复合软骨修复材料支架的构建

目的：构建一种脱细胞软骨/GelMA复合材料支架用于修复软骨缺损。方法：对α-1，3-半乳糖苷转移酶基因敲除（GTKO）猪肋软骨脱细胞处理，得到低免疫原性脱细胞软骨基质，进行4，6-脒基-2-苯基吲哚（DAPI）染色、DNA含量检测评估脱细胞效果，苏木素-伊红（HE）染色、Masson三色染色分别评估细胞外基质结构及蛋白保留情况。再将脱细胞软骨冷冻研磨成粉末状，选取聚合物甲基丙烯酰化明胶（GelMA）溶液与脱细胞软骨粉末按不同比例均匀混合，制备一种可注射型生物材料，经紫外交联后获得可生物降解的复合支架，通过溶胀性能、储能模量检测对支架进行表征，并观察支架上细胞增殖情况。结果：天然软骨DNA含量为161.2 ng/mg，脱细胞软骨DNA含量仅为15.27 ng/mg，该种脱细胞方法效果良好。HE染色显示细胞外基质结构完整，Masson三色染色显示脱细胞组织保留了大部分细胞外基质成分。与纯GelMA相比，脱细胞软骨/GelMA复合支架溶胀率低，力学强度稍强。骨髓间充质干细胞能在支架上黏附并增殖。结论：通过可注射型脱细胞软骨/GelMA复合材料制备的支架具有低免疫原性，适宜的储能模量，良好的生物相容性，适用于软骨修复。

Construction of composite scaffolds for cartilage repair

Objective: To construct decellularized cartilage/GelMA composite scaffolds for repairing the cartilage defects. Methods: The α-1, 3-galactosidase gene knockout (GTKO) porcine costal cartilage was decellularized to obtain low immunogenic decellularized cartilage matrix. DAPI staining and DNA content detection were used to evaluate the effect of decellularization. HE staining and Masson trichrome staining were used to evaluate the extracellular matrix structure and protein retention. The decellularized cartilage was frozen and ground into powder, the polymer gelatin methacryloyl (GelMA) solution and decellularized cartilage powder were uniformly mixed in different proportion to prepare an injectable biomaterial, then obtained biodegradable composite scaffolds by ultraviolet crosslinking. Finally, the scaffolds were characterized by swelling performance, storage modulus and cell proliferation. Results: The DNA content of natural cartilage was 161.2 ng/mg, and the decellularized cartilage was only 15.27 ng/mg, so this method had a good effect on decellularization. HE staining showed that the extracellular matrix structure was intact, and Masson trichromatic staining showed that most of the extracellular matrix components were retained in decellularized cartilage. Compared with pure GelMA, the decellularized cartilage/GelMA composite scaffolds had lower swelling ratio, stronger mechanical strength. Bone marrow mesenchymal stem cells could adhere to scaffolds and proliferate. Conclusion: The scaffolds prepared by injectable decellularized cartilage/GelMA composite have low immunogenicity, suitable storage modulus, good biocompatibility, and are suitable for repairing cartilage defects.