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While it is known that the degenerated intervertebral disc (IVD) is one of the primary reasons for low-back pain and subsequent need for medical care, there are currently no established effective methods for direct treatment. Nuclear factor-κB (NF-κB) is a transcription factor that regulates various genes’ expression, among which are inflammatory cytokines, in many tissues including the IVD. NF-κB decoy is an oligodeoxynucleotide containing the NF-κB binding site that entraps NF-κB subunits, resulting in suppression of NF-κB activity. In the present preclinical study, NF-κB decoy was injected into degenerated IVDs using the rabbit anular-puncture model. In terms of distribution, NF-κB decoy persisted in the IVDs up to at least 4 weeks after injection. The remaining amount of NF-κB decoy indicated that it fit a double-exponential-decay equation. Investigation of puncture-caused degeneration of IVDs showed that NF-κB decoy injection recovered, dose-dependently, the reduced disc height that was associated with reparative cell cloning and morphological changes, as assessed through histology. Gene expression, by quantitative real-time polymerase chain reaction (qRT-PCR), showed that NF-κB decoy attenuated inflammatory gene expression, such as that of interleukin-1 and tumor necrosis factor-α, in rabbit degenerated IVDs. NF-κB decoy also reduced the pain response as seen using the “pain sensor” nude rat xenograft-radiculopathy model. This is the first report demonstrating that NF-κB decoy suppresses the inflammatory response in degenerated IVDs and restores IVD disc height loss. Therefore, the intradiscal injection of NF-κB decoy may have the potential as an effective therapeutic strategy for discogenic pain associated with degenerated IVDs.

PURPOSE: To elucidate the effects of growth differentiation factor-6 (GDF6) on: (i) gene expression of inflammatory/pain-related molecules and structural integrity in the rabbit intervertebral disc (IVD) degeneration model, and (ii) sensory dysfunction and changes in pain-marker expression in dorsal nerve ganglia (DRGs) in the rat xenograft radiculopathy model. METHODS: Forty-six adolescent rabbits received anular-puncture in two non-consecutive lumbar IVDs. Four weeks later, phosphate-buffered saline (PBS) or GDF6 (1, 10 or 100 μg) was injected into the nucleus pulposus (NP) of punctured discs and followed for four weeks for gene expression analysis and 12 weeks for structural analyses. For pain assessment, eight rabbits were sacrificed at four weeks post-injection and NP tissues of injected discs were transplanted onto L5 DRGs of 16 nude rats to examine mechanical allodynia. The rat DRGs were analyzed immunohistochemically. RESULTS: In GDF6-treated rabbit NPs, gene expressions of interleukin-6, tumor necrosis factor-α, vascular endothelial growth factor, prostaglandin-endoperoxide synthase 2, and nerve growth factor were significantly lower than those in the PBS group. GDF6 injections resulted in partial restoration of disc height and improvement of MRI disc degeneration grades with statistical significance in rabbit structural analyses. Allodynia induced by xenograft transplantation of rabbit degenerated NPs onto rat DRGs was significantly reduced by GDF6 injection. Staining intensities for ionized calcium binding adaptor molecule-1 and calcitonin gene-related peptide in rat DRGs of the GDF6 group were significantly lower than those of the PBS group. CONCLUSION: GDF6 injection may change the pathological status of degenerative discs and attenuate degenerated IVD-induced pain.

Study design The axonal growth potential of dorsal root ganglion (DRG) neurons in an organ culture system was investigated. Objective To examine the effects of neuronal injury and tumor necrosis factor-alpha (TNF-alpha) on the axonal growth potential of 2 types of nociceptive DRG neurons: nerve growth factor (NGF)-sensitive and glial cell line-derived neurotrophic factor (GDNF)-sensitive neurons. Summary of background data Nerve ingrowth into the disc is recognized to be one of the causes of discogenic pain. Almost all of these disc-innervating neurons are NGF-sensitive. The axonal growth potential of NGF-sensitive neurons has not been investigated. Methods Adult Sprague-Dawley rats were used for immunohistochemistry (n = 7) and cell viability studies (n = 6). Bilateral L3-L5 DRGs, which were successfully removed without damage, were noncultured or cultured in serum-free medium containing TNF-alpha at 0, 0.01, 0.1, and 1 ng/mL for 48 hours (n = 5, each treatment). The DRGs were then immunostained for activating transcription factor 3 (ATF3, a marker for injured neurons) or double-stained for growth-associated protein 43 (GAP-43, a marker for axonal growth) with calcitonin gene-related peptide (CGRP, a marker for NGF-sensitive neurons) or isolectin B4 (IB4, a marker for GDNF-sensitive neurons). Cell viability was assessed by a lactate dehydrogenase (LDH) assay and an MTS assay (n = 6, each treatment). Results Immunoreactive evidence of injured neurons (ATF3 positive) was frequently observed in cultured DRGs, but never in noncultured DRGs. The percentage of neurons exhibiting axonal growth potential (GAP-43 immunoreactive) was significantly higher for NGF-sensitive neurons than for GDNF-sensitive neurons at any concentration of TNF-alpha. More than 95% of the cultured neurons were viable. Conclusions The results suggest that the cultured DRG neurons exhibit pathologic changes similar to those found in injured neurons. NGF-sensitive neurons, which include disc-innervating neurons, may have a greater potential to extend their axons in response to neuronal injury under pathologic conditions in the presence of TNF-alpha than GDNF-sensitive neurons.

SummaryObjectivePlatelet-rich plasma (PRP) is a fraction of plasma that contains high levels of multiple growth factors. The purpose of this study was to examine the effects of PRP on cell proliferation and matrix synthesis by porcine chondrocytes cultured in alginate beads, conditions that promote the retention of the chondrocytic phenotype, in order to determine the plausibility of using this plasma-derived material for engineering cartilage.DesignPRP and platelet-poor plasma (PPP) were prepared from adult porcine blood. Adult porcine chondrocytes were cultured in the presence of 10% PRP, 10% PPP or 10% fetal bovine serum (FBS) for 3 days. Cell proliferation, proteoglycan (PG) and collagen synthesis were quantified, and the structure of newly synthesized PG and collagen was characterized.ResultsTreatment with 10% PRP resulted in a small but significant increase in DNA content (+11%, vs FBS; P

Study design In vitro assessment of the effects of platelet-rich plasma on the extracellular matrix metabolism of porcine intervertebral disc cells. Objectives To determine whether platelet-rich plasma is effective in stimulating cell proliferation and extracellular matrix metabolism by porcine disc cells cultured in alginate beads. Summary of background data Platelet-rich plasma is used to accelerate wound healing and tissue regeneration. Activated platelets release multiple growth factors that regulate cell proliferation, differentiation, and morphogenesis. Individual growth factors present in platelet-rich plasma have been demonstrated to affect the metabolism of intervertebral disc cells. Methods Platelet-poor and platelet-rich plasma was isolated from fresh porcine blood using a commercially available platelet concentration system. After preculture for 7 days and serum starvation for 24 hours, the beads containing nucleus pulposus and anulus fibrosus cells were then cultured for another 72 hours in serum-free medium, 10% fetal bovine serum, 10% platelet-poor plasma, or 10% platelet-rich plasma. The synthesis of proteoglycans and collagen, the accumulation of proteoglycans, and the DNA content were biochemically assessed. Results Platelet-rich plasma had a mild stimulatory effect on cell proliferation of intervertebral disc cells. Platelet-rich plasma treatment significantly upregulated proteoglycan and collagen synthesis and proteoglycan accumulation when compared with platelet-poor plasma. Conclusions Platelet-rich plasma was effective in stimulating cell proliferation and extracellular matrix metabolism. The response to platelet-rich plasma was greater in the case of anulus fibrosus cells than of nucleuspulposus cells. The local administration of platelet-rich plasma might stimulate intervertebral disc repair. In addition, given the risks of using animal serum for tissue engineering, autologous blood may gain favor as a source of growth factors and serum supplements needed for stimulating cells to engineer intervertebral disc tissues.

Study design: This is a prospective study with a follow-up period of 4 years. Objectives: The study aimed to evaluate the possible clinical utility of three biomarkers [i.e., keratan sulfate (KS), hyaluronan, and cartilage oligomeric matrix protein] measured in peripheral blood in severe acute sciatica at intake and follow-up. Summary of background: Our previous study and others have pointed out the interest of different laboratory tests in the acute phase of sciatica. Several blood biomarkers have been reported useful in the long-term follow-up of patients with osteoarthritis. We have found no information about the potential interest of these tests in spinal disorders. Methods: Patients were admitted to the hospital for intensive conservative management of acute sciatica (n=82). A subgroup of patients (n=33) was selected based on the duration of symptoms at visit 1, and included those with the shortest (n=24) as well as those with the longest (n=9) duration of sciatica. Blood samples were drawn, centrifuged, and the plasma frozen. Antigenic KS, hyaluronan, and cartilage oligomeric matrix protein were measured by ELISA. Patients were re-evaluated at an average of 4.3 years (range: 2.1–6.8 years). Results: Thirty-three subjects with an average age of 49.2±10.2 years participated. At intake, levels of the three biomarkers evaluated were within the range of normal values. No significant differences were found between the results of patients with a short history of sciatica (≤3 weeks) and those with a long duration of symptoms (>20 weeks). At follow-up, a significant increase (P

OBJECTIVE: To evaluate whether and how moderate physical activity following a night of rest influences serum levels of matrix metalloproteinase 3 (MMP-3), tissue inhibitor of metalloproteinases 1 (TIMP-1), antigenic keratan sulfate (Ag KS), and hyaluronan (HA) in 10 normal subjects and 38 patients with rheumatoid arthritis (RA). METHODS: Blood was obtained from 20 RA patients before they arose from a night's sleep, and again 1 and 4 hours after they had begun to perform moderate physical activity. Another 18 RA patients remained in bed and blood was sampled at the same time periods. Serum levels of MMP-3, TIMP-1, Ag KS, and HA were measured by enzyme-linked immunosorbent assay. Clinical activity was evaluated by the Lansbury index. RESULTS: Both in normal subjects and in RA patients who did not remain in bed throughout the period of blood sampling, levels of HA, Ag KS, and MMP-3 increased significantly during the first hour after the subjects arose: the increase in HA and Ag KS correlated with the Lansbury index in the RA group. Three hours later, levels of Ag KS had dropped to baseline values in both groups of subjects. Levels of HA remained significantly and moderately elevated in the RA group but not in the control group, while levels of MMP-3 did not drop significantly in either group. In contrast, levels of HA, Ag KS, and MMP-3 did not change significantly in RA patients who had remained in bed. Unlike the other markers, the levels of TIMP-1 remained unchanged at the different time periods in all 3 groups studied. CONCLUSION: Significant changes in serum levels of some metabolic markers occur during the first hour after one arises from a night of sleep, especially in patients with RA. Measurement of the magnitude of these changes at different times in individual patients provides very different information about metabolic changes occurring in joint tissue than does measurement of the level of the markers at a single time point, as is usually currently reported.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Objective The adduction moment at the knee during gait is the primary determinant of medial-tolateral load distribution. If the adduction moment contributes to progression of osteoarthritis (OA), then patients with advanced medial tibiofemoral OA should have higher adduction moments. The present study was undertaken to investigate the hypothesis that the adduction moment normalized for weight and height is associated with medial tibiofemoral OA disease severity after controlling for age, sex, and pain level, and to examine the correlation of serum hyaluronan (HA) level with disease severity and with the adduction moment in a subset of patients. Methods Fifty-four patients with medial tibiofemoral OA underwent gait analysis and radiographic evaluation. Disease severity was assessed using the Kellgren-Lawrence (K-L) grade and medial joint space width. In a subset of 23 patients with available sera, HA was quantified by sandwich enzyme-linked immunosorbent assay. Pearson correlations, a random effects model, and multivariate regression models were used. Results The adduction moment correlated with the K-L grade in the left and right knees (r = 0.68 and r = 0.60, respectively), and with joint space width in the left and right knees (r = -0.45 and r = -0.47, respectively). The relationship persisted after controlling for age, sex, and severity of pain. The partial correlation between K-L grade and adduction moment was 0.71 in the left knees and 0.61 in the right knees. For every 1.0-unit increase in adduction moment, there was a 0.63-mm decrease in joint space width. In the subset of patients in whom serum HA levels were measured, HA levels correlated with medial joint space width (r = -0.55), but not with the adduction moment. Conclusion There is a significant relationship between the adduction moment and OA disease severity. Serum HA levels correlate with joint space width but not with the adduction moment. Longitudinal studies will be necessary to determine the contribution of the adduction moment, and its contribution in conjunction with metabolic markers, to progression of medial tibiofemoral OA.

Keratan sulfate (KS) is a highly-negatively charged glycosaminoglycan which is found principally in aggrecan, the most abundant proteoglycan in the extracellular matrix of hyaline, fibrous and elastic cartilage. In 1985, we discovered that small peptidoglycans bearing antigenic KS (agKS) were present in small amounts in human serum and postulated that the great majority of these were derived from the degradation of cartilage aggrecan (Thonar et al. 1985). During the past decade, studies of antigenic KS in joint fluid and serum have provided strong evidence in support of this hypothesis. By asking questions which could not have been addressed previously, these studies have shed new light upon the metabolism of aggrecan in vivo. In this manuscript, we describe some of these findings and put in perspective their contribution to our understanding of the metabolism of aggrecan in joint diseases.

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. However, the stability of serum levels with time and the relationship between cartilage mass and SF levels may be useful for monitoring the individual patient's response to therapy, both systemically (serum KS) and in single joints (SF KS).

Plasma levels of hyaluronate (HA) in patients with rheumatoid arthritis (RA) and patients with osteoarthritis (OA), measured by enzyme-linked immunosorbent assay, were compared with levels in a healthy, age-matched non-arthritic control group, in a retrospective study. Compared with the controls, the mean level of plasma HA was sevenfold higher in the RA group and twofold higher in the OA group. There was no statistically significant correlation between HA levels and 7 other clinical and biochemical parameters in patients with RA. In the OA group, however, plasma HA levels were found to correlate with an objective functional capacity score and with an articular index based on the total amount of cartilage in involved joints. In a retrospective longitudinal study of 6 patients with RA, plasma levels of HA did not show a significant correlation with plasma levels of elastase or with the erythrocyte sedimentation rate. These data support in part the contention that plasma HA may be unique as a marker, in that it may be a reflection of synovial involvement and inflammation, rather than only of inflammation, in arthritis.

Keratan sulfate is a glycosaminoglycan; in man, most KS is present in cartilage proteoglycans, 1 but small amounts have been identified in blood.l, 2 Recent evidence indicates that this KS is almost exclusively derived from catabolism of cartilage proteoglycans.l-3 As a result, it has been postulated that the concentration of KS in the blood is directly proportional to the rate of degradation of cartilage proteoglycans.I, 2 Linear growth results from bone formation on a cartilage scaffold (endochondral ossification) and depends on adequate function of the hypothalamic-pituitary-somatomedin-insulin-like growth factor I axis and the genetic ability of cartilage to respond to these secretions. 4, 5 In the growing child, serum levels of KS are elevated, which is not unexpected because growing cartilages are constantly undergoing degradation and replacement by new bone formation. We previously reported that serum levels of keratan sulfate rise from low levels in infancy and reach a plateau by the age of 4 to 5 years. Serum concentrations of KS appear to remain high until age 12 years, when they rapidly decline toward the levels found in normal adult sera. Serum levels of KS in a growing child correlate with the individual child's age-adjusted percentile height. 6 In our investigation, we measured the serum concentrations of KS in two groups of children with short stature: one group with Constitutional delay of maturity and the other with growth hormone deficiency. In the latter group, we compared KS levels before and after treatment with growth

Objective—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. Animals—12 clinically normal adult mixed-breed dogs. Procedure—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. Results—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. Conclusions and Clinical Relevance—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.

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.

To compare concentrations of joint biomarkers in synovial fluid (SF) between idiopathic osteonecrosis of the femoral head (ION) and osteoarthritis (OA) of the hip joint.Levels of the joint biomarkers cartilage oligomeric matrix protein (COMP), antigenic keratan sulfate (AgKS), and hyaluronan (HA) in SF samples from 21 cases of ION and their relationship to disease stage and history of steroid use were assessed and compared to the result of 29 cases of hip OA.In both the ION and hip OA groups, levels of COMP and AgKS in SF showed a significant positive correlation. The ION group had significantly higher levels of AgKS in SF than the hip OA group. In the ION group, stage II patients had significantly higher SF levels of both COMP and AgKS than those in stage III patients. No difference in level of HA in hip joint SF was found between steroid and non-steroid treated ION patients or between the stage II and III subgroups.SF levels of COMP and AgKS may serve as useful joint biomarkers that reflect cartilage metabolism not only in hip OA but also in ION.

OBJECTIVE: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

The alginate bead culture system has unique properties that make it possible to study the accumulation and turnover of macromolecules in two distinct matrix compartments of the cartilage matrix: the cell-associated matrix (CM) and the further removed matrix (FRM). Taking advantage of this culture system, the purpose of this study was to examine age-related changes in the metabolism of hyaluronan (HA) in these two compartments. Bovine chondrocytes, isolated from fetal, young adult, and old adult articular cartilage, were cultured in alginate beads. On Days 7 and 14 of culture, the alginate gel was solubilized, the CM and FRM were separated and macromolecules in both compartments were analyzed. When compared to the cells from fetal and old adult animals, the young adult cells proliferated at the fastest rate. Fetal cells produced a more abundant CM that was richer in proteoglycans (PGs) than the CM of young or old adult cells. With increasing age, there was an increased tendency for PG, collagen, and HA to escape incorporation into the CM and to become immobilized in the FRM. Very striking changes also were observed in the ratio of HA to PG, which increased markedly with age, and in the size of the HA molecules, which decreased markedly with age. The results suggest that the metabolism of HA in cartilage undergoes pronounced age-related changes, some of which are retained during culture in alginate gel. The findings also suggest that the previously documented age-related decrease in the size of HA in native bovine cartilage reflects, at least in part, a biochemical process occurring at the time or at least soon after the glycosaminoglycan chain is synthesized. It does not appear to simply be the result of age-related changes occurring slowly with time after synthesis, as was previously suggested to be the case for human articular cartilage.

Osteoarthritis (OA) may be described as a syndrome in which biochemical and biomechanical abnormalities combine to cause pain and functional deterioration associated with structural degradation of the joint. The end of the 20th century saw the emergence of assays capable of measuring molecules whose concentrations in body fluids (joint fluid, blood and urine) provide information about specific metabolic processes occurringin vivoin the joint tissues of patients with OA and other joint diseases [1].The ability to quantify with precision these molecules, referred to below as metabolic markers, is helping scientists and clinicians address questions they had not been able to ask before. While the field of research on body fluid markers of joint metabolism is still in its infancy, there already is clear evidence that the measurement of some of these markers is going to shed more light on the pathogenesis of both the pre-clinical and clinical stages of joint disease, including OA; that will prove helpful in developing effective therapeutic treatments. For example, it is now relatively simple to rapidly determine if a drug, with documented effects upon the metabolism of aggrecan in cartilage or chondrocytes cultured in vitro, has the same effect in vivo [2].The ability to measure, in a small volume of blood, metabolic markers that will help monitor or prognosticate OA-related changes remains a longterm goal of investigators in the field. There is good agreement that, over the shorter term, this may be achieved by measuring a panel of a few markers. Indeed, recent observations that the quantification of the level of hyaluronan (HA) [3],cartilage oligomeric matrix protein (COMP) [4] and C-reactive protein [5]in serum may have prognostic value in OA suggest that sensitive assays capable of measuring markers of specific metabolic processes may soon be used in the clinical assessment of patients with this condition.

Cartilage oligomeric matrix protein (COMP) is a high-molecular-weight glycoprotein found at a high concentration in articular cartilage. Recent studies have shown that the joint fluid and serum levels of antigenic COMP, measured by an enzyme-linked immunosorbent assay (ELISA) which uses a polyclonal antiserum raised against bovine COMP, provide important information about metabolic changes occurring in the cartilage matrix in joint disease. In this report, we describe the specificity of three monoclonal antibodies (mAbs) to human COMP and their usefulness in quantifying antigenic COMP fragments in body fluids. Two of the mAbs (16-F12 and 18-G3) recognized both oligomeric and monomeric forms of COMP, but the third (17-C10) reacted positively only with the former. Immunoblots of human COMP, predigested with trypsin for up to 6 h, showed that the three mAbs are directed against different epitopes identified on small tryptic fragments of 30 kDa (16-F12), 25 kDa (17-C10), and 40 kDa as well as 30 kDa (18-G3), respectively. The antibodies also recognized a different pattern of fragments in human pathological synovial fluids. This was particularly striking in the case of the medium size fragments (16-F12: 90 and 110 kDa; 17-C10: 70 and 90 kDa; 18-G3: up to five bands from 70 to 130 kDa). Competitive indirect inhibition ELISAs developed with mAbs 16-F12 and 17-C10 revealed further differences in the specificities of these antibodies. Thus, while mAb 16-F12 can be used only to quantify antigenic COMP in human synovial fluid and serum, mAb 17-C10 is useful in addition when analyzing canine and horse synovial fluid as well as canine serum. The results of analyses of synovial fluid samples from patients with osteoarthritis and rheumatoid arthritis provided preliminary evidence in support of the contention that measurement of the different COMP epitopes recognized by these mAbs in body fluids could prove useful in the clinical assessment of patients with joint disease.

In der jungsten Vergangenheit hat die Zahl der Untersuchungen uber die Beteiligung des Immunsystems an destruktiven Prozessen in rheumatischen Erkrankungen rapide zugenommen. Zum einen wird zunehmend deutlich, wie spezifische T-Zellpopulationen aktiv im Gelenk zur Zerstorung der Knorpelzellen beitragen [3]. Weiterhin deuten Untersuchungen der Knorpelzellen selbst darauf hin, das diese Zellen durch Immunmodulatoren wie Interleukine und Interferon γ in ihrem Metabolismus gestort werden und auf starke katabole Stoffwechselwege umschalten, ihre eigene Matrix abbauen und moglicherweise uber neu exprimiertes HLA-DR (Ia) selbst dem Immunsystem als Neoantigene prasentieren [6]. Somit wird deutlich, das eine eventuelle humorale, uber (Auto-)Antikorper und Komplement laufende Immunreaktion komplementar zu erwarten ist.

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.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

PURPOSE: Synchrotron x-ray diffraction experiments indicate that corneas with macular corneal dystrophy (MCD) contain unusual 4.6-A periodic repeats thought to reside in proteoglycans or glycosaminoglycans. Recently the 4.6-A x-ray reflection was found to be significantly diminished after incubation of MCD specimens in buffer containing chondroitinase ABC or N-glycanase. We examined the sulfated proteoglycans in these glycosidase-digested MCD corneas. METHODS: Transmission electron microscopy was used in conjunction with cuprolinic blue-staining for sulfated proteoglycans. RESULTS: Incubation of an MCD specimen in enzyme buffer left both small and large proteoglycan filaments in the stromal matrix, whereas incubation in the presence of chondroitinase ABC removed these molecules from the tissue. Incubation in buffer containing N-glycanase, on the other hand, removed the large proteoglycan filaments from the MCD stroma but left unaffected the small collagen-associated proteoglycans. CONCLUSION: These results are consistent with the interpretation that 4.6-A periodic repeats in MCD corneas reside in large sulfated proteoglycan filaments (or aggregates thereof) that may contain chondroitin/dermatan sulfate and keratan sulfate or keratan components.

We have developed an enzyme-linked immunosorbent assay (ELISA) with an inhibition step to quantify hen egg white lysozyme (HEWL) on a weight basis. The assay is relatively insensitive to changes in ionic strength or pH. Quantification is not affected by the presence or large amounts of mammalian lysozymes or inhibitors of enzymatic activity such as soluble chitin oligomers. We used this ELISA to show that the HEWL content of chick cartilages increases progressively between days 14 and 18 of embryonic life. The maturation-related increase does not appear to be the result of increased synthesis by the chondrocytes for the latter did not synthesize detectable amounts of lysozyme in vitro. Evidence was obtained to suggest that most, if not all, the lysozyme in cartilage is derived from the surrounding fluids which come in contact with the matrix.

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.

An ELISA assay using a monoclonal antibody (ET-4-A-4) that recognizes a sulfated carbohydrate epitope in both keratan sulfate type I (corneal) and type II (skeletal) was employed to quantify keratan sulfate in serum and corneal tissue from patients with macular corneal dystrophy (MCD). This assay disclosed significant quantities of keratan sulfate in the serum in 45 healthy individuals (251 +/- 78 ng/ml), and in 66 patients with various corneal diseases (273 +/- 101 ng/ml). In contrast keratan sulfate was not detected (less than 2 ng/ml) in the serum of 16 patients with histopathologically confirmed MCD. Keratan sulfate was also detected in extracts of normal corneas and corneal tissue with a variety of pathologic conditions, but was virtually absent in corneal tissue from five patients with MCD. In corneas with MCD the chondroitin sulfate/keratan sulfate ratio was considerably higher than that of all normal and pathologic corneas studied. Since keratan sulfate in the serum appears to be derived predominantly from the normal turnover of cartilage these studies strongly suggest that the defect in keratan sulfate synthesis in MCD is not restricted to corneal cells and that MCD is one manifestation of a systemic disorder of keratan sulfate. The cartilage changes, however, do not have clinical significance. Moreover, since keratan sulfate can be detected in the blood of newborns it should be possible to diagnose MCD prior to corneal opacification.

Sensitive measurements of serum keratan sulfate (KS), a glycosaminoglycan found in large quantities in the proteoglycans of human cartilage, can be obtained using an enzyme-linked immunosorbent-inhibition assay. Patients who are undergoing chemonucleolysis (CN) provide a clinical opportunity to monitor the large-scale proteolytic degradation of cartilage. By measuring serum KS levels both pre- and post-CN, we have demonstrated that serum KS levels rise predictably after CN, in a manner that reflects major catabolic events of cartilage.

Summary Objective To determine the effects of exercise and weight loss interventions on serum levels of four biomarkers and to examine if changes in biomarker levels correlate with clinical outcome measures in obese and overweight adults with knee osteoarthritis (OA). Methods Serum was obtained at baseline, 6 and 18 months from 193 participants in Arthritis, Diet and Activity Promotion Trial. This was a single-blind 18-month trial with subjects randomized to four groups: healthy-lifestyle (HL), diet (D), exercise (E) and diet plus exercise (D+E). Serum levels of cartilage oligomeric matrix protein (COMP), hyaluronan (HA), antigenic keratan sulfate (AgKS), and transforming growth factor-β1 (TGF-β1) were measured by enzyme linked immunosorbent assay. Results At baseline there were no significant differences in biomarker levels between intervention groups. When results for all the intervention groups were combined, the levels of HA were found to be negatively correlated with medial joint space width and positively correlated with Kellgren–Lawrence scores (K–L scores) while TGF-β1 levels negatively correlated with K–L scores. When biomarker levels measured at 6 and 18 months were adjusted for baseline values, age, gender, and body mass index, weak but significant differences between intervention groups were present for mean levels of COMP and TGF-β1. Furthermore, AgKS levels averaged over all groups tended to decrease over time. There were no significant associations of baseline biomarkers and the follow-up outcomes. Weak associations were noted between change in the biomarkers at 18 months and change in outcome measures that included change in weight with AgKS and COMP and change in Western Ontario and McMaster Universities Osteoarthritis Index pain with AgKS. Conclusion Overall, the E and D interventions did not show a consistent effect on levels of potential OA biomarkers. The four biomarkers showed differences in correlations with outcome measures suggesting that they may measure different aspects of disease activity in OA. The strongest correlations were between serum HA and radiographic measures of OA at baseline.

A sensitive and highly specific enzyme-linked immunosorbent assay with an inhibition step was used to monitor the concentration of keratan sulfate, a cartilage-related glycosaminoglycan, in the serum of three adult male deer. During the course of one complete annual antler regeneration cycle, keratan sulfate levels were found to fluctuate predictably in relation to the growth and maturation of the antlers: levels are substantially elevated during the growth phase and drop precipitously when growth ceases and the antlers become fully mineralized. In addition, an unanticipated elevation of serum keratan sulfate was observed in early spring prior to casting of the preceding year's antlers and the initiation of regrowth. This suggests that changes in cartilage metabolism occur concomitantly, with this phase of the antlerogenic cycle. These results show predictable and physiologically regulated variation in serum keratan sulfate levels which correlate directly with specific phases of the antler regeneration cycle. Furthermore, the findings provide additional support for the assertion that measurements of keratan sulfate levels in serum can provide useful information about changes in cartilage metabolism in normal as well as diseased states.

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.

Serum was obtained from 155 children at the time of admission to hospital for elective surgery. The concentration of serum keratan sulphate was determined by an ELISA which uses an antibody specific for keratan sulphate, a molecule found predominantly in cartilage. Concentrations of keratan sulphate rise progressively during the first four years of life (0-2: mean = 357 micrograms/l; 2-4: mean = 422 micrograms/l) and then remain high until 12 years of age (mean = approx. 500 micrograms/l). At this time, concentrations drop markedly (13-year olds: mean = 377 micrograms/l; 14-year olds: mean = 318 micrograms/l). After age 15, concentrations continue to fall toward the concentrations found in normal adults. Serum concentrations did not show significant differences with respect to disease category, sex or race but were found to vary, sometimes markedly, from child to child at any one age. The results suggest human cartilage undergoes significant changes in metabolic activities during maturation. Measurements of keratan sulphate concentration in serum may prove useful in studying the biochemical and physiological bases of these changes and in monitoring growth or endochondral ossification during maturation.

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.

We measured levels of sulfated keratan sulfate in serum using a monoclonal antibody in an enzyme-linked immunosorbent assay. Sulfated keratan sulfate was not detected in the serum of 16 patients with macular corneal dystrophy, but was present at normal levels in 66 patients with other corneal diseases. There were no differences with respect to age, sex, and other ocular findings. This monoclonal antibody recognizes a sulfated carbohydrate epitope present in both corneal and skeletal keratan sulfate. Since most serum keratan sulfate is derived from the cartilages, the defect in keratan sulfate synthesis in macular corneal dystrophy may not be restricted to corneal cells. This assay should prove useful in the diagnosis of macular corneal dystrophy, particularly in children at risk before the appearance of opacification.