双向精准募集程序性诱导B-MSC和S-MSC内源性再生修复全层关节软骨的实验研究

The repair with high quality for full-thickness articular cartilage defect always is a challenge. Endogenous regeneration of cartilage is a novel concept to repair cartilage defect. But how to recruit sufficient effective stem cells continuously, a key problem, is still waiting to be resolved. Based on previous research, a hypothesis of “double gradient release, precise recruitment and programming differentiation” by growth factor is proposed in the present project. The platelet-derived growth factor (PDGF) and BMP-1，which can recruit and induce bone marrow derived mesenchymal stem cells（B-MSC）into bone differentiation respectively, are to be assembled on the bone phase (contacting with bone defect side) of the elastic chitosan/gelatin scaffold with layer-by-layer(LbL) electrostatic assembly technique. The stromal cell-derived factor (SDF-1) and TGF-β3, which can recruit and induce synovial membrane-derived MSC(S-MSC) into chondrogenous differentiation respectively, are to be assembled on the cartilage phase (contacting with the cartilage defect side) of the scaffold. The double continuous release to bone marrow and joint space is to be established. B-MSC with potent bone differentiation and S-MSC with potent cartilage differentiation are recruited simultaneously. The two type stem cells are to be induced into bone and cartilage differentiation respectively and programically. The full-thickness articular cartilage defect is to be regenerated endogenously. The cross talk between TGFβ3 and SDF-1 during recruiting and inducing S-MSC is to be investigated. The coordination and additional protective effect of TGFβ3 will be explored. A novel “one-step” repair method for full-thickness articular cartilage defect with high quality will be explored. The experiment foundation for its practical application will be established.

全层关节软骨高质量修复是个难题，“内源性再生”修复是一新思路，但持续募集诱导足量有效干细胞之关键问题尚待解决，本申请拟在以往研究基础上，提出“双向梯度释放、精准募集、程序性诱导干细胞”假说，应用静电层层自组装技术，在自身弹性固定三维支架的骨相（支架与骨接触层），组装对骨髓来源B-MSC有强募集作用的PDGF和定向诱导其成骨分化的BMP-1，在该支架软骨相（支架与软骨接触层）组装对滑膜来源S-MSC具有强募集作用的SDF-1和定向诱导其成软骨分化的TGFβ3，实现向骨髓和关节腔双向、长时释放，精准募集成骨潜能强的B-MSC 和成软骨潜能强的S-MSC，同期程序性诱导两类干细胞分别成骨、成软骨分化，修复全层关节软骨缺损；并就TGFβ3对SDF-1 是否具有募集协同作用，是否具有软骨修复保护作用的信号通路深入研究，探索关节软骨“一站式”修复新途径，并为其实际应用奠定实验基础。

关节软骨修复是临床科研中的难题，关节软骨原位再生目前被认为是最有前景的修复方法，而缺损修复部位的机械力、支架材料的选择及小分子药物可能影响软骨修复效果。..本项目中收集因骨关节炎行膝关节置换患者膝关节标本，提取人膝关节正常软骨细胞，利用不同时长不同浓度IL-1β诱导关节炎软骨细胞，发现IL-1β刺激后，软骨细胞增殖活性较刺激前增加，二型胶原蛋白含量在刺激3天时明显下降，RNA水平有所增加，蛋白聚糖RNA水平较刺激前下降；细胞骨架染色提示正常软骨细胞中微丝成束状平行排列，而关节炎软骨细胞骨架紊乱，疏散成网状分布；对调控细胞骨架的关键蛋白cofilin行免疫荧光染色，发现cofilin荧光强度及面积增多且伴有明显的核转位现象。进一步借助甲基丙烯酰化明胶（Gelma）行 3D培养，利用Flexcell-5000c对软骨细胞行压力刺激，发现较小压力刺激下，细胞外基质二型胶原和蛋白聚糖仍有下降，基质金属蛋白酶各亚型含量升高，且伴随整合素-Rho-CFL通路的激活。..为探索高效软骨修复的材料，本项目中采用高能球磨法制备纳米级β-TCP颗粒，采用溶液冷冻干燥法制备PLA/纳米级β-TCP多孔支架，同时制备丝蛋白混合水凝胶，通过化学键交联构建双相支架，有较强的力学性能。利用负载药物的复合支架（PLLA/β-TCP/Gelatin-RSF）修复兔膝关节软骨缺损，载药组3D骨相及软骨相修复更佳。..小分子化合物甲基巴多索酮(Bardoxolone methyl，BM在软骨修复中通过抑制软骨细胞氧化应激而发挥作用，研究发现BM能部分缓解氧化应激下软骨细胞增殖能力、细胞内活性氧自由基水平、下调线粒体膜电位、抑制凋亡。小分子BM能明显抑制氧化应激引起的软骨基质丢失，促进软骨细胞及软骨下骨内间充质干细胞分泌细胞外基质二型胶原及蛋白聚糖。