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Objectives To investigate cartilage tissue turnover in response to a supervised 12-week exercise-related joint loading training program followed by a 6-month period of unsupervised training in patients with knee osteoarthritis (OA). To study the difference in cartilage tissue turnover between high- and low-resistance training. Method Patients with knee OA were randomized into either high-intensity or low-intensity resistance supervised training (two sessions per week) for 3 months and unsupervised training for 6 months. Blood samples were collected before and after the supervised training period and after the follow-up period. Biomarkers huARGS, C2M, and PRO-C2, quantifying cartilage tissue turnover, were measured by ELISA. Changes in biomarker levels over time within and between groups were analyzed using linear mixed models with baseline values as covariates. Results huARGS and C2M levels increased after training and at follow-up in both low- and high-intensity exercise groups. No changes were found in PRO-C2. The huARGS level in the high-intensity resistance training group increased significantly compared to the low-intensity resistance training group after resistance training (p = 0.029) and at follow-up (p = 0.003). Conclusion Cartilage tissue turnover and cartilage degradation appear to increase in response to a 3-month exercise-related joint loading training program and at 6-month follow-up, with no evident difference in type II collagen formation. Aggrecan remodeling increased more with high-intensity resistance training than with low-intensity exercise. These exploratory biomarker results, indicating more cartilage degeneration in the high-intensity group, in combination with no clinical outcome differences of the VIDEX study, may argue against high-intensity training.

The association between structural changes and pain sensation in osteoarthritis (OA) remains unclear. Joint deterioration in OA leads to the release of protein fragments that can either systemically (serum) or locally (synovial fluid; SF) be targeted as biomarkers and describe structural changes and potentially pain. Biomarkers of collagen type I (C1M), type II (C2M), type III (C3M), type X (C10C), and aggrecan (ARGS) degradation were measured in the serum and SF of knee OA patients. Spearman’s rank correlation was used to assess the correlation of the biomarkers’ levels between serum and SF. Linear regression adjusted for confounders was used to evaluate the associations between the biomarkers’ levels and clinical outcomes. The serum C1M levels were negatively associated with subchondral bone density. The serum C2M levels were negatively associated with KL grade and positively associated with minimum joint space width (minJSW). The C10C levels in SF were negatively associated with minJSW and positively associated with KL grade and osteophyte area. Lastly, the serum C2M and C3M levels were negatively associated with pain outcomes. Most of the biomarkers seemed to mainly be associated with structural outcomes. The overall biomarkers of extracellular matrix (ECM) remodeling in serum and SF may provide different information and reflect different pathogenic processes.

Purpose of the review Osteoarthritis (OA) is a painful disease for which drug development has proven difficult. One major reason for this is the heterogeneity of the disease and the current lack of operationalized means to distinguish various disease endotypes (molecular subtypes). Biomarkers measured in blood or urine, reflecting joint tissue turnover, have been developed and tested during the last decades. In this narrative review, we provide highlights on biomarkers derived from the two most studied and abundant cartilage proteins - type II collagen and aggrecan. Recent findings Multiple biomarkers assessing type II collagen degradation and formation, and aggrecan turnover have been developed. Several markers, such as uCTX-II, have been validated for their association with disease severity and prognosis, as well as pharmacodynamically used to describe the mode of action and efficacy of drugs in development. There is a great need for biomarkers for subdividing patients (i.e., endotyping) and recent scientific advances have not yet come closer to achieving this goal. Summary There is strong support for using biomarkers for understanding OA, reflecting degradation and formation of the joint tissues, focused on type II collagen and aggrecan. There is still a lack of in vitro diagnostics, in all contexts of use.

The type II collagen C-terminal pro-peptide is one of the most abundant polypeptides in cartilage. The purpose of this study was to develop a competitive chemiluminescence enzyme-linked immunosorbent assay, CALC2, targeting this pro-peptide as a marker of cartilage formation. Technical assay parameters were evaluated. CALC2 level was measured after in vitro cleavage of recombinant type II collagen with bone morphogenetic protein-1 (BMP-1) and treatment of ex vivo human osteoarthritis (OA) cartilage explant model (HEX) with insulin-like growth factor-1 (IGF-1). Serum CALC2 levels were assessed in 18 patients with rheumatoid arthritis (RA), 19 patients with ankylosing spondylitis (AS), and 18 age- and sex-matched controls in cohort 1 and 8 patients with OA and 14 age- and sex-matched controls in cohort 2. Type II collagen cleavage with BMP-1 increased the CALC2 level. IGF-1 treatment increased the CALC2 levels in HEX compared with the untreated explants (p < 0.05). Results were confirmed using Western blot analysis. CALC2 levels were decreased in the patients with RA and AS compared with the healthy controls (p = 0.01 and p = 0.02, respectively). These findings indicate that CALC2 may be a novel biomarker of type II collagen formation. However, further preclinical and clinical studies are required to validate these findings.

Introduction: Plasma volume (PV) changes in response to physical activity, possibly as a consequence of adrenergic activation. We estimated changes in PV in response to common exercise modalities; cycling and running as well as adrenaline infusion and control at rest.Methods: On separate days, forty circulatory healthy subjects [aged 60 years (range: 42–75)] with knee osteoarthritis underwent moderate-high intensity cycling, running, and intravenous adrenaline infusion to mimic the circulatory response to exercise. Blood samples were obtained from peripheral veins taken at several pre-defined time points before, during, and after the interventions. PV changes were estimated using venous hemoglobin and the derived hematocrit. The temporal associations between PV and selected biomarkers were explored.Results: Changes in PV were observed during all four interventions, and the response to cycling and running was similar. Compared to rest, PV decreased by -14.3% (95% CI: -10.0 to -18.7) after cycling, -13.9% (95% CI: -10.9 to -17.0) after running, and -7.8% (95% CI: -4.2 to -11.5) after adrenaline infusion.Conclusion: PV decreased in response to moderate-high intensity running and cycling. Adrenaline infusion mimicked the PV change observed during exercise, suggesting a separate influence of autonomic control on blood volume homeostasis. In perspective, a temporal association between PV and biomarker dynamics suggests that consideration of PV changes could be relevant when reporting plasma/serum constituents measured during exercise, but more research is needed to confirm this.

To preclinically characterize a mutant form of growth and differentiation factor 5, R399E, with reduced osteogenic properties as a potential disease-modifying osteoarthritis (OA) drug.Cartilage, synovium, and meniscus samples from patients with OA were used to evaluate anabolic and antiinflammatory properties of R399E. In the rabbit joint instability model, 65 rabbits underwent transection of the anterior cruciate ligament plus partial meniscectomy. Three intraarticular (IA) R399E doses were administered biweekly 6 times, and static incapacitance was determined to assess joint pain. OA was evaluated 13 weeks after surgery. In sheep, medial meniscus transection was performed to induce OA, dynamic weight bearing was measured in-life, and OA was assessed after 13 weeks.Intermittent exposure to R399E (1 week per month) was sufficient to induce cell proliferation and release of anabolic markers in 3-dimensional chondrocyte cultures. R399E also inhibited the release of interleukin-1β (IL-1β), IL-6, and prostaglandin ER399E influences several pathologic processes contributing to OA, highlighting its potential as a disease-modifying therapy.

To investigate acute changes in biochemical markers of bone and cartilage turnover in response to moderate intensity exercise with and without joint impact in healthy human subjects. A randomized, cross-over, exploratory, clinical study was conducted. Twenty healthy subjects with no history of joint trauma completed 30 min interventions of standardized moderate intensity cycling and running as well as a resting intervention 1 week apart. Blood samples were taken immediately before, four times after exercise and again the next day. Urine was sampled, before, after and the next day. On the day of rest, samples were taken at timepoints similar to the days of exercise. Markers of type I (CTX-I), II (C2M, CTX-II) and VI (C6M) collagen degradation, cartilage oligomeric matrix protein (COMP) and procollagen C-2 (PRO-C2) was measured. Trial registration number: NCT04542655, 02 September 2020, retrospectively registered. CTX-I was different from cycling (4.2%, 95%CI: 0.4–8.0%, p = 0.03) and resting (6.8%, 95%CI: 2.9–10.7%, p = 0.001) after running and the mean change in COMP was different from cycling (10.3%, 95%CI: 1.1–19.5%, p = 0.03), but not from resting (8.6%, 95%CI: − 0.7–17.8%, p = 0.07) after running. Overall, changes in other biomarkers were not different between interventions. In this exploratory study, running, but not cycling, at a moderate intensity and duration induced acute changes in biomarkers of bone and cartilage extra-cellular matrix turnover.

Objective To examine the association of body mass index (BMI) with pain in people with hand osteoarthritis (OA), and explore whether this association, if causal, is mediated by systemic inflammatory biomarkers. Methods In 281 Nor-Hand study participants, we estimated associations between BMI and hand pain, as measured by the Australian/Canadian Osteoarthritis Hand Index (AUSCAN; range 0–20) and Numerical Rating Scale (NRS; range 0–10); foot pain, as measured by NRS (range 0–10); knee/hip pain, as measured by the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC; range 0–20); painful total body joint count; and pain sensitization. We fit natural-effects models to estimate natural direct and natural indirect effects of BMI on pain through inflammatory biomarkers. Results Each 5-unit increase in BMI was associated with more severe hand pain (on average increased AUSCAN by 0.64 [95% confidence interval (95% CI) 0.23, 1.08]), foot pain (on average increased NRS by 0.65 [95% CI 0.36, 0.92]), knee/hip pain (on average increased WOMAC by 1.31 [95% CI 0.87, 1.73]), generalized pain, and pain sensitization. Mediation analyses suggested that the effects of BMI on hand pain and painful total body joint count were partially mediated by leptin and high-sensitivity C-reactive protein (hsCRP), respectively. Effect sizes for mediation by leptin were larger for the hands than for the lower extremities, and were statistically significant for the hands only. Conclusion In people with hand OA, higher BMI is associated with greater pain severity in the hands, feet, and knees/hips. Systemic effects of obesity, measured by leptin, may play a larger mediating role for pain in the hands than in the lower extremities. Low-grade inflammation, measured by hsCRP, may contribute to generalized pain in overweight/obese individuals.

To examine the effect of dynamic compressive loading applied intermittently on bovine cartilage explants stimulated with proinflammatory cytokines over 21 days.Cartilage explants were cultured for 21 days with Oncostatin M and TNFα (O ​+ ​T) [10/5 ​ng/mL] or in culture medium alone (w/o). The explants were either left free-swelling or subjected to dynamic compressive loading of 20 ​min, at 1 ​Hz, with loads ranging between 0.1 and 1 ​MPa, 5 times weekly. Metabolic activity was measured once weekly using Alamar Blue and cartilage turnover was assessed with biomarkers targeting degradation fragments of aggrecan (AGNx1) and type II collagen (C2M). Glycosaminoglycan degradation was quantified was the DMMB assay. Furthermore, explant weight and histological analysis was used to assess the cartilage degradation.Dynamic compression of cartilage explants attenuated the O ​+ ​T-mediated C2M release on day 21 with 40% (p ​= ​0.0068) compared to the unloaded explants. Additionally, the change in explant weight from day -1 to day 21 showed that O ​+ ​T stimulation alone mediated a cartilage loss of 11%, whereas O ​+ ​T-stimulated explants subjected to compressive loading demonstrated a decreased cartilage weight loss of 6%, which was supported by the histological analysis. However, loading had no effect on aggrecan degradation.In cartilage explants cultured in a proinflammatory milieu, dynamic compressive loading confers anti-catabolic effects, inhibiting type II collagen degradation and reducing explant cartilage loss. These results demonstrate that compressive loading alters cartilage tissue turnover and enforces the need to include mechanical loading in a translation

Featured Application This work shows the use of an ex vivo cartilage compression model to assess the effect of combining compressive loading and growth factors on cartilage remodeling. (1) Background: Mechanical loading is an essential part of the function and maintenance of the joint. Despite the importance of intermittent mechanical loading, this factor is rarely considered in preclinical models of cartilage, limiting their translatability. The aim of this study was to investigate the effects of intermittent dynamic compression on the extracellular matrix during long-term culture of bovine cartilage explants. (2) Methods: Bovine articular cartilage explants were cultured for 21 days and subjected to 20 min of 1 Hz cyclic compressive loading five consecutive days each week. Cartilage remodeling was investigated in the presence of IGF-1 or TGF-beta 1, as well as a TGF-beta receptor 1 (ALK5) kinase inhibitor and assessed with biomarkers for type II collagen formation (PRO-C2) and fibronectin degradation (FBN-C). (3) Results: Compression of cartilage explants increased the release of PRO-C2 and FBN-C to the conditioned media and, furthermore, IGF-1 and compression synergistically increased PRO-C2 release. Inhibition of ALK5 blocked PRO-C2 and FBN-C release in dynamically compressed explants. (4) Conclusions: Dynamic compression of cartilage explants increases both type II collagen formation and fibronectin degradation, and IGF-1 interacts synergistically with compression, increasing the overall impact on cartilage formation. These data show that mechanical loading is important to consider in translational cartilage models.

To investigate the association between markers of synovial inflammation and matrix turnover (MRI-based and serum biomarkers) and knee symptoms in established knee osteoarthritis (KOA). This cross-sectional study utilised data from a randomised, multicentre placebo-controlled trial (UK-VIDEO) of vitamin D therapy in symptomatic KOA. Data on serum biomarkers, type III collagen degradation (C3M), metabolite of C-reactive protein (CRPM) and cartilage oligomeric matrix protein (COMP), were available at baseline whilst contrast-enhanced (CE) MRI data were acquired in a subsample at baseline and annually. Knee symptoms were assessed using WOMAC at all visits. We examined the cross-sectional association between knee symptoms and three MRI-based and three serum markers of synovitis and matrix turnover, respectively. A total of 447 participants were included in the serum and 136 participants in the MRI analyses. MRI-defined medial perimeniscal synovitis was positively associated with knee pain and, suprapatellar and medial perimeniscal synovitis with knee function in multivariate analysis. We observed a statistically significant, negative association between a higher concentration of serum C3M and CRPM and knee pain, respectively. Furthermore, the highest CRPM quartile was negatively associated with knee function. Our findings suggest that, in established painful radiographic KOA, MRI-defined medial perimeniscal and suprapatellar synovitis were positively associated with knee symptoms. Serum-based C3M and CRPM markers were negatively associated with knee symptoms. Pain fluctuations are common in KOA and a better understanding of the relationship between markers of synovitis and matrix turnover and knee symptoms would facilitate a more accurate assessment of temporal changes in disease progression.

Objective Currently, there are no disease-modifying osteoarthritis drugs (DMOADs) approved for osteoarthritis. It is hypothesized that a subtype of OA may be driven by inflammation and may benefit from treatment with anti-inflammatory small molecule inhibitors adopted from treatments of rheumatoid arthritis. This study aimed to investigate how small molecule inhibitors of intracellular signaling modulate cartilage degradation and formation as a pre-clinical model for structural effects. Design Bovine cartilage explants were cultured with oncostatin M (OSM) and tumour necrosis factor α (TNF-α) either alone or combined with the small molecule inhibitors: SB203580 (p38 inhibitor), R406 (Spleen tyrosine kinase (Syk) inhibitor), TPCA-1 (Inhibitor of κB kinase (Ikk) inhibitor), or Tofacitinib (Tofa) (Janus kinases (Jak) inhibitor). Cartilage turnover was assessed with the biomarkers of degradation (AGNx1 and C2M), and type II collagen formation (PRO-C2) using ELISA. Explant proteoglycan content was assessed by Safranin O/Fast Green staining. Results R406, TPCA-1 and Tofa reduced the cytokine-induced proteoglycan loss and decreased AGNx1 release 3.7-, 43- and 32-fold, respectively. SB203580 showed no effect. All inhibitors suppressed C2M at a concentration of 3 µM. TPCA-1 and Tofa increased the cytokine reduced PRO-C2 3.5 and 3.7-fold, respectively. Conclusion Using a pre-clinical model we found that the inhibitors TPCA-1 and Tofa inhibited cartilage degradation and rescue formation of type II collagen under inflammatory conditions, while R406 and SB203580 only inhibited cartilage degradation, and SB203580 only partially. These pre-clinical data suggest that TPCA-1 and Tofa preserve and help maintain cartilage ECM under inflammatory conditions and could be investigated further as DMOADs for inflammation-driven osteoarthritis.

C-telopeptide of type II collagen (CTX-II) has been shown to be a highly relevant biomarker of cartilage degradation in human rheumatic diseases, if measured in synovial fluid or urine. However, serum or plasma CTX-II have not been demonstrated to have any clinical utility to date. Here, we describe the GPDPLQ1237 ELISA which targets the EKGPDPLQ↓ neo-epitope, an elongated version of the CTX-II neo-epitope (EKGPDP↓), speculated to be a blood-precursor of CTX-II generated by the cysteine protease cathepsin K. Human osteoclast cartilage resorption cultures as well as oncostatin M and tumour necrosis factor α-stimulated bovine cartilage explant cultures were used to validate GPDPLQ1237 biologically by treating the cultures with the cysteine protease inhibitor E-64 and/or the matrix metalloproteinase (MMP) inhibitor GM6001 to assess the potential contributions of these two protease classes to GPDPLQ1237 release. Cartilage resorption-derived GPDPLQ1237 release was inhibited by E-64 (72.1% inhibition), GM6001 (75.5%), and E-64/GM6001 (91.5%), whereas CTX-II release was inhibited by GM6001 (87.0%) but not by E-64 (5.5%). Cartilage explant GPDPLQ1237 and CTX-II release were both fully inhibited by GM6001 but were not inhibited by E-64. No clinically relevant GPDPLQ1237 reactivity was identified in human serum, plasma, or urine from healthy donors or arthritis patients. In conclusion, the GPDPLQ1237 biomarker is released during osteoclast-derived cysteine protease- and MMP-mediated cartilage degradation in vitro, whereas CTX-II release is mediated by MMPs and not by cysteine proteases, as well as from MMP-mediated cartilage degradation under a pro-inflammatory stimulus. These findings suggest that GPDPLQ1237 may be relevant in diseases with pathological osteoclast activity and cartilage degradation. Further studies are required to validate the neo-epitope in human samples.

Osteoarthritis (OA) and rheumatoid arthritis (RA) are serious and painful diseases. Protease-activated receptor 2 (PAR2) is involved in the pathology of both OA and RA including roles in synovial hyperplasia, cartilage destruction, osteophyogenesis and pain. PAR2 is activated via cleavage of its N-terminus by serine proteases. In this study a competitive ELISA assay was developed targeting the 36-amino acid peptide that is cleaved and released after PAR2 activation. Technical assay parameters including antibody specificity, intra- and inter-assay variation (CV%), linearity, accuracy, analyte stability and interference were evaluated. PAR2 pro-fragment release was confirmed after in vitro cleavage of PAR2 recombinant protein and treatment of human synovial explants with matriptase. Serum levels of 22 healthy individuals, 23 OA patients and 15 RA patients as well as a subset of RA patients treated with tocilizumab were evaluated. The PAR2 antibody was specific for the neo-epitope and intra-inter assay CV% were 6.4% and 5.8% respectively. In vitro cleavage and matriptase treated explants showed increased PAR2 pro-fragment levels compared to controls. In serum, PAR2 pro-fragment levels were increased in RA patients and decreased in RA patients treated with tocilizumab. In conclusion, PAR2 may be a potential biomarker for monitoring RA disease and pharmacodynamics of treatment.

Biomarkers, especially biochemical markers, are important in osteoarthritis (OA) research, clinical trials, and drug development and have potential for more extensive use in therapeutic monitoring. However, they have not yet had any significant impact on disease diagnosis and follow-up in a clinical context. Nevertheless, the development of immunoassays for the detection and measurement of biochemical markers in OA research and therapy is an active area of research and development. The evaluation of biochemical markers representing low-grade inflammation or extracellular matrix turnover may permit OA prognosis and expedite the development of personalized treatment tailored to fit particular disease severities. However, currently detection methods have failed to overcome specific hurdles such as low biochemical marker concentrations, patient-specific variation, and limited utility of single biochemical markers for definitive characterization of disease status. These challenges require new and innovative approaches for development of detection and quantification systems that incorporate clinically relevant biochemical marker panels. Emerging platforms and technologies that are already on the way to implementation in routine diagnostics and monitoring of other diseases could potentially serve as good technological and strategic examples for better assessment of OA. State-of-the-art technologies such as advanced multiplex assays, enhanced immunoassays, and biosensors ensure simultaneous screening of a range of biochemical marker targets, the expansion of detection limits, low costs, and rapid analysis. This paper explores the implementation of such technologies in OA research and therapy. Application of novel immunoassay-based technologies may shed light on poorly understood mechanisms in disease pathogenesis and lead to the development of clinically relevant biochemical marker panels. More sensitive and specific biochemical marker immunodetection will complement imaging biomarkers and ensure evidence-based comparisons of intervention efficacy. We discuss the challenges hindering the development, testing, and implementation of new OA biochemical marker assays utilizing emerging multiplexing technologies and biosensors.

Background Tissue released blood-based biomarkers can provide insight into drug mode of action and response. To understand the changes in extracellular matrix turnover, we analyzed biomarkers associated with joint tissue turnover from a phase 3, randomized, placebo-controlled study of baricitinib in patients with active rheumatoid arthritis (RA). Methods Serum biomarkers associated with synovial inflammation (C1M, C3M, and C4M), cartilage degradation (C2M), bone resorption (CTX-I), and bone formation (osteocalcin) were analyzed at baseline, and weeks 4 and 12, from a subgroup of patients (n = 240) randomized to placebo or 2-mg or 4-mg baricitinib (RA-BUILD, NCT01721057). Mixed-model repeated measure was used to identify biomarkers altered by baricitinib. The relationship between changes in biomarkers and clinical measures was evaluated using correlation analysis. Results Treatment arms were well balanced for baseline biomarkers, demographics, and disease activity. At week 4, baricitinib 4-mg significantly reduced C1M from baseline by 21% compared to placebo (p p p p Conclusion Baricitinib treatment resulted in reduced circulating biomarkers associated with joint tissue destruction as well as concomitant RA clinical improvement. Trial registration ClinicalTrials.gov NCT01721057; date of registration: November 1, 2012

To investigate acute changes in biochemical markers of bone and cartilage turnover in response to moderate intensity exercise with and without joint impact in healthy human subjects.A randomized, cross-over, exploratory, clinical study was conducted. Twenty healthy subjects with no history of joint trauma completed 30 min interventions of standardized moderate intensity cycling and running as well as a resting intervention 1 week apart. Blood samples were taken immediately before, four times after exercise and again the next day. Urine was sampled, before, after and the next day. On the day of rest, samples were taken at timepoints similar to the days of exercise. Markers of type I (CTX-I), II (C2M, CTX-II) and VI (C6M) collagen degradation, cartilage oligomeric matrix protein (COMP) and procollagen C-2 (PRO-C2) was measured.NCT04542655, 02 September 2020, retrospectively registered.CTX-I was different from cycling (4.2%, 95%CI: 0.4-8.0%, p = 0.03) and resting (6.8%, 95%CI: 2.9-10.7%, p = 0.001) after running and the mean change in COMP was different from cycling (10.3%, 95%CI: 1.1-19.5%, p = 0.03), but not from resting (8.6%, 95%CI: - 0.7-17.8%, p = 0.07) after running. Overall, changes in other biomarkers were not different between interventions.In this exploratory study, running, but not cycling, at a moderate intensity and duration induced acute changes in biomarkers of bone and cartilage extra-cellular matrix turnover.

Osteoarthritis (OA) is associated with cartilage breakdown, brought about by ADAMTS-5 mediated aggrecan degradation followed by MMP-derived aggrecan and type II collagen degradation. We investigated a novel anti-ADAMTS-5 inhibiting Nanobody&reg, (M6495) on cartilage turnover ex vivo. Bovine cartilage (BEX, n = 4), human osteoarthritic - (HEX, n = 8) and healthy&mdash, cartilage (hHEX, n = 1) explants and bovine synovium and cartilage were cultured up to 21 days in medium alone (w/o), with pro-inflammatory cytokines (oncostatin M (10 ng/mL) + TNF&alpha, (20 ng/mL) (O + T), IL-1&alpha, (10 ng/mL) or oncostatin M (50 ng/mL) + IL-1&beta, (10 ng/mL)) with or without M6495 (1000&minus, 0.46 nM). Cartilage turnover was assessed in conditioned medium by GAG (glycosaminoglycan) and biomarkers of ADAMTS-5 driven aggrecan degradation (huARGS and exAGNxI) and type II collagen degradation (C2M) and formation (PRO-C2). HuARGS, exAGNxI and GAG peaked within the first culture week in pro-inflammatory stimulated explants. C2M peaked from day 14 by O + T and day 21 in co-culture experiments. M6495 dose dependently decreased huARGS, exAGNxI and GAG after pro-inflammatory stimulation. In HEX C2M was dose-dependently reduced by M6495. M6495 showed no effect on PRO-C2. M6495 showed cartilage protective effects by dose-dependently inhibiting ADAMTS-5 mediated cartilage degradation and inhibiting overall cartilage deterioration in ex vivo cartilage cultures.

Sprifermin, recombinant human fibroblast growth factor 18 (rhFGF18), induces cartilage regeneration in knees of patients with osteoarthritis (OA). We hypothesized that a temporal multiphasic process of extracellular matrix (ECM) degradation and formation underlie this effect. We aimed to characterize the temporal ECM remodeling of human knee OA articular cartilage in response to sprifermin treatment. Articular cartilage explants from patients with knee OA (npatients = 14) were cultured for 70 days, with permanent exposure to sprifermin (900, 450, 225 ng/mL), FGF18 (450 ng/mL), insulin-like growth factor-1 (100 ng/mL, positive control) or vehicle (nreplicates/treatment/patient = 2). Metabolic activity (AlamarBlue) and biomarkers of type IIB collagen (PIIBNP) formation (Pro-C2 enzyme-linked immunosorbent assay [ELISA]) and aggrecanase-mediated aggrecan neo-epitope NITEGE (AGNx1 ELISA) were quantified once a week. At end of culture (day 70), gene expression (quantitative reverse transcription polymerase chain reaction) and proteoglycan content (Safranin O/Fast green staining) were quantified. The cartilage had continuously increased metabolic activity, when treated with sprifermin/FGF18 compared to vehicle. During days 7–28 PIIBNP was decreased and NITEGE was increased, and during days 35–70 PIIBNP was increased. At end of culture, the cartilage had sustained proteoglycan content and relative expression of ACAN

Osteoarthritis (OA) is the most common form of joint disease. This presents the OA research community and pharmaceutical companies developing disease-modifying osteoarthritis drugs (DMOADs) with great opportunities.. The different OA subtypes complicates the traditional approaches for developing new treatments. If we can identify new markers that can distinguish different subtypes this can greatly facilitate drug development from early discovery to late clinical development. Nevertheless, the current approaches result in poorly targeted treatments and the inability to recruit the right patients for clinical trials. Thus, there is an urgent medical need for objective biomarker tools for patient phenotyping.

There is an increasing demand for noninvasive and descriptive biochemical markers (biomarkers) in osteoarthritis; for enabling early drug development (including translational research), evaluating clinical trial at an early stage and for subtyping. Purpose of the review is to review and comment on current availability of such biomarkers.Many different biomarkers have been tested in the last 18 months. The main focus has been on testing whether the biomarkers, whether is reflect joint tissue turnover or inflammatory status, can differentiate osteoarthritis patients from healthy controls or whether the biomarkers are associated with progression. Less than a handful of studies, investigate the role of the biomarkers as response markers. Thus, there is still a great need for developing biomarkers that reflect disease activity and thereby can be used for treatment response or patient phenotyping.Osteoarthritis is the most common form of joint disease. This presents the osteoarthritis research community and pharmaceutical companies developing disease-modifying osteoarthritis drugs (DMOADs) with great opportunities. There are different osteoarthritis subtypes, which complicates the traditional approaches for developing new treatments. If we can identify new markers that can distinguish different subtypes, this can greatly facilitate drug development from early discovery to late clinical development.

Objectives: There is an unmet medical need for biomarkers in OA which can be applied in clinical drug development trials. The present study describes the development of a specific and robust assay measuring type II collagen degradation (T2CM) and discusses its potential as a noninvasive translational biomarker. Methods: A type II collagen specific neoepitope (T2CM) was identified by mass spectrometry and monoclonal antibodies were raised towards the epitope, employed in a chemiluminescence immunoassay. T2CM was assessed in bovine cartilage explants with or without MMP-13 inhibitor, and explant supernatants were analyzed by Western blot. T2CM was measured in plasma samples from one study (n ​= ​48 patients) where OA patients were referred to total knee replacement (TKR). Additionally, T2CM was quantified in serum from OA patients receiving salmon calcitonin treatment (sCT) (n ​= ​50) compared to placebo (n ​= ​57). Results: The T2CM assay was technically robust (13/4 ​% inter/intra-variation) and specific for the type II collagen fragment cleaved by MMP-1 and -13. The MMP-13 inhibitor reduced the T2CM release from bovine cartilage explants receiving catabolic treatment. These results were confirmed by Western blot. In human end-stage OA patients (scheduled for TKR), the T2CM levels were elevated compared to moderate OA (p

Objectives Here we tested the hypothesis that human M-CSF (hM-CSF) overexpressed in CB CD34+ cells would induce differentiation and survival of monocytes and osteoclasts in vitro and in vivo. Methods Human M-CSF was overexpressed in cord blood CD34+ cells using a lentiviral vector. Results We show that LV-hM-CSF-transduced CB CD34+ cells expand 3.6 and 8.5 fold more with one or two exposures to the hM-CSF expressing vector, respectively, when compared to control cells. Likewise, LV-hM-CSF-transduced CB CD34+cells show significantly higher levels of monocytes. In addition, these cells produced high levels of hM-CSF. Furthermore, they are able to differentiate into functional bone resorbing osteoclasts in vitro. However, osteoclast differentiation and bone resorption was blunted compared to control CD34+ cells receiving exogenous hM-CSF. NSG mice engrafted with LV-hM-CSF-transduced CB CD34+ cells have physiological levels of hM-CSF production that result in an increase in the percentage of human monocytes in peripheral blood and bone marrow as well as in the spleen, lung and liver. Conclusion In summary, ectopic production of human M-CSF in CD34+ cells promotes cellular expansion and monocyte differentiation in vitro and in vivo and allows for the formation of functional osteoclasts, albeit at reduced levels, without an exogenous source of M-CSF, in vitro. This article is protected by copyright. All rights reserved.

Purpose: Exercise therapy with a focus on muscle strengthening has proven to be effective for the management of knee osteoarthritis (OA). Yet it is not known whether high-intensity resistance training (RT) is more effective in improving upper leg muscle strength and physical performance than low-intensity RT. Still, there is some controversy regarding the effectiveness of high-intensity RT and whether or not it is harmful, for instance by accelerating cartilage degeneration, osteophyte formation, or increasing synovitis. Any catabolic or anabolic response of musculoskeletal tissue to RT might first be visible on a biochemical level before changes in clinical symptoms are measurable. Serum biomarkers can objectively measure early biochemical changes and assess whether RT leads to a more anabolic or catabolic response. The aim of this study is to assess (i) whether high-intensity RT elicits a different response (e.g. catabolic) on systemic inflammation and musculoskeletal tissues in and surrounding the joint, including bone, cartilage, muscle, and synovial tissue compared to low-intensity RT; and (ii) whether there is an association between changes in serum levels of inflammatory and musculoskeletal tissue-derived biomarkers and improvements in clinical outcomes (performance-based tests and self-reported outcomes on pain and activity limitations).Methods: In a randomized controlled trial, 177 participants with knee OA conducted a high-intensity (70%-80% of the Repetition Maximum (1RM)) or low-intensity (40%-50% 1RM) RT program 3 times a week for 12 weeks. Measures of clinical outcomes and serum samples were collected at the start of RT (pre-intervention), after 3 months at the end of RT (post-intervention), and 6 months after RT (follow-up). As a reflection of systemic inflammation (CRP), synovitis (CRPM, C3M), bone turnover (OC, CTX-I), cartilage turnover (PRO-C2, C2M, huARGS), muscle turnover (PRO-C3, PRO-C6), and cell behaviour (col10neo) a total of eleven serum biomarkers were analysed. With the exception of CRP, which was determined with an immunoturbidimetric assay, ELISA assays were used to quantify serum levels of the other 10 serum biomarkers. The primary outcome measures are the changes in serum biomarker levels. Other outcome measures include upper leg muscle strength, performance-based tests, and self-reported outcomes on pain and activity limitations.Results: High-intensity RT resulted in greater improvements in muscle strength compared to low-intensity RT when measured by the estimated 1RM. No significant differences between groups were found for upper leg muscle strength (Nm/kg) when measured with an isokinetic dynamometer. Both groups showed similar improvements in pain and physical functioning. Although there is no difference between groups in clinical outcomes, except for the estimated 1RM, we expect that participants in the high-intensity RT group are more likely to have enhanced serum levels of catabolic biomarkers than participants in the low-intensity RT group. Since both the high-intensity RT group and low-intensity RT group improved over time, we expect that changes in serum biomarker levels are associated with overall improvements in clinical outcomes. Almost all participants had normal CRP values (Conclusions: The work to date on CRP serum levels suggests that RT did not influence CRP levels. This result may be explained by the high percentage of participants with normal CRP levels (

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique tissue important for proper function of the synovial joint and is constituted by a dense extracellular matrix (ECM), rich in proteoglycan and type II collagen. Chondrocytes are the only cell type present within cartilage and are widespread and relatively low in number. Altered external stimuli and cellular signalling can lead to changes in ECM composition and deterioration, which are important pathological hallmarks in diseases such as osteoarthritis (OA) and rheumatoid arthritis. Ex vivo cartilage models allow 1) profiling of chondrocyte mediated alterations of cartilage tissue turnover, 2) visualizing the cartilage ECM composition, and 3) chondrocyte rearrangement directly in the tissue. Profiling these alterations in response to stimuli or treatments are of high importance in various aspects of cartilage biology, and complement in vitro experiments in isolated chondrocytes, or more complex models in live animals where experimental conditions are more difficult to control. Cartilage explants present a translational and easily accessible method for assessing tissue remodeling in the cartilage ECM in controllable settings. Here, we describe a protocol for isolating and culturing live bovine cartilage explants. The method uses tissue from the bovine knee, which is easily accessible from the local butchery. Both explants and conditioned culture medium can be analyzed to investigate tissue turnover, ECM composition, and chondrocyte function, thus profiling ECM modulation.

Summary Objective Links between pain and joint degradation are poorly understood. We investigated the role of activation of Toll-like receptors (TLR) by cartilage metabolites in initiating and maintaining the inflammatory loop in OA causing joint destruction. Methods Synovial membrane explants (SMEs) were prepared from OA patients’ synovial biopsies. SMEs were cultured for 10 days under following conditions: culture medium alone, OSM + TNFα, TLR2 agonist - Pam2CSK4, Pam3CSK4 or synthetic aggrecan 32-mer, TLR4 agonist - Lipid A. Release of pro-inflammatory and degradation biomarkers (acMMP3 and C3M) were measured by ELISA in conditioned media along with IL-6. Additionally, human cartilage was digested with ADAMTS-5, with or without the ADAMTS-5 inhibiting nanobody - M6495. Digested cartilage solution (DCS) and synthetic 32-mer were tested for TLR activation in SEAP based TLR reporter assay. Results Western blotting confirmed TLR2 and TLR4 in untreated OA synovial biopsies. TLR agonists showed an increase in release of biomarkers - acMMP3 and C3M in SME. Synthetic 32-mer showed no activation in the TLR reporter assay. ADAMTS-5 degraded cartilage fragments activated TLR2 in vitro. Adding M6495 – an anti-ADAMTS-5 inhibiting nanobody®, blocked ADAMTS-5-mediated DCS TLR2 activation. Conclusion TLR2 is expressed in synovium of OA patients and their activation by synthetic ligands causes increased tissue turnover. ADAMTS-5-mediated cartilage degradation leads to release of aggrecan fragments which activates the TLR2 receptor in vitro. M6495 suppressed cartilage degradation by ADAMTS-5, limiting the activation of TLR2. In conclusion, pain and joint destruction may be linked to generation of ADAMTS-5 cartilage metabolites.

Background Osteoarthritis (OA) leads to joint failure and total joint replacement (TJR, either hip (H) or knee (K)). Worsening of pain and joint space narrowing are believed to be surrogates for joint failure; however, we hypothesize that TJR, as a reflection of joint failure, can be used as an endpoint in event-driven clinical trials within a reasonable duration. We explored the incidence of TJR in the Prospective Epidemiologic Risk Factor (PERF I) study. Methods A total of 5855 Danish postmenopausal women aged 49–88 enrolled in the PERF I study during 1999–2001 (baseline). Three-, six- and twelve-year follow-up data from the Danish National Patient Registry was collected, including occurrence of TJR and OA diagnosis. At baseline the women were asked whether they had OA. Results The women with a TJR diagnosis before or after baseline were on average 1 year older (p

Background: Patients with joint diseases such as osteoarthritis (OA) are believed to have an abundance of endogenous Toll like receptor (TLR) ligands in their joints, which might be responsible for activating TLRs that may ultimately initiate a self-perpetuating inflammatory loop in the disease. TLR ligands may be generated from the breakdown of articular cartilage, which in turn arises from the activity of two key enzymes: A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5) and Matrix metalloproteinases (MMPs). Objectives: In this study, we investigate the effect of M6495, a novel anti ADAMTS-5 inhibiting nanobody® on TLR2 activation by ADAMTS-5 derived cartilage cleaved fragments Methods: Human cartilage biopsies were retrieved from OA patients undergoing total knee replacement. The tissue cartilage was snap frozen in liquid nitrogen to eliminate underlying metabolic activity and then digested with recombinant human ADAMTS-5 (4 μg/ml) for 24, 48 and 72 hours respectively at 37 oC. The undigested remaining cartilage was discarded after each timeframe and the digested cartilage solution (DS) was used for further testing. Tissue cleavage was assessed by measuring the release of aggrecan degradation biomarkers: Aggrecanase mediated aggrecan degradation (AGNx1) and MMP mediated aggrecan degradation (FFGV). DS was tested for TLR activation in a secreted embryonic alkaline phosphatase (SEAP) reporter gene based HEK hTLR2 (human Toll like receptors) cell line. Cartilage tissue in buffer alone was used as control at each time point. Results: Aggrecan degradation in cartilage was confirmed by increased release of AGNx1 (p Conclusion: ADAMTS-5-mediated cartilage degradation leads to release of aggrecan fragments, which activate the TLR2 receptor in vitro in a specialised reporter system. Anti-ADAMTS-5 inhibiting nanobody®, M6495, showed a suppression in release of degradation biomarkers leading to limited activation of TLR2. The data suggest a potential chondro-protective effect by M6495. Disclosure of Interests: Neha Sharma: None declared, Christian Thudium Employee of: I am a full time employee of Nordic Bioscience, Morten Karsdal Shareholder of: I own shares of Nordic Bioscience, Employee of: I am a full-time employee in Nordic Bioscience, Anne-Christine Bay-Jensen Shareholder of: I own shares of Nordic Bioscience, Employee of: I am a full-time employee in Nordic Bioscience, Thorbjorn Gantzel: None declared, Martin Michaelis Employee of: Employee of Merck KGaA, Darmstadt, Germany, Christoph Ladel Employee of: Employee of Merck KGaA, Darmstadt, Germany, Daniela Werkmann Employee of: Employee of Merck KGaA, Darmstadt, Germany, Sven Lindemann Employee of: Employed by Merck KGaA

Summary Objective A hallmark of osteoarthritis (OA) is degradation of articular cartilage proteoglycans. In isolated human OA chondrocytes, the anti-inflammatory cytokine Interleukin-37 (IL-37) lowers the expression of the proteolytic MMP and ADAMTS enzymes, which mediate this degradation. Therefore, we investigated if IL-37 protects against proteoglycan loss in freshly obtained human OA explants. Material and methods Human OA cartilage explants were incubated with IL-37. Release of sulphated proteoglycans (sGAGs) was measured with the dimethylmethylene-blue assay. Production and degradation of newly synthesized proteoglycans was measured using 35S-sulphate. Proteoglycan and proteolytic enzyme expression were analyzed by qPCR and Western Blot. Proteolytic activity was determined by measuring MMP- and ADAMTS-generated aggrecan neo-epitopes with ELISA and by using MMP-3-, MMP-13- or ADAMTS-5-inhibitors. Results Over time, a linear release of sGAGs from OA cartilage was measured. IL-37 reduced this release by 87 μg/ml (24%) 95%CI [21.04–141.4]. IL-37 did not affect 35S-sulphate incorporation or proteoglycan gene expression. In contrast, IL-37 reduced loss of 35S-sulphate labeled GAGs and reduced MMP-3 protein expression, indicating that IL-37 inhibits proteoglycan degradation. Remarkably, we observed two groups of patients; one group in which MMP-3-inhibition lowered sGAG release, and one group in which ADAMTS5-inhibition had this effect. Remarkably, IL-37 was only functional in the group of patients that responded to MMP-3-inhibition. Conclusion We identified a relationship between IL-37 and reduced sGAG loss in OA cartilage. Most likely, this effect is mediated by inhibition of MMP-3 expression. These results suggest that IL-37 could be applied as therapy in a subgroup of OA patients, in which cartilage degradation is mediated by MMP-3.

Background Pain is a debilitating symptom of rheumatoid arthritis (RA), caused by joint inflammation and cartilage and bone destruction. Nonsteroidal anti-inflammatory drugs (NSAIDs) are used to treat pain and inflammation in RA, but are not disease-modifying and do not prevent joint destruction when administered alone. KBPs (Key Bioscience peptides) are synthetic peptides based on salmon calcitonin and are expected to inhibit bone resorption and to be chondroprotective. In this study, we investigated if combining a standard of care NSAID (naproxen) with a KBP resulted in improvement in pain scores, as well as disease activity and structural damage in a rat model of RA. Methods Collagen-induced arthritis (CIA) was induced in 40 female Lewis rats by immunization with porcine type II collagen; 10 rats were given sham injections. CIA rats were treated with KBP and/or naproxen. Health scores and joint scores were evaluated daily. Mechanical and cold allodynia tests and burrowing tests were used to assess pain-like behaviors. Blood samples were collected for biomarker testing, and paws were collected for histology and microcomputed tomography. Results Naproxen monotherapy increased the time until humane endpoints was reached, and improved health score, pain assessments, and trabecular thickness, while KBP monotherapy did not result in improvements. Combination therapy had improved efficacy over naproxen monotherapy; combination therapy resulted in improved health scores, and importantly reduced mechanical and cold allodynia assessment. Furthermore, protection of articular cartilage structure and preservation of bone structure and bone volume were also observed. Conclusions This study demonstrates that combining KBP and naproxen may be a relevant therapeutic strategy for RA, resulting in improvements to the overall health, pain, inflammation, and joint structure. Electronic supplementary material The online version of this article (10.1186/s13075-019-1819-9) contains supplementary material, which is available to authorized users.

Little is known about local and systemic biomarkers in relation to synovitis and pain in end-stage osteoarthritis (OA) patients. We investigated the associations between the novel extracellular matrix biomarker, C1M, and local and systemic interleukin 6 (IL-6) with synovitis and pain. Serum C1M, plasma and synovial fluid IL-6 (p-IL-6, sf-IL-6) were measured in 104 end-stage knee OA patients. Contrast-enhanced magnetic resonance imaging (MRI) was used to semi-quantitatively assess an 11-point synovitis score; pain was assessed by the Western Ontario & McMaster Universities Osteoarthritis Index (WOMAC) and the Neuropathic Pain Questionnaire (NPQ). Linear regression was used to investigate associations between biomarkers and synovitis, and biomarkers and pain while controlling for age, sex and body mass index. We also tested whether associations between biomarkers and pain were confounded by synovitis. We found sf-IL-6 was associated with synovitis in the parapatellar subregion (B=0.006; 95% CI 0.003-0.010), and no association between p-IL-6 and synovitis. We also observed an association between C1M and synovitis in the peri-ligamentous subregion (B=0.013; 95% CI 0.003-0.023). Further, sf-IL-6, but not p-IL-6, was significantly associated with pain, WOMAC (B=0.022; 95% CI 0.004-0.040) and NPQ (B=0.043;95% CI 0.005-0.082). There was no association between C1M and WOMAC pain but we did find an association between C1M and NPQ (B=0.229; 95% CI 0.036-0.422). Lastly, synovitis explained both biomarker-NPQ associations, but not the biomarker-WOMAC association. These results suggest C1M and IL-6 are associated with synovitis and pain, and synovitis is an important confounding variable when studying biomarkers and neuropathic features in OA patients., 19 pages, 2 figures, 5 tables and 2 supplementary figures

Spondyloarthritis (SpA) is an umbrella term describing a family of chronic inflammatory rheumatic diseases. These diseases are characterised by inflammation of the axial skeleton, peripheral joints, and entheseal insertion sites throughout the body which can lead to structural joint damage including formation of axial syndesmophytes and peripheral osteophytes. Genetic evidence, preclinical and clinical studies indicate a clear role of interleukin (IL)- 23 and IL-17 as mediators in SpA pathogenesis. Targeting the IL-23/−17 pathways seems an efficient strategy for treatment of SpA patients, and despite the remaining challenges the pathway holds great promise for further advances and improved therapeutic opportunities. Much research is focusing on serological markers and imaging strategies to correctly diagnose patients in the early stages of SpA. Biomarkers may facilitate personalised medicine tailored to each patient's specific disease to optimise treatment efficacy and to monitor therapeutic response. This narrative review focuses on the IL-17 pathway in SpA-related diseases with emphasis on its role in pathogenesis, current approved IL-17 inhibitors, and the need for biomarkers reflecting core disease pathways for early diagnosis and measurement of disease activity, prognosis, and response to therapy.

Background:Altered extracellular matrix (ECM) remodelling is an important part of the pathology seen in joint degenerative diseases. Type II collagen is the most abundant ECM protein in the cartilage and provides the tissue with essential tensile strength in order to withstand high compressive loading. During cartilage erosion, type II collagen is cleaved by matrix metallopeptidases (MMPs) which generates new protein fragments called neo-epitopes. These fragments are released into circulation and may potentially serve as biomarkers by indicating the degree of cartilage destruction.Objectives:The aim of this study is to develop a highly specific immunoassay targeting a neo-epitope fragment of type II collagen cleaved, named T2CM. Moreover, we investigated the assays potential to evaluate type II collagen degradation in anex vivobovine full-depth cartilage explants model (BEX) with catabolic treatment and in healthy controls and osteoarthritis (OA) patients.Methods:A monoclonal antibody was raised in mouse against the C-terminus from protease cleavage site of type II collagen and a direct competitive ELISA was developed and technically validated. The assay specificity was evaluated for the standard peptide excluding cross-reactivity with elongated and truncated peptides, and a non-sense coating peptide. Human OA cartilage was cleaved with MMP-1, -2, -9 and -13 and measured with the T2CM-assay to investigate which MMPs generated the neo-epitope. T2CM levels were measured in supernatant from BEX explants cultured for 21 days in serum free DMEM/F12 medium with six different doses of OSM+TNF-α (O+T) treatment (20/10, 20/20, 20/40, 10/10, 10/20, 10/40 ng/mL) including a control group without (w/o) treatment. The supernatant was harvested 3 times weekly and replaced with new culture medium with O+T treatment. Biomarker results were confirmed by western blot, where T2CM was measured in supernatant from explants with O+T treatment 20/20 ng/mL and 20/40 ng/mL harvested on day 14 and day 21. To confirm the preclinical data, serum samples from 23 healthy controls (age range from 44-59 years with mean 51.4 ± SD 5.1, gender distribution was 56% female and 44% male, and 100% Caucasian) and 23 OA patients (age range from 41-77 years with mean 57.7 ± SD 13.7, gender distribution was 61% female and 39% male, and 100% Caucasian) were measured by T2CM.Results:A technically robust and T2CM-specific assay was developed. The assay linearity and spike-recovery were accepted with percentage of 99.69% and 93.15%. The assay showed no cross-reaction with the elongated, truncated or non-sense coating peptide. In addition, it was demonstrated that the T2CM neo-epitope was derived from MMP-1 and MMP-13 cleavage of type II collagen. O+T treatment induced the T2CM release in BEX compared to the untreated (Figure 1-2). Moreover, the western blot confirmed the T2CM results by the presence of two T2CM bands on day 21 from O+T treated explant compared to day 14 where no bands appeared. T2CM showed to be significantly elevated in patients with OA compared to controls (p=0.036; mean 3.262 ng/mL ± SD 1.065 vs 2.698 ng/mL ± SD 1.118).Conclusion:The newly developed assay was specific for the T2CM neo-epitope and was determined to be generated by MMP-1 and MMP-13. Additionally, the assay detected elevated levels of T2CM in supernatant from explants treated with O+T after 19 days of treatment compared to untreated. This was further confirmed in human OA patients, where the level of T2CM was elevated compared to healthy controls. This suggests that T2CM may have potential as biomarker for type II collagen degradation. Future preclinical and clinical studies are needed to validate these findings.Figure 1-2.T2CM measurements in BEX model. OSM + TNF-a (O+T) ng/mL.Disclosure of Interests:Solveig Skovlund Groen Employee of: Nordic Bioscience, Dovile Sinkeviciute Grant/research support from: Industrial PhD Student, Employee of: Industrial PhD Student, Christian Thudium Employee of: Employee at Nordic Bioscience A/S., Patrik Önnerfjord: None declared, Morten Karsdal Shareholder of: Nordic Bioscience A/S., Employee of: Full time employee at Nordic Bioscience A/S., Anne-Christine Bay-Jensen Shareholder of: Nordic Bioscience A/S, Employee of: Full time employee at Nordic Bioscience A/S., Signe Holm Nielsen Employee of: Full time employee at Nordic Bioscience

Summary Objectives Pain and disability are the main clinical manifestations of osteoarthritis, for which only symptomatic therapies are available. Hence, there is a need for therapies that can simultaneously alter disease progression and provide pain relief. KBP is a dual amylin- and calcitonin-receptor agonist with antiresorptive and chondroprotective properties. In this study we investigated the effect of KBP in a rat model of osteoarthritis. Methods Medial meniscectomy (MNX) was performed in 39 rats, while 10 underwent sham surgery. Rats were treated with KBP and/or naproxen. Nociception was assessed by mechanical and cold allodynia, weight bearing asymmetry, and burrowing behavior. Blood samples were collected for biomarker measurements, and knees for histology. Cartilage histopathology was evaluated according to the advanced Osteoarthritis Research International (OARSI) score and KBPs in vitro antiresorptive effects were assessed using human osteoclasts cultured on bone. Results The MNX animals displayed an increased nociceptive behavior. Treatment with KBP attenuated the MNX-induced osteoarthritis-associated joint pain. The cartilage histopathology was significantly lower in rats treated with KBP than in MNX animals. Bone and cartilage degradation, assessed by CTX-I and CTX-II plasma levels, were decreased in all KBP-treated groups and KBP potently inhibited bone resorption in vitro. Conclusions Our study demonstrates the effectiveness of KBP in ameliorating osteoarthritis-associated joint pain and in protecting the articular cartilage, suggesting KBP as a potential drug candidate for osteoarthritis.

Knee osteoarthritis (KOA) is commonly defined by radiographic changes and knee pain. KOA has a multifactorial aetiology, but there are several well-recognised strong risk factors for the incidence of KOA. These include age, sex, body mass index (BMI), previous injuries, bone density, smoking and pre-existing hand osteoarthritis (HOA). Factors associated with mild systemic and synovial inflammation are potential markers for KOA incidence. The hypothesis for this research was that selected markers associated with synovial inflammation and/or variables linked with metabolic syndrome (MetS) are prognostic for KOA incidence. Self-reported knee stiffness, serum markers associated with knee synovitis, and variables associated with MetS were selected as potential risk factors for KOA incident.

Background: Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by progressive inflammation systemically and local joint deterioration. Chronic inflammation leads to elevated levels of tissue remodeling and release of extracellular matrix (ECM) metabolites into circulation. Type VI collagen (Col-VI) is found at the interface between the interstitial- and the basement membrane where it binds other matrix proteins and support cell-cell interactions. Blood levels of Col-VI metabolites (C6M) have been associated with increased tissue turnover and disease activity in animal models and clinical studies of lung 1 and liver2 fibrosis and ankylosing spondylitis3. These studies indicate that systemic levels of C6M are released ubiquitously from many organs and may be modulated by anti-inflammatory treatments. Treatment with the αIL-6 receptor agent Tocilizumab (TCZ) results in decreased ECM remodeling Objectives: Investigate the modulation of Col-VI metabolites in circulation by TCZ treatment and its association with treatment response. Methods: Systemic Col-VI metabolism was measured in the LITHE study (n=740), a one year double blind, placebo controlled phase III parallel group study in patients receiving placebo, 4 mg/kg or 8 mg/kg TCZ in combination with MTX. Col-VI degradation was measured using the C6M assay, measuring a specific MMP generated metabolite at bl, w4 and w16. The odds ratio (OR) of treatment response (ACR20, 50 or 70) at >median decrease was calculated using logistic regression, adjusting for age, sex, BMI, dis. duration (model 1) and treatment (model 2). Results: TCZ treatment dose dependently reduced of Col-VI metabolites in serum at w4 in both 4 and 8 mg/kg (9% ns and 46% p Conclusions: Suppression of Col-VI turnover was significantly associated with a higher level of response to TCZ treatment in RA patients after already 4 weeks. We hypothesize that there is an increased systemic connective tissue turnover in RA and connective tissue metabolites may be used as indicators of treatment response. References 1. Jenkins RG, Simpson JK, Saini G, et al. Lancet Respir Med. 2015;2600(15):1–11. 2. Leeming DJ, Byrjalsen I, Jimenez W, Christiansen C, Karsdal MA, Liver Int. 2013;33(3):439–447. 3. Bay-Jensen AC, Leeming DJ, Kleyer A, Veidal SS, Schett G, Karsdal MA., Rheumatol Int. 2012;32(11):3565–3572. Disclosure of Interest: C. Thudium Employee of: Nordic Bioscience, N. Gudmann Employee of: Nordic Bioscience, M. Karsdal Shareholder of: Nordic Bioscience, Employee of: Nordic Bioscience, A.-C. Bay-Jensen: None declared

Background Glycosaminoglycans (GAGs) are essential for the pressure-resistant function of cartilage. During osteoarthritis (OA), GAGs are lost from cartilage. This loss impairs the functional and structural integrity of cartilage, thereby accelerating further cartilage damage. Proteoglycan degradation is mediated by enzymes such as MMP3, MMP13 and ADAMTS5. Objectives Recently we discovered that interleukin 37 (IL37) lowers the expression of these enzymes in human OA chondrocytes. The goal of this study was to investigate if IL37 protects against GAG loss in freshly obtained human OA explants. Methods Human cartilage was obtained from ten OA patients undergoing total knee or hip arthroplasty. Per condition 6 cartilage explants of 4 mm in diameter were used. Explants were incubated with recombinant IL37 (rhIL37) for up to 6 days. Every other day new rhIL37 was added. Additionally, an MMP3-inhibitor, or MMP13-inhibitor or ADAMTS5-inhibitor was added in the same protocol. Sulfated GAGs (sGAGs) were visualised by histology, and sGAG release in culture medium was quantified using 1,9-dimethylmethylene blue. To study sGAG synthesis, explants were incubated with rhIL37 followed by 4 hour labelling with 35SO4. Alternatively, explants were pre-labelled for 4 hour with 35SO4 followed by incubation with rhIL37 to study sGAG degradation. Additionally, expression levels of proteoglycans and cartilage matrix degrading enzymes were measured by qPCR and Western Blot. Activity of MMP and ADAMTS enzymes was determined by measuring FFGV, ARGS and NITEGE neo-epitope levels in the supernatant of the cultures, using ELISA. Results Culturing human OA explants for up to 6 days, caused a reduction in GAG content as observed by loss of Safranin O staining. In addition, a release of sGAGs was measured in the supernatant of on average 17,5 µg/ml per mg cartilage. Incubation of cartilage explants with rhIL37 significantly reduced the release of GAGs. A maximal reduction of 24% was already observed with the lowest dose of rhIL37 (1 ng/ml). No effect of rhIL37 on the amount of incorporated 35SO4 was observed, indicating that rhIL37 does not alter the synthesis of GAGs. This is supported by the observation that rhIL37 does not affect the mRNA expression of the large proteoglycans and SLRPS. In contrast, we did find that rhIL37 significantly reduced the loss of 35SO4-labelled GAGs from cartilage explants. In addition, rhIL37 reduced MMP3 and MMP13 protein expression and lowered both MMP and ADAMTS mediated degradation of proteoglycan fragments: FFGV neo-epitope and ARGS and NITEGE neo-epitopes, confirming that rhIL37 reduces active degradation of sGAGs. Lastly, to investigate which proteolytic enzyme contributes to the sGAG release in our culture system, a MMP3-, MMP13- or ADAMTS5-inhibitor was added to the explants. Strikingly, we found that only MMP3 inhibition mimicked IL37 function, suggesting that the effects of rhIL37 run via MMP3. Conclusions Our data show that rhIL37 reduces sGAG release of cartilage explants, indicating that IL37 supports cartilage matrix integrity. To our knowledge this is the first report demonstrating this anti-destructive effect of IL37 on freshly obtained human OA cartilage explants. Possible these effects run via MMP3 because IL37 reduced MMP3 expression and only MMP3 inhibition results in similar effects as rhIL37 addition. Disclosure of Interest None declared

Background The innate immune system is important for the initiation and development of joint diseases such as rheumatoid arthritis (RA) and Osteoarthritis (OA). Exogenous TLR (Toll like receptor) ligands are abundant in the synovial joints of RA and OA patients and induce the first immune response. Stimulation of TLRs results in an increased immune response and is characterised by induction of cytokines expression which in turn causes tissue damage and results in generation of more DAMPs. Hence, resulting in a self-perpetuating loop of TLR activation and TLR mediated tissue damage. Objectives The aim of this study was to investigate the effect of degradation fragments of human cartilage generated ex vivo or in vitro, on TLR-2 activation in a TLR-2 reporter cell system. Methods Human cartilage biopsies retrieved from OA patients undergoing total knee replacement at Gentofte hospital were used to generate human explants (HEX). HEX were cultured for 21 days either without (w/o) treatment or in the presence of IL-1α (10 ng/ml, 5 ng/ml and 2.5 ng/ml). Unconditioned media incubated without cartilage tissue was used as control. Tissue degradation was verified by measuring the release of aggrecan neo-epitope biomarkers AGNx1 and FFGV into the conditioned media (CM). Correspondingly, human cartilage was digested with ADAMTS5 for different time intervals (16 hour, 24 hour and 88 hour) at 37°C. The digested cartilage was removed by centrifugation and the supernatant was stored. Crushed cartilage in buffer alone was used as a control set up for each time point and buffer alone was subtracted as background. The CM and digested media (DM) was tested in the SEAP (secreted embryonic alkaline phosphatase) reporter gene based HEK hTLR2 cell line. The HEK null 1 parent cell line was used as a control. Pam3CSK4 was used as a positive control for the hTLR2 cell line. Results IL-1α induced aggrecan degradation was confirmed by increased exAGNx1 and exFFGV release in the conditioned media compared to w/o (p Conclusions IL-1α induced cartilage degradation leads to the release of aggrecan fragments into the CM, and this CM as well as in vitro cleavage products from ADAMTS-5 digestion of human cartilage was able to activate the TLR2 receptor in vitro in a specialised reporter cell system. These data indicate that DAMPs may be released from human cartilage in the presence of pro-inflammatory cytokines and proteolytic enzymes. The released fragments can lead to TLR2 activation and cause further inflammation. These data suggest that DAMPs may play a role in the onset and maintenance of inflammation in diseases such as OA and RA. Disclosure of Interest None declared

Background Osteoarthritis (OA) patients suffer from progressive degradation of articular cartilage, to which there is no available treatment to block or reverse the catabolic mechanisms. Articular cartilage confers a biomechanical function in the joints, and in concordance chondrocytes have shown to be mechanosensitive. The effect of dynamic compression on cartilage extracellular matrix (ECM) could prove to be crucial for translational research in development and screening of new OA drug candidates. Objectives This study investigates the effect of long-term dynamic compression of a bovine articular cartilage ex vivo model through quantification of cartilage associated biomarkers. Methods Full depth bovine cartilage explants were cultured for 2 weeks. The explants were treated 3 times a week with either OSM [10 ng/mL] and TNF-a [2 ng/mL] (O+T), or TGF-B1 [50 ng/mL]. Untreated samples were included as negative controls (w/o). For each condition two groups were established; an unloaded group and a group compressed 3 times a week. Compression was applied using Electroforce 5500 (TA Instruments), in a sine wave with a maximum load per cycle of 1 MPa, at 1 Hz frequency for 1200 cycles. Metabolic activity was measured once a week using AlamarBlue. Biomarkers released to the supernatant were assessed using the following well-described ELISAs: formation of type II collagen was measured by ProC2, and MMP-degradation of type II collagen was measured by C2M. Sulfated glycosaminoglycans (sGAG) released to media, and sGAG extracted from the explants on the last day using a 4 mM GuHCl solution were quantified using a 1.9-dimethylmethylene blue (DMB) assay. Results Compression of bovine explants significantly decreased release of C2M compared to unloaded samples in O-T and w/o groups with 42% and 48% respectively. ProC2 release was not affected by compression. Compression of w/o or TGF-b treated explants increased sGAG release with 195% and 152% relative to their unloaded controls. However, the total amount of sGAG extracted from the explants on the last day remained unchanged. Compression of the explants did not affect the metabolic activity. Conclusions Compression significantly reduced the degradation of type II collagen, which in combination with unaltered formation as measured by ProC2, may suggest suppression of the involved catabolic processes and increased deposition of type II collagen in the cartilage matrix. Furthermore, compression elevated the sGAG release without reduction in the explant GAG content, indicating an increased turnover of GAG. In conclusion, the cartilage ECM turnover is significantly modulated by dynamic compression. This method adds essential translational value to the continuous research addressing OA. Disclosure of Interest None declared

Background Osteoarthritis (OA) is a heterogeneous disease described by a combination of joint pain, physical disability and radiographic alterations leading to joint failure and total joint replacement (TJR). Commonly used endpoints in OA trials are worsening of pain and joint space narrowing. TJR is normally not considered an endpoint. Age and female gender are considered as major risk factors for developing OA. Objectives We hypothesise that TJR can be used as an endpoint in OA outcome studies within reasonable time frame. To investigate the basis for this hypothesis, we explored the prevalence and incidence of TJR as a reflection of joint failure in the Prospective Epidemiologic Risk Factor (PERF I) study. Methods A total of 5,855 Danish postmenopausal women aged 49–88 enrolled in the Prospective Epidemiologic Risk Factor (PERF I) study during 1999–2001 (baseline). Three, six and twelve year follow-up data from the Danish National registry was collected in end of 2014, including occurrence of TJR, OA and other relevant diagnosis. Also, women where at baseline and in 2014 asked whether they had a TJR or OA. The biomarker C1M was measured in baseline serum samples. The PERF I study was carried out in accordance with ICH-GCP and the study protocol was approved by the local ethics committees. Results There were 798 women that had their first TJR between baseline and 12 year follow-up; giving an incidence proportion of 13.6%. The TJR women were on average 1 year older (p=0.010) and heavier (1.7 kg/cm2, p 40 ng/mL, median) that after baseline underwent there first ever TJR (841). Group 1 is as described above. The 3, 6 and 12 incidence rates were 9.0, 17.3% and 29.7% for the prior TJR group, 6.2, 12.1% and 23.3% for the OA group, and 7.6, 13.6% and 25.1% for the OA +C1M group. The age-dependent prevalence and incidence for the first TJR. The prevalence was insignificant for the age group younger than 60 years old ( Conclusions Within a timeframe of 3, 6 or 12 years TJR incidence for women with an OA diagnosis reached 6, 12% and 23%, which was a doubling compared to the All population. The incidence increased by adding a single diagnostic measure. This reflects that TJRs are frequent amongst elderly women and that if designed minutiously, such as including specific diagnostic criteria (e.g. biomarker, OA diagnose), it may be feasible to conduct clinical studies with TJR as an endpoint. However, special attention much be directed to the objectiveness of the criteria for TJR. This may build a case for design of outcome studies (joint failure) for developing drugs in OA. Disclosure of Interest A. Bay-Jensen Shareholder of: Nordic Bioscience, Grant/research support from: ApproacH (IMI support), Employee of: Nordic Bioscience, C. Bager Employee of: Proscion, A. Bihlet Shareholder of: Nordic Bioscience, Employee of: Nordic Bioscience, C. Thudium Employee of: Nordic Bioscience, I. Byrjalsen Employee of: Nordic Bioscience, H. Nielsen Employee of: Nordic Bioscience, J. Andersen Shareholder of: Nordic Bioscience, Employee of: Nordic Bioscience, B. Riis Shareholder of: Nordic Bioscience, C. Christiansen Shareholder of: Nordic Bioscience, M. Karsdal Shareholder of: Nordic Bioscience, Grant/research support from: ApproacH (IMI support), Employee of: Nordic Bioscience

Background Response to any treatment in rheumatoid arthritis (RA) is assessed by symptomatic changes, such as swollen joint count. Such assessments does not provide information regarding the effect of the treatment at tissue level. Chronic inflammation has a detrimental effect resulting in elevated levels of tissue remodelling and the release of extracellular matrix (ECM) metabolites into the circulation. The tissues consist mainly of interstitial matrix, basement membrane and cells, which are all affected by auto-immune disorders. Tissue metabolites can be measured in serum as biomarkers of tissue remodelling. Objectives The purpose was to investigate if tissue remodelling is differently modulated by tocilizumab (TCZ) and methotrexate (MTX). Methods The AMBITION study, a phase III RCT with tocilizumab vs. Methotrexate in which TCZ monotherapy (8 mg/kg every 4 weeks) was compared to methotrexate monotherapy over 24 weeks in patients with moderate-severe RA (AMBITION, NCT00109408). TCZ is a compound that inhibits the IL-6 receptor. Tissue metabolites were measured in baseline and 8 weeks sera (n=319) by ECM specific ELISAs: Connective tissue remodelling was measured by C3M (type III collagen degradation), basement membrane remodelling by C4M (type IV collagen), inflammation by C-reactive protein (CRP) and its metabolite CRPM. Comparison between groups were done by ANCOVA adjusting for age, gender, BMI and disease duration. Results Tissue remodelling was increased by 10% in the placebo group and significantly (p Conclusions Chronic inflammation results in an increased amount of tissue remodelling. There was a significant difference in the magnitude of effect MTX and TCZ on tissue remodelling. In addition there was a disconnect between tissue remodelling and change in disease activity, which was treatment dependent. Disclosure of Interest A. Bay-Jensen Shareholder of: Nordic Bioscience, Employee of: Nordic Bioscience, A. S. Siebuhr Employee of: Nordic Bioscience, C. Thudium Employee of: Nordic Bioscience, M. Karsdal Shareholder of: Nordic Bioscience, Employee of: Nordic Bioscience

Background: Osteoclasts have been strongly implicated in osteoarthritic cartilage degradation, at least indirectly via bone resorption, and have been shown to degrade cartilage in vitro. The osteoclast resorption processes required to degrade subchondral bone and cartilage—the remodeling of which is important in the osteoarthritic disease process—have not been previously described, although cathepsin K has been indicated to participate. In this study we profile osteoclast-mediated degradation of bovine knee joint compartments in a novel in vitro model using biomarkers of extracellular matrix (ECM) degradation to assess the potential of osteoclast-derived resorption processes to degrade different knee joint compartments.Methods: Mature human osteoclasts were cultured on ECMs isolated from bovine knees—articular cartilage, cortical bone, and osteochondral junction ECM (a subchondral bone-calcified cartilage mixture)—in the presence of inhibitors: the cystein protease inhibitor E-64, the matrix metalloproteinase (MMP) inhibitor GM6001, or the vacuolar-type H+-ATPase (V-ATPase) inhibitor diphyllin. Biomarkers of bone (calcium and C-terminal type I collagen (CTX-I)) and cartilage (C2M) degradation were measured in the culture supernatants. Cultures without osteoclasts were used as background samples. Background-subtracted biomarker levels were normalized to the vehicle condition and were analyzed using analysis of variance with Tukey or Dunnett's T3 post hoc test, as applicable.Results: Osteochondral CTX-I release was inhibited by E-64 (19% of vehicle, p = 0.0008), GM6001 (51% of vehicle, p = 0.013), and E-64/GM6001 combined (4% of vehicle, p = 0.0007)—similarly to bone CTX-I release. Diphyllin also inhibited osteochondral CTX-I release (48% of vehicle, p = 0.014), albeit less than on bone (4% of vehicle, p p = 0.07) and GM6001 (14% of vehicle, p = 0.006), with complete abrogation when combined (0% of vehicle, p = 0.004). Cartilage C2M release was non-significantly inhibited by E-64 (69% of vehicle, p = 0.98) and was completely abrogated by GM6001 (0% of vehicle, p = 0.16).Conclusions: Our study supports that osteoclasts can resorb non-calcified and calcified cartilage independently of acidification. We demonstrated both MMP-mediated and cysteine protease-mediated resorption of calcified cartilage. Osteoclast functionality was highly dependent on the resorbed substrate, as different ECMs required different osteoclast processes for degradation. Our novel culture system has potential to facilitate drug and biomarker development aimed at rheumatic diseases, e.g. osteoarthritis, where pathological osteoclast processes in specific joint compartments may contribute to the disease process.

Background: Osteoarthritis (OA) leads to joint failure and total joint replacement (TJR, either hip (H) or knee (K)). Worsening of pain and joint space narrowing are believed to be surrogates for joint failure; however, we hypothesize that TJR, as a reflection of joint failure, can be used as an endpoint in event-driven clinical trials within a reasonable duration. We explored the incidence of TJR in the Prospective Epidemiologic Risk Factor (PERF I) study. Methods: A total of 5,855 Danish postmenopausal women aged 49-88 enrolled in the PERF I study during 1999-2001 (baseline). Three-, six- and twelve-year follow-up data from the Danish National Patient Registry was collected, including occurrence of TJR and OA diagnosis. At baseline the women were asked whether they had OA. Findings: The TJR women were on average 1 year older (p