Experimental Study On The Effect Of Human Bone Marrow Mesenchymal Stem Cell-Derived Exosomes Pretreated With DMOG On Bone Repair

BACKGROUND : Exosomes derived by mesenchymal stem cell(MSC)have been considered as a new and effective drug candidate for the treatment of critical size bone tissue defects.The exosomes are generally considered as an alternative for cell therapy by promoting angiogenesis.The way to improve the function and efficiency of exosomes has been a focus which needs further research in the field of orthopaedics.OBJECTIVES: Bone marrow-derived mesenchymal stem cells(BMSCs)were pretreated with low-dose Dimethyloxaloylglycine(DMOG)to observe the role of exosomes(DMOG-BMSC-Exos)on bone tissue neovascularization and bone repair,and to explore its related mechanism and signal pathway.This study in order to provide a new technical method for the better use of mesenchymal stem cell-derived exosome to repair bone defect.Methods: We pretreated BMSCs with low dose DMOG,then we got and identified the DMOG-BMSC-Exos.At the same time,exosomes secreted by normal BMSCs(BMSCs-Exos)were extracted for control.In order to verify the role of exosomes,we observed the effects of pretreated exosomes on the proliferation rate,migration and tube formation assays of human umbilical vein endothelial cells(HUVECs)in vitro,and verified its cellular pathway.In vivo,the critical-sized calvarial defect rat model was established.The defect was pretreated with DMOG-BMSC-Exos and eight weeks after the operation the bone tissue regeneration was measured by histological analysis,the bone tissue repair and neovascularization were measured by micro-computerized tomography,Microfil and sequential fluorescent labeling.RESULTS:(1).BMSCs-Exos and DMOG-BMSC-Exos were isolated and extracted successfully and the animal model was established successfully.(2).Through the detection of transmission electron microscope,fluorescence microscope and surface protein markers detection,it was proved that the exosomes were extracted successfully.(3).In cell experiment,it was observed that both BMSCs-Exos and DMOG-BMSCExos could promote cell proliferation,migration and tube formation,while DMOG-BMSC-Exos promoted cell migration and angiogenesis faster than the control group.(4).In the rat model of calvarial defect,Micro CT,Microfil perfusion and fluorescent staining were used to verify that DMOG-BMSCExos can promote bone tissue healing and angiogenesis.(5).The mechanism of DMOG-BMSC-Exos promoting angiogenesis and bone regeneration:DMOG-BMSC-Exos activated the Akt/mTOR pathway to stimulate angiogenesis in HUVECs,which contributed to bone regeneration and angiogenesis in the critical-sized calvarial defect rat model in vivo.CONCLUSIONS:(1).DMOG-BMSC-Exos promotes HUVECs migration and tube formation more significantly than BMSC-Exos.(2).DMOG-BMSC-Exos can promote bone tissue rapair and angiogenesis.(3).DMOG-BMSC-Exos promote neovascularization through Akt/mTOR pathway,and thus enhances bone tissue regeneration in critical-sized calvarial defect rat model;(4).Low dose DMOG stimulation can enhance the angiogenic activity of exosomes,so as to further strengthen its application in cell-free therapy.