Effects Of Graphene Oxide On Osteogenic Differentiation And Repairing Bone Defects

The formation of bone defect is mainly caused by trauma,pathology and surgery.Due to the existence of bone defect,overgrowth of fibrous tissue in the defect area often causes delayed healing or non-healing,and local dysfunction,which seriously affects the quality of life of patients.With the increase of various high-energy injuries in daily life,bone defects caused by severe open comminuted fractures are becoming more and more common in clinical practice.Due to the poor state of the whole body and the delayed healing of bone tissue,the recovery period of patients is longer.Currently available strategies for the treatment of bone defects mainly include autologous cancellous bone transplantation,vascularized bone transplantation,allograft bone transplantation,traction osteogenesis technology,combination of scaffold materials and bioactive factors or cells,etc.However,there is still no effective method to solve this problem that has been perplexing clinical orthopedic surgeons.Graphene Oxide(GO)can promote the proliferation of mesenchymal stem cells(MSC),and in vitro studies have confirmed that 0.1 ?g/mL GO can regulate macrophage differentiation.However,there is no report about the porous scaffold formed by the combination of GO and bioactive glass(BG)and applied to the repair of bone defects and the related mechanism.The aims of this study are to investigate the effects of GO on the proliferation and osteogenic differentiation of MSCs;GO and BG composite scaffolds promote angiogenesis and bone formation to repair bone defects;and explore the possible immune mechanism of GO on the surface of BG in promoting MSCs to differentiate into osteoblasts.rBMSCs were extracted and cultured in conditioned medium with different concentrations of GO.The toxicity and proliferation effects of GO on rBMSCs were detected by CCK-8 method.The osteogenic differentiation of rBMSCs was detected by alkaline phosphatase staining(ALP)and alzarin red staining(ARS)and real-time quantitative polymerase chain reaction(RT-q PCR).Then,0,2.5 mg and 25 mg of GO powder were mixed with 4 mL of BG solution,respectively,and sintered at high temperature under the protection of nitrogen to prepare porous scaffolds and implanted into the critical skull defect model of SD rats.Twelve weeks after the operation,Micro-CT analysis,fluorescence confocal method,Microfil vascular perfusion,hard histomorphological analysis and immunohistochemical method were used to detect the effect of GO on the repair of critical bone defects in the skull of rats.Finally,porous BG scaffolds containing 0-2.5%GO were prepared by 3D printing,and the effects of GO on macrophage polarization on angiogenesis of endothelial cell and osteogenic differentiation of rBMSCs were detected by co-culture with macrophages in vitro.The results confirmed that 0.1 ?g/mL GO could promote the proliferation and osteogenic differentiation of rBMSCs without significant cytotoxicity in vitro.The porous BG scaffold containing GO can significantly enhance the regeneration of blood vessels and new bone in the cranial bone defect area of SD rats,and the repair effect of the porous bioactive glass scaffold containing high amount of GO is more obvious.3D printed porous BG scaffolds with 0.5%GO can promote the polarization of RAW264.7 cells from M0 to M2,and the conditioned medium obtained by co-culture with macrophages can effectively promote endothelial cell migration and tubular formation,and significantly enhance the osteogenic differentiation of rBMSCs.In summary,0.1 ?g/mL GO can significantly promote the proliferation and osteogenic differentiation of rBMSCs;the porous BG scaffolds containing GO can effectively repair the skull bone defects of SD rats by promoting vascular regeneration and bone regeneration;GO may promote osteogenic differentiation of rBMSCs and endothelial cell angiogenesis by regulating macrophage polarization.