Bioburden Increases Heterotopic Ossification Formation in an Established Rat Model

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 × 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 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 ; 95% CI, 13.61–28.14; p < 0.001) or vehicle (16.3 ± 3.2 mm ³ ; 95% CI, 10.06–22.47; p < 0.001). Soft tissue and marrow from the residual limb of rats inoculated with A baumannii tested negative for A baumannii infection but were positive for other strains of bacteria (1.33 × 10 ²  ± 0.89 × 10 ² ; 95% CI, −0.42 × 10 ² –3.08 × 10 ² and 1.25 × 10 ⁶  ± 0.69 × 10 ⁶ ; 95% CI, −0.13 × 10 ⁶ –2.60 × 10 ⁶ colony-forming units in bone marrow and muscle tissue, respectively), whereas tissue from MRSA-infected rats contained MRSA only (4.84 × 10 ¹  ± 3.22 × 10 ¹ ; 95% CI, −1.47 × 10 ¹ –11.1 × 10 ¹ and 2.80 × 10 ⁷  ± 1.73 × 10 ⁷ ; 95% CI, −0.60 × 10 ⁷ –6.20 × 10 ⁷ in bone marrow and muscle tissue, respectively). Conclusions: Our findings demonstrate that persistent infection with MRSA results in a greater volume of ectopic bone formation, which may be the result of chronic soft tissue inflammation, and that early wound colonization may be a key risk factor. Clinical Relevance: Interventions that mitigate wound contamination and inflammation (such as early débridement, systemic and local antibiotics) may also have a beneficial effect with regard to the mitigation of HO formation and should be evaluated with that potential in mind in future preclinical studies.