Cultured keratinocyte grafting on various biologic matrices

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Objective: To make attempts to use cell constructs from subconfluent keratinocyte cultures, which contain a much larger percentage of epidermal colony forming “stem cells”. Methods: Subconfluent keratinocytes were suspended in fibrin glue (TissucollR ) and directly applied onto full thickness wounds in athymic mice or combined with allogenic split thickness overgrafts and compared with cultured sheet grafts. This keratinocyte fibrin glue suspension (KFGS) has also been used in burns up to 88% burned TBSA as well as in chronic wounds. Keratinocytes were also seeded onto various biomaterials (BiobraneR, HYAFF LaserskinR, IntegraTM, TissuFascieR) as carriers. Results: Human suspended keratinocytes were effective to reorganize to skin in vivo both in nude mice and in patients and superior if compared to sheet grafts. 3-5 d after seeding onto various biomaterials, cell reached subconfluence and were ready for grafting. These cell-membrane constructs were always grafted onto the wound “upside down” permitting a good primary nutrition of the cultured cells in vivo. Keratinocytes cultured on microspheres in spinner cultures could increase the cell yield, and the subconfluently covered microspheres were directly grafted onto the wound. Conclusion: These experiments demonstrated that keratinocytes can grow on a variety of carrier materials in vitro and these cell constructs were able to spontaneously reform a multilayer neoepithelium in vivo. The current screening looks for the ideal carrier for keratinocytes that also would serve as a temporary wound cover and induce dermis formation by tissue conduction which further may be enhanced by gene therapy.