Ada A. Cole

Prevalence of degenerative morphological changes in the joints of the lower extremity

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.

Differential patterns of response to doxycycline and transforming growth factor β1 in the down-regulation of collagenases in osteoarthritic and normal human chondrocytes

OBJECTIVE To investigate the ability of doxycycline, transforming growth factor beta1 (TGFbeta1), and phorbol myristate acetate (PMA) to modulate collagenase synthesis in osteoarthritic (OA) chondrocytes. METHODS Levels of fibroblast collagenase (matrix metalloproteinase 1 [MMP-1]), neutrophil collagenase (MMP-8), and collagenase 3 (MMP-13) proteins and messenger RNA (mRNA) were measured in chondrocytes isolated from involved and uninvolved areas of OA cartilage and from normal human chondrocytes, after treatment with doxycycline, TGFbeta1, and PMA. RESULTS Chondrocytes isolated from cartilage immediately adjacent to the OA lesion had, on average, 1.8-3.9-fold higher basal levels of MMP mRNA. These cells down-regulated collagenase proteins and mRNA upon incubation with TGFbeta1. In contrast, chondrocytes from areas located more distant from the macroscopic lesion increased MMP-13 mRNA, while MMP-1 and MMP-8 decreased after stimulation with TGFbeta1. Discoordinate regulation was observed after stimulation with PMA, with an increase in MMP-1 and MMP-8 but a decrease in MMP-13. Incubation of OA chondrocytes with doxycycline (1-10 microg/ml), at pharmacologically achievable levels, decreased levels of mRNA of all 3 collagenases, but not G3PDH. In addition, doxycycline inhibited the increase in mRNA for these enzymes in normal chondrocytes stimulated with tumor necrosis factor alpha. CONCLUSION These findings suggest that regulation of MMP-1, MMP-8, and MMP-13 in OA chondrocytes, although mediated by differing pathways, can be decreased by treatment with doxycycline at low concentrations. Our data provide a rationale for the use of doxycycline in the treatment of OA.

Molecular basis of osteoarthritis: biomechanical aspects.

Abstract. The unique biomechanical properties of healthy cartilage ensure that articular cartilage is able to transmit force between the joints while maintaining almost friction-free limb movement. In osteoarthritis, the biomechanical properties are compromised, but we still do not understood whether this precedes the onset of the disease or is a result of it. This review focuses on the physical changes to cartilage with age, disease, and mechanical loading, with specific reference to the increased collagen cross-linking that occurs with age (nonenzymatic glycation), and the response of chondrocytes to physiological and pathological loads. In addition, the biomechanical properties and matrix biosynthesis of cartilage from various joint surfaces of the knee and ankle are compared to elucidate reasons why the ankle is less affected by progressive osteoarthritis than the knee.

Human knee and ankle cartilage explants: catabolic differences

The prevalence of osteoarthritis (OA) is lower in some joints, i.e., the ankle, than in the knee. We have compared the cartilages from these two joints of the same limb in adult donors (matched pairs). Our data to date suggest that there are metabolic, biochemical and biomechanical differences between the cartilages of the two joints. The current study has focused on extending the metabolic studies comparing the response of chondrocytes to Interleukin‐1β (IL‐1β) and osteogenic protein 1 (OP‐1) by analyzing changes in sulfate incorporation into glycosaminoglycans (GAGs) as a measure of proteoglycan (PG) synthesis. Human adult chondrocytes from normal knees (tibiofemoral) and ankles (talocrural) joints cultured as explants both responded to IL‐1β after 72 h by decreasing PG synthesis; however, the IC50 for the knee chondrocytes was 6.2 pg/ml, while that for the ankle was 35 pg/ml. When the explants were incubated for 72 h with IL‐1β and allowed to rebound without IL‐1β, synthesis of PG was significantly elevated by ankle chondrocytes within five days; knee chondrocytes were unable to significantly increase synthesis even after eight days. However, in both knee and ankle, application of OP‐1 enhanced PG synthesis in the rebound phase. In response to IL‐1, an upregulation of proteinase activity was detectable by an increase in the neoepitopes proteolytically‐generated by both aggrecanase and matrix metalloproteinases (MMPs), in the deep zone of the knee cartilage. Stromelysin and collagenase were upregulated as well. The data emerging from these studies confirm that the ankle is less responsive to catabolic stimulation and more responsive to anabolic stimulation following IL‐1 removal. These differences in metabolic activity between the cartilages of the two joints could in part help to explain their differences in susceptibility to OA.

Experimental haemophilic arthropathy in a mouse model of a massive haemarthrosis: gross, radiological and histological changes.

Summary.  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.

Horizontally oriented clusters of multiple chondrons in the superficial zone of ankle, but not knee articular cartilage.

Osteoarthritis is a progressive disease that is initiated at the surface of articular cartilage and proceeds to destroy the entire depth of the cartilage. The prevalence of osteoarthritis varies in different joints; e.g., the ankle joint has a very low prevalence of the disease compared to the knee joint. To better understand any inherent differences between the articular cartilage of the ankle and that of the knee that would account for the difference in occurrence of osteoarthritis, studies were undertaken to examine differences between the superficial zones in these two joint cartilages obtained from human donors. Chondrocytes in the superficial zones of the normal ankle (talocrural) and the normal knee (tibiofemoral) joints were identified with a monoclonal antibody specific for the superficial zone protein (SZP). When the chondrocytes from both joints were compared in serial horizontal sections, the chondrocytes in the superficial zone of the knee cartilage were seen either as isolated single cells or as doublets. However, the chondrocytes within the superficial zone of normal ankle cartilage were arranged in planar clusters containing multiple chondrons composed of 2–13 cells. There were no detectable differences in the chondrocyte clusters in the superficial zone of the ankle with respect to age, gender, or site on the cartilage surface. Adjacent to a lesion in an ankle joint with degenerative changes, the clusters were larger, containing up to 22 chondrocytes. This is the first report documenting the presence of multiple chondrons in the superficial zone of normal human adult articular cartilage. Anat Rec 266:241–248, 2002.

Gene Expression by Human Articular Chondrocytes Cultured in Alginate Beads

Culture of articular chondrocytes in alginate beads offers several advantages over culture in monolayer; cells retain their phenotype for 8 months or longer. Earlier studies of chondrocytes cultured in alginate concentrated on collagen and proteoglycan synthesis. However, gene expression by in situ hybridization (ISH) has not been investigated. The purposes of the present study on human chondrocytes were (a) to modify the ISH procedure for the alginate beads to examine the mRNA expression of α1 (II) procollagen, aggrecan, and two matrix metalloproteinases (MMP-3 and MMP-8) thought to be involved in cartilage matrix degradation, and (b) to compare expression in cultured chondrocytes with that in chondrocytes of intact human cartilage. The modifications made for ISH include the presence of CaCl2 and BaCl2 in the fixation and washing steps and exclusion of cetyl pyridinium chloride. By ISH we show that aggrecan, MMP-3, and MMP-8 are continuously expressed during 8 months of culture. The α1 (II) procollagen gene is expressed only during the first 2 months of culture and after 3 months its expression is undetectable, which is consistent with its absence in adult articular cartilage. By Western blotting, Type II collagen protein had been synthesized and deposited in both the cell-associated and further-removed matrix compartments at 7 and 14 days of culture. These data indicate that chondrocytes cultured in alginate beads could be preserved for immunohistochemistry and ISH and that culture of human chondrocytes in alginate beads may serve as a good model for studying cartilage-specific phenotype as well as factors that influence cartilage matrix turnover.

Expression of anchorin CII (cartilage annexin V) in human young, normal adult, and osteoarthritic cartilage.

In its tissue-specific function as a collagen receptor of chondrocytes, cartilage annexin V (anchorin CII) occupies a key position in the organization of the cell-extracellular matrix (ECM) junction for the tissue. The general role of annexin V (Anx V) in other tissues suggests involvement in cellular secretory processes and in regulation of apoptosis. Immunohistochemical analysis of Anx V in growth plate cartilage, confirmed by in situ hybridization, suggests that Anx V is prominently expressed and forms a major constituent of growth plate chondrocytes. Anx V epitopes are also located in the pericellular matrix of hypertrophic cartilage. In adult articular cartilage the expression is downregulated, with the highest levels of immunostaining found in the upper third of the articular cartilage layers and almost no antigen found in the deep layers. Osteoarthritic (OA) cartilage is characterized by a significant upregulation of message and protein throughout the entire depth of the tissue, an accumulation of cytoplasmic annexin V epitopes, and a release of epitopes into the pericellular and interterritorial matrix, in part co-localized with granular structures. Therefore, Anx V expression and tissue distribution may serve as a histological marker for metabolic alterations and for changes in the cellular phenotype associated with OA.

Tumor necrosis factor-? induced DNA cleavage in human articular chondrocytes may involve multiple endonucleolytic activities during apoptosis

Apoptosis has been documented in chondrocytes both in the growth plates of young, healthy cartilages and in osteoarthritic cartilages; little, however, is known about apoptosis in chondrocytes of normal adult articular cartilage. For the current study, apoptosis in adult chondrocytes was evaluated by labeling DNA fragments using the ISEL in situ end labeling of 3′‐recessed strand breaks) or TUNEL (5′‐recessed or blunt‐ended strand breaks with terminal deoxynucleotidyl transferase‐mediated nick end labeling) techniques in primary cultures of chondrocytes in monolayer. Apoptosis was induced in the chondrocytes by either Tumor Necrosis Factor α (TNFα), Interleukin 1‐β (IL‐1β), or anti‐Fas antibody but only after 48 hours in culture. At 4 and 24 hours, there was no detectable DNA fragmentation. With TNFα, IL1β, and anti‐Fas antibody, chondrocytes show evidence of at least two types of DNA strand breaks within the same cell (as assessed by simultaneous labeling with ISEL and TUNEL). Therefore, some pathways leading to apoptosis in chondrocytes appear to involve more than one type of endonuclease activity. When the chondrocytes were cultured as explants with the articular matrix intact (ex vivo), neither IL‐1β, TNFα, the anti‐Fas antibody, nor fibronectin fragments were able to induce apoptosis in the chondrocytes. In normal human adult cartilage that was untreated and uncultured (in situ), DNA fragmentation was undetectable; however, a significant number of chondrocytes in osteoarthritic cartilage did contain strand breaks. These data suggest that apoptosis occurs in chondrocytes in which the matrix has been disrupted experimentally or destroyed by the osteoarthritic disease process. The results of these studies suggest that the ECM may be an essential survival factor for chondrocytes. Microsc. Res. Tech. 50:236–242, 2000.

Differential Calcium Independent Regulation of Matrix Metalloproteinases and Tissue Inhibitors of Matrix Metalloproteinases by Interleukin-1β and Transforming Growth Factor-β in Peyronie's Plaque Fibroblasts

PURPOSE Peyronies disease is a fibrotic disorder of the tunica albuginea characterized by the localized formation of an inelastic plaque. We characterized matrix metalloproteinases and TIMPs (tissue inhibitors of matrix metalloproteinase) in Peyronies disease tissue. MATERIALS AND METHODS Matrix metalloproteinases and TIMPs were investigated in Peyronies disease plaque tunica removed from patients with stable Peyronies disease. Immunological methods were used to characterize the matrix metalloproteinases and TIMPs produced by cell cultures stimulated with transforming growth factor-beta or interleukin-1beta (PreproTech, Rocky Hill, New Jersey). Enzyme activity was quantified with a fluorescent substrate and correlated with mRNA levels using real-time polymerase chain reaction. RESULTS Interleukin-1beta significantly induced (p <0.01) matrix metalloproteinase-1, 3, 10 and 13 protein production, endogenous matrix metalloproteinase-13 activity (12-fold) and matrix metalloproteinase-13 mRNA expression (11.2-fold) through a Ca(2+) independent mechanism in cultured fibroblasts. Transforming growth factor-beta stimulation failed to induce any detectable matrix metalloproteinase protein production or activity and conditioned culture medium even had the capacity to inhibit (p <0.01) the activity of purified recombinant human matrix metalloproteinase-13. Intact Peyronies disease plaques were highly enriched with TIMP-1 to 4 compared to donor matched perilesional tunica. CONCLUSIONS These data show that, while interleukin-1beta strongly induces matrix metalloproteinase expression, transforming growth factor-beta strongly induces TIMP expression without any effect on matrix metalloproteinases and may represent an important downstream biochemical mechanism that leads to the progression of Peyronies disease. The localized accumulation of TIMPs together with decreased matrix metalloproteinase activity in the Peyronies disease lesion may be the biochemical consequence of the transforming growth factor-beta over expression that has been reported in many fibrotic disorders, including Peyronies disease.