Full Length ArticleTargeted stimulation of retinoic acid receptor-γ mitigates the formation of heterotopic ossification in an established blast-related traumatic injury model☆
Introduction
Heterotopic ossification (HO) consists of ectopic bone formation within non-skeletal tissues and sites and can be classified as genetic, neurogenic or traumatic in origin [1], [2], [3], [4], [5]. Post-traumatic HO is most commonly encountered following high-energy trauma, spinal cord and brain injury, severe burns, [6], [7] and in orthopaedic practice following hip or elbow surgery [8], [9]. Recent studies have revealed that HO is actually most prevalent in severe combat-related extremity wounds and within residual limbs after combat-related amputations [3], [10], [11], [12]. Blast and other severe polytraumatic injuries elicit complex and often dysregulated inflammatory and physiological responses, and the pathological interactions amongst diverse tissues, cell types, inflammatory mediators and local and systemic signaling pathways provide fertile ground for HO development. The clinical complications of HO include activity-limiting pain, primary (ankylosis) and secondary (muscle-tendon tethering) joint stiffness, skin ulceration and poor tolerance of prosthesis wear, wherein conservative interventions such as multi-modal pain regimens, physiotherapy and prosthetic modification fail to alleviate symptoms in up to 41% of patients [10]. Surgical excision of HO, although the standard of care, often fails to relieve the underlying joint contractures [13], can be fraught with complications and, even when successful, can further delay rehabilitation because of its invasive nature; in some cases, it can even lead to recurrent HO [3], [11], [14]. Surgical excision is also unable to address the chronic symptoms associated with HO including joint contracture, chronic pain, and non-healing wounds. In addition, current prophylaxis strategies used in the general population -such as administration of non-steroidal anti-inflammatory drugs (NSAIDs) and external beam radiotherapy- are generally contraindicated in the setting of multi-system combat trauma because they have deleterious systemic and local side effects and may not be logistically feasible in forward deployed war settings [15]. Furthermore, it is not clear how effective these strategies actually are given that penetrance and severity of HO in the general population vary significantly. Thus, it would be highly desirable to have a simple, effective and safe prophylactic pharmacologic therapy that could be easily administered systemically post-injury, would avoid adverse complications and outcomes, and would minimize treatment duration. Such treatment would have immense impact on the burden of HO disease and resulting pathogenic sequelae, and the search for such novel and non-invasive means of primary HO prevention continues to attract substantial interest and effort.
Heterotopic ossification, when triggered by traumatic injury or other major insults most often arises via endochondral ossification, one of the two essential processes by which the native skeleton forms and grows during embryogenesis and early postnatal life [16], [17], [18], [19]. Skeletal elements including long bones, ribs and vertebrae are initially laid down as entirely cartilaginous structures in early embryogenesis. The cartilaginous tissue then undergoes a process of maturation and hypertrophy, and the resulting hypertrophic cartilage is invaded by vascular and osteopotent cells and is replaced by endochondral bone and marrow. This complex series of events and differentiation steps is governed by equally complex mechanisms that include local and systemic hormones and cytokines, transcription factors and multiple signaling proteins including members of the bone morphogenetic protein (BMP) family. Experimental prophylactic therapies targeting the BMP signaling pathway and its regulatory networks have shown some degree of efficacy against HO in murine models [20], [21]. However, it is not known how effective and safe these treatments would be in patients and in particular those suffering from combat injuries and whether they would have unwanted side effects because of the multiple roles BMPs and BMP signaling have in body physiology and function.
The retinoid signaling pathway and nuclear retinoic acid receptors (RARs) are important regulators of skeletal development and growth [22]. In particular, chondrogenesis was shown to require a steep decrease in endogenous retinoid action during skeletogenesis [23], and exogenous retinoid agonists were in fact found to strongly inhibit chondrogenesis and skeletal development [22], [24]. Because of their ability to block condensation of skeletal progenitor cells and their differentiation into chondrocytes that represent the initial stages of HO, we previously asked whether synthetic retinoid agonists could block HO. We used mouse models of skeletogenic protein- or genetic mutation-induced HO and found that the RAR-γ agonist Palovarotene was amongst the most potent anti-HO agents [25], [26]. Analysis of mechanisms showed that this and other retinoids did, in fact, act by significantly decreasing chondrogenesis and in turn osteogenesis and vascularization. At the cellular level, the drugs dampened canonical BMP signaling -an effect involving ubiquitination and proteasome degradation of Smad proteins- and concurrently increased Wntβ-catenin signaling, a potent endogenous inhibitor of chondrogenesis [27], [28]. While the above studies are quite promising, it remains unclear whether a retinoid-based strategy would be equally effective in the far more complex setting of HO developing after combat injuries in which the affected site is usually large, the wounds are extremely severe, multiple tissues are damaged, and bacterial infections are common.
We recently developed a rat model that incorporates the multiple pathogenic insults sustained by combat casualties with severe orthopaedic extremity injuries which we believe is significantly more representative than non-physiologic models of excess BMP ligand administration or genetic mutations. These insults include exposure to blast overpressure, femur fracture, quadriceps crush injury followed by limb amputation through the zone of injury, and finally inoculation of the myodesis with a highly virulent isolate of methicillin-resistant Staphylococcus aureus (MRSA) obtained from combat wound infections [13], [29]. Previously, we reported on the timing and kinetics of ectopic bone development in this trauma induced HO model. Using molecular and histological tools, we found that chondrogenesis became apparent approximately 3 days following injury and was subsequently followed by cartilage hypertrophy and endochondral bone formation within soft tissues surrounding the amputation site starting around post-injury day 10 [16]. Understanding the timing of these key developmental phases and events was quite important as it now provides a window for prophylactic administration of candidate therapies that could target mechanisms and pathways involved in endochondral ossification. Herein, we sought to determine whether and how Palovarotene may be able to inhibit trauma-induced endochondral HO formation in our rat blast model. To evaluate possible unwanted side effects, we monitored and assessed wound healing as well.
Section snippets
Animals
A total of 110 young adult pathogen-free male Sprague Dawley rats (Rattus norvegicus; 400–600 g) were purchased from Taconic Farms (Germantown, NY). All animals were housed individually in plastic cages and kept on a 12-hour light/dark cycle with unlimited access to food (standard rodent chow) and fresh water ad libitum. They were acclimated for at least one week before experimentation. Prior to experimentation, animals were randomized into experimental and control groups. The study protocol
Palovarotene mitigates post traumatic ectopic bone formation
To test the effectiveness of Palovarotene against blast-induced HO, rats were subjected to whole body blast exposure, femur fracture and crush injury that were followed by amputation of affected limb site and inoculation (or not) of local MRSA. Rats were then administered the drug or vehicle starting every other day for 14 days starting on either postoperative day 1 (POD-1) or POD-5 and were then monitored up to 84 days post-injury. Volumetric assessment of bone formation using μCT demonstrated
Discussion
Herein, we report for the first time that Palovarotene is a strong inhibitor of HO in blast-associated rat model of combat-related extremity injury. This model is both severe and encompassing and includes not only the initial blast injury, but also compounding complications often occurring in combat situations, including bacterial infections and/or limb amputation. In each permutation, Palovarotene was able to counteract HO and did so in a significant manner, with an average of 50 to nearly
Conflict of interest
M.P., M.I., and J.F. are consultants for Clementia Pharmaceuticals. The other authors have no financial or personal conflicts of interest to disclose.
Acknowledgments
We like to thank Dr. Matthew Wagner, Dr. John Tra and Mr. Ying Cao for their assistance with statistical analysis. This work was supported by CDMRP grant W81XWH-13-2-0077 (to JAF) and grant H81XWH-13-2-0076 (to MP). B.L. received funding from NIH/NIGMS - K08GM109105-0, Plastic Surgery Foundation National Endowment Award, the Association for Academic Surgery Roslyn Award American Association for the Surgery of Trauma Research & Education Foundation Scholarship, and American Association of
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Some of the authors are employees of the US Government. This work was prepared as part of their official duties. Title 17 U.S.C. §105 provides that “Copyright protection under this title is not available for any work of the United States Government.” Title 17 U.S.C. §101 defined a US Government work as a work prepared by a military service member or employees of the US Government as part of that person’s official duties. The opinions or assertions contained in this paper are the private views of the authors and are not to be construed as reflecting the views, policy or positions of the Department of the Navy, Department of Defense nor the US Government.
Each author certifies that his or her institution approved the animal protocol for this investigation and that all investigations were conducted in conformity with ethical principles of research.
This work performed at the Naval Medical Research Center, Silver Spring, MD, USA.
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These authors contributed equally.