Effects of carprofen and dexamethasone on canine chondrocytes in a three-dimensional culture model of osteoarthritis

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.     

Effects of proinflammatory cytokines on canine articular chondrocytes in a three-dimensional culture

OBJECTIVE: To determine the effects of interleukin (IL)-1 and tumor necrosis factor (TNF)-alpha on canine chondrocytes cultured in an agarose-based 3-dimensional (3-D) system. SAMPLE POPULATION: Humeral head articular cartilage chondrocytes obtained from 6 adult dogs. PROCEDURE: Chondrocytes were cultured in a 3-D system for < or = 12 days in serum-free medium with IL 1alpha, IL-1beta, or TNF-alpha at concentrations of 20, 50, or 100 ng/mL. After 1, 3, 6, and 12 days, glycosaminoglycan (GAG) concentrations in 3-D constructs; nitric oxide and prostaglandin E2 (PGE2) concentrations in media samples; and relative expressions of selected genes, including metalloproteinase (MMP)-13 and tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2, were evaluated. Control specimens were comprised of chondrocytes cultured without proinflammatory cytokines. RESULTS: In control 3-D constructs, GAG content was significantly higher than for all other constructs. Compared with control values, relative expressions of MMP-13, TIMP-1, and TIMP-2 genes in the IL-1beta (50 ng/mL) group were significantly higher at day 1; at all evaluations, media concentrations of nitric oxide were significantly higher in all TNF-alpha-treated cultures; and concentrations of PGE2 in media samples were significantly higher in the IL-1beta (50 ng/mL) and IL-1beta (100 ng/mL) groups at days 1 and 3, in the IL-1beta (100 ng/mL) group at day 6, and in all TNF-alpha groups at days 1, 3, and 6. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that TNF-alpha more readily induces production of nitric oxide and PGE2 by canine chondrocytes, compared with IL-1beta. In vitro, IL-1alpha appeared to have a minimal effect on canine chondrocytes.     

Effects of interleukin-1beta and tumor necrosis factor-alpha on expression of matrix-related genes by cultured equine articular chondrocytes

OBJECTIVE: To determine the effects of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) on expression and regulation of several matrix-related genes by equine articular chondrocytes. SAMPLE POPULATION: Articular cartilage harvested from grossly normal joints of 8 foals, 6 yearling horses, and 8 adult horses. PROCEDURE: Chondrocytes maintained in suspension cultures were treated with various doses of human recombinant IL-1beta or TNF-alpha. Northern blots of total RNA from untreated and treated chondrocytes were probed with equine complementary DNA (cDNA) probes for cartilage matrix-related genes. Incorporation of 35S-sulfate, fluorography of 14C-proline labeled medium, zymography, and western blotting were used to confirm effects on protein synthesis. RESULTS: IL-1beta and TNF-alpha increased steady-state amounts of mRNA of matrix metalloproteinases 1, 3, and 13 by up to 100-fold. Amount of mRNA of tissue inhibitor of metalloproteinase-1 also increased but to a lesser extent (1.5- to 2-fold). Amounts of mRNA of type-II collagen and link protein were consistently decreased in a dose-dependent manner. Amount of aggrecan mRNA was decreased slightly; amounts of biglycan and decorin mRNA were minimally affected. CONCLUSIONS AND CLINICAL RELEVANCE: Treatment of cultured equine chondrocytes with IL-1beta or TNF-alpha resulted in marked alterations in expression of various matrix and matrix-related genes consistent with the implicated involvement of these genes in arthritis. Expression of matrix metalloproteinases was increased far more than expression of their putative endogenous inhibitor. Results support the suggestion that IL-1beta and TNF-alpha play a role in the degradation of articular cartilage in arthritis.     

In vitro effects of glucosamine and acetylsalicylate on canine chondrocytes in three-dimensional culture

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.     

Phenotypic expression of equine articular chondrocytes grown in three-dimensional cultures supplemented with supraphysiologic concentrations of insulin-like growth factor-1

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.     

Effects of tissue inhibitor of metalloproteinases on canine chondrocytes cultured in vitro with tumor necrosis factor-alpha

OBJECTIVE: To elucidate tissue inhibitor of metalloproteinase (TIMP)-mediated effects on chondrocytes. SAMPLE POPULATION: Articular cartilage from humeral heads of 6 dogs. PROCEDURE: Chondrocytes from harvested specimens were cultured in 3-dimensional (3-D) agarose at 10(6) cells/mL. We prepared 3-D constructs exposed to only tumor necrosis factor (TNF)-alpha (50 ng/mL). Recombinant human TIMP-1 (255nM), -2 (285nM), or -3 (250nM) was added to liquid media bathing 3-D constructs cultured with TNF-alpha. Chondrocytes cultured without TIMP or TNF-alpha served as control samples. Samples of liquid media were collected on days 6, 9, 15, and 21 of culture for evaluation of glycosaminoglycan (GAG) and nitric oxide concentrations. The 3-D constructs were collected on days 9, 15, and 21 for evaluation of GAG, hydroxyproline (HP), and DNA contents. RESULTS: GAG content in control samples increased significantly during the study, whereas GAG content in 3-D constructs cultured with TNF-alpha or TNF-alpha plus TIMP did not increase. On day 9, GAG release from 3-D constructs cultured with TNF-alpha was significantly higher than that in other constructs. The HP content in control samples increased during the study and was significantly higher than that in all other constructs on day 21. Concentrations of nitric oxide were significantly lower in control samples on day 6, compared with concentrations for all other constructs. CONCLUSIONS AND CLINICAL RELEVANCE: Addition of TIMPs did not counteract suppression of GAG and HP accumulation in 3-D constructs exposed to TNF-alpha. Apparently, adverse effects on chondrocytes exposed to TNF-alpha cannot be prevented by addition of TIMP alone.     

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 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.     

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.     

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.     

Evaluation of the influence of prostaglandin E2 on recombinant equine interleukin-1beta-stimulated matrix metalloproteinases 1, 3, and 13 and tissue inhibitor of matrix metalloproteinase 1 expression in equine chondrocyte cultures

OBJECTIVE: To determine the effects of prostaglandin E2 (PGE2) on recombinant equine interleukin (IL)-1beta-stimulated expression of matrix metalloproteinases (MMP 1, MMP 3, MMP 13) and tissue inhibitor of matrix metalloproteinase 1 (TIMP 1) in vitro. SAMPLE POPULATION: Cultured equine chondrocytes. PROCEDURE: Stationary monolayers of first-passage chondrocytes were exposed to graduated concentrations of PGE2 with or without a subsaturating dose (50 pg/ml) of recombinant equine IL-1beta (reIL-1beta) to induce expression of MMP 1, MMP 3, MMP 13, and TIMP 1, followed by RNA isolation and northern blotting. In subsequent experiments, gene expression was similarly quantified from mRNA isolated from cultures pretreated with phenylbutazone to quench endogenous PGE2 synthesis, followed by exposure to reIL-1beta and exogenous PGE2 (5 mg/ml) with appropriate controls. RESULTS: Exogenous PGE2 (10 mg/ml) significantly reduced reIL-1beta-induced expression of MMP 1, MMP 3, MMP 13, and TIMP 1. Abrogation of cytokine induction with this dose of PGE2 was comparable to that for dexamethasone (10(-5) M) control. Similarly, pretreatment with phenylbutazone, followed by exposure to relL-1beta and PGE2 (5 mg/ml), was associated with a reduced expression of the genes of interest, an effect that was significant for MMP 1, MMP 13, and TIMP 1. CONCLUSIONS AND CLINICAL RELEVANCE: The MMP and TIMP 1 are important mediators in the pathophysiologic events in osteoarthritis. The potential for physiologically relevant regulation of expression of these genes by PGE2 is a consideration in the use of drugs that inhibit prostanoid synthesis in the treatment of equine arthropathies.     

Differential regulation of the GLUT1 and GLUT3 glucose transporters by growth factors and pro-inflammatory cytokines in equine articular chondrocytes

Glucose serves as the major energy substrate for articular chondrocytes and as the main precursor for the synthesis of extracellular matrix glycosaminoglycans in cartilage. Chondrocytes have been shown to express several glucose transporter (GLUT) isoforms including GLUT1 and GLUT3. The aim of this investigation was to determine the effects of endocrine and cytokine factors on the capacity of equine articular chondrocytes for transporting 2-deoxy-d-[2,6-3H] glucose and on the expression levels of GLUT1 and GLUT3. Chondrocytes maintained in monolayer culture were stimulated for 24 h with TNF-alpha (100 ng mL(-1)), IL-1beta (100 ng mL(-1)), IGF-I (20 ng mL(-1)), TGF-beta (20 ng mL(-1)) and insulin (12.5 microg mL(-1)) before measuring uptake of non-metabolizable 2-deoxyglucose in the presence and absence of the glucose transport inhibitor cytochalasin B. Polyclonal antibodies to GLUT1 and GLUT were used to compare GLUT1 and GLUT3 expression in stimulated and un-stimulated alginate encapsulated chondrocytes by Western blotting. Results indicated that 2-deoxyglucose uptake was inhibited by up to 95% in the presence of cytochalasin B suggesting that glucose uptake into equine chondrocytes is GLUT-mediated. Insulin had no effect on glucose uptake, but treatment with IGF-I, TGF-beta, IL-1beta and TNF-alpha resulted in a significant increase (>65%) in 2-deoxyglucose uptake compared to control values. GLUT1 was found to be increased in chondrocytes stimulated with all the growth factors and cytokines but GLUT 3 was only upregulated by IGF-I. The data presented support a critical role for glucose in the responses of equine articular chondrocytes to pro-inflammatory cytokines and anabolic endocrine factors.     

铜对体外仔猪软骨细胞增殖和细胞骨架的影响

体外分离、培养仔猪关节软骨细胞,在细胞培养液中分别添加铜0、7.8、15.6、31.2、62.5μmol/L。结果表明,软骨细胞在4种铜浓度中可存活并增殖,但随铜浓度的增加,其存活率、增殖率、3H-TdR掺入率有明显的差异,且能破坏软骨细胞骨架。培养液中添加铜31.2μmol/L,对软骨细胞的增殖作用最强,增殖率、3H-TdR掺入数显著高于对照组(P<0.01),软骨细胞形态及骨架均正常。表明31.2μmol/L铜浓度是促进体外软骨细胞增殖的最适浓度。     

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.     

Assessment of cellular, biochemical, and histologic effects of bipolar radiofrequency treatment of canine articular cartilage

OBJECTIVE: To assess the cellular, biochemical, and histologic effects of bipolar radiofrequency-generated heat on canine articular cartilage. SAMPLE POPULATION: Articular cartilage explants (n = 72) from 6 canine cadavers and cultured articular chondrocytes from 5 canine cadavers. PROCEDURE: Cartilage explants were randomly assigned to receive no treatment or treatment with focal (3 seconds) or diffuse bipolar radiofrequency. Following treatment, methylene blue permeability assay was performed (n = 12) and remaining samples (60) were cultured. Immediately and 5, 10, and 20 days after treatment, cultured explants were assessed for glycosaminoglycan (GAG) and collagen contents, type II collagen and matrix metalloproteinase (MMP)-13 immunoreactivity, and modified Mankin histologic scores. Liquid culture media were collected every 4 days and GAG content measured. Additionally, cultured chondrocytes were exposed for 3 seconds to media preheated to 37 degrees, 45 degrees, or 55 degrees C. Cell viability was determined via 2 different assays immediately and 24 hours after treatment. RESULTS: Radiofrequency-treated cartilage had reduced permeability and considerable histologic damage, compared with control samples; most treated samples had reduced collagen II staining and increased MMP-13 immunostaining. Compared with other treatments, less GAGs were released from cartilage after diffuse radiofrequency treatment throughout the study period. Cell viability was significantly different between controls and cells treated at 55 degrees C immediately and 24 hours after heat treatment. CONCLUSIONS AND CLINICAL RELEVANCE: In this study, bipolar radiofrequency treatment had detrimental effects on normal articular cartilage cells and extracellular matrix with probable long-term clinical consequences. The usefulness of radiofrequency for treatment of osteoarthritic articular cartilage requires further investigation.     

In vitro characterization of chondrocytes isolated from naturally occurring osteochondrosis lesions of the humeral head of dogs

OBJECTIVE: To characterize chondrocytes from naturally occurring osteochondrosis (OC) lesions of the humeral head of dogs. SAMPLE POPULATION: 15 cartilage specimens from 13 client-owned dogs with humeral head OC and 10 specimens from the humeral head of healthy dogs (controls). PROCEDURE: Chondrocytes were isolated and cultured in a 3-dimensional system. On days 7, 10, 15, 20, and 25, glycosaminoglycan and hydroxyproline content and cytologic characteristics were evaluated. Expression of collagen types I, II, and X was assessed by use of immunohistochemistry. RESULTS: Chondrocytes from OC lesions were less viable, compared with control chondrocytes. Glycosaminoglycan content in the OC group was significantly less than in the control group on all days except day 20. Hydroxyproline content was also significantly less in the OC group on days 10, 20, and 25. Expression of collagen type II was significantly less in the OC group, compared with the control group on all days, whereas expression of collagen type I was significantly greater in the OC group on days 20 and 25. Expression of collagen type X was significantly less in the OC group on all days except day 25. CONCLUSIONS AND CLINICAL RELEVANCE: Chondrocytes from naturally occurring OC lesions of the humeral head of dogs cultured in a 3-dimensional system were less viable and less capable of producing appropriate extracellular matrix molecules than chondrocytes from unaffected dogs. Alterations in the synthetic capabilities of chondrocytes from OC-affected cartilage may be a cause or an effect of the disease process.     

Cytokine and chemokine gene expression of IL-1beta stimulated equine articular chondrocytes

OBJECTIVE: To evaluate mRNA expression of several proinflammatory and anti-inflammatory cytokines and chemokines in equine unstimulated and interleukin-1beta (IL-1beta)-stimulated chondrocytes. STUDY DESIGN: In vitro experiment using equine chondrocyte cultures. SAMPLE POPULATION: Whole articular cartilage from metacarpophalangeal joints (n=5 horses; 10 fetlocks). METHODS: Chondrocyte monolayer cultures were established from digested adult equine articular cartilage and stimulated with 5 ng/mL of recombinant human IL-1beta. RNA was extracted from the cells 24 hours after stimulation. IL-1beta, IL-4, IL-6, IL-8, tumor necrosis factor-alpha (TNF-alpha), and ubiquitin (house keeping gene) mRNA expression were investigated by real-time RT-PCR. RESULTS: IL-1beta, IL-6, and IL-8 mRNA were expressed in unstimulated chondrocytes from macroscopically normal joints and were significantly up-regulated after stimulation (5/5 horses). IL-4 mRNA was not detected in any samples (0/5 horses). TNF-alpha mRNA, by comparison, was expressed in 2/5 unstimulated samples and in all stimulated samples but a considerable sample variation in response to IL-1beta stimulation was observed. CONCLUSIONS: Equine chondrocytes express mRNA for several proinflammatory cytokines and chemokines and IL-1beta modulates their expression. CLINICAL RELEVANCE: Chondrocytes express proinflammatory cytokines and chemokines capable of modulating a local inflammatory cascade in articular cartilage, which could potentially lead to focal degradation and osteoarthritis.     

Hypertrophy and physiological death of equine chondrocytes in vitro

REASON FOR PERFORMING STUDY: Equine osteochondrosis results from a failure of endochondral ossification during skeletal growth. Endochondral ossification involves chondrocyte proliferation, hypertrophy and death. Until recently no culture system was available to study these processes in equine chondrocytes. OBJECTIVE: To optimise an in vitro model in which equine chondrocytes can be induced to undergo hypertrophy and physiological death as seen in vivo. METHODS: Chondrocytes isolated from fetal or older (neonatal, growing and mature) horses were cultured as pellets in 10% fetal calf serum (FCS) or 10% horse serum (HS). The pellets were examined by light and electron microscopy. Total RNA was extracted from the pellets, and quantitative PCR carried out to investigate changes in expression of a number of genes regulating endochondral ossification. RESULTS: Chondrocytes from fetal foals, grown as pellets, underwent hypertrophy and died by a process morphologically similar to that seen in vivo. Chondrocytes from horses age >5 months did not undergo hypertrophy in pellet culture. They formed intramembranous inclusion bodies and the cultures included cells of osteoblastic appearance. Pellets from neonatal foals cultured in FCS resembled pellets from older horses, however pellets grown in HS underwent hypertrophy but contained inclusion bodies. Chondrocytes from fetal foals formed a typical cartilage-like tissue grossly and histologically, and expressed the cartilage markers collagen type II and aggrecan mRNA. Expression of Sox9, collagen type II, Runx2, matrix metalloproteinase-13 and connective tissue growth factor mRNA increased at different times in culture. Expression of fibroblast growth factor receptor-3 and vascular endothelial growth factor mRNA decreased with time in culture. CONCLUSIONS: Freshly isolated cells from fetal growth cartilage cultured as pellets provide optimal conditions for studying hypertrophy and death of equine chondrocytes. POTENTIAL RELEVANCE: This culture system should greatly assist laboratory studies aimed at elucidating the pathogenesis of osteochondrosis.