keratan-sulfate and Osteoarthritis

Animal origin chondroitin sulfate is employed as anti-inflammatory drug and food supplement against anti-osteoarthritis, but also as antioxidant, antitumor, anticoagulant, and immune-regulatory agent or as biomaterial in tissue engineering scaffolds and in drug-delivery systems. As its biological properties depend on the structural characteristics, multi-analytical approaches are necessary to correlate specific features of its heterogenic composition to the different bioactivities. This is of paramount importance to assess the efficacy of pharmaceuticals and food supplements, beyond safety quality control. This review would address the issue of chondroitin sulfate characterization according to the Pharmacopeia testing monograph point of view giving an update of the analytical novelties reported in the last ten years that might be employed for the product testing and releasing on the market. Not-instrumental (e.g. colorimetric assays) and instrumental techniques, most of them coupling diverse chromatographic separation methods with spectroscopic and spectrometry detection techniques, mono and bi-dimensional NMR approaches, are compared as tools to evaluate identity, titer, purity grade, monosaccharide and disaccharide composition, averaged molecular weight and viscosity, charge and sulfate content, impurities and related substances including the presence of other glycosaminoglycans.

Topics: Animals; Anticoagulants; Chondroitin Sulfates; Dietary Supplements; Glycosaminoglycans; Keratan Sulfate; Osteoarthritis

Osteoarthritis (OA) is characterized by increased cartilage degradation and wearing. The principal disease hallmarks for assessment of OA are radiographic aspects. However, laboratory markers of joint fluid, serum or urine have received growing attention in recent years. Biomarkers should be useful for improvement of diagnosis, assessment of disease progression and evaluation of therapeutic effects in OA. Here we described the outline of biomarkers in cartilage including utility and weakness.

Topics: Biomarkers; Cartilage; Chondroitin Sulfates; Collagen Type I; Collagen Type II; Early Diagnosis; Humans; Hyaluronic Acid; Keratan Sulfate; Matrix Metalloproteinases; Osteoarthritis; Peptides; Proteoglycans

Joint cartilage is a dynamic tissue that reacts to trauma, inflammation, and other insults by attempting to repair its matrix. This reaction results in the release of cartilage macromolecules into the body fluids. Analysis of these fluids has identified a limited number of at least somewhat tissue-specific markers of altered cartilage metabolism. Analyses of serum are less specific and less sensitive than analyses of synovial fluid, but their use as research tools in clinical studies, drug development, and experimental work in animal models is increasing.

Topics: Aggrecans; Animals; Biomarkers; Cartilage Oligomeric Matrix Protein; Cartilage, Articular; Collagen; Disease Progression; Extracellular Matrix Proteins; Glycoproteins; Humans; Hyaluronic Acid; Keratan Sulfate; Lectins, C-Type; Matrilin Proteins; Osteoarthritis; Proteoglycans

A review of the literature.. To investigate the potential of serum levels of keratan sulfate as a biomarker of the effects of loading of the spine.. Exposure to mechanical loading of the spine causes changes in metabolism of intervertebral discs, eventually leading to accelerated disc degeneration. This process is characterized by the degradation of proteoglycans, which is reflected by an increase in the blood level of proteoglycan components. The serum level of keratan sulfate, an epitope present on these proteoglycan components, has been suggested as a marker of changes in metabolism of cartilaginous tissues.. A review of the literature on serum keratan sulfate levels in relation to degenerative changes in cartilaginous tissue.. In a number of studies keratan sulfate in serum was reported to be related to degeneration of articular cartilage in patients with osteoarthritis. In addition, massive and rapid degradation of intervertebral discs was determined to result in a large rise in serum keratan sulfate levels. Whether degenerative changes of intervertebral discs induced by mechanical stress also cause a detectable increase in serum keratan sulfate should be subjected to further investigation.. Quantification of keratan sulfate in serum offers a promising measure for the early effects of mechanical loading of the spine, but research is needed for validation.

Topics: Biomarkers; Biomechanical Phenomena; Cartilage, Articular; Humans; Intervertebral Disc; Keratan Sulfate; Osteoarthritis; Spinal Diseases; Stress, Mechanical

Osteoarthritis (OA) is a common joint deterioration initiated by multiple factors. To better understand related factors in the development of this disease, we focused on the mechanical stress loaded on articular cartilage.. The anterior cruciate ligaments of rabbit knee joints were transected, and expression of protein kinase C (PKC) examined immunohistochemically. The PKC activator 12-o-tetradecanoyl-phorbol-13-acetate (TPA) was then administered intraarticularly. To determine the involvement of gas mediators, a cartilage defect was made on the medical femoral condyle of rabbit knee joints. Hydrostatic pressure was loaded on the cartilage taken from the surrounding defects, and levels of superoxide anion and nitric oxide (NO) were measured. Bovine chondrocytes were subjected to cyclic mechanical stretch using a Flexercell Strain Instrument. Proteoglycan synthesis and PKC activity were measured. Expression of matrix metalloproteinase (MMP)-3 and tissue inhibitor of metalloproteinase (TIMP)-1 in articular cartilages obtained from OA patients were examined using Northern blots.. Chondrocytes from experimentally induced OA were stained positively with anti-alpha-PKC antibody. Intraarticular administration of TPA prevented the development of OA changes. Cyclic tensile stretch loaded on chondrocytes decreased proteoglycan synthesis and PKC activity. Thus, PKC is involved in the stress-mediated degradation of articular cartilage. Cartilage defects led to degradation of surrounding cartilage and to enhanced superoxide anion and NO synthesis. We also noted increased and decreased expressions of MMP-3 and TIMP-1 mRNA in human OA cartilage, respectively.. PKC, gas mediators (superoxide anion, NO), and proteinases are all involved in OA.

Topics: Animals; Anterior Cruciate Ligament; Cartilage, Articular; Cattle; Disease Models, Animal; Humans; Keratan Sulfate; Knee Joint; Matrix Metalloproteinase 3; Nitric Oxide; Osteoarthritis; Protein Kinase C; Rabbits; Stress, Mechanical; Superoxides; Tensile Strength; Tetradecanoylphorbol Acetate; Tissue Inhibitor of Metalloproteinase-1

Topics: Adult; Aggrecans; Arthritis, Rheumatoid; Body Fluids; Cartilage; Child; Chondroitin Sulfate Proteoglycans; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix Proteins; Humans; Joint Diseases; Keratan Sulfate; Lectins, C-Type; Osteoarthritis; Proteoglycans; Synovial Fluid

Topics: Animals; Cartilage, Articular; Chondroitin Sulfates; Enzyme-Linked Immunosorbent Assay; Epitope Mapping; Humans; Joint Diseases; Keratan Sulfate; Osteoarthritis; Synovial Fluid

Fragments of high density cartilage proteoglycan (aggrecan) are released during either the normal or pathological turnover of cartilage proteoglycans, which fragments diffuse into the synovial fluids and then appear in the serum. The keratan sulphate (KS; a glycosaminoglycan side chain of aggrecan) is resistant to enzymatic degradation, it has a relatively low clearance and has a "standard" serum level indicating the actual level of cartilage (proteoglycan) breakdown. Using anti-KS monoclonal antibody in ELISA (enzyme-linked immunosorbent assay), we measured serum KS levels in patients with different joint diseases. The highest KS content (595 ng/ml) was measured in the sera of patients with articular chondrocalcinosis (calcium pyrophosphate crystal deposition disease/pseudogout). Slightly lower KS levels were determined in osteoarthrosis (OA; 578 ng/ml) and much less in rheumatoid arthritis (RA; 421 ng/ml). All these patient groups (either with degenerative or inflammatory joint diseases) expressed slightly higher KS levels compared to control blood donors (295 ng/ml). However, there were remarkable variations between these diseased groups, i. e., KS levels in patients with RA were significantly lower than in patients with OA (p < 0.001) and this difference was more pronounced in rheumatoid patients with I-II Steinbrocker stage (370 ng/ml) or in those treated with non-steroid anti-inflammatory drugs (NSAIDs) (382 ng/ml). Keratan sulphate levels in RA patients chronically treated with corticosteroid (460 ng/ml) or auro-thiomalat (473 ng/ml) indicate that these drugs may influence the cartilage metabolism more effectively than the NSAIDs.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; Aged; Arthritis; Cartilage Diseases; Cartilage, Articular; Chondrocalcinosis; Female; Humans; Joint Diseases; Keratan Sulfate; Male; Middle Aged; Osteoarthritis

We have used an ELISA to quantify a highly sulfated epitope present on keratan sulfate, a carbohydrate chain found principally in cartilage proteoglycans. The serum level of the epitope provides an indirect measure of the rate of degradation of cartilage proteoglycans during normal turnover and can be used to diagnose specific abnormalities in keratan sulfate metabolism. Serum levels of the epitope are elevated in a high percentage of patients with osteoarthritis and correlate with the number of joints involved. The elevated rate of proteoglycan turnover in these patients appears to be systemic, affecting not only the degenerating articular surfaces but apparently normal articular cartilages as well. We have postulated that this acceleration in the rate of proteoglycan turnover precedes clinical evidence of degenerative changes; and we discuss the rationale for the contention that this elevation may predispose adult humans to polyarticular osteoarthritis.

Topics: Aggrecans; Animals; Biomarkers; Cartilage, Articular; Extracellular Matrix Proteins; Humans; Keratan Sulfate; Lectins, C-Type; Osteoarthritis; Proteoglycans

The serum level of a highly sulfated epitope present on long keratan sulfate chains provides a direct measure of the rate of catabolism of cartilage proteoglycans. Levels of the keratan sulfate epitope are elevated in patients with generalized osteoarthritis (OA), indicating these individuals have elevated rates of cartilage proteoglycan catabolism. In the Pond-Nuki model of canine OA, the serum level of the keratan sulfate epitope rises rapidly after the transection of the anterior cruciate ligament, long before OA lesions can be detected, and remains high for at least 13 weeks.

Topics: Animals; Biomarkers; Cartilage; Disease Models, Animal; Dogs; Keratan Sulfate; Osteoarthritis; Proteoglycans

The article elucidates the new laboratory tests for chronic joint diseases, with special attention to quantitative and qualitative serum markers for metabolic and immunologic processes. The assays include the quantification of keratan sulphate, hyaluronan and beta-D-xylosyltransferase in serum and pyridinolin in serum and urine as markers for cartilage catabolism, as well as the detection of autoantibodies to cartilage matrix and cell components and cartilage-directed T-cell reactions in the patients. Taken together, these assays represent models for future laboratory diagnostic tools for the characterization of arthritic diseases. So far, however, it remains unclear whether they will prove to be adequate assays for the practicing physician.

Topics: Autoantibodies; Cartilage, Articular; Collagen; Humans; Hyaluronic Acid; Keratan Sulfate; Osteoarthritis; Phospholipases A; Proteoglycans

The diagnosis of osteoarthritis (OA) is usually based on clinical and radiologic findings and is usually made only after the patient presents with joint pain. There is today much interest in development of an immunologic "marker" of OA, to detect subclinical disease and/or monitor therapy. The approach employs measurement of serum or synovial fluid levels of articular cartilage macromolecules, such as proteoglycans or glycosaminoglycans, or fragments of these. The data, however, raise questions about interpretation and utility of such tests. What causes egress of such macromolecules from OA cartilage? Overproduction? Hypercatabolism? Leakage from an excessively permeable matrix? Does the marker reflect the rate of cartilage breakdown? Or of repair? How reliable are the quantitative immunologic methods in tests of sera from patients with OA? Data show, for example, that serum keratan sulfate levels may be influenced by the mode of presentation of the antigen, i.e., single vs multiple chains, and by the degree of sulfation, etc. To what extent might serum levels of a marker reflect release from degenerating but asymptomatic joints, rather than from painful joints? Also, since all putative marker molecules studied to date are widely distributed throughout the connective tissue of the body, they could be released from an asymptomatic degenerating intervertebral disc rather than, e.g., a painful osteoarthritic hip or knee. In the present climate of "marker mania," it should be emphasized that no marker exists today which can confidently be used for diagnosis of subclinical OA or for monitoring therapeutic response.

Topics: Animals; Biomarkers; Cartilage, Articular; Extracellular Matrix; Glycosaminoglycans; Humans; Keratan Sulfate; Osteoarthritis; Prognosis; Proteoglycans; Synovial Fluid

Topics: Animals; Cartilage, Articular; Chemical Phenomena; Chemistry; Chondroitin Sulfates; Collagen; Dogs; Humans; Keratan Sulfate; Osteoarthritis; Proteoglycans

To examine longitudinal changes in serum and synovial fluid concentrations of keratan sulfate (KS) and hyaluronan (HA) after cranial cruciate ligament (CCL) transection in dogs.. 12 clinically normal adult mixed-breed dogs.. Following CCL transection in the right stifle joint, KS and HA concentrations were determined in serum and neat (undiluted) synovial fluid prior to and 1, 2, 3, and 12 months after surgery. Postsurgical dilution of synovial fluid was corrected by use of urea as a passive marker.. Synovial fluid KS and HA concentrations decreased at 1, 2, and 3 months after surgery in operated stifle joints, compared with baseline values. Synovial fluid KS concentration decreased in unoperated stifle joints at 1 month. A decrease in synovial fluid KS concentration was found in operated stifle joints, compared with unoperated stifle joints, at 2 and 3 months, and a decrease in synovial fluid HA concentrations was also found in operated stifle joints, compared with unoperated stifle joints, at 1, 2, and 3 months. Serum KS concentrations increased from baseline values at 3 months after surgery. Hyaluronan concentrations in operated stifle joints were lower than baseline values at 1, 2, and 3 months. Urea-adjusted synovial fluid concentrations revealed that dilution did not account for the decline in biomarker concentrations.. The initial decrease and subsequent increase in synovial fluid concentrations of HA and KS may be caused by an acute inflammatory response to surgical intervention that negatively affects cartilage metabolism or an increase in production of immature proteoglycans.

Topics: Animals; Dog Diseases; Dogs; Hindlimb; Hyaluronic Acid; Keratan Sulfate; Osteoarthritis; Synovial Fluid

The aim of this study was to assess the clinical, radiological and biological efficacy and tolerability of the SYSADOA, chondroitin 4- and 6-sulfate (CS, Condrosulf, IBSA, Lugano, Switzerland), in patients suffering from knee osteoarthritis. This was a 1-year, randomized, double-blind, controlled pilot study which included 42 patients of both sexes, aged 35-78 years with symptomatic knee OA. Patients were treated orally with 800 mg chondroitin sulfate (CS) per day or with a placebo (PBO) administered in identical sachets. The main outcome criteria were the degree of spontaneous joint pain and the overall mobility capacity. Secondary outcome criteria included the actual joint space measurement and the levels of biochemical markers of bone and joint metabolism. This limited study confirmed that chondroitin sulfate was well-tolerated and both significantly reduced pain and increased overall mobility capacity. Treatment with CS was also associated in a limited group of patients with a stabilization of the medial femoro-tibial joint width, measured with a digitized automatic image analyzer, whereas joint space narrowing did occur in placebo-treated patients. In addition, the metabolism of bone and joint assessed by various biochemical markers also stabilized in the CS patients whereas it was still abnormal in the PBO patients. These results confirm that oral chondroitin 4- and 6-sulfate is an effective and safe symptomatic slow-acting drug for the treatment of knee OA. In addition, CS might be able to stabilize the joint space width and to modulate bone and joint metabolism. This is the first preliminary demonstration that a SYSADOA might influence the natural course of OA in humans.

Topics: Administration, Oral; Adult; Aged; Arthralgia; Chondroitin Sulfates; Double-Blind Method; Female; Humans; Keratan Sulfate; Knee Joint; Male; Middle Aged; Movement; Osteoarthritis; Osteocalcin; Pain Measurement; Pilot Projects; Radiography; Treatment Outcome

To study the effects of piroxicam on cartilage metabolism in vivo, a three phase (placebo/piroxicam 20 mg/day by mouth/placebo) double blind controlled trial was conducted in patients with osteoarthritis of the knee joint. Twenty one patients were recruited, 19 of whom (11 women, eight men, median age 70 years) completed the treatment schedule. The knee joint under study was aspirated to dryness at four week intervals. Treatment with piroxicam was accompanied by a decrease in the pain score, an improvement in the functional index, and an increased range of movement. Reductions in the concentration (mean (SEM) 120 (6) to 110 (8) micrograms/ml) and the total amount (1.22 (0.34) to 0.99 (0.37) mg) of keratan sulphate, but not the effusion volume (9.4 (2.5) to 8.3 (2.6) ml) were observed during treatment with piroxicam. These findings are consistent with decreased proteoglycan catabolism during treatment with piroxicam. Neither depressed synthesis nor enhanced clearance of degraded proteoglycan fragments can be excluded, however.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Double-Blind Method; Female; Humans; Keratan Sulfate; Knee Joint; Male; Middle Aged; Osteoarthritis; Piroxicam; Proteoglycans; Synovial Fluid

Chondroitin sulfate and glucosamine, commercialized as anti-osteoarthritis food supplements, do not undergo the strict quality controls of pharmaceuticals. In this paper a systematic multi-analytical approach was designed to analyse 25 food supplements from 8 European countries compared to 2 pharmaceuticals by using high performance anion-exchange chromatography with pulsed amperometric detection, size exclusion chromatography with triple detector array, capillary electrophoresis, mono and bi-dimensional NMR. Furthermore the biological activity was assessed on in vitro human synoviocyte and chondrocyte primary cell models. Most of the samples (over 19 out of 25) showed lower condroitin sulfate and glucosamine contents than the declared ones (up to -60.3%) while all of them showed a KS contamination (up to 47.1%). Mixed animal origin chondroitin sulfate and multiple molecular weight species were determined in more than 32% of the samples. Only 1 on 5 biologically screened samples had an effective action in vitro almost comparable to the pharmaceuticals.

Topics: Cells, Cultured; Chondrocytes; Chondroitin Sulfates; Dietary Supplements; Drug Contamination; Europe; Glucosamine; Humans; Keratan Sulfate; Osteoarthritis; Synoviocytes

Several epidemiological studies have reported that temporomandibular disorders (TMDs) are more prevalent in women than in men. It has recently been proposed that sex hormones such as estrogen, testosterone and dehydroepiandrosterone (DHEA) are involved with the pathogenesis of TMDs. Although studies have investigated the relationship between estrogen and testosterone and the restoration of TMDs, the relationship between DHEA and TMDs is unknown. The synovial tissue of the temporomandibular joint (TMJ) is made up of connective tissue with an extracellular matrix (ECM) composed of collagen and proteoglycan. One proteoglycan family, comprised of small leucine-rich repeat proteoglycans (SLRPs), was found to be involved in collagen fibril formation and interaction. In recent years, the participation of SLRPs such as lumican and fibromodulin in the internal derangement of TMJ has been suggested. Although these SLRPs may contribute to the restoration of the synovium, their effect is still unclear. The purpose of this study was to investigate the effect of DHEA, a sex hormone, on the expression of lumican and fibromodulin in human temporomandibular specimens and in cultured human TMJ fibroblast-like synovial cells in the presence or absence of the pro-inflammatory cytokine interleukin-1beta (IL-1beta). In the in vivo study, both normal and osteoarthritic (OA) human temporomandibular synovial tissues were immunohistochemically examined. In the in vitro study, five fibroblast-like synoviocyte (FLS) cell lines were established from human TMJ synovial tissue of patients with osteoarthritis. The subcultured cells were then incubated for 3, 6, 12 or 24 h with/without IL-1beta (1 ng/mL) in the presence or absence of DHEA (10 μM). The gene expression of lumican and fibromodulin was examined using the real-time polymerase chain reaction (PCR) and their protein expression was examined using immunofluorescent staining. We demonstrated that the expression of lumican significantly differs from that of fibromodulin in synovial tissue in OA and furthermore, that IL-1beta induced a significant increase in lumican mRNA and immunofluorescent staining in FLS compared to cells without IL-1beta. DHEA plus IL-1beta induced a significant increase in fibromodulin, but not in lumican mRNA, compared to DHEA alone, IL-1beta alone and in the absence of DHEA and IL-1beta. In immunofluorescent staining, weaker fibromodulin staining of FLS cells was observed in cells cultured in the absence of

Topics: Adjuvants, Immunologic; Adult; Aged; Cells, Cultured; Chondroitin Sulfate Proteoglycans; Dehydroepiandrosterone; Extracellular Matrix Proteins; Female; Fibroblasts; Fibromodulin; Gene Expression Regulation; Humans; Immunohistochemistry; Interleukin-1beta; Keratan Sulfate; Lumican; Male; Middle Aged; Osteoarthritis; Proteoglycans; Real-Time Polymerase Chain Reaction; Synovial Membrane; Temporomandibular Joint

The aim of this study was to investigate the correlations of severity of osteoarthritis (OA) and serum biomarkers including keratan sulfate (KS), hyaluronic acid (HA) and chondroitin sulfate (CS) 846 epitope. We also investigated the effect of glucosamine and fish collagen peptide (FCP) on OA. OA was induced in 12 rabbits (12 weeks of age) by anterior cruciate ligament transection (ACLT). After the surgery, the rabbits were orally administered FCP (F group), glucosamine (G group) or FCP and glucosamine (FG group) for 4 weeks. The control group was provided water ad libitum (C group). Blood was collected before surgery (pre-ACLT) and before euthanasia (post-ACLT) for serum marker measurement. Biomarker levels were measured by using commercial kits. We evaluated OA severity both macroscopically and histologically. Macroscopic evaluation showed mildly eroded condylar surfaces in the C group. Histological findings were significantly different from the FG and other groups. There were no significant differences between each group at post-ACLT in terms of serum KS, HA and CS 846. Histological assessment and serum biomarker measurements performed at post-ACLT showed a significant correlation between HA concentration and OA severity. Variations in the CS 846 concentration at pre-ACLT and post-ACLT were significantly correlated with OA severity. Administration of glucosamine and FCP had chondroprotective effects in the ACLT model. Serum biomarker concentrations were significantly correlated with cartilage injury. Serum biomarker measurement would be useful for monitoring articular cartilage damage in the clinical setting.

Topics: Animals; Anterior Cruciate Ligament; Biomarkers; Cartilage, Articular; Chondroitin Sulfates; Collagen; Disease Models, Animal; Glucosamine; Histocytochemistry; Hyaluronic Acid; Keratan Sulfate; Osteoarthritis; Rabbits; Statistics, Nonparametric

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.

Topics: ADAM Proteins; ADAMTS4 Protein; Adolescent; Adult; Aged; Aged, 80 and over; Aggrecans; Cartilage, Articular; Chondroitin Sulfates; Extracellular Matrix; Humans; Keratan Sulfate; Knee Injuries; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Middle Aged; Osteoarthritis; Peptide Fragments; Procollagen N-Endopeptidase; Protein Interaction Domains and Motifs; Proteolysis; Recombinant Proteins; Substrate Specificity; Synovial Fluid; Young Adult

Strenuous running of rats enhances mechanical stress on the knee, thereby inducing degeneration of articular cartilage. Bone morphogenetic protein-7 (BMP-7) has an inhibitory effect on cartilage degeneration, suggesting its usefulness for human osteoarthritis patients. However, its mode of administration should be investigated. We examined whether weekly knee injections of BMP-7 delayed the progression of cartilage degeneration. Wistar rats were forced to run 30 km in 6 weeks on a rodent treadmill, and BMP-7 was injected weekly into the knee. Macroscopically and histologically, this strenuous running regimen induced cartilage degeneration. Weekly injections of 250 ng BMP-7 delayed the progression of cartilage degeneration. Immunohistochemically, in the control knee, type II collagen expression decreased, while BMP-7 expression in chondrocytes slightly increased. Interestingly, weekly injection of BMP-7 increased BMP-7 expression even 9 days after the final injection. Disulfate disaccharide keratan sulfate in serum transiently increased in the control group, while it remained at a low level in the BMP-7 group. Weekly BMP-7 injection increased BMP-7 expression in chondrocytes and its effect seemed to last more than 7 days. The effect of BMP-7 could be monitored by serum keratan sulfate concentration. Periodical injections of BMP-7 delayed progression of cartilage degeneration induced by excessive running in rats.

Topics: Animals; Bone Morphogenetic Protein 7; Cartilage, Articular; Chondrocytes; Collagen Type II; Injections, Intra-Articular; Keratan Sulfate; Knee Joint; Osteoarthritis; Rats; Rats, Wistar; Running

To optimize sampling and to understand sources of variation in biomarkers for osteoarthritis (OA), we evaluated variation due to activity and food consumption.. Twenty participants, with radiographic knee OA, provided serial serum and urine samples at 4 time points: before arising in the morning; after 1 h of light activity; 1 h after eating breakfast; and in the evening. Five serum (s) and 2 urinary (u) analytes were measured: hyaluronan (sHA); cartilage oligomeric matrix protein (sCOMP); keratan sulfate (sKS-5D4); transforming growth factor beta (sTGF-ss1); and collagen II-related epitopes (sCPII, uCTXII, and uC2C). Activity was monitored by an accelerometer.. All serum biomarkers increased and one of the urinary biomarkers decreased after 1 h of non-exertional activity. Food consumption following activity was associated with a return of biomarker concentrations to baseline levels. Accelerometers proved to be a novel way to monitor protocol compliance and demonstrated a positive association between the mean level of activity and sCOMP concentration. Urinary CTXII varied the least but demonstrated both true circadian variation (peak in the morning and nadir in the evening) and the most robust correlation with radiographic knee OA.. We confirm activity related variation in these markers. These data suggested that biomarkers also varied due to upright posture, glomerular filtration rate stimulated by food intake, and circadian rhythm in the case of uCTXII.

Topics: Aged; Aged, 80 and over; Biomarkers; Cartilage Oligomeric Matrix Protein; Circadian Rhythm; Collagen Type II; Eating; Epitopes; Extracellular Matrix Proteins; Female; Glycoproteins; Humans; Hyaluronic Acid; Keratan Sulfate; Knee; Male; Matrilin Proteins; Middle Aged; Motor Activity; Osteoarthritis; Radiography; Transforming Growth Factor beta

Animal shapes are maintained by connective tissue extracellular matrices (ECMs). ECM shapes depend on keeping collagen fibrils in the right places, held by regular frequent proteoglycan (PG) bridges attached at specific sites. The PGs carry anionic glycosaminoglycan (AGAG) 'strings' that span and determine interfibrillar distances, thus holding us together. I called these repeating structures 'shape modules'. The strings are aggregated antiparallel chains of dermochondan, keratan and chondroitan sulphates (DS, KS and CS); stabilised by hydrophobic and H-bonds. Shape modules are elastic. AGAG/AGAG interactions break under stress and reform when the stress is removed and/or they contain an elastic sugar, L-iduronate (in DS). Cartilage ECMs are also based on shape modules. Depots therein of aggrecan, the large PG which carries many chains of CS and KS, imbibe water, thereby exerting swelling pressure. External pressure forces this water into the elastic shape modules, from whence it is returned post compression. Cartilage anisotropic responses (along and at right angles to shape module axes) to compressive and tensile stresses are now interpretable. Degradation of shape modules in osteoarthrosis reduces these responses. Inability to hold collagen fibrils together results in imbibition of excess water, fissuring and erosion, characteristic of this condition.

Topics: Aggrecans; Animals; Cartilage; Chondroitin Sulfate Proteoglycans; Compressive Strength; Elasticity; Extracellular Matrix; Glycosaminoglycans; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Keratan Sulfate; Osteoarthritis; Rabbits; Tensile Strength

Osteoarthritis is influenced by genetic and environment factors, including mechanical stress; however, the relationship between running and the development of osteoarthritis remains a matter of controversy. We investigated whether osteoarthritic change could be obtained in a rat strenuous running model, whether serum keratan sulfate in rats could be detected by HPLC and was associated with onset or progression of osteoarthritis, and whether hyaluronan injection suppressed development of osteoarthritis and elevation of serum keratan sulfate.. Wistar rats were forced to run 30 km in 6 weeks on a treadmill machine. Articular cartilage of the knees was evaluated macroscopically and immunohistologically. Serum keratan sulfate was examined every week by HPLC. The effect of weekly knee injection of hyaluronan was also investigated.. Cartilage surfaces stained with India ink became irregular, metachromasia by safranin-O staining appeared to be almost lost, and Mankin's score significantly worsened after 30 km of running. Serum keratan sulfate in rats was detected by HPLC and transiently increased (peaked at 3 to 4 weeks) along with depletion of keratan sulfate in cartilage tissue. Hyaluronan treatment suppressed morphological progression of osteoarthritis and elevation of serum keratan sulfate.. Rat strenuous running induced osteoarthritis. Serum keratan sulfate was associated with progression of osteoarthritis. Weekly intraarticular injection of hyaluronan controlled the development of osteoarthritis, and the effect was reflected by serum keratan sulfate.

Topics: Animals; Cartilage, Articular; Chromatography, High Pressure Liquid; Disease Progression; Hindlimb; Hyaluronic Acid; Immunohistochemistry; Injections, Intra-Articular; Keratan Sulfate; Motor Activity; Osmolar Concentration; Osteoarthritis; Physical Endurance; Rats; Rats, Wistar; Time Factors

Topics: Acupuncture Therapy; Chest Pain; Fibromyalgia; Humans; Keratan Sulfate; Osteoarthritis; Serotonin; Substance P

To elucidate the specific function of m-calpain in the metabolism of aggrecan in human articular cartilage, the prevalence and localization of a large glycosaminoglycan-bearing aggrecan product generated by m-calpain in human osteoarthritis (OA) cartilage were investigated. Extracts of human OA articular cartilage were analysed by immunostaining using new polyclonal anti-VPGVA antiserum that detects the COOH terminal neoepitope IVTQVVPGVA(709) generated by m-calpain-related cleavage within the keratan sulphate rich region of human aggrecan. Immunoblotting analyses of aggrecan populations in guanidine hydrochloride-extracts showed that OA cartilages contained anti-VPGVA positive aggrecan products with the COOH terminal neoepitope ... VPGVA(709), resulting from truncation between the Ala(709)-Ala(710) m-calpain-related cleavage site. This aggrecan product consisted of two NH(2) terminal globular domain (G1 and G2) and KS side chains. Immunohistochemical staining showed that anti-VPGVA positive staining was localized within chondrocytes and spread to the surrounding interterritorial matrix. Confocal microscopic analysis showed subcellular colocalization of anti-VPGVA and anti m-calpain. These results indicate that the aggrecan product with the COOH terminal neoepitope VPGVA(709) is synthesized regularly by intracellular processing in chondrocytes, and is present abundantly as a limited form of aggrecan. M-calpain is the major candidate of the proteinase to generate this aggrecan product during the intracellular aggrecan processing.

Topics: Aggrecans; Animals; Blotting, Western; Calpain; Cartilage, Articular; Chondrocytes; Epitopes; Humans; Keratan Sulfate; Osteoarthritis; Swine

The measurement of body fluid levels of biochemical markers in joint tissues has begun to provide clinically useful information. Synovial fluid (SF) plays an important role in articular joint lubrication, nutrition, and metabolism of cartilage and other connective tissues within the joint. The purpose of our study was to identify and characterize osteogenic protein 1 (OP-1) in SF from patients with rheumatoid arthritis (RA) or with osteoarthritis (OA) and to correlate levels of OP-1 with those of hyaluronan (HA) and antigenic keratan sulfate (AgKS). SF was aspirated from the knees of patients with either RA or OA and from the knees of asymptomatic organ donors with no documented history of joint disease. The presence of detectable OP-1 in SF was demonstrated by western blots with specific anti-pro-OP-1 and anti-mature OP-1 antibodies. Measurement of levels of OP-1, HA and AgKS was performed using ELISAs. OP-1 was identified in human SF in two forms, pro-OP-1 and active (mature) OP-1--mature OP-1 being detected only in SF from OA patients and RA patients. Levels of OP-1 and HA were higher in RA patients than in OA patients and asymptomatic donors, while the level of AgKS was highest in SF from asymptomatic donors. Statistically significant differences were found between SF levels of OP-1 in RA and OA patients and between SF levels of AgKS among the three groups tested. The SF content of OP-1 tended to correlate positively with HA levels, but negatively with AgKS concentrations. In conclusion, the results of this study suggest that measurement of OP-1 in joint fluid may have value in the clinical evaluation of joint disease processes.

Topics: Arthritis, Rheumatoid; Autoantigens; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Humans; Hyaluronic Acid; Keratan Sulfate; Knee Joint; Osteoarthritis; Reference Values; Synovial Fluid

To identify biochemical markers of osteoarthritis (OA) in the guinea pig, we characterized four biomarkers and 17 cytokines for age- and strain-related differences.. Two guinea pig strains were examined in this study: (1) the Hartley (OA-prone) and (2) Strain 13 (OA-resistant). Levels of synovial fluid keratan sulfate (KS) and cartilage oligomeric matrix protein (COMP), as well as levels of serum C2C, CPII, and a panel of cytokines and chemokines were quantified in both guinea pig strains. These included: IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12p40, IL-12p70, IL-17, G-CSF, GM-CSF, IFN-gamma, KC, MIP-1 alpha, RANTES, and TNF-alpha.. Synovial fluid concentrations of KS and COMP increased coincident with histological OA and correlated positively with the severity of histological damage in both strains. Synovial fluid concentrations of these biomarkers were elevated in the knees of the Hartley compared to the Strain 13 animals, as early as 2 months of age. From as early as 4 months of age, the levels of serum C2C/CPII, representing the ratio of type II collagen degradation and synthesis, were elevated in the OA-prone Hartley compared with Strain 13 animals. Also, at 12 months of age, strain-related differences were apparent for 11 of the 16 cytokines and chemokines. Using multiple linear regression, serum IL-6 and TNF-alpha concentrations were each strongly associated with strain, weight, and their interaction (r2 = 0.80, P = 0.0002 for IL-6; r2 = 0.55, P = 0.02 for TNF-alpha).. Biomarkers derived from synovial fluid are reflective of histological severity in the spontaneous model of OA in the guinea pig. The synovial fluid biomarker profiles indicated accelerated cartilage matrix turnover in the Hartley strain as early as 2 months of age, prior to evidence of histological damage. The Hartley strain also exemplified an imbalance in type II collagen metabolism and a serum cytokine/chemokine profile indicative of a pro-inflammatory state. These findings elucidate additional disease-related features in the guinea pig that have relevance to OA in humans.

Topics: Age Factors; Animals; Biomarkers; Cartilage Oligomeric Matrix Protein; Cartilage, Articular; Chemokines; Collagen; Cytokines; Disease Progression; Extracellular Matrix Proteins; Glycoproteins; Guinea Pigs; Keratan Sulfate; Linear Models; Matrilin Proteins; Osteoarthritis; Sensitivity and Specificity; Species Specificity; Synovial Fluid

The aim of this study was to assess whether enzymatically isolated chondrons from normal adult articular cartilage could be used as a model for the onset of osteoarthritis, by comparison with mechanically extracted chondrons from osteoarthritic cartilage. Enzymatically isolated chondrons (EC) were cultured for 4 weeks in alginate beads and agarose gel constructs. Samples were collected at days 1 and 2, and weekly thereafter. Samples were immunolabelled for types II and VI collagen, keratan sulphate and fibronectin and imaged using confocal microscopy. Mechanically extracted chondrons (MC) were isolated, immunohistochemically stained for type VI collagen and examined by confocal microscopy. In culture, EC showed the following characteristics: swelling of the chondron capsule, cell division within the capsule and remodelling of the pericellular microenvironment. This was followed by chondrocyte migration through gaps in the chondron capsule. Four types of cell clusters formed over time in both alginate beads and agarose constructs. Cells within clusters exhibited quite distinct morphologies and also differed in their patterns of matrix deposition. These differences in behaviour may be due to the origin of the chondrocytes in the intact tissue. The behaviour of EC in culture paralleled the range of morphologies observed in MC, which presented as single and double chondrons and large chondron clusters. This preliminary study indicates that EC in culture share similar structural characteristics with MC isolated from osteoarthritic cartilage, confirming that some processes that occur in osteoarthritis, such as pericellular remodelling, take place in EC cultures. The study of EC in culture may therefore provide an additional tool to investigate the mechanisms operating during the initial stages of osteoarthritis. Further investigation of specific osteoarthritic phenotype markers will, however, be required in order to validate the value of this model.

Topics: Alginates; Animals; Cartilage, Articular; Cell Movement; Chondrocytes; Collagen Type II; Collagen Type IV; Dogs; Fibronectins; Gels; Immunohistochemistry; Keratan Sulfate; Microscopy, Confocal; Microspheres; Models, Animal; Osteoarthritis; Sepharose; Tissue Culture Techniques

To evaluate the hypothesis that the concentration of the 1/20/5D4 epitope of keratan sulphate, cartilage oligomeric matrix protein and total sulphated glycosaminoglycans in synovial fluids from dogs with cranial cruciate ligament disease would be affected by tibial plateau levelling osteotomy. In addition, to evaluate the hypothesis that medial meniscal release or meniscal injury would alter the expression of these candidate biomarkers.. Forty-one dogs with naturally occurring cranial cruciate ligament disease were recruited prospectively. Synovial fluids were collected from the index joint before surgery and six weeks and six months postsurgery. Following tibial plateau levelling osteotomy, synovial fluids were assayed for 1/20/5D4 epitope of keratan sulphate and cartilage oligomeric matrix protein concentration using an inhibition ELISA and for sulphated glycosaminoglycans using a direct dye-binding assay.. The sulphated glycosaminoglycans ratio did not change significantly during the study. Medial meniscal injury at entry was associated with lower concentrations of synovial fluid cartilage oligomeric matrix protein (P<0.05, unpaired t test). There was no association between medial meniscal release and the changes in marker concentrations, either from 0 to six weeks or 0 to six months.. Tibial plateau levelling osteotomy did not significantly alter the expression of the named candidate biomarkers. These findings reflect the limited nature of the arthrotomy or indicate that tibial plateau levelling osteotomy does not influence the progression of osteoarthritis (OA). From these studies, there is no evidence that tibial plateau levelling osteotomy affects cartilage metabolism.

Topics: Animals; Anterior Cruciate Ligament; Anterior Cruciate Ligament Injuries; Biomarkers; Dog Diseases; Dogs; Enzyme-Linked Immunosorbent Assay; Epitopes; Female; Glycosaminoglycans; Keratan Sulfate; Male; Osteoarthritis; Osteotomy; Prospective Studies; Rupture; Synovial Fluid; Tibia

Macromolecules of the articular cartilage extracellular matrix released into synovial fluid, blood, or urine can serve as potentially useful biomarkers of the severity of osteoarthritis (OA). Biomechanical factors play an important role in OA pathogenesis, yet their influence on biomarker production is not well understood. The goal of this study was to examine the hypothesis that dynamic mechanical stress influences the release of these biomarkers from articular cartilage.. Explants of porcine cartilage were subjected to dynamic compression at 0.5 Hz for 24h at stresses ranging from 0.006 to 0.1 MPa. The concentrations of cartilage oligomeric matrix protein (COMP), keratan sulfate (KS measured as the 5 D 4 epitope), total sulfated glycosaminoglycan (S-GAG), and the KS (keratanase-digestible) and chondroitin sulfate (CS) (chondroitinase-digestible) fractions of S-GAG were measured. Radiolabel incorporation was used to determine the rates of proteoglycan and protein synthesis.. The magnitudes of mechanical stress applied in this study induced nominal tissue strains of 4-23%, consistent with a range of physiological to hyperphysiologic strains measured in situ. COMP release increased in proportion to the magnitude of dynamic mechanical stress, while KS, CS and total S-GAG release increased in a bimodal pattern with increasing stress. Protein and proteoglycan synthesis were significantly decreased at the highest level of stress.. Mechanical stress differentially regulates the turnover of distinct pools of cartilage macromolecules. These findings indicate that mechanical factors, independent of exogenous cytokines or other stimulatory factors, can influence the production and release of OA-related biomarkers from articular cartilage.

Topics: Animals; Biomarkers; Cartilage, Articular; Chondroitin Sulfates; Extracellular Matrix Proteins; Female; Glycoproteins; Glycosaminoglycans; In Vitro Techniques; Keratan Sulfate; Matrilin Proteins; Osteoarthritis; Radiopharmaceuticals; Stress, Mechanical; Swine

To study 3 body fluids for changes in the levels of 5 biomarkers of cartilage metabolism during the early phases of experimental osteoarthritis (OA).. Twenty skeletally mature mixed-breed canines underwent unilateral surgical transection of the anterior cruciate ligament. Samples of joint fluid, serum, and urine were obtained preoperatively and just before necropsy (3 weeks or 12 weeks postoperatively). Biomarkers included 2 markers of cartilage matrix synthesis/turnover (aggrecan 846 epitope and C-propeptide of type II collagen) and 3 markers of cartilage degradation (keratan sulfate proteoglycan epitope, the collagenase-generated cleavage epitope of type II collagen [Col2-3/4C(long mono), or CIIC], and crosslinked peptides from the C-telopeptide domain of type II collagen [Col2CTx]). Significant changes in the levels of these biomarkers were determined by paired analyses.. Joint pathology was more severe in the 12-week group compared with the 3-week group. In joint fluid, due to limited volume, only Col2-3/4C(long mono) and Col2CTx were measured. Significant elevations in the levels of both of these markers were observed in experimental joints in both the 3-week group and the 12-week group. In serum, the level of aggrecan 846 epitope was elevated at both 3 weeks and 12 weeks, the level of Col2-3/4C(long mono) was elevated at 12 weeks, and the level of Col2CTx was elevated at both 3 weeks and 12 weeks. In urine, the level of Col2-3/4C(long mono) was elevated at 12 weeks after surgery.. Levels of biomarkers of intact aggrecan proteoglycan (aggrecan 846 epitope) and type II collagen degradation (Col2-3/4C(long mono) and Col2CTx) were elevated early after unilateral stifle joint injury, suggesting that these markers are sensitive and specific for early cartilage changes associated with isolated joint injury in this established model of experimental OA.

Topics: Aggrecans; Animals; Anterior Cruciate Ligament; Biomarkers; Calcium-Binding Proteins; Cartilage, Articular; Chondroitin Sulfate Proteoglycans; Collagen; Collagen Type II; Disease Models, Animal; Dogs; Epitopes; Extracellular Matrix Proteins; Female; Keratan Sulfate; Lectins, C-Type; Lumican; Osteoarthritis; Proteoglycans; Stifle; Synovial Fluid

Mechanical stress is an essential factor in the pathogenesis of osteoarthrosis. We sought to determine whether the strain-mediated alteration in proteoglycan (PG) synthesis was modulated by nitric oxide (NO) synthesis.. Cyclic tensile strain was applied to bovine articular chondrocytes. PG and NO synthesis were determined by [35S] sulfate incorporation and chemiluminescence analysis, respectively. To determine the expression of inducible NO synthase (iNOS), quantitative RT-PCR was used.. Enhanced PG and NO synthesis were evident when cyclic tensile strain was applied to chondrocytes seeded on fibronectin-coated plates. When NO production was inhibited, PG synthesis was further enhanced.. Cyclic tensile strain loaded on the chondrocytes enhanced NO synthesis and this enhanced NO inhibited PG synthesis.

Topics: Animals; Cartilage; Cattle; Cell Adhesion; Chondrocytes; DNA; DNA Primers; Dose-Response Relationship, Drug; Fibronectins; Keratan Sulfate; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oligonucleotides; Osteoarthritis; Proteoglycans; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stress, Mechanical; Sulfates; Time Factors

To evaluate the effect of calcitonin (CT) on the histology and biochemistry of articular cartilage from unstable operated and nonoperated knee in a canine model of experimental osteoarthritis (OA).. Eighteen dogs underwent anterior cruciate ligament transection (ACLT) of the right knee and were randomly distributed into three groups of six dogs each. From day-1 after surgery until sacrifice 84 days post-ACLT, each dog received a daily nasal spray that delivered the placebo, 100 units of CT or 400 units of CT. Histologic lesions were scored. Hyaluronan (HA) and antigenic keratan sulfate (AgKS) were quantified by enzyme-linked immunosorbent assays (ELISAs), whereas aggrecan molecules extracted under nondissociative conditions were characterized by velocity gradient centrifugation.. All canine cruciate-deficient knees developed OA. At a daily dose of 400 units, CT had no effect on the size of osteophytes but significantly reduced the severity of cartilage histologic lesions in unstable knees. CT also enhanced the HA content as well as the size distribution and relative abundance of fast-sedimenting aggrecan aggregates in cartilage from both operated and nonoperated knees. On the other hand, in the CT-treated group, the cartilage content of AgKS increased in operated joints, but not in nonoperated joints.. Because CT delivered as a nasal spray markedly reduced the severity of most OA changes, both at the histological and biochemical level, this form of therapy may have benefits for humans who have recently experienced a traumatic knee injury, and as well as for dogs who spontaneously rupture their ACL.

Topics: Aggrecans; Animals; Anterior Cruciate Ligament; Calcitonin; Cartilage, Articular; Centrifugation, Density Gradient; Collagen; Dogs; Extracellular Matrix Proteins; Hindlimb; Hyaluronic Acid; Keratan Sulfate; Lectins, C-Type; Osteoarthritis; Proteoglycans

Results from our previous studies suggest that surgical induction of anterior disk displacement (ADD) in the rabbit craniomandibular joint (CMJ) leads to histopathological alterations consistent with osteoarthritis. In addition, molecular changes in collagens and glycosaminoglycans (GAGs) were observed using immunohistochemistry. The purpose of the present study was to further characterize those molecular changes in collagens and GAGs using immuno-electron microscopy.. The right joint of 15 rabbits was exposed surgically and all discal attachments were cut except for the posterior attachment (the bilaminar zone). The disc was then repositioned anteriorly and sutured to the zygomatic arch. The left joint was used as a sham-operated control. Ten additional joints were used as non-operated controls. Mandibular condyles were removed 2 weeks following surgery and processed for light and immuno-electron microscopy using colloidal gold-labeled antibodies against collagen type I, II, VI and IX and against keratan sulfate, chondroitin-4 and -6-sulfate, and link protein.. Light microscopic results showed osteoarthritic changes. Immuno-electron microscopy of osteoarthritic cartilage demonstrated a decline in type II collagen, the abnormal presence of type I collagen and loss of type VI and IX collagens. Quantitative colloidal gold immuno-electron microscopy confirmed the depletion of keratan sulfate, chondroitin-4 and -6-sulfate, and link protein in osteoarthritic cartilage.. Anterior disk displacement leads to molecular alterations in both the collagen and the proteoglycans of rabbit condylar cartilage characteristic of osteoarthritis in other synovial joints. These alterations are consistent with loss of the shock absorber function of the cartilage and injury of the underlying bone.

Topics: Animals; Antibodies; Cartilage; Chondroitin Sulfates; Collagen; Collagen Type I; Collagen Type II; Collagen Type IX; Collagen Type VI; Extracellular Matrix Proteins; Gold Colloid; Immunohistochemistry; Joint Dislocations; Keratan Sulfate; Male; Mandibular Condyle; Microscopy, Immunoelectron; Osteoarthritis; Proteins; Proteoglycans; Rabbits; Temporomandibular Joint Disc; Temporomandibular Joint Disorders

To explore the levels of matrix metalloprotease-3 (MMP-3), tissue inhibitor of metalloproteases-1 (TIMP-1), 5D4 keratan sulfate, and two 3B3 chondroitin-sulfate epitopes in several canine osteoarthritic and inflammatory arthropathies.. Blood and synovial fluid were obtained from 103 dogs with rupture of the anterior cruciate ligament (ACLR), osteochondritis dissecans (OCD), fragmented coronoid process (FPC), patella luxation (PL), hip dysplasia (HD) or infectious arthritis. Dogs with non-musculosceletal disorders were used as controls. The biomarkers were measured by immunoassays.. Median levels of synovial MMP-3, TIMP-1 and molar ratios of MMP/TIMP-1 were significantly higher in the arthritis than in the control group. The release of 5D4 keratan sulfate epitope and serum 3B3 neoepitope was reduced in arthritis patients. Increases in synovial TIMP-1 in OA were less pronounced and the molar ratio of MMP-3/TIMP-1 remained far below 1.0, demonstrating a surplus of the protease inhibitor. In osteoarthritic patients median levels of synovial 5D4 keratan sulfate were up-regulated after ACLR and PL and were inversely correlated with increasing duration of lameness. Serum TIMP-1 levels were significantly reduced in the joint disorder group when compared with the control group.. Our observations present the TIMP-1 serum level as a potential marker for the detection of degenerative changes in cartilage and also indicate that in canine OA, the MMP-3 mediated matrix destruction is not of major importance. However MMP-3 seems to be a sensitive marker for the local inflammation in canine arthritis.

Topics: Animals; Anterior Cruciate Ligament Injuries; Arthritis, Infectious; Chondroitin Sulfates; Dogs; Epitopes; Hip Dysplasia, Canine; Keratan Sulfate; Matrix Metalloproteinase 3; Osteoarthritis; Osteochondritis Dissecans; Patella; Rupture; Tissue Inhibitor of Metalloproteinase-1; Ulna Fractures

This study was designed to assay and compare cartilage oligomeric matrix protein (COMP) in horse sera, in samples from normal and joint diseased horses, and to investigate the relationships between COMP in sera and synovial fluids (SF) with keratan sulphate (KS) data. Sera from 38 horses free of any joint pathology (controls) and from horses with aseptic joint disease (AJD horses, n = 40) were assayed for COMP and KS concentrations. Of the 78 horses in the study, 53 were also assayed for COMP and KS concentrations in SF. COMP and KS were measured by inhibition ELISA, using monoclonal antibodies 12C4 and 5D4, respectively. The COMP concentration in sera from AJD horses (mean +/- s.d. 10.7 +/- 7.4 microg/ml) was significantly (P<0.02) lower than in control sera (14.8 +/- 7.8 microg/ml). The joint disease sera also had significantly lower (P<0.01) KS levels (180.5 +/- 61.8 ng/ml) than controls (237.1 +/- 116.1 ng/ml). A significant correlation (r = 0.52, n = 53, P<0.001) was seen between serum and SF in COMP levels; no such relationship was seen in KS levels. It is possible that serum COMP concentration could be a more specific marker of equine joint disease than any other described to date.

Topics: Animals; Antibodies, Monoclonal; Biomarkers; Blotting, Western; Case-Control Studies; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix Proteins; Glycoproteins; Horse Diseases; Horses; Joint Diseases; Keratan Sulfate; Matrilin Proteins; Osteoarthritis; Synovial Fluid

To elucidate variations in tissue ultrastructure and incidence of pathology between fibromodulin (FM)-null mice and wild-type (WT) animals.. FM-null and WT siblings from different age groups were compared. Serial sections were made through paraffin-embedded whole knees and investigated histologically. Additionally, medial femoral condyle peaks from sibling pairs were investigated ultrastructurally using transmission electron microscopy.. Histological findings demonstrated a clear and increasing disparity between tissue degeneration in WT and FM-null animal knees with progressing age. Distinct differences were apparent by 36 weeks. Around the 80 week period and onward these differences became profound. However, qualitative ultrastructural investigation did not indicate either any aberrant tissue ultrastructure or any abnormal collagen fibril forms in FM-null articular cartilage compared with WT. Biochemical and immunohistochemical investigation of FM-null articular cartilage showed a significant increase in tissue levels of lumican (LUM). Conversely, the cruciate ligaments of the knee showed both an increase in LUM content and considerable structural abnormalities including the tendency towards rupture.. This report indicates for the first time that FM-null mice have a higher propensity towards degenerative changes in their knee joints than comparable WT animals. Interestingly, no underlying ultrastructural or fibril abnormalities within the articular cartilage could be identified to explain why FM-null cartilage is more prone to pathological changes than wild-type tissue. We conclude that alterations in ligaments, and possibly other tissues within the knee, are of considerable importance in the pathogenesis of the observed articular cartilage degeneration.

Topics: Animals; Blotting, Western; Carrier Proteins; Cartilage, Articular; Chondroitin Sulfate Proteoglycans; Collagen; Extracellular Matrix Proteins; Fibromodulin; Keratan Sulfate; Knee Joint; Ligaments, Articular; Lumican; Mice; Mice, Inbred Strains; Microscopy, Electron; Osteoarthritis; Proteoglycans

To evaluate the interaction of bone and cartilage in knee osteoarthritis (OA) pathogenesis in two guinea-pig strains with appreciable differences in bone metabolism.. Two guinea-pig strains were evaluated for their susceptibilities to OA using semi-quantitative histological grading of knee joints and quantification of biomarkers including urinary excretion of hydroxylysyl-pyridinoline (HP) and lysyl-pyridinoline (LP) collagen cross-links, serum osteocalcin (OC), and synovial fluid levels of keratan sulfate (KS).. At 12 months of age, Strain 13 guinea-pigs had minimal to mild histological evidence of OA compared to the Hartley strain guinea-pigs. The Hartley strain, with more severe OA, had a higher rate of bone formation (serum osteocalcin) and bone resorption (HP and LP) evident at a young age with persistence of a greater rate of bone formation at 12 months of age. The Strain 13 possessed much thicker subchondral bone at the outset (2 months) compared to the Hartley; however, the Hartley strain showed the greatest increase in subchondral bone thickness coincident with the development of cartilage degeneration. Thus, the process of subchondral bone thickening, in contrast to the absolute initial subchondral bone thickness, was a hallmark of OA in the guinea-pig. Moreover, Strain 13 had lower intraarticular proteoglycan turnover. Levels of synovial fluid keratan sulfate were positively correlated with the severity of histological OA.. This pilot study represents the first evidence of differential susceptibility to OA in guinea-pigs. Comparison of these two strains of guinea-pig has revealed that increased metabolism within the affected tissues, cartilage and bone, is associated with the development and progression of OA. This work demonstrates that the Strain 13 is a viable age-matched control to the Hartley strain and merits a more in depth evaluation of the contribution of bone and bone metabolism to OA.

Topics: Amino Acids; Animals; Bone and Bones; Bone Density; Cartilage, Articular; Guinea Pigs; Hindlimb; Joints; Keratan Sulfate; Male; Microscopy, Electron; Osteoarthritis; Osteocalcin; Pilot Projects; Synovial Fluid

To investigate the relationship between biochemical markers in the synovial fluid of osteoarthritic and contralateral equine joints and gross articular cartilage pathology.. Twenty-two horses underwent bilateral arthroscopy of their carpal or metacarpophalangeal joints following recent onset lameness. The degree of cartilage damage in each joint was scored and synovial fluid, from both the clinically affected and the contralateral joint, was collected. Bone specific alkaline phosphatase (BAP), 5D4 epitope of keratan sulphate (KS), total glycosaminoglycans (GAG) and hyaluronan (HA) were measured.. The mean age of the horses was 4.1 years and the maximum duration of lameness was three months. Joints examined were midcarpal, antebrachiocarpal and metacarpophalangeal. The median concentration (semi-interquartile range) of BAP was significantly higher in the clinically active joint than in the contralateral joint, 21.75 (6.22) vs. 12.35 (4.07) units, while the other biomarkers measured were significantly lower in the clinically active joint than in the contralateral joint, i.e. KS 8.79 (1.96) microg/ml vs. 16.39 (5.65) microg/ml, KS:GAG ratio 0.19 (0.04) vs. 0.31 (0.10) and HA 741.6 (222) microg/ml vs. 1061.75 (325) microg/ml. BAP was positively (R=0.57), and KS (R=-0.57) and KS:GAG ratio (R=-0.49) were negatively correlated to the degree of cartilage damage within the joint.. The correlation between articular cartilage damage and synovial fluid BAP and KS imparts validity to their potential use as non-invasive diagnostic aids in equine osteoarthritis (OA). The positive correlation between BAP and cartilage damage suggests that there is a link between bone turnover and cartilage damage in OA.

Topics: Alkaline Phosphatase; Animals; Biomarkers; Cartilage, Articular; Cross-Sectional Studies; Glycosaminoglycans; Horse Diseases; Horses; Hyaluronic Acid; Keratan Sulfate; Osteoarthritis; Synovial Fluid

Although none of these markers currently have any clinical applications, researchers have made strong advances in understanding molecular markers of OA and remain optimistic that the goal of identifying clinically useful markers of OA is attainable.(3-6,8,9) Much of this positive sentiment arises from the large array of molecules that have been identified.(8) Molecular markers of the greatest potential clinical use will be those that allow early detection of OA, permit disease progression to be monitored, or allow efficacy of various treatment regimens to be assessed. Earlier and more sensitive detection of OA changes may increase effective opportunities for treatment intervention during reversible phases of the OA disease process and allow more objective assessment of treatment efficacy and prognosis.(6)

Topics: Animals; Biomarkers; Cartilage, Articular; Chondroitin Sulfates; Disease Models, Animal; Dog Diseases; Dogs; Femur; Keratan Sulfate; Matrix Metalloproteinase 3; Osteoarthritis; Synovial Fluid

To compare synovial fluid (SF) levels of oncostatin M (OSM), tumor necrosis factor alpha (TNFalpha), and interleukin-6 (IL-6) in patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to determine which correlate best with SF levels of antigenic keratan sulfate (Ag KS), a marker of aggrecan catabolism, and pyridinium crosslinks, markers of the degradation of mature collagen molecules.. SF was drawn from the knee joints of patients with RA (n = 31) or OA (n = 31). Levels of Ag KS, D-pyridinoline (D-Pyr), pyridinoline (Pyr), OSM, TNFalpha, and IL-6 were measured by enzyme-linked immunosorbent assay.. RA patients had higher median SF levels of OSM, TNFalpha, IL-6, and Pyr, but a lower median level of D-Pyr, than OA patients. In both groups, IL-6 levels correlated positively with those of OSM and TNFalpha. However, the correlation between levels of OSM and TNFalpha was only significant in the RA group. Ag KS and Pyr levels correlated positively in RA but not in OA. The correlation between TNFalpha and Ag KS was positive in RA and negative in OA. Further, in RA, OSM and IL-6 levels correlated strongly with Pyr and Ag KS levels but not with D-Pyr levels, while there were no strong correlations in OA for OSM or IL-6 levels with Pyr, Ag Ks, or D-Pyr levels.. This in vivo study suggests that TNFalpha and other proinflammatory cytokines are involved in the up-regulation of the coordinated degradation of cartilage aggrecan and collagen in RA. Further, OSM may act synergistically with other proinflammatory cytokines in up-regulating the production of metalloproteinases by chondrocytes in rheumatoid joints.

Topics: Adult; Aged; Aggrecans; Antigens; Arthritis, Rheumatoid; Biodegradation, Environmental; Biomarkers; Cartilage; Collagen; Cross-Linking Reagents; Cytokines; Extracellular Matrix Proteins; Female; Growth Inhibitors; Humans; Keratan Sulfate; Lectins, C-Type; Male; Middle Aged; Oncostatin M; Osteoarthritis; Peptides; Proteoglycans; Synovial Fluid; Tumor Necrosis Factor-alpha

To investigate the relationships between serum and urinary molecular markers (MM) used to monitor osteoarthritis.. Forty osteoarthritis patients had blood and urine collected at baseline and 1, 3, 6 and 12 months later. Specimens from 20 controls were obtained twice at a one month interval. The concentration of 14 different markers was determined at each time point and the data were analyzed by statistical methodology.. The markers could be divided by the method of principal components analysis into five clusters of related markers: inflammation markers (C-reactive protein, tumor necrosis receptor type I and tumor necrosis receptor type II, interleukin 6, eosinophilic cationic protein), bone markers (bone sialoprotein, hydroxylysyl pyridinoline, lysyl pyridinoline), putative markers of cartilage anabolism (carboxypropeptide of type II procollagen, hyaluronan, epitope 846) and catabolism (keratan sulfate, cartilage oligomeric matrix protein), and transforming growth factor beta. Three markers (tumor necrosis factor receptor II, cartilage oligomeric matrix protein and epitope 846) from independent clusters discriminated osteoarthritis patients from controls. Inflammation was not a confounding factor in measurement, but a recognizable distinguishing factor in osteoarthritis.. The markers separated into rational groups on the basis of their covariance, a finding with independent biochemical support. The covariance of markers from the same cluster suggests the use of a representative marker from the cluster to reflect changes in osteoarthritis. If multiple markers are being measured within a single cluster, then the use of a weighted cluster 'factor' may be preferable to the separate use of individual markers.

Topics: Amino Acids; Biomarkers; Blood Proteins; C-Reactive Protein; Carboxypeptidases; Case-Control Studies; Epitopes; Extracellular Matrix Proteins; Female; Humans; Hyaluronic Acid; Interleukin-6; Keratan Sulfate; Male; Middle Aged; Osteoarthritis; Osteoarthritis, Hip; Osteoarthritis, Knee; Procollagen; Receptors, Tumor Necrosis Factor; Sialoglycoproteins; Transforming Growth Factor beta

Damage to the meniscus can lead to posttraumatic osteoarthritis. Early markers of joint injury and tissue disease may be useful in developing and administering clinical treatment. We investigated the effects of total medial meniscectomy on biomarkers measured serially in synovial lavage fluid each month for 3 months. Following meniscectomy in dogs, four biomarkers were evaluated: cartilage oligomeric matrix protein, keratan sulfate epitope (5D4), the 3B3(-) neoepitope of chondroitin-6-sulfate, and the 3B3(+) chondroitinase-generated epitope of chondroitin-6-sulfate. Meniscectomy led to statistically significant elevations of all four biomarkers, with levels peaking at 4 weeks. By 12 weeks, the level of the 5D4 epitope returned to the preoperative baseline level whereas that of cartilage oligomeric matrix protein, 3B3(-), and 3B3(+) remained above the baseline. Concentrations of these biomarkers in the knees not operated on did not change significantly from the baseline. The levels of cartilage oligomeric matrix protein and 3B3(-) relative to 3B3(+) remained constant in all knees. In contrast, the level of 5D4 relative to 3B3(+) declined over time in the knee operated on but remained constant in the knee not operated on. These results demonstrate a quantitative change in the molecular components of synovial fluid after meniscectomy, as well as a qualitative change evinced by an alteration in the relative proportions of these epitopes. Extensive analyses showed a strong correlation between serum levels of 3B3(-) from the femoral and cephalic veins; however, serum 3B3(-) was not correlated with synovial fluid 3B3(-). These findings support the hypothesis that the concentrations of select cartilage biomarkers in synovial fluid are altered following meniscectomy and are promising tools for objectively monitoring the induction of osteoarthritis in this model system.

Topics: Animals; Bacterial Proteins; Biomarkers; Cartilage, Articular; Chondroitin Sulfates; Disease Models, Animal; Dogs; Epitopes; Keratan Sulfate; Male; Membrane Proteins; Menisci, Tibial; Osteoarthritis; Synovial Fluid; Transferases

Topics: Biomarkers; Cartilage, Articular; Humans; Hyaluronic Acid; Keratan Sulfate; Osteoarthritis

To relate the rate of bone resorption to serum levels of both hyaluronan (HA) and antigenic keratan sulfate (KS) in canine experimental osteoarthritis (OA) and to evaluate the effects of calcitonin on these parameters and the OA lesions of the unstable knee.. Twenty-two dogs underwent anterior cruciate ligament transection (ACLT) and 6 dogs underwent sham operation. Urinary pyridinium crosslinks were quantified by high-performance liquid chromatography. Immunoassays quantified hyaluronan (HA) and antigenic KS. Macroscopic and histologic OA lesions were scored. Calcitonin treatment was started on day 14 postsurgery and stopped on either day 49 or day 104 postsurgery. Control dogs and all treated dogs were killed on day 105.. All ACLT joints developed OA. In contrast to sham-operated animals, all operated dogs exhibited an early and sustained rise in the levels of their urinary and serum markers. Calcitonin markedly reduced the levels of these markers and the severity of OA lesions. Furthermore, the longer the period of calcitonin therapy, the lower the score of the OA lesions.. Bone, synovium, and articular cartilage all appear to be involved in the state of hypermetabolism that develops in unstable joints. Furthermore, the rate of bone resorption increases markedly in the early stages of this OA model and is likely to contribute to cartilage breakdown. Since calcitonin reduced the severity of OA changes, this form of therapy may have benefits for humans who have recently experienced a traumatic knee injury.

Topics: Amino Acids; Animals; Anterior Cruciate Ligament; Biomarkers; Bone and Bones; Bone Resorption; Calcitonin; Cartilage, Articular; Chromatography, High Pressure Liquid; Disease Models, Animal; Dogs; Hyaluronic Acid; Keratan Sulfate; Osteoarthritis; Synovial Membrane

To investigate longitudinal changes in concentrations of the 1/20/5D4 epitope (5D4) of keratan sulfate and total sulfated glycosaminoglycans (S-GAG) in synovial fluid and serum of dogs with cranial cruciate ligament (CCL) rupture that was repaired via intra-articular surgery.. 58 dogs with a ruptured CCL and osteoarthritis of the affected (index) joint.. Prior to surgical repair of the ruptured CCL, 5D4 concentration was measured in serum and synovial fluid samples by use of an inhibition ELISA, and total S-GAG concentration was measured in synovial fluid samples by use of a direct dye-binding assay. Ruptured CCL were repaired surgically, using an intra-articular fascial graft. Dogs were reexamined 1.5, 7, and 13 months after surgery, and 5D4 and S-GAG concentrations in synovial fluid and serum were measured again.. Serum 5D4 concentrations did not change significantly during the study. Concentrations of 5D4 in synovial fluid (expressed as a ratio of S-GAG concentration) did change significantly with time. In the index joint, the 5D4:S-GAG decreased from 0.19 at the beginning of the study to 0.09 1.5 months after surgery, but 7 months after surgery, the ratio increased again to 0.20.. Results support the hypothesis that serum concentration of 5D4 is not a useful marker of osteoarthritis in dogs. Surgical intervention transiently reduced the concentration of 5D4 in synovial fluid but had no effect on S-GAG concentration.

Topics: Animals; Anterior Cruciate Ligament; Anterior Cruciate Ligament Injuries; Biomarkers; Cohort Studies; Dog Diseases; Dogs; Enzyme-Linked Immunosorbent Assay; Epitopes; Female; Glycosaminoglycans; Joints; Keratan Sulfate; Longitudinal Studies; Male; Osteoarthritis; Rupture; Synovial Fluid

The specific aim of this investigation was to assess differences between primary and secondary osteoarthritis (OA) of the temporomandibular joint (TMJ) using clinical evaluation and synovial fluid analysis for proteoglycans.. Arthroscopic surgery was performed on 101 TMJs from patients with significant pain or dysfunction and who had failed to respond to treatment. Joints were assessed for primary and secondary osteoarthritis. Synovial fluid aspirates were obtained and analyzed to determine the levels of keratan sulfate (KS) epitope and a novel 3B3(-) epitope by enzyme-linked immunosorbent assay (ELISA).. Fifty-four patients and 67 joints had OA diagnosed by both clinical examination and arthroscopy. Primary OA was diagnosed in 14 joints (20%), and the remaining 53 joints were regarded as having secondary OA. No differences were detected in the levels of KS in the synovial fluid from the primary and secondary OA joints. Furthermore, the 3B3(-) epitope was not detectable in the synovial fluid aspirates of any TMJ.. Secondary OA is a common disorder of the TMJ. However, there is no apparent difference in the metabolism of the joints with primary and secondary OA as assessed by proteoglycans in the synovial fluid. The apparent absence of the 3b3(-) epitope, in contrast to its presence in OA of other major synovial joints, suggests that there are some differences between the cartilage metabolism of the TMJ and these other joints during OA.

Topics: Adult; Arthroscopy; Cartilage; Chondroitin Sulfates; Enzyme-Linked Immunosorbent Assay; Epitopes; Female; Humans; Keratan Sulfate; Male; Osteoarthritis; Proteoglycans; Statistics, Nonparametric; Synovial Fluid; Temporomandibular Joint Disc; Temporomandibular Joint Disorders

Keratan sulphate (KS) concentration and anticollagen type II antibody levels were measured in the serum and synovial fluid (SF) of clinically normal horses and horses with osteoarthritis (OA). Serum KS in OA was significantly higher than that in normal horses, while no significant difference was found in KS levels of SF between normal and OA. Assays of antibody to collagen type II showed no significant increase in sera and SF of OA. It was suggested that levels of serum KS would be of value in the pathological detection of OA in the joint, although there was no evidence that the measurable autoimmunity to collagen antigens would reflect the process of OA.

Topics: Animals; Autoantibodies; Collagen; Horse Diseases; Horses; Keratan Sulfate; Osteoarthritis; Synovial Fluid

To investigate the hypothesis that concentrations of the keratan sulfate epitope, 1/20/5D4 (5D4) and total sulfated glycosaminoglycans (S-GAG) in synovial fluids, and of 5D4 in serum of dogs with naturally acquired osteoarthritis of the genual joint (stifle), secondary to cranial cruciate ligament deficiency, are associated with other disease parameters of osteoarthritis.. 58 dogs with stifle osteoarthritis secondary to naturally acquired cranial cruciate ligament (CCL) deficiency.. All dogs were examined clinically, radiographically and, in some instances, scintigraphically. Serum and synovial fluid from both stifles were assayed for 5D4 concentration, using an inhibition ELISA, and for S-GAG, using a direct dye-binding assay.. Serum 5D4 concentration was not significantly associated with other disease features. Total S-GAG values were high, and 5D4 values were low, in synovial fluid from clinically active, compared with contralateral joints. The S-GAG concentration in synovial fluid from clinically active joints was negatively correlated with radiographic severity score (rs = -0.389, P = 0.004, Spearman's rank correlation). Other associations between marker concentrations and disease parameters could not be detected.. Serum 5D4 concentration is not a useful marker of stifle osteoarthritis in dogs; however, synovial fluid S-GAG and 5D4 values may hold more promise as disease measures.

Topics: Animals; Biomarkers; Dog Diseases; Dogs; Epitopes; Female; Glycosaminoglycans; Keratan Sulfate; Male; Osteoarthritis; Regression Analysis; Stifle; Synovial Fluid; Time Factors

The specific aims of this investigation were to determine if there is a relationship between an arthroscopic diagnosis of synovitis and osteoarthritis, and if the presence of synovitis influences the level of cartilage degradation, as evidenced by keratan sulfate levels in the synovial fluid.. Arthroscopic surgery was performed on 114 temporomandibular joints in 88 patients who had significant pain or dysfunction and whose condition had failed to improve with conservative treatment. Synovial fluid aspirates were obtained immediately before arthroscopy and used for the determination of keratan sulfate levels. Arthroscopic examination included assessment of the presence or absence of osteoarthritis and synovitis.. Synovitis was present in 90% of joints, and osteoarthritis was present in 62% of joints examined arthroscopically. Both osteoarthritis and synovitis existed in 57% of the joints. Joints with an arthroscopic diagnosis of synovitis had significantly lower levels of keratan sulfate in the synovial fluid aspirates than joints with osteoarthritis. Synovial fluid aspirates from temporomandibular joints with osteoarthritis had significantly higher levels of keratan sulfate than synovial fluids from joints without osteoarthritis.. Osteoarthritis and synovitis are common diagnoses and are often present concurrently in patients with symptomatic temporomandibular joints. Osteoarthritis is associated with elevated keratan sulfate levels; however, the elevation of keratan sulfate is less in patients with concomitant synovitis.

Topics: Analysis of Variance; Arthroscopy; Cartilage, Articular; Humans; Keratan Sulfate; Osteoarthritis; Paracentesis; Synovial Fluid; Synovitis; Temporomandibular Joint; Temporomandibular Joint Disorders; Temporomandibular Joint Dysfunction Syndrome

To determine if a single time point estimation of chondroitin sulphate (CS) or keratan sulphate (KS) epitopes, hyaluronan (HA), or total glycosaminoglycans (GAG) in knee synovial fluid at time of hospital referral can predict subsequent radiographic progression of knee osteoarthritis.. Two groups of hospital referred patients with knee osteoarthritis were compared: (1) a "progressive" group (n = 45), showing further reduction in radiographic joint space of at least one grade (0-3) in at least one compartment; and (2) a "non-progressive" group (n = 25) in whom radiographs showed no change during the mean follow up period of 2.3 years (median 2, range 1 to 5 years). Knee synovial fluid obtained at the first visit was examined by ELISA for: CS epitopes, using monoclonal antibodies 3B3 and 7D4; KS epitope, using monoclonal antibody 5D4; and HA, using biotinylated HA binding region of cartilage proteoglycan. Total sulphated GAG were measured by dye binding with 1:9 dimethylmethylene blue.. In patients with bilateral synovial fluid data right and left knee values were closely correlated for all variables. There were no significant differences between CS and KS epitopes, HA, total sulphated GAG, or ratios of individual CS or KS epitopes to total GAG, between progressive and non-progressive groups.. Single time point estimation of CS, KS, HA, or total GAG in synovial fluid does not distinguish radiographically progressive and non-progressive knee osteoarthritis patients followed for two years.

Topics: Aged; Aged, 80 and over; Biomarkers; Chondroitin Sulfates; Disease Progression; Epitopes; Female; Follow-Up Studies; Glycosaminoglycans; Humans; Hyaluronic Acid; Keratan Sulfate; Knee Joint; Male; Middle Aged; Osteoarthritis; Synovial Fluid

To determine concentrations of chondroitin sulphate (CS) and keratan sulphate (KS) epitopes, glycosaminoglycans (GAGs) and hyaluronan (HA) in knee synovial fluid (SF) from normal subjects and patients with osteoarthritis (OA) or rheumatoid arthritis (RA), to test whether these variables may be used as markers of the OA process.. OA was subdivided into large joint OA (LJOA), nodal generalised OA (NGOA), and OA with calcium pyrophosphate crystal deposition (CPA). Clinical assessment of inflammation (0-6) was undertaken on OA and RA knees. Knee SF was examined by enzyme linked immunosorbent assay for: CS epitopes, using monoclonal antibodies 3-B-3 and 7-D-4; KS epitope using monoclonal antibody 5-D-4; and HA, using biotinylated HA binding region of cartilage proteoglycan. Total sulphated GAGs were measured by dye binding with 1:9 dimethylmethylene blue.. Increased SF 3-B-3 concentrations and 3-B-3/GAG ratio were found in OA, compared with RA or normal knees, with higher 3-B-3 and 3-B-3/GAG in LJOA and NGOA than in CPA. SF 7-D-4 and 7-D-4/GAG were reduced in RA, compared with normal and OA; SF 5-D-4 was reduced in OA compared with normal. GAG and HA concentrations were decreased in both OA and RA. No correlations with radiographic scores were observed, but SF 7-D-4 was lower in 'inflamed' compared with 'non-inflamed' RA and OA knees. In patients with bilateral samples there were strong correlations between right and left knees for all SF variables.. Changed concentrations of SF CS and KS can be detected in OA with a profile that differs from that seen in RA. Clinical subgrouping and local joint inflammation may influence these measures, supporting different pathogenesis within OA subgroups and requirement for careful patient characterisation in SF studies.

Topics: Adult; Aged; Arthritis, Rheumatoid; Biomarkers; Chondroitin Sulfates; Enzyme-Linked Immunosorbent Assay; Epitopes; Female; Glycosaminoglycans; Humans; Hyaluronic Acid; Keratan Sulfate; Knee Joint; Male; Middle Aged; Osteoarthritis; Synovial Fluid

To determine the effects of low intensity weight-bearing exercise on osteoarthritis (OA) of the knee.. Synovial fluid keratan sulfate (KS) and hydroxyproline were measured as markers of cartilage degradation. The Arthritis Impact Measurement Scales (AIMS) were used to measure health status, and a visual analog scale for pain assessment was used before and after intervention. An exercise (EX) group (n = 15) received a thrice-weekly 12-week low intensity exercise program and a weekly educational program, and a minimal treatment (Min RX) group (n = 15) received only the education program.. Pain levels declined in the EX group, and the Min RX group showed improvement on the AIMS. Synovial fluid was obtained in 11 subjects before and after the intervention. Levels of KS and hydroxyproline did not change.. Further study of exercise effects should include both clinical and biologic parameters to examine the outcome of exercise as a therapeutic intervention in OA of the knee.

Topics: Aged; Biomarkers; Exercise Therapy; Female; Humans; Hydroxyproline; Keratan Sulfate; Knee Joint; Male; Middle Aged; Osteoarthritis; Patient Education as Topic; Pilot Projects; Synovial Fluid

To determine if osteocalcin (OC) is locally produced in the joint and to study the relation between markers of bone, cartilage, and synovial tissue turnover.. The concentrations of OC, keratan sulphate epitope (5D4), and hyaluronate (HA) were measured in paired serum and synovial fluid in 10 healthy volunteers and 15 patients with osteoarthritis (OA) and 16 with rheumatoid arthritis (RA). OC was measured with a commercial immunoradiometric assay and concentrations of 5D4 and HA were measured using enzyme linked immunosorbent inhibition assays.. Synovial fluid OC was found to be significantly lower than serum (p < 0.001) in all patients and controls. Synovial fluid OC concentrations were directly correlated with serum concentrations (r = 0.63, p < 0.001) and with age (r = 0.48, p < 0.01). There were also some relations between OC, HA, and 5D4 in patients with OA and RA. The OC concentrations were directly correlated with HA (r = 0.68, p < 0.01) in OA serum and there was a similar correlation in RA synovial fluid (r = 0.69, p < 0.01). A weak negative correlation was found between OC and 5D4 in OA serum (r = -0.55, p = 0.035) while a weak positive correlation was found in RA serum (r = 0.53, p = 0.034).. These results show that more OC is present in the circulation than in knee joint fluids suggesting that synovial fluid OC may be derived from the blood.

Topics: Adult; Aged; Aged, 80 and over; Arthritis, Rheumatoid; Biomarkers; Bone Remodeling; Cartilage, Articular; Female; Humans; Hyaluronic Acid; Immunoradiometric Assay; Keratan Sulfate; Knee Joint; Male; Middle Aged; Osteoarthritis; Osteocalcin; Rheumatic Diseases; Statistics, Nonparametric; Synovial Fluid

Topics: Biomarkers; Cartilage Oligomeric Matrix Protein; Cartilage, Articular; Extracellular Matrix Proteins; Glycoproteins; Humans; Keratan Sulfate; Matrilin Proteins; Osteoarthritis; Synovial Fluid; Synovitis

The objective of this study on the glenohumeral joint was to assess the (1) accuracy of clinical diagnosis of osteoarthritis compared with arthroscopic diagnosis, and (2) the ability of biochemical markers in synovial fluid to detect osteoarthritis. Patients (96) were examined clinically and the preoperative diagnosis of osteoarthritis was recorded. At surgery (arthroscopy or arthroplasty), the glenohumeral joint was inspected for signs of osteoarthritis, and the joint osteoarthritis grade (I-IV) was recorded. At surgery, synovial fluid lavage was obtained from the joint, and later analyzed to determine levels of aggrecan components: total sulfated glycosaminoglycan and keratan sulfate epitope, link protein and the chondroitin sulfate epitope recognized by antibody 3B3 (3B3(-)). Compared with arthroscopic diagnosis of osteoarthritis, the results showed that the clinical diagnosis did not wrongly identify joints without osteoarthritis, and was always able to identify joints with advanced (Grade IV) osteoarthritis. Grade II osteoarthritis was rarely identified (10% of the time), and Grade III osteoarthritis was identified 50% of the time. Biochemical assessment of the synovial fluid showed that the catabolic markers (sulfated glycosaminoglycan, keratan sulfate and link protein) were elevated in fluids from joints with moderate (Grade III) and advanced osteoarthritis (Grade IV), and the 3B3(-) epitope was elevated in Grades II, III, and IV. These results show that arthroscopic diagnosis for osteoarthritis, of the glenohumeral joint is particularly useful for early and moderate osteoarthritis, where clinical (nonarthroscopic) diagnosis is poor, and that biochemical analysis of the synovial fluids corresponds well to arthroscopic diagnosis of shoulder osteoarthritis.

Topics: Arthroscopy; Biomarkers; Cross-Sectional Studies; Glycosaminoglycans; Humans; Keratan Sulfate; Osteoarthritis; Predictive Value of Tests; Rotator Cuff Injuries; Rupture; Sensitivity and Specificity; Shoulder Joint; Synovial Fluid

This study investigated the synovial fluid concentrations of glycosaminoglycan (GAG), keratan sulphate (KS) epitope 5D4 and chondroitin sulphate (CS) sulphation patterns in healthy volunteers and patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Synovial fluids were collected from knee joints of healthy volunteers (n = 24), and patients with OA (n = 28) and RA (n = 29). Concentrations of GAG and the keratan sulphate epitope 5D4 were measured in 15 of the healthy volunteers, and all of the OA and RA synovial fluids. Total GAG was measured using a dye-binding method and 5D4 by an ELISA. The unsaturated CS disaccharides delta C4 and delta C6 were measured by capillary electrophoresis in all synovial fluids. The concentrations of GAG, 5D4 and delta C6 in the normal synovial fluid were higher but that of delta C4 lower than those of the disease groups. The delta C6:delta C4 ratios correlated with age (r = -0.437, P < 0.001) and the mean value was lower in females than males (2.92 compared with 5.22, P < 0.001). After allowing for age and sex, the delta C6:delta C4 ratio in the control group was significantly elevated (P < 0.001) compared to both OA and RA. The ratio was also related to proteoglycan markers (r = 0.383 for 5D4 and r = 0.357 for GAG). The finding that 5D4 and delta C6:delta C4 ratios are higher in synovial fluid from healthy volunteers compared to OA and RA suggests that they may be markers of the susceptibility of articular cartilage to early damage in arthritis.

Topics: Adult; Age Factors; Aged; Analysis of Variance; Arthritis, Rheumatoid; Biomarkers; Cartilage; Chondroitin; Disaccharides; Epitope Mapping; Female; Glycosaminoglycans; Humans; Keratan Sulfate; Linear Models; Male; Middle Aged; Osteoarthritis; Sex Factors; Sulfur; Synovial Fluid

We studied the effects of intraarticular (ia) administration of hyaluronic acid (HA) (Mw approximately 9 x 10(5)) (Artz) on cartilage integrity and release into synovial fluid (SF) of keratan sulfate peptides (KS-pep) in an ovine model of early osteoarthritis (OA) induced by meniscectomy. Five consecutive weekly injections of HA (2 ml, 10 mg/ml) or saline (2 ml) were initiated 16 weeks after meniscectomy, and animals were sacrificed 5 weeks after the last injection. SF was sampled 8, 16, 23, and 26 weeks postoperation. In the saline injected animals KS-pep levels increased progressively in SF, relative to nonoperated controls (p < 0.05). KS-pep levels in SF of the HA treated group also increased, but were not statistically different from controls. Using a modified Mankin histological scoring system, cartilage at necropsy of HA injected joints showed less damage than similar regions of saline treated animals. A new mechanism for the protective effects of HA on cartilage is proposed.

Topics: Animals; Cartilage, Articular; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Hyaluronic Acid; Injections, Intra-Articular; Keratan Sulfate; Knee Joint; Osteoarthritis; Sheep; Synovial Fluid

These studies seek to define the gene expression of proteoglycans and collagens in the developing hypertrophic phase of an experimental model of osteoarthritis (OA). Total RNA was extracted from articular cartilage of nonoperated and operated dog knees 10 weeks after induction of OA by transection of the anterior cruciate ligament. The relative amounts of mRNA for type II collagen and the core proteins for the aggrecan, biglycan, decorin, and fibromodulin were analyzed by Northern blotting. Total RNA in OA vs nonoperated knees was statistically significantly elevated 2.5x, the mRNA for type II collagen was elevated 8x, aggrecan 2x, biglycan 4x, fibromodulin 2x. The level for decorin was increased 1.6x, but this difference was not statistically significant. Chondrocytes respond actively to joint injury. Gene expression of proteoglycans and type II collagen is discoordinate in early experimental OA, and may contribute to the development of cartilage abnormalities.

Topics: Aggrecans; Animals; Biglycan; Carrier Proteins; Collagen; Decorin; Disease Models, Animal; Dogs; Extracellular Matrix Proteins; Fibromodulin; Gene Expression; Keratan Sulfate; Lectins, C-Type; Male; Osteoarthritis; Proteoglycans

To investigate the prognostic value of serum hyaluronic acid (HA) and keratan sulfate (KS) levels in relation to tibiofemoral osteoarthritis (OA) of the knee.. Clinical and demographic data were collected on 94 patients. Radiographs were obtained at study entry and at 5-year followup. Disease progression was defined as 2 mm of joint space narrowing of any tibiofemoral compartment, and/or knee joint surgery during the study period. Serum HA and KS were measured and levels were correlated with entry data and disease progression.. At entry, HA levels were significantly related to disease duration (P = 0.036), minimum joint space (P = 0.049), and previous surgery (P = 0.001). After these variables were taken into account, patients whose disease had progressed were shown to have had significantly higher levels of HA at baseline compared with those whose disease had not progressed (P = 0.019). However, there were no significant differences in levels of serum KS between those with and those without disease progression, at entry (P = 0.779) or at subsequent visits.. These results suggest that serum HA levels predict disease outcome in OA of the knee and confirm that a single measurement of the serum level of KS is not useful as a prognostic marker in OA.

Topics: Age Factors; Aged; Biomarkers; Cross-Sectional Studies; Disease Progression; Enzyme-Linked Immunosorbent Assay; Female; Follow-Up Studies; Humans; Hyaluronic Acid; Keratan Sulfate; Knee Joint; Male; Middle Aged; Osteoarthritis; Prognosis; Radiography; Regression Analysis

To test the hypothesis that scintigraphic evidence of bone activity will correlate with biochemical evidence of increased matrix turnover in osteoarthritis (OA).. Keratan sulfate epitope (5D4), chondroitin sulfate epitope (3B3), and osteocalcin (OC) were measured in synovial fluid (SF) from 35 patients with knee OA, within 1 month of bone scan.. SF OC levels correlated with 5D4 levels (r = 0.32, P = 0.047) and with abnormalities on scintigraphic scan. Mean OC levels were 47% higher (P = 0.016) in patients with severely abnormal findings on scans compared with levels in patients with mildly abnormal scan findings. No significant association of 5D4 or 3B3 levels and perfusion- or late (bone)-phase scintigraphic abnormalities was found.. These data support the hypothesis that there is an association between late-phase bone scan abnormalities and SF biochemical markers of bone turnover in OA.

Topics: Aged; Aged, 80 and over; Biomarkers; Bone and Bones; Cartilage, Articular; Chondroitin Sulfates; Female; Humans; Keratan Sulfate; Knee Joint; Male; Middle Aged; Osteoarthritis; Osteocalcin; Radionuclide Imaging; Synovial Fluid

Topics: Cartilage, Articular; Chondroitin Sulfate Proteoglycans; Collagen; Cryopreservation; Humans; Keratan Sulfate; Lumican; Microscopy, Immunoelectron; Osteoarthritis; Pressure

The metabolism of the cartilage proteoglycan aggrecan was studied in patients with osteoarthritis (OA, n = 83), rheumatoid arthritis (RA, n = 127), and in controls (n = 117) using monoclonal antibody-based radioimmunoassays for glycosaminoglycans in the serum and synovial fluid (SF) to detect epitope 846 on chondroitin sulfate (probably only on recently synthesized molecules) and a keratan sulfate (KS) epitope AN9PI, present on intact and degraded molecules. Epitope 846 levels were always elevated in SF over serum (mean 38-fold in OA and 8.6-fold in RA) being highest in OA patients with the longest disease duration and greatest loss of cartilage, and lowest in RA joints with high leucocyte counts. Serum levels were more often elevated in RA (56%) than in OA (19%) and probably reflect increased aggrecan synthesis in diseased joints. KS levels were higher in SF than in serum in 69% of patients (up to 2.3-fold); levels were inversely (OA) and directly (RA) related to SF leucocyte counts. Serum KS was reduced in both diseases and in RA was inversely related to both systemic and joint inflammation markers. SF 846 levels were inversely related to SF KS in both diseases. These epitopes may provide a measure of the balance between cartilage synthesis and degradation in these diseases.

Topics: Adult; Aged; Aged, 80 and over; Aggrecans; Arthritis, Rheumatoid; Cartilage; Chondroitin Sulfates; Epitopes; Extracellular Matrix Proteins; Female; Humans; Keratan Sulfate; Lectins, C-Type; Male; Middle Aged; Osteoarthritis; Proteoglycans; Synovial Fluid

The levels of proteoglycan aggregate components (link protein, keratan sulfate epitope, and total sulfated glycosaminoglycan) were determined in the synovial fluid lavages of dogs with experimental osteoarthritis or disuse atrophy. A model of experimental osteoarthritis was created by transection of the anterior cruciate ligament of the right knee; studies were carried out 6 and 12 weeks after surgery. Joint disuse was studied at 4 and 8 weeks after initiation of the disuse. Recovery after disuse also was studied in joints that had 3 weeks of remobilization after 4 or 8 weeks of disuse. Synovial fluid lavages from the right knee joints of untreated animals were used as controls. The concentrations of keratan sulfate epitope, sulfated glycosaminoglycan, and link protein in the synovial fluid lavages at 6 and 12 weeks after transection of the anterior cruciate were elevated compared with the control values. Similar analysis of the fluid after disuse showed that the levels of keratan sulfate epitope and sulfated glycosaminoglycan were increased compared with the control levels and the levels after transection. However, the concentration of link protein in the fluid after disuse was not significantly different from the control level. The levels of keratan sulfate epitope and sulfated glycosaminoglycan in the synovial fluid lavages after disuse with recovery were high, but the levels of link protein remained low. The results indicate that the catabolism of proteoglycan aggregates in articular cartilage during early osteoarthritis and disuse is different. The determination of keratan sulfate epitope in synovial fluid lavages appears to provide a relatively general indication of proteoglycan catabolism, whereas increased levels of link protein may be more indicative of cartilage degeneration.

Topics: Aggrecans; Analysis of Variance; Animals; Bone Matrix; Cartilage, Articular; Disease Models, Animal; Dogs; Enzyme-Linked Immunosorbent Assay; Epitopes; Extracellular Matrix Proteins; Glycosaminoglycans; Joint Diseases; Keratan Sulfate; Knee Joint; Lectins, C-Type; Osteoarthritis; Proteoglycans; Radioimmunoassay; Synovial Fluid

To measure serum levels of collagenase (MMP-1), stromelysin-1 (MMP-3), and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) in normal subjects and in patients with osteoarthritis (OA), and to assess how these correlate with biochemical and clinical indicators of disease activity in OA.. Specific immunoassays were used to measure MMPs, TIMP-1, and antigenic keratan sulfate (KS). The total area of cartilage affected by the disease was measured (expressed as an articular index).. In the normal population (n = 118), the serum concentration of MMP-3, but not of MMP-1 or TIMP-1, increased with age and was approximately 2 times higher in males than in females. In the OA patients (n = 33), the serum levels of MMP-3, but not of MMP-1 or TIMP-1, were significantly elevated and correlated strongly with the articular index but poorly with objective and subjective functional capacity scores as well as with serum levels of antigenic KS and systemic parameters of inflammation.. These findings illustrate the importance of matching patients and normal controls for age and sex in further studies of MMP-3 and are consistent with the hypothesis that MMP-3 might play an important role in the degradation of joint cartilage in OA. Further, serum levels of MMP-3 may prove useful for monitoring therapy for OA.

Topics: Aged; Anti-Inflammatory Agents, Non-Steroidal; Antigens; Blood Sedimentation; Collagenases; Female; Glycoproteins; Humans; Keratan Sulfate; Male; Matrix Metalloproteinase 3; Metalloendopeptidases; Middle Aged; Osteoarthritis; Tissue Inhibitor of Metalloproteinases

Glycosaminoglycans (GAG) and keratan sulphate (KS) were measured in sera and synovial fluids from dogs with either osteoarthritis (OA) or rupture of the cranial cruciate ligament (CCL) and normal dogs. The dogs with OA had higher synovial fluid GAG levels (P < 0.002) and serum KS (P < 0.03) compared to the normal dogs. No significant differences in serum GAG were found in either group. In both OA and rupture of the CCL, GAG levels were increased in the synovial fluid from the affected joint compared with the clinically normal (inactive) contralateral joint. Neither GAG nor KS measurements correlated with serum and synovial fluid antibodies to collagen type II, synovial fluid white cell count or age of dog. It is unlikely that the measurement of these cartilage breakdown products is of value for diagnostic or prognostic use in canine arthropathies.

Topics: Analysis of Variance; Animals; Dog Diseases; Dogs; Enzyme-Linked Immunosorbent Assay; Glycosaminoglycans; Joint Diseases; Keratan Sulfate; Ligaments, Articular; Osteoarthritis; Reference Values; Rupture, Spontaneous; Synovial Fluid

The present study was undertaken to identify the cartilage matrix molecules that are bound with intermolecular disulfide bonds to IgG and serum albumin molecules recovered from the articular cartilage of patients with rheumatoid arthritis (RA) or osteoarthritis (OA). The cartilage specimens were extracted sequentially with three changes of neutral buffer, three changes of 6 M guanidine hydrochloride and then partially degraded with bacterial collagenase. The extracted IgG and albumin, along with matrix molecules bound to these proteins, were isolated with affinity chromatography using antibodies to IgG or human serum albumin conjugated to agarose beads. The isolated materials were characterized with sodium dodecyl sulfate polyacrylamide gel electrophoresis and transfer blotting, using specific antibodies to IgG, albumin, and proteoglycans. In the isolated materials, heteropolymers with IgG or albumin were identified. These polymers contained keratan sulfate and less frequently chondroitin-4-sulfate and chondroitin-6-sulfate. These findings identified the keratan sulfate rich proteoglycans, prevalent at the surface of joint cartilage, as the most common cartilage matrix molecules that are covalently bound to IgG or to serum albumin by disulfide bonds in the articular cartilage of patients with RA or OA.

Topics: Arthritis, Rheumatoid; Cartilage, Articular; Chondroitin Sulfates; Humans; Immunoglobulin G; Keratan Sulfate; Osteoarthritis; Serum Albumin

Canine experimental models of osteoarthritis (OA) and disuse atrophy were used to study cartilage metabolism. The synovial fluids from the OA joints showed elevated levels of keratan sulfate (KS) epitope and link protein, indicating increased catabolism. Analysis of fluids from joints with disuse atrophy showed high levels of KS epitope, but no increase in link protein. Quantitation of a novel chondroitin sulfate (3B3) epitope showed it to be present only in the synovial fluids and articular cartilage of the OA joints. The results indicate that these may be important indicators, or markers, of degenerative joint disease.

Topics: Animals; Atrophy; Cartilage, Articular; Chondroitin Sulfates; Disease Models, Animal; Dogs; Keratan Sulfate; Osteoarthritis; Proteoglycans; Synovial Fluid

Human chondrocyte strains were derived from explant outgrowth of nonarthritic or osteoarthritic human cartilage. Chondrocytes radiolabeled with [35SO4] or [35S]-methionine were used to measure the biosynthesis of proteoglycans recovered from the most buoyant fraction (A4) of a CsCl density gradient centrifugation performed under associative conditions. The proteoglycans isolated from the A4 fraction (rho < 1.47 g/ml) were hydrodynamically small and contained both large and small glycosaminoglycan chains. When assessed by SDS/PAGE using 3-16% gradient gels, two subpopulations of small proteoglycans (smPG) were identified. The larger of the two species (smPG-I) migrated slower than the 200 kDa marker protein; when reassessed on 3-5% acrylamide gels, its apparent molecular mass was larger than the 480 kDa and 440 kDa alpha and beta heavy chains of dynein. We estimated the apparent molecular size of this smPG to be approximately 520 kDa. The smaller smPG (smPG-II) had an apparent average molecular mass of 180 kDa (range 170-210 kDa) after 3-16% SDS/PAGE. Three monoclonal antibodies, 1C6, 5D4, and S103L, reactive with the hyaluronic acid binding region of the aggregating proteoglycan core protein, keratan sulfate, and a core protein domain in the chondroitin sulfate attachment region, respectively, reacted with a single protein (apparent molecular mass, 180 kDa) that was similar in size to smPG-II.

Topics: Antibodies, Monoclonal; Binding Sites; Cartilage, Articular; Cells, Cultured; Centrifugation, Density Gradient; Chondroitin Sulfates; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Humans; Hyaluronic Acid; Keratan Sulfate; Molecular Weight; Osteoarthritis; Proteoglycans

Serum concentrations of antigenic keratan sulphate determined by an enzyme linked immunosorbent assay (ELISA) with a monoclonal antibody were studied in patients with rheumatoid arthritis (RA), osteoarthritis, ankylosing spondylitis, other inflammatory diseases, and a large control group of women without arthritis. Mean keratan sulphate concentrations were low in 117 women with RA compared with 227 female control subjects matched for age drawn from a community survey. There were significant correlations between serum keratan sulphate concentrations in patients with RA and serum C reactive protein and the erythrocyte sedimentation rate. Serum keratan sulphate concentrations were also low in 29 men and women with ankylosing spondylitis and 29 patients with arthritis and high concentrations of C reactive protein. In 98 women undergoing an operation for benign breast disease there were decreases in serum keratan sulphate concentrations after the operation which correlated with doses in serum C reactive protein. No differences were found in keratan sulphate concentrations in 137 women with osteoarthritis compared with controls. Within the group with osteoarthritis there were no differences for the various joint groups and there was no obvious correlation with radiographic severity or progression. These findings suggest serum keratan sulphate is unlikely to be useful as a diagnostic marker in osteoarthritis or RA but indicate a role for inflammation in the regulation of cartilage loss.

Topics: Acute-Phase Reaction; Aged; Arthritis, Rheumatoid; Biomarkers; Blood Sedimentation; C-Reactive Protein; Cartilage, Articular; Enzyme-Linked Immunosorbent Assay; Female; Humans; Keratan Sulfate; Male; Middle Aged; Osteoarthritis; Rheumatic Diseases; Spondylitis, Ankylosing

The release rates of specific components of the proteoglycan aggregates (G1 domain, the chondroitin sulfate and keratan sulfate containing portion of the protein core, and link protein) of the articular cartilage of mature beagles were studied at early stages of canine experimental osteoarthritis (OA), generated by transection of the anterior cruciate ligament. Analysis of cartilage explants and synovial fluids indicates that at early stages of experimental OA, there is increased release of the proteoglycan aggregates of the articular cartilage. This involves a release from the tissue of the components of the proteoglycan that are specifically involved with aggregation together with the glycosaminoglycans of the proteoglycan. These components were detected at elevated levels in the media of explants of cartilage from the operated joint, and in the synovial fluids of the operated joints.

Topics: Animals; Cartilage, Articular; Chondroitin; Culture Techniques; Disease Models, Animal; Dogs; Extracellular Matrix Proteins; Female; Keratan Sulfate; Osteoarthritis; Proteoglycans; Synovial Fluid

The metabolism of newly-synthesised and total ("resident") proteoglycans was examined in control and osteoarthritic cartilage explants obtained from an experimental model (Pond and Nuki, 1973) of canine osteoarthritis. The following findings were obtained: (i) Non-labelled proteoglycans extracted from normal cartilage with 4 M guanidine HCl showed two bands visualised by staining with toluidine blue. The electrophoretic mobilities of proteoglycans from osteoarthritic cartilage were unchanged but the relative abundance of the slower migrating band increased with time after surgery. (ii) There were qualitative differences in the proteoglycan breakdown products released into the medium of explant cultures of osteoarthritic compared with control cartilage. This was apparent for both labelled and total unlabelled proteoglycans. (iii) There were similarities in the electrophoretic mobilities of the major labelled and non-labelled proteoglycan breakdown products suggesting that total ("resident") proteoglycans and newly-formed proteoglycans were degraded by similar mechanisms. There were however some differences in the labelled and non-labelled proteoglycans, suggesting that the mechanisms of breakdown were not identical. (iv) Immunoblotting techniques showed differences in the distribution of various glycosaminoglycans in proteoglycan breakdown products from control compared with osteoarthritic cartilage explant cultures. (v) Monoclonal antibodies 7-D-4 and 3-B-3 (which recognise unusual native chondroitin sulphate epitopes) showed greatly increased expression on proteoglycans from osteoarthritic cartilage compared with controls.

Topics: Animals; Antibodies, Monoclonal; Binding Sites; Cartilage, Articular; Chondroitin Sulfates; Dogs; Electrophoresis, Polyacrylamide Gel; Female; Glycosaminoglycans; Hyaluronic Acid; Keratan Sulfate; Osteoarthritis; Proteoglycans

Glycosaminoglycans in normal and osteoarthrotic temporomandibular joint disks were studied by means of high-performance liquid chromatography methods. Normal disk tissue contains galactosaminoglycans (chondroitin sulfate and dermatan sulfate) as the main polysaccharides and with smaller amounts of hyaluronate and heparan sulfate. The galactosaminoglycans are mainly sulfated in 6-position, and some of the disaccharides contain iduronic acid. There was a slight general variation in glycosaminoglycan concentration with increasing age. In the severely arthrotic disks the content of glycosaminoglycans was considerably lower than in normal disk tissue. This decrease was far more extensive than that observed in relation to age in normal tissue. The 4/6-sulfate ratio of the galactosaminoglycans was increased, whereas the proportion of iduronic acid was markedly decreased.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Cartilage, Articular; Chondroitin Sulfates; Chromatography, High Pressure Liquid; Dermatan Sulfate; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Hyaluronic Acid; Keratan Sulfate; Male; Middle Aged; Osteoarthritis; Temporomandibular Joint; Temporomandibular Joint Disorders

In order to evaluate the effect of calcium pyrophosphate dihydrate (CPPD) deposition on articular cartilage catabolism, the proteoglycans released into normal synovial fluid were compared with those in synovial fluid obtained from patients with osteoarthritis (OA), chronic pyrophosphate arthropathy (CPA) and acute pyrophosphate arthropathy (APA). Keratan sulphate (KS) was measured by the modified 1,9-dimethylmethylene blue (DMB) assay in synovial fluids treated with chondroitin ABC lyase. This enzyme was found to eliminate all of the sulphated glycosaminoglycans in synovial fluid except KS. In OA, CPA and APA the concentrations of KS were found to be significantly higher than in normal synovial fluid (NSF) (P less than 0.01). Similar KS concentrations were observed in CPA and APA. In CPA they were significantly higher than in OA (P less than 0.02). The size distribution of proteoglycan fragments varied between different patients with the same disease, but only minor differences were observed in patients with OA and CPA who were matched for age, sex and disease severity. Furthermore, the size distribution of proteoglycan fragments in the acute and chronic phases of pyrophosphate arthritis was similar. Thus although in pyrophosphate arthritis the rate at which proteoglycans are released from the cartilage may be greater than in OA or normal joints, the fundamental processes governing the release of these macromolecules may be the same.

Topics: Adult; Aged; Aged, 80 and over; Arthritis; Calcium Pyrophosphate; Chromatography, Gel; Diphosphates; Female; Humans; Keratan Sulfate; Male; Middle Aged; Osteoarthritis; Proteoglycans; Synovial Fluid

The levels of serum keratan sulfate (KS) were measured in 9 dogs who underwent anterior cruciate ligament transection. They rose in some animals as early as 7 days after surgery, long before any decrease in the articular cartilage content of KS containing proteoglycans could be measured; and they reached their maximum in most dogs by Day 21 and remained elevated for at least 13 weeks. In contrast, the serum levels of the KS epitope showed no increase in sham operated control animals. Analysis of synovial fluid suggested an increase in the catabolism of cartilage proteoglycans in the operated joint contributed significantly to the increased levels of serum KS.

Topics: Analysis of Variance; Animals; Anterior Cruciate Ligament; Dogs; Epitopes; Keratan Sulfate; Knee Joint; Osmolar Concentration; Osteoarthritis; Synovial Fluid; Time Factors

Synovial fluid (SF) was obtained from 40 patients with varying grades of osteoarthritis (OA) of the knee and examined by transmission electron microscopy to ascertain how frequently hydroxyapatite crystals (HA) were present and whether they were related to disease severity or putative markers or promoters of cartilage resorption. HA crystals were conspicuous and abundant in specimens from 21 of the 40 patients studied. Patients in whom HA was present had significantly larger effusions (13.0 +/- 8.9 vs 8.7 +/- 6.1 ml, p less than 0.05). They also tended to have radiologically more severe disease (radiological grade: 2.91 +/- 0.92 vs 2.39 +/- 0.85, p = 0.056). No difference in keratan sulfate (KS) concentrations was observed. Moreover, despite the presence in some specimens of numerous free histiocytes which were actively phagocytosing HA aggregates, the concentrations of interleukin 1 beta (IL-1 beta), a monocyte product with cartilage and bone resorbing activity, were below the limit of detection (20 pg/ml). Our results confirm that HA crystals are a common finding in patients with OA of the knee and show that HA is associated with larger effusions, but not increased SF concentrations of cartilage proteoglycan substituents (KS) or IL-1 beta.

Topics: Aged; Crystallization; Female; Humans; Hydroxyapatites; Interleukin-1; Keratan Sulfate; Knee Joint; Macrophages; Male; Microscopy, Electron; Osteoarthritis; Synovial Fluid; Synovial Membrane

Horse articular cartilage glycosaminoglycans (GAGs) were measured in synovial fluids from 48 joints affected with osteoarthritis (OA), 22 normal joints, four joints with osteochondritis, three joints with traumatic arthritis and seven joints infected with bacteria. Serum and urine from individual horses were also examined for the presence of GAGs. High levels of GAGs were found in synovial fluids (SF) from horses with OA. In each case, the level was higher in the synovial fluid than in the serum or urine from the same horse. Horses with OA showed high GAG levels in SF, serum and urine compared to horses with normal and infected joints. High levels were also found in horses with osteochondritis and traumatic arthritis. Levels of synovial fluid GAG reflect cartilage destruction in arthritis and may be useful for monitoring disease progression in the equine species.

Topics: Animals; beta-Galactosidase; Chondroitin Lyases; Chondroitin Sulfates; Glycosaminoglycans; Glycoside Hydrolases; Hyaluronic Acid; Hyaluronoglucosaminidase; Keratan Sulfate; Osteoarthritis; Synovial Fluid

We examined the relationship between serum and synovial fluid (SF) levels of antigenic keratan sulfate (KS) and the clinical, laboratory, and radiologic features of disease in 125 well-characterized patients with knee osteoarthritis (OA). KS was quantified by enzyme-linked immunosorbent assay, using an antibody specific for a highly sulfated epitope on KS chains; the results were calculated as equivalents of an international standard of KS from human costal cartilage. The mean level of serum KS (393 ng/ml) was significantly higher than those previously reported for populations of adults without OA. There was a wide scatter of serum KS values (range 156-912 ng/ml), with little correlation with clinical or radiologic features. Men had significantly higher levels than women (456 +/- 135 ng/ml versus 368 +/- 110 ng/ml, mean +/- SD), and there was a statistically significant but weak association with indicators of polyarticular involvement (number of symptomatic joints, Heberden's nodes, hip symptoms) in women. Despite the wide scatter of results in the population as a whole, individual levels of KS were stable for up to 4 consecutive years in the 9 patients studied. Levels of KS were much higher in SF (n = 25) than in serum, but the two were not correlated. There was an inverse correlation between radiographic evidence of cartilage loss and the level of KS in SF. The large variations in serum KS values suggest that this measure may not be of diagnostic significance among populations of patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adolescent; Adult; Body Height; Body Weight; Child; Child, Preschool; Cross-Sectional Studies; Female; Humans; Infant; Keratan Sulfate; Knee Joint; Male; Middle Aged; Osteoarthritis; Prospective Studies; Radiography; Sex Factors; Synovial Fluid

The concentration of keratan sulfate (KS) epitope was measured in the serum of patients with osteoarthritis (OA) or rheumatoid arthritis (RA) by enzyme-linked immunosorbent assay and compared with that in the serum of patients with primary fibromyalgia syndrome (PFS) and of controls who had no joint disease. By Student's tau-test, the mean serum KS concentrations in OA and RA patients measured with monoclonal antibodies (MAb) 5-D-4 and 2-D-3 were significantly increased over those in the PFS and normal groups; similar findings were observed using a nonparametric test, except that levels in RA patients showed no difference from those in PFS patients and normal subjects. There was no significant correlation between joint scores or disease duration and KS levels in OA or RA patients. Gel filtration of sera revealed mainly large, polydisperse KS-bearing fragments which eluted in a broad profile. KS purified from sera by immunoaffinity chromatography consisted mainly of high-density proteoglycans. Electrophoresis of pooled high-density KS fractions in polyacrylamide-agarose gels followed by Western blotting with MAb 5-D-4 showed diffuse bands with relative mobilities corresponding to large proteoglycans. These findings are consistent with attachment of KS to protein core fragments of various sizes; KS in patient sera is comparable in size with that in normal sera. Elevations of serum KS levels occur in the presence of cartilage degradation, but do not quantitatively define the extent or duration of articular involvement.

Topics: Arthritis, Rheumatoid; Blotting, Western; Chromatography, Affinity; Chromatography, Gel; Enzyme-Linked Immunosorbent Assay; Fibromyalgia; Humans; Joints; Keratan Sulfate; Molecular Structure; Osteoarthritis

The role of osteoarthrosis (OA) and proteoglycan degradation in the pathogenesis of temporomandibular joint (TMJ) disorders has not been well established. The orthopaedic literature has demonstrated that proteoglycan degradation plays a significant role in the pathology of many joints. The purpose of this investigation was to determine if levels of immunoreactive keratan sulfate (an important component of cartilage proteoglycans) present in synovial fluid aspirates from TMJs correlated with arthroscopically demonstrated OA. Temporomandibular joint arthroscopy was performed on 25 joints in 20 patients and synovial fluid aspirates were obtained just prior to the insertion of arthroscopic cannulas. The results showed that synovial fluid aspirates from joints that arthroscopically demonstrated OA had significantly higher levels of keratan sulfate than synovial fluid aspirates from those joints that showed no evidence of OA (NON-OA). This study gives support to the theory that the pathogenesis of OA of the TMJ is similar to that of chondromalacia of other synovial joints. The combination of TMJ arthroscopy and synovial fluid analysis is an important model that can be used for investigation of the pathogenesis of TMJ disorders.

Topics: Adult; Arthroscopy; Female; Humans; Keratan Sulfate; Male; Osteoarthritis; Proteoglycans; Synovial Fluid; Temporomandibular Joint Disorders

Degradation of cartilage in osteoarthritis of man results in the release of sulphated glycosaminoglycans, particularly keratan sulphate, into tissue fluids. A study was made to evaluate these markers for osteoarthritis in the horse. Synovial fluid and serum levels of keratan sulphate, measured by an ELISA-inhibition technique, and sulphated glycosaminoglycans measured by specific dye binding assay, were found to be significantly increased (P less than 0.001) in joints from horses with osteoarthritis, compared with normal joints. Synovial fluids from joints with infective arthritis also showed high keratan sulphate levels, but statistically were not significantly different from osteoarthritis. Measurement of serum/synovial fluid levels of proteoglycan may enable cartilage degeneration to be detected and monitored and help more effective treatments to be developed in the equine species.

Topics: Animals; Antibodies, Monoclonal; Arthritis, Infectious; Cartilage, Articular; Enzyme-Linked Immunosorbent Assay; Epitopes; Glycosaminoglycans; Horse Diseases; Horses; Keratan Sulfate; Osteoarthritis; Proteoglycans; Synovial Fluid

A new sandwich-ELISA for the determination of keratan sulphate peptides in biological fluids is described. The technique involves binding a commercially available monoclonal antibody against keratan sulphate to microtitre plates, adding the unknown keratan sulphate antigen then interacting the keratan sulphate-antibody complex with a biotin-monoclonal antibody conjugate which was also specific for keratan sulphate peptides. Alkaline phosphatase conjugated streptravidin was then added and the amount which bound to the biotin was determined by measuring the release of chromogen from an added chromogenic substrate. Using this assay, keratan sulphate peptides in biological fluids within the range 10-1000 ng/ml could be quantitated. This method was found to be more sensitive than presently used techniques. The intra- and inter-assay coefficients of variation were 11% and 13%, respectively.

Topics: Adult; Arthritis, Rheumatoid; Enzyme-Linked Immunosorbent Assay; Humans; Keratan Sulfate; Middle Aged; Osteoarthritis; Peptides; Proteoglycans; Synovial Fluid

In order to clarify the cause and mechanism of joint degeneration in osteoarthritis (OA), a histopathological study and ultrastructural-histochemical analysis were performed on the articular cartilage of the C57 Black mouse, a model of spontaneous osteoarthritis. Using transmission electron microscopy (TEM), in all stages of light microscopically recognized OA, we found that Golgi apparatus++ were poorly developed, intracellular microfilaments markedly increased, proteoglycan granules decreased and collagen networks broken. Observing histologically normal models of different ages by TEM, we found Golgi apparatus++ and other organelles to be well developed regardless of age. Many proteoglycan granules were seen, mainly consisting of keratan sulphate in the later months. Collagen networks were maintained. These results suggest that disturbed protein transport and sugar synthesis in chondrocytes due to the deficient development of Golgi apparatus++ caused the degenerative change in articular cartilage and that, with aging, the structure and function of the matrix were maintained mainly due to the continued presence of keratan sulphate.

Topics: Aging; Animals; Cartilage, Articular; Female; Golgi Apparatus; Keratan Sulfate; Male; Mice; Mice, Inbred C57BL; Microscopy, Electron; Osteoarthritis; Proteoglycans

The effects of the chondroprotective agents (Arteparon, SP-54 and DH40J) on the release of proteoglycan degradation products (as keratan sulphate peptide fragments) from articular cartilage implanted into rat subcutaneous air pouches have been investigated by using an enzyme-linked immunosorbent-inhibition assay (ELISIA). The ELISIA technique was capable of quantitating the keratan sulphate peptides (KS peptides) in fluids within the range of 100-2,000 ng/ml by using the monoclonal antibody line 1/20/5-D-4 and human articular cartilage KS peptides as standard reagents. It was found that the levels of KS peptides present in the air-pouch fluid of rats treated with the chondroprotective drugs was significantly less than in fluid aspirated from the pouches of non-drug-treated control animals. On the basis of these findings we suggest that the assessment of KS peptide by ELISIAs may provide a useful means of monitoring proteoglycan breakdown products in biological fluids (e.g. synovial fluids or blood) and for evaluating the effects that antiarthritic drugs may have on this process.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arthritis, Rheumatoid; Cartilage, Articular; Chondroitin Sulfate Proteoglycans; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Glycosaminoglycans; Keratan Sulfate; Lumican; Male; Osteoarthritis; Pentosan Sulfuric Polyester; Peptide Fragments; Rats; Rats, Inbred Strains

To determine whether the serum keratan sulfate (KS) concentration reflected the status of degenerating articular cartilage in a commonly used model of osteoarthritis (OA), serum KS levels were measured in 9 dogs prior to transection of the anterior cruciate ligament, 4 weeks later, and when the dogs were killed 8-14 weeks after surgery, at which time mild OA was present. In all cases, the serum KS levels were within the normal range. Values were not related to the cartilage uronic acid concentration, the rate of net 35SO4 glycosaminoglycan synthesis, or the histopathologic changes of OA. Although the serum KS concentration was not helpful as an indicator of the current status of the articular cartilage abnormality in the OA knee, serial samples from 6 dogs showed an increase of at least 10% over the baseline KS level at both timepoints following surgery (P = 0.031 and 0.027). This presumably reflects changes in proteoglycan metabolism in the unstable knee, although the possibility of a systemic change in proteoglycan metabolism following cruciate ligament transection cannot be excluded.

Topics: Animals; Cartilage, Articular; Dogs; Glycosaminoglycans; Keratan Sulfate; Knee Joint; Ligaments, Articular; Male; Osteoarthritis

Serum keratan sulfate (KS) levels were measured with an enzyme-linked immunosorbent assay using monoclonal antibody (MAb) 5-D-4 (anti-KS) in a rabbit model of osteoarthritis (OA) induced by partial meniscectomy. The partial medial meniscectomy produced pathologic changes of OA in the joints of the rabbits, which were seen when the animals were killed at 3, 6, 9, or 12 weeks postsurgery. Tibial or femoral osteophytes were seen in up to 90% of the operated joints; pitting and ulceration of medial femoral condyles were also frequently noted (77% of cases). Rabbits that underwent sham surgery, back-skin-operated rabbits, or nonoperated normal rabbits served as controls; the joints of these animals were normal at the time of killing. A rise in the level of serum KS was recorded in 50% of rabbits following partial meniscectomy, but this was matched by similar changes in the control groups. The mean serum KS level of the OA animals at serial intervals (3, 6, 9, or 12 weeks) following surgery was not significantly different from that in the control groups. When measured with a second MAb, 2-D-3, KS levels showed similar trends as with MAb 5-D-4, although lower assay values were obtained. These findings indicate that experimentally induced OA in rabbits is not associated with a significant rise in serum KS levels. KS levels did not differentiate OA from non-OA animals, nor did they parallel disease progression.

Topics: Animals; Cartilage, Articular; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Glycosaminoglycans; Keratan Sulfate; Osteoarthritis; Rabbits; Time Factors

Newly synthesized and endogenous proteoglycan was isolated from human femoral head osteochondrophytic spurs. 35SO4-containing keratan sulphate was measured by its susceptibility to endo-beta-D-galactosidase (keratanase) and comprised 15-17% of the two subpopulations of a proteoglycan monomer fraction (D1) resolved by Sepharose CL-2B chromatography (Kav (I), 0.22; (II), 0.78). The size of the newly synthesized keratan sulphate in these fractions was large (Mr greater than 7,000). The hydroxylamine cleavage product of a proteoglycan aggregate fraction (A1) which eluted in the void volume of a Sepharose CL-2B column was immunoreactive with an anti-keratan sulphate monoclonal antibody, 5-D-4. Unlike the proteoglycan aggregate A1 fraction from bovine nasal cartilage, immunoreactivity against 5-D-4 was also found in chromatographic fractions retarded by Sepharose CL-2B. These results lend additional support to our assertion that the osteophyte extracellular matrix consists of hyaline cartilage-type proteoglycan. Stimulation of osteophyte proliferation may be useful as a repair mechanism for resurfacing denuded areas of osteoarthritic femoral heads.

Topics: Aged; Antibodies, Monoclonal; Cartilage, Articular; Chromatography, Gel; Femur Head; Glycosaminoglycans; Humans; In Vitro Techniques; Keratan Sulfate; Middle Aged; Osteoarthritis; Proteoglycans

Very thin slices of the superficial and deep layers of osteophytic and apparently normal articular cartilage from six human osteoarthritic femoral heads were digested with papain. The digests were analyzed for keratin sulfate content using an enzyme-linked immunosorbent assay (ELISA) with inhibition step, and for chondroitin sulfate and collagen contents using biochemical assays. Although there were marked differences in Safranin-O staining of the superficial and deep layers of osteophytic cartilage, these two layers had identical high ratios of chondroitin sulfate/collagen. Keratan sulfate was present only in small amounts in osteophytic cartilage. However, the deeper layer contained significantly more of this glycosaminoglycan. The deeper layer of articular cartilage contained approximately twice as much chondroitin sulfate and six times more keratan sulfate relative to collagen than the superficial layer. The results of this study showed that this new sensitive approach, which requires as little as 200 micrograms wet cartilage as starting material, provides important qualitative and quantitative information about the major constituents of the matrix.

Topics: Cartilage, Articular; Chondroitin Sulfates; Collagen; Femur Head; Glycosaminoglycans; Humans; Keratan Sulfate; Osteoarthritis; Papain

Serum levels of keratan sulfate (KS), measured by an enzyme-linked immunosorbent-inhibition assay, were found to be significantly higher in 31 patients with hypertrophic osteoarthritis (OA) than those in 41 adults without joint disease. Seventy-seven percent of patients with OA, but only 12% of control subjects, had serum levels which were more than 1 SD above the mean of the control group. Following replacement of a single osteoarthritic hip joint, serum KS levels decreased, at first, in all patients. Subsequently, the concentration of serum KS progressively increased; 6 months following surgery, KS levels were similar or close to the preoperative levels in virtually all patients. The results suggest that patients with hypertrophic OA may have a generalized imbalance of cartilage proteoglycan metabolism. Measurements of serum KS are likely to prove most useful in studying this particular subset of patients with generalized OA.

Topics: Aged; Female; Glycosaminoglycans; Hip Joint; Hip Prosthesis; Humans; Keratan Sulfate; Male; Middle Aged; Osmolar Concentration; Osteoarthritis; Postoperative Period

The intensity of safranin 'O' staining is directly proportional to the proteoglycan content in normal cartilage. Safranin 'O' has thus been used to demonstrate any changes that occur in articular disease. In this study, staining patterns obtained using monoclonal antibodies against the major components of cartilage proteoglycan chondroitin sulphate (anti CS) and keratan sulphate (anti KS), have been compared with those obtained with safranin 'O' staining, in both normal and arthritic tissues. In cartilage where safranin 'O' staining was not detectable, the monoclonal antibodies revealed the presence of both keratan and chondroitin sulphate. Thus, safranin 'O' is not a sensitive indicator of proteoglycan content in diseases where glycosaminoglaycan loss from cartilage has been severe.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Arthritis; Arthritis, Rheumatoid; Cartilage, Articular; Chondroitin Sulfates; Extracellular Matrix; Humans; Immunohistochemistry; Keratan Sulfate; Middle Aged; Osteoarthritis; Phenazines; Proteoglycans

Antibodies were used in radioimmunoassays with gel chromatography to detect the hyaluronic acid-binding region, core protein, and keratan sulfate of human cartilage proteoglycan in the synovial fluids of patients with rheumatoid arthritis, juvenile rheumatoid arthritis, and osteoarthritis. All fluids contained proteoglycan that was mainly included on Sepharose CL-4B; this result indicates cleavage of proteoglycan (which is normally excluded). The hyaluronic acid-binding region was the smallest and most commonly detected fragment. It was relatively free of keratan sulfate and core protein, and it could sometimes bind to hyaluronic acid. Other larger fragments containing core protein and/or keratan sulfate were detected in every fluid.

Topics: Aggrecans; Arthritis; Arthritis, Juvenile; Arthritis, Rheumatoid; Carrier Proteins; Cartilage, Articular; Extracellular Matrix Proteins; Glycoproteins; Humans; Hyaluronan Receptors; Keratan Sulfate; Lectins, C-Type; Osteoarthritis; Proteoglycans; Radioimmunoassay; Synovial Fluid

Concentrations of circulating cartilage-derived keratan sulfate (KS) are significantly higher in patients with osteoarthritis (OA) than in adults without cartilage disease. The increases in serum KS levels in the OA group are likely the result, at least in part, of cartilage degradation in affected joints. If the appearance of elevated levels of serum KS do indeed correlate with the extent of cartilage erosion or destruction in individuals with OA, measurements of serum KS levels will prove extremely useful in the assessment and diagnosis of this joint disease.

Topics: Adult; Aged; Cartilage; Glycosaminoglycans; Humans; Keratan Sulfate; Middle Aged; Osteoarthritis; Proteoglycans

In the cartilage-pannus junction of 14 patients with rheumatoid arthritis (RA) and seven patients with osteoarthritis (OA), monoclonal antibodies to keratan sulphate (KS) and chondroitin sulphate (CS) stained a transitional fibroblastic zone (TFZ) within the pannus in nine RA patients and one OA patient. In three patients this was clearly localised to the cytoplasm of cells in this zone, but in all remaining cases KS and CS could be demonstrated in the surrounding matrix. This area was distinguished from adjacent pannus which contained many blood vessels and cells positive for MHC Class II antigen. Specific markers for glycosaminoglycans have been employed to demonstrate that chondrocyte-derived cells and matrix contribute to the changes seen at the cartilage-pannus junction in RA-affected joints.

Topics: Adult; Aged; alpha 1-Antichymotrypsin; Antibodies, Monoclonal; Arthritis, Rheumatoid; Cartilage, Articular; Chondroitin Sulfates; Female; Histocompatibility Antigens Class II; Humans; Keratan Sulfate; Male; Middle Aged; Osteoarthritis

Articular cartilage contains at least 2 proteoglycans (PGs) which aggregate with hyaluronate: one larger, richer in chondroitin sulphate (CSRPG); the other smaller, relatively richer in keratan sulphate (KSRPG). With maturation the ratio of CSRPG/KSRPG decreases. In order to test the hypothesis that osteoarthritic cartilage contains an increased amount of proteoglycans characteristic of immature cartilage, experimental osteoarthritis (OA) was induced in 11 dogs by transection of the anterior cruciate ligament. Proteoglycan populations were assessed by composite agarose polyacrylamide gel electrophoresis (CAPAGE). The OA cartilage had more proteoglycan and an increased proportion of the slower migrating band on CAPAGE, which corresponds to the CSRPG, supporting the hypothesis that OA cartilage contains an increased amount of proteoglycans characteristic of immature cartilage.

Topics: Animals; Cartilage, Articular; Chondroitin Sulfate Proteoglycans; Disease Models, Animal; Dogs; Keratan Sulfate; Lumican; Male; Osteoarthritis; Proteoglycans

We have developed an enzyme-linked immunosorbent-inhibition assay which makes use of a monoclonal antibody specific for keratan sulfate to quantify keratan sulfate present as single chains in adult human serum. In adults hospitalized with conditions not thought to affect the turnover of keratan sulfate-containing tissues, the serum levels varied from individual to individual (53-1,009 ng/ml) but did not show significant differences with respect to age, sex, or disease category. There were no significant differences between the serum levels of adult hospitalized patients and those of nonhospitalized normal adults. In contrast, the concentration of keratan sulfate in the sera of children aged 5-12 was significantly higher. No keratan sulfate was detected in the sera of 3 adult patients with macular corneal dystrophy, an inherited disorder of the cornea. This may indicate that individuals with macular corneal dystrophy have no keratan sulfate-containing proteoglycans in their cartilage. Adult patients with osteoarthritis have significantly higher concentrations of circulating keratan sulfate. This suggests that the assay could prove valuable in monitoring increased cartilage catabolism in joint diseases.

Topics: Adolescent; Adult; Aged; beta-Galactosidase; Borohydrides; Cartilage; Child; Chondroitinases and Chondroitin Lyases; Chromatography, Gel; Chromatography, Ion Exchange; Corneal Diseases; Enzyme-Linked Immunosorbent Assay; Female; Glycosaminoglycans; Glycoside Hydrolases; Humans; Keratan Sulfate; Macular Degeneration; Male; Middle Aged; Osteoarthritis; Papain

We studied changes in subchondral bone and articular cartilage in an animal model of osteoarthrosis. In this model we applied repetitive impulsive loads to rabbits' knees. Their legs were held in short leg splints so the rabbits were unable to dampen the peak applied load with ankle flexion. After sacrifice, at 1 day to 6 weeks, we studied proximal tibial load-bearing cartilage histologically, biochemically, and with radioactive sulfate uptake. We also studied the subchondral bone under that cartilage histologically, histomorphometrically, with bone scan (99mTc pyrophosphate), and by tetracycline labeling. An increase in 99mTc labeling of the subchondral bone was the first reliable change observed. This was followed by an increase in tetracycline labeling, bone formation, and a decrease in porosity, which has been associated with relative stiffening of bone. Horizontal splitting and deep fibrillation of the overlying articular cartilage followed the early bone changes. All of these changes preceded changes in content and characterization of cartilage proteoglycans or increased chondrocyte activity as manifested by incorporation of radioactive sulfate. In this model the early bone changes preceded changes in the articular cartilage. The deep splitting of articular cartilage occurred prior to metabolic alteration of that tissue.

Topics: Animals; Calcium Pyrophosphate; Cartilage, Articular; Chondroitin Sulfates; Disease Models, Animal; Female; Glycosaminoglycans; Keratan Sulfate; Knee Joint; Osteoarthritis; Proteoglycans; Rabbits; Synovial Membrane

The glycosaminoglycans in the menisci of beagles 5--7 years old were analyzed at various times after osteoarthritis was induced by sectioning the anterior cruciate ligament of one knee; the unoperated knee served as control. In the first month after induction, there were signs of inflammation in the operated joint. After 1 week, the water content was elevated and the glycosaminoglycan content (per dry weight) was reduced. The content of keratan sulfate decreased more than that of chondroitin sulfate, but the hyaluronic acid content did not change consistently. The relative proportions of chondroitin-4-sulfate, chondroitin-6-sulfate, and dermatan sulfate remained unchanged. After 3--18 months, the glycosaminoglycan levels reverted to normal, and there was some evidence that after 15--18 months, they were elevated above normal. These results, together with results obtained from single examples of mild and severe osteoarthritis in working foxhounds, suggest that, in contrast to articular cartilage, the meniscus is capable of some regeneration in response to injury.

Topics: Animals; Body Water; Chondroitin Sulfates; Dermatan Sulfate; Dogs; Female; Glycosaminoglycans; Hyaluronic Acid; Keratan Sulfate; Knee; Menisci, Tibial; Osteoarthritis; Uronic Acids

The compositional profiles of meniscal glycosaminoglycans (GAG) at different ages and in the presence of osteoarthritis were determined. The major components of meniscal GAG were chondroitin sulphates and dermatan sulphate. Other major elements were keratan sulphate and hyaluronic acid. Chondroitin sulphate-4 and -6 decreased with age. Dermatan sulphate content remained unchanged with age whereas keratan sulphate and hyaluronic acid increased. GAG compositional profiles of meniscus from osteoarthritis were similar to those of comparable age group. The results indicate that GAG compositional make-up of the meniscus changes with age. These changes may make it more susceptible to horizontal tears in the elderly.

Topics: Adolescent; Adult; Age Factors; Aging; Child; Chondroitin; Glycosaminoglycans; Humans; Hyaluronic Acid; Keratan Sulfate; Menisci, Tibial; Middle Aged; Osteoarthritis

Proteoglycans were extracted from normal and degenerate cartilage of the human tibial plateau. Both areas possessed proteoglycans of similar chemical composition, though the degenerate cartilage contained a greater proportion of molecules of lower buoyant density and enriched in keratan sulfate. There was no evidence for the changes in glycosaminoglycan synthesis that have been described for clinically osteoarthritic cartilage, or for changes in the ability to aggregate with hyaluronic acid.

Topics: Aged; Amino Acids; Cartilage Diseases; Cartilage, Articular; Chromatography; Electrophoresis, Polyacrylamide Gel; Humans; Keratan Sulfate; Knee Joint; Microscopy, Electron, Scanning; Middle Aged; Osteoarthritis; Proteoglycans; Tibia

Topics: Adult; Aged; Bone Matrix; Cartilage, Articular; Chondroitin; Chondroitin Sulfates; Femur Head; Glycosaminoglycans; Humans; Hydrogen-Ion Concentration; Keratan Sulfate; Middle Aged; Osteoarthritis; Staining and Labeling

Proteoglycan subunits (PGS) were isolated from bovine articular cartilage of calves and from cows, 18 months and 8 years old respectively. From the latter cartilage of osteoarthrotic and of non-osteoarthrotic sites was taken. PGS were characterized by gel-chromatography on Sepharose 2B columns and subjected to digestion with chondroitinase ABC and with papain. The isolated keratan sulphate-protein cores obtained from chondroitinase digestion were characterized on Sepharose 4B and the chondroitin sulphate chains on Sephadex G-200 gels. A larger molecular size of PGS was found in calf cartilage than in the other samples. This was attributed to the larger molecular size of chondroitin, whereas no change was observed in the keratan sulphate-protein cores. No change was observed in molecular size of PGS, isolated chondroitin sulphates or keratan sulphate-protein cores in osteoarthrosis in comparison with non-osteoarthrotic cartilage from the same joint or from younger adult animals.

Topics: Age Factors; Animals; Cartilage, Articular; Cattle; Centrifugation, Density Gradient; Chondroitin Sulfates; Chromatography, Gel; Keratan Sulfate; Molecular Weight; Osteoarthritis; Proteoglycans

An experimental model of osteoarthritis resulting from laxity of the joint was induced in eighteen mature dogs (at least two years old) by sectioning the anterior cruciate ligament of the right knee (stifle) with a stab incision, the left knee providing a control. A sham operation was also performed in three other dogs, in which a stab incision was made but the ligament left intact. The dogs were killed at various intervals from one to forty-eight weeks later. Morphological changes in bone, cartilage, synovial membrane and joint capsule were examined in all the joints and biochemical changes in the cartilage of three dogs killed after two, eight, and sixteen weeks. All the changes resulting from the operation progressed with time and became indistinguishable from those found in three dogs with natural osteoarthritis of the knee. There were no changes in the joints which had sham operations. As the time of onset is known, this experimental model in a larger species enables a study to be made of the biochemical as well as the morphological changes in the early stages of osteoarthritis.

Topics: Animals; Bone Development; Cartilage, Articular; Chondroitin Sulfates; Disease Models, Animal; Dogs; Female; Femur; Galactosamine; Glucosamine; Keratan Sulfate; Knee Joint; Ligaments, Articular; Male; Menisci, Tibial; Osteoarthritis; Proteoglycans; Stress, Physiological; Synovial Membrane; Tibia; Uronic Acids

Radiochemical and biochemical methods were used to characterize post-mortem and osteoarthrotic femoral head cartilage. Fixed charge density measurements were correlated with glycosaminoglycan content as estimated by uronic acid and hexosamine analyses. In post-mortem cartilage water content decreased from a maximum at the surface to a minimum in the deep zones. In the osteoarthrotic specimens water content was greatest in the middle zones. Glycosaminoglycan content increased with depth and in the osteoarthrotic specimens was reduced throughout the depth of the cartilage. With increasing degeneration there was an increase in water content and decrease in glycosaminoglycan content. The difference in the water content profile in osteoarthrotic cartilage was explained in terms of damage to the collagen network. In osteoarthrosis the latter is no longer capable of restraining the swelling pressure produced by the glycosaminoglycans and swelling is greatest in the midzones, where glycosaminoglycan content is highest.

Topics: Body Water; Cartilage, Articular; Chondroitin Sulfates; Collagen; Femur Head; Glycosaminoglycans; Humans; Keratan Sulfate; Middle Aged; Osteoarthritis

Glycosaminoglycan turn-over has been studied both in vivo and in vitro, by using sodium [35S]sulphate as a precursor. The in vivo experiments were performed on rabbits and dogs, taking special care to monitor the 35S radioactivity in the serum throughout the experiment and to measure the radioactivity due to unincorporated inorganic [35S]sulphate in cartilage at the end of each experiment, in addition to that due to incorporated sulphate. The inorganic sulphate content of the serum was also determined as well as the distribution coefficient for the inorganic sulphate ion between cartilage and serum. From this information it was possible to calculate accurately the rate of sulphate uptake by cartilage in vivo and hence the turn-over rate. Experiments were then performed in vitro on cartilage from rabbits and dogs and the in vivo and in vitro results were compared. A very good agreement was obtained between the two sets of results. Studies were then carried out under exactly the same in vitro conditions on human articular cartilage and it was thus possible to obtain a turn-over rate for the latter which one could trust was close to the actual in vivo value. The mean half-lives thus obtained varied from 45 days for the young rabbit to 150 days for the adult dog and 800 days for the human femoral head. In human cartilage there were considerable variations in turn-over rate within a single joint as a function of depth below the surface, and between different joints. Thus, while the mean half-life for the human femoral head is 800 days, that for the femoral condyle is 300 days. Cartilage from osteoarthrosic femoral heads did not appear to differ much with respect to sulphate uptake from the normal specimens although the turn-over rates were somewhat higher.

Topics: Adult; Animals; Cartilage, Articular; Chondroitin; Dogs; Femur Head; Glycosaminoglycans; Hip; Humans; Keratan Sulfate; Kinetics; Knee; Models, Biological; Osteoarthritis; Rabbits; Sulfates; Synovial Fluid

Tissue contents of small, easily extracted, proteoglycans, relatively poor in keratan sulphate, were compared in normal and osteoarthrotic cartilage. Although the amounts of small proteoglycans were similar in each tissue, as were the collagen contents, some proteoglycans in the diseased cartilage were much more readily extracted than those in the normal tissue.

Topics: Animals; Cartilage, Articular; Cattle; Cattle Diseases; Chromatography; Collagen; Femur; Glycosaminoglycans; Immunodiffusion; Keratan Sulfate; Osteoarthritis; Proteoglycans