Jürgen Steinmeyer

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.

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.

Pharmacological effect of tetracyclines on proteoglycanases from interleukin-1 -treated articular cartilage

Based on previous in vivo and in situ studies showing that tetracyclines possess antidegenerative effects on cartilage in conjunction with a reduced proteoglycan (PG) loss from the extracellular matrix, we investigated the effects of doxycycline, minocycline and tetracycline on the degradation and biosynthesis of PGs by bovine articular cartilage explants, both in vitro and in situ. Doxycycline, minocycline and tetracycline dose dependently, although weakly, inhibited PG degrading matrix metalloproteinases (MMPs) in vitro, when tested at concentrations ranging from 1 to 100 microM. Ro 31-4724 proved to be a potent inhibitor of MMP proteoglycanases (IC50 value 1.5 nM). Only at a concentration of 100 microM did doxycycline and minocycline significantly inhibit the interleukin-1 (IL-1)-induced augmentation of PG loss from cartilage explants into the nutrient media. The tetracyclines did not modulate the IL-1-mediated reduced aggregability of PGs, whereas 10 microM Ro 31-4724 partially restored the aggregability of PGs ex vivo. Tetracycline even at this high concentration was ineffective. Compared to the effects of the MMP inhibitor Ro 31-4724, treatment with tetracyclines at therapeutic serum levels of 1 or 10 microM was minimal, with little or no effect on cartilage proteoglycanases and PG biosynthesis. In our experiments, tetracyclines and Ro 31-4724 at doses evaluated had no cytotoxic effects on chondrocytes.

The sulfation pattern of chondroitin sulfate from articular cartilage explants in response to mechanical loading

Chondrocytes within articular cartilage experience complete unloading between loading cycles thereby utilizing mechanical signals to regulate their own anabolic and catabolic activities. Structural alterations of proteoglycans (PGs) during aging and the development of osteoarthritis (OA) have been reported; whether these can be attributed to altered load or compression is largely unknown. We report here on experiments in which the effect of intermittent loading on the fine structure of newly synthesized chondroitin sulfate (CS) in bovine articular cartilage explants was examined. Tissues were subjected for 6 days to cyclic compressive pressure using a sinusoidal waveform of 0.1, 0.5 or 1.0 Hz frequency with a peak stress of 0.5 MPa for a period of 5, 10 or 20 s, followed by an unloading period lasting 10, 100 or 1000 s. During the final 18 h of the culture, cartilage explants were radiolabeled with 50 microCi/ml D-6-[3H]glucosamine, and newly synthesized as well as endogenous CS chains were isolated after proteinase solubilization of the tissue. CS chains were depolymerized with chondroitinase ABC and ACII, and the 3H-digestion products were quantified after fractionation by high-performance anion-exchange chromatography using a CarboPac PA1 column. Intermittently applied cyclic mechanical loading did not affect the proportion of 4- and 6-sulfated disaccharide repeats, but caused a significant decrease in the abundance of the 4,6-disulfated nonreducing terminal galNAc residues. In addition, loading induced elongation of CS chains. Taken together, these data provide evidence for the first time that long-term in vitro loading results in marked and reproducible changes in the fine structure of newly synthesized CS, and that accumulation of such chains may in turn modify the physicochemical and biological response of articular cartilage. Moreover, data presented here suggest that in vitro dynamic compression of cartilage tissue can induce some of the same alterations in CS sulfation that have previously been shown to occur during the development of degenerative joint diseases such as OA.

Effects of polysulfated glycosaminoglycan and triamcinolone acetonid on the production of proteinases and their inhibitors by IL-1α treated articular chondrocytes

In this study we determined the in vitro effects of polysulfated glycosaminoglycan (PSGAG) and the glucocorticoid triamcinolone acetonid (TA) on the IL-1 altered expression and activity of matrix metalloproteinases (MMP-1, MMP-3), tissue inhibitor of metalloproteinases-1, the plasminogen activators tPA and uPA and plasminogen activator inhibitor 1 by articular chondrocytes. Bovine chondrocytes were cultured in alginate gel beads. Cells were treated with interleukin-1alpha (IL-1alpha) in the presence of vehicle or drugs at various concentrations. After 48hr mRNA expression of MMP-1, MMP-3, TIMP-1, uPA, tPA and PAI-1 was analyzed by RT-PCR-ELISA. The protein synthesis of TIMP-1 and MMP-3 was determined by immunoprecipitation, PAI-1 protein was quantitated by ELISA. The activity of enzymes and inhibitors was measured by functional assays. Treating chondrocytes with IL-1 induced the expression of MMPs and downregulated TIMP-1 but stimulated both the expression of PAs and PAI-1. Both drugs significantly reduced collagenase and proteoglycanase activities which was accompanied by inhibition of the expression of MMP-1 and MMP-3. The IL-1 decreased expression of TIMP-1 was further reduced by TA, which resulted in a significant loss of TIMP activity. No effects on TIMP activity or TIMP-1 biosynthesis were observed after treatment of chondrocytes with PSGAG. Both drugs inhibited the IL-1-induced mRNA expression of tPA, whereas expression of uPA was only mildly reduced by PSGAG, which also induced PAI-1 above IL-1 stimulated levels. As inhibition of collagenase activities and tPA expression by PSGAG occurred at physiological concentrations it might be of clinical relevance, indicating that PSGAG could help reducing cartilage degradation and has a strong anti-fibrinolytic potential. Due to their co-regulation of MMPs and TIMP(s) glucocorticoids should be carefully studied for their overall effect on extracellular matrix proteolysis.

A new pressure chamber to study the biosynthetic response of articular cartilage to mechanical loading.

A prototype chamber was used to apply a precise cyclic or static load on articular cartilage explants under sterile conditions. A variable pressure, pneumatic controller was constructed to power the chambers air cylinder, capable of applying, with a porous load platen, loads of up to 10 MPa at cycles ranging from 0 to 10 Hz. Pig articular cartilage explants were maintained successfully in this chamber for 2 days under cyclic mechanical loading of 0.5 Hz, 0.5 MPa. Explants remained sterile, viable and metabolically active. Cartilage responded to this load with a decreased synthesis of fibronectin and a small but statistically significant elevation in proteoglycan content. Similar but less extensive effects on fibronectin synthesis were observed with the small static load (0.016 MPa) inherent in the design of the chamber.

Pharmacological influence of antirheumatic drugs on proteoglycanases from interleukin-1 treated articular cartilage

The purpose of this study was to examine whether drugs used in the treatment of arthritic disorders possess any inhibitory potential on the proteoglycanolytic activities of matrix metalloproteinases (MMPs), and to determine whether drugs which inhibit these enzymes also modulate the biosynthesis and release of proteoglycans (PGs) from interleukin-1-(IL-1) treated articular cartilage explants. The cartilage-bone marrow extract and the glycosaminoglycan-peptide complex (DAK-16) dose-dependently inhibited MMP proteoglycanases in vitro when tested at concentrations ranging from 0.5 to 55 mg/mL, displaying an IC50 value of 31.78 mg/mL and 10.64 mg/mL (1.9 x 10[-4] M) respectively. (R,S)-N-[2-[2-(hydroxyamino)-2-oxoethyl]-4-methyl-1-oxopentyl++ +]-L-leucyl-L-phenylalaninamide (U-24522) proved to be a potent inhibitor of MMP proteoglycanases (IC50 value 1.8 x 10[-9] M). None of the other tested drugs, such as possible chondroprotective drugs, nonsteroidal anti-inflammatory drugs (NSAIDs), disease modifying antirheumatic drugs (DMARDs), glucocorticoids and angiotensin-converting enzyme inhibitors tested at a concentration of 10(-4) M displayed any significant inhibition. Only U-24522, tested at a concentration ranging from 10(-4) to 10(-6) M, significantly inhibited the IL-1-induced augmentation of PG loss from cartilage explants into the nutrient media, whereas DAK-16 and the cartilage-bone marrow extract were ineffective. DAK-16 and the cartilage-bone marrow extract did not modulate the IL-1-mediated reduced biosynthesis and aggregability of PGs by the cartilage explants. The addition of 10(-5) M U-24522, however, partially maintained the aggregability of PGs ex vivo. In our experiments, both possible chondroprotective drugs as well as U-24522 demonstrated no cytotoxic effects on chondrocytes.

Modulation of the Synthesis and Activation of Matrix Metalloproteinases in IL‐1‐Treated Chondrocytes by Antirheumatic Drugs

The destruction of articular cartilage during osteoarthritis and rheumatoid arthritis is characterized by the degradation and loss of collagen and proteoglycans. Matrix metalloproteinases (MMP) such as collagenase (MMP-1) and stromelysin (MMP-3) belong to the key enzymes of this proteolytic destruction. They are secreted as inactive proforms and must be activated before they can degrade their substrates. The serine protease plasmin, generated from the proenzyme plasminogen by the plasminogen activators u-PA (urokinase type) and t-PA (tissue type), is involved in the activation of these MMPs. Within the extracellular matrix, control over this enzymatic cascade is exerted by specific inhibitors, called tissue inhibitor of metalloproteinases (TIMP) and plasminogen activator inhibitor (PAI). Identification of agents that might inhibit or slow down the degradation of extracellular matrix in vivo has long been a therapeutic goal. Our study was therefore designed to determine the potential of some commercially available antirheumatic drugs to reduce the IL-1–mediated increase in proteolytic activities by either inhibiting the biosynthesis of the enzymes MMP-1, MMP-3, t-PA, and u-PA and/or by stimulating the biosynthesis of the specific inhibitors TIMP-1 and PAI-1.

Effects of the hydroxamic acid derivate Ro 31-4724 on the metabolism and morphology of interleukin-1-treated cartilage explants.

Matrix metalloproteinases (MMPs) belong to the key enzymes of the proteolytic destruction of cartilage matrix during chronic rheumatic diseases. Our work focused on the inhibitory potential of the hydroxamate Ro 31-4724 on the activity of MMP-proteoglycanases as well as on the viability, morphology and proteoglycan metabolism of interleukin-1 (IL-1)-treated bovine articular cartilage explants. The in vitro activity of MMP-proteoglycanases as well as the release of proteoglycans from IL-1-treated cartilage explants were significantly and concentration-dependently inhibited by Ro 31-4724 tested at concentrations ranging from 1 nmol/l to 10 mumol/l. Histopathological evaluation of sections from cartilage explants treated with this drug revealed no microscopically discernible alterations, and did not show any cytotoxic effects of Ro 31-4724. In addition, Ro 31-4724 had no effect on the rate of proteoglycan biosynthesis by IL-1-treated cartilage explants and increased the percentage of newly synthesized proteoglycans to form macromolecular aggregates. In conclusion, Ro 31-4724 displayed MMP-proteoglycanase inhibitory activity both in vitro and ex vivo and proved to be not harmful to the morphology, viability and proteoglycan biosynthesis of bovine articular cartilage explants.