In tooth eruptions, the presence of apoptotic epithelial cells at the eruption site has been reported, but the factors that induce apoptosis in these cells remain to be elucidated, as do the induction pathways. In this study, we focused our attention on transforming growth factor beta (TGF-beta), which is known to induce apoptosis during embryonic development. Oral epithelium and dental lamina of maxillary first molars in 8- and 15-day-old rats were used to investigate the induction pathway of apoptosis by performing the immunohistochemical tests outlined below and assessing the characteristics of cells that undergo apoptosis by transmission electron microscopy in rats 8 and 15 days after birth. We examined TGF-beta-receptor 1, TGF-beta inducible transcription factor 1 (TIEG1), NADPHoxidase 4 (Nox4), cytochrome c, caspase-3 (active form and pro-enzyme), apoptosis-inducing protein Daxx, apoptosis signal-regulating kinase 1 (ASK1), glycogen synthase kinase-3 beta phosphorylated on serine 9 (p-GSK-3beta), and beta-catenin. We also performed periodic acid Schiff (PAS) reaction and terminal deoxynucleotidyl transferase-mediated dUTD nick end labeling (TUNEL) staining. At eruption sites 8 days after birth, reactions to TGF-beta-receptor 1, TIEG1, Nox4, cytochrome c, caspase-3, p-GSK-3beta, and beta-catenin, and PAS-positive cells were observed in areas close to the basal layer of oral epithelium through to the center of the dental lamina, but no reaction to Daxx or ASK1 was noted at these sites. Electron microscopy revealed the accumulation of glycogen granules in the cells that showed reactions to the above-mentioned markers as well as in the spaces among them. In the rats 15 days after birth (immediately before tooth eruption), the PAS-positive cells that showed reactions to the above antibodies remained on the buccal side of the epithelium, and high-electron-density apoptotic bodies and TUNEL-positive bodies were noted. Therefore, during tooth eruption, TGF-beta may induce apoptosis of cells rich in glycogen granules, and cytochrome c and caspase-3 may function to induce apoptosis. In addition, reactive oxygen species may be involved in this induction pathway via TIEG1 and Nox4 without involvement of Daxx and ASK1. Moreover, overexpression of p-GSK-3beta and beta-catenin may also contribute to apoptosis of oral epithelium at the eruption site and dental lamina cells. Glycogen storage mediated by p-GSK-3beta and crosstalk between the TGF-beta and Wnt signaling pathways may participate in the formation of tooth eruption passage.

In the Wnt/β-catenin signaling pathway, Wnt signal is transmitted to glycogen synthase kinase-3β (GSK-3β) through Dishevelled (Dvl), GSK-3β activity is inhibited, β-catenin phosphorylation is inhibited by the inactive-type GSK-3β, and β-catenin is transferred to the nucleus where it interacts with lymphoid enhancing factor (LEF)/T-cell factor (TCF), a transcription factor, which is considered to induce and regulate gene expression. In tooth development, it has been reported that Wnt and LEF are expressed at the earliest stage and are related to tooth development, but there are few reports on the situation at a later stage, and there have been no reports on Dvl and GSK-3β. In this study, we immunohistochemically examined the distribution of factors related to the Wnt/β-catenin signaling pathway, Wnt10, Dvl, GSK-3β, p-GSK-3β (inactive GSK-3β), and β-catenin, using serial sections of rat first molar germ to investigate the role of the Wnt signaling pathway in tooth germ development and tooth morphogenesis. Immunostaining for anti-Wnt10, anti-Dvl, anti-GSK-3β, anti-p-GSK-3β, and anti-β-catenin showed positive reactions at the inner enamel epithelium of tooth germ and weakly positive reactions at the dental papilla cells in contact with the inner enamel epithelium at embryonic day 19. At 8 days after birth, immunostaining for every antibody showed positive reactions for preameloblasts and preodontoblasts and more clearly positive reactions for secretory ameloblasts and odontoblasts. These results suggest that Wnt10, Dvl, GSK-3β, p-GSK-3β, and β-catenin are distributed in inner enamel epithelium, secretory ameloblasts, and odontoblasts, and that the Wnt/β-catenin signaling pathway via Dvl and p-GSK-3β is involved in these cells. In addition, for each of the factors, differentiated secretory cells showed more clearly positive reactions than undifferentiated cells; therefore, we conclude that the Wnt10 signaling pathway may be involved in differentiation to ameloblasts and odontoblasts, as well as secretory functions of ameloblasts and odontoblasts.

In tooth development, transforming growth factor beta (TGF-β) and bone morphogenetic protein (BMP) are involved in cell differentiation and matrix protein production. TGF-β and BMP have two signaling pathways: the Smad pathway and the non-Smad pathway. However, only a few studies have focused on the non-Smad pathway in tooth development. TGF-β-activated kinase 1 (TAK1) is activated by TGF-β or BMP and binds to TAK1-binding protein (TAB1), activating p38 or c-Jun N-terminal kinase (JNK), forming the non-Smad signaling pathway. In this study, we examined the distribution of these kinases, TGF-β receptor 1 (TGF-β-R1), BMP receptor-1B (BMPR-1B) and Smad4 in cells of the rat molar germ histochemically, in order to investigate the signaling pathway in each type of cell. Immunostaining for TGF-β-R1, BMPR-1B, Smad4, TAK1, TAB1 and phosphorylated-p38 (p-p38) showed similar reactions. In the cervical loop, reactions were clearer than in other enamel epithelium. In the inner enamel epithelium, signal increased with differentiation into ameloblasts, became strongest in the secretory stage, and decreased rapidly in the maturation stage. Signal also increased upon differentiation from preodontoblasts to odontoblasts. In Hertwig's epithelial sheath, with the exception of BMPR-1B, reactions were stronger in the later stage, showing more enamel protein secretion than in the early stage. However, no clear reaction corresponding to phosphorylated-JNK was observed in any type of cell. These results suggest that TGF-β or BMP is involved in the induction of differentiation of inner enamel epithelium cells into ameloblasts, and preodontoblast differentiation into odontoblasts, the regulation of cervical loop cell proliferation, the elongation or regulation of the epithelial sheath, and the secretion of enamel protein and dentin matrix protein through the non-Smad signaling pathway via TAK1, TAB1 and p38 as well as Smad signaling pathways in the rat molar germ.

The incidence of root caries and hyperesthesia in elderly people tend to increase with increasing age. This can be prevented by enhancing the root surfaces against aciduric bacteria. In order to prevent root caries, a chewing gum containing calcified seaweed rich in Ca, Mg, Na, F and P was created. A dentin block was made from the cervical part of the third molar after removing the cementum. The dentin blocks were immersed in remineralizing solution with extract of the gum with or without calcified seaweed for 2 weeks. The dentin blocks were immersed in demineralizing solution for 6 hours and acid resistance was evaluated after. Demineralization was evaluated by CMR image, demineralization depth (Ld) and mineral loss quantity ( Z) by image analysis, qualitative analysis by EPMA and the surface structure with the SEM. CMR image analysis revealed that the region immersed in aciduric solution had less quantity of demineralization than the region which was not immersed. A significant difference in Ld and Z of the demineralization area after the aciduric treatment between the solution with calcified seaweed and the solution without aciduric treatment (p<0.05) was observed. Levels of Ca, P and Mg were detected in EPMA except for F. In SEM, the dentinal tubules of the demineralized region without aciduric treatment was enlarged which was not seen in demineralized region after aciduric treatment. The results indicate that the chewing gum with calcified seaweed is effective in increasing dentinal acid resistance.Journal of Hard Tissue Biology 20[2] (2011) p87-92

Keratan sulfate proteoglycan and dermatan sulfate proteoglycan have been reported to inhibit collagen fibrillogenesis. We investigated t heir distribution in order to evaluate the role of proteoglycan in dentinogenesis.Specimens of porcine tooth-germ dentin and erupted teeth were the materials, on which antibodies of keratin sulfate and dermatan sulfate proteoglycan were used. Predentin was found to be positive for both antibodies, and the reaction ceased in the calcification front. Uniformly thick collagen fibrils (30-70 nm in diameter) were distributed in the predentin matrix, which would become intertubular dentin in the future.Both antibodies reacted positively along these fibrils. On the other hand, the surface layer of dentin in the tooth germ and that in erupted teeth, collagen fibrils of 10-300 nm in diameter were occasionally noted in dentinal tubules whose odontoblastic processes had disappeared, and these heterogeneous fibrils were negative for both antibodies.Our findings suggest that keratan sulfate proteoglycan and dermatan sulfate proteoglycan distributed in the predentin inhibit calcification of collagen fibrils in the uncalcified matrix and disappear in the calcification 3 front. It was further suggested that keratin sulfate proteoglycan and dermatan sulfate proteoglycan distributed along collagen fibrils in the predentin matrix maintain uniform thickness, whereas collagen fibrils in dentinal tubules varied in thickness because of the absence of involvement of both proteoglycans. Keratan sulfate proteoglycan and dermatan sulfate proteoglycan were therefore thought to be involved in both calcification and matrix formation.