Keratinocyte-fibroblast Cocultures on A Bi-layered Gelatin Scaffold for Skin Equivalent Tissue Engineering

The skin is composed of three parts: epidermis, dermis and subcutaneous fatty layers. In order to develop a biomimetic material for more realistic tissue regeneration, we fabricated a bi-layered gelatin sponge with different pore sizes by freeze-drying at different temperatures -20 < degrees >C and -196 < degrees >C, respectively. A rete ridges-like topographic microporous structure, which provided the paracrine crosstalk in epithelial-mesenchymal interactions similar to the basement membrane in epidermal-dermal junction, was formed between these two compartments. 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 1-ethyl-3 (3-dimethyl aminopropryl) carbodiimide (EDC). The lattice structure and pore sizes were evaluated by SEM to confirm the interconnected porous structures retained. We chose a dynamic spinner flask seeding method for more evenly distribution followed by a culture system in the air-liquid interface and cultured for 21 days. Differentiation and phenotypic expression of keratinocytes were investigated by histological analysis. In this study, we found a multiple epidermis-like layers constructed by cultured keratinocytes. It is suggested that the bi-layered scaffolds have the potential to be used as skin equivalents for the application in burn patients. In the future, the in vivo animal model tests will be evaluated.