The objectives of our study were to investigate the roles of mTORC1 in odontoblast proliferation and mineralization and to determine the mechanism by which mTORC1 regulates odontoblast mineralization. In vitro, MDPC23 cells were treated with rapamycin (10 nmol/L) and transfected with a lentivirus for short hairpin (shRNA)-

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.

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Background Dental pulp stem cells (DPSCs) play a crucial role in dentin-pulp complex regeneration. Further understanding of the mechanism by which DPSCs remain in a quiescent state could contribute to improvements in the dentin-pulp complex and dentinogenesis. Methods TSC1 conditional knockout (DMP1-Cre+; TSC1f/f, hereafter CKO) mice were generated to increase the activity of mechanistic target of rapamycin complex 1 (mTORC1). H&E staining, immunofluorescence and micro-CT analysis were performed with these CKO mice and littermate controls. In vitro, exosomes were collected from the supernatants of MDPC23 cells with different levels of mTORC1 activity and then characterized by transmission electron microscopy and nanoparticle tracking analysis. DPSCs were cocultured with MDPC23 cells and MDPC23 cell-derived exosomes. Alizarin Red S staining, ALP staining, qRT‒PCR, western blotting analysis and micro-RNA sequencing were performed. Results Our study showed that mTORC1 activation in odontoblasts resulted in thicker dentin and higher dentin volume/tooth volume of molars, and it increased the expression levels of the exosome markers CD63 and Alix. In vitro, when DPSCs were cocultured with MDPC23 cells, odontoblastic differentiation was inhibited. However, the inhibition of odontoblastic differentiation was reversed when DPSCs were cocultured with MDPC23 cells with mTORC1 overactivation. To further study the effects of mTORC1 on exosome release from odontoblasts, MDPC23 cells were treated with rapamycin or shRNA-TSC1 to inactivate or activate mTORC1, respectively. The results revealed that exosome release from odontoblasts was negatively correlated with mTORC1 activity. Moreover, exosomes derived from MDPC23 cells with active or inactive mTORC1 inhibited the odontoblastic differentiation of DPSCs at the same concentration. miRNA sequencing analysis of exosomes that were derived from shTSC1-transfected MDPC23 cells, rapamycin-treated MDPC23 cells or nontreated MDPC23 cells revealed that the majority of the miRNAs were similar among these groups. In addition, exosomes derived from odontoblasts inhibited the odontoblastic differentiation of DPSCs, and the inhibitory effect was positively correlated with exosome concentration. Conclusion mTORC1 regulates exosome release from odontoblasts to inhibit the odontoblastic differentiation of DPSCs, but it does not alter exosomal contents. These findings might provide a new understanding of dental pulp complex regeneration.

Human periodontal ligament stem cells (hPDLSCs) are promising cells for dental and periodontal regeneration. Objective This study aimed to develop novel alginate-fibrin fibers that encapsulates hPDLSCs and metformin, to investigate the effect of metformin on the osteogenic differentiation of hPDLSCs, and to determine the regulatory role of the Shh/Gli1 signaling pathway in the metformin-induced osteogenic differentiation of hPDLSCs for the first time. Methodology CCK8 assay was used to evaluate hPDLSCs. Alkaline phosphatase (ALP) staining, alizarin red S staining, and the expression of osteogenic genes were evaluated. Metformin and hPDLSCs were encapsulated in alginate-fibrinogen solutions, which were injected to form alginate-fibrin fibers. The activation of Shh/Gli1 signaling pathway was examined using qRT-PCR and western blot. A mechanistic study was conducted by inhibiting the Shh/Gli1 pathway using GANT61. Results The administration of 50 μM metformin resulted in a significant upregulation of osteogenic gene expression in hPDLSCs by 1.4-fold compared to the osteogenic induction group (P < 0.01), including ALP and runt-related transcription factor-2 (RUNX2). Furthermore, metformin increased ALP activity by 1.7-fold and bone mineral nodule formation by 2.6-fold (P<0.001). We observed that hPDLSCs proliferated with the degradation of alginate-fibrin fibers, and metformin induced their differentiation into the osteogenic lineage. Metformin also promoted the osteogenic differentiation of hPDLSCs by upregulating the Shh/Gli1 signaling pathway by 3- to 6- fold compared to the osteogenic induction group (P<0.001). The osteogenic differentiation ability of hPDLSCs were decreased 1.3- to 1.6-fold when the Shh/Gli1 pathway was inhibited, according to ALP staining and alizarin red S staining (P<0.01). Conclusions Metformin enhanced the osteogenic differentiation of hPDLSCs via the Shh/Gli1 signaling pathway. Degradable alginate-fibrin hydrogel fibers encapsulating hPDLSCs and metformin have significant potential for use in dental and periodontal tissue engineering applications. Clinical Significance Alginate-fibrin fibers encapsulating hPDLSCs and metformin have a great potential for use in the treatment of maxillofacial bone defects caused by trauma, tumors, and tooth extraction. Additionally, they may facilitate the regeneration of periodontal tissue in patients with periodontitis.