Serum ARGS-aggrecan in a phase 2 clinical trial targeting osteoarthritis.

OBJECTIVE: To monitor serum concentrations of the aggrecan alanine-arginine-glycine-serine (ARGS) neoepitope in a clinical trial of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 inhibition as disease-modifying therapy of knee osteoarthritis, and to investigate relationships between reduction in ARGS and change in cartilage thickness, knee-related pain and function. DESIGN: ROCCELLA trial participants received once-daily oral S201086 75, 150 or 300 mg, or placebo, for 52 weeks. Serum was collected at baseline, 4, 12, 28 and 52 weeks, and 2 weeks post-treatment with ARGS measured by an in-house immunoassay. Change from baseline to week 52 in central medial femorotibial compartment cartilage thickness was measured by magnetic resonance imaging, function and pain by Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) subscores. Associations between cumulative change in ARGS and change in cartilage thickness or WOMAC subscores were evaluated by linear regression. RESULTS: S201086 reduced serum levels of ARGS in a dose-dependent manner throughout the treatment period. Maximal reduction was at 4 weeks with a 58.5% [95% CI 60.8%, 56.2%] reduction of ARGS compared to baseline for 300 mg S201086. Two weeks post-treatment, ARGS concentrations rebounded with a dose-dependent overshoot compared to baseline levels. Cumulative change of ARGS concentration from baseline to week 52 had no effect on change in cartilage thickness (slope -0.8×10-6 [-2.9×10-6, 1.3×10-6]) or change in WOMAC pain and function (slopes -30×10-6 [-64×10-6, 5.2×10-6] and -97×10-6 [-214×10-6, 20×10-6], respectively) at week 52. CONCLUSION: Systemic inhibition of ADAMTS-5 resulted in markedly reduced serum ARGS, but change in serum ARGS concentrations showed no association with the progression of cartilage thinning, or patient reported pain and function.

Osteoarthritis endotype discovery via clustering of biochemical marker data.

OBJECTIVES: Osteoarthritis (OA) patient stratification is an important challenge to design tailored treatments and drive drug development. Biochemical markers reflecting joint tissue turnover were measured in the IMI-APPROACH cohort at baseline and analysed using a machine learning approach in order to study OA-dominant phenotypes driven by the endotype-related clusters and discover the driving features and their disease-context meaning. METHOD: Data quality assessment was performed to design appropriate data preprocessing techniques. The k-means clustering algorithm was used to find dominant subgroups of patients based on the biochemical markers data. Classification models were trained to predict cluster membership, and Explainable AI techniques were used to interpret these to reveal the driving factors behind each cluster and identify phenotypes. Statistical analysis was performed to compare differences between clusters with respect to other markers in the IMI-APPROACH cohort and the longitudinal disease progression. RESULTS: Three dominant endotypes were found, associated with three phenotypes: C1) low tissue turnover (low repair and articular cartilage/subchondral bone turnover), C2) structural damage (high bone formation/resorption, cartilage degradation) and C3) systemic inflammation (joint tissue degradation, inflammation, cartilage degradation). The method achieved consistent results in the FNIH/OAI cohort. C1 had the highest proportion of non-progressors. C2 was mostly linked to longitudinal structural progression, and C3 was linked to sustained or progressive pain. CONCLUSIONS: This work supports the existence of differential phenotypes in OA. The biomarker approach could potentially drive stratification for OA clinical trials and contribute to precision medicine strategies for OA progression in the future. TRIAL REGISTRATION NUMBER: NCT03883568.

The role of cartilage glycosaminoglycan structure in gagCEST.

Glycosaminoglycan (GAG) chemical exchange saturation transfer (gagCEST) is a potential method for cartilage quality assessment. The aim of this study was to investigate how the gagCEST effect depends on the types and molecular organization of GAG typically found in articular cartilage. gagCEST was performed on different concentrations of GAG in various forms: free chains of chondroitin sulfate (CS) of different types (-A and -C) and GAG bound to protein in aggregated and nonaggregated aggrecan extracted from calf articular cartilage. The measured magnetization transfer ratio asymmetry (MTRasym ) was compared with known GAG concentrations or GAG concentrations determined through biochemical analysis. The gagCEST effect was assessed through the linear regression coefficient with 95% confidence interval of MTRasym per GAG concentration. We observed a lower gagCEST effect in phantoms containing a mixture of CS-A and CS-C compared with phantoms containing mainly CS-A. The difference in response corresponds well to the difference in CS-A concentration. GAG bound in aggrecan from calf articular cartilage, where CS-A is assumed to be the major type of GAG, produed a similar gagCEST effect as that observed for free CS-A. The effect was also similar for aggregated (ie, bound to hyaluronic acid) and nonaggregated aggrecan. In conclusion, our results indicate that the aggrecan structure in itself does not impact the gagCEST effect, but that the effect is strongly dependent on GAG type. In phantoms, the current implementation of gagCEST is sensitive to CS-A while for CS-C, the main GAG component in mature human articular cartilage, the sensitivity is limited. This difference in gagCEST sensitivity between GAG types detected in phantoms is a strong motivation to also explore the possibility of a similar effect in vivo.

Juvenile idiopathic arthritis patients have a distinct cartilage and bone biomarker profile that differs from healthy and knee-injured children.

OBJECTIVES: Joint destruction is a hallmark of juvenile idiopathic arthritis (JIA). Clinical evaluation and radiographic imaging are current methods to identify destruction. Biomarkers could aid an earlier and more sensitive diagnosis. Our aim was to investigate levels of bone and cartilage degradation biomarkers in JIA patients, compared to healthy children or juveniles with knee injuries. METHODS: Triple-paired synovial fluid, plasma and urine samples from 29 JIA patients were compared to 61 plasma samples from healthy children and synovial fluid from 41 knee-injured juveniles. Cartilage biomarkers ARGS neoepitope of aggrecan (ARGS), cartilage oligomeric matrix protein (COMP), type II collagen epitope (C2C), bone biomarkers N-terminal type I collagen cross-linked telopeptide (NTX-I) and tartrate-resistant acid phosphatase 5b (TRAP5b) were analysed by immunoassays. RESULTS: Plasma levels of ARGS, C2C, COMP and TRAP5b were increased in JIA compared to healthy children. Compared to knee-injured juveniles, synovial fluid C2C and TRAP5b were increased in JIA, while ARGS and COMP were decreased. For JIA patients, local (synovial fluid) and systemic (plasma/urine) levels of bone biomarkers correlated positively; age correlated negatively to plasma levels of C2C and TRAP5b; no correlation was found between biomarkers and gender, affected joint count, disease duration or medication. CONCLUSIONS: Elevated levels of destruction biomarkers in JIA compared to healthy children indicate a potential to serve as clinical tools for destructive joint disease. High levels of TRAP5b, NTX-I and collagen II in JIA in contrast to more pronounced aggrecan and COMP degradation in juvenile knee injuries, suggests that JIA patients have a unique biomarker pattern, different from healthy and knee-injured children.

Higher aggrecan 1-F21 epitope concentration in synovial fluid early after anterior cruciate ligament injury is associated with worse knee cartilage quality assessed by gadolinium enhanced magnetic resonance imaging 20 years later.

BACKGROUND: To investigate if cartilage related biomarkers in synovial fluid are associated with knee cartilage status 20 years after an anterior cruciate ligament (ACL) injury. METHODS: We studied 25 patients with a complete ACL rupture without subsequent ACL reconstruction or radiographic knee OA. All had a delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) 20 years after the ACL injury, using the T1 transverse relaxation time in the presence of gadolinium (T1Gd) which estimates the concentration of glycosaminoglycans in hyaline cartilage. Synovial fluid samples were aspirated acutely (between 0 and 18 days) and during 1 to 5 follow up visits between 0.5 and 7.5 years after injury. We quantified synovial fluid concentrations of aggrecan (epitopes 1-F21 and ARGS), cartilage oligomeric matrix protein, matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 by immunoassays, and sulfated glycosaminoglycans by Alcian blue precipitation. Western blot was used for qualitative analyses of aggrecan fragments in synovial fluid and cartilage samples. RESULTS: Western blot indicated that the 1-F21 epitope was located within the chondroitin sulfate 2 region of aggrecan. Linear regression analyses (adjusted for age, sex, body mass index and time between injury and sampling) showed that acute higher synovial fluid 1-F21-aggrecan concentrations were associated with shorter T1Gd values 20 years after injury, i.e. inferior cartilage quality (standardized effects between - 0.67 and - 1.0). No other statistically significant association was found between molecular biomarkers and T1Gd values. CONCLUSION: Higher acute synovial fluid 1-F21-aggrecan concentrations in ACL injured patients, who managed to cope without ACL reconstruction and were without radiographic knee OA, were associated with inferior knee cartilage quality assessed by dGEMRIC 20 years after injury.

Activation of Complement by Pigment Epithelium-Derived Factor in Rheumatoid Arthritis.

The aim of this study was to identify molecules that trigger complement activation in rheumatic joints. C4d, the final cleavage product of C4 activation, is found in the diseased joint and can bind covalently to complement-activating molecules. By using a highly specific Ab against a cleavage neoepitope in C4d, several molecules that were specifically bound to C4d were identified from pooled synovial fluid (SF) from four rheumatoid arthritis (RA) patients. One of these molecules, pigment epithelium-derived factor (PEDF), is a broadly expressed multifunctional member of the serine proteinase inhibitor family. Using ELISA, we confirmed the presence of various amounts of complexes between PEDF and C4d in the SF from 30 RA patients, whereas none were detected in SF from control subjects. Correlation analyses suggested that, in arthritis patients, C4d-PEDF complexes found in sera arise from the joints, as well as from other tissues, and levels of the complexes did not differ in sera of RA patients and healthy controls. When immobilized, recombinant PEDF expressed in eukaryotic cells activated the classical complement pathway but not the alternative or lectin pathways. C1q protein was demonstrated to bind immobilized PEDF, and PEDF was shown to bind to immobilized C1q, in particular its head regions, which are known to interact with other activators of the classical pathway. Our results call for further investigation into the role of PEDF in inflammatory processes in the joint, which, in combination with classical complement activation, appears to be part of a (patho-)physiologic response.

Plasma pro-inflammatory cytokines, IgM-uria and cardiovascular events in patients with chest pain: A comparative study.

BACKGROUND: Risk stratification of patients presenting with acute chest pain is crucial for immediate and long-term management. Traditional predictors are suboptimal; therefore inflammatory biomarkers are studied for clinical assessment of patients at risk. Recently, we reported the association of IgM-uria with worse cardiovascular outcome in patients with acute chest pain. In this study, in the same cohort of patients with chest pain, we compared the value of IgM-uria to pro-inflammatory cytokines in predicting the occurrence of subsequent cardiovascular events. METHODS: A total of 178 consecutive patients presenting with acute chest pain to the emergency department at the University Hospital of Lund, were recruited. Twenty-seven of 57 patients with acute coronary syndrome (ACS), and 18 of 118 patients with non-specific chest pain at baseline developed a subsequent major cardiovascular event during the 18 months follow-up. Urinary proteins (IgM-uria and Microalbuminuria) and plasma inflammatory markers (IL-6, Il-8, IL-10, IFN-γ and TNF-α) were measured at time of admission. RESULTS: Using the receiver operating characteristic curves, the area under the curve for predicting cardiovascular events was 0.71 (95%CI 0.61-0.81) for IgM-uria, 0.61 (95%CI 0.51-0.71) for IL-6, 0.63 (95%CI 0.53-0.72) for IL-8, 0.65 (95%CI 0.56-0.74) for IL-10, and 0.64 (95% CI 0.54-0.74) for TNF-α. In multivariate Cox-regression analysis adjusted for age, microalbuminuria, IgM-uria, IL-10, TNF-α, troponin T, hsCRP and ACS at baseline; IgM-uria was the only biomarker that remained an independent predictor of outcome (HR = 4.2, 95%CI 2.2-7.8, p < 0.001). CONCLUSION: In patients with chest pain with or without acute coronary syndrome, IgM-uria could better predict the occurrence of cardiovascular events than plasma pro-inflammatory cytokines.

From biochemical markers to molecular endotypes of osteoarthritis: a review on validated biomarkers.

INTRODUCTION: Osteoarthritis (OA) affects over 500 million people worldwide. OA patients are symptomatically treated, and current therapies exhibit marginal efficacy and frequently carry safety-risks associated with chronic use. No disease-modifying therapies have been approved to date leaving surgical joint replacement as a last resort. To enable effective patient care and successful drug development there is an urgent need to uncover the pathobiological drivers of OA and how these translate into disease endotypes. Endotypes provide a more precise and mechanistic definition of disease subgroups than observable phenotypes, and a panel of tissue- and pathology-specific biochemical markers may uncover treatable endotypes of OA. AREAS COVERED: We have searched PubMed for full-text articles written in English to provide an in-depth narrative review of a panel of validated biochemical markers utilized for endotyping of OA and their association to key OA pathologies. EXPERT OPINION: As utilized in IMI-APPROACH and validated in OAI-FNIH, a panel of biochemical markers may uncover disease subgroups and facilitate the enrichment of treatable molecular endotypes for recruitment in therapeutic clinical trials. Understanding the link between biochemical markers and patient-reported outcomes and treatable endotypes that may respond to given therapies will pave the way for new drug development in OA.

A missense mutation in the aggrecan C-type lectin domain disrupts extracellular matrix interactions and causes dominant familial osteochondritis dissecans.

Osteochondritis dissecans is a disorder in which fragments of articular cartilage and subchondral bone dislodge from the joint surface. We analyzed a five-generation family in which affected members had autosomal-dominant familial osteochondritis dissecans. A genome-wide linkage analysis identified aggrecan (ACAN) as a prime candidate gene for the disorder. Sequence analysis of ACAN revealed heterozygosity for a missense mutation (c.6907G > A) in affected individuals, resulting in a p.V2303M amino acid substitution in the aggrecan G3 domain C-type lectin, which mediates interactions with other proteins in the cartilage extracellular matrix. Binding studies with recombinant mutated and wild-type G3 proteins showed loss of fibulin-1, fibulin-2, and tenascin-R interactions for the V2303M protein. Mass spectrometric analyses of aggrecan purified from patient cartilage verified that V2303M aggrecan is produced and present in the tissue. Our results provide a molecular mechanism for the etiology of familial osteochondritis dissecans and show the importance of the aggrecan C-type lectin interactions for cartilage function in vivo.

Aggrecanase cleavage in juvenile idiopathic arthritis patients is minimally detected in the aggrecan interglobular domain but robust at the aggrecan C-terminus.

OBJECTIVE: To investigate aggrecan degradation in juvenile idiopathic arthritis (JIA). METHODS: The pattern and abundance of aggrecan fragments in synovial fluid (SF) aspirates from JIA patients were analyzed and compared with aggrecan fragments in SF from patients with other arthritides, children with knee injury, and a knee-healthy reference group. Concentrations of sulfated glycosaminoglycan (sGAG) in SF were measured by Alcian blue precipitation assay. Aggrecan fragments were purified by dissociative CsCl density-gradient centrifugation, deglycosylated, and analyzed by Western blot using antibodies specific for either aggrecanase-derived ARGS, SELE, and KEEE neoepitopes or the aggrecan G3 domain. RESULTS: The concentration of sGAG in SF from patients with JIA was significantly lower compared with that in SF from patients with osteoarthritis (OA) (P < 0.001), patients with juvenile knee injury (P = 0.006), and knee-healthy controls (P = 0.022). Western blot analysis revealed KEEE, SELE, and G3 fragments generated by aggrecanase cleavage in the chondroitin sulfate-rich region of aggrecan in patients with JIA. The pattern of aggrecan fragments in JIA patients was not identical to that in pooled OA SF, although there were notable similarities. Surprisingly, aggrecanase-derived ARGS fragments were barely detectable in JIA SF, in marked contrast to levels in OA SF. CONCLUSION: Aggrecanases appear to cleave minimally in the interglobular domain of aggrecan in JIA patients despite robust levels of cleavage in the chondroitin sulfate-rich region. These results suggest that in JIA, unlike other arthritides, aggrecanase cleavage in the aggrecan interglobular domain might not be a major pathogenic event.

MMP proteolysis of the human extracellular matrix protein aggrecan is mainly a process of normal turnover.

Although it has been shown that aggrecanases are involved in aggrecan degradation, the role of MMP (matrix metalloproteinase) aggrecanolysis is less well studied. To investigate MMP proteolysis of human aggrecan, in the present study we used neoepitope antibodies against MMP cleavage sites and Western blot analysis to identify MMP-generated fragments in normal and OA (osteoarthritis/osteoarthritic) cartilage, and in normal, knee injury and OA and SF (synovial fluid) samples. MMP-3 in vitro digestion showed that aggrecan contains six MMP cleavage sites, in the IGD (interglobular domain), the KS (keratan sulfate) region, the border between the KS region and CS (chondroitin sulfate) region 1, the CS1 region, and the border between the CS2 and the G3 domain, and kinetic studies showed a specific order of digestion where the cleavage between CS2 and the G3 domain was the most preferred. In vivo studies showed that OA cartilage contained (per dry weight) 3.4-fold more MMP-generated FFGV fragments compared with normal cartilage, and although aggrecanase-generated SF-ARGS concentrations were increased 14-fold in OA and knee-injured patients compared with levels in knee-healthy reference subjects, the SF-FFGV concentrations did not notably change. The results of the present study suggest that MMPs are mainly involved in normal aggrecan turnover and might have a less-active role in aggrecan degradation during knee injury and OA.

Quantification of chondroitin sulfate, hyaluronic acid and N-glycans in synovial fluid - A technical performance study.

Objective: To validate a quantitative high performance liquid chromatography (HPLC) assay for chondroitin sulfate (CS) and hyaluronic acid (HA) in synovial fluid, and to analyze glycan-patterns in patient samples. Design: Synovial fluid from osteoarthritis (OA, n â€‹= â€‹25) and knee-injury (n â€‹= â€‹13) patients, a synovial fluid pool (SF-control) and purified aggrecan, were chondroitinase digested and together with CS- and HA-standards fluorophore labelled prior to quantitative HPLC analysis. N-glycan profiles of synovial fluid and aggrecan were assessed by mass spectrometry. Results: Unsaturated uronic acid and sulfated-N-acetylgalactosamine (ΔUA-GalNAc4S and ΔUA-GalNAc6S) contributed to 95% of the total CS-signal in the SF-control sample. For HA and the CS variants in SF-control the intra- and inter-experiment coefficient of variation was between 3-12% and 11-19%, respectively; tenfold dilution gave recoveries between 74 and 122%, and biofluid stability test (room temperature storage and freeze-thaw cycles) showed recoveries between 81 and 140%. Synovial fluid concentrations of the CS variants ΔUA-GalNAc6S and ΔUA2S-GalNAc6S were three times higher in the recent injury group compared to the OA group, while HA was four times lower. Sixty-one different N-glycans were detected in the synovial fluid samples, but there were no differences in levels of N-glycan classes between patient groups. The CS-profile (levels of ΔUA-GalNAc4S and ΔUA-GalNAc6S) in synovial fluid resembled that of purified aggrecan from corresponding samples; the contribution to the N-glycan profile in synovial fluid from aggrecan was low. Conclusions: The HPLC-assay is suitable for analyzing CS variants and HA in synovial fluid samples, and the GAG-pattern differs between OA and recently knee injured subjects.

Aggrecan accumulates at sites of increased pulmonary arterial pressure in idiopathic pulmonary arterial hypertension.

Expansion of extracellular matrix occurs in all stages of pulmonary angiopathy associated with pulmonary arterial hypertension (PAH). In systemic arteries, dysregulation and accumulation of the large chondroitin-sulfate proteoglycan aggrecan is associated with swelling and disruption of vessel wall homeostasis. Whether aggrecan is present in pulmonary arteries, and its potential roles in PAH, has not been thoroughly investigated. Here, lung tissue from 11 patients with idiopathic PAH was imaged using synchrotron radiation phase-contrast microcomputed tomography (TOMCAT beamline, Swiss Light Source). Immunohistochemistry for aggrecan core protein in subsequently sectioned lung tissue demonstrated accumulation in PAH compared with failed donor lung controls. RNAscope in situ hybridization indicated ACAN expression in vascular endothelium and smooth muscle cells. Based on qualitative histological analysis, aggrecan localizes to cellular, rather than fibrotic or collagenous, lesions. Interestingly, ADAMTS15, a potential aggrecanase, was upregulated in pulmonary arteries in PAH. Aligning traditional histological analysis with three-dimensional renderings of pulmonary arteries from synchrotron imaging identified aggrecan in lumen-reducing lesions containing loose, cell-rich connective tissue, at sites of intrapulmonary bronchopulmonary shunting, and at sites of presumed elevated pulmonary blood pressure. Our findings suggest that ACAN expression may be an early response to injury in pulmonary angiopathy and supports recent work showing that dysregulation of aggrecan turnover is a hallmark of arterial adaptations to altered hemodynamics. Whether cause or effect, aggrecan and aggrecanase regulation in PAH are potential therapeutic targets.

The Effects of Different Management Strategies or Rehabilitation Approaches on Knee Joint Structural and Molecular Biomarkers Following Traumatic Knee Injury: A Systematic Review of Randomized Controlled Trials for the OPTIKNEE Consensus.

OBJECTIVE: To summarize the effectiveness of management strategies and rehabilitation approaches for knee joint structural and molecular biomarker outcomes following anterior cruciate ligament (ACL) and/or meniscal tear. DESIGN: Intervention systematic review. LITERATURE SEARCH: We searched the MEDLINE, Embase, CINAHL, CENTRAL, and SPORTDiscus databases from their inception up to November 3, 2021. STUDY SELECTION CRITERIA: We included randomized controlled trials (RCTs) investigating the effectiveness of management strategies or rehabilitation approaches for structural/molecular biomarkers of knee joint health following ACL and/or meniscal tear. DATA SYNTHESIS: We included 5 RCTs (9 papers) with primary ACL tear (n = 365). Two RCTs compared initial management strategies (rehabilitation plus early vs optional delayed ACL surgery), reporting on structural biomarkers (radiographic osteoarthritis, cartilage thickness, meniscal damage) in 5 papers and molecular biomarkers (inflammation, cartilage turnover) in 1 paper. Three RCTs compared different post-ACL reconstruction (ACLR) rehabilitation approaches (high vs low intensity plyometric exercises, accelerated vs nonaccelerated rehabilitation, continuous passive vs active motion), reporting on structural biomarkers (joint space narrowing) in 1 paper and molecular biomarkers (inflammation, cartilage turnover) in 2 papers. RESULTS: There were no differences in structural or molecular biomarkers between post-ACLR rehabilitation approaches. One RCT comparing initial management strategies demonstrated that rehabilitation plus early ACLR was associated with greater patellofemoral cartilage thinning, elevated inflammatory cytokine response, and reduced incidence of medial meniscal damage over 5 years compared to rehabilitation with no/delayed ACLR. CONCLUSION: Very low-certainty evidence suggests that different initial management strategies (rehabilitation plus early vs optional delayed ACL surgery) but not postoperative rehabilitation approaches may influence the incidence of meniscal damage, patellofemoral cartilage loss and cytokine concentrations over 5 years post-ACL tear. J Orthop Sports Phys Ther 2023;53(4):1-22. Epub: 20 February 2023. doi:10.2519/jospt.2023.11576.

Sustained delivery of a heterodimer bone morphogenetic protein-2/7 via a collagen hydroxyapatite scaffold accelerates and improves critical femoral defect healing.

Despite the glimmer of hope provided by the discovery and commercialization of bone morphogenetic protein-2 (BMP-2) as a bone graft substitute, side effects related to the use of supraphysiological doses have hindered its clinical usage. In this study, we compared the osteoinductive potential of BMP-2 homodimer with a heterodimer of BMP-2/7, both delivered via a collagen-hydroxyapatite (CHA) scaffold delivery system, with the aim to reduce the overall therapeutic BMP doses and the associated side-effects. We first show that the incorporation of hydroxyapatite in collagen-based BMP delivery systems is pivotal for achieving efficient BMP sequestration and controlled release. Using an ectopic implantation model, we then showed that the CHA+BMP-2/7 was more osteoinductive than CHA+BMP-2. Further evaluation of the molecular mechanisms responsible for this increased osteoinductivity at an early stage in the regeneration process indicated that the CHA+BMP-2/7 enhanced progenitor cell homing at the implantation site, upregulated the key transcriptomic determinants of bone formation, and increased the production of bone extracellular matrix components. Using fluorescently labelled BMP-2/7 and BMP-2, we demonstrated that the CHA scaffold provided a long-term delivery of both molecules for at least 20 days. Finally, using a rat femoral defect model, we showed that an ultra-low dose (0.5 µg) of BMP-2/7 accelerated fracture healing and performed at a level comparable to 20-times higher BMP-2 dose. Our results indicate that the sustained delivery of BMP-2/7 via a CHA scaffold could bring us a step closer in the quest for the use of physiological growth factor doses in fracture healing. STATEMENT OF SIGNIFICANCE: • Incorporation of hydroxyapatite (HA) in a collagen scaffold dramatically improves bone morphogenic protein (BMP) sequestration via biophysical interactions with BMP, thereby providing more controlled BMP release compared with pristine collagen. • We then investigate the molecular mechanisms responsible for increased osteoinductive potential of a heterodimer BMP-2/7 with is clinically used counterpart, the BMP-2 homodimer. • The superior osteoinductive properties of BMP-2/7 are a consequence of its direct positive effect on progenitor cell homing at the implantation site, which consequently leads to upregulation of cartilage and bone related genes and biochemical markers. • An ultra-low dose of BMP-2/7 delivered via a collagen-HA (CHA) scaffold leads to accelerated healing of a critical femoral defect in rats while a 20-times higher BMP-2 dose was required to achieve comparable results.

Biological variation of human aggrecan ARGS neoepitope in synovial fluid and serum in early-stage knee osteoarthritis and after knee injury.

Objective: To determine biological variation of the aggrecanase-generated aggrecan ARGS neoepitope in serum (sARGS) and synovial fluid (sfARGS) within and between patients with osteoarthritis (OA) or anterior cruciate ligament (ACL) injury. Design: Matched samples of serum and synovial fluid were available, as parts of clinical trials, from i) 16 subjects with early-stage OA on 8 occasions over 1 year, and ii) 120 subjects with acute ACL injury with samples available from at least 2 of 6 visits over 5 years. We used an in-house immunoassay to quantify ARGS and one-way ANOVA for statistical analyses. Results: Variability in ARGS was higher in synovial fluid than in serum in both patient groups. Subjects with OA had the lowest variability both within and between patients and showed no variation over time in the degree of variability or in the cross-sectional mean, neither in serum nor in synovial fluid. After ACL injury, the concentration and the variability of ARGS was highest immediately after injury, with a subsequent decline both in concentration and in variability with time. In both patient groups there was a positive correlation between sfARGS and sARGS both within and between individuals (correlation coefficients between 0.16 and 0.20). Conclusions: The biological variation of ARGS is lower in serum than in synovial fluid, and lower in OA than after ACL injury. Serum ARGS is a measure of the total release of ARGS aggrecan from the whole body and a poor reflection of the release of ARGS aggrecan within the affected joint.

Associations Between Physical Activity, Self-reported Joint Function, and Molecular Biomarkers in Working Age Individuals With Hip and/or Knee Osteoarthritis.

Objective: Previous research has suggested an association between physical activity (PA), joint function, and molecular biomarkers, but more studies are needed. The aim of this study was to explore the associations between PA or self-reported joint function and molecular biomarkers of cartilage and inflammation in individuals with hip and/or knee osteoarthritis (OA). Specific objectives were to explore the correlations between (1) the change over 3 months in self-reported PA/joint function and the change in molecular biomarkers (2) objectively measured PA and molecular biomarkers measured at 3-month follow-up. Design: Working age participants (n = 91) were recruited from a cluster randomized controlled trial. Self-reported PA, joint function, and serum samples were collected at baseline and after 3 months. Serum concentrations of the inflammatory marker C-reactive protein (CRP) and the cartilage markers Alanine-Arginine-Glycine-Serine (ARGS)-aggrecan, cartilage oligomeric matrix protein (COMP), and type II collagen C2C were analyzed by immunoassays. Objectively measured PA (steps/day) was collected during 12 weeks from activity trackers used by 53 participants. Associations were analyzed with Spearman's rank correlation. Results: There was a weak negative correlation between the change in self-reported PA and the change in COMP (r s = -0.256, P = .040) but not for the other molecular biomarkers. There were no correlations between the change in self-reported joint function and the change in molecular biomarkers or between the average steps/day and the molecular biomarkers at follow-up (r s ⩽ -0.206, P ⩾ .06). Conclusion: In general, no or only weak associations were found between PA/joint function and molecular biomarkers. Future research recommends including participants with lower PA, extend the follow-up, and use a design that allows comparisons.

Inflammatory cytokines and mechanical injury induce post-traumatic osteoarthritis-like changes in a human cartilage-bone-synovium microphysiological system.

BACKGROUND: Traumatic knee injuries in humans trigger an immediate increase in synovial fluid levels of inflammatory cytokines that accompany impact damage to joint tissues. We developed a human in vitro cartilage-bone-synovium (CBS) coculture model to study the role of mechanical injury and inflammation in the initiation of post-traumatic osteoarthritis (PTOA)-like disease. METHODS: Osteochondral plugs (cartilage-bone, CB) along with joint capsule synovium explants (S) were harvested from 25 cadaveric distal femurs from 16 human donors (Collin's grade 0-2, 23-83years). Two-week monocultures (cartilage (C), bone (B), synovium (S)) and cocultures (CB, CBS) were established. A PTOA-like disease group was initiated via coculture of synovium explants with mechanically impacted osteochondral plugs (CBS+INJ, peak stress 5MPa) with non-impacted CB as controls. Disease-like progression was assessed through analyses of changes in cell viability, inflammatory cytokines released to media (10-plex ELISA), tissue matrix degradation, and metabolomics profile. RESULTS: Immediate increases in concentrations of a panel of inflammatory cytokines occurred in CBS+INJ and CBS cocultures and cultures with S alone (IL-1, IL-6, IL-8, and TNF-α among others). CBS+INJ and CBS also showed increased chondrocyte death compared to uninjured CB. The release of sulfated glycosaminoglycans (sGAG) and associated ARGS-aggrecan neoepitope fragments to the medium was significantly increased in CBS and CBS+INJ groups. Distinct metabolomics profiles were observed for C, B, and S monocultures, and metabolites related to inflammatory response in CBS versus CB (e.g., kynurenine, 1-methylnicotinamide, and hypoxanthine) were identified. CONCLUSION: CBS and CBS+INJ models showed distinct cellular, inflammatory, and matrix-related alterations relevant to PTOA-like initiation/progression. The use of human knee tissues from donors that had no prior history of OA disease suggests the relevance of this model in highlighting the role of injury and inflammation in earliest stages of PTOA progression.

Novel missense ACAN gene variants linked to familial osteochondritis dissecans cluster in the C-terminal globular domain of aggrecan.

The cartilage aggrecan proteoglycan is crucial for both skeletal growth and articular cartilage function. A number of aggrecan (ACAN) gene variants have been linked to skeletal disorders, ranging from short stature to severe chondrodyplasias. Osteochondritis dissecans is a disorder where articular cartilage and subchondral bone fragments come loose from the articular surface. We previously reported a missense ACAN variant linked to familial osteochondritis dissecans, with short stature and early onset osteoarthritis, and now describe three novel ACAN gene variants from additional families with this disorder. Like the previously described variant, these are autosomal dominant missense variants, resulting in single amino acid residue substitutions in the C-type lectin repeat of the aggrecan G3 domain. Functional studies showed that neither recombinant variant proteins, nor full-length variant aggrecan proteoglycan from heterozygous patient cartilage, were secreted to the same level as wild-type aggrecan. The variant proteins also showed decreased binding to known cartilage extracellular matrix ligands. Mapping these and other ACAN variants linked to hereditary skeletal disorders showed a clustering of osteochondritis dissecans-linked variants to the G3 domain. Taken together, this supports a link between missense ACAN variants affecting the aggrecan G3 domain and hereditary osteochondritis dissecans.