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

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

The Effect of Glucosamine and Chondroitin on Stressed Equine Cartilage Explants

Effective prevention and treatment of osteoarthritis for horses is still needed. This research tests the ability of glucosamine and chondroitin sulfate (GLN/CS) to mitigate inflammatory and mechanical stress in vitro. In this study, GLN/CS mediate this effect by a decrease of the synthesis of nitric oxide (NO) and a decrease of proteoglycan release from the extracellular matrix in stressed cartilage explants. Explants were cultured with interleukin-1 (IL-1) + mechanical trauma with and without GLN/CS. NO and prostaglandin E2 were measured as indicators of an inflammatory response. Glycosaminoglycans were measured as an indicator of cartilage breakdown. NO levels in the stressed explants with GLN/CS treatment were lower than the IL-1 + mechanical impact treatment alone and did not differ from control group. The glycosaminoglycan release was also lower in the GLN/CS treatment than the IL-1 + mechanical impact treatment, although the prostaglandin E2 concentration was not affected. This study offers some evidence that GLN/CS treatment can partially mitigate the catabolic response to inflammatory stress and mechanical trauma in equine cartilage explants. These results provide additional support for the continued study on the benefit of GLN/CS for horses with cartilage degeneration.     

Comparison of proteoglycan and collagen in articular cartilage of horses with naturally developing osteochondrosis and healing osteochondral fragments of experimentally induced fractures

OBJECTIVE: To compare articular cartilage from horses with naturally developing osteochondrosis (OC) with normal articular cartilage and healing cartilage obtained from horses with experimentally induced osteochondral fractures. SAMPLE POPULATION: 109 specimens of articular cartilage from 78 horses. PROCEDURE: Morphologic characteristics, proteoglycan (PG), and type II collagen were analyzed in articular cartilage of OC specimens (group 1), matched healing cartilage obtained 40 days after experimentally induced osteochondral fractures (group 2), and matched normal cartilage from the same sites (group 3). RESULTS: 79 specimens of OC cartilage were obtained from horses. Ex vivo PG synthesis was significantly greater in the femoral cartilage, compared with synthesis in the tibial cartilage, and significantly greater for groups 1 and 2, compared with group 3. For groups 1 and 2, femoral fragments had significantly greater PG content, compared with PG content in tibial fragments. Keratan sulfate content was significantly less in group 3, compared with groups 1 and 2. Cartilage from the OC specimens had loss of structural architecture. The OC tissue bed stained positive for chondroitin sulfate and type II collagen, but the fracture bed did not. CONCLUSIONS AND CLINICAL RELEVANCE: Our analyses could not distinguish articular cartilage from horses with OC and a healing fracture. Both resembled an anabolic, reparative process. Immunohistochemical analysis suggested a chondromyxoid tissue in the OC bed that was morphologically similar to fibrous tissue but phenotypically resembled hyaline cartilage. Thus, tissue in the OC bed may be degenerative cartilage, whereas tissue in the fracture bed may be reparative fibrous callus.     

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.     

Morphologic and biochemical study of sternal cartilage autografts for resurfacing induced osteochondral defects in horses.

Using biodegradable pins, sternal cartilage autografts were fixed into osteochondral defects of the distal radial carpal bone in ten 2 to 3-year-old horses. The defects measured 1 cm2 at the surface and were 4 mm deep. Control osteochondral defects of contralateral carpi were not grafted. After confinement for 7 weeks, horses were walked 1 hour daily on a walker for an additional 9 weeks. Horses were euthanatized at 16 weeks. Half of the repair tissue was processed for histologic and histochemical (H&E and safranin-O fast green) examinations. The other half was used for the following biochemical analyses: type-I and type-II collagen contents, total glycosaminoglycan content, and galactosamine-to-glucosamine ratio. On histologic examination, the repair tissue in the grafted defects consisted of hyaline-like cartilage. Repair tissue in the nongrafted defects consisted of fibrocartilaginous tissue, with fibrous tissue in surface layers. On biochemical analysis, repair tissue of grafted defects was composed predominantly of type-II collagen; repair tissue of non-grafted defects was composed of type-I collagen. Total glycosaminoglycan content of repair tissue of grafted defects was similar to that of normal articular cartilage. Total glycosaminoglycan content of nongrafted defects was 62% of that of normal articular cartilage (P less than 0.05). Repair tissue of all defects was characterized by galactosamine-to-glucosamine ratio significantly (P less than 0.05) higher than that of normal articular cartilage. These results at 16 weeks after grafting indicate that sternal cartilage may potentially constitute a suitable substitute for articular cartilage in large osteochondral defects of horses.     

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

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

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 oral administration of phenylbutazone to horses on in vitro articular cartilage metabolism.

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.     

In vivo kinetic study on uptake and distribution of intramuscular tritium-labeled polysulfated glycosaminoglycan in equine body fluid compartments and articular cartilage in an osteochondrial defect model

The uptake and distribution of intramuscularly (IM) administered tritium-labeled polysulfated glycosaminoglycan (³H-PSGAG) in serum, synovial fluid, and articular cartilage of eight horses was quantitated, and hyaluronic acid (HA) concentration of the middle carpal joint was evaluated in a pharmacokinetic study. A full-thickness articular cartilage defect, created on the distal articular surface of the left radial carpal bone of each horse served as an osteochondral defect model. ³H-PSGAG (500 mg) was injected IM, between 14 and 35 days after creation of the defects. Scintillation analysis of serum and synovial fluid, collected from both middle carpal joints at specific predetermined times up to 96 hours post-injection, revealed mean ³H-PSGAG concentrations peaked at 2 hours post-injection. ³H-PSGAG was detected in cartilage and subchondral bone 96 hours post-injection in samples from all eight horses. There were no statistically significant differences in ³H-PSGAG concentration of synovial fluid or cartilage between cartilage defect and control (right middle carpal) joints.

HA assay of synovial fluid revealed concentrations significantly increased at 24, 48, and 96 hours post-injection in both joints. The concentration nearly doubled 48 hours post-injection. However, no statistically significant differences were found between synovial concentrations of HA in cartilage defect and control joints.

³H-PSGAG administered IM to horses, was distributed in the blood, synovial fluid, and articular cartilage. HA concentrations in synovial fluid increased after IM administration of polysulfated glycosaminoglycan.     

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

The effect of intramuscular polysulfated glycosaminoglycan (PSG) on repair of cartilage injury was evaluated in eight horses. In each horse, one middle carpal joint had both a partial-thickness and a full-thickness articular cartilage defect created. In the contralateral middle carpal joint, chemical articular cartilage injury was created by intra-articular injection of 50 mg sodium monoiodoacetate (MIA). Horses were divided into two groups for treatment. Group 1 horses (control) received an intramuscular injection of normal saline every four days for a total of seven injections starting seven days after cartilage injury. Group 2 horses received 500 mg of PSG intramuscularly every four days for seven treatments starting seven days after cartilage injury. Horses were maintained for 12 weeks. Horses were evaluated clinically, and their middle carpal joints were evaluated radiographically and arthroscopically at the end of the study. Joint tissues were also collected and examined microscopically. The only significant difference between groups was slightly greater matrix staining intensity for glycosaminoglycans in the radiate articular cartilage layer in MIA injected and PSG treated joints. Partial-thickness defects had not healed and the predominant repair tissue in full-thickness defects was fibrous tissue. It was concluded that using this joint injury model, 500 mg PSG administered intramuscularly had no effect on the healing of articular cartilage lesions, and minimal chondroprotective effect from chemically induced articular cartilage degeneration.     

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

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

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

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.     

Evaluation of the effects of intra-articular injection of dimethylsulfoxide on normal equine articular tissues

To evaluate the effects of intra-articular injection of dimethylsulfoxide (DMSO) on normal equine articular structures, 7 adult horses with clinically normal carpi were allotted to 2 treatment groups (group A, n = 4; group B, n = 3). In each horse after collection of synovial fluid samples, the right antebrachial carpal and middle carpal joints were aseptically injected with 2 ml of a 40% solution of 90% medical grade DMSO in lactated Ringer solution, and the corresponding joints of the left forelimb (controls) were injected with 2 ml of lactated Ringer solution. In group-A horses, 2 ml of synovial fluid was obtained prior to injections of 40% DMSO at 24 hours and 72 hours, for a total of 3 injections. At necropsy, synovial fluid, synovial membrane, and articular cartilage specimens were obtained. Group-B horses were injected with 40% DMSO in the same sequence; however, the series was repeated following a 1-week interval. Clinical evaluation of these horses revealed no evidence of carpal inflammation associated with any injection in any group. Synovial fluid analysis of DMSO-injected and control joints revealed insignificant differences in leukocyte counts and total protein content. There was no evidence of cartilage degradation on gross, histologic, or histochemical evaluation of any of the joints. Intercellular matrix staining of the articular cartilage failed to reveal any observable difference in glycosaminoglycan content between injection with DMSO or lactated Ringer solution.     

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.     

Effect of Dimethylsulfoxide on Articular Cartilage Proteoglycan Synthesis and Degradation, Chondrocyte Viability, and Matrix Water Content

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

Effects of three antiarthritic drugs on fibronectin and keratan sulfate synthesis by cultured canine articular cartilage chondrocytes.

Because articular chondrocytes are a target for drugs that can influence the integrity of cartilage, we investigated the effects of 3 antiarthritic drugs, glycosaminoglycan polysulfate, diclofenac-Na, and S-adenosylmethionine sulfate p-toluenesulfonate on total protein, fibronectin, and DNA synthesis, as well as on extradomain-A fibronectin and keratan sulfate content. Glycosaminoglycan polysulfate stimulated dose-dependent incorporation of [35S]methionine into protein and fibronectin, whereas incorporation of [3H]thymidine into DNA was unaffected. Total fibronectin, extradomain-A fibronectin, and keratan sulfate content were high in chondrocyte cultures treated with glycosaminoglycan polysulfate. In contrast, fibronectin and DNA synthesis, as well as extradomain-A fibronectin and keratan sulfate content were unaffected by diclofenac-Na. S-Adenosyl-methionine decreased dose-dependently the synthesis of fibronectin, as well as the content of fibronectin and keratan sulfate. At the highest concentration of S-adenosyl-methionine tested, findings suggest that cell viability was impaired as assessed by the release of lactate dehydrogenase into the media.     

Influence of polysulfated glycosaminoglycan on equine articular cartilage in explant culture.

Articular cartilage explants from 3 horses were maintained in tissue culture to test the effects of a polysulfated glycosaminoglycan on proteoglycan biosynthesis. Cultures were exposed to concentrations of 0, 50, or 200 micrograms of the drug/ml for either 2 days or 6 days, and labeled with 35S, before measuring the content of sulfated proteoglycan in the culture media and in extracts of cartilage. In a second experiment, the explants were incubated with the isotope and subsequently exposed to the same concentrations of the polysulfated glycosaminoglycan for 4 days. Subsequently, the amount of remaining labeled proteoglycan was determined. Gel filtration chromatography was used to compare the hydrodynamic size of proteoglycans from the cartilage explants in each experiment. Polysulfated glycosaminoglycan caused a dose-dependent depression of sulfated proteoglycan synthesis, which was statistically significant after 6 days of exposure. Radioactive proteoglycan content in explants was similar in the experiment involving isotopic labeling prior to exposure to the drug. Proteoglycan monomer size was similar in all treatment groups. It was concluded that polysulfated glycosaminoglycan caused a modest depression in proteoglycan synthesis, had little effect on endogenous proteoglycan degradation, and did not influence the size of sulfated proteoglycans synthesized by normal equine chondrocytes in explant culture.     

Evaluation of avocado and soybean unsaponifiable extracts for treatment of horses with experimentally induced osteoarthritis

OBJECTIVE: To evaluate the use of a combination of avocado and soybean unsaponifiable (ASU) extracts for the treatment of experimentally induced osteoarthritis in horses. ANIMALS: 16 horses. PROCEDURES: Osteoarthritis was induced via osteochondral fragmentation in 1 middle carpal joint of each horse; the other joint underwent a sham operation. Horses were randomly allocated to receive oral treatment with ASU extracts (1:2 [avocado-to-soybean] ratio mixed in 6 mL of molasses; n = 8) or molasses (6 mL) alone (placebo treatment; 8) once daily from days 0 to 70. Lameness, response to joint flexion, synovial effusion, gross and histologic joint assessments, and serum and synovial fluid biochemical data were compared between treatment groups to identify effects of treatment. RESULTS: Osteochondral fragmentation induced significant increases in various variables indicative of joint pain and disease. Treatment with ASU extracts did not have an effect on signs of pain or lameness; however, there was a significant reduction in severity of articular cartilage erosion and synovial hemorrhage (assessed grossly) and significant increase in articular cartilage glycosaminoglycan synthesis, compared with placebo-treated horses. CONCLUSIONS AND CLINICAL RELEVANCE: Although treatment with ASU extracts did not decrease clinical signs of pain in horses with experimentally induced osteoarthritis, there did appear to be a disease-modifying effect of treatment, compared with findings in placebo-treated horses. These objective data support the use of ASU extracts as a disease-modifying treatment for management of osteoarthritis in horses.