PROJECT SUMMARY Heterotopic ossification (HO) is the pathologic formation of ectopic bone in extra-skeletal tissues. Approximately 20% of patients who suffer some type of musculoskeletal injury develop HO. These injuries can be traumatic in nature or controlled tissue damage that occurs as a part of routine orthopaedic surgeries like joint replacement surgery or surgical amputations. The risk of HO disproportionately affects the Veteran population compared to civilians, especially due to higher risk of experiencing traumatic blast, brain, or spinal cord injury. Indeed, 64% of military blast injuries from the recent conflicts and Iraq and Afghanistan have resulted in HO. The presence of HO is associated with chronic pain, chronic infection, ulceration, impaired wound healing, and other related health complications. These complications from HO often preclude regaining mobility and function in the injured limb and substantially limit the use of prosthetics, impeding Veteran independence and return to duty or integration into civilian life. These difficulties can lead to opioid addiction, depression, and suicide, which are all major concerns to overall Veteran health. Current treatment options are limited due to the lack of understanding of the molecular mechanisms that drive ectopic bone formation during soft tissue healing following damage. Thus, the proposed work seeks to elucidate the mechanisms driving bone formation in HO in order to identify new, targeted therapeutic approaches for preventing and treating ectopic bone formation. The extracellular matrix (ECM) plays an essential role in regulating many biological processes including tissue repair and maintenance. While upregulated inflammation due to injury is known to significantly increase ECM synthesis, mechanisms linking aberrant ECM deposition to ectopic bone formation remain largely unexplored. We have carried out preliminary experiments in mouse models that consistently form ectopic bone that is histologically similar to HO in patients. Importantly, the soft tissue changes that are observed in these mice leading up to HO, particularly the aberrant and progressive accumulation of ECM molecules like collagens (COLs) and glycosaminoglycans (GAGs), closely recapitulate the changes observed in patients. Therefore, we hypothesize that abnormal overproduction of COL I and chondroitin sulfate (CS) GAGs creates an ECM environment capable of activating aberrant osteogenic signals in soft tissue in HO. The overall objective of this proposal is to elucidate the role of COL I and CS GAG accumulation in ectopic bone formation in order to identify potential therapeutic strategies and diagnostic markers using mouse models of HO. In Aim 1, we will determine the critical concentration and chemical composition of COL I and CS GAGs in the ECM that is able to establish a microenvironmental niche conducive for osteogenic differentiation and verify the concomitant upregulation of associated bone formation mar...