PROJECT SUMMARY The incidence of acute and chronic wounds from surgery, traumatic injury, diabetes, and metabolic syndrome is on the rise, affecting over three million people in the US alone and costing tens of billions of dollars a year. Successful wound healing progresses through coagulation/inflammation, proliferation, and remodeling phases, culminating in tissue regeneration and/or contraction and scarring. Macrophages are essential mediators of progression through these stages, and responsive to external therapeutic modulation. Yet, the soluble and mechanical factors mediating macrophage interactions with these materials and other wound effectors remain largely unknown. I propose to use immunology and bioengineering approaches to improve our tools and work to fill this unmet clinical need. Our previous work demonstrated that fibrin, a major component of the provisional extracellular matrix, skews murine bone marrow derived macrophages (BMDM) towards an anti-inflammatory phenotype in vitro, suppressing the stereotyped cytokine response to potent inflammatory stimuli, including LPS. Preliminary data from 5 mm full-thickness skin wounds in mice showed anti-inflammatory skew in the wound, measured by Arginase, iNOS, and YAP expression in macrophages, and diminished myofibroblast marker alpha smooth muscle actin (aSMA) with fibrin treatment, suggesting a role for macrophage-myofibroblast interactions in the wound. Additionally, both soft (1.2 kPa) and stiff (840kPa) gels have been shown to increase macrophage secretion of inflammatory cytokines seen in the stereotyped foreign body response (e.g. TNFa), with intermediate stiffness showing lower secretion, indicating a mechanical sweet spot for immunomodulation. I propose to engineer fibrin-PEG hydrogels, mechanically optimized for macrophage immunomodulatory capacity, and apply them to in vitro cocultures to determine macrophage-fibroblast-fibrin interaction dynamics and test the hypothesis that fibrin hydrogels of moderate stiffness (~140 kPa) will best promote anti-inflammatory macrophage phenotype, facilitating decreased myofibroblast contractility. I also aim to define the mechanisms by which fibrin hydrogels modulate macrophage phenotype to achieve wound healing and regeneration in vivo, using a full thickness skin wound model in wild type, and selective immune cell depletions models (macrophage- MAFIA, clodrosome;; neutrophil- anti-GR1, B-cell-anti-CXCL13). I will use histology, flow cytometry, and RNA-seq to test the hypothesis that fibrin-PEG hydrogels of moderate stiffness (~140 kPa) will accelerate wound resolution and tissue regeneration by promoting anti-inflammatory macrophage phenotype. Understanding the mediators of fibrin-enhanced wound healing will help us better understand physi...