胶原凝胶人工皮肤的体外构建

Construction of a Composite Skin Composed of Cultured Epithelium and Fibroblasts and Collagen-gel in Vitro

目的探讨以胶原凝胶为支架材料构建组织工程皮肤的方法。方法体外分离、培养人皮肤表皮细胞和成纤维细胞,利用本研究所自制胶原蛋白,制备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,构建人工复合皮肤,运用HE染色与免疫组织化学进行组织学检测。结果HE染色可见构建的人工复合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行气-液界面培养可构建出具有类似正常皮肤结构的组织工程皮肤。

Objective To investigate an approach of constructing tissue-engineered skin substitute. Methods Epithelium and fibroblast were isolated and cultured in vitro. The collagen protein extracted from rat-tail tendons was used to construct collagen-gel scaffold. Fibroblasts were seeded onto scaffold during logarithmic growth phase and cultured for one week, then 4.0 ×10^5 cells/cm^2 of epithelium were seeded on the surface of the collagen-gel and cultured for another 3 days, then the skin graft substitutes were elevated to air-liquid interface to allow the maturation of the epidermis, and cultured for 6 weeks, and observed by HE staining and immunohistochcmistry under microscope. Results The cells harvested were pure human fibroblasts and epithelium which could be used as seeding cells for the construction of tissue-engineered artificial skin. The epidermal histological appearance and the constitution of composite skin were found to be similar to that of normal skin with IV type collagen-, fibroncctin- and laminin-pesitive reaction. Conclusion The composite skin constructed with human epithelium and fibroblast and collagen gel was similar to normal human skin.