Undifferentiated pleomorphic sarcoma (UPS) is one of the most common and aggressive adult soft tissue sarcomas (STS). Once metastatic, UPS is rapidly fatal. Most STS, including UPS, are resistant to conventional immunotherapies as these tumours have low numbers of spontaneous tumour infiltrating lymphocytes (TILs) and are densely populated with immune suppressive macrophages. Intra-tumoural activation of the STimulator of INterferon Genes (STING) pathway is a novel immunotherapeutic strategy to recruit anti-tumour TILs into the tumour microenvironment. In a murine model of UPS, we have demonstrated that intra-tumoural injection of a murine-specific STING agonist, DMXAA, results in profound immune mediated tumour clearance. Recently, molecules capable of activating both human and mouse STING pathways have been developed. In pursuit of clinically relevant therapeutic opportunities, the purpose of this study is to evaluate the anti-tumour potential of two agonists of the human and murine STING receptors: ADU-S100 and MSA-2 as monotherapies and in combination with the immune checkpoint inhibitor, anti-PD1 in a murine model of UPS.

Immune competent mice were engrafted with murine UPS cells in the hindlimb muscle. Once palpable, mice in the monotherapy group were treated with a single intra-tumoural dose of 1) ADU-S100 or 2) MSA-2 or 3) DMXAA. In additional experimental groups, mice were treated with the different STING agonists and monoclonal anti-PD1. Tumour volume measurements and tumour bioluminescence were measured over time. To quantify dynamic changes in immune populations and in the tumour immune microenvironment, STING treated UPS tumours were evaluated using flow cytometry and mRNA quantification at various timepoints after therapy.

DMXAA monotherapy produced complete tumour eradication in 50% of mice, whereas both ADU-S100 or MSA-2 monotherapy only extended survival but did not result in complete tumour clearance. Flow cytometry and transcriptional profiling of tumours at multiple timepoints post-treatment showed similar inflammatory changes and increased TILs numbers across all STING agonists. The addition of anti-PD1 treatment to STING therapy significantly extended survival times with both ADU-S100 and MSA-2, and resulted in 14% complete tumour clearance with ADU-S100. No complete survivors were observed with MSA-2-anti-PD1 combinations therapy.

STING activation is a promising immunotherapeutic strategy for UPS. Recently developed human STING agonists are not as effective as DMXAA despite similar immunologic responses to treatment. STING and anti-PD-1 treatment were therapeutically synergistic for both human STING agonists. These results justify further research around STING activation as a therapeutic modality for STS. DMXAA may possess additional off-target therapeutic properties beyond STING activation which warrants further investigation. Elucidating these differences may be critical to further optimize STING therapy for human STS.

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.

Soft tissue sarcomas (STS) are rare, aggressive malignancies derived from connective tissues such as muscle and fat. Undifferentiated pleomorphic sarcoma (UPS) is one of the most common STS in adults. UPS is an aggressive, highly metastatic sarcoma, and is resistant to chemotherapy. New therapies for UPS are desperately needed. STS have an immune desert tumour immune microenvironment (TIME), characterized by a paucity of tumour infiltrating lymphocytes and subsequent resistance to immunotherapies such as immune checkpoint inhibitors. Strategies capable of creating an immune-rich, inflamed TIME may improve immunotherapy efficacies for sarcoma. Activation of the STING (stimulator of interferon genes) receptor can induce potent innate and adaptive immune responses within immunogenic solid tumours. However, this approach has never been attempted in immune-inert sarcomas.

Purpose: To determine the therapeutic anti-tumour effects of STING activation in UPS tumours.

We have developed an inducible, immune-competent mouse model of UPS. We evaluated intra-tumoural injection of the murine STING receptor agonist, DMXAA, into UPS-bearing immune-competent mice. DMXAA was injected into palpable UPS tumours of the hindlimb. Tumour volume and bioluminescence imaging was recorded bi-weekly. DMXAA treated UPS tumours were also evaluated for necrosis and immune infiltration at defined time points.

UPS tumours developed necrosis and lymphocytic infiltration 72 hours after DMXAA treatment. A single intra-tumoural dose of DMXAA into UPS tumours resulted in durable cure in 50% of mice. All survivors rejected a re-challenge of the UPS tumours in both the contralateral hindlimb and lung, suggesting adaptive immunity. The therapeutic effects of DMXAA were mitigated in lymphocyte deficient Rag2 knockout mice.

STING therapy is a promising immunotherapeutic opportunity for immune-inert sarcomas. Our data warrants further preclinical investigations in other sarcoma models and in combination with other immune-based therapies.

Impaired bone healing biology secondary to soft tissue deficits and chemotherapy contribute to non-union, fracture and infection following limb salvage surgery in Osteosarcoma patients. Approved bone healing augments such as recombinant human bone morphogenetic protein-2 (rhBMP-2) have great potential to mitigate these complications. rhBMP-2 use in sarcoma surgery is limited, however, due to concerns of pro-oncogenic signalling within the tumour resection bed. To the contrary, recent pre-clinical studies demonstrate that BMP-2 may induce Osteosarcoma differentiation and limit tumour growth. Further pre-clinical studies evaluating the oncologic influences of BMP-2 in Osteosarcoma are needed. The purpose of this study is to evaluate how BMP-2 signalling affects Osteosarcoma cell proliferation and metastasis in an active tumour bed.

Two Osteosarcoma cell lines (143b and SaOS-2) were assessed for proliferative capacity and invasion. 143b and SaOS-2 cells were engineered to upregulate BMP-2. In vitro proliferation was assessed using a cell viability assay, motility was assessed with a scratch wound healing assay, and degree of osteoblastic differentiation was assessed using qRT-PCR of Osteoblastic markers (CTGF, ALP, Runx-2 and Osx). For in vivo evaluation, Osteosarcoma cells were injected into the intramedullary proximal tibia of immunocompromised (NOD-SCID) mice and local tumour growth and metastases were assessed using weekly bioluminescence imaging (BLI) and tumour volume measurements for 4–6 weeks. At the experimental end point we assessed radiographic tumour burden using ex-vivo micro-CT, as well as tibial and pulmonary gross and histologic pathology.

SaOS-2 was more differentiated than 143b, with increased expression of Runx-2 (p = 0.009), Osx (p = 0.004) and ALP (p = 0.035). BMP-2 upregulation did not stimulate an osteoblast differentiation response in 143b, but stimulated an increase in Osx expression in SaOS-2 (p = 0.002). BMP-2 upregulation in 143b cells resulted in increased proliferation in vitro (p = 0.014), faster in vitro wound healing (p = 0.03), significantly increased tumour volume (p = 0.001) with enhanced osteolysis detected on micro-CT, but did not affect rates of lung metastasis (67% vs. 71%, BMP-2 vs. Control). BMP-2 over-expression in SaOS-2 cells reduced in vitro proliferation when grown in partial osteogenic-differentiation media (p < 0.001), had no effect on in vitro wound healing (p = 0.28), reduced in vivo SaOS-2 tumour burden at 6 weeks (photon counts, p < 0.0001), decreased tumour-associated matrix deposition as assessed by trabecular thickness (p = 0.02), and did not affect rates of lung metastasis (0% vs. 0%).

Our results indicate BMP-2 signalling incites a proliferative effect on a poorly differentiated Osteosarcoma cell line (143b), but conditionally reduces proliferative capacity and induces a partial differentiation response in a moderately-differentiated Osteosarcoma cell line (SaOS-2). This dichotomous effect may be due to the inherent ability for Osteosarcoma cells to undergo BMP-2 mediated terminal differentiation. Importantly, these results do not support the clinical application of BMP-2 in Osteosarcoma limb salvage surgery due to the potential for stimulating growth of poorly differentiated Osteosarcoma cells within the tumour bed. Additional studies assessing the effects of BMP-2 in an immune-competent mouse model are ongoing.

Impaired bone healing biology secondary to soft tissue deficits and chemotherapy contribute to non-union, fracture and infection following limb salvage surgery in Osteosarcoma patients. Approved bone healing augments such as recombinant human bone morphogenetic protein-2 (rhBMP-2) have great potential to mitigate these complications. rhBMP-2 use in sarcoma surgery is limited, however, due to concerns of pro-oncogenic signalling within the tumour resection bed. To the contrary, recent pre-clinical studies demonstrate that BMP-2 may induce Osteosarcoma differentiation and limit tumour growth. Further pre-clinical studies evaluating the oncologic influences of BMP-2 in Osteosarcoma are needed. The purpose of this study is to evaluate how BMP-2 signalling affects Osteosarcoma cell proliferation and metastasis in an active tumour bed.

Two Osteosarcoma cell lines (143b and SaOS-2) were assessed for proliferative capacity and invasion. 143b and SaOS-2 cells were engineered to upregulate BMP-2. In vitro proliferation was assessed using a cell viability assay, motility was assessed with a scratch wound healing assay, and degree of osteoblastic differentiation was assessed using qRT-PCR of Osteoblastic markers (CTGF, ALP, Runx-2 and Osx). For in vivo evaluation, Osteosarcoma cells were injected into the intramedullary proximal tibia of immunocompromised (NOD-SCID) mice and local tumour growth and metastases were assessed using weekly bioluminescence imaging and tumour volume measurements for 4–6 weeks. At the experimental end point we assessed radiographic tumour burden using ex-vivo micro-CT, as well as tibial and pulmonary gross and histologic pathology.

SaOS-2 was more differentiated than 143b, with significantly increased expression of the Osteoblast markers Osx (p = 0.004) and ALP (p = 0.035). BMP-2 upregulation did not stimulate an osteoblast differentiation response in 143b, but stimulated an increase in Osx expression in SaOS-2 (p = 0.002). BMP-2 upregulation in 143b cells resulted in increased proliferation in vitro (p = 0.014), faster in vitro wound healing (p = 0.03), significantly increased tumour volume (p = 0.001) with enhanced osteolysis detected on micro-CT, but did not affect rates of lung metastasis (67% vs. 71%, BMP-2 vs. Control). BMP-2 over-expression in SaOS-2 cells reduced in vitro proliferation when grown in osteogenic-differentiation media (p < 0.001), had no effect on in vitro wound healing (p = 0.28), reduced in vivo SaOS-2 tumour burden at 6 weeks (photon counts, p < 0.0001), decreased tumour-associated matrix deposition as assessed by trabecular thickness (p = 0.02), but did not affect rates of lung metastasis (0% vs. 0%).

Our results indicate BMP-2 signalling incites a proliferative effect on a poorly differentiated Osteosarcoma cell line (143b), but conditionally reduces proliferative capacity and induces a partial differentiation response in a moderately-differentiated Osteosarcoma cell line (SaOS-2). This dichotomous effect may be due to the inherent ability for Osteosarcoma cells to undergo BMP-2 mediated terminal differentiation. Importantly, these results do not support the clinical application of BMP-2 in Osteosarcoma limb salvage surgery due to the potential for stimulating growth of poorly differentiated Osteosarcoma cells within the tumour bed. Additional studies assessing the effects of BMP-2 in an immune-competent mouse model are ongoing.

Soft tissue sarcomas (STS) have not demonstrated favourable clinical responses to emerging immunotherapies such as checkpoint inhibitors. Studies in carcinomas and melanoma have demonstrated that tumours lacking T-cell infiltrates are associated with poor responses to immunotherapies. It is postulated that STS lack tumour asscoiated lymphocytes which renders these tumours insensitive to checkpoint inhibitors. Our objective was to develop a novel syngeneic mouse model of STS and characterize the immune phenotype of these tumours. Additionally, we sought to evaluate the therapeutic responses of these sarcomas to checkpoint inhibitors and a Type I interferon agonist.

K-ras mutagenesis and p53 deletion was induced using a Lenti-Cre-recombinase injection into the hindlimb of 3 week old C57BL/6 mice. Tumours were harvested and characterized using standard histopathology techniques and whole trascriptome sequencing (RNAseq). Full body necrospy and histopathology was performed to identify metastases. Flow cytometry and immunohistochemistry was used to evaluate tumour immune phenotypes. Tumours were implanted into syngeneic C57BL/6 mice and the therapeutic responses to anti-CTLA4, anti-PD1 and DMXAA (Type I interferon agonist) were performed. Tumour responses were evaluated using bioluminescent imaging and caliper measurements.

Soft tissue sarcomas developed in mice within 2–3 months of Lenti-Cre injection with 90% penetrance. Histologic analyses of tumours was consistent with a high-grade myogenic sarcoma characterized by smooth muscle actin, Desmin and Myogenin D positive immunostaining. Using crossplatform normalization protocols, geneexpression signatures of the mouse tumours most closely correlated with human undifferentiated pleomorphic sarcoma (UPS). Collectively, gene expression signatures of this murine sarcoma correlated with all muscle-derived human sarcomas (ERMS, ARMS, Synovial sarcoma, UPS). No lung or other visceral metastases were observed in all mice who developed spontaneous tumours. Immune phenotyping demonstrated a paucity of tumour-infiltrating lymphocytes (TILs, (TAMs). 50% of identified TILs in these murine sarcomas expressed PD-1, yet tumours were not responsive to anti-PD1 therapy or anti-CTLA4 therapy. A single intra tumoural (i.t.) injection of the Type I interferon agonist, DMXAA resulted in 80–90% tumour necrosis 72 hrs post-injection, decreased tumour viability up to 2 weeks post-injection and a marked infiltration of CD8+ T-cells and anitgen presenting dendritic cells and macrophages. Additional longitudinal experiments demonstrate a sustained and progressive anti-tumour effect in 83% (5/6) mice up to 6weeks following a single i.t. injection of DMXAA. All control treated mice (6/6) reached humane endpoint within 14 days. At 3 months post-DMXAA treatment, 4/6 mice were free of disease. We re-injected UPS tumours into these mice and tumours did not grow, suggesting abscopal effects after DMXAA treatment of primary tumours.

We have characterized a new orthotopic and syngeneic mouse model of a myogenic soft tissue sarcoma. Like most human STS sub-types, these tumours have an immune inert tumour microenvironment and are not sensitive to checkpoint inhibitors. This model, syngeneic to C56BL/6 mice will enable future opportunities to investigate how various branches of the immune system can be targetted or manipulated to unearth new immunotherapeutic strategies for sarcoma. Using this model we have demonstrated that a single, intra-tumoural injection of a Type I interferon agonist can result in anti-tumour effects, recruit cytotoxic lymphocytes and antigen presenting cells with into the the tumour microenvironment. Abscopal tumour rejection after DMXAA treatement suggest adaptive T-cell responses against UPS are active in this model. Future work is needed to determine if upregulation of Type I inferferon pathways can be used as a therapeutic strategy for sarcoma or as a sensitization strategy for checkpoint inhibitors.

Total Elbow Arthroplasty (TEA) is a procedure to treat a number of conditions including rheumatoid arthritis (RA), post-traumatic arthritis, and osteoarthritis. To date, there has been minimal literature published on the Latitude since its release in 2001. There is one study reporting outcomes from the Latitude, a German study published in 2010. The purpose of this study was to analyse outcomes from primary Latitude TEAs.

We performed a retrospective case series of 23 TEAs performed on 20 patients. 6 patients required revision surgery and were not included in the analysis. One patient was lost to follow up, resulting in 17 patients included for ROM analysis. All patients received Latitude TEA through a posterior approach and underwent a standard rehab protocol. 11 Patients were recalled at least two years post-op and were administered DASH and MAYO questionnaires. Complications such as triceps insufficiency, ulnar nerve dysfunction, infection, and aseptic loosening were recorded. Outcomes were compared using the Wilcoxon Signed-Rank test in STATA. Immediate post-op radiographs and patients most recent radiographs were analysed by a blinded upper-extremity surgeon not involved in the initial operation and analysed for loosening and implant malpostioning.

Mean follow up was 4.8 years (range 2.6–7.5 years). Analysis of 17 TEAs in 16 patients revealed no difference in pre-operative ROM and post-operative ROM for flexion (121°±20 vs 129°±16, p=0.13) extension (40°±27 vs 27°±15, p=0.19), pronation (73°±13 vs 75°±24, p=0.55) or supination (64°±22 vs 68°±14, p=0.52). Patients who underwent TEA for RA had a significant improvement in flexion (121°±15 vs 135°±10, p<0.02). There was a statistically significant improvement in flexion-extension arc post-operatively (101°±28) compared to pre-operative scores (83±23 degrees, p<0.02). DASH and MAYO scores were calculated from 11elbows in 11 non-revision patients able to return for examination. The average MAYO score was 87.9 with nine patients in the “excellent” category, two patients in the “good” category, one patient in the “fair” category, and one in the “poor” category. The average DASH score was 32.9. Two patients underwent revision for periprosthetic fractures, two patients underwent revision for infection, one underwent revision for aseptic loosening and two for radial head dissociation (rate of 30%).

This is one of the first studies examining the outcomes of the Latitude TEA. This retrospective case series demonstrates that the Latitude TEA has promising outcomes with respect to improving patient pain and functioning as assessed by the MAYO. Treatment using the Latitude TEA results in favorable functional outcomes for a majority of patients and offers an improvement in flexion-extension arc. Furthermore, our results are comparable to the MAYO scores reported by other studies analysing different prosthesis designs. The complication rate in our series was comparable to published rates of 20–40%.

Summary

Previous work in a rabbit model of post-traumatic joint contractures shows that the mast cell stabilizer ketotifen decreases contracture severity. We show here that ketotifen decreases collagen gel contraction mediated by rabbit joint capsule fibroblasts when mast cells are present.

Purpose: To determine if mast cell activity is vital to the induction of joint capsule fibrosis and contracture formation in a rabbit model of posttraumatic joint contracture.

Method: To reproducibly induce joint contractures, we used a model of surgical injury and immobilization of the knee in skeletally mature New Zealand white rabbits. Four animals groups were studied: a non-operative control group (CON), an operative contracture group (ORC) and two-operative groups treated with a mast cell stabilizer, Ketotifen fumarate at doses of 0.5mg/kg (KF0.5) and 1.0mg/kg (KF1.0) twice daily subcutaneously, respectively. Animals were sacrificed after 8 weeks of immobilization. Flexion contractures (biomechanics), cellular counts of myofibroblasts and mast cells within the joint capsule (immunohistochemistry) and the joint capsule protein expression of TGF-β1, collagen I and III were quantified (western blots). Biomechanical data was interpreted using a linear regression analysis of repeated measures and an ANOVA analysis of variance was used for molecular data. Significance was defined at p< 0.05 for all statistical tests.

Results: Flexion contractures were most severe in the ORC group and treatment with Ketotifen (both KF0.5 and KF1.0) significantly reduced contracture severity by 52% and 42%, respectively (p< 0.03). Joint capsule myofibroblast and mast cell hyperplasia was a prominent feature of the more severely contracted ORC group and myofibroblast and mast cell numbers were dramatically reduced in both Ketotifen groups (p< 0.001). The expression of TGF-β1 and collagen I was also increased in the ORC group and significantly reduced in both Ketotifen groups (p< 0.01).

Conclusion: Joint capsule fibrosis, characterized by hyperplasia of myofibroblasts and mast cells and enhanced collagen deposition, is a prominent feature of posttraumatic joint contractures in this animal model. Treatment with a mast cell stabilizer reduced the molecular markers of joint capsule fibrosis and the resultant biomechanical severity of contracture formation. These results suggest mast cell activity may be an important process in the development of posttraumatic contractures and future work is needed to determine if pharmacological inhibition of mast cell activity has a preventative or therapeutic role in humans.

Purpose: To determine if cells isolated from the rabbit joint capsule in post-traumatic joint contractures have altered responses to stimuli and inhibitors.

Method: The right knee of three rabbits had cortical windows removed from the femoral condyles followed by 4 weeks of immobilization, a recently developed model of post-traumatic contractures. The contralateral knee served as an unoperated control. Primary cells (2.5 × 105 cells/ml) isolated from the posterior capsule were mixed with neutralized bovine collagen solution and then cast into tissue culture plate wells. Gelation occurred overnight and then the cells were treated with 1% serum replacement (control), 10 ng/mL TGF-beta1, and the TGF-beta1 receptor kinase inhibitor SB431542 at 10 microM or 0.1 microM. Gel contraction was measured at 24 post release from the edges of the well using captured images of the gels and computer software.

Results: In all groups the contracture (injured) knees displayed significantly greater collagen gel contraction than control capsule cells. The addition of the TGF-beta1 increased, while SB431542 at the higher dose decreased, the extent of contraction by contracture and control cells. The TGF-beta1 contraction effect was neutralized by the addition of the inhibitor at the higher dose. In terms of sensitivity to the manipulations, the contracture capsule cells had greater responses to the stimulant and inhibitor.

Conclusion: This work is significant as this is the first description of joint capsule cell properties. In post-traumatic contractures, these cells have an intrinsically increased contraction ability at baseline conditions (serum replacement), and these cells also have heightened responses to TGF-beta1 and the TGF-beta1 receptor kinase inhibitor SB431542, a known pathway associated with fibrosis. These results support future work modifying this pathway directly, or by manipulating cells that liberate TGF-beta1. One such strategy would be to prevent mast cells from degranulating as they are a source of TGF-beta1 and other profibrotic growth factors, cytokines and enzymes.

The hypothesis is that cells isolated from capsules of joints with contractures will contract collagen gels at a faster rate when compared to cells obtained from capsules of joints free of contractures.

Post-traumatic joint contractures were produced by removing cortical bone windows from the femoral condyles of three skeletally mature rabbits and immobilizing the knees for four weeks with a K-wire. The contralateral knees served as an unoperated control. At sacrifice, the posterior capsules were immediately placed in medium and the tissue was minced. Upon confluence, cells were trypsinised and gel contraction studies were carried out on passage four cells. Five x 105 cells/ml were mixed with 58% neutralised bovine collagen solution and five hundred microlitres of collagen gel/cells solution were then cast into wells of a tissue culture plate. Gelation occurred overnight at 37C in a humidified incubator containing 5% CO2. At cultured day zero, day one, day three, the gels were released from the well walls. The areas of the gel were measured using an image analyzer immediately after release (zero hour), and one hour, two hour, three hour and four hour post-release.

The amount the collagen gels were contracted depended on the time of preincubation of cells and collagen before release and the source of the joint capsule cells. In general, increasing the time of preincubation heightened the contractile response of the cells. The collagen gel contraction was small for the day zero groups over the first four hours, but for the day three groups the rate of contraction was markedly increased. In all cases the collagen gel contraction was larger for the contracture capsule cells when compared to the control capsule cells. The patterns of the contraction over the four hours post release were similar for contracture and control groups.

Cells from capsules of joints with post-traumatic contractures have intrinsically heightened in vitro contractile properties when compared to normal cells. Future work will determine whether the response is exaggerated to fibrotic stimuli such as TGF-beta1 in these capsule cells from post-traumatic joint contractures.

The hypothesis is that mast cell numbers and neuropeptide containing nerve fibres are increased in the elbow joint anterior capsule of patients with post-traumatic contractures when compared to normal capsules.

Capsules were obtained from two patients with chronic contractures following radial head fractures and two organ donor elbows free of contractures. Four sections from each capsule were double-labelled with specific antibodies to the mast cell marker chymase and the neuropeptide calcitonin gene-related peptide (CGRP). Species specific secondary fluorescent antibodies were used to detect the marker antibodies and cells were identified with a fluorescent nuclear marker (DAPI). Images were captured using a microscope (200x magnification) and five randomly selected areas were sampled for each section obtained from all joint capsules. Chymase positive cell numbers and numbers of nerve fibers (minimum length fifty micrometres) were gathered.

The number of chymase positive mast cells was 6x greater in the contracture capsules when compared to normal capsules. In the contracture capsule, chymase positive mast cells represented 39% of total cells while in control capsules they represented 7% of total cells. Total cell numbers were similar in the capsules of both groups. The number of CGRP positive nerve fibres was increased 3x in the contracture capsule when compared to normal capsule.

Mast cell numbers and neuropeptide positive fibre numbers are increased in the elbow joint anterior capsule of patients with post-traumatic contractures when compared to normal tissues. Neuropeptides such as CGRP can induce mast cell degranulation. Mast cells release profibrotic molecules such as transforming growth factor beta1 (TGF-b1), a myofibroblast upregulator. It has been described that TGF-b1 and myofibroblast numbers are elevated in human elbow joint capsules in post-traumatic contractures. While these trends are encouraging, more subjects are needed to determine whether the mast cell and neuropeptide nerve fibre findings can be generalised to larger numbers. If future work supports a myofibroblast - mast cell - neuropeptide - fibrosis axis in the joint capsule in post-traumatic contractures, then methods to modulate this axis, such as mast cell stabilisers, may be evaluated in animal models.

Ligaments, menisci and joint capsules were obtained from experimental knees with post-traumatic joint contractures and their unoperated contralateral controls in 6 rabbits. Relative mRNA expression was altered for six of seven matrix molecules, growth factors and _-SMA (myofibroblast marker) in the joint capsule, four of seven molecules in the ACL, and two of seven molecules in the MCL and medial meniscus. The joint capsule had the most molecules with altered expression corresponding to it’s acknowledged key role in joint contracture development. Changes in molecular expression of several joint structures in post-traumatic contractures is similar to changes seen following ligament injury.

To evaluate alteration of mRNA expression in ligaments, meniscus and joint capsules in post-traumatic contractures. mRNA expression was altered most frequently in the joint capsule.

The mRNA expression alterations in the joint capsule reflect it’s significant contribution to contractures.

The right knee had a stable intraarticular fracture coupled with Kirschner wire immobilization while the left knee was not surgically manipulated. The rabbits (n=6) were sacrificed two weeks later, and the ACL, MCL, posterior joint capsule and medial meniscus were obtained from both knees. Semiquantitative RT-PCR was used to evaluate relative mRNA expression of selected matrix molecules, growth factors and _-smooth muscle actin (_-SMA), a myofibroblast marker. Glyc-eraldehyde-3-phosphate dehydrogenase, a housekeeping gene, served as a normalization. Optical density measures of the gels were used for analysis. Statistical comparisons were made with a paired t-test. Statistical significance was p< 0.05.

Relative mRNA expression was altered for six of seven molecules in the joint capsule, four of seven molecules in the ACL, and two of seven molecules for the MCL and meniscus. For the joint capsule, relative mRNA expression in the contracture capsule was 2-4x greater than the expression in the control capsules, except for TIMP one where the expression in the contracture capsule was 1/3 of the control capsules. As has been noted with other joint injuries (ligament instability), several structures in the joint display altered molecular expression as was found in this model of joint injury, post-traumatic joint contractures.

Please contact author for tables and/or graphs.

Purpose: To evaluate the role of myofibroblasts in post-traumatic contractures, studies were performed on the myofibroblast marker & #945;-SMA and myofibroblast up-regulators TGF-& #946;1 and the ED-A domain of fibronectin (ED-A) in joint capsules during early stages of post-traumatic contractures. Our hypotheses are mRNA expression of & #945;-SMA, TGF-& #946;1, and ED-A, and myofibroblast numbers, would increase in joint capsules of post-traumatic contractures when compared to contralateral and normal capsule.

Methods: Post-traumatic joint contractures were stimulated in right knees of 24 skeletally mature female rabbits by injury and immobilization. They were equally divided based on time of immobilization: 0-weeks, 2-weeks, 4-weeks, or 6-weeks. Contralateral limbs served as unoperated controls. Normal knee capsules were obtained from three age and gender matched rabbits. Posterior joint capsules were collected for semi-quantitative RT-PCR and mRNA levels of & #945;-SMA, TGF-& #946;1, and ED-A were evaluated in all four groups. Primers were normalized to GAPDH. Myofibroblasts were counted in the 4-weeks immobilization group. Immunohistochemistry was employed using a double labeling technique: monoclonal antibodies to & #945;-SMA and affinity purified antibodies to laminin. DAPI was applied to label nuclei. Statistical analysis was completed. Paired t-tests examining intragroup comparisons and ANOVA with posthoc tukey analyzing changes over time were used (significant if p& #8804;0.05).

Results: There was a significant increase in & #945;-SMA and TGF-& #946;1 mRNA expression in the posterior joint capsule of contracture knees when compared to contralateral control knees in all four groups. The mRNA levels for ED-A were significantly increased in the contracture group compared to the control group at 0-weeks. At 4-weeks immobilization, myofibroblasts were present in control and contracture tissue. Absolute myofibroblast numbers and percentage of myofibroblasts to total cells were significantly increased in contracture tissue compared to control tissue. There was no difference between total cells obtained from contracture and control knees.

Conclusions: Immediately upon injury (0-weeks), mRNA expression of & #945;-SMA, TGF-& #946;1, and ED-A increased in contracture knees compared to control knees. Myofibroblast numbers and percentage of myofibroblasts were elevated in contracture tissue compared to control tissue. It would appear mRNA changes occur immediately and are associated with increased numbers of myofibroblasts at 4-weeks.

Funding : Other Education Grant

Funding Parties : Alberta Heritage Foundation for Medical Research, Health Research Foundation, and Canadian Institutes of Health Research.

We describe the natural history of a rabbit knee model of permanent post-traumatic joint contractures. Twenty-four skeletally mature female NZW rabbits had five-mm-squares of cortical bone removed from both femoral condyles. An extra-articular K-wire immobilized the knee joint in flexion. The K-wire was removed eight weeks later and the rabbits were divided into four groups depending on the remobilization time. The average extension loss for the experimental knees in the zero, eight, sixteen and thirty-two weeks remobilization groups was thirty-eight, thirty-two, twenty-one and twenty degrees, respectively. The motion loss stabilized in the later time intervals suggesting permanent contractures had developed. The contralateral unoperated knees average extension loss was nine degrees.

The purpose of this study was to develop a rabbit knee model of post-traumatic contractures.

A simulated intra-articular fracture plus eight weeks of immobilization leads to a permanent joint contracture even after thirty-two weeks of remobilization.

This animal model of human post-traumatic joint contractures will allow further studies investigating mechanisms underlying the process.

Twenty-four skeletally mature female NZW rabbits had 5 mm squares of cortical bone removed from both femoral condyles. An extra-articular Kirschner wire (K-wire) immobilized the knee in flexion. A second operation was performed eight weeks later to remove the K-wire. The rabbits were divided into four groups. Hind limbs were dissected, preserving the joint capsule. A device allowing six degrees-of-freedom coupled to a material testing system which applied a 0.2 Nm torque measured joint angles. Statistical analysis was performed using ANOVA with a posthoc Student-Newman-Keuls test. Data are presented as mean ± SD.

The loss of extension for the experimental knees in the zero and eight weeks remobilization groups was significantly greater than the values of all contralateral unoperated knees. The loss of extension for the experimental knees in the sixteen and thirty-two weeks remobilization groups was also greater than the contralateral knees, although it was not statistically significant (p = 0.07). With this model, the severity of the contracture decreased with time of remobilization. However, the degree of contracture stabilized between sixteen and thirty-two weeks of remobilization, suggesting that the joints had developed a permanent contracture. This mimics the human scenario of permanent post-traumatic joint contractures.

Funding: has not been received from a commercial party. This work was supported by The Alberta Heritage Foundation for Medical Research.

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Purpose: Classification systems for orthopaedic injuries are used in clinical care and research to allow for communication about the diagnosis, prognosis, treatment, and outcome of fractures and dislocations. The inter-observer reliability of the Hotchkiss modification of the Mason classification system and the AO classification system were evaluated to assess their reliability in classifying radial head fractures.

Methods: Forty-three consecutive fractures with and without other associated injuries were identified from the practice of a single sub-specialist orthopaedic surgeon. Their radiographs were compiled and all identifying marks were hidden. Five observers classified the radiographs according to each classification system. Percent agreement was calculated pair-wise between observers for each system. The mean percent agreement and 95% confidence intervals were calculated for each system. Additionally, the systems were collapsed, with types II and III combined for the Hotchkiss system, and the final digit dropped for the AO system. The mean percent agreement and 95% confidence intervals were then recalculated.

Results: The percent agreement for the Hotchkiss modification of the Mason classification system was 72.3% (95% CI 65.8% to 78.9%). The percent agreement for the AO classification system was 37.7% (95% CI 30.5% to 44.9%). The percent agreement for the collapsed Hotchkiss system was 89.3% (95% CI 86.6% to 92.0%). The percent agreement for the collapsed AO system was 67.4% (95% CI 54.6% to 80.3%).

Conclusions: The inter-observer reliability for the AO classification system had a low percent agreement, possibly reflecting the complexity of the system. Collapsing the AO system improved the percent agreement, however it still could be considered fair. The inter-observer reliability for the Hotchkiss modification of the Mason classification system was higher. However, when the lower end of the 95% confidence interval is taken into consideration, the reliability of the system could be considered fair. Collapsing this system did improve the percent agreement into what could be considered a good range, suggesting that this system may be able to reliably identify fractures requiring operative treatment.

Funding : Other Education Grant

Funding Parties : Alberta Heritage Foundation for Medical Research

The objective of this report was to evaluate myofibroblast numbers in human elbow anterior joint capsules. Joint capsules were obtained from six patients with post-traumatic contractures and from six elbow joints of age-matched organ donors. Frozen sections were labeled with α-smooth muscle actin (α-SMA), a marker of myofibroblasts. Myofibroblasts were identified in both experimental and control tissues. Myofibroblast numbers and percentage of total cells were significantly elevated in the capsules of patients (919 ± 187; 36 ± 0.04%) when compared to organ donor control tissue (485 ± 335; 9 ± 0.04%). Future work will look at the expression of myofibroblast modulators in human elbow joint contractures.

The purpose of this study was to determine whether myofibroblasts are associated with human elbow joint contractures.

Myofibroblast numbers and percentage of myofibroblasts to total cells were significantly increased in anterior elbow joint capsules of patients with post-traumatic contractures.

Methods to alter myofibroblast expression may be strategies to prevent or treat post-traumatic elbow joint contractures.

Joint capsules were obtained from six patients (age 33±13 yrs, preoperative flexion-extension arc range of motion 58°±15°) and from six elbow joints of organ donors free of contractures (age 26±15 yrs). Frozen sections were double labeled using monoclonal antibodies to α-smooth muscle actin (α-SMA) with peroxidase conjugated secondary antibodies, and affinity purified antibodies to laminin with Elexa Fluor 488 conjugated secondary antibodies. The laminin antibodies label components of blood vessels, to differentiate between α-SMA expression associated with blood vessels or myofibroblasts. Endogenous peroxidases were quenched and 10% normal goat serum was used as a blocking agent. DAB/peroxide substrate was added for thirteen minutes. DAPI was applied to label nuclei. Cell nuclei associated with α-SMA and not with laminin were counted as myofibroblasts.

Myofibroblast numbers and percentage of total cells were significantly increased (t-test, p < 0.05) in the joint capsules of the patients when compared to organ donor control tissue. Total cell numbers were not significantly different in the patient and control tissue.

Modulators of α-SMA expression and myofibroblast formation include growth factors and matrix molecule components. Future work will look at the expression of these modulators in human elbow joint contractures.

Funding: Funding has not been received from a commercial party. This work was supported by The Alberta Heritage Foundation for Medical Research.

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