Therapies to repair or regenerate damage to the musculoskeletal system often involve the implantation of synthetic materials to stabilize tissues or promote regrowth. However, synthetic materials induce a foreign body response (FBR), which can lead to adverse outcomes. Our ability to design effective therapeutic strategies to mitigate the FBR is hampered by an incomplete understanding of the molecular mechanisms that trigger the FBR. The current dogma of the FBR assumes that serum proteins adsorb to biomaterial surfaces and unfold, leading to irreversible adsorption and creating damage-associated molecular patterns (DAMPs) that initiate inflammation. Our recent studies suggest that this view is insufficient and instead that proteins interact with surfaces dynamically and that DAMPs may arise from multiple different sources. To this end, this proposal aims to test the hypothesis that the adsorption of proteins onto implanted biomaterials is dynamic (turning over continually and changing in time), and that the FBR is maintained by DAMPs derived from serum and by the continuous generation of DAMPs that are produced by recruited myeloid cells. Two specific aims were developed to test this hypothesis. Specific Aim #1 will determine the identity of surface-adsorbed proteins over time in the FBR using bioorthogonal tagging. This aim will incorporate the methionine (Met) analog azidohomoalanine to ubiquitously tag newly synthesized proteins at different times during the FBR in wildtype mice with implants. The tagged and untagged newly synthesized proteins will be quantified and the proteins identified with LC-MS/MS to determine the transient nature of the surface-adsorbed proteins. Specific Aim #2 will determine the origin of surface-adsorbed proteins and their identity in the FBR using cell-specific bioorthogonal tagging. This aim will use a recently created mouse line that has a point mutation in methionyl- tRNA synthetase (MetRS*) that enables cell-specific loading (via Cre drivers) of the Met analog azidonorleucine into newly synthesized proteins. Albumin-Cre and LysM-Cre drivers will be used to determine the origin of the adsorbed proteins from serum and myeloid cells, respectively. When combined with LC-MS/MS, the identity of the adsorbed proteins from each source will also be determined. Each aim will investigate silicone as a model implant, having a surface chemistry that is either hydrophobic (native surface) or hydrophilic (plasma-treated), to study the role of hydrophobicity on the dynamics of surface-adsorbed proteins. In addition, a subset of proteins from the LC-MS/MS results will be tested for their ability to activate macrophages in vitro and act as DAMPs. In summary, this exploratory project will utilize recently developed in vivo protein labeling techniques to answer fundamental questions about the events that trigger the FBR. Through this understanding, this project will generate new hypotheses and inform the rational design of biomate...