Induction of human stem cells into ameloblasts by reaggregation strategy.

BACKGROUND: Human epithelium-derived stem cells and induced pluripotent stem cells (hiPSCs) possess the capability to support tooth formation and differentiate into functional enamel-secreting ameloblasts, making them promising epithelial-component substitutes for future human tooth regeneration. However, current tissue recombination approaches are not only technically challenging, requiring precise induction procedures and sophisticated microsurgery, but also exhibit low success rates in achieving tooth formation and ameloblastic differentiation. METHODS: Suspended human keratinocyte stem cells (hKSCs) or cells from three hiPSC lines were directly mixed with dissociated embryonic mouse dental mesenchymal cells (mDMCs) that possess odontogenic potential in different proportions and reaggregated them to construct bioengineered tooth germs. The success rates of tooth formation and ameloblastic differentiation were confirmed after subrenal culture. The sorting capability, sequential development, and ameloblastic differentiation of stem cells were examined via GFP tracing, RT-PCR, and histological analysis, respectively. RESULTS: Our reaggregation approach achieved an impressive success rate of more than 90% in tooth formation and 100% in ameloblastic differentiation when the chimeric tooth germs contained 1%~10% hKSCs or 5% hiPSCs. In addition, we observed that hiPSCs, upon exposure to mDMCs, initially transformed into epidermal cells, as indicated by KRT14 and CD29 expression, before progressing into dental epithelial cells, as indicated by SP6 and SHH expression. We also found that epithelial-derived hiPSCs, when reaggregated with mDMCs, were more favorable for tooth formation than their mesenchymal-derived counterparts. CONCLUSIONS: This study establishes a simplified yet highly effective cell-cell reaggregation strategy for inducing stem cells to support tooth formation and differentiate into functional ameloblasts, paving the way for novel approaches for the development of stem cell-based tooth organoids and bioengineered tooth germs in vitro.

Six1 Regulates Mouse Incisor Development by Promoting Dlx1/2/5 Expression.

Tooth development is a complex process orchestrated by intricate gene regulatory networks, involving both odontogenic epithelium and ectomesenchyme. Six1, a pivotal transcription factor (TF), is involved in the development of the lower incisor. However, its precise role during incisor development and the molecular mechanisms underpinning its regulatory functions remain poorly understood. This study employs Six1 deletion mouse models to elucidate the critical regulatory role of Six1 in governing dental mesenchyme development. By performing single-cell RNA sequencing, we constructed a comprehensive transcriptome atlas of tooth germ development from the bud to bell stage. Our analyses suggest that the dental follicle and the dental papilla (DP) are differentiated from dental ectomesenchyme (DEM) and identify the key TFs underlying these distinct states. Notably, we show that Dlx1, Dlx2, and Dlx5 (Dlx1/2/5) may function as the key TFs that promote the formation of DP. We further show that the deletion of Six1 perturbs dental mesenchyme development by impeding the transitions from DEM to DP states. Importantly, SIX1 directly binds to the promoters of Dlx1/2/5 to promote their co-expression, which subsequently leads to widespread epigenetic and transcriptional remodeling. In summary, our findings unveil Six1's indispensable role in incisor development, offering key insights into TF-driven regulatory networks that govern dental mesenchyme cell fate transitions during tooth development.

Exploring the role of DNMT1 in dental papilla cell fate specification during mouse tooth germ development through integrated single-cell transcriptomics and bulk RNA sequencing.

OBJECTIVES: This study aimed to investigate the regulatory mechanisms governing dental mesenchymal cell commitment during tooth development, focusing on odontoblast differentiation and the role of epigenetic regulation in this process. METHODS: We performed single-cell RNA sequencing (scRNA-seq) of dental cells from embryonic day 14.5 (E14.5) mice to understand the heterogeneity of developing tooth germ cells. Computational analyses including gene regulatory network (GRN) assessment were conducted. We validated our findings using immunohistochemistry (IHC) and in vitro loss-of-function analyses using the DNA methyltransferase 1 (DNMT1) inhibitor Gsk-3484862 in primary dental mesenchymal cells (DMCs) isolated from E14.5 mouse tooth germs. Bulk RNA-seq of Gsk-3484862-treated DMCs was performed to identify potential downstream targets of DNMT1. RESULTS: scRNA-seq analysis revealed diverse cell populations within the tooth germs, including epithelial, mesenchymal, immune, and muscle cells. Using single-cell regulatory network inference and clustering (SCENIC), we identified Dnmt1 as a key regulator of early odontoblast development. IHC analysis showed the ubiquitous expression of DNMT1 in the dental papilla and epithelium. Bulk RNA-seq of cultured DMCs showed that Gsk-3484862 treatment upregulated odontoblast-related genes, whereas genes associated with cell division and the cell cycle were downregulated. Integrated analysis of bulk RNA-seq data with scRNA-seq SCENIC profiles was used to identify the potential Dnmt1 target genes. CONCLUSIONS: Dnmt1 may negatively affect odontoblast commitment and differentiation during tooth development. These findings contribute to a better understanding of the molecular mechanisms underlying tooth development and future development of hard-tissue regenerative therapies.

The temporal protein signature analyses of developing human deciduous molar tooth germ.

The tooth serves as an exemplary model for developmental studies, encompassing epithelial-mesenchymal transition and cell differentiation. The essential factors and pathways identified in tooth development will help understand the natural development process and the malformations of mineralized tissues such as skeleton. The time-dependent proteomic changes were investigated through the proteomics of healthy human molars during embryonic stages, ranging from the cap-to-early bell stage. A comprehensive analysis revealed 713 differentially expressed proteins (DEPs) exhibiting five distinct temporal expression patterns. Through the application of weighted gene co-expression network analysis (WGCNA), 24 potential driver proteins of tooth development were screened, including CHID1, RAP1GDS1, HAPLN3, AKAP12, WLS, GSS, DDAH1, CLSTN1, AFM, RBP1, AGO1, SET, HMGB2, HMGB1, ANP32A, SPON1, FREM1, C8B, PRPS2, FCHO2, PPP1R12A, GPALPP1, U2AF2, and RCC2. Then, the proteomics and transcriptomics expression patterns of these proteins were further compared, complemented by single-cell RNA-sequencing (scRNA-seq). In summary, this study not only offers a wealth of information regarding the molecular intricacies of human embryonic epithelial and mesenchymal cell differentiation but also serves as an invaluable resource for future mechanistic inquiries into tooth development.

The Essential Role of Proteoglycans and Glycosaminoglycans in Odontogenesis.

Tooth development and regeneration are regulated through a complex signaling network. Previous studies have focused on the exploration of intracellular signaling regulatory networks, but the regulatory roles of extracellular networks have only been revealed recently. Proteoglycans, which are essential components of the extracellular matrix (ECM) and pivotal signaling molecules, are extensively involved in the process of odontogenesis. Proteoglycans are composed of core proteins and covalently attached glycosaminoglycan chains (GAGs). The core proteins exhibit spatiotemporal expression patterns during odontogenesis and are pivotal for dental tissue formation and periodontium development. Knockout of core protein genes Biglycan, Decorin, Perlecan, and Fibromodulin has been shown to result in structural defects in enamel and dentin mineralization. They are also closely involved in the development and homeostasis of periodontium by regulating signaling transduction. As the functional component of proteoglycans, GAGs are negatively charged unbranched polysaccharides that consist of repeating disaccharides with various sulfation groups; they provide binding sites for cytokines and growth factors in regulating various cellular processes. In mice, GAG deficiency in dental epithelium leads to the reinitiation of tooth germ development and the formation of supernumerary incisors. Furthermore, GAGs are critical for the differentiation of dental stem cells. Inhibition of GAGs assembly hinders the differentiation of ameloblasts and odontoblasts. In summary, core proteins and GAGs are expressed distinctly and exert different functions at various stages of odontogenesis. Given their unique contributions in odontogenesis, this review summarizes the roles of proteoglycans and GAGs throughout the process of odontogenesis to provide a comprehensive understanding of tooth development.

Spatiotemporal expression profiles of c-Mpl mRNA in the tooth germ: Comparative expression dynamics of vascularization-related genes.

BACKGROUND: Vascularization is an essential event for both embryonic organ development and tissue repair in adults. During mouse tooth development, endothelial cells migrate into dental papilla during the cap stage, and form blood vessels through angiogenesis. Megakaryocytes and/or platelets, as other hematopoietic cells, express angiogenic molecules and can promote angiogenesis in adult tissues. However, it remains unknown which cells are responsible for attracting and leading blood vessels through the dental papilla during tooth development. METHODS: Here we analyzed the spatiotemporal expression of c-Mpl mRNA in developing molar teeth of fetal mice. Expression patterns were then compared with those of several markers of hematopoietic cells as well as of angiogenic elements including CD41, erythropoietin receptor, CD34, angiopoietin-1 (Ang-1), Tie-2, and vascular endothelial growth factor receptor2 (VEGFR2) through in situ hybridization or immunohistochemistry. RESULTS: Cells expressing c-Mpl mRNA was found in several parts of the developing tooth germ, including the peridental mesenchyme, dental papilla, enamel organ, and dental lamina. This expression occurred in a spatiotemporally controlled fashion. CD41-expressing cells were not detected during tooth development. The spatiotemporal expression pattern of c-Mpl mRNA in the dental papilla was similar to that of Ang-1, which preceded invasion of endothelial cells. Eventually, at the early bell stage, the c-Mpl mRNA signal was detected in morphologically differentiating odontoblasts that accumulated in the periphery of the dental papilla along the inner enamel epithelium layer of the future cusp region. CONCLUSION: During tooth development, several kinds of cells express c-Mpl mRNA in a spatiotemporally controlled fashion, including differentiating odontoblasts. We hypothesize that c-Mpl-expressing cells appearing in the forming dental papilla at the cap stage are odontoblast progenitor cells that migrate to the site of odontoblast differentiation. There they attract vascular endothelial cells into the forming dental papilla and lead cells toward the inner enamel epithelium layer through production of angiogenic molecules (e.g., Ang-1) during migration to the site of differentiation. C-Mpl may regulate apoptosis and/or proliferation of expressing cells in order to execute normal development of the tooth.

Single-cell RNA-seq of in vitro expanded cells from cranial neural crest reveals a rare odontogenic sub-population.

Ecto-mesenchymal cells of mammalian tooth germ develops from cranial neural crest cells. These cells are recognised as a promising source for tooth development and regeneration. Despite the high heterogeneity of the neural crest, the cellular landscape of in vitro cultured cranial neural crest cells (CNCCs) for odontogenesis remains unclear. In this study, we used large-scale single-cell RNA sequencing to analyse the cellular landscape of in vitro cultured mouse CNCCs for odontogenesis. We revealed distinct cell trajectories from primary cells to passage 5 and identified a rare Alx3+/Barx1+ sub-population in primary CNCCs that differentiated into two odontogenic clusters characterised by the up-regulation of Pax9/Bmp3 and Lhx6/Dmp1. We successfully induced whole tooth-like structures containing enamel, dentin, and pulp under the mouse renal capsule using in vitro cultured cells from both cranial and trunk neural crests with induction rates of 26.7% and 22.1%, respectively. Importantly, we confirmed only cells sorted from odontogenic path can induce tooth-like structures. Cell cycle and DNA replication genes were concomitantly upregulated in the cultured NCCs of the tooth induction groups. Our data provide valuable insights into the cell heterogeneity of in vitro cultured CNCCs and their potential as a source for tooth regeneration.

Connexin 43 expression in tooth germ and benign odontogenic tumors.

OBJECTIVE: The aim of this study was to investigate and compare the immunohistochemical expression of connexin 43 (Cx43) in tooth germs (TGs), ameloblastic fibromas (AFs), ameloblastic fibro-odontomas (AFOs), and conventional ameloblastomas (AMs). STUDY DESIGN: Nine TGs, 12 AFs, 12 AFOs, and 27 AMs were evaluated for Cx43 expression by immunohistochemistry. RESULTS: Most of the TGs expressed Cx43 in the mesenchyme (77.6%) and in the late stages of odontogenesis. Cx43 was more highly expressed (P < .05) in the mesenchymal layer of all groups than in the epithelial layer except for the AFOs. When comparing the expression of Cx43 in the different layers of the analyzed groups, statistically significant differences were observed between AFO vs AM (*P = .0158) in the epithelial layer and between AF vs AFO (P** = .0046) in the mesenchymal layer. CONCLUSIONS: The results obtained in this study showed that Cx43 is a protein with important expression in the mesenchymal layer of the embryonic and odontogenic tissues studied. It could be speculated that Cx43 participates in mineralization events based on the relationship of the expression of this protein between the epithelial and mesenchymal layers of odontogenic tissues.

mTORC1 signaling pathway regulates tooth repair.

Tooth germ injury can lead to abnormal tooth development and even tooth loss, affecting various aspects of the stomatognathic system including form, function, and appearance. However, the research about tooth germ injury model on cellular and molecule mechanism of tooth germ repair is still very limited. Therefore, it is of great importance for the prevention and treatment of tooth germ injury to study the important mechanism of tooth germ repair by a tooth germ injury model. Here, we constructed a Tg(dlx2b:Dendra2-NTR) transgenic line that labeled tooth germ specifically. Taking advantage of the NTR/Mtz system, the dlx2b+ tooth germ cells were depleted by Mtz effectively. The process of tooth germ repair was evaluated by antibody staining, in situ hybridization, EdU staining and alizarin red staining. The severely injured tooth germ was repaired in several days after Mtz treatment was stopped. In the early stage of tooth germ repair, the expression of phosphorylated 4E-BP1 was increased, indicating that mTORC1 is activated. Inhibition of mTORC1 signaling in vitro or knockdown of mTORC1 signaling in vivo could inhibit the repair of injured tooth germ. Normally, mouse incisors were repaired after damage, but inhibition/promotion of mTORC1 signaling inhibited/promoted this repair progress. Overall, we are the first to construct a stable and repeatable repair model of severe tooth germ injury, and our results reveal that mTORC1 signaling plays a crucial role during tooth germ repair, providing a potential target for clinical treatment of tooth germ injury.

Establishing protein expression profiles involved in tooth development using a proteomic approach.

Various growth and transcription factors are involved in tooth development and developmental abnormalities; however, the protein dynamics do not always match the mRNA expression level. Using a proteomic approach, this study comprehensively analyzed protein expression in epithelial and mesenchymal tissues of the tooth germ during development. First molar tooth germs from embryonic day 14 and 16 Crlj:CD1 (ICR) mouse embryos were collected and separated into epithelial and mesenchymal tissues by laser microdissection. Mass spectrometry of the resulting proteins was carried out, and three types of highly expressed proteins [ATP synthase subunit beta (ATP5B), receptor of activated protein C kinase 1 (RACK1), and calreticulin (CALR)] were selected for immunohistochemical analysis. The expression profiles of these proteins were subsequently evaluated during all stages of amelogenesis using the continuously growing incisors of 3-week-old male ICR mice. Interestingly, these three proteins were specifically expressed depending on the stage of amelogenesis. RACK1 was highly expressed in dental epithelial and mesenchymal tissues during the proliferation and differentiation stages of odontogenesis, except for the pigmentation stage, whereas ATP5B and CALR immunoreactivity was weak in the enamel organ during the early stages, but became intense during the maturation and pigmentation stages, although the timing of the increased protein expression was different between the two. Overall, RACK1 plays an important role in maintaining the cell proliferation and differentiation in the apical end of incisors. In contrast, ATP5B and CALR are involved in the transport of minerals and the removal of organic materials as well as matrix deposition for CALR.

Promotion effect of apical tooth germ cell-conditioned medium on osteoblastic differentiation of periodontal ligament stem cells through regulating miR-146a-5p.

BACKGROUND: MicroRNAs (miRNAs) play an important role in gene regulation that controls stem cells differentiation. Periodontal ligament stem cells (PDLSCs) could differentiate into osteo-/cementoblast-like cells that secretes cementum-like matrix both in vitro and in vivo. Whether miRNAs play key roles in osteoblastic differentiation of PDLSCs triggered by a special microenviroment remains elusive. In this study, we aimed to investigate potential miRNA expression changes in osteoblastic differentiation of PDLSCs by the induction of apical tooth germ cell-conditioned medium (APTG-CM). METHODS AND RESULTS: First, we analyzed the ability of APTG-CM to osteogenically differentiate PDLSCs. The results exhibited an enhanced mineralization ability, higher ALP activity and increased expression of osteogenic genes in APTG-CM-induced PDLSCs. Second, we used miRNA sequencing to analyze the miRNA expression profile of PDLSCs derived from three donors under 21-day induction or non-induction of APTG-CM. MiR-146a-5p was found to be up-regulated miRNA in induced PDLSCs and validated by RT-qPCR. Third, we used lentivirus-up/down system to verify the role of miR-146a-5p in the regulation of osteoblastic differentiation of PDLSCs. CONCLUSIONS: In conclusion, our results demonstrated that miR-146a-5p was involved in the promotion effect of APTG-CM on osteoblastic differentiation of PDLSCs, and suggested that miR-146a-5p might be a novel way in deciding the direction of PDLSCs differentiation.

Ep-CAM (MOC-31) expression in tooth germ and ameloblastoma.

BACKGROUND: Ep-CAM, a transmembrane glycoprotein expressed in most epithelium in normal conditions, has diverse roles in these tissues, including in cell adhesion, proliferation, differentiation, cell cycle regulation, migration and intracellular signaling. It is also over-expressed in most malignant neoplasia, participating in the initiation, progression, and metastatic dissemination of the tumor. The expression and roles of this protein in oral neoplasia, particularly in odontogenic tumors, remain unestablished. The objective of this study consisted in analyzing the expression of this protein in ameloblastoma and tooth germ. MATERIAL AND METHODS: Ep-CAM (MOC-31) expression was evaluated by immunohistochemistry in tooth germs (TG) (n = 16) ameloblastomas (AM) (n = 60) and 2 ameloblastic carcinomas. Sections were visualized in their totality with an optical microscope, and positivity observed in cell membrane and cytoplasm was graded according to the following semi-quantitative scale: Neg, "essentially unstained", for negative sections or staining <5% of cells; + for staining of 5-50% of cells; ++ for staining >50% of cells. RESULTS: Most tooth germs expressed MOC-31 (81.3%), strong staining was observed both in the inner epithelium of the enamel organ and in the adjacent stellate reticulum. 16.7% of the AM cases showed MOC-31 expression, the immunoexpression expression was diffuse at the cytoplasmic and membrane level. The only two cases of ameloblastic carcinoma included were strong positive to MOC-31. No correlation was observed between protein expression and gender, age, clinical variants, or histological subtypes. CONCLUSIONS: Overexpression was found in TG and ameloblastic carcinoma compared to AM; further studies with different experimental strategies are suggested to clarify the biological significance of this finding.

MSX1 Drives Tooth Morphogenesis Through Controlling Wnt Signaling Activity.

Tooth agenesis is a common structural birth defect in humans that results from failure of morphogenesis during early tooth development. The homeobox transcription factor Msx1 and the canonical Wnt signaling pathway are essential for "bud to cap" morphogenesis and are causal factors for tooth agenesis. Our recent study suggested that Msx1 regulates Wnt signaling during early tooth development by suppressing the expression of Dkk2 and Sfrp2 in the tooth bud mesenchyme, and it demonstrated partial rescue of Msx1-deficient molar teeth by a combination of DKK inhibition and genetic inactivation of SFRPs. In this study, we found that Sostdc1/Wise, another secreted Wnt antagonist, is involved in regulating the odontogenic pathway downstream of Msx1. Whereas Sostdc1 expression in the developing tooth germ was not increased in Msx1-/- embryos, genetic inactivation of Sostdc1 rescued maxillary molar, but not mandibular molar, morphogenesis in Msx1-/- mice with full penetrance. Since the Msx1-/-;Sostdc1-/- embryos exhibited ectopic Dkk2 expression in the developing dental mesenchyme, similar to Msx1-/- embryos, we generated and analyzed tooth development in Msx1-/-;Dkk2-/- double and Msx1-/-;Dkk2-/-;Sostdc1-/- triple mutant mice. The Msx1-/-;Dkk2-/- double mutants showed rescued maxillary molar morphogenesis at high penetrance, with a small percentage also exhibiting mandibular molars that transitioned to the cap stage. Furthermore, tooth development was rescued in the maxillary and mandibular molars, with full penetrance, in the Msx1-/-;Dkk2-/-;Sostdc1-/- mice. Together, these data reveal 1) that a key role of Msx1 in driving tooth development through the bud-to-cap transition is to control the expression of Dkk2 and 2) that modulation of Wnt signaling activity by Dkk2 and Sostdc1 plays a crucial role in the Msx1-dependent odontogenic pathway during early tooth morphogenesis.

An in situ hybridization study of syndecan family during the late stages of developing mouse molar tooth germ.

Expression of syndecan-1, 2, 3, and 4 mRNAs during the late stages of tooth germ formation was investigated by in situ hybridization, using [35S]-UTP-labeled cRNA probes. Syndecan-1 mRNA was mainly expressed in the stellate reticulum and stratum intermedium as well as at the cervical region of dental papilla/dental follicle during E18.5-P3.0. Expression in the dental epithelium was enhanced during the postnatal periods, which was supported by real-time RT-PCR analysis. These spatiotemporal expression patterns may suggest specific roles of syndecan-1 in tooth formation such as tooth eruption or root formation. Syndecan-3 mRNA expression became evident in odontoblasts at E18.5, but compared to collagen type I mRNA, which was strongly expressed at this stage, syndecan-3 expression in odontoblast was restricted in mature odontoblasts beneath the cusps during the postnatal periods. This result was also supported by real-time RT-PCR analysis, and indicated that syndecan-3 may be involved in the progress of dentinogenesis rather than in the initiation of it. Syndecan-4 mRNA roughly showed comparable expression patterns to those of syndecan-3. Syndecan-2 mRNA did not show significant expression during the experimental period, but real-time RT-PCR analysis suggested that syndecan-2 expression might be enhanced with hard tissue formation.

Identification of novel candidate genes implicated in odontogenic potential in the developing mouse tooth germ using transcriptome analysis.

BACKGROUND: In tooth bioengineering for replacement therapy of missing teeth, the utilized cells must possess an inductive signal-forming ability to initiate odontogenesis. This ability is called odontogenic potential. In mice, the odontogenic potential signal is known to be translocated from the epithelium to the mesenchyme at the early bud stage in the developing molar tooth germ. However, the identity of the molecular constituents of this process remains unclear. OBJECTIVE: The purpose of this study is to determine the molecular identity of odontogenic potential and to provide a new perspective in the field of tooth development research. METHODS: In this study, whole transcriptome profiles of the mouse molar tooth germ epithelium and mesenchyme were investigated using the RNA sequencing (RNA-seq) technique. The analyzed transcriptomes corresponded to two developmental stages, embryonic day 11.5 (E11.5) and 14.5 (E14.5), which represent the odontogenic potential shifts. RESULTS: We identified differentially expressed genes (DEGs), which were specifically overexpressed in both the E11.5 epithelium and E14.5 mesenchyme, but not expressed in their respective counterparts. Of the 55 DEGs identified, the top three most expressed transcription factor genes (transcription factor AP-2 beta isoform 3 [TFAP2B], developing brain homeobox protein 2 [DBX2], and insulin gene enhancer protein ISL-1 [ISL1]) and three tooth development-related genes (transcription factor HES-5 [HES5], platelet-derived growth factor D precursor [PDGFD], semaphrin-3 A precursor [SEMA3A]) were selected and validated by quantitative RT-PCR. Using immunofluorescence staining, the TFAP2B protein expression was found to be localized only at the E11.5 epithelium and E14.5 mesenchyme. CONCLUSIONS: Thus, our empirical findings in the present study may provide a new perspective into the characterization of the molecules responsible for the odontogenic potential and may have an implication in the cell-based whole tooth regeneration strategy.

Transcriptional regulation of the basic helix-loop-helix factor AmeloD during tooth development.

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-ß1 induced AmeloD expression and ameloblast differentiation in the dental epithelial cell line, SF2. TGF-ß1 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-ß1 stimulation. These results suggested that TGF-ß1 activates AmeloD transcription via ERK1/2 phosphorylation. Our findings provide new insights into the mechanisms that govern tooth development.

Immunohistochemical localization of vascular factors in tooth germ of amphibian (Cynops pyrrhogaster) / Localización inmunohistoquímica de factores vasculares en germen dentario de anfibios (Cynops pyrrhogaster)

SUMMARY: Vascular endothelial growth factor (VEGF) and its receptor, VEGFR-2, are known to regulate blood vessel endothelium growth. They play important role in human and rodents teeth development. In newt jaws, there are sequential developmental teeth germs following behind the mature teeth. We examined the immunohistochemical localization of VEGF and its receptor and showed the specific expression pattern of VEGF and VEGF receptor in Cynops pyrrhogaster sequential tooth development. The intensity of immunoreactivity for VEGF in the inner enamel epithelium was weaker than that in the outer enamel epithelium in the dentine matrix formation and mineralization stages. Finally, at the enameloid maturation and enamel-like matrix formation stage, immunoreactivity for VEGF in inner enamel epithelium was stronger than in the outer enamel epithelium. The intensity of immunoreactivity for VEGFR-2 was positive for the outer enamel epithelium throughout tooth development. The crown sides of the odontoblasts were stained especially strongly for VEGF and VEGFR-2 during the dentine matrix formation and mineralization stage of the enameloid maturation and enamel- like matrix formation stage. We postulate that the expression of VEGF in the inner enamel epithelium and odontoblast widely effects tooth development in newts, as well as in human and rodents.

RESUMEN: Se sabe que el factor de crecimiento endotelial vascular (VEGF) y su receptor, VEGFR-2, regulan el crecimiento del endotelio de los vasos sanguíneos. Desempeñan un papel importante en el desarrollo de los dientes humanos y de los roedores. En las mandíbulas de tritón, hay gérmenes dentales de desarrollo secuenciales que siguen a los dientes maduros. Examinamos la localización inmunohistoquímica de VEGF y su receptor y mostramos el patrón de expresión específico de VEGF y receptor de VEGF en el desarrollo secuencial de dientes de Cynops pyrrhogaster. La intensidad de la inmunorreactividad para VEGF en el epitelio interno del esmalte era más débil que en el epitelio externo del esmalte en las etapas de formación y mineralización de la matriz de dentina. Finalmente, en la etapa de maduración del esmalte y de formación de la matriz similar al esmalte, la inmunorreactividad para VEGF en el epitelio interno del esmalte fue más fuerte que en el epitelio externo del esmalte. La intensidad de la inmunorreactividad para VEGFR- 2 fue positiva para el epitelio externo del esmalte durante el desarrollo del diente. Los márgenes de la corona de los odontoblastos se tiñeron especialmente para VEGF y VEGFR-2 durante la etapa de formación de la matriz de dentina y mineralización de la etapa de maduración del esmalte y la etapa de formación de la matriz similar al esmalte. Postulamos que la expresión de VEGF en el epitelio interno del esmalte y odontoblastos afecta ampliamente el desarrollo de los dientes en tritones, así como en humanos y roedores.

C-Jun N-terminal kinase (JNK) pathway activation is essential for dental papilla cells polarization.

During tooth development, dental papilla cells differentiate into odontoblasts with polarized morphology and cell function. Our previous study indicated that the C-Jun N-terminal kinase (JNK) pathway regulates human dental papilla cell adhesion, migration, and formation of focal adhesion complexes. The aim of this study was to further examine the role of the JNK pathway in dental papilla cell polarity formation. Histological staining, qPCR, and Western Blot suggested the activation of JNK signaling in polarized mouse dental papilla tissue. After performing an in vitro tooth germ organ culture and cell culture, we found that JNK inhibitor SP600125 postponed tooth germ development and reduced the polarization, migration and differentiation of mouse dental papilla cells (mDPCs). Next, we screened up-regulated polarity-related genes during dental papilla development and mDPCs or A11 differentiation. We found that Prickle3, Golga2, Golga5, and RhoA were all up-regulated, which is consistent with JNK signaling activation. Further, constitutively active RhoA mutant (RhoA Q63L) partly rescued the inhibition of SP600125 on cell differentiation and polarity formation of mDPCs. To sum up, this study suggests that JNK signaling has a positive role in the formation of dental papilla cell polarization.

Tooth Repair and Regeneration: Potential of Dental Stem Cells.

Tooth defects are an extremely common health condition that affects millions of individuals. Currently used dental repair treatments include fillings for caries, endodontic treatment for pulp necrosis, and dental implants to replace missing teeth, all of which rely on the use of synthetic materials. By contrast, the fields of tissue engineering and regenerative medicine and dentistry (TERMD) use biologically based therapeutic strategies for vital tissue regeneration, and thus have the potential to regenerate living tissues. Methods to create bioengineered replacement teeth benefit from a detailed understanding of the molecular signaling networks regulating natural tooth development. We discuss how key signaling pathways regulating natural tooth development are being exploited for applications in TERMD approaches for vital tooth regeneration.

Perilipin 1 and adipophilin immunoexpression suggests the presence of lipid droplets in tooth germ, ameloblastoma, and ameloblastic carcinoma.

BACKGROUND: Increased lipogenesis and lipid droplet accumulation are observed in diverse tumors, and these processes are associated with poor prognosis in several tumors, representing potential therapeutic targets. The presence of lipid droplets in odontogenic tissues and/or tumors is unknown. METHODS: Immunohistochemistry for perilipin 1 and adipophilin was performed in 12 human tooth germs (TG), 27 conventional ameloblastoma (AM), and 8 ameloblastic carcinoma (AC) samples. Cytoplasmic staining was analyzed using an immunoreactive score (IRS), and the results were compared for the TG, AM, and AC samples by Kruskal-Wallis test followed by Dunn's post-test and confirmed by Mann-Whitney U test. RESULTS: Perilipin 1 was negative in 91.7% of the TG samples, positive in 48.2% of the AM samples, and positive in 87.5% of the AC samples. Adipophilin was positive in 100% of the TG samples, 92.6% of the AM samples, and 100% of the AC samples. The perilipin 1 and adipophilin IRS revealed statistically significant differences between the TG, AM, and AC samples (p = .007 and p = .018, respectively). The perilipin 1 levels among the TG and AC samples were statically significant (**p = .0085), as well as the adipophilin levels when TG and AM samples were compared (**p < .0029). CONCLUSIONS: Adipophilin exhibits significant activity in human tooth development. The immunoexpression of perilipin 1 and adipophilin in the AM and AC samples suggests the presence of lipid droplets, providing further evidence of metabolic alterations in these tumors. Additional studies with larger samples and alternative techniques are necessary to confirm these findings.