Biomaterials are clinically used to replace lost or damaged tissue, but the surgical implantation procedure initiates an inflammatory response, which is orchestrated by macrophages. The long-term goal is to understand the signaling pathways used by macrophages to recognize biomaterial features and the resulting inflammatory processes that lead to tissue healing and regeneration. The objective of this proposal is to determine the contribution of cell-biomaterial interactions and danger-associated molecular patterns (DAMPs) in the regulation of Wnt ligands, and to elucidate the effect of macrophage-secreted Wnt ligands on T cell activation and stem cell recruitment and differentiation. The central hypothesis is that macrophages interact with biomaterials and DAMPs, triggering Wnt release that regulates bone healing by modulating T cell polarization and MSC proliferation. Our rationale is that macrophage activation is dependent on the physicochemical properties of biomaterials and the local environment. Macrophages respond to biomaterials by secreting inflammatory molecules, including Wnt ligands. Ablating Wnt ligand secretion in macrophages decreases their activation and disrupts the inflammatory response and bone-biomaterial integration. By identifying key signaling pathways that trigger Wnt ligand secretion, we can identify biomaterial surface modifications and signaling pathways that can be targeted to modulate and ameliorate the inflammatory process after biomaterial implantation to improve clinical outcomes. The central hypothesis will be tested in three aims: 1) Elucidate the role of integrin signaling in triggering Wnt secretion by macrophages in response to biomaterial physicochemical cues.; 2) Establish the effect of DAMPs in altering macrophage-secreted Wnt ligands; 3) Determine the effect of macrophage-secreted Wnt ligands on T cell polarization and MSC proliferation and differentiation after biomaterial implantation. We will pursue these aims using a combination of in vitro and in vivo studies with conditional transgenic knockouts in macrophages, pharmacological inhibitors, and macrophage adoptive transfer and CRISPR-mediated transcriptional activation to the activation of endogenous Wnt3a and Wnt5a expression. The study of Wnt signaling on macrophage behavior and as an orchestrator of the inflammatory response after biomaterial implantation is innovative and has yet to be studied. The proposed work is significant because it will determine the role of Wnt proteins in macrophage activation and will provide evidence of the importance of Wnt signaling in the inflammatory milieu that can be translated into other areas like chronic inflammatory diseases and cancer. The near-term expected outcome of this work is the understanding of Wnt signaling in macrophage activation and its contribution to the inflammatory and healing process in response to biomaterial surface characteristics. Results from this proposal will have an immediate positive im...