无细胞真皮支架与同种细胞相容性的动态观察

Cytocompatibility of acellular dermal scaffolds with allogenic cells

背景:无细胞真皮作为真皮替代材料已广泛应用于临床皮肤缺损的修复,能否将其应用范围扩展使其作为真皮支架用于组织工程皮肤的构建呢?目前国内外对这一方面都在进行着大量的研究。目的:动态评价同种无细胞真皮基质的细胞相容性,验证其作为皮肤组织工程支架材料的可行性。设计、时间及地点:单一样本观察,于2007-10/2008-03在中国辐射防护研究院组织工程实验室完成。材料:试验中皮肤样品源自武警山西总队医院行包皮环切术的健康儿童切除的包皮。方法:采用高渗盐-SDS-胰蛋白酶法制备同种无细胞真皮,组织块贴壁法培养人成纤维细胞,使其接种于支架材料上,通过苏木精-伊红染色以及扫描电镜观察成纤维细胞在支架材料上的贴附、生长、增殖以及迁移情况。主要观察指标:①同种无细胞真皮基质的形态结构。②无细胞真皮基质的细胞相容性。结果:同种无细胞真皮基质肉眼观察为乳白色,柔韧有弹性,苏木精-伊红染色和扫描电镜显示表皮以及真皮乳头层和网状层细胞以及细胞成分已经完全去除,且真皮的胶原网状支架保留完整;接种该支架材料上的成纤维细胞生物学行为活跃,不仅能够在其表面贴附、生长、增殖,并沿细胞间隙向真皮内部浸润生长,穿过乳头层达到网状层,而且分泌细胞外基质,填充细胞间隙形成膜片样结构。结论:通过高渗盐-SDS-胰蛋白酶法制备的无细胞真皮具有良好的细胞相容性,可作为真皮支架材料用于组织工程皮肤的构建。

BACKGROUND： Acellular dermal matrix （ADM）, as a dermal substitute, has been widely applied to repair skin defects in clinics. However, whether it is feasible for the ADM to be developed as the dermal scaffolds？ This question has been concerned by the domestic and overseas scholars but without conclusion. OBJECTIVE： To dynamically study the cytocompatablhty of the allogemc ADM, and to verify the feasibility of being as the skin tissue engineering scaffolds. DESIGN, TIME AND SETTING： Single sample study was performed in Tissue Engineering Laboratory of China Institute for Radiation Protection from October 2007 to March 2008. MATERIALS： The foreskin was donated from the healthy children who were hospitalized in Shanxi Provincial Corps Hospital, Chinese People＇s Armed Police Forces. METHODS： Hyper tonic salt-SDS-trypsin treatment was adopted to prepare the allogenic ADM, and the explants attached methods were conducted to culture the human fthroblasts on the scaffolds. Hematoxylin-eosin staining and scanning electronic microscopy examination were involved to observe the adhesion, growth, proliferation and migration of fibroblasts on scaffolds. MAIN OUTCOME MEASURES： The morphological structure of allogenic ADM and the cytocompatibility of ADM were both detected. RESULTS： The allogenic ADM appeared white color and elasticity macroscopically, the cells and cellular components in the epidermis, papillary layer and reticular layer in dermis had been removed completely while the dermal collagen remained intact mesh stent microscopically. The co-culturing process of the ADM and the fibroblasts showed that the fibroblasts not only could attach, grow and proliferate, but also functioned actively and secreted the extracellular matrix, which formed the membrane-like layer on the scaffold. At the same time the fibroblasts penetrated along intercellular space from the papillary layer into the reticular layer of ADM. CONCLUSION： The ADM prepared by hyper tonic saline-SDS-trypsin is well-cytocompatible to the fibroblasts, and can be used as the dermal scaffolds of the skin tissue engineering.