Reconstruction Of Human Skin Research Articles

Reconstruction of human skin from glycerol-preserved allodermis and cultured keratinocyte sheets

The aim of this project was to reconstruct human skin from glycerol-preserved dermis and layers of cultured keratinocytes for use in the treatment of deep burns and ulcers. Glycerol-preserved cadaver skin from the Euro Skin Bank was treated with Dispase II or PBS, under various conditions, to find the best method of retaining the physical structure of the dermis while removing the epidermis and remnants of dead dermal cells which might provoke an allogeneic reaction in a graft recipient. Monoclonal antibodies LH39 and LH7.2, with specificity for basement membrane determinants, showed that treatment with Dispase II resulted in separation of the epidermis from the dermis with concomitant loss of all cellular elements from the dermal layer (as judged by Hand E staining). However, when sheets of cultured keratinocytes were applied to the treated dermis and cultured for several days, the keratinocytes attached and regenerated a new basement membrane.

Immunochemistry of a keratinocyte-fibroblast co-culture model for reconstruction of human skin.

Our purpose was to determine differentiation markers of an in vitro co-culture model in which fibroblasts grown in a three-dimensional nylon mesh were recombined with human keratinocytes. The cultures were kept for 5 weeks and then processed for electron microscopy and immunochemistry. The specimens revealed an epidermis, a basal lamina, an anchoring zone, and a dermis. Epidermal differentiation was confirmed by the presence of K10-keratin, trichohyalin, and filaggrin. The basal lamina contained Type IV collagen, laminin, nidogen, and heparan sulfate. Type IV collagen, laminin, and nidogen were also noted in the extracellular matrix. Type VI collagen was present in the anchoring zone and also gave a reticulated pattern in the rest of the dermis. There was a heavy signal for tenascin and fibronectin throughout the dermis. Osteonectin was restricted to the epidermis and dermal fibroblasts. Fibrillin stained at the anchoring zone and dermis but elastin and vitronectin were negative, suggesting early formation of elastic fibrils. Collagen fibrils stained for Types I, III, and V, as well as the amino propeptide of Types I and III procollagen, suggesting newly synthesized collagen. Decorin was present throughout the dermis. The model described appears suitable for in vitro reconstruction of the skin and may be useful to study the development of various supramolecular skin structures.

In vitro reconstruction of human skin: The use of skin equivalents as potential indicators of cutaneous toxicity

A three-dimensional reconstruction of living skin—the skin equivalent—has been modified to accept materials that can be applied topically to the skin. Using an irritant gel containing 10% benzoyl peroxide, changes in the skin equivalent model were investigated. Histologically, epidermal necrosis and vacuolar change were observed within 4–6 hr after topical application. Using skin equivalents prelabelled with [ 3H]arachidonic acid, studies involving direct measurement using HPLC and radioimmunoassay have indicated the release of potent lipoxygenase products, such as leukotriene B 4 and 15-hydroxyeicosa-tetraenoic acid. These preliminary results suggest that the skin equivalent may prove to be a useful in vitro model for the prediction of cutaneous toxicity of topically applied substances.