The development of a rat model to investigate the formation of blast-related post-traumatic heterotopic ossification

Clinical Orthopaedics &Amp; Related Research

Qureshi

Hope

et al. 2015

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

Section: Rat Model Of Trauma-induced Ho and Bacterial Inoculationsupporting

confidence: 75%

Section: Rat Model Of Trauma-induced Ho and Bacterial Inoculationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Bioburden Increases Heterotopic Ossification Formation in an Established Rat Model

Clinical Orthopaedics &Amp; Related Research

Qureshi

Hope

et al. 2015

“…First, clinically relevant animal models have only recently been developed [21,30]. Second, clinical diagnostic methods are unable to accurately predict the site of HO development [5,20].…”

Section: Introductionmentioning

confidence: 99%

“…We developed a rat model of combat-related HO that incorporates the critical elements associated with combat injury, specifically a blast injury, femur fracture-crush, and transfemoral amputation, through the zone of injury wherein all animals develop radiographic evidence of HO within 2 months postinjury [21]. In this report, we use our model to address (1) the timing of early chondrogenesis, cartilage formation, and radiographic ectopic bone development; and (2) the early cartilage and bone-related gene and protein patterns in traumatized soft tissue subsequent to calcium deposition, tissue mineralization, and ectopic bone formation.…”

Section: Introductionmentioning

confidence: 99%

Early Characterization of Blast-related Heterotopic Ossification in a Rat Model

Qureshi

Crump

Clinical Orthopaedics &Amp; Related Research

et al. 2015

Background Heterotopic ossification (HO) affects the majority of combat-related lower extremity wounds involving severe fracture and amputation. Defining the timing of early osteogenic-related genes may help identify candidate prophylactic agents and guide the timing of prophylactic therapy after blast and other combat-related extremity injuries. Questions/purposes Using a recently developed animal model of combat-related HO, we sought to determine (1) the timing of early chondrogenesis, cartilage formation, and radiographic ectopic bone development; and (2) the early cartilage and bone-related gene and protein patterns in traumatized soft tissue. Methods We used an established rat HO model consisting of blast exposure, controlled femur fracture, crush injury, and transfemoral amputation through the zone of injury. Postoperatively, rats were euthanized on Days 3 to 28. We assessed evidence of early ectopic bone formation by micro-CT and histology and performed proteomic and gene expression analysis. Results All rats showed radiographic evidence of HO within 28 days. Key chondrogenic (collagen type I alpha 1 [COL1a1], p = 0.016) and osteogenic-related genes (RuntThis work was supported by CDMRP (W81XWH-14-2-0010; PI-JAF) and BUMED (602115HP.3720.001.A1014; PI-JAF). 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. All ICMJE Conflict of Interest Forms for authors and Clinical Orthopaedics and Related Research 1 editors and board members are on file with the publication and can be viewed on request. Clinical Orthopaedics and Related Research 1 neither advocates nor endorses the use of any treatment, drug, or device. Readers are encouraged to always seek additional information, including FDA-approval status, of any drug or device prior to clinical use. 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. Conclusions We found that genes that regulate early chondrogenic and osteogenic signaling and bone development (COL1a1, RUNX-2, OCN, PHEX, and POU5F1) are induced early during the tissue reparative/healing phase in a rat model simulating a combat-related extremity injury. Clinical Relevance The ability to correlate molecular events with histologic and morphologic changes will assist re...

Early local delivery of vancomycin suppresses ectopic bone formation in a rat model of trauma‐induced heterotopic ossification

Seavey

Wheatley

Journal Orthopaedic Research

et al. 2017

Heterotopic ossification (HO) is a debilitating sequela of high-energy injuries. It frequently requires surgical excision once symptomatic and there is no practical prophylaxis for combat-injured patients. In this study, we examined the effect of local vancomycin powder on HO formation in a small animal model of blast-related, post-traumatic HO. Male Sprague-Dawley rats were subjected to a polytraumatic extremity injury and amputation with or without methicillin-resistant Staphylococcus aureus infection. Animals were randomized to receive a single local application of vancomycin (20 mg/kg) at the time of injury (POD-0, n = 34) or on postoperative day-3 (POD-3, n = 11). Quantitative volumetric measurement of ectopic bone was calculated at 12-weeks post-injury by micro-CT. Bone marrow and muscle tissues were also collected to determine the bacterial burden. Blood for serum cytokine analysis was collected at baseline and post-injury. Vancomycin treatment on POD-0 suppressed HO formation by 86% and prevented bone marrow and soft tissue infections. We concurrently observed a marked reduction histologically in nonviable tissue, chronic inflammatory cell infiltrates, bone infection, fibrous tissue, and areas of bone necrosis within this same cohort. Delayed treatment was significantly less efficacious. Neither treatment had a marked effect on the production of pro-inflammatory cytokines. Our study demonstrates that local vancomycin treatment at the time of injury significantly reduces HO formation in both the presence and absence of infection, with decreased efficacy if not given early. These findings further support the concept that the therapeutic window for prophylaxis is narrow, highlighting the need to develop early treatment strategies for clinical management. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2397-2406, 2017.