Metabolic and mitogenic activities of insulin-like growth factor-1 in interleukin-1-conditioned equine cartilage

OBJECTIVE: To determine response of interleukin-1alpha (IL-1alpha)-conditioned equine articular cartilage explants to insulin-like growth factor-1 (IGF-1). Sample Population-Cartilage from the trochlea and condyles of the femur of a clinically normal 4-year-old horse. PROCEDURE: Effects of IGF-1 (0 to 500 ng/ml) after addition of IL-1alpha were evaluated by assessing matrix responses, using a sulfated glycosaminoglycan (GAG) assay, matrix 35SO4 GAG incorporation, and release of GAG. Mitogenic response was assessed by 3H-thymidine incorporation into DNA and fluorometric assay of total DNA concentration. RESULTS: Human recombinant IL-1alpha (40 ng/ml) increased the amount of labeled GAG released and decreased labeled and total GAG remaining in explants, and IL-1alpha decreased mitogenic response. Addition of IGF-1 counteracted effects seen with IL-1alpha alone. In general, IGF-1 decreased total and labeled GAG released into the medium, compared with IL-1alpha-treated explants (positive-control sample). Values for these variables did not differ significantly from those for negative-control explants. A significant increase in total and newly synthesized GAG in the explants at termination of the experiment was observed with 500 ng of IGF-1/ml. Labeled GAG remaining in explants was greater with treatment at 50 ng of IGF-1/ml, compared with treatment with IL-1alpha alone. Concentrations of 200 ng of IGF-1/ml abolished actions of IL-1alpha and restored DNA synthesis to values similar to those of negative-control explants. CONCLUSIONS AND CLINICAL RELEVANCE: IGF-1 at 500 ng/ml was best at overcoming detrimental effects associated with IL-1alpha in in vitro explants. These beneficial effects may be useful in horses with osteoarthritis.     

Effects of dosage titration of methylprednisolone acetate and triamcinolone acetonide on interleukin-1-conditioned equine articular cartilage explants in vitro

REASONS FOR PERFORMING STUDY: Osteoarthritis is a frequent sequela of joint disease, especially with severe injuries or if attempts at therapy are unsuccessful. Negative and positive effects of corticosteroid treatment of articular cartilage have been demonstrated by in vitro and in vivo studies. OBJECTIVES: To assess the metabolic effects of varying dosages of methylprednisolone acetate (MPA) and triamcinolone acetonide (TA) on interleukin-1alpha (IL-1) conditioned equine cartilage explants. Our hypothesis was that lower dosages of corticosteroids would be less detrimental to cartilage metabolism than higher dosages. TA would be less detrimental to cartilage metabolism than MPA. METHODS: Treatment groups included articular cartilage explants with no IL-1 (control), IL-1 alone, and IL-1 plus 10, 5, 1 and 0.5 mg/ml MPA or 1.2, 0.6, 0.12 and 0.06 mg/ml TA. Explants were labelled with 35SO4 prior to the beginning and end of the experiment to assess glycosaminoglycan (GAG) degradation and synthesis, respectively. Total GAG content in media and explants and total cartilage DNA were also analysed. RESULTS: MPA and TA reduced GAG synthesis compared to control and IL-1 alone. The highest dosage of MPA (10 mg/ml) reduced GAG synthesis less than lower dosages of MPA and all dosages of TA. Compared to IL-1 alone, all dosages of TA and lower dosages of MPA increased GAG degradation. MPA at 10 mg/ml reduced GAG degradation. Both MPA and TA increased media GAG content compared to control and IL-1 explants. Total cartilage GAGs were unchanged with MPA, but reduced with TA, compared with IL-1 alone. Total cartilage DNA was decreased with MPA and increased with TA compared to IL-1 and control explants. CONCLUSIONS: MPA and TA did not counteract the negative effects of IL-1 and did not maintain cartilage metabolism at control levels. Lower dosages of MPA and TA were not less detrimental to cartilage metabolism than higher dosages. TA did not appear to be less harmful than MPA on cartilage metabolism. The results of this study differ from the findings of comparable in vivo studies. POTENTIAL RELEVANCE: The low numbers of horses used in this study limits extrapolation of these findings to the equine population; however, this study also questions the clinical relevance of this in vitro model.     

Effects of triamcinolone acetonide, sodium hyaluronate, amikacin sulfate, and mepivacaine hydrochloride, alone and in combination, on morphology and matrix composition of lipopolysaccharide-challenged and unchallenged equine articular cartilage explants

OBJECTIVE: To evaluate the effects of triamcinolone acetonide (TA), sodium hyaluronate (HA), amikacin sulfate (AS), and mepivacaine hydrochloride (MC) on articular cartilage morphology and matrix composition in lipopolysaccharide (LPS)-challenged and unchallenged equine articular cartilage explants. Sample POPULATION: 96 articular cartilage explants from 4 femoropatellar joints of 2 adult horses. PROCEDURES: Articular cartilage explants were challenged with LPS (100 ng/mL) or unchallenged for 48 hours, then treated with TA, HA, AS, and MC alone or in combination for 96 hours or left untreated. Cartilage extracts were analyzed for glycosaminoglycan (GAG) content by dimethyl-methylene blue assay (ng/mg of dry wt). Histomorphometric quantification of total lacunae, empty lacunae, and lacunae with pyknotic nuclei was recorded for superficial, middle, and deep cartilage zones. RESULTS: LPS induced a significant increase in pyknotic nuclei and empty lacunae. Treatment with TA or HA significantly decreased empty lacunae (TA and HA), compared with groups without TA or HA, and significantly decreased empty lacunae of LPS-challenged explants, compared with untreated explants. Treatment with AS or MC significantly increased empty lacunae in unchallenged explants, and these effects were attenuated by TA. Treatment with MC significantly increased empty lacunae and pyknotic nuclei and, in combination with LPS, could not be attenuated by TA. Content of GAG did not differ between unchallenged and LPS-challenged explants or among treatments. CONCLUSIONS AND CLINICAL RELEVANCE: Treatment with TA or HA supported chondrocyte morphology in culture and protected chondrocytes from toxic effects exerted by LPS, AS, and MC.     

Effects of sodium hyaluronate and methylprednisolone acetate on proteoglycan synthesis in equine articular cartilage explants

OBJECTIVE: To determine effects of sodium hyaluronate (HA) on corticosteroid-induced cartilage matrix catabolism in equine articular cartilage explants. SAMPLE POPULATION: 30 articular cartilage explants from fetlock joints of 5 adult horses without joint disease. PROCEDURE: Articular cartilage explants were treated with control medium or medium containing methylprednisolone acetate (MPA; 0.05, 0.5, or 5.0 mg/mL), HA (0.1, 1.0, or 1.5 mg/mL), or both. Proteoglycan (PG) synthesis was measured by incorporation of sulfur 35-labeled sodium sulphate into PGs, and PG degradation was measured by release of radiolabeled PGs into the medium. Total glycosaminoglycan (GAG) content in media and explants and total explant DNA were determined. RESULTS: Methylprednisolone acetate caused a decrease in PG synthesis, whereas HA had no effect. Only the combination of MPA at a concentration of 0.05 mg/mL and HA at a concentration of 1.0 mg/mL increased PG synthesis, compared with control explants. Methylprednisolone acetate increased degradation of newly synthesized PGs into the medium, compared with control explants, and HA alone had no effect. Hyaluronate had no effect on MPA-induced PG degradation and release into media. Neither MPA alone nor HA alone had an effect on total cartilage GAG content. Methylprednisolone acetate caused an increase in release of GAG into the medium at 48 and 72 hours after treatment. In combination, HA had no protective effect on MPA-induced GAG release into the medium. Total cartilage DNA content was not affected by treatments. CONCLUSIONS AND CLINICAL RELEVANCE: Our results indicate that HA addition has little effect on corticosteroid-induced cartilage matrix PG catabolism in articular cartilage explants.     

Proteoglycan metabolism of equine articular cartilage and its modulation by insulin-like growth factors

The effect of human recombinant insulin-like growth factor 1 (rhIGF-1) on proteoglycan (PG) metabolism of full thickness equine articular cartilage explants was investigated. PG synthesis was stimulated at all ages, but higher concentrations of rhIGF-1 were required for maximal stimulation of adult cartilage. There were no changes in the hydrodynamic size, electrophoretic heterogeneity or composition of proteoglycans isolated from rhIGF-1-stimulated cartilage. rhIGF-1 reduced the rate of turnover of both newly synthesized and endogenous proteoglycans in all ages of cartilage investigated. The structure of proteoglycan fragments retained within the matrix and those released into the culture medium was unaffected by IGF-1 stimulation, suggesting that this peptide is a key regulator of the proteoglycan composition of equine articular cartilage extracellular matrix.     

Effects of sodium hyaluronate and methylprednisolone acetate on proteoglycan metabolism in equine articular chondrocytes treated with interleukin-1

OBJECTIVE: To determine the effects of sodium hyaluronate (HA) in combination with methylprednisolone acetate (MPA) on interleukin-1 (IL-1)-induced inflammation in equine articular cartilage pellets. Sample POPULATION: Chondrocytes collected from 7 horses euthanatized for problems unrelated to the musculoskeletal system. PROCEDURES: Chondrocyte pellets were treated with medium (negative control); medium containing IL-1 (positive control); or medium containing IL-1 with MPA only (0.05 or 0.5 mg/mL), HA only (0.2 or 2 mg/mL), or MPA (0.05 or 0.5 mg/mL) and HA (0.2 or 2 mg/mL) in combination. Proteoglycan (PG) synthesis was determined by incorporation of sulfur 35-labeled sodium sulfate into PGs. Glycosaminoglycan (GAG) content of the media and the pellets and total pellet DNA content were determined. RESULTS: Methylprednisolone acetate at 0.5 mg/mL caused an increase in PG synthesis, whereas HA had no effect alone. The combination of MPA, both 0.05 mg/mL and 0.5 mg/mL, with HA at 2 mg/mL increased PG synthesis, compared with IL-1-treated control. All treatment groups containing the high concentration of MPA (0.5 mg/mL) and the high concentration of HA (2.0 mg/mL) had pellets with increased GAG content. The addition of HA caused an increase in total GAG content in the media, regardless of MPA treatment. Cyclooxygenase-2 mRNA and aggrecan mRNA expression was significantly reduced with MPA treatment. Total pellet DNA content was unchanged by any treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Our results indicate that MPA in combination with HA has beneficial effects on PG metabolism of IL-1-treated equine chondrocytes.     

Effects of insulin-like growth factor-II on the mitogenic and metabolic activities of equine articular cartilage with and without interleukin 1-beta

OBJECTIVE: To investigate the effects of insulin-like growth factor-II (IGF-II) on DNA and glycosaminoglycan (GAG) synthesis and the expression of matrix-related genes in equine articular cartilage explants and chondrocytes, respectively, with and without interleukin 1-beta (IL1-beta). SAMPLE POPULATION: Articular cartilage from 12 adult horses. PROCEDURE: Articular cartilage was incubated in standard media with and without equine IL1-beta (10 ng/mL) containing various concentrations of IGF-II for 72 hours. Synthesis of DNA and GAG was determined by incorporation of thymidine labeled with radioactive hydrogen (3H) and sulfate labeled with radioactive sulfur (35S), respectively. Total GAG content of the explants and spent media was determined by use of the 1,9-dimethylmethylene blue assay. Northern blots of RNA from cultured equine articular cartilage chondrocytes were hybridized with cDNA of major matrix molecules. RESULTS: Insulin-like growth factor-II stimulated DNA and GAG synthesis at concentrations of 25 and 50 ng/mL, respectively. In cartilage explants conditioned with IL1-beta, IGF-II stimulated DNA and GAG synthesis at concentrations of 500 and 50 ng/mL, respectively. Insulin-like growth factor-II had no effect on total GAG content as determined by the 1,9-dimethylmethylene blue assay. No specific effects on steady-state levels of messenger RNAs were observed. CONCLUSIONS AND CLINICAL RELEVANCE: Insulin-like growth factor-II stimulated DNA and GAG synthesis in equine adult cartilage and may have potential application in vivo.     

Consideration of the role of antigenic keratan sulphate reacting to a 1/14/16H9 antibody as a molecular marker to monitor cartilage metabolism in horses

The role of keratan sulphate (KS) as a marker of cartilage metabolism was evaluated by using an in vitro model of equine articular cartilage. Articular cartilage was harvested from clinically healthy 6-month-old foals (n=3). Chondrocytes were centrifuged and cultured as pellets. Chondrocyte pellets were stimulated by insulin-like growth factor (IGF)-Ialpha or interleukin (IL)-1alpha for 2 weeks. The sulfated glycosaminoglycans (GAG) and antigenic KS concentrations in the culture media were measured by a 1,9-dimethyl-methylene blue (DMMB) colorimetric assay and an inhibition ELISA using a 1/14/16H9 antibody, respectively. Concentration of GAG was significantly increased in the media of pellets stimulated by both IGF-Ialpha and IL-1alpha. Antigenic KS concentration was significantly increased in those stimulated by IL-1alpha, while no significant change was found in those stimulated by IGF-Ialpha. A high correlation between GAG and antigenic KS concentrations was found in the media of pellets stimulated by IL-1alpha (r=0.87), but not in those stimulated by IGF-Ialpha (r=0.43). The results suggest that the concentration of antigenic KS reacting to 1/14/16H9 mirrors the GAG concentration during the stage of cartilage catabolism, but not during the cartilage anabolic stage. The concentration of antigenic KS reacting to 1/14/16H9 antibody in biological fluids could therefore be a useful marker to further understand principally the catabolic and slightly the anabolic process of articular cartilage metabolism.     

Assessment of the catabolic effects of interleukin-1beta on proteoglycan metabolism in equine cartilage cocultured with synoviocytes

OBJECTIVE: To evaluate the effects of interleukin (IL)-1beta on proteoglycan metabolism in equine cartilage explants when cultured in the presence of synoviocytes. SAMPLE POPULATION: Samples of cartilage and synovium collected from the femoropatellar joints of three 2- to 3-year-old horses. PROCEDURES: 3 experimental groups were established: cartilage explants only, synoviocytes only, and cartilage explants-synoviocytes in coculture. In each group, samples were cultured with or without IL-1beta (10 ng/mL) for 96 hours. Glycosaminoglycan (GAG) content of cartilage and medium samples was measured by use of a spectrophotometric assay; RNA was isolated from synoviocytes and cartilage and analyzed for expression of matrix metalloproteinases (MMP)-3 and -13 (cartilage and synoviocytes), aggrecan (cartilage), collagen type IIB (cartilage), and 18S as a control (cartilage and synoviocytes) by use of quantitative PCR assays. Cartilage matrix metachromasia was assessed histochemically. RESULTS: IL-1beta-induced GAG loss from cartilage was significantly less in cocultures than in cartilage-only cultures. Cartilage aggrecan gene expression was also significantly less downregulated and synoviocyte MMP-3 expression was less upregulated by IL-1beta in cocultures, compared with cartilage- and synoviocyte only cultures. Histochemical findings supported the molecular and biochemical results and revealed maintenance of matrix metachromasia in cocultured cartilage treated with IL-1beta. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that synoviocytes secrete 1 or more mediators that preferentially protect matrix GAG metabolism from the degradative effects of IL-1beta. Further studies involving proteomic and microarray approaches in similar coculture systems may elucidate novel therapeutic targets for the treatment of osteoarthritis.     

Effects of glucosamine hydrochloride and chondroitin sulphate, alone and in combination, on normal and interleukin-1 conditioned equine articular cartilage explant metabolism

REASONS FOR PERFORMING STUDY: Clinical trials in human and veterinary literature have documented the benefits of oral nutraceutical joint supplements containing glucosamine (GU) and chondroitin sulphate (CS) to treat mild to moderate osteoarthritis, but the effects of these components have not yet been conclusively determined. OBJECTIVES: To assess varying dosages of GU and CS on normal and interleukin-1alpha (IL-1) conditioned equine cartilage explants and rationalise the use of these products. HYPOTHESIS: Treatment would not be detrimental to cartilage metabolism and higher dosages and the combination of GU and CS would be more beneficial than lower dosages and. GU or CS alone. METHODS: Articular cartilage explants collected from the femoral trochlea and condyles were cultured in normal and IL-1 conditioned media. Treatment groups included 0, 12.5, 25,125 and 250 microg/ml concentrations of GU alone, CS alone, or GU+CS in combination. Glycosaminoglycan (GAG) synthesis and total GAG content in the explants and media were analysed. RESULTS: There were no detrimental effects of GU, CS or GU+CS on cartilage metabolism. High dosages of GU+CS reduced total GAG release into the media (degradation). CONCLUSIONS: Our results suggests that GU+CS may prevent cartilage GAG degradation. POTENTIAL RELEVANCE: The combination of GU and CS may be more effective in preventing or treating osteoarthritis in horses than either product alone.     

Effects of equine recombinant interleukin-1alpha and interleukin-1beta on proteoglycan metabolism and prostaglandin E2 synthesis in equine articular cartilage explants

OBJECTIVES: To evaluate the effects of equine recombinant interleukin-1alpha (rEqIL-1alpha) and recombinant interleukin-1beta (rEqIL-1beta) on proteoglycan metabolism and prostaglandin E2 (PGE2) synthesis by equine articular chondrocytes in explant culture. SAMPLE POPULATION: Near full-thickness articular cartilage explants (approx 50 mg) harvested from stifle joints of a 3-year-old and a 5-year-old horse. PROCEDURE: Expression constructs containing cDNA sequences encoding EqIL-1alpha and EqIL-1beta were generated, prokaryotically expressed, and the recombinant protein purified. Near full-thickness articular cartilage explants (approx 50 mg) harvested from stifle joints of a 3-year-old and a 5-year-old horse were separately randomized to receive rEqIL-1alpha or rEqIL-1beta treatments 10 to 500 ng/ml). Proteoglycan release was evaluated by 1,9-dimethylmethylene blue spectrophotometric analysis of explant media glycosaminoglycan (GAG) concentration and release of 35S-sulfate-labeled GAG to explant media. Proteoglycan synthesis was assessed by quantification of 35S-sulfate incorporation into proteoglycan. Explant media PGE2 concentrations were evaluated using a PGE2-specific enzyme-linked immunoassay. Data were collected at 48-hour intervals and normalized by DNA content. RESULTS: Proteoglycan release was induced by rEqIL-1alpha and rEqIL-1beta at concentrations > or =0.1 ng/ml, with 38 to 76% and 88 to 98% of total GAG released by 4 and 6 days, respectively. Inhibition of proteoglycan synthesis (42 to 64%) was observed at IL-1 concentrations > or = 0.1 ng/ml at 2 and 4 days. Increased PGE2 concentrations were observed at IL-1 concentrations > or = 0.1 ng/ml at 2 and 4 days. CONCLUSIONS AND CLINICAL RELEVANCE: The rEqIL-1 induced potent concentration-dependent derangement of equine chondrocyte metabolism in vitro. These findings suggest this model may be suitable for the in vitro study of the pathogenesis and treatment of joint disease in horses.     

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

OBJECTIVE: To evaluate the effects of dimethyl sulfoxide (DMSO) on equine articular cartilage matrix metabolism. STUDY DESIGN: 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. SAMPLE POPULATION: Cartilage harvested from metacarpophalangeal and metatarsophalangeal joints of 12 horses (age range, 1 to 10 years). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. CLINICAL RELEVANCE: Equine clinicians adding DMSO to intraarticular lavage solutions should be aware that DMSO may have deleterious effects on equine articular cartilage matrix metabolism.     

Differential anti-inflammatory and chondroprotective effects of simulated digests of indomethacin and an herbal composite (Mobility) in a cartilage explant model of articular inflammation

Herbs are an increasingly popular treatment option for horses with cartilage inflammation, despite a relative paucity of research demonstrating efficacy. The research objective was to evaluate the differential anti-inflammatory and chondroprotective efficacy of a simulated digest of indomethacin and a commercially available herbal product in a cartilage model of osteoarthritis. Cartilage explant was integrated with simulated digestion of indomethacin and the herbal product in order to account, at least in part, for the actions of major digestive enzymes and pH. The resulting digests were ultrafiltrated (50 kDa), to account for absorption from the GI tract and movement into the cartilage matrix. We hypothesized that (i) a simulated digest of indomethacin would block interleukin 1 beta-(IL-1) dependent formation of prostaglandin E2 (PGE2) and nitric oxide (NO) without protecting cartilage against IL-1-induced glycosaminoglycan (GAG) release, and (ii) the herbal product would reduce PGE2 and NO in IL-1-stimulated explants, and inhibit release of GAG, in IL-1-stimulated explants. Results showed that indomethacin is an effective anti-inflammatory, evidenced by strong inhibition of IL-1-induced PGE2 and NO from cartilage explants. However, indomethacin provided no protection against IL-1-induced GAG release. Simulated digest of the herbal extract significantly inhibited IL-1-induced NO production and GAG release, while having a slight increase in PGE2. These data provide evidence for the anti-inflammatory effect of indomethacin on IL-1-stimulated cartilage explants, and the herbal product Mobility may be a useful adjunct in arthritis because of its chondroprotective properties in IL-1-stimulated cartilage.     

Evaluation of the role of keratan sulphate as a molecular marker to monitor cartilage metabolism in horses

The role of keratan sulphate (KS) as a metabolic marker of cartilage was evaluated using an in vitro model of equine articular cartilage. Articular cartilage was harvested from clinically healthy 6-month-old foals (n = 3). Chondrocytes were centrifuged and cultured as pellets. Chondrocyte pellets were stimulated by insulin-like growth factor-I alpha (IGF-I alpha) or interleukin-1 alpha (IL-1 alpha) for 2 weeks. The concentrations of sulphated glycosaminoglycans (GAG) and KS in the culture media were measured by a 1,9-dimethyl-methylene blue (DMMB) colorimetric assay and an inhibition enzyme-linked immunosorbent assay using a 1/20/5D4 antibody, respectively. The concentration of GAG was significantly increased both in the media of pellets stimulated by IGF-I alpha and in those stimulated by IL-1 alpha. KS concentration was significantly increased in those stimulated by IL-1 alpha, while no significant change was found in those stimulated by IGF-I alpha. A high correlation between GAG and KS concentrations was found in the media of pellets stimulated by IL-1 alpha (r = 0.84), but not in those stimulated by IGF-I alpha (r = 0.59). The results suggest that the concentration of KS reacting to 1/20/5D4 mirrors the GAG concentration during the stage of cartilage catabolism, but not during the cartilage anabolic stage. The KS concentration in biological fluids could therefore be a useful marker to understand further the cartilage catabolic process. It may also represent some aspects of the cartilage anabolic process.     

The effects of radial shock waves on the metabolism of equine cartilage explants in vitro

AIM: To investigate, in vitro, the effects of radial shock waves on the release of nitric oxide (NO) and synthesis of prostaglandin E2 (PGE2) and glycosaminoglycan (GAG), and liberation of GAG, from equine articular cartilage explants. METHODS: Equine cartilage from normal metacarpophalangeal and metatarsophalangeal joints was exposed to radial shock waves at various impulse doses and then maintained as explants in culture for 48 h. Shock waves were delivered at 1,876 Torr pressure and a frequency of 10 Hz. Treatment groups consisted of a negative control group, or application of 500, 2,000, or 4,000 impulses by use of either a convex handpiece (Group A) or concave handpiece (Group B). Synthesis of GAG was measured using incorporation of 35S-labelled sodium sulphate. Additionally, the synthesis of NO and PGE2, and content of GAG of the explants and media were determined. RESULTS: No significant effects (p>0.05) of radial shock-wave treatment were evident on the synthesis of NO or PGE2, or release of GAG by cartilage explants. However, radial shock waves decreased synthesis of GAG measured 48 h after exposure for all treatment groups other than the 500-impulse Group-A explants (p<0.05). CONCLUSIONS: Radial shock waves impact the metabolism of GAG in chondrocytes in equine articular cartilage. Further studies will be required to fully investigate the impact of this effect on the health of joints, and to elucidate the clinical impact.     

Effects of enrofloxacin and magnesium deficiency on matrix metabolism in equine articular cartilage

OBJECTIVE: To investigate the effects of enrofloxacin and magnesium deficiency on explants of equine articular cartilage. SAMPLE POPULATION: Articular cartilage explants and cultured chondrocytes obtained from adult and neonatal horses. PROCEDURE: Full-thickness explants and cultured chondrocytes were incubated in complete or magnesium-deficient media containing enrofloxacin at concentrations of 0, 1, 5, 25, 100, and 500 microg/ml. Incorporation and release of sulfate 35S over 24 hours were used to assess glycosaminoglycan (GAG) synthesis and degradation. An assay that measured binding of dimethylmethylene blue dye was used to compare total GAG content between groups. Northern blots of RNA from cultured chondrocytes were probed with equine cDNA of aggrecan, type-II collagen, biglycan, decorin, link protein, matrix metalloproteinases 1, 3, and 13, and tissue inhibitor of metalloproteinase 1. RESULTS: A dose-dependent suppression of 35S incorporation was observed. In cartilage of neonates, 35S incorporation was substantially decreased at enrofloxacin concentrations of 25 mg/ml. In cartilage of adult horses, 35S incorporation was decreased only at enrofloxacin concentrations of > or =100 microg/ml. Magnesium deficiency caused suppression of 35S incorporation. Enrofloxacin or magnesium deficiency did not affect GAG degradation or endogenous GAG content. Specific effects of enrofloxacin on steady-state mRNA for the various genes were not observed. CONCLUSION AND CLINICAL RELEVANCE: Enrofloxacin may have a detrimental effect on cartilage metabolism in horses, especially in neonates.     

The effect of sodium heparin on equine articular cartilage.

This study compared the effect of sodium heparin and gentamicin sulphate on equine articular cartilage (AC) explants in order to investigate the possible use of sodium heparin in the treatment of infectious arthritis. Six concentrations of sodium heparin and gentamicin sulphate were tested. The supernatant and explant digest were assayed for glycosaminoglycan (GAG) content with the dimethyl-methylene blue assay and the per cent loss of GAG was calculated. A significant (P< 0.001) increase in percentage GAG loss was noted for the sodium heparin groups when compared to the control, whilst no significant increase was found among the treatment groups (P =0.782). For gentamicin, no significant difference in percentage GAG loss was found between the control and three of the five treatment groups (P =0.667). The percentage GAG loss in the sodium heparin treated AC explants was greater than for any of the gentamicin-treated AC explants. It can be concluded that sodium heparin sulphate stimulates an increase in GAG release from equine articular cartilage explants, though no firm conclusions can be drawn on its use in treating equine infectious arthritis. Copyright Harcourt Publishers Ltd.     

Development of an in vitro model of feline cartilage degradation

Osteoarthritis is the most common arthropathy of mammalian species including cats. Cartilage degradation is central to the disorder and here we present, for the first time, an in vitro model of feline cartilage degradation which will be useful for further studies in this target species. Feline articular cartilage explant cultures were maintained for 28 days and in the presence of oncostatin M with and without interleukin (IL)-17, tumour necrosis factor (TNF), IL-1α, or IL-1β. Media samples and digested cartilage explants were analysed for glycosaminoglycan (GAG) and collagen content. The combination of IL-1β and OSM, both at 20 ng/ml, was able to promote GAG release to the greatest extent at 14 days. At 28 days, all groups showed relatively high release of GAG. At 14 days, only IL-1β and OSM in combination were associated with a statistically significant increase in collagen release over and above control tissue. IL-1β dose-response studies showed that an IL-1β dose of 10 ng/ml promotes a statistically significant increase in GAG breakdown when used with OSM, and higher doses of IL-1β did not result in significantly greater response. The model demonstrated both GAG and collagen degradation and will be of use for further understanding of feline cartilage metabolism and for screening of potential structure-modifying agents to be used in cats.     

Assessment of cartilage degradation effects of matrix metalloproteinase-13 in equine cartilage cocultured with synoviocytes

OBJECTIVE: To determine the effects of matrix metalloproteinase (MMP)-13, compared with interleukin (IL)-1alpha, on cartilage matrix molecule gene expression in a coculture system of equine cartilage explants and synoviocytes. SAMPLE POPULATION: Articular cartilage and synovium specimens harvested from femoropatellar joints of 4 horses, aged 3 to 5 years. PROCEDURES: Synoviocytes were isolated and cocultured with cartilage explants. Cultures were treated with human recombinant MMP-13 (1, 25, or 100 ng/mL) or IL-1alpha (0.01, 0.1, 1.0, or 10 ng/mL) for 96 hours, with medium exchange at 48 hours. Cartilage extracts and media were analyzed for glycosaminoglycan (GAG) content, and results were adjusted to cartilage DNA content. Quantitative PCR was performed on mRNA from cartilage (MMP-3, MMP-13, aggrecan, and collagen type IIB [COL2A1]) and synoviocytes (MMP-3 and MMP-13), and results were adjusted to 18S ribosomal subunit mRNA expression. Treatments were performed in triplicate, and the experiment was repeated 4 times. RESULTS: Cultures treated with MMP-13 or IL-1alpha had increased media GAG concentration at 48 and 96 hours. Aggrecan and COL2A1 mRNA expression were increased by application of MMP-13 or IL-1alpha. Gene expression of the catabolic mediator, MMP-3, in cartilage and synoviocytes was increased in cultures treated with MMP-13 or IL-1alpha. Expression of MMP-13 mRNA in cartilage was increased by IL-1alpha, but decreased in synoviocytes by MMP-13 treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Results support the use of recombinant MMP-13 in a coculture system of synoviocytes and cartilage explants for the study of osteoarthritis.