In Vitro Analysis of VEGF and HGF Production by Fibroblast in Cultured Dermal Substitute Combined with EGF-Incorporating Top Dressing

Abstract

This study aimed to investigate the potential of cultured dermal substitute (CDS) to release angiogenic growth factors when laminated with a membrane containing epidermal growth factor (EGF) as a top dressing. Membranes were prepared by air-drying a solution of hyaluronic acid (HA) and collagen (Col) with or without EGF. Membranes were designed to contain EGF at concentrations of 0, 0.1, 0.2 or 0.5 μg/cm2. CDS was prepared by incorporating fibroblasts into a collagen gel combined with a cross-linked HA spongy matrix, followed by culturing for 5 days. CDS was designed to contain fibroblasts at a density of 2 × 105 (Group I) or 4 × 105 cells/cm2 (Group II). CDS was elevated at the interface between air and culture medium, on the top of which each membrane was placed. This culture system was employed as a wound surface model. Metabolic activity of the fibroblasts in the CDS cultured for 7 days on a wound surface model was measured by MTT assay. The amounts of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) after 7 days of cultivation were measured by using ELISA. Membranes containing EGF ranging from 0.1 to 0.5 μg/cm2 facilitated production of both VEGF and HGF, as compared with control membranes without EGF. However, a membrane containing EGF at a concentration of 0.5 μg/cm2 failed to facilitate fibroblast cytokine production in Group I. These results demonstrated that the EGF-incor- porating membrane was able to stimulate fibroblasts in the CDS to synthesize an increased amount of VEGF and HGF in a dose-dependent manner.