Cartilage Proteoglycan Synthesis Research Articles

Accumulation of advanced glycation endproducts reduces chondrocyte-mediated extracellular matrix turnover in human articular cartilage

Objective The prevalence of osteoarthritis (OAs) increases with age and coincides with the accumulation of advanced glycation endproducts (AGEs) in articular cartilage, suggesting that accumulation of glycation products may be involved in the development of OA. This study was designed to examine the effects of accumulation of AGEs on the turnover of the extracellular matrix of human articular cartilage.Design Chondrocyte mediated cartilage degradation (GAG release, colorimetric) was measured in human articular cartilage of donors aged 19–82 years (N=30, 4-day culture). In addition, to mimic the age-related increase in AGE levels in vitro, cartilage was cultured in the absence or presence of glucose, ribose or threose. Cartilage degradation and proteoglycan synthesis (35SO2−4 incorporation) were measured and related to the degree of cartilage AGE levels (fluorescence at 360/460nm).Results Chondrocyte-mediated degradation of articular cartilage (i.e. GAG release) decreased with increasing age of the cartilage donor (r=−0.43, P< 0.02). In vitro incubation of cartilage with glucose, ribose or threose resulted in a range of AGE levels that was highly correlated to the chondrocyte-mediated cartilage degradation (r=−0.77, P< 0.001, N=26). In addition, in these in vitro glycated cartilage samples, a decrease in proteoglycan synthesis was observed at increasing AGE levels (r=−0.54, P< 0.005,N =25).Conclusions This study shows that an increase in AGE levels negatively affects the proteoglycan synthesis and degradation of articular cartilage. In combination, these two effects reduce the turnover of the cartilage and thereby the maintenance and repair capacity of the tissue. By this mechanism, the age-related increase in cartilage AGE levels may contribute to the development of OA.

In vitro generation of scaffold independent neocartilage.

A novel serum-free culture system was developed in an attempt to generate a three-dimensional hyalinelike neocartilage independent of polymer scaffolds. Neocartilage disks as much as 1.5 mm thick were produced, which were characterized by synthesis of the normal articular cartilage collagens and proteoglycans. In contrast to growth in serum-containing media, chondrocytes from juveniles maintained in static culture under defined serum-free conditions deposited an extracellular matrix that accumulated in the form of tissue disks. Electron microscopic evaluation of neocartilage disks revealed collagenous matrices characteristic of articular cartilage from human infants. The neocartilage did not show terminal chondrocyte differentiation as shown by the absence of Type X collagen production and lack of cellular hypertrophy. Although chondrocytes from preadolescent donor cartilage recapitulated embryonic development in the absence of exogenous factors, chondrocytes from articular cartilage from adults failed to produce neocartilage when grown under identical conditions. This is the first demonstration that autocrine morphogens are sufficient to guide production of hyaline cartilage in vitro. In addition to providing a unique model system to compare the healing response of mature and immature articular chondrocytes, this technology may be of clinical importance in the development of new biomaterials for repair of articular cartilage defects.

The effects of methylprednisolone on normal and monocyte-conditioned medium-treated articular cartilage from dogs and horses.

To study in vitro (1) the dose-response relationships between proteoglycan metabolism in normal and corticosteroid-treated articular cartilage; (2) long-term proteoglycan metabolism after treatment of articular cartilage with corticosteroids; and (3) the effect of corticosteroids on proteoglycan metabolism in articular cartilage treated with monocyte-conditioned medium (MCM). Equine and canine articular cartilage explants were treated with corticosteroids and MCM. Proteoglycan synthesis and degradation were measured by radioactive labeling in short-term culture, and the long-term effect of corticosteroid treatment on proteoglycan metabolism was studied in normal explants. Two young cross-breed horses and 3 young Labrador retrievers. Equine articular cartilage explants were incubated in medium containing methylprednisolone sodium succinate (MPS) at 0, .001, .01, .1, 1, and 10 mg/mL (final concentration) for 1 day and then in fresh medium without MPS. Proteoglycan synthesis was measured by incorporation of sodium [35S]sulfate at 1, 3, 7, 10, and 13 days after initial treatment with MPS. Proteoglycan release was measured from separate explants prelabeled with sodium [35S]sulfate and treated similarly. Equine articular cartilage explants were treated with equine MCM simultaneously with, and 24 hours before MPS, at 0, 0.01, 0.1, 1, or 5 mg/mL for 72 hours. Proteoglycan synthesis and degradation in these explants was compared. Proteoglycan synthesis and degradation were measured similarly in canine articular cartilage explants treated simultaneously with canine MCM and MPS at 0, 0.001, 0.01, 0.1, 1 and 10 mg/mL for 72 hours. Equine articular cartilage explants treated with 0, 0.01, 0.1, 1, and 5 mg/mL of MPS for 72 hours were evaluated histologically. Proteoglycan synthesis in normal equine articular cartilage was severely depressed by 10 mg/mL MPS for 24 hours, and proteoglycan synthesis failed to recover after 13 days of culture in medium without MPS. Cartilage treated with 5 mg/mL MPS had pyknotic chondrocyte nuclei and empty lacunae. Concentrations of 1 and 0.1 mg/mL MPS depressed proteoglycan synthesis in normal equine cartilage explants. For these 2 concentrations, proteoglycan synthesis recovered 2 days after MPS removal and increased significantly (P < .05) 7 days after treatment with MPS compared with controls without MPS. Concentrations of 0.001 and 0.01 mg/mL MPS did not significantly affect proteoglycan synthesis in normal equine cartilage explants. Cumulative proteoglycan loss over 13 days in culture from normal equine explants treated for 24 hours with different concentrations of MPS was not significantly different between treatment groups at any time point. MCM significantly depressed proteoglycan synthesis in both canine and equine articular cartilage explants and significantly increased proteoglycan release. These effects were prevented in the canine explants by simultaneous treatment with MPS at 1 and 0.1 mg/mL, and proteoglycan release induced by MCM in equine articular cartilage was inhibited by 1 mg/mL MPS. Concentrations of 1.0 and 0.1 mg/mL MPS alleviated articular cartilage degradation in MCM-treated articular cartilage in vitro. These concentrations of MPS in contact with normal cartilage explants for 24 hours are unlikely to be detrimental in the long term to proteoglycan synthesis. The response of articular cartilage to MPS was affected by treatment with MCM so that results of experiments with normal articular cartilage explants may not reflect results obtained with abnormal cartilage. It may be possible to find an intraarticular concentration of corticosteroid that protects articular cartilage against cytokine-induced matrix degradation yet not have prolonged or permanent detrimental effects on chondrocyte matrix synthesis.

Nitric oxide inhibits chondrocyte response to IGF-I: inhibition of IGF-IRbeta tyrosine phosphorylation.

Chondrocytes in arthritic cartilage respond poorly to insulin-like growth factor I (IGF-I). Studies with inducible nitric oxide synthase (iNOS) knockout mice suggest that NO is responsible for part of the cartilage insensitivity to IGF-I. These studies characterize the relationship between NO and chondrocyte responses to IGF-I in vitro, and define a mechanism by which NO decreases IGF-I stimulation of chondrocyte proteoglycan synthesis. Lapine cartilage slices, chondrocytes, and cartilage from osteoarthritic (OA) human knees were exposed to NO from the donors S-nitroso-N-acetylpenicillamine (SNAP) or (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1, 2-diolate] (DETA NONOate), by transduction with adenoviral transfer of iNOS (Ad-iNOS), or by activation with interleukin-1 (IL-1). NO synthesis was estimated from medium nitrite, and proteoglycan synthesis was measured as incorporation of (35)SO(4). IGF-I receptor phosphorylation was evaluated with Western analysis. SNAP, DETA NONOate, endogenously synthesized NO in Ad-iNOS-transduced cells, or IL-1 activation decreased IGF-I-stimulated proteoglycan synthesis in cartilage and monolayer cultures of chondrocytes. OA cartilage responded poorly to IGF-I; however, the response to IGF-I was restored by culture with N(G)-monomethyl-L-arginine (L-NMA). IGF-I receptor phosphotyrosine was diminished in chondrocytes exposed to NO. These studies show that NO is responsible for part of arthritic cartilage/chondrocyte insensitivity to anabolic actions of IGF-I; inhibition of receptor autophosphorylation is potentially responsible for this effect.

Type II interleukin-1beta receptor: a candidate for gene therapy in human arthritis.

Interleukin-1 plays a pivotal role in the pathophysiologic process of arthritis. It is released in an autocrine fashion in joints affected with arthritis. Human cartilage affected with arthritis (but not normal cartilage) showed upregulation of interleukin-1beta messenger ribonucleic acid and protein in ex vivo conditions. Type II interleukin-1 receptor, interleukin-1 receptor antagonist, and Type I soluble interleukin-1 receptor has potent interleukin-1 neutralizing activity. In view of these observations, the role of type II interleukin-1 receptor in chondrocyte function was examined. Human interleukin-1beta (5-10 ng/mL) induced nitric oxide, prostaglandin E2 and matrix metalloprotease production in bovine or human chondrocytes, which could be inhibited by 500 pg/mL of Type II interleukin-1 receptor. Interleukin-1 inhibited proteoglycan synthesis that could be reversed by Type II soluble interleukin-1 receptor. Similarly, 1 ng/mL human interleukin-1 induced (or spontaneously produced) nitric oxide and prostaglandin E2 production in human osteoarthritis-affected cartilage could be inhibited by 50% or greater with 100 pg/mL Type II interleukin-1 receptor in ex vivo conditions. Type II interleukin-1 receptor transfected chondrocytes were immune to the insults of exogenous interleukin-1. These experiments showed that endogenous or exogenous Type II interleukin-1 receptor can attenuate the effects of interleukin-1 with respect to induction of inflammatory mediators, matrix metalloprotease activity and, proteoglycan synthesis in human chondrocytes and cartilage.

Effect of polysulfated glycosaminoglycan on DNA content and proteoglycan metabolism in normal and osteoarthritic canine articular cartilage explants.

To study the effect of polysulfated glycosaminoglycan (PSGAG) on proteoglycan metabolism and DNA content of control and osteoarthritic (OA) cartilage. An in vitro study comparing the effects of PSGAG on articular cartilage explants from canine stifle joints with and without chronic OA after transection of the left cranial cruciate ligament. Five large cross-breed dogs. Cartilage explants (6 to 13 per treatment group) from the medial side of the femoral trochlea and medial femoral condyle from both stifles of each dog were incubated in a defined medium containing 0, 0.05, 0.5, or 5 mg/mL of PSGAG. After 72 hours in culture, explants were pulsed for 6 hours with sodium [35S]sulfate. Aminophenylmercuric acetate (APMA) was used to activate endogenous neutral matrix metalloproteinases (MMPs) and induce proteoglycan degradation in the radiolabeled explants. DNA content and radioactivity were measured in papain-digested explants, and radioactivity was measured in the medium by liquid scintillation counting. Proteoglycan synthesis and degradation were calculated. Cartilage was examined histologically for signs of OA. A mixed model analysis of variance and linear contrasts were used to test for significant (P < .05) effects of OA and treatment with PSGAG. Transection of the cranial cruciate ligament produced OA in operated joints. DNA content and proteoglycan synthesis of OA cartilage were significantly lower than in cartilage from control joints. For both DNA content and proteoglycan synthesis, significant interactions occurred between the concentration of PSGAG and whether the articular cartilage was from OA or control joints. The two lower concentrations of PSGAG (0.05 and 0.5 mg/mL) predominantly increased DNA content in OA cartilage (7 and 18%, respectively, compared with 0 mg/mL PSGAG) while the highest concentration (5 mg/mL) predominantly increased DNA content in control cartilage (30% compared with 0 mg/mL PSGAG). PSGAG at .05 mg/mL predominantly decreased proteoglycan synthesis in OA cartilage (19% reduction compared with 0 mg/mL PSGAG) while PSGAG at .5 and 5 mg/mL predominantly decreased proteoglycan synthesis in control cartilage (17 and 55% reduction, respectively, compared with 0 mg/mL PSGAG). Following activation of MMPs, PSGAG caused a dose-dependent decrease in degradation of radiolabeled proteoglycan in both OA and control cartilage. OA cartilage was responsive to treatment with PSGAG at 100-fold lower concentration than control cartilage. When treated with PSGAG, articular cartilage explants maintained or increased DNA content at the expense of proteoglycan synthesis. Following MMP activation, proteoglycan degradation was inhibited in OA and control explants in a dose-dependent manner. If the results of this study extend to in vivo use, treatment with PSGAG may modify the progression of OA in articular cartilage by maintaining chondrocyte viability or stimulating chondrocyte division as well as protecting against extracellular matrix degradation.

In vitro evaluation of the effect of dimethyl sulfoxide on equine articular cartilage matrix metabolism.

To evaluate the effects of dimethyl sulfoxide (DMSO) on equine articular cartilage matrix metabolism. Using a cartilage explant culture system, proteoglycan (PG) synthesis, PG release, lactate metabolism, chondrocyte viability, and metabolism recovery were determined after cartilage exposure to DMSO. Cartilage harvested from metacarpophalangeal and metatarsophalangeal joints of 12 horses (age range, 1 to 10 years). Explants were exposed to concentrations of DMSO (1% to 20%) for variable times (3 to 72 hours). PG synthesis and release were determined by a radiolabel incorporation assay and dimethylmethylene blue (DMMB) dye assay, respectively. Lactate released into culture media was measured, and chondrocyte viability was assessed using the Formizan Conversion Assay and a paravital staining protocol. Metabolism recovery was assessed in explants that were allowed to recover in maintenance media after exposure to DMSO. PG synthesis and lactate metabolism were inhibited in a dose- and time-dependent manner after exposure to DMSO concentrations > or = 5%; there was no significant alteration in PG release. No change in chondrocyte viability was detected after incubation with DMSO. PG synthesis and lactate metabolism returned to baseline rates when allowed a recovery period after exposure to DMSO. DMSO concentrations > or = 5% suppress equine articular cartilage matrix metabolism. Suppression of PG synthesis and lactate metabolism is reversible and does not appear to be the result of chondrocyte death. Equine clinicians adding DMSO to intraarticular lavage solutions should be aware that DMSO may have deleterious effects on equine articular cartilage matrix metabolism.

Reduction of interleukin-17-induced inhibition of chondrocyte proteoglycan synthesis in intact murine articular cartilage by interleukin-4.

To investigate the role of interleukin-4 (IL-4) and IL-10 in basal and IL-1- and IL-17-mediated inhibition of chondrocyte metabolism. Cartilage explants of patellae from naive mice were incubated with IL-17 and/or IL-1 alone or were pretreated with IL-4 and IL-10. In addition, knee joints of naive mice were injected intraarticularly with IL-4 and IL-10 alone or were coinjected with IL-1. Chondrocyte proteoglycan (PG) synthesis was measured in intact murine articular cartilage. Levels of nitric oxide (NO) were measured using the Griess reagent. IL-17, a novel cytokine secreted by CD4+ activated memory T cells, inhibited chondrocyte PG synthesis in intact murine articular cartilage, although the suppressive effect was less potent than that of IL-1. The suppressive effect of IL-17 was completely abolished in the presence of L-NIO (L-NS-[1-iminoethyl]ornithine), an inhibitor of NO metabolism, and IL-17 only slightly induced inhibition of PG synthesis in cartilage explants of patellae from iNOS (inducible NO synthase) knockout mice. This indicates that the suppressive effect of IL-17 was mediated by NO. Pretreatment with IL-4, but not IL-10, significantly reduced the inhibition of chondrocyte PG synthesis induced by IL-1 or IL-17. The IL-4-induced reduction in the inhibitory effects of IL-1 and IL-17 on chondrocyte PG synthesis was accompanied by decreased NO formation in the culture supernatants. IL-17 plays a role in the inhibition of chondrocyte PG synthesis. IL-4 and IL-10 have no effect on basal chondrocyte metabolism. However, IL-4-pretreated cartilage is less sensitive to the suppressive effect of IL-1 as well as IL-17. This suggests that IL-4 is protective in T cell-driven cartilage disturbances, probably through reduction of iNOS.

Role of nitric oxide in the inhibition of BMP-2-mediated stimulation of proteoglycan synthesis in articular cartilage

Objective Bone morphogenetic protein-2 (BMP-2)-mediated stimulation of articular cartilage proteoglycan (PG) synthesis is suppressed in arthritic murine knee joints and by interleukin-1 (IL-1). The goal of this study was to investigate whether the gaseous mediator nitric oxide (NO) plays a crucial role in the inhibition of BMP-2 effects by IL-1.Methods Bone morphogenetic protein-2 alone or in combination with IL-1 was injected into the right knee joint of wild-type and NOS2 deficient C57Bl/6×129/Sv mice. Proteoglycan synthesis was measured ex vivo by incorporation of35S-sulfate on day 1, 2 and 3 after injection. To study the role of NO in the inhibition BMP-2-mediated stimulation of PG synthesis in arthritic joints, BMP-2 was injected intra-articularly in the joints of wild-type and NOS2 deficient mice with zymosan-induced arthritis. To check for NOS2 deficiency, NO production was measured in conditioned medium after challenge of patellae with surrounding tissue with IL-1.Results BMP-2 potently stimulated proteoglycan synthesis in articular cartilage of normal knees (up to 4-fold) but not in arthritic knees. Co-injection of BMP-2 with tumor necrosis factor α had no effect on BMP-2-mediated stimulation of PG synthesis but co-injection with IL-1 α resulted in a nearly total inhibition of BMP-2-mediated stimulation. In contrast, in NOS2 deficient mice IL-1 had no effect on BMP-2-mediated stimulation of PG synthesis. However, injection of BMP-2 into arthritic knee joints of NOS2 knock out mice did not result in significant stimulation of PG synthesis.Conclusions In this study we show that NO plays a role in the inhibition of BMP-2-mediated stimulation of PG synthesis by IL-1. However, NO, or at least NOS2, plays no dominant role in the inhibition of BMP-2 effects in arthritic knee joints.

Osteoarthritis-like changes in the murine knee joint resulting from intra-articular transforming growth factor-β injections

Objective To examine the impact of prolonged TGF-β exposure on cartilage and ligamentous joint structures in vivo, to investigate involvement of TGF-β in osteoarthritis pathology.Methods TGF-β was injected into murine knee joints once or repeatedly, whereafter articular cartilage proteoglycan (PG) synthesis and content, and histological changes in knee joints were studied over a 2-month period.Results A single injection of TGF-β stimulated patellar cartilage PG synthesis for 3 weeks and PG content for 2 weeks. Triple TGF-β injections prolonged the increase in PG content to 3 weeks. Patellar cartilage showed no histological abnormalities at 1 and 2 months after the last injection. In contrast, 2 months after triple TGF-β injections the superficial layer of tibial cartilage still had an increased proteoglycan content, while severe PG depletion was found in deeper layers of the posterior part of the lateral tibia in particular. Eventually, lesions occurred at the level of the tide-mark, exactly the site where cartilage is torn off in experimental and spontaneous osteoarthritis in mice. Additionally, multiple TGF-β injections induced formation of chondroid structures along the margins of articular cartilage. These chondroid structures were transformed into osteophytes via endochondral ossification. Formation of chondroid tissue was also observed in collateral ligaments.Conclusion Multiple intra-articular injections of TGF-β induce changes in articular cartilage and surrounding tissues that have strong resemblance to features of experimental and spontaneous osteoarthritis in mice, suggesting a role for TGF-β in the OA process.

Enhancement of Toluidine Blue Staining by Transforming Growth Factor-β, Insulin-Like Growth Factor and Growth Hormone in the Temporomandibular Joint of Aged Mice

Osteoarthritic lesions appear in the articular cartilage of the temporomandibular joint of mice aged 7 months and older. Reduced rate of proteoglycan (PG) synthesis leading to destruction of the articular cartilage was observed in this joint. The purpose of the present study was to test the ability of transforming growth factor-β1 (TGF-β1), insulin-like growth factor-1 (IGF-1) and growth hormone (GH) to induce PG synthesis in joint cartilage of aged animals and to compare it with the effect of interleukin-1α (IL-1α). Mandibular condyle explants from 18-month-old mice were cultured up to 72 h in serum-free medium, supplemented with IL-1α (TGF-β1 (0.1–5.0 ng/ml), TGF-β1 (1.0 ng/ml) + IGF-1 (2 ng/ml) or GH (10 ng/ml). The incorporation of ³⁵S-SO₄ into sulfated PG was tested. Cartilage samples were processed for histomorphometry using sections stained with 0.1% toluidine blue (TB), pH 1.8. Results indicated that in cultures supplemented (48 h) with either TGF-β, TGF-β + IGF-1 or with GH, an increased height and area of TB-positive staining as well as increased incorporation of ³⁵S-SO₄ into sulfated PG were observed. In contrast, the cytokine IL-1α exerted an inhibitory effect on TB staining and on ³⁵S-SO₄ incorporation. The present study demonstrated that in vitro supplementation of IL-1α to mandibular condyle cartilage reduced the height and area of TB staining and incorporation of ³⁵S-SO₄, whereas TGF-β1, TGF-β1 + IGF-1 or GH increased the height and area of TB staining and increased incorporation of ³⁵S-SO₄. The two parameters used to identify increased PG synthesis were shown to reveal similar results and were useful for studying the dynamic events taking place in cartilage destruction and repair in osteoarthritis.

Pleiotrophin inhibits chondrocyte proliferation and stimulates proteoglycan synthesis in mature bovine cartilage.

Pleiotrophin (PTN) is a secreted heparin-binding, developmentally regulated protein that is found in abundance in fetal, but not mature, cartilage. SDS-page and glycosaminoglycan (GAG) analysis of sulfate-radiolabeled proteoglycans isolated from the medium of mature cultured chondrocytes treated with PTN showed a threefold increase in the levels of proteoglycan synthesis. In contrast, in cultures of fetal chondrocytes, no changes in proteoglycan synthesis were observed. Thymidine incorporation experiments showed a dose-dependent decrease in proliferation of treated cells compared with control cultures, suggesting that pleiotrophin had an inhibitory effect on growth of chondrocytes. Neither FGF or heparin reversed the inhibitory effect of PTN. Capillary electrophoresis of chondroitinase ABC-digested proteoglycans isolated from mature chondrocytes showed 2-4-fold increases in the amounts of the 4S- and 6S-substituted GAG chains for the PTN-treated chondrocytes. Northern analysis showed a twofold upregulation in the mRNA levels of biglycan and collagen type II, but no difference in the message levels for decorin and aggrecan. These results establish that PTN inhibits cell proliferation, while stimulating the synthesis of proteoglycans in mature chondrocytes in vitro, suggesting that PTN may act directly or indirectly to regulate growth and proteoglycan synthesis in the developing matrix of fetal cartilage.

Hyaluronan suppresses fibronectin fragment-mediated damage to human cartilage explant cultures by enhancing proteoglycan synthesis.

Hyaluronic acid, recently renamed hyaluronan, has been used as a therapeutic intervention in the treatment of osteoarthritis. We have reported that high-molecular-weight (800 kDa) hyaluronan is effective in blocking the catabolic action of fibronectin fragments in explant cultures of bovine cartilage and in an experimental in vivo model of damage to the rabbit knee joint. The fibronectin fragments induce catabolic cytokines in human cartilage, which, in turn, suppress proteoglycan synthesis and induce matrix metalloproteinases to decrease the proteoglycan content. Since the clinical target of high-molecular-weight hyaluronan is human cartilage, which may differ in certain ways from bovine cartilage, we tested the effect on human knee cartilage. We found that 1 mg/ml hyaluronan completely blocked fibronectin fragment-mediated decreases in proteoglycan content in five of five specimens of cartilage from the human knee. This was associated with binding of exogenous hyaluronan to the superficial surface, suppressed penetration of the fibronectin fragment into the cartilage, decreased expression for the first week in culture of one of the matrix metalloproteinases involved in cartilage degradation, matrix metalloproteinase-3, and proteoglycan synthesis rates that increased to supernormal levels. However, the appearance of the NITEGE and VDIPEN neoepitopes, indices of cartilage degradation, was not blocked but was delayed by 1 week. The addition of hyaluronan to cartilage previously damaged by the fibronectin fragments or to osteoarthritic cartilage fully restored the proteoglycan content to control levels. We conclude that hyaluronan blocked damage at least partly by blocking penetration of the fibronectin fragments and slowing matrix metalloproteinase expression. However, the major effect on blocking damage and promoting repair may be through enhanced proteoglycan synthesis, a mechanism that requires further study. Nonetheless, these data clearly demonstrate that hyaluronan completely protected human cartilage in explant culture and facilitated a full restoration of proteoglycan in damaged cartilage.

In vitro stimulation of equine articular cartilage proteoglycan synthesis by hyaluronan and carprofen

The effects of hyaluronan and carprofen (both racemic mixture and separate R and S enantiomers) on proteoglycan (PG) synthesis by equine cultured chondrocytes and cartilage explants were examined. Hyaluronan stimulatedPG synthesis in both cell and explant cultures. The concentration–response curve of the latter was bell-shaped. Racemic carprofen and R and S enantiomers also stimulated PG synthesis, although concentration–response relationships varied for each preparation and high concentrations inhibited synthesis. It was concluded that (a) hyaluronan exerts a stimulatory effect on PG synthesis at low concentrations and (b) stimulatory effects of carprofen on PG synthesis are, to some degree, enantioselective with the carprofen S-enantiomer exerting the greatest effect. Hyaluronan and carprofen are used clinically despite incompletely understood mechanisms of action. These results suggest (a) hyaluronan and carprofen might exert an anti-arthritic action through stimulation of PG synthesis and (b) there is possible justification for therapeutic administration of enantiomeric rather than racemic carprofen.

Parathyroid Hormone-(1–34) Enhances Aggrecan Synthesis via an Insulin-like Growth Factor-I Pathway

During endochondral bone formation, the growth plate chondrocytes proliferate, become hypertrophic, lose the cartilage phenotype, undergo mineralization, and provide a scaffold upon which subsequent longitudinal bone growth occurs. Parathyroid hormone (PTH), a calcium-regulating hormone, and parathyroid hormone-related peptide (PTHrP), which shares several properties with PTH, have profound effects on skeletal growth and new bone formation. In order to define further the mechanism by which PTH/PTHrP promotes the cartilage phenotype, chondrocytes isolated from the rib cages of developing rat embryos were evaluated for the biosynthesis of aggrecan. Cells treated with PTH-(1-34) for a 4-h period followed by a 20-h recovery period showed a significant increase in cartilage proteoglycan (aggrecan) synthesis in a dose-dependent manner. Only N-terminally intact PTH and PTHrP were effective in stimulating aggrecan synthesis. Addition of a neutralizing antibody to insulin-like growth factor-I (IGF-I) during PTH treatment resulted in the inhibition of PTH-stimulated aggrecan synthesis, whereas the addition of a neutralizing antibody to insulin-like growth factor-binding protein-2 (IGFBP-2) resulted in an increase in synthesis in both the control and PTH-treated cells. In addition, PTH treatment resulted in an increase in the mRNA for aggrecan, a reduction in IGFBP-3 mRNA, and no discernible changes in IGF-I mRNA levels, which was complemented by quantitative changes in IGFBP-3 and free IGF-I levels. The reciprocal relationship in the expression of aggrecan and IGFBP was further confirmed in chondrocytes from various gestational stages during normal development. Collectively, our results indicate that the effect of PTH may be mediated at least in part through the regulation of the IGF/IGFBP axis, by a decrease in the level of IGFBP-3, and an increase in free IGF-I levels. It is likely that the local increase in IGF-I may lead to an increase in cartilage type proteoglycan synthesis and maintenance of the cartilage phenotype. The consequence of the prolonged maintenance may be to halt mineralization while a new scaffolding is created.

Effects of the NSAIDs meloxicam and indomethacin on cartilage proteoglycan synthesis and joint responses to calcium pyrophosphate crystals in dogs.

NSAIDs are a major cause for concern for their propensity to cause joint deterioration in canine, as in human, patients receiving these drugs for treatment of pain in osteoarthritis and other acute and chronic painful conditions. To determine the potential effects of the new NSAID meloxicam on cartilage integrity, the effects of this drug on proteoglycan biosynthesis in vitro and ex vivo were compared with those of indomethacin, a known inhibitor of sulphated proteoglycans that accelerates joint injury in human osteoarthritis. In vitro cartilage proteoglycan synthesis from a radiosulphate precursor was unaffected by 0.5-10.0 micromol/L meloxicam but was significantly inhibited by 50 micromol/L indomethacin after 6 or 24 h incubation of femoral or tibial cartilage explants in organ culture. This is in accord with previous observations in human or porcine articular cartilage under the same culture conditions. Studies were performed in vivo to establish the effects of the NSAIDs on joint integrity. This involved determining cartilage proteoglycan synthesis ex vivo, leukocyte, fluid and protein accumulation, as well as pain relief. Thus, meloxicam (0.2 mg/kg i.v. x 3 doses) or indomethacin (0.5 mg/kg i.v. x 3 doses) was given for 26 h and the effects were compared with a control (1.0 ml saline i.v. x 3 doses) in dogs in which acute inflammation had been induced by intra-articular (i.a.) injection of calcium pyrophosphate dihydrate (CPPD) crystals into the right stifle joint, an equivalent volume of saline being injected into the left stifle joint as a control. No effects were observed of the treatment with the NSAIDs on ex vivo sulphated proteoglycan synthesis. The lack of the expected inhibitory effects of indomethacin may be related to the relatively low plasma concentrations of this drug obtained during the 26 h period of treatment. The pain response, which was elicited up to 6 h following i.a. injection of CPPD crystals, was totally prevented by the treatment with meloxicam and to a lesser extent with indomethacin. There were no effects from the drug treatment on synovial inflammatory reactions (fluid and cell accumulation), although the protein concentration of the exudate was reduced by meloxicam. This indicates that, at the doses given, it was possible to discriminate the analgesic action from the anti-inflammatory action of the two NSAIDs, this being achieved at relatively low plasma concentrations of these drugs. In conclusion, while relatively high therapeutic concentrations of indomethacin inhibit cartilage proteoglycan synthesis, this is not an effect seen even at high concentrations of meloxicam. Furthermore, the lack of effects on proteoglycan synthesis was evident when these two drugs were given in vivo to dogs. However, the signs of pain, but not the inflammation in the joint, were relieved by low plasma concentrations of the drugs. Meloxicam may thus be safely employed for acute analgesia without the potential risks of joint cartilage damage that occurs with indomethacin given at antiinflammatory doses for long periods of time.

Differential effects of local application of BMP-2 or TGF-β1 on both articular cartilage composition and osteophyte formation

Objective: The related molecules bone morphogenetic protein-2 (BMP-2) and transforming growth factor beta-1 (TGF-β1) have both been shown to stimulate chondrocyte proteoglycan (PG) synthesisin vitro. We investigated thein-vivoeffects of these factors on articular cartilage PG metabolism.Design: Several doses of BMP-2 or TGF-β1 were injected into the murine knee joint, once or repeatedly. Patellar cartilage PG synthesis was measured by [35S]-sulfate incorporation and reverse transcriptase polymerase chain reaction (RT-PCR). PG content was analyzed by measuring safranin O staining intensity on histologic sections.Results: A single injection of 200 ng BMP-2 induced a much earlier and more impressive stimulation of articular cartilage PG synthesis, than 200 ng TGF-β1. RT-PCR revealed that both factors upregulated mRNA of aggrecan more than that of biglycan and decorin. However, 21 days after a single injection of 200 ng TGF-β1 PG synthesis still was significantly increased, while stimulation by BMP-2 only lasted for 3 to 4 days. Stimulation by BMP-2 could be prolonged to at least 2 weeks by triple injections of 200 ng each, at alternate days. Remarkably, even after this intense exposure to BMP-2, stimulation of PG synthesis was not reflected in long-lasting enhancement of PG content of articular cartilage. In contrast, even a single injection with 200 ng of TGF-β1 induced prolonged enhancement of PG content. After repeated injections, both BMP-2 and TGF-β1 induced chondrogenesis at specific sites. ‘Chondrophytes’ induced by BMP-2 were found predominantly in the region where the growth plates meet the joint space, while those triggered by TGF-β1 originated from the periosteum also at sites remote from the growth plates.Conclusions: BMP-2 and TGF-β stimulate PG synthesis and PG content with different kinetics, and these factors have different chondro-inductive properties.

Effect of dimethylsulfoxide on articular cartilage proteoglycan synthesis and degradation, chondrocyte viability, and matrix water content.

To determine the effects of dimethylsulfoxide (DMSO) exposure on cartilage proteoglycan (PG) synthesis, PG degradation, chondrocyte viability, and matrix water content. Using a cartilage explant culture system, PG synthesis, PG degradation, matrix water content, and chondrocyte viability were determined for cartilage exposed to DMSO daily for selected periods of time. Juvenile bovine (calf) carpometacarpal joint cartilage explants. PG synthesis: Explants (n = 30/group) were separated into 10 groups based on the time of daily exposure to 10% DMSO. Exposure time was repeated daily for 3 days. The control group was incubated in basal medium alone for 3 days, with daily medium changes. Once all DMSO exposure times were complete for the third day, PG synthesis was determined by analysis of incorporation of radiolabelled sulfate. Cell viability: Explants (n = 3/group) were subjected to an identical DMSO exposure protocol, and examined histologically. The percentage of viable cells/high power field (hpf) was calculated for each group. PG degradation: Explants (n = 21/group) were preincubated with radiolabelled sulfate, then subjected to a similar DMSO exposure protocol. The medium was collected from all explants daily and assayed for PG content. After 3 days, the explants were digested and total labelled PG content determined. Percent of total explant labelled PG content released into the medium daily was determined for each group. Water content: Explants (n = 21/group) were separated into three treatment groups, one of which had no treatments performed, whereas the other two groups were incubated in basal medium for 72 hours, one with, and one without, 10% DMSO. Wet and dry weights were determined, and percent water calculated, for all three groups. Separate 1-way ANOVA were performed, with appropriate post hoc tests (P < .05). PG synthesis was significantly lower than control for all time periods of DMSO exposure except for 1 and 3 hours, and decreased in a time-dependent manner after the 1-hour exposure time. The mean percentage of viable cells/hpf was significantly lower than control for the 1-, 3-, 9-, 12-, and 24-hour treatment groups. There was no significant difference in PG degradation for any group compared with control for the first 2 days of incubation. All groups except the 24-hour group had a significantly higher degradation compared with control for the third day of incubation. Cartilage exposed to DMSO for 72 hours had a significantly lower water content, and cartilage incubated in basal medium alone for 72 hours had a significantly higher water content than cartilage that received no DMSO and no incubation. DMSO, in relatively low concentration, is detrimental to articular cartilage PG synthesis in a time-dependent manner. Dehydration of the cartilage and chondrocyte death also occur with increasing time of DMSO exposure. Significant PG degradation occurs on the third day of culture with daily DMSO exposure. As a joint lavage solution, DMSO has potentially deleterious effects on the metabolism of chondrocytes.

Enhanced synovial production of hyaluronic acid may explain rapid clinical response to high-dose glucosamine in osteoarthritis

Anecdotal reports of rapid symptomatic response to high-dose glucosamine in osteoarthritis are not credibly explained by the traditional view that glucosamine promotes synthesis of cartilage proteoglycans. An alternative or additional possibility is that glucosamine stimulates synovial production of hyaluronic acid (HA), which is primarily responsible for the lubricating and shock-absorbing properties of synovial fluid. Many clinical and veterinary studies have shown that intra-articular injections of high-molecular-weight HA produce rapid pain relief and improved mobility in osteoarthritis. HA has anti-inflammatory and analgesic properties, and promotes anabolic behavior in chondrocytes. The concentration and molecular weight of synovial fluid HA are decreased in osteoarthritis; by reversing this abnormality, high-dose glucosamine may provide rapid symptomatic benefit, and in the longer term aid the repair of damaged cartilage.

Load-Controlled Compression of Articular Cartilage Induces a Transient Stimulation of Aggrecan Gene Expression

The effects of short- and long-term load-controlled compression on the levels of aggrecan mRNA have been determined. Results show that a compressive stress of 0.1 MPa on bovine articular cartilage explants for 1, 4, 12, and 24 h produces a transient up-regulation of aggrecan mRNA synthesis. At 1 h, aggrecan mRNA levels in loaded explants were increased 3.2-fold compared to control explants. At longer times (≥4 h), the levels of aggrecan mRNA returned to baseline values or stayed slightly higher. There is a dose dependence in the response of the explant to increasing levels of compressive stress (0–0.5 MPa) for 1 h. Aggrecan mRNA levels increased 2- to 3-fold at 0–0.25 MPa. At 0.5 MPa, the level of aggrecan mRNA was lower than those at 0.1 and 0.25 MPa. This dose-dependent effect suggests a reversal of the stimulatory effects of compression on aggrecan gene expression at higher loads. After 24 h of compression, the levels of aggrecan mRNA in explants subjected to any of the stress levels were not significantly different from those in control explants. The stimulatory effect of 0.1 MPa compressive stress on aggrecan mRNA levels was blocked by Rp-cAMP and U-73122, indicating the involvement of the classical signal transduction pathways in the mechanical modulation of aggrecan gene expression. The responses of link protein mRNA to compression paralleled those of aggrecan, while there was no significant change in expression of the gene for the housekeeping protein elongation factor-1α. The results indicate that articular cartilage chondrocytes can respond to short-term compressive loads by transiently up-regulating expression of the aggrecan gene. The fact that long-term compression did not significantly alter aggrecan mRNA levels suggests that previously observed inhibitory effects of prolonged static compression on proteoglycan synthesis in articular cartilage may be, for the most part, mediated through mechanisms other than suppression of aggrecan mRNA levels.