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Objective: Chondromodulin I (ChM-I), a cartilage matrix protein, promotes the growth and proteoglycan synthesis of chondrocytes. However, it also inhibits angiogenesis. Since ChM-I is expressed not only in cartilage, but also in the thymus, we investigated the modulation of T cell function by ChM-I to assess its therapeutic potential in rheumatoid arthritis (RA)., Methods: The localization of ChM-I expression in mouse thymus tissue was examined by in situ hybridization. The proliferative response of peripheral blood T cells and synovial cells obtained from patients with RA was evaluated by (3)H-thymidine incorporation assay. The effects of ChM-I were examined using recombinant human ChM-I (rHuChM-I). Modulation of the antigen-specific immune response was evaluated by the recall response of splenic T cells and the delayed-type hypersensitivity response induced in the ear of mice primed with ovalbumin (OVA). Antigen-induced arthritis (AIA) was induced in mice by injecting methylated bovine serum albumin into the ankle joints 2 weeks after the priming., Results: ChM-I was expressed in the cortex of the thymus. Recombinant human ChM-I suppressed the proliferative response of mouse splenic T cells and human peripheral blood T cells stimulated with anti-CD3/CD28 antibodies, in a dose-dependent manner. Production of interleukin-2 was decreased in rHuChM-I-treated mouse CD4 T cells. Ten micrograms of rHuChM-I injected intraperitoneally into OVA-primed mice suppressed the induction of the antigen-specific immune response. Finally, rHuChM-I suppressed the development of AIA, and also suppressed the proliferation of synovial cells prepared from the joints of patients with RA., Conclusion: These results suggest that ChM-I suppresses T cell responses and synovial cell proliferation, implying that this cartilage matrix protein has a therapeutic potential in RA.

Objective: To use noninvasive magnetic resonance imaging (MRI), biochemical analyses, and mechanical testing of engineered neocartilage grown in a hollow- fiber bioreactor (HFBR) to establish tissue properties, and to test the hypothesis that MRI can be used to monitor biochemical and biomechanical properties of neocartilage., Methods: Chondrocytes from day 16 embryonic chick sterna were inoculated into an HFBR and maintained for up to 4 weeks with and without exposure to chondroitinase ABC. The fixed-charge density (FCD) of the cartilage was determined using the MRI gadolinium exclusion method. The sulfated glycosaminoglycan (S-GAG), hydroxyproline, and DNA contents were determined using biochemical procedures, while dynamic and equilibrium moduli were determined from mechanical indentation tests., Results: S-GAG content, tissue cross-sectional area, and equilibrium modulus of the neocartilage increased with development time. There was a gradient of S-GAG content across the length of control neocartilage at the 4-week time point, with higher values being found toward the inflow region. Exposure to chondroitinase ABC resulted in a decrease in tissue area, negative FCD, proteoglycan content, and equilibrium and dynamic moduli. The treated bioreactors displayed a lengthwise variation in S-GAG content, with higher values toward the outflow end. Linear correlations were established among FCD, proteoglycan content, and biomechanical properties., Conclusion: HFBR-derived neocartilage showed regional variation in S-GAG content under control conditions, and in the decrease of S-GAG in response to enzyme treatment. In addition, the results support the hypothesis that tissue parameters derived from MRI can be used to noninvasively monitor focal neocartilage formation and biochemical and biomechanical properties.

Objective: To assess the role of matrix metalloproteinases (MMPs) in cartilage and bone erosions in Lyme arthritis, Methods: We examined synovial fluid from 10 patients with Lyme arthritis for the presence of MMP-2, MMP-3, MMP-9, and "aggrecanase" activity using gelatinolytic zymography and immunoblot analysis. We developed an in vitro model of Lyme arthritis using cartilage explants and observed changes in cartilage degradation in the presence of Borrelia burgdorferi and/or various protease inhibitors., Results: Synovial fluid from patients with Lyme arthritis was found to contain at least 3 MMPs: gelatinase A (MMP-2), stromelysin (MMP-3), and gelatinase B (MMP-9). In addition, there was evidence in 2 patients of "aggrecanase" activity not accounted for by the above enzymes. Infection of cartilage explants with B. burgdorferi resulted in induction of MMP-3, MMP-9, and "aggrecanase" activity. Increased induction of these enzymes by B. burgdorferi alone was not sufficient to cause cartilage destruction in the explants as measured by glycosaminoglycan (GAG) and hydroxyproline release. However, addition of plasminogen, which can act as an MMP activator, to cultures resulted in significant GAG and hydroxyproline release in the presence of B. burgdorferi. The MMP inhibitor batimastat significantly reduced the GAG release and completely inhibited the collagen degradation., Conclusion: MMPs are found in synovial fluids from patients with Lyme arthritis and are induced from cartilage tissue by the presence of B. burgdorferi. Inhibition of MMP activity prevents B. burgdorferi-induced cartilage degradation in vitro.

Objective: To test the hypothesis that Fourier transform infrared (FTIR) spectral imaging, coupled with multivariate data processing techniques, can image the spatial distribution of matrix constituents in native and engineered cartilage samples., Methods: Tissue sections from native and trypsin-digested bovine nasal cartilage (BNC) and from engineered cartilage, generated by chick sternal chondrocytes grown in a hollow fiber bioreactor, were placed either on calcium fluoride windows for FTIR analysis or gelatinized microscope slides for histologic analysis. Based on the assumption that cartilage is predominantly chondroitin sulfate (CS) and type II collagen, chemical images were extracted from FTIR spectral imaging data sets using 2 multivariate methods: the Euclidean distance algorithm and a least-squares approach., Results: Least-squares analysis of the FTIR data of native BNC yielded a collagen content of 54 +/- 13% and a CS content of 37 +/- 16% (mean +/- SD). Euclidean distance analysis of measurements made on trypsin-digested BNC demonstrated only trace amounts of CS. For engineered cartilage, the CS content was significantly lower (15 +/- 5%), while the collagen content (73 +/- 6%) was significantly higher than biochemically determined values (CS 34%, collagen 5%, protein 61%). These differences are due to the fact that the dimethylmethylene blue assay overestimated the CS content of the tissue because it is not specific for CS, while the FTIR spectral imaging technique overestimated the collagen content because it lacks specificity for different proteins., Conclusion: FTIR spectral imaging combines histology-like spatial localization with the quantitative capability of bulk chemical analysis. For molecules with a unique spectral signature, such as CS, the FTIR technique coupled with multivariate analysis can define a unique spatial distribution. However, for some applications, the lack of specificity of this technique for different types of proteins may be a limitation.

Objective: To test the hypothesis that magnetic resonance imaging (MRI) results correlate with the biochemical composition of cartilage matrix and can therefore be used to evaluate natural tissue development and the effects of biologic interventions., Methods: Chondrocytes harvested from day-16 chick embryo sterna were inoculated into an MRI-compatible hollow-fiber bioreactor. The tissue that formed over a period of 2-4 weeks was studied biochemically, histologically, and with MRI. Besides natural development, the response of the tissue to administration of retinoic acid, interleukin-1beta (IL-1beta), and daily dosing with ascorbic acid was studied., Results: Tissue wet and dry weight, glycosaminoglycan (GAG) content, and collagen content all increased with development time, while tissue hydration decreased. The administration of retinoic acid resulted in a significant reduction in tissue wet weight, proteoglycan content, and cell number and an increase in hydration as compared with controls. Daily dosing with ascorbic acid increased tissue collagen content significantly compared with controls, while the administration of IL-1beta resulted in increased proteoglycan content. The water proton longitudinal and transverse relaxation rates correlated well with GAG and collagen concentrations of the matrix as well as with tissue hydration. In contrast, the magnetization transfer value for the tissue correlated only with total collagen. Finally, the self-diffusion coefficient of water correlated with tissue hydration., Conclusion: Parameters derived from MR images obtained noninvasively can be used to quantitatively assess the composition of cartilage tissue generated in a bioreactor. We conclude that MRI is a promising modality for the assessment of certain biochemical properties of cartilage in a wide variety of settings.

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.

Objective: To investigate the relationship between occurrence of the matrix metalloproteinase-generated neoepitope VDIPEN and proteoglycan (PG) loss in arthritis, and to examine the role of interleukin-1 (IL-1) in VDIPEN expression., Methods: VDIPEN expression was investigated in murine antigen-induced arthritis by immunolocalization studies on joint sections. The involvement of IL-1 in VDIPEN expression was studied by blocking of IL-1 using IL-1 receptor antagonist (IL-1Ra)., Results: Profound PG loss was evident early in arthritis, without significant VDIPEN expression. Full expression of the neoepitope appeared after a few days, when PG depletion was severe, and disappeared at late stages when cartilage showed recovery from PG depletion. At sites where chondrocyte death occurred and cartilage did not recover from the initial cartilage depletion, VDIPEN expression remained present. Prophylactic IL-1Ra treatment of arthritic mice resulted in almost complete prevention of VDIPEN expression. However, IL-1Ra had only a minor effect on PG depletion, emphasizing that there is no correlation between VDIPEN and early PG depletion., Conclusion: This study indicates that IL-1 is involved in VDIPEN expression. Although VDIPEN-inducing metalloproteinases do not seem to be involved in early PG depletion during antigen-induced arthritis, metalloproteinase neoepitopes are present when PG depletion is severe.

Objective: To study the effects of recombinant human osteogenic protein-1 (rHuOP-1; bone morphogenetic protein-7) on proteoglycan and collagen synthesis by human articular chondrocytes., Methods: Articular chondrocytes from fetal, adolescent, and adult human donors were cultured in alginate beads for 4 days in a mixture of Ham's F-12, Dulbecco's modified Eagle's medium, 10% fetal bovine serum (FBS), then for an additional 3-10 days in the presence and absence of rHuOP-1, with and without FBS. Chondrocyte synthetic activity was measured as the amount of incorporation of 35S-sulfate into proteoglycans and 3H-proline into hydroxyproline. Sieve chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were performed to identify specific proteoglycans and collagens., Results: Recombinant human OP-1 markedly stimulated the synthesis of proteoglycans (mostly aggrecan) and collagens (predominantly type II) by all chondrocyte preparations. This did not require the presence of FBS and was associated with continued expression of the chondrocyte phenotype., Conclusion: Recombinant human OP-1 is a more potent stimulator of the synthesis of cartilage-specific molecules by human articular chondrocytes than are other factors tested for comparison, including TGF beta 1 and activin A.

Objective: To evaluate the utility of computed tomography (CT) of the knee joint for detecting intraarticular tophaceous deposits., Methods: A prospective study of 16 patients with gout affecting the knee was conducted. A condition for inclusion in the study was the presence of needle-shaped crystals with negative birefringence in the knee joint synovial fluid. Conventional radiography and CT were performed in each case., Results: Intraarticular opacities in the capsule and the synovium, consistent with the presence of tophaceous deposits, were found in 5 of the 16 patients (9 knee joints). The mean duration of gout was longer in the patients with intraarticular tophi than in those without tophi, and 2 of the patients with tophi had poor tolerance to antihyperuricemic therapy., Conclusion: Intraarticular opacities considered to represent tophi were observed in approximately one-third of the patients. The presence of tophi correlated with a longer duration of the disease and a poor tolerance to medication. We therefore suggest that CT of the knees could be useful in the assessment and followup of certain patients with gout.

Objective: To investigate mechanisms of cartilage matrix destruction by a study of the effects of a specific inactivator of cathepsin B and an inhibitor of several matrix metalloproteinases (MMP) on cartilage proteoglycan release., Methods: Cartilage explants were treated with either recombinant human interleukin-1 alpha (rHuIL-1 alpha) or retinoic acid in the presence or absence of the inhibitors, and proteoglycan release was quantitated. Tests for nonspecific effects of the inhibitors included reversibility, rates of protein synthesis and glycolysis, and effects on other rHuIL-1 alpha-mediated events., Results: The cathepsin B inactivator inhibited rHuIL-1 alpha-stimulated proteoglycan release at nanomolar concentrations, but failed to significantly inhibit retinoic acid-stimulated proteoglycan release. An inhibitor of MMP was inhibitory to both rHuIL-1 alpha-stimulated release and retinoic acid-stimulated release., Conclusion: Cathepsin B is implicated in rHuIL-1 alpha-stimulated loss of cartilage proteoglycan. Its lack of involvement in retinoic acid-stimulated proteoglycan release suggests the existence of at least 2 pathways of cartilage proteoglycan breakdown, which may converge at the activation of a matrix prometalloproteinase.

Objective: Extensive changes in articular cartilage metabolism occur during the acute phase of reactive arthritis, as indicated by altered release of cartilage macromolecules into synovial fluid (SF) demonstrated immunochemically. Nevertheless, permanent cartilage lesions are rare in this disease. To monitor specific events during the evolution of reactive arthritis, we investigated the content of cartilage macromolecules in sequentially obtained SF samples from 22 patients., Methods: Two groups of proteoglycan epitopes, the glycosaminoglycan-rich region of aggrecan (referred to as proteoglycan) and its hyaluronan-binding region (HABr), as well as one matrix protein, cartilage oligomeric matrix protein (COMP), were quantified by immunoassay., Results: SF proteoglycan concentrations, which were initially elevated, decreased significantly with prolonged arthritis, whereas COMP levels changed less markedly and levels of HABr remained stable. There was a positive correlation between SF and serum concentrations of COMP in samples obtained during the early phase of the disease., Conclusion: Cartilage involvement in reactive arthritis is transient, in contrast to findings in rheumatoid arthritis. Reactive arthritis should therefore be a suitable model for studies of repair processes in cartilage, which will facilitate understanding of the pathophysiology of cartilage involvement in arthritis.