具有BMSC原位募集和BMP-2可控释放功能的可注射骨修复材料的制备及生物学性能研究

Bone defect remains a big challenge for clinical therapy. It is a simple and effective way to treat the bone defect by recruiting target cells to the site of injury and then promote them to differentiate into bone tissue. However, the efficiency of repair is restricted by the currently used approaches of recruitment for the relatively low specificity. In recent years, studies found that the CD271 antigen was specifically expressed by a population of bone marrow mesenchymal stem cells (BMSCs) with great capability of osteogenic differentiation in bone marrow. What’s more, CD271 relevant signaling could promote the migration of cells, indicating the high-osteogenic BMSCs recruiting potential of the anti-CD271 antibody. In previous research we have successfully applied the anti-CD271 modified microspheres to capture BMSCs with specificity. However the regenerative capacity of these microspheres was unsatisfactory. So in this study we attempt to combine the anti-CD271 antibody with bone morphogenetic protein-2 (BMP-2), and hope to improve the efficiency of regeneration by promoting the osteogenic differentiation of the recruited cells through the controllably released BMP-2. In this study the anti-CD271 antibody and BMP-2 are loaded to the porous silk fibroin microspheres to fabricate functional building blocks, which are further encapsulated by the fast degradable alginate-fibrin hydrogel to fabricate the novel injectable material which can match the irregular shape of defect. The effect of this novel component will be tested in vitro and in vivo to evaluate its ability in bone formation. We hope to provide an effective way for treating bone defect by this novel material, and further broaden the vision in the field of in situ repair.

目前骨缺损仍是临床治疗的一大难题。在缺损部位募集目标细胞并促其向骨组织分化是一种有效的治疗方法。但目前常用的募集途径特异性不强，影响修复效果。近来研究发现骨髓中一群高成骨潜能骨髓间充质干细胞（BMSC）特异性表达CD271抗原，同时CD271相关信号还可介导细胞迁移，预示着CD271抗体拥有高成骨性BMSC募集潜能。申请人预实验中已成功通过CD271抗体修饰微球特异性捕获BMSC，但体系修复效果尚不理想。因此，本项目拟进一步引入可控释放的骨形态发生蛋白-2（BMP-2）诱导募集后干细胞成骨分化以提升修复效能。项目拟采用多孔丝素微球作为支架装载CD271及BMP-2构成功能主体构件，并封装于快速降解性藻酸钠-纤维蛋白水凝胶中，从而制备出可注射且能适应不规则缺损的新型骨修复材料。分别于体外及体内考察该材料的成骨能力，希望借助这种新型修复材料为骨缺损提供一条高效的治疗途径，更为原位修复拓展思路。

项目背景：目前骨缺损仍是临床治疗的一大难题。在缺损部位募集目标细胞并促其向骨组织分化是一种有效的治疗方法。但目前常用的募集途径特异性不强，且众多材料忽视了被募集细胞向骨组织转变的过程，亦难以很好地适应骨缺损部位不规则的形状，限制了材料的修复效能。因此，目前的挑战在于构建出一种可特异性募集高成骨潜能细胞并诱导其成骨分化、同时可高度适应缺损部位形状的原位骨修复材料。.主要研究内容与重要结果：我们首先制备出表面较为光滑的亚微米级CS微球，但发现BMSC难以在其表面粘附，故对其进行聚多巴胺（PDA）涂覆以改善细胞相容性。随后在微球表面接枝CD271抗体，并验证了抗体接枝的微球能够从周围环境中捕获BMSC。接着我们利用BMSC及人外周血单个核细胞的混合细胞群证明了CD271抗体修饰微球可从复杂细胞环境中特异性募集大量BMSC至表面。我们还通过大鼠股骨髁缺损部位观察到了CD271抗体修饰微球于体内募集大量的CD271+干细胞，最终促进了缺损的修复。随后，我们对微球进行改进，制备出了多孔CS微球，且BMSC可直接于微球表面粘附增殖，细胞相容性大幅度提升。我们对微球进行CD271抗体接枝后还利用其多孔结构负载了BMP-2，验证了新型微球同样可以有效介导BMSC的迁移和募集。同时，适当浓度的BMP-2可以显著提高成骨相关蛋白的表达，并促使细胞矿化形成钙结节。最后，我们采用鱼明胶包裹微球，构建出具有良好注射性可注射原位骨修复材料，其注射后能高度适应局部形状。.关键数据及意义：制备了CD271抗体修饰微球并验证了CD271抗体特异性募集BMSC的能力，引入BMP-2有效促进募集后细胞成骨分化，并最终构建出可注射原位骨修复材料，有望为骨缺损修复提供一种新的高效、安全的治疗方法。.项目执行期间，共发表SCI论文2篇，北大核心期刊论文1篇，撰写发明专利1项，参加学术会议1次。