Organotypic keratinocyte-fibroblast cocultures on a bilayer gelatin scaffold as a model of skin equivalent

The skin consists of epidermis and dermis. In order to develop a biomimetic material for more realistic tissue regeneration, we fabricated a double layer gelatin sponge with different pore sizes by freeze-drying at different temperatures -20 < square > and -196 < square >, respectively. A pseudo microporous structure, which provided the paracrine crosstalk in epithelial-mesenchymal interactions similar to the basement membrane in epidermal-dermal junction was formed between this two compartment. Chrondroitin-6-Sulphate (C-6-S) and hyaluronic acid (HA) were incorporated within the gelatin scaffold to create an appropriate microenvironment for cell proliferation and migration. The mechanical strength of gelatin sponges was improved by cross-linking of gelatin with carbodiimide in 95% ethanol solution two times. The lattice structure and pore sizes were evaluated by SEM to confirm that the interconnected porous structures were retained. We chose a dynamic spinner flask seeding method for more even distribution followed by a culture system in the air-liquid interface cultured for 21 days. Differentiation and phenotypic expression of keratinocytes were investigated by histological analysis and immunohistochemical identification. In this study, we found multiple epidermis-like layers constructed by cultured keratinocytes. It is suggested that the bi-layer scaffolds have the potential to be used as skin equivalents for application in burn wounds. In the future, the qualitative of chemical reagents and in vivo animal model tests will be evaluated.