The Effect Of Treated Dentin Matrix Material On The Differentiation Of Dental Pulp Stem Cells By Inhibiting The Expression Of GSK3β

ObjectiveTreated dentin matrix material(TDM)is an ideal scaffold material containing a variety of growth factors and biologically active substances,which can induce tooth differentiation of dental stem cells.In the process of tooth development and stem cell self-renewal,Wnt/β-catenin signaling pathway plays an important role.GSK3βis a very important negative regulator in the Wnt/β-catenin signaling pathway.It can continuously phosphorylate β-catenin,thereby making β-catenin ubiquitinated degradation.In the process of the differentiation of hDPSCs,this experiment found that the expression of GSK3β decreased,but whether it is involved in this process needs further research.This study intends to use TDM extract to induce hDPSCs to study the tooth-forming ability and specific mechanism changes of hDPSCs,so as to provide a new method and theoretical basis for tooth regeneration.MethodsTDM is prepared by gradient demineralization,and the extract of TDM is prepared by soaking method.The normal culture medium was used as the control group,which was divided into 4 groups according to the concentration of the extract:100%group,80%group,60%group,and 40%group.Each group was induced and cultured hDPSCs.Real-time Quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression level of genes related to odontogenesis in cells;GSK3βand Wnt were detected by Western Blot Assay(WB)The protein expression level of the Wnt/β-catenin signaling pathway and the expression level of odontogenesis-related proteins;interfering with the expression of GSK3β in hDPSCs by lentiviral transfected cells,and the expression level of odontogenesis-related genes in cells were detected by qRT-PCR,and GSK3β was detected by WB As well as the protein expression level of Wnt/β-catenin signaling pathway;use the processed dentin matrix material to cover the pulp in situ to explore the feasibility of the dentin matrix material as a pulp capping agent;confirm the induction of TDM extract by mandibular bone culture experiment The expression changes of GSK3β during tooth formation and the location of its expression;finally,the dentin matrix materials were compounded with Ad-NC,Ad-GSK3β,Sh-NC,GSK3β-RNAi hDPSCs through the nude mouse ectopic experiment,and it was clear that TDM induced The mechanism of tooth differentiation of hDPSCs.ResultsBy comparing the differences in gene and protein expression of hDPSCs induced by normal culture medium and extracts,it was found that hDPSCs induced by TDM extracts had an enhanced ability to differentiate into teeth,and during this process the expression of GSK3β decreased;Lentivirμs was used to construct hDPSCs that knock down and overexpress GSK3P,and compare the expression levels of Ad-NC,Ad-GSK3β,Sh-NC,GSK3β-RNAi tooth formation-related genes and protein expression levels,and found that GSK3β-RNAi hDPSCs related to tooth formation And protein expression is the strongest,while Ad-GSK3β has the weakest expression of hDPSCs.Comparing the ability of using TDM and calcium hydroxide for in situ pulp capping,it is found that the restorative dentin formed by TDM pulp capping is more regular;by mandibular culture the experiment confirmed that the TDM extract induced changes in the expression of GSK3β during tooth formation and localized its expression position.The results showed that GSK3β was expressed in both enamel and dentin and the thickness of the restorative dentin formed in the TDM induction group was greater.Thick;TDM was compounded with Ad-NC,Ad-GSK3β,Sh-NC,GSK3β-RNAi hDPSCs through the experiment of ectopic tooth formation in nude mice,and the results of HE staining of GSK3β-RNAi group showed newly formed irregular dentin No similar structures appeared in other groups;in addition,the immunohistochemical results showed that the hDPSCs of GSK3β-RNAi had the strongest expression of dental related proteins(DSPP,DMP-1,ALP,RUNX2,etc.),while the expression of hDPSCs of Ad-GSK3β Weaken.ConclusionsTDM activates the Wnt/β-catenin signaling pathway by inhibiting the expression of GSK3β to enhance the differentiation ability of hDPSCs.This result provides new inspiration for the differentiation of dental pulp stem cells and the regeneration of tooth roots.