OBJECTIVES:After total knee arthroplasty (TKA), ∼30% of knee osteoarthritis (KOA) patients show little symptomatic improvement. Earlier studies have correlated urinary (u) type 2 collagen C terminal cleavage peptide assay (C2C-HUSA), which detects a fragment of cartilage collagen breakdown, with KOA progression. This study determines whether C2C levels in urine, synovial fluid, or their ratio, are associated with post-surgical outcomes. METHODS:From a large sample of 489 subjects, diagnosed with primary KOA undergoing TKA, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) pain and function scores were collected at baseline (time of surgery) and one-year post-TKA. Baseline urine (u) and synovial fluid (sf) were analysed using the IBEX-C2C-HUSA assay, with higher values indicating higher amounts of cartilage degradation. For urine, results were normalised to creatinine. Furthermore, subjects' changes in WOMAC scores were categorised based on percent reduction in pain or improvement in function, compared to baseline, such that >66.7%, >33.3 to ≤66.7%, and ≤33.3% denoted "strong", "moderate" and "mild/worse" responses, respectively. Associations of individual biofluid C2C-HUSA levels, or their ratio, with change in WOMAC pain and function scores up to one-year post-TKA, or category of change, were analysed by linear, logistic, or cumulative odds models. RESULTS:Higher baseline uC2C-HUSA levels or a lower ratio of baseline sfC2C-HUSA to uC2C-HUSA were associated with improvements in WOMAC pain by linear multivariable modelling [odds ratio -0.40 (95% confidence interval -0.76, -0.05) p = 0.03; 0.36 (0.01, 0.71), p = 0.04, respectively], while sfC2C-HUSA alone was not. However, lower ratios of sfC2C-HUSA to uC2C-HUSA were associated with improvements in WOMAC function [1.37 (0.18, 2.55), p = 0.02], while sfC2C-HUSA and uC2C-HUSA alone were not. Lower ratios of sfC2C-HUSA to uC2C-HUSA were also associated with an increased likelihood of a subject being categorised in a group where TKA was beneficial in both univariable [pain, 0.81 (0.68, 0.96), p = 0.02; function, 0.92 (0.85, 0.99), p = 0.035] and multivariable [pain, 0.81 (0.68, 0.97) p = 0.02; function, 0.92 (0.85, 1.00), p = 0.043] ordinal modelling, while sfC2C-HUSA and uC2C-HUSA alone were not. CONCLUSIONS:Overall, ratios of baseline sfC2C-HUSA to uC2C-HUSA, and baseline uC2C-HUSA, may play an important role in studying post-TKA surgical outcomes.

Purpose: Osteoarthritis (OA) is the most common form of arthritis with the knee being the most commonly affected joint. Total knee arthroplasty (TKA) is a cost-effective treatment for end-stage OA; however, ∼30% of patients show little improvement in pain or function up to 12 months post-TKA. C2C a fragment of type II collagen generated from its enzymatic degradation that can be detected in various biofluids. How levels of C2C in urine and other biofluids, like synovial fluid, relate to post-surgical patient reported pain and function outcomes remains to be determined.

The influence of vitamin D metabolites (at 1 X 10(-10) M) on the calcification of cartilage matrix (measured by 45Ca2+ uptake) and the C-propeptide of type II collagen (measured by radioimmunoassay) has been studied using organ cultures and chondrocytes isolated from growth plates of vitamin D-deficient and -sufficient 11-day-old rats. Vitamin D-deficient rats had reduced amounts of C-propeptide in their serum and freshly isolated growth plate chondrocytes. In all chondrocytes cultured from vitamin D-deficient animals, the C-propeptide content was maximal at 24 hr whereas calcification continued to increase for up to 72 hr. In organ and chondrocyte cultures of tissue from vitamin D-sufficient rats, both 1,25-dihydroxycholecalciferol (1,25(OH)2D3) and 24,25-dihydroxycholecalciferol (24,25(OH)2D3) were required for maximal stimulation of calcification and maximal increases in C-propeptide content. In these D-replete tissues, 24,25-(OH)2D3 had a less stimulatory effect on both calcification (organ and cell cultures) and C-propeptide (organ cultures only), while 1,25(OH)2D3 alone had no effect in cell cultures but an inhibitory effect in organ cultures. Studies of cells or tissue from growth plates of vitamin D-deficient rats demonstrated that 24,25(OH)2D3 alone produced maximal calcification and maximal increases in the C-propeptide content. 1,25(OH)2D3 generally had an inhibitory effect on both calcification and C-propeptide when used alone. In the presence of 1,25(OH)2D3, the stimulatory effect of 24,25(OH)2D3 was partly abrogated. Maximal stimulation of calcification and increases in C-propeptide by 24,25(OH)2D3 were observed at 1 X 10(-9) M and 1 X 10(-10) M. In none of these studies was there any effect on proteoglycan content.(ABSTRACT TRUNCATED AT 250 WORDS)

An early detection of Osteoarthritis is urgently needed and still not possible until today. The aim of the study was to assess whether molecular biomarkers of cartilage turnover are associated with longitudinal change in knee cartilage thickness during a 2 year period in individuals with increased risk of developing knee osteoarthritis. A secondary aim was to assess whether prior knee injury or subjective patient-reported outcomes at baseline (BL) were associated with articular cartilage changes. Nineteen volleyball players (mean age 46.5 ± 4.9 years, 47% male) with a 30-year history of regular high impact training were recruited. The serum biomarkers Cpropeptide of type II procollagen (CPII), cartilage oligomeric matrix protein (COMP), collagenase generated carboxy-terminal neoepitope of type II collagen (sC2C), cartilage intermediate layer protein 2 (CILP-2), and the urine biomarkers C-telopeptide of type II collagen (CTX-II) and collagenase-generated peptide(s) of type II collagen (C2C-HUSA) were assessed at BL and at 2 year follow up (FU). Femorotibial cartilage thinning, thickening and absolute thickness change between BL and FU was evaluated from magnetic resonance imaging. Subjective clinical status at BL was evaluated by the International Knee Documentation Committee Subjective Knee Form and the Short-Form 36 Physical Component Score. CILP-2 was significantly higher at FU and linearly associated with the absolute cartilage thickness change during the experimental period. Prior injury was a predictor of increased absolute cartilage thickness change. Measuring the change in the cartilage biomarker CILP-2 might be a valid and sensitive method to detect early development of knee osteoarthritis as CILP-2 appears to be related to cartilage thickness loss in certain individuals with increased risk of developing knee osteoarthritis. Prior knee injury may be predictive of increased articular cartilage thickness change.

BACKGROUND:This study aimed the feasibility to assess longitudinal changes in biomarkers of cartilage turnover and to determine their relationship with patient-rated outcomes over 2 years in volleyball athletes.METHODS:Thirty-seven athletes were studied: 18 adolescents (age 15.9 ± 0.64 years) in a 2-year intensive volleyball training program and 19 adult recreational volleyball players (age 46.5 ± 4.9 years). Blood and serum samples were taken at baseline (BL) and 2-year follow-up (FU). Subjects completed the International Knee Documentation Committee (IKDC) Subjective Knee Form and the Short-Form 36 (SF-36) at BL.RESULTS:Thirteen adolescents (72%) had open growth plates at BL (BL open adolescents), the rest had closed growth plates at BL (BL closed adolescents), and all but one adolescent had closed growth plates at FU as assessed by MRI. BL open and closed adolescents had greater levels of the cartilage degradation-based biomarkers 45 mer collagenase peptide of type II collagen (C2C-HUSA) and C-telopeptide of type II collagen (CTX-II) than adults. BL open adolescents showed decreases in C2CHUSA, collagen synthesis marker C-propeptide of type II procollagen (CPII), and CTXII, and adults showed increases in cartilage intermediate layer protein 2 (CILP-2) and C2C-HUSA. In adolescents, IKDC scores were correlated with CPII changes. In adults, SF-36 Physical Component Scores were correlated with cartilage oligomeric matrix protein (COMP) changes.CONCLUSION:Significant differences in biomarker levels over time show the feasibility to assess their changes. Greater levels of C2C-HUSA and CTX-II in adolescents than in adults may reflect increased cartilage turnover in response to higher joint loading. CPII and COMP may be more reflective of subjective patient outcomes. These biomarkers may thus be useful in assessing mechanical loading-induced cartilage changes, their associated symptoms, and Osteoarthritis risk in athletes.

We aimed to determine whether synovial fluid (SF) biomarkers can predict the progression of articular cartilage damage as determined by arthroscopic evaluation during and after anterior cruciate ligament (ACL) reconstruction.

Muscle dysfunction is the most important modifiable mediating factor in primary osteoarthritis (OA) because properly contracting muscles are a key absorber of forces acting on a joint. However, the pathological features of disuse muscle atrophy in OA patients have been rarely studied. Vastus medialis muscles of 14 female patients with OA (age range, 69 to 86 years), largely immobile for 1 or more years, were obtained during arthroplastic surgery and analyzed histologically. These were compared with female patients without arthritis, two with patellar fracture and two with patellar subluxation. Areas occupied by myofibers and adipose tissue were quantified. Large numbers of myofibers were lost in the vastus medialis of OA patients. The loss of myofibers was a possible cause of the reduction in muscle strength of the operated on knee. These changes were significantly correlated with an increase in intramuscular ectopic adipose tissue, and not observed in knees of nonarthritic patients. Resident platelet-derived growth factor receptor α-positive mesenchymal progenitor cells contributed to ectopic adipogenesis in vastus medialis muscles of OA patients. The present study suggests that significant loss of myofibers and ectopic adipogenesis in vastus medialis muscles are common pathological features of advanced knee OA patients with long-term loss of mobility. These changes may be related to the loss of joint function in patients with knee OA.

Objective.To evaluate the association of a sandwich assay for cartilage collagenase-mediated degradation, the C2C human urine sandwich assay (IB-C2C-HUSA), with early and late knee cartilage pathology and with progression of cartilage damage.Methods.A population-based cohort with knee pain, age 40–79 years, was evaluated at baseline (n = 253) and after mean 3.3 years (n = 161). We evaluated the IB-C2C-HUSA and a related competitive inhibition assay (C2C). The C2C assay was applied to serum (sC2C) and urine (uC2C). Based on knee radiographs and magnetic resonance imaging (MRI), 3 subgroups [no cartilage pathology, preradiographic cartilage pathology, and radiographic osteoarthritis (ROA)] were evaluated cross-sectionally for association with biomarker levels. Longitudinally, we evaluated whether baseline assays predict subsequent progression of cartilage degeneration, defined by MRI cartilage loss.Results.Cross-sectionally, statistically significant differences were seen in the 3 subgroups for IB-C2C-HUSA (p < 0.001), with the highest levels seen in ROA, and for sC2C (p = 0.023), while no differences were seen for uC2C (p = 0.501). Baseline IB-C2C-HUSA levels were higher in progressors vs nonprogressors (p = 0.003). In logistic regression analysis, only baseline IB-C2C-HUSA was associated with an increased risk of progression of cartilage damage (OR 1.78, 95% CI 1.03–3.09).Conclusion.The IB-C2C-HUSA degradation assay detects the generation of a pathology-related cartilage collagen peptide(s) that increase(s) with onset of degeneration of knee articular cartilage. The baseline values are associated with progression of cartilage degeneration over 3 subsequent years. This assay may have value in clinical OA trials. Further, it points to collagenase activity as a therapeutic target for controlling degeneration of articular cartilage.

There has been important recent progress in our understanding of the molecular pathology of osteoarthritis (OA) and how it might be treated. New technologies have been developed and others refined to identify patients for recruitment in clinical trials who exhibit measurable progression. Combined with the ability to determine more effectively short-term efficacy of treatment, significant obstacles are being removed that have negated or led to the failure of earlier trials. The future for disease-modifying osteoarthritis drug (DMOAD) development and more effective pain control is therefore much more encouraging. But it is extremely important that these new therapeutic and clinical trial opportunities receive timely recognition and support from regulatory authorities. The importance and clearly demonstrated value of the coordination of clinical research and private/public initiatives, such as the OA Initiative and the European APPROACH project, and involvement of informed patients in research and policy decision making cannot be over emphasised.

This study reports the effects of the iron chelator deferoxamine (DFO) on collagen cleavage, inflammation, and chondrocyte hypertrophy in relation to energy metabolism-related gene expression in osteoarthritic (OA) articular cartilage. Full-depth explants of human OA knee articular cartilage from arthroplasty were cultured with exogenous DFO (1–50 μM). Type II collagen cleavage and phospho-adenosine monophosphate-activated protein kinase (pAMPK) concentrations were measured using ELISAs. Gene expression studies employed real-time PCR and included AMPK analyses in PBMCs. In OA explants collagen cleavage was frequently downregulated by 10–50 μM DFO. PCR analysis of 7 OA patient cartilages revealed that 10 μM DFO suppressed expression of MMP-1, MMP-13, IL-1β, and TNFα and a marker of chondrocyte hypertrophy, COL10A1. No changes were observed in the expression of glycolysis-related genes. In contrast, expressions of genes associated with the mitochondrial Krebs cycle (TCA), AMPK, HIF1α, and COL2A1 were upregulated. AMPK gene expression was reduced in OA cartilage and increased in PBMCs from the same patients compared to healthy controls. Our studies demonstrate that DFO is capable of suppressing excessive collagenase-mediated type II collagen cleavage in OA cartilage and reversing phenotypic changes. The concomitant upregulation of proanabolic TCA-related gene expressions points to a potential for availability of energy generating substrates required for matrix repair by end-stage OA chondrocytes. This might normally be prevented by high whole-body energy requirements indicated by elevated AMPK expression in PBMCs of OA patients.

Combined individually tailored methods for diagnosis and therapy (theragnostics) could be beneficial in destructive diseases, such as rheumatoid arthritis. Nanoparticles are promising candidates for theragnostics due to their excellent biocompatibility. Nanoparticle modifications, such as improved surface coating, are in development to meet various requirements, although safety concerns mean that modified nanoparticles require further review before their use in medical applications is permitted. We have previously demonstrated that iron oxide nanoparticles with amino-polyvinyl alcohol (a-PVA) adsorbed on their surfaces have the unwanted effect of increasing human immune cell cytokine secretion. We hypothesized that this immune response was caused by free-floating PVA. The aim of the present study was to prevent unwanted immune reactions by further surface modification of the a-PVA nanoparticles. After cross-linking of PVA to nanoparticles to produce PVA-grafted nanoparticles, and reduction of their zeta potential, the effects on cell viability and cytokine secretion were analyzed. PVA-grafted nanoparticles still stimulated elevated cytokine secretion from human immune cells; however, this was inhibited after reduction of the zeta potential. In conclusion, covalent cross-linking of PVA to nanoparticles and adjustment of the surface charge rendered them nontoxic to immune cells, nonimmunogenic, and potentially suitable for use as theragnostic agents.

Background When I arrived in Canada in 1977 to establish and direct the Joint Diseases Laboratory at the Shriners Hospital, McGill University, I came from a strong background in connective tissue research in Britain. Originally, in the 1960s, we had national Collagen,Mucopolysaccharide and the Bone and Tooth interest groups that each met regularly and separately. Eventually, the first two groups merged to form the British Society for Matrix Biology. This was and continues to be a very important society as it provides a common meeting ground for students and scientists interested in connective tissue research where progress is shared and collaborations are forged and new training and job opportunities explored. But on arrival in Canada there was very little research in this fundamentally important area. Tony Melchers MRC group in periodontal physiology, that included Jaro Sodek, based in Toronto, was the only group of its kind at that time. There were researchers working on small soft tissue proteoglycans, in Edmonton, Alberta, namely Paul Scott and Carl Pearson; C.P Leblond and his colleagues at McGill who had contributed so much to our understanding of connective tissues and developmental biology, their work inspiring us all. Michel van der Rest was working on collagen in the adjacent Genetics Laboratory at the Shriners Hospital. Dick Cruess, chair of orthopaedics at McGill and Chief Surgeon at Shriners Hospital, who had invited me to start a group at the Shriners Hospital, and his colleague Nelson Mitchell, both based at the Royal Victoria Hospital, were involved in research on joint tissues. They were excellent early role models for the surgeon/ scientist. A number of arthritis researchers were scattered across Canada, mainly with interests in inflammatory arthritis. Connective tissues were never mentioned. Following my arrival, my contacts were mainly with Tony Melcher’s group and laboratories in the USA where there already existed a very strong community of outstanding connective tissue researchers, especially those working on cartilage. These included Larry Rosenberg in NewYork, who gave up orthopaedics to pursue his passion-research on proteoglycans, and Vince Hascall at NIH (proteoglycans) and David Howell and Fred Woessner in Miami (proteases and proteoglycans). Meanwhile Peter Roughley (proteoglycans), John Mort and Anneliese Recklies (proteases) and Elaine Golds (inflammation/cytokines) had joined me in Montreal. Interest in connective tissues had grown progressively in Toronto, with Ken Pritzker working on osteoarthritis, the arrival of Marc Grynpas (bone) from Boston and Rita Kandel (cartilage). Meanwhile, in the early 1980s, Cy Frank, a young orthopaedic surgeon, also with a passion for research, had established a research program in Calgary that would involve orthopaedics, rheumatology, cartilage, tendon, ligament, proteases and bioengineering. By the beginning of the 1990s, Canada had many connective researchers. But they rarely met. Our main meetings were south of the border, such as the Orthopaedic Research Society, or in Europe. There were no opportunities for Canadian scientists and young researchers to meet on a regular basis, and for trainees to seek guidance and explore further training and job opportunities in Canada. I knew of many young researchers moving to the states, often never to return, often because of the lack of a Canadian meeting place.

The gene expression of mTOR, autophagy-related ULK1, caspase 3, CDK-inhibitor p21, and TNF α was measured in the peripheral blood of osteoarthritic (OA) patients at different stages of the disease aiming to establish a gene expression profile that might indicate the activity of the disease and joint destruction. Whole blood of 65 OA outpatients, 27 end-stage OA patients, 27 healthy volunteers, and knee articular cartilages of 28 end-stage OA patients and 26 healthy subjects were examined. OA outpatients were subjected to clinical testing, ultrasonography, and radiographic and WOMAC scoring. Protein levels of p70-S6K, p21, and caspase 3 were quantified by ELISA. Gene expression was measured using real-time RT-PCR. Upregulation of mTOR gene expression was observed in PBMCs of 42 OA outpatients ("High mTOR expression subset") and in PBMCs and articular cartilages of all end-stage OA patients. A positive correlation between mTOR gene expression in PBMCs and cartilage was observed in the end-stage OA patients. 23 OA outpatients in the "Low mTOR expression subset" exhibited significantly lower mTOR gene expression in PBMCs compared to healthy controls. These "Low mTOR" subset subjects experienced significantly more pain upon walking, and standing and increased total joint stiffness versus "High mTOR" subset, while the latter more often exhibited synovitis. The protein concentrations of p70-S6K, p21, and caspase 3 in PBMCs were significantly lower in the "Low" subset versus "High" subset and end-stage subjects. Increases in the expression of mTOR in PBMCs of OA patients are related to disease activity, being associated with synovitis more than with pain.

INTRODUCTION:The intra-helical cleavage of type II collagen by proteases, including collagenases and cathepsin K, is increased with aging and osteoarthritis (OA) in cartilage as determined by immunochemical assays. The distinct sites of collagen cleavage generated by collagenases and cathepsin K in healthy and OA human femoral condylar cartilages were identified and compared.METHODS:Fixed frozen cartilage sections were examined immunohistochemically, using antibodies that react with the collagenase-generated cleavage neoepitopes, C2C and C1,2C, and the primary cleavage neoepitope (C2K) generated in type II collagen by the action of cathepsin K and possibly by other proteases, but not by any collagenases studied to date.RESULTS:In most cases, the staining patterns for collagen cleavage were similar for all three epitopes: weak to moderate mainly pericellular staining in non-OA cartilage from younger individuals and stronger, more widespread staining in aging and OA cartilages that often extended from the superficial to the mid/deep zone of the tissue. In very degenerate OA specimens, with significant disruption of the articular surface, staining was distributed throughout most of the cartilage matrix.CONCLUSIONS:Cleavage of collagen by proteases usually arises pericellularly around chondrocytes at and near the articular surface, subsequently becoming more intense and extending progressively deeper into the cartilage with aging and OA. The close correspondence between the distributions of these products suggests that both collagenases and cathepsin K, and other proteases that may generate this distinct cathepsin K cleavage site, are usually active in the same sites in the degradation of type II collagen.

So, there can be little doubt that the whole joint is involved in OA, resulting in changes in function and proprioception and onset of altered nociception and pain that compound the problem. So, functional assessments of joint function should also be included in any measures of joint changes in OA. Many additional parameters might help in detecting and measuring disease onset and progression instead of relying on traditional measures of loss of and/or damage to articular cartilage. Most, if not all, joint tissues potentially offer important new opportunities as targets for therapeutic intervention.

This ‘Biochemical Journal Classic’ paper reflects a remarkable example of how a biochemist and a biologist came together in the 1920s to solve a biological problem that set the stage for bone and cartilage research today, and demonstrates the great strengths and importance of interdisciplinary collaborations.

Purpose: To examine associations between biomarkers of joint tissue metabolism and whole blood lead (Pb), separately for men and women in an African American and Caucasian population, which may reflect an underlying pathology.Methods: Participants in the Johnston County Osteoarthritis Project Metals Exposure Sub-Study (329 men and 342 women) underwent assessment of whole blood Pb and biochemical biomarkers of joint tissue metabolism. Urinary cross-linked N telopeptide of type I collagen (uNTX-I) and C-telopeptide fragments of type 11 collagen (uCTX-II), serum cleavage neoepitope of type II collagen (C2C), serum type 11 procollagen synthesis C-propeptide (CPII), and serum hyaluronic acid (HA) were measured using commercially available kits; the ratio of [C2C:CPII] was calculated. Serum cartilage oligomeric matrix protein (COMP) was measured by an in-house assay. Multiple linear regression models were used to examine associations between continuous blood Pb and biomarker outcomes, adjusted for age, race, current smoking status, and body mass index.Results are reported as estimated change in biomarker level for a 5-unit change in Pb level. Results: The median Pb level among men and women was 2.2 and 1.9 mu g/dL, respectively. Correlations were noted between Pb levels and the biomarkers uNTX-I, uCTX-II, and COMP in women, and between Pb and uCTX-II, COMP, CPII, and the ratio [C2C:CPII] in men. In adjusted models among women, a 5-unit increase in blood Pb level was associated with a 28% increase in uCTX-II and a 45% increase in uNTX-I levels (uCTX-II: 1.28 [95% Cl: 1.04-1.58], uNTX-I: 1.45 [95% CI:1.21-1.74]). Among men, levels of Pb and COMP showed a borderline positive association (8% increase in COMP for a 5-unit change in Pb: 1.08 [95% Cl: 1.00-1.181]); no other associations were significant after adjustment.Conclusions: Based upon known biomarker origins, the novel associations between blood Pb and biomarkers appear to be primarily reflective of relationships to bone and calcified cartilage turnover among women and cartilage metabolism among men, suggesting a potential gender-specific effect of Pb on joint tissue metabolism that may be relevant to osteoarthritis. (C) 2011 Elsevier Inc. All rights reserved.