Aims. Acquired heterotopic ossification (HO) is a debilitating disease characterized by abnormal extraskeletal bone formation within soft-tissues after injury. The exact pathogenesis of HO remains unknown. It was reported that BRD4 may contribute to osteoblastic differentiation. The current study aims to determine the role of BRD4 in the pathogenesis of HO and whether it could be a potential target for HO therapy. Methods. Achilles tendon puncture (ATP) mouse model was performed on ten-week-old male C57BL/6J mice. One week after ATP procedure, the mice were given different treatments (e.g. JQ1, shMancr). Achilles tendon samples were collected five weeks after treatment for RNA-seq and real-time quantitative polymerase chain reaction (RT-qPCR) analysis; the legs were removed for micro-CT imaging and subsequent histology. Human bone marrow mesenchymal stem cells (hBMSCs) were isolated and purified bone marrow collected during surgeries by using density gradient centrifugation. After a series of interventions such as knockdown or overexpressing BRD4, Alizarin red staining, RT-qPCR, and Western Blot (Runx2, alkaline phosphatase (ALP), Osx) were performed on hBMSCs. Results. Overexpression of BRD4 enhanced while inhibition of Brd4 suppressed the osteogenic differentiation of hBMSCs in vitro. Overexpression of Brd4 increased the expression of mitotically associated long non-coding RNA (Mancr). Downregulation of Mancr suppressed the osteoinductive effect of BRD4. In vivo, inhibition of BRD4 by JQ1 significantly attenuated pathological bone formation in the ATP model (p = 0.001). Conclusion. BRD4 was found to be upregulated in HO and Brd4-Mancr-Runx2 signalling was involved in the modulation of new bone formation in HO. Cite this article: Bone Joint Res 2021;10(10):668–676

Aims. Dystrophic calcification (DC) is the abnormal appearance of calcified deposits in degenerating tissue, often associated with injury. Extensive DC can lead to heterotopic ossification (HO), a pathological condition of ectopic bone formation. The highest rate of HO was found in combat-related blast injuries, a polytrauma condition with severe muscle injury. It has been noted that the incidence of HO significantly increased in the residual limbs of combat-injured patients if the final amputation was performed within the zone of injury compared to that which was proximal to the zone of injury. While aggressive limb salvage strategies may maximize the function of the residual limb, they may increase the possibility of retaining non-viable muscle tissue inside the body. In this study, we hypothesized that residual dead muscle tissue at the zone of injury could promote HO formation. Methods. We tested the hypothesis by investigating the cellular and molecular consequences of implanting devitalized muscle tissue into mouse muscle pouch in the presence of muscle injury induced by cardiotoxin. Results. Our findings showed that the presence of devitalized muscle tissue could cause a systemic decrease in circulating transforming growth factor-beta 1 (TGF-β1), which promoted DC formation following muscle injury. We further demonstrated that suppression of TGF-β signalling promoted DC in vivo, and potentiated osteogenic differentiation of muscle-derived stromal cells in vitro. Conclusion. Taken together, these findings suggest that TGF-β1 may play a protective role in dead muscle tissue-induced DC, which is relevant to understanding the pathogenesis of post-traumatic HO. Cite this article: Bone Joint Res 2020;9(11):742–750

Aims. In recent conflicts, most injuries to the limbs are due to blasts resulting in a large number of lower limb amputations. These lead to heterotopic ossification (HO), phantom limb pain (PLP), and functional deficit. The mechanism of blast loading produces a combined fracture and amputation. Therefore, to study these conditions, in vivo models that replicate this combined effect are required. The aim of this study is to develop a preclinical model of blast-induced lower limb amputation. Methods. Cadaveric Sprague-Dawley rats’ left hindlimbs were exposed to blast waves of 7 to 13 bar burst pressures and 7.76 ms to 12.68 ms positive duration using a shock tube. Radiographs and dissection were used to identify the injuries. Results. Higher burst pressures of 13 and 12 bar caused multiple fractures at the hip, and the right and left limbs. Lowering the pressure to 10 bar eliminated hip fractures; however, the remaining fractures were not isolated to the left limb. Further reducing the pressure to 9 bar resulted in the desired isolated fracture of the left tibia with a dramatic reduction in the fractures to other sites. Conclusion. In this paper, a rodent blast injury model has been developed in the hindlimb of cadaveric rats that combines the blast and fracture in one insult, necessitating amputation. Experimental setup with 9 bar burst pressure and 9.13 ms positive duration created a fracture at the tibia with total reduction in non-targeted fractures, rendering 9 bar burst pressure suitable for translation to a survivable model to investigate blast injury-associated diseases. Cite this article: Bone Joint Res 2021;10(3):166–173

Heterotopic ossification (HO) is defined as aberrant bone formation in extraskeletal locations. In this process, local stromal cells of mesenchymal origin abnormally differentiate, resulting in pathologic cartilage and bone matrix deposition. However, the specific cell type and mechanisms beyond this process are not well understood, in part due to the heterogeneity of progenitor cells involved. Here, a combination of single cell RNA sequencing (scRNA-Seq) and lineage tracing, defined the extent to which synovial / tendon sheath progenitor cells contribute to HO. For this purpose, a Tppp3 (tubulin polymerization-promoting protein family member 3) inducible reporter model was used, in combination with either Scx (Scleraxis) or Pdgfra (Platelet derived growth factor receptor alpha) reporter animals. Both arthroplasty-induced and tendon injury-mouse experimental HO models were utilized. ScRNA-Seq of tendon-induced traumatic HO suggested that Tppp3 is a progenitor cell marker for either osteochondral or tendon or cells. After HO induction, Tppp3 reporter+ cell population expanded in number and contributed to cartilage and bone formation in tendon and joint-associated HO. Using double reporter animals, we found that both Pdgfra+Tppp3+ and Pdgfra+Tppp3- progenitor cells produced HO-associated cartilage. Finally, the examination of human samples showed a significant population of TPPP3+ cells overlapping with osteogenic markers in areas of HO. Overall, these results provide novel observations that peritenon and synovial progenitor cells undergo abnormal osteochondral differentiation and contribute to heterotopic bone formation after trauma

Aims. Calcium sulphate has traditionally been used as a filler of dead space arising during surgery. Various complications have been described following the use of Stimulan bio-absorbable calcium sulphate beads. This study is a prospective observational study to assess the safety profile of these beads when used in revision arthroplasty, comparing the complication rates with those reported in the literature. Methods. A total of 755 patients who underwent 456 revision total knee arthroplasties (TKA) and 299 revision total hip arthroplasties (THA), with a mean follow-up of 35 months (0 to 78) were included in the study. Results. A total of 32 patients (4.2%) had wound drainage, and this was higher with higher bead volumes and in McPherson grade C patients. There was also a significantly higher bead volume in the 41 patients who developed hypercalcaemia, two of which were symptomatic (p < 0.0001). A total of 13 patients (1.7%) had heterotopic ossification (HO). There was no statistically significant relationship between the development of HO and bead volume (p > 0.05). Conclusion. The strength of this study lies in the large number of patients and the detailed data collection, making it the most comprehensive report available in the literature on the use of calcium sulphate-based bone substitutes. Cite this article: R. Kallala, W. Edwin Harris, M. Ibrahim, M. Dipane, E. McPherson. Use of Stimulan absorbable calcium sulphate beads in revision lower limb arthroplasty: Safety profile and complication rates. Bone Joint Res 2018;7:570–579. DOI: 10.1302/2046-3758.710.BJR-2017-0319.R1

This meta analysis address the relationship between infection developing after total hip arthroplasty (THA) and heterotopic ossification (HO). To identify the gaps in available knowledge, we screened for full-length peer-reviewed research articles listed in PubMed, Embase, and Web of Science over the past 20 years. The following search terms and Boolean operators were used: heterotopic ossification AND infection AND (hip replacement OR hip arthroplasty). The search resulted in the identification of as few as 14 articles describing periprosthetic joint infection (PJI) and HO after THA. Data summarized from 6 studies suitable for further meta-analysis yielded a cumulative sample size of 753 observations, with 186 recorded events of HO. The pooled RR was estimated at 2.22 (95% CI: 1.00 to 4.91, p = 0.0497), suggesting a more than twofold risk of HO compared to the group without PJI. In conclusion, there is a clear association between a higher risk of HO and PJI. Basic research findings support the hypothesis that bacterial pathogen-associated molecular patterns (PAMPs) can lead to osteogenesis through a toll-like receptor (TLR) and nuclear factor kappa B (NF-κB) pathway in the course of HO development. Together, these results suggest that HO prophylaxis should always be prescribed in PJI after THA. Moreover, during revisions following THA for presumed non-septic reasons, the presence of HO warrants consideration for infection, as there is a potential heightened risk of pathologic ossification induced by PAMPs. Keywords: heterotopic ossification; total hip arthroplasty; total hip replacement; periprosthetic joint infection; bacteria. Authors Ł. Pulik and P. Łęgosz contributed equally to this work

Severe heterotopic ossification (grade III and IV) after contemporary total hip arthroplasty (THA) requiring excision is very uncommon. We performed a systematic review of the literature, and report a new case series with operative treatment after primary uncemented THA. A systematic review identified papers describing patients who had excision of heterotopic ossification (HO) after contemporary THA, defined as performed after 1988. Concepts of hip arthroplasty, heterotopic ossification, and surgical excision were searched in MEDLINE, Embase, and Scopus, from database inception to November 2022. Inclusion criteria were: articles that included specific patient data on grade of heterotopic ossification, operative procedure, and prophylaxis. Studies were screened for inclusion by two independent reviewers. Extracted data included demographic data, interval from index surgery to excision, clinical results, and complications. One surgeon performed reoperation for ankylosis of primary THA in three patients with severe pain and deformity. Seven case series or case report studies were included. There were 41 patients, with grade III or IV HO, that had excision, and in five patients, revision of a component was also performed. Perioperative prophylaxis was irradiation alone in 10 patients, irradiation and indomethacin in 10, and indomethacin alone in 21 patients. At a mean follow-up time of 14.8 months, definition of the results was not uniform, and range of motion was improved, but relief of pain was inconsistent. There was one dislocation, one gastrointestinal complication, and two recurrences. Treatment of the three patients, with wide excision of peri-articular bone, selective exchange of components, and peri-operative irradiation prophylaxis, was successful in improving motion and deformity. There is insufficient data on the treatment of severe symptomatic HO after contemporary THA. Prophylaxis with low-dose irradiation was successful to prevent recurrence. Multicenter studies will be needed to determine the optimum timing and prognosis for treatment

Heterotopic ossification (HO) is perhaps the single most significant obstacle to independence, functional mobility, and return to duty for combat-injured veterans of Operation Enduring Freedom and Operation Iraqi Freedom. Recent research into the cause(s) of HO has been driven by a markedly higher prevalence seen in these wounded warriors than encountered in previous wars or following civilian trauma. To that end, research in both civilian and military laboratories continues to shed light onto the complex mechanisms behind HO formation, including systemic and wound specific factors, cell lineage, and neurogenic inflammation. Of particular interest, non-invasive in vivo testing using Raman spectroscopy may become a feasible modality for early detection, and a wound-specific model designed to detect the early gene transcript signatures associated with HO is being tested. Through a combined effort, the goals of early detection, risk stratification, and development of novel systemic and local prophylaxis may soon be attainable.

Heterotopic ossification (HO) is a well-known complication of traumatic elbow injuries. The reported rates of post-traumatic HO formation vary from less than 5% with simple elbow dislocations, to greater than 50% in complex fracture-dislocations. Previous studies have identified fracture-dislocations, delayed surgical intervention, and terrible triad injuries as risk factors for HO formation. There is, however, a paucity of literature regarding the accuracy of diagnosing post-traumatic elbow HO. Therefore, the purpose of our study was to determine the inter-rater reliability of HO diagnosis using standard radiographs of the elbow at 52 weeks post-injury, as well as to report on the rate of mature compared with immature HO. We hypothesized inter-rater reliability would be poor among raters for HO formation. Prospectively collected data from a large clinical trial was reviewed by three independent reviewers (one senior orthopedic resident, one senior radiology resident, and one expert upper extremity orthopedic surgeon). Each reviewer examined anonymized 52-week post-injury radiographs of the elbow and recorded: 1. the presence or absence of HO, 2. the location of HO, 3. the size of the HO (in cm, if present), and 4. the maturity of the HO formation. Maturity was defined by consensus prior to image review and defined as an area of well-defined cortical and medullary bone outside the cortical borders of the humerus, ulna, or radius. Immature lesions were defined as an area of punctate calcification with an ill-defined cloud-like density outside the cortical borders of the humerus, ulna or radius. Data were collected using a standardized online data collection form (CognizantMD, Toronto, ON, CA). Inter-rater reliability was calculated using Fleiss’ Kappa statistic and a multivariate logistic regression analysis was performed to identify risk factors for HO formation in general, as well as mature HO at 52 weeks post injury. Statistical analysis was performed using RStudio (version1.4, RStudio, Boston, MA, USA). A total of 79 radiographs at the 52-week follow-up were reviewed (54% male, mean age 50, age SD 14, 52% operatively treated). Inter-rater reliability using Fleiss’ Kappa was k= 0.571 (p = 0.0004) indicating moderate inter-rater reliability among the three reviewers. The rate of immature HO at 52 weeks was 56%. The multivariate logistic regression analysis identified male sex as a significant risk factor for HO development (OR 5.29, 1.55-20.59 CI, p = 0.011), but not for HO maturity at 52 weeks. Age, time to surgery, and operative intervention were not found to be significant predictors for either HO formation or maturity of the lesion in this cohort. Our study demonstrates moderate inter-rater reliability in determining the presence of HO at 52 weeks post-elbow injury. There was a high rate (56%) of immature HO at 52-week follow-up. We also report the finding of male sex as a significant risk factor for post traumatic HO development. Future research directions could include investigation into possible male predominance for traumatic HO formation, as well as improving inter-rater reliability through developing a standardized and validated classification system for reporting the radiographic features of HO formation around the elbow

Abstract. Objectives. The term heterotopic ossification (HO) describes lamellar bone formation within soft tissues following injury. A genome-wide scan of patients after hip arthroplasty has identified that variation within the lncRNA CASC20 is associated with HO susceptibility. Previous findings in our lab have demonstrated upregulation of CASC20 during BMP2-induced osteodifferentiation of adipose-derived stem cells (hMAD) alongside osteodifferentiation markers, RUNX2 and OSX. We hypothesize that CASC20 is a novel regulator of bone formation and aim to investigate CASC20 function in bone formation. Methods. 1) We used miRanda prediction algorithm and the ENCORI database to respectively predict which miRNAs CASC20 interacts with and to select for experimentally validated miRNAs. 2) We characterized the expression and functional role of CASC20-interacting miRNAs by respectively analyzing publicly available datasets (GSE107279 and pubmed.ncbi.nlm.nih.gov/26175215/) and by using Gene Ontology (GO) analysis. 3) We overexpressed CASC20 in hMAD using a lentiviral system and tested the effect of CASC20 overexpression in osteodifferentiation and expression of putative CASC20-interacting miRNAs. Results. 1) We identified 64 experimentally validated miRNAs that are predicted to interact with CASC20. 2) GO analysis revealed that the most frequently targeted molecular functions included SMADs, MAPKK and other kinase activities known to play a central role in osteo and chondrogenesis. We found 10 miRNAs including hsa-miR-485-3p that demonstrated down-regulation in both osteo- and chondrogenesis. 3) We found that CASC20-overexpression augmented the osteodifferentiation of hMAD measured in mineralization using Alizarin Red S. CASC20 overexpression increased the expression of osteogenic marker ALP and decreased the expression of hsa-miR-485-3p. Conclusion. Here we show how CASC20 may regulate bone formation by acting as a competitive endogenous RNA (ceRNA). We are currently using CASC20 overexpression model in osteo- and chondrogenesis, and testing CASC20-miRNA interaction to establish the underlying mechanism for the observed associations

Introduction and Objective. Interest for direct anterior approach (DAA) in hip hemiarthroplasty (HHA) has greatly increased in recent years, however which is the best surgical approach in hip replacement treating femoral neck fractures (FNFs) is already unclear. The aim of this study is to perform a radiographic and perioperative complications analysis by comparing the direct anterior approach (DAA) with the direct lateral approach (DLA) in patients treated with hemiarthroplasty for FNFs. Materials and Methods. Patients with FNFs surgically treated between 2016–2020 with HHA were enrolled. The radiographical outcomes of DAA and DLA are compared. Several peri-operative and post-operative variables were evaluated: mean surgery time, complications as periprosthetic fractures or episodes of dislocation, the average of post-operative diaphyseal filling of the stem (Canal Fill Index, CFI), the extent of heterotopic ossification (HO) (simplified Broker classification) and metadiaphiseal bone loss (Paprosky classification) within one year from surgery. Results. 86 patients underwent HHA by DAA and 80 patients by DLA. The two groups are qualitatively comparable. No statistically significate differences were showed in all variables analyzed (p>0.05). The average of surgical time of DAA were 61 minutes compared to 67 of DLA. No differences were showed in the post-operative CFI (DAA 0.71 ± 6.1; DLA 0.76 ± 13.5), the extent of the HO (DAA 79.07% low; DLA 75% low) and metadiaphiseal bone loss (DAA Grade I 91.86%; DLA Grade I 93.75%). Regarding perioperative complications, we have discovered only one periprosthetic fracture each group. Although there was no statistically significant difference, we highlighted a higher number of dislocations in the group of DLA (2 episodes vs no one). Conclusions. In this study we have shown that the DAA is an adequate surgical choice comparing with the classical DLA for FNFs treated with HHA. The analysis of our radiographic parameters and perioperative complications have not shown a significant difference between the two surgical approach. This study is limited by a purely radiographic analysis without addition of clinical parameters

Heterotopic Ossification (HO) is a known complication that can arise after total elbow arthroplasty (TEA). In most cases it is asymptomatic, however, in some patients it can limit range of motion and lead to poor outcomes. The objective of this review was to assess and report incidence, risk factors, prophylaxis, and management of HO after TEA. A systematic search was conducted using MEDLINE, EMBASE, and PubMed to retrieve all relevant studies evaluating occurrence of HO after TEA. The search was performed in duplicate and a quality assessment was performed of all included studies. A total of 1907 studies were retrieved of which 45 studies were included involving 2256 TEA patients. HO was radiographically present in 10% of patients and was symptomatic in 3%. Less than 1% of patients went on to surgical excision of HO, with outcomes following surgery reported as good or excellent as assessed by range of motion and Mayo Elbow Performance Scores (MEPS). TEA due to ankylosis, primary osteoarthritis, and posttraumatic arthritis are more likely to develop symptomatic HO. HO is an uncommon complication following TEA with the majority of patients developing HO being asymptomatic and requiring no surgical management. Routine HO prophylaxis for TEA is not supported by the literature. The effectiveness of prophylaxis in high risk patients is uncertain and future studies are required to clarify its usefulness. The strength of these conclusions are limited by inconsistent reporting in the available literature

Electron Microscopy and Synchrotron analysis of Heterotopic Ossification (HO) from blast-related amputees' has shown that HO is bone with a disorganised structure and altered remodelling. This research performs mechanical testing of HO to understand its biomechanical properties in an attempt to create an accurate model to predict its morphological appearance. The hypothesis of this work is that HO is mechanically mediated in its formation. Synchrotron mechanical analysis of HO samples was performed to measure Young's modulus, ultimate strength and density distribution. A novel algorithm based on Wolf's law was implemented in a Finite Element (FE) analysis model of HO to take into account the differing mechanical and biological properties measured and the presence of HO outside the skeletal system. An HO modeling factor, which considers boundary conditions, and regulates recruitment of the soft tissue into bone formation, results in a re-creatable formation of HO within the soft tissues, comparable to the appearance of HO seen in military amputees. The results and model demonstrates that certain types of HO are under the control of endogenous and exogenous mechanical stimulus. HO can thus be mechanically exploited in the casualty management and rehabilitation process to achieve better clinical outcomes

Heterotopic ossification (HO) is a relatively common complication of total hip arthroplasty (THA), but is rather rare after total knee arthroplasty (TKA). In both cases, it is usually asymptomatic and is most commonly identified as an incidental finding on post-operative radiographs. However, in severe cases it can result in decreased range of motion and pain. There are several risk factors that have been shown to be associated with development of HO. These include male gender, ceramic-on-ceramic bearings, prior stroke, and hypertrophic osteoarthritis. Heterotopic ossification can be treated with physical therapy during the maturation phase (12 to 24 weeks), but surgical intervention is required if the stiffness persists. All heterotopic bone should be excised with careful attention to neurovascular structures. Patients should begin prophylaxis following HO excision and prior to any subsequent surgeries. Heterotopic ossification prophylaxis consists of NSAIDs, radiotherapy, or a combination of both modalities. These therapies are not without complications, therefore, routine administration of prophylaxis for all patients is not indicated. Several new pathways of inhibiting extra-skeletal bone formation in HO are under investigation (retinoid acid receptor agonists, apyrase, and LDN-193189). Future studies should focus on identification of patients at risk for HO as well as better therapeutic options with less side effects

The incidence of clinically significant (Brooker stage 3–4) heterotopic ossification (HO) after THA is 3–7%. Risk factors include male gender, old age, a history of HO, Paget's disease, post-traumatic arthritis, osteonecrosis and rheumatoid arthritis. Prophylaxis for high-risk patients consists of 1) radiotherapy given as one dose of 7–8 Gy either pre-operatively (<4 hours) or post-operatively (within 72 hours) or 2) NSAIDS. Treatment of clinically significant HO includes intensive physiotherapy during the maturation phase of the disease and surgical excision in conjunction with a combination of radiotherapy and indomethacin once the HO has matured. Less invasive surgical approaches may be associated with a reduced incidence of HO

Heterotopic ossification (HO) is lamellar bone formation that occurs within tissues that do not normally have properties of ossification. The pathoaetiology of HO is poorly understood. We conducted a genome wide association study to better understand the genetic architecture of HO. 891 patients of European descent (410 HO cases) following THA for primary osteoarthritis were recruited from the UK. HO was assessed from plain AP radiographs of the pelvis. Genomic DNA was extracted, genotyped using the Illumina 610 beadchip and referenced using the 1000 Genome Project panel. HO susceptibility case-control analysis and an evaluation of disease severity in those with HO was undertaken using SNPTESTv2.3.0 on>10 million variants. We tested variants most strongly associated with HO in an independent UK THA replication cohort comprising 209 cases and 211 controls. The datasets were meta-analysed using PLINK. In the discovery cohort 70 signals with an index variant at p<9×10–5 were suggestively associated with HO susceptibility. The strongest signal lay just downstream of the gene ARHGAP18 (rs59084763, effect allele frequency (EAF) 0.19, OR1.87 [1.48–2.38], p=2.48×10–8), the second strongest signal lay within the long non-coding (LNC) RNA gene CASC20 (rs11699612, EAF 0.25, OR1.73 [1.1.40–2.16, p=9.3×10–8). In the discovery cohort 73 signals with an index variant at p<9×10–5 were associated with HO severity. At replication, 12 of the leading 14 susceptibility signals showed a concordant direction of allelic effect and 5 replicated at nominal significance. Following meta-analysis, the lead replicating susceptibility signal was the CASC20 variant rs11699612 (p=2.71×10–11). We identify consistent replicating association of variation within the LNC RNA CASC20 with HO susceptibility after THA. Although the function of CASC20 is currently unknown, possible mechanisms include transcriptional, post-transcriptional and epigenetic regulation of downstream target genes. The work presented here provides new avenues for the development of novel predictive and therapeutic approaches towards HO

The incidence of clinically significant (Brooker stage 3–4) heterotopic ossification (HO) after THA is 3–7%. Risk factors include male gender, old age, a history of HO, Paget's disease, post-traumatic arthritis, osteonecrosis and rheumatoid arthritis. Prophylaxis for high-risk patients consists of 1) radiotherapy given as one dose of 7–8 Gy either pre-operatively (< 4 hours) or post-operatively (within 72 hours) or 2) NSAIDS. Treatment of clinically significant HO includes intensive physiotherapy during the maturation phase of the disease and surgical excision in conjunction with a combination of radiotherapy and indomethacin once the HO has matured. Less invasive surgical approaches may be associated with a reduced incidence of HO

Previous reports of the prevalence of Heterotopic Ossification (HO) in limbs from UK blast-related amputees from Afghanistan, is demonstrated to be 57.1%. With the end of UK military operations in Afghanistan in 2014 the aim of this study is establish the rate of HO, assess causality demographics and ascertain risk factors for the formation of HO during the entire period of operations in Afghanistan. Military databases, case notes and radiographs were scrutinised to quantify and qualify the prevalence and risk factors for the formation of HO. 256 servicemen sustained 398 military trauma related amputations. The overall prevalence of HO was 65.9%. Significant (p<0.05) risks identified for the formation of HO included a blast mechanism of injury, a zone of injury the same as the subsequent amputation, and an increased number of debridements prior to closure. Positive correlation existed between the number of amputations and the presence and grade of HO (p=0.04). HO presents clinical problems to military blast injury patient populations. This study demonstrates that both a blast mechanism of injury and an increased injury load are key factors in the increased prevalence of HO seen in military trauma

Heterotopic ossification (HO) is the formation of bone in extraskeletal sites. It is a major problem for combat-related casualties with 64% of such patients showing radiological evidence of the disease. Of these, 19% require surgical excision. Current prophylaxis is problematic due to poor efficacy and unsuitability in a military setting. Our novel anti-HO strategy is to use an inorganic reagent to inhibit the deposition of HA and disperse any pre-formed mineral. Literature review identified several potentially effective agents. These were tested for their ability to disperse solid monoliths of HA. In addition, a standard HA synthetic reaction was performed in the presence of each agent to establish their inhibiting activity. One reagent (a condensed phosphate) dispersed a solid monolith of HA by 38% (mass loss) over 30 days. This reagent was also shown to inhibit HA crystal synthesis yield by 28%. Early work on a hydrogel delivery system has produced favourable results. These preliminary data demonstrate proof of concept that HA may be dispersed and its formation inhibited by a non-toxic polyphosphate. This work will form the justification for development into in vitro osteogenic cell culture models and animal HO models

Background. Heterotopic ossification (HO) is lamellar bone formation in the soft tissues following trauma or joint replacement for osteoarthritis (OA). A genome wide association study of HO patients after total hip arthroplasty for OA has identified Kinesin Family Member 26B (KIF26B) as a gene associated with HO severity. KIF26B has previously been associated with HO in mice. Hypothesis and aims: We hypothesised that Kif26b regulates the osteogenic trans-differentiation of myoblasts; a possible mechanism of HO. Using an in vitro model, we wished to establish whether Kif26b is involved in HO formation and to explore the molecular mechanism. Methods. We developed CRISPR/Cas9 mediated Kif26b knockout (KO) C2C12 myoblasts. Wild type (WT) and KO cells were transdifferentiated towards an osteogenic lineage using BMP-2 for 24 days. The effect of Kif26b KO on mineralisation was quantified by calcium staining. The mean difference (±SEM) in gene expression between WT and KO lines was compared with ANOVA. Results. qPCR and western blotting confirmed Kif26b knockout. Kif26b deficient cells produced substantially less mineral versus WT in response to BMP-2 (34.71% ±3.62%, n=12, P<0.0001). At day 8 of osteogenic differentiation, loss of Kif26b abrogated Osterix (113.6 ±6.781 n=5, P<0.0001), Osteocalcin (737.9 ±84.25, n=5, P<0.0001) and Alkaline phosphatase (6989 ±365.7, n=5, P<0.0001) expression, and down regulated Runx2 (2.725 ±0.7724, n=5, P<0.0052) and Collagen type I (7.25 ±1.154, n=5, P<0.0001) expression relative to WT. The knockout cells also appeared morphologically different. Compared to WT, the Kif26b KO cells displayed a less osteoblast-like morphology during transdifferentiation. Conclusion. Our findings demonstrate an undescribed function for Kif26b as a critical regulator of pathological ossification, with a putative role in HO pathogenesis after THA