以液态胶原为支架构建组织工程化心肌组织的实验研究

目的 探索以液态胶原作为支架材料于体外构建组织工程化心肌组织。方法 将分离并培养的新生大鼠心肌细胞与添加了基质因子的液态Ⅰ型胶原混合,在环形槽中铸成环状心肌细胞／胶原条带。构建的条带在环形槽中培养7d后取出,在对其施加10％的静态拉伸的情况下,再培养7d。通过光学显微镜、常规HE染色、免疫组织化学染色和透射电镜观察等对构建的组织工程化心肌组织进行评价。结果 构建的工程化心肌组织在施加静态拉伸的第2天可观察到局部跳动的区域,随时间的延长跳动的区域逐渐增多,强度逐渐增大,并最终形成一致跳动。组织学和免疫组织化学染色研究结果显示,在构建的工程化心肌组织中,心肌细胞比较均匀地分布在整个条带中,且多数沿拉伸方向伸展,胞核呈长梭形,其形态类似于天然成熟心肌组织。透射电镜观察显示,条带中心肌细胞含有排列整齐的肌原纤维,并沿着细胞的长轴方向伸展,肌小节结构和Z带清晰可见。结论 用液态Ⅰ型胶原可以构建类似天然心肌组织的工程化心肌组织。

Experimental study of tissue-engineered heart tissue using type I collagen as scaffold

OBJECTIVE: To construct tissue-engineered heart tissue (EHT) using liquid collagen as scaffold. METHODS: Neonatal rat cardiac myocytes were isolated, cultured, and mixed with liquid collagen type I and matrix factors and then cast in circular molds to construct circular cardiac myocytes/collagen strand. After a 7-day culture in circular molds, the strands were removed, and subjected to 10% static stretch for another 7 days. Microscopy and transmission electron microscopy, routine HE staining and immunohistochemical staining were used to analyze the engineered heart tissue. RESULTS: Beating areas could be seen on the surface of the EHTs at the second day after stretching; more beating areas could be seen thereafter. These areas beat stronger and stronger, and finally came to synchronzation. Histological and immunohistochemical analyses showed that the cardiac myocytes in the EHTs distributed evenly in the whole strand and the majority of the cells, with elongated nuclei, stretched along the stretching direction. The morphology of EHTs resembled that of the native adult cardiac tissue. Transmission electron microscopy revealed that the cardiac myocytes in EHTs contained arranged myofibrils oriented parallel to the longitudinal cell axis. Clearly defined sarcomeres and Z lines were observed. CONCLUSION: Liquid type I collagen is a good scaffold for generation of EHTs similar to the native heart tissue.