The link between bone and blood vessels is regulated by hypoxia and the hypoxia-inducible transcription factor, HIF, which drives both osteogenesis and angiogenesis. The recent clinical approval of PHD enzyme inhibitors, which stabilize HIF protein, introduces the potential for a new clinical strategy to treat osteolytic conditions such as osteoporosis, osteonecrosis, and skeletal fracture and nonunion. However, bone-resorbing osteoclasts also play a central role in bone remodeling and pathological osteolysis, and HIF promotes osteoclast activation and bone loss in vitro. It is therefore likely that the result of PHD enzyme inhibition in vivo would be mediated by a balance between increased bone formation and increased bone resorption. It is essential that we improve our understanding of the effects of HIF on osteoclast formation and function and consider the potential contribution of inhibitory interactions with other musculoskeletal cells. The PHD enzyme inhibitor FG-4592 stabilized HIF protein and stimulated osteoclast-mediated bone resorption, but inhibited differentiation of human CD14+ monocytes into osteoclasts. Formation of osteoclasts in a more physiologically relevant 3D collagen gel did not affect the sensitivity of osteoclastogenesis to FG-4592, but increased sensitivity to reduced concentrations of RANKL. Coculture with osteoblasts amplified inhibition of osteoclastogenesis by FG-4592, whether the osteoblasts were proliferating, differentiating, or in the presence of exogenous M-CSF and RANKL. Osteoblast coculture dampened the ability of high concentrations of FG-4592 to increase bone resorption. These data provide support for the therapeutic use of PHD enzyme inhibitors to improve bone formation and/or reduce bone loss for the treatment of osteolytic pathologies and indicate that FG-4592 might act in vivo to inhibit the formation and activity of the osteoclasts that drive osteolysis.

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Aims The lack of disease-modifying treatments for osteoarthritis (OA) is linked to a shortage of suitable biomarkers. This study combines multi-molecule synovial fluid analysis with machine learning to produce an accurate diagnostic biomarker model for end-stage knee OA (esOA). Methods Synovial fluid (SF) from patients with esOA, non-OA knee injury, and inflammatory knee arthritis were analyzed for 35 potential markers using immunoassays. Partial least square discriminant analysis (PLS-DA) was used to derive a biomarker model for cohort classification. The ability of the biomarker model to diagnose esOA was validated by identical wide-spectrum SF analysis of a test cohort of ten patients with esOA. Results PLS-DA produced a streamlined biomarker model with excellent sensitivity (95%), specificity (98.4%), and reliability (97.4%). The eight-biomarker model produced a fingerprint for esOA comprising type IIA procollagen N-terminal propeptide (PIIANP), tissue inhibitor of metalloproteinase (TIMP)-1, a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4), monocyte chemoattractant protein (MCP)-1, interferon-γ-inducible protein-10 (IP-10), and transforming growth factor (TGF)-β3. Receiver operating characteristic (ROC) analysis demonstrated excellent discriminatory accuracy: area under the curve (AUC) being 0.970 for esOA, 0.957 for knee injury, and 1 for inflammatory arthritis. All ten validation test patients were classified correctly as esOA (accuracy 100%; reliability 100%) by the biomarker model. Conclusion SF analysis coupled with machine learning produced a partially validated biomarker model with cohort-specific fingerprints that accurately and reliably discriminated esOA from knee injury and inflammatory arthritis with almost 100% efficacy. The presented findings and approach represent a new biomarker concept and potential diagnostic tool to stage disease in therapy trials and monitor the efficacy of such interventions. Cite this article: Bone Joint Res 2020;9(9):623–632.

Purpose: Healthy cartilage homeostasis depends on an intact collagen scaffold and high aggrecan content. ADAMTS-5 (A Disintegrin And Metalloprotease with ThromboSpondin-motifs-5) is critically involved in arthritic diseases because of its direct role in cleaving aggrecan. Several studies indicate that inhibition of ADAMTS-5 may have the potential to stop progression of osteoarthritis (OA). In the present study, we investigated the in vitro efficacy of M6495, an inhibitor of ADAMTS-5, including assessment of affinity, potency, specificity, and its effect on the levels of glycosaminoglycan (GAG) and the ADAMTS-5 generated neoepitope of aggrecan (huARGS) in bovine and human cartilage explant assays. Furthermore, we studied the effect of M6495 on cartilage derived markers from aggrecan (GAG) and type II collagen (C2M), as well as type III collagen (C3M) in a cartilage-synovium co-culture model. Methods: Binding kinetics of M6495: The affinity of M6495 for human ADAMTS-5 was determined via Sapidyne's 'in solution' affinity platform Kinetic Exclusion Assay (KinExA). To determine the binding region of M6495 within ADAMTS-5, binding experiments were performed using SPR (Surface plasmon resonance). Inhibition of enzymatic activity of ADAMTS-5 by M6495 was analyzed with a fluorescence resonance energy transfer (FRET)-based enzymatic activity assay. The specificity of M6495 towards ADAMTS-5 was assessed by binding experiments to homologous metalloproteinases. Explant assay and co-culture: The effect of M6495 (half maximal inhibitory concentration [IC 50 ]) in bovine and human cartilage explants stimulated with pro-inflammatory cytokines was determined. Biomarkers of cartilage turnover, including GAG and huARGS, were investigated in the supernatant after culture had ended. Bovine cartilage explants and synovial membranes were coincubated. GAG as a measure for aggrecan turnover, C2M as a marker for MMP (matrix metalloproteinase) mediated type II collagen degradation and C3M as a marker for MMP-mediated type III collagen degradation were analyzed in the supernatant of the cultures. Results: M6495 is a bifunctional Nanobody® of 28.1 kDa (i.e., one ADAMTS-5-neutralizing moiety and one [HSA]-binding moiety for in vivo half-life extension). M6495 binds ADAMTS-5, but not ADAMTS-1, ADAMTS-4 and ADAMTS-15. In-vitro binding studies revealed that M6495 binds to the catalytic and/or disintegrin domain of ADAMTS-5 with high affinity. Results from FRET assays indicate a concentrationdependent and complete inhibition of the enzymatic activity of ADAMTS-5 by M6495. M6495 dose-dependently inhibited GAG release in bovine-, and GAG and huARGS release in human cartilage explant assays. In the co-culture model, matrix degradation was induced in the presence of synovium. First aggrecan was degraded (GAG release) followed by type II collagen degradation (C2M release). M6495 inhibited both. In addition, C3M was determined in the supernatant of the coculture system. Our data revealed a significant induction of C3M levels in co-cultures compared to t...

Conclusions: Pro-inflammatory cytokines are elevated in SF, but not in serum, immediately after knee ACL injury. SF TNF-a level stay elevated over the first 5 years after ACL injury and this high TNF-a concentration may contribute to a later development of OA.

Aims To determine whether platelet-rich plasma (PRP) injection improves outcomes two years after acute Achilles tendon rupture. Methods A randomized multicentre two-arm parallel-group, participant- and assessor-blinded superiority trial was undertaken. Recruitment commenced on 28 July 2015 and two-year follow-up was completed in 21 October 2019. Participants were 230 adults aged 18 years and over, with acute Achilles tendon rupture managed with non-surgical treatment from 19 UK hospitals. Exclusions were insertion or musculotendinous junction injuries, major leg injury or deformity, diabetes, platelet or haematological disorder, medication with systemic corticosteroids, anticoagulation therapy treatment, and other contraindicating conditions. Participants were randomized via a central online system 1:1 to PRP or placebo injection. The main outcome measure was Achilles Tendon Rupture Score (ATRS) at two years via postal questionnaire. Other outcomes were pain, recovery goal attainment, and quality of life. Analysis was by intention-to-treat. Results A total of 230 participants were randomized, 114 to PRP and 116 to placebo. Two-year questionnaires were sent to 216 participants who completed a six-month questionnaire. Overall, 182/216 participants (84%) completed the two-year questionnaire. Participants were aged a mean of 46 years (SD 13.0) and 25% were female (57/230). The majority of participants received the allocated intervention (219/229, 96%). Mean ATRS scores at two years were 82.2 (SD 18.3) in the PRP group (n = 85) and 83.8 (SD 16.0) in the placebo group (n = 92). There was no evidence of a difference in the ATRS at two years (adjusted mean difference -0.752, 95% confidence interval -5.523 to 4.020; p = 0.757) or in other secondary outcomes, and there were no re-ruptures between 24 weeks and two years. Conclusion PRP injection did not improve patient-reported function or quality of life two years after acute Achilles tendon rupture compared with placebo. The evidence from this study indicates that PRP offers no patient benefit in the longer term for patients with acute Achilles tendon rupture. Cite this article: Bone Joint J 2022;104-B(11):1256–1265.

from pre-1-to-End-tests, except for LPA (+2.3%) and total-SED-time (and À2.7%). Total-daily-counts in the End-test increased for controls (+6,7%) but significantly more for the exercise group (+17,3%). Between the exercise groups´two pre-tests, no significant difference was observed, except for total-PA (+4.6%) and SED-total (À2.4%). Conclusion Elderly participating in supervised exercise increase PA-and decrease SED-time, which is of importance because it is linked with improved health for elderly, longevity and potent socioeconomic gains.

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