In tissue engineering, structure control of biodegradable membrane scaffolds to shape developing tissues is critical for balanced organogenesis requirements. It is currently challenging to control a three-dimensional (3-D) membrane scaffold with beneficial growth of skin cells during wound-healing process. Here we report a new dual-layer fibrous structure made of electrospun chitosan/poly(D,L-lactic acid) (PDLLA) membrane for human skin fibroblast culture. In this work, the electrospinning process incorporated with rotating control was introduced to prepare the random PDLLA microfibrous matrix covered with aligned chitosan nanofibers, providing an increased specific interface area to promote cell attachment, migration and infiltration. The effects of electrospining parameters on the reduction of beads and the uniformity of fibers were determined. The morphology, proliferation and penetration of cells seeded on each electrospun fibrous membrane were observed using laser scanning confocal microscope (LSCM). A dual-layer fibrous membrane prepared with a highly uniform matrix layer of PDLLA microfibers and a well-defined cover layer of chitosan nanofibers without fibrous beads result in aligned migration and directional penetration of skin fibroblasts into 3-D domain. The chitosan nanofibers which covered on PDLLA microfibers promote the skin cells growth and improve their infiltration into the target scaffolds in vitro. Wound-healing test demonstrated that the dual-layer structure significantly improves the rat tissue regeneration in vivo. The results suggest that the dual-layer chitosan/PDLLA structure can mimic the interface of natural extracellular matrices and has the potential for 3-D tissue-engineering applications. (C) 2013 Elsevier B.V. All rights reserved