Smad7 Participate In Regulating BMP2-induced Chondrogenic Differentiation Of Human Synovial Derived Mesenchymal Stem Cells

Background: Bone morphogenetic protein 2(BMP2)is a promising chondrogenic growth factor for cartilage tissue-engineering,but it also induces robust endochondral ossification.Human synovial-derived mesenchymal stromal cells(h SMSCs)have attracted great interest due to their poor potential for differentiation into osteogenic lineages.Smad7 plays a significant in the endochondral ossification.In this study,we explored a new method to amplify the BMP2-induced chondrogenic differentiation of h SMSCs by downregulating Smad7 and applying a cellular scaffold.Methods: h SMSCs were isolated from human knee joint synovium from 3 donors through adhesion growth.In vitro and in vivo models of the chondrogenic differentiation of h SMSCs were established.Transgenic expression of BMP2,and silencing of Smad7 and Smad7 were achieved by adenoviral vectors.The Osteogenic differentiation was detected by alkaline phosphatase staining,alizarin red staining and RT-PCR analysis of the osteogenic genes Runx2,Osterix and Osteocalcin.The Chondrogenic differentiation was detected by alcian blue staining and RT-PCR analysis of the chondrogenic genes Sox9,Col2 and Aggrecan.Hypertrophic differentiation was detected by the markers Col10 and MMP13.A subcutaneous stem cell implantation model was established with polyethylene glycol citrate-co-N-isopropylacrylamide(PPCNg)scaffolds and athymic nude mice(3/group,4–6 week-old female)and evaluated by micro-CT,H&E staining and alcian blue staining.An immunohistochemistry assay was used to detected Col1 and Col2,and an immunofluorescence assay was used to detect Col10 and MMP13.Results: These h SMSCs identified by flow cytometry.These h SMSCs exhibited lower osteo-differentiation potential than i Mads and C3H10T1/2-cells.When Smad7 was silenced in BMP2-induced h SMSCs,the chondrogenic differentiation genes Sox9,Col2 and aggrecan were enhanced in vitro.Additionally,it silencing Smad7 led to a decrease in the hypertrophic differentiation genes Col10 and MMP13.In subcutaneous stem cell implantation assays,immunofluorescence and immunohistochemical staining demonstrated that silencing Smad7 increased the number of Col2-positive cells and decreased the expression of Col1,Col10 and MMP13.Conclusion: This study suggests that the application of h SMSCs,cell scaffolds and silencing Smad7 can potentiate BMP2-induced chondrogenic differentiation and inhibit endochondral ossification.Thus,inhibiting the expression of Smad7 in BMP2-induced h SMSCs differentiation may be a new strategy for cartilage tissue-engineering.