Purpose: Traumatic heterotopic ossification (tHO) has become a signature pathology affecting wounded military personnel who have sustained blast-associated traumatic amputations during the recent conflicts in Iraq and Afghanistan. Heterotopic ossification is characterized by the abnormal development of mature bone depositions in extra-skeletal sites such muscle, tendon, and soft tissues, leading to impaired wound healing, pain, reduced range of motion, and limited use of prostheses. While many factors influence the formation of tHO, the extended use of tourniquets to limit catastrophic hemorrhage during prolonged field care (PFC) has not been explored. Herein, we investigate the impact of tourniquet use following blast-related injury on ectopic bone formation. Methods: Utilizing an established pre-clinical model of blast-associated complex lower limb injury and traumatic amputation, we evaluated the effects of extended tourniquet use on tHO formation. Male rats (11-12-week-old) were subjected to blast overpressure exposure, femur fracture, and soft tissue crush injury. A pneumatic tourniquet (250-300mmHg) was applied to the injured limbs for either 90- and 150-minutes followed by trans-femoral amputation. Limbs were assessed for HO formation using microCT. Analysis of muscle/soft-tissue osteogenesis-related gene transcripts and multiple serum inflammatory mediators were measured by using qRT-PCR and Luminex multiplex assays, respectively. Results: At 12 weeks, volumetric analysis with microCT imaging revealed an 70% increase in total bone formation (P=0.007, n=11) near the site of injury in rats subjected to 150-minutes of tourniquet time compared to rats with no tourniquet time in the setting of blast-injuries. Rats subjected to 150-minute tourniquet usage and blast injury had increased expression of osteochondrogenic genes including Bmp2 (5.4-fold increase, P=0.01) as early as 6 hours post-injury while Hif1α (4.2-fold increase, P
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