Virginia B. Kraus

Serum cartilage oligomeric matrix protein reflects osteoarthritis presence and severity: The Johnston county osteoarthritis project

OBJECTIVE To characterize serum cartilage oligomeric matrix protein (COMP) levels by age and gender for a radiographically defined population free of hip and knee osteoarthritis (OA), and to examine the potential utility of COMP as a diagnostic biomarker for knee OA. METHODS Serum samples and knee and hip radiographs were obtained at a baseline evaluation as part of the Johnston County Osteoarthritis Project, a population-based study of OA in rural North Carolina. A total of 291 Caucasian participants were randomly selected for COMP analysis, 143 patients with radiographic knee OA (Kellgren/Lawrence [K/L] grade > or = 2) and 148 controls with neither hip nor knee OA (K/L grade 0), evenly distributed by age and gender. COMP was quantified by competitive enzyme-linked immunosorbent assay with monoclonal antibody 17-C10. The natural log-transformed COMP data were analyzed using general linear models. RESULTS Serum COMP levels were significantly elevated (P = 0.0001) in the age > or = 65 group (mean +/- SD 1,302.1 +/- 496.7 ng/ml) versus the age 45-54 and age 55-64 groups (1,058.1 +/- 432.4 and 1,038.6 +/- 313.3, respectively). Serum COMP levels of the OA group were significantly higher than those of the control group (1,208.57 +/- 487.47 ng/ml versus 1,061.83 +/- 370.58 ng/ml; P = 0.0093). Serum COMP levels also increased significantly with knee OA K/L grade (P = 0.0047), knee OA laterality (P = 0.0043), and number of knee and hip joints involved (P = 0.0001). There was no significant difference in serum COMP levels by gender or obesity. CONCLUSION We demonstrate that in a population-based sample, serum COMP levels can distinguish an OA-affected subgroup from an unaffected subgroup and can reflect disease severity and multiple joint involvement in OA.

Application of biomarkers in the development of drugs intended for the treatment of osteoarthritis

OBJECTIVE Osteoarthritis (OA) is a chronic and slowly progressive disease for which biomarkers may be able to provide a more rapid indication of therapeutic responses to therapy than is currently available; this could accelerate and facilitate OA drug discovery and development programs. The goal of this document is to provide a summary and guide to the application of in vitro (biochemical and other soluble) biomarkers in the development of drugs for OA and to outline and stimulate a research agenda that will further this goal. METHODS The Biomarkers Working Group representing experts in the field of OA biomarker research from both academia and industry developed this consensus document between 2007 and 2009 at the behest of the Osteoarthritis Research Society International Federal Drug Administration initiative (OARSI FDA initiative). RESULTS This document summarizes definitions and classification systems for biomarkers, the current outcome measures used in OA clinical trials, applications and potential utility of biomarkers for development of OA therapeutics, the current state of qualification of OA-related biomarkers, pathways for biomarker qualification, critical needs to advance the use of biomarkers for drug development, recommendations regarding practices and clinical trials, and a research agenda to advance the science of OA-related biomarkers. CONCLUSIONS Although many OA-related biomarkers are currently available they exist in various states of qualification and validation. The biomarkers that are likely to have the earliest beneficial impact on clinical trials fall into two general categories, those that will allow targeting of subjects most likely to either respond and/or progress (prognostic value) within a reasonable and manageable time frame for a clinical study (for instance within 1-2 years for an OA trial), and those that provide early feedback for preclinical decision-making and for trial organizers that a drug is having the desired biochemical effect. As in vitro biomarkers are increasingly investigated in the context of specific drug treatments, advances in the field can be expected that will lead to rapid expansion of the list of available biomarkers with increasing understanding of the molecular processes that they represent.

Value of biomarkers in osteoarthritis: current status and perspectives.

Osteoarthritis affects the whole joint structure with progressive changes in cartilage, menisci, ligaments and subchondral bone, and synovial inflammation. Biomarkers are being developed to quantify joint remodelling and disease progression. This article was prepared following a working meeting of the European Society for Clinical and Economic Aspects of Osteoporosis and Osteoarthritis convened to discuss the value of biochemical markers of matrix metabolism in drug development in osteoarthritis. The best candidates are generally molecules or molecular fragments present in cartilage, bone or synovium and may be specific to one type of joint tissue or common to them all. Many currently investigated biomarkers are associated with collagen metabolism in cartilage or bone, or aggrecan metabolism in cartilage. Other biomarkers are related to non-collagenous proteins, inflammation and/or fibrosis. Biomarkers in osteoarthritis can be categorised using the burden of disease, investigative, prognostic, efficacy of intervention, diagnostic and safety classification. There are a number of promising candidates, notably urinary C-terminal telopeptide of collagen type II and serum cartilage oligomeric protein, although none is sufficiently discriminating to differentiate between individual patients and controls (diagnostic) or between patients with different disease severities (burden of disease), predict prognosis in individuals with or without osteoarthritis (prognostic) or perform so consistently that it could function as a surrogate outcome in clinical trials (efficacy of intervention). Future avenues for research include exploration of underlying mechanisms of disease and development of new biomarkers; technological development; the ‘omics’ (genomics, metabolomics, proteomics and lipidomics); design of aggregate scores combining a panel of biomarkers and/or imaging markers into single diagnostic algorithms; and investigation into the relationship between biomarkers and prognosis.

Pain Catastrophizing and Pain-Related Fear in Osteoarthritis Patients: Relationships to Pain and Disability

This study examined the degree to which pain catastrophizing and pain-related fear explain pain, psychological disability, physical disability, and walking speed in patients with osteoarthritis (OA) of the knee. Participants in this study were 106 individuals diagnosed as having OA of at least one knee, who reported knee pain persisting for six months or longer. Results suggest that pain catastrophizing explained a significant proportion (all Ps < or = 0.05) of variance in measures of pain (partial r(2) [pr(2)] = 0.10), psychological disability (pr(2) = 0.20), physical disability (pr(2) = 0.11), and gait velocity at normal (pr(2) = 0.04), fast (pr(2) = 0.04), and intermediate speeds (pr(2) = 0.04). Pain-related fear explained a significant proportion of the variance in measures of psychological disability (pr(2) = 0.07) and walking at a fast speed (pr(2) = 0.05). Pain cognitions, particularly pain catastrophizing, appear to be important variables in understanding pain, disability, and walking at normal, fast, and intermediate speeds in knee OA patients. Clinicians interested in understanding variations in pain and disability in this population may benefit by expanding the focus of their inquiries beyond traditional medical and demographic variables to include an assessment of pain catastrophizing and pain-related fear.

Changes in serum and synovial fluid biomarkers after acute injury (NCT00332254)

IntroductionAcute trauma involving the anterior cruciate ligament is believed to be a major risk factor for the development of post-traumatic osteoarthritis 10 to 20 years post-injury. In this study, to better understand the early biological changes which occur after acute injury, we investigated synovial fluid and serum biomarkers.MethodsWe collected serum from 11 patients without pre-existing osteoarthritis from a pilot intervention trial (5 placebo and 6 drug treated) using an intra-articular interleukin-1 receptor antagonist (IL-1Ra) therapy, 9 of which also supplied matched synovial fluid samples at presentation to the clinic after acute knee injury (mean 15.2 ± 7.2 days) and at the follow-up visit for reconstructive surgery (mean 47.6 ± 12.4 days). To exclude patients with pre-existing osteoarthritis (OA), the study was limited to individuals younger than 40 years of age (mean 23 ± 3.5) with no prior history of joint symptoms or trauma. We profiled a total of 21 biomarkers; 20 biomarkers in synovial fluid and 13 in serum with 12 biomarkers measured in both fluids. Biomarkers analyzed in this study were found to be independent of treatment (P > 0.05) as measured by Mann-Whitney and two-way ANOVA.ResultsWe observed significant decreases in synovial fluid (sf) biomarker concentrations from baseline to follow-up for sfC-Reactive protein (CRP) (P = 0.039), sflubricin (P = 0.008) and the proteoglycan biomarkers: sfGlycosaminoglycan (GAG) (P = 0.019), and sfAlanine-Arginine-Glycine-Serine (ARGS) aggrecan (P = 0.004). In contrast, we observed significant increases in the collagen biomarkers: sfC-terminal crosslinked telopeptide type II collagen (CTxII) (P = 0.012), sfC1,2C (P = 0.039), sfC-terminal crosslinked telopeptide type I collagen (CTxI) (P = 0.004), and sfN-terminal telopeptides of type I collagen (NTx) (P = 0.008). The concentrations of seven biomarkers were significantly higher in synovial fluid than serum suggesting release from the signal knee: IL-1β (P < 0.0001), fetal aggrecan FA846 (P = 0.0001), CTxI (P = 0.0002), NTx (P = 0.012), osteocalcin (P = 0.012), Cartilage oligomeric matrix protein (COMP) (P = 0.0001) and matrix metalloproteinase (MMP)-3 (P = 0.0001). For these seven biomarkers we found significant correlations between the serum and synovial fluid concentrations for only CTxI (P = 0.0002), NTx (P < 0.0001), osteocalcin (P = 0.0002) and MMP-3 (P = 0.038).ConclusionsThese data strongly suggest that the biology after acute injury reflects that seen in cartilage explant models stimulated with pro-inflammatory cytokines, which are characterized by an initial wave of proteoglycan loss followed by subsequent collagen loss. As the rise of collagen biomarkers in synovial fluid occurs within the first month after injury, and as collagen loss is thought to be irreversible, very early treatment with agents to either reduce inflammation and/or reduce collagen loss may have the potential to reduce the onset of future post-traumatic osteoarthritis.Trial registrationThe samples used in this study were derived from a clinical trial NCT00332254 registered with ClinicalTrial.gov.

Extreme obesity due to impaired leptin signaling in mice does not cause knee osteoarthritis

OBJECTIVE To test the hypothesis that obesity resulting from deletion of the leptin gene or the leptin receptor gene results in increased knee osteoarthritis (OA), systemic inflammation, and altered subchondral bone morphology. METHODS Leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) female mice compared with wild-type mice were studied, to document knee OA via histopathology. The levels of serum proinflammatory and antiinflammatory cytokines were measured using a multiplex bead immunoassay. Cortical and trabecular subchondral bone changes were documented by microfocal computed tomography, and body composition was quantified by dual x-ray absorptiometry. RESULTS Adiposity was increased by approximately 10-fold in ob/ob and db/db mice compared with controls, but it was not associated with an increased incidence of knee OA. Serum cytokine levels were unchanged in ob/ob and db/db mice relative to controls, except for the level of cytokine-induced neutrophil chemoattractant (keratinocyte chemoattractant; murine analog of interleukin-8), which was elevated. Leptin impairment was associated with reduced subchondral bone thickness and increased relative trabecular bone volume in the tibial epiphysis. CONCLUSION Extreme obesity due to impaired leptin signaling induced alterations in subchondral bone morphology without increasing the incidence of knee OA. Systemic inflammatory cytokine levels remained largely unchanged in ob/ob and db/db mice. These findings suggest that body fat, in and of itself, may not be a risk factor for joint degeneration, because adiposity in the absence of leptin signaling is insufficient to induce systemic inflammation and knee OA in female C57BL/6J mice. These results imply a pleiotropic role of leptin in the development of OA by regulating both the skeletal and immune systems.

Collagen gene expression and mechanical properties of intervertebral disc cell–alginate cultures

Cells of the intervertebral disc have a limited capacity for matrix repair that may contribute to the onset and progression of degenerative disc changes. In this study, the biosynthetic capacity of cells isolated from specific regions of the porcine intervertebral disc was evaluated in vitro. Using a competitive reverse transcription‐polymerase chain reaction technique, gene expression levels for types I and II collagen were quantified in cells cultured for up to 21 d in a three‐dimensional alginate culture system and compared to levels obtained for cells in vivo. The mechanical properties of cell‐alginate constructs were measured in compression and shear after periods of culture up to 16 weeks. Cells from the anulus fibrosus expressed the most type I collagen mRNA in vivo and in vitro, while cells from the transition zone expressed the most type II collagen mRNA in vivo and in vitro. Mechanical testing results indicate that a mechanically functional matrix did not form at any time during the culture period; rather, decreases of up to 50% were observed in the compressive and shear moduli of the cell–alginate constructs compared to alginate with no cells. Together with results of prior studies, these results suggest that intervertebral disc cells maintain characteristics of their phenotype when cultured in alginate, but the molecules they synthesize are not able to form a mechanically functional matrix in vitro.

Basic methods in histopathology of joint tissues.

Histological and histochemical methods are important tools in the evaluation of joint tissue samples for degenerative joint diseases, both in humans and in animal models. In this respect, standardized, simple, and reliable techniques are mandatory. This chapter describes five basic staining procedures appropriate for macroscopic (Indian ink) and histologic (HE/hematoxylin - eosin) visualization and scoring of cartilage proteoglycan and collagen content (toluidine blue/safranin O and picrosirius red/Goldners trichrome).

Uric acid is a danger signal of increasing risk for osteoarthritis through inflammasome activation

Uric acid (UA) is known to activate the NLRP3 (Nacht, leucine-rich repeat and pyrin domain containing protein 3) inflammasome. When activated, the NLRP3 (also known as NALP3) inflammasome leads to the production of IL-18 and IL-1β. In this cohort of subjects with knee osteoarthritis (OA), synovial fluid uric acid was strongly correlated with synovial fluid IL-18 and IL-1β. Synovial fluid uric acid and IL-18 were strongly and positively associated with OA severity as measured by both radiograph and bone scintigraphy, and synovial fluid IL-1β was associated with OA severity but only by radiograph. Furthermore, synovial fluid IL-18 was associated with a 3-y change in OA severity, on the basis of the radiograph. We conclude that synovial fluid uric acid is a marker of knee OA severity. The correlation of synovial fluid uric acid with the two cytokines (IL-18 and IL-1β) known to be produced by uric acid-activated inflammasomes and the association of synovial fluid IL-18 with OA progression, lend strong support to the potential involvement of the innate immune system in OA pathology and OA progression.

Intervertebral disc cells exhibit differences in gene expression in alginate and monolayer culture.

Study Design. The mRNA levels of aggrecan and collagen were quantified in intervertebral disc cells cultured under three conditions: primary alginate culture, monolayer culture, and re-encapsulation in alginate after monolayer culture. Objectives. To compare the phenotype of intervertebral disc cells under different culture conditions and to investigate the reversibility of cell phenotype after re-encapsulation in alginate after monolayer culture. Summary of Background Data. The intervertebral disc contains heterogeneous populations of cells that vary with anatomic region. These cells possess significant differences in phenotype that can be preserved in vitro, although the effect of culture conditions on the phenotype of these cells is poorly understood. Methods. The intervertebral disc cells of 4–5-month-old pigs were isolated enzymatically from three anatomic zones: anulus fibrosus (AF), transition zone (TZ), and nucleus pulposus (NP). Gene expression levels of aggrecan and collagen Types I and II were measured using a quantitative reverse transcriptase–polymerase chain reaction. Results. Gene expression levels of anulus fibrosus and transition zone cells were shifted in monolayer compared with alginate, although the shift was partially reversed when re-encapsulated in alginate. However, NP cells appeared to be insensitive to culture conditions. Furthermore, characteristic patterns of gene expression among AF, TZ, and NP cells in primary alginate culture did not exist in monolayer culture, but they were also observed after re-encapsulation in alginate. Conclusion. The findings of this study suggest that anulus fibrosus and transition zone cells undergo a reversible shift in phenotype when cultured in monolayer compared with alginate. These differences suggest that the culture system exerts a strong influence on cell phenotype and may play a role in the response of these cells to biophysical and biochemical stimuli in vitro.