Construction of chitosan-gelatin-hyaluronic acid artificial skin in vitro.

To further enhance the properties of chitosan (Cs)-gelatin (Gel) scaffolds for skin tissue engineering, hyaluronic acid (HA) is introduced to the Cs-gel complex. Porous scaffolds composed of Cs, Gel, and HA are prepared using the freeze-drying method. The scaffold has an interconnected pore structure with two different pore size layers. The water uptake ability, flexibility, and biocompatibility of the scaffold are greatly increased with the incorporation of HA. To construct an artificial skin in vitro, fibroblasts and keratinocytes are co-cultured in Cs-Gel-HA scaffolds at an air-liquid interface. After 2 weeks of co-culture, the epithelial layer becomes progressively stratiform, including cubic perpendicularly oriented cells and a superficial layer of flattened cells. Immunohistochemical analyses confirmed the presence of laminin and type IV collagen, typical molecules of the basement membrane. The results of this study suggest that it is possible to construct a functional artificial skin in vitro and the Cs-Gel-HA scaffold is a promising matrix for skin tissue engineering.