A simple material model to generate epidermal and dermal layers in vitro for skin regeneration

There is an urgent need for a rationally-designed, cellularized skin graft capable of reproducing the micro-environmental cues necessary to promote skin healing and regeneration. To address this need, we developed a composite scaffold, namely, CA/C-PEG, composing of a porous chitosan-alginate (CA) structure impregnated with a thermally reversible chitosan-poly(ethylene glycol) (C-PEG) gel to incorporate skin cells as a bi-layered skin equivalent. Fibroblasts were encapsulated in C-PEG to simulate the dermal layer while the keratinocytes were seeded on the top of CA/C-PEG composite scaffold to mimic the epidermal layer. The CA scaffold provided mechanical support for the C-PEG gel and the C-PEG gel physically segregated the keratinocytes from fibroblasts in the construct. Three different tissue culture micro-environments were tested: CA scaffolds without C-PEG cultured in cell culture medium without air-liquid interface (-gel-interface), CA scaffolds impregnated with C-PEG and cultured in cell culture medium without air-liquid interface (-gel-interface), and CA scaffolds impregnated with C-PEG cultured in cell culture medium with air-liquid interface (-gel- interface). We found that the presence of C-PEG increased the cellular proliferation rates of both keratinocytes and fibroblasts, and the air-liquid interface induced keratinocyte maturation. This CA/C-PEG composite scaffold design is able to recapitulate micro-environments relevant to skin tissue engineering, and may be a useful tool for future skin tissue engineering applications.