Joint Tissue Damage Research Articles

A spectroscopic diagnostic for rheumatoid arthritis using liquid biopsies

Rheumatoid arthritis (RA) possess not only a substantial degree of clinical heterogeneity but is diagnosed on a diverse array of clinical criteria. The lack of a single marker predictive methodology means that the timely diagnosis and treatment of these patients proves challenging. With the advent of targeted therapies, it is becoming increasingly important to accurately diagnose RA at an early stage of disease in order to ensure effective and timely disease management which can minimise long term sequelae such as joint tissue damage. Raman spectroscopy has recently gained increasing clinical recognition as a non-invasive and label-free method for obtaining a complete biochemical fingerprint of the content of biological samples. This study explored the application of Raman spectroscopy coupled with multivariate data analysis, as an adjunct or alternative tool for the differential diagnosis of RA using peripheral blood mononuclear cells (PBMCs) and purified primary immune cell subsets. High performance partial least square discriminant analysis (PLSDA) classification models constructed in this study enabled identification of spectroscopic discrimination of RA patients and healthy controls without influence from confounding factors. Spectral fitting analysis identified potential spectral biomarkers such as Proteinase K and TNF-α that elucidate the spectral classification between healthy controls and RA patients. These results demonstrate the capability of Raman Spectroscopy in RA diagnosis.

Erythroid Differentiation Regulator 1 Ameliorates Collagen-Induced Arthritis via Activation of Regulatory T Cells.

Erythroid differentiation regulator 1 (Erdr1) has been identified as an anti-inflammatory factor in several disease models, including collagen-induced arthritis (CIA), but its exact mechanisms are still not fully understood. Here, the involvement of regulatory T (Treg) cells in Erdr1-improved CIA was investigated. In the CIA model, Erdr1 was confirmed to reduce collagen-specific IgM in plasma and plasma cells in draining lymph nodes. Importantly, the downregulated Treg cell ratio in draining lymph nodes from CIA mice was recovered by Erdr1 treatment. In addition, administration of Erdr1 improved the CIA score and joint tissue damage, while it revealed no effect in Treg cell-depleted CIA mice, indicating that Treg cells mediate the therapeutic effects of Erdr1 in the CIA model. Results from in vitro experiments also demonstrated that Erdr1 significantly induced Treg cell differentiation and the expression of Treg activation markers, including CD25, CD69, and CTLA4 in CD4+Foxp3+ cells. Furthermore, Erdr1-activated Treg cells dramatically suppressed the proliferation of responder T cells, suggesting that they are functionally active. Taken together, these results show that Erdr1 induces activation of Treg cells and ameliorates rheumatoid arthritis via Treg cells.

Sirt3 Promotes Chondrogenesis, Chondrocyte Mitochondrial Respiration and the Development of High-Fat Diet-Induced Osteoarthritis in Mice.

Understanding how obesity-induced metabolic stress contributes to synovial joint tissue damage is difficult because of the complex role of metabolism in joint development, maintenance, and repair. Chondrocyte mitochondrial dysfunction is implicated in osteoarthritis (OA) pathology, which motivated us to study the mitochondrial deacetylase enzyme sirtuin 3 (Sirt3). We hypothesized that combining high-fat-diet (HFD)-induced obesity and cartilage Sirt3 loss at a young age would impair chondrocyte mitochondrial function, leading to cellular stress and accelerated OA. Instead, we unexpectedly found that depleting cartilage Sirt3 at 5 weeks of age using Sirt3-flox and Acan-CreERT2 mice protected against the development of cartilage degeneration and synovial hyperplasia following 20 weeks of HFD. This protection was associated with increased cartilage glycolysis proteins and reduced mitochondrial fatty acid metabolism proteins. Seahorse-based assays supported a mitochondrial-to-glycolytic shift in chondrocyte metabolism with Sirt3 deletion. Additional studies with primary murine juvenile chondrocytes under hypoxic and inflammatory conditions showed an increased expression of hypoxia-inducible factor (HIF-1) target genes with Sirt3 deletion. However, Sirt3 deletion impaired chondrogenesis using a murine bone marrow stem/stromal cell pellet model, suggesting a context-dependent role of Sirt3 in cartilage homeostasis. Overall, our data indicate that Sirt3 coordinates HFD-induced changes in mature chondrocyte metabolism that promote OA. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Validity of Quantitative Measurements By the Joint Tissue Examination and Damage Exam (JADE) with Musculoskeletal Ultrasound for the Longitudinal Assessment of Hemophilic Arthropathy

Introduction: Clinical scoring systems, such as the Hemophilia Joint Health Score (HJHS), and imaging modalities (radiographs and magnetic resonance) are important instruments for the evaluation of hemophilic joint health. However, they are semi-quantitative scales, exhibit ceiling effects, and may not capture subtle dynamic changes of intra-articular structures. We developed the Joint tissue Activity and Damage Exam (JADE) protocol to quantitatively evaluate the extent of hemophilic arthropathy at the tissue level, individualize management, and objectively assess the efficacy of novel therapies. JADE is an economical, convenient, and radiation free point-of-care musculoskeletal ultrasound protocol. JADE uses quantitative numerical measurements (1/10th of millimeter) for osteochondral alterations, cartilage thickness and soft tissue expansion in hemophilic joints and is validated per international OMERACT guidelines for pathological tissue recognition with high intra/inter-rater and inter-operator reliability. Furthermore, we previously reported JADE association with clinical and functional joint assessments at baseline for patients with hemophilia. Herein, we examined JADE protocol ability to capture tissue changes over time and their associations with HJHS, a clinical joint assessment. We assumed the regression lines for joint HJHS on each JADE measurement at each time point would lie parallel to each other. Verifying this assumption would enhance JADE protocol validity and provide confidence that it could be used to monitor hemophilic arthropathy progression by quantifying specific tissue abnormalities. Methodology: We recruited adult patients (≥ 18 years) with congenital hemophilia and arthropathy in a prospective study performed at 3 sites in North America. We assessed joint HJHS and JADE parameters for each patient (n=44; 264 joints [bilateral elbows, ankles, and knees]) at study entry (baseline), at ~12-18 months (midpoint), and ~24-36 months (final). JADE measurements included osteochondral alterations, cartilage thickness, and soft tissue expansion at sentinel positions for each joint. The association between joint HJHS and each JADE variable was examined by fitting random intercept models with outcomes transformed to ensure normal residuals. We examined the assumption of parallel regression lines for the three time points by applying a test for homogeneity of slopes. If no difference between slopes was found, then we applied an analysis of covariance to test whether the lines for each joint were coincident. Results: Joint HJHSs deteriorated measurably during the study period, reflecting clinically worsening arthropathy over time. Importantly, clinical assessment of deterioration for the elbows, knees, and ankles was associated with JADE measurements that changed in the expected direction, namely increased length of osteochondral alterations, decreased cartilage thickness, and increased soft tissue expansion. As an example, figure 1 shows the association of deteriorating HJHS and osteochondral alterations for elbows, knees, and ankles at baseline, midpoint, and final visits. In each graph, the regression lines were similar in shape. Lines were parallel for the elbow (midpoint vs baseline: p = 0.185; final vs baseline: p = 0.398), and the ankle (midpoint vs baseline: p = 0.225; final vs baseline: p = 0.293). But, the assumption of parallel lines was rejected for the knee (midpoint vs baseline: p = 0.047; final vs baseline: p = 0.024). Conclusions: This study demonstrates a tight association between JADE direct ultrasonographic measurements and clinical deterioration (HJHSs) during several assessments over 3 years. Importantly, the associations are consistent across time, despite clinical joint health examinations and ultrasound measurements being performed at three different centers by multiple providers, with long intervals between assessments. Altogether, these findings constitute a major milestone for the clinical relevance validity of the JADE protocol as a precise instrument for tissue-specific alterations measurements over time. These findings support the use of the JADE protocol to monitor the progression of hemophilic arthropathy longitudinally, permit prompt identification of potentially reversible osteochondral and/or soft tissue changes, and study early interventions that could curb arthropathic progression. Disclosures Kruse-Jarres: F. Hoffmann-La Roche Ltd: Speakers Bureau; Biomarin, Chugai Pharmaceutical Co., CSL Behring, CRISPR Therapeutics, Genentech, Inc.: Honoraria; CSL Behring, Genentech, Inc., Spark: Research Funding; Biomarin, Chugai Pharmaceutical Co., CSL Behring, CRISPR Therapeutics, Genentech, Inc.: Consultancy. Steiner:Uniqure: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi/Genzyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees. Quon:Bayer: Honoraria; Orthopaedic Institute for Children: Current Employment; Biomarin: Honoraria, Speakers Bureau; Novo Nordisk: Honoraria, Speakers Bureau; Bioverativ/Sanofi: Honoraria, Speakers Bureau; Genentech, Inc./F. Hoffmann-La Roche Ltd: Honoraria, Speakers Bureau; Octapharma: Honoraria; Shire/Takeda: Speakers Bureau. von Drygalski:Biomarin: Honoraria; Hematherix LLC: Membership on an entity's Board of Directors or advisory committees, Other: co-founder; Uniqure: Honoraria; Bioverativ/Sanofi: Honoraria, Research Funding; Pfizer: Research Funding; Novo-Nordisk: Honoraria; Takeda: Honoraria.

Ultrasonography findings in knee osteoarthritis: a prospective observational cross-sectional study of 100 Patients

Introduction: Worldwide, knee osteoarthritis (KOA) accounts for 2.2% of total years lived with disability. There is low correlation between joint tissue damage and pain intensity. Periarticular structures may be involved and cannot be identified in X-rays. Objectives: To describe the main ultrasonography (USG) changes in symptomatic patients with primary KOA; to correlate number of USG findings with KOA severity assessed by Kellgren and Lawrence (K&L) radiological scores, with pain intensity measured by a visual analogue scale (VAS) and with functioning scores assessed with Timed up and go test (TUG) and Western Ontario and McMaster Universities (WOMAC) questionnaire. Methods: 100 patients with primary symptomatic KOA were assessed with X-ray and USG. Quantitative and qualitative analyses were evaluated in a systematic manner. Results: The most frequent findings were joint effusion, pes anserinus bursitis, quadriceps tendon enthesopathy, popliteal cyst, iliotibial band tendinitis and patellar tendinitis. Pearson’s correlation analysis demonstrated a significant moderate positive association between VAS scores and number of USG findings (r=0.36; p<0.0001). The number of USG findings was different between K&L grades I and III (p=0.041), I and IV (p<0.001), and II and IV (p=0.001, ANOVA with Bonferroni correction). There was significant association between number of USG findings and TUG (r=0.18; p=0.014) and WOMAC scores for pain (r=0.16; p<0.029) and physical function domains (r=0.16; p<0.028). Conclusion: The most frequent USG finding was joint effusion. Periarticular structures should be explored as potential sources of pain and disability.

Metformin pretreatment suppresses alterations to the articular cartilage ultrastructure and knee joint tissue damage secondary to type 2 diabetes mellitus in rats

ABSTRACT Osteoarthritis (OA) secondary to diabetes affects millions of people worldwide and can lead to disability. The protective effect of metformin pretreatment against alterations to the articular cartilage ultrastructure induced by type 2 diabetes mellitus (T2DM) associated with the inhibition of oxidative stress and inflammation has not been investigated before. Therefore, we induced T2DM in rats (the model group) using high carbohydrate and fat diet and a single injection of streptozotocin (50 mg/kg body weight). The protective group of rats started metformin (200 mg/kg body weight) treatment 14 days before diabetic induction and continued on metformin until the end of the experiment at week 12. Harvested tissues obtained from knee joints were prepared for staining with hematoxylin and eosin (H&E), safranin o staining, and electron microscopy. Histology images showed that OA was developed in the T2DM rats as demonstrated by a substantial damage to the articular cartilage and profound chondrocyte and territorial matrix ultrastructural alterations, which were partially protected by metformin. In addition, metformin significantly (p < .05) reduced hyperglycemia, glycated hemoglobin (HbA1 c), malondialdehyde (MDA), high sensitivity C-reactive protein (hs-CRP), and interleukin-6 blood levels induced by diabetes. Furthermore, a significant (p ≤ 0.015) correlation between either OA cartilage grade score or the thickness of the articular cartilage and the blood levels of HbA1 c, hs-CRP, MDA, superoxide dismutase (SOD) were observed. These findings demonstrate effective protection of the articular cartilage by metformin against damage induced secondary to T2DM in rats, possibly due to the inhibition of hyperglycemia and biomarkers of oxidative stress and inflammation.

Expression of growth and differentiation factor 5 during joint repair and osteoarthritis

Purpose: Growth and differentiation factor 5 (GDF5) is a key risk locus for osteoarthritis (OA), with single-nucleotide polymorphisms that result in decreased GDF5 expression contributing to OA risk. Gdf5, together with Wnt9a, is well known for its critical roles during joint formation. However, little is known regarding the role of Gdf5 following adult joint tissue damage. Regulatory sequences that control Gdf5 expression are distributed over a hundred kilobases, including regions both upstream and downstream of its coding exons.

Regulation of Gdf5 expression in joint remodelling, repair and osteoarthritis

Growth and Differentiation Factor 5 (GDF5) is a key risk locus for osteoarthritis (OA). However, little is known regarding regulation of Gdf5 expression following joint tissue damage. Here, we employed Gdf5-LacZ reporter mouse lines to assess the spatiotemporal activity of Gdf5 regulatory sequences in experimental OA following destabilisation of the medial meniscus (DMM) and after acute cartilage injury and repair. Gdf5 expression was upregulated in articular cartilage post-DMM, and was increased in human OA cartilage as determined by immunohistochemistry and microarray analysis. Gdf5 expression was also upregulated during cartilage repair in mice and was switched on in injured synovium in prospective areas of cartilage formation, where it inversely correlated with expression of the transcriptional co-factor Yes-associated protein (Yap). Indeed, overexpression of Yap suppressed Gdf5 expression in chondroprogenitors in vitro. Gdf5 expression in both mouse injury models required regulatory sequence downstream of Gdf5 coding exons. Our findings suggest that Gdf5 upregulation in articular cartilage and synovium is a generic response to knee injury that is dependent on downstream regulatory sequence and in progenitors is associated with chondrogenic specification. We propose a role for Gdf5 in tissue remodelling and repair after injury, which may partly underpin its association with OA risk.

Epigenetic Regulation in the Pathogenesis of Sjögren Syndrome and Rheumatoid Arthritis.

Autoimmune rheumatic diseases, such as Sjögren syndrome (SS) and rheumatoid arthritis (RA), are characterized by chronic inflammation and autoimmunity, which cause joint tissue damage and destruction by triggering reduced mobility and debilitation in patients with these diseases. Initiation and maintenance of chronic inflammatory stages account for several mechanisms that involve immune cells as key players and the interaction of the immune cells with other tissues. Indeed, the overlapping of certain clinical and serologic manifestations between SS and RA may indicate that numerous immunologic-related mechanisms are involved in the physiopathology of both these diseases. It is widely accepted that epigenetic pathways play an essential role in the development and function of the immune system. Although many published studies have attempted to elucidate the relation between epigenetic modifications (e.g. DNA methylation, histone post-translational modifications, miRNAs) and autoimmune disorders, the contribution of epigenetic regulation to the pathogenesis of SS and RA is at present poorly understood. This review attempts to shed light from a critical point of view on the identification of the most relevant epigenetic mechanisms related to RA and SS by explaining intricate regulatory processes and phenotypic features of both autoimmune diseases. Moreover, we point out some epigenetic markers which can be used to monitor the inflammation status and the dysregulated immunity in SS and RA. Finally, we discuss the inconvenience of using epigenetic data obtained from bulk immune cell populations instead specific immune cell subpopulations.

Quantitative Measurements of Hemophilic Joint Tissues By Point-of-Care Musculoskeletal Ultrasound Are Associated with Clinical and Functional Joint Outcome Parameters

Introduction: Recurrent joint bleeding in patients with congenital hemophilia causes progressive arthropathy, characterized by reduced range of motion, synovial proliferation and osteochondral damage. Chronic hemophilic arthropathy remains a leading disabling complication affecting activity and quality of life. Clinical scoring systems, such as the Hemophilia Joint Health Score (HJHS), along with semi-quantitative imaging modalities (radiographs, magnetic resonance, ultrasound) can aid in the evaluation of joint health. However, scores exhibit ceiling effects and lack the opportunity to provide tissue measurements reflecting subtle dynamic changes of intra-articular structures that may be important in monitoring progression of hemophilic arthropathy. Point-of-care (POC) musculoskeletal ultrasound (MSKUS) has emerged as a rapid and sensitive imaging modality to evaluate not only acute bleeding episodes but also the extent of hemophilic arthropathy at the tissue level in the clinic setting. The Joint Tissue Activity and Damage Exam (JADE) has been developed as a concise, quantitative (not semi-quantitative) MSKUS protocol, validated per Outcomes Measures in Rheumatology guidelines for accurate tissue recognition, with high intra/inter-rater and inter-operator reliability, to quantify (1/10th of millimeter) soft tissue expansion, osteochondral alterations and cartilage thickness in hemophilic joints. We aimed to explore to what extent measurable structural intra-articular tissue changes, evaluated by JADE, correlate with clinical and functional joint assessments. If the value of JADE can be established in this context, such information would enable the use of JADE as a POC precision imaging tool to guide management of hemophilic arthropathy. Methodology: From May 2016 to April 2019, we assessed baseline joint characteristics in a prospective multicenter study in patients with congenital hemophilia and arthropathy (age ≥18 years; n=44 patients; n = 264 joints [bilateral elbows, ankles and knees]). HJHS (a clinical assessment tool) and total arc (a functional assessment tool measuring the sum of flexion and extension degrees of motion) were also evaluated. Each joint was examined using the MSKUS JADE protocol. We analyzed the association between joint HJHS and each JADE variable (comprising measurements of soft tissue expansion, osteochondral alterations and cartilage thickness in validated sentinel positions), and between total arc and each JADE variable, by fitting random intercept models that accounted for intra-individual correlation. The outcomes were transformed to ensure normal residuals, and when necessary polynomial terms were added to fit the curve associations between outcomes and JADE variables. Results: A wide range of joint HJHS values (Elbow: 0 - 13, Knee: 0 - 14; Ankle: 0 - 18 [normal=0; highest abnormal 20]) and total arc values (Elbow: 48 - 158, Knee: 22 - 160; Ankle: 5 - 76) were recorded in the cohort, enabling analyses over a broad range of arthropathic changes. Increasing HJHS and declining total arc were associated with increasing age which is consistent with deteriorating joints. Worsening arthropathy (increasing HJHS or declining total arc) of each joint were associated with JADE measurements in the expected direction including increasing soft tissue expansion, increasing length of osteochondral alterations and decreasing cartilage thickness. This is exemplified for the elbow in Figure 1, showing the associations of worsening HJHSs and arcs with increasing osteochondral alterations and decreasing cartilage thickness. Conclusions: This study demonstrated convincing relationships between direct ultrasonographic measurements using the JADE protocol, clinical HJHS and functional arc in a broad range of arthropathic manifestations in hemophilic patients. Interestingly, changes in arc, limiting joint flexion and extension, appeared often to be closer related to ultrasound tissue measurements than HJHS, highlighting the relevance of JADE towards joint function, a parameter of particular patient interest. These findings confirm the clinical value of POC ultrasonographic direct joint tissue measurements as a precision tool to guide timely changes in the management of hemophilic arthropathy. Disclosures von Drygalski: University of California San Diego: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties; UniQure, Bayer, Bioverativ/Sanofi, Pfizer, Novo Nordisk, Biomarin, Shire, CSL Behring: Consultancy; Hematherix Inc.: Membership on an entity's Board of Directors or advisory committees, Other: Founder.

Trajectories of strenuous physical activity and extensive sitting and 10-year incident radiographic knee OA

Purpose: Participation in exercise, sport or recreational activities offers multiple health benefits, promotes wellbeing and weight management, and preserves function and quality of life. Staying physically active is particularly important for persons at higher risk for knee OA, before disease development, to help prevent a downward spiral of function decline and disability associated with OA development and progression. While individuals with knee symptoms or past knee injury/surgery recognize the health benefits of regular physical activity, uncertainty about whether strenuous physical activity (PA) will accelerate the development of joint tissue damage has been identified as a common concern. Alternatively, extensive sitting could also be detrimental to joint health. Understanding how activity levels evolve over time and their association with risk of incident radiographic knee OA will help illuminate the impact of engagement in strenuous PA or extensive sitting on joint health and motivate healthy PA behavior. Our objective was to identify distinct trajectories of (1) weekly hours spent in strenuous PA and (2) extensive sitting over 8 years among persons at higher risk for knee OA, and evaluate the association of membership in each trajectory group with incident radiographic knee OA identified during the baseline-to-10-year follow-up period. Methods: The OAI (Osteoarthritis Initiative) is a prospective, observational cohort study of persons with or at higher risk for knee OA. Weekly hours engaged in strenuous PA, such as jogging, swimming, cycling, singles tennis, aerobic dance, skiing (downhill or cross-country) or other similar activities were estimated using items in the PASE (Physical Activity Scale for the Elderly) questionnaire over the baseline-to-8-year follow-up interval. Sitting behaviors, such as reading, watching TV, or doing handicrafts, were assessed using PASE items; extensive sitting was defined by frequent prolonged sitting (5-7 days/week and > 4 hours/day). OAI participants who had baseline K/L grade 0 in both knees and PASE data at baseline and at least 2 follow-up visits were included in the analysis. We used latent mixture modeling to identify subgroups of participants with similar underlying trajectories of weekly hours spent in strenuous PA, and separately used similar methods to identify extensive sitting subgroups. Model fit was assessed using the Bayesian Information Criterion (BIC). Final subgroup assignment was based on the maximum posterior probability. Incident radiographic knee OA was defined as K/L grade ≥ 2 in either knee by the 10-year visit. Person-based logistic regression was used to model associations of membership in strenuous PA trajectories (predictor) with incident radiographic knee OA (outcome), unadjusted and adjusted for age, sex, and BMI. Results are reported as odds ratios (ORs) and 95% confidence intervals (CIs). We used similar analytic approaches for extensive sitting trajectories. Results: Among 1194 OAI participants [age: mean 58.4 (SD 8.9) years, BMI: 26.8 (4.5) kg/m2, 697 (58.4%) women], we identified 4 distinct trajectories of weekly hours spent in strenuous PA (Figure 1) and 3 distinct extensive sitting trajectories (Figure 2). For strenuous PA, nearly 50% of the participants were classified into the “persistently no” subgroup (0 hour/week) and 30% into the “low, slightly improving” subgroup (1-2 hours/week). Over 40% were classified into the “moderate to high frequency of extensive sitting” subgroup. One hundred fifty-five participants (13%) developed radiographic knee OA by the 10-year visit. As shown in Figure 3, compared to persistently no strenuous PA, participating in any (adjusted OR 0.75, 95% CI 0.53-1.07) and low-to-moderate (adjusted OR 0.69, 95% CI 0.48-1.01) strenuous PA over 8 years were each marginally non-significantly associated with a reduced likelihood of incident knee OA over the baseline-to-10-year follow-up period. There was no statistical evidence of an association of extensive sitting trajectories with incident knee OA (data not shown). Conclusions: In persons at higher risk of developing knee OA, 4 distinct trajectories of weekly hours spent in strenuous PA were identified over 8 years. Engagement in strenuous PA assessed over an 8-year period was associated with a reduced risk of incident knee OA, however, after adjustment for age, sex and BMI, the association was marginally non-significant. This suggests the possibility that some level of strenuous PA may be protective. Three distinct trajectories of extensive sitting behavior were identified; trajectory membership was not associated with risk of incident knee OA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Attenuated Joint Tissue Damage Associated With Improved Synovial Lymphatic Function Following Treatment With Bortezomib in a Mouse Model of Experimental Posttraumatic Osteoarthritis.

To investigate the roles of the synovial lymphatic system in the severity and progression of joint tissue damage and functional responses of synovial lymphatic endothelial cells (LECs) to macrophage subsets, and to evaluate the therapeutic potential of the proteasome inhibitor bortezomib (BTZ) in a mouse model of experimental posttraumatic osteoarthritis (OA). C57BL/6J wild-type mice received a meniscal ligamentous injury to induce posttraumatic knee OA. Lymphangiogenesis was blocked by a vascular endothelial growth factor receptor 3 (VEGFR-3) neutralizing antibody. Synovial lymphatic drainage was examined by near-infrared imaging. Joint damage was assessed by histology. RNA-sequencing and pathway analyses were applied to synovial LECs. Macrophage subsets in the mouse synovium were identified by flow cytometry and immunofluorescence staining. M1 and M2 macrophages were induced from mouse bone marrow cells, and their effects on LECs were examined in cocultures in the presence or absence of BTZ. The effects of BTZ on joint damage, LEC inflammation, and synovial lymphatic drainage were examined. Injection of a VEGFR-3 neutralizing antibody into the joints of mice with posttraumatic knee OA reduced synovial lymphatic drainage and accelerated joint tissue damage. Synovial LECs from the mouse OA joints had dysregulated inflammatory pathways and expressed high levels of inflammatory genes. The number of M1 macrophages was increased in the knee joints of mice with posttraumatic OA, thereby promoting the expression of inflammatory genes by LECs; this effect was blocked by BTZ. Treatment with BTZ decreased cartilage loss, reduced the expression of inflammatory genes by LECs, and improved lymphatic drainage in the knee joints of mice with posttraumatic OA. Experimental posttraumatic knee OA is associated with decreased synovial lymphatic drainage, increased numbers of M1 macrophages, and enhanced inflammatory gene expression by LECs, all of which was improved by treatment with BTZ. Intraarticular administration of BTZ may represent a new therapy for the restoration of synovial lymphatic function in subjects with posttraumatic knee OA.

Recent developments in advanced imaging in gout.

The plain radiographic features of gout are well known; however, the sensitivity of plain radiographs alone for the detection of signs of gout is poor in acute disease. Radiographic abnormalities do not manifest until late in the disease process, after significant joint and soft tissue damage has already occurred. The advent of dual-energy computed tomography (DECT) has enabled the non-invasive diagnosis and quantification of gout by accurately confirming the presence and extent of urate crystals in joints and soft tissues, without the need for painful and often unreliable soft tissue biopsy or joint aspiration. Specific ultrasound findings have been identified and may also be used to aid diagnosis. Both ultrasound and magnetic resonance imaging (MRI) may be used for the measurement of disease extent, monitoring of disease activity or treatment response, although MRI findings are nonspecific. In this article we summarize the imaging findings and diagnostic utility of plain radiographs, ultrasound, DECT, MRI and nuclear medicine studies in the assessment as well as the implications and utility these tools have for measuring disease burden and therapeutic response.

Detection of Early Deterioration of Joints in Children with Haemophilia — Results of the German Energy Study

Background:Prophylactic substitution of clotting factor concentrates in haemophilia patients starting in early childhood, defined as primary or secondary prophylaxis, is nowadays the gold standard in developed countries. Prophylaxis compared to on-demand treatment is known to reduce bleeding frequency and joint damage. The occurrence of bleeds, especially joint bleeds leads to joint arthropathy in haemophilia patients, even in young children.Aims: To determine the impact of bleeding events on the joint health of children with haemophilia.Methods: Children with haemophilia aged 0-17 years of any type of severity and treatment regimen from a single HTC centre in Duisburg, Germany were compared to healthy children recruited from the environment of the Duisburg HTC. Joint health was measured using ultrasound (US), Clinician-Reported Outcomes (ClinRO) and Patient-Reported Outcomes (PRO). Joint physical structure and joint function were measured with the ClinRO Haemophilia Joint Health Score (HJHSv2.1); subjective physical functioning was assessed with the PRO HEP-Test-Q. US assessment used the Joint Tissue Examination and Damage Exam (JADE) Protocol. Ultrasound images were obtained with a Fujifilm Sonosite Edge II scanner using 13 - 6 MHz transducers. Evaluations were performed in all children by the same person who is experienced in musculoskeletal US scanning.Clinical data of haemophilia patients in terms of treatment regime, total and joint bleeding rates in the past 12 months and target joints were collected from patient records.Differences in the median or in the mean, respectively, were calculated between children with haemophilia and healthy controls and between haemophilia patients across different clinical subgroups.Results: Forty-six children with haemophilia (mean age 9.1±5 years) and 59 healthy children (mean age 8.5±4.7 years) participated in the Energy Study. The majority of the haemophilic children had haemophilia A (91.3%), were severely affected (60.9%) and received prophylactic treatment (69.6%), most of them were on secondary prophylaxis (53.1%). Haemophilia patients had in average 1.57±1.9 total bleeds (range 0-9) and 0.65±1.2 joint bleeds (range 0-6) in the past 12 months, a HJHS of 2.39±3.8 (range 0-13). 43.5% had a target joint, mainly in the ankles. Most of the children with haemophilia participated in physical education in school (58.7%), mainly 1x/week (72.4%). Almost one quarter received physiotherapy (23.9%); only 2 patients had limitations in their mobility.Only children ≥ 6 years of age completed the self-rated HEP-Test-Q, resulting in 33 haemophilia patients and 45 controls. Patients treated on prophylaxis (p<.015), severely affected (p<.001), with joint impairments in the HJHS ≥ 1 (p<.022), number of joint bleeds ≥ 1 (p<.029), number of total bleeds ≥ 2 (p<.0001), showed a worse total score in the HEP-Test-Q compared to children on-demand, with moderate/mild haemophilia, no impairment in HJHS, no joint bleeds, less < 2 total bleeds, respectively. No difference was found for type of prophylaxis and target joints.Compared to controls, patients reported significantly worse subjective physical functioning (MP=77.16±9.5 vs. MC=86.0±7.1; p<0.0001), had a worse HJHS (p<.0001) and showed in the US a significant difference (p<.02) of the longitudinal gap been tibia and talus.Conclusion: Children with haemophilia showed higher deterioration in their joint health of the ankle measured with US and reported higher impairments in their subjective physical functioning than healthy controls.Although 96.4% of severe patients were on regular prophylaxis, early signs of synovitis were detected, especially in the ankle. Patients with haemophilia should be monitored early for arthropathy using US and standardized ClinROs and PROs.Conflict of interest: The Energy Study was sponsored by an unrestricted educational grant from Biotest, Germany. DisclosuresHalimeh:Bayer healthcare, Baxalta Innovations, Biotest, CSL Behring, Novartis, Novo Nordisk, Octapharma, LFB, Pfizer: Honoraria; Bayer Healthcare, Baxalta Innovations, Biotest, CSL Behring, Novo Nordisk, Octapharma, Pfizer: Research Funding. Rott:Biotest AG: Research Funding. Von Mackensen:LFB: Membership on an entity's Board of Directors or advisory committees.

Development and Reliability of the Joint Tissue Activity and Damage Examination for Quantitation of Structural Abnormalities by Musculoskeletal Ultrasound in Hemophilic Joints.

Musculoskeletal ultrasound (US) is used increasingly to examine hemophilic arthropathy. However, quantitative algorithms to document findings are lacking. We developed and sought to validate a protocol quantifying hemophilic joint abnormalities. Thirty-one patients with hemophilia were examined serially for 2 years with musculoskeletal US (≈600 joint examinations and ≈6000 images). Based on the spectrum of pathologies, a quantitative algorithm, named Joint Tissue Activity and Damage Examination (JADE), was developed for soft tissue and osteochondral measurements, including power Doppler, using nominal group techniques. To study intra- and inter-rater reliability, 8 musculoskeletal US-experienced hemophilia providers performed anatomic landmark recognition and tissue measurements on 86 images with arthropathic changes, with repetition 1 month later. Twenty-three musculoskeletal US-inexperienced providers performed similar assessments. Inter-operator reliability was established by 6 musculoskeletal US-experienced hemophilia providers, each acquiring images and JADE assessments of 3 hemophilic arthropathic joints. A radiologist and musculoskeletal sonographer functioned as adjudicators. The statistical analysis was performed with the intraclass correlation coefficient (ICC), Fleiss κ, and Cohen κ where appropriate. The musculoskeletal US-experienced providers showed excellent intra-and inter-rater reliability for tissue measurements (ICCs, 0.94-0.96). Agreement was good to excellent for landmark recognition (Fleiss κ, 0.87-0.94). Inter-operator reliability was excellent for measurements and landmark recognition (ICC, 0.90; Fleiss κ, 1.0). Agreement with adjudicators was mostly good to excellent. Musculoskeletal US-inexperienced providers showed excellent inter-rater reliability for measurements (ICC, 0.96) and moderate agreement for landmark recognition (Fleiss κ, 0.58). The JADE protocol appears feasible for quantifying hemophilic intra-articular abnormalities. Musculoskeletal US-trained hemophilia providers showed high intra-rater, inter-rater, and inter-operator reliability, supporting JADE as a protocol for clinical management and research.

Novel rabbit model of moderate knee contracture induced by direct capsular damage.

The treatment of joint contracture continues to represent a challenging problem in orthopedic surgery and rehabilitation medicine. Existing animal models of knee contracture for evaluating new treatments are mostly created by extensive joint tissue damage with Kirschner-wire immobilization which requires a second surgery to remove Kirschner-wires. This study aimed to develop a less invasive rabbit model of moderate knee contracture through a single surgery. Skeletally mature New Zealand White rabbits had their right knee operated to create surgical damage to the posterior capsule under direct visualization. Operated knees were then held in a flexed position by suturing the superficial flexion muscles with absorbable sutures. The flexion contracture (net extension loss) was determined by comparing the extension angles between the operated and non-operated knees from 8 to 24 weeks post-surgery. The flexion contracture of the operated knees was significantly greater (p < 0.01/0.001) than the non-operated knees at each weekly measurement. The mean flexion contractures were 22° at 8 weeks, 19° at 16 weeks, and 18° at 24 weeks. No significant differences in the severity of flexion contracture were observed between 8-week and each of the following weeks, suggesting that the flexion contracture was essentially stabilized by 8 weeks post-surgery. Histopathologic analyses demonstrated intra-articular and peri-articular scar formation. This less invasive rabbit model of moderate knee contracture is more quickly established through a single surgery with lower risk of surgical complications compared to the previously reported invasive models, and could be an alternative animal model for joint contracture research. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2687-2695, 2018.

The chemokine fragment CXCL9(74-103) diminishes neutrophil recruitment and joint inflammation in antigen-induced arthritis.

This study investigates if treatment with a peptide corresponding to the 30 C-terminal amino acids of CXCL9, CXCL9(74-103), ameliorates joint inflammation in a murine model of antigen-induced arthritis (AIA). AIA was induced in male C57BL/6J mice. Intravenous injection of CXCL9(74-103), simultaneously performed with a tibiofemoral challenge with methylated BSA (mBSA) as antigen in mice immunized with mBSA, diminished the accumulation of leukocytes, in particular neutrophils, in the synovial cavity. The levels of the chemokines CXCL1, CXCL2, and CXCL6 and of the cytokine IL-6 were decreased in inflamed periarticular tissue of mice treated with the CXCL9-derived peptide compared to non-treated AIA mice. In addition, CXCL9(74-103) treatment substantially reduced joint and cartilage damage. CXCL9(74-103) competes with CXCL6 and CCL3 for binding to the glycosaminoglycans heparan sulfate and chondroitin sulfate in vitro. In vivo, CXCL9(74-103) quickly binds to blood vessels in joints as observed by confocal microscopy. Next, we evaluated if later treatment with CXCL9(74-103) had a beneficial impact on joint inflammation. CXCL9(74-103) injection 6h after mBSA challenge still reduced neutrophil accumulation in the joint, although it did not reduce chemokine and IL-6 concentrations. However, a delay of treatment until 12h after challenge had no effect on cell recruitment and chemokine and IL-6 levels. Taken together, we demonstrated that treatment with a peptide, which interferes with the interaction between chemokines and glycosaminoglycans, from the beginning of the disease controlled the massive accumulation of neutrophils in the joint of AIA mice, greatly impacting on joint inflammation and tissue damage.

Suitability of four injectable anesthetic protocols for percutaneous synovial fluid aspiration in healthy swine under field conditions and assessment of lameness seven days post procedure

Objective: To compare the suitability of four anesthetic protocols for ante-mortem percutaneous synovial fluid aspiration from healthy swine in field conditions. A supplemental objective was to assess the iatrogenic impact of ante-mortem joint sampling by monitoring lameness and joint swelling after the procedure and assessing synovium histology at day seven post treatment. Materials and methods: Twenty-four finisher pigs (mean weight 86.1 kg ± 10.6) were each randomly allocated to receive one of four intramuscularly administered anesthetic protocols: telazol-ketamine-xylazine (TKX); telazol-ketamine-acepromazine (TKA); ketamine-acepromazine with lidocaine epidural (KAL); or telazol-acepromazine with lidocaine epidural (TAL). Synovial fluid was collected aseptically from one carpus and tarsus joint per anesthetized pig. The anesthetic protocols were evaluated in terms of successful general anesthesia, time to sternal recumbency and time to standing recovery, and protocol cost. Joint swelling and lameness assessments were completed on days two, four, and seven post sampling. On day seven, pigs were euthanized and synovium was collected from each sampled joint for histologic evaluation. Results: The TKX and TAL treatments were the only anesthetic combinations that provided an adequate anesthesia depth for fluid collection to occur. Mean (SD) time to sternal recumbency for TKX was 125 (26) minutes and for TAL was 198 (28) minutes. There was no evidence of post-aspiration infection in any sampled joints. Implications: The TKX treatment was the most effective anesthetic protocol for ante-mortem joint fluid collection. Ante-mortem joint fluid collection was not associated with significant joint tissue damage and can be a useful diagnostic tool for infectious arthritis.

Sustained intra-articular delivery of cyclin-dependent kinase 9 inhibitors protects against proteolytic activity in an ACL rupture rat model of PTOA

Purpose: Pathogenesis of PTOA starts with a joint injury, which rapidly induces a cellular response that establishes a highly catabolic and inflammatory environment within the joint. This catabolic environment is itself a “secondary injury” that irreversibly degrades joint tissues and greatly accelerates PTOA. The initial step in creating the catabolic environment is the transcriptional activation of primary response genes (PRGs). PRGs are rapidly induced without de novo protein synthesis. Although the complete set of joint injury-induced PRGs is poorly defined, a well-characterized subset of PRGs is the inflammatory genes and lytic enzymes such as MMPs. In our ACL-rupture mouse PTOA model, we've shown that PRG activation is dependent on cyclin-dependent kinase 9 (Cdk9). We postulate that targeting Cdk9 might be more effective than targeting individual inflammatory pathways, cytokines, or matrix-degrading proteinases. Systemic administration of a small-molecule Cdk9 inhibitor (Flavopiridol) effectively prevented early molecular signatures of OA, including inflammatory cytokine and MMP production. However, because of the short in vivo half-life of flavopiridol, multiple injections were required. To address this limitation, we fabricated a sustained-release formulation of flavopiridol by encapsulating it in poly (lactic-co-glycolic acid) (PLGA) microparticles (MPs). Here we characterize these microparticles, and test whether intra-articular injection would reduce signs of joint degradation in an ACL-rupture rat model of PTOA. Methods: Fabrication of flavopiridol-PLGA MPs was performed using a single emulsion-solvent evaporation technique. Briefly, PLGA was dissolved in methylene chloride (5% w/v), flavopiridol added, and the solution added to a bulk volume of a polyvinyl alcohol in DiH2O whilst homogenizing (35,000 rpm for 2 min) to form an emulsion. MPs, thus formed, were stirred for 24 h to evaporate residual methylene chloride. MPs were washed, lyophilized and stored at −20°C. MP size distribution was measured by a Microtrac Nanotrac Dynamic Light Scattering Particle Analyzer, and confirmed by scanning electron microscopy. Flavopiridol release from the MPs was quantified over 42 days in PBS-Tween, by absorbance at 247 nm. To induce PTOA in rats (IACUC approved), 4 rats (Sprague Dawley) were anesthetized and a single mechanical overload applied to the knee joint to rupture the ACL. 5 mg of MPs were suspended in 50ul saline and administered into the intra-articular space using a 23-gauge needle, with 2 rats receiving flavopiridol-PLGA and 2 rats receiving blank-PLGA. To assess OA development and joint degradation, we performed longitudinal (up to 3 weeks) in vivo imaging of MMP activity using intra-articular injections of MMPSense750-FAST on an IVIS-200. Results: Flavopiridol was successfully encapsulated in PLGA microparticles at a mass concentration of ∼8 μg/mg PLGA. The average MP diameter was ∼6.8 μm. The in vitro release kinetics of flavopiridol from the PLGA MPs was rapid for the first 5 days, then linear with approximately 70% of the flavopiridol released by day 42. In the rat PTOA model, we observed a strong increase in the in vivo MMP activity 3 days after injury. However, intra-articular injection of flavopiridol-PLGA microparticles markedly reduced the in vivo MMP activity at all time points tested. Conclusions: Intra-articular delivery of a sustained release formulation of flavopiridol-PLGA microparticles provided long-term protection against injury-induced MMP activity in a rat PTOA model. These results indicate the therapeutic potential of sustained release formulations for locally targeting Cdk9 to effectively suppress injury-induced cellular response that leads to secondary joint tissue damage and potentiates PTOA pathogenesis.

Peptide-based electrochemical biosensor for juvenile idiopathic arthritis detection

Juvenile idiopathic arthritis (JIA) is a wide group of diseases, characterized by synovial inflammation and joint tissue damage. Due to the delay in the implementation of biomarkers into clinical practice and the association with severe sequels, there is an imperative need for new JIA diagnosis strategies. Electrochemical biosensors based on screen-printed electrodes and peptides are promising alternatives for molecular diagnosis. In this work, a novel biosensor for detecting juvenile idiopathic arthritis (JIA) was developed based on the immobilization of the PRF+1 mimetic peptide, as recognition biological element, on the surface of screen-printed carbon electrode. This biosensor was able to discriminate the JIA positive and negative serum samples from different individuals using differential pulse voltammetry, presenting limits of detection and quantification in diluted samples of 1:784 (v/v) and 1:235 (v/v), respectively. Evaluation by electrochemical impedance spectroscopy showed RCT 3 times higher for JIA positive sample than for a pool of human serum samples from healthy individuals. Surface analysis of the biosensor by atomic force microscopy, after contact with JIA positive serum, presented great globular clusters irregularly distributed. The long-term stability of the biosensor was evaluated, remaining functional for over 40 days of storage (after storage at 8°C). Therefore, a simple, miniaturized and selective biosensor was developed, being the first one based on mimetic peptide and screen-printed carbon electrode, aiming at the diagnosis of the juvenile idiopathic arthritis in real serum samples.