A new approach to the production of a biovital skin graft based on human acellular dermal matrix produced in-house, in vitro revitalized internally by human fibroblasts and keratinocytes on the surface.

Patients with extensive and deep burns who do not have enough donor sites for autologous skin grafts require alternative treatment methods. Tissue engineering is a useful tool to solve this problem. The aim of this study was to find the optimal method for the production of a biovital skin substitute based on acellular dermal matrix (ADM) and in vitro cultured fibroblasts and keratinocytes. In this work, nine methods of ADM production were assessed. The proposed methods are based on the use of the following enzymes: Dispase II, collagenase I/ethylenediaminetetraacetic acid (EDTA), collagenase II/EDTA, and mechanical perforation using DermaRoller and mesh dermatome. The obtained ADMs were examined (both on the side of the basement membrane and on the "cut-off" side) by means of scanning electron microscopy, immunohistochemistry tests and strength tests. ADM was revitalized with human fibroblasts and keratinocytes. The ability of in-depth revitalization of cultured fibroblasts and their ability to secrete collagen IV was examined. The obtained results indicate that the optimal method of production of live skin substitutes is the colonization of autologous fibroblasts and keratinocytes on the scaffold obtained using two-step incubation method: Trypsin/EDTA and dispase II.