| Background:Progress in the development of novel biomaterials and advances in our understanding of pulp biology,vital pulp therapy(VPT)and regenerative endodontic procedures(REPs)have been proposed for treatment of dental pulp diseases.The purpose of VPT and REPs is to preserve pulp vitality and regenerate a new pulp-dentin complex.Repair procedures are based on regulating cell fate,including their proliferation,migration,and differentiation.Within the pulp it has been shown that several cell populations participate in tissue regeneration.Dental pulp stem cells(DPSCs)were the first stem cells to be isolated and identified from the oral cavity.DPSCs display multilineage differentiation capability,differentiating into odontoblast-like cells and undertaking dentin formation after stimulation.Therefore,regulating the behavior of DPSCs is crucial for REPs and VPT.Growth factors(GFs)are directly involved in modulating cellular events via the induction of specific intracellular signaling pathways.Transforming growth factor(TGF)-β1 is a ubiquitous multifunctional growth factor stored as a latent complex in the extracellular matrix(ECM).TGF-β1 can be activated following caries or pulpitis.TGF-β1is also embedded in the dentin matrix and can be released under specific conditions to direct cellular activity.In addition,given that exogenous TGF-β1 exhibits the same biological effect as its endogenous counterpart,it can be combined with biomaterials to control cell fate by direct pulp capping or transplantation into root canals.In summary,elucidating how TGF-β1 regulates DPSC differentiation could help improve RET.Based on results obtained from recent studies,the effect of TGF-β1 on dentinogenesis and mineralization remains controversial and the reason is not clear.Furthermore,as TGF-β1can activate multiple signalling pathways,its functional differences may be related to the interactions and joint activities of these intracellular signalling networks,but the specific regulatory mechanisms are not fully understood.This study preliminarily explores the role of TGF-β1 in differentiation of DPSCs and the mechanisms,and the elucidation of these scientific questions has important implications for the application of TGF-β1 in clinical regenerative therapy.Aim:The aim of the study is to identify the effect of TGF-β1 on the DPSCs odontogenic differentiation in vitro and vivo and explore the molecular mechanism of the procedure.Methods:DPSCs were isolated and cultured by enzyme digestion method and limited dilution method.After stimulating the cells with a range concentration of TGF-β1,the changes of cell proliferation,cycle and apoptosis were observed by using CCK8 and flow cytometry.ALP assay,alizarin red staining and Western blot were used to observe DPSCs differentiation at different stages.Meanwhile,H&E staining,Masson staining and Von Kossa staining were used to evaluate the effect of TGF-β1 on the tissue regeneration ability of DPSCs in vivo.To investigate the mechanism of action of TGF-β1,Western blot assays were used to determine which signalling pathways were activated by TGF-β1,and cells were treated with pathway-specific inhibitors to block the pathway inhibitors.The functions of the pathway were determined by changes in ALP,alizarin red staining and protein expression at different stages of differentiation.Co-application of TSA to confirm whether TGF-β1inhibits the differentiation of DPSCs at late stages through deacetylation.Western blot and immunofluorescence staining were used to determine the changes in HDAC5 expression after TGF-β1 stimulation of cells in vitro,and the spatial and temporal changes in TGF-β1and HDAC5 expression were observed in vivo using a rat pulp injury-repair model,followed by lentiviral transfection of sh RNA to determine the regulatory function of HDAC5.Finally,western blot,Co-IP and immunofluorescence staining were used in combination to determine whether TGF-β1 influences the transcriptional activity of Runx2 and thus the late differentiation of DPSCs through the regulation of HDAC5 by Smad3.Results:1.TGF-β1 promotes early dentinogenic differentiation and inhibits late differentiation of DPSCs.In vitro experiments showed that TGF-β1 inhibited the proliferation of DPSCs in a concentration-and time-dependent manner.Cycle and apoptosis assays showed that TGF-β1 inhibited cell proliferation by blocking the cycle at the G1/G0 phase,without affecting apoptosis.The results of differentiation assay showed that TGF-β1 enhanced ALP activity and inhibited alizarin red staining in a concentration-dependent manner.While levels of COL1 A,DMP-1 and BSP were significantly increased in the early stages of differentiation by TGF-β1,while expression of DSPP and OCN were decreased.In vivo experiments showed that compared to the control group the TGF-β1-combined group produced a large amount of collagen,but no hard tissue was produced.2.TGF-β1 promotes adult dentin differentiation through the AKT pathway,the P38 pathway and the Erk1/2 pathway,while the Smad3 pathway inhibits the mineralization of DPSCs.The activation of AKT pathway,P38 pathway,Erk1/2 pathway and Smad3 was observed within two hours after stimulation of DPSCs with TGF-β1.The results showed that the blockage of AKT pathway and Erk1/2 pathway completely inhibited the differentiation of DPSCs and the regulation of DPSCs by TGF-β1;the P38 pathway inhibited the early stage of TGF-β1;while blocking the Smad3 pathway reversed the inhibitory effect of TGF-β1 on the late differentiation of DPSCs.3.TGF-β1 inhibited late differentiation of DPSCs through upregulation of HDAC5 expression.TSA combined with TGF-β1 stimulation reversed the inhibitory effect of TGF-β1 on mineralization formation and DSPP,suggesting that TGF-β1 may regulate late differentiation and mineralization through deacetylation modification.q RT-PCR results showed a significant increase in HDAC5 expression at day 7 of mineralization under TGF-β1 stimulation.Western blot results showed that HDAC5 expression continued to increase after TGF-β1 stimulation of DPSCs in vitro,and in vivo experiments showed that HDAC5 expression increased and then decreased during pulp-dentin repair,and was consistent with spatial and temporal changes in TGF-β1.HDAC5 was determined by lentiviral transfection experiments to play a role in TGF-β1 inhibition of DPSCs.4.TGF-β1 reduced the transcriptional activity of Runx2 through the Smad3/HDAC5 pathway.The Western blot assay showed that DSPP protein expression was reduced while Runx2 was not significantly affected,while the IP assay showed that Runx2 protein was altered by deacetylation.The Co-IP assay confirmed that TGF-β1 promoted the binding of Runx2 to HDAC5 and HDAC5 to p-Smad3.Immunofluorescence staining showed that HDAC5 and Runx2 were co-expressed in the nucleus of the TGF-β1-treated group,while blocking the Smad3 pathway affected HDAC5 expression.Conclusion:TGF-β1 promotes early differentiation and inhibits late differentiation and mineralization of DPSCs,probably through cycle blockade and activation of AKT pathway,Erk1/2 pathway and P38 pathway to promote early differentiation,and through activation of Smad3 pathway to promote increased recruitment of HDAC5 to deacetylate Runx2 to affect its transcriptional activity,thereby inhibiting the expression of DSPP and late differentiation and mineralization.This study describes the stage-dependent role of TGF-β1 in the regulation of dentinogenic differentiation of DPSCs and the possible regulatory mechanism,which provides important theoretical implications for the application of TGF-β1 in clinical pulpal dentin regeneration therapy. |
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