仿生学组织工程软骨支架材料的制备及关节软骨缺损修复的实验研究

In cartilage tissue engineering, artificial synthetic material exist some problems such as degradation products with cytotoxicity, irretention cell phenotype etc. Now, natural scaffold materials were prepared by mixing one or several components of cartilage extracellular matrix. The chondrocyte microenvironment can not be completely simulated. The scaffolds prepared by freeze-dried acellular cartilage matrix-present in a particle state had poor mechanical properties, large pore size, and easily formed hollow phenomenon. In this study, we would use acellular cartilage matrix as raw material, the main components of them were present in a molecular state after enzymolysis, add the right amount of type II collagen, prepare nanofibrous bionic scaffold for cartilage tissue engineering by electrospinning technique, and optimize the physical properties through cross-linking. Then, we would seed BMSCs and chondrocytes in different proportions into the scaffold, construct tissue engineering cartilage in vitro, and repair articular cartilage defects in vivo. A small amount of chondrocytes and the nanofibrous scaffolds prepared by acellular cartilage matrix and collage typeⅡprovided a microenvironment. It could induce BMSCs into chondrogenic phenotype cells efficiently and steadily, and construct tissue engineering cartilage. With the completion of the research, we hope to provide a good scaffold material for cartilage tissue engineering, and promote the clinical application process.

在软骨组织工程中，以人工合成材料制备的支架存在降解产物具有细胞毒性、难以维持细胞表型等问题。以天然生物材料制备支架，主要是以一种或几种细胞外基质成分混合，无法完全模拟软骨的微环境，而以软骨脱细胞基质经低温冷冻制备的支架，其细胞外基质为微粒状态，机械性能欠佳，材料孔径较大，易形成"空心现象"。本研究拟用软骨脱细胞基质为原料，经酶解后使其主要成分以分子状态存在，再加入适量的Ⅱ型胶原，通过静电纺丝技术制备纳米级的仿生学组织工程软骨支架，经过交联优化物理性能。再将不同比例BMSCs/软骨细胞种植于该支架上，在体外构建组织工程化软骨及动物体内修复关节软骨缺损。本研究希望通过软骨脱细胞基质和Ⅱ型胶原构建的纳米支架与少量软骨细胞所形成的微环境，能高效稳定地将BMSCs定向诱导为软骨表型细胞，构建出组织工程化软骨。该计划的完成，有望为软骨组织工程提供一种良好的支架材料，并推动其临床应用的进程。

在软骨组织工程中，以人工合成材料制备的支架存在降解产物具有细胞毒性、难以维持细胞表型等问题。以天然生物材料制备支架，主要是以一种或几种细胞外基质成分混合，无法完全模拟软骨的微环境，而以软骨脱细胞基质经低温冷冻制备的支架，其细胞外基质为微粒状态，机械性能欠佳，材料孔径较大，易形成"空心现象"。本研究将兔肋软骨脱细胞、脱脂、酶解提纯后低温冷冻干燥获得软骨脱细胞基质（CAEM），再将CAEM和Ⅱ型胶原（COLⅡ）按一定质量比混合，利用静电纺丝技术制备 组织工程纳米支架，通过测定其吸水率、降解率等检测支架的理化性能，用CCK8法评价其细胞毒性及粘附性情况。将第二代骨髓基质干细胞(BMSCs)种植到该支架上培养箱内静置2小时。12只日本大耳白兔随机分为实验组和对照组，再将细胞支架复合物植入实验组兔膝关节软骨缺损处，对照组仅行膝关节软骨缺损造模。12周后处死实验动物取材，大体观察修复效果，并进一步行HE染色、Ⅱ型胶原染色观察。研究结果显示：该纳米支架纤维直径为627nm±165.4nm，吸水率为623.0%±27.4%，35天降解率为45.6%±5.8%，CCK8法检测结果显示CAEM-COLⅡ复合支架对软骨细胞具有良好的粘附性。大体观察实验组软骨缺损处修复良好，对照组软骨缺损处肉芽样组织充填。HE染色实验组关节软骨缺损处可见软骨陷窝形成，对照组关节软骨缺损处仅有纤维组织充填。实验组修复区Ⅱ型胶原染色为阳性，对照组为阴性。研究结果表明：该支架具有一定的机械强度、良好的空间结构和满意的生物学性能，具有用于软骨组织工程重建的潜力。该支架-BMSCs复合物对软骨缺损基本达到了组织学修复的效果，具有重要的应用前景。