利用聚羟基乙酸构建组织工程皮肤的实验研究

In vitro skin engineering using polyglycolic acids

目的 以聚羟基乙酸 (PGA)为培养支架 ,用组织工程方法在体外构建双层、有活性皮肤。方法 应用酶消化法分别获取人成纤维细胞 (FB)和表皮角质形成细胞 (KC) ,体外扩增培养。接种HF与PGA上 1周后 ,在复合物表面接种KC ,共培养 1周将复合物置于气 液界面继续培养。于不同时间段取材 ,行组织学、超微结构及免疫组织化学检测。结果 接种表皮角质形成细胞在“FB PGA”复合物表面 1周后可见 2～ 3层连续的HK在复合物上生长良好 ;4周时PGA已部分降解 ,表皮分层分化较完善 ,抗involucrin染色阳性 ,透射电镜观察可见表皮细胞分层分化良好 ,细胞的相邻面有桥粒连接。结论 采用组织工程技术可以在体外构建双层、有活性的人工皮肤 ;组织工程皮肤具有和正常皮肤相似的组织学特征及生化成分。

Objective To investigate the possibility of engineering full-thickness skin in vitro using polyglycolic acids (PGA) fibers.Methods Human keratinocytes (KC) and dermal fibroblasts (FB) were isolated from foreskin and cultured in vitro.FB were first seeded on woven PGA fibers and co-cultured for one week.The KC were then seeded on the top of FB-PGA construct and cultured in a submerged fashion.The artificial skin was then further cultured at air-liquid interface for another 2 or 3 weeks.Engineered skin was evaluated by H&E and immunohistochemical staining and electron microscope at different time points.Results Histology demonstrated that seeded KC started to form an epidermal structure after one-week submerged culture,which was composed of single or double cell layers and evenly covered the construct.At 4th week,more PGA fibers were degraded than before and a neo-dermis rich in newly formed collagen fibers was formed.On the top,a more matured epidermis was formed.This maturation was also confirmed by the positive staining of involucrin.Transmission electron microscopy also revealed the well-differentiated KC and desmosome was observed between the individual KC.Conclusion Double layers and viable full-thickness skin can be in vitro engineered with synthetic biomaterial polyglycolic acids.The engineered skin possesses histological structure and biochemical components similar to native skin tissue.