Cameron Brownley

Inhibition of Hif1α prevents both trauma-induced and genetic heterotopic ossification

Significance Heterotopic ossification (HO) is a debilitating condition in which bone forms inappropriately within soft tissues. Two vastly different patient populations are at risk for developing HO: those with musculoskeletal trauma or severe burns and those with a genetic mutation in the bone morphogenetic protein receptor ACVR1 (Activin type 1 receptor). In this study, we demonstrate that both forms of HO share a common signaling pathway through hypoxia inducible factor-1α, and that pharmacologic inhibition or genetic knockout of this signaling pathway can mitigate and even abolish HO formation. These findings pave the way for pharmacologic inhibitors of hypoxia inducible factor-1α as therapeutic options for heterotopic ossification. Pathologic extraskeletal bone formation, or heterotopic ossification (HO), occurs following mechanical trauma, burns, orthopedic operations, and in patients with hyperactivating mutations of the type I bone morphogenetic protein receptor ACVR1 (Activin type 1 receptor). Extraskeletal bone forms through an endochondral process with a cartilage intermediary prompting the hypothesis that hypoxic signaling present during cartilage formation drives HO development and that HO precursor cells derive from a mesenchymal lineage as defined by Paired related homeobox 1 (Prx). Here we demonstrate that Hypoxia inducible factor-1α (Hif1α), a key mediator of cellular adaptation to hypoxia, is highly expressed and active in three separate mouse models: trauma-induced, genetic, and a hybrid model of genetic and trauma-induced HO. In each of these models, Hif1α expression coincides with the expression of master transcription factor of cartilage, Sox9 [(sex determining region Y)-box 9]. Pharmacologic inhibition of Hif1α using PX-478 or rapamycin significantly decreased or inhibited extraskeletal bone formation. Importantly, de novo soft-tissue HO was eliminated or significantly diminished in treated mice. Lineage-tracing mice demonstrate that cells forming HO belong to the Prx lineage. Burn/tenotomy performed in lineage-specific Hif1α knockout mice (Prx-Cre/Hif1αfl:fl) resulted in substantially decreased HO, and again lack of de novo soft-tissue HO. Genetic loss of Hif1α in mesenchymal cells marked by Prx-cre prevents the formation of the mesenchymal condensations as shown by routine histology and immunostaining for Sox9 and PDGFRα. Pharmacologic inhibition of Hif1α had a similar effect on mesenchymal condensation development. Our findings indicate that Hif1α represents a promising target to prevent and treat pathologic extraskeletal bone.

BMP signaling mediated by constitutively active Activin type 1 receptor (ACVR1) results in ectopic bone formation localized to distal extremity joints

BMP signaling mediated by ACVR1 plays a critical role for development of multiple structures including the cardiovascular and skeletal systems. While deficient ACVR1 signaling impairs normal embryonic development, hyperactive ACVR1 function (R206H in humans and Q207D mutation in mice, ca-ACVR1) results in formation of heterotopic ossification (HO). We developed a mouse line, which conditionally expresses ca-ACVR1 with Nfatc1-Cre(+) transgene. Mutant mice developed ectopic cartilage and bone at the distal joints of the extremities including the interphalangeal joints and hind limb ankles as early as P4 in the absence of trauma or exogenous bone morphogenetic protein (BMP) administration. Micro-CT showed that even at later time points (up to P40), cartilage and bone development persisted at the affected joints most prominently in the ankle. Interestingly, this phenotype was not present in areas of bone outside of the joints - tibia are normal in mutants and littermate controls away from the ankle. These findings demonstrate that this model may allow for further studies of heterotopic ossification, which does not require the use of stem cells, direct trauma or activation with exogenous Cre gene administration.

Wound healing after thermal injury is improved by fat and adipose-derived stem cell isografts.

Patients with severe burns suffer functional, structural, and esthetic complications. It is important to explore reconstructive options given that no ideal treatment exists. Transfer of adipose and adipose-derived stem cells (ASCs) has been shown to improve healing in various models. The authors hypothesize that use of fat isografts and/or ASCs will improve healing in a mouse model of burn injury. Twenty 6 to 8 week old C57BL/6 male mice received a 30% surface area partial-thickness scald burn. Adipose tissue and ASCs from inguinal fat pads were harvested from a second group of C57BL/6 mice. Burned mice received 500 &mgr;l subcutaneous injection at burn site of 1) processed adipose, 2) ASCs, 3) mixed adipose (adipose and ASCs), or 4) sham (saline) injection (n = 5/group) on the first day postinjury. Mice were followed by serial photography until being killed at days 5 and 14. Wounds were assessed for burn depth and healing by hematoxylin and eosin (H&E) and immunohistochemistry. All treated groups showed improved healing over controls defined by decreased wound depth, area, and apoptotic activity. After 5 days, mice receiving ASCs or mixed adipose displayed a non-significant improvement in vascularization. No significant changes in proliferation were noted at 5 days. Adipose isografts improve some early markers of healing postburn injury. The authors demonstrate that addition of these grafts improves specific structural markers of healing. This improvement may be because of an increase in early wound vascularity postgraft. Further studies are needed to optimize use of fat or ASC grafts in acute and reconstructive surgery.

Surgical Excision of Heterotopic Ossification Leads to Re‐Emergence of Mesenchymal Stem Cell Populations Responsible for Recurrence

Trauma‐induced heterotopic ossification (HO) occurs after severe musculoskeletal injuries and burns, and presents a significant barrier to patient rehabilitation. Interestingly, the incidence of HO significantly increases with repeated operations and after resection of previous HO. Treatment of established heterotopic ossification is challenging because surgical excision is often incomplete, with evidence of persistent heterotopic bone. As a result, patients may continue to report the signs or symptoms of HO, including chronic pain, nonhealing wounds, and joint restriction. In this study, we designed a model of recurrent HO that occurs after surgical excision of mature HO in a mouse model of hind‐limb Achilles’ tendon transection with dorsal burn injury. We first demonstrated that key signaling mediators of HO, including bone morphogenetic protein signaling, are diminished in mature bone. However, upon surgical excision, we have noted upregulation of downstream mediators of osteogenic differentiation, including pSMAD 1/5. Additionally, surgical excision resulted in re‐emergence of a mesenchymal cell population marked by expression of platelet‐derived growth factor receptor‐α (PDGFRα) and present in the initial developing HO lesion but absent in mature HO. In the recurrent lesion, these PDGFRα+ mesenchymal cells are also highly proliferative, similar to the initial developing HO lesion. These findings indicate that surgical excision of HO results in recurrence through similar mesenchymal cell populations and signaling mechanisms that are present in the initial developing HO lesion. These results are consistent with findings in patients that new foci of ectopic bone can develop in excision sites and are likely related to de novo formation rather than extension of unresected bone. Stem Cells Translational Medicine 2017;6:799–806

Diminished Chondrogenesis and Enhanced Osteoclastogenesis in Leptin-Deficient Diabetic Mice (ob/ob) Impair Pathologic, Trauma-Induced Heterotopic Ossification

Diabetic trauma patients exhibit delayed postsurgical wound, bony healing, and dysregulated bone development. However, the impact of diabetes on the pathologic development of ectopic bone or heterotopic ossification (HO) following trauma is unknown. In this study, we use leptin-deficient mice as a model for type 2 diabetes to understand how post-traumatic HO development may be affected by this disease process. Male leptin-deficient (ob/ob) or wild-type (C57BL/6 background) mice aged 6-8 weeks underwent 30% total body surface area burn injury with left hind limb Achilles tenotomy. Micro-CT (μCT) imaging showed significantly lower HO volumes in diabetic mice compared with wild-type controls (0.70 vs. 7.02 mm(3), P < 0.01) 9 weeks after trauma. Ob/ob mice showed evidence of HO resorption between weeks 5 and 9. Quantitative real time PCR (qRT-PCR) demonstrated high Vegfa levels in ob/ob mice, which was followed by disorganized vessel growth at 7 weeks. We noted diminished chondrogenic gene expression (SOX9) and diminished cartilage formation at 5 days and 3 weeks, respectively. Tartrate-resistant acid phosphatase stain showed increased osteoclast presence in normal native bone and pathologic ectopic bone in ob/ob mice. Our findings suggest that early diminished HO in ob/ob mice is related to diminished chondrogenic differentiation, while later bone resorption is related to osteoclast presence.

Abstract 89: Novel Targeting of the Alk-2 Receptor Using the Cre/lox System to Enhance Osseous Regeneration by Mesenchymal Stem Cells.

PurPose: There is a significant need for readily available autogenous tissue to aid in bone regeneration without causing a donor-site defect. Most studies focus on the ability of bone morphogenetic protein ligands (BMP-2 and 7) to stimulate the Bmpr1b receptor (ALK-6). The ACVR1 gene is often overlooked, but provides instructions for making the activin receptor type I protein (ALK-2), a protein member of the Bmpr1 family. When the ALK-2 receptor is overexpressed globally, patients develop fibrodysplasia ossificans progressive. We believe this highly osteogenic phenotype can be harnessed in adipose derived stem cells (ASCs) to improve bone tissue engineering. Our goal here was to demonstrate that ALK-2 may serve as a novel target to 1) improve in vitro ASC osteogenic differentiation and 2) enhance in vivo bone regeneration and calvarial healing.

Abstract 156: Optimizing Timing and Targeting of Bone Morphogenetic Protein Receptor One to Treat Heterotopic Ossification with Novel Kinase Inhibitors and Ligand Traps.

www.PRSJournal.com 111 Sarday, M ay 16 RESULTS: It was found that, relative to the normoxic baseline, hypoxia significantly affected the expression of a range of angiogenesis-related factors including VEGF, ANG and TSP-1. HPP obtained after 5 days culture had a five-fold higher concentration of the angiogenesis-stimulator VEGF, and a two-fold lower concentration of the angionesis-inhibitor TSP-1, compared to fresh plasma (p<0.05). While both factors eventually underwent down-regulation over time under hypoxia, there was significant variation in their temporal expression profile. Releasates of HPP factor-loaded gels increased endothelial cell tubule formation and directional migration relative to control medium, while microvessel sprouting was found to be significantly improved compared to recombinant VEGF (p<0.05). Two patients with non-healing wounds treated with application of 5% HPP factor-containing emulsion showed complete wound closure within 20 and 50 days, respectively.

Abstract 20: Defining the Role of the Vasculogenic Niche in Trauma-induced Heterotopic Ossification using Novel Imaging and Lineage-tracing Mice.

PURPOSE: Heterotopic ossification (HO) is extra-skeletal lamellar bone in soft tissues following trauma. Prior to HO deposition, patients develop inflammation and hypervascularization. Endothelial progenitor cells contribute to neo-angiogenesis by differentiating into mature endothelium. We hypothesized that angiogenesis establishes the niche necessary for HO, and that endothelial cells contribute to HO thereby providing a target for future inhibition.