Temporo-spatial distribution of matrix and microfilament components during odontoblast and ameloblast differentiation

Summary Several extracellular matrix components (procollagen type III, fibronectin, collagen type IV, laminin and nidogen) and microfilament constituents (actin, α-actinin and vinculin) were localized by indirect immunofluorescence microscopy in frozen sections of embryonic mouse molars. Nidogen was present at the epithelio-mesenchymal junction during polarization and initial steps of functional differentiation of odontoblasts. Nidogen disappeared at a stage where direct contacts between preameloblasts and predentin were required to allow the initiation of ameloblast polarization. Our observations concerning the distribution of procollagen type III and fibronectin during odontoblast differentiation add to current knowledge. Procollagen type III and fibronectin surrounding preodontoblasts accumulated at the apical part of polarizing and functional odontoblasts secreting “initial” predentin. Procollagen type III, but not fibronectin, disappeared in front of functional odontoblasts synthesizing “late” predentin and dentin. Fibronectin, present in “initial” predentin, was no longer detected in “late” predentin and dentin but was found between odontoblasts secreting “late” predentin and dentin. Actin, α-actinin and vinculin were concentrated in the peripheral cytoplasm of preameloblasts and accumulated at the apical and basal poles of functional ameloblasts. During differentiation of odontoblasts, the three proteins accumulated at the apical pole of these cells. Time and space correlations between matrix and microfilament modifications during odontoblast and ameloblast differentiation are documented. The possibility is discussed that there is transmembranous control of the cytoskeletal activities of odontoblasts and ameloblasts by the extracellular matrix.     

Distribution of calcium-ATPase in developing teeth of embryonic American alligators (Alligator mississippiensis)

Light microscopic and ultrastructural observations were carried out to evaluate the cell morphology and histochemistry (calcium-ATPase activity) of developing teeth in embryonic American alligators (Alligator mississippiensis). Ca-ATPase activity was observed in the distal and lateral cell membranes, rough endoplasmic reticulum (rER), mitochondria, vacuoles, and other organelles of the ameloblast, but only in the distal cell membrane and process of the odontoblast. Enzyme activity in the ameloblasts increased gradually during development. These sites of enzyme activity are related to mineralization of the enamel layer, similar to that in mammalian tooth development. Alligator teeth are heavily mineralized like mammalian teeth; however, alligator ameloblasts have undeveloped distal processes during mineralization in contrast to mammalian ameloblasts in which Tomes' processes are found near the distal portion of ameloblasts at maturation stage. The localization of intense enzyme activity in the distal and lateral ameloblast cell membrane suggests that these regions are the site of accumulation of calcium as enamel differentiates in the developing tooth. © 1993 Wiley-Liss, Inc.     

Development of larval and transformed teeth in Ambystoma mexicanum (Urodela, Amphibia): an ultrastructural study

Odontogenesis of early larval non-pedicellate teeth, late larval teeth with a more or less distinct dividing zone and fully transformed pedicellate teeth in Ambystoma mexicanum (Urodela) was studied to obtain insights into the development of differently structured teeth in lower vertebrates. Using transmission electron microscopy we investigated five developmental stages: (1) papilla; (2) bell stage (secretion of the matrix begins); (3) primordium (mineralization and activity of ameloblasts starts); (4) replacement tooth (young, old); and (5) established, functional tooth. Development of the differently structured teeth is largely identical in the first three stages. Mineralization takes place in apico-basal direction up to the (prospective) pedicel (early and some late larvae) or up to the zone that divides the late larval and transformed tooth in pedicel and dentine shaft (pedicellate condition). Mineralization starts directly at the collagen and by means of matrix vesicles. First odontoblasts develop small processes that extend to the basal lamina of the inner epithelial layer of the enamel organ. The processes are small and lack organelles in early larval teeth, but become larger, arborescent, and contain some organelles in late larval and transformed teeth. The processes are surrounded by unmineralized matrix (predentine). Odontoblasts at the basis of the teeth, at the pedicel, and in the zone of division do not develop significant cytoplasmic processes that extend into the matrix. Cells of the inner enamel epithelium differentiate to ameloblasts that secrete the enamel. In the early larval tooth they show an extensive basal labyrinth that becomes regressive when the enamel layer is completed. In late larval and transformed teeth, however, a large cavity arises between the basal ruffled border of ameloblasts and their basal lamina. This cavity appears to mediate amelogenesis. A small apical zone in early, but not in late larval teeth directly below the thin enamel layer consists of enameloid and is free of dentine channels.     

Immunodetection of osteoadherin in murine tooth extracellular matrices

An antiserum was generated from synthetic peptides highly conserved between different mammalian species to immunolocalise the small leucine-rich proteoglycan osteoadherin (OSAD) in murine teeth. In 19-day-old embryos of rats and mice, a positive staining was found in incisor predentin and alveolar bone surrounding developing incisors and molars. In newborns, OSAD was detected at the tip of the first molar cusp where it accumulated in predentin concomitantly with odontoblast differentiation. In 2-day-old rats and mice, in the first molar, immunostaining revealed positive predentin, enamel matrix close to the apical pole of ameloblasts and a strong signal in dentin. At this stage, OSAD was detected in predentin in the second molar. Ultrastructural immunocytochemistry showed gold particles associated with collagen fibres in predentin and in foci at the dentin mineralisation front. Gold particles were also detected near the secretory pole of ameloblasts where enamel crystallites elongate. No staining was detected in pulp tissue and dental follicle. Restriction of OSAD expression to the extracellular matrix of bone, dentin and enamel suggests a role of this proteoglycan in the organisation of mineralised tissues.     

Epithelial cytodifferentiation and extracellular matrix formation in enamel-free areas of the occlusal cusp during development of mouse molars: light and electron microscopic studies

Mandibular first molars in mice ranging in age from 18 days prenatal to 5 days postnatal were used for light and electron microscopic examinations of the enamel-free area (EFA) during development of the occlusal cusp (mesiobuccal cusp). Notable morphological changes in the inner enamel epithelium and the cells of the stratum intermedium were observed. At prenatal age of 18 days, the inner enamel epithelium of the EFA (EFA epithelium) was composed of a layer of columnar cells and covered by the cells of the stratum intermedium. Two days after birth, the EFA epithelium was made up largely of preameloblasts, with mitochondria located in the proximal side of the cells toward the stratum intermedium. The cells of the stratum intermedium were irregularly shaped, with wide intercellular spaces between them. At a postnatal age of 3 days, most of the EFA epithelial cells resembled maturation-stage ameloblasts, being short and columnar in shape and having nuclei located in their proximal side. Distal cell membranes were folded, and mitochondria were scattered throughout the cytoplasm. In 4-day-old mice, the EFA epithelium was found to be formed of short columnar or cuboidal cells with distinct intercellular spaces. The cells of the stratum intermedium could no longer be detected, and cells of the EFA epithelium could not be distinguished from those of the stellate reticulum. Odontoblasts of the EFA were arranged and polarized parallel to the basal lamina, and odontoblastic processes extended toward the cusp tip. The orientation of thin and thick collagen fibers within predentin and dentin was also parallel to the basal lamina. Even after dentin mineralization, disrupted basal lamina and long, aperiodic, fine fibrils were found between the epithelium and the dentin. Following the disappearance of the basal lamina and fine fibrils, stippled material and crystals appeared on the dentin surface. The mineralized matrix, which x-ray microanalytical energy peaks identified as containing calcium and phosphorus, was continuous with enamel in the distal slope of the cusp at the cusp tip. Thus, the inner enamel epithelium of the EFA differentiated into secretory cells capable of enamel-like matrix formation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fine structure of the secretory and non-secretory ameloblasts in the frog. II. Fine structure of the non-secretory ameloblast.

The non-secretory ameloblasts present at the enamel-free surfaces of maxillary teeth in the frog Rana pipiens were examined by electron microscopy at different stages of tooth development. Their main fine structural features seem to reflect a transport function. During early tooth development, the non-secretory ameloblasts adjacent to odontoblasts and predentin exhibit extensive lateral surface specializations and numerous cytoplasmic vesicles. During late tooth development, the non-secretory ameloblasts adjacent to mineralizing dentin show numerous cellular junctions, well-developed intercellular channels with numerous interdigitating processes and labyrinthine configurations at their distal surfaces. An intact basal lamina is present between the non-secretory ameloblasts and the dentin surface until the dentin becomes fully mineralized. At this stage the adjacent cells no longer exhibit surface specializations. It is suggested that the non-secretory ameloblasts may participate in the mineralization of adjacent dentin at the enamel-free surfaces. This surface dentin becomes fully mineralized at a later stage of development than the underlying dentin.     

Acellular dental matrices promote functional differentiation of ameloblasts

EDTA treatment of post-natal mouse molars made possible the isolation of cell-free dental matrices composed of basal lamina, predentin, dentin and enamel. Trypsin-isolated dental papillae and enamel organs from embryonic-mouse mandibular molars were combined with isolated matrices and cultured in vitro. In such recombinations, functional odontoblasts were never observed. On the other hand, competent preameloblasts in contact with the epithelial side of occlusal predentin overtly differentiated. Matrices treated with guanidine-EDTA or acetic acid were unable to promote the functional differentiation of ameloblasts. These data are discussed in terms of the epitheliomesenchymal interactions involved in odontogenesis.     

Access of horseradish peroxidase (HRP) to the extracellular spaces of the maturation zone of the rat incisor enamel organ

Adult rats received a single dose of HRP intravenously and were killed from 10 min to 6 hr after injection. Following fixation with glutaraldehyde, the enamel organs were treated with a Graham-Karnovsky-type procedure for peroxidase activity, post-osmicated, and embedded in plastic. Sections were studied with light and electron microscopes. Ten minutes after injection, reaction product was found in all extra-cellular spaces of the enamel organ, at the enamel-ameloblast interface over smooth-ended and intermediate ameloblasts, and in apical surface invaginations and vesicles of the latter cell types. The enamel-ameloblast interface over the ruffle-ended aemlo-blasts and the extracellular spaces within the ruffled border were free of reaction product and remained so for up to 6 hr. The apical terminal bars of the ruffle-ended ameloblasts functioned as a barrier to HRP. The basal terminal bars of the smooth-ended ameloblasts likewise seemed to prevent the passage of the HRP. Possibly, HRP flows in a lateral direction from groups of ruffle-ended into groups of smooth-ended ameloblasts. Between 10 min and 6 hr, HRP was cleared more rapidly from the extra-cellular spaces of the papillary layer than from those of the ameloblast layer, and there was little backflow of tracer from the ameloblast into the papillary layer. Eventually, tracer was cleared also from the extracellular spaces of the ameloblast layer, probably mainly through micropinocytosis by the ameloblasts. A working model is proposed regarding the handling of large molecules by the enamel organ in the maturation zone.     

The distribution and ultrastructure of class II MHC-positive cells in human dental pulp

The distribution and ultrastructure of class II major histocompatibility complex (MHC)-positive cells were investigated in human dental pulp, employing immunohistochemistry using an anti-human leukocyte antigen (HLA)-DR-monoclonal antibody. HLA-DR-immunopositive cells, appearing spindle-like or dendritic in profile, were densely distributed throughout the dental pulp. Under the electron microscope, these cells exhibited various sizes of vesicles containing clear or opaque contents, multivesicular bodies and characteristic fine tubulovesicular structures in their cytoplasm. Some reactive cells possessed coated pits and vesicles including electron-dense materials, indicating an active endocytosis. At the periphery of the pulp tissue, the HLA-DR-immunopositive cells were predominantly situated in the subodontoblastic layer, with some located in the odontoblast layer and/or predentin and extending their cytoplasmic processes into the dentinal tubules. Cell processes of these cells occasionally made contact with several odontoblast processes in the same way as the nerve fibers in the predentin. These cells never contained the typical phagosomes frequently observed in the HLA-DR-immunoreactive macrophages in the subodontoblastic layer and the pulp core. The results suggest that the HLA-DR-immunopositive cells in the odontoblast layer and/or predentin have some regulatory function on the odontoblasts under physiological conditions, in addition to their involvement in the initial defense reaction after tooth injury.     

Carbonic anhydrase II regulates differentiation of ameloblasts via intracellular pH‐dependent JNK signaling pathway

Differentiation of ameloblasts from undifferentiated epithelial cells is controlled by diverse growth factors, as well as interactions between epithelium and mesenchyme. However, there is a considerable lack of knowledge regarding the precise mechanisms that control ameloblast differentiation and enamel biomineralization. We found that the expression level of carbonic anhydrase II (CAII) is strongly up‐regulated in parallel with differentiation of enamel epithelium tissues, while the enzyme activity of CA was also increased along with differentiation in ameloblast primary cultures. The expression level of amelogenin, a marker of secretory‐stage ameloblasts, was enhanced by ethoxzolamide (EZA), a CA inhibitor, as well as CAII antisense (CAIIAS), whereas the expression of enamel matrix serine proteinase‐1 (EMSP‐1), a marker for maturation‐stage ameloblasts, was suppressed by both. These agents also promoted ameloblast proliferation. In addition, inhibition of ameloblast differentiation by EZA and CAIIAS was confirmed using tooth germ organ cultures. Furthermore, EZA and CAIIAS elevated intracellular pH in ameloblasts, while experimental decreases in intracellular pH abolished the effect of CAIIAS on ameloblasts and triggered the activation of c‐Jun N‐terminal kinase (JNK). SP600125, a JNK inhibitor, abrogated the response of ameloblasts to an experimental decrease in intracellular pH, while the inhibition of JNK also impaired ameloblast differentiation. These results suggest a novel role for CAII during amelogenesis, that is, controlling the differentiation of ameloblasts. Regulation of intracellular pH, followed by activation of the JNK signaling pathway, may be responsible for the effects of CAII on ameloblasts. J. Cell. Physiol. 225: 709–719, 2010. © 2010 Wiley‐Liss, Inc.     

Tooth development in a scincid lizard, Chalcides viridanus (Squamata), with particular attention to enamel formation

Comparative analysis of tooth development in the main vertebrate lineages is needed to determine the various evolutionary routes leading to current dentition in living vertebrates. We have used light, scanning and transmission electron microscopy to study tooth morphology and the main stages of tooth development in the scincid lizard, Chalcides viridanus, viz., from late embryos to 6-year-old specimens of a laboratory-bred colony, and from early initiation stages to complete differentiation and attachment, including resorption and enamel formation. In C. viridanus, all teeth of a jaw have a similar morphology but tooth shape, size and orientation change during ontogeny, with a constant number of tooth positions. Tooth morphology changes from a simple smooth cone in the late embryo to the typical adult aspect of two cusps and several ridges via successive tooth replacement at every position. First-generation teeth are initiated by interaction between the oral epithelium and subjacent mesenchyme. The dental lamina of these teeth directly branches from the basal layer of the oral epithelium. On replacement-tooth initiation, the dental lamina spreads from the enamel organ of the previous tooth. The epithelial cell population, at the dental lamina extremity and near the bone support surface, proliferates and differentiates into the enamel organ, the inner (IDE) and outer dental epithelium being separated by stellate reticulum. IDE differentiates into ameloblasts, which produce enamel matrix components. In the region facing differentiating IDE, mesenchymal cells differentiate into dental papilla and give rise to odontoblasts, which first deposit a layer of predentin matrix. The first elements of the enamel matrix are then synthesised by ameloblasts. Matrix mineralisation starts in the upper region of the tooth (dentin then enamel). Enamel maturation begins once the enamel matrix layer is complete. Concomitantly, dental matrices are deposited towards the base of the dentin cone. Maturation of the enamel matrix progresses from top to base; dentin mineralisation proceeds centripetally from the dentin–enamel junction towards the pulp cavity. Tooth attachment is pleurodont and tooth replacement occurs from the lingual side from which the dentin cone of the functional teeth is resorbed. Resorption starts from a deeper region in adults than in juveniles. Our results lead us to conclude that tooth morphogenesis and differentiation in this lizard are similar to those described for mammalian teeth. However, Tomes processes and enamel prisms are absent.     

Expression of clock proteins in developing tooth

Morphological and functional changes during ameloblast and odontoblast differentiation suggest that enamel and dentin formation is under circadian control. Circadian rhythms are endogenous self-sustained oscillations with periods of 24h that control diverse physiological and metabolic processes. Mammalian clock genes play a key role in synchronizing circadian functions in many organs. However, close to nothing is known on clock genes expression during tooth development. In this work, we investigated the expression of four clock genes during tooth development. Our results showed that circadian clock genes Bmal1, clock, per1, and per2 mRNAs were detected in teeth by RT-PCR. Immunohistochemistry showed that clock protein expression was first detected in teeth at the bell stage (E17), being expressed in EOE and dental papilla cells. At post-natal day four (PN4), all four clock proteins continued to be expressed in teeth but with different intensities, being strongly expressed within the nucleus of ameloblasts and odontoblasts and down-regulated in dental pulp cells. Interestingly, at PN21 incisor, expression of clock proteins was down-regulated in odontoblasts of the crown-analogue side but expression was persisting in root-analogue side odontoblasts. In contrast, both crown and root odontoblasts were strongly stained for all four clock proteins in first molars at PN21. Within the periodontal ligament (PDL) space, epithelial rests of Malassez (ERM) showed the strongest expression among other PDL cells. Our data suggests that clock genes might be involved in the regulation of ameloblast and odontoblast functions, such as enamel and dentin protein secretion and matrix mineralization.     

Fate of horseradish peroxidase in the secretion zone of the rat incisor enamel organ

Adult CDF albino rats were killed from 10 min to 6 hr after a single intravenous dose of HRP. Experimental and control tissues were reacted for peroxidase activity and processed for light and electron microscopy. At 10 min, all extracellular spaces of the secretion zone showed reaction product. A reaction was also seen around Tomes' processes and in a layer of enamel spaces in the region of thin enamel. At later time intervals, reactions around Tomes' processes were also seen in regions of thicker enamel. Tracer was located preferentially at the growth fronts of rod and interrod enamel, and also diffused for some distance into enamel. From 2 to 6 hr, the enamel over the transition zone became heavily labeled. The tracer penetrated for more than 90 μm into the enamel and was localized mainly in the interrod enamel. Droplets of dense stippled material in the extracellular spaces between Tomes' processes did not mix with tracer, but sites which contain a light stippled material in the controls (extracellular spaces, vesicles within ameloblasts) showed a reaction. It is concluded that (1) the basal terminal bars of secretory ameloblasts do not impede the flow of large molecules, (2) the apical terminal bars are permeable in early secretion, become increasingly tight as secretion progresses, and are again permeable in the transition zone, (3) ameloblasts can shuttle large extracellular molecules towards the enamel growth fronts, (4) large molecules can diffuse into enamel; rod and interrod enamel differ with regard to the diffusion of large molecules, (5) ameloblasts phagocytose significant amounts of light stippled material. The possibility is considered that extracellular enamel precursor molecules move preferentially towards the enamel growth fronts, perhaps by a mechanism involving membrane flow, and diffuse through enamel in similar fashion as HRP.     

Expression and localization of laminin 5, laminin 10, type IV collagen,and amelotin in adult murine gingiva

The biochemical composition of the internal and external basal laminae in the junctional epithelium differs significantly, and the precise cellular origin of their respective molecules remains to be determined. In the present study, the expression and localization of three basement membrane-specific molecules—laminin 5 (γ2 chain), type IV collagen (α1 chain), and laminin 10 (α5 chain)—and one tooth-specific molecule, amelotin, was analyzed in adult murine gingiva by using in situ hybridization and immunohistochemistry. The results showed that the outermost cells in junctional epithelium facing the tooth enamel strongly expressed laminin 5 mRNA, supporting the immunohistochemical staining data. This suggests that laminin 5 is actively synthesized in junctional epithelial cells and that the products are incorporated into the internal basal lamina to maintain firm epithelial adhesion to the tooth enamel throughout life. Conversely, no amelotin mRNA signals were detected in the junctional epithelial cells, suggesting that the molecules localized on the internal basal lamina are mainly derived from maturation-stage ameloblasts. Weak and sporadic expression of type IV collagen in addition to laminin 10 in the gingiva indicates that these molecules undergo turnover less frequently in adult animals.     

Epithelial-Directed Mesenchyme Differentiation in vitro

To assess the requirement for specific or possibly non-specific epithelial instructions for mesenchymal cell differentiation, we designed studies to evaluate and compare homotypic with heterotypic tissue recombinations across vertebrate species. These studies further tested the hypothesis that determined dental papilla mesenchyme requires epithelial-derived instructions to differentiate into functional odontoblast cells using a serumless, chemically-defined medium. Theiler stage 25 C57BL/6 or Swiss Webster cap stage mandibular first molar tooth organs or trypsin-dissociated, homotypic epithelial-mesenchymal tissue recombinants resulted in the differentiation of odontoblasts within 3 days. Epithelial differentiation into functional ameloblasts was observed within 7 days. Trypsin-dissociated and isolated mesenchyme did not differentiate into odontoblasts under these experimental conditions. Heterotypic recombinants between quail Hamburger-Hamilton stages 22–26 mandibular epithelium and Theiler stage 25 dental papilla mesenchyme routinely resulted in odontoblast differentiation within 3 days in vitro. Odontoblast differentiation and the production of dentine extracellular matrix continued throughout the 10 days in organ culture. Ultrastructural observations of the interface between quail and mouse tissues indicated the reconstitution of the basal lamina as well as the maintenance of an intact basal lamina during 10 days in vitro. Quail epithelial cells did not differentiate into ameloblasts and no enamel extracellular matrix was observed. These results show that quail mandibular epithelium can provide the required developmental instructions for odontoblast differentiation in the absence of serum or other exogenous humoral factors in a chemically-defined medium. They also suggest the importance of reciprocal epithelial-mesenchymal interactions during epidermal organogenesis.     

Localization of calcium in differentiating odontoblasts and ameloblasts before and during early dentinogenesis and amelogenesis in hamster tooth germs

Potassium pyroantimonate-osmium tetroxide cytochemistry has been used to study the distribution of ionic calcium in hamster tooth germs during cell differentiation and during early dentinogenesis and amelogenesis. Before the onset of mineralization, pyroantimonate (PA) reaction product was found in the nucleus of differentiating preameloblasts and preodontoblasts. In the predentin, it was preferentially located along striated collagen fibrils, lying perpendicular to the basal lamina. At the onset of mineralization, a pronounced increase of PA reaction product was evident in the predentin and on the plasma membrane and in mitochondria of both preodontoblasts and preameloblasts opposite the mineralizing mantle dentin. During early enamel mineralization, PA reaction product was present in the "growing" crystal ends, while in the secretory ameloblasts, most of the PA reaction product was localized on the cytoplasmic side of the apical plasma membranes and in mitochondria. When Tomes' processes developed, PA reaction product, both cytoplasmic and membrane bound, was low or absent deep in the processes, but gradually increased toward the apical terminal web. A corresponding gradient of PA reaction product was observed on the opposing enamel crystallites. From this study we conclude that both preodontoblasts and preameloblasts seem to be involved in calcium acquisition necessary for the early stages of mantle dentin mineralization. Tomes' processes seem to regulate the entry of calcium into the enamel mineralization front.     

Ultrastructural distribution of sulfated glycosaminoglycans in epithelial-mesenchymal interface of developing rat tooth germs

We investigated the ultrastructural distribution of sulfated glycosaminoglycans in the epithelial-mesenchymal interface of tooth germs by use of the high-iron diamine thiocarbohydrazide silver proteinate (HID-TCH-SP) staining and enzymatic digestion method. At an early stage in odontoblast differentiation, HID-TCH-SP stain deposits were sparsely distributed in the basement membrane and in the intercellular spaces. Subsequently, as formation of the initial predentin matrix began, HID-TCH-SP stain deposits were densely distributed in the interfibrillar spaces and the basement membrane. Testicular hyaluronidase digested most of those in the progenitor pre-dentin, whereas those in the region of basal lamina resisted enzymatic digestion. Testicular hyaluronidase-resistant HID-TCH-SP stain deposits were susceptible to heparitinase, indicating that the sulfated glycosaminoglycan in the basal lamina is heparan sulfate. Furthermore, the heparan sulfate tended to be regularly arranged at the sites of internal and external lamina densa. However, as progenitor pre-dentin matrix formation proceeded, the numbers of stain deposits temporarily increased and their distribution pattern became irregular, finally tending to disappear with the disruption of basal lamina.     

GTPases RhoA and Rac1 are important for amelogenin and DSPP expression during differentiation of ameloblasts and odontoblasts

Morphogenesis and cytodifferentiation are distinct processes in tooth development. Cell proliferation predominates in morphogenesis; differentiation involves changes in form and gene expression. The cytoskeleton is essential for both processes, being regulated by Rho GTPases. The aim of this study was to verify the expression, distribution, and role of Rho GTPases in ameloblasts and odontoblasts during tooth development in correlation with actin and tubulin arrangements and amelogenin and dentin sialophosphoprotein (DSPP) expression. RhoA, Rac1, and Cdc42 were strongly expressed during morphogenesis; during cytodifferentiation, RhoA was present in ameloblasts and odontoblasts, Rac1 and its effector Pak3 were observed in ameloblasts; and Cdc42 was present in all cells of the tooth germ and mesenchyme. The expression of RhoA mRNA and its effectors RockI and RockII, Rac1 and Pak3, as analyzed by real-time polymerase chain reaction, increased after ameloblast and odontoblast differentiation, according to the mRNA expression of amelogenin and DSPP. The inhibition of all Rho GTPases by Clostridium difficile toxin A completely abolished amelogenin and DSPP expression in tooth germs cultured in anterior eye chamber, whereas the specific inhibition of the Rocks showed only a partial effect. Thus, both GTPases are important during tooth morphogenesis. During cytodifferentiation, Rho proteins are essential for the complete differentiation of ameloblasts and odontoblasts by regulating the expression of amelogenin and DSPP. RhoA and its effector RockI contribute to this role. A specific function for Rac1 in ameloblasts remains to be elucidated; its punctate distribution indicates its possible role in exocytosis/endocytosis.     

Interodontoblastic collagen (von Korff fibers) and circumpulpal dentin formation: an ultrathin serial section study in the cat

The collagenous fibers of von Korff pass from the dentin matrix between the odontoblasts into the dental pulp. Although collagen fibrils are known to be present between odontoblasts, the existence of von Korff fibers has remained controversial. This may be because their continuity between the dentin matrix and the pulp has not been demonstrated ultrastructurally. In this study we have examined the odontoblast layer in the middle to apical regions of perfusion-fixed permanent canine teeth of cats by using transmission electron microscopy. Ultrathin sections of demineralized specimens revealed frequent bundles of collagen fibrils 1) entering the odontoblast layer from the predentin, 2) present between odontoblast cell bodies, and 3) passing from between the odontoblasts into the pulp. The question of continuity of these bundles from the predentin, across the odontoblast layer into the pulp was examined in ultrathin serial sections. Unbroken continuity of a collagen bundle from the predentin between the odontoblasts into the pulp was established in a reconstruction of one series of 22 serial sections and was very strongly suggested by a number of other series in which the numbers of available sections restricted their full visibility. This investigation has shown, therefore, that classical von Korff fibers are present and that these fibers are present in fully erupted teeth with closed apices, i.e., at a time when secondary circumpulpal dentinogenesis is in progress. The findings call for a reexamination of the question of von Korff fibers during mantle dentinogenesis and primary circumpulpal dentinogenesis. Resolution of their existence at the earlier stages of dentinogenesis should be possible by using the ultrathin serial-sectioning technique.     

Fibromodulin-deficient mice display impaired collagen fibrillogenesis in predentin as well as altered dentin mineralization and enamel formation.

To determine the functions of fibromodulin (Fmod), a small leucine-rich keratan sulfate proteoglycan in tooth formation, we investigated the distribution of Fmod in dental tissues by immunohistochemistry and characterized the dental phenotype of 1-day-old Fmod-deficient mice using light and transmission electron microscopy. Immunohistochemistry was also used to compare the relative protein expression of dentin sialoprotein (DSP), dentin matrix protein-1 (DMP 1), bone sialoprotein (BSP), and osteopontin (OPN) between Fmod-deficient mice and wild-type mice. In normal mice and rats, Fmod immunostaining was mostly detected in the distal cell bodies of odontoblasts and in the stratum intermedium and was weaker in odontoblast processes and predentin. The absence of Fmod impaired dentin mineralization, increased the diameter of the collagen fibrils throughout the whole predentin, and delayed enamel formation. Immunohistochemistry provides evidence for compensatory mechanisms in Fmod-deficient mice. Staining for DSP and OPN was decreased in molars, whereas DMP 1 and BSP were enhanced. In the incisors, labeling for DSP, DMP 1, and BSP was strongly increased in the pulp and odontoblasts, whereas OPN staining was decreased. Positive staining was also seen for DMP 1 and BSP in secretory ameloblasts. Together these studies indicate that Fmod restricts collagen fibrillogenesis in predentin while promoting dentin mineralization and the early stages of enamel formation.