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1.
A non-arthropathic dose and disposition of ofloxacin, a potent new quinolone antimicrobial agent, were assessed in male juvenile (3-month-old) dogs, when administered orally at 5, 10 and 20 mg/kg/day once daily for 8 consecutive days. Ofloxacin concentrations in sera and articular cartilages were analyzed by high-performance liquid chromatography (HPLC). Macroscopically, arthropathy characterized by fluid-filled vesicles in articular surface of the humerus and femur was observed in animals receiving 10 and 20 mg/kg/day of ofloxacin, but not in those given 5 mg/kg/day. At 20 mg/kg/day, arthropathy of comparable severity also occurred on day 2. Microscopically, the cavity formation in the middle zone of the articular cartilage was first identified and then necrotic chondrocytes were found numerous around the cavity, followed by appearance of chondrocyte clusters. In pharmacokinetics, peak serum concentration (Cmax) and area under the concentrations (AUC0-24) were increased in a dose-dependent manner. However, no remarkable differences in these two parameters were noted between a single and repeated treatments, suggesting no accumulation of the drug. The articular ofloxacin concentration 2 hr after treatment was approximately 1.8 (day 2) to 2.0 times (day 8) higher than the serum concentration. Based on these results, a non-arthropathic dose of ofloxacin in male juvenile dogs following an 8-day treatment is considered to be 5 mg/kg/ day, and its Cmax, AUC0-24 and articular cartilage concentrations 2 hr after treatment were 3.4 microg/ml, 35.1 microg-hr/m/ and 7.0 microg/g, respectively, under these experimental conditions. Thus, arthropathy due to ofloxacin may be predicted by monitoring serum drug concentration.  相似文献   

2.
OBJECTIVE: To determine whether enrofloxacin has detrimental, dose-dependent effects on equine articular cartilage in vitro. ANIMALS: Cartilage explants were developed from 6 healthy horses between 0 and 96 months old. PROCEDURE: Patellar cartilage explants were incubated in 5 concentrations of enrofloxacin (2 microg/ml, 10 microg/ml, 1,000 microg/ml, 10,000 microg/ml, and 50,000 microg/ml) for 72 hours. Proteoglycan synthesis (Na35SO4 incorporation for 24 hours), proteoglycan degradation (Na35SO4 release for 72 hours), endogenous proteoglycan content (dimethylmethlene blue assay), and total protein content were determined. Cartilage explants were evaluated by use of histomorphologic and histomorphometric techniques (toluidine blue stain) for cytologic and matrix characteristics. Quantitative data were analyzed with a one-way ANOVA to compare results among various enrofloxacin concentration groups and the control group. A general linear model was used to determine whether age had an effect. RESULT: Proteoglycan synthesis was excellent in control specimens and in specimens incubated in low concentrations of enrofloxacin (2 microg/ml and 10 microg/ml). High concentrations of enrofloxacin (> 1,000 microg/ml) effectively eliminated proteoglycan synthesis regardless of horse age. Proteoglycan degradation at low concentrations (2 microg/ml and 10 microg/ml) was not different than control. High concentrations of enrofloxacin (> 1,000 microg/ml) caused significant degradation. Different concentrations of enrofloxacin did not affect endogenous proteoglycan. High concentrations of enrofloxacin were associated with a significant increase in number of pyknotic nuclei. CONCLUSION: Concentrations of enrofloxacin that might be achieved following systemic administration did not suppress chondrocyte metabolism in vitro. High concentrations of enrofloxacin (> 1,000 microg/ml) were toxic to chondrocytes.  相似文献   

3.
The concentration-effect relationships of phenylbutazone, indomethacin, betamethasone, pentosan polysulphate (PPS) and polysulphated glycosaminoglycan (PSGAG), on proteoglycan synthesis by equine cultured chondrocytes grown in monolayers, and articular cartilage explants were measured. The effect of PSGAG on interleukin-1beta induced suppression of proteogycan synthesis was also investigated. Proteoglycan synthesis was measured by scintillation assay of radiolabelled sulphate (35SO4) incorporation. Polysulphated glycosaminoglycan and PPS stimulated proteoglycan synthesis in chondrocyte monolayers in a concentration-related manner with maximal effects being achieved at a concentration of 10 microg/mL. Polysulphated glycosaminoglycan reversed the concentration-related suppression of proteoglycan synthesis induced by interleukin-1beta. Neither PSGAG nor PPS exerted significant effects on radiolabel incorporation in cartilage explants. Betamethasone suppressed proteoglycan synthesis by both chondrocytes and explants at high concentrations (0.1-100 microg/mL), but the effect was not concentration-related. At low concentrations (0.001-0.05 microg/mL) betamethasone neither increased nor decreased proteoglycan synthesis. Phenylbutazone and indomethacin increased radiolabel incorporation in chondrocyte cultures but not in cartilage explants at low (0.1, 1 and 10 microg/mL), but not at high (20 and 100 microg/mL) concentrations. These findings may be relevant to the clinical use of these drugs in the treatment of equine disease.  相似文献   

4.
OBJECTIVE: To study the effect of polysulfated glycosaminoglycan (PSGAG) on proteoglycan metabolism and DNA content of control and osteoarthritic (OA) cartilage. STUDY DESIGN: 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. SAMPLE POPULATION: Five large cross-breed dogs. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. CLINICAL RELEVANCE: 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.  相似文献   

5.
OBJECTIVE: To evaluate the effects of orally administered phenylbutazone on proteoglycan synthesis and chondrocyte inhibition by IL-1beta in articular cartilage explants of horses. ANIMALS: 11 healthy 1- to 2-year-old horses. PROCEDURE: Horses were randomly assigned to the control (n = 5) or treated group (4.4 mg of phenylbutazone/kg of body weight, p.o., q 12 h; n = 6). Articular cartilage specimens were collected before treatment was initiated (day 0), after 14 days of treatment, and 2 weeks after cessation of treatment (day 30). Proteoglycan synthesis and stromelysin concentration in cartilage extracts were assessed after 72 hours of culture in medium alone or with recombinant human interleukin-1beta (IL-1beta; 0.1 ng/ml). RESULTS: On day 0, proteoglycan synthesis was significantly less in cartilage explants cultured in IL-1beta, compared with medium alone. Mean proteoglycan synthesis in explants collected on days 14 and 30 was significantly less in treated horses, compared with controls. However, incubation of explants from treated horses with IL-1beta did not result in a further decrease in proteoglycan synthesis. Significant differences in stromelysin concentration were not detected between or within groups. CONCLUSIONS AND CLINICAL RELEVANCE: Oral administration of phenylbutazone for 14 days significantly decreased proteoglycan synthesis in articular culture explants from healthy horses to a degree similar to that induced by in vitro exposure to IL-1beta. Phenylbutazone should be used judiciously in athletic horses with osteoarthritis, because chronic administration may suppress proteoglycan synthesis and potentiate cartilage damage.  相似文献   

6.
OBJECTIVE: To assess the effects of supraphysiologic concentrations of insulin-like growth factor-1 (IGF-1) on morphologic and phenotypic responses of chondrocytes. SAMPLE POPULATION: Articular cartilage obtained from 2 young horses. PROCEDURE: Chondrocytes were suspended in fibrin cultures and supplemented with 25, 12.5, or 0 mg of IGF-1/ml of fibrin. Chondrocyte morphology and phenotypic expression were assessed histologically, using H&E and Alcian blue stains, immunoreaction to collagen type I and II, and in situ hybridization. Proteoglycan content, synthesis, and monomer size were analyzed. The DNA content was determined by bisbenzimide-fluorometric assay, and elution of IGF-1 into medium was determined by IGF-1 radioimmunoassay. RESULTS: Both 12.5 and 25 kg of IGF-1/ml enhanced phenotypic expression of chondrocytes without inducing detrimental cellular or metabolic effects. Highest concentration of IGF-1 (25 microg/ml) significantly increased total DNA content, glycosaminoglycan (GAG) content, GAG synthesis, and size of proteoglycan monomers produced, compared with cultures supplemented with 12.5 microg of IGF-1/ml or untreated cultures. Histologic examination confirmed these biochemical effects. Matrix metachromasia, type-II collagen in situ hybridization and immunoreaction were increased in cultures treated with 25 microg of IGF-1/ml, compared with cultures supplemented with 12.5 microg of IGF-1/ml or untreated cultures. CONCLUSIONS AND CLINICAL RELEVANCE: Chondrocytes exposed to high concentrations of IGF-1 maintained differentiated chondrocyte morphology and had enhanced synthesis of matrix molecules without inducing apparent detrimental effects on chondrocyte metabolism. These results suggest that application of such composites for in vivo use during cartilage grafting procedures should provide an anabolic effect on the grafted cells.  相似文献   

7.
The histologic and histochemical features of quinolone-induced arthropathy were studied using 14 skeletally immature Beagle dogs (3 to 4 months old) dosed orally with difloxacin at 300 mg/kg body weight once daily for 1, 2, 5, or 7 days. A placebo was given to eight other age-matched Beagle dogs that served as controls. A scoring technique that included lesion size and histologic features was used to determine the progression of lesions. Articular-epiphyseal cartilage complexes on the femoral and humeral heads and tibial tarsal bone were identified as predilection sites. Within predilection sites on femoral and humeral heads, lesions developed in specific areas. Lesions appeared within 2 days of the onset of treatment, and lesion scores increased with time. Grossly, the lesions were raised, fluid-filled vesicles on the articular surface. Histologic changes included vesicle formation with loss of proteoglycan, clumping of unmasked collagen, and degeneration and necrosis of chondrocytes. In lesions with higher scores, chondrocytes were often in clusters or they were undergoing metaplasia toward spindle-shaped cells. Although dissolution of matrix and necrosis of chondrocytes were typical of all lesions, smaller lesions had histologically normal chondrocytes adjacent to small vesicles. In sections stained with toluidine blue, proteoglycan was aggregated with collagen fibrils or was absent from the matrix adjacent to vesicles. Unique features, such as biomechanical forces, may predispose specific areas of articular cartilage to develop lesions.  相似文献   

8.
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.  相似文献   

9.
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.  相似文献   

10.
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11.
OBJECTIVE: To study chondrotoxic effects of enrofloxacin (ENR) and ciprofloxacin hydrochloride (CFX) on canine and equine articular chondrocytes in culture and to compare the effects with that of cultivation in Mg2+-free medium. SAMPLE POPULATION: Chondrocytes from articular cartilage of 4- and 6 -month old dogs and 2- to 4- year-old horses. PROCEDURE: Chondrocytes were cultivated with 10, 40, 80, and 160 microg of CFX/ml, 10, 50, 100, and 150 microg of ENR/ml, or in Mg2+-free medium. A live-to-dead test was performed to test cytotoxic effects. Morphologic changes were evaluated by electron microscopy. An attachment assay was used to test the ability of chondrocytes to adhere to collagen type-II coated-chamber slides in the presence of CFX and with Mg2+-free medium. RESULTS: Chondrocytes cultivated in quinolone-supplemented medium or Mg2+-free medium had a decreased ability to adhere to culture dishes. Cell shape and the actin and vimentin cytoskeleton changed in a concentration-dependent manner. These effects were not species-specific and developed with both quinolones. On day 1 of culture, adhesion of chondrocytes to collagen type II was reduced to 70 and 45% of control values in the CFX treatment and Mg2+-free treatment groups, respectively. On day 5 of culture, adhesion of chondrocytes was reduced to 45 and 40% of control values in the CFX treatment and Mg2+-free treatment groups, respectively. CONCLUSION AND CLINICAL RELEVANCE: In vitro, chondrotoxic effects of quinolones appear to be the result of irregular integrin signaling and subsequent cellular changes. Drug concentrations leading to morphologic changes in vitro may be achieved in articular cartilage in vivo.  相似文献   

12.
OBJECTIVE: 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). STUDY DESIGN: 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. ANIMALS: Two young cross-breed horses and 3 young Labrador retrievers. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. CLINICAL RELEVANCE: 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.  相似文献   

13.
Dexamethasone (Dexa) has been commonly used in humans and domestic animals, particularly in the treatment of tendon injuries and cartilage degeneration. However, it is often associated with tendon rupture and impaired tendon and cartilage healing. In the present study, we investigated Dexa's in vitro effects on the growth of cell proliferation and the induction of apoptosis in canine Achilles tendon cells and chondrocytes. Cell proliferation after treatment with Dexa for two to six days was quantified by a 2,3-bis{2-methoxy-4-nitro-5-sulfophenyl}-2H-tetrazolium-5-carboxyanilide inner salt assay (XTT). The results showed that Dexa could inhibit the proliferation of tendon cells and chondrocytes at increasing concentrations (0.1-50 microg/ml) compared with untreated cells. Cell apoptosis was induced by Dexa, as evidenced by the typical nuclear apoptosis using Hoechst 33258 staining. Dexa increased the apoptosis of canine tendon cells and chondrocytes in a time-dependent manner. In canine tendon cells and chondrocytes that were treated with 25 and 50 microg/ml concentration of Dexa, the number of condensed apoptotic nuclei was significantly increased. In addition, culturing with Dexa and the glucocorticoid receptor blocker, mifepristone, significantly arrested apoptosis of tendon cells and chondrocytes. Based on our in vitro data, we hypothesized that in vivo treatment with glucocorticoids may diminish the proliferation of tendon and cartilage cells by increasing apoptosis and suppressing the proliferation. Our findings suggest that Dexa could be used with caution in dogs with articular or tendon problems.  相似文献   

14.
OBJECTIVE: To assay concentrations of cartilage oligomeric matrix protein (COMP) in canine sera and synovial fluid (SF), to compare COMP concentrations in clinically normal dogs and dogs with joint disease, and to analyze changes in COMP concentrations in dogs with experimentally induced acute synovitis. ANIMALS: 69 control dogs without joint disease, 23 dogs with naturally occurring aseptic arthropathy, and 6 dogs with experimentally induced synovitis. PROCEDURE: Serum (n = 69) and SF (36) were obtained from control dogs. Samples of serum (n = 23) and SF (13) were obtained from dogs with naturally occurring aseptic arthropathy with or without radiographic features of osteoarthritis (OA). Serum and SF were obtained before and 1, 2, 3, and 7 days after induction of synovitis. The COMP concentrations were determined by use of an inhibition ELISA that had canine cartilage COMP and monoclonal antibody against human COMP. RESULTS: Concentrations of COMP in serum and SF of control dogs were 31.3+/-15.3 and 298.7+/-124.7 microg/ml, respectively. In naturally occurring OA, COMP concentrations in serum (44.9+/-177 microg/ml) and SF (401.7+/-74.3 microg/ml) were significantly higher than corresponding concentrations in control dogs. The COMP concentration in SF peaked 24 and 48 hours after induction of synovitis, whereas concentration in serum peaked on day 3. CONCLUSIONS AND CLINICAL RELEVANCE: These results supported the hypothesis that COMP concentration in serum and SF of dogs may be altered after cartilage degradation or synovitis. Measurement of COMP concentrations can be useful when differentiating arthropathies in dogs.  相似文献   

15.
OBJECTIVE: To determine effects of glucosamine and acetylsalicylate on canine chondrocytes in 3-dimensional culture. SAMPLE POPULATION: Chondrocytes isolated from articular cartilage of 2 adult female dogs recently euthanatized for reasons unrelated to orthopedic abnormalities. PROCEDURE: Chondrocytes were cultured in a 3-dimensional agarose-based medium alone (control), with glucosamine (100 microg/ml; GL), or with acetylsalicylate (18 microg/ml; AS). Supernatant and agarose plugs from 4 wells/group/d were collected on days 3, 6, and 12 of culture. Agarose plugs were evaluated for percentage of viable cells, percentage of cells producing pericellular or territorial matrix, glycosaminoglycan (GAG) concentration, and type-II collagen production. Prostaglandin E2 concentration in supernatants was determined. RESULTS: Chondrocytes in all groups had characteristics indicative of viability and differentiation; however, on day 12, a lower percentage of viable cells was detected in the AS group, compared with the other 2 groups. On day 6, GAG concentration in the AS group was significantly greater than concentrations in the other 2 groups. On day 12, GAG concentrations in the GL and AS groups were significantly less than in the control group. Within the GL and AS groups, cell viability was significantly less on day 12, compared with day 3. Significant differences in PGE2 concentration among or within groups and evidence of type II collagen production were not detected. CONCLUSIONS: 3-dimensional culture of canine chondrocytes allows for production of hyaline cartilage matrix constituents and growth of cells with morphologic characteristics similar to those of articular cartilage. Acetylsalicylate and glucosamine, at the single concentration evaluated, had detrimental effects on chondrocyte viability, GAG production, or both.  相似文献   

16.
The effect of exogenous hyaluronate on normal cartilage metabolism and interleukin-1 (IL-1)-induced cartilage matrix degradation was investigated in a bovine cartilage explant culture system. Addition of hyaluronate at a concentration of 1.5 mg/ml to cartilage culture explants consistently decreased normal proteoglycan release from the matrix to a value less than that found in control cultures. Addition of 1.5 mg of hyaluronate/ml to IL-1 stimulated cartilage culture systems reduced proteoglycan release from the matrix by 83 to 113%. The reduction in control and IL-1-stimulated proteoglycan degradation by hyaluronate had a concentration-dependent trend. Evaluation of alterations in protein (enzyme) release by IL-1-stimulated chondrocytes after introduction of hyaluronate was evaluated by use of sodium dodecyl sulfate agar gel electrophoresis of cartilage-conditioned media. The quantity or the molecular weight profile of IL-1-induced proteins did not differ after introduction of hyaluronate into the culture system. Results indicate that introduction of high molecular weight hyaluronate into cartilage culture systems results in a decrease in proteoglycan release from the matrix in control systems, as well as in cultures incubated with IL-1. Because IL-1-stimulated protein synthesis by chondrocytes remains unchanged after addition of exogenous hyaluronate, the mechanism of inhibition of matrix degradation does not appear to be interference with binding of IL-1 to chondrocytes or to be inhibition of the production of neutral metalloproteases, including stromelysin.  相似文献   

17.
OBJECTIVE: To determine effects of carprofen and dexamethasone on chondrocytes in a culture model of osteoarthritis (OA). SAMPLE POPULATION: Chondrocytes isolated from articular cartilage of the humeral head of 5 adult dogs. PROCEDURE: Chondrocytes were harvested, cultured and subcultured in monolayer, and then cultured in a 3-dimensional (3-D) medium. Cells from each dog were distributed into 6 groups with differing content of liquid medium for each 3-D construct (agarose [AG], AG plus interleukin [IL]-1beta, AG plus carprofen [4 microg/mL], AG plus dexamethasone [1 mg/mL], AG plus IL-1beta [20 ng/mL] plus carprofen [4 microg/mL], and AG plus IL-1beta (20 ng/mL) plus dexamethasone (1 mg/mL). On days 3, 6, 12, and 20 of culture, samples from all groups were collected. Liquid media were assayed for glycosaminoglycan, prostaglandin (PG)E2, matrix metalloprotease (MMP)-3, and MMP-13 concentrations. All 3-D constructs were evaluated for viability, cell morphology, proteoglycan staining, and collagen type-II concentration. Total glycosaminoglycan content in each 3-D construct was quantitated by spectrophotometric assay. RESULTS: Addition of IL-1beta caused a significant loss of cell viability and matrix production. Addition of carprofen or dexamethasone caused significant decreases in PGE2 in the liquid media, and each was minimally effective in protecting chondrocytes against negative effects of IL-1beta. CONCLUSIONS AND CLINICAL RELEVANCE: Human recombinant IL-1beta resulted in loss of cell viability, alterations in extracellular matrix components, and production of PG and MMP Carprofen and dexamethasone had little effect on cell and matrix variables but did decrease PGE2 concentrations and primarily affected the inflammatory pathway of osteoarthritis.  相似文献   

18.
Commonly used diagnostic tools used to evaluate articular cartilage lack the sensitivity, specificity, and objectivity to measure early changes associated with osteoarthritis. Two techniques using magnetic resonance (MR) imaging have been developed to detect the biology of articular cartilage are delayed gadolinium-enhanced MR imaging of cartilage (dGEMRIC) and T2 mapping. Both techniques have been validated and are used to study the degenerative and adaptive nature of articular cartilage in people. The use of these techniques as a diagnostic tool in dogs has not been well described. We evaluated articular cartilage in the region of the medial coronoid process (MCP) of six healthy dogs free of detectable orthopedic disease using both MR imaging techniques. Histology and proteoglycan (PG) content of the MCP were used to confirm normal articular cartilage. All dogs had ground reaction forces consistent with normal function. Mean dGEMRIC index (T1 value) was 400 +/- 47 ms and mean T2 value was 56 +/- 8 ms. Intra- and interobserver variability was low. dGEMRIC and T2 values for normal cartilage in the elbow of the dog can be generated reproducibly using 3T MR imaging. Using these techniques as objective outcome measures for clinical studies in dogs with OA conditions should help delineate the efficacy of some disease interventions.  相似文献   

19.
OBJECTIVE: To determine the morphologic and phenotypic effects of transforming growth factor beta1 (TGFbeta1) on cultured equine mesenchymal stem cells (MSC) and articular chondrocytes. SAMPLE POPULATION: Bone marrow aspirates and articular cartilage samples from a 2-year-old and two 8-month-old horses. PROCEDURE: After initial isolation and culture, MSC and chondrocytes were cultured in Ham's F-12 medium supplemented with TGF-beta1 at a concentration of 0, 1, 5, or 10 ng/ml. Medium was exchanged on day 2, and cells were harvested on day 4. Medium was assayed for proteoglycan (PG) content. Total RNA was isolated from cell cultures, and expression of aggrecan, decrin, collagen type-I, and collagen type-II mRNA was assessed by means of Northern blot analyses. Cell cultures were stained with H&E or toluidine blue and examined histologically. Additional cultures were examined after immunohistochemical staining for type-I and -II collagen. RESULTS: MSC cultures exposed to TGF-beta1 had an increased cellular density with cell layering and nodule formation that was most pronounced in cultures treated with 5 ng of TGF-beta1/ml. Expression of collagen type-II mRNA in MSC cultures exposed to 5 ng of TGF-beta1/ml was 1.7 times expression in control cultures, and expression of collagen type-I mRNA was 2.8 times expression in control cultures. Treatment of MSC with TGF-beta1 led to dose-related increases in area and intensity of type-II collagen immunoreaction. CONCLUSION: Results suggest that TGF-beta1 enhances chondrogenic differentiation of bone marrow-derived MSC in a dose-dependent manner.  相似文献   

20.
Transarticular external skeletal (TES) fixators were applied unilaterally to the stifle joints of 10 young adult dogs. After 4 weeks, the fixators were removed from all dogs. Two dogs were not allowed a remobilization period, whereas 8 dogs were provided with 4 additional weeks of weight-bearing activity in a kennel run. Four dogs were given high-molecular weight hyaluronic acid by intra-articular injection weekly during the remobilization period. Clinical gait evaluations and range of motion were determined during the remobilization period. Articular cartilage samples from both stifle joints of all dogs were evaluated histologically and histochemically. No significant differences in gait scores or range of motion were noted between treated and untreated dogs. Articular cartilage proteoglycan content was reduced after 4 weeks of trans-stifle external skeletal fixation as determined by loss of alcian blue (AB) histochemical staining. Improved homogeneity of histochemical staining was observed after remobilization. However, remobilization was associated with histological damage to the surface and tangential layers of articular cartilage. Remobilization combined with hyaluronic acid (HA) therapy improved histochemical staining and reduced structural damage to articular cartilage when compared with remobilization alone.  相似文献   

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