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Objective: At this time, there is no validated tool for describing heterotopic ossification (HO) severity or measuring efficacy of therapies after amputation. This study aims to validate the Walter Reed classification system for reliable characterization of HO burden in residual limbs., Design: Descriptive, Level IV., Setting: US Military Trauma Referral Center., Study Population: Five orthopaedic surgeon raters with varying experience levels assessed orthogonal radiographs of 100 randomly selected residual upper and lower limbs after combat-related amputation., Intervention: A standardized education session on the Walter Reed HO classification system followed by 2 grading sessions, separated by a 2-week waiting period., Main Outcome Measures: Scores were analyzed for interobserver and intraobserver reliability using weighted Cohen's kappa, intraclass correlation coefficient, and Krippendorff's alpha for ordinal variables., Results: After the waiting period, 89% of HO was rated the same by observers with an intraobserver kappa = 0.83 (95% confidence interval [CI] 0.75-0.92). Interobserver reliability was evaluated using group-wise comparison among observers, resulting in an overall Krippendorff's alpha = 0.78 (95% CI 0.76-0.80). Using intraclass correlation coefficient, interrater consistency was 0.94 (95% CI 0.91-0.96)., Conclusions: The Walter Reed HO classification system is substantially reliable for use by raters with any level of orthopaedic training. In addition, it is reliable for evaluation of both through bone and disarticulation type amputations in the upper and lower extremities. However, in 11% of cases, a secondary read may vary, suggesting that this system is useful for understanding HO and developing interventions but may benefit from further refinement, including advanced imaging and clinical correlation., Competing Interests: The authors report no conflict of interest. Each author certifies that he or she has no commercial associations (eg, consultancies, stock ownership, equity interest, and patent/licensing arrangements) that might pose a conflict of interest in connection with the submitted article. Each author certifies that all investigations were conducted in conformity with ethical principles of research., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Amputations sustained owing to combat-related blast injuries are at high risk for deep infection and development of heterotopic ossification, which can necessitate reoperation and place immense strain on the patient. Surgeons at our institution began use of intrawound antibiotic powder at the time of closure in an effort to decrease the rate of these surgical complications after initial and revision amputations, supported by compelling clinical evidence and animal models of blast injuries. Antibiotic powder may be useful in reducing the risk of these infections, but human studies on this topic thus far have been inconclusive., Purpose: We sought to determine whether administration of intrawound antibiotic powder at the time of closure would (1) decrease the risk of subsequent deep infections of major lower-extremity combat-related amputations, and (2) limit formation and decrease severity of heterotopic ossification common in the combat-related traumatic residual limb., Methods: Between 2009 and 2015, 252 major lower extremity initial and revision amputations were performed by a single surgeon. Revision cases were excluded if performed specifically to address deep infection, leaving 223 amputations (88.5%) for this retrospective analysis. We reviewed medical records to collect patient information, returns to the operating room for subsequent infection, and microbiologic culture results. We also reviewed radiographs taken at least 3 months after surgery to determine the presence and severity of heterotopic ossification using the Walter Reed classification system. We grouped cases according to whether limbs underwent initial or revision amputations, and whether the limbs had a history of a prior infection. Apart from the use of antibiotic powder and duration of followup, the groups did not differ in terms of age, mechanism of injury, or sex. We then calculated the absolute risk reduction for infection and heterotopic ossification and the number needed to treat to prevent an infection., Results: Overall, administration of antibiotic powder resulted in a 13% absolute risk reduction of deep infection (14 of 82 [17%] versus 42 of 141 [30%]; p = 0.03; 95% CI, 0.20%-24.72%). In revision amputation surgery, the absolute risk reduction of infection with antibiotic powder use was 16% overall (eight of 58 versus 17 of 57; 95% CI, 1.21%-30.86%), and 25% for previously infected limbs (eight of 46 versus 14 of 33; 95% CI, 4.93%-45.14%). The number needed to treat to prevent one additional deep infection in amputation surgery is eight in initial amputations, seven in revision amputations, and four for revision amputation surgery on previously infected limbs. With the numbers available, we observed no reduction in the risk of heterotopic ossification with antibiotic powder use, but severity was decreased in the treatment group in terms of the number of residual limbs with moderate or severe heterotopic ossification (three of 12 versus 19 of 34; p = 0.03)., Conclusions: Our findings show that administration of intrawound antibiotic powder reduces deep infection in residual limbs of combat amputees, particularly in the setting of revision amputation surgery in apparently aseptic residual limbs at the time of the surgery. Furthermore, administration of antibiotic powder for amputations at time of initial closure decreases the severity of heterotopic ossification formation, providing a low-cost adjunct to decrease the risk of two complications common to amputation surgery.Level of Evidence Level III, therapeutic study.

Post-traumatic heterotopic ossification (HO) is the formation of ectopic bone in non-osseous structures following injury. The precise mechanism for bone development following trauma is unknown; however, early onset of HO may involve the production of pro-osteogenic serum factors. Here we evaluated serum from a cohort of civilian and military patients post trauma to determine early induction gene signatures in orthopaedic trauma induced HO. To test this, human adipose derived stromal/stem cells (hASCs) were stimulated with human serum from patients who developed HO following trauma and evaluated for a gene panel with qPCR. Pathway gene analysis ontology revealed that hASCs stimulated with serum from patients who developed HO had altered gene expression in the activator protein 1 (AP1) and AP1 transcriptional targets pathways. Notably, there was a significant repression in FOS gene expression in hASCs treated with serum from individuals with HO. Furthermore, the mitogen-activated protein kinase (MAPK) signaling pathway was activated in hASCs following serum exposure from individuals with HO. Serum from both military and civilian patients with trauma induced HO had elevated downstream genes associated with the MAPK pathways. Stimulation of hASCs with known regulators of osteogenesis (BMP2, IL6, Forskolin, and WNT3A) failed to recapitulate the gene signature observed in hASCs following serum stimulation, suggesting non-canonical mechanisms for gene regulation in trauma induced HO. These findings provide new insight for the development of HO and support ongoing work linking the systemic response to injury with wound specific outcomes., (© 2018 Wiley Periodicals, Inc.)

Objectives: To determine what proportion of residual limbs formed heterotopic ossification (HO) in amputations sustained by US service members, the injury profile of these amputations, and what effect the number of limb amputations sustained has on resource utilization., Design: Retrospective review., Setting: A tertiary military medical center., Patients: Four-hundred seventy-one consecutive patients with 714 combat-related amputations were treated at our institution between September 2009 and August 2014. Four-hundred thirty-nine amputations had radiographic follow-up beyond 2 months of injury and met the criteria for study inclusion., Main Outcome Measure: Formation and grade of HO., Results: HO was present in 399 of 439 (91%) residual limbs, including 211 of 216 (98%) transfemoral amputations. Dismounted improvised explosive device blast injury resulted in HO development in 346 of 372 (93%) residual limbs compared with 36 of 44 (82%) in mounted improvised explosive device blast injury [P = 0.014; odds ratio (OR) 2.96, 95% confidence interval (CI), 1.25-7.04]. As the number of amputations per patient increased, so too did blood product utilization [including packed red blood cells (P < 0.001), fresh frozen plasma (P < 0.001), and platelets (P < 0.001)]; the number of days on a ventilator (P < 0.001), in the intensive care unit (P < 0.001), and in the hospital (P = 0.007)., Conclusions: HO prevalence in the traumatic amputations of war wounded has increased compared with earlier studies, which is temporally associated with higher rates of increasingly severe injuries due to dismounted blast., Level of Evidence: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Heterotopic ossification (HO) develops in the extremities of wounded service members and is common in the setting of high-energy penetrating injuries and blast-related amputations. No safe and effective prophylaxis modality has been identified for this patient population. Palovarotene has been shown to reduce bone formation in traumatic and genetic models of HO. The purpose of this study was to determine the effects of Palovarotene on inflammation, progenitor cell proliferation, and gene expression following a blast-related amputation in a rodent model (n = 72 animals), as well as the ability of Raman spectroscopy to detect early HO before radiographic changes are present. Treatment with Palovarotene was found to dampen the systemic inflammatory response including the cytokines IL-6 (p = 0.01), TNF-α (p = 0.001), and IFN-γ (p = 0.03) as well as the local inflammatory response via a 76% reduction in the cellular infiltration at post-operative day (POD)-7 (p = 0.03). Palovarotene decreased osteogenic connective tissue progenitor (CTP-O) colonies by as much as 98% both in vitro (p = 0.04) and in vivo (p = 0.01). Palovarotene treated animals exhibited significantly decreased expression of osteo- and chondrogenic genes by POD-7, including BMP4 (p = 0.02). Finally, Raman spectroscopy was able to detect differences between the two groups by POD-1 (p < 0.001). These results indicate that Palovarotene inhibits traumatic HO formation through multiple inter-related mechanisms including anti-inflammatory, anti-proliferative, and gene expression modulation. Further, that Raman spectroscopy is able to detect markers of early HO formation before it becomes radiographically evident, which could facilitate earlier diagnosis and treatment. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1135-1144, 2018., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Heterotopic ossification, the formation of bone in soft tissues, is a common complication of the high-energy extremity trauma sustained in modern armed conflict. In the past 15years, military treatment facilities and aligned laboratories have been in a unique position to study and treat this process due to the high volume of patients with these injuries secondary to blast trauma. The devastating nature of these wounds has limited traditional therapeutic options, necessitating alternative solutions to prophylaxis and initial treatment producing substantial advances in modeling, prophylaxis, detection, and therapy. Specific developments include establishment of an animal model that reproduces the systemic and local tissue injury of blast injuries, the use of molecular assays and predictive modeling in clinical decision making, advances in early detection including Raman spectroscopy, and investigation of prophylactic and therapeutic pharmacotherapy targeting the molecular pathways of aberrant bone formation. In this review article, we will present the literature to date, ongoing studies, and future directions for investigation of heterotopic ossification, with a focus on military-specific research., (Copyright © 2018. Published by Elsevier Inc.)

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., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Heterotopic ossification (HO), the abnormal formation of bone within soft tissues, is a major complication after severe trauma or amputation. Transient brown adipocytes have been shown to be a critical regulator of this process in a mouse model of HO. In this study, we evaluated the presence of brown fat within human HO lesions. Most of the excised tissue samples displayed histological characteristics of bone, fibroproliferative cells, blood vessels, and adipose tissue. Immunohistochemical analysis revealed extensive expression of uncoupling protein 1 (UCP1), a definitive marker of brown adipocytes, within HO-containing tissues but not normal tissues. As seen in the brown adipocytes observed during HO in the mouse, these UCP1 + cells also expressed the peroxisome proliferator-activated receptor γ coactivator 1α. However, further characterization showed these cells, like their mouse counterparts, did not express PR domain containing protein 16, a key factor present in brown adipocytes found in depots. Nor did they express factors present in beige adipocytes. These results identify a population of UCP1 + cells within human tissue undergoing HO that do not entirely resemble either classic brown or beige adipocytes, but rather a specialized form of brown adipocyte-like cells, which have a unique function. These cells may offer a new target to prevent this unwanted bone., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Studies presented here, using a murine model of bone morphogenetic protein type 2 (BMP2)-induced heterotopic ossification (HO) show that the protein initiates HO by signaling through progenitors in the endoneurium of peripheral nerves. In the mouse, these cells were identified in the endoneurium one day after BMP2 induction using antibody against phosphoSMAD (PS) 1, 5, and 8. Studies conducted in a tracking mouse that contains a tamoxifen-regulated Wnt1-Cre recombinase crossed with a td Tomato red (TR) reporter (Wnt1 CreErt :Ai9Tm) confirmed their neural origin. In this model both BMP2 induction and tamoxifen are absolutely required to induce TR. SP7 + (osterix + )TR + cells were found in the endoneurium on day 1 and associated with bone on day 7. Quantification of TR + and TR - cells isolated by fluorescence-activated cell sorting showed that all SP7 + cells were found in the TR + population, whereas only about 80% of the TR + cells expressed SP7. Pre-chondrocytes (Sox 9 + ) and transient brown fat (tBAT, UCP1 + ) also coexpressed TR, suggesting that the progenitor in nerves is multi-potential. The endoneurium of human nerves near the site of HO contained many PS + cells, and SP7 + cells were found in nerves and on bone in tissue from patients with HO. Control tissues and nerves did not contain these PS + and SP7 + cells. Some osteoblasts on bone from patients with HO were positive for PS, suggesting the continued presence of BMP during bone formation. The data suggests that the progenitors for HO are derived from the endoneurium in both the mouse model of HO and in humans with HO. Stem Cells Translational Medicine 2017;6:1109-1119., (© 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Extremity amputation or traumatic injury can often lead to the formation of heterotopic ossification (HO). Studies to induce HO in rat muscle using cell-based gene therapy show that this process appears to be location dependent. In the present study, HO was induced in mice and rats through injection of immunologically matched cells transduced with either a replication-defective adenovirus possessing bone morphogenetic protein 2 (BMP2) or an empty adenovirus vector (control). Injection in rat near the skeletal bone resulted in HO, whereas cells injected into the same muscle group but distal from the bone did not result in bone formation. When cells were injected in the same limb at both locations at the same time, HO was formed at both sites. Characterization of the bone formation in rats versus mice demonstrated that different sources of osteogenic progenitors were involved, which may account for the location dependent bone formation observed in the rat. Further experimentation has shown that a potential reason for this difference may be the inability of rat to activate matrix metalloproteinase 9 (MMP9), an essential protease in mice necessary for recruitment of progenitors. Inhibition of active MMP9 in mice led to a significant decrease in HO. The studies reported here provide insight into the mechanisms and pathways leading to bone formation in different animals and species. It appears that not all animal models are appropriate for testing HO therapies, and our studies also challenge the conventional wisdom that larger animal models are better for testing treatments affecting bone. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1894-1904, 2016., (© 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Heterotopic ossification (HO) involves formation of endochondral bone at non-skeletal sites, is prevalent in severely wounded service members, and causes significant complications and delayed rehabilitation. As common prophylactic treatments such as anti-inflammatory drugs and irradiation cannot be used after multi-system combat trauma, there is an urgent need for new remedies. Previously, we showed that the retinoic acid receptor γ agonist Palovarotene inhibited subcutaneous and intramuscular HO in mice, but those models do not mimic complex combat injury. Thus, we tested Palovarotene in our validated rat trauma-induced HO model that involves blast-related limb injury, femoral fracture, quadriceps crush injury, amputation and infection with methicillin-resistant Staphylococcus aureus from combat wound infections. Palovarotene was given orally for 14days at 1mg/kg/day starting on post-operative day (POD) 1 or POD-5, and HO amount, wound dehiscence and related processes were monitored for up to 84days post injury. Compared to vehicle-control animals, Palovarotene significantly decreased HO by 50 to 60% regardless of when the treatment started and if infection was present. Histological analyses showed that Palovarotene reduced ectopic chondrogenesis, osteogenesis and angiogenesis forming at the injury site over time, while fibrotic tissue was often present in place of ectopic bone. Custom gene array data verified that while expression of key chondrogenic and osteogenic genes was decreased within soft tissues of residual limb in Palovarotene-treated rats, expression of cartilage catabolic genes was increased, including matrix metalloproteinase-9. Importantly, Palovarotene seemed to exert moderate inhibitory effects on wound healing, raising potential safety concerns related to dosing and timing. Our data show for the first time that Palovarotene significantly inhibits HO triggered by blast injury and associated complications, strongly indicating that it may prevent HO in patients at high risk such as those sustaining combat injuries and other forms of blast trauma., (Published by Elsevier Inc.)

Heterotopic ossification (HO) is the pathologic formation of bone separate from the normal skeleton. Although several models exist for studying HO, an understanding of the common in vitro properties of cells isolated from these models is lacking. We studied three separate animal models of HO including two models of trauma-induced HO and one model of genetic HO, and human HO specimens, to characterize the properties of cells derived from tissue containing pre-and mature ectopic bone in relation to analogous mesenchymal cell populations or osteoblasts obtained from normal muscle tissue. We found that when cultured in vitro, cells isolated from the trauma sites in two distinct models exhibited increased osteogenic differentiation when compared to cells isolated from uninjured controls. Furthermore, osteoblasts isolated from heterotopic bone in a genetic model of HO also exhibited increased osteogenic differentiation when compared with normal osteoblasts. Finally, osteoblasts derived from mature heterotopic bone obtained from human patients exhibited increased osteogenic differentiation when compared with normal bone from the same patients. These findings demonstrate that across models, cells derived from tissues forming heterotopic ossification exhibit increased osteogenic differentiation when compared with either normal tissues or osteoblasts. These cell types can be used in the future for in vitro investigations for drug screening purposes.

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 [COL1α1], p = 0.016) and osteogenic-related genes (Runt-related transcription factor 2 [RUNX-2], p = 0.029; osteoclacin [OCN], p = 0.032; phosphate-regulating neutral endopeptidase, X-linked [PHEX], p = 0.0290, and POU domain class 5 transcription factor [POU5F], p = 0.016) and proteins (Noggin [NOG], p = 0.04, OCN, p = 0.02, RUNX- 2, p = 0.04, and substance P-1 [SP-1], p = 0.01) in the injured soft tissue, normalized to the contralateral limb and/or sham-treated naïve rats, increased on Days 3 to 14 postinjury. By 14 days, foci of hypertrophic chondrocytes, hyaline cartilage, and woven bone were present in the soft tissue surrounding the amputation site., Conclusions: We found that genes that regulate early chondrogenic and osteogenic signaling and bone development (COL1α1, 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 researchers and clinicians to understand HO and hence formulate therapeutic interventions.

Background: Heterotopic ossification (HO) is common after combat-related amputations and surgical excision remains the only definitive treatment for persistently symptomatic HO. There is no consensus in the literature regarding the timing of surgery, and recurrence frequency, reexcision, and complications have not been reported in large numbers of patients., Questions/purposes: (1) What are the rates of symptomatic recurrence resulting in reexcision and other complications resulting in reoperation in patients with HO? (2) Is either radiographic or symptomatic recurrence dependent on timing and type of initial surgery, the experience of the surgeon in performing the procedure, the severity of preexcision HO, the presence of concomitant neurologic injury, or the use of postoperative HO prophylaxis?, Methods: Between March 2005 and March 2013 our institution treated 994 patients with 1377 combat-related major extremity amputations; of those, 172 amputations underwent subsequent excision of symptomatic HO. The mechanism of injury resulting in nearly all amputations (n = 168) was blast-related trauma. We reviewed medical records and radiographs to collect initial grade of HO, radiographic recurrence, complete compared with partial excision, concomitant neurologic injury, timing to initial surgery, surgeon experience, and use of postexcision prophylaxis with our primary study outcome being a return to the operating room (OR) for repeat excision of symptomatic HO. All 172 combat-related amputations were considered for this study irrespective of followup, which was noted to be robust, with 157 (91%) amputations having at least 6 months clinical followup by an orthopaedic surgeon or physiatrist (median, 20 months; range, 0-88 months)., Results: Eleven of 172 patients (6.5%) underwent reexcision of HO, and 67 complications resulting in return to the OR occurred in 53 patients (31%) of patients. Multivariate analysis of our primary outcome measure showed more frequent symptomatic recurrences requiring reexcision when initial excision was performed as a partial excision (p = 0.03; odds ratio [OR], 5.0; 95% confidence interval [CI], 1.2-29.6) or when the initial excision was performed within 180 days of injury (p = 0.047; OR, 4.1; 95% CI, 1.02-16.6). There was no association between symptomatic recurrence and HO grade, central nervous system injury, experience of the attending surgeon, or postoperative prophylaxis. Radiographic recurrence was observed when partial excisions (eight of 30 [27%]) were done compared with complete excisions (five of 77 [7%]; p = 0.008)., Conclusions: HO is common after combat-related amputations, and patients undergoing surgical excision of HO for this indication often have complications that result in repeat surgical procedures. Partial excisions of immature lesions more often resulted in both symptomatic and radiographic recurrence. The likelihood of a patient undergoing reexcision can be minimized by performing a complete excision at least 180 days from injury to surgery with no evidence of a reduced risk of reexcision by waiting longer than 270 days., Level of Evidence: Level III, therapeutic study.

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.

Background: To prevent symptomatic heterotopic ossification (HO) and guide primary prophylaxis in patients with combat wounds, physicians require risk stratification methods that can be used early in the postinjury period. There are no validated models to help guide clinicians in the treatment for this common and potentially disabling condition., Questions/purposes: We developed three prognostic models designed to estimate the likelihood of wound-specific HO formation and compared them using receiver operating characteristic (ROC) curve analysis and decision curve analysis (DCA) to determine (1) which model is most accurate; and (2) which technique is best suited for clinical use., Methods: We obtained muscle biopsies from 87 combat wounds during the first débridement in the United States, all of which were evaluated radiographically for development of HO at a minimum of 2 months postinjury. The criterion for determining the presence of HO was the ability to see radiographic evidence of ectopic bone formation within the zone of injury. We then quantified relative gene expression from 190 wound healing, osteogenic, and vascular genes. Using these data, we developed an Artificial Neural Network, Random Forest, and a Least Absolute Shrinkage and Selection Operator (LASSO) Logistic Regression model designed to estimate the likelihood of eventual wound-specific HO formation. HO was defined as any HO visible on the plain film within the zone of injury. We compared the models accuracy using area under the ROC curve (area under the curve [AUC]) as well as DCA to determine which model, if any, was better suited for clinical use. In general, the AUC compares models based solely on accuracy, whereas DCA compares their clinical utility after weighing the consequences of under- or overtreatment of a particular disorder., Results: Both the Artificial Neural Network and the LASSO logistic regression models were relatively accurate with AUCs of 0.78 (95% confidence interval [CI], 0.72-0.83) and 0.75 (95% CI, 0.71-0.78), respectively. The Random Forest model returned an AUC of only 0.53 (95% CI, 0.48-0.59), marginally better than chance alone. Using DCA, the Artificial Neural Network model demonstrated the highest net benefit over the broadest range of threshold probabilities, indicating that it is perhaps better suited for clinical use than the LASSO logistic regression model. Specifically, if only patients with greater than 25% risk of developing HO received prophylaxis, for every 100 patients, use of the Artificial Network Model would result in six fewer patients who unnecessarily receive prophylaxis compared with using the LASSO regression model while not missing any patients who might benefit from it., Conclusions: Our findings suggest that it is possible to risk-stratify combat wounds with regard to eventual HO formation early in the débridement process. Using these data, the Artificial Neural Network model may lead to better patient selection when compared with the LASSO logistic regression approach. Future prospective studies are necessary to validate these findings while focusing on symptomatic HO as the endpoint of interest., Level of Evidence: Level III, prognostic study.

Background: Heterotopic ossification most commonly occurs after burn injury, joint arthroplasty, and trauma. Male gender has been identified as a risk factor for the development of heterotopic ossification. It remains unclear why adult male patients are more predisposed to this pathologic condition than adult female patients. In this study, the authors use their validated tenotomy/burn model to explore differences in heterotopic ossification between male and female mice., Methods: The authors used their Achilles tenotomy and burn model to evaluate the osteogenic potential of mesenchymal stem cells of male and female injured and noninjured mice. Groups consisted of injured male (n = 3), injured female (n = 3), noninjured male (n = 3), and noninjured female (n = 3) mice. The osteogenic potential of cells harvested from each group was assessed through RNA and protein levels and quantified using micro-computed tomographic scan. Histomorphometry was used to verify micro-computed tomographic findings, and immunohistochemistry was used to assess osteogenic signaling at the site of heterotopic ossification., Results: Mesenchymal stem cells of male mice demonstrated greater osteogenic gene and protein expression than those of female mice (p < 0.05). Male mice in the burn group formed 35 percent more bone than female mice in the burn group. This bone formation correlated with increased pSmad and insulin-like growth factor 1 signaling at the heterotopic ossification site in male mice., Conclusions: The authors demonstrate that male mice form quantitatively more bone compared with female mice using their burn/tenotomy model. These findings can be explained at least in part by differences in bone morphogenetic protein and insulin-like growth factor 1 signaling.

Currently, there is no animal model in which to evaluate the underlying physiological processes leading to the heterotopic ossification (HO) which forms in most combat-related and blast wounds. We sought to reproduce the ossification that forms under these circumstances in a rat by emulating patterns of injury seen in patients with severe injuries resulting from blasts. We investigated whether exposure to blast overpressure increased the prevalence of HO after transfemoral amputation performed within the zone of injury. We exposed rats to a blast overpressure alone (BOP-CTL), crush injury and femoral fracture followed by amputation through the zone of injury (AMP-CTL) or a combination of these (BOP-AMP). The presence of HO was evaluated using radiographs, micro-CT and histology. HO developed in none of nine BOP-CTL, six of nine AMP-CTL, and in all 20 BOP-AMP rats. Exposure to blast overpressure increased the prevalence of HO. This model may thus be used to elucidate cellular and molecular pathways of HO, the effect of varying intensities of blast overpressure, and to evaluate new means of prophylaxis and treatment of heterotopic ossification., (©2015 The British Editorial Society of Bone & Joint Surgery.)

Over 60% of combat-wounded patients develop heterotopic ossification (HO). Nearly 33% of them require surgical excision for symptomatic lesions, a procedure that is both fraught with complications and can delay or regress functional rehabilitation. Relative medical contraindications limit widespread use of conventional means of primary prophylaxis, such as nonspecific nonsteroidal anti-inflammatory medications and radiotherapy. Better methods for risk stratification are needed to both mitigate the risk of current means of primary prophylaxis as well as to evaluate novel preventive strategies currently in development. We asked whether Raman spectral changes, measured ex vivo, could be associated with histologic evidence of the earliest signs of HO formation and substance P (SP) expression in tissue biopsies from the wounds of combat casualties. In this pilot study, we compared normal muscle tissue, injured muscle tissue, very early HO lesions (< 16 d post-injury), early HO lesions (> 16 d post-injury) and mature HO lesions. The Raman spectra of these tissues demonstrate clear differences in the Amide I and III spectral regions of HO lesions compared to normal tissue, denoted by changes in the Amide I band center (p < 0.01) and the 1340/1270 cm(-1) (p < 0.05) band area and band height ratios. SP expression in the HO lesions appears to peak between 16 and 30 d post-injury, as determined by SP immunohistochemistry of corresponding tissue sections, potentially indicating optimal timing for administration of therapeutics. Raman spectroscopy may therefore prove a useful, non-invasive and early diagnostic modality to detect HO formation before it becomes evident either clinically or radiographically.

Heterotopic ossification--a complication of severe burns, head or blast injuries, and orthopaedic trauma--can result from altered adenosine metabolism in mesenchymal stem cells in response to elevated extracellular ATP (Peterson et al., this issue)., (Copyright © 2014, American Association for the Advancement of Science.)

Background: After a decade of war in Iraq and Afghanistan, we have observed an increase in combat-related injury survival and a paradoxical increase in injury severity, mainly because of the effects of blasts. These severe injuries have a devastating effect on each patient's immune system resulting in massive upregulation of the systemic inflammatory response. By examining inflammatory mediators, preliminary data suggest that it may be possible to correlate complications such as wound failure and heterotopic ossification (HO) with distinct systemic and local inflammatory profiles, but this is a relatively new topic., Questions/purposes: We asked whether systemic or local markers of inflammation could be used as an objective means, independent of demographic and subjective factors, to estimate the likelihood of (1) HO and/or (2) wound failure (defined as wounds requiring surgical débridement after definitive closure, or wounds that were not closed or covered within 21 days of injury) in patients sustaining combat wounds., Methods: Two hundred combat wounded active-duty service members who sustained high-energy extremity injuries were prospectively enrolled between 2008 and 2012. Of these 200 patients, 189 had adequate followups to determine the presence or absence of HO, and 191 had adequate followups to determine the presence or absence of wound failure. In addition to injury-specific and demographic data, we quantified 24 cytokines and chemokines during each débridement. Patients were followed clinically for 6 weeks, and radiographs were obtained 3 months after definitive wound closure. Associations were investigated between these markers and wound failure or HO, while controlling for known confounders., Results: The presence of an amputation (p < 0.001; odds ratio [OR], 6.1; 95% CI. 1.63-27.2), Injury Severity Score (p = 0.002; OR, 33.2; 95% CI, 4.2-413), wound surface area (p = 0.001; OR, 1.01; 95% CI, 1.002-1.009), serum interleukin (IL)-3 (p = 0.002; OR, 2.41; 95% CI, 1.5-4.5), serum IL-12p70 (p = 0.01; OR, 0.49; 95% CI, 0.27-0.81), effluent IL-3 (p = 0.02; OR, 1.75; 95% CI, 1.2-2.9), and effluent IL-13 (p = 0.006; OR, 0.67; 95% CI, 0.50-0.87) were independently associated with HO formation. Injury Severity Score (p = 0.05; OR, 18; 95% CI, 5.1-87), wound surface area (p = 0.05; OR, 28.7; 95% CI, 1.5-1250), serum procalcitonin ([ProCT] (p = 0.03; OR, 1596; 95% CI, 5.1-1,758,613) and effluent IL-6 (p = 0.02; OR, 83; 95% CI, 2.5-5820) were independently associated with wound failure., Conclusions: We identified associations between patients' systemic and local inflammatory responses and wound-specific complications such as HO and wound failure. However, future efforts to model these data must account for their complex, time dependent, and nonlinear nature., Level of Evidence: Level II, prognostic study. See the Instructions for Authors for a complete description of levels of evidence.

Background: Heterotopic ossification (HO) is a frequent complication of modern wartime extremity injuries. The biological mechanisms responsible for the development of HO in traumatic wounds remain elusive., Question/purposes: The aims of our study were to (1) characterize the expression profile of osteogenesis-related gene transcripts in traumatic war wounds in which HO developed; and (2) determine whether expression at the mRNA level correlated with functional protein expression and HO formation., Methods: Biopsy specimens from 54 high-energy penetrating extremity wounds obtained at the initial and final surgical débridements were evaluated. The levels of selected osteogenic-related gene transcripts from RNA extracts were assessed by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. As a result of its key role in osteogenesis, the concentration of BMP-2 in the effluent of 29 wounds also was determined., Results: The transcripts of 13 genes (ALPL [p = 0.006], BMP-2 [p < 0.001], BMP-3 [p = 0.06], COL2A1 [p < 0.001], COLL10A1 [p < 0.001], COL11A1 [p = 0.006], COMP [p = 0.02], CSF2 [p = 0.003], CSF3 [p = 0.012], MMP8 [p < 0.001], MMP9 [p = 0.014], SMAD1 [p = 0.024], and VEGFA [p = 0.017]) were upregulated greater than twofold in wounds in which HO developed compared with wounds in which it did not develop. Gene transcript expression of BMP-2 also correlated directly with functional protein expression in the wounds that formed HO (p = 0.029)., Conclusions: Important differences exist in the osteogenic gene expression profile of wounds in which HO developed compared with wounds in which it did not develop. The upregulation of multiple osteogenesis-related gene transcripts indicates the presence of a proosteogenic environment necessary for ectopic bone formation in traumatic wounds., Clinical Relevance: Understanding the osteogenic environment associated with war wounds may allow for the development of novel therapeutic strategies for HO.

Background: Heterotopic ossification is the ectopic formation of mature lamellar bone in nonosseous tissue. The prevalence of heterotopic ossification following combat injuries is much higher than civilian data would suggest. In certain cases, the aberrant bone formation can envelop major neurovascular structures in the lower extremity, leading to symptomatic neurovascular entrapment., Methods: We describe five consecutive cases of heterotopic ossification leading to symptomatic neurovascular entrapment in the lower extremity as a result of blast trauma and present our method of patient assessment, preoperative planning, and surgical excision., Results: Heterotopic bone was successfully excised without neurovascular injury in all patients. At a mean of twenty months (range, eight to forty-five months) postoperatively, all patients demonstrated continued improvement of their pre-excision function. All patients who had neuropathic pain had a decrease in the pain. Those with decreased joint motion regained motion once their wounds were stable. Sensory deficits resolved before motor deficits did. There was no recurrence of clinically relevant heterotopic ossification in this series., Conclusions: Excision of heterotopic bone, particularly with concurrent neurovascular entrapment, can be associated with major short-term and long-term complications. With use of our treatment algorithm, involving careful preoperative planning and meticulous operative excision, heterotopic bone entrapping major neurovascular structures following severe extremity trauma can be safely excised with subsequent clinical improvement.

Over 60% of our severely combat-injured patient population develops radiographically apparent heterotopic ossification. Nearly a third of these require surgical excision of symptomatic lesions, a procedure that is fraught with complications, and delays or regresses functional rehabilitation in many cases. Unfortunately, for the combat injured, medical contraindications and logistical limitations limit widespread use of conventional means of primary prophylaxis. Better means of risk stratification are needed to both mitigate the risk of current means of primary prophylaxis as well as to evaluate novel preventive strategies currently in development. We asked whether Raman spectral changes, measured ex vivo, correlated with histologic evidence of the earliest signs of HO formation using tissue biopsies from the wounds of combat casualties. In doing so, we compared normal muscle tissue to injured muscle tissue, unmineralized HO tissue, and mineralized HO tissue. The Raman spectra of these tissues demonstrate clear differences in the amide I and amide III spectral regions of HO tissue compared to normal tissue, denoted by changes in the 1640/1445cm(-1)(p<0.01), and 1340/1270cm(-1) (p<0.01) band area ratios (BARs). Additionally, analysis of the bone mineral in HO by Raman spectroscopy appears capable of determining bone maturity by measuring both the 945/960cm(-1) and the 1070/1445cm(-1) BARs. Raman may therefore prove a useful, non-invasive, and early diagnostic modality to detect HO formation prior to it becoming evident clinically or radiographically. This technique could ostensibly be utilized as a non-invasive means to risk stratify individual wounds at a time thought to be amenable to various means of primary prophylaxis., (© 2013.)

Combat-related heterotopic ossification (HO) has emerged as a common and problematic complication of modern wartime extremity injuries, contributing to substantial patient morbidity and loss of function. We have previously reported that HO-forming patients exhibit a more pronounced systemic and local inflammatory response very early in the wound healing process. Moreover, traumatized muscle-derived mesenchymal progenitor cells from these patients have a skewed differentiation potential toward bone. Here, we demonstrate that HO lesions excised from this patient population contain highly vascularized, mature, cancellous bone containing adipogenic marrow. Histologic analysis showed immature hematopoietic cells located within distinct foci in perivascular regions. The adipogenic marrow often contained low numbers of functional erythroid (BFU-E), myeloid (CFU-GM, CFU-M) and multilineage (CFU-GEMM) colony-forming hematopoietic progenitor cells (HPCs). Conversely, tissue from control muscle and non-HO traumatic wound granulation tissue showed no evidence of hematopoietic progenitor cell activity. In summary, our findings suggest that ectopic bone can provide an appropriate hematopoietic microenvironment for supporting the proliferation and differentiation of HPCs. This reactive and vibrant cell population may help maintain normal hematopoietic function, particularly in those with major extremity amputations who have sustained both massive blood loss, prompting systemic marrow stimulation, as well as loss of available native active marrow space. These findings begin to characterize the functional biology of ectopic bone and elucidate the interactions between HPC and non-hematopoietic cell types within the ectopic intramedullary hematopoietic microenvironmental niche identified., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: Heterotopic ossification (HO) develops frequently after modern high-energy penetrating war injuries. The purpose of this prospective study was to identify and characterize the unique cytokine and chemokine profile associated with the development of HO as it pertained to the systemic inflammatory response after penetrating combat-related trauma., Methods: Patients with high-energy penetrating extremity wounds were prospectively enrolled. Surgical debridement along with the use of a pulse lavage and vacuum-assisted-closure device was performed every 48-72 hours until definitive wound closure. Wound bed tissue biopsy, wound effluent, and serum were collected before each debridement. Effluent and serum were analyzed for 22 relevant cytokines and chemokines. Tissue was analyzed quantitatively for bacterial colonization. Correlations between specific wound and patient characteristics were also analyzed. The primary clinical outcome measure was the formation of HO as confirmed by radiographs at a minimum of 2 months of follow-up., Results: Thirty-six penetrating extremity war wounds in 24 patients were investigated. The observed rate of HO in the study population was 38%. Of the 36 wounds, 13 (36%) demonstrated HO at a minimum follow-up of 2 months. An elevated injury severity score was associated with the development of HO (P = 0.006). Wound characteristics that correlated with the development of HO included impaired healing (P = 0.005) and bacterial colonization (P < 0.001). Both serum (interleukin-6, interleukin-10, and MCP-1) and wound effluent (IP-10 and MIP-1α) cytokine and chemokine bioprofiles were individually associated and suggestive of the development of HO (P < 0.05)., Conclusions: A severe systemic and wound-specific inflammatory state as evident by elevated levels of inflammatory cytokines, elevated injury severity score, and bacterial wound colonization is associated with the development of HO. These findings suggest that the development of HO in traumatic combat-related wounds is associated with a hyper-inflammatory systemic response to injury., Level of Evidence: Prognostic Level II. See Instructions for Authors for a complete description of levels of evidence.

Background: The complications of bone-bridging amputations remain ill defined. The purpose of this study was to compare the early and intermediate-term complications leading to reoperation between the modified Burgess and modified Ertl tibiofibular synostosis in combat-related transtibial amputations., Methods: We conducted a retrospective review of consecutive, contemporaneous cohorts of thirty-seven modified Ertl bone-bridge and 100 modified Burgess combat-related transtibial amputations. The primary outcome measure was the need for reoperation following definitive closure., Results: At a mean follow-up of two years (range, nine to forty-eight months), there was a 53% overall reoperation rate. The overall complications included infection (34%), neuroma excision (18%), heterotopic ossification excision (15%), myodesis failure (4%), and scar revision (7%). A significantly higher rate of overall complications (p = 0.008) was noted in the bone-bridge group. Additionally, there was an increased rate of noninfectious complications in the bone-bridge group (p = 0.02). A positive selection bias was also noted for performing bone-bridge amputations late (p = 0.0002) and outside the zone of injury (p < 0.0001). Bone-bridge-specific complications occurred in 32% of the modified Ertl group. Delayed union or nonunion of the synostosis (11%) and implant-related complications (27%) predominated. Three bone bridges were ultimately removed., Conclusions: Reoperations were needed at a significantly greater rate overall and for noninfectious complications following bone-bridge synostosis compared with modified Burgess transtibial amputations. Additionally, despite the positive selection bias favoring the bridge synostosis cohort, infection rates were not lower in that group. Detailed patient counseling and careful patient selection are indicated prior to performing modified Ertl amputations, particularly in the absence of convincing evidence regarding objective functional benefits from the procedure.

Heterotopic ossification (HO) refers to the formation of mature lamellar bone in nonosseous tissue. In the setting of high-energy wartime extremity wounds, HO is expected to complicate up to 64% of patients, has a predilection for the residual limbs of amputees, and remains a significant source of disability. Although the inciting events and the definitive cell(s) of origin continue to remain elusive, animal models and human histology samples suggest that HO formation follows a predictable sequence of events culminating in endochondral ossification. Primary prophylaxis is not medically or logistically practical in most cases because patients have generally sustained massive wounds and are undergoing serial debridements during an intercontinental aeromedical evacuation. Surgical excision of symptomatic lesions is warranted only after an appropriate trial of conservative measures and is associated with low recurrence rates in appropriately selected patients. Future research regarding prognostication and defining the early molecular biology of ectopic bone may permit individualized prophylaxis and development of novel targeted therapies.

Background: Heterotopic ossification in the extremities remains a common complication in the setting of high-energy wartime trauma, particularly in blast-injured amputees and in those in whom the definitive amputation was performed within the zone of injury. The purposes of this cohort study were to report the experience of one major military medical center with high-energy wartime extremity wounds, to define the prevalence of heterotopic ossification in these patients, and to explore the relationship between heterotopic ossification and other potential independent predictors., Methods: We retrospectively reviewed the records and radiographs of all combat-wounded patients admitted to this institution between March 1, 2003, and December 31, 2006. Patients with a minimum of two months of radiographic follow-up who underwent at least one orthopaedic procedure on an extremity constituted our study group; those who underwent at least one orthopaedic procedure but had not had heterotopic ossification develop constituted the control group. Variables recorded for each study subject included age and sex, location and mechanism of injury, method(s) of fracture fixation, number of débridement procedures, duration of negative pressure therapy, location of heterotopic ossification, presence and severity of traumatic brain injury, and Injury Severity Scores., Results: During the study period, 1213 war-wounded patients were admitted. Of those patients, 243 (157 in the heterotopic ossification group and eighty-six controls) met the inclusion criteria. The observed rate of heterotopic ossification was 64.6%. A significant relationship was detected between heterotopic ossification and the presence (p = 0.006) and severity (p = 0.003) of a traumatic brain injury. Risk factors for the development of heterotopic ossification were found to be an age of less than thirty years (p = 0.007, odds ratio = 3.0), an amputation (p = 0.048, odds ratio = 2.9), multiple extremity injuries (p = 0.002, odds ratio = 3.9), and an Injury Severity Score of >or=16 (p = 0.02, odds ratio = 2.2)., Conclusions: The prevalence of heterotopic ossification in war-wounded patients is higher than that in civilian trauma. Although trends associated with local wound conditions were identified, the risk factors for the development of heterotopic ossification found in this study suggest that systemic causes predominate.