Experimental Studies On The Effect Of ADSCs-derived Matrices And MiR-146a In Osteogenesis Of MSCs

PurposeIn order to better study the effect of cell microenvironment and gene modification on osteogenesis differentiation and to explore the application of tissue engineering scaffolds in bone repair,this study will study the effect of decellularized matrices(ADM)of human adipose mesenchymal stem cells(hADSCs)on the osteogenic differentiation of rat bone marrow mesenchymal stem cells(rBMSCs),and ADM coated PSeD(poly(sebacoyl diglyceride)scaffolds will be used to repair bone defects in rats.In addition,the regulation mechanism of miR-146a on osteogenic differentiation of ADSCs will also be investigatedmaterials and methods1.rBMSCs were seeded on the prefabricated ADM.The effect of ADM on the proliferation of rBMSCs was detected by CCK-8 assay and immunohistochemistry of BrdU and ki-67 assays.The effect of ADM on the osteogenesis of rBMSCs were evaluated by qPCR and Western Blot assays.The bone defect of rat skull was repaired by ADM coated PSeD scaffolds,and the new bone formation was detected by micro-CT and immunohistochemical staining2.Construction of lentiviral vectors encoding miR-146a or inhibitor.After rADSCs were tracsfected by lentiviral vectors,quantitative real time PCR(qPCR),western blot and alkaline phosphatse were used to determine the effecs of miR-146a on osteogenic differentiation of rADSCs.Dual luciferase reporter assay was used to detect the regulation mechanisms of miR-146a' s effect on rADSCs osteogenesisresults1.ADM displayed strong osteogenic differentiation,and significantly promoted the proliferation of rBMSCs and expression levels of osteogenesis-related genes and proteins as well as bone matrix mineralization.ADM sustained released growth factors(BMP2 and IGF-1),which played a key role in the proliferation and osteogenic differentiation of rBMSCs.ADM coated PSeD scaffolds can significantly promote the repair of critical-sized calvarial defects in rats.2.Overexpression of miR-146a inhibits osteogenic differentiation of rADSCs,and miR-146a inhibits SMAD4 protein expression by direct binding to the 3 'UTR region of SMAD4 mRNA.conclusion1.ADM promoted the proliferation and osteogenesis of rBMSCs,and PSeD/ADM composite scaffolds could significantly repair the bone defects in vivo.2.MiR-146a inhibited the osteogenic differentiation of rADSCs with SMAD4 as the target gene,and miR-146a inhibitor could promoted osteoblast differentiation of rADSCs.