培养基钙浓度对复方壳多糖组织工程皮肤基底膜构建的形态学研究

目的寻找最适于组织工程皮肤生长及基底膜构建的体外培养基钙浓度。方法按照鲁元刚等建立的方法制备复方壳多糖组织工程皮肤，根据培养基钙浓度不同，分为1．00、1．45、1．65和1．95mmol／L四组。在37℃、5％CO2及90％以上湿度孵箱内浸没培养3d后，将培养物置于不锈钢筛网上进行气一液界面三维培养，每天换液1次。培养7、15d，行HE、PAS染色观察组织工程皮肤的组织学特性；并利用免疫组织化学染色对基底膜的主要成分——Ⅳ型胶原进行观察，评价基底膜构建情况。结果各组组织工程皮肤组织学观察类似正常皮肤，有分化良好的表皮和致密真皮，但1．95mmol／L组表皮分化较其他组更好；1．00、1．45及1．65mmol／L组角化层外侧常伴未角化的角质形成细胞，1．95mmol／L组表皮细胞角化完全，且与颗粒层细胞结合牢固。PAS染色显示各组真、表皮间有紫红色带状结构；免疫组织化学染色显示真皮与表皮间Ⅳ型胶原反应呈阳性，且1．95mmol／L组阳性反应强于1．00mmol／L组。结论钙浓度为1．95mmol／L的培养基最适合于复方壳多糖组织工程皮肤生长及基底膜构建。

EFFECT OF CALCIUM ON RECONSTRUCTION OF BASEMENT MEMBRANE IN COMPOSITE CHITOSAN TISSUE ENGINEERING SKIN

OBJECTIVE: To search the most suitable concentration of calcium in the medium for the basement membrane reconstruction in tissue engineering skin in vitro. METHODS: Composite chitosan tissue engineering skin was prepared according to previous studies. Four groups were included according to the concentration of calcium (1.00, 1.45, 1.65 and 1.95 mmol/L respectively). After 7 days and 15 days of culture, the histological manifestation of basement membrane in tissue engineering skin was observed by hematoxylin & eosin staining and PAS staining, and collagen IV of basement membrane was detected immunohistochemically at the dermal-epidermal junction. RESULTS: This tissue engineering skin shared some histological features of normal skin, including a well-differentiated stratified epidermis and a dense dermis. The epithelium in the group of 1.95 mmol/L. Llcium differentiated better than those in other groups. PAS staining showing a regularly red dying strap domain at the dermal-epidermal junction. Collagen IV was positively stained immunohistochemically at the dermal-epidermal junction in the tissue engineering skin. CONCLUSION: The above results suggest that the medium with 1.95 mmol/L calcium should be suitable for the growth of composite chitosan tissue engineering skin and the reconstruction of basement membrane.