应用胶原海绵构建组织工程皮肤的实验研究

Experimental Study of the Reconstruction of Tissue-Engineered Skin by Collagen-Sponge

目的探讨以胶原海绵为支架材料构建组织工程皮肤的方法.方法体外分离、培养、鉴定角朊细胞和成纤维细胞,制备胶原海绵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,通过大体观察、HE染色与免疫组织化学进行组织学检测.结果制备的胶原海绵孔径平均100～150 μm,孔隙率89%,组织相容性良好;以胶原海绵构建的皮肤替代物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,HE 染色可见均具有表皮和真皮双层结构,在形态结构上与正常皮肤相似.结论培养的人角朊细胞和成纤维细胞种植于胶原海绵支架上培养可构建出具有类似天然皮肤结构的组织工程皮肤.

Objective To investigate a approach of constructing tissue-engineered skin substitute consisting of epidermal keratinocytes seeded on a fibroblast-coutaining collagen matrix layer. Methods Collagen Ⅰ extracted from rat-tail tendons was used to construct scaffold-collagen-sponge and collagen-gel onto which the fibroblasts were seeded during logarithmic growth phase and cultured for one week, then 4.0 × 10^5cells/cm^2 of keratinocytes were seeded on the surface of the collagen-scaffold and cultured for another 3 days, then the skin graft substitutes were elevated to air-liquid interface to allow the maturation of the epidermis cultured for 6 weeks. All specimens underwent the gross, HE staining, histological, immuno-histochemistric and electron microscope evaluation. Results The cells we harvested were pure human fibroblasts and keratinocytes could be used as seeding ceils for the construction of tissueengineered artificial skin. The epidermal histological appearance and the constitution of composite skin was found to be similar to that of normal skin. The autologous skin substitute was constructed successfully with positive collagen Ⅳ ,fibronectin and laminin staining. Conclusion This method supplies tissue engineered skin with enough seeding cells simply and suitably, the constitution of the composite skin was similar to human being＇s skin.