| Bone defects repair is a difficult problem of bone repair filed in clinical, and the development of bone tissue engineering provides a new research direction for bone repair. Medical titanium materials have good mechanical properties and biocompatibility, so, it as bone tissue engineering scaffolds related reports has been widely reported. However, the titanium material surface does not have biological activity, and it is easy to lead the receptor bone tissue to fall from the surface of implanted titanium material. Therefore, exploring how to improve the biological activity of titanium material has become a scientific problem to be solved in this area. Bone morphogenetic protein-2(BMP-2) plays an important role in regulating the process of bone formation and occurrence, and can induce bone mesenchymal stem cells(BMSCs) to differentiate into osteoblast. Thus, it is widely used to enhance the biological activity of bone tissue engineering scaffold material. Some reports show that 3,4-L-dihydroxyphenylalanine(DOPA) can adhere to the surface of Au, Ag and Ti O2. Therefore, DOPA as the medium fixed the BMP-2 to the surface of titanium material has an important significance for improving the biological activities of titanium material.In this project, through genetic engineering method the repeat sequences of tyrosine(Tyrosine-lysine-lyrosine-lysine-lyrosine, YKYKY) are integrated into the terminal genome sequence of BMP-2 through genetic engineering method, and Escherichia coli(E. coli) is used to express the protein. Fast protein liquid chromatography(FPLC) is used to purify the protein expressed by E. Coli, and tyrosine hydroxylation is used to complete the conversion of DOPA. The protein size, purity and specificity are identified by the methods of polyacrylamide gel electrophoresis, matrix-assisted Laser desorption/ ionization time of flight mass spectrometry(MALDI-TOF-MS) and western blot. Liquid chromatography-tandem mass spectrometry(LC-MS/MS) is used to determine whether YKYKY tyrosine is oxidized into DOPA. Circular Dichroism Spectrum(CDS) and ELISA are used to analysis and detect the biological activity of the protein. The surface of the immobilized material, carbon peak, changes in growth factor levels are analyzed through the method of scanning electron microscope(SEM), energy dispersive spectrometer(EDS), and quartz crystal microbalance with dissipation(QCM-D). The co-culture experiments of mouse myoblast cells and human bone mesenchymal stem cells with immobilized titanium metal material are used to determine the osteogenic ability of titanium metal material. Finally, the calcium cumulation of immobilized titanium metal material surface is further determined by in vivo experiments.The results showed that: BMP-2-YKYKY plasmid could be expressed in E.coli, and the target protein had a higher purity after purification. Molecular weight and specific identification were used to confirm the target protein BMP-2-YKYKY. After hydroxylation, the 3 tyrosine of BMP-2-YKYKY have been became DOPA, and the protein solution still had a high purity. Through biological activity identification, we found that the addition of YKYKY and DOPA had no impact on the biological activity of BMP-2 functional areas. Further, the modified BMP-2 not only had almost no change in biological activity, but also had DOPA adhesive capacity(It had the strongest adhesive capacity when p H=8.5). BMP-2-XKXKX could significantly changes the roughness of the titanium material surface, and enhance the amount of BMP-2(Growth factor content of the surface reached 631.43 ng/cm2) adhered to the titanium material surface. After BMP-2-XKXKX modification, the titanium material had strong osteoinducitive ability, and not only could significantly promote Dualluciferase expression of mouse myoblasts, and also could significantly promote the alkaline phosphatase(ALP) and the osteoblastic gene RUNX2, OSTERIX and ALP expression of human bone mesenchymal stem cells, and inhibit the stemness gene OCT4, NANOG and SOX2 expression of human mesenchymal stem cells. Further in vivo experiments showed that BMP-2-XKXKX immobilized titanium metal material could promote the calcium accumulation.In summary, in this project, DOPA is used as a medium to fix the BMP-2 to the titanium material surface, then to explore the biological activity changes of titanium metal material. We found that indert DOPA could not only keep the biological activity of BMP-2, also could promote the adhesive ability of BMP-2 on the titanium material surface. It could improve the biological activity of titanium metal material surface, and enhance osteogenic ability of titanium metal material surface; all of this will provide an experiment basis for the clinical application of titanium materials. |
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