可控式SDF-1α过表达介导体内BMSCs定向趋化的组织工程骨构建策略及其修复骨缺损的实验研究

Autologous bone marrow-derived mesenchymal stem cells (BMSCs) remains as one of the most effective source cells for tissue-engineered bones, however, its use is limited by the requirement of long-term cultivation in vitro and high expense. Recent researches have demonstrated that SDF-1α plays an important role in the migration process of BMSCs. Therefore, this study aims to design a novel tissue-engineered bone that can automatically capture autologous BMSCs in vivo, and to evaluate its effect on repairing bone defects. Xenogenous rat BMSCs transected with SDF-1α gene are used as source cells and transplanted onto microlized deproteinized bone matrix to form a novel 3-D composite bone substitute, using a rotary cell culture system. Through the signal pathway of SDF-1α/CXCR4, this new bone substitute can automatically recruit the autologous BMSCs in the peripheral circulation by the over-expression of SDF-1α, and thus efficiently promoting the process of bone regeneration after transplanted into bone defects. And then the SDF-1α gene will be equipped with hypoxia-induced promoter elements as a switch, by which the cells can secret high concentration of SDF-1α in low-oxygen conditions and shut it up when in normal-oxygen conditions. This site-directed recruitment of autologous BMSCs in vivo to accelerate bone healing by timely over-expression of SDF-1α will provide a beneficial way for bone tissue engineering, and greatly contribute to the eventual design of an clinically usable tissue-engineered bone.

自体骨髓间充质干细胞(BMSCs)是骨再生关键参与者，也是组织工程骨最有效的种子细胞之一，但由于其体外分离培养时间长，临床运用一直受限。本项目设想构建一种能在体内主动捕获BMSCs参与骨再生的组织工程骨，并观察它对骨缺损的修复作用。将转染基质细胞衍生因子SDF-1α基因的大鼠异体BMSCs作为种子细胞，借助三维旋转培养种植于脱蛋白骨微载体上形成复合型骨替代物，使之移植后能快速血管化以保证种子细胞成活并分泌SDF-1α，而近期研究已证实SDF-1α具有趋化BMSCs作用，因此可以通过SDF-1α/CXCR4信号轴介导主动趋化外周血中的BMSCs参与骨再生。在此基础上将低氧反应元件导入SDF-1α基因上游作为调控开关，使其在低氧条件下高表达，而在恢复常氧后基因过表达及时终止。这种利用SDF-1α适时适度表达来诱导体内BMSCs定向迁移和成骨的策略，将为构建临床可用的组织工程骨提供新的理论依据。

自体骨髓间充质干细胞(BMSCs)是骨再生关键参与者，也是组织工程骨最有效的种子细胞之一，但由于其体外分离培养时间长，临床运用一直受限。本项目设想构建一种能在体内主动捕获BMSCs参与骨再生的组织工程骨，并观察它对骨缺损的修复作用。.实验内容及结果：.1.成功通过PCR技术构建 pBOBi-9HRE-SDF-1α质粒载体，转染兔BMSCs后能够在体外低氧环境下实现高表达SDF-1α，常用环境下则正常表达SDF-1α。.2.筛选并比较分析骨质来源MSCs和骨髓来源MSCs的增殖和成骨分化能力，发现骨质和骨髓混合培养的MSCs增殖能力稳定，在成骨诱导分化能力上更有优势，更适合作为组织工程骨的种子细胞。.3.利用静电纺丝技术将PLGA联合万古霉素作为涂层附着在脱蛋白松质骨支架表面，通过体外抑菌和体内感染性骨缺损移植实验证实，该新型抗感染骨支架可以在体内外缓慢释放万古霉素，28天内维持有效抑菌浓度，是较理想的组织工程骨支架材料。.4.利用3D旋转培养技术将脱蛋白松质骨支架和转染9HRE-SDF-1α的BMSCs进行培养，成功构建可控式高表达SDF-1α的组织工程骨，并通过兔胫骨干骨缺损移植实验证实，该新型组织工程骨可以有效修复大段骨缺损。.5.通过局部注射SDF-1α给药的方式，证实SDF-1α联合支架材料Integra可以有效修复全层皮肤缺损创面，改善创面修复效果。.本项目创新性提出利用可调控的SDF-1α过表达来诱导自体内源性BMSCs定向迁移参与组织工程骨移植修复骨缺损的策略，以解决种子细胞分离扩增缓慢和体内移植后难以成活的难题，经过实验研究证实有效，为将来的组织工程骨研究提供了非常好的思路和方法。