Novel Anti-Fibrotic Strategies In The Targeted Treatment And Prevention Of Post-Traumatic Ho And Enhancement Of Post-Traumatic Tissue Regeneration.

Background: Neuromusculoskeletal injuries sustained in recent military conflicts have been notable for their number and complexity. Post-traumatic heterotopic ossification (HO) is the development of bone in the soft tissues, and it is a significant sequela of these traumatic wounds occurring in approximately 60%-70% of the war wounded. HO is the end product of a deranged fibroproliferative healing response and can render the extremity disfigured, painful, and nonfunctional. Our group has studied this condition in combat-related injuries at the cell and molecular level for the greater part of the last decade. In addition to identifying a progenitor cell population involved in this healing response, we have also identified TGF-beta1-mediated tissue fibrosis to be one of the key initial steps in the pathogenesis of HO and that dysregulation of the SMAD3 intracellular signaling protein in conjunction with a fibrotic microenvironment to be a central feature of the bone forming process.Hypothesis: We hypothesize that interrupting this TGF-beta1 mediated signaling process at the level of Smad protein activation will suppress the fibroproliferative response and reduce or eliminate the formation of HO. We propose a preclinical trial to investigate the effect of specifically blocking the activation of the TGF-beta1 intracellular signaling proteins SMAD2 and SMAD3 on the development of post-traumatic HO using the inhibitors SB431542, Galunisertib, SIS3, and Halofuginone.Specific AimsAim 1: To assess the efficacy of SB431542, Galunisertib, SIS3, and Halofuginone treatment in preventing fibrosis in a cell culture model.Aim 2: To assess the efficacy of SB431542, Galunisertib, SIS3, and Halofuginone-based therapy in preventing fibrosis and ectopic bone formation in an animal model.Aim 3: To assess the effectiveness of SB431542, Galunisertib, SIS3, and Halofuginone in promoting muscle regeneration.Study DesignAim 1: Our lab has developed an in vitro model of tissue fibrosis as it relates to wound healing. Using this model, we will assess the ability of the SMAD-specific inhibitors SB431542, Galunisertib, SIS3, and Halofuginone to modulate the TGF-beta1-induced fibrotic response of MPCs derived from war-traumatized wounds. These data will enhance our interpretation of animal model data and provide an in-depth understanding of the mechanisms of action of these inhibitors that will be necessary to transition to human clinical trials.Aim 2: Using a rat-blast model of post-traumatic HO, we will assess the efficacy of the TGF-beta1 inhibitors SB431542, Galunisertib, SIS3, and Halofuginone to reduce tissue fibrosis and prevent the development of HO. Animals will be given intraperitoneal injections of each inhibitor for 2 weeks after injury. The amount of scar and bone formation will then be assessed.Aim 3: Using an established murine model of cardiotoxin-induced muscle injury and regeneration, we will assess the ability of the SMAD-specific inhibitors to improve the quality and quantity of muscle fibers. Cardiotoxin will be introduced into the tibialis anterior muscle of C57/Black6 mice. Test groups will include intraperitoneal delivery of SB431542, Galunisertib, SIS3, and Halofuginone. Histological and immunohistological analysis will be used to compare test groups to untreated controls and evaluate improvement in muscle regeneration.Military Relevance: Neuromusculoskeletal trauma is intimately associated with war-related injuries as is the subsequent tissue fibrosis and HO that develops during the wound healing response. The preclinical trial provides the data necessary to translate recently gained basic science data into a novel clinical therapeutic strategy to prevent the development of HO and reduce the formation of scar tissue.