Osteogenic protein-1 promotes the synthesis and retention of extracellular matrix within bovine articular cartilage and chondrocyte cultures

OBJECTIVE: We have used recombinant osteogenic protein-1 to investigate our hypothesis that proper repair and maintenance of cartilage requires not only enhanced biosynthesis and replenishment of the extracellular matrix but also the enhancement of components necessary for matrix retention. DESIGN: The effects of osteogenic protein-1 were examined on bovine articular cartilage slices as well as isolated chondrocytes grown in alginate beads. Cartilage slices were examined for accumulation of proteoglycan by incorporation of 35S-sulfate and staining using Safranin O or, a biotinylated probe specific for hyaluronan. Bovine chondrocytes were characterized by use of a particle exclusion assay, in-situ hybridization, quantitative-competitive RT-PCR and a hyaluronan-binding assay. RESULTS: Osteogenic protein-1 treatment substantially enhanced the accumulation of hyaluronan and proteoglycan within cartilage tissue slices. As with the tissue, osteogenic protein-1 enhanced the size of cell-associated matrices assembled and retained by chondrocytes in vitro. This enhanced matrix assembly was paralleled by an increased expression of mRNA for aggrecan, hyaluronan synthase-2 and CD44. Of the two hyaluronan synthase genes expressed by chondrocytes, only hyaluronan synthase-2 was upregulated by osteogenic protein-1. Coupled with the increase in the CD44 mRNA was an increase in functional hyaluronan binding activity present at the chondrocyte cell surface. CONCLUSIONS: These results demonstrate that osteogenic protein-1 stimulates not only the synthesis of the major cartilage extracellular matrix component aggrecan, but also two associated molecules necessary for the retention of aggrecan, namely hyaluronan and CD44.     

Evaluation of chondrocyte cell-associated matrix metabolism by flow cytometry

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.     

Aggrecanolysis in human osteoarthritis: confocal localization and biochemical characterization of ADAMTS5-hyaluronan complexes in articular cartilages

OBJECTIVE: Human osteoarthritis (OA) is characterized by aggrecanase-mediated depletion of cartilage aggrecan. We have examined the abundance, location and some biochemical properties of the six known aggrecanases (A disintegrin and metalloproteinase with thrombospondin-like motifs 1 (ADAMTS1) 4, 5, 8, 9 and 15) in normal and OA human cartilages. METHODS: Formalin-fixed, ethylenediamine tetraacetic acid (EDTA)-decalcified sections of full-depth cartilage from human OA tibial plateaus and normal control samples were studied by confocal imaging. Probes included specific antibodies to aggrecanases and two aggrecan epitopes, as well as biotinylated hyaluronan binding protein (HABP) for hyaluronan (HA) visualization. Cartilage extracts were analyzed by Western blot for the individual proteinases and aggrecan fragments. RESULTS: ADAMTS5 was present in association with cells throughout normal cartilage and was markedly increased in OA, particularly in clonal groups in the superficial and transitional zones, where it was predominantly co-localized with HA. Consistent with the confocal analysis, a high molecular weight complex of ADAMTS5 and HA was isolated from human OA cartilage by isotonic salt extraction and chromatography on Superose 6. The complex eluted with an apparent molecular size of about 2x10(6) and contained major ADAMTS5 forms of 150, 60, 40 and 30kDa. The yield of most forms on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was markedly enhanced by prior digestion of the complex with either Streptomyces hyaluronidase or chondroitinase ABC. CONCLUSION: ADAMTS5 abundance and distribution in human OA cartilages is consistent with a central role for this enzyme in destructive aggrecanolysis. HA-dependent sequestration of ADAMTS5 in the pericellular matrix may be a mechanism for regulating the activity of this proteinase in human OA cartilage.     

Glucosamine sulfate modulates the levels of aggrecan and matrix metalloproteinase-3 synthesized by cultured human osteoarthritis articular chondrocytes

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.     

Effect of hylan on cartilage and chondrocyte cultures.

The protective role of hylan, a hyaluronan [hyaluronic acid (HA)] derivative, was studied in explanted bovine cartilage and isolated chondrocytes. Cartilage and chondrocytes were exposed to degradative enzymes (lysate from activated polymorphonuclear leukocytes), oxygen-derived free radicals (ODFR), conditioned media from mononuclear cells (MCCM), and interleukin-1 (IL-1), in the presence and absence of hylan. The effect of HA was also studied. In cartilage explants susceptibility to pertubation was evaluated in terms of 35S release and proteoglycan depletion and was compared to control cultures; high viscosity hylan was found to reduce 35S release in cartilage explants caused by degradative enzymes, ODFR, MCCM, and IL-1. The hylan effect was reversible and viscosity-dependent. In chondrocyte cultures, high viscosity hylan was effective in reducing cell injury caused by degradative enzymes and ODFR. The data suggest that the glycosaminoglycan hylan, as well as native HA, may mediate exposure to and/or response to stimuli associated with initiation of degenerative processes in cartilage tissues.     

In-vitro-Studie zum Einfluss von Fibrin in Knorpelkonstrukten auf der Basis von PGA-Vliesstoffen

BACKGROUND: The matrix component in autologous chondrocyte implantation plays an important role. In this study the influence of an additional fibrin component in cartilage constructs based on polyglycolide polymers (PGA) was investigated. METHODS: Human chondrocytes of femoral heads were isolated and cultured using a serum-free technique. The cells were seeded on PGA-91 scaffolds with and without an additional fibrin component; the constructs were cultured for 2 weeks in vitro. Besides cell viability, DNA content, pH, aggrecan production, mRNA expression of aggrecan, and collagen types I and II were determined by real-time PCR. Furthermore, cartilage grafts were histologically analyzed. RESULTS: All constructs contained viable, metabolically active cells in the investigated time period. There was no cell proliferation within the graft, and the DNA content was decreased over time. The pH level constantly remained within a physiologic range. The Alcian blue staining of the constructs showed the homogeneous cell distribution and a cell-associated proteoglycan production. Aggrecan concentration in the supernatants of fibrin-containing constructs was significantly lower compared to fibrin-free grafts (-24%), a result that correlated with diminished aggrecan mRNA expression (-80%). mRNA expression of collagen type II increased in the fibrin-free constructs over time and was 57% higher than in the fibrin-containing grafts. The immunohistochemical detection of collagen type II was possible in all constructs. CONCLUSION: Cartilage constructs based on carbohydrate matrices are suitable for matrix-associated chondrocyte implantation. The results of this study suggest a partially inhibitory effect of an additional fibrin component in PGA constructs for chondrogenic differentiation.     

Gene expression and proliferation analysis in young, aged, and osteoarthritic sheep chondrocytes effect of growth factor treatment.

Cartilage is a support tissue with a poor capacity to self-repair. Its cells, chondrocytes, are responsible for synthesizing and renewing the matrix that surrounds them in a constant turnover mechanism. Autologous chondrocyte implantation (ACI) is one of the techniques that promises to be an alternative to common strategies for chondral lesions. To apply this technique, a large amount of cells must be obtained. In our work, we studied the state of cells from different cartilage (young, aged, and osteoarthritic sheep) cultured in monolayer by analyzing their proliferation rate using bromodeoxyuridine and their gene expression profile by RT-PCR. A decrease was found in expression of type II collagen and aggrecan in aged, osteoarthritic, and passaged chondrocytes. Treatment of cells with growth factors aFGF, IGF-I, TGF-beta, and OP-1 improved the proliferation rate in all cells studied and stimulated gene expression of type II collagen, aggrecan, and TGF-beta. Osteoarthritic cells showed a poor response according to matrix gene expression, while young cells responded properly, and aged chondrocytes showed a moderate response. These results suggest that the state of cartilage may affect the behavior of cultured chondrocytes.     

Effects of high molecular weight hyaluronan on the distribution and movement of proteoglycan around chondrocytes cultured in alginate beads

OBJECTIVE: To evaluate the effects of high molecular weight hyaluronan (HA) on the distribution and movement of proteoglycan (PG) formed around rabbit chondrocytes cultured in alginate beads. DESIGN: Rooster comb-derived HA (MW 8x10(5) Da) was co-polymerized in alginate gel to study the direct effects of extrinsic HA on chondrocytes. PG metabolism of rabbit chondrocytes cultured in alginate beads was examined by measuring the incorporation of [(35)S]sulfate into glycosaminoglycan in two distinct regions, the cells with their cell-associated matrix (CM) and the further-removed matrix (FRM). Immunohistochemical analysis was performed using monoclonal antibodies against chondroitin sulfate and keratan sulfate. Autoradiography using degenerated cartilage tissue from the rabbit osteoarthritis (OA) model was performed to discover the effect of HA on the distribution of newly-synthesized PG in the cartilage tissue. RESULTS: The incorporation of [(35)S]sulfate into newly-synthesized PG in the cells with CM decreased with the addition of 0.125-1.0 mg/ml HA, while the incorporation in the FRM increased. These effects of HA on the distribution of newly-synthesized PG were the same either in chondrocytes with CM or chondrocytes without CM. Immunohistochemical analysis showed that staining of PG in the CM was decreased and staining in the FRM was increased in the HA treated group compared to the control group. Autoradiography using degenerated cartilage tissue from the rabbit OA model indicated that [(35)S]-labeled macromolecules showed a more diffuse distribution in the HA treated group compared with the control group. CONCLUSION: These results indicate that extrinsic HA could affect the movement of newly-synthesized PG from the CM to the FRM in both alginate beads and cartilage tissue.     

Different response of articular chondrocyte subpopulations to surface motion

OBJECTIVE: To investigate the effect of surface motion on the gene expression of proteoglycan 4 (PRG4), hyaluronan synthases (HAS1, HAS2) and on the hyaluronan (HA) and proteoglycan 4 (PRG4) release of chondrocytes from different zones of bovine articular cartilage. DESIGN: Superficial zone, deep zone, full thickness, and superficial/deep 1:1 mixed chondrocytes were seeded into 3D polyurethane scaffolds and stimulated using our bioreactor that approximates kinematics and surface motion characteristics of natural joints. One hour of surface motion superimposed on cyclic compression was applied twice a day over 3 consecutive days. Scaffolds were cut into top and bottom sections and analyzed for gene expression of PRG4, HAS1, and HAS2. RESULTS: Depending on the cell population, the gene expression levels increased within 8 days of culture in unloaded scaffolds, with a stronger increase in the top compared to the bottom sections. Mechanical loading further enhanced the messenger RNA (mRNA) levels in all cell types, with most pronounced up-regulations observed for the PRG4 expression in deep zone and the HAS2 expression in superficial zone cells. The effect of the biochemical and biomechanical environment appeared to be additive, resulting in highest mRNA levels in the top sections of loaded constructs. Bioreactor stimulation also enhanced the HA release in all cell populations. Full thickness chondrocytes experienced the greatest effect on HAS1 mRNA expression and HA release, indicating that the interaction between cell populations may promote HA synthesis compared to subpopulations alone. CONCLUSIONS: Reciprocating sliding can be an efficient tool for generating tissue-engineered constructs from various chondrocyte populations by providing a functional cartilage-synovial interface.     

Mechanism of proteoglycan aggregate degradation in cartilage stimulated with oncostatin M

OBJECTIVE: To investigate the potential synergistic and differential effects of cytokine combinations on proteoglycan aggregate catabolism in cartilage. METHODS: Bovine articular cartilage explants were maintained in organ culture and subjected to stimulation with cytokine combinations including interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, IL-17, tumor necrosis factor-alpha (TNFalpha) and oncostatin M (OSM). Aggrecan, link protein and hyaluronan (HA) release and degradation were analyzed, and the effect of the hyaluronidase inhibitor apigenin was investigated. RESULTS: For all cytokine mixtures studied cleavage of aggrecan only by aggrecanase action was apparent. However, OSM acting synergistically with IL-1 or TNFalpha produced a rapid release of all proteoglycan aggregate components due to both aggrecan and HA degradation. This was abolished by the hyaluronidase inhibitor, apigenin. In addition, in the presence of OSM a low molecular weight aggrecan G3 product was observed, suggesting altered aggrecanase cleavage activity is induced by this cytokine. CONCLUSIONS: Under cytokine stimulation, aggrecan release from cartilage may take place via proteolysis of the aggrecan core protein or via depolymerization of HA, with the latter mechanism being induced by OSM. OSM is associated with joint inflammation and its participation may account for the more rapid loss of aggrecan from articular cartilage in the inflammatory arthritides, compared to osteoarthritis.     

Cartilage degradation independent of MMP/aggrecanases

OBJECTIVE: To identify and characterize a cartilage degradation mechanism that is independent of the proteolytic cleavages by matrix metalloproteinases (MMPs) and aggrecanases. METHODS: The sensitivity of glycosaminoglycan (GAG) release and collagen release to an MMP/aggrecanase inhibitor, AG3340, was compared using a bovine nasal cartilage explant culture. The release of matrix proteins and hyaluronan (HA) from the culture was analyzed by immunoblotting and radioimmunoassay, respectively. Induction of HA-degrading activity by retinoic acid was examined using the cartilage explant culture and a primary culture of chondrocytes. Degradation of the matrix components of cartilage was also characterized in vivo using an acute arthritis model induced by an intra-articular injection of interleukin 1alpha (IL-1alpha). RESULTS: AG3340 did not effectively inhibit GAG release at a concentration of more than 10muM, while 10nM of the inhibitor completely suppressed collagen degradation. Retinoic acid induced the release of the aggrecan G1 domain, link protein and HA into the culture medium, and the release of these molecules was not completely inhibited by 10muM of AG3340. The molecules were released as ternary complexes. Retinoic acid induced HA degradation in the explant culture and hyaluronidase activity in the primary culture of chondrocytes. The release of the G1 domain of aggrecan and link protein into the synovial fluid was also observed in the IL-1alpha-induced acute arthritis model. CONCLUSION: A novel mechanism by chondrocyte-derived hyaluronidase(s) is involved in the release of the matrix components from cartilage, and the hyaluronidase(s) and MMPs/aggrecanases act in a coordinated manner in cartilage degradation.     

Effects of transforming growth factor-beta on aggrecanase production and proteoglycan degradation by human chondrocytes in vitro

OBJECTIVE: Aggrecan is degraded by Aggrecanases (ADAMTS-4 and -5) and MMPs, which cleave its core protein at different sites. Transforming growth factor (TGF)beta is known to stimulate matrix formation in cartilage, and ADAMTS-4 production in synoviocytes. The aim of this in-vitro study was to examine the effects of TGFbeta on aggrecanase production in human cartilage. DESIGN: Expression of ADAMTS-4 and -5 in chondrocyte cultures from normal or osteoarthritic cartilage was studied at mRNA level by RT-PCR. Aggrecanase activity was examined by western blot of aggrecanase-generated neoepitope NITEGE, and by measure of proteoglycan degradation in cartilage explants. RESULTS: TGFbeta strongly increased mRNA levels of ADAMTS-4, while ADAMTS-5 was expressed in a constitutive way in chondrocytes from normal and osteoathritic cartilage. TGFbeta also increased NITEGE levels and proteoglycan degradation. Addition of an aggrecanase inhibitor blocked the increase of NITEGE, and partially inhibited proteoglycan degradation. CONCLUSIONS: TGFbeta stimulates ADAMTS-4 expression and aggrecan degradation in cartilage. This catabolic action seems to be partially mediated by aggrecanases. It is, therefore, proposed that the role of TGFbeta in cartilage matrix turnover is not limited to anabolic and anti-catabolic actions, but also extends to selective degradation of matrix components such as aggrecan.     

Intra-articular injection of interleukin-4 decreases nitric oxide production by chondrocytes and ameliorates subsequent destruction of cartilage in instability-induced osteoarthritis in rat knee joints

OBJECTIVE: To investigate the in vitro and in vivo effects of interleukin (IL)-4 on mechanical stress-induced nitric oxide (NO) expression by chondrocytes, and destruction of cartilage and NO production in an instability-induced osteoarthritis (OA) model in rat knee joints, respectively. MATERIALS AND METHODS: Cyclic tensile stress (CTS; 0.5Hz and 7% elongation) was applied to cultured normal rat chondrocytes with or without pre-incubation with recombinant rat IL-4 (rrIL-4). Inducible NO synthase (iNOS) mRNA expression and NO production were examined with real-time polymerase chain reaction and the Griess reaction, respectively. OA was induced in rat knee joints by transection of the anterior cruciate and medial collateral ligaments and resection of the medial meniscus. rrIL-4 (10, 50, and 100 ng/joint/day) was injected intra-articularly, and knee joint samples were collected 2, 4, and 6 weeks after surgery. Cartilage destruction was evaluated by the modified Mankin score and Osteoarthritis Research Society International scoring system on paraffin-embedded sections stained with safranin O. Cleavage of aggrecan and NO production were examined by immunohistochemistry for aggrecan neoepitope (NITEGE) and of nitrotyrosine (NT), respectively. RESULTS: rrIL-4 down-regulated CTS-induced iNOS mRNA expression and NO production by chondrocytes. The intra-articular injection of rrIL-4 gave rise to a limited, but significant amelioration of cartilage destruction, prevention of loss of aggrecan, and decrease in the number of NT-positive chondrocytes, an effect that was not dose-dependent. CONCLUSION: The present study suggests that IL-4 may exert chondroprotective properties against mechanical stress-induced cartilage destruction, at least in part, by inhibiting NO production by chondrocytes.     

Control of extracellular matrix homeostasis of normal cartilage by a TGFbeta autocrine pathway. Validation of flow cytometry as a tool to study chondrocyte metabolism in vitro

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.     

Statin stimulates bone morphogenetic protein-2, aggrecan,and type 2 collagen gene expression and proteoglycan synthesis in rat chondrocytes

Statins increase bone morphogenetic protein-2 (BMP-2) mRNA expression and subsequently increase new bone formation in vitro. However, the action of statins on the BMP-2 mRNA regulation of cartilage matrix synthesis by chondrocytes is unknown. We evaluated regulation of BMP-2, aggrecan, and type II collagen (COL2) mRNA and ³⁵S-labeled proteoglycan (PG) synthesis by mevastatin using cultured chondrocytes obtained from articular cartilage of fetal rats. Expression of BMP-2, aggrecan, and COL2 mRNAs were increased in the presence of 2µM mevastatin on day 2. However, longer (10 day) culture in the presence of the drug decreased the expression of these mRNAs. PG synthesis was increased 3 days after treating the cells with mevastatin, which was also decreased with longer (10 day) mevastatin treatment. These results suggest that mevastatin increases mRNA expression of BMP-2, aggrecan, and COL2 as well as PG synthesis by fetal rat chondrocytes early in the treatment period. We suggest that statins have implications for fracture and cartilage repair.     

Reduced NO accumulation in arthrotic cartilage by exposure to methylene blue

Nitric oxide (NO) appears to be a final common inflammation mediator of cartilage degradation. Halting the pathological formation of excessive NO, by suppressing the inducible NO synthase (iNOS) activity, may help to preserve cartilage integrity. We used fresh ex-vivo human articular cartilage explants from normal and arthrotic joints for assessment of NO levels, as determined by its nitrite degradation products and nitric oxide synthase expression. We measured matrix proteoglycan content, assessed by image analysis of alcian blue staining, and proteoglycan synthesis, assessed by sulfate incorporation into proteoglycans. The effect of methylene blue, a nitric oxide synthase inhibitor, on matrix preservation was evaluated. Cartilage discs in vitro, derived from normal appearing joints, secreted about one tenth as much NO compared to discs derived from arthrotic cartilage. Cartilage explants showed a time-dependent reduction in the amount of aggrecan within the cartilaginous matrix. Addition of methylene blue to the growth medium lowered nitric oxide accumulation and prevented matrix degradation in the cultured cartilage discs. The cartilage matrix preservation effect was mediated through downregulation of all three isoforms of NOS, i.e., the neuronal NOS, endothelial NOS and inducible NOS and upregulation of TGF beta receptor in the chondrocytes. Our findings indicate that inhibition of NOS activity preserves cartilage matrix in vitro.     

Reduced NO accumulation in arthrotic cartilage by exposure to methylene blue

Nitric oxide (NO) appears to be a final common inflammation mediator of cartilage degradation. Halting the pathological formation of excessive NO, by suppressing the inducible NO synthase (iNOS) activity, may help to preserve cartilage integrity. We used fresh ex-vivo human articular cartilage explants from normal and arthrotic joints for assessment of NO levels, as determined by its nitrite degradation products and nitric oxide synthase expression. We measured matrix proteoglycan content, assessed by image analysis of alcian blue staining, and proteoglycan synthesis, assessed by sulfate incorporation into proteoglycans. The effect of methylene blue, a nitric oxide synthase inhibitor, on matrix preservation was evaluated. Cartilage discs in vitro, derived from normal appearing joints, secreted about one tenth as much NO compared to discs derived from arthrotic cartilage. Cartilage explants showed a time-dependent reduction in the amount of aggrecan within the cartilaginous matrix. Addition of methylene blue to the growth medium lowered nitric oxide accumulation and prevented matrix degradation in the cultured cartilage discs. The cartilage matrix preservation effect was mediated through downregulation of all three isoforms of NOS, i.e., the neuronal NOS, endothelial NOS and inducible NOS and upregulation of TGF beta receptor in the chondrocytes. Our findings indicate that inhibition of NOS activity preserves cartilage matrix in vitro.     

Reduced NO accumulation in arthrotic cartilage by exposure to methylene blue

Nitric oxide (NO) appears to be a final common inflammation mediator of cartilage degradation. Halting the pathological formation of excessive NO, by suppressing the inducible NO synthase (iNOS) activity, may help to preserve cartilage integrity. We used fresh ex-vivo human articular cartilage explants from normal and arthrotic joints for assessment of NO levels, as determined by its nitrite degradation products and nitric oxide synthase expression. We measured matrix proteoglycan content, assessed by image analysis of alcian blue staining, and proteoglycan synthesis, assessed by sulfate incorporation into proteoglycans. The effect of methylene blue, a nitric oxide synthase inhibitor, on matrix preservation was evaluated. Cartilage discs in vitro, derived from normal appearing joints, secreted about one tenth as much NO compared to discs derived from arthrotic cartilage. Cartilage explants showed a time-dependent reduction in the amount of aggrecan within the cartilaginous matrix. Addition of methylene blue to the growth medium lowered nitric oxide accumulation and prevented matrix degradation in the cultured cartilage discs. The cartilage matrix preservation effect was mediated through downregulation of all three isoforms of NOS, i.e., the neuronal NOS, endothelial NOS and inducible NOS and upregulation of TGF beta receptor in the chondrocytes. Our findings indicate that inhibition of NOS activity preserves cartilage matrix in vitro.     

Effect of link protein concentration on articular cartilage proteoglycan aggregation

Previous work has shown that alterations in proteoglycan aggregates are among the first changes detected with aging, disuse, and degeneration of articular cartilage, yet the cause or causes of these alterations remain unknown. To determine if differences in link protein concentration can explain alterations in the assembly, size, and stability of articular cartilage proteoglycan aggregates, we isolated proteoglycan monomer (aggrecan) and link protein from adult bovine articular cartilage and then assembled proteoglycan aggregates from aggrecan and 0.8% hyaluronan relative to aggrecan weight, in the presence of 0, 2, 4, 6, 8, 10, 15, and 20% concentrations of link protein relative to aggrecan weight. We determined the amount, sedimentation coefficient, and stability of the aggregates by analytical ultracentrifugation and measured their dimensions by electron microscopy with use of the monolayer technique. Increased aggregate size, as determined by ultracentrifugation, was directly correlated with an increased number of aggrecans per aggregate and with increased hyaluronan length, as determined by electron microscopy. The concentration of link protein significantly influenced aggregation: concentrations of 6–8% produced maximum aggregation, aggregate stability, and uniformity of aggrecan spacing; concentrations greater than 10% led to the formation of superaggregates (aggregates with sedimentation velocities greater than 100 S that may result from linking two or more hyaluronan filaments) but decreased aggregate stability; and concentrations of less than 4% link protein significantly decreased aggregation, the size and stability of aggregates, and the regularity of aggrecan spacing. The latter observations suggest that a decline in the concentration of link protein could decrease the organization and stability of the articular cartilage matrix.     

Synergistic effect of IGF-1 and OP-1 on matrix formation by normal and OA chondrocytes cultured in alginate beads

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.