Effects Of MicroRNA And Graphene Oxide Incorporated Scaffold On Mesenchymal Stem Cell Mediated Bone Repair

Objectives To enhance mesenchymal stem cell mediated bone repair,we explore the micro RNA's regulatory effects and the mechanisms governing the osteogenic differentiation of mesenchymal stem cells,together with the development of grapheme oxide incorporated scaffold favoring the osteogensis and angiogenesis,hoping to achieve both anatomical and functional repair,so that to provide experimental and theoretical basis for their applications in repairing bone defects of orbital wall and other parts of the body.Materials and Methods 1.Gene Chip,q PCR,western blot and dual luciferase reporter assay were used to detect the regulatory patterns and underlying mechanisms of miR-135 and miR-146 a on ADSC osteogenic differentiation.Gene-modified MSCs were transplanted into rat critical-sized calvarial defect,micro-CT,hard tissue sections,sequence fluorescent labeling and immunohistochemical methods was used to evaluate new bone formation.2.FTIR,XRD,Raman spectra and SEM were used to evaluate the characterization of the scaffold.Cell and protein adsorption experiments,cell immunofluorescence were used to detect cell attachment.q PCR,western blot were used to detect in vitro osteogensis and angiogenesis.micro-CT,hard tissue sections,sequence fluorescent labeling,immunohistochemical were used to evaluate the neovascularization and bone regeneration.Results 1.Mi R-135 targeted the 3 'untranslated region of Hoxa2 to inhibit Hoxa2 protein translation process,the overexpression of miR-135 significantly increased the expression of osteogenic differentiation markers in ADSCs and promoted the mineralization of extracellular matrix.ADSCs overexpressing miR-135 combined with PSe D porous scaffold significantly enhanced the bone repair.2.BMP-2 transcriptionally repressed miR-146 a,miR-146 a inhibited SMAD4 protein translation by targeting the 3 'untranslated region,the knockdown of miR-146 a enhanced the sensitivity of ADSCs to BMP-2 induced osteogenesis,the expression of osteogenic marker genes and extracellular matrix calcification were significantly increased,which further led to enhanced bone repair.3.Grapheme oxide incorporated scaffold significantly increase the adsorption of extracellular matrix proteins and elevated integrin receptor expression.The enhanced ligand-receptor binding activated FAK,ERK and Akt signaling pathways,which subsequently increased the expression of osteogenesis and angiogenesis marker genes in BMSCs,further led to better neovascularization and bone regeneration.Conclusions 1.Mi R-135 positively regulates osteogenic differentiation of ADSCs and ADSCs-mediated new bone formation through a miR-135/Hoxa2/ Runx2 pathway.2.Mi R-146 a negatively regulates BMP-2 induced ADSC osteogenic differentiation and bone regeneration by modulating SMAD4 expression post-transcriptionally.3.Grapheme oxide incorporated scaffold promotes the osteogenesis and angiogenesis both in vitro and in vivo,it holds great potential in repairing large bone defects.