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Abstract 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.

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. [ABSTRACT FROM AUTHOR]

Objective: To investigate the biological regulatory function of Gremlin1 (GREM1) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein eta (YWHAH) in dental pulp stem cells (DPSCs), and determine the underlying molecular mechanism involved. Methods: Alkaline phosphatase (ALP) activity, alizarin red staining, scratch migration assays and in vitro and in vivo osteo-/dentinogenic marker detection of bone-like tissue generation in nude mice were used to assess osteo-/dentinogenic differentiation. Coimmunoprecipitation and polypeptide microarray assays were employed to detect the molecular mechanisms involved. Results: The data revealed that knockdown of GREM1 promoted ALP activity, mineralisation in vitro and the expression of osteo-/dentinogenic differentiation markers and enhanced osteo-/ dentinogenesis of DPSCs in vivo. GREM1 bound to YWHAH in DPSCs, and the binding site was also identified. Knockdown of YWHAH suppressed the osteo-/dentinogenesis of DPSCs in vitro, and overexpression of YWHAH promoted the osteo-/dentinogenesis of DPSCs in vitro and in vivo. Conclusion: Taken together, the findings highlight the critical roles of GREM1-YWHAH in the osteo-/dentinogenesis of DPSCs. [ABSTRACT FROM AUTHOR]

Most tooth‐bearing non‐mammalian vertebrates have the capacity to replace their teeth throughout life. This capacity was lost in mammals, which replace their teeth only once at most. Not surprisingly, continuous tooth replacement has attracted much attention. Classical morphological studies (e.g. to analyse patterns of replacement) are now being complemented by molecular studies that investigate the expression of genes involved in tooth formation. This review focuses on ray‐finned fish (actinopterygians), which have teeth often distributed throughout the mouth and pharynx, and more specifically on teleost fish, the largest group of extant vertebrates. First we highlight the diversity in tooth distribution and in tooth replacement patterns. Replacement tooth formation can start from a distinct (usually discontinuous and transient) dental lamina, but also in the absence of a successional lamina, e.g. from the surface epithelium of the oropharynx or from the outer dental epithelium of a predecessor tooth. The relationship of a replacement tooth to its predecessor is closely related to whether replacement is the result of a prepattern or occurs on demand. As replacement teeth do not necessarily have the same molecular signature as first‐generation teeth, the question of the actual trigger for tooth replacement is discussed. Much emphasis has been laid in the past on the potential role of epithelial stem cells in initiating tooth replacement. The outcome of such studies has been equivocal, possibly related to the taxa investigated, and the permanent or transient nature of the dental lamina. Alternatively, replacement may result from local proliferation of undifferentiated progenitors, stimulated by hitherto unknown, perhaps mesenchymal, factors. So far, the role of the neurovascular link in continuous tooth replacement has been poorly investigated, despite the presence of a rich vascularisation surrounding actinopterygian (as well as chondrichthyan) teeth and despite a complete arrest of tooth replacement after nerve resection. Lastly, tooth replacement is possibly co‐opted as a process to expand the number of teeth in a dentition ontogenetically whilst conserving features of the primary dentition. That neither a dental lamina, nor stem cells appear to be required for tooth replacement places teleosts in an advantageous position as models for tooth regeneration in humans, where the dental lamina regresses and epithelial stem cells are considered lost. [ABSTRACT FROM AUTHOR]

Research on tooth regeneration using human-induced pluripotent stem cells (hiPSCs) is valuable for autologous dental regeneration. Acquiring mesenchymal and epithelial cells as a resource for dental regeneration is necessary because mesenchymal–epithelial interactions play an essential role in dental development. We reported the establishment of hiPSCs-derived dental epithelial-like cell (EPI-iPSCs), but hiPSCs-derived dental mesenchymal stem cells (MSCs) have not yet been reported. This study was conducted to establish hiPSCs-derived MSCs and to differentiate them into dental cells with EPI-iPSCs. Considering that dental MSCs are derived from the neural crest, hiPSCs were induced to differentiate into MSCs through neural crest formation to acquire the properties of dental MSCs. To differentiate hiPSCs into MSCs through neural crest formation, established hiPSCs were cultured and differentiated with PA6 stromal cells and differentiated hiPSCs formed neurospheres on ultralow-attachment plates. Neurospheres were differentiated into MSCs in serum-supplemented medium. Neural crest-mediated MSCs (NC-MSCs) continuously showed typical MSC morphology and expressed MSC markers. After 8 days of odontogenic induction, the expression levels of odontogenic/mineralization-related genes and dentin sialophosphoprotein (DSPP) proteins were increased in the NC-MSCs alone group in the absence of coculturing with dental epithelial cells. The NC-MSCs and EPI-iPSCs coculture groups showed high expression levels of amelogenesis/odontogenic/mineralization-related genes and DSPP proteins. Furthermore, the NC-MSCs and EPI-iPSCs coculture group yielded calcium deposits earlier than the NC-MSCs alone group. These results indicated that established NC-MSCs from hiPSCs have dental differentiation capacity with dental epithelial cells. In addition, it was confirmed that hiPSCs-derived dental stem cells could be a novel cell source for autologous dental regeneration. [ABSTRACT FROM AUTHOR]

Tooth caries and loss are frequent clinical diseases in dentistry. Tissue engineering is a new therapeutic choice for the complete biological regeneration of pulpal and dental tissues in regenerative dentistry. The aim of this study was to establish a protocol for in situ regeneration of a dental bud in the extracted socket. The current study examined tooth bud regeneration with dental pulp stem cells induced by a dentin derivative signal in a rabbit's jaw. A tooth bud was regenerated; the morphology and structure of it were typical, and it was post–Bell stage. In our study, a real tooth bud was formed in the post–Bell stage with complete morphologic and biological features. However, the application of this method for tooth regeneration in humans necessitates further research. [ABSTRACT FROM AUTHOR]

Introduction: Although there have been many studies on stem cells, few have investigated how neurotransmitters and stem cell proliferation interact to regenerate dental pulp. Dental pulp regeneration is an innovative procedure for reviving dental pulp, if feasible for the entire tooth. Upon tooth injury, activated platelets release serotonin and dopamine in bulk to mobilize dental pulp stem cells to mediate natural dental repair. This has induced research on the role of neurotransmitters in increasing the proliferation rate of stem cells. This review also covers prospective future treatments for dental pulp regeneration. Methods: A literature search was performed via PubMed and ScienceDirect from 2001 to 2022, using the keywords “neurotransmitter,” “stem cell,” “tooth regeneration,” “tooth repair,” “regenerative dentistry,” and “dental pulp.” Different inclusion/exclusion criteria were used, and the search was restricted to English articles. Results: Nine publications reporting neurotransmitter interactions with stem cells for tooth and pulp regeneration were selected. Conclusion: Neurotransmitters were found to interact with dental stem cells. Evidence pointing to neurotransmitters as a factor in the increased proliferation of stem cells was found. This review thus gives hope for tooth pulp regeneration and repair.

Abstract Background Human dental pulp stem cells (hDPSCs) may be the best choice for self-repair and regeneration of teeth and maxillofacial bone tissue due to their homogeneous tissue origin, high proliferation and differentiation rates, and no obvious ethical restrictions. Recently, several studies have shown that extracellular matrix (ECM) proteins can effectively regulate the proliferation and differentiation fate of mesenchymal stem cells (MSCs). However, the role of elastin microfibril interface-located protein-1 (EMILIN-1), a new ECM glycoprotein, in osteo/odontogenic differentiation of hDPSCs has not been reported. The aim of this study was to explore the effect of EMILIN-1 during osteo/odontogenic differentiation of hDPSCs. Methods hDPSCs were cultured in osteo/odontogenic induction medium. qPCR and Western blot analysis were performed to detect osteo/odonto-specific genes/proteins expression as well as the expression of EMILIN-1. After knockdown of Emilin-1 in hDPSCs with small interfering RNA and exogenous addition of recombinant human EMILIN-1 protein (rhEMILIN-1), Cell Counting Kit-8 assay, alkaline phosphatase staining, alizarin red S staining, qPCR and Western blot were performed to examine the effect of EMILIN-1 on proliferation and osteo/odontogenic differentiation of hDPSCs. Results During the osteo/odontogenic induction of hDPSCs, the expression of osteo/odonto-specific genes/proteins increased, as did EMILIN-1 protein levels. More notably, knockdown of Emilin-1 decreased hDPSCs proliferation and osteo/odontogenic differentiation, whereas exogenous addition of rhEMILIN-1 increased them. Conclusions These findings suggested that EMILIN-1 is essential for the osteo/odontogenic differentiation of hDPSCs, which may provide new insights for teeth and bone tissue regeneration.

The lack of treatment options for congenital (0.1%) and partial (10%) tooth anomalies highlights the need to develop innovative strategies. Over two decades of dedicated research have led to breakthroughs in the treatment of congenital and acquired tooth loss. We revealed that by inactivating USAG-1, congenital tooth agenesis can be successfully ameliorated during early tooth development and that the inactivation promotes late-stage tooth morphogenesis in double knockout mice. Furthermore, Anti- USAG-1 antibody treatment in mice is effective in tooth regeneration and can be a breakthrough in treating tooth anomalies in humans. With approximately 0.1% of the population suffering from congenital tooth agenesis and 10% of children worldwide suffering from partial tooth loss, early diagnosis will improve outcomes and the quality of life of patients. Understanding the role of pathogenic USAG-1 variants, their interacting gene partners, and their protein functions will help develop critical biomarkers. Advances in next-generation sequencing, mass spectrometry, and imaging technologies will assist in developing companion and predictive biomarkers to help identify patients who will benefit from tooth regeneration.

Although there have been many studies on stem cells, few have investigated how neurotransmitters and stem cell proliferation interact to regenerate dental pulp. Dental pulp regeneration is an innovative procedure for reviving dental pulp, if feasible for the entire tooth. Upon tooth injury, activated platelets release serotonin and dopamine in bulk to mobilize dental pulp stem cells to mediate natural dental repair. This has induced research on the role of neurotransmitters in increasing the proliferation rate of stem cells. This review also covers prospective future treatments for dental pulp regeneration. A literature search was performed via PubMed and ScienceDirect from 2001 to 2022, using the keywords "neurotransmitter," "stem cell," "tooth regeneration," "tooth repair," "regenerative dentistry," and "dental pulp." Different inclusion/exclusion criteria were used, and the search was restricted to English articles. Nine publications reporting neurotransmitter interactions with stem cells for tooth and pulp regeneration were selected. Neurotransmitters were found to interact with dental stem cells. Evidence pointing to neurotransmitters as a factor in the increased proliferation of stem cells was found. This review thus gives hope for tooth pulp regeneration and repair. [ABSTRACT FROM AUTHOR]

The odontogenic differentiation of dental pulp stem cells (DPSCs), which is vital for tooth regeneration, was regulated by various functional molecules. In recent years, a growing body of research has shown that miRNAs play a crucial role in the odontogenic differentiation of human dental pulp stem cells (hDPSCs). However, the mechanisms by which miRNAs regulated odontogenic differentiation of hDPSCs remained unclear, and the application of miRNAs in reparative dentin formation in vivo was also rare. In this study, we first discovered that miR-3074-3p had an inhibitory effect on odontogenic differentiation of hDPSCs and antagomiR-3074-3p-conjugated PEI-AuNPs effectively promoted odontogenic differentiation of hDPSCs in vitro. AntagomiR-3074-3p-conjugated PEI-AuNPs was further applied to the rat pulp-capping model and showed the increased formation of restorative dentin. In addition, the results of lentivirus transfection in vitro suggested that FKBP9 acted as the key target of miR-3074-3p in regulating the odontogenic differentiation of hDPSCs. These findings might provide a new strategy and candidate target for dentin restoration and tooth regeneration. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Since 1967, when the osteoinduction properties of autogenous demineralized dentin matrix were discovered, autologous tooth grafts have been advocated as a viable option to autologous or heterologous bone graft. Tooth graft materials may be extracted from the patient’s whole tooth using a granulating device. The aim of this study was to examine the size of granules obtained by the Tooth Transformer (TT)® device, using a laser instrument with high precision. MATERIALS AND METHODS: The TT® device can obtain bone graft material in a short period from an extracted tooth. The resulting material can act as an osteoconductive scaffold, providing a mineral substrate during resorption, including platelet growth factors and morphogenetic proteins. Different studies have investigated the dimension and behavior of various graft material particles, since the size of the grafted particles may play a role in osteogenesis and bone regeneration. RESULTS: Different dimensions of granules are available: small (< 400 µm), medium (400 µm1,000 µm) and large (1,000 µm-2,000 µm). From 4.03 µm to 100 µm the percentage of granules was 14.52 ± 1.93%. A larger part of the granules was up to 100 µm, while 85.47 ± 1.93% of the granules were from 100 µm to 1,000 µm. CONCLUSIONS: 85% of the granules produced were in accordance with the dimensions suggested in the literature. [ABSTRACT FROM AUTHOR]

Background: Human dental pulp stem cells (hDPSCs) may be the best choice for self-repair and regeneration of teeth and maxillofacial bone tissue due to their homogeneous tissue origin, high proliferation and differentiation rates, and no obvious ethical restrictions. Recently, several studies have shown that extracellular matrix (ECM) proteins can effectively regulate the proliferation and differentiation fate of mesenchymal stem cells (MSCs). However, the role of elastin microfibril interface-located protein-1 (EMILIN-1), a new ECM glycoprotein, in osteo/odontogenic differentiation of hDPSCs has not been reported. The aim of this study was to explore the effect of EMILIN-1 during osteo/odontogenic differentiation of hDPSCs. Methods: hDPSCs were cultured in osteo/odontogenic induction medium. qPCR and Western blot analysis were performed to detect osteo/odonto-specific genes/proteins expression as well as the expression of EMILIN-1. After knockdown of Emilin-1 in hDPSCs with small interfering RNA and exogenous addition of recombinant human EMILIN-1 protein (rhEMILIN-1), Cell Counting Kit-8 assay, alkaline phosphatase staining, alizarin red S staining, qPCR and Western blot were performed to examine the effect of EMILIN-1 on proliferation and osteo/odontogenic differentiation of hDPSCs. Results: During the osteo/odontogenic induction of hDPSCs, the expression of osteo/odonto-specific genes/proteins increased, as did EMILIN-1 protein levels. More notably, knockdown of Emilin-1 decreased hDPSCs proliferation and osteo/odontogenic differentiation, whereas exogenous addition of rhEMILIN-1 increased them. Conclusions: These findings suggested that EMILIN-1 is essential for the osteo/odontogenic differentiation of hDPSCs, which may provide new insights for teeth and bone tissue regeneration. [ABSTRACT FROM AUTHOR]

Tooth defect and loss are common clinical diseases in stomatology. With the extension of life expectancy, there is an increasing demand for tooth tissue and whole tooth regeneration. Compared with traditional oral prosthetic treatment, tooth regeneration has unique advantages and has become one of the hotspots towards oral biomedical treatment. In this review, we discussed the development and challenges of tooth regeneration based on cells and materials, including tooth enamel, dentin, dental pulp, cementum, dentin-pulp complex, and the whole tooth regeneration, in order to provide a comprehensive, up-to-date, illustrative overview of tooth regeneration issues. The mechanisms of the regeneration were also summarized and discussed. Moreover, this review hints the future perspective and research direction of tooth regeneration in the challenging field of regenerative dentistry.

Psoralea corylifolia L. (P. corylifolia) has been used as an oriental phytomedicine to treat coldness of hands and feet in bone marrow injury. Hydroxyapatite is usually used for tooth regeneration. In this study, the role of P. corylifolia and bakuchiol, a compound originated from P. corylifolia as differentiation-inducing substances for tooth regeneration, was determined by monitoring odontogenic differentiation in human dental pulp stem cells (hDPSCs). We confirmed that P. corylifolia extracts and bakuchiol increased the odontogenic differentiation of hDPSCs. In addition, the expression of the odontogenic differentiation marker genes alkaline phosphatase (APL), Runt-related transcription factor 2 (RUNX-2), osteocalcin (OC), and dentin matrix acidic phosphoprotein-1 (DMP-1) was proved by real-time polymerase chain reaction, and protein expression of dentin matrix acidic phosphoprotein-1 (DMP-1) and dentin sialophosphoprotein (DSPP) was proved by western blotting. Further, by confirming the increase in small mothers against decapentaplegia (SMAD) 1/5/8 phosphorylation, the SMAD signaling pathway was found to increase the differentiation of odontoblasts. This study confirmed that P. corylifolia L. extracts and bakuchiol alone promote odontogenic differentiation in hDPSCs. These results suggest that bakuchiol from P. corylifolia is responsible for odontogenic differentiation, and they encourage future in vivo studies on dentin regeneration.

The development of direct pulp-capping materials with favorable biological and structural properties is an important goal in restorative dentistry. Fucoidan is a sulfated, fucose-containing polysaccharide obtained from brown seaweed, with a wide range of applications; however, its use as a direct pulp-capping material has not been examined. This study aimed to evaluate the mechanical, physical, and biological effects of fucoidan combined with conventional mineral trioxide aggregate (MTA) for direct pulp capping. The capping materials were created using Portland cement (80 wt%) and zirconium oxide (20 wt%) as base components, compared with base components plus 5 wt% fucoidan (PZF5) and base components plus 10 wt% fucoidan (PZF10). The initial and final setting time, compressive strength, chemical components, cell viability, adhesion, migration, osteogenesis, and gene expression were analyzed. Fucoidan significantly reduced the initial and final setting time, regardless of quantity. However, the compressive strength was lower for PZF5. Sulfur levels increased with fucoidan. The biological activity improved, especially in the PZF5 group. Cell migration, Alizarin Red S staining, and alkaline phosphatase activity were upregulated in the PZF5 group. Fucoidan is a useful regenerative additive for conventional pulp-capping materials because it reduces the setting time and improves cell migration and osteogenic ability. [ABSTRACT FROM AUTHOR]

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–E17.0 by promoting cell proliferation. In addition, mRNA‐seq, reverse transcription plus polymerase chain reaction (RT‐qPCR), and immunohistochemical analyses indicated that diphyodontic dentition formation might be related to FGF signal up‐regulation and the Sostdc1‐Wnt negative feedback loop. Conclusions: 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. Key Findings: RSDL, Shh signal, tooth regeneration, Wnt/β‐catenin, mouse molar. [ABSTRACT FROM AUTHOR]

Abstract Background Vertebrate 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. Results In 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, and pitx2), while active Wnt signaling and Lef1 expression 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, and Pitx2) 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 express Lef1 and 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. Conclusions We 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.

Abstract 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.

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. [ABSTRACT FROM AUTHOR]

Tooth is a vital organ in the oral cavity that has functions for chewing, talking, swallowing, and giving facial aesthetics. Tooth decay or loss can reduce the quality of life (QOL). Nowadays, tooth decay treatment is restored through a conservative approach, which uses dental inorganic materials and their supporting tools. Besides, the loss of a tooth can be replaced by a dental prosthetic or artificial tooth. The advanced and greatly promising technology in the future is to apply the concept of regenerative dentistry as a biological repair of lost and damaged tissue using stem cells. Regenerative medicine using stem cells is promising therapeutic tool in the future to restore or replace the damage tissue into normal function by utilized their potency to differentiate into specialized cells type. This review aims to report the potential of stem cells for tooth regeneration and tissue repair in dental field. [ABSTRACT FROM AUTHOR]

Abstract 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.

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.

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.

Tooth defects are an extremely common health condition that affects millions of individuals. Currently used dental repair treatments include fillings for caries, endodontic treatment for pulp necrosis, and dental implants to replace missing teeth, all of which rely on the use of synthetic materials. By contrast, the fields of tissue engineering and regenerative medicine and dentistry (TERMD) use biologically based therapeutic strategies for vital tissue regeneration, and thus have the potential to regenerate living tissues. Methods to create bioengineered replacement teeth benefit from a detailed understanding of the molecular signaling networks regulating natural tooth development. We discuss how key signaling pathways regulating natural tooth development are being exploited for applications in TERMD approaches for vital tooth regeneration. The importance of oral health, and its essential link to systemic health, is a serious health concern worldwide. Current therapies used to repair craniomaxillofacial (CMF) defects largely rely on the use of synthetic materials that lack many essential features of natural CMF tissues. The ability to regenerate living, hard and soft dental tissues to repair dental tissues damaged by disease, trauma, genetic, and other disorders would be a significant improvement over the currently used methods. Recent progress in the TERMD field has helped to devise more effective methods to regenerate vital dental tissues. Novel TERMD therapies are anticipated to significantly improve our ability to effectively repair CMF tissues. These new therapies are anticipated to have a significant impact on the dental therapeutics market. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

Background: Vertebrate 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. Results: In 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, and pitx2), while active Wnt signaling and Lef1 expression 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, and Pitx2) 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 express Lef1 and 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. Conclusions: We 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

Abstract Background Hertwig’s epithelial root sheath (HERS) is important in guiding tooth root formation by differentiating into cementoblasts through epithelial–mesenchymal transition (EMT) and inducing odontoblastic differentiation of dental papilla through epithelial–mesenchymal interaction (EMI) during the tooth root development. Thus, HERS cells are critical for cementum and dentin formation and might be a potential cell source to achieve tooth root regeneration. However, limited availability and lifespan of primary HERS cells may represent an obstacle for biological investigation and therapeutic use of tooth tissue engineering. Therefore, we constructed, characterized, and tested the functionality of immortalized cell lines in order to produce a more readily available alternative to HERS cells. Methods Primary HERS cells were immortalized via infection with lentivirus vector containing the gene encoding simian virus 40 Large T Antigen (SV40LT). Immortalized HERS cell subclones were isolated using a limiting dilution method, and subclones named HERS-H1 and HERS-C2 cells were isolated. The characteristics of HERS-H1 and HERS-C2 cells, including cell proliferation, ability of epithelial–mesenchymal transformation and epithelial–mesenchymal interaction, were determined by CCK-8 assay, immunofluorescence staining, and real-time PCR. The cell differentiation into cementoblast-like cells or periodontal fibroblast-like cells was confirmed in vivo. And the inductive influence of the cell lines on dental papilla cells (DPCs) was also confirmed in vivo. Results HERS-H1 and HERS-C2 cells share some common features with primary HERS cells such as epithelial-like morphology, positive expression of CK14, E-Cadherin, and Vimentin, and undergoing EMT in response to TGF-beta. HERS-C2 cells showed the EMT characteristics and could differentiate into cementum-forming cells in vitro and generate cementum-like tissue in vivo. HERS-H1 could induce the differentiation of DPCs into odontoblasts in vitro and generation of dentin-like tissue in vivo. Conclusions We successfully isolated and characterized novel cell lines representing two key features of HERS cells during the tooth root development and which were useful substitutes for primary HERS cells, thereby providing a biologically relevant, unlimited cell source for studies on cell biology, developmental biology, and tooth root regeneration.

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. [ABSTRACT FROM AUTHOR]

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Tooth defect and tooth loss are common clinical diseases in stomatology. Compared with the traditional oral restoration treatment, tooth regeneration has unique advantages and is currently the focus of oral biomedical research. It is known that dozens of cytokines/growth factors and other bioactive factors are expressed in a spatial-temporal pattern during tooth development. On the other hand, the technology for spatial-temporal control of drug release has been intensively studied and well developed recently, making control release of these bioactive factors mimicking spatial-temporal pattern more feasible than ever for the purpose of tooth regeneration. This article reviews the research progress on the tooth development and discusses the future of tooth regeneration in the context of spatial-temporal release of developmental factors. [ABSTRACT FROM AUTHOR]

Tooth regeneration is an important issue. The purpose of this study was to explore the feasibility of using adult dental pulp stem cells on polylactic acid scaffolds for tooth regeneration. Three teeth were extracted from each side of the lower jaws of two adult dogs. In the experimental group, dental pulp stem cells were isolated and seeded in the 3D-printed hydroxyapatite/polylactic acid (HA/PLA) scaffolds for transplantation into left lower jaw of each dog. The right-side jaw of each dog was transplanted with cell-free scaffolds as the control group. Polychrome sequentially labeling was performed for observation of mineralization. Dental cone beam computed tomography (CBCT) irradiation was used for assessment. Nine months after surgery, dogs were euthanized, and the lower jaws of dogs were sectioned and fixed for histological observation with hematoxylin and eosin staining. The results showed that the degree of mineralization in the experimental group with cells seeded in the scaffolds was significantly higher than that of the control group transplanted with cell-free scaffolds. However, the HA/PLA scaffolds were not completely absorbed in both groups. It is concluded that dental pulp stem cells are important for the mineralization of tooth regeneration. A more rapid absorbable material was required for scaffold design for tooth regeneration.

Then, like much of the world, International Association for Dental Research (IADR) paused in-person meetings. Craniofacial anomalies, craniofacial biology/genetics, stem cell(s), morphogenesis, tooth regeneration Keywords: craniofacial anomalies; craniofacial biology/genetics; stem cell(s); morphogenesis; tooth regeneration EN craniofacial anomalies craniofacial biology/genetics stem cell(s) morphogenesis tooth regeneration 1078 1079 2 09/01/23 20230901 NES 230901 Thank you, President O'Connell, for the kind introduction. [Extracted from the article]

Abstract Background A number of studies have shown that tooth-like structures can be regenerated using induced pluripotent stem cells and mouse embryonic stem (mES) cells. However, few studies have reported the regeneration of tooth–periodontium complex structures, which are more suitable for clinical tooth transplantation. We established an optimized approach to induce high-odontogenic potential dental epithelium derived from mES cells by temporally controlling bone morphogenic protein 4 (BMP4) function and regenerated tooth–periodontium complex structures in vivo. Methods First, immunofluorescence and quantitative reverse transcription-polymerase chain reaction were used to identify the watershed of skin and the oral ectoderm. LDN193189 was then used to inhibit the BMP4 receptor around the watershed, followed by the addition of exogenous BMP4 to promote BMP4 function. The generated dental epithelium was confirmed by western blot analysis and immunofluorescence. The generated epithelium was ultimately combined with embryonic day 14.5 mouse mesenchyme and transplanted into the renal capsules of nude mice. After 4 weeks, the tooth–periodontium complex structure was examined by micro-computed tomography (CT) and hematoxylin and eosin (H&E) staining. Results Our study found that the turning point of oral ectoderm differentiation occurred around day 3 after the embryoid body was transferred to a common culture plate. Ameloblastin-positive dental epithelial cells were detected following the temporal regulation of BMP4. Tooth–periodontium complex structures, which included teeth, a periodontal membrane, and alveolar bone, were formed when this epithelium was combined with mouse dental mesenchyme and transplanted into the renal capsules of nude mice. Micro-CT and H&E staining revealed that the generated tooth–periodontium complex structures shared a similar histological structure with normal mouse teeth. Conclusions An optimized induction method was established to promote the differentiation of mES cells into dental epithelium by temporally controlling the function of BMP4. A novel tooth–periodontium complex structure was generated using the epithelium.

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. [ABSTRACT FROM AUTHOR]

Regenerating the periodontal ligament (PDL) is a crucial factor for periodontal tissue regeneration in the presence of traumatized and periodontally damaged teeth. Various methods have been applied for periodontal regeneration, including tissue substitutes, bioactive materials, and synthetic scaffolds. However, all of these treatments have had limited success in structural and functional periodontal tissue regeneration. To achieve the goal of complete periodontal regeneration, many studies have evaluated the effectiveness of decellularized scaffolds fabricated via tissue engineering. The aim of this study was to fabricate a decellularized periodontal scaffold of human tooth slices and determine its regeneration potential. We evaluated two different protocols applied to tooth slices obtained from human healthy third molars. The extracellular matrix scaffold decellularized using sodium dodecyl sulfate and Triton X-100, which are effective in removing nuclear components, was demonstrated to preserve an intact structure and composition. Furthermore, the decellularized scaffold could support repopulation of PDL stem cells near the cementum and expressed cementum and periodontal-ligament-related genes. These results show that decellularized PDL scaffolds of human teeth are capable of inducing the proliferation and differentiation of mesenchymal stem cells, thus having regeneration potential for use in future periodontal regenerative tissue engineering. [ABSTRACT FROM AUTHOR]

The tooth and its supporting tissues are organized with complex three‐dimensional (3D) architecture, including the dental pulp with a blood supply and nerve tissues, complex multilayer periodontium, and highly aligned periodontal ligament (PDL). Mimicking such 3D complexity and the multicellular interactions naturally existing in dental structures represents great challenges in dental regeneration. Attempts to construct the complex system of the tooth and tooth‐supporting apparatus (i.e., the PDL, alveolar bone, and cementum) have made certain progress owing to 3D printing biotechnology. Recent advances have enabled the 3D printing of biocompatible materials, seed cells, and supporting components into complex 3D functional living tissue. Furthermore, 3D bioprinting is driving major innovations in regenerative medicine, giving the field of regenerative dentistry a boost. The fabrication of scaffolds via 3D printing is already being performed extensively at the laboratory bench and in clinical trials; however, printing living cells and matrix materials together to produce tissue constructs by 3D bioprinting remains limited to the regeneration of dental pulp and the tooth germ. This review summarizes the application of scaffolds for cell seeding and biofabricated tissues via 3D printing and bioprinting, respectively, in the tooth and its supporting tissues. Additionally, the key advantages and prospects of 3D bioprinting in regenerative dentistry are highlighted, providing new ideas for dental regeneration. Complex three‐dimensional architectures, multilayer and multicellular interactions are naturally existing in tooth and its supporting tissues. 3D printing biotechnology is driving major innovations in regenerative dentistry. In this work, Ma and coworkers summarizes current application of 3D printing/bioprinting scaffolds for cell seeding and biofabricated modules in dental tissues, and highlight the key advantages and prospects of 3D bioprinting in regenerative dentistry, which provide new ideas for dental regeneration. [ABSTRACT FROM AUTHOR]

Copyright of West China Journal of Stomatology is the property of Sichuan University, West China College of Stomatology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Recent studies have successfully generated tooth-like structure by mimicking the reciprocal interaction between dental epithelial and mesenchymal cells in tooth organogenesis. However, clinical applications of these methods are limited primarily due to the lack of appropriate sources for dental epithelial cells. Induced pluripotent stem cells (iPSCs) are attractive as a source for dental epithelial cells due to their unique characteristics. In this study, we examined the effect of neurotrophin-4 (NT-4) on the differentiation of mouse iPSCs (miPSCs) into dental epithelial cells. Our results showed that the addition of NT-4 during the formation of embryoid body significantly triggered the upregulation of epithelial markers such as p63 and CK14, suggesting that NT-4 provides an inductive condition for the differentiation of miPSCs into epithelial cells. Expansion of the NT-4-treated cells under serum-free culture conditions improves the formation of cells with cobblestone-like morphology and significantly downregulated the expression of pluripotent and ectodermal markers. Phenotypic analysis revealed that a dental epithelial surface marker, CD49f, was highly expressed on these cells. Formation of miPSCs-derived dental epithelial-like cells was further confirmed by the expression of ameloblast-specific markers. These results suggest that the addition of NT-4 during the formation of embryoid body together with the serum-free culture condition promoted the differentiation of miPSCs into dental epithelial-like cells. [ABSTRACT FROM AUTHOR]

Stem cells are undifferentiated cells that can differentiate into specialized cells. Recently, enormous growth has been seen in the recognition of stem cell-based therapies, which have the potential to ameliorate the life of patients with conditions that span from Parkinson's disease to cardiac ischemia to bone or tooth loss. This research has produced new but unexplored possibilities in the regeneration of different organs and tissues. Presently, research is focused on the proficiency of stem cells and their utilization in dentistry, which is gaining interest. The tooth is nature's “esteem” for these precious stem cells and there are a number of these cells in permanent and primary teeth, as well as in the wisdom teeth. Dental stem cells are easy, convenient, and affordable to collect. They hold promise for a range of very potential therapeutic applications, such as in the treatment of cancer, spinal cord injury, brain damage, myocardial infarction, hearing loss, diabetes, wound healing, baldness, etc. Since these cells were used to regenerate damaged tissue in medical therapy successfully, it is possible that the dentist in future might use stem cell to regenerate lost or damaged dental and periodontal structures. This paper reviews the current concepts, characteristics of stem cells in regeneration, and its subsequent uses in dentistry.

Over the last several decades, a wealth of information has become available regarding various sources of stem cells and their potential use for regenerative purposes. Given the intense debate regarding embryonic stem cells, much of the focus has centered around application of adult stem cells for regenerative engineering along with other relevant aspects including use of growth factors and scaffolding materials. The more recent discovery of tooth-derived stem cells has sparked much interest in their application to regenerative dentistry to treat and alleviate the most prevalent oral diseases—i.e., dental caries and periodontal diseases. Also exciting is the advent of induced pluripotent stem cells, which provides the means of using patient-derived somatic cells for their creation, and their eventual application for generation of the dental complex. Thus, evolving developments in the field of regenerative dentistry indicate the prospect of constructing "custom-made" tooth and supporting structures thereby fostering the realization of "personalized dentistry." On the other hand, others have explored the possibility of augmenting endogenous regenerative capacity through utilization of small molecules to regulate molecular signaling mechanisms that mediate regeneration of tooth structure. This review is focused on these aspects of regenerative dentistry in view of their relevance to personalized dentistry. [ABSTRACT FROM AUTHOR]

Traumatic injuries, genetic diseases, and external harmful agents such as bacteria and acids often compromise tooth integrity. There is an unmet medical need to develop alternative, innovative dental treatments that complement traditional restorative and surgery techniques. Stem cells have transformed the medical field in recent years. The combination of stem cells with bioactive scaffolds and nanostructured materials turns out to be increasingly beneficial in regenerative dental medicine. Stem cell-based regenerative approaches for the formation of dental tissues will significantly improve treatments and will have a major impact in dental practice. To date there is no established and reliable stem cell-based treatment translated into the dental clinics, however, the advances and improved technological knowledge are promising for successful dental therapies in the near future. Here, we review some of the contemporary challenges in dental medicine and describe the benefits and future possibilities of certain novel approaches in the emerging field of regenerative dentistry. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Currently regeneration of tooth and periodontal damage still remains great challenge. Stem cell‐based tissue engineering raised novel therapeutic strategies for tooth and periodontal repair. Stem cells for tooth and periodontal regeneration include dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs), stem cells from the dental apical papilla (SCAPs), and stem cells from human exfoliated deciduous teeth (SHEDs), dental follicle stem cells (DFSCs), dental epithelial stem cells (DESCs), bone marrow mesenchymal stem cells (BMMSCs), adipose‐derived stem cells (ADSCs), embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). To date, substantial advances have been made in stem cell‐based tooth and periodontal regeneration, including dentin–pulp, whole tooth, bioroot and periodontal regeneration. Translational investigations have been performed such as dental stem cell banking and clinical trials. In this review, we present strategies for stem cell‐based tissue engineering for tooth and periodontal repair, and the translational studies. [ABSTRACT FROM AUTHOR]