Effects of adenosine on bacterial lipopolysaccharide- and interleukin 1-induced nitric oxide release from equine articular chondrocytes

OBJECTIVE: To determine whether adenosine influences the in vitro release of nitric oxide (NO) from differentiated primary equine articular chondrocytes. SAMPLE POPULATION: Articular cartilage harvested from the metacarpophalangeal and metatarsophalangeal joints of 11 horses (3 to 11 years old) without history or clinical signs of joint disease. PROCEDURE: Chondrocytes were isolated, plated at a high density (10(5) cells/well), and treated with adenosine, the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), bradykinin, or other agents that modify secondary messenger pathways alone or in combination with bacterial lipopolysaccharide (LPS) or recombinant human interleukin-1alpha (rhIL-1alpha). Nitric oxide release was measured indirectly by use of the Griess reaction and was expressed as micromol of nitrite in the supernatant/microg of protein in the cell layer. Inducible nitric oxide synthase (iNOS) activity was determined by measuring the conversion of radiolabeled arginine to radiolabeled citrulline. RESULTS: Treatment of chondrocytes with adenosine alone had no significant effect on NO release. However, adenosine and NECA inhibited LPS- and rhIL-1alpha-induced NO release. This response was mimicked by forskolin, which acts to increase adenylate cyclase activity, but not by the calcium ionophore A23187 Treatment of chondrocytes with phorbol myristate acetate, which acts to increase protein kinase C activity, potentiated LPS-induced NO release. Adenosine treatment also significantly inhibited the LPS-induced increase in iNOS activity. CONCLUSIONS AND CLINICAL RELEVANCE: Adenosine and the nonspecific adenosine receptor agonist NECA inhibited inflammatory mediator-induced release of NO from equine articular chondrocytes. Modulation of adenosine receptor-mediated pathways may offer novel methods for treatment of inflammation in horses with joint disease.     

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.     

Isolation, propagation, and cryopreservation of equine articular chondrocytes.

Equine articular chondrocytes were isolated from explant cartilage cultures by digestion in a 0.075% collagenase solution for 15 to 19 hours. Cartilage from late-term fetal and neonatal foals resulted in mean chondrocyte yield of 51.99 x 10(6) cells/g of cartilage (wet weight), compared with a yield of 17.83 x 10(6) cells/g for foals 3 to 12 months old. Propagation of chondrocytes in monolayer and 3-dimensional culture was accomplished, using Ham's F-12 as the basal medium, with supplements of fetal bovine serum (10%), ascorbic acid, alpha-ketoglutarate, and L-glutamine. The medium was buffered with HEPES, and penicillin and streptomycin were added for microorganism control. In primary monolayer cultures of freshly isolated chondrocytes, the population doubling time was approximately 6 days. Dedifferentiation of chondrocytes toward a more fibroblastic-appearing cell was observed after the fifth passage (subculture), but was hastened by lower cell-plating density. Chondrocytes were frozen for periods of up to 9 months, using 10% dimethyl sulfoxide as the cryoprotectant. Cell viability of late-term fetal and neonatal foal chondrocytes after storage at -196 C decreased from 86% at 3 weeks to 31% at 12 weeks. Viability of cells derived from older foals and young adult horses was considerably better than that of cells from neonatal foals. Frozen chondrocytes can be stored for extended periods and thawed for immediate implantation or can be sustained in vitro in monolayer or 3-dimensional culture. Such cultures would be suitable for cartilage resurfacing experiments or in vitro assessment of various pharmaceuticals.     

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.     

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.     

Effects of radial shock waves on membrane permeability and viability of chondrocytes and structure of articular cartilage in equine cartilage explants

OBJECTIVE: To investigate in vitro effects of radial shock waves on membrane permeability, viability, and structure of chondrocytes and articular cartilage. SAMPLE POPULATION: Cartilage explants obtained from the third metacarpal and metatarsal bones of 6 horses. PROCEDURE: Equine cartilage was subjected to radial shock waves and then maintained as explants in culture for 48 hours. Treatment groups consisted of a negative control group; application of 500, 2,000, and 4,000 impulses by use of a convex handpiece (group A); and application of 500, 2,000, and 4,000 impulses by use of a concave handpiece (group B). Effects on explant structure were evaluated by use of environmental scanning electron microscopy (ESEM). Membrane permeability was determined by release of lactate dehydrogenase (LDH). Chondrocyte viability was assessed by use of vital cell staining. Comparisons of LDH activity and nonviable cell percentages were performed by ANOVA. RESULTS: Cell membrane permeability increased significantly after application of 2,000 and 4,000 impulses in groups A and B. A significant decrease in cell viability was observed for application of 4,000 impulses in explants of group A. There was no detectable damage to integrity of cartilage explants observed in any treatment group by use of ESEM. CONCLUSIONS AND CLINICAL RELEVANCE: Radial shock waves do not appear to structurally damage articular cartilage but do impact chondrocyte viability and membrane permeability. Caution should be exercised when extremely high periarticular pulse doses are used until additional studies can determine the long-term outcome of these effects and appropriate periarticular treatment regimens can be validated.     

Effects of methylprednisolone acetate and glucosamine on proteoglycan production by equine chondrocytes in vitro

OBJECTIVE: To evaluate the effects of methylprednisolone acetate (MPA) on proteoglycan production by equine chondrocytes and to investigate whether glucosamine hydrochloride modulates these effects at clinically relevant concentrations. SAMPLE POPULATION: Articular cartilage with normal gross appearance from metacarpophalangeal and metatarsophalangeal joints of 8 horses (1 to 10 years of age). PROCEDURES: In vitro chondrocyte pellets were pretreated with glucosamine (0, 1, 10, and 100 microg/mL) for 48 hours and exposed to MPA (0, 0.05, and 0.5 mg/mL) for 24 hours. Pellets and media were assayed for proteoglycan production (Alcian blue precipitation) and proteoglycan content (dimethylmethylene blue assay), and pellets were assayed for DNA content. RESULTS: Methylprednisolone decreased production of proteoglycan by equine chondrocytes at both concentrations studied. Glucosamine protected proteoglycan production at all 3 concentrations studied. CONCLUSIONS AND CLINICAL RELEVANCE: Methylprednisolone, under noninflammatory conditions present in this study, decreased production of proteoglycan by equine chondrocytes. Glucosamine had a protective effect against inhibition of proteoglycan production at all 3 concentrations studied. This suggested that glucosamine may be useful as an adjunct treatment when an intra-articular injection of a corticosteroid is indicated and that it may be efficacious at concentrations relevant to clinical use.     

Viability of equine articular chondrocytes in alginate beads exposed to different oxygen tensions

Ischaemia and reperfusion are suspected to alter chondrocyte metabolism. Here, we studied the effects of three oxygen (O2) tensions on the viability of equine articular chondrocytes isolated from the cartilage of the distal interphalangeal joint of horses. Chondrocytes were cultured in alginate beads under 1%, 5% or 21% gas phase O2 concentration for 14 days, cellular growth kinetics were measured (n=6), and the cells were observed by light microscopy after staining for necrotic and apoptotic cell detection. For information about the metabolic status, the intracellular adenosine triphosphate (ATP) content was measured. The number of chondrocytes remained stable for the first eight days, then decreased especially at 1% and 21% O2. At 21% O2, normal cells decreased and necrotic cells increased at the end of the 14 day-period. No significant variations were found at 5% O2 except for a decrease in necrotic cells at day 14. Most apoptotic cells were found at 1% O2 from days 5 to 11, and normal cells decreased during the same period. But an unexpected increase in normal cells and decrease in apoptotic cells were observed at day 14. The intracellular ATP content remained stable. It was concluded that, in a three-dimensional culture model of equine articular chondrocytes, O2 tension affected the viability of the cells after an 11-day period, with the most important effects observed at 21% and 1% O2 conditions.     

Aquaporin water channels AQP1 and AQP3, are expressed in equine articular chondrocytes

Chondrocytes exist in an unusual and highly variable ionic and osmotic environment in the extracellular matrix of articular cartilage. Alterations to the ionic and osmotic environment of chondrocytes influence the volume and ionic content of the cells, which, in turn, modifies the rate at which extracellular matrix macromolecules are synthesized and degraded. Thus, regulation of the water and solute content of chondrocytes will profoundly affect their anabolic and catabolic functions. The water content of cells is effectively influenced by the abundance of aquaporin (AQP) water channels. Recent studies have shown that several AQP water channel isoforms are expressed in chondrocytes from Meckel's cartilage, developing teeth and other orofacial tissues. The aim of the present investigation was to determine if chondrocytes from equine articular cartilage express AQP water channels. Polyclonal antibodies to AQP1, AQP2 and AQP3 were used in conjunction with immunohistochemistry, immunoblotting and quantitative flow cytometry to determine if AQP1, AQP2 and AQP3 are expressed in equine articular chondrocytes. Our studies show that AQP1 and AQP3 are expressed by chondrocytes in articular cartilage in situ and in isolated chondrocytes. We found no evidence for expression of AQP2, the vasopressin-regulated water channel in chondrocytes. AQP1 and AQP3 may be involved in the transport of water and small solutes and osmotically active metabolites across the chondrocyte plasma membrane during volume regulatory behaviour. AQP1 may be involved in transporting metabolic water. AQP3 may participate in the transport of glycerol and structurally related molecules.     

Comparison of the cytotoxic effects of bupivacaine, lidocaine, and mepivacaine in equine articular chondrocytes

Objective To compare the chondrotoxicity of bupivacaine, lidocaine, and mepivacaine in equine articular chondrocytes in vitro. Study design Prospective, experimental study. Study material Equine articular chondrocytes. Methods Primary cultured equine chondrocytes were exposed to 0.5% bupivacaine, 2% lidocaine, or 2% mepivacaine for 30 or 60 minutes. After treatment, cell viability was evaluated by trypan blue exclusion and the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) colorimetric assay in a dose dependent manner. Apoptosis and necrosis of chondrocytes were analyzed with the double staining of Hoechst 33258 and propidium iodide using fluorescence microscopy, and the results were confirmed using flow cytometry. Results After 30‐minute exposure, trypan blue exclusion assay revealed that cell viability of 0.5% bupivacaine group was 28.73 ± 8.44%, and those of 2% lidocaine and 2% mepivacaine were 66.85 ± 6.03% and 86.27 ± 2.00%, respectively. The viability of chondrocytes after saline treatment was 95.95 ± 2.75%. The results of MTT assay and fluorescence microscopy had similar tendency with trypan blue assay. Each result showed that bupivacaine was the most toxic of the three local anaesthetics. Mepivacaine was less toxic than lidocaine. The results of the viability test suggest that bupivacaine and lidocaine exhibit a marked chondrotoxicity, and that this is mainly due to necrosis rather than apoptosis. Conclusions and clinical relevance Bupivacaine may induce detrimental chondrotoxicity when administered intra‐articularly, especially in patients with joint disease, and we suggest that it should be used cautiously in equine practice. Mepivacaine may be an alternative to both bupivacaine and lidocaine.     

Effects of enrofloxacin and ciprofloxacin hydrochloride on canine and equine chondrocytes in culture

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.     

Effects of lesion size and location on equine articular cartilage repair.

The mechanisms and completeness of equine articular cartilage repair were studied in ten horses over a nine month period. Large (15 mm square) and small (5 mm square) full-thickness lesions were made in weight bearing and nonweight bearing areas of the radiocarpal, middle carpal and femoropatellar joints. The horses were euthanized in groups of two 1, 2.5, 4, 5 and 9 months later. Gross pathology, microradiography, and histopathology were used to evaluate qualitative aspects of articular repair. Computer assisted microdensitometry of safranin-O stained cartilage sections was used to quantitate cartilage matrix proteoglycan levels. Structural repair had occurred in most small defects at the end of nine months by a combination of matrix flow and extrinsic repair mechanisms. Elaboration of matrix proteoglycans was not complete at this time. Statistically better healing occurred in small weight bearing lesions, compared to large or nonweight bearing lesions. Synovial and perichondrial pannus interfered with healing of osteochondral defects that were adjacent to the cranial rim of the third carpal bone. Clinical and experimental experience suggests that these lesions are unlikely to heal, whereas similar lesions in the radiocarpal and femoropatellar joints had satisfactory outcomes. Observations made in this study support the use of early postoperative ambulation, passive flexion of operated joints, and recuperative periods of up to a year for large cartilage defects.     

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.     

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.     

Effects of polysulfated glycosaminoglycan on chemical and physical defects in equine articular cartilage

The effect of intra-articular polysulfated glycosaminoglycan (PSG) on repair of chemical and physical articular cartilage injuries was evaluated in 8 horses. In each horse, a partial- and a full-thickness articular cartilage defect was made on the distal articular surface of the radial carpal bone. In the contralateral middle carpal joint, a chemical articular cartilage injury was induced by injecting 50 mg of Na monoiodoacetate (MIA). Four of the 8 horses were not treated (controls), and 4 horses were treated by intra-articular injection of 250 mg of PSG into both middle carpal joints once a week for 5 treatments starting 1 week after cartilage injury. Horses were maintained for 8 weeks. There was less joint circumference enlargement in PSG-treated horses in MIA-injected and physical defect carpi, compared with that in controls. In MIA-injected joints, there was less articular cartilage fibrillation and erosion, less chondrocyte death, and greater safranin-O staining for glycosaminoglycans in PSG-treated horses. Evaluation of joints in which physical defects were made revealed no differences between control and PSG-injected joints. None of the partial-thickness defects had healed. Full-thickness defects were repaired with fibrous tissue (which was more vascular and cellular in PSG-injected joints) and occasionally small amounts of fibrocartilage. Seemingly, PSG had chondroprotective properties in a model of chemically induced articular cartilage damage, whereas PSG had no obvious effect in a physical articular cartilage-defect model.     

Effects of intra-articular administration of methylprednisolone acetate on normal equine articular cartilage

The effects of the corticosteroid 6-alpha-methylprednisolone acetate on normal equine articular cartilage were evaluated, using the middle carpal joint in 4 clinically normal young horses. One middle carpal joint of each horse was injected 3 times with 100 mg of 6-alpha-methylprednisolone acetate, at 14-day intervals. The opposite middle carpal joint (control) was injected with 2.5 ml of lactated Ringer solution at the same intervals. Effects were studied until 8 weeks after the first injection. Evaluation included clinical and radiographic examination, and gross, microscopic, and biochemical evaluation of joint tissues. Horses remained clinically normal during the study, and significant radiographic changes were not observed. Safranin-0 matrix staining intensity and uronic acid content were significantly (P less than 0.05) lower and hydroxyproline content was significantly (P less than 0.05) higher in articular cartilage of corticosteroid-injected joints vs control joints.     

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 glucosamine and chondroitin sulfate on mediators of osteoarthritis in cultured equine chondrocytes stimulated by use of recombinant equine interleukin-1beta

OBJECTIVE: To determine whether glucosamine and chondroitin sulfate (CS) at concentrations approximating those achieved in plasma by oral administration would influence gene expression of selected mediators of osteoarthritis in cytokine-stimulated equine articular chondrocytes. SAMPLE POPULATION: Samples of grossly normal articular cartilage obtained from the metacarpophalangeal joint of 13 horses. PROCEDURE: Equine chondrocytes in pellet culture were stimulated with a subsaturating dose of recombinant equine interleukin (reIL)-1beta. Effects of prior incubation with glucosamine (2.5 to 10.0 microg/mL) and CS (5.0 to 50.0 microg/mL) on gene expression of matrix metalloproteinase (MMP)-1, -2, -3, -9, and -13; aggrecanase 1 and 2; inducible nitric oxide synthase (iNOS); cyclooxygenase (COX)-2; nuclear factor kappaB; and c-Jun-N-terminal kinase (JNK) were assessed by use of a quantitative real-time polymerase chain reaction assay. RESULTS: Glucosamine at a concentration of 10 microg/mL significantly reduced reIL-1beta-induced mRNA expression of MMP-13, aggrecanase 1, and JNK. Reductions in cytokine-induced expression were also observed for iNOS and COX-2. Chondroitin sulfate had no effect on gene expression at the concentrations tested. CONCLUSIONS AND CLINICAL RELEVANCE: Concentrations of glucosamine similar to those achieved in plasma after oral administration in horses exerted pretranslational regulation of some mediators of osteoarthritis, an effect that may contribute to the cartilage-sparing properties of this aminomonosaccharide. Analysis of results of this study indicated that the influence of CS on pretranslational regulation of these selected genes is limited or lacking.