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Organ development is dictated by the regulation of genes preferentially expressed in tissues or cell types. Gene expression profiling and identification of specific genes in organs can provide insights into organogenesis. Therefore, genome-wide analysis is a powerful tool for clarifying the mechanisms of development during organogenesis as well as tooth development. Single-cell RNA sequencing (scRNA-seq) is a suitable tool for unraveling the gene expression profile of dental cells. Using scRNA-seq, we can obtain a large pool of information on gene expression; however, identification of functional genes, which are key molecules for tooth development, via this approach remains challenging. In the present study, we performed cap analysis of gene expression sequence (CAGE-seq) using mouse tooth germ to identify the genes preferentially expressed in teeth. The CAGE-seq counts short reads at the 5′-end of transcripts; therefore, this method can quantify the amount of transcripts without bias related to the transcript length. We hypothesized that this CAGE data set would be of great help for further understanding a gene expression profile through scRNA-seq. We aimed to identify the important genes involved in tooth development via bioinformatics analyses, using a combination of scRNA-seq and CAGE-seq. We obtained the scRNA-seq data set of 12,212 cells from postnatal day 1 mouse molars and the CAGE-seq data set from postnatal day 1 molars. scRNA-seq analysis revealed the spatiotemporal expression of cell type–specific genes, and CAGE-seq helped determine whether these genes are preferentially expressed in tooth or ubiquitously. Furthermore, we identified candidate genes as novel tooth-enriched and dental cell type–specific markers. Our results show that the integration of scRNA-seq and CAGE-seq highlights the genes important for tooth development among numerous gene expression profiles. These findings should contribute to resolving the mechanism of tooth development and establishing the basis for tooth regeneration in the future.

Background Induced pluripotent stem cell (iPSC)-based technology provides limitless resources for customized development of organs without any ethical concerns. In theory, iPSCs generated from terminally differentiated cells can be induced to further differentiate into all types of organs that are derived from the embryonic germ layers. Since iPSC reprogramming technology is relatively new, extensive efforts by the researchers have been put together to optimize the protocols to establish in vitro differentiation of human iPSCs (hiPSCs) into various desirable cell types/organs. Highlights In the present study, we review the potential application of iPSCs as an efficient alternative to primary cells for modulating signal molecules. Furthermore, an efficient culture system that promotes the differentiation of cell lineages and tissue formation has been reviewed. We also summarize the recent studies wherein tissue engineering of the three germ layers has been explored. Particularly, we focus on the current research strategies for iPSC-based tooth regeneration via molecular modulation. Conclusion In recent decades, robust knowledge regarding the hiPSC-based regenerative therapy has been accumulated, especially focusing on cellular modulation. This review provides the optimization of the procedures designed to regenerate specific organs.

Different stem cell-based strategies, especially induced pluripotent stem cells (iPSCs), have been exploited to regenerate teeth or restore biological and physiological functions after tooth loss. Further research is needed to establish an optimized protocol to effectively differentiate human iPSCs (hiPSCs) into dental epithelial cells (DECs). In this study, various factors were precisely modulated to facilitate differentiation of hiPSCs into DECs, which are essential for the regeneration of functional teeth. Embryoid bodies (EBs) were formed from hiPSCs as embryo-like aggregates, retinoic acid (RA) was used as an early ectodermal inducer, and bone morphogenic protein 4 (BMP4) activity was manipulated. The characteristics of DECs were enhanced and preserved after culture in keratinocyte serum-free medium (K-SFM). The yielded cell population exhibited noticeable DEC characteristics, consistent with the expression of epithelial cell and ameloblast markers. DECs demonstrated odontogenic abilities by exerting an inductive effect on human dental pulp stem cells (hDPSCs) and forming a tooth-like structure with the mouse tooth mesenchyme. Overall, our differentiation protocol provides a practical approach for applying hiPSCs for tooth regeneration.

Tooth-related diseases and tooth loss are widespread and are a major public health issue. The loss of teeth can affect chewing, speech, appearance and even psychology. Therefore, the science of tooth regeneration has emerged, and attention has focused on tooth regeneration based on the principles of tooth development and stem cells combined with tissue engineering technology. As undifferentiated stem cells in normal tooth tissues, dental mesenchymal stem cells (DMSCs), which are a desirable source of autologous stem cells, play a significant role in tooth regeneration. Researchers hope to reconstruct the complete tooth tissues with normal functions and vascularization by utilizing the odontogenic differentiation potential of DMSCs. Moreover, DMSCs also have the ability to differentiate towards cells of other tissue types due to their multipotency. This review focuses on the multipotential capacity of DMSCs to differentiate into various tissues, such as bone, cartilage, tendon, vessels, neural tissues, muscle-like tissues, hepatic-like tissues, eye tissues and glands and the influence of various regulatory factors, such as non-coding RNAs, signaling pathways, inflammation, aging and exosomes, on the odontogenic/osteogenic differentiation of DMSCs in tooth regeneration. The application of DMSCs in regenerative medicine and tissue engineering will be improved if the differentiation characteristics of DMSCs can be fully utilized, and the factors that regulate their differentiation can be well controlled.

Oral organoids are complex 3-dimensional structures that develop from stem cells or organ-specific progenitors through a process of self-organization and re-create architectures and functionalities similar to in vivo organs and tissues in the oral and maxillofacial region. Recently, striking advancements have been made in the construction and application of oral organoids of the tooth, salivary gland, and tongue. Dental epithelial and mesenchymal cells isolated from tooth germs or derived from pluripotent stem cells could generate tooth germ–like organoids by self-organization in a specific culture system. Tooth organoids can also be constructed based on tissue engineering principles by seeding stem cells on a scaffold with the bioregulatory functions of odontogenic differentiation. Two main approaches have been used to construct salivary gland organoids: 1) incubation of salivary gland–derived stem/progenitor cells in a 3-dimensional culture system to form the structure of the gland through mimicking regenerative processes and 2) inducing of pluripotent stem cells to generate embryonic salivary glands by replicating the development process. Taste bud organoids can be generated by embedding isolated circumvallate papilla tissue in Matrigel with a mixture of growth factors, while lingual epithelial organoids have been constructed using lingual stem cells in a suitable culture system containing specific signaling molecules. These oral organoids usually maintain the main functions and characteristic structures of the corresponding organ to a certain extent. Furthermore, using cells isolated from patients, oral organoids could replicate specific diseases such as maxillofacial tumors and tooth dysplasia. Until now, oral organoids have been applied in the study of mechanisms of tooth development, pathology and regeneration of the salivary gland, and precision therapeutics for tongue cancer. These findings strongly demonstrate that the organoid technique is a novel paradigm for the study of the development, pathology, and regeneration of oral and maxillofacial tissue.

The establishment of a method to derive dental epithelial cells seems to be an important challenge toward realizing the whole tooth regeneration. In order to obtain a source of dental epithelial-like cells, a new methodology has been previously developed by our research group. In the method, induced pluripotent stem cells are cultured in suspension in the presence of neurotrophin-4 to form embryoid bodies followed by further adherent culture of the embryoid bodies in DMEM basal nutrient medium. The present study was directed to improve the efficiency of dental epithelial-like cell production, by focusing on the optimization of initial cell number for the formation of embryoid bodies and the addition of epidermal growth factor as well as its timing. Our results demonstrated that an initial cell number of 1000 cells/drop gives the highest efficiency of dental epithelial-like cell production. It appears that, under this condition, medium deterioration is moderated, and that cell-cell interactions are optimized within embryoid bodies. On the other hand, epidermal growth factor serves to increase the abundance of dental epithelial-like cells when added to the medium together with neurotrophin-4 during embryoid body formation. The promotive effect of epidermal growth factor may involve the transactivation of TrkB, mediated by the effectors of epidermal growth factor receptor signaling.

Analysis of various genetically modified mice, with supernumerary teeth, has revealed the following two intrinsic molecular mechanisms that increase the number of teeth. One plausible explanation for supernumerary tooth formation is the rescue of tooth rudiments. Topical application of candidate molecules could lead to whole tooth formation under suitable conditions. Congenital tooth agenesis is caused by the cessation of tooth development due to the deletion of the causative gene and suppression of its function. The arrest of tooth development in Runx2 knockout mice, a mouse model of congenital tooth agenesis, is rescued in double knockout mice of Runx2 and Usag-1. The Usag-1 knockout mouse is a supernumerary model mouse. Targeted molecular therapy could be used to generate teeth in patients with congenital tooth agenesis by stimulating arrested tooth germs. The third dentition begins to develop when the second successional lamina is formed from the developing permanent tooth in humans and usually regresses apoptotically. Targeted molecular therapy, therefore, seems to be a suitable approach in whole-tooth regeneration by the stimulation of the third dentition. A second mechanism of supernumerary teeth formation involves the contribution of odontogenic epithelial stem cells in adults. Cebpb has been shown to be involved in maintaining the stemness of odontogenic epithelial stem cells and suppressing epithelial-mesenchymal transition. Odontogenic epithelial stem cells are differentiated from one of the tissue stem cells, enamel epithelial stem cells, and odontogenic mesenchymal cells are formed from odontogenic epithelial cells by epithelial-mesenchymal transition. Both odontogenic epithelial cells and odontogenic mesenchymal cells required to form teeth from enamel epithelial stem cells were directly induced to form excess teeth in adults. An approach for the development of targeted therapeutics has been the local application of monoclonal neutralizing antibody/siRNA with cationic gelatin for USAG-1 or small molecule for Cebpb.

Functional tooth regeneration has shown promising therapeutic strategy. Tooth regeneration is possible by combine use of adult stem cells, growth factors, and scaffold. In recent years, researchers have explored tooth regeneration. Significant effort has been made in recent decades to identify and characterize tooth stem cells and to unravel the developmental programs which these cells follow to generate a tooth.

Over the past 40 years, studies on tooth regeneration have been conducted. These studies comprised two main flows: some focused on epithelial–mesenchymal interaction in the odontogenic region, whereas others focused on creating a supernumerary tooth in the non-odontogenic region. Recently, the scope of the research has moved from conventional gene modification and molecular therapy to genome and transcriptome sequencing analyses. However, these sequencing data have been produced only in the odontogenic region. We provide RNA-Seq data of not only the odontogenic region but also the non-odontogenic region, which loses tooth-forming capacity during development and remains a rudiment. Sequencing data were collected from mouse embryos at three different stages of tooth development. These data will expand our understanding of tooth development and will help in designing developmental and regenerative studies from a new perspective.

Background Tooth regeneration depends on the longevity of the dental epithelial lamina. However, the exact mechanism of dental lamina regression has not yet been clarified. To explore the role of the sonic hedgehog (Shh) signaling pathway in regression process of the rudimentary successional dental lamina (RSDL) in mice, we orally administered a single dose of a Shh signaling pathway inhibitor to pregnant mice between embryonic day 13.0 (E13.0) and E17.0. Results We observed that the Shh signaling pathway inhibitor effectively inhibited the expression of Shh signaling pathway components and revitalized RSDL during E15.0-17.0 by promoting cell proliferation. In addition, mRNA-seq, RT-qPCR, and immunohistochemical analyses indicated that diphyodontic dentition formation might be related to FGF signal upregulation and the Sostdc1-Wnt negative feedback loop. Conclusion Overall, our results indicated that the Shh signaling pathway may play an initial role in preventing further development of mouse RSDL in a time-dependent manner. This article is protected by copyright. All rights reserved.

Similar to the mesenchymal stem cells (MSCs), dental pulp stem cells (DPSCs) also have pluripotent differentiation characteristic and may be more ideal for tissue regeneration, especially in tooth regeneration engineering. However, bacterial infection may be a powerful obstacle. Berberine (BBR), known with antibacterial effects, was recently found to play functions in bone formation through promoting osteogenic differentiation from pluripotent stem cells. However, whether BBR also function in DPSCs osteogenic differentiation has not yet been reported. Primary DPSCs were isolated from dental pulp tissues extracted from human impacted mandibular third molars, and identified by flow cytometry for cell surface antigen molecules. A dexamethasone osteogenic medium was used to induce DPSCs osteogenic differentiation. BBR (1 μM and 5 μM) was pre-added to into medium, and then cell proliferation, spheroid formation and osteogenic differentiation capacities of DPSCs were analyzed, as well as the underlying molecules modulation mechanism. Flow cytometry identified that CD44, CD90, CD81 and CD105 positively expressed in the isolated hDPSCs, with CD34 and CD45 negetively expressed. BBR enhanced the cell proliferation of hDPSCs in a dose-dependent pattern, and promoted dexamethasone-induced osteogenic differentiation via enhancing Runx2 transcription factor activity followed by upregulating osteogenesis markers expression, whereas the adipogenic differentiation of hDPSCs was suppressed dramatically by BBR. The EGFR and MAPK pathways were activated by BBR, and inhibitors for these pathways significantly suppressed the osteogenic differentiation promotion of BBR. These results have revealed a novel mechanism that berberine might promote hDPSCs osteogenic differentiation through activating EGFR-MAPK-Runx2 signaling pathways.

Although mouse models of epithelial–mesenchymal interaction-based whole-tooth regeneration have been generated, whether this strategy can be applied to humans remains unknown and preclinical invest...

Dental pulp stem cells (DPSCs) are a source of postnatal stem cells essential for maintenance and regeneration of dentin and pulp tissues. Previous in vivo transplantation studies have shown that DPSCs are able to give rise to odontoblast-like cells, form dentin/pulp-like structures, and induce blood vessel formation. Importantly, dentin formation is closely associated to blood vessels. We have previously demonstrated that DPSC-induced angiogenesis is VEGFR-2-dependent. VEGFR-2 may play an important role in odontoblast differentiation of DPSCs, tooth formation and regeneration. Nevertheless, the role of VEGFR-2 signaling in odontoblast differentiation of DPSCs is still not well understood. Thus, in this study we aimed to determine the role of VEGFR-2 in odontoblast differentiation of DPSCs by knocking down the expression of VEGFR-2 in DPSCs and studying their odontoblast differentiation capacity in vitro and in vivo. Isolation and characterization of murine DPSCs was performed as previously described. DPSCs were induced by VEGFR-2 shRNA viral vectors transfection (MOI = 10:1) to silence the expression of VEGFR-2. The GFP+ expression in CopGFP DPSCs was used as a surrogate to measure the efficiency of transfection and verification that the viral vector does not affect the expression of VEGFR-2. The efficiency of viral transfection was shown by significant reduction in the levels of VEGFR-2 based on the Q-RT-PCR and immunofluorescence in VEGFR-2 knockdown DPSCs, compared to normal DPSCs. VEGFR-2 shRNA DPSCs expressed not only very low level of VEGFR-2, but also that of its ligand, VEGF-A, compared to CopGFP DPSCs in both transcriptional and translational levels. In vitro differentiation of DPSCs in osteo-odontogenic media supplemented with BMP-2 (100 ng/ml) for 21 days demonstrated that CopGFP DPSCs, but not VEGFR-2 shRNA DPSCs, were positive for alkaline phosphatase (ALP) staining and formed mineralized nodules demonstrated by positive Alizarin Red S staining. The expression levels of dentin matrix proteins, dentin matrix protein-1 (Dmp1), dentin sialoprotein (Dspp), and bone sialoprotein (Bsp), were also up-regulated in differentiated CopGFP DPSCs, compared to those in VEGFR-2 shRNA DPSCs, suggesting an impairment of odontoblast differentiation in VEGFR-2 shRNA DPSCs. In vivo subcutaneous transplantation of DPSCs with hydroxyapatite (HAp/TCP) for 5 weeks demonstrated that CopGFP DPSCs were able to differentiate into elongated and polarized odontoblast-like cells forming loose connective tissue resembling pulp-like structures with abundant blood vessels, as demonstrated by H&E, Alizarin Red S, and dentin matrix staining. On the other hand, in VEGFR-2 shRNA DPSC transplants, odontoblast-like cells were not observed. Collagen fibers were seen in replacement of dentin/pulp-like structures. These results indicate that VEGFR-2 may play an important role in dentin regeneration and highlight the potential of VEGFR-2 modulation to enhance dentin regeneration and tissue engineering as a promising clinical application.

BackgroundVertebrate teeth exhibit a wide range of regenerative systems. Many species, including most mammals, reptiles, and amphibians, form replacement teeth at a histologically distinct location called the successional dental lamina, while other species do not employ such a system. Notably, a ‘lamina-less’ tooth replacement condition is found in a paraphyletic array of ray-finned fishes, such as stickleback, trout, cod, medaka, and bichir. Furthermore, the position, renewal potential, and latency times appear to vary drastically across different vertebrate tooth regeneration systems. The progenitor cells underlying tooth regeneration thus present highly divergent arrangements and potentials. Given the spectrum of regeneration systems present in vertebrates, it is unclear if morphologically divergent tooth regeneration systems deploy an overlapping battery of genes in their naïve dental tissues.ResultsIn the present work, we aimed to determine whether or not tooth progenitor epithelia could be composed of a conserved cell type between vertebrate dentitions with divergent regeneration systems. To address this question, we compared the pharyngeal tooth regeneration processes in two ray-finned fishes: zebrafish (Danio rerio) and threespine stickleback (Gasterosteus aculeatus). These two teleost species diverged approximately 250 million years ago and demonstrate some stark differences in dental morphology and regeneration. Here, we find that the naïve successional dental lamina in zebrafish expresses a battery of nine genes (bmpr1aa, bmp6, cd34, gli1, igfbp5a, lgr4, lgr6, nfatc1,andpitx2), while active Wnt signaling andLef1expression occur during early morphogenesis stages of tooth development. We also find that, despite the absence of a histologically distinct successional dental lamina in stickleback tooth fields, the same battery of nine genes (Bmpr1a,Bmp6,CD34,Gli1,Igfbp5a,Lgr4,Lgr6,Nfatc1, andPitx2) are expressed in the basalmost endodermal cell layer, which is the region most closely associated with replacement tooth germs. Like zebrafish, stickleback replacement tooth germs additionally expressLef1and exhibit active Wnt signaling. Thus, two fish systems that either have an organized successional dental lamina (zebrafish) or lack a morphologically distinct successional dental lamina (sticklebacks) deploy similar genetic programs during tooth regeneration.ConclusionsWe propose that the expression domains described here delineate a highly conserved “successional dental epithelium” (SDE). Furthermore, a set of orthologous genes is known to mark hair follicle epithelial stem cells in mice, suggesting that regenerative systems in other epithelial appendages may utilize a related epithelial progenitor cell type, despite the highly derived nature of the resulting functional organs.

Tissue engineering (TE) is a multidisciplinary research field aiming at the regeneration, restoration, or replacement of damaged tissues and organs. Classical TE approaches combine scaffolds, cells and soluble factors to fabricate constructs mimicking the native tissue to be regenerated. However, to date, limited success in clinical translations has been achieved by classical TE approaches, because of the lack of satisfactory biomorphological and biofunctional features of the obtained constructs. Developmental TE has emerged as a novel TE paradigm to obtain tissues and organs with correct biomorphology and biofunctionality by mimicking the morphogenetic processes leading to the tissue/organ generation in the embryo. Ectodermal appendages, for instance, develop in vivo by sequential interactions between epithelium and mesenchyme, in a process known as secondary induction. A fine artificial replication of these complex interactions can potentially lead to the fabrication of the tissues/organs to be regenerated. Successful developmental TE applications have been reported, in vitro and in vivo, for ectodermal appendages such as teeth, hair follicles and glands. Developmental TE strategies require an accurate selection of cell sources, scaffolds and cell culture configurations to allow for the correct replication of the in vivo morphogenetic cues. Herein, we describe and discuss the emergence of this TE paradigm by reviewing the achievements obtained so far in developmental TE 3D scaffolds for teeth, hair follicles, and salivary and lacrimal glands, with particular focus on the selection of biomaterials and cell culture configurations., Graphical abstract Image 1, Highlights • Developmental tissue engineering (TE) mimics morphogenesis to regenerate organs. • Tooth, hair follicle, and glands successfully regenerated by developmental TE. • 3D cell-seeded/3D cell-laden scaffolds fundamental to regenerate organs by developmental TE. • Cell source, coculture and scaffold design determine success of developmental TE.

Isolation of high-quality human postnatal stem cells from accessible sources is an important goal for dental tissue engineering. Stem cells from developing organs are a better cell source but are hard to obtain. With extensive caries that are difficult to restore, the extracted deciduous tooth with an immature apex is a developing organ for investigation. In the present study, a cell population from the tip of apical pulp of human deciduous teeth with an immature apex was isolated and termed apical pulp-derived cells of deciduous teeth (De-APDCs). De-APDCs expressed STRO-1, CD44, CD90 and CD105 but not CD34 or CD45. Furthermore, De-APDCs demonstrated a significantly higher clonogenic and proliferative ability and osteo/dentinogenic differentiation capacity than dental pulp cells from exfoliated deciduous teeth (De-DPCs) (P < 0.05). Differentiation potential toward adipogenic, neurogenic and chondrogenic lineages was also observed in induced De-APDCs. In addition, after De-APDCs were seeded into hydroxyapatite/tricalcium phosphate (HA/TCP) scaffolds and transplanted into nude mice, they were able to regenerate dentin/pulp-like structures aligned with human odontoblast-like cells. In conclusion, De-APDCs, which are derived from a developing tissue, represent an accessible and prospective cell source for tooth regeneration.

Uterine sensitization–associated gene-1 (USAG-1) deficiency leads to enhanced bone morphogenetic protein (BMP) signaling, leading to supernumerary teeth formation. Furthermore, antibodies interfering with binding of USAG-1 to BMP, but not lipoprotein receptor–related protein 5/6 (LRP5/6), accelerate tooth development. Since USAG-1 inhibits Wnt and BMP signals, the essential factors for tooth development, via direct binding to BMP and Wnt coreceptor LRP5/6, we hypothesized that USAG-1 plays key regulatory roles in suppressing tooth development. However, the involvement of USAG-1 in various types of congenital tooth agenesis remains unknown. Here, we show that blocking USAG-1 function through USAG-1 knockout or anti–USAG-1 antibody administration relieves congenital tooth agenesis caused by various genetic abnormalities in mice. Our results demonstrate that USAG-1 controls the number of teeth by inhibiting development of potential tooth germs in wild-type or mutant mice missing teeth. Anti–USAG-1 antibody administration is, therefore, a promising approach for tooth regeneration therapy., 先天性無歯症に対する分子標的薬の開発 --USAG-1を標的分子とした歯再生治療--. 京都大学プレスリリース. 2021-02-15.

Mesenchymal stem cells (MSCs) could be identified in mammalian teeth. Currently, dental-derived MSCs (DMSCs) has become a collective term for all the MSCs isolated from dental pulp, periodontal ligament, dental follicle, apical papilla, and even gingiva. These DMSCs possess similar multipotent potential as bone marrow-derived MSCs, including differentiation into cells that have the characteristics of odontoblasts, cementoblasts, osteoblasts, chondrocytes, myocytes, epithelial cells, neural cells, hepatocytes, and adipocytes. Besides, DMSCs also have powerful immunomodulatory functions, which enable them to orchestrate the surrounding immune microenvironment. These properties enable DMSCs to have a promising approach in injury repair, tissue regeneration, and treatment of various diseases. This review outlines the most recent advances in DMSCs’ functions and applications and enlightens how these advances are paving the path for DMSC-based therapies.

Craniofacial and dental tissues are unique and contain specialized cell types. The embryonic origins and characteristics of craniofacial bone are different from long bone. Craniofacial tissues are derived from neural crest cells (NCCs). The evidence has shown that MSCs derived from craniofacial tissue behave differently from those of other tissue origin. Thus, using craniofacial MSCs appears to be a better cell source for craniofacial tissue regeneration. While adult stem cells offer various clinical benefits for tissue regeneration, their key drawback is the limited lifespan and differentiation potential for certain applications. Induced pluripotent stem cells (iPSCs) can overcome these deficiencies. Oral and dental stem cells are also a good source of cells to generate iPSCs. Generated iPSCs either from dental and nondental cell sources have been investigated for their capacity in neurogenesis as well as for craniofacial tissue regeneration, including craniofacial bone regeneration, pulp regeneration, periodontal tissue regeneration, and whole tooth regeneration. Before iPSCs can provide significant clinical benefits for patients, issues revolving iPSC biology, their translation, and clinical advancement that pose various challenges need to be resolved.

Runt-related transcription factor 2 (Runx2)-deficient mice can be used to model congenital tooth agenesis in humans. Conversely, uterine sensitization-associated gene-1 (Usag-1)-deficient mice exhibit supernumerary tooth formation. Arrested tooth formation can be restored by crossing both knockout-mouse strains; however, it remains unclear whether topical inhibition of Usag-1 expression can enable the recovery of tooth formation in Runx2-deficient mice. Here, we tested whether inhibiting the topical expression of Usag-1 can reverse arrested tooth formation after Runx2 abrogation. The results showed that local application of Usag-1 Stealth small interfering RNA (siRNA) promoted tooth development following Runx2 siRNA-induced agenesis. Additionally, renal capsule transplantation of siRNA-loaded cationized, gelatin-treated mouse mandibles confirmed that cationized gelatin can serve as an effective drug-delivery system. We then performed renal capsule transplantation of wild-type and Runx2-knockout (KO) mouse mandibles, treated with Usag-1 siRNA, revealing that hindered tooth formation was rescued by Usag-1 knockdown. Furthermore, topically applied Usag-1 siRNA partially rescued arrested tooth development in Runx2-KO mice, demonstrating its potential for regenerating teeth in Runx2-deficient mice. Our findings have implications for developing topical treatments for congenital tooth agenesis.

Dental pulp stem cells (DPSCs) can differentiate into diverse cell lineages, including odontogenic cells that are responsible for dentin formation, which is important in pulp repair and tooth regeneration. While glycolysis plays a central role in various cellular activities in both physiological and pathological conditions, its role and regulation in odontogenic differentiation are unknown. Here, we show that aerobic glycolysis is induced during odontoblastic differentiation from human DPSCs. Importantly, we demonstrate that during odontoblastic differentiation, protein expression levels of phosphofructokinase 1 muscle isoform (PFKM) and PFK2, but not other glycolytic enzymes, are mainly upregulated by AKT activation, resulting in increased total PFK enzyme activity. Increased PFK activity is essential to enhance aerobic glycolysis, which plays an important role in the odontoblastic differentiation of human DPSCs. These findings underscore that PFK activation-induced aerobic glycolysis accompanies, and participates in, human DPSCs differentiation into odontogenic lineage, and could play a role in the regulation of dental pulp repair.

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.

Synopsis In recent years, nonclassical models have emerged as mainstays for studies of evolutionary, developmental, and regenerative biology. Genomic advances have promoted the use of alternative taxa for the study of developmental biology, and the shark is one such emerging model vertebrate. Our research utilizes the embryonic shark (Scyliorhinus canicula) to characterize key developmental and regenerative processes that have been overlooked or not possible to study with more classic developmental models. Tooth development is a major event in the construction of the vertebrate body plan, linked in part with the emergence of jaws. Early development of the teeth and morphogenesis is well known from the murine model, but the process of tooth redevelopment and regeneration is less well known. Here we explore the role of the dental lamina in the development of a highly regenerative dentition in sharks. The shark represents a polyphyodont vertebrate with continuously repeated whole tooth regeneration. This is presented as a major developmental shift from the more derived renewal process that the murine model offers, where incisors exhibit continuous renewal and growth of the same tooth. Not only does the shark offer a study system for whole unit dental regeneration, it also represents an important model for understanding the evolutionary context of vertebrate tooth regeneration.

Background: Autologous platelet concentrates such as platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) have gained overwhelming popularity in regenerative endodontics. Clinical evidence reveals the lack of a particular advantage of using PRP or PRF over an evoked blood clot in promoting canal wall thickening and/or continued root development in immature necrotic teeth. Moreover, despite stimulating tissue repair and repopulating the root canals of immature and mature permanent teeth, the new vital tissue may not possess the functional activity of the native pulp tissue. Methods: To better understand the origin, nature, and long-term fate of the tissue types found within the pulp space, we critically examine all available histo-/morphological evidence for pulp–dentine complex regeneration using PRP and/or PRF, alone or together with an evoked blood clot, specialised or unspecialised primary cells, and other biomaterials. Results: Histological data from clinical studies is scant. Reportedly, the inner dentinal surface supports cementum-like tissue formation, but this interface likely deviates in structure and function from the native cementodentinal junction. Presence of bone-like tissue within the pulp space is intriguing since de novo osteogenesis requires closely coordinated recruitment and differentiation of osteoprogenitor cells. Compared to untreated necrotic teeth, an evoked blood clot (with/without PRF) improves fracture resistance. Tooth regeneration using PRF and dental bud cells is unreliable and the constituent neoformed tissues are poorly organised. Conclusion: PRP/PRF fail to demonstrate a significant advantage over an induced blood clot, alone. The true nature of neoformed tissues remains poorly characterised while their response to subsequent insult/injury is unexplored.

Remarkable breakthroughs in the fields of developmental biology and stem cell biology during the last 15 yr have led to a new level of understanding regarding how teeth develop and how stem cells can be programmed. As a result, the possibilities of growing new teeth and of tooth bioengineering have been explored. Currently, a great deal is known about how signaling molecules and genes regulate tooth development, and modern research using transgenic mouse models has demonstrated that it is possible to induce the formation of new teeth by tinkering with the signaling networks that govern early tooth development. A breakthrough in stem cell biology in 2006 opened up the possibility that a patient's own cells can be programmed to develop into pluripotent stem cells and used for building new tissues and organs. At present, active research in numerous laboratories around the world addresses the question of how to program the stem and progenitor cells to develop into tooth-specific cell types. Taken together, the remarkable progress in developmental and stem cell biology is now feeding hopes of growing new teeth in the dental clinic in the not-too-distant future.

The teeth are highly differentiated chewing organs formed by the development of tooth germ tissue located in the jaw and consist of the enamel, dentin, cementum, pulp, and periodontal tissue. Moreover, the teeth have a complicated regulatory mechanism, special histologic origin, diverse structure, and important function in mastication, articulation, and aesthetics. These characteristics, to a certain extent, greatly complicate the research in tooth regeneration. Recently, new ideas for tooth and tissue regeneration have begun to appear with rapid developments in the theories and technologies in tissue engineering. Numerous types of stem cells have been isolated from dental tissue, such as dental pulp stem cells (DPSCs), stem cells isolated from human pulp of exfoliated deciduous teeth (SHED), periodontal ligament stem cells (PDLSCs), stem cells from apical papilla (SCAPs), and dental follicle cells (DFCs). All these cells can regenerate the tissue of tooth. This review outlines the cell types and strategies of stem cell therapy applied in tooth regeneration, in order to provide theoretical basis for clinical treatments.

The tooth is an intricate composition of precisely patterned, mineralized matrices and soft tissues. Mineralized tissues include enamel (produced by the epithelial cells called ameloblasts), dentin and cementum (produced by mesenchymal cells called odontoblasts and cementoblasts, respectively), and soft tissues, which include the dental pulp and the periodontal ligament along with the invading nerves and blood vessels. It was perceived for a very long time that teeth primarily serve an esthetical function. In recent years, however, the role of healthy teeth, as well as the impact of oral health on general well-being, became more evident. Tooth loss, caused by tooth decay, congenital malformations (tooth agenesis), trauma, periodontal diseases, or age-related changes, is usually replaced by artificial materials which lack many of the important biological characteristics of the natural tooth. Human teeth have very low to almost absent regeneration potential, due to early loss of cell populations with regenerative capacity, namely stem cells. Significant effort has been made in recent decades to identify and characterize tooth stem cells, and to unravel the developmental programs which these cells follow in order to generate a tooth.

Tooth formation during embryogenesis is controlled through a complex interplay between mechanical and chemical cues. We have previously shown that physical cell compaction of dental mesenchyme cells during mesenchymal condensation is responsible for triggering odontogenic differentiation during embryogenesis, and that expression of Collagen VI stabilizes this induction. In addition, we have shown that synthetic polymer scaffolds that artificially induce cell compaction can induce embryonic mandible mesenchymal cells to initiate tooth differentiation both in vitro and in vivo. As embryonic cells would be difficult to use for regenerative medicine applications, here we explored whether compressive scaffolds coated with Collagen VI can be used to induce adult bone marrow stromal cells (BMSCs) to undergo an odontogenic lineage switch. These studies revealed that when mouse BMSCs are compressed using these scaffolds they increase expression of critical markers of tooth differentiation in vitro, including the key transcription factors Pax9 and Msx1. Implantation under the kidney capsule of contracting scaffolds bearing these cells in mice also resulted in local mineralization, calcification and production of dentin-like tissue. These findings show that these chemically-primed compressive scaffolds can be used to induce adult BMSCs to undergo a lineage switch and begin to form dentin-like tissue, thus raising the possibility of using adult BMSCs for future tooth regeneration applications.

Dental stem cell‐based tooth regeneration is the futuristic treatment for missing teeth. Growth differentiation factor 11 (GDF11), a novel member of the TGF‐beta superfamily, has been reported to play a critical role in regulating stem cell differentiation. However, the role of endogenous GDF11 during dental stem cell differentiation remains unknown. Here, we have shown that GDF11 was highly expressed in dental pulp tissues in both mouse and human. Knockdown of endogenous GDF11 in human dental pulp stem cells (hDPSCs) led to comparable proliferation and migration but attenuated odontogenic differentiation as evidenced by alkaline phosphatase and Alizarin Red S staining. In addition, transcriptional levels of odontogenic‐related genes were significantly down‐regulated according to real‐time polymerase chain reaction. Mechanistically, we performed RNA sequencing analysis and found that silencing of endogenous GDF11 compromised the process of ossification and osteoblast differentiation, especially down‐regulated transcription expression of Wnt pathway‐specific genes. Immunofluorescence staining also showed diminished β‐catenin expression and nuclei accumulation after knockdown of endogenous GDF11 in hDPSCs. In summary, our results suggested that endogenous GDF11 positively regulate odontogenic differentiation of hDPSCs through canonical Wnt/β‐catenin signalling pathway.

Stem cell-based developmental engineering has been considered as a promising strategy for tissue/organ regeneration. Tooth is formed by sequential reciprocal interactions between epithelium derived from surface ectoderm and mesenchymal cells derived from cranial neural crest. The neural crest cell is an appealing cell source for tooth development and regeneration research. In this study, we investigated the odontogenic differentiation and dentin-pulp complex regeneration potential of neural crest cells. Our results showed that neural crest cells (O9-1 mouse cranial neural crest cell line) can sequentially differentiate into dentin matrix acidic phosphoprotein 1 (DMP-1)-positive odontoblasts within a developing tooth germ in vitro. Moreover, O9-1 cells and induced pluripotent stem cell (iPSC)-derived neural crest-like cells (iNCLCs) can form well-organized vascularized dentin-pulp complex when transplanted in vivo with tooth scaffold. Furthermore, both O9-1 cells and iNCLCs can be differentiated into odontoblast-like cells, positive staining with odontogenic-related markers DMP-1 and dentin sialophosphoprotein (DSPP), under odontogenic induction with the administration of bone morphogenetic protein 4 (BMP-4). These results demonstrated that neural crest cells, especially the unlimited iNCLCs, are a promising cell source for tooth development and dental tissue/tooth organ regeneration studies.

The human dentition is a typical diphyodont mammalian system with tooth replacement of most positions. However, after dental replacement and sequential molar development, the dental lamina undergoes apoptosis and fragments, leaving scattered epithelial units (dental lamina rests; DLRs). DLRs in adult humans are considered inactive epithelia, thought to possess limited capacity for further regeneration. However, we show that these tissues contain a small proportion of proliferating cells (assessed by both Ki67 and PCNA) but also express a number of common dental stem cell markers (Sox2, Bmi1, β-catenin and PH3) similar to that observed in many vertebrates that actively, and continuously regenerate their dentition. We compared these human tissues with the dental lamina of sharks that regenerate their dentition throughout life, providing evidence that human tissues have the capacity for further and undocumented regeneration. We also assessed cases of human ameloblastoma to characterise further the proliferative signature of dental lamina rests. Ameloblastomas are assumed to derive from aberrant lamina rests that undergo changes, which are not well understood, to form a benign tumour. We suggest that dental lamina rests can offer a potential source of important dental stem cells for future dental regenerative therapy. The combined developmental genetic data from the shark dental lamina and ameloblastoma may lead to the development of novel methods to utilise these rested populations of adult lamina stem cells for controlled tooth replacement in humans.

Background Tooth loss caused by caries or injuries has a negative effect on human health; thus, it is important to develop a reliable method of tooth regeneration. Research on tooth regeneration has mainly focused on mouse pluripotent stem cells, mouse embryonic stem cells, and adult stem cells from various sources in mice, whereas little has examined the differentiation of human embryonic stem (hES) cells into dental epithelium (DE) and odontogenic potential in vivo. Methods In this study, we induced hES cells to differentiate into dental epithelium using different concentrations of bone morphogenetic protein 4 (BMP4). With 1 pM BMP4, the hES cells differentiated into oral ectoderm (OE). These cells were then stimulated with 30 pM BMP4. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunofluorescence showed the differentiation of OE and DE. The DE generated was mixed with embryonic day 14.5 mouse dental mesenchyme (DM) and transplanted into the renal capsules of nude mice. Thirty days later, the resulting tooth-like structures were examined by micro-computed tomography and hematoxylin and eosin staining. Results After 4 days of 1 pM BMP4 stimulation, Pitx1-positive OE formed. qRT-PCR and immunofluorescence revealed that induction with 30 pM BMP4 for 2 days caused the OE to differentiate into Pitx2/Dlx2/AMBN-positive DE-like cells. These cells also expressed β-catenin and p-Smad1/5/8, which are key proteins in the Wnt/β-catenin and Bmp signaling pathways, respectively. Thirty days after in vivo transplantation, six teeth with enamel and dentin had formed on the kidney. Conclusions These results show that hES cells differentiated into DE after sequential stimulation with different concentrations of BMP4, and the DE thus generated showed odontogenic potential. Electronic supplementary material The online version of this article (10.1186/s13287-019-1378-7) contains supplementary material, which is available to authorized users.

Deep understanding of tooth regeneration is hampered by the lack of lifelong replacing oral dentition in most conventional models. Here, we show that the bearded dragon, one of the rare vertebrate species with both polyphyodont and monophyodont teeth, constitutes a key model for filling this gap, allowing direct comparison of extreme dentition types. Our developmental and high-throughput transcriptomic data of microdissected dental cells unveils the critical importance of successional dental lamina patterning, in addition to maintenance, for vertebrate tooth renewal. This patterning process happens at various levels, including directional growth but also gene expression levels, dynamics, and regionalization, and involves a large number of yet uncharacterized dental genes. Furthermore, the alternative renewal mechanism of bearded dragon dentition, with dual location of slow-cycling cells, demonstrates the importance of cell migration and functional specialization of putative epithelial stem/progenitor niches in tissue regeneration, while expanding the diversity of dental replacement strategies in vertebrates., eLife digest All multicellular organisms, from lizards to humans, must be able to repair and regrow damaged tissue. This includes not only healing after an injury, but also replacing parts of the body that suffer wear and tear. For example, many animals shed and replace worn out teeth throughout their life, but the number of times this occurs varies greatly between species. Much of the understanding about how teeth grow and develop has come from researching mice. However, mice only develop one set of teeth, making them a poor ‘model’ for studying how species such as fish and reptiles can re-grow and replace their teeth. Recent studies of these species has shown that regenerating teeth relies on a specialised structure known as the dental lamina. In mice, the dental lamina forms but then quickly disappears, preventing new sets of teeth from developing. In most animals that regrow their teeth, however, the dental lamina keeps growing beyond the most recently produced tooth to create an area where its replacement will emerge. Now, Salomies et al. have identified other strategies involved in tooth replacement from studying the bearded dragon lizard, a rare example of an animal that continuously regenerates some, but not all, of its teeth. Analysing the cells in different parts of the re-growing teeth from bearded dragon lizards revealed new features of the dental lamina. Specifically, Salomies et al. found that a previously uncharacterized set of genes within the dental lamina could determine whether or not a tooth will be replaced. Further experiments using microscope imaging revealed that bearded dragon lizards use two distinct groups of stem cells – specialised cells that have the potential to develop into various cell types in the body – to re-grow their teeth. These experiments demonstrate how the bearded dragon lizard uses a previously unknown mechanism to regenerate its teeth, combining elements used by gecko lizards and sharks. These findings are an important step towards understanding the different strategies animals can use to maintain and regenerate their teeth. The knowledge gained could one day help design better therapies for patients suffering from inherited dental disorders or tooth loss.

Recent developments in the stem cell biology provided new hopes in treatment of diseases and disorders that yet cannot be treated. Stem cells have the potential to differentiate into various cell types in the body during age. These provide new cells for the body as it grows, and replace specialized cells that are damaged. Since mesenchymal stem cells (MSCs) can be easily harvested from the adipose tissue and can also be cultured and expanded in vitro they have become a good target for tissue regeneration. These cells have been widespread used for cell transplantation in animals and also for clinical trials in humans. The purpose of this review is to provide a summary of our current knowledge regarding the important and types of isolated stem cells from different sources of animal models such as horse, pig, goat, dog, rabbit, cat, rat, mice etc. In this regard, due to the widespread use and lot of attention of MSCs, in this review, we will elaborate on use of MSCs in veterinary medicine as well as in regenerative medicine. Based on the studies in this field, MSCs found wide application in treatment of diseases, such as heart failure, wound healing, tooth regeneration etc.

Background Long non-coding RNAs (lncRNAs) have been widely known to have an appreciable effect in physiology and pathology. In tooth regeneration, periodontal ligament stem cells (PDLSCs) are regarded as a key effector, whereas, how lncRNA acts in the osteogenic differentiation of PDLSCs have not been completely understood. This study aims to find out the relationship between lncRNA DANCR and the proliferation and osteogenic differentiation of PDLSCs. Methods Microarray was used to observe the different expression of lncRNAs in differentiated and undifferentiated PDLSCs. And then osteogenic-related lncRNA, DANCR was screened out. Its effects on proliferation and osteogenic differentiation was explored by constructing an overexpression and inhibition model. qRT-PCR was used to detect the mRNA expression of osteogenesis related genes. MTT assay was performed to assess the effects of DANCR on cell growth curve. To quantify the effects of DANCR on osteogenic differentiation of PDLSCs, ALP staining and alizarin red was performed in basic culture medium and osteogenic medium. Data were statistically processed. Results Compared with the undifferentiated PDLSCs, the alizarin red staining level was higher in differentiated PDLSCs. And the expressions of osteogenic differentiation marker genes Runt-related transcription factor 2 (Runx2), osteocalcin (OCN) and bone morphogenetic protein (BMP-2) were significantly increased in the differentiated PDLSCs. Furthermore, we noticed that comparing with control groups, the expression of lncRNA DANCR decreases markedly in osteogenically induced PDLSCs. DANCR promoted proliferation of PDLSCs, as evidenced by cell viability. Further investigation has proven that the downregulation of DANCR shows in the calcium sediment forming, alkaline phosphatase (ALP) activation and some osteogenic-related gene markers’ upregulation including Runx2, OCN and BMP-2, which finally results in the osteogenic differentiation of PDLSCs following the transfection and induction. Conversely, DANCR upregulation was shown to repress the osteogenic differentiation potential of PDLSCs. Conclusions The osteogenic differentiation of PDLSCs has proven to related to the down regulation of lncRNA DANCR. And this paper throws light on the effects of DANCR in the process of PDLSCs’ osteogenic differentiation.

Biologically oriented regenerative dentistry in an attempt to regrow a functional tooth by harnessing the natural healing capabilities of dental tissues has become a recent trend challenging the current dental practice on repairing the damaged or missing tooth. In this review, we outline the conceptual development on the in situ revitalization of the tooth replacement capability lost during evolution, the updated progress in stem-cell-based in vivo repair of the damaged tooth, and the recent endeavors for in vitro generation of an implantable bioengineered tooth germ. Thereafter, we summarize the major challenges that need to be overcome in order to provide the rationale and directions for the success of fully functional tooth regeneration in the near future.

Previous studies have demonstrated that several types of human stem cells of non-dental origin can be induced to differentiate into enamel-secreting ameloblasts after recombined with mouse embryonic dental mesenchyme. However, the successful rate of ameloblastic differentiation is about rather low, which presents a major obstacle for future stem cell-based whole tooth bioengineering. Previous studies have shown that cultures at reduced temperature could improve the differentiation capability of stem cells in tissue engineering. In this study, we systematically investigated the effects of low temperature on the viability, proliferation and stemness of human keratinocytes stem cells (hKSCs) in cell culture and further examined ameloblastic differentiation of the hKSCs in human–mouse recombinant chimeric tooth germs. Our results demonstrated that low temperature indeed reduces growth rate and maintains healthy undifferentiated morphology of hKSCs without any effects on cell viability. Moreover, examination of stemness makers revealed improved stemness of hKSCs cultured at low temperature with increased expression of stemness markers K15, CD29 and p63 and decreased expression differentiation marker K10, as compared to those cultured at 37 °C. These low temperature treated hKSCs, when recombined with mouse embryonic dental mesenchyme, exhibited significantly increased rate (40%) of ameloblastic differentiation, as compared to that (17%) in tissue recombinants with those hKSCs treated at standard temperature. Our studies demonstrate that low temperature cell culture improves the stemness and plasticity of hKSCs, which in turn enhances ameloblastic differentiation capability of the stem cells in bioengineered teeth.

Patterning is a critical step during organogenesis and is closely associated with the physiological function of organs. Tooth root shapes are finely tuned to provide precise occlusal support to facilitate the function of each tooth type. However, the mechanism regulating tooth root patterning and development is largely unknown. In this study, we provide the first in vivo evidence demonstrating that Ezh2 in the dental mesenchyme determines patterning and furcation formation during dental root development in mouse molars. Mechanistically, an antagonistic interaction between epigenetic regulators Ezh2 and Arid1a controls Cdkn2a expression in the dental mesenchyme to regulate dental root patterning and development. These findings indicate the importance of balanced epigenetic regulation in determining the tooth root pattern and the integration of roots with the jaw bones to achieve physiological function. Collectively, our study provides important clues about the regulation of organogenesis and has general implications for tooth regeneration in the future., eLife digest Different teeth have different numbers of roots. Incisors and canines each have one, and molars have two or three. Roots anchor the teeth to the jawbone, and provide a route for blood and nerves to reach the tooth. Getting the shape and number of the roots right during development is important to make sure that each tooth has proper support and function. A protein called Ezh2 helps the bones of the face to develop, but it was not known how it affects how the roots of teeth grow. Teeth form from two layers of tissue; epithelium on the outside and mesenchyme on the inside. Jing et al. have now looked at what happens when Ezh2 is not present in these tissues in the molar teeth of developing mice. The teeth of the mice were affected in different ways depending on which tissue Ezh2 was missing from. When the mesenchyme lacked Ezh2, the roots of the molar teeth did not form properly: the teeth formed too few roots, and the ‘bridge’ region between the roots did not develop correctly. When the epithelium lacked Ezh2, molar teeth formed the correct number of roots but the bridges between the roots developed later than normal. This suggests that signals from the mesenchyme determine how many roots each tooth grows. Further investigation revealed that Ezh2 opposes the activity of another protein called Arid1a, and together they regulate the production of a protein that influences when cells divide. A balanced interaction between Ezh2 and Arid1a is important for tooth root development. This controls how bridges form between tooth roots, and ultimately determines how many roots a tooth grows. Neanderthal teeth show evidence of forming bridges between roots later than modern human teeth, suggesting that similar regulation mechanisms have been important throughout human evolution. In the future, understanding how the roots of teeth form could help researchers to develop ways to regenerate teeth.

Background/Aims: Periapical periodontitis is a common oral disease caused by bacterial invasion of the tooth pulp, which usually leads to local release of pro-inflammatory cytokines and osteolytic lesion. This study is intended to examine the effect of TNF-α on BMP9-induced osteogenic differentiation of the stem cells of dental apical papilla (SCAPs). Methods: Rat model of periapical periodontitis was established. TNF-α expression was assessed. Osteogenic markers and ectopic bone formation in iSCAPs were analyzed upon BMP9 and TNF-α treatment. Results: Periapical periodontitis was successfully established in rat immature permanent teeth with periapical lesions, in which TNF-α was shown to release during the inflammatory phase. BMP9-induced alkaline phosphatase activity, the expression of osteocalcin and osteopontin, and matrix mineralization in iSCAPs were inhibited by TNF-α in a dose-dependent fashion, although increased AdBMP9 partially overcame TNF-α inhibition. Furthermore, high concentration of TNF-α effectively inhibited BMP9-induced ectopic bone formation in vivo. Conclusion: TNF-α plays an important role in periapical bone defect during the inflammatory phase and inhibits BMP9-induced osteoblastic differentiation of iSCAPs, which can be partially reversed by high levels of BMP9. Therefore, BMP9 may be further explored as a potent osteogenic factor to improve osteo/odontogenic differentiation in tooth regeneration in chronic inflammation conditions.

Background The biological events in dentin–pulpf-complex cells have been actively investigated for decades, but the regulation of dentin formation under both physiological and pathological conditions is still unclear. One reason for the limited knowledge of factors that affect dentin formation is the lack of proper in vitro models. Highlight This review summarizes in vitro experiments performed by our group and others. We present the current state of progress made in the field of stem-cell-based odontoblastic differentiation including the mechanism, and the possibility for clinical use of stem cells. Conclusion Mouse embryonic stem cells, mouse induced pluripotent stem cells, and α7 + human skeletal muscle stem cells can differentiate into odontoblast-like cells without an epithelial–mesenchymal transition, and are characterized by the signaling cascade underpinning odontoblastic differentiation, which may be useful for tooth regeneration and dentin sialophosphoprotein (DSPP) studies.

Bio-engineered teeth that can grow and remodel in a manner similar to that of natural teeth have the potential to serve as permanent replacements to the currently used prosthetic teeth, such as dental implants. A major challenge in designing functional bio-engineered teeth is to mimic both the structural and anisotropic mechanical characteristics of the native tooth. Therefore, the field of dental and whole tooth regeneration has advanced towards the molecular and nanoscale design of bio-active, biomimetic systems, using biomaterials, drug delivery systems and stem cells. The focus of this review is to discuss recent advances in tooth tissue engineering, using biomimetic scaffolds that provide proper architectural cues, exhibit the capacity to support dental stem cell proliferation and differentiation and sequester and release bio-active agents, such as growth factors and nucleic acids, in a spatiotemporal controlled manner. Although many in vitro and in vivo studies on tooth regeneration appear promising, before tooth tissue engineering becomes a reality for humans, additional research is needed to perfect methods that use adult human dental stem cells, as opposed to embryonic dental stem cells, and to devise the means to generate bio-engineered teeth of predetermined size and shape. Copyright © 2016 John Wiley & Sons, Ltd.

The presence of nerves is an important factor in successful organ regeneration in amphibians. The Mexican salamander, Ambystoma mexicanum, is able to regenerate limbs, tail, and gills when nerves are present. However, the nerve-dependency of tooth regeneration has not been evaluated. Here, we reevaluated tooth regeneration processes in axolotls using a three-dimensional reconstitution method called CoMBI and found that tooth regeneration is nerve-dependent although the dentary bone is independent of nerve presence. The induction and invagination of the dental lamina were delayed by denervation. Exogenous Fgf2, Fgf8, and Bmp7 expression could induce tooth placodes even in the denervated mandible. Our results suggest that the role of nerves is conserved and that Fgf+Bmp signals play key roles in axolotl organ-level regeneration. The presence of nerves is an important factor in successful organ regeneration in amphibians. The Mexican salamander, Ambystoma mexicanum, is able to regenerate limbs, tail, and gills when nerves are present. However, the nervedependency of tooth regeneration has not been evaluated. Here, we reevaluated tooth regeneration processes in axolotls using a three-dimensional reconstitution method called CoMBI and found that tooth regeneration is nerve-dependent although the dentary bone is independent of nerve presence. The induction and invagination of the dental lamina were delayed by denervation. Exogenous Fgf2, Fgf8, and Bmp7 expression could induce tooth placodes even in the denervated mandible. Our results suggest that the role of nerves is conserved and that Fgf+Bmp signals play key roles in axolotl organ-level regeneration.

Recently, therapeutic strategies for tooth regeneration have shifted to cell-based approaches. Embryologically, teeth are ectodermal organs derived from sequential reciprocal interactions between oral epithelial cells and cranial neural crest–derived mesenchymal cells. Teeth are a unique and complex organ and composed of both hard (dentin and enamel) and soft (pulp and periodontium) tissues. Throughout life, healthy teeth have a number of different types of stem cells that play a key role in the regenerative capacity of this tissue. Several of these stem cell subpopulations have been identified and characterized and include dental pulp stem cells, stem cells from exfoliated deciduous teeth, periodontal ligament stem cells, stem cells from the apical papilla, and dental follicle progenitor cells. This chapter will review the properties of these MSC subpopulations and discuss their potential use in dental pulp and periodontal regenerative procedures.

Tooth tissue engineering through advancements in cell biology and bioengineering has proceeded toward regeneration of entire tooth or individual and surrounding components of tooth. Tooth regenerative therapy is a novel therapeutic concept directing toward restoration of physiological function of tooth such as mastication, periodontal ligament function, and response to noxious stimuli. Tooth regeneration is achieved through two distinctive approaches such as cell transplantation and cell homing. Cell-based strategies are a promising potential for regenerating the whole tooth structure in rodents but rendering obstacles in therapeutics. Cell homing is an under-recognized alternative approach to cell delivery-based tooth regeneration. This approach provides tangible pathway toward clinical translation. Scaffold-based or scaffold-free tissue engineering is considered for tooth regeneration. Scaffold-based approach uses scaffolds planted with cells either in vitro or by cell homing. Scaffold-free approach directly induces development of embryonic tooth formation by appropriate signals to produce tooth structure which mimics natural teeth in morphology and size. The combination of biomaterials and human tooth‐associated with stem cell populations isolated from dental pulp and periodontal ligament tissues shows promising approach to regenerate human dental tissues. Scaffolds provide biophysical support for cell recruitment, adhesion, proliferation, differentiation, and metabolism. The designed scaffolds should be biocompatible, non-toxic, and promote regeneration of single or multiple dental tissues. Different types of biomaterials for constructing scaffolds are available that can regenerate tooth components which successfully improves the treatment outcome.

Continuous growth of the rodent incisor is enabled by epithelial and mesenchymal stem cells (ESCs and MSCs) which unceasingly replenish enamel and dentin, respectively, that wear by persistent animal gnawing. Lineage tracing studies have provided evidence that ESCs contribute to all epithelial lineages of the tooth in vivo. Meanwhile, in the mouse incisor, MSCs continuously contribute to odontoblast lineage and tooth growth. However, in vitro manipulation of ESCs has shown little progress, mainly due to lack of appropriate protocol to successfully isolate, culture, expand, and differentiate ESCs in vitro without using the co-culture system. In this chapter we describe the isolation of the Sox2-GFP+ cell population that is highly enriched in ESCs. Isolated cells can be used for various types of analyses, including in vitro culture, single cell-related analyses, etc. Furthermore, we describe ways to obtain populations enriched in the incisor MSCs using FACS sorting of antibody-labeled cells. Easily accessible FACS sorting enables easy and relatively fast isolation of the cells labeled by the fluorescent protein. © Springer Science+Business Media, LLC, part of Springer Nature 2019.

A preliminary study shows the precedent of quantum regeneration of a dog rsquo s tooth Regeneration was carried out by a special laser technology based on an expanded understanding of the principles of genetic coding One principle predicts the existence of quantum equivalents of working genes This we proved experimentally The multipotent mesenchymal stromal cells MMSCs derived from human adipose tissue were pre treated with human tooth rudiment quantum genetic information MBER The control symmetrical area from the removed tooth was left unchanged After months a complete regeneration of the dog s tooth was observed In the control area there was no regeneration The proposed briefly outlined work is devoted to an attempt to regenerate the dog s tooth using the method of a new direction in biology Linguistic wave genetics The work should be considered a precedent and it will be continued A close experimental work on the regeneration of the diabetic foot was carried out by us recently