Atlas of Oral Histology [2 ed.] 9788131254844, 8131254844

Table of contents :
Cover
Title page
Table of Contents
Copyright
Dedication
Preface to the Second Edition
Preface to the First Edition
List of videos
1. Introduction
Preparing tissues for microscopic study
Staining
Microscopy
Points to remember
Useful hints
2. Development of tooth
Bud stage (figs 2.1 and 2.2)
Cap stage (figs 2.3–2.5)
Early bell stage (figs 2.6 and 2.7)
Advanced bell stage (figs 2.8 and 2.9)
Hertwig epithelial root sheath and cementum formation (figs 2.10 and 2.11)
Some important terminologies
3. Enamel
Enamel rods (figs 3.1 and 3.2)
Striae of retzius (figs 3.3, 3.4, and 3.5)
Enamel lamellae (figs 3.5, 3.6, and 3.7)
Enamel tufts (figs 3.5, 3.6, and 3.7)
Enamel spindles (figs 3.8, 3.9)
Gnarled enamel (figs 3.10 and 3.11)
Hunter–Schreger bands (figs 3.12, 3.13, and 3.14)
Useful hints
4. Dentin
Primary and secondary dentin (figs 4.1 and 4.2)
Dead tracts (figs 4.3 and 4.4)
Tertiary dentin (fig. 4.4)
Interglobular dentin (figs 4.5–4.7)
Tomes’ granular layer (figs 4.8 and 4.9)
Branching of dentinal tubules (figs 4.10–4.12)
Useful hints
5. Pulp
Zones of the pulp (figs 5.1 and 5.2)
Pulp stones (figs 5.3 and 5.4)
Useful hints
6. Cementum
Acellular cementum (figs 6.1 and 6.2)
Cellular cementum (figs 6.3 and 6.4)
Incremental lines of salter (figs 6.5 and 6.6)
Cementoenamel junction (figs 6.7–6.14)
Useful hints
7. Periodontal ligament
Principal fiber groups of periodontal ligament (figs 7.1–7.4)
Cementicles (fig. 7.5)
Useful hints
8. Bone
Compact bone (figs 8.1–8.4)
Cancellous bone (figs 8.5 and 8.6)
Useful hints
9. Salivary glands
Serous salivary glands (figs 9.1–9.3)
Mucous salivary glands (figs 9.4–9.6)
Mixed salivary glands (figs 9.7–9.9)
Ductal system of salivary glands
Useful hints
10. Oral mucous membrane
Keratinized stratified squamous epithelium (figs 10.1–10.5)
Nonkeratinized stratified squamous epithelium (figs 10.6 and 10.7)
Keratinocytes and nonkeratinocytes (figs 10.8 and 10.9)
Papillae of the tongue
Dentogingival junction (figs 10.16 and 10.17)
Useful hints
11. Maxillary sinus
Histology of the sinus lining (figs 11.1 and 11.2)
Goblet cells (figs 11.3–11.5)
Useful hints

Citation preview

ATLAS OF ORAL HISTOLOGY SECOND EDITION

Harikrishnan Prasad, BDS, MDS (Oral Pathol & Microbiol) Professor, Department of Oral and Maxillofacial Pathology, KSR Institute of Dental Science and Research, Tiruchengode, INDIA

Krishnamurthy Anuthama, BDS, MDS (Oral Pathol & Microbiol) Consultant Oral Pathologist, Herndon, Virginia, United States of America

Table of Contents Cover image Title page Copyright Dedication Preface to the Second Edition Preface to the First Edition List of videos 1. Introduction Preparing tissues for microscopic study Staining Microscopy

Points to remember Useful hints 2. Development of tooth Bud stage (figs 2.1 and 2.2) Cap stage (figs 2.3–2.5) Early bell stage (figs 2.6 and 2.7) Advanced bell stage (figs 2.8 and 2.9) Hertwig epithelial root sheath and cementum formation (figs 2.10 and 2.11) Some important terminologies 3. Enamel Enamel rods (figs 3.1 and 3.2) Striae of retzius (figs 3.3, 3.4, and 3.5) Enamel lamellae (figs 3.5, 3.6, and 3.7) Enamel tufts (figs 3.5, 3.6, and 3.7) Enamel spindles (figs 3.8, 3.9) Gnarled enamel (figs 3.10 and 3.11) Hunter–Schreger bands (figs 3.12, 3.13, and 3.14) Useful hints

4. Dentin Primary and secondary dentin (figs 4.1 and 4.2) Dead tracts (figs 4.3 and 4.4) Tertiary dentin (fig. 4.4) Interglobular dentin (figs 4.5–4.7) Tomes’ granular layer (figs 4.8 and 4.9) Branching of dentinal tubules (figs 4.10–4.12) Useful hints 5. Pulp Zones of the pulp (figs 5.1 and 5.2) Pulp stones (figs 5.3 and 5.4) Useful hints 6. Cementum Acellular cementum (figs 6.1 and 6.2) Cellular cementum (figs 6.3 and 6.4) Incremental lines of salter (figs 6.5 and 6.6) Cementoenamel junction (figs 6.7–6.14) Useful hints

7. Periodontal ligament Principal fiber groups of periodontal ligament (figs 7.1–7.4) Cementicles (fig. 7.5) Useful hints 8. Bone Compact bone (figs 8.1–8.4) Cancellous bone (figs 8.5 and 8.6) Useful hints 9. Salivary glands Serous salivary glands (figs 9.1–9.3) Mucous salivary glands (figs 9.4–9.6) Mixed salivary glands (figs 9.7–9.9) Ductal system of salivary glands Useful hints 10. Oral mucous membrane Keratinized stratified squamous epithelium (figs 10.1–10.5) Nonkeratinized stratified squamous epithelium (figs 10.6 and 10.7) Keratinocytes and nonkeratinocytes (figs 10.8 and 10.9)

Papillae of the tongue Dentogingival junction (figs 10.16 and 10.17) Useful hints 11. Maxillary sinus Histology of the sinus lining (figs 11.1 and 11.2) Goblet cells (figs 11.3–11.5) Useful hints

Copyright

RELX India Pvt. Ltd. Registered Office: 818, Indraprakash Building, 8th Floor, 21, Barakhamba Road, New Delhi-110001 Corporate Office: 14th Floor, Building No. 10B, DLF Cyber City, Phase II, Gurgaon-122002, Haryana, India Atlas of Oral Histology, 2e, Prasad Harikrishnan, Krishnamurthy Anuthama Copyright © 2019, 2015 by RELX India Pvt. Ltd. ISBN: 978-81-312-5483-7 e-ISBN: 978-81-312-5484-4 Package ISBN: 978-81-312-5475-2 Previous edition copyrighted, 2015 No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval

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Notice Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds or experiments described herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. To the fullest extent of the law, no responsibility is assumed by Elsevier, authors, editors or contributors for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Although all advertising material is expected to conform to ethical (medical) standards, inclusion in this publication does not constitute a guarantee or endorsement of the quality or the value of such product or the claims made of it by its manufacturer. Content Strategist: Ruchi Mullick Content Project Manager: Anand K Jha Sr Production Executive: Ravinder Sharma Sr Graphic Designer: Milind Majgaonkar Typeset by GW Tech India Printed in India by . . .

Dedication Dedicated to My beloved teachers from the Department of Oral Pathology and Microbiology Mahatma Gandhi Postgraduate Institute of Dental Sciences, Puducherry Harikrishnan Prasad

Dedicated to My dear parents, Usha and Krishnamurthy, my dear husband, Abinandan and to my guru, Dr Pratibha Ramani Krishnamurthy Anuthama

Preface to the Second Edition Harikrishnan Prasad, Krishnamurthy Anuthama

We are humbled and thankful for the support and encouragement we received after the first edition of Atlas of Oral Histology was published 4 years ago. It has motivated us to put in more effort to ensure that this book would find good use among students and faculty alike. Some phase contrast microscopy and polarizing microscopy images have been added. Every chapter now includes a new section that provides Useful Points to Remember. A few errors that had escaped our scrutiny in the first edition have now been corrected. A major addition to this edition is the audiovisual guide for oral histology slides. We have made an attempt to show the various microscopic structures related to oral histology through these videos. An audio narration of the videos and on-screen annotations also point out the structures being described, so that students find it easy to follow and understand the concept. We hope the readers find this useful. Thank you.

Preface to the First Edition Harikrishnan Prasad, Krishnamurthy Anuthama “One look is worth a thousand words”, it is said. And it is so true. Seeing and understanding has always proven to be more effective to retain information than reading and memorizing. It is with this idea that we started preparing the manuscript for this atlas. Oral histology, as a subject, can be very simple and very complex at the same time. Young undergraduate students, who are introduced to oral histology for the first time, can feel overwhelmed with the amount of information, especially so when they have to imagine everything. Although textbooks carry numerous good photomicrographs, it is very difficult to understand which is what in these pictures, unless the student is guided by a good teacher who can patiently explain each. Our atlas aims to be such a good teacher. Almost every photomicrograph in this atlas is accompanied with a schematic diagram that makes identifying different features easy. We have closely adhered to Orban’s Oral Histology and Embryology in terms of

content arrangement. We would advise our readers to use this atlas in conjunction with the textbook to understand better. We have tried to incorporate as many pictures as possible at this point of time. Being in its first edition, we feel there is sufficient scope for improvement, especially in terms of quantity of content. Readers are the best critics, and comments, criticisms or suggestions are always welcome. They will only help us improve and make this book better. It is our hope that staff and students alike will benefit from this book. Thank you.

List of videos Dev of tooth.mp4 Enamel full.mp4 Dentin full.mp4 Pulp and alveolar bone.mp4 Cementum.mp4 Periodontal ligament.mp4 Pulp and alveolar bone.mp4 Salivary glands.mp4 Oral mucous membrane.mp4 Maxillary sinus.mp4

Chapter 2 - Page 10 - Advanced bell stage Video for full Chapter 3 - Enamel Video for full Chapter 4 - Dentin Video for full Chapter 5 - Pulp (same video file also to be used for chapter 8) Video for full Chapter 6 - Cementum Chapter 7 - Page 47 - Principal fiber groups of periodontal ligament Video for full Chapter 8 - Bone (same video file as used for chapter 5) Video for full Chapter 9 - Salivary glands Video for full Chapter 10 - Oral mucous membrane Video for full Chapter 11 - Maxillary sinus

CHAPTER 1

Introduction Oral histology encompasses the microscopic study of tissues that form the oral cavity. It is the basis on which our knowledge of the physiology of oral cavity, and the pathologies that afflict it, are built upon. Therefore, an understanding of the histology of oral tissues becomes very significant.

Preparing tissues for microscopic study Oral cavity is made up of both hard tissues and soft tissues. Each of these tissue types requires a specific method of preparation, so that it can be viewed under a microscope.

Soft tissues Soft tissues do not contain hard mineralized components. Hence, they can be easily cut with a knife. However, to maintain their architecture, they are subjected to a series of processes before being cut into thin sections. After removal for examination, the soft tissue is first fixed to prevent degradation and decomposition. Neutral buffered formalin (10% concentration) is the routinely used fixative for this purpose. This is followed by complete removal of its water content and replacement of the same by alcohol. To achieve this, the tissue is

immersed in a series of increasing grades of ethyl alcohol, so that water in the tissue is gradually replaced by the alcohol. The next step involves removal of the alcohol in the tissue and its replacement by xylene. At the end of this step, the fixed tissue now contains no trace of water in it; instead it is filled with xylene. Following this, the tissue is immersed in molten paraffin wax, which will replace the xylene completely. This step completes tissue processing. The end result is that we now have a tissue that contains wax instead of water; therefore, it is rigid and firm enough to be cut into thin sections using an instrument called microtome. The microtome allows sections as thin as 4 µm (4/1000 of a millimeter) to be cut. These sections are placed on glass slides, the wax removed by heat, and then subjected to different staining processes.

Hard tissues Different methods are employed to study hard tissues like bone and teeth because these cannot be cut into thin sections using routine microtomes. The simplest method to study hard tissues is using ground sections. Another frequently used method is decalcified sections.

I. Ground section Ground sections are made by grinding the specimen into thin slices that can allow light to pass through them. Initially grinding is done on a lathe or similar mechanical device. Later on, it can be done manually on an abrasive stone (like Arkansas stone), and finally polished on fine sandpapers. Such sections, which are about 25–50 micrometers thick, are then dehydrated and mounted directly on glass slides using a mounting medium and then observed under the microscope. It has to be stressed however that the thinner the ground section, the better it is to appreciate many structures without much overlap. One major

disadvantage of ground sections is that most of the tooth or bone is wasted during the grinding process. Ground sections are useful for visualizing the mineralized components and hypomineralized structures of hard tissues. Pulp, however, cannot be seen in ground sections.

II. Decalcified section Hard mineralized structures can also be studied by making decalcified sections. This works on the premise that most mineralized structures also have a substantial organic component. When the mineral portion is removed, these tissues attain properties similar to soft tissues and can be treated akin to them. Bone, dentin, and cementum have a considerable amount of organic matter and hence can be studied after decalcification. Enamel however, being made up of by 96% inorganic substance will be completely lost during decalcification process. Decalcification is usually done using acids like nitric acid, formic acid and ethylenediaminetetraacetic acid (EDTA). Depending on the acid used, the decalcification process can take anywhere between few days and several weeks. Once decalcification is complete, the tissue loses its hardness. It can then be processed and sections can be made just like any other soft tissue. Decalcified sections are used to study hard tissues, as mentioned earlier. Dental pulp, a soft tissue that is safely enclosed inside dentin, also can be studied by decalcified sections only. Although the pulp can be removed separately and processed like a soft tissue, its tiny volume usually makes this extremely difficult.

III. Sectioning using hard tissue microtome Specialized microtomes that can cut hard tissues like bone and teeth into thin sections are available in the market. These offer many

advantages over other methods of studying hard tissues. However, they are very expensive and not readily available.

Staining In general, tissues have very little contrast when viewed unstained. To impart contrast to the tissue, and thereby identify and observe the different structures and cells, sections from soft tissues and decalcified hard tissues are subjected to staining. The commonest used histological stain is hematoxylin and eosin (H&E). Hematoxylin is a basic dye that gives a blue colour to acidic structures like nucleus and rough endoplasmic reticulum. Eosin, being acidic in nature, stains basic structures like cytoplasm and imparts a pink color. Other special stains can also be used to selectively appreciate and identify specific cells and tissues like skeletal muscle, elastic fibers, basement membrane and microorganisms.

Microscopy Study of histology necessitates the use of specialized equipment called microscopes to magnify the tissues several hundred or thousand times. Routine compound light microscope uses a system of lenses and light source to achieve this. Usually light is allowed to pass through the specimen (transmitted light). Certain structures are better visible when using reflected light in which light is allowed to reflect from the top of the specimen being studied. Various other types of microscopes offering specific advantages are also available. This atlas includes photomicrographs obtained from compound light microscopes only, unless otherwise specified.

Points to remember

There are a few important points that need to be carefully considered while viewing histological slides under the microscope: • Setting up the microscope properly is a very essential step that is frequently overlooked. Even a high-end microscope that has been improperly set up will perform worse than a properly set basic microscope. • The various components of the microscope, especially the ones that come in the light path, and the slides have to be clean and free of dust. • Transmitted light source is usually built-in, although some basic microscopes require external light source. In such cases, be sure to orient the reflecting mirror in such a way that adequate light passes through the specimen being observed. • For viewing structures under reflected light, transmitted light source has to be switched off. If the ambient light in the room is inadequate, additional light using a torch or a mobile phone flash can be shone on top of the slide, and viewed. • Although one structure is being highlighted upon in each photomicrograph in this atlas, it is to be remembered than any particular field can show multiple structures of interest. Students appearing in undergraduate practical examinations are usually expected to identify all such features and label them in the diagrams.

Useful hints • Commonly used fixative for tissues—10% neutral buffered formalin. • A microtome is used to cut tissues into thin sections. • Hard tissues can be studied using ground sections, decalcified sections, or sections obtained by hard tissue microtomy. • Ground sections can be helpful to study the histology of enamel, dentin, cementum, bone, and other hard tissues. Staining is not

needed. • Much of the tooth or bone is lost during grinding for ground sections. • Decalcified sections are useful to study the histology of dentin, cementum, bone, and dental pulp. Enamel cannot be observed with decalcified sections. • Routinely used stain in histology and pathology is H&E stain. • Hematoxylin—basic dye—stains acidic structures like nucleus, RNA. • Eosin—acidic dye—stains basic structures like cytoplasm and its organelles.

CHAPTER 2

Development of tooth In early fetal life, basal cells in some areas of the primitive oral cavity proliferate more rapidly and result in the formation of a primary epithelial band in each arch. This band later divides into a buccal vestibular lamina and a lingual dental lamina. It is from this dental lamina that the ectodermal portions of teeth develop. Each tooth arises from a tooth germ, which is made up of three parts: enamel organ, dental papilla, and dental sac. The enamel organ is purely ectodermal in nature and derives from dental lamina. As the name indicates, enamel organ plays the primary role in enamel formation. Dental papilla is mesenchymal in origin, and gives rise to dentin and pulp. Dental sac or dental follicle helps in the formation of cementum, alveolar bone, and periodontal ligament.

Various stages in the development of tooth can be appreciated, based on the morphology or shape of the enamel organ.

Bud stage (figs 2.1 and 2.2) • Due to increased proliferation, tooth buds form at specific areas of the dental lamina corresponding to the location of future deciduous teeth. • These buds grow downward into the underlying ectomesenchyme. • Two types of cells can be recognized at this stage: outer low columnar cells and inner tightly packed polygonal cells. • Cells of the ectomesenchyme surrounding the bud begin to come close together and condense.

FIGURE 2.1 Development of tooth at bud stage (H&E stain). Source: (Image courtesy: Dept of Oral Pathology, Saveetha Dental College and Hospital, Chennai).

FIGURE 2.2 Schematic representation of bud stage of tooth development.

Cap stage (figs 2.3–2.5) • The cells of the tooth bud do not grow equally. Few cells proliferate at a faster rate than the rest, and this results in a change of shape in the bud. • The enamel organ assumes a cap shape, with a convex exterior surface, and an invaginated or concave interior surface. • The cells lining the concave surface also become elongated and obtain a columnar shape. These are the inner enamel epithelial (IEE) cells. • The cells on the exterior are low cuboidal in shape and constitute the outer enamel epithelium (OEE). • Meanwhile, the central cells of enamel organ begin to separate

from each other due to extracellular fluid accumulation and become star shaped. They are attached to each other only at their desmosomes. Hence, they are called stellate reticulum (stellate – star shaped; reticulum – interconnected network). • Sometimes, the inner enamel epithelial cells in the center of the enamel organ become very closely packed. This dense structure is called enamel knot, and is considered to provide signaling that determines and controls the morphology of the crown. • In this stage, condensed ectomesenchyme located within the concavity of inner enamel epithelium is called dental papilla. • Dental papilla is demarcated from the rest of the ectomesenchyme by accumulation of collagen fibers, which are oriented almost in the form of a circle enclosing the dental papilla. This is the dental sac.

FIGURE 2.3 Development of tooth at cap stage (H&E stain). Source: (Image courtesy: Dept of Oral Pathology, Manipal College of Dental Sciences, Mangalore).

FIGURE 2.4 Schematic representation of cap stage of tooth development.

FIGURE 2.5 Enamel knot (H&E stain). Source: (Image courtesy: Dept of Oral Pathology, Manipal College of Dental Sciences, Mangalore).

Early bell stage (figs 2.6 and 2.7) • As the cells continue to proliferate at different rates, the invagination of IEE deepens and begins to assume the shape of the future crown. • OEE and stellate reticulum are seen similar to cap stage. • Between stellate reticulum and IEE, two to three layers of tightly packed flattened cells are noticed. This is called the stratum intermedium. • The region where the IEE is continuous with the OEE is called cervical loop. • The inner enamel epithelial cells, especially near the tip of the future crown, begin to differentiate into ameloblasts. The cells become more columnar in shape, and a reversal of polarity of the nucleus is noticed. • The cells of the dental papilla in the periphery, near the ameloblasts, begin to differentiate into odontoblasts. Elsewhere, a small acellular zone is seen separating the dental papilla from IEE. • Dental sac is more prominently noticed. • The continuity of the tooth bud with the dental lamina gradually begins to disintegrate.

FIGURE 2.6 Development of tooth at early bell stage (H&E stain). Source: (Image courtesy: Dept of Oral Pathology, SDM College of Dental Sciences, Dharwad).

FIGURE 2.7 Schematic representation of early bell stage of tooth development.

Advanced bell stage (figs 2.8 and 2.9) • The tooth bud is completely separated from the dental lamina. • Cells of IEE and dental papilla differentiate into ameloblasts and odontoblasts, respectively. • Odontoblasts begin depositing the matrix for dentin initially. After this, the ameloblasts begin depositing enamel. • Deposition of dentin begins at the region of cusp tips, and then proceeds in a downward and inward direction. • Enamel deposition begins at the region of cusp tips, and proceeds in a downward and outward direction. • Stellate reticulum begins to collapse, and OEE is brought closer

to the ameloblasts.

FIGURE 2.8 (A) Development of tooth at advanced bell stage (H&E stain). (B) Higher magnification of the enamel organ showing enamel and dentin formation. (C) Higher magnification of the cervical region of the newly forming tooth.

FIGURE 2.9 Schematic representation of the advanced bell stage of tooth development.

Hertwig epithelial root sheath and cementum formation (figs 2.10 and 2.11)

• The cervical loop of enamel organ plays an important role in root formation. • Once enamel and dentin formation reach the future cervical line, the cervical loop begins proliferating in a downward direction. • However, this downgrowth is made up of only outer and inner enamel epithelial cells. Stellate reticulum and stratum intermedium are absent. This structure is called Hertwig epithelial root sheath (HERS). • HERS determines the shape of the roots. The cells of HERS induce the dental papilla to differentiate into odontoblasts and deposit root dentin. • Following this, HERS disintegrates, and dental follicle cells come in contact with root dentin. They then differentiate to cementoblasts and deposit cementum. • HERS at the most apical end shows a horizontal extension, called epithelial diaphragm. This structure determines the number of roots and location of the apical foramen. • Remnants of HERS are called cell rests of Malassez.

FIGURE 2.10 Hertwig epithelial root sheath (HERS) (H&E stain). Source: (Image courtesy: Dept of Oral Pathology, Manipal College

of Dental Sciences, Mangalore).

FIGURE 2.11 Schematic representation showing Hertwig epithelial root sheath and cementum formation.

Some important terminologies Tooth germ: It is the term used to collectively denote the various cells that together give rise to the different components of the tooth. It is organized into enamel organ, dental papilla, and dental sac. Dental lamina: It is the lingual portion of the primary epithelial band that later gives rise to the future primary tooth germs.

Vestibular lamina: It is the buccal portion of the primary epithelial band that later gives rise to the future buccal vestibule. Successional lamina: It is the lingual extension from the dental lamina that gives rise to the tooth germs of the future permanent succedaneous teeth. Ectomesenchyme: It is a type of connective tissue which contains neural crest cells, and which is especially important during embryogenesis in head and neck region. It is thought to develop from the ectoderm. It helps in the formation of bone, cartilage, and connective tissue of cranial region, as well as tooth dentin, cementum, and alveolar bone. Dental papilla: It is the condensation of the ectomesenchyme that is present within the invagination of the tooth germ, seen prominently during bell stage of tooth development. It is surrounded by the IEE and the dental follicle/sac. Cells in the periphery near the ameloblasts differentiate into odontoblasts which secrete dentin. Dental sac: It is the condensed ectomesenchyme with circular collagen fibres that enclose the enamel organ and dental papilla together, thus separating them from the rest of the ectomesenchyme. It gives rise to cementum, periodontal ligament, and alveolar bone. Outer enamel epithelium: This refers to the cells that make up the outer convex surface of the enamel organ. The cells of OEE are usually cuboidal in shape. Inner enamel epithelium: This refers to the cells that make up the inner concave surface (the invaginated portion) of the enamel organ. These cells are usually low columnar in shape. They transform into ameloblasts in the late bell stage, and secrete the enamel matrix. Stellate reticulum: The central cells of enamel organ that form a network of star-shaped cells due to intercellular fluid accumulation are called stellate reticulum. They are seen between OEE and IEE in cap stage, and between OEE and stratum intermedium in bell stages.

Stratum intermedium: Two/three layers of flattened cells seen between stellate reticulum and IEE in the bell stages are called stratum intermedium. They are rich in acid phosphatase enzyme. Cervical loop: The region where the OEE folds and becomes the IEE is called cervical loop (refer Figs 2.6 and 2.7). HERS: Hertwig epithelial root sheath is a downward extension of cervical loop, made up of only two layers of cells, namely, OEE and IEE. The HERS determines the shape of roots. Epithelial diaphragm: Horizontal extension of the HERS near the apical end is called the epithelial diaphragm. This structure corresponds to the future apical foramen region of the teeth, and determines the number of roots.

CHAPTER 3

Enamel Enamel is the hardest tissue in the body, with 96% of it being made up of inorganic content. As a result of its predominantly mineralized nature, enamel can be studied under the light microscope only using ground sections. Decalcification will result in complete loss of enamel, and hence decalcified sections are of little value. Various light microscopic structures are appreciable in enamel. For visualizing these structures, a transmitted light source is almost always used, unless mentioned otherwise. Some of the obvious light microscopic structures of enamel include: 1. Enamel rods 2. Striae of Retzius 3. Neonatal line 4. Enamel lamellae 5. Enamel tufts 6. Enamel spindles 7. Gnarled enamel 8. Hunter–Schreger bands

Enamel rods (figs 3.1 and 3.2) • Hydroxyapatite crystals in the enamel are arranged in the form of enamel rods. • These rods are cylindrical structures extending from dentinoenamel junction (DEJ) to enamel surface.

• They are generally arranged perpendicular to the underlying dentin. • During formation, around 4µ of enamel is deposited each day. Dark lines can be noticed between these 4µ deposits, which are called cross-striations.

FIGURE 3.1 Ground section of tooth showing enamel, dentinoenamel junction, and dentin. Dense enamel rods can be seen extending from the dentinal end to the outer surface in a wavy course.

FIGURE 3.2 Schematic diagram showing the orientation of enamel rods in relation to dentinoenamel junction. Inset shows the direction of striae of Retzius in relation to cross-striations in the rods.

Striae of retzius (figs 3.3, 3.4, and 3.5) • Dark brown lines representing a 6- to 11-day rhythm of enamel deposition are also seen in enamel. These are called incremental lines of Retzius (striae of Retzius). • In cross-sections of teeth, these striae are noticed as concentric

rings around the dentin. In longitudinal sections, they are seen surrounding the tip of the dentin. • An accentuated incremental line that separates the prenatal and postnatal enamel is called neonatal line. • These neonatal lines can be observed only in teeth that begin mineralization of crown in utero. Hence, they are seen in all primary teeth and in permanent first molars only.

FIGURE 3.3 Ground section of a molar crown showing incremental lines of Retzius. Note the concentric arrangement of these lines around the tip of the cusp.

FIGURE 3.4 Schematic diagram showing striae of Retzius and neonatal line in enamel.

FIGURE 3.5 Enamel observed under phase contrast microscopy. Notice the concentric arrangement of striae of Retzius. Enamel lamellae and tufts are also visible.

Enamel lamellae (figs 3.5, 3.6, and 3.7) • Enamel lamellae are linear hypocalcified structures seen in enamel, resembling thin leaf-like structures. • These are seen extending from the enamel surface to the DEJ to varying depths, sometimes even crossing into the dentin.

• These are of three types—A, B, and C. • Type A is made up of poorly calcified rods. • Type B contains degenerated cells. • Type C is filled with organic matter from saliva. • Lamellae can be easily confused with cracks that arise during preparation of ground sections. If such ground sections are decalcified carefully, the cracks will disappear, while lamellae persist. • Lamellae act as pathways for entry of bacteria resulting in dental caries.

FIGURE 3.6 Ground section showing multiple enamel tufts and an enamel lamella.

FIGURE 3.7 Schematic diagram showing enamel lamella and tufts.

Enamel tufts (figs 3.5, 3.6, and 3.7) • These are hypocalcified structures that arise from the DEJ and extend to a short thickness of enamel. • These resemble tufts of grass in ground sections. • These are actually ribbon-like structures made up of hypocalcified enamel rods that arise from the DEJ in different planes and curve in different directions. • These are better seen in cross-sections of teeth.

Enamel spindles (figs 3.8, 3.9) • Enamel spindles are hypocalcified structures that result due to extension of odontoblastic processes beyond the DEJ into the enamel. • These are seen in more numbers near the cusp tips. • They are seen as small, dark extensions, sometimes having blunt knob-like ends.

FIGURE 3.8 Ground section of a tooth near the region of cusp tip showing multiple enamel spindles.

FIGURE 3.9 Schematic diagram showing enamel spindles.

Gnarled enamel (figs 3.10 and 3.11) • Enamel in the region of the cusp tips appears to have a more complex arrangement of the rods. • The rods show intertwining and wavy course, probably an optical phenomenon, due to their increased numbers in a very small area. • It is thought that this arrangement helps in withstanding the masticatory forces.

FIGURE 3.10 Ground section of a tooth in the cusp tip region usually shows a wavy and intertwining course of enamel rods, also called gnarled enamel.

FIGURE 3.11 Schematic diagram of gnarled enamel.

Hunter–Schreger bands (figs 3.12, 3.13, and 3.14) • It is an optical phenomenon that can be visualized in oblique reflected light. • Alternating dark and light bands are visible in enamel in longitudinal sections of the teeth. • The dark bands are called diazones, and light bands are called parazones.

• It is considered that this optical appearance is a result of change in the direction of enamel rods.

FIGURE 3.12 Ground section of a tooth observed under reflected light. Note the alternate dark and light bands in the enamel. These represent the Hunter–Schreger bands.

FIGURE 3.13 Schematic diagram showing Hunter–Schreger bands.

FIGURE 3.14 Enamel viewed under polarizing light. Alternating dark and light Hunter–Schreger bands are visible very clearly.

Useful hints • Enamel is the hardest tissue in the body, and can be studied using ground sections. • Enamel rods make up the structural unit of enamel. These are formed by the regular arrangement of hydroxyapatite crystals. • Enamel is deposited in increments. The dark brown lines separating each increment are called incremental lines of Retzius. • The prominent incremental line separating enamel formed before birth and enamel formed after birth is called neonatal

line. It is seen in all deciduous teeth and in permanent first molars only. • DEJ is the junction where enamel and dentin meet. It has a scalloped appearance. • Hypomineralized or hypocalcified structures of enamel include the following: enamel lamellae, enamel tufts, and enamel spindles. • Hunter–Schreger bands are an optical phenomenon, seen best under oblique reflected light microscopy.

CHAPTER 4

Dentin Dentin is a mineralized tissue that forms the bulk of the tooth. It is made up of 65% inorganic substance in the form of hydroxyapatite crystals. Unlike enamel, dentin is not a completely solid component. Rather, hollow cylindrical structures called dentinal tubules are seen throughout its thickness. These tubules contain odontoblastic cell process and dentinal fluid. Peritubular dentin is seen surrounding the walls of dentinal tubules. Between adjacent peritubular dentin, intertubular dentin is present. This forms the major portion of dentin. Adjacent to the pulp, a small layer of unmineralized dentin called predentin, is also noticed. Dentin can be studied using both ground sections and decalcified sections.

Primary and secondary dentin (figs 4.1 and 4.2) • Dentin can be classified into two types based on whether it is formed before or after root completion. • Dentin formed before root completion is called primary dentin. • Dentinal tubules are seen arranged compactly and showing Sshaped curvature in the primary dentin. • Secondary dentin is formed after root completion. • It contains lesser number of dentinal tubules compared to primary dentin.

• A bend or angle in the dentinal tubules is noticed in the region where primary and secondary dentin meet.

FIGURE 4.1 Ground section of tooth showing primary and secondary dentin. Note the angle formed at the junction where primary and secondary dentin meet.

FIGURE 4.2 Schematic diagram showing primary and secondary dentin.

Dead tracts (figs 4.3 and 4.4) • Sometimes odontoblastic processes inside dentinal tubules disintegrate due to dental caries, attrition, etc. • Such tubules are instead filled with air, and in ground sections they appear dark in transmitted light and white in reflected light. These are called dead tracts.

FIGURE 4.3 (A) Ground section of tooth under transmitted light showing dead tract and tertiary dentin. Note the incisal edge of the tooth appears abraded, along with exposure of dentin. (B) Ground section of tooth under reflected light. Dead tract appears light here.

FIGURE 4.4 Schematic diagram showing dead tract and tertiary dentin.

Tertiary dentin (fig. 4.4) • It is deposited as a healing response to some injury to the tooth. • It is seen localized at the site of injury, as a result of deposition of dentin by the surviving odontoblasts or newly differentiated

odontoblasts. • It appears to have fewer and more convoluted dentinal tubules.

Interglobular dentin (figs 4.5–4.7) • The peripheral portion of circumpulpal dentin, just below the mantle dentin, shows mineralization in the form of globules. • These globules fail to fuse occasionally, resulting in small hypomineralized areas called interglobular dentin. • It is seen most commonly in cervical and middle thirds of the crown. • Dentinal tubules are unaltered and pass through the interglobular dentin undisturbed.

FIGURE 4.5 Ground section of dentin near the dentinoenamel junction showing interglobular dentin.

FIGURE 4.6 Schematic diagram showing interglobular dentin.

FIGURE 4.7 Interglobular dentin as observed under phase contrast microscopy. The interglobular dentin appears refractile, and dentinal tubules can be observed passing uninterrupted through the interglobular dentin.

Tomes’ granular layer (figs 4.8 and 4.9) • It is seen in radicular dentin just adjacent to the cementodentinal junction. • It appears as a dark, granular zone in transmitted light.

• It is thought to occur due to looping of dentinal tubules over themselves during early root dentin formation.

FIGURE 4.8 Ground section of tooth. This field shows radicular dentin and adjacent cellular cementum. Dark granules in the dentin near the cementodentinal junction represent Tomes’ granular layer.

FIGURE 4.9 Schematic diagram showing Tomes’ granular layer.

Branching of dentinal tubules (figs 4.10–4.12) • Dentinal tubules show branching, especially near the outer surface. • These terminal branches have been implicated in dentin hypersensitivity.

• Terminal branches are more common in root dentin. • Dentinal tubules also communicate with each other through lateral branches that can occur anywhere along the course of the tubule.

FIGURE 4.10 Ground section of tooth showing terminal branching of dentinal tubules.

FIGURE 4.11 Numerous dentinal tubules are seen branching near the dentinoenamel junction.

FIGURE 4.12 Dentinal tubules showing fine lateral branches.

Useful hints • Dentin makes up the major portion of the tooth, both in the crown and the root. It is less mineralized than enamel. • Dentin can be studied using both ground sections and decalcified sections. • Dentin is made up of millions of hollow dentinal tubules. Imagine them to be a bunch of closely arranged flexible drinking straws. The hollow portions of the straws correspond to the dentinal tubules, and these hollow structures each contain the odontoblastic process and the dentinal fluid. The space between adjacent straws would then be the intertubular dentin, while the plastic wall of the straw would be the peritubular

dentin. • Dentin structure can be studied in two ways. The dentin can be cut along the long axis of the tubules, so that we can follow and appreciate the tubule from the pulpal surface to the dentinoenamel junction. When we study dentin in this aspect, we can appreciate the wavy course of the tubules, including the primary curvature (S-shaped) and secondary curvatures. We can also identify the primary dentin (which is further divided into mantle dentin and circumpulpal dentin), secondary dentin, and tertiary dentin. Other relevant structures like interglobular dentin and Tomes’ granular layer can also be noticed. • Dentin can also be cut across, perpendicular to the long axes of its dentinal tubules. When we see dentin from this aspect, we can identify intertubular dentin and peritubular dentin, and the dentinal tubule.

CHAPTER 5

Pulp Pulp is the only soft tissue component of teeth, found in the center within a space in the dentin called pulp cavity. The pulp cavity in the crown, called pulp chamber, contains the coronal pulp. Radicular pulp is present in the root portion of pulp cavity known as root canal. Pulp is a loose connective tissue which is richly vascular and also innervated. The vitality of a tooth is determined only by the viability of the pulp. Pulp is probably the only soft tissue that is better studied by decalcified sections, because it is safely located within dentin.

Zones of the pulp (figs 5.1 and 5.2) • There are four recognizable areas or zones in the pulp. Starting from the periphery (dentinal side), these are • Odontogenic or odontoblastic zone • Cell-free zone • Cell-rich zone • Core of the pulp • The odontogenic zone contains the cell bodies of the odontoblasts, arranged parallel to each other, immediately subjacent to the predentin. Throughout life, odontoblasts constantly secrete predentin that calcifies into dentin. • Cell-free zone, immediately beneath the odontoblasts, is a zone that is relatively free of cells, and contains only the ground substance. It is also called the cell-free zone of Weil. The purpose of this zone is to provide space for the moving

odontoblasts, as dentin production occurs. • Cell-rich zone, as the name indicates, is more cellular in nature. More number of fibroblasts and undifferentiated mesenchymal cells are noticed in this zone. • The pulp core is the central portion of pulp, and contains the main trunk and branches of the blood vessels and nerve fibers that supply the pulp. In addition, various cells including the inflammatory cells are also noticed in pulp core.

FIGURE 5.1 Decalcified section of tooth showing the dentin–pulp interface (H&E stain).

FIGURE 5.2 Schematic representation of the various zones of pulp.

Pulp stones (figs 5.3 and 5.4) • Pulp stones, also called denticles, are small nodular mineralized structures in the pulp that can be seen as an age change. • They can be classified as true or false denticles, depending on their microscopic structure: • True denticles contain dentinal tubules with odontoblastic processes. • False denticles are seen as concentric calcifications

without any regular architecture. • Both true and false denticles can also be classified into three types based on their location in pulp: • Free denticles are completely surrounded by pulp on all sides. • Attached denticles are partly within dentin and partly within pulp. • Embedded denticles are seen completely within the dentin. • It is considered that all denticles arise as free denticles initially. As secondary dentin keeps depositing, they gradually become attached, and then later completely embedded in dentin. • Sometimes, the calcifications may be more diffuse and widespread throughout the pulp tissue. Eventually, such diffuse calcifications might lead to an almost complete obliteration of the pulp canals.

FIGURE 5.3 Decalcified section showing free false pulp stones in the pulp (H&E stain).

FIGURE 5.4 Schematic representation of free false pulp stones.

Useful hints • Pulp is the only soft tissue component of the tooth. It provides vitality to the tooth. • Pulp is highly vascular and richly innervated. • Pulp is better studied by using decalcified sections of teeth. • The odontogenic zone of pulp constantly produces predentin throughout life that mineralizes to form secondary dentin. • Pulp can show various age-related degenerative changes like fibrosis, discrete calcifications (denticles) and diffuse

calcifications.

CHAPTER 6

Cementum Cementum is a thin mineralized tissue that covers the roots of teeth. It serves as an attachment for the periodontal ligament fibers, thereby helping to hold the teeth in their sockets. It is avascular and noninnervated. The thickness of cementum gradually increases from the cervical line to the apical region. Cementum is made up of 45–50% inorganic substance in the form of hydroxyapatite crystals.

Acellular cementum (figs 6.1 and 6.2) • Acellular cementum is otherwise called primary cementum. • This is a form of cementum in which entrapped cementocytes are not present. • It is more commonly seen in the cervical third of the roots. • Acellular cementum is slowly deposited and the Sharpey’s fibers in it are well mineralized.

FIGURE 6.1 Ground section of tooth showing acellular cementum and adjacent dentin.

FIGURE 6.2 Schematic diagram of acellular cementum.

Cellular cementum (figs 6.3 and 6.4) • Cellular cementum is otherwise called secondary cementum. • Numerous cementocytes are seen entrapped in lacunae. The lacunae have many extensions called canaliculi that house the cell processes of cementocytes. These canaliculi are directed toward the outer surface of the cementum (toward periodontal

ligament). • This type of cementum is more frequently seen in the apical third of root. • It is deposited at a faster rate; therefore, the Sharpey’s fibers in it are partially mineralized.

FIGURE 6.3 Ground section of apical portion of tooth root showing cellular cementum.

FIGURE 6.4 Schematic diagram showing cellular cementum. Inset shows the orientation of canaliculi in cementocytes.

Incremental lines of salter (figs 6.5 and 6.6) • Careful observation of cementum under the microscope reveals

lines parallel to its surface, which represent its periodic deposition. These are called incremental lines of Salter. • These are seen separating the cementum into layers. • Contrary to the incremental lines of enamel and dentin, incremental lines of cementum are hypermineralized areas with less collagen. • Counting the number of incremental lines can be a useful method to estimate the age.

FIGURE 6.5 Ground section of tooth showing incremental lines of Salter in cementum. Note that these lines are almost parallel to the surface of the cementum.

FIGURE 6.6 Schematic diagram showing incremental lines of Salter in cementum. The horizontal band-like structures in cementum represent cracks. They can be mistaken for Sharpey’s fibers, but Sharpey’s fibers usually do not extend up to the dentinal surface.

Cementoenamel junction (figs 6.7–6.14) • Cementum and enamel meet at the cervical line of the tooth. • Depending on their relationship to each other, three major types of cementoenamel junctions (CEJ) are now recognized: i. Cementum overlapping enamel (Figs 6.7 and 6.8)

ii. Edge-to-edge/butt/knife-edge type junction (Figs 6.9 and 6.10) iii. Gap junction (Figs 6.11 and 6.12) • Cementum overlapping enamel is by far the most common type, accounting for about 60% in all the teeth. • This probably occurs because the reduced enamel epithelium in the cervical region degenerates, resulting in connective tissue in that region to contact the enamel and differentiate into cementoblasts forming cementum. • Edge-to-edge junction is noticed in around 30% of teeth. • Gap junction is seen in about 10% of teeth. It is believed that this occurs because enamel epithelium in the cervical region remains viable without undergoing degeneration for a longer period. As a result, cementum is not deposited in that region. • Recently, rare possibility of enamel overlapping cementum (Figs 6.13 and 6.14) has also been acknowledged, although the mechanism behind this phenomenon is not fully understood.

FIGURE 6.7 Ground section showing overlap type of cementoenamel junction.

FIGURE 6.8 Schematic representation of cementum overlapping enamel type of junction.

FIGURE 6.9 Cementum and enamel meeting at a butt type/edge-toedge type of junction.

FIGURE 6.10 Schematic representation of butt/edge-to-edge type of cementoenamel junction.

FIGURE 6.11 Gap type of cementoenamel junction.

FIGURE 6.12 Schematic representation of gap type of cementoenamel junction. Note that enamel and cementum do not meet in this type.

FIGURE 6.13 Ground section showing a rare type of cementoenamel junction where enamel overlaps cementum.

FIGURE 6.14 Schematic representation of cementoenamel junction where enamel overlaps cementum. This type of junction is quite rare in incidence.

Useful hints • Cementum is an avascular and noninnervated structure covering the roots of teeth.

• It can be studied using ground sections or decalcified sections. • It serves to provide attachment to periodontal ligament fibers. Without cementum, a tooth will not stay in the socket for a long time and will exfoliate very soon. • The portions of periodontal ligament fibers that are embedded into cementum are called Sharpey’s fibers. These are extrinsic fibers (originate outside cementum). • Cementum also has intrinsic fibers, which are short collagen fibers produced by cementoblasts during cementum deposition. • Cementum can be classified into different types based on the presence or absence of cementocytes, and the presence or absence of extrinsic and intrinsic fibers. • Incremental lines of Salter are hypermineralized areas, in contrast to the incremental lines of other structures. They run parallel to the cementum surface.

CHAPTER 7

Periodontal ligament Periodontal ligament is a soft connective tissue that helps in retaining a tooth to its socket in the alveolar bone through bundles of collagen fibers. It is attached on one side to the cementum of the tooth, and on the other side, it is inserted into the alveolar bone. It is seen occupying a thin space around the roots of teeth, sometimes referred to as the periodontal space.

Principal fiber groups of periodontal ligament (figs 7.1–7.4) • Five distinct fiber groups can be observed in periodontal ligament. These are • Alveolar crest group • Horizontal group • Oblique group • Apical group • Interradicular group • Alveolar crest bundles extend from the crest of the alveolar bone to the cementum near the cementoenamel junction. They have an obliquely upward course from bone to tooth. • Fibers of the horizontal group run through the shortest course from bone to cementum. These are almost at right angles to both the cemental surface and the bone. • Oblique fiber groups are most numerous and play a major role in resisting occlusal forces. These run an obliquely downward

course from bone to cementum, with the cemental insertion being more apical than bone attachment. • Apical group fibers are seen extending from around the tip of the root to the bone. These are not seen in teeth with incomplete roots, and probably play a role in protecting the blood vessels and nerves entering the apical foramen (Figs 7.3 and 7.4). • Interradicular fibers are seen only in multirooted teeth. These extend from the cementum in the root furcation to the tip of the interradicular septum of bone. • Dentoperiosteal fibers, a group of gingival fibers, are also seen in Figs 7.1 and 7.2. These extend from the cementum of the tooth to the outer surface (periosteum) of the alveolar bone.

FIGURE 7.1 Decalcified section of tooth and adjacent alveolar bone showing the various principal fiber groups of periodontal ligament

(H&E stain).

FIGURE 7.2 Schematic representation of various principal fiber groups of periodontal ligament, showing their orientation and relationship with each other and with the adjacent tissues.

FIGURE 7.3 Decalcified section of the apical portion of tooth root showing the apical group of periodontal ligament fibers (H&E stain).

FIGURE 7.4 Schematic representation of the apical group of periodontal ligament fibers.

Cementicles (fig. 7.5) • Cementicles are calcified structures noticed sometimes within the periodontal ligament. • These are considered to be a regressive change noticed more frequently in older individuals. • The exact nature and origin of these structures are unknown. It

is considered that these masses arise as a result of degeneration and calcification of epithelial rests of Malassez.

FIGURE 7.5 Decalcified section showing a mass of calcified material (cementicle) located freely in the periodontal ligament (H&E stain).

Useful hints • The periodontal ligament is a dense band of connective tissue that connects the tooth to the alveolar bone. • Within this connective tissue, collagen fibres are arranged and

organized into five principal fibre groups. • Each principal fiber group has a specific location and direction within the periodontal ligament. • The terminal ends of the principal fibre groups that are inserted into the bone or cementum are called Sharpey’s fibers. • The periodontal ligament acts like a thick gel that supports the tooth in the socket and absorbs forces.

CHAPTER 8

Bone Alveolar bone is the part of the jaw that supports and holds the roots of teeth. Although functionally different, the histology of alveolar bone is pretty much similar to bone elsewhere. Alveolar bone can be arbitrarily divided into two parts: alveolar bone proper and supporting alveolar bone. Alveolar bone proper forms that part of the jaw which houses the sockets for tooth roots. It consists of bundle bone and lamellar bone. Bundle bone is the portion of socket into which the principal fibers of periodontal ligament are inserted. Lamellated bone immediately surrounds the bundle bone. Supporting alveolar bone lies beneath the alveolar bone proper and is made up of outer cortical plates and inner spongy bone. Histologically, the cortical plates of jaws are made up of compact bone. The spongy bone between these cortical plates is made up of cancellous bone. The difference between these two types of bones (compact and cancellous) lies in their internal structure.

Compact bone (figs 8.1–8.4) • Compact bone is usually found at the outer surface of bones. • It is dense and compactly arranged without any spaces or gaps. • Basic functional units called osteons are seen throughout. • An osteon or Haversian system is composed of a central Haversian canal and surrounding concentric lamellae.

• Haversian canal usually contains blood vessels and nerve fibers. Sometimes, two adjacent Haversian canals are connected by a Volkmann’s canal. • The external and internal surfaces of the bone also contain circumferential lamellae. • Osteocytes are seen within lacunae that contain numerous extensions called canaliculi. Through these canaliculi, cell processes of osteocytes communicate with each other and with the exterior.

FIGURE 8.1 Ground section of compact bone showing different types of lamellae.

FIGURE 8.2 Schematic diagram showing different types of lamellae that form the compact bone.

FIGURE 8.3 Decalcified section of compact bone (H&E stain).

FIGURE 8.4 Schematic diagram showing appearance of compact bone in decalcified section.

Cancellous bone (figs 8.5 and 8.6) • Cancellous bone is made up of small spicules or pieces of bone called trabeculae. • Trabeculae branch and unite and form a delicate network in the center of bone. • Spaces between these trabeculae are called marrow spaces.

These spaces are richly vascular and form the major site of hematopoiesis in young children. • As age progresses, marrow becomes more fatty in nature.

FIGURE 8.5 Decalcified section of cancellous bone (H&E stain).

FIGURE 8.6 Schematic representation of cancellous bone.

Useful hints • Bone is a hard, mineralized structure that can be studied using ground sections or decalcified sections. • Alveolar bone or alveolar process is that part of the maxilla and mandible that contains the tooth sockets. • The alveolar bone undergoes resorption once the teeth are lost. • The alveolar bone is organized as follows:

CHAPTER 9

Salivary glands Salivary glands are compound exocrine glands located within and around the oral cavity. These have epithelial secretory units called acini, and ducts lined by epithelium that serve to modify and conduct the secretions into the oral cavity. These parenchymal structures are supported by connective tissue that encapsulates the gland and also divides it into small lobes and lobules. Salivary glands can be classified based on their function or histological appearance into: 1. Serous salivary glands 2. Mucous salivary glands 3. Mixed salivary glands

Serous salivary glands (figs 9.1–9.3) • These are glands that are predominantly made up of serous salivary acini. • The cells of the serous acini are pyramidal in shape, and have a round nucleus that is present in the basal third region of the cell. • Serous cells, by virtue of their protein content, stain more eosinophilic. • The acinar cells also contain small eosinophilic granules, which appear more dense and concentrated toward the tip of the cell. These are the zymogen granules. • Interlobular and intralobular ducts (discussed later) are also

noticed. • Parotid gland is a predominantly serous salivary gland. • The only minor salivary gland that is serous in nature is von Ebner salivary gland.

FIGURE 9.1 Serous salivary gland under low magnification (H&E stain). Note the distribution of various intralobular and interlobular ducts.

FIGURE 9.2 Serous salivary gland under higher magnification (H&E stain). Note the shape of nuclei in the acinar cells and the staining property of acini.

FIGURE 9.3 Schematic diagram of a serous salivary gland. Zymogen granules, although visible here, are rarely visible under light microscope.

Mucous salivary glands (figs 9.4–9.6) • Mucous type of salivary acini are predominantly noticed in these glands. • Mucous acinar cells are also pyramidal in shape. However, the nucleus is flat and appears pushed toward the bottom of the cell. • The cytoplasm of acinar cells rarely take up any stain, and appear pale or empty on routine hematoxylin and eosin (H&E)

stained sections. • Interlobular and intralobular ducts are present. • Sublingual salivary glands are predominantly mucous in nature. • All minor salivary glands, except von Ebner glands are also of mucous type.

FIGURE 9.4 Mucous salivary gland under low magnification (H&E stain). Note the overall pale staining property of acini.

FIGURE 9.5 Mucous salivary gland under higher magnification (H&E stain). The acini appear empty and pale staining, while the nuclei are apposed to the basal surface of acinar cells.

FIGURE 9.6 Schematic diagram showing the histology of mucous salivary gland. Nuclei in the acinar cells are flat and pushed toward the basal surface.

Mixed salivary glands (figs 9.7–9.9) • Mixed salivary glands contain both serous and mucous type of acini. • A few serous cells are seen arranged as a crescent-shaped structure on top of some mucous acini. These are called serous demilunes (demilunes of Giannuzzi) (demi—half; lune—moon). • It is now known that demilunes are artifacts that arise due to traditional tissue preparation methods.

• Tissues prepared using liquid nitrogen and osmium tetroxide show serous and mucous cells aligned normally within the acinus. • In conventional tissue preparations, mucous cells swell and push the serous cells outside, resulting in the demilune appearance. • Submandibular salivary gland is a mixed salivary gland.

FIGURE 9.7 Mixed salivary gland under low magnification (H&E stain). Note the distribution of two different types of acini with markedly varying staining property.

FIGURE 9.8 Mixed salivary gland under higher magnification (H&E stain). Note the presence of serous demilunes, which are characteristic of mixed glands.

FIGURE 9.9 Schematic diagram of the histology of mixed salivary gland.

Ductal system of salivary glands Salivary glands contain a system of ducts which acts as canals in which saliva flows from the secretory units (acini) to open into the oral cavity. In addition, some parts of the ductal system also modify the salivary composition.

Intercalated ducts (figs 9.10 and 9.13) • Intercalated ducts are usually found in an intralobular location. • These are the smallest in the ductal system, and carry secretions from the acini to the striated ducts. • Intercalated ducts are lined by a layer of low cuboidal cells.

FIGURE 9.10 High magnification of an intercalated duct (H&E stain).

Note the small lumen surrounded by cuboidal cells with spherical nuclei at their center.

Striated ducts (figs 9.11 and 9.13) • Striated ducts form the major component of the ductal system, and are mostly located intralobularly. • These conduct saliva from the intercalated ducts to the excretory ducts, and also play an important role in modifying salivary composition. • These are lined by columnar cells with centrally placed nuclei. • The basal portion of these cells contains fine striations which give them their name. • Electron microscopy reveals that these striations are deep infoldings of the cell membrane in the basal region, with numerous mitochondria in between.

FIGURE 9.11 High magnification of a striated duct (H&E stain). Lumen

is surrounded by columnar cells. Note the position of the nuclei in these cells. The basal portion below the nucleus is made up of striations due to surface infoldings that can be appreciated under electron microscope.

Excretory ducts (figs 9.12 and 9.13) • Excretory ducts are the terminal structures of the ductal system which ultimately open into the oral cavity. • These are usually seen in the interlobular connective tissue septa. • These are lined by stratified cuboidal, stratified columnar, pseudostratified columnar, or stratified squamous epithelium. • These ducts help pour the secretions into the oral cavity, and also contribute to salivary modification to a lesser extent.

FIGURE 9.12 A large excretory duct can be easily appreciated in the fibrous septum, even under low magnification (H&E stain).

FIGURE 9.13 Schematic diagram showing the histology of the different types of ducts in salivary glands.

Useful hints • Salivary glands are compound, tubuloacinar, exocrine glands that produce saliva. • They can be classified based on their location, size, or type of secretion. • Serous salivary glands produce a watery secretion that is rich in proteins and contains little carbohydrates. • Mucous salivary glands produce a thick, viscous secretion that is rich in carbohydrate and poor in proteins. • The morphology of the terminal secretory units (acini) varies according to the type of secretion it produces. • Ducts of salivary glands help to carry the secretions from acini to the oral cavity. They also modify the composition of saliva. • Minor salivary glands are distributed in most parts of the oral cavity in the submucosa. Attached gingiva and the anterior part of hard palate do not contain minor salivary glands.

CHAPTER 10

Oral mucous membrane The oral mucous membrane covers all the surfaces of the oral cavity and performs several important functions in addition to protecting the underlying structures. It aids in mastication, swallowing, speech, and taste sensation. Histologically, oral mucosa comprises epithelium and lamina propria. Epithelium is of stratified squamous type, and may be keratinized or nonkeratinized. Lamina propria is the connective tissue seen immediately beneath the epithelium. Deeper to this, the connective tissue that contains structures like glands and adipocytes is called submucosa. Submucosa helps in attaching the mucosa to underlying structures like muscle or bone. Although it is present in most parts of the oral cavity, submucosa is absent in some areas like attached gingiva and midpalatine raphe.

Keratinized stratified squamous epithelium (figs 10.1–10.5) • Keratinized oral epithelium has several histological features that differentiate it from nonkeratinized epithelium. • Four distinct layers can be appreciated histologically: • Stratum basale (or basal layer) is the single layer of cells that is seen in apposition with the basement membrane. It is made up of cuboidal cells, which actively proliferate and move toward the surface.

• Stratum spinosum (or spinous layer) is made up of polygonal cells that gradually become larger as they progress toward the surface. These cells are also called acanthocytes. During routine tissue processing and preparation for microscopy, the cells shrink slightly and their intercellular bridges (desmosomes) become visible easily; hence the name. • Stratum granulosum (or granular cell layer) contains two to three layers of flattened cells that have very characteristic dark basophilic granules. These are the keratohyaline granules, which play an important role in keratin formation. • Stratum corneum (or corneal layer or keratin layer) is the most superficial layer made up of flattened cells that are devoid of almost all organelles. Two types of keratin can be distinguished: orthokeratin and parakeratin. • Orthokeratin is made up of flattened cells that contain no nuclei in the corneal layer. Orthokeratinized epithelium has a prominent granular cell layer (Figs 10.1 and 10.2). It is mostly seen in the hard palate (Fig. 10.3). • Parakeratin is made up of flattened cells that contain few pyknotic nuclei. The granular cell layer cannot be appreciated clearly in parakeratinized epithelium in light microscopy (Figs 10.4 and 10.5). Parakeratinized epithelium is noticed commonly in the gingiva.

FIGURE 10.1 Orthokeratinized stratified squamous epithelium (H&E stain).

FIGURE 10.2 Schematic diagram of orthokeratinized stratified squamous epithelium.

FIGURE 10.3 Orthokeratinized stratified squamous epithelium as noticed in the posterior glandular zone of hard palate (H&E stain).

FIGURE 10.4 Parakeratinized stratified squamous epithelium (H&E stain). Note the presence of pyknotic nuclei in the corneal layer. Granular layer is not so prominent.

FIGURE 10.5 Schematic diagram of parakeratinized stratified squamous epithelium.

Nonkeratinized stratified squamous epithelium (figs 10.6 and 10.7) • The stratification in nonkeratinized epithelium is less distinct. • A single layer of cuboidal cells (stratum basale or basal layer) is evident. • Above this, there are polygonal cells that gradually flatten toward the surface. These cells can be divided arbitrarily into

two layers: • Stratum intermedium (or intermediate layer) is made up of predominantly polygonal cells. • Stratum superficiale (or superficial layer) contains flattened cells that have nuclei.

FIGURE 10.6 Nonkeratinized stratified squamous epithelium (H&E stain).

FIGURE 10.7 Schematic diagram of nonkeratinized stratified squamous epithelium.

Keratinocytes and nonkeratinocytes (figs 10.8 and 10.9) • Almost all cells of both keratinized and nonkeratinized epithelium contain cytokeratin proteins. These cells are collectively referred to as keratinocytes (cells containing cytokeratin).

• Few cells in oral epithelium, however, do not contain cytokeratin. These are called nonkeratinocytes. • Nonkeratinocytes include melanocytes, Merkel cells, Langerhans cells, and inflammatory cells. • Melanocytes can be identified by their distinct color due to melanin pigment. These are mostly seen in basal layer. • Langerhans cells are antigen-presenting cells that play a role in immunity. These are seen as clear cells in the more superficial layers of the epithelium. • Merkel cells are commonly noticed in the basal layer. These are cells of neural origin that probably mediate the pressure sensation. These are also identified as clear cells that do not take up any cytoplasmic stain.

FIGURE 10.8 (A) Melanocytes in oral epithelium seen in low magnification (H&E stain). (B) Higher magnification showing a single melanocyte in the basal layer (H&E stain). Note the numerous cell processes that extend between adjacent cells in the epithelium.

FIGURE 10.9 Numerous nonkeratinocytes noticed in oral epithelium (H&E stain). These are observed as clear cells and could represent Merkel cell or Langerhans cell.

Papillae of the tongue Dorsal surface of the tongue has numerous tiny projections called papillae which give it the rough texture. Papillae can be easily observed by the naked eye especially when the tongue is dry. They are of three major types:

Filiform papilla (figs 10.10 and 10.11) • Filiform means thread-like. Filiform papillae are pointed, conical structures in the anterior two-third of the tongue, and are arranged in numerous rows parallel to the sulcus terminalis. • These are the most numerous among all papillae. • Histologically, these are made up of stratified squamous keratinized epithelium, containing a connective tissue core. The tip of the papilla usually shows more keratin.

• These papillae do not contain taste buds.

FIGURE 10.10 Section through anterior tongue showing filiform papillae (H&E stain). Note the abundant keratinization of the papillae and lack of any taste buds.

FIGURE 10.11 Schematic diagram showing the histology of filiform papillae.

Fungiform papilla (figs 10.12 and 10.13) • Fungiform means mushroom-like. Fungiform papillae are seen distributed between filiform papillae and are seen mostly near the tip and sides of the tongue. • These are covered by a thin stratified squamous epithelium that appears nonkeratinized, especially on its surface. As a result, the vascularity of the connective tissue shows through, and the papilla appears reddish in color. • Few taste buds are seen on the dorsal surface.

FIGURE 10.12 Section showing fungiform papilla (H&E stain).

FIGURE 10.13 Schematic diagram showing the histology of fungiform

papillae.

Circumvallate papilla (figs 10.14 and 10.15) • Circumvallate means surrounded by a walled trench or depression. • Circumvallate papillae (or vallate papillae) are characteristically seen just in front of the sulcus terminalis, on either side of the midline. These are about 8–10 in number. • Each papilla is surrounded by a circular depression or furrow in the mucous membrane. • Epithelium is of stratified squamous type. The dorsal surface shows keratinization occasionally. The lateral surfaces of the papilla are nonkeratinized and contain numerous taste buds. • Connective tissue core shows many secondary papillae, especially toward the dorsal surface. • Few minor serous salivary glands are noticed in the submucosa beneath these papillae. These glands are the only minor salivary glands that are serous in nature, and they open their secretions into the trough of the circumvallate papillae. These are called von Ebner glands.

FIGURE 10.14 Section showing a circumvallate papilla (H&E stain). Note the presence of numerous taste buds along the lateral walls. Image courtesy: Dept of Oral Pathology, Saveetha Dental College and Hospital, Chennai.

FIGURE 10.15 Schematic diagram showing the histology of a circumvallate papilla.

Dentogingival junction (figs 10.16 and 10.17) • Dentogingival junction (DGJ) is the junction where the gingiva gets attached to the tooth. • A shallow gingival sulcus lined by nonkeratinized stratified squamous epithelium is seen surrounding the tooth on all sides. • Below the floor of this sulcus, the epithelium is attached to the tooth enamel or cementum. This is called the junctional epithelium. • Junctional epithelium does not show the process of keratinization. • DGJ is initially seen completely on the enamel in newly erupted teeth. It gradually shifts toward a more apical direction as age

progresses. (The schematic diagram in Fig. 10.17 shows DGJ that is partly on enamel and partly on cementum.). • The junctional epithelium is unique in that it contains two sets of basal lamina and basal layers of cells—one in apposition with the tooth surface and the other toward the lamina propria of gingiva. • The lamina propria of gingiva shows the gingival groups of fibers (dentogingival fibers, dentoperiosteal fibers, etc.).

FIGURE 10.16 Decalcified section showing the dentogingival junction (H&E stain). Source: (Image courtesy: Dept of Oral Pathology, SDM College of Dental Sciences, Dharwad).

FIGURE 10.17 Schematic diagram showing the dentogingival junction.

Useful hints • Oral mucous membrane is the soft tissue that covers all surfaces of the oral cavity. It is organized as follows:

• Epithelium of the oral cavity is usually stratified squamous in type. It may be keratinized or nonkeratinized in nature. • Almost all epithelial cells in the oral cavity contain cytokeratin filaments. Therefore, they are called keratinocytes. • Few cells of the epithelium do not contain cytokeratin; hence, they do not have the ability to keratinize. They are called nonkeratinocytes, e.g., melanocytes, Merkel cells, Langerhans cells, inflammatory cells. • The oral mucosa shows structural modifications according to the function it performs. • Some unique structural changes include papillae of tongue, vermilion border of lip, and DGJ. • Papillae are projections on the dorsal surface of tongue, some of which contain taste buds. • Vermilion border is the transitional zone of the lip, where the skin of face gradually changes and continues as the mucosa of the lip. It is easily remembered as the lipstick zone (the part of the lips where lipstick is applied). This zone has very thin epithelium, and does not have salivary glands or sweat glands or sebaceous glands in the submucosa. Therefore, it is susceptible to dry out very easily. • DGJ is unique because it is the only site where epithelium

attaches to a hard tissue directly. It has two basal laminas, one on tooth surface side and other on the connective tissue side. DGJ plays a significant role in tooth eruption, and in the health of the gingiva and the periodontal tissues.

CHAPTER 11

Maxillary sinus The maxillary sinus, also known as antrum of Highmore, is the largest among the paranasal air sinuses. Although a structure entirely within the maxilla and having no direct communication with the oral cavity, it is of interest in oral histology because of its close association with the roots of maxillary premolars and molars. The sinus is entirely lined by mucosa that in most places is firmly bound to the underlying bone, with little or no submucosa.

Histology of the sinus lining (figs 11.1 and 11.2) • The sinus lining is made up of a thin epithelium of pseudostratified ciliated columnar type and lamina propria. • Cilia help to propel mucus, microorganisms, or debris from the surface of the sinus lining toward the nasal cavity through their wave-like beating. • In addition to columnar and ciliated cells, the epithelium also contains columnar nonciliated cells, basal type of cells, and goblet cells (described in the following section). • The lamina propria is made up of connective tissue cells and fibers, along with blood vessels. • Subepithelial glands of serous or mucous type are noticed in the lamina propria. These glands pour their secretions on the surface of the sinus lining through ducts.

FIGURE 11.1 Section showing the maxillary sinus lining made up of pseudostratified ciliated columnar epithelium (H&E stain).

FIGURE 11.2 Schematic diagram showing the histology of maxillary sinus mucosa.

Goblet cells (figs 11.3–11.5) • Goblet cells are simple unicellular intraepithelial glands that are interspersed among the other cells of the sinus epithelium. • These are called so because of their unique shape with a wide apical region and a narrow stalk-like basal region, resembling a wine glass. • These cells secrete mucin, which first accumulates in the apical region of the cell resulting in their distention and widening, before being discharged through exocytosis. • The slender basal region rests on the basement membrane, and contains nucleus and other organelles.

FIGURE 11.3 Higher magnification of the sinus lining showing the pseudostratified appearance clearly (H&E stain). Goblet cells and surface cilia are also appreciable in the picture.

FIGURE 11.4 Periodic acid–Schiff-stained section showing the maxillary sinus lining. Note the distinctly visible goblet cells with their unique shape.

FIGURE 11.5 Periodic acid–Schiff-stained section of maxillary sinus lining at higher magnification, distinctly showing the goblet cells.

Useful hints • The maxillary sinus is the largest among all paranasal air sinuses. • It is seen in close relation to the roots of maxillary posterior teeth (most commonly the first molar and second premolar). • Pathologies affecting maxillary posterior teeth might involve the maxillary sinus too, due to the close association.