三维动态培养构建注射型组织工程软骨远期修复效果的实验研究

目的三维诱导自体细胞软骨分化构建注射型组织工程软骨，探讨动物模型的远期修复效果。方法 分离培养兔自体骨髓间充质干细胞，分别三维动态诱导及二维平面培养诱导，诱导软骨细胞分化并鉴定，比较分化效果。分别收获两种方法诱导后的细胞，与蛋白胶混合制备注射型组织工程软骨，注射修复兔关节软骨缺损。随机设定三维动态培养组、二维平面培养组及空白对照组3组，24、48周后，观察大体和组织学形态，组织学评分比较远期效果。结果二维平面培养诱导后只有低水平的蛋白多糖沉积和Ⅱ型胶原等标志物表达，三维动态培养后的分化质量明显提高，大量表达蛋白多糖及Ⅱ型胶原。在动物模型中，三维动态培养组：24、48周后缺损基本修复，表面光滑坚韧，修复组织与周围软骨无界限，仍保持类透明软骨形态。二维平面培养组：24、48周后明显退化，失去软骨组织形态。空白对照组：无明显修复，缺损长期残留。结论三维动态培养显著改善自体MSC注射型组织工程软骨的修复效果，为提高关节软骨的微创修复提供新思路。

Long-term effect of three-dimensional cultured injectable engineering cartilage on repairing of joint cartilage defects

Objective To induce chondrocyte differentiation from autogenous cells by three-dimensional（3D） dynamic culture, and examine the long-term effects of injectable tissue engineering cartilage on reparing the cartilage defect in animal model. Methods The autogenous marrow mesenchymal stem cells （MSC） were separated from rabbits then induced with 3D dynamic and two-dimensional （2D） culture respectively for chondrocyte differentiation. The differentiated cells＇were harvested separately and mixed with fibrin sealant to prepare injectable engineering cartilage. Rabbit femoral trochlea joint cartilage defect model were adopted, and randomly divided into 3 groups ie. 3D culture group, 2D culture group and control blank group. After 24,48 weeks, animals were sacrificed. Gross and histological morphology and histological scores were adopted in comparing the long-term effects. Results In 2D culture group,low-level chondrocyte differentiation were found,but in 3D culture group cells highly differentiated and obviously expressed collagen Ⅱ and proteoglycan were found. In the animal model in vivo,the 3D group showed most defects were repaired with sustaining hyaline-like cartilage appearance and smoothly connected with surrounding tissues after 24,48 weeks,but the 2D culture group showed cartilage degeneration and lose their hyaline cartilage histological appearances after 24,48 weeks. Repairing were not found in control group. Conclusion The 3D culture cells can improve the long term effects of MSC derived injectable engineering cartilage on repairing cartilage defects, and may be a preferable approach for mini-invasive joint surgery.