4. Keratinocyte Growth on BiopadIn the control sample, human keratinocytes were simultaneously secondly cultured on Biopad. After two weeks of cultivation (one week submerged and one week at the air-liquid interface), the cells showed chaotic distribution. They migrated into the Biopad structure composed of fragile collagen fibers. Within the inner structure of this collagen, the cells were terminally differentiated, as shown by staining for involucrin (Figure 7).Figure 7Keratinocytes cultured on Biopad (equine collagen). Note the fragile collagen fibers (black arrow) and the chaotic distribution of the irregular groups of keratinocytes. Immunohistological detection of involucrin shows terminally differentiated keratinocytes …4.

DiscussionXenografts have been used in the Prague Burn Centre since 1973 [13] for treatment of burns and other acute and chronic skin defects [11]. After 32 years of extensive use, the utilization of porcine xenografts was discontinued due to the regulations of the European Union. Since then, various synthetic materials have been used as temporary covers, but they are not comparable to xenografts because they do not display a similar level of biological activity.Cell-free pig dermis was initially developed in our laboratory as a substrate for the cultivation of human keratinocytes [6]. The previously constructed recombined human/pig skin was used as a delivery system for the keratinocytes applied to accelerate the healing process [7�C9]. However, dried xenodermis itself without keratinocytes has shown excellent healing properties.

The bioactivity of decellularized dermis materials can be demonstrated by a cell culture assay [14]. More detailed information on the cell behavior should contribute to the elucidation of the healing mechanism of the skin substitute. Our study has shown that XD, similarly as its laboratory predecessor cell-free pig dermis [6], is an appropriate substrate for keratinocyte attachment, growth, and differentiation. Morphological differentiation of keratinocytes has been achieved in organotypic culture systems grown at the air-liquid interface on various dermal-like substrates [15�C17]. The cultured human keratinocyte/Xe-Derma organotypic skin model was studied using the criteria of tissue morphology and the presence and distribution of selected keratinocyte differentiation markers. The results were compared to the healing of deep dermal wounds after necrectomy covered with the XD dressing. The morphology of the keratinocyte-derived epidermis cultured in vitro at the air-liquid interface was very similar to the neoepidermis formed in vivo in the deep dermal burn Cilengitide from the remnants of keratinocytes in the residual skin adnexa.