Cartilage was obtained from eight matched knee (tibiofemoral and femoropatellar) and ankle (talocrural) joints of five different donors (both left and right from donors 14, 22, and 38 years of age, and left only from donors 31 and 45 years of age) within 24 hours of death. All cartilage was graded as normal by the macroscopic visual Collins' scale and the histological Mankin scale. Cylindrical disks of cartilage were harvested from 10 sites within the tibiofemoral and femoropatellar joint surfaces and four sites within the talocrural joint, and uniaxial confined compression measurements were performed to quantify a spectrum of physical properties including the equilibrium modulus, hydraulic permeability, dynamic stiffness, streaming potential, electrokinetic coupling coefficient, and electrical conductivity. Matched specimens from the same 14 sites were used for complementary measurements of biochemical composition and molecular interaction, including water content, hypotonic swelling behavior, and sulfated glycosaminoglycan and collagen contents. In comparison of the top 1-mm slices of talar cartilage with the top 1-mm of tibiofemoral cartilage, the talar cartilage appeared denser with a higher sulfated glycosaminoglycan content, lower water content, higher equilibrium modulus and dynamic stiffness, and lower hydraulic permeability. The equilibrium modulus increased with increasing sulfated glycosaminoglycans per wet weight and decreased with increasing water content for all joint surfaces. Multiple linear regression showed that greater than 80% of the variation in the equilibrium modulus could be accounted for by variations in the biochemical parameters (water content, sulfated glycosaminoglycans/wet weight, and hydroxyproline content/wet weight) for each joint surface. Nonhomogeneous depth-dependent changes in the physical properties and biochemical composition of full-thickness distal femoral cartilage were consistent with previous reports. Since the compressive deformation of cartilage during cyclic loading is confined to the more superficial regions, the differences in properties of the upper regions of the talar compared with tibiofemoral or femoropatellar cartilage may be important in the etiology of osteoarthritis.

Objective. To assess the presence of fibroblast collagenase (MMP-l), neutrophil collagenase (MMP-8), and collagenase 3 (MMP-13) in osteoarthritic (OA) cartilage, with particular emphasis on areas of macroscopic cartilage erosion.Methods. Messenger RNA (mRNA) levels were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and Northern blot analysis.Results. MMP-1 and MMP-13 were expressed at higher levels by OA chondrocytes than by normal chondrocytes. In addition, mRNA for MMP-8 was present in OA cartilage but not normal cartilage by PCR and Northern blot analyses. Chondrocytes from areas surrounding the OA lesion expressed greater quantities of MMP-1 and MMP-13 compared with normal chondrocytes, suggesting local modulation by mechanical and inflammatory factors. Tumor necrosis factor a stimulated the expression of all 3 collagenases. Retinoic acid, an agent which induces autodigestion of cartilage in vitro, stimulated only the expression of MMP-13.Conclusion. These findings suggest a key role of MMP-13 and MMP-8, as well as MMP-1 in osteoarthritis.The matrix metalloproteinase (MMP) family of enzymes consists of at least 15 distinct entities, including

Information on the prevalence and extent of degenerative morphological changes (DMC) in the joints of the lower extremity, including foot joints is sparse. In the present study, the first and fifth metatarsalphalangeal (MTP), transverse tarsal, subtalar, talocrural, knee and hip joints of 50 cadavers were examined grossly and graded on a five-point scale for signs of DMC. Selected samples were examined histologically. Our results confirm clinical findings that severe DMC in foot joints are uncommon except in the first MTP joint where the plantar aspect is most affected. The knee joint displayed the most numerous and severe signs of DMC followed by the first MTP joint. The hip, talocrural, subtalar and transverse tarsal joints displayed comparatively moderate levels of DMC while the fifth MTP was rarely affected. The only joint to display significantly greater levels of DMC on the distal side of the joint as compared with the proximal side, when a difference was present, was the hip. There were significantly greater levels of DMC on the medial aspect of two or more joints within an extremity than on the lateral aspect. Radiographs often showed few or no signs of DMC even when erosion down to subchondral bone was observed upon gross examination.

Objective. The receptor for advanced glycation end products (RAGE) binds multiple ligands, including S100 proteins, high mobility group box chromosomal protein 1 (HMGB-1), and AGEs, all of which are present in articular cartilage. Stimulation of RAGE signaling can lead to MAP kinase activation and increased NF-B activity. The objective of the present study was to determine if chondrocytes express functional RAGE.Methods. The presence of chondrocyte RAGE was analyzed by immunohistochemistry using normal and osteoarthritic (OA) cartilage from young and old monkeys and humans, immunoblotting of chondrocyte lysates and human cartilage extracts, and reverse transcription-polymerase chain reaction (RT-PCR) analysis of RNA from chondrocytes treated with interleukin-1 (IL-1) and fibronectin fragments. RAGE signaling was evaluated by stimulating chondrocytes with S100B and HMGB-1 and analyzing for activation of the ERK MAP kinase and NF-B. The ability of S100B and HMGB-1 to stimulate matrix metalloproteinase 13 (MMP-13) production was also assessed. A pull-down assay using biotin-labeled S100B was used to demonstrate binding to RAGE.Results. RAGE was detected in sections of monkey knee cartilage and human knee and ankle cartilage. Increased immunostaining for RAGE was noted in cartilage from older adult monkeys and humans and was further increased in OA tissue. RAGE was also detected by immunoblotting and by RT-PCR, where IL-1␤ and fibronectin fragments were found to stimulate RAGE expression. Stimulation of chondrocytes with S100B or HMGB-1 increased phosphorylation of the ERK MAP kinase and the p65 subunit of NF-B and increased the production of MMP-13. This signaling was inhibited in cells pretreated with soluble RAGE, and S100B was shown to bind to chondrocyte RAGE.Conclusion. Articular chondrocytes express functional RAGE. The increase in RAGE noted in OA cartilage and the ability of RAGE ligands to stimulate chondrocyte MAP kinase and NF-B activity and to stimulate MMP-13 production suggests that chondrocyte RAGE signaling could play a role in OA.

Objective. Traumatic joint injury leads to an increased risk of osteoarthritis (OA), but the progression to OA is not well understood. We undertook this study to measure aspects of proteoglycan (PG) degradation after in vitro injurious mechanical compression, including up-regulation of enzymatic degradative expression and cytokine-stimulated degradation.Methods. Articular cartilage tissue explants were obtained from newborn bovine femoropatellar groove and from adult normal human donor knee and ankle tissue. Following injurious compression of the cartilage, matrix metalloproteinase 3 (MMP-3) and MMP-13 messenger RNA (mRNA) expression levels were measured by Northern analysis, and PG loss to the medium after cartilage injury was measured in the presence and absence of added exogenous cytokine (interleukin-1␣ [IL-1␣] or tumor necrosis factor ␣ [TNF␣]).Results. During the first 24 hours after injury in bovine cartilage, MMP-3 mRNA levels increased 10-fold over the levels in control cartilage (n ‫؍‬ 3 experiments), whereas MMP-13 mRNA levels were unchanged. PG loss was significantly increased after injury, but only by 2% of the total PG content and only for the first 3 days following injury. However, compared with injury alone or cytokine treatment alone, treatment of injured tissue with either 1 ng/ml IL-1␣ or 100 ng/ml TNF␣ caused marked increases in PG loss (35% and 54%, respectively, of the total cartilage PG content). These interactions between cytokine treatment and injury were statistically significant. In human knee cartilage, the interaction was also significant for both IL-1␣ and TNF␣, although the magnitude of increase in PG loss was lower than that in bovine cartilage. In contrast, in human ankle cartilage, there was no significant interaction between injury and IL-1␣.Conclusion. The cytokines IL-1␣ and TNF␣ can cause a synergistic loss of PG from mechanically injured bovine and human cartilage. By attempting to incorporate interactions with other joint tissues that may be sources of cytokines, in vitro models of mechanical cartilage injury may explain aspects of the interactions between mechanical forces and degradative pathways which lead to OA progression.

Variations among joints in the initiation and progression of degeneration may be explained, in part, by metabolic, biochemical and biomechanical differences. Compared to the cartilage in the knee joint, ankle cartilage has a higher content of proteoglycans and water, as well as an increased rate of proteoglycan turnover and synthesis, all of which are responsible for its increased stiffness and reduced permeability. Chondrocytes within ankle cartilage have a decreased response to catabolic factors such as interleukin-1 and fibronectin fragments, compared to the chondrocytes of knee cartilage. Moreover, in response to damage, ankle chondrocytes synthesize proteoglycans at a higher rate than that found in knee cartilage chondrocytes, which suggests a greater capacity for repair. In addition to the cartilages of the two joints, the underlying bones also respond differently to degenerative changes. Taken together, these metabolic, biochemical and biomechanical differences may provide protection to the ankle.

Objective. To determine whether oxidative damage to cartilage proteins can be detected in aging and osteoarthritic (OA) cartilage, and to correlate the results with the local production of interleukin-1␤ (IL-1␤) and the responsiveness of isolated chondrocytes to stimulation with insulin-like growth factor 1 (IGF-1).Methods. The presence of nitrotyrosine was used as a measure of oxidative damage. Histologic sections of knee articular cartilage, obtained from young adult and old adult cynomolgus monkeys, which develop agerelated, naturally occurring OA, were evaluated. Each cartilage section was graded histologically on a scale of 0-7 for the presence of OA-like changes, and serial sections were immunostained using antibodies to nitrotyrosine and IL-1␤. Chondrocytes isolated and cultured from cartilage adjacent to the sections used for immunostaining were tested for their response to IGF-1 stimulation by measuring sulfate incorporation in alginate cultures. For comparison with the monkey tissues, cartilage sections from human tissue donors and from tissue removed at the time of OA-related joint replacement surgery were also immunostained for nitrotyrosine and IL-1␤.Results. The presence of nitrotyrosine was associated with aging and with the development of OA in cartilage samples from both monkeys and humans. All sections that were highly positive for IL-1␤ also showed staining for nitrotyrosine. However, in a few sections from older adult monkeys and humans, nitrotyrosine was present but IL-1␤ was absent, suggesting that some age-related oxidative damage is independent of IL-1␤. In chondrocytes that were isolated from monkey cartilage positive for nitrotyrosine or IL-1␤, the response to stimulation with IGF-1 was significantly reduced. In some samples from older adult monkeys, IGF-1 resistance was seen in cells isolated from tissue that did not stain for nitrotyrosine or IL-1␤.Conclusion. Oxidative damage due to the concomitant overproduction of nitric oxide and other reactive oxygen species is present in both aging and OA cartilage. This damage can contribute to the resistance of chondrocytes to IGF-1 stimulation, but it is unlikely to be the sole cause of IGF-1 resistance in these chondrocytes.

Recurrent haemarthrosis results in chronic synovitis and destructive arthropathy. The long-term effect of a single haemorrhage is not known. To investigate the histopathological changes following a single, but major joint haemorrhage, an animal model of massive haemarthrosis without mechanical trauma was developed and is described in this manuscript. The knee joint capsule of mice deficient in coagulation factor VIII or IX and non-haemophilic wild type mice was punctured to induce a one time, but massive haemorrhage. The single joint puncture resulted in acute haemarthrosis in both types of haemophilic mice but not in wild type mice. Subsequent to injury, the changes in the knee joints were analysed using gross, histological and radiographic assessments and compared with the uninjured knee. In addition, a novel imaging modality, micro-computed tomography, was used to document the structural damage to the joint. Our results indicate that the long-term changes classically observed in patients with advanced haemophilic arthropathy are evident following a single massive haemarthrosis. This model will allow a thorough investigation of the pathobiology of blood-induced joint disease and will be useful to test the efficacy of innovative therapeutic strategies to prevent haemophilic synovitis and arthropathy.