Study On The ?-catenin Regulating Osteogenesis Of Human Periodontal Ligament Stem Cells Under Mechanical Tension

Periodontal ligament stem cells(PDLSCs),which have the potential of cloning,replicating,and multiple-differentiation ability,can differentiate to Osteoblast,fibroblast-like cells and adipocytes.h PDLSCs have high potential application value in dental tissue engineering.However,the mechanism in the differentiation of hPDLSCs remains unclear still,and it critically limited the clinic application of tissue engineering of h PDLSCs.The previous studies showed that the Wnt signal pathway involves in the process of osteogenic differentiation of stem cells,promotes the differentiation of stem cells into osteoblasts and inhibits the mineralization of osteoblasts.we have conf irmed that dynamic tensile stress can promote the osteogenic differentiation of hPDLSCs,but whether Wnt/?-catenin involves in the process of osteogenic differentiation of hPDLSCs under mechanical tension is still unknown.This study first proved Wnt/?-catenin pathway is activated in the osteogenic differentiation of hPDLSCs under mechanical tension.The small molecule agonist and inhibitor of Wnt/?-catenin pathway were applied for the activation intervening.Effects of mechanical tension on hPDLSCs osteogenic differentiation were observed.The role of Wnt/?-catenin signaling pathway in the regulation of hPDLSCs osteogenesis under mechanical tension was investigated,in order to explore the treatment of orthodontic tooth movement and provide theoretical basis fo r hPDLSCs tissue engineering at the molecular level.Objective: To investigate the role of Wnt/?-catenin signaling pathway in the regulation of osteogenesis of h PDLSCs under mechanical tension,and to explore the molecular mechanism of hPDLSCs osteogenic differentiation under mechanical tension.Methods1.hPDLSCs primary cells were isolated,and optimized concentration of agonist WAY262611 and inhibitor XAV939 were screened on 3-6 generation of hPDLSCs.The half maximal effect concentration(EC50)were calculated based on the cell viability measured by CCK-8,after the incubation abuout 1-4 days.In addition,the expression of ?-catenin protein in hPDLSCs osteogenesis under mechanical tension was detected by Western blot.2.The FX-4000 T stress loading system was applied for promoting the osteogenesis of h PDLSCs.The expression of osteogenic markers protein BMP2,Runx2,ALP,and ?-catenin in h PDLSCs under mechanical tension were determined by Western blot.3.The agonist group,inhibitor group,DMSO group(control group)and normal groups were applied for tension loading,then the expression of protein BMP2,Runx2 and ALP compared the bone marker was determined by Western blot.Results1.hPDLSCs primary cells were obtained successfully.With the positive expressi on of Vimentin,CD146 and STRO-1,and the negative expression of pan-cytokeratin,due to the cells have the osteogenic / adipogenic ability,the cells we obtained was identified as h PDLSCs.2.The EC50 of agonist WAY262611 and inhibitor XAV939 were 2.310 umol/L and 22.99 umol/L respectively.The agonist and inhibitor can up-regulate and down-regulate the expression of ?-catenin in hPDLSCs.The CCK-8 essay showed agonist WAY262611 and inhibitor XAV939 decreased the cell viability.3.The expression of osteogenic markers ALP and Runx 2 in h PDLSCs increased under exogenous mechanical tension.The expression level of ?-catenin increased significantly(P<0.05)during the osteogenic differentiation in h PDLSCs,suggesting that ?-catenin participates in the process of osteogenesis.4.Compared with DMSO group,in the agonist group,the expression of osteogenic markers ALP increased significantly in 0-12 h,and the difference was statistically significant.In the inhibitor group,the expression ?-catenin and osteogenic markers showed no significant trend.Conclusion1.The selected concentration agonist WAY262611 and inhibitor XAV939 reduced h PDLSCs proliferation activity.2.After the up-regulation of Wnt/beta-catenin signaling pathway activation level by the agonist,the early-stage osteogenic process in the of h PDLSCs under mechanical tension was enhanced.The further research on osteogenic regulation mechanism in h PDLSCs will provide protection for the orthodontic movement.From the perspective of enhanced osteogenic differentiation,it is also expected to shorten the time of oral orthodontic,improve the correction efficiency and optimize the treatment process.