Prompt closure of wounds is critical to prevention of infection and sepsis in patients suffering massive burn injuries. The most common challenge associated with treating these patients is the lack of available donor skin. Although new technologies are emerging to treat deep partial thickness burns, only one is commercially available for the treatment of full-thickness burns (cultured epithelial autografts, CEAs). CEAs are a life-saving treatment option; however, they are extremely fragile, prone to damage and require > 3 weeks to manufacture. As early wound closure reduces the risk of infection, fluid loss, mortality and scarring, strategies to quickly and permanently close full-thickness wounds are needed to increase survival and improve outcomes. The current obstacles to rapid in situ regeneration of full-thickness wounds using cell sprays include the lack of viable dermis, low engraftment efficiency and variable survivability of spray-on cells. Our team recently developed allogeneic dermal substitutes with laser ablative dermal papillae that significantly enhanced keratinocyte proliferation. This regenerative platform, consisting of freshly isolated autologous cell sprays and an off-the-shelf, allogeneic dermal template with laser micropatterned dermal papillae and growth factor loaded rapid release nanoparticles, is proposed to facilitate rapid, permanent wound closure via enhanced adhesion and survival of spray-on skin cells and rapid angiogenesis. In Aim 1, spray-on skin cell engraftment and survivability will be examined as a function of the form of the laser micropatterned dermal papillae (width, length, angle). Aim 2 seeks to further enhance spray-on skin cell survivability and epidermal regeneration via enhanced angiogenesis. The role of vascular endothelial growth factor (VEGF)/ platelet-derived growth factor (PDGF)-releasing, high surface area to volume polydopamine (PDA) nanoparticles on the rate and extent of angiogenesis and downstream epidermal regeneration will be assessed in a mouse model followed by a highly translational porcine model. In Aim 3, gradient collagen-silk scaffolds will be fabricated to reduce contraction of the wounds while providing a physical and chemical environment that promotes epidermal regeneration. Finally, the efficacy of the fully optimized laser micropatterned dermal template (dermal papillae form, VEGF/PDGF-PDA loading and concentration and scaffold mechanics) will be examined in a porcine burn model compared to standard autografting and spray-on skin cells alone. The proposed studies leverage the expertise in regenerative medicine, vascularization and large animal models to develop a novel, immediate use technology that can dramatically transform treatment for patients suffering from massive burn injuries and improve outcomes and quality of life.