SEQUENTIAL VERSUS COMBINED MEDICAL THERAPIES AS A NOVEL HETEROTOPIC OSSIFICATION PREVENTION STRATEGY

Background/Readiness: The physiological insults produced by blast overpressure resulting in severe extremity injury trauma account for approximately 70% of all combat-related war injuries. Compared to previous conflicts, there is a relative decrease in lower extremity and torso wounds, with a concomitant increase in the head, neck, and upper extremity wounds. Heterotopic ossification (HO), the development of ectopic bone within non-skeletal tissues, affects two-thirds of patients with high-energy blast-related injuries and more specifically HO develops in 30% of injured Service members with complex upper extremity wounds wherein the residual limb is amputated. The clinical complications of HO include life-limiting pain, skin ulceration, and poor tolerance of prosthetic wear, where conservative interventions such as multi-modal pain regimens, physiotherapy, and prosthetic modification fail to alleviate symptoms and prevent surgical excision in approximately 41% of patients. HO prevention in upper extremity combat trauma has garnered a greater focus as current prophylaxis strategies used in civilian trauma, such as post-operative administration of non-steroidal anti-inflammatory drugs and external-radiation beam therapy, can be contraindicated in patients who sustain systemic combat or blast-related trauma. The microenvironment that supports ectopic bone development contains a dynamic composite of cells within an inflammatory, osteogenic, and angiogenic milieu and scaffold of extracellular matrix, where osteogenic stem/progenitor cells thrive. Therefore, we propose that a reasonable approach would be to shift from therapeutic and prevention strategies focused on identifying the origin and targeting progenitor cells towards altering the ectopic bone microenvironment that supports ectopic bone development and progression.Hypothesis/Objectives: The proposal will test the overarching hypothesis that a concurrent or sequential prophylactic treatment strategy using the retinoic acid receptor-gamma agonist Palovarotene and/or the mTOR inhibitor Rapamycin (Sirolimis), Food and Drug Administration-approved drugs, that target the regulation of key micro-environmental molecular signaling components/inductive factors that are critical in the development of the extra skeletal bone tissue formation in damaged/traumatized ischemic tissue will prevent HO formation.Specific Aims (SA): The proposal will determine the effects of Palovarotene and Rapamycin on muscle-derived progenitor cell chondrogenic, angiogenic, and osteogenic differentiation in vitro (SA1), on ectopic bone development in a physiologic rat model of trauma-induced HO (SA2), and on normal fracture healing (SA3).Study Design: In vitro culture studies will be conducted to determine the optimal dosage of Rapamycin and/or Palovarotene that inhibit the functional proliferation and/or differentiation of rat muscle-derived mesenchymal stem/stromal cells (rMSC) and to define their potential underlining anti-chondrogenic, angiogenic and osteogenic effects. Using our established blast-related combat-injury model of HO, adult male Sprague Dawley rats will sustain a blast from a pneumatic shock tube, followed by a controlled, comminuted fracture and crush injury of the thigh. After injury, either limb salvage with extremity fixation or transfemoral amputation through the fracture site will be performed. Closed wounds from limb amputated rats will be inoculated with 106 CFU of Methicillin-resistant Staphylococcus aureus (MRSA). Rats will be randomly assigned to one of four daily treatment groups: (1) Rapamycin treatment for days 1-7 followed by Palovarotene treatment on days 8-14 post-injury, (2) Palovarotene treatment for days 1-7 followed by Rapamycin treatment on days 8-14 post-injury, (3) concurrent treatment with both drugs for the first 14 days post-injury, and (4) vehicle control treatment for 14 days post-injury. Longitudinal assessment of wound healing/repair in addition to the timing and volume of ectopic bone development will be monitored at pre-determined time points using micro-CT imaging, histology, and gene transcript analysis using low density microarray RT-PCR technology. To functionally assess targeted inhibition of MSC proliferation and differentiation, the prevalence of osteogenic, angiogenic, and chondrogenic colony-forming cell connective tissue progenitor cells (CFU/CTPs) tissue per gram of injured/healing muscle will be measured.Military Benefit: HO is a frequent complication in severely traumatized limbs. In the recent conflicts, HO has occurred in ~30% of major combat injuries involving upper extremities. HO can severely compromise both early wound care as well as the process of fitting and effective use of a prosthetic limb. During the treatment and recovery from severe limb trauma, HO often results in the need for additional surgery and can be the determining factor in late-stage amputations or revision of amputations at a higher level. The current proposal will test the utility and feasibility of Palovarotene and Rapamycin to address this challenge to military medicine.