Oral Biochemistry 9789819935956

Table of contents :
Cover
Half Title
Oral Biochemistry
Copyright
Dedication
Preface
Contents
Author and Contributors
1. Characteristics and Importance of the Oral Cavity
What Is Oral Biochemistry?
Characteristics and Importance of Oral Cavity
Digestive Function
Antibacterial Function
Buffering Action
Lubrication
Hygienic Action
Taste
Repair
Control of Water Balance
References
2. Enamel
Microstructure of Enamel
Unit Cell
Crystal
Enamel Rod (Prism)
Inorganic Constituents of Enamel
Isolation of Enamel, Dentine, and Cementum from Teeth
Mechanical Method
Flotation Technique
Chemical Method
Chemical Composition of Enamel
Major Inorganic Components
Calcium and Phosphate
Bicarbonate (CO32-)
Bicarbonate and Dental Caries
Fluoride and Dental Caries
Trace Elements
Distribution of Fluoride in Tooth and Its Uptake Ratio
Fluoride As an Inhibitor of Dental Caries
Fluoride and Mottled Teeth
Effects of Fluoride on Enamel Remineralization
Ionic Substitution in Enamel Apatite
Comparison of Synthetic Hydroxyapatite and Human Enamel Apatite
Changes in Crystallinity After Heterogeneous Ionic Substitutions in Hydroxyapatite
Precursors of Enamel Apatite
Octacalcium Phosphate [Ca8H2(PO4)65H2O]
Amorphous Calcium Phosphate
Tricalcium Phosphate [Whitlockite, Ca3(PO4)2]
Dicalcium Phosphate Dihydrate (Brushite, CaHPO4.2H2O)
Organic Materials in Enamel
Amelogenin
Enamelin
Ameloblastin (Amelin or Sheathlin)
Tuftelin
References
3. Dentine
Dentine Structure
Chemical Composition of Dentine
Inorganic Components of Dentine
Organic Constituents of Dentine
Physicochemical Properties and Biosynthesis of Collagen
Physicochemical Properties of Collagen
Amino Acid Composition of Collagen
Protein Structure of Collagen
Basic Structure of Tropocollagen
Gelatinization of Collagen
Collagen Biosynthesis
Biosynthesis of Pro-α-Chain
Hydroxylation of Proline and Lysine Residues in Pro-α-Chain
Glycosylation of Certain Hydroxylysine Residues
Procollagen Formation
Transport and Secretion of Procollagen
Procollagen-Collagen Conversion (Cleavage of Pro-peptide)
Fiber Formation and Polymerization
Formation of Cross-Links
Collagen Degradation
Collagenase
Gelatinase
Diseases Related to Collagen Synthesis
Elastic Fibers
Non-collagenous Proteins
Non-collagenous Proteins in Bones and Dentine
Osteocalcin (Bone Gla Protein)
Osteonectin (SPARC, Culture Shock Protein, BM-40)
Bone Sialoprotein (BSP)
Osteopontin (Spp-I, BSP-I)
Bone Sialoprotein-II (BSP-II)
Bone Acidic Glycoprotein-75 (BAG-75)
Dentine Matrix Protein-1 (Dmp-1)
Tetranectin
Thrombospondin
Fibronectin
Vitronectin
Proteoglycan
Large Proteoglycans: Aggrecan and Versican
Small Proteoglycans: Decorin and Biglycan
Hyaluronic Acid (Hyaluronan)
Dentine-Specific Non-collagenous Proteins
Phosphophoryn (Dentine Matrix Protein-1)
Dentine Sialoprotein
Extrinsic Non-collagenous Proteins
Serum Protein
Soluble Factors
References
4. Pulp
Composition of Pulp
Pulp Cells
Odontoblasts
Fibroblasts
Immune Cells
Undifferentiated Cells
Changes in the Distribution of Pulp Cells
Extracellular Matrix of the Dental Pulp
Fibrous Proteins
Ground Substance
Metabolism of the Pulp
Pulp Repair and Related Signaling
References
5. Calcification of Bones and Teeth
Calcified Biological Tissue
Function and Structure of Bone
Chemical Composition of Bone and Bone Tissue Cells
Inorganic Components of Bone
Organic Components of Bone
Cells in Bone Tissue
Osteoblasts
Osteocytes
Osteoclasts
Bone Lining Cells
Growth and Remodeling of Bone
Development and Growth of Bone
Bone Remodeling
Age-Dependent Bone Changes
Local Factors Causing Bone Remodeling
Local Factors Regulating Osteoblast Differentiation
Local Factors Regulating Osteoclast Differentiation
Calcium and Phosphorus Metabolism
Calcium Metabolism
Homeostasis of Plasma Calcium
Calcium Absorption
Factors Affecting Calcium Absorption
Phosphorus Metabolism
Hormones That Affect Calcium and Phosphorus Metabolism
Parathyroid Hormone
Regulation of the Activity of Parathyroid Hormone
Action of Parathyroid Hormone
Calcitonin
1,25-Dihydroxycholecalciferol [1,25(OH)2D3]
Biosynthesis and Activation of Vitamin D3
Action of 1,25-Dihydroxycholecalciferol [1,25(OH)2D3]
Sex Hormones
Growth Hormones
Glucocorticoids
Thyroid Hormone
Factors Affecting Bone Metabolism
Disorders of Calcium and Phosphorus Metabolism
Hyperparathyroidism
Hypoparathyroidism
Mechanism of Calcification
Simple Spontaneous Precipitation Hypothesis
Alkaline Phosphatase Hypothesis
Glycolysis and Alkaline Phosphatase Hypothesis
Seeding Theory
Hole-Zone Theory
Matrix Vesicle-Induced Calcification Theory
Generation of Matrix Vesicles
Mechanism of Matrix Vesicle-Induced Calcification
Current Concepts Regarding the Calcification Mechanism
Growth and Maturation of Apatite Crystals
Characteristics of Calcification in Teeth
References
6. Oral Mucosa and Gingiva
Structure and Function of the Oral Mucosa and Gingiva
Epithelial Structure of Oral Mucosa and Gingiva
Connective Tissue Structure of Oral Mucosa and Gingiva
Function of the Oral Mucosa
Metabolism of Gingiva
Composition of Gingiva
Collagen
Proteoglycan
Non-collagenous Proteins
Lipid
Keratin
Metabolism of the Gingiva
Gingivitis
Matrix Metalloproteinases (MMPs)
Changes in Gingival Epithelium and Connective Tissue
Destruction of Gingival Tissue
References
7. Saliva
Structure and Vascular System of Salivary Glands
Structure of Salivary Glands
Vascular System of Salivary Glands
The Nervous System of the Salivary Gland
Brain Functions Involved in Saliva Secretion (Central Autonomic Network)
Peripheral Autonomic Nervous System of Salivary Glands
Mechanism of Salivary Secretion
Secretion of Inorganic Substances in Primary Saliva
Secretion of Organic Substances in Primary Saliva
Transportation Through Intercellular Junctions
Autonomous Properties of Myoepithelial Cells
Modification of Primary Saliva by the Duct System
Properties of Saliva
Functions of Saliva
Constituents of Saliva
Inorganic Constituents
Hydrogen Ion
Calcium and Phosphate
Bicarbonate
Fluoride
Thiocyanate
Sodium, Potassium, Magnesium, and Chloride
Other Ions
Organic Constituents
α-Amylase
Mucin
Antibacterial Salivary Proteins
Lactoferrin (Red Iron-Binding Protein)
Lactoperoxidase
Lysozyme
Immunoglobulin
sIgA
IgM
IgG
Salivary Proteins Associated with Calcification
Anionic Proline-Rich Proteins
Statherin
Other Salivary Proteins
Histatin
Cystatin
Buffering Capacity of Saliva
Bicarbonate Buffer System
Phosphate Buffer System
Salivary Protein Buffer System
Ammonia Buffer System
Factors Affecting Saliva Composition
Relative Contributions of the Salivary Glands
Salivary Secretion Rate
Nature of Stimuli
References
8. Acquired Enamel Integuments: Pellicle, Plaque, and Calculus
Acquired Pellicle
Formation of Acquired Pellicle
Mechanism of Acquired Pellicle Formation
Function of Acquired Pellicle
Dental Plaque (Oral Biofilm)
Mechanism of Dental Plaque Formation
Adhesion and Colonization of Oral Bacteria on Acquired Pellicle
Formation of Plaque Matrix
Formation of Plaque Matrix Without Food Intake
Formation of Plaque Matrix After Food Intake
Dextran
Mutan
Levan
Intracellular Glycogen-Like Polysaccharides
Sugar As a Cariogenic Factor
Characteristics of Mature Dental Plaque
Dental Plaque Metabolism and Oral Diseases
Sugar Metabolism and Acid Production
Sugar Metabolism
Stephan Curve
Nitrogen Metabolism and Base Production
Decarboxylation Reaction
Composition of Dental Plaque
Chemical Composition of Dental Plaque
Function of Fluoride in Dental Plaque
Effect of Fluoride on Sugar Metabolism of Plaque Bacteria
Plaque Bacteria
Dental Plaque and Periodontal Disease
Control of Dental Plaque
Mechanical Method (Tooth Brushing)
Chemical Method
Stannous Fluoride (SnF2)
Lanthanum Ion (La3+)
Dextranase and Mutanase
Chlorhexidine
Antibiotics
Lectin
Dental Calculus
Mechanism of Dental Caries
Theories of Caries Development
The Chemicoparasitic Theory
The Proteolytic Theory
The Proteolysis-Chelation Theory
The Endogenous Theory
The Glycogen Theory
The Organotropic Theory
The Biophysical Theory
Alternative Sweeteners
Xylose
Palatinose
Sorbitol
Maltitol
Xylitol
Fructooligosaccharides and Coupling Sugars
Stevioside
Monellin
Aspartame
Saccharin
References
9. Functions of Fluoride in the Oral Cavity
Fluoride
Effects of Fluoride on Enamel Remineralization
Inhibition of Caries by Fluoride
Mottled Teeth
Effects of Fluoride on Plaque Bacterial Metabolism
Uses of Fluoride for the Prevention of Dental Caries
References
Index

Citation preview

Byung-Moo Min

Oral Biochemistry

Oral Biochemistry

Byung-Moo Min

Oral Biochemistry

Byung-Moo Min Oral Biochemistry and Program in Cancer and Developmental Biology Seoul National University School of Dentistry Seoul, Korea (Republic of)

ISBN 978-981-99-3595-6 ISBN 978-981-99-3596-3 https://doi.org/10.1007/978-981-99-3596-3

(eBook)

Jointly published with DaehanNarae Publishing, Inc Translation from the Korean language edition: “Oral Biochemistry 2nd ed” by Byung-Moo Min, # DaehanNarae Publishing, Inc. 2020. Published by DaehanNarae Publishing, Inc.. All Rights Reserved. # The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publishers, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publishers nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publishers remain neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

To my teachers and my students

Preface

I am very pleased to publish Oral Biochemistry and hope it will help students understand oral biochemistry, which is the study of the molecular basis of life specific to the oral and maxillofacial areas. Oral Biochemistry covers the basic structures and metabolic processes of the hard and soft tissues that comprise the oral and maxillofacial areas, and how metabolic abnormalities are related to the development of oral diseases. In writing the first edition of Oral Biochemistry, I have balanced the desire to present the latest advances with the need to make oral biochemistry as clear and engaging as possible for students studying this subject for the first time. To this end, I have tried to clarify concepts for students, in addition to introducing new discoveries. Dental education has changed rapidly. In particular, integrated courses have been developed and implemented, and problem-based learning has been introduced, giving students more opportunities to study on their own. Oral Biochemistry is designed to help students study efficiently by themselves. It is expected to be a good textbook and reference book for students. I hope this book will provide students and dental workers with an easy means to access oral biochemistry and will facilitate student education and dental advancements. I especially thank the contributors to this book. Their contributions, thoughtful comments, suggestions, and encouragement have been a great help. I also owe a great debt of gratitude to Yong-Won Choi, President of Daehan Narae Publishing, Inc. His vision for dental books makes working with Daehan Narae Publishing, Inc. truly pleasurable. I also deeply appreciate Springer Nature Singapore Pte Ltd. and the enthusiastic support provided by the production staff of the publishing companies. Finally, I owe a debt of gratitude to my family, including my wife Ok-Joo, Seung-Ki, Hong-Ki, ChaeYoon, Hyoeun, and Seo-Yul. Without their support, comfort, and understanding, this endeavor could never have been undertaken, let alone successfully completed. Seoul, Korea (Republic of)

Byung-Moo Min

vii

Contents

1

Characteristics and Importance of the Oral Cavity . . . . . . . . What Is Oral Biochemistry? . . . . . . . . . . . . . . . . . . . . . . . . . . . Characteristics and Importance of Oral Cavity . . . . . . . . . . . . . . Digestive Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antibacterial Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Buffering Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lubrication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hygienic Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Taste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Control of Water Balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 1 1 1 2 2 2 3 4 4 5 5

2

Enamel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microstructure of Enamel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Unit Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Crystal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enamel Rod (Prism) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inorganic Constituents of Enamel . . . . . . . . . . . . . . . . . . . . . . . Isolation of Enamel, Dentine, and Cementum from Teeth . . . . Chemical Composition of Enamel . . . . . . . . . . . . . . . . . . . . . Major Inorganic Components . . . . . . . . . . . . . . . . . . . . . . . . . . Calcium and Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bicarbonate (CO32-) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bicarbonate and Dental Caries . . . . . . . . . . . . . . . . . . . . . . . . . Fluoride and Dental Caries . . . . . . . . . . . . . . . . . . . . . . . . . . . . Trace Elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Distribution of Fluoride in Tooth and Its Uptake Ratio . . . . . . Fluoride As an Inhibitor of Dental Caries . . . . . . . . . . . . . . . . Fluoride and Mottled Teeth . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of Fluoride on Enamel Remineralization . . . . . . . . . . . Ionic Substitution in Enamel Apatite . . . . . . . . . . . . . . . . . . . . . Comparison of Synthetic Hydroxyapatite and Human Enamel Apatite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Changes in Crystallinity After Heterogeneous Ionic Substitutions in Hydroxyapatite . . . . . . . . . . . . . . . . . . . . . . .

7 7 7 9 9 10 10 12 13 14 15 16 16 16 17 18 19 19 19 19 20 ix

x

Contents

Precursors of Enamel Apatite . . . . . . . . . . . . . . . . . . . . . . . . . . Octacalcium Phosphate [Ca8H2(PO4)6
5H2O] . . . . . . . . . . . . . Amorphous Calcium Phosphate . . . . . . . . . . . . . . . . . . . . . . . Tricalcium Phosphate [Whitlockite, Ca3(PO4)2] . . . . . . . . . . . Dicalcium Phosphate Dihydrate (Brushite, CaHPO4.2H2O) . . . Organic Materials in Enamel . . . . . . . . . . . . . . . . . . . . . . . . . . . Amelogenin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enamelin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ameloblastin (Amelin or Sheathlin) . . . . . . . . . . . . . . . . . . . . Tuftelin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

20 22 22 23 23 23 24 25 27 27 28

3

Dentine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dentine Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemical Composition of Dentine . . . . . . . . . . . . . . . . . . . . . . . Inorganic Components of Dentine . . . . . . . . . . . . . . . . . . . . . Organic Constituents of Dentine . . . . . . . . . . . . . . . . . . . . . . Physicochemical Properties and Biosynthesis of Collagen . . . . . . Physicochemical Properties of Collagen . . . . . . . . . . . . . . . . . Collagen Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Collagen Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diseases Related to Collagen Synthesis . . . . . . . . . . . . . . . . . Elastic Fibers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Non-collagenous Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Non-collagenous Proteins in Bones and Dentine . . . . . . . . . . . Dentine-Specific Non-collagenous Proteins . . . . . . . . . . . . . . Extrinsic Non-collagenous Proteins . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

29 29 30 30 31 32 32 36 41 42 42 44 44 51 52 52

4

Pulp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Composition of Pulp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pulp Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Changes in the Distribution of Pulp Cells . . . . . . . . . . . . . . . . Extracellular Matrix of the Dental Pulp . . . . . . . . . . . . . . . . . Metabolism of the Pulp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pulp Repair and Related Signaling . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

53 53 54 55 57 58 59 60

5

Calcification of Bones and Teeth . . . . . . . . . . . . . . . . . . . . . . Calcified Biological Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . Function and Structure of Bone . . . . . . . . . . . . . . . . . . . . . . . Chemical Composition of Bone and Bone Tissue Cells . . . . . . Growth and Remodeling of Bone . . . . . . . . . . . . . . . . . . . . . Local Factors Causing Bone Remodeling . . . . . . . . . . . . . . . . Calcium and Phosphorus Metabolism . . . . . . . . . . . . . . . . . . . . Calcium Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phosphorus Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hormones That Affect Calcium and Phosphorus Metabolism . . . Parathyroid Hormone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

61 61 62 62 66 67 68 68 70 71 71

Contents

xi

Calcitonin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1,25-Dihydroxycholecalciferol [1,25(OH)2D3] . . . . . . . . . . . . Sex Hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Growth Hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glucocorticoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Thyroid Hormone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Factors Affecting Bone Metabolism . . . . . . . . . . . . . . . . . . . . Disorders of Calcium and Phosphorus Metabolism . . . . . . . . . . . Hyperparathyroidism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hypoparathyroidism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanism of Calcification . . . . . . . . . . . . . . . . . . . . . . . . . . . Simple Spontaneous Precipitation Hypothesis . . . . . . . . . . . . Alkaline Phosphatase Hypothesis . . . . . . . . . . . . . . . . . . . . . Glycolysis and Alkaline Phosphatase Hypothesis . . . . . . . . . . Seeding Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hole-Zone Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Matrix Vesicle–Induced Calcification Theory . . . . . . . . . . . . . Current Concepts Regarding the Calcification Mechanism . . . . Growth and Maturation of Apatite Crystals . . . . . . . . . . . . . . Characteristics of Calcification in Teeth . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

73 74 76 76 77 77 77 77 77 78 78 79 80 80 80 82 82 84 84 85 85

6

Oral Mucosa and Gingiva . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure and Function of the Oral Mucosa and Gingiva . . . . . . . Epithelial Structure of Oral Mucosa and Gingiva . . . . . . . . . . Connective Tissue Structure of Oral Mucosa and Gingiva . . . . Function of the Oral Mucosa . . . . . . . . . . . . . . . . . . . . . . . . . Metabolism of Gingiva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Composition of Gingiva . . . . . . . . . . . . . . . . . . . . . . . . . . . . Metabolism of the Gingiva . . . . . . . . . . . . . . . . . . . . . . . . . . Gingivitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

87 87 87 90 91 91 91 93 94 97

7

Saliva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure and Vascular System of Salivary Glands . . . . . . . . . . . Structure of Salivary Glands . . . . . . . . . . . . . . . . . . . . . . . . . Vascular System of Salivary Glands . . . . . . . . . . . . . . . . . . . The Nervous System of the Salivary Gland . . . . . . . . . . . . . . . . Brain Functions Involved in Saliva Secretion (Central Autonomic Network) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanism of Salivary Secretion . . . . . . . . . . . . . . . . . . . . . . . Secretion of Inorganic Substances in Primary Saliva . . . . . . . . Secretion of Organic Substances in Primary Saliva . . . . . . . . . Transportation Through Intercellular Junctions . . . . . . . . . . . . Autonomous Properties of Myoepithelial Cells . . . . . . . . . . . . Modification of Primary Saliva by the Duct System . . . . . . . . Properties of Saliva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functions of Saliva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

99 99 99 99 100 100 103 103 103 104 104 106 107 107

xii

8

9

Contents

Constituents of Saliva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inorganic Constituents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Organic Constituents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Buffering Capacity of Saliva . . . . . . . . . . . . . . . . . . . . . . . . . . . Bicarbonate Buffer System . . . . . . . . . . . . . . . . . . . . . . . . . . Phosphate Buffer System . . . . . . . . . . . . . . . . . . . . . . . . . . . Salivary Protein Buffer System . . . . . . . . . . . . . . . . . . . . . . . Ammonia Buffer System . . . . . . . . . . . . . . . . . . . . . . . . . . . Factors Affecting Saliva Composition . . . . . . . . . . . . . . . . . . . . Relative Contributions of the Salivary Glands . . . . . . . . . . . . Salivary Secretion Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nature of Stimuli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

108 109 113 120 121 122 123 123 123 123 123 124 124

Acquired Enamel Integuments: Pellicle, Plaque, and Calculus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acquired Pellicle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Formation of Acquired Pellicle . . . . . . . . . . . . . . . . . . . . . . . Mechanism of Acquired Pellicle Formation . . . . . . . . . . . . . . Function of Acquired Pellicle . . . . . . . . . . . . . . . . . . . . . . . . Dental Plaque (Oral Biofilm) . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanism of Dental Plaque Formation . . . . . . . . . . . . . . . . . Formation of Plaque Matrix . . . . . . . . . . . . . . . . . . . . . . . . . Sugar As a Cariogenic Factor . . . . . . . . . . . . . . . . . . . . . . . . Dental Plaque Metabolism and Oral Diseases . . . . . . . . . . . . . Composition of Dental Plaque . . . . . . . . . . . . . . . . . . . . . . . . Control of Dental Plaque . . . . . . . . . . . . . . . . . . . . . . . . . . . Dental Calculus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanism of Dental Caries . . . . . . . . . . . . . . . . . . . . . . . . . . . Theories of Caries Development . . . . . . . . . . . . . . . . . . . . . . The Proteolytic Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alternative Sweeteners . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xylose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Palatinose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sorbitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Maltitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xylitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fructooligosaccharides and Coupling Sugars . . . . . . . . . . . . . Stevioside . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Monellin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aspartame . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Saccharin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

125 125 125 125 126 126 126 128 132 134 141 145 147 148 148 149 149 150 150 150 150 150 151 151 151 152 152 152

Functions of Fluoride in the Oral Cavity . . . . . . . . . . . . . . . . Fluoride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of Fluoride on Enamel Remineralization . . . . . . . . . . . . . Inhibition of Caries by Fluoride . . . . . . . . . . . . . . . . . . . . . . . .

153 153 153 154

Contents

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Mottled Teeth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of Fluoride on Plaque Bacterial Metabolism . . . . . . . . . Uses of Fluoride for the Prevention of Dental Caries . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . .

154 154 155 155

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

Author and Contributors

About the Author Byung-Moo Min is Professor of Oral Biochemistry, Emeritus, in the School of Dentistry at Seoul National University (SNU), where he has been on the faculty since 1986. He received his D.D.S. from SNU School of Dentistry in 1980. He received his M.S. and Ph.D. degrees (1989) from SNU College of Medicine, where he performed research with Seung-Won Kimm. In addition, he conducted research with Dong-Kyun Cheong at SNU School of Dentistry. He then completed a postdoctoral fellowship with No-Hee Park at UCLA School of Dentistry. His research focused on oral carcinogenesis, keratinocyte biology, and peptidomics for tissue engineering. He served as the Chair of the Department of Oral Biochemistry (1991–2009), the Program in Maxillofacial Regenerative Medicine (2003–2009), and the Program in Cancer and Developmental Biology (2009–2013). He also showed outstanding leadership as a Director of the SNU Dental Research Institute (2011–2013) and the Brain Korea 21 Craniomaxillofacial Life Sciences Research Program (2008–2013), and as the Department Chair of Life Sciences in Dental Medicine (2012–2015). He was a member of the SNU Senate from 2011 to 2019 and served as the Senate Vice-Chair from 2014 to 2015. He has been a lifetime member of the Korean Academy of Science and Technology since 2006. He served the International Association for Dental Research (IADR), including as the Councilor (2007–2012), and represented the Asia/Pacific Region as the Regional Board Member of the IADR (2012–2015). He has received numerous awards, including the Hatton Award in the IADR Korean Division (1989), an Award for Outstanding Professor of the Year from SNU (2003), an award for outstanding achievement at the 8th World Congress on Advances in Oncology and 6th International Symposium on Molecular Medicine (Greece, 2003), the Korean Dental Association Award of Merit (2005), an Outstanding Research Award from SNU (2008), and Korean Dental Association Grand Prize (Academic Award, 2022). Professor Min was awarded the Order of Science and Technology Merit (2018) and the Order of Service Merit (2020) from the government of the Republic of Korea.

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Contributors Je Yoel Cho Department of Veterinary Biochemistry, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea Je-Yong Choi Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Daegu, Republic of Korea Won-Yoon Chung Department of Oral Biology, College of Dentistry, Yonsei University, Seoul, Republic of Korea Young-Joo Jang Department of Nanobiomedical Science, College of Dentistry, Dankook University, Cheonan, Republic of Korea Jung Sook Kang Department of Oral Biochemistry and Molecular Biology, College of Dentistry (past), Pusan National University, Pusan, Republic of Korea Jeong Hee Kim Department of Oral Biochemistry, College of Dentistry, Kyung Hee University, Seoul, Republic of Korea Joong-Ki Kook Department of Oral Biochemistry, College of Dentistry, Chosun University, Gwangju, Republic of Korea Seon-Yle Ko Department of Oral Biochemistry, College of Dentistry, Dankook University, Cheonan, Republic of Korea Gene Lee Department of Oral Biochemistry, School of Dentistry, Seoul National University, Seoul, Republic of Korea Tae-Hoon Lee Department of Oral Biochemistry, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea Byung-Ju Park Department of Oral Biochemistry, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea Kwang-Kyun Park Department of Oral Biology, College of Dentistry (past), Yonsei University, Seoul, Republic of Korea Soon-Ho Park Department of Biochemistry and Molecular Biology, College of Dentistry, Gangneung-Wonju National University, Gangneung, Republic of Korea Fyo-Jae Rhie Department of Oral Physiology, School of Dentistry (past), Jeonbuk National University, Jeonju, Republic of Korea Hyun-Mo Ryoo Department of Pharmacology and Dental Therapeutics, School of Dentistry, Seoul National University, Seoul, Republic of Korea Yong-Ouk You Department of Oral Biochemistry, College of Dentistry, Wonkwang University, Iksan, Republic of Korea

Author and Contributors

1

Characteristics and Importance of the Oral Cavity

What Is Oral Biochemistry? Biochemistry is the study of the molecular basis of life, and oral biochemistry is the application of the same concept specifically to the oral and maxillofacial areas. Human tissues can be largely classified as hard or soft, and the same principle applies to the oral and maxillofacial areas: soft tissues include the skin, cheek, oral mucosa, tongue, and gingiva, and hard tissues comprise the bones and teeth. The bones in these areas include the maxilla, mandible, and hard palate, whereas teeth can be subclassified into three different entities: enamel, dentine, and cementum. Oral biochemistry covers the basic structures and metabolic processes of the hard and soft tissues that comprise the oral and maxillofacial areas and how metabolic abnormalities are related to the development of oral diseases.

Characteristics and Importance of Oral Cavity Understanding the oral and maxillofacial areas, especially the oral cavity, enables students to learn dentistry. Therefore, let us consider the characteristics and importance of the oral cavity. One of the hallmarks of the oral cavity is that it is an open organ that can be observed directly (Fig. 1.1). Therefore, in contrast to other organs, the oral cavity can be visually examined, making

diagnoses easier and more accurate. Moreover, disorders in other organs are often expressed in the oral cavity (Fig. 1.2). Inversely, disorders in the oral cavity may cause lesions in other organs and tissues. Accordingly, by carefully examining the oral cavity, clinicians can predict disorders in other parts of the body; this is a major benefit of the availability of the oral cavity for visual examination. In addition, the oral cavity is the only organ that contains all of the types of hard tissues in the human body, i.e., bones and teeth (Fig. 1.3). A unique hallmark of teeth is their remarkably constant chemical composition (Lefevre and Hodge, 1937; Lazzari, 1976). Bone tissue is used to store minerals, including calcium and phosphate, and these ions are transported actively in response to the body’s needs. By contrast, calcium and phosphate in teeth are not mobilized in this manner. In other words, bone tissues are in a dynamic state, whereas teeth are in a static state. The oral cavity contains saliva, which serves unique and important functions. Let us now briefly examine the role of saliva.

Digestive Function Saliva contains α-amylase, a carbohydrase that catalyzes the hydrolysis of internal α-1,4 glycosidic linkages of carbohydrates. Thus, α-amylase acts as an endoamylase to break down carbohydrates in food into maltotriose, maltose, or glucose. Consequently, even foods that do not

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contain monosaccharides and disaccharides can cause dental caries as a result of hydrolysis by α-amylase. In addition, food debris can act as a substrate for α-amylase, contributing to the development of dental caries.

Antibacterial Function Saliva contains lysozyme, lactoferrin, lactoperoxidase, and secretory IgA (sIgA), all of which have antibacterial functions. These salivary proteins play significant roles in protecting the oral cavity, an open organ, against oral bacteria. Lysozyme was first identified in nasal secretions by Fleming in 1922 and was soon detected in saliva, tears, gastric juice, and sputum. Lysozyme is an enzyme that selectively hydrolyzes β-1,4 glycosidic linkages between N-acetylmuramate and N-acetylglucosamine in peptidoglycan (Fig. 1.4), the basic structural component of most bacterial cell walls (specifically, those of bacteria that lack a capsule) (Fig. 1.5). Thus, lysozyme digests certain bacteria that enter the oral cavity, thereby helping to maintain the balance among oral bacteria. Lactoferrin, an iron-binding protein synthesized in the acinar cells of salivary glands and neutrophils, is secreted into the oral cavity in the apoprotein form. Because the enzyme requires ferric ion (Fe3+) and bicarbonate as cofactors, apolactoferrin in the oral cavity binds and thereby depletes Fe3+, which is required for bacterial growth. Thus, lactoferrin provides a form of nutritional immunity. Lactoperoxidase was first discovered in milk and was subsequently detected in the acinar cells of the parotid and submandibular glands, saliva, tears and lacrimal glands, acquired pellicles, and dental plaque. Because lactoperoxidase requires a thiocyanate ion (SCN–) as a cofactor, it is referred to as the lactoperoxidase–thiocyanate system. This system exerts its antibacterial effect through a reaction with H2O2 produced by oral bacteria. The reaction products include toxic materials such as hypothiocyanite (OSCN–), hypothiocyanous acid (HOSCN), and

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Characteristics and Importance of the Oral Cavity

thiocyanogen (SCN)2, which inhibit bacterial activity. Secretory IgA (sIgA) exerts antibacterial activity by binding to antigens associated with bacterial attachment, thereby preventing bacterial colonization. Lysozyme, lactoferrin, lactoperoxidase, and secretory IgA will be discussed further in Chapter 7 (Saliva).

Buffering Action Buffering action can be defined as the ability to prevent sudden pH changes when acids or bases are added. In the oral cavity, reductions in pH are caused by acids in food and drink, or by organic acids produced metabolically by plaque bacteria. A decrease in the pH of the oral cavity contributes to dental caries by promoting the dissolution of tooth enamel. However, saliva contains bicarbonate and phosphate buffer systems that function above pH 6, as well as a salivary protein buffer system that functions below pH 5. Buffering prevents dramatic pH reduction in the oral cavity and thus serves not only to prevent dental caries but also to protect soft tissues against chemical damage.

Lubrication The surface of the oral cavity, including teeth and soft tissues, has a total area of approximately 200 cm2 and is covered by a salivary layer at least 100 μm thick. Consequently, as foods are chewed and swallowed, saliva acts as a lubricant to protect the oral mucosa by decreasing friction. A major salivary lubricant is a large (>1 MDa) glycoprotein called MG-1 (high molecular weight mucin-type glycoprotein 1). In addition, MG-2 (low molecular weight mucin-type glycoprotein 2), with a molecular weight of 200–250 kDa, contributes to salivary viscosity. Via the action of these glycoproteins, salivary coverage of the epithelia of the gastrointestinal tract provides mechanical and chemical protection of epithelial tissues.

Hygienic Action

Fig. 1.1 Normal and diseased human oral tissues. (a) Normal human gingival tissue. (b) Carious tooth. (c) Inflamed human gingival tissue affected by periodontal

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disease. (d) Protrusion of a portion of the gingival epithelium. (Courtesy of Dr. B. S. Park)

Stimulated saliva has a water content of 99.5%, whereas unstimulated saliva is 94% water. In the

resting state, approximately 0.3 mL of saliva is secreted per minute, whereas in the stimulated state, 2.5–5.0 mL of saliva is secreted per minute, a dramatic increase. The total amount of saliva secreted per day is ~700–800 mL. As discussed, saliva plays a significant role in removing bacteria and bacterial toxins. In an adult, 1.0–2.5 g of

Fig. 1.2 Human palate infected by varicella zoster virus. Chickenpox or varicella is a highly infectious disease, usually occurring in children, that is associated with blisters and pustules on the skin and mucosa, along with oral lesions

Fig. 1.3 A dental panoramic radiograph showing the highest radiographic density of any part of the body. The oral and maxillofacial regions contain many structures, which exhibit various degrees of calcification. (Courtesy of Dr. B. S. Park)

Hygienic Action

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Characteristics and Importance of the Oral Cavity

Except for the teeth, the oral cavity is covered by soft tissues. Due to their high turnover rate, epithelial cells are constantly removed. Exfoliated epithelium and food debris are also removed by saliva, thus maintaining the hygiene of the oral cavity.

Taste

Fig. 1.4 Lysozyme hydrolyzes the β-1,4 glycosidic bond between N-acetylmuramate (NAM) and N-acetylglucosamine (NAG) in the peptidoglycan structure of the bacterial membrane

bacteria are removed by saliva (i.e., swallowed) each day.

Parotid saliva contains gustin, a zinc-binding protein with a molecular weight of 37 kDa, which plays a role as a taste sensor. Zinc depletion decreases the amount of functional gustin and thereby reduces the sensation of taste, sometimes resulting in anorexia. Conversely, patients who lose the sense of taste or develop pathological structures on the taste buds have reduced levels of gustin in parotid saliva. In such patients, the addition of zinc increases the amount of functional gustin and restores the sensation of taste.

Repair

Fig. 1.5 Diagram showing the structure of peptidoglycan in Staphylococcus aureus. Yellow balls indicate sugars, red triangles indicate tetrapeptides, and blue balls indicate pentaglycine bridges

Early dental caries lesions are caused by acid attack, which dissolves parts of the crystals constituting the tooth. If the acid attack continues, the early lesions are converted into clinically detectable lesions. Precipitation of salivary layers with abundant calcium and phosphate ions is essential for effective remineralization of early dental caries lesions (Cole and Eastoe, 1988). In addition, fluoride keeps the surface of the teeth intact by promoting remineralization. However, saliva contains proteins such as anionic prolinerich proteins, a group of saliva-specific phosphoproteins that inhibit crystal growth of calcium phosphate salts, as well as statherin, a 43-residue tyrosine-rich phosphoprotein that inhibits both spontaneous precipitation and crystal growth of calcium phosphate salts. Therefore, clinically detectable caries lesions into which proline-rich proteins and statherin have penetrated cannot be remineralized.

References

5

Control of Water Balance

References

Our body maintains internal water levels under dehydrating conditions, including consumption of alcohol, by reducing the rate of salivary secretion. Under normal conditions, this causes the individual to drink water by stimulating the sense of thirst, ultimately restoring water balance.

Cole, A.S., Eastoe, J.E.: Biochemistry and Oral Biology, 2nd edn. Wright, Bristol (1988) Lefevre, M.L., Hodge, H.C.: Chemical analysis of tooth samples composed of enamel, dentine and cementum. J. Dent. Res. 16, 279–287 (1937) Lazzari, E.P.: Dental Biochemistry, 2nd edn. Lea & Febiger, Philadelphia (1976)

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Enamel

The chief complaints of patients visiting dental hospitals or private dental clinics are in decreasing order of frequency, dental caries, periodontal disease, and malocclusion. However, the proportion of patients with malformations in the oral and maxillofacial region is expected to increase over time. The primary cause of dental caries and periodontal disease is dental plaque, whereas malocclusion and malformation are primarily the result of genetic factors. Given this background, we will discuss dental caries first. Dental caries can be defined as the local and gradual demineralization of teeth by organic acids, including lactic, acetic, propionic, butyric, and formic acids, which are metabolic products of plaque bacteria. Contact with organic acids decreases the pH of tooth surfaces below 5.5–5.6, eventually causing local demineralization. This demineralization ceases as soon as the pH increases above pH 5.5–5.6 due to various phenomena including the buffering action of saliva. However, demineralization will continue as soon as the pH again decreases below the critical value, ultimately resulting in dental caries. The outer enamel surface of the teeth is always exposed to acid attack. Early dental caries lesions occur within 20 μm of the enamel surface, and continuous acid attack expands the lesion, leading to clinically detectable dental caries. However, the integrity of the enamel surface can be maintained if the early lesion is remineralized by other factors. Thus, the chemical composition of teeth, especially the enamel at the outermost part

of the crown and the outer enamel surface, makes a major contribution to the development and progression of dental caries. To understand the development and prevention of dental caries, it is necessary to be familiar with tooth structure. Accordingly, we will look at the microstructure of enamel, which is closely associated to the development of dental caries. In addition, by comparing the microstructure and chemical composition of enamel with those of dentine, we will obtain deeper insight into dental caries.

Microstructure of Enamel The unit cell is the smallest spatial unit (or building block) that constitutes enamel apatite, which grows along three axes and forms crystals. Tubules are formed by arranging crystals in the direction of the major axis, and enamel rods or prisms are formed from assemblages of tubules. Enamel rods formed in this way constitute enamel by orienting their round heads in the direction of the occlusal surface of the teeth.

Unit Cell The unit cell, the smallest spatial unit of apatite, has a parallelepiped shape containing 10Ca2+, 6PO43-, and 2OH- (Fig. 2.1). Thus, the

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2 Enamel

Fig. 2.1 Structure of a unit cell. (a) Side view of a unit cell, showing the spatial arrangement of the ions constituting the unit cell. (b) Top view of a unit cell.

White balls represent oxygen atoms; blue balls represent hydrogen atoms; red balls represent calcium atoms; and yellow balls represent phosphorus atoms

composition can be expressed by the formula Ca10(PO4)6(OH)2. A unit cell has three axes: two horizontal axes (a1 and a2) and a vertical axis (c). In apatite, the angle between the a1- and a2-axes is 120°; the dimensions of the a1- and a2-axes are 9.42 Å; and the dimension of the c-axis is 6.88 Å (Fig. 2.2). The structure of the unit cell can be understood by considering the spatial arrangement of the 10Ca2+, 6PO43-, and 2OH- ions: the hydroxyl ions are located at the 1/4 and 3/4 positions of the c-axis (where the bottom is defined as 0, and the ceiling is set as 1) (Fig. 2.3). Around each hydroxyl ion, three Ca2+ ions are arranged in an inverse triangular shape. That is, the Ca2+ ions near the c-axis are stacked one on top of the other as shown in Fig. 2.4. Each of the remaining four Ca2+ ions located away from the c-axis is located at 1/3 and 2/3 positions of a diagonal line (equivalent to 0 and 1/2 positions of the c-axis) formed by connecting each endpoint of the a1- and a2-axes (Fig. 2.3). Six PO43- ions are located in the space of the parallelepiped-shaped unit cell, i.e., the volume

not occupied by the 10Ca2+ and 2OH- ions. In other words, the PO43- ions, which occupy most of the space, form the framework of apatite. Examination of the atomic arrangement of PO43demonstrates that each phosphorus atom is located at the center of a regular tetrahedron, and the four oxygen atoms are located at the apices. One oxygen atom of PO43- is connected to a calcium ion.

Fig. 2.2 Three axes of a unit cell and their dimensions

Fig. 2.3 Diagram showing the spatial arrangement of ions constituting the unit cell. Considering the bottom of the c-axis as 0 and its ceiling as 1, two hydroxyl ions are located at 1/4 and 3/4 of the c-axis when it is divided into four parts. Three calcium ions are located near the hydroxyl ions. Of the ten calcium ions, the remaining four calcium ions located away from the c-axis are located at the 1/3 and 2/3 positions of a diagonal line (equivalent to 0 and 1/2 positions of the c-axis) formed by connecting each endpoint of the a1- and a2-axes. Blue circles represent calcium ions

Microstructure of Enamel

Fig. 2.4 Triangles of Ca2+ ions as they would appear superimposed when viewed along the c-axis of the crystal lattice. Three calcium ions are in the vicinity of the hydroxyl ions located at the 1/4 and 3/4 positions of the c-axis. The calcium ions are arranged in an inverted triangle and are stacked when viewed along the c-axis of a unit cell. The red dot in the center represents the c-axis

Crystal These small unit cells form a crystal by growing along three different axes (a1, a2, and c). As noted above, the unit cell is the smallest spatial unit that constitutes the structure of apatite, but a crystal is the real structural entity that constitutes the enamel. A crystal, which consists of numerous unit cells that have grown along the a1-, a2-, and c-axes, has a ribbonlike morphology in the longitudinal section and a hexagonal pattern in the cross section. Overall, a crystal has a pencil-like shape (Fig. 2.5). The primary apatite crystal that is formed during the formation of hard tissues, Fig. 2.5 Diagram of an ideal crystal of hydroxyapatite, showing the principal or c-axis and the three equal a-axes normal to it

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including enamel, is called the primary crystal or seed crystal and has long, ribbonlike morphology. The crystal matures as the enamel matures: its width and thickness increase, with no change in length. In addition, as the enamel matures, some organic constituents and water are lost and replaced by inorganic constituents. An enamel crystal is 30–40 nm (or at least 15–25 nm) wide and 120–210 nm long; it is thicker and longer than the crystals in dentine, cementum, and bones (Table 2.1) and consequently has a 200-fold greater volume than the crystals in the other materials. This large volume contributes to the low acid solubility of enamel: given a constant volume, it takes more crystals of a smaller size to fill the space, increasing the total surface area and making the crystals more vulnerable to acid attack. Consequently, dentine is more vulnerable to acid attack than enamel.

Enamel Rod (Prism) Crystals arranged along the major axis constitute tubules, which in turn make up enamel rods or prisms. Figure 2.6 shows a schematic diagram of an enamel rod in cross and longitudinal sections. A tubule is formed by arranging crystals along the major axis in various patterns. That is, arrangement other than the long axis of the crystals in a tubule occurs in various ways. The tubules formed in this way gather to form an enamel rod. In the case of enamel, the diameter of a tubule is approximately 350–1000 Å, and the tubule wall is 48 Å thick. The sheath of enamel rods has a high concentration of noncalcified organic materials (Fig. 2.6). The space between each crystal in an enamel rod is filled with proteins, lipids, and water; the intercrystalline space consists of a protein–lipid matrix filled with water. Even though enamel is the densest tissue in the human body, with a density of 2.92, 14% of its volume consists of this matrix, which acts as an aqueous channel for the passage of acids during an acid attack. When acids formed in dental plaque get into this passage and make contact with the crystal surface, followed by a reduction of pH below the critical

10 Table 2.1

2 Enamel Width, length, and relative volume of the crystals that constitute tooth and bone tissue

Calcified tissues Enamel Dentine, cementum, bone

Width 30–40 nm (15–25 nm) 2–3.5 nm

value (pH 5.5–5.6), crystals are solubilized and dental caries occurs. Inspection of the cross section of an enamel rod reveals a keyhole-shaped outline (Fig. 2.7a, b). The head-to-tail dimension of an enamel rod is 9 μm, whereas the diameter of the head is 5 μm. The head is directed toward the occlusal surface of the tooth, and the tail is oriented toward the cervical region (Fig. 2.7a). This arrangement provides the enamel rod with a high degree of structural stability, making it durable with respect to external forces. Figure 2.7c illustrates the three-dimensional structure of an enamel rod; dots or oblique lines signify crystals. Because crystals at the head portion of enamel rods run parallel to the major axis, crystals are shown as dots in the cross-section. However, closer to the tail portion, crystals run in a diagonal direction relative to the major axis; accordingly, these crystals are shown as facets in the cross-section (Fig. 2.7c).

Length 120–210 nm

Relative volume 200

20–30 nm

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Inorganic Constituents of Enamel Historical records about the constituents of hard tissues date back to the fifth century BC, indicating that hard tissues have been of major concern throughout human history. In the late nineteenth century, the basic chemical composition of human teeth was determined, and further details were obtained in the early twentieth century. In particular, the analysis of the chemical composition of human teeth performed by Lefevre and Hodge (1937) demonstrated that in comparison with permanent teeth, deciduous teeth have higher water content, lower inorganic constituents, and similar carbonate content. Also, even though the water content of sound teeth is lower than that of carious teeth, they both have similar chemical compositions because the space in the latter is displaced by water. In addition, the chemical constitution of teeth does not differ by age or sex. Finally, Lefevre and Hodge demonstrated that the chemical composition of teeth is remarkably constant, a hallmark of this tissue. As noted previously, bone tissues act as reservoirs of calcium and phosphate, which are actively transported to meet the demand of the body. On the other hand, the chemical composition of teeth is constant, and the calcium and phosphate they contain are not actively transported. In other words, the chemical composition of bones is dynamic, whereas that of teeth is static.

Isolation of Enamel, Dentine, and Cementum from Teeth Fig. 2.6 Diagram showing simultaneous horizontal and vertical sections of an enamel rod, showing the spatial arrangement and orientation of the apatite crystallites. (Source: E. P. Lazzari, Handbook of Experimental Aspects of Oral Biochemistry, (CRC Press, 1983), p. 163)

The chemical composition of teeth, especially that of the outer enamel surface, has a major effect on the development and progression of dental caries. Because teeth are composed of three

Inorganic Constituents of Enamel

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Fig. 2.7 Diagrams showing cross sections and the threedimensional structure of enamel rods. (a and b) Schematic diagrams showing cross-sections of enamel rods, the shapes of the enamel rods, and the orientations of the

crystallites within them. (c) Three-dimensional structure of an enamel rod. The dots or oblique lines indicate crystallites and show their arrangement within the enamel rod

different tissues, enamel, dentine, and cementum must be isolated to accurately determine the chemical composition of each material. Currently, the three tissues can be isolated from teeth by (a) mechanical method, (b) flotation, or (c) chemical method.

2.14, respectively, and the two tissues neighbor each other, with the dentinoenamel junction as a boundary. Consequently, the application of a large external force near the dentinoenamel junction using a blunt dental chisel can separate the enamel portion. Although this is a primitive method, it is one of the few techniques that can separate enamel from dentine without major drawbacks.

Mechanical Method Mechanical isolation involves the separation of enamel and dentine using tools such as a blunt dental chisel, diamond saw, grinding wheel, and cutting disc. For example, when preparing a grinding specimen, one can use these tools to prepare a thin tissue fragment. However, this method has the drawback that pyrolysis may occur during the slicing of high-density tissues such as enamel and dentine. Furthermore, additional contamination may occur during the process of cooling with water. Another mechanical method is to utilize the density difference between enamel and dentine. The average densities of enamel and dentine are 2.92 and

Flotation Technique The mechanical method is very laborious and tedious. Therefore, an easier method was invented to separate enamel, dentine, and cementum. Manly and Hodge (1939) developed a method called the flotation technique, which can separate the tissues by exploiting the differences in their densities. The average densities of enamel, dentine, and cementum are 2.92, 2.14, and 2.03, respectively. Briefly, the originally reported flotation technique worked as follows: Manly and Hodge

12

separated crown and root after completely removing the attached deposits on the surface of teeth and pulp. The crown portion was used to obtain enamel and dentine, whereas the root portion was utilized to obtain dentine and cementum. This method involves pulverization of the crown or root in micropulverizer, followed by separation of enamel, dentine, and cementum by the addition of solvents with appropriate specific gravities. In place of a micropulverizer, mortar, and pestle can be also used to pulverize the crown and root. Even though the density of enamel is relatively constant, 2.92 ± 0.1, the density of dentine (average, 2.14) varies depending on its structure. Peritubular dentine has the highest density (2.40) among dentines. When separating enamel and dentine from the crown, Manly and Hodge used a mixed bromoform–acetone solution with a specific gravity of 2.7. The specific gravity of bromoform is 2.902, whereas that of acetone is 0.788; therefore, it is possible to produce a solution with the desired specific gravity by mixing the two solvents in a particular ratio. When pulverized enamel and dentine powders are added to a solution with a specific gravity of 2.7, the enamel powder (with a density of 2.92) will precipitate, whereas the dentinal powder (with a density of 2.4 or lower) will float on the upper portion of the solution (Fig. 2.8). Centrifugation is used to accelerate this process, which is thus also referred to as the “centrifugal flotation technique.” On the other hand, to separate dentine

2 Enamel

and cementum from the root portion, a bromoform–acetone solution with specific gravity between 2.14 (dentine) and 2.03 (cementum) should be used. This technique can separate enamel and dentine to 99% or greater purity. On the other hand, a disadvantage of this method is the discoloration of the sample due to the use of an organic solution (bromoform–acetone).

Chemical Method Just as teeth are dissolved in organic acids, causing dental caries, all kinds of calcified tissues are etched when they come in contact with an acid. The chemical method of isolation uses acids to separate enamel and dentine. The degree of calcified tissue etching is proportional to the acid strength and time of exposure; consequently, it is possible to separate enamel from dentine by carefully manipulating the conditions. The resultant enamel and dentine will either be dissolved in the acid or remain in a purely residual state. For instance, when attempting to chemically separate enamel and dentine from the tooth, we must obtain the crown portion from the extracted tooth with the pulp removed and then block the dentine on the pulpal side so that it does not come in contact with acids. Exposure of the sample to acids will then cause the dissolution of enamel in the remaining dentinal portion. This method yields the pure residual portion of dentine. As can be seen from the morphology and anatomical structure of teeth, the thickness of enamel and dentine is not constant; consequently, this method requires a high level of manipulation and skill. Using the methods described above, it is possible to obtain pure enamel, dentine, and cementum from a tooth.

Chemical Composition of Enamel

Fig. 2.8 Diagram of Manly and Hodge’s method of separating enamel and dentine

Two types of important biomolecules that the human body cannot synthesize are vitamins and minerals. We must obtain all minerals from external sources such as food and drink. Individual people eat and drink different things, resulting in large inter-individual differences in the composition of minerals consumed. The chemical

Major Inorganic Components Table 2.2

13

Composition of mature human enamel, dentine, and bones (% by weight)

Chemical composition Total inorganic matter Ash (700°C) Carbonate Total organic matter Total proteinaceous material Low molecular weight material Enamelins, etc. Citrate Lactate Carbohydrate Fatty acid Total water Free water, lost at 100°C, in vacuo Bound water, lost between 300°C and 400°C

Enamel

Dentine 70–75

Bone 65

0.6

20

25

4.0

10

10

95.0 91.7 2.9 0.35 0.06 0.29 0.02 0.01 0.0016 0.01 2.2 0.83

Source: S. Cole and J. E. Eastoe, Biochemistry and Oral Biology, 2nd ed. (Wright, 1988), Table 32.1

composition of teeth is significantly affected by factors such as diet, geological location of the residence, location of teeth in the oral cavity, age, status of the teeth, and medical history. Therefore, the chemical composition of teeth must be given as the average value or range. Table 2.2 refers to the chemical composition of mature human enamel and compared it with that of dentine and bones. The total mineral content of enamel, dentine, and bone tissue is 95%, 70–75%, and 65%, respectively, whereas the total amount of organic materials is 0.6%, 20%, and 25% and the amount of water is 4%, 10%, and 10%. Therefore, mature human enamel is mainly composed of inorganic materials, with minor organic components and water content. When the organic components of enamel are examined, more than half (about 58%) consist of protein, mostly enamelin (~48%). Otherwise, the organic fraction includes citrates, organic acids such as lactates, carbohydrates, and fatty acids. A primary difference between the chemical compositions of mature human enamel and those of dentine and bone tissue is that enamel contains very little water. Water can be subclassified as free or bound. “Free” water constitutes approximately 55% of the total water content and is located in the intercrystalline spaces. On the other hand, “bound” water constitutes approximately 20% of the total and

exists in association with enamel crystals. “Free” water can be removed by boiling up to 100°C, whereas the “bound” water must be brought to 300–400°C in order to evaporate. “Bound” water is not present in dentine or bone. The composition of mature human enamel can be expressed as volume %: 86% inorganic materials, 2% organic materials, and 12% water. Thus, 14% of the total volume, representing the combination of organic materials and water, can act as a bypass channel. Enamel crystals are surrounded by a water-filled protein–lipid matrix called the aqueous channel. When organic acids penetrate through this channel and dissolve the crystals, dental caries occurs. Also, the dissolution of crystals causes the replacement of the regions with water, causing a carious tooth to contain a slightly higher amount of water.

Major Inorganic Components The smallest spatial unit that comprises the apatite structure in teeth and bone tissues is the unit cell, which can be expressed by the chemical formula Ca10(PO4)6(OH)2. Thus, the major inorganic components that constitute all types of calcified tissues, including enamel, are calcium and phosphate. In addition, bicarbonate (CO32-), an intrinsic component present in all types of

14

2 Enamel

Table 2.3

Concentration of major inorganic components in human enamel

Component Ca P CO3 Na Mg Cl K

Table 2.4

Ca P Ca/P

Concentration range in percentage (% by dry weight) 33.6–39.4 16.1–18.0 2.7–5.0 0.25–0.90 0.25–0.90 0.19–0.30 0.05–0.30

Comparison of calcium, phosphorus, and Ca/P ratio in sound and carious enamel

Sound enamel (% by dry weight) 36.75 17.4 2.09

Carious enamel (% by dry weight) 35.95 17.01 2.08

human calcified tissues from development onward, is also found in apatite. Therefore, calcium, phosphate, and bicarbonate are the major inorganic components of teeth.

Calcium and Phosphate Table 2.3 displays the composition of sodium, magnesium, chloride, and potassium, which are present in large quantities within the trace elements as well as major inorganic components (including calcium, phosphate, and bicarbonate) in human enamel. The proportions of calcium, phosphate, and bicarbonate are 33.6–39.4% (average, 36.75%), 16.1–18% (17.4%), and 2.7–5% (4%). Table 2.4 provides a comparison of calcium and phosphate contents and Ca/P ratios in sound enamel, carious enamel, and dentine. In carious enamel, the amounts of calcium and phosphate are slightly smaller than in sound enamel but still much higher than in dentine. The mineral contents of calcified tissues can be analyzed by the electron microprobe method. This technique enables scientists to compare changes in amount and density over different portions of enamel in the premolar. This analysis revealed that as one gets closer to the surface enamel from the dentinoenamel junction, the

Sound and carious dentine (% by dry weight) 28.2 13.5 2.05

amount and density of calcium and phosphate increase (Fig. 2.9). In other words, among all types of enamels, surface enamel is the most calcified. The Ca/P ratio, an index of the degree of calcification, can be calculated by molar basis or weight basis. For instance, when calculating the Ca/P ratio of pure hydroxyapatite using a molar basis, the result is 10/6, or 1.67. On a weight basis, the calculation is 10 × 40 (atomic weight of calcium ion) divided by 6 × 31 (atomic weight of phosphorus ion), yielding a result of 2.15. Tables 2.4 and 2.5 display the Ca/P ratios of enamel, sound and carious dentine, and hydroxyapatite precursors on a weight basis. The Ca/P ratios of sound enamel, carious enamel, and dentine are 2.09, 2.08, and 2.05, respectively (Table 2.4). Thus, dentine is less dense than enamel because it is less calcified. Additionally, this indicates that neither enamel nor dentine is composed of pure hydroxyapatite, which has a Ca/P ratio of 2.15. The Ca/P ratios of hydrated tricalcium phosphate [Ca3(PO4)2
H2O] and octacalcium phosphate [Ca8H2(PO4)6
5H2O] are 1.94 and 1.72, respectively (Table 2.5). Therefore, Winand proposed that a chemical formula of apatitic calcium phosphate can be written as Ca10-xHx(PO4)6(OH)2-x (x:0 ~ 2). x = 2 corresponds to octacalcium

Major Inorganic Components

15

Fig. 2.9 Distribution of calcium (a) and phosphorus (b) contents (% by weight), and contour map of density (c) in the same section of adult premolar enamel

Table 2.5

Comparison of Ca/P ratios of hydroxyapatite, its precursors, sound enamel, and dentine

Hydroxyapatite, its precursors, sound enamel, and dentine Hydroxyapatite Sound enamel Sound dentine Hydrated tricalcium phosphate Octacalcium phosphate

phosphate, x = 1 to hydrated tricalcium phosphate, and x = 0 to hydroxyapatite. Tricalcium phosphate and octacalcium phosphate are both precursors of hydroxyapatite. Therefore, hydroxyapatite in calcified tissues originates from the conversion of precursor molecules. During this process, impurity ions from the oral cavity are incorporated into the enamel, causing it to contain various trace elements. Next, let us consider the reasons for changes in the Ca/P ratio in calcified tissues, including enamel. First, take the case in which the calcium or phosphate ion in hydroxyapatite is replaced by heterogeneous ionic substitution. Calcium can be replaced by other divalent or monovalent cations, whereas phosphate ions can be replaced by bicarbonate (CO32-), HPO42-, citric acid (C6H5O73-), vanadate (VO43-), or pyrophosphate (P2O74-), causing changes in the Ca/P ratios. In particular, when trivalent phosphate anions are replaced by divalent anions, including CO32- or HPO42-, less calcium ions are required to maintain an electroneutral balance, thus altering the Ca/P ratio. Second, calcium ions can be adsorbed

Ca/P ratio 2.15 2.09 2.04–2.05 1.94 1.72

onto the crystal surface. Third, calcium-deficient apatite can develop during enamel development, and calcium ions are incorporated into this material to achieve stoichiometric balance. Fourth, precursors of hydroxyapatite can be present in enamel. Together, all of these factors can change the C/P ratio.

Bicarbonate (CO32-) The bicarbonate content within enamel ranges from 2.7% to 5% (% dry wt), with an average of 4%. In enamel, bicarbonate content varies depending on the location within the enamel: 2–3% within 20–50 μm of surface enamel and 5% within body enamel. During an acid attack, bicarbonate can be easily lost, causing its content in surface enamel to decrease over the course of aging; however, the bicarbonate content in body enamel remains stable. From development onward, bicarbonate is an intrinsic component of the apatite structure of enamel. Trace elements present in the enamel

16

are impurity ions incorporated during the conversion of hydroxyapatite precursors into hydroxyapatite. By contrast, bicarbonate is an intrinsic component present in all human calcified tissues, including enamel. For this reason, MaConnell claimed that it is accurate to call hydroxyapatite either carbonate hydroxyapatite or dahllite, with a chemical formula of 3Ca3(PO4,CO3OH)2
Ca (OH)2. Most bicarbonate in enamel apatite is substituted either at the location of the phosphate ions or in their vicinity, and a small amount is substituted with hydroxyl ions. The average amount of bicarbonate in enamel is approximately 4%, equivalent to the substitution of 1/7 of phosphate ions with bicarbonate ions. However, because the phosphate anion is trivalent and the bicarbonate ion is divalent, changes in ionic composition are necessary to maintain electroneutrality. Specifically, calcium ions must be either lost or replaced by monovalent ions. Within enamel apatite, phosphate ions should be replaced by bicarbonate ions, whereas calcium ions should be substituted by monovalent cations such as sodium ions, to preserve electroneutrality (coupled substitution). For this reason, sodium ions constitute most of the trace elements in enamel.

2 Enamel

as the bicarbonate content increases; (d) the crystallinity decreases as the bicarbonate content increases; (e) the amount of phosphate ions within apatite decreases as the bicarbonate content increases; (f) in the infrared absorption spectrum, the resolution of PO43- vibration bands decreases as the intensity of CO32- absorption bands increases; and (g) the amount of apatite dissolution increases as the bicarbonate content increases. Such microstructural changes in bicarbonate-substituted hydroxyapatite eventually increase the development of dental caries by increasing the acid solubility of enamel. That is, an increase in the bicarbonate content in enamel apatite will decrease the crystallinity and crystallite size, ultimately increasing the total surface area of the crystals and increasing their reactivity to acids. For this reason, the amount of dissolution increases during an acid attack, thereby increasing the risk of dental caries. In practical terms, it is notable in this regard that bicarbonate is preferentially lost during dental caries. Specifically, bicarbonate is abundant at the crystal core of enamel apatite and is preferentially dissolved during acid attack (Fig. 2.10).

Fluoride and Dental Caries Trace Elements

Bicarbonate and Dental Caries The chemical composition of enamel greatly affects the development and progress of dental caries. By considering the effect of bicarbonate on changes in the microstructure of enamel apatite, it is possible to examine the influence of bicarbonate on the development of dental caries. Comparing the microstructural changes between pure hydroxyapatite and bicarbonate-substituted hydroxyapatite, we find (a) the dimension of the a-axis decreases, whereas that of the c-axis increases, as the bicarbonate content increases; (b) the size of the crystallite decreases as the bicarbonate content increases; (c) the shape of the crystal becomes more rod-shaped (as opposed to either needle- or pencil-shaped)

Trace elements can be defined as minerals other than calcium, phosphate, and bicarbonate that constitute minor portions of the enamel. Ions of many elements from the periodic table (except for radioactive elements) are present in enamel. Na, Cl, and Mg are present at more than 1000 ppm, and K, S, Zn, Si, Sr, and F are present at 100–1000 ppm. The contents of trace elements differ with aging. Also, the contents of trace elements vary geographically. Because minerals are not synthesized in the human body, they must be obtained from the diet. Differences in the mineral content of food and drink in various geographical areas may contribute to variations in the trace element content of human enamel.

Fluoride and Dental Caries

17

Fig. 2.10 Schematic representation of the pattern of dissolution of enamel apatite crystals substituted with carbonate. During an acid attack, the crystal or central core, which is rich in carbonate, is preferentially dissolved, and dissolution progresses along the c-axis. (From J. C. Voegel and R. M. Frank, Calcif. Tissue Res. 24:19–27, 1977)

Distribution of Fluoride in Tooth and Its Uptake Ratio One of the most notable trace elements in dentistry is fluoride, which is the only inorganic ion that can inhibit the development of dental caries. Therefore, we will look deeply at the nature of fluoride, even though only a small amount of this element is present in the teeth. The content of fluoride within enamel is 100–1000 ppm, depending on the location within the enamel. On the enamel surface, the content of fluoride is high relative to the body enamel (i.e., the concentration decreases closer to the dentinoenamel junction). This is because fluoride is incorporated into the enamel surface from external sources such as saliva, food, or drinks. The distribution of different minerals, including fluoride, in human enamel is shown in Figs. 2.11 and 2.12. Also, the content of fluoride within enamel decreases with aging, which is attributable to continuous attrition or erosion of the enamel surface with high fluoride content. On the other hand, the content of fluoride within the dentine dramatically increases from the dentinoenamel junction to the crown dentine. As aging progresses, the fluoride content in the

crown dentine dramatically increases; this process is closely related to blood supply. By contrast, no change in the content of fluoride is observed in other locations of dentine during aging. The uptake ratio of fluoride is much higher before eruption than post-eruption; in addition, it

Fig. 2.11 Distribution of trace element contents in human enamel by region. Surface enamel contains more inorganic ions than inner enamel. Values represent mineral contents, with % by weight

18

2 Enamel

Fig. 2.12 Changes in the Na+, Mg2+, and Cl- content in different parts of enamel and dentine. Mineral contents in enamel and dentine were analyzed by electron microprobe analysis. EE, enamel edge; DEJ, dentinoenamel junction; PDJ, pulp–dentin junction. (From F. C. Bestic et al., J. Dent. Res. 48:131– 139, 1969)

is higher on the smooth surface of teeth, where saliva and drinks are easily accessible than in pits and grooves. Continuous fluoride uptake continues even after tooth eruption. Saliva, foods, drinks, and fluoride-containing toothpastes and gargles are the major sources of fluoride intake.

Fluoride As an Inhibitor of Dental Caries As described above, an appropriate concentration of fluoride inhibits dental caries. Specifically, approximately 1 ppm fluoride (i.e., a low concentration) inhibits dental caries by converting hydroxyapatite into fluorapatite, which can be expressed by the chemical formula Ca10(PO4)6(OH)2 + 2F- → Ca10(PO4)6F2 + 2OH-. Fluoride replaces hydroxyl ions in hydroxyapatite either partially [Ca10(PO4)6(F,OH)] or completely [Ca10(PO4)6F2]. Microstructural comparison of hydroxyapatite and fluorapatite can provide insight into the development of dental caries. Such a comparison reveals (a) the a-axis dimension decreases from 9.42 Å in hydroxyapatite to 9.36 Å in fluorapatite, whereas the c-axis dimension is unchanged; (b) the crystallite of fluorapatite is larger, (c) the

crystallite strain of fluorapatite is smaller, and (d) the crystallinity of fluorapatite is greater. These features are all reflected by the narrower diffraction peak of fluorapatite in the X-ray diffraction pattern. In addition, for fluorapatite (e) the intensity of fluoride absorption bands in the infrared absorption spectrum is greater, (f) the dissolution rate and acid solubility of fluorapatite is lower, and (g) the thermal stability is greater. Conversion of hydroxyapatite to fluorapatite by fluoride ion substitution leads to changes in microstructure that eventually decrease acid solubility. Specifically, when hydroxyapatite is converted into fluorapatite, the crystallinity and crystallite size increase. Consequently, the total surface area of the crystals decreases, reducing the reactivity and therefore the acid solubility of apatite. Ultimately, these features inhibit the development of dental caries. Because fluoride inhibits dental caries, water fluoridation and topical application of fluoride solution to teeth have been used for this purpose. Based on daily water intake, some developed countries add fluoride to drinking water: 1 ppm of fluoride in temperate zones, 0.7 ppm in tropical zones, and 1.3 ppm in cold regions. These efforts decrease the incidence of dental caries by 40–60%. On the other hand, direct application

Ionic Substitution in Enamel Apatite

involves the topical administration of NaF or SnF2 solution to the surface of teeth, locally. The topical application of fluoride solution is one of the most common clinical and preventive treatments known to yield beneficial effects. Although an appropriate concentration of fluoride may inhibit dental caries, a high concentration risks adverse effects. When a high concentration of fluoride reacts with hydroxyapatite, calcium fluoride is formed, eventually dissolving the enamel. This reaction can be expressed by the chemical equation Ca10(PO4)6(OH)2+20F-→10CaF2+6PO43-+2OH-. Calcium fluoride produced in this way is relatively insoluble and therefore precipitates. However, the precipitant can be easily removed by external forces, such as tooth brushing, and is also slowly dissolved by saliva. This process can be expressed by the chemical equation CaF2 → Ca2++2F-. Fluoride ions released by calcium fluoride through dissolution react with hydroxyapatite and may produce fluorapatite or hydroxyapatite. However, this is a minor reaction, and the majority of the products are removed, resulting in the dissolution of the teeth.

Fluoride and Mottled Teeth Infants born in areas with high fluoride levels in the drinking water have a higher incidence of conditions such as mottled teeth or dental fluorosis. Mottled teeth are a chronic endemic form of hypoplasia of the enamel caused by drinking water with high fluoride content during the period of tooth formation. It is characterized by defective calcification that gives a white chalky appearance to the enamel, which gradually undergoes brown discoloration. Mottled teeth occur because of matrix defects between enamel rods and an accumulation of brown pigment that colors the teeth. The degree of brown pigmentation varies from brown to black in severe cases. Tooth mottling is a type of enamel hypoplasia that is attributable to excessive intake of fluoride during tooth development and is prevalent in specific regions (e.g., regions with high levels of granite, springs,

19

volcanoes, mines) with high fluoride content in the drinking water.

Effects of Fluoride on Enamel Remineralization Organic acids such as lactic acid, acetic acid, propionic acid, butyric acid, and formic acid produced by the metabolism of plaque bacteria contact the surfaces of the teeth, decreasing their pH below 5.5–5.6 and causing acid dissolution. Enamel within 20 μm of the surface is constantly exposed to acid attack. As noted above, this process causes early dental caries lesions to progress to the clinically detectable stage. The presence of fluoride promotes remineralization of the enamel, keeping the surfaces of teeth intact. However, as noted above, saliva contains anionic proline-rich proteins and statherin, both of which inhibit the growth of calcium phosphate crystals and thus interfere with remineralization, except in regions where these proteins cannot penetrate. Also, during the process of enamel remineralization, fluoride is preferentially incorporated into early lesions, where it converts hydroxyapatite to fluorapatite, making the enamel more resistant to continuous acid attack. This is the natural protective mechanism of fluoride against dental caries. During remineralization of enamel of early lesions, Sr and Zn are also incorporated, but their functions remain elusive.

Ionic Substitution in Enamel Apatite Comparison of Synthetic Hydroxyapatite and Human Enamel Apatite Pure hydroxyapatite can be synthesized in a nonaqueous system at high temperature, and hydroxyapatite can also be synthesized in an aqueous system at low temperature. Table 2.6 provides a comparison of the characteristics of hydroxyapatite and enamel apatite, including composition, Ca/P ratio, lattice parameters

20 Table 2.6

2 Enamel Comparison of properties of synthetic hydroxyapatites and human enamel apatite

Properties Composition

Syn Aa Ca10(PO4)6(OH)2

Syn Bb Ca10(PO4)6(OH)2, HPO42–, H2O

Stoichiometry (Ca/P) Lattice parameters a-axis c-axis Infrared spectra absorption bands Ignition products (800°C)

1.67

1.60–1.64

Enamel apatite Ca10(PO4)6(OH)2 Na+, CO32–, F–, K+, HPO42–, Cl–, Mg2+, H2O Trace elements 1.55–1.64

9.422 6.880 PO43–, OH–

9.433 6.882 PO43–, OH–, HPO42–

9.441 6.882 PO43–, OH–, CO32–

Apatite

TCP + Apatite

TCPc + Apatite

Source: E. P. Lazzari, Handbook of Experimental Aspects of Oral Biochemistry, (CRC Press, 1983), p. 165 Syn A (from nonaqueous systems) b Syn B (from aqueous systems, by precipitation or hydrolysis) c Mg-substituted tricalcium phosphate a

(dimension of a-axis and c-axis), absorption bands in the infrared spectrum, and ignition products. Pure hydroxyapatite synthesized in a nonaqueous system also exhibits the characteristics of pure hydroxyapatite. For pure hydroxyapatite, the dimensions of the a-axes and c-axis are 9.42 Å and 6.88 Å, respectively. By contrast, hydroxyapatite that is synthesized in an aqueous system contains HPO42-, and consequently, its parameters are intermediate between those of pure hydroxyapatite and enamel apatite.

(C6H5O73-), vanadate (VO43-), and pyrophosphate (P2O74-), can be substituted for the phosphate ion position in hydroxyapatite. Also, F- and Cl- ions can be substituted at the hydroxyl ion position. The changes in crystallinity that are most strongly associated with the development of dental caries are those that occur when F- is substituted at the hydroxyl ion position: only in this case does the crystallinity increase, whereas in the other cases, crystallinity remains the same or even decreases.

Changes in Crystallinity After Heterogeneous Ionic Substitutions in Hydroxyapatite

Precursors of Enamel Apatite

Table 2.7 shows heterogeneous ions that can substitute for the calcium, phosphate, and hydroxyl ions in hydroxyapatite, as well as the resultant changes in the axis dimensions and crystallinity. The calcium ion position of hydroxyapatite can be substituted by either divalent or monovalent cations. Also, when the radius of substituting ions is larger than that of calcium ion, the dimensions of the a-axes and c-axis tend to increase; inversely, the dimensions tend to decrease when the radius is smaller, although Mg2+ is an exception. Either divalent, trivalent, or quadrupole anions, such as CO32-, HPO42-, citrate

The major component of minerals included in teeth and bones of vertebrates is calcium phosphate, which has a basic structural unit of [Ca10(PO4)6(OH)2]. Apatite is widely distributed in nature and is present in most types of rocks. Over the course of evolution, apatite has been selected as an important player in the mineralization process. By considering the relationship between enamel apatite and other calcium phosphates, we can examine the precursors of enamel apatite. First, let us examine the evidence that enamel apatite is formed from other types of calcium phosphates through intermediate steps, which can be thought of as precursor molecules.

Precursors of Enamel Apatite

21

Table 2.7 Effects of heterogeneous ionic substitutions for calcium, phosphate, and hydroxyl ions in hydroxyapatite on lattice parameters and crystallinity of apatite Substituent For Ca2+ Strontium, Sr2+ Barium, Ba2+ Lead, Pb2+ Potassium, K+ Sodium, Na+ Magnesium, Mg2+ Cadmium, Cd2+ Manganese, Mn2+ Tin, Sn2+ For PO43– Carbonate, CO32– Citrate, C6H5O73– HPO42– Vanadate, VO43– Pyrophosphate, P2O74– For OH– Fluoride, F– Chloride, Cl–

Ionic radius (Å) 0.99 1.12 1.34 1.20 1.33 0.97 0.66 0.97 0.80 0.93

Lattice parameter (±0.003 Å) (a-axis) (c-axis) 9.433 6.882 (+)a (+) (+) (+) (+) (+) (+) (nc)a (nc) (nc) (nc) (nc) (-) (-) (-) (-) (am)a (am)

Crystallinity – (nc) (-)a (-) (nc) (nc) (-) (-) (-) (am)

– – – – –

(-) (+) (+) (-) ?

(+) (+) (nc) (-) ?

(-) (-) (nc) (-) (-)

1.36 1.81

(-) (+)

(nc) (-)

(+) (nc)

a

(+), increase; (-), decrease; (nc), no change; (am), amorphous. (Source: R. C. Weast, CRC Handbook of Chemistry and Physics, 45th ed. (CRC Press, 1965), F89)

Initially, an enamel apatite crystal is ribbonshaped. Three facts provide indirect evidence that enamel apatite originated from various types of other calcium phosphates through a series of steps: (a) the stoichiometry of an enamel apatite is different from that of hydroxyapatite; (b) the structure of enamel apatite is similar to those of other calcium phosphates, such as octacalcium phosphate [Ca8H2(PO4)6.5H2O]; and (c) tricalcium phosphate [Ca3(PO4)2] is formed as a product of enamel apatite combustion. Secondly, dental calculus and urinary calculus in pathologic states contain types of calcium phosphate that are distinct from hydroxyapatite. That is, mature dental calculus contains 55.3% hydroxyapatite, 24.2% tricalcium phosphate, 20.0% octacalcium phosphate, and 8.9% amorphous calcium phosphate. Third, in vitro synthesis of apatite can produce other types of calcium phosphate via modulation of pH, temperature, and composition of the solution, making mutual

conversion possible. Also, ions other than calcium, phosphate, and hydroxyl ions are present in an enamel apatite, suggesting that it is formed via other calcium phosphates. Figure 2.13 explains the interactions between hydroxyapatite and its known precursor molecules, including octacalcium phosphate, amorphous calcium phosphate, tricalcium phosphate, and dicalcium phosphate dihydrate. Octacalcium phosphate can hydrolyze and convert into hydroxyapatite; alternatively, in the presence of magnesium, it can hydrolyze and convert into tricalcium phosphate. Tricalcium phosphate in biological systems is always partially substituted with magnesium ions and is expressed as (Ca,Mg)9(PO4)6. That is, pure tricalcium phosphate is not found in biological systems. Pure tricalcium phosphate can be converted into hydroxyapatite, whereas magnesium-substituted tricalcium phosphate cannot. Amorphous calcium phosphate can be

22

2 Enamel

2

Fig. 2.13 Interactions among hydroxyapatite and related calcium phosphates, octacalcium phosphate (OCP), tricalcium phosphate (TCP), amorphous calcium phosphate, and dicalcium phosphate dihydrate (DCPD). The processes involved are ① hydrolysis, ② hydrolysis in the

presence of Mg2+, ③ dissolution and reprecipitation, and ④ no reaction. [Source: E. P. Lazzari, Handbook of Experimental Aspects of Oral Biochemistry, (CRC Press, 1983), p. 174]

hydrolyzed and converted into hydroxyapatite and into carbonate-substituted hydroxyapatite in the presence of carbonate. However, the conversion of amorphous calcium phosphate to hydroxyapatite is inhibited in the presence of magnesium due to the stabilizing effect of this ion. Thus, only when magnesium is lost can amorphous calcium phosphate be converted into hydroxyapatite. Dicalcium phosphate dihydrate hydrolyzes and converts into octacalcium phosphate and hydroxyapatite, whereas the presence of magnesium promotes the conversion of dicalcium phosphate dihydrate into magnesiumsubstituted tricalcium phosphate. In addition, hydroxyapatite can hydrolyze and convert into magnesium-substituted tricalcium phosphate in the presence of magnesium. Furthermore, hydroxyapatite converts into either dicalcium phosphate dihydrate or amorphous calcium phosphate through dissolution/reprecipitation processes. Thus, hydroxyapatite can be produced from other calcium phosphates that serve as its precursors. Moreover, hydroxyapatite and its precursors can be reciprocally converted.

Octacalcium Phosphate [Ca8H2(PO4)6
5H2O] The chemical formula and composition of octacalcium phosphate are very similar to those of hydroxyapatite; therefore, this compound is the most plausible precursor of hydroxyapatite. Brown claimed that octacalcium phosphate can be hydrolyzed and converted into hydroxyapatite, although impurity ions may be incorporated during the conversion process.

Amorphous Calcium Phosphate Amorphous calcium phosphate is not present in mature enamel, although it constitutes mature human dentine, and the bones of newborn (65%) and mature rats (35%). Amorphous calcium phosphate containing bicarbonate hydrolyzes and converts into carbonate-containing hydroxyapatite. Because magnesium ion stabilizes amorphous calcium phosphate, it inhibits the

Organic Materials in Enamel

conversion of amorphous calcium phosphate into hydroxyapatite.

Tricalcium Phosphate [Whitlockite, Ca3(PO4)2] Tricalcium phosphate in biological systems is always partially substituted by magnesium ions and should therefore be written as (Ca, Mg)9(PO4)6. Magnesium-substituted tricalcium phosphate does not convert to hydroxyapatite. Pure tricalcium phosphate can be synthesized in a nonaqueous system at high temperature (700–1000°C), but not in an aqueous system at low temperature (25–100°C). In an aqueous system, magnesium ions facilitate the production of tricalcium phosphate. Pure synthesized tricalcium phosphate can be converted into hydroxyapatite through hydrolysis. In addition, tricalcium phosphate is found in carious enamel, carious dentine, and sound peritubular dentine.

Dicalcium Phosphate Dihydrate (Brushite, CaHPO4.2H2O) When the tooth surface is exposed to acids, with a pH below 5.5–5.6, the enamel crystals begin to dissolve. One of the calcium phosphates present during this phase is dicalcium phosphate dihydrate. When an extracted tooth is immersed in an acid solution, dicalcium phosphate dihydrate crystals form at the surface of the tooth, with the crystal size and quantity dependent on the pH: the lower the pH of the solution, the larger and more abundant the crystals. Enamel crystals partially dissolve to produce HPO42- in the presence of acid; this process can be expressed by the chemical equation Ca10(PO4)6(OH)2 + 8H+ → 10Ca2+ + 6HPO42- + 2H2O. The product, 10Ca2+ + 6HPO42- + 2H2O, will be converted into 4Ca2+ + 6CaHPO4
2H2O, which will in turn yield dicalcium phosphate dihydrate crystals. Under acidic conditions, dicalcium phosphate dihydrate is a more stable form of calcium phosphate than hydroxyapatite in acidic conditions. Even under

23

such acidic conditions, however, the presence of fluoride ions will cause hydrolysis of dicalcium phosphate dihydrate, converting it into fluorapatite. Additionally, when the pH of the oral cavity recovers, dicalcium phosphate dihydrate is hydrolyzed and converted into hydroxyapatite and octacalcium phosphate. However, continuous exposure to acidic conditions will eventually dissolve enamel apatite, causing dental caries.

Organic Materials in Enamel As described above, the total content of organic materials in mature human enamel is less than 0.6%. Therefore, mature enamel is composed mostly of inorganic materials and an extremely small amount of organic matter. More than half (~58%) of the organic content of mature enamel consists of protein, mostly enamelin (~48%). In addition, extremely small quantities of citrate, lactate, carbohydrate, and fatty acids are present. In terms of development, dentine and bones are of mesodermal origin, whereas enamel is of ectodermal origin. Ameloblasts, enamel-forming cells, disappear from the enamel when the tooth erupts into the oral cavity, explaining why enamel does not regenerate after tooth eruption. The chemical compositions of mature enamel and developing enamel (or fetal enamel) differ significantly: in the early stage of amelogenesis, developing enamel consists of 32% inorganic material, 30% organic material, and 38% water. Also, as enamel develops, the amount of organic material and water decreases, while the levels of inorganic material continue to increase, resulting in a dramatic change in composition (Fig. 2.14). To obtain insight into enamel formation, it is necessary to characterize the enamel proteins. Due to the large amount of inorganic materials in mature enamel, however, it is more effective to isolate protein from developing enamel. To isolate matrix proteins, samples of developing enamel can be treated with 4 M guanidine-HCl (pH 7.4) in order to obtain amelogenin. Under these conditions, enamel crystals cannot be dissolved. Thus, amelogenin usually exists in

24

Fig. 2.14 Changes in the contents of inorganic, water, and organic constituents in enamels with different densities during maturation of enamel. The full lines represent Deakins’ original values, including his correction for carbonate. The dotted lines include an additional correction for 1.2% bound water. (Source: S. Cole and J. E. Eastoe, Biochemistry and Oral Biology, 2nd ed. (Wright, 1988), Fig. 32.2)

the intercrystalline space. After amelogenin is isolated from developing enamel, the sample can be treated with 4 M guanidine-HCl (pH 7.4) containing 0.5 M EDTA to isolate enamelin. This condition dissolves enamel crystals, implying that enamelin is bound to enamel crystals. Developing enamel also contains other types of matrix proteins and enzymes.

Amelogenin Amelogenin, a protein synthesized by ameloblasts, constitutes 90% of the matrix protein in developing enamel. Amelogenin exists in the intercrystalline space, which can be relatively easily dissolved and isolated. Expression of amelogenin increases during the early stage of enamel formation, gradually decreases as enamel matures, and is completely lost in mature enamel (Fig. 2.15). Amelogenin, a hydrophobic protein

2 Enamel

that consists largely of proline (24%), glutamine (14%), leucine, and histidine residues, has a low molecular weight (6.5–27 kDa). The amelogenin gene is encoded on both the X and Y chromosomes, giving rise to sex-dependent differences in its expression. Mutations in the amelogenin gene cause X-linked amelogenesis imperfecta, which represents 5% of all cases of amelogenesis imperfecta (Stephanopoulos et al., 2005). The phenotypes of mutations in the amelogenin gene suggest that this protein plays important roles in enamel formation. The amelogenin gene is expressed as a single transcript, which is processed into multiple isoforms by alternative splicing (Fig. 2.16). In porcine, the molecular weights of amelogenin are 27 kDa (0.5%), 25 kDa (92.7%), 18 kDa (6.6%), and 6.5 kDa (0.1%); 25 kDa amelogenin is the major prototypic protein. The 25 kDa amelogenin isolated from porcine tissue consists of 173 residues and is localized at the outermost region of developing enamel, within 40 μm of the surface enamel. From the 25 kDa form, proteases remove 23 or 12 residues from the hydrophilic C-terminus, yielding the 20 kDa and 23 kDa amelogenins; the former is more abundant. When the hydrophilic residues are removed, the hydrophilicity of the protein is reduced, resulting in the formation of a gel-like structure after coagulation. With this gel-like structure, insoluble calcium phosphate salts accumulate, promoting its calcification. Calcification starts soon after the synthesis and secretion of organic materials. Amelogenin forms aggregates that surround the major axis of enamel crystals and may restrict the growth of thickness and width of crystals. Therefore, it must be removed to allow the growth of crystals. Consequently, when calcification has progressed to a certain extent, 20 kDa amelogenin is dissolved and removed. Specifically, the N-terminal one-third of 20 kDa amelogenin is cleaved by a serine protease in the inner layer of developing enamel. Cleavage of this region increases the solubility of amelogenin at physiological pH and temperature, facilitating its removal from the maturing enamel. In other words, 20 kDa amelogenin provides the site for the accumulation of calcium phosphate

Organic Materials in Enamel

25

Fig. 2.15 Changes in amelogenin and enamelin contents during maturation of enamel, compared with the increasing thickness of enamel and mineral content. The horizontal axis represents changes in enamel thickness and mineral content during enamel maturation. (From L. W. Fisher and J. D. Termine, Clin. Ortho. Relat. Res. 200:362–385, 1985)

Fig. 2.16 Mammalian amelogenin gene structure. (a) Gene structure. Exons 1–5 are small, and exons 6–7 are relatively large. (b) Coding and noncoding exons. Exon 1 is not expressed, most of exon 2 encodes a signal

peptide, and exon 7 encodes only the first three nucleotides. (c) Linear representation of the newly synthesized protein. ex, exon; in, intron; bp, base pair; aa, amino acid

salts during amelogenesis, but it is removed upon accumulation of calcium phosphate salt.

Some enamelin is expressed from the early stage of amelogenesis and is maintained at a constant level throughout the process. After amelogenesis terminates, the level of enamelin decreases; however, enamelin remains very stable in the process of enamel maturation, and a small amount exists in mature enamel (Fig. 2.15). Therefore, the variation in the protein content in enamel is quite and depends on its level of maturity. Enamelin is encoded by a tooth-specific gene that is expressed at high levels in ameloblasts and to a lesser extent in odontoblasts. Enamelin

Enamelin Enamelin, a protein synthesized by ameloblasts, constitutes 1–5% of enamel matrix proteins. Enamelin has a high affinity toward apatite crystals; therefore, it is bound to the apatite crystal surface and can be isolated only under conditions in which enamel crystals are dissolved.

26

2 Enamel

Fig. 2.17 Mutation sites in the human enamelin gene identified in a relative with amelogenesis imperfecta. Square boxes represent exons, and straight lines represent introns. The number under each exon indicates the amino acid sequence of the protein encoded by the exon, and the

line indicates where the mutation occurs in the enamelin gene. X represents the site where the stop codon was generated by a mutation. Mutations affecting the splice junctions are indicated by ‘Spl jctn’

cDNA has been cloned and characterized from mice, porcine, and humans. The human enamelin gene is located at chromosome 4q13.3 and consists of nine exons. The enamelin genes of mouse and porcine each contain 10 exons; the human gene lacks the equivalent of mouse exon 2. In contrast to amelogenin and ameloblastin, alternative splicing has not been reported in enamelin. Amelogenesis imperfecta is a genetic disease that gives rise to defects in enamel formation (Dong et al., 2000). Mutations in the enamelin gene cause a severe form of autosomal-dominant smooth hypoplastic amelogenesis imperfecta, which represents 1.5% of all cases of this disease. Mutations in the enamelin gene can also cause a mild type of autosomal-dominant local hypoplastic amelogenesis imperfecta, which represents 27% of cases (Hu and Yamakoshi, 2003) (Fig. 2.17). The phenotypes of mutations in the enamelin gene mutation indicate that this protein plays important roles in proper enamel formation. Enamelin contains 1103 residues and is synthesized with a signal peptide of 39 residues. Mature enamelin is a hydrophilic protein with abundant glutamic acid, aspartic acid, serine, and glycine residues; porcine-developing enamel contains enamelin proteins ranging in size from 25 to 155 kDa enamelin. Originally, enamelin was isolated from unerupted porcine teeth, and its characteristics were subsequently examined. Porcine enamelin is very similar to human enamelin. The enamelins of mouse, human, and porcine each have three phosphorylation sites and

three N-linked glycosylation sites. In mice, enamelin is expressed at the early stage of amelogenesis, but its expression terminates as soon as amelogenin is expressed. The degradation of enamelin by proteases is essential for the proper development of enamel. Prototypic enamelin is degraded by proteases to yield a 155 kDa intermediate product, which is eventually processed to 142 kDa enamelin. The 142 kDa enamelin is continuously degraded into the 89 kDa and 34 kDa enamelins. In developing porcine enamel, 89 kDa enamelin, which contains 627 residues, is the major enamelin, and it is identical to the N-terminal larger fragment of 142 kDa enamelin. The 89 kDa enamelin again degraded, yielding 32 kDa and 25 kDa of enamelins. The 32 kDa enamelin, the major enamelin in the inner layer of developing enamel, consists of 106 residues and is absorbed to enamel apatite crystals with high affinity. To date, the 32 kDa has been most extensively studied, and its phosphorylation site and asparaginelinked glycosylation site have been identified. During the amelogenesis process, enamelin is localized primarily at the dentinoenamel junction, but it disappears at the early stage of maturation. Enamelins with low molecular weights, including the 25 kDa, 32 kDa, 45 kDa, and 56 kDa enamelins, are found in the inner layer of developing enamel, whereas enamelins with relatively high molecular weights, including the 89 kDa, 142 kDa, and 155 kDa enamelins, are only found in the thin outer layer of developing enamel. However, undegraded enamelin is

Organic Materials in Enamel

usually found near the Tomes’s process of surface enamel. Most enamelin degradation products are rapidly destroyed and are found only in the surface enamel. The stable enamelin degradation product, the 32 kDa form, is found through the whole enamel but is mostly found in enamel rods and portions of inter-rod enamel. Enamelin has a high affinity for hydroxyapatite and delays the seeded growth of hydroxyapatite crystals. Amelogenin and enamelin regulate the morphology of hydroxyapatite crystals. According to recent in vitro experiments, 32 kDa porcine enamelin cooperatively interacts with amelogenin to promote the nucleation of apatite crystals.

27

proteins in developing enamel and is usually localized in sheaths of enamel rods. As soon as ameloblastin is synthesized and secreted, it is degraded into a small N-terminal peptide and a relatively large peptide C-terminal peptide. The N-terminal peptide is relatively stable: although it is gradually degraded, it does not completely disappear at the matrix-formation stage. On the other hand, the large C-terminal peptide is degraded rapidly and soon disappears. Prototypic ameloblastin and the C-terminal peptide are only found on the enamel surface. However, ameloblastin containing the N-terminus exists in every layer and is particularly concentrated on sheaths of enamel rods. Ameloblastin is found at the sites of earliest calcification and is thus thought to act as a nucleator of enamel formation.

Ameloblastin (Amelin or Sheathlin) The ameloblastin gene is expressed primarily in ameloblasts, to a lesser extent in odontoblasts and preodontoblasts, and at intermediate levels in Hertwig’s epithelial root sheath and ameloblastoma. Ameloblastin was first discovered in developing porcine enamel through the discovery of 13–17 kDa non-amelogenin, which refers to the degradation products of amelogenin. In 1996, ameloblastin was first cloned from rats by two different research groups, who named it ameloblastin or amelin. In 1997, ameloblastin was cloned from porcine by the other group, who called it sheathlin. The human ameloblastin gene is located at chromosome 4q21 and consists of 13 exons. Ameloblastin forms multiple isoforms from a single primary transcript via alternative splicing and degradation by proteases. Comparison of the amino acid sequences of ameloblastin reveals extensive similarities among humans, porcine, calves, rats, and mice. Furthermore, the phosphorylation sites, hydrophilicity, and high proline (15.2%), leucine (10.2%), and glycine (9%) contents are shared among these species. Human ameloblastin, a 421-residue protein with a molecular weight of 65 kDa, is synthesized with a signal peptide of 26 residues. Ameloblastin constitutes approximately 5% of enamel matrix

Tuftelin Tuftelin, composed of 389 residues with a molecular weight of 44 kDa, is an acidic protein that is synthesized and secreted from ameloblasts (Deutsch et al., 1997). The tuftelin cDNA was originally cloned from bovine in 1989 and sequenced in 1991. The human tuftelin gene, which is located at chromosome 1q21, is expressed in various organs including the kidney, liver, lung, testis, and tooth. Therefore, the tuftelin gene is not expressed in a tooth-specific manner. Tuftelin is mainly found in the dentinoenamel junction. Based on this localization, and the fact that tuftelin is expressed before amelogenin in ameloblasts, it is presumed to act as a nucleator for apatite crystal formation. The developing enamel expresses multiple additional proteins and contains organic materials and proteins derived from other genes, such as amelogenin, enamelin, ameloblastin (amelin/ sheathlin), tuftelin, dentine sialophosphoprotein, and enzymes. Based on their overall functions, these proteins are thought to participate in de novo mineral nucleation/initiation (dentine sialophosphoprotein, tuftelin); interactions of proteins with inorganic ions, as precursors in crystal formation (amelogenin, enamelin);

28

regulation of crystal growth (amelogenin, enamelin, ameloblastin); support of growing crystals (amelogenin, enamelin); determination of the structure of enamel rods (ameloblastin), cellular signal transduction (ameloblastin, tuftelin); regulation of secretion protein degradation products; and protection of mineral phases (amelogenin, enamelin) (Robinson et al., 1998).

References Deutsch, D., Dafni, L., Palmon, A., Hekmati, M., Young, M.F., Fisher, L.W.: Tuftelin: enamel mineralization and amelogenesis imperfecta. Ciba Found. Symp. 205, 135–147 (1997) Dong, J., Gu, T.T., Simmons, D., MacDougall, M.: Enamelin maps to human chromosome 4q21 within

2 Enamel the autosomal dominant amelogenesis imperfecta locus. Eur. J. Oral Sci. 108, 353–358 (2000) Hu, J.C., Yamakoshi, Y.: Enamelin and autosomal -dominant amelogenesis imperfecta. Crit. Rev. Oral Biol. Med. 14, 387–398 (2003) Lefevre, M.L., Hodge, H.C.: Chemical analysis of tooth samples composed of enamel, dentine and cementum. J. Dent. Res. 16, 279–287 (1937) Manly, R.S., Hodge, H.C.: Density and refractive index studies of dental hard tissues: I. Methods for separation and determination of purity. J. Dent. Res. 18, 133–141 (1939) Robinson, C., Brookes, S.J., Shore, R.C., Kirkham, J.: The developing enamel matrix: nature and function. Eur. J. Oral Sci. 106, 282–291 (1998) Stephanopoulos, G., Garefalaki, M.E., Lyroudia, K.: Genes and related proteins involved in amelogenesis imperfecta. J. Dent. Res. 84, 1117–1126 (2005) Voegel, J.C., Frank, R.M.: Stages in the dissolution of human enamel crystals in dental caries. Calcif. Tissue Res. 24, 19–27 (1977)

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Dentine

The dentine, which constitutes most of the tooth, has a resilient structure because it is less dense than enamel. Dentine is synthesized by odontoblasts, which originate from the mesoderm. The odontoblastic process protrudes into the dentine, and the cell body is located on the inner surface of the pulp. Odontoblasts play roles in synthesizing or repairing dentine and are nourished from the pulp. Unlike enamel, which is static, dentine exhibits cellular activity, such as that of odontoblasts. Like connective tissues, the dentine largely consists of the extracellular matrix and contains a relatively small proportion of cellular components. The extracellular matrix, in turn, consists of dense calcified collagen fibers and mostly surrounds the tubular structure. The dentine forms the body of the tooth, protects the pulp, and supports the enamel and cementum. Dentine can be subclassified as calcified dentine and predentine, which have a low degree of calcification. Because predentine is less calcified than mature dentine, the enzymatic activities in this tissue (e.g., alkaline phosphatase, calcium-dependent ATPase) are relatively high.

Dentine Structure The cross section of mature dentine contains several structures: the intertubular dentine, outer hypomineralized layer, peritubular dentine, inner hypomineralized layer, and odontoblastic process (Fig. 3.1). However, not all of these structures can

be easily observed in one specimen. In particular, the inner hypomineralized layer, a less calcified inner layer, cannot be easily observed in the cross section. In the intertubular dentine, the collagen fibers run tangentially to the dentinal tubules and form a trellis-like framework (Fig. 3.2). The calcified product of this structure is the intertubular dentine. The collagen fibers of intertubular dentine have their own typical structure, with repeated cross-bands or cross-striations at 640 Å. The inner part of the dentinal tubule of mature dentine is composed of the odontoblastic process and is separated from the peritubular dentine by the inner hypomineralized layer, a less calcified inner layer. The peritubular dentine is distinguished from the intertubular dentine by the outer hypomineralized layer, a less calcified outer layer. The odontoblastic process is an extension of the odontoblast cytoplasm that enters through the dentinal tubules. Small amounts of cytoplasmic organelles, such as endoplasmic reticulum and mitochondria, are present at the boundary between the predentine and pulp. Because the inorganic content increases from the predentine–dentine boundary to the mature dentine, the microstructure is not observed in the odontoblastic process. The diameter of the odontoblastic process is about 5 μm in the predentine area and about 2 μm in the mature dentine region. The peritubular dentine, which is located between the outer hypomineralized layer and the inner hypomineralized layer, is the most densely

# The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 B.-M. Min, Oral Biochemistry, https://doi.org/10.1007/978-981-99-3596-3_3

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3

Dentine

calcified region in the dentine. The density of the peritubular dentine is about 2.40 g/cm3, much higher than the average density of the dentine overall, 2.14 g/cm3. In addition, the collagen fibers of the peritubular dentine are narrower than those of the intertubular dentine (250–500 Å vs. 600–700 Å). Densely calcified peritubular dentine is distinguished from the odontoblastic process by the presence of the inner hypomineralized layer, a less calcified layer. Also, because the intertubular dentine is synthesized before the peritubular dentine, the two tissues are divided by the outer hypomineralized layer, a thin layer with less calcification. Fig. 3.1 Ground transverse section of human dentine stained with silver nitrate and reduced in sunlight (magnification, 1500×). OPr, dentinal process of the odontoblast; Ho, outer hypomineralized layer; PtD, peritubular dentine; ItD, intertubular dentine. (Source: R. C. Weast, Handbook of Chemistry and Physics, (Academic Press, 1967), p. 20)

Fig. 3.2 Electron micrograph showing an array of collagen fibrils in matrix obtained from the vicinity of the predentine–dentine border. The collagen fibrils appear in longitudinal, oblique, and cross-sectional views in the matrix. A membranous structure (MS) surrounds the lumen (L) of the canal, stained with phosphotungstic acid. (Source: R. W. Fearnhead and M. V. Stack, Tooth Enamel, (John Wright & Sons, 1965))

Chemical Composition of Dentine Inorganic Components of Dentine The inorganic content of dentine is about 70–75% (wt%), lower than that of enamel but higher than that of bone or cementum. Vertebrate bone and minerals in teeth consist of calcium phosphate in the form of hydroxyapatite crystals [Ca10(PO4)6(OH)2]. Dentine minerals are also closely related to hydroxyapatite, although the chemical composition is not consistent. Specifically, the Ca and P contents are very diverse: the average Ca/P ratio of dentine is 2.04–2.05, whereas that of hydroxyapatite is 2.15. In addition, dentine contains inorganic ions such as CO32-, F–, and Na+ that are not present in pure hydroxyapatite (Table 3.1). Many inorganic ions present in apatite in vivo influence its stability. The inorganic ions present in dentine are either incorporated during the process of conversion of hydroxyapatite precursors to hydroxyapatite or are introduced by heterogeneous ionic substitution. The chemical composition of hard tissue is different from that of pure hydroxyapatite. That is, non-apatite phases such as amorphous calcium phosphate or carbonate are present, and additional ions or molecules are adsorbed on the surface of the apatite crystal. This is either due to heterogeneous ionic substitution or the presence of a space

Chemical Composition of Dentine Table 3.1

31

Contents of major inorganic constituents and several trace elements in dentine and bone

Constituent Ca P (present as PO43- and HPO42-) P (present as pyrophosphate) CO2 (present as carbonate) Na Mg Cl K F Fe Zn Sr Ca/P ratio (by weight) Ca/P ratio (by molar)

Dentine (human dried) Percentage by weight (wt%) 26–28 12.2–13.2 Approximately 0.05 3.0–3.5 0.7 0.8–1.0 0.4 0.02–0.04 Part per million (ppm) 50–10,000 60–150 200–700 100–600 2.10–2.20 1.6–1.7

in the apatite. In particular, dentine contains amorphous calcium phosphate, octacalcium phosphate [Ca8H2(PO4)6∙5H2O], tricalcium phosphate [Ca9(PO4)6], and dicalcium phosphate dihydrate [CaHPO4∙2H2O], and conversion to hydroxyapatite is possible under certain conditions. Of the minerals present in the dentine, 65–70% are present in the crystalline apatite form, and the remaining 35% are in the form of amorphous calcium phosphate. The crystal size of dentine is smaller than that of enamel: the crystal is 2.0–3.5 nm thick and 20–30 nm long, so the volume of the dentine crystal is about 1/200th of that of the enamel crystal. In general, chemical reactions take place on the surface of apatite crystals, so with its small crystal size, dentine has a larger surface area than enamel and is consequently less stable. Moreover, the Ca/P ratio of dentine is 2.04–2.05, smaller than that of enamel.

Organic Constituents of Dentine In normal human dentine, the organic content is about 20% (wt%), the inorganic content is about 70% (wt%), and the remaining 10% consists of water. Approximately 90% of the organic components present in the dentine are collagen

Bone (typical values) 24.0 11.2 Approximately 0.05 3.9 0.5 0.3 0.01 0.2 5000

2.15 1.66

fibers, which play important roles in the calcification process. The collagen in dentine consists mainly of type I collagen. Other organic components are non-collagenous organic materials, including non-collagenous proteins, biopolymers including carbohydrates, small amounts of lipids, and other organic materials such as citrate and lactate (Table 3.2). The non-collagenous proteins include phosphophoryn, which is exclusively found in dentine; dentine sialoprotein; dentine-specific extracellular matrix proteins; and other proteins such as glycoprotein, proteoglycan, and Gla protein. Phosphophoryn accounts for about 60% of the non-collagenous proteins in the dentine, and it is localized mainly at the predentine–dentine boundary. Lipid accounts for about 1.65% of total organic components in dentine and about 0.33% by dry weight. The lipids in dentine include free fatty acids, monoglycerides, diglycerides, triglycerides, cholesterol, and phospholipids. Acidic phospholipids and some of the cholesterol that can be isolated after acidic demineralization of dentine are thought to be bound to minerals or buried in the matrix; these compounds are deeply involved in calcification as components of matrix vesicle membranes. The lipid content of predentine constitutes 6% of total organic components, much higher than in

32 Table 3.2

3

Dentine

Chemical composition of normal human dentine Percentage by weight (wt%)

Inorganic matter Ash Carbon dioxide Organic matter Collagen Resistant protein Citrate Lactate Chondroitin sulfate Lipid Unaccounted for (water retained at 100°C, errors, etc.)

dentine. Other organic components include citrate, lactate, and other metabolites. Citrate is a common ingredient of calcified tissues, and indeed about 70% of total citrate in vivo exists in calcified tissues. The dentine contains citrate levels similar to other calcified tissues; the content is about 0.8–0.9% (Table 3.3). Citrate can be incorporated into calcified tissue in multiple ways: due to coprecipitation with calcium phosphate, as a constituent of the argininerich peptide containing citrate, or as a component of phosphocitrate or pyrophosphoric citrate. With the exception of collagen fibers, the functions of most organic constituents in calcified tissues are not well understood.

Physicochemical Properties and Biosynthesis of Collagen Collagen, the major component of connective tissue, is the most abundant protein in mammals, accounting for one-fourth to one-third of the total protein. Collagen is the main protein of the skin, bones, tendons, cartilage, and teeth and is present in almost all tissues, as well as in connective tissue. One of the hallmarks of collagen is that it forms fibers that are insoluble in water.

Physicochemical Properties of Collagen Collagen fiber, the major protein component of extracellular connective tissue, is a structural

75 72 3 20 18 0.2 0.89 0.15 0.4 0.2 5

protein that serves primarily to maintain the mechanical function of the tissue. The connective tissue consists of cellular components (primarily fibroblasts), fibrous proteins such as collagen fibers, and ground substance (Table 3.4).

Amino Acid Composition of Collagen The amino acid composition of collagen is very different from that of other proteins. As shown in Fig. 3.3, glycine accounts for about one-third of the total amino acid composition of collagen. In addition, collagen has a high proportion of alanine (about 11%), and the contents of proline and hydroxyproline, which are rarely present in other proteins, are also very high (about 22%). The amino acid sequence of the collagen is very regular, with glycine at every third position, i.e., the primary structure consists largely of Gly-X-Y repeats. Although amino acids can be present at the X and Y positions, about 10% (100/1000) of residues at the X position are proline, and about 10% of residues at the Y position are hydroxyproline. Proline and hydroxyproline residues impart strength to the collagen. Hydroxyproline residues are produced by posttranslational hydroxylation of proline residues. Vitamin C (ascorbic acid) is required as a cofactor for the hydroxylation reaction. Deficiency of vitamin C can lead to scurvy, which results in abnormal collagen biosynthesis. In addition to hydroxyproline, collagen also contains the noncanonical amino acid hydroxylysine (Fig. 3.4). Moreover, collagen is also a glycoprotein, and monosaccharides or disaccharides are linked to

Physicochemical Properties and Biosynthesis of Collagen

33

Table 3.3 Comparison of organic constituents of dentine and bone Composition Organic matter Collagen Resistant protein Citrate Lactate Lipid Chondroitin sulfate Sialoprotein Acidic glycoprotein (non-sialic acid containing) Peptides Water (and other minor constituents not accounted for) Inorganic matter Ash Carbon dioxide

Table 3.4

Dentine(dried at 100°C) Weight % 17.5–18.5 0.2 0.86–0.89 0.15 0.044–0.36 0.2–0.6

Bone(whole bovine cortical, dry) 21.2 0.24 0.8–0.9 0.1 0.19 0.19–0.28

0.074–0.105 5

0.13 3–4

72.4–74.5 3.0

70 4.0

Components of connective tissues

Cells

Interfibrillar matrix or ground substances (molecules, often high polymers)

Protein fibers

Fibroblasts in more or less differentiated forms, e.g., chondroblasts, odontoblasts, osteocytes, osteoblasts Fat cells, macrophages, plasma cells, mast cells, and leukocytes Proteoglycans (glycosaminoglycans) Glycoproteins Phospholipids Water Collagen Reticulin Elastin

hydroxylysine residues via O-glycosidic linkages. Overall, collagen is a basic protein consisting of hydrophilic amino acid residues.

α2(V), and α3(V) (Table 3.7). The various types of collagen have distinct physical properties due to differences in their amino acid sequences.

Protein Structure of Collagen Collagen has a unique protein structure. The basic unit of the collagen fibril is tropocollagen, with a molecular weight of about 300 kDa, which has a unique structure in which three polypeptide chains are twisted in a screw shape. In tropocollagen, the individual polypeptide chains are called the α-chains and consist of nearly 1000 residues (Table 3.5). So far, 28 types of collagen have been identified and described; the most important ones are shown in Table 3.6. Type I collagen, which is mainly found in bone, skin (dermis), tendon, cornea, dentine, and cementum, consists of two identical α1(I) chains and one α2(I) chain. On the other hand, type V collagen consists of three different chains: α1(V),

Basic Structure of Tropocollagen Tropocollagen, the basic unit that constitutes the collagen fiber, has a unique arrangement in which the three polypeptide chains are twisted in a threaded manner. Each polypeptide chain (α-chain) consists of about 1000 residues. Tropocollagen is a rod-shaped molecule, about 3000 Å long and only 15 Å in diameter. Because of its high proportion of proline residues, the collagen α-chain cannot form α-helix or β-pleated sheet secondary structure and instead adopts a unique structure. Each α-chain forms a left-handed, non-α-helical chain with an initial 8.7 Å cycle, requiring three amino acid residues to make one turn. That is, the inner diameter is smaller than that of an α-helix, whereas the length for a given

34

3

Fig. 3.3 Composition of collagen in human dentine. The area of the sectors corresponds to the relative abundances of the indicated amino acids

number of residues is greater. In this small helical structure, the three-stranded α-chain forms a unique triple helix: a loose right-handed helical shape with a period of 104 Å (Fig. 3.5). Looking down the axis of the α-chain, the amino acid residues are oriented toward the corners of a triangle (Fig. 3.5d). Glycine must be at every third residue because the interior of the triplehelix structure, where the side chain of the glycine residue is located, cannot accommodate amino acid residues other than glycine (Brodsky and Shah, 1995).

Fig. 3.4

Dentine

The three α-chains do not form hydrogen bonds within the same chain. Instead, the helix is stabilized by steric repulsion of the pyrrolidine rings of proline and hydroxyproline residues. The pyrrolidine rings keep out of each other’s way when the polypeptide chain assumes its helical form, which has about three residues per turn. Three α-chains wind around one another to form a superhelical structure that is stabilized by hydrogen bonds between chains. Hydrogen bonds form between the peptide NH groups of glycine residues and the CO groups of residues on the other chains. The hydroxyl groups of hydroxyproline residues also participate in hydrogen bonding, and the hydrogen bonds among the chains further stabilize the triple-helical structure. This structure is well suited to the role of collagen in muscle and bone. However, in type I collagen, the triple-helical structure of the fibers is long, and no such structure is present at the N- and C-terminus. These triple helices also form a staggered structure in which the glycine residue located on one chain is located next to the X residue of the second chain, and the X residue is located next to the Y residue of the third chain. Due to this staggering, cross-striations are observed in electron microscopy as alternating dark and bright bands (Figs. 3.6 and 3.7). The period of this banding pattern is 64 nm in the dehydrated state and about 70 nm in the hydrated state. Because

Chemical structure of hydroxyproline and hydroxylysine, the specific amino acid residues found in collagen

Physicochemical Properties and Biosynthesis of Collagen Table 3.5

35

Comparison of amino acid composition ratios of collagen, elastin, and typical globular proteins

Collagen fiber (human skin) Amino acids Amino acid (%) Ala 11 Arg 5 Asn Asp 5 Cys 0 Glu 7 Gln Gly 33 His 0.5 Ile 1 Leu 2 Lys 3 Met 0.6 Phe 1 Pro 13 Ser 4 Thr 2 Trp 2 Tyr 0.3 Val 2 Modified amino acid (%) Cystine 0 3-Hydroxyproline 0.1 4-Hydroxyproline 9 5-Hydroxylysine 0.6 Desmosine and 0 Isodesmosine

Elastic fiber (mammal)

Ribonuclease (bovine)

Hemoglobin (human)

22 0.9

31 0.1 2 6 0.8 0.2 3 11 1 1 1 2 12

8 5 8 15 0 12 6 2 4 3 2 11 4 4 4 11 9 9 8 8

9 3 3 10 1 6 1 4 9 0 14 10 1 7 5 4 5 2 3 10

0 0 1 0 1

7 0 0 0 0

0 0 0 0 0

1 0 2

the length of one tropocollagen is about 300 nm, one gap is created for every five tropocollagen transverses. The bright band is called the overlap zone, whereas the dark band is called the hole zone (Fig. 3.8). The hole plays an important role in the calcification process of hard tissue: it is the space where the apatite crystals are first deposited.

Gelatinization of Collagen Collagen is insoluble in its natural state, but when it reaches a certain temperature in an aqueous solution, the regular structure of the collagen fiber is destroyed, and the protein shifts to a

random-coil structure called gelatin (Fig. 3.9). The structural change in the collagen fiber by heating occurs suddenly at a particular temperature, and the temperature at which half of the triple-helical structure is unwound is called the melting temperature (Tm). At neutral pH, the denaturation temperature of collagen fibers of warm-blooded animals is around 40°C, but human collagen fibers have a much higher Tm, 55–60°C or higher. Because most of the collagen fibers in vivo are fibrous, gelatinization due to heat deformation does not occur even when animals are exposed to high temperatures or hot water at 40°C or higher. In general, the

36

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Table 3.6 Amino acid composition of human collagen a-chains (values are given as the number of residues per 1000 total residues) Amino acid Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Hydroxylysine Lysine Histidine Arginine Gal-Hyl Glc-Gal-Hyl

α1(I) 109 42 16 34 73 124 333 115 0 21 7 6 19 1 12 9 26 3 50 1 1

α2(I) 94 44 19 30 68 113 338 102 0 35 5 14 30 4 12 12 18 12 50 1 2

α1(II) 99 43 23 25 89 120 333 103 0 18 10 9 26 2 13 20 15 2 50 4 12

α1(III) 125 42 13 39 71 107 350 96 2 14 8 13 22 3 8 5 30 6 46 – –

α1(IV) 123 45 19 38 78 85 334 30 0 33 15 32 52 5 27 50 6 6 22 2 44

α2(IV) 111 49 30 30 65 73 324 47 2 27 14 38 56 7 36 36 7 6 42 2 29

α1(V) 115 49 21 23 100 130 332 39 0 17 9 17 36 4 12 36 14 6 40 5 29

α2(V) 109 50 29 34 89 107 331 54 0 27 11 15 37 2 11 23 13 10 48 3 5

α3(V) 92 42 19 34 97 98 330 49 1 29 8 20 56 2 9 43 15 14 42 7 17

Gal-Hyl, galactosylhydroxylysine; Glc-Gal-Hyl, glucogalactosylhydroxylysine. (Source: S. Cole and J. E. Eastoe, Biochemistry and Oral Biology, 2nd ed. (Wright, 1988), Table 27.5)

denaturation temperature of collagen is proportional to the amount of proline and hydroxyproline it contains.

Collagen Biosynthesis Collagen is synthesized on ribosomes and then undergoes various processes mediated by intracellular and extracellular enzymes (Fig. 3.10).

Biosynthesis of Pro-a-Chain Like other secretory proteins, collagen is biosynthesized in ribosomes bound to the endoplasmic reticulum (Fig. 3.10). Cells that synthesize collagen include fibroblasts, osteoblasts, odontoblasts, and chondrocytes. The collagen α-chain is biosynthesized in pre-pro-α-chain form. The “pre” portion is a signal peptide that acts to transfer the pro-α-chain to the endoplasmic reticulum. This signal peptide is removed by signal peptidases in the endoplasmic reticulum.

Hydroxylation of Proline and Lysine Residues in Pro-a-Chain Proline or lysine at the Y position in the Gly-X-Y repeating structure of the pro-α-chain is hydroxylated and converted to hydroxyproline or hydroxylysine, respectively. These hydroxylation reactions are catalyzed by prolyl 4-hydroxylase, prolyl 3-hydroxylase, and lysyl hydroxylase (Figs. 3.11 and 3.12), which require molecular oxygen, Fe2+, α-ketoglutarate, and vitamin C as cofactors. Molecular oxygen provides one of its oxygen atoms to the proline or lysine residue, and the other oxygen atom is taken up by α-ketoglutarate (Figs. 3.11 and 3.12). Fe2+ is bound to the enzyme and activates oxygen. Over the course of this process, a Fe3+-Ocomplex is formed, which inactivates the enzyme. Vitamin C reactivates the enzyme by reducing Fe3+ in the inactivated enzyme to Fe2+. Vitamin C is oxidized and converted to dehydroascorbic acid during this process, i.e., it acts as an antioxidant or reducing agent. Therefore, under vitamin

Physicochemical Properties and Biosynthesis of Collagen Table 3.7 Collagen type I

37

Molecular forms and tissue distributions of major types of collagen a-chains α-Chain composition [α1(I)]2α2(I)

I-trimmer [α1(I)]3

Distribution Skin dermis, tendons, bones, cornea, dentine, cementum, periodontal ligament, fascia, blood vessels, wall of the uterus

Mature dentine, inflamed periodontal tissues, tumor cell, cultured cell Cartilage, intervertebral disc, vitreous body of the eye

II

[α1(II)]3

III

[α1(III)]3

Blood vessel wall, periodontal tissue, periodontal membrane, newborn infant skin dermis, wall of the uterus, reticular fiber

IV

[α1(IV)]3 [α1(IV)]2α2(IV) [α2(IV)]3

Basement membranes, lens capsule

V

[α1(V)]3 Cell surface, blood vessels, periodontal tissues [α1(V)]2α2(V) α1(V)α2(V)α3(V)

Fig. 3.5 (a–c) Arrangement of polypeptide chains in tropocollagen. (d) Cross section of a model of tropocollagen. Each strand is hydrogen-bonded to the other two strands. The α-carbon atoms of glycine residues are labeled G. Every third residue must be glycine because there is no space in the center of the helix

Characteristic Low-carbohydrate content Less than 10 hydroxylysine per chain

10% carbohydrate Less than 20 hydroxylysine per chain Low-carbohydrate content High glycine and hydroxyproline content Containing cysteine High 3-hydroxyproline content More than 40 hydroxylysine per chain Low alanine and arginine content Containing cysteine High-carbohydrate content High-carbohydrate content High glycine and hydroxylysine content

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Fig. 3.6 Cross-bands or cross-striations seen in the collagen fibril. Polymerized tropocollagen forms a striated fibril with a quarter-staggered arrangement

C deficiency, Fe2+ is oxidized to Fe3+, and the enzyme is inactivated, the hydroxylation process does not occur efficiently, and protocollagen (unhydroxylated collagen) is produced. These defects in collagen biosynthesis result in defects in connective tissue, resulting in a disease called scurvy.

Glycosylation of Certain Hydroxylysine Residues Glucosylgalactose, a disaccharide, is incorporated into synthesized proteins by conjugation of

Fig. 3.7 Electron micrograph of collagen fibrils isolated from human skin

Fig. 3.8 Features of collagen structure in decreasing order of size

Physicochemical Properties and Biosynthesis of Collagen

39

per chain, whereas type IV and V collagens are much more extensively glycosylated, with 30–50 sugars per chain.

Fig. 3.9

Thermal denaturation of collagen

galactose to the δ-carbon of a specific hydroxylysine residue via an O-glycosidic linkage; subsequently, a glucosyl residue is transferred to the galactosylhydroxylysine residue. This process is referred to as glycosylation. The first reaction is catalyzed by galactosyltransferase, and the second by glucosyltransferase. UDP-galactose and UDPglucose are the sources of saccharides for glycosylation. In general, the number of sugars in the α-chain is proportional to the number of hydroxylysine residues in the chain. Therefore, the degree of glycosylation varies depending on the tissue. Type I collagen has one or two sugars Fig. 3.10 Schematic diagram illustrating the steps involved in the biosynthesis of functional collagen

Procollagen Formation After glycosylation, three pro-α-chains are selfassembled to form a unique triple-helical structure called procollagen. An additional amino acid sequence at the N-terminus plays an important role in the arrangement of the pro-α-chain for helix formation. After procollagen is formed, it is stabilized by interchain disulfide bonds between the C-terminal peptides. Collagen contains many hydrogen bonds: the amino group of a glycine residue acts as a hydrogen donor, and the carboxyl group of a residue in the other chain acts as the hydrogen acceptor. The hydrogen bond is perpendicular to the long axis of collagen. Hydrogen bonds also form between the hydroxyl groups of hydroxyproline residues and water molecules. Transport and Secretion of Procollagen After the intracellular events involved in collagen formation are complete, the molecule is secreted from the cells into the interstitial space. Procollagen produced in the endoplasmic reticulum migrates to the Golgi apparatus to form

40 Fig. 3.11 Hydroxylation reaction of proline

Fig. 3.12 Hydroxylation reaction of lysine

3

Dentine

Physicochemical Properties and Biosynthesis of Collagen

41

secretory granules, which move to the cell surface and fuse with the cell membrane, where they discharge their contents through exocytosis (Fig. 3.10).

Procollagen–Collagen Conversion (Cleavage of Pro-peptide) The physiological events leading to the formation of a functional collagen molecule include the enzymatic removal of the N-terminal and C-terminal extensions of procollagen. During the process of secreting soluble procollagen out of the cell, pro-peptides located at the N- and C-termini are cleaved by procollagen peptidases (the N- and C-peptidases, respectively), followed by conversion of the molecule to insoluble tropocollagen, the functional unit of collagen synthesis. Fiber Formation and Polymerization When procollagen is secreted into the extracellular space and simultaneously converted to tropocollagen, polymerization into microfibrils begins. Each tropocollagen is spontaneously polymerized in the extracellular matrix to form a collagen fibril (Fig. 3.10). At this time, an appropriate sequence at the N-terminus, formed by the action of procollagen peptidase, is necessary for polymerization. Once the process is initiated, polymerization occurs spontaneously by the direction of the specific interactions of charged side groups of collagen α-chains. As described above, the collagen molecules are arranged in a staggered arrangement and stabilized by cross-links. When the collagen fibrils are formed, a space of about 40 nm is created between the tropocollagen units. This space, called a hole, plays an important role in the calcification process. Formation of Cross-Links After the collagen fibril polymerizes, a cross-link (a kind of covalent bond) is formed, which gives the collagen fibril stability (Fig. 3.10). Some of the lysine or hydroxylysine residues of the collagen molecule are oxidatively deaminated at the ε-carbon in a reaction catalyzed by lysyl oxidase, yielding an aldehyde derivative (allysine or hydroxyallysine, respectively) containing an

Fig. 3.13 Representative reactions of collagen crosslink formation. P and P′ represent the polypeptide portions of the collagen α-chain. Hydroxylysine may replace lysine in these reactions

aldehyde group at the δ-carbon (Fig. 3.13). The resultant allysine is aldol-condensed with another allysine to form an aldol cross-link. In addition, Schiff base formation occurs between lysine and allysine or between hydroxylysine and hydroxyallysine (Fig. 3.14). The formation of Schiff bases between hydroxylysine and hydroxyallysine is the major source of crosslinking in dentine, bone, and cartilage, as depicted in Fig. 3.15.

Collagen Degradation Collagen is a stable protein with a half-life of several months. The collagen of the extracellular matrix is degraded by collagenase, a matrix metalloprotease. Type I collagen is further cleaved by other proteases after collagenase has degraded about three-quarters of the protein from the N-terminus.

Collagenase Two types of collagenases, cellular collagenase and bacterial collagenase, have been identified. Matrix metalloprotease-1, which is produced in

42

Fig. 3.14

3

Dentine

Locations of cross-links in the collagen fiber

cells such as fibroblasts or osteoblasts, is involved in tissue remodeling, whereas matrix metalloprotease-8 is produced in neutrophils or macrophages during tissue inflammation. These collagenases cleave the fibrillar structure threequarters of the way down from the N-terminus. Bacterial collagenases hydrolyze the peptide bond immediately N-terminal to the glycine residue in the Gly-X-Y collagen repeat. Consequently, collagen is cleaved at more than 200 positions, generating many peptide fragments.

Diseases Related to Collagen Synthesis

Gelatinase Two types of gelatinase have been identified: 72 kDa gelatinase (also known as gelatinase A or MMP-2) and 92 kDa gelatinase (also known as gelatinase B or MMP-9). MMP-2 is expressed in mesenchymal cells, whereas MMP-9 is expressed in epithelial cells or inflammatory cells. Gelatinases hydrolyze gelatinized collagen fragments generated by collagenase, followed by denaturation at body temperature. Gelatinase is also referred to as type IV collagenase because it cleaves type IV collagen, which constitutes the basement membrane.

Elastic Fibers

Fig. 3.15 Main cross-link that forms Schiff base formation in dentine, bone, and cartilage

If any of the steps of collagen biosynthesis are defective, then normal collagen will not form properly, resulting in disease. Several genetic disorders have been identified in the collagen gene, including Ehlers–Danlos syndrome and Marfan syndrome. In patients with these diseases, the skin is excessively stretched and joints are weakened.

Elastic fibers, bundles of proteins (elastin) present in the extracellular matrix of connective tissue, are produced by fibroblasts and smooth muscle cells in arteries. These fibers can stretch like a rubber band up to 1.5 times their original length

Fig. 3.16

Three-dimensional structure of elastin

Elastic Fibers

and then snap back to their initial size length when relaxed. Elastic fibers include elastin, elaunin, and oxytalan. Elastic fibers are abundant in organs and tissues that regularly stretch and return to their original shapes when tension is released: e.g., lungs, blood vessel walls, and ligaments. However, the absolute amount of elastic fibers in the skin, tendons, and loose connective tissues is relatively small. The main component of elastic fibers is a protein called elastin, in which covalent bonds form between lysine residues (Fig. 3.16). Mature elastin is insoluble and has an unusual amino acid composition: like collagen, elastin is about 30% glycine and contains abundant proline. However, it does not contain hydroxylysine, and polar amino acid residues are uncommon. The rest of the protein consists primarily of alanine and other amino acid residues with nonpolar aliphatic side chains. In collagen, glycine appears regularly in every third residue, whereas elastin does not

Fig. 3.17 Cross-link structure of elastin. (a) Lysinonorleucine. (b) Desmosine cross-link. Desmosine consists of four lysine residues

43

exhibit such regularity, and the hydroxyproline content is low. These features distinguish elastin from collagen. As with collagen, elastic fibers have many cross-links, primarily of two types (Fig. 3.17). Desmosine or isodesmosine, which consists of four lysine side chains, is a unique cross-link found only in elastic fiber. As in collagen, the formation of allysine residues, which serve as cross-link precursors in elastin, is catalyzed by lysyl oxidase. However, in contrast to collagen, no cross-links are derived from hydroxylysine or histidine residues. Instead, the cross-links in elastic fibers are mostly formed by lysinonorleucine or desmosine (Fig. 3.17). Desmosine cross-links consist of three allysine residues and one lysine residue, and 25–30 desmosines are present in each elastin. In other words, elastin contains one cross-link per 28–34 amino acid residues, and many glycine, proline, and valine residues are present between these cross-links. Molecules connected to each other by covalent bonds form a skeleton connected by cross-links. This crosslinked network has elasticity, and consequently has the same properties as a rubber band: it stretches when pulled and shrinks when tension is released (Fig. 3.18). Elastic fibers are fibrous proteins found in most connective tissues along with collagen fibers and proteoglycans, which confer elasticity and

Fig. 3.18

Elasticity of an elastic fiber

44 Table 3.8

3

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Main differences between collagen and elastin

Collagen Many different genetic types Triple helix (Gly-X-Y)n repeating structure Presence of hydroxylysine Carbohydrate-containing Intramolecular aldol cross-links Presence of extension peptides during biosynthesis

Elastin One genetic type No triple helix; random coil conformations permitting stretching No (Gly-X-Y)n repeating structure No hydroxylysine No carbohydrate Intramolecular desmosine cross-links No extension peptides present during biosynthesis

scalability. The major differences between collagen and elastin are outlined in Table 3.8.

Non-collagenous Proteins Among the organic components of hard tissues, such as bones and dentine, proteins other than collagen are collectively referred to as non-collagenous proteins. Quantitatively, non-collagenous proteins constitute less than 10% of total protein in hard tissue and include many acidic proteins. Non-collagenous proteins are intimately involved in calcification because they interact with hydroxyapatite. Approximately 10% of the matrix proteins in the dentine are non-collagenous proteins; of these, 70% are soluble when demineralized with ethylenediaminetetraacetic acid (EDTA), and the remaining 30% are bound to collagen. The non-collagenous proteins found in hard tissues can be divided into those found in both bone and teeth and those found only in dentine (Table 3.9). They can also be classified as endogenous proteins synthesized in the hard tissue itself and extrinsic proteins derived from the blood or liver. When odontoblasts are mature, they secrete a form of extracellular matrix called predentine, without calcification. This extracellular matrix consists largely of collagen fibril, which is converted to primary dentine by calcification. At the same time, uncalcified predentine continues to be synthesized. Conversion of predentine to dentine occurs when the amount of extracellular matrix changes or apatite crystals accumulate inside or around the collagen fibril. The series of events involved in dentinogenesis is very similar

to those involved in osteogenesis: postmitotic osteoblasts secrete an extracellular matrix, generating an uncalcified osteoid, and then apatite crystals accumulate in the type I collagen and convert the osteoid to the bone. Mineral accumulation during dentinogenesis and osteogenesis is precisely controlled, and well-arranged, plate-shaped crystals are deposited in the holes of the collagen fibril. Mineral accumulation involves not only collagen, an extracellular matrix protein, but also non-collagenous proteins of bone or dentine secreted at the calcification front. For example, at the onset of calcification, a phosphorylated extracellular matrix protein attaches to the hole in the collagen fibril, followed by the binding of calcium to phosphate in the protein to form apatite crystals. The matrix protein bound to the growing apatite affects the deposition of new minerals on the well-arranged, platelike crystals, and also affects the rate of ion accumulation, thereby determining the shapes and properties of the crystals. To understand the molecular mechanisms of dentinogenesis and osteogenesis, dentine and bone matrix proteins were isolated and characterized. These studies revealed not only the chemical and physical properties of the matrix protein but also its gene structure and its regulatory mechanisms, as well as its biological activity.

Non-collagenous Proteins in Bones and Dentine The properties of proteins in the extracellular matrices of bone and dentine have been studied extensively. In general, dentine matrix proteins

Non-collagenous Proteins Table 3.9

45

Non-collagenous proteins in bone and dentine

Non-collagenous proteins found in both bone and dentine

Protein Gla protein Glycoprotein Phosphoprotein Proteoglycan Blood protein

Non-collagenous proteins found only in dentine

and bone matrix proteins have similar characteristics. Consistent with this, extracellular matrix proteins isolated from dentine are similar to those isolated from bone, although the two materials differ quantitatively. As in bone, the main extracellular matrix protein of dentine is type I collagen, but it is present at a lower level in dentine than in bone. Osteonectin, osteocalcin, osteopontin, bone sialoprotein, decorin, and biglycan are common in bones and dentine. Dentine matrix protein-1 was first found in dentine but was subsequently also detected in bones. The extracellular matrix proteins of both bone and dentine contain a variety of cytokines, including growth factors, as well as albumin, a bloodderived protein.

Osteocalcin (Bone Gla Protein) Osteocalcin, or bone Gla protein, was originally detected in decalcified solutions of bone tissue treated with EDTA. It is found specifically in the hard tissues of vertebrates and is synthesized and secreted in osteoblasts, odontoblasts, and chondrocytes. The protein is characterized by the presence of three γ-carboxyglutamic acid (Gla) residues, which enable it to bind calcium. The Gla residues are present at amino acid positions 17, 21, and 24 of pre-pro-osteocalcin (Fig. 3.19). In vertebrate bone tissues, osteocalcin counts for 1–2% of total protein and 10–20% of non-collagenous protein. The human osteocalcin gene maps to chromosome 1q25-31, and is expressed in bones, heart, lung, and kidney, as well as other tissues. The amounts of osteocalcin in dentine and cementum are 1/4–1/2 of those in

Phosphoprotein

Example Bone Gla protein Matrix Gla protein Osteonectin Bone sialoprotein Osteopontin PG-I PG-II Albumin Immunoglobulin Phosphophoryn Dentine sialoprotein

bone tissue but not in the enamel. During bone growth, osteocalcin attaches after mineral deposition occurs. The Gla residues in osteocalcin are synthesized by fixing CO2 to the γ-carbon of glutamic acids in a reaction catalyzed by a vitamin K–dependent carboxylase (Fig. 3.20). Human osteocalcin is a 49-residue protein with a molecular weight of 5930 Da (Fig. 3.19). The three Gla residues are spaced 0.54 nm from one another, which is noteworthy given that the spacing of calcium ions in hydroxyapatite is 0.545 nm. Osteocalcin is an acidic protein with an isoelectric point of 4.0 and can bind calcium in apatite crystals through the two carboxyl groups of its Gla residues. In quantitative terms, 1 mg of osteocalcin can bind to 17 mg of apatite (Kd = 0.1 mM). Upon binding to apatite crystals, osteocalcin changes its steric structure and increases its α-helical content. Osteocalcin synthesized by osteoblasts is locally deposited within apatite crystals, but it is also present in blood at 6–7 ng/mL. The blood concentration of osteocalcin is a marker of bone metabolism, and it is elevated when bone metabolism is accelerated, potentially indicating bone disease. Blood osteocalcin concentration is elevated in bone tumors such as Paget’s disease, higher in men than in women, and also elevated during the immature period when bone formation is vigorous. Osteocalcin synthesis is markedly promoted by 1,25-dihydroxycholecalciferol [1,25 (OH)2D3], an active form of vitamin D. On the other hand, in a rat model of rickets, osteocalcin

46

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Fig. 3.19 Primary structure of osteocalcin. Five amino acid residues differ between the human and bovine proteins

Fig. 3.20 Vitamin K– dependent γ-carboxylation reaction. γ-Carboxyglutamates, which are present at residues 17, 21, and 24 of osteocalcin, form as a result of the γ-carboxylation reaction after incorporation of glutamic acid during protein synthesis

concentration in bone tissue is about 60% of normal. Initially, osteocalcin was believed to promote calcification of bone tissue, but it was later shown to inhibit excessive calcification. In addition, osteocalcin promotes chemotaxis by monocytes and macrophages. Because monocytes are precursor cells of osteoclasts, osteocalcin can be considered to promote bone resorption by attracting osteoclast precursor cells. Consistent with this, 1,25(OH)2D3 induces osteocalcin expression. Recently, matrix Gla protein, another protein containing Gla residues, was detected among the

organic components of decalcified bone tissue. It is the second vitamin K–dependent protein found in bones. Unlike osteocalcin, matrix Gla protein, is a water-insoluble protein of 79 residues (10 kDa). The hydroxyproline residue at position 9 of osteocalcin is not present in matrix Gla protein, although the latter does contain five Gla residues and one disulfide bond. Matrix Gla protein and osteocalcin do not exhibit immunological cross-reactivity, but it is likely that the two proteins originate from the same protein because they both share sequence homology near the Gla residues. Matrix Gla protein is synthesized and

Non-collagenous Proteins

excreted by vascular smooth muscle cells and chondrocytes and subsequently accumulates on bones, dentine, and cartilage. In contrast to osteocalcin, matrix Gla protein is expressed at the immature stage, indicating that it acts at the early stage of osteogenesis because it, and its expression, is promoted by 1,25(OH)2D3.

Osteonectin (SPARC, Culture Shock Protein, BM-40) Osteonectin can bind calcium, hydroxyapatite, and collagen, and was named based on its ability to convert hydroxyapatite deposits into crystals. The osteonectin gene maps to chromosome 5q31-33. Osteonectin is a non-collagenous protein of bone and dentine, with a molecular weight of about 35,000–45,000, that acts as a nucleator in the calcification process and promotes calcification in vitro. Osteonectin is abundant in bone tissue and is also expressed in a variety of connective tissues during development, maturation, and repair. It is present at high levels in bone, constituting 20–25% of non-collagenous proteins. In terms of amino acid composition, osteonectin contains large quantities of aspartic acid and glutamic acid. In vitro, the osteonectin– collagen complex acts as a nucleator, but osteonectin in the free state actually inhibits calcification. Osteonectin coexists with bony bridges undergoing calcification. It is synthesized and secreted by osteoblasts, binds to collagen fibers, and continuously captures calcium and phosphate ions to act as a nucleator in the calcification process. In addition, osteonectin is present in non-calcified tissues: SPARC (secreted protein, acidic, rich in cysteine; also called culture shock protein or BM-40), which was isolated from cells forming the basement membrane, is identical to osteonectin. Several structurally different types of osteonectins exist, but all are characterized by the presence of two EF-hands, which are high-affinity calcium binding sites. Bone Sialoprotein (BSP) Many of the matrix proteins that constitute bone tissue contain considerable amounts of sialic acid, and the corresponding genes mostly map to chromosome 4q21-23. Among them, osteopontin,

47

bone sialoprotein, and dentine matrix protein-1 (Dmp-1) have been extensively characterized (Ganss et al., 1999). In addition, dentine sialoprotein and dentine phosphoprotein, which are expressed only in dentine, are also produced by the same gene. Another bone sialoprotein, bone acidic glycoprotein 75, has also been described. Osteopontin (Spp-I, BSP-I) Osteopontin is an important phosphoprotein of bones that is also classified as a bone sialoprotein because it contains sialic acid (Dodek et al., 2000). The osteopontin gene maps to chromosome 4q13-21. Among the extracellular matrix proteins of bone, osteopontin is the main sialoprotein and was independently identified at the same time as bone sialoprotein. Consequently, osteopontin and bone sialoprotein were initially named bone sialoprotein-I (BSP-I) and bone sialoprotein-II (BSP-II), respectively. Osteopontin was subsequently renamed to reflect the potential role of bridges between cells and hydroxyapatite mediated by RGD (Arg-GlyAsp) and polyaspartate motifs found throughout the amino acid sequence. However, osteopontin has also been shown to be identical to Eta-1 (early T-lymphocyte activation gene 1), a novel lymphokine expressed in activated lymphocytes and macrophages. Accordingly, in recognition of the multiple functions of this protein, a new name has been proposed: phosphoprotein-I (Spp-I). However, the term osteopontin is still widely used. Rat osteopontin consists of 295 residues and contains an RGD sequence with cell-binding ability. Osteopontin is synthesized mainly by osteoblasts in immature bone tissues, but in mature bone tissues, it is synthesized mainly by osteoclasts, and it is present in both bones and osteoids. In addition to tissues, osteopontin is expressed in various tissues, including dentine, cementum, cartilage, kidney, brain, bone marrow–derived metrial gland cells, and vascular tissue. Osteopontin mediates the attachment of minerals and cells to the bone and hence appears before osteocalcin during the calcification process. On the other hand, osteopontin is expressed at the late stage of osteoblast maturation,

48

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corresponding to the matrix formation stage just before calcification occurs, and also induces an intracellular signaling pathway in osteoclasts. 1,25(OH)2D3 promotes the biosynthesis of osteopontin.

capacity is higher: ranked by binding activity, the three proteins are ordered bone acidic glycoprotein-75 > bone sialoprotein > osteopontin. Bone acidic glycoprotein-75 is present largely in the calcified region of new bone.

Bone Sialoprotein-II (BSP-II) The bone sialoprotein-II gene maps to chromosome 4q13-21 and is genetically linked to osteopontin. Bone sialoprotein-II has a molecular weight of 46–75 kDa; about 50% of its molecular weight is carbohydrate, with sialic acid accounting for 12%. Unlike osteopontin, it contains polyglutamic acid; overall, it is an acidic protein containing a small amount of phosphate and many aspartic acid and glutamic acid residues. In osteopontin, the RGD is located near the center of the protein, but bone sialoprotein-II has an RGD sequence at its C-terminus, and both Oglycoside and N-glycosides are present in its oligosaccharide chains. Bone sialoprotein-II accounts for about 12% of the non-collagenous protein found in bone and about 1% of that in dentine. Many sulfated tyrosine residues are present in the region adjacent to the RGD motif. The expression of bone sialoprotein-II is more limited than that of osteopontin and is closely associated with calcification. In the skeleton, bone sialoprotein-II is less widely expressed in chondrocytes and osteoblasts. Bone sialoproteinII is expressed at the end of osteoblast differentiation and the beginning of calcification. It acts as a nucleator for apatite formation, has various functions in the metabolism of osteoblasts, and has a strong affinity for calcium.

Dentine Matrix Protein-1 (Dmp-1) Dentine matrix protein-1 exists only in dentine, but it is synthesized by osteoblasts. It is also known as dentine-specific extracellular matrix protein and is expressed at lower levels than phosphophoryn. In addition, it is expressed along with phosphophoryn by odontoblasts at the time of odontogenesis. The protein consists of 473 residues. In addition, dentine matrix protein-1 is hydrophilic overall, except for a short portion at the C-terminus.

Bone Acidic Glycoprotein-75 (BAG-75) Bone acidic glycoprotein-75, with a molecular weight of 75,000, is expressed in developing bone tissues. It is a sialoprotein, and thus similar to osteopontin or bone sialoprotein-II, and has cell-attachment activity. Bone acidic glycoprotein-75 is a phosphorylated acidic protein synthesized by osteoblasts and expressed exclusively in bones and calcified cartilage. Its sialic acid content is 7%, and its phosphate content is 8%. Structurally, it is related to osteopontin and bone sialoprotein, but its calcium binding

Tetranectin Tetranectin is a trimeric protein found in woven bone and tumors that undergo calcification. The three identical polypeptide chains in this protein are noncovalently bound (Fig. 3.21). One chain consists of 202 residues, including a 21–amino acid signal peptide, with a molecular weight of about 20 kDa. This protein has high sequence

Fig. 3.21 Schematic diagram of tetranectin, which is present in both extracellular matrix and blood. Tetranectin, a homotrimer, is a plasminogen-binding protein. It is strongly expressed during chondrogenesis

Non-collagenous Proteins

similarity to the globular domains of aggrecan and asialoglycoprotein receptors.

Thrombospondin Thrombospondin is a protein with diverse functions. It was initially identified as an abundant protein in platelets but was later found in various tissues, including bone. Subsequently, four types of thrombospondin were identified. All four are present in bones, but they are synthesized by different types of cells, depending on the maturation process and developmental stage. Thrombospondin can bind to a variety of matrix and cell-surface proteins. Fibronectin Fibronectin is synthesized in various types of cells in connective tissue and is an important constituent of serum. Several isoforms are synthesized from the same gene via alternative splicing. As a result, the bone matrix contains both endogenous fibronectin and exogenous fibronectin. Fibronectin is produced at an early stage of osteogenesis and is highly expressed in the osteoblast layer. Vitronectin Vitronectin was first discovered as an S-protein because of its cell-spreading activity. As an abundant multifunctional glycoprotein found in serum, the extracellular matrix, and bones, vitronectin is involved in various physiological processes and promotes cell attachment, spreading, and migration. Integrin αvβ3, a cell-surface receptor of vitronectin, is distributed widely throughout the bone tissue. Proteoglycan Proteoglycans are proteins covalently bonded to glycosaminoglycans, long unbranched polysaccharides consisting of a repeating disaccharide unit. Glycosaminoglycans occupy as much as 95% of a proteoglycan by weight and thus resemble a polysaccharide rather than a protein. Proteoglycan properties are determined primarily by the glycosaminoglycan component. Many glycosaminoglycans consist of repeating units of disaccharides containing a derivative of

49

amino sugar. At least one of the two sugars in the repeating unit has a negatively charged carboxylate or sulfate group. The major glycosaminoglycans in animals are chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, heparin, and hyaluronate. Large Proteoglycans: Aggrecan and Versican Two large chondroitin sulfate proteoglycans are present in bone tissue, aggrecan and versican (Fig. 3.22). The core protein has globular domains at the N- and C-termini and binds to hyaluronan to form a large proteoglycan aggregate. Aggrecan has cartilage specificity, but its mRNA is also expressed in developing bone. The chick model of nanomelia harbors a mutation in the core protein gene of aggrecan and does not produce cartilage. The proteoglycan of soft connective tissue associated with intramembranous ossification is called versican. Versican is thought to play a role in securing the space that ultimately becomes a bone, and gradually breaks down as bone is formed. As osteogenesis progresses, versican is replaced by a series of other small proteoglycans. Small Proteoglycans: Decorin and Biglycan Decorin and biglycan are small proteoglycans found in the bone matrix (Embery et al., 2001); bone has chondroitin sulfate chains, whereas soft connective tissue has dermatan sulfate chains (Fig. 3.23). The core proteins contain a leucinerich repeat structure. As bone formation progresses, the large proteoglycan versican is replaced by smaller proteoglycans. The core proteins of decorin and biglycan are very similar, but their expression patterns are very different. In the cartilage, decorin is found in the interterritorial matrix far from chondrocytes and is widely expressed throughout the cell; the expression pattern is almost indistinguishable from that of type I collagen during endochondral ossification. Decorin is maintained in mature osteoblasts after first being expressed in preosteoblasts but subsequently becomes embedded in the extracellular matrix until the osteoblasts become osteocytes, at which time its levels gradually decrease. By contrast, biglycan is found in the intraterritorial

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Fig. 3.22 Structures of aggrecan and versican. Aggrecan has multiple chondroitin sulfates bound to the core protein, as well as keratan sulfate. By contrast, versican has a smaller number of chondroitin sulfates

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Dentine

and keratan sulfates. Both proteins contain binding sites for hyaluronan (HABR), glycosaminoglycan (GAG), liver endothelial cells (LEC), and epidermal growth factor (EGF)

Fig. 3.23 Structures of biglycan (a) and decorin (b). CRD, carbohydrate recognition domain; CBP, cAMP-response element-binding protein (CREB)-binding protein; G1, G3, globular domain; CS, chondroitin sulfate

matrix and is distributed only around cells. The level of biglycan increases until preosteoblasts mature into osteoblasts, and the protein is also expressed in the lacunae of osteoclasts. Decorinknockout mice have thin skin, whereas biglycanknockout mice fail to reach peak bone mass and exhibit osteopenia. Decorin and biglycan are present in noncalcified soft connective tissue, but they are also found in osteoids, in which they act as nucleators for the deposition of hydroxyapatite. Biglycan has a low affinity for calcium, which aids in the deposition of hydroxyapatite at low concentration but hinders deposition at high

concentration. With a molecular weight of 74,600 Da, biglycan is a major small proteoglycan in bone. Decorin is expressed in bone, osteoids, osteoblasts, and preosteoblasts. Dentine also contains small proteoglycans, including decorin and biglycan. The glycosaminoglycan component of proteoglycans of dentine consists mainly of chondroitin 4-sulfate, and the tissue also contains a large proteoglycan similar to cartilage proteoglycan. The total amount of proteoglycan is higher in predentine than in dentine.

Non-collagenous Proteins

Hyaluronic Acid (Hyaluronan) Hyaluronic acid (also called hyaluronan) is unique among glycosaminoglycans in that it does not contain any sulfate and is not found covalently attached to proteins that form a proteoglycan. Unlike other glycosaminoglycans, it is synthesized in the extracellular environment by a series of enzymes located on the outer cellular membrane. Large quantities of hyaluronic acid are synthesized at the early stage of osteogenesis, and hyaluronic acid forms a high molecular weight proteoglycan aggregate with versican. However, this large aggregate is not found in developing bone. Therefore, it is likely that the necessary space is secured before osteogenesis, and subsequently, the huge proteoglycan aggregate is degraded and replaced by bone during osteogenesis.

Dentine-Specific Non-collagenous Proteins The proteins present in the extracellular matrix of dentine have been extensively studied. Phosphophoryn (dentine phosphoprotein) and dentine sialoprotein are found exclusively in dentine. These proteins are synthesized and secreted by preameloblasts, as well as immature or mature odontoblasts. Given that bone matrix protein is synthesized solely by osteoblasts, it is noteworthy that a specific protein is expressed only in dentine. Phosphophoryn and dentine sialoprotein are synthesized from the same gene, which maps to human chromosome 4. Initially, it was thought that phosphophoryn and dentine sialoprotein were unrelated, but cloning experiments revealed that they originate from the same precursor, dentine sialophosphoprotein.

Phosphophoryn (Dentine Matrix Protein-1) Phosphophoryn, first discovered by Veis et al. 1972, is the second most abundant protein in dentine following collagen. It has an isoelectric point of 1.1 and is composed of 35% aspartic acid and 45–50% phosphoserine. The molecular

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weight of phosphophoryn varies from species to species: 72 kDa in mice, 155 kDa in bovines, 90–95 kDa or 38 kDa in rats, and 140 kDa in humans. Analysis of rat phosphophoryn cDNA revealed that it has a molecular weight of 33 kDa and 240 residues, in which 87% of its serine residues are phosphorylated. The primary structure of phosphophoryn is (Asp-X)n (X is mostly serine or phosphoserine), (Asp)n, and (p-Ser)n. Due to a large number of negative charges and shortage of hydrophobic acids, phosphophoryn has a disordered threedimensional structure, but it forms a disc-shaped aggregate in the presence of calcium ions. Because phosphophoryn is a strongly acidic protein, it can bind most divalent cations, but its binding to calcium is particularly important in the context of calcification. Binding to calcium involves the phosphate group of phosphoserine and the carboxyl groups of acidic amino acids such as aspartate and glutamate. Calcium–phosphate or calcium–carboxyl binding causes the protein to precipitate. In addition to calcium, phosphophoryn also has an affinity for apatite. The demineralization process releases soluble phosphophoryn, of which some may form crosslinks through covalent bonding to collagen. Synthesis and secretion of phosphophoryn are performed by odontoblasts. Phosphophoryn synthesized by odontoblasts accumulates on the dentine side of the predentine–dentine boundary. Consequently, phosphophoryn is present only in calcified dentine, but not in uncalcified predentine. This is in contrast to collagen or osteonectin, which is present in both dentine and predentine, and proteoglycan, which is present only in predentine.

Dentine Sialoprotein Dentine sialoprotein, first isolated from dentine extracts, is a glycoprotein that contains a high level of sialic acids. It has a molecular weight of 35 kDa and contains large proportions of aspartic acid, glutamic acid, serine, and glycine residues; 30% of its mass is carbohydrate. Dentine sialoprotein is present in immature odontoblasts, mature odontoblasts, and dentine but not bones, cartilage, or soft connective tissue (Butler, 1998).

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It is also found in preameloblasts, but not in ameloblasts. These observations suggest that dentine sialoprotein plays an important role in dentinogenesis. Dentine sialoprotein contains six Asn-X-Ser/Thr motifs that serve as recognition sites for N-linked glycosylation. In addition, it has 13 Ser/Thr phosphorylation sites at the C-terminus. Dentine sialoprotein promotes crystal growth, but its effect is weaker than that of osteopontin or phosphophoryn.

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Dentine

unknown. Cytokines, including growth factors, are soluble proteins that have the ability to alter the activities of other cells. Like bone, dentine contains insulin-like growth factor (IGF)-1, IGF-2, platelet-derived growth factor, epidermal growth factor, vascular endothelial growth factor, platelet growth factor, fibroblast growth factor, metalloproteinase, members of the transforming growth factor-β superfamily, and other factors. In general, these proteins promote either bone formation or bone resorption, but recent studies have reported conflicting results.

Extrinsic Non-collagenous Proteins Because hydroxyapatite is present in the bone matrix, proteins that are synthesized in places other than bone and enter through the blood can also be adsorbed onto apatite. Most of these proteins are synthesized in the liver or hematopoietic tissue, including immunoglobulin, carrier protein, cytokines, chemokines, and growth factors. Interestingly, some of these proteins are also synthesized in osteoblast-lineage cells.

Serum Protein Albumin is synthesized in the liver and is present at a higher concentration in bone than in blood. This protein hinders the growth of hydroxyapatite by binding to the surface of seed crystals in vitro. Albumin also inhibits the aggregation of crystals. Soluble Factors Enzymes and cytokines have been detected in bones and dentine, but their function is still

References Brodsky, B., Shah, N.K.: The triple helix motif in proteins. FASEB J. 9, 1537–1546 (1995) Butler, W.T.: Dentin matrix proteins. Eur. J. Oral Sci. 106(suppl. 1), 204–210 (1998) Cole, A.S., Eastoe, J.E.: Biochemistry and Oral Biology, 2nd edn. Wright, Bristol (1988) Dodek, J., Ganss, B., McKee, M.D.: Osteopontin. Crit. Rev. Oral Biol. Med. 11, 279–303 (2000) Embery, G., Hall, R., Waddington, R., Septier, D., Goldberg, M.: Proteoglycans in dentinogenesis. Crit. Rev. Oral Biol. Med. 12, 331–349 (2001) Fearnhead, R.W., Stack, M.V.: Tooth Enamel. John Wright and Sons (1965) Ganss, B., Kim, R.H., Sodek, J.: Bone sialoprotein. Crit. Rev. Oral Biol. Med. 10, 79–98 (1999) Veis, A., Spector, A.R., Zamoscianyk, H.: The isolation of an EDTA-soluble phosphoprotein from mineralizing bovine dentin. Biochim. Biophys. Acta. 257, 404– 413 (1972) Weast, R.C.: Handbook of Chemistry and Physics. Academic Press (1967)

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Pulp

Pulp is a connective tissue surrounded by a dental hard tissue, dentine, and is unique in that it does not permit volume change. Unlike enamel, dentine, and cementum, pulp contains dense concentrations of blood vessels, nerve fibers, various cell types, and extracellular matrix. Therefore, the pulp is the most metabolically active part of a tooth. From the histological point of view, pulp is divided (starting from the boundary with the dentine) into the odontoblast zone, the cellpoor zone, the cell-rich zone, and the pulp proper zone. Thus, the pulp is located in a limited space in which changes in volume cannot be allowed, but the components of the pulp are highly organized (Fig. 4.1). Odontoblasts layered at the boundary between the pulp and dentine synthesize primary dentine at the developmental stage and continue to synthesize secondary dentine after the tooth erupts. Pulp can restore damaged teeth by making reactive dentine or reparative dentine in response to external influences such as dental caries or trauma. In addition, pulp can nourish odontoblasts maintaining dentine, sense stimuli applied to the teeth, and participate in immune defense against impurities that penetrate through damaged dentine or dentinal tubules. Thus, the structural and functional aspects of the pulp are important for the maintenance of healthy teeth, and it is correspondingly important to understand the physiochemical properties of the pulp. Pulp and dentine are closely related to each other in developmental and functional terms and

tend to be described as the dentine–pulp complex rather than as independent tissues. However, the two tissues are distinguished from each other by the clear difference between soft and hard tissue. Odontoblasts synthesize dentine during the developmental stage, but serve to maintain and repair dentine after the development is complete; this characteristic leads us to classify odontoblasts as a type of dentinal cell. Because odontoblasts are closely related to pulp, in the sense that they obtain nutrition and oxygen from pulp and carry out their functions, this chapter also describes the physiochemical characteristics of odontoblasts.

Composition of Pulp Pulp is mainly composed of cells and an extracellular matrix and is thus considered a connective tissue. The odontoblasts form a layer at the boundary between dentine and pulp. Pulp contains fibroblasts, which are connective tissue cells responsible for the synthesis, secretion, and turnover of the extracellular matrix. In addition, pulp includes cells responsible for immune function, as well as a well-developed vascular system that provides nutrition and oxygen and helps to remove waste. In addition, a nervous system that senses stimulation is also distributed throughout the pulp. Table 4.1 shows the cells and extracellular matrix distributed in pulp and dentine.

# The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 B.-M. Min, Oral Biochemistry, https://doi.org/10.1007/978-981-99-3596-3_4

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Pulp Cells Odontoblasts Odontoblasts are formed by the differentiation of ectomesenchymal cells at the boundary between the dental papilla and the epithelial layer, and they are layered at the interface between the dentine and the pulp. During tooth development, ectodermal mesenchymal cells (preodontoblasts) near the basement membrane differentiate into odontoblasts under the control of growth factors secreted from inner enamel epithelial cells into the basement membrane (Fig. 4.2). It remains unclear which growth factors are involved in the differentiation of odontoblasts in vivo. However, in vitro experiments have revealed that transforming growth factor-beta 1 (TGF-β1), TGF-β3, bone morphogenetic protein2 (BMP-2), and insulin-like growth factor-1 (IGF-1), are involved in odontoblast differentiation. In preodontoblasts, only cells in contact with the basement membrane differentiate into odontoblasts with spatial specificity. This is likely because growth factors secreted from inner enamel epithelial cells bind to the basement membrane and consequently do not diffuse into the dental papilla. Fully differentiated odontoblasts can no longer divide. Morphologically, odontoblasts are shaped like elongated cylinders at the crown but are more cuboidal at the root. Within the dentinal tubule, the odontoblast has an odontoblastic process that consists of actin fibers and microtubules and does not contain any organelles. The odontoblastic process is the extension of the odontoblastic cytoplasm into the dentine and performs functions such as the transport of materials and structural support of the dentine. Cell junctions (tight junctions, gap junctions, and desmosomes) similar to those in epithelial cells also exist between odontoblasts. After differentiation, the nuclei of odontoblasts, which do not undergo further cell division, are biased toward the pulp. In addition, intracellular organelles such as the endoplasmic reticulum and Golgi apparatus, which are involved in synthesis and secretion, are

4 Pulp

distributed throughout these cells, and secretory granules can be also observed (Fig. 4.3). The cell bodies of odontoblasts are primarily involved in the synthesis and regulation of secretory substances, and the secretion and reabsorption of the extracellular matrix occur in the odontoblastic process. The primary function of odontoblasts is the production of dentine. Odontoblasts secrete type I collagen, which is involved in the calcification of dentine; small amounts of type III and type V collagen; and other non-collagenous proteins such as dentine sialophosphoprotein, dentine matrix protein, osteopontin, bone sialoprotein, osteonectin, and osteocalcin. Fully differentiated odontoblasts secrete growth factors, which can bind to decorin or biglycan, the proteoglycans constituting the dentine, and these growth factors are released when the dentine is damaged. Growth factors either activate pre-existing odontoblasts or promote the differentiation of undifferentiated cells in the pulp into odontoblast-like cells, which then form tertiary dentine.

Fibroblasts Fibroblasts are major cellular constituents of the pulp, and they exhibit varying degrees of differentiation. Mature pulp fibroblasts are elongated, assume a fusiform morphology, and have relatively large nuclei relative to the size of the cytoplasm. In pulp fibroblasts, the Golgi apparatus is near the nucleus, and the rough endoplasmic reticulum is well-developed. Secretory granules are also observed in pulp fibroblasts. Consequently, pulp fibroblasts with well-developed intracellular organelles, which can secrete materials into the extracellular space, synthesize and secrete collagen and the other matrix materials that comprise pulp. Pulp fibroblasts are connected to other fibroblasts by desmosome-like junctions and exchange substances between cells through gap junctions. Immune Cells The healthy pulp contains immune cells such as dendritic cells, macrophages, and lymphocytes. Most of these cells express type II major histocompatibility complex protein (MHC II).

Composition of Pulp

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Fig. 4.1 (a) Photograph showing the dentine–pulp border. (b) Schematic diagram showing several layers of pulp

Dendritic cells act as antigen-presenting cells to stimulate T cell–dependent immune responses. Dendritic cells are mainly distributed around the blood vessels of the pulp and in the boundary between pulp and dentine. Antigens that penetrate following dentine injury are firstly processed by dendritic cells, which express them on the cell surface bound to MHC II. These antigenpresenting dendritic cells activate T cells in the lymph nodes (Fig. 4.4). Pulp macrophages are mainly distributed near blood vessels. Macrophages absorb dead cells and tissue-excreted substances by endocytosis and degrade these materials in the lysosome. In addition to fibroblasts, pulp macrophages help to replace cells and materials that comprise the pulp tissue. They also eliminate bacteria and engage in antigen presentation when an infection is present. Thus, dendritic cells and macrophages perform an immunosurveillance function in the pulp. Macrophages synthesize and secrete interleukin1, tumor necrosis factor (TNF), growth factor, and cytokines. The lymphocytes in pulp are mainly T, T3, and T8 cells, whereas B cells are rare. Mast cells are uncommon in healthy pulp, but they are detectable when the pulp is inflamed. When inflammation occurs, mast cells secrete histamine to cause vasodilation and increase the permeability of blood vessels, causing

lymphocytes and neutrophils to migrate into the pulp from capillaries or venules.

Undifferentiated Cells Among the mesenchymal cells present in the dental papilla during the early developmental stage, the pulp contains some cells that are not completely differentiated. These undifferentiated cells are distributed throughout the pulp, especially in the cell-rich zone. They include a type of fibroblast that can be dedifferentiated by appropriate stimulation and then redifferentiated. However, relative to fully differentiated fibroblasts, the ability of these cells to synthesize and secrete collagen is considerably reduced, and they are not considered to be mature fibroblasts. These undifferentiated cells can differentiate into odontoblast-like cells that are involved in the repair of damaged dentine. In particular, undifferentiated cells distributed near the blood vessels of the pulp differentiate into mast cells or odontoclasts upon inflammation.

Changes in the Distribution of Pulp Cells The lifespan of pulp cells is not known, but as dental pulp ages, the total number of cells decreases. This phenomenon is related to the

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4 Pulp

Table 4.1 Cells and extracellular matrix components found in dentine and pulp Cells

Collagens

Non-collagenous proteins (10% of the dentine ECM)

Dentine Odontoblasts exclusively

Types I and I trimer (98%) Types III (1–2%) and V (1%) (90% of the dentine ECM) Phosphorylated matrix proteins (SIBLINGs): DSPP > DSP and DPP DMP-1, BSP, OPN, MEPE Non-phosphorylated matrix proteins: Matrix GLA protein, osteocalcin, osteonectin Proteoglycans (SLRPs) CS/DS PGs: Decorin-biglycan (CS-4 81%, CS-6 14%, CS/DS 2%) KS PGs: lumican, fibromodulin, osteoadherin Amelogenin 5–7 kDa Growth factors: TGF-β, ILGF-I and -II, FGF-2, VEGF, PDGF Metalloproteinases: collagenase (MMP-1), gelatinases (MMP-2 and -9), stromelysin-1 (MMP-3), enamelysin (MMP-20), MT1-MMP, TIMP-1 to -3 Alkaline phosphatase Serum-derived proteins: αHS2-glycoprotein, albumin, lipoproteins Phospholipids: Membrane phospholipids (66%) Extracellular-mineral-associated phospholipids (33%)

Pulp Fibroblasts (pulpoblasts), vascular cells, pericytes, neural cells, histiocytes/ macrophages, dendritic cells, lymphocytes, mast cells Type I (56%) Types III (41%) and V (2%); Types VI (0.5%) associated with microfibrillin

None BSP, OPN Fibronectin Osteonectin (in tooth germs) Versican CS-4 and -6, 60%; DS, 34%; KS, 2% Hyaluronic acid

BMPs Types IA and II receptors for TGF-β, activin, and BMPs MMPs: collagenases, gelatinases, stromelysin1

TIMPs Fibronectin of serum origin

Membrane and ECM phospholipids

DSPP dentine sialophosphoprotein, DSP dentine sialoprotein, DPP dentine phosphoprotein, DMP-1 dentine matrix protein-1, BSP bone sialoprotein, OPN osteopontin, MEPE matrix extracellular phosphorylated protein, TGF-β transforming growth factor-β, IGF-I insulin-like growth factor-I, FGF-2 fibroblast growth factor-2, VEGF vascular endothelial growth factor, PDGF platelet-derived growth factor, MMP matrix metalloprotease, TIMP tissue inhibitor of matrix metalloprotease, CS chondroitin sulfate, DS dermatan sulfate, PG proteoglycan, CS-4 chondroitin 4-sulfate, CS-6 chondroitin 6-sulfate, KS keratan sulfate, BMP bone morphogenetic protein

formation of secondary dentine by odontoblasts even after tooth eruption, which decreases the volume of the pulp and the area of the interface between dentine and pulp. In particular, the number of odontoblasts and other cells that are just below the odontoblasts decreases as aging progresses. In these regions, the cell fragmentation characteristic of apoptosis can be observed. However, considering that pulp is trapped in a

limited space, the change in the number of pulp cells can be affected not only by cell death but also by cell differentiation. Therefore, changes in the distribution of pulp cells in relation to their differentiation should also be studied.

Composition of Pulp

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collagen. The elastic fibers found in the pulp are distributed only on the outer wall of the arteriole.

Fig. 4.2 Schematic diagram showing secretion of growth factors that induce differentiation of odontoblasts. Growth factors secreted from the inner enamel epithelial cells promote the differentiation of ectomesenchymal cells of dental papilla, which reside in the basement membrane and remain in contact with it, into odontoblasts

Extracellular Matrix of the Dental Pulp Fibrous Proteins The majority of fibrous proteins present in the pulp are collagen and elastin, which are synthesized and secreted mainly by the fibroblasts of the pulp. Collagen fibers of the pulp are mostly type I (56%) and type III (41%) with small amounts of type V (2%) and type VI (0.5%). By contrast, in dentine the fibrous protein is more than 90% type I collagen. The collagen fibers of the pulp have a loose reticular arrangement that supports the structure of the pulp. In addition, microfibrils of 10–14 nm in diameter, including fibrillin, are sometimes combined with type VI

Fig. 4.3 Schematic diagram of a fully differentiated odontoblast

Ground Substance In addition to interstitial fluid, the ground substance of the pulp consists of a gel with no consistent structure. It is composed of various glycoproteins, proteoglycans, glycosaminoglycans, and other molecules. A typical glycoprotein of pulp is fibronectin, which is derived from both pulp cells and serum. Fibronectin, along with collagen fibers, forms a fibrous network that affects cell attachment, migration, growth, and differentiation. The principal glycosaminoglycans of the pulp are chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid. Except for hyaluronic acid, most are present as proteoglycans. A representative proteoglycan of pulp is versican. The ground substance serves not only to maintain the structure of the pulp but also to act as a molecular sieve for molecules such as proteins and urea. Because the ground substance contains many charged molecules, it has a high waterretention capacity and therefore contains a large volume of interstitial fluid. Consequently, it has a strong ability to withstand pressure and serves to buffer the pressure of the pulp cells and vascular system. The composition of the interstitial fluid in the pulp is similar to that of plasma but contains a smaller amount of protein. The interstitial fluid provides an aqueous environment in which

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4 Pulp

Fig. 4.4 Interaction between pulpal dendritic cells and T-lymphocytes in the induction of a primary immune response. The dendritic cells are stained by immunohistochemistry and anti-HLA-DR antibodies. Scale bar, 20 μm. [From M. Jontell et al., Crit. Rev. Oral Biol. Med. 9:179–200, 1998]

water-soluble molecules can move between pulp cells and blood vessels, thus serving as a link between pulp cells and the plasma and the lymphatic fluid. Therefore, the interstitial fluid of the pulp can be considered as an expanded plasma. The interstitial fluid in the pulp, plasma, and lymphatic fluid are exchanged by diffusion driven by the pressure difference between the blood and tissue (Fig. 4.5).

pulp cells can maintain their metabolism and perform their functions, at least to some extent, even under ischemic conditions.

Metabolism of the Pulp The metabolism of the pulp has been studied in in vitro experiments aimed at measuring oxygen consumption, carbon dioxide production, and lactic acid production in this tissue. Pulp metabolism is more active in the developmental phase, when dentinogenesis is active, after the dentine is completely formed. Accordingly, the ATP level is higher in developing pulp than in mature pulp cells. Moreover, the formation of secondary dentine in fully erupted teeth is consistent with the active metabolic activity of odontoblasts. Both aerobic and anaerobic metabolism occurs in the pulp. When the pulp is severely inflamed, anaerobic metabolism is predominant, indicating that

Fig. 4.5 Schematic diagram showing the movement of interstitial fluid. Diffusion due to differences in blood and tissue pressure causes the movement of plasma and interstitial fluid. Because pulp cells are surrounded by interstitial fluid, the interstitial fluid can be thought of as expanded plasma. At steady state, the blood pressure of the pulp and the pressure of the interstitial fluid is 5–20 mmHg

Pulp Repair and Related Signaling

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Collagen metabolism of the pulp is thought to be correlated with odontogenesis; in other words, the turnover of collagen occurs more rapidly in the teeth during their development and eruption than in mature teeth. In addition, collagen degradation in inflamed pulp occurs faster than in normal pulp, likely due to collagenase secreted by cells involved in the inflammatory response. The level of glycosaminoglycan in pulp decreases with aging, concomitant with changes in the glycosaminoglycan composition. In particular, the content of the glycosaminoglycan containing sulfate groups in the tooth germ was about 60% of total glycosaminoglycan but decreased to about 13% during the period of root formation and was only about 2% in the erupted tooth. Thus, the protein and nonprotein components in the pulp are replaced, and this phenomenon is also related to odontogenesis and repair of the teeth.

Pulp Repair and Related Signaling One of the most interesting features of the pulp is its role in dentine restoration (Cohen and Burns, 2002; Goldberg and Smith, 2004). The restoration of damaged dentine can be categorized as reactionary or reparative dentine, depending on the degree of injury (Fig. 4.6). Formation of reactionary dentine is observed in situations such as superficial dental caries, in which damage to the dentine does not reach the pulp. Growth factors, which are secreted from odontoblasts during the developmental stage, are associated with the extracellular matrix of the dentine (Smith, 2003). When the dentine is damaged, these molecules are released, stimulating pre-existing undamaged odontoblasts to form reactionary dentine by promoting the secretion of dentineforming substances. Odontoblasts promote the release of dentine-restorative materials by secreting growth factors such as TGF-β1, TGF-β3, and BMP-7. The formation of reparative dentine implies restoration by dentine-forming substances secreted by odontoblast-like cells after some of the pulp cells newly differentiate into odontoblast-like cells. This occurs in cases of

Fig. 4.6 Schematic diagram showing signal transduction related to the formation of reactionary dentine and reparative dentine

severe damage, including loss of odontoblasts. The fibroblasts distributed directly below the odontoblasts or undifferentiated mesenchymal cells, which reside in the center of the pulp and migrate to the interface between pulp and the dentine when the dentine is damaged, differentiate into odontoblast-like cells. As in the formation of reactionary dentine, growth factors adhered to the dentine matrix are released by injury, and they stimulate the aforementioned cells to differentiate into odontoblast-like cells. In cells stimulated by growth factors, expression of the transcription factor activator protein-1 (AP-1) increases and in turn drives the expression of osteocalcin, alkaline phosphatase, and collagen. At this time, the expression of proteins such as the Notch receptor and Delta 1 ligand, which are normally expressed during tooth development but not in mature teeth, recurs. Type I collagen is expressed in the odontoblast-like cells involved in the synthesis

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of reparative dentine, whereas type III collagen and dentine sialoprotein are not expressed. Identification of the genes expressed in the pulp at the time of tooth injury will improve our understanding of the differentiation of odontoblast-like cells and the regulation of tooth repair.

References Cohen, S., Burns, R.C.: Pathway of the pulp, 8th edn. Mosby, St. Louis (2002)

4 Pulp Goldberg, M., Smith, A.J.: Cells and extracellular matrices of dentin and pulp: a biological basis for repair and tissue engineering. Crit. Rev. Oral Biol. Med. 15, 13– 27 (2004) Jontell, M., Okiji, T., Dahlgren, U., Bergenholtz, G.: Immune defense mechanism of the dental pulp. Crit. Rev. Oral Biol. Med. 9, 179–200 (1998) Smith, A.J.: Vitality of the dentin-pulp complex in health and disease: growth factors as key mediators. J. Dent. Educ. 67, 678–689 (2003)

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Calcification of Bones and Teeth

Calcification (mineralization) refers to a series of processes by which insoluble calcium salts are deposited on the extracellular matrix, which is secreted from unique cells such as fibroblasts, osteoblasts, and odontoblasts. However, many pathological conditions are associated with abnormal calcification of soft tissues, including blood vessels (arteriosclerosis), kidney, and brain. Calcification occurs in a variety of organisms ranging from unicellular organisms to plants and vertebrates and involves various levels of organic and inorganic components. Table 5.1 describes the calcified tissues that are found in different organisms and their major organic and inorganic components.

Calcified Biological Tissue The earliest observation of the calcification phenomenon was reported in a small cone-shaped calcium carbonate fossil of the Precambrian Cloudina (Grant, 1990). The ocean in the Cambrian period had a high mineral content, and organisms needed a protective mechanism against calcification. One of these protective mechanisms consisted of the secretion of charged polymers such as mucus, carbohydrates, and proteins. These organic constituents are believed to be formed the first biominerals together with minerals. The calcification inhibition strategy is well preserved in ocean organisms with shells. Many marine organisms evolved to have shells

only in the form of exoskeletons, which have a protective function against predators. In many organisms, calcified tissues such as the shell of mollusks and the exoskeleton of crustaceans were retained even after the evolution of movement and adaptation to land. Calcium stabilizes proteins and nucleic acids involved in cellular metabolism, and it functions as a second messenger in signal transduction pathways. In addition, calcium plays an important role in muscle contraction and tissue stabilization through strong coupling in epithelial cells. Phosphate is a component of nucleic acids and plays an important role in energy metabolism and signal transduction pathways. However, under certain conditions, calcium and phosphate are converted into insoluble calcium phosphate salts through the process of calcification. Because the solubility of ions in the tissue fluids of organisms depends on the metastable concentrations of calcium and phosphate, precipitation of these ions can occur in the extracellular matrix. Organisms have evolved the ability to retain minerals to adapt to the land environment. Therefore, bone formation in vertebrates can be considered as a mechanism for the maintenance of calcium homeostasis. The skeleton of animals was established from approximately 544 million years ago (early Precambrian) to 300 million years ago. The soft tissues of organisms inhibit unnecessary calcification to maintain their unique capabilities. In this chapter, we explain the

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mechanism by which certain cells of calcified tissues, including bones and teeth, induce calcification.

Function and Structure of Bone Bone tissues have several functions: they (a) protect the internal organs of the human body (heart, lung, and brain among others) from an external force, (b) maintain hematopoietic function by protecting the bone marrow, (c) maintain form and motion by providing support, and (d) function as a reservoir for minerals such as calcium and phosphate to regulate blood acid–base equilibrium and remove toxic heavy metals from the blood. Bone tissues with various functions can be classified according to their structure as cortical bone and trabecular bone (spongy bone). Cortical bone forms the outer part of the body in the long bones, whereas spongy bone provides mechanical support at the proximal and distal ends of the bone. X-ray images show the organized pattern of the long bone containing thick cortical bone and spongy bone (Fig. 5.1). Magnification of the cross-sectional images of cortical bone demonstrates that it is composed of an osteon or Haversian system (Fig. 5.2). Osteon refers to a concentric structure around the central canal through which blood vessels pass; a newly formed osteon shows perfectly preserved concentric circles. Osteons demonstrate the continuous process of bone remodeling. The small dots in the concentric circles indicate the space occupied by osteocytes (Fig. 5.2).

Chemical Composition of Bone and Bone Tissue Cells Inorganic Components of Bone The inorganic components of bones account for 65% (wt%) of bone weight and have the basic crystal structure of hydroxyapatite [Ca10(PO4)6(OH)2]. Bone tissues in the human body serve as a reservoir for minerals, containing 99% of the calcium and 85% of the phosphorus of

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the whole body. In addition, bone tissues contain small amounts of trace minerals and heavy metals that are absorbed by the body. Table 5.2 shows heterogeneous ions that can be substituted by calcium, phosphate, and hydroxyl ions in hydroxyapatite.

Organic Components of Bone Bones are composed of approximately 25% (wt %) of organic components and 10% (wt%) of water. More than 90% of the organic matrix of bones consists of collagen, which plays an important role in calcification. Collagen in bone tissues is mainly composed of type I collagen. The basic unit, a collagen monomer called tropocollagen, forms the collagen fibril via polymerization, and collagen fibrils associate to form the collagen fiber. Collagen functions in a manner similar to iron bars in a concrete building. Non-collagenous organic materials are described in Table 5.3. Loss of the genetic function of these materials in knockout mice suggests the function of each non-collagenous protein. Cells in Bone Tissue Bones and teeth are representative calcified tissues that are physically hard, and their study is therefore challenging. However, advances in imaging techniques such as X-ray, computerized tomography (CT), and magnetic resonance imaging (MRI), and histological analysis techniques such as hard tissue microtome, as well as transgenic animals generated by gene targeting and gene transfer have stimulated the development of bone research. Bone disorders are classified into three categories: (a) alterations in bone size during development and growth (dwarfism and gigantism), (b) defects in the calcification of bones (rickets and osteomalacia), and (c) defects in bone remodeling (osteoporosis and osteopetrosis) (Fig. 5.3). Alveolar bone resorption, a symptom of periodontal disease, refers to defects in bone remodeling caused by inflammation (Lee et al., 2022). The promotion of osteogenesis by orthodontic force and pulling aims to induce intentional bone remodeling via a physical stimulus. Bone disorders occur when the dynamic equilibrium between cells that function in bone

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Table 5.1 Some mineralized tissues and their major inorganic and organic constituents found in various groups of living organisms Organism Diatoms

Mineralized tissue Shell

Inorganic components SiO2

Higher plants

Cell wall

CaCO3 (Calcite)

Radiolaria Brachiopods (Lingula)

Exoskeleton Shell

SrSO4 Hydroxyapatite

Molluscs Arthropods (Squilla)

Exoskeleton Chellae

CaCO3 Calcite Aragonite Hydroxyapatite

Organic components Polyuronic acids Amino acids Polyamines Cellulose Pectins Lignins ? Chitin Protein Chitin Protein Chitin

Vertebrates

Epithelia Balleen Claws, Nails, Feathers Tooth Enamel Dentine Cementum Skeleton Bone Cartilage Pathological Renal calculi

Hydroxyapatite Hydroxyapatite

Keratins Intracellular mineralization

Hydroxyapatite Hydroxyapatite Hydroxyapatite

Amelogenins Collagen Collagen

Hydroxyapatite Hydroxyapatite

Collagen Collagen + Chondroitin sulphate

Ca salts

Glycoproteins

[Source: S. Cole and J. E. Eastoe, Biochemistry and Oral Biology, 2nd ed. (Wright, 1988), Table 28.1]

formation and resorption is altered. Therefore, improving our understanding of the formation, growth, differentiation, and death of these cells may help design treatments for bone disorders (Bilezikian et al., 2001).

Fig. 5.1 Structure of cortical bone and trabecular bone. C cortical bone, T trabecular bone

Osteoblast lineage cells differentiate from mesenchymal stem cells of the bone marrow, whereas osteoclast lineage cells differentiate

Fig. 5.2 Histological structure of cortical bone. The image shows the structure of the Haversian system (osteon), a unit of bone remodeling, which includes a central canal, concentric cement lines surrounding the central canal, and osteocytes surrounded by the matrix

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Table 5.2 Several heterogeneous ions that can substitute for calcium, phosphate, or hydroxyl ions of hydroxyapatite in bone, listed below each of the ions they replace Location Ions and molecules

Ca2+ Sr2+, Ba2+, Pb2+, Zn2+, Cd2+, Mg2+, Fe2+, Mn2+, Na+, K+, H+, Al3+, Nd3+

from hematopoietic stem cells, and these two types of cells are the components of bone. Mesenchymal stem cells differentiate into osteoblasts through osteoblast precursor cells, followed by differentiation into either bone lining cells or osteocytes. Hematopoietic stem cells differentiate into osteoclasts through the fusion of osteoclast precursor cells. Most of the osteoclasts that finish bone resorption are removed through apoptosis (Fig. 5.4). Osteoblasts Osteoblasts are cuboidal- or columnar-shaped cells that surround the surface of bone tissues (Fig. 5.5). These cells are tightly attached to one another and exchange signals via gap junctions. Osteoblasts and adipocytes differentiate from precursor cells in the bone marrow through the action of hormones and growth factors. The transcription factors Runx2 and Osterix are important for osteoblast differentiation from bone marrow precursor cells. Osteoblasts synthesize the organic components of bone tissue, namely, proteins, and regulate bone calcification. Osteoblasts have

PO43SO42-, CO32-, HPO42-, AsO43-, VO43-, CrO43-

OHF-, Cl-, Br-, CO32-, O2-, H2O

receptors for vitamin D, estrogen, and parathyroid hormone, and these molecules induce the synthesis of bone. This process involves the secretion of osteoclast-activating factors such as the receptor activator of NF-κB ligand (RANKL), which plays a central role in bone remodeling. In addition, osteoblasts synthesize and secrete proteins such as phosphate-regulating endopeptidase homolog, X-linked to regulate phosphate metabolism in the kidney. Activated osteoblasts that have completed their bone-forming function are destined to different fates: (a) they can be buried in the bone matrix after differentiating into osteocytes, (b) they can differentiate into bone lining cells that cover the bone surface, and (c) they can be removed through apoptosis. Osteocytes Osteocytes, which reside inside the bone matrix, project long processes involved in tight interactions with other osteocytes or bone lining cells (Fig. 5.5). Therefore, the location of osteocytes is important for their role in detecting mechanical stresses on bone tissue, which leads to

Table 5.3 Major non-collagenous proteins constituting bone tissues and their functions Name Comments Fibronectin Relatively abundant, may help regulate osteoblast differentiation Osteonectin “Bone connector” may regulate mineralization Thrombospondin May inhibit bone cell precursors Osteocalcin Binds calcium Matrix-GlaInhibits mineralization protein SIBLINGS—small integrin-binding ligand, N-linked glycoprotein family Bone sialoprotein Binds to integrins, may assist cancer cells Osteopontin Increases angiogenesis (makes new blood vessels) which enhances bone resorption in some situations May induce a bone disease called osteomalacia Matrix extracellular protein Proteoglycans—proteins with many attached sugars Biglycan Function uncertain

Effect of ‘knockout’ Lethal Osteoporosis Dense bones Bones seem normal Normal bones but calcified blood vessels – Resistance to PTH and removal of ovaries –

Osteopenia

Calcified Biological Tissue

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Fig. 5.5 Osteoblasts with cuboidal or columnar shapes surround the surface of the bone tissue. Osteocytes are present inside the bone matrix and form long protrusions to come in close contact with other osteocytes or bone lining cells Fig. 5.3 Normal bone formation process and related bone diseases caused by an abnormality of each process

the transmission of signals for the secretion of growth factors that stimulate the differentiation of bone lining cells into osteoblasts. Although research on the function of osteocytes is ongoing, they are considered an important cell type that senses mechanical stress and directs bone remodeling for the recovery of damaged bone tissues.

Fig. 5.4 Bone cells in bone tissue and their origin

Osteoclasts Osteoclasts are large cells with multiple nuclei that originated from hematopoietic stem cells and can differentiate from monocytes or macrophages. Osteoclast precursor cells are present in the blood or bone marrow and fuse together to form mature osteoclasts. The precursor cells contain RANK, the receptor for RANKL, which is secreted by osteoblasts or their precursor cells; binding of RANKL to RANK activates signal transduction pathways that induce the differentiation of osteoclast precursor cells into osteoclasts and their activation. Osteoprotegerin, a type of

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decoy receptor, is located in the bone marrow. RANKL binds to osteoprotegerin and inhibits osteoclast differentiation and activation. Osteoclasts mediate bone resorption by attaching to the bone surface, forming a segregated space at the lower part of the osteoclast. Acids and enzymes that are secreted into the segregated space dissolve bone minerals and degrade matrix proteins to induce bone resorption. The peripheral part of osteoclast that is close to the bone is called the ruffled border. After the completion of bone resorption, osteoclasts are removed by apoptosis, which is regulated by proteins secreted by other cells. Bone Lining Cells Bone lining cells are a modified form of osteoblasts characterized by a flat, dish-like shape. Bone lining cells cover the surface of bone tissues and modulate the release of calcium from bones to regulate blood calcium concentration. In addition, bone lining cells protect bone tissues from chemicals in the blood. They have various hormone receptors that receive signals from the blood for the initiation of bone remodeling. However, unlike osteoblasts, bone lining cells have weak bone formation-related cellular activities. Bone cells, such as bone lining cells, osteoblasts, and osteoclasts, cover bone surfaces completely, thereby separating bone fluid from other types of body fluid. Moreover, since bone lining cells are connected to the cell processes of osteocytes, they respond cooperatively to physical stimuli or hormones secreted from the body. Therefore, bone lining cells inhibit the movement of calcium and phosphate from bone to blood or in the opposite direction. For this reason, bone cells form a functional unit. This concept of a functional unit is comparable to that of muscle cells, which fuse to form a large cellular unit called the syncytium.

Growth and Remodeling of Bone Development and Growth of Bone Bone is produced during development through endochondral ossification and intramembranous

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bone formation. Flat bones such as calvaria, mandible, and clavicle are formed by intramembranous ossification through the direct differentiation of mesenchymal stem cells into osteoblasts. Chondrocytes differentiated from mesenchymal stem cells preferentially determine the shape of bones, such as limbs, vertebrae, and the cranial base. After cartilage growth, chondrocytes increase in size, and the enlarged chondrocytes are converted into osteoblasts or undergo apoptosis. Osteoclasts then absorb the cartilage parts, and cartilage is substituted by bone through the formation of osteoblasts in the process of endochondral ossification. After their formation, bones undergo continuous growth; in intramembranous ossification, the edge of two intramembranous bones, namely, the suture, is the major site of intramembranous bone growth, whereas the epiphyseal growth plate becomes the major site of endochondral bone growth.

Bone Remodeling Adult bones are remodeled continuously, although the process is somewhat slow. Old bone tissues are substituted by new ones through a continuous bone remodeling process. Ordinary physical activities can cause micro-fissures, and these tissues are substituted by new bone tissue through bone remodeling. When bone remodeling is suppressed, micro-fissures in the bone tissues accumulate and result in bone tissue aging, which can lead to osteonecrosis. For example, osteonecrosis can be induced by long-term intake of bisphosphonate, which suppresses bone resorption and can also inhibit bone remodeling leading to the accumulation of micro-fissures. In addition, bone remodeling also occurs in response to physical stimuli; orthodontic tooth movement utilizes this feature. If bone remodeling could be suppressed exogenously, it may prevent the recurrence of malocclusion after orthodontic treatment. Bone remodeling refers to a series of processes, by which existing bone is resorbed by osteoclasts, and the space is filled by new bone synthesized by osteoblasts; this process requires the balanced proliferation and differentiation of osteoclasts and osteoblasts from stem cells. The therapeutic regulation of bone remodeling

Calcified Biological Tissue

requires the establishment of methods for the modulation of the growth and differentiation of osteoclasts and osteoblasts.

Age-Dependent Bone Changes Since density is mass per volume, bone density can be measured by dividing bone mass by bone volume. However, this method cannot be directly applied to the human body. Therefore, bone density measurement devices are commonly used. DEXA (dual-energy X-ray absorptiometry), QCT (quantitative CT), and ultrasonography are used for the clinical measurement of bone density. Bone density decreases with age regardless of sex and race. White women show a more dramatic decrease in bone density than black men. In women, bone density decreases considerably starting at menopause. After menopause, estrogen levels decrease substantially, and estrogen is important for bone metabolism and the occurrence of osteoporosis (Fig. 5.6a). Osteoporosis can be caused by (a) menopause, (b) aging, or (c) drugs such as steroids. Osteoporosis is caused by defects in bone remodeling resulting in increased bone resorption and decreased bone formation (Fig. 5.6b). Current drugs used to treat osteoporosis address one aspect of bone remodeling, either suppressing bone resorption or increasing bone formation. Bisphosphonates are widely used for the treatment of osteoporosis; however, they have important limitations regarding their effect on decreasing bone remodeling and undesirable side-effects, including osteonecrosis of the jaw and atypical femoral fracture. The long-term usage of bisphosphonates leads to bone aging by decreasing the bone remodeling rate, and they can cause bone necrosis. Therefore, the development of effective therapeutic agents for the treatment of osteoporosis should focus on maintaining the bone remodeling rate at a certain level by harmonically modulating bone resorption and formation. The level of alveolar bone also decreases with age. The resorption of alveolar bone is caused by the release of lysosomal enzymes in response to toxic materials inside the dental plaque,

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hydrolytic enzymes originating from plaque bacteria, or the release of endotoxins formed by plaque bacteria into the gingiva, which triggers an immune response; these processes destroy periodontal tissue. Studies suggest that osteoporosis of vertebrae and the appendicular skeleton is related to the degree of alveolar bone resorption or bone density, although a causal relationship has not been demonstrated.

Local Factors Causing Bone Remodeling Systemically, the neuroendocrine system regulates the homeostasis of body bone mass. Information received by the brain stimulates the hypothalamic–pituitary axis to regulate hormones or affect bones through the sympathetic system. Various factors such as physical stimuli, exercise, and drug intake can affect systemic bone metabolism; however, these stimuli mainly affect local bone tissue cells. As a result, these stimuli induce secondary local factors, and these factors regulate local bone metabolism.

Local Factors Regulating Osteoblast Differentiation Bone morphogenetic protein (BMP) is synthesized in pre-pro-peptide form by osteocytes, osteoblasts, and mesenchymal stem cells. It is stored in pro-peptide form in the bone matrix. When bone tissues are damaged or bone remodeling occurs, osteoclasts absorb the bone matrix and the amino-terminus of the pro-peptide is cleaved to form a short active form of BMP containing the carboxyl-terminus. The released BMP stimulates mesenchymal matrix cells and regulates the expression and activity of transcription factors essential for ossification, such as Runx2 and Osterix, to promote new bone formation. Osteoblasts stimulated by parathyroid hormone, estrogen, and BMP synthesize insulin-like growth factor (IGF) which is stored in the bone matrix. During the bone remodeling process, the active form of IGF is released by osteoclasts. IGF increases the number of osteoblasts by promoting the proliferation of osteoblast precursor cells and affecting osteoblast

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Fig. 5.6 Decreased bone density with increasing age. (a) Bone density is significantly reduced as age increases in individuals of different races and gender. In addition, bone density is lower in white individuals than in black

differentiation. On the other hand, local factors such as fibroblast growth factor, transforming growth factor-β, platelet-derived growth factor, and Wnt are involved in the regulation of osteoblast proliferation and differentiation.

Local Factors Regulating Osteoclast Differentiation Interleukin (IL)-1, IL-6, tumor necrosis factor, and RANKL are synthesized and secreted by osteoblasts in response to hormones or other systemic stimuli or are present on the cell surface to regulate osteoclast differentiation. These factors are involved in the regulation of cell fusion, differentiation, activity, and apoptosis of osteoclasts. On the other hand, macrophage-colony stimulating factor (M-CSF) is released from mesenchymal stem cells or osteoblast precursor cells to regulate the early stage of osteoclast differentiation. A genetic mutation in M-CSF can cause defects in osteoclast differentiation and tooth eruption disorder. In this case, although tooth development is normal, eruption does not occur and the tooth remains inside the jaw bone.

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individuals, and in females than in males, in all age groups. (b) Photograph of a normal bone from a young individual and bone from a patient with osteoporosis

Calcium and Phosphorus Metabolism Calcium Metabolism Homeostasis of Plasma Calcium The total concentration of calcium in the blood, more specifically in plasma, is maintained within a narrow range. Plasma calcium concentration is strictly maintained in the range of 8.8–10.4 mg/ dL (2.2–2.6 mM). Plasma calcium exists in various forms: (a) ionic calcium, the physiologically active form, is found at approximately 5 mg/dL, accounting for approximately 50% of the total calcium content; (b) approximately 4 mg/dL calcium exists in combination with plasma proteins such as albumin (3 mg/dL) or globulin (1 mg/dL) that are nondiffusible; (c) approximately 1 mg/dL calcium is bound to small anions such as citrate, forming diffusible ionic calcium (Fig. 5.7). Similar to other human proteins, the isoelectric point of plasma proteins is 4.6–4.8; therefore, the amount of calcium bound to plasma proteins increases as the pH increases. The regulation of calcium homeostasis in plasma is important because ionic calcium regulates neuromuscular excitability. A decrease in plasma calcium concentration causes wrist and ankle flexion, muscular twitches, cramps in the

Calcium and Phosphorus Metabolism

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Fig. 5.7 Relationship among plasma calcium compartments

hands and feet, and even tetany accompanied by convulsions. By contrast, an increase in plasma calcium concentration is associated with muscle weakness, impaired mentation, and in severe cases, coma. This may lead to ectopic calcification, with causes nephrolithiasis, urinary stones, or the formation of calcified matter at the cornea of the eyes. Therefore, calcium homeostasis in plasma is directly related to the maintenance of life phenomena and needs to be precisely regulated. Calcium homeostasis is regulated as follows: (a) via calcium reabsorption in the kidney for the rapid regulation of calcium concentration, (b) via bones as calcium storage, bone release, or calcium deposit according to plasma calcium levels, and (c) the coordination of various hormones for the regulation of plasma calcium concentration in the human body.

Calcium Absorption The absorption of calcium mainly occurs at the proximal part of the small intestine, and the rate of absorption decreases toward the distal part; calcium absorption actively occurs in the duodenum. Calcium absorption is usually high in growing children and pregnant and lactating women and decreases with aging. In adults, less than 50% of calcium in food is absorbed. Calcium in food exists in the form of calcium phosphate; therefore, most of the calcium salts taken in via food consist of calcium phosphate. Calcium

phosphates are easily dissolved at the pH of the stomach, whereas they exist as CaHPO4 or Ca(H2PO4)2 at the pH of the duodenum. Because most calcium salts are not soluble, calcium absorption in the small intestine is limited from a nutritional perspective. Calcium is insoluble even after being absorbed, which can lead to ectopic calcification. Therefore, excessive calcium supplementation for the treatment or prevention of osteoporosis can cause ectopic calcification. The intestinal absorption of calcium occurs through two different mechanisms: active transport and diffusion. When the calcium concentration in the small intestine is high, calcium absorption mostly occurs via diffusion. The two mechanisms are physiologically important and regulated by 1,25-dihydroxycholecalciferol [1,25(OH)2D3], which is an active form of vitamin D. The regulation of calcium absorption by 1,25(OH)2D3 is related to the synthesis and regulation of specific calcium-binding proteins involved in the active transport of calcium across the epithelial cells of the intestinal mucosa, such as Ca2+-dependent ATPase. When the calcium intake is low or in the presence of hypocalcemia, the production of 1,25(OH)2D3 increases, followed by increased calcium absorption after a certain time. This latency period is the time required to synthesize 1,25(OH)2D3 via a two-step hydroxylation process from

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cholecalciferol and calcium-binding protein in the small intestine. Therefore, vitamin D deficiency limits calcium absorption in the small intestine despite increased calcium intake. Glucocorticoids that are secreted by the adrenal cortex inhibit calcium absorption; therefore, glucocorticoids are used for the treatment of hypercalcemia.

Factors Affecting Calcium Absorption Factors that affect calcium absorption can be classified into two types: factors that affect small intestinal mucosal cells and the calcium form present in the intestinal tract. The factors that affect small intestinal mucosal cells are as follows: (a) 1,25(OH)2D3, an active form of vitamin D that promotes calcium absorption in the small intestine; (b) an increasing tendency toward calcium absorption when the amount of calcium in the food is small; (c) an increase in calcium absorption with increased requirements for calcium; and (d) pregnancy and growth, which increase calcium absorption at late stages. (a) The pH in the intestinal tract can be considered as a factor affecting the calcium form in the intestinal tract. In an acidic environment, calcium is easily absorbed because it exists in a dissolved state. (b) The Ca/P ratio in food also affects calcium absorption; ratios that are too high or too low alter calcium absorption. (c) Phytic acid (inositol hexaphosphate), which is abundant in the husks of certain grains such as wheat, and its salts inhibit calcium absorption because they form an insoluble salt by binding to the calcium present in food. (d) Oxalate, an organic acid, inhibits calcium absorption by forming insoluble precipitates. (e) Fatty acids also reduce calcium absorption by forming insoluble calcium salts. Additionally, (f) high protein intake increases calcium absorption, and carbohydrates increase calcium absorption in the ileum by preventing the suppression of calcium diffusion. Therefore, the degree of calcium absorption in the intestinal tract varies considerably and many factors are involved in the process.

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Phosphorus Metabolism Phosphorus is widely distributed throughout the human body and is present in large amounts in all organisms. Therefore, phosphorus deficiency is not common with a normal diet. Normal adults contain approximately 1 kg of phosphorus, of which 85% is in the skeleton. Phosphorus is not absorbed in the stomach, and 70–90% of the phosphorus ingested is absorbed by active transport and electrochemical gradient in the small intestine, especially in the jejunum. A normal adult has 12–14 mg/dL phosphorus in the plasma, of which 3–4 mg/dL is in inorganic phosphate form, 0.5 mg/dL in phosphate ester form, and 5–10 mg/dL in phospholipid form. Unlike calcium, approximately 85% of the phosphorus in the plasma can be excreted by diffusion through the glomerulus. Most of the inorganic phosphate in the plasma exists in orthophosphate form, with an approximately 4 to 1 ratio of HPO42- to H2PO4-. Unlike calcium in plasma, which is tightly regulated in a very narrow range, phosphorus concentration varies up to 50% depending on body conditions. However, plasma phosphorus concentrations are also subject to homeostatic regulation by hormones. Phosphorus is stored in bone tissues, and when the phosphorus concentration in the serum is low, it is released from the bone. Conversely, a high phosphorous concentration in serum leads to storage in bone. Phosphorus is excreted mainly through the kidney. The amount of phosphorus normally excreted in the urine is equal to the amount of total phosphorus absorbed from the gastrointestinal tract. In general, approximately 85% of inorganic phosphorus exiting the kidney glomeruli is reabsorbed in the proximal and distal tubules of the kidney by an active transport process. The regulation of homeostasis in this excretory pathway strongly inhibits the resorption of phosphorus in the proximal tubules through the parathyroid hormone, which may lead to phosphaturia. However, the parathyroid hormone does not inhibit the reabsorption of phosphorus in the distal tubules.

Hormones That Affect Calcium and Phosphorus Metabolism

Hormones That Affect Calcium and Phosphorus Metabolism Since plasma calcium and phosphorus concentrations are regulated by the maintenance of dynamic equilibrium in the bone, kidney, and intestine, these organs can be regarded as regulators of calcium and phosphorus. The parathyroid gland monitors the calcium concentration in the blood and adjusts the plasma calcium concentration by modulating the secretion rate of parathyroid hormone. Calcium and phosphorus concentrations in the blood are precisely regulated by hormones. The main hormones involved are parathyroid hormone, calcitonin, and 1,25(OH)2D3, the active form of vitamin D. Other hormones such as sex hormones, growth hormones, and glucocorticoids are also involved in maintaining calcium and phosphate homeostasis. Therefore, these hormones may play a role in modulating calcium and phosphate concentrations in blood and body fluids to ensure normal bone formation.

Parathyroid Hormone The parathyroid glands located on the posterior side of the thyroid are endocrine organs originating from the third and fourth branchial arches. There are usually four glands, two on the left and two on the right. Parathyroid hormone is synthesized in the parathyroid glands, and its biosynthesis is regulated by the number and size of chief cells (principal cells), which produce parathyroid hormone. The parathyroid hormone is composed of 84 residues and is a peptide hormone with a molecular weight of approximately 9500 Da. This hormone is synthesized in parathyroid cells as a pre-pro-parathyroid hormone composed of 115 amino acid residues, and 25 amino acid residues at the N-terminus are cleaved from the ribosome during passage through the endoplasmic reticulum to form the 90 amino acid residue pro-parathyroid hormone (-6 to 84). The pro-parathyroid hormone moves to the Golgi apparatus, where six residues at the

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N-terminus are further cleaved, converting it into the active parathyroid hormone (1–84), which is stored in the secretory granules (Fig. 5.8). Peptides composed of the N-terminal 1–34 amino acids of the parathyroid hormone in humans were artificially synthesized, and their activity was confirmed. These peptides showed the same hormonal activity as the complete parathyroid hormone (1–84). This result suggests that the biological activity of the parathyroid hormone resides in the N-terminal region and that peptides up to the N-terminal 1–34 residues of the parathyroid hormone are the minimum active units.

Regulation of the Activity of Parathyroid Hormone Low calcium levels in the blood promote the secretion of parathyroid hormone; however, the biosynthesis and degradation of the pro-parathyroid hormone are not significantly affected by blood calcium levels. In fact, 80–90% of the synthesized pro-parathyroid hormone is degraded rapidly. The degradation rate of this pro-parathyroid hormone decreases when the serum calcium level is low and increases when it is high. These results suggest that calcium homeostasis in blood is not achieved through the biosynthesis of pro-parathyroid hormone, but rather by regulating the rate of degradation of the pre-existing pro-parathyroid hormone. The secretion of parathyroid hormone from the parathyroid gland is inversely proportional to the calcium concentration in plasma and is regulated by a feedback mechanism. When the plasma calcium level is reduced to approximately 9 mg/dL, the secretion of parathyroid hormone gradually increases, and when it falls below 9 mg/dL, it increases rapidly. When plasma calcium levels increase above normal, parathyroid hormone secretion is suppressed. However, the basal level of parathyroid hormone in the blood is maintained. The half-life of parathyroid hormone is 1–5 min, whereas the physiological activity of a parathyroid hormone fragment (1–34 amino acid residues) is maintained for a longer period. In the bone, kidney, and intestine, parathyroid hormone binds to its receptors on the surface of target cells and induces the synthesis of cAMP, a

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Fig. 5.8 Structure of parathyroid hormone. It is synthesized as a pre-pro-parathyroid hormone of 115 amino acid residues. The N-terminus (31 residues) is cleaved from the pre-pro-hormone to yield mature parathyroid hormone (84 residues). The arrows indicate the peptide bonds that are cleaved during metabolic modification of the precursor: (1) The two methionine residues at the N-terminus are separated, (2) the leader (signal) sequence is cleaved from the newly synthesized peptide,

and (3) the pro-parathyroid hormone is transported to the Golgi apparatus and released. (4) Parathyroid hormone secreted from the cell is cleaved into a biologically active N-terminal fragment and an inactive C-terminal fragment. Residues 1–34 are necessary for full biological activity. The most important region for activity is Ala–Glu (residues 1–6), residues 7–34 are the inhibitory domain, and residues 25–34 are the main binding domain

second messenger, to mediate hormone action in cells. cAMP synthesis is catalyzed by adenylate cyclase, which is present in the membrane of target cells. The cAMP produced in renal tubular cells is excreted in the urine, and excretion is significantly increased by the administration of parathyroid hormone. Osteoblasts and osteocytes

present in bone tissues have a receptor for parathyroid hormone, and binding of the hormone to its receptor increases the intracellular cAMP concentration.

Hormones That Affect Calcium and Phosphorus Metabolism

Action of Parathyroid Hormone The parathyroid gland monitors the concentration of calcium in the blood passing through the parathyroid gland and adjusts the plasma calcium concentration by modulating the rate of secretion of the parathyroid hormone. Therefore, a low plasma calcium concentration induces the secretion of parathyroid hormone, whereas a high calcium concentration suppresses its secretion. Low plasma calcium levels increase parathyroid hormone secretion and promote bone resorption. Parathyroid hormone increases the activity of osteoclasts, promotes osteolysis by osteoclasts, and promotes the production of new osteoclasts. In addition, acid hydrolase is secreted from osteoclasts to degrade organic matter. As a result, calcium and phosphate are released into the blood from the bone tissue. However, studies show that osteoclasts do not have a receptor for parathyroid hormone, suggesting that molecules that are secreted by osteoblasts indirectly affect osteoclasts. Plasma calcium levels are antagonized by parathyroid hormone and calcitonin (Fig. 5.9). Vitamin D is activated by hydroxylation at carbons 25 and 1 in the liver and kidney, respectively. Parathyroid hormone increases the synthesis of 1α-hydroxylase in the proximal tubular cells of the kidney and contributes to the activation of vitamin D. Since 1,25(OH)2D3, the active form of vitamin D, induces the synthesis of calcium-binding proteins in intestinal epithelial cells, parathyroid hormone indirectly acts on the small intestine to promote calcium absorption. The effect of parathyroid hormone on the kidney is as follows: (a) it increases the synthesis of 1α-hydroxylase in the renal proximal tubule, (b) it inhibits the reabsorption of phosphate in the renal proximal tubule to help excretion, and (c) it promotes the reabsorption of calcium in the distal tubule. Calcium in the kidney glomeruli is reabsorbed at a rate of 60–70% in the proximal nephron and 30–40% in the distal nephron, and only 1–2% of total calcium is excreted in the urine.

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Calcitonin The primary structure of calcitonin was first identified in porcine in 1968, and it was subsequently isolated from cows, sheep, humans, rats, mice, salmon, eels, and chickens, and the respective primary structures were determined. The mRNAs for calcitonin and calcitonin gene-related peptide, a neurotransmitter, are produced from the calcitonin gene through alternative splicing, resulting in completely different proteins in terms of amino acid sequence and function. Calcitonin is a peptide hormone consisting of 32 residues with a molecular weight of approximately 3500 Da. This hormone is mainly secreted from the C cells (or parafollicular cells) of the thyroid gland, and calcitonin-secreting cells are also present in the parathyroid and thymus glands. Calcitonin induces the synthesis of cAMP by binding to its receptor on the surface of target cells. The calcitonin receptor is composed of 482 residues and is a 7-membered serpentine receptor with a domain that penetrates the cell membrane seven times similar to the parathyroid hormone receptor. Calcitonin is a powerful hormone that significantly affects plasma calcium concentration even at low levels. The half-life of normally released calcitonin is 2–15 min. One characteristic of human calcitonin is that the N-terminal seven amino acid residues form a ring linked by a disulfide bridge, and a proline-amide is present at the C-terminus. Rat calcitonin differs from human calcitonin by only two amino acid residues. In general, peptide hormones are composed of an active core sequence, and calcitonin is necessary for the activity of all amino acid residues. There is also a disulfide bond between cysteines 1 and 7 of calcitonin. When this bond is cleaved, the activity is completely lost. Plasma calcium concentration is the strongest regulator of calcitonin secretion. High plasma calcium levels inhibit parathyroid hormone secretion and promote calcitonin secretion. Various gastrointestinal hormones such as glucagon, gastrin, and cholecystokinin secondarily regulate calcitonin secretion. Additionally, β-adrenergic

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Fig. 5.9 Complementary roles of parathyroid hormone, calcitonin, and 1,25dihydroxycholecalciferol [1,25(OH)2D3] in controlling calcium and phosphate metabolism. [Source: S. Cole and J. E. Eastoe, Biochemistry and Oral Biology, 2nd ed. (Wright, 1988), Fig. 30.4]

catecholamine (adrenaline) stimulates calcitonin secretion. In bone tissue cells, osteoclasts are the only cells containing calcitonin receptors, which are absent in osteoblasts. Calcitonin inhibits bone resorption and prevents further calcium and phosphate from being released into the blood. Therefore, calcitonin acts directly on osteoclasts to reduce the number of activated osteoclasts, thereby inhibiting bone resorption. Calcitonin induces the excretion of Ca2+, PO43-, Mg2+, Na+, and Cl- by acting directly on the proximal renal tubule and promoting the reabsorption of phosphate. Calcitonin inhibits calcium reabsorption in the distal renal tubule and increases its excretion. Calcitonin acts as an antagonist to parathyroid hormone and decreases the calcium concentration in the blood. The calcium-lowering effect of calcitonin is secondary to the inhibition of calcium release in the blood through the suppression of bone resorption and promotion of calcium excretion in the kidney. Calcitonin suppresses bone pain. It acts on pain induced by bone metastasis of malignant tumors and postoperative pain, although the underlying mechanism remains unclear. Calcitonin could serve as a therapeutic agent for the treatment of osteoporosis

because of its effects on suppressing bone resorption and relieving bone pain.

1,25-Dihydroxycholecalciferol [1,25(OH)2D3] The active form of vitamin D, 1,25(OH)2D3, plays an important role in controlling plasma calcium and phosphate levels. Parathyroid hormone and calcitonin are peptide hormones that bind to receptors on the cell membrane, whereas 1,25(OH)2D3 acts as a steroid hormone. Vitamin D3 is a hormone rather than a vitamin because 1,25(OH)2D3 acts through a steroid hormone-like mechanism. In addition, it is synthesized in the human body and its synthesis is strictly controlled by various factors. 1,25(OH)2D3 is also called calcitriol. In addition to bone, kidney, and small intestine cells, other targets of vitamin D include parathyroid glands, renal tubular cells, pancreatic β cells, placental cells, neuronal cells, thymus, avian Fallopian tube cells, bone marrow cells, skin epidermal cells, and certain tumor cells.

Hormones That Affect Calcium and Phosphorus Metabolism

Biosynthesis and Activation of Vitamin D3 Vitamin D is a comprehensive term for all antirachitic factors that can be obtained by irradiating provitamin D with ultraviolet light. The vitamin D group includes vitamin D2, which is obtained through food, and vitamin D3, which is synthesized by ultraviolet irradiation. We will first look at the biosynthesis process of vitamin D2 and vitamin D3 and their activation process before examining the action of 1,25 (OH)2D3. Vitamin D3 is synthesized when the skin is exposed to sunlight. Exposure of skin to sunlight leads to the photodegradation of 7-dehydrocholesterol (provitamin D3) present in subcutaneous tissues and its conversion to previtamin D3, which is spontaneously isomerized to form cholecalciferol (vitamin D3). Vitamin D2 is biosynthesized by the conversion of ergosterol (provitamin D2) contained in plants (crops) into ergocalciferol (vitamin D2) through a process similar to that of vitamin D3 synthesis. The difference between biosynthesized vitamin D3 and vitamin D2 is that vitamin D2 has one

Fig. 5.10 Biosynthesis and activation process of vitamin D3 in the human body. Additionally, the schematic diagram shows the mechanism of the action of 1,25dihydroxycholecalciferol [1,25(OH)2D3] on the absorption of calcium through the intestinal wall. CaBP calciumbinding protein

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additional double bond at carbons 22 and 23 and a methyl group at carbon 24. Vitamin D3 synthesized in the skin by ultraviolet irradiation and vitamin D2 obtained from food are transported to the liver and converted to 25-hydroxycholecalciferol via hydroxylation of the 25th carbon atom by the catalytic action of 25-hydroxylase in the microsomes of hepatocytes. 25-Hydroxycholecalciferol binds to vitamin D-binding protein and is transported to the proximal renal tubule after being excreted in the blood. The carbon at position 1 is re-hydroxylated by 1α-hydroxylase in the kidney mitochondria. This activation process converts vitamin D3 and vitamin D2 into 1,25(OH)2D3 and 1,25(OH)2D2, respectively (Fig. 5.10). The processes of hydroxylation at carbon 1 and carbon 24 in the kidney are strictly controlled by plasma calcium concentration. A plasma calcium concentration of 9 mg/dL inhibits 1,25(OH)2D3 production and activates 24,25(OH)2D3. This implies that vitamin D is activated in the kidney only in the presence of hypocalcemia, and this regulatory mechanism is important for maintaining plasma calcium homeostasis. The main factors that regulate the production of 1,25 (OH)2D3 are the plasma levels of calcium and phosphorus, parathyroid hormone, and 1,25 (OH)2D3, itself. The enzymatic activity of 1α-hydroxylase in the kidney is the most important factor for the production of 1,25(OH)2D3, whereas the enzymatic activity of 25-hydroxylases is not as important for the regulation of calcium and phosphate concentrations in the body.

Action of 1,25-Dihydroxycholecalciferol [1,25(OH)2D3] The in vivo action of 1,25(OH)2D3 is similar to that of a steroid hormone with a comparable structure. The active form of vitamin D3, 1,25 (OH)2D3, enters the cytoplasm through the membrane of the target cell and binds to its receptor in the cytoplasm to form the 1,25(OH)2D3-receptor complex. This complex moves to the nucleus and promotes the transcription of specific genes,

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thereby increasing the expression of calciumbinding proteins. The synthesized calciumbinding proteins are transferred to the plasma membrane villi of intestinal epithelial cells, where they are involved in calcium absorption through the intestinal wall (Fig. 5.10). Calciumbinding proteins are expressed in the mesial and distal regions of the small intestine and in the renal cortex. A 12.5 kDa calcium-binding protein with a high affinity for calcium is expressed in the mesial part of the small intestine and renal cortex, and a 25 kDa calcium-binding protein with a low affinity for calcium is expressed in the distal region of the small intestine. Therefore, calcium absorption in the small intestine occurs mainly in the mesial region, suggesting that calcium is also resorbed in the kidney. Low plasma calcium or phosphate levels increase serum 1,25(OH)2D3 levels and increase plasma calcium and phosphate levels by acting on bone, the intestine, and the kidney. 1,25(OH)2D3 promotes the uptake of calcium, phosphorus, and magnesium in the intestine (duodenum and jejunum), and in the absence of 1,25(OH)2D3, calcium ingested as food is not absorbed in the intestine. 1,25(OH)2D3 promotes the synthesis of calcium-binding proteins and facilitates calcium absorption in the intestine (Fig. 5.10). Its representative protein is calbindin, which promotes phosphorus uptake in the intestine and mobilizes phosphate present in soft tissues to increase plasma phosphate concentration. 1,25 (OH)2D3 cooperates with parathyroid hormone to induce bone resorption, liberating calcium and phosphate from bone tissue and promoting osteoclast differentiation. 1,25(OH)2D3 receptors are expressed in osteoblasts, but not in osteoclasts. Thus, 1,25(OH)2D3 activates osteoblasts that secrete osteoclast-activating RANKL, which in turn activates osteoclasts, resulting in bone resorption. In addition, 1,25 (OH)2D3 induces osteocalcin expression, whereas it inhibits the synthesis of type I collagen. Alterations in the absorption and activation of vitamin D lead to the development of rickets in children and osteomalacia in adults. In the kidney, 1,25(OH)2D3 increases calcium reabsorption in proximal tubules and distal tubules and

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promotes phosphate reabsorption in proximal tubules. It also inhibits the activity of 1α-hydroxylase through feedback inhibition. In response to hypocalcemia as the primary stimulus, parathyroid hormone levels increase and the extra phosphate is eliminated by the phosphaturic effect. Conversely, in the presence of hypophosphatemia, extra calcium is lost to urine because of a decrease in parathyroid hormone, and calcium reabsorption in the kidney is not stimulated.

Sex Hormones Sex hormones affect bone growth and metabolism. Testosterone, a type of male hormone (androgen), and estrogen, a female hormone, promote epiphyseal growth during puberty. Male hormones also promote calcification. Estrogen inhibits osteoclast production and activity, thereby inhibiting bone resorption. In postmenopausal women, estrogen deficiency abolishes its effect on the suppression of bone resorption. In this case, the rate of bone resorption exceeds that of osteogenesis, resulting in osteoporosis. Osteoporosis in postmenopausal women can be delayed or partially prevented by estrogen administration, suggesting that estrogen is essential for the maintenance of normal bone mass in women. In addition, estrogen increases the activity of 1α-hydroxylase, which catalyzes the activation of vitamin D.

Growth Hormones Growth hormones, which are secreted by the pituitary gland, promote the growth of the human body during specific growth stages such as infancy and puberty. Growth hormones can promote bone growth directly or stimulate growth in length at the epiphysis of the bone apex of long bones indirectly by inducing the secretion of IGF-1 from osteoblasts, osteocytes, and bone lining cells. Growth hormones also increase bone mass in adults. Since growth hormones are involved in determining the length of the

Disorders of Calcium and Phosphorus Metabolism

mandible by regulating the growth of mandibular condyles, defects such as prognathism may be caused by the administration of growth hormones.

Glucocorticoids Glucocorticoids are synthesized and secreted from the adrenal cortex and have the basic structure of steroids. Bone cells have a glucocorticoid receptor in the nucleus. Glucocorticoids, also called stress hormones, are synthesized in response to a stressful situation and play a role in protecting the body. The different pharmacological actions of glucocorticoids have promoted the use of synthesized glucocorticoids as medicine. They are used to inhibit inflammation and restore balance in the body after exposure to various stresses. However, the long-term use of excessive amounts of glucocorticoids causes various problems in the skeletal system, including inhibition of calcium absorption in the intestine, inhibition of osteogenesis, promotion of bone resorption, promotion of calcium excretion in the kidney, and inhibition of sex hormone production, resulting in an overall decrease in bone mass. The long-term use of glucocorticoids increases the likelihood of fracture, especially in patients with autoimmune diseases, skin disorders, arthritis, and chronic fatigue.

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addition to vitamin D, vitamin A and vitamin C (ascorbic acid) are important for the normal growth and development of bone. In young animals lacking vitamin A, skeletal growth stops before the growth of soft tissues. Conversely, excessive use of vitamin A may cause multiple fractures in the ilium, and bone abnormalities may be observed in children treated with excessive amounts of vitamin A. Vitamin C is essential for normal skeletal growth and development. Vitamin C deficiency blocks collagen synthesis in mesenchymal cells, resulting in inadequate calcification. Skeletal growth can stop in response to a deficiency of nutrients, even if energy is insufficient. Therefore, the intake of balanced nutrients is essential for the normal development and remodeling of bone. Prostaglandin, a local hormone, and particularly prostaglandin E, stimulates bone resorption. Prostaglandin mediates bone resorption in inflammatory diseases such as rheumatoid arthritis. In particular, bone remodeling modulated by prostaglandin plays an important role in periodontal disease or orthodontic treatment through physical stimulation. Aspirin, a prostaglandin biosynthesis inhibitor, inhibits bone remodeling or bone resorption.

Disorders of Calcium and Phosphorus Metabolism Hyperparathyroidism

Thyroid Hormone Thyroid hormone is secreted from the thyroid gland, and bone cells have receptors for thyroid hormone. Basically, the thyroid hormone promotes the growth and maturation of bone. Excessive secretion of thyroid hormone, such as in hyperthyroidism, can lead to osteoporosis by promoting osteoclast-induced bone resorption.

Factors Affecting Bone Metabolism A wide variety of hormones and nutrients regulate bone metabolism, remodeling, and growth. In

In patients with hyperparathyroidism, excessive secretion of parathyroid hormone causes hypercalcemia, hypophosphatemia, and increased bone resorption. Accumulation of calcium in the kidney parenchyma due to hypercalcemia (nephrocalcinosis) leads to kidney damage and kidney stones (nephrolithiasis). In addition, increased osteoclast activity results in the appearance of characteristic bone changes termed osteitis fibrosa cystica. This can be associated with the occurrence of osteopenia and fractures, and bone formation is slower than bone resorption. Certain cancers, vitamin D toxicity, hyperthyroidism, and

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adrenal cortical insufficiency are among the factors associated with hypercalcemia.

Hypoparathyroidism Hypoparathyroidism is a relatively rare disease in humans. However, genetic defects of the parathyroid gland, careless surgical removal of the parathyroid gland during thyroidectomy, deliberate removal to compensate for blood supply during thyroid surgery, or damage to the parathyroid gland can cause hypoparathyroidism. Autosomal recessive inheritance can also cause hypoparathyroidism. Parathyroid functional disorders can be classified into idiopathic, acquired, and pseudohypoparathyroidism. Idiopathic and acquired hypoparathyroidism is caused by parathyroid hormone deficiency, whereas pseudohypoparathyroidism is caused by functional defects in parathyroid hormone (mutation of the parathyroid hormone receptor gene). One of the main characteristics of hypoparathyroidism is a deficiency of parathyroid hormone production. Parathyroid hormone deficiency causes hypocalcemia and lowers the cell depolarization threshold, which can induce weakness, muscle cramps, headache, or tetany as a result of contractures of specific muscles of the hand, foot, arm, and face. Parathyroid hormone deficiency causes hypocalcemia; one of the main clinical symptoms of hypocalcemia is tetany of the extremities, which is caused by excessive excitability of the central and peripheral nervous systems. Anxiety associated with hypoparathyroidism is induced by hyperpnea and compensating respiratory alkalosis, which exacerbate hypocalcemia; it also causes increased secretion of epinephrine, which causes tachycardia, perspiration, and paleness near the oral cavity. The treatment of hypoparathyroidism focuses on maintaining proper levels of calcium and phosphate in the blood through the intake of large amounts of calcium and vitamin D. When tetany occurs, an intravenous injection of calcium is needed to prevent laryngospasm and convulsions.

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Calcification of Bones and Teeth

Mechanism of Calcification Calcification or mineralization consists of a series of processes resulting from the deposition of insoluble calcium salts on the organic matrix produced by certain cells, such as fibroblasts, osteoblasts, and odontoblasts. Calcium salts found in mammalian calcified tissues are present in the form of minerals similar to hydroxyapatite. At physiological pH (pH 7.4), approximately 81% of the phosphate ions dissociate into HPO42-, 19% dissociate into H2PO4-, and small amounts (0.008%) dissociate into PO43-; therefore, most of the ions are present as HPO42-. Calcium and phosphate (HPO42-) concentrations in tissue and body fluids are in a metastable state, and calcification does not occur spontaneously. However, in the presence of biological catalysts or seed crystals, the deposition of calcium phosphate salts is possible in a metastable state (Fig. 5.11). Because calcium and phosphate in tissue and body fluids are in a metastable state, a local increase in the ionic products of calcium and phosphate to point B or higher in Fig. 5.11 may lead to the spontaneous deposition of calcium phosphate salts. In the presence of a catalyst, deposition of calcium phosphate salts in the metastable state of calcium and phosphate can occur if the activation barrier (Gibbs free energy of activation) of the apatite formation reaction is decreased (Fig. 5.11). Once hydroxyapatite is formed, it reaches a state of physicochemical equilibrium with the calcium and phosphate ions in blood. The processes described above suggest that the initial stage of calcification is mediated by two processes. The first is a homogeneous nucleation process in which calcium and phosphate concentrations are locally increased in tissues in which calcification occurs, leading to the spontaneous deposition of apatite crystals. The second process is heterogeneous nucleation. In this process, the deposition of apatite crystals occurs in the presence of metastable concentrations of calcium and phosphate through a decrease in the activation energy required for apatite formation.

Mechanism of Calcification

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Fig. 5.11 Schematic diagram showing the possible path of deposition of calcium phosphate salts in metastable calcium and phosphate (HPO42-) concentrations in tissue fluid and body fluids (blood). The metastable state of Ca2+ x HPO42- is at point A, and the solid (hydroxyapatite) state is at point C. To form hydroxyapatite, either the level of the ionic product must be increased to point B, at which spontaneous precipitation would occur, or a catalyst must be introduced, which would lower the energy required to

form apatite crystals. Once hydroxyapatite is formed, it is in physical and chemical equilibrium with the ion product of the fluid phase, indicated by the double arrow between A and C. The dotted line represents the ionic product of calcium and phosphate, which can cause spontaneous precipitation of calcium phosphate salts. [Source: E. P. Lazzari, Dental Biochemistry, 2nd ed. (Lea & Febiger, 1976), Fig. 6.1]

This occurs in the presence of biological catalysts in the tissues undergoing calcification (Lazzari, 1976). To date, the most important factor in the calcification process is the generation of the nuclei of the first inorganic crystals. The nucleus of a primary crystal promotes the formation of the primary crystal, which facilitates crystal growth because the calcium and phosphate concentrations in tissue and body fluids are in a metastable state. Therefore, to elucidate the mechanism underlying the calcification process, the generation of the nuclei of primary crystals needs to be understood, and early calcification theories are focused on homogeneous nucleation. Several hypotheses have been published about these processes, and these hypotheses will be described in this section.

presented the simple spontaneous precipitation theory by which hard tissues are formed by spontaneous deposition of supersaturated calcium and phosphate, with the existing hard tissues acting as nuclei for crystal formation. However, later research showed that, even if calcium and phosphate are supersaturated in tissue fluids or blood, it is not sufficient for the spontaneous formation of apatite crystal deposits. Therefore, other factors were thought to be involved.

Simple Spontaneous Precipitation Hypothesis Calcium and phosphate are present in a supersaturated state in tissue fluids or blood surrounding hard tissues. Based on the supersaturation state around hard tissues, Krammer first

Fig. 5.12 To explain the local increase in phosphate concentration in calcifying tissues, a reaction that utilizes organic phosphate esters as substrates to provide inorganic phosphate by alkaline phosphatase is shown. Also, the predicted process by which calcium and phosphate ions form apatite is shown

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Alkaline Phosphatase Hypothesis In 1923, Robison et al. reported the alkaline phosphatase theory based on the finding that alkaline phosphatase activity is high in tissues such as bones and teeth, which undergo calcification. This theory suggests that, in the early stage of calcification, organic phosphate esters such as hexose phosphate that are present in tissues act as substrates for alkaline phosphatase, producing inorganic phosphate, which locally increases the phosphate concentration. Phosphate reacts with calcium ions in tissue fluids, causing precipitation of insoluble calcium phosphate salts (Fig. 5.12). Since this theory was proposed, various counterarguments have been raised. One of these arguments is that the activity of alkaline phosphatase is not only high in tissues that undergo calcification but also in tissues in which calcification does not occur. Evidence suggests that calcification occurs in the absence of alkaline phosphatase activity in vitro. Another argument is that the amount of organic phosphate esters is not sufficient in the tissues in which calcification occurs. In addition, Robison et al. pointed out that the authors did not identify the specific organic phosphate ester that acts as a substrate for alkaline phosphatase in the tissues that undergo calcification. Although Robison et al. proposed a secondary booster mechanism that could act as a substrate for alkaline phosphatase, the additional explanation was not sufficient to overcome the weaknesses of the alkaline phosphatase hypothesis.

Glycolysis and Alkaline Phosphatase Hypothesis Gutman and Yu (1949) investigated potential secondary booster mechanisms, similar to Robison, and their research uncovered several facts. When calcification occurs in cartilage, glycogen is degraded, and the addition of monoiodoacetate and fluoride, which are glycolysis inhibitors, inhibits cartilage calcification. The inhibition of cartilage calcification is restored by

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Calcification of Bones and Teeth

the addition of glycolytic intermediates, and glycolysis is active in tissues in which calcification occurs. Based on these results, Gutman and Yu suggested that the glycolytic intermediates, namely, sugar phosphates, act as substrates for alkaline phosphatase, which serves as a secondary booster. This theory was cited as a mechanism to explain the early stages of calcification until the late 1950s. However, this theory did not answer the following questions. Although tissues such as the intestinal mucosa have high rates of alkaline phosphatase activity and glycolysis, calcification does not occur in these tissues. In fact, the organic phosphate esters present in the tissue fluid and the sugar phosphate glycolytic intermediates do not provide sufficient amounts of ions for spontaneous deposition of apatite. This indicates that cellular activity alone does not provide sufficient amounts of ions for the spontaneous deposition of apatite. These facts led to a change in paradigm. Alkaline phosphatase activity or glycolysis occurs within the cells, whereas calcification is a reaction that takes place in the extracellular matrix. Therefore, a direct link between the two reactions is difficult. In particular, despite supersaturated calcium and phosphate concentrations in plasma, apatite crystals are not formed, and this finding challenged the previous concept explaining the early stages of calcification. The theory of calcification, which was initially adapted to homogeneous nucleation, gradually began to shift to heterogeneous nucleation.

Seeding Theory The seeding theory is also called the nucleation catalysis theory or epitaxy theory, and it was introduced by Neuman and Neuman in 1953. According to this theory, a substance that can act as a nucleus for apatite crystals is pre-existing or generated in situ and mediates the formation of hard tissues by epitaxy. Epitaxy refers to the deposition of crystal layers around other crystals with a similar lattice structure. Investigation of the materials that can act as a

Mechanism of Calcification

seed for apatite crystal formation identified collagen fibers, lipids, proteoglycan, and phosphoproteins as candidates. Collagen fibers have been studied extensively in relation to the nucleation catalysis theory. In the early stage of calcification, the relationship between collagen fibers and apatite crystal deposits can be explained by electron microscopic findings, and the initial deposition site of apatite crystals is morphologically coincident with the cross-striation of collagen fibrils. This fact demonstrates that collagen fibers are important for the deposition of apatite crystals, and in particular, cross-striations that occur repeatedly in the collagen fibril are likely to promote calcification. Proteoglycans act as a nucleus for crystal formation by binding to calcium ions because of their strong negative charge at physiological pH. In fact, proteoglycan synthesis is detected histologically before the deposition of minerals in calcifying tissues, and the acidic groups of proteoglycans such as chondroitin sulfate, which shows the highest acidity, can bind to calcium ions and act as nuclei for crystal formation. Although proteoglycan synthesis is active in tissues undergoing calcification, the synthesized proteoglycans are rapidly degraded before the onset of calcification. The rapid degradation of proteoglycans results in the release of bound calcium ions, leading to a local increase in calcium concentration followed by the initiation of calcification. However, the rapid production of proteoglycans may cause a reduction of the surrounding calcium concentration, which may inhibit calcification. Therefore, although proteoglycans are closely related to calcification, their role in nucleation remains unclear. Proteoglycans may therefore modulate calcification by inhibiting or promoting this process. The role of lipids in the calcification process is not clear; however, lipids are present in the calcifying areas of bones and teeth and calcium is strongly associated with lipids, suggesting that they play an important role in calcification. Osteocalcin is an acidic protein contained in bones that contains γ-carboxyglutamic acid (Gla) residues. This Gla residue has two carboxyl

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groups that are negatively charged, suggesting that it binds to the divalent calcium ion. Osteocalcin accounts for 1–2% of total bone protein and 0.5–1% of dentine, although it is not present in enamel. Osteocalcin is strongly expressed during calcification; since osteocalcin is strongly bound to apatite, it was thought to be a factor promoting calcification. However, osteocalcin knockout mice show no abnormalities in calcification, and in aging mice, osteoclastinduced bone resorption is inhibited, and bone mass increases. Therefore, the current theory is that osteocalcin is not involved in calcification.

Fig. 5.13 Schematic diagram showing the locations of apatite crystals deposited in holes created when tropocollagens polymerize to form a collagen fibril in the early stage of the calcification process, according to the hole-zone theory

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Hole-Zone Theory Individual tropocollagen molecules polymerize in the extracellular matrix to form a collagen fibril, which has a gap of approximately 40 nm between the tropocollagen that is called a hole. The holezone theory, first proposed by Glimcher (1959), states that the first calcification and the deposition of apatite crystals occur in this gap (Fig. 5.13). The hydroxyl group of the serine residue in the α-chain of the collagen fibril is phosphorylated by protein phosphokinase to produce a protein (collagen fibril α-chain)-serine-OPO32- complex. Calcium ions bind to the complex, acting as the nucleus for crystal formation and causing the deposition of calcium phosphate salts. Therefore, according to this theory, a hole in the collagen fibril acts as a nucleation site. However, the holezone theory fails to explain the fact that collagen fibrils isolated from the skin, tendons, and ligaments show nucleation ability, whereas calcification does not occur in these tissues. The absence of calcification in collagen fibers isolated from tissues such as the skin, tendons, and ligaments is thought to be due to differences in the structure of collagen fibers and the presence of calcification inhibitors. X-ray diffraction measurements indicate that the distance between the tropocollagens in collagen fibrils isolated from calcifying bones and dentine is 6 Å, whereas that of tendons, in which calcification does not occur, is 3 Å. The size of the HPO42- ion is approximately 4 Å, suggesting that HPO42- ions are not accessible to the collagen fibril of tendons and apatite deposition cannot occur. In addition, tissue and body fluids contain approximately 1 μM pyrophosphate, which acts as a calcification inhibitor. Pyrophosphate acts as an inhibitor of calcification by suppressing the formation of crystals through absorption to the apatite surface at the site of calcification. Calcification does not occur in tissues that contain calcification inhibitors such as pyrophosphate. However, in tissues that undergo calcification, alkaline phosphatase and inorganic pyrophosphatase degrade pyrophosphate and convert it into a calcification inducer.

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Calcification of Bones and Teeth

Davis and Walker (1972) argued that the carboxyl groups of acidic amino acid residues such as aspartate and glutamate in the α-chain of collagen fibers may act as nuclei for crystal formation by binding to calcium ions.

Matrix Vesicle–Induced Calcification Theory In 1967, Anderson first reported the presence of matrix vesicles in cartilage undergoing endochondral ossification. In addition, he reported the presence of apatite crystals in matrix vesicles (Anderson, 1969) and demonstrated that the matrix vesicle is the initial site of calcification in trabecular bone and predentine and in abnormal calcification. The discovery of these matrix vesicles has become a major turning point in the study of calcification. Matrix vesicles are 30–300 nm in diameter and are surrounded by biological membranes. They vary in shape and internal structure from low to high electron density. Matrix vesicles are present in cartilage, trabecular bone, and dentine, but not in cortical bone and enamel. In the initial calcification stage, apatite crystals appear inside matrix vesicles or surrounding them, gradually filling the inside and extruding from the matrix vesicle membrane.

Generation of Matrix Vesicles Matrix vesicles are pinched off or bud from the plasma membrane of bone-forming cells. This hypothesis was verified by transmission electron microscopy analysis of the process of budding of matrix vesicles from chondrocytes. In addition, the plasma membrane and matrix vesicle membrane show similar biochemical characteristics, supporting the fact that the matrix vesicle originates from the plasma membrane (Table 5.4). However, the matrix vesicle membrane has a higher cholesterol/phospholipid ratio and phosphatidyl serine content than the plasma membrane, suggesting that the biochemical characteristics of the two membranes are not identical. Therefore, matrix vesicles are believed to be produced by the pinching off of certain

Mechanism of Calcification

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Table 5.4 Biochemical similarity of the matrix vesicle membrane and plasma membrane Presence of marker molecules: 5′-Nucleotidase Sphingomyelin Membrane-associated NTP-pyrophosphohydrolase H2 histocompatibility antigen High levels of: Alkaline phosphatase Cholesterol Cholesterol:phospholipid Actin Low levels of: Esterase (lysosomal marker) NADP-cytochrome C reductase (ER marker)

regions of the plasma membrane with a high level of acidic phospholipids. Studies suggest that matrix vesicles are produced by exocytosis or necrosis of cells.

Mechanism of Matrix Vesicle–Induced Calcification The mechanism of calcification induced by matrix vesicles can be divided into two stages. The first step is the initial production of apatite crystals in the matrix vesicle, followed by the growth and maturation of apatite crystals in response to exposure to extravesicular fluid (Fig. 5.14).

In the first step, acidic phospholipids such as phosphatidyl serine and phosphatidyl inositol, which are constituents of the matrix vesicle membrane and have a high affinity for calcium, bind easily to calcium ions and concentrate calcium ions on specific areas of the matrix vesicle inner membrane. As the matrix vesicles approach the calcification front, phosphatases such as alkaline phosphatase, inorganic pyrophosphatase, and ATPase present in the matrix vesicle membrane are activated. The activated phosphatases hydrolyze the phosphate esters in the matrix vesicle sap or outside the matrix vesicle, thereby releasing phosphate ions and locally increasing the concentration of phosphate adjacent to the matrix vesicle

Fig. 5.14 Electron micrographs showing the mechanism of calcification induced by matrix vesicles. [From H. C. Anderson, R. Garimella and S. E. Tague. Front. Biosci. 10:822–837, 2005]

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membrane. As the concentration of phosphate increases locally around the phospholipid attached to calcium, a calcium-phospholipidphosphate complex is formed. This complex acts as the nucleus of the primary or embryo crystal to form the seed crystal (Fig. 5.14). Since the matrix vesicle sap has metastable calcium and phosphate concentrations, deposition of hydroxyapatite occurs around the seed crystal, and crystals are continuously formed. The second step is the growth and maturation of the apatite crystals in response to exposure to the outside of the matrix vesicle (Fig. 5.14). Since the extracellular environment of matrix vesicles contains metastable calcium and phosphate concentrations, apatite crystals exposed to the outside of matrix vesicles continue to undergo mineral deposition, leading to continuous crystal growth. In addition, the precursors of hydroxyapatite contained in the crystals are converted to hydroxyapatite, resulting in crystal maturation.

Current Concepts Regarding the Calcification Mechanism To summarize the theories related to the calcification mechanism, in the hole-zone theory proposed by Glimcher, the hole created when collagen fibrils are formed is the first site of calcification, and calcium binds to the protein– serine–OPO32- complex to act as a nucleus for crystal formation. Therefore, this theory mainly explains calcification in terms of structure. By contrast, in the matrix vesicle–induced calcification theory proposed by Anderson et al., a calcium-phospholipid-phosphate complex that is formed around the phospholipids in the matrix vesicle membrane acts as the nucleus of the primary crystal and causes calcification. This theory suggests that the matrix vesicle is the first site of calcification. Therefore, the matrix vesicle– induced calcification theory explains calcification mainly in terms of function. In a comprehensive review of the functional aspects of calcification, the matrix vesicle–induced calcification theory is considered as the most appropriate theory, whereas from a structural point of view, the hole-zone theory is more reliable. However,

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Calcification of Bones and Teeth

existing theories do not explain all of the calcification processes occurring in hard tissues in vivo. Thus, it will be necessary to determine whether calcification can be explained by theories based on what is known about in vivo hard tissues or by a combination of both of these theories, or whether a new theory is required.

Growth and Maturation of Apatite Crystals The growth of crystals occurs when the ion clusters reach a suitable size to become a seed, and ions attach to the surface of the ion clusters continuously to develop the proper structure and shape (Ferguson, 1999). Once crystals are formed, crystal growth occurs at a rapid rate, followed by slow crystal growth resulting in a 10–20-fold increase in size. In the case of multiple crystal formation, the generation of new crystals begins with crystals that act as nuclei (secondary nucleation), and multiple crystals are formed simultaneously during the conversion of hydroxyapatite precursors such as octacalcium phosphate to hydroxyapatite. Once the crystals begin to grow, the shape, size, and final arrangement of the crystals are adjusted according to each tissue and the specific organic components of the extracellular matrix of each tissue. For example, the shape and size of bone tissue, dentine, and cementum are determined by collagen fibers, whereas enamel is controlled by enamelin. The microenvironment adjacent to the growing crystal is particularly important for the growth of crystals. One example is the action of non-collagenous proteins that determine the shape of a crystal by selectively attaching to the surface of the crystal to inhibit its growth. Pyrophosphate, which accumulates on the crystal surface, also inhibits crystal growth. The maturation process of in vivo tissues undergoing calcification is somewhat different for each tissue, although the following common features have been detected. (a) During the maturation of calcified tissues, organic components that predominate over non-collagenous proteins are lost, as is water. (b) Mineral deposits occur in the space where the organic components and

References

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water escaped, and (c) precursors of hydroxyapatite such as amorphous calcium phosphate are converted to hydroxyapatite. The presence of Mg2+ promotes the formation of amorphous calcium phosphate and is deeply involved in the stabilization of the amorphous calcium phosphate. Therefore, amorphous calcium phosphate can be converted to hydroxyapatite when Mg2+ is lost during maturation.

the process of dentine growth, organic components are constantly deposited at a rate of approximately 4 μm per day and are calcified in a 12 h cycle. Calcification of cementum is similar to that of bone tissue. The cells involved in calcification are cementoblasts, which are located in a thin layer between the border of the periodontal membrane and the uncalcified precementum.

Characteristics of Calcification in Teeth

References

The calcification process of enamel is unique compared with that of other oral calcified tissues. Instead of collagen fiber, the organic component is a protein called amelogenin, and the size of the apatite crystal, which is the structural entity constituting the calcified tissue, is relatively large. The cells involved in enamel calcification are ameloblasts, and sufficient calcium ions are found in the matrix at the initial calcification stage. Apatite crystals are initially deposited side by side on the collagen fibers over the dentine, forming a plate or ribbon with a thickness of 15 Å that grows in width and length as the crystals mature. Mature hexagonal crystals have a thickness of 500–1200 Å and a length of 3000–5000 Å. The average size of the enamel crystals is 1600 × 400 × 170 Å. Unlike in other calcified tissues, amorphous calcium phosphate is not present. During maturation, the cell volume decreases and the number of intracellular organelles decreases. Calcification of dentine occurs in two stages. First, collagen fibers from predentine are arranged from the pulp tissue. Then, calcification occurs in confined areas between predentine and dentine. The cells involved in the calcification of predentine are odontoblasts, and an amorphous calcium phosphate is a form of the initial calcium phosphate salts similar to those of bone tissues. A peritubular matrix is involved in the odontoblastic process, and it is altered prior to mineral deposition. Small apatite crystals with the same orientation along the collagen fibers of the peritubular matrix fuse until the entire matrix is calcified. Apatite crystals are smaller than enamel and form small plates with a length of 1000 Å. In

Anderson, H.C.: Electron microscopic studies of induced cartilage development and calcification. J. Cell Biol. 35, 81–101 (1967) Anderson, H.C.: Vesicles associated with calcification in the matrix of epiphyseal cartilage. J. Cell Biol. 41, 59– 72 (1969) Anderson, H.C., Garimella, R., Tague, S.E.: The role of matrix vesicles in growth plate development and biomineralization. Front. Biosci. 10, 822–837 (2005) Bilezikian, J.P., Raisz, L.G., Rodan, G.A.: Principles of bone biology, 2nd edn. Academic Press, Washington (2001) Cole, A.S., Eastoe, J.E.: Biochemistry and oral biology, 2nd edn. Wright, Bristol (1988) Davis, N.R., Walker, T.E.: The role of carboxyl groups in collagen calcification. Biochem. Biophys. Res. Commun. 48, 1656–1662 (1972) Ferguson, D.B.: Oral bioscience. Churchill Livingstone, Edinburgh (1999) Glimcher, M.J.: Molecular biology of mineralized tissues with particular reference to bone. Rev. Mod. Phys. 31, 359–393 (1959) Grant, S.W.: Shell structure and distribution of Cloudina, a potential index fossil for the terminal Proterozoic. Am. J. Sci. 290-A, 261–294 (1990) Gutman, A.B., Yu, T.F.: Further studies of the relation between glycogenolysis and calcification in cartilage. Trans. Macy Conf. Metab. Interrelat. 1, 11 (1949) Gutman, A.B., Yu, T.F.: A concept of the role of enzymes in endochondral calcification. Trans. Macy Conf. Metab. Interrelat. 2, 167 (1950) Lazzari, E.P.: Dental biochemistry, 2nd edn. Lea & Febiger, Philadelphia (1976) Lee, J., Min, H.K., Park, C.Y., Kang, H.K., Jung, S.Y., Min, B.M.: A vitronectin-derived peptide prevents and restores alveolar bone loss by modulating bone remodeling and expression of RANKL and IL-17A. J. Clin. Periodontol. 49, 799–813 (2022) Neuman, W.F., Neuman, M.W.: The nature of the mineral phase of bone. Chem. Rev. 53, 1–45 (1953) The possible significance of Robison, R.: hexosephosphoric esters in ossification. Biochem. J. 17, 286–293 (1923)

6

Oral Mucosa and Gingiva

The mucosa, which is composed of epithelium and connective tissue (Fig. 6.1), is a wet membrane covering the inner surface of the body cavity that is connected to the external surface of the body. The mucosa acts as a functional unit and is thus considered to be an organ. Briefly, the function of the mucosa is as follows. (a) The membrane covering the surface protects the tissues and organs beneath it from the external environment. (b) Stimuli outside the mucosa are detected by receptors that sense temperature, touch, or pain; thus, the mucosa is responsible for sensory function. (c) Secretions from sweat, sebaceous, and salivary glands maintain their moist surface. Finally, (d) the mucosa plays a key role in thermoregulation.

Structure and Function of the Oral Mucosa and Gingiva In general, a mucosa is composed of an epithelium, corresponding to the epidermal layer and underlying connective tissues; the oral mucosa is no exception (Fig. 6.1). Depending on the site, a submucosa (also called the hypodermis) is present beneath oral mucosa. Even though epithelial tissue is innervated, it contains no blood vessels. Therefore, the epithelial cells must be nourished by substances that diffuse from blood vessels in the underlying connective tissue. In addition, epithelial cells, which are the main cells constituting the epithelial tissue, have a higher turnover rate

than other cells. These cells contain desmosomes, tonofilaments, and keratohyalin granules that are generally not present in other cell types and consequently have a distinct morphology. Oral mucosa can be classified as lining, masticatory, or specialized mucosa. The lining mucosa is located in the cheek, lip, soft palate, and below the undersurface of the tongue, and the epithelium of this mucosa is usually composed of nonkeratinized stratified squamous epithelium. The lining mucosa is somewhat wavy on the side adjacent to the connective tissue (Fig. 6.2a). The masticatory mucosa is located on the inner surface of the attached gingiva and the hard palate, and the epithelium of this mucosa is composed of keratinized stratified squamous epithelium (Fig. 6.2b). Finally, the specialized mucosa refers to the upper part of the tongue, which is composed of a specialized papilla (so-called taste buds). The stratified squamous epithelium of specialized mucosa has a range of thickness and degree of keratinization; in addition, its lower connective tissue is abundant and contains the specialized tongue papilla. Moreover, no submucosa is present beneath specialized mucosa (Fig. 6.2c).

Epithelial Structure of Oral Mucosa and Gingiva The epithelium of the oral mucosa is divided into four cell layers depending on the degree of

# The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 B.-M. Min, Oral Biochemistry, https://doi.org/10.1007/978-981-99-3596-3_6

87

88

Fig. 6.1 Different types of tissue that constitute oral mucosa. [Source: T. Cate, Oral Histology, 6th ed. (Mosby, 2003), Fig. 12.4]

keratinocyte differentiation (Fig. 6.3). That is, in order from the surface adjacent to the basement membrane, the epithelial tissue is divided into the stratum basale, stratum spinosum, stratum granulosum, and stratum corneum. The keratinocytes of the basal layer are composed of monolayer cuboidal cells and are the only cells in the epithelial tissue that are capable of cell division. Accordingly, they are responsible for the continuous supply of new cells to the epithelium (Fig. 6.3). The keratinocytes of the prickle-cell layer, which itself consists of multiple layers of keratinocytes, have a spine-like shape formed by cellular shrinkage during the histological fixation process; consequently, this layer is also called the spinous layer. This cell layer accounts for approximately one-half to two-thirds of the epithelial

6 Oral Mucosa and Gingiva

layer, and is the thickest layer in the epithelial tissue (Fig. 6.3). This layer also contains intercellular connective structures called desmosomes. The cells themselves contain a tonofibril, a bundle of fine fibers about 10 nm thick, which is responsible for their mechanical function. The basal and prickle-cell layers are present in the epithelium of all three types of oral mucosa, i.e., lining, masticatory, and specialized. However, the granular and cornified layers have different structures in different regions of the oral mucosa. The granular layer is composed of flat epithelial cells on the prickle-cell layer, and the cytoplasm is filled with keratohyalin granules that stain black. These granules contain the chemical precursors of keratin. The granular layer consists of 2–4 layers of cells, with the concentration of granules increasing toward the top (Fig. 6.3). The cornified layer, which is the outermost cell layer of the epidermis, consists of flat, disc-like cells. The cells that constitute the cornified layer are non-vital: i.e., they lack a nucleus and most organelles (Fig. 6.3). Instead, the cells in the cornified layer are filled with cytokeratin bundles in a filaggrin matrix: i.e., they are keratinized and non-viable. In the epithelial tissue, the marker molecules of the basal layer are keratins 5 and 14, whereas the markers of the prickle-cell layer are keratins 1 and 10. The markers of the granular layer are involucrin, loricrin, filaggrin, and transglutaminase, and the marker of the cornified layer is the cornified cell envelope. More than 95% of the oral mucosal epithelium consists of keratinocytes, and the remaining cells include melanocytes, Langerhans cells, and Merkel cells. Melanocytes, located in the basal layer, produce melanin pigment and often appear black. Langerhans cells are located in the upper layer of melanocytes and are morphologically similar to these cells. Langerhans cells have immune receptors on their surfaces and are largely responsible for the immune defense of the mucosa. Merkel cells, also present in the basal layer, are associated with nerve terminals present in the epithelium. In the oral mucosal epithelium, migrated inflammatory cells such as leukocytes, monocytes, and neutrophils are also

Structure and Function of the Oral Mucosa and Gingiva

89

Fig. 6.2 Schematic diagrams showing the general characteristics of three types of oral mucosa: lining mucosa (a), masticatory mucosa (b), and specialized mucosa (c). The thickness of the epithelium, degree of epithelial keratinization, thickness of connective tissue, shape of connective tissue papilla, and presence or absence of submucosal tissue can be confirmed

present, and sometimes mast cells are also observed. The gingiva also consists of epithelium and connective tissue, and the gingival epithelium can be divided into three parts. The oral

epithelium faces the oral cavity, whereas the oral sulcular epithelium faces the teeth but is not directly attached to them. Junctional epithelium occupies the area where the gingiva and teeth are attached (Fig. 6.4a, b). The interface between the

Fig. 6.3 Schematic diagram showing the structure of the oral mucosal epithelium. Succession of cells in lightly keratinizing stratified squamous epithelium. Keratinized squames are shown in red, and the widths of intercellular spaces are exaggerated. From the basement membrane, the

epithelial tissue contains the stratum basale, stratum spinosum, stratum granulosum, and stratum corneum. [Source: S. Cole and J. E. Eastoe, Biochemistry and Oral Biology, 2nd ed. (Wright, 1988), Fig. 26.1]

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6 Oral Mucosa and Gingiva

Fig. 6.4 Schematic diagrams showing the structure of the gingiva. (a) The gingiva is divided into the oral epithelium (OE), oral sulcular epithelium (OSE), and junctional epithelium (JE). E enamel. (b) Photograph showing the structure of free gingiva, including epithelium and connective

tissue, located longitudinally at the horizon of the cementum–enamel junction (CEJ) (actual picture of a). (c) Histological image showing the structure of the oral epithelium. OE oral epithelium, CT connective tissue, CTP connective tissue papillae, ER epithelial pegs

oral epithelium and underlying connective tissues is a tangled, wavy structure (Fig. 6.4c). The portion of the connective tissue that protrudes toward the epithelium is called the connective tissue papillae, whereas the portion that penetrates into the underlying connective tissue is called the epithelial pegs (rete ridges or rete pegs). Connective tissue papillae and epithelial pegs are present in the oral epithelium and oral sulcular epithelium but are not found between the junctional epithelium of normal gingiva and the underlying connective tissue.

Among these, fibroblasts (spindle- or stellateshaped cells with one or more elliptical nuclei) account for about 65% of the total. These cells produce various kinds of fibers found in connective tissue, including collagen, reticulin, oxytalan, and elastic fibers. Collagen fibers are the most abundant fibers in the gingival connective tissue. When they are observed by electron microscopy, cross-bands or cross-striations appear as alternating dark and bright bands. The period of this banding pattern is 640 Å in the dehydrated form and 700 Å in the hydrated form. The collagen monomer, tropocollagen, is synthesized and polymerized in the extracellular matrix to form collagen fibrils. Cross-links, a type of covalent bond, provide stability to collagen fibrils, thereby decreasing their solubility. Reticular fibers, found at the epithelium–connective tissue and endothelial cell–connective tissue interface, contain more carbohydrates than collagen fibers and thus stain differently than collagen fibers. Oxytalan fibers are elongated fibers with a diameter of 150 Å that lie parallel to the long axis of the tooth. They are abundant in the periodontal ligament but are present at lower levels in gingival tissue. To date, their function has not yet been elucidated. Finally, the

Connective Tissue Structure of Oral Mucosa and Gingiva The major component of the oral mucosa and gingiva is the connective tissue called the lamina propria. This connective tissue consists largely of collagen fibers (40–60% of its volume), blood vessels and nerves buried in an amorphous matrix (35%), and fibroblasts (5%). The fibroblasts reside between the collagen fibrils in the connective tissue. In addition, the lamina propria contains various kinds of cells, including mast cells, macrophages, and inflammatory cells.

Metabolism of Gingiva

elastic fibers in gingival connective tissue and periodontal ligament are associated with blood vessels and are also abundant in the connective tissue of the alveolar mucosa. Most of the matrix constituting the connective tissue originates from substances that are synthesized and secreted from fibroblasts, but some of the components are derived from mast cells or blood. The major components are macromolecules such as proteins and carbohydrates, which can be classified as proteoglycan and glycoproteins. In proteoglycans, glycosaminoglycans are covalently attached to one or more protein cores. Via the matrix, water, electrolytes, nutrients, and metabolites migrate to the cells in the connective tissue.

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the matrix and thus is an important determinant of tissue fluid content and osmotic pressure. In addition, proteoglycan acts as a molecular sieve and thus regulates cell migration within the tissue. Due to the characteristic structure of proteoglycan and its association with water, this macromolecule is resistant to deformation and consequently serves as a regulator to maintain the homeostasis of connective tissue. When pressure is applied to the gingiva, proteoglycan is deformed, but when the pressure is removed, it retains its original shape. Therefore, proteoglycan plays important roles in imparting resilience to the gingiva.

Metabolism of Gingiva Composition of Gingiva

Function of the Oral Mucosa Based on its function, the epithelium can be classified into protective, secretory, and absorptive epithelium. Oral mucosa is a membrane covering the tissue surface, such as skin, and serves as a shield to protect tissues and organs from the external environment (Bartold et al., 2000). In addition to mechanical shielding to protect the underlying tissue, the connective tissue is also essential for the maintenance of vulnerable tissues such as blood vessels and nerves. Mucous and salivary glands embedded in the epithelium secrete glandular substances that maintain the moist surface of the oral mucosa. In addition, the oral mucosa contains Merkel cells bearing receptors that sense temperature, tactile sensation, and nociceptive stimuli that occur outside the mucosa and thus serve as a sensory receptor. It also plays a key role in thermoregulation. The normal resilience of connective tissue is typically mediated by proteoglycans and glycosaminoglycans. The glycosaminoglycan of proteoglycan is a large, anionic molecule with a disordered structure that occupies a relatively large space. In this space, relatively small molecules, such as water and electrolytes, are mixed with substances of high molecular weight, which impedes their movement. In other words, proteoglycan regulates diffusion and fluid flow in

Because gingival tissue is mixed with epithelium and connective tissue at various ratios, the chemical composition of the gingiva varies greatly depending on the sampling site. Gingival connective tissue consists of collagen, proteoglycan, glycoprotein, and lipid, whereas the epithelium is composed mainly of non-collagenous proteins, lipids, nucleic acids, and basement membrane components. Representative constituents of human and porcine gingiva are shown in Table 6.1. The major chemical constituent of gingiva is collagen.

Collagen Because the gingival epithelium is composed of tightly connected cells, little intercellular space is available, and no fibrous proteins are present in the extracellular matrix. The basement membrane between the epithelium and connective tissue consists mainly of type IV collagen, and the anchoring fibril contains type VII collagen. Type IV collagen is present in all basement membranes of the oral, oral sulcular, and junctional epithelia of the gingiva. Gingival connective tissue consists predominantly of type I collagen, which accounts for more than 80% of the collagen in the gingiva. Two types of collagen fibers are present in the connective tissue: one with big and dense bundles of thick collagen fibers and another that is

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Table 6.1 Composition of the whole gingiva Componenta Collagen

Non-collagenous proteins Totald Structural glycoproteins Lipid Glycosaminoglycans Hexosamine Glucuronic acid DNA RNA Plasma proteins RBC Uncharacterized

Human 45.00

Humanb 49.05 31.00 27.60

Humanc 37.70

Porcined 45.00

– 20.00 7.20 1.40

62.00 – 7.35 0.32 0.37 0.20 0.20 1.90 1.80

47.70 – 7.20 –

28.60 – 7.20 –

1.42 1.52 – – –

0.72 2.20 3.80 3.00 –

0.85 2.12 – – 24.43

[Source: E. P. Lazzari, Handbook of Experimental Aspects of Oral Biochemistry, (CRC Press, 1983), p. 212] Expressed as percentage of dry weight of tissue b Water content estimated as 80% c Water content estimated as 74% d Measured by the biuret reaction a

interwoven with short and thin collagen fibers; the latter can assume the form of a thin reticular web or a loose (i.e., less ordered) form. Type I collagen is primarily distributed in thicker fibers, rather than in the lamina propria, whereas type III collagen is distributed in the reticular-shaped thin fibers near the basement membrane. Gingival connective tissue also contains small amounts of type V and type VI collagen. A histidinohydroxymerodesmosine bond constitutes most of the cross-links in gingival collagen fiber. One of the characteristics of the collagen in the gingiva is its low solubility: the salt solubilities of the periodontal ligament and gingiva are 2.8% and 3.0%, very low compared with the value for skin (6.3%).

Proteoglycan Hyaluronan, decorin, syndecan, CD-44, and other molecules are present on the surfaces of gingival epithelial cells and between epithelial cells. The gingival basement membrane is mainly composed of perlecan, a type of heparan sulfate proteoglycan, and also includes other proteoglycans containing heparan sulfate and chondroitin.

Decorin, biglycan, and versican are also present in gingival connective tissue. Glycosaminoglycan composition varies greatly depending on the species. Human gingiva contains high levels of hyaluronic acid, dermatan sulfate, and chondroitin 4-sulfate, in that order. By contrast, in animal gingiva, the order of abundance is chondroitin 4-sulfate, dermatan sulfate, and hyaluronic acid; heparan sulfate is present in small amounts, and chondroitin 6-sulfate may or may not be present.

Non-collagenous Proteins β1, β4, and α6 integrins are present on gingival epithelial cells and basement membrane. β1 integrins, including α2β1, α3β1, and α9β1, and the intercellular adhesion molecule (ICAM)-1 (CD54) are involved in cell adhesion. ICAM-1 is expressed in the oral and junctional epithelia, but not in the oral sulcular epithelium. Integrin α6β4 is a major component of the hemidesmosome, which is involved in the adhesion of cells to the basement membrane, as well as in intracellular signal transduction. Laminin, a major non-collagenous protein of the basement membrane, forms a complex with nidogen-1

Metabolism of Gingiva

(formerly known as entactin) and interacts with type IV collagen. Laminin is also involved in cell adhesion through integrin (mainly α6β1) on the cell surface. Fibroblasts of gingival connective tissue express α1, α2, α5, αv, and αvβ3 integrins, which act as receptors for vitronectin, fibronectin, collagen, and other proteins. Gingival fibroblasts synthesize large amounts of fibronectin. Other non-collagenous proteins of gingiva include osteonectin, vitronectin, elastin, and tenascin.

Lipid The lipid comprises 7.35% of the dry weight of the gingiva; the specific lipid composition is shown in Table 6.2. In particular, gingival epithelial tissue is rich in phospholipid, and the connective tissue contains high levels of triacylglycerol and cholesterol. Almost all of the arachidonic acid is in the connective tissue, implying that fibroblasts are the source of prostaglandin synthesis. Phospholipids, free fatty acids, and cholesterol stain strongly in the epithelium, especially in the cornified layer. Keratin The oral sulcular and junctional epithelia are nonkeratinized stratified squamous epithelia, and keratinocytes are the primary cells of the oral cavity and gingival epithelium. Keratin, a protein expressed only in keratinocytes, consists of several polypeptides with different isoelectric points and molecular weights and is numbered according to the molecular weights of its component proteins. In general, keratins with small molecular weights are expressed in keratinocytes of the basal layer, whereas those with higher molecular weights are expressed closer to the surface. Others, including carbohydrates and free amino acids, are contained in the gingiva. Oral epithelial cells also produce cationic antimicrobial peptides, commonly referred to as β-defensin, including human β-defensin-1 (hBD-1), hBD-2, and cathelicidin LL-37.

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Metabolism of the Gingiva The respiratory rate (Qo2) of normal human gingiva is shown in Table 6.3. The average respiratory rate of normal human gingiva is 1.49, lower than those of other tissues. The respiratory rate decreases with age, but does not differ significantly by gender or race. The respiratory rate of the gingiva is slightly lower than that of the skin but higher than that of uterine tissue or the smooth muscle of the stomach. The respiratory rate of the gingiva depends on the thickness and volume of the sample, the concentration of the reactant, the ratio of epithelium and connective tissue within the sample, and the degree of inflammation. Because the respiration rate of the epithelium is higher than that of connective tissue, samples with a greater proportion of epithelial tissue have higher respiratory rates. Moreover, when the inflammation of the gingival tissue is mild or moderate, the respiratory rate rises, but when the inflammation becomes severe, the respiration rate falls. The biochemical activity of the gingiva was measured by determining the turnover rate of connective tissue components. To this end, rats received feed containing 15N-glycine, and the amount of 15N-glycine remaining in the proteins of gingival mucosa, palate mucosa, tongue mucosa, and incisor pulp was measured. All oral tissues of rats absorbed 15N relatively quickly, and the release rate of 15N from the proteins of the gingival, palate, and tongue mucosa was also relatively rapid. The release rate of incisor pulp protein was the slowest, comparable with that of liver protein. This observation demonstrates that gingival protein turns over more rapidly than liver protein. The turnover half-life of mature collagen was 5 days in rat gingiva, 1 day in periodontal ligament, 6 days in alveolar bone, and 15 days in skin dermis (corium). Therefore, the gingiva and periodontal ligament exhibit faster protein turnover rates than other connective tissues. The half-life of glycosaminoglycan is shorter in the gingiva than in the skin and cartilage. Therefore, gingiva has a low respiratory rate but

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6 Oral Mucosa and Gingiva

Table 6.2 Lipids in the gingiva Total lipida Nonpolar lipids Free fatty acids Triacylglycerols Mono- and diacylglycerols Cholesterol esters Cholesterol Total phospholipids Phosphatidylcholine Phosphatidylethanolamine Sphingomyelin Other

Human 5.65 70.00 17.00 14.34 4.14 11.60 10.60 30.00 15.90 8.20 1.30 4.60

Human 5.65 67.00 19.20 17.60 10.30 12.90 7.00 33.00 13.40 6.30 4.70 8.60

Porcine 7.12 69.80 21.00 17.20 8.20 16.30 8.20 30.20 11.30 10.40 3.80 5.10

Bovine 7.67 70.70 17.00 16.90 7.00 22.80 7.00 29.30 10.00 9.40 8.80 Trace

a

Total lipids are expressed as percentage of dry weight of tissue. The remaining values are expressed as percentage of total lipids. [Source: E. P. Lazzari, Handbook of Experimental Aspects of Oral Biochemistry, (CRC Press, 1983), p. 222]

a relatively rapid turnover of protein and glycosaminoglycan. The enzymatic activities of glycolytic enzymes such as phosphofructokinase, pyruvate dehydrogenase complex, and glucose 6-phosphate dehydrogenase, an enzyme of the pentose phosphate pathway, in rat gingiva are low. However, lactate dehydrogenase is much more active than the other enzymes, demonstrating that anaerobic glycolysis is occurring. In the rat gingiva, energy metabolism by the citric acid cycle or oxidative phosphorylation, which can occur only under aerobic conditions, is minimal. In gingival epithelium, the levels of mitochondria are high in the cells of the basal layer but decrease gradually closer to the surface. Because the basal layer and its surrounding cells are adjacent to the connective tissue, the activities of mitochondrial enzymes such as succinate

dehydrogenase and cytochrome oxidase are high, facilitating ATP synthesis through normal energy metabolism. Conversely, the activity of enzymes involved in the pentose phosphate pathway, such as glucose 6-phosphate dehydrogenase, became more active closer to the surface layer.

Gingivitis Matrix Metalloproteinases (MMPs) Collagenase, gelatinase, and stromelysin, which belong to matrix metalloproteinases (MMPs), degrade extracellular matrix proteins (Table 6.4). All MMPs contain Zn2+ in the active site and are stabilized by Ca2+ (Birkedal-Hansen, 1993). MMPs are secreted as zymogens in an inactive form, which is subsequently activated

Table 6.3 Endogenous Qo2 values for a normal human gingiva Number of samples 8 5 10 36 75 21 36 18 Average endogenous respiratory rate

State of tissue Normal Normal Normal Normal Normal

Age

0–20 21–40 41–60

Endogenous Qo2 1.60 1.90 1.36 1.39 1.43 1.64 1.39 1.26 1.49

[Source: E. P. Lazzari, Handbook of Experimental Aspects of Oral Biochemistry, (CRC Press, 1983), p. 234]

Metabolism of Gingiva

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Table 6.4 Classification of metalloproteinases (MMPs) present in the matrix

MMP Collagenases

No. MMP-1

Common name Collagenase 1, interstitial collagenase

Molecular weighta (kDa) 55

75 Collagenase 2, neutrophil collagenase MMP-13 Collagenase 3 65 MMP-8

Gelatinases

Stromelysins

54

Aggrecan, gelatins, fibronectin, laminin, tenascin, vitronectin, collagens III, IV, IX, and X Aggrecan, fibronectin, laminin, collagen IV Fibroblast Fibronectin, laminin, collagen IV, aggrecan, gelatins Fibroblast, epithelial Aggrecan, fibronectin, laminin, cell collagen IV, gelatins, elastin, entactin, tenascin, vitronectin Macrophage Elastin

63

Fibroblast

69

Ameloblast, odontoblast

Collagens I, II, and III, fibronectin, laminin, vitronectin, proteoglycan, ProMMP-2, ProMMP-13 Amelogenin

Gelatinase A, type IV collagenase

72

MMP-9

Gelatinase B, type V collagenase

92

MMP-3

Stromelysin 1, transin

57

Pump-1

Metalloelastase MMP-12 Macrophage elastase MMP-14 Membranetype-1-MMP (MT1-MMP) Enamelysin MMP-20 a

Collagens I > II > III, VII, and X, gelatins, entactin, aggrecan, tenascin Collagens II > III > I, VII, and X, gelatins, entactin, aggrecan, tenascin Gelatins, elastin, fibronectin, laminin, aggrecan, vitronectin, collagens I, IV, V, VII, X, and XI

MMP-2

MMP-7

Matrix Collagens III > I > II, VII, X, gelatins, entactin, aggrecan, tenascin

Fibroblast, epithelial cell Fibroblast, keratinocyte, macrophage, endothelial cell Neutrophil, keratinocyte, macrophage, endothelial cell Fibroblast, macrophage, endothelial cell Keratinocyte

MMP-10 Stromelysin 57 2, transin 2 MMP-11 Stromelysin 3 51 Matrilysin

Origin Fibroblast, keratinocyte, macrophage, endothelial cell Neutrophil

28

Gelatins, elastin, fibronectin, laminin, aggrecan, vitronectin, collagens I, IV, V, VII, X, and XI

Expressed as average molecular weight

by the removal of the pro-peptide. The sequence of the Zn2+ binding site is HEXGHXXGXXHS (T). The enzymatic activities of MMPs are inhibited by tissue inhibitors of metalloproteinases (TIMPs) or α-macroglobulin, and MMPs selectively degrade substrates with hydrophobic residues at the N-terminus. Interstitial collagenase (MMP-1) cleaves triple-helical regions of collagen about threequarters of the way down its N-terminus. However, the bacterial collagenase produced from Clostridium histolyticum, which causes gas gangrene, hydrolyzes the peptide bond between

X-Gly in the -X-Gly-Pro-Y- sequence of collagen, causing each polypeptide chain to be cleaved into more than 200 fragments. TIMPs, including TIMP-1, TIMP-2, TIMP-3, and TIMP-4, have six conserved disulfide bonds. TIMP-1, a 30 kDa glycoprotein that is synthesized in macrophages and most connective tissue cells, is most abundant. TIMP-2 inhibits gelatinases more efficiently than TIMP-1. In particular, TIMP-2 binds to progelatinase A at a 1:1 ratio, but does not bind to progelatinase B.

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Changes in Gingival Epithelium and Connective Tissue At the beginning of the inflammatory response, the gingival epithelium expresses cytokines involved in neutrophil transport, such as interleukin (IL)-1 and IL-8, as well as factors involved in neutrophil binding, such as ICAM-1 and E-selectin. When cytokines such as IL-1 and IL-8 and adhesion molecules are expressed in the epithelium, the intercellular gaps in the junctional epithelium widen, a chemotactic gradient is formed as the substance moves from the exterior toward the connective tissue, and neutrophils move further inward. As the plaque is maintained, the movement of neutrophils continues, and macrophages and lymphocytes in gingival connective tissue are activated. Simultaneously, the junctional epithelium of the gingiva moves toward the root apex, forming early gingival pockets. In addition, gingival epithelial cells are stimulated by TNF-α, transforming growth factor (TGF)-β, and keratinocyte growth factor to produce collagenase-3 (MMP-13). Gingival connective tissues are destroyed within 3–4 days after the early inflammatory responses, beginning with the perivascular collagen fibers, and about 70% of collagen fibers at sites of inflammation sites are lost. In inflamed gingiva, the amount of type III collagen decreases, and the amount of type V collagen increases, with the level of the α1-chain increasing three- to fourfold. In addition, fibroblasts from inflamed gingiva secrete type I trimer [α1(I)]3, which is not present in normal gingiva. Although the proteoglycan content of inflamed gingiva does not change as much as that of collagen, the amounts of dermatan sulfate and chondroitin sulfate decrease and increase, respectively. In addition, the levels of the core protein of proteoglycan and hyaluronic acid also decrease: a hallmark of inflamed gingival connective tissue. Destruction of Gingival Tissue Gingivitis and periodontitis are the result of immune responses induced by plaque bacteria in

6 Oral Mucosa and Gingiva

supragingival and subgingival plaques. Bacterial antigens, including endotoxins or lipopolysaccharides of the plaque bacteria, diffuse into the gingival tissue, where they activate complement directly or indirectly by forming an antigen–antibody complex. When chemotaxin is produced by activation of complement, neutrophils and macrophages gather. Polymorphonuclear leukocytes (PMNs) and macrophages are activated by plaque bacterial antigens entering cells by endocytosis, and the activated cells release neutrophil collagenase (MMP-8), gelatinase B (MMP-9), elastase, cathepsin G, and other lysosomal enzymes. These enzymes work cooperatively to degrade components of the gingival matrix such as collagen fibers or proteoglycan. In addition, neutrophils and macrophages secrete inflammatory cytokines such as IL-1, which stimulate the surrounding gingival fibroblasts to produce collagen. However, the production of TIMP, a collagenase inhibitor, remains unchanged or is even suppressed, leading to matrix degradation (Fig. 6.5). IL-1α and IL-1β are the chief inducers of MMP expression, which is also upregulated by TNF-α, prostaglandin E2, TGF-α, epidermal growth factor, and platelet-derived growth factor. By contrast, TGF-β and interferon-γ inhibit MMP expression. In addition, hyaluronidase, chondroitin sulfatase, glycosidase, collagenase, and other proteolytic enzymes synthesized by oral bacteria contribute to the degradation of collagen fibers or proteoglycan in periodontal tissue. In addition, reactive oxygen species (ROS) are produced by the respiratory bursts of PMNs during inflammation, and also as metabolic byproducts of the normal respiratory processes of cells. When the body’s reducing power is insufficient, ROS destroy collagen, proteoglycan, and glycosaminoglycan and damage DNA, proteins, and unsaturated fats, leading to cell death (Waddington et al., 2000).

References

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Fig. 6.5 Mechanism of destruction of gingival tissue. LPS lipopolysaccharide, PMN polymorphonuclear leukocyte, Ag–Ab antigenantibody complex, MΦ macrophages, IL interleukin, TNF tumor necrosis factor

References Bartold, P.M., Walsh, L.J., Narayanan, A.S.: Molecular and cell biology of the gingiva. Periodontol. 24, 28–55 (2000) Birkedal-Hansen, H.: Role of matrix metalloproteinases in human periodontal diseases. J. Periodontol. 64, 474–484 (1993)

Cate, T.: Oral Histology: Development, Structure, and Function, 6th edn. Mosby (2003) Cole, A.S., Eastoe, J.E.: Biochemistry and oral biology, 2nd edn. Wright, Bristol (1988) Lazzari, E.P.: Handbook of experimental aspects of oral biochemistry. CRC Press, Boca Raton (1983) Waddington, R.J., Moseley, R., Embery, G.: Reactive oxygen species: a potential role in the pathogenesis of periodontal diseases. Oral Dis. 6, 138–151 (2000)

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Structure and Vascular System of Salivary Glands Structure of Salivary Glands Salivary glands are classified, based on their size, as major or minor salivary glands. More than 90% of the saliva is secreted from the major salivary glands of which there are three pairs: the parotid, submandibular, and sublingual glands (Fig. 7.1). The numerous minor salivary glands include the palatinal, buccal, labial, lingual (von Ebner’s), and retromolar glands. Minor salivary glands are located in the submucosa throughout the oral cavity and are named depending on their anatomical locations. The serous acinar cells of the parotid gland, which is innervated by the glossopharyngeal nerve (cranial nerve [CN] IX), secrete profuse watery saliva. The submandibular and sublingual glands, which are innervated by the facial nerve (CN VII), are composed of acinar cells, consisting of mixed serous and mucous cells (Fig. 7.1). When these glands are stimulated by the sympathetic nerve, they produce a thick viscous secretion (α-receptor type) and amylase (β-receptor type). The palatinal, buccal, and labial glands, as well as part of the retromolar gland, which are innervated by the facial nerve, are composed entirely of mucous cells and thus secrete mucous saliva. The glossopharyngeal nerve innervates the lingual gland and the other part of the retromolar

gland, which secretes lipase-rich saliva. This salivary lipase plays an important role when the level of pancreatic lipase is low. Salivary glands are made up of large numbers of individual secretory functional units, which consist of acinar cells, an intercalated duct, a striated duct, an excretory duct, and a main collecting duct. (Note that the sublingual and minor salivary glands do not have intercalated ducts.) Acinar cells are saliva-producing cells with a pyramidal shape; the basolateral side of an acinar cell contains a broad surface area with complex functions, whereas the apical or luminal side of the acinar cell membrane has a small surface area and relatively simple functions (Fig. 7.2). They contain ionic channels, which transport electrolytes, and perform exocytotic functions involved in protein release. Striated duct cells actively modify primary salivary compositions by absorbing Na+ and Cl- or secreting K+ and HCO3-.

Vascular System of Salivary Glands The blood supply is essential for producing saliva. The parotid gland receives its blood supply from the external carotid artery; the submandibular gland from the submental artery, which arises from the facial artery; and the sublingual gland from the sublingual and submental arteries. These arteries contain two arteriolar capillary systems arranged in series that are necessary for the

# The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 B.-M. Min, Oral Biochemistry, https://doi.org/10.1007/978-981-99-3596-3_7

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transportation of substances: one arteriolar capillary network around the duct cells, and another around the acinar cells (Fig. 7.2c). Consequently, ions that are absorbed by duct cells are secreted from the acinar cells, and the reverse phenomenon can also be observed, i.e., ions are secreted primarily from acinar cells and subsequently reabsorbed by duct cells. The distribution of the arteriolar capillary network is denser around the duct cells than around the acinar cells. The blood vessel runs parallel to the ductal system, but blood flow and salivary flow run in opposite directions to each other (Fig. 7.2c).

The Nervous System of the Salivary Gland Brain Functions Involved in Saliva Secretion (Central Autonomic Network) Salivary secretion, which normally occurs when foods are placed in the mouth, is observed even in anesthetized and decerebrate animals. This reflex secretion results from the stimulation of oral mechanoreceptors, especially periodontal mechanoreceptors, as well as taste buds. Afferent impulses from these organs travel along sensory fibers to the brain. The human brain is in some sense an “anticipation machine,” and predicting the future is one of its most important functions. Anticipatory cognitive, affective, and behavioral processes are executed to avoid or reduce the impacts of potential threats or stresses. Salivation also occurs adaptively under the control of the brain. In general, parasympathetic activation is the main driver of secretion of fluid by the salivary glands, whereas sympathetic stimulation leads to secretion of proteins. The cholinergic and α-adrenergic receptors are coupled to phospholipase C. One substance linked to phospholipase C is an inositol 1,4,5-trisphosphate, which releases Ca2+ from the endoplasmic reticulum, and this Ca2+ evokes the opening of Ca2+-activated K+ channels in the basolateral membrane of the acinar cell and activation of the Cl- channel for Clefflux in the luminal membrane.

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Parasympathetic effects are augmented by activation of the left insula and anterior cingulate cortex, whereas the right insula, which has an interoceptive awareness function, is more likely to produce sympathetic responses. The insular cortex, along with the anterior cingulate cortex and subcortical amygdala nucleus, comprises the central autonomic network that mediates higherorder autonomic control. Especially, cognitive dissonance can influence central and peripheral autonomic responses.

Peripheral Autonomic Nervous System of Salivary Glands Mental and physical efforts during goal-directed behaviors (i.e., stress) are associated with changes in the central autonomic network and the peripheral autonomic nervous system. The magnitude of stress and its physiological consequences are strongly influenced by an individual’s perception of their own ability to control the presence or intensity of the stimulation. In the salivation system, peripheral autonomic nerve fibers are responsible for the control of salivary secretion associated with the acinar and ductal cells, blood flow, and contraction of myoepithelial cells; so these autonomic nerve fibers control both the volume and type of saliva unconsciously. The peripheral autonomic nervous system, which is directly regulated by the hypothalamus, has two branches: the sympathetic nervous system, often referred to as the “fight or flight” system, and the parasympathetic nervous system, the “rest and digest” system. Furthermore, in the salivary glands, cholinergic parasympathetic stimulation dramatically boosts saliva secretion by increasing blood flow and oscillations in cytoplasmic Ca2+ concentration, which drive oscillations of acinar cell volume that reflect changes in solute content and fluid volume. Also, Ca2+ oscillations modulate the activities of ion transport and granule exocytosis in acinar and ductal cells. On the other hand, salivary glands are also innervated by sympathetic nerves that release noradrenaline, which evokes protein synthesis and release of stored proteins, mostly from acinar and ductal cells. The sympathetic spinal neural bodies innervating the salivary glands are located

The Nervous System of the Salivary Gland

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Fig. 7.1 Overview of anatomical location and histological structure of the major salivary glands and the nervous system that governs them. Shown are the salivary nuclei (upper and lower), the cell bodies of parasympathetic fibers; in the medulla, the facial nerve (VII) innervates the submandibular gland and the sublingual gland, and

the glossopharyngeal nerve (IX) innervates the parotid gland. The sympathetic ganglion cells are located in the nucleus of the pleural fluid (mainly T2), and the efferent nerves enter the salivary glands of the cervical ganglion and travel along the blood vessels

primarily in the intermediolateral nucleus of the second thoracic segment in the spinal cord. Starting from the intermediolateral nucleus, the preganglionic fibers synapse with the superior cervical sympathetic ganglion and become postganglionic fibers. The postganglionic fibers then travel through the facial artery and enter the submandibular and sublingual glands, and some postganglionic fibers travel through the middle meningeal artery and enter the parotid gland (Fig. 7.1). At the terminus of a sympathetic preganglionic fiber, acetylcholine is released as a neurotransmitter for nicotinic receptors, whereas the terminus of a sympathetic postganglionic fiber releases noradrenaline (norepinephrine), which acts on the α- and β-adrenergic receptors located on the basolateral membrane of acinar and ductal cells. In the parotid gland, α-adrenergic receptors are less abundant than parasympathetic muscarinic receptors in the basolateral membrane of

acinar cells; therefore, sympathetic nerve stimulation has little effect on serous saliva secretion. Consequently, humans experience dryness in their oral cavity due to insufficient fluid salivation. However, in the submandibular and sublingual glands, the basolateral membrane contains a large number of β-adrenergic receptors, which result in abundant protein and mucin secretion by these glands. The parasympathetic nerve functions normally in relaxed states and acts as a secretomotor to the salivary glands. The glossopharyngeal nerve (IX), which innervates the parotid glands and some minor salivary glands, is a general visceral efferent neuron that starts in the inferior salivatory nucleus. The tympanic nerve, a preganglionic ninth parasympathetic nerve, travels through the tympanic plexus and synapses with a postganglionic parasympathetic nerve at the otic ganglion near the parotid gland. The postganglionic fiber

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Fig. 7.2 Intracellular ion and water transport in various gland segments. Salivary glands consist of many thousands of secretory units comprising acini (arrangement of acinar cells), an intercalated duct segment, and a striated duct segment. A large number of such secretory units converge into a main excretory duct that drains the saliva into the mouth. (a, b) Main membrane transport mechanisms of acinar and striated duct cells. In both cell types, specific neurotransmitters bind to specific receptor complexes and, via a number of intracellular biochemical events, activate plasma membrane ion transporters, leading to the formation of saliva. For the purpose of simplicity, two acinar cells are used to illustrate the combination of transporters that are activated in single acinar cells. The upper acinar cell illustrates the Ca2+-activated loss of K+ and Cl- that occurs initially upon stimulation/receptor activation, whereas the lower acinar cell illustrates the transporters involved in the re-establishment of the pre-stimulatory ion gradients across the plasma

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membrane. Note that the cells have two transport mechanisms: the parallel operating exchange systems for Na+/H+ and Cl-/HCO3-, as well as the Na+/K+/2Clcotransporter, which both result in cellular uptake of Na+ and Cl-. Notably, an important mechanism involved in intracellular pH regulation is the conversion of CO2 (arising from the metabolic turnover) and H2O into equal amounts of HCO3- and H+, catalyzed by the presence of cytosolic carbonic anhydrase. (c) Although the duct system is branched and thus serves several secretory units, this illustration includes only a simplified schematic drawing of a major gland and its blood supply. The blood flows in the opposite direction relative to the flow of saliva. To increase saliva secretion upon autonomic stimulation, vasodilatation occurs, increasing the rate of blood flow. Ach acetylcholine; adr adrenaline. [From J. L. McManaman, M. E. Reyland and E. C. Thrower. J. Mammary Gland Biol. Neoplasia 11:249–268, 2006]

Mechanism of Salivary Secretion

then enters the parotid gland after merging with the auriculotemporal nerve of the trigeminal nerve branch. On the other hand, the facial nerve (VII), which innervates the submandibular and sublingual glands as well as some minor salivary glands, is also a general visceral efferent neuron that starts in the superior salivatory nucleus. The fiber of the chorda tympani, a preganglionic parasympathetic nerve, travels through the facial nerve and synapses with a postganglionic parasympathetic nerve at the submandibular ganglion near the submandibular and sublingual glands and then joins the lingual nerve and enters the submandibular and sublingual glands (Fig. 7.1). The preganglionic parasympathetic nerve fiber terminal primarily releases acetylcholine, which binds nicotinic receptors. On the other hand, acetylcholine released from the postganglionic fiber terminal of the parasympathetic nerve binds muscarinic receptors and participates in the secretion of electrolytes and water, which constitute 99% of the stimulated saliva. Vasoactive intestinal polypeptides released from the postganglionic fiber terminal of the parasympathetic nerve participate in the secretion of proteins and mucins, as well as vasodilation around the salivary gland. As much as 80–90% of average daily salivary production is the result of parasympathetic nerve stimulation.

Mechanism of Salivary Secretion Secretion of Inorganic Substances in Primary Saliva Saliva fluid secretion takes place primarily from the salivary acinar cells, where electrolyte transporters and ion channels on the basolateral membrane (extracellular space) and water transporters and ion channels on the apical (lumen) membrane play key roles. The autonomic nervous system is functionally coupled to the processes of ion (Na+ and Cl-) and granule secretion. In the sustained presence of cholinergic agonists, changes in cell volume are tightly coupled to dynamic levels of Ca2+. Cholinergic and

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α-adrenergic stimulation are initially associated with a rapid tenfold increase in Ca2+ concentration via activation of phospholipase C, followed by a production of inositol 1,4,5-trisphosphate and substantial cell shrinkage. Subsequently, the cholinergic-evoked Ca2+-activated Cl- channel (CaCC) secretes Cl- apically. The native CaCC expressed in the salivary glands is encoded by the TMEM16A gene, and the TMEM16A protein serves as the apical Cl- channel for Cl- efflux. The efflux of Cl- across the apical membrane with K+ efflux across the basolateral membrane results in a transepithelial potential difference. This augments the driving force of Cl- efflux, and the released Cl- is neutralized by Na+ transport across tight junctions. The neutralized solute (NaCl) generates the transepithelial osmotic gradient, which drives the movement of water, and produces plasma-like isotonic primary saliva, which is also associated with changes in acinar cell volume. The acinar cell volume depends on shrinkage following cholinergic muscarinic stimulation, swelling following β-adrenergic stimulation, and an imbalance between the efflux and influx of ions (specifically Cl-) between the luminal and basolateral membranes. The major regulator of water permeability in acinar cells is aquaporin 5, which is expressed in the apical membrane. The main Cl- concentrative component in the acinar cells is derived from the basolateral Na+/K+/2Cl- cotransporter NKCC1, which is encoded by the Slc12a2 gene. More than 70% of primary saliva secretion is carried out by NKCC1 cotransporter. The remaining 30% of primary saliva secretion is HCO3--dependent, suggesting that a second mechanism is involved in concentrating Cl-. This alternative pathway of Cl- uptake is dependent on the coordinated activities of the basolateral Na+/H+ and Cl-/ HCO3- exchangers (Fig. 7.2).

Secretion of Organic Substances in Primary Saliva The stimulation of β-adrenergic receptors by the sympathetic nerve and vasoactive intestinal polypeptide receptors by the parasympathetic nerve

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result in G-protein activation followed by adenylate cyclase activation, ultimately causing an increase in cAMP production. Elevated levels of cAMP activate protein kinase A. Subsequently, target proteins are activated by a phosphorylation cascade, resulting in transport of the corresponding protein granules towards the lumen, where they are transiently fused with the outer cell membrane and their contents are secreted into the extracellular environment. That is, transport of secretory granules is microtubuleand F-actin–dependent; therefore, the delivery of secretory granules from the trans-Golgi network to the luminal membrane area is mediated via the fast microtubule-dependent transport system, and 70–80% of secretory granules are conveyed to the subplasmalemmal region by this system. Because the subplasmalemmal region is rich in F-actin and the microtubules do not extend significantly over this area, it is likely that transport of secretory granules in this F-actin–rich subplasmalemmal region (luminal cell surface) requires other types of myosin motors (molecular motors or molecular machines) along actin filaments (actin track). Molecular motors are molecules that are capable of unidirectional rotation motion powered by external energy input. They include motor proteins, such as myosin, which is responsible for muscle contraction; kinesin, which moves cargo inside cells away from the nucleus along microtubules; and dynein, which also transports cargo along microtubules towards the cell nucleus. Myosin I proteins, which contain a secondary actin-binding site or a membrane-binding site in their tails, are involved in intracellular organization and the protrusion of actin-rich structures at the cell surface (Fig. 7.3).

Transportation Through Intercellular Junctions When substances move across epithelia, a variety of substances must be transported via the intercellular space because water-soluble substances are difficult to transport across cell membranes. Accordingly, the acinar cells of the salivary glands utilize intercellular junctions as pathways

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for transporting water and sodium ions from the plasma interstitial fluid (extracellular space) to the acinar cell lumen. In salivary glands, those intercellular junctions consist of tight junctions (occluding junction), gap junctions (communicating junction), desmosomes, hemidesmosomes (anchoring junction), and others. Tight junctions act as a gate to close or seal intercellular space in order to block the transportation of substances between the basolateral or basal membrane and lumen. Tight junctions are distributed on acinar and ductal cells but are not present on myoepithelial cells. Tight junctions in the acinar cells are shallower and simpler than those in the ductal cells. Consequently, the gate function of tight junctions is weak in acinar cells but strong in ductal cells (Fig. 7.3). Ions and low-molecularweight molecules can be transported through gap junctions, which connect the cytoplasmic compartments of neighboring cells. Gap junctions are distributed in acinar cells and myoepithelial cells, but are not present in ductal cells. In acinar cells, gap junctions control the secretion of salivary proteins, whereas in myoepithelial cells, they control pressure around the acinar cells and intercalated duct. Because gap junctions are not present between ductal cells, especially striated ductal cells, information is not transferred between these cells. Additionally, desmosomes and hemidesmosomes, which are present between acinar cells, ductal cells, and myoepithelial cells, provide mechanical bonds between epithelial cells and connective tissues. Desmosomes of acinar cells are less well developed than those in ductal cells.

Autonomous Properties of Myoepithelial Cells Myoepithelial cells are sympathetically innervated, and their squeezing function is mediated by myoepithelial contractions. Acinar cells synthesize proteins and secrete saliva fluid, and ductal cells serve as a pathway for the transport of secreted primary saliva to the oral cavity. Myoepithelial cells exert a contractile force to

Mechanism of Salivary Secretion

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Fig. 7.3 The mechanism of salivary secretion of serous and mucous saliva in salivary acinar cells during stimulation. (a) Secretion of serous saliva, which contains ions, by salivary acinar cells. Secretion of serous saliva is mediated by multiple factors, including autonomic neurotransmitters (mainly acetylcholine), muscarinic receptors, G-proteins, phospholipase C, inositol 1,4,5trisphosphate, calcium ions, the opening of chlorine ion channels, etc. Chloride ions are secreted because their intracellular concentrations increase due to the action of sodium–potassium–chloride transporters. Sodium ions follow a gradient of charge down the resultant osmotic gradient. Sodium ions are thought to be transported via an

intercellular route. Water may be transported via an intercellular route, but aquaporins may transport water transcellularly. (b) Secretion of mucous saliva, which contains proteins and mucins by salivary acinar cells. The secretion of mucous saliva is mediated by multiple factors, including autonomic neurotransmitters (mainly noradrenaline), β-adrenergic receptors, G-protein, adenylate cyclase activity, cAMP formation, protein kinase activity, rough endoplasmic reticulum biosynthesis (1), enrichment and fractionation in Golgi apparatus (2,3), migration of secretory granules (4), and extracellular release (exocytosis) (5). NA noradrenaline, Ach acetylcholine

drive the transport of stagnant primary saliva in the lumen into the duct. Myoepithelial cells contain a large number of actomyosin filaments, which are arranged into two groups, actin and myosin, that run in opposite directions.

Myoepithelial cells localized around the acinar cells and on the basal side of intercalated duct cells pressurize the terminal of the acinar lumen via actomyosin contraction.

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Modification of Primary Saliva by the Duct System The isotonic primary saliva produced in acinar cells starts to undergo changes in composition as it passes through the ductal system. The ductal systems consist of several different cell types, and their compositions differ between salivary glands. Essentially, there are three main types of ducts: intercalated, striated, and excretory. Intercalated and striated ducts are intralobular, whereas excretory ducts are primarily extralobular. Absorption of Na+ and Cl- takes place in both intralobular and extralobular ducts. The epithelial Na+ channel, ENaC, which is localized to the luminal membrane, plays a key role in ductal Na+ absorption. In addition, a Na+/H+ exchanger, also localized to the luminal membrane, participates in the Na+ absorption process. Activation of these Na+ channels depolarizes the luminal membrane and enhances Cl- influx across the luminal Clchannel. A Cl-/HCO3- exchanger, encoded by Slc26a6, is located at the luminal membrane, and the activity of this protein is functionally coupled to the activity of a luminal Na+/H+ exchanger, most likely the Nhe3 isoform. These paired exchangers furnish an alternative route for Na+ and Cl- absorption from the ductal lumen. Therefore, the combination of Na+ and Cl- channels in the luminal membrane provides an effective influx mechanism for Na+ and Cl- absorption. Here, the Na+/K+ ATPase provides the primary pathway for Na+ efflux across the basal membrane of duct cells. Also, Cl- efflux occurs via Cl- channels located on the basal membrane of duct cells and is driven by the Cl- electrochemical gradient. That is, depolarization of the luminal membrane following activation of the Na+ channel promotes Cl- influx across the luminal membrane, generating a negative membrane potential during the period of cholinergic stimulation, which drives Cl- efflux from the duct cells towards the extracellular space. Therefore, it is necessary for membrane potential to oscillate in such a manner as to favor Cl- uptake at the luminal membrane and Cl- exit across the basal membrane.

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The K+ concentration is higher in saliva than in plasma, primarily because K+ is secreted by intralobular and extralobular ducts in response to secretagogues. The luminal K+/H+ exchangers and K+/HCO3- cotransporters have been proposed to play a role in K+ secretion. A basal membrane K+ channel recycles K+ to the extracellular space, and the opening of such a channel hyperpolarizes the membrane, thereby increasing the driving force for Cl- efflux. Two different K+ channels have been characterized in salivary glands: IK1 or SK4, encoded by the Kcnn4 gene, which is a Ca2+-activated K+ channel, and maxi K or Slo, encoded by the Kcnma1 gene, which is both Ca2+- and voltage-activated. Most K+ secretion requires the activity of the Slo channel. In particular, the ductal system, including striated and excretory ducts, has relatively low permeability to water, but absorption of electrolytes is steadily maintained. In summary, the luminal side membrane of striated ductal cells contains channels for Na+ and Cl-, and exchangers for Cl-/HCO3-, Na+/H+, and K+/H+. The integrated functions of those transporters cause Na+ and Cl- in the primary saliva to be selectively absorbed into the striated ductal cells. On the other hand, K+ and HCO3- are selectively secreted into the striated ductal lumen from the striated duct cells. However, because the water steadily remains in the striated ductal lumen due to the system’s low permeability to water, the saliva is hypotonic. Also, Na+-K+ pumps, Na+/ H+ exchangers, Cl- channels, and K+ channels contribute to pumping Na+ ions outward to the extracellular space, providing a continuous Na+ concentration gradient between the striated ductal lumen and striated ductal cells. For this reason, Na+ can be easily transported into striated ductal cells from the striated ductal luminal saliva. Additionally, Cl- ions that are absorbed into striated ductal cells diffuse into the extracellular space and follow Na+ in order to maintain electrical neutrality. The increase in Ca2+ concentration, which is necessary for these activities of striated ductal cells, is evoked by acetylcholine receptors and α-adrenergic receptors, which are located at the basal membrane. The intercalated duct cell is involved in the secretion of kallikrein, which is

Functions of Saliva

necessary for vasodilation in salivary glands. Lastly, the micro-regulation of ions takes place in the excretory duct before the saliva is transported into the collecting duct (Fig. 7.2).

Properties of Saliva The mixed secretion that is secreted into the oral cavity from the major and minor salivary glands is called saliva. Saliva secreted from the major salivary glands is called parotid, submandibular, or sublingual saliva, depending on its origin. The combined saliva that is secreted into the oral cavity is called either mixed saliva or whole saliva, whereas the combined secretions produced from both the salivary glands and the gingival sulcus or gingival crevice are called the oral fluid (Table 7.1). The saliva that is secreted from the major salivary glands constitutes more than 90% of the whole saliva, with the remaining 10% provided by the minor salivary glands. The pH of saliva is closely related to the salivary secretion rate: the pH of mixed saliva is 6.2–7.6 (average, 6.7). The specific gravity of saliva, which is hypotonic relative to other types of body fluids, is 1.002–1.008. The physical properties of saliva differ markedly depending on the gland that produces it: parotid saliva is serous (or watery), whereas submandibular and sublingual saliva is highly viscous with high protein content. If the viscosity of water at 37°C is defined as 1, the viscosities of parotid, submandibular, and sublingual saliva are 1.3, 2.6, and 11.0, respectively. The salivary secretion rate is 0.05–1.8 mL/min (average, 0.3 mL/min) in the resting state, increases dramatically to 2.5–5.0 mL/min under stimulation, and falls dramatically during sleep. The amount of saliva produced in a healthy person per day remains controversial: estimates range from 0.75 to 1.5 L/day, whereas it is generally accepted that during sleep the rate of production drops to nearly zero. On the other hand, when an excessive amount of saliva is present in the oral cavity, the swallowing reflex occurs; approximately 1.1 mL saliva is sufficient to induce this response. The

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amount of saliva that is swallowed by the swallowing reflex is about 0.3 mL, with the rest remaining in the oral cavity. The amount of saliva remaining varies among individuals, with an average of 0.8–1 mL. The swallowing reflex occurs frequently, allowing effective removal of food debris remaining in the oral cavity and preventing excessive accumulation of bacteria. Since saliva is always present in the oral cavity, the whole surface of the oral tissues is covered by a thin layer of saliva with a thickness of approximately 0.1 mm. The total surface area of the oral cavity, including the soft tissue and teeth together, is approximately 200 cm2. Various reactions can occur between the surfaces of the oral tissues and the saliva, and these processes are important for maintaining homeostasis in oral tissues.

Functions of Saliva The inner surface of the digestive tract, including the oral cavity, is covered by mucus, distinguishing its contents from those of the intestinal tract. Saliva covers the whole surface of the oral tissue and exerts multiple complex functions, mediated both by its characteristics as a liquid and the activities of its various components: (1) Through constant secretion and swallowing, the saliva removes food debris and bacteria. (2) Certain types of proteins included in saliva have antibacterial activity, and serve to eliminate oral bacteria and toxins, thereby protecting the oral tissue. (3) Salivary glycoproteins protect both the soft and hard tissues of the oral cavity from mechanical and chemical damage. In other words, salivary glycoproteins function as lubricant, protecting the oral tissues from physical damage caused by various external materials and friction between the tissues themselves. (4) Salivary buffering capacity protects the oral tissues from chemical damage caused by organic acids, which are produced as metabolic end products by oral bacteria. (5) When teeth are damaged physically or chemically, calcium and phosphate ions present at supersaturating concentrations in the saliva restore the damaged teeth. For instance,

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Table 7.1 Components of oral fluid Cells Bacteria Leukocytes Buccal squames Secretions Parotid glands Submandibular glands Sublingual glands >600 minor salivary glands Gingival crevicular fluid

60–70 × 106/L 25–650 × 103/L 6–600 × 103/L Varying proportions according to the degree of stimulation

Varying in amount according to the degree of gingival inflammation

when fluoride ions are present in the oral cavity, early dental caries lesions can undergo remineralization, a process involving calcium and phosphate. On the other hand, the oral cavity, which is the entrance to the gastrointestinal tract, takes in food and begins its digestion by both physical and chemical means, after which the food is swallowed and transported to the stomach through the esophagus. (6) α-Amylase, a digestive enzyme in saliva, hydrolyzes α-1,4 glycosidic bonds of carbohydrates, aiding in the digestion and removal of food debris. (7) Saliva is also important to pronunciation, and thus communication. Thus, saliva is involved in supplementary actions of mastication, bolus formation, swallowing, tasting, digestion, and pronunciation. In other words, saliva makes ingested foods softer, promoting bolus formation and facilitating swallowing. The lubricating effect on the lips and tongue makes pronunciation more precise, thereby facilitating speaking. By acting as a solvent for food, saliva also aids the sense of taste. In addition, saliva has functions such as excretion of heavy metals into the oral cavity. Also, saliva can help maintain denture balance. When an individual is dehydrated, the salivary secretion rate decreases to retain water within the body, causing the individual to feel thirst and inducing them to drink water. Moreover, the oral cavity is constantly exposed to various external materials, and one of the important functions of saliva is to dilute and remove materials taken into the mouth. In particular, saliva plays very important roles related to the ingestion of carbohydrates and the development of dental caries. Ingestion of food

stimulates salivary secretion, inducing the swallowing reflex when the saliva volume reaches a certain level. At that point, food debris can be removed by swallowing, along with the saliva; the unremoved food debris will be further diluted by saliva and can be removed by a subsequent route of swallowing. Through these repetitive processes, ingested foods are diluted and removed from the oral cavity. Accordingly, the salivary secretion rate and saliva volume are the important determinants of the rate at which foods are cleared. In general, when the salivary secretion rate is high, the clearing effect occurs rapidly. Consequently, when the salivary secretion rate changes, the clearance rate is greatly affected. Moreover, the salivary clearance rate is related to the pH of dental plaque: when the salivary clearance rate is low, the pH decreases dramatically, and the recovery to a normal pH is delayed. On the other hand, when the salivary clearance rate is high, the pH of dental plaque decreases slowly, speeding the recovery of its pH to normal levels.

Constituents of Saliva Saliva mostly consists of water with small amounts of inorganic and organic constituents. In addition, saliva contains a mixture of exfoliated epithelial cells, degraded leukocytes, oral bacteria, yeast, protozoa, food debris, and gingival crevicular fluid. Consequently, the contents of saliva can vary widely at different times. Also, because saliva is produced by active secretion processes, it differs greatly from

Constituents of Saliva

plasma: the constituents and contents of mixed saliva differ greatly depending on various factors such as the salivary secretion rate, the relative contributions of the salivary glands, and the nature of stimulation of salivary secretion. Water constitutes 94% of resting saliva but up to 99.5% of stimulated saliva. Mixed saliva contains 0.5% (stimulated saliva) to 6% (unstimulated) solid materials, of which about 2/3 are organic and 1/3 are inorganic. The chemical composition of mixed saliva is shown in Table 7.2. Table 7.3 shows a chemical comparison of human mixed saliva and normal plasma.

Inorganic Constituents The inorganic constituents of saliva are similar to those in blood plasma, although the levels of individual components differ greatly. In particular, the levels of calcium, magnesium, sodium, chloride, bicarbonate, and fluoride are smaller in saliva than in blood plasma, whereas the levels of potassium and phosphate are higher. In addition, saliva contains thiocyanate, iodine, copper, iron, manganese, cobalt, molybdenum, and other ions. Among the most physiologically important inorganic ions are calcium, phosphate, and bicarbonate. Calcium and phosphate are involved in remineralization of enamel, whereas bicarbonate is the most important factor in salivary buffering capacity. Carbon dioxide, phosphate, ammonia, and proteins in saliva are also involved in salivary buffering capacity. The fluoride in saliva increases resistance to acids by converting hydroxyapatite into fluorapatite, whereas thiocyanate exerts antibacterial activity as a cofactor of lactoperoxidase. Resting saliva is usually composed of parotid and submandibular saliva with slightly acidic pH; when the salivary secretion rate is increased by stimulation, the pH increases, and the saliva becomes alkaline. The pH of parotid saliva ranges from 7.85 to 8.30 (salivary secretion rate of 1–3 mL/min), the largest increase; that of sublingual saliva from 7.5 to 8.0 (0.01–0.1 mL/min); and that of submandibular saliva from 6.85 to 7.60 (1–4 mL/min).

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Hydrogen Ion The concentration of hydrogen ions in saliva determines the pH of the oral cavity and thus affects most of the chemical reactions that occur there. In particular, hydrogen ion concentrations strongly influence the calcium and phosphate balance between dental hard tissues and their surrounding environments (i.e., saliva). In addition, hydrogen ions are important for the solubility of macromolecules (e.g., enzymes) within saliva, as well as their activities. Hydrogen ions in saliva originate from the salivary gland, oral bacteria, drinks, and other sources. The concentration of hydrogen ions may also vary depending on the location within the oral cavity. For instance, in regions where food debris is present, the pH may be lower due to ongoing bacterial metabolism, whereas regions in which the saliva supply is sufficient, including the orifices of the salivary gland ducts, maintain a normal pH. For this reason, pH can vary within the same oral cavity. One of the most important factors that determine the pH of the oral cavity is bicarbonate, which is present in resting saliva at 1/10th of the level in plasma; therefore, reabsorption occurs during the process of salivary secretion. On the other hand, when the salivary secretion rate increases due to stimulation, the concentration of bicarbonate increases up to or above the level in plasma. In addition, sialin (glycine-glycinelysine-glycine), a tetrapeptide in parotid saliva, increases the pH of saliva by increasing the baseline production of oral bacteria and stimulates glycolysis, increasing pH. The pH of the oral cavity can also be increased by bacterial synthesis of urea from carbon dioxide and ammonia. Calcium and Phosphate Calcium is a divalent cation that is secreted into the saliva by active transport, and phosphate commonly exists as a monovalent or divalent anion in saliva secreted from salivary gland ducts. In resting saliva, the concentrations of calcium and phosphate in saliva are 1.4 mmol/L (1–3 mmol/ L) and 6.0 mmol/L, respectively; when the salivary secretion rate is increased by stimulation, the calcium concentration increases slightly to

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Table 7.2 Composition of whole saliva (mixed saliva) Unstimulated Flow rate (mL/min) pH Inorganic Sodium Potassium Calcium Magnesium Chloride Hydrogen carbonate Phosphate Thiocyanate Iodide (μmol/L) Fluoride (μmol/L) Organic Protein (g/L) Serum albumin (mg/L) Gamma globulins (mg/L) Mucoproteins (g/L) MG1 MG2 Amylase (g/L) Lysozyme (g/L) Proline-rich proteins (mg/L) Histidine-rich proteins Lactoferrin Carbonic anhydrase Fibronectin (mg/L) Statherin (mg/L) Carbohydrate (g/L) Blood group substances (mg/L) Glucose Lipids (mg/L) Cortisol (nmol/L) Amino acids (mg/L) Urea Ammonia

Stimulated

0.25–0.35

1.0–3.0

6.0 Mean ± SD

5.7–6.2 Range

Mean ± SD

Up to 8.0 Range

7.7 ± 3.0 21 ± 4 1.35 ± 0.45 0.31 ± 0.22 24 ± 8 2.9 ± 2.4 5.5 2.5 5.5 ± 4.2 1.5

2–26 13–40 0.5–2.8 0.15–0.6 8–40 0.1–8.0 2–22 0.4–5.0 2–22 0.2–2.8

32 ± 20 22 ± 12 1.70 ± 1.0 0.18 ± 0.15 25 ± 18 20 ± 8 10 1.2 10 ± 7 5.0

13–80 13–38 0.2–4.7 0.2–0.6 10–56 4–40 1.5–25 0.4–3.0 2–30 0.8–6.3

1.75 25 50 0.45

1.0–6.4

0.42 0.14 0–80

0.2–2.0 16–147 0.27–0.40 10–20 0.02–0.17 20 2–20 40 2.0–4.20 0.6–7.0

All concentrations are given in mmol/L unless otherwise designated. [Source: D. B. Ferguson, Oral Bioscience, (Churchill Livingstone, 1999), Table 6.3]

1.7 mmol/L, but the phosphate concentration decreases to 4.0 mmol/L. The calcium concentration is approximately twofold higher in submandibular and sublingual saliva than that in the parotid saliva; this is because the salivary secretion rate of the parotid gland increases the most when stimulated, causing the calcium concentration of mixed saliva to increase. Moreover, the

calcium concentration of saliva is strongly affected by circadian rhythm, with the minimum and maximum concentrations differing by a factor of 2. The distribution of calcium and phosphate ions between their ionic and bound forms depends on the pH of the saliva. Ionic calcium and phosphate concentrations strongly influence the balance of calcium phosphate salts between

Constituents of Saliva

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Table 7.3 Comparison of chemical compositions of whole saliva and plasma in humans Water Solids Specific gravity pH (average) pH (range) Inorganic Ca2+ Mg2+ Na+ K+ NH4+ H2PO4- + HPO42ClHCO3FSNOrganic Urea (adults) Urea (children) Uric acid Amino acids (free) Glucose (free) Lactate Fatty acids (mg/L) Macromolecules (mg/L) Proteins Glycoprotein sugars Amylase Lysozyme Peroxidase IgA IgG IgM Lipid

94.0–99.5% 0.5 (stimulated) to 6.0% (unstimulated) 1.002–1.008 6.7 6.2–7.6 Saliva (mM)

Plasma (mM)

1–2 0.2–0.5 6–26 14–32 1–7 2–23 17–29 2–30 0.001–0.005 0.1–2.0

2.5 1.0 140 4 0.03 2 103 27 0.01 –

2–6 1–2 0.2 1–2 0.05 0.1 10

5 – 3 2 5 1 3000

1400–6400 110–300 380 109 3 194 14 2 20–30

70,000 1400 – – – 1300 13,000 1000 5500

[Source: S. Cole and J. E. Eastoe, Biochemistry and Oral Biology, 2nd ed. (Wright, 1988), Table 33.1]

dental hard tissues and their surrounding local environments and are thus important determinants of tooth remineralization following damage due to dental caries. More than 90% of calcium in saliva is in ionic form, with the remainder bound to proteins or small-molecule anions. However, when the pH in the oral cavity drops below 4, most of the calcium in saliva is in ionic form. Of bound calcium, 10–20% is associated with inorganic ions (phosphate, bicarbonate, etc.), less than 10% with organic ions such as citrate, and 10–30% with macromolecules

such as proteins. Almost all phosphate exists in ionic form, but a small proportion exists in organic phosphate or pyrophosphate form. On the other hand, acquired pellicle and dental plaque can be formed on the surface of dental hard tissues, and these substances have a high binding capacity for calcium, promoting the precipitation of calcium phosphate salts. In reality, the calcium concentration in dental plaque (6.5 mM) is much higher than that of saliva (1.5 mM). When the pH within dental plaque decreases, bound calcium is released, but the

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calcium in saliva and the released calcium in dental plaque return to a balanced state when the pH within dental plaque increases back to the normal level. As shown, more than 90% of calcium and phosphate in saliva exist in ionic form; however, they are not present at sufficiently high levels to allow spontaneous precipitation of calcium phosphate salts. Because calcium and phosphate in saliva are supersaturated at the normal pH of the oral cavity, salivary calcium and phosphate levels can promote the calcium phosphate crystal growth when a nucleator of calcification is present. Salivary proteins, including anionic prolinerich proteins and statherin, inhibit the growth of calcium phosphate crystals and spontaneous precipitation of calcium phosphate salts and thus participate in the maintenance of calcium and phosphate supersaturation in saliva. By maintaining the calcium and phosphate in a supersaturated state, these factors can inhibit the remineralization of early dental caries lesion and improper calcification in the salivary gland ducts. Regarding the relationship between calcium and phosphate concentrations in saliva and the solubility of hydroxyapatite, at pH 6.0 (the pH of the oral cavity in the resting state), hydroxyapatite essentially does not dissolve. However, when the pH increases or the concentrations of calcium and phosphate increase, dental calculus can form through precipitation of calcium phosphate salts. In addition, as the salivary secretion rate increases, the calcium concentration in mixed saliva increases slightly. At the same time, however, the phosphate concentration in mixed saliva decreases slightly: because phosphate is mainly secreted from the salivary gland ducts, an increase in the salivary secretion rate causes the saliva to spend less time passing the ducts. The phosphate concentration in saliva secreted from the minor salivary glands is very low. On the other hand, the high calcium concentration in submandibular saliva is strongly associated with the high level of dental calculus formation on the lingual side of the lower anterior teeth. However, this relationship cannot explain why dental calculus forms largely on the buccal side of upper anterior teeth. For this reason, even though it is

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difficult to properly explain the exact relationship between calcium and phosphate concentrations in saliva and the formation of dental calculus, these observations are consistent with the fact that people with higher pyrophosphate concentration in saliva have less dental plaque formation, indicating that pyrophosphate is an inhibitor of mineralization. Moreover, calcium plays a role as a prosthetic group for α-amylase in saliva and thus helps to maintain its enzymatic activity.

Bicarbonate Bicarbonate, the most important determinant of salivary buffering capacity, is synthesized by carbonic anhydrase from carbon dioxide, which forms as a metabolic end-product in salivary glands and other types of tissues. The concentration of bicarbonate is low in the resting saliva but rises in stimulated saliva as the salivary secretion rate increases (Fig. 7.4): when the metabolic activity of the salivary glands increases, the rate of carbon dioxide production also rises, boosting the bicarbonate concentration. Bicarbonate increases the pH of stimulated saliva to pH 8.0 and has buffering capacity at pH 6.1 ± 1.0, which includes the critical pH range of 5.5–5.6. In other words, the bicarbonate buffer system effectively protects the teeth against organic acids produced by cariogenic (acid-producing) bacteria. For this reason, an individual with a higher salivary secretion rate has a lower chance of developing dental caries. Fluoride The fluoride content in saliva is relatively low, similar to that in plasma and extracellular fluid. Ingestion of fluoride causes an increase in the fluoride concentration in plasma and therefore in

Fig. 7.4 Production of bicarbonate in saliva

Constituents of Saliva

the saliva as well. In the resting saliva, the fluoride concentration is slightly higher but varies slightly depending on the salivary secretion rate. Although the saliva contains very little fluoride, this ion plays a critical role in dental caries. The fluoride concentration is higher in dental plaque than in saliva, although saliva remains the primary source of fluoride. As the salivary secretion rate increases, the fluoride concentration in dental plaque increases as well, which helps to prevent dental caries.

Thiocyanate Thiocyanate is secreted into saliva from the salivary gland ducts, and its concentration decreases as the salivary secretion rate increases. The thiocyanate concentration in saliva is approximately 13 mg/dL. Thiocyanate is a cofactor of lactoperoxidase in saliva, and the lactoperoxidase–thiocyanate system reacts with H2O2 produced by bacteria, forming hypothiocyanite (OSCN-), an oxidative derivative of thiocyanate that is highly toxic to many bacteria. Hypothiocyanite not only affects H2O2producing bacteria but also the bacteria near them, decreasing their activity. For this reason, the presence of thiocyanate in saliva is associated with a low incidence of dental caries. In smokers, the thiocyanate concentration in saliva is about twice as high as in non-smokers. Sodium, Potassium, Magnesium, and Chloride Sodium, potassium, magnesium, and chloride are important for the production and secretion of saliva, rather than for their functions within saliva. In the plasma, the sodium content is much higher than the potassium content, but this relationship is reversed in parotid saliva. The salivary secretion rate strongly affects the concentrations of sodium and potassium: as the salivary secretion rate increases, the sodium concentration increases dramatically, whereas the potassium concentration decreases. Sodium usually originates from serum, but potassium originates from the acinar cells of the salivary glands. Magnesium is less abundant than calcium, and the magnesium concentration decreases as

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the salivary secretion rate increases. Magnesium mostly exists in a protein-bound form, and only some of it is present as a free ion. The major function of chloride in saliva is to serve as an allosteric activator of the digestive enzyme α-amylase. Additionally, the chloride concentration dramatically increases as the salivary secretion rate increases.

Other Ions Saliva also contains trace elements, including lead, iron, copper, zinc, manganese, and cadmium, in cationic form, and their concentrations reflect the concentrations in plasma, making them diagnostically useful. In addition, saliva contains bromine and iodine; the latter accumulates in the salivary glands and is secreted with the saliva.

Organic Constituents The major organic constituents of saliva are various proteins that are secreted mainly by the salivary glands and oral bacteria (Fig. 7.5, Table 7.4). Most of the enzymes found in mixed saliva are secreted by oral bacteria. Other organic constituents, including fats, urea, vitamins, and amino acids, are present in saliva. The most abundant amino acids are alanine, glycine, and glutamate, in that order, whereas only trace amounts of proline and sulfur-containing amino acids are present.

Fig. 7.5 Types and functions of salivary proteins

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Table 7.4 Distribution of several salivary proteins in various body fluids

Mucins Acidic PRPs α-amylase Basic PRPs Basic PRG slgA

Saliva ++++ ++++ ++++ +++ +++ +++

Cystatins Statherin lgG EP-GP VEGH

++ ++ + + +

Histatins Lysozyme

+ +

Kallikrein Lactoferrin

+ +

Lactoperoxidase Haptocorrin β-microseminoprotein lgM Albumin Zn-α2 glycoprotein

+ + + + + +

Tear + + +++ + + + + + +++ + +++ + +++ + + + + + + +

Nasal mucus ++++ + +

Bronchial mucus ++++ + +

++++ + + + + -

+ +

++++

+ ++++

++++

Cervical mucus ++++ + -

Sweat ++++ + -

Plasma + -

+

+

-

+

+

++

-

+ +

++++ -

+

+

+

+

+ +

+ +

++++

+ + ++ + +

Semen + + -

-

+ +

+ +

+++ + +

+ ++++

+

+

+

+ +

+ + ++ ++++ +

-, not detected; +, detected but not measurable, or less than 1% of total protein; ++, 1–5% of total protein; +++, 5–15% of total protein; ++++, over 15% of total protein; blank, not measured. [From L. C. Schenkels, E. C. Veerman, A. V. Nieuw Amerongen, Crit. Rev. Oral Biol. Med. 6:161–175, 1995]

a-Amylase α-Amylase is a type of carbohydrase, a digestive enzyme, and is found in saliva at a concentration of 38–42 mg/dL. The enzyme consists of a single polypeptide chain with a molecular weight of 56–59 kDa (glycosylated form: 62–63 kDa) and an isoelectric point of 5.70–6.88. α-Amylase is an allosteric enzyme in which chloride ion acts as an allosteric activator, causing the protein’s affinity for calcium to increase dramatically. Ca2+, in turn, functions as a prosthetic group to maintain the molecular arrangement and activity of the enzyme. For this reason, the removal of Ca2+ from α-amylase reversibly decreases enzymatic activity and stability, facilitating protein degradation. α-Amylase is found in the digestive tract, including the salivary glands and pancreas, as well as in tears, serum, urine, milk, and genital

secretions. Amylase is also found in plants and bacteria, although that enzyme is β-amylase, an exoamylase that hydrolyzes the nonreducing end of carbohydrates to units of maltose. Another enzyme, γ-amylase, is present in the human liver. α-Amylases help to break down ingested carbohydrates by hydrolyzing α-1,4 glycosidic bonds; thus it is an endoamylase. When the activity of salivary α-amylase is high, it hydrolyzes carbohydrate into glucose and maltose, but when its activity is low, it hydrolyzes carbohydrate into maltose and maltotriose. α-Amylase retains its digestive function from the oral cavity to the digestive tract until it is inactivated by gastric juice in the stomach. For this reason, even though the contact time of α-amylase to foods in the oral cavity is short, its high concentration makes it possible to partially digest some carbohydrates

Constituents of Saliva

while within the oral cavity. In newborn babies, the concentration of salivary α-amylase is low but increases dramatically at the age of 1–2 years and is similar to the adult level by the age of 5 or 6. Because salivary α-amylase is a digestive enzyme, it serves to cleanse the oral cavity of carbohydrate debris. In addition, α-amylase has antibacterial activity against Neisseria gonorrhoeae. By contrast, animals such as dogs, cats, and horses do not have salivary α-amylase and also have a lower incidence of dental caries than humans because the ingested carbohydrates are not hydrolyzed into monosaccharides in the oral cavity. When these animals ingest foods containing monosaccharides or disaccharides, however, they have a higher chance of developing dental caries (Lazzari, 1983).

Mucin Mucin is a type of glycoprotein that is secreted from the submandibular, sublingual, and minor salivary glands. It is classified into two forms: MG-1 (high-molecular-weight mucin-type glycoprotein 1), with a molecular weight greater than 1000 kDa, and MG-2 (low-molecular-weight mucin-type glycoprotein 2), with a molecular weight lower than 120 kDa. MG-1 constitutes 30% of the mucin in human saliva and MG-2 the remaining 70% (Rayment et al., 2000). As with other glycoproteins, mucin has a protein core structure connected to neutral or acidic oligosaccharide chains. These oligosaccharide chains consist mainly of fucose, galactose, Nacetylglucosamine, N-acetylgalactosamine, etc., with chains of 1–20 sugars connected to either serine or threonine residues via O-glycosidic linkages. Sialic acid is the terminal sugar of such chains. Hydroxyl groups, which are abundant in mucin, attract water, thereby increasing and maintaining the viscosity of saliva. Additionally, mucin gives saliva its lubricating properties, which protect the oral mucosa and aid in mastication, pronunciation, and swallowing. Moreover, mucin exerts antibacterial activity by binding to sIgA and improving its binding affinity for bacteria. Mucin can also directly bind to surface components of oral bacteria, including

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Streptococcus mutans. Bacteria that are bound to mucin cannot attach to the surface of the oral cavity and therefore can be easily removed. The oligosaccharide chains in mucin are structurally similar to tissue receptors for bacterial attachment to the oral cavity and thus inhibit bacterial attachment to the surface of the oral cavity by competing with tissue receptors, ultimately preventing bacterial colonization. Mucin also binds to bacterial toxins, inhibiting their toxic effects.

Antibacterial Salivary Proteins Lactoferrin (Red Iron-Binding Protein) Lactoferrin, an iron-binding protein synthesized by acinar cells of salivary glands and neutrophils, is present in saliva at concentrations less than 1 mg/dL. This red protein was initially found in human milk in 1939 by Sorensen and Sorenson, who named it lactoferrin. Early research on lactoferrin was done mostly in human milk; however, its analog was subsequently separated from saliva. Lactoferrin is red because it binds to iron: as with transferrin, one molecule of lactoferrin binds spontaneously to two atoms of Fe3+ and two molecules of bicarbonate. The molecular weights of lactoferrin and transferrin are equal (76,000 Da) and are composed of a single polypeptide chain. However, unless the two proteins denature, they do not exhibit immunological cross-reactivity. Moreover, lactoferrin maintains its iron-binding capacity below pH 4, whereas transferrin does not. Furthermore, the isoelectric points of lactoferrin and transferrin differ (9 and 5.9, respectively). Lactoferrin binds to iron ion, an essential bacterial nutrient, and thus exerts antibacterial activity, a phenomenon termed “nutritional immunity” (Murdoch and Skaar, 2022). Lactoferrin is secreted as an apoprotein without bound iron and then binds Fe3+ in the oral cavity, where it suppresses bacterial growth by depleting iron ions. This antibacterial activity has been demonstrated for multiple bacterial species, including S. mutans. Lactoferrin exhibits a bactericidal effect even at a low concentration; however, after it is saturated by iron ions, it no longer has antibacterial activity. The exact mechanism of

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this bactericidal effect is still elusive, but it may be mediated by binding the apoprotein to the bacterial surface; consistent with this, mature lactoferrin does not bind bacteria. Some bacteria are resistant to lactoferrin; these species synthesize iron-binding enterochelins, which compete with lactoferrin for iron ions. Lactoperoxidase Lactoperoxidase (hydrogen peroxide oxidoreductase; EC 1,11,1,7), first found in milk, inhibits the growth and metabolic activity of Lactobacillus. This finding inspired research on this enzyme’s role in saliva. Initially, this enzyme was shown to consist of a high-molecular-weight thermolabile molecule and a smaller, thermostable cofactor: lactoperoxidase protein and thiocyanate, respectively. Lactoperoxidase is present in the acinar cells of the parotid and submandibular glands, saliva, Harderian gland, lacrimal gland, acquired pellicle, and dental plaque; human saliva contains 0.3 mg/dL lactoperoxidase. Lactoperoxidase and thiocyanate in the saliva react with H2O2, which is produced by bacteria, to form hypothiocyanite (OSCN-), an oxidative derivative of thiocyanate ion that is toxic to various bacterial species. Hypothiocyanite not only decreases the bacterial activity of H2O2-producing bacteria but also that of bacteria in the vicinity. In fresh saliva, the concentration of hypothiocyanite is approximately 10 μM but can rise as high as 60 μM. The concentration of hypothiocyanite increases to an average of 35 μM when saliva is incubated in vitro and results from the conversion of thiocyanate to hypothiocyanite. Human salivary glands contain three different forms of lactoperoxidase with isoelectric points of 6.4, 7.1, and 9.7. The antibacterial activity of the lactoperoxidase–thiocyanate system is induced by hypothiocyanite, which is produced by lactoperoxidase from thiocyanate. Additionally, thiocyanogen [(SCN)2] formed from thiocyanate, which can be converted to either hypothiocyanous acid (HOSCN) or hypothiocyanite by hydrolysis, also exerts antibacterial activity. The lactoperoxidase–thiocyanate system also strongly inhibits bacterial hexokinase activity, and this effect is induced by

7

Saliva

hypothiocyanite. Hexokinase is involved in glycolysis and serves as an essential enzyme for obtaining the energy and building blocks necessary for the synthesis of biomolecules. In addition, the lactoperoxidase–thiocyanate system inhibits the activities of other enzymes in bacteria, including phosphotransferase, which is involved in sugar transportation. In particular, hypothiocyanite has high reactivity with proteins such as hexokinase and free sulfhydryl groups in low-molecular-weight compounds. In other words, hypothiocyanite is able to oxidize sulfhydryl groups: first, it forms sulfenyl thiocyanate by binding to the sulfhydryl group, a reversible reaction. The next step is the release of thiocyanate from sulfenyl thiocyanate to irreversibly oxidize the sulfhydryl group to sulfenic acid. Thiocyanate released at this point can once again participate in the lactoperoxidase–thiocyanate system. Under weakly acidic conditions (pH 5–6), the ionic form hypothiocyanite converts into hypothiocyanous acid (HOSCN), which can easily cross the bacterial cell membrane. Because hypothiocyanite without a sulfhydryl group is very stable, it accumulates in saliva. In vitro, peroxide is necessary for the antibacterial effect of lactoperoxidase, but is not essential for the activation of the enzyme. Nevertheless, given that catalase, which degrades H2O2, inhibits antibacterial activity in both saliva and milk, it is likely that H2O2 produced by bacteria plays an important role in the antibacterial activity of lactoperoxidase. The addition of H2O2 greatly increases the range of bacteria affected by the lactoperoxidase–thiocyanate system. S. sanguis and S. mitis, which are very abundant in the oral cavity and on tooth surfaces, produce and secrete H2O2; consequently, the lactoperoxidase–thiocyanate system is highly active in the oral cavity. Furthermore, the lactoperoxidase–thiocyanate system represses the growth of bacteria, including S. mutans, and activation of the system by the addition of H2O2 decreases plaque accumulation, dental caries, and gingivitis. Moreover, lactoperoxidase is rapidly absorbed on the surfaces of bacteria, enamel, and hydroxyapatite, and the absorbed lactoperoxidase retains its activity even in the

Constituents of Saliva

absorbed state. For this reason, lactoperoxidase serves as a host defense mechanism that blocks the early bacterial colonization of dental hard tissues. Lysozyme Lysozyme (muramidase or N-acetylmuramide glycanohydrolase; EC 3,2,1,17), first identified in nasal secretions by Fleming 1932, is a type of antibacterial protein found in nasal secretions, tears, saliva, gastric juice, sputum, breast milk, and most tissues. The concentration of lysozyme is higher in sublingual and submandibular saliva than in parotid saliva, and stimulated saliva contains 10.9–14.0 mg/dL lysozyme. Although the activity of lysozyme was first detected in nasal secretion, most of the early studies of this enzyme were performed on avian lysozyme obtained from egg white. These investigations revealed the biochemical structure, physicochemical characteristics, and action mechanism of lysozyme. Avian lysozyme consists of a single polypeptide chain that is composed of 129 residues with four disulfide bonds. It is a strongly basic protein, with an isoelectric point of pH 11.0 and a molecular weight of approximately 14,000. Lysozyme selectively hydrolyzes β-1,4 glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine in the peptidoglycan layer of the bacterial cell wall. Therefore, bacteria that are sensitive to lysozyme will eventually die after being solubilized. Additionally, lysozyme induces aggregation of some bacteria, resulting in the selective removal of oral bacteria. However, resident bacteria that are adapted to the oral environment have a capsule that protects them from the action of lysozyme. Thus, the role of lysozyme in the oral cavity is to maintain the balance of oral bacteria through selective lysis of bacteria without capsules. Plants have an enzyme that is similar to lysozyme enzymatically, but chemically distinct. Lysozyme concentrations do not differ significantly as a function of individual susceptibility to dental caries. Also, resident oral bacteria are resistant to higher concentrations of lysozyme than are present in saliva.

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Immunoglobulin Immunoglobins that exist in saliva include secretory immunoglobulin A (sIgA), immunoglobulin G (IgG), immunoglobulin M (IgM), and others (Table 7.5). sIgA

sIgA, an immunoglobulin commonly found in exocrine secretions, including tears, saliva, nasal secretions, bronchial mucus, and intestinal mucus, is responsible for the immunity of the mucosal surface. Among several types of immunoglobulin in saliva, sIgA is the most abundant. Although the amount of sIgA produced in salivary glands varies among individuals, each parotid gland produces 10–80 mg of sIgA per day. The salivary glands produce 50–60-fold more sIgA than the lacrimal gland and twice as much as in nasopharyngeal secretion. However, the salivary level is 2–5% of the amount secreted by the small intestine. sIgA is secreted from the lumen of salivary glands mainly in a dimer form; each monomer consists of two heavy chains and two light chains. The variation in salivary sIgA concentration is closely related to the salivary secretion rate: when the salivary secretion rate decreases, the sIgA concentration increases, whereas when the rate increases due to chemical or psychological stimuli, the sIgA concentration decreases. However, although the sIgA concentration decreases in stimulated saliva, the amount of saliva secreted per unit of time under stimulation increases dramatically; consequently, the total amount of sIgA is higher in stimulated than unstimulated saliva. Compared to skin, mucous membranes are more susceptible to bacterial and viral infection due to their very thin keratinized layers. However, antimicrobial proteins in mucus provide a barrier against microorganisms. sIgA inhibits microbial infection by binding to antigens, a mechanism termed “local immunity” (Naik et al., 2012). sIgA found in breast milk contributes to local immunity in the mucous membrane of the upper respiratory tract and digestive tract of the nursing infant. sIgA also blocks the entrance of various types of bacteria into the intestine and inhibits

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Table 7.5 Immunoglobulin concentration in saliva (mg/mL) Fluida Major gland saliva (parotid)b Minor gland saliva (lower labial)b Minor gland saliva (palatine)b Resting whole saliva

IgA 69.4 99.4 82.9 192.5 (102.9)c

IgG 0.6 5.1 10.3 8.9

IgM 0.4