reading

Cover image illustrating the innervation of normal human dental pulp using antibodies for neurons (N52, green; PGP, blue) and the receptor TRPA1 (red). Image courtesy of Michael A. Henry, 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

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

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

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

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19 Differential Diagnosis of Toothache: Odontogenic Versus Nonodontogenic Pain

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.

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.

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

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.

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.

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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.)

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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).

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

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.

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.

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.

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.

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.

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

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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).

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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.)

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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).

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

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.

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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).

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.

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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).

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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.)

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.

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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).