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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.

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.

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.

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.

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.

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.

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.