Biomechanical, histological and immunohistological studies of patellar cartilage in an ovine model of osteoarthritis induced by lateral meniscectomy.
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OBJECTIVE: To evaluate the biomechanical, histological and immunohistochemical changes induced in patellar articular cartilage (AC) in ovine stifle joints 3 months after bilateral lateral meniscectomy, a procedure known to induce experimental osteoarthritis (OA) in the femoro-tibial joint (FTJ). METHODOLOGY: Fifteen mature adult Merino female sheep were used in this study. Ten were subjected to bilateral-lateral meniscectomy, while the remaining five were used as 'non-operated controls' (NOC). All animals were killed 3 months post-surgery. Topographical biomechanical indentation tests were performed on each patellae using a UMIS-2000 micro-indentation system. Initial load, relaxed and unload shear moduli were determined using an elastic analytical model, while the permeability was assessed by comparing the indentation response to a simulated indentation test conducted using a poroelastic finite element model. Immunohistochemical, normal and polarized histological studies were performed on each specimen after biomechanical testing. RESULTS: Patellar AC from meniscectomized joints exhibited an overall decrease in initial (-34%), relaxed (-32%) and unload shear modulus (-22%), and an increase in the permeability (+72%) relative to NOC cartilage (P< 0.01). The most significant differences in mechanical properties occurred on the lateral and central aspects of the patellae. There were no significant histological difference in staining between sections from NOC and meniscectomized joint AC using Toluidine Blue, a dye which binds to proteoglycans. However immunohistochemical staining with monoclonal antibody MAb 3B3(-), a putative marker of early OA change in PGs, demonstrated increased binding in the lateral and central regions of patellar sections from meniscectomized joints relative to the same regions of NOC AC. Moreover polarized light microscopy of Picro Sirius red stained sections revealed a significant decrease in birefringence intensity in the superficial-middle zones of the lateral and central regions of the patellar cartilage derived from the meniscectomized joints. CONCLUSION: This study has demonstrated that lateral meniscectomy is a procedure which was known to induce classical OA like changes in AC and subchondral bone of the FTJ also produced an early pathological response in the patellar AC. (+info)
Sulfation of chondroitin sulfate in human articular cartilage. The effect of age, topographical position, and zone of cartilage on tissue composition.
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The chondroitin ABC lyase digestion products of normal human femoral condyle articular cartilage and of purified aggrecan were analyzed for their mono- and nonsulfated disaccharide composition. Changes in the total tissue chemistry were most pronounced during the period from birth to 20 years of age, when the -[GlcAbeta,3GalNAc6]- disaccharide content increased from approximately 50% to 85% of the total disaccharide content and there was a concomitant decrease in the content of the 4-sulfated disaccharide. In general, the disaccharide content of the deeper layers of immature cartilage were richer in the 4-sulfated residue than the upper regions of the tissue. As the tissue aged and decreased in thickness, the disaccharide composition became more evenly 6-sulfated. The newly synthesized chondroitin sulfate chains had a similar composition to the endogenous chains and also underwent the same age and zonal changes. The monoclonal antisera 3B3(+) and 2B6(+) were used to immunolocalize the unsaturated 6- and 4-sulfated residues generated at the reducing termini of the chondroitin sulfate chains by digestion with chondroitin ABC lyase, and these analyses indicated that the sulfation pattern at this position did not necessarily reflect the internal disaccharide composition of the chains. In summary, the sulfation pattern of chondroitin sulfate disaccharides from human normal articular cartilage varies with the age of the specimen, the position (topography) on the joint surface, and the zone of cartilage analyzed. Furthermore, these changes in composition are a consequence of both extracellular, post-translational processing of the core protein of aggrecan and changes in the sulfotransferase activity of the chondrocyte. (+info)
Gene therapy of arthritis.
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Genes encoding anti-arthritic products can be transferred to intra-or extraarticular sites where their expression suppresses various aspects of the pathophysiology of arthritis. A variety of viral and non-viral vectors can be used for the in vivo or ex vivo delivery of such genes. Promising pre-clinical data have resulted from the application of these strategies in several animal models of disease. Genes showing efficacy in this way include these encoding interleukin (IL) -1Ra, IL-1sR, TNFsR, transforming growth factor beta (TGF-beta), IL-13, Fas L, IL- 10 and vIL-10. Two human arthritis gene therapy protocols are underway in the USA and Germany. Both studies involve the ex vivo transfer of an IL-1Ra cDNA to the metacarpophalangeal joints of patients with rheumatoid arthritis. Progress in developing gene treatments for arthritis has been rapid, and permits optimism about their ability eventually to improve the treatment of this group of diseases. (+info)
Increased vulnerability of postarthritic cartilage to a second arthritic insult: accelerated MMP activity in a flare up of arthritis.
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OBJECTIVE: Murine antigen induced arthritis (AIA) is a chronic, smouldering inflammation. Flares of arthritis can be induced by antigen rechallenge or exposure to inflammatory mediators like interleukin 1 (IL1). These flares are characterised by a fast and marked proteoglycan (PG) depletion if compared with the initial arthritis. This study investigated the involvement of metalloproteinases in both the initial and the flare phase of arthritis. METHODS: Murine AIA was induced and a flare up of arthritis was induced by injection of 10 ng of IL1beta. Messenger RNA levels of MMP-1 and -3 were studied by RT-PCR. MMP activity in cartilage, during both primary AIA as well as the flare up of arthritis, was studied by immunodetection of MMP specific neoepitopes in aggrecan (VDIPEN). Cartilage just before flare induction was analysed for presence of MMPs at the mRNA level as well as at the protein level by zymography. RESULTS: At the onset of AIA, a fast upregulation of mRNA for stromelysin and collagenase was noted. However, no VDIPEN epitopes were detected during this early phase of arthritis. They appeared when PG depletion was severe at day 7 of arthritis and disappeared when cartilage was repaired. IL1 injection into a knee joint at week 4 of AIA caused a flare up of arthritis, coinciding with a fast and marked PG degradation. This degradation was characterised by accelerated expression of VDIPEN epitopes if compared with the expression in primary AIA. Analysis of cartilage at week 4 of AIA showed still increased mRNA levels of MMP-1 and -3. Moreover, increased levels of latent MMPs were present as well, as APMA activation induced profound VDIPEN epitope. In vitro exposure to IL1 did show increased PG breakdown but no VDIPEN expression, suggesting that factors in addition to IL1 are needed to cause the in vivo VDIPEN expression. CONCLUSIONS: The fast and marked PG depletion seen in a flare up of AIA coincides with accelarated expression of MMP induced neoepitopes compared with expression during primary AIA. This accelerated expression is probably linked to increased levels of latent enzyme, which were found to be present in the cartilage before induction of a flare up. (+info)
In situ zymographic localisation of type II collagen degrading activity in osteoarthritic human articular cartilage.
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OBJECTIVES: Chondrocytic matrix metalloproteinases (MMPs) are believed to be important in osteoarthritic cartilage degradation. The cartilage lesion of osteoarthritis (OA) is focal and often progressive. During its development chondrocytes differentially up and down regulate production of mRNA for individual MMPs. This observation has potential implications for understanding the disease processes that lead to progressive cartilage loss in OA and designing appropriate targeted treatment. The complex regulation of MMP mediated effects means there is a pressing need to establish whether visualisation of MMP mRNA or protein equates to enzyme activity. The technique of in situ zymography (ISZ) offers a way of examining diseased human tissue for in vivo production of an excess of degrading enzyme over inhibitor. The primary objective of this study was to assess, and if positive follow, collagen II degrading activity in cartilage during development of the OA lesion. A secondary objective was to assess whether there was any correlation between sites of collagen II degrading activity and expression of the collagenase (MMP-13), recently implicated in type II collagen degredation in this lesion. METHODS: Biopsied human normal and osteoarthritic cartilage, showing various degrees of damage, was examined by in situ zymography, with and without enzyme inhibitors, to establish sites of type II collagenase activity. Paired samples were probed for MMP-13 mRNA using 35S-labelled oligonucleotide probes. Comparative analyses were performed. RESULTS: In situ zymography showed collagen II degrading activity over chondrocytes only in osteoarthritic cartilage. Distribution and amount varied with the extent of cartilage damage and position of chondrocytes, being greatest in deep cartilage and in cartilage lesions where fissuring was occurring. The enzyme causing the degradation behaved as a matrix metalloproteinase. MMP-13 mRNA expression codistributed with the type II collagenase activity. CONCLUSION: In OA, chondrocytes can degrade type II collagen. The type II collagen degrading activity varies in site and amount as the cartilage lesion progresses and throughout codistributes with MMP-13 mRNA expression. (+info)
The 'instantaneous' compressive modulus of human articular cartilage in joints of the lower limb.
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METHODS: The instantaneous compressive modulus of articular cartilage was surveyed in 11 sets of human lower limb joints obtained from the ipsilateral side. The average modulus for the entire joint surface of each joint and the topographical variations in the modulus within each joint were examined for all 11 sets, and subjected to statistical analysis. RESULTS: Within each set of joints (hip, knee and ankle), the ankle always had a significantly greater mean compressive modulus than the hip and knee (P < 0.001-P < 0.05). In seven sets of joints, there was no significant difference between the mean compressive moduli of the knee and hip joints. In three sets of joints, the compressive modulus of the knee was significantly greater than that of the hip (P < 0.001-P < 0.01), while in only one set of joints was the compressive modulus of the hip significantly greater than that of the knee (P < 0.01). CONCLUSION: The topographical variations in the cartilage instantaneous compressive modulus over the surfaces of the lower limb joints were matched by differences in the stresses occurring in different areas of each joint. The results of the present study corroborate previous findings and show that the site-specific stresses and corresponding values of the instantaneous cartilage compressive modulus over the surfaces of lower limb joints were correlated (r = 0.82 at P < 0.01), thus adding credence to the conditioning hypothesis of cartilage by prevalent stress. (+info)
Destruction of articular cartilage by alpha 2 macroglobulin elastase complexes: role in rheumatoid arthritis.
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OBJECTIVE: Neutrophil elastase accounts for the ability of some fresh rheumatoid synovial fluids to degrade cartilage matrix in vitro. The aim of this study was to determine if enzyme activity could result from depletion of synovial fluid inhibitors or protection of the enzyme from inhibition. METHODS: The ability of synovial fluids to inhibit porcine pancreatic elastase was investigated together with chemical pretreatments capable of inactivating alpha 1 protease inhibitor (alpha 1PI) or preventing formation of alpha 2 macroglobulin (alpha 2M) elastase complexes. Subsequently, complexes of human neutrophil elastase with alpha 2M were prepared and applied to frozen sections of cartilage. Proteoglycan loss was quantified by alcian blue staining and scanning and integrating microdensitometry. Parallel studies were carried out using a low molecular weight chromogenic elastase substrate. The effects of alpha 1PI and SF on these systems were investigated. Finally, synovial fluids were subjected to gel filtration and the fractions assayed for elastase activity. High molecular weight fractions were pooled, concentrated, and tested for their ability to degrade cartilage sections. RESULTS: All synovial fluids reduced the activity of porcine pancreatic elastase, the inhibition mainly being attributable to alpha 1PI, whereas remaining activity resulted from complexes of elastase with alpha 2M. Complexes of human neutrophil elastase with alpha 2M were shown to cause proteoglycan degradation in frozen sections of human articular cartilage. Alpha 1PI prevented alpha 2M elastase complexes from degrading cartilage but not the chromogenic substrate. The data suggested that alpha 1PI does not inhibit elastase bound to alpha 2M but sterically hinders the complex. However, only one of five synovial fluids was able to completely block the actions of alpha 2M elastase complexes against cartilage. Gel filtration of rheumatoid synovial fluids showed elastase and cartilage degrading activity to be associated with fractions that contained alpha 2M, and not with fractions expected to contain free enzyme. CONCLUSIONS: The data suggest that synovial fluid alpha 2M elastase complexes can degrade cartilage matrix in rheumatoid arthritis. (+info)
Bone scintigraphy in chronic knee pain: comparison with magnetic resonance imaging.
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OBJECTIVE: To compare increased bone uptake of 99Tcm-MDP and magnetic resonance (MR) detected subchondral lesions, osteophytes, and cartilage defects in the knee in middle aged people with long-standing knee pain. METHODS: Fifty eight people (aged 41-58 years, mean 50) with chronic knee pain, with or without radiographic knee osteoarthritis, were examined with bone scintigraphy. The pattern and the grade of increased bone uptake was assessed. On the same day, a MR examination on a 1.0 T imager was performed. The presence and the grade of subchondral lesions, osteophytes, and cartilage defects were registered. RESULTS: The kappa values describing the correlation between increased bone uptake and MR detected subchondral lesions varied between 0.79 and 0.49, and between increased bone uptake and MR detected osteophytes or cartilage defects the values were < 0.54. The kappa values describing the correlation between the grade of bone uptake and the grade of the different MR findings was < 0.57. CONCLUSIONS: Good agreement was found between increased bone uptake and MR detected subchondral lesion. The agreement between increased bone uptake and osteophytes or cartilage defects was in general poor as well as the agreement between the grade of bone uptake and the grade of the MR findings. (+info)