胶原凝胶人工皮肤的体外构建

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.

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

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 4.0×105 cells/cm2 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 immunohistochemistry 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-,fibronectin-and laminin-positive reaction.Conclusion The composite skin constructed with human epithelium and fibroblast and collagen gel was similar to normal human skin.