Exosomes Derived From MicroRNA-764 Overexpressing Mesenchymal Stem Cells Promote Bone Regeneration

DBM derived from natural cancellous bone has become ideal scaffold for bone repair due to its natural porous structure,low immunogenicity,good biocompatibility and adjustable mechanical properties.However,the bone regeneration of DBM was restricted as the bone inducing growth factors were often eliminated during the production process.Scaffolds modified with MSCs-Exo enhanced bone regeneration by promoting vascularization.In this paper,genetic modification of MSCs were proposed to overexpressing osteogenesis-related miR-764.Exo^miR-764 with both pro-angiogenesis and osteogenesis abilities were obtained and Exo^miR-764/DBM was fabricated to improve the bone repair capacity of DBM.The main research and experimental results are as follows:?1?Construction of recombinant lentiviral vector encoding miR-764 and functional identification of miR-764 in rBMSCs.The recombinant plasmids pCMV-miR-764 were constructed.Subsequently,lentivirus were producted through three plasmids packaging system,and the titer of lentiviral vectors was 23.5×10⁸ TU/mL.The relative expression of miR-764 in rBMSCs up-regulated by 2.85±0.20 times?p<0.05?after lentiviral vectors transfected.The ALP activities and calcium deposition in miR-764overexpressing rBMSCs were enhanced after osteogenic induction in vitro.?2?Osteogenesis capacity of Exo^miR-764/DBM evaluation in vitro.Firstly,MSCs-Exo derived from rBMSCs were harvested by ultracentrifugation,and the results showed that MSCs-Exo possessed saucer shape with bilayer membrances,with particle size range from 87.87 to 191.69 nm,and expressed CD9 and CD63.After incubated with Exo^miR-764,the ALP activities and calcium deposition of rBMSCs were increased significantly.DBM from porcine cancellous bone with 5 mm diameter and 2 mm high was successfully prepared by decellularized and demineralized.In order to modify DBM with MSCs-Exo,both of them were incubated at 4?for 9 h.The release rate of MSCs-Exo from DBM were 87.25±0.28%over 6 d,most MSCs-Exo can released from DBM.After cultured with Exo^miR-764/DBM,the expression of extracellular matrix,collagen deposition,OC and OPN of rBMSCs were all increased when compared with DBM.?3?In vivo osteogenesis effect of Exo^miR-764/DBM evaluation.Compared with DBM group,the cell infiltration,collagen deposition,OC and OPN expression were increased in rat cranial defect model after Exo^miR-764/DBM implanted for 4 weeks,and CD34 positive cells could be observed.In conclusion,these results confirmed that Exo^miR-764/DBM promoted the osteogenic differentiation of rBMSCs in vitro,and also promoted bone regeneration in rat cranial defects model.