应用胶原凝胶构建组织工程化皮肤的研究

Study of the use of collagen-gel to construct tissue-engineered skin

目的探讨以胶原凝胶为支架材料构建组织工程化皮肤的可行性。方法体外分离、培养人皮肤表皮细胞和成纤维细胞;利用自制的胶原蛋白制成胶原凝胶作为组织工程支架材料;在成功构建人工真皮的基础上种植表皮细胞,构建复合人工皮肤;采用HE染色与免疫组织化学的方法对复合人工皮肤进行组织学检测。结果HE染色可见,构建的复合人工皮肤具有表皮和真皮双层结构;免疫组织化学染色显示,Ⅳ型胶原、纤维连接蛋白和层粘连蛋白阳性,在形态结构上与正常皮肤相似。结论培养的人表皮细胞和成纤维细胞种植于胶原凝胶支架上,气-液界面培养可构建出具有类似正常皮肤结构的组织工程化皮肤。

Objective To investigate an approach for constructing a tissue- engineered skin substitute. Methods Epidermal cells and fibroblasts were isolated and cultured in vitro. We used type I collagen extracted from rattail tendons by our laboratory to construct a scaffold for tissue- engineering. Fibroblasts during logarithmic growth phase were seeded onto the scaffold and cultured for one week, and 4.0 × 10^5 cells/cm^2 d keratinocytes were seeded on the surface of the collagen- gel and cultured for another 3 days. The skin graft substitutes were then devated to an air- liquid interface to an allow the maturation of the epidermis, after culture for 6 weeks. All specimens we evaluated by HE staining and immunohistochemistry. Results The results showed that the cells we harvested were pure human fibroblasts and keratinocytes which could be used as seeding cells for the construction of tissue - engineered artificial skin. The epidermal histological appearance and the constitution of skin were found to be similar to those of normal skin. Results also showed that the skin substitute had been constructed successfully simultaneously with collagen- gel. Conclusion Tissue engineered skin can be constructed by using cultured human keratino eytes and fibroblasts seeded onto collagen- gel. The constitution of this skin composite was similar to normal human skin.