ABSTRACT
BACKGROUND:Study confirmed that the de-epidermized dermis(DED)can be used as dermal substitute and may form epidermal structure after incubating keratinocytes.However,the cell biological activity,tissue structure characteristics and the basement membrane component analysis of dermal substitute have been reported less.OBJECTIVE:To investigate the cell activity and the tissue structure characteristics of DED.METHODS:Skin flap was treated with 56℃ phosphate buffered solution to remove the epidermis,and the dermal cell components were deleted by freezing and thawing with liquid nitrogen to obtain DED.The DED cell activity was detected with tissue culture method,hematoxylin nuclear staining was used to determine the DED cell nuclei,and vimentin immunohistochemistry was applied for fibroblast determinations.The basement membrane and its components were detected using Periodic Acid-Schiff staining and collagen type Ⅳ immunohistochemistry.Van Gieson stain,Weigart stain and those double staining were respectively used to determine DED collagen fibers and elastic fibers.The DED ultrastructure was observed under transmission and scanning electron microscope.RESULTS AND CONCLUSlON:Using tissue culture method,the cultured DED did not exhibit cell growth at 2 weeks.Hematoxylin-eosin staining showed no nuclear in DED,vimentin immunohistochemistry showed no vimentin expressed in DED.Van Gieson staining showed DED collagen fibers were stained as rose red,Weigert staining showed DED elastic fibers were stained as pu rplish black double staining further demonstrated uniform arrangement of collagen fibers and elastic fibers.DED surface and the remaining appendages were strongly positive for Periodic Acid-Schiff staining,and type Ⅳ collagen expression was significant.Transmission and scanning electron microscope results showed that,the DED elastic fibers and collagen overlap arranged with pore intervals,they intercrossed into a network.There is no living cell component in DED,dermal matrix surface and appending organ luminal wall still retain glycogen,type Ⅳ collagen and other basement membrane components,dermal matrix is rich in collagen and elastic fibers.it is a three-dimensional collagen matrix similar to in vivo dermis.
ABSTRACT
Objective To establish a reconstructed human epidermis model in vitro, and to study the process of re-epithelialization. Methods A dermal substrate devoid of epidermis was prepared; a 2 mm skin biopsy explant was transplanted onto the dermal substrates. Visualization of epidermal cell migration was carried out by fluorescence imaging. The proliferation and differentiation of the new epithelial cells were observed using histopathological and immunohistochemical staining (Ki67). EGF was added to the culture medium of the experimental samples but not to that of the controls. Results After 3 days of culture, re-epithelialization was observed on the surface of the dermal substrate. A complete structure resembling regular epidermis was noted in 10 days. As compared to control samples, EGF-treated samples had larger area of re-epithelialization (t= 3.02, P