A preliminary in vitro study on the fabrication and tissue engineering applications of a novel chitosan bilayer material as a scaffold of human neofetal dermal fibroblasts

The bilayer structure of chitosan film and sponge was designed as a scaffold of skin tissue engineering and a dermis substitute. It was processed successively via the formation of a dense chitosan film by casting method and a porous chitosan sponge by lyophilization. The dry thickness of the film layer was 19.6 microm and that of the sponge layer was controlled at 60-80 microm. Porogens such as sodium chloride, glucose, and sucrose were used to create large pores of the chitosan sponge layer. Human neofetal dermal fibroblasts were seeded in the chitosan sponge layer and cultured for 4 weeks. It was found that the cells could grow and proliferate well in an extended shape on the flat bottom of large pores with 15-100 microm width and in spherical form on the rough pore walls or at the edges of micropores less than 5 microm. Fibroblasts after the culture could bind tightly with the sponge layer via newly formed extracellular matrices to give a living cell-matrix-chitosan composite. The bilayer chitosan material remained stable in shape and size during the cell culture. The results suggested that the bilayer chitosan material would be an alternative of collagen materials which was obviously contracted during cell culture.