Assessment of transformed properties in vitro and of tumorigenicity in vivo in primary keratinocytes cultured for epidermal sheet transplantation.

Epidermal keratinocytes are used as a cell source for autologous and allogenic cell transplant therapy for skin burns. The question addressed here is to determine whether the culture process may induce cellular, molecular, or genetic alterations that might increase the risk of cellular transformation. Keratinocytes from four different human donors were investigated for molecular and cellular parameters indicative of transformation status, including (i) karyotype, (ii) telomere length, (iii) proliferation rate, (iv) epithelial-mesenchymal transition, (v) anchorage-independent growth potential, and (vi) tumorigenicity in nude mice. Results show that, despite increased cell survival in one keratinocyte strain, none of the cultures displayed characteristics of cell transformations, implying that the culture protocol does not generate artefacts leading to the selection of transformed cells. We conclude that the current protocol does not result in an increased risk of tumorigenicity of transplanted cells.

Grafting of large pieces of human reconstructed skin in a porcine model.

Present techniques can save about 25% of patients burnt over more than 90% of their body surface. However, problems of functional and aesthetic repair arise, which are often resolved only by major therapeutic procedures. Current advances in skin substitutes permit the cultivation, from a skin biopsy, of large surfaces of in vitro human reconstructed skin (HRS). Our model, obtained by the co-culture of fibroblasts and keratinocytes on a dermal substrate composed of collagen-glycosaminoglycan-chitosan, reproduces, in vitro, a tissue close to human skin, which could play a role in reconstructive surgery. The objectives of this experiment were to assess whether it is possible to perform large HRS grafts and to evaluate the preliminary cosmetic results. We used four immunosuppressed female pigs. Full-thickness skin resections of 50-100 cm(2)were performed on the dorsa of the animals. The defects were grafted with between one and six pieces of HRS under tied-over dressings. At day 14, we found a soft and smooth surface of good transparent healthy pink skin, which was very easy to distinguish from the surrounding tissues. The junctions between different pieces of living skin were not visible. Immunohistological studies with specific anti-human keratin 14 antibodies confirmed the graft take: 7 days after grafting the human epidermis was attached to the living dermis and showed good organisation with a basal cell layer and suprabasal cells; 28 days after grafting the human epidermis seemed to be replaced by pig epidermis. This study highlights the possibility of grafting large surfaces with HRS using a routine operating technique.