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The epithelial-mesenchymal interactions are essential for the initiation and regulation of the development of teeth. Following the initiation of tooth development, numerous growth factors are secreted by the dental epithelium and mesenchyme that play critical roles in cellular differentiation. During tooth morphogenesis, the dental epithelial stem cells differentiate into several cell types, including inner enamel epithelial cells, which then differentiate into enamel matrix-secreting ameloblasts. Recently, we reported that the novel basic-helix-loop-helix transcription factor, AmeloD, is actively engaged in the development of teeth as a regulator of dental epithelial cell motility. However, the gene regulation mechanism of AmeloD is still unknown. In this study, we aimed to uncover the mechanisms regulating AmeloD expression during tooth development. By screening growth factors that are important in the early stages of tooth formation, we found that TGF-β1 induced AmeloD expression and ameloblast differentiation in the dental epithelial cell line, SF2. TGF-β1 phosphorylated ERK1/2 and Smad2/3 to induce AmeloD expression, whereas treatment with the MEK inhibitor, U0126, inhibited AmeloD induction. Promoter analysis of AmeloD revealed that the proximal promoter of AmeloD showed high activity in dental epithelial cell lines, which was enhanced following TGF-β1 stimulation. These results suggested that TGF-β1 activates AmeloD transcription via ERK1/2 phosphorylation. Our findings provide new insights into the mechanisms that govern tooth development., (© 2021 Wiley Periodicals LLC.)

Objective: This study evaluated the angiogenesis-enhancing potential of a tricalcium silicate-based mineral trioxide aggregate (ProRoot MTA), Biodentine, and a novel bioceramic root canal sealer (Well-Root ST) in human dental pulp stem cells (hDPSCs), human periodontal ligament stem cells (hPLSCs), and human tooth germ stem cells (hTGSCs)., Methodology: Dulbecco's modified Eagle's medium was conditioned for 24 h by exposure to ProRoot MTA, Biodentine, or Well-Root ST specimens (prepared according to the manufacturers' instructions). The cells were cultured in these conditioned media and their viability was assessed with 3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfo-phenyl)-2H tetrazolium (MTS) on days 1, 3, 7, 10, and 14. Angiogenic growth factors [platelet-derived growth factor (PDGF), basic fibroblast growth factor (FGF-2), and vascular endothelial growth factor (VEGF)] were assayed by sandwich enzyme-linked immunosorbent assay (ELISA) on days 1, 7, and 14. Human umbilical vein endothelial cell (HUVEC) migration assays were used to evaluate the vascular effects of the tested materials at 6-8 h. Statistical analyses included Kruskal-Wallis, Mann-Whitney U, and Friedman and Wilcoxon signed rank tests., Results: None of tricalcium silicate-based materials were cytotoxic and all induced a similar release of angiogenic growth factors (PDGF, FGF-2, and VEGF) (p>0.05). The best cell viability was observed for hDPSCs (p<0.05) with all tricalcium silicate-based materials at day 14. Tube formation by HUVECs showed a significant increase with all tested materials (p<0.05)., Conclusion: The tricalcium silicate-based materials showed potential for angiogenic stimulation of all stem cell types and significantly enhanced tube formation by HUVECs.

Chondroitin sulfate proteoglycan (CSPG) is an important component of extracellular matrix (ECM), it is composed of a core protein and one or more chondroitin sulfate glycosaminoglycan side chains (CS-GAGs). To investigate the roles of its CS-GAGs in dentinogenesis, the mouse mandibular first molar tooth germs at early bell stage were cultivated with or without β-xyloside. As expected, the CS-GAGs were inhibited on their incorporation to CSPGs by β-xyloside, accompanied by the change of morphology of the cultured tooth germs. The histological results and the transmission electron microscopy (TEM) investigation indicated that β-xyloside exhibited obvious inhibiting effects on odontoblasts differentiation compared with the control group. Meanwhile the results of immunohistochemistry, in situ hybridization and quantitative RT-PCR for type I collagen, dentin matrix acidic phosphoprotein 1 and dentin sialophosphoprotein, the products of differentiated odontoblasts, further proved that odontoblasts differentiation was inhibited. Collagen fibers detected in TEM decreased and arranged in disorder as well. Thus we conclude that the inhibition of CS-GAGs incorporation to CSPGs can affect odontoblast differentiation in cultured embryonic mouse molars.

Objective: F-spondin is an extracellular matrix (ECM) protein that belongs to the thrombospondin type I repeat superfamily and is a negative regulator of bone mass. We have previously shown that f-spondin is specifically expressed in the dental follicle (DF), which gives rise to the periodontal ligament (PDL) during the tooth root formation stage. To investigate the molecular mechanism of PDL formation, we investigated the function of f-spondin in DF differentiation., Design: The expression patterning of f-spondin in the developing tooth germ was compared with that of periodontal ligament-related genes, including runx2, type I collagen and periostin, by in situ hybridization analysis. To investigate the function of f-spondin during periodontal ligament formation, an f-spondin adenovirus was infected into the bell stage of the developing tooth germ, and the effect on dental differentiation was analyzed., Results: F-spondin was specifically expressed in the DF of the developing tooth germ; by contrast, type I collagen, runx2 and periostin were expressed in the DF and in the alveolar bone. F-spondin-overexpresssing tooth germ exhibited a reduction in gene expression of periostin and type I collagen in the DF. By contrast, the knockdown of f-spondin in primary DF cells increased the expression of these genes. Treatment with recombinant f-spondin protein functionally inhibited periostin expression induced by transforming growth factor-β (TGF-β)., Conclusion: Our data indicated that f-spondin inhibits the differentiation of DF cells into periodontal ligament cells by inhibiting TGF-β. These data suggested that f-spondin negatively regulates PDL differentiation which may play an important role in the immature phenotype of DF., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Mutations in MSX1 cause craniofacial developmental defects, including tooth agenesis, in humans and mice. Previous studies suggest that Msx1 activates Bmp4 expression in the developing tooth mesenchyme to drive early tooth organogenesis. Whereas Msx1 -/- mice exhibit developmental arrest of all tooth germs at the bud stage, mice with neural crest-specific inactivation of Bmp4 (Bmp4 ncko /ncko ), which lack Bmp4 expression in the developing tooth mesenchyme, showed developmental arrest of only mandibular molars. We recently demonstrated that deletion of Osr2, which encodes a zinc finger transcription factor expressed in a lingual-to-buccal gradient in the developing tooth bud mesenchyme, rescued molar tooth morphogenesis in both Msx1 -/- and Bmp4 ncko/ncko mice. In this study, through RNA-seq analyses of the developing tooth mesenchyme in mutant and wildtype embryos, we found that Msx1 and Osr2 have opposite effects on expression of several secreted Wnt antagonists in the tooth bud mesenchyme. Remarkably, both Dkk2 and Sfrp2 exhibit Osr2-dependent preferential expression on the lingual side of the tooth bud mesenchyme and expression of both genes was up-regulated and expanded into the tooth bud mesenchyme in Msx1 -/- and Bmp4 ncko/ncko mutant embryos. We show that pharmacological activation of canonical Wnt signaling by either lithium chloride (LiCl) treatment or by inhibition of DKKs in utero was sufficient to rescue mandibular molar tooth morphogenesis in Bmp4 ncko/ncko mice. Furthermore, whereas inhibition of DKKs or inactivation of Sfrp2 alone was insufficient to rescue tooth morphogenesis in Msx1 -/- mice, pharmacological inhibition of DKKs in combination with genetic inactivation of Sfrp2 and Sfrp3 rescued maxillary molar morphogenesis in Msx1 -/- mice. Together, these data reveal a novel mechanism that the Bmp4-Msx1 pathway and Osr2 control tooth organogenesis through antagonistic regulation of expression of secreted Wnt antagonists., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: High fluoride can induce stress-mediated apoptosis and degradation of ameloblasts. Fas ligand (FasL) has been regarded as a key regulator in intracellular responses for stress-induced apoptosis in reproductive or cancerous cell lineages. The objective of this study is to explore the role of FasL in the regulation of ameloblast ultrastructure damage., Design: Primary ameloblasts were isolated from the molar tooth germ of 4-day-old SD rats. The ameloblasts were incubated with 3.2mM NaF or nothing. After incubation for different time arranging from 12h to 72h, ELISA was used to detected the secretion levels of FasL in the medium. Then at 48h post treatment, the ameloblast ultrastructure was detected with Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM), and expression of apoptotic proteins and peroxidative enzymes/products were examined. Finally, a specific FasL inhibitor was applied to co-treat the ameloblasts with NaF, and the ameloblast ultrastructure was detected with TEM and SEM., Results: The secretion of FasL was notably increased by 3.2mM NaF treatment, and the increase reached to the peak after incubation for 48h. High fluoride incubation damaged the ameloblast untrastructure manifesting a series of intracelluar stress responsing cell organelle destruction, and a marked increase in expression of apoptotic genes and oxidative stress. The FasL inhibitor treatment partially mitigated the untrastructure damage caused by high dose NaF., Conlusion: High-fluoride leads to damage of the ameloblast ultrastructure through paritially acitivating the FasL signalling pathway., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Although several heat shock proteins have been investigated in relation to tooth development, no available information is available about the spatial and temporal expression pattern of heat shock protein 60 (Hsp 60). To characterize Hsp 60 expression in the structures of the developing tooth germ, we used Western blotting, immunohistochemistry and in situ hybridization. Hsp 60 was present in high amounts in the inner and outer enamel epithelia, enamel knot (EK) and stratum intermedium (SI). Hsp 60 also appeared in odontoblasts beginning in the bell stage. To obtain data on the possible effect of Hsp 60 on isolated lower incisors from mice, we performed in vitro culturing. To investigate the effect of exogenous Hsp 60 on the cell cycle during culturing, we used the 5-bromo-2-deoxyuridine (BrdU) incorporation test on dental cells. Exogenously administered Hsp 60 caused bluntness at the apical part of the 16.5-day-old tooth germs, but it did not influence the proliferation rate of dental cells. We identified the expression of Hsp 60 in the developing tooth germ, which was present in high concentrations in the inner and outer enamel epithelia, EK, SI and odontoblasts. High concentration of exogenous Hsp 60 can cause abnormal morphology of the tooth germ, but it did not influence the proliferation rate of the dental cells. Our results suggest that increased levels of Hsp 60 may cause abnormalities in the morphological development of the tooth germ and support the data on the significance of Hsp during the developmental processes.

Objective: The aim of this work is to investigate the possible role of Toll-like receptor 4 (TLR4) during the development of mouse tooth germ. TLR4 is well known to inhibit mineralization and cause inflammation in mature odontoblasts and dental pulp cells. However, unlike these pathological functions of TLR4, little is known about the developmental function(s) of TLR4 during tooth development., Materials and Methods: TLR4 expression was studied via Western blot in developing lower mouse incisors from E13.5 to E18.5. To generate functional data about the effects of TLR4, a specific agonist (LPS) was applied to the medium of in vitro tooth germ cultures, followed by Western blot, histochemical staining, ELISA assay, in situ hybridization and RT-qPCR., Results: Increased accumulation of biotin-labelled LPS was detected in the enamel organ and in preodontoblasts. LPS treatment induced degradation of the inhibitor molecule (IκB) of the NF-κB signalling pathway. However, no morphological alterations were detected in cultured tissue after LPS addition at the applied dosage. Activation of TLR4 inhibited the mineralization of enamel and dentin, as demonstrated by alizarin red staining and as decreased levels of collagen type X. mRNA expression of ameloblastin was elevated after LPS administration., Conclusion: These results demonstrate that TLR4 may decrease the mineralization of hard tissues of the tooth germ and may trigger the maturation of ameloblasts; it can give valuable information to understand better congenital tooth abnormalities.

Our previous research has shown that the extracellular matrix metalloproteinase inducer (EMMPRIN) is expressed during and may function in the early development of tooth germs. In the present study, we observed the specific expression of EMMPRIN in ameloblasts and odontoblasts during the middle and late stages of tooth germ development using immunohistochemistry. Furthermore, to extend our understanding of the function of EMMPRIN in odontogenesis, we used an anti-EMMPRIN function-blocking antibody to remove EMMPRIN activity in tooth germ culture in vitro. Both the formation and mineralisation of dental hard tissues were suppressed in the tooth germ culture after the abrogation of EMMPRIN. Meanwhile, significant reductions in VEGF, MMP-9, ALPL, ameloblastin, amelogenin and enamelin expression were observed in antibody-treated tooth germ explants compared to control and normal serum-treated explants. The current results illustrate that EMMPRIN may play a critical role in the processing and maturation of the dental matrix.

The dentition in zebrafish is extremely and richly vascularized, but the function of the vasculature, in view of the continuous replacement of the teeth, remains elusive. Through application of SU5416, a vascular endothelial growth factor receptor inhibitor, we studied the role of the blood vessels in the dentition of the zebrafish. We were unable to show an effect on the development of first-generation teeth as well as first tooth replacement. However, in juvenile fish, a delay was observed in the developmental state of the replacement tooth compared with what was expected based on the maturation state of the functional tooth. Furthermore, we observed a difference between treated and nontreated fish in the distance between blood vessels and developing replacement teeth. In conclusion, our results provide support for a nutritive, rather than an inductive, function of the vasculature in the process of tooth development and replacement., (© International & American Associations for Dental Research 2014.)

BMP and Wnt signaling pathways play a crucial role in organogenesis, including tooth development. Despite extensive studies, the exact functions, as well as if and how these two pathways act coordinately in regulating early tooth development, remain elusive. In this study, we dissected regulatory functions of BMP and Wnt pathways in early tooth development using a transgenic noggin (Nog) overexpression model (K14Cre;pNog). It exhibits early arrested tooth development, accompanied by reduced cell proliferation and loss of odontogenic fate marker Pitx2 expression in the dental epithelium. We demonstrated that overexpression of Nog disrupted BMP non-canonical activity, which led to a dramatic reduction of cell proliferation rate but did not affect Pitx2 expression. We further identified a novel function of Nog by inhibiting Wnt/β-catenin signaling, causing loss of Pitx2 expression. Co-immunoprecipitation and TOPflash assays revealed direct binding of Nog to Wnts to functionally prevent Wnt/β-catenin signaling. In situ PLA and immunohistochemistry on Nog mutants confirmed in vivo interaction between endogenous Nog and Wnts and modulation of Wnt signaling by Nog in tooth germs. Genetic rescue experiments presented evidence that both BMP and Wnt signaling pathways contribute to cell proliferation regulation in the dental epithelium, with Wnt signaling also controlling the odontogenic fate. Reactivation of both BMP and Wnt signaling pathways, but not of only one of them, rescued tooth developmental defects in K14Cre;pNog mice, in which Wnt signaling can be substituted by transgenic activation of Pitx2. Our results reveal the orchestration of non-canonical BMP and Wnt/β-catenin signaling pathways in the regulation of early tooth development., (© 2015. Published by The Company of Biologists Ltd.)

Periodontal ligament stem cells (PDLSCs) have recently been proposed as a novel option in periodontal regenerative therapy. However, one of the issues is the difficulty of stably generating PDLSCs because of the variation of stem cell potential between donors. Here, we show that Semaphorin 3A (Sema3A) can induce mesenchymal-stem-like properties in human periodontal ligament (PDL) cells. Sema3A expression was specifically observed in the dental follicle during tooth development and in parts of mature PDL tissue in rodent tooth and periodontal tissue. Sema3A expression levels were found to be higher in multipotential human PDL cell clones compared with low-differentiation potential clones. Sema3A-overexpressing PDL cells exhibited an enhanced capacity to differentiate into both functional osteoblasts and adipocytes. Moreover, PDL cells treated with Sema3A only at the initiation of culture stimulated osteogenesis, while Sema3A treatment throughout the culture had no effect on osteogenic differentiation. Finally, Sema3A-overexpressing PDL cells upregulated the expression of embryonic stem cell markers (NANOG, OCT4, and E-cadherin) and mesenchymal stem cell markers (CD73, CD90, CD105, CD146, and CD166), and Sema3A promoted cell division activity of PDL cells. These results suggest that Sema3A may possess the function to convert PDL cells into mesenchymal-stem-like cells.

MicroRNAs (miRNAs) are known to regulate a variety of gene functions in many tissues and organs, but their expression and function in tooth development are not well-understood. A specific miRNA, miR-135a, was determined to be highly expressed at the bud stage. Interestingly, after the cap stage, miR-135a was expressed in the epithelium and mesenchyme but not in the inner enamel epithelium. To identify the relationship between miR-135a and its putative target genes, Bmpr-Ia and Bmpr-Ib, in early tooth development, miR-135a was ectopically overexpressed with a lentivirus. This overexpression resulted in the repression of Bmpr-Ia and Bmpr-Ib. Furthermore, miR-135a inhibited both Bmpr-Ia and Bmpr-Ib transcription. BMP2 proteins were expressed ectopically in tooth germs during the cap stage to determine the relationship between miR-135a and BMP signaling in early tooth development. When miR-135a was ectopically expressed, no tooth formation was observed after 4 wk of incubation in the kidney capsule. This study suggested that Bmp signaling, specifically Bmpr-Ia and Bmpr-Ib, regulates tooth formation via miR-135a., (© International & American Associations for Dental Research.)

Vascularization is essential for organ and tissue development. Teeth develop through interactions between epithelium and mesenchyme. The developing capillaries in the enamel organ, the dental epithelial structure, occur simultaneously by mechanisms of vasculogenesis and angiogenesis at the onset of dentinogenesis. The vascular neoformation in the dental mesenchyme has been reported to start from the cap stage. However, the mechanisms of vascularization in the dental mesenchyme remain unknown. In the hope of understanding the mechanisms of the formation of dental mesenchymal vasculature, mouse lower molar germs from embryonic day (E) 13.5 to E16.5 were processed for immunostaining of CD31 and CD34, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and transmission electron microscopy (TEM). In addition, the role of apoptosis for the vascularization in dental mesenchyme was examined by in vitro culture of E14.0 lower molars in the presence of the apoptosis inhibitor (z-VAD-fmk) and a subsequent subrenal culture. Our results showed that CD31- and CD34-positive cells progressively entered the central part of the dental papilla from the peridental mesenchyme. For TEM, angioblasts, young capillaries with thick endothelium and endothelial cells containing vacuoles were observed in peripheral dental mesenchyme, suggesting vasculogenesis was taking place. The presence of lateral sprouting, cytoplasmic filopodia and transluminal bridges in the dental papilla suggested angiogenesis was also occurring. Inhibition of apoptosis delayed the angiogenic vascularization of the dental papilla. Therefore, these data demonstrated that molar mesenchyme is progressively vascularized by mechanisms of both vasculogenesis and angiogenesis and apoptosis partially contributes to the vascularization of the dental papilla.

Unlabelled: Dental amalgam is the most common restorative material used in dentistry. It was reported that amalgam might constitute potential toxic hazards to pregnant patients and foetuses through mercury release and absorption. The present study aimed to investigate the vital tissue response in contact to dental amalgam plus determination of blood mercury levels in mother and offspring Wistar strain albino rats. Pregnant mothers were divided into two main groups each had dental amalgam implanted into either an oral mucosa incision or a bony socket following extraction. Third and fourth groups included the offspring rats of mothers from the first and second groups, respectively. The blood mercury levels and histopathology of oral tissues were analyzed in mothers at one and six months post-implantation and in offspring rats one day after birth. The blood mercury levels of mothers increased significantly at six months (P<0.01) as compared to levels at one month. However, blood mercury levels were not significant (P>0.05) when the two offspring (third and fourth) groups were compared. Histopathology results from mothers showed inflammatory response at the bottom of the socket, one month after amalgam implantation. At six months, teeth germs showed vacuolation of the abnormal odontoblasts with globular dentine formation. Degenerated periodontal fibres and thin trabeculae forming the bony sockets with large marrow spaces were evident. A fibrous connective tissue capsule surrounded the amalgam mass inside the mucosa of mothers at one month and was evident also at 6 months with a huge inflammatory cell infiltrate. Teeth germs showed elongated odontoblasts with intercellular oedema, thinner dentine and bony trabeculae with wider marrow spaces. Offspring rats showed comparable oral tissue response., Conclusions: There is a positive correlation between blood mercury levels and oral tissue response in mothers, however, the negative impact of mercury on oral tissues of offspring rats was due to high mercury levels in their mothers' blood during pregnancy. We would recommend that women should - as far as possible - postpone having dental amalgam filling placed or removed during pregnancy to avoid its harmful effect on the foetus. Further clinical studies are recommended to test our findings in man., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We have previously demonstrated that tooth size is determined by dental mesenchymal factors. Exogenous bone morphogenetic protein (BMP)4, Noggin, fibroblast growth factor (FGF)3 and FGF10 have no effect on tooth size, despite the expressions of Bmp2, Bmp4, Fgf3, Fgf10 and Lef1 in the dental mesenchyme. Among the wingless (Wnt) genes that are differentially expressed during tooth development, only Wnt5a is expressed in the dental mesenchyme. The aims of the present study were to clarify the expression pattern of Wnt5a in developing tooth germs and the role of Wnt5a in the regulation of tooth size by treatment with exogenous WNT5A with/without an apoptosis inhibitor on in vitro tooth germs combined with transplantation into kidney capsules. Wnt5a was intensely expressed in both the dental epithelium and mesenchyme during embryonic days 14-17, overlapping partly with the expressions of both Shh and Bmp4. Moreover, WNT5A retarded the development of tooth germs by markedly inducing cell death in the non-dental epithelium and mesenchyme but not widely in the dental region, where the epithelial-mesenchymal gene interactions among Wnt5a, Fgf10, Bmp4 and Shh might partly rescue the cells from death in the WNT5A-treated tooth germ. Together, these results indicate that WNT5A-induced cell death inhibited the overall development of the tooth germ, resulting in smaller teeth with blunter cusps after tooth-germ transplantation. Thus, it is suggested that Wnt5a is involved in regulating cell death in non-dental regions, while in the dental region it acts as a regulator of other genes that rescue tooth germs from cell death.

Regression of vestigial tooth buds results in the formation of the toothless diastema, a unique feature of the mouse dentition. Revitalization of the diastemal vestigial tooth bud provides an excellent model for studying tooth regeneration and replacement. It has been previously shown that suppression of fibroblast growth factor (FGF) signaling in the diastema results in vestigial tooth bud regression. In this study, we report that application of exogenous FGF8 to the mouse embryonic diastemal region rescues diastemal tooth development. However, this rescue of diastemal tooth development occurs only in an isolated diastemal regions and not in the mandibular quadrant, which includes the incisor and molar germs. FGF8 promotes cell proliferation and inhibits apoptosis in diastemal tooth epithelium, and revitalizes the tooth developmental program, as evidenced by the expression of genes critical for normal tooth development. Our results also support the idea that the adjacent tooth germs contribute to the suppression of diastemal vestigial tooth buds by means of multiple signals., (Copyright © 2011 Wiley-Liss, Inc.)

Introduction: Biocompatibility of pulp capping materials is important for successful use in dentistry. These materials should be nontoxic and permissive for proliferation and induction of odontogenic differentiation of pulp cells. The aim of our study was to evaluate the effects of enamel matrix derivative (EMD), mineral trioxide aggregate (MTA), and calcium hydroxide-containing cement (DYCAL) on proliferation and odontogenic differentiation of human tooth germ stem cells (hTGSCs) in which cells belonging to both pulp tissue and dental follicle exist., Methods: The 96-well plates, 24-well plates, and special chamber slides were coated with biomaterials for cell proliferation, differentiation, and scanning electron microscopy analysis. Odontogenic differentiation of hTGSCs was evaluated by analyzing mRNA expression of dentin sialophosphoprotein (DSPP) by real-time polymerase chain reaction expression analysis, measurement of alkaline phosphatase activity, and visualization of calcium depositions by von Kossa staining., Results: Our results demonstrate that EMD is the best material in terms of inducing differentiation and proliferation of hTGSCs. DYCAL was found to be toxic to hTGSCs; however, EMD-coated DYCAL showed less toxicity. EMD-coated MTA was not efficient at inducing proliferation and differentiation., Conclusions: Pulp capping materials come in direct contact with dental pulp cells; thus, they require comprehensive evaluation of interactions between cells and biomaterials. Therefore, we cultured hTGSCs, capable of odontogenic differentiation, on pulp capping materials directly. Our results suggest that combination of capping materials with EMD would increase the quality of capping by increasing biocompatibility of capping materials., (Copyright © 2011 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

The mutation of phosphate-regulating gene with homologies to endopeptidases on the X-chromosome (PHEX) can lead to human X-linked hypophosphatemic rickets which displays hypo-mineralization in bone and dentin. To study its possible roles in teeth, PHEX antibody was injected into pregnant mice on E15 to explore its roles on the formation of enamel and dentin. Mallory trichrome staining results showed that arrangements of ameloblasts and odontoblasts were irregular after PHEX antibody treatment. Differentiation of odontoblasts and the formation of dentin were inhibited. Spatiotemporal distribution of PHEX protein was observed in various stages of tooth germ. Immunohistochemical results showed positive PHEX signals appeared in the inner enamel epithelium on E16 and became stronger on E18. Ameloblasts and odontoblasts showed much higher PHEX expression on P1 and P3. Expression of PHEX in odontoblasts decreased accordingly. However, enamel formation was only slightly affected. The findings proved that a decrease in PHEX expression could suppress dentin formation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

White opacities and pits are developmental defects in enamel caused by high intake of fluoride (F) during amelogenesis. We tested the hypothesis that these enamel pits develop at locations where F induces the formation of sub-ameloblastic cysts. We followed the fate of these cysts during molar development over time. Mandibles from hamster pups injected with 20mg NaF/kg at postnatal day 4 were excised from 1h after injection till shortly after tooth eruption, 8 days later. Tissues were histologically processed and cysts located and measured. Cysts were formed at early secretory stage and transitional stage of amelogenesis and detected as early 1h after injection. The number of cysts increased from 1 to almost 4 per molar during the first 16h post-injection. The size of the cysts was about the same, i.e., 0.46±0.29×10(6)μm(3) at 2h and 0.50±0.35×10(7)μm(3) at 16h post-injection. By detachment of the ameloblasts the forming enamel surface below the cyst was cell-free for the first 16h post-injection. With time new ameloblasts repopulated and covered the enamel surface in the cystic area. Three days after injection all cysts had disappeared and the integrity of the ameloblastic layer restored. After eruption, white opaque areas with intact enamel surface were found occlusally at similar anatomical locations as late secretory stage cysts were seen pre-eruptively. We conclude that at this moderate F dose, the opaque sub-surface defects with intact surface enamel (white spots) are the consequence of the fluoride-induced cystic lesions formed earlier under the late secretory-transitional stage ameloblasts., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Objective: To explore a novel nanometer biomaterial which could induce the regeneration of tooth tissues intelligently, and to evaluate the feasibility of using this kind of biomaterial as the scaffold for tooth tissue engineering by investigating the role it plays in tooth tissue engineering., Methods: The scaffold for tooth tissue engineering containing recombinant human bone morphogenetic protein 2 (rhBMP-2) was prepared by mixing nanoscale I tricalcium phosphate (beta-TCP)/collagen particles. Forty-six 8-10 weeks old specific pathogen free Sprague Dawley (SD) rats, including 34 females and 12 males, weighing 250-300 g, were involved in this study. Tooth germs were removed under a stereomicroscope from the mandible of newborn SD rat, then digested and suspended. Scanning electronic microscope (SEM), adhesion rate of cells, and MTT assay were used to evaluate the effects of the scaffold on the tooth germ cells cultured in vitro. The tissue engineered tooth germ which was constructed by tooth germ cells and scaffold was transplanted under SD rat's kidney capsule as the experimental group (n=12); the tooth germ cells (cell-control group, n=12) or scaffold without cells (material-control group, n=4) were transplanted separately as control groups. Specimens were harvested to perform general and histological observations at 4 and 8 weeks after transplantation., Results: beta-TCP/collagen showed a loose and porous appearance with soft texture and excellent hydrophilicity. Tooth germ cells grew well and could attach to the scaffold tightly 3 days after coculture. The adhesion rates of tooth germ cells were 27.20% +/- 2.37%, 44.52% +/- 1.87%, and 73.81% +/- 4.15% when cocultured with scaffold for 4, 8, and 12 hours, respectively. MTT assay showed that the cell proliferation status of experimental group was similar to that of the control group, showing no significant difference (P > 0.05). Some white calcified specimens could be harvested at 4-8 weeks after transplantation. At 4 weeks after transplantation, some typical structures of dental cusp and enamel-dentin like tissues could be seen in the experimental group. Enamel-dentin like tissues also formed in some specimens of cell-control group, but they arranged irregularly. At 8 weeks after transplantation, the enamel-dentin like tissue of experimental group exhibited a mature appearance and organized structure in comparison with that at 4 weeks. And mature enamel or dentin like tissue also could be seen in cell-control group. In contrast, there was no enamel or dentin like tissue in material-control group at 4 or 8 weeks after transplantation., Conclusion: rhBMP-2 decorated beta-TCP/collagen scaffold has good biocompatibility and can be used as a novel nanometer biomaterial, so it is a good choice in scaffolds for tooth tissue engineering.

Using in vitrotooth germ cultures and analysis by confocal microscopy, ameloblasts treated with sodium fluoride were found to have elevated amounts of filamentous actin. Because this response is reduced by inhibitors of the Rho/ROCK signaling pathway, we generated mice that express dominant negative RhoA (RhoA(DN)) in ameloblasts for in vivo analysis. Expression of the EGFP-RhoA(DN) fusion protein was evaluated by RT-PCR and immunohistochemistry, and teeth were analyzed by scanning electron microscopy. The 3 strains expressed at either low (TgEGFP-RhoA(DN)-8), intermediate (TgEGFP-RhoA(DN)-2), or high (TgEGFP-RhoA(DN)-13) levels, and the molar teeth from the 3 strains had enamel hypoplasia and surface defects. We conclude that RhoA(DN) expressed in ameloblasts interferes with normal enamel development through the pathway that is induced by sodium fluoride., (Copyright © 2011 S. Karger AG, Basel.)

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., (© 2010 Wiley-Liss, Inc.)

Excessive fluoride ingestion has been identified as a risk factor for fluorosis and oxidative stress. The oxidative stress results from the loss of equilibrium between oxidative and antioxidative mechanisms that can produce kinase activation, mitochondrial disturbance and DNA fragmentation, resulting in apoptosis. Actually many people are exposed to no-adverted fluoride consumption in acute or chronic way. The aim of this study was to determine the effect of sodium fluoride on first molar germ in relation to its effect on antioxidative enzymes immunoexpression and apoptosis. Thirty first molar germs from 1-day-old Balb/c mice were cultured for 24 h with sodium fluoride (0 mM, 1 mM and 5 mM). Immunoexpression determination of CuZnSod, MnSod, catalase, Bax, Bid, caspase 8, caspase 9, caspase 3 and TUNEL assay were performed. Cellular disorganization in ameloblast and odontoblast-papilla zones was observed. CuZnSod and MnSod immunoexpression decrease in experimental groups. Caspase 8, caspase 3, Bax, Bid increase expression and more TUNEL positive cells in both experimental groups than control, suggest that apoptosis induced by fluoride is related to oxidative stress due to reduction of the enzymatic antioxidant., (© 2010 John Wiley & Sons A/S.)

Intrauterine hypoxia decreased the nucleolar organizator quantity in enameloblasts of newborn albino rats tooth germ. Influence the leu-encepgalin synthetic analog dalargin and non-opiate synthetic leu-encephalin on newborn rats from 2 to 6 day abolished the intrauterine hypoxia affect. The main mechanism of peptide antioxidant effect may be NO system stimulation.

Limitations of conventional regeneration modalities underscore the necessity of recapitulating development for periodontal tissue engineering. In this study, we proposed a novel three-dimensional pellet cultivation system for periodontal ligament stem cells (PDLSCs) to recreate the biological microenvironment similar to those of a regenerative milieu. Monodispersed human PDLSCs were cultured in medium with ascorbic acid and conditioned medium from developing apical tooth germ cells and were subsequently harvested from culture plate as a contiguous cell sheet with abundant extracellular matrix. The detached cell-matrix membrane spontaneously contracted to produce a single-cell pellet. The PDLSCs embedded within this cell-matrix complex exhibited several phenotypic characteristics of cementoblast lineages, as indicated by upregulated alkaline phosphatase activity, accelerated mineralization, and the expression of bone sialoprotein and osteocalcin genes. When this PDLSC pellets were transplanted into immunocompromised mice, a regular aligned cementum/PDL-like complex was formed. These results suggest that the combination of apical tooth germ cell-conditioned medium and endogenous extracellular matrix could maximally mimic the microenvironment of root/periodontal tissue development and enhance the reconstruction of physiological architecture of a cementum/PDL-like complex in a tissue-mimicking way; on the other hand, such PDLSC pellet may also be a promising alternative to promote periodontal defect repair for future clinical applications.

The purpose of this study was to evaluate the behaviors of rat tooth germ (TG) cells cultured on poly(vinyl alcohol) (PVA). It was found that TG cells suspended and aggregated to form three-dimensional spheroids on PVA. Compared with traditional monolayered cells on tissue culture polystyrene, TG cell spheroids on PVA obviously increased the alkaline phosphatase activity, the degree of mineralization, and upregulated both osteopontin and dentin matrix protein 1 genes, regardless of the seeding density. Surprisingly, PVA appears to activate the alkaline phosphatase activity and mineralization effects on TG cell spheroids in the absence of a differentiation medium. Furthermore, the present study indicates that integrins may play an important role in the mineralization on TG cell spheroids by adding Arg-Gly-Asp (RGD) peptides. Therefore, the information presented here should help to clarify the role of PVA in the regulation of the mineralization, differentiation and integrin-mediation of TG cells.

Objective: To detect expression of ClC chloride channel mRNA in tooth germ and odontoblasts, and explore the affect of chloride channel function on cell proliferation and cell cycle., Design: We extracted total RNA of tooth germ from newborn C57BL mice and mouse odontoblast-like cells (MDPC-23), then detected mRNA expression of chloride channel genes Clcn1-7 with RT-PCR. We used chloride channel blocker 5-nitro-2-(3- phenylpropylamino)benzoic acid (NPPB) to interfere with chloride channel function of MDPC-23 cells. Cell proliferation rate and cell cycle were detected with MTT assay and flow cytometry, respectively. Student's t-test was used to determine statistical significance between control and treatment groups., Results: The mRNA of Clcn1-7 chloride channel genes was expressed in tooth germ of newborn mice. Clcn3, Clcn5 and Clcn7 mRNAs were expressed in MDPC-23 cells. NPPB slowed down the proliferation rate of MDPC-23 cells from day 2 to day 4 (P<0.01), and also changed the proportion of cell cycle phase. Comparing to the control, the proportion of G2/M phase cells reduced from 3.93+/-2.62% to 0.54+/-0.25% (P<0.05). The ratio of G1/G2 increased from 1.86+/-0.01 to 1.95+/-0.02 (P<0.05)., Conclusions: There is abundant chloride channel gene expression in tooth germ. Some of these chloride channels may regulate tooth development through effects on cell proliferation and cell cycle signal pathway.

Background: Langerhans cell histiocytosis (LCH, histiocytosis X, ICD-O 9751/1) refers to a neoplastic proliferation of Langerhans cells. The course of the disease determines the treatment and prognosis. Solitary forms (eosinophilic granuloma) and limited multilocational lesions may be treated successfully with local surgical intervention and intralesional corticosteroid injection., Purpose: Presentation of our own case will review LCH, a very rare disease entity in oral-facial surgery and will document that intralesional corticosteroid injection is a less invasive alternative to the classical surgical curettage and local radiation therapy., Case Report: In a 10-year-old boy, a progressive, pressure-sensitive swelling had developed within 1 week in the left paramandibular and submandibular area. Dental status was good. Diagnostic imaging demonstrated a diffusely contoured osteolysis caudal to tooth germ 37 with infiltration of the adjacent chewing muscles. Histological evaluation of the intraoral biopsy sample established an LCH. Having excluded a multifocal form of LCH, treatment with intralesional injection of methylprednisolone 200 mg was chosen. Symptoms of pain were quickly relieved and the swelling receded. Follow-up visits 6 weeks, 3 months, and 6 months after corticosteroid injection revealed continuous regression of mandibular osteolysis. OPG and MRI after 17 months demonstrated a good osseous consolidation in the left mandibular angle area, but a still discernible bone marrow edema. Development of the intralesional tooth germ 37 appeared normal., Conclusion: Local surgical interventions continue to be central to the range of accepted therapeutic measures. However, the increasing numbers of reports on the successful treatment of solitary LCH by intralesional corticosteroid injection suggest that this treatment option should be considered especially in children to preserve tooth germs.

Introduction: An excellent model system that links evolutionary biology and developmental biology in seeking to understand evolutionary diversity is the study of tooth development in mammals. These studies reflect the diversity of mammalian radiations which bear on the interpretation of South African fossil hominids. Tooth development occurs via epithelio-mesenchymal interactions and involves the production of many substances, including alkaline phosphatase, which is necessary for dentine and enamel formation. Retinoic acid is a known morphogen and is important in tooth development. In excess, retinoic acid has been found to alter the formation of teeth., Objectives: In order to determine whether retinoic acid has any effect on tooth morphology, exogenous retinoic acid was administered to developing mouse molar teeth in vitro, and alkaline phosphatase was utilized as an indicator of differentiation., Methods: Molars were microdissected from 15.5 day mouse embryo mandibles and cultured at the air: medium interface with or without retinoic acid for seven days. Following fixation and embedding, the explants were sectioned for morphological analysis. Alkaline phosphatase activity was detected using a modified Gomori's histochemical method., Results and Conclusion: Retinoic acid appeared to retard the growth and differentiation of the molar explants. This was coincident with reduced alkaline phosphatase.

Dioxins are ubiquitous environmental poisons that cause disturbances in developing organs, including the teeth. Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at the cap stage leads to reduced tooth size and deformation of cuspal morphology. Our hypothesis was that TCDD affects the expression of genes specific for tooth development, which leads to these aberrations. Mouse embryonic E14 tooth germs were cultured for 24 hrs with/without 1 microM TCDD. Analysis of total RNA on Affymetrix arrays showed that TCDD altered the expression of 31 known genes by a fold factor of at least 2. Genes implied in tooth development expressed only slight changes. Genes active at the cap stage were selected for quantitative PCR analysis. Of these, the most highly up-regulated were Follistatin and Runx2, while TGFbeta1 and p21 were the most down-regulated genes. Incomplete tooth morphogenesis caused by TCDD may thus result from modified expression of developmentally regulated genes.

Purpose: Culture of the whole organ and regulation of its development using biologic and engineering principles can be used to produce structures and organs for reconstructing defects. The application of these bioengineering approaches in artificial tooth development may be the alternative way to replace missing dentition., Materials and Methods: For the artificial bioengineering of a mouse tooth, tooth buds were dissected and transplanted into the diastema of the developing mandible. The mandiblular primordia containing transplanted tooth buds were culture in vitro and in vivo using a bioengineering method. In addition, to regulate the development of tooth germs, bone morphogenetic protein 4 (BMP4) or its antagonist, Noggin was administered., Results: After the period of in vitro and in vivo culture, the transplanted tooth germ in the diastema showed tooth development with supportive structure formation. In the BMP-treated group, the bioengineered tooth was observed with increased maturation of cusp and enamel matrix. However, in the Noggin-treated tooth germs, the developing molar had a crater-like appearance with the immature development of the cusp and suppressed formation of the enamel matrix., Conclusions: This study confirmed that tooth germ transplantation in the diastema and culture with administration of BMP4 could lead to the mature development of the dental structures. In addition, these results suggest the possibility of bioengineering the tooth in morphogenesis and differentiation even in the toothless area.

Fluoride alters the expression and post-translational modifications of extracellular matrix proteins in dentin. The aim of our study was to determine the effects of fluoride on type I collagen expression during the early stages of tooth germ development in rats. Pregnant dams were divided into three groups and fed a standard diet. From the fifth day of pregnancy the three groups received tap water with, respectively, trace amounts of fluoride (C), a low fluoride concentration (FL) or and a high fluoride concentration (FH). Changes in type I collagen expression and distribution were evaluated. The expression of type I collagen was restricted to the extracellular spaces of cells of mesenchymal origin. In the youngest animals the most intense immunoreactivity for type I collagen was detected in predentin of the FL group. Although the intensity of immunostaining increased in proportion to the age of the animals, the largest increase in the groups investigated was detected in the FL group. We concluded that a low concentration of fluoride can act as a stimulator of type I collagen deposition in the extracellular matrix of dentin, while high concentrations of fluoride have an opposite effect, acting as an inhibitor of type I collagen formation in dentin.

Apoptosis plays important roles in various stages of organogenesis. In this study, we hypothesized that apoptosis would play an important role in tooth morphogenesis. We examined the role of apoptosis in early tooth development by using a caspase inhibitor, z-VAD-fmk, concomitant with in vitro organ culture and tooth germ transplantation into the kidney capsule. Inhibition of apoptosis at the early cap stage did not disrupt the cell proliferation level when compared with controls. However, the macroscopic morphology of mice molar teeth exhibited dramatic alterations after the inhibition of apoptosis. Crown height was reduced, and mesiodistal diameter was increased in a concentration-dependent manner with z-VAD-fmk treatment. Overall, apoptosis in the enamel knot would be necessary for the proper formation of molar teeth, including appropriate shape and size.

Dental epithelium comprises different cell populations, including ameloblasts and stratum intermedium cells. Ameloblasts are vitamin D targets, and at least five proteins undergo specific modulation of their expression following the addition of 1alpha,25(OH)2 vitamin D3[1alpha,25(OH)2D3]. Stratum intermedium cells have not been studied in any great detail regarding vitamin D impact. Interestingly, in these cells, the tissue non-specific alkaline phosphatase (TNAP) is overexpressed. On the other hand, TNAP is a reliable bone marker of vitamin D action, similar to calbindins in kidney and intestine, previously used for studies of vitamin D activity in ameloblasts. Here, TNAP expression and activity were investigated in vivo in the microdissected epithelium and mesenchyme of mandible incisors. Physiological doses of 1alpha,25(OH)2D3 injected in control rats failed to modify TNAP activity in both dental epithelium and mesenchyme. No significant differences were observed in the steady-state levels of TNAP mRNAs of dental tissues from wild-type and vitamin D nuclear receptor (VDRnuc)-deficient mice of the same litters. These data suggest that, in contrast to ameloblasts, stratum intermedium cells are not sensitive to 1alpha,25(OH)2D3. An explanation for such a responsiveness of stratum intermedium cells to 1alpha,25(OH)2D3 is proposed based on the respective expressions of both vitamin D receptors (VDRnuc and 1,25D3-[MARRS]) and the Dlx2 homeobox gene.

We tested the hypothesis that high-calcium medium given prior to or immediately after exposure to fluoride (F) reduces the negative effects of F on secretory amelogenesis. Hamster molar tooth germs were grown in organ culture in media with different calcium levels. Deposition of enamel matrix and matrix mineralization were monitored by incorporation of [3H]proline and uptake of 45Ca and acid-soluble 32PO4. Ameloblast structure and the occurrence of a fluorotic enamel matrix were examined by light and electron microscopy. A preculture of explants in high-calcium medium partially prevented the formation of fluorotic (non-mineralizing) enamel matrix, increased matrix secretion but could not prevent F-induced hypermineralization of the pre-exposure enamel. High-calcium medium, applied after F insult, accelerated the recovery of fluorotic matrix, improved ameloblast structure, enhanced amelogenin secretion, and increased enamel thickness. The data indicate that it might be the balance between the amount of mineral deposition and that of matrix secretion which is critical for the mineralization of newly secreted enamel. Exposure to F disturbs this balance by enhancing mineralization of the pre-exposure enamel, probably generating an excess of protons. High calcium may protect against F exposure by enhancing amelogenin secretion into the enamel space, thereby increasing the local buffering capacity at the mineralization front.

We tested the hypothesis that the sensitivity of forming dental enamel to fluoride (F-) is ameloblast developmental stage-dependent and that enamel mineralization disturbances at the surface of fluorotic enamel are caused by damage to late-secretory- and transitional-stage ameloblasts. Four-day-old hamsters received a single intraperitoneal dose of 2.5-20 mg NaF/kg body weight and were examined, 24 h later, by histology and histochemistry. A single dose of >or=5 mg of NaF/kg induced the formation of a hyper- followed by a hypomineralized band in the secretory enamel, without changing the ameloblast structure. At 10 mg of NaF/kg, cystic lesions became apparent under isolated populations of distorted late-secretory- and transitional-stage ameloblasts. Staining with von Kossa stain showed that the enamel under these lesions was hypermineralized. At 20 mg of NaF/kg, cystic lesions containing necrotic cells were also found in the early stages of secretory amelogenesis and were also accompanied with hypermineralization of the enamel surface. We concluded that the sensitivity to F- is ameloblast developmental stage-dependent. Groups of transitional ameloblasts are most sensitive, followed by those at early secretory stages. These data suggest that a F-induced increase in cell death in the transitional-stage ameloblasts accompanies the formation of cystic lesions, which may explain the formation of enamel pits seen clinically in erupted teeth.

Fluoride is associated with a decrease in the incidence of dental caries, but excessive fluoride intake during tooth enamel formation can result in enamel fluorosis. Fluorosed enamel has increased porosity, which has been related to a delay in the removal of amelogenin proteins as the enamel matures. This delay in protein removal suggests that fluoride may affect either the amount or the activity of enamel matrix proteinases. In this study, we investigated the role of fluoride in the synthesis and secretion of matrix metalloproteinase-20 (MMP-20), the proteinase primarily responsible for the initial hydrolysis of amelogenin during the secretory stage of enamel formation. Cultured human fetus tooth organ ameloblast-lineage cells were exposed to 10 microM fluoride and analyzed for synthesis of MMP-20. Immunoblotting showed that 10 microM NaF down-regulated the synthesis of MMP-20 by 21% compared with control cells, but did not alter the amount of amelogenin or kalikrein-4 (KLK-4) synthesized by the cells. Real-time polymerase chain reaction (PCR) showed that 10 microM NaF down-regulated MMP-20 mRNA expression to 28% of the levels found in the non-treated cells. These in vitro results suggest that fluoride can alter the expression of MMP-20 by ameloblasts, resulting in a disturbance of the balance between MMP-20 and its substrate that may contribute to the retention of amelogenins in the formation of fluorosed enamel.

Unlabelled: Fluoride is known to alter expression of dentin matrix proteins and affect their posttranslational modifications., Objective: The objective of our study was to examine dentin sialoprotein (DSP) expression in the early and late bell stages of development of the first molar tooth germs in rats treated with fluoride., Design and Methods: Pregnant dumps were divided into three groups. They were fed a standard diet and from the fifth day of pregnancy, each group received either tap water (with trace amounts of fluoride), tap water with a low concentration of fluoride, or tap water with a high concentration of fluoride. Changes in DSP expression and distribution were visualized by immunohistochemistry., Results: Immunoreactivity for DSP was detected in the cervical regions of the early bell stage in tooth germs of the 1-day-old animals. The earliest reaction was visible in the control group and the group supplemented with the low fluoride concentration (F(L)) but not in the group supplemented with the high fluoride concentration (F(H)). In early bell stages across all experimental groups, the immunoreactivity to DSP was observed in the cusp tip regions and was localized to preameloblasts, young and mature odontoblasts, dental pulp cells, predentin, and dentin. Generally, more intense positive staining for DSP was detected in animals supplemented with the high fluoride concentration. In the late bell stage found in the 4-day-old control group and the group supplemented with the low fluoride concentration, immunoreactivity for DSP was less intense compared with younger animals. However, immunoreactivity was greater in the group treated with the high dose of fluoride. In this group, the positive immunostaining for DSP, especially in young ameloblasts, was prolonged and relatively strong., Conclusions: Fluoride supplementation causes changes in the developmental pattern of DSP expression and its distribution in rat tooth germs.

Background: Ethanol consumption during pregnancy leads to changes in murine dental morphogenesis, dental size, cellular differentiation, enamel mineralization, and delayed eruption. It has been proposed that glycoproteins play a role during embryonic dental development that may determine the dental morphological pattern and extracellular matrix secretion. O-glycosylation and sialylation appear to actively participate in the differentiation and maturation processes. Because glycosylation may be affected by teratogens that can alter the maturation of several organisms, in this work we describe the main modifications of the sialylation pattern in prenatal day (PD) 18.5 murine tooth germs exposed to ethanol., Methods: Pregnant female mice were divided into groups that were given 15% or 20% ethanol solutions, or water as a control. The histochemistry of tooth germs from PD 18.5 fetuses was revealed with lectins specific for sialic acid (Neu5Ac), such as Sambucus nigra (SNA), Maackia amurensis (MAA), and Machrobrachium rosenbergii (MRL), and for sialylated-O-glycosidically linked glycans, such as Amaranthus leucocarpus (ALL)., Results: The basement membrane, preameloblasts, inner-enamel epithelium, preodontoblasts, and subodontoblastic cells of the test groups showed changes in labeling according to the 4 lectins used. Intranuclear staining was observed with SNA (specific for Neu5Acalpha2,6Gal/GalNAc) in the control group, but this was reduced in the test groups. The nuclei of dental papillary cells under the experimental conditions were stained with MAA (Neu5Acalpha2,3Gal)., Conclusions: Dental development involves different types of sialylated O-glycosidically linked glycans that are likely to regulate cell-to-cell and cell-to-matrix interactions. Our results suggest that ethanol consumption during pregnancy alters the sialylation pattern during murine dental morphogenesis.

Objective: A polyclonal antibody was used to investigate the effects of ethanol ingestion before and during pregnancy, in the expression of EGF on dentinogenesis and amelogenesis of rat mandibular first molar., Design: Ethanol was administered to drinking water (treated group) starting at concentrations of 1% and increasing weekly to 5, 10, 15, 20 and 25% (v/v). During week 7, these rats were mated and continued to receive the 25% alcoholic solution, up to delivery. The control group received tap water. On postnatal days 0, 4 and 9, two offspring of each litter were killed, their hemimandibles removed and prepared for paraffin processing and immunohistochemistry., Results: At postnatal day 0 the EGF immunoreactivity of the inner enamel epithelium and presecretory ameloblasts was weak when compared to controls. At postnatal day 4 EGF immunoreactivity of the secretory ameloblasts and odontoblasts was only moderate compared to controls. At postnatal day 9 EGF staining of the ameloblasts was weak when compared to controls., Conclusions: These results suggest that, maternal alcoholism interferes with EGF expression during initial dentinogenesis and amelogenesis and in the secretion and maturation of the dentin and enamel, therefore, which may cause a reduction of dentin and enamel formation.

Vitamin A, 1,25-dihydroxyvitamin D3 and dexamethasone are well-characterized hydrophobic molecules whose biological actions are mediated via different members of the nuclear hormone receptor family. We report here their actions on tooth formation at the molecular level. We have tested the effects of these compounds on osteopontin (OPN), dentin sialoprotein (DSP-PP), and collagen type I expression in pre-mineralization and mineralization stage rat tooth organ cultures which mirror in vivo developmental patterns. These proteins are all believed to participate in the mineralization of dentin. 1,25-Dihydroxyvitamin D3 up-regulated OPN, but had no effect on DSP-PP mRNA expression. Vitamin A up-regulated DSP-PP expression as did dexamethasone. Dexamethasone also up-regulated collagen type I expression. Our results suggest that 1,25-dihydroxyvitamin D3 does not modulate dentin mineralization by directly affecting DSP-PP expression. Vitamin A likely contributes to dentin mineralization by up-regulating DSP-PP expression. Finally, the up-regulation of DSP-PP expression in tooth germ cultures treated with dexamethasone suggests that its application to patient's dental pulp might promote increased extracellular matrix synthesis and mineralization in the pulp and may explain the narrowing of the dental pulp cavity in patients undergoing long-term dexamethasone administration.

Background: Parathyroid hormone-related protein (PTHrP) promotes osteoclastogenesis by inhibiting expression of osteoprotegerin (OPG), a decoy receptor for the receptor activator of nuclear factor kappa B (RANK), and by enhancing production of RANK ligand (RANKL) by osteoblasts. However, little is known regarding the role of PTHrP in regulating cementoblast-mediated osteoclastogenesis., Methods: This study determined the impact of PTHrP on osteoclastogenesis using: 1) OCCM-30 (immortalized murine cementoblasts), 2) RAW 264.7 cells (murine myeloid cells), or 3) OCCM-30 plus RAW 264.7 cells. Cells were treated with PTHrP (1-34), RANKL, or PTHrP and RANKL combined. Enzyme-linked immunosorbent assays (ELISAs) for OPG and RANKL were performed on media and cell lysates, and tartrate-resistant acid phosphatase (TRAP) and mRNA detection for the osteoclast associated receptor (OSCAR) were performed., Results: The highest numbers of TRAP-positive cells and cells expressing OSCAR were found in the RAW cell group treated with either RANKL alone or RANKL and PTHrP. TRAP-positive cells were fewer when OCCM cells were co-cultured with RAW, but the greatest numbers were still with both PTHrP and RANKL. OPG levels were highest from OCCM cells and PTHrP decreased these levels. In contrast, RANKL levels were low in OCCM cell lysates and PTHrP increased RANKL. In vivo studies also revealed high osteoclastic activity surrounding developing teeth in mice administered PTH., Conclusions: These results demonstrate that PTHrP influences the balance of OPG and RANKL production by cementoblasts, and further indicate that this effect, in the context of surrounding cells, might have a significant impact on osteoclastogenesis, root resorption, and tooth eruption.

In order to study the involvement of matrix metalloproteinases (MMPs) on dentin formation and mineralization, day 18 embryonic mouse tooth germs were cultured for 10 d in the presence or absence of Marimastat, a general MMP inhibitor, or CT(1166), a more selective inhibitor of gelatinases (MMP-2 and MMP-9) and stromelysin-1 (MMP-3). With Marimastat a dose-dependent increase in thickness of the predentin layer and a decreased mineralization of dentin were observed. At the highest concentration of the inhibitor used, enamel formation had ceased. With CT(1166), these effects were already apparent at the lowest concentration used. Western blot analyses demonstrated that the two inhibitors inhibited the expression of enamelysin (MMP-20). These observations indicate that MMPs (possibly MMP-2, -3, -9 and/or -20) play a role in the onset of dentin mineralization. The lack of enamel formation was possibly due to diffusion of amelogenin from its normal site of apposition. The protein clearly was not retained at the surface of the non-mineralized dentin layer, and immunopositive amelogenin accumulated in the odontoblast compartment. The diffusion of enamel proteins and the accumulation revealed by immunolabeling of two small leucine-rich proteoglycans, decorin and biglycan, in the predentin may have contributed to impaired dentin mineralization.

To accommodate developing tooth germ in the alveolar bone, active bone resorption and the recruitment of numerous osteoclasts are essential. Recently, the signaling of receptor activator of nuclear factor-KappaB (RANK) and its ligand (RANKL) was reported to play a pivotal role in osteoclast formation and activation. The aim of this study was to examine the expression of RANKL and the contribution of RANK-RANKL signaling to the process of tooth germ and alveolar bone development. In situ hybridization showed RANKL was expressed in dental follicle cells and osteoblasts on the alveolar bone surface surrounding developing tooth germs. To elucidate the function of RANKL, mouse mandibular explants on embryonic day 14 were subjected to organ culture with osteoprotegerin (OPG), an inhibitor of RANK-RANKL signaling as a decoy receptor of RANKL. Many tooth germs were compressed with the surrounding bone tissue in the OPG-treated explants, whereas these abnormalities were not seen in untreated explants. The numbers of tartrate-resistant acid phosphatase (TRAP)-positive osteoclastic cells aligning on the alveolar bone surface were significantly decreased in OPG-treated explants compared with untreated explants. Moreover, TRAP-positive osteoclastic cells were not observed along the alveolar bone surfaces depressing tooth germs. These observations suggest that osteoclastogenesis in the alveolar bone, which is essential for the accommodation of normal tooth development, is mediated by RANK-RANKL signaling.

We examined the effects of basic fibroblast growth factor (FGF-2) on cultured lower molar tooth germ at the differentiative (bell) stage. Although FGF-2 has been detected in odontogenesis, its roles in biological activities, such as cell proliferation, differentiation and extracellular matrix mineralization are unclear. We assayed mRNA levels of the differentiation markers, dentine sialophosphoprotein (DSPP), amelogenin and alkaline phosphatase (ALP) using reverse transcription-polymerase chain reaction (RT-PCR), and histological methods. Tooth germs dissected from 17-day-old embryonic mice were cultured for 4 days with either recombinant human FGF-2 or specific antisense phosphorothioate oligodeoxynucleotide (antisense ODN) for FGF-2. Exogenous FGF-2 decreased the gene expression of differentiation markers in molars at the bell stage. Abrogation of endogenous FGF-2 by antisense ODN increased the gene expression of differentiation markers, and also significantly enhanced enamel and dentine formation. This histological change was recovered by adding exogeneous FGF-2. These findings suggest that FGF-2 at the bell stage regulates cell differentiation and matrix secretion.

In the developing tooth, 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) causes hypoplasia and hypomineralization of enamel and dentine. The present study was undertaken to clarify the effects of HEBP on the formation of dental tissues of tooth germs in an organ culture system. Mandibular first molars from 17.5-day-old mouse embryos were cultured with or without 250 microM HEBP in culture medium. Cultured tooth germs were analyzed by histological examination and by immunohistochemical localization using anti-amelogenin antibody. In cultured tooth germs treated with HEBP before the commencement of calcification in dentine, calcification of dentine matrix was inhibited completely and enamel formation was not observed. Ameloblasts were directly adjacent to dentine matrix. However, immunohistochemical data indicated that these ameloblasts secreted amelogenin. In the experiments of adding HEBP to cultured tooth germs on culture day 13, calcified dentine and enamel had formed before the administration of HEBP, but the dentine matrix newly formed after the administration of HEBP had not calcified. It was confirmed by immunohistochemical observations that enamel matrix-like material had penetrated into uncalcified dentine matrix and accumulated in dental papilla of tooth germs. However, no enamel matrix-like material was observed in calcified dentine and predentine underneath the calcified dentine by immunohistochemical staining. From these results, it might be concluded that ameloblasts secreted enamel matrix in the presence of HEBP and diffused through uncalcified dentine matrix into dental papilla. These findings suggests the calcification of dentine might be essential for the physical barrier to accumulate the enamel matrix and form a distinct layer of enamel as enamel.

dHAND/Hand2 is a basic helix-loop-helix transcription factor required for the development of the heart, pharyngeal arches, and vasculature and is expressed during embryogenesis. However, there are no reports on the involvement of the dHAND gene in tooth development. In the present study, the expression of dHAND was examined in developing tooth germs of mice. The dHAND gene was expressed in the mesenchyme of the presumptive incisor region of the lower jaw at an early stage and in the mesenchyme of the lower incisor tooth germ at a later stage. However, the dHAND gene was not expressed in the upper incisor region or the upper and lower molar regions during jaw development. Treatment of tooth germ explants of lower incisors with antisense oligodeoxinucleotide (ODN) against dHAND prevented the differentiation of tooth germ cells, including ameloblasts and odontoblasts, the formation of dentin and enamel, and the proliferation of tooth germ cells and increased the apoptosis of tooth germ cells, suggesting that dHAND is essential for these cells during development. On the other hand, the treatment of tooth germ explants of upper incisor and upper or lower molars did not induce severe effects on their development. Treatment of the explants with basic fibroblast growth factor in association with antisense ODN partially rescued them from the effects of antisense ODN. The present results suggest that the dHAND gene plays important roles in type-specific development of lower incisors, and that basic fibroblast growth factor is involved downstream of the dHAND pathway in tooth germ cells.

Shh expression is highly restricted to the future sites of tooth development during the initiation of odontogenesis. This suggests a role for Shh as a proliferative factor, as localized epithelial thickenings invaginate to form a tooth bud. We have investigated this role by blocking Shh signaling between E10.5 and E12.5 in murine mandibular processes using a 5E1 blocking antibody and the PKA activator Forskolin. This results in down-regulation of Ptc, a principle target of Shh signaling. The effects of inhibition varied with developmental time. At E10.5, tooth development was arrested as epithelial thickenings and the numbers of teeth developing were considerably reduced. Inhibition at E12.5 produced localized apoptosis in the epithelium at the tip of the tooth buds, although some teeth were able to develop. Thus, Shh has dual roles in early odontogenesis, first in bud formation by stimulating epithelial proliferation, and second in the development of cap-stage tooth germs by increasing epithelial cell survival.