PURPOSE: Thermal burns to the face or extremities are highly morbid injuries risking severe disfigurement, functional and psychosocial impairment. These injuries commonly require debridement and prolonged or staged reconstruction often with multiple grafts, flaps, and need for delayed-touch ups. This standard-of-care is costly, time-consuming, deeply frustrating, and puts patients at risk of extensive donor-site and surgical complications. Currently there are no simple, scalable, and single-stage procedures available for extensive or multifocal burns able to address full-thickness trilaminar defects. Our team has previously demonstrated the viability of an adipose-first reconstruction to address hypodermal defects and provide a well-vascularized base reconstruction of complex burns. In this study, we demonstrate the efficacy of a combined fat plus finely minced skin (pixel-grafted) to achieve a single-stage trilaminar skin reconstruction with minimal donor site morbidity. METHODS: Female Yorkshire swine sustained 16 full-thickness circular burns with a custom burn device of 7 cm2 in surface area for 16 seconds at 100oC. After 48-hours, escharectomies were performed to the level of the fascia. Adipose from female Yorkshire swine was used on the wound as the initial layer of the reconstruction upon removal of the eschars. In one group, autologous split-thickness skin grafts were cut into pixel size (0.3x0.3 mm) grafts and applied on top of the adipose grafts. Pigs were maintained for 4-weeks with weekly photography, ultrasound, and biopsies, followed by sacrificed for histology and tension measurements. RESULTS: At the end of the 4-week period, adipose combined with pixel graft demonstrated improved epithelialization and less contracture (p<0.01). Thickness and mobility measurements were consistent in both groups and were similar to our previous approaches using adipose following surgical debridement. Tissue pliability in the pixel grafting group was maintained to a high degree. On histologic analyses the presence of distinct, viable epidermal, dermal, and hypodermal elements was noted on cross-sections, suggesting the reconstitution of full-thickness trilaminar cutaneous architecture. CONCLUSION: We found that a basal layer of particulate fat provided enough nutrient exchange to support immediate pixel-grafted skin. This particulate skin-plus-fat approach allowed us to generate a single-stage trilaminar reconstruction in complex burn defects in a highly translatable swine model. Immediate, single-stage trilaminar reconstruction of full-thickness complex burns reduces contracture, mitigates adhesion, and restores normal soft-tissue thickness, therefore, presenting a paradigm changing approach in the current practice of burn injuries to the mobile surfaces.

and thawed to room temperature prior to testing and half of which were freshly excised and at room temperature, were loaded onto a 100N load cell force-displacement apparatus. Samples were stretched at a constant strain rate (1mm/ sec) until failure. Durometer, stress-strain curves, Young’s Modulus (YM), and ultimate tensile burst strength were analyzed. Wilcoxon Mann-Whitney tests were performed and p-value<0.05 were considered statistically significant.

INTRODUCTION: Radiation-induced skin fibrosis is one of the main adverse effects of radiation therapy for cancer treatment. Radiation fibrosis syndrome is caused by the overactivation of TGF-B that promotes fibroblast that induces collagenases and breaks down type III collagen and replaces it with type I collagen. The inhibition of P53 and the accumulation of ROS are the main mechanisms of radiation-induced damage. To fully understand these mechanisms, we tested the use of our novel human skin perfusion model to recreate the damage caused by radiation and as a platform to test possible therapeutic and/or prophylactic treatments to prevent fibrosis. METHODS: We use our perfusion model, which consists of a human tissue sample recovered from abdominoplasty. We dissect the superficial inferior epigastric artery and cannulated it and perfuse it with special culture media. On the first day after cannulation, we exposed the skin to a single targeted irradiation dose of 20gy and 40gy and took punch biopsies on days 3, 6, 12, and 16 for histological and gene expression analyses. The histological samples were stained with H&E, and Masson´s Trichrome stain to determine the morphology changes and extracellular matrix deposition respectively. TUNEL and DAPI immunofluorescent staining was performed to analyze for apoptotic changes in the epidermis/dermis. Expression of inflammatory, fibrotic and apoptotic genes was analyzed by real-time quantitative PCR. RESULTS: The morphological changes in the skin were significant. The radiation-exposed skin started peeling compared to the control group. The H&E staining showed an increase of inflammation in the dermis along with epidermis/dermis separation as well as papillary dermis containing fibrin deposition, accumulation of inflammatory cells, reactive changes in the endothelial cells, and abundant necrotic keratinocytes. Masson’s Trichrome staining revealed a increased deposition of extracellular matrix between the papillary and reticular dermis in the irradiated skin. The TUNEL and DAPI stain shows an increase in apoptotic cells in the radiation group that correlates with the damage induced by radiation. Gene expression analyses revealed upregulation of inflammatory and anti-apoptotic genes expression. CONCLUSIONS: Our perfusion model was stable for 19 days and was able to recreate the radiation-induced deposition of collagen shown in the H&E and Masson´s trichrome as well as the radiation-induced damage in the TUNEL and DAPI staining. Our system is reliable and can be used as a platform for developing therapeutics to mitigate the effects of radiation on the skin.

BACKGROUND: There is a delicate balance between physiologic healing and formation of unfavorable scars. Homeostasis between extracellular matrix (ECM) deposition and remodeling is maintained by an array of inter-connected signaling networks with situationally-dependent functions. We previously demonstrated that systemic mineralocorticoid receptor (MR) inhibition improves epithelialization and diminishes collagen deposition without eroding scar strength. MR-inhibition, however, has a range of off-target effects when applied systemically. Consequently, herein we evaluated the role of local MR-inhibition in wound healing and hypothesize that signaling through the MR on macrophages contributes to a pro-fibrotic phenotype in wound healing. METHODS: Female C57Bl/6 mice sustained bilateral 6 mm full-thickness biopsies with stenting and were stratified into either a) vehicle control, b) 5% topical spironolactone, or c) systemic spironolactone. Systemic spironolactone was delivered intraperitoneally every three days, and topical cream formulations of spironolactone and vehicle control were reapplied every three days. Tegaderm was placed over the mice to allow topical cream to remain over wound. Mice were followed photographically for 6 weeks for time to re-epithelialization and terminal scar area. Wound biopsies were collected for gross architectural analysis and assess collagen, fibrin, and elastic fibers with Movat’s pentachrome. RESULTS: By day 5, both spironolactone groups demonstrated epithelization with minimal contracture. Scar area was noted to be diminished in both the spironolactone groups versus the control group. In mice receiving systemic spironolactone, all wounds had epithelialized by day 14, while wounds with topical application continued to remain open. All wounds had epithelialized by day 17 in mice receiving topical spironolactone and by day 21 receiving the topical control agent. Skin elasticity was improved with topical spironolactone application as compared to topical control. As opposed to human wounds, murine injures begin to contract immediately post-epithelialization. This was not impaired by MR-inhibition and consequently we noted a significant reduction in scar area in both spironolactone treatment groups. Histologic evaluation demonstrated persistence of inflammation, wound edema, and immature ECM. CONCLUSION: These results corroborate our prior findings of the efficacy of MR-inhibition in improving scar resolution with a systemic delivery. Mice receiving systemic and topical spironolactone healed their wounds quicker than mice receiving a topical control agent. Topical application further highlighted the promising role of ECM-modifying mechanism involved with MR manipulation, with the benefit to minimize side effects and maximize the treatment efficacy. Given that wounds in the systemic spironolactone group healed the earliest, next steps would be to test different concentrations of topical Spironolactone to determine how this enhances wound healing.

BACKGROUND: Free tissue transfer stands at the apex of the reconstructive ladder, however, despite decades of surgical innovation, free flaps remain limited by physiologic need for rapid and sustained reperfusion to maintain viability. This need can cause a particular limitation in cases of prolonged ischemia periods and restricted availability of recipient vessels. To overcome this limitation, paradigm shifts towards device-assisted and/or ex vivo perfusion supported flap technology are necessary to expand capabilities of free flap surgery. However, in developing these technologies, there is a need for a replicable, docile, and anatomically relevant large-animal model from which we could easily assess and modify free tissue transfer with external analytic and/or treatment devices. Here we aim to describe the novel ovine model of fasciocutaneous free flap transfer, which we have developed for this purpose. METHODS: Female Suffolk sheep cadavers weighing 50-65 kg were studied to identify a saphenous system-based fasciocutaneous free flap model. Anatomical measurements and photography were performed to standardize flap harvest and feasibility of cadaveric flap perfusion was assessed via ex vivo fluorescein angiography. Following flap identification, the autologous microsurgical transfer of the flap to the neck was performed in a female Hampshire sheep weighing 65 kg. The sheep was followed for 2 weeks with daily photography. Extracellular tissue lactate and glucose levels were characterized during the transfer and weekly with a custom microdialysis probe placed in the flap tissue. Doppler ultrasonography was performed weekly to assess pedicle viability. Fluorescein angiography was used to assess flap viability at the initial surgery as well as the endpoint. Tissues were collected for histology at the endpoint. RESULTS: Saphenous vessels branching off the femoral system were identified to have up to 2.5 mm vein and 2.0 mm artery diameter, with the total pedicle length reaching up to 6 cm. The cadaveric flap tissue demonstrated feasible inflow and outflow with fluorescein angiography. Upon autologous transfer, the transferred flap of 9x6 cm provided complete coverage of the neck defect and demonstrated viability for the entire duration of the experiment. Viability of the free flap was confirmed with doppler ultrasonography and fluorescein angiography. The donor site was covered with a skin graft from the neck with bolster placement. CONCLUSION: We identified a novel model of free flap transfer to serve as a testbed for clinically translatable approaches to improve outcomes in reconstructive microsurgery. Ovine docility, relative to other quadrupeds, improves daily flap accessibility and supports survival. These initial studies serve to set the foundation for the use of complex extracorporeal membrane oxygenation (ECMO)-like devices for enhancing uses of the free flaps.

PURPOSE: Rapid functional recovery after facial nerve injury is critical to restoring expression. Tissue engineered nerve guides support enhanced recovery, acting as regenerative scaffolds. This can be improved by addition of exogenous neuro-supportive agents such as glial-derived neurotrophic factor (GDNF), which improves axonal growth and pathfinding. In this study, we evaluated efficacy of a composite poly(caprolactone) nerve guide containing double-walled GDNF microspheres on functional, electrophysiological, and histological outcomes in a rat facial nerve injury model. METHODS: Male Lewis rats underwent transection and repair of the buccal branch of the facial nerve and were divided as follows: a) transection and repair only, b) empty guide, c) GDNF-guide. Weekly measurements of the whisking movements for protraction and retraction angles were recorded. At the endpoint of 12-weeks, compound muscle action potentials at the whisker pad were assessed and nerve, muscle, and whisker pad were collected for histomorphometric analysis, including Schwann cell analysis. RESULTS: GDNF-guide treated rats displayed earliest peak and highest recovery in normalized whisking amplitude (p<0.001). Compound muscle action potentials were significantly higher after GDNF-guide placement vs. all others (p<0.001). Mean muscle fiber surface area was the highest (p<0.01) and the axonal integrity loss was less prominent with GDNF-guides. Gross morphology of the whisker pad was not different across the groups. CONCLUSION: The novel tissue engineered nerve guide containing GDNF microspheres enhances recovery after facial nerve transection. Results support the clinical viability of these guides to enhance recovery after nerve injury and hold promise to facilitate recovery in defects with larger gaps.

Purpose: Injury to the facial nerve and the resulting facial nerve palsy lead to devastating functional, psychological, and cosmetic challenges. Rapid functional recovery after facial nerve injury is critical to prevent muscle atrophy and restore expression. Bioengineering plays an important role to create artificial materials that are able to mimic the nerve itself without the need for a donor nerve. Tissue engineered nerve guides support enhanced recovery, by reducing axonal sprouting, minimizing neural scar formation, and acting as regenerative scaffolds. This can be improved by addition of exogenous neuro-supportive agents such as glial-derived neurotrophic factor (GDNF). GDNF is a promoter of axonal elongation and branching and has been shown to promote Schwann cell proliferation and migration. In this study, we evaluated efficacy of a composite poly(caprolactone) nerve guide containing double-walled GDNF microspheres on functional, electrophysiological, and histological outcomes in a rat facial nerve injury model. Methods: GDNF was encapsulated within double-walled poly(lactic-co-glycolic acid)/poly(lactide) microspheres and embedded in the walls biodegradable poly(caprolactone) nerve guides. This nerve guide capable of providing a sustained release of GDNF for >50 days was used to repair a facial nerve injury model in male Lewis rats. After transection and primary repair of the buccal branch of the facial nerve, the rats were divided as follows: a) transection and repair only, b) empty guide, c) GDNF-guide. Marginal mandibular branch of the facial nerve was also transected and ligated to prevent innervation of the whiskers. Weekly measurements of the whisking movements for protraction, retraction and amplitude angles were recorded. At the endpoint of 12-weeks, compound muscle action potentials at the whisker pad were assessed and nerve, muscle, and whisker pad were collected for histomorphometric analysis, including Schwann cell analysis. Results: GDNF-guide treated rats displayed earliest peak and achieved the highest whisking amplitude with 36% recovery compared to the baseline. Weekly whisking amplitude measurements demonstrated both time and the treatment groups were independently associated with the recovery (p<0.001) and GDNF treatment had the highest impact versus all others (p<0.05). Compound muscle action potentials were significantly higher after GDNF-guide placement versus all others (p<0.001). Mean muscle fiber surface area at the levator labii superioris muscle was the highest (p<0.01). The axonal integrity loss was less prominent within the GDNF-guides, and the nerves demonstrated the highest mean axonal count (p<0.05). Gross morphology of the whisker pad was not different across the groups. Conclusion: The novel tissue engineered nerve guide containing double-walled GDNF microspheres enhances recovery after facial nerve transection. Results support the clinical viability of these guides to enhance recovery after nerve injury and hold promise to facilitate recovery in defects with larger gaps.

PURPOSE: Mechanical tension is a central determinant of the size, strength, and physiology of scars formed after cutaneous injury. (1) During post-traumatic proliferation and remodeling, supraphysiologic tension modulates cell signaling and differentiation as well as angiogenic and inflammatory mediators. (2,3) Platelet-rich fibrin (PRF) is an autologous, patient-derived biologic scaffold generated from the blood that maintains a locally high concentration of growth factors, previously demonstrated to enhance angiogenesis and mitigate inflammation. (4,5) Here, we sought to evaluate the possible therapeutic relationship between PRF and cutaneous wounds in a model of variable-tension murine injury. METHOD: 60 Wistar Hannover rats were stratified to receive either high, medium, or low tension injuries via controlled dorsal skin incision/excision. Each cohort received a) isotonic solution injection (sham) or b) PRF emplacement. Wounds were followed for 28 days andTracked visually utilizing the Vancouver Scar Scale (VSS). On the 28th-day scar, the width was measured by caliper, and skin samples were collected for mechanical testing and/or histologic evaluation via H&E and Type I collagen immunochemistry. RESULTS: Wound healing was appropriately delayed under high tension conditions with the formation of more proliferative scars as assessed by the VSS. Scar width increased in direct correlation to the magnitude of tension applied. Under conditions of PRF treatment, scar/wound scores were improved vs. controls at all levels of tensions assessed. Scar width was noticeably and statistically thinner vs. control in all groups. High-tension scars retained tensile characteristics consistent with lower-tension injuries in the presence of PRF but not control treatment. PRF-treated wounds additionally demonstrated more robust Type I Collagen expression in PRF-treated high-tension wounds. CONCLUSION: PRF-treatment improved scar and wound healing characteristics vs. control. This effect was amplified in the high-tension wound environment. REFERENCES: 1. Morin G, Rand CPTM, P.A. Burgess MAJL, Voussoughi J, M. Graeber COLG. Wound healing: relationship of wound closing tension to tensile strength in rats. The Laryngoscope. 1989;99(8). doi:10.1288/00005537-198908000-00003 2. Wilkinson HN, Hardman MJ. Wound healing: Cellular mechanisms and pathological outcomes. Open Biology. 2020;10(9):200223. doi:10.1098/rsob.200223 3. Shaw TJ, Martin P. Wound repair: A showcase for cell plasticity and Migration. Current Opinion in Cell Biology. 2016;42:29-37. doi:10.1016/j.ceb.2016.04.001 4. Strauss F-J, Nasirzade J, Kargarpoor Z, Stähli A, Gruber R. Effect of platelet-rich fibrin on cell proliferation, migration, differentiation, inflammation, and osteoclastogenesis: A systematic review of in vitro studies. Clinical Oral Investigations. 2019;24(2):569-584. doi:10.1007/s00784-019-03156-9 5. Dohle E, El Bagdadi K, Sader R, Choukroun J, James Kirkpatrick C, Ghanaati S. Platelet-rich fibrin-based matrices to improve angiogenesis in an in vitro co-culture model for Bone Tissue Engineering. Journal of Tissue Engineering and Regenerative Medicine. 2017;12(3):598-610. doi:10.1002/term.2475

PURPOSE: Post-operative reabsorption of engrafted adipose results in up to 50% tissue loss in the first postoperative year and is limits reliable volumetric results. We previously demonstrated that Vitamin D (VD3) enhances adipose retention in xenograft fat transfer and enhances adipose stem cell (ASC) viability ex vivo. To approach a mechanism, we evaluated how VD3 affected the metabolism and proliferative indices of ASCs and adipocytes.

INTRODUCTION: Hypodermal restoration via fat grafting after burn trauma to the face provides padding for the overlying skin, helps restore native features, and enhance contour and texture. While powerful, this technique is limited by graft retention often requires multiple rounds of grafting. Here we utilize a cryopreserved allogeneic fat transfer model to demonstrate the efficacy of cryopreserved fat in bolstering skin thickness and dermal-epidermal architecture after burn debridement and skin graft reconstruction. METHODS: Female Yorkshire swine received 16 4 × 4 cm full-thickness burns. After 48 hours escharectomy was performed to fascia. Wounds were allocated to the following treatment groups: (a) No Treatment; (b) Fat Grafting Only; (c): Skin Grafting Only; (d) Skin then Fat Grafting. Split-thickness skin autografts (0.012 in.) were collected from the lateral thighs, pie crusted at back table, and grafted directly to the wound base. After 10 days, cryopreserved allogeneic adipose from female Yorkshire swine were grafted immediately deep to the graft or eschar depending on group allocation. Subjects were maintained for 8 weeks with interval ultrasound and biopsy for histologic analysis. RESULTS: On ultrasonic evaluation total skin thickness was noted to be significantly greater in Skin + Fat group when compared with Skin Only (P < 0.05). On histologic assessment, dermal thickness was increased in both Fat Only versus Untreated (P = 0.0395) and Skin + Fat versus Skin Only groups samples (P = 0.0016). When compared with Skin Only samples, Skin + Fat groups demonstrated significantly increased epidermal depth (P = 0.0011). Both groups receiving skin grafts demonstrated significantly greater presence and depth of dermal papillae verus groups without skin graft (P < 0.052). CONCLUSIONS: Facial burns are highly morbid injuries affecting quality of life and psychosocial well-being. Debridement and reconstruction can require extensive and repeat surgical interventions often with significant soft tissue deficit and obliteration of native facial architecture with long-lasting disfigurement. Fat grafting may address this; however, uncertain retention and need for multiple surgeries provides a barrier to some patients. Cryopreservation of adipose at initial liposuction addresses that limitation and here we demonstrate the efficacy of this technique in enhancing the thickness and structure of reconstructed skin.

Purpose: Autologous fat grafting is a widely used technique in aesthetic and reconstructive surgery, however, unpredictable volume reabsorption may lead to unsatisfactory outcomes. Previously, we demonstrated that a fat-soluble Vitamin D3 analogue, calcitriol, significantly improved fat retention in a xenograft mouse model by 25% across multiple donors when injected systemically (p < 0.05). While calcitriol has minimal toxicity, is FDA approved, and has positive immunomodulatory and antioxidant properties, systemic administration bypasses key Vitamin D synthesis regulatory steps, thus increasing risk with high-dose use. We hypothesize that systemic supplementation with Vitamin D3 (cholecalciferol) will likewise improve fat-graft retention similar to calcitriol while avoiding potential regulatory and iatrogenic risk. In this study we compared in vivo human fat graft retention in mice treated with systemic cholecalciferol, calcitriol or vehicle control in a mouse xenograft model. In vitro adipose lipoaspirate culture was used to interrogate the therapeutic mechanism of action. Methods: Lipoaspirate was harvested from 6 unique donors using a 2mm cannula and used in parallel for both in vitro and in vivo studies. In vivo: 0.3mL of lipoaspirate was injected bilaterally on dorsal flanks of homozygous Foxn1nu immunocompromised mice. Calcitriol (50ng), cholecalciferol (50ng, 500ng, 5000ng) or vehicle control was administered thrice weekly by IP injection. Graft volume retention was measured at 12 weeks. In vitro: 1mL of lipoaspirate was submerged in phenol-red free DMEM (10% FBS) containing calcitriol or cholecalciferol (15.6nM, 62.5 nM or 250 nM) for 7 days with one media change. Terminal analyses include tissue weight, stromal cell viability, concentration of active vitamin D metabolite (1,25(OH)2D3), and gene upregulation by qRT-PCR. Results: Previously, we demonstrated that systemic administration of 50ng calcitriol thrice weekly significantly improved human fat graft retention across multiple donors in a mouse xenograft model. Our current in vivo data suggest 5000ng cholecalciferol is similarly effective. In-vitro assays show 62.5nM and 250nM cholecalciferol significantly increased adipose stromal cell viability compared to controls (85.3+/-2.9% and 87.7+/-3.7 versus 77.6+/-2.8%, respectively p<0.05;). Analysis of final adipose 1,25(OH)2D3 concentration by ELISA showed both calcitriol and cholecalciferol treatments equally increased Vitamin D metabolite concentrations in all donors. qRT-PCR analysis of gene expression show that pro-survival autophagy is significantly increased by both cholecalciferol and calcitriol, though increased concentration of calcitriol was required to induce significant increases from controls. Conclusion: Cholecalciferol (Vitamin D3) is a highly promising therapeutic for improving fat grafting outcomes. Our in vitro data suggests that in the context of hypoxia, nutrient depletion, or growth factor deprivation, such as occurs immediately following fat grafting, vitamin D3 promotes stromal cell autophagy. In this context, autophagy is crucial for maintaining cellular ATP production and macromolecular synthesis and, therefore, represents an essential pro-survival pathway which allows grafted cells to survive. The results herein provide evidence to incorporate vitamin D3 as a safe, cost-effective nutritional supplement into the perioperative workflow to improve fat viability after grafting.

Purpose: Traumatic heterotopic ossification (HO) is a debilitating condition where aberrant bone is formed outside the skeleton due to a fate switch of tissue resident mesenchymal/progenitor cells (MSCs). HO can occur after extremity trauma, burns, and extremity surgeries including amputations and joint replacements. No effective preventive strategies exist as the underlying mechanisms have not been elucidated. Though HO forms at sites of mechanical stress, the role of joint mobilization during extremity trauma, healing, and HO formation has not been clearly defined. We hypothesize that movement is central to HO formation via mechanotransductive signaling, and can provide a basis for improving post-trauma guidelines to prevent HO. Methods: HO was induced in mice via a dorsal partial thickness burn with concomitant Achilles tenotomy (B/T). Single cell RNA (scRNA) sequencing was performed prior to injury and on 3, 7, and 21 days post-B/T tissue using 10X genomics and downstream analysis with Seurat R package. scRNA sequencing was performed on immobilized mice 7 days post B/T and compared to that of mobile mice at the same time point. Scores were generated for each cell based on correlations with either osteogenic or adipogenic gene signatures in MSCs from mobile or immobilized mice. B/T was performed in mice of 4 groups (n=3/group): forced run, exercised passive range of motion (ROM), ambulated normally (mobile), or immobilized, and hindlimb bone volume was assessed at 9 weeks post-B/T by MicroCT (uCT). Immunofluorescent (IF) labeling for PDGFRα and pFAK, TAZ, or Perilipin-1 was done on 1 week cross sections and quantified (n=3/group). Results: Single cell clustering showed there are 15 unique clusters, 3 of which are MSC populations with increased expression of mechanotransductive markers such as Ptk2 (FAK), Yap1 (YAP) and Wwtr1 (TAZ). Joint immobilization of the ankle completely inhibited HO formation, therefore, the comparison of mobile and immobile mice was explored. Histology of immobilized mice demonstrated there is decreased mechanotransductive signaling (pFAK and nuclear TAZ) compared to mobile group. Interestingly, we noted increased adipocytes in the immobilized group at 1 week. Comparing scRNA sequencing revealed that MSCs (clusters 2, 3, and 14) from immobile mice correlated with an adipogenic signature compared to mobile MSCs that favored osteogenesis. This finding suggests a cell fate shift towards adipogenesis with joint immobilization. Conclusion: Hindlimb immobilization plays a significant role altering mechanotransductive pathways which we demonstrate results in an shift in MSC differentiation programming from endochondral ossification to adipogenesis. Immobilization protocols should be considered in patients at high HO risk.

PURPOSE: Musculoskeletal extremity injury is the most common trauma seen in civilians and combat casualties. Despite its high frequency, little is known about how to tune the immune response to maximize wound healing and prevent complications. Here, we assess the circulatory monocyte/macrophage recruitment to abnormal extremity wound healing as seen with heterotopic ossification (HO) and evaluate the role of macrophage derived Tgfβ1 in the pathogenesis and therapeutic targeting. We hypothesize that macrophage phenotype and cytokine secretion can be tuned to improve traumatic extremity wound healing. METHODS: A proven musculoskeletal extremity trauma model of HO (30% total body surface area dorsal burn and Achilles tenotomy) was used. Inflammation at the injury site was followed with bioluminescent in-vivo imaging of myeloperoxidase activity and recruited inflammatory cell subpopulations were quantified using flow cytometry. Circulatory monocyte depletion was performed with intravenous injection of clodronate. Early HO was assessed histologically at 3 weeks. In-vitro cultured bone marrow derived macrophages were polarized to M1 and M2 phenotypes and expression of Tgfβ1 was analyzed on RNA and protein levels. In-vivo, TGFβ1 in macrophages and downstream signaling in HO progenitor cells was visualized with immunostaining of HO anlagen in human and mouse samples. Furthermore, a CD47 receptor activating peptide was used to attenuate macrophage Tgfβ1 activity. RESULTS: Extremity trauma resulted in a profound inflammatory response at the injury site with initial recruitment of circulatory inflammatory Ly6Chi monocytes peaking 3 days post injury comprising over 30% of cells. These were replaced by regenerative Ly6Clow macrophages by day 14 which remained the prevalent macrophage population indicating a role in HO. Chemical ablation of circulatory monocytes with clodronate reduced recruitment of Ly6Clow monocytes and F480+/MHCII+ macrophages and attenuated HO. Expression of profibrotic Tgf-β1, a cytokine commonly secreted by regenerative macrophages, was highly expressed at the injury site and co-localized with recruited macrophages in human early HO anlagen. These findings were confirmed in-vitro where Tgfβ1 expression was significantly increased in M2 polarized macrophages compared to M1. Interestingly, treatment with CD47 receptor activating peptide decreased macrophage Tgfβ1 expression in macrophages in vitro and systemic administration showed attenuation of early HO anlagen in vivo. CONCLUSION: Our data demonstrate that recruited circulatory macrophages play an instrumental role in the inflammatory response and aberrant musculoskeletal wound healing through contribution from distinct macrophage sub-populations. Furthermore, we identify macrophage secreted TGF-β1 as a key cytokine driving HO. Systemic administration of therapeutics targeting macrophage phenotype and TGF-β1 secretion have a high potential to reduce aberrant musculoskeletal wound healing. These results uncover a novel role of macrophages in musculoskeletal wound healing and allow for new cell specific HO prevention. This macrophage targeted therapy can also be translated to additional wound healing disorders. M. Sorkin: None. C. Hwang: None. S. Ucer: None. J. Li: None. D. Cholok: None. M.T. Chung: None. S. Agarwal: None. S. Loder: None. C. Pagani: None. K. Vasquez: None. S. Li: None. A. James: None. Y. Mishina: None. B. Levi: None.

PURPOSE: Patients who sustain mechanical trauma, spinal cord injury, burns, or extremity surgeries are at risk for developing heterotopic ossification (HO), the pathologic formation of extraskeletal bone. HO is formed through a process of endochondral ossification initiated by acute inflammation. Vascular endothelial growth factor (VEGF) has been shown to be critical for both normal bone development and for fracture repair. We hypothesized that VEGF plays a central role during ossification of the cartilaginous matrix present during pathologic HO formation and that therapeutic targeting of the vascular niche is sufficient to prophylax against traumatic HO. METHODS: Male C57BL/6J mice underwent Achilles’ tendon transection and 30% of total body surface area (TBSA) dorsal burn injury to induce HO. Mice underwent Microfil CT and near infra-red imaging with intravascular injection of Angiosense to survey local vascularity. CDH5 endogenous reporter lines were imaged with confocal microscope. Hindlimb sections from injured mice were immunostained and injury site was harvested for flow cytometry and PCR. To further validate these findings and define the source of VEGF, co-staining with PDGFRa and VEGF was performed. Mice with genetic loss of Vegf in cells of mesenchymal lineage (Vegf cKO: Prx-cre/Vegffl/fl) and their littermate controls underwent burn/tenotomy (n=4–7). Human histological sections from analogous early HO sites were also examined via immunohistochemistry to confirm the translational value of these findings. Finally, a subset of mice after burn/tenotomy were treated with bevacizumab (biweekly injections of drug 10mg/kg) or vehicle control and were analyzed by histology (3 weeks) and MicroCT (9 weeks, n=5–6) for therapeutic testing. RESULTS: In wild-type mice, vascular density was intimately associated with the HO anlagen as demonstrated by near infrared imaging with Angiosense, Microfil imaging 5 weeks after burn/tenotomy, and endogenous CDH5 signal. Immunostaining of early human HO specimens with VEGFa and PDGFRa confirmed co-localization, corroborated by observations in mouse histology. Mice treated with bevacizumab, a potent anti-VEGF antibody, formed significantly less HO when compared at 9 weeks with vehicle control (2.64 v. 6.85 mm3, p=.0013). Genetic targeting produced parallel findings, with Vegf cKO mice forming significantly less HO near the distal tibia when compared with littermate controls (2.52 v. 4.53 mm3/mm, normalized to tibial cortical thickness compared to littermate control). CONCLUSIONS: Leveraging our knowledge of normal bone development, these findings suggest that VEGF from the mesenchymal niche plays a critical role in the formation of HO and can be successfully targeted to attenuate this process via bevacizumab, an FDA-approved pharmacologic agent used for its anti-VEGF properties. Given the importance of VEGF and angiogenesis during normal post-injury healing, future studies will focus on identifying the ideal treatment timing to prevent ossification of the cartilage anlagen and minimize off target effects. C. Hwang: None. S. Ucer: None. M. Sorkin: None. S. Loder: None. M.T. Chung: None. C. Pagani: None. J. Li: None. C. Priest: None. C. Breuler: None. K. Vasquez: None. S. Li: None. J. Xu: None. A. James: None. S. Agarwal: None. B. Levi: None.