Effects Of Silk Fibroin/Graphene Oxide/Simvastatin Composite Films On The Expression Of BMP-2 And ALP In Rat Calvarial Defects

Objective: To evaluate the effect of bone tissue repair by implanting silk fibroin / graphene oxide / simvastatin composite films(SF/GO/Sim)and expression of BMP-2 and ALP in a critical size calvarial defect rat model,and the feasibility of inducing osteogenesis in vivo when using silk fibroin / graphene oxide composite films(SF/GO)as biological scaffolds combined with simvastatin.Methods: A total of 48 healthy male SD rats were randomly divided into 4 groups according to the different implanted membrane materials,with 12 in each.After the preparation of 5mm diameter circular defect on both sides of the rat calvarial bone under anesthesia,the materials were implanted in different groups :(A)SF only,(B)SF/GO,(C)SF/Sim,(D)SF/GO/Sim which A was the control group.At 4 weeks and 8 weeks after implantation,6 rats from each group randomly selected were sacrificed by using 4% of paraformaldehyde for systemic perfusion.The collected bone specimens containing implants for general observation,HE staining and immunohistochemical staining to observe the morphological changes of bone tissue,and the expression level of BMP-2 and ALP in bone defect area.The experimental data was calculated through factorial design ANOVA and LSD by SPSS19.0 statistics software to evaluate the bone regeneration.Results: General observation: There was no loss of the implantation and no obvious rejection in the specimens after surgery at 4 and 8 weeks.Histological observation: HE staining showed that at 4 weeks and 8 weeks after operation,there were various bone formation in each group,the D group showed the best condition.And the osteoblasts,angiogenesis and osteoid in scaffolds of D group were much more than that of other groups,even the scaffolds showed various degradation.There was less new bone formation in A group but filled with more fibrous connective tissue.Immunohistochemical detection: the results of IHC staining showed that the expression of osteogenic markers BMP-2 and ALP had significant difference in different time(P<0.001);The expression of BMP-2 and ALP had significant difference in different groups(P<0.001);There was no interaction between different groups and different time(P>0.05);The expression of BMP-2 and ALP in different groups were compared by LSD,it showed that,there were significant differences between different groups(P<0.001)except for B and C groups(P>0.05),so it can be regarded as the expression of BMP-2 and ALP in D group>B group,C group >A group.Conclusion: 1.Silk fibroin / graphene oxide / simvastatin composite films can promote the formation of new bone in the rat calvarial defect and obviously increase the expression of BMP-2 and ALP;2.Silk fibroin / graphene oxide composite films can promote osteogenic differentiation by increasing the expression of osteogenic factors such as BMP-2 and ALP as a scaffold material for repairing of bone defects.