A bioprinted vascularized skin substitute with fibroblasts, keratinocytes, and endothelial progenitor cells for skin wound healing

Skin substitutes are highly demanded by patients with extensive burns and full-thickness skin wounds. Bioprinting offers a promising technology to fabricate customized cell-laden skin substitutes. In this study, a hydrogel of biodegradable polyurethane (PU)-gelatin (4:1) laden with human fibroblasts, endothelial progenitor cells (EPCs), and keratinocytes was used as the bioink for building a bi-layer dermo-epidermal skin substitute. The seven-layer cell-laden constructs with stack thickness of 1.4 mm were precisely deposited through a 210 μm nozzle with an air pressure of 0.055–0.175 MPa and nozzle temperature of 19 °C. When grown in vitro, three types of cells in the bioprinted constructs showed good cell viability (>99%) in 24 h and reached a high proliferation rate (>220%) in 14 days. Significantly, EPCs were successfully differentiated into endothelial-like cells in the constructs and expressed the vasculogenesis-related proteins (CD31+ and eNOS+). When implanted in vivo, the bi-layer constructs attained ∼90% wound healing ratio and ∼76% re-epithelialization after 28 days in the nude mice model. Histological analyses revealed that skin wounds treated with the bi-layer constructs achieved high degrees of tissue integration and collagen production after 28 days. Vasculogenesis and angiogenesis of the wound treated with bi-layer constructs was significantly greater (∼300%) than those of the untreated wounds. The in vitro and in vivo findings indicate that the bioprinted skin substitutes with fibroblasts, EPCs, and keratinocytes embedded in PU-gelatin hydrogel may offer a promising strategy for clinical wound treatment and development of bioprinted skin.