| Bone morphogenetic protein 2 (BMP-2) belongs to transforming growth factor beta (TGF-β) superfamily. BMP-2 plays an important role in the stimulating osteoblast differentiation and the development of bone. In recent years, BMP-2 has been wildly used for the research of bone tissue engineering. HA, as the main inorganic component of bone, is able to induce bone formation and revascularization. HA will not cause inflammatory response in vivo, and is one of commonly used bone repair materials. Silk fibroin (SF) has good biocompatibility, biodegradability and the unique mechanical properties. It is widely used in biomedical field. In this study, the bioactive rhBMP-2 was produced by a prokaryotic expression system. On this basis, we have prepared rhBMP2-loaded HA/SF composite scaffolds, examined their biocompatibility, and studied their ability to repair bone defects. This study has three main parts:1. Expression, purification, and refolding of rhBMP-2 in vitroThe rhBMP-2 gene was cloned from MG-63 cells by RT-PCR, and inserted into the corresponding site of expression vector pET-30a (+) to generate pET30a-rhBMP2. Then pET30a-rhBMP2 was transformed into E. coli strain BL21 (DE3). The proteins were expressed, purified by Ni-NTA column under denaturing conditions and refolded at 4℃by urea gradient dialysis (different pH value). MG-63 cells were used to value the bioactivity of rhBMP-2. The results showed that rhBMP-2 was well purified and the yield was 58 mg per liter of medium (4.8 mg per gram of wet cells). The protein refolding yield at pH 6.0 was only 3.4%, while the yield was 42% and 96% at pH 7.4 and pH 9.0, respectively. In vitro experiments showed that rhBMP-2 refolded at pH 7.4 had higher biological activity for cell proliferation and differentiation than that refolded at pH 9.0. This is due to the fact that pH 7.4 is closer to the conditions in vivo leading to the formation of dimers through the interchain disulfide bond. 2. Preparation of rhBMP-2 loaded HA/SF scaffoldsIn this study, HA was prepared by precipitation. The HA/SF scaffolds were prepared by freeze-drying. Properties of scaffolds were investigated by SEM, XRD, FTIR and TGA, etc. Then HA/SF scaffolds were modified by soaked into rhBMP-2 solution of different protein concentration. The adsorption and release of BMP-2 were tested in vitro. The results showed that HA/SF scaffolds had high porosity, interconnected pores. HA was uniformLy distributed in the scaffolds. XRD and FTIR showed that SF was in (3-sheet conformation. The adsorption of rhBMP-2 showed that amount of rhBMP-2 loaded onto the scaffolds was controlled by varying the initial protein concentration. The rhBMP-2 was sustainablely released from scaffolds for a long time, and maintained its biological activity.3. Biocompatibility of rhBMP-2 loaded HA/SF composite scaffoldsIn this study, MG-63 cells were seeded onto HA/SF scaffolds to assess cell biocompatibility of the scaffolds. The rat skull defect model was used to value the effect of composite scaffolds on repair of bone. The impact on ectopic bone formation was detected by implanting scaffolds into a hind limb muscle. The results showed that HA/SF scaffolds carrying rhBMP-2 promoted cell proliferation and differentiation. In vivo experiment showed that composite scaffolds enhanced new bone formation, and also induced ectopic bone formation. It means that HA/SF scaffolds carrying rhBMP-2 could be a kind of potential materials for the bone tissue repair.In summary, rhBMP-2 with biological activity was successfully expressed in E.coli, and refolded in vitro. Then, a new type of bone repair materials was prepared by HA/SF scaffolds loaded with rhBMP-2. Our experimental studies showed that this material has good biocompatibility and could promote the formation of new bone. It is a potential bone repair materials.
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