Use of stacked layers of electrospun L-lactide/glycolide co-polymer fibers for rapid construction of skin sheets

Abstract We report on the development of a method using multiple layers of electrospun mesh for the rapid construction of layered tissues, demonstrated by construction of skin,. Human skin cells were cultured initially for 48 hrs on multiple separate thin layers of electrospun mesh composed of biodegradable L-lactide/glycolide co-polymer fibers attached peripherally to thin stainless steel layer frames. Dermal fibroblasts were seeded on both sides of the mesh of each layer frame except for one frame on which keratinocytes were seeded on one side only. All layers were then stacked between basal and upper support frames using guide pins and screws, with the keratinocyte cells uppermost, to instantly form a multi-layered ‘dermis’ and a nascent epidermis. Over 5 further days of culture the cell-covered layers adhered to form a mechanically coherent tissue. Fiber direction influenced fibroblast elongation, with fibroblasts extending in the dominant fiber direction. Selected placement of frames to alternate the orientation of the co-polymer fibers in adjacent layers mimicked the alternating orientation of fibroblasts and matrix fibers in native dermis. The co-polymer fibers were compatible with cell proliferation and attached fibroblasts exhibited a normal extended morphology and were able to exert tensional forces on individual fiber elements. This novel approach could potentially be used to engineer a range of layered tissues for grafting over comparatively short culture times and where gradual degradation of the initial electrospun fiber substrate allows for mechanical integrity to be transferred to synthesised extracellular components