Increase in degraded collagen type II in synovial fluid early in the rabbit meniscectomy model of osteoarthritis.

OBJECTIVE: The objective of this study was to determine whether collagen type II breakdown products in synovial fluid (SF), detected by an enzyme-linked immunoassay, represent a useful marker for early events in osteoarthritis (OA) in the rabbit medial meniscectomy model. DESIGN: Complete medial meniscectomy was performed on the right knee joints of 32 rabbits. Balanced groups of rabbits were then sacrificed at 2, 4, 8, and 12 weeks post-surgery. An additional 8 unoperated and 11 sham-operated animals served as controls. SF lavages were performed on right and left knee joints of the same animals at sacrifice. The proteolytic epitope of type II collagen was monitored using an enzyme-linked immunoassay. RESULTS: Macroscopically visible surface fibrillation and focal erosions appeared as early as 2 weeks after meniscectomy in the femorotibial joint (P<0.01). OA developed gradually during the later observation period, and then predominantly on the medial tibial plateau and medial femur. Significant histological alterations in cartilage, including a loss of proteoglycans, surface irregularities, and clefts, were detected at 2 weeks after meniscectomy (P<0.01). Collagen type II epitope levels in SF lavage samples were elevated peaking at 2 weeks after meniscectomy (P<0.02). Levels decreased at later time points, but they were still raised at 12 weeks (P< or =0.05). Highly significant correlations were found between the SF collagen type II epitope levels and the macroscopic and microscopic scoring results (Spearman rho correlation coefficient, macroscopy-collagen type II epitope r=0.222, P=0.025; microscopy-collagen type II epitope r=0.436, P< or =0.01). CONCLUSION: In this rabbit model of medial meniscectomy, levels of type II collagen fragments in SF appear to provide a useful marker of the early degenerative changes.

Proteoglycan metabolism and viability of articular cartilage explants as modulated by the frequency of intermittent loading.

OBJECTIVE: This study was designed to systematically determine whether and to what extent the frequency of intermittent loading modulates the biosynthesis and release of proteoglycans (PGs), and to assess chondrocyte viability within mature bovine articular cartilage explants exposed to different loading patterns. METHODS: Cultured full-thickness cartilage explants from the weight-bearing area of healthy bovine fetlock joints were exposed to intermittently applied, uniaxial cyclic loads by introducing a sinusoidal waveform of 0.1, 0.5 or 1.0Hz, frequency and a peak stress of 0.5MPa for a period of 6 days. The cyclic loads were applied for 5, 10 or 20s followed by a period of unloading lasting 10, 100 or 1000s. The incorporation of radiolabeled sulfate into glycosaminoglycans (GAGs) during the final 18h, the content of GAGs and DNA, the deformation of loaded explants as well as the viability of chondrocytes within the different zones of explants were determined. RESULTS: PG synthesis and loss of endogenous PGs were non-linearly and independently regulated by the frequency of the chosen intermittent load, whereas the release of newly synthesized PGs remained unaffected. The viability of chondrocytes within the superficial zone decreased drastically under intermittent loading in a manner independent of the frequency applied. CONCLUSIONS: Our results confirm the hypothesis that the frequency of intermittent loading is an important mechanical factor controlling the metabolic activities of chondrocytes. They also implicate that an initially healthy cartilage explant can be mechanically manipulated to generate an in vitro model of degenerative, osteoarthritic-like cartilage.

Effects of intermittently applied cyclic loading on proteoglycan metabolism and swelling behaviour of articular cartilage explants.

OBJECTIVE: The aim of this study was to evaluate the effect of tissue load, frequency and load duration on the biosynthesis and release of proteoglycans (PGs) as well as on the swelling behaviour of cultured mature bovine articular cartilage superimposed with intermittent loads. METHODS: Cyclic compressive pressure was introduced for 1, 3 or 6 days using a sinusoidal waveform of 0.5 Hz-frequency with a peak stress of 0.1, 0.5 or 1.0 MPa. The loads were applied for a period of 10 seconds (s) followed by a load-free period of 10, 100 or 1000 s. The incorporation of [35S]-SO4 into glycosaminoglycans (GAGs) during the final 18 h, the content of GAGs and DNA as well as the deformation of loaded explants were determined. RESULTS: The PG synthesis is sensitive to changes in the loading conditions applied, whereas the release of newly synthesized PG is not. A maximum PG synthesis is observed at day 3, and under load-free intervals of 100 s. After 6 days of loading the release of endogenous PGs is significantly elevated, the viability of superficial chondrocytes decreased, and cartilage swelling is observed. CONCLUSIONS: Considering numerous reports of elevated PG levels synthesized as well as released from human and experimental osteoarthritic cartilage, our results implicate that degenerative processes can also be mimicked by applying well-defined mechanical conditions as described here.

Can magnetization transfer magnetic resonance imaging follow proteoglycan depletion in articular cartilage?

In this study we determined the efficiency of magnetization transfer magnetic resonance imaging (MT-MRI) to differentiate native and enzymatically degraded cartilage, using bovine sesamoid bones from the metacarpophalangeal joint as a model system. Gradual proteoglycan (PG) depletion was achieved by increasing incubation periods with testicular hyaluronidase. For native cartilage a Ms/Mo ratio of 0.303 +/- 0.09 (mean +/- SEM) was measured. Biochemically determined PG diminution up to 50% correlated strongly (r = 0.953) with changes in the Ms/Mo ratio. Further PG loss is not reflected in an equally drastic Ms/Mo increase, whereas subsequent treatment of PG-depleted cartilage samples with collagenase led to an additional rise in the Ms/Mo ratio. Proteoglycan depletion and the beginning destruction of the collagen structure were also assessed histochemically. Our study confirms that collagen contributes to the baseline MT effect observed in articular cartilage. However, the changes in the MT ratio in gradually PG-depleted cartilage with a largely intact collagen network indicate that PG contributes to the MT effect as well. Therefore MT-MRI might become a sensitive technique for the monitoring of subtle degradational changes in articular cartilage, the still inaccessible process in osteoarthritis.

Intermittent cyclic loading of cartilage explants modulates fibronectin metabolism.

OBJECTIVE: The aim of this study was to evaluate systematically the effect of tissue load, its amplitude, time of intermittence and duration of loading on the biosynthesis and release of fibronectin by intermittently loaded mature bovine articular cartilage explants. METHODS: Cyclic compressive pressure was introduced using a sinusoidal waveform of 0.5 Hz-frequency with a peak stress of 0.1, 0.5 or 1.0 MPa for a period of 10 s followed by an unloaded period lasting 10, 100 or 1000 s. Fibronectin and total proteins were radiolabeled with 10 microCi/ml [3H]-phenylalanine during the final 18 h of the 1, 3 or 6 day experiments. The content of endogenous fibronectin was determined using enzyme-linked immunosorbant assay (ELISA), whereas the viability of explants was measured using sections of cartilage explants stained with fluorescein diacetate and propidium iodide. The deformation of loaded explants was determined using a load-displacement transducer system. RESULTS: The mechanical factor time of intermittence significantly altered the synthesis and release of fibronectin by cartilage explants, whereas the tested range of load magnitudes, as well as the duration of loading, seemed to be of subordinate importance. Loading affected the viability of the superficial zone in the cartilage, whereas the chondrocytes of the intermediate and deep zone remained viable. The compression of loaded explants was dependent on the magnitude of stress, as well as on the duration of unloading between each loading cycle. Synthesis of fibronectin, the retention of newly synthesized fibronectin within the extracellular matrix, and the portion of newly synthesized proteins that were fibronectin was significantly increased in cartilage explants which were cyclically compressed with 0.5 MPa for 10 s followed by a period of unloading lasting 100 s. CONCLUSIONS: Previous studies reporting that cartilage explants of human and animal osteoarthritic joints synthesize and retain elevated amounts of fibronectin imply that in our experiments mechanical stimuli can induce a fibronectin metabolism in vitro which mimics some of the osteoarthritic characteristics.

Chondrocytes and antirheumatic drugs.

Effects of antirheumatic drugs upon cartilage matrix metabolism have been studied in a variety of chondrocyte in vitro systems. When compared longterm in 60 experiments under standardized conditions, articular chondrocytes cultured in agarose exhibit variability in proteoglycan synthesis, and its suppression by interleukin 1 (IL-1), but a high reproducibility in the modulation of these effects by antirheumatic drugs. Pentosan polysulfate, tenidap, tiaprofenic acid, and RO 31-9790 all compensated to a certain extent the IL-1 induced suppression of matrix synthesis in bovine chondrocytes, but only for tiaprofenic acid could this be confirmed using chondrocytes of human origin. Culture conditions as well as species differences should therefore be considered carefully when chondrocyte cultures are used as pharmacological models.

Influence of interleukin-1 on the morphology and proteoglycan metabolism of cultured bovine articular chondrocytes.

Bovine articular chondrocytes cultured in agarose gel in the presence of serum elaborated a highly organized extracellular matrix rich in proteoglycans and collagens. The cultures were evaluated quantitatively by radiosulfate labeling of proteoglycans, and by densitometry following staining with alcian blue. In addition, immunohistochemical methods were used to demonstrate the presence of several components of cartilage proteoglycan molecules. Treatment with Interleukin-1 (Il-1) or retinol resulted in diminished synthesis and enhanced catabolism of matrix proteoglycans, but the chondrocytes were more sensitive to human recombinant Il-1 alpha than to Il-1 beta. Treatment with Il-1 alpha or retinol resulted in a profound disorganization of the residual matrix around the majority of the chondrocytes, while Il-1 beta caused much less severe changes. Some variation in cellular response to Il-1 alpha may result from the heterogeneity previously reported among articular chondrocytes.

Responses of normal and rheumatic human articular chondrocytes cultured under various experimental conditions in agarose.

The purpose of the present study was to test if agarose could support the maintenance of normal and arthritic human chondrocytes in culture, and under which experimental conditions they could be successfully grown. Cultures of chondrocytes isolated from articular cartilage from patients with rheumatoid arthritis (RA), juvenile rheumatoid arthritis (JRA), and healthy controls were assessed by light microscopy, alcian blue staining, formazan uptake and incorporation of radiosulfate into the extracellular matrix. The results showed that both normal and arthritic chondrocytes proliferated, and synthesized proteoglycan (PG) in agarose in short term and long term culture. Proliferation and PG synthesis occurred at a slower rate in chondrocytes from adult rheumatic patients than from healthy controls. Supplements to the medium influenced chondrocyte proliferation, PG synthesis and release into the medium. Serum from RA patients stimulated chondrocyte responses more than normal human serum (NHS), and NHS promoted PG synthesis more than fetal calf serum (FCS). Exposure to inflammatory synovial fluid (SF) enhanced PG synthesis of healthy chondrocytes, but suppressed it in arthritic chondrocytes. We conclude that species-specific serum is optimal for chondrocyte cultures, and that disease related culture conditions change the chondrocyte response. As metabolic responses of human chondrocytes are maintained in agarose, this culture system appears as a suitable in vitro tool for further studies of human joint disease.

Development of autoimmunity in MRL/lpr mice and the effects of drugs on this murine disease.

MRL/lpr mice spontaneously develop a systemic Lupus erythematosus (SLE)-like disease with a wide range of clinical and serological characteristics that mimic not only human SLE but other autoimmune disorders. such as Sjögren's syndrome, and rheumatoid arthritis (RA). Unlike other murine SLE-like disorders, these mice have circulating rheumatoid factor (RF) and develop histological changes in their joints. Therapy of this disease with cyclophosphamide (CY), cyclosporin A, prednisolone, or leflunomide (HWA 486) resulted in very differing effects. Treating these mice with HWA 486 or cyclophosphamide (CY) resulted in a decrease in the amount of autoantibodies, and immune complex deposits on the glomeruli. HWA 486 therapy led to restoration of the depressed immune response of MRL/lpr mice. In the established disease, prednisolone (Pr), cyclosporin A (CSA), and HWA 486 could inhibit the proteinuria and return the urine-protein values to normal levels, but, unlike HWA 486, neither PR nor CSA could extend the longevity of these animals. The MRL/lpr mouse should prove to be very useful as a model for SLE, RA, and for discovering novel drugs to combat such disorders.

Dose related effects of dexamethasone treatment on the ultrastructure of articular cartilage in rats.

Corticosteroid administration is known to cause degenerative changes in articular cartilage interfering with the cell metabolism of chondrocytes. The present study analyzes the ultrastructural changes in chondrocytes after systemic dexamethasone acetate treatment in relation to dosage, using a standardized morphometrical method. Five male Wistar rats of 300 g body weight in each experimental group were subjected to 3, 4 and 5 mg dexamethasone acetate by intramuscular injections of 1 mg per week. 1000 electron micrographs of single chondrocytes in the middle zone of hyaline cartilage from the knee joints were evaluated with standardized morphometry and nonparametric statistics. With increasing dexamethasone dosage the amount of rough endoplasmic reticulum and Golgi apparatus decreased. Considerably increased glycogen granula and clusters indicated a severe change in glycolytic pathways. Lysosomes duplicated in number. Degenerative changes were also manifested in lipid droplets and myeloid bodies, which, like the amount of microfilaments, exhibited a clear dosage-dependent increase under dexamethasone treatment. The ratio of dead versus living chondrocytes increased in relation to dosage up to 25% cell mortality.

[The effect of steroidal and nonsteroidal anti-inflammatory agents on the ultrastructure of chondrocytes in the rat. Electron microscope and morphometric study]. / Einfluss steroidaler und nichtsteroidaler Antiphlogistika auf die Ultrastruktur von Chondrozyten der Ratte. Elektronenoptische und morphometrische Studie.

In order to obtain indications regarding the different influences of antiinflammatory drugs on the articular cartilage, the effects of a steroidal drug (dexamethasone) and of nonsteroidal antiinflammatory substances (indometacin and phenylbutazone) on the ultrastructure of the chondrocyte were examined in comparison with tiaprofenic acid (Surgam). Using an equiefficient dosage of the active principles, referred to the ED50 in the model of adjuvant arthritis in the rat, the synthetic apparatus of the chondrocytes was stimulated by tiaprofenic acid while it was diminished by indometacin and phenylbutazone and particularly by dexamethasone.