J Dent Res 83(12):896-902, 2004
© 2004 International and American Associations for Dental Research
Parallels between Tooth Development and Repair: Conserved Molecular Mechanisms following Carious and Dental Injury
T.A. Mitsiadis1,*, and
C. Rahiotis2
1 Department of Craniofacial Development, Floor 27, GKT Dental Institute, King’s College, Guy’s Hospital, London Bridge, London SE1 9RT, UK; and 2 Department of Oral Biology, Dental Institute, University of Athens, Thivon 2 Street, 115 27, Goudi, Athens, Greece;
* corresponding author, thimios.mitsiadis{at}kcl.ac.uk
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ABSTRACT
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The reparative mechanisms that operate following carious and traumatic dental injury are critical for pulp survival and involve a series of highly conserved processes. It appears that these processes share genetic programs—linked to cytoskeletal organization, cell movement, and differentiation—that occur throughout embryogenesis. Reactionary dentin is secreted by surviving odontoblasts in response to moderate stimuli, leading to an increase in metabolic activity. In severe injury, necrotic odontoblasts are replaced by other pulp cells, which are able to differentiate into odontoblast-like cells and produce a reparative dentin. This complex process requires the collaborative efforts of cells of different lineage. The behavior of each of the contributing cell types during the phases of proliferation, migration, and matrix synthesis as well as details of how growth factors control wound cell activities are beginning to emerge. In this review, we discuss what is known about the molecular mechanisms involved in dental repair.
KEY WORDS: tooth • injury • odontoblast • dentin • nestin • Notch • cadherin
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INTRODUCTION
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Tooth development involves a series of reciprocal interactions between the oral epithelium and cranial neural-crest-derived mesenchymal cells. These interactions progressively lead to transformation of the tooth primordia into complex mineralized structures. Mesenchymal cells form the dental follicle and dental pulp. The last division of pulp cells coming into contact with the basement membrane gives rise to two daughter cell populations: odontoblasts and cells of the sub-odontoblastic layer (Höhl cells). Signaling molecules expressed in dental epithelium and captured in the basement membrane control the differentiation of pulp cells into odontoblasts (Ruch et al., 1995). Odontoblasts produce the extracellular matrix components found in dentin and are implicated in dentin mineralization.
The hard tissues of the tooth provide a barrier against bacteria. When a traumatic injury or a caries lesion breaks down this barrier, repair takes place to prevent invasion of the pulp chamber by bacteria. The capacity for pulp cells to resist and repair injuries is fundamental to maintenance of the integrity and homeostasis of the dental organ. In the adult pulp, cell division and the secretory activity of odontoblasts are limited, but these processes may be re-activated after injury. The most common and well-known feature of pulp repair is the formation of tertiary dentin, which is distinguished by reactionary and reparative dentin (reviewed in both of these two cited reviews, Tziafas et al., 2000; Goldberg and Smith, 2004). Reactionary dentinogenesis generally follows a mild injury (i.e., caries lesions of only moderate progression), when odontoblasts survive after the stimuli. Reparative dentinogenesis occurs after more intense injury (e.g., deep cavity preparation) that leads to odontoblast death. The formation of reparative dentin results from the recruitment and proliferation of pulp cells, which can have stem cell properties (Gronthos et al., 2000). Pulp cells are attracted to the injury site and differentiate into a second-generation of odontoblasts or odontoblast-like cells. Signaling molecules that are expressed by pulp cells (e.g., Bone Morphogenetic Proteins or BMPs, Transforming Growth Factors-beta or TGFß) could play a role in both the homeostasis of a healthy pulp and the pulp healing during dental repair (reviewed in Smith and Lesot, 2001).
Little is known of the molecular mechanisms involved in dental healing and, most notably, the recruitment and differentiation of pulp cells into odontoblast-like cells. To obtain a better understanding of the molecular cascades controlling dental repair, we will probably need to use information from genetic studies on the control of tissue migration and differentiation during embryonic tooth development. Recent findings have shown that some molecular mechanisms are common to dental development and repair (About et al., 2000, 2002; Smith and Lesot, 2001; Heymann et al., 2002; Mitsiadis et al., 2003), indicating that parallels exist between wound repair and morphogenesis in the embryo.
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MOLECULAR REGULATION OF NORMAL ODONTOBLAST DIFFERENTIATION
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To provide a clear understanding of pulp regeneration and dental repair in humans, we will review mainly recent findings where human teeth were used as the experimental model. These studies focus on molecules, other than growth factors, that are involved in cell activities as diverse as cell fate specification, cell adhesion, and cytoskeletal organization, which may influence pulp cell behavior from the initial stages of tissue repair to the terminal phase of cell differentiation and dentin deposition.
The Notch pathway is an evolutionarily conserved signaling mechanism that enables adjacent cells to adopt different fates (Artavanis-Tsakonas et al., 1999) and participates in the molecular cascade of events governing embryonic tooth development in rodents and humans (Mitsiadis et al., 1998, 2003). Notch transmembrane receptors interact with membrane-bound ligands encoded by the Delta and Serrate/Jagged genes. Upon binding, the Notch receptor undergoes activation (a process involving proteolytic cleavage of the protein), release, and translocation of the intracellular Notch domain to the nucleus (Artavanis-Tsakonas et al., 1999). Notch receptors expressed by bipotential progenitors are activated by neighboring cells bearing Notch ligands, leading to inhibition of differentiation of the Notch-expressing cells along a fate-specific pathway (Artavanis-Tsakonas et al., 1999; Frisén and Lendahl, 2001). These cells either remain undifferentiated or differentiate along an alternate pathway in the presence of appropriate stimuli. Signals exchanged between neighboring cells through the Notch receptors influence differentiation, proliferation, and apoptotic events (Artavanis-Tsakonas et al., 1999). During primary dentinogenesis, Delta1 and Notch have complementary expression patterns: Delta1 is expressed in differentiating odontoblasts, whereas Notch expression is confined to sub-odontoblastic cells (Fig. 1A
), suggesting a role for Delta/Notch signaling in the control of odontoblast differentiation (Mitsiadis et al., 1998). Notch expression in the pulp follows a cytodifferentiation gradient: Expression in sub-odontoblastic cells of the apical area becomes progressively down-regulated in the cuspal area, where pulp cells are mature. Both Notch and Delta1 are absent from adult dental tissues (Mitsiadis et al., 1999).
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Figure 1. Patterns of Notch2 (A), nestin (B), and N-cadherin (C,D) immunostaining in pulp cells of deciduous human teeth (About et al., 2000; Heymann et al., 2002; Mitsiadis et al., 2003). Abbreviations: d, dentin; o, odontoblasts; p, pulp; soc, sub-odontoblastic cells. Scale bars: 50 µm. Fig. 1B is reprinted from Am J Pathol 157:287–295, 2000, with permission from the American Society for Investigative Pathology. Figs. 1C and 1D are reprinted from Am J Pathol 160:2123–2133, 2002, with permission from the American Society of Investigative Pathology. Fig. 1A is reprinted from Exp Cell Res 228:101–109 (Mitsiadis et al., "Notch 2 protein...", 2003), with permission from Elsevier.
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Intermediate filaments (i.e., keratins, desmin, vimentin, peripherin) are filamentous structures of the cytoskeleton that are important for the organization and function of cells and tissues (Fuchs and Weber, 1994). Nestin is an intermediate filament most related to neurofilaments and expressed predominantly in the developing nervous system and muscles. Nestin is expressed in odontoblasts and pulp fibroblasts of the cusp area during dentinogenesis (About et al., 2000). Expression of nestin becomes progressively restricted to odontoblasts and localized to both the cell bodies and the odontoblast processes (Fig. 1B
). Similarly to Delta1, nestin expression is down-regulated in odontoblasts of permanent human teeth.
Differentiating odontoblasts of developing permanent teeth, but not odontoblasts of adult teeth, express the cell-surface glycoprotein N-cadherin (Figs. 1C, 1D; Heymann et al., 2002). Cadherins function through a Ca2+-dependent homophilic binding mechanism, and belong to a large family of cell adhesion molecules (CAMs) involved in diverse biological processes, such as cell adhesion, cell recognition, control of cell division, migration, and differentiation (Yagi and Takeichi, 2000). In their cytoplasmic domain, cadherins interact with the catenins that connect them to the actin filaments of the cytoskeleton. The connection of cytoskeletal elements to cadherins is crucial for efficient cell-cell adhesion (Yamada and Geiger, 1997). Changes in the expression of cadherins in dental tissues correlate with the onset of processes that control cell proliferation and differentiation (Heymann et al., 2001, 2002). Selective expression patterns of various cadherins point to a role for cadherins in cellular organization and segregation of different cell populations during odontogenesis (Heymann et al., 2001, 2002).
Adjacent cells can directly exchange ions and small regulatory molecules through channels (gap junctions) interconnecting their cytoplasms. Gap junction channels are exclusively formed from a variety of cell membrane proteins, connexins, which are, to some degree, tissue- and cell-type-specific (Wei et al., 2004). Connexin 43 (Cx43) is expressed in pulp cells and in differentiating odontoblasts in developing teeth (About et al., 2002).
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MECHANISMS CONTRIBUTING TO TERTIARY DENTIN DEPOSITION IN PATHOLOGICAL CONDITIONS
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The cellular responses to various degrees of injury are wide-ranging. In mild injury (e.g., slowly progressing caries), transiently damaged odontoblasts do survive, their morphology remains normal, and their activity is stimulated, leading to the production of reactionary dentin (reviewed in Tziafas et al., 2000). Therefore, odontoblasts must have evolved some protective mechanisms, one component of which is the intermediate filament cytoskeletal network. Indeed, it has been reported that molecules of the cytoskeleton—such as kinesin, myosin, and actin—play a fundamental role in wound-healing processes (Martin, 1997). Increased protein synthesis in the injured odontoblasts targets the re-organization of the cytoskeleton (Magloire et al., 1992). Expression of the fibronectin-binding protein and nestin in the apical areas of odontoblasts located beneath caries lesions has been associated with odontoblast cytoskeletal re-organization (Farges et al., 1995; About et al., 2000). Vimentin is a major structural component of intermediate filaments found also in odontoblasts (Ruch et al., 1995). While vimentin knock-out mice are phenotypically normal (Colucci-Guyon et al., 1994), wound healing in these mice is delayed, because the contractile capacity of the fibroblasts is reduced. Intermediate filaments may be important for repair as well as for the normal development and differentiation of organs. Wounding leads to a dramatic re-organization of the cytoskeleton, since a dense cytoskeletal network is rapidly localized to the region of the cell membrane adjacent to the wound site (Woolley and Martin, 2000).
Injury of greater intensity—such as chronic deep dentinal caries lesions, tooth cavity preparation, treatment, and restoration—can invoke pulp responses that are accompanied by reparative dentin deposition (Figs. 2A–2C). Teeth from different species (i.e., human, mouse, rat, ferret, and dog) have been used as models for the study of dental repair. However, caution is required in the interpretation of the results, since the pulp responses may differ between and among species. For example, it has been suggested that the rat dental pulp is capable of a much stronger reparative response following injury than the human pulp (Smith, 2002). During cavity preparation in human teeth, the nuclei of the primary odontoblasts may be aspirated into the dentinal tubules under more extreme conditions. Tunnel-staining techniques have shown that damaged odontoblasts can become apoptotic (Kitamura et al., 2001; Mitsiadis et al., unpublished results), and cell renewal may then occur to form a new generation of odontoblasts. The origin of these cells is unclear: It has been suggested that cells of the immune system, or cells of the sub-odontoblastic layer, or pulp fibroblasts may give rise to odontoblast-like cells (Mitsiadis et al., 1999; Tziafas et al., 2000). Differences in the embryonic derivation of these cells could influence the resulting cell phenotype and its relationship to that of the odontoblast.
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Figure 2. Comparison between the patterns of Notch2 (D), nestin (E), and N-cadherin (F) immunostaining in human dental tissues after dental injury. (A) Schematic illustration showing reparative dentin production (asterisk) after a cavity preparation in an incisor. The blue line indicates an equivalent wounding area of a premolar shown in panel B. (B) Hematoxylin-eosin staining showing reparative dentin production (asterisk) 9 wks after class V cavity preparation (About et al., 2000). The deep blue line indicates an equivalent area shown in panel C. (C) Hematoxylin-eosin staining. Higher magnification of the reparative dentin area (asterisk) (Mitsiadis et al., 2003). (D-F) Immunostaining showing expression of Notch2 protein in sub-odontoblastic cells and blood vessels (Mitsiadis et al., 2003) (D), nestin protein in odontoblasts (About et al., 2000) (E), and N-cadherin in newly formed odontoblasts (Heymann et al., 2002) (F), after cavity preparation. Abbreviations: ab, alveolar bone; c, cementum; cav, cavity; d, dentin; e, enamel; nfo, newly formed odontoblasts; o, odontoblasts; oe, oral epithelium; p, pulp; rd, reparative dentin; soc, sub-odontoblastic cells. Scale bars: 50 µm. Figs. 2B and 2E are reprinted from Am J Pathol 157:287–295, 2000, with permission from the American Society for Investigative Pathology. Fig. 2F is reprinted from Am J Pathol 160:2123–2133, 2002, with permission from the American Society of Investigative Pathology. Figs. 2C and 2D is reprinted from Exp Cell Res 228:101–109 (Mitsiadis et al., "Notch 2 protein...", 2003), with permission from Elsevier.
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SIGNALS INFLUENCING THE RECRUITMENT AND DIFFERENTIATION OF PULP CELLS DURING DENTAL REPAIR
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Fundamental to our understanding of the dental repair process is knowledge of the signals that trigger pulp cells of various lineages at the injury site to proliferate, migrate, and then to lay down a new collagen-rich matrix. Once a reparative dentin matrix has been formed, with a layer of odontoblast-like cells covering the injury site, migration of pulp cells ceases. Studies in the last decade have identified several of the growth factors and matrix components that are potential candidates to provide the initial signals (reviewed in Smith and Lesot, 2001). In contrast, nothing is known of the signals that prevent further pulp cell differentiation into odontoblast-like cells, except that they may include contact inhibition from adjacent undamaged pulp cells.
Growth factors present at the injury site may act as both mitogens and chemotactic factors for pulp cells. TGFß, BMPs, fibroblast growth factors (FGFs), and insulin growth factors (IGFs) are secreted by functional odontoblasts and pulp fibroblasts, and are captured in the dentin matrix, which serves as a reservoir of growth factors (Ruch et al., 1995). These molecules are released from the dentin following dental injury (reviewed in both of these cited reviews, Tziafas et al., 2000; Goldberg and Smith, 2004). Furthermore, damaged odontoblasts may deliver paracrine signals to undamaged neighbor cells. This early ‘cocktail’ of growth factors initiates the healing process, providing chemotactic cues to recruit inflammatory cells and undifferentiated pulp cells to the injury site, stimulating the angiogenic response, and initiating the subsequent tissue movements for repair (Martin, 1997). TGFß-1 and BMPs have been found to participate in the processes of both reactionary and reparative dentinogenesis (reviewed in both of these cited reviews, Tziafas et al., 2000; Goldberg and Smith, 2004). In vitro experiments and genetic studies in knock-out animals have demonstrated that TGFß-1 is a very important factor involved in dental repair events (D’Souza et al., 1998). TGFß-1 is up-regulated at the injury site during the healing process and act as a signal directing tissue regeneration (Martin, 1997; Farges et al., 2003). TGFß-1 and some pro-inflammatory cytokines appear to stimulate expression of some of the integrin subunits that facilitate cell migration (Woolley and Martin, 2000). Because nerve growth factor (NGF) is up-regulated in odontoblasts after dental injury and after exposure to TGFß-1 (Mitsiadis and Magloire, in preparation), it is tempting to consider nerves as another indirect target for TGFß-1 at the wound site. The sensory nerve endings are sensitive to signals released during dental injury, resulting in nerve sprouting at the site of the lesion (Mitsiadis and Magloire, in preparation). NGF is one of the wound-induced signals controlling neuronal outgrowth and sprouting (Sano and Iwanaga, 1996). Sprouting nerves may have a stimulatory role in the healing process by delivering neuropeptides and other factors to the wound site (Nilsson et al., 1985). Indeed, transgenic mice lacking the low-affinity NGF receptor p75 suffer from impaired wound healing (Lee et al., 1992). FGF2 and other growth factors released at the injury site from the dentin matrix, as well as by endothelial and damaged cells, promote angiogenesis (Nugent and Iozzo, 2000) and may be mitogenic for progenitor pulp cells. Inflammatory cells (i.e., macrophages) that act at the injury site to clear any remaining pathogenic organisms and cell debris amplify earlier wound signals by the release of further cytokines and growth factors (Martin, 1997).
The induction of reparative dentinogenesis by growth factors has been studied in vivo in different animal models. Dentin matrix extracts are capable of inducing odontoblast-like cell differentiation when implanted in vivo within exposed cavity preparations (reviewed in Goldberg and Smith, 2004). Similarly, the distribution of TGFß and BMP molecules during pulp healing promotes cell differentiation into odontoblast-like cells (reviewed in Tziafas et al., 2000).
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MOLECULAR COORDINATION OF CELLULAR DECISIONS DURING DENTAL REPAIR
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Growth factors are not the only relevant influences during dental repair. Calcium, which is released by injured cells, may serve as a mediator signaling undamaged cells to coordinate their migration and junction re-arrangement and, furthermore, to regulate cytoskeletal re-organization (Martin, 1997). Immediate early genes (i.e., c-jun, c-fos) operate by linking the damage stimuli to long-term cellular responses and, subsequently, act as activators of cell division to help replenish adjacent tissues that may have suffered further cell loss (Woolley and Martin, 2000; Chai and Tarnawski, 2002). The induction of early genes could activate transcription of genes encoding proteases and other direct regulators of cell behavior. Growth factors and cytokines activate the expression of early response genes in odontoblast-like cells. Expression of c-jun and junB, two nuclear proto-oncogenes, is induced in odontoblast-like cells of injured teeth (Kitamura et al., 1999). The gene products of c-jun and junB are components of the activator protein-1 (AP-1) family of transcription factors. AP-1 are heterodimeric proteins formed between a fos member (i.e., c-fos, fosB, fra-1, and fra-2) and a jun member (i.e., c-jun, junB, and junD) and are key regulators of cellular proliferation, migration, and differentiation processes. AP-1 proteins are up-regulated within minutes of wounding, and enhance the expression of TGFß-1, osteocalcin, alkaline phosphatase, and type I collagen (Woolley and Martin, 2000; Yates and Rayner, 2002). Thus, it is possible that the production of the main dentin matrix components could be under the control of the AP-1 family of proteins.
Notch, nestin, cadherins, and connexins are also involved in the dynamic processes triggered by pulp injury and contribute to the signaling cascade, resulting in odontoblast-like cell differentiation. Re-expression of these molecules at the injured pulp may help to coordinate cell fate decisions as well as proliferative, migratory, and differentiation activities.
Notch up-regulation in pulp cells represents one of the earlier molecular events in the process of dental tissue repair (Mitsiadis et al., 1999 , 2003). Since Notch signaling plays an essential role in fate determination, its re-activation during pulp healing might permit cells either to differentiate to an alternative pathway or to self-renew. In injured human teeth, Notch receptors are most likely expressed by early odontoblast precursors and intermediate-stage cells of the sub-odontoblastic layer, but not by terminally differentiating odontoblasts, suggesting a role for the Notch pathway in enhanced survival of uncommitted precursors, while preserving multi-lineage potential. Notch2 expression is activated in pulp cells close to the injury site, as well as in cells located at the apex of the roots (Fig. 2D), suggesting that these sites represent important pools of cells from which different cell types will derive after injury (Mitsiadis et al., 1999 HREF="#MITSIADIS-ETAL-2003">, 2003). This is in accordance with recent findings, in skin, showing that Notch receptors are present in transiently amplifying cells, and that the cell-cell signaling system promotes both expansion and differentiation of these cells (Lowell et al., 2000). Bone marrow stem cells express Notch receptors (Calvi et al., 2003), suggesting that the Notch-positive cells of the pulp have at least some stem cell properties. Activation of the Notch molecules in endothelial cells after injury (Fig. 2D) may reflect ingrowths of new blood vessels in the regenerating pulp (Mitsiadis et al., 2003).
Wounding causes severe disruption to the cytoskeleton of cells adjacent to the injury site, and, while not so urgent, these cells must be remodeled to re-establish a normal cyto-architecture. During tooth repair, nestin is re-expressed in odontoblasts facing the irritation front, as well as in odontoblasts at a distance from the injury site (Fig. 2E), suggesting that nestin may be involved in the remodeling and/or the secretory activity of the odontoblasts after dental injury (About et al., 2000; Mitsiadis, unpublished results).
Cell-cell communication and cell adhesion are fundamental to the tooth repair process. Changes in gap-junctional connections and adhesion between pulp cells may help to coordinate proliferative, migratory, and differentiation activities at the injury site. Recent studies in a skin wound-healing model have shown that direct cell-cell communication through Cx43 gap junction channels plays a major role in repair (Qiu et al., 2003). In teeth, Cx43 is up-regulated in odontoblasts around the wound site (About et al., 2002). The physiological significance and mechanism for this up-regulation is not clear. Formation of gap junctions between odontoblasts that have an intense period of synthesis and secretion during reactionary dentin formation may facilitate an exchange of metabolites which otherwise become less available due to disturbances of the local blood flow. Furthermore, gap junctions may be important in maintaining odontoblast position and polarity during dentin repair by allowing for spatially graded distributions of molecules (Fried et al., 1996). Similarly, the expression of the adhesion molecule N-cadherin is activated in odontoblasts during tooth repair processes (Fig. 2F; Heymann et al., 2002), suggesting that Cx43 and N-cadherin may have functions that govern pulp tissue re-organization after injury.
Taken together, these findings suggest that molecules involved in cell fate specification, cell adhesion and communication, and cytoskeletal organization are instrumental in tooth homeostasis. Notch expression is activated in undifferentiated cells that are engaged in a differentiation pathway, leading to nestin- and N-cadherin-positive odontoblasts. In this way, activation of Notch, N-cadherin, and nestin during pulp regeneration may ensure a continuous balance between odontoblasts and progenitors committed to becoming odontoblasts.
In vitro models with cultured human tooth slices, pulp explants, and pulp cells mimic in vivo conditions and provide valuable experimental models for the study of molecular events and the characterization of cell phenotypes during dental repair (Fig. 3). Studies performed in the human tooth-slice model have shown that TGFß-1 induces proliferation and migration of sub-odontoblastic cells and pulp fibroblasts at the injury area (Melin et al., 2000), and also stimulates odontoblast differentiation and reparative dentinogenesis (reviewed in Smith and Lesot, 2001). To influence cell fate decisions, Notch interacts with other signaling pathways, such as TGFß molecules: TGFß-1 down-regulates Notch2 expression in pulp cells in cultured human tooth slices (Figs. 3D, 3E; Mitsiadis et al., 2003), while other isoforms of TGFß and BMP stimulate the expression of nestin in cultured dental pulp explants (Fig. 3F; About et al., 2000). Similarly, pulp cells cultured in the presence of ß-glycerophosphate form dentin mineralization nodules and express nestin (Fig. 3G; About et al., 2000) and Cx43 (About et al., 2002).
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Figure 3. Patterns of Notch2 (D,E) and nestin (F,G) immunostaining in human dental tissues in different culture conditions. (A) Schematic representation showing ink diffusion (blue) from the cavity area to the dental pulp (asterisk) in a human tooth. (B) The human tooth-slice culture system. The dark blue spot shows the area where the ink-containing tube is placed, thus permitting diffusion of the ink to the dental pulp (asterisk) (Melin et al., 2000). Growth factors can be used instead of the ink to influence pulp cell activities (i.e., migration, proliferation, differentiation). (C) Diffusion of the ink (blue) through the dentin to the dental pulp (asterisk) (Melin et al., 2000). (D) In a cultured human tooth slice, Notch2 immunostaining is localized in odontoblasts and sub-odontoblastic cells (Mitsiadis et al., 2003). (E) Under the influence of TGFß-1, Notch2 immunostaining is localized only in sub-odontoblastic cells (Mitsiadis et al., 2003). (F) Nestin immunostaining is localized around BMP4-releasing beads in cultured pulp explants (About et al., 2002). (G) Nestin immunostaining is detected in pulp cells cultured in the presence of ß-glycerophosphate (About et al., 2000). Abbreviations: b, bead; cav, cavity; d, dentin; e, enamel; n, mineralized nodule; nf, nerve fibers; o, odontoblasts; p, pulp; pc, pulp chamber; pe, pulp explant; soc, sub-odontoblastic cells. Scale bars: 50 µm. Figs. 3F and 3G are reprinted from Am J Pathol 157:287–295, 2000, with permission from the American Society for Investigative Pathology. Figs. 3B and 3C are reprinted from J Dent Res 79:1689–1696, 2000, with permission from the International Association for Dental Research. Figs. 3D and 3E are reprinted from Exp Cell Res 228:101–109 (Mitsiadis et al., "Notch 2 protein...", 2003), with permission from Elsevier.
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EXPERIMENTAL APPROACHES FOR DENTAL REPAIR IN THE FUTURE
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Pulp regeneration may be greatly improved by the supplementation of growth factors, cell adhesion molecules, and supportive extracellular matrix components. Growth factors (i.e., TGFßs, BMPs, FGFs, and IGFs), dentin matrix, and enamel matrix proteins have been evaluated for their potential contributions to dental tissue repair (reviewed in Tziafas et al., 2000). TGFß-1, BMP, and FGF molecules have opposite effects on the regulation of nestin and Notch expression, thus providing a possible approach for obtaining either more Notch-positive (proliferative) or nestin-positive (differentiated) pulp cell populations during regenerative processes. However, it is difficult to deliver active growth factors over the entire duration of pulp regeneration. Scaffolds may provide a finite reservoir of molecules that can be used for controlled-release devices, which can release these molecules in a spatially and temporally controlled manner. Cell aggregates may be effective and appropriate vehicles for supplying bioactive factors. Furthermore, cell aggregates of either Notch- or nestin-positive populations could be used for transplantation in the wounded dental tissue. Local transplantation of Notch-positive pulp cells for therapeutic applications may permit a more extensive reconstruction than in those wounds that would heal spontaneously. Finally, the potential of pulp stem cells in dental repair applications is under investigation. These experimental approaches show that the requirements for functional tooth regeneration are complex. Yet a single approach has not allowed for effective clinical therapy. The increased understanding of the molecular events of tooth development and regeneration will provide us with new therapeutic strategies and approaches in tooth repair.
In conclusion, strong parallels exist between early developmental events and repair processes. Molecules involved in cell fate determination, cell adhesion, cell-cell communication, and cytoskeletal organization, which are expressed in dental tissues during odontogenesis, are re-expressed in dental tissues after injury. Molecules of the Notch signaling pathway, cadherins, connexins, and intermediate filament proteins may influence more than one pulp cell population during tooth repair (Fig. 4). Notch expression is related to proliferative and migratory activities, while expression of N-cadherin, Cx43, and nestin is related to differentiation processes in the pulp of both developing and injured teeth. Taking into account the molecular similarities and differences between tooth development and repair should probably help to develop more biological strategies for dental tissue repair in the clinic.
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Figure 4. Schematic representation of the molecular events occurring during reparative dentinogenesis. (A) Illustration of the reparative dentin area showing expression of AP-1, nestin, N-cadherin, and connexin 43 (Cx43) in odontoblasts, and of Notch2 in sub-odontoblastic cells. (B) Diagram showing the sequence of molecular events during tooth repair. Shorter arrows indicate early events, longer arrows late events. FGF and TGFß molecules that are released from dentin and pulp cells activate wound healing. Abbreviations: d, dentin; o, odontoblasts; p, pulp; rd, reparative dentin; soc, sub-odontoblastic cells.
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ACKNOWLEDGMENTS
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This review was prepared with support from institutional grants from the University of Athens, by specific grants from the Guy’s & St Thomas’ Charitable Foundation (R040234), and by the Friends of Guy’s Hospital (356).
Received June 22, 2004;
Last revision October 8, 2004;
Accepted October 13, 2004
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ABSTRACT
INTRODUCTION
