Seltzer and Bender's Dental Pulp E-Book

Kenneth M Hargreaves

• Herausgeber: Quintessence Publishing Co, Inc
• Kategorie: Fachliteratur
• Sprache: Englisch

Seitenzahl: 1632

Veröffentlichungsjahr: 2019

Beschreibung

This comprehensive update of a classic text presents the latest research on the dental pulp and its interaction with other tissues, highlighting its central role in both local and systemic health. The second edition has been completely revised to incorporate new chapters featuring the most topical issues in research and clinical practice, including developments in stem cell research and pulpodentin regeneration, the effects of the aging process on the pulp, and the interdependent relationship of the pulp and restorative dental procedures. New contributors bring fresh perspective to topics such as pulpal infections, odontalgia, and the relationship between the pulp and periodontal disease. Each chapter provides an introduction to its major themes for the busy clinician or dental student as well as up-to-date, biologically based clinical recommendations for restorative and endodontic procedures. Practicing clinicians will find this information to be essential to providing accurate diagnoses and effective treatment.

Leseprobe

Edited by

Kenneth M. Hargreaves, DDS, PhD

Professor and Chair Department of Endodontics Professor Departments of Pharmacology, Physiology, and Surgery University of Texas Health Science Center at San Antonio San Antonio, Texas

Harold E. Goodis, DDS

Professor Emeritus Department of Preventive and Restorative Dental Sciences University of California at San Francisco San Francisco, California Professor Department of Endodontics Boston University Institute for Dental Research and Education–Dubai Dubai, United Arab Emirates

Franklin R. Tay, BDSc (Hons), PhD

Associate Professor Departments of Endodontics and Oral Biology Georgia Health Sciences University Augusta, Georgia

Library of Congress Cataloging-in-Publication Data

Seltzer and Bender's dental pulp. -- 2nd ed. / edited by Kenneth M. Hargreaves, Harold E. Goodis, Franklin R. Tay.       p. ; cm.    Dental pulp    Includes bibliographical references.    ISBN 978-0-86715-582-2 (ebook)   1. Dental pulp. 2. Endodontics. I. Hargreaves, Kenneth M. II. Goodis, Harold E. III. Tay, Franklin R. IV. Seltzer, Samuel, 1914- Dental pulp. V. Title: Dental pulp.    [DNLM: 1. Dental Pulp. 2. Dental Pulp--physiology. 3. Dental Pulp Diseases. 4. Endodontics--methods. WU 230]    RK351.S4 2011   617.6'342--dc23                                           2011023692

© 2012 Quintessence Publishing Co, Inc

Quintessence Publishing Co Inc 4350 Chandler Drive Hanover Park, IL 60133www.quintpub.com

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All rights reserved. This book or any part thereof may not be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, or otherwise, without prior written permission of the publisher.

Editors: Lisa C. Bywaters and Leah Huffman Design: Sue Robinson Production: Angelina Sanchez

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Table of Contents

Preface

Contributors

1 Development of the Pulpodentin Complex

Rena D’Souza and Chunlin Qin

2 Formation and Repair of Dentin in the Adult

Anthony J. Smith

3 Pulpodentin Complex

David H. Pashley and Franklin R. Tay

4 Pulp as a Connective Tissue

Takashi Okiji

5 Stem Cells and Regeneration of the Pulpodentin Complex

Peter E. Murray and Franklin García-Godoy

6 Circulation of the Pulp

Hideharu Ikeda and Hideaki Suda

7 Dental Innervation and Its Responses to Tooth Injury

Margaret R. Byers, Michael A. Henry, and Matti V. O. Närhi

8 Pain Pathways and Mechanisms of the Pulpodentin Complex

Anibal Diogenes and Michael A. Henry

9 Pharmacologic Control of Dental Pain

Asma Khan and Kenneth M. Hargreaves

10 Pulpal Infections, Including Caries

José F. Siqueira, Jr

11 Molecular Mediators of Pulpal Inflammation

Ashraf F. Fouad

12 Interrelationship of the Pulp and Apical Periodontitis

Hajime Sasaki and Philip Stashenko

13 Repair of Pulpal Injury with Dental Materials

Harold E. Goodis, Sally Marshall, Franklin R. Tay, and Grayson W. Marshall, Jr

14 Caries, Restorative Dentistry, and the Pulp

Franklin R. Tay, Harold Messer, and Richard Schwartz

15 Effects of Thermal and Mechanical Challenges

Harold E. Goodis and David H. Pashley

16 Interrelationship of Pulpal and Periodontal Diseases

Ilan Rotstein and James H. Simon

17 Root Resorption

Linda G. Levin

18 Aging and the Pulp

Harold E. Goodis, Arnold Kahn, and Stéphane Simon

19 Differential Diagnosis of Toothache: Odontogenic Versus Nonodontogenic Pain

Jeffrey P. Okeson

20 Interrelationship of Pulp and Systemic Disease

Michaell A. Huber

Preface

Welcome to the second edition of Seltzer and Bender’s Dental Pulp. Like the first edition, this book focuses on the dental pulp and its interaction with other tissues during health and disease, with each chapter providing the latest information on the biologic principles and the basis for clinical treatment procedures. As such, the book is ideally suited for practicing dentists as well as residents and dental students. This newly revised second edition includes entirely new topics (eg, regenerative endodontics) as well as greatly expanded reviews on dental implications of biofilms, immune interactions, pain mechanisms, the interactions between restorative dental procedures and pulpal health, and neuroanatomy, among other topics. We welcome many new and returning authors to this edition who have shared their incredible expertise with you, our reader.

The central theme of this book—a fundamental theme of dentistry in our opinion—is the critical role that pulp tissue plays in dental health. Both local (eg, caries, periodontitis) and systemic (AIDS, hyperparathyroidism) disease can contribute to pulpal pathosis. In turn, pulpal pathosis can contribute to both local (eg, root resorption, periodontitis) and systemic (eg, referred pain) conditions. The astute clinician needs this information to provide accurate diagnoses and effective treatment. Accordingly, we have focused on the biology of dental pulp and its interaction with other tissues during health and disease in order to provide comprehensive, biologically based clinical recommendations for practicing dentists.

We have been gratified by the support and encouragement generated from the first edition of this text, and we were thrilled that both I. B. Bender and Sam Seltzer lived to enjoy its publication. We have now lost many of the pioneering giants of endodontics and pulp biology. Their early contributions laid the foundation for generations of dentists to deliver biologically based dental care. In this age of gene arrays, signal transduction pathways, novel restorative materials, and computerized data retrieval, it is difficult to appreciate the magnitude of their contributions based entirely upon intellectual rigor and using relatively simple tools. To their memories, we dedicate this second edition.

Contributors

Margaret R. Byers, PhD

Professor Emeritus

Department of Anesthesiology and Pain Medicine

University of Washington

Seattle, Washington

Anibal Diogenes, DDS, MS, PhD

Assistant Professor

Department of Endodontics

University of Texas Health Science Center at San Antonio

San Antonio, Texas

Rena D’Souza, DDS, MS, PhD

Professor and Chair

Department of Biomedical Sciences

Baylor College of Dentistry

Texas A&M Health Science Center

Dallas, Texas

Ashraf F. Fouad, BDS, DDS, MS

Professor and Chair

Department of Endodontics, Prosthodontics, and Operative Dentistry

Director

Postgraduate Endodontics

University of Maryland Dental School

Baltimore, Maryland

Franklin García-Godoy, DDS, MS

Professor and Senior Executive Associate Dean of Research

University of Tennessee Health Science Center

Memphis, Tennessee

Harold E. Goodis, DDS

Professor Emeritus

Department of Preventive and Restorative Dental Sciences

University of California at San Francisco

San Francisco, California

Professor

Department of Endodontics

Boston University Institute for Dental Research and Education–Dubai

Dubai, United Arab Emirates

Kenneth M. Hargreaves, DDS, PhD

Professor and Chair

Department of Endodontics

Professor

Departments of Pharmacology, Physiology, and Surgery

University of Texas Health Science Center at San Antonio

San Antonio, Texas

Michael A. Henry, DDS, PhD

Professor

Department of Endodontics

University of Texas Health Science Center at San Antonio

San Antonio, Texas

Michaell A. Huber, DDS

Associate Professor

Oral Medicine Subject Expert

Department of Comprehensive Dentistry

University of Texas Health Science Center at San Antonio

San Antonio, Texas

Hideharu Ikeda, DDS, PhD

Associate Professor

Department of Pulp Biology and Endodontics

Graduate School of Medical and Dental Sciences

Tokyo Medical and Dental University

Tokyo, Japan

Arnold Kahn, PhD

Senior Scientist

California Pacific Medical Center Research Institute

Professor Emeritus and Former Chair

Department of Cell and Tissue Biology

School of Dentistry

University of California at San Francisco

San Francisco, California

Asma Khan, BDS, PhD

Assistant Professor

Department of Endodontics

University of North Carolina

Chapel Hill, North Carolina

Linda G. Levin, DDS, PhD

Former Chair

Department of Endodontics

University of North Carolina

Chapel Hill, North Carolina

Private practice

Durham, North Carolina

Sally Marshall, PhD

Professor

Division of Biomaterials and Bioengineering

Department of Preventive and Restorative Dental Sciences

University of California at San Francisco

San Francisco, California

Grayson W. Marshall, Jr, DDS, MPH, PhD

Professor and Chair

Division of Biomaterials and Bioengineering

Department of Preventive and Restorative Dental Sciences

University of California at San Francisco

San Francisco, California

Harold Messer, MDSc, PhD

Professor Emeritus

Melbourne Dental School

The University of Melbourne

Melbourne, Australia

Peter E. Murray, BSc (Hons), PhD

Professor

Department of Endodontics

College of Dental Medicine

Nova Southeastern University

Fort Lauderdale, Florida

Matti V. O. Närhi, DDS, PhD

Professor, Chair, and Research Director

Department of Physiology

Institute of Biomedicine

University of Eastern Finland

Kuopio, Finland

Jeffrey P. Okeson, DMD

Professor and Chair

Department of Oral Health Science

Director

Orofacial Pain Program

College of Dentistry

University of Kentucky

Lexington, Kentucky

Takashi Okiji, DDS, PhD

Professor

Division of Cariology, Operative Dentistry, and Endodontics

Department of Oral Health Sciences

Graduate School of Medical and Dental Sciences

Niigata University

Niigata, Japan

David H. Pashley, DMD, PhD

Emeritus Regents’ Professor

Department of Oral Biology

College of Dental Medicine

Georgia Health Sciences University

Augusta, Georgia

Chunlin Qin, DDS, MS, PhD

Associate Professor

Department of Biomedical Sciences

Baylor College of Dentistry

Texas A&M Health Science Center

Dallas, Texas

Ilan Rotstein, DDS

Professor and Chair

Division of Endodontics, Oral and Maxillofacial Surgery, and Orthodontics

University of Southern California

Los Angeles, California

Hajime Sasaki, DDS, PhD

Assistant Member of the Staff

Department of Cytokine Biology

The Forsyth Institute

Cambridge, Massachusetts

Richard Schwartz, DDS

Clinical Assistant Professor

Department of Endodontics

University of Texas Health Science Center at San Antonio

San Antonio, Texas

James H. Simon, DDS

Director

Advanced Endodontic Program

Wayne G. and Margaret L. Bemis Endowed Professor

Division of Endodontics, Oral and Maxillofacial Surgery, and Orthodontics

University of Southern California

Los Angeles, California

Stéphane Simon, DDS, MPhil, PhD

Senior Lecturer

Department of Endodontics

University of Paris 7, Diderot

Paris, France

José F. Siqueira, Jr, DDS, MSc, PhD

Professor and Chair

Department of Endodontics

Faculty of Dentistry

Estácio de Sá University

Rio de Janeiro, Brazil

Anthony J. Smith, BSc, PhD

Professor

Department of Oral Biology

School of Dentistry

University of Birmingham

Birmingham, United Kingdom

Philip Stashenko, DMD, PhD

President and CEO

The Forsyth Institute

Cambridge, Massachusetts

Associate Professor

Department of Oral Medicine, Infection, and Immunity

Harvard University

Cambridge, Massachusetts

Hideaki Suda, DDS, PhD

Professor

Department of Pulp Biology and Endodontics

Graduate School of Medical and Dental Sciences

Tokyo Medical and Dental University

Tokyo, Japan

Franklin R. Tay, BDSc (Hons), PhD

Associate Professor

Departments of Endodontics and Oral Biology

Georgia Health Sciences University

Augusta, Georgia

Development of the Pulpodentin Complex

Rena D’Souza, DDS, MS, PhD Chunlin Qin, DDS, MS, PhD

Dentin is a unique, avascular mineralized connective tissue that forms the bulk of the tooth. It underlies enamel in the crown and cementum in the roots, providing these tissues structural support and the tooth its resilience. In a mature tooth, dentin encloses a richly innervated and highly vascularized soft connective tissue, the dental pulp. Dentin and pulp are derived from the dental papilla, whose cells migrate to the first branchial arch from within the ectomesenchyme of the cranial neural crest. The tissues remain closely associated during development and throughout the life of an adult tooth and are hence most commonly referred to as the pulpodentin complex. It is this biologic intimacy that dictates the response of the pulpodentin complex to physiologic and pathologic stimuli.

Because the practice of endodontics involves manipulation of both the dentin and pulp, learning about the mechanisms that lead to their formation is crucial and will create a better understanding of the response to and treatment of pulpal injuries. The purpose of this chapter is to provide a background for the succeeding chapters that discuss the biology of the mature pulpodentin complex during health and disease and in aging. The chapter has two goals: The first goal is to review classic and current knowledge of the events in tooth development that lead to odontoblast differentiation and to convey the excitement of this flourishing field. Attention is focused on the common themes that have emerged and what is known about the influence of tooth-signaling molecules and transcription factors on the development and homeostasis of the pulpodentin complex. The second goal of this chapter is to describe the general principles of dentin matrix formation, particularly the synthesis and secretion of extracellular matrix molecules and their postulated roles in the biomineralization of dentin. Examples of how basic information about the normal biology of the pulpodentin complex can be applied toward solving clinical problems are integrated throughout the chapter. The overarching goal is to emphasize how fundamental theories about development and homeostasis of differentiated and undifferentiated or stem cell populations can be translated to regenerative approaches targeted at restoring the integrity of the adult pulpodentin complex.

Tooth Development (Odontogenesis)

For several decades, the developing tooth organ has served as a valuable paradigm to study the fundamental processes involved in organogenesis. These processes are (1) the determination of position, through which the precise site of tooth initiation is established; (2) the determination of form or morphogenesis, through which the size and shape of the tooth organ are set; and (3) cell differentiation, through which organ-specific tissues are formed by defined cell populations, each with unique properties. The dental literature is enriched with excellent reviews on tooth development, and the reader is encouraged to study the topic in further detail.1–4

General features

Although the tooth is a unique organ, the principles that guide its development are shared in common with other organs such as the lung, kidney, heart, mammary glands, and hair follicles.5,6 The most important among developmental events are those guiding epithelial-mesenchymal interactions, which involve a molecular crosstalk between the ectoderm and mesenchyme, two tissues that have different origins. Although only vertebrates have teeth, their development involves genetic pathways that are also active in invertebrates. This conservation of a “molecular toolbox” for organogenesis throughout evolution proves that certain master regulatory molecules are critical to all tissue interactions during development. New studies have also shown that tooth-signaling molecules are repeatedly used at various stages of development. Both tooth morphogenesis and cell differentiation occur as a result of sequential interactions. Hence, it is not one biologic event involving a single molecule but a series of interactions involving several molecules that leads to the development of the pulpodentin complex.3,4

Importantly, signaling is reciprocal, whereby an exchange of information occurs in both directions, from dental epithelium to mesenchyme and from dental mesenchyme to epithelium. For example, in experiments where dental epithelium was separated from mesenchyme, cusp patterning failed to occur. Similarly, in the absence of dental epithelium, odontoblasts are unable to differentiate from dental mesenchyme.7–9

It is only logical to apply these basic developmental principles to the current understanding of how the adult or mature pulpodentin complex responds to injury and repair. The latter clearly involves a series of molecules that operate in concert to dictate the outcome of pulpal disease and therapies. In the adult situation, whether certain cells and molecules can mimic the inductive influence of dental epithelium during development has yet to be definitively proven and remains a subject of interest in pulpal biology research.

Stages of tooth development

Teeth develop in distinct stages that are easily recognizable at the microscopic level. These stages in odontogenesis, described by the histologic appearance of the tooth organ, are termed, from early to late, the lamina, bud, cap, and bell (early and late) stages of tooth development.10–12 Although the following descriptions use these common terms, the modern literature uses functional terminology to describe odontogenesis in four phases: (1) initiation, (2) morphogenesis, (3) cell differentiation or cytodifferentiation, and (4) matrix apposition (Fig 1-1). The photomicrographs in Fig 1-2 depict the morphologic stages of tooth development.

Fig 1-1 Stages of tooth development. Note the sequential transformation from the dental lamina to a distinctly shaped dental organ. The transient appearance of the enamel knot in the region of the forming cusp tips precedes the terminal differentiation of cells and the formation of specialized matrices. (Reprinted from Thesleff and Sharpe12 with permission.)

Fig 1-2 Histologic survey of odontogenesis in a pig embryo. (Courtesy of the University of Texas Health Science Center at Houston, Dental Branch.) (a) Lamina stage: A, nasal septum; B, tongue; C, palatal shelves; D, dental lamina (hematoxylin-eosin [H&E] stain; original magnification ×4). (b) Bud stage: A, ectodermal outgrowth; B, dental mesenchyme; C, tongue; D, oral cavity space; E, oral ectoderm (H&E stain; original magnification ×10). (c) Cap stage or transition to early bell stage: A, outer dental epithelium; B, internal dental epithelium; C, stellate reticulum; D, dental papilla ectomesenchyme; E, dental lamina (H&E stain; original magnification ×10). (d) Late bell stage: A, nerve bundle; B, alveolar bone; C, vas-culature; D, oral ectoderm; E, tongue. Note the extension of the dental lamina on the right aspect of the dental organ that will form the succeda-neous incisor (H&E stain; original magnification ×10). (e) Onset of dentinogenesis: A, dental pulp; B, cluster of odontoblasts that appear crowded at the tip; C, odontoblast process; D, dentin (H&E stain; original magnification ×20).

Lamina stage

The dental lamina is the first morphologic sign of tooth development and is visible around 5 weeks of human development and at embryonic day 11 (E11) in mouse gestation. This thickening of the oral epithelium lining the frontonasal, maxillary, and mandibular arches occurs only at sites where tooth organs will develop. At the lamina stage, cells in the dental epithelium and underlying ectomesenchyme are dividing at different rates, more rapidly in the latter. As explained later, the dental lamina has the full potential to induce tooth formation by dictating the fate of the underlying ectomesenchyme.13

Bud stage

As the dental lamina continues to grow and thicken to form a bud, cells of the ectomesenchyme proliferate and condense to form the dental papilla. At this stage, the inductive or tooth-forming potential is transferred from the dental epithelium to the dental papilla.

Cap stage

At this stage, the tooth bud assumes the shape of a cap that is surrounded by the dental papilla. The ectodermal compartment of the tooth organ is referred to as the dental or enamel organ. The enamel organ and dental papilla become encapsulated by another layer of mesenchymal cells, called the dental follicle, that separates the tooth organ papilla from the other connective tissues of the jaws.

The transition from the bud to the cap stage is an important step in tooth development because it marks the onset of crown formation. Recent studies have pointed to the role of the enamel knot as an important organizing center that initiates cuspal patterning.14,15 Formally described as a transient structure with no ascribed functions, the enamel knot is formed by the only cells within the central region of the dental organ that fail to grow. As described later, the enamel knot expresses a unique set of signaling molecules that influence the shape of the crown as well as the development of the dental papilla. Similar to the fate of signaling centers in other organizing tissues, such as the developing limb bud, the enamel knot undergoes programmed cell death, or apoptosis, after cuspal patterning is completed at the onset of the early bell stage. In incisors, the enamel knot initiates the first folding of dental epithelium. Secondary enamel knots determine the site of new cusps in molars.

Early bell stage

The dental organ assumes the shape of a bell as cells continue to divide but at different rates. A single layer of cuboidal cells called the external or outer dental epithelium lines the periphery of the dental organ, while cells that border the dental papilla and are columnar in appearance form the internal or inner dental epithelium. The latter gives rise to the ameloblasts, cells responsible for enamel formation. Cells located in the center of the dental organ produce high levels of glycosaminoglycans that are able to sequester fluids as well as growth factors that lead to its expansion. This network of star-shaped cells is named the stellate reticulum. Interposed between the stellate reticulum and the internal dental epithelium is a narrow layer of flattened cells, termed the stratum intermedium, that express high levels of alkaline phosphatase. The stratum intermedium is believed to influence the biomineralization of enamel. In the region of the apical end of the tooth organ, the internal and external dental epithelial layers meet at a junction called the cervical loop.16–18

At the early bell stage, each layer of the dental organ has assumed special functions. The reciprocal exchange of molecular information between the dental organ and dental papilla influences the important events that lead to cell differentiation at the late bell stage.

Late bell stage

The dental lamina that connects the tooth organ to the oral epithelium gradually disintegrates at the late bell stage. The cells of the internal dental epithelium continue to divide at different rates to determine the precise shape of the crown. Shortly after, cells of the internal dental epithelium at the sites of the future cusp tips stop dividing and assume a columnar shape. The most peripheral cells of the dental papilla enlarge and become organized along the basement membrane at the tooth’s epithelial-mesenchymal interface. These newly differentiated cells are called odontoblasts, cells that are responsible for the synthesis and secretion of dentin matrix. At this time, the dental papilla is termed the dental pulp.

After odontoblasts deposit the first layer of predentin matrix, cells of the internal dental epithelium receive their signal to differentiate further into ameloblasts, or enamel-producing cells. As enamel is deposited over dentin matrix, ameloblasts retreat to the external surface of the crown and are believed to undergo programmed cell death. In contrast, odontoblasts line the inner surface of dentin and remain metabolically active throughout the life of a tooth.

In summary, development of the tooth rudiment from the lamina to the late bell stages culminates in the formation of the tooth crown. As root formation proceeds, epithelial cells from the cervical loop proliferate apically and influence the differentiation of odontoblasts from the dental papilla as well as cementoblasts from follicle mesenchyme, leading to the deposition of root dentin and cementum, respectively. The dental follicle that gives rise to components of the periodontium, namely the periodontal ligament fibroblasts, the alveolar bone of the tooth socket, and the cementum, also plays a role during tooth eruption, which marks the end phase of odontogenesis.

Experimental Systems

In the last decade, basic understanding of the molecules that control the events that lead to odontoblast differentiation and the formation of dental pulp has advanced significantly.19 Most of the contemporary experimental approaches used in these studies have taken advantage of the mouse model because of its availability and ease of accessibility. The development of dentition in mice closely parallels that in humans. Mice are the predominant system for genetic engineering approaches that have generated a volume of exciting data on tooth development.

Before the families of signaling molecules are discussed, it is important to understand modern experimental approaches and key techniques that are available for use in studies on tooth development. The intention is to provide a simple description of these modern scientific tools as the basic framework of reference for the dental student or endodontic resident interested in pursuing research in the area of pulpal biology.

Tooth organ culture systems

Over the years, researchers have utilized various approaches to study and manipulate developing tooth organs.20–22 In vitro systems include whole mandibular and maxillary explants as well as individually dissected molar organs that can be cultured in enriched serum by means of a Trowell-type system. The system involves placing the tooth organ in the correct orientation on a filter that is supported by a metal grid at the gas-liquid interface within a culture well23 (Fig 1-3).

Another in vitro approach is the use of functional tooth organ recombination assays. Dental epithelium is separated from papilla mesenchyme using enzymes that degrade the basement membrane at the interface.24,25 Isolated epithelium and mesenchyme can be cultured separately or recombined and then transplanted in vivo to study the effects on tooth development. Modifications of this approach include heterotypic recombinant cultures of epithelium and mesenchyme, each derived from a different organ system, and heterochronic recombinations where the tissues used are from the same organ system but at different stages in development26 (Fig 1-4). Researchers interested in studying the effects of various molecules add these reagents in soluble form to the culture and then transplant the treated culture to the anterior chamber of the eye or the subcapsular region of the kidney in mice.27 Overall, in vivo tooth organ explants that are cultured at in vivo ectopic sites advance farther than in vitro systems (Fig 1-5). In vivo culture systems are also better suited for tooth organ dissections and recombinations that are performed at early stages of development.

The availability of mice strains with spontaneous mutations and genetically engineered knockout mice has further refined the use of the bead implantation assay.30,31 When the reactions of mutant dental mesenchyme and wild-type (normal) mesenchyme are compared, it is possible to determine whether a certain molecule is needed for the expression of a second gene. This approach has led to new information about the relationships of tooth-signaling molecules within a genetic pathway.4,32

Fig 1-3 Tooth organ culture system. (Courtesy of Dr Richard Finkelman.) (a) Trowell method showing two molar organs at the early cap stage placed on a filter on top of a metal grid within a culture dish. (b) Molar organ after 12 days in culture. Note the formation of distinct cusps. (c) Histologic view of Fig 1-3b shows fully differentiated odontoblasts (od) and a layer of mineralized dentin (arrows) (Von Kossa stain; original magnification ×10).

Fig 1-4 Strategy used for homotypic and heterotypic recombination assays. E11–12, days 11 to 12 of mouse embryonic development; e, epithelium; m, mesenchyme; h, hours in culture; d, days in culture. (Reprinted from Mitsiadis et al26 with permission.)

Fig 1-5 Microscopic view of a fully formed tooth that developed from the early cap stage after placement beneath the renal capsule. od, odontoblasts; p, dental pulp; k, kidney (H&E stain; original magnification ×4).

An elegant experimental approach that has yielded important information on the nature of the signaling interactions between tooth epithelium and mesenchyme is the use of bead implantation assays.28 Briefly, either heparin or agarose beads that are soaked in a known concentration of a growth factor are placed on separated dental mesenchyme. After approximately 24 hours in culture, the mesenchyme is analyzed for changes in gene or protein expression in the region surrounding the bead29 (Fig 1-6).

Fig 1-6 Principles of tooth tissue recombination and bead assays. (Modified from Thesleff and Sahlberg29 with permission.)

Odontoblast and dental pulp cell lines

While tooth organ cultures have facilitated elegant studies of early tooth development, the recent availability of odontoblast and pulp cell culture systems have made it possible to study late events that involve cell differentiation and matrix synthesis. An interesting approach is to utilize hemisectioned human teeth from which dental pulp has been carefully extirpated. The remaining layer of intact odontoblasts can then be cultured within the native pulp chamber, to which nutrient media and various growth factors or cytokines are added. Thick slices of human teeth with the odontoblastic layer left intact offer another useful approach to study the behavior of odontoblasts under conditions that simulate dental caries.33,34 The use of primary odontoblast cultures has been limited because intact cells are difficult to isolate in sufficient numbers and become phenotypically altered after several passages in culture. The recent development of cell-immortalization procedures has made it possible to generate two established odontoblast-like cell lines. The M06-G3 cell line was derived from an established murine odontoblast monolayer cell culture system that was infected with a temperature-sensitive simian virus 40 (SV40).35 MDPC-23 cells are a spontaneously immortalized cell line derived from mouse embryonic dental papilla that expresses dentin matrix proteins.36 Dental pulp clones, the RPC-C2A and RDP 4-1 cell lines, which exhibit characteristics ranging from pulpal fibroblasts to preodontoblasts, are also available.37,38

Tooth-derived stem cell lines

Central to the development of new regenerative strategies involving tissue engineering is the use of stem cells. These cells are classically defined as clonogenic and self-renewing cells that have the capacity to differentiate into multiple cell lineages. The two major types are embryonic stem cells and postnatal stem cells. Embryonic stem cells are obtained from embryos in the blastocyst stage of development. At this stage, these stem cells are pluripotent with the ability to differentiate into any cell type. In contrast, postnatal stem cells reside within niches in adult organs and retain the capacity to differentiate into a limited number of cell types.

Recently, stem cell characteristics were detected in cells isolated from primary39 as well as permanent teeth,40,41 periodontal ligament,42,43 periapical follicle, and apical papilla mesenchyme.43–45 Mesenchymal stem cells, which represent less than 10% of the total cell population, can be tagged with stem cell markers such as STRO-1 or CD 146 in fluorescence-activated cell-sorting analysis.39 Stem cells isolated from dental tissues are capable of differentiating into adipocytes, neurons, and odontoblast-like cells.39

They form mineralized nodules in vitro43 and are capable of creating boneor pulpodentin-like complexes after transplantation into immunocompromised mice.39,41 These cells appear to reside in a perivascular niche and are likely to represent the population of cells that were shown by Fitzgerald et al46,47 to migrate to the site of dentin injury to form reparative dentin. Tooth-derived stem cells are essential for the development and application of regenerative approaches to treat an injured pulpodentin complex (see chapter 5).

Figure 1-7 depicts the activity of two tooth-derived cell lines: stem cells from human exfoliated deciduous teeth (SHED) and periodontal ligament–derived stem cells (PDLSC). When seeded in a fibrin hydrogel system, both cell lines are capable of proliferation and further differentiation, as indicated by the deposition of a collagen-enriched matrix.

Fig 1-7 Osteogenic induction of stem cells increases the production of extracellular matrix such as collagen (blue staining). (a) SHED cells in a fibrin patch, treated with bone morphogenetic protein 4 (BMP-4) (Masson trichrome stain; original magnification ×60). (b) PDLSCs (cells provided by Dr Songtao Shi) in fibrin patch, treated with BMP-4 (Masson trichrome stain; original magnification ×30).

Transgenic and knockout mice

The modern era of recombinant DNA technology and genetic engineering has made it possible to alter or mutate a gene of interest in vitro and then inject it into the pronucleus of a fertilized mouse egg.48,49 The transgene, if successfully integrated into the host genome, can be transmitted through the germ line to the progeny. Transgenic and knockout mice thus offer a powerful means to study the role of molecules in their natural in vivo environment.

Transgenic mice generated using conventional technology can be designed to overexpress the gene of interest in cells or tissues where it is normally expressed50 (Fig 1-8). When it is desirable to study the behavior of a gene at an ectopic site, the transgene of interest is placed behind the promoter of another gene that will drive expression in tissues where it is not normally expressed. In the case of a gene that is expressed in multiple tissues or organs, it is now possible to study activity at one particular site. This is achieved by driving the expression of the transgene using a tissue- or cell-specific promoter.

The following example illustrates the usefulness of a tissue-specific transgenic mouse model. As a means of assessing the precise role of transforming growth factor β1 (TGF-β1) in odontoblast differentiation, transgenic mice that overexpressed active TGF-β1 were generated. Because TGF-β1 is also highly expressed in bone and other tissues, TGF-β1 over-expression was restricted to odontoblasts alone by using the promoter for dentin sialophosphoprotein (Dspp), a gene that is highly tooth specific. TGF-β1 overexpressors have defects in dentin that closely resemble dentinogenesis imperfecta (DI), an inherited disorder of dentin. This model permitted the analysis of the direct role of TGF-β1 in dentin formation without confounding its effect in bone and other tissues.51 The results indicated an important role for the growth factor in dentin formation, a subject that is discussed in further detail in chapter 2.

Knockout technology is used to generate lines of mice that lack a functional gene of interest throughout their life span. The targeted deletion of the gene is performed in embryonic stem cells that are derived from the inner cell mass of an early embryo. After these cells are cultured in a petri dish, to select for the desired deletion, they are implanted in the cavity of a fertilized egg to generate a percentage of offspring that inherit the mutation. Knockout mice offer a powerful means to assess the biologic roles of a molecule in the context of a normal mouse. Certain knockout strains appear completely unaffected, indicating that the functions of the gene that are eliminated in vivo can be shared by other genes within the family, a phenomenon of biologic (functional) redundancy.

Several knockout mice strains die during gestation or shortly after birth, indicating the importance of these genes in developmental processes. As a result, these mice have not proven to be informative about the role of the gene later in postnatal and adult life. Examples of this include the bone morphogenetic proteins 2 (Bmp2) and 4 (Bmp4) gene knockout mice that undergo embryonic lethality during early gestation. This problem is overcome by the use of conditional knockout technology, where the inactivation of the gene occurs at a specific time and location.

Of relevance to pulpal biology is the phenotype of the Tgfβ1-mutant mouse. Teeth develop fully in Tgfβ1–/– mice and show no pathologic conditions at birth or through the first 7 to 10 days of postnatal life compared to those of Tgfβ1+/– or Tgfβ1+/+ littermates. By the end of the second week, Tgfβ1–/– mice develop a rapid wasting syndrome that is characterized by multifocal inflammatory lesions with dense infiltration of lymphocytes and macrophages in major organs such as the heart and lungs; these eventually lead to death by the third week of life. These observations support a vast volume of research documenting a critical role for TGF-β1 as a potent immunomodulator.

To study the state of the adult dentition in Tgf-β1–/– mice, their survival was prolonged with dexamethasone treatment. The absence of a functional Tgfβ1 gene resulted in significant destruction of pulp and periradicular tissues as well as the hard tissues of the crown52 (Fig 1-9). These data prove that no other TGF-β family members can substitute for the loss of TGF-β1. Clearly, the dual role of this growth factor as a key modulator of pulpal inflammation and its potent effects on extracellular matrix (ECM) production are significant. Chapter 2 discusses in further detail the role of TGF-β1 in reparative dentinogenesis.

Fig 1-8 (a) Whole-mount view of a developing transgenic mouse embryo in which expression of the LacZ reporter gene (blue staining) is driven by a type I collagen promoter. Expression of ß-galactosidase is seen in all areas of the embryo that express type I collagen. mn, meninges (ß-gal stain). (Reprinted from Niederreither et al50 with permission.) (b) Section through the developing incisor at the neonatal stage showing activity of the transgene in differentiating odontoblasts (od) and some cells of the dental pulp (dp). Note the complete absence of staining for type I collagen in ameloblasts (am) (H&E stain; original magnification ×10).

Fig 1-9 (a) Normal mandibular first molar in a Tgfβ1+/+ mouse at 50 days of postnatal life. d, dentin; dp, dental pulp (H&E stain; original magnification ×10). (Reprinted from D’Souza et al52 with permission.) (b) In a Tgfβ1–/– animal whose survival was prolonged with dexamethasone, there is extensive damage to the hard and soft tissues of the molar. arrowheads, periapical inflammatory infiltrates; star, calcification within the pulp chambers and canals (H&E stain, original magnification ×10). (Reprinted from D’Souza et al52 with permission.)

Laser capture microdissection

Among the new in vivo approaches available to analyze the behavior of cells under normal and diseased conditions, laser capture technology stands out as being most innovative. Laser capture microdissection (LCM) was developed and applied in cancer biology to detect a mutant protein or gene in a single malignant cell53 and to monitor in vivo differential gene expression levels in normal and malignant breast cell populations.54 LCM is being used in the Cancer Genome Anatomy Project to catalog the genes that are expressed during solid tumor progression and to construct complementary DNA (cDNA) libraries from normal and premalignant cell populations. Microarray panels55 containing these index genes are being used to obtain gene-expression patterns in human tissue biopsies. The fluctuation of expressed genes that correlate with a particular stage of disease is compared within or between individual patients. Such a fingerprint of gene-expression patterns will provide important clues about etiology and contribute to diagnostic decisions and therapy.

Applications in this area will be particularly useful for oral tissues, such as dental pulp, oral mucosa, and periodontal ligament, where individual cell populations are difficult to access. The progress in understanding odontoblast differentiation has been slow because of serious limitations inherent to both in vivo and in vitro approaches. Pure populations of differentiating or mature odontoblasts are technically difficult to obtain from heterogenous dental papilla and pulp. Furthermore, immortalized odontoblast-like cell lines fail to fully reflect the molecular events that occur in the complex milieu of the tooth organ from which they are derived. The terminology used to describe odontoblast differentiation is sketchy because it is unclear how morphologic change is reflected at the molecular cytogenetic level.

Initial studies of dentin ECM gene expression in differentiating odontoblasts have been promising (Fig 1-10). Data from the future use of LCM will provide a correlation between the morphologic changes and the expression of known ECM genes during odontoblast differentiation. Information generated from this approach will also be valuable in developing a nomenclature that can be consistently used by researchers. Moreover, known and unknown genes will be identified from the developmentally staged, odontoblast-specific cDNA libraries. Genes that are defined for each stage of primary dentin formation will provide important clues about temporal patterns of gene expression and the potential functions of encoded protein products in dentin mineralization. Such fundamental information will be useful in characterizing cells within the cell-rich zone of dental pulp, identifying the replacement population of pulpal cells involved in reparative dentin formation, and developing vital pulp therapies aimed at hastening the healing of the injured pulpodentin complex.

Fig 1-10 Preliminary reverse transcriptase–polymerase chain reaction (RT-PCR) amplification of type I collagen from odontoblasts retrieved by laser capture microdissection. (a) Thick, stained frozen section through the mesial cusp tip of a first molar from a newborn mouse prior to laser capture. od, odontoblasts (H&E stain; original magnification ×10). (b) Outline of a zone of odontoblasts cut by the laser beam (H&E stain; original magnification ×10). (c) Hole created in tissue after catapulting of cells into PCR tube (H&E stain; original magnification ×10). (d) RT-PCR reaction showing type I collagen gene expression in odontoblasts at embryonic day 15.5 (E15.5), day 0 (D0), and day 3 (D3) of development. M, DNA markers; Control, MDPC-23 odontoblast-like cells.

Signaling Interactions that Influence Odontoblast Differentiation and Dental Pulp Formation

The combined use of conventional tooth organ culture and recombination techniques, as well as the application of modern molecular and genetic approaches, has significantly advanced current understanding of the genes responsible for tooth initiation and morphogenesis. Readers can access an informative internet site56 for a current cataloging of all the molecules that are expressed in tooth organs.

The two principal groups of molecules that are involved in the reciprocal exchange of information between tooth epithelium and mesenchyme are transcription factors and growth factors. Transcription factors are proteins that bind to DNA near the start of transcription of a gene. They regulate gene expression by either facilitating or inhibiting the enzyme RNA polymerase in the initiation and maintenance of transcription. Transcription factors are rarely found in high amounts and are not secreted outside the cell. In general, they perform critical cell or tissue-specific functions. Mutations involving transcription factors often result in defects of tooth formation.

Growth factors are secreted proteins that are capable of binding to specific receptors on the cell surface. Subsequent interaction with both membrane and cytoplasmic components leads to a complex series of intracellular events (signal transduction) that result in altered gene expression. These changes activate cell growth and differentiation. Most growth factors are synthesized at higher levels than transcription factors and perform versatile functions. In many instances, the functions of one growth factor overlap with those of a related family member so that loss of function can be compensated for by biologic redundancy.

Molecular changes in dental mesenchyme are affected by the following families of molecules (Fig 1-11): the bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and WNT family; sonic hedgehog (SHH) as well as transcriptional molecules such as the Msx1 and Msx2 homeobox genes; lymphoid enhancer-binding factor 1 (Lef1); and Pax9, a member of the paired-box–containing transcription factor gene family. The actions and interactions of these molecules are complex and described eloquently in recent reviews.1,4,12 The following discussion captures selected highlights.

The BMPs are among the best-characterized signals in tooth development. In addition to directly influencing morphogenesis of the enamel organ (see the discussion on enamel knots later in the chapter), epithelial BMP-2 and BMP-4 are able to induce expression of Msx1, Msx2, and Lef1 in dental mesenchyme, as shown in bead implantation assays.28,30,57,58 The shift in Bmp4 expression from epithelium to mesenchyme occurs around E12 and is coincident with the transfer of inductive potential from dental epithelium to mesenchyme.28 In mesenchyme, BMP-4 in turn requires Msx1 to induce its own expression.30Figure 1-12 summarizes the experiments performed on the role of the BMPs in dental mesenchyme.

The FGFs, in general, are potent stimulators of cell proliferation and division both in dental mesenchyme and epithelium. Expression of FGF-2, -4, -8, and -9 is restricted to dental epithelium and can stimulate Msx1 but not Msx2 expression in underlying mesenchyme. Fgf8 is expressed early in odontogenesis (E10.5 to E11.5), in presumptive dental epithelium, and can induce the expression of Pax9 in underlying mesenchyme. Interestingly, BMP-4 prevents this induction and may share an antagonistic relationship with the FGFs similar to that observed in limb development.59

The expression of SHH, a member of the vertebrate family of hedgehog signaling proteins, is limited to presumptive dental epithelium. Recent studies by Hardcastle et al60 have shown that SHH protein in beads cannot induce Pax9, Msx1, or Bmp4 expression in dental mesenchyme but is able to stimulate other genes that encode patched (Ptc), a transmembrane protein, and Gli1, a zinc finger transcription factor. Because neither FGF-8 nor BMP-4 can stimulate the genes Ptc or Gli1, it can be assumed at the present time that the SHH signaling pathway is independent of the BMP and FGF pathways during tooth development.60

Several Wnt genes are expressed during tooth development and may be required for the formation of the tooth bud.12 These genes are believed to play a role in activating the intracellular pathway involving frizzled receptors, β-catenin, and nuclear transport of LEF-1. Other signaling molecules, including the Notch genes and the epidermal growth factor, hepatocyte growth factor, and platelet-derived growth factor families, may also influence tooth development, although the exact nature of their involvement remains to be elucidated.61,62

Mice genetically engineered with targeted mutations in transcription factor genes such as Msx1, Lef1, and Pax9, as well as activin-βA, a member of the TGF-β superfamily, have revealed important information. Knockouts of Bmp2, Bmp4, and Shh have proven less informative, largely because death occurs in utero prior to the onset of tooth development. In Msx1-, Lef1-, Pax9-, and activin-βA–mutant strains, tooth development fails to advance beyond the bud stage. Thus, these molecules are important in directing the fate of the dental mesenchyme and its ability to influence the progress of epithelial morphogenesis to the cap stage.63–66 Exciting discoveries in the field of human genetics have shown that mutations in MSX1 and PAX9 are associated with premolar and molar agenesis, respectively67–70 (Frazier-Bowers and D‘Souza, unpublished data; Fig 1-13). These findings illustrate the importance of animal models in studies of human disease (Figs 1-14 and 1-15).

More recently, mice lacking an important osteoblast-specific transcription factor, Runx2 (formerly known as core binding factor a1[Cbfa1]), were shown to completely lack osteoblast differentiation and bone formation.71,72 Of interest to clinicians is the fact that mutations in Runx2 cause cleidocra-nial dysplasia, an inherited disorder in humans that is characterized by open fontanels in the skull, defective clavicles, multiple supernumerary teeth that fail to erupt, and various tooth matrix defects.73Runx2–/– molar organs arrest at the late cap to early bell stage of development and appear hypoplastic and misshapen. Runx2-mutant incisors showed defective odontoblasts and highly dysplastic dentin74 (see Fig 1-14).

One theory is that this transcription factor serves multiple functions in mineralizing tissue organs.75–77 In addition to playing an essential role in osteoblast differentiation, it is likely that Runx2 conditions the dental papilla mesenchyme to become responsive to epithelial signals. Once the molecular trigger from the enamel organ reaches the peripheral zone of dental papilla cells, Runx2 is downregulated in dental papilla and odontoblast differentiation ensues. Thus, the presence of Runx2 in dental papilla can be viewed as a limiting factor of odontoblast differentiation; its presence in dental papilla at the bud and cap stages of odontogenesis has an osteogenic-like influence, while its removal from dental papilla triggers terminal events in odontoblast differentiation.

More recent studies have shown that the ability of Runx2 to activate osteoblast differentiation is regulated by an antagonistic partner, Twist-1, a basic helix-loop-helix–containing nuclear protein factor.78 Twist-1 functions as a cell survival factor and an inhibitor of cell differentiation, mineralization, and apoptosis.79,80 Through its interaction with Runx2, a cell differentiation factor, Twist-1 controls the onset of osteoblast differentiation.81 Decreased levels of Twist-1 in mice result in premature odontoblast differentiation as measured by the earlier onset of expression of ECM gene markers and the formation of dentin matrix (see Fig 1-15).

Our results also indicate that Twist-1 deficiency distinctly affects odontoblast-like cells, a population known to exist in adult dental pulps. These cells become more responsive to Runx2 when Twist-1 levels are decreased, leading to the formation of pulp stone–like deposits within the pulp core. In mice that lack both Runx2 and Twist-1 concurrently, the pulp appears free of ectopic calcifications (see Fig 1-15). Taken together, these findings support the hypothesis that Twist-1 is important in the control of the terminal events that lead to odontoblast differentiation and in maintaining homeostasis in dental pulp.

Multiples lines of evidence suggest that the process of terminal differentiation of odontoblasts is dependent on a vertical gradient of positional cues from overlying dental epithelium. Hence a morphogenetic gradient exists, so that the most differentiated cells align at the tooth epithelial-mesenchymal interface and less-differentiated cells toward the core of the papilla mesenchyme. This theory can explain why adult dental papilla cells have the capacity to differentiate into mineralizing cells when provided with the appropriate signal.

While the precise nature of the molecular events leading to the terminal differentiation of odontoblasts remains unknown, data from dentin repair studies have shown that the matrix deposited after injury to dentinal tubules and pulp resembles osteoid rather than tubular dentin. This can be explained by the fact that cells of the dental papilla share a common developmental niche with surrounding osteogenic mesenchyme and that the presence of specific dental epithelial signals results in further lineage diversification of osteoblast-like cells into odontoblasts. Because the absence of the permissive influences required for differentiation of “true” odontoblasts is lacking in an adult tooth, cells engaged in the process of reparative dentin formation retain the osteoblastic phenotype and secrete a matrix that more closely resembles bone than dentin.82,83

Fig 1-11 Molecules (transcription factors, growth factors, and other proteins) involved in epithelial-mesenchymal signaling interactions during tooth development. Little is known about molecules that influence the latest stages of terminal differentiation and tooth eruption. Note the time of arrest of tooth development in knockout mice that lack important transcription factors. (Reprinted from Jernvall and Thesleff4 with permission.)

Fig 1-12 BMP, as shown by expression within the developing tooth organ in situ hybridization pictures that have been digitized and processed. Red dots represent messenger RNA transcripts. (Reprinted from Thesleff and Sharpe12 with permission.) (a) Bmp2 gene expression is highly restricted to the dental lamina. (b) Bmp7 is coexpressed in the thickened dental epithelium. (c) A bead that releases BMP-2 protein is capable of stimulating Msx1 expression in dental mesenchyme. (d) A control bead that has been soaked in bovine serum albumin is not capable of stimulating Msx1 expression.

Fig 1-13 Role of PAX9 in the formation of the posterior human dentition. (a) Panoramic radiograph of a normal dentition in an unaffected family member. (b) Panoramic radiograph of an affected individual who is missing molars (arrows). (c) Insertion mutation of a single nucleotide, guanine, at residue 219. This defect caused a frameshift and a premature truncation site that resulted in a defective protein (compare the bottom drawing to the normal protein in the center) that could not function like a normal PAX9 protein.

Fig 1-14 Bone and tooth phenotype in mice genetically engineered to lack a functional Runx2 (Cbfa1) gene. (a) Coronal section through the molar region of a Cbfa1+/+ newborn mouse, revealing normal molar development (arrows) and formation of the alveolus (H&E stain; original magnification ×4). (b) Extremely hypoplastic molar organs and a complete lack of bone in a Cbfa1–/– mouse. arrows, knockout tooth organs (H&E stain; original magnification ×4). (c) High-magnification view of a normal Cbfa1+/+ first molar (H&E stain; original magnification ×10). (d) High-magnification view of a Cbfa1-mutant molar, which lacks normal cusp formation (H&E stain; original magnification ×10). (e) Newborn Cbfa1–/– mouse with incisor organs that show defects in odontoblast differentiation and dentin formation. The dentin (asterisk) resembles an osteodentin matrix (H&E stain; original magnification ×10). (f) Whole-mount staining reveals the absence of bone (red staining) in the mutant (top) compared to a normal littermate (bottom) (alizarin red stain for bone; alcian blue stain for cartilage; original magnification ×2). (Reprinted from the cover of Cell, volume 89, May 1997, with permission.)

Fig 1-15 Genetic rescue experiments where Twist+/– animals at later stages show matrix deposits (arrows) and dentin-like structures within the pulpal tissues in incisors (a) and molars (b); these cannot be found in Twist+/– × Runx2+/– compound double heterozygous animals in incisors (c) and molars (d) (H&E stain; original magnification ×10). (Reprinted from Galler et al71 with permission.)

Enamel knots as signaling centers for cuspal morphogenesis

For more than a century, enamel knots were described as histologically distinct clusters of epithelial cells located first in the center of cap stage tooth organs (primary) and then at the sites of future cusp tips (secondary). For years, it was speculated that these structures, appearing only transiently in odontogenesis, controlled the folding of the dental epithelium and hence cuspal morphogenesis. Recently, the morphologic, cellular, and molecular events leading to the formation and disappearance of the enamel knot have been described, thus linking its role to that of an organizing center for tooth morphogenesis. 15,84,85

The primary enamel knot appears at the late bud stage, grows in size as the cap stage is reached, and is no longer visible at the early bell stage (Fig 1-16). Cells of the enamel knot are the only cells within the enamel organ that stop proliferating14 and eventually undergo programmed cell death.86 Another intriguing finding has linked p21, a cyclin-dependent kinase inhibitor that is associated with terminal differentiation events, to apoptosis of the enamel knot.85

Although morphogens such as BMP-2, -4, and -7,74 FGF-9,87 and SHH are expressed variantly throughout tooth morphogenesis, their colocalization within the primary enamel knot is strongly suggestive of its role as an organizing center for tooth morphogenesis. Notably, Fgf4 is exclusively expressed in the enamel knot,14,88 either singly or in concert with Fgf9, to influence patterning or to regulate expression of downstream genes such as Msx1 in underlying papilla mesenchyme. Because the instructive signaling influence lies with the dental mesenchyme prior to the development of the primary enamel knot, it is reasonable to assume that the dental mesenchyme is involved in the regulation of enamel knot formation. It is highly likely that signals from the enamel knot area influence gene expression in an autocrine and paracrine fashion, thus influencing the fate of the enamel organ and the dental papilla.

Fig 1-16 (a) Role of the enamel knot as a signaling center for morphogenesis of the tooth cusp. (Courtesy of Dr I. Thesleff.) (b) 5-Bromo-2’-deoxyuridine (BrdU) labeling identifies cells in the putative enamel knot (ek) that are negative for the stain and have left the cell cycle. (Reprinted from Thesleff and Sharpe12 with permission.) (c) Same cell within the enamel knot expresses high levels of SHH. (Reprinted from Thesleff and Sharpe12 with permission.) (d) Same cell within the enamel knot expresses high levels of FGF-4. Three-dimensional reconstructions of serial sections (b to d) illustrate the shape of the enamel knot. (Reprinted from Thesleff and Sharpe12 with permission.)

Role of the extracellular matrix in tooth morphogenesis and cytodifferentiation

Remodeling of the ECM is an important feature of epithelial morphogenesis, especially in branching organs such as salivary and mammary glands.5,89 The ECM also regulates morphogenetic functions in a variety of craniofacial tissues.90 Results of several functional in vitro studies have shown that the integrity of the ECM, in particular the basement membrane, influences the budding and folding of dental epithelium during morphogenesis and the spatial ordering of cells that undergo terminal differentiation.9,91,92 Molecules such as collagen types I, III, and IV, along with laminin and various proteoglycans, are differentially expressed in the basement membrane at the epithelial-mesenchymal interface of the tooth.9,93,94 The precise nature of the molecular interactions that influence morphogenesis at this dynamic interface are unknown.

The presence of matrix metalloproteinases (MMPs) has been linked with the morphogenesis of several epithelial-mesenchymal organs, including teeth.95,96 Studies by Sahlberg et al97 showed that gelatinase A (ie, MMP-2), an MMP that cleaves type IV collagen and increases in odontoblasts shortly after cuspal morphogenesis, contributes to the degradation of the basement membrane. The expression of protease inhibitors, tissue inhibitors of metalloproteinases (TIMPs) 1, 2, and 3, also correlates with tooth morphogenesis.98

Odontoblast Differentiation

Odontoblast differentiation is initiated at the cusp tip in the most peripheral layer of dental papilla cells that align the epithelial-mesenchymal interface and follows three steps: (1) induction, (2) competence, and (3) terminal differentiation. Inductive signals from the internal epithelial cells most likely involve members of the TGF-β family (BMP-2, BMP-4, and TGF-β1) that become partially sequestered in the basal lamina to which peripheral cells of the dental papilla become aligned. Competence is achieved after a predetermined number of cell divisions are completed and cells express specific growth factor receptors. In the final round of cell division, only the most peripheral layer of cells subjacent to the basal lamina responds to the signals from the internal dental epithelium to become fully differentiated into odontoblasts. The subodontoblastic layer of cells thus represents dental papilla cells that are competent cells exposed to the same inductive signals as differentiated odontoblasts except the final one (Fig 1-17).

Based on the information presented so far (see Fig 1-11), it is clear that considerable progress has been made in understanding the molecular events preceding the terminal differentiation of odontoblasts. However, the final determinants of odontoblast differentiation remain to be characterized.

As is well documented in the literature, fully differentiated odontoblasts are postmitotic cells that are morphologically distinct from cells of the dental pulp. As differentiation proceeds in an apical direction, these cells change their shape, which ranges from round to cuboidal, to a tall columnar appearance. On the subcellular level, cells acquire a synthetic and secretory apparatus by developing an extensive rough endoplasmic reticulum and Golgi apparatus along with numerous lysosomes. To accommodate these organelles and to prepare for the secretion of dentin matrix components in an apical and unidirectional manner, the nucleus moves to the opposite pole of the cell in a position opposite to the inner dental epithelial cells. Nuclear repolarization is one of the hallmarks of terminal odontoblast differentiation.

Fig 1-17 Terminal events in odontoblast differentiation. (a) Undifferentiated mesenchymal cells. (b) Committed dental mesenchymal cells that are in a state of mitosis or cell division. (c) Inner dental epithelium, which is important for driving the differentiation of cells nearest the basement membrane. (d) Basement membrane. (e) Daughter cells that are competent to become odontoblasts remain in the peripheral zone of the dental papilla. (f) Differentiated odontoblasts with a polarized nucleus and cytoplasmic extensions. (g) Subodontoblastic cells.

Dentin Matrix Proteins and the Biomineralization of Dentin

The formation of dentin follows the same principles that guide the formation of other hard connective tissues in the body, namely, cementum and bone. In dentinogenesis, the first requirement is the presence of highly specialized cells, termed odontoblasts, that are capable of synthesizing and secreting type I collagen–rich unmineralized ECM, referred to as predentin, which is subsequently mineralized when apatite crystals are deposited. As the odontoblasts build this type I collagen–rich fibrillar matrix, they recede in a pulpal direction and leave behind odontoblastic processes, through which these cells remain connected to the mineralized matrix.

The formation of predentin and its transformation to dentin as mineralization takes place are highly controlled, orderly processes. For example, under normal conditions of growth, the predentin width is rather uniform, not random, indicating that the rates of synthesis of unmineralized matrix and its conversion to dentin at the predentin-dentin border must be equal (Fig 1-18). On the other hand, in pathologic conditions such as DI, the widening and disorganization of the predentin layer indicate an incapacitation of this process.

The overall process of dentinogenesis involves a series of events that apparently begin at the boundary of the odontoblast cell body with the matrix and continue until the mineralization process is complete. In the past five decades, researchers in the biomineralization field have attempted to answer the following questions:

• What is the exact composition of dentin matrix and what biochemical features distinguish dentin from bone and cementum?

• Are there dentin-specific markers and can they be used to characterize the nature of the replacement cells responsible for forming reparative dentin?

• How do the physical features and conformational structures of ECM molecules facilitate the calcification of dentin?

• Do these macromolecules interact with each other during the mineralization of dentin?

• Do these macromolecules form supramolecular complexes that promote the deposition of hydroxyapatite crystals?

• What is the nature of the ECM molecules that modulate the initiation, rate, and extent of dentin deposition?

• What is the nature of the genes that encode for dentin ECM molecules?

• Are defects in these genes responsible for the inherited dentin disorders, namely DI and dentin dysplasia?

• What genes regulate the expression of key dentin ECM molecules?

Based on new research advances made in understanding dentinogenesis, valuable insights have been gained about the unique roles of these proteins in controlling the process of biomineralization. For example, ultrastructural studies on the collagen fibrils formed by rat incisor odontoblasts demonstrated that collagen fibrils progressively thicken from the time they are secreted until they are mineralized at the predentin-dentin border100; these observations along with many others101–103 indicate that the transition from the zone of predentin immediately outside the bodies of the odontoblasts to the area adjacent to dentin represents a gradient of events, a maturation process that is dynamic. Another example is the important discovery that mutations in dentin sialophosphoprotein (DSPP) genes are responsible for the underlying dentinal defects seen in DI type II and DI type III.104–107 These conditions affect a large number of individuals worldwide and are characterized by severe defects in dentin mineralization in both primary and permanent dentitions (Fig 1-19).

More than 90% of the organic component in dentin matrix is type I collagen (Fig 1-20). The importance of the correct collagen structure in dentin formation is clearly seen in patients with DI type I, caused by mutations in the type I collagen gene,108 which clinically resemble DI types II and III, caused by DSPP mutations.

In addition to type I collagen, the ECM of dentin contains a number of noncollagenous proteins (NCPs) and proteoglycans (Table 1-1). One category of the NCPs that are principally found in dentin and bone and are secreted into the ECM during the formation and mineralization of these tissues is termed the small integrin-binding ligand, N-linked glycoprotein (SIBLING) family, which includes dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), bone sialoprotein (BSP), osteopontin (OPN), and matrix extracellular phosphoglycoprotein (MEPE).109