ABSTRACT INVESTIGATORS: Rhima Coleman, PhD (PI) is an Associate Professor of Biomedical Engineering focused on genetic engineering of stem cells for the treatment of joint pathology. Tristan Maerz, PhD (Co-PI) is an Assistant Professor of Orthopaedic Surgery with expertise in PTOA pathophysiology and synovial inflammation. RESEARCH CONTEXT: Persistent intra-articular inflammation is a recognized driver of joint pathology during post-traumatic osteoarthritis (PTOA) progression. Mesenchymal stem/multipotent stromal cells (MSCs) possess potent anti-inflammatory properties, and MSC-derived chondrocytes (MdChs) have shown promise as a regenerative intra-articular treatment. Successful implementation of MdChs as a cellular therapy is currently hindered by rapid hypertrophic maturation of MdChs driven by the transcription factor RUNX2, which is exacerbated by cytokines known to be enriched in the PTOA joint. We have genetically engineered MdChs to autonomously suppress RUNX2 (sRX2-MdChs), and, therefore, chondrocyte hypertrophy, which supports cartilage formation, even under inflammation. Critically, our preliminary data also indicate that the secretome of these genetically modified cells markedly reduce the local pro-inflammatory environment by inducing repolarization of pro-inflammatory macrophages (M1) into the anti-inflammatory (M2) phenotype. OBJECTIVE: To test the disease-modifying and anti-inflammatory efficacy of genetically engineered RUNX2- suppressing MdChs as a cellular treatment in a murine model of PTOA. SPECIFIC AIMS: 1). Demonstrate that a single sRX2-MdCh injection blocks pathological inflammation and PTOA progression following joint injury. 2). Characterize the mechanisms that mediate the autocrine and paracrine effects of the sRX2-MdChs secretome on cartilage formation and the anti-inflammatory cellular crosstalk between sRX2-MdChs and macrophages in vitro. RESEARCH PLAN: In Aim 1, we will employ a noninvasive murine joint injury model of PTOA to test the anti- inflammatory and PTOA-mitigating effects of a single sRX2-MdCh injection following injury. Male and female mice will be randomized to 1) sRX2-MdChs; 2) Scramble-MdChs; 3) Unmodified/WT MSCs; or 4) Vehicle. We will use a multifaceted set of live, in vivo and ex vivo outcomes to assess joint pain, intra-articular inflammation and protease activity, bone remodeling, and articular cartilage damage to assess PTOA severity. We will track intra-articular retention of injected cells via live fluorescent monitoring of luciferase activity and a near-infrared tag. In Aim 2, we will test the hypothesis that the observed autocrine and paracrine effects of the sRX2-MdCh secretome are mediated by IL-4 and assess the mechanism by which IL-4 effects MdCh cartilage formation and M1-to-M2 macrophage polarization.