Abstract Body: An impaired microcirculatory network due to injury, ischemia, or disease stimulates maladaptive remodeling and disrupts broader tissue function. Additionally, innate regenerative models in zebrafish and neonatal mice have demonstrated that supplemental vascularization occurs first in heart regeneration and is critical for subsequent cell proliferation and remuscularization, yet clinical approaches to small vessel regeneration remain elusive. In previous work, we employed an iterative fractional factorial design of experiments (DOE) approach with 10 angiogenic proteins to identify 5 high-performing factors, VEGF, bFGF, Shh, PDGF, and IGF-1. In this work, we optimized a fully defined and proangiogenic biomaterial delivery system using type 1 collagen and heparin-modified alginate to prolong co-release of multiple proteins up to 21 days in vitro. Using an in vivo subcutaneous gel plug assay in the WT immune competent rat, we have empirically evaluated 1-, 2-, and 3-protein combinations of the top five factors after 1 and 2 weeks of release. Custom, automated, high-fidelity quantification of vascular size, density, and morphology enabled identification of promising cocktails based on target tissues and desired vascular kinetics. A combination of VEGF, IGF-1, and PDGF (VIP) was identified for rapid, local vascularization, increasing perfused vascular density by 6-fold vs. the blank biomaterial at 1 week, with over 2/3 of vessels stable and persisting at 2 weeks. We employed a rat model of ischemic wound healing as a severe example of ischemic injury requiring a rapid vascular flourish for wound closure. Over the course of 2 weeks, the VIP-loaded biomaterial enhanced vascularization, increased both the rate and magnitude of wound closure, and improved skin regeneration when compared to the blank material and no treatment controls. This work has demonstrated both the promise of revascularization to facilitate regeneration in ischemic injury and the success of the VIP-loaded biomaterial in directing it. In conclusion, we identified a high-performing proangiogenic protein cocktail and optimized its controlled, local release from a defined biomaterial to develop a promising revascularization therapy for local microvascular regeneration.