The Study Of Osteogenic Differentiation Of RBMSCs Induced By DOPA-IGF1 Coating On Pure Titanium Surface

Objective:The recombinant human growth factor DOPA-IGF1 coating on the surface of pure titanium was prepared to construct a single material coating which promoted the early adhesion state and osteogenic activity of the titanium sheet.The study was to investigate the effect of DOPA-IGF1 on the osteogenic activity of rabbit bone marrow mesenchymal stem cells on the surface of titanium,which may provide a new research direction for the surface modification of implants.Methods:1.The strain carrying the recombinant human growth factor DOPA-IGF1 gene was expressed and purified,and the purified protein DOPA-IGF1 was identified by SDS-PAGE electrophoresis.2.The target protein DOPA-IGF1 was hydroxylated and immobilized on the surface of titanium sheet to construct a coating system.The surface morphology of each group of titanium sheets was observed by scanning electron microscopy(SEM),and the surface element composition of each group was determined by X-ray energy dispersive X-ray analysis(EDX).All groups of titanium sheets were characterized.3.Rabbit bone marrow mesenchymal stem cells(rBMSCs)were extracted by whole bone marrow adherence method.The proliferation curve was measured by cell proliferation assay(CCK-8)at the third generation.After osteogenesis induction,the osteogenic differentiation potential was verified by alizarin red staining and osteogenesis-related gene detection.4.CCK-8 test and fluorescence staining were used to detect the effects of each group of coated titanium on the proliferation and early adhesion of rBMSCs after co-culture.5.To investigate the effect of DOPA-IGF1 to rBMSCs on the osteogenesis-related activity,the expression of ALP were measured by alkaline phosphatase(ALP)assay and the relative expression of osteogenesis-related genes Runx2,BMP2 and Col-a1 were tested after co-culture osteogenesis induction.Results:1: Rabbit bone marrow mesenchymal stem cells were successfully extracted and cultured,and the cells were observed to be arranged in a long fusiform order under the microscope.And the osteogenic differentiation potential was verified after osteogenic induction.2.The results of SEM and X-ray energy spectrum analysis showed that DOPA-IGF1 coating was successfully coated on the surface of titanium sheet in this experiment with excellent characterization.3.CCk-8 proliferation experiment results showed that the proliferation rate of cells was DOPA-IGF1 group > IGF-1 group >TI group,and there was no significant difference in the proliferation rate between TI group and CG group.The results showed that the titanium sheet used in this study had no effect on the growth of rBMSCs,and DOPA-IGF1 coating could significantly promote the early proliferation of rBMSCs on the surface of titanium sheet.4.The results of fluorescence staining showed that the cell morphology of IGF-1 group was similar to that of TI group,with clumps and unclear kinase filaments.The DOPA-IGF1 group showed more pseudopodia than the other two groups,and the actin microfilaments were clearly visible and the cells were more malleable.5.The results of ALP experiments showed that the alkaline phosphatase activity of the four groups of cells gradually increased with the prolongation of culture time.Alkaline phosphatase activity in DOPA-IGF1 group>IGF-1 group>TI group.There was no significant difference between TI group and CG group.The difference between DOPA-IGF1 group and IGF-1 group,DOPA-IGF1 group and TI group were both significant(P<0.05).6.The results of osteogenic-related gene test showed that the expression of three osteogenic related genes in DOPA-IGF1 group was enhanced at 4 and 7 days after osteogenic induction.And the difference was significant compared with other groups(P<0.05).Conclusion1.Rabbit bone marrow mesenchymal stem cells were successfully extracted,cultured and identified.The results showed that the biological characteristics of the cells extracted in this experiment were consistent with the stem cells.2.The experimental components covering DOPA-IGF1 and IGF-1 coating were successfully constructed,and the coating was characterized by scanning electron microscopy and X-ray energy spectrum analysis.3.The effect of DOPA-IGF1 on the early proliferation of rBMSCs was confirmed by cell experiments.It was confirmed that it can promote bone formation,but its osteogenic mechanism and effectiveness still need to be further studied.