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

Abstract

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, composed 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 the 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 an air–liquid interface (−gel −interface), CA scaffolds impregnated with C–PEG and cultured in cell culture medium without an air–liquid interface (+gel −interface), and CA scaffolds impregnated with C–PEG cultured in cell culture medium with an 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 capable of recapitulating micro-environments relevant to skin tissue engineering, and may be a useful tool for future skin tissue engineering applications.