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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., (© 2024. The Author(s).)

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., (Copyright © 2024 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.)

Understanding the cellular composition and trajectory of human tooth development is valuable for dentistry and stem cell engineering research. Previous single-cell studies have focused on mature human teeth and developing mouse teeth, but the cell landscape of human embryonic dental development is still unknown. In this study, tooth germ tissues were collected from aborted foetus (17-24 weeks) for single-cell RNA sequence and spatial transcriptome analysis. The cells were classified into seven subclusters of epithelium, and seven clusters of mesenchyme, as well as other cell types such as Schwann cell precursor and pericyte. For epithelium, the stratum intermedium branch and the ameloblast branch diverged from the same set of outer enamel-inner enamel-ALCAM+ epithelial cell lineage, but their spatial distribution of two branches was not clearly distinct. This trajectory received spatially adjacent regulation signals from mesenchyme and pericyte, including JAG1 and APP. The differentiation of pulp cell and pre-odontoblast showed four waves of temporally distinct gene expression, which involved regulation networks of LHX9, DLX5 and SP7, and these genes were regulated by upstream ligands such as the BMP family. This provides a reference landscape for the research on early human tooth development, covering different spatial structures and developmental periods., (© 2024 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

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., (© 2024 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

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.)

Signaling centers, or organizers, regulate many aspects of embryonic morphogenesis. In the mammalian molar tooth, reiterative signaling in specialized centers called enamel knots (EKs) determines tooth patterning. Preceding the primary EK, transient epithelial thickening appears, the significance of which remains debated. Using tissue confocal fluorescence imaging with laser ablation experiments, we show that this transient thickening is an earlier signaling center, the molar initiation knot (IK), that is required for the progression of tooth development. IK cell dynamics demonstrate the hallmarks of a signaling center: cell cycle exit, condensation and eventual silencing through apoptosis. IK initiation and maturation are defined by the juxtaposition of cells with high Wnt activity to Shh-expressing non-proliferating cells, the combination of which drives the growth of the tooth bud, leading to the formation of the primary EK as an independent cell cluster. Overall, the whole development of the tooth, from initiation to patterning, is driven by the iterative use of signaling centers., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Teeth arise from the tooth germ through sequential and reciprocal interactions between immature epithelium and mesenchyme during development. However, the detailed mechanism underlying tooth development from tooth germ mesenchymal cells (TGMCs) remains to be fully understood. Here, we investigate the role of Wnt/β-catenin signalling in BMP9-induced osteogenic/odontogenic differentiation of TGMCs. We first established the reversibly immortalized TGMCs (iTGMCs) derived from young mouse mandibular molar tooth germs using a retroviral vector expressing SV40 T antigen flanked with the FRT sites. We demonstrated that BMP9 effectively induced expression of osteogenic markers alkaline phosphatase, collagen A1 and osteocalcin in iTGMCs, as well as in vitro matrix mineralization, which could be remarkably blunted by knocking down β-catenin expression. In vivo implantation assay revealed that while BMP9-stimulated iTGMCs induced robust formation of ectopic bone, knocking down β-catenin expression in iTGMCs remarkably diminished BMP9-initiated osteogenic/odontogenic differentiation potential of these cells. Taken together, these discoveries strongly demonstrate that reversibly immortalized iTGMCs retained osteogenic/odontogenic ability upon BMP9 stimulation, but this process required the participation of canonical Wnt signalling both in vitro and in vivo. Therefore, BMP9 has a potential to be applied as an efficacious bio-factor in osteo/odontogenic regeneration and tooth engineering. Furthermore, the iTGMCs may serve as an important resource for translational studies in tooth tissue engineering., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

The gene expression and protein synthesis of small leucine-rich proteoglycans (SLRPs), including decorin, biglycan, fibromodulin, and lumican, was analyzed in the context of the hypothesis that they are closely related to tooth formation. In situ hybridization, immunohistochemistry, and organ culture with metabolic labeling of [35S] were carried out in mouse first molar tooth germs of different developmental stages using ICR mice at embryonic day (E) 13.5 to postnatal day (P) 7.0. At the bud and cap stage, decorin mRNA was expressed only in the surrounding mesenchyme, but not within the tooth germ. Biglycan mRNA was then expressed in the condensing mesenchyme and the dental papilla of the tooth germ. At the apposition stage (late bell stage), both decorin and biglycan mRNA were expressed in odontoblasts, resulting in a switch of the pattern of expression within the different stages of odontoblast differentiation. Decorin mRNA was expressed earlier in newly differentiating odontoblasts than biglycan. With odontoblast maturation and dentin formation, decorin mRNA expression was diminished and localized to the newly differentiating odontoblasts at the cervical region. Simultaneously, biglycan mRNA took over and extended its expression throughout the new and mature odontoblasts. Both mRNAs were expressed in the dental pulp underlying the respective odontoblasts. At P7.0, both mRNAs were weakly expressed but maintained their spatial expression patterns. Immunostaining showed that biglycan was localized in the dental papillae and pulp. In addition, all four SLRPs showed clear immunostaining in predentin, although the expressions of fibromodulin and lumican mRNAs were not identified in the tooth germs examined. The organ culture data obtained supported the histological findings that biglycan is more predominant than decorin at the apposition stage. These results were used to identify biglycan as the principal molecule among the SLRPs investigated. Our findings indicate that decorin and biglycan show spatial and temporal differential expressions and play their own tissue-specific roles in tooth development.

Tooth formation is accomplished under strict genetic control procedures. Therefore, exploring the gene network system of tooth development has a very positive practical significance for the study of tooth tissue regeneration and the prevention and treatment of tooth abnormalities. Early bell stage is the initial phase of odontoblast formation and dentin matrix deposition in the process of tooth development. Through RNA sequencing and differential gene analysis of the rat tooth germ samples at cap stage and early bell stage, we found that the bile secretion pathway was the most significant difference signal pathway during the development between cap stage and bell stage, which mainly included ABCC3, AQP4, SLC10A1, SLC2A1, SLC4A4, ADCY5, AQP9, CFTR, ATP1A2, ATP1B1 and ATP1A1, totally 11genes. Immunostaining revealed that SLC2A1, SLC4A4, ADCY5 and ATP1B1were mainly expressed in epithelium in bud stage and inner and outer enamel epithelium during the embryonic phase. In the postnatal 1 and postnatal 7, SLC2A1, SLC4A4 and ABCC3 were highly expressed in ameloblasts and odontoblasts while ADCY5, ATP1B1 and SLC10A1was expressed moderately only in odontoblasts. This finding illustrated that the bile secretion pathway related genes may participate in the development of tooth germ.

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.

Bone defects due to trauma or diseases still pose a clinical challenge to be resolved in the current tissue engineering approaches. As an alternative to traditional methods to restore bone defects, such as autografts, bone tissue engineering aims to achieve new bone formation via novel biomaterials used in combination with multipotent stem cells and bioactive molecules. Mesenchymal stem cells (MSCs) can be successfully isolated from various dental tissues at different stages of development including dental pulp, apical papilla, dental follicle, tooth germ, deciduous teeth, periodontal ligament and gingiva. A wide range of biomaterials including polymers, ceramics and composites have been investigated for their potential as an ideal bone scaffold material. This article reviews the properties and the manufacturing methods of biomaterials used in bone tissue engineering, and provides an overview of bone tissue regeneration approaches of scaffold and dental stem cell combinations as well as their limitations.

Objectives: The successional dental lamina is the distinctive structure on the lingual side of the vertebrate tooth germ. The aim of this study was to investigate the relationship among Sox2, Claudin10 and laminin5 and the role of Sox2 in successional dental lamina proliferation during vertebrate tooth development., Materials and Methods: To understand the successional dental lamina, two types of successional tooth formation, that in geckos (with multiple rounds of tooth generation) and that in mice (with only one round of tooth generation), were analysed., Results: Unique coexpression patterns of Sox2 and Claudin10 expression were compared in the successional dental lamina from the cap stage to the late bell stage in the mouse tooth germ and in juvenile gecko teeth to support continuous tooth replacement. Furthermore, Laminin5 expression was shown in the cap stage and decreased after the bell stage. Upon comparing the epithelial cell cycles and cell proliferation in successional dental lamina regions between mouse and gecko molars using BrdU and IdU staining and pulse-chase methods, distinctive patterns of continuous expression were revealed. Moreover, Sox2 overexpression with a lentiviral system resulted in hyperplastic dental epithelium in mouse molars., Conclusions: Our findings indicate that the regulation of Sox2 in dental lamina proliferation is fundamental to the successional dental lamina in both species., (© 2019 The Authors. Cell Proliferation published by John Wiley & Sons Ltd.)

Dental epithelial‑mesenchymal signaling is crucial for tooth development, but the detailed mechanism is not fully understood. Using microarray analysis, it was revealed that the expression of osteoprotegerin, an important factor regulating bone remodeling, significantly increased after removal of the dental epithelium. Immunohistochemical staining revealed that osteoprotegerin expression within the dental mesenchyme was quite low during the prenatal period, but significantly increased after birth. To investigate the influence of osteoprotegerin upon tooth development, first‑molar tooth germs from embryonic day 14.5 (E14.5) Chinese Kunming mice were treated with different concentrations of osteoprotegerin. It was revealed that osteoprotegerin could inhibit the expression of odontogenic markers while promoting the expression of osteogenic markers, thereby disrupting tooth morphogenesis. These findings were further supported by in vitro and in vivo cultures. Finally, quantitative reverse transcription‑polymerase chain reaction and immunofluorescence studies revealed that, after osteoprotegerin treatment, the activity of the wingless/integrated (Wnt)/β‑catenin pathway increased, indicating that increased osteoprotegerin expression in prenatal tooth development could lead to uncontrolled upregulation of the Wnt/β‑catenin pathway.

The TGFβ superfamily of proteins participates in tooth development. TGFβ1 and TGFβ3 regulate odontoblast differentiation and dentin extracellular matrix synthesis. Although the expression of TGFβ family member ligands is well-characterized during mammalian tooth development, less is known about the TGFβ receptor, which is a heteromeric complex consisting of a type I and type II receptors. The molecular mechanism of ALK5 (TGFβR1) in the dental mesenchyme is not clear. We investigated the role of ALK5 in tooth germ mesenchymal cells (TGMCs) from the lower first molar tooth germs of day 15.5 embryonic mice. Human recombinant TGFβ3 protein or an ALK5 inhibitor (SD208) was added to the cells. Cell proliferation was inhibited by SD208 and promoted by TGFβ3. We found that SD208 inhibited TGMCs osteogenesis and dentinogenesis. Both canonical and noncanonical TGFβ signaling pathways participated in the process. TAK1, P-TAK1, p38 and P-p38 showed greater expression and SMAD4 showed less expression when ALK5 was inhibited. Our findings contribute to understanding the role of TGFβ signaling for the differentiation of mesenchymal stem cells derived from dental germ and suggest possible targets for optimizing the use of stem cells of dental origin for tissue regeneration.

Junctional epithelium (JE), which is derived from odontogenic epithelial cells immediately after eruption, is believed to be gradually replaced by oral gingival epithelium (OGE) over a lifetime. However, the detailed process of replacement remains unclear. The aim of the present study was to clarify the process of JE replacement by OGE cells using a green fluorescent protein (GFP)-positive tooth germ transplantation method. GFP-positive JE was partly replaced by OGE cells and completely replaced on day 200 after transplantation, whereas there was no difference in the expression of integrin β4 (Itgb4) and laminin 5 (Lama5) between JE before and after replacement by OGE cells. Next, GFP-positive JE was partially resected. On day 14 after resection, the regenerated JE consisted of GFP-negative cells and also expressed both Itgb4 and Lama5. In addition, the gene expression profile of JE derived from odontogenic epithelium before gingivectomy was partly different from that of JE derived from OGE after gingivectomy. These results suggest that JE derived from the odontogenic epithelium is gradually replaced by OGE cells over time and JE derived from the odontogenic epithelium might have specific characteristics different to those of JE derived from OGE.

Odontomas, developmental anomalies of tooth germ, frequently occur in familial adenomatous polyposis patients with activated Wnt/β-catenin signaling. However, roles of Wnt/β-catenin signaling in odontomas or odontogenic cells are unclear. Herein, we investigated β-catenin expression in odontomas and functions of Wnt/β-catenin signaling in tooth germ development. β-catenin frequently accumulated in nucleus and/or cellular cytoplasm of odontogenic epithelial cells in human odontoma specimens, immunohistochemically. Wnt/β-catenin signaling inhibited odontogenic epithelial cell proliferation in both cell line and tooth germ development, while inducing immature epithelial bud formation. We identified Semaphorin 3A (Sema3A) as a downstream molecule of Wnt/β-catenin signaling and showed that Wnt/β-catenin signaling-dependent reduction of Sema3A expression resulted in suppressed odontogenic epithelial cell proliferation. Sema3A expression is required in appropriate epithelial budding morphogenesis. These results suggest that Wnt/β-catenin signaling negatively regulates odontogenic epithelial cell proliferation and tooth germ development through decreased-Sema3A expression, and aberrant activation of Wnt/β-catenin signaling may associate with odontoma formation.

Most mammals have two sets of teeth (diphyodont) - a deciduous dentition replaced by a permanent dentition; however, the mouse possesses only one tooth generation (monophyodont). In diphyodonts, the replacement tooth forms on the lingual side of the first tooth from the successional dental lamina. This lamina expresses the stem/progenitor marker Sox2 and has activated Wnt/β-catenin signalling at its tip. Although the mouse does not replace its teeth, a transient rudimentary successional dental lamina (RSDL) still forms during development. The mouse RSDL houses Sox2-positive cells, but no Wnt/β-catenin signalling. Here, we show that stabilising Wnt/β-catenin signalling in the RSDL in the mouse leads to proliferation of the RSDL and formation of lingually positioned teeth. Although Sox2 has been shown to repress Wnt activity, overexpression of Wnts leads to a downregulation of Sox2, suggesting a negative-feedback loop in the tooth. In the mouse, the first tooth represses the formation of the replacement, and isolation of the RSDL is sufficient to induce formation of a new tooth germ. Our data highlight key mechanisms that may have influenced the evolution of replacement teeth.This article has an associated 'The people behind the papers' interview., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The extracellular matrix (ECM) contains a variety of complex macromolecules including proteoglycans (PGs) and glycosaminoglycans (GAGs). PG consists of a protein core with covalently attached carbohydrate side chains called GAGs. Several PGs, including versican, biglycan, decorin and syndecan are involved in odontogenesis while the role of GAGs in those PGs in this process remains unclarified. The purpose of this study was to investigate the influence of GAGs on tooth development. The mandibular first molars at early bell stage were cultivated with or without 4-methylumbelliferyl-β-D-xyloside (Xyl-MU). The cultured tooth germs were metabolically labelled with [ 35 S] Na 2 SO 4 , then PGs in tooth germs and cultured medium were extracted separately and analyzed by gel filtration. Morphological changes were evaluated on days 2, 4, 6, and histological changes were examined by hematoxylin-eosin (HE) staining and transmission electron microscope (TEM). Related proteins and genes of cytodifferentiation were further examined by immunohistochemistry (IHC) and quantitive real-time PCR (qPCR) respectively. Meanwhile, BrdU incorporation assay was used to explore the effect of Xyl-MU on the cell proliferation of cultured tooth germs. The results demonstrated that the incorporation of GAGs to PGs in cultured tooth germs was heavily inhibited by Xyl-MU. Accompanied by the inhibition of GAGs incorporation, Xyl-MU altered tooth morphogenesis and delayed the differentiation of ameloblasts and odontoblasts. Proliferation of inner enamel epithelium (IEE) was also inhibited. Therefore, we draw a conclusion that the inhibition of GAGs incorporation influences the cell proliferation and cytodifferentiation in cultured embryonic mouse molars.

Stem cells are undifferentiated cells located in different parts of the body. The major role of stem cells is to restore of injured tissues. Since the discover of stem cells, they gained a big attention due to their differentiation and regeneration capacity. The main source of stem cells was known as bone marrow. However, different sources for obtaining stem cells were discovered. Dental tissues, a new source for stem cells, provide cells having mesenchymal stem cell characteristics such as fibroblast-like structure, expression of surface antigens specific for mesenchymal stem cells, regeneration ability, multilineage differentiation capacity and immunomodulatory features. Dental pulp stem cells (DPSCs), dental follicle progenitor cells (DFPCs), stem cells from apical papilla (SCAP), tooth germ stem cells (TGSCs) and periodontal ligament stem cells (PDLSCs) are stem cells derived from dental tissues as well as stem cells from exfoliated deciduous teeth (SHED). Dental stem cells express mesenchymal stem cell markers like Stro-1, CD146, CD106, CD90, CD73 CD29 and CD13. However, they do not express hematopoietic stem cell markers such as CD11b, CD45 and CD34. Dental stem cells are able to undergo myogenic, chondrogenic, adipogenic, neurogenic, osteogenic and odontogenic differentiation. Thanks to these differentiation ability of dental stem cells, they can easily be manipulated in regenerative medicine. Dental stem cells, that can effortlessly be transfected, can also be used in cell therapy application. Immunomodulatory features of dental stem cells make them suitable candidates for the therapy of immune-related disorders. Dental stem cells with high potentials such as ability of self-renewal, mesenchymal stem cell characteristics, multilineage differentiation and immunomodulation are promising tool for in vitro and in vivo differentiation studies as well as the therapy of immune-related diseases.

In this study, we attempted to localize the immunoreactivities of podoplanin/E11/gp38 and CD44, a counterpart possessing a high affinity to podoplanin/E11/gp38, as well as endomucin-immunoreactive blood vessels in the regions of odontoblast layers and the underlying sub-odontoblastic layers in murine tooth germs. Endomucin-reactive small blood vessels were scattered throughout the dental papillae of the tooth germs at postnatal day 1 but came to be localized close to the odontoblast/sub-odontoblastic layers until day 3. After postnatal day 5, small blood vessels were seen in odontoblast cell layers, while blood vessels with relatively larger diameters were seen forming in sub-odontoblastic layers. Immunoreactivities of podoplanin/E11/gp38 and CD44 were not detectable in the cells of dental papillae facing the inner enamel epithelium at postnatal day 1. However, at around postnatal days 3-5, podoplanin/E11/gp38 was localized in the odontoblast layer but not in the sub-odontoblastic layer, whereas CD44 was observed in the sub-odontoblastic layer but not in the odontoblast layer. The exclusive immunolocalization of podoplanin/E11/gp38 and CD44 in the odontoblast layers and sub-odontoblastic layers was seen after postnatal day 3 of the tooth germs, when the mesenchymal cells of dental papillae have already differentiated into mature odontoblasts at the cusp tip. Taken together, it seems likely that endomucin-reactive small blood vessels extended to the podoplanin/E11/gp38-positive odontoblast layers, whereas endomucin-reactive large blood vessels were already present in CD44-immmunopositive sub-odontoblastic layer, indicating the cellular regulation on the vascularization of endomucin-reactive endothelial cells during odontogenesis of the tooth germs.

Objectives: Whole-tooth regeneration for tooth loss has long been a goal of dentistry. There is also an increasing demand to carry out pre-clinical in vitro and in vivo research methods in large animal model similar to human. The miniature pig has proven to be an alternative as a large mammal model owing to its many similarities to human. However, whole-tooth regeneration in large animal remains a challenge. Here, we investigated the feasibility of cell re-association-based whole-tooth regeneration in miniature pigs., Materials and Methods: Single cells from the forth deciduous molar germs (p4) of pig were reconstituted to bioengineered tooth bud using different treatment for in vitro culture and in vivo transplantation in mouse subrenal capsules and jawbones., Results: The bioengineered tooth bud from re-aggregated epithelial to mesenchymal single cells with and without compartmentalization restored the morphogenesis, interactions or self-sorting between 2 cells in vitro culture. The pig bioengineered tooth bud transplanted in mouse subrenal capsules and jawbones restored odontogenesis and developed into large size tooth., Conclusions: We characterized the morphogenesis and interaction of single-tooth germ cells in vitro, and first addressed efficient long-term survival and growth through transplantation of pig bioengineered tooth bud under mouse subrenal capsules or in mouse jawbones, where it can develop into large size tooth. Our study extends the feasibility of whole-tooth regeneration in large animal., (© 2018 John Wiley & Sons Ltd.)

Homeobox protein MSX-1 (hereafter referred to as MSX-1) is essential for early tooth-germ development. Tooth-germ development is arrested at bud stage in Msx1 knockout mice, which prompted us to study the functions of MSX-1 beyond this stage. Here, we investigated the roles of MSX-1 during late bell stage. Mesenchymal cells of the mandibular first molar were isolated from mice at embryonic day (E)17.5 and cultured in vitro. We determined the expression levels of β-catenin, bone morphogenetic protein 2 (Bmp2), Bmp4, and lymphoid enhancer-binding factor 1 (Lef1) after knockdown or overexpression of Msx1. Our findings suggest that knockdown of Msx1 promoted expression of Bmp2, Bmp4, and Lef1, resulting in elevated differentiation of odontoblasts, which was rescued by blocking the expression of these genes. In contrast, overexpression of Msx1 decreased the expression of Bmp2, Bmp4, and Lef1, leading to a reduction in odontoblast differentiation. The regulation of Bmp2, Bmp4, and Lef1 by Msx1 was mediated by the Wnt/β-catenin signaling pathway. Additionally, knockdown of Msx1 impaired cell proliferation and slowed S-phase progression, while overexpression of Msx1 also impaired cell proliferation and prolonged G1-phase progression. We therefore conclude that MSX-1 maintains cell proliferation by regulating transition of cells from G1-phase to S-phase and prevents odontoblast differentiation by inhibiting expression of Bmp2, Bmp4, and Lef1 at the late bell stage via the Wnt/β-catenin signaling pathway., (© 2017 Eur J Oral Sci.)

Undifferentiated odontogenic epithelium and dental papilla cells differentiate into ameloblasts and odontoblasts, respectively, both of which are essential for tooth development. These differentiation processes involve dramatic functional and morphological changes of the cells. For these changes to occur, activation of mitochondrial functions, including ATP production, is extremely important. In addition, these changes are closely related to mitochondrial fission and fusion, known as mitochondrial dynamics. However, few studies have focused on the role of mitochondrial dynamics in tooth development. The purpose of this study was to clarify this role. We used mouse tooth germ organ cultures and a mouse dental papilla cell line with the ability to differentiate into odontoblasts, in combination with knockdown of the mitochondrial fission factor, dynamin related protein (DRP)1. In organ cultures of the mouse first molar, tooth germ developed to the early bell stage. The amount of dentin formed under DRP1 inhibition was significantly larger than that of the control. In experiments using a mouse dental papilla cell line, differentiation into odontoblasts was enhanced by inhibiting DRP1. This was associated with increased mitochondrial elongation and ATP production compared to the control. These results suggest that DRP1 inhibition accelerates dentin formation through mitochondrial elongation and activation. This raises the possibility that DRP1 might be a therapeutic target for developmental disorders of teeth., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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.

Mesenchymal stem cells derived from the human tooth germ (hTGSCs) are a heterogeneous cell population that can differentiate into osteogenic, neurogenic, and adipogenic lineages. The aim of this study was to compare the osteogenic differentiation capacity of STRO-1 positive (STRO-1+) hTGSCs and unsorted heterogeneous hTGSCs and to establish if STRO-1+ cells are more committed to osteogenic differentiation. HTGSCs were isolated from impacted third molar tooth germ tissues of adolescents, and a subpopulation of STRO-1+ hTGSCs was obtained by fluorescence-activated cell sorting. STRO-1+, STRO-1 negative (STRO-1-), and unsorted cells were cultured in osteogenic and standard culture media to compare their capacity to differentiate towards osteoblastic lineage. Cells were tested for proliferation rates, alkaline phosphatase activity, and amounts of accumulated calcium. Gene expression levels of the RUNX2, osteocalcin, and osteonectin genes were analyzed with real time PCR. Mineralization and osteogenic protein expression were examined by using von Kossa staining and confocal microscopy. Our results indicated that osteogenically induced cell populations showed greater mineralization capacity than non-induced cells. However, expression levels of early and late osteogenic markers were not significantly different between STRO-1+ and unsorted cells. In conclusion, the selection by STRO-1 expression does not yield cells with osteogenic capacity higher than that of the heterogeneous hTGSC population. Cell sorting using osteogenic markers other than STRO-1 might be beneficial in obtaining a more sensitive osteogenic sub-population from unsorted heterogenous hTGSCs., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objective: The RNA-binding proteins Lin28 and Lin28b are expressed in many developing tissues and are involved in the biosynthesis of the microRNA let-7 family and embryogenesis processes. However, their roles in mammalian tooth development remain ill-defined., Design: The spatiotemporal expressions of Lin28 and Lin28b during mouse molar odontogenesis from day E11.5 to P21 were examined through immunohistochemistry and western blot analysis., Results: Both Lin28 and Lin28b were initially expressed in dental epithelium, but the expression patterns varied thereafter. Lin28 was expressed in tooth germ from early embryonic stages and was consistently expressed in the ameloblasts and odontoblasts throughout all stages of tooth development. However, positive staining of Lin28b gradually faded out with tooth germ development, before finally disappearing in tooth organ cells after birth., Conclusions: These results indicate that Lin28 was spatiotemporally expressed in tooth germ throughout tooth development progression and may play an active role in ameloblast and odontoblast differentiation, as well as matrix secretion and the mineralization of enamel and dentin. Its paralogue Lin28b may have a distinct function in tooth germ formation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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.)

Whole tooth regeneration approaches currently are limited by our inability to bioengineer full-sized, living replacement teeth. Recently, decellularized organ scaffolds have shown promise for applications in regenerative medicine by providing a natural extracellular matrix environment that promotes cell attachment and tissue-specific differentiation leading to full-sized organ regeneration. We hypothesize that decellularized tooth buds (dTBs) created from unerupted porcine tooth buds (TBs) can be used to guide reseeded dental cell differentiation to form whole bioengineered teeth, thereby providing a potential off-the-shelf scaffold for whole tooth regeneration. Porcine TBs were harvested from discarded 6-mo-old pig jaws, and decellularized by successive sodium dodecyl sulfate/Triton-X cycles. Four types of replicate implants were used in this study: 1) acellular dTBs; 2) recellularized dTBs seeded with porcine dental epithelial cells, human dental pulp cells, and human umbilical vein endothelial cells (recell-dTBs); 3) dTBs seeded with bone morphogenetic protein (BMP)-2 (dTB-BMPs); and 4) freshly isolated nondecellularized natural TBs (nTBs). Replicate samples were implanted into the mandibles of host Yucatan mini-pigs and grown for 3 or 6 mo. Harvested mandibles with implanted TB constructs were fixed in formalin, decalcified, embedded in paraffin, sectioned, and analyzed via histological methods. Micro-computed tomography (CT) analysis was performed on harvested 6-mo samples prior to decalcification. All harvested constructs exhibited a high degree of cellularity. Significant production of organized dentin and enamel-like tissues was observed in dTB-recell and nTB implants, but not in dTB or dTB-BMP implants. Micro-CT analyses of 6-mo implants showed the formation of organized, bioengineered teeth of comparable size to natural teeth. To our knowledge, these results are the first to describe the potential use of dTBs for functional whole tooth regeneration.

In this study, fibrous scaffolds based on poly(γ-benzyl-l-glutamate) (PBLG) were investigated in terms of the chondrogenic differentiation potential of human tooth germ stem cells (HTGSCs). Through the solution-assisted bonding of the fibres, fully connected scaffolds with pore sizes in the range 20-400 µm were prepared. Biomimetic modification of the PBLG scaffolds was achieved by a two-step reaction procedure: first, aminolysis of the PBLG fibres' surface layers was performed, which resulted in an increase in the hydrophilicity of the fibrous scaffolds after the introduction of N 5 -hydroxyethyl-l-glutamine units; and second, modification with the short peptide sequence azidopentanoyl-GGGRGDSGGGY-NH 2 , using the 'click' reaction on the previously modified scaffold with 2-propynyl side-chains, was performed. Radio-assay of the 125 I-labelled peptide was used to evaluate the RGD density in the fibrous scaffolds (which varied in the range 10 -3 -10 pm/cm 2 ). All the PBLG scaffolds, especially with density 90 ± 20 fm/cm 2 and 200 ± 100 fm/cm 2 RGD, were found to be potentially suitable for growth and chondrogenic differentiation of HTGSCs. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

The main objective was to study cartilage regeneration through differentiation of human tooth germ stem cells (HTGSCs) into chondrocytes on different three-dimensional (3D) scaffolds (PCL, PLLA and PCL-PLLA). Scaffold topographies were studied by scanning electron microscopy and it was found that the scaffolds had interconnected macroporous structures. HTGSCs were isolated from impacted third molar tooth germs of young adult patients and grown for 3 weeks on the scaffolds in chondrogenic differentiation medium. Cell proliferation on the scaffolds was determined by MTS assay and it was observed that all scaffolds supported cell proliferation. Immunostaining was carried out for morphological and differentiation analyses. Immunohistochemical analyses revealed that the cells attached onto the scaffolds and deposited cartilage-specific extracellular matrix (ECM). Real-time PCR was performed to determine the expression levels of cartilage-specific genes. After 21 days of incubation in cartilage differentiation medium, expression of collagen type II increased only in the cells seeded onto PCL-PLLA blend scaffolds. Similarly, aggrecan expression was the highest on PCL-PLLA scaffolds after 3 weeks. These results suggest that all the scaffolds, and especially PCL-PLLA, were suitable for chondrogenic differentiation of HTGSCs. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

Vangl2, one of the core components of the planar cell polarity (PCP) pathway, has an important role in the regulation of morphogenesis in several tissues. Although the expression of Vangl2 has been detected in the developing tooth, its role in tooth morphogenesis is not known. In this study, we show that Vangl2 is expressed in the inner dental epithelium (IDE) and in the secondary enamel knots (SEKs) of bell stage tooth germs. Inhibition of Vangl2 expression by siRNA treatment in in vitro-cultured tooth germs resulted in retarded tooth germ growth with deregulated cell proliferation and apoptosis. After kidney transplantation of Vangl2 siRNA-treated tooth germs, teeth were observed to be small and malformed. We also show that Vangl2 is required to maintain the proper pattern of cell alignment in SEKs, which maybe important for the function of SEKs as signaling centers. These results suggest that Vangl2 plays an important role in the morphogenesis of teeth.

Tissue engineering and regenerative medicine technologies offer promising therapies for both medicine and dentistry. Our long-term goal is to create functional biomimetic tooth buds for eventual tooth replacement in humans. Here, our objective was to create a biomimetic 3D tooth bud model consisting of dental epithelial (DE) - dental mesenchymal (DM) cell sheets (CSs) combined with biomimetic enamel organ and pulp organ layers created using GelMA hydrogels. Pig DE or DM cells seeded on temperature-responsive plates at various cell densities (0.02, 0.114 and 0.228 cells 10(6)/cm(2)) and cultured for 7, 14 and 21 days were used to generate DE and DM cell sheets, respectively. Dental CSs were combined with GelMA encapsulated DE and DM cell layers to form bioengineered 3D tooth buds. Biomimetic 3D tooth bud constructs were cultured in vitro, or implanted in vivo for 3 weeks. Analyses were performed using micro-CT, H&E staining, polarized light (Pol) microscopy, immunofluorescent (IF) and immunohistochemical (IHC) analyses. H&E, IHC and IF analyses showed that in vitro cultured multilayered DE-DM CSs expressed appropriate tooth marker expression patterns including SHH, BMP2, RUNX2, tenascin and syndecan, which normally direct DE-DM interactions, DM cell condensation, and dental cell differentiation. In vivo implanted 3D tooth bud constructs exhibited mineralized tissue formation of specified size and shape, and SHH, BMP2 and RUNX2and dental cell differentiation marker expression. We propose our biomimetic 3D tooth buds as models to study optimized DE-DM cell interactions leading to functional biomimetic replacement tooth formation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Gingival-derived mesenchymal stem cells (GMSCs) have recently been harvested; however, the use of GMSCs in periodontal tissue engineering requires further study. The present study established an indirect co‑culture system between rat apical tooth germ‑conditioned medium (APTG‑CM) and GMSCs, in order to determine the effects on periodontal tissue differentiation in vitro and in vivo. Using the limiting dilution technique, single‑colony derived human GMSCs and periodontal ligament stem cells (PDLSCs) were isolated and expanded to obtain homogeneous populations. PDLSCs were used as a positive control group. Cell cycle distribution, alkaline phosphatase (ALP) activity, mineralization behavior, expression of genes associated with a cementoblast phenotype (osteocalcin, bone sialoprotein, ALP, type I collagen, cementum‑derived protein 23), and in vivo differentiation capacities of GMSCs/PDLSCs co‑cultured with APTG‑CM were evaluated. Flow cytometry indicated that GMSCs and PDLSCs were positive for STRO‑1 and CD105, whereas CD45 expression was negative. The cell types were capable of forming colonies, and of osteogenic and adipogenic differentiation in response to appropriate stimuli. The induced GMSCs and PDLSCs exhibited numerous characteristics associated with cementoblast lineages, as indicated by increased proliferation and ALP activity, and upregulated expression of cementum‑associated genes in vitro. In vivo, cementum/periodontal ligament‑like structures were shown to form along the dentin surface and ceramic bovine bone in GMSCs and PDLSCs induced by APTG‑CM group. Conversely, vertical fibers could not insert in the control group, which was not co‑cultured with APTG‑CM. In conclusion, GMSCs are likely to have a role in periodontal tissue regeneration. In addition, APTG‑CM was able to provide a cementogenic microenvironment and promote differentiation of GMSCs along the cementoblastic lineage.

Objectives: The purpose of this study is to identify the lncRNAs that are associated with the odontogenic potential in mouse dental mesenchymal cells., Design: The odontogenic potential of dental mesenchymal cells was found to be lost in the course of in vitro culture, so the lncRNA profiles were subsequently compared between freshly-isolated and cultured dental mesenchymal cells using RNA-sequencing. A co-expression analysis of differentially expressed lncRNAs and coding RNAs was performed to understand their potential functions. The expression of several selected lncRNAs was also examined in developing tooth germs., Results: Compared with cultured dental mesenchymal cells, 108 lncRNAs were upregulated and 36 lncRNAs were downregulated in freshly-isolated dental mesenchymal cells. Coding genes correlated with the lncRNAs were mainly associated with DNA and protein metabolic processes and cytoskeletal anchorage. Meg3, Malat1, Xist, and Dlx1as were significantly downregulated in cultured dental mesenchymal cells but were upregulated in odontogenic dental mesenchymal tissues. Moreover, the levels of Dlx1as were negatively correlated with that of Dlx1 in dental mesenchymal cells and dental mesenchymal tissues., Conclusions: The lncRNA profiles of dental mesenchymal cells are significantly changed during culturing, and the dysregulation of lncRNAs is associated with the loss of odontogenic potential., (Copyright © 2016. Published by Elsevier Ltd.)

The purpose of this study was to demonstrate the feasibility of whole-tooth regeneration using a tooth germ-like construct. Dental pulp from upper incisors, canines, premolars, and molars were extracted from sexually mature miniature pigs. Pulp tissues were cultured and expanded in vitro to obtain dental pulp stem cells (DPSCs), and cells were differentiated into odontoblasts and osteoblasts. Epithelial cells were isolated from gingival epithelium. The epithelial cells, odontoblasts, and osteoblasts were seeded onto the surface, upper, and lower layers, respectively, of a bioactive scaffold. The lower first and second molar tooth germs were removed bilaterally and the layered cell/scaffold constructs were transplanted to the mandibular alveolar socket of a pig. At 13.5 months postimplantation, seven of eight pigs developed two teeth with crown, root, and pulp structures. Enamel-like tissues, dentin, cementum, odontoblasts, and periodontal tissues were found upon histological inspection. The regenerated tooth expressed dentin matrix protein-1 and osteopontin. All pigs had regenerated molar teeth regardless of the original tooth used to procure the DPSCs. Pigs that had tooth germs removed or who received empty scaffolds did not develop teeth. Although periodontal ligaments were generated, ankylosis was found in some animals. This study revealed that implantation of a tooth germ-like structure generated a complete tooth with a high success rate. The implant location may influence the morphology of the regenerated tooth., (Copyright © 2015 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.)

Dental enamel formation, known as "amelogenesis," is initiated by cytodifferentiation of the ectodermally derived dental epithelium. Enamel cannot regenerate itself because once it is completely formed, ameloblasts are lost as the tooth erupts. Rodent teeth have been useful for studying the mechanisms of amelogenesis because ameloblast cell lines can be derived from the ever-growing incisors. However, higher mammals such as humans have no growing teeth, and cell lines derived from larger animals that are more similar to humans are required for higher fidelity studies. Here, we isolated embryonic enamel epithelium-derived epithelial cells from fetal swine. The explant culture of the developing deciduous molars that had been removed from the dental papilla-derived mesenchymal tissue and cells inside the tooth buds provided the epithelial cell population for the primary culture. To isolate the cell population, we performed a unique cell isolation technique called cell fishing. The isolated cells showed clear embryonic-stage ameloblast characteristics with appropriate gene/protein expressions of enamel matrix and proteinases, abundant glycogen pools, and secretory granular materials. They could be continuously subcultured several times and are presently being maintained. This cell population will facilitate the establishment of a stable cell line and allow us to characterize the definitive phenotype and functional behavior of porcine ameloblasts, which, in turn, promises to yield useful and practical findings that are more relevant than those provided by rodent studies. Finally, analysis of in vitro enamel formation will be important for engineering "bio-enamel" as a new dental therapy to restore enamel defects.

Tooth replacement in polyphyodont is a well-organized system for maintenance of homeostasis of teeth, containing the dynamic structural change in skeletal tissues such as the attachment bone, which is the supporting element of teeth. Histological analyses have revealed the character of tooth replacement, however, the cellular mechanism of how skeletal tissues are modified during tooth replacement is largely unknown. Here, we showed the important role of osteoblasts for controlling osteoclasts to modify the attachment bone during tooth replacement in medaka pharyngeal teeth, coupled with an osterix-DsRed/TRAP-GFP transgenic line to visualize osteoblasts and osteoclasts. In the turnover of the row of attachment bones, these bones were resorbed at the posterior side where most developed functional teeth were located, and generated at the anterior side where teeth were newly erupted, which caused continuous tooth replacement. In the cellular analysis, osteoclasts and osteoblasts were located at attachment bones separately, since mature osteoclasts were localized at the resorbing side and osteoblasts gathered at the generating side. To demonstrate the role of osteoclasts in tooth replacement, we established medaka made deficient in c-fms-a by TALEN. c-fms-a deficient medaka showed hyperplasia of attachment bones along with reduced bone resorption accompanied by a low number of TRAP-positive osteoclasts, indicating an important role of osteoclasts in the turnover of attachment bones. Furthermore, nitroreductase-mediated osteoblast-specific ablation induced disappearance of osteoclasts, indicating that osteoblasts were essential for maintenance of osteoclasts for the proper turnover. Taken together, our results suggested that the medaka attachment bone provides the model to understand the cellular mechanism for tooth replacement, and that osteoblasts act in the coordination of bone morphology by supporting osteoclasts., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The histone demethylase, lysine (K)-specific demethylase 2A (Kdm2a), is highly conserved and expressed ubiquitously. Kdm2a can regulate cell proliferation and osteo/dentinogenic, adipogenic and chondrogenic differentiation of mesenchymal stem cells (MSCs) derived from dental tissue. We used quantitative real-time RT-PCR analysis and immunohistochemistry to detect Kdm2a expression during development of the murine molar at embryonic days E12, E14, E16 and E17 and postnatal days P3 and P14. Immunohistochemistry results showed no positive staining of Kdm2a at E12. At E14, Kdm2a was expressed weakly in the inner enamel epithelium, stellate reticulum cells and dental sac. At E16, Kdm2a was expressed mainly in the inner and outer enamel epithelium, stratum intermedium and dental sac, but weaker staining was found in cervical loop and dental papilla cells adjacent to the basement membrane. At E17, the strongest Kdm2a staining was detected in the ameloblasts and stronger Kdm2a staining also was detected in the stratum intermedium, outer enamel epithelium and dental papilla cells compared to the expression at E16. Postnatally, we found that Kdm2a was localized in secretory and mature ameloblasts and odontoblasts, and dentin was unstained. Real-time RT-PCR showed that Kdm2a mRNA levels in murine germ cells increased from E12 to E14 and from E14 to E16; no significant change occurred at E16, E17 or P3, then the levels decreased at P14 compared to P3. Kdm2a expression may be closely related to cell proliferation, to ameloblast and odontoblast differentiation and to the secretion of extracellular enamel and dentin during murine tooth development.

Maturation-stage ameloblasts are firmly bound to the tooth enamel by a basal lamina-like structure. The mechanism underlying this adhesion, however, remains to be fully clarified. The goal of this study was to investigate the mechanism underlying adhesion between the basal lamina-like structure and the enamel in monkey tooth germ. High-resolution immunogold labeling was performed to localize amelotin and laminin 332 at the interface between ameloblasts and tooth enamel. Minute, electron-dense strands were observed on the enamel side of the lamina densa, extending into the degrading enamel matrix to produce a well-developed fibrous layer (lamina fibroreticularis). In un-demineralized tissue sections, mineral crystals smaller than those in the bulk of the enamel were observed adhering to these strands where they protruded into the surface enamel. Immunogold particles reactive for amelotin were preferentially localized on these strands in the fibrous layer. On the other hand, those for laminin 332 were localized solely in the lamina densa; none were observed in the fibrous layer. These results suggest that the fibrous layer of the basal lamina-like structure is partly composed of amelotin molecules, and that these molecules facilitate ameloblast-enamel adhesion by promoting mineralization of the fibrous layer during the maturation stage of amelogenesis.

In the early stage of tooth germ development, the bud of the dental epithelium is invaginated by the underlying mesenchyme, resulting in the formation of a cap-like folded shape. This bud-to-cap transition plays a critical role in determining the steric design of the tooth. The epithelial-mesenchymal interaction within a tooth germ is essential for mediating the bud-to-cap transition. Here, we present a theoretical model to describe the autonomous process of the morphological transition, in which we introduce mechanical interactions among cells. Based on our observations, we assumed that peripheral cells of the dental epithelium bound tightly to each other to form an elastic sheet, and mesenchymal cells that covered the tooth germ would restrict its growth. By considering the time-dependent growth of cells, we were able to numerically show that the epithelium within the tooth germ buckled spontaneously, which is reminiscent of the cap-stage form. The difference in growth rates between the peripheral and interior parts of the dental epithelium, together with the steric size of the tooth germ, were determining factors for the number of invaginations. Our theoretical results provide a new hypothesis to explain the histological features of the tooth germ., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cell differentiation is essential for maxillaries and tooth development. Facial mesenchymal tissue is formed by neural crest cells (NC). These cells are highly migratory, giving rise to various cell types, considered with a high level of plasticity, indicating that they contain progenitor cells with a great power of differentiation. In this study, it was analyzed the presence of NC cell progenitors and mesenchymal stem cells (MSC) during maxillaries osteogenesis and odontogenesis in rats. Histological slides were collected in two phases: embryonic age of 15 and 17 days; 2, 4 and 7 days after birth. Immunohistochemistry for MSC markers (Osterix) and NC cells (Sox10, HNK1) was performed. The results showed positive expression for Osterix and HNK1 in undifferentiated ectomesenchymal cells in early and late stages; Sox10 was present only in early stages in undifferentiated cells. All markers were present in differentiated cells. Although the experiments performed do not allow us to explain a possible role for Osx, HNK1 and Sox10 in both differentiated and undifferentiated cells during osteogenesis and odontogenesis, it had shown important results not yet described: the presence of HNK1 and Sox10 in osteoblasts and odontoblasts in late development stages and in the tooth germ epithelial cells and ameloblasts., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Aims: To analyze expression patterns of IGF-1, caspase-3 and HSP-70 in human incisor and canine tooth germs during the late bud, cap and bell stages of odontogenesis., Materials and Methods: Head areas or parts of jaw containing teeth from 10 human fetuses aged between 9th and 20th developmental weeks were immunohistochemically analyzed using IGF-1, active caspase-3 and HSP-70 markers. Semi-quantitative analysis of each marker's expression pattern was also performed., Results: During the analyzed period, IGF-1 and HSP-70 were mostly expressed in enamel organ. As development progressed, expression of IGF-1 and HSP-70 became more confined to differentiating tissues in the future cusp tip area, as well as in highly proliferating cervical loops. Few apoptotic bodies highly positive to active caspase-3 were observed in enamel organ and dental papilla from the cap stage onward. However, both enamel epithelia moderately expressed active caspase-3 throughout the investigated period., Conclusions: Expression patterns of IGF-1, active caspase-3 and HSP-70 imply importance of these factors for early human tooth development. IGF-1 and HSP-70 have versatile functions in control of proliferation, differentiation and anti-apoptotic protection of epithelial parts of human enamel organ. Active caspase-3 is partially involved in formation and apoptotic removal of primary enamel knot, although present findings might reflect its ability to perform other non-death functions such as differentiation of hard dental tissues secreting cells and guidance of ingrowth of proliferating cervical loops., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Aim: To investigate the expression of CD56 in dog odontogenesis in order to elucidate the expression found in ameloblastomas., Materials and Methods: Immunohistochemical analysis of CD56 expression of developing dog teeth in the bud, cap and bell stages including the remnants of the dental lamina., Results: Weak CD56 expression was observed in the dental epithelium during the bud stage with intense staining of certain peripheral epithelial cells. Positive staining of epithelial cells was also observed in the cap stage with intense staining of the inner enamel epithelium at this stage. During the bell stage the staining was concentrated on the cervical loop areas. The dental papilla revealed positive staining throughout the cap and bell stages while the dental follicle stained intensely positive throughout all the phases examined. The dental lamina and Serres rests also stained positive for CD56., Conclusions: The expression of CD56 in dog odontogenic tissue varies according to the stage of tooth development. There is a positive correlation between the positive staining observed in ameloblastomas and their odontogenic cells of origin., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena are not well understood yet. In particular, the role of innervation in tooth development and regeneration is neglected. Several in vivo studies have provided important information about the patterns of innervation of dental tissues during development and repair processes of various animal models. However, most of these approaches are not optimal to highlight the molecular basis of the interactions between nerve fibres and target organs and tissues. Co-cultures constitute a valuable method to investigate and manipulate the interactions between nerve fibres and teeth in a controlled and isolated environment. In the last decades, conventional co-cultures using the same culture medium have been performed for very short periods (e.g., two days) to investigate the attractive or repulsive effects of developing oral and dental tissues on sensory nerve fibres. However, extension of the culture period is required to investigate the effects of innervation on tooth morphogenesis and cytodifferentiation. Microfluidics systems allow co-cultures of neurons and different cell types in their appropriate culture media. We have recently demonstrated that trigeminal ganglia (TG) and teeth are able to survive for a long period of time when co-cultured in microfluidic devices, and that they maintain in these conditions the same innervation pattern that they show in vivo. On this basis, we describe how to isolate and co-culture developing trigeminal ganglia and tooth germs in a microfluidic co-culture system.This protocol describes a simple and flexible way to co-culture ganglia/nerves and target tissues and to study the roles of specific molecules on such interactions in a controlled and isolated environment.

Objectives: The boundary where inner and outer enamel epithelia meet is referred to as the cervical loop (CL) in molar tooth germs. In contrast, rodent incisors are continuously growing: the labial side of the teeth is covered with enamel (crown-analog), and the lingual side is covered with cementum (root-analog). These results in the appearance of CL in the frontal sections apart from the apical end. However, many researchers have used the term "labial CL" to indicate the apical end of incisors., Design: This study investigated the gene expression patterns for the enamel knot signaling center in tooth morphogenesis to clarify the difference between "labial CL" and molar CL. We examined the three-dimensional expression patterns of markers for the enamel knot including fibroblast growth factor 4 (Fgf4), sonic hedgehog (Shh), Msx2, and P21 in frontal sections of murine mandibular incisors., Results: Serial frontal sections from the apical end of the postnatal incisor clearly demonstrated the existence of enamel knot-like structures composed of supra-inner enamel epithelium and stellate reticulum in the "labial CL". This structure includes the expression of all examined markers for enamel knots such as Fgf4, Shh, Msx2, and P21., Conclusions: The molar tooth germ-like structure is maintained indefinitely in the "labial CL". Therefore, the "labial CL" is not equivalent to the molar CL. The existence of an EK-like structure in the apical end of incisors implies that the usage of "labial CL" may be inappropriate for indicating this region. The "apical bud" could be used as an alternative term., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objectives: Due to the rarity of human embryonic samples and limited proliferating capability of primary human dental mesenchymal cells, it is valuable to create an immortalized human dental mesenchymal cell line for studying dental mesenchymal cell differentiation and signalling pathways during dentinogenesis in humans., Methods: In this study, dental mesenchymal cells from human molar tooth germs at 19-week gestation were isolated and immortalized with pSV40. Single cell colonies were then selected by 96-well plate dilution. The immortalized cell line was characterized using immunofluorescent microscopy, RT-PCR and Western blot for the expression of SV40 large T antigen and five genes specific for the mesenchymal stage during tooth development. The differentiation and mineralization activities of the immortalized and primary cells were compared using adipogenic and calcifying induction., Results: The immortalized dental mesenchymal cell line displayed a higher proliferation rate, expressed several tooth-specific markers including Msx1, Pax9, Lhx6, Barx1, and Runx2, and maintained the ability to differentiate and form mineralized nodules., Conclusions: Our results demonstrated that the immortalized human mesenchymal cell line retained the characteristics similar to primary human dental mesenchymal cells and can be used for studying the mechanisms of human dental mesenchymal cell differentiation and signalling pathways involved in human odontogenesis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The goal of the study was to characterize the transcriptome profiles of human ameloblasts and odontoblasts, evaluate molecular pathways and advance our knowledge of the human "dentome". Laser capture microdissection was used to isolate odontoblasts and ameloblasts from human tooth buds (15-20week gestational age) from 4 fetuses. RNA was examined using Agilent 41k whole genome arrays at 2 different stages of enamel formation, presecretory and secretory. Probe detection was considered against the array negative control to control for background noise. Differential expression was examined using Significance Analysis of Microarrays (SAM) 4.0 between different cell types and developmental stages with a false discovery rate of 20%. Pathway analysis was conducted using Ingenuity Pathway Analysis software. We found that during primary tooth formation, odontoblasts expressed 14,802 genes, presecretory ameloblasts 15,179 genes and secretory ameloblasts 14,526 genes. Genes known to be active during tooth development for each cell type (eg COL1A1, AMELX) were shown to be expressed by our approach. Exploring further into the list of differentially expressed genes between the motile odontoblasts and non-motile presecretory ameloblasts we found several genes of interest that could be involved in cell movement (FN1, LUM, ASTN1). Furthermore, our analysis indicated that the Phospholipase C and ERK5 pathways, that are important for cell movement, were activated in the motile odontoblasts. In addition our pathway analysis identified WNT3A and TGFB1 as important upstream contributors. Recent studies implicate these genes in the development of Schimke immuno-osseous dysplasia. The utility of laser capture microdissection can be a valuable tool in the examination of specific tissues or cell populations present in human tooth buds. Advancing our knowledge of the human dentome and related molecular pathways provides new insights into the complex mechanisms regulating odontogenesis and biomineralization. This knowledge could prove useful in future studies of odontogenic related pathologies.

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.

Current clinical techniques in dental practice include stem cell and tissue engineering applications. Dental stem cells are promising primary cell source for mainly tooth tissue engineering. Interaction of mesenchymal stem cell with epithelial and endothelial cells is strictly required for an intact tooth morphogenesis. Therefore, it is important to investigate whether human tooth germ stem cells (hTGSCs) derived from wisdom tooth are suitable for endothelial and epithelial cell transformation in dental tissue regeneration approaches. Differentiation into endothelial and epithelial cell lineages were mimicked under defined conditions, confirmed by real time PCR, western blotting and immunocytochemical analysis by qualitative and quantitative methods. HUVECs and HaCaT cells were used as positive controls for the endothelial and epithelial differentiation assays, respectively. Immunocytochemical and western blotting analysis revealed that terminally differentiated cells expressed cell-lineage markers including CD31, VEGFR2, VE-Cadherin, vWF (endothelial cell markers), and cytokeratin (CK)-17, CK-19, EpCaM, vimentin (epithelial cell markers) in significant levels with respect to undifferentiated control cells. Moreover, high expression levels of VEGFR1, VEGFR2, VEGF, CK-18, and CK-19 genes were detected in differentiated endothelial and epithelial-like cells. Endothelial-like cells derived from hTGSCs were cultured on Matrigel, tube-like structure formations were followed as an indication for functional endothelial differentiation. hTGSCs successfully differentiate into various cell types with a broad range of functional abilities using an in vitro approach. These findings suggest that hTGSCs may serve a potential stem cell source for tissue engineering and cell therapy of epithelial and endothelial tissue., (© 2014 International Federation for Cell Biology.)

Members of the Sox gene family play roles in many biological processes including organogenesis. We carried out comparative in situ hybridization analysis of seventeen sox genes (Sox1-14, 17, 18, 21) during murine odontogenesis from the epithelial thickening to the cytodifferentiation stages. Localized expression of five Sox genes (Sox6, 9, 13, 14 and 21) was observed in tooth bud epithelium. Sox13 showed restricted expression in the primary enamel knots. At the early bell stage, three Sox genes (Sox8, 11, 17 and 21) were expressed in pre-ameloblasts, whereas two others (Sox5 and 18) showed expression in odontoblasts. Sox genes thus showed a dynamic spatio-temporal expression during tooth development.