Heterotopic ossification (HO) is an aberrant regenerative process with ectopic bone induction in response to musculoskeletal trauma, in which mesenchymal stem cells (MSC) differentiate into osteochondrogenic cells instead of myocytes or tenocytes. Despite frequent cases of hospitalized musculoskeletal trauma, the inflammatory responses and cell population dynamics that regulate subsequent wound healing and tissue regeneration are still unclear. Here we examine, using a mouse model of trauma-induced HO, the local microenvironment of the initial post-injury inflammatory response. Single cell transcriptome analyses identify distinct monocyte/macrophage populations at the injury site, with their dynamic changes over time elucidated using trajectory analyses. Mechanistically, transforming growth factor beta-1 (TGFβ1)-producing monocytes/macrophages are associated with HO and aberrant chondrogenic progenitor cell differentiation, while CD47-activating peptides that reduce systemic TGFβ levels help ameliorate HO. Our data thus implicate CD47 activation as a novel therapeutic approach for modulating monocyte/macrophage phenotypes, MSC differentiation and HO formation during wound healing.

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.

Existing clinical approaches and tools to measure burn tissue destruction are limited resulting in misdiagnosis of injury depth in over 40% of cases. Thus, our objective in this study was to characterize the ability of short-wave infrared (SWIR) imaging to detect moisture levels as a surrogate for tissue viability with resolution to differentiate between burns of various depths. To accomplish our aim, we constructed an imaging system consisting of a broad-band Tungsten light source; 1,200-, 1,650-, 1,940-, and 2,250-nm wavelength filters; and a specialized SWIR camera. We initially used agar slabs to provide a baseline spectrum for SWIR light imaging and demonstrated the differential absorbance at the multiple wavelengths, with 1,940 nm being the highest absorbed wavelength. These spectral bands were then demonstrated to detect levels of moisture in inorganic and in vivo mice models. The multiwavelength SWIR imaging approach was used to diagnose depth of burns using an in vivo porcine burn model. Healthy and injured skin regions were imaged 72 hours after short (20 seconds) and long (60 seconds) burn application, and biopsies were extracted from those regions for histologic analysis. Burn depth analysis based on collagen coagulation histology confirmed the formation of superficial and deep burns. SWIR multispectral reflectance imaging showed enhanced intensity levels in long burned regions, which correlated with histology and distinguished between superficial and deep burns. This SWIR imaging method represents a novel, real-time method to objectively distinguishing superficial from deep burns.

Inflammation after trauma is both critical to normal wound healing and may be highly detrimental when prolonged or unchecked with the potential to impair physiologic healing and promote de novo pathology. Mechanical strain after trauma is associated with impaired wound healing and increased inflammation. The exact mechanisms behind this are not fully elucidated. Neutrophil extracellular traps (NETs), a component of the neutrophil response to trauma, are implicated in a range of pro-inflammatory conditions. In the current study, we evaluated their role in linking movement and inflammation. We found that a link exists between the disruption and amplification of NETs which harbors the potential to regulate the wound's response to mechanical strain, while leaving the initial inflammatory signal necessary for physiologic wound healing intact.

Heterotopic ossification (HO) is a debilitating condition characterized by the pathologic formation of ectopic bone. HO occurs commonly following orthopedic surgeries, burns, and neurologic injuries. While surgical excision may provide palliation, the procedure is often burdened with significant intra-operative blood loss due to a more robust contribution of blood supply to the pathologic bone than to native bone. Based on these clinical observations, we set out to examine the role of vascular signaling in HO. Vascular endothelial growth factor A (VEGFA) has previously been shown to be a crucial pro-angiogenic and pro-osteogenic cue during normal bone development and homeostasis. Our findings, using a validated mouse model of HO, demonstrate that HO lesions are highly vascular, and that VEGFA is critical to ectopic bone formation, despite lacking a contribution of endothelial cells within the developing anlagen.

Aberrant wound healing presents as inappropriate or insufficient tissue formation. Using a model of musculoskeletal injury, we demonstrate that loss of transforming growth factor-β activated kinase 1 (TAK1) signaling reduces inappropriate tissue formation (heterotopic ossification) through reduced cellular differentiation. Upon identifying increased proliferation with loss of TAK1 signaling, we considered a regenerative approach to address insufficient tissue production through coordinated inactivation of TAK1 to promote cellular proliferation, followed by reactivation to elicit differentiation and extracellular matrix production. Although the current regenerative medicine paradigm is centered on the effects of drug treatment (“drug on”), the impact of drug withdrawal (“drug off”) implicit in these regimens is unknown. Because current TAK1 inhibitors are unable to phenocopy genetic Tak1 loss, we introduce the dual-inducible COmbinational Sequential Inversion ENgineering (COSIEN) mouse model. The COSIEN mouse model, which allows us to study the response to targeted drug treatment (“drug on”) and subsequent withdrawal (“drug off”) through genetic modification, was used here to inactivate and reactivate Tak1 with the purpose of augmenting tissue regeneration in a calvarial defect model. Our study reveals the importance of both the “drug on” (Cre-mediated inactivation) and “drug off” (Flp-mediated reactivation) states during regenerative therapy using a mouse model with broad utility to study targeted therapies for disease. Stem Cells 2019;37:766–778

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.

Patients sustaining mechanical trauma, burns, or orthopedic procedures can develop heterotopic ossification (HO) or pathologic development of extra-skeletal bone. Vascularization, mainly mediated by VEGFa, is required for different stages of endochondral ossification, namely, establishment of the primary osseous center. Moreover, VEGFa is crucial for bone repair by also promoting bone turnover signaling. We hypothesize that VEGFa is required for HO and cells of the mesenchymal lineage are the major contributors for this signal. First, C57BL/6J male mice underwent through Achilles tendon transection and 30% of TBSA dorsal burn injury. Mice underwent Microfil CT scans to survey local vascular structures. Hindlimb sections from injured mice were immunostained, and injury site was harvested for flow cytometry and PCR. Mice in bevacizumab v. control treatment arms received biweekly injections of drug (10mg/kg) or PBS. Separate cohorts underwent MicroCT analysis at 9 weeks. Five weeks after induction surgery, vascular density was higher, as demonstrated by Microfil. These findings were consistent with near infrared imaging using Angiosense. Furthermore, VEGFa expression was increased in both protein and mRNA levels in tissue isolated from HO and surrounding regions. To understand whether direct actions of VEGFa derived from the mesenchymal cells play a role, we deleted the VEGFa gene from cells of mesenchymal lineage by crossing Vegff/f mouse with Prx1-Cre mice. Both VEGFf/f,Prx1-Cre male mice and their littermate control underwent burn/tenotomy as previously described. MicroCT scans demonstrated that conditional knockout mice exhibit less HO formation near the distal tibia but not proximal tibia, after normalized to tibial cortical thickness. To translate this clinically, we next performed our traumatic HO model in C57BL/6J mice treated with VEGFa inhibitor bevacizumab vs. PBS. Bevacizumab treated mice formed significantly less HO than PBS injection controls at 800HU (2.64 v. 6.85 mm3, p=.0013). This data suggests that acute local trauma causes alterations in vascular signaling. Additionally, VEGFa derived from mesenchymal cells are a major source for VEGFa which is required for HO formation. Attenuation of local VEGFa signaling via existing, FDA-approved monoclonal antibodies might be an effective therapeutic treatment to reduce or prevent HO formation in burn and polytrauma patients.

PURPOSE: Heterotopic ossification (HO) is a debilitating formation of ectopic bone restricting joint mobility and causing chronic pain. Fibrodysplasia Ossificans Progressiva (FOP) is a congenital variant of HO caused by a genetic mutation in a bone morphogenetic protein receptor that causes severe, progressive lesions resulting in immobility and often fatal mechanical respiratory failure at a premature age that currently lacks a cure. With increased proclivity for osteogenesis at baseline, surgical excision of bony lesions is contraindicated in FOP patients due to universal recurrence. We have previously shown that rapamycin is a powerful drug which can eliminate lesions in mouse models of FOP. Here, we demonstrate that combination therapy with low-dose rapamycin and the BMP receptor inhibitor LDN-212854 presents an option to prevent both primary HO and post-excision HO in an FOP mouse model. METHODS: Within the recurrence study arm, ACVR1R206H/+ P21 mice received inductive Ad.cre and cardiotoxin (CTX) injection in bilateral hindlimbs. Three weeks later, mice were live-scanned with in vivo µCT, reinjected with Ad.cre bilaterally, and underwent HO excision at the 3 week timepoint. These mice were randomized to daily rapamycin (5 mg/kg, n=12) or PBS (n=8) for 3 weeks with endpoint µCT. For low-dose rapamycin and LDN adjunctive therapy, 16 mice were stratified into low dose rapamycin (0.5 mg/kg, n=10, both hindlimbs of 5 mice), rapamycin and LDN212854 (0.5mg/kg and 0.6mg/kg respectively, n=8), and injection control (PBS, n=14) cohorts for daily injections. They were subsequently induced as previously described, with additional second and third CTX booster series on post-operative day 3 and 7. Mice were scanned with µCT 3 weeks later. Contours were drawn manually by blinded experts around HO to compute total volumes (800HU). Recurrence cohorts were analyzed by log-transform/ANOVA/post-hoc Hochberg and low-dose results were analyzed similarly with restriction to right legs only. RESULTS: In post-surgical mice, PBS injection showed statistically similar HO volumes to baseline volumes of pre-excision mice (p=0.054). However, rapamycin treatment significantly reduced the post-surgical HO volume 11-fold (p=.044,). In the primary HO model treated with low-dose rapamycin/LDN, mice treated with rapamycin for 21 days showed 17-fold less ectopic bone (p=0.001) compared to PBS vehicle injection, paralleling a 9-fold reduction (p=.003) when treated with rapamycin plus LDN212854 adjunct. CONCLUSIONS: These studies demonstrate that rapamycin prevents primary development of HO and is also effective in preventing post-surgical recurrence in a FOP mouse model. Furthermore, rapamycin with and without LDN adjunct remains effective at preventing primary development of HO even at lower concentrations. This study further corroborates rapamycin as a promising candidate for primary and post-surgical HO prophylaxis in children with FOP, with opportunities for subsequent dosing studies and adjunct therapies to minimize prohibitive adverse effects. Existing literature implicates similar molecular mechanisms among various etiologies of HO, suggesting a putative role for rapamycin even beyond FOP in post-traumatic and post-surgical HO patients. C. Hwang: None. M. Sorkin: None. S. Ucer: None. S. Kader: None. A. Vaishampayan: None. M.T. Chung: None. C. Breuler: None. C. Priest: None. N. Patel: None. J. Li: None. K. Vasquez: None. H. Pan: None. A. Economides:; Regeneron Pharmaceuticals Inc. S. Agarwal: None. Y. Mishina: None. B. Levi:; patent holder; I currently have a use patent for rapamycin to prevent heterotopic ossification. It has not been licensed by any company.

Purpose: Early treatment of heterotopic ossification (HO) is currently limited by delayed diagnosis due to limited visualization at early time points. In this study, we validate the use of spectral ultrasound imaging (SUSI) in an animal model to detect HO as early as one week after burn tenotomy. Methods: Concurrent SUSI, micro CT, and histology at 1, 2, 4, and 9 weeks post-injury were used to follow the progression of HO after an Achilles tenotomy and 30% total body surface area burn (n = 3-5 limbs per time point). To compare the use of SUSI in different types of injury models, mice (n = 5 per group) underwent either burn/tenotomy or skin incision injury and were imaged using a 55 MHz probe on VisualSonics VEVO 770 system at one week post injury to evaluate the ability of SUSI to distinguish between edema and HO. Average acoustic concentration (MC) and average scatterer diameter (ASD) were calculated for each ultrasound image frame. Micro CT was used to calculate the total volume of HO. Histology was used to confirm bone formation. Results: Using SUSI, HO was visualized as early as 1 week after injury. HO was visualized earliest by 4 weeks after injury by micro CT. The average acoustic concentration of HO was 33% more than that of the control limb (n = 5). Spectroscopic foci of HO present at 1 week that persisted throughout all time points correlated with the HO present at 9 weeks on micro CT imaging. Conclusion: SUSI visualizes HO as early as one week after injury in an animal model. SUSI represents a new imaging modality with promise for early diagnosis of HO. (C) 2018 Elsevier Inc. All rights reserved.

Heterotopic ossification (HO) occurs secondary to trauma, causing pain and functional limitations. Identification of the cells that contribute to HO is critical to the development of therapies. Given that innate immune cells and mesenchymal stem cells are known contributors to HO, we sought to define the contribution of these populations to HO and to identify what, if any, contribution circulating populations have to HO. A shared circulation was obtained using a parabiosis model, established between an enhanced green fluorescent protein-positive/Luciferase(+) donor and a same-strain nonreporter recipient mouse. The nonreporter mouse received Achilles tendon transection and dorsal burn injury to induce HO formation. Bioluminescence imaging and immunostaining were performed to define the circulatory contribution of immune and mesenchymal cell populations. Histologic analysis showed circulating cells present throughout each stage of the developing HO anlagen. Circulating cells were present at the injury site during the inflammatory phase and proliferative period, with diminished contribution in mature HO. Immunostaining demonstrated that most early circulatory cells were from the innate immune system; only a small population of mesenchymal cells were present in the HO. We demonstrate the time course of the participation of circulatory cells in trauma-induced HO and identify populations of circulating cells present in different stages of HO. These findings further elucidate the relative contribution of Local and systemic cell populations to HO.

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.

PURPOSE: Heterotopic ossification (HO) can commonly occur after severe trauma, burn injuries, and is a debilitating consequence of the congenital disease fibrodysplasia ossificans progressive (FOP). The etiology remains poorly understood, however, it is presumed that inflammation plays a critical role with several inflammatory cell types being recruited to the site of HO development. While the role of aberrant BMP signaling is established in HO formation, there is recent indication that macrophage secreted transforming growth factor beta 1 (TGFβ1) is also involved through propagation of chondrogenesis and endochondral ossification. Here we explore the effect of Tgfβ1 inhibition utilizing a near clinical Tgfβ1 receptor ligand trap to attenuate HO formation. METHODS: Bone marrow derived macrophages were isolated and polarized into M2 phenotype in-vitro. Secreted TGFβ was measured in conditioned medium using ELISA. A model of traumatic heterotopic ossification involving a 30% dorsal burn and Achilles tenotomy was utilized in-vivo and 6-week old male C57BL/6 mice were randomized into receiving treatment with the pre-clinical pharmaceutical grade TGFβR-Fc ligand trap (n=10) or PBS control (n=10). This was administered subcutaneously twice weekly for 3 weeks. At 3 weeks, histology samples were collected, decalcified and stained with Safranin O to assess formation of HO anlagen (n=3/group). Volume of mature formed HO was quantified using micro CT analysis and imaging reconstruction at 9 weeks (n=6/group). RESULTS: Recruited macrophages are a known source of cytokines that influence the inflammatory microenvironment. We therefore initially assessed the secretion of Tgfβ1 in cultured macrophages. Interestingly, we observed that while M0 macrophages secrete only minimal TGFβ1, the regenerative M2 polarized macrophages, which are known to be recruited to inflammatory sites, had a 500-fold increase in Tgfβ1 secretion. Furthermore, this increase in TGFβ1 levels was completely abrogated when the TGFβR-Fc ligand trap was present in the culture media. We next aimed to assess the effect of systemically administered TGFβR-Fc on HO formation. Following treatment for 3 weeks, we observed a substantially attenuated development of HO anlagen in mice treated with TGFβR-Fc with decreased osseous deposition and marrow space formation on histologic examination. Furthermore, the volume of mature HO was significantly decreased in the treatment group. CONCLUSIONS: Heterotopic ossification, from trauma or congenital FOP, severely limits mobility, function and quality of life of affected patients and currently, no prevention strategies exist. In this study, we demonstrate that Tgfβ1 signaling plays a critical role in HO formation and treatment with TGFβR-Fc is effective in attenuating the early stages of ectopic bone deposition resulting in decreased HO volume. This therapeutic approach reveals a novel avenue for the treatment of this condition that may translate into effective clinical applications. N. Patel: None. M. Sorkin: None. C. Hwang: None. J. Li: None. S. Ucer: None. S. Kader: None. K. Vasquez: None. S. Li: None. R. Kumar:; Senior Vice President and Chief Scientific Officer; Acceleron Pharm, Inc.. Y. Mishina: None. B. Levi: None.

PURPOSE: Severe musculoskeletal trauma as observed in crush injuries and prolonged ischemia as seen in flap reconstruction and composite tissue allotransplantation is associated with ischemia-reperfusion (IR) injury leading to an aberrant inflammatory response. While smaller muscle injuries heal by complete muscle regeneration, complex injuries result in aberrant healing with muscle fibrosis. However, the inflammatory cascade that is governing this process remains incompletely understood. Recently, neutrophil extracellular traps (NETs), extracellular DNA structures released by neutrophils, have been implicated in the initial phases of ischemic inflammation. In this study, we develop a mouse extremity polytrauma model of combined muscle trauma and IR injury to assess the initial inflammatory response. METHODS: Male C57BL/6 mice were randomized into three treatment groups: 1. Fibrosis model: Cardiotoxin (CTX) injection into the tibialis anterior (known fibrosis model); 2. IR model: hindlimb ischemia by occluding the femoral artery using a microvascular clamp for 3.5 hours followed by reperfusion for up to 5 days 3. Polytrauma model: IR plus CTX injection (n=4 mice/group). Bioluminescent imaging for myeloperoxidase as a readout of inflammation was analyzed as well as neutrophil elastase. Additionally, lower extremity muscle was harvested for flow cytometry to assess inflammatory cell subpopulations and histology. RESULTS: Macroscopic evaluation of muscle specimens revealed an evident area of necrosis and apparent muscle changes while the CTX alone and IR alone muscles appeared normal. In-vivo imaging revealed significantly increased inflammation as measured by myeloperoxidase and neutrophil elastase activity in mice undergoing IR+CTX than either of the other groups. This was further corroborated by flow cytometry where we found differences in inflammatory cell subpopulations with more inflammatory monocytes/macrophages and neutrophils recruited to the muscle injury site in the IR+CTX group (p(Monocytes: IR+CTX vs. CTX only)=.000002 and p(Neutrophils: IR+CTX vs. CTX only)=.037). CONCLUSIONS: Our findings demonstrate that combined IR+muscle injury (CTX) results in significantly increased muscle injury than either ischemia or muscle injury alone. This was characterized by an elevated inflammatory response with amplified recruitment of pro-inflammatory macrophages and neutrophils. This model can be utilized to examine the effects of musculoskeletal polytrauma on muscle regeneration and fibrosis. A better understanding and description of the inflammatory cascade after extremity IR injury will enable early intervention and prevention of post-traumatic muscle fibrosis. M. Sorkin: None. C. Hwang: None. J. Li: None. N. Patel: None. S. Li: None. K. Vasquez: None. S. Agarwal: None. B. Levi: None.

PURPOSE: The prevention of heterotopic ossification (HO) is limited by inadequate detection of ectopic bone formation at early time points during which prophylaxis must be initiated in order to be effective. Current imaging modalities are costly, invasive, and unable to visualize HO prior to six weeks after bone formation. Therefore, these technologies are not utilized, and patients receive treatment in a delayed fashion as a result of these diagnostic inadequacies. In this study, we validate the use of spectroscopic ultrasound imaging (SUSI) to diagnose HO as early as one week after injury, and five weeks earlier than microCT imaging which is the current gold standard. METHODS: Concurrent SUSI and microCT were performed after an Achilles tenotomy and 30% total body surface area burn (C57BL6 mice; n=4 per time point). Imaging of the injured limb and skin incision only contra-lateral limb (control) was performed weekly (1–9 weeks post-injury) in a longitudinal fashion. Acoustic concentration (10*log(mm-3)) was calculated for each ultrasound frame. Houndsfield units were used to calculate HO volume on microCT imaging. Histology was used to confirm the presence of HO and to correlate with imaging findings at each time point. RESULTS: Using SUSI, the acoustic concentration of bone was significantly different than that of muscle, cartilage, and tendon (61.3 ± 7.3 vs. 36.5 ± 8.6 vs. 49.5 ± 5.4 vs. 39.8 ± 0.67; p<0.05). HO was visualized on SUSI as early as 1 week after injury and 5 weeks prior to detection by MicroCT. The acoustic concentration of HO was significantly greater than that of the control limb (56.9 ± 10.9 vs. 29.0 ± 10.7; p<0.05) at all time points. The surrounding edema also had a significantly lower acoustic concentration than the foci of HO (28.9 ± 5.6 vs. 56.9 ± 10.9; p<0.05), allowing for clear anatomic and structural delineation within this region. Spectroscopic foci of HO present at 1 week within the left limb correlated with the HO present at 9 weeks on microCT and histology. CONCLUSION: Spectroscopic ultrasound visualizes HO as early as one week after injury. As prophylaxis must be initiated within 1–3 weeks of initial HO formation to prevent the need for surgery, SUSI represents the ideal imaging modality to guide treatment. Additionally, SUSI can be used to monitor the progression of HO to measure when growth has halted in preparation for surgical excision.

PURPOSE:Heterotopic ossification (HO) occurs in the setting of persistent systemic inflammation. The identification of reliable biomarkers can serve as an early diagnostic tool for HO, especially given the current lack of effective treatment strategies. Although serum biomarkers have great utility, they can be inappropriate or ineffective in traumatic acute injuries and in patients with fibrodysplasia ossificans progressiva (FOP). Therefore, the goal of this study is to profile the cytokines associated with HO using a different non-invasive source of biomarkers.METHODS:Serum and saliva were collected from a model of trauma-induced HO (tHO) with hind limb Achilles' tenotomy and dorsal burn injury at indicated time points (pre-injury, 48 h, 1 week, and 3 weeks post-injury) and a genetic non-trauma HO model (Nfatc1-Cre/caAcvr1fl/wt ). Samples were analyzed for 27 cytokines using the Bio-Plex assay. Histologic evaluation was performed in Nfatc1-Cre/caAcvr1fl/wt mice and at 48 h and 1 week post-injury in burn tenotomy mice. The mRNA expression levels of these cytokines at the tenotomy site were also quantified with quantitative real-time PCR. Pearson correlation coefficient was assessed between saliva and serum.RESULTS:Levels of TNF-α and IL-1β peaked at 48 h and 1 week post-injury in the burn/tenotomy cohort, and these values were significantly higher when compared with both uninjured (p < 0.01, p < 0.03) and burn-only mice (p < 0.01, p < 0.01). Immunofluorescence staining confirmed enhanced expression of IL-1β, TNF-α, and MCP-1 at the tenotomy site 48 h after injury. Monocyte chemoattractant protein-1 (MCP-1) and VEGF was detected in saliva showing elevated levels at 1 week post-injury in our tHO model when compared with both uninjured (p < 0.001, p < 0.01) and burn-only mice (p < 0.005, p < 0.01). The Pearson correlation between serum MCP-1 and salivary MCP-1 was statistically significant (r = 0.9686, p < 0.001) Similarly, the Pearson correlation between serum VEGF and salivary VEGF was statistically significant (r = 0.9709, p < 0.05).CONCLUSION:In this preliminary study, we characterized the diagnostic potential of specific salivary cytokines that may serve as biomarkers for an early-stage diagnosis of HO. This study identified two candidate biomarkers for further study and suggests a novel method for diagnosis in the context of current difficult diagnosis and risks of current diagnostic methods in certain patients.

PURPOSE: Patients with fibrodysplasia ossificans progressiva (FOP) carry a genetic mutation in the type I bone morphogenetic protein (BMP) receptor ACVR1 leading to hyperactivity. This mutation leads to pathologic cartilage and bone formation at extra-skeletal sites of even mild soft tissue injury which cause severe pain, restrict function and motion, and ultimately lead to early death. Until now, strategies to treat patients with FOP have focused on candidate small molecule agents with unproven safety profiles or translational potential. Here we show that an FDA-approved drug with a previously proven record of safety is able to completely inhibit ectopic lesions in a mouse model of FOP carrying the same mutation. METHODS: Mice carrying the floxed FOP mutation (ACVR1 R206H) received a simultaneous hindlimb injection of Ad.cre to induce gene transformation and cardiotoxin to induce local injury (Ad.cre/CTX). Mice were treated with either daily vehicle control or rapamycin (5 mg/kg) administered i.p. (n=10/group). The presence of mesenchymal cells at the injury site was determined using immunofluorescent staining for PDGFRa and Sca-1 five days after injury. Ectopic cartilage and bone were determined using histology and microCT imaging 21 days after injury. PLGA microparticles were synthesized to deliver rapamycin as a slow-release; flow cytometry was used to quantify release time profile. Finally, a separate set of mice underwent Ad.cre/CTX injection with resection of formed HO 3 weeks after injury and subsequent treatment with or without rapamycin to eliminate recurrence. RESULTS: While mice which received Ad.cre/CTX without rapamycin produced ectopic bone consistently, treatment with rapamycin nearly eliminated HO based on both microCT imaging (34.0 mm3 v. 1.0 mm3, p<0.01). Furthermore, histologic imaging showed elimination of ectopic cartilage with rapamycin treatment based on H&E, pentachrome staining (red arrows) and SOX9 immunofluorescence. Finally, rapamycin reduced the presence of mesenchymal cells (PDGFRa+ or aSMA+) at the injury site. PLGA microparticles released rapamycin during the first week after injury based on flow cytometry analysis. Mice that had resection of HO and were treated with rapamycin did not recur, while 100% of mice which had resection of HO without rapamycin developed new lesions at the resection site. CONCLUSION: These findings demonstrate that rapamycin, an FDA-approved drug, eliminates ectopic cartilage and bone in a mouse model of FOP. This may occur through a reduction in the presence of activated mesenchymal cells at the injury site. These findings have prompted the initiation of clinical studies to assess efficacy of this potential therapeutic in humans. A slow-release microparticle may obviate repeated treatments in these patients. Rapamycin may also have a role in the management of FOP patients with ossified lesions, making surgical resection a reality for the first time.

PURPOSE: Lymphedema is a common, life-long complication of cancer treatment that currently has no cure. Patients with lymphedema have decreased quality of life and suffer recurrent infections, while current treatments are merely palliative and designed to prevent disease progression. Accumulating evidence indicates that T cells play a key role in the pathology of lymphedema by inhibiting lymphangiogenesis and promoting tissue fibrosis. Because the pathophysiology of lymphedema involves primarily the skin and subcutaneous tissues, it may be possible to target T cells locally using topical medications such as tacrolimus without inducing systemic immunosuppression. The purpose of this study was therefore to study the efficacy of topical tacrolimus for prevention and treatment of lymphedema using preclinical mouse models.

PURPOSE: Inflammation following trauma is a critical step during both normal and pathologic wound healing. Recently, we have identified Scleraxis, a transcription factor with conserved topoisomerase homology, as a mediator of pathologic wound healing. Here we demonstrate that ciprofloxacin, a known topoisomerase inhibitor, reduces exhibits anti-inflammatory properties during injury through a reduction in Scleraxis signaling. METHODS: Mesenchymal cells were isolated from mice with genetic loss of Scleraxis (Prx-cre/Scxfl/fl) and wild type mice. Separately, wild type cells were treated with ciprofloxacin (10 mg/kg) or vehicle control. In vitro assays were performed to quantify proliferation and Scleraxis signaling. Furthermore, mutant or wild type mice underwent hindlimb tendon transection and subsequent flow cytometry and histologic analysis during the first week after injury. This was similarly performed for mice treated with ciprofloxacin or vehicle control. RESULTS: Flow cytometry demonstrated that genetic loss of scleraxis among mesenchymal cells significantly reduced the presence of macrophages (F4/80+) and neutrophils (Cd11b+Ly6G+) at the injury site within 48 hours after injury. The presence of PDGFRa+ mesenchymal cells was also significantly reduced based on both flow cytometry and histologic analysis. These findings were confirmed with ciprofloxacin treatment. Furthermore, genetic loss of Scleraxis and ciprofloxacin both corresponded with a significant reduction in mesenchymal cell proliferation. Ciprofloxacin treatment led to reduced chondrogenic differentiation and aggrecan expression. Genetic loss of Scleraxis reduced ectopic cartilage formation when compared with wild type controls. CONCLUSION: These findings indicate that Scleraxis is a potent target to prevent mesenchymal cell proliferation and inflammation. Ciprofloxacin, an FDA-approved drug, has therapeutic efficacy as an anti-inflammatory agent with translational potential to prevent pathologic wound healing.

PURPOSE: The ability to modulate signaling pathways at the site of injury represents a novel paradigm in wound healing with implications for regenerative medicine and tissue engineering. Transforming growth factor-beta (TGF-β) activating kinase 1 (TAK1) is a key regulator in the TGF-β and bone morphogenetic protein (BMP) signaling pathways with central roles in proliferation, differentiation, survival, and apoptosis of myriad tissues during development and in a range of pathologic processes. Here we examine the effect of TAK1 inhibition both in vitro and in the in vivo wound environment. We introduce a novel dual inducible Cre/loxP and Flp/FRT recombinase system to precisely control the expression of TAK1 during wound healing and identify a role for TAK1 as a molecular switch between mesenchymal cell proliferation and differentiation. METHODS: We developed a novel dual recombinase system (Cre/loxP; Flp/FRT) allowing TAK1 to be specifically inactivated (Ad.Cre) and sequentially re-activated afterwards (Ad.Flp). We first evaluated the effect of this system in vitro mesenchymal cells harvested from sites of musculoskeletal injury. Osteogenic differentiation was assayed via alizarin red and alkaline phosphatase staining. SMAD protein signaling was analyzed directly via immunocytochemistry and western blot analysis. Proliferation was assayed directly via BrdU and cell counting. In vivo, critical sized-calvarial defects (4mm) were performed and mice received either: 1. Ad.LacZ (control) or 2. Ad.Cre (inactivation) or 3. Ten days Ad.Cre (inactivation) followed by Ad.FLP (reactivation) to modulate Tak1 expression. Calvarial tissue was harvested to assay for gene expression and cellular proliferation and differentiation were quantified histologically. RESULTS:In vitro analysis of mesenchymal cells carrying the Ad.Flp/Ad.Cre construct demonstrated a significant loss in osteogenic potential (p<0.05) and pSMAD1/5 signaling in the absence of Tak1. Reactivation of Tak1 was sufficient to restore pSMAD1/5 signaling and osteogenic differentiation. Tak1 knockout demonstrated an opposite effect on cell proliferation with immediate and significant (p<0.05) increases in cell growth upon knockout and normalization of cell proliferation on gene reactivation. Loss of Tak1 in the calvarial wound environment resulted in increased presence of mesenchymal cells and increased expression of proliferative genes including Ccnd1, E2f1, and Ki67, an effect reversed by Ad.Flp reactivation of Tak1. Consistent with our in vitro data, loss of Tak1 in calvarial tissue led to diminished osteogenic differentiation genes including Bmp2, Tgfβ1, Col1, Ocn, and Runx2. Again this effect was reversed by Ad.Flp reactivation of Tak1. CONCLUSION: We demonstrate that precise control of Tak1 can be used to modulate a switch between proliferation and osteogenic differentiation in mesenchymal cells. These findings are possible due to a novel dual-recombinase system with applications in other animal models studying TGF-B signaling and TAK1. Our in vivo data suggests that therapeutic modulation of Tak1 may provide a target to control the proliferation/differentiation switch required during tissue regeneration.