This application seeks funding to support a NIAMS K08 recipient during her transition to research independence. The broad objective is to investigate the role of mitochondrial Damage-Associated Molecular Patterns (mDAMPs) after cartilage injury and in the development of posttraumatic osteoarthritis (PTOA). In many tissues, injury-induced mitochondrial dysfunction causes cells to release mDAMPs into the extracellular environment, which perpetuates inflammation and leads to ongoing tissue damage. Recent work, performed during the applicant’s K08 award, demonstrated that mitochondrial dysfunction is an acute response of chondrocytes to cartilage injury, and importantly, mitoprotective therapy prevents cell death and cartilage degeneration. However, the basic mechanisms whereby mitochondrial dysfunction leads to PTOA are not well understood, and mDAMPs have not been studied in cartilage. To test the hypothesis that mDAMPs are released from chondrocytes undergoing injury-induced mitochondrial dysfunction, and that these damage signals are associated with clinical joint trauma, we will measure mDAMPs in four different model systems. The goal of Aim 1 is to identify specific signals that initiate mDAMP release. First, chondrocytes will be stressed in vitro using several stimuli known to induce mDAMP release in other cell types. Inhibitors of mitochondrial dysfunction and mitophagy will also be tested for their ability to block mDAMP release. Next, we will determine if mechanical injury to cartilage explants results in mDAMP release ex vivo. The results of Aim 1 will provide insight into how mDAMP release is triggered, and how it may be manipulated therapeutically. Aim 2 will analyze mDAMPs in equine synovial fluid to determine if articular injury results in mDAMP release in vivo. mDAMP concentration will be measured in banked synovial fluid from previous studies, where horses had experimental joint injury, with and without intraarticular mitoprotective therapy. To determine if mDAMP release is associated with naturally occurring disease, mDAMPs will be measured in equine synovial fluid from clinical patients with acute joint trauma. Results of Aim 2 will determine if mitoprotection can prevent mDAMP release in vivo, and if synovial fluid mDAMPs are useful indicators of early cartilage/bone injury. This proposal builds on the applicant’s K08 research, and utilizes samples generated during those studies, allowing the proposed aims to be accomplish within the 2-year time frame. These studies represent a new line of inquiry, independent from the applicant’s mentor. Dr. Delco solely generated the research concept, designed the experiments, wrote the proposal, and will be responsible for project oversight. By establishing a new area of research within the field, this award will allow Dr. Delco to build an independent research program and continue to develop a niche in mitochondrial biology in osteoarthritis and regenerative medicine. Understanding the role of mDAMPs has the near-term potential to change the way we diagnose and treat joint injury.