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A previous study suggested that fibroblast growth factor (FGF) signaling plays an important role in dentin formation during tooth development. In this study, to examine dentin formation after tooth eruption involving secondary and tertiary dentin, we analyzed the expression patterns and expressing cells of Fgfr1, -2c, and -3c in mouse maxillary first molars (M1). Since it is difficult to recover the mRNAs from mineralized tissues, we tested methods for extraction after fixation and decalcification of teeth. We successfully obtained consistent results with quantitative real-time PCR (qPCR) using β-actin transcripts for validation. qPCR for Dentin sialo phosphoprotein (Dspp), Fgfr1, -2c, and -3c transcripts was performed on mice at ages of 2–20 weeks. The results showed that the highest expression levels of Dspp and Fgfr2c occurred at 2 weeks old followed by lower expression levels after 4 weeks old. However, the expression levels of Fgfr1 and Fgfr3c were constant throughout the experimental period. By in situ hybridization, Dspp, Fgfr1, and Fgfr3c transcripts were detected in odontoblasts at ages of 2 and 4 weeks. In addition, Dspp and Fgfr1 transcripts were detected in odontoblasts facing reactionary dentin at 8 weeks old. These results suggest that FGF-FGFR signaling might be involved in the regulation of odontoblasts even after tooth eruption, including secondary and tertiary dentin formation. Moreover, our modified method for extracting mRNA from mineralized tissues after fixation and decalcification successfully produced consistent results.

The dentin-pulp complex is a unique structure in teeth that contains both hard and soft tissues. Generally, deep caries and trauma cause damage to the dentin-pulp complex, and if left untreated, this damage will progress to irreversible pulpitis. The aim of this study was to fabricate a layered cell sheet composed of rat dental pulp (DP) cells and odontogenic differentiation of pulp (OD) cells and to investigate the ability to regenerate the dentin-pulp complex in a scaffold tooth. We fabricated two single cell sheets composed of DP cells (DP cell sheet) or OD cells (OD cell sheet) and a layered cell sheet made by layering both cells. The characteristics of the fabricated cell sheets were analyzed using light microscopy, scanning electron microscope (SEM), hematoxylin-eosin (HE) staining, and immunohistochemistry (IHC). Furthermore, the cell sheets were transplanted into the subrenal capsule of immunocompromised mice for 8 weeks. After this, the regenerative capacity to form dentin-like tissue was evaluated using micro-computed tomography (micro-CT), HE staining, and IHC. The findings of SEM and IHC confirmed that layered cell sheets fabricated by stacking OD cells and DP cells maintained their cytological characteristics. Micro-CT of layered cell sheet transplants revealed a mineralized capping of the access cavity in the crown area, similar to that of natural dentin. In contrast, the OD cell sheet group demonstrated the formation of irregular fragments of mineralized tissue in the pulp cavity, and the DP cell sheet did not develop any hard tissue. Moreover, bone volume/tissue volume (BV/TV) showed a significant increase in hard tissue formation in the layered cell sheet group compared with that in the single cell sheet group (

Hyaluronan (HA) is a major constituent molecule in most extracellular matrices and is synthesized by Hyaluronan synthase (Has). In the present study, we examined expression patterns of Has1, -2, -3 mRNA in developing mouse molar and incisor tooth germs from embryonic day (E) 11.5 to postnatal day (P) 7, focusing on Hertwig's epithelial root sheath (HERS) and the apical bud in particular. Has1 mRNA expression was not detected in all tooth germs examined. Has2 mRNA was expressed in the surrounding mesenchyme from E12.0 to 18.0 in both molar and incisor tooth germs, but disappeared after birth. Meanwhile, Has3 mRNA was exclusively expressed within the enamel organ, especially in the inner enamel epithelium (IEE), stellate reticulum (SR), and stratum intermedium (SI) until the early bell stage at E16.0. Has3 mRNA disappeared as IEE differentiated into differentiating ameloblasts (dABs), but remained in SI until the root developmental stage of the molar tooth germ at P7. Has3 mRNA was also expressed in HERS until P7. In incisors, Has3 mRNA was expressed in the apical bud, especially in the transit-amplifying (TA) cell region from E16.0 to P7, and in the papillary layer (PL) adjacent to the mature enamel. These gene expression patterns suggested that Has3 is the main control factor for prenatal and postnatal HA synthesis of the tooth germ, and may in part regulate crown and root formation of the tooth germ, maintenance of stem cell niches in the apical bud as well as mineral transport in PL.

The small-sized teleost fish medaka, Oryzias latipes, has as many as 1000 pharyngeal teeth undergoing continuous replacement. In this study, we sought to identify the tooth-forming units and determine its replacement cycles, and further localize odontogenic stem cell niches in the pharyngeal dentition of medaka to gain insights into the mechanisms whereby continuous tooth replacement is maintained. Three-dimensional reconstruction of pharyngeal epithelium and sequential fluorochrome labeling of pharyngeal bones and teeth indicated that the individual functional teeth and their successional teeth were organized in families, each comprising up to five generations of teeth and successional tooth germs, and that the replacement cycle of functional teeth was approximately 4 weeks. BrdU label/chase experiments confirmed the existence of clusters of label-retaining epithelial cells at the posterior end of each tooth family where the expression of pluripotency marker Sox2 was confirmed by in situ hybridization. Label-retaining cells were also identified in the mesoderm immediately adjacent to the posterior end of each tooth family. These data suggest the importance of existence of slow-cycling dental epithelial cells and Sox2 expressions at the posterior end of each tooth family to maintain continuous tooth formation and replacement in the pharyngeal dentition of medaka.

Purpose To examine the effect of occlusal hypofunctional conditions on orthodontic tooth movement and its relation to the structure and quality of alveolar bone using the rat model. Materials and methods Twelve-week-old male Sprague-Dawley rats were divided into 4 groups of 8 animals each: normal occlusion (N) group, normal occlusion with tooth movement (M) group, occlusal hypofunction (H) group, and occlusal hypofunction with tooth movement (HM) group. In H and HM groups, the anterior bite plate and metal cap were attached to the maxillary and mandibular incisors using a light-curing composite resin to induce the occlusal hypofunctional condition. In M and HM groups, an orthodontic force was applied in a palatal direction to the buccal surface of the maxillary first molar (M1) using a nickel–titanium alloy wire. Micro-CT imaging and histomorphometric analysis using fluorescent bone labeling of the alveolar bone surrounding the M1s were performed in each group. Results Tooth movement of M1 in HM group, was rather accelerated with enhanced tipping than in M group. Micro-CT analysis revealed significant decrease in bone volume fraction, bone mineral density and trabecular thickness of the interradicular bone in HM group among the experimental groups. The fluorescent labeling lines in the interradicular bone were decreased in number in H and M groups compared with N group. A few discontinuous irregular dotted lines-like labeling was observed in HM group. Conclusion The occlusal hypofunctional condition accelerates orthodontic tooth movement of the respective teeth, while it results in severe bone loss in the surrounding alveolar bone.

Objective The C-terminal end of nascent amelogenin is dissociated immediately after secretion and rapidly re-absorbed by ameloblasts, presumably by endocytosis. The purpose of this study was to test whether organic anion transporters (OATs) are also involved in the re-absorption process of enamel matrix proteins via non-endocytotic pathways. Materials and methods Localization of OAT1, OAT2, and OAT3 in rat tooth germs was examined by immunohistochemistry using specific antibodies. Actual translocation of organic anions through the ameloblast layer was further tested by systemic tracer experiments in rats in which Lucifer Yellow (LY), a fluorescent organic anion, was used as a tracer. Results In rat tooth germs, OAT2 was associated exclusively with the distal cell membranes of secretory ameloblasts where Tomes' processes were developed and disappeared when matrix formation was terminated. On the other hand, OAT1 was absent in secretory ameloblasts and was colocalized with the ruffled border of ruffle-ended ameloblasts in the maturation stage. OAT3 was undetectable in ameloblasts and located instead only in the stratum intermedium cells. Systemic administration of LY resulted in intense labeling of immature enamel and also a transient labeling of the cytosol of secretory ameloblasts immunopositive for OAT2. In the maturation stage, cytosolic labeling of LY was negligible in all cells of the enamel organs, including ameloblasts. Conclusions These data suggest the existence of OATs in rat tooth germs and their possible involvement in matrix re-absorption at least in the secretory stage of amelogenesis.

There are many reports on the variation of origin site of the lingual artery branching from the external carotid artery. However, there are few reports systematically investigating the course of the lingual artery in detail from branching site to the body of tongue. The purpose of this study is to classify systematically the courses of the lingual artery including variations. Using 111 body sides of 63 Japanese cadavers for dissection practices, the lingual artery and the surrounding structures were investigated gross anatomically. The courses of the lingual artery were classified into five types based on the positional relationships with the hyoglossus and the mylohyoid as follows; type M: coursing medial to the hyoglossus (normal course, 104 sides), type L: coursing lateral to the hyoglossus (2 sides), type T: transferring its course from lateral to medial to the hyoglossus (2 sides), type P: penetrating the mylohyoid (2 sides), and type C: the coinciding of types M and P (1 side). Types L, T, P, and C were considered to be variant lingual arteries. Types M and T, type L, and type P arose from the external carotid, facial, and submental arteries, respectively. In types L and P, adding to the variant lingual artery, the remnant of the normal lingual artery was also observed. The present study provides detailed information on the courses of lingual artery which will be of clinical importance especially in the super-selective arterial angiography.

Mineralization of circumpulpal dentin has been interpreted in such a way that predentin matrix is abruptly converted to almost fully mineralized dentin at the mineralization front. A group of investigators pointed out the existence of intermediary layer along the mineralization front of rat incisor dentin and claimed that dentin mineralization is a rather transient process. Owing to a paucity of information, however, the entity of transient mineralization of dentin has remained elusive. Here we confirmed the existence of a lightly mineralized layer (LL) along the mineralization front of rat incisor dentin, recognizable by both light and electron microscopy, in routinely processed specimens. LL less than 3 µm thick was shown to be located along the mineralization front of crown-analog dentin and tapered out toward the root analog of the incisor. Electron microscopy revealed that mineral deposition first occurred in the non-collagenous matrix of LL and that mineralization of collagen fibers took place sometime later at the conventional mineralization front. Microscopic appearance of the mineral phase of LL varied considerably depending on the histological processing of ultrathin sections, thus explaining the inconsistent interpretation of dentin mineralization in previous studies. These data suggest that mineralization of circumpulpal dentin in rat incisors proceeds in a stepwise or a transient manner, initiated by crystal deposition in the non-collagenous matrix followed by massive mineral deposition in collagen fibers at the mineralization front. The thickness of LL where only the non-collagenous matrix is mineralized may vary in relation to differences in the local non-collagenous matrix and also the rate of collagen mineralization in the respective portions of circumpulpal dentin.

C-src deficiency is characterized by osteopetrosis due to impaired bone resorption by hypofunctional osteoclasts and the resultant failure of tooth eruption. In preliminary observations, we frequently encountered erupted molars in c-src deficient mice unlike in other osteopetrotic animals. Here we examine the effects of c-src deficiency on the development of molar teeth with an emphasis on the spatial relation of growing teeth with the surrounding bones. In c-src deficient mice, the magnitude of tooth impaction differed considerably among the types of molars; all maxillary 1st molars were totally impacted deep in the alveolar sockets, whereas most mandibular 1st molars fully erupted into oral cavity. Distribution of osteoclasts in the alveolar bone was identical among all types of molars, and electron microscopy revealed signs of bone resorbing activity in these osteoclasts despite the absence of a ruffled border. From early development, the alveolar space was much narrower in the upper molar tooth germs than in the lower ones in both wild type and homozygous animals, and particularly so in the upper 1st molars. Current observations thus indicate a significant contribution of "hypofunctional osteoclasts" in c-src deficient mice in molar tooth development except for the upper 1st molars, which appear to require highly functional osteoclasts to gain sufficient space for them to grow normally. Taken together, these findings on the seemingly tooth-type specific effects of c-src deficiency on the development and eruption of molar teeth in c-src deficient mice can be attributed to the given differential spatial relation of the respective tooth germs with the surrounding bones in the presence of hypofunctional osteoclasts.

The extracellular matrix (ECM) of bone and dentin contains several non-collagenous proteins. One category of non-collagenous protein is termed the SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein) family, that includes osteopontin (OPN), bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), and matrix extracellular phosphoglycoprotein (MEPE). These polyanionic SIBLING proteins are believed to play key biological roles in the mineralization of bone and dentin. Although the specific mechanisms involved in controlling bone and dentin formation are still unknown, it is clear that some functions of the SIBLING family members are dependent on the nature and extent of post-translational modifications (PTMs), such as phosphorylation, glycosylation, and proteolytic processing, since these PTMs would have significant effects on their structure. OPN and BSP are present in the ECM of bone and dentin as full-length forms, whereas amino acid sequencing indicates that DMP1 and DSPP exist as proteolytically processed fragments that result from scission of X-Asp bonds. We hypothesized that the processing of DMP1 and DSPP is catalyzed by the PHEX enzyme, since this protein, an endopeptidase that is predominantly expressed in bone and tooth, has a strong preference for cleavage at the NH2-terminus of aspartyl residue. We envision that the proteolytic processing of DMP1 and DSPP may be an activation process that plays a significant, crucial role in osteogenesis and dentinogenesis, and that a failure in this processing would cause defective mineralization in bone and dentin, as observed in X-linked hypophosphatemic rickets.

Cloning and sequencing of the cDNA indicates that dentin sialophosphoprotein (DSPP) is a precursor of both dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Dentin sialophosphoprotein must be proteolytically processed to form these two extracellular matrix (ECM) proteins. Numerous studies led us to conclude that DSP (and DSPP) are exclusively expressed by odontoblasts and preameloblasts. However, recent observations suggest a wider distribution. To test this hypothesis, we conducted systematic studies on rat first molar during root formation with immunohistochemical techniques using specific anti-DSP polyclonal and monoclonal antibodies. We also performed in situ hybridization, using high-stringency RNA probes to detect DSP transcripts. Immunohistochemical studies demonstrated that DSP is not only localized in odontoblasts, dentin ECM and preameloblasts, but also in alveolar bone, cellular cementum, osteocytes, cementocytes, and their matrices. The results of in situ hybridization were consistent with those from immunohistochemistry, showing the expression of DSP transcripts in osteoblasts of alveolar bone, fibroblasts in periodontal ligament and cementoblasts in cellular cementum. Together, these observations suggest that DSP is involved in formation of the periodontium as well as tooth structures.

Dentin sialoprotein (DSP) is a glycoprotein accounting for 5-8% of the dentin non-collagenous proteins. The cDNA sequence predicts that rat DSP has 13 potential casein kinase phosphorylation sites and six potential N-linked glycosylation sites. However, its total phosphorylation level, as well as the nature and locations of the carbohydrate moieties, are unknown. Our findings in the present study show that rat DSP has 6.2 phosphates per molecule and that the majority of carbohydrates are attached to the protein through N-linked glycosylations. During our separation of dentin non-collagenous proteins with ion-exchange chromatography, we observed high molecular weight components eluting late in the salt gradient that were recognized by anti-DSP antibodies. We have purified these high molecular weight components using a monoclonal anti-DSP antibody affinity column. Data from amino acid analysis, phosphate level measurements and Edman degradation of tryptic peptides unequivocally proved that the very acidic, high molecular weight components are isoforms of DSP (designated HMW-DSP). Deglycosylation analysis indicates that the slower migration rate of HMW-DSP on SDS-PAGE results from its higher level of carbohydrate modifications.

The distribution of matrix vesicles and its role in biological mineralization were examined in bone and dental hard tissues of the rat after daily administrations of 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP), a potent inhibitor of mineralization, for 7 or 14 days. Newly formed, nonmineralized matrices of the HEBP-affected bone and mesodermal dental hard tissues other than circumpulpal dentin contained numerous mineral-filled matrix vesicles (MV), randomly distributed throughout the collagenous matrix. The distribution density of the mineral-filled MV in the HEBP-affected matrices of calvaria, metaphyseal trabecular bone, alveolar bone, and cellular cementum ranged from 60 to 70 per 100 microm(2), and no statistically significant differences were noted among the values. In the HEBP-affected dentin, however, MV were located only in the nonmineralized matrix of mantle dentin and totally absent in the circumpulpal dentin layers. Instead, the HEBP-affected circumpulpal dentin contained a dense meshwork of noncollagenous matrix enriched with calcium and phosphorus. Comparable meshwork structures were undetectable in nonmineralized matrices of the other hard tissues affected by HEBP. These observations suggest that a certain population of MV (60-70 per 100 microm(2)) is involved in the process of appositional mineralization in most of the mesodermal hard tissues, in addition to their well-known role in initial mineral induction in these tissues. Circumpulpal dentin appears to be an exception, where MV are not required for the appositional mineralization process. Exclusive localization of dentin phosphoproteins in circumpulpal dentin layers must take place to facilitate appositional mineralization at the calcification front, in the absence of MV.

Fibroblast growth factors (FGFs) regulate the proliferation and differentiation of various cells via their respective receptors (FGFRs). During the early stages of tooth development in fetal mice, FGFs and FGFRs have been shown to be expressed in dental epithelia and mesenchymal cells at the initial stages of odontogenesis and to regulate cell proliferation and differentiation. However, little is known about the expression patterns of FGFs in the advanced stages of tooth development. In the present study, we focused on FGF18 expression in the rat mandibular first molar (M1) during the postnatal crown and root formation stages. FGF18 signals by RT-PCR using cDNAs from M1 were very weak at postnatal day 5 and were significantly up-regulated at days 7, 9 and 15. Transcripts were undetectable by in situ hybridization (ISH) but could be detected by in situ RT-PCR in the differentiated odontoblasts and cells of the sub-odontoblastic layer in both crown and root portions of M1 at day 15. The transcripts of FGFR2c and FGFR3, possible candidate receptors of FGF18, were detected by RT-PCR and ISH in differentiated odontoblasts throughout postnatal development. These results suggest the continual involvement of FGF18 signaling in the regulation of odontoblasts during root formation where it may contribute to dentin matrix formation and/or mineralization.

Ectodermal contribution to the induction of pharyngeal teeth that form in the endodermal territory of the oropharyngeal cavity in some teleost fishes has been a matter of considerable debate. To determine the role of ectodermal cell signaling in scale and tooth formation and thereby to gain insights in evolutionary origin of teeth, we analyzed scales and teeth in rs-3 medaka mutants characterized by reduced scale numbers due to aberrant splicing of the ectodysplasin-A receptor (edar). Current data show that, in addition to a loss of scales (83% reduction), a drastic loss of teeth occurred in both oral (43.5% reduction) and pharyngeal (73.5% reduction) dentitions in rs-3. The remaining scales of rs-3 were irregular in shape and nearly 3 times larger in size relative to those of the wild-type. In contrast, there was no abnormality in size and shape in the remaining teeth of rs-3. In wild-type medaka embryos, there was a direct contact between the surface ectoderm and rostral endoderm in pharyngeal regions before the onset of pharyngeal tooth formation. However, there was no sign of ectodermal cell migration in the pharyngeal endoderm and hence no direct evidence of any ectodermal contribution to pharyngeal odontogenesis. These data suggest differential roles for Eda-Edar signaling in the induction and growth of scales and teeth and support the intrinsic odontogenic competence of the rostral endoderm in medaka.

The enamel organ engaged in enamel matrix formation in tooth germs comprises four different cell types: the ameloblasts, the cells of the stratum intermedium, stellate reticulum, and the outer enamel epithelium, each characterized by distinct structural features. In ordinary primary cultures of tooth-derived cells, these cells generally become flat in profile and hardly regain their original profiles comparable to those in vivo, even under conditions that can induce the expression of functional markers from these cells. To overcome this limitation inherent to the cell culture of tooth-derived cells, we introduced a novel co-culture method, a "three-dimensional and layered (TDL) culture", a three-dimensional (3D) culture of dental pulp-derived cells dispersed in type I collagen gel combined with a layered culture of enamel epithelial cells seeded on top of the gel to establish thereby a culture condition where the functional tooth-derived cells regain their original structures and spatial arrangements. We subjected the TDL gels thus prepared to floating cultures and found that, in the layered epithelial cells, those facing the 3D gel became cuboidal/short columnar in shape, showed cell polarity and well-developed intercellular junctions, had PAS positive material in their cytoplasm, and expressed a distinct immunoreactivity for cyotokeratin 14 and amelogenins. Pulpal cells in the gel displayed a strong ALP activity throughout the 3D gel. The current observations have clearly shown that the structural and functional features reminiscent of early secretory ameloblasts could be restored in the enamel organ-derived cells in a TDL culture.

Ectopic tooth transplants are known to receive rich innervation of local neurons, but the precise location and structural features of neurites in the pulp and periodontal ligament (PDL) of such transplants are unclear. In this experiment, the molar tooth germs of rat embryos and incisors of young rats were subcutaneously transplanted into the dorsal regions of rats and processed, at various time intervals, for immunohistochemical demonstration of neural elements. Teeth with periodontal tissue elements developed in most of the molar transplants in 6 or 8 wk and received rich innervation, including some autonomic fibres, in the pulp. Nerve elements were also confirmed to be present in the PDL of these transplants, including specialized nerve ending-like structures reminiscent of the periodontal Ruffini endings. Mechanoreceptor-like structures were also induced in the regenerated PDL of similarly transplanted incisors, although the success rate was low. We conclude that rich and highly ordered innervation of the pulp, and occasional development of mechanoreceptors in the regenerated PDL of ectopic dental transplants, imply a high probability of successful induction of teeth with both nociceptive and mechanical sensations in the ectopic tooth and/or tooth germ transplant systems, although differentiation of mechanoreceptor-like nerve endings occurred in only a few rare cases.

Degradation of enamel matrix proteins, and their removal during early maturation, is critical for the growth of large enamel crystals in the subsequent processes of enamel maturation. In this study, we sought to demonstrate, using in vivo zymography, the exact sites of proteolysis in maturing enamel and its relationship to the overlying ameloblasts. The maturing enamel surfaces of rat and bovine incisors were exposed and painted either with pre-exposed autoradiographic emulsion or with densely fluorescein-conjugated (DQ) gelatin. After a few hours, photographic development of the emulsion revealed alternate black and white banding patterns over the maturing enamel surface. DQ gelatin also revealed similar banding patterns of fluorescent and non-fluorescent regions. White, powdery areas of emulsion and fluorescent bands of DQ gelatin both corresponded to the areas of ruffle-ended ameloblasts, at least up to the mid stages of enamel maturation, implicating a predominant contribution of ruffle-ended ameloblasts in the degradation of enamel matrix proteins. Powdery white bands in autoradiographic emulsion shifted from the areas of ruffle-ended to smooth-ended ameloblasts in late maturation in both bovine and rat incisors and were not influenced by proteinase inhibitors or heat inactivation, implicating non-enzymatic interactions. DQ gelatin, in fact, did not generate any fluorescence in such smooth-ended ameloblast regions.

Here we present the developmental progression of bioengineered pig teeth from 1 to 25 weeks of development. We demonstrate that 2-25 week implants contained embryonic tooth bud- and cap-stage tooth structures consisting of dental epithelium expressing the sonic hedgehog gene and condensed dental mesenchyme. Implants harvested at 18-25 weeks also contained tooth bud-like structures, as well as mature tooth structures containing enamel, dentin and pulp tissues. Immunohistochemical analyses confirmed the expression of dentin- and enamel-specific proteins in differentiated bioengineered tooth tissues. Three-dimensional computer modelling further demonstrated a spatial organization of enamel, dentin and pulp tissues resembling that of natural teeth. We conclude that bioengineered teeth commonly exhibit morphological stages characteristic of naturally forming teeth. Furthermore, the presence of immature tooth buds at all times assayed and increased numbers of bioengineered tooth structures over time suggests that porcine dental progenitor cells maintain the ability to form teeth for at least 25 weeks.

Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) are acidic proteins found in the extracellular matrices of bones and teeth. Recent data from gene knockouts, along with those of gene mutations, indicate that these two phosphoproteins are critical for bone and tooth development and/or maintenance. However, the precise functions of the two proteins have not been elucidated. In order to gain insights into their functions in tooth formation, we performed systematic, comparative investigations on the immunolocalization of DMP1 and dentin sialoprotein (DSP, a cleaved fragment of DSPP), using the rat first molar at different developmental stages as a model. Immunohistochemistry (IHC) was performed with specific, monoclonal antibodies against the COOH-terminal fragments of DMP1 and against DSP. In 1-day- and 1-week-old rats, weak immunoreactions for DMP1 were observed in dentinal tubules while stronger reactions for DSP were seen in the tubules and predentin. In rats older than 2 weeks, immunoreactions for DMP1 were found in dentinal tubules, predentin and odontoblasts. In 5-week- and 8-week-old rats, strong immunoreactions for DMP1 were widely distributed in odontoblasts and predentin. The distribution pattern of DSP was strikingly similar to that of DMP1 after 2 weeks and the localization of each was distinctly different from that of bone sialoprotein (BSP). The unique colocalization of DMP1 and DSPP in tooth development suggests that the two proteins play complementary and/or synergistic roles in formation and maintenance of healthy teeth.

In addition to seven known exons of the amelogenin gene, recent studies have identified two exons downstream of amelogenin exon 7 in genomic DNA of mouse and rat. Here the spatial and temporal expression of mRNAs and of the translated proteins derived from alternative splicing of the amelogenin gene ending with exon 8 and exon 9 were examined by in situ hybridization (ISH) and immunohistochemistry (IHC). RNA signals for exons 8 and 9 were expressed in the ameloblast layer extending from early presecretory to postsecretory transitional stages of amelogenesis. IHC of amelogenin proteins that include sequences encoded by these exons demonstrated identical localization of these proteins in the ameloblast layer corresponding to RNA signals identified by ISH. There was intense immunostaining of the enamel matrix secreted by these cells. Western blotting analysis of rat enamel proteins revealed three distinct protein bands with sequences encoded by the new exons. These data confirmed the existence of the transcripts of alternatively spliced mRNAs coding for exons 8 and 9 of the amelogenin gene in rat tooth germs and suggest that the translated proteins contribute to the heterogeneity of amelogenins and have some significant roles in enamel formation and mineralization.

In order to clarify the initial process of dentin mineralization, the inhibitory effect of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) on dentin mineralization was investigated. Rats (100 g) were subcutaneously injected with HEBP (8 mg P/kg) for 7 or 14 d, and the incisors were processed for Ca histochemistry and/or electron microscopy. HEBP-treated incisors demonstrated ladder-like alternate rows of mineralized and non-mineralized dentin at the apical end. GBHA revealed moderate Ca reactions in the non-mineralized circumpulpal dentin matrix where electron microscopy revealed rich distribution of fine mesh-like electron-dense material. Non-mineralized mantle dentin matrix was negative for Ca but contained numerous matrix vesicles (MVs) filled with crystalline and/or amorphous mineral deposits. Mineralization of circumpulpal dentin occurred independently of MV-rich mantle dentin layer in affected specimens. Our data provide histochemical evidence of possible Ca-binding property of the circumpulpal dentin matrix and its absence in the mantle dentin where MV-mediated mineralization occurs. In the mantle dentin, HEBP does not interfere with crystal growth in MVs but inhibits its outgrowth after membrane rupture. It is proposed that circumpulpal dentin matrix has a potential to mineralize independently of MV-mediated mineralization of mantle dentin, although MVs determine the initial site and timing of dentin mineralization.

Glucose is an essential source of energy for body metabolism and is transported into cells by glucose transporters (GLUTs). Well-characterized class I GLUT is subdivided into GLUTs1–4, which are selectively expressed depending on tissue glucose requirements. However, there is no available data on the role of GLUTs during tooth development. This study aims to clarify the functional significance of class I GLUT during murine tooth development using immunohistochemistry and an in vitro organ culture experiment with an inhibitor of GLUTs1/2, phloretin, and Glut1 and Glut2 short interfering RNA (siRNA). An intense GLUT1-immunoreaction was localized in the enamel organ of bud-stage molar tooth germs, where the active cell proliferation occurred. By the bell stage, the expression of GLUT1 in the dental epithelium was dramatically decreased in intensity, and subsequently began to appear in the stratum intermedium at the late bell stage. On the other hand, GLUT2-immunoreactivity was weakly observed in the whole tooth germs throughout all stages. The inhibition of GLUTs1/2 by phloretin in the bud-stage tooth germs induced the disturbance of primary enamel knot formation, resulting in the developmental arrest of the explants and the squamous metaplasia of dental epithelial cells. Furthermore, the inhibition of GLUTs1/2 in cap-to-bell-stage tooth germs reduced tooth size in a dose dependent manner. These findings suggest that the expression of GLUT1 and GLUT2 in the dental epithelial and mesenchymal cells seems to be precisely and spatiotemporally controlled, and the glucose uptake mediated by GLUT1 plays a crucial role in the early tooth morphogenesis and tooth size determination.