. (From A.-G. Gustafson, 1959.)

14. M I C R O A N A T O M Y

AND

Fig. 21. Microradiograph of a Retzius line in human enamel showing an appearanc e similar to Fig. 19 with widening of interprismatic substanc e and narrowing of prisms, x 305.

3. Retzius Lines in Decalcified

Sections

Retzius lines are also seen in decalcified sections (Fig. 22). Usually it is possible to see only a somewhat denser organic material in the Retzius lines but in other instances it is possible to find a deformation in the shape of the segments (Fig. 23) to the extent that they are no longer regular squares with sharp corners. Whether or not this is due to shrinkage in the preparation of the sections cannot be decided. The interprismatic substanc e is broader than normal between the affected prism segments . There are thus many variations in the form and extent of pathologic Retzius lines. Some of the least pronounced may be confused with incremental Retzius lines while others are so broad that it would be better to denote them as areas of hypomineralization. However, as long as they are more or less sharply delineated we prefer to call them Retzius lines.

H I S T O C H E M I S T RY

OF

ENAMEL

97

Fig. 22. Decalcified section of developing human enamel. Relatively broad Retzius lines (dark) show a greater amount of organic matter than the rest of the enamel. Decalcified in EDTA. Haematoxylin and eosin. χ 600.

4. Neonatal

Lines

A s pathologic Retzius lines are caused by disturbances during formation of the prisms and their segments , it is natural that disturbance s in cell activity occurring during and shortly after birth can result in a specific line, the so-called neonatal line. Fundamentally there is no difference between a neonatal line and other pathologic lines. I n Fig. 24 the enamel external to the neonatal line (postnatal enamel) shows disturbance s but this is not always the case. Sometimes the neonatal line is very insignificant and the postnatal enamel is as well mineralized as the prenatal, sometimes even better. C.

DISCUSSION

The incremental lines of Retzius are due to the successiv e apposition of layers of enamel matrix during formation of the crown (Sicher, 1962) and

98

G. G U S T A F S ON

AND

Fig. 23. Decalcified section of developing human enamel. A narrow Retzius line with two rows of segments (arrows). Between these segments relatively wide interprismatic substance and a broad cross-striation . The segments are not square but round, which may be due to shrinkage. Decalcified in EDTA. Haematoxylin and eosin. χ 890. (From A.-G. Gustafson, 1959.)

represent pauses or physiological disturbance s in the processe s of enamel formation (Darling, 1964). Sognnaes (1949b) considered that the striae of Retzius and the neonatal lines owe their appearanc e to the pattern of the organic framework. According to Hanazawa (1957), these lines are due to periods of incomplete mineralization and are rich in organic matter, a view supported by the work of Jansen and Visser (1950), who found that Retzius lines in ground sections had an affinity for fluorescent dyes, and by that of Berke (1961), who showed that striae of Retzius are evident in decalcified preparations in which much of the organic phase of enamel is preserved . The findings of Engfeldt et al. (1954) on tooth germs of dogs using microradiograph y are very similar. This view

A . - G.

G U S T A F S ON

receives support from Grimbert and Pigeat (1961), who studied replicas with phase-contras t microscopy. Berggren (1947) in his work on the staining in vivo of enamel found that the striae of Retzius had a pronounced affinity for methylene blue. He considered them to be acting as barriers for further penetration of dyes. Besides Retzius lines due to pathological influences he described lines which he considered to be due to normal nutritive channels. According to Schmidt and Keil (1958), Retzius lines are already preformed in the enamel matrix but become more pronounced after mineralization because the precipitation of the crystallites is dependen t on the organic matrix. Kérébel (1961a) has drawn attention to the great variation that exists in the appearanc e of prisms within the striae of Retzius and neonatal line, some showing thickening of the prisms sheaths and some thickening of the interprismatic substance. Hals (1953) realized the great variations in striae of Retzius when investigating them with both the fluorescence microscope and the polarizing microscope. He found that some of the striae were fluorescent whereas others appeare d as black streaks. The fluorescent striae were low in negative birefringence or were isotropic, whereas the nonfluorescent were highly negatively biréfringent. He concluded that the fluorescent striae were less mineralized than the nonfluorescen t in comparison to their surroundings . Alla n (1959b, 1963) found in polarized light a well-defined variation in mineral content along the Retzius lines. Negative lines were seen entering the isotropic and positive zones, and positive lines penetrated negative zones. The fundamenta l cause of the variations was found to be differences in the organic matrix; that is, differences in ability to accept minerals. Control microradiograph s of the same section showed lines of low absorption and also lines of high absorption and there was agreement between the results obtained with the two methods. The widely accepted theory that the striae of Retzius are caused by disease s in childhood was

14. M I C R O A N A T O M Y

AND

H I S T O C H E M I S T RY

not confirmed by G o dt (1963), who found that the width and the sites of the striae showed no relation to constitutional disorders or other disease s in infancy. This is contrary to the view of Bouyssou, Bouyssou and Teulie (1957) who found that each incremental layer was separate d from the other by a line the degree of accentuatio n of which was dependen t upon the influence of certain factors such as birth, puberty and seasona l changes . Lines due to substantia l disturbance s of structure were produced by kernicterus, birth injury, premature birth and by disease of the mother during pregnancy (prenatal syphilis, rubella, chronic nephritis, chronic fluorosis). The neonatal line is considered to be the result of trauma at birth and of consequen t changes in

OF

ENAMEL

99

nutrition (Harndt, 1955; Markert, 1958). Postnata l enamel is less mineralized and more rich in organic matter than the prenatal enamel. Sicher (1962) explained the more full mineralization of prenatal enamel in terms of the foetus developing in a well-protected environment with an adequate supply of all the essentia l materials. Hals (1953, p. 110) found it easy to identify the neonatal line in ground sections because it showed a particularly strong primary fluorescence . It also exhibited strong positive double-refraction in polarized light. I n the specimen figured by Hals the prenatal enamel appears as a uniformly dark area, while the enamel immediately beyond the neonatal line has a certain amount of fluorescence . This seems to indicate that the prenatal enamel in this

Fig. 24. Pronounce d neonatal line in the enamel of a human deciduous tooth. The enamel on the dentine side (to the left) is homogeneousl y mineralized whereas the postnatal enamel is uneven and in particular shows well-defined interprismatic areas. There is a broad band of highly negatively biréfringent enamel close to the enamel-dentin e junction x 300. (From A.-G. Gustafson, 1959.)

100

G. G U S T A F S ON

AND

specimen was more highly mineralized than the postnatal enamel. Hammarlund-Essle r (1958) on the other hand, wit h the aid of a microradiographic-microphoto metric method, found a significantly higher degree of mineralization in postnatal enamel. According to Vogel (1959), postnatal enamel is sometimes less mineralized and sometimes more mineralized than prenatal. He considers the term "Assimilationsstreifen" more appropriate than the names more commonly used for the neonatal line, because it is not so much due to a disturbance as to a process of acclimatization to the new conditions after birth. In microradiograph s of postnatal enamel, Crabb (1959) found radiopaque and radiolucent bands lying parallel to the striae of Retzius and external to the neonatal line. Structural defects were more common in postnatal than in prenatal enamel. I n conclusion, one must agree with Klees and Brabant (1962) who wrote: " L a question de l'interprétation des stries de Retzius est loin d'être entièrement résolue".

A . - G.

G U S T A F S ON

Fig. 25. Ground longitudinal section through a cusp of a human tooth in polarized light. The inner half of the thickness of enamel shows alternating dark and white Hunter-Schrege r bands, x 40. Compare with Fig. 52.

V. HUNTER-SCHREGER LINES If a tooth is cut longitudinally, lines or bands are visible running from the enamel-dentin e junction outwards, through about the inner two-thirds of the thickness of the enamel. In the tips of cusps they curve from one side of the cusp to the other. I n ground surfaces the bands are dark or light according to the direction of the incident light. Even in ground sections viewed with transmitted light they may alter their characteristic s if the direction of the light is changed. These HunterSchreger lines are most easily seen in ground sections placed between crossed filters in the polarizing microscope (Fig. 25). I t is clear that the bands are absent in the outer third of the thickness of the enamel because there the prisms are practically all parallel to each other whereas within the inner two-thirds of the enamel the prisms are arranged in bundles; within each

bundle the prisms tend to be parallel to each other. That the bands are caused by the changing direction of the prisms can be seen in decalcified sections (Fig. 26) where the cross-cut and the longitudinally cut prisms are easily visible. A s the prisms deviate in this way only on the sides of the tooth crown they are not as a rule seen in crosssection in transverse section through the crown (Fig. 27). If a ground section is investigated in polarized light with the vibration planes of the polarizing filters crossed, as in Fig. 28A, the appearanc e is obscured by the changing direction of the prisms (see section II , p. 76). The dark and light alternating bands are not related to differences in mineralization, but to the changing direction of the prisms. Thus the photograph does not tell very much about the real nature of this specimen. If the filters are rotated during the exposure (Fig. 28B) all bire-

14. M I C R O A N A T O M Y

AND

H I S T O C H E M I S T RY

Fig. 26. Longitudinal decalcified section of human developing enamel showing alternating bands of transversel y cut and longitudinally cut prisms. The bands appear almost to reach the enamel surface. However, the full thickness of enamel is not yet formed. Decalcified in EDTA. Haematoxyli n and eosin. χ 270.

fringent parts in the specimen light up whereas nonbirefringent parts do not. Cross-cut prisms do not light up. The fact that the Hunter-Schrege r lines are visible in microradiograph s (Fig. 29) should indicate a real difference in mineral content; that is, something more than just a difference in prism direction. That this may not be the case is demonstrated diagrammatically in Fig. 30 which indicates the different possibilities. Even with the same amount of organic material in a piece of enamel the X-rays may penetrate more easily in one direction than in another. The penetrability, and thus the density of the image on the photographic

OF

ENAMEL

101

Fig. 27. Transverse decalcified section of an erupted human tooth, showing the deviation of the prisms. The changing direction of the prisms can be seen. Note the more compact and relatively structureles s surface layer. Decalcified in EDTA. Haematoxylin and eosin. X 216.

plate, is dependen t on the direction of the X-rays i n relation to that of the organic prism sheaths . The effect of the presence or absence of enamel tufts wil l be discusse d in the next section. The possibility of preferential loss of tissue during the grinding of the specimens must also be considered. I n particular the polishing of sections on soft surfaces tends to produce sections which are uneven and not piano-parallel (G. Gustafson, 1945). A.

DISCUSSION

The bands of Schreger correspond to a horizontal double curvature of the prisms in the inner twothirds of the enamel. This double curvature

102

G. G U S T A F S ON

AND

Fig. 28. (A) Ground section of human enamel in polarized light. Hunter-Schrege r bands show as alternating dark and white bands. Some of the dark areas are associate d with changes in direction of the prisms in relation to the vibration planes of the polarizing filters, x 230.

involves a complete ring of enamel in transverse sections of the crown and in different rings the deviation is to the left or to the right alternately. Erausquin (1949) has produced some very clear diagrams showing the disposition of these bands. Fusayama , Higaki and Uehara (1956) have described the patterns of bands and inclination of the prisms in the enamel on the various surfaces of the h u m an permanen t teeth. Most authorities take the view that HunterSchreger bands are due to more or less regular changes in direction of groups of prisms (Staz, 1946; D. B. Scott and Wyckoff, 1947; Westin, 1952; Manley et al, 1955; Klees, 1964) and not to quantitative differences in the structure or composition of the enamel (Kriiger and Rakuttis, 1952). Sicher (1962) and others regard the pattern of arrangemen t of prism bundles as a functional

A . - G.

G U S T A F S ON

(B) The same section photographe d with rotating polars during the exposure. Clearer definition is obtained. Note Retzius lines which are not clearly visible in (A), χ 230.

adaptation minimizing the risk of cleavage in the axial direction under the influence of occlusal masticatory stresses . The appearanc e of the Schreger bands changes according to the direction of the illuminating light (Held, 1947). Klees (1964) is of the opinion that Schreger bands represen t an optical phenomenon resulting from groups of prisms running in different directions which reflect oblique incident light in various directions. Therefore the picture of diazones and parazone s is not produced by any particular portion of the prisms. This observation must be regarded as an objection against the concept of different degrees of mineralization in the zones. Berke (1961), however, found differences in the organic content of zones of enamel correspondin g wit h bands of Schreger as seen in reflected light. Baud and Held (1956) made a comparison between microradiography and staining with silver. The Schreger bands of low X-ray absorption were seen

14. M I C R O A N A T O M Y

AND

H I S T O C H E M I S T RY

Fig. 29. A microradiograph of a longitudinal section of human tooth showing Hunter-Schrege r bands of alternating radiolucency and radiopacity. The radiolucent bands are associate d with cross-cut prisms, χ 75. (From Sundstrom, 1966b.)

ENAMEL

103

on microradiographs in places where the silver stain penetrate d easily. Thus they concluded that the Schreger bands were caused by alternations in mineral content. Amprino and Camanni (1956a,b) could not always detect Hunter-Schrege r bands on microradiographs and were disinclined to believe that they represen t differences of mineralization. Wislocki and Sognnae s (1950) found that Schreger's bands wil l not take up stains unless the enamel is first partially demineralized and its organic matter unmasked, as occurs when ground sections are stained with toluidine blue at pH 5 or 4. The Schreger's bands have then a greater affinity for dyes than the rest of the enamel. This appeare d to confirm the findings of Sognnae s (1949a), who found in decalcified sections of enamel some evidence of a greater amount of organic material in Hunter-Schrege r bands. Berggren (1947) in his in vivo staining experiments found the dye distributed unevenly in the Hunter-Schrege r bands, the diazones being well stained whereas the parazone s were either stained very slightly or not at all. By microhardnes s tests Avery (1963) found that bands which in ground sections present crosssections of enamel prisms were softer than those presenting the longitudinal aspects of prisms.

1.00 Prism>—core

0.25 D

OF

.00 1.00 1

0.0

1.00

A

Β

0.37

Core

Fig. 30. Diagram showing the differential absorption of X-rays in relation to the amount of organic material passed through. If there is total (here designate d 1.00) absorption when the rays pass along the prism core {A) less will be absorbed if the rays pass areas with more organic material, e.g. in the prism sheaths . There will be virtually no absorption if the rays pass along the prism sheath (B). In other areas the proportion of X-rays absorbed depends on the relation of the rays to the prism axis (C). If the X-rays are perpendicula r to the prisms there will be a certain degree of absorption (D) which will be the same over the whole area.

104

G. G U S T A F S ON

AND

A . - G.

G U S T A F S ON

The widely held opinion that Schreger's lines are caused by differences in the direction of the prisms in successiv e layers is considered only partly correct by de Boer and Stiebeling (1958). They consider that undulations of prisms contribute as well as crossing of groups. They point out that Hunter did not give a clear description of these bands although it is customary to name them partly after him. Schreger gave the first definitive description of them in 1800 and thus strictly they should bear only his name. Mortell and Peyton (1956) also hold the view that to base an explanation entirely on changes in direction of the prisms may be an oversimplification. They claim that bands may be seen in areas of the enamel where a change in prism direction is not apparent. In contrast to most investigators, these authors, using replicas, could trace the bands from the enamel-dentin e junction right out to the periphery of the enamel in many teeth.

VI. ENAMEL TUFTS AND LAMELLAE I n transverse sections of teeth there are seen, spreading from the enamel-dentin e junction and distributed round the tooth at approximately equal distances from each other, the so-called enamel tufts. In ordinary transmitted light they have the appearanc e of narrow bands. When the same area is seen in polarized light (Fig. 31) there are, on both sides of the narrow bands, areas of different character from the rest of the enamel which, in balsam, are isotropic or positively biréfringent. I n decalcified sections (Fig. 32) of developing enamel the tufts stand out as relatively broad bands of prisms, well-stained in comparison to the rest. Hardness measurement s (G. Gustafson and Kling , 1948) show that the enamel tufts are relatively soft areas and that the areas of softness are broader than the tufts seen in ordinary light and correspond more with their extent as seen in polarized light. There is a gradient of degree of hardness from the normal enamel to the centres of the tufts.

Fig. 31. A ground section transverse to the long axis of a human tooth showing tufts arising from the enameldentine junction and traversing about one-fourth of the thickness of the enamel. When viewed in polarized light, as here, the tufts are broader than in ordinary light. They are positively biréfringent and the enamel close to the enamel-dentin e junction is highly negatively biréfringent. In the top-left corner are some negatively biréfringent Retzius lines, x 52. (From G. Gustafson, 1945.)

A direct comparison can be made between tufts seen in transmitted light (Fig. 33a) and the same tufts on a microradiograph (Fig. 33b). Al l these observations show that the tufts are less well-mineralized areas affecting a number of prisms. N ot only the prism sheath but the whole prism is involved (G. Gustafson, 1945). The tufts run as continuous bands in the long axis of the tooth (Fig. 34). The part nearest to the dentine is practically straight but farther out the tufts have a wave-like appearanc e which can be seen in planes tangential to the enamel-dentin e junction. The difference in mineralization manifest in the longitudinal soft layers, i.e. the tufts, and the variation in the direction of the prisms, i.e. the

14. M I C R O A N A T O M Y

AND

H I S T O C H E M I S T RY

OF

ENAMEL

105

section of the tooth. If the section is relatively thin, it may pass between two tufts and thus wil l appear to be evenly mineralized throughout. On the other hand, if the section includes a tuft area and at the same time is relatively thin, it may pass through several undulations of the tuft and so wil l appear to be very patchily mineralized. In sections cut obliquely to the long axis of the tooth the tufts wil l likewise appear as patches of low mineralization but more widely spaced, depending on the obliquity. If the section is thick, several tufts are likely to be included in it and microradiographs wil l thus show variations of mineralization dependen t on the presence of tufts. In very thick sections there may be so many tufts distributed over the inner part of the enamel in various planes that microradiographs wil l tend deceptively to show an evenly mineralized inner zone of enamel.

Fig. 32. An EDTA-decalcified section, about 4-5 μ thick, of enamel of an adult human tooth, showing a deeply stained tuft apparently composed of interprismatic substanc e although close to the dentine the whole prism appears to be stained. Haematoxylin and eosin. χ 380.

Hunter-Schrege r bands, are obviously connected problems so that only with difficult y can it be decided which structures are being observed in any particular area of the enamel. Differences in hardness between various Hunter-Schrege r bands are almost certainly due to the impingement of tufts on the Hunter-Schrege r bands (G. Gustafson and Kling, 1948). The relative proportion of organic material to mineral within any area of a ground section depends upon whether the section happens to pass through tufts or not. Depending on the thickness of the section and on the direction of the tufts, the following possibilities may be encountere d in a longitudinal

The possibility of the presence of tufts must always be borne in mind in interpreting any appearanc e in enamel. As the tufts tend to follow Hunter-Schrege r bands it is obvious that in microradiographs tufts wil l closely resemble HunterSchreger bands of a low degree of mineralization. Some tufts, particularly near the neck of the tooth, can be traced through the whole thickness of the enamel and to end on the surface. Such penetrating tufts in our view are the so-called lamellae. I n high magnification the finer details of tufts and lamellae are similar (Fig. 35) and it is usually not possible to distinguish between them. I t is our opinion that lamellae are tufts which for one reason or another exceed the usual length and reach the enamel surface. This does not, however, exclude the possibility of cracks occurring, during formation of the enamel or after eruption, which may be filled secondarily either by ingrowing epithelium or by material from the saliva. Such defects may be found on the surface of enamel with an extension of the epithelium penetrating the enamel. It is possible that this is an ingrowth into a crevice in the already formed enamel or a defect that occurred during amelogene sis.

106

G. G U S T A F S ON

AND

A . - G.

G U S T A F S ON

Fig. 33. (a) Ground section of human tooth in ordinary transmitted light showing tufts extending from the enamel-dentin e junction, χ 230. (b) Microradiograph of the same area showing that the tufts are radiolucent as compared with the surrounding enamel, x 230.

14. M I C R O A N A T O M Y

AND

HISTOCHEMISTRY

Fig. 34. The tufts follow a straight line close to the dentine but have a wavy course further out. This variation in direction gives rise to the Hunter-Schrege r bands perpendicular to the tufts. Al l prisms, both in the tufts and in the areas between them, are more or less parallel to one another. (From G. Gustafson and Kling, 1948.)

A.

DISCUSSION

The most commonly accepted view is that both tufts (Manley, 1948) and lamellae (Gottlieb, 1947) are due to failure of calcification. Manley found that in the tuft the organic prism sheaths stain deeply, whereas the interior of the prisms and the interprismatic substanc e do not. When seen in ultraviolet light there is a region of high fluorescence in the vicinity of the enamel tufts (Dickson et al, 1952). Darling and C r a bb (1956) and Istock, Mille r and Losee (1957) support the view that enamel tufts are hypocalcified areas. Amprino and Camanni (1956a,b) from microradiographs , Lenz (1961) from electron microscopy and Boyde and Stewart (1962), from the fact that tuft enamel eroded away more rapidly than adjacent enamel wit h the argon ion beam, reached similar conclusions.

OF

ENAMEL

107

Fig. 35. Microradiograph of a lamella and a tuft. The lamella (left) actually reaches the enamel surface but in this situation, close to the enamel-dentin e junction, there is no detectable difference between the two structures, χ 219.

Whether all the components of tufts are equally hypomineralized, or only certain parts, is not agreed by all authorities; for example, according to Meyer (1951) and Hanazawa (1957) tufts consist entirely of incompletely mineralized interprismatic substance . Although lamellae typically extend to the surface of the enamel and are continuous with the substanc e of the enamel cuticle, they occasionally do not reach the surface (Kérébel, 1952, 1959a,b) so that those who maintain that they are different from tufts or have a different origin face a very real difficulty . Klees and Brabant (1959) consider that tufts are composed of some sort of membrane which nearer the surface divides into numerous fibrils of various sizes and lengths. They consider that the organic

108

G. GUSTAFSON AND A.-G. GUSTAFSON

substance of the tufts is localized between the prisms, but that it is probable the area of tufts is not limited to the tufts as they are usually seen. I n any case the tufts do not consist of simple spaces between prisms but have a content which is resistant to acids and which both in vitro and in vivo is receptive of dyes introduced into the pulp cavity. Berggren (1947) found that dyes penetrate d deeply into enamel in vivo and that there was a considerable spreading of the dye, clearly visible as fine thin lines, beyond the limit s of the tufts. Lamellae and tufts have an affinity for silver nitrate (Frank, Deluzarche and Minck, 1950). , found Losee et al. (1957), using fluorescence that the base of the tufts was composed of fibril s consisting of interprismatic material or prism sheaths. They found that tufts were intimately related to the terminal bifurcations of dentinal tubules. Vill a (1955, 1958), who described a membrane e junction, considered the tufts at the enamel-dentin to be continuations of this membrane, each tuft being related to a "scallop" on the dentine surface. Awazawa (1959), using electron microscopy, concluded that lamellae consist of organic enamel e which are to prisms and interprismatic substanc various degrees homogeneous . I n later investigations (1960, 1959-1961) he concluded that the tufts are composed of hypomineralized enamel, i.e. enamel prisms and interprismatic substanc e which are poorly mineralized, and distinguished between tufts and lamellae on the basis of their location in the enamel and their respective widths. Berke (1961) from the residual matrix after decalcification of the enamel of erupted teeth concluded that lamellae are poorly calcified enamel prisms, extending from the dentine or enameldentine junction right up to Nasmyth's membrane. The components of the lamellae were identical in size to those of adjacent enamel prisms and followed the same course. Extensions of the dentinal processe s or tubules into lamellae were not observed. The difficulties involved in referring to "normal d by Hodson (1951) in the enamel" are discusse

course of reporting a study of structures such as lamellae and tufts in carefully decalcified and microdissecte d preparations of whole tooth crowns. He pointed out that writers who have claimed that the lamellae are mainly post-eruption phenomena or cracks have made a major error since the crowns of unerupted teeth can be shown to contain lamellae of similar diverse character and distribution to those in erupted teeth. Most of the cracktype of lamellae appear to originate during, or immediately after, the final stages of matrix formation and are, according to Hodson, probably l processe s of related to the physico-chemica maturation. Sometimes lamellar cracks were associated with a maturation defect of a wide area of the surrounding matrix. Hodson ( 1953a,b) found that the most common type of lamella is one which penetrates the enamel without any particular regard l pattern. It passes through, to its developmenta around and between rods and segments of rods. He confirmed that most lamellae occur in the cervical region of the crown. Chase (1948) put forward the view that most, if not all, lamellae have their origin in cracks which may become permeate d by organic matter from the environment of the enamel before or after eruption. Wildbolz (1950) considered that the lamellae found in the enamel could be classified into three different types: (a) true lamellae of developmenta l origin, (b) crevices artificially produced by grinding, and (c) crevices in the livin g tooth. The problem of lamellae and cracks was taken s (1950), who pointed out that most up by Sognnae of the discussion has been based upon observations of ground sections in which a lamella cannot be satisfactorily distinguished from a crack or fracture line. Sognnae s therefore based his discussion of the gross morphology, distribution and histological relationship of the lamellae to the surrounding organic framework of the enamel entirely upon the study of decalcified specimens . He found that lamellae were situated between the prism sheaths and interprismatic substanc e and pursued straight courses, not following any one prism or group of

14. M I C R O A N A T O M Y

AND

H I S T O C H E M I S T RY

prisms. This seems to indicate the independenc e of the lamella from the process of prism development . For these reasons he later (Sognnaes , 1955) suggeste d that lamellae may represen t artificial separations or cracks between the enamel prisms occurring after the tooth had erupted, the cracks becoming secondarily filled with organic matter originating, at least in part, from the saliva; that is, they could be regarded as the result of a "reparative" process. Contrary to Hodson, Sognnae s (1950) found that there were no lamella-like elements in unerupted teeth and claimed that lamellae and similar organic membranes are found where functional stresse s of one kind or another have caused discontinuity between the enamel prisms. Illustrations which show epithelial cells and hornified epithelium inside the enamel of developing teeth he regarded as examples of a rare phenomenon . I n order to clarify the situation, Hutton and Nuckolls (1953) proposed several new terms for varieties of what hitherto had been called, indiscriminately, enamel lamellae, microscopic or macroscopic developmenta l organic tracts, infiltrated cracks and regions of bacterial invasion. The matter was discusse d in a symposium by Bodecker et al (1951), who suggeste d that the best criterion for identifying a lamella was to demonstrate its continuity with the keratinized cuticle. Sognnaes in this discussion suggeste d that lamellae could not be differentiated from cracks unless specific histochemica l reactions were applied. The latter view is supported by Lavina (1956), who found that histochemically many so-called lamellae do not show the characteristic s of the organic matrix of enamel but are composed of organic substance s originating from outside. Many workers agree that lamellae and cracks cannot be distinguished from each other in ground sections (Sauerwein, 1962) while others have maintained that it is possible to distinguish between them (Brabant, 1960) especially if the sections are observed while being decalcified. Bornet (1953), pointing out that there is no relation between the number of lamellae and the

OF

ENAMEL

109

age of the tooth, took the view that lamellae consist of the organic matrix and are developmenta l in origin. Bornet, who took the view that lamellae and tufts are quite different structures, claimed that lamellae extend from the enamel-dentin e junction to the surface cuticle of enamel; but, according to Klees (1958), not all lamellae extend through the whole thickness: many extend only a short distance into the enamel from the surface, an observation which supports the view that these at least are cracks produced during the functional lif e of the tooth and repaired by an acid-resistan t substance . That they are cracks is further supported by the fact that, according to Klees, about 10 % of all lamellae extend for short distances into the dentine. Brabant and Klees (1958a, b, 1960) acknowledge d the existence of different kinds of lamellae and attributed the varieties to (1) incompletely calcified parts of the enamel; (2) cracks during development , which they consider to be rare; and (3) cracks produced after the eruption of the tooth. Lamellae of the first category are found in both unerupted and erupted teeth, contain fibrillar material and are therefore analogous to enamel tufts. This accords well with the view expresse d by Sicher (1962). I n order to detect even finer details in the enamel, Losee (1956) treated tooth sections with ethylenediamine which removes the organic component. Under the polarizing microscope Losee could then observe what he called "microlamellae" running from the surface of the enamel-dentin e junction. He was of the opinion that these microlamellae were essentially the same as the classical lamellae, the only difference being one of size, both the lamellae and the microlamellae having the same filamentous or fibrous type of structure. Very few investigators have considered the relationship between Hunter-Schrege r bands and enamel tufts (G. Gustafson and Kling, 1948). Sognnaes (1949a), however, found that the corrugated pattern of the tufts reflects itself in the grossly visible Schreger's bands. Klees and Brabant (1959) found that the tufts presente d a wave-like appearanc e where they were related to the bands of

110

G.

G U S T A F S ON

AND

Schreger. Macia (1954) regarded lamellae, or structures similar to the lamellae of other writers, as responsible for the appearance s known as the Hunter-Schrege r bands. The experimenta l demonstration by Bergman (1963) of a fluid flow from the dentine across to the enamel surface, which appears to be related to tufts and lamellae, accords well with the view of many histologists that these structures constitute the most important pathways for the passag e of substance s into the enamel both from within and from without (Kérébel, 1961b).

VII. ENAMEL SPINDLES Enamel spindles are expanded continuations of dentinal tubules which extend into the enamel for a certain distance (Fig. 36) independen t of the

Fig. 36. Ground section longitudinally through the cusp of a human tooth, showing many enamel spindles. In general they are disposed at angles to the axes of the prisms. Transmitted ordinary light, χ 228.

A . - G.

G U S T A F S ON

direction of the prisms (Fig. 37). The angle at which the spindles are disposed to the enameldentine junction is variable; at the extremities of cusps they are nearly perpendicular , but on the sides of cusps they tend to be directed towards the cervix of the tooth (A.-G. Gustafson, 1949). We have observed that if a freshly extracted tooth is fractured through the tip of a cusp a contraction of the contents of the spindles can be seen under the microscope as the preparation dries. This observation suggests that the contents have characteristic s similar to cytoplasm. A.

DISCUSSION

Concepts concerning the nature of spindles has changed very littl e in the past 20 years (G. Gustafson, 1945) but a littl e light has been thrown on their ontogenesis . Takahashi (1959), for example, found dentinal processe s projecting into the ameloblast layer before either dentine or enamel was laid down. He believes that dentinal processe s

Fig. 37. High magnification of enamel spindles in a ground section of human enamel. The air-filled spindles cross the prisms and do not at all conform to the direction of the prisms, x 425.

14.

MICROANATOMY

AND

H I S T O C H E M I S T RY

in this situation are invested with a delicate organic membrane formed probably by the ameloblasts and therefore continuous morphologically with the organic matrix of the enamel. Schmidt (1963b), on the other hand, suggests that the dentinal processe s penetrate into the enamel during amelogenesi s and are transformed into enamel spindles by the resorptive expansion of the lumen at the expense of the adjoining enamel prisms. According to Hodson (1952) there are two kinds of spindle, one due to prolongations of the dentinal processe s and one due to defects in amelogenesis .

VIII . E N A M E L

CUTICLE

The surface layer of enamel, i.e. the enamel cuticle and the enamel immediately underneath , is of the greatest importance in the initial carious attack. Nevertheles s it has been the subject of very few investigations. Replica studies have demonstrated great variation in the actual surface but the cause of these variations in enamel structure is incompletely understood ( D . B. Scott and Wyckoff, 1946a,b). The extreme surface layer (the enamel cuticle) is difficul t to examine because of the contamination of the original layer by later-acquired layers of different noncellular origin. However, on the enamel a thin layer can sometimes be found, estimated to be about 1 μ thick, which in fluorescence microscopy is the same colour as the prism sheaths (G. Gustafson, 1945). It is extremely difficul t to distinguish or study with the ordinary light microscope because there are always optical phenomena at surfaces which obscure the appearances , especially if the sections are not extremely thin. A.

DISCUSSION

When the ameloblasts have finished producing the enamel prisms, they produce a thin continuous layer which covers the surface of the enamel (Sicher, 1962). Later on during the eruption of the 9

OF

ENAMEL

111

tooth, the reduced enamel epithelium produces on the surface of this primary cuticle a keratinous secondary cuticle which varies in thickness up to about 10 μ. Ussing (1955), using electron microscopy, found that cuticular isolates obtained from the enamel surface by acid flotation consisted of a continuous structureles s membrane, the enamel cuticle, about 200-500 A thick, to the outer surface of which was attached a thicker membrane composed of epithelial cells which in sections were identified as cells of the reduced enamel epithelium. These cells were attached by submicroscopi c fibril s which enter the inner enamel cuticle. These two layers together appear to constitute Nasmyth's membrane. Turner (1958) found that the inner primary enamel cuticle was not biréfringent and, although it did not stain readily, was slightly eosinophil and, in this respect and others, resembled enamel matrix. Lenz (1961), with electron micrography, was unable to identify a primary cuticle produced by ameloblasts and reported that the enamel surface of full y developed unerupted teeth was covered only by disrupted remnants of enamel epithelium. Baud et al. (1962) were unable to support the view that Nasmyth's membrane is composed of two layers. They regard it as a single membrane, in accord with a view expresse d by Frank (1949) and Johnson and Bevelander (1958). Klees and Klees (1958) found that shortly after the destruction of the original cuticle by wear a new membrane is regularly formed on the enamel surface of both livin g and dead teeth. In their view the membrane normally present may represen t in part the embryonic cuticle and in part an acquired cuticle or post-eruptive deposit on the enamel surface. Vallotton (1945a,b,c) described these acquired cuticles as light-brown to black in colour and took the view that they represente d the initial step in the formation of plaque (see also Grimbert and Pigeat, 1961). Waerhaug (1956) found that a cuticle about 1 μ thick, comparable to the primary cuticle, formed not only on enamel surfaces which had been ground away but also on acrylic crowns which had

112

G. G U S T A F S ON

AND

been worn in the mouth. A membrane of the same thickness was also found between the enamel and the epithelial attachment and was presumably formed by these epithelial cells. The histochemistry of enamel cuticle has been studied by Johnson and Bevelander (1958). They found a well-formed cuticle staining deeply with orange G which they regard as formed by the actual conversion of the ameloblasts into a cuticle rather than by a process of secretion. Strong sulphydryl reactions indicative of keratins were given both by the cuticle and the interprismatic substance . The most extensive investigation of the enamel cuticle is that of Schule (1962) according to whom the cuticle, although no distinct boundary exists between it and the enamel, is about 0.6 μ thick, whereas after eruption it becomes 3-6 μ thick. The increase in thickness is due to the addition to the outer surface of the structureles s enamel cuticle of layers of epithelial cells. Dawes, Jenkins and Tonge (1963) point out the considerable confusion in the literature regarding the nomenclature of the organic structures on the enamel surface. They propose that the acellular inner layer, the final product of the ameloblasts , should be called the "primary enamel cuticle", that the cellular layer should be termed the "reduced enamel epithelium" and the term "acquired pellicle" should be used for the cuticle of salivary origin acquired after eruption.

A . - G.

G U S T A F S ON

structures have been named the perikymata of Preiswerk and they have long been considered to be the termination of the Retzius lines at the surface. I n sections of teeth they are seen to be slight grooves where Retzius lines reach the surface (Fig. 39). The width between Retzius lines diminishes as the lines approach the enamel surface due to a reduction in height of the prism segments present between the Retzius lines (Fig. 40). Very often, at the surface, the individual segments lose their

IX. SURFACE LAYER OF ENAMEL

If replicas are taken from the enamel surface (Fig. 38) and studied after shadowing or viewed i n the phase-contras t microscope, they reveal a pattern in which, at higher magnification, prisms md the interprismatic substanc e can easily be seen. I n addition there are more pronounced structures running horizontally lik e ribs. These are closer to each other near the cervix and at a greater distance from each other near the cusps. These

Fig. 38. Replica of a human enamel surface (in transmitted ordinary light) showing perikymata as alternating light and dark bands. In the darker bands prism ends are to be seen. In the light bands no particular structure is visible. The distance between the perikymata is smaller near the cervix (below) than towards the occlusal surface (above), χ 216.

Fig. 39. Longitudinal ground section in polarized light of human enamel, showing the relation between a perikyma (arrow) and Retzius lines. The zone of enamel at A between two Retzius lines gradually tapers as it reaches the enamel surface and finally ends in an edge at the base of the perikyma. The cervical region is below, χ 1100. (From A.-G. Gustafson, 1959.)

114

G.

G U S T A F S ON

AND

A . - G.

G U S T A F S ON

graphy (Fig. 44b), in which two Retzius lines meet producing an unusually marked depressio n on the surface of the enamel. Variations in the structure of the surface layer may be even more severe and complex (Figs. 45-47) and these variations are also encountere d in developing enamel (Figs. 48-50). In some sections the surface may present a homogene ous surface layer which is highly negatively biréfringent and hard to distinguish from the enamel cuticle (Figs. 46 and 47). Al l these details of the surface layer could be important with respect to caries, and not enough is known about them. A.

Fig. 40. Diagram showing that the decreasin g thickness of the zones between two Retzius lines as they end at the enamel surface and constitute perikymata (a) is the result of the continuous lessening in size of the prism segments . Usually there is about the same number of segment s in both the thick areas (at b) and the thin areas (at a). Sometimes the cross-striatio n is more marked in the thin areas than in the thick ones.

identity (Figs. 27 and 39) forming a nonprismatic zone. On the other hand, in other places the crossstriations may be accentuate d (A.-G. Gustafson, 1962) and the distinction between individual prisms may be less evident (Fig. 41). Alternatively the final prism segments may deviate at an angle which results in a smoother enamel surface (Fig. 42). Occasionally quite flat enamel surfaces without perikymata, and not due to wear, are met with, unassociate d with anything unusual in the internal structure of the enamel (Fig. 43). Some defects of mineralization occur, detected by polarized light (Fig. 44a) or by microradio-

DISCUSSION

The close correspondenc e between striae of Retzius and surface perikymata was clearly demonstrate d by Pantke (1959), who compared the Retzius pattern in ground longitudinal sections wit h the pattern of perikymata on replicas of the natural enamel surface made before sectioning. According to Sicher (1962), in deciduous teeth, there are no perikymata in the occlusal, antenatally formed, part of the crown, whereas they are present in the postnatal cervical part. The absence of perikymata is presumably due to the relatively undisturbed conditions in utero. The different directions of the prisms in human deciduous teeth, particularly in relation to the enamel surface, have been recorded by Miake and Higashi (1961). The prisms of the cervical third of the labial and lingual surfaces run almost at right angles to the enamel surface. The direction of the prisms near the incisai edges or cusps changes making acute angles with the enamel surface. Changes on the tooth surface occur in relation to age and other influences (Mannerberg, 1960) and D. B. Scott, Kaplan and Wyckoff (1949) point out the danger of interpreting electron micrographs of randomly selected tooth surfaces before an adequate preliminary survey has been made at lower magnification. It is necessar y to take account of the age, morphological type of tooth and position of tooth surface selected.

Fig. 41. Groundsectio n of human enamel in polarized light. There is a defective area of low mineralization to the lower left with pronounced "incremental lines", ÷ 960. (From A.-G. Gustafson, 1959.)

Fig. 42. Ground section of human enamel showing perikymata associate d with zones of prism segments of reduced height associate d with bending of prisms. x600. (From A.-G. Gustafson, 1959.)

116

G. G U S T A F S ON

AND

A . - G.

G U S T A F S ON

X. DEVELOPMENTAL HYPOMINERALIZATIONS

Fig. 43. Ground section of unworn human enamel showing an exceptionally flat enamel surface without perikymata. ÷ 459. (From A.-G. Gustafson, 1959.)

The variations in structure and mineralization mentioned so far all conform to a more or less orderly pattern. Nearly every tooth, however, presents some degree of variation, particularly in the form of hypomineralization (G. Gustafson and Gustafson, 1962). It may be said that " n o r m a l" enamel does not exist. I t is characteristic of these developmenta l areas of hypomineralization that, unlike Schreger lines and Retzius lines, they do not have sharply defined margins. They also vary very much in shape and extent. There are, however, some areas of predilection. The most common form, for instance, is a band-like zone immediately beneath the surface layer showing up in polarized light (Fig. 51). Sometimes large areas in the middle of the enamel

Fig. 44. (a) Ground section of human enamel in polarized light showing a defective hypomineralized isotropic area where a layer of enamel between two Retzius lines reaches the surface, ÷ 540.

(b) A microradiograph of the same enamel surface. The surface zone, which was isotropic in polarized light, shows a low degree of mineralization. The arrow indicates the perikyma shown in (A), x 150.

14. M I C R O A N A T O M Y

AND

Fig. 45. Ground section of human enamel in polarized light showing a surface layer of atypical structure. No prisms can be seen but there are many lines more or less parallel to the enamel surface, ÷ 540. (From A.-G. Gustafson, 1959.)

H I S T O C H E M I S T RY

OF

ENAMEL

117

Fig. 47. Surface layer of human enamel without visible prisms but with pronounced continuous crossstriation lines. The whole area is strongly negatively biréfringent, x 1100. (From A.-G. Gustafson, 1959.)

Fig. 48. Surface layer of developing human enamel. The layer contains no visible prisms but instead continuous lines parallel with the surface. There is a defect in the outermost layer. Decalcified in EDTA. Haematoxylin and eosin. χ 1600. (From A.-G. Gustafson, 1959.)

14. M I C R O A N A T O M Y

AND

Fig. 49. Surface layer of human developing enamel. There is bending of the prisms and at the actual surface no prisms are visible. Decalcified in EDTA. Haematoxylin and eosin. ÷ 350.

Fig. 50. Surface layer of developing human enamel. Three pronounced Retzius lines reach the surface. The overprinted lines help to emphasize that the area between the Retzius lines is diminishing in height (formation of perikymata). Decalcified in EDTA. Haematoxylin and eosin. ÷ 600. (From A.-G. Gustafson, 1959.)

H

120

G.

G U S T A F S ON

AND

(Fig. 52) show positive birefringence surrounded by isotropy, or can be demonstrate d in microradiographs (Fig. 53). In these areas cross-striation s are very clearly distinguishable , in contrast with areas of normal or high mineralization. Hypomineralized areas are particularly frequent near the neck of the tooth. They are softer than the surrounding enamel when investigated with the microhardnes s tester (G. Gustafson and Kling, 1948). I n many instances the defect in mineralization (Craig and Peyton, 1958) is associate d with irregularities in prism direction making it necessar y when investigating ground sections in polarized light to use rotating polarizers. Pronounce d Retzius lines (Figs. 54 and 55) tend to occur in relation to irregular areas of low mineralization. Defective areas may occur very close to the enamel surface (Fig. 56) and they then, both clinically and histologically, can be confused with

A . - G.

G U S T A F S ON

early carious lesions. On the dried tooth in situ they stand out as white spots. A.

DISCUSSION

Hals (1953) noted that hypomineralized areas showing greater primary fluorescence than the rest of the enamel are particularly common at the cervix. Atkinson and Saunsbur y (1953), Swartz and Phillips (1952) and Craig and Peyton (1958) confirmed the earlier work of G. Gustafson and Klin g (1948) which showed that these areas are softer than the surrounding enamel. The softest tooth in the study of Swartz and Phillips had a hardness number of 155, while the hardest was 229, a difference of more than 40 %. Darling and Crabb (1956) found zones of hypomineralization close to the enamel-dentin e junction but also extending over nearly the whole

Fig. 51. Ground section of human enamel in polarized light showing a zone of isotropy (pseudoisotropy ) or hypomineralizatio n at a distance from the enamel surface but parallel with it. x 250. (From A.-G. Gustafson , 1959.)

14. M I C R O A N A T O M Y

AND

H I S T O C H E M I S T RY

Fig. 52. Ground longitudinal section through the top of a human tooth cusp in polarized light. There is an extensive area of developmenta l hypomineralization affecting a large part of the middle of the thickness of the enamel. At the centre the area shows positive birefringence and, at the sides, isotropy. Hunter-Schrege r bands can be seen nearer the dentine and Retzius lines sweep over the cusp, x 34.

width of the enamel. Some differences in mineralization appeare d to correspond with bands of Schreger. Schmidt and Keil (1958) have shown that these presumed areas of hypomineralization lend themselves to study by polarized light. They found that they usually show positive birefringence which cannot be changed by any form of imbibition. Alla n (1959a) on the other hand found areas of enamel exhibiting negative birefringence. These had a central positively biréfringent zone which was bordered by isotropy. With control microradiographs he was able to show that the central part had a low mineral content. Kruger and Rakuttis (1952) also demonstrate d hypomineralized areas by microradiography. Frank (1959) studied sections of non-deminer-

OF

ENAMEL

121

Fig. 53. Microradiograph of human enamel showing radiolucent Retzius lines and diffuse radiolucent areas, ÷ 175. (From G. Gustafson and Gustafson, 1961.)

alized enamel with the electron microscope and showed that the degree of mineralization of enamel was not uniform, even in the same prism. He confirmed that differences in birefringence seen wit h polarized light represen t differences in mineralization. This is a view that was summarized by Sognnaes et al. (1960) as follows, "The mineralization in the enamel of adult human teeth may be quite variable as suggeste d by the differences in birefringence observed in the polarizing microscope by G, Gustafson (1945), G. Gustafson and Payen (1957) and Schmidt and Keil (1958). Our electron microscopic observations of nondecalcified sections indicate that these variations are morphologically related to demonstrabl e dif-

122

G. G U S T A F S ON

AND

Fig. 54. Ground section of human tooth in polarized light showing diffuse areas of developmenta l hypomineralization with characteristicall y the lowest degree of mineralization along Retzius lines (pseudo-isotropi c or positively biréfringent), ÷ 58. (From A.-G. Gustafson, 1959.)

ferences in the inorganic crystallization of enamel withi n small localized submicroscopica l areas of an enamel rod. Such differences have been detected from one rod to another and even in limited zones wit h the same rod." Kostlân and Plackovâ (1962) described three categories of developmenta l hypomineralization , segmental, central, and superficial, based upon a polarized light study of ground sections. The areas were either diffuse or circumscribed. The diffuse areas were faintly opaque, had ill-defined outlines and frequently were scattered over the greater part of the thickness of the enamel. Contrary to the findings of Schmidt and Keil (1958), they found

A . - G.

G U S T A F S ON

Fig. 55. Ground section of human tooth from an area with high fluoride content in the drinking water (4.3 ppm). Nearly all the enamel is hypomineralized . Negatively biréfringent Retzius lines alternate with isotropic or positively biréfringent lines. The boundary between enamel and dentine shows some pronounced arcades , characteristic of fluorosed enamel, χ 81. (From A.-G. Gustafson, 1961.)

that the developmenta l hypomineralized areas could easily imbibe watery solutions. Bhussry (1958) found that areas of enamel of the "whit e spot" kind show primary fluorescence and that in them histological features such as crossstriations and incremental lines are more prominent than usual. Decalcification of the sections under continuous observation showed that these areas of enamel were relatively acid-resistant . Furthermore, density and nitrogen determination showed them to be of lower specific gravity and to have a higher nitrogen content than the rest of the enamel.

14. M I C R O A N A T O M Y

AND

H I S T O C H E M I S T RY

OF

ENAMEL

123

Fig. 57. Ground section of human tooth in polarized light showing a highly negatively biréfringent zone of enamel at the enamel-dentin e junction. The enamel is at the left and dentine to the right. Compare with Figs. 24, 31, and 54. ÷ 360.

Fig. 56. Ground section of fluorosed human enamel in polarized light. The surface zone shows high negative birefringence and beneath it is an irregular zone of defective enamel, χ 170. (From A.-G. Gustafson, 1959.)

been made on this zone in which prism sheaths , segmentatio n of prisms and interprismatic substance are seldom visible with methods of light microscopy. A.

XI. INNERMOST PART OF THE ENAMEL The zone of enamel immediately next to the enamel-dentin e junction often shows high negative birefringence (Figs. 24 and 31) and there is evidence that this indicates a high mineral content (see below). It is often impossible to distinguish individual prisms in this zone which has therefore a more or less homogeneou s structure (Fig. 57). This is of some interest as many of the investigations carried out with the electron microscope have

DISCUSSION

The highly mineralized enamel at the enameldentine junction was demonstrate d on microradiographs by Engfeldt et al (1954) and Engfeldt and Hammarlund-Essle r (1956) and in polarized light by Allan (1959b) and by Hammarlund-Essle r (1958). Brudevold, Steadman and Smith (1960), however, were unable to confirm this. A band of highly mineralized enamel appeare d along the enamel-dentin e junction during early matrix formation (Allan, 1959b; Crabb, 1959; Avery, 1963). However, according to Carlstrom (1964) the special optical character of this zone

124

G.

G U S T A F S ON

AND

A . - G.

G U S T A F S ON

must depend on factors other than its degree of mineralization. Dickson et al. (1952) found in their fluorescence investigations a narrow band of very low fluorescence at the enamel-dentin e junction. This was confirmed by Losee et al. (1957), who found a hypermineralized zone, 20-30 μ thick, which exhibited low fluorescence , a characteristic birefringence and low ultraviolet light absorption. Vill a (1949, 1955, 1958) and Land (1950) described a membrane-like structure in the enamel but very close to the dentine. It appears as a mere line and is related to the enamel tufts.

XII . SIMILARIT Y OF S T R U C T U R ES I N C O N T R A L A T E R AL T E E TH Variations in the character and distribution of Retzius lines are characteristic for an individual and are duplicated quite exactly in the correspond ing areas of contralatera l teeth (Fig. 58), that is, i n areas of teeth formed at the same time. For this reason experiments on teeth should as far as possible be carried out on contralatera l teeth, because of the frequent duplication of disturbance s and variations (A.-G. Gustafson, 1955). N ot all variations are similar in contralatera l teeth: there are some exceptions, for example in many fluorosed teeth and in teeth that have been affected by local disturbance s such as trauma during development , the hypomineralized areas are not bilaterally symmetrical in distribution.

XIII . H I S T O C H E M I S T RY OF T H E A D U L T E N A M EL The relative impermeability of adult enamel, associate d with its degree of mineralization, constitutes a serious obstacle to a study of its histochemistry. Weill (1963) observed that the localization of stains is more dependen t on the density of the structures than on their chemical composition.

Fig. 58. One above the other are ground sections from contralateral human teeth as seen with a comparison microscope. The close correspondenc e between the pattern of Retzius lines is demonstrated . Polarized light, ÷ 266. (From A.-G. Gustafson, 1955.)

The reason that histochemica l investigations are carried out on undemineralize d enamel in spite of this, is that decalcification interferes with the specificity of histochemica l reactions (Wislocki and Sognnaes , 1950). In general, however, enamel can be stained histochemically only if there has been a slight demineralization on the surface such as occurs with some of fixatives, e.g. Zenker's fluid, or with some reagents , such as in the periodic acid-Schiff reaction, or can be carried out deliberately with weak acids or chelating agents. If cracks are present in the sections, coloured reagents can aggregate in them and simulate a histochemical reaction. Even small irregularities on the polished surface of a ground section may be enough to " a t t a c h" the dyes (Weill, 1963). I t is clear that the structures which are most

14. M I C R O A N A T O M Y

AND

H I S T O C H E M I S T RY

likely to react with histochemica l reagents belong to the organic matrix of the enamel and therefore investigations have been made on fully demineialized enamel. It is, however, obvious that demineralization with complete removal of the inorganic substance s must be harmful both mechanically and chemically and that it is possible for organic substance s to be so removed (Weill, 1963). There is naturally a complete removal of the soluble proteins and, most likely, changes even in the insoluble ones. According to Sognnae s (1955), the enamel matrix shares certain staining reactions with cornifying epidermis; for example, with the standard Masson trichrome method, dentine, bone and other connective tissues stained green, whereas the enamel stained reddish lik e other epithelial structures. A distinction could be drawn between the deeply staining pre-ename l matrix and the less readily staining calcifying portion. Similarly, Wislocki and Sognnae s (1950), using orange II for keratin, showed that both the organic matter of demineralized enamel and the outer stratum of skin stained a deep orange. A sulphydryl positive reaction, indicative of keratinization, can be observed in the ameloblasts and in the pre-ename l matrix (Sognnaes , 1955). Later on, when the enamel calcifies, this reaction is replaced by a reaction for disulphide bonds. In an earlier piece of work, Wislocki and Sognnae s (1950) used the Prussian blue method of Chèvremont and Frederic in which potassium cyanide as a reducing agent reconverts the disulphide groups into sulphydryls which then give the Prussian blue reaction. In developing enamel prisms, there is a well-defined greenish-blue positive reaction of the organic matter similar to, though less intense than, that of the keratinizing outer zones of epidermis. I n accord with the view that Schreger's bands are particularly rich in organic matter and specially indicative of the presence of acid mucopolysac charides, in developing enamel these bands, according to Wislocki and Sognnae s (1950), are metachromatic with toluidine blue at pH 4 - 5. In adult enamel, however, the bands do not stain or show metachromasi a until the organic matter is

OF

ENAMEL

125

unmasked by partial demineralization . Then both tufts and lamellae show metachromasia . Fullmer and Alpher (1958) found very littl e acid mucopolysaccharid e in the organic enamel matrix after demineralization of nearly completed crowns. The matrix reacted strongly with aldehyde fuchsin, but the chemical significance of this is unknown. The metachromatic substance s present appear to be ones which are susceptible to digestion with testicular hyaluronidase ; that is, chondroitin sulphate A and C and hyaluronic acid. Wislocki and Sognnaes (1950), however, found that some metachromasi a remains after hyaluronidase digestion, suggesting the presence of some more highly sulphated acid mucopolysaccharide . According to Weill (1960) and Weill and Tassin (1961), staining with both toluidine blue (at pH 4.6) and alcian blue (at pH 2.9) shows that the enamel matrix is relatively rich in acid mucopolysac charides. Weill (1960), using metachromasi a with toluidine blue, confirmed the presence of acid mucopolysac charides in the enamel cuticle and lamellae and concluded from this that both these structures are of salivary origin. The basophilia of the enamel matrix is controlled wit h buffered solutions of methylene blue or toluidine blue. In ground sections of teeth stained wit h methylene blue at low pH (about 2.4), the organic matter of the enamel, including the prism sheaths, is intensely stained (Wislocki and Sognnaes , 1950). This basophilia is consistent with the presence of acid mucopolysaccharide . It must be borne in mind, however, that treatment with dyes at such low pH produces some demineralization of the section. Wislocki and Sognnae s (1950) point out also that it is perhaps significant that basophilia is encountere d in the enamel prisms where the sulphydryl reaction occurs, as well as in the tufts and prism sheaths where the metachromatic reaction is localized. Thus, it is possible that the basophilia observed in demineralized enamel results from the presence of two different components . I n general, normal adult enamel does not give a periodic acid-Schiff reaction (Wislocki and

126

G. G U S T A F S ON

AND

Sognnaes , 1950) but in some cases lamellae give an intensely positive reaction and in some areas the interprismatic substanc e may show some reactivity. A n indistinct sudanophilic reaction is found in the prism sheaths of developing enamel (Wislocki and Sognnaes , 1950) but in ground sections of erupted teeth lipi d cannot be demonstrate d histochemically. Verne and Weill (1953) stated that the enamel was very difficult to stain because it contained very littl e organic matter. Only the prism sheaths were coloured with the Schiff method. Weill and Tassin (1961), using the tetrazonium reaction of Danielli and alcian blue, demonstrate d a protein fibrilla r network within the prism core as well as in the sheaths of immature enamel which was not present, except faintly, in the sheaths of full y matured enamel. They suggeste d that the proteins undergo some change so that they are no longer reactive wit h this reagent. These workers also found that young enamel, including the prism cores, gave a positive Barnett and Seligman sulphydryl reaction and that this reaction was diminished in older enamel. I n a later investigation, Weill (1963) found a faintly positive PAS reaction in sections of adult enamel decalcified by a special unspecified technique whereby the organic matrix was well preserved. In his view it is not possible to deduce whether the reaction is faint because of smallness of the quantity of glycoprotein or because the glycoprotein was of a kind unreactive to PAS. A. histochemica l study was carried out by Wertheimer and Fullmer (1962) on the enamel cuticle in a number of demineralized specimens . The cuticles showed acidophilia and reacted strongly with the dinitrofluorobenzene-H-aci d method for protein but was unreactive with the ninhydrinSchiff method. Evidence was found of the presence of a peculiar type of carbohydrate , which did not react with the periodic acid-Schiff method, and of a possibly protein-bound lipid. The results obtained from the relatively few histochemical investigations on adult enamel con-

A . - G.

G U S T A F S ON

firm the observations made with the light microscope with ordinary staining or with other methods such as polarized light. The different parts of the enamel which have been considered less mineralized show with the various histochemica l methods such distinct properties that they cannot be due to differences in optical behaviour only or to particular arrangement s of the submicroscopi c crystallites. To the results from the mineralized enamel must be added those from the developing or mineralizing enamel. In those stages all the details later found i n the enamel can be followed clearly. It has not been proved that the difference in staining reactivity between forming and adult enamel depends on the removal of certain ingredients. It is more likely that there is a transformation into less reactive combinations. It must, however, be expected that the histochemical investigation of the enamel wil l continue to add to our present knowledge of this tissue, and it is to be hoped that more workers wil l turn their attention to these aspects .

XIV. ENAMEL STRUCTURES IN S O M E MAMMALS Al l the foregoing is confined to a consideration of h u m an enamel and, although in broad outline the enamel of other mammals is very similar, there are certain differences in detail which it is important to point out. The morphology of the prisms in different animals has been extensively investigated by Shobusawa (1952), according to whom the morphology is characteristic or "specific" in many species. The interprismatic substance , especially its degree of development , breadth and shape, contributes essentially to the characteristic enamel structure. According to Shobusawa , the pattern of the prisms is constant for each order of mammals. Thus, in the Primates the prisms are arcade-shape d i n cross-section , with the sheath confined to one side, and the interprismatic substanc e is not sharply delineated. I n the Carnivora the prisms are hexagonal or polygonal in cross-sectio n and the

14. M I C R O A N A T O M Y

AND

H I S T O C H E M I S T RY

sheaths are well developed and surround the entire circumference of the prisms. The interprismatic substance is well demarcated . I n the Ungulata the interprismatic region is wide and prisms are ovoid or oblong in cross-section . According to Shobusawa , in the Rodentia the prisms are oval or rhomboid i n section and are arranged in lines or in closely packed zones. The prisms are very thin and the interprismatic substanc e is barely demonstrable . The Cetacea have prisms which are circular on section. Many other detailed differences are recorded by Shobusawa . A . T HE D OG The enamel of the dog has in general a structure similar to that of man. The difference is related only to the arrangemen t of the prisms, those in the dog being much more regularly arranged. For instance, the Hunter-Schrege r bands are much more pronounced and sharply defined with abrupt changes in the direction of the prisms. The interprismatic substanc e is more abundant making the prisms stand out more clearly. B . RODENTS AND LAGOMORPHS

The enamel of the continuously growing incisors of rodents is divided into three layers, of which only the middle one shows distinct prisms. The inner and the outer layers are devoid of distinct rod structure but instead show the appearanc e of fibres which are in continuity with the prisms of the middle layer and contain similarly orientated apatite crystals. It would appear also that rat enamel, in addition to its complicated microscopic architecture, is devoid of prism sheaths and interprismatic substanc e (Frank and Sognnaes , 1960). The enamel fibres in the outer thii d li e more or less parallel to one another while those of the inner enamel li e in bundles which are distributed so as to give the characteristic herring-bone appearanc e (Watson and Avery, 1954). In the intermediate layer of the enamel columns formed of single rows of prisms in cross-section , which therefore IO

OF

ENAMEL

127

have a beaded appearance , alternate with single prisms in longitudinal section. Although the outer fibrous layer of enamel is probably the most important portion of the rodent enamel, it has been the subject of but few investigations. Butcher (1956) found that the prisms in the fibrous layer are not only nearly at right angles to the transverse plane, but they are also bent about 30 degrees towards the apical end of the tooth. The complicated structure of the prisms in rat incisors leads to great difficulties of interpretation. The inter-crossing arrangemen t of prisms in the middle layer and consequen t beaded rows of prisms has led to the view that cross-striation s of prisms lik e those in h u m an enamel exist in the rat. Helmcke and R au (1962), however, could find no trace of cross-striation or of lamellae or organic substance s corresponding to such striations and thus were unable to accept the hypothesis that rhythmic growth of prisms accounted for the pattern of beaded columns. Instead, lik e Watson and Avery, they interpreted this appearanc e as due to the two-ply arrangemen t of the layers of fibrillar bundles. The most extensive investigations into the architecture of the enamel of the rat (Rattus norvegicus) have been carried out by Bouyssou, Guilhem and Viall e (1962), according to whom "L'ignorance ou la méconnaissanc e des caractère s anatomiques normaux peut conduire à des erreurs d'observation et surtout d'interprétation d'une portée considérable" . Bouyssou et al. included some comparative observations on the guinea pig (Cavia cobaya) which also appears to posses s enamel prisms which are devoid of cross-striations . Bouyssou et al. (1962) and Bouyssou (1963) described two principal layers in rat enamel, one inner and one outer. It is only in the inner layer that the criss-cross appearanc e is obvious and this has given rise to the name "honeycomb layer". I n his view the inner zone is formed by long rods (prisms) orientated in two directions and the rods i n the two layers cross each other at an angle of about 90 degrees . The rods continue into the outer layer forming fibres and here it is difficult to

128

G.

G U S T A F S ON

AND

recognize individual prisms. Bouyssou et al. confirm the opinion of Schmidt and Keil (1958) that there is no single definitive type of enamel structure among rodents. Instead there are a number of variations in structure. Yamakawa (1959) noted distinct differences i n the details of enamel structure between the Myomorpha, Sciuromorpha , Hystricomorpha and the Lagomorpha. The enamel of the Lagomorphs (rabbits and hares) do not show the division into two layers but instead the arrangemen t of groups of rods produces the appearanc e of Schreger's bands. I n the Lagomorphs each band is composed of a number of rows of prisms; in the rat and other true rodents the bands are much narrower and are composed of the alternation of single rows of prisms. C. COMPARATIVE STUDIES

Kawai (1955) has drawn attention to the different appearanc e of Hunter-Schrege r bands in various groups of mammals and placed them in four main categories, namely: (1) Carnivora, (2) Primates, (3) Ungulata and (4) Rodentia. He found that the Primate type is characteristicall y relatively broad, each band being 5-15 prisms in width. According to Held (1947), Hunter-Schrege r bands are more accentuate d in carnivores than in man. The surfaces of teeth from various carnivores were studied by Heuser and Pantke (1964). They found that the intervals between perikymata were notably different from those in man. F or instance, whereas in m an the intervals are of the order of 35 μ in the dog they were about 90 μ and in the fox about 100 μ. The cat appeare d to have a very thick enamel cuticle which obscured the surface markings, but where perikymata could be discerned they were, though irregularly distributed, often as close together as 7-18 μ. I n the bear the perikymata were more easily visible than in any of the other species mentioned and were present all over the enamel surfaces. Bodingbauer (1951) found tufts and lamellae in the enamel of the majority of a wide variety of

A . - G.

G U S T A F S ON

species he studied but noted that both of these features were absent from the enamel of the rat. Y a m a da and Ohazama (1961) were unable to find any enamel spindles in the enamel of dogs and cats but there were a few examples of dentinal tubules ending as fine points in the enamel. However, these did not pass as far into the enamel as is the case i n human enamel. I t seems likely that the diverse structure of the enamel in various mammals has evolved as an adaptation to a variety of circumstances .

XV . C O N C L U D I NG R E M A R KS The foregoing description of enamel structures is based on the long-accepte d idea that the prisms are individual, more or less rod-like structures with a core and a sheath at the periphery, and which supposes that they are separate d from each other by interprismatic substance . A s has been pointed out, there are great differences of opinion about practically every detail and the ideas arrived at from different investigations cannot yet be united to form a common concept. The so-called prism sheaths show such confusing d and variable appearance s that it may be suspecte that they may not be precise structures ensheathin g a prism core. A s they are sometimes missing entirely, sometimes missing on one side, sometimes contiguous with one another with nothing intervening and sometimes separate d by the interprismatic substance , they can hardly be an essentia l functional feature of enamel structure. These remarks apply also to the so-called interprismatic substanc e and it is equally hard to be precise about the real existence or nature of this feature. It may be abundant, totally missing or partially missing. The crystallites in the interprismatic region appear to be arranged differently from those of the prisms themselves but no one has been able to explain the formation of either prism sheaths or interprismatic substanc e during amelogenesis . I t is obvious that concepts of the structure and

14. M I C R O A N A T O M Y

AND

H I S T O C H E M I S T RY

processe s of formation of enamel must arise from consideration of all the evidence obtainable by every type of investigation.

References Allan, J. H. (1959a). The polarized light appearance s of human dental enamel. J. dent. Res. 38, 60-66. Allan, J. H. (1959b). Investigations into the mineralization pattern of human dental enamel. / . dent. Res. 38, 10961128. Allan, J. H. (1960a). Birefringence of enamel organic matrix. Nature, Lond. 185, 402. Allan, J. H. (1960b). Crystallite orientation in dental enamel. Naturwissenschaften 47, 376-377. Allan, J. H. (1963). Observations on the developmen t of dental enamel in acute experimenta l fluorosis. Proc. 9th ORCA Congr. dent. Caries, Paris, 1962 pp. 41-51. Pergamon Press, Oxford. Amprino, R. and Camanni, F. (1956a). Historadiographic and autoradiographi c researche s on hard dental tissues. Acta anat. 28, 217-258. Amprino, R. and Camanni, F. (1956b). Applicazione del metodo istoradiografico alio studio dei tessuti mineralizzati del dente. Minerva stomat. 5, 1-24. Atkinson, H. F. (1950). A high speed section cutting machine. Brit. dent. J. 88, 29-31. Atkinson, H. F. and Saunsbury , P. (1953). An investigation into the hardness of human enamel. Brit. dent. J. 94, 249-253. Avery, J. K. (1963). Microradiographic and microhardnes s studies of developing enamel. Proc. 9th ORCA Congr. dent. Caries, Paris, 1962 pp. 245-256. Pergamon Press, Oxford. Awazawa, Y. (1959). Electron microscope studies of the tissue structure of the enamel lamella. Arch, histol. jap. 16, 467-485. Awazawa, Y. (1959-1961). Optic and electron microscope observation of the tissue composition of enamel lamella. /. Nihon Univ. Sch. Dent. 2, 24-34, 91-99, 99-105 and 145-162; 3, 108-118. Awazawa, Y. (1960). Microscopie électronique des buissons de Témail dentaire humain. Bull. Grp. int. Rech. sci. Stomat. 3, 1-26. Baud, C. A. and Held, A. J. (1956). Silberfârbung, Rôntgenmikrographie und Mineralgehalt der Zahnhartgewebe . Dtsch. zahnàrztl. Z. 11, 309-314. Baud, C. Á., Bradford, E., Frank, R. M., Gustafson, G., Held, A.-J., Helmcke, J.-G. and Perdok, W. G. (1962).

OF

ENAMEL

129

Quelques aspects structuraux des tissus dentaires. Proc. 6th ORCA Congr. dent. Caries, Pavia, 1959 pp. 289-304. Edition Clin. Dent. Univ. Pavia, Pavia. Berggren, H. (1947). Experimental studies on the permeability of enamel and dentine. Svensk tandlàk. Tidskr. 40, 5-110. Bergman, G. (1963a). Microscopic demonstratio n of liquid flow through human dental enamel. Arch, oral Biol. 8, 233-234. Bergman, G. (1963b). Techniques for microscopic study of the enamel fluid in vitro. Odont. Revy, Lund 14, 1-7. Berke, J. D. (1961). Further studies on the nature of the organic matrix of human enamel. N.Y. St. dent. J. 27, 59-66. Bernick, S., Baker, R. F., Rutherford, R. L. and Warren, O. (1952). Electron microscopy of enamel and dentin. / . Amer. dent. Ass. 45, 689-696. Bhussry, B. R. (1958). Chemical and physical studies of enamel from human teeth. III . Specific gravity, nitrogen content, and histologic characteristic s of opaque white enamel. / . dent. Res. 37, 1054-1059. Bodecker, C. F., Gottlieb, B., Robinson, H. B. G., Schour, I. and Sognnaes , R. F. (1951). Enamel lamellae. Oral Surg. 4, 787-798. Bodingbauer, J. (1951). Vergleichende Untersuchunge n uber die Schmelzlamelle n und Buschel. Dtsch. Zahn-, Mund- u. Kieferheilk. 14, 303-324. Bornet, A. (1953). Weitere Untersuchunge n uber die Schmelzlamellen. Inaugural-Dissertation , University of Bern, 16 pp. Buchdruckere i DUVAG , Ostermundigen . Bouyssou, M. (1963). Quelques notions topographique s indispensable s à l'étude de l'amélogenès e chez le rat. Proc. 9th ORCA Congr. dent. Caries, Paris, 1962 pp. 209-221. Pergamon Press, Oxford. Bouyssou, M., Bouyssou,H. and Teulie,S.(1957) . L'influence de la naissanc e et de la période néo-natale sur les structures dentaires à l'état normal et pathologique. Cah. odonto-stomat. 7, 15-53. Bouyssou, M., Guilhem, A. and Vialle, P. (1962). Contribution à l'étude optique de la matrice fibrillair e de l'émail dans des dents à croissance continue et à croissance limitée. Bull. Grp. int. Rech. sci. Stomat. 5, 199-241. Boyde, A. (1965). The structure of developing mamalian dental enamel. In "Tooth Enamel. Its Composition, Properties, and Fundamenta l Structure" (M. V. Stack and R. W. Fearnhead , eds.), pp. 103-167. John Wright & Sons, Bristol. Boyde, A. and Stewart, A. D. G. (1962). A study of the etching of dental tissues with argon ion beams. / . Ultrastruct. Res. 7, 159-172. Boyde, Á., Switsur, V. R. and Stewart, A. D. G. (1963). A n assessmen t of two new physical methods applied to the study of dental tissues. Proc. 9th ORCA Congr. dent. Caries, Paris, 1962 pp. 185-193. Pergamon Press, Oxford.

130

G. G U S T A F S ON

AND

Brabant, H. (1960). Contribution l'étude des parties organiques de l'émail dentaire humain et de leurs rapports avec la carie et le métabolisme de la dent. Mém. Acad. R. Méd. Belg. [2] 4, 138-186. Brabant, H. and Klees, L. (1958a). Histological contribution to the study of lamellae in human dental enamel. Int. dent. J. 8, 539-551. Brabant, H. and Klees, L. (1958b). Contribution histologique l'étude des "lamelles transparentes " de l'émail. Rev. Stomat., Paris 59, 385-406. Brabant, H. and Klees, L. (1960). propos de quelques travaux récents concernan t la cuticule et les lamelles de l'émail dentaire humain. Rev. belge Sci. dent. 15, 33-59. Brain, Å. B. (1962). A new method for the preparation of decalcified sections of human enamel in situ. Arch, oral Biol. 7, 757-760. Brudevold, F., Steadman , L. T. and Smith, F. A. (1960). Inorganic and organic components of tooth structure. Ann. N.Y. Acad. Sci. 85, 110-132. Butcher, E. O. (1956). Enamel rod matrix formation in the rat's incisor. / . Amer. dent. Ass. 53, 707-712. Caldwell, R. C, Muntz, M. L., Gilmore, R. W. and Pigman, W. (1957). Microhardness studies of intact surface enamel. /. dent. Res. 36, 732-738. Carlstrom, D. (1964). Polarization microscopy of dental enamel with reference to incipient carious lesions. Advanc. oral Biol. 1, 255-296. Chase, S. (1948). The development , histology and physiology of enamel and dentine. Their significance to the caries process. / . dent. Res. 27, 87-92. Crabb, H. S. M. (1959). The pattern of mineralization of human dental enamel. Proc. R. Soc. Med. 52, 118-122. Crabb, H. S. M. and Darling, A. I. (1962). "The Pattern of Progressive Mineralization in Human Dental Enamel, Int. Ser. Monographs Oral Biol. No. 2, 99 pp. Pergamon Press, Oxford. Craig, R. G. and Peyton, F. A. (1958). The microhardnes s of enamel and dentin. J. dent. Res. 37, 661-668. Darling, A. I. (1963). Resistanc e of the enamel to dental caries. / . dent. Res. 42, 488-496. Darling, A. I. (1964). The structure of human dental enamel (Summary). In "Bone and Tooth" (H.J.J. Blackwood, ed.), pp. 129-133. Pergamon Press, Oxford. Darling, A. I. and Crabb, H. S. M. (1956). X-ray absorption studies of human dental enamel. Oral Surg. 9, 995-1009. Dawes, C , Jenkins, G. N. and Tonge, C. H. (1963). The nomenclature of the integuments of the enamel surface of teeth. Brit. dent. J. 115, 65-68. de Boer, J. G. and Stiebeling, G. (1958). Ein Beitrag zur Erklarung der Schregersche n Streifen. Stoma 11, 157-164. Dickson, G., Forziati, A. F., Lawson, M. E. and Schoonover , I. C. (1952). Fluorescenc e of teeth: a means of investigating their structure. / . Amer. dent. Ass. 45, 661-667. Engfeldt, B. and Hammarlund-Essler , E. (1956). Studies

A . - G.

G U S T A F S ON

on mineralized dental tissues. IX . A microradiographic study of the mineralization of developing enamel. Acta odont. scand. 14, 273-289. Engfeldt, B., Bergman, G. and Hammarlund-Essler , E. (1954). Studies on mineralized dental tissues. I. A microradiographic and autoradiographi c investigation of teeth and tooth germs of normal dogs. Exp. Cell Res. 7, 381-392. Erausquin, J. (1949). The aspect of the bands of Schreger in the horizontal sections of the enamel. / . dent. Res. 28, 195-200. Erausquin, J. (1952). Arquitectura del esmalte. Rev. odont., B. Aires 40, 181-188. Erausquin, J. (1953). "Histologia dentaria humana", 281 pp. Editado Progrental, Buenos Aires. Erausquin, J. (1961). "Histologia y embriologia dentaria", 285 pp. Editado Progrental, Buenos Aires. Fearnhead , R. W. (1963). Recent observations on the structure of developing enamel. Proc. 9th ORCA Congr. dent. Caries, Paris, 1962 pp. 257-264. Pergamon Press, Oxford. Frank, R. M. (1949). Recherche s sur la membrane de Nasmyth. C.R. Soc. Biol., Paris 143, 1243-1245. Frank, R. M. (1952). Sur les images de la trame organique de l'émail fournies par le dispositif à contraste de phase. C. R. Soc. Biol., Paris 146, 498-500. Frank, R. M. (1957). Contributions à l'étude au microscope électronique des tissus calcifiés normaux et pathologiques , pp. 1-97. Thèse de Doctorat, Fac. Méd. Université de Strasbourg. Frank, R. M. (1959). Étude au microscope électronique de l'émail humain adulte. Rapports existant entre le réseau fibrillair e organique et les cristaux d'apatite. Actualités odontostomat. No. 45, 13-35. Frank, R. M. and Deluzarche, A. (1950). Technique nouvelle de préparation d'émail humain par décalcification sous vide conduite de façon ménagée . Bull. Histol. Tech, micr. No. 2, 35-38. Frank, R. M., Deluzarche, A. and Minck, R. (1950). Sur la perméabilité de la trame organique de l'émail humain. C.R. Soc. Biol, Paris 144, 1121-1122. Fremlin, J. H. and Mathieson, J. (1962). Some preliminary observations of dental enamel by phase-contras t microscopy. Brit. dent. J. 112, 323-327. Fremlin, J. H., Mathieson, J. and Hardwick, J. L. (1961). The preparation of thin sections of dental enamel. Arch, oral Biol. 5, 55-60. Fujita, T. (1953). Uber die Gestalt der Schmelzprisme n menschliche r Zàhne. Z. Zellforsch. 38, 237-258. Fullmer, H. M. and Alpher, N. (1958). Histochemical polysaccharid e reactions in human developing teeth. Lab. Invest. 7, 163-170. Fusayama , T., Higaki, T. and Uehara, C. (1956). The directions of the enamel rods in central longitudinal sections throughout all kinds of permanen t teeth. Bull. Tokyo med. dent. Univ. 3, 43-49.

14. M I C R O A N A T O M Y

AND

HISTOCHEMISTRY

Godt, H. (1963). Uber das Auftreten und die Breite der Retziussche n Parallelstreifen im pr - und postnatale n Schmelz. Dtsch. zahnàrztl. Z. 18, 1148-1153. Gottlieb, B. (1947). "Dental Caries. Its Etiology, Pathology, Clinical Aspects and Prophylaxis". Lea & Febiger, Philadelphia, Pennsylvania . Grimbert, L. and Pigeat, G. (1961). Examen des répliques de la surface de l'émail au moyen du microscope contraste interférentiel. Arch, oral Biol. 6, 139-148. Gustafson, A.-G. (1949). Emaljkolvarnas riktning mot emaljdentingrnsen och mot prismorna. Odont. Tidskr. 57, 123-125. Gustafson, A.-G. (1955). The similarity between contralatera l pairs of teeth. Odont. Tidskr. 63, 245-248. Gustafson, A.-G. (1959). A morphologic investigation of certain variations in the structure and mineralization of human dental enamel. Odont. Tidskr. 67, 361-472. Gustafson, A.-G. (1961). The histology of fluorosed teeth. Arch, oral Biol. 4, 67-69. Gustafson, A.-G. (1962). The outermost layer of the enamel. Proc. 6th ORCA Congr. dent. Caries, Pavia, 1959 pp. 173-177. Edition Clin. Dent. Univ. Pavia, Pavia. Gustafson, G. (1945). The structure of human dental enamel. A histological study by means of incident light, polarized light, phase contrast microscopy, fluorescenc e microscopy and micro-hardnes s tests. Odont. Tidskr. 53, Suppl., 150 pp. Gustafson, G. (1957). The histopathology of caries of human dental enamel. Acta odont. scand. 15, 13-55. Gustafson, G. and Gustafson, A.-G. (1961). Human dental enamel in polarized light and contact micro-radiography . Acta odont. scand. 19, 259-287. Gustafson, G. and Gustafson, A.-G. (1962). Imperfections in enamel structure and their possible relation to caries susceptibility. Proc. 6th ORCA Congr. dent. Caries, Pavia, 1959 pp. 27-33. Edition Clin. Dent. Univ. Pavia, Pavia. Gustafson, G. and Kling, Ï . (1948). Micro-hardness measure ments in the human dental enamel. Odont. Tidskr. 56, 24-44. Gustafson, G. and Payen, J. (1957). Intérêt de l'utilisation de la lumière polarisée en histopathologic Application à l'étude des tissus durs de la dent. Pr. méd. 65, 315-318. Hallén, O. and Rockert, H. (1958). A new method for obtaining thin plane parallel sections of mineralized specimens . Nature, Lond. 182, 1225-1226. Hallén, Ï . and Rockert, H. (1960). The preparation of plane parallel sections of desired thickness of mineralized tissues. Proc. 2nd int. Symp. X-ray Micr. and X-ray Microanal., Stockholm, 1960 pp. 169-176. Elsevier, Amsterdam. Hals, E. (1953). "Fluorescenc e Microscopy of Developing and Adult Teeth. Supplemente d by Investigations with Ordinary, Polarizing and Phase-Contras t Microscope",

OF

ENAMEL

131

130 pp. Norwegian Academic Press, Oslo (and Odont. Tidskrift 61 Suppl.) Hammarlund-Essler , E. (1955). A method of preparing ground sections for microradiograph y and autoradiography. Acta odont. scand. 13, 167-179. Hammarlund-Essler , E. (1958). Comparison between degree of mineralization of prenatal and postnatal enamel. Trans. R. Sch. Dent., Stockh. and Umea No. 4, 7-13. Hanazawa, K. (1957). "Hanazawa's Atlas of the Normal Histology of Human Teeth" (rev. by S. Matsumiya, T. Matsui and S. Takuma). Dental College Press, Tokyo (in Japanese) . Hardwick, J. L., Martin, C. J. and Davies, T. G. H. (1965). The microstructure of mature dental enamel as observed under the optical microscope. In "Tooth Enamel. Its Composition, Properties and Fundamenta l Structure" (M . V. Stack and R. W. Fearnhead , eds.), pp. 168-171. John Wright & Sons, Bristol. Harndt, E. (1955). Die Bedeutung der Schmelzstruktu r in der Kariesprophylaxe . Dtsch. zahnàrztl. Z. 10, 874-883. Held, A.-J. (1947). "Structure microscopique de l'organe dentaire," 195 pp. F. Roth, Lausanne . Helmcke, J.-G. (1955). Elektronenmikroskopisch e Strukturuntersuchunge n an gesunde n und kranken Zàhnen. Dtsch. zahnàrztl. Z. 10, 1461-1478. Helmcke, J.-G. (1959a). Grenzen der licht- und polarisationsmikroskopische n Methoden in der Erforschung des Zahnschmelzes . Mikroskopie 13, 313-320. Helmcke, J.-G. (1959b). Eine Betrachtung iiber die Grenzen polarisationsmikroskopische r Methoden in der Zahnhistologie. Naturwissenschaften 46, 233-234. Helmcke, J.-G. (1959c). Anatomie und Pathologie der Zahnhartsubstanze n in kritischer Betrachtung neuerer mikroskopische r Forschungsmethoden . Dtsch. Zahn-, Mund- u. Kieferheilk. 30, 353-366. Helmcke, J.-G. (1960a). Bau und Struktur der Zahnhartsubstanzen . Ergebnisse licht-, polarisations- und elektronmikroskopische r Forschung. Dtsch. zahnàrztl. Z. 15, 155-168. Helmcke, J.-G. (1960b). Le phénomèn e des gaines prismatiques et des substance s interprismatique s de l'émail vu en microscopie optique et électronique. Bull. Grp. int. Rech. sci. Stomat. 3, 7-23. Helmcke, J.-G. (1963). Querstreifung der menschliche n Schmelzprismen . Dtsch. zahnàrztl. Z. 18, 569-575 and 626-637. Helmcke, J.-G. (1964). Kombination von elektronenmikroskopische n und neuen lichtmikroskopische n Untersuchungsmethode n fii r Strukturen des Zahnschmelzes . Proc. Wth ORCA Congr. dent. Caries, Geneva, 1963 Vol. 2, pp. 127-139. Pergamon Press, Oxford. Helmcke, J.-G. and Rau, R. (1962). La structure de l'émail des rongeurs (souris et rat). Bull. Grp. int. Rech. sci. Stomat. 5, 177-198.

132

G. G U S T A F S ON

AND

Helmcke, J.-G., Schulz, L. and Scott, D. B. (1961). Fine structure of cross-striation s in prisms of human enamel. /. dent. Res. 40, 668 (Abstract). Helmcke, J.-G., Schulz, L. and Scott, D. B. (1963). Querstreifung der menschliche n Schmelzprismen . Dtsch. zahnàrztl. Ζ. 18, 569-637. Heuser, Ç. (1956). Oberflchenhistologische Untersuchunge n uber die Grosse der Schmelzprisme n in den einzelnen Schmelzschichte n am menschliche n Zahn. Dtsch. zahnàrztl. Ζ. 11, 705-711. Heuser, Ç. (1961). Die Struktur des menschliche n Zahnschmelzes im oberflâchenhistologische n Bil d (Replicatechnik). Arch, oral Biol. 4, 50-58. Heuser, H. and Pantke, H. (1964). Oberflchenhistologische Untersuchunge n auf Perikymatien am Schmelz der Carnivore. Zahnàrztl. Praxis 15, 125-126. Hodson, J. J. (1951). A new presentatio n of some common features of the structure of human enamel. Dent. Practit. 2, 3-15 and 34-44. Hodson, J. J. (1952). The removal and replacemen t of air in enamel spindles. Dent. Practit. 3, 18-20. Hodson, J. J. (1953a). The structure and histogenesi s of certain wedge and other shaped defects in the surface of human enamel, with a note on their relation to the carious process. Brit. dent. J. 94, 141-142. Hodson, J. J. (1953b). An investigation into the microscopic structure of the common forms of enamel lamellae with special reference to their origin and contents. I. General consideration s and development . II . The form and contents of lamellae in unerupted but calcified crowns with special reference to hypoplastic lesions. III . Lamellae in erupted teeth with special reference to the form and contents of the classical type. Oral Surg. 6, 305-317, 383-398 and 495-515. Hodson, J. J. (1955). Identification of hypocalcified and demineralized areas in sections of human enamel. Nature, Lond. 175, 261-262. Hurst, V., Nuckolls, J. and Conlon, D. (1953). A simple technique for the histologic preparation of the organic matrix of tooth enamel. / . dent. Res. 32, 432-434. Hutchinson, A. C. W., Rowland, R. E. and Fosdick, L. S. (1963). Microradiographic examination of thin sections of sound dental enamel. / . dent. Res. 42, 1040. Hutton, W. E. and Nuckolls, J. (1953). Organic tracts in the enamel of completely unerupted human third molars. Oral Surg. 6, 1015-1019. Istock, J. F., Miller , C. W. and Losee, F. L. (1957). Simplified microradiograph y of bone and teeth. U.S. Forces med. J. 8, 991-997. Jansen , M. T. (1950). The grinding of very thin sections of enamel. / . dent. Res. 29, 633-636. Jansen , M. T. and Visser, J. B. (1950). Permeable structures in normal enamel. / . dent. Res. 29, 622-632. Johnson, P. L. and Bevelander, G. (1958). Histogenesis

A . - G.

G U S T A F S ON

of the keratinized enamel cuticle. Oral Surg. 11, 10551063. Kawai, N. (1955). Comparative anatomy of the bands of Schreger. Okajimas Folia anat. jap. 27, 115-131. Kérébel, Â. (1952). Le métabolisme et les prétendus canaux de l'émail. Schweiz. Mschr. Zahnheilk. 62, 507-520. Kérébel, B. (1955). Les méthodes histologiques modernes utilisées dans les recherche s sur la carie dentaire. ( l'exception du microscope électronique. ) Rev. belge Stomat. 52, 209-227. Kérébel, B. (1959a). Note sur les lamelles de l'émail dentaire humain. Schweiz. Mschr. Zahnheilk. 69, 159-161. Kérébel, B. (1959b). Recherche s sur les lamelles de l'émail dentaire humain. Rev. franc. Odonto-stomat. 6, 317-338. Kérébel, B. (1961a). Connaissance s actuelles sur la structure organique de l'émail dentaire. Actualités odontostomat. 54, 223-235. Kérébel, B. (1961b). Les structures organiques de l'émail et les théories protéolytiques des caries. Arch, oral Biol. 4, 107-119. Klees, L. (1958). Contribution histologiques l'étude des lamelles de l'émail dentaire et de leur importance pratique. Cah. odonto-stomat. 8, 17-69. Klees, L. (1961). Les lamelles, les buissons et le développement de la carie dans l'émail dentaire humain. Bull. Grp. int. Rech. sci. Stomat. 4, 1-53. Klees, L. (1964). Contribution histologique l'étude des bandes de Schreger de l'émail dentaire humain et de leur influence sur le developmen t de la carie. Bull. Grp. int. Rech. sci. Stomat. 7, 311-352. Klees, L. and Brabant, H. (1959). Contribution histologique l'étude des buissons de l'émail dentaire humain. Bull. Grp. int. Rech. sci. Stomat. 2, 1-71. Klees, L. and Brabant, H. (1962). Les stries de Retzius de l'émail dentaire humain et leur rôle dans le développemen t de la carie. Bull. Grp. int. Rech. sci. Stomat. 5, 401-459. Klees, L. and Klees, K. (1958). Uber die Regenerations fâhigkeit des Schmelzoberhutchens . Stoma 11, 58-76. Knapp, D. E., Avery, J. K. and Costich, E. R. (1958). A technique for the study of the internal structure of calcified tissues. / . dent. Res. 37, 880-885. Kostlân, J. and Plackovâ, A. (1962). The histological investigation of the developmenta l hypomineralized areas of the enamel and their comparison with the carious lesion. Arch, oral Biol. 7, 317-326. Kruger, V. and Rakuttis, G. (1952). Das Rôntgenbild der Hartsubstanze n des normalen und kariôsen Zahnes. Dtsch. zahnàrztl. Ζ. 7, 141-155. Land, M. (1950). Normal structures versus pathosis. Dent. Radiogr. 23, 32-36. Lavina, J. C. (1956). Sobre fisuros y laminillos del esmalte. An. Fac. Odont. Univ. Urug. 2, 123-138. Leicester, H. M. (1949). "Biochemistry of the Teeth". Mosby, St. Louis, Missouri.

14. M I C R O A N A T O M Y

AND

HISTOCHEMISTRY

Leimgruber, C. (1949). Schmelzbusche l und Prismenquerstreifung. Beitrag zur submikroskopische n Schmelzhistologie. Schweiz. Mschr. Zahnheilk. 59, 227-242. Lenz, H. (1961). Recherche s au microscope électronique sur la structure de l'émail et spécialemen t sur les buissons et la cuticule. Bull. Grp. int. Rech. sci. Stomat. 4, 1-23. Losee, F. L. (1956). Microlamellae in enamel demonstrate d by the use of ethylenediamine . Dent. Radiogr. 29, 23-29. Losee, F. L., Jennings, W. H., Lawson, M. E. and Forziati, A. F. (1957). Microstructure of the human tooth. A. Investigation of the dentino- enamel junction by polarization, fluorescence , micro-radiographic , and ultraviolet absorption techniques . Proj. NM 008 012.05.01, Nav. med. Res. Inst., Bethesda, Md., Res. Rep. No. 15, pp. 229-252. Lyon, D. G. and Darling, A. I. (1957). Orientation of the crystallites in human dental enamel (I). Brit. dent. J. 102, 483-488. Macia, G. (1954). Evidencia de materia organica en el esmalte. Rev. odont., B. Aires 42, 105-107. Maj, G. (1947). Ricerche statistiche sulla grossezz a dei prismi dello smalto del dente umano. Arch. ital. Anat. Embriol. 52, 186-192. Manley, Å. B. (1948). The organic structure of enamel. Brit. dent. J. 84, 183-189. Manley, Å. B., Brain, Å. B. and Marsland, E. A. (1955). "A n Atlas of Dental Histology", pp. 1-91. Blackwell, Oxford. Mannerberg, F. (1960). Appearance of tooth surface as observed in shadowed replicas. Odont. Revy 11, Suppl. 6, 1-116. Markert, Ê. H. (1958). Der Geburtsstreife n im Milchzahnschmelz. Zahnàrztl. Prax. 9, 75-76. Mathieson, J. and Fremlin, J. H. (1963). Phase-contras t illumination of the surface structure of thin tooth sections. Brit. dent. J. 114, 364-369. Meyer, W. (1951). "Lehrbuch der normalen Histologie und Entwicklungsgeschicht e der Z hn e des Menschen", pp. 3-32. Carl Hanser, Munchen. Miake, K. and Higashi, S. (1961). Microscopic investigation of the directions of enamel rods on human deciduous teeth. Bull. Tokyo dent. Coll. 2, 89-97. Mortell, J. F. and Peyton, F. A. (1956). Observations of Hunter-Schrege r bands. / . dent. Res. 35, 804-813. Myers, H. M. (1955). A study of the ultrastructure of chalky white enamel. / . dent. Res. 34, 38-43. Nalbandian, J. and Frank, R. M. (1962). Microscopie électronique des gaines, des structures prismatiques et interprismatique s de l'émail foetal humain. Bull. Grp. int. Rech. sci. Stomat. 5, 523-542. Pantke, H. (1959). Das Zahnschmelzoberh utche n im oberflchenhistologischen Bild. Stoma 12, 59-65. Poole, D. F. G. and Brooks, A. W. (1961). The arrangemen t of crystallites in enamel prisms. Arch, oral Biol. 5, 14-26.

OF

ENAMEL

133

Ryge, G., Foley, D. E. and Fairhurst, C. F. (1961). Microindentation hardness . / . dent. Res. 40, 1116-1126. Sauerwein, E. (1962). Sind die Schmelzlamelle n Artefakte? Dtsch. zahnàrztl. Ζ. 17, 1098-1103. Schmidt, W. J. (1963a). Bemerkunge n zu J.-G. Helmcke's Betrachtunge n uber die Grenzen polarisationsoptische r Methoden in der Zahnhistologie. Z. wiss. Mikr. 65, 301-317. Schmidt, W. J. (1963b). Bemerkunge n uber die Schmelzkolben beim Menschen. Dtsch. zahnàrztl. Ζ. 18, 852-855. Schmidt, W. J. and Keil, A. (1958). "Di e gesunde n und die erkrankten Zahngeweb e des Menschen und der Wirbeltiere im Polarisationsmikroskop" , 386 pp. Carl Hanser, Munchen. Schule, H. (1962). "Das Schmelzoberhautchen . Untersuchunge n uber die morphologischen , chemische n und physikalischen Eigenschaften" . 143 pp. Thieme, Stuttgart. Scott, D. B. (1955). The electron microscopy of enamel and dentin. Ann. N.Y. Acad. Sci. 60, 575-584. Scott, D. B. and Nylen, M. U. (1960). Changing concepts in dental histology. Ann. N.Y. Acad. Sci. 85, 133-144. Scott, D. B. and Wyckoff, R. W. G. (1946a). Shadowed replicas of tooth surfaces. Publ. Hlth. Rep., Wash. 61, 697-700. Scott, D. B. and Wyckoff, R. W. G. (1946b). Typical structures on replicas of apparently intact tooth surfaces. Publ. Hlth. Rep., Wash. 61, 1397-1400. Scott,D. B. and Wyckoff, R. W. G. (1947). Shadowed replicas, of ground sections through teeth. Publ. Hlth. Rep., Wash. 62, 422-425. Scott, D. B., Ussing, M . J., Sognnaes , R. F. and Wyckoff, R. W. G. (1952). Electron microscopy of mature human enamel. / . dent. Res. 31, 74-84. Scott, D. B., Kaplan, H. and Wyckoff, R. W. G. (1949). Replica studies of changes in tooth surfaces with age. /. dent. Res. 28, 31-47. Scott, J. H. and Symons, Í . Â. B. (1958). "Introduction to Dental Anatomy", pp. 131-157. Livingstone, Edinburgh and London. Shobusawa , M. (1952). Yergleichende Untersuchunge n uber die Form der Schmelzprisme n der Sâugetiere . Okajimas Folia anat. jap. 24, 371-392. Sicher, H., ed. (1962). "Orban's Oral Histology and Embryology", 5th ed. Mosby, St. Louis, Missouri. Sognnaes , R. F. (1948). The organic elements of the enamel. I. A study of the principal factors involved in the histological preservation of the organic elements of enamel and other highly calcified structures. / . dent. Res. 27, 609-622. Sognnaes , R. F. (1949a). The organic elements of enamel. II . The organic framework of the internal part of the enamel, with special regard to the organic basis for the so-called tufts and Schreger's bands. / . dent. Res. 28, 549-557.

134

G. G U S T A F S ON

AND

Sognnaes , R. F. (1949b). The organic elements of the enamel. III . The pattern of the organic framework in the region of the neo-natal and other incremental lines of the enamel. /. dent. Res. 28, 558-564. Sognnaes , R. F. (1950). The organic elements of the enamel. IV . The gross morphology and the histological relationship of the lamellae to the organic framework of the enamel. /. dent. Res. 29, 260-269. Sognnaes , R. F. (1955). Microstructure and histochemica l characteristic s of the mineralized tissues. Ann. Ν. Y. Acad. Sci. 60, 545-572. Sognnaes , R. F., Frank, R. M. and Kern, R. (1960). Calcification of dental tissues with special reference to enamel ultrastructure. In "Calcification in Biological Systems", Publ. No. 64, pp. 163-202. Amer. Ass. Advanc. Sci., Washington, D. C. Staz, J. (1946). Bands of Schreger. / . dent. Res. 25, 373-380. Sullivan, H. R. (1953). The composition and structure of human dental enamel, Dent. J. Aust. 25, 83-95. Sundstrôm, B. (1966a). A technique of preparing thinground sections of hard tissues: tooth and bone. Acta odont. scand. 24, 159-178. Sundstrôm, Â. (1966b). Schreger bands and their appearance s in microradiograph s of human dental enamel. Acta odont. scand. 24, 179-194. Sundstrôm, Â. (1966c). A technique for decalcifying thin ground specimens of adult human enamel. Arch, oral Biol. 11, 1221-1231. Swartz, M. L. and Phillips, R. W. (1952). Solubility of enamel on areas of known hardness . / . dent. Res. 31, 293-300. Syrrist, A. (1949). En orientering i elektronmikroskopie n med noen resultater fra histologiske undersokelse r av emalje og dentin. (An introduction in electron microscopy with some results from histological investigations of enamel and dentine). Odont. Tidskr. 57, 79-105. Syrrist, A. and Gustafson, G. (1951). A contribution to the technique of the electron microscopy of dentine. Odont. Tidskr. 59, 500-513. Takahashi, K. (1959). Supplementa l studies on the developmental process and histological structure of human enamel spindles. / . Japan stomat. Soc. 26, 1975-1993 (in Japanese , English summary). Turner, E. P. (1958). The integument of the enamel surface of the human tooth. Dent. Practit. 8, 341-348 and 373382. Ussing, M. J. (1955). The developmen t of the epithelial attachment. Acta odont. scand. 13, 123-154. Vallotton, C. F. (1945a). An acquired pigmented pellicle of the enamel surface. I. Review of the literature. / . dent. Res. 24, 161-169. Vallotton, C. F. (1945b). An acquired pigmented pellicle

A . - G.

G U S T A F S ON

of the enamed surface. II . Clinical and histologic studies. J. dent. Res. 24, 171-181. Vallotton, C. F. (1945c). An acquired pigmented pellicle of the enamel surface. III . Chemical studies. / . dent. Res. 24, 183-187. Verne, J. and Weill, R. (1953). Données histologiques nouvelles sur la genèse de la matrice protéique des tissus durs de la dent. Pr. méd. 61, 427-428. Villa , V. G. (1949). Dentino-ename l cuticle present in adult human teeth. J. dent. Res. 28, 565-568. Villa , V. G. (1955). Further evidence on the presence of dentino-ename l cuticle in adult human teeth. Oral Surg. 8, 1315-1317. Villa , V. G. (1958). Further evidence tending to show that enamel tufts are extensions of the dentino-ename l cuticle into the substanc e of the enamel. Aust. dent. J. 3, 331-333. Vogel, H. (1959). 1st der in utero gebildete Schmelz stets besser mineralisiert als der extrauterine? Dtsch. zahnàrztl. Ζ. 14, 815-823. Waerhaug, J. (1956). Enamel cuticle. / . dent. Res. 35, 313-322. Watson, M. L. and Avery, J. K. (1954). The developmen t of the hamster lower incisor as observed by electron microscopy. Amer. J. Anat. 95, 109-162. Weill, R. (1960). Note sur l'histochimie de la cuticule de l'émail. Ann. Histochim. 5, 147-151. Weill, R. (1963). Histochimie et autoradiographi e de la dent normale et pathologique. Proc. 9th ORCA Congr. dent. Caries, Paris, 1962 pp. 111-123. Pergamon Press, Oxford. Weill, R. and Tassin, M. T. (1961). Détection simultanée de polysaccharide s acides et de certains protides. Ann. Histochim. 6, 145-152. Wertheimer, F. W. and Fullmer, H. M. (1962). Morphologie and histochemica l observations on the human dental cuticle. / . Periodont. 33, 29-39. Westin, G. (1952). Some details in the histology of the enamel. Acta odont. scand. 10, 29-62. Wildbolz, G. (1950). Die Schmelzlamelle n und ihre Beziehung zur Karies. Schweiz. Mschr. Zahnheilk. 60, 1177-1196. Wislocki, G. B. and Sognnaes , R. F. (1950). Histochemical reactions of normal teeth. Amer. J. Anat. 87, 239-275. Wustrow, F. (1951). Das organische Grundgerus t im menschliche n Zahnschmelz . Z. Anat. EntwGesch. 116, 115-133. Yamada, M. and Ohazama , H. (1961). Observations on the enamel spindle found in dogs and cats. / . Nihon Univ. Sch. Dent. 3, 95-100. Yamakawa, K. (1959). Comparative-anatomica l studies on the enamel structure of the rodents. Acta anat. Nipponica 34, 852-866.

CHAPTER

15

ULTRASTRUCTURE OF ENAMEL J.-G.

HELMCKE

I . Methods for the Investigation of the Ultrastructure of Mature Tooth Enamel A . Ultrathin Sectioning B. Replica Technique C. Pseudo-replic a Technique D. Photogrammetr y of Electron Micrographs II . Units of Construction of Enamel A . Organic Components B. Inorganic Components

135 136 136 138 139 140 140 141

III . Ultrastructure of Human Enamel and That of Some Other Primates . . . . A . The Inner Structure of the Prisms B. The "Prism Sheaths " C. Cross-Striation of Prisms D . The "Interprismatic Substance " E. The Course of Prisms F. Retzius Lines and Hunter-Schrege r Bands G. The Enamel-Dentine Junction H. The Surface Layer

145 145 146 150 152 158 159 160 160

IV . Conclusion

161

Reference s

162

I. METHODS FOR THE INVESTIGATION OF THE ULTRASTRUCTURE OF MATURE TOOTH ENAMEL There are several possible methods of investigating the ultrastructure of tooth enamel: polarization microscopy, X-ray diffraction, electron microscopy, electron diffraction and so forth. Although none of these methods alone can explore exhaustively all stages of enamel formation as well as the full y mineralized state, each has something to contribute as a means of unravelling the problems of ultrastructure. The full potentiality of each is, however, realized only when used in combination, at least to the extent that the results of one method

are utilized in the interpretation of the results of another. The electron microscope, which studies structure more or less directly, at the present time appears to offer the widest opportunities of increasing our understandin g of ultrastructural detail, and this chapter deals mainly with evidence obtained by this means. Although this chapter is concerned with the ultrastructure of fully formed enamel, full comprehension of the structure of any tissue requires 135

J . - G.

136

HELMCKE

a consideration of the way it came into existence. Therefore, although amelogenesi s is the primary concern of other chapters, some reference to this process is necessar y here. Most aspects of amelogenesis, certainly at an ultrastructural level, are not yet beyond controversy; therefore the views expresse d here do not in all particulars agree with those expresse d in Chapter 10 (Volume I) and Chapter 14 (this volume). The techniques of preparation of enamel for study by electron microscopy are described in the following sections (A-C). A . ULTRATHI N

SECTIONING

This has been discusse d briefly in Chapter 10 (Volume I). Unmatured enamel lends itself very well to this technique, and sections can be cut on the ultramicrotome with glass and the other types of knives without prior demineralization so that, providing fixation is adequate , the structure of tissue is available for examination in a relatively undisturbed state. For mature enamel, unless demineralization is resorted to, sections must be cut with a diamond knife (Fig. 1). However, shattering of the enamel and compressio n artifacts cannot at present be avoided. Not only does the structure of the prisms tend to be disturbed to a varying extent but shattering of individual crystals may occur. Demineralization of the enamel before sectioning can be performed by special techniques which appear to preserve much of the organic matter and possibly some of the mineral (see page 140). However, it is almost certain that some of the organic matter is unavoidably lost during demineralization . What remains is denatured and tends to be displaced. Single electron micrographs cannot provide complete enough information about the relationship of various ultrastructures to one another; for example, some prisms may be in cross-sectio n and others cut obliquely or longitudinally. However, now that it is possible to cut and examine serial ultrathin sections, wax models can be made from tracings of serial electron micrographs,

Fig. 1. Electron micrograph of a section of mature full y mineralized human enamel, cut with diamond knife Three prisms are cut longitudinally, ÷ 23,000.

using the classical methods of making reconstructions. In this way the spatial relationships of various structures in enamel can be clarified (Helmcke, 1964; Boyde, 1965). B. REPLICA

TECHNIQUE

This method was first introduced, in light microscopy, by Wolf (1940) and in electron microscopy by Gerould (1944). The methods have

15. U L T R A S T R U C T U RE

OF

ENAMEL

137

Fig. 2. Electron micrograph of pseudo-replic a of the fractured surface of a fully mineralized human tooth. Shadowed . The fracture has laid open the two prisms in the centre with silicon monoxide. Parts of four enamel prisms are represented of the field approximately through the middle of their longitudinal axes. These prisms are not constructed axio-symmetrically, because the longitudinal axis is lying almost entirely to the right side of the prism. The small black spots are caused by single crystals, the larger ones by crystal aggregate s that had been torn out of the enamel and are adherent to the replica film , χ 7,400.

138

J . - G.

been later elaborated upon and good accounts of the varieties of replica technique are available in Reimer (1959), Kay (1961) and Muller (1962). I t is applicable to the outer intact surface of enamel, to artificially fractured surfaces, or to ground surfaces or the surfaces of ground sections. For large-scale examples Helmcke (1953), Matsumiya and Takuma (1954) and Awazawa (1963) may be consulted. The replica technique avoids many of the artifacts associate d with other methods and, providing meticulous care is used, it provides one of the best methods at the present time for the investigation of mature human enamel (Fig. 2). Nevertheless , misleading artifacts can easily arise from misuse. The limitation of the resolution is dependen t on the molecular size of the replica material. It is possible that on account of the molecular size of the materials used it does not go into all of the smallest of the submicroscopi c

HELMCKE

crevices of surfaces and thus may not record the finest detail of structure, at least as far as the level of resolution of the electron microscope is concerned . C . PSEUDO-REPLICA

TECHNIQUE

This is similar to the replica technique and is a term employed when crystals or other structures become attached to the replica fil m so that, when the fil m is removed, they remain on the replica surface and are removed with it (Figs. 2 and 3). When this happens it is possible to study with the electron microscope single crystals and their relation to one another. Only planes of ultrastructure can be studied wit h replicas and pseudo-replicas , especially if fractured surfaces are employed. However, it is possible to build up an appreciation in several planes, as with sections, by taking a series of

Fig. 3. Stereo electron micrographs of the border of two contiguous prisms of human enamel. The fracture surface was slightly etched. After drying, a replica was made with Triafol to which the single crystals of the etched surface are adhering in their original arrangemen t (pseudo-replica) . The crystals of both prisms interdigitate with one another at the border line, x 27,000.

15. U L T R A S T R U C T U RE

replicas at intervals during successiv e grinding into the enamel. Grinding, however, damages and transforms the structure in the ground surface itself (Berlin 1962). Short periods of etching may help to get rid of this damaged layer but at the same time etching introduces chemical alteration of the remaining enamel. The principal disadvantag e of the fracture technique is that it is not possible to select the area of enamel to be studied. Fracture is a random

OF

ENAMEL

139

process although, strictly speaking, it is selective in the sense that the planes of fracture tend to follow preferential planes of mechanica l weakness in the enamel. D . PHOTOGRAMMETRY OF ELECTRON M I C R O G R A P HS

The three-dimensiona l spatial relationships of structures in enamel can be studied in stereo-pairs of electron micrographs by applying to them

y J

ί

3 λ η

/ y '4 5 °/

4t/

-7//

f

'

\

2 5°

!

Ê ^

.

L—\

L*z

Fig. 4. (a) Stereo electron micrographs of fracture surface through Hunter-Schrege r bands of completely mineralized human enamel. The three-dimensiona l view shows the spatial arrangemen t of the enamel structural elements, ÷ 3,800. (b) Photogrammetri c representatio n of (a): The mean slope within the defined areas is indicated by a Τ ad n an angular measurement . The cross-line indicates the direction of a horizontal line in the plane of the slope in terms of points of the compass . The perpendicula r drawn to that line indicates the angle of elevation of the slope. The slope falls in the direction of its free end at an angle indicated by the number.

J . - G.

140

techniques of photogrammetr y similar to those which were evolved for the analysis of aerial photographs . Fractured surfaces on the whole provide the more favourable conditions for the use of this method in which the ultrastructure is revealed in relief rather lik e geological strata as seen in the sides of a quarry. Exact measurement s of the height of structures seen in relief can be made (Fig. 4) and in this way an appreciation of the mutual arrangemen t of the various structural elements can be obtained.

II. UNITS OF CONSTRUCTION OF ENAMEL A.

O R G A N IC COMPONENTS

1. Fibrils Organic fibrils can be regarded as unequivocably demonstrate d with the replica technique in mature, full y mineralized enamel, if the stereo photograph shows that the one end of the fibri l projects freely out of the replica while the other part of the fibri l lies within the substanc e of the replica. Organic fibril s of mature enamel that survive demineralization can be stained with contrast substance s used in electron microscopic preparative techniques and compare closely with the fibril s of immature enamel. Nevertheless , whether those in mature enamel are identical in their chemical structure with those of developing enamel cannot be determined by electron microscopy at present. Artificia l demineralization as well as natural mineralization can produce changes in the nature of the organic material. 2. Lamellae, Tufts and Spindles Lamellae, tufts, spindles and other apparently organic structures have been described at great length in the literature of light microscopy and are mentioned in a few works dealing with the electron microscopy of enamel. Awazawa (1963),

HELMCKE

for instance, used the replica technique and his figures show narrow, elongated structureles s areas surrounded by typical enamel structures. It may well be that these structures are lamellae but it has to be borne in mind that occasionally with the replica technique replica material does not adhere everywhere to the specimen. Air-fille d spaces are then enclosed by the fil m and faulty appearance s result. The matter could only be resolved if ultrathin sections could be prepared at right angles to the plane of the critical area (Fig. 5). Lenz (1961b) observed lamellae and tufts with the replica technique and described them as areas of less calcification. Boyde and Stewart (1962) found tuft regions in the scanning electron microscope by etching polished enamel surfaces with an argon ionic beam. They found that certain areas correspondin g with tufts etched away more rapidly than the rest, from which they deduced that these areas were less mineralized than the rest of the enamel. In a later work Boyde (1964, p. 217) says that these tuft areas are several whole prisms wide and, contrary to the classical view, do not consist simply of areas where there is more "prism sheath" or more interprismatic substance . It is something that affects the whole prism or "whole enamel". M y own efforts to demonstrat e these structures i n electron micrographs have met with slight success . Thin ground sections of human enamel i n which lamellae could be identified with the light microscope were coated on both sides with film s of collodion such as are used in the preparation of grids in electron microscopy. By this means the loss of organic substanc e could be expected to be avoided during subsequen t artificial demineralization of the section with dilute hydrochloric acid. Lamella-like structures were repeatedly observed but further work is required before the exact nature of these structures can be regarded as established . Some promising results were also obtained by demineralizing portions of similar thin ground-section s on film-coated grids and then shadowing with metal before examining in the electron microscope.

15. U L T R A S T R U C T U RE

OF

ENAMEL

141

Fig. 5. Stereo electron micrographs of a replica of a fracture surface through the enamel-dentin e junction of the completely mineralized human tooth. An odontoblast process can be seen to reach, or possibly just penetrate into, the enamel, ÷ 8,000. Â . INORGANIC COMPONENTS

1. Crystals The inorganic crystals of apatite (Figs. 6 and 7) are smaller than half the wavelength of visible light and, therefore, are not visible with the optical microscope. They are, of course, directly visible wit h the electron microscope and information about them can be obtained indirectly by such means as X-ray diffraction and polarized light studies. I t is not always possible to decide with complete certainty whether the radiation images seen on the fluorescent screen of the electron microscope represent mineral crystals or structures of similar morphology but of different chemical composition.

Proof of their crystalline character can, however, be obtained by the use of electron diffraction or the electron dark field technique. Detailed comparative investigation by electron microscopy of the enamel of many vertebrates (Helmcke, unpublished observations since 1949) suggests that the mineral crystals do not differ essentially in different species. Nearly all are individual crystals. Johanse n (1965) has recently demonstrate d the range of variation in the shape of individual crystals of human enamel. Studies of minutiae of this sort in a range of vertebrate enamels might be rewarding by revealing, if only in statistical terms, species differences in enamel crystal morphology. The best electron micrographs of the interior

142

J . - G.

HELMCKE

Fig. 6. Electron micrograph of a pseudo-replic a of a fracture surface through mature human enamel. The replica was shadowed with silicon monoxide producing the white shadows of the dark adherent crystals. In the rest of the replica the impressions of the surfaces of similar crystals can be distinguished, ÷ 36,000.

Fig. 7. Electron micrograph of carbon replica of the fracture surface through a human enamel prism. The fracture surface was directly shadowed with carbon and then the enamel was removed with acid. The replica shows the mutual arrangemen t of the crystals, ÷ 13,500.

of enamel crystals are those of enamel from incisors of 76-day-old rats published by Nylen and Omnell (1962). Transverse sections through the crystals show a series of dark stripes with a periodic distance of 8.2 Â which are interpreted as diffraction images (Fig. 8). Knowledge of this amount of detail of the ultrastructure of the human enamel is still lacking, but it seems reasonabl e to assume that wherever enamel crystals occur their structure is very similar. I n spite of their usually simple shape, enamel crystals do not in electron micrographs always show the same electron density throughout. Darker stripes are often present but cannot be taken to indicate structural features such as incorporation of organic substance s or disturbance s of the crystal structure since such "extinction contours" are common features of electron micrographs of single crystals. Such systems of lines are due to diffraction phenomena (Bragg condition). The atomic structure of the single enamel crystal

15. U L T R A S T R U C T U RE

OF

ENAMEL

143

Fig. 8. Thin section through an early stage of enamel developmen t of a rat incisor. The hexagona l structures are transverse sections through apatite crystals and show parallel striations with a periodicity of 8.2 Â. (From Nylen and Omnell, 1962.) ÷ 1,100,000.

cannot in any case be concluded from electron micrographs alone. For this the aid of electron fine beam diffraction and other techniques is needed (see Chapter 16). Occasionally, lines are seen in enamel crystals that contrast with the otherwise very uniform crystal image and generally run from one crystal edge across to adjacent ones. Such lines have been erroneously interpreted as being enclosed protein fibrils. Nylen (personal communication, 1964) however, has demonstrate d with enamel crystals of the mouse that these lines are optical artifacts caused by imprecise focussing. The possibility that the crystals grow upon pre-existent organic fibril s (epitaxy) cannot yet be excluded. Fearnhea d and Elliott (1962), however, conclude from a study of the needle-like crystals of the mineralization front of rat incisors II

that "enamel crystals are not deposited on to a fibrous matrix". I n electron micrographs of replicas taken from the ground surface of human enamel after short treatment with ethylenediamine , Scott (1960) has described fine structures which could be composed of unit crystals of the order of 300 Â in length. In regions where disintegration of the enamel by ethylenediamin e was in an early stage, the typical ribbon-like crystals of varying length appeared to be composed of narrower parallel bands. Scott believed that, if these smaller units are the actual crystal units of enamel, a fibrilla r organic matrix may be intimately involved in the orderly arrangemen t that gives rise to the larger ribbon-like structures. Since matrix formation precedes mineralization, axial fibres within the prisms could form a mould in which the crystals

144

J . - G.

grow, or nucleation could occur at periodic intervals on the fibrils themselves . A t the present time there is not full agreemen t about the overall or general shape of the crystals. They are described by some as hexagona l prisms, by others as ribbons, needles etc. Hohling (1960) has shown human enamel crystals which normally run parallel to the longitudinal axes of the prism, from which he concludes that the enamel crystals are hexagona l prisms terminating in pyramid-shape d points. This observation of Hohling needs further investigation. Johanse n (1965) has described the variety of shapes of crystal seen in ultrathin sections of full y formed human enamel cut with the diamond knife (Fig. 9). Irregular hexagona l shapes are common but individual crystals may be of more irregular shape and width and often the borders of adjacent crystals are in complementar y apposition. Some crystals appear to be homogeneou s in structure while others exhibit regularly spaced

Fig. 9. Electron micrograph of a section of sound human enamel from an erupted permanen t tooth. Crystals show great variation in morphology and possibly junctions between crystals. Boundaries of crystals are in complementary apposition with those of adjacent ones. (From Johansen , 1965.) X 184,500.

HELMCKE

areas of reduced electron density, particularly in the centre of crystals sectioned longitudinally. I n others the areas of reduced density are irregularly distributed. Our own work tends to confirm Johansen' s findings. 2. Amorphous Inorganic

Components

I t has been suggeste d (Chapter 16) that X-ray diffraction has provided evidence of the existence in enamel of an amorphous inorganic component, and chemical analysis suggests that this may be calcium carbonate . However, the direct demonstration of such a component by electron microscopy has yet to be accomplished . Lenz (1961a) was of the opinion that thin layers of amorphous calcium carbonate exist between the apatite crystals in full y developed enamel and that this carbonate is dissolved faster than the apatite crystals both when enamel is etched with weak acids and in the initial stage of dental caries. F r om the observation that with the replica technique usually no crystals are lifted off enamel surfaces that have not been etched whereas, after etching and in early caries, many isolated crystals are found adhering to the replica surface (pseudo-replicas) , he concluded that the amorphous C a C 03 serves to bind the apatite crystals and that etching removes the carbonate differentially and so loosens the apatite crystals. The evidence for the existence of such amorphous inorganic substance s in enamel is indirect and for the present must be regarded as hypothetical. Sometimes in electron micrographs two crystals may be found lying with their longitudinal edges parallel as pairs side by side. The degree of density i n the micrographs would indicate that the interstices between the crystals are not filled with amorphous, inorganic substances . It may, however, be possible that thin layers of organic matter are present which in the unstained specimens do not project an electron image. Scott's (1960) views on 300 Â cross-marking s on crystals have already been referred to. Lenz

15. U L T R A S T R U C T U RE

(1961a) interprets his own findings in similar terms. Frank, Sognnae s and Kern (1960) on the other hand put forward the suggestion that such an amorphous material is contained within the substance of the crystals in such a way that the unit crystals are cemented together in the direction of the o a x i s. Al l these postulated relationships between apatite crystals and other inorganic material (Torell, Zelander and Gerdin, 1964) are theoretically possible and in accord with present evidence.

OF

ENAMEL

145

the arrangemen t is fan-shape d or icefern-like (Fig. 10). In electron micrographs, a small divergency of crystal orientation can be detected; that is, the crystals li e at a slight angle to each other, an angle which is open towards the tooth surface. Hohling (1961) produced a purely hypothetical schematic representatio n to show that submicroscopic spaces can theoretically exist between the crystals of prisms, indeed must exist if the axes of the crystals are not exactly parallel to one another, and to show how the spaces formed by the angles between crystals could at first be relatively large

III. ULTRASTRUCTURE OF H U M A N ENAMEL AND THAT OF S O M E OTHER PRIMATES The majority of the earlier studies of the structure and ultrastructure of enamel were made on human teeth. Researcher s naturally tend to be interested in their own species and furthermore much of the work done on tooth structure has been motivated by a desire to comprehen d and control the dental caries with which the h u m an race is afflicted. The choice of h u m an enamel as the principal variety for study proved unfavourable because structures were observed with the optical microscope, for instance prisms sheaths and interprismatic substance , which are not well differentiated, or at least not so easily seen, as in some other mammals. Moreover, the choice of h u m an enamel was unfortunate because researcher s have tended to assume that this is the ideal model and have sought for, or assume d the presence of, the characteristic s of human enamel in every other type they have studied. H ad the first observations been made on the simpler enamel of nonmammalian vertebrates or even of some primitive mammalian forms, the more highly organized forms could have been approache d with greater understanding . A.

T H E INNER STRUCTURE OF THE PRISMS

I n human enamel the apatite crystals within the prisms are not exactly parallel to each other but

Fig. 10. Much schematize d reconstructio n of a prism of human enamel (Kurt Bogen). The outer surface is drawn round and smooth, whereas in reality it varies according to d its relationship with neighbouring prisms. The funnel-shape end of the cross section representatio n indicates a fracture surface which follows the direction of crystallization of the prism and hence of stratification of the mineral and organic . The angles which the crystals take with structural elements the prism axis vary in individual prisms. (From Helmcke, 1953.)

146

J . - G.

and then filled in as a second stage by a "secondary" deposition of other crystals between the " p r i m a r y" ones and so reduce the size of the spaces . According to Hôhling's schematic representation , owing to the divergence of the crystals within prisms, microgaps between them could occur, particularly at the borders of contiguous prisms. However, all the evidence of electron microscopy of amelogenesis at present suggests that crystals are deposited only at the mineralizing front and increases in the density of mineralization of enamel (e.g. enamel maturation) occur by an increase in size of the primary crystal, not by the later addition of new crystals. Hohling and Erwig (1960) believe that they can discern the prismatic shape of enamel crystals from stereo micrographs. They believe that the crystals posses s a pyramidal-shape d point at one end (of the oaxis) and a correspondin g hollow at the opposite one and that the point of one crystal fits into the hollow of the other. They expressly point out, however, that these schemes are hypothetical and not proven. Observations are still too meagre for firm conclusions to be reached. On the basis of some new work, Hohling (1966) is of the opinion that the hexagona l outlines as well as the dense packing need not be a universal principle i n the enamel structure. The greatest factor opposed to the acquisition of knowledge of the structure of the fan-shape d arrangement s of crystals in enamel is the fact that it has not been so far possible to secure sufficiently thin sections of completely mineralized enamel, that is without prior demineralization , in an uninterrupted sequenc e so as to be able to trace the three-dimensiona l continuity of the ultrastructure of individual prisms in three dimensions. Boyde (1965) is of the opinion that electron microscopy combined with the use of stereo techniques and electron diffraction is the only method which can relate crystal orientation to morphological position in the enamel with high enough lateral resolution in depth. F or example, polarized light used to determine extinction directions and X-ray diffraction techniques used

HELMCKE

to determine crystal orientation both use substantially thick specimens ; that is, a certain mass of enamel must be present in order to obtain any information at all. B . T H E " P R I SM

S H E A T H S"

Withi n each prism the degree of order in the arrangemen t of the crystals is relatively great. However, where any two things, each having its own ordered structure, are in contact a boundary is bound to exist; thus there is a boundary between contiguous enamel prisms which is not determined predominantly by one or the other of them. Furthermore, because at the boundary the slightly differently aligned systems of crystals do not fit together perfectly, or gear together as exactly fitting joints, structural gaps of extremely small dimensions exist within this boundary zone with greater frequency than within the substanc e of the prisms themselves (Figs. 1 and 3). The phenomeno n known as the prism sheath can be most distinctly observed with two special preparation techniques : in undemineralize d thin ground sections viewed with the light microscope, and in sections of artificially demineralized enamel viewed with either the light or electron microscope. The investigation of human enamel with the optical microscope was prosecute d almost without exception with these two techniques . Therefore it can be understood how the belief in the existence of a prism sheath arose in dental histology. Although in both cases the same optical phenomenon occurs, the appearance s are based on quite different conditions. The course of a beam of light transmitted through a microcrystalline mixed system of optically anisotropic material, such as enamel, is always influenced by the direction of the crystals; in fact each ground section of an enamel prism, in which the apatite crystals are arranged lik e a comet tail, can be regarded as a minute optical lens. If the borders of such lenses are joined together without perceptible joints, the borders stand out as dark zones against the lighter centres because of sudden changes in

15. U L T R A S T R U C T U RE

OF

ENAMEL

147

Fig. 11. Electron micrograph of a section, 20 m^ thick, of demineralized enamel from a rhesus monkey one and a half years old. The section is cut obliquely to the long axes of the prisms. In the centre and in the contact zones between prisms the organic components consist of fibrils, and in the border zones fibril s of the same kind have aggregate d during demineralization . x 25,500.

148

J . - G.

crystal orientation at the junctions of adjacent prisms (Helmcke, 1953; Meckel, Griebstein and Neal, 1965b). The planes of discontinuity in the gradual change of crystal orientation from one prism to the other are related to abrupt changes i n the orientation of the mineralizing front in these prisms and are equivalent in position to the "prism sheath" of adult enamel (Boyde, 1965). This is observed to be the case for spheritic crystal systems (e.g. chalcedonies ) as well as thin ground sections of enamel. Similar phenomena have been described in other biological microcrystalline systems such as in walls of Foraminifera (Wood, 1949). When sections of demineralized enamel are examined, these optical phenomena do not appear but instead a change is wrought in the enamel which likewise gives rise to the appearanc e of a prism sheath. The fragile organic matrix, lacking

Fig. 12. Diagram of the contact zone between two contiguous prisms. The optical effect of a prism sheath is easily created at those zones where the crystals are adjacent and are orientated differently. This effect disappear s where, in the lower part of the diagram, the prisms are parallel to one another.

HELMCKE

the support of the crystals, tends to collapse towards the junction zones between prisms and the result is an apparent concentration of organic matter at the peripheries of the prisms. The phenomenon of a prism sheath always appears after demineralization as an artifact when the crystals of two contiguous prisms are joined obliquely to each other (Fig. 11) but is absent in areas where the crystals of adjacent prisms are parallel with one another (Fig. 12). Boyde (1964) and other authors maintain that there really is more organic matter in the periphery of the prism than in the rest of the prism but this "prism sheath" region is in fact, as seen with the electron microscope, only about 1000 Â wide. It appears to be very much wider (0.5-1 μ) in ground sections viewed with light microscope because of the thickness of section and optical artifacts in general, and wider in demineralized specimens because the width of the " s h e a t h" and the quantity of organic matter in it is increased by various types of artifacts. Electron microscopic investigations on carefully orientated specimens have confirmed that the axis of the prism from which the crystals radiate is eccentrically situated; it lies to the occlusal side of the centre of the prism. According to the plane through which a prism is divided longitudinally in a ground section or section surface so the structural appearanc e varies (Figs. 13 and 14). Only if this section lies by chance

Fig. 13. Simplified diagram (K. Bogen) of three prisms. Only the centre prism is laid open in the plane of the long axis. Both the others are cut through peripheral areas. Different structural patterns produced in electron micrographs are represente d on the right.

Fig. 14. (A) Electron micrograph of replica of a fracture surface of human enamel in a plane parallel to the long axes of the prisms. The various structural patterns are due to the fact that the fracture surface has laid open the prisms partly in the plane of the longitudinal axis and partly through the peripheral areas, x 9,000. (B) Another part of the same specimen at a higher magnification. To the left the crystals are in cross-sectio n and to the right they are seen longitudinally, ÷ 10,600.

150

J . - G.

i n the plane of the "crystal orientation axis", as rarely happens , is the characteristic fan-shape d arrangemen t distinctly seen. But if the section surface lies to one side of the axis, the crystals of the prisms wil l appear partly in cross section and partly in oblique section. Consequentl y zones with band-like patterns and zones with punctate patterns alternate. Withi n one single prism a variety of structural patterns may be revealed according to the plane of section; enough to induce the cursory observer to attribute each pattern to a special substanc e ("prism", "prism sheath" or "interprismatic substance"). Of course it is easy to associate different phenomena with correspondingl y different substances and to provide them with correspondingl y separate terms. C.

CROSS-STRIATION OF PRISMS

On the whole there are four hypothese s for the appearanc e of cross-striations . a. Cross-striation due to optical effects: rhythmical sequenc e of constriction of the prism (Helmcke, Schulz and Scott, 1961, 1963). b. Cross-striation resulting from rhythmical mineralization: in the area of cross-striation poorer mineralization and consequentl y increased organic substanc e (Gustafson, 1959). c. Cross-striation preformed in the initiall y la d down organic matrix: thus insufficient space for mineralization (Wustrow, 1951). d. Cross-striation by fine spaces within the substance of the prism: on the evidence of polarization-microscopic and X-ray diffraction investigations the opinion was expresse d (Schmidt and Keil , 1958; Darling, 1961a; Poole and Brooks, 1961) that these spaces may be filled with saliva, wit h air or water, according to the medium in which the tooth is placed. In preparative procedures for histological investigations the spaces may become filled with various embedding media. Cross-striation is nearly always observed in ground sections examined with the optical microscope but seems to be restricted principally to the

HELMCKE

outward half of the enamel layer, usually disappearing near the Hunter-Schrege r bands. I t must be assume d that cross-striation s have a real structural basis because the effect can also be transmitted to replicas (Fig. 15). Hence the phenomeno n of cross-striation is to be seen not only with the optical, but also with the electron microscope and with X-rays. A n explanation may be advanced to account for the appearanc e of cross striations but the explanation may hold good only for the electron micrographs under consideration and may not have universal applicability (Helmcke et ai, 1961, 1963). In the area of each cross-striation of our specimens the size of the prism was increased (Fig. 16). The regular cross-striation s on a prism correspond to a rhythmical sequenc e of enlargements and constrictions of the diameter that determine the external form of the prism. The interior structure of the prism remains fan-shaped and unchange d where there is a crossstriation; that is, the arrangemen t of the crystals is

Fig. 15. Electron micrograph showing two human enamel prisms. Triafol replica, shadowe d with palladium, of a thin ground section which had been etched for 30 seconds with 0.1 7VHC1. The rhythmical alternation between elevations and troughs of waves suggest s cross-striation . χ 4,250.

15. U L T R A S T R U C T U RE

OF

ENAMEL

151

Fig. 16. Electron micrograph of replica of an etched ground section of mature human enamel. Shadowed with silicon monoxide. In the photograph on the right the borders of two contiguous prisms have been indicated by lines. The prisms are laid open in the plane of the longitudinal axis. Note the feather-like arrangemen t of crystals. The rhythmical sequenc e of the waves at the border line correspond s to the cross-striation . (From Helmcke et ai, 1963.) × 9,600.

the same as elsewhere in the prisms, slightly diverging in the direction of the enamel surface. In the area of the crystal orientation axis of a prism the crystals are lying approximately in the direction of this axis. The greater the distance of the crystals from that axis the greater the angle becomes between the crystals and the axis (Fig. 17). Naturally the expanded portion of one prism always correspond s to a constriction of the contiguous prism, and there are correspondin g reciprocal conditions in the angulation of the crystals in contiguous prisms. The optical influences produced by these conditions would neutralize each other where the axes of rays of light are

transverse to the axes of the prisms. Generally, however, it can be observed that the unilaterally developed extension of one prism protrudes nearly to the crystal orientation axis of the contiguous prism. Therefore different angles in the arrangemen t of the crystals wil l appear to either side of the boundary area so that no neutralization of the optical effects of rays of light traversing such a pair of prisms is to be expected. In this case different orientations of crystals are responsible for the phenomeno n of cross-striations . A s a result of the scattering of light by the manifold sub-microcrystalline patterns of enamel, diverse dispersions , diffractions and other optical phenomena occur which often produce appearance s

152

J . - G.

HELMCKE

than the relatively large molecules of replicating materials so far used, and are therefore not reproduced on the replica surface. D . T H E "INTERPRISMATIC SUBSTANCE"

Fig. 17. Diagram of a prism laid open in the longitudinal axis. The angles of inclination of the crystals in various regions are indicated. In the upper part of the prism the angle of inclination of some crystals towards the longitudinal axis is indicated. For simplicity the crystals are shown in tetragonal and not hexagona l cross-section . (From Helmcke et al, 1963.)

suggestive of the existence of real chemical or structural differences. Different sub-microcrystalline patterns can produce by scattering, dispersion, diffraction, interference and other optical defects the same appearances as would be expected if structures composed of different materials were present. Another hypothesis assume s that the crossstriations are caused by rhythmic variations of higher and lower mineralization (Gustafson, 1959). However, it has not been possible to detect with the electron microscope such rhythmic variations either of the mineral or of the organic fraction. Furthermore, preformed organic structures (Wustrow, 1951) have not been observed with the electron microscope either in developing or in full y mineralized enamel. N or have the microspace s that have been postulated on the basis of imbibition experiments (Darling, 1961b; Darling et al., 1961) as the cause of the phenomeno n of cross-striation yet been demonstrate d with the electron microscope. It may be that these microspace s are smaller

The outline of prisms has been described in earlier times as uniform, regular, triangular, roundish, oval, hexagonal, polygonal, irregular or arcade-like. It has been left to the electron microscope to clarify the details of prism morphology and structure. It has been establishe d that there is no special interprismatic substanc e (Helmcke, 1953). What was regarded as such has been shown to be projections or processe s from the prisms themselves and an integral part of them. This knowledge was gained using the combined light and electron microscopic technique of uninterrupted section series (Helmcke, 1964) as well as by ultrathin sections through the mature, undemineralize d enamel (Hohling, 1961, 1963). Moreover, a carefully orientated reconstruction from sections of completely mature enamel using the three-dimensiona l system of coordinates has been made by Meckel et al. (1965a,b) (Fig. 18). F r om sections in which the prisms were cut in a variety of planes, including planes exactly perpendicular to the long axes of the prisms, Meckel and associate s came to the conclusion that the cross-sectio n of each prism is shaped lik e a keyhole (Fig. 19) with a more or less round head, about 5 μ in diameter, and a narrower tail-lik e extension, about 5 μ in length, which passes between the heads of adjoining prisms (Fig. 20). The heads are orientated towards the occlusal surface of the tooth and the tails towards the cervix of the crown. A s the plane of sectioning deviates from being perpendicular to the long axis of the prism, its cross-sectio n appears broadene d or lengthened . The borders of the "keyholes" are demarcate d by sudden changes in crystal orientation. In the front parts of the head region of the prism crosssection, the crystals appear to li e in the long axis of the prism but towards the tail end there is a gradual change in the orientation of the prisms

15. U L T R A S T R U C T U RE

OF

ENAMEL

153

Fig. 18. Solid model of enamel structure constructed of prisms of extruded Play-Doh and cut with a wire cutting tool. (From Meckel et al.y 1965a.)

Fig. 19. Cardboard model of enamel structure corresponding to that of Fig. 18. (From Meckel et al.9 1965a.)

until within the tail itself the crystals li e with their long axes nearly perpendicula r to the longitudinal axis of the prism and fan out towards the extremity of the tail from the midline of the "keyhole" in a herringbone fashion. N o differences in the density of enamel structure were observed by Meckel et al. between prism borders and main body of the prism, nor between the head and tail regions of the prism cross-sections . N o evidence has been obtained for the existence of any interprismatic material; in other words, all of the crystals observed in enamel can be clearly assigned to specific enamel prisms. Carlstrom (1964) came to similar conclusions from the evidence of X-ray diffraction and polarization microscopic investigations (Fig. 21). This concept corresponds , with certain reservations, to the model (pattern 3 of Primates, Proboscidea and Carnivora) of Boyde (1965) (see Fig. 34 of Chapter 3, Volume I). In this arrangement the cervical, open side of the horsesho e (arcade) cross-sectio n of the prism sheath faces a " g a p" between two prisms sheaths of the next row cervically. This means that there is no abrupt

change in the crystal orientation in the passag e from the coronally situated prisms to the cervical ones. The substanc e of the "winged process" is therefore in direct continuity with the main body of the prism and it is therefore permissible to say that there is no region which can be called interprismatic. Boyde (1965) has also described other patterns which occur in other groups of mammals. These observations are very important to comparative dental histology and have phylogenetic implications for a discussion of which the reader is referred to Chapter 3 (Volume I). The models of Meckel et al. (1965a,b) and Boyde (1965) are very convincing but they do not represent the only conditions found in human enamel. F r om their light microscopic study of very thin ground sections prepared by the Fremlin, Mathieson and Hardwick (1961) method, Hardwick, Martin and Davies (1965) have expresse d the opinion that the condition of the prism borders is so diverse in human enamel that the keyhole pattern cannot be accepted as the generalized form.

HELMCKE

Fig. 20. Electron micrographs of sections of mature human enamel. (A) Prisms viewed in cross-section , ÷ 6,000. (Â) Prisms viewed in longitudinal section; plane of sectioning perpendicula r to the head-tail direction of prisms, x 5,000. C. Prisms viewed in longitudinal section; plane of sectioning passes through the head-tail direction of prisms, ÷ 7,200. (From Meckel et al, 1965a,b.)

15. U L T R A S T R U C T U RE

Fig. 21. Schematic drawing of the crystal orientation in enamel based in accord with X-ray diffraction and polarization microscopy data. (From Carlstrom, 1964.)

OF

ENAMEL

155

Microradiographic examinations (Glas and Nylen, 1965) in which enamel specimens were radiated correspondin g to the three dimensions of a cube confirm these findings. If the X-ray beam penetrates the enamel in the direction of the long axes of the prisms, then the typical cross-sectio n images of the prism bodies and prism processe s appear, equivalent to the keyhole-like figures. If the beam penetrate s the enamel perpendicula r to the longitudinal axes of the prisms from one direction then the outline of the prisms changes , and "interprismatic substance " appears . If the beam penetrate s the enamel in the third dimension, namely perpendicula r to the prism axes but at right angles to the last mentioned direction, then no alteration in appearanc e occurs. Wit h a combined light and electron microscopic technique the outward form of several prisms could be exactly traced in all their details and for

Fig. 22. Solid reconstruction of an enamel prism of rhesus monkey based upon electron micrographs ( ÷ 10,000) of a series of 161 sections. Outlines of the cross-section s of the prisms were transferred to sheets of plastic of appropriate thickness, cut out and assembled . Total length of prism = 80 μ. (From Helmcke, 1964.)

156

J . - G.

as much as 80 μ of the length of a prism (Helmcke, 1964) or, in one instance (Helmcke, unpublished observations , 1965) for 120 μ. The results show how inadmissible are generalization s about the shape of the prisms (Fig. 22). Each prism has a shape of its own. Its external outline can remain nearly uniform for an appreciable length and this is particularly so in the surface layers of enamel where the prisms tend to run comparatively regularly parallel to each other. But in the main body of the enamel there is a great mutual distortion of the external morphology of the prisms. In consequenc e they often change in shape a great deal and very abruptly along their length and it

Fig. 23. Electron micrograph of replica (shadowed with silicon monoxide) of a fracture surface of mature human enamel passing approximately perpendicula r to the longitudinal axes of the prisms. At the centre of the lower part there is the body of a prism. Immediately above it is the tail process of another prism, the body of which lies beyond the field. This process passes to either side of the body of the prism below, embracing it. In the upper left is part of the tail process of yet another prism which joins the one to the right of it without any definable boundary, ÷ 11,200.

HELMCKE

is difficult to identify and trace them through the sections. The simplest sort of change is one of diameter, of increases i n width and constrictions, or of change in direction. A prism may change along its length from being round in cross-sectio n to being polygonal or angular in outline. Contiguity wit h adjacent prisms may produce a flat surface several microns in length which may then become a bulge or become intensified as a concavity. Generally speaking each prism has one "tail " which extends between a pair of adjacent prisms but sometimes there may be more than one (Fig. 23). The number of prisms with which each prism is contiguous varies and depends upon the closenes s wit h which they are packed together (Fig. 24). It is unusual to find the theoretically closest possible packing manifested by regular hexagona l crosssectional forms. Serial sections show that, although a pair of prisms may be closely contiguous for a certain part of their length, with consequen t effects on their shape, they may then draw apart so that tails of other prisms, or even the bodies of prisms, may come to li e between them.

Fig. 24. Relief-diagram of prisms in human enamel based on electron microscopy of sections and replicas of fracture preparations . The prisms are represente d laid open partially through their longitudinal axes, and partially through the peripheral zones. The course of the lateral processe s of prisms which slip between other prisms is seen. Prism bodies with processe s are represente d at the front corners. (Drawing by Kurt Bogen.) (After Helmcke et ai, 1963.)

15. U L T R A S T R U C T U RE

By marking individual prisms through a series of photographs of serial sections, the general effect of a cinematograp h is obtained and the complete details of the shape and structure of selected prisms can be studied (Figs. 25 and 26). The tails or winged processe s pass in and out of the spaces between adjacent prisms rather lik e the pseudopodia of amoebae (Figs. 23 and 24). The sharp contour-lines of these tails disappea r in places because their shape within the thickness of the section changes a great deal and frequently. Wit h the light microscope, therefore, what is seen is a cumulative effect of different configurations of these tails. This summation effect has led to the assumption that interprismatic substanc e is involved which, it was believed, has a chemical composition quantitatively, if not qualitatively, different from that of the prisms themselves . Precise proof of such a difference has, however, never been produced. The traditional concept of amelogenesi s is that prisms and interprismatic substanc e are formed i n different ways, the prisms by the ameloblasts themselves and the interprismatic substanc e from some intercellular product (Sicher, 1962, p. 89; Meyer, 1951). This concept was never full y accepted even before the evidence of electron microscopy became available. Certainly now, when we take account of this new evidence, it can be seen that prisms and their winged processe s or tails, which produce the appearanc e of an interprismatic substance , are composed of the same ingredients and are the same product of the ameloblast (Helmcke, 1952 unpublished communication to the 80th Congress of the Deutsche Gesellschaf t fur Zahn-, Mund- und Kieferheilkunde, Munchen; Helmcke, 1953; Hohling, 1961, 1966; Boyde, 1965; Meckel et al, 1965a,b). Prisms and their winged processe s differ only in the orientation of the crystals. Between the prism and its winged process there is a continuous transition (Fig. 23) (Helmcke, 1953; Hohling and Erwig, 1960; Hohling, 1961, 1966; Boyde, 1965; Meckel et al, 1965a,b). The farther away the crystals are from the

OF

ENAMEL

157

crystal orientation axis of the prism, the greater the angle of inclination they have to the axis (Fig. 17). The maximum value of this angle is still unknown, but quite certainly it does not reach 90°. I n the literature the following values are quoted: 2 0 ° - 4 0° (Kennedy, Teuscher, and Fosdick, 1953; Scott, 1955); up to 90° (Meckel et al, 1965a). Furthermore, it is to be observed that with increasing distance also the initiall y radially diverging arrangemen t of the crystals changes more and more to a parallel arrangemen t (Meckel etal, 1965b; Hohling and Erwig, 1960; Hohling, 1961, 1966). This effect has contributed to the erroneous idea that the winged processe s or tails constitute a special interprismatic substanc e different from the prisms. Usually in the literature the expression "prism" is used for cases where the comet-like structure of diverging lines can be perceived; that is, where the prism has been laid open by the preparation surface within the direction of its axis (Figs. 13 and 14). The designation "interprismatic substance " is usually employed for those punctiform patterns that are formed when the plane of the ground section reveals only the periphery of the prism and the crystals are sectioned obliquely (Figs. 13 and 14). Another phenomeno n must be emphasized . In serial sections (Figs. 10-12, 19, and 21) not only one but two adjacent prisms often simultaneousl y dispatch their tails or winged processe s into the gap between two other prisms which are diverging but which, at another level, may be contiguous. A s long as these tails are not very far away from their origin, the prism to which they belong may be recognized. After demineralization of such specimens a conglomeration of fibril s produced by the effect of acid (Fig. 11) occurs at those places. However, by following carefully the ultrastructures of the winged processe s through serial sections it can be observed how by degrees ones which were originally separate run for a distance side by side i n the same direction and then fuse to form one structural unit (Fig. 12). I n such a case no aggregation of fibril s occurs after demineralization .

158

J . - G.

The above account represent s a synthesis of opinions concerning the interprismatic substanc e which are at first sight contradictory. It attempts to unify many observations into a comprehensiv e picture. Seen in this light many contradictory observations and views are explained.

HELMCKE E. T HE COURSE OF PRISMS

By the method described, of marking individual or groups of prisms on serial phase-contras t micrographs and on serial low-power electron micrographs, it is possible to trace their path

Fig. 25. Course of some prisms in rhesus monkey enamel. From a series of 144 sections ( = 72 μ in length). Three selected rows of prisms are shown in their course through the series. A few prisms are numbered or lettered from the left ends of the row at the 13 ì level so that their course can be more easily followed. By the 72 μ level some of the prisms have migrated out of the field represented . (From Helmcke, 1964.)

15.

U L T R A S T R U C T U RE

Fig. 26. Perspectiv e model of the prism movement represente d in Fig. 25. The six levels and the letters and numbers correspon d with those of that figure. (From Helmcke, 1964.)

through the enamel (Fig. 25). Some taper off and disappear entirely and other new ones appear, both processe s appearing to be independen t of each other; that is, it is not the same prism that has appeared again. Each prism appears to pursue an independen t course; that is, prisms do not appear to "behave" as groups; for instance, single prisms may wander quite far away in terms of prism dimensions. Occasionally evidence of division of prisms into two, as well as fusion of originally separate prisms, is found. Sometimes only the branches of prisms may unite and not the whole prism. A n IZ

OF

ENAMEL

159

instance has been met with in which one prism divided into two, and a contiguous one into three, branches and then one branch of each prism fused into a new prism. In this way a complex of four prisms was formed. Occasionally a small island forms within a prism and, as the serial sections are followed through, grows gradually; as the cross-sectiona l area of the prism gets larger, the island grows to a small prism within the larger one. Such a prism within a prism can either then disappea r or branch off and become independent . Prisms within prisms in pig enamel have been described by Boyde (1964). A similar configuration in human enamel prisms was mentioned by Scott (1955). The concept of the structure of a prism, shown in the relief-diagrams of Meckel et al. (1965a,b) (Figs. 18 and 19), as well as Carlstrom (1964) (Fig. 21), and Boyde (1965) has been ascertaine d on the evidence of many electron microscopic and microradiographic observations . They apply to enamel in general but they are to some extent a simplification because in certain situations, especially near the enamel-dentin e junction, prisms of even more irregular morphology and arrangemen t are to be found (Fig. 27). To make a final assess ment of form and shape of the prisms, it wil l be necessar y to take account of these special areas. F. RETZIUS LINES AN D HUNTER-SCHREGER BAND S

For many years the view has been widely held, based upon light microscopic observations , that Retzius lines and Hunter-Schrege r bands are caused by the bending and twisting of enamel prisms. Preliminary personal observations with the electron microscope tend to confirm this view. However, further work, including the timeconsuming reconstruction of wax models from serial ultrathin sections, is needed before this can be regarded as definitely established . Study of electron micrographs suggests that the character of prisms in the Retzius lines and Hunter-Schrege r bands in most cases is similar to that elsewhere (Fig. 28).

160

J . - G.

HELMCKE

Fig. 27. Electron micrograph of section through only slightly mineralized developing enamel from a rhesus monkey embryo. The shape and course of the prisms does not conform at all to the scheme and concepts described in the literature. Appearance s of this kind occur commonly in human enamel also, x 3,000. G . T H E ENAMEL-DENTIN E JUNCTION

Many electron micrographs have been published which show the zone between dentine and enamel not only in ultrathin sections of immature, partially mineralized enamel but also in replicas of mature, full y mineralized enamel. The photographs available have been taken, without exception, of preparations where the plane of section or of grinding was perpendicula r to the enamel-dentin e junction. N o ne of the electron micrographs of mature human enamel shows an organic junctionmembrane between dentine and enamel; both tooth tissues merge into each other imperceptibly. Ultrastructural evidence of the extension of odontoblast processe s into the enamel is rare (Fig. 5 ).

I t seems that directly next to the dentine there is a thin zone, about 40 μ thick, of enamel which, although composed of typical enamel crystals, shows no prism structure. Much more study of this junction region is required before it can be said to be full y understood either in terms of light microscopy or electron microscopy. H . T H E SURFACE LAYE R

So far no adequate electron microscopic investigation of the surface layer has been made. The reason for this is that in this region there are problems of surface film , of bacterial plaques and other phenomena . It is to be hoped that, nevertheless , in due course these problems can be overcome and information about the ultrastructure

15. U L T R A S T R U C T U RE

OF

ENAMEL

161

Fig. 28. Solid reconstruction of five prisms prepared from a 90-section series through rhesus monkey enamel. Three prisms have sharp bends associate d with Hunter-Schrege r bands.

of this very important surface layer wil l be forthcoming. The latest electron microscopic observations indicate that the crystals in the bulk of the enamel have the ordered arrangemen t that gives rise to the well-known prisms, but that in the zone of transition to the surface layer the crystals are distributed more parallel to one another so that prism structure is entirely lost.

IV. CONCLUSION Since the introduction of the electron microscope for the investigation of ultrastructure of h u m an enamel many accepted beliefs about the structure of enamel have been called into question. This is mainly because the structural elements of which enamel is composed are smaller than the half-wave length of visible light and can, therefore, only be made visible by electron microscopy. Withi n the relatively short period since the introduction of

this new instrument, fundamentally new ideas about enamel structure have emerged but we are far from answering all questions unequivocably and confidently. I t is known that X-rays pass through, or are absorbed by, a specimen in ways quite different from visible light, and it is, therefore, understand able that the images formed differ accordingly. Similar consideration s are applicable to the electron microscope as well as to the polarization microscope. It must be the goal of future research to explain all effects by a uniform and close order of perception even if the observations have been gained by different methods of investigation. The fundamenta l difficulties that over and over again have led to differences of views are firstly that the enamel consists for the most part of crystals that are optically anisotropic, and, therefore, effect the formation of the image, and secondly that most of the preparation methods are unsuitable for revealing both the organic and inorganic structural elements of the mature enamel reliably

162

J . - G.

and at the same time. These difficulties probably provide the reason why final agreemen t between all workers concerning the interpretation of the structure of enamel still seems far off. This is especially true for the question whether there is a differentiation in h u m an enamel into prisms, prism sheaths and "interprismatic substance". Those authors who have investigated ultrathin sections and replicas of mature, full y mineralized enamel by electron microscopy have subscribed to the view that we do find appearance s consistent wit h those of light microscopy. Other workers, however, who have investigated immature, partially mineralized enamel, have observed structures which, they believe, could be brought into accord wit h the concept of prisms, prism sheaths and interprismatic substance . Therefore diverse concepts wil l be found in the various chapters of this book. We are far from having complete knowledge of the ultrastructure and the manifold patterns of ultrastructure of enamel within a single species . But much less do we know about the many possible differences that may exist in the structure of enamel between various groups of vertebrate s (Boyde, 1964, 1965). Until comparative microanatomic research has m a de good this deficiency of knowledge it is not worthwhile discussing which ideas are true and which are false.

References Awazawa, Y. (1963). "The Electron Microscope Atlas of Dental Diseases" , 1st ed. Dept. Pathol., Nihon Univ. Sch. Dent., Tokyo. Berlin, V. (1962). Elektronenmikroskopisch e Untersuchunge n mechanische r und chemische r Verletzungen des Schmelzes . Proc. 6th ORCA Congr. dent. Caries, Pavia, 1959 pp. 207214. Edition Clin. Dent., Univ. Pavia, Pavia. Boyde, A. (1964). The structure and developmen t of mammalian enamel. Ph.D. Thesis, 298 pp. University of London. Boyde, A. (1965). The structure of developing mammalian dental enamel. In "Tooth Enamel, Its Composition, Properties and Fundamenta l Structure" (M. V. Stack and R. W. Fearnhead , eds.), pp. 163-167. John Wright, Bristol.

HELMCKE Boyde, A. and Stewart, A. D. G. (1962). A study of the etching of dental tissues with argon ion beams. / . Ultrastruct. Res. 7, 159-172. Carlstrôm, D. (1964). Polarization microscopy of dental enamel with reference to incipient carious lesions. Advanc. oral Biol. 1, 255-296. Darling, A. I. (1961a). The structure of the enamel revealed in dental disease . Proc. 7th ORCA Congr. dent. Caries, Hamburg, 1960 Vol. 4, pp. 80-85. Pergamon Press, Oxford. Darling, A. I. (1961b). The selective attack of caries on the dental enamel. Ann. R. Coll. Surg. Engl. 29, 354-369. Darling, A. I., Mortimer, Ê. V., Poole, D. F. G. and Ollis, W. D. (1961). Molecular sieve behaviour of normal and carious human dental enamel. Arch, oral Biol. 5, 251-273. Fearnhead , R. W. and Elliott, J. C. (1962). Observations on the relationship between the inorganic and organic phases in dental enamel. Proc. 5th int. Congr. Electron Micr., Philadelphia, 1962 Vol. 2, art. QQ-7. Academic Press, New York. Frank, R. M., Sognnaes , R. F. and Kern, R. (1960). Calcification of dental tissues with special reference to enamel ultrastructure. In "Calcification in Biological Systems", Publ. N o. 64, pp. 163-202. Amer. Ass. Advanc. Sci., Washington, D.C. Fremlin, J. H., Mathieson, J. and Hardwick, J. L. (1961). The preparation of thin sections of dental enamel. Arch, oral Biol. 5, 55-60. Gerould, C. H. (1944). Ultramicrostructure s of the human tooth as revealed by the electron microscope. / . dent. Res. 23, 239-245. Glas, J.-E. and Nylen, M. U. (1965). A correlated electron microscopic and microradiographic study of human enamel. Arch, oral Biol. 10, 893-908. Gustafson, A.-G. (1959). A morphologic investigation of certain variations in the structure and mineralization of human dental enamel. Odont. Tidskr. 67, 361-472. Hardwick, J. L., Martin, C. J. and Davies, T. G. H. (1965). The microstructure of mature dental enamel as observed under the optical microscope. In "Tooth Enamel, Its Composition and Fundamenta l Structure" (M. V. Stack and R. W. Fearnhead , eds.), pp. 168-171. John Wright, Bristol. Helmcke, J.-G. (1953). "Atlas des menschliche n Zahnes im elektronenmikroskopische n Bild" , Part I: Histologie des normalen Zahnes. Transmare Photo, Berlin. Helmcke, J.-G. (1964). Kombination von elektronenmikroskopische n und neuen lichtmikroskopische n Untersuchungsmethode n fur Strukturen des Zahnschmelzes . Proc. 10th ORCA Congr. dent. Caries, Geneva, 1963 Vol. 2, pp. 127-139. Pergamon Press, Oxford. Helmcke, J.-G., Schulz, L. and Scott, D. B. (1961). Fine structures of cross-striation s in prisms of human enamel. /. dent. Res. 40, 668 (Abstract). Helmcke, J.-G., Schulz, L. and Scott, D. B. (1963).

15. U L T R A S T R U C T U RE Querstreifung der menschliche n Schmelzprismen . Dtsch. zahnàrztl. Ζ. 18, 569-637. Hôhling, H. J. (1960). Elektronenmikroskopisch e Untersuchunge n an Ultradunnschnitte n vom kompakten, nicht vorbehandelte n Zahnschmelz . Z. Naturf. 15b, 59-61. Hôhling, H. J. (1961). Elektronenmikroskopisch e Untersuchunge n an gesunde m und kariosem Zahnschmel z unter besondere r Berucksichtigung der UltramikrotomschnittTechnik an nicht entmineralisierte r Substanz . Dtsch. zahnàrztl. Ζ. 16, 694-705. Hôhling, H. J. (1963). Beispiele zur Herstellung und Untersuchung von Ultradunnschnitte n biologischer Hartsubstanzen. Leitz-Mitt. Wiss. Technik 2, 164-167. Hôhling, H. J. (1966). Die Bauelement e von Zahnschmel z und Dentin aus morphologischer , chemische r und struktureller Sicht. Thesis for habilitation, TônningEiderstedt. Med. F a c, University of Munster. Carl Hanser, Munchen. Hôhling, H. J. and Erwig, R. (1960). Licht- und elektronenmikroskopische Untersuchunge n am kompakten, nicht vorbehandelte n Zahnschmelz . Dtsch. zahnàrztl. Z. 15, 1193-1201. Johansen , E. (1965). Comparison of the ultrastructure and chemical composition of sound and carious enamel from human permanen t teeth. In "Tooth Enamel, Its Composition, Properties and Fundamenta l Structure" (M. V. Stack and R. W. Fearnhead , eds.), pp. 177-181. John Wright, Bristol. Kay, D. (1961). "Techniques for Electron Microscopy". Blackwell, Oxford. Kennedy, J. J., Teuscher, G. W. and Fosdick, L. S. (1953). The ultra-microscopic structure of enamel and dentin. /. Amer. dent. Ass. 46, 423-431. Lenz, H. (1961a). Elektronenmikroskopisch e Untersuchunge n der Mineralisation des Zahnschmelze s und der beginnende n Schmelzkaries . Proc. 7th ORCA Congr. dent. Caries, Hamburg, 1960 Vol. 4, pp. 34-39. Pergamon Press, Oxford. Lenz, H. (1961b). Recherche s au microscope électronique sur la structure de l'émail et spécialemen t sur les buissons et la cuticule. Bull. Gr. int. Rech. sci. Stomat. 4, 189-211. Matsumiya, S. and Takuma, S. (1954). "Atlas of Electron Micrographs of the Human Dental Tissues", pp. 1-91. Dental College Press, Tokyo.

OF

ENAMEL

163

Meckel, A . H., Griebstein, W. J. and Neal, R . J. (1965a). Ultrastructure of fully calcified human dental enamel. In "Tooth Enamel, Its Composition, Properties and Fundamental Structure" (M. V. Stack and R. W. Fearnhea d eds.), pp. 160-162. John Wright, Bristol. Meckel, A. H., Griebstein, W. J. and Neal, R. J. (1965b). Structure of mature human dental enamel as observed by electron microscopy. Arch, oral Biol. 10, 775-784. Meyer, W. (1951). "Lehrbuch der normalen Histologie und Entwicklungsgeschicht e der Z hn e des Menschen", 2nd ed. Carl Hanser, Munchen. Millier , H. (1962). "Prâparation von technisch-physikali schen Objekten fur die elektronenmikroskopisch e Untersuchung". Akad. Verlagsges. , Leipzig. Nylen, M. U. and Omnell, K.-A . (1962). The relationship between the apatite crystals and the organic matrix of rat enamel. Proc. 5th int. Congr. Electron Micr., Philadelphia, 1962 Vol. 2, art. QQ-4. Academic Press, New York. Poole, D. F. G. and Brooks, A. W. (1961). The arrangemen t of crystallites in enamel prisms. Arch, oral Biol. 5, 14-26. Reimer, L. (1959). "Elektronenmikroskopisch e Untersuchungs - and Prâparationsmethoden" . Springer, Berlin. Schmidt, W. J. and Keil, A. (1958). "Di e gesunde n und die erkrankten Zahngeweb e des Menschen und der Wirbeltiere im Polarisationsmikroskop" . Carl Hanser, Munchen. Scott, D. B. (1955). The electron microscopy of enamel and dentin. Ann. N.Y. Acad. Sci. 60, 575-585. Scott, D. B. (1960). The crystalline component of dental enamel. Proc. 4th int. Congr. Electron Micr., Berlin, 1958 Vol. 2, pp. 348-351. Springer, Berlin. Sicher, H., ed. (1962). "Orban's Oral Histology and Embryology", 5th ed. Mosby, St. Louis, Missouri. Torell, P., Zelander, T. and Gerdin, P.-O. (1964). Surface crystals in human dental enamel from Alingsâs. Odont. Revy, Lund 15, 366-369. Wolf, J. (1940). Plastische Histologie der Zahngewebe . I, II , III . Dtsch. Zahn-, Mund- u. Kieferheilk. 7, 265-284, 507-538 and 678-690. Wood, A. (1949). The structure of the wall of the test in the foraminifera; Its value in classification. Quart. J. geol. Soc, Lond. 101, 229-255. Wustrow, F. (1951). Das organische Grundgeriist im menschliche n Zahnschmelz . Z. Anat. Entw.Gesch. 116, 115-133.

This page intentionally left blank

SECTION 4

Physical and Chemical Organization of the Tooth

This page intentionally left blank

CHAPTER

16

CRYSTALLINE ORGANIZATION OF DENTAL MINERAL O T T O R.

TRAUTZ

I. Introduction A . Mineral Content of the Tissues B. Identity of the Mineral

165 165 166

II . Crystallographic Investigations and Methods A. Crystallograph y of Apatite B. Physical Properties of Apatite C. X-ray Diffraction and the Apatite Structure D . Substitutions in the Apatite Structure E. Crystallinity

166 166 168 169 184 185

III . The Mineral Phase in Dental Hard Tissues. Results of Investigations . . . A . Degree of Mineralization B. Composition of the Mineral Phase and Structure of Its Apatite . . . . C. Crystallinity of the Mineral Phase in Hard Tissues D. Texture of the Mineralized Tissues and the Effect of the Organic Matrix. E. Influence of the Tissue Fluid upon the Type of Mineral F. Separatio n of the Mineral Phase from the Organic Matrix

188 188 189 193 194 196 197

IV . Conclusion

197

Reference s

197

I. INTRODUCTION The mineralized or hard tissues of the teeth consist of an organic matrix and an inorganic material. The organic matrix contains fibres and a ground substance . The inorganic material, which includes the crystallized and the noncrystallized inorganic salts, is often called "the mineral". I n this chapter we wil l be concerned with the crystalline structure and texture of the mineral and with its chemical composition insofar as this influences structure and texture. It is chiefly the composition of the flui d dispersed through the organic matrix which determines the composition

of the mineral precipitated in the matrix. The structure and texture of the precipitated mineral in turn greatly influence the physical and chemical behaviour of the hard tissue. A.

MINERAL

CONTENT OF THE TISSUES

The various types of hard tissues, namely enamel, dentine, cementum and bone, differ in their mineral content. Considerable differences are noted even among specimens of the same tissue type. Because the X-ray absorption in a sample of hard tissue is chiefly due to the mineral, 165

166

O T TO

variations in the degree of mineralization can be demonstrate d with X-ray radiography. Microradiographs of thin sections show areas of lower and higher mineralization on a microscopic scale: for instance, the younger and older osteones in bone or the consecutive incremental layers in enamel and dentine. Measuring the X-ray absorption in a tissue sample is a nondestructive method for determining its mineral content. The mineral fraction can also be isolated by extracting the solubilized organic matrix or determined by chemical analysis for calcium and phosphate . B. IDENTITY OF THE MINERA L

F r om early analyses it was known that the mineral matter in the tooth enamel is calcium phosphate . The hardness of the enamel and its density and optical properties were found to be almost the same as those of the well-known mineral apatite. Thus, it seemed highly probable that the enamel mineral was indeed apatite. Definite proof of this identity was furnished by Gross (1926) when he examined a slice of enamel by X-ray diffraction. He also showed that a preferred orientation of the apatite existed in the enamel. X-ray diffraction also verified the apatite nature of the mineral components of bone, cementum and dentine (Gross, 1926; DeJong, 1926). A s the principles of the crystallographic methods employed in the investigations of the crystalline organization of the dental mineral are not common knowledge, an attempt wil l be made in section I I , below, to explain the general principles, using, where appropriate, apatite as test sample. Section I I I is reserved for a detailed description of the results from current research on the mineral phases in the dental hard tissues. II. CRYSTALLOGRAPHIC INVESTIGATIONS AND METHODS A . CRYSTALLOGRAPHY OF APATIT E

The name apatite (from Greek απατάειν = to deceive) was given by the chemist Alfred Werner

R.

TRAUTZ

to a calcium phosphate mineral, C a1 0( P O4) 6F 2, which is found in various rocks as colourless, green, yellow, pink or purple gem-clear crystals, because their appearanc e is similar to other harder and more valuable gem stones, with which they were confused. Many natural apatite crystals grow in the form of hexagona l prisms. Figure 1 shows a combination of the prism faces m with the pyramid faces χ and the basal pinacoids c.

i l i ! m

Fig. 1. A crystal habit of natural apatite showing the faces of the hexagona l prism (m = 100), pyramid (x = 101), and base (c = 001).

The crystal structure of the apatite was established in 1930 by Nâray-Szabo . Simultaneously , Mehmel (1930) arrived at almost the same structure. A refinement of the atomic positions has been reported by Beevers and Maclntyre (1946) and by Posner, Perloff and Diori o (1958). Figure 2 is a stereoscopi c view of a model of the apatite structure, and Fig. 3 shows the projection of the structure upon the basal plane in a direction parallel to the c-axis. The name apatite is now used for a whole group of similar crystalline structures rather than for a compound of definite chemical composition. However, in this chapter, the calcium hydroxyapatite, C a1 0( P O4) 6( O H ) 2, is selected as the basic structure for the explanation of the chemistry and crystallography of apatites. Crystallographically, apatite belongs to the hexagonal system, which is characterize d by a sixfold oaxis perpendicula r to three equivalent á-axes (αλ, a2, a3) at angles of 120° to each other. The crystal habit (i.e. combination of faces) of the naturally occurring apatite crystals (Fig. 1)

16. C R Y S T A L L I N E

ORGANIZATION

OF DENTAL

MINERAL

167

l axis down upon the basal plane. Fig. 2. Stereopicture of a model of apatite structure viewed parallel to the hexagona The hexagona l unit cell is outlined by the taped rhombus. The unit cell contains 10 Ca, 6 P 04 (tetrahedra) , 2 OH on the c-axes at the corners of the rhombus). The depth of the unit cell extends from the first to the third OH. The taped hexagon indicates the hexagona l symmetry around the oaxis. The P 04 tetrahedra have one vertical and one horizontal edge and two vertical and two tilted faces. Two symmetry planes perpendicula r to the c-axes pass through the horizontal edges of the P 04 tetrahedra , and through the OH and the Ca ions not lying on the threefold rotation axes. Wit h a littl e practice one may succeed , without the use of a stereoscope , in seeing the stereoscopi c picture in space. Place the well-illuminated pictures symmetrically before you. Alig n your eyes parallel by viewing "through the pictures" an imaginary distant spot. While the eyes accommodat e to the closer distance of the pictures (25-30 cm) the two pictures fuse into a single three-dimensiona l view. A piece of cardboard placed vertically between the two pictures may be of help at the beginning. (This model was built by Dr. Edward Klein using data of Beevers and Mac-Intyre, 1946.) together with their physical properties (e.g. uniaxial birefringence, lack of piezoelectric behaviour) and the symmetry of the etch figures demonstrat e that the c-axis is a nonpolar sixfold axis. Accordingly, of the 32 symmetry classes, the class 6/m is the one which represent s the symmetry of the apatite crystal. This symbol indicates the presence of a symmetry plane (m for mirror) perpendicula r to the sixfold c-axis. Further details of the symmetry of the apatite structure are obtained wit h the help of X-ray diffraction.

Fig. 3. Apatite structure projected parallel to the c-axis upon the basal plane. The symbols indicate the height (in fractions of c) of the various ions above the base. F indicates fluoride or hydroxyl ions at 1/4 and 3/4c. (After Posner, 1955.)

I n the crystal we can visualize a smallest building unit of parallelepipe d shape, the "unit cell", which contains a complete representatio n of the crystal and which by indefinite repetition in the direction of the three axes can generate the whole crystal. The edges of the unit cell are identical

168

OTTO R. TRAUTZ

wit h the lengths and directions of the (horizontal) ax- and a2-axes and of the (vertical) c-axis. The lengths of the a- and c-axes are derived from the positions of the X-ray "reflections" on the fil m in the diffraction camera. Of the 230 possible space groups, the one which represent s the combination of symmetry elements exhibited by the apatite structure is selected by considering which reflections are systematically absent. This space group has the symbol P 63/ m. Ñ indicates a primitive cell, i.e. there is only one assemblag e of atoms consistent with the symmetry of the cell. 6 3 indicates that the c-axis is a sixfold screw axis. This means that, after rotating the structure about c through 277/6 = 60° and simultaneousl y shifting it along the c-axis through c X 3/6, i.e. c/2, the appearanc e of the structure wil l be identical to what it was originally. The positions of the symmetry elements in the unit cell projected upon its base are shown in Fig. 4. Between the sixfold screw axes at the vertical

Fig. 4. Positions of the symmetry elements in the apatite unit cell. They are the sixfold and twofold screw axes, with inversion centres at c = 0 and 1/2; and the threefold rotation axes. The horizontal symmetry planes at c = 1/4 and 3/4 are indicated at the edge.

edges of the cell are the vertical twofold screw axes. The vertical threefold rotation axes are in the centre of the two half-cells. The symbol on the side of the figure refers to the horizontal mirror planes at heights 1/4 and 3/4 along the c-axis. In order to determine the positions of the atoms in the structure, it is necessar y to take into

account the intensities of the X-ray reflections as well as the crystal symmetry. B. PHYSICAL PROPERTIES OF APATIT E

The hardness , cleavage and optical properties of apatite are of particular importance because these physical properties provide convenient tests for the investigations of the mineralization and demineralization of the hard tissues. 1. Hardness The hardness of well-mineralized enamel tissue is approximately that of its apatite. Enamel is the hardest tissue in the vertebrate body and therefore is best suited as a surface cover for chewing tools (teeth), aggressive weapons (e.g. boar's tusks) and armour [e.g. the "mesoderma l enamel" of the placoid scales of sharks (see Glas, 1962)]. On the Molls' scratch hardness scale, apatite has hardness 5. Another hardness test, the microindentation test, provides a more sensitive and meaningful hardness number for a material (Knoop, Peters and Emerson, 1939). A flat diamond pyramid is pressed into the polished surface of the test material. The ratio of load to indentation-are a (in kg/mm2) is defined as the K n o op hardness number ( K H N ) . For apatite, this ratio is about 430 k g / m m2 (Newbrun and Pigman, 1960). The hardness of an apatite crystal depends on its chemical composition. For example, a lead chlorophosphat e apatite, P b1 0( P O4) 6C l 2, is much softer ( K H N about 150) than the common calcium fluorapatite, C a1 0( P O4) 6F 2 ( K H N about 430). In tooth enamel, the apatite is present in the form of submicroscopi c crystallites elongated parallel to their c-axes. Since in human enamel they are preferentially aligned parallel to the direction of the enamel rods, hardness values are somewhat different on sections cut parallel to the enamel rods from those of sections cut perpendicula r to the rods. By keeping errors due to these differences minimized by proper experimenta l design, hardness tests have become a useful tool for laboratory investigations of the effect of various

16. C R Y S T A L L I N E O R G A N I Z A T I O N O F D E N T A L

agents which cause demineralization and remineralization (Newbrun, Triberlake and Pigman, 1959). 2. Cleavage Large fluorapatite crystals exhibit a cleavage parallel to the basal planes. Cleavage is defined as the tendency of a crystal to split along certain crystallographic planes across which the cohesion in the crystal is minimal. When fluorapatite is ground in a mortar to a fine powder, minute platelets perpendicula r to c are formed. The hydroxyapatite and carbonate apatites, on the other hand, exhibit prismatic cleavage and form needles parallel to c. The cleavage of apatite is not very pronounced . However, in certain measure ments the results may be influenced by the cleavage behaviour (cf. section II , C, 4 f, p. 179). 3. Optical

MINERAL

169

Precipitated apatites are usually submicroscopi c and only a mean refractive index, n, can be determined. This mean value is related to the principal refractive indices of the individual crystals by η = (2ω + e)/3 (Larsen and Berman, 1934, p. 31). The ratio (n — l)/d is a constant (specific refractivity ) and can be used to calculate the change in the refractive index «, when the density d is changed (e.g. due to change in temperature) . Changes in refractivity due to substitution in the structure can also be calculated by considering the atomic refractivities. Precipitated calcium phosphate s often contain water (either adsorbed or as water of crystallization or as H + and O H - or O 2 - ions). Such materials are expected to have considerably lower refractive indices than the water-free materials, the lowering of the index running parallel with the water content.

Properties

Apatite, in accordanc e with its hexagona l structure, is optically uniaxial and characterize d by two refractive indices: ω for the ordinary ray vibrating perpendicula r to c, and e for the extraordinary ray vibrating parallel to c. The birefringence, i.e. the difference between these refractive indices (e — ω), is weak and negative (—0.004). Crystals with tetrahedra l ion groups lik e P 0 4 , S 0 4 , S i 04 usually have a low birefringence whereas crystals with flat groups, lik e the parallel-aligned C 0 3 groups in calcite, exhibit a large negative birefringence (e — ω = —0.172 for calcite). A change of the orientation of the flat groups in a material from a parallel to a r a n d om one causes this contribution to the birefringence of the material to decreas e to zero. I n the natural carbonate hydroxyapatites (dahllites) and carbonate fluorapatites (francolites) a more negative birefringence is observed than in the correspondin g carbonate free apatites. This markedly increased negative birefringence can be explained by assuming that the carbonate groups are not randomly orientated but have some degree of preferred orientation, their planes being more or less tilted parallel to the basal plane (Trautz, 1960).

C.

X - R A Y DIFFRACTION AN D THE APATIT E S T R U C-

TURE

The chief tool for the investigation of crystal structure is X - r ay diffraction, made possible by the similarity of the X - r ay wavelengths and the atomic distances in the crystals. A simple examination of enamel with X - r ay diffraction gives the following information: that the mineral is an apatite, that in enamel the mineral is fairly well crystallized and that the crystallites have a preferential orientation. X - r ay diffraction of other hard tissues, such as bone and dentine, shows that in these materials the apatite is less crystallized and less orientated than in enamel. The use of X - r ay diffraction is not limited to the above observations . I n many studies on the precipitation of calcium phosphates , X - r ay diffraction is the most important tool for the characterizatio n of the solid phase. I n order to provide a background for understandin g the research reports in this field, a brief description is given of the X - r ay diffraction principles and of the techniques that have been employed. [Detailed information can be obtained from Cullity (1956), Bunn (1949), Buerger (1956), Henry, Lipson and Wooster (1960), Klu g and

170

O T TO

Alexander (1954) and "International Tables for X-ray Crystallography" (1952-1962).] 1. Production of X-rays X-rays are electromagneti c waves with wavelengths about 1/1000 of those in the range of visible light. For crystal structure analysis, the commonly used wavelength range extends from 0.7 to 2.3 Â (1 Angstrom unit = 1 0- 8 cm). When high speed electrons are emitted from a hot filament cathode, they are accelerate d by a high direct current voltage (10-70 kv). These electrons are focussed upon a small area ("focal spot", sometimes 1 X 10 mm) of a water-cooled metal target and cause this target to emit X-rays with wavelengths characteristic of the target metal. In addition to these rays, a noncharacteristi c continuous spectrum is emitted. The most prominent and commonly used characteristic radiation from a target is the Ir-

R.

TRAUTZ

radiation. This radiation consists of three principal lines, an and a 2 doublet and a β1 line. Since for many applications monochromatic radiation is preferred, the β line is eliminated by an appropriate filter. The filter has its absorption edge between the wavelengths of the K a doublet and Κβ line. The β line is selectively absorbed in the filter while the á doublet is relatively unaffected. A great part of the continuous spectrum is also absorbed by this filter (Fig. 5). The higher the atomic number of the target material, the shorter the characteristic wavelength (Table 1). The minim um voltage necessar y to excite the Ê radiation is given by the equation:

ν =

ψ

(D

(where is the Ê absorption edge of the target metal in  , and V is in kilovolts). The optimum operating voltage of the X-ray tube, in order to

Fig. 5. Monochromatizatio n of X-radiation by use of filter. Radiation from Cu target at 40 kv. Intensity (counts per second) vs. wavelength (A) before, after, passing through 0.021 mm thick Ni filter. Mass absorption coefficient μ/ρ of Ni, showing absorption discontinuity at 1.488 A. The intensity of the C u K a line is reduced to 40%, of the CuKjs line to 0.6%, of their original intensities. The original intensity ratio of Ê ( áé + «2) : Êâ is 7.5 : 1. Filtration improves it to 530 : 1.

16.

CRYSTALLINE

ORGANIZATION

OF

DENTAL

171

MINERAL

Table 1 TARGETS, WAVELENGTHS AND FILTERS FOR K-RADIATIO N

Target

Wavelengths (A)

Atomic No.

Ê

Molybdenum

42

0.709

1.713

0.632

Copper0

29

1.540

1.544

1.392

20.0 8.98

Zirconium

35

Nickel

1.488 1.896 2.269

Iron

26

1.936

1.940

1.756

7.11

30

Chromium

24

2.290

2.294

2.085

5.99

25

Vanadium

Relative intensities (Cu target) á2 : á÷ = 0.50; β 1 :

0.689

55

Manganes e

á

Absorpti edge (A )

Filter

Excitation Optimum voltage voltage (kv) (kv)

= 0.20.

get sufficient intensity of the characteristic radiation and yet not too much intensity of the continuous spectrum radiation, is about 3 to 4 times this minimum excitation voltage. Equation (1) also relates the operating voltage to the minimum wavelength of the continuous spectrum. A n increase in the tube voltage above the optimum value unfavourably affects monochromatizatio n because the intensity of the continuous radiation is more strongly increased than that of the Ê radiation (Fig. 6). The target material most frequently used in diffraction work is copper. Its high heat conductivit y permits the tube to be operated with a comparatively high load (operating voltage times tube current).

response of the flow proportional counter is linear up to rates as high as 104 cps and the response of the scintillation counter is linear up to 105 cps. I n this respect, for the measuremen t of high intensities the scintillation counter is to be preferred. However, the electronic circuitry of the scintillation counter is more complex than that for the Geiger counter. The flow proportional and the scin-

50kv

2. Detection of X-rays X-rays are detected either by photographic or counter devices. The emulsions of the X-ray films are loaded with more silver salts than ordinary photographic emulsions in order to increase their absorption of the X-rays, and they are developed with higher contrast developer. The common counter detectors are the Geiger counter, the flow proportional counter and the scintillation counter. The Geiger counter can be used for counting rates up to 103 counts per second (cps). The

0.2 0.3 0.4

0.5

0.6

0.7

0.8

0.9

1.0

Wavelength (A)

Fig. 6. Intensity distribution in continuous spectrum as function of X-ray tube voltage (Ulrey).

O T TO

172

tillatio n counters have the additional advantage that, when used with a pulse height analyzer, a better monochromatizatio n of the detected X-rays is achieved. 3. Diffraction Effect in a Crystal X-rays falling upon matter are scattered by the electrons of the atoms, each atom serving as a scattering centre. If the atoms are arranged in a geometrical order, as they are in a crystal, the X-rays which are coherently scattered by the atoms produce a diffraction pattern by additive and subtractive interference. This pattern is characteristic of the arrangemen t and nature of the atoms in the crystal.

100

210

321

001

Fig. 7. Unit cell of apatite, showing the orientation of the axes and of four lattice planes, identified by their Mille r indices.

The shape of the hydroxyapatite unit cells is shown in Fig. 7. The lengths of the axes of the unit cell are αλ = a2 = 9.42 Â and c = 6.88 Â. The axial ratio (c/a) is 0.732. The apatite unit cell contains 10 Ca, 6 P 04 and 2 OH ions. Crystallographic planes in a crystal are named using "Mille r indices." These indices, A, k, /, are the reciprocals of the fractional intercepts which the plane makes with the respective crystallographic axes, a, b9 c. For example, a plane with the Mille r

R.

TRAUTZ

indices hkl intersects the axes at a/h, b/k, c/l (Fig. 7). I n a crystal the atoms can be visualized arranged on or near families of equidistant parallel planes. When the entering X-ray beam makes certain specific glancing angles with such a set of planes, scattering interference produces sharp maxima of the diffracted intensity. These maxima are usually called reflections. The Bragg law describes this reflection condition with the equation: n\ = 2 d sin θ

(2)

I t relates the glancing angles θ of a reflection (diffraction peak) to the wavelength ë of the X-ray and the spacing d between the "reflecting planes" (family of equidistant parallel planes in the crystal). The integer η denotes the order of reflection. The smaller the spacing, the larger the diffraction angle 0; however, θ cannot exceed 90°. The Bragg equation, rearranged , shows that from one family of planes (d constant) a series of reflections is obtained whose sin θ are whole multiples (n) of X/2d (Fig. 8). Symmetry conditions determine which orders of reflections are extinguished by destructive interference; in the apatite, the odd orders of the 00/ reflections are systematically missing due to the symmetry planes perpendicula r to the c-axis. A s mentioned in section II , A , p. 166, the systematic absence of certain orders of reflections is an important criterion for locating the symmetry elements in the crystal lattice and thus determining its space group. The d-spacings of any crystallographic plane in the crystal can be calculated from the unit cell dimensions of the crystal and the Mille r indices of the plane. In the hexagona l system, for instance, 1

4 / h2 + hk + k2

(3)

Combination of Eqs (2) and (3) gives the diffraction angles θ of all possible reflections of the apatite: sin^

ë2 r4 / h2 + hk + k2

(4)

16. C R Y S T A L L I N E

ORGANIZATION

4. Diffraction Methods and Apparatus Various methods of obtaining diffraction patterns of crystalline materials are available. In some of them, single crystals are used, in others polycrystalline materials and crystal powders are examined. The single crystal methods are discusse d below. a. The Laue method. This method can demonstrate the Laue symmetry of a crystal and its orientation. Wit h this method (Fig. 9A) a stationary thin crystal is irradiated, preferably parallel to a crystallographic axis, by a narrow X-ray beam of the continuous spectrum. A diffraction pattern obtained on a flat fil m some distance behind the crystal (transmission Laue) represent s the symmetry parallel to the respective crystal axis. Since the crystal is stationary, the various reflections come from planes of different spacings and are produced by X-rays of different wavelengths . Figure 9Â is a Laue pattern of apatite, with the incident beam parallel to the c-axis. It demonstrate s the hexagona l symmetry. There is no symmetry plane parallel to the c-axis. Each elliptical array of reflections in the photograph represent s a " z o n e" of planes. The planes in a crystal which belong to a " z o n e" intersect each other in mutually parallel lines, 13

OF

DENTAL

MINERAL

173

e.g., the prism faces in Fig. 1. The faces 100 (m), 101 (x), 001 (c), TOI, TOO. TOT, 00T, 10T form another zone. b. The rotating crystal method. This method employs a tiny crystal, bathed in a monochromatic X-ray beam (Fig. 10A). A n axis of the crystal (e.g. c-axis) is adjusted to be coincident with the rotation axis of the crystal holder and perpendicula r to the X-ray beam. During the exposure the crystal is rotated about its axis and the diffracted X-rays are recorded on a fil m cylinder whose axis also coincides with the rotation axis. Wit h the above alignment the diffraction spots of the apatite crystal li e on horizontal "layer lines". Indexing the reflections (assigning to them the hkPs of their reflecting planes) is comparatively easy. For example in Fig. 10B, the middle line ( = zero layer line, or equatorial line) contains all reflections of the prismatic zone hkO, with / = 0. The next line above and below contains the reflections (hkl) and (hkl) with 1 = 1 and T, respectively; the next layer line shows the (hkl) reflections; and so forth. The spots can also be lined up from planes with the same hk, but different f s, in more or less curved vertical " r ow lines". If necessary , superimpose d reflections can be separate d by letting

O T TO

Film Β

R.

TRAUTZ

Film A

Fig. 9. (A) Laue camera for single crystal patterns (Barrett). The transmission pattern is recorded on fil m A. The back reflection pattern is recorded on fil m Β. (B) Laue transmission pattern of apatite showing the hexagona l symmetry of the c-axis and the absence of a symmetry plane parallel to it. (C) Laue back reflection pattern of apatite; same crystal as for Fig. 9B.

16. C R Y S T A L L I N E

ORGANIZATION

OF

DENTAL

MINERAL

175

Fig. 9C. For legend, see opposite page.

the crystal oscillate through only a few degrees (oscillating crystal method). Then a series of six consecutive films, each one a recording of 10° oscillation, wil l contain all the apatite reflections of the rotation pattern without superimposition so that their intensities can be individually assessed . The reflection intensities are essentia l data for determining the atomic positions within the unit cell. c. The Weissenberg method. This method is more desirable for the analysis of complex crystals because normally superimpose d spots are separate d in a single recording. However, only one layer lin e at a time is recorded on the film . Lik e the previous method, this one employs a monochromatic beam. The cylinder axis and the crystal axis are coincident with the crystal rotation axis, but

the fil m cylinder is shifted along the cylinder axis by a gear. In this way the superimposition s of the reflections are avoided and the intensities can be individually assessed . The arrangemen t of the spots on the fil m makes their indexing easy (Fig. 11). d. The automatic single crystal diffractometer. I n recent years the electronic computer has been put into service for crystal structure analysis. In the experimenta l part of the task, after the unit cell dimensions of a crystal have been determined and the crystal has been mounted on the goniometer head of a modified Weissenber g goniometer, the computer is instructed to operate the goniometer. It rotates the crystal to a reflecting position and moves the counter tube to the correspondin g receiving position, scanning over the reflection

O T TO

176

R.

TRAUTZ

 Fig. 10.

(A) Rotating crystal camera (Barrett). (B) Rotation pattern of an apatite crystal. The c-axis is the rotation axis.

16. C R Y S T A L L I N E

ORGANIZATION

OF

DENTAL

MINERAL

177

•lie

Fig. 11. Fig. 10B.

Weissenber g pattern of an apatite crystal.

layer; c-axis photograph. Same crystal was used as for

and recording its intensity profile. It then automatically proceeds to scan over and record the intensity of the next and all other reflections of the same layer line, and then also of the other layer lines. Thus with one crystal a few hundred to a few thousand reflection profiles and their integrated intensities are recorded. In the computational part, the computer is programmed to calculate from these intensities by Fourier analysis contour maps of the electron density on various sections through the unit cell. On a cathode-ra y screen these maps can be observed. The position of the various atoms are indicated by spots with increased electron density (crowding of the contour

lines) correspondin g to the number of electrons in the respective atoms. The hydrogen atoms are not shown by this technique, as their scattering power for X-rays is exceedingly low. Neutron diffraction on the other hand may be of help, as the scattering of a neutron beam by hydrogen atoms is appreciable . For the determination of crystal structure, wit h the exception of the very simplest structures, it is necessar y to work with a single crystal in order to record the individual intensities of all reflections. F or many other crystallographic studies, methods using polycrystalline materials give sufficient information.

178

O T TO

R.

TRAUTZ

A

Â

Fig. 12. (A) Debye-Scherre r camera (Norelco). The X-rays enter from the left through a collimator tube. The powdered sample in a narrow glass capillary is mounted in the cylinder axis of the camera. The diffracted rays produce lines concentric with the exit hole on a fil m strip placed along the cylinder. (B) Powder pattern of well crystallized F apatite. Cu K a radiation. The pattern, i.e. the positions and relative intensities of the lines, is singularly characteristic for apatite. It is an easy means of identification.

16. C R Y S T A L L I N E

ORGANIZATION

e. The Debye-Scherrer method. The DebyeScherrer method allows quick identification of powder samples. It uses a very small amount of finely powdered material (0.02 m m3 or even less) in a Lindeman glass (beryllium-boron glass) capillary of 0.2 mm inside diameter. This capillary is placed in the centre of a cylindrical camera lined with a film strip (Fig. 12A). The collimated monochromatic X-ray beam passes through holes in the cylinder at right angles to the cylinder axis. The X-rays diffracted by the specimen form cones with apex angles of 2 θ with the primary beam. The camera diameter is usually made to be 57.3 or 114.6 mm so that the ring diameter of a reflection measure d in millimeters is numerically equal to 2 Θ or 4 θ, respectively. A normal film exposure time is 6-8 hours. Figure 12B is a powder pattern of fluorapatite obtained with a Debye-Scherre r camera. /. The diffractometer method. If sufficient powder material is available (5-20 m m3) the X-ray diffractometer can be used for the indentification of materials. This method has the advantage of being much faster than the Debye-Scherre r method. Monochromatic X-rays, collimated by a

r

OF

DENTAL

MINERAL

179

slit, irradiate the sample which is spread out over a 2 c m2 flat surface. A Geiger or scintillation counter scans over a selected range of 2 θ values (Fig. 13A). The intensity of the diffracted rays is recorded on a strip chart and plotted against the diffraction angle 2 Θ. Figure 13B shows the diffractometer curve of a well crystallized hydroxyapatite. The diffraction peaks in the curve correspond to the diffraction lines of the DebyeScherrer film strip (Fig. 12B). The diffractometer method is the most convenient method when changes in peak positions (2 0), peak intensities or peak profiles are important considerations . Wit h well crystallized materials, the angle 2 Θ can be read with a precision of ± 0 . 0 1 °. For the correct interpretation of the recorded intensities it is essentia l that the surface of the test specimen is flat and that the powder grains (size C a10 ( P 04) 6 F 2

R.

TRAUTZ

A partial substitution is indicated by C a1 0( P O4) 6 ( O H , F )2. If 10% of the OH positions are occupied by F i.e. 10 mole % substitution, then the formula may be written C a1 0( P O4) 6( O H 0 9F 0 Ë) 2 . Chloride can also replace the OH to form a chlorapatite. The natural chlorapatites are usually not completely substituted, e.g. C a1 0( P O4) 6( C l , O H ) 2 or C a1 0( P O4) 6( C l , F , O H )2. Calcium can also be substituted by other divalent cations such as 2_ j_

2-f-

2-)-

2+

Sr , Ba , Pb and Ra which are larger than C a2 +; also to a limited extent by M g 2 +, Z n 2 +, C d 2+ and M n 2+ which are smaller than C a2 +. I n nature, apatite minerals are found which contain A s 04~ or V 0 4~ in place of P O j - . In cases of poisoning with lead, radium, strontium90 (fall-out strontium), or with arsenate , the ions are first deposited in the mineral phase of the hard tissues and then only slowly released . The various apatites form crystals that are isomorphous and isostructural. Perhaps " iso " in this case is synonymous with "similar" or "analogous", as the structures of all these substances have somewhat different cell dimensions. Two isomorphous substance s can crystallize together and form solid solutions, also called "mixed crystals". In such crystals, the ions which substitute for each other are randomly distributed among the available positions. When the unit cell dimensions of the two isomorphous crystals are similar, as in fluor- and hydroxyapatite, the two substance s usually mix in any proportion and form a continuous series of solid solutions. When the cell dimensions of the two isomorphous crystals are more than about 10% different, the substitution may be limited and a gap in the series may result. Generally, substitutions are accompanied by changes in the unit cell dimensions. The changes usually follow Vegard's law, which states that the changes are proportional to the mole per cent substitution. When several substitutions occur simultaneously, the dimensional changes are usually additive. Many of the natural apatite crystals are examples of such simultaneous substitutions, e.g. (Ca, Pb, Mg, M n ) 1 0( P O4, A s 04) 6 (F, CI, O H ) 2 . Some apatites contain N a 1+ and

16. C R Y S T A L L I N E

ORGANIZATION

K 1 + which have substituted for C a2 +. A deficiency i n positive charges may be compensate d by simultaneous substitution of A l 3+ for C a2+ or by substitution of S O2 - or CO*r for P O j" . In ellestadite, C a1 0( S i O4, S 04, P 04) 6( C 1, F, OH), the S i 04~ and S 04~ are present in almost equal amounts and replace most of the P 04 ~ (Dana and Dana, 1951, p. 906; McConnell, 1937). A synthetic sulphate apatite, N a6C a4( S 04) 6F 2 has unit cell dimensions and X-ray patterns very similar to those of the calcium fluorapatite (Trautz, unpublished data, 1955). These few examples demonstrate the great versatility and comparative stability of the apatite structures. These structures have the six oxygen tetrahedra as a common feature. The carbonate ion, C O2. - , on the other hand, has a flat triangular shape and at first sight has no evident relation to the apatite structure. In spite of this, carbonate-containin g apatites are found as macroscopic natural crystals and also can be synthesize d (Ames, 1959; Trautz and Zapanta, 1960; Zapanta-LeGeros , 1965). The role which the carbonate ion plays in the mineral phase of hard tissues wil l be discusse d in section III , B, p. 189. E. CRYSTALLINIT Y

1. Crystal Size and Shape The physical properties of the hard tissues and the chemical behaviour of the mineral embedde d in the organic matrix are highly influenced by the state of crystallinity and by the degree of subdivision of this mineral. Very finely divided apatite has a higher solubility, higher reactivity and greater specific surface than larger particles of the same composition. Wit h greater specific surface, the surface adsorption of ions from a solution is greater and may lead to erroneous values for the composition of the crystals. For this reason the problem of determining the size of the crystals is of great interest. The crystals of all biological apatites are of submicroscopi c size. This also is true of the synthetic precipitated apatites. Special thermal treatments in the auto-

OF

DENTAL

MINERAL

185

clave are necessar y for growing crystals of microscopic size. I n spite of their smallness , several methods are available for estimating the size of the crystals. These methods include (a) nitrogen adsorption, (b) electron microscope observations and (c) broadening of the X-ray diffraction lines. (a) In the nitrogen adsorption method, the BET method, the amount of N 2 held in a monomolecula r layer on the surface of the material, is determined (Brunauer, Emmet and Teller, 1938). F r om the amount of nitrogen, the specific surface (surface area per gram of the material) is calculated and, assuming a spherical particle shape, an average particle diameter is obtained. This method gives the particle size rather than the crystal size since it is quite possible that several crystals have grown together and function as one particle. (b) Wit h the electron microscope, particularly the modern high resolution instrument, the size and shape of particles with dimensions from 10,000 to 10Â can be observed. One of the diffi culties in deriving the average particle size from the electron microscope observations is that a limited range of sizes are observable at a particular magnification. Therefore, systematic measurement s must be made at various magnifications in order to derive a true average particle size. (c) The X-ray diffraction method of determining crystal size is based upon the internal structure of the crystals rather than their external shape. A sharp X-ray reflection results from the constructiv e interference of coherent X-rays scattered simultaneously by a set of many parallel equidistant hkl planes in the crystal. When the number of planes in such a set is less than about 100, the reflection line loses its sharpnes s and becomes broader and more diffuse. F r om this line broadening, the number Ν of planes in a set can be determined; and by multiplying this number by the d spacing, the dimension L of the crystal in the direction normal to these reflecting planes can be calculated: L = Nd. By applying this technique to the various reflections, e.g. the basal planes and the prismatic planes, one can obtain

186

O T TO

an idea of the shape of the crystals. Line broadening information is obtained by scanning over the specific diffraction line to get its intensity profile (diffracted intensity vs. diffraction angle 2 Θ). The line width B 1 /2 measure d at half-maximum intensity is expresse d in terms of the angle 2 Θ. This width must be corrected for the broadening caused by the instrument to give a " p u r e" line width. This instrument broadening is determined by examining a well crystallized material of comparatively large crystal size (over 2000 Â) . For strain-free material, the corrected line broadening, â 1 / ,2 is due to the smallness of the crystals and is related to the crystal dimension L by the Scherrer equation:

R.

TRAUTZ

sufficiently well distinguished from the background. I n cases where the particles are composed of several crystals having lattice discontinuities between them, the X-ray method allows the determination of the crystal size rather than the particle size. The Scherrer formula has been derived for homodispers e systems with uniform crystal sizes. In multidisperse systems with crystals of greatly differing sizes, the Scherrer formula wil l give an "average" crystal size. The size distribution in a multidisperse system cannot be evaluated from the line width alone but requires consideration of the whole profile. 2. Lattice

β1/2 is expresse d in radians. The angle θ is the diffraction angle of the line at its maximum intensity, ë is the wavelength of the radiation; and Κ is a constant which is approximately equal to 0.9. Instead of the width at half-maximum, the "integral" width β{ is often used. This width is defined as the area of the line profile divided by the maximum intensity. In a strain-free material, the widths of the diffraction lines increase in proportion to (1/cos θ). When different groups of reflections exhibit different degrees of broadening, the shape of the crystals can be deduced from this information. F or example, if the crystals are hexagonal needles with a thickness (perpendicula r to c) which is one-fifth of their length (parallel to c), then the broadening effect on the hkO reflections would be 5 times the effect on the 00/ reflections after applying the (1/cos θ) corrections. Wit h ordinary instrumentation , the useful range of this method is limited to crystal sizes between 1000 and 10 Â (Guinier, 1963). The upper limi t is set by the almost unmeasurabl y small broadening effect of 0.1 mm (at 20° 2 θ with C u K a in a powder camera of 60 mm radius), and the lower limi t is set by the great broadening (8 mm) which makes the line so weak that it cannot be

Distortion

Aside from the broadening due to the smallness of the crystals, any distortion of the crystal lattice wil l also affect the diffraction profile, and this effect must be considered before the crystal size can be correctly determined. In one kind of distortion, the lattice is compresse d in one direction and dilated in another direction. As a result, the spacings of the lattice planes show local variations. Another cause of lattice distortion is the inclusion of impurities in the structure. If the substituting ions are of equal charge and similar size and are randomly distributed, a shift in the line positions wil l be observed which correspond s to the change in the average spacing of the lattice planes. If, however, the substitutions are such that there are some domains with higher and other domains wit h lower concentration s of the substituents , then the profiles of the lines are broadened . This broadening is proportional to the tangent of the diffraction angle θ rather than to (1/cos Θ) as in broadening due to size. Ions which have a different charge and are considerably different in size and shape from those for which they substitute cause considerable local warping of the lattice planes. A n example of this is HPO^", and particularly C O2, - substituting for P O ^- . If one measure s the line broadening of various

16. C R Y S T A L L I N E

ORGANIZATION

orders of a reflection and finds more broadening at the higher order than can be explained by the (1/cos Θ) relation, then strain is considered to be a contributing factor.

3. Crystallinity

Value

Wit h poorly crystallized materials where the reflections are insufficiently separate d or defined for the determination of line broadening, the combined effect of small size and strain is considered in a diffractometer method which numerically describes the state of crystallinity of the material (LeGeros, Zapanta-LeGero s and Trautz, 1963). The diffraction peaks are lower and the background is higher with poorly crystallized materials than with those better crystallized (Fig. 16). If within a given range of diffraction angles the intensity of the coherently scattered radiation ( = area of the diffraction peaks) is given by Ρ and the intensity of the incoherently scattered radiation ( = area of background) is given by B, the numer-

~2Ϊ

'

'

'

36

'

1

'

3'4°



Fig. 16. DifFractometer curves and crystallinity value X of calcium phosphates . Curve A, human enamel; curve B, human bone; curve C, amorphous calcium carbonate phosphate precipitate. The poorly crystallized state of the mineral phase in bone is well demonstrate d by these curves.

OF

DENTAL

MINERAL

187

ical value for crystallinity, X9 is proportional to the ratio P:B. The diffraction range should include strong reflections of both the 00/ and the hkO planes. A convenient range for apatite is from 24° to 36° 2 0. This range includes the strong 002, 211, 112, 300 and 202 reflections as well as the weaker 201, 102, 210 reflections. The correction for instrumental broadening, monochromatization , geometric and electronic factors is made by normalizing the P:B ratio of the apatitic materials and arbitrarily assigning a value of 100 to the P:B ratio from the powdered sample of a very well crystallized natural apatite. The numerical crystallinity, X, of an apatite material is then given i n terms of percentag e of this value. The fluorapatite from Durango, Mexico, and hydroxyapatite from Holly Springs, Georgia, U.S.A., are suitable standards for comparison. This crystallinity scale is suitable for both the well crystallized and also the very poorly crystallized materials. The use of this method allows description and comparison of the effects of various agents (e.g. carbonate, fluoride, magnesium ) and conditions (concentration , p H, temperature ) on the crystallinity of precipitated apatites. In general, the presence of fluoride ions in the solution slightly raises the crystallinity value of the precipitate whil e the coprecipitation of carbonate and magnesium lowers this value. Precipitates made at temperature of 60-100°C instead of 25-37°C have dramatically increased crystallinity. Coprecipitation of increasing amounts of carbonate from solutions with carbonate : phosphate ratios of 15 and higher reduces the crystallinity of the precipitate to such an extent that an amorphous calcium carbonate phosphate is precipitated. The amorphous state is characterize d by a diffraction pattern in which any recognizable diffraction peak is lacking (Fig. 16, curve C). Of all the apatite peaks, the (002) peak at 25.9° is the last one to disappear while no trace of a crystallized calcium carbonate (i.e. no calcite peak at 29.4° or aragonite peak at 26.2°C) is yet observed. Magnesium tends to stabilize the amorphous state of calcium phosphate .

188

O T TO

R.

TRAUTZ

III. THE MINERAL P H A S E IN DENTAL HARD TISSUES. RESULTS OF INVESTIGATIONS

WEIGHT %

1. Chemical

Analysis

Chemical analysis gives the average value of the mineral content in a specimen. This analysis is usually accomplishe d by determining the calcium and phosphorus content in the ash or the acid extract of the tissue. Figure 17 compares representative values for the mineral, organic and water content of the various tissue types. However, considerable differences of these contents may be encountere d within each type of tissue. When considering processe s dependen t on diffusion, as the mineralization or demineralization (e.g. in caries) of the enamel, it is often useful to visualize the relative volumes occupied by these three components . The values have been calculated from the weight percentag e values of Fig. 17A using per millilite r 3.1 gm and 1.33 gm as the respective densities of the mineral and of the dry organic matter (Fig. 17B). H u m an enamel is one of the most highly mineralized dental tissues. There is nevertheles s an appreciable volume of water in this tissue which enables the enamel, after eruption of the tooth, to pick up calcium and phosphate ions from its environment. The water also permits acid ions to enter the enamel and permits dissolved mineral to leave the tissue (e.g. in caries).

%

IP IBl

A . DEGREE OF MINERALIZATIO N

The extent to which tissues are mineralized (degree of mineralization) influences their hardness , strength and resistance to chemical attack. Because the apatite crystals in the dental hard tissues are very small, the mineral phase cannot be differentiated from the organic matrix by direct microscopic inspection. Therefore, indirect methods are used to determine what fraction of these tissues is represente d by the mineral phase. Two such methods are given below.

VOLUME

H al -

:

...

E

D

E=Enamel Mineral

C

B

D = Dentine

11111 1

E

D

C= Cementum

Organic WWM

Water

C

B

B=Bone 1

I

Fig. 17. Composition of various hard tissues. Mineral, organic and water content expresse d (A) in weight per cent; (B) in volume per cent.

2. X-ray

Microradiography

Local variations in mineral content can be observed on X-ray microradiograph s of thin sections of the hard tissue. X-ray microradiograph s are in reality X-ray shadowgraphs , where the absorption is produced chiefly by the calcium and phosphate in the mineral of the tissue. Microscopic enlargemen t of these radiographs allows a resolution of about 1 μ. In this way the different degree of mineralization of younger and older osteones can be demonstrate d in thin sections of bone. Similarly, microradiograph s of thin sections of enamel cut perpendicula r to the growth surfaces show alternate zones of higher and lower mineralization (Retzius lines) caused by disturbance s in the process of mineral deposition. Superimpose d upon these coarse irregular variations is a fine rhythmic variation which correspond s to the diurnal variation in tissue apposition. In order to obtain sufficient contrast between variations in shadow density on the X-ray film , the thickness of the section is made such that about 80% of the radiation is absorbed in the section. F or Mo, Cu or Cr radiation, the optimum thickness of a section of well calcified enamel is about 575, 66 or 22 μ, respectively. Thicker sections give higher contrast for areas that are less mineralized.

16.

CRYSTALLINE

ORGANIZATION

Table 3 COMPOSITION OF HUMAN ENAMEL, DENTINE AND BONE Constituents Calcium Phosphoru s

co2 Sodium Magnesium Chloride Fluorided Potassium Ca : Ñ(weight) Ca : Ñ (molar)

Enamela

Dentine*

Bone&

36.0C 17.5 2.5 0.77 0.42 0.25 0.01 0.05 2.08 1.64

27.0 13.0 3.3 0.3 0.84 traces 0.04 0.05 2.07 1.64

24.0 10.5 3.9 0.5 0.3 0.01 0.03 0.2 2.28 1.77

a

Leicester (1949, pp. 14-25). Calculated from values given by Morse a nd Looney (1927, pp. 357-359). c Values are percentag e dry weight. d Depends on F content of drinking water; see McClure and Likin s (1951). b

B . COMPOSITION

OF THE M I N E R A L

PHASE A N D

STRUCTURE OF I T S APATIT E

The mineral phase in the dental hard tissues is an apatite. However, the composition of this phase does n ot exactly agree wit h that of the standard OH apatite. T he Ca : Ñ ratio is usually lower than that of standard apatite (10 : 6) a nd there are various other constituents present (Table 3). There are lesser amounts of the various "impurities" present in the well crystallized apatite of the enamel than in the poorly crystallized apatite of dentine a nd bone. I n the X-ray diffraction patterns of the vertebrate hard tissues, there is no indication of the presence of any crystallin e compound other than the apatite. Therefore one of the problems with the biological apatites is to determine where the impurities are positioned and h ow they affect the apatite properties. 1. Chloride and Fluoride Ions Studies with the chlorapatite a nd fluorapatite have shown that CI a nd F ions substitute for the OH ion a nd cause a change in the unit cell 14

OF D E N T A L

189

MINERAL

dimensions which is proportional to the mole percent substitution. Table 4 gives the unit cell dimensions of some natural, biological and syntheti c apatites. Although an á-axis increase of only 0.01 Â is expected from the 0.33 wt. % CI (4.6 mole % substitution) found in enamel (Leicester, 1949, p. 50), an increase of 0.02 Â is observed and therefore must be a result of additional factors. T o o th enamel collected from areas where the F content of the water supply is 1 p pm contains enough fluoride so that one o ut of each 300 OH Table 4 UNI T CELL DIMENSIONS ( A ) OF SOME SYNTHETIC AND BIOLOGICAL APATITES A . Synthetic apatites

Apatite Calcium" Strontium^ Barium0

ao

CI

OH

F Co

9.373 6.882 9.720 7.286

ao

Co

9.422 6.883 9.745 7.256 10.19 7.70

ao

Co

9.634 6.778 9.880 7.19c

a

Trautz (1955), Trautz et al. (1964a). Suchow (1963). c Trautz and Zapanta-LeGero s (unpublished 1964). b

results,

B. Biological apatites^ Apatite

a0

Co

Enamel Man, permanen t Man, permanent , 900°C Sea cow Hippopotamus * Shark, Galeocerdof Shark, Galeocerdo, 900°C Shark, Lamna

9.442 9.425 9.442 9.450 9.385 9.38! 9.385

6.883 6.884

6.883 6.879 6.885 6.884 6.886

Dentine Man Shark, Galeocerdo a e f

9.42 9.42

Glas (1962). Glas and Omnell (1960). Contains 3.77% F; 0.55% c o 2 .

6.88 6.84

190

O T TO

R.

TRAUTZ

groups can be replaced by F. When the F content of the water is 6 ppm, enough F is found to replace one out of each 100 OH groups. This 1 mole % substitution shortens the á-axis by 0.005%, which is too littl e to be detected by X-ray diffraction. A change in the length of the á-axis is most readily observed in terms of the shift of either the 300 or 410 diffraction lines. The minimum shift of these lines that can reliably be detected from an enamel pattern is about 0.03° 20, which correspond s to an á-axis shortening of 0.05% or 10 mole % F substitution.

that magnesium substitution is manifested by a detectable change in the unit cell dimensions of the apatite, no evidence of M g substitution in synthetic apatites has yet been observed (Trautz, Zapanta-LeGero s and Klein, 1964a). This lack of evidence for substitution suggests that M g in hard tissues is assigned to the organic matrix or to the surface layers of the crystals. The tendency of the M g ions to disturb the crystallization of apatite is consistent with the latter assignmen t (Trautz, Zapanta-LeGero s and LeGeros, 1964b).

The situation is less favorable for the X-ray detection of F substitution in bone than in enamel because the diffraction peaks are less sharp. However, in bone, in which metabolism continues throughout life, considerably more F can accumulate. The F content of h u m an bone from areas with 4 p pm F in the water supply was found to rise up to 0.9 wt. % (Zipkin, McClure and Lee, 1960) correspondin g to about 30 mole % substitution of F for OH in the bone mineral. Simultaneously, a considerable improvement of the crystallinity of the apatite and a shortening of it s á-axis was observed (Posner et al.9 1963). Still higher F contents have been found in the bones of cattle which had grazed on pasturage contaminated by falling fluoride dust (Baud and Slatkine, 1965; Baud and Moghissibuchs , 1965). The F content rose up to 2% of the ash weight, which indicates an F substitution of 50%. Also in these bone samples the apatite á-axis become shorter with increasing F content and the crystallinit y had improved as shown by the sharpening of the diffraction peaks.

3. Monovalent Cations:

2. Divalent

Cations

Strontium, barium, radium, lead and some other divalent cations can readily substitute for calcium. Lik e all other substitutions, these are manifested by changes in the unit cell dimensions. The content of magnesium in well crystallized natural apatite is very small, less than about 0.2 wt. % (Dana and Dana, 1951, p. 883). Assuming

Sodium

The sodium content of dry bone is about 0.7 wt. %. Half of the sodium is easily exchangeabl e withi n 4 hours and is assume d to be part of the organic matrix. The other half is fixed in the mineral phase and amounts to about 0.5 wt. % of this phase (Edelman et al, 1954). Also the apatitic calcium carbonate-phosphat e precipitates which have been prepared from sodium phosphate and carbonate solutions may contain up to 5 wt. % sodium (Zapanta-LeGero s et al, 1964b). Considering that divalent H P O * - or Codions possibly substitute for P O 4- , it can be assumed simultaneousl y that N a 1+ would sub2 + stitute for C a . If this is so, then 0.5 and 5wt. % sodium in the apatite correspond s to 2 and 20 mole % substitution for calcium. Such a substitution of N a C 03 for C a P 04 raises the Ca : Ñ ratio of the precipitate. This is just one example showing that a molar Ca : Ñ ratio of 1.67 is not sufficient to characterize a " p u r e" hydroxyapatite. 4. Secondary Phosphate:

HPO\~

The growing apatite crystal strongly discriminates against the inclusion of H P O ^- ions. Nonetheless, some of these ions get included and have to be accommodate d in the apatite structure. The presence of H P O 4- lowers the Ca : Ñ ratio in the tissue mineral. Phosphat e solutions between pH 7 and 8 contain chiefly HPO*" ions, but some Ç 2Ñ è 4~ ions and a very few P O 4- ions are also present. Increasing the pH and increasing the

16. C R Y S T A L L I N E O R G A N I Z A T I O N O F

temperature reduces the H P O2" content of the precipitates. The coprecipitation of H P O2" wit h apatite from solutions has been demonstrate d i n many laboratory investigations and is assume d to occur also in the precipitation of the biological apatites (Bjerrum, 1958; Zapanta-LeGeros , LeGeros and Trautz, 1964a). Most likely the proton forms a hydrogen bond between the oxygens of two adjacent P 04~ groups. Simultaneously , for each two HPO4- groups, either one C a2+ must be omitted from the structure (Posner and Perloff, 1957) or two C a2+ must be replaced by two N a 1+ to maintain the electroneutrality of the structure. a. Pyrophosphate formation. The presence of HPO4 - in precipitates is not only deduced from the low Ca : Ñ ratio, but also is more definitely deduced from the formation of pyrophosphat e which is found when the precipitates are heated at temperature s between 300 and 500°C. Pyrophosphate is also found in heated biological apatites, thereby giving evidence for the presence of HP0 4~ groups. This pyrophosphat e is not detected by X-ray diffraction, as the large P 2 O 7groups cannot at these temperature s congregate and crystallize as C a2P 2 0 7 in its own structure. However, by chemical analysis (Gee and Deitz, 1955) and by infrared analysis (Winand, 1961) the pyrophosphat e content can be determined. The quantitative relationship between the amount of HP0 4~ present in the precipitated material and the amount of pyrophosphat e found after ignition has not been satisfactorily established . One difficult y is that during the heating the amount of pyrophosphat e may be diminished by a reaction wit h some other coprecipitated salts such as C a C 03 and Ca ( O H ) 2. The simultaneous coprecipitation of these salts with the secondar y phosphate must be postulated since it happens that even precipitates with a Ca : Ñ ratio of 1.67 or higher may upon ignition yield some pyrophosphate. b. Crystallographic effects. Two crystallographic effects of the H P O |~ ion on the precipitated apatite must be considered : (1) effect on crystallinit y and (2) effect on the unit cell dimensions.

DENTAL

MINERAL

191

That H P O2" disturbs the crystallization of the apatite can be explained perhaps because its bonding characteristic s differ from that of the trivalent P 04~ ions. Precipitates made at 100°C are better crystallized and also contain less H P 04 than those made at lower temperatures . The disturbing effect of H P O2 - in synthetic apatite can be avoided by growing the crystals from strongly alkaline solutions ( N a3P 04) in an autoclave at 400°C. " L a r g e" crystals of microscopic size have been grown in this way (Hayek, Lechtleiner and Bohler, 1955; Perloff and Posner, 1956). The second effect of H P O2 - inclusion in the apatite is the slight lengthening of the á-axis. A n apatite precipitated at 100°C has an á-axis 0.01 Â longer than that of the standard OH apatite. During ignition of the precipitate at 900°C, its á-axis regains its standard length of 9.42 Â . These results suggest that some of the á-axis lengthening of the h u m an enamel apatite can be ascribed to its HPO*T content. c. Octocalcium phosphate (OCP). Under suitable conditions, particularly at pH less than 7 at 40°C, the calcium phosphate precipitates contain appreciable amounts of octocalcium phosphate , C a8H 2( P 04) 6 · 5 H 2 0 . The crystal structure of this substanc e is closely related to that of the apatite (Brown, 1962). On heating, it hydrolyzes and also loses its water and converts to an apatitic structure. Brown (1962; Brown et a l . , 1962) suggests that the biological apatites are nucleated as O CP which then grows by lamellar apposition and subsequen t dehydration to give the lath-shape d apatite crystals observed with the electron microscope. Octocalcium phosphate can be recognized wit h X-ray diffraction by its strong characteristic reflection at a value of 2 θ of 4.8° for Cu K a radiation, which correspond s to its exceptionally large 100 spacing (d100 = 18.4 Â) . This reflection has never been observed in the patterns of the hard tissues. Furthermore, experiments have shown that the presence of carbonate or magnesium in a solution suppresse s the crystallization of O CP (Zapanta-LeGero s et a l . 9 1964a).

192

5. Carbonate:

O T TO

CO:

Chemical analyses of dental enamel and bone give a C 0 2 content of their respective mineral phases of about 2.5 and 3.5 wt. %. The C 0 2 can only be present as carbonate ions. This carbonate greatly influences the solubility of the mineral phase in weak acids (Gron et ai, 1963), and by this effect it also influences the caries susceptibility of the enamel. The form in which the carbonate is contained in enamel, dentine and bone has been discusse d for some years (McConnell, 1952a,b; Trautz, 1953, 1960). Three general ideas are (1) that it occurs as a separate phase; (2) that it is adsorbed on the surface of the crystals; or (3) that it is contained as a part of the apatite structure. Chemical and physical examinations of the biological apatites are not sufficient to explain how the carbonate is contained. Instead, it is necessar y first to study the chemical and physical behaviour of the synthetic calcium carbonate phosphate s precipitated under various conditions and then to extrapolate the results to the biological carbonate phosphates . A few results which bear on the issue are discusse d below. (a) No separate phase of crystallized calcium carbonate has been detected in the sound hard tissues of the vertebrates , except in otoconia and otoliths and in egg shells. For this reason, either the calcium carbonate must be present as an amorphous calcium carbonate phosphate phase or adsorbed on the surface of the apatite crystals, or it must be present as an integral part of, and therefore within, the apatite structure. (b) In the natural dahllite and francolite single crystals (carbonate-containin g hydroxy-and fluorapatites) and in other carbonate-containin g fluorapatite deposits, the á-axes are measurabl y shorter than in carbonate-fre e apatites. In some of these carbonate apatites, the c-axes are slightly longer than the carbonate-fre e varieties. Such effects upon the unit cell dimensions can come about only when the carbonate groups are incorporated into the apatite structure, and not when they are

R.

TRAUTZ

relegated to the surface of the crystals. Carbonate containing apatites made in the laboratory exhibit shortening of the á-axes with increasing carbonate content (Zapanta-LeGeros , 1965). Synthetic carbonate apatites made in the autoclave at 360°C or in the muffle at 500°C also exhibit a short á-axes (Trautz and Zapanta, 1960). The most attractive assumption is that carbonate substitutes for P 0 4 because there are no sufficiently large interstitial spaces in the apatite structure to accommodate the carbonate groups and it is less likely that carbonate substitutes for OH since the space available to an OH group is probably too small to accommodat e the C 0 3 group. Moreover, in some francolites, the full quota of fluoride (substitution for all the OH groups) is present together wit h carbonate . Because the carbonate groups can form sufficiently strong bonds with the calcium ions, they are able to compete with the phosphate ions for the link with the calcium ions. In spite of the fact that the growing crystal discriminates against carbonate inclusion, an increasing carbonate : phosphate concentration ratio in the precipitating solution causes an increasing number of carbonate groups to become included in the apatite structure (Zapanta-LeGeros , 1965). The triangular plane C 0 3 group is somewhat smaller than the triangular face of the P 04 tetrahedron for which it is assume d to substitute, and therefore enough space is available for adjustment of the C 0 3 to a favourable orientation. The increased negative birefringence measure d on relatively large natural single crystals of francolite demonstrates that the orientation of the plane of the C 0 3 group is not random, but is, on the average, inclined at an angle less than 56° (perhaps 40-43°) wit h the basal plane (Trautz, 1960). The substituting carbonate groups do not form an ordered "superstructure " with the phosphate and do not increase the stability of the apatite. Quite to the contrary, the carbonate-containin g apatitic precipitates made at room temperature and biological temperature s exhibit a decreas e in the crystalline order, which is indicated by a decreas e in the peak intensity and by a broadening

16. C R Y S T A L L I N E

ORGANIZATION

of the X-ray diffraction lines. Wit h an increasing carbonate content of the precipitates, their crystallinit y deteriorates progressively until a material results which gives a smooth diffraction curve devoid of any line and which therefore is described as an amorphous calcium carbonate phosphate (Trautz and Zapanta, 1961; LeGeros et al, 1963). The crystals in this material must be less than about 20 unit cells in size or this material may even be a completely unorganized heap of calcium, carbonate and phosphate ions. Such amorphous materials have been found also as normal biological products in the tissues of tapeworms and crustaceans (Fig. 16C; Trautz, 1960). (c) The structure of the biological apatites of the mammalian vertebrate hard tissues may be assumed to be similar to that of some synthetic apatite precipitates. However, most of the biological apatites are poorly crystallized, and therefore their X-ray diffraction peaks are so broad and diffuse that no sufficiently precise axis measurement s can be made to give information regarding the substitution in their structure. The apatite in dental enamel, on the other hand, is better crystallized. Its á-axis is 0.02 Â longer than that of the standard hydroxyapatite. A n elongation of 0.01 Â can be attributed to the CI content (see section III , Â, 1, p. 189). If the carbonate is assume d to be substituting in the apatite, as it does in other well crystallized carbonate apatites, then a shortening of 0.02 Â should be expected. The difference of 0.03 Â between the observed and expected length of the á-axis is still unexplained. A part of this difference can be assume d to be due to some H P 04 groups substituting for P 0 4 groups, as has been observed in many synthetic apatite precipitates (section III , B, 4, p. 190). C. CRYSTALLINIT Y HARD

OF THE MINERA L

PHASE

IN

TISSUES

1. Crystal Size and Shape I t is important to know the crystal size in bone and in enamel because , as mentioned in section II ,

OF

DENTAL

MINERAL

193

E, p. 185, the crystal size has an influence on the chemical and physical behaviour of these tissues. The diffraction patterns of bone are so diffuse that only the 002 reflection is sufficiently sharp and isolated for line profile measurements . A detailed study of the crystal size of fish bone has been reported by Carlstrom and Glas (1959) and of hippopotamus enamel by Glas and Omnell (1960). These tissues were selected because they possesse d a high degree of orientation of the apatite crystals. Thus, the specimens could be orientated to give very strong 00/ and weak hkO reflections, while in the other position, the hkO reflections are intensified and the 001's subdued. The average dimensions of the crystals listed in Table 5 were Table 5 AVERAGE SIZE (Â) OF CRYSTALS IN FISH BONE AND IN HIPPOPOTAMUS ENAMEL" Perpendicula r to c Tissue hkl: (210)

(310)

(702)

Parallel to c (002)

(004)

(006)

(008)

(a) As calculated with Scherrer formula Bone Enamel

410

42.5 410

413

215 741

129 460

361

56 296

(b) Corrected for strain Bone Enamel

a

40-45 410 (370-450)

600-700 1600 (1040-2700)

Carlstrom and Glas (1959), Glas and Omnell (1960).

obtained by inserting the " p u r e" line width in the Scherrer formula. The hkO line width of enamel was found to increase linearly with (1/cos Θ) indicating that this broadening is caused by crystal size limitation. The 00/ line width increased at a higher rate indicating that a strain factor, which increases the line width proportional to tan Θ, has also to be taken into account. Thus, after correction for strain, the average crystal size in fish bone becomes approximately 43 Â by 600-700 Â and in hippopotamus enamel, 410 Â by 1600 Â.

194

O T TO

I n both cases, the long direction is parallel to the c-axis. I n human bone and enamel crystal sizes of the same magnitude can be expected. 2. Crystallinity

Value

The apatite examined must be rather well crystallized or well orientated in order that the line-broadening effects of strain may be separate d from the broadening effects due to small size and to allow the determination of the crystal size with some practical certainty. In the majority of cases where crystal size determination is not possible i t is still of value to indicate the general state of crystallinity. The apatite crystallinity value, X, of a tissue is calculated from the diffractometer curve by dividing the peak intensity Ρ by the background intensity Β and expressing the ratio as a percentag e of the Ñ : Â ratio of a well crystallized apatite (cf. section II , E, 3, p. 1 8 7 ). The l v a l u es of a few tissues have been determined and are reported here: hydroxyapatite from Holly Springs, Georgia = 1 0 0; human enamel = 7 0; human dentine and bone = 3 5; amorphous calcium carbonate phosphate = 1 7. The numerical crystallinity value, X, of different hard tissues can be compared and the effects of various factors lik e age, fluoridated water and pathological conditions upon crystallinity of the tissues can be studied. On account of the large amount of organic matrix present in bone type tissues, the apatite crystallinity value, X, of a tissue is lower than that which is obtained with the matrix-free mineral. The diffuse X-ray scattering of the matrix tends to increase Β and the absorption by the matrix lowers P9 thus giving a lower X. D.

TEXTURE OF THE MINERALIZE D TISSUES AN D THE

EFFECT OF THE O R G A N IC M A T R I X

The strength of bone and other mineralized tissues is dependen t not only upon the degree of mineralization, but also upon the orientation of the fibres as well as of their mineral crystals.

R.

TRAUTZ

1. Texture of the Enamel A n approximate distribution of the orientation of the crystals of a tissue section can be determined by taking an X-ray diffraction pattern of this section. In order to determine the orientation in directions other than that in the plane of this section, another section preferably cut 9 0° to the first one must be examined. The crystal orientation (texture) of a single enamel rod from a human incisor has been examined by Carlstrom ( 1 9 6 0) and the orientation in a bundle of parallel straight enamel rods ( 24 χ 29 μ across, 1 0 50 μ long) has been establishe d by Glas ( 1 9 6 2 ). This orientation was determined by a pole figure analysis of the 0 02 reflection. [For details of the description of textures by pole figure diagrams, see Barrett ( 1 9 5 2, p.

1 7 0) or

Cullit y ( 1 9 5 6, p. 2 7 2 ) .]

The fibre axis, which is defined as the integrated mean direction of the c-axes, is inclined at an angle with the rod axis towards the neck of the tooth. This angle varies from about 2 3° near the enamel surface to 6° near the enamel-dentin e junction (Fig. 1 8 ). The c-axes of the majority of the crystals did not deviate by more than 3 0° from the fibre axis. A smaller fraction deviated up to 5 0 °. The deviation is always greater in the sagittal plane than in the mesio-distal plane. A double fibre pattern, i.e. two intensity maxima on the 0 02 arc separate d by about 3 5 °, is often obtained from the region near the enamel surface. The specimen used in the above investigation was selected for its comparatively "perfect" straight rods. However, certain disturbances , lik e kinking of the rods and twisting of the rod bundles, often occur in the enamel. A less regular and more disperse orientation of the crystals is found in such specimens . 2. Texture of Bone Bone usually gives an X-ray diffraction pattern which shows apparent random orientation of the crystals within the irradiated volume element ( 0 . 0 01 m m3) . However, there exist bony tissues in which a fibre structure can be demonstrated .

16. C R Y S T A L L I N E O R G A N I Z A T I O N O F D E N T A L

Fig. 18. Sagittal section through enamel of human incisor. Orientation of enamel rods (R) and of the fibre axis (FA) of the crystallite aggregate within the rods. C indicates the spread in the orientation of the individual crystallites.

Withi n the cylindrical lamellae of the osteones , a spiral orientation of the crystals may be observed which alternates from one lamella to the next. The best orientation is found in the thin (0.1 mm thick) trabeculae of the spongiosa where the crystals are orientated more or less parallel with the spicule axis and where none are orientated in the radial or circumferential direction (Fig. 19). Where the trabeculae joi n a connecting thin platelet the orientation of the crystal spreads out lik e a fan, but remains more or less parallel to the plane of the platelet. Calcifying tendons and tendon attachment s also give good examples of a fibre pattern. 3. Effect of Matrix Crystals

on the Orientation

MINERAL

195

of the fibre directions may also be derived from the X-ray patterns if conditions are favourable. Such favourable conditions are that the fibres themselves , lik e chitin but unlike collagen, give sharp X-ray reflections and that their degree of mineralization is low so that the fibres can produce on the film diffraction spots of sufficient intensity to be recognizable beside those produced by the mineral crystals (Trautz and Bachra, 1963.) The preformed fibres exert a directional influence upon the crystallizing apatite. Earlier investigators, recognizing the similarity in the c-axis periodicity of the apatite (6.88 Â ) and the periodicity along the keratin type fibre (7 Â) in the enamel, explained this orientation as the effect of epitaxy (Neuman and N e u m a n, 1958; Ambady, 1959). Epitaxy may be defined as the orientated overgrowth of a crystal upon the surface of another. This phenomeno n is well known in the field of mineralogy. However, many examples of such parallel alignment of fibre and crystal exist where no "harmonizing" relationship between the peri-

of the

Generally, when the matrix fibres are orientated then the crystals associate d with them are also orientated. In these cases, the fibre axis of the crystal aggregate coincides with the fibre axis of the organic matrix. The angular spread of the crystal axes, however, is greater than the angular spread of the fibre directions. F r om the X-ray pattern the angular spread of the crystal c-axis direction can be derived. The angular spread

Fig. 19. Fibre pattern of the apatite crystals in a bone trabecula. The spicule axis is vertical. In spite of the very small size of the crystals, their preferential orientation parallel to the collagen fibres in the spicule is remarkable.

196

O T TO

odicity along the fibre and along the crystal c-axis could be established . For instance, while in bone the periodicity along the collagen fibre (28.6 Â) is nearly 4 times the periodicity along the apatite c-axis (6.88 Â) (Glimcher, 1959, 1960), the periodicity along the collagen fibres cannot be made to agree with any multiple of the periodicity (5.11 Â) of thallous phosphate , T 1 3P 04, which also orientates itself parallel to the collagen fibres. Apatite crystals (periodicity 6.88 Â) have also been observed to be orientated parallel to the chitin fibres which have a periodicity of 10.3 Â (Trautz and Bachra, 1963). Blocking of the active groups does not destroy the effect of collagen upon the parallel alignment of the apatite. It has also been observed that collagen can act as a nucleation catalyst for the precipitation of apatite from metastable solutions. Metastable solutions are supersaturate d solutions which do not spontaneousl y precipitate apatite, but give a precipitate when they are seeded with apatite crystals or with a nucleation catalyst lik e collagen. These catalysts may act in such a way that they hold groups of calcium and phosphate ions together for a long enough time for the attachment of additional ions to form a "viable" crystal nucleus. The parallel orientation of the crystals may be imparted to the nucleus through its initial attachment to the fibre; or the crystals, as they grow into needle or ribbon shape, turn and orientate themselves in the elongated spaces between the fibres. Turkey leg tendons, which progressively calcify from their ends towards their middle region, allow one to study the earliest stages of the calcification of the collagen fibrils. Nylen and Scott (1960; Nylen et al, 1960) have demonstrate d on remarkable electron micrographs that the first mineral deposit occurs in the "interband" region of the fibrils. Selected area electron diffraction on the same specimens revealed the first mineral to be apatite crystals and to be preferentially aligned parallel to the fibrils. It seems that special groupings in the interband region of the fibrils are particularly suited for the attachmen t of the nuclei.

R.

TRAUTZ

Thus, on the basis of the results discusse d above, the idea of an "epitactic overgrowth" has been modified to an "epitactic nucleation", whereby the collagen fibrils function as nucleation catalysts. E. INFLUENCE OF THE TISSUE F L U I D

UPON

THE

T Y PE OF MINERAL

Except for a very few cases, the hard tissues of the invertebrates are mineralized with calcium carbonates while those of the vertebrates are generally mineralized with apatite. The composition of the tissue flui d determines the type of precipitate which is formed. In vitro studies of precipitation of calcium carbonate s and phosphates from solutions containing sodium bicarbonate and phosphate have shown that the phosphate is precipitated preferentially, i.e. the phosphate : carbonate molar ratio (P : C) in the precipitate is always considerably higher than the Ñ : C in the solution. Slow precipitation of calcium carbonate s and phosphate s from metastable solutions (at pH 7.3, 37°C) have shown that even at very low concentrations the phosphate ions can strongly interfere with the crystallization of the carbonate . The Ñ : C of the solution must be below 1/300 before the carbonate can crystallize as calcite or aragonite. On the other hand, although the carbonate disturbs the crystallization of the apatite, apatitic precipitates are obtained from solutions with a P : C ratio of as low as 1/40 (Bachra, Trautz and Simon, 1963). Higher concentrations and fast precipitation (pH 8) allow the formation of crystallized carbonate and of amorphous carbonate phosphate at Ñ : C ratios of about 1/100 and about 1/20, respectively (Trautz and Zapanta, 1961; Zapanta-LeGero s et al., 1964a). In the intermediate regions, amorphous calcium carbonate phosphate precipitates. A few biological occurrence s of such an amorphous material have been observed, as for instance, the calcareous concretions in tapeworms and the mineral which impregnates the tendon plates

16. C R Y S T A L L I N E

ORGANIZATION

and other tissues of the crustaceans . The presence of magnesium in all these biological amorphous calcium carbonate phosphate s seems to be essentia l for the stabilization of the amorphous state, as has been shown with synthetic materials (Trautz et al, 1964a). The h u m an serum ultrafiltrate has a Ñ : C ratio in the neighbourhood of 1 : 22. It is undersaturated with respect to calcite and metastable wit h respect to apatite. In this flui d it cannot be expected that crystallized carbonate or amorphous carbonate phosphate wil l be precipitated. Also the pathological calcifications observed in the walls of blood vessels and in tendons, muscles and skin consist of apatite lik e that in bone. On the other hand, crystallized carbonate is found in the otoliths of fishes, in the otoconia of amphibia, birds and mammals (Carlstrom and Engstrom, 1955) and in the egg shells of birds. I n these cases it must be assume d that the phosphate ions at the site of the mineral deposition are kept at very low concentrations . In necrotic cartilage the presence of calcite crystals together wit h comparatively large apatite crystals has been observed (Taylor and Little, 1964). These may be secondary alterations occurring under conditions quite different from those in normal cartilage. F. SEPARATION OF THE M I N E R A L PHASE FROM THE ORGANIC M A T R I X

I n the investigations of the structure and composition of the mineral phase of the hard tissues, it is necessar y to separate the mineral phase from the organic matrix. Unfortunately, some methods are quite unreliable because they change the crystalline state of the mineral to a considerable extent. For instance, when a bone sample is heated with aqueous K O H in order to destroy the protein matrix, the poorly crystallized bone apatite recrystallizes into a much better crystallized carbonate apatite. This recrystallization takes place only by dissolution and reprecipitation which is likely to be accompanie d by a change in the composition of the mineral.

OF

DENTAL

MINERAL

197

The same changes are experience d when bone is heated with 6% K O H in glycerol at 290°C (Gabriel, 1894) or with 3% K O H in ethylene glycol at 200-205°C (Crowell, Hodge and Line, 1934). A n improved method is to reflux the sample or to extract it in a soxhlet apparatus with constantboiling 80% ethylenediamin e at 93°C or with water-free ethylenediamin e (b.p. 118°C) which solubilizes the protein by aminolysis (Williams and Irvine, 1954; Peckham, Losee and Ettleman, 1956). Even this latter method gives a slight increase in the crystallinity of the bone mineral (Zapanta and Trautz, 1961).

IV. CONCLUSION X-ray diffraction is the most important tool for investigating the crystalline structure of solid materials. The description of its methods and their application in unravelling the crystalline organization of the dental hard tissues comprise a large part of this chapter. The information obtained by the direct examination of the tissues wit h this and other tools is comparatively meagre and the results can be described in a few sentences . However, their interpretation requires a great deal more experimentation . It is necessar y to study individually the effects which each of the many factors has on the composition, crystalline state and chemical behaviour of the calcium phosphate precipitates. It is in these investigations that X-ray diffraction is successfully employed to a very large extent. It is hoped that thereby further knowledge wil l be gained which will enable us to prolong the useful lif e of our teeth by improving the amount, composition and crystalline state of their mineral and by interfering with the destructive effects of caries. References Ambady, G. K. (1959). Studies on collagen. III . Oriented crystallization of inorganic salts on collagen. Proc. Ind. Acad. Sci. 49A, 136-143.

198

O T TO

Ames, L. L., Jr. (1959). The genesis of carbonate-apatites . Econ. Geol. 54, 829-841. Amer. Soc. Testing Materials (1965). "ASTM Powder Dif fraction File" (latest éd.). A.S.T.M., Philadelphia, Pennsylvania (yearly additions and corrections). Bachra, Â. N., Trautz, O. R. and Simon, S. L. (1963). Precipitation of calcium carbonate s and phosphates . I. Spontaneou s precipitation of calcium carbonate s and phosphate s under physiological conditions. Arch. Biochem. Biophys. 103, 124-138. Barrett, C. S. (1952). "Structure of Metals", 2nd ed. McGraw-Hill, New York. Baud, C. A. and Moghissibuchs , M. (1965). Données radiocrystallographique s sur les changement s de cristallinit é en rapport avec le taux de fluor incorporé in vivo dans la substanc e minérale osseuse . C. R. Acad. Sci., Paris 260, 1793-1794. Baud, C. A. and Slatkine, S. (1965). Fluoride metabolism and bone mineral substance . Congr. Coll. Univ. Liège 31, 89-91. Beevers, C. A. and Maclntyre, D. B. (1946). The atomic structure of fluorapatite and its relation to that of tooth and bone material. Miner. Mag. 27, 254-259. Bjerrum, N. (1958). Calciumorthophosphate . I. Die festen Calciumorthophosphate . II . Komplexbildung in Losungen von Calcium- und Phosphate-Ionen . Mat. Fys. Medd. K. danske vidensk. Selsk. 31, No. 7. Brown, W. E. (1962). Octocalcium phosphate and hydroxyapatite. Nature 196, 1048-1050. Brown, W. E., Smith, J. P., Lehr, J. R. and Frazier, A. W. (1962j. Crystallographic and chemical relations between octocalcium phosphate and hydroxyapatite. Nature, Lond. 196, 1050-1055. Brunauer, S., Emmet, P. H., and Teller, E. (1938). Adsorption of gases in multimolecular layers. / . Amer, chem. Soc. 60, 309-319. Buerger, M. J. (1956). "Elementary Crystallography". Wiley, New York. Bunn, C. W. (1949). "Chemical Crystallography. An Introduction to Optical and X-ray Methods". Oxford Univ. Press, London and New York. Carlstrom, D. (1960). Micro-X-ray diffraction on biological materials. Advanc. biol. med. Phys. 1, 77-106. Carlstrom, D. and Engstrom, A. (1955). The ultrastructure of statoconia. Acta Otolaryngol. 45, 14-18. Carlstrom, D. and Glas, J.-E. (1959). The size and shape of the apatite crystallites in bone as determined from linebroadening measurement s on oriented specimens . Biochim. biophys. Acta 35, 46-53. Crowell, C. D., Jr., Hodge, H. C. and Line, W. R. (1934). Chemical analysis of tooth samples composed of enamel, dentin and cementum. / . dent. Res. 14, 251. Cullity, B. D. (1956). "Elements of X-ray Diffraction". Addison-Wesley, Reading, Massachusetts .

R.

TRAUTZ Dana, J. D. and Dana, E. S. (1951). "The System of Mineralogy" (C. Palache et al., eds.), Vol. II . Wiley, New York. DeJong, W. F. (1926). Le substanc e mineral dans les os. Rec. Trav. chim. Pays-Bas 45, 445-448. Edelman, 1. S., James, A. H., Baden, H. and Moore, F. D. (1954). Electrolyte composition of bone. J. clin. Invest. 33, 122-129. Gabriel, S. (1894). Chemische Untersuchunge n uber die Mineralstoffe der Knochen und Zàhne. Hoppe-Seyl. Z. 18, 257. Gee, A. and Deitz, V. (1955). Pyrophosphat e formation upon ignition of precipitated basic calcium phosphates . J. Amer. chem. Soc. 11, 2961-2964. Glas, J.-E. (1962). Studies on the ultrastructure of dental enamel. II . The orientation of the apatite crystallites as deduced from X-ray diffraction. Arch, oral Biol. 1, 91-104. Glas, J.-E. and Omnell, K.-A. (1960). Studies on the ultrastructure of dental enamel. I. Size and shape of apatite crystallites as deduced from X-ray diffraction data. /. Ultrastruct. Res. 3, 334-344. Glimcher, M. J. (1959). Molecular biology of mineralized tissues with particular reference to bone. Rev. mod. Phys. 31, 359-393. Glimcher, M. J. (1960). Specificity of molecular structure of organic matrices in mineralization. In "Calcification in Biological Systems", Publ. No. 64, pp. 421-487. Amer. Ass. Advanc. Sci., Washington, D. C. Gron, P., Spinelli, M., Trautz, O. R. and Brudevold, F. (1963). The effect of carbonate on solubility of hydroxylapatite. Arch, oral Biol. 8, 251-263. Gross, R. (1926). "Di e kristalline Struktur von Dentin und Zahnschmelz" , Festschr. Zahnàrztl. Inst. Univ. Greifswald, Berlin. Guinier, A. (1963). "X-ray Diffraction in Crystals, Imperfect Crystals and Amorphous Bodies". Freeman, San Francisco, California. Hayek, E., Lechtleiner, J. and Bohler, W. (1955). Hydrothermalsynthes e von Hydroxyapatit. Angew. Chem. 61, 326-327. Henry, N. F. M., Lipson, H., and Wooster, W. A. (1960). "Interpretation of X-ray Diffraction Photographs" , 2nd ed. Macmillan, New York. "International Tables for X-ray Crystallography" (19521962). Vol. I (1952): Symmetry Groups; Vol. II (1959): Mathematical Tables; Vol. Il l (1962): Physical and Chemical Tables. Kynoch Press, Birmingham, England. Kay, M. I., Young, R. A. and Posner, A. S. (1964). Crystal structure of hydroxyapatite, Nature, Lond. 204, 10501052. Klein, E., Trautz, O. R., and Addelston, H. K. and Fankuchen, I. (1951). An X-ray diffraction microcamera and the specimen preparation for the the study of tooth structure. / . dent. Res. 30, 439-444.

16. C R Y S T A L L I N E

ORGANIZATION

Klug, H. P. and Alexander, L. E. (1954). "X-ray Diffraction Procedure s for Polycrystalline and Amorphous Materials". Wiley, New York. Knoop, F., Peters, G. C. and Emerson, W. B. (1939). A sensitive pyramidal diamond tool for indentation measurements . J. Res. nat. Bur. Stand. 23, 39-61. Larsen, Å. K. and Berman, H. (1934). "The Microscopic Determination of the Non-opaque Minerals", Bull. No. 848, 2nd ed. U.S. Govt. Printing Office, Washington, D. C. Le Geros, J. P., Zapanta-Le Geros, R. and Trautz, O.R (1963). Crystallinity measurement s on poorly crystallized materials such as precipitated apatites. Abstr. ann. Meet. Amer, cryst. Ass., Cambridge, Mass., p. 59. Leicester, H. M. (1949). "Biochemistry of the Teeth". Mosby, St. Louis, Missouri. McClure, F. J. and Likins, R. C. (1951). Fluorine in human teeth studied in relation to fluorine in the drinking water. J. dent. Res. 30, 172. McConnell, D. (1937). The substitution of S i 04 and S 04 groups for P 04 groups in the apatite structure; ellestadite, the end member. Amer. Min. 22, 977-986. McConnell, D. (1952a). The problem of the carbonate apatites. IV . Structural substitutions involving C 0 3 and OH. Bull. Soc. franc. Miner. 75, 428-445. McConnell, D. (1952b). The crystal chemistry of carbonate apatites and their relationship to the composition of calcified tissues. / . dent. Res. 31, 53-63. Mehmel, M. (1930). Uber die Struktur des Apatits. Z. Krist. 75, 323-331. Morse, W., and Looney, J. M. (1927). "Applied Biochemistry", 2nd ed. Saunders , Philadelphia, Pennsylvania . Nâray-Szabo , S. (1930). The structure of apatite. Z. Krist. 75, 387-398. Neuman, W. F. and Neuman, M. W. (1958). "The Chemical Dynamics of Bone Mineral". Univ. of Chicago Press, Chicago, Illinois. Newbrun, E., Triberlake, P. and Pigman, W. (1959). Changes in microhardnes s of enamel following treatment with lactate buffer. / . dent. Res. 38, 293-300. Newbrun, E. and Pigman, W. (1960). The hardness of enamel and dentine. Aust. dent. J. 5, 210-217. Nylen, M. U. and Scott, D. B. (1960). Basic studies in calcification. J. dent. Med. 15, 80-84. Nylen, M. U., Scott, D. B. and Mosley, M. V. (1960). Mineralization of turkey leg tendon. II . Collagen-minera l relation revealed by electron and X-ray microscopy. In "Calcification in Biological Systems", Publ. No. 64, pp. 129-142. Amer. Ass. Advanc. Sci., Washington, D. C. Nylen, M. U., Eanes, E. D. and Omnell, K.-Â . (1963). Crystal growth in rat enamel. / . Cell Biol. 18, 109-123. Peckham, S. C, Losee, F . L. and Ettleman, I. (1956). Ethylenediamine vs. KOH glycol in the removal of the organic matter of dentin. J, dent. Res. 35, 947-949.

OF

DENTAL

MINERAL

199

Perloff, A. and Posner, A. S. (1956). Preparation of pure hydroxyapatite. Science 124, 583-584. Posner, A. S. (1955). X-ray diffraction studies of tooth structure. Norelco Repr. 2, 26-30. Posner, A. S., and Perloff, A. (1957). Apatites deficient in divalent cations. / . Res. nat. Bur. Stand. 58, 279-286. Posner, A. S., Perloff, A. and Diorio, A. F. (1958). Refinement of hydroxyapatite structure. Acta cryst. 11, 308-309. Posner, A. S., Eanes, E. D., Harper, R. A. and Zipkin, I. (1963). X-ray diffraction analysis of the effect of fluoride on human bone apatite. Arch, oral Biol. 8, 549-570. Suchow, L. (1963). X-ray diffraction in solid state inorganic chemistry. Norelco Repr. 10, 119-120. Taylor, T. K. F. and Little, K. (1964). Prolapsed calcified thoracic invertebral disc. J. Path. Bact. 88, 153-157. Trautz, O. R. (1953). The use of X-ray diffraction in dental research . Ann. Dentist. 12, 47-54. Trautz, O. R. (1955). X-ray diffraction of biological and synthetic apatites. Ann. N.Y. Acad. Sci. 60, 696-712. Trautz, O. R. (1960). Crystallographic studies of calcium carbonate phosphate . Ann. N.Y. Acad. Sci. 85, 145-160. Trautz, O. R. and Bachra, Â. N. (1963). Oriented precipitation of inorganic crystals in fibrous matrices. Arch, oral Biol. 8, 601-613. Trautz, O. R. and Zapanta, R. (1960). Synthetic carbonate apatites. J. dent. Res. 39, 664. Trautz, O. R. and Zapanta, R. (1961). Experiments with calcium carbonate phosphate s and the effect of topical application of sodium fluoride. Arch, oral Biol (Spec. Suppl.) pp. 122-133. Trautz, O. R., Klein, E., Fessenden , E. and Addelston, H. K. (1953). The interpretation of the X-ray diffractograms obtained from human dental enamel. J. dent. Res. 32, 420-431. Trautz, O. R., Zapanta-LeGeros , R. and Klein, E. (1964a). X-ray diffraction in dental research . Norelco Repr. 11, 29-33. Trautz, O. R., Zapanta-LeGeros , R. and LeGeros, J. P. (1964b). Effect of magnesium on calcium phosphate s II . J. dent. Res. 43, 751 (Abstract). Williams, J. B. and Irvine, J. W., Jr. (1954). Preparation of the inorganic matrix of bone. Science 119, 771. Winand, L. (1961). Étude physio-chimique des phosphate s de calcium de structure apatitique. Ph.D. Thesis, University of Liège. Zapanta, R. R., and Trautz, O. R. (1961). The separation of the mineral phase in mineralized tissues. / . dent. Res. 40, 702. Zapanta-LeGeros , R. (1965). Effect of carbonate on the lattice parameter s of apatite. Nature, Lond. 206, 403-404. Zapanta-LeGeros , R., LeGeros, J. P. and Trautz, O. R. (1964a). The effect of various ions on unit cell dimensions and crystallinity of the apatites. J. dent. Res. 43, Suppl., 775-776 (Abstract).

200

O T TO

Zapanta-LeGeros , R., LeGeros, J. P., Trautz, O. R. and Klein, E. (1964b). Infra-red investigation of the carbonate substitution in the apatite. / . dent. Res. 43, Suppl., 750-751 (Abstract).

R.

TRAUTZ Zipkin, I., McClure, F. J. and Lee, W. A. (1960). Relation of the fluoride content of human bone to its chemical composition. Arch, oral Biol. 2, 190-195.

CHAPTER

17

CHEMISTRY OF THE MINERAL PHASE OF DENTINE S.

L.

ROWLES

I. Introduction

201

II . Preparation of Dentine for Analysis A . Separation of Dentine from Other Dental Tissues B. Separation of Dentine Mineral from the Organic Phase

202 202 203

III . Analytical Data on the Inorganic Composition of Dentine A . Basis of Expression of Results B. Water Content C. The Inorganic Composition of Human Dentine D. The Inorganic Composition of Dentine from Other Mammalian Teeth E. Trace Inorganic Constituents in Dentine

206 206 206 207 214 215

IV . Nutrition and the Inorganic Composition of Dentine A . Man B. Other Mammals C. Special Topics

222 222 223 228

V. Inorganic Composition of Dentine in Diseased and Other Abnormal States A . Dental Caries B. Secondar y Dentine C. Altered Dentine D. Hereditary Opalescen t Dentine E. Periodontal Disease F. Dentine under Fillin g Materials VI . Nature of the Mineral Phase in Dentine A . Homogeneity of the Inorganic Fraction B. Number of Inorganic Phases Present C. The Calcium : Phosphoru s Ratio D. State of the Minor Constituents

230 230 231 231 232 233 233 233 233 234 235 235

VII . Summary

237

Reference s

238

I. INTRODUCTION The chemical composition of dentine mineral is of fundamenta l interest in relation to the nature of the mineral phase of calcified tissues. A s a meso-

dermal tissue dentine is properly compared with bone but, in contrast to b o n e, it is not normally subject to cellular remodelling and in this respect 201

202

S.

L.

is more comparable with ectodermally derived enamel. The composition of dentine mineral is therefore likely to be determined largely at the time of formation and provides a partial record of the nutritional status of the body at that time. Once formed, however, the mineral phase is not completely inert and, as shown by radioisotope studies, such processe s as ionic exchange and recrystallization can affect its properties and to some extent its composition. I t is of some importance to determine whether the composition of dentine mineral is related to the susceptibility of the tissue to disease , and many attempts have been made to demonstrat e a relationship between the levels of either major or minor constituents in dentine and the incidence of disease . Study of the affected tissue can also provide information on the nature of the tissue reaction to injurious stimuli. The earlier literature on this subject was comprehensivel y reviewed by Leicester in 1949. As he and others have pointed out, much of the early analytical work utilized whole teeth and is of littl e significance with regard to the individual constituent dental tissues. Unfortunately, even today, many reports appear in the literature of analyses made on whole teeth. More recently the chemistry of dentine mineral has been reviewed briefly by Hartles (1960) and lucidly discusse d by Jenkins (1966) in a more general context. Shaw (1955) and Irving (1957) have considered dietary aspects of dentine composition while evidence on the distribution of minor inorganic constituents within the dentinal structure has been summarized by Brudevold, Steadma n and Smith (1960). Miles (1961) has discusse d the alterations in composition of human dentine with age in a general review of the effect of ageing on dental tissues. Determination of the chemical composition of any material requires an adequate sampling technique. In view of the variation in composition withi n the dentine of a single tooth which has been demonstrate d by chemical analysis and by microradiography, the method of obtaining tissue

R O W L ES

samples for analysis is of considerable importance in determining the significance of the results. Strictly, a distinction must be made between the dentine mineral phase and the inorganic fraction of dentine, since the latter includes any inorganic material associate d with the organic or tissue flui d components . Various methods have been employed in attempts to isolate the mineral phase before analysis. Many analyses , however, have been performed on whole dentine. Comparison of the data therefore requires some consideration of the effect of the various treatments on the composition of the dentine and of the basis used for expression of the results.

II. PREPARATION OF DENTINE FOR ANALYSIS A.

SEPARATION OF DENTINE FROM O T H ER DENTAL TISSUES

For analysis by chemical methods it is necessar y to separate dentine from the associate d enamel, cementum and pulp tissues. This has been achieved by various methods: 1. Grinding, chipping or flaking. These methods can give a very good separation from enamel but do not yield an average sample. Furthermore, it is not possible to recover all the dentine. Grinding, coupled with a suitable technique for the recovery of the powder (Sognnae s and Volker, 1941 ; Yoon et al.9 1960a), is, however, particularly useful for analyses of different layers of the dental tissues (Brudevold et al, 1960). Microdissection of a tooth section has been used for a similar purpose (Selvig and Selvig, 1962). Incineration of the whole tooth facilitates the separation of enamel from dentine. Most simply this can be effected by heating the teeth in a gas flame, and the method has been employed for analytical purposes by Lowater and Murray (1937) and Dam, Granados and Maltesen (1950), using rat teeth, and by Steadman , Hodge and Horn (1941) using human teeth.

17.

C H E M I S T RY

OF

THE

MINERAL

2. Differential flotation. Because of their different degrees of calcification, the dental hard tissues have differing densities (mature human enamel, 2.9-3.0; dentine, 2.14; cementum, 2.03). Powdered teeth can consequentl y be separate d into their component tissues by flotation in liquids of appropriate density (Brekhus and Armstrong, 1935; Manly and Hodge, 1939). Since the tissues in fact show a range of densities, the efficiency of the separation depends upon the choice of density for the flotation liquid and upon the proportion of junction particles present. Separation of dentine from enamel can be made with almost complete recovery. Moreover the enamel impurity can be estimated by use of optical methods (Manly and Hodge, 1939; Hutton, 1953). Separation of dentine from cementum is more difficul t owing to the similarity of their mean densities. It is rarely stated in the descriptions of the preparation of dentine samples by flotation recorded in the literature whether or not cementum has been separated . The behaviour of secondar y dentine, which is often less calcified than primary dentine, in flotation separation does not appear to have been determined. I n tissues where there is a considerable variation of density within individual samples, e.g. in developing tissues, it may not be possible to separate the whole of each individual tissue by this method. Application of the flotation method to teeth from non-human species requires some modification of the technique (Gilda, 1951; Hartles, 1951a) because of the different densities of the component tissues and diverse shapes of the teeth in various species. Gilda pointed out the desirability of adapting the method to the species studied and recommende d that purity tests should be carried out on the separate d fractions. Differential analysis of dentine can be carried out without the necessity of separating it from the other hard tissues by utilizing certain physical methods of analysis with piano-parallel sections of teeth. The most widely used of these are the quantitative development s of the radiographic

P H A SE O F

DENTINE

203

method (e.g. Thewlis, 1940; Amprino and Camanni, 1956; Omnell et al, 1960). Other methods introduced in recent years which have so far given largely qualitative results but may be expected to produce useful quantitative data in the near future, are X-ray microscope study (Rockert, 1958), electron probe analysis and its developmen t as the X-ray scanning microanalyzer (Boyde, Switsur and Fearnhead , 1961) and activation analysis coupled wit h autoradiograph y (Ntiforo and Fremlin, 1964). B. SEPARATION OF DENTINE M I N E R A L FROM THE O R G A N IC PHASE

Removal of the organic fraction of dentine can be accomplishe d either by dry ashing, in which the organic matter is burnt off at high temperature , or by wet ashing, in which the organic matter is rendered soluble by the action of a hot alkaline solution in which the mineral is insoluble. 1. Dry Ashing s of 500°C or This is carried out at temperature higher. The actual temperature can, however, affect the amount of ash recovered, and the progress of the ashing may also be affected by the conditions used. Complete removal of the organic material may not occur in the lower temperature range unless an adequate supply of oxygen from the air or from an added oxidant is available. I n the author's experience powdered dentine is not completely ignited even after 40 hours' ashing at 600°C as a thin layer in a covered crucible. Peckham, Losee and Ettelman (1956b) found 1.6 % nitrogen remaining in dentine samples ashed by heating for 3 hours at 550°C. Citrate present in dentine as calcium citrate is converted into calcium carbonate at 500-550°C and wil l augment the carbonate fraction present. The mineral phase is also affected by dry ashing. Nordback, Johanse n and Parks (1961) studied the effect on the carbonate content of heating root dentine at various temperatures . About 30 % of the carbonate content was lost at 400°C, 80 % at 600°C

204

S. L .

R O W L ES

and only a trace remained at 700°C or 800°C. These results suggest that ashing below 700°C causes only partial loss of the carbonate in dentine. Experimental conditions are important here also; Vogel (1961) recommend s that a temperature of 1100°C or more be used to ensure quantitative decomposition of calcium carbonate , and it may be noted that Losee, Leopold and Hess (1951) found it necessar y to ignite dentine at 1100-1150°C in order to achieve constant weight. A further source of variation in weight loss in the lower ashing temperature range is the formation of pyrophosphate from secondar y phosphate groups initiall y present on igniting dentine at temperature s up to about 650°C (Herman and Dallemagne, 1961). A t 700°C and above, the pyrophosphat e reacts with the hydroxyapatite phase to form â-tricalcium phosphate (whitlockite) with elimination of water. I t may be concluded that simple dry ashing at relatively low temperature s gives results of dubious significance. F r om the work of Nordback et al. (1961) it is evident that it is not possible to retain the carbonate fraction during dry ashing. Satisfactory dry ashing would appear to be best carried out at temperature s of 1000°C or higher to ensure rapid and complete ignition with loss of carbon dioxide and constitutional water.

b. Potassium hydroxide I ethylene glycol. Crowell, Hodge and Line (1934) replaced the glycerol of Gabriel's method by ethylene glycol in order to avoid certain experimenta l difficulties encountere d in the wet ashing of dentine using glycerol. The ashing temperature is reduced to 205°C (the boiling point of ethylene glycol), and boiling water is used for removal of the reagent. The use of boiling water undoubtedly introduces the possibility of structural modification of the mineral through recrystallization (Beaulieu et al, 1950; Zapanta and Trautz, 1961). A number of investigators have compared this method with dry ashing. Bird et al. (1948a) found consistently low recoveries by the glycol ashing method, the loss averaging 2 % of the dry weight of dentine. Burnett and Zenewitz (1957) observed similar losses with hamster dentine but much smaller losses in the case of human material (Burnett and Zenewitz, 1958b). Peckham et al. (1956b) obtained low and inconsistent recovery of mineral by the glycol ashing method, although it would seem that their results were due i n part at least to the experimenta l technique employed. Stack (1951) on the other hand found reasonable agreemen t between dry ashing and glycol ashing methods after allowing for loss of an assumed 3 % carbon dioxide content during dry ashing.

2. Wet Ashing

The losses observed by Bird et al. (1948a) and by Burnett and Zenewitz (1957, 1958b) were in different proportions for calcium, phosphorus and magnesium and thus affected the ratios of these elements found in the ashed residue. In both cases relatively more phosphorus was lost than calcium or magnesium . c. Ethylenediamine. Introduced for the preparation of "anorganic" bone (Williams and Irvine, 1954), this reagent has been investigated as a wet ashing reagent for dentine by Peckham et al. (1956b) and by Losee and Hurley (1956b). Digestion is carried out in a Soxhlet extraction apparatus using a constant boiling mixture of ethylenediamin e and water (ethylenediamin e hydrate) which is subsequentl y removed by hot (Peckham et al., 1956b) or cold (Losee and Hurley, 1956b) water

a. The Gabriel method. The original wet ashing method of Gabriel (1894), in which the calcified tissue is heated at 250-300°C with a solution of potassium hydroxide in glycerol, was advocated by Beaulieu et al. (1950) who considered that the anhydrous reagents involved were unlikely to dissolve the mineral elements and would produce only minor structural modifications. However, in this procedure calcium citrate is decomposed , giving calcium carbonate (Dallemagne, 1952), and dehydration of secondar y phosphate groups with formation of pyrophosphat e may also occur. Zapanta and Trautz (1961), moreover, observed recrystallization of a synthetic calcium carbonate phosphate precipitate treated by the Gabriel method.

17. C H E M I S T RY

OF

THE

MINERAL

rinses. The temperature of the extracting flui d (boiling point 118°C) is much lower than that employed in the earlier methods, with consequen t lessene d possibility of alteration in the mineral phase. The use of water rinses may cause such changes, however. Zapanta and Trautz (1961) found no evidence of recrystallization of calcium carbonate phosphate precipitates after ethylenediamine treatment, although it is not clear whether they used the hydrate or employed subsequen t water rinses to remove the reagent. Losee and Hurley (1956a) used hot ethanol to remove the reagent from digested bone, thus avoiding the use of aqueous media, but this refinement of the technique has apparently not been applied in studies on dentine mineral. Fabry (1959) found that various synthetic calcium phosphate s were decomposed by ethylenediamine , although the calcium:phosphat e ratio of bone was littl e altered. The present author (unpublished observations , 1960) has observed the disappearanc e of octocalcium phosphate from dental calculus samples during ethylenediamin e hydrate ashing followed by ethanol rinses. Although there is no direct evidence that this phase occurs in normal calcified tissues, its presence cannot be ruled out (Brown et al., 1962). Takuma (1960a), using electron microscopy, has observed changes in the appearanc e of the crystals in intact mature dentine after treatment with ethylenediamine . Losee and Hurley (1956b) obtained complete recovery of phosphorus in dentine samples extracted by this method but obtained an unexplained loss of 3 % of the dentine calcium. The behaviour of bone samples was stated to be similar. The citrate content of dentine, and of bone also, was reduced to one-fifth of that of the orginal tissue whereas calcium citrate was found to be unaffected by ethylenediamin e treatment, an observation suggesting that only part of the tissue citrate is present as calcium citrate, the remainder presumably being linked with organic matter. A retention of 2-4 % nitrogen was also observed by these authors. However, in view of the nitrogenous nature of the ashing reagent it was uncertain 15

P H A SE O F

DENTINE

205

whether the residual nitrogen content represente d incomplete ashing or insufficient washing to remove the ethylenediamine . d. Hydrazine. Filson and Hope (1957) used a mixture of hydrazine and ethanol for the solubilization of keratin. This reagent converts proteins into hydrazides of low molecular weight (Akabori, Ohno and Narita, 1952). The present author (unpublished observations , 1960) has used a mixture of hydrazine hydrate and ethanol (boiling point 93°C) for wet ashing bone and dentine. Changes in the X-ray diffraction pattern were less marked than with ethylenediamin e hydrate while dissolution of the organic fraction was complete. e. Enzymic digestion. Removal of organic material by digestion with enzymes, such as the tryptic digestion method employed by Bell, Chambers and Dawson (1947) with bone, does not appear to have been used preparatively with dentine. This method requires an aqueous medium and thus is open to the objection of possible recrystallization and leaching out of more soluble salts. /. Autoclaving. Robinson and Bishop (1950) employed a combination of autoclaving, blending and ultrasonic vibration in order to separate the mineral phase from bone and teeth for electron microscopy. This method has not been used for analytical purposes and is open to the same criticisms as the enzymic digestion method. Of the wet ashing methods used, none effect removal of the organic fraction without producing structural or compositional changes in, or loss of, the inorganic material. Methods using anhydrous reagents would appear preferable from the point of view of minimizing compositional changes and loss of inorganic material and of these ethylenediamine or hydrazine are less likely to produce structural changes because of the relatively low temperature at which the extraction is carried out. The relative efficacy of the ethylenediamin e and hydrazine reagents for wet ashing calcified tissues has not been investigated. Addendum A n elegant microdissection method for obtaining

S. L .

206

R O W L ES

samples of calcified tissues from selected areas of ground sections has been described by Cooper (1965). Shapiro and Hartles (1965) have developed a modification of the flotation method which allows separation of individual samples into fractions of differing densities with very littl e loss of material. Ferguson and Hartles (1964), however, found the flotation method in its present form useless for separation of poorly mineralized tissues from rat teeth formed on deficient diets. I n studies of citrate in human dentine, Hartles and Leaver (1960) observed that all of the citrate was retained wit h the mineral after extraction with boiling ethylenediamine followed by ethanol washing. Nitrogen (0.9 %) was also retained with the mineral. The loss of 70 % of the citrate on further washes wit h boiling water may be compared with the 8 0% loss observed by Losee and Hurley (1956b).

atmospheric pressure or in vacuo, have been employed by some; others have used desiccants such as calcium chloride or phosphorus pentoxide. I t has been shown by Losee et al. (1951) and Burnett and Zenewitz (1958a) that the amount of water removed during drying varies with the conditions and time allowed, and with the particle size of the sample. Burnett and Zenewitz found that considerably more water was lost at 197°C than at 100°C. However, Stack (1951) found that the water content obtained by drying at 100°C was equal to that estimated using the Karl Fischer reagent. Hence it would seem that drying to constant weight at the conventional temperature of 100°C, or just above, gives the most significant dry weight value. Any further loss of water at higher temperature is probably related to the organic or inorganic content.

III . ANALYTICA L DAT A ON T H E I N O R G A N IC C O M P O S I T I ON OF D E N T I NE

A s mentioned earlier, various conditions have been employed for both dry and wet ashing and have resulted in varying yields and degrees of alteration of the solid phase, so affecting its apparent composition. I n conclusion, it is evident that comparison of analytical figures quoted in the literature is valid only when either the same basis of expression is used and the effect of preliminary treatment on the sample is precisely known. Extraction of fat is sometimes carried out before analysis of dentine samples. The a m o u nt of fat in dentine, however, is very small (Stack, 1955) and its removal has no significant effect on analytical figures.

2. Ashed

A.

BASIS OF EXPRESSION OF RESULTS

Analyses of dentine are usually presente d as a proportion of the weight of dentine examined. Ideally the weight of dentine as it exists in vivo would be the most suitable basis of expression for analyses of whole dentine. However, since dentine is composed of hydrophilic organic matter and finely divided mineral, its water content is very sensitive to variations in humidity (LeFevre and Manly, 1938; Burnett and Zenewitz, 1958a), and thus it is virtually impossible to obtain the true weight in vivo. The water content of dentine varies wit h its method of preparation, the humidity of the atmosphere with which it is in contact, and the particle size in powdered samples. Consequentl y the use of fresh or moist weight as a basis of expression is unreliable. 1. Dry

Weight

Various workers have used different methods of drying dentine samples previous to analysis. Elevated temperature s up to 197°C, either at

Weight

B. W A T ER CONTENT

A s noted earlier, the "free" water content of dentine samples varies according to their previous treatment. LeFevre and Manly (1938) found an average of 1 3 . 2% moisture in powdered dentine samples separate d by flotation after rehydration at room temperature under 100 % humidity (dried at 110°C in vacuo). Burnett and Zenewitz (1958a) found similar values for similarly prepared samples: incisor and molar dentine, 13.2% H aO ; molar

17.

C H E M I S T RY

OF

THE MINERA L

dentine, 14.5 % H 2 0 (dried at 100°C in vacuo). For fresh whole dentine, however, Burnett and Zenewitz found a loss of only 10.0% at 100°C in vacuo, while Losee et al. (1951) found only 7.8 % water in coronal dentine samples obtained by drillin g (dried at 111°C in vacuo over calcuim chloride). The presence of " b o u n d" water in dentine is shown by comparison of the organic + water content calculated from ashed weight with the organic content obtained by summation of the protein, citrate and chondroitin sulphate contents (Stack, 1951). This gives a value of about 5 % water. It is usually assume d that this water is associate d with the organic fraction of dentine largely because of the smaller loss from enamel which contains very littl e organic matter. However, dentine mineral has a much smaller crystallite size than enamel mineral and such calcium phosphate precipitates always contain water, part of which is not removed by drying at 100°C. N o determination of the bound water content of anorganic dentine appears to have been made.

C.

THE

INORGANIC

COMPOSITION

OF

HUMA N

DENTINE

1. The Principal Inorganic Constituents Dentine

of Human

Tables l a and l b are collected analyses of human dentine for ash, calcium, phosphorus , magnesium, carbon dioxide, sodium and chlorine for permanent and deciduous material respectively. Certain details regarding the type and preparation of material are also included. Where possible, data have been expresse d as percentage s of dry or dry fat-free dentine since this is the most widely used expression . Although expression on an ash basis would be more useful for comparison with the composition of other calcified tissues, the variety of methods used and the variation in composition of the ash in consequenc e would make such comparison of littl e value. Further, in a number of cases no ash determinations are quoted.

P H A SE O F

DENTINE

207

The mean levels of calcium, phosphorus , magnesium and carbon dioxide found in the more recent analyses are in general similar to and show the same variation among investigators as seen in the results of earlier studies. I n view of the various types of teeth employed and of preparative treatments the differences are of dubious significance. Differences in the conditions of drying the samples, often not described, may affect the elemental concentration s but cannot be the cause of the variation in the calcium:phosphoru s ratio. Analyses of dentine mineral obtained by wet ashing show no greater consistency . The mean sodium content of pooled dentine found by McCann and Bullock (1955) is in agreemen t with the recent determination by Sôremark and Samsah l (1962) but both values are considerably higher than the figures given by earlier workers (Logan, 1935; Bowes and Murray, 1935). A n intermediate value of 0.48 % Na on a fresh weight basis was obtained by Forbes in 1958 (unpublished observations quoted by Forbes, 1962) on unspecified material. Sôremark and Samsah l also found a significant concentration of chlorine in sound dentine whereas Bowes and Murray (1935) could not detect it in sound dentine and found a much lower level in disease d teeth (Bowes and Murray, 1936; Murray and Bowes, 1936). The differences in sodium and chlorine contents found by these groups are almost certainly related to the ease with which the ions can be washed out by water from whole dentine. The specimens studied by Bowes and Murray were intentionally washed before analysis to remove such soluble constituents , which were considered to be derived from the "dental lymph". McCann and Bullock (1955) also observed that half of the dentine sodium could be leached out during water washes. 2. Comparison with Other Calcified Tissues Dentine is always less highly mineralized than enamel but has a rather higher mineral content than bone or cementum on a dry, fat-free basis. The Ca : Ñ ratio in h u m an dentine has usually, but

208

S.

L.

R O W L ES Table THE INORGANIC COMPOSITION

Reference

Subjects

No. Wainwrightb (1933) Wainwrightb (1933) Logan (1935) Logan (1935) Bowes and Murray (1935) Bowes and Murray (1936) Bowes and Murray (1936) Bowes and Murray (1936) Murray and Bowes (1936) Murray and Bowes (1936) Murray and Bowes (1936) Murray and Bowes (1936) Murray and Bowes (1936) Armstrong and Brekhus(1937 ) French et al. (1938)e French et al. (1938)e Dragiff and Karshan(1943 ) Dragiff and Karshan (1943) Ockerse (1943) Ockerse (1943) Bird et al. (1948a) Bird etal. (1948b) McClure (1950)

Age

Teeth

Region

Sex

No.

Type

U.S.A.

Dentine

State

Fraction

Method of separation

D , FE

Unerupted







U.S.A.





1 1 LN

15 55 14

— — —

1 1 LN

Mol . Inc. PM

LN

14



LN

LN

14



LN

14





14





Ad.





14





14



Ad.



20





U.S.A. U.S.A. London, England London, England London, England London, England London, England London, England London, England London, England London, England U.S.A.

3

17-41



3

17-41



av. 34



av. 28

— —

— —

— —





1

44



16-60

Treatment

Erupted

D , FE





Sound Carious0 Sound, Hy

Crown Crown Crown

Drillin g Drillin g Drillin g

None None D(105°)

PM

Sound, Hy+

Crown

Drillin g

D(105°)

LN

PM

Sound, H y ++

Crown

Drillin g

D(105°)

LN

PM

Sound, Hy gross Crown

Drillin g

D(105°)

c. 20

PM

Carious

Crown

Drillin g

D(105°)

Crown

Drillin g

D(105°)

c. 20

PM, Inc. Pyorrhetic

c. 20

PM

Sound

Root

Drillin g

D(105°)

c. 20

PM

Carious

Root

Drillin g

D(105°)

Root

Drillin g

D(105°)

Sound -f carious Al l

Flotn.

D(60°), FE

c. 20

PM, Inc. Pyorrhetic

20



U.S.A.

6

Var.

Sound

Al l

Flotn.

D(110°) FE



U.S.A.

54

Var.

Carious

Al l

Flotn.

D(110°) FE

F

U.S.A.

21

Var.

Sound + carious Root*7

Grinding

D(105°) FE

U.S.A.

31

Var.

Sound + carious Root*7

Grinding

D(105°) FE



S. Afric a S. Afric a U.S.A.

13 9 c. 50

Var. Var. Var.

Crown Sound Crown Carious Sound + carious Al l

Flotn. Flotn. Flotn.

D, FE D, FE D(105°)

M

U.S.A.

WM

WM

Sound + carious Al l

Flotn.

D, FE

M + F U.S.A.

28

Var.

Sound + fluorosed

Flotn.

D , FE

NOTE: See pages 210 and 211 for footnotes.

Crown

17. C H E M I S T RY

OF

THE

MINERAL

P H A SE O F D E N T I N E

209

OF PERMANENT HUMAN DENTINE0

Ashing

Basis of expression

Ash

(%)

Ca (%)

Ñ (%)

Mg (%)

co2 (%)

Na (%)

CI (%)

Ca:P (molar)



D , FF



29.0

13.3



3.54





1.68



D , FF



28.5

13.0



3.54





1.69

— Dry

Fresh Fresh D

— 71.09

26.3 25.1 27.79

12.7 12.3 13.81

0.83 0.85 0.83

3.17 3.00 3.18

0.31 0.30 0.19 d

— Nil d

1.60 1.57 1.55

Dry

D

70.64

27.54

14.35

0.88

3.38



Nil d

1.48

Dry

D

70.17

27.27

13.61

0.80

3.05



Nil d

1.55

Dry

D

70.28

26.96

13.5

0.73

3.10



0.023d

1.54

Dry

D

72.46

27.98

13.87

1.26

3.19



0.04d

1.56

Dry

D

73.97

27.71

14.09

0.93

3.41



0.025d

1.51

Dry

Ash



37.01

18.47

1.33







1.55

Dry

Ash



39.22

19.09

1.35







1.58

Dry

Ash

37.76

18.50

2.01







1.58

Glycol

D, FF





1.58

Glycol

D, FF

Glycol

D, FF

Glycol

D, FF

Glycol

D, FF

— — D (500°) Glycol Glycol

D, FF D, FF D D D , FF

Dry

D, FE

26.18 ± 0.34 12.74 ± 0.48 0.83 ± 0.08 3.57 ± 0.13 80.2 (74.0-82.5) 79.8 (72.2-83.0) 70.7 (68.2-74.4) 70.3 (66.8-74.9)



— 69.4 69.8 81.9 (74.6-87.9) 77.2 ± 0A0j

27.8 (26.3-28.8) 28.2 (25.7-30.6) 26.5 (25.7-27.6) 26.5 (25.3-27.9) 27.33 27.45 26.4 24.5 27.5 (24.8-31.4) 29.3 ± 0.18

13.8 (12.9-14.5) 13.6 (12.1-14.8) 12.7 (12.5-13.2) 12.7 (12.1-13.4) 13.03 13.39 13.1 11.5 13.2 (10.8-16.2) 14.0 ± 0.07

0.85' (0.64-1.07) 1.01' (0.58-1.49) —

2.97 (2.70-3.05) 3.22 (2.19-3.76)



















0.96 0.90 1.05 0.96 0.97 (0.63-1.39) —

— —

— — — —

— —



— — — — —









1.57 (1.57-1.58) 1.61 (1.49-1.75) 1.61 (1.55-1.67) 1.61 (1.55-1.74) 1.62 1.58 1.57 1.66 1.61 (1.42-1.77) 1.62 ± 0.01

(continued)

210

S.

L.

R O W L ES Table la THE INORGANIC COMPOSITION

Reference

Subjects

No.

16-60

McClure (1950) McCann and Bullock (1955) Peckham et al. (1956b)

Soremark and Samsah l (1962)

a

Sex

Region

M + F U.S.A.

No.

Type

45

Var.

Dentine

State

Fraction

Method of separation

Treatme







U.S.A.

VL N

Var.

Carious + fluorosed Sound + carious

LN

17-21

M

U.S.A.

LN

M3

Sound

Crown

Drillin g

D, RT

Sound

All *

Flotn.

D(65°)

Asgar (1956) Asgar (1956) Verbraeck (1958a) Verbraeck (1958a) Verbraeck (1958a) Verbraeck (1958a) Burnett and Zenewitz (1958b) Burnett and Zenewitz (1958b) Burnett and Zenewitz (1958b) Ohmori (1961)

Age

Teeth

LN LN LN LN -

New York, U.S.A. — Greece 20 30 Negro Congo, Afric a 20-30 Albino Congo, Afric a 20-30 European, Congo, White Afric a 15-20 White Congo, Afric a — — U.S.A.

10

Crown

Flotn.

D, FE

Al l

Flotn.

D, FE

10 LN

M 1 >2

Sound Sound + carious

All 3 All 3

Flotn. Grinding

D(65°) —

LN

M 1 >ss

Sound + carious

All 3

Grinding



LN

M 1 >2

Sound + carious

All 3

Grinding



LN

M 1 >2

Sound + carious

All 3

Grinding



96

Inc.

Sound

Al l

Flotn.

D









U.S.A.

48

Can.

Sound

Al l

Flotn.

D





- -

U.S.A.

96

Mol .

Sound

Al l

Flotn.

D

6

10-12



Japan

6

Var.

Al l

Flotn.

D, Ac.

15

14-16

15

MP

Crown

Chipping

D(105°)

M + F Sweden

_ Sound

Analytical data are mean values. Where analyses of individual teeth were made ranges (in parentheses ) or expression s of standard deviation are quoted. Abbreviations: Ac., Acetone; Ad., adult; Can. = canine;/) = dried (temperature in parentheses) ; ED, ethylenediamine ; F, female; FE, fat extracted; FF, fat-free; Flotn., flotation; Hy, very mild hypoplasia; Hy+, Hy++, Hy gross, increasing degrees of hypoplasia; Inc., incisor; LN, large number; M, male; MoL, molar; PM, premolar; Var., various; VLN, very large number; WM, whole mouth. b Data quoted from Leicester (1949) c Analyses of carious teeth quoted throughout Tables l a and l b refer to the sound dentine of carious teeth from which the carious material was previously removed. d Sodium and chlorine values determined after washing dentine sample with water.

17. C H E M I S T RY

OF

THE

MINERAL

P H A SE

OF

DENTINE

211

( continued) OF PERMANENT HUMA N DENTINE0

Ashing

Basis of expression

D, FE

Dry —

D, FE

Ash (%)

Ca (%)

76.3 ± 0.37' 29.2 ± 0 . 18

Ñ (%)

Mg (%)

co2 (%)

Na (%)

CI (%)

Ca: Ñ (molar)

14.0 ± 0.07









1.62 ± 0.01

0.71



1.58

— — — —

— — — —

1.61 1.55 1.45 1.54 1.76



26.7

12.9

0.79

3.2

73.8 56.4 78.0



27.0 25.6 18.9 26.1 30.2

13.0 12.7 10.1 13.1 13.2

— — — 0.75*

— 3.54

None D (550°) Glycol ED Glycol

D D D D D

Glycol D(1300°)

D Fresh

26.8 33.03

12.5 16.08

0.42*

3.51

74.0









1.69 1.58

D(1300°)

Fresh

66.9

30.16

15.83









1.48

D(1300°)

Fresh

72.0

31.24

14.63







1.65

D(1300°)

Fresh

64.1

28.05

14.10







1.54

D (900°) Glycol

D D

72.25 77.75

27.12 24.51

12.48 12.64

0.90 1.00







1.68 1.50

D (900°) Glycol

D D

71.80 77.60

26.54 24.48

11.57 12.14

0.87 0.93







1.77 1.56

D (900°) Glycol

D D

72.00 78.15

26.86 24.71

12.51 12.44

0.84 0.88







1.66 1.53



25.9 (24.6-27.0) 28.2 ± 1.2

12.2 (11.5-12.8) 13.5 ± 2.5

D



e

D



— —



1.65 (1.58-1.68) 0.75 ± 0.21 0.39 ± 0 . 11 1.62

Analyses discarded by the authors on the basis of the standard deviation have also been omitted here. Magnesium analyses taken from Tefft et al. (I94l). Dry weight percentage s calculated from moisture and organic contents quoted by LeFevre and Manly (1938). 9 Cementum separated . h Teeth from pregnant females. *' Magnesium determination by difference of (Ca + Mg) and Ca shows a relatively large analytical error. i Standard error of the mean. 1

212

S.

L.

R O W L ES Table THE INORGANIC COMPOSITION

Reference

Teeth

Subjects

No.

Age

Sex

Region

No.

Murray (1936) Bird et ah (1940)

— —

— 5-12

— England M + F U.S.A.

— 45

Ohmori (1961)

7

7-10

M + F Japan

7

not always, been found to be lower than that of the corresponding enamel and is more variable; Bale, LeFevre and Hodge (1936) observed that the X-ray diffraction patterns of ignited dentine samples showed mixtures of apatite (Ca : Ñ = 1.67) and â-tricalcium phosphate ( C a :P = 1.50) whereas enamel samples gave only an apatite pattern upon ignition. The magnesium content of human dentine is considerably greater than that of enamel on a dry weight basis, and about three times that of the enamel on an ash basis. This difference has been correlated with the smaller crystallite size in dentine. Bone, however, contains only about half as much magnesium as dentine in spite of its much greater surface area (Wood, 1947). The magnesium content of dentine varies, in general, in the same direction as the calcium content (Table 1) and does not appear to show the inverse relationship to calcium suggeste d by Murray (1936). The carbonate content of dentine is rather higher than that of enamel and fairly similar to, though less variable than, that of bone. Comparison of the analyses of the sodium and chlorine contents of the tissues is made difficult by the variation in concentration s reported, probably due to the preanalytical treatment of the samples. Harrison (1937) considered that the sodium content of washed bone and dental enamel paralleled the calcium content, and the analyses of washed enamel and dentine by Bowes and Murray (1935)

Type

Dentine

State

— Sound Mol . Sound + carious (mainly) var. —

Fraction

Method of separation

Treatment

— Al l

Drillin g Flotn.

D(105°) D(110°)

Al l

Flotn.

D, Ac.

and of enamel and dentine after treatment with very dilute fluoride solution by McCann and Bullock (1955) are in agreemen t with this hypothesis. Bowes and Murray (1935) also found that enamel contained about 0.3 % chlorine which, in contrast to that in dentine, was not extracted by water. 3. Variation of Composition Dentine

in Different Types of

a. Type of tooth. Bird et al. (1948b) found a considerable variation in the total ash, calcium, phosphorus and magnesium contents and Ca : Ñ ratios of dentine specimens from individual teeth of the same mouth, with no definite trend according to position in the mouth. Bird et al. (1948a) also found no significant differences in similar analyses on fift y teeth separate d into similar categories . Stack (1951) found similar ash contents in pooled dentine from large numbers of incisor, canine, premolar and molar teeth respectively. The slight differences between the calcium, phosphorus and magnesium contents of similar material from incisor, canine and molar teeth reported by Burnett and Zenewitz (1958b) are probably not significant. b. Sex. No significant sex differences in dentine composition have been reported (Bird et al, 1940; Sôremark and Samsahl, 1962). c. Race. Asgar (1956) found higher concentrations of calcium, magnesium and phosphorus in

17.

C H E M I S T RY

OF

THE MINERA L

P H A SE

OF

DENTINE

213

lb OF DECIDUOUS HUMAN DENTINE

Ashing

Dry Glycol —

Basis of expression

Ash D D

Ash (%)

Ca (%)

Ñ (%)

Mg (%)

C 02 (%)

Na (%)

CI (%)

Ca: Ñ (molar)

— 76.0 (58.0-94.9) —

37.6 26.1 (17.8-36.5) 26.5 (25.5-27.0)

18.2 12.9 (9.0-17.3) 12.1 (11.5-12.7)

1.39 —

— —

— —

— —

1.60 1.61 * (1.45-1.70) 170 (1.63-1.77)

pooled dentine obtained from American (New York) residents than in similar material obtained from inhabitants of Greece, although the carbonate contents were similar in both samples. The Ca : Ñ ratio for the American sample is unusually high in comparison with mean values reported by other workers for samples from the same region. Verbraeck (1958a) has reported differences in the inorganic composition of pooled dentine samples from Congolese natives, native albinos and whites, and whites of European origin. While dentine from the native negroes differed from that of the European whites principally in the degree of calcification, material from the native albinos gave a relatively low calcium content and an unusually low Ca : Ñ ratio. The high calcium and phosphorus concentrations for dentine found in this study must be regarded with some reserve since the ash contents are not high and the CaO + P 2 0 5 content, calculated from the percentag e calcium and phosphorus values, is well over 100 %. The comparative values have, however, been confirmed radiographically (Verbraeck, 1958b). d. Age. The data of Wainwright (1933) show no differences in the calcium, phosphorus and carbon dioxide contents between unerupted and erupted teeth. McClure (1950) found no difference in the mean ash, calcium and phosphorus contents and Ca : Ñ ratios of individual sound and carious teeth from two groups aged 16-40 years (average 27) and 40-60 years (average 53) respectively. In

_

_

_

_

_

agreemen t with these observations , Burnett and Zenewitz (1958a) state that the water content of dentine does not decreas e with age in the way that happens in bone. Moreover, Stack (1951) found no change in the organic (nitrogen) content of premolar dentine between groups of average age 13 and 33 years respectively. Forster and Happel (1959), however, claim to have obtained a weak correlation between the degree of mineralization of dentine and age based on the dry weight:ignition loss ratio. A n increase in the mineral content of dentine with age has also been observed by histological (e.g. Bradford, 1958), microradiographic (Amprino and Camanni, 1956; Rockert, 1956b) and electron microscopic studies (e.g. Nalbandian, Gonzales and Sognnaes , 1960). The failure of the chemical studies to detect changes with age may be due to the use of the whole dentine of a tooth indiscriminately in analyses with the proportion of relatively poorly calcified secondar y dentine balancing the increased mineral content of the mature dentine. Selvig and Selvig (1962), using a microdissection technique, were able to show a slight increase in the calcium + magnesium, and phosphorus contents of apical root dentine, although the (Ca + Mg) : Ñ ratio showed littl e change. e. Differential composition. Microradiographic observations on sections of dentine show that the mineral content varies in different anatomical parts of the tooth. Thus Van Huysen (1936), Thewlis

S.

214

L.

(1940), McCauley (1942), Rockert (1956a) and Amprino and Camanni (1956) have shown the higher degree of mineralization in the crown as compared with the root dentine, the existence of a circumpulpal layer of rather higher calcification than the main body of the dentine, and a radial increase in mineralization from the pulp outwards. N o chemical determinations of the principal constituents of dentine in the various parts (other than the different fractions noted for the analyses in Table 1) appear to have been made. High resolution microradiograph y (Miller , 1954; Rockert, 1956b; Symons, 1961; Atkinson and Harcourt, 1961) has shown an uneven distribution of mineral on a smaller scale, the mineral of dentine being more concentrate d round the tubules (peritubular zone). Electron microscopic observations also demonstrat e a highly mineralized peritubular zone (Frank, 1959; Nalbandian et al., 1960; Nalbandian, 1962; Scott and Nylen, 1960; Takuma, 1960a, b; Johanse n and Parks, 1962). Blake (1958) considers that the high calcium content of the peritubular region may account for the greater mineral level of dentine in comparison with bone. D.

T HE

INORGANIC

COMPOSITION

OF

DENTINE

FROM O T H ER MAMMALIA N TEETH

Leicester (1949) tabulated the available data on the composition of the teeth of a variety of mammals. Dentine analyses were given only for monkey, elephant, horse, dog and pig. Apart from some phosphorus analyses on dentine from the unerupted incisor teeth of the rhesus monkey quoted by Sognnaes , Shaw and Bogoroch (1955), no analyses of dentine from these species appear to have been published since that time. Some analyses of root dentine from the seal have been reported recently by Selvig and Selvig (1962). Leicester also reviewed chemical analyses of rodent teeth, the data in all cases referring to whole teeth. In recent years, largely through the application of the flotation separation method, many analyses of rat dentine have been made, and these, together with

R O W L ES

some analyses of Syrian hamster dentine, are summarized in Table 2. N o data for dentine from the teeth of the guinea pig or hare (for which whole tooth analyses are quoted by Leicester) appear to have been reported. The data in Table 2, which are for calcium, phosphorus , magnesium and carbon dioxide, have been selected to include only those in which the dietary intake can be considered adequate . The analyses refer to dentine and cementum combined. The hamster data are for material obtained from the same stock and analyzed after dry ashing (Lobene and Burnett, 1954) or after glycol ashing (Burnett and Zenewitz, 1957). The two sets of analyses show notable differences in all three constituents for which analyses were made. Further correction of the glycol-ashed values for the inorganic material extracted by the reagent did not give figures consistent with the dry-ashed dentine concentrations . Until the reason for this discrepancy is ascertaine d it would appear that the hamster dentine data must be treated with some caution. Concentrations of calcium and phosphorus and Ca : Ñ ratios found in rat molar teeth are mostly comparable with those in human dentine. Magnesium contents, however, are only about half those found in human material, while the carbon dioxide content of rat molar dentine is rather variable but tends to be less than that of human dentine. The analytical values of Elli s and Dwyer (1960) and Elli s (1963) for rat molar dentine are anomalous and more analogous to the values obtained by other workers for incisor dentine. The incisor teeth of the rat are continuously growing during lif e and their dentine shows distinct compositional differences compared to molar dentine. The incisor dentine contains less calcium, rather more phosphorus , considerably more magnesium but less carbon dioxide than the molar dentine. The Ca : Ñ ratio reported for rat incisor dentine is lower than that of the corresponding molar dentine and is often remarkably small in comparison with values found in other normal calcified tissues. Sobel et al. (1949) found a

17.

C H E M I S T RY

OF

THE

MINERAL

ratio as low as 1.2 for rats on a stock diet. The low incisor ratio has been found consistently by several investigators and is not paralleled in the comparable enamel. Murray (1936) first drew attention to the low Ca : Ñ ratio of whole rat incisor teeth, in which the higher enamel ratio is outweighted by the greater proportion of dentine in the tooth, and showed that similarly low ratios occurred in the continuously growing teeth of other rodents such as rabbit, hare and guinea pig. The relative calcium and magnesium contents of rat incisor and molar dentine, and the hamster dentine data, support the inverse relation between the concentration s of these elements suggeste d by Murray (1936). d by Analyses of dentine and enamel separate flotation from the incisor and molar teeth of rabbits have been reported by Herman (1964). For incisor and molar dentine, respectively, the followin g data were obtained (% dry weight): Ca 26.98, 28.15; Ñ 14.43, 14.66; C 0 2 1.89, 1.97; C a :P (molar) 1.45, 1.48. Magnesium contents of 1.16 % and 1.32 %, respectively, can be calculated from the results quoted in this paper (estimation by difference). While the low C a :P ratio, and the M g and C 0 2 contents are in accord with the data for the rat incisor quoted in Table 2, it is of interest to note that the greater mineralization of the continuously growing rabbit molar dentine is similar to that of dentine from the rat molar tooth which is of limited growth. E. TRACE INORGANIC CONSTITUENTS I N DENTINE

Earlier analyses of dentine for trace inorganic constituents, made by the spectrographi c method (Drea, 1936; Lowater and Murray, 1937), gave littl e more than a qualitative indication of the presence of a large number of elements. Drea detected Al,Ba,Cu, F, M n, Fe, P b, Si, Ag, Sr, Ti, V and Zn in all samples of h u m an dentine and K, Cr and Li occasionally. Lowater and Murray reported also Sn and Ni , but no Li . Some of the elements were also determined by chemical methods, particularly fluorine. I n recent years more

P H A SE

OF

DENTINE

215

reliable chemical methods have been developed for the determination of trace constituents , the spectrographi c method has been improved (Burnett and Lobene, 1955 ; Mansell and Hendershot , 1960)to yield quantitative results, and valuable new methods such as activation analysis (Soremark and Samsahl, 1962) have been introduced. The use of radioisotopes is also advantageous , coupled with autoradiography for investigating the distribution of trace constituents (e.g. Jee and Arnold, 1960). 1. Fluorine The importance of fluoride as a caries-protectiv e agent has stimulated a vast amount of research into its concentration in dental hard tissues and its effect on their chemical properties. In vitro basic calcium phosphate precipitates readily take up fluoride from solution. A similar phenomeno n is evident for the calcified tissues of the body both in vitro (e.g. McCann and Bullock, 1955; Leach, 1959) and in vivo (e.g. Ericsson, Ullberg and Appelgren, 1960). Peckham, Leopold and Hess (1956a) showed that fluorine in dentine was associate d with the inorganic fraction only. The concentration of fluorine in the dentine appears to depend both on the level of dietary intake and on the time of exposure to fluoride. A connection between the fluoride concentration in the drinking water and the level in the dental hard tissues was suggeste d by Boissevain and Drea (1933) and has since been demonstrate d by a number of investigations (Table 3). Other dietary components may also be involved. Shaw, Gupta and Meyer (1956) found relatively high dentine fluorine contents in teeth from Delhi and Bombay in India although the drinking water levels were very low. Furthermore, Alkalaev (1959) has reported lower fluorine concentration s in teeth from meat-eater s compared with vegetarians who were using the same drinking water. Elliott and Smith (1960) found nearly twice as much fluorine in dentine from children's teeth obtained from St. John's, Newfoundland, where water fluoride was low but dietary fluoride was high, as in dentine from similar teeth obtained from Toronto-Sarnia

Table THE INORGANIC COMPOSITION

Reference White rat: Lund and Armstrong (1942)

Number of animals

10

11 months + 5 weeks

Dam et al. (1950)

12

Weaning -f- 70 days

Hartles (1951a)

22

21 days + 70 days

Hartles (1951a)

28

21 days -f 70 days

McClure, Folk and Rust (1956)

38

Weaning + 90 days

Teeth

Diet

Age and dietary period

Bulk

Stock

High refined sucrose Stock High refined sucrose High carbohydrate

% Ca

ï/ ñ /ï

Adequate Adequate

Inc.

Sound

Mol .

Sound

Inc. (U)

Sound

1.41

0.80

Inc.

Sound

0.87

0.50

Inc.

Sound

0.42

0.38

Mol . (U)

Sound

0.5

0.5

Mol . (L) Mol . (U) Mol . (L) Mol .

Sound Carious Carious Sound

0.5 —

0.5 —

0.63

0.74

Inc. Mol . Inc. Mol . Inc. Mol . Inc. Mol . (L)

Sound Sound Sound Sound + carious Sound Sound Sound Sound

Mol .

Sound

Mol .

Sound

Wynn et al. (1957) McCann and Bullock (1957) McClure (1958)

12 118

21 days + 70 days Weaning + 100 days

40

Weaning + 60 days

Haldi et al. (1959)

40

Weaning + 70 days

High sucrose

0.29

0.5

Elli s and Dwyer (1960) Wynn et al. (1962)

8

Weaning + 8 weeks

0.45

0.25

50

Weaning + 60 days

High carbohydrate High sucrose

Wynn et al. (1962)

50

Weaning -f 60 days

High sucrose

Syrian hamster. Lobene and Burnett (1954)

116

Adult

Stock

Burnett and Zenewitz (1957)

Condition



16

High sucrose High sucrose High carbohydrate High carbohydrate

Type



Wynn et al. (1956)

21 days + 70 days

r

0.25 0.5 (Emoryd) 0.25 0.5 (Harvard**)





Mol . (male)

Sound

Mol . (female) Inc. (male) Inc. (female) Mol . (male)

Sound Sound Sound Sound

Inc. (male) Mol . (female) Inc. (female)

Sound Sound Sound

106

Adult

Stock





105

Adult

Stock





a Analytical data are mean values. Where analyses were made on pooled teeth from individual animals expression s for the variation of individual values are also quoted. Abbreviations: Inc., incisor; Mol., molar; Flotn., flotation; M - H , Manly-Hodge; G, Gilda; B-A, Brekhus-Armstrong ; D, dry; D-A , dry ashed; Gl-A , glycol ashed; FF = fat-free; U = upper; L = lower.

216

2 OF RODENT DENTINE0

Method of separation

Weight basis of expression

Flotn. (M-H )

D, FF

Flotn. (M-H )

D , FF

Heat



Flotn. (mod. M-H )

D(110°), FF

Flotn. (mod. M-H )

D(110°), FF D



Mg

co2

(%)

Ñ (%)

26.35 ( ± 1.08)b 30.24 ( ± 0.6) 22.6

14.84 ( ± 0.61) 13.79 ( ± 0.79) 12.99

26.00 ( ± 0.29)b 26.75 ( ± 0.23) 29.3

13.54 ( ± 0.16) 14.57 ( ± 0.12) 13.9







Ca

(%)

(%)

Ca: Ñ (molar)

P 04: 2C (molai

1.38 —



1.70



1.43



1.35









1.486 ( ± 0.012) 1.425 ( ± 0.014) 1.62



— — Flotn. (G)

D D D D, FF

28.1 28.6 28.6 26.7

13.5 13.7 13.9 13.3

— — 0.28

— — 3.85

1.61 1.62 1.59 1.56

— 2.45

Flotn. (G) Flotn. (M-H ) Flotn. (M-H ) —

D, FF D, FF D, FF D

24.7 28.03 26.80 28.36

14.2 14.04 15.41 14.46

1.28 0.37 1.49 —

2.03 2.57 1.92 —

1.35 1.55 1.35 1.52

4.96 3.88 5.70 —

Flotn. (G) Flotn. (G) Flotn. (B-A)

D D D D , FF

0.31 1.59 1.06 ( ± 0.11) 0.29

2.67 1.93 2.75 ( ± 0.08) 2.64

3.65 5.40 4.19

D, FF

15.35 13.8 14.6 12.5 ( ± 0.2) 14.0

1.34 1.51 1.35 1.22

Flotn. (G)

26.61 26.7 25.6 19.7 ( ± 0.6)c 29.3

1.62

3.73

Flotn. (G)

D, FF

28.9

13.8

0.27

2.65

1.61

3.75

Flotn. (G) D -A

D (100°), FF

22.94

15.42

0.97



1.15



Flotn. Flotn. Flotn. Flotn.

D(100°), D(100°), D (100°), D(100°),

FF FF FF FF

19.25 23.72 23.10 28.3

14.12 16.17 15.42 13.1

0.81 1.48 0.92 0.37

— — — —

1.06 1.13 1.15 1.67

— — —

D (100°), FF D (100°), FF D(100°), FF

22.95 25.5 25.1

12.0 12.05 12.95

1.12 0.29 1.45

— —

1.48 1.64 1.50

— —

(G), (G), (G), (G),

D -A D -A D -A Gl-A

Flotn. (G), Gl-A Flotn. (G), Gl-A Flotn. (G), Gl-A b c d

Standard error of the mean. Mean deviation. Diets differ only in the composition of the salt mixture. 217



218

S.

L.

R O W L ES

Table 3 THE FLUORINE CONCENTRATION IN HUMAN DENTINE0

Reference Shaw et al. (1956) McClure and Likin s (1951) McClure and Likin s (1951) C. G. Elliott and Smith (1960) McClure and Likin s (1951) Boissevain and Drea (1933) McClure and Likin s (1951) Tempestini (1953) McClure and Likin s (1951) Bang (1961) Shaw et al. (1956) Shaw et al. (1956)

Region Boston, Mass., U.S.A. Oslo, Norway Aurora, Illinois, U.S.A. Brantford, Ontario, Canada Galesburg, Illinois, U.S.A. Colorado Springs, U.S.A. Campagnan o di Roma, Italy Catenanuova , Italy Chetopa, Kansas, U.S.A. Alaska (Eskimo) Bombay, India Delhi, India

Water F level (ppm)

Teeth

Dentine F (ppm)

Dentine F: enamel

0.12 0.2-0.4 1.1-1.2 1.3 1.9 2.5-2.6 3.5 4.3 7.6 0.02-0.11 0.04 0.25

Sound Sound + carious Carious Sound -f carious — Sound + carious Sound Sound Sound — —

85 86 385 325 425 1120 1589 1337 1277 243 456 710

2.1 1.1 2.9 2.5 — 2.4 2.7 1.9 1.9 2.1 2.5 2.4

a The values quoted in the table illustrate the range of fluorine levels found in dentine. Additional data can be found in analytical tables given by McClure (1948), McClure and Likin s (1951), Shaw, Resnick and Sweene y (1959), D. Jackson and Weidmann (1959), Bang (1961).

where fluoride intake was low from both water and diet. The uptake of fluoride may also be affected by the levels of calcium and phosphate in the diet. Wagner and Muhler (1960) found that simultaneous administration of calcium or calcium + phosphate wit h fluoride reduced the incorporation of fluoride into the teeth of rats, while phosphate alone taken wit h the fluoride increased the uptake of the latter. S.H. Jackson and Train (1955) demonstrate d that the fluoride retention in rats was increased with a diet low in calcium. The effect of calcium and phosphate on the uptake of fluoride is probably due to interference with the absorption of the fluoride ion in the digestive tract. Fluoride is taken up by the dentine not only during the formation of the teeth but also subsequentl y both pre- and posteruptively. The increase in the fluorine concentration in dentine wit h age has been shown by D. Jackson and Weidmann (1959) and by Yoon et al. (1960b). Jackson and Weidmann found that the fluorine concentration in sound premolar dentine from persons over 50 years of age was three to four times greater than that of similar dentine from

children of 6-12 years of age. The comparable increase in enamel fluorine was approximately a doubling of the concentration in the older age group. A t a given level of fluoride intake the dentine content reached a maximum concentration at about 55 years. The determination of the fluorine content of successiv e layers of dentine and enamel (Jenkins and Speirs, 1954b; Yoon et al., 1958, 1960b) has provided further insight into the uptake of fluoride by the tooth. Considerable differences exist between the concentration of fluorine at the surface of the dentine and that in the interior dentine. The highest dentinal fluorine concentration s are found at the pulpal surface where concentration s of 0.12 % fluorine (water level 1.0 ppm F) to 0.88 % fluorine (water level 5.2 ppm F) were recorded on an ashed weight basis (Yoon et al., 1960b). Further out from the pulp the fluorine concentration drops and the lowest content is found in the central crown and root regions. In the root the fluorine concentration increases again towards the cémentai surface. In the cementum itself concentration s are found similar to those in the pulpal dentine. In

17. C H E M I S T RY

OF

THE

MINERAL

the crown the increase in concentration towards the enamel-dentin e junction is relatively slight; in the latter region the fluorine content is greater than that of the adjacent enamel. Yoon et al. found that all the tissue fluorine concentrations increased with the level of fluorine in the drinking water, but increase with advancing age was much more marked in the pulpal layer than in the main body of the coronal dentine. Jenkins and Speirs considered that the increment of fluorine wit h age was largely due to the deposition of secondary dentine at the pulpal surface with a small contribution from increases of fluorine in the primary dentine close to the enamel. Y o on et al. consider that the fluorine concentration in dentine is directly related to the time of exposure of the crystallites to fluoride and inversely related to the rate of appositional growth. Thus the relatively rapidly formed primary dentine has a low fluorine content while the slowly deposited secondar y dentine and cementum acquire high concentrations . The increase in the fluorine content of the inner layers of dentine is attributed to the diffusion of fluoride via the tubules. This process, however, must be limited since the amount of fluorine in the dentine at the enamel-dentin e junction did not increase with age and sometimes decreased . The decreas e was attributed to the effect of secondar y deposition of mineral relatively free from fluoride, the latter ions having been preferentially deposited near to the pulp. Weidmann (1962) was unable to find any significant increase in the fluorine content of dentine from adult cats after 60 days on a diet containing 500 ppm F, although half-grown cats did show such an increase. He concluded that the fluorine content was largely dependen t upon the formation and mineralization of new tissue. Driak (1952) claimed to have shown differences in the concentration of fluorine between molar and incisor dentine. His observations were, however, based on a very small number of teeth. McClure (1948) found considerable variation in the fluorine content of dentine from the teeth of the same mouth. I n permanent teeth the average fluorine con-

P H A SE

OF

DENTINE

219

centration in dentine has always been found to be greater than that of the enamel. This difference has been attributed to the smaller crystal size in dentine (Brudevold et al, 1960). The reported ratios of dentine F to enamel F are usually about 2 : 1 but vary approximately from 1 :1 to 4 : 1 or even higher. The variation of the ratio may be related to the observation of D. Jackson and Weidmann (1959) that the ratio increases with age. They found ratios of about 2 : 1 in teeth of recent eruption, increasing to about 4 : 1 in the oldest age groups examined. The fluorine content in bone is even higher than that of the dentine (e.g. Singer and Armstrong, 1962), and there is a reasonable correlation between the concentration s in the two tissues. Douglas (1949) found a concentration of fluorine in the dentine from deciduous teeth similar to that i n permanent teeth. Since the concentration of fluorine was higher in the permanent enamel than in the deciduous enamel he concluded that the dentine: enamel ratio was lower in permanent than in deciduous teeth. On the other hand, Harness and Smith (1951) found a lower concentration of fluorine in deciduous dentine than in permanent dentine of similar age, and a higher dentine:ename l ratio in the permanent teeth. 2.

Potassium

Bowes and Murray (193.5) reported 0.07 % or less potassium in human dentine. No recent analyses have been reported, but Burnett and Lobene (1955) found 0.04-0.05% in hamster incisor dentine ash and a smaller content (0.02 %) in molar dentine ash. Both the studies cited record less potassium in enamel than in dentine. 3. Iron Murray, Glock and Lowater (1939), using a colorimetric method, observed concentration s of iron in the ash of dentine from h u m an teeth ranging from 0.006 to 0.015 % with an average of 0.0087 %. N o significant differences in iron content according to type or condition of the teeth could be demonstrated.

220

S. L .

Analyses of rat dentine indicate higher concentrations of iron than that reported for human dentine. Murray et al. obtained values of 0.0214 % and 0.0142 % for the ash of upper and lower incisor dentine respectively, using a simple incineration technique for the separation of dentine from enamel. The correspondin g enamel samples contained nearly seven times as much iron in both types of teeth. These analyses are in agreemen t with the iron content reported for whole rat incisors by Ratner (1935) and Pindborg and Plum (1946), although a more recent determination by a spectrographi c method has given a somewhat lower value (Mansell and Hendershot , 1960). D am et al. (1950), however, also using heat to facilitate separation of dentine from enamel and a colorimetric method of analysis, recorded concentration s of iron ten times as great for the ash of dentine and enamel from upper rat incisor teeth, namely 0.23 % and 1.8 % respectively. The relatively high concentration of iron in the rat incisor enamel appears to be connected with the presence of an iron-rich pigmented surface layer (Lowater and Murray, 1937; Murray et al, 1939; D am et al, 1950). Boyde et al. (1961) have shown that this layer may contain as much as as 7.6 % Fe. Mansell and Hendersho t (1960) found that the iron content of rat molar enamel, which is not pigmented, averaged only 0.0094 % Fe in the ash. Although the pigmentation does not appear to be associate d with the dentine in the rodent incisor, Burnett and Lobene (1955) found a higher iron content in the ash from hamster incisor dentine, separate d by flotation, than in that from the molar dentine, the values being 0.006-0.010 % and less than 0.003 % respectively. Dietary conditions which reduce the pigmentation of the rat upper incisor teeth cause a reduction in the iron content of the enamel and also of the dentine. In fluorosis, Murray et al. (1939) observed a much greater reduction in the iron content of the dentine from the rat upper incisors than in the enamel from the same teeth. In vitamin Å deficiency D am et al. (1950) observed a considerable reduction in the iron content of both enamel and dentine.

R O W L ES

The lowered iron concentration in the dentine does not appear to be due to the anaemia associate d wit h the conditions. Thus, in the experiments of Murray et al, the iron concentration in the lower incisor dentine was unaffected by the feeding of fluoride. Iron is not known to play any part in the calcification process and the significance of the iron content of dentine remains obscure. 4. Zinc Cruickshank (1936, 1940) reported mean concentrations of 0.024% and 0.018% Zn in two investigations into the zinc content of permanen t human dentine. Later (1949), he reported an average concentration of 0.02 % Zn in sound premolar root dentine + cementum from persons 10-17 years old. The enamel content was similar or slightly less. F r om frequency distribution curves he concluded that the population was heterogeneou s as regards the concentration s of zinc in dentine and enamel, the distribution correspondin g to that of the alkaline phosphatas e levels in the serum. Cruickshank suppose d that the population subgroup with higher zinc content of the dentine might be exhibiting preclinical manifestations of tuberculosis since his earlier investigations had indicated a higher mean zinc content in the dentine of tuberculous patients. Brudevold et al. (1963), however, considered that such a distribution could arise from ingestion of zinc at different levels in the food. Cruickshank also found an indication that the zinc content of the teeth increased with age. Brudevold et al. (1963) were unable to demonstrat e an increase in zinc in the dentine with age. They concluded that the main uptake of zinc occurred at the time of calcification. They found that the main body of dentine contained 0.02-0.07 % zinc but the cémentai and pulpal surfaces showed much higher concentrations , the greatest, up to 0.14%, being found at the pulpal surface. In contrast dentine at the enamel-dentin e junction showed similar or slightly less zinc than the central mass of the tissue. In this region the zinc concentration was similar to that in the adjacent enamel. Soremark and Samsah l (1962) found a con-

17. C H E M I S T RY

OF

THE

MINERAL

centration of 0.02 ± 0.008 % zinc in sound premolar dentine, the large standard deviation indicating considerable variation in individual levels. Burnett and Lobene (1955) were unable to detect zinc in hamster dentine by spectrographi c methods, presumably because of the insensitivity of the technique. 5.

Strontium

Considerable interest has been aroused in the association of strontium with calcified tissues because of the formation of the radioactive isotope S r90 during nuclear fission and its presence in fall-out material. The presence of S r90 in the dentine has been demonstrate d in several studies (e.g. Holgate, 1959). Soremark and Samsah l (1962) found an average concentration of 0.0085 % strontium in sound premolar dentine. The concentration of strontium in various layers of dentine and cementum was investigated by Steadman , Brudevold and Smith (1958), who found littl e variation in the strontium level through the tissue. Samples from different geographica l areas varied in their average strontium content from 0.01 to 0.06 %. The main uptake occurs during formation of the teeth, further deposition occurring only where further calcification occurs in secondar y dentine or cementum. Consequentl y the strontium content of dentine varies littl e with age. Values of the same order were reported by Burnett and Lobene (1955) for hamster dentine. 6. Lead Lead is a constant constituent of teeth. The concentration increases with age and is dependen t on the level of intake (Pfrieme, 1934; Maulbetsch and Rutishauser , 1936; Campbell et ai, 1950; Wyss, 1951; Altshuller et al, 1962; Storozheva , 1963). In whole teeth from persons without special exposure to the element, the average lead content ranges from 0.001 to 0.004 % Pb on a dry weight basis. Maulbetsch and Rutishause r (1936) concluded that practically all of the lead was present in the dentine, particularly in the root dentine. Brudevold and co-workers, however, confirming i6

P H A SE

OF

DENTINE

221

the earlier analyses of Pfrieme, showed that enamel contained significant concentration s of lead, particularly in the outer layer (Brudevold and Steadman , 1956; Brudevold et al, 1960). The distribution of lead in the teeth was found to be similar to that of fluorine. The central mass of the dentine contained about 0.02 % Pb but near the enamel-dentin e junction the concentration fell to about half this figure, being similar to that in the adjacent enamel. In the circumpulpal layers the concentration of lead was higher than in the centre of the dentine, the maximal content, about 0.035 % Pb on a dry weight basis, being found in the pulpal surface of the root dentine. The concentration of lead recorded for the central mass of dentine by Brudevold and his colleagues is, however, considerably higher than that observed by Pfrieme (about 0.004 %) or those reported for whole teeth after allowing for some dilution in the latter by the enamel portion. 7.

Manganese

Soremark and Samsah l (1962) found concentrations of 0.19 ± 0.06 p pm M n in dried, sound premolar dentine from children aged 14-16 years, whereas enamel from the same teeth contained three times as much manganes e (Soremark et al, 1962). H u m an bone appears to be much richer in this element than dentine, with concentration s up to 3 p pm M n even on a fresh weight basis (Kehoe, Cholak and Story, 1940), similar concentration s being found in animal bones (Fore and Morton, 1952). I n contrast to the h u m an tissue, rodent teeth have been reported to contain relatively large quantities of manganese . By spectrographi c analysis Burnett and Lobene (1955) recorded concentration s of 10-15 p pm and 41-49 p pm M n in the ash of hamster incisor and molar dentine respectively, wit h enamel concentration s about twice as great. Mansell and Hendershot (1960), who found concentrations of the same order in rat molar enamel, observed an average of only 1.8 p pm for the whole incisor teeth ash. D am et al (1950), on

S.

222

L.

the other hand, have reported concentration s of as much as 250 p pm M n in the ash of rat incisor dentine and even higher concentration s in the enamel. The great variation in manganes e content reported for dental tissues may, in part at least, be related to differences in the amount of manganes e present in the diet. A correlation between the dietary level and the amount of the element in bone was demonstrate d by G . H. Smith and Elli s (1947) while an increased concentration in rat molar enamel was observed by Mansell and Hendershot (1960) when the diet was supplemente d wit h manganese . 8. Other

Elements

Littl e information regarding the concentration in dentine of other trace elements is available. Sôremark and Samsah l (1962) and Soremark et al. (1962) have reported analyses of sound premolar dentine from children aged 14-16 years (see tabulation).

Element Bromine Tungsten Copper Gold Cobalt Chromium Silver

Concentration in dentine (ppm dry weight ± S.D.) 4.0 2.6 0.21 0.03 0.006 0.005 0.004

± ± ± ± ± ± ±

2.0 1.1 0.1 0.01 0.002 0.003 0.002

The concentration of tungsten found in dentine is remarkable in being nearly eleven times greater than that in the enamel from the same teeth. Concentration s of trace constituents in hamster dentine have been reported by Burnett and Lobene (1955). Hadjimarkos and Bonhorst (1959) found 0.52 p pm selenium in permanent h u m an dentine 4.50 p pm in deciduous dentine. In both tissues the enamel content was somewhat lower.

R O W L ES

Soremark and Lundberg (1964) have presente d further analyses for several minor components of dentine from sound erupted human teeth, employing a more sensitive technique. The mean concentrations of Cr and A g are in agreemen t wit h the earlier figures given by Soremark and co-workers but the concentration of Co, 0.00034 ppm, is considerably lower than the earlier determination. The mean iron concentration , 110 ppm, is in agreemen t with the chemical determination of Murray et al. (1939). Rubidium was found present in a mean concentration of 5.6 ppm. Comparable analyses of unerupted (impacted) teeth have been reported by Lundberg, Soremark and Thilander (1965). These show no significant differences in concentration of Na, Zn or Sr in dentine from erupted teeth. Rubidium, however, was found at a concentration of 69 ppm, whil e at 0.56 p pm M n shows a mean concentration three times that in the tissue after eruption. Gold and cadmium could not be detected in dentine from unerupted teeth.

IV. NUTRITION AND THE INORGANIC COMPOSITION OF DENTINE A.

MA N

The mineral pattern of human dentine varies greatly among individuals and this has usually been attributed to nutritional disturbance s at the time of formation of the teeth (see e.g. Bergman, Gothe and Welander, 1961). The chemical composition of teeth showing varying degrees of hypoplasia was studied by Bowes and Murray (1936). These workers were unable to find any significant differences in the calcium, phosphorus , magnesium or carbon dioxide contents or in the Ca : Ñ ratios between dentine from normal teeth or from teeth showing hypoplasia. In washed, grossly hypoplastic dentine a small concentration of chlorine, 0.025 %, was observed which was not present in normal or moderately hypoplastic dentine after similar washing.

17. C H E M I S T RY

OF

THE

MINERAL

Jenkins and Speirs (1954a) compared the composition of English schoolchildren's teeth, which had been formed on the relatively high-sugar diet consumed before the Second World W ar (1939-1945), with the composition of similar teeth formed during the War on a diet containing restricted amounts of sugar. There were no differences in the phosphorus , carbonate or fluoride levels in the dentine or enamel. B . OTHER MAMMAL S

The early experiments on the effects of dietary modification on the composition and structure of teeth were carried out largely from a nutritional point of view and relied mainly on histological methods of analysis. I n many instances observations on the mineral phase were restricted to the assess ment of the degree of calcification from determinations of the ash content. Chemical analyses , where made, were performed on whole teeth without separation of the constituent tissues, which in view of the differences in their individual composition reduces, or may obscure, the significance of the analyses . Most of the more recent studies have been performed on separate d dentine and enamel. Practically all studies involving chemical analyses have utilized the rat. While the dog, the guinea pig and other species have been used in nutritional experiments, there appear to be few data available on the effect of the diet on the chemical composition of the teeth of these species. 1. Variation in the Bulk Constituents

of the Diet

The apparent relation between the sugar content of the diet and the incidence of dental caries has led a number of investigators to study the effect of increasing the proportion of sugar in the diet on the inorganic composition of the teeth. In the albino rat Losee et al. (1957) found no difference in the whole incisor concentration s of calcium and phosphorus after 6 weeks on diets containing various casein : sucrose ratios. In the femur a

P H A SE

OF

DENTINE

223

slight increase of calcium content was noted with an increasing proportion of carbohydrate in the diet. Cremer et al. (1953) also found no change in the calcium and phosphorus content of whole teeth from rats maintained for three generations on a high carbohydrate diet compared with controls on a normal diet. Hartles (1951a), however, found slightly, but significantly, higher concentrations of calcium and phosphorus in both incisor dentine and enamel from rats fed a high sucrose diet in comparison with dental tissues from animals fed a stock cubed diet. I n both cases the diets provided adequate nutritive levels of calcium and phosphorus , although these were considerably higher in the stock than in the experimenta l diet. I n contrast, femur calcium and phosphorus levels showed no differences in relation to diet. Hartles considered these results indicative of differences in the mineralization processe s in bones and in teeth, and suggeste d that the high sugar diet might stimulate the calcification of the teeth by maintaining a high level of glycogen in the developing dentine and enamel. 2. Variation[in Levels

Dietary

Calcium

and

Phosphorus

G a u nt and Irving (1940) showed that, provided the level of each element was above 0.3 % in the diet, the rat incisor dentine was normal histologically and in ash content even if the calcium:phosphorus ratio in the diet varied between 4.0 and 0.5. Where one element was below the optimal level the degree of calcification of the dentine increased with the dietary Ca : Ñ ratio. In contrast to bone, greater interference with the formation and calcification of dentine was noted at low Ca : Ñ ratios. The effect of deficiency was much less marked in dentine than in bone, as first noted by Erdheim (1914), but was more readily seen histologically than from the degree of mineralization determined by chemical analysis. Lund and Armstrong (1942) demonstrate d by chemical analysis that the calcium and phosphorus contents of both incisor and molar dentine of

224

S. L .

mature rats were unaffected by a diet containing adequate phosphorus but only 0.007 % calcium and lacking vitamin D. During the dietary period of 220 days the calcium and phosphorus concentrations in the humerus, by contrast, fell by 17 %. Similar observations were later made by McClure (1958) using weanling rats which were fed on deficient diets supplemente d with minerals to give Ca : Ñ ratios varying from 0.11 to 1.57. The results with a low calcium-high phosphorus diet tended, however, to show reduced mineralization i n accordanc e with the observations of G a u nt and Irving. Sobel and H a n ok (1948, 1958; see also Sobel, 1955, 1960) investigated the relation between the composition of incisor dentine and enamel and that of the blood following an earlier demonstration of a relation between the compositions of bone and blood (Sobel, Rockenmache r and Kramer, 1945). Albin o rats (Sobel and Hanok, 1948) and cotton rats (Sobel and Hanok, 1958) were given a low mineral stock diet supplemente d to give Ca : Ñ ratios varying from 0.029 to 7.67 (molar). In spite of the wide variation in dietary ratios and resulting variation in the serum ratios, the dentine calcium and phosphorus contents and Ca : Ñ ratios showed littl e variation. The ratio of phosphorus to carbonate in the dentine, however, did show some variation, largely due to changes in carbonate levels, which could be correlated with the same ratio for serum. The authors concluded that there is a relationship between the composition of the teeth and that of the flui d from which the tooth salts are deposited and that the composition of this flui d is i n turn related to that of the blood serum. The teeth of animals fed on a high-calcium lowphosphate diet contained more carbonate in both enamel and dentine than did the teeth of those given low-calcium high-phosphat e diet and the high-carbonate teeth showed a greater caries susceptibility than those containing relatively littl e carbonate (Sobel, 1955). Al l the diets used by Sobel and H a n ok contained either calcium or phosphorus below the level of 0.3 % which was considered by G a u nt and Irving (1940) to be necessar y for normal tooth formation.

R O W L ES

I n a series of experiments with the albino rat, employing adequate but varying levels of calcium and phosphorus in conjunction with a high-sugar diet, Wynn and colleagues (Wynn et al, 1956, 1957; Haldi et al, 1959) failed to find significant alterations in the calcium, phosphorus , carbon dioxide or magnesium contents of the enamel or dentine of either incisor or molar teeth. The elemental ratios showed no significant variation although the dietary Ca : Ñ ratio varied from 0.3 to 2.0 irrespective of whether the calcium level was kept constant and the phosphorus level varied or vice versa. These workers concluded that the findings of Sobel and H a n ok reflect the use of a suboptimal dietary level of calcium or phosphorus rather than alteration in Ca : Ñ ratio. Alterations in the inorganic composition of the blood also occur during the acidosis induced by a high respiratory level of carbon dioxide. Under these conditions dentine formation is affected (Stanmeyer et al., 1958) and caries susceptibility may be increased (King, Wil k and McClure, 1962). The effect of the acidosis on dentine mineral composition has not been determined. Because of their limited growth, the molar teeth of rats would appear less likely to show compositional changes in response to dietary alterations, particularly since these teeth, with the possible exception of the third molar, are usually already full y formed prior to the period of experimenta l observation, usually commence d at weaning. However, although enamel formation is probably complete at this stage, deposition of dentine continues for a considerable period (Hoffman and Schour, 1940). Thus, McCann and Bullock (1957) observed a 50 % increase in the weight of molar teeth of rats from weaning to the end of a 100-day period. Sobel and Hanok (1958) and Elli s (1963) have reported changes in the inorganic composition of rat molar teeth in relation to the Ca : Ñ ratio of the diet. Sobel and Hanok found that the dentine calcium and phosphorus levels were not significantly affected by variation in the dietary ratio of these elements, but Elli s obtained a lowered phosphorus concentration in dentine from rats fed

17. C H E M I S T RY

OF

THE

MINERAL

upon a diet deficient in calcium. F r om their analyses Sobel and H a n ok concluded that the phosphate : carbonate ratio in the molar teeth of cotton rats was related to the phosphate : carbonate ratio of the serum, and to the Ca : Ñ ratio of the diet. Surprisingly, in view of the relatively smaller amount of mineralization occurring in the enamel during the experimenta l period, they found that the phosphatexarbonat e ratio of enamel was more sensitive than that of the dentine to changes in the Ca : Ñ ratio of the diet. Possibly the composition of the enamel may be altered posteruptively through contact with the oral fluids as has been observed in human enamel (Littl e and Brudevold, 1958). In view of the differences in serum composition produced by the different diets used by Sobel and Hanok, differences in salivary composition probably occurred also. I t is evident that, although the composition of the dentine may be affected to some extent by extreme fluctuations in the serum composition as a result of the use of diets deficient in calcium or phosphorus or both, much greater variations in the mineral composition of dentine are produced by the action of "local factors" in the tissue itself. Thus, as can be seen from the data in Table 2, the Ca : Ñ ratio of molar dentine is much higher than that of incisor dentine from the same animals, resulting from differences in the concentration s of both calcium and of phosphorus between the two types of teeth. Similarly the magnesium and carbon dioxide contents differ considerably between molar and incisor dentine. Practically nothing is known of the nature and mode of operation of these "local factors". Significant differences in dentine composition have been reported between different experiments with the same strain of rat. A s an example, the carbonate levels given by Wynn et al. (1956) for molar dentine can be compared with those given by Wynn, Haldi and Law (1962). Failure of the conventional flotation separation technique when applied to the teeth of rats maintained on diets deficient in calcium or phosphorus has been reported by Ferguson and Hartles (1964). This observation indicates that data obtained for such material by previous workers must be

P H A SE

OF

DENTINE

225

interpreted with some caution. Using incisai and apical portions of whole rat incisors for ash determinations , Ferguson and Hartles confirmed the slight effect of vitamin D deficiency on the mineralization of the teeth provided that the calcium and phosphorus contents of the diet were adequate . I n the absence of the vitamin supplement, however, these workers observed a considerable reduction in mineralization, particularly in the apical portion of the tooth, on the calcium deficient diet, while the phosphorus deficient diet caused less marked reduction in ash content. Whil e the elevation of the serum calcium level accompanying vitamin supplementatio n of the calcium deficient diet may be responsible in part for the increased mineralization of the incisor, these workers consider that a direct action of the vitamin on the formation of the matrix may also be a contributory factor. 3. Magnesium

Deficiency

Acute magnesium deficiency produces conconsiderable histological changes in the teeth of rats (H. Klein, Orent and McCollum, 1935; Irving, 1940; Becks and Furuta, 1942). Chemical studies have been limited to observations on whole rat incisor teeth, although, in view of the greater magnesium content and proportion of the dentine i n the teeth, the figures probably represen t a fairly accurate reflection of the changes in dentine. I n short term experiments Duckworth and Godden (1940) induced acute deficiency by administering a diet to weanling rats in which the magnesium content was reduced to 6 ppm. The rate of incisor growth and increase in tooth weight were only slightly reduced in animals on the deficient diet in comparison with those maintained on a diet containing adequate magnesium (700 ppm). In spite of loss of tooth material through attrition, the total amount of magnesium in the whole incisor teeth remained roughly constant, indicating continued deposition of the element, albeit at a reduced level, in the newly formed tissue. In contrast, the total magnesium content of bone fell by about onethird during the same period and, after prolonged deprivation, to a minimum of approximately one-

226

S.

L.

third of the normal level (Duckworth, Godden and Warnock, 1940; McAleese and Forbes, 1961; similar findings for calves bone by Blaxter, 1956; R.H. Smith, 1959). Thus, part of the magnesium in existing bone forms a mobilizable pool on which the body draws during hypomagnesaemi a in an attempt to maintain the plasma level. No mobilization of magnesium from the teeth occurs. Calcification occurring during magnesium deficiency still results in the incorporation of magnesium into the mineral phase, not only in teeth but also in newly formed bone (Duckworth and Godden, 1943) at a level depending on the existing plasma concentration. Magnesium does not appear to be an essentia l constituent of the mineral crystals, being probably surface bound, and its level in the solid is related to that in the supernatan t fluid. Restitution of magnesium to the diet, or injections of magnesium , causes a rapid repletion of the magnesium content of the incisor teeth, but repletion of the bone magnesium occurs more slowly (Duckworth and Godden, 1943; McAleese, Bell and Forbes, 1961), probably because of competition from calcium ions for positions on the surface of the bone crystals. The diminished level of magnesium in bone in acute magnesium deciency is associate d with an increase in the calcium concentration correspondin g to the exchange of one atom of calcium for one atom of magnesium (Duckworth and Godden, 1941; Blaxter, 1956; McAleese and Forbes, 1961). I n longer-term experiments using 40-day-old rats Watchorn and McCance (1937) induced subacute magnesium deficiency by administration of a diet containing 40 ppm magnesium . Though the ash content of the teeth and bones was not significantly affected by the deficient diet, the calcium concentration in the incisors increased while that of phosphorus decrease d relative to the control animals. The resulting increase in Ca : Ñ ratio was apparent even after allowing for the decrease d magnesium concentration. The concentration of magnesium in ash of the incisor teeth fell more (48 %) than that in the bone (36 %) of the deficient animals in comparison with the controls. On a fresh weight basis the bone lost more than the teeth; however,

R O W L ES

there appears to be some discrepanc y in the figures given in the paper since the bone magnesium concentration on a fresh weight basis is much lower relative to the ash weight concentration than in the case of the respective calcium and phosphorus concentrations . Nevertheles s the actual magnesium concentration in the incisor teeth remained higher than that in bone in the deficient animals. During the experimenta l period the incisor teeth would have been completely replaced and the concentration of magnesium represent s the rate at which it is being laid down in the teeth. The magnesium content of the teeth was reduced in the same proportion as the concentration of the element in the serum. Watchorn and McCance concluded that the teeth required a higher concentration of magnesium than bone for normal functioning. I t is possible that the relatively small reduction in bone magnesium observed by these authors in comparison with that found in acute deficiency may reflect a decrease d availability of bone magnesium in the older rats used in their experiments. Buchanan and N a k ao (1952) have observed a decrease d exchangeabilit y of bone carbonate with increasing age in rats and mice, and an analogous decrease for bone magnesium has been suggeste d by Blaxter (1956). It may be noted that in the experiments of McAleese and Forbes (1961), using weanling rats, even a diet containing 80 ppm magnesium resulted in a fall of about two-thirds in the concentration in the bone in comparison with the adequately fed controls after a 4-week period. O'Dell, Morri s and Regan (1960) observed erosion, darkening and decay of the incisor teeth of guinea pigs maintained on a diet containing 50 ppm magnesium but made no chemical analyses . The molar teeth were not affected in this way but, as observed by earlier workers (H. Klein et al, 1935), showed heavy deposits of calculus-like material. The nature of the disturbance s causing the histological changes observed in dentine formation during magnesium deficiency remains obscure. The element does not appear to be necessar y for the mineralization process, but is known to be essentia l for the functioning of several enzyme

17. C H E M I S T RY

OF

THE

MINERAL

systems, in particular for alkaline phosphatase , an enzyme which is concerned in the elaboration of the fibrous protein collagen, the principal constituent of the organic matrix of dentine. It would seem likely, therefore, that the disturbance occurs in the metabolism of the matrix in which the mineral is deposited. However, soft tissues in general appear to be littl e affected by magnesium deficiency, maintaining a normal concentration of the element (numerous observations , e.g. Watchorn and McCance, 1937; McAleese and Forbes, 1961). Whether this also applies to the matrices of hard tissues before calcification occurs, which according to Blaxter (1956) may have a high magnesium content, is not known. 4. Influence of Vitamins on the Inorganic tion of Dentine

Composi-

I n general, there is remarkably littl e information available concerning the effect of hypo- or hypervitaminosis on the inorganic composition of dentine. a. Vitamin A. Deficiency of vitamin A causes characteristic changes in the naked-eye appearanc e and histology of the rat incisor. Interference with the odontoblasts causes the formation of interglobular dentine. M . C. Smith and Lantz (1933) found a reduced ash content in incisors from rats maintained on a vitamin A deficient diet. The calcium content was higher and the phosphorus content lower on an ash weight basis, so that the Ca : Ñ ratio was greater than normal. b. Vitamin C. Ascorbic acid is not an essentia l dietary factor for certain experimenta l animals, including the rat and the dog, but is required for the normal formation of hard tissues in man and in the guinea pig. The formation of dentine in the continuously growing teeth of the guinea pig is particularly sensitive to the level of vitamin C in the blood (Kuether, Telford and Roe, 1944) and, in acute deficiency, apposition of dentine ceases . Abnormal deposition of amorphous calcified material occurs in the pulp during chronic or subscurvy, and in treated scurvy, and is considered on histological grounds to resemble secondar y dentine (Fish and Harris, 1935).

P H A SE

OF

DENTINE

227

I n chemical studies on teeth during hypovitaminosis C, Toverud (1923) claimed to have obtained a decrease d concentration of calcium, increased magnesium content, roughly equivalent to the decrease in calcium, and slightly decrease d ash content in guinea-pig incisor teeth. Nishi (1939) on the other hand reported an increase in both calcium and ash contents of incisors in similar experiments on guinea pigs. Several factors may contribute towards an explanation of these apparently contradictory observations . The effective replacemen t of dentine by amorphous deposits through continued growth and attrition could result in an increase or decreas e in the tooth mineral content according to whether the deposits are more or less highly calcified than the dentine. Similarly, replacemen t of pulp tissue by such deposits may affect the mineral content of the teeth. The amount of dentine formed during ascorbic acid deficiency is inversely related to the severity of the deficiency (Boyle, Bessey and Howe, 1940) but enamel formation is not so affected. Hence, the enamekden tine ratio increases with the severity of the deficiency and, by virtue of the greater degree of mineralization in the enamel relative to the dentine, the mineral content may also be expected to increase, that is, on a dry weight basis, where the increased amount of pulp tissue has a negligible effect on the concentration. c. Vitamin D. The only chemical studies which have been reported in connection with vitamin D and tooth formation were made on the rat. A s Irvin g (1957) has pointed out, this is unfortunate since the requirement for vitamin D is rather different in the rat than in m an and most other species. For the rat, vitamin D appears to be unnecessar y unless the dietary levels of calcium and phosphorus are below about 0.3 %. Boyle and Wesson (1943) compared the calcium and ash contents of incisor and molar teeth with those of bone from rats maintained on calciumdeficient, high-protein, or high-carbohydrat e diets, wit h or without vitamin D supplement . Incisor calcification increased on both high protein and high carbohydrate diets when supplemente d with vitamin D, but the mineral content of bone was only

228

S.

L.

increased on the high carbohydrate diet. The molar teeth were not significantly affected although these workers observed that any secondar y dentine formed was not calcified when the vitamin D supplement was lacking. Sobel and H a n ok (1948) found that the inclusion of vitamin D in diets of widely varying Ca : Ñ ratio had littl e effect on the calcium and phosphorus contents of the incisor dentine. Their figures suggest, in accord with the findings of Boyle and Wesson, that calcification was slightly increased wit h vitamin D supplementatio n on the lowcalcium high-phosphat e diet. The inclusion of vitamin D in the diet did, however, produce an alteration in the factor correlating the phosphate : carbonate ratio of serum with the same ratio for dentine and enamel, apparently making the composition of the tooth minerals more sensitive to changes in the serum ratio. The effect of vitamin D deficiency on the distribution of mineral in the dentine of the teeth of young dogs was investigated by Engfeldt and Hammarlund-Essle r (1956) using microradiography and autoradiography . The characteristic globular mineral deposits produced on the deficient diet appeare d to be normally mineralized, but evenly hypomineralized areas were evident in some teeth. A n uneven calcification was also observed at the enamel-dentin e junction. In contrast to normal teeth, in which radiocalcium was taken up primarily in a narrow band at the pulpal edge of the dentine, the isotope was taken up throughout the defective dentine of the teeth from the vitamin D-deficient animals. d. Vitamin E. This vitamin appears to play no part in the calcification process, and deficiency causes no alteration in the calcium and phosphorus contents of the teeth (Dam et ah, 1950; Irving, 1957). According to D am et al. the vitamin plays an important part in the maintenanc e of normal concentrations of some metals in the teeth. These workers observed a marked decreas e in the iron content of both dentine and enamel from the upper incisor teeth of rats maintained on a diet deficient in vitamin E, while the manganes e

R O W L ES

content of these tissues was substantially increased . Moore and Mitchell (1955) in similar experiments could confirm only the reduction in enamel iron content, which probably related to the depigmentation of the enamel surface occurring in the deficient animals. 5. Effect of Hormones on the Inorganic of Dentine

Composition

The absence , or presence in excess, of the secretions of most endocrine glands has a disturbing effect on the formation and calcification of dentine (for reviews see Leicester, 1949; Irving, 1957; Jenkins, 1966). Possible changes in the composition of the mineral associate d with the disturbance s in calcification do not appear to have been investigated, although Livrea, De Stefano and Picciotto (1958) could find no effect on the calcium and phosphorus contents of whole incisor teeth from rats with deficiency or excess of sex hormones. C.

SPECIAL TOPICS

1. Fluorosis Symptoms of fluorosis are observed in human teeth even at levels of intake of a few parts fluorine per millio n in the drinking water. The fluorine concentrations in dentine in general parallel the level of intake. In districts where the water fluorine content is high, as in regions of endemic fluorosis, the dentine fluorine concentration may reach 0.2 % (McClure and Likins, 1951). Fluorosis has also been reported among workers in industries using fluorine compounds and is characterize d by considerable skeletal accumulation of fluorine. Roholm (1937) examined the teeth and other tissues of persons who had worked for periods up to 10 years in a factory using cryolite (sodium aluminium fluoride). The ash of dentine separate d by drillin g from a mixed batch of teeth from one worker was found to contain 0.083 % F whil e the correspondin g enamel contained only 0.029 %. However, much greater fluorine concentrations were observed in analyses of whole teeth from other workers. The ash of molar teeth

17. C H E M I S T R Y

OF

THE

MINERAL

contained up to 0.53 % F, and of the incisor teeth up to 0.25 % F. In view of the age of the workers examined, the uptake of fluorine must have occurred in the teeth post-formatively, and by analogy with the observations of Y o on et al. (1960a, b) the concentration s of fluorine in the dentine at the pulpal surface might be expected to have been considerably greater than those reported for the whole teeth. I n grazing animals fluorosis occurs where the pasture is contaminated from the presence of rock phosphate , which may contain up to 3.5 % F, in the soil or by the effluent fumes from industrial processe s involving fluorine. A concentration of 0.76 % F was found in the dentine of teeth from an 8-year-old cow reared on pasture containing up to 61 ppm F due to the proximity of an aluminium factory using cryolite (Boddie, 1949). The corresponding enamel contained 0.48 % F while the mandibular bone had an even higher concentration of 0.92 % F on a dry weight basis. In sheep similarly exposed, mandibular bone concentration s as great as 1.7 % F were observed. Similarly high concentration s of fluorine in skeletal tissues have been reported in laboratory animals fed experimentally diets containing sodium fluoride or other fluorine compounds. Roholm (1937) found 0.78 % F in the dentine ash from deciduous molar teeth of calves after administering a diet containing 20 mg F as N aF per kilogram body weight per day for 7 months from birth. The teeth were considered to have been full y formed at the start of the experiment. In contrast, the enamel contained only 0.051 % F, but the bone ash contained up to 1.93 % F. Roholm also found concentrations of up to 0.74 % F in the molar teeth of pigs fed for 6 months with a diet supplemente d wit h sodium fluoride, and of 0.6 % F in molar teeth from dogs maintained for 19-21 months on diets containing fluorine compounds. In the rabbit Weidmann (1962) obtained a concentration of 0.75 % F in the molar root dentine after feeding sodium fluoride at 500 ppm F in the diet for 98 days. I n these experiments the enamel of the continuously growing teeth contained 0.66 % F.

PHASE

OF

DENTINE

229

I t has been suppose d from histological observations that calcium fluoride is deposited in dentine during fluorosis (e.g. Irving and Neinaber, 1946). The only definite chemical evidence in support of this conjecture is the report of Reynolds et al. (1938) in which C a F2 is stated to have been detected by X-ray diffraction examination of incisor dentine from rats maintained on a diet containing sodium fluoride. The identification, however, is based essentially on one line in the diffraction pattern, with d = 3.36 A, which, in view of the limited resolution of the technique employed, seems more assignable to the apatite phase. It may be noted that the standard spacings for C a F2 quoted by these authors are in distinct disagreemen t with those given in the A.S.T.M. index for this compound in which the strong, and only, line in this region has d = 3.15 Â (Swanson and Tatge, 1953). The observed variation in intensity of the line at 3.36 Â may have been due to the orientation effects which Reynolds et al. noted in rat incisor dentine but which does not occur in human dentine. Lindemann (1956) using an X-ray diffraction technique capable of detecting as littl e as 0.5 % C a F2 was unable to detect this phase in fluorosed rat bones or teeth. Complete replacemen t of the hydroxyl ions in the apatite lattice by fluoride, producing fluorapatite, gives a fluorine content of 3.77 % which seems unlikely to be exceede d even in the pulpal regions of fluorotic dentine. Roholm (1937) obtained a concentration of 3 . 1 2% F in the ashed ribs of fluorosed dogs, while the highest concentration reported, also in fluorosed dogs, 3.69 % F for bone ash (Brandi and Tappeiner, 1891), is still within the limit s of the fluorapatite composition. I n a recent study of teeth from residents of parts of Sweden where the drinking water contains moderate amounts of fluoride (1.6-2.0 ppm) Zelander and Torell (1964) observed by electron microscopy several non-apatitic crystal types. I n an exposed dentine surface layer, cubic crystals of K C a F3 were identified by electron diffraction. Thin plate-like crystals giving a hexagona l diffrac-

230

S. L. ROWLES

tion pattern were believed to consist of an ironcontaining phosphate . C a F2 was not observed. 2. Heavy Metal

Poisoning

A number of heavy metals which are toxic to the animal body have been found to accumulate in the skeletal tissues. Few analyses of the teeth in such cases have been reported. Most of the available data concern lead poisoning. Pfrieme (1934) reported abnormally high concentration s of lead in the teeth and bones in cases of lead poisoning. Altshuller et al. (1962) found ten times the normal lead concentration in whole teeth from acute fatal cases of lead poisoning and seven times the normal concentration in the teeth of patients surviving the intoxication. The fatal cases showed an average content of 0.016 % Pb in whole deciduous teeth. Campbell et al. (1950) have reported a tendency for increased uptake of lead in the teeth in cases of disseminate d sclerosis. They found whole tooth concentrations of up to 0.02 % in chronic lead poisoning.

V. INORGANIC COMPOSITION OF DENTINE IN DISEASED AND OTHER ABNORMAL STATES A.

D E N T AL

CARIES

The inorganic composition of the unaffected portion of dentine from teeth showing carious lesions has been compared with that of dentine from sound teeth by several investigators. Their results, which are quoted in Table 1, indicate that no significant differences in the concentration s of calcium and phosphorus exist between normal and carious teeth. A n increased concentration of magnesium in unaffected dentine from carious teeth was found by Murray and Bowes (1936), particularly in the root dentine. A similar, but not significant, trend was found by Tefft, French and Hodge (1941) while Cremer et al. (1953) found a slight increase in the magnesium content of whole teeth from rats reared on a caries-producin g diet. The data of Ockerse (1943), however, show no

difference in the magnesium content of dentine from normal and carious teeth. Murray and Bowes (1936) also reported the presence of a small quantity, 0.04 %, of chlorine in carious dentine, whereas this element could not be demonstrate d in normal dentine. I n view of the improved caries resistance of teeth formed in the presence of low fluorine concentrations , attempts have been made to demonstrate a difference in the fluorine concentrations of sound and carious teeth. Armstrong and Brekhus (1937) could find no difference between the fluorine content of unaffected dentine from carious teeth and that of dentine from sound teeth. A similar result was obtained by McClure (1948) using large numbers of teeth. This worker pointed out the difficult y of attempting to compare the fluorine content of sound and carious teeth taken from different persons owing to the possibility that individual differences in the degree of exposure to this element might have caused differences in the uptake of fluorine. However, using teeth of the same type from the same individual, he was still unable to find any significant difference in the fluorine content of sound and carious teeth. Whether the fluoride concentration at the time of mineralization affects the size of the crystals, as has been shown for bone by Posner et al. (1963), remains to be demonstrated . I n contrast to the composition of the unaffected dentine of carious teeth, the composition of the carious dentine itself shows marked changes . Manly and Deakins (1940) concluded that the net effect of the carious process was the removal of inorganic material and its replacemen t by water. The studies of Johanse n and his colleagues (Johansen , 1962; Nordback and Johansen , 1962), however, have shown that, while littl e change in the calcium and phosphorus contents and Ca : Ñ ratio can be detected in the ashed tissue, notable changes in the concentration s of other mineral constituents occurred. The magnesium content of the mineral from carious regions of the dentine was reduced to less than one-quarte r of that of the mineral from sound dentine of the same teeth, and

17.

C H E M I S T RY

OF

THE

MINERAL

the carbonate content was reduced to about half that of the sound dentine. Araiche, Nordback and Johanse n (1962) further observed that, while the 100°-300°C labile carbonate was reduced in carious dentine, the residual carbonate after ignition at 600°C was not significantly affected. A considerable increase in the fluorine content of the mineral of the carious dentine was observed (Johanse n and Nordback, 1962), presumably a result of the smaller solubility of the crystals containing fluoride. Whereas the ash of the sound dentine from carious teeth contained 60-500 p pm F, the ash of the carious material contained 700-4000 p pm F. B. SECONDARY DENTINE

Secondary dentine may be formed as a normal ageing process or in response to external stimuli such as attrition or caries. Few chemical data regarding its composition have been reported. Most observations have been made by radiographic techniques demonstratin g only the degree of mineralization. Thewlis (1940) found that secondar y dentine was similarly or less mineralized than primary dentine and that the latter was separate d from the secondar y dentine by a thin hypercalcified circumpulpar layer. Secondar y dentine formed in response to acute carious attack may contain so littl e mineral that it is undetectabl e with conventional radiographic techniques (Kruger and Rakuttis, 1952) but in chronic caries Amprino and Camanni (1956) found 6 - 1 0% greater mineralization in the secondar y dentine than in the dentine proper. Posteruptive uptake of several trace constituents occurs through deposition in the secondar y dentine. This has been demonstrate d in the cases of fluorine (Jenkins and Speirs, 1954b; Yoon et al, 1960b), strontium (Steadma n et al., 1958), zinc (Brudevold et al, 1963) and lead (Brudevold et al., 1960). C. ALTERED DENTINE

I n addition to the formation of secondar y dentine, external stimuli can also evoke changes in the primary dentine on the pulpal side of the region

P H A SE

OF

DENTINE

231

in which the stimulus occurs. The nature of the changes depends on the severity of the stimulus and is also affected to some extent by the age of the tooth. In response to an acute stimulus the odontoblast process in the dentinal tubules disintegrates (Fish, 1932) or may possibly be withdrawn towards the odontoblast by an amoeboid movement (Bradford, 1960). The resultant "dead tract" dentine is characterize d by its opaque appearanc e when viewed by transmitted light in ground section. Less severe stimuli cause sclerosis of the dentine with the formation of transparen t (translucent) zones due to the occlusion of the dentinal tubules with material having the same refractive index as the calcified dentinal matrix. A similar process also occurs as a result of the natural ageing changes of the teeth. 1. Dead Tract

Dentine

Ellison and Halpert (1947) compared the chemical composition of dentine from the opaque zone with that of normal dentine from the same teeth by the use of microdissecte d samples which were pooled for analysis. They found an increased ash content i n the opaque zones but, surprisingly, a decrease d content of both calcium and phosphorus . Their conclusion that a mineral containing neither calcium or phosphorus was deposited in these regions has not been substantiated . Miles (1961) has suggeste d that the increased ash content in dead tract dentine is simply the result of the absence of the odontoblast process with consequen t reduction in organic content of the tissue. While this factor may contribute to the increased ash content on a weight basis it does not seem to be capable of explaining the magnitude of the increase observed by Ellison and Halpert, which averaged 3.4 % on a dry weight basis (maximum 12 % ). Assuming complete loss of the organic content of the tubular material, that the water content of this material is 90 % and that the volume of the dentine occupied by tubules is 1 4% (Manly and Brooks, 1947) then the expected increase in ash content would be only about 1.5 %.

S.

232

2. Sclerotic

L.

Dentine

The increased mineral content of sclerotic dentine has been demonstrate d radiographically (e.g. Warren et á/., 1934; Amprino and Camanni, 1956). Nalbandian et al. (1960) observed deposition of apatite crystals in the lumen of dentinal tubules i n the root region of adult teeth by electron microscopy. Since this region was considered less liable to irritation from external stimuli, the deposition was attributed to an ageing process. Nalbandian (1962) observed a similar intratubular deposit in crown dentine and in this case concluded that it was a response to caries or erosion, or an effect of ageing. A n attempt to detect chemically a difference in composition of dentine between unworn teeth and teeth showing severe attrition was made by Bird et al. (1948b), who compared the dentine composition of teeth from a mouth showing unilateral wear. No significant differences were found between the dentine samples obtained from opposite sides of the mouth. Simon and Armstrong (1941) compared the ash content of samples from normal root dentine (middle third) with that of translucent root dentine (apical third) from the same teeth. They found a higher ash content in the normal root dentine of adult teeth but found a similar reduced ash content in the apical third root dentine of teeth from young persons where the translucenc y was only slight. There was a slight reduction (0.3 %) wit h age in the apical third region samples while the ash content of the normal dentine was not significantly different in the two age groups. While these workers considered that the reduced ash content of the translucent zone indicated decalcification, Manly and Brooks (1947), who found a similar reduction (0.4 %) in the ash content of translucent crown dentine, pointed out that replacemen t of the tubule material by calcified matrix would only produce a significant increase in mineral concentration on a volume basis, as in radiographic examination, but not on a weight basis. Assuming that the material deposited in the tubules had the same composition as cementum,

R O W L ES

they calculated that the expected change in ash content on a weight basis would be a reduction similar to that observed. The assumption that the tubular deposits have a lower degree of mineralization than the dentine itself may be questioned in view of the high degree of mineralization indicated for the deposits formed both as a result of ageing (Rockert, 1956b; Nalbandian et al, 1960) and in response to caries (Bergman and Engfeldt, 1955; Wyckoff and Croissant, 1963). Frank (1955) observed large polyangular crystals in the transparen t dentine under a carious lesion. These "caries crystals" were also described by Helmcke (1955), Lenz (1955), Torell (1958) and Hohling (1960). By electron diffraction the crystals have been identified as whitlockite (/8-Ca3(P04) 2) (Hôhling and Newesely, 1961; Vahl, Hohling and Frank, 1964). D . Jackson (1956) had previously detected by X-ray diffraction the presence of whitlockite in areas of "metamorphosed " dentine in teeth showing arrested dental caries. The precipitation of whitlockite in these regions may be related to the relatively high concentration of magnesium in the solution phase resulting from the preferential solution of magnesium ions during the carious process. Such a preferential solution is indicated by the reduced level of magnesium found in carious dentine (Johansen , 1962). The presence of magnesium ions stabilizes the whitlockite lattice (normally a high-temperatur e form of tricalcium phosphate ) sufficiently for it to exist at physiological temperature s (Trautz, 1955; Jensen and Rowles, 1957). Keil (1955, 1958), from observations with the polarizing microscope, claimed to have detected calcite crystals in the dentinal tubules of transparen t zones under caries. D . HEREDITARY OPALESCENT DENTINE

The chemical analyses of Hodge (Hodge, 1936; Skillen, 1937; Hodge and Finn, 1938; Hodge et al, 1940) demonstrate d that the calcium, phosphorus and carbonate concentration s of the mineral in hereditary opalescen t dentine were normal although the tissue was relatively poorly mineralized and

17. C H E M I S T RY

OF

THE

MINERAL

contained more water than normal dentine. The low mineral content in such tissue has been confirmed by McCauley (1942) and Bergman, Engfeldt and Sundvall-Haglan d (1956). E. PERIODONTAL DISEASE

Murray and Bowes (1936) found no differences in the calcium, phosphorus , magnesium or carbon dioxide contents between normal crown dentine and similar material from mouths showing periodontal disease (pyorrhea alveolaris). In contrast to normal dentine, however, the tissue from disease d mouths retained after washing with water 0.025 % of chloride ion. In the root dentine a slightly increased calcium concentration compared wit h normal dentine was observed while the magnesium concentration was twice the level of normal dentine. Kaushansky (1932) reported increased magnesium levels in both crown and roots of whole teeth in periodontal disease . Sharaevskay a (1958) has also reported modifications of the magnesium content of dentine in periodontal disease . Selvig and Zander (1962) compared the mineral content of normal root dentine and root dentine affected by periodontal disease by microradiography and by chemical analyses on microdissecte d samples. They observed a slight increase in calcium + magnesium content, but not in phosphorus content, principally in the outer layer of the root. This was also found in cases where the dentine was exposed; in those cases some subsurface decalcification was also observed microradiographically. Reduction in the levels of copper (Nezhivenko, 1961) and of nickel (Vikhm, 1962) in whole teeth from mouths with periodontal disease have been reported although their significance with regard to dentine is unknown.

P H A SE

OF

DENTINE

233

stimulated under cavity linings containing calcium hydroxide (Sowden, 1956). A.I . Klein (1961) also observed the increased mineralization under a calcium hydroxide-methylcellulos e base material but considered it to be a sclerosis rather than recalcification of existing carious dentine. Eda (1961) studied the reaction of the odontoblasts to calcium hydroxide, magnesium oxide, "triozinc" and calcium fluoride pastes and observed the deposition of mineral in which calcium, magnesium and phosphorus could be detected histochemically. The discoloration of dentine under amalgam filling s was studied by Massler and Barber (1953) who confirmed an earlier observation of Schoonover and Sonder (1941)—namely that small amounts of zinc, silver, tin and copper were present i n the discoloured areas. Massler and Barber, however, also found relatively large (0.5-5 %) quantities of mercury. A similar discoloration was produced by applying a voltage between the pulp and an amalgam fillin g in sound dentine after immersion in ammonium sulphide solution. Nixon (1961), using an amalgam fillin g material containing H g 2 0 ,3 could find mercury only within 1 mm of the cavity. Soremark et al. (1962) made a thorough study of the effect of various dental restorative materials on the composition of the dentine both in vitro and in vivo. Their results showed that the elements present in the restoration and clasp materials were in higher concentration s in the treated dentine than in normal dentine. Platinum and mercury were found in the treated dentine but not in normal dentine.

VI. NATURE OF THE MINERAL P H A S E IN DENTINE Since this aspect of dentine mineral is discusse d full y in Chapter 16 only a brief review wil l be undertaken here.

F . DENTINE UNDER F I L L I N G MATERIAL S

A.

The rehardening of carious dentine in open carious lesions ( D . Jackson, 1956; Kothe, 1960) is

Before considering the nature of the mineral phase it is necessar y to consider whether the

HOMOGENEITY OF THE INORGANIC FRACTION

234

S.

L.

analytical figures actually represen t the composition of the mineral fraction. The postulated existence of a flui d fraction in dentine, the so-called dental lymph, appears to have received some confirmation from the studies of von Kreudenstein and Stuben (1955), and this flui d presumably has some inorganic content. A number of workers have assumed that the inorganic ions present in aqueous extracts of dentine are parts of the dental lymph (Karshan, Weiner and Stofsky, 1934; Bowes and Murray, 1936; Tokunaga, 1960). While this assumption is scarcely justified, since ions adsorbed on the crystallite surfaces would be similarly leached out, the removal of these ions, under conditions where extensive recrystallization seems unlikely, does indicate that they are not part of the main mineral phase. This fraction includes much of the sodium content, virtually all the chlorine, part of the magnesium but very littl e of the carbonate . The amounts of calcium and phosphate are relatively small. The mobility of sodium in dentine has also been demonstrate d by the isotopic experiments (unpublished, quoted in Sognnaes , 1962) of R. Bogoroch, J.H. Shaw, R . F. Sognnaes , and P. Yen, who showed that the level of sodium in monkey and rat dentine could be depleted by a low-sodium diet. Tokunaga (1960) has identified pyrophoshate in extracts of dentine. Herman and Dallemagne (1961) found 0.57 % pyrosphosphat e in whole dentine dried at 105°C. They concluded that this pyrophosphate is associate d with the organic fraction. Other inorganic radicals are also present in the organic fraction of dentine, sulphate in chondroitin sulphate, phosphate in phospholipid. The behaviour of these non-mineral phase inorganic compounds would vary according to the pre-analytical treatment and the effect of some ashing procedures on them is uncertain. The available evidence suggests that relatively littl e of the calcium and carbonate are associated with the non-mineral phase but further studies on the amount of phosphate in the fraction would appear desirable. It seems unlikely, however, that the amount of phosphorus so bound would significantly affect the mineral calciumiphosphoru s ratio. Recent experiments by Herman (1964) using

R O W L ES

synthetic "tricalcium p h o s p h a t e " gelatin mixture suggest that at least part of the pyrophosphat e found in dentine may be formed artifactually during the drying process at 105°C. Hoppe (1965) has determined the extracellular water content of dentine in vivo in dogs. He found that one quarter of the total water content was present as an extracellular phase. The extracellular liqui d represente d 3-4 % of the dentine tissue. B. NUMBER OF INORGANIC PHASES PRESENT

The analytical data for dentine can give no direct information as to the nature of the mineral phase but must be considered in conjunction with the evidence of physical methods of analysis. The methods of X-ray diffraction and electron diffraction applied to normal dentine have revealed the presence of only one crystalline phase—a structure resembling that of hydroxyapatite which has the formula C a1 0( P O4) 6( O H ) 2 (X-ray diffraction: Cape and Kitchin 1930; Roseberry, Hastings and Morse 1931; Clark, 1931; Bredig, 1933; Gruner, McConnell and Armstrong, 1937; Bale, 1940; Sobel et al, 1949; Bergman and Engfeldt, 1954; Trautz, 1955; Torell, 1958; Tonogai, Minami and Sakurada, 1962. Electron diffraction: Frank, 1959; Nalbandian et al., 1960; Takuma, 1960a). This structure has been found both for human dentine, where the Ca : Ñ ratio is fairly close to the theoretical figure of 1.67 for hydroxyapatite, and for the rat incisor (Sobel et al., 1949), where the ratio may be as small as 1.2. The limited sensitivity of diffraction methods should, however, be emphasized, particularly where poorly crystallized materials are concerned . Further, these methods cannot detect amorphous (noncrystalline) substances . The presence of another phase or phases , either below the detectable limi t or as an amorphous phase, has been suggeste d by some workers (e.g. Thewlis, Glock and Murray, 1939). Posner and Duyckaerts (1954) claimed to have detected the presence of calcium carbonate and magnesium carbonate phases from the infrared absorption curve but the detailed shape of the curve (Emerson and Fischer, 1962) has not confirmed this identifi-

17.

C H E M I S T RY

OF

THE

MINERAL

cation. The fact that weak-acid treatment wil l preferentially remove carbonate from dentine (Cartier, 1948; Posner, 1954) has also been used to support the existence of a second phase. Buchanan and N a k ao (1958), however, consider that the differential solution of carbonate may be explained in terms of solution and reprecipitation of a carbonate-fre e phase. y in dentine Demonstration of heterogeneit mineral has been made by Herman and Dallemagne (1961), who showed that upon ignition at temperatures up to 600°C formation of pyrophosphat e occurred in excess of that expected from the overall Ca : Ñ ratio and by determinations based on synthetic apatitic calcium phosphate s having various Ca : Ñ ratios. A t higher temperature s further reaction occurred with the disappearanc e of pyrophosphat e and, in accordanc e with the initial Ca : Ñ ratio, formation of an apatitewhitlockite mixture. The dentine mineral behaved similarly to mixtures of synthetic "tricalcium phosphate " and calcium carbonate except that the maximum yield of pyrosphosphat e was obtained at a much lower temperature (325°C) with the dentine. Herman and Dallemagne concluded that the two mineral phases were kept apart by the organic matrix and could react together when this was destroyed either by heating or with potassium hydroxide-glycerol. C. T H E CALCIU M : PHOSPHORUS R A T I O

The theoretical Ca : Ñ ratio for hydroxyapatite is 1.67. The ratio for dentine mineral may be slightly higher or much lower. This variation has been explained (a) in terms of mixtures of two or more phases (e.g. Dallemagne and Melon, 1946; Cartier, 1948; Sobel, 1955), (b) adsorption of ions on the surface of apatite crystallites (Neuman and Neuman, 1953), (c) the existence of solid solutions of various types (Eisenberger , Lehrman and Turner, 1940; McConnell, 1952; Trautz, 1955), or the occurrence of "defect" apatite lattices (Posner, Fabry and Dallemagne, 1954; Posner and Perloff, 1957; Winand, 1960; Winand, Dallemagne and Duyckaerts, 1961).

P H A SE

OF

DENTINE

2 35

The presence of a substantia l excess of ions on the surface of the crystallites is not in accord with the isotopic exchange experiments of Francois (1958). Assumption of a separate carbonate phase is sufficient to explain Ca : Ñ ratios greater than that of hydroxyapatite; the explanation of ratios lower than 1.67 is difficult unless the presence of a calcium phosphate less basic than apatite is assumed . The existence of a tricalcium phosphate phase of similar structure to the apatite lattice but constituting a distinct phase (Dallemagne and Melon, 1946; Hendricks and Hill , 1950) has not been satisfactorily established , but the presence of secondar y phosphate ions(HPC>4) in dentine has been demonstrate d by the formation of pyrophosphat e upon ignition at intermediate temperature s (Herman and Dallemagne, 1961, Dallemagne, 1964). These observations support the existence of a defect apatitic structure in which calcium ions are missing from the lattice, the charge imbalance being made up by the presence of secondar y phosphate ions. The limit s of composition of such an apatitic structure formed under physiological conditions are not known. Trautz (1955) was unable to prepare apatitic calcium phosphate s with Ca : Ñ ratio lower than 1.33 and a similar limi t is given by Winand et al. (1961). This type of structure would therefore appear adequate to account for the variation in Ca : Ñ ratios encountere d in h u m an dentine. Whether it can also account for the remarkably low ratio reported for some samples of rat incisor dentine has yet to be demonstrated . D.

STATE OF THE M I N O R CONSTITUENTS

1.

Carbonate

By infrared absorption studies, Underwood, Toribara and N e u m an (1955) claimed to have shown that the carbonate in calcified tissues was present as carbonate ion, not as bicarbonate ion as might reasonabl y have been expected at physiological p H. The detailed absorption curves of Emerson and Fischer (1962) show that the carbonate ion exists in two different types of environment, and the similarity of the curves to those of enamel may indicate that some of the

236

S.

L.

carbonate in dentine can substitute for hydroxyl ions inside the apatite lattice, as has been demonstrated in the case of enamel by J.C. Elliott (1963). Earlier workers had concluded that such a substitution was not feasible on steric grounds (Gruner and McConnell, 1937; Thewlis et al, 1939), while McConneli (1952, 1960) and Trautz (1960) considered that carbonate ions might substitute for phosphate ions in the lattice. The isotopic exchange experiments of Francois (1958) show that there is very littl e adsorbed carbonate on accessible surfaces. The experiments of Herman and Dallemagne (1961) suggest that some carbonate may be present as calcium, or magnesium , carbonate . Trautz (1955, 1960) has suggeste d that carbonate as an ion rather than as calcium carbonate may be coprecipitated with the calcium phosphate , and is included in the lattice as an anomalous substituent which, owing to its size, is a source of strain in the lattice. F r om detailed studies of synthetic and natural apatites(Dallemagne , 1964; Herman, 1964; Herman and Dallemagne, 1964), the Belgian workers maintain that the infra-red absorption curves of calcified tissues indicate the presence of calcite as a separate phase. While confirming the existence of carbonate ions in two types of environment they conclude that these are a separate calcite phase and a surface replacemen t of phosphate ions in the micro-crystals of the apatite phase. J. C. Elliott (1964) considers that the similarity of the infra-red absorption curves for enamel and dentine indicates a similarity of the carbonate environments in the two tissues. In enamel he concludes that about 15 % of the carbonate ions replace hydroxyl groups inside the apatite lattice, the remainder being largely adsorbed on the crystal surfaces. However, he points out that in the case of dentine the current observations do not rule out the possibility of the presence of an amorphous carbonate phase. 2.

Magnesium

There is some indication from the inverse relationship of calcium and magnesium concentra-

R O W L ES

tion in dentine of various mammalian species that magnesium can replace calcium in the mineral structure (Murray, 1936). It has often been assumed that the magnesium ion can substitute for calcium in the apatite lattice (Thewlis et al, 1939; Posner, 1960). There is, however, littl e evidence for this (Trautz, 1955; Jensen and Rowles, 1957). Hendricks and Hil l (1950) suppose d that magnesium is adsorbed on the crystal surface as the MgOH+ ion. Possibly it is coprecipitated with the apatite in the same way that Trautz (1955, 1960) has suggeste d for carbonate . 3. Fluorine Chemical studies with powdered dentine (McCann and Bullock, 1955; Leach, 1959) have demonstrate d that fluoride ions react with dentine in vitro to form fluorapatite by exchange with the hydroxyl ion. Since this reaction occurs at concentrations likely to exist under physiological conditions, it seems reasonabl e to suppose that a similar process occurs in vivo. By virtue of the greater stability of fluorapatite relative to hydroxyapatite, concentration of fluoride occurs during precipitation and recrystallization of dentine mineral, particularly during slow deposition as in secondary dentine. 4.

Strontium

Strontium is coprecipitated with hydroxyapatite in vitro and has been shown to form a continuous series of solid solutions. The composition of these ranges between that of calcium hydroxyapatite and that of strontium hydroxyapatite (Collin, 1959, 1960), in which calcium and strontium occupy similar lattice positions. In dentine the radioisotopic experiments of Likin s et al. (1959) have shown that calcium is incorporated in preference to strontium. Discrimination occurs during the equilibration of the initiall y formed crystals with the tissue fluid, resulting in partial elimination of strontium ions. Consequently , postformative uptake occurs only at sites of new dentine formation. A similar discrimination against radium during recrystallization in dentine was observed by

17. C H E M I S T RY

OF

THE

MINERAL

Stover, Atherton and Arnold (1957) in the teeth of dogs.

VII. SUMMARY Chemical examination of dentine mineral usually entails separation of the dentine from associate d tissues and removal of the organic and tissue fluid fractions. Separation from associate d tissues has been achieved in general by mechanica l grinding, or microdissection , or by differential flotation of the powdered dental tissues. The flotation method is to be preferred where recovery of all of the dentine is required and is particularly convenient for use with the teeth of small laboratory animals. Separation of the mineral phase has been effected by a variety of methods for destructive removal of the organic fraction. N o ne of the ashing procedures appear to yield a mineral residue without some structural or compositional change or some loss of material. The behaviour of inorganic components associate d with the organic fraction or present in extracellular fluid during such procedures is often unknown. Published data on the inorganic composition of human dentine include analyses made both on separate d mineral fractions and on whole dentine. The most generally applicable and satisfactory basis for comparison of analytical data appears to be in terms of dry, whole dentine. Mature dentine contains approximately 75 % inorganic material, of which the mineral phase constitutes the greater part. The inorganic material is composed principally of calcium phosphate with smaller amounts of carbonate, magnesium , sodium and chlorine and trace amounts of a large number of other elements. A small part of the inorganic material, including much of the sodium and chlorine, and some of the magnesium, is relatively loosely bound and may be associate d with the organic or tissue fluid fractions. The inorganic composition of dentine varies among individual human teeth, partly as a result of differences in the degrees of mineralization, but also because of a definite variation in the Ca : Ñ i7

P H A SE

OF

DENTINE

237

ratio. Values for the mean composition for dentine found by different investigators also show some variation but the significance of this is uncertain in view of the differences in materials and methods employed. Direct comparison, in individual investigations, has shown no significant differences in the mean calcium and phosphorus contents of dentine according to type or condition of tooth, or with the sex of the subject. Sclerotic age changes in the dentine observed by histological methods are not paralleled by significant changes in the gross inorganic composition although localized increases i n mineralization may occur. Some recent analyses have indicated differences in the inorganic composition of dentine in teeth from persons of different racial origin. Certain ions, particularly those with chemical affinity for the apatite lattice or its component ions, may be incorporated in the mineral through coprecipitation or adsorption. The levels of these are related to the concentration in the blood and hence to the dietary levels. Ions, such as fluoride and lead, which react with the apatite lattice to form stable (insoluble) compounds tend to accumulate with age, although the greater part of the posteruptive uptake occurs in the secondar y dentine. Few analyses of dentine from the teeth of other mammals, apart from the rat and the hamster, have been reported. I n general the calcium and phosphorus contents of dentine from n o n h u m an teeth of limited growth are similar to those of human dentine, while the magnesium and carbonate contents vary between the various species. The dentine of continuously growing rodent teeth, however, shows a remarkably low Ca : Ñ ratio, and markedly different magnesium and carbonate contents from the dentine of teeth of limited growth. Although the sensitivity of dentine formation to nutritional changes has frequently been demonstrated histologically, the effect of such changes on the inorganic composition of dentine has been investigated in relatively few cases, virtually limited to the rat. The mineralization of dentine is relatively insensitive, in comparison with bone, to low levels

238

S. L .

of calcium or phosphorus , or both, in the diet. The carbonate content of the dentine formed during mineral deficiency, however, varies with the Ca : Ñ ratio and thus reflects alterations in the composition of the blood. Dentine mineral consists principally of calcium phosphate in the crystal form of an apatite. No other crystalline phase has been demonstrate d in normal dentine, but there is some evidence for a heterogeneou s composition of the mineral, particularly in regard to the carbonate . The observed Ca : Ñ ratio variations in human dentine can be reconciled with the apatite structure, but explanation of the remarkably low ratios reported for dentine from continuously growing rodent teeth on this basis is less satisfactory.

References Akabori, S., Ohno, K. and Narita, K. (1952). Hydrazinolysis of proteins and peptides: method for the characterizatio n of carboxyl terminal amino acids in proteins. Bull. chem. Soc. Japan. 25, 214-218. Alkalaev, Ê. K. (1959). Effect of type of nutrition on fluoride concentration in teeth. Sb. trudov. Stomat. Fak. Posvyashch. 40-Cetiyu Irkutsk med. Inst. 1919-1959 pp. 51-59; see Chem. Abstr. 56, 2749e (1962). Altshuller, L. F., Halak, D. B., Landing, Â. H. and Kehoe, R. A. (1962). Deciduous teeth as index of body burden of lead. / . Pediat. 60, 224-229. Amprino, R. and Camanni, F. (1956). Historadiographic and autoradiographi c researche s on hard dental tissues. Acta anat. 28, 217-258. Araiche, M., Nordback, L. G. and Johansen , E. (1962). The chemistry of carious lesions II . The carbonate content of carious and sound human dentin as affected by heat treatment. Preprint. Abstr. Intern. Ass. dent. Res. 40th gen. Meet., St. Louis, 1962 No. 142, p. 39. Armstrong, W. D. and Brekhus, P. J. (1937). Chemical constitution of enamel and dentin. I. Principal components . /. biol. Chem. 120, 677-687. Asgar, K. (1956). Chemical analysis of human teeth. / . dent. Res. 35, 742-748. Atkinson, H. F. and Harcourt, J. K. (1961). Peritubular translucent zones in human dentine. Aust. dent. J. 6. 194-197.

R O W L ES Bale, W. F. (1940). A comparative Roentgen-ra y diffraction study of several natural apatites and the apatite-like constituent of bone and tooth substances . Amer. J. Roentgenol. 43, 735-747. Bale, W. F., LeFevre, M. L. and Hodge, H. C. (1936). Uber den anorganische n Auf bau der Z hne . Naturwissenschaften 40, 636-637. Bang, G. (1961). Developmenta l microstructure and fluorine content of Alaskan Eskimo tooth samples. / . Amer, dent. Ass. 63, 67-75. Beaulieu, M. M., Dallemagne, M. J., Brasseur, H. and Melon, J. (1950). Critique des méthodes de minéralisation de l'os. Arch. int. Physiol. 57, 411-418. Becks, H. and Furuta, W. J. (1942). The effects of magnesium deficient diets on oral and dental structures. III . Changes in dentin and pulp tissues. Amer. J. Orthodont. 28, 1-14. Bell, G. H., Chambers , J. W. and Dawson, I. M. (1947). The mechanica l and structural properties of bone in rats on a rachitogenic diet. / . Physiol. 106, 286-300. Bergman, G. and Engfeldt, B. (1954). Studies on mineralized dental tissues. IV . Biophysical studies on teeth and tooth germs in osteogenesi s imperfecta. Acta path, microbiol. scand. 35, 537-548. Bergman, G. and Engfeldt, B. (1955). Studies on mineralized dental tissues. VI . The distribution of mineral salts in the dentine with special reference to the dentinal tubules. Acta odont. scand. 13, 1-7. Bergman, G., Engfeldt, B. and Sundvall-Hagland , I. (1956). Studies on mineralized tissues. VIII . Histologic and microradiographic investigation of hereditary opalescen t dentine. Acta odont. scand. 14, 103-117. Bergman, G., Gothe, G. and Welander, E. (1961). Studies on mineralized tissues. XV . Mineral pattern in dentine. Arch, oral biol. 4, 6-23. Bird, M. J., French, E. L., Woodside, M. R., Morrison, M . I. and Hodge, H. C. (1940). Chemical analysis of deciduous enamel and dentin. / . dent. Res. 19, 413-423. Bird, M. J., Gallup, H., Gaudino, J. and Hodge, H. C. (1948a). A comparison of two methods of ashing enamel and dentin. J. dent. Res. 27, 693-704. Bird, M. J., Kelman, E., Lerner, H., Rosenfeld, D., Totah, V. and Hodge, H. C. (1948b). Chemical analysis of teeth showing unusual wear. / . dent. Res. 27, 629-634. Blake, G. C. (1958). The peritubular translucent zone in human dentine. Brit. dent. J. 104, 57-64. Blaxter, K. L. (1956). The magnesium content of bone in hypomagnesaemi c disorders of livestock. Ciba Fdn. Symp., Bone Struct. Metab. pp. 117-134. Boddie, G. F. (1949). Effect of fluorine compounds on animals in Fort Willia m area. M.R.C. Memor. 22, 32-46 Boissevain, C. H. and Drea, W. F. (1933). Spectroscopi c

17. C H E M I S T RY

OF

THE

MINERAL

determination of fluoride in bones and teeth and other organs in relation to fluorine in drinking water. J. dent. Res. 13, 495-500. Bowes, J. H. and Murray, M . M. (1935). The chemical composition of teeth. II . The composition of human enamel and dentine. Biochem. J. 29, 2721-2727. Bowes, J. H. and Murray, M . M. (1936). The chemical composition of teeth. III . The variations in chemical composition in relation to dental structure. Biochem. J. 30, 977-984. Boyde, Á., Switsur, V. R. and Fearnhead , R. W. (1961). Application of the scanning electron-probe X-ray microanalyse r to dental tissues. / . Ultrastruct. Res. 5, 201-207. Boyle, P. E. and Wesson, L. G. (1943). Influence of vitamin D on the structure of the teeth and of the bones of rats on low calcium diets. Arch. Path. 36, 243-252. Boyle, P. E., Bessey, O. A. and Howe, P. R. (1940). Rate of dentin formation in incisor teeth of guinea-pigs on normal and on ascorbic acid deficient diets. Arch. Path. 30, 90-107. Bradford, E. W. (1958). The maturation of dentine. Brit. dent. J. 105, 212-216. Bradford, E. W. (1960). The dentine, a barrier to caries. Brit. dent. J. 109, 387-393. Brandi, J. and Tappeiner, H. (1891). Uber die Ablagerung von Fluorverbindunge n im Organismus nach Futterung mit Fluornatrium. Z. Biol. 28, 518-539. Bredig, M. A. (1933). Zur Apatitstruktur der anorganische n Knochen- und Zahnsubstanz . Hoppe-Seyl. Z. 216, 239-243. Brekhus, P. J. and Armstrong, W. D. (1935). A method for the separation of enamel, dentin and cementum. / . dent. Res. 15, 23-29. Brown, W. E., Smith, J. P., Lehr, J. R. and Frazier, A. W. (1962). Crystaliographic and chemical relations between octacalcium phosphate and hydroxyapatite. Nature, Lond. 196, 1050-1055. Brudevold, F. and Steadman , L. T. (1956). The distribution of lead in human enamel. / . dent. Res. 35, 430-437. Brudevold, F., Steadman , L. T. and Smith, F. A. (1960). Inorganic and organic components of tooth structures. Ann. N.Y. Acad. Sci. 85, Art . 1, 110-132. Brudevold, F., Steadman , L. T., Spinelli, Ì . Á., Amdur, Â. H. and Gron, P. (1963). A study of zinc in human teeth. Arch, oral Biol. 8, 135-144. Buchanan, D. L. and Nakao, A. (1952). Turnover of bone carbonate. / . biol. Chem. 198, 245-257. Buchanan, D. L. and Nakao, A. (1958). Studies on the nature of bone carbonate . Arch. Biochem. Biophys. 11, 168-180. Burnett, G. W. and Lobene, R. R. (1955). Studies of the composition of teeth. II . Spectrochemica l analysis of

P H A SE

OF

DENTINE

239

enamel and dentin from Syrian hamsters . J. dent. Res. 34, 814-819. Burnett, G. W. and Zenewitz, J. A. (1957). Studies of the composition of teeth. VI . The composition of Syrian hamster enamel and dentin extracted with KOH-ethylene glycol. dent. Res. 36, 684-689. Burnett, G. W. and Zenewitz, J. A. (1958a). Studies of the composition of teeth. VII . The moisture content of calcified tissues. / . dent. Res. 37, 581-589. Burnett, G. W. and Zenewitz, J. A. (1958b). Studies of the composition of teeth. VIII . The composition of human teeth. / . dent. Res. 37, 590-600. Campbell, A. M. G., Herdan, G., Tatlow, W. F. T. and Whittle, E. G. (1950). Lead in relation to disseminate d sclerosis. Brain 73, 52-71. Cape, A. T. and Kitchin, P. C. (1930). Histologic phenomen a of tooth tissues as observed under polarized light; with a note on the Roentgen-ra y spectra of enamel and dentin. /. Amer. dent. Ass. 17, 193-227. Cartier, P. (1948). Les constituants minéraux des tissus calcifiés. I. La structure minérale de l'os, de la dentine et du cement. Bull. Soc. Chim. biol., Paris 30, 65-73. Clark, J. H. (1931). A study of tendons, bones and other forms of connective tissue by means of X-ray diffraction patterns. Amer. J. Physiol. 98, 328-337. Collin, R. L. (1959). Strontium-calcium hydroxyapatite solid solutions. Preparation and lattice constant measure ments. J. Amer. chem. Soc. 81, 5275-5278. Collin, R. L. (1960). Strontium-calcium hydroxyapatite solid solutions precipitated from basic aqueous solutions. J. Amer. chem. Soc. 82, 5067-5069. Cooper, W. E. G. (1965). A new method of sampling sections of calcified tissues. Arch, oral Biol. 10, 193-194. Cremer, H. D., Buttner, W., Dittmann, G. and Voelker, W. (1953). Ern hrungsfaktoren bei Zahn- und Knochenbildung. II . Der Mineralgehalt der Zâhne bei Carieskost. Biochem. Z. 324, 83-88. Crowell, C. D., Hodge, H. C. and Line, W. R. (1934). Chemical analysis of tooth samples composed of enamel, dentin and cementum. / . dent. Res. 14, 251-268. Cruickshank, D. B. (1936). The natural occurrence of zinc in the teeth. Brit. dent. J. 61, 530-531. Cruickshank, D. B. (1940). The natural occurrence of zinc in the teeth. III . Variations in tuberculosis. Brit. dent. J. 68, 257-27º. Cruickshank, D. B. (1949). Frequency distribution of the zinc concentration s in dental tissues of the normal population. Biochem. J. 44, 299-302. Dallemagne, M. J. (1952). Some recent facts about the properties of tricalcium phosphate and the composition of bone salt. Trans. Macy Conf. metab. Interrelations 4, 154-168. Dallemagne, M. J. (1964). Phosphat e and carbonate in

240

S.

L.

bone and teeth. In "Bone and Tooth" (H. J. J. Blackwood, éd.), pp. 171-174. Pergamon Press, Oxford. Dallemagne, M. J. and Melon, J. (1946). La localisation de l'apatite et du phosphate tricalcique dans l'émail dentaire. Arch. Biol., Paris 57, 79-98. Dam, H., Granados , H. and Maltesen, L. (1950). Changes in minerai composition of enamel and dentine of the incisors in vitamin Å deficient rats. Acta physiol. scand. 21, 124-130. Douglas, T. H. J. (1949). The dental conditions of adults and schoolchildren in the Fort Willia m area. M.R.C. Memor. 22, 76-84. Dragiff, D. A. and Karshan, M. (1943). Effect of pregnancy on the chemical composition of human dentin. / . dent. Res. 22, 261-265. Drea, W. F. (1936). Spectrum analysis of dental tissues for "trace" elements. / . dent. Res. 15, 403-406. Driak, F. (1952). Der Fluorgehalt der Hartsubstanze n menschliche r Z hne . Ζ. Stomat. 49, 38-47. Duckworth, J. and Godden, W. (1940). The influence of diets low in magnesium upon the chemical composition of the incisor tooth of the rat. J. Physiol. 99, 1-7. Duckworth, J. and Godden, W. (1941). The lability of skeletal magnesium reserves . The influence of rates of bone growth. Biochem. J. 35, 816-823. Duckworth, J. and Godden, W. (1943). The replenishmen t of depleted skeletal reserves of magnesium . Biochem. J. 37, 595-598. Duckworth, J., Godden, W. and Warnock, McG. M. (1940). The effect of acute magnesium deficiency on bone formation in rats. Biochem. J. 34, 97-108. Eda, S. (1961). Histochemical analysis on the mechanism of dentine formation in dog's pulp. Bull. Tokyo dent. Coll. 2, 59-88. Eisenberger , S., Lehrman, A. and Turner, W. D. (1940). The basic calcium phosphate s and related systems. Some theoretical and practical aspects . Chem. Rev. 26, 257-296. Elliott , C. G. and Smith, M. D. (1960). Dietary fluoride related to fluoride content of teeth. / . dent. Res. 39, 93-98. Elliott , J. C. (1963). Interpretation of carbonate bands in infrared spectrum of dental enamel. / . dent. Res. 42, 1081-1082 (Abstract). Elliott, J. C. (1964). The crystallographic structure of dental enamel and related apatites. Ph.D. Thesis, University of London. Ellis, L. N. (1963). Influence of dietary calcium upon composition of mineralized tissues and upon susceptibility of enamel to erosion in vivo. J. dent. Res. 42, 973-980. Ellis, L. N. and Dwyer, E. J. (1960). The influence of dietary factors upon the composition of mineralized tissues and upon the susceptibility of enamel to erosion in vivo. I. Phosphorus . / . Nutr. 72, 224-232.

R O W L ES Ellison, S. A. and Halpert, W. (1947). Chemical changes in the dentin following ageing and peripheral irritation. /. dent. Res. 26, 79-82. Emerson, W. H. and Fischer, Å. E. (1962). Infra-red absorption spectra of carbonate in calcified tissues. Arch, oral Biol. 7, 671-683. Engfeldt, B. and Hammarlund-Essler , E. (1956). Studies on mineralized dental tissues. X. A microradiographic , autoradiographi c and histochemica l investigation on dental hard tissues in dogs with experimentally produced vitamin D deficiency. Acta odont. scand. 14, 293-311. Erdheim, J. (1914). Rachitis und Epithelkorperchen . Denkschr. Akad. Wiss., Wien 90, 363-688. Ericsson, Y., Ullberg, S. and Appelgren, L. E. (1960). Autoradiographic localization of radioactive fluorine (F 1 8) in developing teeth and bones. Acta odont. scand. 18, 253-261. Fabry, C. (1959). La composition des sels osseux dans les tissus en voie d'ossification. I. Influence des traitements de minéralisation de l'os sur les phosphate s de calcium synthétiques . Biochim. biophys. Acta 34, 58-67. Ferguson, H. W. and Hartles, R. L. (1964). The effect of vitamin D on the dentine of the incisor teeth and on the alveolar bone of young rats maintained on diets deficient in calcium or phosphorus . Arch, oral Biol. 9, 447-460. Filson, A. and Hope, J. (1957). Isolation of melanin granules. Nature, Lond. 179, 211. Fish, E. W. (1932). The pathology of the dentine and the pulp. Brit. dent. J. 53, 351-363. Fish, E. W. and Harris, L. J. (1935). The effects of vitamin C deficiency on tooth structure in guinea-pigs. Brit. dent. J. 58, 3-20. Fôrster, A. and Happel, G. (1959). Untersuchunge n zur Altersbestimmun g des Menschen auf Grund des Mineralisationsgrade s des Zahndentine . Dtsch. Z. ges. gerichtl. Med. 48, 195-201. Forbes, G. B. (1962). Sodium. In "Mineral Metabolism" (C. L. Comar and F. Bronner, eds.), Vol.2, Part B, Chapter 25. Academic Press, New York. Fore, H. and Morton, R. A. (1952). Manganes e in rabbit tissues. Biochem. J. 51, 600-603. Francois, C. (1958). La localisation de l'anhydride carbonique dans les sels osseux et dentaires, étudiée par la méthode des échange s isotopiques. Bull. Soc. Chim. biol., Paris 40, 1341-1347. Frank, R. M. (1955). Contributions apportées par le microscope électronique a l'étude de la carie dentaire. Rev. belge Stomat. 52, 228-246. Frank, R. M. (1959). Electron microscopy of undecalcified sections of human adult dentine. Arch, oral Biol. 1, 29-32. French, E. L., Welch, Å. Á., Simmons, E. J., LeFevre, M. L. and Hodge, H. C. (1938). Calcium, phosphorus and carbon

17. C H E M I S T RY

OF

THE

MINERAL

dioxide determination on all the dentin from sound and carious teeth. / . dent. Res. 17, 401-410. Gabriel, S. (1894). Chemische Untersuchunge n iiber die Mineralstoffe der Knochen und Z hne . Hoppe-Seyl. Z. 18, 257-303. Gaunt, W. E. and Irving, J. T. (1940). Influence of calcium and phosphorus intake on tooth formation. / . Physiol. 99, 18-29. Gilda, J. E. (1951). Studies on the physical properties of rodent enamel. I. The Manly-Hodge separation method as applied to the teeth of the rat and hamster. / . dent. Res. 30, 445-452. Gruner, J. W. and McConnell, D. (1937). The problem of the carbonate apatites. The structure of francolite. Z. Krist. 97A, 208-215. Gruner, J. W., McConnell, D. and Armstrong, W. D. (1937). The relationship between crystal structure and chemical composition of enamel and dentin. / . biol. Chem. 121, 771-781. Hadjimarkos, D. M. and Bonhorst, C. W. (1959). The selenium content of human teeth. Oral Surg. 112,113-116. Haldi, J., Wynn, W., Bentley, Ê. D. and Law, M. L. (1959). Dental caries in the albino rat in relation to the chemical composition of the teeth and of the diet. IV . Variations in the calcium : phosphorus ratio of the diets induced by changing the calcium content. / . Nutr. 67, 645-653. Harness, S. R. and Smith, M. D. (1951). Examination of the fluoride content of teeth from some Ontario districts. /. Canad. dent. Ass. 17, 20-24. Harrison, H. E. (1937). The sodium content of bone and other calcified material. / . biol. Chem. 120, 677-687. Hartles, R. L. (1951a). The effect of a high-sucrose diet on the calcium and phosphorus content of enamel and dentine of rat incisors. Biochem. J. 48, 245-249. Hartles, R. L. (1951b). The influence of a high-sucrose diet on the calcium and phosphorus percentag e of the rat femur, and a comparison with its effect on the enamel and dentine of the rat incisor teeth. Biochem. J. 49, 574-577. Hartles, R. L. (1960). Chemistry of bone and tooth minerals. Chem. Soc. (Lond.) ann. Rep. 56, 322-331. Hartles, R. L. and Leaver, A. G. (1960). Citrate in mineralized tissues. I. Citrate in human dentine. Arch, oral Biol. 1, 297-303. Helmcke, J.-G. (1955). Elektronenmikroskopisch e Strukturuntersuchunge n an gesunde n und kranken Z hnen . Dtsch. zahnarztl. Z. 10, 1461-1478. Hendricks, S. B. and Hill , W. L. (1950). The nature of bone and phosphate rock. Proc. nat. Acad. Sci., Wash. 36, 731-737. Herman, H. (1964). La composition des dents du lapin: étude chimique et spectrométriqu e dans l'infra-rouge. Bull. Soc. Chim. biol., Paris 46, 385-394. Herman, H. and Dallemagne, M. J. (1961). The main

P H A SE

OF

DENTINE

241

minerai constituent of bones and teeth. Arch, oral Biol. 5, 137-144. Herman, H. and Dallemagne, M. J. (1964). Les carbonatohydroxyl-apatites et le carbonate des os et des dents étudiés par la spectrophotométri e dans l'infra-rouge. Bull. Soc. Chim. biol., Paris 46, 373-383. Hodge, H. C. (1936). Correlated clinical and structural study of hereditary opalescen t dentin. / . dent. Res. 15, 316-317 (Abstract). Hodge, H. C. and Finn, S. B. (1938). Hereditary opalescen t dentin. A dominant hereditary tooth anomaly in man. /. Hered. 29, 359-364. Hodge, H. C , Finn, S. B., Robinson, H. B. G., Manly, R. S., Manly, M. L., Van Huysen, G. and Bale, W. F. (1940). Hereditary opalescen t dentin. III . Histological, chemical and physical studies. / . dent. Res. 19, 521-536. Hohling, H.-J. (1960). Untersuchunge n elektronenmikros kopischer Schnittbilder am gesunde m und krankem Dentin. Z. Naturforsch. 15, 753-755. Hohling, H.-J. and Newesely, H. (1961). Kristallisationsversuche und Elektronenbeugunguntersuchunge n zur Aufklârung der chemische n Verbindung der "Karieskristalle". Dtsch. Zahnàrztebl. 5, 706-710. Hoffman, M. M. and Schour, I. (1940). Quantitative studies in the developmen t of the rat molar. I. The growth pattern of the primary and secondar y dentin (from birth to 500 days of age). Anat. Rec. 78, 233-249. 0 Holgate, W. (1959). Incorporation and retention of 9Sr in teeth. Brit. dent. J. 107, 131-143. Hoppe, W. F. (1965). Tierexperimentelle Untersuchunge n iiber den Gehalt an extrazellulârem Wasser im Dentin und in der Kompakta verschiedene r Knochen. Dtsch. zahnârztl. Z. 20, 128-134. Hutton, W. E. (1953). A test for the purity of powdered enamel. / . dent. Res. 32, 626-627. Irving, J. T. (1940). The influence of diets low in magnesium upon the histological appearanc e of the incisor tooth of the rat. / . Physiol. 99, 8-17. Irving, J. T. (1957). Comparison of the influence of hormones, vitamins and other dietary factors upon the formation of bone, dentine and enamel. Vitam. & Horm. 15, 291-323. Irving, J. T. and Neinaber, M. W. P. (1946). The effect of sodium fluoride on the teeth and blood calcium and inorganic phosphorus levels of rachitic rats. / . dent. Res. 25, 327-335. Jackson, D. (1956). Initial consideration s on the phenomeno n of arrested dental caries. / . dent. Res. 35, 956 (Abstract). Jackson, D. and Weidmann, S. M. (1959). The relationship between age and fluorine content of dentine and enamel. A regional survey. Brit. dent. J. 107, 303-306. Jackson, S. H. and Train, D. (1955). Stabilization of the fluorine concentration of the total ash of rats. Canad. J Biochem. Physiol. 33, 93-98.

242

S. L .

Jee, W. S. S. and Arnold, J. S. (1960). Radioisotope s in the teeth of dogs. I. The distribution of plutonium, radium, radiothorium, mesothorium and strontium and the histopathologic changes in teeth containing plutonium. Arch, oral Biol. 2, 215-238. Jenkins, G. N. (1966). "The Physiology of the Mouth", 3rd ed. Blackwell, Oxford. Jenkins, G. N. and Speirs, R. L. (1954a). An investigation of possible differences between the chemical composition of deciduous teeth calcified before and during the 1939— 1945 war. / . dent. Res. 33, 736 (Abstract). Jenkins, G. N. and Speirs, R. L. (1954b). Some observations on the fluoride concentration of dental tissues. / . dent. Res. 33, 734 (Abstract). Jensen , A. T. and Rowles, S. L. (1957). Magnesian whitlockite, a major constituent of dental calculus. Acta odont. scand. 15, 121-139. Johansen , E. (1962). The nature of the carious lesion. Symp. dent. Caries. Dent. Clin. N. Amer. pp. 305-320. Johansen , E. and Nordback, L. G. (1962). Chemistry of carious lesions. III . The fluoride content of carious dentin. Preprint. Abstr. Int. Ass. dent. Res., 40th gen. Meet., St. Louis, 1962 No. 143, p. 39. Johansen , E. and Parks, H. F. (1962), Electron-microscopi c observations on sound human dentine. Arch, oral Biol. 7, 185-193. Karshan, M., Weiner, R. and Stofsky, N. (1934). Biochemical studies of aqueous extracts of enamel and dentin in relation to dental caries. / . dent. Res. 14, 445-454. Kaushansky , L. I. (1932). Chemical analysis of teeth, roots and crowns affected by pyorrhea alveolaris and dental caries. II . Analysis of phosphorus and magnesium content. Dent. Cosmos 74, 468-473. Kehoe, R. Á., Cholak, J. and Story, R. V. (1940). A spectrochemica l study of the normal ranges of concentration of certain trace metals in biological materials. / . Nutr. 19, 579-592. Keil, A. (1955). Polarisationmikroskopisch e Untersuchunge n am kariosen Dentine. Dtsch. zahnàrztl. Ζ. 10, 1525-1529. Keil, A. (1958). Histopathology of dental caries. Int. dent. J. 8, 171-173. King, C. T. G., Wilk , A. and McClure, F. J. (1962). Carbon dioxide induced acidosis in pregnant rats and the caries susceptibility of their progeny. Proc. Soc. exp. Biol., N.Y. I l l , 486-489. Klein, A. I. (1961). Association between deciduous dentin sclerosis and calcium hydroxide methyl cellulose base material. / . Amer. dent. Ass. 63, 76-84. Klein, H., Orent, E. R. and McCollum, Å. V. (1935). The effects of magnesium deficiency on the teeth and their supporting structures. Amer. J. Physiol. 112, 256-262. Kothe, J. (1960). Untersuchunge n uber Vernderungen der Zusammensetzun g des kariosen Dentins gegenube r dem normalen Dentin und uber den Hartnungsmechanismu s

R O W L ES des im Rahmen der Caries-profunda-Therapi e in der Kavit t belassene n erweichten kariosen Dentins. Dtsch. Zahn-, Mund- u. Kieferheilk. 34, 10-18. Kriiger, V. and Rakuttis, G. (1952). Das Rontgenbild der Hartsubstanze n des normalen und kariosen Zahnes. Dtsch. zahnàrztl. Ζ. 7, 141-155. Kuether, C. Á., Telford, I. R. and Roe, J. H. (1944). The relation of the blood level of ascorbic acid to the tissue concentration s of this vitamin and to the histology of the incisor teeth in the guinea pig. / . Nutr. 28, 347-358. Leach, S. A. (1959). Reactions of fluoride with powdered enamel and dentine. Report of a chemical study over a range of concentration s of sodium fluoride. Brit. dent. J. 106, 133-142. LeFevre, M. L. and Manly, R. S. (1938). Moisture, inorganic and organic contents of enamel and dentin from carious teeth. / . Amer. dent. Ass. 25, 233-242. Leicester, H. M . (1949). "Biochemistry of the Teeth". Mosby, St. Louis, Missouri. Lenz, H. (1955). Elektronenmikroscopische r Nachweis der Dentinver nderungen durch Karies. Dtsch. Zahn-, Mund- u. Kieferheilk. 22, 24-33. Likins, R. C, Posner, A. S., Kunde, M. L. and Craven, D. L. (1959). Comparative metabolism of strontium and calcium in the rat. Arch. Biochem. Biophys. 83, 472-481. Lindemann, G. (1956). An X-ray diffraction study of osseous and dental tissues from rats with experimenta l chronic fluorosis. Acta odont. Scand. 14, 33-46. Little, M. F. and Brudevold, F. (1958). A study of the inorganic carbon dioxide in intact enamel. J. dent. Res. 37, 991-1000. Livrea, G., De Stefano, F. and Picciotto, A. (1958). Sui fattori délia crescita continua e della calcificazione delfincisivo del ratto. I. L'azione degli ormoni sessuali. Biol. latina 11, 407-420. Lobene, R. R. and Burnett, G. W. (1954). Studies of the composition of teeth. I. Chemical analysis for the principal inorganic constituents of enamel and dentin from Syrian hamsters. / . dent. Res. 33, 487-496. Logan, M. A. (1935). The composition of bone, cartilage, dentin and enamel. J. biol. Chem. 110, 375-389. Losee, F. L. and Hurley, L. A. (1956a). Bone treated with ethylenediamin e as a successfu l foundation material in cross species bone grafts. Nature, Lond. Ill, 1032-1033. Losee, F. L. and Hurley, L. A. (1956b). Successfu l crossspecies bone grafting accomplishe d by removal of the donor organic matrix. Nav. med. Res. Inst. Rep. 14, 911-948. Losee, F. L., Leopold, R. S. and Hess, W. C. (1951). Dentinal protein: recovery in pure form. / . dent. Res. 30, 565-574. Losee, F. L., Van Reen, R., Peckham, S. C, Hess, W. C, Henderson , N. and Gerende, L. J. (1957). Dietary caseinsucrose ratios and their effects on mineralized tissues. /. dent. Res. 36, 904-910.

17. C H E M I S T RY

OF

THE

MINERAL

Lowater, F. and Murray, M. M. (1937). Chemical composition of teeth. V. Spectrographi c analysis. Biochem. J. 31, 837-841. Lund, A. P. and Armstrong, W. D. (1942). The effect of low calcium and vitamin D deficient diet on bones and teeth of mature rats. Proc. Soc. exp. Biol., N.Y. 50, 363-365. Lundberg, M., Sôremark, R. and Thilander, H. (1965). Gamma-ray spectrometric analysis of some elements in coronal dentine of unerupted (impacted) teeth. Odont. Revy, Lund 16, 97-100. McAleese, D. M. and Forbes, R. M. (1961). The requirement and tissue distribution of magnesium in the rat as influenced by environmenta l temperature and dietary calcium. / . Nutr. 73, 94-106. McAleese, D. M., Bell, M. C. and Forbes, R. M. (1961). Magnesium-28 studies in lambs. / . Nutr. 74, 505-514. McCann, H. G. and Bullock, F. A. (1955). The reaction of the fluoride ion with powdered enamel and dentin. /. dent. Res. 34, 59-67. McCann, H. G. and Bullock, F. A. (1957). The effect of fluorine ingestion on the composition and solubility of mineralized tissues of the rat. / . dent. Res. 36, 391-398. McCauley, H. B. (1942). A roentgen-ra y absorption study of hereditary opalescen t dentin. / . dent. Res. 21, 107-114. McClure, F. J. (1948). Fluorine in dentin and enamel in sound and carious human teeth. / . dent. Res. 27, 287-294. McClure, F. J. (1950). Fluorine, ash, calcium and phosphorus in human teeth. J. dent. Res. 29, 315-319. McClure, F. J. (1958). Wheat cereal diets, rat caries, lysine and minerals. / . Nutr. 65, 619-631. McClure, F. J. and Likins, R. C. (1951). Fluorine in human teeth studied in relation to fluorine in the drinking water. /. dent. Res. 30, 172-176. McClure, F. J., Folk, J. E. and Rust, J. D. (1956). Smooth surface caries in white rats: effects of fluoride, iodoacetate , penicillin, Crisco butter fat and a salt mixture. / . Nutr. 53, 1-8. McConnell, D. (1952). Crystal chemistry of the carbonate apatites and their relation to the composition of calcified tissues. / . dent. Res. 31, 53-63. McConnell, D. (1960). Recent advances in the investigation of the crystal chemistry of dental enamel. Arch, oral Biol. 3, 28-34. Manly, R. S. and Brooks, E. J. S. (1947). Transparenc y and light scattering of dental hard tissues. J. dent. Res. 26, 427-434. Manly, R. S. and Deakins, M. L. (1940). Changes in the volume percentag e of moisture, organic and inorganic material in dental caries. / . dent. Res. 19, 165-170. Manly, R. S. and Hodge, H. C. (1939). Density and refractive index studies of dental hard tissues. / . dent. Res. 18, 133-141.

P H A SE

OF

DENTINE

243

Mansell, R. E. and Hendershot , L. C. (1960). The spectrochemical analysis of metals in rat molar enamel, femur and incisors. Arch, oral Biol. 2, 31-37. Massler, M. and Barber, T. K. (1953). The action of amalgam on dentin. / . Amer. dent. Ass. 47, 415-422. Maulbetsch, A. and Rutishauser , E. (1936). Le teneur des dents en plomb. Arch. int. Pharmacodyn. 53, 55-64. Miles, A. E. W. (1961). Ageing in the teeth and oral tissues. In "Structural Aspects of Ageing" (G. H. Bourne, ed.), pp. 353-397. Pitman, London. Miller , J. (1954). The microradiographic appearanc e of dentine. Brit. dent. J. 97, 7-9. Moore, T. and Mitchell, R. L. (1955). Dental depigmentatio n and lowered content of iron in the incisor teeth of rats deficient in vitamin A or E. Brit. J. Nutr. 9, 174-180. Murray, M. M. (1936). The chemical composition of teeth. IV . The calcium, magnesium and phosphorus contents of the teeth of different animals. A brief consideration of the mechanism of calcification. Biochem. J. 30, 15671571. Murray, M. M. and Bowes, J. H. (1936). The composition of enamel, dentine and root in caries and pyorrhea. Brit. dent. J. 61, 473-477. Murray, M. M., Glock, G. E. and Lowater, F. (1939). Chemical and spectrographi c determination of iron in tooth material. Brit. dent. J. 66, 345-350. Nalbandian, J. (1962). Ultrastructure of developing and adult human dentin. / . dent. Res. 41, 1270 (Abstract). Nalbandian, J., Gonzales, F. and Sognnaes , R. F. (1960). Sclerotic age changes in root dentine of human teeth as observed with optical, electron and X-ray microscopy. /. dent. Res. 39, 598-607. Neuman, W. F. and Neuman, M. W. (1953). The nature of the mineral phase of bone. Chem. Rev. 53, 1-45. Nezhivenko, L. N. (1961). The copper content in the teeth in health and in alveolar pyorrhea. Stomatologiia, Moskva 40, No. 1, 32-35; see Chem. Abstr. 55, 18957f (1961). Nishi, T. (1939). Experimental studies of calcium content of teeth in C-avitaminous guinea-pigs. Trans. Jap. path. Soc. 29, 294-298. Nixon, G. S. (1961). Estimation of Mercury2 03 in the dental tissues. / . dent. Res. 40, 1283 (Abstract). Nordback, L. G. and Johansen , E. (1962). The chemistry of carious lesions. I. The calcium, magnesium , phosphorus and carbonate content of sound and carious human dentin and enamel. Preprint. Abstr. Int. Ass. dent. Res., 40th gen. Meet., St. Louis, 1962 No. 141, p. 39. Nordback, L. G., Johansen , E. and Parks, H. F. (1961). Effect of heat on the carbonate content of dentin. / . dent. Res. 40, 704 (Abstract). Ntiforo, C. P. and Fremlin, J. H. (1964). Determination of the distribution of carbon in dental enamel by activation analysis. / . dent. Res. 43, 958 (Abstract).

244

S.

L.

Ockerse, T. (1943). Chemical composition of enamel and dentin in high and low caries areas in South Africa. /. dent. Res. 22, 441-446. O'Dell, B. L., Morris, E. R. and Regan, W. O. (1960). Magnesium requirement of guinea-pigs and rats. Effect of calcium and phosphorus and symptoms of magnesium deficiency. / . Nutr. 70, 103-110. Ohmori, I. (1961). Biochemical studies on deciduous tooth substances . Part I. Application of silver nitrate. Bull. Tokyo med. dent. Univ. 8, 83-95. Omnell, Ê. Á., Lindstrôm, B., Hoh, F. C. and Hammarlund-Essler, E. (1960). Method for the non-destructive determination of inorganic and organic material in mineralized tissue. Acta radiol., Stockh. 54, 209-219. Peckham, S. C, Leopold, R. S. and Hess, W. C. (1956a). Determination of fluoride in organic and inorganic fractions of dentin and enamel. / . dent. Res. 35, 205-209. Peckham, S. C, Losee, F. L. and Ettelman, I. (1956b). Ethylenediamine vs. KOH-glycol in the removal of the organic matter of dentin. / . dent. Res. 35, 947-949. Pfrieme, F. (1934). Uber den normalen und pathologische n Bleigehalt der Zâhne von Menschen und Tieren. Arch. Hyg., Berl 111, 232-242. Pindborg, J. J. and Plum, C. M. (1946). Studies in incisor pigmentation in relation to liver-iron and blood-picture in the white rat; gastrectom y as the cause of depigmenta tion of incisors of rats. Acta odont. scand. 7, 105-113. Posner, A. S. (1954). A study of the constitution of certain mineralogical, biological, and synthetic calcium phosphates. Thesis, University of Liège. Posner, A. S. (1960). The nature of the inorganic phase in calcified tissues. In "Calcification in Biological Systems", Publ. No. 64, pp. 373-394. Amer. Ass. Advanc. Sci., Washington, D. C. Posner, A. S. and Duyckaerts, G. (1954). Infra-red study of the carbonate in bone, teeth and francolite. Experientia, Basel 10, 424-425. Posner, A. S. and Perloff, A. (1957). Apatites deficient in divalent cations. / . Res. nat. Bur. Stand. 58, 279-285. Posner, A. S., Fabry, C. and Dallemagne, M. J. (1954). Defect apatite series in synthetic and natural calcium phosphates : the concept of pseudoapatites . Biochim. biophys. Acta 15, 304-305. Posner, A. S., Eanes, E. D., Harper, R. A. and Zipkin, I. (1963). X-ray diffraction analysis of the effect of fluoride on human bone apatite. Arch, oral Biol. 8, 549-570. Ratner, S. (1935). The iron content of teeth of normal and anaemic rats. / . dent. Res. 15, 89-92. Reynolds, L., Corrigan, K. E., Hayden, H. S., Macy, I. G. and Hunscher, H. A. (1938). Diffraction studies of the effect of sodium fluoride and parathormon e upon the incisors and tibiae of rats. Am. J. Roentgenol. 39, 103-126.

ROWLES Robinson, R. A. and Bishop, F. W. (1950). Methods of preparing bone and tooth samples for viewing in the electron microscope. Science 111, 655-657. Rockert, H. (1956a). Variations in the calcification of cementum and dentine as seen by the use of microradiographic technique. Experientia, Basel 12, 16-18. Rockert, H. (1956b). Some observations correlated to obliterated dentinal tubules and performed with microradiographic technique. Acta odont. scand. 13, 271-275. Rôckert, H. (1958). A quantitative microscopical study of calcium in the cementum of teeth. Acta odont. scand. 16, Suppl. 25. Roholm, K. (1937). "Fluorine Intoxication". H. K. Lewis, London. Roseberry, H. H., Hastings, A. B. and Morse, J. K. (1931). X-ray analysis of bone and teeth. / . biol. Chem. 90, 395-406. Schoonover , I. C. and Sonder, W. (1941). Corrosion of dental alloys. J. Amer. dent. Ass. 28, 1278-1291. Scott, D. B. and Nylen, M. U. (1960). Changing concepts in dental histology. Ann. N.Y. Acad. Sci. 85, 133-144. Selvig, K. A. and Selvig, S. K. (1962). Mineral content of human and seal cementum. / . dent. Res. 41, 624-632. Selvig, K. A. and Zander, H. A. (1962). Chemical analysis and microradiograph y of cementum and dentin from periodontally disease d human teeth. / . Periodont. 33, 303-310. Shapiro, I. M. and Hartles, R. L. (1965). An improved method with low material loss for the separation of teeth into fractions of differing density. Arch, oral Biol. 10, 155-159. Sharaevskaya , Æ. N. (1958). Spectral analysis data of hard dental tissues and the alveolar appendag e of man in norm and in paradontitis. Stomatologiia, Moskva 37, No. 2, 12-14; see Chem. Abstr. 53, 5471c (1959). Shaw, J. H. (1955). Effect of nutritional factors on bones and teeth. Ann. N.Y. Acad. Sci. 60, 733-762. Shaw, J. H., Gupta, O. P. and Meyer, M. E. (1956). High fluoride content of teeth from communities with low fluoride water supplies. Amer. J. clin. Nutr. 4, 246-253. Shaw, J. H., Resnick, J. B. and Sweeney, E. A. (1959). Fluoride content of human teeth from the Orient and the Canadian Arctic. / . dent. Res. 38, 129-134. Simon, W. J. and Armstrong, W. D. (194)). Translucent dentin. / . Amer. dent. Ass. 28, 1115-1120. Singer, L. and Armstrong, W. D. (1962). Comparison of fluoride contents of human dental skeletal tissues. / . dent. Res. 41, 154-157. Skillen, W. G. (1937). Histologic and clinical study of hereditary opalescen t dentin. / . Amer. dent. Ass. 24, 1426-1433. Smith, G. H. and Ellis, S. Å. (1947). Studies on the manganes e requirements of rabbits. / . Nutr. 34, 33-41. Smith, M. C. and Lantz, Å. M. (1933). Changes in the

17. C H E M I S T R Y

OF

THE

MINERAL

incisors of albino rats accompanyin g a deficiency of vitamin A. / . Home Econ. 25, 411-415. Smith, R. H. (1959). Calcium and magnesium metabolism in calves. 4. Bone composition in magnesium deficiency and the control of plasma magnesium . Biochem. J. 71, 609-614. Sobel,. A. E. (1955). Local factors in the mechanism of calcification. Ann. N.Y. Acad. Sci. 60, 713-732. Sobel, A. E. (1960). Interrelationship of tooth composition, body fluids, diet and caries susceptibility. Ann. Ν. Y. Acad. Sci. 85, 96^-109. Sobel, A. E. and Hanok, A. (1948). Calcification of teeth. I. Composition in relation to blood and diet. J. biol. Chem. 176, 1103-1122. Sobel, A. E. and Hanok, A. (1958). Calcification. XVI . Composition of bones and teeth in relation to blood and diet in the cotton rat. / . dent. Res. 37, 631-637. Sobel, A. E., Rockenmacher , M. and Kramer, B. (1945). Carbonate content of bone in relation to the composition of blood and diet. / . biol. Chem. 158, 475-489. Sobel, A. E., Hanok, Á., Kirshner, H. A. and Fankuchen I. (1949). Calcification of teeth. III . X-ray diffraction patterns in relation to changes in composition. / . biol. Chem. 179, 205-210. Soremark, R. and Lundberg, M. (1964). Analysis of concentrations of Cr, Ag, Fe, Co, Pt and Rb in normal human dentine. Odont. Revy, Lund 15, 285-289. Soremark, R. and Samsahl, K. (1962). Gamma-ray spectrometric analysis of elements in normal human dentin. /. dent. Res. 41, 603-606. Sôremark, R., Ingels, O., Plett, H. and Samsahl, K. (1962). Influence of some dental restorations on the concentrations of inorganic constituents of the teeth. Acta odont. scand. 20, 215-224. Sognnaes , R. F. (1962). Radioisotope interactions with dental hard tissues. In "Radioisotopes and Bone, a Symposium" (P. Lacroix and A. M. Budy, eds.), pp. 355-370. Blackwell, Oxford, Sognnaes , R. F. and Volker, J. F. (1941). Studies on the distribution of radioactive phosphorus in tooth enamel of experimenta l animals. Amer. J. Physiol. 133, 112-120. Sognnaes , R. F., Shaw, J. H. and Bogoroch, R. (1955). Radiotracer studies on bone, cementum, dentin and enamel of rhesus monkeys. Amer. J. Physiol. 180, 408-420. Sowden, J. R. (1956). A preliminary report on recalcification of carious dentin. / . Dent. Child. 23, 187-188. Stack, M. V. (1951). Organic constituents of dentine. Brit, dent. J. 90, 173-181. Stack, M. V. (1955). The chemical nature of the organic matrix of bone, dentin and enamel. Ann. N.Y. Acad. Sci. 60, 585-595. Stanmeyer , W. R. S., King, C. T. G., Scholfield, H. and Colby, R. (1958). Effect of carbon dioxide toxicity on formation of dentine. / . dent. Res. 37, 7 (Abstract).

PHASE

OF

DENTINE

245

Steadman , L. T., Hodge, H. C. and Horn, H. W. (1941). Spectrochemica l studies of potassium in bone and tooth substance . / . biol. Chem. 140, 71-76. Steadman , L. T., Brudevold, F. and Smith, F. A. (1958). Distribution of strontium in teeth from different geographical areas. / . Amer. dent. Ass. 57, 340-344. Storozheva , Í . N. (1963). Soderzhani e svintsa i olova í zubakh cheloveka í norme i pri kariere [The content of lead and tin in human teeth in health and in dental caries]. Stomatologiia, Moskva 42, No. 1, 44-48; see Chem. Abstr. 59, 988f (1963). Stover, B. J., Atherton, D. R. and Arnold, J. S. (1957). Comparative metabolism of C a45 and R a2 2.6 Proc. Soc. exp. Biol., N.Y. 94, 269-272. Swanson, H. E. and Tatge, E. (1953). Standard X-ray diffraction powder patterns. Circ. nat. Bur. Stand. 539(1), 69-70. Symons, Í . Â. B. (1961). A histochemica l study of the intertubular and peritubular matrices in normal dentin. /. dent. Res. 40, 1279 (Abstract). Takuma, S. (1960a). Preliminary report on the mineralization of human dentin. / . dent. Res. 39, 964-973. Takuma, S. (1960b). Electron microscopy of the structure around the dentinal tubule. / . dent. Res. 39, 973-981. Tefft, H., French, E. L. and Hodge, H. C. (1941). Magnesium determinations on all the dentin from sound and carious teeth. / . dent. Res. 20, 45-48. Tempestini, O. (1953). Studi chemici sui denti fluorotici. 2. Determinazioni di fluoro nello sualto e nella dentina di denti sani nella zona di fluorosi endemica di Catenanuova (Sicilia). Clin, odontoiat. 8, 176-181. Thewlis, J. (1940). The structure of teeth as shown by X-ray examination. M.R.C. Spec. Rep. Ser. 238. Thewlis, J., Glock, G. E. and Murray, M. M. (1939). Chemical and X-ray analysis of dental, mineral and synthetic apatites. Trans. Faraday Soc. 35, 358-363. Tokunaga, M. (1960). Uber die Phosphat e im Gewebssaf t des Zahnbeins bes. des erweichten Zahnbeins. Lymphatologia, Kyoto 4, 29-39. Tonogai, K., Minami, N. and Sakurada , H. (1962). Comparative studies on crystal structures of deciduous and permanent teeth by X-ray diffraction. / . dent. Res. 41, 735 (Abstract). Torell, P. (1958). Rôntgendiffraktion av pulver frân Homodentin. Svensk. Tandlàk. Tidskr. 51, 137-144. Toverud, G. (1923). The influence of diet on teeth and bones. J. biol. Chem. 58, 583-600. Trautz, O. R. (1955). X-ray diffraction of biological and synthetic apatites. Ann. N.Y. Acad. Sci. 60, 696-712. Trautz, O. R. (1960). Crystallographic studies of calcium carbonate phosphate . Ann. N.Y. Acad. Sci. 85, 145-160. Underwood, A. L., Toribara, T. Y. and Neuman, W. F. (1955). An infra-red study of the nature of bone carbonate . /. Amer. chem. Soc. 11, 317-319.

246

S.

L.

Yahl, von J., Hôhling, H. J. and Frank, R. M. (1964). Elektronenstrahlbeugun g an rhomboedrisc h aussehende n Mineralbildungen in kariosen Dentin. Arch, oral Biol. 9, 315-320. Van Huysen, G. (1936). The roentgen-ra y absorption per unit thickness of dentin. Dent. Cosmos 78, 272-281. Verbraeck, L. (1958a). Analyse quantitative du taux de calcium et de phosphore dans la dentine au Congo belge. Ann. Soc. belge Med. trop. 38, 1075-1083. Verbraeck, L. (1958b). Densité de la dentine chez le blanc et le noir en Afrique Centrale. Ann. Soc. belge Med. trop. 38, 1085-1090. Vikhm, N. A. (1962). Soderzhani e nikelia i serebra í zubakh í norme pri kariese i aPreoliarnoï pioree [The content of nickel and silver in healthy teeth, dental caries and alveolar pyorrhea]. Stomatologiia, Moskva 41, No. 6, 13-17; see Intern. Abstr. biol. Sci. 29, 2865 (1963). Vogel, A. I. (1961). " A Textbook of Quantitative Inorganic Analysis", 3rd ed., p. 475. Longmans, Green, London. von Kreudenstein , T. S. and Stiiben, J. (1955). Dentinstoffwechselstudien . III . Die thermische Méthode zum Nachweis des Dentinliquors. Dtsch. zahnârztl. Z. 10, 1178-1182. Wagner, M. J. and Muhler, J. C. (1960). The effect of calcium and phosphorus on fluorine absorption. / . dent. Res. 39, 49-52. Wainwright, W. W. (1933). Dissertation, University of California; quoted by Leicester (1949), p. 18. Warren, S. L., Bishop, F. W., Hodge, H. C. and Van Huysen, G. (1934). A quantitative method for studying the roentgen-ray absorption of tooth slices. Amer. J. Roentgenol. 31, 663-672. Watchorn, E. and McCance, R. A. (1937). Subacute magnesium deficiency in rats. Biochem. J. 31, 1379-1390. Weidmann, S. M. (1962). Uptake and retention of fluoride by teeth of animals under experimenta l fluorosis. Arch, oral Biol. 7, 63-72. Williams, J. B. and Irvine, J. W., Jr. (1954). Preparation of the inorganic matrix of bone. Science 119, 771 — 772. Winand, L. (1960). Étude physico-chimique des phosphate s

R O W L ES de calcium de structure apatitique. Thesis for doctorate in chem., University of Liège. Winand, L., Dallemagne, M . J. and Duyckaerts, G. (1961). Hydrogen bonding in apatitic calcium phosphates . Nature, Lond. 190, 164-165. Wood, Í . V., Jr. (1947). Specific surfaces of bone, apatite, enamel and dentin. Science 105, 531-532. Wyckoff, R. W. G. and Croissant, O. (1963). Microradiography of dentine using characteristic X-rays. Biochim. biophys. Acta 66, 137-143. Wynn, W., Haldi, J., Bentley, Ê. D. and Law, M. L. (1956). Dental caries in the albino rat in relation to the chemical composition of the teeth and the diet. II . Variations in the calciumrphosphoru s ratio of the diet induced by changing the phosphorus content. J. Nutr. 58, 325-333. Wynn, W., Haldi, J., Bentley, Ê. D. and Law, M. L. (1957). Dental caries in the albino rat in relation to the chemical composition of the teeth and the diet. III . Composition of incisor teeth of animals fed diets with different calcium: phosphorus ratios. / . Nutr. 63, 57-64. Wynn, W., Haldi, J. and Law, M. L. (1962). Chemical composition of the molar teeth of albino rats fed two diets with different cariogenicity. J. dent. Res. 41, 41-45. Wyss, V. (1951). II disaggio spettografico del plombo in denti di individui normali e di individui esposti al rischio saturnino. Rass. Med. industr. 20, 40-47. Yoon, S. H., Brudevold, F., Gardner, D. E. and Smith, F. A. (1958). Fluorine in enamel, coronal dentin and roots of human teeth. / . dent. Res. 37, 25 (Abstract). Yoon, S. H., Brudevold, F., Smith, F. Á., Gardner, D. E. and Soni, N. (1960a). Distribution of fluorine in teeth and alveolar bone. / . Amer. dent. Ass. 61, 565-570. Yoon, S. H., Brudevold, F., Gardner, D. E. and Smith, F. A. (1960b). Distribution of fluorine in the teeth and fluorine levels in water. J. dent. Res. 39, 845-856. Zapanta, R. R. and Trautz, O. R. (1961). The separation of the mineral phase of mineralized tissues. / . dent. Res. 40, 702 (Abstract). Zelander, T. and Torell, P. (1964). Crystalline ferric compounds in dental enamel and dentine. Odont. Revy, Lund 15, 271-278.

C H A P T ER

CHEMISTRY

18

OF THE

F. B R U D E V O L D

MINERAL

AND R .

PHASE

OF

ENAMEL

SOREMARK

I. Introduction

247

II . Concentration s of Inorganic and Organic Material and Water

248

III . The Mineral Phase

249

A . Crystalline Nature B. Major and Minor Constituents

249 249

IV . Surface Chemistry—Distribution in Depth of Various Constituents A . Distribution of Mineral, Organic Matter and Water B. Constituents Decreasin g in Gradient from Surface C. Constituents Evenly Distributed D. Constituents Increasing in Gradient from Surface V. Topical Treatment of the Enamel Surface

251 251 253 259 262 265

VI . Enamel Caries

269

VII . Concluding Remarks

271

Reference s

I.

. . .

272

I N T R O D U C T I ON

i n appreciable concentrations . I n contrast, some elements, such as strontium, are fairly evenly distributed throughout the enamel, while a third group, which includes carbonate, is sparse in the surface and increases in concentration towards the dentine. The distribution of various mineral constituents, the mechanism of their deposition, and the significance of their presence wil l be discusse d to the extent that available knowledge and space permit. The reactions between topically applied agents and the enamel, and the changes in composition which occur in carious lesions wil l also be considered in so far as they concern the mineral phase.

The mineral of mammalian enamel is crystalline and has a lattice structure characteristic of hydroxyapatite. It contains a great number of elements which may be acquired during or after mineralization. The chemistry of the mineral phase is governed l properties of the apatite by the physical-chemica crystals and by the water which is located in the intercrystalline spaces and serves as a medium for diffusion. Due to the restricted diffusion, chemical activity is limited almost entirely to the outer portion of the enamel after its formation has been completed. As a result, a number of ionic species, e.g. fluoride, wil l accumulate in the surface enamel 247

248

F.

B R U D E V O LD

II. CONCENTRATIONS OF INORGANIC AND ORGANIC MATERIAL AND WATER

AND

R.

S Ô R E M A RK Table 1

PERCENTAGE OF MINERAL , ORGANIC MATTER AND WATER IN HUMA N ENAMEL BY WEIGHT AND BY VOLUM E

Enamel is unique among body tissues for its high mineral content and its low content of organic matter and water. Chemical analyses have usually shown an inorganic concentration between 95 and 96 % for the enamel of human permanent teeth (Armstrong and Brekhus, 1937; LeFevre and Manly, 1938) and between 92 and 93 % for the enamel of deciduous teeth (Bird et al, 1940). The amount of organic material varies considerably in enamel from different teeth but has been reported to be within the range of 0.5-2 % by most investigators (Armstrong and Brekhus, 1937; LeFevre and Manly, 1938; Deakins and Volker, 1941), with slightly higher values in deciduous than in permanen t teeth (Stack and Williams, 1952; Savory and Brudevold, 1959). Lack of a suitable method has prevented the accurate measuremen t of the water content. The usual approach has been to equate the amount of water present with the loss in weight sustained by enamel after heating at 100°C for several hours. This method has given concentration s slightly above 2 % for permanen t enamel (LeFevre and Manly, 1938; Burnett and Zenewitz, 1958) and nearly 3 % for deciduous enamel (Bird et al., 1940). Recent studies have shown that full y mineralized enamel contains a greater concentration of water than was indicated by earlier work, since appreciable amounts are firml y bound and wil l be released only after heating to temperature s considerably in excess of 100°C (Little, Cueto and Rowley, 1962a; Carlstrom, Glas and Angmar, 1963). A total of at least 4.0 % by weight may therefore be present in enamel of human permanent teeth. Table 1 shows the approximate percentag e of mineral, organic material and water in enamel by weight and by volume, as calculated from their respective densities. The density of the mineral was assumed to be 3.15, that of organic matter 1.2 and that of water 1. A recent quantitative microradiographic study (Angmar et al., 1963) showed the average space occupied by the mineral ranged from

Phase

Weight (%)

Mineral Organic Water

95.0 1 4.0

Volume

(%)

87 2 11

84.4 to 87.2 %, while the volume of water was about 1 2 %, a somewhat higher figure than the 1 1% given in Table 1. It was estimated that a portion of the water, roughly one quarter of the total, is loosely bound and associate d primarily with the organic material, while the remainder, the major portion, is firml y bound to the mineral. The latter fraction may be present as a hydration shell adjacent to individual apatite crystals, and/or be associate d with the mineral salt in some unknown manner. Current and future studies by means of X-ray and neutron diffraction, as well as by nuclear magnetic resonanc e determinations on single crystals, may be expected to increase our knowledge regarding the mechanism by which water is bound to the crystals and the organic matter. Since the water serves the important function of mediating diffusion of molecules and ions through the enamel, such knowledge wil l undoubtedly aid in elucidating the chemical behaviour of the enamel. I t is interesting that there is a relatively free flow of water through human enamel (Bergman, 1963; Poole, Tailby and Berry, 1963), probably involving the loosely bound fraction. The velocity of the flow has been found in vitro to be approximately 4 m m3/ c m2/ 24 hr, suggesting that significant quantities of water may pass through intact, mature enamel (Bergman and Siljestrand, 1963). Other liquids, including methyl, ethyl, and propyl alcohols also diffuse through the enamel, but passage of certain ions is impeded (Poole et al., 1963). Enamel thus appears to act as an ion exchanger but, because of many uncontrollable variables, e.g. lamellae or cracks which greatly

18.

C H E M I S T RY

OF

THE

MINERAL

facilitate the " n o r m a l" fluid movements , it has not been possible to characterize this behaviour in detail. The restricted passag e of ions in the enamel wil l be considered later with the surface chemistry of the enamel. III .

THE MINERAL

P H A SE

A . CRYSTALLINE N A T U RE

Under the electron microscope, enamel appears to consist of a mass of rod-like crystals, orientated essentially with their long axes parallel to the direction of the enamel prisms and separate d by exceedingly narrow spaces . X-ray diffraction studies have establishe d that the lattice structure of the crystals is that of apatite, the same as is found in dentine and bone. The largest and best developed enamel crystals average 1600 A (1200-2100) in length and 200 Â (150-250) in width, as measure d by X-ray diffraction (Glas and Omnell, 1960) and by electron microscopy (Ronnholm, 1962). Somewhat larger values based upon electron microscopy have also been reported (Frank, Sognnae s and Kern, 1960). It has to be borne in mind, however, that the observations of Glas and Omnell were made on hippopotamus enamel, those of Rônnholm on human enamel and those of F r a nk et al. on rat and human enamel. There is no doubt that the size of the crystals in enamel is greater than in dentine and bone. The volume of individual enamel crystals has been estimated to average more than 200 times that of dentine crystals (Glas, 1962). Since chemical reactions are confined to the surface of the apatite crystals, the larger size of the enamel crystal, associate d with a correspondingl y smaller surface area, gives enamel a greater stability or chemical inertia than other calcified tissues. The stability is the greater because the crystals are closely packed and the movement of water-borne ions in the spaces between them is restricted. Evidently the reactive capacity of the mineral depends on both the surface area of the crystals and the volume, content and character of the submicroscopi c spaces between them. Chemi-

P H A SE

OF

ENAMEL

249

cally, enamel may be regarded as an inorganic colloidal fibre structure composed of apatite crystals and water, "contaminated" with small amounts of organic as well as inorganic matter (Glas, 1962). Attempts to determine the internal accessible surface area of intact, full y mineralized enamel have been unsuccessful . It is apparently of small magnitude, since gas absorption measurement s of powdered enamel and dentine have shown that surface areas increase as the size of the particles is decrease d (McCann and Bullock, 1955), whereas the surface area in bone appears to be less dependent upon particle size (Hendricks and Hill , 1950). Since enamel mineral can exchange ions with body fluids only by diffusion, and since the passag e of many ions and molecules is highly restricted, the composition of surface enamel deviates from that of subsurface enamel in many respects . Before we elaborate on this statement , the chemistry of the total enamel and its relation to the chemical behaviour of apatite crystals wil l be briefly considered. B. M A J OR AN D M I N O R CONSTITUENTS

The concentration s of principal mineral constituents in intact enamel are listed in Table 2. Table 2 MAJOR INORGANIC CONSTITUENTS'1 OF HUMA N ENAMEL Constituent

Ca Ñ Ca: Ñ (by weight)

co2 Na Mg CI Ê a b c d

Thermal neutron activation analysis6 37.4 ± 18.3 ± 2.04 — 1.16 ± 0.36 ± 0.65 ± —

1.0 2.2

0.40 0.04d 0.30

Percentag e dry weight. Sôremark and Samsah l (1961). Compiled from the literature. Soremark (1964a).

Chemical analysis0 33.6 -39.4 16.1 -18.0 1.92-2.17 1.95-3.66 0.25-0.90 0.25- 0.56 0.19- 0.30 0.05- 0.30

250

F.

B R U D E V O LD

These values have been derived both from recent thermal neutron activation analyses (Soremark and Samsahl, 1961) and from the numerous chemical analyses reported in the literature. Although the considerable variation in concentrations seen in Table 2 to some extent reflects the different chemical and sampling procedures , there is no doubt that there are real and sometimes marked differences in the composition of enamel mineral. In one study, enamel from individual teeth of a single person varied as much in composition as that of teeth from different persons; perhaps having to do with the fact that mineralization occurs at different ages and hence under different metabolic conditions (Armstrong and Brekhus, 1937). X-ray diagrams of pure, synthetic hydroxyapatit e have shown that the unit cell, the smallest repeating unit, can be expresse d by the formula C a1 0( P O4) 6( O H ) 2 (e.g. Posner, PerlofT and Diorio, 1958). The presence in enamel apatite of constituents other than calcium, phosphate and hydroxyl ions is due to the capacity of the crystal lattice to substitute other ionic species of appropriate size and charge for these ions. Thus, for example, strontium and radium may replace calcium, vanadium or carbonate may exchange wit h phosphate , and fluoride with hydroxyl groups. I n addition there are many other ionic species, e.g. sodium, magnesium , bicarbonate and citrate, which may be located at the crystal surface. The ability to retain foreign ions is further enhance d by the ability of hydroxyapatite crystals to bind water and hydrated ions (Neuman and Neuman, 1958). Apparently, electrical asymmetry on the crystal surface sets up a powerful electrical field which pulls charged ions and water molecules towards it. In vitro radioactive studies have shown that layers of water adjacent to the crystal, termed the hydration shell, must be considered part of the crystal from a chemical point of view. The hydration shell probably represent s the principal portion of the water which is firml y held by the mineral phase. Exchange of ions between apatite and surrounding fluids can involve: (1) only the hydration

AND

R.

S Ô R E M A RK

shell, (2) the crystal surface, and (3) the body of the crystal (Neuman and Neuman, 1958). This versatile system permits the presence of a host of elements in the enamel without necessaril y being incorporated in the crystal lattice. The concentrations of a number of minor constituents of enamel mineral, determined by neutron activation or by chemical analysis, are given in Table 3. The exact location of many of these elements in enamel crystals is not known. The mechanism of acquisition of some constituents which are known to affect the physical properties of the enamel, and the significance of their presence , wil l be discusse d in the section of this chapter dealing with the surface chemistry of the enamel. I t wil l be noticed from Table 2 that the Ca : Ñ ratio of enamel mineral has been found to vary between 1.92 and 2.17 (by weight). With few exceptions, the ratio is below that of pure hydroxyapatite (2.15). A number of factors may contribute to the low ratio, including absorption Table 3 MINO R INORGANIC CONSTITUENTS" OF HUMA N ENAMEL

Constituent

F Fe Zn Sr Rb Br W Cu Mn Au Ag Cr Co V a

Parts per Soremark c Compiled d Soremark e Soremark b

Thermal neutron activation analysis^

388 ± I09d 276 ± 106 94 ± 22 4.9 ± \.6d 4.6 ± 1.1 0.24 ± 0 . 12 0.26 ± 0 . 11 0.54 ± 0.08 0.02 ± 0.01 0.0049 ± 0.0012" 0.0027 ± 0.0016 d 0.00024 ± 0.00009* < io- 5* million, dry weight. and Samsah l (1961). from the literature. and Lundberg (1964). (1964b).

Chemical analysis0 62-650 8-218 152-227 50-400



— — 10-100 0-18



0-100

— — —

18.

C H E M I S T RY

OF

THE

MINERAL

of excess phosphate on the crystal surfaces, or substitution of sodium, magnesium and other ions for calcium. I t has been suggeste d that hydrogen ions in the form of H 3 0 + can replace up to two calcium ions in the lattice (Neuman and Neuman, 1958). This exchange may take place when enamel is exposed to acid. Low Ca : Ñ ratios may also result from defects in the crystal structure, with calcium ions missing from certain positions in the lattice (Posner and Perloff, 1957). Such defective apatites can take up calcium, and this "repair" renders the apatite more resistant to acid dissolution (Likins, Posner and Steere, 1958). The low Ca : Ñ ratio in tooth and bone mineral has been discusse d by a number of workers (e.g. P. W. Arnold, 1950; Neuman and Neuman, 1953, 1958; Posner, 1960; McConnell, 1965).

IV. SURFACE CHEMISTRY—DISTRIBUTION IN DEPTH OF VARIOUS CONSTITUENTS A . DISTRIBUTION OF M I N E R A L , O R G A N IC M A T T E R AN D W A T ER

There is good evidence to show that surface enamel differs markedly from subsurface enamel. I t is harder (Caldwell et ah, 1957; Newbrun, Timberlake and Pigman, 1959) and less reactive to fluoride than underlying enamel (Brudevold et al., 1957a). Moreover, surface enamel has a reduced acid solubility as compared to subsurface enamel (Isaac et al, 1958a). It is also more resistant to the carious process. The greater resistance of surface enamel to dental caries is evident from the finding that in initial carious lesions the outermost enamel is only slightly affected, while marked decalcification takes place under the surface layer (Bergman and Engfeldt, 1954; Darling, 1956; Soni and Brudevold, 1959). Al l these findings are likely to be related to the many differences between the composition of the surface and the rest of the enamel. Briefly stated, surface enamel tends to contain a higher concentration of mineral (Thewlis, 1940; Soni and Brudevold, 1959; Angmar, Carl-

P H A SE

OF

ENAMEL

251

strom and Glas, 1963) and a lower concentration of water (Brudevold, Steadman and Smith, 1960). In addition, at least in human enamel, certain constituents, including fluoride, zinc and lead, accumulate in the surface enamel, while other constituents, including carbonate and magnesium , are sparse in surface as compared with subsurface enamel (Brudevold et al., 1960). The organic material is also unevenly distributed. It decrease s from the surface inwards for a fraction of a millimeter and then gradually increases to reach a maximum at the dentine-ename l junction (Savory and Brudevold, 1959). The approximate distribution of mineral, organic matter and water in enamel can be seen in Fig. 1. The gradual decreas e in mineral from the enamel surface to the dentine-ename l junction is usually observed by X-ray absorption studies. According to recent studies (Soni and Brudevold, 1959; Angmar et al, 1963), there is no marked zone of hypermineralization in the outermost enamel, as was suggeste d previously (e.g. Thewlis, 1940), nor is there an increase in mineral adjacent to the dentine, as was concluded on the basis of the strong negative birefringence found in this region (e.g. Keil, 1937). The difference in degree of mineralization between the outermost and innermost human enamel has been found to average 2.2 % by weight and 5.7 % by volume, and to range between 0.7 and 4.4 % by weight (Angmar et al, 1963). The distribution curve of the organic matter, superimpose d upon the inorganic curve in Fig. 1, was calculated from data on the concentration s of nitrogen (Savory and Brudevold, 1959) and on citrate and lactate (Brudevold et al, 1960) in successiv e layers of enamel. The remaining fraction needed to make up 100 % was assume d to be composed of water. Although the absolute values of mineral, organic matter and water may deviate significantly in individual teeth from the levels indicated in Fig. 1, it is believed that the distribution pattern shown occurs in the majority of cases. The distribution of water, with relatively small amounts in the surface enamel, is particularly

252

F.

B R U D E V O LD

Fig. 1. Distribution of water and organic and inorganic material in human enamel from surface to the dentineenamel junction (D.E.J).

interesting. As previously mentioned, the water is present in both loosely bound and firml y bound forms. Measurement s of the birefringence of enamel after drying at different temperature s have shown that most of the loosely bound water is located in the inner portion of human enamel (Carlstrom et al, 1963), possibly as a result of the lower concentration s of mineral and higher concentrations of organic matter in the inner enamel. Deakins (1942) has shown that the process of mineralization in the enamel involves the displacement of water by minerals, suggesting an inverse relationship between the extent of mineralization and concentration of water. According to this concept, the nearly complete displacemen t of loosely bound water from the outer enamel is the result of its particularly high content of mineral. A high degree of mineralization may indeed be expected in the superficial enamel because of its direct exposure to calcium and phosphate ions in

AND

R.

S Ô R E M A RK

tissue fluids before eruption and in saliva after eruption. Physical consideration s suggest that the process of crystal formation and growth can occur only as long as there is free passag e of ions. Charges on the surface of the crystal would increasingly restrict movement of ions as the spaces between the crystals diminish in size (Neuman and Neuman, 1958). The rate of mineralization, which is initiall y high, would gradually slow down. Although the body of the enamel is well calcified the possibility of further mineralization by ingress of ions from the enamel surface becomes less as the ionic pathways in the surface zone diminish in size through mineralization from the oral fluids to which the outer enamel is continuously exposed. The following discussion wil l show that many changes occur in the composition of the surface enamel with age. It is of interest that the concentrations of phosphate (dry weight) remain relatively constant after mineralization has been completed (Littl e and Brudevold, 1958). This finding is consistent with the theory that the chemical changes that occur mainly involve the surface of the enamel crystals and that the inner part of the crystal lattice is not affected. Measurements of successiv e layers of a great number of human teeth have shown a trend toward a decreas e in density of the external enamel with age, suggesting a slight loss of mineral (Table 4). Since Table 4 DENSITY OF SUCCESSIVE LAYERS OF HUMA N ENAMEL DRIED AT

100° Ca Erupted

Layerb

Unerupted

1 2 3 4 5

3.00 3.04 3.04 3.09

a b



Under 20 20-29 3.05 3.10 2.98 3.06 2.98

2.94 3.00 3.00 2.98 3.00

From Brudevold (1957). Numbered from surface inwards.

30-49

over 50

3.12 3.09 3.08 3.02

2.89 2.86 2.97 3.00 3.03

18.

C H E M I S T RY

OF

THE

MINERAL

a slight increase in enamel nitrogen has also been observed in the surface enamel of old teeth, it appears that small amounts of mineral, very likely the most soluble constituents , e.g. carbonate , are replaced in part by organic matter from saliva. The brownish pigment frequently seen in the enamel surface of the teeth of old people may well be associate d with the inclusion of such material. B.

CONSTITUENTS DECREASING I N FROM

G R A D I E NT

SURFACE

Several works have shown that radioactive isotopes administered intravenously to experimenta l animals rapidly accumulate in the surface layers of bone and teeth (e.g. Sognnaes , Shaw and Bogoroch, 1955). While the rate of diffusion of isotopes is relatively rapid in soft tissues, it is inversely related to the extent of mineralization in teeth and bone, as has been discusse d previously. In the highly mineralized enamel a number of elements with great affinity for the mineral phase accumulate in appreciable concentration s in the surface layer, but penetrate only to a limited extent into the body of the tissue. The uptake of various ions in the outermost enamel begins before eruption, as soon as calcification has been completed, by means of exchange reactions between the enamel surface and tissue fluids. This build-up continues after eruption because of similar reactions with oral fluids. The elements involved include fluorine, zinc, lead, radium, uranium and chlorine. 1. Fluoride Because of its caries-inhibiting effect, fluorine has been studied most extensively and wil l be considered first. Analyses of total h u m an enamel show low concentration s of fluoride (Armstrong and Brekhus, 1938) and a small but fairly consistent increase in levels with increase of fluoride in the drinking water (McClure and Likins, 1951). Some of the findings relating fluoride in the enamel and the drinking water are summarized in Table 5. The difficulty of correlating the generally low concentrations shown in Table 5 with the caries18

P H A SE

OF

ENAMEL

253

Table 5 FLUORIDE IN HUMA N ENAMEL AND DENTINE IN RELATION TO FLUORIDE IN

DRINKIN G WATER"

Fluoride (ppm) Source of sample

Water

Enamel

Dentine

Norway Washington, D.C., U.S. Illinois, U.S. Texas, U.S. Italy

0.2-0.4 0.0-0.2 1.1-1.2 2.5-5.0 3.5

79 102 135 345 599

86 240 360 762 1589

a

From McClure and Likin s (1951).

inhibiting effect of fluoride was resolved when surface and subsurface enamel was subjected to analysis, and it was found that several times greater concentration s occurred in surface than subsurface enamel (Jenkins and Speirs, 1953; Brudevold, Gardner and Smith, 1956a). The fluoride concentration s in successiv e enamel layers in teeth from persons of different ages from a low-fluoride area are shown in Table 6. The level of fluoride in the outermost layer (about 1/20 mm thick) is 20 or more times greater than the concentrations in the inner portion in some tooth groups. Among the permanent teeth, the lowest fluoride concentration s in the external enamel (331 ppm) occur in the teeth that are not fully formed (group 1). There is a continuous increase in the surface levels as the unerupted teeth mature. I n group 3, which includes impacted third molars of young adults, the concentration s in the surface are higher than those of erupted teeth, which include many first permanent molars from persons under 20 years of age. The greatest concentration s are found in erupted teeth from persons over 50 years of age, clearly demonstratin g posteruptive fluoride acquisition. The fluoride level decrease s wit h increasing enamel depth to a fairly constant level of 30-60 p pm in all tooth groups. The enamel of deciduous teeth contains less fluoride than that of permanent teeth, but the gradient from the surface inwards is similar. Only

254

F.

BRUDEVOLD

Table 6 CONCENTRATION OF FLUORINE (PPM) OF A SH IN SUCCESSIVE LAYERS OF ENAMEL OF HUMA N TEETH FROM A LOW-FLUORIDE AREA"

Unerupted0 &

ayer Deciduous (1)

1 2 3 4 5 6 7 8 9

448 126 43 56

— — — — —

(2)

(3)

Erupted Under Over 20 20-29 30-39 50

331 528 847 571 1205 101 232 391 269 531 57 150 201 113 324 33 96 172 90 233 — — 88 48 180 — — 64 — 77 — — 40 — 67 — — 26 — 77 — — 32 — 105

1070 553 368 249



— —

— —

1247 667 404 315 176 147 97 58



a

F r om Brudevold et al. (1956a). Numbered from surface inwards. c Group (1): not fully formed from Buffalo, New York; (2): fully formed from Buffalo, New York; (3): fully formed from Rochester , New York.

h

small amounts of human enamel are mineralized in utero (Reiss, 1961). Since the enamel surface of deciduous teeth continues to be mineralized postnatally, the surface accumulation of fluoride which is important for caries resistance is due to fluoride exposure after birth. The deposition of fluoride in the enamel occurs i n three stages (Brudevold et al, 1956a): 1. Low concentrations , reflecting the low levels of fluoride present in tissue fluids, are incorporated i n the apatite crystals during their formation. These concentrations , deposited during the period of calcification, correspond to those found in the inner layers of the enamel. 2. After calcification is complete, but before eruption, more fluoride is taken up by the external enamel which is in contact with tissue fluids. There is only slight penetration of fluoride into the underlying enamel, which is virtually blocked off by the high mineralization (minute intercrystalline spaces ) of the outer layer.

AND

R.

S Ô R E M A RK

3. After eruption and throughout life, the surface enamel continues to take up fluoride from the oral environment. It is evident from the low concentrations of fluoride in the innermost portion of the enamel (layers 8 and 9) that there is no significant diffusion of fluoride from the pulp via the dentine. Whil e the foregoing findings demonstrat e the effect of time of exposure in the uptake of fluoride, the concentration of fluoride in the environment of the tooth is also important, as has already been suggeste d on the basis of the data in Table 5. The marked effect of fluoride in the drinking water on the level of fluoride in surface enamel can be seen i n Table 7. The distribution of fluoride in enamel of persons of comparable ages (under 20 years) who had continuously used drinking water with 0.1, 1.0, 3.0, and 5.0 p pm of fluoride is shown here. While the concentration of fluoride in surface enamel increases by 390 p pm when the level in the water is raised from 0.1 to 1.0 ppm, the correspondin g increase in the 6th layer is only 87 ppm. The relatively slight increments of fluoride seen in the subsurface enamel must be related to the finding that the concentration of fluoride in serum is not significantly different for persons who drink water containing only traces or up to 3 p pm of fluoride (Singer and Armstrong, 1960). The increase in blood level which has been shown by means of Table 7 PARTS PER MILLIO N OF FLUORIDE IN SUCCESSIVE LAYERS OF ASHED ENAMEL OF PERSONS UNDER 20 YEARS OF AGE, FROM COMMUNITIES WITH 0.1, 1.0, 3.0 AND 5.0 PPM FLUORIDE I N THE WATER SUPPLY*

Layerb

0.1

1.0

3.0

5.0

1 2 3 4 5 6

499 162 108 63 76 42

889 363 255 171 133 129

1930 1010 992 262 353 152

3370 1710 1124 926 811 570

a 6

From Isaac et al. (1958b). Numbered from the surface inwards.

18. C H E M I S T RY

OF

THE

MINERAL

5 0 00 4 0 00 3 0 00 1

2 0 00

t:

1000

e

8 00

?

6 00

CL CL

^

4 00 300 2

0

00

10

20

30

40

Tooth age in years

Fig. 2. Rate of F uptake by human surface enamel at different levels of F in water supply.

radioactive isotope techniques (Carlson, Armstrong and Singer, 1960) to occur after ingestion of fluoride is too minute and transient to be detectable by chemical analyses at these low concentration s of fluoride in the drinking water, but it is nevertheles s of sufficient magnitude significantly to influence the deposition of fluoride in the enamel. Analyses of enamel from a great number of human teeth have shown that the pre-emptive uptake of fluoride by surface enamel increases markedly with increase of fluoride in the drinking water (Brudevold et al., 1960). These findings, summarized in Fig. 2, also demonstrate d that the more fluoride-saturate d the surface enamel is at the time of eruption, the less tendency it has to accumulate additional amounts. A t the levels of 0.1 and 1.0 ppm fluoride in the water supply, the posteruptive uptake of fluoride is particularly apparent. The posteruptive acquisition of fluoride by the enamel is mostly due to direct contact of the drinking water, since salivary fluoride, lik e serum fluoride, is only to a slight extent increased by ingestion of water-borne fluoride at these levels (McClure, 1941). I t is interesting that the surface enamel of normal teeth has an equivalent or higher amount of fluoride than the subsurface enamel of mottled teeth

P H A SE

OF

ENAMEL

255

(Table 7). This suggests that mottling is not caused by the actual amounts of fluoride deposited in the enamel, but rather by an increase in concentration s present in the enamel organ during matrix formation or mineralization. Perhaps the toxic effect of fluoride on the ameloblast is most marked at the time the last portion of the enamel matrix is laid down, since histologically the deficiencies in the interprismatic substanc e of mottled enamel is most marked in the outer third of the enamel (Newbrun and Brudevold, 1960). The reduction in the prevalence of dental caries when the fluoride content of the water is increased from 0.1 to 1.0 p pm must be related to the difference, amounting to over 300 ppm, in the fluoride content of the external enamel from such areas (Table 7). Since caries begins at the enamel surface i t is very likely that the caries-reducin g effect of water fluoridation at the level of 1 p pm is due mostly to the increase in the fluoride content of the enamel surface. This is in accord with the concept of Armstrong and Brekhus (1938) that there is a direct relationship between the fluoride content of human enamel and caries susceptibility. However, since many factors in addition to fluoride influence the carious process, the fluoride-caries relationship is not a simple one, and teeth with the same fluoride content do not necessaril y show the same incidence of dental caries. This is clearly demonstrate d by the fact that there are no differences in the fluoride contents of sound enamel from carious and noncarious teeth from equivalent fluoride areas. Similar conclusions were drawn by G. E. Harrison et al. (1959) and McClure and Likin s (1951). Wit h regard to the mechanism of fluoride acquisition by calcified tissues, the following three steps have been postulated: (1) diffusion into and exchange with anions in the tightly held water adjacent to the crystal, (2) fixation in the surface of the hydroxyapatite by replacing hydroxyl or bicarbonate ions, (3) migration into the body of the crystal and incorporation in the interior lattice structure (Neuman and Neuman, 1958). According to studies with P3 2, which by analogy would apply to fluoride, the first step is very rapid, the second

256

F.

B R U D E V O LD

is slower and the third step is so slow that significant incorporation of fluoride in the body of enamel crystals is unlikely during the lif e span of the tooth. This concept of fluoride fixation explains the extreme rapidity with which fluoride reacts with the enamel (Brudevold et al, 1957b). It also explains the fact that the concentration of fluoride in enamel, even from high fluoride areas, is far below the theoretical saturation level of pure fluorapatite (about 3.6 % ). Conceivably, higher concentration s are present in the outer molecular layers of the enamel than those shown for the surface layers in Table 7, which had a thickness of about 0.05 mm. If the reasonabl e assumption is made that the fluoride gradient continues in these surface layers, the maximal concentration at the ultimate surface can be shown to be of the order of 0.7-0.8 %. Evidently pure fluorapatite cannot be formed under physiological conditions. 2. Zinc Zinc is the only other micro-element which so far has been found to occur in h u m an enamel in concentrations approaching those of fluoride. It also accumulate s in surface enamel, and the level can be over ten times greater in the outermost enamel than in the enamel adjacent to the dentine (Brudevold et á/., 1962b). The distribution of zinc in enamel of teeth of different ages obtained from one community is shown in Table 8. The high concentrations present in the enamel of unerupted teeth show that most of the zinc is deposited before eruption. Pre-eruptive deposition is undoubtedly favoured by the high concentration s of zinc known to be present in extracellular fluids of young individuals. Plasma zinc decrease s with age, the level being three times greater in human foetuses than in adults (Berfenstam, 1952). Although erupted teeth show greater concentration s than unerupted teeth from the same geographica l area, a gradual increase in concentration with age has not been found. It is nevertheles s possible that zinc is acquired by the enamel surface throughout life, since this is known to be the case with bone in rats (Taylor, 1961). Foods differ in zinc content, and

A N D

R.

S Ô R E M A RK

Table 8 ZIN C (PPM) IN SUCCESSIVE LAYERS OF ENAMEL OF HUMA N TEETH

FROM AUGUSTA,

MAINE "

Erupted Layer6

Unerupted

Under 20

30-49

1 2 3 4 5 6 7 8 9 10 11 12 13

1300 110 930 930 610 —

2100 1200 1100 970 700 730 430 370 300 270 230 180 —

1500 1100 900 770 730 670 570 510 470 400 250 230 210





— — — —



" F r om Brudevold et al. (1963b). b Numbered from the surface inward.

thus the age effect may be masked by differences in zinc ingestion in different age groups. The level in saliva is known to increase after ingestion of zinc, but quantitative values are not available. There are marked differences in the zinc concentration of teeth from different regions. Table 9 gives the level of zinc in the outer, middle and inner portions of enamel from a number of communities in the United States and from Greenland. The fivefold difference observed in surface conTable 9 ZIN C (PPM) IN HUMA N ENAMEL FROM DIFFERENT AREAS"

Big Doland, Schenectady , Augusta, Lake, South Maine Texas Dakota Greenland Enamel New York Surface Middl e Inner

1700 400 350

2100 400 180

900 400 200

" F r om Brudevold et al. (1963b).

1800 700 230

430 190 —

18. C H E M I S T RY

OF

THE

MINERAL

centrations of Eskimo teeth and some of the American tooth groups undoubtedly reflect dietary differences. Daily adult consumption of zinc is normally in the range of 10-15 mg in N o r th America, but may be as high as 24 mg on an exclusively meat diet (Underwood, 1956). It has been postulated that persons with tuberculosis had higher concentration s of zinc in the teeth than healthy individuals (Cruickshank, 1940). However, the residential and dietary background of the subjects was not considered in that study and, since the differences noted occur in normal populations, they are more likely to be related to differences in ingestion of zinc than to disease processes . Although zinc is readily acquired by hydroxyapatit e and competes with Ca++ for positions on the crystal surface (Brudevold et al, 1963b), it also complexes readily with proteins and may, therefore, be partially organically bound in the enamel (Graig and Sergei, 1960). Unlik e fluoride, zinc occurs in appreciable concentration s in many soft tissues. The significance of the presence of zinc in teeth is uncertain. Enamel exposed to zinc has decrease d acid solubility (Brudevold et al, 1963b), but there is no evidence that the application of zinc to the enamel surface reduces caries in m an (Ast, Bushel and Chase, 1950; Pelton, 1950; Anderson and Knutson, 1951) or in experimenta l animals (McClure, 1948; Mansell and Hendershot , 1960). H a u m o nt (1961) found high concentration s at points of calcification in bone, but was unable to prove that zinc was necessar y for the calcification process. However, the effect of zinc on tooth formation and mineralization, and the relation between the concentration s of zinc in surface enamel and caries susceptibility, must be clarified before this element can be dismissed as a factor in dental health. 3. Lead The distribution of lead in enamel from persons of different ages who were lifelong residents of one community is shown in Table 10. Both unerupted and erupted teeth show relatively high concentra-

P H A SE

OF

257

ENAMEL

tions in the external layer and rapidly decreasin g values in underlying successiv e layers. The concentration of lead increases with age, and this increase is more marked in the external than in the internal enamel. Although much less lead than fluoride is present in the enamel, it is evident that the pattern of distribution of the two elements is similar. Both are acquired by the external enamel throughout life, and the reaction with the enamel appears to be primarily a surface reaction with littl e penetration into the body of the enamel. Pre-eruptive deposition of lead in human deciduous teeth is insignificant, suggesting low exposure to lead during infancy (Altshuller et al, 1962). Posteruptive deposition increases with time and is greatly augmente d in cases of lead poisoning. Although these findings are concerned with whole teeth, and not specifically with enamel, they are mentioned because they demonstrate the relationship between ingestion of lead and deposition in dental tissue. The significance of the presence of lead in teeth has not been established . Workers in lead plants have about the same caries experience as other industrial workers (Aston, 1952). Topical applications of lead fluoride failed to reduce caries in two studies (Bibby, DeRoche and Wilkins, 1947; Galagan and Knutson, 1947) and, in a third study Table 10 CONCENTRATION OF LEAD (PPM) IN SUCCESSIVE LAYERS OF HUMA N ENAMEL 0

Erupted Layer6

Unerupted

Under 20

20-29

30-49

Over 50

1 2 3 4 5 6

210 130 67 60 55 47

350 220 180 74 35 —

360 260 105 85 65 54

520 430 280 200 — —

550 460 420 310 156 152

á b

From Brudevold and Steadma n (1956a). Numbered from surface inwards.

258

F.

B R U D E V O LD

(Klinkenberg and Bibby, 1950), no greater effect was found than has been observed with sodium fluoride. I n spite of these negative findings, it is possible that the presence of lead may render the teeth more resistant to acid decalcification. This is suggeste d by the observation that powdered human enamel exposed to lead salts has a decrease d solubility (Buonocore and Bibby, 1945; Manly and Bibby, 1949). However, the solubility-reducing effect of lead needs further investigation because of the contradictory findings on intact enamel which have been reported. Thus, exposure of the enamel surface to lead has been found to reduce acid solubility effectively (Ericsson, 1950-1951), moderately (Massler, 1954) or not at all (Brudevold and Little , 1951). Nothing is known about the process of deposition of lead in enamel, but studies on bone suggest that the lead exchange s with calcium (Aub et al, 1925). More recent work indicates that lead may first be absorbed on the surface of the apatite crystallites and that it may eventually assume a position withi n the crystal lattice (MacDonald et al, 1951a). Lead apatite can be produced under laboratory conditions (Posner and Perloff, 1957). N o studies have been done on the mobility of lead in the enamel, but studies on bone indicate that lead may be withdrawn from calcified structures. For instance, citrate, acid, or alkaline agents deplete bone of lead, as shown by increased urinary output (Kety and Letonoff, 1941) and, under certain conditions, lead may be mobilized from bone more easily than calcium (Gulberg and Swenson, 1943). The oral environment must be generally unfavourable for the mobilization of lead from the tooth surface, for otherwise the high concentrations found in the surface of intact enamel would be inconceivable. I n view of the findings cited above, however, it is likely that lead may be lost from surface areas where acid conditions prevail and it seems doubtful whether the presence of lead contributes materially towards making the enamel more resistant to the action of acid. There is some evidence that lead leaches from enamel

AND

R.

S O R E M A RK

faster than phosphate at pH 4 (Brudevold and Steadman , 1956a). This was concluded from the finding that the lead : phosphorus ratio of the outer layer of enamel, etched in an acetate buffer, was higher than would be expected. A t least at this low p H, lead is loosely bound in the enamel and goes into solution faster than some of the other constituents of the enamel. 4. Iron This mineral also accumulate s in the outermost portion of the enamel. Concentration s varying from 200 to 280 ppm, and from 120 to 220 ppm, have been reported in surface and subsurface enamel respectively of newly erupted human premolars (Torell, 1957). Correspondin g values for teeth of 30- to 40-year-old persons were 420-640 p pm and 200-310 ppm. These findings suggest an increase in iron with age in the superficial portion of the enamel. Considerably lower levels of iron (25-60 ppm) have been found in surface enamel of teeth from various communities in the United States, but approximately three times greater concentration s were consistently present in surface than in subsurface enamel of both unerupted and erupted teeth (Steadma n and Brudevold, unpublished observations , 1963). Analyses of total enamel have shown considerable variation in the iron content of individual human teeth (Lowater and Murray, 1937), probably due to differences in iron ingestion. Human enamel from areas with 5 ppm and 0.1 ppm of fluoride have been found to contain similar levels of iron (Steadman and Brudevold, unpublished data, 1963). The form in which iron is deposited in the enamel and the mechanism of its acquisition are not known. There is some evidence that iron is a component of the organic matrix of the enamel (Roseburg, 1934) and it has been postulated that it may serve as a bridge between the matrix and the mineral phase (Torell, 1955). However, in view of the low concentration of iron in enamel, such a mechanism would be of limited significance. Although iron forms insoluble phosphates , there is

18. C H E M I S T RY

OF

THE

MINERAL

P H A SE

OF

ENAMEL

259

Table 11 PIGMENTATION OF R AT INCISOR TEETH AND IRON CONTENT OF THE ENAMEL"

Pigment score F e%

2.62 ± 0 . 12

1.98 ± 0.11

1.47 ± 0.11

1.20 ± 0.10

0.38 ± 0.09

0.19

0.18

0.13

0.14

0.07

" From McClure and McCann (1960).

no indication in the literature that its presence in enamel is associate d with resistance to caries. I n rat incisors an iron-containing pigment is present in the surface layer of the enamel. The pigment is formed within the ameloblasts and incorporated in the enamel just before its formation is completed (Stein and Boyle, 1959). Nutritional deficiencies and metabolic disturbance s which interfere with iron metabolism tend to prevent formation of the pigment (Pindborg, 1953). The close correlation between the extent of pigmentation and the content of iron in the enamel of rat incisors is shown in Table 11 taken from a study by McClure and M c C a nn (1960). I n this study the decrease in pigmentation resulted from ingestion of diets which were high in magnesium and calcium, and low in phosphate . Table 12 CONCENTRATION OF T I N (PPM) IN SUCCESSIVE LAYERS OF HUMA N ENAMEL"

Erupted Uneruptedc

Filled

Layer6

(0

(2)

20

1 2 3 4

0.5 0.5 0.5 0.5

1.5 1.5 1.5 1.0

20.0 4.0 2.5 2.0

Intact

20-29 30-49 65 21 28 17

80 22 Lost 16

" F r om Brudevold and Steadma n (1956b). b Numbered from surface inwards. c Group (1): incompletely formed teeth; (2): fully formed teeth.

50 Mottled 7.0 2.0 0.0 0.0

3.5 2.0 0.5 1.0

5. Tin Lik e fluoride and lead, tin is consistently found i n enamel and dentine (Lowater and Murray, 1937), but only in small concentrations . Table 12 shows the level of tin in successiv e layers of enamel from persons of different ages. There is a tendency towards surface accumulation in the enamel. However, only teeth which had been filled with amalgam contained significant amounts, suggesting that the presence of tin stems more from dental restorations than from food, drinking water or saliva. The low concentration s of tin in enamel parallels the low levels found in bone ( < 1 ppm) and soft tissues (Kehoe, Cholak and Story, 1940). Systemic absorption of tin appears to be minimal as a result of the tendency of tin to form insoluble complexes at physiological pH (Dundon and Hughes, 1950). However, tin is readily deposited in human surface enamel at low pH (Brudevold et al., 1956b). Under this condition tin forms a coating on the enamel surface, but there is also some penetration into the enamel (Cooley, 1961). Tin is probably deposited in the form of tin phosphate and oxide (Scott, 1960; Cooley, 1961). The reactions between stannous fluoride and enamel wil l be considered further in the discussion of topically applied fluoride. C. CONSTITUENTS EVENLY DISTRIBUTED

1. Strontium This occurs those Smith,

element is always present in enamel and in concentration s (dry weight) similar to in dentine (Steadman , Brudevold and 1958). F r om the data in Table 13, which

260

F.

B R U D E V O LD

AND

Table 13 STRONTIUM (PPM) IN SIX SUCCESSIVE ENAMEL LAYERS OF UNERUPTED AND ERUPTED TEETH FROM PERSONS UNDER 2 0 AND 3 0 - 49 YEARS O LD IN AUGUSTA, MAINE "

Layer6

Unerupted

Under 20

30-49

1 2 3 4 5 6

100

120 120 125 140 110 80

105 110 125 112 150 125

— —

100



110

" F r om Steadman , Brudevold and Smith (1958). b Numbered from the surface inwards.

shows the distribution of strontium in enamel of unerupted and erupted teeth from one community, it is evident that strontium is deposited fairly evenly during enamel formation. The level of strontium varies considerably in enamel of different teeth and appears to be correlated with the concentration s present in food and drinking water. Analyses of teeth from different geographica l areas have shown a range of strontium in human enamel from 25 to 600 ppm. Representativ e data are shown in Table 14. Strontium can be deposited in the apatite crystal either by the replacemen t of surface-absorbe d calcium or by substitution of intracrystalline

R.

S Ô R E M A RK

calcium (MacDonald et al, 1951b; G. Å. Harrison et á/., 1959; Christensen , 1962). It has been reported that calcium is favoured over strontium in both these processes , and that the discrimination against strontium is greater in slowly grown, large crystals than in small, rapidly formed crystals (Likin s et al, 1960). The latter explanation could account for the slightly higher concentration of strontium in the dentine than in the enamel measured on an ash basis (dry weight concentrations are similar). I t has been claimed that the fluoride intake from drinking water affects the deposition of strontium in teeth and bone (Kerwin, 1958). This is not so; the acquisition of these elements by calcified structures is independen t and unrelated. The lack of correlation between fluoride in the drinking water and the level of strontium in enamel is shown in Table 15. The presence of strontium in teeth and bone is of particular interest because of the possible fixation of Sr9 0, one of the fission products of atomic explosions, in these tissues. Concentration s of 0.1-0.4 μμC per gram calcium have been observed i n human permanent teeth from Great Britain (Bryant, Henderson and Holgate, 1960). In deciduous teeth from St. Louis, United States, obtained from children born in the years 1951-1952 and 1954, concentration s increased from 0.16-

Table 14 STRONTIUM (PPM) IN SUCCESSIVE ENAMEL LAYERS IN HUMA N TEETH FROM DIFFERENT GEOGRAPHIC AREAS0 IN THE U . S. AN D FROM GREENLAND AND TONGA

Layer6 1 2 3 4 5 6 a b

Big Lake, Texas 270 260 230 260 260 350

Post, Texas 160

Ã-

Clovis, New Mexico

Augusta, Maine

25

105 110 125 112 150 125



É60

42 —

200



F—

From Steadman , Brudevold and Smith (1958). Numbered from the surface inwards.

100

Lewiston, Maine 60 60 —

60

Greenland

Tonga

260 275 295 295

320 400 470 450 600 320









18.

C H E M I S T RY

OF

THE

MINERAL

Table 15 COMPARISON BETWEEN FLUORIDE IN WATER SUPPLY AND DEPOSITION OF STRONTIUM IN HUMA N ENAMEL"

Community Post, Texas Doland, South Dakota Clovis, New Mexico Big Lake, Texas Augusta, Maine Lewiston, Maine Godhavn, Greenland Udsted, Greenland

F in water (ppm)

Sr in enamel (ppm)

5.0 3.6 2.2 2.1 Trace Trace Trace Trace

170 160 50 270 125 60 300 280

" F r om Steadma n et al. (1958).

0.20 /x/xC to 0 . 5 6 - 0 . 73 ì/x C per gram calcium (Rosenthal, Gilster and Bird, 1962). The radiation from the deciduous teeth is indicative of that in bone, since the ratio S r90 : Ca is the same in teeth and bone. 2. Copper I n contradistinction from the micro elements so far considered , the concentration of copper in enamel does not appear to change with age. This is shown in Table 16, where the concentration of copper in four successiv e layers of enamel from teeth of different ages is given. It is evident that copper is fairly evenly distributed. There are small differences from layer to layer within each group, but no definite pattern of distribution. Somewhat lower concentration s of copper are found in enamel of unerupted than of erupted teeth. However, there is no indication that enamel absorbs significant amounts from food, drinking water or saliva, which are known to contain small amounts of copper (Dreizen, Spies and Spies, 1952), since the amounts found in the different age groups of erupted teeth are similar. There is considerable variation in the concentration of copper in enamel from different teeth (Brudevold and Steadman , 1955). Up to 110 ppm has been found in the surface enamel, but subsurface enamel rarely contains more than 5 0-

P H A SE

OF

261

ENAMEL

60 ppm (Steadma n and Brudevold, unpublished data, 1963), and may contain only traces (Table 3). High concentration s may be due to diffusion into the tooth of copper from amalgam, gold, and cement restorations (Soremark et al, 1 9 6 2; Bergenholtz, Hedeg r d and Soremark, 1965). Although it has been reported that certain copper salts reduce the acid solubility of powdered human enamel (Buonocore and Bibby, 1 9 4 5; Muhler, Boyd and Van Huysen, 1950), no such effect was found after application to intact enamel (Ericsson, 1950-1951). The fact that many copper-containin g proteins are known to occur in animals and plants suggests that copper may be associate d with the organic matter in enamel. The significance of its presence is not known. It has been reported that human enamel with a marked brownish colouration has a high copper content (Rygge, 1939), but the ordinary brown stain frequently acquired by enamel of older persons is unrelated to the concentration of copper (Brudevold and Steadman , 1955). As copper is always present in the enamel and traces of copper in protein are known to catalyze enzymic pigment formation, it is possible that copper may be a factor in the natural colouration of teeth. No correlation has been found between the copper content of enamel and enamel hypoplasia or caries (Brudevold and Steadman , 1955). Table 16 DISTRIBUTION OF COPPER (PPM) IN SUCCESSIVE LAYERS OF HUMA N

ENAMEL"

Erupted Mayer*

Unerupted

Under 20

20-29

30-49

Over 50c

1 2 3 4

7 7 7 5

20 15 30 25

17 26 25 26

20 20 20 18

20 22 13 12

" F r om Brudevold and Steadma n (1955). b Numbered from the surface inwards. c Intact teeth only.

F.

262 D.

CONSTITUENTS INCREASING IN FROM

B R U D E V O LD G R A D I E NT

SURFACE

1. Carbonate Whil e many micro elements accumulate in the surface enamel, certain enamel constituents have a reverse pattern of distribution and are present in lower concentration s in surface than in subsurface enamel. These constituents include carbonate, sodium and magnesium . The distribution of carbonate (percentag e of C 0 2 dry weight) in enamel of human unerupted and erupted teeth of different ages from a low fluoride area is given in Table 17. There is a gradual increase in concentration from the surface inwards in all tooth groups. The carbonate gradient continues to the dentineenamel junction (not shown in Table 17), and concentrations are almost twice as great in the innermost enamel as in the surface enamel (Littl e and Brudevold, 1958). The mechanism which brings about these differences has not been established . Since the carbonate level in tooth and bone mineral is related to the C 0 2 : Ñ ratio of serum (Sobel and Hanok, 1948), it is possible that the C O S : Ñ ratio of the fluid in the enamel organ decrease s with Table 17 PERCENTAGE CARBONATE ( C 0 2 D RY WEIGHT) IN SUCCESSIVE LAYERS OF ENAMEL FROM HUMA N PERMANENT TEETH OF DIFFERENT AGES"

Erupted

Unerupted

Layer5 1 2

3 4 5 6

Not fully Fully formed formed Under 20 20-29 30-49 Over 50 1.65 1.80 2.01 2.04

— —

1.72 1.73 1.79 1.90 2.03 2.11

1.66 1.88 2.04 2.17 2.21 2.36

1.51 1.59 1.70 1.77 1.86 1.99

" F r om Littl e and Brudevold (1958). h Numbered from the surface inwards.

1.35 1.46 1.68 1.96 2.21 2.22

1.38 1.57 1.66 1.94 2.10 2.00

AND

R.

S Ô R E M A RK

advance in mineralization. It is also possible that the carbonate is located at the surface of the enamel crystals and that the surface area is greater (i.e. smaller crystal size) in internal than external enamel. N o ne of these possibilities has been supported by experimenta l evidence. Table 17 shows that the carbonate level in human surface enamel tends to increase slightly during the pre-eruptive period. A similar increase has been observed with age in bone, apparently reflecting concomitant increase in the C 0 2 : Ñ ratio of blood (Neuman and Neuman, 1953). After eruption there is a gradual decreas e in carbonate in the surface enamel, which can similarly be attributed to the much lower C 0 2 : Ñ ratio in resting saliva than in serum (Gron et al, 1963). I t has been reported that the level of carbonate in enamel decrease s as a result of drinking fluoridated water (Nikiforuk, 1961). Analysis of the enamel of deciduous teeth from individuals who lived in a community with 0.1 ppm of fluoride in the water supply showed higher concentration s of carbonate than enamel from persons who used water containing 1.0 and 1.6 ppm. The assertion is supported by the observation of several workers that increased deposition of fluoride in bone is associate d with a decreas e in carbonate (e.g. Zipkin, McClure and Lee, 1960). Mottled enamel, however, has been found to have higher concentration s of carbonate (Bowes and Murray, 1936) or similar concentrations (Littl e and Brudevold, unpublished data, 1963) compared to normal enamel. In the teeth of pigs no relation was observed between fluoride and carbonate concentration s (Kick et al., 1955). The location of carbonate in the mineral phase of teeth and bone has been a controversial subject for several years. There is no evidence of the presence of amorphous calcium carbonate in elephant enamel (Elliott, 1961). X-ray diffraction of calcium phosphate precipitates has shown that carbonate can be incorporated in the apatite lattice, but that crystallization is disturbed in proportion to the concentration of carbonate present (Trautz, 1960). When located on the surface of the crystal it may

18. C H E M I S T RY

OF

THE

MINERAL

be present as bicarbonate , and it exchange s with phosphate on a mole for mole basis (Gron et al., 1963). Since presence of carbonate increases the birefringence of apatite (Geiger, 1950) and the intrinsic birefringence of enamel is greater than that of pure apatite (Carlstrom et al., 1963), it is apparent that a substantia l part of the carbonate present is incorporated in the lattice. F r om findings of an increased preferential acid dissolution of carbonate from powdered enamel with decreas e in particle size, it has been concluded that carbonate is evenly distributed in the unit cells of the apatite lattice (Little, 1961), thus supporting McConnelPs concept (1952) that enamel mineral may be considered to be a carbonate apatite. There is still uncertainty about the significance of the presence of carbonate in the enamel. Incorporation of carbonate in synthetic hydroxyapatite does increase its solubility (Gron et al, 1963), which is in accord with the stronger binding of calcium to phosphate than to carbonate in the crystal lattice (Trautz, 1960). It has been reported that low carbonate levels in teeth are associate d wit h caries resistance in rats (Sobel et al., 1960), but other studies do not support this view (Wynn et al., 1956, 1957; McClure and McCann, 1960). 2.

Magnesium

The concentration of magnesium in h u m an enamel has been reported to range from 0.28 to 0.55 % (Bowes and Murray, 1935; Armstrong and Brekhus, 1937; Ockerse, 1943; M c C a nn and Bullock, 1955). Hypoplastic enamel appears to have approximately the same concentration s of magnesium as normal enamel (Bowes and Murray, 1936). Layer analyses of intact enamel have shown increasing concentration s from the surface inwards and approximately three times greater concentrations in the innermost than in the superficial portion. Representativ e figures for surface, middle and inner layers are respectively 0.11, 0.18 and 0.30 % dry weight (Brudevold, A m d ur and Rasmussen , 1962a). Similar findings were obtained in unerupted and erupted teeth, suggesting that the deposition of magnesium is primarily a pre-eruptive

P H A SE

OF

ENAMEL

263

process. No data are available on the effect of age on magnesium in the enamel. Experiments in vitro have shown that magnesium is taken up by hydroxyapatite more readily at high than at low p H. Solution-magnesiu m displaced solid-phase-calciu m by molar ratios of approximately 1 : 1 at pH 6.4, 2 : 1 at pH 7.4, and 7 : 1 at pH 8.4. These findings suggest that the uptake of magnesium is depresse d by the greater concentration s of calcium released from the hydroxyapatite with decreas e in p H. This hypothesis, that magnesium competes with calcium for positions on the crystal surface, has been verified in other experiments (Brudevold et al., 1962a). These investigations also showed that magnesium interfered with the precipitation of hydroxyapatite, and that presence of magnesium in the precipitates increased their solubility both at neutral and acid p H. Several workers have observed that increased fluoride deposition in bone and teeth of experimental animals is accompanie d by an increase in magnesium (Kick et al., 1955; McCann and Bullock, 1957; Weidmann, Weatherell and Whitehead, 1959; Zipkin et al, 1960). For example, ingestion of 100 ppm of fluoride in the drinking water more than doubled the magnesium in the rat incisor enamel (increase from 0.25 to 0.60 %). However, human enamel (and dentine) from high and low fluoride areas have failed to show consistent differences in magnesium (Ockerse, 1943; Bowes and Murray, 1936). This latter work should be repeated with the improved methods of analysis now available. Most of the magnesium in enamel is probably located on the surface of the apatite crystals. In view of the large dissimilarity in ionic radii of calcium and magnesium (1.33 Â and 0.70 Â respectively) it is unlikely that significant amounts are present in lattice positions (Palache, Berman and Frondel, 1960). The finding that magnesium may be leached from powdered h u m an enamel at a greater rate than calcium and phosphate (McCann and Bullock, 1955; Karshan, Weiner and Stofsky, 1934) shows that it is not firmly bound to the mineral phase.

264

F.

B R U D E V O LD

The significance of the presence of magnesium in enamel is uncertain. No difference has been found in the magnesium concentration of sound enamel from caries-free and carious human teeth (Armstrong and Brekhus, 1937; Tefft, French and Hodge, 1941; Ockerse, 1943). 3. Sodium There is littl e information about sodium in enamel. The concentration in total human enamel has usually been reported to be in the range of 0.70-1.16% (Logan, 1935; McCann and Bullock, 1955; Soremark and Samsahl, 1961). H. E. Harrison (1937) found a constant ratio of Ca : Na of approximately 30 : 1 in the mineral of all calcified tissues, which would explain the relatively high concentrations in the highly mineralized enamel. Layers of the outer portion of human enamel of a thickness from 0.18 to 0.59 mm have been found to vary in concentration s from 0.61 to 0 . 8 9 %, 5 of 6 samples being below 0.70 % (Little, Posen and Singer, 1962b). These findings suggest lower levels of sodium in surface than subsurface enamel. Preliminary analyses of successiv e layers of enamel have confirmed this, showing increasing concentrations from the surface inwards (Littl e and Brudevold, unpublished data, 1963). Data are not available concerning the relationship of sodium in enamel to age. The main uptake in developing enamel in the dog is in a zone adjacent to the ameloblasts (Applegren, Ullberg and Soremark, 1962). According to Bauer and Carlsson (1955), the major portion of the sodium present in bone is in the crystal lattice of the apatite crystals. Neuman and N e u m an (1958) have presente d evidence that sodium can be located on the surface of hydroxyapatite, and that it exchange s with calcium on a mole for mole basis. The location of sodium in the enamel crystals has not been established and nothing is known about the significance of its presence . It is dissolved from powdered enamel at a greater rate than calcium, suggesting that at least part of it is loosely bound to the mineral (Karshan et ah, 1934).

AND

R.

S Ô R E M A RK

4. Other

Constituents

A number of other elements, of which very littl e is known, are consistently present in human enamel. These include silicon, observed in concentrations of 5-50 ppm, manganese , traces to 18 ppm, silver, traces to 100 ppm, and aluminium, 40-140 p pm (Steadma n and Brudevold, unpublished data, 1963) (see Table 3). The concentration s of these elements vary in teeth from different geographica l areas, but it is uncertain to what extent this reflects differences in diet or composition of drinking water. Three minor elements in the enamel, vanadium, molybdenum and selenium, deserve some comment because they have been reported to affect dental caries. Increase of vanadium in the drinking water (range in water supplies 0.03-0.22 ppm) has been associate d with a decreas e in the prevalence of caries (Tank and Storvik, 1960), but the findings need to be confirmed because of the small number of children in the study and the presence of variable amounts of other elements in the drinking water which could have affected the results. Vanadium has failed to decreas e caries in most experimenta l animals (e.g. Shaw and Griffiths, 1961). Although the evidence in favour of a caries-reducin g effect of vanadium is not impressive, it is noteworthy that it is a bone-seekin g element that is readily taken up by enamel. The rate of deposition of V 48 by buccal and lingual intact enamel is shown in Fig. 3. It is believed that vanadium is taken up by exchange wit h phosphate (Sôremark and Andersson, 1962). Analyses of total enamel have shown very low values (Soremark, 1964b). Since vanadium competes with phosphate for positions on the hydroxyapatite crystals, the high phosphate levels of saliva may prevent significant accumulation in the surface enamel. High levels of molybdenum in food or drinking water associate d with low prevalence of caries in human communities have been reported by several workers (e.g. Ludwig, Healy and Losee, 1960). It has been reported that molybdenum increases fluoride retention in rats (Stookey and Muhler,

18. C H E M I S T RY

OF T H E M I N E R A L

1959). D a ta on molybdenum in human enamel are lacking. A slight but significant increase in caries with increase in urinary levels of selenium has been reported in children (Hadjimarkos and Bonhorst, 1958; Tank and Storvick, 1960). Since only trace amounts from 0.43 to 1.60 p pm have been found in human enamel (Hadjimarkos and Bonhorst, 1959), it seems unlikely that enamel mineral is sufficiently affected by its presence to account for this effect. The concentration of bromide in enamel is in the order of 5 ppm. According to Tables 2 and 3 the Br : Cl ratio is roughly 1 : 1000. The same ratio has also been reported for dentine (Soremark and Samsahl, 1962), blood, urine and drinking water, as well as for sea water (Soremark, 1960). The surprisingly high concentration of rubidium (approximately 5 ppm) in the enamel cannot be explained. Two organic constituents of enamel, citrate and lactate, wil l be mentioned because they have an

c 10 Ε 9 QJ Cl ΙΛ

r>

00

Lingual surface

> 4 3




P H A SE

2 I I

0

I I 2 4

I 6

I 8

I I I I I é é é é é 10 12 14 16 18 20 22 24 26 28 Hours 48

Fig. 3. Rate of V uptake by intact human enamel, buccal and lingual surfaces, immersed in a V 4 8-labelled vanadium pentoxide solution, correspondin g to a 15 ppm V 20 5 solution. The curves illustrate the mean uptake of V 48 of 50 buccal and 48 lingual surfaces repeatedly immersed in the radioactive solution for various periods of time. The lingual surfaces showed a somewhat slower rate of the mean uptake of V 48 than did the buccal surfaces ; this difference, however, was not significant.

V . T O P I C AL T R E A T M E NT OF T H E E N A M E L S U R F A CE This discussion wil l be confined to the chemistry of topically applied fluoride as it concerns enamel mineral and wil l not deal with the clinical literature and the relative merits of different procedures in the prevention of caries. The aim of research in this area is to find an agent capable of rendering the surface enamel caries resistant after a short period of treatment. There is no clear concept of what chemical changes could take place in enamel to produce this effect. There is general agreemen t that formation of fluorapatite is desirable, since reduction in caries obtained from ingesting water-borne fluoride is

266

F.

B R U D E V O LD

associate d with conversion of hydroxyapatite to fluorapatite. However, with the high concentration s of fluoride used in topical treatments , calcium fluoride is formed in addition to fluorapatite (McCann and Bullock, 1955; Leach, 1959). These reactions may be expresse d by Eqs. (1) and (2). Ca1 0(PO4) 6(OH) 2 + 2 F- -> C a1 0( P O4) 6F 2 + 2 0 H~ 3

Ca1 0(PO4) 6(OH) 2 + 20F- — 1 0 C a F2 + 6P0 -

(1)

+ 2 0 H~ (2)

Fluorapatite may form by ionic exchange (Neuman et al., 1950) or by dissolution of calcium and phosphate and reprecipitation with fluoride as fluorapatite (Kuyper and Kutnerian, 1962). In either case, it becomes an integral part of the enamel crystals. Calcium fluoride forms as a precipitate, presumably to the extent that its solubility product is exceede d (KSp 3 ÷ 1 0- 1 )1 . Its formation is associated with dissolution of the apatite (Eq. 2). Other fluoride reactions may also take place. For example, fluoride may exchange with carbonate, and it may combine with magnesium present in the enamel to form an as yet unidentified complex (McCann and Bullock, 1955). These findings, which were obtained on powdered enamel exposed to fluoride for three or more weeks, also showed that fluorapatite was the chief reaction product wit h concentration s of solution fluoride up to 100 ppm. A t higher concentration s the formation of fluorapatite levels off, while calcium fluoride is formed in increasing amounts. Wit h a shorter exposure time (6 hours), the formation of both fluorapatite and calcium fluoride increases with increase in concentration s tested (Leach, 1959). It is apparent from these findings that the rate of formation of the reaction products is important, and that the formation of fluorapatite is favoured by increase in concentration s of solution fluoride for short periods of fluoride exposure. The reaction products formed from high concentrations of fluoride are influenced by the Ca : Ñ ratio of the apatite. Calcium fluoride is formed more readily at low ratios (McCann, 1953)

AND

R.

S Ô R E M A RK

probably because of the greater solubility (dissolution of calcium) of low ratio apatites. At fluoride concentrations of 0.1 to 0.2 %, littl e or no fluorapatit e was formed until the Ca : Ñ ratio (molar) exceede d 1.62. These findings, which were derived from chemical analyses , have been substantiate d by X-ray diffraction (Trautz, 1960). Only traces of calcium fluoride were detected in well-crystallized hydroxyapatite (Ca : Ñ ratio 1.67) after 1 month's exposure to a solution of 2 % sodium fluoride, whil e appreciable amounts were formed from a material with a Ca : Ñ ratio of 1.5, and large amounts from brushite (Ca : Ñ ratio of 1.0). Calcium carbonate was also rapidly converted to calcium fluoride, suggesting that the carbonate in enamel, as well as the Ca : Ñ ratio, influences the reaction product with fluoride. Although the aforementione d studies employed powdered human enamel, the results undoubtedly apply in principle to intact enamel. A slower reaction rate, due to its limited surface area, and a lesser tendency to form calcium fluoride, due to its low solubility, would be expected in intact enamel (Brudevold, 1948). Minute amounts of calcium fluoride have been detected by electron diffraction and electron microscopy on ground enamel surfaces after only 3 minutes' exposure to 2 % sodium fluoride (Scott, Picard and Wyckoff, 1950). The fate of calcium fluoride in the mouth is uncertain. I t may well wash away, since it has been detected i n enamel only after in vitro fluoride exposures . It has been suggeste d that it may dissolve and provide fluoride for formation of fluorapatite, but this is unlikely since the reaction is exceedingly slow at body temperature (Knappworst, 1949). It is notable that X-ray diffraction of surface enamel of human teeth, which were treated for 20 minutes wit h sodium fluoride and extracted 1 week later, suggeste d the presence of fluorapatite rather than calcium fluoride (Syrrist, 1949). It has been hypothesized that the presence of calcium fluoride i n the enamel may increase its resistance to caries, because calcium fluoride has a low solubility in the pH range of 4-7 (Seidell, 1958) known to occur in carious lesions. This view seems untenable con-

18. C H E M I S T RY

OF

THE

MINERAL

P H A SE

OF

ENAMEL

267

Table 18 CONCENTRATIONS OF FLUORIDE (PPM OF A SH WEIGHT) IN ENAMEL OF HUMA N TEETH TREATED WITH N A F AND UNTREATED CONTROLS

Number of treatments

Length of each treatment (min)

8 4 4 1

10 5 10 20

N aF 0.02%

1

20

N aF 2%

4

4

Agent N aF N aF NaF N aF

á b c

2% 2% 4% 1%

Fluoride



Sampling of enamel

Experimental

Outer enamel Outer portion Outer portion First outer layer Second outer layer First outer layer Second outer layer First outer layer Second outer layer

950 620« 1100« 1440* 561 968 508 165c 60

Control 340 430 570 924 400 777 445 110 50

Investigation

Hord and Elli s (1951) Syrrist (1949) Syrrist (1949) Brudevold et al (1956a) Brudevold et al (1956a) Brudevold et al. (1956a) Brudevold et al. (1956a) Sundvall-Haglan d et ai. (1959) Sundvall-Haglan d et αι. (1959)

Fluoride exposure in situ once a week. Fluoride exposure in vitro. Fluoride exposure in situ within a week.

sidering that topically applied fluoride deposits only a small amount of fluoride and that of this amount only a part is in the form of calcium fluoride. Findings of the uptake of fluoride by intact enamel from different concentration s of sodium fluoride are summarized in Table 18. The fluoride increase was observable only in the outermost enamel and ranged from 750 to 500 ppm. Even if all this fluoride were deposited as calcium fluoride, it is unlikely that such small quantities of a precipitate could form an effective barrier against decalcification of the enamel. Since calcium fluoride is a decomposition product of the enamel, its formation may actually be undesirable . This concept is supported by the clinical finding that topical application of acidulated solutions of sodium fluoride has failed to reduce caries (e.g. Arnold, Dean and Singleton, 1944), since, as can be seen from Eq. 2, formation of calcium fluoride and acid etching may well overshado w this effect. It appears that this problem has been overcome by acidulating fluoride solutions with phosphoric acid rather than with the acetic acid used in previous studies. This is suggeste d by the finding that topical treatment with a solution of 1.2 % fluoride

and 0.1 M phosphate with a pH of about 3 produced substantia l reduction in caries (Wellock and Brudevold, 1963). A s a result of the low pH of this solution (about pH 3) large amounts of fluoride are deposited in the enamel. The presence of phosphate in the solution depresse s formation of calcium fluoride (Eq. 2). Acid etching of the enamel is also counteracte d by the phosphate due to common ion effect (Brudevold et al., 1963a). As a result the fluoride acquired from the solution is deposited primarily as fluorapatite. The marked effect of concentration s of solution fluoride and pH on the acquisition of fluoride by powdered enamel may be seen in Table 19. The solutions employed in this study contained 0.1 M phosphate . Topical applications of stannous fluoride involve stannous as well as fluoride ion reactions. Table 20 shows that appreciable concentration s of tin are acquired by intact enamel surfaces and that there is a slight penetration of tin into the enamel. Tin deposits rapidly in enamel, probably in the form of ti n phosphate s and oxides (Cooley, 1961). In addition a loose fluoride-containing precipitate (Smith et al., 1957) forms on the enamel surface as a result of hydrolysis of the stannous fluoride. The

F.

268

B R U D E V O LD

AND

Table 19 FLUORIDE IN POWDERED SURFACE ENAMEL OF HUMA N PERMANENT TEETH EXPOSED FOR 20 MINUTES TO

1.6%

FLUORIDE AS SODIUM FLUORIDE IN 0.1 M SODIUM PHOSPHATE AT PH 2.8 AND AS STANNOUS FLUORIDE

2.6"

AT PH

Layer

S n F2 (1.6% F)

N a F - P 04 (1.6% F)

Control

1 2

2229 1254

650 370

582 316

1 2

2813 987

1066 623

881 451

" From Brudevold et al. (1963a).

protection to the enamel surface that topical application of stannous fluoride provides is unlikely to be due to this easily removable complex, but probably results from impregnation of the surface enamel with fluoride and possibly tin. The deposition of tin is actually of doubtful significance in regard to caries, since topical application of a fluoride-free tin salt has been reported to be ineffective (Muhler, 1958). The usefulness of the stannous ion may be to decreas e dissolution of enamel by the acid stannous fluoride solution (pH 3) during the treatment procedure, and to

R.

S Ô R E M A RK

permit reaction of fluoride with the enamel at low pH without undue detrimental effects. It is possible that the combination of limited dissolution of the enamel (limited release of C a+ +) and low pH promotes formation of fluorapatite, since in one study no calcium fluoride was detected even after 1 week's exposure to stannous fluoride solution (Fischer, Elsheimer and Muhler, 1954). The decreas e in fluoride deposition from stannous fluoride compared to sodium fluoride, also seen in Table 20, may thus be offset by the fact that fluorapatite is the main fluoride reaction product. However, this point needs clarification. The yellowish-brown discolouration noted in human teeth following stannous fluoride treatments is probably due to the formation of insoluble stannous sulphide, since in vitro experiments have demonstrate d that, this compound wil l readily form in stannous fluoridetreated enamel in the presence of sulphide (Isaac and Brudevold, 1957). The limitin g effect of the stannous ion on fluoride uptake by enamel can be clearly seen from the data given in Table 19 (Brudevold et ai, 1963a). Teeth were exposed to solutions of either stannous fluoride or sodium fluoride dissolved in OA M phosphoric acid. The solutions contained the same concentrations of fluoride and had a similar p H. Analyses of two consecutive thin layers ground off

Table 20 FLUORIDE AND T I N CONCENTRATION IN HUMA N EXTERNAL ENAMEL AFTER 20-MINUTE EXPOSURE TO 1% SODIUM FLUORIDE, 1.86%

STANNOUS FLUORIDE AND 4.12%

STANNOUS CHLORO FLUORIDE"

Enamel Percent in test solution Test agent Sodium fluoride Stannous fluoride Stannous chlorofluoride Control « From Brudevold (1959).

pH 5.6 2.9 1.6 —

Tin 0.0 1.41 2.82 0.0

Tin (ppm) in layer

Fluoride (ppm) in layer

Fluoride 0.452 0.452 0.452 0.0

25 000 640 15

5.6 250 300 10

1440 1200 1031 924

561 580 570 400

18. C H E M I S T RY

OF

THE

MINERAL

the fluoride-exposed teeth showed approximately 3 times greater uptake and a much greater penetration of fluoride into the enamel of the teeth treated wit h phosphate-fluorid e solutions. I t is of considerable interest that the capacity to take up fluoride varies in different teeth. Experiments have shown that, with teeth that had received the same cleaning with an abrasive, some picked up as much as 10 times more fluoride than others after a 3 minutes' exposure to an 0.1 % sodium fluoride solution. By electron microscopy of ground surfaces of human enamel, a difference in reactivity to fluoride has been observed both in different teeth and in different areas on the same tooth (Scott et al, 1950). The reasons for this difference in reactivity is not known. It is possible that aging decrease s the response of enamel to fluoride. This is suggeste d by the observation that water fluoridation is effective i n teeth first fluoride-exposed 1or 2 years after eruption, but apparently not in teeth erupted for several years before the first fluoride exposure. Because of the concentrate d solutions of fluoride used, topical treatment can even benefit teeth of adults, but differences in structural or chemical characteristics of the surface enamel affects the fluoride uptake in a way which is not understood . There is only slight penetration of topically applied fluoride into intact human enamel. For exmple, in the experiments shown in Table 19, the two layers of enamel ground from teeth were estimated to be approximately 0.05 mm thick, and the fluoride increase in the second layer was only a fraction of that in the first layer. The small increments of fluoride and the slight penetration are consistent with the view that fluoride acquisition is limited to the surface of the apatite crystals and that diffusion in the intercrystalline spaces is restricted. Fluoride impregnation of the enamel in depth would presumably materially increase the caries-reducin g effect, but it is doubtful whether this can ever be accomplishe d in full y mineralized enamel. In the partly decalcified enamel of carious lesions, spaces between the crystals are augmented , as has been demonstrate d by polarizing microscopy 19

P H A SE

OF

ENAMEL

269

(Darling, 1961). The increased hydration of carious enamel (Littl e et al, 1962a) would facilitate diffusion of fluoride, a fact verified by radioisotope studies (Myers, Hamilton and Becks, 1952; Brudevold et al., 1957a). But, even when there is appreciable penetration of fluoride, the reactions involved must be limited to the surface of the crystals. Because of the greater uptake of fluoride i n carious than in intact enamel, the beneficial effects of topically applied fluoride may include arresting initial lesions as well as increasing the resistance to caries of intact enamel. The extent to which these two different modes of action of fluoride operate is not known since carious lesions are well advanced before they can be detected with the clinical methods now available. Topical treatment with fluoride-free salts have not been successful . Solutions of silver nitrate, zinc chloride, indium nitrate (Bibby and Brudevold, 1954) and stannous sulphate (Muhler, 1958) have all been clinically ineffective, in spite of the fact that exposure of enamel to these salts reduces its acid solubility. This and other evidence suggests that precipitation of extraneous salts in the enamel does not effectively inhibit the carious process. The fluoride effect, which is one of consolidating the apatite lattice, is the only mechanism involving the mineral phase which is known to inhibit caries.

VI. ENAMEL CARIES Chemical analysis has shown that the main process occurring in caries of the enamel is demineralization and replacemen t of the dissolved mineral with loosely bound water (Littl e et al., 1962b). There is a slight increase in organic material (Savory and Brudevold, 1959; Littl e et al., 1962b), probably resulting from deposition of salivary material and growth of micro-organisms . A s mineral dissolves, fluoride is retained in the residual enamel. This is suggeste d by the fact that when enamel is demineralized by weak acid, the fluoride from the dissolved material is redeposite d in the remaining solid (Isaac et al., 1958a). The fluoride apparently

270

F.

B R U D E V O LD

deposits as fluorapatite, since low concentration s of fluoride wil l precipitate as fluorapatite in the presence of phosphate and calcium in solution (Kuyper and Kutnerian, 1962). In addition to this process, carious enamel is known to acquire extraneous fluoride more readily than intact enamel (Brudevold et al, 1957b). The overall effect is a greater concentration of fluoride in carious than i n adjacent sound enamel (Jenkins, 1960; Littl e et al., 1962b). Whereas fluoride tends to accumulate in carious enamel, the opposite is true of carbonate (Coolidge and Jacobs, 1957; Nordback and Johansen , 1962; Littl e et al., 1962b), sodium (Littl e et al., 1962b) and magnesium (Nordback and Johansen , 1962). This is consistent with the finding of Karshan et al. (1934) and others that in powdered enamel these components are preferentially dissolved. Obviously, demineralization of enamel is not a simple process of dissolution, but an interplay of dissolution and recrystallization. It is significant that the preferential dissolution leads to formation of increasingly insoluble mineral residues (Jenkins, 1960; Posen, 1962).

Fig. 5. Microradiograph of initial carious lesion in human enamel. (From Soni and Brudevold, 1959.)

AND

R.

S Ô R E M A RK

One of the most interesting morphological aspects of initial carious lesions is that the enamel surface may appear intact, while the underlying enamel is significantly demineralized . Clinically these lesions are known as "white spots", the white appearanc e being due to optical phenomena associate d with increased enamel porosity. A microradiograph of a "white spot" is shown in Fig. 5. Densitometric tracings of such mieroradiographs show only a slight decreas e in radiodensity of the affected surface enamel, suggesting diffuse removal of small amounts of mineral (Soni and Brudevold, 1959). The dissolution of mineral causes enlargemen t of the intercrystalline spaces , as is demonstrate d by increase in form birefringence (Darling et al., 1961). Subsurface lesions, indistinguishable from initial carious lesions, can be produced by exposing intact human enamel to weak acids (e.g. Soni and Brudevold, 1960) and by incubating enamel in saliva sugar mixtures or in media containing carbohydrate and inoculated wit h acidogenic bacteria (Francis and Meckel, 1963). It is possible to produce subsurface lesions in enamel which has the outermost layer ground off if the viscosity of the demineralizing acid is significantly increased (Muhlemann, 1960) or if ions which inhibit demineralization (e.g. aspartic acid) are present (Sperber and Buonocore, 1963). However, the resistance to demineralization is not as marked in the ground surface as in the intact surface. The reasons for a greater rate of dissolution below the enamel surface in initial carious lesions are not entirely understood . One important causative factor must be that the acid solubility of enamel increases from the surface towards the dentine. This can be seen in Fig. 6, where the solubility of successiv e layer samples of human enamel, ground and pooled from a great number of teeth, is plotted against the depth from the surface. The slope of the curve depends on the pH of the buffer, but the enamel is consistently more soluble wit h increase in depth. The organic material is of littl e significance in this effect, because the pattern of demineralization was the same after the organic

18. C H E M I S T RY

OF T H E M I N E R A L

50 h 45 h

0

20

40

60

80

100

Depth from surface

Fig. 6. Weight loss of 50-mg portions of successiv e layers of human enamel. Exposure for 5 ml at 0.5 M acetate buffer at room temperature for 1 hour.

material had been extracted with ethylenediamin e (Isaac et al., 1958a). A relationship was found between the concentration of fluoride in the layer samples and the resistance to acid action. This relationship was consistent for surface-subsurfac e enamel from the same tooth group, but did not always apply when different tooth groups were compared. Mottled enamel, with its high fluoride content, was more resistant to acid dissolution than the enamel of teeth from low-fluoride areas; and enamel from deciduous teeth, which has lower levels of fluoride than that of permanent teeth, was also less resistant. On the other hand, enamel of teeth from different areas may be similar in respect of fluoride, and yet have different dissolution rates (Isaac et al, 1958b). Other constituents which occur in different concentration s in surface and subsurface enamel may contribute to the unequal rate of dissolution which occurs in initial carious lesions. Carbonate is probably particularly important because it is present in relatively high concentrations and because of its marked solubilityincreasing effect on apatite (Gron et al, 1963), but unequal distribution of many trace elements in enamel may also contribute to the preferential pattern of demineralization seen in Fig. 5. A recent study has shown that the penetration of acid into

P H A SE

OF

ENAMEL

271

intact enamel is not controlled by the restricted diffusion in the inter-crystalline spaces (Kapur, Fischer and Manly, 1961). It was suggeste d that the rate-limiting factor is the ionic exchange which takes place between the apatite crystals and the fluid medium. I n view of the complexity of, and marked differences in composition between, surface and subsurface enamel, it is evident that the exchange of ions, which takes place in the mineral of carious lesions, is no simple process. The fact that the demineralizing process leaves an increasingly insoluble residual enamel shows that the mineral phase can be consolidated in the oral environment. The compositional changes which occur in the mineral, e.g. increasing in fluoride and decreasin g in carbonate , may give a clue for future approache s to the enhancemen t of caries resistance by topical treatments of the enamel surface.

VII . C O N C L U D I NG R E M A R KS I n this chapter the purpose has been to bring together the available data concerning the composition and anatomy of the mineral phase of the enamel. The significance of the presence of various mineral components and the mechanism of their deposition have been discussed . The chemistry involved in topical fluoride applications, and in the dissolution of enamel mineral associate d with caries, has also been considered . I n summary, it may be mentioned that enamel contains approximately 87,2 and 11 volume per cent of mineral, organic matter and water, respectively. Roughly one fourth of the water is loosely bound. The remainder is firmly bound to mineral. Microradiographic tracings of intact enamel have demonstrate d a slight decreas e in total mineral concentration from the surface to the dentine-ename l junction. The distribution of organic and inorganic constituents suggests that water is most sparse in surface enamel and that it occurs in greater amounts in the body of the enamel. Analysis of layer samples shows greater

F.

272

B R U D E V O LD

concentration s of F, P b, Zn, Sn, Fe, Cl and, to a lesser extent A l a nd Si, in surface t h an in subsurface

enamel.

T he

surface

increasing concentration s of the

subsurfac e

material

enamel

acquires

F wit h age,

remains

while

virtually

u n-

changed . The level of F is closely related to that of drinking water. Certain constituents , including carbonate , N a, a nd M g increase in concentratio n from the surface inward while others, such as Cu and Sr, are evenly distributed. A m o ng the micro elements , C 0 2 , N a, M g, Cl, K , F a nd Zn occur i n relatively large concentrations . Other elements , including Al , Sr, P b, M n, Cu, Si, Ag, Fe and Sn

are

normally

present

in

concentration s

below 10 micromoles per gram and are therefore less likely to affect

the physical

properties

of

dental structures . A m o ng the many challenging u n k n o w ns which await investigation are: the nature of the b o nd between the enamel crystallites a nd the organic matrix; the mechanis m of water binding by enamel mineral; the role of the various mineral constituents i n strengthenin g or weakening the crystal lattice; the relative significance of the firmly loosely

b o u nd

diffusion

fractions

of

water;

b o u nd and the rate

of

of ions and molecules in the enamel,

as contraste d to the rate of reaction which may occur

with

the apatite crystals;

the extent

of

accessibl e internal surface area of intact enamel, and its role in determining the chemical behaviour of the enamel; the nature of caries resistanc e in chemical terms; the p h e n o m e n a which are responsible for

and influence the developmen t of

subsurfac e lesions; the role of

remineralization

i n maintaining the integrity of the enamel, and i n retarding the carious process ; and, last n ot

least,

the solubility

of

enamel mineral

but in

thermodynamic terms and as affected by salivary constituents and caries-inhibiting agents.

ACKNOWLEDGEMENTS

The authors' own studies, both published and unpublished, which are referred to here were supported by U.S. Public Health Service grants D-917, D-1362 and AM-08157.

A N D R.

S Ô R E M A RK

References Altshuller, L. F., Halak, D. B., Landing, Â. H. and Vehoe, R. A. (1962). Deciduous teeth as an index of body burden of lead. / . Pediat. 60, 224-229. Anderson, R. W. and Knutson, J. W. (1951). Effect of topically applied zinc chloride and potassium ferrocyanide on dental caries experience . Publ. Hth. Rep., Wash. 66, 1164-1166. Angmar, B., Carlstrom, D. and Glas, J. E. (1963). Studies on the ultrastructure of dental enamel. IV . The mineralization of normal human enamel. / . Ultrastruct. Res. 8, 12-23. Appelgren, L. E., Ullberg, S. and Soremark, R. (1962). Radiosodium incorporation in developing teeth of dog and rat studied by means of autoradiography . Acta odont. scand. 20, 189-197. Armstrong, W. D. and Brekhus, P. J. (1937). Chemical . constitution of enamel and dentin. Principal components /. biol. Chem. 120, 677-687. Armstrong, W. D. and Brekhus, P. J. (1938). Possible relationship between the fluorine content of enamel and resistance to dental caries. / . dent. Res. 17, 393— 399. Armstrong, W. D. and Singer, L. (1956). Ciba Fdn. Symp., Bone Struct. Metab. pp. 109-116. Arnold, F. Á., Jr., Dean, T. H. and Singleton, D. W., Jr. (1944). The effect on caries incidence of a single topical application of a fluoride solution to the teeth of young adult males of a military population. / . dent. Res. 23, 155-162. Arnold, P. W. (1950). The nature of precipitated phosphates . Trans. Faraday Soc. 46, 1061-1063. Ast, D. B., Bushel, A . and Chase, H. C. (1950). A clinical study of caries prophylaxis with zinc chloride and potassium ferrocyanide. J. Am. dent. Ass. 41, 437-442. Aston, E. R. (1952). Dental study of employees of five lead plants. Industr. Med. Surg. 21, 17-19. Aub, J. C , Fairhall, I. T., Minot, A. S. and Rexonikoff, P. (1925). Lead poisoning. Medicine 4, 1-10. Bauer, G. C. H. and Carlsson, A. (1955). On the availability for exchange of skeletal water, sodium, and calcium. Acta orthop. scand. 24, 275-291. Berfenstam, R. (1952). Studies on blood zinc. A clinical and experimenta l investigation into the zinc content of plasma and blood corpuscles with special reference to infancy. Acta paediat., Stockh. 41, Suppl. 87. Bergenholtz, Á., Hedegârd, Â. and Soremark, R. (1965). Studies of the transport of metal ions from gold inlays into environmenta l tissues. Acta odont. scand. 23, 135— 146. Bergman, G. (1963). Microscopic demonstratio n of liquid flow through human dental enamel. Arch, oral Biol. 8, 233-234.

18.

C H E M I S T RY

OF

THE

MINERAL

Bergman, G. and Engfeldt, B. (1954). Studies on mineralized dental tissues. Acta odont. scand. 12, 99-122. Bergman, G. and Siljestrand, B. (1963). Water evaporation in vitro from human dental enamel. Arch, oral Biol. 8, 37-38. Bibby, B. G. and Brudevold, F. (1954). The external action of fluorides and other agents on the teeth in the prevention of dental decay. In "Fluoridation as a Public Health Measure", Symposium, pp. 148-178. Amer. Ass. Advanc. Sci., Washington, D.C. Bibby, B. G., DeRoche, F. and Wilkins, E. (1947). The effect of topical application of lead fluoride on dental caries. / . dent. Res. 26, 446 (Abstract). Bird, M. J., French, E. L., Woodside, M. R., Morrison, M . I. and Hodge, H. C. (1940). Chemical analysis of deciduous enamel and dentin. / . dent. Res. 19, 413-423. Bowes, J. H. and Murray, M. M. (1935). The chemical composition of teeth. II . The composition of human enamel and dentin. Biochem. J. 29, 2721-2727. Bowes, J. H. and Murray, M. M. (1936). The chemical composition of teeth. III . The variations in chemical composition in relation to dental structure. Biochem. J. 30, 977-984. Brudevold, F. (1948). Study of the phosphate solubility of the human enamel surface. / . dent. Res. 27, 320-329. Brudevold, F. (1957). Changes in enamel with age. Proc. 25th Year Celebration. Univ. Rochester dent. Res. Fellowship Program pp. 185-192. Brudevold, F. (1959). Action of topically applied fluoride. /. Dent. Child. 26, 186-190. Brudevold, F. and Little, M. F. (1951). Chemical studies of the human enamel surface. / . dent. Res. 30, 477 (Abstract). Brudevold, F. and Steadman , L. T. (1955). A study of copper in human enamel. / . dent. Res. 34, 209-216. Brudevold, F. and Steadman , L. T. (1956a). The distribution of lead in human enamel. / . dent. Res. 35, 430-437. Brudevold, F. and Steadman , L. T. (1956b). A study of ti n in enamel. / . dent. Res. 35, 749-752. Brudevold, F., Gardner, D. E. and Smith, F. A. (1956a). The distribution of F in human enamel. J. dent. Res. 35, 420-429. Brudevold, F., Steadman , L. T., Gardner, D. E., Rowley, J. and Little, M . F. (1956b). Uptake of tin and fluoride by intact enamel. / . Amer. dent. Ass. 53, 159-164. Brudevold, F., Hein, J. W., Bonner, J. R., Nevin, R. B., Bibby, B. G. and Hodge, H. C. (1957a). Reaction of tooth surfaces with 1 ppm of fluoride as sodium fluoride. /. dent. Res. 36, 771-779. Brudevold, F., Savory, A. and Bowman, L. (1957b). The effect of fluoride in phosphate buffers on enamel. Abstr. Int. Ass. dent. Res., 35th gen. Meet., 1957. No. 72. Brudevold, F., Steadman , L. T. and Smith, F. A. (1960). Inorganic and organic components of tooth structure. Ann. N.Y. Acad. Sci. 85, 110-132.

P H A SE

OF

ENAMEL

273

Brudevold, F., Amdur, B. and Rasmussen , S. (1962a). Magnesium in human teeth. Abstr. Int. Ass. dent. Res., 40th gen. Meet., St. Louis, 1962 No. 33. Brudevold, F., Gron, P., Speirs, R. L., Spinelli, M. and Gardner, D. E. (1962b). Acquisition and desorption of fluoride. Preprint. Abstr. Int. Ass. dent. Res., 40th gen. Meet., St. Louis, 1962 No. 123. Brudevold, F., Savory, Á., Gardner, D. E., Spinelli, M. A. and Speirs, R. (1963a). A study of acidulated fluoride solutions. I. In vitro effects on enamel. Arch, oral Biol. 8, 167-177. Brudevold, F., Steadman , L. T., Spinelli, Ì . Á., Amdur, Â. H. and Gron, P. (1963b). A study of zinc in human teeth. Arch, oral Biol. 8, 135-144. Bryant, F. J., Henderson , Å. H. and Holgate, W. (1960). Strontium-90 in human teeth. Brit. dent. J. 108, 291-294. Buonocore, M. G. and Bibby, B. G. (1945). The effect of various ions on enamel solubility. / . dent. Res. 24,103-108. Burnett, G. W. and Zenewitz, J. (1958). Studies of the composition of teeth. VII . The moisture content of calcified tooth tissues. / . dent. Res. 37, 581-589. Caldwell, R. C , Muntz, M . L., Gilmore, R. W. and Pigman, W. (1957). Microhardness studies of intact surface enamel. /. dent. Res. 36, 732-738. Carlson, C. H., Armstrong, W. D. and Singer, L. (1960). Distribution and excretion of radiofluoride in the human. Proc. Soc. exp. Biol. N.Y. 104, 235-239. Carlstrom, D., Glas, J. E, and Angmar, B. (1963). Studies on the ultrastructure of dental enamel. V. The state of water in human enamel. / . Ultrastruct. Res. 8, 24-29. Christensen , H. (1962). Isomorphic substitutions of calcium by strontium in calcium hydroxyapatite. AB Atomenergi (Nykôping, Sweden) Rep. No. AE-96. Cooley, W. E. (1961). Reactions of tin (II ) and fluoride ions with etched enamel. / . dent. Res. 40, 1199-1210. Coolidge, T. B. and Jacobs, M. H. (1957). Enamel carbonate in caries. / . dent. Res. 36, 765-768. Cruickshank, D. B. (1940). The natural occurrence of zinc in teeth. III . Variation in tuberculosis. Brit. dent. J. 68, 257-271. Darling, A. I. (1956). Studies of the early lesion of enamel caries with transmitted light, polarised light, and radiography. Brit. dent. J. 101, 289-297. Darling, A. I. (1961). Structure of the enamel revealed in dental lesions. Arch, oral Biol. 4, 80-85. Darling, A. I., Mortimer, Ê. V., Poole, D. F. G. and Ollis, W. D. (1961). Molecular sieve behavior of normal and carious dental enamel. Arch, oral Biol. 5, 251-273. Deakins, M. (1942). Changes in the ash, water, and organic content of pig enamel during calcification. / . dent. Res. 21, 429-435. Deakins, M. and Volker, J. F. (1941). Amount of organic matter in enamel from several types of human teeth. /. dent. Res. 20, 117-121.

274

F.

B R U D E V O LD

Dreizen, S., Spies, H. A. and Spies, T. D. (1952). The copper and cobalt levels of human saliva and dental caries activity. / . dent. Res. 31, 137-142. Dundon, C. C. and Hughes, J. P. (1950). Stannic oxide pneumocomosis . Amer. J. Roentgenol. 63, 797-804. Elliott, J. C. (1961). The infrared spectrum of the carbonate ion in carbonate-containin g apatites. / . dent. Res. 40, 1284 (Abstract). Ericsson, Y. (1950-1951). Reduction of the solubility of enamel surfaces. Acta odont. scand. 9, 60-83. Fischer, R. B., Elsheimer, H. N. and Muhler, J. C. (1954). The effects of several fluoride reagents on the structure of whole teeth. / . dent. Res. 33, 538-554. Francis, M. D. and Meckel, A. H. (1963). The in vitro formation and quantitative evaluation of carious lesions. Arch, oral Biol. 8, 1-12. Frank, R., Sognnaes , R. F. and Kern, R. (1960). Calcification of dental tissues with special reference to enamel ultrastructure. In "Calcification in Biological Systems", Publ. No. 64, pp. 163-202. Amer. Ass. Advanc. Sci., Washington, D.C. Galagan, D. J. and Knutson, J. W. (1947). The effect of topically applied fluorides on dental caries experience . V. The report of findings with two, four and six applications of sodium fluoride and lead fluoride. Publ. Hlth. Rep., Wash. 62, 1477-1483. Geiger, T. (1950). Beitrg e zum Problem der Karbonat apatit. Schweiz. mineral-petrog: Mitt. 30, 161-173. Glas, J. E. (1962). "Studies on the Ultrastructure of Calcified Tissues". Tryclan Balder, Stockholm. Glas, J. E. and Omnell, K.-A. (1960). Studies on the ultrastructure of dental enamel. I. Size and shape of the apatite crystallites as deduced from X-ray diffraction data. J. Ultrastruct. Res. 3, 334-346. Graig, F. A. and Sergei, E. (1960). Distribution in blood and excretion of Z n 65 in man. Proc. Soc. exp. Biol., N.Y. 104, 391-393. Gron, P., Spinelli, M., Trautz, O. R and Brudevold, F. (1963). The effect of carbonate on the solubility of hydroxylapatite. Arch, oral Biol. 8, 251-263. Gulberg, B. and Swenson, A. (1943). Mobilization of lead due to A. T. Svenska Ldkartidn. 45/47, 2263-2269. Hadjimarkos, D. M. and Bonhorst, C. W. (1958). The trace element selenium and its influence on dental caries susceptibility. / . Pediat. 52, 274-278. Hadjimarkos, D. M. and Bonhorst, C. W. (1959). The selenium content of human teeth. Oral surg. 12, 113-116. Harrison, G. E., Lumsden, E., Raymond, W. H. A. and Sutton, A. (Part I), and Boyd, J., Neuman, W. F. and Hodge, H. C. (Part II) . (1959). On the mechanism s of skeletal fixation of strontium. Arch. Biochem. Biophys. 80, 97-113. Harrison, H. E. (1937). The sodium content of bone and other calcified material. / . biol. Chem. 120, 457-462.

AND

R.

S Ô R E M A RK

Haumont, S. (1961). Distribution of zinc in bone tissue. / . Histochem. Cytochem. 9, 141-163. Hendricks, S. B. and Hill , W. L. (1950). The nature of bone and phosphate rock. Proc. nat. Acad. Sci., Wash. 36, 731-737. Hord, A. B. and Ellis, R. G. (1951). The effect of the topical application of sodium fluoride on the calcium fluoride content of vital teeth. / . dent. Res. 30, 360-362. Isaac, S. and Brudevold, F. (1957). Discoloration of teeth by metallic ions. / . dent. Res. 36, 753-758. Isaac, S., Brudevold, F., Smith, F. A. and Gardner, D. E. (1958a). Solubility rate and natural fluoride content of surface and subsurface enamel. / . dent. Res. 37, 254-263. Isaac, S., Brudevold, F., Smith, F. A. and Gardner, D. E. (1958b). The relation of fluoride in the drinking water to the distribution of fluoride in enamel. / . dent. Res. 37, 318-325. Jenkins, G. N. (1960). The biochemical background to the action of fluoride in dental caries. "Lectures on the Scientific Basis of Medicine", No. 8, 1958-1959. Univ. of London Press (Athlone), London. Jenkins, G. N. and Speirs, R. L. (1953). Distribution of fluorine in human enamel. / . Physiol. 121, 21-28. Kapur, Ê. K., Fischer, E. and Manly, R. S. (1961). Effect of surface alterations on the permeability of enamel to a lactate buffer. J. dent. Res. 40, 1174-1182. Karshan, M., Weiner, R. and Stofsky, N. (1934). Biochemical studies of aqueous extracts of enamel and dentin in relation to dental caries. / . dent. Res. 14, 445-454. Kehoe, R. Á., Cholak, J. and Story, R. V. (1940). A spectrochemical study of the normal ranges of concentration of certain trace metals in biological materials. / . Nutr. 19, 579-592. Keil, A. (1937). Beitrg e zur Kenntnis der Doppelbrechun g des menschliche n Zahnschmelzes . Z. Zellforsch. 25, 204-224. Kerwin, J. G. (1958). Possible biologic hazards of strontium90 and fluoridation. Dent. Dig. 64, 58-61. Kety, S. S. and Letonoff, T. V. (1941). Treatment of lead poisoning with sodium citrate. Proc. Soc. exp. Biol. N.Y. 46, 476-477. Kick, C. H., Bethke, R. M., Edington, R. H., Wilder, Ï . H. M., Record, P. R., Wilder, W., Hill , T. J. and Chase, W. W. (1955). Fluorine in animal nutrition. Ohio agric. exp. Sta. Bull. 588. Klinkenberg, E. and Bibby, B. G. (1950). Effect of topical applications of fluoride on dental caries in young adults. /. dent. Res. 29, 4-8. Knappworst, A. (1949). Physikalisch-chemisch e Grundlagen einer lokalen Karietherapie des Dentins mit Fluor-ionen. Dtsch. Zahnàrztl. Ζ. 4, 553-567. Kuyper, A. C. and Kutnerian, K. (1962). Mechanism of incorporation of fluoride into bone salt. / . dent. Res. 41, 345-350.

18. C H E M I S T RY

OF

THE

MINERAL

Leach, S. A. (1959). Reactions of fluoride with powdered enamel and dentine. Brit. dent. J. 106, 133-142. LeFevre, M. L. and Manly, R. S. (1938). Moisture, inorganic and organic contents of enamel and dentin from carious teeth. / . Amer. dent. Ass. 24, 233-242. Likins, R. C, Posner, A. S. and Steere, A. C. (1958). Effect of calcium treatment on solubility and calcium uptake of synthetic hydroxapatite and rat molar enamel. / . Amer. dent. Ass. 57, 385-389. Likins, R. C , McCann, H. G., Posner, A. S. and Scott, D. B. (1960). Comparative fixation of calcium and strontium by synthetic hydroxyapatites . J. biol. Chem. 235, 2152-2156. Little , M. F. (1961). Studies on the inorganic carbon dioxide component of human enamel. II . The effect of acid on enamel C 02. / . dent. Res. 40, 903-914. Little, M. F. and Brudevold, F. (1958). A study of the inorganic carbon dioxide in intact human enamel. / . dent. Res. 37, 991-1000. Little , M. F., Cueto, E. S. and Rowley, J. (1962a). Chemical and physical properties of altered and sound enamel. 1. Ash, Ca, P, C 02, N, water, microradiolucenc y and density. Arch, oral Biol. 7, 173-184. Little , M. F., Posen, J. and Singer, L. (1962b). Chemical and physical properties of altered and sound enamel. 3. Fluoride and sodium content. / . dent. Res. 41, 784-789. Logan, M. A. (1935). Composition of cartilage, bone, dentin, and enamel. / . biol. Chem. 110, 375-389. Lowater, F. and Murray, M. M. (1937). Chemical composition of teeth. V. Spectrographi c analysis. Biochem. J. 31, 837-841. Ludwig, T. G., Healy, W. B. and Losee, F. L. (1960). An association between dental caries and certain soil conditions in New Zealand. Nature 186. 695-696. McCann, H. G. (1953). Reaction of fluoride ion with hydroxyapatite. / . biol. Chem. 201, 247-259. McCann, H. G. and Bullock, F. A. (1955). Reactions of fluoride ion with powdered enamel and dentin. / . dent. Res. 34, 59-67. McCann, H. G. and Bullock, F. A. (1957). The effect of fluoride ingestion on the composition and solubility of mineralized tissues of the rat. / . dent. Res. 36, 391-398. McClure, F. J., (1941). Domestic water and dental caries. III . Fluorine in human saliva. Amer. J. Dis. Child. 62, 512-515. McClure, F. J. (1948). Observations on induced caries in rats. VI . Summary results of various modifications of food and drinking water. / . dent. Res. 27, 34-40. McClure, F. J. and Likins, R. C. (1951). Fluorine in human teeth studied in relation to fluorine in drinking water. /. dent. Res. 30, 172-176. McClure, F. J. and McCann, H. G. (1960). Dental caries and composition of bones and teeth of white rats. Effect of dietary mineral supplements . Arch, oral Biol. 2,151-161.

P H A SE

OF

ENAMEL

275

McConnell, D. (1952). The crystal chemistry of carbonate apatites and their relationship to the composition of calcified tissues. / . dent. Res. 31, 53-63. McConnell, D. (1965). Crystal chemistry of hydolyapatite; its relation to bone mineral. Arch, oral Biol. 10, 4 2 1431. MacDonald, N. S., Ezmirlian, F., Spain, P. and McArthur, C. (1951a). The ultimate site of skeleton deposition of strontinum and lead. / . biol. Chem. 189, 387-399. MacDonald, N. S., Nusbaum, R. E., Stearns, R., Ezmirlian, F., McArthur, C. and Spain, P. (1951b). The skeletal deposition of non-radioactive strontium. / . biol. Chem. 188, 137-143. Manly, R. S. and Bibby, B. G. (1949). Substance s capable of decreasin g the acid solubility of tooth enamel. / . dent. Res. 28, 160-171. Mansell, R. E. and Hendershot , L. C. (1960). The spectrochemical analysis of metals in rat molar enamel femur and incisors. Arch, oral Biol. 2, 31-37. Massler, M. (1954). Is sodium fluoride best for topical applications? / . Dent. Child. 21, 14-19. Muhlemann, H. R. (1960). Experimental modifications of the enamel surface. Helv. odont. acta 4, 5-12. Muhler, J. C. (1958). Effects of fluoride and non-fluoride containing tin salts on the dental caries experience in children. / . dent. Res. 37, 422-426. Muhler, J. C , Boyd, T. M. and Van Huysen, G. (1950). Effect of fluoride and other compounds on the solubility of enamel, dentin and in calcium phosphate in dilute acids. / . dent. Res. 29, 182-193. Myers, H. M., Hamilton, J. G. and Becks, H. (1952). A tracer study of the transfer of F 18 to teeth by topical application. / . dent. Res. 31, 743-750. Neuman, W. F. and Neuman, M. W. (1953). The nature of the mineral phase of bone. Chem. Rev. 53, 1-13. Neuman, W. F. and Neuman, M. W. (1958). "The Chemical Dynamics of Bone Mineral", Chapter 5. Univ. of Chicago Press, Chicago, Illinois. Neuman, W. F., Neuman, M. W., Main, E. R., O'Leary, J. and Smith, F. A. (1950). The surface chemistry of bone. II . Fluoride deposition. J. biol. Chem. 187, 655-661. Newbrun, E. and Brudevold, F. (1960). Studies on the physical properties of fluorosed enamel. I. Microradiographic studies. Arch, oral Biol. 2, 15-20. Newbrun, E., Timberlake, P. and Pigman, W. (1959). Changes in microhardnes s of enamel following treatment with lactate buffer. / . dent. Res. 38, 293-300. Nikiforuk, G. (1961). Carbonate s and fluorides as chemical determenant s of tooth susceptibility to caries. Symp. Present Status Caries Prevent. Fluorine-Containing Dentifrices, Zurich, 1961 pp. 62-69. Nordback, L. G. and Johansen , E. (1962). The chemistry of carious lesions. I. The calcium, magnesium , phosphorus and carbonate content of sound and carious human

276

F.

B R U D E V O LD

dentin and enamel. Abstr. Int. Ass. dent. Res., 40th gen. Meet., St. Louis, 1962 No. 141. Ockerse, T. (1943). The chemical composition of enamel and dentin in high and low caries areas in South Africa. /. dent. Res. 22, 441-446. Palache, C, Berman, H. and Frondel, C. (1960). "Dana's System of Mineralogy", 7th ed., Vol. 2, pp. 877-878. Wiley, New York. Pelton, W. F. (1950). The effect of zinc chloride and potassium ferrocyanide as a caries prophylaxis. / . dent. Res. 29, 756-759. Pindborg, J. J. (1953). The pigmentation of the rat incisor as an index of metabolic disturbances . Oral Surg. 6, 780-789. Poole, D. F. G., Tailby, P. W. and Berry, D. C. (1963). The movement of water and other molecules through human enamel. Arch, oral Biol. 8, 771-772. Posen, J. M. (1962). Chemical and physical properties of altered and sound enamel. II . Relative dissolution and residue light absorption (color) / . dent. Res. 41, 471-475. Posner, A. S. (1960). The nature of the inorganic phase in calcified tissues. In "Calcification in Biological Systems", Publ. No. 64, pp. 373-394. Amer. Ass. Advanc. Sci., Washington, D. C. Posner, A. S. and Perloff, A. (1957). Apatites deficient in devaient cations. / . Res. nat. Bur. Stand. 58, 279-286. Posner, A. S., Perloff, A. and Diorio, A. F. (1958). Refinement of the hydroxyapatite structure. Acta cryst. 11, 308. Reiss, L. F. (1961). Strontium90 absorption by deciduous teeth. Science 134, 1669-1673. Ronnholm, E. (1962). The amelogenesi s of human teeth as revealed by electron microscopy: the developmen t of enamel crystallites. / . Ultrastruct. Res. 6, 249-278. Roseburg, T. (1934). Presenc e of iron in enamel keratin. /. dent. Res. 14, 269-272. Rosenthal, H. L., Gilster, J. E. and Bird, J. T. (1962). Accumulation of strontium 90 in primary teeth of American children. Abstr. Int. Ass. dent. Res., 40th gen. Meet., St. Louis, 1962 No. 41. Rygge, J. (1939). Trois cas de coloration brune de l'émail de toutes les dents chez trois enfants de même famille. Acta odont. scand. 1, 57-74 . Savory, A. and Brudevold, F. (1959). The distribution of nitrogen in human enamel. / . dent. Res. 38, 436-442. Scott, D. B. (1960). Electron microscopic evidence of fluoridereaction. / . dent. Res. 39, 1117 (Abstract). Scott, D. B., Picard, G. and Wyckoff, R. W. G. (1950). Studies of the action of sodium fluoride on human enamel by electron microscopy and electron diffraction. Publ. Hlth. Rep., Wash. 65, 43-56. Seidell, A. (1958). "Solubilities. Inorganic and MetalOrganic Compounds". Van Nostrand, Princeton, New Jersey.

AND

R.

S Ô R E M A RK

Shaw, J. H. and Griffiths, D. (1961). Developmenta l influence on incidence of experimenta l dental caries resulting from dietary supplementatio n by various elements. Arch, oral Biol. 5, 301-322. Singer, L. and Armstrong, W. D. (1960). Regulation of human plasma fluoride concentrations . / . appl. Physiol. 15, 508-510. Smith, F. Á., Gardner, D. E., Leach, S. A. and Hodge, H. C. (1957). Fluoride removal by powdered dental enamel from solutions of stannous or sodium fluoride. Nature 180, 1421-1422. Sobel, A. E. and Hanok, A. (1948). Calcification of teeth. I. Composition in relation to blood and diet. / . biol. Chem. 176, 1103-1112. Sobel, A. E., Shaw, J. H., Hanok, A. and Nobel, S. (1960). Calcification. XXVI . Caries susceptability in relation to composition of teeth and diet. / . dent. Res. 39, 462-472. Soremark, R. (1960). Distribution and kinetics of bromide ions in the mammalian body. Acta radiol., Stockh. Suppl. 190. Soremark, R. (1964a). To be published. Soremark, R. (1964b). Studies on the concentration of vanadium in some biological specimens . 3e Colloque Int. Biol. Analyse par Radioactivation, Saclay, 1963, pp. 223-238. Commissaria t Energie Atomique, Saclay, Paris. Soremark, R. and Andersson, N. (1962). Uptake and release of vanadium from intact human enamel following ν ^ Ï ä applications in vitro. Acta odont. scand. 20, 81-93. Soremark, R. and Lundberg, M. (1964). Gamma-ray spectrometric analysis of the concentration of Cr, Ag, Fe, Co, Pt, and Rb in normal human enamel. Acta odont. scand. 22, 255-259. Soremark, R. and Samsahl, K. (1961). Gamma-ray spectrometric analysis of elements in normal human enamel. Arch, oral Biol. 6, 275-283. Soremark, R. and Samsahl, K. (1962). Gamma-ray spectrometric analysis of elements in normal human dentin. /. dent. Res. 41, 603-606. Soremark, R., Ingels, O., Plett, H. and Samsahl, K. (1962). Influence of some dental restorations on the concentration s of inorganic constituents of the teeth. Acta odont. scand. 20, 215-224. Sognnaes , R. F., Shaw, J. H. and Bogoroch, R. (1955.) Radiotracer studies on bone, cementum, dentin, and enamel of Rhesus monkeys. Amer. J. Physiol. 180, 408-420. Soni, Í . N. and Brudevold, F. (1959). Microradiographic and polarized light studies of initial carious lesions. / . dent. Res. 38, 1187-1194. Soni, Í . N. and Brudevold, F. (1960). Microradiographic and polarized light studies of artificially produced lesions. /. dent. Res. 39, 233-240. Sperber, G. H. and Buonocore, M. G. (1963). Effect of

18. C H E M I S T RY

OF

THE

MINERAL

different acids on character of demineralization of enamel surfaces. / . dent. Res. 42, 707-723. Stack, M. V. and Williams, G. (1952). Quantitative variation in the total organic matter of enamel. Brit. dent. J. 92, 261-267. Steadman , L. T., Brudevold, F. and Smith, F. A. (1958). Distribution of strontium in teeth from different geographical areas. J. Amer. dent. Ass. 57, 340-344. Stein, G. and Boyle, P. E. (1959). Pigmentation of the enamel of albino rat incisor teeth. Arch, oral Biol. 1, 97-105. Stookey, G. K. and Muhler, J. C. (1959). Effect of molybdenum on fluoride retention in the rat. Proc. Soc. exp. Biol, N.Y. 101, 379-380. Sundvall-Hagland , I., Brudevold, F., Armstrong, W. D., Gardner, D. E. and Smith, F. A. (1959). A comparison of the increment of fluoride in enamel and the reduction of dental caries resulting from topical fluoride applications. Arch, oral Biol. 1, 74-79. Syrrist, A. (1949). Histological studies on the effect of sodium fluoride on human dental enamel. Odont. Tidskr. 47, 395-408. Tank, G. and Storvick, C. A. (1960). Effect of naturally occurring selenium and vanadium on dental caries. / . dent. Res. 39, 473-488. Taylor, D. M. (1961). Retention of zinc-65 in the bones of rats. Nature 189, 932-933. Tefft, H., French, E. L. and Hodge, H. C. (1941). Magnesium determinations on all the dentin from sound and carious teeth. / . dent. Res. 20, 45-48. Thewlis, J. (1940). The structure of teeth as shown by X-ray examination. M. R. C. Spec. Rep. Ser. 238.

P H A SE

OF

ENAMEL

277

Torell, P. (1955). Acid resistant substanc e in arrested carious lesions. Odont. Tidskr. 63, 495-508. Torell, P. (1957). Determination of iron in dental enamel. Odont. Tidskr. 65, 20-23. Trautz, O. R. (1960). Crystallographic studies of calcium carbonate phosphate . Ann. N.Y. Acad. Sci. 85, 145160. Underwood, E. J. (1956). "Trace Elements in Human and Animal Nutrition", p. 224. Academic Press, New York (2nd ed., 1962). Weidmann, S. M., Weatherell, J. A. and Whitehead, R. G. (1959). The effect of fluorine on the chemical composition and calcification of bone. / . Path. Bact. 78, 435-x 445. Wellock, W. D. and Brudevold, F. (1963). A study of acidulated fluoride solutions. II . The caries inhibiting effect of single annual topical applications of an acidic fluoride and phosphate solution. A two year's experience . Arch, oral Biol. 8, 179-182. Wynn, W., Haldi, J., Bentley, Ê. D. and Law, M. L. (1956). Dental caries in the albino rat in relation to the chemical composition of the teeth and of the diet. II . Variations in the Ca/Pratio of the diet induced by changing the phosphorus content. / . Nutr. 58, 325-333. Wynn, W., Haldi, J., Bentley, Ê. D. and Law, M. L. (1957). Dental caries in the albino rat in relation to the chemical composition of the teeth and of the diet. III . Composition of the incisor teeth in animals fed diets with different Ca/P ratios. / . Nutr. 63, 57-63. Zipkin, I., McClure, F. J. and Lee, W. A. (1960). Relation of the fluoride content of human bone to its chemical composition. Arch, oral Biol. 2, 190-195.

This page intentionally left blank

CHAPTER

19

CHEMICAL ORGANIZATION OF THE ORGANIC MATRIX OF DENTINE J.

E.

EASTOE

I. Introduction A . The "Chemical Anatomy" of Tissues B. Definition of the Organic Matrix of Dentine C. Microchemical Analysis and Histochemistry D. Preparation of Dentine for Chemical Investigations II . The Organic Components of Dentine A . Collagen B. Other Proteins C. Chondroitin Sulphate D. Mucoproteins and Sialoproteins E. Lipids F. Citrate and Lactate G. Fluorescen t Substance s

279 279 281 281 282 283 283 295 296 296 298 298 299

III . Chemical Balance Sheet for Dentine

300

IV . Changes in the Organic Components of Dentine A. Biological Variation B. Changes with Age C. Changes Accompanying Dental Caries

301 301 301 302

V. Mineralization of the Organic Matrix A . Introduction B. The Alkaline Phosphatas e Theory C. Phase Transformation between Solution and Solid Apatite D. The Epitactic Concept E. Collagen as an Epitactic Agent F. The Role of Mucosubstance s G. The Control of Mineralization—Pyrophosphatase s VI . Concluding Remarks

303 303 304 304 305 306 308 308 309

Addendum

310

Reference s

312

Note Added in Proof

315

I . I N T R O D U C T I ON A . T H E " C H E M I C A L A N A T O M Y " OF TISSUES

of size, ranging from atomic dimensions upwards to those of the entire t o o th (Fig. 1). The broad

Dentine possesse s a highly organized structure,

features of a picture representin g this structural

the various features of which cover several orders

hierarchy of dentine can now be filled in as a result 279

280

J.

E.

E A S T OE

of advances made in the 15 years which have elapsed since the publication of the most recent monograph dealing with chemical aspects of teeth (Leicester, 1949). Some of the details of this structure and of the manner in which it develops, however, still await discovery. Early organic chemistry and biochemistry could deal effectively only with rather small molecules. For this reason biochemistry tended to become identified with physiology in being mainly concerned with the more rapid changes that take place in livin g organisms. These largely involve reactions undergone by metabolites of small molecular size. Many cell components and extracellular tissue elements were too large and complex for analysis by the earlier chemical techniques . Methods for the characterizatio n of macromolecular substance s of biological origin (e.g. carbohyi

drates, nucleic acids and proteins) have been developed only comparatively recently. As a result, it has become possible to work out completely the chemical structures of molecules as complex as insulin (Ryle et ah, 1955) and ribonuclease (Spackman, Stein and Moore, 1960). Chemical studies of tissue macromolecule s and the fibrous aggregate s in which they are often arranged have been supplemente d and extended by X-ray diffraction analysis (Perutz, 1962). This permits the distances and directions in space at which regularly repeating atoms or groups of atoms occur to be measured accurately. Thus, chemical and X-ray diffraction techniques together are reaching up into larger orders of size and are enabling complex biological structures to be described in fundamenta l terms. Over the same period, the developmen t of the

r- 1 0 2 c m

10

L e n g t h of < whole teeth h i L e n g t h of human odontoblast processes

hioioLight microscope

>

Width of odontoblast

h 10"

3

ΙΟ" 4 1μ

10"5

640 Â s p a c i n g of collagen fibril Inorganic crystallites

X-Ray diffraction

U 2. 86 A l e n g t h of a m i n o a c i d unit along m a c r o m o l e e u l e

Fig. 1.

Electron microscope

10"

10~

7

1



Chemical ^> s e q u e n c e studies

— ΙΟ" 8 1 A

Sizes of the components of dentine. The 2.86 A length of an amino acid unit is illustrated in Fig. 4.

19.

CHEMICAL

ORGANIZATION

OF

electron microscope has enabled observations with some of the characteristic s of classical microscopic anatomy to be extended downwards to a lower practical limi t of resolution of perhaps 5 A. Thus, three distinct methods for the investigation of structure overlap for a considerable range (Fig. 1). It is therefore reasonabl e to begin to think in terms of a "chemical a n a t o m y" in which comparatively large organized systems can be considered in terms of their component atoms. There is no absolute distinction between the anatomical and chemical structures of an organism; the apparent difference concerns only the nature of the technique used for revealing structure at a particular order of size. Continuity of structure is particularly clearly shown in the extracellular fibrous proteins of animal tissues. B . DEFINITION OF THE O R G A N IC M A T R I X

THE

O R G A N IC

MATRI X

OF

DENTINE

281

high molecular weight mucopolysaccharide s of the histological "ground substance" . A s developmen t proceeds, fibrous material is laid down which rapidly becomes more abundant and less soluble as it increases in stability and mechanica l strength. The characteristic s of the full y formed dentine matrix are largely attributable to collagen, its most abundant organic constituent. The presence in dentine of a high proportion of this fibrous protein which can be changed into gelatine when "boiled for some time in water acidulated with acetic or weak hydrochloric acid", has been known for many years (Tomes, 1896). However, its full significance has only recently been revealed as a result of extensive investigations on several collagenous tissues. The resulting overall co-ordinated picture of collagen structure has an important bearing on the organization of dentine, and is described in section II , p. 283.

OF DENTINE

The term " m a t r i x" is considered here in relation to dentine in the sense already defined for bone and other mineralized tissues (Eastoe, 1956, 1964). I t is the organic material within which the inorganic crystallites are laid down and which is probably responsible for their genesis. Lik e the matrices of other hard tissues, it comes into existence before the crystallites are formed, it participates in their development and encloses them spatially, as small structures within a more extensive one. When mineralization is complete, the dentine matrix, lik e that of bone, persists comparatively unchanged . The matrix in many ways resembles the predentine which is laid down by the odontoblasts at a stage before mineralization begins. This comparison is only approximate because dentine probably undergoes changes during the course of its development , similar in kind, though perhaps less in degree, to other connective tissues such as skin, tendon and bone which, lik e dentine, arise from mesoderma l cells. A t an early stage of development these tissues have a very high water content and consist largely of thin amorphous gels, the main organic components of which are the

C . MLCROCHEMICAL ANALYSI S AN D HISTOCHEMISTRY

Whil e the recently developed methods for the determination of structure have produced results of outstanding value for the most abundant constituents, their usefulness decrease s for tissue components present only in small amounts. The main techniques for the investigation of minor constituents are those of microchemical analysis and histochemistry, which have been developed from the methods of larger-scale chemical analysis and histology, respectively. The characteristic s of these techniques are compared in Table 1, which shows that they are largely complementar y to each other. They should therefore be used in conjunction, histochemica l localization of substance s within the tissue permitting a study of their distribution and microchemical d more methods enabling them to be characterize precisely and the amounts present to be determined. Unfortunately, this has seldom been done, especially for studies of dentine, where it is often necessar y to compare the results of separate analytical and histochemica l investigations.

282

J.

E.

EASTOE

Table 1 COMPARISON OF FEATURES OF MICROCHEMICAL ANALYSI S AND HISTOCHEMISTRY

Histochemistry

Microchemical analysis 1. Carried out in ordinary chemical apparatus , scaled down as necessar y 2. Specimen destroyed 3. Localization of substance s in terms of structure at a histological level—poor 4. Sensitivity rather low for small specimens 5. Methods available for practically all known substance s 6. Identification of a substanc e is absolute 7. Chemical structure of new substance s can be investigated 8. Quantitative

D.

PREPARATION OF D E N T I NE FOR

CHEMICAL

INVESTIGATIONS

1.

Pulverization

The main criteria for satisfactory pulverization of dentine are the production of a sufficiently finely divided powder and the minimum degradation of the organic components . Degradation results from the generation of heat, either generally throughout the mass of material or locally, at the moment when disintegration occurs. The former effect can be avoided by cooling the apparatus , whereas a slight local rise in temperature is probably unavoidable and difficult even to measure . Stack (1951) found that when dentine was reduced to a powder by filing or grinding, 15 % of the nitrogenous compounds were obtained in water-soluble form. This degree of degradation was equivalent to heating at 150° C for 2 hours. Use of a percussion mortar resulted in a reduction of soluble nitrogen to only 5 % of the total. Preliminary cooling of the specimen and mortar in solid carbon dioxide should decreas e degradation effects still further. A stainless steel reciprocating ball mill fitted with a cooling jacket is suitable for small samples. For larger quantities of material a hammer mill or beater-cros s mill is suitable. It must be fed slowly with small pieces of material

1. Carried out on a tissue section on microscope slide 2. Specimen not destroyed 3. Localization better, but masking and diffusion can occur 4. Sensitivity adequate for microscopical identification 5. Methods are available only for a very restricted range of substance s 6. Identification ultimately depends on chemical analysis in the same or another tissue 7. Chemical structure cannot be investigated in detail 8. Not quantitative, although approximate comparisons of relative amounts can be made

(Eastoe and Courts, 1963). Probably the most satisfactory means of grinding hard tissues for chemical studies is a specially developed beater mill continuously cooled by liquid nitrogen (Herring, 1964a). 2. Removal of Adventitious

Tissues

The dentine occurs in intimate association with enamel, cementum and pulp, which must be removed to permit the isolation of purified dentine. The softer pulp is readily detached by scraping or grinding, if the tooth is first broken, although the process probably also removes odontoblasts . Enamel may be chipped from the intact tooth and the remaining portions be ground away before the dentine is pulverized (Stack, 1951). Alternatively, the whole tooth may be initiall y reduced to powder and the dentine separate d by flotation from the enamel which sinks in a bromoform-aceton e mixture of specific gravity 2.70 (Manly and Hodge, 1939). Subsequently , the lighter cementum is separate d in a mixture of specific gravity 2.07 and the "junction particles" containing enamel at 2.42. A similar method, which avoids centrifugation, has been introduced by Battistone and Burnett (1956). I n studies of the organic components of dentine, the removal of the last traces of enamel and cementum is less crucial than for similar studies on

19. C H E M I C A L O R G A N I Z A T I O N O F

THE O R G A N I C M A T R I X OF D E N T I N E

II. THE ORGANIC COMPONENTS OF DENTINE

enamel. This is because of the very low content of organic matter in enamel and the marked similarity i n composition of cementum and dentine. 3. Demineralization Investigations

of Dentine for

A.

Chemical

NaOOC- C H 2

/

\

NaOOC- C H 9

, Ν · CH 9- CH P- Ν

÷/

Ν

/

\

a. Types of amino acid present. When collagen is heated with strong acid (e.g. 6A^-hydrochloric acid at 100° C for 24 hours) it is eventually broken down completely into its ultimate structural units. These are the amino acids, which also serve as building blocks for the biosynthesis of proteins in the livin g organism. Altogether, eighteen completely different types of amino acid unit are present in collagen; sixteen of these are found widely distributed in most animal and vegetable proteins, while the remaining two, hydroxyproline and hydroxylysine, appear to be confined almost exclusively to collagen (Harkness, 1961). Two other amino acids, commonly found in proteins— cystine, which forms sulphur cross-linkage s between protein chains, and tryptophane, which is a dietary essential—ar e not present in collagen. The amino acids found in proteins have an amino group in the á-position relative to the carboxyl group. This provides the key to those features of their chemical structure which they all hold in common. The structure centres round a particular carbon atom to which four different groups are attached: (1) the á-amino group (2) the carboxyl group, (3) a hydrogen atom and (4) a side-chain group R thus,

R — CH +

Ca2

CH,- COONa

CHXOONa

C H2 Ca CH2 . / \ / COO OOC

Composition

NH0

C H 2- COONa

CH,- C H 2 NaOOC- CH. ,Ν

COLLAGEN

1. Amino Acid

When the inorganic portion of dentine is dissolved by means of acid, it is preferable to use either a weak acid or a dilute solution of a strong acid, in order to minimize degradation and solution of the organic matter. Swelling of the collagen is also avoided if the pH is not allowed to fall too low. Stack (1951) added AMiydrochloric acid to a stirred suspensio n of powdered dentine at such a rate that the solution did not fall below pH 3. Eastoe (1963) obtained rapid demineralization at a somewhat lower pH by means of sulphur dioxide solution. The introduction of organic chelating reagents , notably ethylenediaminetetra-acetat e (EDTA), has enabled demineralization to be carried out avoiding the use of acid altogether. A solution of the disodium salt, initiall y at pH 4.5, is adjusted to pH 7.0-7.4 by means of sodium hydroxide. A t this p H, E D TA demineralizes at a maximum rate, causes minimum breakdown and has a greater solubility than in the original solution (Nikiforuk and Sreebny, 1953). The demineralizing effect results from the substantially complete removal of free calcium ions from solution by the formation of a complex chelate (Greek χηλη = claw) of high stability.

283

+

2 Na

+

^ C O OH

This carbon atom is therefore bound in an asymmetrical configuration, the naturally occurring amino acids existing in the optically active L-form. The four different groups all have important functions in relation to protein structure. The á-amino and carboxyl groups participate in the main polypeptide chain by forming peptide bonds wit h the amino acids (see page 288 and Fig. 3).

284

J.

E.

E A S T OE

Table 2 STRUCTURE OF THE AMIN O ACI D SIDE CHAINS IN COLLAGEN Glycine

Gly

H—

Alanine

Ala

CH 3-

Valine

Val

Leucine

Leu

Isoleucine

lieu

Simple

CH 3 \

CH/ CH, CH 3 \3

CH. CH 9— / CH 3

Hydrocarbon

CH 3 /

Methionine

Met

CH—

CH 3- S- CH 5 •CH 9

Sulphur containing

CH 9—CH

Proline (pyrollidine group)

\

o

r

/

N—

CH 9 - C H 9 CH 9 Hydroxyproline

Hypro

Serine

Ser

I

CH

Imino

\

N— I / HO—CH—CH2 HO- C H 2—

Hydroxy

HO \

CH—

Threonine

Thr

Tyrosine

Tyr

Phenylalanine

Phe

A s p a r t i c acid

Asp

Glutamic acid

Glu

Histidine (imidazole group)

JJ.

" O O C C H 9C H 9— CH- -NH22 \ CH 9 // Ν CH

Lysine

Lys

+

Hydroxylysine

Hylys

Arginine (guanidino group)

.

/

CH 3 HO- C 6H 4- C H 2—

Aromatic

C 6H 5« CH 2 " O O C C H 2—

Acidic

N H 3( C H 2) 4ΝΗ 3· CH 2- CH(OH)(CH 2) 2-

Basic

TNH. g

r

).NH

( C H 2) 3—

NH

The small size of the hydrogen atom, which is invariably present, provides the space necessar y for the accommodatio n of large groups elsewhere in the protein and permits the close approach of different polypeptide chains.

The structure of the side-chain group R is the only feature which distinguishes different amino acids from each other and gives each its characteristic properties. These side chains retain their character when the amino acid units are in-

19. C H E M I C A L

ORGANIZATION

OF

corporated into a protein, and collectively are responsible for the intrinsic properties of each particular protein. The structures of the side chain groups found i n collagen are given in Table 2, where they are subdivided according to their chemical structures. The acidic and basic side chains are normally ionized and carry negative or positive charges, respectively, unless the pH is below or above definite limits. The collagen molecule therefore functions as a complex type of ion with numerous positive and negative charges. The structure of the side-chain group in the imino acids proline and hydroxyproline differs from that of all the other amino acids since it is attached not only to the carbon atom, but also to the adjacent nitrogen atom, so that it forms a closed planar ring (Table 2). The resulting imino group is nevertheles s capable of being incorporated i n the polypeptide chain of proteins. The bulky pyrrolidine ring, however, prevents free rotation about the bond joining the carbon and nitrogen atoms and may thus restrict the configurations which are possible. I n collagen, 4-hydroxyproline is the predominating isomer, but small amounts of 3-hydroxyproline have recently been isolated by several groups of workers (Ogle, Arlinghaus and Logan, 1962; Piez, Eigner and Lewis, 1963). b. Relative numbers of amino acid units. Quantitative amino acid analysis has been greatly facilitated by the introduction of ion-exchange chromatograph y (Moore and Stein, 1951), and it is now possible to analyze a protein completely in 24 hours by automatic means (Spackman , Stein and Moore, 1958). Where material is scarce, analyses may be performed on sub-milligram quantities (Eastoe, 1961). Collagens from a variety of tissues i n several species have been analyzed and the earlier results for vertebrates have been summarized by Eastoe and Leach (1958). The values obtained more recently for the amino acid composition of dentine (human and ox) are given in Table 3a, together with some results of particular interest for collagens from other tissues (Table 3b). F or simplicity, values in the tables are expresse d as the numbers of amino acid units (residues) of each

20

THE

O R G A N IC

MATRI X

OF

DENTINE

285

particular type per 1000 units of all types. This gives a picture of the relative frequency of occurrence of each type of amino acid. The composition of those members of the collagen family of proteins found in the tissues of present day vertebrates is fairly constant. The greatest species variations occur between the collagens of the various groups of fishes, especially the more specialized Actinopterygii and the mammals. Some of the fish collagens have markedly lower shrinkage temperature s and the relative proportions of the hydroxy amino acids are different, there being less hydroxyproline and more serine (Table 3b, see Eastoe, 1957). Piez (1960) has pointed out that the total content of imino acids (proline and hydroxyproline) is reduced in fish collagens. Unfortunately, no data appear to be available for fish dentine collagen except for that from shark dentine, which has been shown to resemble collagens from other elasmobranc h tissues in these respects (Moss, Jones and Piez, 1964). The composition of collagen from the various tissues of different mammalian species is almost constant, variations being of the same order as the experimental errors of the analysis and consequent ly difficult to distinguish. Piez and Likin s (1960) pointed out that dentine collagen appears to be unusually rich in hydroxylysine and corresponding ly poor in lysine when compared with collagens from other tissues .They considered that hydroxylysine may play some part in the mineralization process. When all the published data for dentine collagen are taken into consideration (Eastoe, 1963), the ratio of lysine to hydroxylysine varies from 1.1 to 3.8 although their sum is more constant (29.1-37 residues per thousand). It is not yet altogether clear to what extent these variations in the degree of hydroxylation of lysine reflect true differences between specimens or analytical errors. Apart from these differences for hydroxylysine, the variations between different analyses of dentine collagen are reasonabl y small (Table 3a). Dentine collagen is closely similar to collagen from other mammalian tissues (Table 3b), and shows those

286

J.

E.

E A S T OE

Table 3a COMPOSITION OF DENTINE COLLAGEN"

Human dentine

Bovine

Amino acid Alanine Glycine Valine Leucine Isoleucine Proline Phenylalanine Tyrosine Serine Threonine Methionine Arginine Histidine Lysine Aspartic acid Glutamic acid Hydroxyproline Hydroxylysine Amide

(1) 112 327 22 25 11 116 16 3.2 39 16 4.1 47 3.7 19 52 72 103 13 39

(2) 96 317 20 31 11 124 13 5.8 35 19 3.6 48 5.5 25 46 72 120 8.5 31

(3)

(4)

114 297 24 27 11 130 12 3.2 33 20 5.2 49 7.5 25 58 77 100 6.7

112 329 25 24 9.3 116 16 6.4 33 17 5.3 52 4.7 22 46 74 99 9.6





Permanen t (5)

Deciduous (developing) (6)

112 319 25 26 10 115 14 2.3 38 19 5.2 47 5.3 23 55 73 101 8.4 41

108 308 27 27 12 115 15 4.9 41 20 6.8 51 6.5 25 51 75 98 8.1 54 '

a

Values are expresse d as numbers of units of each amino acid per 1000 units of all types. The values were obtained from the following sources: Column (1) Piez and Likin s (1960); (2) Hess et al. (1961); (3) W. G. Armstrong (1961); (4) Piez (1962); (5) and (6) Eastoe (1963).

striking features of composition which distinguish this protein from all others (Fig. 2). Thus, one in every three amino acid units is accounted for by glycine, which has no side chain, and one in nine by alanine with a very small (methyl group) side chain. The imino acids, proline and hydroxyproline, account for two in every nine units so that two thirds of the available positions are occupied by only four types of amino acid unit. The acidic amino acids occupy one in eight units, although free carboxyl groups account for only one side chain in every thirteen, owing to the presence of amide groups on the remainder. The four kinds of basic group together occupy one position in twelve. The slight preponderanc e of basic groups over

free carboxyls implies that collagen is a basic protein. This is supported by the finding that when it is degraded under conditions which involve negligible loss of amide groups, the resulting gelatin has an isoionic point at pH 9.4 (Eastoe, Long and Willan, 1961). A much lower value of pH 5.8 has been reported for the isoelectric point of collagen suspende d in buffer solutions (Brown and Kelly, 1953); possibly this is a result of ionbinding. Only 11 % of the total positions are available for the remaining eight amino acids. Half of these are occupied by the hydroxy amino acids serine (1 in 27) and threonine (1 in 54). The total number of hydroxyl groups in collagen fill one side chain in six.

19. C H E M I C A L

ORGANIZATION

OF

THE

O R G A N IC

MATRI X

OF

DENTINE

287

Table 3b COMPOSITION OF COLLAGEN FROM VARIOUS TISSUES'1

Human

Amino acid

Alanine Glycine Valine Leucine Isoleucine Proline Phenylalanine Tyrosine Serine Threonine Methionine Arginine Histidine Lysine Aspartic acid Glutamic acid Hydroxyproline Hydroxylysine Amide

Guinea pig granuloma, microsomes (1)

Ox skin,

Kidney

Bone

Cod bone

Acid-soluble (2)

reticulin (3)

(4)

(5)

93.4 263 29.1 41.6 22.4 101.5 18.5 11.3 51.8 31.5 6.0 59.0 9.9 36.1 61.6 76.0 79.6 7.6 —

115.1 341 119.0 24.0 10.4 111.3 11.8 2.8 39.7 18.2 5.1 47.1 1.9 24.0 44.6 73.7 102.3 7.8 36.3

96.5 309 26.8 35.8 18.0 97.2 18.1 3.0 42.8 21.9 8.6 45.3 5.3 21.6 52.9 76.7 107.7 12.2 43.0

113.5 319 23.6 25.5 13.3 123.4 13.9 4.5 35.9 18.4 5.3 47.1 5.8 28.0 47.0 72.2 100.2 3.5 37.3

106.6 349 18.3 23.0 11.6 100.0 11.4 3.3 69.8 23.8 14.0 48.5 7.4 23.3 51.8 72.0 58.6 8.2 44.5

a

Values are expresse d as numbers of units of each amino acid per 1000 units of all types. The values were obtained from the following sources: Column (1) Eastoe (1961); (2) Bowes et al. (1955); (3) Windrum, Kent and Eastoe (1955); (4) Eastoe (1955); (5) Eastoe (1957).

The amino acids with lipophilic (hydrocarbon) side chains—leucine , valine, isoleucine and phenylalanine—togethe r account for only approximately one residue in twenty. The excess of the hydrophilic (hydroxyl and the ionizable acidic and basic groups) over the lipophilic type renders collagen predominantly hydrophilic. Methionine and tyrosine groups are very infrequent; each occurs only once every 200 units. The frequencies quoted here are rounded off to give a broad picture of collagen composition which is illustrated in Fig. 2. The values reported for deciduous dentine collagen (Table 3a) refer to foetal teeth which are developing rapidly. The slight deficiency of amino acids relatively abundant in collagen (glycine,

alanine and hydroxyproline) indicates that a small proportion of another protein may be present. This is probably attributable to the early stage of development rather than a difference between deciduous and permanent dentine. 2. The Polypeptide

Chain

a. Chemical structure. The biosynthesis of a protein such as collagen involves essentially the joining together of the individual amino acids to form a polypeptide chain. The overall process involves the carboxyl group of one amino acid reacting with the á-amino group of a second amino acid molecule, with the elimination of a molecule of water and the formation of a peptide

J.

288

E.

EASTOE „ N H 2

Amino acid

R - C H sC O O H

xn h

Polypeptide chain

^ NH Ro-CH .NH R 3- C H N CO

Macromolecule ( protofibril)

Basic

Imino Amide

}

I Hydroxy

Acidic

[

I Lipophilic

Fig. 2. Relative proportions of amino acids in dentine collagen. The areas are proportional to the number of amino acid units of each type per 1000 total units. N.B., Hydroxyproline is an imino acid with a hydroxy group.

bond. This bond has the structure —CO · N H — and hence is of an amide type. By reaction of the carboxyl group of the second amino unit with the amino group of a third amino acid molecule, a chain of three units (tripeptide) may be formed (see Fig. 3). The process, if continued for a sufficient number of bonds, gives rise to the polypeptide chain of a protein, with a general formula (—NH · C HR · CO— ) n where η may be very large. b. Amino acid sequence. The order in which the different kinds of amino acid unit are arranged i n the polypeptide chain is an important feature of protein structure. The number of arrangement s possible with eighteen different kinds of unit in the collagen chain, which contains upwards of 1000 units altogether, is astronomical. Studies on shorter protein chains, insulin (51 units) and ribonuclease (124 units) have shown that the amino acid sequenc e of these particular proteins is unique and identical i n all their molecules. The complete sequenc e for

Fibril

Fibres in bundles

Dentine and

tendon

Fig. 3. Some features of collagen structure. Structural features at increasing orders of size are represente d at different levels. More than one method of representatio n is used at some levels in order to relate the feature shown, to the next smaller and larger orders of size. The macromolecules can be considered as stiff rods (arrows) which are laid parallel to one another in the fibrils. The bundle of fibril s shown would make up one of the fibres which in turn are also arranged in bundles.

collagen has not yet been determined as the increase in the size of the molecule introduces great practical difficulties. The sequence s of fragments of the collagen chain, however, have been investigated by several workers. Astbury (1940) suggeste d on the basis of early chemical analyses and X-ray diffraction measure ments that collagen had a sequenc e with the amino

19. C H E M I C A L

ORGANIZATION

OF

acid units repeating in groups of three in the order G - P -R [where G is glycine, Ñ is proline or hydroxyproline and R is any residue (unit) other than these]. Figure 2 illustrates that the relative proportions of amino acids as determined by somewhat more accurate methods, while approximately in accord with this concept, show a definite deficiency of Ñ and a correspondin g excess of R types. The earliest direct investigation of the sequenc e i n collagen involved the identification of the more abundant small peptides from partial hydrolyzates. Both Schroede r et al. (1954) and Kroner, Tabroff and M c G a rr (1955) found not only the peptide glycyl proline (Gly · Pro see Table 2), which conforms to Astbury's proposed sequence , but also Hypro · Gly, which does not. The peptide Gly · Hypro, also required by the original suggestion, was missing. Schroede r et al. (1954) therefore proposed instead that the sequenc e Gly · Pro · Hypro · Gly was of frequent occurrence . This was confirmed by the identification of tri- and tetrapeptides with appropriate structures (Kroner et al, 1955). Grassmann , Hannig and Schleyer (1960) have reported on the composition of some fifty larger peptides isolated from collagen, after partial hydrolysis with trypsin. They found that almost all of these contained close to 33 % of glycine, whereas imino acids could either be extremely abundant (28 out of 62 residues) or almost absent. I t therefore seems that sequence s similar to Gly · Pro · Hypro * Gly predominate in some parts of the polypeptide chain of collagen, whereas, in other parts, glycine is associate d with amino acids other than proline and hydroxyproline. Local variations in the distribution of amino acids in different parts of the chain are also suggeste d by the work of Solomons (1960) on dentine collagen. After mild hydrolysis by heating wit h distilled water, it could be separate d into four peptide fractions by paper electrophoresis . One of these contained all the amino acids, a second was lacking in hydroxyproline and a third had no imino acids or phenylalanine . The fourth peptide

THE

O R G A N IC

MATRI X

OF

DENTINE

289

fraction contained glycine and alanine in combination with acidic and basic amino acids only. This finding is consistent with the suggestion of Bear (1952) that certain regions of the collagen fibrils are rich in amino acid units with comparatively bulky side chains which have ionizable groups (see also page 291). 3. The Collagen Macromolecule Tropocollagen Molecule

or

a. Arrangement of polypeptide chains. The individual polypeptide chains of proteins are in themselves comparatively flexible structures, the component atoms of which are all b o u nd together by strong covalent bonds. A variety of configurations can be assume d by a flexible chain, though some may be excluded for steric reasons . In a given environment, one particular configuration wil l usually be preferred, which is compatible with the maximum stability of the system. For proteins, factors such as the forces between charged groups on the side chains and the possibility of forming the maximum number of hydrogen bonds (which are weaker and less permanent than covalent bonds) largely determine the configuration adopted. The polypeptide chains in the collagen macromolecule are arranged in a complex and beautiful pattern, the details of which were gradually worked out by a number of investigators between 1945 and 1960. This was done largely on the basis of the X-ray diffraction spectrum of native collagen from unmineralized tissues (e.g. tendon) together wit h certain broad features of chemical composition already discussed . A rigorous account of this structure and the evidence upon which it is based, is given by Rich and Crick (1961). A brief description of its main features follows. The collagen macromolecule consists of three distinct polypeptide chains each of which is twisted into a left-handed spiral about an individual axis. The helix has a comparatively short pitch of 9.3 Â and there are three amino acid units per turn (Fig. 4a). The macromolecule is made up from three such left-handed helices with each individual axis gently twisted into a right-handed helix about

290

J.

a

b

E.

c

Fig. 4. Triple helical structure of collagen, (a) Polypeptide chain wound in left-handed helix of pitch 9.3 A and three amino acid units per turn, (b) The axis of the simple helix in (a) wound in a right-handed helix of pitch 28.6 A, so the polypeptide chain forms a compound helix, (c) Three units of the type shown in (b), arranged to form a triple helix. For simplicity, only the three axes of the polypeptide chains are shown. Two of the chains are of the al type and the third (black) of the ocl type. The triple helical macromolecule is a stiff rod with a straight axis and is some 60 times longer than the portion shown in the diagram.

the common axis of the macromolecule which is straight (Fig. 4b). The pitch of the right-handed helix is much longer (28.6 A) and there are ten amino acid units for each complete turn. The complete system is thus a "super-helix" consisting of three polypeptide chains, wound into compound helices (Fig. 4b) and arranged symmetrically at 120-degree intervals as shown in Fig. 4c, in which the polypeptide chains have been omitted for

E A S T OE

clarity and only the axes are drawn. Altogether there are thirty residues per turn of the compound helix. b. Interchain hydrogen bonds. Various portions of the amino acid structures on different polypeptide chains are brought into close proximity as a result of interweaving within the super-helix. This enables a high proportion of "hydrogen b o n d s" (1 for every 3 residues) to be formed between the hydrogen atoms of imino (—NH—) groups and the carbonyl (—CO—) oxygens in the backbones of neighbouring polypeptide chains. Though individually hydrogen bonds are weak compared with covalent bonds, they are collectively sufficiently strong to hold the three chains together. If heat is applied sufficient to exceed the "thermal denaturation temperature " of the tropocollagen (or "shrinkage temperature " of a tissue, 37-65°C), the bonds are no longer sufficiently strong to maintain the stability of the stucture, which breaks down and becomes disordered. Partial reorganization may occur, on cooling, as when a gelatine sol sets to a gel. c. Cross-links between polypeptide chains in the tropocollagen molecule. Collagen is more stable to complete disorganization on heating than would be expected if the polypeptide chains were held together by hydrogen bonds alone. Recent chemical evidence suggests that more-stable covalent bonds are also present which link together the individual chains in native collagen. These are not of the well-known disulphide type (involving cystine residues) which exists in many proteins but are of an unusual kind. The various structures which have been proposed include: (i) an " a m i d e" cross-link, - ( C H 2 ) 4 N H · C O ( C H2) w— , in which the e-amino group of lysine in one chain is joined to a carbonyl group formed from the side chain carboxyl of glutamic or aspartic acid in the other (Ames, 1952); (ii ) an ester link, - C H R O C O ( C H2 ) n - , between serine or threonine in one chain and glutamic or aspartic acid in the other (Gustavson , 1952) ;

19.

CHEMICAL

ORGANIZATION

OF

(iii ) a similar linkage to (ii ) but with a single hexose group interposed between the hydroxy and acidic amino acid units (von Hippel et al, 1960);

(iv) hydroxyproline linked via a phosphoric acid unit and a hexose group to serine in dentine collagen (Veis and Schlueter, 1963). d. a l and a l polypeptide chains. Piez et al. (1963) have separate d the single polypeptide chains from tropocollagen, which had been denatured by heat, by means of chromatograph y on carboxymethyl cellulose. They found that there were two kinds, designate d al and á2 , which have small but definite differences in amino acid composition, although both conform to the general collagen pattern. The triple helix (Fig. 4) consists of two al chains and one a2 chain bound by hydrogen bonds. Subsequen t formation of covalent cross-links results in stabilized two-chain systems, β12 or ( al + a2) and the less abundant βη or ( al + a l ), and eventually a full y cross-linked three-chain system. 4. The Collagen Fibril The individual tropocollagen molecules described in section II , A, 3, p. 289, are soluble in salt solutions and behave as stiff, rod-like structures, the length of which (3300 Â) greatly exceeds the diameter (13.5 A) (Doty and Nishihara, 1958). The nextlargest manifestation of collagen structure is the fibril (Fig. 3) which is insoluble, unless broken down, and is of much greater size. Collagen fibrils are of indefinite length and have a width of the order of 1 0 0 0 - 2 0 00 A. The fibrils are made up of a large number of tropocollagen molecules laid down parallel to one another lik e regularly overlapping matchsticks. The organization of tropocollagen molecules into fibrils takes place extracellularly and perhaps involves mainly electrostatic forces between charged groups in the protein side chains, since the pattern of organization in vitro depends mainly on the type and concentration of electrolyte present (Schmitt, 1956). The thermal stability of native collagen

THE ORGANIC

MATRIX

OF D E N T I N E

291

suggests that some cross-links are subsequentl y formed between polypeptide chains in adjacent tropocollagen rods within the fibril. Thus as the collagen grows older it gradually becomes more stable and less soluble, being converted to a giant molecule all parts of which are covalently linked together. The most striking feature of fibril structure is a characteristic periodic striation at intervals of 640 Â. This is revealed by the electron microscope together with finer details within the individual bands. The 640 A spacing also appears in the X-ray diffraction diagram at a very small angle, close to the undiffracted beam (Bear, 1952). Most electron micrographs fail to show fine structure in a direction parallel to the length of the fibrils. Tromans et al. (1963) have used a negative staining technique with potassium phosphotungstat e to reveal minute details of structure in collagen fibrils from tendon. This clearly showed some forty to fifty filaments across the width of the fibril, each running parallel to the fibril axis and discernible in the various subdivisions of the 640 A spacing (Fig. 5). The width of these filaments, 15-20 A, suggests that they represen t the individual macromolecules already described in section II , A,3,p. 289, and provides a striking illustration of the structural relationship between, and the relative sizes of, macromolecuie s and fibrils. The various subdivisions of the periodic structure along a fibril probably correspond to the predominanc e of certain types of amino acid side chains at intervals along the collagen macromolecule (Bear, 1952). (See also Addendum, page 310). 5. Collagen Fibres and Their

Arrangement

The diameter of individual collagen fibrils (0.1—0.2 /x) is somewhat below the limi t of resolution of the optical microscope with visible light. The smallest fibres seen are bundles containing a number of approximately parallel fibrils. The fibres themselves are disposed in larger bundles which are arranged differently in the various tissues, according to the mechanica l function which they perform. The fibre pattern is characteristic for each tissue,

292

J.

E.

EASTOE

Fig. 5. Electron micrograph of negatively stained collagen from tendon. The fine structure within the 640 A spacing is clearly shown. L, longitudinal filamentous units possibly correspondin g to the individual macromolecule s of collagen. (Electron micrograph by courtesy of Dr. A. J. Bailey.)

e.g. parallel to the length of a tendon, which has great strength in tension. The linear nature of a tendon several inches long may thus be traced directly to its component polypeptide chains. In dentine, the beautiful pattern of collagen fibres enables the tissue to provide a tough resilient backing for the enamel, and also damps shocks of impact before they reach the pulp. 6. Soluble Collagen and Biosynthesis of Collagen When collagenous tissues are extracted, first with a neutral or slightly alkaline salt (e.g. phosphate buffer or sodium chloride) solution, followed by weak acid (e.g. dilute acetic acid or citrate buffer of pH 3-4), a limited amount of collagen-like material dissolves at each stage and can be precipitated in fibrous form by dialyzing away the salt or acid (Harkness et al, 1954). Most of the collagen remains undissolved, the amount of soluble collagen being greatest in young growing tissues, and is probably unlikely to exceed the values obtained by Bowes, Elliott and Moss (1955), who found that 5.5 and 4.9 % of the total protein of calf skin dissolved in slightly alkaline phosphate and acid citrate buffer, respectively. The former figure, in

particular, may be much too high owing to the solution of albumins and globulins. Orekhovitch and Shpikiter (1957) consider that the citrate-soluble collagen, which they term procollagen, is a metabolic precursor of collagen. However, both Harkness et al. (1954) and Jackson (1957) have shown that it is the neutral-saltsoluble fraction (this extraction was omitted by Orekhovitch) which shows the most rapid uptake of isotopically labelled amino acids. The activity of this fraction rapidly diminishes in vivo ; the labelled isotope passes into the citrate-soluble fraction, which is probably not fully cross-linked, and finally into the stable insoluble collagen. Citratesoluble collagen probably represent s a stage at which no cross-links have yet formed between the protofibrils. The amino acids which are peculiar to collagen, hydroxyproline and hydroxylysine, are incorporated into the molecule as proline (Stetten and Schoenheimer , 1944) and lysine (Piez and Likins, 1957), respectively. Hydroxylation of these latter amino acids appears to take place as part of the mechanism of collagen biosynthesis . Hydroxyproline and hydroxylysine cannot be incorporated

19. C H E M I C A L

ORGANIZATION

OF

directly. Green and Lowther (1959) demonstrate d collagen biosynthesis in vitro, by slices of tissue from experimentally produced granulomata, which synthesize collagen rapidly in vivo. Later they showed a particularly rapid uptake of radioactive proline by material contained in a neutral-salt extract of the microsomal fraction. This fraction contained no fibrils, but the extract consisted of a protein, the composition of which showed the main features of collagen, but with some 20 % of a protein contaminant present (Eastoe, 1961) (Table 3b). It appears therefore, that collagen biosynthesis takes place in the microsomes of the tissue-forming cells. Nonfibrillar collagen contained in dilated cisternae of the endoplasmic reticulum of cells grown in tissue culture are shown in Fig. 6. These cisternae subsequentl y discharge their contents outside the cell, where fibril formation later occurs. Study of protein synthesis in hard tissues has been hampered by the difficult y of extracting undegrade d soluble collagens (Glimcher, 1959). Recently Araya et al. (1961) have reported the separation of a soluble collagen from bone. 7. Reconstituted

Collagens

When solutions of acid-soluble collagen are dialyzed or treated with sodium chloride, the collagen is converted once more to the form of fibrils and is known as reconstituted collagen (Schmitt, 1956). The manner in which aggregation of the protofibrils occurs within the reconstituted fibril, depends upon the conditions used, and affects the spacing of the striations of these fibrils. I n addition to the native collagen spacing (640 A), it is possible to obtain fibrils with 210 A or 2600 A spacings or with no striations, depending largely on the salt concentration . 8. Special Structural Characteristics Dentine Collagen

of

Until very recently littl e work has been carried out concerning the chemical organization of dentine collagen compared with collagen systems in other tissues. The overall amino acid composition of

THE

O R G A N IC

MATRI X

OF

DENTINE

293

dentine collagen is very similar to that of other collagens, with the possible exception of the hydroxylysine content (see page 285). However, Yeis and Schlueter (1963, 1964) have shown that many of the properties of dentine collagen are quite different from those of collagens from unmineralized tissues and they have interpreted these differences in terms of an additional level of stabilization in the structure of dentine collagen. This concept rests upon a somewhat technical experimental basis but since it is highly relevant to the present chapter, this is considered in some detail, below. Microscopic and electron microscope observations have shown that the fibrils of soft tissue collagens are highly orientated in directions parallel to one another, so as to form bundles at increasing orders of size. I n bone and dentine, however, fibrils are arranged more haphazardl y so as to form more tightly knit structures, in which the individual fibrous elements can be discerned only at high magnification. I n addition, several workers have found that these hard-tissue collagens yield very littl e or none of the soluble fractions, which are readily extracted from soft-tissue collagens by neutral salt or acidic buffer solutions at low temperatures . The intractable nature of collagens from hard tissues suggests that they are unusually stable, probably as the result of enhance d crosslinkin g of the monomer units by covalent bonds to form an extensive three-dimensiona l network. No direct comparisons of dentine and bone collagens appear to have been made but such evidence as there is suggests that dentine collagen is the more highly cross-linked. Veis and Schlueter (1964) compared the collagen from the dentine of unerupted bovine teeth, which had been extracted with 1 5% sodium chloride solution and demineralized with 0.5 M E D TA at pH 7.4, with collagen from the corium layer of bovine skin. Although the calcium content of the dentine collagen was reduced to zero by the demineralization treatment, a small but definite quantity of phosphate (see page 307) remained firmly bound to the collagen. Hexosamine s were

294

J. E. EASTOE

Fig. 6. Electron micrograph showing collagen formation in vitro by fibroblasts from mouse heart tissue. ER, dilated cisternae of endoplasmic reticulum-containing collagen precursor; V, vacuole; C, extracellular collagen fibrils, x 32,000. (Electron micrograph by courtesy of Dr. Susan Heyner.)

19. C H E M I C A L

ORGANIZATION

OF

absent from the dentine collagen, showing that it was not contaminate d by mucopolysaccharide s and the hexose content (2.2 units per 1000 amino acid residues) was almost identical with skin collagen. The two types of collagen showed enormous differences in physical properties—swellin g and solubilization—and these were used to asses s the degree of cross-linking. Whereas corium collagen exhibited maximum swelling, amounting to several hundred per cent, in the range pH 2-2.5, no swelling whatsoeve r could be discerned in the collagen of dentine matrix from pH 0.5 to 5.0. Prolonged standing and addition of urea did not result in swelling of the dentine collagen. No soluble fraction could be extracted from the dentine material by means of neutral salt solution, acid buffers or 8 M urea solution, for periods up to 2 weeks at room temperature . Under conditions designed for extraction of gelatine (60°C and pH 6.5), corium collagen gave a much higher yield (8-10 % in 1 hour) than dentine collagen (none in 7 hours and 2 % in 24 hours). A t 80°C, where hydrolysis of peptide bonds occurs, corium collagen was much more rapidly and completely solubilized than dentine, which required an induction period of several hours before beginning to dissolve. The induction period for dentine collagen was reduced and the rate of solution increased by pretreatmen t at 60°C. The lack of swelling and low degree of thermal solubilization of the collagenous matrix of dentine are consistent with a highly cross-linked network having large numbers of restricting bonds. The induction period at 80°C in particular suggeste d that long chains are joined together by substantia l numbers of comparatively short cross-links. The degree of cross-linking in dentine was demonstrabl y much greater than in the soft tissue collagen. The failure of both E D TA and neutral salt solutions to dissolve any fraction of the dentine collagen represent s another sharp distinction from unmineralized tissues. It suggests that if a neutralsalt-soluble collagen participates in dentine matrix formation, as is believed to occur in soft tissues, then dentine must posses s a very efficient cross-

THE

O R G A N IC

MATRI X

OF

DENTINE

295

linkin g mechanism which rapidly incorporates the collagen macromolecule s into a stable insoluble network. Schlueter and Veis (1964) also found that, followin g pretreatmen t with periodate, both corium and dentine collagen had the same induction period and then dissolved at the same rate, at 30°C. This was interpreted in terms of there being a common set of cross-links in the two types of collagen. This type of cross-linkage was considered to exist in regions which are susceptible to periodate oxidation. Since solubilization by periodate is accompanied by destruction of tyrosine, which has been postulated as occurring in the so-called end-chain or telo-peptide regions of the collagen macromolecule , those cross-linkage s which are common to both types of collagen may also be located in these regions. The additional set of cross-linkages , found only i n dentine collagen, probably remains intact after periodate oxidation. Both phosphate groups and hexose residues may be involved in these. It is probable that the pyranose ring of the hexoses is not oxidized by periodate and that oxidation occurs only at the C-6 position, resulting in the formation of a hexuronic acid unit. The cross-link therefore remains intact, although its nature is slightly altered. There is thus a substantia l body of evidence to show that the collagenous matrix of dentine differs markedly from that of unmineralized tissues. This evidence strongly suggests that, in addition to the types of cross-linkage s found in soft-tissue collagens, dentine collagen possesse s a set of stabilizing linkages, which incorporate both hexose residues and phosphate groups. B. O T H ER PROTEINS

Littl e work has been carried out on the proteins of dentine other than collagen. Stack (1951) found that some 5 % of the total protein of dentine was water soluble, and concluded, from the results of paper chromatography , that this was gelatine. Some of the water-soluble fraction may have arisen from

296

J.

Å.

collagen degradation during preparation of the sample. He also found that 2.5 % of the total protein of dentine fails to dissolve after repeated autoclaving. The residue contained 1 2% of N, 5 % tyrosine and only 0.3-0.6 % of hydroxyproline (Stack, 1955). The amino acid composition of this protein, as indicated by paper chromatography , is quite different from collagen, reticulin and elastin, but is similar to that of correspondin g residues from bone and skin. It appears to come from Neumann's sheaths (Tomes, 1896). Quigley and Zwarych (1963) found that when dentine is treated with a 0.1 % solution of a collagenase preparation from Clostridium histolyticum, which dissolves collagen specifically, only the odontoblasts and their processe s remain. C. CHONDROITIN

SULPHATE

Chondroitin sulphate is a common minor constituent of mesoderma l tissues and occurs in larger amounts in cartilage. It is often found together wit h hyaluronic acid (Kanamori and Yanamoto, 1959) and forms apparently structureles s gels in which extracellular protein fibres are subsequentl y laid down. The amount usually diminishes with age as the tissue becomes more fibrous. Chondroitin sulphate is a polymer of high molecular weight made up of alternating galactosamin e and glucuronic acid units (Fig. 7). The amino groups are acetylated, and the presence of a hydrogen sulphate group on each hexosamine unit renders the polymer strongly acidic, which assists histochemica l identification (Chapter 12). Chondroitin sulphate was first reported in dentine by Pincus (1948), who subsequentl y claimed to have isolated 2 . 6 4% (Pincus, 1950). Rogers (1949) reported the presence of only 0.1 % of hexosamine in dentine hydrolyzates analyzed colorimetrically. This correspond s to approximately 0.2 % of chondroitin sulphate, a value which was confirmed by Stack (1951). Hess and Lee (1952) extracted 0.64 % of chondroitin sulphate from normal human dentine with potassium chloridepotassium carbonate solution, followed by a

E A S T OE

OH

N H C O C H3 (a)

HO OH I I Η H O C H 2- C - C - C Η Η /

—Ο

NHCOCH, I CH \ Η

3

COO"

Fig. 7. Structures of (a) chondroitin sulphate A and c acid (sialic acid) group. (b) a terminal N-acetylneuramini d I n chondroitin sulphate C, the sulphate group is interchange wit h the hydroxy group marked with an asterisk.

purification procedure. They compared the composition of this material with chondroitin sulphate isolated from bovine tracheal cartilage by the same method, and with material isolated from dentine by the method of Pincus (1950). The results (Table 4) suggest that potassium carbonate extraction yields a reasonabl y pure product, whereas the material obtained by Pincus was much more heavily contaminate d by protein. Hess and Lee showed, by means of paper chromatography , that the hexosamine present was galactosamin e and that glucosamine was absent. They obtained a lower value (0.3 %) for chondroitin sulphate calculated from the hexosamine and hexuronic acid content of whole dentine and attributed this to destruction of these substance s during hydrolysis. D . MUCOPROTEINS AND SIALOPROTEINS

Although chondroitin sulphate is the only mucosubstanc e to have been characterize d chemically in dentine, the presence in hydrolyzates of small quantities of neutral sugars, apparently in

19. C H E M I C A L

ORGANIZATION

OF

THE

O R G A N IC

MATRI X

OF

DENTINE

297

Table 4 COMPOSITION OF CHONDROITIN SULPHATE ISOLATED FROM DENTINE AND TRACHEAL CARTILAGE 0

Sample Dentine (human)

Value

Hexosamine

%

Molesc

Trachea (bovine) Dentine Theoretical

/o /o

0/

/o

0

Moles a b c

Hexuronic acid

Í

25.08 0.96 29.20 14.0 31.96 1

2.63 1.21 2.13 5Ab 2.31 1

27.78 1 28.07 15.3 24.49 1

Yield

Method of extraction

0.64

)



\

0.75 —



K C 1 / K 2C 03 solution CaCl2 solution

— —

Hess and Lee (1952). Pincus (1950). Molar proportion relative to hexosamine .

excess of what would be required to form occasiona l cross-links in collagen, suggests that small amounts of other carbohydrate-containin g complexes may be present. The recent finding by Castellani et al. (1960) of sialic acid in dentine matrix, in concentrations similar to those in bone (less than 0.06 %) suggests the presence of a sialoprotein. The general and localized staining of dentine by various histochemical methods (Martens, Bradford and Frank, 1959), especially the periodic acid-Schiff method, which stains carbohydrate-protei n complexes strongly (Leblond, Glegg and Eidinger, 1957), particularly those which contain sialic acid (Herring, 1964b), supports the scanty evidence of chemical analysis. Veis and Schlueter (1964) have reported that a complex of carbohydrate with noncollagenous protein can be extracted from immature dentine by E D T A. This may be similar to the mixture of sialo- and mucoproteins similarly isolated from bone by Herring and Kent (1964) as described below. I n many respects , dentine is similar to bone in chemical composition and, in the absence of other evidence, the mucoproteins and allied substance s found in bone support speculation for the existence of similar high molecular weight substance s in dentine. Considerable advances have been made in the knowledge of bone mucosubstance s during the last 15 years, which have been summarized by Eastoe (1956) and Herring (1964b). I n addition to

chondroitin sulphate, three main types of proteincarbohydrate complexes (mucoproteins or glycoproteins) have been found in small quantities in bone. 1. Alkali-soluble bone mucoprotein. This mucoprotein dissolves, together with chondroitin sulphate when bone is extracted with weak alkali. A complex of the two substance s (osseomucoid ) can be precipitated by addition of a slight excess of acid. The mucoprotein contains galactose , mannose, glucosamine and xylose or possibly fucose, and the protein part of the molecule is rich in the longer aliphatic side chains (leucine, valine, isoleucine) and poor in glycine when compared with collagen (Eastoe and Eastoe, 1954). 2. Alkali-insoluble bone mucoproteins . Some carbohydrate-containin g materials remain firmly attached to the largely collagenous residue after treatment with dilute alkali, and it is difficul t to decide whether they are essentially components of the collagen macromolecula r system or distinct mucoproteins. Dische, Danilczenko and Zelmenis (1958) considered that there are two types of heteropolysaccharid e of this kind in bone which contain respectively, (a) galactose , mannose, hexosamine and fucose, and (b) galactose and glucose. 3. Bone sialoprotein. A sialic acid-containing glycoprotein has recently been isolated from bone (Herring and Kent, 1964) by extraction with E D TA followed by separation from two other soluble

298

J.

E.

mucoproteins and chondroitin sulphate. The sialoprotein from bone is homogeneou s in the electrophoresi s apparatus and ultracentrifuge and, unlike other glycoproteins, it contains both glucosamine and galactosamine . It also contains N-acetylneuraminic acid (Fig. 7), galactose , glucose, mannose and fucose. It differs significantly from serum orosomucoid in composition and may be deposited during bone formation rather than incorporated from the plasma. Being strongly acidic, it has cation-binding properties and it stains very strongly in the periodic acid-Schiff reaction.

E. LIPID S

Dentine has a low but definite content of lipi d substances . The variability in values for total lipi d reported by different investigators suggests that there is probably some difficult y in extracting them completely. Differences in the method of preparing the dentine and in the solvent used for extraction may also influence the value obtained. Stack (1951) quoted a value of 0.2% fat in h u m an dentine, and Hess, Lee and Peckham (1956) found that 0.36 % of lipids were extracted by means of ethanol followed by anhydrous diethyl ether. Ikels and Dirksen (1962) obtained a total lipi d value of only 0.044 % when a mixture of chloroform and methanol was used for extraction. A variety of types of lipi d are present in dentine, some in very small amounts. Leopold, Hess and Carter (1951) investigated the occurrence of cholesterol and found that none was extractable wit h petroleum ether, whereas 95 % ethanol, followed by diethyl ether, removed a total of 0.014 % cholesterol, approximately half of which was in the form of cholesteryl esters. Greater yields, up to 0.024 % of total cholesterol, were obtainable after partially hydrolyzing the protein from dentine wit h hydrochloric acid or potassium carbonate . Hess et al. (1956) found that 0.014 % of phospholipids are present in human dentine, whereas Ikels and Dirksen (1962) reported 0.00007% of lipi d phosphorus . Dirksen (1963) identified

E A S T OE

sphingomyelin, lecithin, phosphatidylethanolamin e and lysocephalin, among the phosphatide s present in sound dentine, by means of paper chromatogra phy. Phosphatidylserin e was extracted from carious dentine, but it is not clear whether this is of microbiological origin or whether it is released from combination with other material by the carious process. A m o ng the nonphosphatidi c lipids extracted from normal dentine by chloroform-methanol , Ikels and Dirksen found cholesterol (0.0031 %) cholesteryl esters (0.0014 %), diglycerides (0.0007 %) and triglycerides (0.0011 % ). It would appear that the greater part of the lipi d fraction of dentine is not yet identified. Irving (1963) considers that a lipi d material, revealed by a specific staining technique, is present at sites of calcification in all mineralized tissues (see page 308).

F. CITRATE AN D LACTAT E

Among the minor organic constituents of dentine, citrate is present in greatest amount, the level in normal human dentine being not far short of 1 %. Results of different investigators—Fre e (1943), 0 . 8 %; Zipki n and Piez (1950), 0.89 ± 0 . 1 0 %; Stack (1951), 0.86-0.89%; Hartles and Leaver (1960), 0.87 %—are in good agreement . The function of citrate in dentine, and also in the skeleton generally, is not yet clear. A small, polyvalent organic ion, known to form complexes of low but definite solubility with calcium, might be expected to be associate d almost exclusively with the inorganic crystallites of mineralized tissues, and undoubtedly its presence must influence their deposition and solubility characteristic s (McGann, 1960). Citrate has long been recognized as playing a central role in the tricarboxylic acid metabolic cycle, and consequentl y is present continuously in the body fluids. W. D. Armstrong and Singer (1956) considered that deposition in the skeleton, in association with calcium, results automatically from the stability of crystals containing calcium, phosphate and citrate in contact with solutions of

19. C H E M I C A L

ORGANIZATION

OF

these ions, and that the presence of citrate is, therefore, adventitious. Hartles and Leaver (1960) showed that citrate from dentine was much less soluble in water than would be expected from the known solubility of calcium citrate. Removal of protein by treatment wit h ethylenediamin e did not dissolve the citrate, but greatly increased its solubility in water. This citrate was subsequentl y found to be associate d wit h a substanc e having a peptide nature. The peptide dissolved, together with the whole of the citrate, when dentine was completely demineralized wit h 2 JV-hydrochloric acid. The peptide, which was contaminated with nearly 30 % of degraded collagen, was found to be very rich in arginine wit h moderate proportions of valine, aspartic acid, leucine and two unidentified substance s (Leaver, Eastoe and Hartles, 1960) (see Table 5). The tyrosine content was much higher than that of collagen, this amino acid being released particularly slowly on hydrolysis. Further investigations with bone showed that peptide material was also associate d with citrate in this tissue, but quantitative variations in composition occur, perhaps reflecting differences in the conditions used for isolation. It is therefore possible that while the bonding between citrate and the peptide material is strong, it may be electrostatic in nature and perhaps arises fortuitously during the isolation process. If this is so, there may be no such special relationship between citrate and any particular peptide fraction in the intact bone or dentine. A thorough investigation of the effect of extreme variations in the calcium, phosphorus and vitamin contents of diet on deposition of citrate in bone has recently been conducted by Hartles and his coworkers, as a result of which it has been establishe d that some citrate is synthesize d in bone itself (Hartles, Leaver and Triffitt , 1963). The association of citrate with both inorganic and organic constituents suggests that it may play an important part in the mineralization process. Leaver, Triffit t and Hartles (1963) have shown that human dentine contains 0 . 1 5% of another hydroxy acid, lactic acid, the ratio of citrate to

THE

O R G A N IC

MATRI X

OF

DENTINE

299

Table 5 AMIN O ACI D COMPOSITION OF A CITRATE-PEPTIDE COMPLEX FROM

Amino acid (Ammonia) Arginine Glycine Aspartic acid Valine Glutamic acid Unknown 11 Leucine Alanine Unknown II I Phenylalanine Isoleucine Serine Tyrosine Threonine Lysine Methionine Hydroxylysine Histidine Unknown I

HUMA N

D E N T I N E 06

Amino acids in hydrolyzate of entire complex

"Non-collagen" amino acids of complexb c,

161 97 53.7 36.6 32.7 23.2 19.4 19.4 19.2 16.3 12.3 10.75 9.24 7.50 6.04 2.60 1.89 1.61 1.49 1.35

153.2 88.0 d

26.1 27.8 9.2 19.4 14.41 d

16.3 9.63 8.75 2.01 7.07 2.36 d

0.90 0.00 0.48 1.35

a

From Leaver et al. (1960). Values are expresse d as number of moles of amino acid per 1000 gram atoms of total N. c Corrected for contamination by collagen on the basis of hydroxylysine content. d The small negative values obtained are meaningles s in t that the correction is valid, within themselves , but sugges experimenta l error, assuming that these amino acids are absent except in the collagen. b

lactate being 5.93 : 1. Rat incisor dentine, however, contains less citrate (0.046 %) than lactate (0.070 % ), a ratio of 0.66 : 1. G.

FLUORESCENT SUBSTANCES

Hartles and Leaver (1953) showed that a number of fluorescent substance s are present in very small amounts in the solution obtained by extracting human dentine with alkaline ethylene glycol. They can be partially separate d from one another by

300

J.

E.

adsorption on charcoal, solvent extraction and chromatograph y on alumina. The fluorescence is associate d mainly with organic compounds present i n low concentration , but not with amino acids. Some of the fluorescence persists after heating with concentrate d sulphuric acid, suggesting that very stable substances , such as pyrimidines, may be responsible. This view is supported by the ultraviolet absorption spectra, which also indicate that the 3-positions are substituted. Thymine (2,6-dioxy-5-methylpyrimidine ) was subsequentl y identified by means of its ultraviolet and infrared absorption spectra in alkaline extracts of cementum from the sperm whale (Hartles and Leaver, 1955). Smaller amounts of thymine are probably present in the dentine, together with an ether-soluble, basic pyrimidine (possibly 5-methylcytosine) which forms an insoluble complex with phosphotungsti c acid. These and other unidentified pyrimidines are probably produced by alkaline degradation of deoxyribonucleic acid ( D N A ) present in the dentine tubules, which fluoresce very strongly in intact dentine.

III .

CHEMICAL

BALANC E

S H E ET

F OR

DENTINE

Attempts to account for as much as possible of the weight of human dentine, as known constituents, have been made by Stack (1951, 1955) and by Burnett and Zenewitz (1958b). The results of these studies, when allowance has been made for slight differences in the method of calculation employed, show very good agreement . Represent ative values, some of which have been rounded off, and ranges of results from various investigators are summarized in Table 6. The dense inorganic phase (specific gravity approximately 3.0) makes up 75 % of the weight of dentine, and the organic material (specific gravity 1.62) some 2 0 %. The volumes occupied by the two phases are approximately 62 and 38 %, respectively. A s far as possible, the values in Table 6 have been selected as having been carried out by methods

E A S T OE Table 6 CHEMICAL BALANC E SHEET FOR THE ORGANIC CONSTITUENTS OF NORMAL HUMA N DENTINE"

Inorganic matter (ash + C 02) (residue from KOH-glycol extraction 77.8) Ash (71.5-72.4) Carbon dioxide Organic matter Collagen, total (17.5-18.5) (water-soluble, 0.5-0.9) Citrate (0.86-0.89) Lactate Resistant protein Lipi d (0.044-0.36) Chondroitin sulphate (0.2-0.6) Other mucosubstance s Unaccounte d for (water retained at 100°C, errors, etc.)

75 72.0 3.0 20 18.0 0.89 0.15 0.2 0.2 0.4 ? 5 100

a Values are given as percentag e by weight of dentine which has been dried at 100°C.

which give a direct measure of each constituent, exclusive of any water which may be associate d wit h it in dentine. Thus, the inorganic value is based on the ash content after heating to 900°C, and the collagen content on total nitrogen values. Despite all values being expresse d in terms of dentine, which has been dried at 100°C, there is a deficiency of some 5 % of the weight of the tissue, left unaccounted for. This was first noted by Tomes (1896), who pointed out that it was probably mainly attributable to firml y bound water which is retained on drying at 100°C, but is lost on heating the inorganic constituents to the temperature of the ash determination. Stack (1951) considered that such water is firml y bound to the collagen, but this would appear unlikely as, when collagen from tendon or demineralized bone is thoroughly dried at 105°C, 98-99 % of its weight can be accounted for as amino acids (Eastoe, 1955). The water would thus appear to be associate d with the inorganic crystallites or else to be in intimate association with the bonds between collagen and the inorganic component.

19.

CHEMICAL

ORGANIZATION

OF

The discrepanc y of 5 % includes not only firml y bound water, but also volatile inorganic matter, unidentified organic components and errors. Al l these, however, are probably small compared with the water content. Stack (1951) made a very careful study of the organic content of dentine in terms of its oxidation by dichromate in hot, acid solution. He reached the conclusion that, as the total capacity of dentine for oxidation by dichromate agreed with the sum of the separate capacities of its known organic constituents , any unidentified organic constituents must be present in very small amounts only. This type of measuremen t helps to overcome the difficulties associate d with the indefinite water content. For proteins, this object can also be achieved by basing results on the total nitrogen content of dentine or any separate d fraction. The total Í content of collagen is 18.5 ± 0.1 % (Stack, 1951), corresponding to a nitrogen-to-protein conversion factor of 5.41. Stack (1951) and Burnett and Zenewitz (1958b) found average total nitrogen contents for whole dentine of 3.38 and 3.43 %, respectively, which correspond to total protein contents of 18.25 and 18.5 %. The value of 21.4 % protein calculated by Burnett and Zenewitz is erroneously high, due to application of a conversion factor of 6.25, which is widely used for proteins but is inappropriate for collagen (Eastoe and Courts, 1963). Stack found that the ratio of total Í to capacity for oxidation was lower for dentine than for a model mixture simulating its known composition. This suggests that any unidentified organic components have lower nitrogen contents than the mean for dentine organic matter. The very slightly higher values obtained by Burnett and Zenewitz for most constituents are probably due to a slightly lower residual moisture content, resulting from their knowledge of the lengthy period required to dry dentine to constant weight at 100°C (Burnett and Zenewitz, 1958a). They found values for the moisture content of dentine from freshly extracted teeth to be 10.0 %, when dried at 100°C in vacuo and 12.2 % at 197°C in vacuo. Thus approximately 2 % of the weight of

21

THE

O R G A N IC

MATRI X

OF

DENTINE

301

dentine is accounted for by water lost between 100° and 200°C. The discrepanc y of weight previously referred to is also narrowed by considering the value of 77.8 % (Table 6) for the largely inorganic residue remaining after extraction with potassium hydroxide and ethylene glycol, instead of the sum of the ash and carbon dioxide contents, 75 %. A similar deficiency in weight of material recovered is found for bone (Eastoe, 1956) but is minimized by considering the inorganic residue after autoclaving instead of the ash value.

IV . C H A N G ES I N T H E O R G A N I C C O M P O N E N TS O F D E N T I N E A . BIOLOGICAL VARIATIO N

Dentine, at least in m a n, is more constant in composition than bone. This is partly due to its greater uniformity of structure; neighbouring Haversian systems in bone vary considerably in age and degree of mineralization. Stack (1951) has shown that variations in both the nitrogen and organic matter content (measure d by dichromate oxidation) of dentine between any individual tooth and its opposite number on the other side of the arch, are only a littl e smaller than those between any of the teeth in the dentition and the general mean. The biological variation is only 2-4 %, so that most samples of h u m an dentine wil l contain 19-21 % of total organic matter. The constancy of composition of different types of tooth is illustrated by the mean values in Table 7. B . CHANGES W I T H A G E

Dentine from individual premolar teeth of children averaging 13 years old, had the same total nitrogen content (3.38 ± 0.18 % ), as that from other types of teeth from adults of average age 33 years (Stack, 1951). N o significant correlation was found between the total Í content of dentine and age between 10 and 65 years. Thus, unlike bone, which decrease s in organic content with age

302

J.

Å.

Table 7 COMPOSITION OF POOLED HUMA N DENTINE FROM TEETH OF F O UR M A I N

Paramete r Number of teeth Ash(%) T o t a l N ( %) N/organic6 Citri c acid (%)

Incisor 80 71.5 3.50 138 0.86

TYPES0

Canine 50 72.4 3.38 140 0.86

Premolar 80 72.2 3.45 140 0.89

° F r om Stack (1951). 6 Expresse d as grams of nitrogen per litr e of solution ( X 100).

Molar 20 72.2 3.45 136 0.87

N - N a2S 20 3

(Rogers, Weidmann and Parkinson, 1952), dentine, once full y mineralized, remains almost constant in its organic content. Dentine is generally regarded as having a lower content of organic matter than bone. Certainly human dentine, with some 19 % of nitrogenous organic matter, is more highly mineralized than compact bone from the h u m an femur, which has 26-27 % organic matter (see Eastoe, 1956). However, compact bone from ox femora has a similar organic content (20-21 %) to human dentine, while that of the rabbit (15.8 %) is significantly lower. Thus, whilst dentine must be regarded as highly mineralized, it may not have reached the maximum level possible for a tissue with a collagenous matrix. C.

CHANGES ACCOMPANYING D E N T AL

CARIES

W. G. Armstrong (1964) has recently carried out a series of investigations into the changes in composition and properties of the organic matrix of dentine, which has been affected by dental caries. A s a result of this work, he has been able to clarify their probable mechanism . The main differences observed in carious dentine, when compared with normal dentine, are as follows: (1) quantitative changes in the amino acid composition of the collagen; (2) a large increase in the content of carbohydrate material; (3) formation of a brown pigment; (4) increased resistance to breakdown by collagenase ; (5) loss or diminution of fluorescence in ultraviolet light.

E A S T OE

The average amino acid composition of "leathery" carious dentine is compared with that of normal human dentine in Table 8 (W. G. Armstrong, 1964). The overall pattern of amino acids present in collagen is still apparent in carious dentine, but the amounts of some (above the line i n the table) are diminished compared with normal dentine, whereas others (below the line) are present i n increased amounts. The proline and hydroxyproline contents are reduced by 30 and 22 %, respectively, possibly as a result of the breakdown of peptide bonds in that portion of the collagen polypeptide chain which is rich in these imino acids Table 8 AMIN O A C I D COMPOSITION OF SOUND AND CARIOUS HUMA N DENTINE AND THE COLLAGENASE-RESISTANT FRACTION

OF CARIOUS DENTINE0

Sound dentine6

Carious dentine6

Collagenase resistant fraction0

Hydroxylysine Histidine Lysine Arginine Proline Hydroxyproline

0.99 1.07 3.31 7.9 13.17 11.79

0.87 0.88 3.28 5.76 9.25 9.01

1.0 1.2 3.63 3.97 11.43 9.67

Aspartic acid Threonine Serine Glutamic acid Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine

6.85 2.08 3.0 10.29 17.54 8.41 2.49 0.71 1.34 3.17 0.54 1.82

8.64 3.00 4.06 11.47 19.52 10.81 3.57 1.12 1.92 4.38 1.54 3.57

8.42 2.66 3.76 11.27 17.64 10.39 3.79 1.43 1.90 3.80 1.45 2.60

Total

96.45

102.65

Amino acid

° F r om W. G. Armstrong (1964). 6 Results calculated as grams amino acid residue per 100 gm of dentine matrix. c Results calculated as grams amino acid residue per t residue 100 gm of total protein. The collagenase-resistan contained 13.6% of carbohydrate .

19. C H E M I C A L

ORGANIZATION

OF

(see page 289). This may result from the action of a proteolytic enzyme, of the collagenas e type, produced by micro-organisms in the carious cavity. The 27 % reduction in the basic amino acid, arginine, is believed to result from modification of its side chain by reaction with substance s of carbohydrate origin. The involvement of basic (positively charged) side-chain groups is supported by the decrease d acid-binding and increased basebinding power of the protein from carious dentine. There is also a 12 % decreas e in the reactivity of e-amino groups of lysine with fluorodinitrobenzene , suggesting that these groups may be involved in less stable complexes with carbohydrate in the carious dentine, from which the lysine can be subsequentl y liberated again by acid hydrolysis. The relative quantities of many of the other amino acids are increased slightly as a direct consequenc e of the losses of the imino acids. However, doubling and trebling of the phenylalanine and tyrosine values, respectively, in carious dentine can probably be explained only in terms of the gain of material rich in these amino acids. The characteristic s of the composition of the collagenase-resistan t fraction of carious dentine (Table 8) are similar to those of entire carious dentine matrix. The content of material giving colorimetric reactions typical of carbohydrate s is increased from 0.4 % (expresse d as glucose) in sound dentine, to 4 % in carious dentine and 1 2 - 1 4% in the collagenase-resistan t fraction. Attempts to identify the carbohydrate have been unsuccessful , probably because it has reacted with the basic groups of the protein to produce the pigmented material characteristic of carious dentine. Such " b r o w n i n g" or "Maillard"-type reactions between carbohydrate s and amino acids are well known (Gottschalk, 1966) and occur readily in almost dry materials at neutral or slightly acid p H. The collagen from normal dentine, which has been demineralized with acid or E D TA (provided that C a+ + ions are present) is, lik e other collagens, completely dissolved by clostridial collagenase . Prophet and Atkinson (1953) found that the matrix of carious dentine is resistant to the action

T H E O R G A N IC

MATRI X

OF D E N T I N E

303

of collagenase . W. G. Armstrong (1958) showed that even carious matrix is initiall y hydrolyzed by the enzyme, but the rate of the reaction continuously diminishes until a completely collagenase resistant residue remains. It was shown that intact groups on the basic side chains of collagen are essentia l for collagenas e activity. If the e-amino groups of lysine or the guanidino groups of arginine are destroyed or modified in collagen from demineralized normal dentine, the protein becomes collagenase-resistant . Similarly, if normal dentine collagen is allowed to react with any of various aldehydes , which are degradation products of carbohydrates , it is converted to a resistant form. The reduction of fluorescence in carious dentine compared with sound dentine is minimized by diluting the solution. It results, not from loss of fluorescent material, but from an "inner filter" effect, whereby material absorbing strongly in the region 230-330 ταμ screens the fluorescent substances from exciting radiation. A similar effect is observed with sound dentine which has been allowed to react with 5-hydroxymethylfurfural. The changes affecting dentine collagen in caries are therefore partly proteolytic, probably involving partial hydrolysis of that portion of the chain which is rich in imino acids and deficient in polar side chains. Reaction of the more polar part of the molecule, particularly the side chains of basic amino acid units, with material of a carbohydrate nature produces a complex of characteristic brown colour, which reduces fluorescence and renders this remaining portion of the collagen resistant to further proteolysis.

V. MINERALIZATION OF THE ORGANIC MATRIX A.

INTRODUCTION

The process by which the organic matrix present i n predentine becomes impregnated with minute crystals of apatite, and the relationship between the collagen fibrils and inorganic phase in mature

304

J.

Å.

dentine, represen t some of the most fascinating aspects of the organization of this tissue, but at the same time are among the least understood. The problems presente d by biological mineralization have received much attention recently, with the result that they can now be considered in fundamental terms. Some of the work in this field has been carried out on bone, but the process of mineralization in dentine appears to be sufficiently similar to intramembranou s or the later stages of endochondra l ossification, that the basic principles may be considered to apply to both tissues. Greater differences are apparent in the processe s by which dentine and enamel become calcified. This section is primarily concerned with the part played by the organic matrix in mineralization, but i t is also necessar y to consider the nature of the phase transformations occurring in the crystallization of an inorganic substanc e with a complex crystal lattice such as hydroxyapatite (see also Chapter 16). The crucial test of any hypothesis proposed to explain mineralization lies in its ability not only to account for the mechanism of the process in tissues which normally become calcified, but also to explain why mineralization fails to occur in apparently similar situations in other tissues, except comparatively rarely in pathological conditions. B . T H E ALKALIN E PHOSPHATASE THEORY

Robison (1923) noticed that increased amounts of enzymes of the alkaline phosphatas e group were frequently present in tissues which were undergoing mineralization. These enzymes are capable of hydrolyzing the phosphate groups from organic esters, such as the hexose phosphates , with the release of inorganic phosphate ions. Robison suggeste d that alkaline phosphatas e activity might be responsible for mineralization, by locally raising the phosphate ion concentration , so that precipitation of calcium phosphate occurred. This concept has since been abandone d as the primary mechanism by which mineralization occurs, although enzymes of the phosphatas e group

E A S T OE

may, nevertheless , play some part in the process (see page 308). The main objection is the very low concentration of the organic phosphate substrate s present in blood and tissue fluids. This would be insufficient to account for the observed rapid rate of mineralization. Further, other organs, such as the kidney, have very high levels of alkaline phosphatas e but do not normally become calcified. Another difficult y concerns the nature of the transition between solutions of calcium and phosphate ions and the solid inorganic phase. C.

PHASE TRANSFORMATION BETWEEN SOLUTION AN D SOLID APATIT E

The alkaline phosphatas e theory depende d upon the idea that increase in the concentration of inorganic phosphate at calcification sites caused the ion product of calcium and phosphate in solution to exceed the solubility product of calcium phosphate, resulting in precipitation of solid. The inorganic phase of dentine approximates to hydroxyapatite (see Chapter 17), the solubility product of which is now known to vary with temperature , pH and the presence of foreign ions, both in solution and in the apatite lattice. There can be littl e doubt, however, that the product of the concentration s of free calcium and phosphate ( H P 04 ) ions, approximately 18 (mg % ) 2 [or 1.5 ( m M ) 2] , normally present in blood serum, considerably exceeds the practical solubility product of the impure form of hydroxyapatite present in calcified tissues (Solomons and Neuman, 1960), which is in the region of 10 (mg % ) 2 (Fleisch and Neuman, 1960). However, if a solution is artificially prepared which has the same concentration s of calcium and phosphate ions as serum, it can be preserved indefinitely without formation of solid crystals, despite the fact that the solubility product for apatite is exceeded . Introduction of crystals of solid apatite into such a solution results in their growth, ions for this purpose being supplied by the solution. This illustrates the important practical distinction between the nucleation of new crystals of hydroxyapatite and the growth of existing crystals.

19. C H E M I C A L

ORGANIZATION

OF

If the concentration s of calcium and phosphate are increased to an ionic product of approximately 50 (mg % ) 2 in the original homogeneou s solution, spontaneou s formation of solid takes place (Fleisch and Neuman, 1960). The first solid to be formed, however, is not hydroxyaparite, 3 C a3( P 04) 2 · C a ( O H )2, but hydrated calcium hydrogen phosphate (brushite), C a H P 04 · 2 H 2 0 , which has a solubility product of approximately 35 (mg % ) 2, decidedly higher than the ionic product for plasma. The solid calcium hydrogen phosphate soon becomes transformed into hydroxyapatite, which is the more stable form in the region of pH 7. Classical ideas on the relative difficult y of the homogeneou s nucleation process for hydroxyapatit e were based on the thermodynamic improbabilit y of a simultaneous collision of the eighteen ions present in the crystallographic unit cell and required by the empirical formula (Neuman and Neuman, 1953). Glimcher (1960) pointed out that this concept is mistaken, since molecules of hydroxyapatite do not exist as such. Instead he considered the crystallisation process as a change in the state of aggregation of ions between the solution and the solid phase, which is similar to the physical change when a liquid freezes. On this basis, the factor which governs the probability of the nucleation of crystallites in a homogeneou s solution is the amount of energy required to form ionic aggregate s of various sizes. I t is probable that for the hydroxyapatite system, there is a certain critical size of aggregate which is particularly unstable, i.e. a great deal of energy is required for its formation from C a+ + and H P 04 ions (Fig. 8). Above this size, the stability of the solid phase increases rapidly and energy is released in its formation from smaller units. Thus a solution of calcium and phosphate ions, such as serum, where the ionic product exceeds the solubility product of apatite, is in a metastable state. Homogeneou s nucleation wil l not occur because of the large energy barrier which must be overcome. This is equivalent to rolling a ball over the h u mp Β from the metastable position M to the stable position S, of lower potential energy. Addition of

THE

O R G A N IC

Å

MATRI X

OF

DENTINE

305

Β

r

Fig. 8. Diagrammatic representatio n of energy relationships between the metastable and stable states of a system containing calcium and phosphate ions at physiological concentrations . E, potential energy of system; r, size of ionic aggregate ; M , metastable equilibrium; 5, stable equilibrium; B, energy barrier; —ΔΕ loss of energy by system on passing from metastable to stable equilibrium.

solid apatite, however, is equivalent to liftin g the ball from position M and replacing it in position S. There is an overall loss of energy by the system, which is thereby rendered stable, ions passing from solution to solid phase in the process of crystal growth. D . T H E EPITACTIC C O N C E PT

Mineralization of dentine and bone does not occur simply as a reaction in homogeneou s solution, but occurs in the presence of previously formed organic matrix. Contact with solid surfaces, e.g. dust, is known to promote crystal nucleation in supersaturate d solutions (heterogeneou s nucleation). Induction by a foreign solid is more probable when the dimensions of its structural features are similar to those of the crystals being formed. A s long ago as 1921, Freudenber g and Gyorgi suggeste d that the organic matrix of calcifiable tissues might play a part in the initiation of crystal formation. Robison, Macleod and Rosenheim (1930) had a similar idea in mind when they

306

J.

Å.

proposed a "second mechanism", which operated i n addition to phosphatase , to promote the smooth deposition of the inorganic phase in calcifying tissues. More recently the idea of heterogeneou s nucleation by some portion of the organic phase to produce "seeds" of hydroxyapatite, has been developed by a number of workers, being clearly formulated by N e u m an and N e u m an (1953, 1958). This process of epitaxy (or epitaxis) in this particular context has been defined as the formation of apatite induced by a seeding agent. The precise structure of that part of the matrix responsible for epitactic nucleation has not yet been determined, but it seems probable that it must act as a kind of template. Ions from solution probably become attracted to the surface of the seeding agent by electrostatic forces which tend to retain them in relative positions similar to those in the apatite lattice. In this way, less energy is required to build up ionic aggregate s in the critical range of size, and once this size is exceede d the apatite seeds wil l grow spontaneously , since energy is released by further growth (Fig. 8). E. COLLAGEN AS AN EPITACTIC A G E NT

The evidence for some portion of the collagen macromolecula r system playing an essentia l part in epitaxy has been thoroughly summarized by Glimcher (1959, 1960). Collagen is the principal constituent of predentine; its highly organized "crystalline" structure maintains charged groups on the amino acid side chains in a precise spatial arrangemen t ready to interact with ions from solution. In mature bone and dentine, the inorganic crystallites appear to be in intimate relationship wit h the larger collagen fibrils since they li e between, on the surface of and possibly even within, the fibrils. I n the early stages of mineralization of embryonic bone, the first detectable apatite crystals have been found to form at definite positions in the fine structure of the collagen fibril, within each 640 A periodicity (FittonJackson, 1957). These sites presumably correspond

E A S T OE

to regions where particular types of reactive side chains predominate. Glimcher (1959) and his co-workers have shown that formation of apatite crystallites can be induced in vitro from metastable solutions containing calcium and phosphate , by reconstituted collagen fibrils. These were prepared from acidsoluble collagen, extracted from nonmineralized tissues (see page 292). Only the fibrils with the natural (640 A ) spacing were found to be capable of inducing crystal formation. Other reconstituted fibrils with different spacings were completely ineffective. Glimcher attributed this not to any direct property of the 640 Â spacing itself, but to the need for a definite spatial relationship between amino acid side chains on neighbouring protofibrils withi n the fibril. This view is supported by a loss of the ability to induce crystal formation, when the regular structure of the 640 A fibrils is disorganized by thermal shrinkage or treatment with acid. Although demineralized native bone was able to induce mineralization, the native soft tissue collagens (from which the reconstituted collagens were derived) were unable to do so. Preliminary treatment of these collagens with salt solutions, known to dissolve mucopolysaccharides , or with enzymes capable of depolymerizing mucopolysaccharides , enabled these native forms to initiate mineralization. On this basis, Glimcher suggeste d that whereas collagen in bone and dentine is capable of epitactic nucleation, in the tissues which do not normally mineralize it is somehow prevented from reacting with ions, by mucopolysaccharide s which are present in somewhat higher concentration s in such tissues. A criticism of these and similar experiments by other workers is that the crystal nucleation by collagens could only be demonstrate d for metastable solutions of calcium and phosphate where the ionic product substantially exceede d the values found in serum. More recently, Solomons and N e u m an (1960) have been able to obtain evidence for the remineralization of dentine (which had been recently demineralized with EDTA) by artificially prepared metastable solutions with ionic

19. C H E M I C A L

O R G A N I Z A T I O N OF

products in the same range as serum [15-25 (mg % ) 2] . This evidence, together with the extreme specificity of the state of aggregation of protofibrils, required for nucleation by collagen in vitro strongly suggests that collagen plays an essentia l part in the mineralization of dentine and bone. The reactive groups responsible for the epitactic behaviour of collagen have not yet been identified wit h certainty. Earlier workers favoured the binding of calcium ions as the primary mechanism of mineralization (see Glimcher, 1960), but recently attention has been directed to the part played by phosphate . I t is not yet clear whether the epitactic surface is formed simply by electrostatic attraction between phosphate ions from solution and the positively charged anions (e.g. e-amino, guanidino etc.) of the collagen side chains, or whether active phosphorylation of collagen by "high energy" phosphate compounds , such as adenosine triphosphate (ATP), is necessary . The former view is not inconsistent with the predominance of positively charged side chains over negative ones in collagen at pH 7, although it is their local distribution and spatial arrangemen t which wil l determine their suitability as sites for effective epitactic ion-binding. Solomons and Irving (1958) found that the e-amino groups of lysine and hydroxylysine of the collagen in ox and h u m an dentine were liberated for reaction with fluorodinitrobenzen e in proportion to the percentag e of demineralization achieved with E D T A. This experiment indicates a direct participation of e-amino groups in the formation of bonds between collagen and the inorganic phase. The evidence is, however, inconclusive since removal of apatite may simply have permitted physical penetration of fluorodinitrobenzene into the 0.25-cm cubes of dentine used. It is possible that both calcium and phosphate ions become attached to suitably disposed amino and carboxyl side chains, respectively, at the epitactic centre of collagen. Veis and Schlueter(l964),unlik e Solomons and Irving(1958), found that about 10 % of the e-amino groups of demineralized dentine did not react with fluorodinitrobenzene . Wuthier, Gron and Irving

THE

O R G A N IC

MATRI X

OF D E N T I N E

307

(1964) found that aqueous fluorodinitrobenzene caused extensive recrystallization and dismutation of the inorganic crystallites, to which the degree of dinitrophenylation was related. They considered that in full y mineralized bone, 25 % of the total e-amino groups were free, 20-25 % were internally bonded within the collagen molecule and the remaining 45-50 % were directly linked with the inorganic material. Half the latter type were electrovalently bonded, probably to the orthophosphate ions in the apatite and the remaining half more or less firmly bonded, perhaps to pyrophosphat e ions. According to the second view, collagen requires activation in the form of addition of covalently bound phosphate groups to appropriate regions of the macromolecule . This concept is based on the idea that the mineralization reaction requires a source of considerable energy which would not be required for a purely ionic epitactic mechanism . Such energy could be provided by A T P, which is known to be produced in calcifying cartilage (Whitehead and Weidmann, 1959). The possible role of A T P in mineralization has been reviewed by Weidmann (1959, 1963), who concluded that it is clearly essentia l for many of the more general metabolic reactions of mineralizing tissue and for the synthesis and sulphation of chondroitin sulphate. The rate of phosphate uptake is decrease d only when the glycolytic pathway has been blocked wit h iodoacetate . This cannot be regarded as conclusive evidence for the phosphorylation of collagen by A T P being the primary stage of epitactic nucleation. Iodoacetate would inhibit most of the synthetic processe s in the tissue, and may thus prevent the formation of other substance s (e.g. pyrophosphatase ) which are essentia l for mineralization. Solomons and N e u m an (1960) and Veis and Schlueter (1963, 1964) noted that a small amount of phosphate (equivalent to 4-6 phosphate residues per 1000 amino acid residues) remained firmly attached to dentine collagen after exhaustive demineralization with E D T A. The latter group of workers consider that the phosphate groups occur

308

J.

Å.

as cross-links, owing to the greater stability of bovine dentine collagen to thermal shrinkage and acid swelling when compared with skin collagen. The finding of such covalently bound phosphate groups, exclusively in hard tissue collagens, would form the most reliable evidence so far available for a phosphorylation mechanism in epitactic mineralization. Weidmann (1963), however, states that failure of soft-tissue collagens to calcify cannot be ascribed to absence of A T P, since this is present in practically all the tissues of the body. F . T H E R O LE OF MUCOSUBSTANCES

The assumption that mucopolysaccharides , especially chondroitin sulphate, play an active part in mineralization is based on histochemica l changes , including metachromasi a and basophilia, which frequently accompan y calcification. Increased uptake of radioactive sulphur at about this stage (Dziewiatkowski et al, 1957) suggests that molecular transformations of chondroitin sulphate occur. Sobel (1952, 1955) considered that a substanc e wit h properties lik e chondroitin sulphate participated as a "local factor" in combination with collagen, and was responsible for the orderly deposition of inorganic crystals. Undoubtedly the strongly acidic sulphate groups wil l associate with calcium ions, but this may be interpreted either as assisting mineralization by providing a temporary store of calcium in regions near calcification sites (e.g. epiphysea l cartilage) or as an inhibitor of mineralization (Glimcher, 1959) by decreasin g the effective calcium concentration at sites where collagen epitaxy might otherwise occur. The acetylated amino groups of chondroitin sulphate are uncharged and therefore are unable to associate wit h phosphate ions. Irvin g (1963) has reported a specific staining reaction at all sites of mineralization. After prolonged extraction with hot pyridine, local staining with Sudan black becomes possible. This is attributed to the unmasking of an unidentified lipi d material. The possible occurrence in dentine of mucopro-

E A S T OE

teins and a sialoprotein similar to those isolated from bone (see page 297) needs to be considered both in relation to the mineralization process and i n interpreting histochemica l staining. Changes in such staining in the region where mineralization is occurring may, however, reflect changes in tissue permeability as well as in chemical composition and reactivity. G.

T H E C O N T R OL OF M I N E R A L I Z A T I O N — PYROPHOSPHATASE S

Whil e the possessio n of a mechanism for mineralization provides vertebrates with an efficient means for building hard tissues, its presence is also a potential danger as regards the spread of calcification to inappropriate sites. A number of independent checks exist to control the process, some of which have already been described. Thus, although the solubility product of apatite is exceede d in blood, nucleation of apatite crystals does not occur spontaneously , since it requires a highly specific macromolecula r system. Nevertheless , accidental introduction of either apatite crystals or the epitactic agent would, in principle, be expected to be dangerous , as extensive crystal growth could result. Solomons and Neuman (1960) found that an ultrafiltrate of plasma would not cause remineralization of collagen from demineralized dentine, whereas an artificial solution having the same ionic product of calcium and phosphate brought about crystal nucleation (page 306). These investigators suggeste d that plasma may contain a specific inhibitor of mineralization. Fleisch and Bisaz (1962a,b) have subsequentl y identified this inhibitor as inorganic pyrophosphate , present in concentrations of 10"5 to 10~4M and of 10~5M in human urine and plasma, respectively. The enzyme, pyrophosphatas e (a member of the alkaline phosphatas e group) has been found at mineralization sites by Perkins and Walker (1958), who considered that its function, in conjunction with A T P, is to synthesize pyrophosphat e from inorganic phosphate as an essentia l step in crystal

19. C H E M I C A L

ORGANIZATION

OF

nucleation. Fleisch and Bisaz (1962a), however, suggeste d that this enzyme acts in the reverse direction and breaks down pyrophosphat e to phosphate O H - + ( H P 2 0 7 ) — -> 2 ( H P 04) — If this occurs, the inhibitory pyrophosphate , present elsewhere in the organism, would be specifically destroyed at the actual mineralization sites, so allowing the epitactic mechanism to proceed there. The influence of citrate ions in modifying the solubility of the inorganic phase is mentioned on page 298. This may also act as a control mechanism , largely independentl y of both epitaxy and inhibition by pyrophosphate . The remodelling of bone represent s another level of control through changes in cellular activity, influenced in turn by the external environment through hormone and vitamin concentrations . Dentine is relatively unaffected by such influences, but renewed tissue formation readily occurs in response to disease or injury affecting the enamel, provided that the odontoblasts remain alive.

VI . C O N C L U D I NG R E M A R KS During the period reviewed in this chapter, considerable advances have been made in our overall knowledge of the organization of the matrix of dentine at an order of size where phenomena are considered as chemical. In so far as the major component of dentine matrix is a member of the collagen family, the progress made in the field of collagen structure in tissues generally, has also contributed to knowledge of dentine. Near the beginning of this period, the improvement of microbiological and chromatographi c methods of amino acid analysis made it possible to establish the quantitative composition of typical collagens with considerable accuracy and to investigate possible variations between organs and species. Mammalian dentine collagen has a composition which is closely similar to collagens from

THE

O R G A N IC

MATRI X

OF

DENTINE

309

other tissues of the same species with the possible exception of the content of the " u n u s u a "l amino acid, hydroxylysine, and more definitely the possessio n by dentine collagen of firmly bound phosphate groups. The refinement of X-ray diffraction analysis has enabled the spatial arrangemen t of the polypeptide chains to be studied and has resulted in the development of the concept of the triple-helical structure of collagen. This in turn, helped to establish the idea of the collagen macromolecule , a very long and narrow structure, lik e a stiff rod, which could be isolated in solution and reconstituted into fibres. Fibrogenesis , in vivo, could thus be considered as involving extracellular aggregation of such macromolecule s into fibrils, in which parallel orientation and the initial stability of the aggregate s result from the action of temporary forces such as electrostatic interaction and hydrogen bonding. Hydrogen bonds are also responsible for holding together the three polypeptide chains withi n the macromolecule . These forces are insufficient for the maintenanc e of a highly stable fibrous system and there is growing evidence that in most tissues collagen undergoes " m a t u r a t i o n" by the formation of a gradually increasing number of more permanent cross-linkages both intramolecularly between neighbouring amino acid side chains on different polypeptide chains within a single helix and intermolecularly between those in adjoining helices. I t is possible that several types of cross-linkage wit h different chemical structures exist, even in soft-tissue collagens. Preliminary evidence has been obtained by Veis and Schlueter suggesting that dentine collagen contains a set of cross-linkages , perhaps associate d with hexose and ester phosphate groups, in addition to other cross-links which it possesse s in common with the collagens from unmineralized tissues. This appears to represen t a basic structural difference which probably accounts for the very high stability of dentine collagen but it awaits further investigation, especially in relation to other mineralized collagens, such as bone. The discovery, by Piez and co-workers, that

310

J.

Å.

different polypeptide chains in a helix differ somewhat in composition, represent s another fundamental advance. The separation of two types of chain and various stable multiple forms from denatured soft-tissue collagens by chromatograph y has provided additional evidence for the existence of both intra- and intermolecular covalent crosslinkages. Dentine collagen would be more difficult to study by these methods because of its high stability. Apart from the identification of chondroitin sulphate in dentine by Pincus and the recent investigations of citrate by Hartles and co-workers, the minor organic components seem to have been largely neglected by the chemist. This particularly applies to the glyco- and sialoprotein group of macromolecula r substances . Such evidence as there is suggests that dentine may contain similar types of substanc e to those present in bone, where considerable progress has recently been achieved by Herring. The possible part played by this type of component in the mineralization process also needs consideration. The relationship between the organic matrix and the inorganic crystallites in dentine, as in other mineralized tissues, is still very far from being completely understood . A synthesis of recent ideas has been attempted in this chapter, in which it is suggeste d that mineralization is a carefully poised process, subject to a number of independen t controls at different levels. However, this may be misleading as, at the present time, the writer finds it impossible to asses s the probable relative importance of the various suggeste d mechanisms . Thus the balance of the arguments given may be wrongly emphasize d or some hithertoo overlooked mechanism may even be paramount. The core of this problem is concerned with the chemistry of the interface between organic and inorganic phases at crystallite initiation, as well as under conditions more nearly at equilibrium, when the crystals have grown to their maximum size. Even if it be assume d that collagen is the main agent in crystallite initiation, it is not yet clear whether this property is possesse d by collagens

E A S T OE

from all tissues (as would be indicated by Glimcher's experiments on the mineralization of reconstituted collagens in vitro) or whether it is a specific property of hard-tissue collagens, as perhaps might be indicated by the possible occurrence of covalently bound phosphate solely in these collagens. Wit h regard to the latter hypothesis, the collagen might either be aggregate d in a form capable of initiating mineralization or, alternatively, it may need to be subsequentl y activated by another component of the system, such as adenosine triphosphate or perhaps even by a specific sialoprotein. So far, none of these more and less intelligent guesse s has been sufficiently clearly demonstrate d to warrant the exclusion of the others. Finally there is need for much more detailed chemical information concerning substance s present in the various regions of dentine, which are discernible at a histological level. In particular, there would seem to be substantia l differences in organization between the intertubular and peritubular matrices but there is littl e comparative information concerning the chemical composition of these regions. Improvements in histochemica l techniques alone may not be sufficient to solve this and similar problems, without parallel studies in microanalytical histological chemistry. In view of its applicability to a wider range of substance s and its potentially quantitative nature, conventional microanalysis in test tubes still offers a great deal towards interpreting the more aesthetic sections of the histochemist.

ADDENDUM

Recently Grant, H o me and Cox ( 1 9 6 5) proposed a new model for the tropocollagen macromolecule , the mode of aggregation of which into native collagen fibrils is capable of explaining their 6 40  ( = D) periodicity. This was based upon electron microscope examination of negatively stained fibrils in which light (i.e. electron transparent) A bands ( 0 .4 D wide) alternated with darker  bands ( 0 .6 D in width). It was noticed that the

19. C H E M I C A L

ORGANIZATION

OF

length of a reconstituted segment-long-spacin g (SLS) crystallite, in which the tropocollagen units were assumed , from its fine structure, to be exactly aligned side by side with their ends in register, spanned exactly five A bands on the native type fibril. Treatment of native collagen with a crosslinkin g agent, such as glutaraldehyde , before negative staining, increased the width and further enhanced the electron transparenc y of the A bands. Since the A bands are electron transparen t without artificial cross-linking, they were thought to correspond with those regions of the fibril where intermolecular bonding occurs. This would probably take the form of electrostatic and hydrogen bonds and would be sufficient to prevent penetration of the electron-dens e staining material between the macromolecules . The new model for a macromolecule consisted of a filament 2800  in length, divided into 9 zones, consisting of 5 bonding zones of length approximately 0.4 D (265 Â) alternating with 4 non-bonding zones 0.6 D (375 Â) in length. The macromolecule was no longer considered as completely rigid, since detailed examination of the electron micrographs revealed fine longitidinal structures, (similar to those shown by Tromans et al, 1963) (Fig. 5), which were not everywhere parallel, but tended to cross over one another in both A and  bands. This would suggest, however, only very slight flexibilit y compared wit h that shown by a single polypeptide chain. Although the precise positions of the ends of filaments were difficult to determine, no end-to-end junctions were observed; instead the filaments appeared to terminate at the edge of an A band, after passing through it. Despite the apparent symmetry of their zone structure, the macromolecules were considered to be polarized along their length, as indicated by asymmetry in the SLS pattern. Formation of a collagen fibril was thus considered to occur by side-by-side aggregation of macromolecule s with their bonding zones in register. This can be regarded as a rather random process because , apart from the need for orienta-

THE

O R G A N IC

MATRI X

OF

DENTINE

311

tion parallel to and in the same direction of polarization as the rest of the macromolecules , which can be achieved comparatively easily for a long structure of defined charge distribution, each macromolecule may overlap its neighbours by from one to five bonding zones. This mechanism necessitate s less critical requirements for the relative positioning of macromolecule s in fibrilogenesis than are needed for the "quarter staggered " arrangemen t suggeste d by Hodge and Schmitt (1960). It is also significant that the model proposed by Grant et al (1965) calls for no systematic arrangemen t of "holes" in the structure, though perhaps rather randomly distributed spaces may be present. These might be expected to be more numerous in the  bands of the fibril, which must contain only four fifths of the number of macromolecule s present in the A bands. The lower degree of intermolecular bonding in the  bands would also facilitate penetration of small molecules between the tropocollagen macromolecules. The complexity of composition, sequenc e and structural arrangemen t of collagen would seem to represent a biological extravaganc e as regards its elaborate demands on chemical synthesis (compared with say cellulose or chitin) for a material which is required in large quantities and is ultimately destined to form only inert, though admittedly varied, mechanica l structures. In the present state of knowledge, it would seem that this is justified by the need for the precise préfabrication of macromolecules , which can be easily assemble d into fibrous structures, at diverse extracellular sites, with the minimum of cellular supervision or enzymic participation. Furthermore, such structures must be capable of demolition, after reaching increasing degrees of permanenc e (Lapiere, 1966), to meet the needs of tissue growth, remodelling and repair. Ease of control of fibrogenesis in a varied extracellular environment, where other chemical systems must continue to function without undue disturbance, is perhaps the most critical of the factors to be satisfied by the architecturally complex collagen system.

312

J.

Å.

References Ames, W. H. (1952). The conversion of collagen to gelatin and their molecular structures. J. Sci. Fd. Agric. 3, 454-463. Araya, S., Saito, S., Nakaniski, S. and Kawanishi, Y. (1961). Soluble collagen in bone. Nature, Lond. 192, 758-759. Armstrong, W. D. and Singer, L. (1956). In vivo uptake and exchange of bone citrate. Ciba Fdn. Symp., Bone Struct. Metab. pp. 103-113. Armstrong, W. G. (1958). Further studies on the action of collagenas e on sound and carious human dentin. / . dent. Res. 37, 1001-1015. Armstrong, W. G. (1961). A quantitative comparison of the amino acid composition of sound dentine, carious dentine and the collagenase-resistan t fraction of carious dentine. Arch, oral Biol. 5, 115-124. Armstrong, W. G. (1964). Modification of the properties and composition of the dentine matrix caused by dental caries. Advanc. oral Biol. 1, 1-24. Astbury, W. T. (1940). The molecular structure of the fibres of the collagen group. / . int. Soc. Leath. Chem. 24, 69-92. Battistone, G. C. and Burnett, G. W. (1956). Studies of the composition of teeth. III . The amino acid composition of human dentinal protein. / . dent. Res. 35, 255-259. Bear, R. S. (1952). The structure of collagen fibres. Advanc. Protein Chem. 7, 69-160. Bowes, J. H., Elliott, R. G. and Moss, J. A. (1955). The composition of collagen and acid-soluble collagen of bovine skin. Biochem. J. 61, 143-153. Brown, G. L. and Kelly, F. C. (1953). In ' T he Nature and Structure of Collagen" (J. T. Randall, ed.), pp. 169-176. Academic Press, New York. Burnett, G. W. and Zenewitz, J. (1958a). Studies of the composition of teeth. VII . The moisture content of calcified tooth tissues. / . dent. Res. 37, 581-589. Burnett, G. W. and Zenewitz, J. (1958b). Studies of the composition of teeth. VIII . The composition of human teeth. / . dent. Res. 37, 590-600. Castellani, Á. Á., Ferri, G., Bolognani, L. and Graziano, V. (1960). Presenc e of sialic acid in connective tissue. Nature, Lond. 185, 37. Dirksen, T. R. (1963). Lipi d components of sound and carious dentin. / . dent. Res. 42, 128-132. Dische, Z., Danilczenko, A. and Zelmenis, G. (1958). Ciba Fdn. Symp., Chem. Biol. Mucopolysaccharides pp. 116-136. Doty, P. and Nishihara, T. (1958). In "Recent advances in Gelatine and Glue Research " (G. Stainsby, ed.), pp. 92-99. Pergamon Press, Oxford. Dziewiatkowski, D. D., DiFerrante, N., Bronner, F. and 5 Okinaka, G. (1957). Turnover of 3S-sulphat e in epiphyses and diaphyses of suckling rats. J. exp. Med. 106, 509-524.

E A S T OE Eastoe, J. E. (1955). The amino acid composition of mammalian collagen and gelatin. Biochem. J. 61, 589-602. Eastoe, J. E. (1956). In ' T he Biochemistry and Physiology of Bone" (G. H. Bourne, ed.), pp. 81-105. Academic Press, New York. Eastoe, J. E. (1957). The amino acid composition of fish collagen and gelatin. Biochem. J. 65, 363-368. Eastoe, J. E. (1961). The composition of collagen from subcellular fractions of guinea-pig granuloma tissue. Biochem. J. 79, 648-656. Eastoe, J. E. (1963). The amino acid composition of proteins from the oral tissues. II . The matrix proteins in dentine and enamel from developing human deciduous teeth. Arch, oral Biol. 8, 633-652. Eastoe, J. E. (1964). In "Bone and Tooth" (H. J. J. Blackwood, ed.), pp. 269-281. Pergamon Press, Oxford. Eastoe, J. E. and Courts, A. (1963). "Practical Analytical Methods for Connective Tissue Proteins". Spon, London. Eastoe, J. E. and Eastoe, B. (1954). The organic constituents of mammalian compact bone. Biochem. J. 57, 453-459. Eastoe, J. E. and Leach, A. A. (1958). In "Recent Advances in Gelatin and Glue Research " (G. Stainsby, ed.), pp. 173-178. Pergamon Press, Oxford. Eastoe, J. E., Long, J. E. and Willan, A. L. D. (1961). The amide nitrogen content of gelatins. Biochem. J. 78, 51-56. Fitton-Jackson , S. (1957). The fine structure of developing bone in the embryonic fowl. Proc. roy. Soc. B146, 270-280. Fleisch, H. and Bisaz, S. (1962a). Mechanism of calcification: Inhibitory role of Pyrophosphate . Nature, Lond. 195, 911. Fleisch, H. and Bisaz, S. (1962b). Isolement du plasma de pyrophosphat e un inhibiteur de la calcification. Helv. physio I. acta 20, C52-C53. Fleisch, H. and Neuman, W. F. (1960). Quantitative aspects of nucleation in calcium phosphate precipitation. J. Amer. chem. Soc. 82, 996. Free, A. H. (1943). The citrate content of whole teeth, dentin and enamel. J. dent. Res. 22, 477-478. Freudenberg , E. and Gyôrgi, P. (1921). Uber Kalkbindung durch tierische Gewebe. III . Biochem. Z. 118, 50-54. Glimcher, M. J. (1959). Molecular biology of the mineralized tissues with particular reference to bone. Rev. mod. Phys. 31, 359-393. Glimcher, M. J. (1960). In "Calcification in Biological Systems", Publ. No. 64, pp. 421-487. Amer. Ass. Advanc. Sci., Washington, D.C. Gottschalk, A. (1966). In "Glycoproteins. Their Composition, Structure and Function" (A. Gottschalk, ed.). Elsevier, Amsterdam. Grant, R. Á., Home, R. W. and Cox, R. W. (1965). New model for the tropocollagen macromolecule and its mode of aggregation Nature, Lond. 207, 822-824. Grassmann , W., Hannig, K. and Schleyer, M. (1960).

19. C H E M I C A L

ORGANIZATION

OF

Zur Aminosâuresequen z des Kollagens. II . Hoppe-Seyl. Z. 322, 71-95. Green, Í . M. and Lowther, D. A. (1959). Formation of collagen hydroxyproline in vitro. Biochem. J. 71, 55-66. Gustavson , Ê. H. (1952). "The Chemistry and Reactivity of Collagen". Academic Press, New York. Harkness, R. D. (1961). Biological functions of collagen. Biol. Rev. 36, 399-463. Harkness, R. D., Marko, A. M., Muir, H. M . and Neuberger, A . (1954). The metabolism of collagen and other proteins of the skin of rabbits. Biochem. J. 56, 558-569. Hartles, R. L. and Leaver, A. G. (1953). The fluorescenc e of teeth under ultraviolet irradiation. Biochem. J. 54, 632-638. Hartles, R. L. and Leaver, A. G. (1955). The identification of pyrimidines in the fluorescing fractions of the teeth of the sperm whale (Physeter macrocephalus). J. dent. Res. 34, 820-830. Hartles, R. L. and Leaver, A. G. (1960). Citrate in mineralized tissues. I. Citrate in human dentine. Arch, oral Biol. 1, 297-303. Hartles, R. L., Leaver, A. G. and Triffitt , J. T. (1963). Citrate in mineralized tissues. VI . The effects of dietary rehabilitation with calcium or with vitamin D or with calcium and vitamin D in rats previously maintained on a diet deficient in both calcium and vitamin D. Arch, oral Biol. 8, 657-671. Herring, G. M. (1964a). In "Bone and Tooth" (H. J. J. Blackwood, ed.), pp. 263-268. Pergamon Press, Oxford. Herring, G. M. (1964b). Chemistry of the bone matrix. Clin, orthopaed. 36, 169-183. Herring, G. M . and Kent, P. W. (1964). Some studies on mucosubstance s of bovine cortical bone. Biochem. J. 89, 405-414. Hess, W. C. and Lee, C. (1952). Isolation of chondroitin sulfuric acid from dentin. / . dent. Res. 31, 793-797. Hess, W. C, Lee, C. Y. and Peckham, S. C. (1956). The lipide content of enamel and dentin. J. dent. Res. 35, 273-275. Hess, W. C, Dhariwal, Á., Chambliss, J. F. and Alba, Z. C. (1961). Effect of dietary casein-sucros e ratios on the amino acid composition of dentinal protein. J. dent. Res. 40, 87-89. Hodge, A. J. and Schmitt, F. O. (1960). The charge profile of the tropocollagen macromolecule and the packing arrangemen t in native-type collagen fibrils. Proc. nat. Acad. Sci., Wash. 46, 186-197. Ikels, K. G. and Dirksen, T. R. (1962). Quantitative determination of some constituent lipids in dentin. Abstr. Int. Ass. dent. Res., 40th gen. Meet. St. Louis, 1962 p. 45. Irving, J. T. (1963). The Sudanophil material at sites of calcification. Arch, oral Biol. 8, 735-745. Jackson, D. S. (1957). Connective tissue growth stimulated

THE

O R G A N IC

MATRI X

OF

DENTINE

313

by carrageenin . 1. The formation and removal of collagen. Biochem. J. 65, 277-284. Kanamori, M. and Yanamoto, N. (1959). Distribution of hyaluronic acid in teeth and the oral mucosa. / . Jap. stomat. Soc. 8, 72-75. Kroner, T. D., Tabroff, W. and McGarr, J. J. (1955). Peptides isolated from a partial hydrolysate of steer hide collagen. II . Evidence for the prolyl-hydroxyproline linkage in collagen. / . Amer. chem. Soc. 11, 3356-3359. Lapiere, C. M. (1966). Mechanism of remodelling of collagenous structures. In "Symposium on the Mechanisms of Tooth Support", Oxford, 1965. Wright, Bristol (in press). Leaver, A. G., Eastoe, J. E. and Hartles, R. L. (1960). Citrate in mineralized tissues. II . The isolation from human dentine of a complex containing citric acid and a peptide. Arch, oral Biol. 2, 120-126. Leaver, A. G., Triffitt , J. T. and Hartles, R. L. (1963). Relative levels of citric and lactic acids in certain mineralized tissues. Arch, oral Biol. 8, 23-26. Leblond, C. P., Glegg, R. E. and Eidinger, D. (1957). Presenc e of carbohydrate s with free 1,2-glycol groups in sites stained by the periodic acid-Schiff technique. /. Histochem. Cytochem. 5, 445-458. Leicester, H. M. (1949). "The Biochemistry of Teeth". Mosby, St. Louis, Missouri. Leopold, R. S., Hess, W. C. and Carter, W. J. (1951). Dentinal protein: Bound cholesterol. J. dent. Res. 30, 837-839. McGann, T. C. A. (1960). The influence of citrate on the composition and form of calcium phosphate precipitates. /. Bone Jt. Surg. B42, 855-856. Manly, R. S. and Hodge, H. C. (1939). Density and refractive index studies of dental hard tissues. I. Methods for separation and determination of purity. / . dent. Res. 18, 133-141. Martens, P. J., Bradford, E. W. and Frank, R. M. (1959). Tissue changes in dentine. Int. dent. J. 9, 330-348. Moore, S. and Stein, W. H. (1951). Chromatograph y of amino acids on sulfonated polystyrene resins. / . biol. Chem. 192, 663-681. Moss, M. L., Jones, S. J. and Piez, K. A. (1964). Calcified ectoderma l collagens of shark tooth enamel and teleost scale. Science 145, 940-942. Neuman, W. F. and Neuman, M. W. (1953). The nature of the mineral phase of bone. Chem. Rev. 53, 1-45. Neuman, W. F. and Neuman, M. W. (1958). "The Chemical Dynamics of the Bone Mineral". Univ. of Chicago Press, Chicago, Illinois. Nikiforuk , G. and Sreebny, L. (1953). Demineralization of hard tissues by organic chelating agents at neutral pH. / . dent. Res. 32, 859-867. Ogle, J. D., Arlinghaus, R. B. and Logan, M. A. (1962). 3-Hydroxyproline, a new amino acid of collagen. / . biol. Chem. 237, 3667-3673.

314

J.

Å.

Orekhovitch, V. Í . and Shpikiter, V. O. (1957). In "Connective Tissue—A Symposium" (R. E. Tunbridge, ed.), pp. 281-289. Blackwell, Oxford. Perkins, H. R. and Walker, P. G. (1958). The occurrence of pyrophosphat e in bone. / . Bone Jt. Surg. B40, 333-339. Perutz, M. F. (1962). The structure of proteins. Biochem. Soc. Symp. 21, 80-87. Piez, K. A. (1960). The relation between amino acid composition and denaturation of vertebrate collagens. J. Amer. chem. Soc. 82, 247. Piez, K. A. (1962). In "Fundamental s of Keratinization— Summaries", Publ. No. 70, pp. 173-184. Amer. Ass. Advanc. Sci., Washington, D. C. Piez, K. A. and Likins, R. C. (1957). The conversion of lysine to hydroxylysine and its relation to the biosynthesis of collagen in the several tissues of the rat. / . biol. Chem. 229, 101-109. Piez, K. A. and Likins, R. C. (1960). In "Calcification in Biological Systems", Publ. No. 64, 411-420. Am. Ass. Advance. Sci., Washington, D. C. Piez, Ê. Á., Eigner, E. A. and Lewis, M. S. (1963). The chromatographi c separation and amino acid composition of the subunits of several collagens. Biochemistry 2, 58-66. Pincus, P. (1948). Relation of enamel protein to dental caries. Nature, Lond. 161, 1014. Pincus, P. (1950). Sulphated mucopolysaccharid e in human dentine. Nature, Lond. 166, 187. Prophet, A. S. and Atkinson, H. F. (1953). The action of collagenas e on carious dentine. Brit. dent. J. 94, 278-281. Quigley, M. B. and Zwarych, P. D. (1963). The preferential removal of bone and tooth collagen. Anat. Rec. 146, 357-363. Rich, A. and Crick, F. H. C. (1961). The molecular structure of collagen. / . mol. Biol. 3, 483-506. Robison, R. (1923). The possible significance of hexosephosphoric esters in ossification. I. Biochem. J. 17, 286-293. Robison, R., Macleod, M. and Rosenheim , A. H. (1930). Calcification in vitro. Biochem. J. 24, 1927-1941. Rogers, H. J. (1949). Concentration and distribution of polysaccharide s in cortical bone and the dentine of teeth. Nature, Lond. 164, 625-626. Rogers, H. J., Weidmann, S. M. and Parkinson, A. (1952). Studies on the skeletal tissues. 2. The collagen contents of bones from rabbits, oxen and humans. Biochem. J. 50, 537-542. Ryle, A. P., Sanger, F., Smith, L. F. and Kitai, R. (1955). The disulphide bonds of insulin. Biochem. J. 60, 541-556. Schlueter, R. J. and Veis, A. (1964). The macromolecula r organization of dentine matrix collagen. II . Periodate degradation and carbohydrate crosslinking. Biochemistry 3, 1657-1665. Schmitt, F. O. (1956). Macromolecular interaction patterns in biological systems. Proc. Amer. phil. Soc. 100, 476-486.

E A S T OE Schroeder , W. Á., Kay, L. M., LeGette, J., Honnen, L. and Green, F. C. (1954). The constitution of gelatin. Separation and estimation of peptides in partial hydrolysates. / . Amer. chem. Soc. 76, 3556-3564. Sobel, A. E. (1952). Studies on the "local factor" of calcification. Trans. Macy Conf. metab. Interrelations 4, 113-129. Sobel, A. E. (1955). Local factors in the mechanism of calcification. Ann. N.Y. Acad. Sci. 60, 713-722. Solomons, C. C. (1960). Peptides obtained from partial hydrolysis of decalcified human dentine collagen. Nature, Lond. 185, 101-102. Solomons, C. C. and Irving, J. T. (1958). The reaction of some hard- and soft- tissue collagens with l-fluoro-2,4, dinitrobenzene . Biochem. J. 68, 499-503. Solomons, C. C. and Neuman, W. F. (1960). On the mechanisms of calcification, the remineralization of dentin. / . biol. Chem. 235, 2502-2506. Spackman, D. H., Stein, W. H. and Moore, S. (1958). Automatic recording apparatus for use in the chromatography of amino acids. Anal. Chem. 30, 1190-1206. Spackman, D. H., Stein, W. H. and Moore, S. (1960). The disulfide bonds of ribonuclease . / . biol. Chem. 235, 648-659. Stack, M . V. (1951). Organic constituents of dentine. Brit. dent. J. 90, 173-181. Stack, M. V. (1955). The chemical nature of the organic matrix of bone, dentin and enamel. Ann. N.Y. Acad. Sci. 60, 585-595. Stetten, M. R. and Schoenheimer , R. (1944). The metabolism of 1-proline studied with the aid of deuterium and isotopic nitrogen. / . biol. Chem. 153, 113-132. Tomes, C. S. (1896). Notes upon dentine and enamel. Trans, odont. Soc, Lond. 28, 114-132. Tromans, W. J., Home, R. W., Gresham, G. A. and Bailey, A . J. (1963). Electron microscope studies on the structure of collagen fibril s by negative staining. Z. Zellforsch. 58, 798-802. Veis, A. and Schlueter, R. J. (1963). Presenc e of phosphate mediated cross-linkage s in hard tissue collagens. Nature, Lond. 197, 1204. Veis, A. and Schlueter, R. J. (1964). The macromolecula r organization of dentine matrix collagen. I. Characteriza tion of dentine collagen. Biochemistry 3, 1650-1657. von Hippel, P. H., Gallop, P. M., Seifter, S. and Cunningham, R. S. (1960). An enzymatic examination of the structure of the collagen macromolecule . / . Amer. chem. Soc. 82, 2774-2786. Weidmann, S. M. (1959). Review of modern concepts on calcification. Arch, oral Biol. 1, 259-264. Weidmann, S. M. (1963). Mechanism of calcificaton. Biological aspects . Proc. 9th ORCA Congr. dent. Caries Paris, 1962 pp. 79-84. Pergamon Press, Oxford. Whitehead, R. G. and Weidmann, S. M. (1959). Oxidative

19. C H E M I C A L

ORGANIZATION

OF

enzyme systems in ossifying cartilage. Biochem. J. 72, 667-672. Windrum, G. M., Kent, P. W. and Eastoe, J. E. (1955). The constitution of human renal reticulin. Brit. J. exp. Path. 36, 49-59. Wuthier, R. E., Gron, P. and Irving, J. T. (1964). Biochem. J. 92, 205-216. Zipkin, I. and Piez, K. A. (1950). The citric acid content of human teeth. / . dent. Res. 29, 498-505.

Note Added in Proof Further progress concerning the chemical structure of collagen has been made during the period while this

THE

O R G A N IC

MATRI X

OF

DENTINE

315

volume has been in the press. One of the most important advances has been the demonstratio n by Bornstein and Piez (1965) that all three á chains in the macromolecule of mammalian collagens probably differ from each other in structure. For a more detailed account of recent studies on this protein, reference may be made to the treatise compiled by Ramachandra n (1967). Bornstein, P. and Piez, K. A. (1965). Collagen: structural studies based on the cleavage of methionyl bonds. Science 148, 1353-1355. Ramachandran , G. N. (1967). "Treatise on Collagen," Vol. I—Chemistry of Collagen. Academic Press, New York.

This page intentionally left blank

C H A P T ER

20

CHEMICAL ORGANIZATION OF THE ORGANIC MATRIX OF ENAMEL M.

V.

STACK

I. Introduction II . Immature Matrix A . Structural Features of Immature Enamel B. Preparation of Immature Enamel for Chemical Analysis C. Solubility of Immature Enamel Matrix Proteins D. Heterogeneit y of Immature Enamel Proteins E. Optical Rotatory Dispersion of Soluble Matrix F. Kératose Fractionation of Enamel Matrix G. X-ray Diffraction of Immature Enamel Matrix

318 318 319 320 321 322 323 323

III . Maturation of the Organic Matrix A. The Role of Serine Phosphat e B. Removal of Organic Matrix during Maturation

324 324 324

IV . Mature Matrix A . Mature Enamel B. Enamel Cuticle C. Preparation of Mature Enamel for Chemical Analysis D. Regional Variations in Enamel Composition E. Organic Content of Mature Enamel F. Water Content of Enamel G. Protein Components H. Lipids I. Carbohydrate s J. Citrate and Lactate

327 327 328 329 329 330 333 334 338 338 339

V. Altered Enamel VI . Concluding Remarks Reference s

I.

339 341 342

I N T R O D U C T I ON

This chapter reviews aspects of the organic matrix of the enamel which have been reported since the publication of Leicester's comprehensiv e account (1949) and his short supplementar y review (1953). Dental researc h workers continued to

22

317

investigate the acid-insoluble matrix in more detail during the intervening period and have found evidence of the existence of acid-soluble matrix which escape s visualization by the conventiona l histological procedures . A t the present time the

318

M.

V.

situation appears to be that the true enamel matrix is to be classified as "soluble", at least on prolonged extraction under neutral conditions; whatever is "insoluble" appears to be collagenous , derived largely from the boundary regions of the enamel. The need to study the matrix as it develops has been seen to be urgent, and the changing emphasis i n studies of the organic matrix is reflected in the sequence of presentatio n adopted here. I t has recently become possible to assemble a major section dealing with the chemistry of the immature matrix. A smaller section on later changes i n the matrix, which wil l doubtless become better defined during the next few years, is followed by the more familiar subject matter concerning the mature matrix. Mention wil l be made of the modifications of the organic matrix in enamel hypoplasia and during early stages in caries. Techniques which have been of value in studying the organic matrix include density distribution analysis, demineralization under neutral conditions, differential solubility in organic solvents, electrophoresis, chromatography , gel filtration, ultracentrifugation, optical rotatory dispersion, and X-ray diffraction. Details of conditions used have not been full y described in the present text. It wil l be noted that the most recent studies deal with enamel from bovine teeth. Much of the earlier work on enamel matrix was confined to h u m an teeth, and littl e attention has been devoted to matrix obtained from monkey and rat enamel.

II. IMMATURE MATRIX A.

STRUCTURAL FEATURES OF IMMATUR E ENAMEL

Studies of immature enamel with the electron microscope have shown the cellular changes associate d with the deposition and mineralization of the matrix (Volume I , Chapter 10). Several studies have been directed towards the growth, shape and size of crystals (Chapter 16), and others to the organization and structure of the matrix and its relationship to mineral (Chapter 16). The latter

S T A CK

aspects are of particular importance, and they require continual reference to stages of enamel development. This is most easily achieved by examining the zones in order of age, from the junction of enamel with the cells which form it to the junction with dentine. A detailed study has been reported by Travis and Glimcher (1964). They confined their observations to enamel at a stage when it formed a band only 60 μ wide, distinguishing a region next to the ameloblast-ename l junction, another next to the enamel-dentin e junction and a third midway between these. They establishe d dimensions for intraprismatic matrix, filaments (and their periodic density spacings), inter-filament spaces and compartments formed by filament systems. They also measured crystal dimensions, taking care to measure only those in which some overlap could be seen. The most striking feature of these was that the thickness of the crystals was nearly ten times greater at the older enamel-dentin e junction than at the younger ameloblast-ename l junction. The intraprismatic filaments (48 Â in diameter) i n demineralized material, which are orientated similarly to the mineral crystallites, tended to be arranged as doublets separate d by intervals of approximately 120 A. A t higher resolution individual filaments were seen to be electron-dens e strands 12 A wide separate d by less electron-dens e spaces of about 17 Â. In cross-section s of prisms, dot-like structures 48 A in diameter, but resolvable into groups of smaller dots, appeare d to be filaments in cross-section . Many filaments, except those just formed at the ameloblast-ename l junction, showed an axial periodicity of approximately 170 A. Travis and Glimcher are at variance with Ronnholm (1962a,b) who considered that the filament-bounded compartments , which are presumably occupied by crystals prior to demineralization, were formed by randomly dispersed sheets or three-dimensiona l networks of cross-bridge d septa. Travis and Glimcher point out that the filamentous appearanc e could be produced by very narrow pleated sheets having the so-called anti-parallel

20.

C H E M I C A L

O R G A N I Z A T I ON

conformation. These are visualized as being extended in a direction perpendicula r to the polypeptide chains, with relatively short distances between the 180° bends, according to the structure proposed by Glimcher et al. (1961a). Additional studies by Glimcher's group, combined with the above study, suggest that prism sheaths and filaments are morphologically distinct. Their chemical investigations showed that the proline- and histidine-rich protein components extractable in neutral buffer were primarily derived from the prismatic filaments (inter- and intra-). Glimcher et al. (1964d) reported the preservation of prism sheaths but loss of prismatic filaments as demineralized matrix from immature bovine enamel was extracted with neutral buffer in the cold. These soluble proteins comprised 70-90 % of the total proteins present in the matrix, all the insoluble residue being soluble in 0.05 % acetic acid. Their electron microscopy indicated that not more than 5 % of the matrix remaining after exhaustive buffer extraction comprised prismatic filaments, the bulk being prism sheaths . Amino-acids recovered by ion exchange chromatography of hydrolyzed prism sheath material were less in amount than was indicated by sample weights. The authors were of the opinion that considerable amounts of substance s other than protein were present in this material. Its composition, especially in regard to proline, histidine, arginine, serine, phenylalanine and glycine, was quite distinct from that of the prismatic filaments (Table 1). Their conclusion that it is the prismatic filaments which are lost during the change to mature enamel is supported by electron microscopy of organic matrix from mature enamel in which chemical cross-linking was used to prevent solubilization; preservation of prism sheaths alone was seen. Structural features can thus be correlated with chemical features at a stage where only a small fraction of the enamel has been formed. Moreover, mil d chemical treatment removes structures which are identifiable with the prismatic filaments and yield a major component, rich in proline and

OF

E N A M E L

319

M A T R I X

Table 1 COMPARISON OF NEUTRAL BUFFER-SOLUBLE AND BUFFER-INSOLUBLE FRACTIONS FROM IMMATUR E BOVINE ENAMEL

MATRIX. 0

Residues of certain amino-acids per 1000 total residues

Soluble matrix (first fraction) Insoluble matrix a

Pro Ser

G l y P h e H is Arg lieu Sum

253

44

48

23

82

14

36

500

176

84

83

48

48

35

24

498

Data of Glimcher et al. (1964d).

histidine, which has a molecular weight exceeding one million. Glimcher, Friberg and Levine (1964a) offer the speculation that the progressive loss of protein during developmen t and maturation of enamel may be associate d with a depolymerization of proteins, since they found that most of the proteins obtained from mature enamel are easily diffusible, their retardation on Sephade x G-25 columns suggesting molecular weights of less than 3500. Although these structural and chemical features are determined by the mineral and protein components, their relationship is moderated by the considerable water content of immature enamel. Both weight and volume changes should therefore be determined as mineralization proceeds . Much of the change takes place with respect to water content, and this is less readily controlled. Theoretically an environment for samples under study can be establishe d with the correct conditions of humidity, so that any processe s applied, such as extraction of proteins by solvents which do not affect the mineral, should not affect the moisture content more than temporarily. B. PREPARATION OF IMMATUR E ENAMEL FOR CHEMICAL ANALYSI S

I n their studies of immature enamel from bovine teeth, Burgess and Maclaren (1965) distinguished one series of samples according to their degree of

320

M.

V.

mineralization, that is according to their specific gravity. Such samples may be prepared by selecting regions of suitable overall density, using blunt dental burs and chisels for less-mineralize d zones and sharp tools for well-mineralized enamel. Flotation procedures may be contraindicate d because samples are thereby exposed to organic compounds liable to affect components of the matrix. I t is difficul t to obtain total samples of immature enamel without using both mechanica l and physical procedures . Soft enamel can be removed by scraping (Burgess and Maclaren, 1965; Eastoe, 1960), and samples of hard enamel can be obtained by pulverizing the scraped teeth and centrifuging from a suitable bromoform mixture. The specific gravity range of total enamel from neonatal h u m an incisors and molars overlaps that of the dentine (Stack, 1955b). Glimcher, Mechanic and Friberg (1964c) prepared immature bovine enamel proteins by E D TA demineralization, first removing the enamel organ layers, and continuing exposure to 0.3 M E D TA at 2°C (pH 7.4) in order to obtain insoluble matrix. E D TA and salts were removed by repeated washing wit h acetone to which one-sixth volume of water had been added. They found that 75-90 % of this material could be dissolved by prolonged treatment wit h 0.05 M tris buffer (2-amino-2-(hydroxymethyl) propane-1 : 3-diol) at the same p H. Most of the remainder was soluble in 0.05 % acetic acid. A similar method is applicable to proteins from the continuously growing lower incisors of rats and rabbits (Levine and Glimcher, 1965). Nondiffusible enamel proteins were prepared by exhaustive treatment of teeth (50 per sample) with 0.5 M E D TA (pH 8.1) at 2°C. Absence of hydroxyproline indicated that there was negligible contamination by collagenous material. Prolonged dialysis in the cold against 0.02 M tris buffer (pH 7.4) or 0.05 M N H 4 H C 0 3 ( p H 7 . 7) yielded neutral-soluble proteins suitable for amino-acid analysis. These were characterize d by their high proline, histidine, leucine and glutamic acid content, as had been shown for the bovine and porcine developing enamel proteins. It is noteworthy that the alanine

S T A CK

levels for calf, rabbit and rat soluble enamel proteins were in the ratios 2 : 1 2: 3, and the /sO-leucine levels 3 : 1 : 3, whereas the correspond ing ratios for methionine were 30 : 1 : 30. There was about 40 % less glutamic acid, but 40 % more leucine and glycine in the rabbit samples than in those from calf and rabbit enamel. The authors are of the opinion that the incorporation of tritiated proline into the developing enamel matrix of rat molars (Greulich, 1963) may be associate d with the intraprismatic region rather than with the prism sheaths (Glimcher et al, 1964d). Greulich suggeste d that the proline-containing component of enamel matrix, which represent s the major constituent, is stable even while maturation proceeds , and during a period when a considerable bulk of the matrix is thought to be removed. C.

SOLUBILITY MATRI X

OF IMMATUR E ENAMEL

PROTEINS

The impression that the proteins of immature enamel are insoluble derives fom the distinction made by histologists between the "soluble enamel" which leaves a scarcely perceptible residue at the definitive stage when treated with dilute mineral acid and the "insoluble enamel" which yields a matrix comparable with that of other mineralized tissues at the developing stage when mineralization is incomplete. Demineralization of immature enamel in E D TA at neutral pH has given access to matrix proteins of relative insolubility (Glimcher et al., 1961a; Eastoe, 1963a). This solubility is, on the one hand, decrease d markedly by demineralizing at room temperature (20-22°C) instead of at the more biochemically "advisable" temperature just above freezing point (Glimcher et al, 1964a) and, on the other hand, demonstrabl y high on prolonged treatment with tris buffer at the same neutral p H. Furthermore, the residue remaining after exhaustive extraction is freely soluble in dilute acid. Some additional properties which can be exploited are referred to by Glimcher et al. (1964a). The acid-soluble fraction is particularly prone to

20. C H E M I C A L

ORGANIZATION

precipitation when the temperature is raised above 15°C, and especially when the ionic strength is raised above 0.1. Glimcher et al. found that both neutral-soluble and acid-soluble fractions are insoluble at ionic strengths greater than 0.5-0.6, even at 2°C. Burgess and Maclaren (1965) made use of this property when they precipitated at chloride levels of 0.25 N. The heterogeneit y of soluble fractions, as shown by electrophoresis , was similar whether chloride, nitrate, or sulphate ions were used. Moreover, the same components were present (Burgess, 1963) whatever the demineralization flui d (hydrochloric, phosphoric, or formic acids at 0.15 or 0.25 M , and 0.25 M E D TA at pH 7.1). The soluble nature of the matrix is also shown by the uptake of labelled amino-acids (see, e.g. Karpishka, Leblond and Carneiro, 1959; Young and Greulich, 1962; Greulich, 1963; Hwang, Tonna and Cronkite, 1963). The previous list of seven amino-acids studied thus has recently been doubled by Greulich and Slavkin, (1965). In each case the amino-acids were spread throughout the thickness of the enamel in a relatively short time, whereas they appeare d as sharp bands in the dentine. Eastoe (1963b) believes that the behaviour of the immature enamel matrix is consistent with its existence in the form of an amorphous gel (see also Eastoe, 1962). Much of the soluble fraction must be accessible because it can be extracted without the use of salts. Eastoe (1963a) remarks that, after enamel has been treated with sodium chloride solution, E D TA removes only an eighth of the more soluble protein. Accessibility may not, however, be directly related to degree of mineralization. Stack (unpublished data, 1962) could not relate tryptic digestibility of immature h u m an enamel powders with specific gravity range; yet EDTA-insoluble protein was readily digested by trypsin. Aci d solubility appears to vary with the degree of mineralization. Stack (1955b) found that acid demineralizing solutions dissolved about one-third of the protein from developing human enamel samples of specific gravity 2.2-2.4 and 2.4-2.6, but that the proportion of acid-insoluble protein was

OF

ENAMEL

MATRI X

321

somewhat less in the more highly mineralized sample and the proportion of diffusible peptide correspondingly greater. Of considerable interest is the solubility of immature enamel matrix proteins in strong urea and in 50 % methanol (Burgess and Maclaren, 1965). A fully alkylated derivative, tetramethylurea , has been found even more effective in extracting proteins from immature h u m an enamel (Stack, unpublished data, 1965). The apparent internal volume of such material (Poole and Stack, 1965, and unpublished data, 1964) has been shown to be doubled when extracted with strong urea solutions. Attention should be directed to the abundant histidine residues in the proteins of developing enamel. Eastoe (1963a) points out that pH changes withi n the physiological range could affect both the charge distribution and the net charge, since the basic side chain of histidine is the only one in which ionization could be modified significantly over a narrow pH range about neutrality. D . HETEROGENEITY OF IMMATUR E ENAMEL PROTEINS

Burgess and Maclaren (1965) noted at least seven electrophoretically distinct zones when processing enamel matrix proteins on starch gels. The same components were evident whether samples were obtained by neutral or acid demineralization or by urea extraction, and the diversity was similar when difficul t gels were employed. They showed that this heterogeneit y was not brought about by contamination with tissue fluids or by the methods of isolation. A scheme of fractionation based on their description appears thus: Immature enamel 0.1 M EDTA pH 11

Precipitate (i.e. insoluble) Dissolve in 0.05 Ν H C O OH Precipitate (C10 C, Å, Ê, ( + Ç and J)

Supernate to pH 7 Precipitate; pH 7-8(CL) Dissolve in 0.05 Ν H C O OH Precipitate (CIO J, H, G, Ê, ( + Å and I)

M.

322

V.

Table 2 COMPARISON OF E D T A pH (BUT INSOLUBLE AT pH

11 INSOLUBLE AND SOLUBLE

7) FRACTIONS FROM IMMATUR E

BOVINE ENAMEL

MATRIX 0

Residues of certain amino-acids per 1000 total residues Proteins

Pro

pH 11-insoluble pH 7-insoluble

291 263

Glu Gly Met Al a Lys Tyr Sum 163 85

47 79

49 65

23 12

6 21

32 95

611 620

a

D a ta of Burgess and Maclaren (1965).

Comparison in terms of amino-acid residues of these two fractions is made in Table 2. It is noteworthy that the E D TA fraction remaining soluble at pH 7 contained more amide residues than the sums derived from the partial list of amino acids. Filtration of fractions through Sephade x G-50 showed a sequenc e of the electrophoretic fractions C-E-I-K-H-G-J, as compared with a migration sequenc e C-E-G-H-I-J-K on formate-urea starch gel at pH 3.7. Component C was the major one of the first elution peak from gel filtration, the smaller peak following at double the elution volume and representing mainly component J. This retardation by Sephade x is not, however, a good indication of molecular weight, for the tyrosine level is high (Table 2), giving rise to anomalous adsorption. Amino-acid analysis of purified component C indicated a basic molecular weight of 12,500, a value which agrees well with the sedimentatio n equilibrium analysis range of 10,000-13,00 0 also reported by Burgess and Maclaren. Ultracentrifugal analysis yielded data on both components C (coefficient 0.84) and J (coefficient 2.45), the amino-acid composition of the latter indicating a molecular weight of 29,000 or a multiple of this figure. Component C, the principal protein of immature enamel matrix at an early stage, was considered to contain 108-114 amino-acid residues, 78 of them being contributed by proline (31), glutamic acid (19), leucine (12), histidine (9) and methionine (7).

S T A CK

Component C is lost at a faster rate than component J during mineralization. A t the lowest level of mineralization there was as much component C present as all other components combined. The proline- and histidine-rich fractions should be differentiated from collagen, keratin, and indeed all other known proteins, as Eastoe (1965a) has suggested . He has designate d such proteins as "amelogenins" and has discusse d restraints imposed upon their configurations arising from their unusual composition. Free rotation about bonds in their polypeptide chains is limited, according to Eastoe, in proteins with such a high proportion of rigi d planar pyrrolidine rings. The chains are therefore subject to bending, thus reducing the possibility of their forming systematic configurations. Structures of this type would be consistent wit h the fibre-less material of low crystallinity proposed by Fearnhea d (1963) on the basis of infrared spectroscop y and X-ray diffraction, and wit h the diffusely staining material examined by Fincham, G r a h am and Pautard (1965). Possibly the neutral-soluble fraction reported by Glimcher et al. (1964a) as being least retarded on Sephade x G-100, with sedimentatio n constant between 8 and 10 (molecular weight 1-4 million), is a more highly polymerized state of the component C described by Burgess and Maclaren (1965). It is unlikely that anomalous retardation due to the moderate tyrosine content (around 40 residues per 1000) needs to be considered in connection with this fraction, for it is the one which is least retarded. Glimcher et al. reported proteins from mature bovine enamel which are retarded on Sephade x G-25 and appear to have molecular weights below 3500 (tyrosine content being usually below 20 residues per 1000). The range of protein polymerization in enamel during its developmen t is thus rather extensive. E.

O P T I C AL

ROTATORY

DISPERSION

OF

SOLUBLE M A T R I X

Attempts to assign a structure to immature enamel matrix on the basis of X-ray diffraction

20.

CHEMICAL

ORGANIZATION

interpretations are made difficul t by the presence of such large amounts of proline. Glimcher's group therefore had recourse to optical rotatory dispersion measurement s (Bonar, 1965). They wished to show whether proline was present as prolylproline and whether a regular or r a n d om distribution was evident. Collagen manifests the Gly · Pro · Hypro · Gly sequenc e frequently, and the specific rotation of —300° to —330° for the pyrrolidines therein is a reflection of the —350° rotation due to prolylproline. The value is about 100° less, however, when the proline in other structures is randomly orientated. Such a value (about —260°) was found when examining a high molecular weight fraction ( M . W . 1-4 million) of sedimentatio n constant 8-10 prepared from the neutral buffer extracts from bovine foetal enamel protein. This was the first third of a first peak appearing when the material x G-100 column, was passed through the Sephade from which a somewhat complex array of components was observed to emerge. The rather modest changes in specific rotation on treatment with urea suggeste d that a r a n d om coil type of structure was present. A s noted above, the orientation of pyrrolidines appeare d r a n d om also, n of proline in every fourth and the accommodatio position along the chain therefore seems to require some frequently reversed anti-parallel pleated sheet configuration resembling the classical type of Pauling and Corey structure only remotely. F . KÉRATOSE FRACTIONATION OF ENAMEL M A T R I X

A refractory protein with numerous disulphide linkages, such as wool, is oxidized at these links by peracetic acid into keratoses which can be fractionated by their solubility in ammonia. The fibrous component of wool keratin yields an alpha-keratos e amounting to some 60 % of the total, and its X-ray analysis shows it to have an alpha-structure . The beta-keratos e comprises some 10 % of the total, probably originating in the cell-wall or cuticle, and giving a cross-beta diffraction pattern. The cement substanc e is thought to

OF

ENAMEL

MATRI X

323

give rise to the gamma-keratose , amounting to 25 % of the total, but giving a very diffuse diffraction pattern. Although it was possible to account for about 90 % of the keratoses prepared from wool and whale baleen, only 40 % of the oxidized keratin from demineralized enamel matrix was assigned to kératose fractions (Fincham et al. 1965): 2-5 % as alpha-keratose , and 36 % as beta-keratose . These workers considered that the high-proline fractions of foetal enamel were concentrate d mainly in the beta- and gamma-keratos e fractions, and that the alpha-componen t would diminish as mineralization became complete. Eastoe (1965b) disagrees with this as it is the proline-rich fractions of enamel which are lost during mineralization, leaving behind a protein . having a composition suggesting an alpha-keratose G. X - R A Y DIFFRACTION OF IMMATUR E ENAMEL M A T R I X

The matrix of the immature enamel from bovine foetuses has been examined by X - r ay diffraction both at the EDTA-insoluble stage, after washing and drying on slides in air, and from gels after exhaustive extraction with neutral buffer. The gel was prepared by centrifugation at 38,000 r pm and picking a portion up between the tips of forceps i n order to achieve orientation (Bonar, 1965, p. 146). Fibres so obtained gave orientated diffraction patterns essentially identical with the pattern of intact demineralized matrix. The meridional spacing was determined accurately to be 4.68 A, with a standard deviation g hydrogen probably below 0.05 A, and representin bonding. Another reflection (9.8 Â, equatorial) representing polypeptide side-chain spacing was found at right angles to this. The pattern was therefore classified as a "cross-beta " diffraction representing , in structural terms, a situation where hydrogen bonding is a right angles to the side-chain direction, and that the "fibr e axis", around which crystallites are orientated, is parallel to the hydrogen bonding direction.

324

M.

V.

I t was possible to obtain the same pattern, though less well-defined, from gels prepared from acetic acid solutions of the small residue ultimately insoluble in neutral buffer. Much less definition was shown in preparations from neonatal bovine enamel, probably because it is much more difficult to obtain pure acellular matrix at this later stage. Hohling, Frank and H a r n dt (1963) noted a diffuse diffraction (4.7 ± 0.5 A) from h u m an foetal enamel before demineralization , and a similar value following removal of mineral (4.3-5.0, mean 4.6 A). G r a h am and Pautard (1963) obtained only a meridional reflection at 4.2 A, and rejected the cross-beta or beta configurations (K. Little, 1958; Pautard, 1961a,b; Glimcher, Bonar and Daniel, 1961b; Hohling et al, 1963). Fearnhea d (1963, 1965) reported infrared absorption measurement s which were inconsistent with the beta or cross-beta configurations. Exclusion of the beta configuration was less certain for the immature enamel tuft components . X-ray diffraction failed to confirm either the cross-beta or alpha-helix configurations. Pautard (1965), applying the method described by Glimcher et al. (1961b) to neonatal bovine incisor enamel, was unable to show cross-beta configurations in specimens which had not been allowed to dry. Denaturation due to drying these fibres was shown by the tendency towards beta-type patterns. Bonar (1965) was unwilling to believe that structures with such a high proportion of proline would be able to form alpha-type patterns. The problem of the configuration of the developing enamel matrix does not therefore seem to be resolved; further study is required of the conditions which could produce material which has not undergone denaturation (see also Pautard, 1963). III. MATURATION OF THE ORGANIC MATRIX A . T H E R O LE OF SERINE PHOSPHATE

Eastoe (Chapter 19, p. 304) has pointed out that the relationship between the inorganic and organic phases of dentine is one of the most fascinating

S T A CK

aspects of the tissue and yet at the same time is among the least understood. The same could be said of enamel. Eastoe's account of the situation i n respect of dentine (Chapter 19) provides a good basis from which to speculate on the mechanism s that may operate during the developmen t of enamel. The finding of protein-bound phosphate in dentine collagen has recently been extended to highly purified collagen from other sources (Glimcher et al, 1965), and to both immature and mature enamel (Glimcher and Krane, 1964). They observed that phosvitin appears to be the only other phosphoprotei n containing significantly more organic phosphate than enamel protein. Enamel protein (also pepsin and ovalbumin) contain this phosphate combined with serine, whereas phosvitin and casein contain serine phosphate polymers. Glimcher and Krane point out that the presence of protein-bound serine phosphate meets a number of the criteria considered necessar y before matrixbound inorganic ions can participate in crystal nucleation. Such phosphate groups remain very reactive despite their covalent links. Present evidence thus favours serine as being of greater importance than hydroxylysine, which Piez (1961) suggeste d has a role in the mineralization process: Glimcher and Krane specifically mention that phosphohydroxylysin e was not found. Krane, Stone and Glimcher (1965) have identified P3 2-labelled serine phosphate in partial acid hydrolyzates of enamel matrix protein after incubation wit h purified protein phosphokinas e derived from muscle. They discusse d their findings in connection wit h substrate specificity and the position of serine in the polypeptide chain, and with the role of enzymic phosphorylation of structural proteins in the mechanism of nucleation of apatite crystals. It was noted that the correspondin g protein phosphokinase from brewers' yeast would not phosphorylate embryonic enamel proteins. B. REMOVAL OF O R G A N IC M A T R I X DURING MATURATIO N

Reith (1965) believes that the removal of organic matrix has actually been observed with the electron

20. C H E M I C A L

ORGANIZATION

microscope. The cells involved are those engaged in the maturation process, and, when viewed in the electron microscope, organic material can be traced through the processe s of a striated border present at the distal end of the ameloblasts and then into the cell substance . Cell granules of variable morphology are seen, resembling lysosomes or multivesiculated bodies. Such structures have been located in macrophage s and other cells implicated in depolymerization . A b u n d a nt mitochondria are evident near the striated borders of these metabolically active cells, and this suggests that the energy requirements for the activities of these cells are considerable . Eastoe (1963b), following the concept of the enamel matrix as a structureles s gel (Fearnhead , 1963), has suppose d that the structure of the matrix protein is such as to provide for its eventual removal. A gel with thixotropic properties might have its rigidity and viscosity reduced locally in

OF E N A M E L

MATRI X

325

regions of pressure such as could result when enamel crystals grow towards each other. Liquefaction of the gel could then result when mineralization was reaching completion. Further speculation (Eastoe, 1963b) relates the features which characterize the composition of the major components of the enamel matrix with the likely behaviour of such a gel. Stress might be transmitted via the bulky pyrrolidine rings of the numerous proline residues to a specially sensitive portion of the structure, causing temporary or permanent rupture (e.g. local collective hydrogen bond systems). Large numbers of histidine residues might also play a part, in altering molecular shape through charge redistribution within a relatively slight pH range. Glimcher et al (1964a) suggest that the progressive loss of matrix is accompanie d and possibly preceded by a depolymerization of proteins of high molecular weight. These may be as much as 1000

0.12 -

0.10

0.08

0.06

0.04

0.02 [

0.2

0.4

0.6

0.8

1.0

Enamel formed per dentition (grams)

1.2

Fig. 1. Gain and loss of enamel proteins during crown growth. Abscissa: grams of enamel formed per dentition; ordinate: grams of enamel proteins present. Data on enamel from 10 developing human dentitions.(From Stack, unpublished data, 1960.)

326

V.

M.

S T A CK

times more complex than the diffusible proteins which they found to be retarded by Sephade x G-25 gel filtration andwhich seem to account forthemajor portion of the residual proteins of mature enamel. The gain and loss of enamel matrix during development can be seen clearly during analysis of samples from the whole of the enamel obtainable from dentitions at various stages of growth (Stack, 1960). Figure 1 shows a fourfold decreas e in the amount of protein present during a phase in growth when there is a twofold increase in dry weight of the enamel. Burgess and Maclaren (1965) have shown aminoacid levels, defined by specific gravity, at several stages of enamel formation. The data in their Table 1 have been transformed into logarithms for

Fig. 2. Amino-acid levels at three stages of bovine enamel formation (complete matrix, early and late mineralization). Abscissa: mean density of wet enamel (gm/ml); ordinate: log micromoles per litre. (From Burgess and Maclaren, 1965.)

72

display in Fig. 2, which has been divided into four in order to accommodat e depictions of groups of amino-acids. There was at least an 80 % decreas e i n amino-acid levels except in the cases of hydroxyproline and hydroxylysine. Some of the rates of loss were similar, but the end result, as Burgess and Maclaren point out, was a protein having littl e similarity to the protein of developing enamel (Eastoe, 1960). Unpublished observations of water content in relation to organic content (Fig. 3) indicate the close dependenc e of hydration on the matrix. One of the points plotted correspond s to the situation where the mineral content had reached one-half the total weight, water content being then about 30 % and organic content 20 %.

1

62

0

2

2

7

72

1

Specific gravity (wet enamel)

62

0

2

2

7

20. C H E M I C A L

ORGANIZATION

Moisture content (per cent)

IV . M A T U R E M A T R I X A . M A T U R E ENAMEL

Definitive enamel, as a biological tissue, may be said to li e between the remnants of the ameloblast layer and the enamel-dentin e junction. F r om the viewpoint of the analyst interested in the chemical composition of such material it is of great importance to avoid contamination from the regions adjacent to the enamel because of their relatively high collagen content (Stack, 1955a). Sufficiently uniform material can certainly be prepared by abrading the outer surface to remove the mineralized cuticular layer and by pulverizing and floating off any particles having a specific gravity less than 2.75. Those having no tendency to float or sink appear by microscopy to be particles fractured at the enamel-dentin e junction and comprise no more than 1 part of dentine to 8-10 of enamel. They amount to no more than 1 % of the

OF

ENAMEL

MATRI X

327

Fig. 3. Organic content in relation to moisture content in developing human enamel. Abscissa: percentag e of water; ordinate: percentag e of organic content. (From Stack, unpublished data, 1960.)

enamel-particle yield. Innermost enamel containing tufts and spindles derived from the dentine therefore seems likely to be retained by this process. Although tufts and spindles may reasonabl y be thought of as structures characteristic of enamel they are liable to confuse analyses of the organic matrix of mature enamel ; in other words, they are liable to have a large effect on the matrix which remains when the immature matrix has undergone the transformation that occurs during development . Interpretation of the analysis of a more-or-less complete sample of enamel would be straightforward if it were necessar y merely to subtract a correction due to collagenous contribution from the adjacent tissues. However, few studies lead to the conclusion that the protein resisting solution during demineralization is wholly collagenous . Present interest wil l focus on the painstaking work of Glimcher's group of workers, who apparently had to use the enamel from no less than 2500 mature bovine incisor teeth before they could

328

M.

V.

prepare sufficient EDTA-insoluble protein for analysis (Glimcher et al., 1964a). Even this small amount did not appear to be significantly collagenous but showed only 12 residues of hydroxyproline per 1000, whereas their demineralized coronal cementum protein (Glimcher, Friberg and Levine, 1964b) contained 91 residues per 1000. The typical matrix of mature enamel is apparently best regarded as completely soluble in E D T A, but to obtain material of this description it is necessar y to retain only that layer which can be obtained safely clear of contamination by the adjacent tissues, namely the middle third of the thickness. The quantity of soluble matrix is surprisingly low i n bovine enamel; Glimcher et al. (1964a) report only 0.06 % by weight of mature enamel was represente d by amino-acids, and of this from 50 to 90 % was lost either on dialysis or desalting. Earlier work had given the impression that 0.6 % is a probable value for the organic content of mature h u m an enamel. It may be of significance that it is this value which may be taken to mark the discontinuity in the relationship between organic content and mineral composition which may be deduced (see page 330) from the work of Bose, Blackwell and Fosdick (I960). B. ENAMEL CUTICLE

Pincus (1948) described some chemical reactions of cuticle obtained by steeping well-brushed (with 0.5 % sodium carbonate ) unerupted human molar teeth in 0.5 M HCi for a day. He identified the cuticle as Nasmyth's membrane, and showed its property of swelling in acetic acid, dissolving in HCI at 37°C, and being somewhat resistant to digestion with pepsin and trypsin. It also resisted cupriammonium and cupriethylenediamin e solutions, dissolving very slowly in thioglycollic acid. The Molisch test was negative and no purple colour was seen during hydrolysis in acid solution; the presence of carbohydrate was therefore not obvious. This author compared this material in parallel tests with the contents of grooves on the occlusal surface of cusped teeth. He was conscious that

S T A CK

developmenta l material might become "trapped" between the cusps, and freed from them by mild acid treatment of unerupted teeth. This material reacted positively in the Molisch test and gave purple solutions in strong acid, indicative of carbohydrate component. It dissolved freely in the basic copper reagents and thioglycollic acid but was somewhat resistant to pepsin and trypsin. More definite demonstration of a continuous collagenous layer all over the crowns of human teeth is due to Levine, Glimcher and Bonar (1964). They examined partially formed premolars from children aged 9-11, after scraping the enamel surface with a scalpel. Prolonged steeping of these teeth in a 2 % solution (pH 6.6) of dichloro-symtriazine (Procion Brilliant Red-M-2BS) enabled them to fix and stain the tenuous covering of the crown. Demineralization for a month in EDTA (pH 8.3) enabled them to remove a dentine core and to examine the amino-acid composition of the layers covering crown and root. The analyses showed typical values for collagen, although not more than two-thirds of the material comprising the layer over the crown was considered to be collagen. Levine et al. noted that Owen had described such a layer over a century ago, but that its presence had not received sufficient attention in recent years. Owen had reported its presence on the teeth of many animals, sometimes at greater thickness. Glimcher et al. (1964b) described such a layer on erupted bovine crowns and compared its amino acid composition with that of better-defined collagen. There it had the appearanc e of cementum, and it was a feature of their work on the proteins of erupted bovine teeth that they took great care to remove this layer (in 0.5 M E D T A, pH 8.3) before isolating the primarily EDTA-soluble protein. They remarked u p on the relatively deep penetration of the enamel by collagenous fibrils from the coronal cementum layer. The amino-acid composition of cuticles from both unerupted and recently erupted teeth was considered by Schule (1961) to be comparable with that of the insoluble enamel matrix. Cuticle protein

20.

CHEMICAL

ORGANIZATION

was found to resist acids, alkalis and proteolytic enzymes and was impermeable to cystine, congo red and trypan blue; however, it allowed passag e of lactic and hydrochloric acids, sodium chloride, amino acids, glucose, methylene blue, calcium, and phosphate . The cuticle on unerupted teeth was more permeable than that from teeth recently erupted, and its acid mucopolysaccharid e component was more evident. C . PREPARATION OF M A T U R E ENAMEL FOR CHEMICAL

ANALYSI S

Microscopy of enamel sections is a useful guide to the selection of a mid-portion unlikely to be contaminated by adjacent tissues, but macroscopic examination is necessar y in the first instance in order to allow the exclusion of developmenta l and environmenta l concentration s of organic material. Losee and Hess (1949) drew attention to the need to describe the types of enamel selected for analysis. Their acid-insoluble protein yields (Table 3) indicate that reproducible analyses are unlikely unless pits, fissures and stains are excluded as far as possible. It was their intention to make use of "enamel proper", a mid-portion free from surface cuticle and from inner regions where the tufts are located. Acid-soluble protein was not detectable , but the Table stresse s the importance of "clean" enamel. Table 3 YIELD S OF ACID-INSOLUBLE PROTEIN FROM ENAMEL"' B

Material demineralize d Total enamel, deep pits and grooves, stained Total enamel, deep pits and grooves, unstained Total enamel, pits and grooves minimal, unstained Enamel "proper", pits and grooves minimal Enamel "proper", pits and grooves removed a b

Data of Losee and Hess (1949). Enamel freed from cuticle by acid treatment.

Yield (%) 0.49 0.39 0.30 0.22 0.21

OF

ENAMEL

MATRI X

329

The importance of developmenta l differences was perhaps not sufficiently appreciate d until Bhussry (1958a) separate d molar cusps and found nitrogen levels to be one-fifth greater in mesio-distal zones than in labio-lingual zones. D.

REGIONAL VARIATION S IN ENAMEL COMPOSITION

I n addition to the study of total enamel, attention has been given to the division of enamel according to arbitrary depth, distinguishable zones and physical properties. Successiv e layers of enamel have been ground away and samples analyzed in terms of "thickness scores" based on surface areas (Savory and Brudevold, 1959). This permits a direct comparison of nitrogen distribution, which was found to be over 100 mg % at the surface, 65 mg % in the mid-zone and 140 mg % at the enamel-dentin e junction. Examination of 0.2-mm sections of unerupted third molars by reflected light reveals zones which have been distinguished by Wachtel (1959) as "clear", "cloudy white" and "opaque yellow". The zones were isolated by fracturing at the interfaces, and were shown to have nitrogen contents in the ratio 1 : 3 : 10. Enamel from clear zones has been found to lose phosphate at the same rate in 0.007-0.009 Ν lactic acid and hydrofluoric acid buffered (sodium) to pH 4.0-4.2 (Wachtel, 1964), but much greater solubility is shown by cloudy enamel, particularly in lactate buffer. Another method, due to Bose et al. (1960), depended upon the projection of previously pulverized enamel at high speed against a refractory target. This appears to separate structurally distinct regions of the enamel. The resultant particles were very much smaller than those usually analyzed and had a wide range of organic content, the lightest fraction (probably interprismatic material) having an organic content ten times greater than that of the heaviest fraction (probably prism-core material). It is of interest to note an apparent discontinuity when their data are displayed graphically (Fig. 4). Sodium, potassium,

330

M.

V.

S T A CK

70 h

65

60

55

Να

50

45

Fig. 4. Na, K, Mg, and C 0 2 contents of enamel particles in relation to organic content. Abscissa: percentag e of organic content; ordinate: Na (per cent χ 100), Ê (per cent χ 1000), Mg (per cent χ 100), C 0 2 (per cent χ 20). (From Bose et al, 1960.)

magnesium and carbonate levels vary with organic content, linear inverse relationships being marked in samples with organic content below 0.6 % and changing rather abruptly to a less marked slope in the remaining samples. E. O R G A N IC CONTENT OF M A T U R E ENAMEL

1. Mean

Values

The determination of the organic content of enamel would seem to be dependen t upon adequate control of water and carbonate levels. Extraction wit h alkaline ethylene glycol removes organic matter from dentine, but less completely from enamel (Burnett and Zenewitz, 1958b); moreover, this reagent extracts a significant proportion of calcium and phosphate . Mineral solubility is not appreciable as a result of ethylenediamin e extraction. However, neither of these reagents extracts all the organic material, and difference-weighing

0.4

0.6

0.8

1.0

Organic content (per cent)

after treatment with them does not lead to any confident estimate of total organic content. Internal spaces are too small to allow adequate penetration of extracting agents (see page 334) and more drastic methods are necessary . A temperature of 200°C, as used in alkaline ethylene glycol extraction, is probably not adequate to remove even loosely bound water, some of which, it was suggested , was prevented from escape by firml y bound water unless the temperature was raised as high as 300°C Emerson (1960) concluded that at this temperature all the protein of mature enamel was pyrolyzed within 15 minutes. Stack (1965) has reported pyrolysis "spectra" from developing enamel held at this temperature for no more than 3 minutes, and littl e organic material remains even i n mature enamel after 5 minutes at this temperature, according to gas chromatographi c evidence (unpublished observations , 1964). Wet-combustion is also a drastic method,

20. C H E M I C A L

ORGANIZATION

resulting in likely access to all organic material. Heating with excess oxidizing reagent (dichromate) and sulphuric acid in high concentration at 100°C gives an estimate for organic content which can be expresse d in terms of equivalents of reagent per unit weight. Components of different chemical nature differ in their susceptibility to this type of reagent. However, Stack and Williams (1952) determined equivalents for the acid-soluble and acid-insoluble protein fractions and for citric acid. I t was noticed that the summated values in oxidizing equivalents numerically matched the percentag e organic matter in terms of the above three components, i.e. 1 /xeq/mg corresponde d to 1 % organic matter. The assumption that the organic components are always present in the same proportion does not seem likely in view of the variable ratios found when comparing wet-ash and nitrogen analyses (Stack and Williams, see below). However, the method is of utilit y for semi-quantitative comparisons. Despite the obvious partial sulphation of enamel samples during treatment with this reagent, it is of interest to record that a substantia l proportion of the proteins of enamel remain relatively intact, judging from their pyrolysis "spectra", when Table 4 NITROGEN AND WET-COMBUSTION DATA " FOR PERMANENT HUMA N ENAMEL IN RELATION TO A GE

Authors

Analysis lst-2nd 3rd 4th 5th 6th decade

Nitrogen Bhussry and Hess (1963) Savory and Nitrogen Brudevold (1959) Nitrogen Pilz& (1959a) Organic Stack (1954b) Organic

46

49

61

72

78

64

61 60 60

61 57 64

59 54 58

7th

" D a ta in milligrams per cent for nitrogen; equivalent per cent for organic. Ages grouped in decades . b Published data divided by 10. c 60 mg Í per cent at age 73, but 120 mg Í per cent at age 59 (means).

OF

ENAMEL

331

MATRI X Table 5

MEA N NITROGEN CONTENT OF ENAMEL SAMPLES (DEFINED ORIGIN)

Authors Anderson (1949) Losee and Hess (1949) Stack and Williams 1952) Burnett and Zenewitz (1958b) Savory and Brudevold (1959) Wachtel (1959) Pilz (1959a) Bhussry and Hess (1963)

Nitrogen (mg%) 64 70 73 57) 64) 65

Sample

Premolars (young) Premolars (older) Molars (minimal pits and grooves) Pooled (range) Incisors, canines, molars

70

Permanen t

99 70 62 58

Deciduous Molars (clear zones) Pooled Pooled (all age groups)

tooth crowns are treated with 50 % (v/v) sulphuric acid at 150°C, conditions sufficiently drastic to result in complete destruction of the dentine while leaving the enamel cap apparently intact, although showing a sulphate " b l o o m" (Stack, unpublished observations , 1964). There is a fair measure of agreemen t among values from a number of laboratories where wet-combustion and nitrogen analyses have been reported (Tables 4 and 5). A s a guide to total organic content, nitrogen analysis also suffers from lack of knowledge about the relative proportions of other components . Values published over a 10 year period lead to an overall mean value for nitrogen of 65 mg % correspondin g to 0.5 % protein (taking nitrogen contents of the acid-soluble and acid-insoluble protein to be 10 and 13 %, as given by Stack, 1954a). The mean value in Table 5 derived from the data tabulated by Anderson (1949) excludes values exceeding these by more than twice the standard deviation. The consistenc y of the above values from various laboratories suggests that the organic content of enamel does not depend as much as might be expected upon origin or method of preparation.

332

M.

V.

Analysis of purified enamel from young premolars collected in various European and American cities but analyzed in the same laboratory (Stack, unpublished data, 1956) confirmed the first point. The mean values for organic content, according to wet-combustion analyses , did not differ by more than 5 %. 2. Variation in Organic

Content

The same methods have been applied to determining factors responsible for variability in individual enamel samples or in selected groups. Enamel from pairs of homologous teeth has not shown less within-pair variability as compared with enamel from random pairs of teeth (Stack and Williams, 1952). However, less variability was noted in two out of five sets where all four homologous teeth were available. Stack and Williams applied the flotation process to purify the enamel from 50 permanent tooth crowns and reported a population variability of between 11 and 14 % for organic content (allowing for analytical replication). Similar variability was apparent when analyzing enamel samples from 50 deciduous teeth (Stack, 1953b), the mean organic content being 0.1 % greater (permanen t 0.6 %, deciduous 0.7 % ). These data are shown in Table 6. Table 6 VARIABILIT Y IN ORGANIC CONTENT ( W E T - A SH ANALYSIS) OF DECIDUOUS AND PERMANENT ENAMEL"

Deciduous enamel

Permanen t enamel

Central value (meq%)

Number of samples

Central value (meq%)

Number of samples

52 61 70 79 88

6 13 15 12 4

45 52 59 66 73

4 12 19 11 6

a Data in milliequivalents per cent from Figs. V and V I , interchanged , of Stack (1954b).

S T A CK

Another aspect of variability was examined by comparing wet-ash and nitrogen analyses . Extreme ratios (equivalents of oxidizing reagent: grams of nitrogen) differed by as much as 25 % among 10 individual samples, although values were all withi n 3 % of a 1 : 1 ratio when ten large pooled samples were compared (Stack, 1954a). Means for both individual and pooled samples were 60 meq % by wet-ash analysis. Bhussry and Hess (1963) and Pilz (1959a,b) were able to detect significant trends with age; organic content, determined by nitrogen and wet-ash analyses, was found by Pilz to reach a maximum in the over-50 age group and to decline thereafter. Savory and Brudevold (1959) observed that the nitrogen content in surface and junction enamel was comparatively greater in the over-50 age group, and the minimum nitrogen level occurred at greater depth than was the case in young teeth. Table 4 does not suggest that there is a clear relationship between age and nitrogen content of the enamel. Variation with type of tooth has been studied by Hattyasy and Hatos (1953), differences in ranges of protein content being significant when enamel from canines (0.56-2.94 %) was compared with that from premolars (0.25-0.38 %). A n improvement in flotation technique has been recently described by Shapiro and Hartles (1965). They were able to achieve separation of rat enamel wit h losses below 2 % on samples of the order of a gram. Previous density distribution of rat enamel by Gilda (1951) was somewhat less efficient. Weidmann (1965) used a density gradient method for evaluating the particle density range of h u m an enamel (2.88-3.02). Rodents and monkeys are used extensively in experimental caries research . Stack (1957) therefore compared enamel from human, rhesus monkey and rat teeth in regard to specific gravity range and organic content. Rat enamel was found to contain twice as much organic matter as human and monkey enamel, the latter being from permanen t teeth and comparable in organic content to human deciduous enamel. The threefold difference in carbohydrate content of rodent and primate teeth

20.

CHEMICAL

ORGANIZATION

was ascribed to the very different surface-volume relationships. Stack and Holloway (1959) compared carbohydrate levels in enamel and dentine from molars of rats on contrasted diets, and noted the dependenc e of carbohydrate levels on growth status rather than dietary carbohydrate : protein ratio. Protein-deficient rats were retarded in growth by about 2 weeks at the end of the sixth week from birth. Wynn, Haldi and Law (1962) compared albino rats on two diets, one being cariogenic, and found 0.11-0.12% nitrogen in the molar enamel from both groups. Higher nitrogen levels were noted by Burnett and Zenewitz (1957) in hamster molar enamel, those in males being less than mean values i n females (0.29 and 0.47 %, respectively). Similar high levels were noted in the enamel from incisors (males, 0.35 %; females, 0.42 % ). Less than one-half of the organic matter determined as weight loss during alkaline ethylene glycol extraction was attributable to protein as calculated from nitrogen levels. The disparity was even greater in the study reported by Wynn et al., ash loss less carbon dioxide being about 2.5 % or some four times the protein content calculated from nitrogen data. F . W A T ER CONTENT OF ENAMEL

Quantitative studies of the organic matrix of enamel have always been hampered by lack of certainty about the water content. Drying periods need to be as long for enamel as for dentine (Chapter 19, p. 301). Burnett and Zenewitz (1958a) found that enamel reached constant weight in a day at 100°C in vacuo, whereas a week was necessar y at normal pressure . Moisture content of "humidified" enamel was 2.2 %, the same value being found on heating at 200°C, although a day at this temperature would be expected to be deleterious to the organic matrix. Greater loss in weight was found on drying smaller particles; this could be ascribed to the dependenc e of organic contents on particle size. Stack and Williams (1952), for example, found two-thirds as much 23

OF

ENAMEL

MATRI X

333

organic matter in coarser particles (0.20-0.35 mm) as in particles of diameter below 0.1 mm. Regional changes have been followed by birefringence measurement s in polarized light microscopy. Carlstrom, Glas and Angmar (1963) noted that the full y hydrated negative value of 0.0020/25 changed on drying to —0.0017 in outer enamel, and to a value of +0.001 in inner enamel; the mid-zone was isotropic. It appeare d that the biréfringent properties were regained after 2 hours at 60, 70 or 80°C, more slowly at 90°C, and only the innermost enamel imbibed after heating at 110°C. Their gravimetric results indicated 0.8 and 3.2 % by weight of loosely bound and firml y bound water. Emerson (1960) noted a reversal of the increase i n birefringence within the range 100-200°C, the maximum value attained being +0.007 at 400°C. He ascribed the 0.83 % weight loss between 300 and 400°C to " b o u n d" water, and commented that no protein could be detected after exposure at 300°C for 15 minutes. Nitrogen adsorption measurement s of surface area have been found to reach a maximum in " a n o r g a n i c" bone at 300°C, a 5 % increase being apparent as the temperature was raised from 100°C (Holmes et al., 1964). These authors suggeste d that some of the loosely bound water might be prevented from escaping from submicroscopi c spaces between crystallites by the presence of firml y bound water until the temperature rose sufficiently. Relatively slow diffusion of nitrogen into some of the void space (totalling 0.4 cc/gm) was likely, according to adsorption microcalorimetry by Holmes and Beebe (1961). This slow phase was not seen to occur in the presence of " a monolayer or two of chemisorbe d methanol" acquired by pre-adsorption . This presumably blocked critically small pores, possibly of diameter below 10 Â. Such pores may be equated wit h boundaries between contiguous apatite crystals where inter-ionic attraction would exist even in dehydrated material, fortified by hydrogen bonding in the hydrated state. I t seems unlikely that more than a fractional percentage of the crystal surface area, which Sognnaes (1965) calculates to be of the order of

334

M.

V.

50 m2/gm, would be accessible to various types of small molecules such as would be involved in ionic exchange . Mercury porosimetry and liquid diffusion (Poole and Stack, 1965; Bergman, 1963; Poole et al., 1963) give some indication of this. Microscopy with imbibable fluids has shown that enamel qualifies to be included among materials having molecular sieve properties (Darling et al., 1961). F r om birefringence measurement s it was calculated that only 0.1 % of the volume of normal enamel is void space. On the assumption that the crystal surfaces could adsorb a double layer of water molecules, by analogy with parameter s suitable for bone mineral (Holmes et al., 1964), the above value of 5 0 m2/ gm would correspond to 25 mg water per gram of enamel. This is equivalent to the content of loosely bound water found in specimens of "humidified" enamel (Burnett and Zenewitz, 1958a). However, allowance should be made for water associate d with the organic components , and it is possible that inter-particulate condensatio n takes place when enamel is "humidified" unless the partial pressure of water vapour is controlled to a value around 0.95 (Poole and Stack, 1965).

G.

PROTEIN COMPONENTS

1. "Soluble" Proteins, Peptides

and

Amino-Acids

Eastoe (1965b) points out the need to define the solvents and conditions under which solubility is demonstrate d in connection with enamel matrix studies. A typical protein which would be soluble in E D TA at neutral pH could be freed from E D TA readily by dialysis. This is not true of EDTA-soluble proteins from mature bovine enamel, which Glimcher et al. (1964a) state to be diffusible and retarded on Sephade x G-25 columns, a finding indicative of molecular weights below 3500. Weidmann and H a mm (1965), however, found sufficient nondiffusible material from E D T Asoluble human enamel to show four peaks when passed through a Sephade x G-25 column, instead of the two evident when a water-soluble fraction

S T A CK

was studied. This suggeste d a greater proportion of large peptides than could be dissolved by water alone. Burrows (1965) distinguished three peaks by Sephade x G-50 filtration of EDTA-soluble matrix from the interior of human enamel, hydroxyproline being evident in the first major peak. Weidmann and Lofthouse (1963) found very similar analyses for the two protein fractions obtained by demineralizing mature enamel, except in regard to hydroxyproline content, which was lower in the soluble fraction in the ratio 2 : 9. Stack (1954a) separate d a collagenous fraction by electrophoresi s of a soluble fraction in acetate buffer, the least mobile component having a hydroxyproline : nitrogen ratio of the same order as collagen. Yields of acid-soluble and acidinsoluble proteins were of the same order, but slightly more insoluble material was recovered by demineralizing with E D T A. Anderson (1951) noted differences in yields depending on the type of acid, at molar strength, used in demineralizing human enamel. The amounts of insoluble protein, expressed as percentage s of the total nitrogen contents, amounted to 51-54 % in hydrochloric acid, 69-70 % in trichloracetic acid and 66-71 % in perchloric acid (cited by Stack and Williams, 1952). The characteristic amino-acids of the E D T Asoluble nondiffusible fractions studied by Glimcher et al. (1964a) were serine, glutamic acid and glycine. High aspartic acid contents were noted in a number of fractions. Each sample was considered to be a mixture of several components . Although 50-90 % of the material containing amino-acid was lost by dialysis or desalting, these authors pointed out that the composition of the original material was not remarkably different from that of the residual nondiffusible fraction. Some of the high serine content was shown to involve serine phosphate , incompletely hydrolyzed by mineral acid under the conditions required for amino-acid analysis. Serine phosphate and cysteic acid have been reported by Gasparini and Gotte (1961) in EDTA-insoluble protein from enamel. They reported only 13 mg EDTA-soluble protein per gram

20. C H E M I C A L

ORGANIZATION

of enamel. Hexosamine was demonstrate d in their preparations . Ninhydrin staining on the surfaces of unerupted human molars was used by Hutton and Nuckolls (1951) as an index of peptide location. Stack (1954a) reported a fivefold increase in ninhydrin reaction products when water-soluble material from mature h u m an enamel was hydrolyzed. He was unable to report (Stack, 1955c) reproducible separations of butanol-soluble peptides from acid solutions of enamel when using Dowex 50 resins (2 and 4 % cross-linked). High-voltage electrophoresi s of concentrate d aqueous extracts of mature human enamel (Weidmann and H a m m, 1965) showed unseparate d peptides as well as two distinct bands both at pH 1.85 and at pH 5.2. Free amino-acids such as alanine, glycine, serine, glutamic and aspartic acids were identified. Hydrolysis sharpene d the aminoacid bands shown on electrophoresis , especially the basic amino-acids, and the bands ascribed to peptides were no longer evident. The percentag e of free amino-acids appeare d to vary from 3 to 7. The same authors showed that two main peaks were eluted when such aqueous extracts were passed through ion-exchange resins (Amberlite CG-50 and D E AE Sephade x A-50). The similar pattern evident when extracts were filtered through Sephade x G-25 was indicative of two groups of peptides. Hydrolysis of filtrates showed 9 aminoacids in the first peak and 13 in the second larger peak, some of these being free amino acids. Larger

OF

ENAMEL

335

MATRI X

peptides were found in a major first peak when EDTA-soluble material was submitted to gel filtration; three less pronounced peaks were noted i n this instance. Sephade x G-25 excludes substance s of molecular weight up to 5000, whereas the G-100 polymer excludes up to 100,000. Recycling the water-soluble extract through G-100 was found to reduce the first peak and enhance the second. The water-soluble peptides were thus thought to range i n molecular weight from below 5000 up to 100,000. Presumably absence of the full range of amino-acids in the material not retarded by G-100 accounts for its being regarded as peptide in nature rather than protein. I n connection with peptides found to be present i n enamel, mention should be made of artificial peptides because of their high serine phosphate content (Glimcher and Krane, 1964). Water-soluble proteins from enamel of erupted bovine teeth (Glimcher et al, 1964a) were partially hydrolyzed i n 12 A^HCl at 37°C for 12 hours after removal of inorganic P, acid-soluble organic Ñ and phospholipids. The peptides were eluted from a Dowex 50 resin column (X-2, H + form) and hydrolyzed further in a 2 TV HC1 at 100° for 2 hours. They were then subjected to high-voltage electrophoresi s at pH 1.9. One positively charged phosphopeptide , of which 86 % of the amino acid residues comprised similar amounts of serine (phosphate ) and aspartic acid, and 3 negatively charged phosphopeptide s were thus separated . The least mobile of these contained

Table 7 ELECTRONEGATIVE PHOSPHOPEPTIDE S PREPARED BY PARTIAL HYDROLYSIS OF WATER-SOLUBLE PROTEINS FROM MATURE BOVINE ENAMEL "

Residues of certain amino-acids per 1000 total residues Sample Fraction 2 Fraction 3

Cys

Asp

Thr

Ser

Glu

Pro

2 5

396 65

5 30

476 100

28 96

5 86

° Data of Glimcher and Krane (1964). b Leu -f lieu; fraction 3 also had 14/1000 Phe residues.

Gly 20 315

Al a

Val

Leu&

Lys

His

Arg

9 96

6 32

5 56

40 37

3 16

4 49

336

M.

V.

few amino-acids other than serine and aspartic acid (59 and 33 %, respectively). M o re variety of amino-acids was found in the more mobile fractions (Table 7). 2. "Insoluble"

Proteins

Studies so far reported on overall amino-acid composition suggest the presence of collagen in the majority of acid-insoluble enamel matrix preparations. This collagen represent s a " c o n t a m i n a n"t from the tufts and spindles of the inner zone or from a cementum-like external layer. Piez (1962) indeed asserte d that this fraction consists primarily of dentine collagen despite the great care taken to avoid contamination. Rodriguez and Hess (1962) offered the opinion that the acid-insoluble protein was a "secreted collagen". Geller (1958) also concluded that it was a collagen. By reference to histochemical identifications by Sullivan (1953) and by Bevelander and Johnson (1955) he suggeste d further that it was calcium chondroitin sulphate collagen. These statement s may be taken as applicable to the collagenous material present in the boundary regions of the enamel but not to the true enamel. Hydroxyproline, presumably a good indicator of collagen contamination in proteins not containing this imino-acid, has been found present in amounts which could account for somewhat less than one-half of the insoluble matrix as collagen, according to data from three laboratories (Hess, Lee and Neidig, 1953; Hess and Lee, 1954; Rodriguez and Hess, 1963; Stack, 1953a; Battistone and Burnett, 1956; see also Piez and Likins, 1960). A hydroxyproline level intermediate between these values and the high value (100 residues per 1000) for collagen was noted by Rodriguez and Hess (1963). Glycine and alanine contents were comparable with those of collagen in all the above studies, except that of Stack, who reported a remarkably low value. Lofthouse (1961) found the same level of alanine as Stack recorded, but the glycine level was between those given by Stack and the remaining authors. Hydroxyproline accounted for between 40 and 50 residues per 1000 in these studies, and a similar

S T A CK

value was found by Lofthouse (1961) in a study where the content was found to be 4 times less in the water-soluble and EDTA-soluble protein. Similar results were obtained by Glimcher et al. (1964a) when they prepared mature bovine enamel by hand microdissection of transverse sections. They selected the outer one-quarte r to one-third of the labial enamel, so that contamination by dentinal protein should have been avoided. However, they did not abrade the outer surface, which was cleaned by mechanica l means. Their finding of a collagenous protein in insoluble fractions from these enamel samples led to their surface study in which they located a mineralized collagenous layer (Glimcher et al, 1964b) which was continuous wit h the cementum covering the roots of the teeth. I n later work, Glimcher et al. (1964a) removed his layer by "chemical dissection" with E D TA before microdissection of the enamel. They continued to detect hydroxyproline until there was deeper penetration of the enamel by the demineralizing solution. Their observation of a surface collagenous layer on mature human enamel (Levine et al., 1964) confirms the observation of Rodriguez and Hess (1963), who used histochemica l means. The question whether hydroxyproline is a true component of enamel matrix has thus been an issue for a number of years. Burrows (1965) studied several types of enamel with this feature in mind. Inner and outer layers of the enamel were rejected and the EDTA-soluble and -insoluble fractions (pH 7.2-7.4) were dialyzed. Soluble fractions were subjected to gel filtration (Sephade x G-50), monitoring by measuring optical density at 280 m/x (dependen t on aromatic amino-acids). A major and two minor peaks were distinguished, the first two being separable equivalently by disc electrophoresis . Hydroxyproline was detectable only in the hydrolyzate of the first peak. This imino-acid was detected in all samples where sufficient material was available for a test to be attempted. Glimcher et al. (1964a) separate d imino-acids from mature bovine enamel on Dowex 50 (X-12) on the microgram scale. They deduced from their

20. C H E M I C A L

ORGANIZATION

analyses that cémentai collagen (Glimcher et al., 1964b) penetrate d the surface of mature enamel relatively deeply. N o more than traces were found in all other samples. They were able, in immature enamel fractions, to distinguish between 3- and 4-hydroxyproline, the ratio being about ten times as high as in the cémentai collagen and 3-4 times as high as in dentine collagen. The proportions in relation to 1000 amino acid residues were 0.3 for the 3-OH and 4.0 for the 4-OH isomer in the last neutral-soluble extract. Corresponding values for the acid-soluble fraction were 0.4 and 5.5 residues of the hydroxyprolines per 1000 total residues. Surface-abrade d enamel purified by flotation had been found by Stack (1954a) to yield 0.13-0.22% acid-insoluble protein, the upper value correspond ing to that reported by Losee and Hess (1949) for their e n a m e l " p r o p e"r (0.21-0.22 % , T a b le 3) from which cuticle had been removed. These authors, together with Hess et al. (1953), found nitrogen content to be 13.6%. Stack gave a slightly lower value (13.3 % ), while Block, Horwitt and Boiling (1949) found only 12 % in purified material prepared by demineralization with 5 % nitric acid. The latter authors showed this acid-insoluble protein to have the histidine : lysine : arginine ratios of the order of the 1 : 4 : 12 ratio found for eukeratins. Battistone and Burnett (1956) stated that only EDTA-insoluble protein contained the basic amino-acids in these ratios. They warned that flotation of particulate enamel appeare d to reduce the glutamic and aspartic acid levels as well as that of an unresolved trio (phenylalanine , leucine and /^-leucine were not separate d in their analyses) . Storage appeare d to have littl e effect on enamel protein composition. Perdok and Gustafson (1961) pointed out that the chain-repea t distances of the insoluble enamel protein as deduced from X-ray diffraction are related to the lattice parameter of hydroxyapatite (6.9 A). They proposed that the protein be termed a delta-keratin as the parameters could not be fitted to those characteristic of alpha- and betakeratin.

OF

ENAMEL

MATRI X

3. Enzyme Susceptibility Enamel Proteins

of

337

Acid-Insoluble

On the assumption that acid-insoluble protein isolated from enamel would be contaminate d with collagen, Stack (1954a) took steps to reduce this by treatment with several enzymes as well as with boiling water. The nitrogen : hydroxyproline ratio of collagen is as low as 1.2 : 1, and the change in this ratio was considered to be of value as a guide to the proportion of collagenous protein in samples analyzed, assuming that the protein which was more characteristic of enamel would be low in hydroxyproline. A s isolated, the acid-insoluble protein was found to contain nitrogen and hydroxyproline in the ratio 2 . 2 : 1, but this hardly differed after chymotrypsin treatment, although values of 2.73.8 : 1 were noted after incubation with pepsin, trypsin, or a purified collagenase . However, at least three-quarter s of the protein was brought into solution by these means. This recalls the pepsin and chymotrypsin treatments to which Block et al. (1949) submitted acid-insoluble protein, when two-thirds of the sample was rendered soluble. Although not markedly susceptible to protease s other than clostridial collagenase (see Chapter 19) any collagen present would probably have become swollen during demineralization, if not during pepsin treatment; in any case relatively large amounts of enzyme were employed in all these digestions. Such amounts are known to produce definite digestion, although this usually comes to a halt in a relatively short time. A s Eastoe showed in Chapter 19, the collagen of carious dentine is initiall y hydrolyzed by collagenase but a resistant residue can be demonstrated. Similar resistance can be brought about by reacting normal dentine collagen with aldehydic degradation products of carbohydrates ; these reaction products are usually brown. Acid-insoluble protein from mature enamel is usually fawn or brown, and it may be assume d that the collagenas e resistance noted was due to reaction of carbohydrate components particularly with the side chains of the basic amino-acid units.

M.

338

V.

I t seems likely that the partial resistance of this material to boiling water was also due to degradative changes . It was possible, by this means, to raise the nitrogen : hydroxyproline ratio of the residue to 6.4 : 1. In case the reactions for hydroxyproline were not sufficiently specific, these treatments were repeated with fingernail and hair samples, which showed hydroxyproline-reactin g material to be 11 and 14 times less abundant, respectively. H.

LIPID S

Hess, Lee and Peckham (1956) reported the lipi d content of a pooled sample of enamel at no less than 0.6 %—equal in amount to all other organic components combined. They determined 0.7 mg cholesterol in a salt-carbonat e extract of enamel, and much less in a petroleum ether extract of the residue. The total was equivalent to only 0.008 %. Fatty acids in the enamel lipi d fraction were found to be less saturated than those in bone lipids, and their (aliphatic) chains longer. I . CARBOHYDRATES

Bélanger (1955) demonstrate d the incorporation of radiosulphate into rodent enamel matrix during maturation at a time when the tissue showed metachromasia . The positive periodic acid-Schiff reaction evident in rodent enamel during maturation has been shown by Fullmer and Alpher (1958) to become markedly weaker during maturation of comparable regions of enamel from human teeth. These observations suggest that sulphated and neutral mucopolysaccharide s are present in enamel during growth. Frank, Sognnae s and Kern (1960) have shown histochemica l staining of material suggesting a strongly acid mucopolysaccharid e at intermediate stages of mineralization, but the reaction disappear s as the full mineral composition is attained. However, some mucopolysaccharid e appears to persist in the mature matrix, since this reacts with methylene blue at pH values below 3. Coolidge

(1951) reported

no

significant

dif-

S T A CK

ferences in hexose analyses when comparing sound enamel from carious teeth with control material. Stack (1954a) indicated the proportions of total hexose, reacting in the anthrone test, associate d wit h the acid-soluble and acid-insoluble protein fractions (3 : 1). Total hexose was equivalent to 2 0 m g% "glucose" (Stack, 1956), but the major component present in enamel was thought to be glycogen because of the resistance of the carbohydrate-reactin g material to hot alkali, as noted by Egyedi and Stack (1956). Two-thirds of the total hexose was associate d with the outer surface of the enamel (see also Speirs, 1959). Stack et al. (1956) analyzed acid- and alkaliseparate d fractions for total hexose and found that acid-soluble and -insoluble material were present to a similar extent, the latter being again divided into roughly equal fractions, as regards hexose level, by heating for 90 minutes at 100°C with 40 % K O H . This treatment destroys monosaccharide s but is without effect on glycogen, for example. The increased carbohydrate level near the surface was supposed by Stack (1957) to account for the considerably greater level found in total rat enamel as compared with human and monkey enamel. Burgess, Nikiforuk and Maclaren (1960) separated acid-soluble and acid-insoluble nondiffusible fractions during demineralization of purified human and bovine enamel. These were hydrolyzed by an acid-resin system, giving a filtrate containing only neutral sugars and uronic acids; amino-acids and hexosamine s remained adsorbed . More carbohydrate was found in the acid-insoluble protein fraction than in the acid-soluble material, but the aldose sugar components were qualitatively similar. Paper chromatograph y accounted for 1.65 mg % as aldose sugars, all but 6 % of the total being aldohexose . Galactose , glucose and mannose were found in the ratio 3 : 2 : 1. Smaller amounts of aldopentoses—fucos e and xylose—were identified, possibly accompanie d by rhamnose. A hexuronic acid fraction was noted, but not identified unequivocally. Hexosamine s eluted from the resin were identified by Nikiforuk, Burgess and Maclaren (1959) as glucosamine and galactosamine , in

20. C H E M I C A L

ORGANIZATION

equivalent amounts and qualitatively similar in the acid-soluble and acid-insoluble fractions. Clark, Smith and Davidson (1965) have determined hexosamine s in acid glycosaminoglyca n fractions isolated from human teeth.

The most recent figures for these organic acids appear to be those of Leaver, Triffit t and Hartles (1963), determined in rat incisor enamel, the amounts being also equal (0.032 and 0.034 % ). Similarity in amount, but on a molar basis (citric acid, M.W. 210; lactic acid, M.W. 90), was evident throughout the thickness of human enamel (Brudevold, Steadma n and Smith, 1960): 3 /xmoles/gm (0.063 and 0.027%) at the enamel surface, 1 jLtmole/gm within the enamel and 2 ^moles/gm at the enamel-dentin e junction (see Chapter 18). The values for citrate are lower than those given by Zipki n and Piez (1950), who studied the range in citri c acid levels in total enamel samples, finding a mean of 0.1 ± 0.02 % independen t of age or tooth type (Table 8). Also lower were the values reported by Peckham, Losee and Hess (1956), who found 0.08 % citrate in intermediate and junction enamel but 0.1 % at the surface. Nikiforu k and Grainger (1965) noted that the citrate content of outer enamel from permanent teeth was lower in fluoride (0.039 %) than in non-fluoride areas (0.048 % ). Citrate is freely acquired by hydroxyapatite (Hartles, 1961) but its association in enamel with a peptide fraction, as recently shown in dentine (see Table 8 FREQUENCY DISTRIBUTION OF CITRATE LEVELS IN ENAMEL SAMPLES0

a

85 14

95

ENAMEL

MATRI X

339

Leaver et al, 1963), has not yet been demonstrated . Lactate does not seem to be taken up by hydroxyapatite in the same way as citrate at physiological pH values (Brudevold et al, 1960).

V . A L T E R ED E N A M EL

J. CITRATE AN D LACTAT E

Central value 65 75 (mg%) Number of 3 4 samples

OF

105

115

9

6

Data of Zipkin and Piez (1950).

125 8

135 6

145 4

2

Clinical observation of the penetration of enamel by the carious process indicates that the subsurface shows the first signs of attack. Points where there are physical or chemical variations in the surface, or imperfections in the cuticle, are thought to be the foci of attack (Darling, 1958; Hodson, 1950). Only the inorganic components appear to be susceptible up to the stage where the initial lesion has progresse d from the subsurface zone to one where the surface is affected over a considerable region. However, Darling (1963) has submitted evidence consistent with an early loss of acid-soluble matrix. Histological changes develop shortly before cavitation, which occurs with destruction of the organic matrix under the attack of micro-organisms (Hardwick andManley, 1952; Sharpenak , Nikolaeva and Magid, 1957). Various structural components of the enamel do not seem to react in the same way during the early stages of the process. The prism cortex, the outermost layer of the enamel and enamel near the striae of Retzius are relatively resistant compared wit h the striae themselves and the interprismatic substance . This recalls the preservation of prism sheaths but loss of prism filaments (Glimcher et al.9 1964d) as demineralized matrix from immature bovine enamel was extracted with neutral buffer (page 319). (See also Sognnaes , 1955, 1962.) Darling (1961) deduced from polarized light studies that spaces in normal enamel capable of imbibing the liquids used in microscopy could not account for more than one-thousandt h of the volume. Translucent zones, which he and others believe to correspond to the first stage of caries, were associate d with 1 % of spaces . A later stage ("dark zone") corresponde d with 2-4 % spaces , and the main body of the carious lesions was

M.

340

V.

thought to be able to imbibe at least 5 % of its volume, sometimes as much as 25-50 %. Darling's view is that soluble matrix is removed at the earliest stage from the intraprismatic region of enamel. Artificiall y altered enamel, prepared by exposure to Brain's demineralizing solution (5 % formic acid "saturated" with tricalcium phosphate ) also appeare d to lack intraprismatic material (see K . Little, 1962). Crystallites were displaced during the preparation of sections for electron microscopy, and were thought to have been washed out when soluble matrix was removed. Nitrogen analyses (Rowles and Little, 1955) showed that an organic fraction dissolved preferentially from powdered enamel subjected to the demineralizing reagent which Brain had found successfu l in histology; demineralization ensued over a longer period. K . Littl e (1962) has distinguished between enamel specimens from caries-prone and caries-free subjects. Only the latter showed intact matrix after treatment with Brain's solution, and fewer crystallites were displaced around sections prepared for electron microscopy. The crystallites were more completely separate d from the matrix in sections from teeth which had been interred for many centuries. Prism cores, walls and adjacent regions were sometimes affected in enamel from fluorotic teeth. Frank (1965) disagrees with Little's statemen t that the rod core matrix of caries-resistan t enamel is more acid-stable. His concept of the ultrastructure of the process of caries is of a scattered apatite crystal destruction, following a definite pattern, which results in a significant broadening of the Table 9 DEPTHS OF TYPES OF ALTERED ENAMEL"

Type of enamel (altered) Stained White-spot Brown-spot

Number of samples

Depth and S.D. (mm)

46 27 29

0.18 ± 0.12 0.35 ± 0.24 0.71 ± 0.36

° Data from M. F. Littl e et al. (1962b).

S T A CK

inter-crystallite spaces filled by organic material of amorphous appearance , rather than destruction of the rod core in caries-prone enamel. The superficial appearanc e of altered enamel varies among a collection of teeth in which there has been no loss of surface contour. Macroscopic blemishes are sometimes developmental , such as areas of hypomineralization or fluorotic mottling. A n early carious lesion is most usually seen as a white spot which later becomes brown, and a diffusely stained area is often to be seen. The organic content of another type of altered enamel, on the inner surface adjacent to grossly carious dentine in an advanced lesion, has been shown to have ten times the organic content of sound enamel, whereas the altered enamel on the outer surface shows a threefold increase (Stack, 1954c). Certain types of altered enamel differ significantly in degree of penetration of enamel, according to M . F. Little, Posen and Singer (1962b), as shown in Table 9 based on a footnote to their study of fluoride and sodium levels. A n extensive study by M . F. Little, Cueto and Rowley (1962a) deals with comparative values for ash, Ca, P, C 0 2 , water, radiolucency and density Table 10 SPECIFIC GRAVITY ( S. G .) AND NITROGEN CONTENT OF NORMAL

AND

ALTERED

ENAMEL

SAMPLES"

Dissected samples Ground samples Age group

Í S.G. (mg/cm3)

Stained Control White-spot

> 30 > 30 < 30

2.23 2.91 1.90

Control

< 30

2.86

Brown-spot

< 30 > 30 < 30 > 30

2.44 2.49 2.82 2.71

Type of enamel

Control

a

2.88 1.31 3.04

30 > 30 1.46 < 30 > 30 2.15 1.94 1.41 1.84

Data from M. F. Littl e et al. (1962a).

Í S.G. (mg/cm3: I A4 2.90 1.65 1.81 3.10 2.85 1.80 1.50 3.10 3.08

4.35 1.28 3.78 3.51 0.96 1.51 3.62 4.18 0.94 0.95

20. C H E M I C A L

ORGANIZATION

in samples of sound, stained, white-spot and brown-spot enamel (Table 10). Some of the altered enamel contained only one-half as much mineral as sound enamel, most of the deficiency being occupied by "loosely b o u n d" water. Calculated protein content was inversely proportional to density, even in normal enamel, whereas there was no obvious relationship between density and loss on incineration. These authors exposed an interesting inverse relationship between Ca : Ñ ratio and weight loss not ascribed to C 0 2 or protein on heating to 900°C. The curve was noted to correspond to one showing substitution of "tightly b o u n d" water for calcium i n synthetic hydroxyapatite lattices. They point out the implication of hydrogen bonding as the common denominator in the relationship between nitrogenous components and "tightly b o u n d" water, and between "tightly b o u n d" water and mineral. Diffuse staining in teeth had already been shown to be associate d with a reduction in refractive index (M . F. Littl e and Cueto, 1959). Further study indicated that the discoloration was accompanie d by subsurface demineralization (density decrease , nitrogen increase, loss of C 02) . The C 0 2 content of sound enamel from the same teeth decrease d towards the surface, as had been found for enamel from erupted teeth (M . F. Littl e and Brudevold, 1958). They suggeste d that there was a similar unstained but invisible zone under the surface of the enamel in younger teeth. The inverse relationship between nitrogen content and specific gravity (2.1 : 1) was shown by M . F. Little , Cueto and Rowley (1962a) on the basis of their own results combined with those of Bhussry (1958b). This enabled them to derive values on a volumetric basis for inorganic, "total organic" (ash loss less C 02) and " p r o t e i n" which showed that volume decreas e per unit density was equivalent to absolute volume increase in "loosely b o u n d" water. Brown-spot and white-spot enamel, substantially free of supporting radiopaque enamel, were found to have densities of 1.3-1.8 and lost 30-25 % "loosely b o u n d" water on drying.

OF

ENAMEL

341

MATRI X Table 11

SPECIFIC GRAVITY AND NITROGEN CONTENT OF ALTERED ENAMEL IN RELATION TO AGE01

Type of enamel Hypocalcified White-spot Fluorosed Brown-spot Control

a

Age group Specific gravity —

Nitrogen (mg/cm:



2.62 2.44 2.88

1.94 1.97 2.17

< 30 > 30 < 30 > 30

2.50 2.39 2.82 2.77

2.00 3.66 1.45 1.93



Data from Bhussry (1958a,b).

Increased nitrogen and decrease d specific gravity were noted by Bhussry (1958a,b,c) in zones of enamel characterize d by brown and white spots. These are shown in Table 11 on a volume basis in relation to similar data on sound enamel. These effects were tentatively ascribed to diffusion of extraneous material into the outer third of the enamel. Earlier workers had suggeste d a deficiency of interprismatic substance . The histological structures seen in opaque white enamel of the early carious white-spot lesions and developmentall y defective zones were similar, but the latter lacked the "zone of consolidation" (see also Bhussry, 1959). Jenkins (1961) draws attention to the possibility, evident from the analyses of Hess and Lee (1954), that the various proteins of enamel might be present in different proportions in carious and non-carious teeth. Recent analyses by Sawant (1965) reveal significant variations between amino-acid levels in enamel from genetically resistant and susceptible rats (see also Jacobs, Coolidge and Besic, 1958). VI . C O N C L U D I NG R E M A R KS Interest is likely to focus during the next few years on the high molecular-weight proteins of the immature matrix. The elucidation of their transformation into the very much less complex protein

342

M.

V.

or peptide units characterizin g the m a t u re matrix is of major interest. Is there a depolymerizatio n brought about by thixotropic effects during the completion of mineralization, as Eastoe suggests ? D o these complex proteins have built-in mechanisms for their simplification? Does the hydroxyapatite have a preferential affinity for one type of subunit, thus leading to a directed breakdown of the molecular complex ? Ar e previously undisclose d enzymes implicated ? The filling by hydroxyapatite of space vacated by easily diffusible depolymerize d protein is a process which may be amenable to demonstratio n by physico-chemica l technology. Such spaces , unlikely to extend beyond small fractions of a micron, are already demonstrabl e by microscopy, and their "molecular sieve" properties are revealed also by vapor uptake "porosimetry". The newer continuous-flow techniques for microcalorimetry and sorptometry may be found suitable for revealing changes in the internal structural pattern as mineralization is completed. Here there may be a need to apply what may be described as preparativ e gradient density separation . Whatever the newer techniques employed, the greatest advance may be expected from those workers who are willin g to utilize structural and chemical methods together (e.g. Hohling, 1965).

References Anderson, D. J. (1949). Nitrogen in human dental enamel. Biochem. J. 45, 31. Anderson, D. J. (1951). Persona l communication cited by Stack and Williams (1952). Battistone, G. C. and Burnett, G. W. (1956). Studies of the composition of teeth. V. Variations in the amino-acid composition of dentine and enamel. / . dent. Res. 35, 263-272. Bélanger, L. F. (1955). Autoradiographic determination of radiosulphate incorporation by the growing enamel of rats and hamsters . J. dent. Res. 34, 20-27. Bergman, G. (1963). Microscopic demonstratio n of liquid flow through human dental enamel. Arch, oral Biol. 8, 233-234. Bevelander, G. and Johnson, P. L. (1955). The level of

S T A CK polysaccharid e in developing teeth. / . dent. Res. 34, 123-131. Bhussry, B. R. (1958a). Specific gravity and nitrogen content of enamel from different surfaces. / . dent. Res. 37, 832-836. Bhussry, B. R. (1958b). Specific gravity, nitrogen content, and hardness rating of discolored enamel. / . dent. Res. 37, 1045-1053. Bhussry, B. R. (1958c). Specific gravity, nitrogen content, and histologic characteristic s of opaque white enamel. /. dent. Res. 37, 1054-1059. Bhussry, B. R. (1959). Density and nitrogen content of mottled enamel. / . dent. Res. 38, 369-373. Bhussry, B. R. and Hess, W. C. (1963). Aging of enamel and dentin. / . Geront. 18, 343-344. Block, R. J., Horwitt, M. K. and Boiling, D. (1949). Comparative protein chemistry: The composition of the protein of human teeth and fish scales. / . dent. Res. 28, 518-524. Bonar, L. C. (1965). In "Tooth Enamel, Its Composition, Properties, and Fundamenta l Structure" (M. .V. Stack and R. W. Fearnhead , eds.). pp. 147-155. Wright, Bristol. Bose, A. K., Blackwell, R. Q. and Fosdick, L. S. (1960). Fractionation of human enamel on the basis of density. /. dent. Res. 39, 141-149. Brudevold, F., Steadman , L. T. and Smith, F. A. (1960). Inorganic and organic components of tooth structure. Ann. N.Y. Acad. Sci. 85, 110-132. Burgess, R. C. (1963). Proteins in developing bovine enamel. Preprint. Abstr. int. Ass. dent. Res., 41st gen. Meet., Pittsburgh, 1963 No. 179. Burgess, R. C. and Maclaren, C. (1965). Proteins in developing bovine enamel. In "Tooth Enamel, Its Composition, Properties, and Fundamenta l Structure" (M. V. Stack and R. W. Fearnhead , eds.), pp. 74-82 and 109-110. Wright, Bristol. Burgess, R. C , Nikiforuk, G. and Maclaren, C. (1960). Chromatographi c studies on carbohydrate components in enamel. Arch, oral Biol. 3, 8-14. Burnett, G. W. and Zenewitz, J. A. (1957). The composition of Syrian hamster enamel and dentine extracted with KOH-ethylene glycol. / . dent. Res. 36, 684-689. Burnett, G. W. and Zenewitz, J. A. (1958a). The moisture content of calcified tooth tissues. / . dent. Res. 37, 581-590. Burnett, G. W. and Zenewitz, J. A. (1958b). The composition of human teeth. / . dent. Res. 37, 590-600. Burrows, L. R. (1965). An investigation of proteins of human enamel matrix with special reference to the imino-acid hydroxyproline. In "Tooth Enamel, Its Composition, Properties, and Fundamenta l Structure" (M. V. Stack , eds.), pp. 59-62. 99-101. Wright, and R. W. Fearnhead Bristol. Carlstrom, D., Glas, J.-E. and Angmar, B. (1963). Studies on the ultrastructure of enamel. V. The state of water in human enamel. / . Ultrastruct. Res. 8, 24-29. Clark, R. D., Smith, J. D., Jr. and Davidson, E. A. (1965).

20. C H E M I C A L

ORGANIZATION

Hexosamine and acid glycosaminoglycan s in human teeth. Biochim. biophys. Acta 101, 267-272. Coolidge, T. B. (1951). Carbohydrate of carious enamel. J. dent. Res. 30, 480 (Abstract). Darling, A. I. (1958). Study of the early lesion of enamel caries. Its nature, mode of spread, and points of entry. Brit. dent. J. 105, 119-135. Darling, A. I. (1961). The selective attack of caries on the dental enamel. Ann. R. Coll. Surg. Engl. 29, 354-369. Darling, A. I. (1963). Resistanc e of the enamel to dental caries. / . dent. Res. 42, 488-496. Darling, A. I., Mortimer, Ê. V., Poole, D. F. G. and Ollis, W. D. (1961). Molecular sieve behaviour of normal and carious human dental enamel. Arch, oral Biol. 5, 251-273. Eastoe, J. E. (1960). Organic matrix of tooth enamel. Nature, Lond. 187, 411-412. Eastoe, J. E. (1962). Characteristic s of proteins from the matrix of developing human enamel. / . dent. Res. 41, 1258 (Abstract). Eastoe, J. E. (1963a). The amino acid composition of proteins from the oral tissues. II . The matrix proteins in dentine and enamel from developing human deciduous teeth. Arch, oral Biol. 8, 633-652. Eastoe, J. E. (1963b). Recent studies on the organic matrices of bone and teeth. In "Bone and Tooth" (H. J. J. Blackwood, ed.), pp. 269-281. Pergamon Press, Oxford. Eastoe, J. E. (1965a). The chemical composition of bone and tooth. Proc. 11th Ο RCA Congr. dent. Caries, Sandefjord, Norway, 1964 pp. 5-16. Pergamon Press, Oxford. Eastoe, J. E. (1965b). In "Tooth Enamel, Its Composition, Properties, and Fundamenta l Structure" (M. V. Stack and R. W. Fearnhead , eds.), pp. 91-98. Wright, Bristol. Egyedi, H. and Stack, M. V. (1956). The carbohydrate content of enamel. TV. Y. State dent. J. 22, 486. Emerson, W. H. (1960). Relation of thermolabile components to optical properties of dental enamel. / . dent. Res. 39, 864. Fearnhead , R. W. (1963). Recent observations on the structure of developing enamel. Arch, oral Biol. Suppl. 257-264. Fearnhead , R. W. (1965). The insoluble organic component of human enamel. In "Tooth Enamel, Its Composition, Properties, and Fundamenta l Structure" (M. V. Stack and R. W. Fearnhead , eds.), pp. 127-131. Wright, Bristol. Fincham, A. G., Graham, G. N. and Pautard, F. G. E. (1965). The matrix of enamel and related calcified keratins. In "Tooth Enamel, Its Composition, Properties, and Fundamenta l Structure" (M. V. Stack and R. W. Fearnhead, eds.), pp. 117-121 and 136-137. Wright, Bristol. Frank, R. M. (1965). Ciba Fdn. Symp., Caries-Resistant Teeth pp. 180 and 227 . Frank, R. M., Sognnaes , R. F. and Kern, R. (1960). Calcification of dental tissues with special reference to enamel ultrastructure. In "Calcification in Biological Systems", Publ. No. 64, pp. 163-202. Amer. Ass. Advanc. Sci., Washington, D. C.

OF

ENAMEL

MATRI X

343

Fullmer, H. M. and Alpher, N. (1958). Histochemical polysaccharid e reactions in human developing teeth. Lab. Invest. 7, 163-170. Gasparini, F. and Gotte, P. (1961). Chimia dello matrice organice dello smalto. Biochim. Biol. sper. 1, 119-125. Geller, J. H. (1958). Metabolic significance of collagen in tooth structure. / . dent. Res. 37, 276-279. Gilda, J. E. (1951). Studies on the physical properties of rodent enamel. II . Density distribution and X-ray diffraction pattern. / . dent. Res. 30, 828-836. Glimcher, M. J. and Krane, S. M. (1964). The identification of serine phosphate in enamel proteins. Biochim. biophys. Acta 90, 477-483. Glimcher, M. J., Mechanic, G. L., Bonar, L. C. and Daniel, E. J. (1961a). Amino acid composition of bovine fetal enamel matrix. / . biol. Chem. 237, 3210-3213. Glimcher, M. J., Bonar, L. C. and Daniel, E. J. (1961b). Molecular structure of the protein matrix of bovine dental enamel. / . mol. Biol. 3, 541-546. Glimcher, M. J., Friberg, U. A. and Levine, P. T. (1964a). The isolation and amino acid composition of the enamel proteins of erupted bovine teeth. Biochem. J. 93, 202-210. Glimcher, M. J., Friberg, U. A. and Levine, P. T. (1964b). Identification and characterizatio n of a calcified layer of coronal cementum in erupted bovine teeth. / . Ultrastruct. Res. 10, 76-86. Glimcher, M. J., Mechanic, G. L. and Friberg, U. A. (1964c). The amino acid composition of the organic matrix and the neutral soluble and acid soluble components of embryonic bovine enamel. Biochem. J. 93, 198— 202. Glimcher, M. J., Travis, D. F., Friberg, U. A. and Mechanic, G. L. (1964d). The electron microscopic localization of the neutral soluble proteins of developing bovine enamel. /. Ultrastruct. Res. 10, 362-376. Glimcher, M. J., François, C, Richards, L. and Krane, S. M. (1965). The presence of organic phosphorus in collagens and gelatins. Biochim. biophys. Acta 93, 585-602. Graham, G. N. and Pautard, F. G. E. (1963). Mature enamel matrix. / . dent. Res. 42, 1100 (Abstract). Greulich, R. C. (1963). Autoradiographic observations on proline-Ç3 in dentin and enamel matrices. Preprint. Abstr. int. Ass. dent. Res., 41st. gen. Meet., Pittsburgh 1963 No. 298. Greulich, R. C. and Slavkin, H. C. (1965). Amino acid utilization in the synthesis of enamel and dentin matrices as visualized by autoradiography . Symp. int. Soc. Cell Biol. 4, 199-214. Hardwick, J. L. and Manley, Å. B. (1952). Caries of the enamel. II . Acidogenic caries. Brit. dent. J. 92, 225-236. Hartles, R. L. (1961). Role of citric acid in mineralized tissues. Brit. dent. J. I l l , 322-331. Hattyasy, D. and Hatos, G. (1953). Die Bestimmung des N-Gehaltes des Zahnschmelzes . Ôst. Z. Stomat. 50, 1-7.

344

M.

V.

Hess, W. C. and Lee, C. Y. (1954). The amino acid composition of proteins isolated from the healthy enamel and dentin of carious teeth. / . dent. Res. 33, 62-64. Hess, W. C , Lee, C. Y. and Neidig, B. A. (1953). The amino acid composition of enamel protein. / . dent. Res. 32, 585-587. Hess, W. C, Lee, C. Y. and Peckham, S. C. (1956). Lipide content of enamel and dentin. / . dent. Res. 35, 273275. Hodson, J. J. (1950). Study of some of the developmental , structural, and pathological aspects of tubular hypoplasia in human enamel. Brit. dent. J. 89, 6-13 and 34-38. Hôhling, H. J. (1965). Combined infra-red absorption, X-ray diffraction studies and amino-acid analysis of the organic enamel substanc e of unfixed, fully developed teeth. In "Tooth Enamel, Its Composition, Properties, and Fundamenta l Structure" (M. V. Stack and R. W. Fearnhead , eds.), pp. 122-126, 142-143. Wright, Bristol. Hohling, H. J., Frank, R. M. and Harndt, R. (1963). Rontgenographisch e Untersuchunge n an der organische n Matrix von foetalen und jugendlichen, menschliche n Schmelz. Dtsch. zahnàrztl. Ζ. 17, 77-84. Holmes, J. M. and Beebe, R. A. (1961). Adsorption studies on bone mineral—heats of adsorption of nitrogen and argon on bone mineral at —195°C. Advanc. Chem. Ser. 33, 291-300. Holmes, J. M., Davies, D. H., Meath, W. J. and Beebe, R. A. (1964). Gas adsorption and surface structure of bone mineral. Biochemistry 3, 2019-2024. Hutton, W. E. and Nuckolls, J. (1951). Frequency of ninhydrin staining in the enamel surfaces of completely unerupted undecalcified human third molars in relation to the problem of caries susceptibility. Oral Surg. 4, 1451-1456. Hwang, W. S. S., Tonna, Å. A. and Cronkite, Å. P. (1963). A n autoradiographi c study of the mouse incisor using tritiated histidine. Arch, oral Biol. 8, 377-385. Jacobs, M. H., Coolidge, T. B. and Besic, F. C. (1958). Changes in organic constituents of enamel in clinical and artificial caries. Brit. dent. J. 104, 275-280. Jenkins, G. N. (1961). The biochemistry of enamel in relation to caries resistance . Arch, oral Biol. 6, 305-314. Karpishka, L, Leblond, C. P. and Carneiro, J. (1959). Radioautograph y of uptake of labelled methionine by dentine and enamel matrix. Arch, oral Biol. 1, 23-28. Krane, S. M., Stone, M. J. and Glimcher, M. J. (1965). The presence of protein phosphokinas e in connective tissues and the phosphorylation of enamel proteins in vitro. Biochim. biophys. Acta 97, 11-81. Leaver, A. G., Triffitt , J. T. and Hartles, R. L. (1963). Relative levels of citric and lactic acids in certain mineralized tissues. Arch, oral Biol. 8, 23-26. Leicester, Ç. M. (1949). "Biochemistry of the Teeth". Mosby, St. Louis, Missouri.

S T A CK Leicester, Ç. M. (1953). The biochemistry of the teeth. Ann. Rev. Biochem. 22, 341-351. Levine, P. T. and Glimcher, M. J. (1965). The isolation and amino acid composition of the organic matrix and neutral soluble proteins of developing rodent enamel. Arch, oral Biol. 10, 753-756. Levine, P. T., Glimcher, M. J. and Bonar, L. C. (1964). Collagenous layer covering crown enamel of unerupted permanent human teeth. Science 146, 1676-1677. Little, K. (1958). Electron microscope studies on human dental enamel. / . R. micr. Soc. 78, 58-67. Little, K. (1962). The matrix in caries-resistan t teeth. / . R. micr. Soc. 80, 199-208. Little , M. F. and Brudevold, F. (1958). A study of the inorganic carbon dioxide in intact human enamel. I. /. dent. Res. 37, 991-1000. Little, M. F. and Cueto, E. S. (1959). Some chemical and physical properties of "altered" enamel. / . dent. Res. 38. 674-675 (Abstract). Little, M. F., Cueto, E. S. and Rowley, J. (1962a). Chemical and physical properties of altered and sound enamel. I. (Ash, calcium, phosphorus , carbon dioxide, nitrogen, water, radiolucency, and density.) Arch, oral Biol. 7, 173-184. Little , M. F., Posen, J. and Singer, L. (1962b). Chemical and physical properties of altered and sound enamel. III . Fluoride and sodium content. J. dent. Res. 41, 784789. Lofthouse, R. W. (1961). M.Sc. Thesis, University of Leeds. Losee, F. L. and Hess, W. C. (1949). The chemical nature of the proteins of human enamel. J.dent. Res. 28, 512517. Nikiforuk , G. and Grainger, R. M. (1965). Fluoride-carbonate-citrate interrelations in enamel. In "Tooth Enamel, Its Composition, Properties, and Fundamenta l Structure" (M . V. Stack, and R. W. Fearnhead , eds.), pp. 26-31. Wright, Bristol. Nikiforuk , G., Burgess, R. C. and Maclaren, C. (1959). Amino sugars in dentine and enamel. / . dent. Res. 38, 675 (Abstract). Pautard, F. G. E. (1961a). Diffraction studies of enamel and baleen. / . dent. Res. 40, 1285-1286 (Abstract). Pautard, F. G. E. (1961b). X-ray diffraction pattern from human enamel matrix. Arch, oral Biol. 3, 217-220. Pautard, F. G. E. (1963). Mineralization of keratin and its comparison with the enamel matrix. Nature, Lond. 199, 531-535. Pautard, F. G. E. (1965). In "Tooth Enamel, Its Composition, Properties, and Fundamenta l Structure" (M. V. Stack and R. W. Fearnhead , eds.), p. 155. Wright, Bristol. Peckham, S. C, Losee, F. L. and Hess, W. C. (1956). Serial determination of the carbonate , citrate, phosphate , calcium, and nitrogen content of enamel. Abstr. int. Ass. dent. Res., 34th gen. Meet., St. Louis, 1956 No. 83.

20. C H E M I C A L

ORGANIZATION

Perdok, W. G. and Gustafson, G. (1961). X-ray diffraction of the insoluble protein in mature human enamel. Arch, oral Biol. 4, 70-75. Piez, K. A. (1961). Amino acid composition of soluble proteins. Science 134, 841-842. Piez, K. A. (1962). Chemistry of the protein matrix of enamel. In "Fundamental s of Keratinization—Summaries " Publ. No. 70, pp. 173-184. Amer. Ass. Advanc. Sci., Washington, D. C. Piez, K. A. and Likins, R. C. (1960). The nature of collagen. II . Vertebrate collagens. In "Calcification in Biological Systems", Publ. No. 64, pp. 411-420. Amer, Ass. Advanc. Sci., Washington, D. C. Pilz, W. (1959a). Beitrg e zur Biomorphose der Zahnartgewebe. I. Uber Alternsverânderunge n organische r Schmelz- und Dentinanteile w hren d der Funktionsperiode. Dtsch. Zahn-, Mund-, u. Kieferheilk. 30, 381— 392. Pilz, W. (1959b). Zur Analyse physiologische r Wandlungen der Zusammensetzun g organische r Zahnhartgewebestand teile. Dtsch. Zahn-, Mund-, u. Kieferheilk. 30, 393-404. Pincus, P. (1948). Further tests on human enamel protein. Biochem. J. 42, 219-221. Poole, D. F. G. and Stack, M . V. (1965). The structure and physical properties of enamel. In "Tooth Enamel, Its Composition, Properties, and Fundamenta l Structure" (M . V. Stack and R. W. Fearnhead , eds.), pp. 172-176 and 205-208. Wright, Bristol. Poole, D. F. G., Tailby, P. W. and Berry, D. C. (1963). The movement of water and other molecules through human enamel. Arch, oral Biol. 8, 771-772. Reith, E. J. (1965). In "Tooth Enamel, Its Composition, Properties, and Fundamenta l Structure" (M. V. Stack and R. W. Fearnhead , eds.), p. 108. Wright, Bristol. Rodriguez, M. S. and Hess, W. C. (1962). The chemical nature of human enamel protein. Abstr. int. Ass. dent. Res., 40th gen. Meet. St. Louis, 1962 No. 57. Rodriguez, M. S. and Hess, W. C. (1963). The nature of mature human enamel protein. Abstr. int. Ass. dent. Res., 41st gen. Meet. Pittsburgh, 1963 No. 64. Ronnholm, E. (1962a). The amelogenesi s of human teeth as revealed by electron microscopy: The developmen t of the enamel crystallites. / . Ultrastruct. Res. 6, 249-303. Ronnholm, E. (1962b). The structure of the organic stroma of human enamel during amelogenesis . / . Ultrastruct. Res. 6, 368-389. Rowles, S. L. and Little, K. (1955). Some observations on histological technics. / . dent. Res. 34, 778 (Abstract). Savory, A. and Brudevold, F. (1959). Distribution of nitrogen in human enamel. / . dent. Res. 38, 436-442. Schule, H. (1961). Chemische Zusammensetzun g und physikalische Eigenschafte n des Schmelzoberhâutchens . Arch, oral Biol. 4, 40-49. Sawant, A. (1965). Amino acids characterizatio n of dental

OF

ENAMEL

MATRI X

345

enamel of genetically caries-resistan t and caries-susceptibl e rats. / . dent. Res. 44, 869-872. Shapiro, I. M . and Hartles, R. L. (1965). A n improved method with low material loss for the separation of teeth into fractions of differing density. Arch, oral Biol. 10, 155-159. Sharpenak , A. E., Nikolaeva, Í . V. and Magid, E. A. (1957). Osobennost i khimicheskogo sostava emali í oblasti belego karioznogo pyatna, po dannum gistokhimicheskog o issledovaniya [Histochemical study of enamel composition in the region of white carious spots]. Stomatologiia, Moskva 35, No. 2, 8-10. Sognnaes , R. F. (1955). Microstructure and histochemica l characteristic s of the mineralized tissues. Ann. N.Y. Acad. Sci. 60, 572-595. Sognnaes , R. F. (1962). Microstructure and histochemistry of caries. In "Chemistry and Prevention of Dental Caries" (R. F. Sognnaes , ed.), pp. 3.-31. Thomas, Springfield, Illinois. Sognnaes , R. F. (1965). Ciba Fdn. Symp., Caries-Resistant Teeth p. 147. Speirs, R. L. (1959). The nature of surface enamel. Brit. dent. J. 107, 209-217. Stack, M . V. (1953a). Hydroxyproline in enamel keratin. /. Bone Jt. Surg. 35B, 267 (Abstract). Stack, M. V. (1953b). Variation in the organic content of deciduous enamel and dentine. Biochem. J. 54, xv (Abstract). Stack, M. V. (1954a). Organic constituents of enamel. J. Amer. dent. Ass. 48, 297-306. Stack, M. V. (1954b). La variabilité de la teneur en matière organique de l'os, de la dentine, et de rémail. Biotypologie 15, 23-32. Stack, M. V. (1954c). The organic content of 'chalky' enamel. Brit. dent. J. 96, 73-76. Stack, M . V. (1955a). The chemical nature of the organic matrix of bone, dentine, and enamel. Ann. N.Y. Acad. Sci. 60, 585-595. Stack, M. V. (1955b). Protein and peptide components of human enamel at birth. J. dent. Res. 34, 780 (Abstract). Stack, M. V. (1955c). Soluble protein and peptide fractions from human dental enamel. Abstr. 3rd Int. Congr. Biochem., Brussels, 1955 No. 2-14. Seer. Gén., Liège. Stack, M. V. (1956). The carbohydrate content of human dental enamel. / . dent. Res. 35, 966 (Abstract). Stack, M. V. (1957). Mineral and protein levels in enamel from human, monkey, and rat molars. Odont. Revy, Lund 8, 89-93. Stack, M. V. (1960). Changes in the organic matrix of enamel during growth. / . Bone Jt. Surg. 42B, 853 (Abstract). Stack, M. V. (1965). Differentiation of proteins from developing enamel by pyrolysis and gas chromatography . /. dent. Res. 44, 1175 (Abstract).

346

M.

V.

Stack, M. V. and Holloway, P. J. (1959). Carbohydrate levels in dental tissues of young rats on contrasted diets. /. dent. Res. 38, 1223 (Abstract). Stack, M. V. and Williams, G. (1952). Quantitative variation in the total organic matter of enamel. Brit. dent. J. 92, 261-267. Stack, M. V., van Daatselaar , J. J., de Vries, L. A. and Egyedi, H. (1956). A study of carbohydrate levels in British and Dutch enamel samples. Tijdschr. Tandheelk. 63, 833-841. Sullivan, H. R. (1953). Composition and structure of human dental enamel. Dent. J. Aust. 25, 83-95. Travis, D. F. and Glimcher, M. J. (1964). The structure and organization of and the relationship between the organic matrix and the inorganic crystals of embryonic bovine enamel. / . Cell Biol. 23, 447-497. Wachtel, L. W. (1959). Nitrogen content of zones distinguished by reflected light in enamel sections from unerupted third molars. / . dent. Res. 38, 3-8. Wachtel, L. W. (1964). Chemical and visual changes in

S T A CK tooth enamel caused by lactic acid and hydrofluoric acid. / . dent. Res. 43, 237-245. Weidmann, S. M. (1965). Enamel density measurement s using density gradients. / . dent. Res. 44, 1170 (Abstract). Weidmann, S. M. and Hamm, S. M. (1965). Studies on the enamel matrix of mature teeth. In "Tooth Enamel, Its Composition, Properties, and Fundamenta l Structure" (M . V. Stack and R. W. Fearnhead , eds.), pp. 83-90 and 111-113. Wright, Bristol. Weidmann, S. M. and Lofthouse, R. W. (1963). Studies on the enamel matrix. / . dent. Res. 42, 1101 (Abstract). Wynn, W., Haldi, J. and Law, M. L. (1962). Chemical composition of the molar teeth of albino rats fed two diets with different cariogenicities. / . dent. Res. 41, 4 145. Young, R. W. and Greulich, R. C. (1962). Utilization of glycine in rat enamel matrix. Abstr. int. Ass. dent. Res., 40th gen. Meet., St. Louis, 1962 No. 98. Zipkin, I. and Piez, K. A. (1950). The citric acid content of human teeth. dent. Res. 29, 498-505.

SECTION 5

The Organization of the Dental Supporting Tissues

This page intentionally left blank

CHAPTER

21

CONNECTIVE TISSUE COMPONENTS OF THE PERIODONTIUM H. M.

FULLMER

I . General Introduction

349

II . Development A . Introduction B. Periodontal Membrane Histogenesi s C. Cementum Histogenesi s D. Alveolar Bone Histogenesi s E. Tooth Eruption

349 349 350 352 354 354

III . Organization of the Definitive Supporting Tissues A . Introduction B. Collagen Fibres C. Reticular Fibres D. Acid Mucopolysaccharide s E. Oxytalan Fibres F. Elastic Fibres G. Connective Tissue Cells H. Mast Cells I . Epithelial Cell Rests J. Cementum K . Alveolar Bone

358 358 359 365 365 368 376 376 381 382 384 385

IV . Pathology of the Supporting Tissues

401

V. Concluding Remarks

402

Reference s

404

I. GENERAL INTRODUCTION

II. DEVELOPMENT

The supporting tissues of the teeth consist of the periodontal membrane, the cementum, the alveolar process and the gingiva, known collectively as the periodontium. This chapter concerns the connective tissue components of the periodontium leaving the gingiva, especially its epithelium, to be dealt wit h in Chapter 22.

A.

The alveolar process is the part of the jaw bones which forms the sockets of the teeth and develops, of course, as an integral part of the jaw bones. The alveolar processes , however, show a degree of specialization and are certainly functionally, and 349

24

INTRODUCTION

350

H.

M.

possibly genetically, closely associate d with the dentition because , in general, if the dentition fails to develop so does the alveolar process, and when the dentition is lost through disease or accident there is a marked tendency for the alveolar process to disappear . The periodontal membrane and the tissue by which it is attached to the tooth, namely the cementum, develop from the dental follicle. G o od descriptions of the processe s of development of the supporting tissues are available in textbooks (e.g. Sicher, 1962) and there seems no point in repeating them here. The text wil l be confined, therefore, largely to certain aspects which represen t new knowledge concerning these structures.

FULLMER

have indicated the close association and probable importance of fibroblastic cell membrane s to the development of collagen fibres intracellularly as well as extracellularly. After preliminary intracellular formation, the collagen precursor is secreted where it undergoes a type of extracellular maturation which results in a fibri l with a typical 640 Á-banded appearanc e under the electron microscope, a typical low angle diffraction pattern (Fig. 1) and staining qualities first of reticulin and

B. PERIODONTAL MEMBRAN E HISTOGENESIS

1. Collagen Fibre Formation The dominant fibres in the periodontal membrane are the collagen fibres. Fibrogenesis of the periodontal tissues has received scant study but data from some other sources are directly relevant. Collagen formation involves the synthesis of appropriate amino acids with subsequen t proper alignment and linkage by peptide bonds. Hydroxyproline is a constituent characteristic of the protein collagen, and in some manner is probably linked to an ascorbic acid requirement. Hydroxyproline i n the collagen molecule derives from proline rather than from free hydroxyproline (Stetten and Schoenheimer , 1944; Stetten, 1949) and hydroxy-, lysine comes from lysine (Sinex and Van Slyke, 1955, 1957; K ao and Boucek, 1958). Peterkofsky and Udenfriend (1961, 1963) have secured a cell free system (microsomal fraction) from chick embryo homogenate s capable of forming collagen. Some type of collagen molecule (tropocollagen ?) is believed to form intracellularly, although collagen fibres with their* characteristic extracellular appearance have not been observed intracellularly. Porter and Pappas (1959), Yardley et al. (1960), Karrer (1960), Chapman (1961, 1962) and Zelickson (1963)

Fig. 1. Low angle X-ray diffraction pattern of dried rat-tail tendon having a periodicity of 638.5 A. Variations in intensity of the lines is related to the electron density distribution of the atoms within the collagen molecule. (Courtesy of P. D. Frazier.)

later collagen. Maturation of characteristic fibril s extracellularly in the vicinity of other connective tissue cells tends to foster the assumption that connective tissue cells in any particular region control environmenta l conditions therein. 2. Mucopolysaccharide

Formation

A characteristic feature of growing connective tissues, both in the course of embryogenesi s and i n repair, is the production of abundant mucopolysaccharide s (Fig. 2) (Wislocki, Singer and Waldo, 1948; Wislocki and Sognnaes , 1950; Bevelander and Johnson, 1955; Engel, 1948, 1951; Holmgren,

21. C O N N E C T I VE

T I S S UE

C O M P O N E N TS

OF

P E R I O D O N T I UM

351

Fig. 3. Section through the upper jaw and first molar of a child aged 3 1/2 months. Arrow indicates region from which Figs. 2 and 4 were taken. Oxytalan fibres are situated in the connective tissues peripheral to the enamel organ and Hertwig's epithelial root sheath and in the oral mucosa over the erupting teeth. Peracetic acid-aldehyde fuchsin-Halmi stain. x8.6.

Fig. 2. Connective tissues over an erupting deciduous molar (area indicated by arrow in Fig. 3) from a child aged 3 1/2 months. Dark amorphous stained material (arrow) is mucopolysaccharide . The same substanc e exhibited metachromasia with azure A at pH 4. Broad bands between are collagen (c). Paracetic acid-aldehyde fuchsin-Halmi stain. x435.

1940; Sylvén, 1938, 1941, 1945; Stearns , 1940a,b; Bensley, 1934; Bunting, 1950; D u n p hy and Udupa, 1955). The hydroxyproline : hexosamin e ratio increases as connective tissues undergo progressive maturation (Dunphy and U d u p a, 1955). The precise function of the mucopolysaccharide s during the course of developmen t has not been delineated . 3. Oxytalan Fibre

Formation

When the formation of about half of the crown of the tooth is completed, developmen t of oxytalan

Fig. 4. Connective tissues over a developing tooth (area indicated by arrow in Fig. 3). Oxytalan fibres (dark) appear to arise from regions containing mucopolysaccharides , such as those indicated by the arrow in Fig. 2. c, collagen. Peracetic acid-aldehyde fuchsin-Halmi stain, ÷ 435. fibres can be discerned in the tooth germ (Figs. 3 and 4). Perhaps significantly, mucopolysac -

352

H.

M.

FULLMER

ment of the peracetic acid-orcein-Halmi stain (Fullmer, 1959a) for identification of the oxytalan fibres at this stage of developmen t is sometimes useful, because this method stains many oxytalan fibres but littl e of the investing mucopolysaccharide .

C.

CEMENTUM

HISTOGENESIS

Proliferation of Hertwig's epithelial root sheath apically, and the commencemen t of the formation of the dentine of the root is followed by the disengagemen t and fragmentation of sheath cells (Figs. 5-7) (Selvig, 1963a,b). Connective tissue cells in this region infiltrate between the fragmented sheath cells and differentiate into cementoblast s (Fig. 6). Collagen and oxytalan fibres in the vicinity appear to be incorporated into cementum simultaneously and in a similar manner (Fig. 5).

Fig. 5. Developing upper first deciduous molar from child aged 1 year. Note the incorporation of oxytalan (dark-staining fibres indicated by arrow) fibres into cementum simultaneous with collagen fibres. PM, periodontal membrane; P, pulp. Peracetic acid-aldehyde fuchsin-Halmi stain. X900.

charide and collagen formation precedes oxytalan fibre formation. Oxytalan fibres originate in the follicl e surrounding the developing teeth external to Hertwig's epithelial root sheath and the enamel organ (Figs. 3-5). They appear between bundles of collagen fibres in regions that display abundant mucopolysaccharide s (Fullmer, 1959b, 1963). They are identified by the peracetic acid-aldehyde fuchsin-Halmi stain (Fullmer and Lillie , 1958), which also stains the amorphous mucopolysac charides that frequently adhere to the fibres, making identification difficult at this stage. Employ-

Fig. 6. Electron micrograph of commencing cementum formation in a rat molar. Hertwig's sheath has fragmented and cementoblast s (Cmb) lie next to dentine (D). Fine fibrils which are probably collagen are visible between the cementoblasts . Potassium dichromate-osmiu m stain, ÷ 9000. (From Selvig, 1963a.)

21. C O N N E C T I V E

TISSUE

COMPONENTS

Fig. 7. Electron micrograph of later stage of cementum formation than Fig. 6. Cementum (Cm) is now present on dentine (D). Fibrils next to cementoblast s (Cmb) are also incorporated into new cementum. Potassium dichromateosmium stain, ÷ 9500. (From Selvig, 1964.)

Fig. 8. Apical end of an upper incisor from a young rat stained for TPN-diaphoras e and counterstaine d with carmalum for nuclei. Note intense staining (dark) of odontoblasts (o), and lesser activities in cementoblast s (Cmb), cells of the periodontal membrane (PM) and pulp (P). D, dentine, ÷ 243.

OF

PERIODONTIUM

353

Thus, collagen and oxytalan fibres are initiall y incorporated into cementum at the cervix; this is followed by incorporation into cementum progressively more distant from the cervix. Figures 8-10 depict TPN-diaphorase , succinic dehydrogenase and glucose-6-phosphat e dehydrogenas e activities of cementoblast s associate d with cementogenesis at the apical ends of the upper incisors of young rats. The lesser enzymic activities of cementoblast s as contrasted to odontoblasts are to be noted, a difference between these cell types which is frequently observed with a variety of enzymic systems. During cementogenesis , cementoblasts also manifest acid and alkaline phosphatases , nonspecific esterase , DPN-malic, isocitric, glutamic, lactic, ^-glycerophosphat e and D( —)-j3-hydroxy butyric dehydrogenase s and DPN-diaphorases . The dehydrogenase s mentioned are undoubtedly

Fig. 9. Apical end of an upper incisor from a young rat stained for succinic dehydrogenas e and counterstaine d with carmalum for nuclei. Note intense staining (dark) of odontoblasts (o), and lesser activities in cementoblast s (Cmb), cells of the periodontal membrane (PM) and pulp (P). D, dentine. X 2 5 5.

354

H.

M.

Fig. 10. Apical end of an upper incisor from a young e dehydrogenas e and rat stained for glucose 6-phosphat counterstaine d with carmalum for nuclei. Note intense staining (dark) in odontoblasts and lesser activities in cementoblast s (Cmb), cells of the periodontal membrane (PM) and pulp (P). D, dentine, ÷ 243.

associate d with the production of energy as well as with fatty acid metabolism and glycolysis.

D . ALVEOLA R BONE HISTOGENESIS

Alveolar bone grows in relation to developing teeth to form their bony crypts. During embryogenesis, cells adjacent to Hertwig's epithelial root sheath differentiate into cementoblasts , those slightly more distant differentiate into specialized cells which form periodontal membrane, and those at a greater distance differentiate into osteoblast s which produce alveolar bone (Fig. 11). The structure of alveolar bone is never static; it is constantly being modified, first in relation

FULLMER

Fig. 11. Electron micrograph of fibrous elements of the periodontal membrane of a molar from a young rat entering alveolar bone (AB). c, collagen fibres. Potassium dichromate-osmiu m tetroxide stain, ÷ approx. 33,600. (From Selvig, 1964.)

to growth and developmen t of the teeth and then, in the adult, in response to the ever changing demands of the dentition as well as in response to certain systemic circumstances , such as calcium needs.

E. T O O TH

ERUPTION

1. Introduction The nature of the forces or processe s that bring about the eruption of teeth remains unknown notwithstanding many investigations designed to reveal them. Constant (1902) and Massler and Schour (1941) attributed eruptive forces to blood pressure. Sicher (1942a,b) described a cushioned

21. CONNECTIVE TISSUE COMPONENTS OF PERIODONTIUM hammock ligament at the apices of developing teeth, of both those which erupt continuously d that cell and those which do not. He assume d proliferation in the pulp within the funnel-shape open ends of growing teeth provides the pressure which results in the eruption of the teeth. Scott (1953) largely confirmed Sicher's findings. However, Eccles (1959, 1961), mainly in the rat, and Main (1965), in a variety of mammalian species, failed t resembling a to find any structural arrangemen hammock in developing teeth and therefore discount Sicher's concept of tooth eruption. Likewise, a hammock ligament was not found in rabbits by Ness and Smale (1959) or in guinea-pig molars by Hunt (1959). It is interesting that eruption of rat incisors continues despite (1) mechanical occlusion of pulpal vessels which resulted in pulpal necrosis (Taylor and Butcher, 1951), (2) removal of pulps (Herzberg and Schour, 1941; Kostlân, Thofova and Skach, 1960), (3) administration of the hypotensive drugs guanethidine and hydralazine (Main and Adams 1966a,b,c), (4) administration of the nucleotoxic drugs demecolcine and triethylene melamine (Main and Adams 1966a,b,c) and (5) the irradiation of teeth of monkeys sufficient to curtail odontogenesi s (Gowgiel, 1961). The reader is referred to papers by Main (1965) and Main and Adams (1966a,b,c) for excellent discussions of the subject.

355

which the erupting teeth must pass undergoes dissolution. It seems that the process of proteolysis cond with cerned in this dissolution is not accomplishe the aid of inflammatory cells (Figs. 12, 13 and 14). A few lymphocytes can occasionally be seen in the centre of such degeneratin g regions, but not in the peripheral areas where the initial changes occur. Microscopic observations would suggest that the enzymic systems required to promote or permit dissolution of the regional connective tissues are already present within the connective tissue cells in the area. The pattern of degradation cannot l be explained simply on the basis of mechanica pressure exerted by the erupting teeth, because a gradient of effect extending from the tooth in its direction of movement is not observed. A search among the tissues surrounding erupting teeth for collagenase such as that found in cultures of gingiva excised for the treatment of periodontal disease s (Fullmer and Gibson, 1966) and Gibson and Fullmer (1966) should be undertaken.

2. Changes in the Supporting Tissues during Eruption d with resorption Eruption of teeth is associate of bone and degradation of connective tissues in the region later occupied by the teeth. Resorption of bone is effected by osteoclastic activity which continues throughout odontogenesis . Processe s effecting the degradation of fibrous connective tissues in advance of erupting teeth are more subtle and difficult to delineate. I n general, the connective tissue of the follicl e which is destined to contribute to formation of the periodontal membrane appears to be preserved intact. By contrast, that of the oral mucosa and regional bone through

Fig. 12. Mesio-distal section through two upper deciduous molars and erupting first permanen t molar in a child. Pale area (arrow) where connective tissues are undergoing dissolution in advance of the erupting tooth. Connective tissues immediately adjacent to the tooth are not undergoing change. Van Gieson stain, ÷ 7.7.

356

H.

M.

FULLMER

Fig. 14. Higher magnification of region of the arrow in Fig. 13. Connective tissue less dense. Haematoxylin and eosin. ÷ 160.

21. C O N N E C T I VE

T I S S UE

C O M P O N E N TS

Histochemically, in regions of degradation , collagen bundles undergo a loss of density and appear purplish-grey in peracetic acid-aldehyde fuchsin-Halmi stained sections, suggesting the presence of mucopolysaccharide s (Fullmer, 1960a, 1961). Further confirmation of the presence of mucopolysaccharide s in these regions is provided by the fact that they stain blue in this region with the Hale and Rinehart methods. They also stain intensely red with the periodic acid-Schiff method, indicating the presence of abundant carbohydrates . Engel (1948, 1951) has noted changes in the glycoproteins of the connective tissues in connection wit h eruption of the teeth. These regions have not yet been studied with the aid of enzymic histochemical or microchemical methods. A knowledge

OF

P E R I O D O N T I UM

357

of the factors associate d with the degradation and formation of connective tissues is sorely needed and could be provided, at least in part, by employment of these methods. Bernick (1960) and Trott (1962) have provided a detailed description of collagen fibre rearrange ment during the course of tooth eruption in rats as observed with the light microscope. Selvig (1964) includes a few electron microscopic observations of collagen fibre readjustment s associate d with eruption of teeth in rats, in connection with his study of cementum formation (Fig. 15). In accord wit h the observations of light microscopists , he noted a change in orientation of periodontal fibres following the eruption of molars in rats. Previous to eruption, the fibres were orientated

Fig. 15. Electron micrograph of periodontal membrane of rat molar at the time of eruption. Note the high degree of organization of the collagen fibres (c). Potassium dichromate-osmiu m tetroxide stain, χ approx. 8000. (From Selvig, 1964.)

358

H.

M.

Fig. 16. Mesio-distal section in the mid-root region of a human upper deciduous molar. Many oxytalan (dark) fibres are disposed apico-occlusally. Cm, cementum; AB, alveolar bone. Peracetic acid-aldehyde fuchsin-Halmi stain. x298.

principally in an apico-occlusa l direction, whereas at the time of eruption the major orientation became perpendicula r to the root surface. Fullmer (1959b) described the developmen t of oxytalan fibres. Goggins (1965) described the distribution of oxytalan fibres in periodontal membranes of deciduous, permanen t and mixed dentitions. Periodontal membrane s about the apical and middle thirds of the roots of deciduous teeth have more oxytalan fibres apico-occlusally orientated than do comparable regions of permanent teeth (Figs. 16 and 17). Oxytalan fibres are larger and more numerous in the bifurcation or trifurcation areas of deciduous teeth than in similar regions of permanent teeth. Otherwise, comparable distributions of oxytalan fibres occur in both dentitions.

FULLMER

Fig. 17. Mesio-distal section through a deciduous molar in a monkey. Dark oxytalan fibres (arrow) are here continuous with the endothelium of vessels and then pass into cementum. Cm, cementum. Peracetic acid-aldehyde fuchsinHalmi stain. x294.

III . O R G A N I Z A T I O N

OF THE

D E F I N I T I V E S U P P O R T I NG A.

T I S S U ES

INTRODUCTION

The chemical and physicochemica l properties of products of connective tissue cells vary with anatomic site and with disease processe s (Engel, Joseph and Catchpole, 1954; Catchpole, Joseph and Engel, 1956). The relative proportions, and kinds, of fibrous elements, as well as mucopolysaccharides , globulins, glycoproteins, albumin, mineral and other constituents , results in different types of connective tissues in various parts of the body. Thus, normal hydroxyproline: hexosamine

21.

C O N N E C T I VE

T I S S UE

C O M P O N E N TS

ratios of 2.8 for cartilage, 12.2 for skin and 30 for tendon depict the broad range of two major constituents of three normal connective tissues (Engel et al., 1960). These values are subject to change with age and disease . The workers quoted view connective tissues as immobile charged electrolytes with a composition that varies with anatomic site, and subject to change with growth, development, age and disease . The periodontal membrane is a fibrous connective tissue that unites the teeth to the alveolar bone. I t is composed principally of connective tissue cells, intercellular substances , blood and lymphatic vessels and nerves. The intercellular substance s include collagen, oxytalan fibres, a few elastic fibres and acid mucopolysaccharide s (chondroitin sulphates and hyaluronic acid). The formation and maintenanc e of the periodontal membrane is the function of the connective tissue cells therein. The periodontal membrane is a highly specialized functional organ developed to meet the special needs of this part of the body. Evidence indicating the highly specialized nature of periodontal membrane cells is the production and maintenanc e of oxytalan fibres, and the intricate arrangemen t of the intercellular substances . Each of the various intercellular substance s wil l be considered separately . B. COLLAGEN FIBRES

1. Collagen Orientation in the Periodontal Membrane Although many of the collagen fibres in the periodontal membrane are orientated indiscriminately, several specially orientated groups generally may be discerned. A gingival group is inserted into the cementum of the necks of teeth, extends occlusally following the general contour of the epithelial attachment , and thereafter is distributed as a broad band within the gingiva. Subjacent to the gingival group is the transsepta l group which bridges adjacent teeth. The fibres of this group are inserted into the cementum of the necks of adjacent

OF

P E R I O D O N T I UM

359

teeth, extend in the direction of approximal teeth over the alveolar crest and interdigitate with one another. Another defined group in this region, the alveolar crest group, is inserted into cementum at the necks of teeth and passes to the alveolar crests. Beginning at the alveolar crest and extending for variable distances to be inserted over the coronal third of the roots of teeth, the collagen fibres are principally horizontal and so are known as the horizontal group. Nearer the root apex, the collagen fibres are orientated predominantly in an oblique fashion. Finally, there is the apical group of collagenous fibres which radiate in all directions. I n addition to the above groups, which conform to classical descriptions, Arni m and Hagerman (1953) have described a circular band of connective tissue fibres which encircles the tooth and extends from the epithelial attachmen t to the alveolar crest. These fibres, which Arni m and Hagerman point out probably correspond with the circular ligament of earlier writers, intermingle with fibres of the free gingival groups, the transsepta l groups and with groups of fibres axially orientated in the buccal and lingual regions adjoining the interdental regions. The latter groups composed of coarse fibres arise from the alveolar bone and cementum and extend axially to the tips of the interdental papillae. Arni m and Hagerman describe the early destruction of the circumferential fibres wit h the progress of periodontal disease . N o mention wil l be made here of the numerous interruptions in the periodontal membrane utilized for vascular and nervous contributions, for this topic has been discusse d elsewhere (Volume I, Chapters 5 and 6). The arrangemen t and organization of the collagenous fibres, as well as the oxytalan fibres and that of the supporting bone described below, is a manifestation of the remarkable differentiation of the periodontal membrane for function. 2. Soluble Collagen

Chemistry

Collagen is classified as one of the scleroproteins which are categorized generally as being insoluble in organic solvents, salt solutions, dilute alkalies and acids, and in cold water. However, as wil l

360

H.

M.

be seen later, newly formed collagen does undergo solution in cold dilute acids, salts and alkalies as well as in cold water. Collagen swells more in weak acids than in strong acids even at the same p H. Collagen is converted into gelatin by boiling water. However, before it is transformed, shrinkage occurs at a temperature which is characteristic for the collagen source and amino acid content (Piez, 1960; Piez and Gross, 1960). Collagen and gelatin are only weakly antigenic. Most of the information about the chemical behaviour of collagen has been obtained from reconstituted collagen. Zacharides (1900) appears to have been the first to note that collagen could be extracted with weak acid. Nageotte (1927) later demonstrate d that fibrous precipitates that stained lik e collagen could be obtained by the neutralization of an acid solution of collagen. Later workers have shown that the acid-soluble collagen possesse s an amino-acid composition, wide-angle diffraction appearanc e and the 640 Â periodicity under the electron microscope which characterize insoluble collagen (Wyckoff and Corey, 1936; Schmitt, Hall and Jakus, 1942; Bahr, 1950; N o da and Wyckoff, 1951; Gross, Schmitt and Highberger, 1952; Bowes, Elliot and Moss, 1955; Eastoe, 1956b; Jackson, Leach and Jacobs, 1958; Gross and Kirk , 1958; and subsequen t workers). Soluble collagen is obtained by placing specimens containing collagen in cold weak buffers, acids, alkalis or even in distilled water. After a day or more, the extracted collagen may be precipitated by a variety of conditions including heat, neutralization and the addition of mucopolysaccharides , nucleic acids or even ATP. Results from several experiments indicate that collagen extractable with cold water and weak salts is newly formed. C1 4-labelled collagen appeare d withi n 24 hours in collagen of carageenin-induce d granulomas extractable with 0.2 M NaCl (Jackson, 1957). The rate of incorporation into citrateextractable collagen was much slower. Likewise, Gross (1958) found a direct correlation between collagen extractable with 0.45 M NaCl and the rate of growth of young guinea pigs, and an inverse

FULLMER

relationship between this fraction and starvation and the scorbutic state. Recent evidence indicates that 5 M guanidine extracts a more highly crosslinked collagen than either salts or acids (Bornstein, Martin and Piez, 1964). Extracted collagens denatured by heating at a pH less than 5, by urea or other salts form two components of unequal size (Orekhovitch et al., 1960; Piez et al, 1961) which have been designate d oc- and j3-components on the basis of their rates of sedimentation . Piez et al. (1961) observed that salt-extracted , heat-denature d collagen sedimented largely (but not exclusively) in one boundary whereas acid-extracted , heat-denature d collagen sedimente d with two boundaries . Two fractions designate d oc-l and oc-2 were separate d from the oc-, and two fractions designate d β-l and β-2 were separate d from the ^-fraction. 3. Lathyritic

Collagen

Chemistry

The administration of certain compounds, such as /3-aminoproprionitrile, to rats and certain other animals induces a condition called lathyrism characterize d by a loss of tensile strength and an increased solubility of collagen (Levene and Gross, 1959; Gross, Levene and Orloff, 1960; Martin et al, 1961a; Martin, Piez and Lewis, 1963). Martin and his colleagues noted that acid-extracte d collagen from rats fed â-aminoproprionitrile for 34 days exhibited a chromatographi c pattern analogous to that of salt-extracte d collagens from normal animals in that it sedimente d in a major single boundary with slight amount of faster moving material. On the other hand, acid-extracted , heat-denature d collagen from the skin of animals that received /3-aminoproprionitrile for 4 days sedimented with two major components characteristic of the collagen fraction obtained from normal animals. Data indicate an increase in the weight ratio of the single chains (oc-l and oc-2) to the chain pairs (β-l and β-2), signifying a decrease d formation of chain pairs in chronic lathyrism. The amino-acid compositions of collagens from lathyritic animals were identical with those from normal animals. The results suggest

21. C O N N E C T I VE

T I S S UE

C O M P O N E N TS

that lathyrism is a condition in which an inhibition of collagen chain-pair formation prevails, resulting in a failure of maturation and loss of tensile strength. 4. Amino-Acid Composition of Collagen The amino-acid composition of collagen is peculiar in that approximately one-third of the total residues are glycine and another third of the residues are proline and hydroxyproline (Table 1).

OF

P E R I O D O N T I UM

361

5. Structure of Collagen The present generally accepted model for the structure of collagens is that of Rich and Crick (1958). The models in general fit chemical, infrared, X-ray and physiochemica l data. Rich and Crick have designate d the models collagen I and collagen I I (see A and  in Diagram 1). The structures are

Table 1 THE AMINO-ACI D COMPOSITION OF GELATINE, COLLAGENS, AND ELASTINS"

Amino-acid Nitrogen Alanine Glycine Valine Leucine Isoleucine Proline Phenylalanine Tyrosine Tryptophane Serine Threonine Cystine Methionine Arginine Histidine Lysine Aspartic acid Glutamic acid Hydroxyproline a

Gelatin from calfskin

Cattle hide collagen

17.4 8.7 26.9 2.6 3.1 1.9 14.0 1.9 0.14

17.8 8.8 27.5 2.7 3.2 2.2 15.8 2.3 0.89



2.9 2.2 0.05 0.85 6.4 0.63 5.2 6.9 12.1 14.4



2.8 2.2



0.89 8.7 0.62 5.3 7.0 11.4 13.2

Cattle ligamentum Cattle nuchae aorta 16.7 18.4



18.4 8.4 4.3



5.7 1.85 0.01 1.0 1.15 0.25 0.03 1.05 0.05 0.40



2.2 1.84

17.1 19.3 30.2 17.6 8.7 3.9 16.8 5.1 1.63



0.80 1.01 0.12 0.0 0.87



0.47 0.64 2.1 1.91

From Tristram (1953).

The aromatic amino-acids and cystine are present i n low proportions (Bowes et al., 1955; Eastoe, 1956a,b). The smallest units of collagen have been called tropocollagen (Boedeker and Doty, 1955; Highberger, 1961).

Diagram 1. A and  are suggeste d molecular structures of collagens I and II , respectively, by Rich and Crick (1958, p. 20), which they propose as compatible with X-ray, infrared, chemical and physicochemica l date. Numbers 1, 2 and 3 represen t the three types of side-chain positions. Broken lines represen t hydrogen bonds. C shows in a general way how the structures A and  are deformed to give the collagen models. The solid lines represen t the axes of the three polypeptide chains as they spiral in a left-hand manner. The broken line depicts the common axis around which the three chains wind.

identical, except in the manner in which the three constituent polypeptides are phased, resulting in OH groups extending peripherally in collagen II and extending internally in collagen I. The OH groups in collagen II are available for linkage with neighbouring molecules whereas those in collagen I may unite internally. The molecule is assume d to be composed of three polypeptide strands which slowly spiral each other in a left-hand manner with a pitch which displaces one residue from the next

362

H.

M.

approximately —120°, with a displacemen t of about 3 Â in the fibre direction. Each of the three polypeptides is approximately 5 Â apart around an axis. The major helix has a pitch of approximately 28.6 A. The three strands are phased in such a fashion as to permit every third carbonyl group to link with every third —NH— group. The collagen molecule is approximately 2900 A in length, 14 A in diameter and has a molecular weight of about 340,000 (D. A. Hall, 1961). Under the electron microscope, collagen is identified by a characteristic cross-ban d periodicity of 640 A although less definite and shorter periods are frequently observed. Collagen is biréfringent, and the birefringence is abolished in specimens treated with phenolic compounds (Baer, 1952). Although collagenous cross-band s are probably due to periods of alternating greater and lesser densities, the significance of the characteristic 640 A periodicity as well as the less definite periods is unknown. Hodge and Schmitt (1960) have postulated that linear collagen molecules align themselves in a staggere d manner so that each molecule extends beyond the other by one-fourth of its length, resulting in the 640 Â periodicity observed under the electron microscope. N o da and Wyckoff (1951), Highberger, Gross and Schmitt (1951), Randall et al. (1955), Martin, Mergenhage n and Scott (1961b) and others have noted that the structure of reconstituted collagen can be altered by modifications of precipitating conditions. Figure 18A, B, C, taken from Martin et al. (1961b) illustrate the various morphologies of collagen fibres reconstituted at three different pH levels. Gross and Schmitt (1948) noted that the width of collagen fibres from human skin ranged from 700 A to 1000 A. Wood and Keech (1960) noted that the width of reconstituted collagenous fibres decrease d as the rate of precipitation of acidsoluble collagen increased and vice versa. However, the increased rate of fibre formation induced by a depression of pH resulted in increased fibre width. Although all fibres manifested typical 640 A periodicity, the observations encourage d Wood and

FULLMER

Keech to speculate that electrostatic forces significantly influence collagen fibre formation. Peach, Williams and Chapman (1961) and Fernando and Movat (1963) studied collagen formation during the course of tendon repair in rabbits. Fernando and Movat recognized typical collagen fibres with 640 A bandings in fibres that ranged in width from 200 A to 350 A, and in others that ranged from 600 A to 700 A. There were no other sizes of characteristic collagenous fibres observed. However, other fibres that ranged in width from 80 A to 90 A, and others that ranged from 50 A to 60 A with ill-defined cross-ban d periodicities were observed. Peach et al. (1961) also noted the 100 A width fibres with ill-defined periods. The size and width of fibres observed under the electron microscope varies with preparative procedures . 6. Staining Reactions of Collagen Collagen is identified histochemically by the Van Gieson stain and various trichrome stains of the Mallory or Masson types. With the Van Gieson procedure, collagen stains red with the acid fuchsin component of the stain. Preliminary work by Lilli e (1958) intimated that acid fuchsin reacts wit h amino or hydroxyl groups in collagen inasmuch as suppressio n of collagen staining was observed in sections previously acetylated [Acetylated collagen stains avidly with most elastic tissue stains (Fullmer and Lillie , 1956, 1957a)]. Later, Lilli e (1964) noted that nitrosation (deamination) of tissue sections prior to the Van Gieson staining procedure was without effect on the intensity of the staining of collagen. This suggeste d that amino groups of collagen were not reactive wit h the stain. In the same study, he also noted that sulphation of sections completely prevented the Van Gieson staining reaction of collagen. Inasmuch as methanolysis (a desulphation procedure) of sulphated sections resulted in restoration of the Van Gieson staining of collagen, Lilli e concluded that the participation of hydroxyl groups in the Van Gieson staining procedure appears probable.

Fig. 18. Modification of collagen structure by pH during reconstitution. (A) Reconstitute d at 37°C at pH 5.5. (B) Reconstitute d at 37°C at pH 6.2. (C) Reconstitute d at 37°C at pH 7. The typical 640 A periodicity is evident in (C) and less evident on (B) and appears to be absent from (A), ÷ 40,000. (From Martin et al.9 1961b.)

364

H.

M.

The trichrome stains use amphoteric dyes such as aniline blue in combination with either phosphotungstic or phosphomolybdi c acids as mordants. Sections may be mordanted with the phosphotungstic or phosphomolybdi c acid either prior to or concurrently with the stain. Evidence which tends to suggest that the basic groups in collagen molecules are operating with the trichrome stains for collagen has been contributed by Bolduan, Salo and Baet (1951) and Puchtler and Isler (1958). Their data are compatible with the assumption that part of the anionic groups of either phosphotungstic or phosphomolybdi c acids link to basic groups of collagen and the remainder of the same acidic molecules unites with the amphoteric dye resulting in stained collagen. Additional data are required for proof of this hypothesis. Collagen may also be stained yellow to brownishred with phosphotungsti c acid haematoxylin, and deep violet with the Lindner-Thomas phosphomolybdic acid haematoxylin method (Lillie , 1954). Collagen in the periodontal membrane stains lightly to moderately with the periodic acid-Schiff method for carbohydrates . On the basis of the works of Glegg, Eidinger and Leblond (1953, 1954) and Leblond, Glegg and Eidinger (1957), it is probable that galactose , fucose, hexosamine , glucose and sialic acid associate d with collagen are the stained components rather than collagen itself. The workers mentioned secured a positive correlation between the degree of staining of several connective tissues and bones with the periodic acid-Schiff method and the quantity of the above-mentione d monosaccharide s extracted. Periodontal membranes were not tested. SchultzHaudt (1957) and Schultz-Haudt, Paus and Assev (1961) identified glucuronic acid, glucosamine , glucose, mannose and ribose in fraction I, and glucuronic acid, glucosamine , galactose , mannose, possibly fucose and ribose in fraction II of hydrolysates from gingiva which stained with the periodic acid-Schiff method. Readers are referred to papers by Lilli e (1953), to Bangle and Alfor d (1954), and to Graumann (1954) for detailed analyses of the periodic acid-Schiff staining reaction with collagen.

FULLMER

A useful variant of the periodic acid-Schiff reaction is the allochrome stain developed by Lilli e (1951). The stain is particularly helpful for the delineation of the gingival and oral mucosal basemen t membranes . The staining method consists essentially of the periodic acid-Schiff procedure followed by a counterstain of picric acid-aniline blue. The smaller collagen fibres stain blue and denser collagen stain blue to red due to the persistence of the red periodic acid-Schiff stain through the counterstain . Collagen is also stained with methods that identify proteins, i.e., the dinitrofluorobenzene-H acid method with which it stains intensely red, and the ninhydrin-Schiff or the alloxan-Schiff method wit h which it stains pink. Although the above methods stain collagen, collagen is not specifically stained, for other tissue components are coloured under identical staining conditions. A specific stain for collagen is sorely needed in order to asses s the formation, maturation and degradation of collagen in growth, health and disease . The requirements of histochemica l methods do not appear to lend themselves to the detection of collagen because collagen does not contain a sufficient proportion of any amino-acid for which a histochemica l method is available. Furthermore, the biochemical methods for collagen determinations require solubilization, thereby abrogating their utilit y for a histochemica l procedure. It is possible that a specific immunohistochemica l method for collagen can be developed although the very weak antigenicity of collagen is a handicap to the developmen t of such a method (Maurer, 1955; Battista, 1949; Waksman and Mason, 1949; Loiseleur and Urbain, 1930; Watson, Rothbard and Vaname, 1954). 7. Effect of Enzymes on Collagen Unfixed collagen is readily digested by pepsin but only slightly attacked by trypsin. Grant and Alburn (1960) noted that collagen could be digested by trypsin, chymotrypsin, elastase and an endopeptidas e from procarboxypeptidas e provided salicylates and calcium salts were present.

21.

C O N N E C T I VE

T I S S UE

C O M P O N E N TS

Solubilization was enhance d by the presence of arginine, creatinine and guanidine, and inhibited by lysine. Collagen is readily digested by the collagenase produced by certain strains of Clostridium. Fullmer and Gibson (1966) and Gibson and Fullmer e derived (1966) recently described a collagenas from a nonmicrobial source. The collagenolytic factor is produced by cultures of gingiva excised s (see for the treatment of periodontal disease section V, p. 402). C. RETICULAR FIBRES

A t least three types of reticulins are known to exist: precollagenou s reticulin, basemen t membrane reticulin and stromal reticulins (Robb-Smith, 1958; Fullmer, 1965b). The first two are present in the periodontium. Newly formed collagenous fibres blacken with silver impregnation methods and are therefore sometimes known as argyrophilic fibres. The argyrophilia is abolished in sections extracted with 0.2 M NaCl and at the same time there is the acquisition of the affinity for the acid fuchsin of the Van Gieson stain for collagen (Jackson and Williams, 1956; Robb-Smith, 1958). Complete solubilization of the fibres occurs in citrate buffer pH 3.6. Citrate buffer pH 3.6 does not solubilize basemen t membrane reticulin or stromal reticulin. Basement membrane reticulin stains red with the periodic acid-Schiff reaction and the red stain is sustained in the allochrome which contrasts with the subjacent blue collagen. Basemen t membrane reticulin is also argyrophilic with silver impregnation methods. I t is readily digested with pepsin, and less readily with trypsin. D . A C I D MUCOPOLYSACCHARIDES

1. Kinds of

Mucopolysaccharides

There are eight known acid mucopolysaccharides : namely, according to Meyer, Hoffman and Linker (1959),hyaluronic acid (glucosamine and glucuronic acid), chondroitin sulphate A (glucuronic acid 25

OF

P E R I O D O N T I UM

365

and galactosamine) , chondroitin sulphate  (galactosamin e and iduronic acid), chondroitin sulphate C (galactosamin e and glucuronic acid), chondroitin (galactosamin e and glucuronic acid), heparin (glucosamine and glucuronic acid), keratosulphate (glucosamine and galactose) , and heparin monosulphuric acid (glucosamine and glucuronic acid?). They are disaccharide polymers with the components indicated, and comprise but one constituent of the complex ground substance s of connective tissues. Acid mucopolysaccharide s are produced by all connective tissue cells, but the kinds and quantities produced are peculiar to the type of connective tissue, age, anatomic site and disease (Loewi, 1953 ; Dorfman and Mathews, 1956; Meyer, 1957; Loewi and Meyer, 1958; Hallén, 1958). The types of acid mucopolysaccharide s present in the human periodontal membrane are unknown. Schultz-Haudt (1957) secured 175.7 mg of crude polysaccharide mixture from 13.995 gm dried acetone-defatte d h u m an gingiva. Approximately 27% of the crude polysaccharid e mixture was acid mucopolysaccharide , within which hyaluronic acid and chondroitin sulphate were identified. Sialic acid (neuraminic acid) is an aminosuga r that has been isolated from several connective tissue sources (Anderson, 1961; Courts, 1960; Czerkawski, 1962), and stains with methods that identify acid mucopolysaccharides . 2. Stains for

Mucopolysaccharides

Several stains are used for the histochemica l demonstration of acid mucopolysaccharides . However, none is specific; none stains a particular mucopolysaccharide . Furthermore, the mechanism s by which the stains identify the acid mucopolysaccharide s are for the most part unknown. Useful stains for the general detection of acid mucopolysaccharide s are those capable of showing metachromasi a (examples are azure A , toluidine blue O, thionine, etc.) the Hale (and R i n e h a r tAbul-Haj modification), alcian blue and aldehyde fuchsin. F or many years mucicarmine was widely used as a stain for the acid mucopolysaccharides ,

366

H.

MTFULLME R

but other methods are now regarded as more reliable. The periodic acid-Schiff reaction was formerly believed to stain acid mucopolysaccharide s in connective tissue structures. However, the works of Bradfield and Kodicek (1951), Glegg et al. (1954), Leblond et al. (1957) and Dische, Danilczenko and Zelmenis (1958) suggest that hexosamines , galactose , mannose, glucose fucose and possibly sialic acid are reactive with the method rather than the acid mucopolysaccharides . Gersh and Catchpole (1960) used intravenous injections of Evans blue which they found localized in regions of depolymerized mucopolysaccharide s and vascular damage. The methylene blue extinction test is perhaps one of the more precise methods for the detection of acid mucopolysaccharides . The method consists of staining sections in dilute solutions of methylene blue buffered to various acid pH levels. Weak acids cannot stain with a basic dye at low p H; however, the pH can be raised sufficiently for reactivity with the stain. The pH just below that which the object stains is called the extinction value, and is a measure of basophilia. The method is a test for degree of acidity, and depends upon the number, kind and availability of anionic groups. Some acid mucopolysaccharide s in connective tissues may be detected with stains that exhibit metachromasi a (Fig. 19). This is accomplishe d simply by the treatment of sections with dilute solutions of a suitable metachromati c dye. In the presence of certain substances , such as the acid mucopolysaccharides , the absorption spectrum is shifted to a shorter wavelength. This phenomeno n is called metachromasia . Al l the essentia l factors contributing to this process are unknown, but a certain density of appropriately arranged anionic groups (not necessaril y sulphate) are known requirements . In addition, concentrate d solutions of dyes suppress recognition of metachromasi a because large accumulations of dye particles may cover up already adhered metachromatic molecules resulting in obscurity of the initial metachromasia . Extremely dilute solutions do not permit the development of metachromasia , thereby indicating an essentia l concentration requirement.

Fig. 19. Gingiva distal to human upper third molar stained with azure A pH 4. Dark stained material (arrow) devoid of cells is stained metachromaticall y (red). Bundles of collagen (c) remain unstained, ÷ 459.

Some mucopolysaccharide s not naturally metachromatic may be made metachromati c by a sulphation procedure. Bignardi (1940a,b) appears to have been the first to sulphate tissue sections. Later, McManus and Mowry (1952) used the procedure, and Kramer and Windrum (1953, 1954) developed the method to the status of a histochemical procedure. I n practice, sections are treated with strong solutions of a sulphuric acid in anhydrous media which results in the sulphation of the hydroxyl groups of carbohydrates . These compounds thereby exhibit metachromasi a when stained as usual with a suitable dye. There is no reason to suspect that hydroxyl groups on molecules other than the mucopolysaccharide s are unreactive with this procedure, and reservations on this aspect must be registered. The staining reaction is prohibited in sections acetylated prior to sulphation due to a blockade of the hydroxyl groups. The Hale method for the demonstratio n of acid mucopolysaccharide s prescribes the treatment of sections with an acid solution of dialyzed iron

21. C O N N E C T I VE

T I S S UE

C O M P O N E N TS

(iron hydroxide), thorough rinsing and then conversion of the remaining iron to Prussian blue by immersion of sections in an acid solution of potassium ferrocyanide. The blue iron is adherent to the acid mucopolysaccharides . Although fibrin, casein, albumin and globulin stain with the Hale method under the same conditions as the acid mucopolysaccharide s stain (Davies, 1952; Braden, 1955; Craig, 1956; Wagner and Shapiro, 1957), the method is useful especially if it can be associate d wit h other evidence for the presence of acid mucopolysaccharides . A very useful modification of the method was developed by Rinehart and Abul-Haj (1951), who applied the Van Gieson picric acid fuchsin after the Hale reaction for mucopolysaccharides . The result is blue acid mucopolysaccharides , yellow muscle and red collagen, dentine, cementum and bone. Alcian blue was introduced as a histochemica l stain for acid mucopolysaccharide s by Steedma n (1950). It is a copper phthalocyanine dye which stains acid mucopolysaccharide s in an unknown manner. The specificity of the staining reaction wit h acid mucopolysaccharide s is relative and depends, in part, upon brief staining periods at low p H. Spicer (1960) suggests that alcian blue generally does not stain sulphated mucins. I n contrast, Zugibe (1962a,b) claims that all acid mucopolysaccharide s are stained with alcian blue. The alcian blue stain has been combined with the periodic acid-Schiff method (Mowry and Winkler, 1956). Proponents of the stain claim that neutral mucopolysaccharide s and mucoproteins stain red with the periodic acid-Schiff whereas acid mucopolysaccharide s stain blue with alcian blue. A recent account of the mechanism of alcian blue staining is relevant here (Quintarelli, Scott and Dellovo, 1964a,b; Scott, Quintarelli and Dellovo, 1964). Aldehyde fuchsin is a transitory dye formed at room temperature from basic fuchsin and paraldehyde in an acidic alcoholic solution (Gomori, 1950). Effective concentration s of the stain in the mixture are present for about one week, and thereafter the staining solution must be discarded. Gomori (1950) initiall y recommende d the stain for elastic fibres;

OF

P E R I O D O N T I UM

367

however, he noted that mucins also stained. The mechanism whereby it stains either mucins or elastic fibres remains unknown. The possibility that the aldehyde fuchsin-stainabl e component of elastic fibres is a mucopolysaccharid e has not been excluded. Aldehyde fuchsin is one of the most important stains for use in investigations of the supporting tissues of the teeth because it stains oxytalan fibres as well as amorphous ground substance s in the connective tissues of the periodontium in sections previously oxidized with peracetic acid, performic acid or K M n 0 4 (Fullmer and Lillie , 1958; Fullmer, 1960a,b, 1961), or oxone (Rannie, 1963). The basis for the presumption that the amorphous intercellular substanc e is stained by aldehyde fuchsin after strong oxidation is that the stainable component can be digested with ^-glucuronidase , and the digestion can be prevented by the inclusion of an inhibitor (saccharic acid) in the incubation medium (Fullmer, 1960a). The stained amorphous material is less effectively removed by testicular hyaluronidase digestions, although hyaluronidase regularly digests all material that exhibits metachromasia. D a ta from several experiments suggest that aldehyde fuchsin stains some mucopolysaccharide s other than those exhibiting metachromasia in sections oxidized with peracetic acid (Fullmer, 1960a). Confirmatory chemical experiments on the reactivity of the various mucopolysaccharide s with aldehyde fuchsin after oxidation should be conducted. Supplementa l proof of the nature of stained amorphous substance s may sometimes be secured by digestion experiments . Prior to the conduct of the staining procedure, sections may be digested wit h enzymes such as a diastase , an amylase, a hyaluronidase , ^-glucuronidase , lysozyme, nuclease s and various proteolytic enzymes. The absence of the stained substanc e under investigation under appropriately controlled conditions can lend supporting evidence for the chemical composition of the stained material. Several reservations must always be kept in mind during the conduct of these experiments: (1) enzyme preparations are rarely pure,

H.

368

M.

thereby permitting other enzymes, if present, to act; (2) enzymes may adhere to the substrate under observation without effecting enzymatic activity, thereby either preventing the subsequen t staining reaction (French and Benditt, 1953; Morri s and Krikos, 1958) or supplying additional groups for staining (Eidinger and Ghosh, 1956); (3) enzymes may remove more than the desired substrate , i.e. i n a hypothetical protein-mucopolysaccharide lipi d complex, cleavage of the mucopolysaccharid e component may result in the removal of the lipi d ; and also (4) the tissues must be properly prepared for the enzyme to be used, i.e. collagenas e is ineffective against formalin-fixed collagen. Examples of the difficulties enumerate d above include those encountere d by Lilli e (1949), who found a ribonuclease component in malt diastase , and by French and Benditt (1953), who reported the presence of a proteolytic component in their sample of diastase . Theoretically, testicular hyaluronidase may be expected to cleave hyaluronic acid, and chondroitin sulphates A and C. Sialidase may also be helpful in some cases to effect digestion of sialic acid (Spicer and Warren, 1960; Spicer, Wollman and Warren, 1960).

E.

OXYTALA N FIBRES

1. Description and Distribution Until a few years ago it was believed that white collagenous fibres constituted the sole type of fibrous element in human periodontal membranes except for a few elastic fibres associate d principally wit h vascular structures. I n a series of papers, Fullmer (1958, 1959a,b, 1960a,b, 1961, 1962, 1963) and Fullmer and Lilli e (1958) demonstrate d a previously undescribe d connective tissue fibre in the periodontal membrane of man, mice, rats, guinea pigs, swine, dogs and certain other animals (Fig. 20). The fibre was named oxytalan (oks-ifa-lan) in recognition of its resistance to acid solution in contrast with collagen. Its morphology resembles that of elastic fibres in the periodontal membrane of several mammalian species.

FULLMER

Fig. 20. Periodontal membrane in the transsepta l region distal to the upper first molar of middle-aged individual. Oxytalan (dark fibres indicated by arrows) as well as collagen fibres (c) are inserted into cementum and extend outward. Cm, cementum. Peracetic acid-aldehyde fuchsin-stain. X353.

The major distributions of oxytalan fibres are i n general those of the collagenous fibre groups (Fullmer, 1961, 1962, 1963, 1964a, 1965a,b). Largest oxytalan fibres are observed at the cervices of teeth. They are inserted into cementum, extend outward for varying distances and may or may not arborize prior to termination. Single fibres have not been observed to span the entire distance from tooth to bone. Many small oxytalan fibres are continuous with the basemen t membrane of the epithelial attachmen t and extend in a rootward direction into the connective tissues. Others of the free gingiva course in many directions, including i n association with the free gingival collagenous group and the circumferential group. In the transsepta l region, oxytalan fibres are inserted into

21. C O N N E C T I VE

T I S S UE

C O M P O N E N TS

OF

P E R I O D O N T I UM

369

cementum at the necks of the teeth and extend outward toward adjacent teeth. Others in the vicinity may be unattache d to the teeth; however, a small zone of variable size in the transsepta l region near the bone frequently remains devoid of oxytalan fibres. I n the substanc e of the periodontal membrane oxytalan fibres may arise from either the alveolar bone or cementum of teeth (Fig. 21). Some follow the course of major collagenous groups, and some do not. A t the apex of the tooth, oxytalan fibres frequently form a complex network which unites the tooth to bone. Moura(1964) has also described the distribution of oxytalan fibres. The fnuction of oxytalan fibres has not yet been

clarified. They are larger at the cervices of teeth than in other regions of the periodontium of the same tooth. Sections from upper first premolars and the first molars used as abutment teeth for fixed bridges taken at autopsy from two individuals revealed larger than usual oxytalan fibres about the necks of these teeth, particularly in the region distal to the premolars and mesial to the molars (Fullmer, 1964a, 1965a,b). Other observations have revealed that sections from autopsy specimens of teeth in occlusion and therefore functional appear to have more and larger oxytalan fibres than nonfunctional teeth (Fig. 22). These data permit the assumption that the degree of development of oxytalan fibres is related to functional demands. The fact that more and larger oxytalan fibres were present about the necks of teeth used for abutments presumes that new fibres may develop in response to functional demands , and that a mechanism exists for their growth in fibres of increased size.

Fig. 21. Mesio-distal section through the apical third of the periodontal membrane of a human upper permanen t molar. Oxytalan fibres (dark fibres indicated by arrow) arise from either alveolar bone (AB) or cementum, and course with the collagenous groups. Others are apicoocclusally disposed. Peracetic acid-aldehyde fuchsin-Halmi stain. X300.

Fig. 22. Mesio-distal section through the trifurcation region of an upper second molar from a woman aged 48 years. The tooth was subjected to hard stress because the adjacent first molar was missing. Oxytalan fibres (dark fibres indicated by arrow) are larger and more numerous than normal. AB, alveolar bone. Peracetic acid-aldehyde fuchsin-Halmi stain, χ 250.

370

H.

2. Staining

M.

Reactions

Oxytalan fibres are demonstrabl e with the peracetic acid-aldehyde fuchsin-Halmi stain, and this accounts for the failure to detect them earlier. Sections of periodontal membrane s had not been oxidized with peracetic or performic acids and then stained with an elastic tissue stain previous to our description of the method (Fullmer and Lillie , 1958). More recently, Rannie (1963) noted that oxytalan fibres oxidized with 10% aqueous oxone for 1 hour stain with several basic dyes in neutral aqueous solution. Oxone is a monopersulphate bleach made by the Electrochemical s Division, Å. I. duPont Co., Wilmington, Delaware. Among the elastic tissue stains, aldehyde fuchsin serves best, and fewer fibres are demonstrable with resorcin fuchsin or orcein. Aldehyde fuchsin appears to be the only stain, known at the present time, which reliably stains all oxytalan fibres. Verhoeff's stain and orcinol new fuchsin Table 2 STAINING REACTIONS OF OXYTALA N AND ELASTIC FIBRES

Reactions The five elastic tissue stains Azure A pH 4 Hale stain Schiff's reagent Peracetic acid oxidation/ then: aldehyde fuchsin resorcin fuchsin orcein Verhoeff's stain orcinol-new fuchsin azure A pH 4 Hale reaction Schiff's reagent

Oxytalan fibres

Elastic fibres

_

+

-







-

+ -

Pinka

+ + + + +

-

-







Pinka

° Elastic fibres in rodents, but not in man, give a direct Schiff reaction. b Incomplete and inadequate stain ; some are stained and others are not. c Best oxidant is 10% aqueous oxone (Å. I. duPont Co., Electrochemical s Division, Wilmington, Delaware) for 1 hour 25°C (Rannie, 1963).

FULLMER

(Fullmer and Lillie , 1957b) are ineffective as stains for oxytalan fibres. Likewise, all other stains for mucopolysaccharides , as well as Schiff's reagent, have failed to stain them (Table 2). It is important to point out here that not only oxytalan fibres but amorphous ground substanc e is stained by aldehyde fuchsin after oxidation, and that peracetic acid (or oxone) oxidation engender s susceptibility of the ground substanc e and the stainable component of oxytalan fibres to ^-glucuronidase digestion (see below). These observations foster the assumption that the stainable component of oxytalan fibres is mucopolysaccharide . This hypothesis is strengthene d by the observation that digestion of ground substanc e and the stainable component of oxytalan fibres is prevented by the presence of an inhibitor of ^-glucuronidase (saccharid acid) in the incubation medium (Fullmer, 1960a). Although oxytalan fibres are stained by three out of the five elastic tissue stains, strong oxidation is required. This oxidative requirement distinguishes elastic from oxytalan fibres. Oxidants that have proven successfu l are peracetic acid, performic acid, potassium permanganate , bromine and oxone. Ineffective oxidants tested are periodic acid, hydrogen peroxide, ferric chloride and chromic acid. Treatments with acetic, nitric and hydrochloric acids, and alkaline treatments with sodium and potassium hydroxides likewise fail to induce the staining reaction. Data suggest that a strong oxidation is required for induction of the staining reaction. Enzymic digestions may also serve to distinguish oxytalan from elastic fibres (Table 3). Formalinfixed oxytalan fibres are generally refractory to digestion by elastase , although they are remarkably susceptible to elastase after oxidation with peracetic acid (Fullmer, 1960a). Any special significance of the acceleration of oxytalan fibre digestion by elastase after peracetic acid oxidation is not readily apparent because the same sections revealed a remarkable acceleration in the digestion of keratin, striated muscle, epithelial cells and some collagen. It was also noted that elastic fibres are more readily digested by elastase if they have been

21. C O N N E C T I VE

T I S S UE

C O M P O N E N TS

Table 3 EFFECT OF ENZYMI C HYDROLYSIS ON STAINING

OF

P E R I O D O N T I UM

trypsin, the concept of a nonspecific effect entertained.

371

is

REACTIONS OF FORMALIN-FIXED OXYTALA N AND ELASTIC FIBRES WITH ALDEHYD E FUCHSIN

Enzymic reactions Undigested Elastase Peracetic-elastas e Testicular hyaluronidase Peracetic-hyaluronidas e Lysozyme** rf Peracetic-lysozyme ^-Glucuronidase Peracetic-glucuronidas e

Oxytalan fibres0 Elastic fibres6 Purple Purple Dissolved Purple Unstained0 Purple Unstained0 Purple Unstained0

Purple Dissolved Dissolved Purple Purple Purple Purple Purple Purple

a

Stained by the peracetic-aldehyd e fuchsin sequence . Stained by aldehyde fuchsin. c Here the fibres slowly lose their stainability in serially exposed preparations ; meanwhile unstained portions of fibres adjacent to stained portions are still visible. d Fresh frozen sections of human gingiva and tendon were used with lysozyme. Formalin and alcohol-fixed oxytalan and elastic fibres were resistant to lysozyme. b

pre-oxidized with peracetic acid. The working out of the significance of elastase digestions awaits further purification of the enzyme. Table 3 also shows that the stainable component of oxytalan but not of elastic fibres is digestible wit h ^-glucuronidase , testicular hyaluronidase and lysozyme provided sections are preoxidized with peracetic acid. Of these, ^-glucuronidase is the most effective and informative. Sections removed periodically from the incubation mixtures with ^-glucuronidase and subsequentl y stained with the peracetic acid-aldehyde fuchsin-Halmi method revealed many partially digested oxytalan fibres. Frequently, parts of individual fibres stained and other parts did not, indicating that the structure of oxytalan fibres is composed of at least two components . Again, the mechanism whereby peracetic acid engender s substrate susceptibility to these mucopolysaccharase s is unknown. Inasmuch as peracetic acid oxidation fosters nonspecific as well as specific elastase activity, and engender s susceptibility of oxytalan fibres to digestion with

3. Comparative Studies of Oxytalan, Reticulin and Elastic Fibres Oxytalan fibres are readily distinguished from reticular fibres. Reticular fibres are demonstrate d by several silver impregnation stains which do not delineate oxytalan fibres. Furthermore, the peracetic acid-aldehyde fuchsin-Halmi stain which identifies oxytalan fibres stains neither the precollagenou s reticulins nor the supportive stromal reticulins of organs such as the lymph nodes and spleen, although the basemen t membrane between epithelial attachmen t and subjacent connective tissues is generally well stained. The study of oxytalan and elastic fibres in the periodontal membrane s of several mammals has shed considerable light on the nature of oxytalan fibres (Fullmer, 1960b). The distribution of oxytalan fibres in the periodontium of mice, rats and monkeys approximates to that of man (Table 4). However, deer, cattle, swine and dogs have elastic fibres in the cervical and middle portions of the periodontal membrane which display an arrangement and morphology analogous to oxytalan fibres in man (Fig. 23). The largest elastic fibres are in the coronal thirds of their roots, namely in the region where oxytalan fibres achieve their greatest size in man. The noncollagenou s fibrous elements of the periodontal membrane about the apices of teeth from cattle, deer and dogs are usually oxytalan rather than elastic (Table 4). Other noncollagenous , nonelastic fibres with an elasticoxytalan fibre morphology, which stain only with the peracetic acid-aldehyde fuchsin-Halmi method, are distributed throughout the periodontal membranes of these animals (Fig. 24). Furthermore, parts of some non-collagenou s fibres stain with elastic tissue stains, and other parts of the same fibre reject the elastic tissue stain and colour with the oxytalan fibre stain. These data suggest that oxytalan fibres may represen t elastic-like fibres, perhaps partially developed or incompletely differentiated ones.

H.

372

M.

FULLMER Table 4

APPROXIMATE PROPORTIONS OF ELASTIC AND OXYTALA N FIBRES IN THE PERIODONTIUM OF SEVERAL MAMMAL S

Portion of the periodontium"

Type

Order Primate

Man Monkey

Rodentia

Guinea pig Rat Mouse

Lagomorpha Rabbit Carnivora Dog Artiodactyla Sheep Deer Swine Steer

Tooth Typea I M I M I M I M I M M M I M I M I M I M

Gingiva Fibre Type Å

Ï

++++ +/+/++++ +/++++ +/+ +++ ++ + + + /- + + + + /+ +++ + +++ + +++ + +++ ++ ++ ++ ++ + +++ +++ + + +++ ++ +++ + +++ + ++ ++

Coronal 3rd Fibre Type Å

Ï

++++ + +++ + +++ ++++ + +++ +/- + + + + + /- + + + + +/- + + + + + /- + + + + +/++++ ++ ++ + +++ ++ ++ + +++ ++ ++ ++ ++ +++ + +++ + ++ —



Middl e 3rd Fibre Type Å

Ï

++++ + +++ ++++ ++++ — + +++ + +++ ++++ + +++ +/- ++++ +/- ++++ + +++ +/- ++++ + ++++ +/- ++++ + +++ ++++ + +++ ++ ++ + +++ + +++ —

Apical 3rd Fibre Type Å

— —

-



-

+/-

+/-

+/-

+/-'

+

+/+/+/-

+ ++

+/+/-

Ï

+ +++ ++++ ++++ + +++ + +++ ++++ ++++ ++++ + + ++ ++++ ++++ + +++ +++ ++++ ++++ ++++ +++ ++++ ++++

a I, incisor; M, molar; Å, elastic fibres; Ï , oxytalan fibres. Coronal, middle and apical thirds refer respectively to those portions of the periodontal membrane s surrounding the coronal, middle and apical thirds of the roots of teeth.

A study of the embryogenesi s of elastic fibres and of the structure of human periodontal membranes involved in scleroderma have served to clarify further the nature of oxytalan fibres (Fullmer and Witte, 1962). During human embryogenesis , developing elastic fibres in the periosteum can be selected at a time when they fail to stain with elastic tissue stains but do so brilliantly with the peracetic acid-aldehyde fuchsin method. A t later stages of development , elastic fibres at the same sites are readily stained and identified. A t the time that developing elastic fibres can be stained with the peracetic acid-aldehyde fuchsin method and are not yet revealed with elastic tissue stains, these pre-elastic fibres exhibit all the staining and

digestion reactions that characterize oxytalan fibres. In other words, pre-elastic fibres cannot be distinguished from oxytalan fibres with histochemical methods at the present time. I n scleroderma the periodontal membrane of individual teeth may or may not be affected. The periodontal membrane is enlarged, there is an apico-occlusa l arrangemen t of the collagen fibres in the apical and middle regions of the roots and marked irregular resorption of the lamina dura, as well as an irregular distribution of calcified deposits throughout the periodontal membrane (Fig. 25). The interesting observation in connection wit h oxytalan fibres in the case described (Fullmer and Witte) is that they were proportionately

21. C O N N E C T I VE

T I S S UE

C O M P O N E N TS

Fig. 23. Transsepta l region of incisor from a 5-montha old Chester White hog stained with the Taenzer-Unn orcein method for elastic fibres. Some elastic fibres (dark) stain intensely in some regions and lightly or not at all in other regions. Cm, Cementum. χ 315.

increased in number in association with the collagen fibres which accounted for the thickening of the membrane. In addition, some elastic fibres were also present (Fig. 26). Their distribution followed those of the collagenous and oxytalan fibres, and their greatest numbers were in regions in association wit h the most mature and sclerotic collagen. The proportionate increase of oxytalan and collagenous fibres with the progress of scleroderma suggests that connective tissue cells of the periodontal membranes posses s the enzymic and other requisites to produce oxytalan fibres, and that the sclerodermatous process excites the production of both. The observation that some elastic fibres are formed in regions of sclerotic collagen is interpreted to suggest that the sclerodermatou s process fosters

OF

P E R I O D O N T I UM

373

Fig. 24. Comparable section of same incisor as that of Fig. 23 stained with the peracetic acid-aldehyde fuchsinHalmi method. All noncollagenou s (dark) fibres stain uniformly densely and are much more numerous than the elastic fibres of Fig. 23. Dark very thick fibres indicated by arrows are probably elastic fibres ; those stained less intensely are oxytalan. C, collagen; Cm, cementum. ÷ 3 1 5.

not only the increased formation and maturation of collagen, but also the overmaturation of the elastic-like fibrous system which in this region is manifested by the transformation or maturation of oxytalan fibres to the elastic fibre stage. 4. Electron Microscopic Studies of Oxytalan

Fibres

Preliminary electron microscopic investigations of oxytalan fibres have been conducted by Goggins (1965) and Carmichael and Fullmer (1966). Oxytalan fibres appear to be composed of long filaments approximately 100 Â to 150 Â in diameter, together with an interfilamentous amorphous substance of approximately the same diameter (Fig. 27). Some oxytalan fibres appear to be more dense and homogeneou s (Goggins, 1965). Electron microscopic photographs of elastic fibres taken by Thomas (1963) and others serve

374

H.

M.

Fig. 25. Mesio-distal section of cervical third of the root of an upper first molar involved with scleroderma in a 21-year-old Negro woman. Although the transsepta l fibre group is normally orientated, the principal direction of fibre groups higher up is apico-occlusal . The periodontal membrane is thickened and contains bone spicules (lower arrow) as well as amorphous calcified and uncalcified masses (upper arrow). There is an increase in dark-staining oxytalan fibres. Peracetic acid-aldehyde fuchsin-Halmi stain. (From Fullmer, 1962.) x 2 7.

to lend further support to the conjecture that oxytalan fibres are related to elastic fibres and perhaps comprise an immature variety. Thomas (see his Figs. 2, 3, and 4) described elastic fibres in epineurium which had a homogeneou s interior associate d with many fine filaments, about 100 Â thick, at the periphery. The structural arrangemen t of this two-componen t system could be interpreted

FULLMER

Fig. 26. Apex of the tooth shown in Fig. 25 stained with orcein. In regions where collagen is most scirrhous (adjacent to the tooth) elastic fibres (dark) are present. Elastic fibres are cementum (Cm); bone spicules (b). (From Fullmer, 1962.) ÷ 205.

as a growth mechanism whereby elastic fibres enlarge by accretion and homogenizatio n of filaments. It is too early to draw conclusions at this stage of our knowledge of oxytalan fibres. However, it could be hypothesize d that oxytalan fibres represen t an arrested developmen t of elastic fibres whereby interference with, or incomplete, homogenization occurs. Other authors who have expresse d the view that elastic fibres are composed of a filamentous and a homogeneou s substanc e include Gross (1949), Bahr (1951), Lansing et al. (1952), Schwarz and Dettmer (1953), Schwarz (1954) and D. A. Hall, Reed and Tunbridge (1955). Cox and Littl e (1961) on the other hand deny the presence of a peripheral fibrillar component.

Fig. 27. Electron micrograph of periodontal membrane of a rat incisor. The structure between the arrows is believed to be an oxytalan fibre composed of long filaments approximately 200-150 A in diameter and an inter-filamentous amorphous substance of approximately the same diameter, c, collagen; Cm, plasma membrane , ÷ 35,400. (From Carmichael and Fullmer, 1966.)

H.

376

M.

F. ELASTIC FIBRES

The normal periodontal membrane in man contains only a few elastic fibres, and these are generally associate d with the vascular system. Rarely, however, elastic fibres may be detected in periodontal membrane s with an arrangemen t analogous, in a somewhat rudimentary fashion, to oxytalan fibres (Fig. 28). Instances where I have found elastic fibres in human periodontal membranes were on teeth subjected to strong occlusal forces (Fig. 28). Trifurcation regions of upper molars appear to respond initiall y to these forces by the formation of rudimentary elastic fibres and well-developed oxytalan fibres.

Fig. 28. Mesio-distal section in the transsepta l region mesial to the upper first molar from a woman aged 38 years. Arrows point to elastic fibres, some of which enter the cementum (Cm). Orcein stain, ÷ 355. G. CONNECTIVE TISSUE CELLS

1. Structure and Histochemical

Staining

Reactions

The connective tissue cells of the periodontal membrane are highly specialized. Manifestations

FULLMER

of this property are (a) the production of oxytalan fibres (elastic fibres are produced by connective tissue cells in most regions); (b) the production, orientation and maintenanc e of collagenous fibres in a manner to withstand normal forces of mastication; (c) the formation and maintenanc e of a structure that differs in various regions, i.e. denser, coarser and larger numbers of mature fibres (collagenous and oxytalan) are present close to the root surface than close to the bone in the same periodontal membrane; (d) the enzymic activity of connective tissue cells of the periodontal membrane is usually greater than that of many other connective tissue cells such as those of the dermis; and (e) the enzymic activity exhibited by connective tissue cells of the periodontal membrane s is not uniform, but is related to the location and function of the cells. Fibroblasts of the human periodontal membrane are generally ovoid or flattened in appearanc e due to compressio n of surrounding fibrous tissue. Nucleic acids may be identified by the use of basic dyes such as azure A, and deoxyribonucleic acids may be specifically identified by application of the Feulgen stain. Nucleoproteins may be identified by either the Fullmer-Lilli e (1962) or Alfert and Geschwind (1953) methods. The Fullmer-Lilli e method is far more selective. Enzymic histochemica l studies of periodontal structures have only recently been undertaken because methods for the demineralization of bones and teeth with the preservation of enzymic activity are currently under developmen t (Mori , Takada and Okamoto, 1962; Balogh, 1962, 1963; Fullmer and Link, 1964). Of the hydrolytic enzymes, fibroblasts of the periodontal membrane have been shown to exhibit alkaline and acid phosphatase s and nonspecific esterase . Other enzymes which can be demonstrate d histochemically in fibroblasts of the periodontal membrane suggest that they posses s functional citric acid cycles [succinic, D P N- and TPN-malic (Fig. 29), D P N- and TPN-isocitric and D P Nglutamic dehydrogenase s (Fig. 30)], pentose shunts (TPN-glucose-6-phosphat e and 6-phosphoglu -

21. C O N N E C T I VE T I S S UE

C O M P O N E N TS O F

Fig. 29. Mesio-distal section through the lower molar region of a rat, stained for malic dehydrogenas e and counterstaine d with carmalum for nuclei. Fibroblasts of the periodontal membrane exhibit considerable activity, but the activity is exceede d by osteoclast s at the alveolar bone margin and by odontoblasts . The specimen was demineralized by the Fullmer-Link method (1964). AB, alveolar bone; r, root dentine; PM, periodontal membrane ; o, odontoblasts . X100.

conate dehydrogenases) , as well as enzymes operative in glycolysis (á-glycerophosphat e and lactic dehydrogenases) , fatty acid metabolism (jS-hydroxybutyric dehydrogenase) , and D P N- and TPN-diaphorase s (Fig. 31). A s noted above, the distribution of many of these enzymes is not uniform. Far greater alkaline phosphatas e activity is evident in osteoblast s and adjacent fibroblasts near the alveolar bone than elsewhere (Fig. 32). Capillaries also generally exhibit alkaline phosphatas e activity. Alkaline phosphatas e activity is greatly increased in inflamed regions where not only fibroblasts but inflammatory cells and intercellular fibrous structures stain. Baratieri (1955, 1958, 1960b) is one of the early investigators of alkaline phosphatas e in the human

P E R I O D O N T I UM

377

Fig. 30. Mesio-distal section through the lower molar region of a rat, stained for glutamic dehydrogenas e and counterstaine d with carmalum for nuclei. Although fibroblasts of the periodontal membrane exhibit great activity, less is evident than manifested by odontoblasts and osteoclast s at the alveolar bone margin. This pattern is observed generally with the various dehydrogenase s and diaphorases . The specimen was demineralized by the Fullmer-Link method (1964). AB, alveolar bone; r, root dentine; PM, periodontal membrane ; o, odontoblasts . x72.

periodontal membrane. Suga, Shimizu and Namie (1959) studied alkaline phosphatas e in the periodontal tissues of the rat. Cabrini and Carranza (1951, 1958), Carranza and Cabrini (1955) and Baratieri (1955, 1958, 1960a,b) studied alkaline phosphatas e in the periodontal tissues of both man and rats. Suga et al. (1959) studied alkaline phosphatas e in periodontal tissues of rats. Lisanti (1960) studied the reactivity of several hydrolytic enzymes in the human gingiva. Aci d phosphatas e activity of fibroblasts of the periodontal membrane is generally uniform except

378

H.

M.

Fig. 31. Mesio-distal section through the lower molar e and counterregion of a rat, stained for TPN-diaphoras stained with carmalum for nuclei. The staining pattern resembles that for glutamic and malic dehydrogenases . The specimen was decalcified by use of the Fullmer-Link method (1964). P M, periodontal membrane ; AB, alveolar bone; r, root dentine, o, odontoblasts , χ 111.

FULLMER

Fig. 32. Mesio-distal section between the lower molars of a rat, stained for alkaline phosphatas e with a naphthol substrate method. Greater activity (dark stain) near alveolar bone than elsewhere . AB, alveolar bone; P M, periodontal membrane; root dentine; o, odontoblasts , ÷ 9 7.

2. I sotopic adjacent to the alveolar bone, where it is greater (Fig. 33). I n the region of the periodontal membrane bordering the bone, osteoblast s generally exhibit slightly greater activity than do adjacent fibroblasts, but, in turn, slightly less than osteoclast s in the same region. The acid phosphatas e activity of fibroblasts of the periodontal membrane generally exceeds that of fibroblasts of dermis. Sections of periodontal membrane stained for nonspecific esteras e reveal the greatest activity in osteoclasts adjacent to bone with lesser amounts in macrophages , osteoblasts , fibroblasts and osteocytes. Nerve fibres also exhibit esterase .

Studies

Isotopic studies of periodontal tissues by Stallard (1963, 1964a,b), Crumley (1964) and Carneiro and Leblond (1959) confirm histochemica l observations that cells of the periodontal membrane are in a highly active metabolic state. Both histochemica l and autoradiographi c studies indicate a rapid metabolic turnover of intercellular substance s in periodontal membranes . Carneiro and de Moraes (1965) have shown by autoradiograph y that tritium-labelled glycine appears within, or in the close vicinity of, fibroblasts in the periodontal membrane of the mouse within 30 seconds of its intravenous injection (Fig. 34A). After about

21. C O N N E C T I VE

T I S S UE

C O M P O N E N TS

OF

P E R I O D O N T I UM

379

teristic constituent of collagen) labelled proline is also a useful tool for the investigation of collagen synthesis. However, both proline and glycine may enter into other constituents . Several other interesting findings have been revealed with the aid of isotopes. Al l the workers just referred to noted the greater metabolic activity of cells of the periodontal membrane adjacent to the bone and in the central zone of the membrane than in regions near the root surface. They also noted considerable activity near the alveolar crest. Further, no evidence was found to substantiat e the existence of an intermediate plexus. (Sicher, 1962, p. 187) in which readjustment s of the bundles of principal fibres are said to take place as an adaptation to tooth movement. Experimental interference with the occlusion of teeth and pressures exerted on teeth resulted in increased utilization of labelled amino acids. 3. Other Metabolic Fig. 33. Mesio-distal section through the lower molar region of a rat, stained for acid phosphatas e with the Barka method. Nuclei have been stained with haematoxylin. Osteoclast s (arrow) at the margins of alveolar bone show the greatest activity; however, great activity is also shown by fibroblasts of the periodontal membrane and a lesser amount by cementoblast s and osteocytes . r, root dentine; PM, periodontal membrane ; AB, alveolar bone, ÷ 132.

7 days the glycine is found distributed extracellularly within what may reasonabl y be assume d to be new collagen (Fig. 34B). Some evidence indicated that collagen may be formed within 4 hours. Fortyfiv e days after injection no labelled glycine remained i n the tissue suggesting that there had been a complete turnover of collagen at that site within that period. Inasmuch as approximately one-third of the amino acid residues in collagen are glycine, it is presumed that much of the labelled material observed first in cells is labelled glycine being formed into collagen, and that which appears later i n the intercellular regions is labelled glycine in formed collagen. Inasmuch as proline is the precursor of hydroxyproline (a major and charac-

Studies

Although the present topic pertains mainly to the periodontal membrane, it seems appropriate at this point to refer to some metabolic studies of gingiva which relate to the discussion at hand. Glickman, Turesky and Hil l (1949) conducted respiration studies of human gingiva and found average QQti values of 1.6 ± 0.4 for normal gingiva, 1.8 ± 0 .1 for specimens with slight inflammation and 2.6 for markedly inflamed gingiva. The figures are on the basis of milligrams per dry weight per hour. Other endogenou s respiration studies on gingiva were conducted by Senter, Eiler and Leek (1959) and Schrader and Schrader (1957). Senter et al. used slices of human gingiva, some of which had been subjected to local anaesthetics . Al l contained gingival epithelial cells as well as connective tissue cells. Ten samples were tested at 24°C. Wet Qo2 for individual samples, averaged over 1 hour, varied from 0.265 to 0.446 wit h a mean value of 0.339 ± 0.068. The mean QQ% ( N ) w as 10.67 ± 1.88. The endogenou s respiration of human gingival tissues is markedly time dependent . Average Qo (N) calculated on the basis of the first 10-minute

380

H.

M.

FULLMER

Fig. 34. Radioautograph s of periodontal membrane in the oblique fibre region of mice injected with glycine-H3. (A) Thirty minutes after injection. Radioactivity (black dots) is principally in fibroblasts. (B) Seven days later it is in the intercellular spaces and presumably in new collagen. Haematoxylin and eosin stain, χ 700. (From Carneiro and de Moraes, 1965.)

determination would amount to 14.1, whereas the value calculated for the last 10 minutes would be 8.1. A total of 6 determinations were made at 10-minute intervals. Person, Stahl and Scapa (1961) and Person (1963) have also conducted respiratory studies of gingiva. Person et al. in a combined histochemical-Warbur g study found Qo2 values (milligrams dry weight per hour) of 2.7-4.0. Leng (1942) appears to have been the first individual to study gingiva with a respirometer. The above respiromete r studies would appear to indicate that cells of gingiva have a low oxygen consumption. Persona l experience s wit h oxidative activity of connective tissue cells of the periodontium manifested in histochemica l procedures do not support this assumption . It is

probable that low figures are secured in respirometer studies because fibrous elements contribute to weight but not to oxidative activity. 4. Research

Perspectives

Studies of periodontal structures have hitherto been directed largely toward the description of inflammatory processe s and regions of loss of collagen fibres and bone. In order to understan d the structure, physiology and the pathology of the supporting tissues of the teeth, attention must be focussed u p on the cells therein which produce and sustain these parts. The view that must be foremost and ever present in the minds of investigators of the periodontal tissues is that the connective tissue cells in their respective locations have

21.

C O N N E C T I VE

T I S S UE

C O M P O N E N TS

the function of forming and maintaining an il — defined area of intercellular substance s (bone, periodontal membrane, etc.) in their vicinity. This function is accomplishe d through enzymic activity which, in turn, is directed by individual cellular genetic endowment, and modified by humoral factors circulating throughout the host at any one time, and by local environmenta l inhibitory and stimulatory factors. The acquisition of meaningful data from periodontal membranes which are comparatively small, and contain heterogeneou s cell populations wit h wide variations in enzymic activities, is technically difficult. The disparity in the degrees of enzymic activity in the several types of bone cells, such as between osteoclast s and some osteocytes , is several orders of magnitude (Fullmer, 1964b). The same is true for alkaline phosphatas e activity in various distinct parts of periodontal membranes . Biochemical procedures which secure data based upon an average of these activities fail to delineate the numerous local prevailing conditions throughout the specimen. Evidence to date is consistent with the view that a measure of the degree and kinds of enzymic activities displayed by cells constitutes a proper assessmen t of their activity or performance at any particular time. Present histochemica l methods only partially provide the essentia l information because (a) current histochemica l methods are only crudely quantitative, and (b) histochemical methods have not yet been developed for the determination of many enzyme and other systems. Combined histochemical, microchemical and other methods employing isotopes wil l be required to delineate the local factors affecting cells that promote the formation and maintenance , or favour degradation of the supporting tissues of the teeth.

H.

M A ST CELLS

The distribution of mast cells in the periodontium is variable and unpredictable . There may be few or many, and they may or may not be associate d 26

OF

P E R I O D O N T I UM

381

wit h inflammation. The function of mast cells is in dispute. They contain heparin (Holmgren and Wilander, 1937; Jorpes, 1937; and others), histamine (Riley and West, 1953, 1955), which has been demonstrate d fairly certainly histochemically (Lagunoff, Phillips and Benditt, 1961), and serotonin (Benditt et al., 1955; Asboe-Hanse n et al, 1959). There are marked species differences in the composition, distribution and various other features of mast cells. They stain in different ways in different species. For example, in mice they stain brilliantly wit h orcein, but orceinophilia is absent in man. In mice and rats mast cells colour intensely with the Hale method, but in man some may fail to stain or stain only slightly. Mast cells in man frequently reject the aldehyde fuchsin stain except after peracetic acid oxidation, which induces intense staining. Mast cells in different species stain with varying intensities with alcian blue. Some human mast cells stain intensely with the periodic acid-Schiff method for carbohydrate , and others may stain only faintly. Mast cells are selectively identified by the metachromasi a which they exhibit after staining wit h certain basic dyes such as azure A at low p H. The metachromasi a frequently appears to be imparted by large cytoplasmic granules in fixed preparations . Many authors have attributed metachromasia to the presence of heparin (which they presumed to be a component of the mast cell granules); however, Julén, Snellman and Sylvén (1950), Sylvén (1951) and Hedbom and Snellman (1955) found heparin exclusively in the microsomal fraction and not in the fraction containing the large cytoplasmic basophilic granules obtained from mast cells in ox liver capsules . These authors concluded that, at least in this species, heparin is distributed entirely with the microsomal fraction and not with the large basophilic granules, although they could visualize the possibility that granules could become coated wit h the heparin fraction during preparation of the tissues for microscopy. The aldehyde fuchsin-stainabl e component of some mast cells may be digested with either lysozyme or

382

H.

M.

^-glucuronidase leaving the intensely metachromatic substanc e (Fullmer, 1959c). Some authors (Radden, 1962; Jorpes and Gardell, 1948) have noted an inverse relationship between staining intensities with the periodic acidSchiff and with metachromasi a methods in mast cells of rats. On this basis they have postulated that newly formed nonsulphate d heparin in mast cells may stain intensely with the periodic acidSchiff method and manifest weak metachromasia , if at all; but they speculate that with progressive sulphation increased metachromasi a and diminished staining with the periodic acid-Schiff method result. Mast cells concentrate large quantities of radioactive sulphate (Jorpes, Odeblad and Bostrom, 1953; Curran and Kennedy, 1955; Guidotti, 1957). The biological half-lif e of sulphur in mast cells was estimated to exceed 18 days (Jorpes et al, 1953). Sulphate in mast cells is presumed to be associate d with mucopolysaccharid e production. Enzymes that have been detected in mast cells include alkaline phosphatas e (Wislocki and Dempsey, 1946; Norback and Montagna, 1946; Dalgaard and Dalgaard, 1948; Riley, 1953), acid phosphatas e (Noback and Montagna, 1946; Wislocki, Bunting and Dempsey, 1947; Montagna and Noback, 1948), cytochrome oxidase (Montagna and Noback, 1948), nonspecific esteras e (Montagna, 1962) and a trypsin-like enzyme (Glenner and Cohen, 1960; Hopsu and Glenner, 1963). Kirkman (1950) and Compton (1952) could not confirm the presence of cytochrome oxidase in mast cells. I . EPITHELIAL CELL RESTS

Epithelial cell rests of Malassez are frequently observed in the periodontal membrane of the deciduous and permanent teeth of man and other mammals (Fig. 35). They are the persistent remains of Hertwig's epithelial root sheath. Reitan (1961) investigated the cell rests of Malassez in m an and dogs during the course of orthodontic procedures . He mentions that, although

FULLMER

Fig. 35. Epithelial rests (B) in the periodontal membrane of a 12-year-old child. Haematoxylin and eosin. χ 260. (From Reitan, 1961.)

credit is usually given to Malassez for the discovery of these cell rests, Malassez states in his paper (1885) that they were observed originally by Serres in 1817. According to Malassez also, Kollike r (1852, 1867) and Legros and Magitot (1879) believed that the epithelial rests were present only in the young. Malassez, however, demonstrate d the existence of these cell rests in the periodontal membrane s of adults. Cell rests have been investigated by Mummery (1921), Noyes (1930), Bruszt (1932), Fischer (1932), Meyer (1932), Orban (1944) and Loe and Waerhaug (1961). The consensu s of opinion is that the number of cell rests diminishes with increasing age. Waerhaug (1958) and Loe and Waerhaug (1961) have suggeste d that the rests in some manner may protect roots from resorption. Black (1887), Robinsohn (1926) and Higaki (1932) entertained the notion that they may posses s a glandular function. Reitan recorded the incidence and location of cell rests in the periodontal membranes of 25 teeth

21. C O N N E C T I VE

T I S S UE

C O M P O N E N TS

OF

P E R I O D O N T I UM

383

taken from 11- and 12-year-old children, and compared them with those in human adults, dogs and monkeys. He found epithelial rests associate d wit h all the teeth of the children. More rests were found in the specimens from children than those from adults and more rests were found in children than in dogs. The effects on the periodontium of pressure s associate d with appliance-induce d movements of teeth have been studied by Reitan (1961). He found that, when the force is considerable , hyalinization of the periodontal membrane occurs on the side of compressio n (Fig. 36). Subsequen t to this type of severe injury, connective tissue cells and the epithelial cell rests die. Regeneratio n of the connective tissues follows, but not of the epithelial cell rests (Figs. 37, 38). Less pressure results in bone resorption, loss of fewer fibroblasts and cell rests and more rapid repair (Fig. 39). A n enzymic histochemica l study of cell rests of Malassez has recently been conducted by Ten Cate

Fig. 37. Initial stage of recovery of human periodontal membrane following continuous pressure for 8 days. Epithelial cell rests are absent; they do not regenerate . Haematoxylin and eosin. χ 150. (From Reitan, 1961.)

Fig. 36. Results of a continuous force of 60 gm for 5 days on the cervical third of a human periodontal membrane. Connective tissue cells have atrophied but cell rests at A have survived longer. There is hyalinization of the membrane. Haematoxylin and eosin. χ 340. (From Reitan, 1961.)

Fig. 38. Later stage of cell proliferation. Centre of hyalinized tissue located at A. Resorption continues in the region B. Haematoxylin and eosin. χ 140. (From Reitan, 1961).

H.

384

M.

FULLMER

part of the roots of teeth is typically acellular. A t the apices of teeth, and to a variable extent beyond, cementum contains cells, situated in lacunae which are interconnecte d by canaliculi. The distribution of the lacunae in cellular cementum is, however, less regular than in bone and furthermore the majority of the canaliculi of cementum are directed towards the outer surface of the tissue. Cementum formation, especially at the apices of teeth, progresse s slowly throughout life, but wit h an irregular rhythm. Cementum formation at the cervices of teeth proceeds more slowly than elsewhere and may cease altogether. Cementum formation in this region is connected with the process of migration of the dento-gingival junction in ways that have been speculate d upon but are by no means understood . The process of migration of the dento-gingival junction in due course, of course, leaves the cervical cementum exposed to the oral environment. Bass (1951) has described alcohol-soluble granules in this exposed cementum. The granules are situated throughout the substanc e of the cementum. Fig. 39. Bone resorption due to slight continuous compressio n of the human periodontal membrane for ; 2 weeks. A, long strand of epithelial cells; B, cementoblast C, osteoclast . Haematoxylin and eosin. ÷ 80. (From Reitan, 1961).

(1965). Staining reactions for D P N- and T P Ndiaphorase s and for succinic, lactic and glucose-6phosphate dehydrogenase s were obtained. The presence of glycogen was determined by application of the periodic acid-Schiff method. The staining reactions indicate that these cells posses s the capacity for glycolysis and the operation of the citric acid cycle and the pentose shunt. Thus the histochemical tests do not suggest that the cells are in a state of metabolic "rest". J.

CEMENTUM

1. Introduction Cementum is a tissue which very much resembles bone in structure and chemical composition. However, the cementum which invests the greater

2.

Histochemistry

By virtue of its collagen content, cementum stains red with the Van Gieson stain and blue wit h the Masson and Mallory trichrome stains. Most cementum stains orange with the phosphotungstic acid haematoxylin and phosphomolybdi c acid haematoxylin methods, but sometimes interlamellar regions stain blue. Cementum is generally eosinophilic, but interlamellar regions may stain blue with haematoxylin, as they frequently do in bone. Cementum stains with methods that identify protein, i.e. red with the dinitrofluorobenzene-H acid and pink with the alloxan-Schiff methods. I t stains intensely with the periodic acid-Schiff method for carbohydrate . Cementum probably also contains acid mucopolysaccharides , for it generally, but not always, stains metachromaticall y after exposure to azure A at pH 4. Furthermore, it stains with aldehyde fuchsin after peracetic acid oxidation and blue with the Hale method. I n sections stained with the Rinehart modification

21.

C O N N E C T I VE

T I S S UE

C O M P O N E N TS

OF

P E R I O D O N T I UM

385

of the Hale method, cementum stains red by virtue of its collagen. Baratieri (1960a,b) studied cementum with the combined PAS-alcian blue technique. Lorber (1951) is one of the earliest investigators of cementum and alveolar bone with histochemica l methods. Parvis and Roncoroni (1950) and Paynter and Pudy (1958) also made histochemica l observations . 3. Cementoblasts and

Cementocytes

Valid histochemica l observations on cementoblasts and cementocyte s used to be difficult to acquire because of their association with a mineralized tissue. Strong mineral acids used previously for demineralization resulted in the loss of nucleic acids and other cellular disturbances . Recently developed methods of demineralization which adequately preserve enzymic activity now permit routine investigations of these important cells (Balogh, 1962; Fullmer and Link, 1964). Baratieri (1955) has noted that cementoblast s and cementocyte s manifest alkaline phosphatase . Cementoblast s and cementocyte s have been shown to exhibit succinic dehydrogenase , DPN-malic, isocitric, glutamic, lactic, ^-glycerophosphat e and D( —)-jS-hydroxybutyric dehydrogenases , TPN-6phosphogluconat e dehydrogenas e and D P N- and TPN-diaphorase s (Fullmer, 1964a, 1965b). Cementoblasts and active cementocyte s also show acid phosphatas e (Fig. 40) and nonspecific esteras e activity. Cementocyte s in the interior of cementum generally exhibit less enzymic activity than those located near the periphery, and, in turn, cementocytes near the periphery generally manifest greater enzymic activity than adjacent cementoblasts . Wit h few exceptions, the enzymic activities displayed by osteoblast s adjacent to the lamina dura exceed that of cementoblast s adjacent to the teeth. No enzyme in cementoblast s has been observed to surpass the activity of the same enzyme i n osteoblast s in comparable regions of the periodontium. Wit h some enzymes, such as the D P N- and TPN-diaphorases , osteoblasts , cementoblast s and fibroblasts of the periodontal

Fig. 40. Apex of a lower molar of an adult rat, stained with the Barka method for acid phosphatase . Activit y (dark stain) in cementoblasts , cementocyte s and cells of the periodontal membrane (PM). Cm, cementum; D, dentine. Demineralized section, ÷ 243.

membrane present a nearly uniform appearanc e (Figs. 41-43). K . ALVEOLA R BONE

1. Introduction I t would be inappropriate to attempt here a comprehensiv e account of the structural and chemical organization of bone in general. For such an account the reader is referred to Bourne (1956). Alveolar bone is essentially the same as bone elsewhere and this text wil l be confined to a consideration of certain aspects of new knowledge which appear to be relevant to the tooth supportive functions of this tissue and relevant to further research in this field.

386

H.

M.

Fig. 41. Mesio-distal section through a lower molar of an adult rat, stained for DPN-diaphorase . Activit y in odontoblasts , cementocytes , osteoblasts , osteocytes and cells of the periodontal membrane (PM). Cm, cementum. Demineralized section, ÷ 170.

A typical periosteum covers the lingual and facial aspects of the alveolar bone—a periosteum which, as elsewhere , contains numerous elastic fibres many of which enter the bone. The fibrous connective tissue which lines the bony sockets and serves to attach the teeth to the bone, namely the periodontal membrane, contrary to the view expresse d in many textbooks, should not be regarded as periosteum. I n the first place, unlike periosteum, the periodontal membrane does not contain elastic fibres; secondly, oxytalan fibres which comprise part of the fibrous structure of periodontal membranes , are not constituents of periosteum; thirdly, the entire fibrous structure of the periodontal membrane is specifically organized for a tooth supportive function and does not resemble periosteal structure.

FULLMER

Fig. 42. Mesio-distal section through a lower molar an adult rat, stained for TPN-diaphorase . Activit y odontoblasts (o), cementocytes , cementoblast s and cells the periodontal membrane (PM). Cm, cementum; dentine. Demineralized section, ÷ 325.

2. Chemical

of in of D,

Composition

Approximately 70% of normal human bone consists of mineral salts with a small amount of water (Strandh, 1961a,b; Strandh and Bengtsson 1961a, b) and the remaining 30% is composed of organic material of which the principal constituent i s collagen. According to Eastoe (1956a), bone contains an "osseomucoid " and an unidentified "resistant protein" in small amounts. Leblond et al. (1957) found hexose 4.5%, hexosamine 3.1%, hexose-hexosamin e 1.4%, sialic acid 4.0%, methylpentose 0.52%, and hexose-methylpentos e 9.0% of dry weight in the bones of Sherman rats. Chondroitin sulphates A and C, hyaluronic acid and keratosulphat e have been isolated from bone (Meyer et al., 1959; Roseman, 1959). Bone also contains variable a m o u n ts of fats.

21. C O N N E C T I VE

T I S S UE

C O M P O N E N TS

OF

P E R I O D O N T I UM

387

1958; Strandh, 1961a,b; Strandh and Bengtsson , 1961a,b). 3.

Microradiography

The distribution of inorganic constituents in bone may be studied by quantitative microradiography, a method developed by Engstrom (1946, 1950, 1953) and Engstrom and Wegstedt (1951). It consists essentially in the passag e of X-rays of appropriate wavelength through a thin bone specimen in contact with a fine-grained photographic emulsion. The following formula expresse s the X-ray absorption of the sample: Ε = In -f- = — ι ÷ mt: + — σ ÷ ma I Ρ Ρ

Fig. 43. Mesio-distal section through a lower molar of an adult rat, stained for glutamic dehydrogenase . Activit y in cementocyte s and cells of the periodontal membrane (PM). Cm, cementum; D, dentine. Demineralized section. X145.

Approximately two-thirds of dried bone, by weight, is inorganic. Carlstrom (1955a) defatted bones, dried them at 100°C and analyzed them quantitatively for minerals. He found the following mean values in percentag e by weight: calcium, 27 ; phosphorus , 12; C O 2 , 4; citrate, 1; Mg, 0.5; Na, 0.5; K, 0.05; F, 0.02. There is evidence to indicate that bone salt composition may vary in different species (Klement, 1929), in population samples of the same species (Cartier, 1949; Klement, 1929; MacDonald, 1954; Weinges, Leppelman and Hartl, 1953) as well as individual variations (Strandh, 1960a,b; Strandh and Bengtsson, 1961a,b). Increased mineralization wit h increased age has also been reported (Baker, Butterworth and Langley, 1946; Cartier, 1949; Strobino and Farr, 1949; Weidman and Rogers,

Ε is the X-ray extinction, I0 is the intensity of the incident and / the intensity of the transmitted X-rays, μ/ρ is the mass absorption coefficient (cm2 · g m -1) and m the mass in gm c m -2. The subscripts / and ο represen t the inorganic and organic fractions of bone, respectively (Carlstrom and Engstrom, 1956). The fil m is processed , enlarged under a standard procedure and the distribution of mineral salts determined on the basis of radiographic density according to the above mathematica l expression . Some objects as small as 0.5 μ can be resolved with the use of this method. Recently, Combee and Engstrom (1954) developed a microradiographic method for the quantitative analysis of organic matter in decalcified specimens . 4. X-ray

Diffraction

The crystalline structure of bone may be studied by X-ray diffraction methods. The two principal types of X-ray diffraction procedures are high-angle diffraction and low-angle diffraction. High-angle diffraction patterns examined in powder cameras disclose data on the size of the unit cell. The study of single crystals with wide-angle diffraction methods yields information about the positions of constituent atoms. The wide-angle diffraction study of bone has been hampered by the small

388

H.

M.

size of the constituent crystallites, which hinders precise resolution. Low-angle diffraction methods are useful in the study of collagen in bone, although collagen also gives a high-angle pattern. If the specimen is highly organized, the low-angle diffraction method can yield data on particle orientation and size. The method consists of the discharge of X-rays to a specimen at low angle. Reflections from X-rays, which are believed to originate from a series of parallel reflecting planes, are recorded on a photographic plate. A n especially efficacious collimation system is required for adequate resolution in low angle diffraction studies. The reader is referred to studies by Bolduan and Baer (1949) and Finean (1953) for descriptions of lowangle cameras and the theory of collimation systems. Texts useful for the interpretation and use of X-ray diagrams are those by Bunn (1946), Henry, Lipson and Wooster (1951), Guinier (1952), Wyckoff (1949) and Klu g and Alexander (1954). 5. Electron

Diffraction

The inorganic constituents of bones can be identified by the use of electron diffraction in the electron microscope, and their crystal orientation can be determined in very small specimens . Limitations are that (a) only relatively short interplanar spacings can be assesse d due to the relatively short wavelengths of electrons, (b) electrons scatter more than X-rays and (c) electron bombardmen t causes heating of the specimen which may foster crystal growth and chemical changes. The reader is referred to reports by Wyckoff (1949), Bretschneide r (1952), Dalton (1953) and C. E. Hall (1953) for technical details. Polarized light may also be useful for the study of mineralized tissues (Bunn, 1946; Burri, 1950; Frey-Wyssling, 1953). Finean and Engstrom (1953), Carlstrom and Finean (1954) and Carlstrom (1954, 1955a,b) estimate that the dimensions of crystallites in bone are of the order of 220 X 65 A. The inorganic crystallites are orientated parallel to the collagen

FULLMER

fibres in such a manner that 3 crystals fil l the 640 A period of collagen. Due to the small size of bone crystals, their surface area is large, and Engstrôm has calculated that 1 gm of bone salt has a surface area of 130 square metres. The large surface area may favour the adsorption of other ions such as carbonate, and may account for the failure of the appearanc e of calcite lines on X-ray diagrams. The difference in organization between highly and lowly mineralized osteons is unknown. 6.

Microchemistry

Strandh (1960a,b; 1961a,b; Strandh and Bengtsson, 1961a,b) combined microradiographic methods (Amprino and Engstrôm, 1952; Engfeldt and Engstrom, 1954) with microchemical determinations of single osteons. He developed a dissection technique which permitted the isolation of single Haversian systems. The procedures consisted of microradiography of cleansed thinly ground bone samples, the identification of individual Haversian systems, the dissection of solitary systems and subsequen t quantitative analyses of each for calcium, phosphorus and nitrogen (Strandh, 1960a, b). He noted that osteons generally contained a uniform distribution of nitrogen although phosphorus varied as much as 25% between regions of high and low mineralization. With refined procedures, it is possible to ascertain some variations in nitrogen distribution in very small regions in individual osteons (Engfeldt, 1958; Engfeldt and Strandh, 1960). Strandh (1961a,b; Strandh and Bengtsson , 1961a,b) could find no significant variation in the calcium : phosphorus ratios in the different types of bone structures, or with deviate degrees of mineralization in samples of the same individual. However, analyses of the calcium : phosphorus , calcium : nitrogen and phosphorus : nitrogen ratios of bone samples from 5 persons aged 9 months, and 6, 17, 52 and 76 years revealed relatively high ratios in the specimen aged 9 months, slightly depresse d ratios in specimens from individuals 6 and 17 years and subsequen t elevation of the ratios in the two specimens of more advanced age

21. C O N N E C T I VE

T I S S UE

C O M P O N E N TS

(Strandh, 1961 a,b ; Strandh and Bengtsson , 1961a, b) (Table 5 and Fig. 44). 7.

Histochemistry

Bone stains with methods that identify collagen, i.e. blue with the Mallory, Masson and other trichrome stains, and red with the Van Gieson stain. It is generally intensely eosinophilic, except between some lamellae where it may stain with haematoxylin. The significance of this haematoxyphili a is unknown. Varying degrees of blackening of bone occurs in sections stained for reticulin fibres. The dinitrofluorobenzene-H-aci d and the alloxan-Schiff stains for protein stain bone intensely. Bone and dentine produced in the scorbutic state fails to stain with the alloxan-Schiff

OF

P E R I O D O N T I UM

389

method (Burns, Fullmer and Dayton, 1959; Fullmer, Martin and Burns, 1961). Bone stains intensely with the periodic acidSchiff method for carbohydrates . The intensity of this staining reaction in bone and other connective tissues has been directly correlated with the amount of hexosamine , galactose , fucose and sialic acid therein (Leblond et al, 1957). The allochrome stain (a periodic acid-Schiff procedure with a subsequen t picro-aniline blue counterstain developed by Lillie , 1951) applied to bone sections results in the retention of the red (periodic acidSchiff reactive) stain in some parts and the total or partial acquisition of the counterstain in other parts resulting in varied colours. Bone stains with methods that identify acid

Table 5 Ca:P,

Ca:N

AND P:N

RATIOS FOR MICROSCOPIC BONE TISSUE STRUCTURES

OF DIFFERENT TYPES AND VARYIN G X - R A Y ABSORPTION, DISSECTED O UT OF TRANSVERSE SECTIONS FROM HUMA N FEMORAL SHAFTS OF VARIOUS A G E S0

Type of bone structure

Degree of X-ray absorption

Haversian systems

Low

Relatively low High

Endostea l bone

Relatively low

Periostea l or High interstitial bone Segment of or whole transverse section

Ratio Ca:P Ca:N P:N Ca:P Ca:N P:N Ca:P Ca:N P:N Ca:P Ca:N P:N Ca:P Ca:N P:N Ca:P Ca:N P:N

9 months 2.42 4.36 1.81 2.44 4.83 1.98 2.42 5.17 2.14 2.46 4.61 1.88 2.42 5.51 2.29 2.37 4.90 2.07

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.03 0.07 0.04 0.03 0.06 0.01 0.02 0.07 0.02 0.04 0.11 0.04 0.09 0.15 0.10

6 years 2.25 4.36 1.94 2.26 5.10 2.28 2.29 5.77 2.52 2.31 5.26 2.27 2.26 5.93 2.61 2.24 5.43 2.42

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.04 0.14 0.07 0.02 0.07 0.04 0.04 0.04 0.04 0.02 0.07 0.02 0.02 0.08 0.02

17 years 2.39 4.32 1.81 2.37 5.25 2.19 2.34 5.67 2.40 2.34 5.48 2.35 2.30 5.98 2.59 2.31 5.65 2.45

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.04 0.08 0.05 0.02 0.06 0.03 0.02 0.07 0.02 0.02 0.06 0.02 0.02 0.03 0.02

52 years 2.39 4.56 1.91 2.41 5.50 2.28 2.43 6.04 2.50 2.46 5.64 2.29 2.42 6.16 2.55 2.42 5.78 2.39

± 0.02 ± 0.12 ± 0.05 ± 0.01 ± 0.14 ± 0.05 ± 0.03 ± 0 . 15 ± 0.05 ± 0.04 ± 0.16 ± 0.05 ± 0.03 ± 0.10 ± 0.04

76 years 2.54 5.23 2.05 2.48 5.69 2.30 2.53 6.22 2.46 2.53 6.00 2.37 2.51 6.52 2.59 2.46 6.03 2.45

± ± ± ± ± ± ± ± ± ± ± ± ±

0.02 0.09 0.03 0.05 0.09 0.02 0.04 0.02 0.04 0.02 0.05 0.02 0.03 dz 0.10 ± 0.01

a Values are means and standard deviations. Each mean is based on 5 or 6 separate analyses . Also recorded are the Ca:P, Ca:N and P:N ratios in correspondin g nitrogen standard solutions. The Ca:N ratio generally increases with increasing age. (From Strandh, 1961b.)

390

H.

M.

FULLMER

Fig. 44. Micro radiograms of ground sections, 3 33 μ thick, from femoral shafts of human subjects of various ages. (A) 9 months; (B) 6 years; ( C) 17 years; (D) 52 years; ( E) 76 years. Many Haversian systems with low X-ray absorption (dark) are evident in specimens from younger individuals, whereas fewer are evident in specimens from older individuals, χ approx. 14. (From Strandh, 1961b.)

21. C O N N E C T I VE

T I S S UE

C O M P O N E N TS O F

mucopolysaccharides , i.e. metachromaticall y with dyes that manifest metachromasia , and blue with the Hale and alcian blue methods. Application of the Van Gieson picric acid fuchsin mixture after the Hale stain, as in the Rinehart-Abul-Haj method, results in the retention of the blue stain (for acid mucopolysaccharide ) in some areas and the acquisition of the counterstain in others in a manner similar to that of the allochrome stain as just mentioned. 8.

Osteoblasts

The specialized connective tissue cells that produce bone have ovoid or spherical nuclei which are frequently vesicular and eccentrically placed. The cytoplasm of active osteoblast s stains intensely wit h basic dyes indicating abundant ribonucleic acid. It is rich in mitochondria and a distinct Golgi apparatus is generally demonstrabl e with the appropriate stains. Osteoblast s may or may not contain glycogen. Alkalin e phosphatas e has been noted in osteoblasts ever since Gomori (1939) first developed the method for the histochemica l detection of this enzyme. Other work in which alkaline phosphatas e is recorded in osteoblast s includes Gomori (1943), K a b at and Furth (1941), Freeman and McLean (1941), Lorch (1947), Pritchard (1952, 1956) and Zorzoli and Nadel (1953). Alkaline phosphatas e has been described in both the nuclei and cytoplasm of osteoblast s (Siffert, 1951; Morse and Greep, 1951; Mâjno and Rouiller, 1951). The consensu s of opinion seems to be that alkaline phosphatas e appears first in the nuclei and later in the cytoplasm of osteoblast s during embryogenesis . Cleland's (1950) demonstratio n of the profound affinity of chromosome s for calcium phosphate , which is used in the Gomori procedure for alkaline phosphatase, should warn histochemists to exercise great caution in their interpretation of nuclear localization of phosphatas e on the basis of this method. A stain which indicates alkaline phosphatase activity in red and succinic dehydrogenas e i n blue has been described (Fullmer, Link and Baer, 1964). It is sometimes useful for the

P E R I O D O N T I UM

391

discrimination of osteoblast s or osteocytes from osteoclasts . The function of alkaline phosphatas e in bone formation is unknown. Great quantities of alkaline phosphatas e is present in osteoblast s as well as in the intercellular substance s that are involved in ossification. However, large amounts of alkaline phosphatas e are evident concomitant with the course of formation of all connective tissue intercellular substances , such as during embryogenesi s and in reparative processe s irrespective of whether or not they later mineralize. These observations discourage the view that alkaline phosphatas e is specifically related to the mineralization process. The presence of acid phosphatas e activity in osteoblasts was first demonstrate d by Cabrini et ai (1962) (Fig. 33). According to Lipp (1959) and Fullmer (1964a), osteoblasts exhibit aminopeptidas e activity, but Burstone (1960) reported that they do not. It has been reported that osteoblast s contain galactosidas e (Schlager, 1959), ^-glucuronidase (Gubisch and Schlager, 1961), cytochrome oxidase (Follis, 1948; Folli s and Berthrong, 1949) and esteras e (Figs. 45 and 46). The presence of DPN-diaphorase , T P Ndiaphorase , succinic dehydrogenase , DPN-malic and lactic dehydrogenases , and TPN-glucose-6 phosphate dehydrogenas e in osteoblast s has been reported by Walker (1961), Balogh, Dudley and Cohen (1961), Herrmann-Erlee (1962) and Fullmer (1964a,b, 1965a,b). Balogh et al (1961), HerrmannErlee (1962) and Fullmer (1964a,b, 1965a,b) noted the presence of D(—)-jS-hydroxybutyric dehydrogenas e in osteoblast s (Fig. 47). Walker (1961) failed to demonstrat e D P N- and T P Nisocitric dehydrogenas e in osteoblasts , Balogh et al (1961) and Herrmann-Erlee (1962) noted the presence of TPN-isocitric in osteoblasts , and Fullmer (1964b, 1965b) reported the presence of both these enzymes in osteoblasts . Osteoblast s also exhibit DPN-glutamic dehydrogenas e (Fig. 48) (Herrmann-Erlee , 1962, Fullmer, 1964b, 1965b), TPN-6-phosphogluconate , malic and á-glycerophosphate dehydrogenase s (Fullmer, 1964b, 1965b).

H.

392

M.

FULLMER

Fig. 46. Mesio-distal section mandible of 5-day-old rat, stained with the Holt method for nonspecific esteras e and counterstaine d with carmalum for nuclei. Arrow indicates osteoblasts with blue (dark) dots of positive reaction. db, developing bone, ÷ 135.

• i

Fig. 45. Distal end of femur of young rat stained with e and counterstaine d the Holt method for nonspecific esteras with carmalum for nuclei. The field shows periosteum just proximal to the metaphysis where there is considerable osteogenic activity. Intense esteras e activity (dark dots) is manifest in large osteoclasts , and less is evident in smaller osteoblast s of the periosteum (P). F, femur. Demineralized section, ÷ 350. These results sugges t the presenc e of active citric acid and pentose cycles in osteoblasts . Recent data sugges t that D(—)-/3-hydroxybutyric dehydrogenas e may play a critical role in osteogenesis and dentinogenesis . Severe depressio n of the activity of this enzyme has been noted in osteoblast s and odontoblasts , but not in osteoclasts , concurrent with the developmen t of the scorbutic state (Fullmer and Martin, 1964). The relation

Fig. 47. Mesio-distal section through mandible from 5-day-old rat, stained for D( — )-/8-hydroxybutyric dehydrod with carmalum for nuclei. genase and counterstaine s (arrow) and Greatest activity is exhibited by osteoclast lesser by osteoblast s which surround the alveolar bone (AB). X225.

21. C O N N E C T I VE T I S S UE C O M P O N E N TS O F P E R I O D O N T I UM

Fig. 48. Margin of mandible from 5-day-old rat, stained for glutamic dehydrogenas e and counterstaine d with carmalum for nuclei. Dark granules in osteoblast s (arrow) indicate glutamic dehydrogenas e activity. AB, alveolar bone. X300.

between the activity of this enzyme and the concentration of ascorbic acid has not yet been defined. However, the observation is in accord wit h the known failure of osteogenesi s and dentinogenesis concomitant with progressive bone resorption that characterize the scorbutic state. The data suggest that either D(—)-/3-hydroxybutyric dehydrogenas e is affected differently in bone and dentine formative cells as opposed to resorptive cells, or osteoclast s may obtain ascorbic acid during the course of bone resorption. The degree of enzymic activity displayed by osteoblasts and osteocytes depends upon the level of function of these cells at the time of the assay. I n adults, osteoblast s situated at the surface of bone, such as the lamina dura, display far less enzymic activity than osteoblast s in the same position during the course of developmen t of the alveolar process. Likewise, recently formed osteocytes situated near the periphery of a bone usually manifest greater enzymic activities than those located more centrally. Mineral metabolism of osteoblast s has been elucidated with the aid of autoradiographi c methods. The earliest investigators used lead

393

isotopes (Lomholt, 1930; Behrens and Baumann, 1933; Copp, Axelrod and Hamilton, 1947). Pecher (1942) used radiostrontium. More recently Leblond et al. (1950) used Ñ32 and Gross et al. (1951) and Tomlin, Henry and Kon (1953) used C a4 5. Use of P32 and C a45 is favoured because they are physiological minerals, and C a45 is more favoured because better radiographic images are obtained. Autoradiographic studies of bone by a number of authors, including Engfeldt and Engstrom (1954), Lacroix (1953, 1956) and Engfeldt and Hjertquist (1954), have disclosed a non-uniform uptake of levels of labelled calcium and phosphorus with greater uptake in the periosteal and endostea l regions and in particular osteons. Haversian systems with a high uptake appear to be newly formed osteons with a low X-ray absorption (Arnold and Jee, 1953, 1956; Cohen et al, 1957; Strandh, 1961a,b; Strandh and Bengtsson , 1961a, b). Although uptake by compact bone was generally related to the degree of mineralization, this relation was not always evident in endostea l and periosteal regions where uptake was always high (Strandh, 1961a,b; Strandh and Bengtsson , 1961a,b). Arnold and Jee (1956, 1957), Cohen et al. (1957) and Strandh (1961a,b; Strandh and Bengtsson , 1961a,b) found that the uptake of isotopes can vary as much as 11- to 35-fold. Also, the epiphysea l uptake may be greater than the diaphysea l by 2- to 3-fold (Manly and Bale, 1939; Leblond et al, 1950), and the uptake in cancellous bone may be higher than that in compact bone (Manly, Hodge and Van Voorhis, 1940). Studies of tracer levels of radioactive amino acids and other organic precursors of bone indicate that these organic constituents pass through cells before entering bone. This permits cellular control of intercellular substances . Experiments by Carneiro and Leblond (1959), Carneiro and de Moraes (1965), Young (1962) and Tonna (1962) disclose this action. In the exemplary experiments of Carneiro and Leblond, autoradiograph s of sections of bones from adult mice sacrificed 30 minutes after the injection of glycine-Ç3 revealed the label

Fig. 49A and Β.

For legend see opposite page.

21. C O N N E C T I VE

T I S S UE

C O M P O N E N TS

OF

P E R I O D O N T I UM

395

Fig. 49. Autoradiographs of sections of bones from adult mice sacrificed periodically after the injection of glycine-H3. Nuclei have been stained with haematoxylin. (A) Thirty minutes after injection. Activit y principally in osteoblasts . (B) Four hours after injection. Major activity in bone adjacent to osteoblasts . (C) Seven days after injection. (D) Forty-five days after injection, ÷ 200. (From Carneiro and Leblond, 1959.)

396

H.

M.

principally in osteoblast s (Fig. 49A). Four hours later the labelled product was located principally in bone adjacent to osteoblast s (Fig. 49B). Seven and forty-fiv e days later, the labelled product was situated progressively farther from osteoblast s due to continued osteoblastic activity (Figs. 49C and D). Recently Greulich (1953a,b) and Greulich and Leblond (1953) inferred that C1 4-bicarbonate probably entered the organic portion of bone inasmuch as it remained in demineralized bone. Bélanger (1954, 1956) found that S3 5-sulphate likewise entered the organic portion of bone. Bélanger reasone d that radioactive sulphate probably entered the mucopolysaccharid e moiety of bone inasmuch as it was removed by digestion wit h hyaluronidase . Dziewiatowsky (1951) also studied sulphur metabolism with the aid of isotopes. 9.

Osteocytes

Osteocytes exhibit a wide variety of enzymic activities, and the kind and degree of activity displayed depends largely upon local factors. For example, osteocytes newly formed from osteoblasts in the metaphyse s of growing animals may evince all the enzymes manifested by productive osteoblasts , though more often to a slightly less degree, whereas those in the diaphyses of older animals, or even sometimes in the younger ones, may not exhibit activity of this enzyme even in sections incubated for prolonged periods. Likewise, in alveolar bones, osteocytes near the periosteum and the lamina dura frequently exhibit more enzymic activity than do those situated farther internally. Osteocytes have been noted to exhibit succinic dehydrogenase , D P N- and TPN-linked malic and isocitric dehydrogenases , DPN-linked lactic, D(—)-j8-hydroxybutyric á-glycerophosphat e and glutamic dehydrogenases , TPN-linked glucose-6phosphate and 6-phosphogluconat e dehydrogenase s and T P N- and DPN-diaphorase s (Fullmer, 1964b, 1965b). Dehydrogenas e histochemica l studies of bone have also been conducted by Walker (1961), Balogh et al. (1961) and Herrmann-Erlee (1962).

FULLMER

Balogh et al. (1961) did not find 6-phosphogluconic, D(—)-/3-hydroxybutyric and glutamic dehydrogenase s in osteocytes , and Walker (1961) failed to obtain reactions for TPN-diaphorase , glucose-6-phosphate , 6-phosphogluconate , succinic, malic, isocitric and glutamic dehydrogenase s in osteocytes . Discrepancie s between these findings and my own may well be due to the use of higher coenzyme and substrate concentration s in my investigations. The failure of Herrmann-Erlee to obtain activities of succinic and á-glycerophosphat e dehydrogenase s in osteocytes can be explained only by the presumption the inhibitions were due to the conditions of tissue culture for 24 hours and the omission of menadione , which generally enhances the staining reaction, from the á-glycerophosphate dehydrogenas e incubation mixture. Newly formed osteocytes manifest esteras e activity (Fig. 46), whereas later they generally do not. Schlager (1959) has noted that osteocytes exhibit â-galactosidase , and Gubisch and Schlager (1961) observed ^-glucuronidase activity in osteocytes. ^-Glucuronidase activity, as visualized by the Hayashi method, is generally least in osteocytes , more in osteoblast s and most in osteoclasts . Osteocytes may exhibit acid phosphatas e (Fig. 33). Bélanger and Migicovsky (1963) have also demonstrated protease activity of osteocytes in chick and rat bones. 10.

Osteoclasts

Osteoclasts are large mobile cells that may have as many as 50 to 100 nuclei. They have been found in all places where bone is found, including the bony fish (Stephan, 1900; Jordan, 1925) and birds (Fell, 1925; Bloom, Bloom and McLean, 1941; Benoit and Calvert, 1952). They are always associate d with bone resorption, although bone resorption may occasionally be observed in the absence of recognizable osteoclasts . Howship (1817) first recognized a cavity surrounding osteoclasts , and since then these spaces have been called Howship's lacunae. Megakaryocyte s were later distinguished from osteoclast s (Robin, 1849, 1864), and Kolliker (1873) recognized a relation

Fig. 50. Electron micrograph of an active osteoclast . Very dark region in lower left is bone. Abundant mitochondria (m) are evident. Active regions of octsoclasts exhibit numerous cytoplasmic folds (arrow) which correspond to the striated border observed by light microscopy, x 14,000. (Courtesy of D. A. Cameron.) 27

Fig. 51. Higher magnification of the active area of an osteoclast , showing the mineral and collagen fibres sometimes observed between the cytoplasmic folds, ÷ 56,000. (Courtesy of D. A. Cameron.)

21. C O N N E C T I VE

T I S S UE

C O M P O N E N TS

between osteoclast s and bone resorption. KirbySmith (1933) and Goldhaber (1960) have described the activities of livin g osteoclast s resorbing bone kept under observation in tissue culture. Osteoclasts have a characteristic striated border adjacent to bone (Figs. 50 and 51). The cytoplasm is generally acidophilic although occasiona l cells may posses s basophilic cytoplasm. Arey (1919) and Benoit and Calvert (1952) believe that the cytoplasm of newly formed osteoclast s may be basophilic and that of older cells acidophilic. This view is in accord with the belief that osteoclast s originate by a fusion of pre-existing cells. Many authors (Hancox, 1949, 1956; Bloom et al, 1941; Tonna and Cronkite, 1961a, b; Fishman and Hay, 1962; Schmidt, 1963) have postulated that histiocytes, macrophages , reticular cells, fibroblasts, osteoblasts and osteocytes unite to form osteoclasts . Tonna and Cronkite (1961a, b) studied the origin of osteoclast s by the use of tritiated thymidine and concluded that they may originate from osteoblasts inasmuch as osteoclast s were not initiall y labelled after injection of the radioactive tracer, but later became labelled after a time interval correspondin g to the process of fusion of osteoblasts which had become labelled earlier. The ultimate fate of osteoclast s is unknown. Mitochondria are abundant in osteoclasts . Arey (1919) described many granules in the cytoplasm of osteoclast s for which Barnicot (1947) accords a secretory function and which Fell (1925) and Hyslop (1952) suggest may be residues of mitochondria. The periodic acid-Schiff method for carbohydrate s identifies some granules in the cytoplasm of osteoclast s (Heller-Steinberg , 1951). Vacuoles may also be evident in the cytoplasm, and Barnicot (1947) noted the accumulation of neutral red in these vacuoles within a few minutes after the immersion of calvaria of mice into the dye promptly after sacrifice. Although alkaline phosphatas e has been noted i n occasiona l osteoclast s by a few authors, including Lorch (1947), Greep, Fischer and Morse, (1948) and Mâjno and Rouiller (1951), most osteoclast s do not manifest alkaline phosphatas e activity.

OF

P E R I O D O N T I UM

399

r

Fig. 52. Mesio-distal section through lower first molar of 5-day-old rat, stained with the Holt method for nonspecific esteras e and counterstaine d with carmalum for nuclei. The granules in osteoclast s (arrow) indicate esteras e activity, x 100.

The reason for this is unknown. A n investigation of the relationship, if any, between alkaline phosphatas e activity, basophilia and newly formed osteoclasts might prove fruitful . Schajowicz and Cabrini (1957), Burstone (1959, 1960), Simaski and Yagi (1960) and Fullmer (1964a,b, 1965a,b) have reported acid phosphatas e in osteoclasts . Other hydrolytic enzymes displayed by osteoclast s include esteras e (Figs. 45 and 52), /3-glucuronidas e (Cabrini and Schajowicz, 1959; Gubisch and Schlager, 1961), â-galactosidas e (Schlager, 1959) and aminopeptidas e (Schlager, 1959). Burstone (1960) reported the presence of

400

H.

M

Fig. 53. Mesio-distal through lower first molar of adult rat, stained for DPN-linked glutamic dehydrogenase . Intense activity is displayed by osteoclast s (arrow), and odontoblasts (o), with lesser amounts by osteoblasts , osteocytes , fibroblasts, of the periodontium and cementocytes. Cm, cementum; AB, alveolar bone; D, dentine, x 124.

cytochrome oxidase, and Hancox (1956) noted that osteoclast s could liquefy a plasma clot. Fullmer (1964b) noted that osteoclast s manifest succinic dehydrogenase , DPN-malic, isocitric, glutamic (Fig. 53), lactic, /3-hydroxybutyric, and á-glycerophosphat e dehydrogenases , TPN-malic, isocitric, glucose-6-phosphat e and 6-phosphat e and 6-phosphogluconat e dehydrogenases , and D P Nand TPN-diaphorases . Previous workers had noted the presence of succinic dehydrogenas e (Schajowicz and Cabrini, 1960; Goldhaber, 1960; Yoshiki, 1962). Walker (1961) noted the intense activity of many enzymes in osteoclast s but failed to secure reactions for D P N- and TPN-isocitric dehydrogenases , TPN-malic dehydrogenas e and T P N-

FULLMER

diaphorase . The studies of Balogh et al. (1961) are generally in accord with the foregoing but they failed to elicit DPN-glutamic and TPN-6-phospho giuconate dehydrogenase s in osteoclasts . I have used higher coenzyme and substrate concentration s in incubating solutions and this probably accounts for the lack of accord with the findings of the other workers. Two impressive invariable findings evident throughout the course of investigations of the enzymic activities of bone cells were that (a) the qualitative enzymic activities of osteocytes , osteoblasts and osteoclast s were relatively uniform, and (b) the enzymic activities of osteoclast s generally greatly exceede d all other bone cells with osteoblasts exhibiting next greatest activity and osteocytes least. The gradations of reactions are interpreted as a reflection of a common cellular heritage, the differences in degree of activity being attributable to an adaptation to functional requirements . Bone formation and resorption is in equilibrium i n the adult. Both processe s can occur simultaneously at various sites. The mechanism s that operate to generate osteoclasts , and those which activate individual osteoclast s in an adult or during embryogenesi s are unknown. Parathormon e is known to produce increased bone resorption, and this is believed to be mediated through osteoclastic activity. However, the circulation of parathormone cannot explain the formation of osteoclasts at specific sites and the induction of osteoclastic activity at specific time intervals at these sites during embryogenesis , an action which, in conjunction with osteoblastic activity, results in a skeletal morphology characteristic of a species. Pressure on bone, such as occurs during the course of orthodontic tooth movement (Fig. 39), as well as inactivity of bone, such as occurs while bones are splinted, initiate osteoclastic resorption. Neuman and Neuman (1958) postulate that an increase in concentration of parathormone results in the formation and/or accumulation of lactic and citric acids in osteoclasts , which in turn effects demineralization of bone. Secondly, proteolysis

21. C O N N E C T I VE

T I S S UE

C O M P O N E N TS

and phagocytosis is presumed to consummate the process. Barnicot (1948) and Chang (1951) favour the view that parathormone has a direct effect on bone resulting in dissolution because bone resorption occurred in sites where parathyroid glands were transplante d close to bone. Other workers have postulated that the level of blood calcium affects activity of the parathyroid gland and thereby the parathormone level. Using mouse calvaria in tissue culture as an experimenta l system, Mecca, Martin and Goldhaber (1963) found that the addition of parathormone to the media resulted in an increased utilization of glucose, a marked increase in citrate production and a slight increase of lactate production concomitant with bone resorption. Sometimes giant multinucleated cells, foreign body giant cells, develop in relation to the presence of materials or tissues alien to the host. Several authors have noted that the degree and quality of enzymic activity manifested by these cells is remarkably similar to that displayed by osteoclast s (Schajowicz, Cabrini and Merea, 1961; Cabrini, Schajowicz and Merea, 1962; Irving and Handelman, 1963; Silverman and Shorter, 1963).

IV. PATHOLOGY OF THE SUPPORTING TISSUES Periodontal disease is a major cause of the loss of teeth in mankind generally. The disease is characterize d by the progressive loss of mineralized and nonmineralized connective tissues investing the teeth; and for this reason may be categorized as a connective tissue disorder. There is no valid evidence to support the view that periodontal disease is a specific disease in the sense that scurvy is due to a deficiency of ascorbic acid and tuberculosis is caused by the Mycobacterium tuberculosis. On the other hand, there is ample evidence to sustain the view that the loss of the supporting tissues surrounding the teeth is provoked, and associate d with, a variety of factors including

OF

P E R I O D O N T I UM

401

multitudes of microorganisms of various types, deposits of salivary calculus on the teeth, abnormalities in the arrangemen t and occlusion of the teeth, dietary imbalances and deficiencies, as well as a number of disease s of the host which may contribute to the condition. On this basis, a concept that provides a suitable basis for investigations of periodontal disease is one which conceives periodontal health to be an equilibrium and periodontal disease to be a disturbance of that equilibrium; that there can be either an increase of aggressive forces or a decreas e of host resistance favouring the generation of periodontal disease . Periodontal disease results in the destruction of bone, ravage of the original highly organized fibrous elements and partial replacemen t by a less specifically organized tissue which cannot possibly function as a periodontal membrane. The association of inflammation with periodontal disease is establishe d beyond question but, although this process contributes to a major degree toward the loss of the tooth-supporting tissues, the loss of periodontal structures cannot be completely explained on this basis. Evidence indicates that degradation of connective tissues and bone supporting the teeth can be effected in the absence of inflammatory cells, as during eruption of the teeth (Figs. 12-14) as well as in periodontal disease (Fullmer, 1961). The morphological appearanc e of the connective tissues is indistinguishable in both circumstances . The significance of this observation in relation to periodontal disease is that degradation of tissue may result in a loss of bone and fibrous connective tissues far in advance of the inflammatory lesion (Fullmer, 1961). Even though new collagen and bone may be formed in these regions, chemical and other evidence indicates that the newly formed collagen is more susceptible to enzymic digestion and solution in a variety of mixtures than is older collagen (Keech, 1955; Jackson and Williams, 1956; Jackson, 1957, 1958; Gross, 1958) and newly formed Haversian systems are more labile and subject to physiological demands than are older systems. Inasmuch as

H.

402

M.

degradation of connective tissues of the periodontium is not uniform, and a widely variable degree of maintenanc e is evident frequently between areas of microscopic proportions, investigations of factors promoting the formation, maintenance and degradation of mineralized and nonmineralized connective tissues of the periodontium is very difficult. However, an understanding of periodontal disease awaits the results of research that identifies such factors, because the status of either the mineralized or nonmineralized intercellular substance s in any region constitutes an account of the cells therein. A very promising clue which warrants systematic and exhaustive investigations is the recent finding of a collagenolytic factor produced by cultures of gingiva excised for the treatment of periodontal disease s (Fullmer and Gibson, 1966; Gibson and Fullmer, 1966).

V . C O N C L U D I NG R E M A R KS The foregoing attempts to review recent advances in knowledge in respect of the supporting tissues of the teeth and to review current ideas that are relevant to this subject. Further investigations in this field are likely to be rewarding. The discovery of a tissue collagenas e just referred to opens up certain possibilities of investigation; for example, to reveal (a) which type of cell produces the enzyme, (b) whether epithelial-connectiv e tissue interaction is required for enzyme formation, (c) whether the enzyme is produced by normal gingiva, (d) the possible role of the enzyme in periodontal disease , (e) the possible role of such enzymes in morphogenesis , (f) whether the enzyme is specific for collagen, (g) the more precise nature of the enzyme, (h) the nature of the action of the enzyme on collagen, and (i) whether the collagenas e activity is the result of a single or multiple enzymes. The significance of this collagenas e can only be surmised at present principally because the cellular source of the enzyme is unknown. For many years the existence of a tissue collagenas e has been

FULLMER

assumed and sought for. Collagen, once formed, does not persist indefinitely. There is a metabolic turnover, and metabolic turnovers are enzymically mediated. The major question is whether or not the detected enzyme is the one responsible for the metabolic turnover of collagen or is one produced by epithelial cells with assistanc e or information from connective tissue cells, as is the case with an enzyme isolated from tadpoles by Gross (1964). Methods could be developed for the assay of quantities needed for normal metabolism, and to determine whether alterations occur in disease s such as periodontal diseases , arthritis, scleroderma and vascular diseases . If the enzyme were related to diseases , a search for enzymic inhibitors might well be profitable. During the course of investigations of collagenase, Gross and Lapiere (1962), Lapiere and Gross (1963) and Gross (1964) considered the possibility that earlier attempts to isolate the enzyme failed because of a marked tendency for the enzyme to become attached to the substrate , thus remaining undetected . These workers surmounted the problem by culturing tissues under conditions that promoted the formation of an excess of enzyme, enzymic activity being subsequently detected by lysis of the collagen fil m upon which the tissues were cultured. These ideas and this technique could lead to the developmen t of new histochemica l methods. Existing histochemica l methods detect enzymes or other substance s present in the tissues at the moment the tissue was prepared. Very much needed is a method whereby tissue sections can be cut in which the cells, or at least a proportion of them, are still living, Then, perhaps after tissue culture, the localization of enzyme activity could be determined exactly in respect even of individual cells by lysis of a suitable substrate. Inasmuch as cells of the mineralized and nonmineralized connective tissues are concerned with the formation, maintenanc e and degradation of the intercellular substance s in their vicinity, and these functions are enzymically mediated, a qualitative assay of appropriate enzymes should signify

21. C O N N E C T I VE

T I S S UE

C O M P O N E N TS

theoretically the status of these cells in relation to the status of associate d intercellular substances . Until recently, it was impossible to study the enzymic activity of individual cells of the periodontal membrane and bone because of their association with mineralized tissues. The activity of most enzymes was lost during demineralization . The use of recently developed demineralization procedures now permits detailed analyses of several key enzymes in individual cells of hard tissues. Although large amounts of some of the soluble enzymes are lost during demineralization , thereby precluding quantitative assays , some remain for qualitative histochemica l study. Further studies should define the normal metabolic parameters of cells in relation to their function and location as well as in the altered metabolism associate d with diseases . Significant contributions from microchemical analyzes of the supportive tissues of the teeth may be expected. These tissues are comprised of heterogeneous cell types with sometimes widely varying degrees of enzymic activity, and therefore very small samples are necessar y for meaningful results. Correlations with histochemica l studies should be especially profitable. The accomplishment s of various workers utilizing isotopes and autoradiographi c methods for investigations of the supporting tissues of the teeth provide a basis for the prediction that further great strides wil l be made in these areas in the near future. These methods wil l be particularly useful for shedding light on the intricate metabolic steps concerned in the formation, maintenanc e and degradation of the supporting tissues of the teeth. Correlated autoradiographi c and electron microscopic methods are particularly suitable for such investigations. Several problems remain germane to oxytalan fibres. They are stainable with three (but not with two other) elastic tissue stains. The stainable component is readily digested by /3-glucuronidas e in sections previously oxidized with peracetic acid or, preferably, oxone. Examination of incompletely digested sections, however, shows the existence

OF

P E R I O D O N T I UM

403

of a component other than that digested, from which the deduction is made that oxytalan fibres are composed of at least two components . The elastic fibres in the same sections digested with ^-glucuronidase are largely unaffected. Initial electron microscopic observations (Goggins, 1965; Carmichael and Fullmer, 1966) also indicate the existence of two components in oxytalan fibres, i.e. long filaments 100-150 A in diameter and an interfibrillar amorphous component of approximately the same diameter (Fig. 27). Notwithstanding the data which differentiate oxytalan from elastic fibres, other data serve to associate them; namely (1) the morphology and arrangemen t of oxytalan fibres in the human periodontal membrane is identical with that of elastic fibres in periodontal membranes of some animals such as swine and cattle, (2) oxytalan rather than elastic fibres are usually found in the apical and middle thirds of periodontal membranes of cattle and swine, (3) the morphology and arrangement of oxytalan fibres at the apical and middle thirds of roots of teeth of cattle and swine are identical to that of elastic fibres situated elsewhere in the same periodontal membrane and to that of oxytalan fibres in the h u m an periodontal membrane, (4) developing (pre-elastic) fibres in man are readily identified with the oxytalan fibre stain prior to the stage when they can be stained with elastic tissue methods, (5) pre-elastic fibres have staining characteristic s and a susceptibility to ^-glucuronidase indistinguishable from those of oxytalan fibres, (6) elastic fibres develop in human periodontal membranes affected with scleroderma and (7) rarely, typical elastic fibres may develop in human periodontal membranes subjected to heavy occlusion. Further electron microscopic studies are needed to characterize the fine structure of oxytalan fibres in comparison with that of other connective fibres, including pre-elastic fibres. Furthermore, we need information concerning the elasticity of oxytalan fibres, their normal function and the nature of their participation in orthodontic tooth movements and i n various disease s of the periodontium.

404

H.

M.

References Alfert, M. and Geschwind, L I. (1953). A selective staining method for the basic protein and of cell nuclei. Proc. nat. Acad. Sci., Wash. 39, 991-999. Amprino, R. and Engstrôm, A. (1952). Studies on X-ray absorption and diffraction of bone tissue. Acta anat. 15, 1-22. Anderson, A. J. (1961). Some studies on the occurrence of sialic acid in human cartilage. Biochem. J. 78, 399-415. Arey, L. B. (1919). The origin, growth and fate of osteoclast s and their relation to bone resorption. Am. J. Anat. 26, 315-337. Arnim, S. S. and Hagerman, D. A. (1953). The connective tissue fibers of marginal gingiva. / . Amer. dent. Ass. 47, 271-281. Arnold, J. S. and Jee, W. S. S. (1953). Haversian system growth and formation in rabbits. Anat. Rec. 115, 276. Arnold, J. S. and Jee, W. S. S. (1956). Observations and quantitative radioautographi c studies of calcium45 deposited in vivo in forming Haversian systems and old bone of rabbit. Amer. J. Anat. 99, 291-308. Arnold, J. S. and Jee, W. S. S. (1957). Bone growth and osteoclastic activity as indicated by radioautographi c distribution of plutonium. Amer. J. Anat. 101, 367-417. Asboe-Hansen , G., Dyrbye, M. O., Moltke, E. and Wegelius, O. (1959). Tissue edema—A stimulus of tissue regenera tion. / . invest. Derm. 32, 505-507. Baer, R. S. (1952). The structure of collagen fibrils. Advanc. Protein Chem. 7, 69-160. Bahr, G. F. (1950). The reconstruction of collagen fibril s as revealed by electron microscopy. Exp. Cell Res. 1, 603-606. Bahr, G. F. (1951). Die Feinstruktur elastische r Fasern. Z. Anat. EntwGesch. 116, 134-138. Baker, S. L., Butterworth, E. C. and Langley, F. A. (1946). The calcium and nitrogen content of human bone tissue cleaned by micro-dissection . Biochem. J. 40, 391-396. Balogh, K. (1962). Decalcification with versene for histochemical study of oxidative enzyme systems. / . Histochem. Cytochem. 10, 232-233. Balogh, K. (1963). Histochemical study of oxidative enzyme systems in teeth and periodontal tissues. / . dent. Res. 42, 1457-1466. Balogh, K., Dudley, H. R. and Cohen, R. B. (1961). Oxidative enzyme activity in skeletal cartilage and bone. Lab. Invest. 10, 839-845. Bangle, R. and Alford, W. C. (1954). The chemical basis of the periodic acid SchifT reaction of collagen fiber with reference to periodate consumption by collagen and by insulin. / . Histochem. Cytochem. 2, 62-76. Baratieri, A. (1955). Ricerche istochimiche sulle localization della iosfatasi alcalina negli elementi del periodonto e del cemento. Riv. ital. Stomat. 10, 3-15.

FULLMER Baratieri, A. (1958). Ricerche istochimiche sulle localizzazioni della fosfatasi acida nella gingiva. Rass. trimest. Odont. 40, 285-307. Baratieri, A. (1960a). Alcuni aspetti istochimici del cemento radicolare e dello strato granulare di Tomes. Arch. ital. Biol. orale 1, 39-52. Baratieri, A. (1960b). Histochemical research on radicular cement. In "Les Parodontopathies" , Proc. 16th ARPA Congr. (R. M. Rohrer, ed.), pp. 170-175. Vienna. Barnicot, N. A. (1947). The supravital staining of osteoclast s with neutral red: their distribution on the parietal bone of normal growing mice, and a comparison with the mutants grey-lethal and hydrocephalus-3 . Proc. R. Soc. B134, 467-485. Barnicot, N. A. (1948). The local action of the parathyroid and other tissues on bone in intracerebra l grafts. / . Anat., Lond. 82, 233-247. Bass, C. C. (1951). A previously undescribe d demonstrabl e pathologic condition in exposed cementum and the underlying dentine. Oral Surg. 4, 641-652. Battista, A. F. (1949). The reaction of various tissues to implants of a collagen derivative. Canad. J. Res. E27, 94-104. Behrens, B. and Baumann, A. (1933). Zur Pharmakologie des Bleis. IX . Mitteilung. Weitere Untersuchunge n uber die Verteilung des Bleis mit Hilf e der Méthode der Autohistoradiographie . Z. Ges. exp. Med. 92, 241-263. Bélanger, L. F. (1954). Autoradiographic visualization of the entry and transit of S35 in cartilage, bone and dentine of young rats and the effect of hyaluronidase in vitro. Canad. J. Biochem. Physiol. 32, 161-169. Bélanger, L. F. (1956). Autoradiographic visualization of the entry and transit of S35 methionine and cystine in the soft and hard tissues of the growing rat. Anat. Rec. 124, 555-572. Bélanger, L. F. and Migicovsky, Â. B. (1963). Histochemical evidence of proteolysis in bone: The influence of parathormone. / . Histochem. Cytochem. 11, 734-737. Benditt, E. P., Wong, R. L., Arase, M. and Roeper, E. (1955). 5-Hydroxytryptamine in mast cells. Proc. Soc. exp. Biol., N.Y. 90, 303-304. Benoit, J. and Calvert, J. (1952). Sur l'existence de deux types d'ossification nettement distincts chez les oiseaux. /. Physiol., Paris 43, 645-646. Bensley, S. H. (1934). On the presence , properties and distribution of the intercellular ground substanc e of loose connective tissue. Anat. Rec. 60, 93-108. Bernick, S. (1960). The organization of the periodontal membrane fibres of the developing molars of rats. Arch, oral Biol. 2, 57-63. Bevelander, G. and Johnson, P. L. (1955). The localization of mucopolysaccharide s in developing teeth. J. dent. Res, 34, 123-131. Bignardi, C. (1940a). Sulla probabile presenza di un

21. C O N N E C T I V E

TISSUE

COMPONENTS

polisaccaride non esterificato nelle cellule mucose. Boll. Soc. ital. Biol. sper. 15, 593-594. Bignardi, C. (1940b). Cellule mucose e cellule mucoidi. IV . Esterificazione solforica della sostanza mucoide e sua dimonstrazione istochimica. Atti Soc. Nat. Mat. Modena 71, 59-61. Black, G. V. (1887). In "Dental Histology and Embryology" (F. B. Noyes, ed.), p. 274. Lea & Febiger, Philadelphia, Pennsylvania . Bloom, W., Bloom, M. A. and McLean, F. C. (1941). Calcification and ossification. Medullary bone changes in the reproductive cycle of female pigeons. Anat. Rec. 81, 443^75. Boedeker, H. and Doty, P. (1955). On the nature of the structural element of collagen. / . Amer. chem. Soc. 11. 248-249. Bolduan, E. A. O. and Baer, R. S. (1949). Effective use of collimating apertures in small-angle X-ray diffraction cameras . / . Appl. Phys. 20, 983-992. Bolduan, E. A. O., Salo, T. P. and Baer, R. S. (1951). X-ray diffraction studies of the penetration of stains and tans into collagen fibrils. / . Amer, heath. Chem. Ass. 46, 124-138. Bornstein, P., Martin, G. R. and Piez, K. A. (1964). Intermolecular cross linking of collagen and the identification of a new β component. Science 144, 1220-1222. Bourne, G. H., ed. (1956) ."The Biochemistry and Physiology of Bone." Academic Press, New York. Bowes, J. H., Elliot, R. G. and Moss, J. A. (1955). The composition of collagen and acid-soluble collagen of bovine skin. Biochem. J. 61, 143-150. Braden, A. W. (1955). The reactions of isolated mucopolysac charides to several histochemica l tests. Stain Tech. 30, 19-26. Bradfield, J. R. G. and Kodicek, E. (1951). Abnormal mucopolysaccharid e and "precollagen" in vitamin Cdeficient skin wounds. Biochem. J. 49, xvii-xviii . Bretschneider , L. H. (1952). The electron microscopic investigation of tissue sections. Int. Rev. Cytol. 1, 305-321. Bruszt, P. (1932). Uber die netzartige Anordnung des paradentale n Epithels. Z. Stomat. 30, 679-682. Bunn, C. W. (1946). "Chemical Crystallography". Oxford Univ. Press, London and New York. Bunting, H. (1950). The distribution of acid mucopolysac charides in mammalian tissues as revealed by histochemical methods. Ann. N.Y. Acad. Sci. 52, 977-982. Burns, J. J., Fullmer, H. M. and Dayton, P. G. (1959). Observations on vitamin C activity of D-ascorbic acid. Proc. Soc. exp. Biol, N.Y. 101, 46-49. Burri, C. (1950). "Das Polarisationsmikroskop" . Birkhuser , Basel. Burstone, M. S. (1959). Histochemical demonstratio n of acid phosphatas e activity in osteoclasts . / . Histochem. Cytochem. 7, 39-41.

OF

PERIODONTIUM

405

Burstone, M. S. (1960). Hydrolytic enzymes associate d with osteogenesi s and dentinogenesis . In "Calcification in Biological Systems". Publ. No. 64, pp. 217-243. Amer. Ass. Advanc. Sci., Washington, D. C. Cabrini, R. L. and Carranza, F. Á., Jr. (1951). Histochemical study on alkaline phosphatas e in normal gingivae varying the pH and the substrate . / . dent. Res. 30, 28-32. Cabrini, R. L. and Carranza, F. A. Jr. (1958). Histochemical distribution of acid phosphatas e in human gingiva. Periodont. 29, 34-37. Cabrini, R. L. and Schajowicz, F. (1959). Distribution histoquinica de la beta-glucuronidas a en el Tejido oseo y cartilageinoso . Rev. Or top. Traum., B. Aires 5, 193-198. Cabrini, R. L., Schajowicz, F. and Merea, C. (1962). Histoenzymologic behavior of the giant cell of foreign body granuloma as compared with the osteoclasts . Experientia 18, 322-325. Carlstrom, D. (1954). Micro X-ray diffraction techniques for use in histochemistry. / . Histochem. Cytochem. 2, 149-160. Carlstrom, D. (1955a). X-ray crystallographic studies on apatites and calcified structures. Acta radiol., Stockh. Suppl. 121. Carlstrom, D. (1955b). Particle size and chemical composition of the crystallites in bone and synthetic apatites. Biochim. biophys. Acta 17, 603-604. Carlstrom, D. and Engstrom, A. (1956). Ultrastructure and distribution of mineral salts in bone tissue. In "Biochemistry and Physiology of Bone" (G. H. Bourne, ed.), pp. 149-176. Academic Press, New York. Carlstrom, D. and Finean, J. B. (1954). X-ray diffraction studies on the ultrastructure of bone. Biochim. biophys. Acta 13, 183-191. Carmichael, G. G. and Fullmer, H. M. (1966). The fine structure of the oxytalan fiber. / . Cell Biol. 28, 33-36. Carneiro, J. and de Moraes, F. F. (1965). Radioautographi c visualization of collagen metabolism in the periodontal tissues of the mouse. Arch, oral Biol. 10, 833-848. Carneiro, J. and Leblond, C. P. (1959). Role of osteoblast s and odontoblasts in secreting the collagen of bone and dentin as shown by radioautograph y in mice given tritiu m labelled glycine. Exp. Cell Res. 18, 291-300. Carranza, F. Á., Jr. and Cabrini, R. L. (1955). Estudio histoquimico de la fosfatasa alcalina en los tejidos periodontales . Rev. Asoc. Odont. Argent. 43, 206-211. Cartier, P. (1949). Variations des anions organiques de l'os (citrates et lactates) au cours de la croissance . C.R. Soc. Biol., Paris 143, 37-39. Catchpole, H. R., Joseph, N. R. and Engel, M. B. (1956). Homeostasis of connective tissues. III . Magnesium-sodiu m equilibrium and interaction with strontium and lead. Arch. Path. 61, 503-511. Chang, H. Y. (1951). Grafts of parathyroid and other tissues to bone. Anat. Rec. I l l , 23-47.

406

H.

M.

Chapman, J. Á. (1961). Morphological and chemical studies of collagen formation. / . biophys. biochem. Cytol. 9, 639-651. Chapman, J. A. (1962). Fibroblasts and collagen. Brit. med. Bull. 18, 233-237. Cleland, K. (1950). A study of the alkaline phosphatas e reaction in tissue sections. Part II . Proc. Linn. Soc. N.S. W. 75, 55-69. Cohen, J., Maletskos, C. J., Marshall, J. H. and Williams, J. B. (1957). Radioactive calcium tracer studies in bone grafts. / . Bone Jt. Surg. 39A, 561-577. Combee, B. and Engstrom, A. (1954). A new device for microradiograph y and a simplified technique for the determination of the mass of cytological structures. Biochim. biophys. Acta 14, 432-434. Compton, A. S. (1952). A cytochemical and cytological study of the connective tissue mast cell. Am. J. Anat. 91, 301-326. Constant, T. E. (1902). The eruption of the teeth. C.R. 3e Congr. dent, int., Paris, 1900 Vol. 2, pp. 180-192. L'Odontologie, Paris. Copp, D. H., Axelrod, D. J. and Hamilton, J. G. (1947). The deposition of radioactive metals in bone as a potential health hazard. Amer. J. Roentgenol. 58, 10-16. Courts, A. (1960). Structural changes of collagen. The action of alkalis and acids in the conversion of collagen into eucollagen. Biochem. J. 74, 238-247. Cox, R. W. and Little, K. (1961). An electron microscope study of elastic tissue. Proc. R. Soc. B155, 232-242. Craig, J. M. (1956). Hale colloidal-iron procedure in certain metabolic storage diseases . Lab. Invest. 5, 62-71. Crumley, P. J. (1964). Collagen formation in the normal and stresse d periodontium. Periodontics 2, 53-61. Curran, R. C. and Kennedy, J. S. (1955). The distribution of the sulphated mucopolysaccharide s in the mouse. /. Path. Bact. 70, 449-457. Czerkawski, J. W. (1962). Elastin a sialoprotein. Nature, Lond. 194, 869. Dalgaard, E. and Dalgaard, J. A. (1948). Om masteeller og deres indhold af fosfatase en histologisk og histokemisk undersogelse . Ugeskr. Laeg. 110, 513-518. Dalton, A. J. (1953). Electron microscopy of tissue sections. Int. Rev. Cytol. 2, 403-416. Davies, D. V. (1952). Specificity of staining methods for mucopolysaccharide s of the hyaluronic acid type. Stain Tech. 21, 65-70. Dische, Z., Danilczenko, A. and Zelmenis, G. (1958). The neutral heteropolysaccharide s in connective tissue. Ciba Fdn. Symp., Chem. Biol. Mucopolysaccharides pp. 116-136. Dorfman, A. and Mathews, M. B. (1956). Physiology of connective tissue, Ann. Rev. Physiol. 18, 69-88. Dunphy, J. E. and Udupa, Ê. N. (1955). Chemical and histochemica l sequence s in the normal healing of wounds. New Eng. J. Med. 253, 847-851.

FULLMER Dziewiatkowski, D. D. (1951). Radioautographi c visualization of sulfur-35 disposition in the articular cartilage and bone of suckling rats following injection of labelled sodium sulfate. / . exp. Med. 93, 451-458. Eastoe, J. E. (1956a). The organic matrix of bone. In "The Biochemistry and Physiology of Bone" (G. H. Bourne, ed.), pp. 81-103. Academic Press, New York. Eastoe, J. E. (1956b). The amino acid composition of mammalian collagen and gelatin. Biochem. J. 61, 589-600. Eccles, J. D. (1959). Studies on the developmen t of the periodontal membrane . The principal fibres of the molar teeth. Dent. Practit. dent. Rec. 10, 31-35. Eccles, J. D. (1961). Studies in the developmen t of the periodontal membrane . The apical region of the tooth. Dent. Practit. dent. Rec. 11, 153-157. Eidinger, D. and Ghosh, A. (1956). The effect of mucolytic enzymes on tissues stained with the periodic acid Schiff technique. Histochem. Cytochem. 4, 200-207. Engel, M. B. (1948). Glycogen and carbohydrate protein complex in developing teeth of the rat. / . dent. Res. 21, 681-692. Engel, M. B. (1951). Some changes in the connective tissue ground substanc e associate d with the eruption of the teeth. / . dent. Res. 30, 322-300. Engel, M. B., Joseph, N. R. and Catchpole, H. R. (1954). Homeostasis of connective tissues. I. Calcium-sodium equilibrium. Arch. Path. 58, 26-39. Engel, M. B., Joseph, N. R., Laskin, D. M. and Catchpole, H. R. (1960). A theory of connective tissue behaviour: its implications in periodontal disease . Ann. N.Y. Acad. Sci. 85, 399-420. Engfeldt, B. (1958). Recent observations on bone structure. /. Bone Jt. Surg. 40A, 698-706. Engfeldt, B. and Engstrom, A. (1954). Biophysical studies on bone tissue. XII . Experimentally produced ectopic bone tissue. Acta orthopaed. scand. 24, 85-100. Engfeldt, B. and Hjertquist, S. O. (1954). Biophysical studies on bone tissue. X. The in vivo and in vitro uptake of radioactive isotopes and ionic exchange reactions in bone tissue. Acta path, microbiol. scand. 35, 205-216. Engfeldt, B. and Strandh, J. (1960). Microchemical and biophysical studies of normal human compact bone tissue. Clin. Orthoped. 17, 63-68. Engfeldt, B., Engstrôm, A. and Zetterman, R. (1954). Biophysical studies of bone tissue. III . Osteopetrosi s (marble bone disease) . Acta paediat. 43, 152-162. Engstrôm, A. (1946). Quantitative micro- and histochemica l elementary analysis by Roentgen absorption spectrography. Acta radiol., Stockh. Suppl. 63. Engstrôm, A. (1950). Use of soft X-rays in the assay of biological material. Progr. Biophys. biophys. Chem. 1, 164-196. Engstrôm, A. (1953). X-ray methods in histochemistry. Physiol. Rev. 33, 190-201.

21. C O N N E C T I V E

TISSUE

COMPONENTS

Engstrom, A. and Wegstedt, L. (1951). Equipment for microradiography with soft Roentgen rays. Acta radiol., Stockh. 35, 345-355. Fell, H. B. (1925). The histogenesi s of cartilage and bone in the long bones of the embryonic fowl. / . Morph. 40, 417-451. Fernando, Í . V. P. and Movat, H. Z. (1963). Fibrillogenesis in regeneratin g tendon. Lab. Invest. 12, 214-229. Finean, J. B. (1953). A versatile X-ray camera for low-angle diffraction studies. / . sci. Instr. 30, 60-61. Finean, J. B. and Engstrom, A. (1953). The low-angle scatter of X-rays from bone tissue. Biochim. biophys. Acta 11, 178-189. Fischer, G. (1932). Uber die Bedeutung des Epithels im periodontalen Raum menschliche r und tierischer Z hne . Vjschr. Zahnheilk. 48, 413-425. Fishman, D. A. and Hay, E. D. (1962). Origin of osteoclast s from mononuclea r leucocytes in regeneratin g newt limbs. Anat. Rec. 143, 329-338. Follis, R. H., Jr. (1948). Histochemical studies on cartilage and bone. Amer. J. Path. 24, 685. Follis, R. H., Jr. and Berthrong, M. (1949). Histochemical studies on cartilage and bone. I. The normal pattern, 1, 2. Bull. Johns Hopkins Hosp. 85, 281-298. Freeman, S. and McLean, F. C. (1941). Experimental rickets. Arch. Path. 32, 387-408. French, J. E. and Benditt, E. P. (1953). The histochemistry of connective tissue. II . The effect of proteins on the selective staining of mucopolysaccharide s by basic dyes. /. Histochem. Cytochem. 1, 321-325. Frey-Wyssling, A. (1953). "Submicroscopic Morphology of Protoplasm", 2nd ed. Elsevier, Amsterdam. Fullmer, H. M. (1958). Differential staining of connective tissue fibers in areas of stress. Science 127, 1240. Fullmer, H. M. (1959a). The peracetic-orcein-Halm i stain: A stain for connective tissue. Stain Tech. 34, 81-84. Fullmer, H. M. (1959b). Observations on the developmen t of oxytalan fibers in the periodontium of man. / . dent. Res. 38, 510-518. Fullmer, H. M. (1959c). Differences in mechanism in staining reactions for mast cells. Nature, Lond. 183, 1274-1275. Fullmer, H. M. (1960a). Effect of peracetic acid on the enzyme digestion of carious mucopolysaccharides : Reversal of the PAS staining reaction of mucin. J. Histochem. Cytochem. 8, 113-121. Fullmer, H. M. (1960b). A comparative histochemica l study of elastic, pre-elastic and oxytalan connective tissue fibers. /. Histochem. Cytochem. 8, 290-295. Fullmer, H. M. (1961). A histochemica l study of periodontal disease in the maxillary alveolar processe s of 135 autopsies. / . Periodont. 32, 206-218. Fullmer, H. M. (1962). A critique of normal connective tissues of the periodontium and some alterations with periodontal disease . / . dent. Res. 41, 223-229.

OF

PERIODONTIUM

407

Fullmer, H. M. (1963). The oxytalan connective tissue fiber in health and disease . Ann. Histochim. 8, 51-54. Fullmer, H. M. (1964a). The use of histochemistry in oral histology. In "Oral Histology; Inheritance and Development" (D. V. Provenza, ed.), pp. 497-529. Lippincott, Philadelphia, Pennsylvania . Fullmer, H. M. (1964b). Dehydrogenase s in developing bone in the rat. / . Histochem. Cytochem. 12, 210-214. Fullmer, H. M. (1965a). The use of histochemistry in oral pathology. In "Oral Pathology" (R. W. Tiecke, ed.), pp. 749-785. McGraw-Hill, New York. Fullmer, H. M. (1965b). The histochemistry of the connective tissue. Int. Rev. Connective Tissue Res. 3, 1-76. Fullmer, H. M. and Gibson, W. (1966). Collagenolytic activity in gingivae of man. Nature, Lond. (in press). Fullmer, H. M. and Lillie , R. D. (1956). Some aspects of the mechanism of orcein staining. / . Histochem. Cytochem. 4, 64-68. Fullmer, H. M. and Lillie , R. D. (1957a). The staining of collagen with elastic tissue stains. J. Histochem. Cytochem. 5, 11-14. Fullmer, H. M. and Lillie , R. D. (1957b). A selective stain for elastic tissue (Orcinol-New Fuchsin). Stain Tech. 31, 27-29. Fullmer, H. M. and Lillie , R. D. (1958). The oxytalan fiber: A previously undescribe d connective tissue fiber. / . Histochem. Cytochem. 6, 425-430. Fullmer, H. M. and Lillie , R. D. (1962). Dilute unmordante d hematoxylin as a stain for basic nuclear protein. / . Histochem. Cytochem. 10, 502-503. Fullmer, H. M . and Link, C. C. (1964). A demineralization procedure for enzymatic histochemica l use. A quantitative succinic dehydrogenas e assay. Stain Tech. 39, 387-396. Fullmer, H. M. and Martin, G. R. (1964). Activit y of D(-)beta-hydroxybutyric dehydrogenas e in scurvy. Nature, Lond. 202, 302. Fullmer, H. M. and Witte, W. E. (1962). Periodontal membrane affected by scleroderma . Arch. Path. 73, 184-189. Fullmer, H. M., Martin, G. R. and Burns, J. J. (1961). Role of ascorbic acid in the formation and maintenanc e of dental structure. Ann. N.Y. Acad. Sci. 92, 286-294. Fullmer, H. M., Link, C. C. and Baer, M. (1964). A stain for bone—illustrating apposition and absorption in two colors. Stain Tech. 39, 71-73. Gersh, I. and Catchpole, H. R. (1960). The nature of ground substanc e of connective tissue. Persp. Biol. Med. 3, 282319. Gibson, W. and Fullmer, H. M. (1966). Collagenolytic activity of gingival tissues in vitro. J. dent. Res. (in press). Glegg, R. E., Eidinger, D. and Leblond, C. P. (1953). Some carbohydrate components of reticular fibers. Science 118, 614-616.

408

H.

M.

Glegg, R. E., Eidinger, D. and Leblond, C. P. (1954). Presenc e of carbohydrate s distinct from mucopolysac charides in connective tissue. Science 120, 839-840. Glenner, G. G. and Cohen, L. H. (1960). Histochemical demonstratio n of a species-specifi c trypsin-like enzyme in mast cells. Nature, Lond. 185, 846-847. Glickman, I., Turesky, S. and Hill , R. (1949). Determination of oxygen consumption in normal and inflamed human gingiva using the Warburg manometric technic. J. dent. Res. 28, 83-94. Goggins, J. F. (1965). The oxytalan fiber: Its fine structure and its distribution in the periodontal membrane . M.S. Thesis, pp. 1-56. Marquette University. Goldhaber, P. (1960). Behavior of bone in tissue culture. In "Calcification in Biological Systems", Publ. No. 64, pp. 349-372. Amer. Ass. Advanc. Sci., Washington, D.C. Gomori, G. (1939). Micro technical demonstratio n of phosphatas e in tissue sections. Proc. Soc. exp. Biol., N.Y. 42, 23-26. Gomori, G. (1943). Calcification and phosphatase . Amer. J. Path. 19, 197-209. Gomori, G. (1950). Aldehyde-fuchsin: A new stain for elastic tissue. Am. J. clin. Path. 20, 665-666. Gowgiel, J. M. (1961). Eruption of irradiation-produce d rootless teeth in monkeys. J. dent. Res. 40, 538-547. Grant, Í . H. and Alburn, H. E. (1960). Collagen solubilization by mammalian proteinases . Arch. Biochem. Biophys. 89, 262-270. Graumann, W. (1954). Die histochemisch e Periodatreaktio n der Reticulin- und Kollagenfasern . Acta histochem. 1, 116-125. Greep, R. O., Fischer, C. J. and Morse, A. (1948). Alkaline phosphatas e in odontogenesi s and osteogenesi s and its histochemica l demonstratio n after demineralization . J. Amer. dent. Ass. 36, 427-442. Greulich, R. C. (1953a). Entry of radiocarbon from labelled bicarbonate into the organic matrix of growing bones and teeth. Anat. Rec. 115, 312-313. Greulich, R. C. (1953b). Radioautographi c localization of C 14 in tissues of rats following administration of C 14 labelled carbonate . Doctoral Thesis, McGill University. Greulich, R. C. and Leblond, C. P. (1953). Radioautographi c visualization of radiocarbon in the organs and tissues of newborn rats following administration of CI4 labelled bicarbonate . Anat. Rec. 115, 559-580. Gross, J. (1949). Structure of elastic tissue as studied with the electron microscope. / . exp. Med. 89, 699-708. Gross, J. (1958). Studies on the formation of collagen. II . The influence of growth rate on neutral salt extracts of guinea pig dermis. J. exp. Med. 107, 265-277. Gross, J. (1964). Studies on the biology of connective tissues: remodelling of collagen in metamorphosis . Medicine 43, 291-303. Gross, J. and Kirk , D. (1958). The heat precipitation of

FULLMER collagen from neutral salt solutions: Some rate regulating factors. / . biol. Chem. 233, 355-360. Gross, J. and Lapiere, C. M. (1962). Collagenolytic activity in amphibian tissues: A tissue culture assay. Proc. nat. Acad. Sci., Wash. 48, 1014-1022. Gross, J. and Schmitt, F. O. (1948). The structure of human skin collagen as studied with the electron microscope. /. exp. Med. 88, 555-568. Gross, J., Bogoroch, R., Nadler, N. J. and Leblond, C. P. (1951). The theory and methods of the radioautogra phic localization of radioélément s in tissues. Amer. J. Roentgenol. 65, 420-458. Gross, J., Schmitt, F. O. and Highberger, J. H. (1952). In vitro fibrogenesis of collagen. Trans. Macy Conf. metab. Interrelations 4, 32-57. Gross, J., Levene, C. I. and Orloff, S. (1960). Fragility of extractable collagen in the lathyritic chick embryo. An assay for lathyrogenic agents. Proc. Soc. exp. Biol., N.Y. 105, 148-151. Gubisch, W. and Schlager, F. (1961). Fermente im Knochenund Knorpelgewebe . III . Mitteilung: β-Ό Glucuronidase . Acta histochem. 12, 69-74. Guidotti, G. (1957). Turnover of the sulfate group in mast cells of young and old adult rats. Exp. Cell Res. 12, 659-661. Guinier, A. (1952). "X-ray Crystallography". Hilger & Watts, London. Hall, C. E. (1953). "Introduction to Electron Microscopy". McGraw-Hill, New York. Hall, D. A. (1961). "The Chemistry of Connective Tissues". Thomas, Springfield, Illinois. Hall, D. Á., Reed, R. and Tunbridge, R. E. (1955). Electron microscope studies of elastic tissue. Exp. Cell Res. 8, 35-48. Hallén, A. (1958). Hexosamine and ester sulphate content of the human nucleus pulposus a tdifferent ages. Acta chem. scand.12,1869-1872. Hancox, Í . M. (1949). The osteoclast . Biol. Rev. 24, 448-471. Hancox, Í . M. (1956). In "Biochemistry and Physiology of Bone" (G. Bourne, ed.), pp. 213-247. Academic Press, New York. Hedbom, A. and Snellman, O. (1955). Isolation and analysis of the large cytoplasm granules of tissue mast cells. Exp. Cell Res. 9, 148-156. Heller-Steinberg , M. (1951). Ground substance , bone salts, and cellular activity in bone formation and destruction. Amer. J. Anat. 89, 347-380. Henry, N. F. M., Lipson, H. and Wooster, W. A. (1951). "The Interpretation of X-ray Diffraction Photographs" . Macmillan, New York. Herrmann-Erlee , M. P. M. (1962). A histochemica l investigation of embryonic long bones. The effect of parathyroid hormone on the activity of a number of enzymes. Proc. Acad. Sci. Amst. C65, 22-40.

21. C O N N E C T I V E

TISSUE

COMPONENTS

Herzberg, F. and Schour, I. (1941). Effects of the removal of the pulp and Hertwig's sheath on the eruption of the incisors in the albino rat. J. dent. Res. 20, 264 (Abstract). Higaki, R. (1932). Besondere Befunde von Epithelnester n ausserhal b des Periodontium. Dtsch. Mschr. Zahnhedk. 8, 337-341. Highberger, J. H. (1961). Recent advances in knowledge of the structure of the collagen fibril s and of the properties of the tropo-collagen macromolecule . J. Amer. Leath. Chem. Ass. 56, 422-456. Highberger, J. H., Gross, J. and Schmitt, F. O. (1951). The interaction of mucoprotein with soluble collagen. An electron microscope study. Proc. nat. Acad. Sci., Wash. 37, 286-291. Hodge, A. J. and Schmitt, F. O. (1960). The charge profile of the tropocollagen macromolecule and the packing arrangemen t in native-type collagen fibrils. Proc. nat. Acad. Sci., Wash. 46, 186-197. Holmgren, H. (1940). Studien uber Verbreitung und Bedeutung der chromotropen Substanz . Z. mikr.-anat. Forsch. 47, 489-521. Holmgren, H. and Wilander, Ï . T. (1937). Beitrag zur Kenntnis der Chemie und Funktion der Ehrlichschen Mastzellen. Z. mikr.-anat. Forsch. 42, 242-278. Hopsu, V. K. and Glenner, G. G. (1963). Further observations on histochemica l esteras e and amidase activities with similarities to trypsin. / . Histochem. Cytochem. 11, 520-528. Howship, J. (1817). Experiments and observations in order to ascertain the means employed by the animal economy in the formation of bone. Med.-chir. Trans., Lond. 6, 263-295. Hunt, A. M. (1959). A description of the molar teeth and investing tissues of normal guinea pigs. / . dent. Res. 38, 216-231. Hyslop, D. B. (1952). The effect of supravital dyes on osteoblast s in tissue culture. M.S. Thesis, University of Liverpool. Irving, J. T. and Handelman, C. S. (1963). Bone destruction by multinucleated giant cells. In "Mechanisms of Hard Tissue Destruction", Publ. No. 75, pp. 515-530. Amer. Ass. Advanc. Sci., Washington, D.C. Jackson, D. S. (1957). Connective tissue growth stimulated by carrageenin . I. The formation and removal of collagen. Biochem. J. 65, 277-284. Jackson, D. S. (1958). Some biochemical aspects of fibrogenesis and wound healing. New Eng. J. Med. 259, 814-820. Jackson, D. S. and Williams, G. (1956). Nature of reticulin. Nature, Lond. 178, 915-916. Jackson, D. S., Leach, A. A. and Jacobs, S. (1958). The amino acid composition of the collagen factions of rabbit skin. Biochim. biophys. Acta 27, 418-420. Jordan, H. E. (1925). The experimenta l production of

OF

PERIODONTIUM

409

osteoclast s in the frog, Rana pipiens. Anat. Rec. 30, 107-121. Jorpes, J. E. (1937). Heparin: A mucoitin polysulfuric acid. J. biol. Chem. 118, 447-457. Jorpes, J. E. and Gardell, S. (1948). On heparin monosulfuric acid. J. biol. Chem. 176, 267-276. Jorpes, J. E., Odeblad, E. and Bostrom, H. (1953). An autoradiographi c study on the uptake of S3 5—labelled sodium sulphate in the mast cells. Acta haemat. 9, 273-276. Julén, C, Snellman, O. and Sylvén, Â. (1950). Cytological and fractionation studies on the cytoplasmic constituents of tissue mast cells. Acta physiol. scand. 19, 289-305. Kabat, E. A. and Furth, J. (1941). A histochemica l study of the distribution of alkaline phosphatas e in various normal and neoplastic tissues. Am. J. Path. 17, 303-318. Kao, Ê. Y. T. and Boucek, R. J. (1958). Incorporation and conversion of lysine 2-C14 in rat biopsy connective tissue. Proc. Soc. exp. Biol., N.Y. 98, 526-530. Karrer, Ç. H. (1960). Electron microscope study of developing chick embryo aorta. J. Ultrastruct. Res. 4, 420-454. Keech, M. K. (1955). Human skin collagen from different age groups before and after collagenas e digestion. An electron microscope study. Ann. Rheum. Dis. 14, 19-50. Kirby-Smith, H. T. (1933). Bone growth studies: a miniature bone fracture observed microscopically in a transparen t chamber introduced into the rabbit's ear. Am. J. Anat. 53, 377-402. Kirkman, H. (1950). A comparative morphological and cytochemical study of globule leucocytes (Schollenleukocyten) of the urinary tract and of possibly related cells. Am. J. Anat. 86, 91-132. Klement, R. (1929). Die Zusammensetzun g der Knochenstiitzsubstanz . Hoppe-Seyl. Z. 184, 132-142. Klug, H. P. and Alexander, L. E. (1954). "X-ray Diffraction Procedures" . Wiley, New York. Kolliker, A. (1852). "Mikroskopische Anatomie oder Gewebelehr e des Menschen". Engelmann, Leipzig. Kolliker, A. (1867). "Handbuch der Gewebelehr e des Menschen fur Ârzte und Studierende" . Engelmann, Leipzig. Kolliker, A. (1873). "Di e normale Resorption des Knochengewebes in ihrer Bedeutung fur die Entstehung der typischen Knochenformen". Vogel, Leipzig. Kostlân, J., Thorova, J. and Skach, M. (1960). Erupce hlodavého zubu po resekci jeho rûstové zony [Eruption of rodent tooth after resection of its zone of growth]. Cesk. Stomat. 60, 401-410. Kramer, H. and Windrum, G. M. (1953). Metachromasi a after treating tissue sections with sulfuric acid. / . clin. Path. 6, 239-240. Kramer, H. and Windrum, G. M. (1954). Sulphation techniques in histochemistry with special reference s to metachromasia . / . Histochem. Cytochem. 2, 196-208. Lacroix, M. P. (1953). Sur le métabolisme du calcium dans

410

H.

M.

l'os compact du chien adulte. Bull. Acad. R. Méd. Belg. [6] 18, 489-496. Lacroix, M. P. (1956). The histological remodeling of adult bone. An autoradiographi c study. Ciba Fdn. Symp., Bone Struct. Metab. pp. 36-46. Lagunoff, D., Phillips, M. and Benditt, E. P. (1961). The histochemica l demonstratio n of histamine in mast cells. /. Histochem. Cytochem. 9, 534-541. Lansing, A. I., Rosenthal, J. B., Alex, M. and Dempsey, E. W. (1952). The structure and chemical characterizatio n of elastic fibers as revealed by elastase and by electron microscopy. Anat. Rec. 114, 555-575. Lapiere, C. M. and Gross, J. (1963). Animal collagenas e and collagen metabolism. In "Mechanisms of Hard Tissue Destruction", Publ. No. 75, pp. 663-694. Amer. Ass. Advanc. Sci., Washington, D.C. Leblond, C. P., Wilkinson, G. W., Bélanger, L. F. and Robichon, J. (1950). Radioautographi c visualization of bone formation in the rat. Amer. J. Anat. 86, 289327. Leblond, C. P., Glegg, R. E. and Eidinger, D. (1957). Presenc e of carbohydrate s with free 1,2-Glycol groups in sites stained by the periodic acid-Schiff technique. / . Histochem. Cytochem. 5, 445-458. Legros, C. and Magitot, E. (1879). Morphologie de follicule dentaire dans les mammifères. / . Anat., Paris 15, 248-293; p. 286 cited in Malassez (1885). Leng, A. (1942). Contribucion al estudie del metabolismo de paradencio. Rev. Odont., B. Aires 30, 911-917. Levene, C. I. and Gross, J. (1959). Alterations in state of molecular aggregation of collagen induced in chick embryos by /8-aminopropionitrile (Lathyrus factor). / . exp. Med. 110, 771-790. Lillie , R. D. (1949). On the destruction of cytoplasmic basophilia (ribonucleic acid) and of the metachromati c basophilia of cartilage by the glycogen splitting enzyme malt diastase : A histochemica l study. Anat. Rec. 103, 611-633. Lillie , R. D. (1951). The allochrome procedure. A differential method segregatin g the connective tissues collagen, reticulum and basemen t membrane s into two groups. Am. J. clin. Path. 21, 484-488. Lillie , R. D. (1953). Factors influencing periodic acid Schiff reaction of collagen fibers. / . Histochem. Cytochem. 1, 353-361. Lillie , R. D. (1954). "Histopathologic Technic and Practical Histochemistry". McGraw-Hill (Blakiston), New York. Lillie , R. D. (1958). Acetylation and nitrosation of tissue amines in histochemistry. / . Histochem. Cytochem. 6, 352-362. Lillie , R. D. (1964). Histochemical acylation of hydroxyl and amino groups. Effect on the periodic acid Schiff reaction, anionic and cationic dye and Van Gieson collagen stains. /. Histochem. Cytochem. 12, 821-841.

FULLMER Lipp, W. (1959). Aminopeptidase in bone cells. / . Histochem. Cytochem. 7, 205. Lisanti, V. F. (1960). Hydrolytic enzymes in periodontal tissues. Ann. N.Y. Acad. Sci. 85, 461-466. Loe, H. and Waerhaug, J. (1961). Experimental replantation of teeth in dogs and monkeys. Arch, oral Biol. 3, 176-184. Loewi, G. (1953). Changes in the ground substanc e of ageing cartilage. / . Path. Bact. 65, 381-388. Loewi, G. and Meyer, K. (1958). The acid mucopolysac charides of embryonic skin. Biochim. biophys. Acta 27, 453-456. Loiseleur, J. and Urbain, A. (1930). Sur les propriétés antigéniques du collagène et leur modification sous l'action de l'émanation du radium. C.R. Soc. Biol., Paris 103, 776-778. Lomholt, S. (1930). Investigations into the distribution of lead in the organism on the basis of photographic (radiochemical) methods. / . Pharmacol. 40, 235-245. Lorber, M. (1951). A study of the histochemica l reactions of the dental cementum and alveolar bone. Anat. Rec. I l l , 129-144. Lorch, I. J. (1947). Localization of alkaline phosphatas e in mammalian bones. Quart. J. micr. Sci. 88, 367-382. MacDonald, I. (1954). Chemical analysis of human foetal skull bones. Biochem. J. 57, 437-439. McManus, J. F. A. and Mowry, R. W. (1952). Sulfuric acid hematoxylin stain for basemen t membranes . Lab. Invest. 1, 208-209. Mâjno, G. and Rouiller, C. (1951). Die alkalische Phosphatase in der Biologie des Knochengewebes . Histochemische Untersuchungen . Virchows Arch. 321, 1-61. Main, J. H. P. (1965). A histological survey of the hammock ligament. Arch, oral Biol. 10, 1-16. Main, J. H. P. and Adams, D. (1966a). Measuremen t of the rate of eruption of the rat incisor. I. Investigations into the cellular proliferation and blood pressure theories of tooth eruption. Arch, oral Biol. 11 (in press). Main, J. H. P. and Adams, D. (1966b). Measuremen t of the rate of eruption of the rat incisor. II . The effects of demecolcine and tri-ethylene melamine on the dental tissues of the rat. Arch, oral Biol. 11 (in press). Main, J. H. P. and Adams, D. (1966c). Measuremen t of the rate of eruption of the rat incisor. III . The effect of guanethidine , hydralazine, demecolcine and tri-ethylene melamine on the rate of eruption of the rat incisor. Arch, oral Biol. 11 (in press). Malassez, L. (1885). Sur l'existence d'amas épithéliaux auto de la racine des dents chez l'homme adulte et à l'état normal (Débris épithéliaux paradenta l res). Arch. Physiol, norm. path. [3] 5, 129-148. Manly, M. L. and Bale, W. F. (1939). The metabolism of inorganic phosphorus of rat bones and teeth as indicated by the radioactive isotope. / . biol. Chem. 129, 125-134. Manly, M. L., Hodge, H. C. and Van Voorhis, S. N. (1940).

21. C O N N E C T I VE

T I S S UE

C O M P O N E N TS

Distribution of ingested phosphorus in bone and teeth of a dog, shown by radioactive isotope. Proc. Soc. exp. Biol., N.Y. 45, 70-72. Martin, G. R., Gross, J., Piez, K. A. and Lewis, M. S. (1961a). On the intramolecular cross-linking of collagen in lathyritic rats. Biochim. biophys. Acta 53, 599-601. Martin, G. R., Mergenhagen , S. E. and Scott, D. B. (1961b). Relation of ionizing groups to the structure of the collagen fibril . Biochim. biophys. Acta 49, 245-250. Martin, G. R., Piez, K. A. and Lewis, M. S. (1963). The 4 glycine into the subunits of collagens incorporation of 1 C from normal and lathyritic animals. Biochim. biophys. Acta 69, 472-479. Massler, M. and Schour, I. (1941). Studies in tooth development: theories of eruption. Amer. J. Orthodont. 27, 552576. Maurer, P. H. (1955). Immunochemica l comparisons of various gelatins and their derivatives. Arch. Biochem. Biophys. 58, 205-213. Mecca, C. E., Martin, G. R. and Goldhaber, P. (1963). Alteration of bone metabolism in tissue culture in response to parathyroid extract. Proc. Soc. exp. Biol., N.Y. 113, 538-540. Meyer, K. (1957). The chemistry of the mesoderma l ground substance . Harvey Lect. 51, 88-112. Meyer, K., Hoffman, P. and Linker, A. (1959). Chemistry of the ground substance . In "Connective Tissue, Thrombosis and Atherosclerosis " (T. H. Page, ed.), pp. 181-191. Academic Press, New York. Meyer, W. (1932). "Lehrbuch der normalen Histologie und Entwicklungsgeschicht e der Zàhne des Menschen". Mùnchen. Montagna, W. (1962). "The Structure and Function of Skin", 2nd ed., p. 148. Academic Press, New York. Montagna, W. and Noback, C. R. (1948). Localization of lipids and other chemical substance s in the mast cells of man and laboratory mammals. Anat. Rec. 100, 535-545. Mori , M., Takada, K. and Okamoto, K. (1962). Histochemical studies on the localization and activity of acid phosphatas e in calcifying tissues. Effects of decalcification on the enzymatic activity. Histochemie 2, 427-434. Morris, A. L. and Krikos, G. A. (1958). Artifacts in M.C. metachromasi a produced by hyaluronidase preparations . Proc. Soc. exp. Biol., N.Y. 97, 527-529. Morse, A. and Greep, R. O. (1951). Effect of abnormal metabolic states upon the histochemica l distribution of alkaline phosphatas e in the tibia of the albino rat. Anat. Rec. I l l , 193-219. Moura, C. S. (1964). "Contribuicao ao estudo do paradencio do sagui", pp. 1-34. Private publication, Fac. Odont., Bahia, Brazil. Mowry, R. W. and Winkler, C. H. (1956). The coloration of acidic carbohydrate s of bacteria and fungi in tissue sections with special reference to capsules of Cryptococcus

OF

P E R I O D O N T I UM

411

neofor mans, pneumococci , and staphylococci. Amer. J. Path. 32, 628-629. Mummery, J. H. (1921). Studies in dental histology. II . The sheath of Hertwig and the epithelial debris. Dent. Cosmos 63, 1207-1215. Nageotte, J. (1927). Action des sels neutres sur la formation du caillot artificial de collagène. C.R. Soc. Biol, Paris 96, 828-830. Ness, A. R. and Smale, D. E. (1959). The distribution of mitosis and cells in the tissues bounded by the socket wall of the rabbit mandibular incisor. Proc. R. Soc. B151, 106-128. Neuman, W. F. and Neuman, M. W. (1958). "The Chemical Dynamics of Bone Mineral". Univ. of Chicago Press, Chicago, Illinois. Noda, H. and Wyckoff, R. W. G. (1951). The electron microscopy of reprecipitated collagen. Biochim. biophys. Acta 1, 494-506. Noback, C. R. and Montagna, W. (1946). Some histochemical aspects of the mast cell with special reference to alkaline phosphatas e and cytochrome oxidase. Anat. Rec. 96, 279-287. Noyés, F. B. (1930). " A Textbook of Dental Histology and Embryology", 4th ed., p. 274. Lea & Febiger, Philadelphia, Pennsylvania . Orban, B. (1944). "Oral Histology and Embryology", p. 181. Mosby, St. Louis, Missouri. Orekhovitch, V. N., Chpikiter, V. O., Mazourov, V. I. and Kounina, Ï . V. (1960). Procollagènes . Classification, métabolisme , action des proteinases . Bull. Soc. Chim. biol., Paris 42, 505-518. Parvis, P. V. and Roncoroni, G. (1950). Ricerche istochimiche e morfologiche sul cemento condroide e sulle connession e alveolodentale dei molari della cavia. Biol. latina 3, 121-220. Paynter, K. J. and Pudy, G. (1958). A study of the structure, chemical nature and developmen t of cementum in the rat. Anat. Rec. 131, 233-251. Peach, R., Williams, G. and Chapman, J. A. (1961). A light and electron optical study of regeneratin g tendon. Am. J. Path. 38, 495-513. Pécher, C. (1942). Biological investigations with radioactive calcium and strontium; preliminary report on the use of radioactive strontium in the treatment of metastatic bone cancer. Univ. Calif. Publ. Pharmacol. 2, 111. Person, P. (1963). Relation of metabolism of oral tissues to periodontal disease . / . dent. Res. 1, 497-501. Person, P., Stahl, S. S. and Scapa, S. (1961). Cytochemical and biochemical studies of aerobic oxidative metabolism of human gingiva. / . dent. Res. 40, 304-309. Peterkofsky, B. and Udenfriend, S. (1961). Conversion of proline—C14 to peptide-boun d hydroxyproline—C14 in a cell free system from chick embryo. Biochem. Biophys. Res. Comm. 6, 184-190.

412

H.

M.

Peterkofsky, B. and Udenfriend, S. (1963). Localization of the site of proline hydroxylation during the cell-free biosynthesis of collagen. Biochem. Biophys. Res. Comm. 12, 257-262. Piez, K. A. (1960). The relation between amino acid composition and denaturation of vertebrate collagen. / . Amer, chem. Soc. 82, 247. Piez, K. A. and Gross, J. (1960). The amino acid composition of some fish collagens: The relation between composition and structure. / . biol. Chem. 235, 995-998. Piez, Ê. Á., Lewis, M. S., Martin, G. R. and Gross, J. (1961). Subunits of the collagen molecule. Biochim. biophys. Acta 63, 596-598. Porter, Ê. E. and Pappas , G. D. (1959). Collagen formation by fibroblasts of the chick embryo dermis. / . biophys. biochem. Cytol. 5, 153-161. Pritchard, J. J. (1952). A cytological and histochemica l study of bone and cartilage formation in the rat. / . Anat., Lond. 86, 259-277. Pritchard, J. J. (1956). In "Biochemistry and Physiology of Bone" (G. H. Bourne, ed.), pp. 179-211. Academic Press, New York. Puchtler, H . and Isler, H. (1958). The effect of phosphomolybdic acid on the stainability of connective tissues by various dyes. / . Histochem. Cytochem. 6, 265-270. Quintarelli, G., Scott, J. E. and Dellovo, M. C. (1964a). The chemical and histochemica l properties of Alcian Blue. II . Dye binding of tissue polyanions. Histochemie 4, 86-98. Quintarelli, G., Scott, J. E. and Dellovo, M. C. (1964b). The chemical and histochemica l properties of Alcian Blue, III . Chemical blocking and unblocking. Histochemie 4. 99-112. Radden, B. G. (1962). Cytochemical variations in the granules of rat mast cells, and their relation to mast cell maturation. Aust. J. exp. Biol. med. Sci. 40, 9-16. Randall, J. T., Booth, F., Burge, R. E., Fitton-Jackson , S. and Kelly, F. C. (1955). Abservations on native and precipitated collagen. In "Fibrous Proteins and Their Biological Significance," Symp. Soc. exp. Biol., pp. 127147. Academic Press, New York. Rannie, I. (1963). Observations on the oxytalan fibre of the periodontal membrane . Trans. Eur. Orthodont. Soc. 39, 127-136. Reitan, K. (1961). Behavior of Malassez' epithelial rests during orthodontic tooth movement. Acta odont. scand. 19, 443-468. Rich, A. and Crick, F. H. C. (1958). In "Recent Advances in Gelatin and Glue Research " (G. Stainsby, ed.), pp. 20-25. Pergamon Press, Oxford. Riley, J. F. (1953). The relationship of the tissue mast cells to the blood vessels in the rat. J. Path. Bact. 65, 461-480. Riley, J. E. and West, G. B. (1953). The presence of histamine in tissue mast cells. / . Physiol. 120, 528-537. Riley, J. E. and West, G. B. (1955). Tissue mast cell studies

FULLMER with a histamine liberator of low toxicity (compound 48/80). J. Path. Bact. 69, 269-282. Rinehart, J. F. and Abul-Haj, S. K. (1951). Acid mucopolysaccharide s in tissue. Arch. Path. 52, 189-191. Robb-Smith, Á. H. T. (1958). The relationship of reticulin to other collagen. In "Recent Advances in Gelatin and Glue Research " (G. Stainsby, ed.), pp. 38-44. Pergamon Press, Oxford. Robin, C. R. (1849). Sur l'existence de deux espèce s nouvelles d'éléments anatomique s qui se trouvent dans le canal médullaire des os. C.R. Soc. Biol., Paris [11] 1, 149-150. Robin, C. R. (1864). Note sur les éléments anatomique s appelés myéloplaxes. / . Anat., Paris 1, 88-109. Robinsohn, I. (1926). Weitere Beitràge zur Théorie der hormonalen Morphogenes e der Zàhne. Z. Stomat. 24, 1-34. Roseman, S. (1959). Metabolism of connective tissue. Ann. Rev. Biochem. 28, 545-578. Schajowicz, F. and Cabrini, R. L. (1957). Estudio histoquimico de la fosfatasa acids en la osification endocondral . Rev. Soc. argent. Biol. 33, 257-261. Schajowicz, F. and Cabrini, R. L. (1960). Histochemical distribution of succinic dehydrogenas e in bone and cartilage. Science 131, 1043-1044. Schajowicz, F., Cabrini, R. L. and Merea, C. E. (1961). Comportamiento histoquimico del granuloma giganto— cellular de cuerpo extrano experiental. Rev. Ortop. Traum., B. Aires 6, 179-188. Schlager, F. (1959). Vorkommen und Lokalisation der â-Galactosidas e in Knochen, Knorpel und in benachbarte n Geweben der weissen Maus. Acta histochem. 8, 176-184. Schmidt, A. J. (1963). Multinuclearity of osteoclasts . Nature, Lond. 199, 1113-1114. Schmitt, F. O., Hall, C. E. and Jakus, M. A. (1942). Electron microscope investigations of the structure of collagen. J. cell. comp. Physiol. 20, 11-33. Schrader, H. K. and Schrader, R. (1957). Oxygen uptake by normal and inflamed gingiva and saliva. Helv. odont. Acta 1, 13-16. Schultz-Haudt, S. D. (1957). "Observations on the Acid Mucopolysaccharide s of Human Gingivae". Oslo Press, Oslo. Schultz-Haudt, S. D., Paus, S. and Assev, S. (1961). Periodic acid Schiff reactive components of human gingiva. / . dent. Res. 40, 141-148. Schwarz, W. (1954). Elektronmikroskopisch e Untersuchunge n der Altersvernderungen in der Media der menschliche n Aorta. Virchows Arch. 324, 612-628. Schwarz, W. and Dettmer, N. (1953). Elektronenmikros kopische Untersuchun g des elastische n Gewebes in der menschliche n Aorta. Virchows Arch. 323, 243-268. Scott, J. E., Quintarelli, G. and Dellovo, M. C. (1964). The chemical and histochemica l properties of Alcian Blue.

21. C O N N E C T I V E

TISSUE

COMPONENTS

1. The mechanism of Alcian Blue staining. Histochemie 4, 73-85. Scott, J . H. (1953). How teeth erupt. Dent. Practit. 3, 345-350. Selvig, K. A. (1963a). Elektronmikroskopet—e t hjelpe middel i odontologisk forskning. Norske Tandlaegeforen. Tid. 73, 387-393. Selvig, K. A. (1963b). Electron microscopy of Hertwig's epithelial sheath and of early dentin and cementum formation in the mouse incisor. Acta odont. scand. 21, 175-186. Selvig, K. A. (1964). A n ultrastructural study of cementum formation. Acta odont. scand. 22, 105-120. Senter, A. D., Eiler, J. J. and Leek, H. (1959). Endogenou s respiration of human gingival tissue. Proc. Soc. exp. Biol., N.Y. 100, 323-324. Serres, A. (1817). "Essai sur l'anatomie et la physiologie des dents ou nouvelle théorie de la dentition," pp. 1-183. Méquignon-Marvis, Paris; p. 28 cited in Malassez (1885). Sicher, H. (1942a). Tooth eruption: the axial movement of continuously growing teeth. / . dent. Res. 21, 201-210. Sicher, H. (1942b). Tooth eruption: axial movement of teeth with limited growth. / . dent. Res. 21, 395-402. Sicher, H., ed. (1962). "Orban's Oral Histology and Embryology", 5th ed. Mosby, St. Louis, Missouri. Siffert, R. S. (1951). The role of alkaline phosphatas e in osteogenesis . / . exp. Med. 93, 415-426. Silverman, L. and Shorter, R. G. (1963). Histogenesis of the multinucleated giant cell. Lab. Invest. 12, 985-990. Simaski, M. and Yagi, T. (1960). Histochemistry of carbonic anhydrase with special reference to the osteoclast . Dent. Bull. Osaka Univ. 1, 89-98. Sinex, F. M. and Van Slyke, D. D. (1955). The source and state of hydroxylysine of collagen. J. biol. Chem. 216, 245-250. Sinex, F. M. and Van Slyke, D. D. (1957). Role of hydroxylysine in the synthesis of collagen. Fed. Proc. 16, 250. Spicer, S. S. (1960). A correlative study of the histochemica l properties of rodent acid mucopolysaccharides . /. Histochem. Cytochem. 8, 18-33. Spicer, S. S. and Warren, L. (1960). The histochemistry of sialic acid containing mucoproteins . /. Histochem. Cytochem. 8, 135-137. Spicer, S. S., Woliman, S. H. and Warren, L. (1960). Histochemical demonstratio n of sialomucin in transplantable thyroid carcinoma. Amer. J. Path. 37, 599-610. Stallard, R. E. (1963). The utilization of H 3-proline by the connective tissue elements of the periodontium. Periodontics 1, 185-188. Stallard, R. E. (1964a). Tissue changes in the supporting structures in occlusal traumatism using radioisotopes . Periodontics 2, 143-144. Stallard, R. E. (1964b). The effect of occlusal alterations on collagen formation within the periodontium. Periodontics 2, 49-52. 28

OF

PERIODONTIUM

413

Stearns, M. L. (1940a). Studies on the developmen t of connective tissue in transparen t chambers in the rabbit's ear. I. Amer. J. Anat. 66, 133-176. Stearns, M. L. (1940b). Studies on the developmen t of connective tissue in transparen t chambers in the rabbit's ear. IL Amer. J. Anat. 67, 55-97. Steedman , H. F. (1950). Alcian blue 8 GS: A new stain for mucin. Quart. J. micr. Sci. 91, 477-479. Stephan, P. E. (1900). Recherche s histologiques sur la structure du tissu osseux des poissons. Bull. sci. Fr. Belg. 33, 281-429. Stetten, M. R. (1949). Some aspects of the metabolism of hydroxyproline, studied with the aid of isotopic nitrogen. /. biol. Chem. 181, 31-37. Stetten, M. R. and Schoenheimer , R. (1944). The metabolism of L(-)-proline studied with the aid of deuterium and isotopic nitrogen. / . biol. Chem. 153, 113-132. Strandh, J. (1960a). Microchemical studies on single Haversian systems. I. Methodological consideration s with special reference to variations in mineral content. Exp. Cell Res. 19, 515-530. Strandh, J. (1960b). Microchemical studies on single Haversian systems. II . Methodological consideration s with special reference to the Ca/P ratio in microscopic bone structures. Exp. Cell Res. 21, 406-413. Strandh, J. (1961a). "Chemical and Biophysical Studies of Microscopic Structures in Compact Bone", pp. 1-16. Almqvist & Wiksell, Uppsala. Strandh, J. (1961b). Microchemical studies on single Haversian systems. Biochim. Biol. sper. 1, 60-65. Strandh, J. and Bengtsson , A. (1961a). The uptake of phosphorus in microscopic bone structures in compact bone. Acta soc. med. Upsala 66, 49-64. Strandh, J. and Bengtsson , A. (1961b). The uptake of calcium in microscopic bone structures in compact bone. Acta soc. med. Upsala 66, 95-103. Strobino, L. J. and Farr, L. E. (1949). The relation to age and function of regional variations in nitrogen and ash content of bovine bones. / . biol. Chem. 178, 599-609. Suga, S., Shimizu, M. and Namie, K. (1959). The histochemical localization of alkaline phosphatas e in tooth supporting tissues of rat. Arch, histol. jap. 17, 305-322. Sylvén, Â. (1938). Uber das Vorkommen von metachromatische r Substanz in wachsende m Gewebe und ihre Bedeutung. Klin. Wochschr. 17, 1545-1547. Sylvén, Â. (1941). Uber das Vorkommen von hochmolekularen Esterschwefelsuren im Granulationsgeweb e und bei der Epithelregeneration . Acta chir. scand. 86, Suppl. 66, 1-151. Sylvén, Â. (1945). Ester sulphuric acids of high molecular weight and mast cells in mesenchyma l tumors. Acta radiol., Stockh. Suppl. 59, 1-99. Sylvén, Â. (1951). On the cytoplasmic constituents of normal tissue mast cells. Exp. Cell Res. 2, 252-255.

414

H.

M.

Taylor, A. C. and Butcher, Å. Ï . (1951). The regulation of eruption rate in the incisor teeth of the white rat. / . exp. Zool. 117, 165-188. Ten Cate, A. R. (1965). The histochemica l demonstratio n of specific oxidative enzymes and glycogen in the epithelial cell rests of Malassez. Arch, oral Biol. 10, 207-213. Thomas, P. K. (1963). The connective tissue of peripheral nerve: an electron microscopic study. / . Anat., Lond. 97, 35-44. Tomlin, D. H., Henry, Ê. M. and Kon, S. K. (1953). Autoradiographic study of growth and calcium metabolism in the long bones of the rat. Brit. J. Nutr. 1, 235-252. Tonna, Å. Á. (1962). An autoradiographi c examination of the utilization of tritiated histidine by cells of the skeletal system. Nature, Lond. 193, 1301-1302. Tonna, Å. A. and Cronkite, Å. P. (1961a). Cellular response to fracture studied with tritiated thymidine. / . Bone Jt. Surg. 43A, 352-362. Tonna, Å. A. and Cronkite, Å. P. (1961b). Use of tritiated thymidine for the study of the origin of the osteoclast . Nature, Lond. 190, 459-460. Tristram, G. R. (1953). The amino acid composition of proteins. In "The Proteins" (H. Neurath and K. Bailey, eds.), 1st ed., Vol. I, Part A. Academic Press, New York. Trott, J. R. (1962). The developmen t of the periodontal attachment in the rat. Acta anat. 51, 313-328. Waerhaug, J. (1958). Effect of C-avitaminosis on the supporting structures of the teeth. / . Periodont. 29, 87-97. Wagner, Â. M . and Shapiro, S. H. (1957). Application of alcian blue as a histochemica l method. Lab. Invest. 6, All-All.

Waksman, Â. H. and Mason, H. L. (1949). The antigenicity of collagen. / . Immunol. 63, 427-433. Walker, D. G. (1961). Citric acid cycle in osteoblast s and osteoclasts . A histochemica l study of normal and parathormone-treate d rats. Bull. John Hopkins Hosp. 106, 80-99. Watson, R. F., Rothbard, S. and Vaname, P. (1954). The antigenicity of rat collagen. / . exp. Med. 99, 535-550. Weidmann, S. M. and Rogers, Ç. J. (1958). Studies on the skeletal tissues. 5. The influence of age upon the degree of calcification and the incorporation of 3P2 in bone. Biochem. J. 69, 338-343. Weinges, K. F., Leppelman, H. J. and Hartl, F. (1953). Uber den Calcium-, Phosphat, und Carbonatgehal t menschliche r Skeletteille im Zusammenhan g mit Fragen der Knochenstruktu r und ihres Umbaues unter normalen und krankhaften Bedingungen . Klin. Wochschr. 31, 10571059.

FULLMER Wislocki, G. B. and Dempsey, E. W. (1946). Observations on the chemical cytology of normal blood and hemopoietic tissues. Anat. Rec. 96, 249-277. Wislocki, G. B. and Sognnaes , R. F. (1950). Histochemical reactions of normal teeth. Amer. J. Anat. 87, 239-275. Wislocki, G. B., Bunting, H. and Dempsey, E. W. (1947). Further observations on the chemical cytology of megakaryocytes and other cells of hemopoietic tissues. Anat. Rec. 98, 527-537. Wislocki, G. B., Singer, M. and Waldo, C. M. (1948). Some histochemica l reactions of mucopolysaccharides , glycogen, lipids and other substance s in teeth. Anat. Rec. 101, 487-513. Wood, G. C. and Keech, M. K. (1960). The formation of fibril s from collagen solutions. I. The effect of experimenta l conditions: Kinetic and electron microscope studies. Biochem. J. 75, 588-598. Wyckoff, R. W. G. (1951). "Crystal Structures". Wiley (Interscience) , New York. Wyckoff, R W. G. and Corey, R. B. (1936). X-ray diffraction patterns from reprecipitated connective tissue. Proc. Soc. exp. Biol., N.Y. 34, 285-287. Wyckoff, W. G. (1949). "Electron Microscopy". Wiley (Interscience) , New York. Yardley, J. H., Heaton, M. W., Gaines, M . and Shulman, L . E. (1960). Collagen formation by fibroblasts. Preliminary electron microscope observations using thin sections of tissue cultures. Bull. Johns Hopkins Hosp. 106, 381-393. Yoshiki, S. (1962). Histochemistry of various enzymes in developing bone cartilage and tooth of rat. Bull. Tokyo dent. Coll. 3, 14-28. Young, R. W. (1962). Autoradiographic studies on postnatal growth of the skull in young rats injected with tritiated glycine. Anat. Rec. 143, 1-13. Zacharides , P. A. (1900). Recherche s sur la structure du tissu conjonctif, sensibilité du tendon aux acides. C.R. Soc. Biol., Paris 52, 182-184. Zelickson, A. (1963). Fibroblast developmen t and fibrogenesis. Arch. Derm. 88, 497-509. Zorzoli, A. and Nadel, Å. M. (1953). Alkaline phosphatas e activity in the bones of normal and scorbutic guinea pigs. /. Histochem. Cytochem. 1, 362-371. Zugibe, F. T. (1962a). The demonstratio n of the individual acid mucopolysaccharide s in human aortas, coronary arteries and cerebral arteries. I. The methods. / . Histochem. Cytochem. 10, 441-447. Zugibe, F. T. (1962b). The demonstratio n of the individual acid mucopolysaccharide s in human aortas, coronary arteries and cerebral arteries. II . Identification and significance with ageing. J. Histochem. Cytochem. 10, 448-461.

CHAPTER

22

THE STRUCTURE AN D PHYSIOLOGY OF THE DENTO-GINGIVAL JUNCTION HARALD

LÔE

I. The Histogenesis of the Dento-gingival Junction

415

II . The Nature of the Connection between Epithelium and Enamel A . The Enamel Surface B. The Epithelial Attachment Concept C. The Epithelial Cuff Concept D. Epithelial Attachment versus Epithelial Cuff

417 418 421 422 428

III . The Apical Shift of the Dento-gingival Junction

432

IV . The Structure and Chemistry of the Crevicular Epithelium

437

V. The Physiology of the Gingival Crevice A . The Bacteriology of the Gingival Crevice B. The Crevicular Fluid C. The Defence Mechanism of the Gingiva

446 446 448 450

VI . Conclusion

451

Reference s

452

I. THE HISTOGENESIS OF THE DENTO-GINGIVAL JUNCTION

Most of the information on the structure and physiology of the dento-gingival junction is derived from the study of man, monkeys and the dog. I n these species the relevant tissues as well as their mutual relationship appear to be essentially identical. Although some data are available on other species and, although the analogous tissues of rat, mouse, guinea pig and hamster may to some extent resemble those of man, there are known differences in what appear to be minutiae but which in some respects might be highly important. For this reason and because no comprehensive comparative anatomical studies of the dento-gingival junction have as yet been reported, the following description wil l mostly refer to studies on man, monkeys and the dog.

A s soon as formation of the enamel is completed, and the ameloblasts (Fig. 1) no longer discharge their primary function, they seem to undergo degenerative changes . The Tomes processe s disappear and a thin cuticle, the primary cuticle, marks the boundary between the enamel and the surrounding soft tissues. The ameloblasts are still cylindrical in shape, but they are shorter, less regular in arrangemen t than during the phase of enamel synthesis and the nuclei are more centrally situated (Fig. 2). A t their peripheries the ameloblasts are connected with the cells which formerly made up the 415

416

HARAL D

Fig. 1. Enamel epithelium during matrix formation: enamel (E), ameloblasts (A) with the Tomes processe s (Pr), stratum intermedium and outer layer (OL) derived from the outer enamel epithelium and stellate reticulum. Rhesus monkey (Macaca mulatto) Mallory-azan. x 100. (From McHugh, 1963.)

LÔE

stratum intermedium and the other layers of the enamel organ, and which have fused. The degeneratin g ameloblasts and the fused layers of the enamel organ at this stage prior to the eruption of the tooth constitute the reduced enamel epithelium which covers the entire crown of the tooth. It s cells resemble those of the basal layers of the stratified squamous epithelium of epidermis. The reduced enamel epithelium is composed of 6-12 layers of cells which are arranged with their long axes parallel to the enamel surface (Fig. 3). Soon after the completion of the enamel, important changes occur in the area over the tip of the crown. The ameloblasts become flattened, the nuclei disintegrate and the basal cells of the reduced enamel epithelium show mitotic activity (Fig. 4). As the tooth moves towards the surface, increased mitotic activity also occurs in the oral epithelium correspondin g to the occlusal aspect of the tooth. A s tooth eruption proceeds , these two actively proliferating epithelia are brought into contact with each other and, at the moment of tooth eruption, where the tip of the crown breaks through the oral epithelium, the most coronally located part of the reduced enamel epithelium fuses with the oral epithelium (Fig. 5). A s the tooth moves towards occlusion, the crown is gradually uncovered until the tooth has reached

Fig. 2. The reduced enamel epithelium of a permanent molar of rhesus monkey (M. mulatto) after enamel maturation. The Tomes processe s have disappeare d and the primary cuticle (pointer) marks the border to the enamel. The ameloblasts (A) are still cylindrical, but shorter, and the nuclei are more centrally located. Peripherally the ameloblasts are connected with a layer of squamous type cells (SC). Van Gieson. χ 200. (From McHugh, 1961.)

22. THE DENTO-GINGIVAL

JUNCTION

417

by Skillen (1931) and Baume (1952, 1953). This is to the effect that, as the tooth erupts, the enamel epithelium degenerate s and is replaced by a downgrowth of oral (marginal) epithelium. A similar view emerges from recent investigations by M c H u gh (1961, 1963), who maintains that the proliferative processe s which are responsible for the substitution of the reduced enamel epithelium take place both in the oral epithelium and in the basal layer of the formerly quiescent cells of the reduced enamel epithelium (Fig. 6) and that the ameloblasts and the rest of the reduced enamel epithelium may still for some time continue to cover the cervical parts of the enamel (Fig. 7). The exact time required for the total substitution of the reduced enamel epithelium is not known. There are, however, observations which indicate that this is subject to individual and species variation. M c H u gh estimates that for human t of the reduced permanent teeth the replacemen l enamel epithelium down to the cementum-ename junction is completed 2-4 years after the exposure of the ti p of the crown to the oral cavity.

II . T H E N A T U R E O F T H E

C O N N E C T I ON

B E T W E EN E P I T H E L I U M A N D E N A M E L Fig. 3. The reduced enamel epithelium overlying the incisai edge of a lower incisor rhesus of monkey (M. mulatto) The epithelial layer at the tip of the crown is thin, and the cells are orientated with their long axes parallel to the enamel surface. Haematoxylin and eosin. ÷ 13.5. (From McHugh, 1961.)

the plane of occlusion. A t that time about onefourth of the enamel surface is still covered by gingival epithelium. U p to this point in the process t among various authorthere is general agreemen ities. However, the further stages in the process, and in particular what happens in that part of the gingiva which faces the tooth, are the subject of a great deal of discussion and controversy. According to Gottlieb (1921a, b, 1927), the reduced enamel epithelium persists in that part of the tooth which is not exposed to the oral cavity at the time in question. Another view has been advanced by Becks (1929) and later supported

The amelo-gingival junction is composed of two different types of tissues, tooth enamel on one side and gingival epithelium on the other. These show major dissimilarities in both structural and chemical composition, the main difference being that mature enamel is an acellular, highly mineralized tissue, whereas gingival epithelium is a cellular soft tissue. Naturally basic differences in structure and chemistry of this magnitude give rise to equally profound differences in the physiology of the tissues, which in turn must characterize the relationship between them. The nature of this relationship has been the subject of discussion for many years and a great deal of research has been done in this field without actually arriving at a clear-cut definition of this relationship. The essence of the problem may be expresse d in the

HARAL D

418

LÔE

Fig. 4. Area of epithelium from over the cusp of a tooth of rhesus monkey (M. mulatto) at about the same stage of eruption as that in Fig. 3. Above: To the left columnar ameloblasts are still present. Nearer to the tip of the cusp to the right, they have become flattened and cells of the outer layer are starting to divide, x 250 Below: Higher magnification of mitotic activity in the outer layer, ÷ 400 Haematoxylin and eosin. (From McHugh, 1961.)

followin g question: Is the gingival epithelium i n structural continuity with the enamel, or are they simply in contact with one a n o t h e r? A . T H E ENAMEL

SURFACE

Refined methods of specimen preparation and investigation have disclosed a definite organic stroma within the enamel, the most conspicuous parts of which are the primary cuticle, the prism

sheaths and enamel lamellae. Together they amount to 0.5% of the total weight of the enamel, and have been estimated to correspond to 1-2% of the volume. Although the primary cuticle and prism sheaths are derivatives of the ameloblasts and are formed during amelogenesis , there is still some disagreemen t on whether the enamel lamellae are formed during the initial deposition of enamel (Awazawa, 1959) or whether they originate as cracks which later are filled by organic

Fig. 5. Section through the gum tag overlying an erupting molar of rhesus monkey (M. mulatto). The oral epithelium (OEp) and the most coronally located part of the reduced enamel epithelium (pointers) have fused. The overlap between the paler reduced enamel epithelial cells and the darker oral epithelial cells can be seen in the depths of the crevice (lower pointer). Haematoxylin and eosin. ÷ 40. (From McHugh, 1961.)

Fig. 6. Diagrammatic representatio n of the epithelial changes associate d with tooth eruption. (1) The crown is covered by a cap of enamel epithelium without connection with the oral epithelium. (2) The tip of the tooth has entered the oral cavity and the gingival cuff has formed from oral epithelium and the outer layer of enamel epithelium. (3) The inner layers of the enamel epithelium degenerat e and are replaced by the cuff which grows apically down to the cementum-ename l junction. (FromjMcHugh, 1961.)

420

HARAL D

LÔE

Fig. 7. Erupting canine of a green monkey (Cercopithecus aethiops). The cuff epithelium (Cf Ep) proliferates apically and gradually replaces the reduced enamel epithelium (EEp). ÷ 2 0. Higher power views (x 1 0 0) of these two epithelia appear on the right. Haematoxylin and eosin.

material from saliva (Sognnaes , 1950). The prism sheaths extend from the enamel-dentin e junction to the enamel surface, where they end in a thin continuous membrane approximately 1 micron in thickness (Fig. 8). This is the primary cuticle. The staining characteristic s of the organic structures, including the primary cuticle, suggest that chemically they are closely related to keratin. For instance, enamel matrix reacts positively for sulphydryl groups before mineralization and for disulphide groups after mineralization (Wislocki and Sognnaes , 1950). This mode of reaction is in complete agreemen t with the histochemica l changes which take place in keratinizing tissues. Recent chemical analyses and crystallographic data, however, tend to show that the organic

matrix of enamel is not a keratin (see Chapter 20) and that the enamel protein may be of a very special nature (Glimcher, Bonar and Daniel, 1961a; Glimcher et al, 1961b). The a m o u nt of organic material in the surface layer is greater than in the main mass of the enamel (Loe and Ravnik, 1961), and the content of inorganic matter and the degree of mineralization may to some extent vary from the surface into the deeper parts of the enamel (Brudevold, Steadman and Smith, 1960). In this context, however, the important fact is that, once enamel has been formed, no regularly occurring physiological change has been reported to take place in the organic matrix of enamel. In normal mature enamel the organic elements at or near the surface,

22. T H E

DENTO-GINGIVAL

d Fig. 8. Longitudinal section through demineralize enamel of dog. The prism sheaths and a thin, continuous membrane (the primary cuticle) at the enamel surface. Haematoxylin and eosin. χ 1100. (From Loe, 1962.)

including the primary cuticle, are mineralized and an unmasking of these structures does not occur under physiological conditions. B. T H E EPITHELIAL ATTACHMEN T

CONCEPT

Investigations with the light microscope (Gottlieb, 1921a,b) and the electron microscope (Ussing, 1955) show that the ameloblasts are in structural continuity with the enamel through the medium of their organic components for some time after enamel formation has ceased . This relationship, however, is limited to a short period of time before the ameloblasts atrophy and disappear . According to Gottlieb, after the disappearanc e of the ameloblasts the remaining cells of the reduced enamel epithelium secrete a noncellular cuticle, which

J U N C T I ON

421

he named the secondar y enamel cuticle, which unites or comes into structural continuity with the primary cuticle by, as it were, "melting into it " and keratinizing. It was suggeste d by Gottlieb that under ideal conditions the entire length of the epithelium from the gingival margin to the cementum-ename l junction comes to be attached to the tooth surface in this way. This concept was known as "der Epithelansatz " in German literature, and was introduced into English and American literature as "the epithelial attachment". Another view, slightly different from that of Gottlieb, was put forward by Becks (1929) and Skillen (1931), who postulated that the reduced enamel epithelium does not persist, but degenerate s and becomes keratinized to form an acellular secondary cuticle which is in organic continuity wit h the primary cuticle. I n their view, at the same time epithelial cells of the oral epithelium proliferate and migrate alongside the secondar y cuticle to take the place of the reduced enamel epithelium. Thoughts along this line have also been expresse d by Glickman and Bibby (1943) and Ussing, Scott and Kaplan (1951). Baume (1952, 1953) on the other hand, rejected the concept of a secondar y cuticle as a medium of structural continuity between the two tissues and took the view that the new epithelial cells arising from the oral epithelium become attached to the primary cuticle by means of tonofibrils. Cohen (1962) has produced some evidence for the at least occasiona l existence of connections between crevicular epithelium and enamel in the form of prolongations of the cells extending into the substance of the enamel which resemble enamel lamellae. According to these schools of thought, in the absence of disease no crevice exists between gingiva and the tooth surface. A shallow groove produced by degeneratio n of the cells at the coronal border of the epithelial attachment is considered to be a physiological reaction to continuous bacterial and mechanica l irritation. This groove is known as the gingival sulcus and extends from the gingival margin to the reflection

422

HARAL D

of the secondar y enamel cuticle as it is commonly seen in histological sections (Fig 10.). The average depth of this sulcus varies according to the estimates of different authors. According to Kronfeld (see Boyle, 1955) the depth of the sulcus as it appears in histological preparations averages 0.05 mm, whereas Orban and Kohler (1924), also on sections, found that under normal conditions the depth may vary between zero and 6 mm, but that 50% of all preparations measure d were close to zero. The concept of the epithelial attachment as a true structural union between gingiva and enamel is derived from the study of routine histological preparations of autopsy material and seems to be confirmed by the common finding of epithelial cells adhering to the tooth surface of extracted teeth. Moreover, various attempts to detach the gingiva from the tooth in vivo (Weinreb, 1960) or in histological preparations have tended to show that the epithelium tears rather than peels off the enamel surface. Such findings have been taken as an indication of the strength of the connection between enamel and epithelium (Sicher, 1962). C . T H E EPITHELIAL C U FF CONCEPT

Lately, the research into this problem has changed from descriptive morphological studies towards a more dynamic approach. This new phase of enquiry was introduced by the experiments of Waerhaug (1952). His working hypothesis was based on the consideration that the histological concept of the epithelial attachment did not coincide full y with the clinical observations . 1. Clinical and Experimental

Evidence

By inserting thin steel blades (0.05 mm thick and 1 mm wide) into the gingival sulci of children, it was found by radiography that the steel blades in all cases ended at the cementum-ename l junction (Fig. 9). Histologic examination subsequen t to such manipulations showed that the epithelium did not differ morphologically from that facing

LÔE

Fig. 9. Radiograph of upper premolars of a 12-year-old girl after the insertion of thin steel blades to the bottom of the gingival crevice. The blades reach to the cementum enamel junction (arrows). (From Waerhaug, 1952.)

untreated teeth (Fig. 10). The force which was required to bring the steel blades down to the cementum-ename l junction was measure d and rarely exceede d 1 gm. On the basis of these and a variety of other experiments , Waerhaug (1952) restated the view, which was held to be valid prior to Gottlieb, that no structural continuity exists between gingiva and enamel (see Black, 1915). Furthermore, Waerhaug produced evidence that the tears in the epithelium seen in histological preparations , and which served as a basis for the assessmen t of the bottom of the gingival sulcus, were artifacts, and that the bottom of the gingival crevice is located at the cementum-ename l junction. The objection has been raised that the steel blades in question acted as knives and that during insertion these might have cut through the epithelial attachment (Orban et al., 1956). Repetition of the experiments by several researcher s (Zander, 1955; Waerhaug, 1960; Cohen, 1962) using cellulose strips, where the strips could hardly have any cutting effect and where the force which could be applied was negligible, has confirmed the results obtained with steel strips. In Fig. 11 the cellulose strip was inserted into the gingival crevice and cemented to the crown above the gingival margin 3 days before the animal was

22. T H E

DENTO-GINGIVAL

Fig. 10. Gingival crevice of dog 1 hour after insertion of steel blade to the cementum-ename l junction (CEJ). ÷ 7. Higher magnifications ( x 315) shows that cuff epithelium does not deviate morphologically from the normal. Cu indicates the cuticle. BS indicates the bottom of the gingival sulcus according to the epithelial attachmen t concept. Haematoxylin and eosin. (From Waerhaug, 1952.)

sacrified. The edge of the strip is at the cementumenamel junction. According to the theory of the epithelial attachment, this would be regarded as a crevice of zero depth, whereas this experimenta l measuremen t suggests a depth of approximately 3 mm. I n a series of experiments it has been demonstrated, as far as the light microscope can reveal, that the epithelial cells come to li e closely against artificial crowns (Waerhaug, 1953) and other nonirritating materials (Waerhaug, 1956a, 1957a,b; Waerhaug and Zander, 1957; Waerhaug and

J U N C T I ON

423

Fig. 11. Gingival crevice of a dog 3 days after the insertion of a cellulose strip which has reached the cementumenamel junction. Haematoxylin and eosin. ÷ 15. (From Waerhaug, 1960.)

Lôe, 1958a) in the same way as the epithelial cells li e against the enamel. The sum total of these research activities indicates that the generally accepted theory concerning the epithelial attachment and its practical implications are open to serious question. In fact, it has been suggeste d that no true attachment exists and that the epithelial cells facing the tooth normally are kept in contact with the enamel surface by adhesion (Waerhaug, 1960) and the tissue tonus of the free gingiva (Arni m and Hagerman, 1953). Accordingly, Waerhaug

424

HARAL D

(1952) proposed the substitution of the term epithelial cuff for the term epithelial attachment. Hitherto, the means by which the superficial cells were kept in close contact with the enamel were not considered in detail. It was suggeste d that the superficial cells may adhere to the enamel surface by the same mechanism s as those by which cells from the outer layer of the mucous membrane attach themselves to a microscopic slide when pressed against the inside of the li p (Waerhaug, 1952). A similar pressure of the crevicular epithelium against the tooth surface may be brought about by the tissue tonus of the free gingiva. I t seems unlikely that the special arrangemen t of the fibres of the free gingiva contribute as an active force in this direction because the circular fibres (Arni m and Hagerman, 1953) and the fan-shaped system of free gingival fibres (Ainamo and Loe 1966) are composed of collagen which possesse s no intrinsic elasticity. However, these fibre systems constitute the greater part of the free gingiva, and it is therefore reasonabl e to assume that the state and the organization of the collagen fibres are essentia l for the developmen t and the maintenance of a proper dento-gingival relationship. Some attempts have been made to characterize more specifically the nature of the contact between the epithelium and enamel surface. It is well known from tissue culture work that many types of cells tend to stick to glass and that cellular stickiness is dependen t on the coating on the cell surface or on the surface to which the cell sticks. It has recently been shown in tissue culture experiments that the squamous epithelial cells of oral mucosa exhibit sticky properties and stick to siliconized glass (Berwick and Coman, 1962). Experiments with various enzymes in this model system suggest that the stickiness is probably related to a mucopolysaccharid e protein substance produced by the cells themselves . Some experiments performed by Schultz-Haudt et al. (1963) show even more directly that crevicular epithelium can stick to glass as well as to enamel. Cells obtained by scraping the inside of the

LOE

human gingival crevice were transferred to the surface of fragments of glass or dental enamel which were then centrifuged at various speeds . The stickiness of the cells was measure d in terms of the centrifugal force that the adhesion between the cells and the glass or enamel surface would survive. The effect of various enzymes on the adhesion was assesse d in this way. They inferred from the results that the cells of the crevicular epithelium stick to the enamel surface in vivo and that the stickiness is mainly dependen t on the serum proteins of the crevicular fluid. Quite recently it has been shown, however, that strictly normal gingiva exhibits no flow of fluid (see p. 449). It has, therefore, been suggeste d (Loe and Holm-Pedersen , 1965) that in the absence of disease the stickiness is mediated by the intercellular cement substanc e of the epithelium. This implies that the superficial cells on the surface facing the enamel are coated with intercellular substance . Recent investigations tend to show that this is the case and that the intercellular substance is of a polysaccharide-protei n nature (Stallard, Diab and Zander, 1965; Toto and Sicher, 1964). 2. Renewal Rate of the Crevicular

Epithelium

Counts of mitotic figures (Hirth, Hartl and Muhlemann, 1955; Trott, 1962; Trott and Gorenstein, 1963) indicate that the mitotic activity in the crevicular epithelium is as great as or greater than in any other oral epithelium. These observations suggest that new cells are constantly formed. If so, and the crevicular epithelium is not to increase progressively, it follows that the surface cells must be desquamate d at a correspondin g rate. If, furthermore, cells are desquamate d from the crevicular epithelium, they would most likely be released from the gingival crevice into the oral cavity. Based on this statemen t of the problem, an investigation was undertaken of cellular elements emanating from the gingival crevice (Loe, 1961). Forty-nine clinically normal gingival crevices of dogs were sealed by painting a solution of colo-

22. T H E

DENTO-GINGIVAL

J U N C T I ON

425

d epithelial Fig. 12. Dog canine 1 hour after the gingival crevice had been sealed at the margin. A cluster of desquamate cells and polymorphonuclea r leucocytes has formed in the marginal part of the pocket at Α . ÷ 10. Higher power views ( x 450) of the areas A , B, and C showing leucocytes within the crevicular epithelium appear on the right. Haematoxylin and eosin. (From Loe, 1961.)

phony upon and around the marginal parts of the gingiva and tooth. The animals were sacrificed after intervals of 1-48 hours. During the subsequen t procedures , great care was taken to produce microscopical preparations which preserved the possible contents of the gingival crevices. The histological preparations showed in all cases small and larger aggregation s of cells within the crevice. In the teeth observed for short periods (1-6 hours) the cells were clustered near the gingival margin (Figs. 12 and 13). In the long-term experiments (12-48 hours) the gingival crevices were fille d with cells to such an extent that the free

gingiva was forced away from the enamel (Fig. 14). Close examination showed that the cells were desquamate d epithelial cells and neutrophil leucocytes in varying stages of degeneration . In other words, this investigation showed that epithelial cells are desquamate d from the crevicular epithelium as a continuous process. Wit h this method it was impossible to asses s the rate of renewal of the crevicular epithelium. However, research on the deoxyribonucleic acid ( D N A ) synthesis in various oral epithelia by means of tritiated thymidine has demonstrate d that the synthesis of D N A , and hence the mitotic activity

426

HARAL D

LÔE

Fig. 13. Dog canine 6 hours after the crevice was sealed, ÷ 20. (Â) Desquamate d epithelial cells and altered polymorphonuclea r leucocytes have aggregate d at the gingival margin, ÷ 450. (C) Leucocytes within otherwise normal crevicular epithelium, ÷ 400. (A) Mitoses at the apical end of the gingival cuff, x 1150. Haematoxylin and eosin. (From Loe, 1961.)

22. T H E

Fig. 14. Dog canine cells and leucocytes are material has forced the stages of degeneratio n at

DENTO-GINGIVAL

J U N C T I ON

427

18 hours after closure of the gingival crevice. (A) Heavy aggregation s of desquamate d epithelial present between the crevicular epithelium and the enamel surface. The pressure of the trapped gingiva away from the enamel surface, x 9.5. (B) Desquamate d cells and leucocytes in varying the surface of the crevicular epithelium, x 1045. Haematoxylin and eosin. (From Lôe, 1961.)

in this part of the gingiva, is as great as, or greater than that of squamous epithelium elsewhere in the mouth, and that the epithelium from the gingival margin to the cementum-ename l junction is constantly renewed (Greulich, 1961; Beagrie and Skougaard, 1962). By observations and calculation, the renewal time in rhesus monkeys has been found to be 3-6 days (Skougaard and Beagrie, 1962). In other words, these epithelial cells have a lif e cycle the length of which corresponds to the time required for the cells to move or be moved from the basal layer to the surface. Naturally, this state of flux has a decisive bearing on the question of whether or not the superficial epithelial cells are in permanent structural continuity with the enamel or are otherwise attached to it. Fig. 15. Gingival crevice of dog. An escharotic (camphor and phenol mixture) was inserted one hour previously. The epithelial cuff and parts of the subjacent connective tissue have necrotized. Haematoxylin and eosin. χ 15. (From Waerhaug and Lôe, 1958b.)

428

3. Regeneration of the Crevicular

HARAL D

Epithelium

I t is a corollary of the concept of the epithelial attachment as a static primary union between the epithelium and the mineralized primary cuticle (Gottlieb, 1921a) that if the attachment is damaged, e.g. if the epithelium is torn away from the enamel, the damage wil l be permanent. That the concept is invalid in this respect has been shown by the following. A n escharotic (a mixture of phenol and camphor) was inserted into the gingival crevices of dogs (Waerhaug and Loe, 1958b). Histological examination of controls showed that the epithelium of the crevice and parts of the subjacent connective tissue were destroyed (Fig. 15). However, examination after intervals showed that new epithelial cells proliferated from the gingival margin, or

LÔE

from crevicular epithelial cells which survived, and after 3 or 4 weeks a new, in every respect normal, epithelium was formed (Fig. 16). If the entire free gingiva is excised (Fig. 17) in experimenta l gingivectomy (Waerhaug and Lôe, 1957; Loe and Silness, 1961), new epithelial cells grow from the epithelium of the attached gingiva and meet the tooth surface more or less at right angles so that there is virtually no gingival crevice. However, the free gingiva then tends to grow in height so that 6 weeks later a new epithelial cuff has formed with a new crevicular epithelium which histologically and histochemically can be characterize d as normal (Fig. 18A). By careful histological processing the organic matter of the enamel can be preserved to show the close contact between the new epithelium and the enamel (Fig. 18B).

Fig. 16. Crevicular epithelium in a dog 32 days after treatment with escharotic. ÷ 15. The higher power view shows new epithelial cells which have the normal relationship to the enamel surface, ÷ 400. Haematoxylin and eosin. (From Waerhaug and Loe, 1958b.)

22. T H E

DENTO-GINGIVAL

J U N C T I ON

429

Fig. 17. The free gingiva of this tooth of a dog was removed surgically. Twenty-three days later new epithelial cells, proliferating from the wound edge on top of granulation tissue, have met the tooth in a linear contact. The mass d is the dressing. Haematoxylin and eosin. ÷ 8. (From Loe and Silness, 1961.)

Fig. 18. (A) Gingival margin of a dog 6 weeks after gingivectomy to the level of the bottom of the gingival crevice (notch). A new epithelial cuff of normal morphology has formed, x 13. (B) A higher power view of the area indicated by the pointer in (A). By carefully histological processing the organic structure of enamel has been preserved , showing the contact relationship between the new epithelium and the enamel. Haematoxylin and eosin. ÷ 400. (From Lôe and Silness, 1961.) 29

430

HARAL D

On the basis of this and similar experimenta l evidence it is reasonabl e to conclude that if the gingival crevice is probed (Waerhaug, 1952), if medicament s (Waerhaug and Loe, 1958b; Lôe and Silness, 1963) and other foreign bodies (Waerhaug, 1957b; Zander, 1956) are brought in between the epithelium and the tooth, or even if the entire epithelial cuff is removed (Waerhaug and Loe, 1957; Loe and Silness, 1961), complete healing wil l occur provided that no irritating material is retained between the new epithelium and the tooth surface. D . EPITHELIAL ATTACHMEN T VERSUS EPITHELIAL C U FF

A completely adequate description or demonstration of the structural relationship between the surface layer of the gingival epithelium and the enamel surface does not exist. However, as already noted, according to the concept of epithelial attachment , this union could be mediated by the secondar y cuticle (Weski, 1922), by tonofibrils from the surface cells (Baume, 1953) or occasionally by cellular prolongations into the enamel (Cohen, 1962). The secondar y cuticle, also called the dental cuticle or the epithelial attachment cuticle, was originally thought to be the product of the reduced enamel epithelium. Recent research has shown that the reduced enamel epithelium does not persist, but that it is replaced by new epithelial cells from its basal layers and from the oral epithelium. Accordingly, the opinion is held that the secondar y cuticle is a derivative of this new epithelium (Sicher, 1962), both when the epithelial attachment is confined to the enamel surface and when this has proliferated beyond the cementum-enamel junction. The secondar y cuticle has been studied in scrapings and in jaw specimens subjected to various histological and histochemica l techniques . Cuticles which have been designate d secondar y cuticles have been described on natural teeth (Weski, 1922; Waerhaug, 1952; Wertheimer and

LÔE

Fullmer, 1962), on artificial crowns (Waerhaug, 1953), on prothèses (Turner, 1954) and on various other artificial implants in the oral cavity (Zander, 1956; Voreadis and Zander, 1958). They have been observed in relation to both normal and chronically inflamed gingiva and upon salivary calculus and nonmineralized debris on the tooth surface. The morphological and chemical characteristic s of these so-called secondar y cuticles described in the literature are very diverse so that it is by no means certain that they are comparable structures or are all formed in the same way. The secondar y cuticle described by Cape and Kitchin (1930) was biréfringent in polarized light while others have commented on its nonbirefringence (Rushton, 1954; Wertheimer and Fullmer, 1962). In haematoxyli n and eosin stained sections it may stain pink, or may not stain at all (Waerhaug, 1956b). It reacts positively to the periodic acid-Schiff procedure according to some authorities (Mandel and Levy, 1957), and does not according to others (Wertheimer and Fullmer, 1962). Conflicting accounts exist also of other tinctorial properties of the cuticles. The secondar y cuticle has for a long time been commonly regarded as a hornified or keratinized product of the surface cells of the epithelial attachment (Weski, 1922). Others have regarded it as a product of degeneratio n of these cells. A s wil l be shown in a subsequen t section, there is no histochemica l basis for the assumption that keratinized cells, viable or degenerated , are present i n this area under normal conditions (McHugh, 1964), and in any case the view that keratinization is caused by cell degeneratio n and decay has been replaced by the more recent concept of keratinization as a form of cytodifferentiation involving specific active biochemical processe s (Rothman, 1954). The main objection to the results of many studies of the secondar y cuticle derives from the fact that very few workers have distinguished between normal and pathological material. The thickest membranes are found in connection with calculus and soft deposits (bacterial plaque) and patholo-

22. T H E

DENTO-GINGIVAL

gically changed pocket epithelium. In uninflamed gingiva the cuticle tends to be thinner, and may even be absent. Therefore it seems possible that the secondar y cuticle actually represent s the high molecular weight organic constituents of the crevicular fluid which are caught between the tooth surface and the adjacent epithelium on the way to the orifice of the pocket and in histological preparations have been coagulated by fixatives. In the presence of different degrees of inflammation the amount of fluid varies. It increases with the severity of the inflammation and may also be changed in chemical composition. This quantitative and qualitative difference of the gingival exudate may account for the varying width and staining reaction of the so-called secondar y cuticle. A n organic union between epithelium and enamel by means of tonofibrils into the primary cuticle, as suggeste d by Baume, has not been demonstrated . N or has it in any way been explained how such

...

.

J U N C T I ON

431

an attachment of fibril s into an already mineralized cuticle is brought about. On the contrary, investigations with the electron microscope have disclosed that tonofibrils do not pass from one cell to another but are intracellular components (Figs. 25-27). Whether extensions of epithelial cells into the enamel of the kind described by Cohen (Fig. 19) can maintain this biological continuity after the enamel epithelium is replaced is not known. Most of the work supporting the concept of the epithelial attachment is based on decalcified preparations , in which not only is the enamel dissolved, but in which also the organic structures have probably been the subject of changes during the preparative procedures . A few workers (Toller, 1939; Weinreb, 1960) have used undecalcified material. However, ground sections of the thickness commonly obtained by this method are of doubtful value in this context. As yet, no one has

©

Fig. 19. (A) Erupting incisor of green monkey ( C. aethiops) showing extension of the reduced enamel epithelium into the enamel (enamel lamella) close to the area of the cementum-ename l junction. The lamella is composed of squamous epithelial cells which are continuous with the basal cells of the reduced enamel epithelium, x 8.3. (B) Higher power view of the connection between lamella and reduced enamel epithelium, ÷ 83. ( C) Higher power view of the lamella, ÷ 332. Haematoxylin and eosin.

432

HARAL D

furnished concrete evidence that a structural continuity exists between epithelium and the tooth. Indeed, the most severe handicap in discussing this issue is that so far no demonstratio n of the submicroscopica l relations between the cells at the surface of the crevicular epithelium and the intact enamel surface has been made. Until such evidence is available the finer arrangemen t of this relationship must continue to be a matter of conjecture. A t the present time the concept of the dentogingival junction as an epithelial cuff seems to be in accord with clinical experience and with the histological evidence. A t any rate, it is clear that if there is some form of attachment by structural continuity it is mechanically very weak. The most convincing evidence against the existence of structural continuity and in favour of the concept of the dento-gingival junction as a contact relationship is derived from the study of the dynamic processe s taking place in this area. The continuous loss at the surface and the renewal of the epithelium imply that the union between the surface cells and the enamel would have to be continuously re-established , irrespective of whether the attachmen t is mediated by a secondary cuticle or in some other way. Thus, with reference to the data presente d as well as on the existing knowlege of the morphology, chemistry and physiology of the dento-gingival junction which wil l appear in the following sections, we may justifiably conclude that, following the atrophy and disappearanc e of the ameloblasts , the epithelium facing the tooth surface is not in structural continuity with it but is kept in close contact with it by the stickiness of the intercellular substance of the superficial cells and the tonus exerted by the blood pressure and the connective tissue fibres of the marginal gingiva. This relationship is adequatel y expresse d by the term epithelial cuff. The gingival crevice extends from the gingival margin to the deepes t point of the epithelial cuff, which at any rate at first is located at the cementumenamel junction. The depth of the gingival crevice in conditions of health rarely exceeds 3 mm.

LÔE

III. THE APICAL SHIFT OF THE DENTO-GINGIVAL JUNCTION A t the stage where the tooth has reached the occlusal plane the bottom of the gingival crevice is located at the cementum-ename l junction. Over the entire surface of the root Sharpey fibres are anchored into the cementum, constituting the attachment of the teeth to the jaws. This is the fundamenta l arrangemen t of the periodontium. I t is held by many authorities, starting with Gottlieb (1920), that this state of affairs is merely a transitory one and that the epithelium, as age progresses , tends to proliferate in the direction of the root apex, and, in so doing, establishe s a new fir m union (epithelial attachment ) with the cémentai surface. This apical shift of the dentogingival junction has been termed passive eruption and is believed to continue at varying rates throughout life. The migration towards the root apex of the bottom of the gingival crevice may be unaccompanied by an increase in pocket depth or only shallow pockets may be found, in which case recession of the gingival margin has taken place concomitantly with the downgrowth of the epithelium. Why marginal atrophy occurs sometimes, and in other instances a deepening of the pocket takes place, has not been explained. In any event, downgrowth of the crevicular epithelium below the cementum-ename l junction, with or without gingival recession , represent s significant changes in the periodontium, changes which from a functional point of view are associate d with loss of fibrous attachmen t and functional support of the teeth. According to the Viennese school of thought (Kronfeld, 1936; Sicher, 1962), passive eruption was considered to be a physiological process that continues throughout lif e at a rate correspondin g to continuous axial movement, or active eruption, of the teeth as compensatio n for attrition. Active and passive eruption were thought to occur simultaneously, the purpose of the latter being

22. T H E

DENTO-GINGIVAL

to keep the exposed functional or "clinical" crown to a more or less constant length. Any apical proliferation of cells of the crevicular epithelium along the cémentai surface presuppose s a breakdown of the adjacent Sharpey fibres. According to Gottlieb (1920), loss of fibre attachment occurs as a result of a devitalization of the cementum. In his opinion, attached fibres exist and function for a certain length of time and then they degenerat e and disappea r leaving behind devitalized cementum. According to Gottlieb, the survival of cementum is age-related . This view has never been substantiated , however. A s wil l be shown in the following, the fate of the fibre attachment seems to be entirely dependen t on the supra-alveola r connective tissue, and whether or not apical migration of the epithelial cells can take place is determined by the state of that part of the fibre system which is not anchored in the cementum. A simple degeneratio n of these fibres as part of a physiological process has not been demonstrated and seems very unlikely. On the other hand, collagen wherever it is situated in the body has a certain turnover; that is, collagen fibrils and fibres are dissolved and replaced by new ones. The turnover of periodontal collagen has not been studied sufficiently, but there seems to be no reason to believe that this collagen differs in this respect from similar collagen elsewhere in the body (Stallard, 1963). These processes , however, are physiological ones which aim at the maintenanc e of the tissues and cannot be responsible for the permanen t destruction of the principal fibres in question. Aisenberg and Aisenberg (1948) have suggeste d that a permanent dissolution of the collagen fibres may be brought about by an enzymic lysing effect of the epithelial cells. There is, however, as yet no evidence to substantiat e such an activity on the part of the crevicular epithelium. Rushton, in a series of experiments on hamsters , explored the possibility that the endocrine systems and general nutritional factors, by influencing

J U N C T I ON

433

collagen formation and maintenance , or in some other way, may play a part in the removal of gingival collagen fibres and migration of the crevicular epithelium. Rushton (1951) first noted that in the hamster the rate of migration of the epithelium, as measure d on sections of the jaws of animals over a range of age, varied according to a constant pattern in different sites which were not related to the presence of bacterial plaque. He noted that the addition of charcoal to the diet, though not increasing attrition, reduced the amount of bacterial plaque and of epithelial migration. In a later series of experiments , Rushton (1955) found that epithelial migration was retarded by the addition of Aureomycin to the diet. It is well known that endocrine function affects connective tissue physiology, and that especially the sexual hormones have specific effects on the synthesis and maintenanc e of fibrous collagen of the reproductory organs during the pregnancy and post p a r t um (Fainstat, 1963a,b). There is, however, no evidence to suggest that a clinical or subclinical imbalance of the female or male sexual hormones during or outside pregnancy produces appreciable changes of the periodontal collagen fibres (Lôe, 1965). Rushton (1952) conducted an experiment to determine whether hormonal agents believed to produce and maintain collagenous matrix would affect the rate of epithelial downgrowth in hamsters . He demonstrate d that administration of methyltestosteron e retarded migration of the crevicular epithelium though it by no means abolished it. Deficiencies of special dietary components do not seem to cause downgrowth of crevicular epithelium. N ot even in vitamin C deficiency, where connective tissue metabolism is seriously altered, is the gingival connective tissue destroyed to the extent that a downgrowth of epithelium can take place (Waerhaug, 1958) (Fig. 20). The concept of passive eruption as a physiological process seems to derive mainly from microscopic examination of h u m an teeth (Gottlieb, 1920) of different ages and in which it was regularly seen that the bottom of the pocket in the teeth of adults

434

HARAL D

LÔE

Fig. 20. Marginal periodontium of upper canine of green monkey (C. aethiops) fed on a vitamin C-free diet for 6 months. There is a notable absence of acid fuchsin staining collagen in the connective tissue subjacent to the crevicular epithelium (CrEp), except in the area CEJ, in comparison with the connective tissue (CT) subjacent to the oral epithelium, ÷ 9.5. A higher power view ( x 95) of the area CEJ appears on the right. Van Gieson stain.

was situated at varying levels below the cementumenamel junction. I n view of the fact that it is wellknown that nearly all h u m an subjects who liv e on contemporar y sophisticate d diets suffer in some degree from chronic inflammation of the gingiva (Loe, 1963), such selected h u m an specimens, and even haphazard preparations of the teeth of animals, without a known history (Kronfeld and Ullik , 1928) are of doubtful value as a basis for investigating this problem. The most obvious cause of apical migration of the cells of the crevicular epithelium is that the uppermost fibres have been destroyed as a result of gingival inflammation. I t is true that histological pre-

parations may be encountere d where the crevicular epithelium is below the cementum-ename l junction and where histological signs of inflammation are absent. However, appearance s lik e these alone do not exclude the prior existence of inflammatory reactions at the moment of dissolution of the fibres and apical migration of epithelium. Histological sections represen t static pictures of the morphological relationship at the moment of death of the individual and, unless the specimens are parts of an experimenta l series, such preparations do not usually demonstrat e the events that lead up to the situation that is seen at the moment of examination.

Fig. 21. Lower first molar of a green monkey (C. aethiops) 9 months after having been ground out of occlusion. (A) The alveolar margin showing that the bottom of the gingival crevice is still located at the cementum-ename l junction, ÷ 10. (B) The apical area, ÷ 100. (C) The bifurcation, ÷ 100. Arrows point to new bone and cementum laid down in association with the eruption of the tooth into occlusion. (From Loe, 1966.) Haematoxylin and eosin.

436

HARAL D

LÔE

A certain amount of evidence, from man and from observations on a variety of mammals under both natural and experimenta l conditions, has been adduced in support of Gottlieb's view that apical migration of an "epithelial attachment" is a physiological age change and probably integrated with wear of the teeth (Miles, 1961). On the whole it is unconvincing and, having mind to the difficulties of separating the influence of disease processes , this is not very surprising. Much of the evidence is conflicting and in fact there appears to be an almost equal weight of evidence against Gottlieb's idea. For instance, Belting, Schour, Weinmann and Shepro (1953) found some evidence of progressive migration of the crevicular epithelium wit h age in two different strains of rat, but noted that there were notable differences between the two strains in respect of rates of attrition and of cementum formation. On the other hand, in laboratory-bred mice, Baer and Bernick (1957) found that the amount of gingival recession was related to the severity of local inflammatory processe s and was not in any way related to age. Preliminary results in our laboratory show that active eruption may take place without any movement of the epithelial cuff below the cementumenamel junction. Figure 21 shows one of several teeth of three green monkeys. These teeth were ground out of occlusion so that approximately 2 mm separate d the opposed teeth. In the course of nine months occlusal movement brought these teeth into occlusion again. New cementum and bone were formed to compensat e for the eruption. The bottom of the gingival crevice is still located at the cementum-ename l junction, and the relationship between this and the margin of the alveolar bone is normal, due to apposition of bone tissue at the alveolar margin. In other words, occlusal movement of teeth does not necessaril y imply an apical shift of the dento-gingival junction. The observation that in the dentition of Eskimos (Waugh, 1937; C. H. M . Williams, personal communication, 1964) the gingival margin usually covers the cementum-ename l junction even in the presence

Fig. 22. Anterior teeth of a man aged 64 years without periodontal disease . Although there is a great deal of occlusal wear there is no recessio n of the gingival margin and examination with a probe showed the bottom of the crevice to be still at the cementum-ename l junction; that is, passive eruption had not occurred pari passu with the shortening of the clinical crowns.

22. T H E

DENTO-GINGIVAL

of extreme occlusal wear may corroborate these experimental findings. Similar conditions may occasionally be found in Europeans (Fig. 22) who have escape d periodontal disease . I n summarizing this section it may be stated that passive eruption, or more accurately, the apical shift of the dento-gingival junction is not a physiological progress. Migration of the crevicular epithelium beyond the cementum-ename l junction is possible only after dissolution of the adjacent Sharpey fibres. Commonly, this destruction is effected at the stage where the marginal inflammation has extended into the supra-alveola r connective tissue. It should be noted, however, that in either case the process can be arrested by therapy or naturally. Providing there is no continuous ingress of bacteria a normal crevicular epithelium wil l re-form and wil l become adapted to the root surface in the same relationship as existed in respect of enamel when the tooth first erupted.

IV. THE STRUCTURE A N D CHEMISTRY OF THE CREVICULAR EPITHELIUM I t is widely held that clinically normal gingiva always exhibits a low degree of chronic inflammation and that the distinction between a normal and a pathologically changed gingiva is vague. Recent, but still unpublished, results from our laboratory show, however, that a strictly healthy gingiva at the clinical level also shows absence of inflammation when examined in microscopical preparations . Accordingly, in the following discussion efforts wil l be made to deal with a crevicular epithelium which is literally free of inflammatory cells and which covers a connective tissue showing no leucocytes or other blood cells in an extracellular location. Only in this way, it is felt, is it possible to arrive at a description of the normal morphology and histochemistry which may contribute to the understandin g of the physiology of this region and which may serve

J U N C T I ON

437

as a basis for a more precise attitude towards the pathological deviations. I n full y developed and erupted teeth the crevicular epithelium extends from the gingival margin to the cementum-ename l junction. Usually the h u m an free gingiva, instead of forming a knife edge termination on the tooth surface, presents a rounded margin. The gingival crevice then terminates in a V-shaped orifice, the soft tissue wall of which is composed of marginal epithelium. The crevicular epithelium is bounded by the tooth surface on one side and by the subepithelial connective tissue on the other and consists of a thin layer of stratified squamous epithelium of varying thickness. Ordinarily, it is made up of 5-15 rows of cells, the number of layers decreasin g nearer the cervix. A t the cementum-enamel junction it is not unusual to find that it consists of a single cell layer. The histological appearanc e of the crevicular epithelium exhibits certain differences from the oral parts of gingival epithelium. It is thinner and lacks the connective tissue papillae and their corresponding epithelial ridges. Consequently , the basemen t membrane bordering the subepithelial connective tissue is smooth and does not undulate (Fig. 23). In the normal gingiva of adults, however, epithelial ridges and connective tissue papillae may be encountere d in the marginal part of the crevicular epithelium. Provided that no signs of inflammation or other pathological conditions are present, this should be regarded as normal. It may merely indicate that the gingiva previously has been subject to irritation, but that healing has occurred. The crevicular epithelium may be divided into two layers, a basal cell layer and a prickle cell layer. The basal cells do not differ from those of other types of stratified squamous epithelium. They are cuboidal cells resting on a basemen t membrane. The cells of the prickle cell layer are somewhat smaller than in other types of squamous epithelium. The cells of the first row may have the ordinary polygonal appearanc e but, in passing

438

HARAL D

Fig. 23. Crevicular epithelium of dog. The epithelium (Ep) consists of 12-15 rows of cells, and basal and prickle cell layers can be recognized. The prickle cells are orientated parallel to the enamel surface (E). The junction between epithelium and connective tissue is flat. The connective tissue (CT) is relatively dense and compact. Between the fibre bundles there are fibroblasts and small blood vessels . Haematoxylin and eosin. ÷ 96.

towards the surface, the cells become more flattened and arranged with their long axes parallel to the surface of the tooth. Between the cells of both layers intercellular regions occur (Fig. 24). The cytoplasm of the cells is generally filled with cytoplasmic granules and mitochondria are usually numerous. No basic difference in cytoplasmic content of the basal and spinous cells seems to exist, except for the relatively higher number of tonofibrils in the spinous layer (Fig. 25). I n the deeper parts of the crevicular epithelium the tonofibrils and their finer elements, the tonofilaments, are clearly visible in electron micrographs (Figs. 25 and 26). Tonofilaments are found throughout the cytoplasm and do not seem to have any preferential orientation. In the peripheral region of the cells, however, thick bundles of these

LÔE

Fig. 24. Cells of the human epithelial cuff after staining with periodic acid-Schiff subsequen t to diastase digestion. Intercellular substanc e is PAS positive, ÷ 500.

filaments are orientated perpendicula r to the cell surface and appear to end in the attachmen t plaques of the desmosome s (Fig. 29) (Listgarten, 1964). A s yet, electron microscopic evidence of these fibrils in the superficial cells of the crevicular epithelium has not been published. However, polarization microscopy reveals that the cells are biréfringent towards the surface (Fig. 27A,B) and, if intracellular birefringence can be taken to indicate the presence of microscopic or submicroscopi c arrangemen t of filamentous structures, this would tend to confirm the phase contrast findings reported by Baume and Frandsen (1953) of tonofibrils within the superficial cells of crevicular epithelium. In the attached and free gingiva, as in epidermis generally, a change in the appearanc e of the tonofibrils takes place as the cells move from the prickle cell layer towards the surface. It is believed that the cytomorphologica l characteristic s

22. T H E

DENTO-GINGIVAL

J U N C T I ON

439

Fig. 25. Electron micrograph showing prickle cells of the marginal part of epithelial cuff in man. The cells are irregular in shape and numerous cytoplasmic processe s give the cell periphery an uneven appearance . The intercellular space is narrow. Numerous desmosome s are seen regularly distributed around the periphery of the cells. Osmiophilic fibril s (tonofibrils) in the cytoplasm, χ 3750. (From Thilander, 1963.)

of the cells of the granular layer is due to a specific alteration of the tonofibrils as a first step in the process of keratinization which is completed i n the superficial cornified layer. The mechanism s by which cells of the prickle cells are altered to form keratin are only partly understood. It is believed, however, that the chemical changes

connected with keratinization involve the oxidation of sulphydryl groups of cysteine to disulphide groups (Van Scott and Flesch, 1954). The crevicular epithelium does not contain a granular layer, nor a cornified layer, as can readily be demonstrate d by routine stains (Fig. 23). Staining for sulphydryls and disulphides also

HARAL D

440

Fig. 26.

LÔE

Higher magnification of the cell in centre of Fig. 25. χ 10,700. (From Thilander, 1963.)

shows that cells of the normal crevicular epithelium of primates, unlike the attached and free gingiva and the gingival margin, do not keratinize, nor show any tendency to do so (McHugh, 1964). When doubts have been expresse d concerning the nonkeratinized state of this epithelium (Dewar, 1955), they are probably explained by the fact that, unless great caution is exercised during the removal of the tissue at biopsy, the apical part of the thin crevicular epithelium breaks and is lost. Consequently , such a specimen wil l show only the marginal portion of the tissue, and most likely only that part which covers the orifice of crevice and which is keratinized. Corresponding to the changes in polarity and morphology of the cells in passing from the basal

layer into the prickle cell layer and further towards the surface, the areas between them become less dominant, and the intercellular bridges are less conspicuous . Electron micrographs (Thilander, 1963) of the spatial interrelationship of crevicular cells show that numerous cytoplasmic processe s from the entire cell periphery project into the intercellular space (Fig. 28), giving the cell surface an irregular or jagged pattern. A s already mentioned, no visible structure bridging the space between the cells is demonstrabl e by electron microscopy. The connection of one cell to another seems to depend on adhesivenes s and as yet not understood chemico-physica l forces mediated by the desmosomes . The desmosome s consist of two adjacent attachmen t plaques (one

22. T H E

DENTO-GINGIVAL

J U N C T I ON

Fig. 27. (A) Epithelial cuff of dog viewed in polarized light, ÷ 10. birefringence of the entire thickness of the cuff epithelium, x 60.

from each cell) separate d by an interval of approximately 200-300 Â. A t these sites the intercellular space contains a finely granular material (Fig. 29). Electron microscopic studies show that in the intercellular space between two attachment plaques there are multiple layers of different density arranged parallel to one another (Listgarten, 1964). Desmosome s in the deeper parts of the crevicular epithelium are plentiful (Thilander, 1963) and appear to be fairly evenly distributed at the cell periphery. A s yet, there is no evidence to show that desmosome s of the cells of the crevicular epithelium undergo changes similar to those taking place before desquamatio n of keratinized epithelial cells occurs (Listgarten, 1964).

441

(Â) Higher power view of the intracellular

Histochemical procedures have revealed (Thonard and Scherp, 1962) that the intercellular substance reacts positively to periodic acidSchiff reagents after treatment with amylase and bacterial hyaluronidase . The PAS-positive substance is only slightly affected by testicular hyaluronidase or bacterial chondroitinase . It stains also with Hale's iron absorption technique. After treatment with testicular and bacterial hyaluronidase this method fails to stain. Intense metachromasi a is apparent in the intercellular regions after staining with toluidine blue at low p H, and metachromatic reactivity persists after bacterial and testicular hyaluronidase digestion. This indicates that the intercellular substanc e

442

HARAL D

contains high molecular weight carbohydrates , possibly in the form of acid mucopolysaccharide s and heteropolysaccharides . These observations were made on keratinizing gingival epithelium, but it is reasonabl e to believe that the intercellular regions of the crevicular epithelium behave similarly. I n fact, recent investigations on the crevicular epithelium to some extent corroborate these findings (Toto and Sicher, 1964). I t is well known that epithelium disintegrates after exposure to proteolytic enzymes. The intercellular substanc e also reacts positively to brom-

LÔE

phenol blue (Thilander, 1963). In combination these observations indicate that the substanc e also contains proteins. Quite recently autoradiographi c studies using labelled proline (Stallard et al, 1965) have disclosed the presence of this amino-acid between the cells of the crevicular epithelium. F r om these and other observations on the histochemistry of pathological material (SchultzHaudt, 1953) it can be inferred that the intercellular substanc e of the crevicular epithelium contains carbohydrate s and proteins in a combination which may resemble the intercellular substance of the connective tissue.

22. T H E

DENTO-GINGIVAL

J U N C T I ON

443

Fig. 29. Above: Electron micrograph of region between two adjacent epithelial cells of the prickle cell layer of human crevicular epithelium (x 5600). The plasma membrane s (PM) form numerous projections into the intercellular region (IR). Some of these are cut transversel y and appear as islands. The cells are connected by attachmen t plaques (API) in which the plasma membrane is more dense and the intercellular substanc e appears granular. Two opposing attachmen t plaques and the interjacent substanc e constitute a desmosom e (Des). The tonofilaments (Tf) attach to the intracellular aspect of the desmosomes . In the cytoplasm the following organelles are seen: mitochondria (Mi), tonofilaments (Tf), ribosomes (small dark particles), and endoplasmic reticulum (ER). Below: Higher magnification of the desmosome s reveal multiple layers of varying electron density of the intercellular substanc e (arrows), ÷ 90,000. (From Theilade, 1966.)

444

HARAL D

Conflicting views have been expresse d concerning the glycogen content of the cells of the crevicular epithelium. Some authors report that small deposits of glycogen are regularly seen in spinous cells (Wislocki and Sognnaes , 1950; Engel, Ray and Orban, 1950; Schultz-Haudt and From, 1961). Others have found scattered granules only (Turesky, Glickman and Litvin , 1951) or state that glycogen is occasionally observed (Turesky, Glickman and Fischer, 1959), whereas others deny the presence of glycogen in any of the cell layers of normal gingiva (Dewar, 1955). A critical appraisal of the material examined reveals that all these studies deal with glycogen in keratinizing gingival epithelium. Only two studies (Dewar, 1955; Schultz-Haudt and From, 1961) are concerned wit h the distribution of PAS-reactive substance s in the crevicular epithelium and, as emphasize d in both papers, in a slightly inflamed state. The presence or absence of demonstrabl e amounts of glycogen in the cytoplasm of epithelial cells is a fundamenta l question as far as cell physiology is concerned . Normally, glycogen and glucose are not stored but are transformed into high energy compounds which can be used by the cells for their normal functions (Rothman, 1954). According to this view, storage of glycogen in epithelial cells may reflect an upset metabolic activity. The fact that glycogen accumulate s in epidermal cells during experimenta l inflammation (Lobitz and Holyoke, 1954) seems to be in keeping wit h this view. Comparisons between the localization of glycogen and the degree of keratinization of attached gingiva have shown that there seems to be an inverse relationship between the presence of surface keratinization and the occurrence of glycogen (Turesky et al., 1959; Weiss, Weinmann and Meyer, 1959). This relation has led to much speculation on the possible role of glycogen in the mechanism of keratinization (Schultz-Haudt and From, 1962). Since most of the studies on the occurrence of glycogen in the gingiva are based on examination of inflamed tissues or on keratinizing epithelium,

LÔE

it may be pertinent to summarize the results from our own investigations on biopsies from absolutely normal h u m an gingiva including an uninflamed crevicular epithelium. The specimens were immediately after removal immersed in liqui d nitrogen and subsequentl y cut in a cryostat at —20°C. The sections were then directly, or after treatment with diastase , subjected to the periodic acid-Schiff procedure according to Pearse (1960). The epithelial cells of normal gingiva contained no PAS-positive material. This pertains to the keratinized epithelium of the attached and free gingiva, as well as the nonkeratinized crevicular epithelium. It seems, therefore, that, under physiological conditions, gingival epithelial cells do not store glycogen. Phosphorylase , amylo-1.4 -> 1.6 transglucosidas e ("branching enzyme") and uridine-diphosphoglu cose (UDPG)-glycogen transglucosidas e are enzymes which are essentia l for the synthesis and breakdown of glycogen. The distribution in tissue sections of such activities may accordingly be considered as an indirect evidence of glycogen metabolism. High phosphorylas e (Quintarelli and Cheraskin, 1961) and "branching enzyme" activities (From and Schultz-Haudt, 1963) have been demonstrated in cells of the spinous layer and boundary layers of inflamed, keratinizing gingival epithelium. This distribution correspond s to that of glycogen, as revealed by the PAS method (see above). UDPG-glycogen transglycosidas e activity has been demonstrate d only in a few sections and seems to occur with some irregularity in spinous cells (From and Schultz-Haudt, 1963). Whether or not these enzymes can be demonstrate d in normal crevicular epithelium is not known. Phospho-esterase s are enzymes which hydrolyse monophosphori c esters with liberation of inorganic phosphate . Acid phosphatases , which are active in an acid environment, are found in an increased concentration in the granular layer of keratinizing epithelium (Cabrini and Carranza, 1958). Due to this distribution, the suggestion has been made that this enzyme plays a part in the process of keratinization (Carranza and Cabrini, 1962).

22. T H E

DENTO-GINGIVAL

Aci d phosphatas e activity has not been demonstrated in the crevicular epithelium. Numerous studies have been made on the occurrence of alkaline phosphatas e in gingival tissues (see Carranza and Cabrini, 1960). This enzyme acts in an alkaline medium. Alkaline phosphatas e activity is regularly found in relation to the connective tissue fibres (Danielli, 1946), during fibrogenesis (Follis, 1952) and the formation of the organic matrix of bone (Loe, 1959). New methods for the demonstration of alkaline phosphatase activity in gingival epithelium have failed to stain both keratinized gingival epithelium and crevicular epithelium, but reactivity is regularly found among collagen fibres, in blood cells and blood vessels in the adjacent connective tissue (Fig. 30). Intermediary metabolism or energy metabolism consists of a series of chemical reactions within the cell, the majority of which are governed by the activity of specific enzymes, for example, cytochrome oxidase and the dehydrogenase s which,

Fig. 30. Frozen section of human marginal gingiva stained for alkaline phosphatas e activity. Enzyme activity is chiefly confined to blood vessels and blood cells (BC) although at higher magnification some activity could be seen in fibroblasts. The epithelium (E) is devoid of alkaline phosphatas e activity. C r, connective tissue. As-MX salt and Fast Blue RR. ÷ 148. 30

J U N C T I ON

445

alone or in association with coenzymes , catalyze the anaerobic and aerobic oxidation of the breakdown products of carbohydrates , fats and proteins. I t follows that knowledge of the oxidative enzyme activities of the cells may form a basis for estimating the metabolic activity of the tissues. Until recently, littl e was known about oxidative enzymes of gingival tissues and most studies were made with biochemical techniques (Eichel, 1960). However, new histochemica l methods for the direct visualization of the products of the enzyme activity (Pearse , 1960), offer a precise localization of these activities within the cell cytoplasm. It should be stressed , however, that histochemica l demonstration s of enzyme activity are to be considered only as qualitative measure s and that such preparations only to some degree may permit relative quantitation. Cytochrome oxidase has been demonstrate d in the basal layer of attached and free gingiva, whereas spinous cells and the surface layers of keratinizing epithelium show littl e or no activity. The crevicular epithelium, on the other hand, according to Burstone (1960), seemed to display a high cytochrome oxidase activity. It is likely that the contrast in activity of oral and crevicular epithelium may have been due to inflammatory changes of the crevicular epithelium. Shahrik et al. (1963) found that succinic acid dehydrogenas e activity seems to be high in the basal and spinous layer of the attached gingival epithelium and decrease s towards the surface. The enzyme activity is mainly localized within the perinuclear cytoplasm. Lactic acid dehydrogenase has also been studied in keratinizing gingiva and shows a distribution essentially similar to that of succinic dehydrogenas e (Shahrik, Eichel and Lisanti, 1964). N o r m al crevicular epithelium has quite recently been tested for D P NH and T P NH diaphorases , succinic, lactic, glutamic, isocitric, malic and glucose-6-phosphat e dehydrogenase s using NitroBT as the hydrogen acceptor (Loe and Nuki, 1966). A s revealed by the deposition of formazan, all the oxidative enzymes studied, with the

HARAL D

446

exception of glueose-6-phosphat e dehydrogenase , show enzyme activity in the basal and spinous cells, comparable to the correspondin g layers of the keratinizing oral epithelium, whereas the superficial cells at the surface of the crevicular epithelium show low oxidative enzyme activity. Glucose-6-phosphat e dehydrogenas e activity, however, exhibits a reverse trend with increased deposition of formazan in the surface cells. The occurrence of formazan deposition in gradually decreasin g amounts towards the surface when incubated for aerobic enzyme activity may be an expression of decreasin g citric acid cycle activity. Conversely, the increase in the glucose-6phosphate dehydrogenas e activity as an expression of anaerobic enzyme activity towards the surface layers would indicate an increase in the glycolytic activity via the pentose shunt. This type of interpretation correlates well with the morphological appearanc e of the surface cells. It would seem reasonable to suppose that these cells, which are i n the process of desquamation , would show increased anaerobic glycolytic activity. I t should be realized that much still remains to be learned about the crevicular epithelium and its individual cells. This is particularly true as regards the ultrastructural changes of the cells and the intercellular regions as they relate to the continuous process of desquamatio n and to the dynamic relationship to the tooth surface.

V. THE PHYSIOLOGY OF THE GINGIVAL CREVICE The concept of the dento-gingival junction as a contact relationship raises the question of the gingival crevice and its relation to the bacterial flora of the oral cavity. Indeed, this is of importance, because the developmen t of periodontal disease is intimately connected with the bacterial activity in this area, and because the supporters of the epithelial attachmen t concept contend that the incompletenes s of such a " b r e a k" in the

LÔE

epithelial continuity implied by the contact relationship concept is not in accord with sound biological principle. A.

T H E BACTERIOLOGY OF THE GINGIVA L CREVICE

I n 1952 Waerhaug and Steen devised a method for the bacterial investigation of the gingival crevice. I n order to avoid contamination at the entrance of the crevice or from the neighbouring tissues, the gingival margin was painted with iodineglycerine. The area was dried with a blast of air and sampling was accomplishe d by inserting a thin steel blade down to the bottom of the gingival crevice. After removal, the steel blade was transferred to appropriate media for incubation. This method was tried on two dogs and the results show that, out of 46 "absolutely clinically s o u n d" gingival crevices, 43 yielded no growth. After this initial sampling, pure cultures of different pathogenic microorganisms were inserted into the crevices. The application of identical methods of sampling revealed that the microorganisms could invariably be recovered in pure cultures. F r om this pilot study it appeare d that although the iodine-glycerine did, as desired, kil l the microorganisms at the entrance of the crevice it spared those located subgingivally. It was therefore concluded that the method of sampling was satisfactory. Further investigations showed that out of 48 clinically healthy gingival crevices not one yielded growth, whereas all but one of the gingival pockets associate d with subgingival plaque or calculus formation gave positive cultures. The overwhelming majority of 240 pockets, from which calculus and bacterial plaque had been removed, yielded no growth. Finally, negative cultures were obtained from 61 out of 85 crevices related to teeth bearing artificial crowns. On the basis of these results, Waerhaug and Steen (1952) concluded that in clinically healthy gingiva the gingival crevices are sterile. This view is not universally held. Boyd and Rosenthal (1958) claimed, on the basis of a similar investigation, that painting the gingival margin

22. T H E

DENTO-GINGIVAL

J U N C T I ON

447

wit h iodine-glycerine prior to sampling killed the microorganisms within the crevice as well. So, instead of disinfecting the marginal areas before sampling, they tried to avoid contamination by pulling the gingival margin away from the tooth by means of a small hook while inserting the loop into the crevice. By this method 9 3% of the crevices of clinically healthy gingiva yielded growth. Accordingly, they concluded that the gingival crevice is not sterile, and other workers have supported this view. Schultz-Haudt, Bruce and Bibby (1954) found the same types of bacteria subgingivally as on the clinical crowns of the teeth, and Gavin and Collins (1961) also obtained positive cultures in the majority of the crevices investigated. N o ne of these studies involved attempts to sterilize the marginal areas before sampling. Gavin and Collins, however, used a sterile tube through which a sterile paper point could be inserted to the bottom of the crevice without touching the marginal tissues. In all cases where iodine-glycerine had been painted upon the marginal gingiva and adjacent tissues this sampling method gave negative cultures.

whereas the approximal and palatal regions regularly contained bacteria. Thus, conflicting evidence exists and, in any event, before forming an opinion, several points may be considered. Firstly, it should be noted that the gingival crevice does not lend itself to bacterial tests, and that several critical objections may be raised to the techniques hitherto used. The iodineglycerine method of Waerhaug and Steen has been shown to be imperfect. The results from studies which did not include a sterilization of the orifice of the crevices, on the other hand, cannot positively exclude contamination from adjacent infected areas. The positive cultures obtained by Bervel and by Egelberg and Cowley after the use of iodine varnish tend to show that under physiological conditions gingival crevices harbour microorganisms . It is conceivable, however, that the iodine varnish hardens and forms a bridge across the orifice of the crevice leaving some of the superficially situated microorganisms untouched. It is still an open question whether the positive growth which is obtained derives from this area or from the deeper parts of the crevice.

Bervel (1964) has re-examined the iodineglycerine method in experiments in vitro and in vivo and by the use of test bacteria. He concluded that iodine-glycerine, although it is an effective disinfectant, is not suitable for the sterilization of the marginal areas because it has a tendency to diffuse into the crevices. He accordingly introduced an iodine varnish which hardens and forms a membrane across the orifice of the crevice, leaving the presumptive micro-organisms within the crevice untouched. Wit h this method 59 out of 60 clinically healthy gingival crevices of 20 young individuals showed growth and, as a consequence , Bervel concluded that gingival crevices are always infected. Most studies have dealt with the bacteriology of the gingival crevice as a whole. Recently, Egelberg and Cowley (1963) found that the presence and absence of microorganisms varies in different areas of the crevice. A low incidence of positive cultures was obtained from the buccal region,

Another important point is the concept of the cervical extension of the gingival crevice. Some investigators mentioned above (Boyd and Rosenthal, 1958; Gavin and Collins, 1961) hold the opinion that the bottom of the gingival crevice is at "the top of the epithelial attachment, " whereas others (Waerhaug and Steen, 1952) believe that the bottom of the crevice coincides with "the base of the epithelial attachment" (see sections II , p. 417 and III , p. 432). It is thus only to be expected that some disagreemen t would exist regarding the bacterial state at the bottom of the crevice as long as complete agreemen t cannot be reached as to its location. Thirdly, it is obvious that, unless the gingiva is scrutinized very carefully indeed to establish the clinical criteria of a healthy state, the results obtained may be valueless. It may even be that, not only the actual area of gingiva from which samples are taken should be deemed healthy, but that the entire circumference of the gingiva of the tooth concerned

448

HARAL D

should be without signs of inflammation so as to avoid the risk of lateral contamination of the sampled area. Therefore, until more adequate methods, and especially methods of control, are devised, the bacterial status of the gingival sulcus is subject to conjecture. However, no one conceives of the healthy gingival pocket, as it appears clinically, being an open crevice between the gingiva and the tooth surface. I t may be opened by inserting an instrument, but it closes again as soon as the instrument is removed. The physiological gingival pocket is only a potential pocket (Waerhaug, 1960). Because of anatomical features of the dentogingival junction, some organisms of the oral flora (Lôe, Theilade and Jensen , 1965) are always located at the orifice or marginal part of the gingival pocket. It may also be that during chewing, toothbrushing etc. oral microorganisms are forced into the crevice. On the other hand, there seems to be no doubt that they are rapidly removed, provided that no special conditions leading to their retention are present. B. T H E CREVICULAR F L U I D

Flow of tissue flui d through the crevicular epithelium and the possible biological function of this flui d have been the object of considerable research since Waerhaug and Steen (1952) put forward the suggestion that there is a constant flow of tissue flui d within the gingival crevice. After the intravenous injection or oral administration of fluorescein, which binds with plasma proteins, Bril l and co-workers (Bril l and Krasse, 1958; Bril l and Bjorn, 1959) recovered the dye at the orifices of gingival crevices on filter paper strips which were inserted into the crevices. The results indicated that a flui d containing small molecules might pass from the subepithelial tissues into the gingival crevice and out into the oral cavity. Other epithelial surfaces of the mouth did not show such a penetration of tissue fluid. A series of similar experiments tended to show that the flow of flui d into the gingival crevices

LÔE

is intimately related to capillary permeability (Bril l and Krasse, 1958; Brill , 1959a,b). Passag e of flui d has been shown to occur in dogs (Bril l and Krasse, 1958), in m an (Bril l and Bjorn, 1959; Salkind, Oshrain and Mandel, 1963) and in rabbits (Browne, 1962, 1964). Immunoelectrophoreti c analyses have disclosed that at least seven different plasma proteins are present in this flui d (Bril l and Bronnestam , 1960; Mann, 1963a). Both a r and a2-globulins, as well as β- and y-globulins, were identified. Heavy precipitation lines of the y-globulin, transferrin and albumin fractions suggeste d a relatively high concentration of these components (Fig. 31). A s yet, no study has been made of the mechanisms involved in the passag e of the flui d through the crevicular epithelium, but, as pointed out by Weinstein and Mandel (1964) in a comprehensiv e appraisal of current research on this subject, there are several theoretical possibilities. There is reason to believe that the gingival flui d basically is of the same composition as blood plasma and that it originates from the blood and passes from the subepithelial connective tissues between or through the cells of the crevicular epithelium. The theory that the flui d may be a product of secretion does not seem tenable as there are no glands in this region. The most attractive explanation for the mechanism of gingival flui d production seems to be that it is a simple filtration of tissue fluid modified by the cells of the crevicular epithelium (Browne, 1962; Krasse and Egelberg, 1962; Weinstein and Mandel, 1964). A filtration of fluid through the crevicular epithelium could be explained (Loe, 1962) by the slightly higher hydrostatic pressure of tissue flui d (approximately 10 mm Hg: Evans, 1952) as compared to atmospheric pressure . The absence of keratinized cells in this epithelium facilitates such a process. The amount of flui d from healthy gingiva is scanty. It increases after mechanica l stimulation of the gum (Bril l and Krasse, 1958) or after intravenous injection of histamine (Brill , 1959b). If bacteria or other particulate matter are introduced into the healthy crevice they are expelled

22. T H E

DENTO-GINGIVAL

J U N C T I ON

449

Fig. 31. Precipitation lines of normal serum (above) and of fluid from human gingival pockets (below). The prominen lower curves occupy (from left to right) positions correspondin g to those of y-globulin, transferrin and albumin precipitated from human serum. (From Bril l and Bronnestam , 1960.)

wit h the fluid within minutes provided that they are not mechanically retained (Waerhaug and Steen, 1952; Brill , 1959a). Also, under these circumstance s the flow of fluid is increased , and the suggestion has been made that the scouring effect which is so produced may form an important part of the local defence mechanism . It has been held that the outward flow can normally prevent the penetration of foreign particulate matter into the gingival crevice (Waerhaug, 1955). When there is gingival inflammation the rate of outward flow is markedly increased (Bril l and Bjorn, 1959; M a n n, 1963b; Salkind et al, 1963). Obviously, this fluid must be considered not simply as a filtrate from tissues having a normal metabolism, but as an inflammatory exudate. I n view of the almost invariable presence of some degree of inflammatory reaction at the h u m an gingival margin it is of interest that neutrophil leucocytes are regularly found in the crevicular fluid (Loe, 1961; Egelberg, 1963b) and that the relative concentration of different inorganic ions seems to resemble that of an inflammatory exudate (Krasse and Egelberg, 1962). On this account it

has been difficul t to accept the crevicular fluid as part of the normal, non-inflamed gingiva (Mann, 1963b). A study was, therefore, undertaken, to investigate this problem (Loe and HolmPedersen , 1965). This investigation showed that strictly healthy h u m an gingiva did not exhibit a flow of fluid to the extent that it could be collected on paper strips placed over the orifices of gingival crevices or when strips were gently placed subgingivally. Furthermore, mechanica l stimulation of the periodontium did not produce flow of fluid from such healthy crevices. Inflamed gingiva, on the other hand, regularly showed the presence of fluid, the a m o u nt of which varied with the severity of the inflammation. These results tend to show that the fluid which oozes out between the gingiva and the tooth is closely related to tissue changes in the area. This relationship has been confirmed in a longitudinal study of the developmen t of h u m an gingivitis (Loe et al, 1965). During this experiment, gingiva that at the start did not exhibit a flow of fluid started to do so as soon as increased bacterial activity developed in the region. The amount of

450

HARAL D

fluid increased steadily throughout the experimental period and maximal flow occurred shortly before clinically observable gingivitis developed. A s soon as gingival inflammation became less as a result of local treatment a correspondin g decrease in flow of fluid occurred. Finally, a few days after gingival inflammation had resolved, the flow of fluid ceased . Together with the fact that inflammatory cells are regularly present in this fluid, that its chemical composition differs from tissue fluid proper and that the passag e of fluid is closely related to the area of inflammation (Cowley, 1964), this strongly suggests that the gingival fluid is an inflammatory exudate rather than a part of a physiological mechanism . The flow of fluid regularly starts before inflammatory changes can be detected clinically and persists some time after clinical inflammation has subsided. It is possible, therefore, that absence or presence of fluid may represen t the best available clinical means of establishing the distinction between healthy and inflamed gingiva. C . T HE DEFENCE MECHANISM OF THE G I N G I V A

Recent periodontal research has furnished substantial evidence to the effect that bacterial irritation is essentia l for the developmen t and maintenance of marginal periodontal inflammation. A s in any other infection the clinical manifestations of the disease are dependen t on the aggressive properties of the micro-organisms and the capabilit y of the host to withstand the aggression . I t has been emphasize d that an uninterrupted structural continuity or some kind of organic union (an epithelial attachment ) is a necessar y lin k in the preservation of a continuous and intact integument over the whole of the body surface, protecting the underlying tissues against external damage. Thus, the concept of the dento-gingival junction as a contact relationship between a nonkeratinized epithelium and the tooth surface may superficially seem to conflict with basic biological requirements . However, the fact that

LÔE

the crevicular epithelium is of the nonkeratinized type does not imply that the continuity of the surface cover is literally broken. There are many other parts of the surface of the body, within the oral cavity and elsewhere , that are covered wit h nonkeratinized squamous epithelium. Obviously, these epithelia cannot be characterize d as breaches of the continuity or be considered inferior just because they show no keratinization. The weakest aspect of the dento-gingival junction is its morphology. The niche which is created between the gingival margin and the tooth crown provides excellent opportunities for the retention of bacterial aggregation s and, when bacterial accumulations are well establishe d in these crevices, the crevicular epithelium, lik e any epithelium under similar circumstances , seems to offer littl e protection against the microorganisms or their products. Accordingly the initial lesion in periodontal inflammation is located at the marginal part of the crevicular epithelium rather than in the oral aspects of the gingiva which are keratinized. Besides structural protection the local defence against exogenous attack generally rests on other mechanical, chemical and cellular mechanisms . The efficacy of mechanica l factors is probably best illustrated by the lubricating and scouring action of saliva which tends to prevent the bacteria from settling on the nonkeratinized oral mucosa. However, recent experiments (Lôe et al., 1965) suggest that an effective self-cleansing of the marginal parts of h u m an teeth does not occur, not even during prolonged mastication of a diet composed of hard food (Arnim, 1963). Some cleansing effect has been ascribed to the crevicular fluid in as much as it has been shown that the fluid is able to expel bacteria and particles which have gained entrance into the crevice. F r om this it has been inferred that the fluid to some extent may also resist the introduction of foreign material into the gingival crevice. The presence of y-globulins in the crevicular fluid may indicate that it possesse s antibacterial properties, but how effective this immunological

22. T H E

DENTO-GINGIVAL

factor is needs to be elucidated. Moreover, it has been demonstrate d that polymorphonuclea r leucocytes are consistently found in this fluid. There can be littl e doubt about the phagocytic properties of these cells as long as they are lodged in the epithelium or the connective tissue. However, the microorganisms are usually located outside the gingival tissues, on the tooth surface or on the top of the gingiva, and whether leucocytes are capable of exercising phagocytic activity after having passed through the epithelium into the gingival pocket, and if it occurs how long it lasts, is not known. I t should be emphasized , however, that the production of a crevicular flui d and the presence of phagocytic cells are associate d with the tissue changes occurring in marginal inflammation, and that the flui d is most likely an inflammatory exudate. This means that the crevicular flui d cannot be considered as arising from the normal healthy gingiva, but that the gingival exudate comes into effect as part of the total tissue changes that occur subsequen t to irritation. Thus, it follows that the defence mechanism of the dento-gingival junction rests on the inflammatory process, the essentia l parts of which are represente d by the immunological properties of the crevicular exudate and the phagocytic activity. It is possible that variations in the efficacy of these mechanism s may explain why some subjects are more liable to suffer periodontal disease than others.

VI. CONCLUSION

I n the course of this survey an attempt has been made to form an estimate of the genesis, the morphology and the physiology of the dentogingival junction. Efforts have been made to show that our knowledge in many respects is still inadequate , and that there is a great deal of research to be done. A n explanation of the submicroscopi c relationship is necessary , the question of the

J U N C T I ON

451

bacteriology of the gingival crevice is unsettled, the significance of the crevicular flui d should be further elucidated, and there are many other similar problems which still await answer. The lack of information is particularly marked in the field of comparative anatomy as much of the recent research on gingival disease has been performed on laboratory animals. It is therefore highly important that as much as possible should be learned about the normal status of gingival tissues in these and as many other species as possible, and about such species differences as exist and the extent to which these tissues in all their details resemble or differ from the human state. I t should also be noticed that many of the available results may not refer to normal structure or physiology in the proper sense of the word, but are probably quite commonly based on investigations of pathologically changed tissues. The term "clinically healthy gingiva" appears to be a highly arbitrary concept, and what appears to be " n o r m al gingiva" to one investigator may not fulfi l the requirements of another. This is, of course, very unfortunate and means that complete confidence cannot be placed in all data presented . M a ny areas of marginal gingiva are probably always subject to some irritation from the environment and some response on the part of the tissues is inevitable. On the other hand, it should be demanded that, for the study of the normal structure and physiological process of the dento-gingival junction, areas should be selected which at least satisfy the clinical criteria of healthy gingiva. Problems concerned with the developmen t and nature of the dento-gingival junction are not merely academic ones. These problems have also great practical implications since lack of specific knowledge of the anatomy and physiology of this area necessaril y wil l influence the understandin g of the aetiology and pathogenesi s of h u m an gingival disease and consequentl y bring about an attitude of uncertainty in the practical aspects of its treatment.

452

HARAL D

References Ainamo, J. and Lôe, H. (1966). Anatomical characteristic s of gingiva. I. A clinical and microscopic study of the free and attached gingiva. J. Periodont. 37, 5-13. Aisenberg, M. S. and Aisenberg, A. D . (1948). New concept of pocket formation. Oral Surg. 1, 1047-1055. Arnim, S. S. (1963). The use of disclosing agents for • measuring tooth cleanliness . J. Periodont. 34, 227245. Arnim, S. S. and Hagerman, D. A. (1953). Connective tissue fibers of the marginal gingiva. / . Amer. dent. Ass. 47, 271-281. Awazawa, Y. (1959). Optic and electron microscope observation of the tissue composition of enamel lamella. /. Nihon Univ. Sch. Dent. 2, 24-32. Baer, P. N. and Bernick, S. (1957). Age changes in the periodontium of the mouse. Oral Surg. 10, 430-436. Baume, L. J. (1952). Observations concerning the histogenesis of the epithelial attachments . / . Periodont. 32, 71-84. Baume, L. J. (1953). Structure of the epithelial attachmen t revealed by phase contrast microscopy. / . Periodont. 24, 99-110. Baume, L. J. and Frandsen , A. M. (1953). Phase contrast microscope study of oral epithelium of normal and vitamin Á-deficient rats. Proc. Soc. exp. Biol., N.Y. 83, 356-360. Beagrie, G. S. and Skougaard , M. R. (1962). Observations on the lif e cycle of the gingival epithelial cells of mice as revealed by autoradiography . Acta odont. scand. 20, 15-31. Becks, H. (1929). Normal and pathologic pocket formation. /. Amer. dent. Ass. 16, 2167-2188. Belting, C. M., Schour, I., Weinmann, J. P. and Shepro, M . J. (1953). Age changes in the periodontal tissues of the rat molar. J. dent. Res. 32, 332-353. Bervel, S. F. A. (1964). The bacteriology of physiological gingival pockets. Acta odont. scand. 22, 167-183. Berwick, L. and Coman, D. R. (1962). Some chemical factors in cellular adhesion and stickiness. Cancer Res. 22, 982-986. Black, G. V. (1915). " A Work on Special Dental Pathology". Medico-Dental Publ. Co., Chicago, Illinois. Boyd, W. S. and Rosenthal, S. L. (1958). The presence of bacteria in the healthy gingival sulcus. J. dent. Res. 37, 288-291. Boyle, P. E., ed. (1955). The structure and function of the tooth-supporting tissues. In "Kronfeld's Histopathology of the Teeth and Their Surrounding Structures", 4th ed., pp. 297-319. H. Kimpton, London. Brill , N. (1959a). Removal of particles and bacteria from gingival pockets by tissue fluid. Acta odont. scand. 17, 432-440.

LÔE Brill , N. (1959b). Influence of capillary permeability on flow of tissue fluid into gingival pocket. Acta odont. scand. 17, 23-33. Brill , N. and Bjôrn, H. (1959). Passag e of tissue fluid into human gingival pockets. Acta odont. scand. 11, 11-21. Brill , N. and Bronnestam , R. (1960). Immuno-electrophoretic study of tissue fluid from gingival pockets. Acta odont. scand. 18, 95-100. Brill , N. and Krasse, B. (1958). The passag e of tissue fluid into the clinically healthy gingival pocket. Acta odont. scand. 16, 233-245. Browne, R. M. (1962). A preliminary study of the fluid flow from the gingival sulcus. Proc. R. Soc. Med. 55, 486-488. Browne, R. M. (1964). Some observations on the fluid from the gingival crevice. Dent. Practit. dent. Rec. 14, 470-474. Brudevold, F., Steadman , L. T. and Smith, F. A. (1960). Inorganic and organic components of tooth structure. Ann. N.Y. Acad. Sci. 85, 110-132. Burstone, M. S. (1960). Histochemical study of cytochrome oxidase in normal and inflamed gingiva. Oral Surg. 13, 1501-1505. Cabrini, R. L. and Carranza, F. Á., Jr. (1958). Histochemical distribution of acid phosphatas e in human gingiva. Periodont. 29, 34-37. Cape, A. T. and Kitchin, P. C. (1930). Histologic phenomen a of tooth tissues as observed under the polarized light: with a note on the roentgenra y spectra of enamel and dentin. J. Amer. dent. Ass. 15, 193-227. Carranza, F. Á., Jr. and Cabrini, R. L. (1960). Histochemical reactions of periodontal tissues: A review of the literature. /. Amer. dent. Ass. 60, 464-470. Carranza, F. Á., Jr. and Cabrini, R. L. (1962). Histochemical distribution of acid phosphatas e in healing wounds. Science 135, 672. Cohen, B. (1962). A study of the periodontal epithelium. Brit. dent. J. 112, 55-68. Cowley, G. C. (1964). Application of fluorescent protein tracing to the study of gingival inflammation. / . dent. Res. 43, 947 (Abstract). Danielli, J. F. (1946). A critical study of techniques for determining the cytological position of alkaline phosphatase. / . exp. Biol. 22, 110-117. Dewar, M. R. (1955). Observations on the composition and metabolism of normal and inflamed gingivae. / . Periodont. 26, 29-39. Egelberg, J. (1963a). Diffusion of histamine into the gingival crevice and through the crevicular epithelium. Acta odont. scand. 21, 271-282. Egelberg, J. (1963b). Cellular elements in gingival pocket fluid. Acta odont. scand. 21, 283-287. Egelberg, J. and Cowley, G. C. (1963). The bacterial state of different regions within the clinically healthy gingival crevice. Acta odont. scand. 21, 289-296.

22. T H E

DENTO-GINGIVAL

Eichel, B. (1960). Oxidative enzymes of gingiva. Ann. N.Y. Acad. Sci. 85, 479-489. Engel, M. B., Ray, H. G. and Orban, B. (1950). The pathogenesis of desquamativ e gingivitis: a disturbance of the connective tissue ground substance . / . dent. Res. 29, 410-478. Evans, C. L. (1952). "Principles of Human Physiology", 2nd ed. Lea & Febiger, Philadelphia, Pennsylvania . Fainstat, T. (1963a). Extracellular studies of uterus. I. Disappearanc e of the discrete collagen bundles in endometriae stroma during various reproductive states in the rat. Amer. J. Anat. 112, 337-369. Fainstat, T. (1963b). Extracellular studies of uterus. II . Regeneratio n of collagen bundles in uterine stroma after parturition. Amer. J. Anat. 112, 371-387. Follis, R. H., Jr. (1952). Cartilage and bone matrix. Chemical structure, formation and destruction. Trans. Macy Conf. metab. Interrelations 4, 11. From, S. H. and Schultz-Haudt, S. D. (1963). Histochemical studies of the metabolism of glykogen in human gingival epithelium. Periodontics 1, 63-69. Gavin, J. B. and Collins, A. A. (1961). Occurrence of bacteria within the clinically healthy gingival crevice. J. Periodont. 32, 198-202. Glickman, I. and Bibby, B. G. (1943). The existence of cuticular structures on human teeth. / . dent. Res. 22, 91-96. Glimcher, M. J., Bonar, L. C. and Daniel, E. J. (1961a). The molecular structure of the protein matrix of bovine dental enamel. / . mol. Biol. 3, 541-546. Glimcher, M. J., Mechanic, G. L., Bonar, L. C. and Daniel, E. J. (1961b). The amino acid composition of the organic matrix of decalcified foetal bovine dental enamel. / . Biol. Chem. 236, 3210-3213. Gottlieb, B. (1920). Zur Âtiologie und Thérapie der Al veolarpyorrhôe . Ost. Z. Stomat. 18, 59-82. Gottlieb, B. (1921a). Der Epithelansat z am Zahne. Dtsch. Mschr. Zahnheilk. 39, 142-127. Gottlieb, B. (1921b). thologi e und Prophylaxe der Zahnkaries. Z. Stomat. 19, 129-132. Gottlieb, B. (1927). Tissues changes in pyorrhea. / . Amer. dent. Ass. 14, 2178-2207. Greulich, R. C. (1961). Epithelial D NA and RNA synthetic activities of the gingival margin. Preprint. Abstr. Int. Ass. dent. Res., 39th gen Meet. Boston, No. 118. Hirth, C. M., Hartl, S. and Muhlemann, H. R. (1955). Distribution of mitosis in the epithelium of the interdental papillae of the rat molar. / . Periodont. 26, 229-232. Krasse, B. and Egelberg, J. (1962). The relative proportions of sodium, potassium and calcium in gingival pocket fluid. Acta odont. scand. 20, 143-152. Kronfeld, R. (1936). Increase in size of the clinical crown of human teeth with advancing age. / . Amer. dent. Ass. 18, 382-392.

JUNCTION

453

Kronfeld, R. and Ullik , R. (1928). Brechen auch bei wilden Tieren die Z hn e kontinuierlich durch? Z. Stomat. 26, 84-102. Listgarten, M. A. (1964). The ultrastructure of human gingival epithelium. Amer. J. Anat. 114, 49-69. Lobitz, W. C , Jr. and Holyoke, J. B. (1954). The histochemical respons of the human epidermis to controlled injury: Glykogen. / . invest. Derm. 22, 189-198. Lôe, H. (1959). Bone tissue formation. A morphological and histochemica l study. Acta odont. scand. 17, 311417. Loe, H. (1961). Physiological aspects of the gingival pocket. A n experimenta l study. Acta odont. scand. 19, 387-395. Lôe, H. (1962). Nyere undersôkelse r over emaljens organiske komponent [Recent investigations on the organic component of dental enamel]. Odont. Tidskr. 70, 483-502. Loe, H. (1963). Epidemiology of periodontal disease . An evaluation of the relative significance of the aetiological factors in the light of recent epidemiological research . Odont. Tidskr. 71, 479-503. Lôe, H. (1965). Periodontal changes in pregnancy. / . Periodont. 36, 209-217. Lôe, H. (1966). Passive eruption. An experimenta l study. (In preparation. ) Lôe, H. and Holm-Pedersen , P. (1965). Absence and presence of fluid from normal and inflamed gingiva. Periodontics 3, 171-177. Lôe, H. and Nuki, K. (1966). Oxidative enzyme activity in keratinizing and non-keratinizing epithelium of normal and inflamed gingiva. J. periodont. Res. 1, 43-50. Lôe, H. and Ravnik, C. (1961). A morphological study of the surface layer of dog tooth enamel. Acta odont. scand. 19, 483-493. Lôe, H. and Silness, J. (1961). Tissue reactions to a new gingivectomy pack. Oral Surg. 14, 1305-1314. Lôe, H. and Silness, J. (1963). Tissue reactions to string packs used in fixed restorations . / . prosth. Dent. 13, 318-323. Lôe, H., Theilade, Å. and Jensen , S. B. (1965). Experimental gingivitis in man J. Periodont. 36, 177-187. McHugh, W. D. (1961). The developmen t of the gingival epithelium in the monkey. Dent. Practit. dent. Rec. 11, 314-324. McHugh, W. D. (1963). Some aspects of the developmen t of gingival epithelium. Periodontics 1, 239-244. McHugh, W. D. (1964). The keratinization of gingival epithelium. / . Periodont. 35, 338-348. Mandel, I. D. and Levy, Â. M. (1957). Studies on salivary calculus. I. Histochemical and chemical investigations of supra- and subgingival calculus. Oral Surg. 10, 874884. Mann, W. V., Jr. (1963a). Electrophoresi s of tissue fluid from gingival pockets. Preprint. Abstr. Int. Ass. dent. Res, 41st gen. Meet., Pittsburgh No. 63.

454

HARAL D

Mann, W. V., Jr. (1963b). The correlation of gingivitis, pocket depth and exudate from the gingival crevice. /. Periodont. 34, 379-387. Miles, A. E. W. (1961). Aging in the teeth and oral tissues. In "Structural Aspects of Ageing" (G. H. Bourne, ed.), Chapter 21. Pitman, London. Orban, B. and Kôhler, J. (1924). Die physiologische Zahnfleischtasche . Epithelansat z und Epitheltiefenwucherung . Z. Stomat. 22, 353-425. Orban, B. J., Bathia, H., Kollar, J. A. and Wentz, F. M. (1956). Epithelial attachmen t (the attached epithelial cuff). J. Periodont. 27, 167-180. Pearse , A. G. E. (1960). "Histochemistry. Theoretical and Applied". Little, Brown, Boston, Massachusetts . Quintarelli, G. and Cheraskin, E. (1961). Histochemistry of gingiva. VI . Distribution and localization of phosphorylase. J. Periodont. 32, 338-342. Rothman, S. (1954). "Physiology and Biochemistry of Skin". Univ. of Chicago Press, Chicago, Illinois. Rushton, M. A. (1951). The epithelial downgrowth on the molar roots of golden hamsters . Brit. dent. J. 90, 87-93. Rushton, M. A. (1952). Epithelial downgrowth: Effect of methyl testosterone . Brit. dent. J. 93, 27-31. Rushton, M. A. (1954). Acquired enamel cuticle. Brit. dent. J. 97, 64-66. Rushton, M. A. (1955). Dental effects of dietary aureomycin. Brit. dent. J. 98, 313-317. Salkind, Á., Oshrain, Ç. I. and Mandel, I. D. (1963). Observations on gingival pocket fluid. Periodontics 1, 196-198. Schultz-Haudt, S. D. (1953). An exploration of the role of bacteria in chronic marginal gingivitis. Ph. D. Thesis, University of Rochester . Schultz-Haudt, S. D. and From, S. (1961). PerjodsyreSchiff metoden, anvendels e og fortolkning nâr det gjelder menneskelig gingiva. Odont. Tidskr. 69, 226-246. Schultz-Haudt, S. D. and From, S. (1962). Dynamics of periodontal tissues. I. The epithelium. Odont. Tidskr. 70, 430-460. Schultz-Haudt, S. D., Bruce, M. A. and Bibby, B. G. (1954). Bacterial factors in non-specific gingivitis. / . dent. Res. 33, 454-458. Schultz-Haudt, S. D., Waerhaug, J., From, S. H. and Attramadal, A. (1963). On the nature of contact between the gingival epithelium on the tooth enamel surface. Periodontics 1, 103-108. Shahrik, A. H., Eichel, B., Lisanti, V. F., Yacovone, J. A. and Klinkhamer, J. M. (1963). Histochemical study of succinic dehydrogenas e in human epithelium. Periodontics 1, 28-33. Shahrik, A. H., Eichel, B. and Lisanti, V. F. (1964). Cytochemical localization and distribution of a lactate utilizing enzyme system in human gingiva. Arch, oral Biol. 9, 1-15. Sicher, H. ed. (1962). In "Orban's Oral Histology and

LÔE Embryology," 5th ed., pp. 220-271. Mosby, St. Louis, Missouri. Skillen, W. G. (1931). A contribution to the anatomy and pathology of the human gingiva. / . dent. Res. 11, 727-744. Skougaard, M. R. and Beagrie, G. S. (1962). The renewal of gingival epithelium in marmosets (Callithrix-jacchus) as determined through autoradiograph y with thymidineH 3. Acta odont. scand. 20, 467-484. Sognnaes , R. F. (1950). The organic elements of the enamel. IV . The gross morphology and the histological relationship of the lamellae to the organic framework of the enamel. / . dent. Res. 29, 260-269. Stallard, R. E. (1963). The utilization of H 3-Proline by the connective tissue elements of the periodontium. Periodontics 1, 185-188. Stallard, R. E., Diab, M. A and Zander, H. A. (1965). The attaching substanc e between enamel and epithelium— a product of the epithelial cells. J. Periodont. 36, 130-132. Theilade, J. (1966). The ultrastructure of the gingival crevicular epithelium. / . Ultrastruct. Res. 14, 420-421. Thilander, H. (1963). The effect of leukocytic enzyme activity of the structure of the gingival pocket epithelium in man. Acta odont. scand. 21, 431-451. Thonard, J. C. and Scherp, H. W. (1962). Histochemical demonstration of acid mucopolysaccharide s in human gingival epithelial intercellular spaces . Arch, oral biol. 7, 125-136. Toller, J. R. (1939). The organic continuity of the dentine, the enamel and the epithelial attachmen t in dogs. Brit, dent. J. 67, 443-449. Toto, P. D. and Sicher, H. (1964). The epithelial attachment . Periodontics 2, 154-156. Trott, J. R. (1962). A desquamativ e cytological study of healthy oral mucosa. Acta anat. 49, 289-296. Trott, J. R. and Gorenstein, S. L. (1963). Mitoti c rates in the oral and gingival epithelium of the rat. Arch, oral Biol. 8, 425-434. Turesky, S., Glickman, I. and Litvin , T. (1951). Histochemical evaluation of normal and inflamed human gingiva. / . dent. Res. 30, 792-798. Turesky, S., Glickman, I. and Fischer, B. (1959). The effect of physiologic and pathologic processe s upon certain histochemically detectable substanc e in the gingiva. /. Periodont. 30, 116-123. Turner, E. P. (1954). The cuticles covering the enamel surface of erupted teeth. / . dent. Res. 33, 732-733 (Abstract). Ussing, M. J. (1955). Development of the epithelial attachment. Acta odont. scand. 13, 123-154. Ussing, M. J., Scott, D. B. and Kaplan, H. (1951). Microstructure of the enamel cuticles and epithelial attachment . /. dent. Res. 30, 478-479. Van Scott, E. J. and Flesch, P. (1954). Sulfhydryl groups and disulfide linkages in normal and pathological keratinization. Arch. Derm. Syph., Chic, 70, 141-154.

22. T H E

DENTO-GINGIVAL

Voreadis, E. G. and Zander, H. A. (1958). Cuticular calculus attachment . Oral Surg. 11, 1120-1125. Waerhaug, J. (1952). The gingival pocket. Odont. Tidsk. 60, Suppl. 1, 5-186. Waerhaug, J. (1953). Tissue reactions around artificial crowns. J. Periodont. 24, 172-185. Waerhaug, J. (1955). The source of mineral salts in subgingival calculus. / . dent. Res. 34, 563-568. Waerhaug, J. (1956a). Observations on replanted teeth plated with gold foil . Oral Surg. 9, 780-791. Waerhaug, J. (1956b). Enamel cuticle. / . dent. Res. 35, 313-322. Waerhaug, J. (1957a). Tissue reaction around acrylic root tips. / . dent. Res. 36, 27-38. Waerhaug, J. (1957b). Tissue reaction to metal wires in healthy gingival pockets. / . Periodont. 28, 239-248. Waerhaug, J. (1958). Effect of C-avitaminosis on the supporting structures of the teeth. J. Periodont. 29, 87-97. Waerhaug, J. (1960). Current concepts concerning gingival anatomy. The dynamic epithelial cuff. Dent. Clin. N.Amer. pp. 715-722. Waerhaug, J. and Lôe, H. (1957). Tissue reaction to gingivectomy pack. Oral Surg. 10, 923-937. Waerhaug, J. and Lôe, H. (1958a). Tissue reaction to selfcuring acrylic resin implants. Dent. Practit. dent. Rec. 8, 234-240. Waerhaug, J. and Lôe, H. (1958b). Effect of phenol camphor on gingival tissue. / . Periodont. 29, 59-66. Waerhaug, J. and Steen, E. (1952). The presence or absence

J U N C T I ON

455

of bacteria in gingival pockets and the reaction in healthy pockets pure cultures. Odont. Tidskr. 60, 1-24. Waerhaug, J. and Zander, H. A. (1957). Reaction of gingival tissues to self-curing acrylic restorations . / . Amer. dent. Ass. 54, 760-768. Waugh, L. M. (1937). Influence of diet on the jaws and face of the American eskimoes. J. Amer. dent. Ass. 24, 16401647. Weinreb, M. M. (1960). Epithelial attachment . J. Periodont. 31, 186-196. Weinstein, E. and Mandel, I. D. (1964). The fluid of the gingival sulcus. Periodontics 2, 147-153. Weiss, M. D., Weinmann, J. P. and Meyer, J. (1959). Degree of keratinization and glykogen content in the uninflamed and inflamed gingiva and alveolar mucosa. /. Periodont. 30, 208-218. Wertheimer, F. W. and Fullmer, H. M. (1962). Morphologic and histochemica l observations on the human dental cuticle. J. Periodont. 33, 29-39. Weski, O. (1922). Rôntgenologische-anatomisch e Studien aus dem Gebiete der Kieferpathologie. Vjschr. Zahnheilk. 38, 1-29. Wislocki, G. B. and Sognnaes , R. F. (1950). Histochemical reaction of normal teeth. Amer. J. Anat. 87, 239-285. Zander, H. A. (1955). Union of the enamel and gingival epithelium. J. Periodont. 26, 138-139. Zander, H. A. (1956). A method for studying "the epithelial attachment". / . dent. Res. 35, 308-312.

This page intentionally left blank

AUTHOR INDEX Numbers in italics refer to pages on which the complete reference s are listed. Arnim, S. S., 404, 423, 424, 450, 452 Arnold, F. Á., Jr., 267, 272 Arnold, J. S., 215, 237, 242, 245, 393, 404 Arnold, P. W., 251, 272 Arwill , T., 49, 72 Asboe-Hansen , G., 381, 404 Asgar, K., 210, 212, 238 Assev, S., 364, 412 Ast, D. B., 257, 272 Astbury, W. T., 288, 312 Aston, E. R., 257, 272 Astrôm, Á., 65, 73 Atherton, D, R., 237, 245 Atkinson, H. F., 27, 32, 11, 120, 129, 214, 238, 303, 314 Attramadal, Á., 424, 454 Aub, J. C., 258, 272 Avery, J. K., 57, 65, 72, 74, 76, 80, 81, 103, 123, 127, 129, 132, 134 Awazawa, Y., 53, 72, 108, 129, 138, 140, 162, 418, 452 Axelrod, D. J., 393, 406

A Abul-Haj, S. K., 367, 412 Adams, D., 355, 410 Addelston, H. K., 55, 57, 74, 180, 181, 190, 198, 199 Adloff , P., 9, 32 Ainamo, J., 424, 452 Aisenberg, A. D., 433, 452 Aisenberg, M. S., 433, 452 Akabori, S., 205, 238 Alba, Z. C , 286, 313 Albright, J. T., 67, 74 Alburn, H. E ., 364, 408 Alex, M., 374, 410 Alexander, L. E ., 170,199, 388, 409 Alfert, M., 376, 404 Alford, W. C , 364, 404 Alkalaev, Ê. K., 215, 238 Allan, J. H., 78, 79, 88, 98, 121, 123, 129 Alpher, N., 125, 130, 338, 343 Altshuller, L. F., 221, 230, 238, 257, 272 Ambady, J. K., 195, 197 Amdur, Â. H., 256, 263, 273, 220, 231, 239 Ames, L. L., Jr., 185, 198 Ames, W. H., 790, 312 Amprino, R., 103, 107, 129, 203, 213, 214, 231, 232, 238, 388, 404 Andersson, A. J., 365, 404 Anderson, D. J., 331, 334, 342 Anderson, R. W., 257, 272 Andersson, N., 264, 276 Angmar, B., 248 , 251, 252, 263 , 272, 273, 333 , 342 Anthony, M. R., 9, 32 Appelgren, L. E ., 764, 272, 215, 240 Araiche, M., 231, 238 Arase, M., 381, 404 Araya, S., 293, 312 Arey, L. B., 399, 404 Arlinghaus, R. B., 285, 313 Armstrong, W. D., 203, 208, 216, 219, 223, 230, 232, 234, 238, 239, 243, 244, 248, 250, 253, 254, 255, 263, 264, 265, 267, 272, 273, 276, 277, 298, 312 Armstrong, W. J., 286, 302, 303, 312

 Bachra, Â. N., 195, 196, 198, 199 Baden, H., 190, 198 Baer, M., 391, 407 Baer, P. N., 436, 452 Baer, R. S., 362, 364, 388, 404, 405 Bahr, G. F., 360, 374, 404 Bailey, A. J., 291,311,314 Baker, R. F., 45, 47, 49, 67, 72, 73, 87, 129 Baker, S. L., 387, 404 Bale, W. F., 55, 57, 72, 212, 232, 234, 238, 241, 393, 410 Balogh, K., 376, 385, 391, 396, 400, 404 Banez, L. Ï . N., 69, 71, 72 Bang, F., 218, 238 Bangle, R., 364, 404 Baratieri, Á., 377, 385, 404 Barber, T. K., 233, 243 Barnicot, Í , Á., 399, 401, 404 457

458

AUTHOR

Barrett, C. S., 194, 198 Bass, C. C, 384, 404 Bathia, H., 422, 454 Battista, A. F., 364, 404 Battistone, F. C, 282, 312, 336, 337, 342 Baud, C. Á., 80, 102, 111, 129, 190, 198 Bauer, F. C. H., 264, 272 Baumann, Á., 393, 404 Baume, L. J., 417, 421, 430, 438, 452 Beagrie, F. S., 427, 452, 454 Bear, R. S., 289, 291, 312 Beaulieu, M . M., 204, 238 Becks, H., 269, 275, 225, 238, 417, 421, 452 Beebe, R. Á., 333, 334, 344 Beevers, C. Á., 166, 167, 198 Behrens, B., 393, 404 Bélanger, L. F., 338, 342, 393, 396, 404, 410 Bell, J. H., 205, 238 Bell, M. C , 226, 243 Belting, C. M., 436, 452 Benditt, E. P., 368, 381, 404, 407, 410 Bengtsson , Á., 386, 387, 388, 389, 393, 413 Benoit, J., 396, 399, 404 Bensley, S. H., 351, 404 Bentley, K. D., 263, 277, 216, 224, 225, 241, 246 Berfenstam, R., 256, 272 Bergenholtz, Á., 261, 272 Berggren, H., 76, 81, 98, 103, 108, 129 Bergman, F., 80, 81, 87, 98, 110, 123, 129, 130, 232, 233, 234, 238, 248 , 251, 272, 273, 334, 342 Berke, J. D., 98, 102, 108, 129 Berlin, V., 139, 162 Berman, H., 169, 199, 263, 276 Bernick, S., 45, 47, 49, 67, 72, 73, 87, 129, 357, 404, 436, 452 Berry, D. C, 248, 276, 334, 345 Bertaud, W. S., 20, 34, 53, 74 Berthrong, M., 391, 407 Bervel, S. F. Á., 447, 452 Berwick, L., 424, 452 Besic, F. C , 341, 344 Bessey, Ï . Á., 227, 239 Bethke, R. M., 262, 263, 274 Bevelander, F., 112, 132, 336, 342, 350, 404 Bhussry, B. R., 29, 32, 129, 329, 331, 332, 341, 342 Bibby, B. G., 251, 257, 258, 261, 269, 273, 274, 275, 421, 447, 453, 454 Bignardi, C., 366, 404, 405 Bird, J. T., 261, 276 Bird, M. J., 204, 208, 212, 232, 238, 248, 273 Bisaz, S., 308, 309, 312 Bishop, F. W., 205, 232, 244, 246 Bjerrum, N., 191, 198 Bjôrn, H., 448, 449, 452

INDEX Black, G. V., 382, 405, 422, 452 Blackwell, R. Q., 328, 329, 330, 342 Blackwood, H. J. J., 24, 32 Blake, G. C , 27, 32, 214, 238 Blaxter, K. L., 226, 227, 238 Block, R. J., 337, 342 Bloom, F., 49, 72 Bloom, M. Á., 396, 399, 405 Bloom, W., 396, 399, 405 Boddie, J. F., 228, 238 Bodecker, C. F., 28, 29, 32, 109, 129 Bodingbauer, J., 128, 129 Boedeker, H., 361, 405 Bogoroch, R., 214, 245, 253, 276, 393, 408 Bohler, W., 191, 198 Boissevain, C. H., 215, 218, 238 Bolduan, E. A. O., 364, 388, 405 Boiling, D., 337, 342 Bolognani, L., 297, 312 Bonar, L. C , 319, 320, 323, 324, 328, 342, 343, 344, 420, 453 Bonhorst, C. W., 222, 241, 265, 274 Bonner, J. R., 251, 269, 273 Booth, F., 362, 412 Bornet, Á., 109, 129 Bornstein, P., 315, 360, 405 Bose, A. K., 328, 329, 330, 342 Bostrom, H., 382, 409 Boucek, R. J., 350, 409 Bourne, J. H., 385, 405 Bouyssou, H., 76, 99, 129 Bouyssou, M., 16, 99, 127, 129 Bowes, J. H., 207, 208, 212, 219, 222, 230, 233, 234, 239, 243, 262, 263, 273, 287, 292, 312, 360, 361, 405 Bowman, L., 256, 270, 273 Boyde, Á., 81, 89, 107, 129, 136, 140, 146, 148, 153, 157, 159, 162, 162, 203, 220, 239 Boyd, J., 255, 260, 274 Boyd, T. M., 261, 275 Boyd, W. S., 446, 447, 452 Boyle, P. E., 227, 239, 259, 277, 422, 452 Brabant, H., 100, 107, 109, 130, 132 Braden, A. W., 367, 405 Bradfield, J. R. F., 366, 405 Bradford, E. W., 4, 9, 14, 15, 17, 18, 19, 20, 26, 28, 30, 31, 32, 33,\\\, 129, 213, 231, 239, 297, 313 Brânnstrôm, M., 65, 73 Brain, Å. B., 76, 88, 102, 130,133 Brandi, J., 229, 239 Brasseur, H., 204, 238 Bredig, M. Á., 234, 239 Brekhus, P. J., 203, 208, 230, 238, 239, 248, 250, 253, 255, 263, 264, 272 Bretschneider , L. H., 388, 405

AUTHOR Brill , N., 448, 449, 452 Brônnestam , R., 448, 449, 452 Bronner, F., 308, 312 Brooks, A . W., 133, 150,163 Brooks, E. J. S., 231, 232, 243 Brown, J. L., 286, 312 Brown, W. E., 191, 198, 205, 239 Browne, R. M., 448, 457 Bruce, Ì . Á., 447, 454 Brudevold, F., 65, 73, 123, 130, 192, 198, 202, 218, 219, 220, 221, 225, 229, 231, 239, 242, 245, 246, 248, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 266, 267, 268, 269, 270, 271, 273, 274, 275, 276, 277, 329, 331, 332, 339, 341, 342, 344, 345, 420, 452 Brunauer, S., 185,198 Bruszt, P., 382, 405 Bryant, F. J., 260, 273 Buchanan, D. L., 226, 235, 239 Buergers, M . J., 169, 198 Biittner, W., 223, 230, 239 Bullock, F. Á., 207, 210, 212, 215, 216, 224, 236, 243, 249, 263, 264, 266, 275 Bunn, C. W., 169, 198, 388, 405 Buonocore, M. F., 258, 261, 270, 273, 276 Bunting, H., 351, 382, 405, 414 Burge, R. E., 362, 412 Burgess, R. C, 319, 320, 321, 322, 326, 338, 342, 344 Burnett, G. W., 204, 206, 210, 212, 213, 214, 215, 216, 219, 220, 221, 222, 239, 242, 248, 273, 282, 300, 301, 312, 330, 331, 333, 334, 336, 337, 342 Burns, J. J., 389, 405, 407 Burri, C, 388, 405 Burrows, L. R., 334, 336, 342 Burstone, M. S., 391, 399, 405, 445, 452 Bushel, Á., 257, 272 Butcher, E. O., 127,130, 355, 414 Butterworth, E. C., 387, 404

C Cabrini, R. L., 377, 391, 399, 400, 401, 405, 412, 444, 445, 452 Caldwell, R. C , 81, 130, 251, 273 Calvert, J., 396, 399, 404 Camanni, F., 103, 107, 129, 203, 213, 214, 231, 232, 238 Campbell, A. M. G., 221, 230, 239 Cape, A. T., 234, 239, 430, 452 Carlson, C. H., 255, 273 Carlsson, Á., 264, 272 Carlstrom, D., 55, 73, 80, 86, 123, 130, 153, 155, 159, 162, 193, 194, 197, 198, 248, 251, 252, 263, 272, 273, 333, 342, 387, 388, 405

INDEX Carmichael, A. F., 37, 39, 73 Carmichael, G. G., 373, 375, 403, 405 Carneiro, J., 321, 344, 378, 380, 393, 395, 405 Carranga, F. Á., Jr., 377, 405, 444, 445, 452 Carter, W. J., 298, 313 Cartier, P., 235, 239, 397, 405 Castellani, Á. Á., 297, 312 Catchpole, H. R., 358, 359, 366, 405, 406, 407 Chambers, J. W., 205, 238 Chambliss, J. F., 286, 313 Chang, Ç. Y., 401, 405 Chapman, J. Á., 350, 362, 406, 411 Chase, H. C , 257, 272 Chase, S., 108, 130 Chase, W. W., 267, 274 Cheraskin, E., 444, 454 Cholak, J., 221, 242, 259, 274 Chpikiter, V. O., 360, 411 Christensen , H., 260, 273 Churchill, H. L., 16, 17, 33 Clark, J. H., 234, 239 Clark, R. D., 339, 342 Cleland, K., 391, 406 Cohen, B., 421, 422, 430, 452 Cohen, J., 393, 406 Cohen, L. H., 382, 408 Cohen, R. B., 391, 396, 400, 404 Colby, R., 224, 245 Collin, R. L., 236, 239 Collins, Á. Á., 447, 453 Coman, D. R., 424, 452 Combee, B. 387, 406 Compton, A. S., 382, 406 Conlon, D., 76, 132 Constant, T. E., 354, 406 Cooiey, W. E., 259, 267, 273 Coolidge, T. B., 270, 273, 338, 341, 343, 344 Cooper, W. E. G., 206, 239 Copp, D. H., 392, 406 Corey, R. B., 360, 414 Corrigan, Ê. E., 229, 244 Costich, E. L., 81, 132 Courts, Á., 282, 301, 312, 365, 406 Cowley, G. C , 447, 450, 452 Cox, R. W., 310, 311, 312, 374, 406 Crabb, H. S. M., 76, 78, 79, 86, 100, 120, 123, 130 Craig, L. G., 107, 120, 130 Craig, J. M., 367, 406 Craven, D. L., 236, 242 Cremer, H. D., 223, 230, 239 Crick, F. H. C, 47, 73, 74, 789, 314, 361, 412 Croissant, O., 232, 246 Cronkite, E. P., 321, 344, 399, 414 Crowell, C. D., Jr., 197, 198, 204, 239

460

AUTHOR

Cruickshank, D. B., 220, 239, 257, 273 Crumley, P. J., 318, 406 Cueto, E. S., 248, 269, 275, 340, 341, 344 Cullity, B. D., 169, 194, 198 Cunningham, R. S., 291, 314 Curran, R. C, 382, 406 Czerkawski, J. W., 365, 406

D Dalgaard, E., 382, 406 Dalgaard, J. Á., 382, 406 Dallemagne, M. J., 204, 234, 235, 236, 238, 239, 240, 241, 244, 246 Dalton, A. J., 388, 406 Dam, H., 202, 216, 220, 221, 228, 240 Dana, E. S. 185, 190, 198 Dana, J. D., 185, 190,198 Daniel, E. J., 319, 320, 324, 343, 420, 453 Danielli, J. F., 445, 452 Danilezenko, Á., 297, 312, 366, 406 Darling, A. I., 76, 78, 79, 80, 86, 88, 95, 98, 107, 120, 130, 133, 150, 152, 162, 251, 269, 270, 273, 334, 339, 343 Davidson, Å. Á., 339, 342 Davies, D. H., 333, 334, 344 Davies, D. V., 367, 406 Davies, T. G. H., 77, 86, 88, 131, 153, 162 Dawes, C , 112, 130 Dawson, I. M., 205, 238 Dayton, P. G., 389, 405 Deakins, M. L., 230, 243, 248, 252, 273 Dean, T. H., 267, 272 de Boer, J. G., 104, 130 Deitz, V., 191, 198 de Jong, W. F., 55, 73, 166, 198 Dellovo, M. C , 367, 412 Deluzarche, Á., 76, 108, 130 de Moraes, F. F., 378, 380, 393, 405 Dempsey, E. W., 374, 382, 410, 414 DeRoche, F., 257, 273 De Stefano, F., 228, 242 Dettmer, N., 374, 412 de Vries, L. Á., 338, 346 Dewar, M. R., 440, 444, 452 Dhariwal, Á., 286, 313 Diat, M. Á., 424, 442, 454 Dickson, G., 107, 124, 130 DiFerrante, N., 308, 312 Diorio, A. F., 166, 199, 250, 276 Dirksen, T. R., 798, 312, 313 Dische, Z., 297, 312, 366, 406

INDEX Dittmann, G., 223, 230, 239 Dorfman, Á., 365, 406 Doty, P., 291, 312, 361, 405 Douglas, T. H. J., 219, 240 Dragiff, D. Á., 208, 240 Drea, W. F., 215, 218, 238, 240 Dreizen, S., 261, 274 Driak, F., 219, 240 Duckworth, J., 225, 226, 240 Dudley, H. R., 391, 396, 400, 404 Dundon, C. C , 259, 274 Dunphy, J. E., 351, 406 Duyckaerts, G., 234, 235, 244, 246 Dwyer, E. J., 214, 216, 240 Dyrbye, M. O., 381, 404 Dziewiatkowski, D. D., 308, 312, 396, 406

E Eanes, E. D., 183, 190, 199, 230, 244 Eastoe, B., 297, 312 Eastoe, J. E., 281, 282, 283, 285, 286, 287, 293, 297, 300, 301, 302, 312, 313, 315, 320, 321, 322, 323, 326, 334, 343, 361, 386, 406 Eccles, J. D., 355, 406 Eda, S. 233, 240 Edelman, L. S., 198 Edington, R. H., 262, 263, 274 Egelberg, J., 447, 448, 449, 452, 453 Egyedi, H., 338, 343, 346 Eichel, B., 445, 453, 454 Eidinger, D., 297, 313, 364, 366, 368, 386, 389, 406, 408, 410 Eigner, Å. Á., 285, 291, 314 Eder, J. J., 379, 413 Eisenberger , S., 235, 240 Elliott , C. G., 215, 218, 240 Elliott , J. C , 143, 162, 236, 240, 262, 274 Elliott , R. G., 287, 292, 312, 360, 361, 405 Ellis, L. N., 214, 216, 224, 240 Ellis, R. G., 267, 274 Ellis, S. E., 222, 244 Ellison, S. Á., 231, 240 Elsheimer, H. N., 268, 274 Emerson, W. B., 168, 199 Emerson, W. H., 234, 235, 240, 330, 333, 343 Emmet, P. H., 185, 198 Emslie, R. D., 29, 33 Engel, M. B., 350, 357, 358, 359, 405, 406, 444, 453 Engfeldt, B ., 80, 87, 98, 123, 130, 228, 232, 233, 234, 240, 251, 273, 388, 393, 406

299, 325,

407,

238,

AUTHOR Engstrom, Á., 55, 73, 197, 198, 387, 388, 393 , 404, 406, 407 Erausquin, J., 76, 102, 130 Erdheim, J., 223, 240 Ericsson, Y., 215, 240, 258, 261, 274 Erwig, R., 146, 157, 163 Ettleman, I., 197, 199, 203, 204, 210, 244 Evans, C. L., 448, 453 Ezmirlian, F., 258, 260, 275

405,

F Fabry, C, 205, 235, 240, 244 Fainstat, T., 433, 453 Fairhall, I. T., 258, 272 Fairhurst, C. F., 81, 133 Fankuchen , I., 180, 198, 214, 234, 245 Farr, L. E., 387, 413 Fearnhead , R. W., 86, 130, 143, 162, 203, 220, 239, 322, 324, 325, 343 Fell, H. B., 396, 399, 407 Ferguson, H. W., 206, 225, 240 Fernando, Í . V. P., 362, 407 Ferri, G., 297, 312 Fessenden , E., 55, 57, 74, 181, 199 Filson, Á., 205, 240 Fincham, A. G., 322, 323, 343 Finean, J. B., 388, 405, 407 Finn, S. B., 232, 241 Fischer, B., 444, 454 Fischer, C. J., 399, 408 Fischer, E., 271, 274 Fischer, Å. E., 234, 235, 240 Fischer, J., 382, 407 Fischer, H., 7, 33 Fischer, R. B., 268, 274 Fish, E. W., 17, 28, 29, 33, 227, 231, 240 Fishman, D. Á., 399, 407 Fitton-Jackson , S., 306, 312, 362, 412 Fleisch, H., 304, 305, 308, 309, 312 Fleischman, L., 15, 16, 33 Flesch, P., 439, 454 Forster, Á., 213, 240 Foley, D. E., 81, 133 Folk, J. E.,216, 243 Follis, R. H., Jr., 391, 407, 445, 453 Forbes, G. B., 207, 240 Forbes, R. M., 226, 227, 243 Fore, H., 221, 240 Forziati, A. F., 107, 108, 124, 130, 133 Fosdick, L. S., 80, 132, 157, 163, 328, 329, 330, 342 Francis, M. D., 270, 274 3i

461

INDEX

François, C., 235, 236, 240, 324, 343 Frandsen, A. M., 438, 452 Frank, R. M., 19, 20, 26, 33, 39, 41, 43 , 45, 51, 53 , 73, 76, 79, 86, 87, 108, 111, 121, 127, 129, 130, 133, 134, 145, 162, 214, 232, 234, 240, 246, 249, 274, 297, 313, 324, 338, 340, 343, 344 Franzier, A. W., 191, 198, 205, 239 Free, A. H., 298, 312 Freeman, S., 391, 407 Fremlin, J. H., 77, 78, 87, 130, 133, 153, 162, 203, 243 French, E. L., 208, 211, 212, 230, 238, 240, 245, 248, 264, 273, 277 French, J. E., 368, 407 Freudenberg , E., 305, 312 Frey,Wyssling, Á., 388, 407 Friberg, U. Á., 319, 320, 322, 325, 328, 334, 335, 336, 337, 339, 343 From, S. H., 424, 444, 453, 454 Frondel, C., 263, 276 Fujita, T., 86, 87, 130 Fullmer, H. M., 125, 126, 130, 134, 338, 343, 352, 355, 357, 358, 362, 365, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 381, 382, 385, 389, 391, 392, 396, 399, 400, 401, 402, 403, 405, 407, 430, 455 Furth, J., 391, 409 Furuta, W. J., 225, 238 Fusayama , T., 102, 130

G Gabriel, S., 197, 198, 204, 241 Gaines, M., 350, 414 Galagan, D. J., 257, 274 Gallop, P. M., 291, 314 Gallup, H., 204, 208, 212, 238 Gardell, S., 382, 409 Gardner, D. E., 202, 218, 229, 231, 246, 251, 253, 254, 256, 259, 266, 268, 269, 271, 273, 274, 276, 277 Gasparini, F., 334, 343 Gaudino, J., 204, 208, 212, 238 Gaunt, W. E., 223, 224, 241 Gavin, J. B., 447, 453 Gee, Á., 191, 198 Geiger, T., 263, 274 Geller, J. H., 336, 343 Gerdin, P.-O., 145, 163 Gerende, L. J., 223, 242 Gerould, C. H., 49, 73, 136, 162 Gersh, 1., 366, 407 Geschwind, I. I., 376, 404 Ghosh, Á., 368, 406 Gibson, W., 355, 365, 402, 407

462

AUTHOR

Gilda, J. E., 203, 241, 332, 343 Gilmore, R. W., 81, 130, 251, 273 Gilster, J. E., 261, 276 Glas, J. E., 155, 162, 168, 189, 193, 194, 198, 248, 249, 251, 252, 263, 272, 273, 274, 333, 342 Glegg, R. E., 297, 313, 364, 366, 386, 389, 407, 408, 410 Glenner, G. G., 382, 408, 409 Glickman, L, 379, 408, All, 444, 453, 454 Glimcher, M. J., 61, 73, 183, 196, 198, 293, 305, 306, 307, 308, 312, 318, 319, 320, 322, 324, 325, 328, 334, 335, 336, 337, 339, 343, 344, 346, 420, 453 Glock, G. E., 219, 220, 222, 234, 236, 243, 245 Godden, W., 225, 226, 240 Godt, H., 99, 131 Gôthe, G., 222, 238 Goggins, J. F., 358, 373, 403, 408 Goldhaber, P., 399, 400, 401, 408, 411 Gomori, G., 367, ?>9\,408 Gonzales, F., 55, 73, 213, 214, 232, 234, 243 Gorenstein, S. L., 424, 454 Gotte, P., 334, 343 Gottlieb, B., 107, 109, 129, 131, AM, 421, 428, 432, 433, 453 Gottschalk, Á., 303, 312 Gowgiel, J. M., 355, 408 Graham, G. N., 322, 323, 324, 343 Graig, F. Á., 257, 274 Grainger, R. M., 339, 344 Granados, H., 202, 216, 220, 221, 228, 240 Grant, Í . H., 364, 408 Grant, R. Á., 310, 311, 312 Grassmann , W., 47, 73, 289, 312 Graumann, W., 364, 408 Graziano, V., 297, 312 Green, F. C., 289, 314 Green, Í . M., 293, 313 Greep, R. O., 391, 399, 408, 411 Gresham, G. Á., 291, 311, 314 Greulich, R. C., 320, 321, 338, 343, 346, 396, 408, All, 453 Griebstein, W. J., 148, 152, 153, 154, 157, 159, 163 Griffiths, D., 264, 276 Grimbert, L., 81, 98, 131 Gron, P., 192, 198, 220, 231, 239, 256, 257, 262, 263, 271, 273, 274, 307, 315 Gross, J., 47, 74, 360, 362, 374, 393, 401, 402, 408, 409, 410, 411, 412 Gross, R., 55, 73, 166, 198 Gruner, J. W., 234, 236, 241 Gubisch, W., 391, 396, 408 Guidotti, G., 382, 408 Guilhem, Á., 127, 129 Guinier, Á., 186, 198, 388, 408

INDEX Gulberg, B., 258, 274 Gupta, O. P., 215, 218, 244 Gustafson, A. G., 76, 79, 80, 84, 85, 89, 90, 91, 92, 93, 94, 96, 98, 99, 110, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 131, 150, 152, 162 Gustafson, G., 28, 33, 43, 49, 51, 74, 16, 79, 80, 83, 85, 86, 92, 93, 101, 104, 105, 107, 109, 110, 111, 116, 120, 121, 129, 131, 134, 337, 345 Gustavson , Ê. H., 290, 313 Gyorgi, P., 305, 312

Ç Hadjimarkos, D. M., 222, 241, 265, 274 Hagerman, D. Á., 359, 404, All, A1A, 452 Halak, D. B., 221, 230, 238, 251, 272 Haldi, J., 216, 224, 225, 241, 246, 263, 277, 333, 346 Hall, C. E., 360, 388, 408, 412 Hall, D. Á., 362, 374, 408 Hall, D. M., 20, 34, 53, 74 Hallén, Á., 365, 408 Hallén, Ï. , 77, 131 Halpert, W., 231, 240 Hals, Å., 76, 80, 87, 98, 99, 120,131 Hamilton, J. G., 269, 275, 393, 406 Hamm, S. M., 334, 335, 346 Hammarlund-Essler , E., 76, 77, 80, 87, 98, 100, 123, 130, 131, 203, 228, 240, 244 Hanazawa, K., 15, 16, 17, 33, 88, 98, 107, 131 Hancox, N. M., 399, 400, 408 Handelman, C. S., 401, 409 Hannig, K., 289, 312 Hanok, Á., 214, 224, 228, 234, 245, 262, 263, 276 Happel, G., 213, 240 Harcourt, J. K., 27, 32, 214, 238 Hardwick, J. L., 77, 86, 88, 130, 131, 153, 162, 339, 343 Harkness, R. D., 283, 292, 313 Harndt, E., 99, 131 Harndt, R., 324, 344 Harness, S. R., 219, 241 Harper, R. Á., 190, 199, 230, 244 Harris, L. J., 227, 240 Harrison, G. E., 255, 260, 274 Harrison, H. E., 212, 241, 264, 274 Hartl, F., 387, 414, 424, 453 Hartles, R. L., 202, 203, 206, 216, 223, 225, 240 241, 244, 298, 299, 300, 313, 332, 339, 343, 344, 345 Hastings, A. B., 55, 74, 234, 244 Hatos, G., 332, 343 Hattyasy, D., 332, 343 Haumont, S., 257, 274

AUTHOR Hay, E. D., 399, 407 Hayden, H. S., 229, 244 Hayek, E., 191, 198 Healy, W. B., 264, 275 Heaton, M. W., 350, 414 Hedbom, Á., 381, 408 Hedegârd, Â., 261, 272 Hein, J. W., 251, 269, 273 Held, A. J., 80, 82, 87, 102, 111, 128,129, 131 Heller-Steinberg , M., 399, 408 Helmcke, J.-G., 49, 51, 73, 79, 82, 86, 87, 88, 111, 127, 129, 131,132,136, 138, 145, 148, 150, 151, 152, 155, 156, 157, 158, 159, 162, 232, 241 Helwig, G., 51, 73 Hendershot , L. C , 215, 220, 221, 222, 243, 257, 275 Henderson , E. H., 260, 273 Henderson , N., 223, 242 Hendricks, S. B., 235, 236, 241, 249, 274 Henry, K. M., 393, 414 Henry, N. F. M., 169, 198, 388, 408 Herdan, G., 221, 230, 239 Herman, H., 204, 215, 234, 235, 236, 241 Hermann-Erlee , M. P. M., 391, 396, 408 Herring, G. M., 282, 297, 313 Herskenov, B., 29, 33 Herting, A. C., 63, 73 Herzberg, F., 355, 409 Hess, W. C., 29, 32, 204, 206, 207, 215, 223, 242, 244, 286, 296, 297, 298, 313, 329, 331, 332, 336, 337, 338, 339, 341, 342, 344, 345 Heuser, H., 81, 85, 128, 132 Higaki, R., 382, 409 Higaki, T., 102, 130 Higashi, S., 114, 133 Highberger, J. H., 47, 74, 360, 361, 362, 408, 409 Hill , R., 379, 408 Hill , T. J., 262, 274 Hill , W. L., 235, 236, 241, 249, 274 Hirth, C. M., 424, 453 Hjertquist, S. O., 393, 406 Hodge, A. J., 311, 313, 362, 409 Hodge, H. C., 55, 57, 72, 197, 198, 202, 203, 204, 208, 211, 212, 230, 232, 238, 239, 240, 241, 243, 245, 246, 248, 251, 255, 260, 264, 267, 269, 273, 274, 276, 282, 313, 393, 410 Hodson, J. J., 79, 108, 111, 132, 339, 344 Hôhling, H. J., 63 , 73, 144, 145, 146, 152, 157, 163, 232, 241, 246, 324, 342, 344 Hoffman, M. M., 224, 241 Hoffman, P., 365, 386, 411 Hofmann, U., 47, 73 Hoh, F. C., 203, 244 Holgate, W., 221, 241, 260, 273 Holloway, P. J., 333, 346

463

INDEX Holmes, J. M., 333, 334, 344 Holmgren, H., 350, 381, 409 Holm-Pederse n P., 424, 449, 453 Holyoke, J. B., 444, 453 Honnen, L., 289, 314 Hope, J., 205, 240 Hopewell-Smith, Á., 24, 33 Hoppe, W. F., 234, 241 Hopsu, V. K., 382, 409 Hord, A. B., 267, 274 Horn, H. W., 202, 245 Home, R. W., 291, 310, 311, 312, 314 Horwitt, M. K., 337, 342 Howe, P. R., 227, 239 Howship, J., 396, 409 Huber, L., 45, 47, 49, 53, 74 Hughes, J. P., 259, 274 Hunscher, H. Á., 229, 244 Hunt, Á. M., 355, 409 Hurley, L. Á., 204, 205, 206, 242 Hurst, V., 76, 132 Hutchinson, A. C. W., 80, 132 Hutton, W. E., 109, 132, 203, 241, 335, 344 Hwang, W. S. S., 321, 344 Hyslop, D. B., 399, 409

I Ikels, K. G., 298, 313 Ingels, O., 221, 222, 233, 245, 261, 276 Irvine, J. W., Jr., 197,199, 204, 246 Irving, J. T., 202, 223, 224, 225, 227, 228, 229, 241, 298, 307, 308, 313, 314, 315, 401, 409 Isaac, S., 251, 254, 268, 269, 271, 274 Isler, H., 364, 412 Istock, J. F., 107, 132

J Jackson, D., 218, 219, 232, 233, 241 Jackson, D. S., 292, 313, 360, 365, 401, 409 Jackson, S. H., 218, 244 Jacobs, M . H., 270, 273, 341, 344 Jacobs, S., 360, 409 Jalm, B., 49, 51, 73 Jakus, Ì . Á., 360, 412 James, A. H., 190, 198 James, W. W., 21, 33 Jansen , M. T., 76, 77, 81, 86, 87, 98, 132

464

AUTHOR

Jee, W. S. S., 215, 242, 393, 404 Jenkins, G. N., 112, 130, 202, 218, 223, 228, 231, 242, 253, 270, 274, 341, 344 Jennings, W. H., 108, 124, 133 Jensen , A. T., 57, 73, 232, 236, 242 Jensen , S. B., 448, 449, 450, 453 Johansen , E., 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63 , 65, 67, 69, 71, 72, 73, 141, 144, 163, 203 , 204, 214, 230, 231, 232, 238, 242, 243, 270, 275 Johnson, P. L., 112, 132, 336, 342, 350, 404 Jones, S. J., 285, 313 Jordan, H. E., 396, 409 Jorpes, J. E., 381, 382, 409 Joseph, N. R., 358, 359, 405, 406 Julén, C, 381, 409

Ê Kabat, Å. Á., 391, 409 Kanamori, M., 296, 313 Kao, K. Y. T., 350, 409 Kaplan, H., 114, 133, 421, 454 Kapur, Ê. K., 271, 274 Karpishka, I., 321, 344 Karrer, H. H., 350, 409 Karshan, M., 208, 234, 240, 242, 263, 264, 270, 274 Kaushansky , L. I., 233, 242 Kawai, N., 128, 132 Kawanishi, Y., 293, 312 Kay, D., 138, 163 Kay, L. M., 289, 314 Kay, M. I., 184, 198 Keech, M. K., 362, 401, 409, 414 Kehoe, R. Á., 221, 230, 238, 242, 259, 274 Keil, Á., 4, 21, 22, 24, 25, 33, 76, 78, 79, 88, 98, 121, 122, 128, 133, 150, 163, 232, 242, 251, 274 Kelly, F. C, 286, 312, 362, 412 Kelman, E., 204, 208, 212, 232, 238 Kennedy, J. J., 157, 163 Kennedy, J. S., 382, 406 Kent, P. W., 287, 297, 313, 315 Kérébel, Â., 76, 86, 98, 107, 110, 132 Kern, R., 79, 121, 134, 145, 162, 249, 274, 338, 343 Kerr, T., 7, 33 Kerwin, J. G., 260, 274 Kety, S. S., 258, 274 Kick , C. H., 262, 263, 274 King, C. T. J., 224, 242, 245 Kirby-Smith, H. T., 399, 409 Kirk , D., 360, 408 Kirkman, H., 382, 409 Kirshner, H. Á., 214, 234, 245

INDEX Kitai , R., 280, 314 Kitchin, P. C, 234, 239, 430, 452 Klees, K., 111, 132 Klees, L., 100, 102, 107, 109, 111, 130, 132 Klein, A. L, 233, 242 Klein, E., 55, 57, 74, 180, 181, 189, 190, 197, 198, 199 Klein, H., 225, 226, 242 Klement, R., 387, 409 Kling , Ô., 80, 93, 104, 105, 107, 109, 120, 131 Klinkenberg, E., 258, 274 Klinkhamer, J. M., 445, 454 Klug, H. P., 169, 199, 388, 409 Knapp, D. E., 81, 132 Knappworst, Á., 266, 274 Knoop, F., 168, 199 Knutson, J. W., 257, 272, 274 Kodicek, E., 366, 405 Kôhler, J., 422, 454 Kôlliker, Á., 15, 33, 382, 396, 409 Kohlenberger, H., 7, 33 Kollar, J. Á., 422, 454 Kon, S. K., 393, 414 Kostlân, J., 122, 132, 355, 409 Kothe, J., 233, 242 Kounina, Ï . V., 360, 411 Kramer, B., 224, 245 Kramer, I. R. H., 18, 20, 33 Kramer, H., 366, 409 Krane, S. M., 324, 335, 343, 344 Krasse, B., 448, 449, 452, 453 Kreshover, S. J., 63, 73 Krikos, G. Á., 368, 411 Kroner, T. D., 289, 313 Kronfeld, R., 432, 434, 453 Kruger, V., 102, 121, 132, 231, 242 Kiihn , K., 47, 73 Kuether, C. Á., 227, 242 Kunde, M. L., 236, 242 Kurahashi, Y., 53, 67, 74 Kutnerian, K., 266, 270, 274 Kuyper, A. C., 266, 270, 274 Kvam, T., 4, 33

L Lacroix, M. P., 393, 409, 410 Lagunofî, D., 381, 410 Land, M., 124, 132 Landing, B. H., 221, 230, 238, 257, 272 Langley, F. Á., 387, 404 Lansing, A. L, 374, 410 Lantz, Å. M., 227, 244

AUTHOR Lapiere, C. M , 311, 313, 402, 408, 410 Larsen, Å. K., 169, 199 Laskin, D. M., 359, 406 Lavina, J. C , 109, 132 Law, M. L., 216, 224, 225, 241, 246, 263, 277, 333, 346 Lawson, M. E., 107, 108, 124, 130, 133 Leach, Á. Á., 285, 312, 360, 409 Leach, S. Á., 215, 236, 242, 266, 267, 275, 276 Leaver, A. G., 206, 241, 298, 299, 300, 313, 339, 344 Leblond, C. P., 297, 313, 321, 338, 343, 344, 364, 366, 378, 386, 389, 393, 395, 396, 405, 407, 408, 410 Lechtleiner, J., 191, 198 Lee, C., 296, 297, 313 Lee, C. Y., 298, 313, 336, 337, 338, 341, 344 Lee, W. Á., 190, 200, 262, 263, 277 Leek, K , 379, 413 Le Fevre, M. L., 206, 208, 211, 212, 238, 240, 242, 248, 275 Lefkowitz, W., 29, 33 L e Geros, J. P., 187, 190, 191, 193, 194, 196, 199, 200 Le Gette, J., 289, 314 Legros, C., 382, 410 Lehr, J. R., 191, 198, 205, 239 Lehrman, Á., 235, 240 Leicester, Ç. M., 29, 33, 16, 132, 189, 199, 202, 210, 214, 228, 242, 280, 313, 317, 344 Leimgruber, C., 89, 133 Leng, Á., 380, 410 Lenz, H., 69, 73, 107, 111, 133, 140, 144, 163, 232, 242 Leopold, R. S., 204, 206, 207, 215, 242, 244, 298, 313 Leppelman, H. J., 387, 414 Lerner, H., 204, 208, 212, 232, 238 Letonoff, T. V., 258, 274 Levene, C. I., 360, 408, 410 Levine, P. T., 319, 320, 322, 325, 328, 334, 335, 336, 337, 343, 344 Levy, Â. M., 430, 453 Lewis, M. S., 285, 291, 314, 360, 411, 412 Likins, R. C , 189, 199, 218, 228, 236, 242, 243, 251, 253, 255, 260, 275, 285, 286, 292, 314, 336, 345 Lillie , R. D., 352, 362, 364, 367, 368, 370, 376, 389, 407, 410 Lindemann, G., 229, 242 Lindstrôm, B., 203, 244 Line, W. R., 197, 198, 204, 239 Link, C. C, 376, 377, 378, 385, 391, 407 Linker, Á., 365, 386, 411 Lipp, W., 391, 410 Lipson, H., 169, 198, 388, 408 Lisanti, V. F., 377, 410, 445, 454 Lison, L., 4, 33 Listgarten, Ì . Á., 438, 441, 453 Little, K., 57, 73, 197, 199, 324, 340, 344, 345, 374, 406 Little, M. F., 225, 242, 248, 252, 258, 262, 263, 264, 269, 270, 273, 275, 340, 341, 344

465

INDEX

Litvin , T., 444, 454 Livrea, G., 228, 242 Lobene, R. R., 214, 215, 216, 219, 220, 221, 222, 239, 242 Lobitz, W. C, Jr., 444, 453 Lôe, H., 382, 410, 420, 421, 423, 424, 425, 426, 427, 428, 429, 430, 433, 434, 435, 445, 448, 449, 450, 452, 453, 455 Loewi, G., 365, 410 Lofthouse, R. W., 334, 336, 344, 346 Logan, Ì . Á., 207, 208 , 242, 264, 275, 285, 313 Loiseleur, J., 364, 410 Lomholt, S., 393, 410 Long, J. E., 286, 312 Looney, J. M., 189, 199 Lorber, M., 385, 410 Lorch, I. J., 391, 399, 410 Losee, F. L., 107, 108, 109, 124, 132, 133, 197, 199, 203, 204, 205, 206, 207, 210, 223, 242, 244, 264, 275, 329, 331, 337, 339, 344 Lowater, F., 202, 215, 219, 220, 222, 243, 258, 259, 275 Lowther, D. Á., 293, 313 Ludwig, F. G., 264, 275 Lumsden, E., 255, 260, 274 Lund, A. P., 216, 223, 243 Lundberg, M., 222, 243, 245, 250, 276 Lyon, D. G., 79, 80, 133

M McAleese, D. M., 226, 227, 243 McArthur, C., 258, 260, 275 McCance, R. Á., 226, 227, 246 McCann, H. G., 207, 210, 212, 215, 216, 224, 236, 249, 259, 260, 263, 264, 266, 275 McCauley, Ç. B., 214, 233, 243 McClure, F. J., 189, 190, 199, 200, 208, 210, 213, 216, 219, 224, 228, 230, 242, 243, 253, 255, 257, 259, 263, 275, 276 McCollum, Å. V., 225, 226, 242 McConnell, D., 185, 192, 199, 234, 235, 236, 241, 251, 263, 275 MacDonald, I., 387, 410 MacDonald, N. S., 258, 260, 275 McGann, T. C. Á., 298, 313 McGarr, J. J., 289, 313 Macgregor, Á., 17, 33 McHugh, W. D., 416, 417, 418, 419, 430, 440, 453 Macia, G. 110, 133 Maclntyre, D. B., 166, 167, 198 Maclaren, C, 319, 320, 321, 322, 326, 338, 342, 344 McLean, F. C, 391, 396, 399, 405, 407

243,

218, 267,

243,

466

AUTHOR

Macleod, M., 305, 314 McManus, J. F. Á., 366, 410 Macy, I. G., 229, 244 Mâjno, G., 391, 399, 410 Magid, Å. Á., 339, 345 Magitot, E., 382, 410 Mahler, D. B., 29, 33 Main, E. R., 266, 275 Main, J. H. P., 355, 410 Maj, G., 85, 133 Malassez, L., 382, 410 Maletskos, C. J., 393, 406 Maltesen, L., 202, 216, 220, 221, 228, 240 Mandel, I. D., 430, 448, 449, 453, 454, 455 Manley, Å. B., 76, 88, 102, 107, 133, 339, 343 Manly, M . L., 231, 232, 241, 393, 410 Manly, R. S., 203, 206, 211, 230, 232, 241, 242, 243, 248, 258, 271, 274, 275, 282, 313 Mann, W. V., Jr., 448, 449, 453, 454 Mannerberg, F., 81, 114, 133 Mansell, R. E., 215, 220, 221, 222, 243, 257, 275 Markest, Ê. H., 99, 133 Marko, A. M., 292, 313 Marshall, J. H., 393, 406 Marsland, Å. Á., 76, 88, 102, 133 Martens, P. J., 297, 313 Martin, C. J., 77, 86, 88, 131, 153, 162 Martin, G. R., 360, 362, 363, 389, 392, 401, 405, 407, 411, 412 Mason, H. L., 364, 414 Massler, M., 233, 243, 258, 275, 354, 411 Mathews, M. B., 365, 406 Mathieson, J., 77, 78, 87, 130, 133, 153, 162 Matsumiya, S., 138, 163 Maulbetsch, Á., 221, 243 Maurer, P. H., 364, 411 Mazourov, V. I., 360, 411 Meath, W. J., 333, 334, 344 Mecca, C. E., 401, 411 Mechanic, G. L., 319, 320, 339, 343, 420, 453 Meckel, A. H., 148, 152, 153, 154, 157, 159, 163, 270, 274 Mehmel, M., 55, 73, 166, 199 Melon, J., 204, 235, 238, 240 Menke, E., 51, 53, 73 Merea, C., 391, 401, 405 Merea, C. E., 401, 412 Mergenhagen , S. E., 362, 363, 411 Meyer, J., 444, 455 Meyer, K., 365, 386, 410, 411 Meyer, M. E., 215, 218, 244 Meyer, W., 107, 133, 157, 163, 382, 411 Miake, K., 114, 133 Migicovsky, Â. B., 396, 404

INDEX Miles, A. E. W., 21, 33, 202, 231, 243, 436, 454 Miller , C. W., 108, 132 Miller , J., 214, 243 Minami, N., 234, 245 Minck, R., 108, 130 Minot, A. S., 258, 272 Mitchell, R. L., 228, 243 Moeller, Á., 57, 73 Moghissibuchs , M., 190, 198 Mohanarakrishnan , V., 29, 33 Maltke, E., 381, 404 Montagna, W., 63, 73, 382, 411 Moore, F. D., 190, 198 Moore, S., 280, 285, 313, 314 Moore, T., 228, 243 Mori , M., 376, 411 Morris, A. L., 368, 411 Morris, E. R., 226, 244 Morrison, M . I., 212, 238, 248, 273 Morse, Á., 391, 399, 408, 411 Morse, J. K., 55, 74, 234, 244 Morse, W., 189, 199 Mortell, J. F., 104, 133 Mortimer, Ê. V., 152, 162, 270, 273, 334, 343 Morton, R. Á., 221, 240 Mosley, M. V., 196, 199 Moss, J. Á., 287, 292, 312, 360, 361, 405 Moss, M. L., 285, 313 Moura, C. S., 369, 411 Movat, Ç. Z., 362, 407 Mowry, R. W., 366, 367, 410, 411 Muhlemann, H. R., 270, 275, 424, 453 Millier , H., 138, 163 Muhler, J. C., 218, 246, 261, 264, 268, 269, 274, 275, 277 Muir , H. M., 292, 313 Mummery, J. H., 7, 23, 33, 382, 411 Muntz, M. L., 81, 130, 251, 273 Murray, M. M., 202, 207, 208, 212, 215, 219, 220, 222, 230, 233, 234, 236, 239, 243, 245, 258, 259, 262, 263, 273, 275 Myers, H. M., 78, 133, 269, 275

Í Nadel, Å. M., 391, 414 Nadler, N. J., 393, 408 Nageotte, J., 360, 411 Nakaniski, S., 293, 312 Nakao, Á., 226, 235, 239 Nalbandian, J., 19, 20, 26, 33, 55, 73, 79, 87, 133, 213, 214, 232, 234, 243 Namie, K., 377, 413 Nâray-Szabo , S., 55, 73, 166, 169

AUTHOR Narita, K., 205, 238 Neal, R. J., 148, 152, 153, 154, 157, 159, 163 Neidig, Â. Á., 336, 337, 344 Neinaber, M. W. P., 229, 241 Ness, A. R., 355, 411 Neuberger, Á., 292, 313 Neuman, M. W., 61, 65, 73, 195, 199, 235, 243, 250, 251, 252, 255, 262,264, 266, 275, 305, 306, 575,400, 411 Neuman, W. F., 61, 65, 75, 195, 199, 235, 243, 245, 250, 251, 252, 255, 260, 262, 264, 266, 274, 275, 304, 305, 306, 307, 308, 572, 575, 314, 400, 477 Neumann, E., 15, 16, 55 Nevin, R. B., 251, 269, 273 Newbrun, E., 168, 169, 199, 251, 255, 275 Newesely, H., 232, 241 Nezhivenko, L. N., 233, 243 Nikiforuk , G., 262, 275, 283 , 575, 338, 339, 342, 344 Nikolaeva, Í . V., 339, 345 Nishi, T., 227, 243 Nishihara, T., 291, 572 Nixon, G. S., 233, 243 Noback, C. R., 382, 411 Nobel, S., 263, 276 Noble, H. W., 37, 39, 75 Noda, H., 360, 362, 411 Nordback, L. G., 203, 204, 230, 231, 238, 242, 243, 270, 275 Noyés, F. B., 17, 55, 382, 411 Ntiforo, C. P., 203, 243 Nuckolls, J., 76, 109, 752, 335, 344 Nuki, K., 445, 453 Nusbaum, R. E., 260, 275 Nylen, M. U., 37, 63, 75, 89, 755, 142, 143, 155, 762, 765, 183, 196, 199, 214, 244

Ï Ockerse, T., 208, 230, 244, 263, 264, 276 Odeblad, E., 382, 409 O'Dell, B. L., 226, 244 Ogle, J. D., 285, 575 Ohazama, H., 128, 134 Okinaka, G., 308, 572 Ohmori, I., 210, 212, 244 Ohno, K., 205, 238 Okamoto, K., 376, 411 O'Leary, J., 266, 275 Ollis, W. D., 152, 762, 270, 273, 334, 343 Omnell, K.-Â., 142, 143, 765, 183, 189, 193, 198, 199, 203, 244, 249, 274 Orban, B. J., 382, 411, All, AAA, 453, 454

INDEX

467

Orekhovitch, V. N., 292, 314, 360, 411 Orent, E. R., 225, 226, 242 Orloff, S., 360, 408 0rvig, T., 4, 55 Oshrain, H. I., 448, 449, 454 Owen, R., 21, 55

Ñ Palache, C, 263, 276 Pantke, H., 114, 128, 752, 755 Pappas, G. D., 350, 412 Parkinson, Á., 302, 314 Parks, H. F., 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63 , 67, 69, 75, 203 , 204, 214, 242, 243 Parvis, P. V., 385, 411 Paus, S., 364, 412 Pautard, F. G. F., 57, 75, 322, 323, 324, 343, 344 Payen, J., 79, 121,757 Paynter, K. J., 385, 477 Peach, R., 362, 477 Pearse , R. G. E., 444, 445, 454 Pecker, C , 393, 477 Peckham, S. C., 197, 199, 203, 204, 210, 215, 223, 242, 244, 298, 575, 338, 339, 544 Pelton, W. F., 257, 276 Perdok, W. G., I l l , 729, 337, 345 Perkins, H. R., 308, 574 Perloff, Á., 166, 191, 799, 235, 244, 250, 251, 258, 276 Person, P., 380, 477 Perutz, M. F., 280, 574 Peterkofsky, B., 350, 477, 472 Peters, G. C., 168, 799 Peyton, F. Á., 29, 55, 104, 120, 130, 133 Pfrieme, F., 221, 230, 244 Phillips, M., 381, 410 Phillips, R. W., 120, 754 Picard, G., 266, 269, 276 Picciotto, Á., 228, 242 Piez, Ê. Á., 285, 286, 291, 292, 298, 575, 574, 575, 324, 336, 339, 545, 546, 360, 405, 411, 412 Pigeat, G., 81, 98, 757 Pigman, W., 81, 750, 168, 169, 799, 251, 273, 275 Pilz, W., 331, 332, 545 Pincus, P., 296, 297, 574, 328, 545 Pindborg, J. J., 220, 244, 259, 276 Plackovâ, Á., 51, 74, 122, 752 Plett, H., 221, 222, 233, 245, 261, 276 Plum, C. M., 220, 244 Poale, D. F. G., 55, 79, 755, 150, 152, 762, 765, 248, 270, 273, 276, 321, 334, 545, 545 Porter, Ê. E., 350, 472

AUTHOR

468

Posen, J. M., 264, 269, 270, 275, 276, 340, 344 Posner, A. S., 166, 167, 184, 190, 191, 198, 199, 230, 234, 235, 236, 242, 244, 250, 251, 258 , 260, 275, 276 Pritchard, J. J., 391, 412 Prophet, A. S., 303, 314 Puchtler, H., 364, 412 Pudy, G., 385, 411

Q Quigby, M. B., 296, 314 Quintarelli, G., 367, 412, 444, 454

R Radden, B. G., 382, 412 Rakuttis, G., 102, 121, 132, 231, 242 Ramachandran , G. N., 315 Ramathan, M., 29, 33 Randall, J. T., 362, 412 Rankine, D. M., 37, 39, 73 Rannie, I., 367, 370, 412 Rapp, R., 65, 72 Rasmussen , S., 263, 273 Ratner, S., 220, 244 Rau, R., 127, 131 Ravnik, C., 420, 453 Ray, H. G., 444, 453 Raymond, W. Ç. Á., 255, 260, 274 Record, P. R., 262, 274 Reed, R., 374, 408 Regan, W. O., 226, 244 Reimer, L., 138, 163 Reiss, L. F., 254, 276 Reitan, K., 382, 383, 384, 412 Reith, E. J., 324, 345 Resnick, J. B., 218, 244 Rexonikoff, P., 258, 272 Reynolds, L., 229, 244 Rich, Á., 47, 73, 74, 289, 314, 361, 412 Richards, L., 324, 343 Riley, J. F., 381, 382, 412 Rinehart, J. F., 367, 412 Robb-Smith, A. H. T., 365, 412 Robichon, J., 393, 410 Robin, C. R., 396, 412 Robinsohn, I., 382, 412 Robinson, H. B. G., 109, 129, 232, 241 Robinson, R. Á., 205, 244 Robison, R., 304, 305, 314

INDEX Rockenmacher , M., 224, 245 Rodriguez, M. S., 336, 345 Roe, J. H., 227, 242 Rôckert, H., 77, 131, 203, 213, 214, 232, 244 Romer, O., 33 Rônnholm, E., 249, 276, 318, 345 Roeper, E., 381, 404 Rose, C. R., 4, 33 Rogers, H. J., 296, 302, 314, 387, 414 Roholm, K., 228, 229, 244 Roncoroni, G., 385, 411 Roseberry, Ç. H., 55, 74, 234, 244 Roseburg, T., 258, 276 Roseman, S., 386, 412 Rosenfeld, D., 204, 208, 212, 232, 238 Rosenheim , A. H., 305, 314 Rosenthal, H. L., 261, 276 Rosenthal, J. B., 374, 410 Rosenthal, S. L., 446, 447, 452 Rothbard, S., 364, 414 Rothman, S., 430, 444, 454 Rouiller, C., 45, 47, 49, 53, 74, 391, 399, 410 Rowland, R. E., 80, 132 Rowles, S. L., 232, 236, 242, 340, 345 Rowley, J., 248, 269, 273, 275, 340, 341, 344 Rushton, Ì . Á., 21, 29, 33, 430, 433, 454 Rust, J. D., 216, 243 Rutherford, R. L., 45, 47, 49, 72, 81, 129 Rutishauser , E., 45, 47, 49, 53, 74, 221, 243 Ryge, G., 81, 133 Rygge, J., 261, 276 Ryle, A. P., 280, 314

S Saito, S., 293, 312 Sakurada, H., 234, 245 Salkind, Á., 448, 449, 454 Salo, T. P., 364, 405 Samsahl, K., 207, 210, 212, 215, 220, 221, 222, 233, 245, 249, 250, 261, 264, 265, 276 Sanger, F., 280, 314 Sauerwein, E., 109, 133 Saunsbury , P., 120, 129 Savory, Á., 248, 251, 256, 267, 268, 269, 270, 273, 276, 329, 331, 332, 345 Sawant, Á., 341, 345 Scapa, S., 380, 411 Schajowicz, F., 391, 399, 400, 401, 405, 412 Scherp, H. W., 441, 454 Schlager, F., 391, 396, 399, 408, 412 Schleyer, M., 289, 312

AUTHOR Schlueter, R. J., 291, 293, 295, 297, 307, 314 Schmidt, A. J., 399, 412 Schmidt, W. J., 4, 21, 24, 25, 33, 76, 78, 79, 88, 98, 111, 121, 122, 128, 133, 150, 163 Schmitt, F. O., 47, 74, 291, 293, 311, 313, 314, 360, 362, 408, 409, 412 Schoenheimer , R., 292, 314, 350, 413 Scholfield, H., 224, 245 Schoonover , I. C, 107, 130, 233, 244 Schour, I., 21, 34, 109, 129, 224, 241, 354, 409, 411, 436, 452 Schrader, H. K., 379, 412 Schrader, R., 379, 412 Schroeder , W. Á., 289, 314 Schiile, H., 76, 112, 133, 328, 345 Schultz-Handt, S. D., 364, 365, 412, 424, 442, 444, 447, 453, 454 Schulz, L., 82, 88, 132, 150, 151, 152, 156, 162 Schwarz, W., 374, 412 Scott, D. B., 37, 43, 45, 49, 51, 63, 67, 73, 74, 81, 82, 85, 87, 88, 89, 102, 111, 114, 132, 133, 143, 144, 150, 151, 152, 156, 157, 159, 162, 163, 196, 199, 214, 244, 259, 260, 266, 269, 275, 276, 362, 363 , 367, 411, 421, 454 Scott, J. E., 412 Scott, J. H., 87, 133, 355, 413 Seidell, Á., 266, 276 Seifter, S., 291, 314 Selvig, Ê. Á., 63, 74, 202, 213, 214, 233, 244, 352, 353, 354, 357, 413 Selvig, S. K., 202, 213, 214, 244 Senter, A. D., 379, 413 Sergei, E., 257, 274 Serres, Á., 382, 413 Shahrik, A. H., 445, 454 Shapiro, I. M., 206, 244, 332, 345 Shapiro, S. H., 367, 414 Sharaevskaya , Æ. N., 233, 244 Sharpenak , A. E., 339, 345 Shaw, J. H., 65, 74, 202, 214, 215, 218 , 244, 245, 253 , 263, 264, 276 Shepro, M. J., 436, 452 Shimizu, M., 377, 413 Shobusawa , M., 126, 133 Shorter, R. G., 401, 413 Shpikiter, V. O., 292, 314 Shroff, F. R., 20, 34, 53, 74 Shulman, L. E., 350, 414 Sicher, H., 76, 88, 97, 99, 102, 109, 111, 114, 133, 157, 163, 350, 354, 379, 413, 422, 424, 430, 432, 442, 454 Siffert, R. S., 391, 413 Siljestrand, B., 248, 273 Silness, J., 428, 429, 430, 453 Silverman, L., 401, 413

INDEX

469

Simaski, M., 399, 413 Simmons, E. J., 208, 240 Simon, S. L., 196, 198 Simon, W. J., 232, 244 Sinex, F. M., 350, 413 Singer, L., 219, 244, 254, 255, 265, 266, 269, 270, 272, 273, 275, 276, 298, 312, 340, 344 Singer, M., 350, 414 Singletar, D. W., Jr., 267, 272 Skach, M., 355, 409 Skillen, W. G., 232, 244, 417, 421, 454 Skougaard , M. R., 427, 452, 454 Slatkine, S. S., 190, 198 Slavkin, H. C., 321, 343 Smale, D. E., 355, 411 Smith, F. Á., 123, 130, 202, 218, 219, 221, 229, 231, 239, 245, 246, 251, 253, 254, 255, 259, 260, 265, 266, 269, 271, 273, 274, 275, 276, 277, 339, 342, 420, 452 Smith, G. H., 222, 244 Smith, J. D., Jr., 339, 342 Smith, J. P., 191,795, 205, 239 Smith, L. F., 280, 314 Smith, M. C., 227, 244 Smith, M. D., 215, 218, 219, 240, 241 Smith, R. H., 226, 245 Snellman, O., 381, 408, 409 Sobel, A. E., 214, 224, 228, 234, 235, 245, 262,2 63, 276, 308, 314 Soremark, R., 207, 210, 212, 215, 220, 221, 222, 233, 243, 245, 249, 250, 261, 264, 265, 272, 276 Sognnaes , R. F., 55, 65, 73, 74, 76, 79, 85, 86, 98, 103, 108, 109, 121, 124, 125, 126, 127, 729, 133, 134, 145, 162, 202, 213, 214, 232, 234, 243, 245, 249, 253, 274, 276, 333, 338, 339, 343, 345, 350, 414, 420, 444, 454, 455 Solomons, C. C., 289, 304, 306, 307, 308, 314 Sonden, W., 233, 244 Soni, N., 202, 229, 246 Soni, Í . N., 251, 270, 276 Sonden, J. R., 233, 245 Spackman, D. H., 280, 285, 314 Spain, P., 258, 260, 275 Speirs, R. L., 218, 223, 231, 242, 253, 256, 267, 268, 273, 274, 345 Sperber, G. H., 270, 276 Spicer, S. S., 367, 368, 413 Spies, Ç. Á., 261, 274 Spies, T. D., 261, 274 Spinelli, Ì . Á., 192, 198, 220, 231, 239, 256, 257, 262, 263, 267, 268, 270, 273, 274 Sreebny, L., 283, 313 Stack, M. V., 29, 33, 204, 206, 207, 212, 213, 245, 248, 277, 282, 283, 295, 296, 298, 300, 301, 302, 314, 320, 321, 325, 326, 327, 330, 331, 332, 333, 334, 335, 336, 337, 338, 340, 343, 344, 345, 346

AUTHOR

470

Stahl, S. S., 380, 411 Stallard, R. E., 378, 413, 424, 433, 442, 454 Stanmeyer , W. R. S., 224, 245 Staz, J., 102, 134 Steadman , L. T., 123, 130, 202, 219, 220, 221, 231, 239, 245, 251, 255, 256, 257, 258, 259, 260, 261, 265, 273, 277, 339, 342, 420, 452 Stearns, M. L., 351, 413 Stearns, R., 260, 275 Steedman , H. F., 367, 413 Steen, E., 446, 447, 448, 449, 455 Steere, A. C, 251, 275 Stein, G., 259, 277 Stein, W. H., 280, 285, 313, 314 Stëpânek , J., 51, 74 Stephan, P. E., 396, 413 Stetten, M . R., 292, 314, 350, 413 Stewart, A. D. G., 81, 107, 129, 140, 162 Stiebeling, G., 104, 130 Stofsky, N., 234, 242, 263, 264, 270, 274 Stone, M. J., 324, 344 Stookey, G. K., 264, 277 Storozheva , Í . N., 221, 245 Storvick, C. Á., 264, 265, 277 Story, R. V., 221, 242, 259, 274 Stover, B. J., 237, 245 Stuben, J., 234, 246 Strandh, J., 386, 387, 388, 389, 390, 393, 406, 413 Strobino, L. J., 387, 413 Suchow, L., 189, 199 Suga, S., 377, 413 Sullivan, H. R., 76,134, 336, 346 Sundstrôm, Â., 76, 78, 134 Sundvall-Hagland , L, 233, 238, 267, 277 Sutton, Á., 255, 260, 274 Swanson, Ç. E., 229, 245 Swartz, M. L., 120, 134 Sweeney, Å. Á., 218, 244 Swenson, Á., 258, 274 Switsur, V. R., 81, 129, 203, 220, 239 Sylvén, Â., 19, 34, 351, 381, 409, 413 Symons, Í . Â. Â., 19, 26, 34, 87, 133, 214, 245 Syrrist, Á., 43, 49, 51, 74, 85, 86, 134, 266, 267, 277

Ô Tabroff, W., 289, 313 Tailby, P. W., 248, 276, 334, 345 Takada, K., 376, 411 Takahashi, K., 110, 134 Takuma, S., 53, 55, 57, 67, 74, 138, 163, 205, 214, 234, 245

INDEX Tank, G., 264, 265, 277 Tappeiner, H., 229, 239 Tassin, M. T., 125, 126, 134 Tatge, E., 229, 245 Tatlow, W. F. T., 221, 230, 239 Taylor, A. C, 355, 414 Taylor, D. M., 256, 277 Taylor, T. K. F., 197, 199 Tefft, H., 211, 230, 245, 264, 277 Telford, I. R., 227, 242 Teller, E., 185, 198 Tempestini, O., 218, 245 Ten Cate, A. R., 383, 414 Teulie, S., 76, 99, 129 Teuscher, G. W., 157, 163 Theilade, J., 443, 448, 449, 450, 453, 454 Thewlis, J., 25, 34, 55, 74, 203, 213, 231, 234, 236, 245, 251, 277 Thilander, H., 222, 243, 439, 440, 441, 442, 454 Thomas, P. K., 373, 414 Thomasett, J. J., 4, 5, 34 Thonard, J. C , 441, 454 Thofova, J., 355, 409 Timberlake, P., 251, 275 Tokunaga, M., 234, 245 Toller, J. R., 431, 454 Tomes, C. S., 6, 8, 34, 781, 796, 300, 314 Tomlin, D. H., 393, 414 Tonge, C. H., 112, 130 Tonna, Å. Á., 321, 344, 393, 399, 414 Tonogai, K., 233, 245 Torell, P., 145, 163, 229, 232, 234, 245, 246, 258, 277 Toribara, T. Y., 235, 245 Totak, V., 204, 208, 212, 232, 238 Toto, P. D., 424, 442, 454 Toverud, G., 227, 245 Train, D., 218, 241 Trautz, O. R„ 55, 57, 74, 169, 180, 181, 185, 187, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 204, 232, 234, 235, 236, 245, 246, 262, 263 , 266, 271, 274, 277 Travis, D. F., 318, 320, 321, 339, 343, 346 Tretjakoff, D. K., 4, 34 Triberlake, P., 169, 199 Triffitt , J. T., 299, 313, 339, 344 Tristram, G. R., 414 Tromans, W. J., 291, 311, 314 Trott, J. R., 357, 414, 424, 454 Tunbridge, R. E., 374, 408 Turesky, S., 379, 408, 444, 454 Turner, E. P., 76, 111, 134, 430, 454 Turner, W. D., 235, 240 Tyldesley, W. R., 29, 30, 34

AUTHOR U Udenfriend, S., 351, 411, 412 Udupa, Ê. N., 350, 351, 406 Uehara, C, 102,130 Ullberg, S., 215, 240, 264, 272 Ullik , R., 434, 453 Underwood, A. L., 235, 245 Underwood, E. J., 277 Urbain, Á., 364, 410 Ussing, M. J., 85, 111, 133, 134, 421, 454

V Vahl, von J., 232, 246 Vallotton, C. F., I l l , 134 Vaname, P., 364, 414 Van Daatselaar , J. J., 338, 346 Van Huysen, G., 213, 232, 241, 246, 261, 275 Van Reen, R., 223, 242 Van Scott, E. J., 439, 454 Van Slyke, D. D., 350, 413 Van Voorhis, S. N., 393, 410 Vehoe, R. Á., 257, 272 Veis, Á., 291, 293, 295, 297, 307, 314 Verbraeck, L., 210, 213, 246 Verne, J., 126, 134 Vialle, P., 127, 129 Vikhm, Í . Á., 233, 246 Villa , V. G., 108, 124, 134 Visser, J. B., 76, 86, 87, 98, 132 Voelker, W., 230, 239 Vogel, A. L, 204, 246 Vogel, H., 100, 134 Volker, J. F., 202, 223, 245, 248, 273 von Beust, T. B., 28, 29, 34 von Ebner, V., 15, 34 von Hippel, P. H., 291, 314 von Korff, K., 17, 34 , T. S., 234, 246 von Kreudenstein von Saal, R., 15, 17, 34 Voreadis, E. G., 430, 455

W Wachtel, L. W., 329, 331, 346 Waerhaug, J., I l l , 134, 382, 410, 414, All, A13, A1A, All, 428, 430, 433, 446, 447, 448, 449, 454, 455 Wagner, Â. M., 367, 414 Wagner, M. J., 218, 246

INDEX

471

Wainwright, W. W., 208, 213, 246 Waksman, Â. H., 364, 414 Waldo, C. M., 350, 414 Walker, D. G., 391, 396, 400, 414 Walker, P. G., 308, 314 Walkhoff, O., 16, 34 Warnock, McG. M., 226, 240 Warren L., 368, 413 Warren, O., 45, 47, 49, 67, 72, 73, 87, 129 Warren, S. L., 55, 57, 72, 232, 246 Watchorn, E., 226, 227, 246 Watson, M. L., 57, 74, 76, 127, 134 Watson, R. F., 364, 414 Waugh, L. M., 436, 455 Weatherell, J. Á., 263, 277 Wegelius, O., 381, 404 Wegstedt, L., 387, 407 Weidenreich, F., 4, 5, 24, 34 Weidmann, S. M., 218, 219, 229, 241, 246, 263, 277, 302, 307, 308, 314, 332, 334, 335, 346, 387, 414 Weill, R., 124, 125, 126, 134 Weiner, R., 234, 242, 263, 264, 270, 274 Weinges, K. F., 387, 414 Weinmann, J. P., 436, 444, 452, 455 Weinret, M. M., 422, 431, 455 Weinstein, E., 448, 455 Weiss, M. D., 444, 455 Welander, E., 222, 238 Welch, Å. Á., 240 Wellock, W. D., 267, 277 Wentz, F. M., 422, 454 Wertheimer, F. W., 126, 134, 430, 455 Weski, O., 430, 455 Wesson, L. G., 227, 239 West, G. B., 381, 412 Westin, G., 102, 134 White, J. W., 21, 33 Whitehead, R. G., 263, 277, 307, 314 Whittle, E. G., 221, 230, 239 Wilander, O. T., 381, 409 Wildbalz, G., 108, 134 Wilder, Ï . H. M., 262, 274 Wilder, W., 262, 274 Wilk , Á., 224, 242 Wilkins, E., 257, 273 Wilkinson, G. W., 393, 410 Willan, A. L. D., 286, 312 Williams, G., 248, 277, 331, 332, 333, 334, 346, 362, 365, 401, 409, 411 Williams, J. B., 197, 199, 204, 246, 393, 406 Williamson, Ê. I., 20, 34, 53, 74 Winand, L., 191, 199, 235, 246 Windrum, G. M., 287, 315, 366, 409 Winkler, C. H., 367, 411

472

AUTHOR

Wislocki, G. B., 86, 103, 124, 125, 126, 134, 350, 420, 444, 455 Witte, W. E., 372, 407 Wolf, J., 136, 163 Wollman, S. H., 368, 413 Wong, R. L., 381, 404 Wood, Á., 148, 163 Wood, G. C, 362, 414 Wood, Í . V., Jr., 212, 246 Woodside, M. R., 212, 238, 248, 273 Wooster, W. Á., 169, 198, 388, 408 Wustrow, F., 82, 88, 134, 150, 152, 163 Wuthier, R. E., 307, 315 Wyckoff, R. W. G., 43, 45, 74, 81, 85, 87, 102, 133, 232, 246, 266, 269, 276, 360, 362, 388, Wynn, W., 216, 224, 225, 241, 246, 263, 277, 333, Wyss, V., 221, 246

Y Yacovone, J. Á., 445, 454 Yaeger, J. Á., 53, 74 Yagi, T., 399, 413 Yamada, M., 128, 134 Yamaguchi, N., 53, 74 Yamakawa, K., 128, 134 Yanamoto, N., 296, 313

382, 414,

INDEX Yardley, J. H., 350, 414 Yoon, S. H., 202, 218, 229, 231, 246 Yoshiki, S., 400, 414 Yoshioka, N., 53, 74 Young, R. Á., 184, 198 Young, R. W., 321, 346, 393, 414

Ζ

111, 114, 411, 414 346

Zacharides, P. Á., 360, 414 Zander, Ç. Á., 233, 244, 422, 423, 424, 430, 442, 455 Zapanta, R. R., 185, 192, 193, 196, 197, 199, 204, 246 Zapanta-Le Geros, R., 185, 187, 189, 190, 191, 192, 194, 196, 197, 199, 200 Zelander, T., 145, 163, 229, 246 Zelickson, Á., 351 Zelmenis, G., 297, 312, 366, 406 Zenewitz, J., 204, 206, 210, 212, 213, 214, 216, 239, 273, 300, 301, 312, 330, 331, 333, 334, 342 Zetterman, R., 406 Zipkin, I., 190, 199, 200, 230, 244, 262, 263, 277, 298, 339, 346 Zorzoli, Á., 391, 414 Zugibe, F. T., 367, 414 Zwarych, P. D., 296, 314

454, 205, 193,

248,

315,

SUBJECT INDEX Numbers in boldtype face refer to pages where the subject is more fully treated. Numbers in italic type refer to pages where the subject is illustrated. A

periodic acid-Schiff, 338 sodium in, 264 sudanophilia, 126 traditional concept, 157 mineralization commencing in prism centres, 86 electron microscopy, 143-144, 146, 318-319, 325 mucopolysaccharides , changes in, 338 physico-chemica l aspects , 325 pyrrolidine rings in, 325 by "secondary" crystal deposition, 146 Amino acids, see under Dentine matrix and Enamel matrix Amphibia, 11, 72 Ankylosis, 11, 32 Apatites in bone, 166, 169, 187, 190, 388 of fishes, 193 orientation, 194-195 size, 388 carbonate apatite, 197, 263 carbonates in relation to, 57, 144-145, 169, 180, 185, 187, 190, 191, 192-193, 235-236, 262, 271 effect on birefringence, 263 solubility increasing effect, 271 in cementum, 166 chemical formulae, 166, 168, 184, 185, 234, 250, 266 chemico-physica l forces in formation of, 196, 197, 304-305 chlorapatite, 168, 184, 189 citrate, 250 comparison of bone, dentine and enamel, 169, 187, 189 crystal lattice, 35, 55, 61, 72, 166, 167, 172, 235, 236, 250, 252, 269 same in bone, dentine, and enamel, 249 crystallinity values, 187, 190, 193, 194 for amorphous calcium carbonate , 187, 194 for bone, 187, 194 for dentine, 194 for enamel, 187, 194 crystallographic methods, 166-184, see also Electron diffraction, Neutron diffraction and X-ray diffraction

Aardvark, dentine, 9, 10, 31, 32 Acid mucopolysaccharides , see Mucopolysaccharides , acid and neutral Acid phosphatase , see also under various tissues in crevicular epithelium, 445 in gingival epithelium, 444 mast cells, 382 in periodontal membrane , 376, 377-378, 385 Age changes in dentine chemistry, 202, 213, 237 in dentine structure, 28, 30, 237 in dento-gingival junction, 432, 433, 436, 437 in enamel chemical composition, 252-253, 331, 332 specific gravity, 252 of enamel surface, 114 Age determination, 28 Agnatha, dentine in skeleton, 3 Alkaline phosphatase , see also under various tissues in crevicular epithelium, 445 in gingiva, 445 in osteoblasts , 391 in periodontal membrane , 376, 377, 385 Alveolar bone, 354, 3S5-386, 392, 393, 399, 400, see also Bone Ameloblasts after maturation, see Reduced enamel epithelium during matrix formation, 415, 416 during maturation, 415-416 electron microscopy of withdrawal of organic matter, 325 Amelogenesis , see also Ameloblasts carbon dioxide: phosphorus ratio in enamel organ fluid, 262 crystal dimensions, 318 matrix formation, 76, 415 electron microscopy, 143, 318-319 histochemistry, 125-126 473

474

S U B J E CT

Bragg's law, 172, 173 crystal morphology, 166, 185-186 i n dentine, 57, 58, 59 in enamel, 141, 143, 144, 146 anatomical regional differences, 141 species differences, 141 crystal size methodology, 185-186 electron microscopy, 185, 249 nitrogen adsorption, 185, 249 X-ray diffraction, 185-186, 249 crystal structure, 167 in dentine, 55, 57, 58, 59, 72, 169, 234, 237 crystal size, 25, 57, 59, 207 in enamel, 78-79, 141-142, 143, 169, 181, 187, 189, 190, 192, 193, 194, 236, 249 crystal size, 141, 193, 249 orientation, 194, 195, 249, 250 water-binding, 250 fluorapatite, 168, 169, 179-180, 184, 185, 187, 189, 236, 256, 266 general properties, 166-168, 184 hydroxyapatite, 166, 169, 180, 184 of dentine, 25, 55, 57, 234, 235, 236 of enamel, 236, 247, 250 water-binding, 250 infrared analysis, 191, 234, 235-236 ionic exchange , 250 lattice distortion, 186-187, 235 magnesium , influence on dentine crystal size, 212 non-biological, 55, 166, 169, 180, 184-185, 187, 189, 236 octocalcium phosphate and, 191 physical properties, 168-169, 193, 250 cleavage, 169 hardness , 168 optical, 169 pyrophosphat e and, 191 formation in dentine on ashing, 204, 234, 235 reactivity dependen t on crystal size, 185, 193, 249 secondar y phosphate , in relation to, 186, 190-191, 234 solubility, 251, 263, 271, 304-305 strontium apatite, 189, 236 substitutions, 184-185, 186, 250, 251, 264 calcium:phosphoru s ratio, effect of, 266 carbonate, 185, 186, 187, 192, 193, 235, 262, 266 chlorine, 184, 189, 193 fluorine, 184, 187, 189-190, 192, 236, 250, 266 hydroxyl, 184, 189, 236, 250 lead, 184, 190, 258 magnesium , 187, 190, 191, 212, 236, 263 radium, 184, 190, 236, 250 sodium, 190, 251, 264 strontium, 184, 190, 236, 250, 260 Vegard's law, 184

INDEX surface absorption, 235, 250, 252, 260 of carbonate , 236, 262 of lead, 258 of magnesium , 190, 263 hydration shell, 248, 250 synthetic, 55, 180, 185, 189, 192, 193, 235, 236, 263 unit cell, 145, 168, 172 definition, 55, 167, 250 dimensions, 172, 184, 189, 190, 192, 193 whitlockite (/3-tricalcium phosphate ) and, in dentine, 204, 212, 232 in arrested caries, 232 Autoradiography, see Isotope labelling

 Bear, enamel, 128 Bone, see also Osteoblast s and Osteocytes alveolar, see Alveolar bone calcium: phosphorus ratio, 251 chemical composition, 386-387 age changes , 387 inorganic constituents , 189, 387 organic constituents , 386 species differences, 302 by volume, 188 by weight, 188 chondroitin sulphate, 298 citrate, 299 electron diffraction, 388 fluorine content, 190 histochemistry, 389, 391 periodic acid-Schiff, 298 isotope labelling, 393-394-395-396 amino acids, 393, 394, 395 14 bicarbonate-C , 396 sulphate-S3 5, 3 96 lead mobilization, 258 matrix, definition, 281 membrane (dermal), relation to dentine, 3-4, 10 microchemistry, 388-389 age differences, 388-559 calcium: phosphorus ratios, 388-559 microradiography , 390 quantitative, 387 mucoproteins, 297, 389, 396 polarization microscopy, 388 pore system, 333 resorption, 396-401, see also Osteoclast s parathormone , 400-401 pressure , 400 in scurvy, 389, 393 sialoproteins, 297-298

S U B J E CT X-ray diffraction, 194-195, 387-388 collagen, 388 orientation of mineral crystals, 194-195, 388 Bony fishes, 4, 5, 6, 7, 8, 10, 11, 12 bone apatite, 193 osteoclasts , 396 Bone of attachment , 4, 32

C Calcification, see Mineralization Cartilaginous fishes, 3, 4, 5, 8, 9, 10-11, 32 apatite, 189 dentine collagen, 285 Cat dentine, 219 enamel, 128 Cattle dentine collagen, 293 enamel, 128 chemical composition, 319, 320, 322, 324, 326, 327, 328, 335, 336, 338 collagenous surface layer, 328 fluorotic, 229 fluorotic bone, 190 Cementum, 384-385 boundary with dentine, 24, see also Dentine-cementu m junction cementoblasts , 385 cementocytes , 385 changes on exposure to mouth, 384 chemical composition, 188 enzyme histochemistry, 385 acid phosphatase , 353, 385 alkaline phosphatase , 353, 385 dehydrogenases , 353-354, 385 fluorescent substances , 300 formation, 352-55i-354, 384 electron microscopy, 352, 353 histochemistry, 353, 354, 384-385 acid mucopolysaccharide , 384-385 histogenesis , 352-354 in reptiles, 11 strontium content, 221 Chemical analysis, see also Bone, Dentine and Enamel microchemical and histochemistry compared, 281, 282 by physical methods, 203 whole teeth, 202, 214, 221, 223 Chitin, X-ray diffraction, 195, 311 Chondroitin sulphates , 365, see also Mucopolysaccharides , acid and neutral in bone, 297-298 in dentine, 296, 297, 310

INDEX

475

Circumpulpar dentine, 4, 5, 7 Cod, dentine, 7 Collagen, amino acid composition, 283-257, 361 compared with gelatin and elastin, 361 in fishes, 285, 287 in mammalian dentine, 285, 286, 287, 288, 293, 309 regional differences, 285-257 in shark dentine, 285 sequenc e in polypeptide chain, 287-289 species differences, 285-257 amino acid side chains, structure, 284 antigenicity weak, 360, 364 basic macromolecule (tropocollagen), 47, 289-291, 309, 310-311, 315, 361 cross linking between, 291, 309 biosynthesis , 292-293, 294 in microsomes , 293, 350 in vitro, electron microscopy, 294 birefringence, 24-25, 362 C1 4-labelled, 360 of dentine, special characteristics , 47, 285, 293, 295 compared with skin collagen, 293, 295 electron diffraction, 360 electron-staining , 47 enzyme digestibility, 364-365 fibril s formation from macromolecule precursor, 291, 311, 350 periodic banding, 47, 291, 292, 311, 362 size, 47, 280, 291,362 lathyritic, chemistry, 360-361 metabolic turnover and collagenase , 402 of mineralized tissues, special characteristics , 293, 295 reconstitution from solution, 293, 311, 360 effect of precipitating conditions, 362 morphology dependen t on conditions, 360, 362, 363 by mucopolysaccharides , 360 solubilities, 292, 359-360 staining, 47 negative, with potassium phosphotungstate , 291, 292, 310 staining-reactio n chemistry, 362, 364 acid fuchsin and hydroxyl groups, 362 allochrome, 364 periodic acid-Schiff, 364 protein methods, 364 trichrome amphoteric, 364 triple helical structure, 47, 289-290-291, 309, 315, 361-362 tropocollagen, see basic macromolecule above ultrastructure dependen t on preparative procedures , 362, 363 X-ray diffraction, 280, 288, 289, 291, 309

S U B J E CT

476 Collagenas e clostridial, 296, 365 resistance of carious dentine to, 303 from gingival cultures, 365, 402 role in collagen turnover, 402 Cotton rat, dentine, chemical composition, 225 Crevicular epithelium, 437-446 adhesive properties, 423-424, 432 apical migration, 432-437, 446 birefringence, 438, 441 cell characteristics , 438-439 desquamation , 424-425, 427, 446 electron microscopy, 438, 439, 440, 442, 443 desmosomes , 440-441, 442, 443 enzyme digestibility, 441, 442 enzyme histochemistry, 444-446 acid phosphatase , 444 alkaline phosphatase , 445 cytochrome oxidase, 445 dehydrogenases , 445-446 phosphorylases , 444 histochemistry, 441-446 glycogen, 444 relation to keratinization, 444 periodic acid-Schiff, 438, 441 metachromasia , 441-442 intercellular substance , 432, 440 histochemistry, 441-442 non-keratinizing, 439-440, 450 regeneratio n after injury, 421-428 renewal rate, 424-426-427 thymidine-H3, 425, 427 two cell layers, 437-438 Crevicular fluid, 448-450 immuno-electrophoresis , 448, 449 leucocytes in, 449 mechanism of production, 448, 450 plasma proteins in, 450 scanty in health, 448 Crocodilia, dentine, 11, 13 Crossopterygii, 12 D

Dental caries, 65, 111 "caries crystals", 69, 232 chemical composition of carious dentine, 230-231, 302-303 resistance to collagenase , 303 of dentine electron microscopy, 66, 67, 68, 70 microradiography , 69 of enamel, 251, 269-271, 272, 339-340 chemistry, 341

INDEX polarized light, 80, 95, 339 fluoride, relation to, 230, 253, 255, 265, 266, 270 microradiography , 69, 270 Dentinal fluid, 234, 237 in ionic exchange , 234, 236, 237 Dentinal osteons, 9 Dentinal tubules branching, 24 electron microscopy, 50, 53, 56 into enamel, 141, 160 fluorescence, 300 primary curvatures, 13-14 in elephant ivory, 21 tooth movements responsible , 14 secondar y curvatures, 14 sheath of Neumann, 16-17 Dentine, see also Dentine matrix, Dentine mineral and Peritubular dentine age changes , 28, 29, 30 in organic content, 301 in agnathans , 3 in amphibians, 10, 11 anorganic, 22, 24-25 arcade patterns, 21-22-23 basophilia, 19 in bony fishes, 4, 5, 6, 7, 8, 10, 11, 12 boundary with cementum, 24 electron microscopy, 63, 64, 12 caries, microradiography , 69 in cartilaginous fishes, 3, 4, 5, 8, 9, 10-11, 32 apatite, 189 collagen chemistry, 285 chemical analysis, 202-206, 237 demineralization , 283 by physical methods, 203 activation analysis with autoradiography , 203 electron probe, 203 X-ray microanalyzer, 203 X-ray microscope, 203 tissue separative techniques , 202-203, 205-206, 237, 282-283 differential flotation, 29, 203, 206, 237 hazards of, 29, 225, 282 microdissection , 205, 237 chemical composition, quantitative, see also Dentine matrix and Dentine mineral mineral by volume, 188, 300 by weight, 188, 300 organic matter changes with age, 301-302 individual variation, 301 by volume, 188, 300 by weight, 188, 300-30Λ

S U B J E CT water by volume, 188 by weight, 188, 300-301 circumpulpar, 24 collagen-crysta l relation, 25, 59-61, 310, see also Dentinogenesis , mineralization crystals, 55,57, 59, see under Apatites and Dentine mineral dead tracts, 28-29 chemical composition, 231 definition of, 3 dermal armour, relation to, 3-4, 10 elasticity, 28, 29, 30 electron microscopy, 45-72 collagen-minera l relation, 26, 49, 59-60-61, 62, 72 collagens, typical and atypical, 45, 46, 47 differences between peritubular and intertubular, 47, 48, 49 mineral phase, 53-61 organic matrix, 45-53 collagen orientation, 49 peritubular dentine, 26, 48, 49, 53-54-55 peritubular (pericanalicular) membrane , 48, 49-50-51, 52, 71 on experimenta l diets, chemical composition, 222-228 under fillings, chemical composition, 233 fluorotic, 229 ground substance , 19, 45, 281 hereditary opalescent , chemical composition, 232 histochemistry, 19, 297 lipid, 53 mucopolysaccharide , 51, 53 acid, 19 periodic acid-Schiff, 297 of horse, see Horse, dentine hyaline layer, 24 hypoplasia, 21, 23, 24, 27, 31, 63 chemical composition, 222 incremental contour lines, 16, 22, 25, 63 interglobular, see Interglobular dentine intertubular, 13, 19, 23, 30, 32 ionic exchange , passive, 65, 202, 234 lipi d content, 53, 298 in lizards, 8, 11-12 mantle, 4, 5, 24 metachromasia , 19 metamorphosed , 28 whitlockite in, 232 microhardness , 29 influence of peritubular dentine, 30 mineral matter, see Dentine mineral neonatal line, 21 organic matrix, see Dentine matrix in periodontal disease chemical composition, 233 32

INDEX

477

peritubular, see Peritubular dentine polarization microscopy, 22, 24-25, 26 post-eruptive changes in, 26-30 ionic exchange , 65 relation to bone, 3-4, 10 in reptiles, 8, 11-12, 25, 30 resorption, pathological, 65 Schreger lines, 14, 21 sclerosis of, 28, 29, 30, 45 chemical composition, 232 and microhardness , 29 whitlockite in, 232 in scorbutic guinea pigs, 227 secondary , see Secondar y dentine sections, misinterpretation s from, 75-17 silver staining, 20, 21, 22 species differences, 30-31 specific gravity, 20, 29 tetracycline deposition, 27 tubules, see Dentinal tubules and Peritubular dentine vitamins, effect on chemical composition, 227-228 water, bound or free, 204, 206-207, 300-301 variability of content, 206 X-ray diffraction, 25, 55, 72, 166, 212, 234 of "altered" dentine, 55, 232 of fluorotic dentine, 229 X-ray microscope study, 203 Dentine-cementu m junction, 24 electron microscopy, 63, 64, 72 Dentine innervation, electron microscopy, 41, 43, 44, 65, 71 Dentine matrix birefringence, 24-25, 79 changes with caries chemical analysis, 302-303 chemical composition, 283-301, 309-310 summary, 300-301 chondroitin sulphate content, 296, 310 citrate content, 298-299, 310 peptide association , 299 collagen characteristics , 285, 293, 295, see also under Collagen amino acid composition, 286 compared with skin collagen, 293 hydroxylysine: lysine ratio, 285, 309 stabilizing linkages, 293, 295, 309 collagen fibre pattern in, 24-25, 49 collagenas e solubility, 296 definition, 281 fluorescent substances , 299-300, 303 lactate content, 299 lipi d content, 53, 298 mucoproteins, 51, 53, 296-297 phospholipid content, 298 protein from Neumann sheaths , 296

478

S U B J E CT

sialoproteins, 296-297, 310 and periodic acid-Schiff reaction, 297 water-soluble protein, 295-296 Dentine mineral, see also Apatites calcium:phosphoru s ratio, 207, 212, 213, 234, 235, 237, 238 in rat, 214-215, 223-225, 234-235, 237 calcite phase, 236 carbonate phase, 192, 234-235, 236, 237, 238 "caries crystals", 69, 232 chemical composition, 206-238 in man, 189, 207-214, 215, 218-223 age changes , 213, 219, 220, 221 anatomical differences, 212, 213-214, 219 in caries, 230-231 changes with attrition, 232 comparison with other tissues, 207, 212 nutritional influences, 222-228 in non-human, 214-215, 223-228, 237 in rodents, 214, 216-217, 237 content as dry or ashed weights, 206, 237 crystal size, 25, 57, 59 smaller than in enamel, 207, 212 crystals, action of ethylenediamine , 205 dead tract dentine, 231 under dentists' fillings, 233 with experimenta l diets, 237 calcium:phosphoru s ratios, 223-225, 238 high carbohydrate , 223 magnesium deficiency, 225-227 vitamins, 227-228 fluorapatite, 236 fluorine content, 215, 218-219, 236, 237 in fluorosis, 228-230 non-apatite crystals, electron diffraction, 229 hereditary opalescent , 232-233 hormonal influences, 228 human deciduous, 212-213 human permanent , 208-212 ionic exchange , 65, 202, 234 iron, 219, 222 in rat, 220 lead, 221, 237 in lead poisoning, 230 magnesium , 212, 232, 233, 236 manganese , 221 in rodents, 221 noncrystalline phase, 234 non-detectability by diffraction methods, 189, 234 non-withdrawal at systemic need, 201 in periodontal disease , 233 phases , number of, 234-235 potassium, 219 pyrophosphate , 204, 234, 235 racial differences, 212-213, 237

INDEX recrystallization, intrinsic, 202 sclerosed dentine, 232, 237 secondar y dentine, 231, 236 secondar y phosphate ions, 204, 235 separation from organic phase, 203-206, 234, 237 dry ashing, 203 wet ashing, 204-205 sodium, 207, 234, 237 strontium, 221, 222, 236 strontium9 0, 221 trace constituents , 215, 222 water content, 206, 234 whitlockite (β-tricalcium phosphate) , 232 on ignition, 204, 212 zinc, 220, 222 Dentine sensation , fluid movement hypothesis, 65 Dentinogenesis , 11-18-20, 24, see also Odontoblasts electron microscopy, 36, 31-40-42-45, 61, 63 enzyme histochemistry D (-)-j3-hydroxybutyric dehydrogenase , 353, 392-393 6-phosphat e dehydrogenase , 354 succinic dehydrogenase , 353 TPN-diaphorase , 353, 378 metabolic disturbance s of, 63 mineralization, 19, 25, 303-309, 310 citrate, role of, 309 electron microscopy, 61 epitaxy, 25, 305-306, 308 collagen in, 25, 306-308, 310 pyrophosphat e as inhibitor, 308-309 inotropic, 25 mucopolysaccharides , role of, 19, 51, 53, 61, 308 spheritic, 25 primary curvatures, factors responsible , 14 pulpodentinal membrane , see Pulpodentina l membrane in scurvy, 393 Von Korff fibres, see Von Korff fibres Dento-gingival junction, see also Gingiva and Gingival crevice absence of inflammatory cells in clinical health, 437 apical shift, 384, 432-437 cementum vitality, role of, 384, 433 connective tissue fibre changes , 433 endocrine experiments , 433 inflammation as cause, 434 non-physiologica l process, 437 nutrition experiments , 433 as physiological age change, 433, 436 connection between epithelium and enamel, 417-432 electron microscopy, 431 epithelial attachmen t concept, 421-422, 430-432 epithelial cuff concept, 422-424, 430-432 dynamic aspects , 432, 433, 434 as contact relationship, 432

S U B J E CT defence mechanism , 450-451 histogenesis , 415^417, 419 normality, criteria of, 451 Dog ascorbic acid inessential, 227 dentine chemistry, 214, 223, 228, 234, 237 fluorotic bone, chemical composition, 229 enamel, 78, 98, 127, 128, 264, 421 gingiva, 423, 425, 426, 427, 428, 429, 438, 441 periodontal membrane , 368, 371, 372 Dugong, dentine, 27 enamel apatite, 189 Durodentine, 4

Å Elasmobranchs , see Cartilaginous fishes Elastic fibres enzyme digestibility, 370 in periodontium, 371-374, 376 staining, 370, 371, 372 ultrastructure, 373-374 Elastin, amino acid composition, 361 Electron diffraction of bone, 388 of collagen, 360 of dentine, 234 non-apatitic crystals, 229-230 whitlockite, 232 of enamel apatite, 146, 183 insensitivity to poor crystallization, 234 methodology, 182-183, 388 selected area, 183, 196 X-ray diffraction, compared with, 183, 388 Electron microscopy, see also various tissues technique dark field, 141 demineralization on grid, 140 fixation, 41, 43 glutaraldehyde , 43 reconstruction s from serial sections, 152, 755, 756, 158, 159, 161 replicas, 81, 136-140, 143 staining, lead, 47 phosphotungsti c acid, 46, 47, 48, 67 uranyl acetate, 47 stereoscopic , 72, 138 photogrammetry , 139-140 ultrathin sectioning, 136 diamond knife, 136, 144 Elephant dentine, 21, 30, 31, 214

INDEX

479

enamel, 153, 262 Enamel, see also Enamel matrix and Enamel mineral aprismatic near dentine, 123 electron microscopy, 160 at surface, 83, 114, 777, 118, 119 electron microscopy, 161 caries, 251, 269-271, 272, 339-340 chemical changes , 341 microradiography, 69, 270 polarized light, 80, 95, 339 specific gravity and nitrogen content, 340, 341 as white spot, 270 chemical analysis, 248, 251 demineralization by acid, 329 by EDTA, 320 tissue separative techniques , 320, 327, 329-330, 332, see also Dentine, chemical analysis differential flotation, 332 chemical composition, quantitative, human, see also Enamel matrix and Enamel mineral age changes , 252-253 mineral layer gradient, 251, 252 by volume, 188, 248, 251, 271, 341 by weight, 188,189, 248, 251 organic matter age relation, 331 anatomical zone variations, 329-330 layer gradient, 251, 252 by volume, 188, 248, 271, 341, 418 by weight, 188, 248, 328, 330-333, 418 water content layer gradient, 252 by volume, 188, 248, 271 by weight, 188, 248 contralatera l teeth, structure, 124 cross-striations , 83-84, 114, see also under Enamel prisms crystals, see under Apatites and Enamel mineral demineralization techniques , 76-77, 340 electron microscopy, 82, 87-88, 140-162 crystals, 141-142-143-144, see also under Apatites internal structure, 142-143, 144 shape, 143-144 on pseudoreplicas , 137, 142, 143 methodology, 78-79, 135-140 demineralization on grid, 140 photogrammetry , 759-140 reconstruction s from serial sections, 152, 755, 756, 158, 159, 161 replicas, 136-139, 143, 144, 150, 151 ultrathin sectioning, 136, 144

480

S U B J E CT

organic fibril s on replicas, 140 replica of EDTA-treated, 143 enamel fluid, 81, 248 mediator of ionic exchange , 248-249 velocity of flow, 248 flui d flow from dentine, 110 fluorescence microscopy, 80, 81, 87, 98, 107, 108, 111 fluorotic, 722, 725, 189-190 acid resistance , 271, 340 chemical composition, 254, 255 ground section techniques , 77-78, 80 surface damage, 81 thickness artifacts, 78, 86, 88, 111 histochemistry, 124-126 demineralization , a prerequisite, 124 simulated reactions, 124 hypomineralized areas, 79, 80, 82, 85, 114, 776-122, 124 acid resistance , 122 chemical composition, 122, 341 electron microscopy, 121 fluorescence , 120, 122 incremental lines in, 120, 727, 122 microhardness , 120 microradiography , 114, 776, 727 in polarized light, 114, 776, 777, 727, 122 subsurface zone, 120 varieties, 122 "whit e spot", 120, 122 acid resistance , 122 chemical composition, 122, 341 imbibition, 79, 152, 333, 334, 339 with air, 93 innermost layer absence of cross-striations , 88 chemical composition, 251-252, 329 structure, 78, 725-124 ion exchange at surface, 252-259 ion passag e into, 188, 249, 272, 342 in Lagomorpha, structure, 128 microhardness , 78, 80-81 microradiography , 77, 78, 79-80, 83, 87, 97, 103, 106, 107, 116, 121, 155, 161 quantitative, 248, 251 neonatal line, 21, 97, 98, 99, 100 fluorescence , 99 organic matrix, see also Enamel matrix replacemen t by plastic, 81 permeability to dyes, 81, 82 phase contrast, 78, 87 of replicas, 81 polarization characteristics , 78-79, 80 crystal orientation, evidence of, 79, 153, 755 drying effects, 252 as measure of mineralization, 78, 79, 80, 333

INDEX position of water deduced from, 252, 333 temperature effects, 252 pore system, 79, 152, 249, 333, 342 pore size, 249, 333, 339, 342 prenatal and postnatal, differences in mineralization, 99100 replicas, 81, 85, 86, 87, 88, 772,139,141, 149,150,151, 156 pseudoreplicas , 137, 138, 142, 143 technique, 81, 136-139 in Rodentia, structure, 127-128 species structural differences, 126-128, 153 specific gravity, 252, 282, 341 age changes , 252 layer gradients, 252 subsurface , unchanging composition, 272 surface features, 112-115 age changes , 114 incremental lines, 772, 113, 114 in replicas, 772 surface layer acid resistance , 251, 271 caries resistance , 251 chemical composition, 251-259, 272 electron microscopy, 160-161 fluoride-reactivity, 251, 269 isotope accumulation 253 microhardness , 251 organic content high, 420 post-eruptive mineralization, 247, 252, 253, 272 structure, 112-116, 777, 118, 119, 421 perikymata, 772, 113, 114, 115, 116 water, bound or free, 247, 248, 252, 271-272, 333 Enamel cuticles, 82, 107, 108, 109 birefringence, 111, 114, 430 chemistry of, 328-329 collagenous layer, 328 electron microscopy, 111 fluorescence , 111 histochemistry, 111, 125, 126, 420 carbohydrate and protein-bound lipid, 126 nomenclature , 112 primary, 111-112, 415, 418, 420, 427, 431 secondary , 421-422, 430-431 Enamel-dentin e junction, 106, 107, 108, 725-124 electron microscopy, 62, 64, 12, 141, 160, genesis, 61, 63 enamel at, 725 fluorescence , 124 microradiography , 106, 107 Enamel-dentin e membrane , 108, 124 electron microscopy, 61, 63, 64 Enamel iron pigment in rat, 220, 259 Enamel lamellae, 82, 105-110, 248, 457 absence in rat, 128

S U B J E CT age, no relation to, 109 in anorganic enamel, 109 "crack" hypothesis, 108-109, 418 electron microscopy, 108, 140 enamel cuticle, relation to, 105, 107, 108, 109, 421, 431 metachromasia , 125 periodic acid-Schiff reaction, 126 in replicas, 140 Enamel matrix acid-insoluble, 317-318, 320, 328, 329, 331, 336-337 chain repeat distances and apatite lattice, 337 as collagen, 318, 327, 328, 336-337 enzyme digestibility, 337-338 acid-soluble, 317-319, 320-321, 328, 331, 334-336 acid solubility, related to degree of mineralization, 321 amorphous or fibrillar , 86, 323-324, 325 basophilia, 125 birefringence, 79 carbohydrate s content, 338 chemistry, 317-342 methods of study, 318 copper, 261 ethylenediamin e extraction, 81, 320, 321 formation glycine-Ç3 incorporation, 321 histidine-H3 incorporation, 321 methionine-C1 4, -H 3, - S35 incorporation, 321 proline-Ç3 incorporation, 320, 321 hexose content, 338 histochemistry, 125-126 acid mucopolysaccharide , 125, 338 metachromasia , 125 periodic acid-Schiff reaction, 125-126, 338 sulphydryl reaction, 125, 126 hydroxyproline content, 336-337 iron, 258 keratin characteristics , staining, 79, 125, 420 X-ray diffraction, 195, 337 during maturation, see also Enamel maturation pyrrolidine rings, role of, 325 serine phosphate , role, 324 water content, 326, 327 withdrawal of organic material chemical aspects , 325-326 electron microscopy, 324-325 of mature enamel chemical composition, 327-339 acid-insoluble protein content, 329 amino acid analysis, 328, 334-336 bovine, 327-328 carbohydrates , 338-339

INDEX citrate, 339 on experimenta l diets, 333 human, 328, 329, 331 age differences, 332 anatomical region differences, 329-330 lactate, 339 lipids, 338 nitrogen analysis, 331, 332, 341 peptides, 335 sample preparation, 327, 329 separative techniques , 327, 329 water, 248, 333-334 wet-ash estimations, 331, 332 histochemistry, 124, 126 disulphide-reactive , 420 specific gravity, 248 non-keratinous nature, 420 prematuration "amelogenins", 322 amorphous gel concept, 321, 325 chemical analysis EDTA preparation, 320 sample separation , 319-320 chemical composition, 319-322 amino acids, 319, 320, 322, 323 bovine, 319, 320 electrophoretic fractions, 321-322 infrared spectroscopy , 322, 324 kératose fraction argument, 322, 323 non-protein substances , 319 optical rotatory dispersion, 322-323 protein heterogeneity , 321 protein solubility, 320-321 species differences, 320 specific gravity, 320, 326 X-ray diffraction configurations, 323-324, 337 histochemistry, 125-126 acid mucopolysaccharide , 125 basophilia, 125 metachromasia , 125 sulphydryl reactive, 420 staining reactions, 125 thixotropic properties, 325, 342 zinc, 257 Enamel maturation displacemen t of water, 252 protein depolymerization , 319, 325, 342 protein withdrawal, 319 reduced enamel epithelium, 416 solubility increase of matrix, 321 Enamel mineral amorphous, 144-145 apatite crystals, 141-144, see also under Apatites morphology, 143, 144

481

482 orientation, 79, 80 by polarized light, 78, 153 by X-ray diffraction, 194, 195 reactivity, 249 size, 141, 249 calcium:phosphoru s ratio, 250-251, 266 carbonate, 144, 247, 262-263 effect on birefringence, 263 chemical composition by chemical analysis, 249-250 by physical methods, 81 electron probe analysis, 81, 203 neutron activation analysis, 249-250 nuclear magnetic resonance , 248 X-microradiography, quantitative, 165-166 in man, 249-262 layer gradients, 253-265 specimen variability, 250 copper, 261 association with organic matrix, 261 fluorapatite, 265, 266 fluoride, 253-256 age, relation to, 253, 255, 272 caries, relation to, 255, 269-271 caries-inhibiting mechanism , 269-270 citrate, effect of, 265 drinking water, relation to, 254-255 in endemic fluorosis, 254, 255, 271 magnesium , relation to, 263 molybdenum, effect of, 264 physico-chemica l mechanisms , 255, 266 prenatal, 254 stages of deposition, 254 strontium, relation to, 260 topical application of, 266-269 uptake in vitro, 265-269 iron, 258-259 association with organic matrix, 258 of rat incisor, 259 lead, 251, 257-258 magnesium , 263-264, 266 molybdenum, 264-265 sodium, 264 surface-laye r chemistry, 249, 251-265, 271-272 carbonate, 247, 262-263 fluoride, 247, 251, 253-256, 272 lead, 251, 257-258, 272 magnesium , 251, 263-264, 272 zinc, 251, 256-257, 272 strontium, 247, 259-261 strontium9 0, 260-261 tin, 259 topical applications, effect of, 265-269 fluoride, 266-268

S U B J E CT

INDEX tin, 267-269 trace constituents , 264-265 vanadium, 264-265 zinc, 256-257 association with organic matrix, 257 Enameloid, 4, 7, 11, 31 Enamel organ, 416 Enamel prisms, 82-55-89 in Carnivora, 126-127, 153 cross-sectio n morphology, 83, 86-87, 152-157 species differences, 153 cross-striations , 83-84, 85, 88, 94, 114, 150 genesis, 88, 150, 152 in microradiographs , 83, 84 in polarized light, 84, 88-89, 94 crystal orientation, 168 by electron microscopy, 145-146, 151 by microradiography , 155 by polarized light, 78-79, 80, 88, 153, 155 by X-ray diffraction, 153, 194, 195 differential hardness , 78, 168 electron microscopy, 145-159 branching, 159 course pursued by, 158-159, 160 cross-striations , 150-151-152 crystal orientation, 145-148-149-150, 151, 152, 155, 157 interprismatic substance , 152-158, 162 morphology of, 152-153-154-155-156, 160 prisms within prisms, 159 "secondary crystal" mineralization, 146 sheaths , 146-747-150, 162 wing processes , 153, 156, 157 in Primates, 126, 153 sheaths , see Prism sheath size, 85, 88 staining, 126 wing processes , 87, 153, 756, 157 Enamel spindles, 28, 770-111, 140 absence in carnivores, 128 electron microscopy, 140, 747, 160 Enamel tufts, 82, 104-105-106-110, 140 in demineralized sections, 104, 705, 108 dentinal tubules, relation to, 108 electron microscopy, 140 fluorescence , 107, 108 Hunter-Schrege r bands, relation to, 105, 109-110, in ion-etched specimens , 107, 140 metachromasia , 125 microhardness , 104 in microradiographs , 104, 106, 107 permeability to dyes, 108 in polarized light, 704 in replicas, 140 staining, 705, 108

S U B J E CT Enzyme histochemistry, see also Acid phosphatas e and Alkaline phosphatas e aminopeptidase , 391, 399 "branching" enzyme, 444 cytochrome oxidase, 382, 391, 400, 445 DPN-diaphorase , 353, 384, 385, 386, 391, 396, 400, 445 DPN-glutamic dehydrogenase , 391, 400 DPN-malic dehydrogenase , 353, 385, 391, 396, 400 esterase , non-specific, 353, 378, 382, 391, 392, 396, 399 galactosidase , 391 â-galactosidase , 396, 399 glucose 6-phosphat e dehydrogenase , 354, 384, iS-glucuronidase , 382, 391, 396, 399 glutamic dehydrogenase , 353, 377, 385, 387, 393, 396, 400, ^-glycerophosphat e dehydrogenase , 353, 385, 391, 396, 400 D (-)-jS-hydroxybutyric dehydrogenase , 353, 385, 391, 392, 393, 396, 400 isocitric dehydrogenase , 353, 385, 396, 400, 445 lactic dehydrogenase , 353, 384, 385, 391, 396, 400, 445 malic dehydrogenase , 377, 391, 396, 445 phospho-esterases , 444 protease , 396 phosphorylase , 444 succinic dehydrogenase , 353, 384, 385, 391, 396, 400, 445 TPN-diaphorase , 353, 378, 384, 385, 386, 391, 396, 400,445 TPN-glucose 6-phosphat e dehydrogenase , 391, 396, 400, 445, 446 TPN-isocitric dehydrogenase , 391, 396, 400 TPN-6-phosphogluconi c dehydrogenase , 385, 391, 396, 400 UDPG-glycogen transglycosidase , 444 Epitaxy, 25, 143, 305-308, 324, see also under Mineralization definition, 195-196 Epithelial attachment , see Crevicular epithelium and Dentogingival junction Eruption, 416 continuous, 355 dissolution of tissue in, 555-356-357 electron microscopy, 357 experimenta l investigations, 355 hypotensive drugs, 355 nucleotoxic drugs, 355 X-irradiation, 355 hammock ligament, 355 histochemica l changes , 357 mucopolysaccharides , 357 "passive", 416-417, 432-437 processe s involved, 354-355 rearrangemen t of fibres, 357-358 union of epithelia, 416, 419 Ethylenediamine dentine crystals, action on, 205 phosphate , action on, 205

483

INDEX F

Fluorapatite, see Apatites Fluorescenc e microscopy, of enamel, 80, 81, 87, 98, 99, 107, 108,111,124 Fluoride caries-inhibition, 253, 255, 271 dentine content, human, 215-219 age, related to, 219 carious teeth, 230 deciduous, 219 drinking water, related to, 219 in endemic fluorosis, 228, 229 in industrial fluorosis, 228, 229 nutritional factors and, 215 permanent, 218 post-eruptive increase, 218 secondar y dentine, 219 dentine reaction in vitro, 236 enamel content, human, 253-256 age, related to, 254, 255 carious enamel, 270 deciduous, 253, 254 drinking water, related to, 253, 254, 255, 265 fluorapatite in, 256, 265 mottled teeth, 255 permanent, 253-254 stages of deposition, 254 in successiv e layers, 219, 254 theoretical, of ultimate outer surface, 256 topical application, 265-269 decrease d response with age, 269 fluorapatite and calcium fluoride as reaction products, 265-267 of stannous fluoride, 267-269 mechanism of uptake by mineralized tissues, 255-256 serum levels, 254-255 isotope study, 255 Frog, dentine, \ \, 12

G Gelatin, amino-acid composition, 361 Gingiva, see also Dento-gingival junction, Gingival crevice and Periodontium acid mucopolysaccharide , 366 alkaline phosphatase , 445 collagenes e prepared from, 365, 402 defence mechanism , 450-451 enzyme histochemistry, 441-446 lactic acid dehydrogenase , 445 oxidative enzymes, 445 oxytalan fibres, 368-369

484

S U B J E CT

recession related to inflammation, 436 "sulcus", 421-422, see also Gingival crevice tissue respiration studies, 379-380 Gingival crevice, see also Crevicular epithelium bacteriology of, 446-448 crevicular fluid, see Crevicular fluid histology, 419, 420 experimental, 423, 425, 426, 427, 428, 429, 430, 434, 435 physiology of, 446-451 tooth grinding, effect of, 436 Granular layer of Tomes, 23-24, 45 Guinea pig dentine, chemical composition, 214, 215, 223, 226, 227 periodontal membrane , 372

H Haddock, vasodentine , 7 Hake, vasodentine , 7 Hammock ligament, 354-355 Hamster dentine, chemical composition, 204, 214, 215, 219, 220, 222, 237 gingiva, 433 periodontium, 415 Hertwig's root sheath, 352, 354 electron microscopy, 63 Hippopotamus, enamel, 249 apatite dimensions, 189, 193 Histochemistry, see various tissues Histological methods for acid mucopolysaccharides , 365-368 alcian blue, 365, 367 Hale method, 365, 366-367 methylene blue extinction test, 366 sulphation, 366 for basophilia, 125 collagen stains, 362, 364 decalcification EDTA, 283 effect on enzymes, 403 effect on histochemica l reactions, 124 for enamel matrix preservation , 76-77 Brain's solution, 340 for elastic fibres, 370, 371, 373 orcein, 373 enzyme digestions, 125, 367-368, 370, 371 collagenase , 365, 368 elastase , 370-371 ^-glucuronidase , 367, 370-371, 382 hyaluronidase , 125, 367, 371 ribonuclease , 368

INDEX ground sections, 15, 17, 77-78 for metachromasia , 19, 125 for oxytalan fibres, 370-373 aldehyde fuchsin, 370, 371 peracetic acid-aldehyde fuchsin, 372 peracetic acid-aldehyde fuchsin-Halmi stain, 351, 352, 358, 370 periodic acid-Schiff of bone, 389 of collagen, 364 in connective tissue during eruption, 357 of enamel, 126, 338 for sulphydryl groups, 125, 420 Horse, dentine, 25, 26, 30, 31 chemical composition, 214 wide peritubular zone, 19 Howship's lacunae, 396 Hunter-Schrege r bands, 78, 80, 82, 100-104, 107, 109 in decalcified sections, 101, 103 electron microscopy, 159, 161 microhardness , 103, 105 microradiography, 101, 103 as mineralization differences, 103 in polarized light, 100-101, 102 as section-grinding artifact, 78, 101 species differences, 128 staining characteristics , 102-103, 125

I

Interglobular dentine, 23, 27, 31, 45 mineralization in vitro, 27 unalterability, 63 in vitamin A deficiency, 227 Intermediate predentine, 19 Interprismatic substance , 79, 81, 83, 85, 87, 128 differential hardness , 78 electron microscopy, 87-88, 152-158, 162 in microradiographs , 87, 155 non-existenc e concept, 152-158 in polarized light, 88 Ion-beam etching, 81, 107, 140 Isotope labelling 14 bicarbonate-C , 396 C a4 5, 393 F 1 8, 255 glycine-C1 4, 360 glycine-H3, 321, 378-379, 380, 393, 394, 395 H g 2 03 , 23 3 histidine-H3, 321 methionine-C1 4, 321 methionine-H3, 321 methionine-S3 5, 321

S U B J E CT Ñ3 2, 255-256, 393 P b2 1,0 393 proline-H3, 320, 321, 379, 442 radiostrontium, 393 32 serine phosphate-P , 3 24 90 Sr , 260, 261 sulphate-S3 5, 338, 382, 396 thymidine-H3, 399 Ivory, elephant, 21, 30, 31, 214 Ê Kangaroo, dentine, 30 Koala bear, dentine, 26, 27

L Lagomorpha, see also Rabbit dentine, 214 enamel, 128 Lathyrism, 360-361, see also Collagen lathyritic Lizards, dentine, 8, 11-12 Lysosomes , 325

M Mammal-like reptiles, dentine, 25, 30 Mantle dentine, 4, 5, 24 Microchemical analysis, compared with histochemistry, 282 Microhardness dentine, 29, 30 enamel, 78, 80-81 Hunter-Schrege r bands, 103, 105 prisms, 78, 168 striae of Retzius, 93 tufts, 104 Microradiography of bone, 166, 389, 390 of dentine, 166, 202, 214 in caries, 69 in periodontal disease , 233 peritubular zone, 214 of enamel, 78, 80, 103 Hunter-Schrege r bands, 102 striae of Retzius, 114, 166 tufts, 104, 105, 106, 107 methodology, 188 quantitative, 387 Mineralization alkaline phosphatas e hypothesis, 304 apatite solubility, chemico-physica l forces, 196, 304-305 calcite in, 197

INDEX

485

citrate, role of, 309 collagen as epitactic agent, 306-308 colllagen-minera l relationship, 59, 61, 72, 306-308 of collagenous tissue inotropic, 22, 25 "globular" in dentine, 25 epitaxy, 195, 305-306 definition, 195-196 in dentine, 25 in enamel, 143, 195, 324 pyrophosphat e as inhibitor of, 308-309 estimation by chemical analysis, 188 in invertebrates , 196, 197 by polarization microscopy, 79, 121 by X-ray absorption, 79, 188, 387 in vitro studies, 196 Liesegang phenomenon , 23 mineral-matrix relationship, 195-196, 303-304 mucopolysaccharides , role of, 51, 53, 308, 310 in non-mammalia n tissues, 192, 193, 197 in pathological, 197 pyrophosphatases , 308 serine phosphate in enamel, role of, 324 tissue fluid, influence of, 196-197 of turkey leg tendon, 196 Monkeys green (Cercopithecus aethiops), gingiva, 420, 431, 434, 435, 436 rhesus (Macaca mulatto) amelogenesis , 416, 417, 418 dentine, chemical composition, 214, 234 enamel, 147, 155, 158, 160 chemical composition, 318, 332, 338 gingiva, 419 irradiation, effect on odontogenesis , 355 Mouse amelogenesis , 63, 143 bone, 226, 393, 394, 395, 399 collagen formation, 294 dentine, chemical composition, 226 dentinogenesis , 37, 63 gingiva, 436 mast cells, 381 periodontal membrane , 368, 371, 372, 378, 380, 415 Mucopolysaccharides , acid and neutral in bone, 389, 391 in cementum, 384-385 in crevicular epithelium, 441-442 in dentine, 19, 51, 53, 72, 234, 281 of enamel matrix, 125, 338 enzyme digestion, 367-368 formation in embryogenesi s and repair, 350-351 increase with connective tissue maturation, 350-351

S U B J E CT

486 of periodontal membrane , 359, 365 sialic acid, 365, 368 stains for, 365-368 alcian blue, 365, 367 enzyme digestion as adjunct to, 367-368 Hale method, 365, 366-367 metachromasia , 365, 366 methylene blue extinction test, 366 periodic acid-Schiff, 366 sulphation-acetylatio n method, 366 varieties, 365

Í Nasmyth's membrane , see Enamel cuticles Neutron diffraction, 177, 182, 183-184, 248 more sensitive than electron and X-ray, 183-184

Ï Odontoblast electron microscopy, 36-41, 42, 71 cytoplasmic organelles, 56, 37, 39 desmosomes , 39 glycogen, 39 Golgi apparatus , 36, 37, 39 nuclear pores, 39 terminal bars, 39 Von Korff fibres, 38, 39, 40 enzyme histochemistry glucose 6-phosphat e dehydrogenase , 354 succinic dehydrogenase , 353 TPN-diaphorase , 353 function non-resorptive , 65 vasodentine , associate d with, 7 Odontoblast process, 14, 15, 16, 17, 19, 20-21 electron microscopy, 41-45 collagen fibres within tubule, 43 cytoplasmic organelles, 41-44 granular secretion, 43, 71 lipid, 43, 71 nerve fibres related to, 41, 43, 44, 65, 71 pericanalicular membrane , 48, 49, 50, 51, 52, 71 perifibrillar circulation, 43 plasma membrane , 41, 51 fixation difficulties, 20-21, 41 length, 280 spiral course, 14, 15 thickness, 19, 20, 280 tubule wall, relation to, 16-17, 20-21 Orthodentine, 4, 5, 9-31, 32 definition, 4, 5

INDEX in mammals, 12-31, see also Dentine in non-mammals , 10-12 Osteoblasts , 391-396 autoradiography , 393-394-395-396 cytoplasmic organelles, 391 enzyme histochemistry alkaline phosphatase , 391 aminopeptidase , 391 dehydrogenases , 391-592, 393 esterase , non-specific, 592 ribonucleic acid, 391 Osteoclasts , 396-401 cytoplasmic granules, 399 electron microscopy, 71, 597, 398, 399 striated border, 597, 399 enzyme histochemistry, acid phosphatase , 579, 399 alkaline phosphatase , 399 aminopeptidase , 399 cytochrome oxidase, 400 dehydrogenases , 400 D (—)-£-hydroxybutyric dehydrogenase , 592, 393 in scurvy, 393 esterase , non-specific, 378 392, 399 foreign body giant cells, compared with, 401 and Howship's lacunae, 396 origin from osteoblasts , 399 in parathormone-induce d resorption, 400-401 in pressure-induce d resorption, 384, 400 thymidine - H 3, 399 Osteocytes , see also Bone and Osteoblast s enzyme histochemistry, 396 Osteodentine , 4, 5-6, 8, 10, 32 relation to vasodentine , 7 Oxytalan fibres compared with other fibres, 570, 371-373 electron microscopy, 373-575, 403 enzyme digestibility, 370-371, 403 to elastase , 370 to ^-glucuronidase , 371, 403 histogenesis , 351-352 in periodontal membrane , 358, 368-369 increase with masticatory function, 569 staining characteristics , 352, 370-371 compared with elastic, 570

Ñ Passive eruption, see Dento-gingival junction, apical shift Periodontal disease , 401-402 collagenolytic factor in gingiva, 402 Periodontal membrane (ligament) acid mucopolysaccharides , 365

S U B J E CT cellular activities, 376-384 collagen fibres, 357-359 electron microscopy, 354, 357, 375 tinctorial characters , 357 turnover, 433 collagen fibre orientation, 359 cytochemistry acid phosphatase , 376, 377-378, 379, 385 alkaline phosphatase , 376, 377, 378, 385 increase with inflammation, 377 dehydrogenases , 376, 377, 384, 385, 387 esterase , non-specific, 378 elastic fibres, 311-372-373-374 by orcein stain, 376 quantitative estimation, 372 species differences, 372 epithelial cell rests, 382-383-384 enzyme reactivity, 383-384 fewer in adults than children, 383 non-regeneratio n after injury, 382, 383 species differences, 383 histogenesis , 350-352, 355 oxytalan fibres, 358 hydroxyprolinerhexosamin e ratio, 350 isotope studies, 378-379 glycine-H3, 379, 380 proline-H3, 379 mast cells, 381-382 enzyme reactivity, 382 histochemistry, 381 species differences, 381 nerve fibres, esteras e reactive, 378 oxytalan fibres, 368-375, see also Oxytalan fibres elastase digestion, 370 electron microscopy, 313-375, 403 increase in scleroderma , 373, 374 as pre-elastic fibres, 372, 374 quantitative estimation, 372 species differences, 372, 403 transseptal , 368, 374, 376 oxytalan fibre formation, 351-352, 358 in response to functional demand, 369 pre-elastic fibres, 372, 373, 374 orientated collagen groups, 359 transseptal , 359, 369, 373 osteoblast s enzyme histochemistry, 376-377 alkaline phosphatase , 377 osteoclast s acid phosphatase , 379 esterase , non-specific, 378 reticular fibres, 365, 371 Periodontium, see also Gingiva and Periodontal membrane alveolar bone, 385-401, see also Alveolar bone

INDEX

487

cementum, 352-354, 384-385, see also Cementum connective tissue elements, 358-403 definition, 349 development , 349-557-355-356-35 8 elastic fibres, 370, 371-373, 374, 376 in eruption, see Eruption isotopic studies, 378-350, 393, 394, 395, 396 mast cells, 381 oxytalan fibres, 357, 352, 368-375, see also Oxytalan fibres pathology, 401-402 tissue respiration studies, 379-380 Peritubular dentine, 13, 76, 17, 19-20, 26-28, 32, 53 absence of in certain mammals, 30 centripetal mineralization of, 26, 27 in dead tracts, 28 formation, 19, 26, 55 in response to injury, 55, 67 electron microscopy, 26, 48, 49, 53-55, 214 of lateral branches , 53, 56 in horse, 19, 25, 26, 30, 37 inner and outer layers, 20, 26 lipi d in, 53 microradiography, 69, 214 mineralization, degree of, 53, 55 related to that of intertubular, 26-27 polarization characteristics , 25 separation for chemical analysis, 19 species differences, 13, 17, 19, 26, 27, 30-31 specific gravity, 19-20, 26 staining reactions, 17, 20, 26 tetracycline, in, 27 Phase contrast microscopy of enamel, 78, 87, 158 of replicas, 81, 98, 112 Phosphatases , see Acid phosphatas e and Alkaline phosphatase Pig enamel, 159, 214 fluorosed dentine, chemical composition, 229 periodontal membrane , 368, 372 Pike, dentine, 5, 6, 7 Placoderms , 10 Plicidentine, 4, 6, 8-9, 12, 32 Polarization microscopy, see also under various tissues of bone, 388 degree of mineralization, in estimation of, 79, 80, 121 of dentine, 6, 24 of enamel, 78-79, 88, 94, 333, 339 of gingiva, 438 Predentine electron microscopy, 40, 41, 42, 51, 63 intermediate, 21, 23 peritubular dentine, absence of, 72

S U B J E CT

488

problem of non-mineralization of, 281, 304, 306, 308 zonation, 19, 20, 23 Prism sheath, 79, 82, 85, 128 in demineralized sections, 747, 148 electron microscopy, 146-747-749-150 , 162, 319 comparison with other crystal systems, 148 fluorescence , 86, 111 histochemistry, 86, 126 insolubility, 319 non-existenc e concept, 86, 146-150 as optical phenomenon , 86, 146 staining characteristics , 86, 126 Pristis, dentine, 8, 9, 32 Pulp, see Tooth pulp Pulpodentinal membrane , 75-19, 23

R Rabbit dentine, chemical composition, 215, 229 enamel, chemical composition, 215, 320 periodontium, 355, 372 Raia dentine, 4, 5, 70-11 tooth attachment , 4 Rat bone, 226, 256, 392, 393, 399, 400 lathyritic, 360 cementum, 64, 352, 353, 385, 386, 400 dentine, chemical composition, 202, 206, 214-216, 218, 220, 222-230, 234, 237, 299, 333 dentinogenesis , 36, 37, 53, 64, 353, 354, 386, 400 enamel, 64, 127, 128, 142, 745, 249 chemical composition, 220, 222, 259, 263, 264, 318, 320, 332, 333, 338, 339 gingiva, 436 periodontal membrane , 354, 355, 357, 368, 371, 372, 375, 377, 378, 379, 385, 386, 387, 399, 400 tail tendon, 350 Reduced enamel epithelium, 476, 477, 418 fate controversy, 417, 479, 420, 421 mitotic activity, 416, 418 Rests of Malassez, see Periodontal membrane , epithelial cell rests Reptiles, dentine, 11, see also Mammal-like reptiles Reticular fibres argyrophilia, 365 varieties, 365 Rodents dentine composition, 216-217, 221 enamel, 127, 128, 215

INDEX S Schreger bands, see Hunter-Schrege r bands Seal, dentine, 214 Secondar y dentine, 28, 45, 231 fluorine content, 236 Shark dentine apatite, 189 enameloid apatite, 189 Sheath of Neumann, 16, see also Dentine Sheep fluorosed bone, chemical composition, 229 periodontium, 372 Striae of Retzius, 83, 85, 89-100 in contralatera l teeth, 124 in decalcified sections, 97, 98 disease processes , relation to, 98-99 electron microscopy, 159 hypermineralized , 93, 95 hypomineralized, 95, 98 microhardness , 93 in microradiographs , 92, 93, 97, 98 perikymata, relation to, 112-114, 115, 116, 119 in polarized light, 90, 91, 92, 93, 94, 95, 96, 98, 113, 115, 116, 124 staining characteristics , 98 Ô Teleosts bone, 4 dentine, 5, 6, 7, 10, 11, 72 Tetracycline, deposition in dentine, 27 Tomes fibre, 16, 17, see also Odontoblast process Tooth attachmen t ankylosis, 11, 32 fibrous, 4, 32 hinged, 11,32 socketted, 11 Tooth movements , see also Eruption responsible for primary curvature of dentinal tubules, 14 Tooth pulp, see also Odontoblasts ground substance , 18 scorbutic, calcification within, 227 V Varanus, dentine, 8, 11-12 Vasodentine, 4, 6, 7, 32 relation to osteodentine , 7 Vitrodentine, 4 Von Korff fibres, 17-18, 19, 49

S U B J E CT W Walrus, dentine, 21 Whale, cementum, 300 Wrasse, dentine, 11, 12

X X-ray diffraction bone, 166, 183, 194-795, 387-388 of collagen, 388 cementum, 166 collagen, 288, 289, 291, 309, 361 compared with electron and neutron, 183, 184 dentine, 25, 55, 72, 166, 212, 234, 280 in caries, 232 fluorosed, 229

INDEX

489

enamel, 78-79, 93, 166, 183, 195, 248, 249 enamel matrix, pre-maturation , 323-324, 337 macromolecules , 280 methodology, 167-168, 169-172, 183, 387-388 Bragg's law, 172, 775 insensitivity to light atoms, 183 insensitivity to poor crystallinity, 234 wavelengths , K-radiation, 170, 171, 172 methods automatic single crystal, 175, 177 Debye-Scherrer , 775, 179 diffractometer, 779-180, 187 Laue, 173, 174, 180 microbeam, 180-757 rotating crystal, 173, 175, 776 Weissenberg , 175, 777 X-ray emission microanalysis, 81, 203 X-ray microscopy of dentine, 203

Printed in Belgium