基于同轴生物打印技术的骨/软骨双相GelMA水凝胶中空套管重建韧带-骨界面及机制的研究

The formation of ordered calcified fibrocartilage as the transition structure in the ligament-bone interface is one of the most difficult and attractive issues for the ligament-bone healing. In our previous study, 3D printed polylactic acid/hydroxyapatite (PLA/HA) screw scaffolds loaded with bone marrow mesenchymal stem cells (BMSCs) improved the ligament-bone healing, the formation of calcified fibrocartilage surrounding the ligament graft was not desirable. In addition, we found that the overexpression of cartilage oligomeric matrix protein (COMP) enhanced the chondrogenic differentiation of BMSCs, and up-regulated the marker of calcified fibrocartilage, collagen X. However, the mechanism has not been established. The present study attempts to fabricate osteochondral biphasic gelatin methacryloyl (GelMA) hydrogel hollow cannulas loaded with BMSCs via coaxial bioprinting. The chondral phase (inner layer) promotes the chondrogenic differentiation of BMSCs via overexpression of cartilage oligomeric matrix protein (COMP); the osteal phase (outer layer) uses bioceramics to induce osteogenic differentiation of BMSCs, and up-regulates the expression of integrin α5β1. We propose a hypothesis that, the binding of the overexpressed COMP and integrin α5β1 switches on integrin α5β1-FAK signaling pathway in the junction area between two layers, inducing the concentric ring formation of calcified fibrocartilage. Moreover, this research attempts to explore and reveal the mechanism of calcified fibrocartilage formation in vitro and in vivo, which could provide new ideas for ligament-bone healing combined with mechanical manufacture.

韧带移植物-骨界面内有序钙化纤维软骨过渡结构的形成是促进界面愈合的研究热点和难点。前期研究中我们利用三维打印聚乳酸/羟基磷灰石支架负载骨髓间充质干细胞（BMSCs）植入骨隧道虽促进了界面整合，但环绕移植物排布的钙化纤维软骨形成不理想。此外，我们发现过表达软骨寡聚基质蛋白（COMP）的BMSCs成软骨分化同时上调钙化纤维软骨标志Collagen X的表达，具体机制尚未阐明。本课题拟同轴生物打印构建骨/软骨双相GelMA水凝胶中空套管：软骨相（内层）载COMP过表达的BMSCs成软骨分化；骨相（外层）由生物陶瓷诱导凝胶内BMSCs成骨分化。由此提出假说：双相交界区内BMSCs过表达的COMP可与整合素α5β1配体受体结合启动整合素α5β1-FAK信号通路，诱导生成同心环形排布的钙化纤维软骨。本研究将通过体内外实验探索并揭示钙化纤维软骨的生成机制，为结合机械制造促进移植物-骨界面整合提供新思路。

韧带移植物-骨界面内有序钙化纤维软骨过渡结构的形成是促进界面愈合的研究热点和难点。本项目通过构建成骨/软骨生物墨水，在原软骨相生物墨水设计的基础上，探究GelMA水凝胶与GDF-5相结合用于成软骨相关研究，探究成软骨相关分子机制。GDF5-GelMA组的成软骨标志基因Col2和ACAN显著升高。免疫荧光显示，搭载GDF5的GelMA表面细胞外基质Col2和ACAN分泌明显增加。ADSCs番红染色明显增强，表明成软骨微球具有显著的成软骨特性。在骨相生物墨水设计的基础上，利用掺镁硅酸盐陶瓷材料CSi-Mg制备成骨髓内钉棒，通过大鼠骨缺损模型探究成骨相关机制。骨相生物墨水能促进细胞黏附和BMSCs的成骨分化，并提高细胞活性，同时能促进p38的磷酸化，调节促炎症因子的合成或释放。此外，还能影响极化相关的分泌和巨噬细胞的表达，通过释放硅离子、镁离子和钙离子，诱导巨噬细胞向M2型极化。最后，将骨/软骨生物墨水结合生物打印骨/软骨一体化植入材料，以界面螺钉形式植入骨隧道，开展韧带移植物-骨界面愈合的修复研究。研究显示骨/软骨组术后MRI显示其韧带移植物重建后再生能力及血管化性能更强，骨隧道内新生骨量显著提升，韧带-骨愈合效果良好，有望转化为ACL重建临床应用的内植物。