development of bilayer gelatin scaffolds for coculture of heterogeneous cell populations

Abstract

The skin consists of epidermis and dermis. In order to develop a biomimetic material for more realistic tissue regeneration, we fabricated bilayer gelatin scaffolds by freeze-drying and electrospinning method. The dry thickness of the upper layer was 24-144㎛ and that of the lower layer was controlled to be at 800㎛. The bottom layer with macropores was fabricated by a conventional freeze-drying method. Subsequently, gelatin was dissolved in 2,2,2-trifluoroethanol at 5 and 10 wt%, and electrospun directly to the top of the macroporous scaffold to produce the macro/nano fibrous top layer with fiber diameters ranging from 400 nm to 3㎛. The polymer concentration, voltage, needle size, and spinning time for the electrospinning were varied to create various porosities and mean diameters. Bilayer gelatin scaffolds were then cross- linked by using 1-ethyl- (3-3-dimethylaminopropyl) carbodiimide hydrochloride. To examine cellular responses to the bilayer scaffolds, fibroblasts and keratinocytes were isolated from child`s foreskin and co-cultured. After 14 days of culture, the fibroblasts and keratinocytes showed good affinity to, and proliferation on, the scaffolds with little evidence of contraction. Furthermore, using cell tracking fluorescent dyes, we observed the formation of basal laminae that separated keratinocytes from fibroblasts. The process to fabricate bilayer structure may be promising to design tissue engineering scaffold with native tissue-like architecture for restoring damaged skin. Further experimental studies are in progress to test its applicability to human skin replacement.