Development of cultured dermal substitute composed of hyaluronic acid and collagen spongy sheet containing fibroblasts and epidermal growth factor.

The present study aimed to develop a two-layered cultured dermal substitute (CDS). The upper layer is a hyaluronic acid (HA) and collagen (Col) spongy sheet with or without epidermal growth factor (EGF). The lower layer is a HA spongy sheet and Col gel containing fibroblasts. The CDS is prepared in serum-free medium, followed by placing on the wound surface. Corresponding to clinical application, CDS was incubated in serum-free medium for a period of 1, 3 or 5 days, followed by placing onto the air and culture medium interface (wound surface model), and culture for 6 days using conventional culture medium supplemented with serum. Metabolic activity and cytokine production were considerably higher in EGF-incorporating CDS, as compared with EGF-free CDS. Metabolic activity of EGF-incorporating CDS was maintained for a period of 3 days, but decreased slightly after 5 days. EGF-incorporating CDS is able to effectively stimulate fibroblasts within CDS to release increased amounts of vascular endothelial growth factor and hepatocyte growth factor, which are essential for wound healing. CDS is promising for wound therapy, because there is no risk of cellular damage caused by cryopreservation, thawing and rinsing processes. The critical issue is how to reduce the cellular damage during a prolonged period of incubation in serum-free medium. EGF-incorporating CDS can be used after a period of 3-5 days incubation in serum-free medium. This period is sufficient for transport of CDS from manufacturing facilities to hospitals.