This paper describes a novel method for the rapid construction of skin, using multiple layers of aligned electrospun fibers as starting scaffolds. Scaffolds were spun from biodegradable L-lactide/glycolide (molar ratio 10:90) with predominantly parallel arrays of fibers attached peripherally to thin 304 stainless steel layer frames. Each layer frame was held between two thicker support frames. Human skin cells were seeded onto multiple (three-nine) scaffolds. Dermal fibroblasts were seeded on both sides of each scaffold except for one on which keratinocytes were seeded on one side only. Following 48 h of culture, the scaffolds and layer frames were unmounted from their support frames, stacked, with keratinocytes uppermost, and securely held in place by upper and lower support frames to instantly form a multilayered "dermis" and a nascent epidermis. The stack was cultured for a further 5 days during which time the cells proliferated and then adhered to form, in association with the spun fibers, a mechanically coherent tissue. Fibroblasts preferentially elongated in the dominant fiber direction and a two-dimensional weave of alternating fiber and cell alignments could be constructed by selected placement of the layer frames during stacking. Histology of the 7-day tissue stacks showed the organized layers of fibroblasts and keratinocytes immuno-positive for keratin. Electron microscopy showed attachment of fibroblasts to the lactide/glycolide fibers and small-diameter collagen fibers in the extracellular space. This novel approach could be used to engineer a range of tissues for grafting where rapid construction of tissues with aligned or woven layers would be beneficial.
Keywords: culture; electrospun mesh; fibroblasts; keratinocytes; layered scaffolds; skin.