ABSTRACT INVESTIGATORS: Tristan Maerz, PhD (PI) is an ESI biomedical engineer focused on developing new treat- ments for post-traumatic osteoarthritis (PTOA). The Co-Investigators Kurt Hankenson, DVM PhD and Craig Duvall, PhD have expertise in Wnt signaling and biomaterial-mediated tissue regeneration, respectively. RESEARCH CONTEXT: Overactivation of canonical Wnt/β-Catenin (cWnt) signaling is increasingly recognized as a driver of joint degeneration in PTOA. A novel cWnt agonist in the context of PTOA is R-spondin 2 (Rspo2), which some literature and our data support as a disease-promoting ligand via cWnt-mediated induc- tion of synovial fibrosis, protease overexpression, chondrocyte hypertrophy, and aberrant bone sclerosis. A recent drug screen identified that the drug Mianserin inhibits Rspo2 signaling by blocking Lgr5 binding. Given high solute clearance rates in the joint, we must engineer new depot-type formulations using biomaterials to deliver small molecules such as Mianserin. Poly(lactic-co-glycolic acid) (PLGA) microspheres are a versatile and biocompatible delivery vehicle well-suited to deliver molecules to the joint, and they can be engineered to deliver Mianserin for sustained intra-articular cWnt pathway inhibition by targeting Rspo2 signaling. OBJECTIVE: To validate the role of Rspo2 in PTOA pathogenesis and test efficacy of sustained intra-articular Rspo2 inhibition using Mianserin-loaded PLGA microspheres. SPECIFIC AIMS: 1). Elucidate the impact of Rspo2 on PTOA progression; 2). Advance the development of PLGA microspheres to achieve sustained in vivo delivery of a small-molecule Rspo2 inhibitor; 3). Test the effi- cacy of sustained intra-articular Rspo2 inhibitor therapy as a novel PTOA treatment. RESEARCH PLAN: Aim 1). We will delineate Rspo2-responsive cell types and characterize the disease-pro- moting effect of Rspo2 in healthy and injured joints of TCF/LEF Wnt reporter mice. Single-cell RNAseq will elu- cidate Rspo2-Lgr signaling cells while unbiasedly assessing transcriptional responses to Rspo2. Rspo2 will then be ablated globally at the time of joint injury using Rosa26-CreERT2; Rspo2f/f mice and PTOA severity will be assessed. Aim 2). We will formulate Mianserin-loaded PLGA microspheres, aiming to achieve >4 weeks of in vitro release with negligible burst. We will confirm 4 weeks of in vivo cWnt signaling suppression and assess safety. Using primary cells, we will examine the effect of sustained Mianserin delivery on cWnt signaling in chondrocytes, synovial fibroblasts, and osteoprogenitor cells. We will further test PLGA-based Mianserin deliv- ery on Rspo2-treated human cartilage explants obtained during total knee arthroplasty. Aim 3). We will admin- ister Mianserin-loaded PLGA microspheres to the joint following injury and comprehensively phenotype PTOA, benchmarking efficacy against repeated Mianserin and repeated β-Catenin inhibitor injections. We will verify that the in vivo effect of Mianserin is mediated thro...