纳米HA/CS支架穿“靴”复合软骨样细胞修复兔关节软骨和软骨下骨缺损

摘要

[Objective] To observe the condition of constructing the tissue engineered chondral composite constructing by co culturing the chondrocyte which were induced from bone marrow stem cells (BMSCs) and nano-hydroxyapatile/chitosan scaffold (nano-HA/CS), under a boot-shaped structure after compositing. The effect of the composite on repairing the osteochondral defect of rabbits' knee were observed. [Methods]The technique of combining the in situ hybridization and freeze-drying was taken to fabricate nano-HA/CS scaffold. The cylindrical boot-shaped structure were made of polytetrafluoroethylene. The chondrocyte were induced from BMSCs by cultivating in the chondrogenic differentiation medium. Then diey were seeded into the bottom of the nano-HA/CS scaffold whose top was coverd by the boot-shaped structure. The composite was cultivated in the chondrogenic differentiation medium for 2 weeks. The structure of nano-HA/CS scaffold and composite was observed by scanning electron microscope. The osteochondral defects in the epicondyle of femur, which was 4 mm in diameter and 8 mm in depth, were constructed in 30 New Zealand white rabbits. The rabbits were divided into three groups, experimental group (composite induced with boot-shaped structure grafted into the defect), control group (composite induced without boot-shaped structure), and blank group (with no implant). Finally the curative effect of specimens after 4 and 12 weeks were evaluated respectively. [Result] BMSCs can be induced to chondrocyte after cultivated in the chondrogenie differentiation medium. The nano-HA/CS scaffold with 92% interstice ratio and 125 (un diameter had a good adhesion with chondrocyte. In general, cartilage-like tissue were found in experimental group and control group, while fibrous tissue were found in blank group where the defect was still obvious. The HE staining showed that the cartilage defect was repaired by chondrocyte partly and the bone defect was repaired by osteoblasts partly in the experimental group. There was a crossing area in the cartilage-bone interface. Thus the repairing effect and coupling status of cartilage-bone interface in the experimental group was superior to the control group and blank group obviously. Statistical analysis of gross specimen and ehondral histology also showed that the experimental group was superior to the other two groups. [Conclusion] BMSCs have the chondrogenie potentiality. Nano-HA/CS scaffold has good adhesion with chondrocyte. By co-culturing chondrocyte and Nano-HA/CS under a boot-shaped structure can construct the tissue engineered ehondral composite, which was proved to be able to repair osteochondral defect in rabbits.%[目的]观察把骨髓间质干细胞(BMSCs)诱导成的软骨细胞与纳米羟基磷灰石/壳聚糖支架(nano-HA/CSscaffold)通过特殊的穿“靴”结构复合构建而成组织工程软骨复合体的可行性,并观察此复合体修复兔膝关节软骨及软骨下骨缺损的能力.[方法]原位复合和冷冻干燥相结合的方法制备nano-HA/CS支架,用聚四氟乙烯制作成圆柱形“靴”结构.把BMSCs经软骨诱导液诱导成软骨细胞后,接种于穿“靴”的nano-HA/CS支架底部,把细胞一支架复合物置入成软骨条件培养液中培养2周.扫描电镜观察nano-HA/CS支架结构及细胞一支架复合体结构.30只新西兰大白兔的股骨髁制造直径4mm、深8mm的骨软骨缺损,随机分为实验组、对照组、空白组,于缺损处分别植入经穿“靴”结构复合的软骨支架复合体、不经穿“靴”结构复合的软骨支架复合体,空白组仅造缺损,4、12周后取材初步观察修复情况.[结果]BMSCs经软骨诱导液诱导后转化成软骨细胞;制备的nano-HA/CS支架支架孔隙率为92％,平均孔径为125μm,与软骨细胞有较好的粘附性.大体观察实验组和对照组缺损均有类软骨样组织生长,空白组缺损明显,见纤维组织生长.苏木精-伊红染色切片观察实验组软骨缺损部分软骨细胞修复,成骨区部分骨样细胞修复,两者耦合处部分交叉,修复缺损程度及成骨区和成软骨区界面耦合情况明显优于对照组和空白组.3组的大体评分和软骨组织学评分统计分析,实验组优于对照组和空白组.[结论]BMSCs具有成软骨潜能,nano-HA/CS支架具有较好的细胞相容性；软骨细胞与nano-HA/CS支架经过穿“靴”结构可成为组织工程软骨复合体,并初步证实有修复兔软骨及软骨下骨缺损的能力.