间充质干细胞复合其细胞外基质在修复骨关节炎软骨缺损中的应用及其机制研究

Osteoarthritis (OA) is a common degenerative disease, which is often accompanied by cartilage defect. Up to date, cell hypertrophy, insufficient mechanical property, and low cartilage adhesion remain big challenges in tissue engineering repair of OA cartilage defect. Applicant’s study revealed that mesenchymal stem cell (MSC) derived extracellular matrix (ECM) could impregnate MSC to form MSC/ECM constructs, which could form engineered cartilage with low hypertrophy, mechanical property similar to that of native articular cartilage, and fill in cartilage explant defect with good adhesion with surrounding tissue in vitro. The low hypertrophic phenotype of MSC/ECM could be maintained subcutaneously in a mice model. Past literature reported that cartilage matrix production and components were correlated with the mechanical property and cartilage adhesion ability of engineered cartilage; Condensation degree was correlated with both cartilage matrix deposition and hypertrophic changes; Neural Ca2+ Dependent Cell Adhesion Molecule (N-cadherin) expression was positively correlated with Condensation degree. From the above, the applicant came to the hypothesis that MSC/ECM constructs could form engineered cartilage with reduced hypertrophy, mechanical properties similar to native cartilage and good integration with surrounding tissue at defect site under OA condition to repair OA cartilage defects and N-cadherin played a very important role in it. This project plans to test the hypothesis both in vitro and in vivo by using overexpression transfection, siRNA interference and atomic force microscopy technique, aiming to explore new strategies for OA cartilage repair via tissue engineering approaches.

骨关节炎（OA）是常见关节退行性疾病，常有软骨缺损。目前细胞肥大、机械强度不足及软骨结合力低是组织工程软骨修复OA软骨缺损的难题。申请人的研究发现间充质干细胞（MSC）复合其细胞外基质（ECM）形成的干细胞-ECM复合体可以在体外形成低肥大、高机械强度、强软骨结合能力的工程软骨并能在小鼠皮下保持低肥大。文献报道软骨基质及其相关组分与工程软骨机械强度、结合能力相关；MSC细胞凝聚（Condensation）与软骨基质生成及肥大相关；同时神经型钙离子依赖的细胞粘附素（N-cadherin）表达与凝聚强度正相关。提出假说：干细胞-ECM复合体可在OA关节中形成低肥大、高强度、强结合的工程软骨修复软骨缺损且N-cadherin在其中发挥重要作用。本项目拟采用过表达转染、siRNA干扰、原子力显微镜检测等技术，在体外与活体动物两个层面证实以上假说，旨在为组织工程修复OA关节软骨缺损提供新思路。

骨关节炎（OA）是最为常见的关节炎类型，常产生软骨缺损，且软骨缺损可进一步加重OA，但目前尚缺乏安全有效的治疗方式。基于水凝胶的软骨组织工程技术目前具备治疗潜力，但仍存在依赖交联剂、组织粘附性有限、缺乏细胞募集能力、种子细胞需手术提取和体外培养等缺陷。针对上述难题，本项目在FDA批准药用辅料F127基础上，创新通过多巴胺（Dopa）修饰引入邻苯二酚基团并利用辣根过氧化物酶（HRP）催化不同剂量过氧化氢（v/v）进行氧化，合成温敏性F127-DA-HRP 水凝胶。红外谱图、核磁谱图及相转变检测：在F127单体基础上，成功枝接了Dopa基团，并在摄氏20度下快速完成相转变；扫描电镜检测：温敏性F127-Dopa-HRP 水凝胶具有清晰明显的三维网络和微观孔隙结构；流变学测试：相比 F127 水凝胶，温敏性F127-Dopa-HRP 水凝胶具有更高的剪切弹性模量；粘附力测试：相比F127水凝胶，温敏性F127-Dopa-HRP 水凝胶具备更强的复合粘附力；体外骨髓间充质干细胞（MSC）相关实验：扫描电镜、 Calcein AM染色显示随着过氧化氢比例提升，F127-Dopa-HRP中出现明确的MSC粘附及聚集现象。体内软骨缺损修复实验：在缺损部位植入F127-Dopa-HRP 水凝胶4周后，番红固绿染色结果显示相比非填充组，水凝胶填充部位产生了连续均匀的新生软骨基质沉积。.本项目首次构建的温敏性F127-Dopa-HRP 水凝胶具有较好的温敏性能、流变学性能，具备一定的机械性能及粘附性，可有效支持MSC粘附及聚集，并可在体内有效修复关节软骨缺损，可望为骨关节炎软骨缺损修复提供潜在新方法。