PROJECT SUMMARY Patient age is the predominant risk factor in the incidence of osteoarthritis (OA), a degenerative joint disease that is thought to result from persistent low-grade inflammation, i.e., “inflamm-aging.” Macrophages (MΦ), immune cells that line the synovial membrane, contribute to cartilage degradation through the secretion of inflammatory cytokines and matrix metalloproteinases (MMPs), adding to the proinflammatory feedback loop and progression of OA symptoms. MΦ functions are highly dependent on environmental cues, such as tissue stiffness, extracellular matrix (ECM) composition, and damage-associated molecular patterns (DAMPs); however, there are conflicting hypotheses as to whether environmental cues directly cause synovial MΦ- inflamm-aging or if aged MΦs inherently obtain intrinsic defects. This knowledge gap has limited our ability to determine and prevent the contribution of MΦ inflamm-aging in OA. However, there are currently no robust in vitro or in vivo models to study the mechanism of MΦ inflamm-aging. Cell lines or short-lived primary synovial MΦs cultures are not useful for elucidating age-specific drivers in vitro, while established murine OA in vivo models, such as destabilization of medial meniscus (DMM) surgery, have not been applied to the study of MΦ-specific inflamm-aging. The dearth of studies in suitable model systems prevents the mechanistic determination of factors driving MΦ inflamm-aging and ultimately hinders development of curative therapeutics. There remains a fundamental need to identify the underlying causes of MΦ inflamm-aging in OA and to develop meaningful preclinical tools that can model them. Utilizing synthetic nanoparticle (NP) systems that can directly interface with synovial MΦ (SMΦs), the objective of this proposal is to establish in vivo and in vitro tools that address our central question: how is SMΦ function impacted specifically by age? Our work will advance knowledge of MΦ inflamm-aging mechanisms in two aims; we will 1) detail SMΦ phenotype and behavior as a function of subject age using SMΦ-selective NP probes in a murine OA model that captures the full complexity of disease to elucidate intrinsic aging effects in SMΦ function, and 2) develop a dynamic hydrogel culture model that recreates in vivo responses in vitro to identify the role of extrinsic environmental cues in MΦ inflamm-aging. Our in vitro approach will be the first attempt in recreating MΦ inflamm-aging through microenvironment mimicry and validated with in vivo findings in an accepted OA model. These combined studies will assess our central hypothesis—that the SMΦ inflamm-aging phenotype is directly regulated by both age (intrinsic) and microenvironment (extrinsic) cues. The overall scientific impact of this project will be generation of new evidence of intrinsic age effects on MΦ response to isolated extrinsic environmental cues and a novel in vitro tool to study MΦ inflamm-aging. Both outcomes will directly support fut...