Interleukin-1ß affects the phospholipid biosynthesis of fibroblast-like synoviocytes from human osteoarthritic knee joints.

OBJECTIVE: Phospholipids (PLs), together with hyaluronan and lubricin, are involved in boundary lubrication within human articular joints. Levels of lubricants in synovial fluid (SF) have been found to be associated with the health status of the joint. However, the biosynthesis and release of PLs within human joints remains poorly understood. This study contributes to our understanding of the effects of cytokines on the biosynthesis of PLs using cultured fibroblast-like synoviocytes (FLS) from human osteoarthritic knee joints. METHODS: Cultured FLS were stimulated with IL-1ß, TNFα, IL-6, or inhibitors of cell signaling pathways such as QNZ, SB203580 and SP600125 in the presence of stable isotope-labeled precursors of PLs. Lipids were extracted and quantified using electrospray ionization tandem mass spectrometry (ESI-MS/MS). RESULTS: Our analyses provide for the first time a detailed overview of PL species being synthesized by FLS. IL-1ß increased the biosynthesis of both phosphatidylethanolamine (PE) and PE-based plasmalogens. We show here that the NF-κB, p38 MAPK and JNK signaling pathways are all involved in IL-1ß-induced PL biosynthesis. IL-6 had no impact on PLs, whereas TNFα increased the biosynthesis of all PL classes. CONCLUSION: The biosynthesis of various PLs is controlled by IL-1ß and TNFα. Our detailed PL species analysis revealed that FLS can partly contribute to the elevated PL levels found in human osteoarthritis (OA) SF. IL-1ß in particular stimulates PE and PE-based plasmalogens which can act as cell-protective antioxidants. These results suggest that during OA progression, FLS undergo alterations in their PL composition to adapt to the new diseased environment.

Comparative lipidomic analysis of synovial fluid in human and canine osteoarthritis.

OBJECTIVE: The lipid profile of synovial fluid (SF) is related to the health status of joints. The early stages of human osteoarthritis (OA) are poorly understood, which larger animals are expected to be able to model closely. This study examined whether the canine groove model of OA represents early OA in humans based on the changes in the lipid species profile in SF. Furthermore, the SF lipidomes of humans and dogs were compared to determine how closely canine lipid species profiles reflect the human lipidome. METHODS: Lipids were extracted from cell- and cellular debris-free knee SF from nine donors with healthy joints, 17 patients with early and 13 patients with late osteoarthritic changes, and nine dogs with knee OA and healthy contralateral joints. Lipid species were quantified by electrospray ionization tandem mass spectrometry (ESI-MS/MS). RESULTS: Compared with control canine SF most lipid species were elevated in canine OA SF. Moreover, the lipid species profiles in the canine OA model resembled early OA profiles in humans. The SF lipidomes between dog and human were generally similar, with differences in certain lipid species in the phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and sphingomyelin (SM) classes. CONCLUSIONS: Our lipidomic analysis demonstrates that SF in the canine OA model closely mimics the early osteoarthritic changes that occur in humans. Further, the canine SF lipidome often reflects normal human lipid metabolism.

Catabolic properties of microdissected human endosteal bone lining cells.

Bone lining cells cover > 80% of endosteal surfaces of human cancellous bone. Current research assigns to them a dual role: (1) as a biological membrane regulating exchange of substrates between the bone fluid compartment and the extracellular fluid of bone marrow and (2) as a signaling link between the osteocytic network as mechanical receptor and the osteoclastic cell pool for local induction of bone resorption. Furthermore, a catabolic role has been considered. We therefore examined the presence of matrix-metalloproteinases (MMPs) and their physiological tissue inhibitors (TIMPs) as putative proteolytic elements. Firstly, human cancellous bone from 60 patients was examined by immunofluorescence with antibodies against MMPs and TIMPs. Secondly, we applied laser-assisted microdissection (LMD) to isolate bone lining cells from frozen sections of human trabecular bone. mRNA analysis was performed using a single-cell PCR protocol. Three laser microdissection systems were tested: the new generation of Leica LMD and P.A.L.M. laser pressure catapulting (LPC) were compared to P.A.L.M. laser microdissection and micromanipulation (LMM). In a few pooled cell profiles, mRNA of MMP13, MMP14, TIMP1, and CBFA-1 was clearly detected. By immunofluorescence MMP13 and -14 as well as TIMP1 and -2 were strongly present in lining cells, while MMP2, TIMP3, and TIMP4 showed weak or negative signals. Although the functional impact of these enzymatic components remains open, there is additional evidence for a catabolic function of lining cells. The new diode-laser microdissection with LMD and LPC proved to be especially suitable to gain new insights into the properties of bone lining cells.

Intermittent mechanical loading of articular cartilage explants modulates chondroitin sulfate fine structure.

OBJECTIVE: Alterations in the sulfation pattern of chondroitin sulfate (CS) chains of proteoglycans have been associated with aging and degeneration of articular cartilage. The purpose of the present study was to investigate systematically the effect of load amplitudes, frequencies and load durations of intermittently applied mechanical pressure on the sulfation of CS chains of cultured bovine articular cartilage explants. METHODS: Using a sinusoidal waveform of 0.5 Hz frequency, cyclic compressive pressure of 0.1-1.0 MPa was applied for 10s followed by a period of unloading lasting 10-1000 s. These intermittent loading protocols were repeated for a total duration of 1-6 days. Newly synthesized as well as endogenous CS chains were isolated, depolymerized and subsequently quantitated after fractionation by high-performance anion-exchange chromatography. RESULTS: Increasing the mechanical demands on cartilage explants by elevating either the duration or the frequency of loading can significantly alter the fine structure of newly synthesized CS in that less chains terminate on galNAc4,6S and, in that simultaneously the ratio of the internal disaccharides DeltaDi6S to DeltaDi4S is increased. Similar results were obtained with explants being slightly mechanically challenged by low magnitudes of loads. CONCLUSION: Our data show for the first time that intermittent loading of articular cartilage explants can significantly alter the sulfation pattern of the terminal CS residues as well as of the internal disaccharides. Furthermore, our results indicate that explants possess a physiological window of stress in which they are able to produce also a normal extracellular matrix.

Collagen biosynthesis of mechanically loaded articular cartilage explants.

OBJECTIVE: The purpose of this study was to investigate systematically the effect of load amplitudes, frequencies and load durations of intermittently applied mechanical pressure on the biosynthesis of collagen and non-collagenous proteins (NCP) as well as on the water content of cultured bovine articular cartilage explants. METHODS: Cyclic compressive pressure was applied 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 10s followed by a load-free period of 10, 100 or 1000s. These intermittent loading protocols were repeated for a total duration of 1, 3 or 6 days. During the final 18 h of experiments, the incorporation of [(3)H]-proline into collagen and NCP, the content of water as well as the deformation of loaded explants were determined. RESULTS: Intermittently applied, cyclic mechanical loading of articular cartilage explants consistently reduced the relative rate of collagen synthesis compared to load-free conditions. This reduced proportion of newly synthesized collagen among newly made proteins was independent of the mechanical stimuli applied. The release of newly synthesized collagen and NCP from loaded explants into the nutrient media was unaffected by any of the loading protocols applied. In addition, quantitative data are provided showing that only high amplitudes of loads and frequencies enhanced the water content of the explants. CONCLUSIONS: Previous studies reporting that osteoarthritic cartilage in vivo can synthesize elevated amounts of collagen imply that the loading protocols chosen were inadequate for simulating in vitro osteoarthritic-like alterations of collagen synthesis. In our experiments the collagen biosynthesis of chondrocytes was only minor responsive to alterations in mechanical stimuli, applied over a wide range. Thus, our results imply that the synthesis of these structural macromolecules is under the strict control of normal chondrocytes enabling them to maintain the shape of this physical demanded tissue.

[MRI in the follow-up of matrix-supported autologous chondrocyte transplantation (MACI) and microfracture]. / Die MRT in der Verlaufskontrolle nach matrixgestützer autologer Chondrozytenimplantation (MACI) und Mikrofrakturierung.

AIM: Matrix-guided autologous chondrocyte implantation (MACI) was compared with microfracture (MFX) to demonstrate the reconstitution of cartilage over a two-year period using the morphological capabilities of MRI. PATIENTS AND METHODS: 27 patients (9 females and 18 males, mean age 33 years) underwent MACI on the knee joint. The defects originated from trauma (15 cases), osteochondritis dissecans (8 cases) and chronic repetitive trauma (4 cases) and were localized at the condyles (24 cases) or patella (3 cases). All patients were examined postoperatively after 1, 3, 6, 12 and 24 months with a 1,5 T unit (Gyroscan, Philips) using proton- and T2w spinecho and T1w fatsuppressed 3D gradientecho sequences. We measured the signal intensities of the implant and neighbouring cartilage to calculate the contrast-to-noise ratio (CNR), and the thickness of cartilage and implant layers to define the defect filling rate. Finally, partial and complete remission was defined on MRI and compared with clinical data and morphology on MRI. Additionally, 7 patients were treated with MFX and, subsequently examined on MRI with the same protocol. RESULTS: After MACI, MRI showed a partial but no complete equilibration of signal intensities of implant and adjacent cartilage over the 1 and 2 year follow-up periods which was shown by reduction of CNR from 21 to 10 on 3D-GE and from 26 to 9 on T2w SE sequences. Continuous growth of the implants resulted in an increased filling of the defects starting at 40% after 0.5 year to 85% after 1 or 2 years. Complete remission was found on MRI in 17/27 cases, and remission rate was influenced by etiology of cartilage defect but not by age and gender of patients or size and location of defects. The Lysholm-Gillquist score improved from 49.7 to 97.3. After MFX equilibration of signal intensities and growth of the regenerating fibrous cartilage was less pronounced and complete remission was found in only 2/7 cases. In addition, the clinical score improved from 45.5 to 74.2. CONCLUSION: Direct imaging of cartilage with MRI and assessment of clinical scores allowed improved documentation of the outcome after MACI and MFX. MRI showed that MACI is superior to MFX concerning rate of complete remissions and filling of the defect with regenerating tissue. Clinical examinations showed better scores for MACI than for MFX.

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.

Differential effects of nonsteroidal antiinflammatory drugs on the IL-1 altered expression of plasminogen activators and plasminogen activator inhibitor-1 by articular chondrocytes.

OBJECTIVE: To determine the in vitro effects of several nonsteroidal antiinflammatory drugs on the IL-1 altered expression and activity of tPA, uPA and PAI-1 by articular chondrocytes. METHODS: Bovine chondrocytes were cultured in alginate gel beads. Cells were treated with IL-1alpha in the presence or absence of drugs at various concentrations. Expression of mRNA for the plasminogen activators (uPA and tPA) and their inhibitor (PAI-1) were analyzed by RT-PCR-ELISA. The protein content of PAI-1 in culture media was deter mined by ELISA. PA activity was measured by a functional assay. RESULTS: All tested NSAIDs dose dependently inhibited the IL-1 induced mRNA expression of tPA, whereas only indomethacin and tiaprofenic acid were also able to reduce the expression of uPA. Expression of PAI-1 was elevated by IL-1 without an accompanying increase in secreted amounts of the inhibitor. Indomethacin, naproxen and tiaprofenic acid stimulated the release of PAI-1 into culture media, whereas meloxicam also induced expression of PAI-1 above IL-1 stimulated levels. CONCLUSION: In conclusion, our studies indicate that NSAIDs preferentially inhibit tPA expression by bovine articular chondrocytes. By increasing the production of PAI-1 at therapeutical concentrations meloxicam could reduce PA activity, whereas the other NSAIDs tested mainly enhanced the release of this inhibitor from the extracellular matrix. In how far this would affect the enzyme-inhibitor balance within cartilage has to be determined in further studies.

Effects of non-steroidal antiinflammatory drugs and dexamethasone on the activity and expression of matrix metalloproteinase-1, matrix metalloproteinase-3 and tissue inhibitor of metalloproteinases-1 by bovine articular chondrocytes.

OBJECTIVE: To determine the in-vitro effects of several non-steroidal antiinflammatory drugs and the glucocorticoid dexamethasone on the IL-1 altered expression and activity of MMP-1, MMP-3 and TIMP-1 by bovine articular chondrocytes. DESIGN: Bovine chondrocytes were cultured in alginate gel beads. Cells were treated with IL-1alpha in the presence of vehicle or drugs at various concentrations. After 48 h mRNA expression of MMP-1, MMP-3, and of the tissue inhibitor of metalloproteinases (TIMP-1) was analysed by RT-PCR-ELISA. The protein synthesis of TIMP-1 and MMP-3 was determined by immunoprecipitation. The activity of enzymes and inhibitors was measured by functional assays. RESULTS: IL-1 increased the expression and activity of MMPs. In contrast, TIMP activity remained unchanged although TIMP-1 expression was down-regulated. All tested NSAIDs and dexamethasone inhibited collagenase activity induced by IL-1. Transcript levels of MMP-1, however, were only reduced by indomethacin, meloxicam, naproxen and dexamethasone. Proteoglycanase activity was only reduced by indomethacin, meloxicam and dexamethasone. These effects were pre-translational as confirmed by immunoprecipitation. The IL-1 decreased expression of TIMP-1 was further reduced by dexamethasone, 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 NSAIDs. CONCLUSION: Our studies clearly demonstrate that marked differences exist between individual NSAIDs with respect to their ability to modulate the imbalance between proteases and inhibitors during OA and RA, suggesting that the respective modes of action are independent of the inhibition of cyclooxygenases. Due to their co-regulation of MMPs and TIMP(s) glucocorticoids should be carefully studied for their overall effect on ECM proteolysis.

Effects of tetracyclines on the production of matrix metalloproteinases and plasminogen activators as well as of their natural inhibitors, tissue inhibitor of metalloproteinases-1 and plasminogen activator inhibitor-1.

OBJECTIVE: Evaluation of tetracycline effects on the expression of MMP-1, MMP-3, tissue inhibitor(s) of metalloproteinase-1 (TIMP-1), plasminogen activators (PA), and PA inhibitor-1, which are all involved in the ultimate regulation of MMP activity could provide new insight into how tetracyclines achieve their cartilage preserving effects. MATERIALS AND METHODS: We used bovine articular chondrocytes cultured in alginate gel beads for our studies which were initially treated with 10 microM tetracyclines in the presence of IL-1. Only significant effects were studied at additional concentrations. Expression of mRNA was analyzed by RT-PCR-ELISA. The activity of enzymes and TIMP was measured by functional assays; whereas, the level of PAI-1 was determined by ELISA. RESULTS: Treating chondrocytes with IL-1 induced the expression of MMPs and downregulated TIMP-1 but stimulated both the expression of PAs and PAI-1. When tested at 10 microM only minocycline reduced collagenase activity and expression of MMP-1. Further pharmacokinetic analysis revealed IC50 values of 26 microM and 16 microM for the inhibition of collagenase activity and mRNA expression, respectively. Production of MMP-3 was only decreased by tetracycline (IC50 = 45.4 microM). No effects of tetracyclines could be observed on proteoglycan degradation, TIMP activity and the production of PAs, PAI-1, and TIMP-1. CONCLUSIONS: We conclude that the inhibition of MMPs by tetracyclines occurs mainly via down-regulation of the respective gene expression.

Minocycline inhibits the production of inducible nitric oxide synthase in articular chondrocytes.

OBJECTIVE: To determine the in vitro effects of tetracyclines and nonsteroidal antiiflammatory drugs on interleukin 1alpha (IL-1alpha) induced NO production and biosynthesis of inducible NO synthase (iNOS) by articular chondrocytes. METHODS: Bovine chondrocytes were cultured in alginate beads. Cells were treated with IL-lalpha in the presence or absence of drugs at various concentrations. Expression of mRNA for iNOS was analyzed by reverse transcription polymerase chain reaction-ELISA. Protein synthesis of iNOS was determined by immunoprecipitation. NO production was taken as a measure for the activity of the enzyme. RESULTS: Minocycline dose dependently reduced IL-1 stimulated NO production by inhibition of the mRNA expression (IC50 = 69.9 microM) and protein synthesis (IC50 = 37.11 microM) of iNOS. Diclofenac-Na at a concentration of 10 microM only weakly reduced nitrite accumulation and mRNA expression of iNOS. No effects were observed for tetracycline, doxycycline, and meloxicam. CONCLUSION: Inhibition of iNOS in articular chondrocytes may be a new mechanism by which minocycline could exert its beneficial effects in the treatment of joint diseases.

[Drug therapy of arthrosis]. / Medikamentöse Therapie der Arthrose.

Osteoarthritis is one of the most common and economically important chronic diseases amongst adults, especially those of a senior age. There now exists a range of effective medications, which either alone or in combination can alleviate the symptoms of the disease and improve the quality of life. Because these medications are not always sufficiently effective and must sometimes be interrupted due to side effects, a large arsenal of active agents is necessary. Alleviation of pain and inhibition of inflammation are the primary goals of pharmacotherapy, whereby the objective is to return an active or transiently painful, decompensated osteoarthritis to a latent (silent, pain-free) condition. This therapeutic goal can almost always be accomplished by using analgesics, nonsteroidal anti-inflammatory drugs (NSAIDs), or intraarticular injection of glucocorticoids. The main problem in administering NSAIDs is their gastrointestinal toxicity,for which a prophylactic medication (e.g., simultaneous application of misoprostol or switching to a COX-2 selective NSAID) should be considered especially with risk groups. The newly developed COX-2 selective NSAIDs represent a true enrichment of our therapeutic options. The spectrum of indications for COX-2 selective NSAIDs should in the future correspond to that of older NSAID preparations, providing that no as yet unknown and serious side effects come to light from their use. Pharmacological results published until now confirm that a clinically relevant analgesic and/or anti-inflammatory effect is associated with the use of SYSA-DOAs (symptomatic slow acting drugs in osteoarthritis). However, no clinical studies exist which can positively confirm prevention of morphologically recognizable cartilage defects in man, or a slowing down or reversal of any progressively developing joint cartilage destruction by any individual medication. Neither the benefits, risks, pharmaceutical quality, nor composition of Orthokin are known, and for this reason its use can not be recommended. Pharmacotherapy should only be considered as one of the three pillars of a long-term,stage-adjusted, and individually customized therapy, the other two of which are represented by nonpharmacological measures and surgical treatment.

Pharmacological basis for the therapy of pain and inflammation with nonsteroidal anti-inflammatory drugs.

Nonsteroidal anti-inflammatory drugs (NSAIDs) belong to the most frequently used drugs. The discovery of an inducible isoform of cyclo-oxygenase (COX-2) has led to an intensive worldwide search and the introduction of selective COX-2 inhibitors. In this review, recent advances in understanding the mechanism of action of NSAIDs and, in this context, clinical findings on NSAID-induced gastrointestinal side effects are summarized. This knowledge is important for the effective treatment of pain and inflammation, as well as for preventing serious and sometimes lethal gastrointestinal side effects.

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.

The effect of continuously applied cyclic mechanical loading on the fibronectin metabolism of articular cartilage explants.

Articular cartilage serves primarily as a load-bearing material able to regulate its own metabolic activity in response to the mechanical stimuli applied. Fibronectin plays a critical role in the organization and function of the cartilage extracellular matrix. The purpose of this study was to investigate systematically the effect of load magnitude, frequency and duration of loading on the synthesis, content and release of fibronectin and proteins by mature bovine articular cartilage explants using a novel mechanical loading system. Increasing the load magnitude, as well as the duration of loading, inhibited the synthesis and content of fibronectin and proteins; the fibronectin synthesis was more specifically affected than the overall protein synthesis indicating that fibronectin is more responsive to pressure than synthesis of other proteins. Reducing the load frequency did not modulate the inhibitory effect of a given cyclic stress on synthesis and content of fibronectin and proteins even though explants were more compressed. The release of endogenous fibronectin was significantly reduced independent of the applied loading protocols when compared with unloaded controls. This study demonstrates that the magnitude and the duration of loading influences the degree of inhibition of fibronectin and protein synthesis, while loaded explants possess an elevated but limited capacity to bind fibronectin. Compared with other studies, our present results show that the applied load function in particular has a profound effect on the metabolism of chondrocytes.

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 proteoglycan metabolism of mature bovine articular cartilage explants superimposed to continuously applied cyclic mechanical loading.

This study describes the effect of load magnitude, frequency and duration on proteoglycan (PG) biosynthesis and loss in mature bovine articular cartilage explants. Cultured full thickness cartilage discs were subjected to a continuously applied, uniaxial compressive cyclic load. The loads were applied using a sinusoidal waveform of 0.001, 0.01, 0.1 or 0.5 Hz-frequency and a peak stress of 0.1, 1.0, 2.5, or 5.0 MPa for a period of 1, 3 or 6 days. Increasing the load magnitude, as well as the duration of loading, reduced the PG biosynthesis. Reducing the load frequency abolished the inhibitory effect of a given load magnitude on PG biosynthesis, even though explants were more compressed. Increasing the load magnitude stimulated the release of newly synthesized PGs from explants, whereas an elevated duration of loading significantly decreased the release of endogenous PGs. Explants loaded for 1 or 3 days were viable as determined biochemically, whereas 6 days of loading resulted in a slightly diminished viability of explants. This study demonstrates that the duration and intensity of loading influences the inhibition of PG biosynthesis, while PG loss is only modulated by the magnitude and duration of loading.

A computer-controlled mechanical culture system for biological testing of articular cartilage explants.

A computer-controlled mechanical culture system was designed to investigate the interaction between mechanical stimuli and the metabolism of articular cartilage. The main features of this system include the following capabilities: (1) Accurately controlled static, permanent cyclic or intermittent cyclic mechanical loads can be applied; (2) a great variety of different functions to load cartilage explants can be chosen; (3) a wide range of selectable forces (1.0-500 N) and frequencies (up to 5.0 Hz) can be used to load explants; (4) cartilage explants can be cultured and loaded within a standard CO2-incubator for extended time periods; (5) similar culture conditions are provided within the loading chambers as in standard tissue culture plates; (6) simultaneously the applied load and the resulting displacement of specimens is measured, and (7) the load chambers are biocompatible, sterilizable, and non-corrosive. We expect that the newly designed mechanical culture system will increase our understanding on the regulatory role of direct mechanical pressure on the metabolic activities of chondrocytes.

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.