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1.
OBJECTIVE: To evaluate the influence of cyclodextrin polysulphate (CDPS) on the extracellular matrix (ECM) metabolism of human articular cartilage chondrocytes. METHODS: Isolated chondrocytes from femoral condyle cartilage of human knee joints were cultured in gelled alginate to maintain their differentiated phenotype. During 1 week of culture, the cells were exposed to different concentrations of CDPS. Synthesis of aggrecans was investigated in these cultures after using Na(2)(35)SO(4) as a radioactive precursor during the last 24h of culture. The artificial matrix was then solubilised with Na-citrate and newly synthesised aggrecan aggregates, accumulated during culture, were liberated and assayed. The isolated chondrocytes were labelled with antibodies against aggrecan and type II collagen to analyse the ECM molecules in the cell-associated matrix (CAM). Plasma membrane levels of receptors for insulin-like growth factor-1 (IGF-1RI) and for interleukin-1 (IL-1RI and IL-1RII), as well as levels of IGF-1, IL-1alpha and -beta were determined after the cells had been permeabilized and stained with the appropriate antibodies. The release of IL-6 in the culture media was used as a variable reflecting auto/paracrine IL-1 activity of the cells in different experimental conditions. RESULTS: CDPS significantly increased total (35)S-incorporation rates in ECM aggrecan. When compared with controls, CDPS-treated chondrocytes expressed significantly higher CAM aggrecan and type II collagen levels. As plasma membrane-bound IGFR1 and intracellular IGF-1 levels remained unchanged, this increase in accumulated CAM compounds may have resulted from suppressed catabolic activities by the chondrocytes in culture. CDPS-treated cells expressed significantly lower amounts of intracellular IL-1alpha and -beta levels. Plasma membrane-bound IL-1RI and decoy IL-1RII remained unchanged. beta-cyclodextrin-treated chondrocytes released significantly less IL-6 in the supernatant culture media. CONCLUSION: CDPS is a novel polysulfated polysaccharide showing cartilage structure modifying effects in vitro as it improves the synthesis of aggrecan and the accumulation of CAM macromolecules. This effect probably resulted in part from the downregulation of IL-1.  相似文献   

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OBJECTIVE: To validate flow cytometry as an experimental technique for the study of the homeostasis of the extracellular matrix (ECM) of human articular cartilage. METHODS: Given the established insights in the relation between the transforming growth factor (TGF)-beta type II Receptor (TGF-betaRII)/TGF-beta auto/paracrine pathway, the intracellular levels of matrix metalloproteinases (MMPs) and their natural inhibitors (TIMPs), and the accumulation of ECM molecules in the ECM of articular cartilage, this metabolic pathway was used as a reference model to fulfill the objective. Chondrocytes were liberated from visually intact femoral condyle cartilage and cultured in gelled agarose to maintain their differentiated phenotype. After 2 weeks of culture, the chondrocytes were isolated from the agarose and flow cytometry was used to analyse the expression of TGF-betaRII on the plasmamembrane, the expression of TGFbeta1, MMP-1, MMP-3, TIMP-1 and TIMP-3 inside the cells, as well as the amounts of aggrecan, type II collagen and hyaluronan in the cell-associated matrix (CAM). The expression of the different substances was analysed with flow cytometry and reported as mean fluorescence intensity (MFI), which is due to the binding of FITC-labeled antibodies to their specific antigens. In addition, the effects of exogenous TGFbeta1 on the expression of these proteins was investigated on chondrocytes cultured in serum-free media. Enzyme Linked Immunosorbent Assay (ELISA) was performed to evaluate the MMP-1, MMP-3, TIMP-1 and MMP-1/TIMP-1 complex in the culture medium collected after the last 3 days of the culture period. The correlations between the data were analysed with the Spearman's test. RESULTS: Exogenous TGF-beta1 increased the accumulation of aggrecan and hyaluronan in the CAM of chondrocytes and down-regulated the intracellular levels of MMP-1 and -3. TIMP-1 and -3 were increased after exposure to TGF-beta1. Baseline expression of TGF-betaRII on the plasmamembrane of normal human articular chondrocytes significantly correlated with the intracellular levels of TGFbeta1, TIMP-1 and TIMP-3. TGFbeta1 was correlated with TIMP-1, TIMP-3 and MMP-1. Aggrecan in the CAM was inversely correlated with the ratio of MMP-1 to TIMPs. In addition, there were correlations between TIMP-1 and TIMP-3, aggrecan and hyaluronan. ELISA also revealed the correlation between MMP-1 and TIMP-1 secreted by the chondrocytes into the nutrient medium. MMP-1/TIMP-1 complex was hardly found in the medium. CONCLUSIONS: Some aspects of ECM metabolism of normal cartilage were evaluated by flow cytometry. Chondrocytes from normal human cartilage, when cultured in gelled agarose, showed correlations between the expression of TGF-betaRII/TGF-beta1 and the intracellular levels of TIMPs, indicating that TGF-beta autocrine pathway may contribute to homeostasis of the ECM in the normal cartilage. The relations between MMPs, TIMPs and the ECM molecules support that a physiological balance between MMPs and TIMPs results in a well-controlled matrix turnover in normal cartilage.  相似文献   

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OBJECTIVE: Growth factor therapy may be useful for stimulation of cartilage matrix synthesis and repair. Thus, the purpose of our study was to further understand the effect of combined insulin-like growth factor-1 (IGF-1) and osteogenic protein-1 (OP-1) treatment on the matrix synthesized by human adult normal and osteoarthritic (OA) chondrocytes. DESIGN: Chondrocytes were isolated post-mortem from articular cartilage from tali of normal human donors and femoral condyles of OA patients undergoing knee replacement surgery. Cells were cultured in alginate beads for 21 days in four experimental groups: (1) "mini-ITS" control; (2) 100 ng/ml IGF-1; (3) 100 ng/ml OP-1; (4) IGF-1+OP-1, each at 100 ng/ml. Beads were processed for histological (Safranin O and fast green), morphometrical and immunohistochemical (aggrecan, decorin, type I, II, VI, and X collagens, and fibronectin accumulation) analyses. RESULTS: Histology showed that IGF-1 alone did not induce substantial matrix production. OP-1 alone caused a considerable matrix formation, but the highest matrix accumulation by normal and OA chondrocytes was found when OP-1 and IGF-1 were added together. Morphometrical analysis indicated larger matrices produced by OA chondrocytes than by normal cells under the combined treatment. All tested matrix proteins were more abundant in the combination group. Type X collagen was detected only under the combined OP-1 and IGF-1 treatment and was present at very low levels. Type I collagen was found only in OA chondrocytes. CONCLUSIONS: The results obtained in the current study suggest that combined therapy with IGF-1 and OP-1 may have a greater potential in treating cartilage defects seen in OA than use of either growth factor alone.  相似文献   

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OBJECTIVE: To analyze human articular chondrocyte cell-associated matrix aggrecan, hyaluronan (HA) and type II collagen metabolism using flow cytometry, and to compare the results obtained for aggrecan with classic(35)Sulfate incorporation methods and an enzyme linked immunosorbent assay (ELISA). DESIGN: Human articular chondrocytes obtained from five donors were cultured in gelled agarose and tested for their response to different concentrations of interleukin-1beta (IL-1beta). Synthesis and distribution of aggrecan in the cell-associated matrix (CAM), in the interterritorial matrix and in the nutrient medium of the chondrocytes in culture were analyzed using(35)Sulfate incorporation. The results were expressed as pg SO(4)incorporated in aggrecan per 1 x 10(6)cells/h. Flow cytometry with FITC-conjugated monoclonal antibodies against aggrecan and type II collagen, and with the biotinylated hyaluronic acid binding protein (b-HABP), was used to investigate the synthesis and accumulation of aggrecan, type II collagen and HA in the CAM of the cultured cells. The packing of these macromolecules in the CAM of the chondrocytes was assessed by measuring the mean fluorescence intensity (MFI) of the cell sample due to the binding of the specific monoclonal antibodies or b-HABP used. ELISA was used in parallel to quantify CAM aggrecans after these macromolecules were brought into solution with guanidinium chloride. Detection of aggrecan by flow cytometry was compared with(35)S-incorporation in chondrocytes from two subjects and with ELISA in a further two donors. RESULTS: IL-1beta suppressed aggrecan synthesis by chondrocytes in agarose. An IL-1beta dose-dependent suppression of(35)S-aggrecan in the CAM reflected the changes in the interterritorial matrix. IL-1beta-induced aggrecan breakdown was followed by a rise in(35)S-aggrecan metabolites in the incubation media of the cells in culture. Flow cytometry and ELISA confirmed this decreased accumulation of aggrecan in the CAM of the chondrocytes. The results obtained with flow cytometry were closely related to those obtained with ELISA.(35)S-incorporation, on the other hand, indirectly measures the glycosaminoglycan content of the aggrecan and does not necessarily reflect the absolute amount of aggrecan molecules. Therefore, the effects of IL-1beta on cell-associated aggrecan, where assessed with(35)S-incorporation, did not correlate with the results of the flow cytometric assays. Flow cytometry enabled the detection of an impaired synthesis and accumulation of HA and of type II collagen in the CAM of the cultured chondrocytes. IL-1beta-induced changes in CAM aggrecan and hyaluronan closely agreed. CONCLUSIONS: Flow cytometry offers an efficient tool to study the metabolism of the chondrocyte CAM. The MFI has been used as a parameter to quantify the ECM molecules in the CAM.  相似文献   

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Osteoarthritis (OA) is closely related to the function of several inflammatory cytokines. It has been reported that older age is associated with higher serum levels of the inflammatory cytokine IL-18. In the present study, we investigated the long-term role of serum IL-18 in cartilage loss in vivo using a new strain of IL-18 transgenic mouse (Tg) in comparison with wild-type (WT) mice. The IL-18 Tg mouse strain we developed constitutively overproduces soluble mature IL-18 in the lungs but not in other tissues, including joints. These Tg mice showed high levels of serum IL-18, but not IL-1beta. No inflammatory cells, fibrillation or synovitis were observed in the knee joints of either IL-18 Tg or WT mice. However, the cartilage cellularity of the femoral and tibial condyles of IL-18 Tg mice was significantly reduced in comparison with control WT mice. Aggrecan was detected in only a few cells in the deep zone of the articular cartilage of Tg mice. The expression of aggrecan mRNA was also significantly decreased in articular chondrocytes from Tg mice when compared with WT mice. In contrast, endogenous IL-18 mRNA was significantly increased in the chondrocytes of Tg mice in comparison with WT mice. Expression of IFN-gamma was also significantly increased in the Tg mice. Moreover, IL-18 transgene-positive caspase-1-deficient mice showed articular cartilage loss that was independent of endogenous IL-1beta. In cultured chondrocytes isolated from WT mice, the expression of aggrecan mRNA was dosage-dependently suppressed by treatment with recombinant IL-18. In contrast, IL-18 stimulated the expression of mRNA for endogenous IL-18 and IFN-gamma. These results suggest that high levels of serum IL-18 promote the overexpression of endogenous IL-18 in articular chondrocytes, resulting in cartilage loss through suppression of aggrecan synthesis. Thus IL-18 may play an important role in the pathogenesis of articular cartilage loss in osteoarthritis.  相似文献   

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OBJECTIVE: Studies on the biology of the human meniscus cell are scarce. The objective of our studies was to assess survival/proliferation of human meniscus cells in different culture conditions and to characterize the extracellular matrix (ECM) produced by these cells in these artificial environments. The composition of this ECM offers a variable to define the distinct meniscus cell phenotype. MATERIALS AND METHODS: Human meniscus cells were isolated enzymatically from visually intact lateral and medial knee menisci. Cells were cultured in monolayer conditions or in alginate gel. The composition of the cell-associated matrix (CAM) accumulated by the isolated cells during culture was investigated and compared to the CAM of articular chondrocytes cultured in alginate using flow cytometry with fluorescein isothiocyanate-conjugated monoclonal antibodies against type I collagen, type II collagen and aggrecan. Additional cell membrane markers analysis was performed to further identify the different meniscus cell populations in the alginate culture conditions and meniscus tissue sections. Proliferation was analyzed using the Hoechst 33258 dye method. In some experiments, the effect of TGFbeta1 on some of these variables was investigated. RESULTS: The CAM of monolayer cultured meniscus cells is composed of high amounts of type I and II collagen and low amounts of aggrecan. A major population of alginate cultured meniscus cells on the other hand synthesized a CAM containing high amounts of type I collagen, low amounts of type II collagen and high amounts of aggrecan. This population is CD44+CD105+CD34-CD31-. In contrast, a minor cell population in the alginate culture did not accumulate ECM and was mainly CD34+. The CAM of alginate cultured articular chondrocytes is composed of low amounts of type I collagen, high amounts of type II collagen and aggrecan. The expression of aggrecan and of type II collagen was increased by the addition of TGFbeta1 to the culture medium. The proliferation of meniscus cells is increased in the monolayer culture conditions. Cell numbers decrease slightly in the alginate culture, but can be increased after the addition of TGFbeta1. CONCLUSION: These results demonstrate that the human meniscus is populated by different cell types which can be identified by a distinct CAM composition and membrane marker expression. Unlike the monolayer culture conditions, the alginate culture conditions appear to favor a more fibrochondrocyte-like cell accumulating a CAM resembling the native tissue composition. This CAM composition is distinctly different from the CAM composition of phenotypically stable articular cartilage chondrocytes cultured in the same alginate matrix.  相似文献   

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Avascularity is important for the unique biomechanical properties of articular cartilage, and normal cartilage actively repels vascular invasion. This study investigated whether the antiangiogenic properties changed in the presence of osteoarthritis (OA) by culturing explants of human articular cartilage on the chorioallantoic membrane (CAM) of chick embryos and investigating the incidence of vascular invasion and the effects of exogenous vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9). The results were compared with those of non-OA cartilage obtained after femoral neck fractures. Altogether, 72% of OA samples but only 5% of non-OA samples were invaded by the CAM, indicating that changes in the antiangiogenic properties represented a fundamental difference between control and OA cartilage. Exogenous VEGF or MMP-9 increased the frequency of invasion to 70%–100%. Invasion most frequently occurred into cartilage matrix from which proteoglycans had been lost, the latter being detectable by sirius red staining of cartilage collagen. VEGF was synthesized by chondrocytes in proportion to the severity of degradation and might exacerbate the loss of resistance to invasion. These results indicate that loss of resistance to vascular invasion distinguishes OA cartilage from normal articular cartilage, which may be important in the pathogenesis of OA.  相似文献   

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OBJECTIVE: Synthesis rates of aggrecans by phenotypically stable human articular chondrocytes and the immobilization of these aggrecans in large aggregates were used as variables reflecting the capability of these cells of restoring the extracellular matrix of articular cartilage in vivo in an aging population. DESIGN: Human articular chondrocytes were isolated from articular cartilage obtained from 33 different donors at autopsy. The chondrocytes were cultured in gelled agarose. Synthesis of aggrecans was investigated using Na(2)(35)SO(4)as a radioactive precursor after a 2-week culture period. Electron microscopic study of aggrecan aggregates was done on the macromolecules accumulated over 3 weeks in culture by the chondrocytes obtained from eight other donors with increasing ages. RESULTS: Sulfate incorporation rates into aggrecans correlated inversely with the age of the donor. The value of sulfate incorporation in aggrecans for chondrocytes obtained from mature cartilage of a 20-year-old individual in this system drops to 50% and 25% for chondrocytes obtained from 45- and 69-year-old individuals respectively. Electron microscopic study of aggrecan aggregates showed that the 'de novo' synthesized hyaluronan molecules were fully loaded with aggrecans. Mature human articular cartilage cells were found to synthesize an aggrecan aggregate which carried an average number of 11.7 to 13.1 aggrecans. Cells obtained from immature donors synthesized aggrecan aggregates of which the hyaluronan chain carried twice the amount of aggrecans. These immature human articular cartilage cells were also found to synthesize significant proportions of large aggrecan aggregates with 20 to over 100 aggrecans immobilized on a single hyaluronan chain. The proportions of these large aggrecan aggregates decreased with increasing age of the donors of the chondrocytes. CONCLUSION: The declining aggrecan synthesis rates and the decreased capability of assembling large molecular size aggregates with increasing age in humans illustrates a progressive failure of the repair function of articular cartilage cells in humans.  相似文献   

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OBJECTIVES: To examine the localization of bone morphogenetic protein (BMP)-2 mRNA and protein in human osteoarthritic (OA) articular cartilage and osteophyte. DESIGN: Five normal, four growing and 14 OA human cartilage samples, graded histomorphologically by Mankin Score, were studied by in situ hybridization and immunohistochemistry for the expression of BMP-2. RESULTS: BMP-2 mRNA was present in chondrocytes in neonatal growing articular cartilage, but was scarcely present in normal adult articular cartilage. In OA articular cartilage, BMP-2 mRNA and protein were detected in both clustering and individual chondrocytes in moderately or severely damaged OA cartilage. In moderately damaged OA cartilage, BMP-2 mRNA was localized in both upper and middle zone chondrocytes, but was not detected in deep layer chondrocytes. In severely damaged OA cartilage, cellular localization of BMP-2 mRNA was extended to the deep zone. In the area of osteophyte formation, BMP-2 mRNA was intensely localized in fibroblastic mesenchymal cells, fibrochondrocytes, chondrocytes and osteoblasts in newly formed osteophytic tissue. The pattern of BMP-2/4 immunolocalization was associated with that of mRNA localization. CONCLUSIONS: BMP-2 mRNA and BMP-2/4 were detected in cells appearing in OA tissues. BMP-2 was localized in cells of degenerating cartilage as well as osteophytic tissue. Given the negative localization of BMP-2 in normal adult articular cartilage, BMP-2 might be involved in the regenerating and anabolic activities of OA cells, which respond to cartilage damage occurring in osteoarthritis.  相似文献   

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OBJECTIVE: The functional integrity of articular cartilage is determined by a balance between chondrocyte biosynthesis of extracellular matrix and its degradation. In osteoarthritis (OA), the balance is disturbed by an increase in matrix degradative enzymes and a decrease in biosynthesis of constitutive extracellular matrix molecules, such as collagen type II and aggrecan. In this study, we examined the effects of the sulfate salt of glucosamine (GS) on the mRNA and protein levels of the proteoglycan aggrecan and on the activity of matrix metalloproteinase (MMP)-3 in cultured human OA articular chondrocytes. DESIGN: Freshly isolated chondrocytes were obtained from knee cartilage of patients with OA. Levels of aggrecan and MMP-3 were determined in culture media by employing Western blots after incubation with GS at concentrations ranging from 0.2 to 200 microM. Zymography (casein) was performed to confirm that effects observed at the protein level were reflected at the level of enzymatic activity. Northern hybridizations were used to examine effects of GS on levels of aggrecan and MMP-3 mRNA. Glycosaminoglycan (GAG) assays were performed on the cell layers to determine levels of cell-associated GAG component of proteoglycans. RESULTS: Treatment of OA chondrocytes with GS (1.0-150 microM) resulted in a dose-dependent increase in aggrecan core protein levels, which reached 120% at 150 microM GS. These effects appeared to be due to increased expression of the corresponding gene as indicated by an increase in aggrecan mRNA levels in response to GS. MMP-3 levels decreased (18-65%) as determined by Western blots. Reduction of MMP-3 protein was accompanied by a parallel reduction in enzymatic activity. GS caused a dose-dependent increase (25-140%) in cell-associated GAG content. Chondrocytes obtained from 40% of OA patients failed to respond to GS. CONCLUSIONS: The results indicate that GS can stimulate mRNA and protein levels of aggrecan core protein and, at the same time, inhibit production and enzymatic activity of matrix-degrading MMP-3 in chondrocytes from OA articular cartilage. These results provide a cogent molecular mechanism to support clinical observations suggesting that GS may have a beneficial effect in the prevention of articular cartilage loss in some patients with OA.  相似文献   

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There have been few reports on the localization of S-100 protein positive chondrocytes in the human articular cartilages. We studied 59 articular cartilages of the aged subjects, 65 osteoarthritic (OA) and 39 rheumatoid arthritic (RA) articular cartilages, to detect the histological localization of S-100 protein using immunoperoxidase method (ABC). The results obtained from normal cartilages demonstrated strongly positive cells representing hypertrophic chondrocytes in the perivascular areas of the neonatal articular cartilage and in the deep zone of the infant articular cartilage. The moderately positive cells were found in the intermediate zone of infant and adult articular cartilages. In mild OA, there were many positive chondrocytes in the intermediate zone with erosion of the surface layer, while in moderate or severe OA many strongly positive cells were found in clusters. The hypertrophic cells in the metaplastic cartilage arising from bone marrow in subjects with severe OA, or from pannus after RA were also positive. It is therefore, suggested that S-100 protein may be correlated with the metabolic activity of the cartilage matrix such as collagen and proteoglycan, as reported in the literature. S-100 protein further, appears to be useful for evaluating histologically the activity of cartilage repair in the pathologic human articular cartilages.  相似文献   

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AIM: Osteoarthritis (OA) is characterized by an irreversible destruction of articular cartilage. This is associated with a multiplicity of factors, causing an increased catabolic metabolism in cartilage. However, the prevalence of the OA is very variable in different joints. Therefore , we conducted a comparative analysis of chondrocytes derived from knee and hip joints with respect to their expression of inflammatory factors, such as IL-1beta, IL-1beta-receptorantagonist, iNOS, components of cartilage matrix (collagen I, II, and VI) as well as vimentin. METHODS: Different cytokines and proteins were detected by immune-histochemical staining of cartilage samples ex vivo. Further, chondrocytes were isolated from OA knee and hip joints, expanded in vitro and gene expression patterns were investigated by quantitative RT-PCR. RESULTS: Chondrocytes from knee and hip joints of OA patients express collagenes I, II and VI, IL-1beta and IL-1beta-RA, iNOS as well as Vimentin. A significant difference in gene expression patterns was not found in chondrocytes from the hip joints versus the knee joint ex vivo or in primary culture cells in vitro. However, in vitro the expression of type I collagen exceeded the expression of type II collagen. The IL-1beta-expression was high ex vivo, remained low during primary culture but was significantly elevated after primary culture in hip chondrocytes. CONCLUSION: Osteoarthritic gene expression patterns in cells derived from hip or knee joints ex vivo and in primary culture were not significantly different. We conclude that the rather frequent occurrence of OA in these joints in comparison to the ankle joint may be associated with a close physiological relation of cells in these joints. However, future studies which will include ankle cartilage must be investigated in further detail.  相似文献   

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