Bioburden Increases Heterotopic Ossification Formation in an Established Rat Model

Abstract

Background Heterotopic ossification (HO) develops in a majority of combat-related amputations wherein early bacterial colonization has been considered a potential early risk factor. Our group has recently developed a small animal model of trauma-induced HO that incorporates many of the multifaceted injury patterns of combat trauma in the absence of bacterial contamination and subsequent wound colonization. Questions/purposes We sought to determine if (1) the presence of bioburden (Acinetobacter baumannii and methicillin-resistant Staphylococcus aureus [MRSA]) increases the magnitude of ectopic bone formation in traumatized muscle after amputation; and (2) what persistent effects bacterial contamination has on late microbial flora within the amputation site. Methods Using a blast-related HO model, we exposed 48 rats to blast overpressure, femur fracture, crush injury, and subsequent immediate transfemoral amputation through the zone of injury. Control injured rats (n = 8) were inoculated beneath the myodesis with phosphate-buffered saline not containing bacteria (vehicle) and treatment rats were inoculated with 1 9 10 6 colony-forming units of A baumannii (n = 20) or MRSA (n = 20). All animals formed HO. Heterotopic ossification was determined by quantitative volumetric measurements of ectopic bone at 12-weeks postinjury using micro-CT and qualitative histomorphometry for assessment of new bone formation in the residual limb. Bone marrow and muscle tissue biopsies The authors are employees of the US Government. This work was prepared as part of their official duties. Title 17 USC §105 provides that ''Copyright protection under this title is not available for any work of the US Government.'' Title 17 USC §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 Departments 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 was performed at the Naval Medical Research Center, Silver Spring, MD, USA. were collected from the residual limb at 12 weeks to quantitatively measure the bioburden load and to qualitatively determine the species-level identification of the bacterial flora. Results At 12 weeks, we observed a greater volume of HO in rats infected with MRSA (68.9 ± 8.6 mm 3 ; 95% confidence interval [CI], 50.52-85.55) when compared with A baumannii (20.9 ± 3.7 mm 3

Targeted stimulation of retinoic acid receptor-γ mitigates the formation of heterotopic ossification in an established blast-related traumatic injury model

“…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.…”

Heterotopic Ossification Following Upper Extremity Injury

“…A group of researchers at the naval academy have developed a rat model of HO in which the hindlimb is exposed to high-energy blast followed by transfemoral amputation and bacterial inoculation. These extensive injuries are not dissimilar to the conditions experienced by wound military personnel; in this rat model, HO forms at the site of the transfemoral amputation(66, 67). …”

Lymphatic Contribution to the Cellular Niche in Heterotopic Ossification

“…Evidence from clinical and animal models suggests that degree of inflammation correlates positively with ectopic bone volume. 5,6 …”

“…Mounting evidence suggests that degree of inflammation correlates with increased bone formation after trauma. 5,6 However, it is unclear which cellular components of the inflammatory response may contribute to HO. Endothelial networks, specifically the lymphatic endothelium provides a logical link between inflammatory/immunologic signals and the local cellular populations at the site of HO development.…”