Project Summary Tissue damage due to injury, disease, or congenital defect continues to be a critical clinical obstacle in human health. Regenerative medicine-based therapies are a promising strategy, yet the critical determinants of success are the immune response and fibrosis. Typically, in tissue repair, inflammation is closely controlled before remodeling by activated fibroblasts, whose presence is also tightly regulated. Immunomodulation is a promising strategy to avoid fibrotic outcomes, yet it is not the immune cells that directly participate in tissue remodeling and collagen production. Therefore, it is critical to understand how immune cells coordinate stromal cells to promote tissue repair or fibrosis. In fibrosis, evidence suggests that chronic immune cell infiltration and proinflammatory cytokines accompany fibroblasts poised to promote fibrosis. Those fibroblasts are characterized by aberrant mechanotransduction (i.e. sensing “normal”/soft as stiff/scarred) leading to the overproduction of ECM. We now know that immune cells and fibroblasts exist as phenotypically distinct subpopulations, including those with a molecular signature tied to fibrosis. Whether inflammatory signals elicit distinct fibroblast subpopulations with aberrant mechanotransduction in fibrosis is unknown and is a key objective. Understanding the relationship between inflammation and fibroblast heterogeneity is essential to predict the regenerative potential of therapies. I hypothesize that fibroblast subpopulations exist during fibrosis with aberrant mechanotransduction that is regulated by inflammatory signaling. During the mentored K99 phase of this application, I will identify the immune and fibroblast subpopulations that emerge during tissue regeneration and fibrosis leveraging non-fibrotic/regenerative microporous annealed particle hydrogels (MAP gels) and fibrotic nonporous bulk hydrogels (NP gels). I will determine the mechanotransductive differences across those subpopulations by purifying them with identified markers and measuring ECM production, myofibroblastic differentiation, integrin activation, and focal adhesion signaling. I will identify signaling pathways necessary for the phenotypes unique to fibroblast subpopulations. I will validate these findings by confirming the identified fibroblast subpopulations and signaling pathways in fibrotic clinical implants. I will continue my scientific and professional development in preparation for the independent phase with consistent guidance from my scientific advisory committee, diversity advisory committee, and collaborators. During the independent phase, I will determine mediators unique to regenerative and fibrotic microenvironments using MAP and NP gels. I determine if IL-1 signaling in fibroblasts is necessary for fibrosis by implanting NP gels in fibroblast-specific IL-1RI knockout mice. I will also determine whether IL-1 signaling is sufficient to promote fibrosis using MAP gels conjugated with IL-1....