Are Hertwig's epithelial root sheath cells necessary for periodontal formation by dental follicle cells?

OBJECTIVE: The role of Hertwig's epithelial root sheath (HERS) cells in periodontal formation has been controversial. This study aimed to further clarify whether HERS cells participate in formation of the periodontium, and the necessity of HERS cells in differentiation of dental follicle cells (DFCs) for periodontal regeneration. DESIGN: HERS cells and DFCs were isolated and identified from post-natal 7-day Sprauge-Dawley rats. In vitro, direct co-culture of HERS cells and DFCs as well as the individual culture of HERS and DFCs were performed and followed by alizarin red staining and the quantitative real-time polymerase chain reaction analysis. For in vivo evaluation, the inactivated dentin matrix (iTDM) was fabricated. HERS cells and DFCs were seeded in combination or alone on iTDM and then transplanted into the rat omentum. Scanning electron microscope and further histological analysis were carried out. RESULTS: In vitro, mineral-like nodules were found in the culture of HERS cells alone or HERS + DFCs either by alizarin red staining or scanning electronic microscope. The mineralization and fiber-forming relevant mRNA expressions, such as bone sialoprotein, osteopontin, collagen I and collagen III in HERS + DFCs were significantly higher than that of the HERS or DFCs alone group. After transplantation in vivo, cementum and periodontal ligament-like tissues were formed in groups of HERS + DFCs and HERS alone, while no evident hard tissues and attached fibers were found in DFCs alone. CONCLUSIONS: Hertwig's epithelial root sheath cells directly participate in the formation of the periodontium, and they are essential for the differentiation of dental follicle cells to form periodontal structures. The combination use of Hertwig's epithelial root sheath cells and dental follicle cells is a promising approach for periodontal regeneration.

Odontoblast ß-catenin signaling regulates fenestration of mouse Hertwig's epithelial root sheath.

The interaction between Hertwig's epithelial root sheath (HERS) and the adjacent mesenchyme is vitally important in mouse tooth root development. We previously generated odontoblast-specific Ctnnb1 (encoding ß-catenin) deletion mice, and demonstrated that odontoblast ß-catenin signaling regulates odontoblast proliferation and differentiation. However, the role of odontoblast ß-catenin signaling in regulation of HERS behavior has not been fully investigated. Here, using the same odontoblast- specific Ctnnb1 deletion mice, we found that ablation of ß-catenin signaling in odontoblasts led to aberrant HERS formation. Mechanistically, odontoblast-specific Ctnnb1 deletion resulted in elevated bone morphogenetic protein 7 (Bmp7) expression and reduced expression of noggin and follistatin, both of which encode extracellular inhibitors of BMPs. Furthermore, the levels of phosphorylated Smad1/5/8 were increased in HERS cells. In vitro tissue culture confirmed that BMP7 treatment disrupted the HERS structure. Taken together, we demonstrated that odontoblast ß-catenin signaling may act through regulation of BMP signaling to maintain the integrity of HERS cells.

[Enamel: a unique self-assembling in mineral world]. / L'émail - Un autoassemblage unique dans le monde du minéral.

Enamel is a unique tissue in vertebrates, acellular, formed on a labile scaffolding matrix and hypermineralized. The ameloblasts are epithelial cells in charge of amelogenesis. They secrete a number of matrix proteins degraded by enzymes during enamel mineralization. This ordered cellular and extracellular events imply that any genetic or environmental perturbation will produce indelible and recognizable defects. The specificity of defects will indicate the affected cellular process. Thus, depending on the specificity of alterations, the teratogenic event can be retrospectively established. Advances in the field allow to use enamel defects as diagnostic tools for molecular disorders. The multifunctionality of enamel peptides is presently identified from their chemical roles in mineralization to cell signaling, constituting a source of concrete innovations in regenerative medicine.

Expression of cytokeratins in enamel organ, junctional epithelium and epithelial cell rests of Malassez.

BACKGROUND AND OBJECTIVE: After tooth formation is complete, it is suggested that continuity exists between the epithelial cell rests of Malassez (ERM), reduced enamel epithelium (REE) and subsequently the junctional epithelium. However, the junctional epithelium was reported to differ from REE and ERM. The developmental relationships between and among them remain controversial. Therefore, in the present study we examined the expression of cytokeratins in the three types of epithelia to investigate the epithelial phenotypes. MATERIAL AND METHODS: The maxillae of Wistar rats, 1, 2, 3 and 7 wk of age, were used, and the expression of CK14, CK17, CK19, CK10/CK13 and AE1/AE3 was detected using immunoperoxidase techniques. RESULTS: There was negative staining for CK10/CK13 in all the epithelia. ERM stained strongly for AE1/AE3, CK14, CK17 and CK19. During the transformation of inner enamel epithelial (IEE) cells into reduced ameloblasts and subsequently into junctional epithelium, strong staining for CK14 was evident in IEE, REE and junctional epithelium, whereas the expression of AE1/AE3 and of CK19 were initially negative in IEE and then strong in REE and junctional epithelium, respectively. In particular, the expression of CK17 was strongly positive in ERM and REE, but was negative in IEE and junctional epithelium. CONCLUSION: ERM are of odontogenic origin and junctional epithelium has an epithelial phenotype different from REE and ERM. This is the first report to demonstrate that CK17 can be used as a marker to distinguish junctional epithelium from ERM.

Effect of interrupted eruption on the enamel organ of the rat incisor.

The aim of this study was to investigate the behavior of rat incisor tissues during the inhibition of tooth eruption. Twenty Sprague-Dawley rats were used in this study, and incisor eruption was inhibited by a screw pin. Animals were sacrificed 1, 3, 7 and 14 days after the start of the experiment. Cross-sections at the mesial point of the mandibular first molar and sagittal sections of the mandibular tooth germ area were examined using immunohistochemical and immunofluorescence methods. For morphometric analysis, numbers of TRAP-positive cells were calculated against the total number of cells. In cross-sections from the experimental group, dentin was thickened and pulp tissue was constricted day by day. On days 1, 3 and 7, nestin-positive cells were observed in all odontoblast cell bodies and processes, while on day 14 fewer nestin-positive cells were seen than in the control group. On day 14, the mesial area of the periodontal ligament was constricted and the number of TRAP-positive cells in the mesial area was significantly higher than in the control group. In sagittal sections, enamel formation was found to be increased on days 7 and 14. Furthermore, in the enamel matrix amelogenin was expressed more strongly than in the control group. PCNA-positive cells were significantly increased in cells of the tooth germ compared with the control group. These results suggest that inhibition of tooth eruption accelerates the apical elongation with resorption of the mesial area of the alveolar bone and stimulates cell proliferation with thickened enamel towards the apical end.

ß-catenin is required in odontoblasts for tooth root formation.

The tooth root is an important part of the tooth that works together with the surrounding periodontium to maintain the tooth in the alveolar socket. The root develops after crown morphogenesis. While the molecular and cellular mechanisms of early tooth development and crown morphogenesis have been extensively studied, little is known about the molecular mechanisms controlling tooth root formation. Here, we show that ß-catenin is strongly expressed in odontoblast-lineage cells and is required for root formation. Tissue-specific inactivation of ß-catenin in developing odontoblasts produced molars lacking roots and aberrantly thin incisors. At the beginning of root formation in the mutant molars, the cervical loop epithelium extended apically to form Hertwig's epithelial root sheath (HERS), but root odontoblast differentiation was disrupted and followed by the loss of some HERS inner layer cells. However, the outer layer of the HERS extended without the root, and the mutant molars finally erupted. The periodontal tissues extensively invaded the dental pulp. These results indicate that there is a cell-autonomous requirement for Wnt/ß-catenin signaling in the dental mesenchyme for root formation.

Enamel pits in hamster molars, formed by a single high fluoride dose, are associated with a perturbation of transitional stage ameloblasts.

Excessive intake of fluoride (F) by young children results in the formation of enamel subsurface porosities and pits, called enamel fluorosis. In this study, we used a single high dose of F administered to hamster pups to determine the stage of ameloblasts most affected by F and whether pit formation was related to F-related sub-ameloblastic cyst formation. Hamster pups received a single subcutaneous injection of either 20 mg or 40 mg NaF/kg body weight, were sacrificed 24 h later, and the number of cysts formed in the first molars were counted. Other pups were sacrificed 8 days after F injection, when the first molars had just erupted, to score for enamel defects. All F-injected pups formed enamel defects in the upper half of the cusps in a dose-dependent way. After injection of 20 mg NaF/kg, an average of 2.5 white spots per molar was found but no pits. At 40 mg NaF/kg, almost 4.5 spots per molar were counted as well as 2 pits per molar. The defects in erupted enamel were located in the upper half of the cusps, sites where cysts had formed at the transition stage of ameloblast differentiation. These results suggest that transitional ameloblasts, located between secretory- and maturation-stage ameloblasts, are most sensitive to the effects of a single high dose of F. F-induced cysts formed earlier at the pre-secretory stage were not correlated to either white spots or enamel pits, suggesting that damaged ameloblasts overlying a F-induced cyst regenerate and continue to form enamel.

Dental follicle cells and cementoblasts induce apoptosis of ameloblast-lineage and Hertwig's epithelial root sheath/epithelial rests of Malassez cells through the Fas-Fas ligand pathway.

Hertwig's epithelial root sheath (HERS), epithelial rests of Malassez (ERM) cells, and reduced ameloblasts undergo apoptosis during tooth development. This study examined the effects of dental follicle cells and cementoblasts on the apoptosis of ameloblast-lineage and HERS/ERM cells derived from the enamel organ. We also elucidated the induction pathways and identified the apoptotic pathway involved in this process. Here, we showed terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling (TUNEL)-positive HERS cells and reduced ameloblasts near dental follicle cells during tooth development. Co-culturing ameloblast-lineage cell line (ALC) ameloblasts and HERS/ERM cells with either dental follicle cells or OCCM-30 cementoblasts markedly enhanced the apoptosis of ameloblasts and HERS/ERM cells compared with cells cultured alone. However, dental follicle cells and cementoblasts did not modulate the apoptotic responses of co-cultured non-odontogenic MCF10A or KB cells. When ameloblasts + HERS and cementoblasts + dental follicle cells were co-cultured, the expression of Fas ligand (FasL) increased in cementoblasts + dental follicle cells, while the expression of Fas increased in ameloblasts + HERS. Interestingly, recombinant FasL induced ameloblast apoptosis while the cementoblast-induced ameloblast apoptosis was suppressed by the Fas/FasL antagonist Kp7-6. These results suggest that during tooth development, dental follicle cells and cementoblasts induce apoptosis of ameloblast-lineage and HERS/ERM cells through the Fas-FasL pathway, but do not induce the apoptosis of non-odontogenic epithelial cells.

Reptilian tooth development.

Dental patterns in vertebrates range from absence of teeth to multiple sets of teeth that are replaced throughout life. Despite this great variation, most of our understanding of tooth development is derived from studies on just a few model organisms. Here we introduce the reptile as an excellent model in which to study the molecular basis for early dental specification and, most importantly, for tooth replacement. We review recent snake studies that highlight the conserved role of Shh in marking the position of the odontogenic band. The distinctive molecular patterning of the dental lamina in the labial-lingual and oral-aboral axes is reviewed. We explain how these early signals help to specify the tooth-forming and non-tooth forming sides of the dental lamina as well as the presumptive successional lamina. Next, the simple architecture of the reptilian enamel organ is contrasted with the more complex, mammalian tooth bud and we discuss whether or not there is an enamel knot in reptilian teeth. The role of the successional lamina during tooth replacement in squamate reptiles is reviewed and we speculate on the possible formation of a vestigial, post-permanent dentition in mammals. In support of these ideas, we present data on agamid teeth in which development of a third generation is arrested. We suggest that in diphyodont mammals, similar mechanisms may be involved in reducing tooth replacement capacity. Finally, we review the location of label-retaining cells and suggest ways in which these putative dental epithelial stem cells contribute to continuous tooth replacement.

Wnt-RhoA signaling is involved in dental enamel development.

Transgenic mice that express dominant-negative RhoA (RhoA(DN) ) in ameloblasts have hypoplastic enamel with defects in molar cusps. ß-catenin and Wnt5a were up-regulated in enamel organs of RhoA(DN) transgenic mice, which indicated that both canonical and non-canonical Wnt pathways are implicated in the process of enamel defect formation. It was hypothesized that expression of RhoA(DN) in ameloblasts interfered with normal enamel development through the pathways that were induced by fluoride. The Wnt and RhoA pathways were further investigated in an ameloblast-lineage cell line (ALC) by treatment with sodium fluoride (NaF). The activities of RhoA and Rho-associated protein kinase (ROCK) II decreased significantly by 8-12 hours, similar to decreased activity in RhoA(DN) transgenic mice. Both canonical and non-canonical Wnt pathways were activated by treatment with NaF, which was verified by western blotting and the ß-catenin-TCF/LEF (T cell factor lymphanoid/enhancer factor) reporter gene (TOPflash) assay. ß-catenin localization to both cytoplasm and nucleus was up-regulated in NaF-treated ALC, while Gsk-3ß, the negative regulator of the Wnt pathway, showed a decreased pattern of expression. The current results indicate that both Wnt and RhoA pathways are implicated in fluoride-induced signaling transductions in the ALC as well as in the development of enamel defects in RhoA(DN) transgenic mice.

Gene-expression analysis of early- and late-maturation-stage rat enamel organ.

Enamel maturation is a dynamic process that involves high rates of mineral acquisition, associated fluctuations in extracellular pH, and resorption of extracellular enamel proteins. During maturation, ameloblasts change from having a tall, thin, and highly polarized organization, characteristic of the secretory stage, to having a low columnar and widened morphology in the maturation stage. To identify potential differences in gene expression throughout maturation, we obtained enamel organ epithelial cells derived from the early- and late-maturation stages of rat incisor and analyzed the global gene-expression profiles at each stage. Sixty-three candidate genes were identified as having potential roles in the maturation process. Quantitative PCR was used to confirm the results of this genome-wide analysis in a subset of genes. Transcripts enriched during late maturation (n = 38) included those associated with lysosomal activity, solute carrier transport, and calcium signaling. Also up-regulated were transcripts involved in cellular responses to oxidative stress, proton transport, cell death, and the immune system. Transcripts down-regulated during the late maturation stage (n =25) included those with functions related to cell adhesion, cell signaling, and T-cell activation. These results indicate that ameloblasts undergo widespread molecular changes during the maturation stage of amelogenesis and hence provide a basis for future functional investigations into the mechanistic basis of enamel mineralization.

Regulation of dental enamel shape and hardness.

Epithelial-mesenchymal interactions guide tooth development through its early stages and establish the morphology of the dentin surface upon which enamel will be deposited. Starting with the onset of amelogenesis beneath the future cusp tips, the shape of the enamel layer covering the crown is determined by five growth parameters: the (1) appositional growth rate, (2) duration of appositional growth (at the cusp tip), (3) ameloblast extension rate, (4) duration of ameloblast extension, and (5) spreading rate of appositional termination. Appositional growth occurs at a mineralization front along the ameloblast distal membrane in which amorphous calcium phosphate (ACP) ribbons form and lengthen. The ACP ribbons convert into hydroxyapatite crystallites as the ribbons elongate. Appositional growth involves a secretory cycle that is reflected in a series of incremental lines. A potentially important function of enamel proteins is to ensure alignment of successive mineral increments on the tips of enamel ribbons deposited in the previous cycle, causing the crystallites to lengthen with each cycle. Enamel hardens in a maturation process that involves mineral deposition onto the sides of existing crystallites until they interlock with adjacent crystallites. Neutralization of acidity generated by hydroxyapatite formation is a key part of the mechanism. Here we review the growth parameters that determine the shape of the enamel crown as well as the mechanisms of enamel appositional growth and maturation.

A multi-scale, discrete-cell simulation of organogenesis: Application to the effects of strain stimulus on collective cell behavior during ameloblast migration.

A multi-scale strategy is presented for simulating organogenesis that uses a single cell response function to define the behavior of individual cells in an organ-scale simulation of a large cell population. The response function summarizes detailed information about the behavior of individual cells in a sufficiently economical way that the organ-scale model can be commensurate with the entire organ. The first application demonstrates the effects of strain stimulus on the migration of ameloblasts during enamel formation. Ameloblasts are an attractive study case because mineralization preserves a complete record of their migratory paths. The response function in this case specifies the motions of cells responding to strain stimuli that propagate through the population. The strain stimuli are related to the curvature of the surface from which the ameloblasts migrate (the dentin-enamel junction or DEJ). A single unknown rate parameter is calibrated by an independent datum from the human tooth. With no remaining adjustable parameters, the theory correctly predicts aspects of the fracture-resistant, wavy microstructure of enamel in the human molar, including wavelength variations and the rate of wave amplitude damping. At a critical value of curvature of the DEJ, a transition in the ordering of cells occurs, from invariant order over the whole population to self-assembly of the population into groups or gangs. The prediction of an ordering transition and the predicted critical curvature are consistent with gnarled enamel in the cusps of the human molar. The calibration of the model using human data also predicts waves in the mouse incisor and an ordering transition at the chimpanzee cingulum. Widespread compressive strain is predicted late in the migration for both the human molar and mouse incisor, providing a possible signal for the termination of amelogenesis.

Odontogenic potential of post-natal oral mucosal epithelium.

A bioengineered tooth would provide a powerful alternative to currently available clinical treatments. Previous experiments have succeeded in bioengineering teeth using tooth germs from animal embryos. However, the ultimate goal is to develop a technology which enables teeth to be regenerated with the use of autologous cells. To pursue this goal, we re-associated the palatal epithelium from young mice with the odontogenic dental mesenchyme and transplanted the re-associated tissues into mouse kidney capsules. Morphologically defined teeth were formed from the re-associated cultured palatal epithelial cell sheets from mice aged up to 4 wks, but no tooth was formed when the palatal epithelium from mice after 2 days of age was directly re-associated. Our results demonstrated that post-natal non-dental oral mucosal epithelium can be used as a substitute for dental epithelium, and that epithelial cell sheet improves the ability of the oral epithelium of older mice to differentiate into dental epithelium.

From conical to spatulate: intra- and interspecific changes in tooth shape in closely related cichlids (Teleostei; Cichlidae: Eretmodini).

The Eretmodini are closely related cichlids endemic to Lake Tanganyika with very divergent oral tooth shapes, ranging from spatulate in Eretmodus to conical in Tanganicodus. To study how closely related cichlids can generate such divergent tooth shapes, we investigated how the enamel organ directs the development of spatulate teeth in Eretmodus cf. cyanostictus (lineage A), both in ontogeny and in adults, and of conical teeth in adult Tanganicodus cf. irsacae, using 3D-reconstructions from serially sectioned tooth germs. The spatulate oral tooth shape that characterizes adult E. cf. cyanostictus (lineage A) is preceded early in ontogeny by a conical tooth shape. We propose two possible hypotheses to account for changes in the folding of the enamel organ (in particular its epithelio-mesenchymal boundary) capable of generating such distinct tooth shapes. Different arguments lead us to favor the hypothesis of an asymmetric growth and differentiation of the enamel organ, such that the tip of a conical tooth corresponds to one "corner" of a spatulate tooth. Applying current molecular models of tooth shape variation, this would imply the existence of asymmetric fields of inhibition. Whether such asymmetric growth reflects the reutilization of a simple mechanism operating in ontogeny has to be clarified.

Apoptosis distribution in the first molar tooth germ of the field vole (Microtus agrestis).

Apoptosis represents an important process in organ and tissue morphogenesis and remodeling during embryonic development. A role for apoptosis in shape formation of developing teeth has been suggested. The field vole is a useful model for comparative studies in odontogenesis, particularly because of its contrasting molar morphogenesis when compared to the mouse. However, little is known concerning apoptosis in tooth development of this species. Morphological (cellular and nuclear alterations) and biochemical (specific DNA breaks--TUNEL staining) characteristics of apoptotic cells were used to evaluate the temporal and spatial occurrence of apoptosis in epithelial and mesenchymal tissues of the developing first molar tooth germs of the field vole. Apoptotic cells were found in non-proliferating areas (identified previously) throughout bud to bell stages, particularly in the epithelium, however, scattered also in the mesenchyme. A high concentration of TUNEL positive cells was evident in primary enamel knots at late bud stage with increasing density of apoptotic cells until ED 16 when the primary enamel knot in the field vole disappears and mesenchyme becomes protruded in the middle axes of the bell forming two shallow areas with zig-zag located secondary enamel knots. Distribution of TUNEL positive cells corresponded with localisation of secondary enamel knots as shown using histological and 3D analysis. Apoptosis was shown to be involved in the first molar development of the field vole, however, exact mechanisms and roles of this process in tooth morphogenesis require further investigation.

Cellular expression of eve1 suggests its requirement for the differentiation of the ameloblasts and for the initiation and morphogenesis of the first tooth in the zebrafish (Danio rerio).

even-skipped-related (evx) genes encode homeodomain-containing transcription factors that are involved in a series of developmental processes such as posterior body patterning and neurodifferentiation. Although evx1 and evx2 were not reported to be expressed during mammalian tooth development, we present here evidence that eve1, the closest paralog of evx1 in the actinopterygian lineage, is expressed during pharyngeal tooth formation in the zebrafish, Danio rerio. We have performed whole-mount in situ hybridization on zebrafish embryos and larvae ranging from 24 to 192 hours postfertilization (hpf). A detailed analysis of serial sections through the pharyngeal region of whole-mount hybridized and control specimens indicates that only dental epithelial cells express eve1. eve1 transcription was activated at 48 hpf, in the placode of the first tooth (i.e., the initiation site of tooth 4V(1)), and maintained in the dental epithelium throughout morphogenesis. Then, by 72 hpf, eve1 expression was restricted to the differentiating ameloblasts of the enamel organ during early differentiation stage, and this expression decreased as soon as matrix was deposited. In subsequent primary teeth (3 V(1) and 5 V(1)) as well as in their successors (replacement teeth 4V(2), 3V(2), and 5V(2)), eve1 expression was restricted to the differentiating ameloblasts and, again, disappeared when matrix was deposited. Therefore, in the zebrafish, eve1 expression in the pharyngeal region is correlated with two key steps of tooth development: initiation and morphogenesis of the first tooth, and ameloblast differentiation of all developing teeth.

The effect of partial damage to the enamel-related periodontium combined with root resection on eruption of the rat incisor eruption.

Previous work has indicated that the enamel-related periodontium (ERP) has a role in the eruptive process of the rat lower incisor. By combining partial damage of this tissue with resection of the odontogenic organ, we examined the effect of the damage on subsequent incisor eruption. The connective tissue of the enamel-related periodontium was regenerated in less than 2 weeks, showing morphology close to normal. The injured part of the enamel organ was neither regenerated nor repaired, and a cement-like tissue, continuous with the true acellular cement, was formed on the denuded enamel. Before tooth exfoliation, the operated teeth erupted at a slower rate compared with root-resected and sham-operated incisors, probably because of the absence of a substantial part of the enamel organ due to surgical damage. As with the coronal dental follicle and the enamel organ in rat molars, the enamel-related periodontium and the enamel organ of rat incisors may have some control on their eruptive process.

The immunohistochemical localization of Bax and Bcl-2 and their relation to apoptosis during amelogenesis in developing rat molars.

Bax and Bcl-2 are members of a family of intracellular, membrane-associated proteins that regulate programmed cell death. It has been suggested that, when Bax predominates, programmed cell death is accelerated and the apoptosis inhibitory activity of Bcl-2 is suppressed. The present study was undertaken to immunohistochemically (IHC) localize Bax and Bcl-2 in the cells of the enamel organ during amelogenesis in rat molars. Also, apoptotic cells were detected by TUNEL staining. The IHC intense localization of Bcl-2 and light staining for Bax in the pre-ameloblasts suggest that apoptosis is inhibited in the proliferating pre-ameloblasts. This is consistent with an absence of TUNEL staining for apoptosis in these cells. However, in the late secretory and transition ameloblasts, and adjacent stratum intermedium, evidence of apoptosis of the ameloblasts was observed. Bax and Bcl-2 were co-localized in the proximal ends of late secretory, transition and early maturation-stage ameloblasts, but immunoreactivity for Bax markedly increased in the proximal ends of late secretory and transition ameloblasts, while the Bcl-2 staining appeared to be lighter. This suggests that Bax antagonized Bcl-2 function, limiting the ability of Bcl-2 to prolong cell survival. In the early maturation stage, Bax staining faded while the immunoreactivity for Bcl-2 increased. Evidence of distinct apoptosis was reduced in the early maturation stage ameloblasts. When related to the occurrence of apoptosis during amelogenesis, the relative intensity of expression of Bax and Bcl-2 changed in a pattern consistent with that observed in other cell lines. This indicates that these proteins play essential roles in the process of amelogenesis, as predicted by their proposed mechanisms of action in the control of apoptosis.

Epigenetic signals during odontoblast differentiation.

Odontoblast terminal differentiation occurs according to a tooth-specific pattern and implies both temporospatially regulated epigenetic signaling and the expression of specific competence. Differentiation of odontoblasts (withdrawal from the cell cycle, cytological polarization, and secretion of predentin/dentin) is controlled by the inner dental epithelium, and the basement membrane (BM) plays a major role both as a substrate and as a reservoir of paracrine molecules. Cytological differentiation implies changes in the organization of the cytoskeleton and is controlled by cytoskeleton-plasma membrane-extracellular matrix interactions. Fibronectin is re-distributed during odontoblast polarization and interacts with cell-surface molecules. A non-integrin 165-kDa fibronectin-binding protein, transiently expressed by odontoblasts, is involved in microfilament reorganization. Growth factors (TGF beta 1, 2, 3/BMP2, 4, and 6), expressed in tooth germs, signal differentiation. Systemically derived molecules (IGF1) may also intervene. IGF1 stimulates cytological but not functional differentiation of odontoblasts: The two events can thus be separated. Immobilized TGF beta 1 (combined with heparin) induced odontoblast differentiation. Only immobilized TGF beta 1 and 3 or a combination of FGF1 and TGF beta 1 stimulated the differentiation of functional odontoblasts over extended areas and allowed for maintenance of gradients of differentiation. Presentation of active molecules in vitro appeared to be of major importance; the BM should fulfill this role in vivo by immobilizing and spatially presenting TGF beta s. Attempts are being made to investigate the mechanisms which spatially control the initiation of odontoblast differentiation and those which regulate its propagation. Analysis of molar development suggested that odontoblast differentiation and crown morphogenesis are interdependent, although the possibility of co-regulation requires further investigation.