脂肪干细胞体内软骨形成过程中细胞材料复合物稳定性欠佳的机制及对策研究

Due to poor regenerative capacity of cartilage in vivo, the repair of cartilage defects is always a great challenge in surgery therapy. Tissue engineering is a promising method to repair cartilage defects in regenerative medicine. The adipose-derived stroma cells (ADSC) are easy to harvest in plastic surgeries with minimal insults to the patient. Their chondrogenic sub-population have also been isolated in our previous studies. Therefore it is an ideal candidate for the cell source of cartilage tissue engineering. However, the currently ADSC- engineered cartilage in vitro is unsatisfactory for clinical application due to the deformation and ectopic ossification after subcutaneous implantation. As to the deformation, we employed the microtubule oriented PLGA scaffold to improve the structure of engineered cartilage.As to the ectopic stability,it has been widely reported that endochondral ossification mainly results from angiogenesis and thus anti-angiogenesis becomes the major obstacle for clinical application of the ADSC-engineered cartilage. Chondromodulin-1 (CHM1), one of the anti-angiogenic factors during embryonic chondrogenesis, plays the most important role in maintaining the cartilage stability. But it remains unclear whether CHM1 could modulate the ectopic chondrogenesis in ADSC-based cartilage engineering. Our preliminary study has already showed that the CHM1 expression might be correlated with ectopic angiogenesis of ADSC-engineered cartilage. Based on the preliminary study, this research project will investigate the indispensability of CHM1 during ADSC-based cartilage engineering through the comparison of both in vitro construction and in vivo fate between the CHM1 knockout and widetype mice. Furthermore, we will attempt to apply the CHM1 controlled release system in microtubule oriented scaffold in ADSC-based cartilage engineering, which might lay the foundation for the clinical application of cartilage engineering.

组织工程技术是软骨再生的一种全新手段。然而，目前细胞材料复合物存在收缩变形和体内皮下稳定性欠佳两个问题，而耳、鼻等软骨的修复均需保持精确外形且位于皮下环境。保持软骨稳定性的关键在于抗血管化，项目组前期研究表明抗血管化因子软骨调节素1(CHM1) 很可能是影响构建软骨异位稳定性的关键因素，本课题拟采用前期研究已证实能对抗收缩变形问题的取向性微管材料为支架，以整复外科来源最为丰富的脂肪组织为细胞来源，根据前期结果分选其成软骨潜能亚群为种子细胞，通过CHM1基因敲除与野生型小鼠脂肪干细胞（ADSC）构建软骨的体内外系统比较，探索CHM1表达与 ADSC构建的软骨稳定性之间的因果关联，阐明CHM1调控ADSC体外软骨形成及体内异位稳定性的重要作用与机制，在此基础上应用缓释CHM1的取向微管材料复合ADSC构建稳定的软骨，以期为临床耳、鼻软骨缺损提供自体移植物，使软骨再生能真正转化为临床应用。

在前期研究中阐明了CHM1对于骨髓间充质干细胞再生软骨异位稳定性作用的理论基础上，我们在探索CHM1对于脂肪干细胞再生软骨异位转归中的作用进行探索，并提取CHM1的人及鼠类蛋白，以外添加的方式，观察对于干细胞构建软骨的异位稳定性的作用。.由于ADSC更广阔的应用前景及其构建的软骨在皮下环境相对与BMSC更容易发生血管化，因此，抗血管化问题对ADSC的软骨再生显得更加关键。在三维度软骨诱导中。虽然 ADSC具有软骨分化潜能，但是与BMSC相比软骨分化效率具有较大差异，阻碍了ADSC应用于软骨再生。产生这种差异的主要原因是脂肪来源细胞是一群混杂的细胞，软骨潜能细胞含量较低。尽管根据前期研究将脂肪来源细胞分为具有和不具有软骨分化潜能的两类克隆群，筛选出具有软骨分化潜能细胞亚群的特异性分子标志CD204。而且应用分选后的ADSC已能在体外构建出均质性的软骨，极大的提高了转化效率。 通过优化脂肪干细胞的筛选及三维软骨构建的流程，有效提高实验效率和降低课题费用的浪费风险，采取磁柱分选而不是流式细胞分选，这样能够有效率的提高分选速度，减少对细胞活力的损害，同时也能够保证较高的分选后细胞数量。.通过无血清加bFGF方案的诱导，体外5周促血管化因子占优势，异位植入后发生血管化及骨化；体外10周抗血管化因子占主导，异位植入后软骨表型稳定，进一步确证了血管化与ADSC异位软骨形成稳定性间之间可能存在关联。据此得出，ADSC构建软骨在皮下环境中发生的血管化进而纤维化，可能与抗血管化因子与促血管化因子的分泌失衡相关。.我们对敲除小鼠的发育软骨形成过程进行分析，找到此蛋白对于软骨发育过程中的必要证据。在具体纯化并提纯蛋白后，我们尝试将材料复合CHM1蛋白，由于蛋白的活性和降解时间的限制，复合时间只能维持在 14天的有效释放浓度，因此我们尝试在体外构建过程中采用外添加的方式，将CHM1重组蛋白加入诱导液，观察其对于ADSC构建软骨的作用，在次过程中调整诱导液CHM1蛋白的添加剂量及添加时间，找出了最佳的CHM1作用时间和作用剂量。