Tissue-Engineered Skin Substitutes

Abstract

Severe burns, chronic diabetic ulcers, and traumatic full-thickness skin defects remain major clinical challenges because autologous split-thickness skin grafts (STSGs) -- the gold standard treatment -- are limited by donor-site availability, morbidity, and inability to regenerate functional dermal appendages. Tissue-engineered skin substitutes (TESSs) aim to overcome these limitations by combining biomaterial scaffolds, autologous or allogeneic cells, and bioactive signalling molecules into constructs that replicate the bilayered architecture of native skin. This study developed and evaluated five TESS formulations -- collagen-glycosaminoglycan (Col-GAG) sponge, electrospun polycaprolactone-gelatin (PCL-Gel) nanofibrous membrane, 3D-bioprinted gelatin methacryloyl (GelMA) hydrogel, decellularised porcine dermis (DPD), and a proposed tri-layered construct combining a PCL-Gel dermal scaffold seeded with human dermal fibroblasts (HDFs) overlaid with a GelMA epidermal layer containing human keratinocytes (HKCs) -- in a validated full-thickness excisional wound model in athymic nude mice (n = 150). The tri-layered construct achieved the highest wound closure rate (94.8 + 3.2% at day 21), significantly outperforming Col-GAG (78.4 +- 5.6%; p < 0.001), DPD (82.6 +- 4.8%; p = 0.002), and acellular PCL-Gel (68.2 +- 6.4%; p < 0.001). Histological analysis at day 28 revealed that the tri-layered construct supported formation of a stratified epidermis with 5-7 cell layers (vs 2-3 in controls), organised collagen deposition in the neo-dermis (collagen alignment index 0.72 +- 0.08 vs 0.34 +- 0.12 in untreated wounds), and nascent vascularisation (CD31+ vessel density 48.2 +- 6.4 vessels/mm2 vs 18.4 +- 4.8 in acellular scaffolds). These results establish the PCL-Gel/GelMA tri-layered construct as a promising bilayered skin substitute and provide a systematic framework for evaluating scaffold composition, cell source, and fabrication strategy in skin tissue engineering.