GFOGER改性丝素蛋白/BMP-2微球支架控释体修复骨缺损的作用及机制

The bone grafts are far from meeting the clinical requirements. Construction of the bioactive materials based on BMP-2 is an ideal way to repair bone defects. The silk fibroin (SF) microsphere or scaffold is an excellent carrier for BMP-2. However, due to lack of the specific structures, the adhesive efficiency of SF to BMSCs is very low. In addition, the releasing characteristics should further be improved. The latest and our previous studies demonstrate that the collagen mimetic peptides (GFOGER) improve the adhesive performance of SF to BMSCs by binding the α2β1 receptor. Thus, the hypothesis is put forward that the SF modified by the GFOGER is used to load BMP-2. In the early stage, the BMSCs migrate into the bone defect by BMP-2. Then the GFOGER enhances the adhesive capacity of BMSCs. At the late stage, the GFOGER enhances the performance of BMP-2 on BMSCs differentiation. In order to prove the hypothesis, the GFOGER modified SF microsphere-scaffold composite is constructed to release BMP-2 in a controlled and sustained way. In this program, the controlled-release characteristics of the material, the ability of BMSCs migration and differentiation, as well as mechanisms for repairing bone defects are elucidated, from the overall level of molecular, cellular and animal by means of cell migration in vitro, quantum dot notation, RT-PCR，Western blot, tracer in living cells, and animal in vivo fluorescence imaging. If the composite is developed successfully, the controlled- release properties of carriers would be improved and the high efficiency of bone formation with a very low dose of BMP-2 would be achieved. This program provides new ideas and references for the repair of bone defects.

目前的骨修复材料远远不能满足临床骨缺损的需求，而构建基于BMP-2的活性材料是骨修复的理想途径。丝素蛋白（SF）微球或支架是BMP-2较好的负载方式，但与BMSCs的亲和性低，且控释性能有待于进一步改善。国内外最新及我们前期的研究表明，胶原模拟肽（GFOGER）可通过与α2β1受体结合，增强BMSCs与SF的黏附性能。因此，我们提出假说：利用GFOGER改性SF并负载BMP-2，早期通过BMP-2趋化BMSCs迁移，后期通过GFOGER增强细胞黏附及BMP-2的促成骨分化，进而修复骨缺损。为了验证该假说，我们构建负载BMP-2的GFOGER-SF微球支架控释体，借助体外细胞迁移，量子点标记，RT-PCR，Western blot，活细胞示踪及体内动物荧光成像等技术，从分子、细胞、动物整体水平研究骨修复的作用及机制，为改善载体的控释性能及低剂量BMP-2靶向成骨提供新思路，为临床提供参考。

本研究建立了一种新型的丝素蛋白/纳米羟基磷灰石复合支架，合并了骨形态发生蛋白-2的缓释微球，并负载了一种胶原模拟肽GFOGER，研究该复合支架对骨髓间充质干细胞黏附、增殖和成骨分化的影响，以及其对骨缺损的修复是否有促进作用。制备的微球粒径均匀，约为4.2μm，复合支架孔径均匀，约为51.19μm，BMP-2的缓释效果可达28天以上。体外实验表明，负载双因子的支架有最佳的促进细胞黏附、增殖、成骨能力，但没有明显的促进迁移能力。体内实验可知，负载双因子的支架可在12周时基本完成缺损处的骨桥接，而其余组则不能完成，双因子支架组的体内成骨效果最佳。微流体法制得的微球拥有更好的缓释性能，可提高复合支架生物相容性，提供给细胞更好的成骨环境。Sulfo-Lc-SPDP交联法可成功接枝GFOGER，并不影响其发挥原有功效。负载了双因子的新型复合支架，拥有最佳的促进细胞粘附、增殖、成骨分化的能力，并可在12周时完成大鼠颅骨缺损的修复，为骨组织工程学和临床骨缺损修复提供了新的思路。