Reconstructed skin from co-cultured human keratinocytes and fibroblasts on a chitosane cross-linked collagen-GAG matrix

Abstract

Reconstruction of skin requires both the dermal and epidermal equivalent of the skin. A reconstructed skin has been developed, composed of two compartments: (1) a dermal equivalent (DE) comprising an acellular dermal substrate (DS) populated by foreskin fibroblasts (FF); (2) an epidermis regenerated from normal human keratinocytes (NHK) seeded on to the DE. The DS contains Types I and III collagen and glycosaminoglycans (GAGs) cross-linked by chitosane. FF seeded into the porous structure of the DS provide a DE suitable to support epidermal cells. NHK attach quickly, exhibit mitotic activity and form a continuous and stratified epidermis. After 2 weeks culture, histological sections of the RS show a basal layer with cuboidal cells attached to the DE and several suprabasal cell layers including the stratum corneum (SC). Transmission electron microscopy revealed the cell membrane densification (hemidesmosomes) at the dermal-epidermal junction; however, the lamina densa was found to be discontinuous at this stage. We noted the presence of lipid vesicles in spinous layer and keratohyalin granules in granular layer. Terminal epidermal differentiation was complete with the SC consisting of several layers of corneocytes filled with tonofilaments. Immunofluorescence studies revealed the presence of bullous pemphigoïde antigen and Type IV collagen at the dermal-epidermal junction as well as the presence of keratins 1/10 and filaggrin in the suprabasal layers characteristic for epidermal differentiation. Reconstructed skin based on our chitosane cross-linked collagen-GAG matrix is morphologically equivalent to normal human skin and should thus provide a useful tool for the treatment of patients with severe burns.