采用温敏性壳聚糖水凝胶体外构建组织工程化软骨的实验研究

Reconstruction of tissue engineered cartilage using temperature-responsive chitosan hydrogel

目的验证自行制备的温敏性壳聚糖水凝胶作为软骨细胞支架材料构建组织工程化软骨的可行性。方法以壳聚糖、β-甘油磷酸钠和羟乙基纤维素为原料,制备温敏性壳聚糖水凝胶,通过SD大鼠肌内注射植入,进行组织相容性检测。在此基础上,将其与软骨细胞复合构建组织工程化软骨,观察软骨细胞在壳聚糖水凝胶中的存活情况,并于体外培养3周后,作相关组织形态学检测。结果制备的壳聚糖水凝胶具有温度敏感性,即室温时为液态,37℃时10～15min可发生交联反应成为固态凝胶。SD大鼠肌内注射不同时间点组织相容性检测表明,该材料具有良好的组织相容性,植入体内2、4周时有少量炎性细胞浸润,6周时材料降解明显,8周时已经基本降解。采用该材料构建的组织工程化软骨体外培养3周后取材,通过组织学检查,HE染色可观察到软骨陷窝样结构,甲苯胺蓝染色、番红O染色及免疫组化染色结果显示软骨细胞具有分泌细胞外基质的功能。结论本研究自行制备的温敏性壳聚糖水凝胶材料具有良好的组织相容性,将其与软骨细胞复合后,可以在体外再造组织工程化软骨,是一种有广泛应用前景的软骨组织工程支架材料。

Objective To reconstruct tissue engineered cartilage using temperature responsive chitosan hydrogel, which were prepared as scaffold materials. Methods Temperature responsive chitosan hydrogel was prepared using chitosan, β-sodium glycerophosphate and cellulose, and the histocompafibility of the hydregel was detected by injecting the hydrogel into rat muscle Then the tissue engineered cartilage was constructed by mixing rabbit chondrocytes expanded in vitro with the chitosan hydrogel. The solidification was very fast when the construction was set on 37℃. Then the construction was cultured in vitro and the survival and growth of the chondrocytes in the construction were observed under microscope The constructs were analyzed 3 weeks after culturing using histological and immunohistochemical methods. Results The chitosan hydrogel was temperature-responsive： it was liquid when be put on room temperature, and became solid hydrogel within 10 -- 15 minutes when be put on 37℃. The histocompatibility test suggested that this material has good histocompatibility. The chondrocytes retained their normal morphology in constructions when cultured in vitro and produced matrix The results of histology and immunohistochemistry confirmed that the chondrocytes in the chitosan hydrogel retained their ability to secrete matrix Conclusion The chitosan hydrogel has good histocompatibility. It may provide a good environment for chondrocytes to survive, growth and secrete matrix when used as tissue engineered scaffold material. Therefore, chitosan hydrogel is a good scaffold in cartilage tissue engineering.