PH And Enzyme Double Response And Time Sequnence Releasing Hydrogen Sulfide And BFP-1 For Osteoporotic Bone Repair

Objective:Design and prepare GelMA hydrogel that releases hydrogen sulfide and BFP-1 polypeptide,to study its biological activity and repairing effect on osteoporotic bone defect.Methods:(1)In vitro experiments: MA-BFP-1 was synthesized by solid component synthesis method,its purity was analyzed by mass spectrometry,and its bone-promoting ability was detected by RT-PCR and ALP staining.Hydrogel composites loaded with BFP-1 and JK2 were prepared by optical crosslinking method using methacrylate gelatin(GelMA)as loading material.The hydrogels were divided into GelMA,GelMA-BFP1,GelMA-JK2 and GelMA-BFP1 /JK2.The pore size of the hydrogels was observed by scanning electron microscopy,and the elements contained in the hydrogels were analyzed by EDS.The hydrogel degradation performance was tested by swelling test.Methylene blue method was used to determine the release rate of hydrogen sulfide in different PH environments.The weight of each hydrogel was measured after adding MMP-9(MMP-9).The biocompatibility of materials in each group was tested by live and dead cell staining and cell proliferation assay.The adhesion,aggregation and growth status of bone marrow mesenchymal stem cells on the cytoskeleton were detected by ghobi cyclic peptide staining and DAPI staining,and the bone-promoting ability of hydrogels in each group was tested from the aspects of osteogenic gene level,specific alkaline phosphatase and calcium nodules formation.(2)In vivo experiment: The osteoporosis model of rats was induced by oophorectomy.After the successful modeling was verified by Micro CT,the osteoporosis SD rats were randomly divided into five groups: blank control group,GelMA,GelMA-BFP1,GelMA-JK2 and GelMA-BFP1/JK2,with 6 rats in each group.The bone defect model of lateral femoral condyle was made,and each group of hydrogels were applied to the bone defect.The repair of the bone defect was evaluated by imaging and histology 8 weeks after surgery.Results:(1)In vitro experiment: staining of live and dead cells and CCK-8 proved that the material had good biocompatibility.Swelling experiments show that the biomaterial has good degradability.The methylene blue detection method proved that the hydrogen sulfide release of JK2 loaded hydrogel was stable.ALP staining and alizarin red staining showed that the hydrogel group loaded with BFP1 could effectively promote osteogenic differentiation of BMSCs.RT-PCR indicated that the expression of BFP1 loaded hydrogel group was higher than that of BFP1 free group,and there was a statistical difference.(2)In vivo experiment: Hydrogels loaded with BFP1 and JK2 had more obvious effect on bone defect repair than other groups.Conclusion: GelMA-BFP-1/JK2 composite hydrogel has good biocompatibility,osteogenic ability and bone defect repair ability.