Porous, nowoven fibrous gelatin scaffolds were prepared using electrospinning. Electrospun scaffolds with varying fiber diameter, interfiber distance, and porosity were fabricated by altering the concentration of the electrospinning solution. Solution concentration was a significant predictor of fiber diameter, interfiber distance, and porosity with higher solution concentration correlated with larger fiber diameters and interfiber distances. The potential of electrospun gelatin as a scaffolding material for dermal and epidermal tissue regeneration was also evaluated. Interfiber distances >5.5 mu m allowed deeper penetration of human dermal fibroblasts into the scaffold, whereas cells in scaffolds with more densely packed fibers were able to infiltrate only into the upper regions. Scaffolds with interfiber distances <= 10 mu m exhibited well-stratified dermal and epidermal layers including a continuous basal keratinocyte layer. These scaffolds were shown to form a keratinized layer like in normal skin, which acts as a barrier to infection and fluid loss. Interfiber distances between 5 and 10 pm appear to yield the most favorable skin substitute in vitro, demonstrating high cell viability, optimal cell organization, and excellent barrier formation. These results demonstrate the feasibility of electrospun gelatin as a scaffold for dermal-epidermal composite skin substitutes. (C) 2007 Wiley Periodicals, Inc.