Effects of sodium hyaluronate and triamcinolone acetonide on glucosaminoglycan metabolism in equine articular chondrocytes treated with interleukin-1

Elysia C. Schaefer Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Allison A. Stewart Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Sushmitha S. Durgam Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Christopher R. Byron Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Matthew C. Stewart Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Abstract

Objective—To determine whether the effects of a high–molecular-weight sodium hyaluronate alone or in combination with triamcinolone acetonide can mitigate chondrocyte glyocosaminoglycan (GAG) catabolism caused by interleukin (IL)-1 administration.

Sample Population—Chondrocytes collected from metacarpophalangeal joints of 10 horses euthanized for reasons unrelated to joint disease.

Procedures—Chondrocyte pellets were treated with medium (negative control), medium containing IL-1 only (positive control), or medium containing IL-1 with hyaluronic acid only (0.5 or 2.0 mg/mL), triamcinolone acetonide only (0.06 or 0.6 mg/mL), or hyaluronic acid (0.5 or 2.0 mg/mL) and triamcinolone acetonide (0.06 or 0.6 mg/mL) in combination. Chondrocyte pellets were assayed for newly synthesized GAG, total GAG content, total DNA content, and mRNA for collagen type II, aggrecan, and cyclooxygenase (COX)-2.

Results—High-concentration hyaluronic acid increased GAG synthesis, whereas high-concentration triamcinolone acetonide decreased loss of GAG into the medium. High concentrations of hyaluronic acid and triamcinolone acetonide increased total GAG content. There was no change in DNA content with either treatment. Triamcinolone acetonide reduced COX-2 mRNA as well as aggrecan and collagen type II expression. Treatment with hyaluronic acid had no effect on mRNA for COX-2, aggrecan, or collagen type II.

Conclusions and Clinical Relevance—Results indicated that high concentrations of hyaluronic acid or triamcinolone acetonide alone or in combination mitigated effects of IL-1 administration on GAG catabolism of equine chondrocytes.

Abstract

Objective—To determine whether the effects of a high–molecular-weight sodium hyaluronate alone or in combination with triamcinolone acetonide can mitigate chondrocyte glyocosaminoglycan (GAG) catabolism caused by interleukin (IL)-1 administration.

Sample Population—Chondrocytes collected from metacarpophalangeal joints of 10 horses euthanized for reasons unrelated to joint disease.

Procedures—Chondrocyte pellets were treated with medium (negative control), medium containing IL-1 only (positive control), or medium containing IL-1 with hyaluronic acid only (0.5 or 2.0 mg/mL), triamcinolone acetonide only (0.06 or 0.6 mg/mL), or hyaluronic acid (0.5 or 2.0 mg/mL) and triamcinolone acetonide (0.06 or 0.6 mg/mL) in combination. Chondrocyte pellets were assayed for newly synthesized GAG, total GAG content, total DNA content, and mRNA for collagen type II, aggrecan, and cyclooxygenase (COX)-2.

Results—High-concentration hyaluronic acid increased GAG synthesis, whereas high-concentration triamcinolone acetonide decreased loss of GAG into the medium. High concentrations of hyaluronic acid and triamcinolone acetonide increased total GAG content. There was no change in DNA content with either treatment. Triamcinolone acetonide reduced COX-2 mRNA as well as aggrecan and collagen type II expression. Treatment with hyaluronic acid had no effect on mRNA for COX-2, aggrecan, or collagen type II.

Conclusions and Clinical Relevance—Results indicated that high concentrations of hyaluronic acid or triamcinolone acetonide alone or in combination mitigated effects of IL-1 administration on GAG catabolism of equine chondrocytes.

Intra-articular injections of corticosteroids rapidly resolve joint inflammation, synovitis, and signs of pain1–7 and are the gold standard of treatment for horses with osteoarthritis. Corticosteroids inhibit inflammation via 2 mechanisms. Corticosteroids suppress arachidonic acid metabolism through lipocortin-induced phospholipase inhibition.3 This inhibition helps to stabilize the phospholipids in the cell membrane, making them unavailable for entrance into the arachidonic cascade. Corticosteroids also block production of proinflammatory cytokines, such as IL-1, by binding to cytoplasmic receptors and modulating inflammatory gene transcription.3 Two corticosteroids that are frequently used are methylprednisolone acetonide and triamcinolone acetonide. High doses of methylprednisolone acetonide can have detrimental effects on normal and abnormal articular cartilage by impairing chondrocyte activity, inhibiting GAG and proteoglycan synthesis in the articular cartilage matrix, and decreasing expression of mRNA for collagen type II.1,2,4–6 In contrast, studies7–9 on triamcinolone acetonide reveal many beneficial effects on abnormal articular cartilage, including decreased IL-1–induced GAG degradation, increased proteoglycan synthesis, and enhanced chondrocyte viability. Six to 18 mg of intra-articularly administered triamcinolone acetonide is recommended for treatment of most equine joints.10

Clinically, hyaluronic acid has been used as a treatment for osteoarthritis. In a clinically normal joint, hyaluronic acid is secreted into the synovial fluid by joint capsule synoviocytes and serves to lubricate the joint.11 It is also an important component of the articular cartilage, where it binds to chondrocyte CD44 receptors12 and serves as a backbone of attachment sites for proteoglycan structures.13 Proteoglycans maintain the hydrostatic pressure of cartilage, allowing resistance to compressive forces during weight bearing,14 and are depleted first in the early stages of osteoarthritis.15 Documented beneficial effects of hyaluronic acid are increased chondrocyte metabolism, increased GAG content in the cartilage matrix, and decreased activity of pro-inflammatory mediators, which results in decreased matrix degradation.2,12,14,16,17 However, it remains uncertain whether the molecular weight of various hyaluronic acid products is an important factor in its efficacy. Several studies18–21 reveal that a high–molecular-weight hyaluronic acid may have longer efficacy and increased metabolic and anti-inflammatory properties, compared with a low–molecular-weight hyaluronic acid. However, results of other studies22,23 indicate little to no difference between high- and low–molecular-weight hyaluronic acid products.

Two recently published studies have evaluated combination therapy with methylprednisolone acetonide and hyaluronic acid on normal equine cartilage explants1 and an IL-1–induced inflammatory model of chondrocyte metabolism.2 The cartilage explant study revealed detrimental effects of methylprednisolone acetonide on normal cartilage and that the addition of a medium–molecular-weight hyaluronic acid had little effect on the corticosteroid-induced proteoglycan catabolism of the cartilage matrix.1 The other study found beneficial effects on proteoglycan metabolism by use of lower doses of methylprednisolone acetonide and a medium–molecular-weight hyaluronic acid on chondrocyte pellets in an inflammatory environment (ie, IL-1–treated).2 The purpose of the study reported here was to evaluate the effects of 2 of the most commonly used intra-articular treatments—a corticosteroid (triamcinolone acetonide) and a high–molecular-weight hyaluronic acid. Our hypothesis was that administration of hyaluronic acid alone or in combination with triamcinolone acetonide would mitigate the chondrocyte GAG catabolism caused by IL-1 administration.

Materials and Methods

Horses and pellet culture—All horses used in this study were euthanized for reasons unrelated to joint disease by use of a lethal dose of sodium pentobarbital administered IV. The Institutional Animal Care and Use Committee at the University of Illinois approved this study. Articular cartilage was aseptically collected from the metacarpophalangeal joints of 10 horses that ranged in age from 2 to 4 years. All joints were evaluated to ensure there was no gross evidence of joint disease prior to cartilage collection. On day 1, the cartilage was placed in chondrogenic medium consisting of Dulbecco modified Eagle medium,a 10% fetal bovine serum,b 1% L-glutamine,c 1% penicillin-streptomycin,d and ascorbic acid (50 μg/mL)e and was digested overnight with 0.2% collagenase.f After digestion, an estimation of total chondrocyte number and viability was made by use of a Reichart hematocytometer and trypan blue stain.g The chondrocytes were suspended at a concentration of 500,000 cells/mL in chondrogenic medium. The medium containing the chondrocytes was transferred to an Eppendorf tube (0.5 mL/tube). The medium was centrifuged to form chondrocyte pellets containing 250,000 cells. The pellets were incubated at 37°C for 7 days to allow formation of an extracellular matrix, and the medium was changed every 2 to 3 days.

Treatments were administered on day 7, and the pellets were incubated for an additional 24 hours. There were 10 treatment groups with a minimum of 10 pellets in each group (Appendix). Treatment groups consisted of fresh medium only (negative control), fresh medium with IL-1h only (10 ng/mL [positive control]), IL-1 (10 ng/mL) and hyaluronic acidi (0.5 mg/mL or 2 mg/mL [2 treatment groups]), IL-1 (10 ng/mL) and triamcinolone acetonidej (0.06 mg/mL or 0.6 mg/mL [2 treatment groups]), and IL-1 (10 ng/mL) with hyaluronic acid (0.5 mg/mL or 2 mg/mL) and triamcinolone acetonide (0.06 mg/mL or 0.6 mg/mL [4 treatment groups]). The concentrations of hyaluronic acid and triamcinolone acetonide were determined from a range of published concentrations likely to be present in the metacarpophalangeal joint 24 to 48 hours after an intra-articular injection.24,25 At the time of treatment, 4 pellets from each treatment group were radiolabeled with medium containing sulfur 35 (35S)–labeled sodium sulfate (10 MCi/mL).k All pellets were removed from the treatment medium after 24 hours of incubation and were washed 3 times with PBS solution. For 7 horses, 4 radiolabeled pellets and exhausted medium were stored at −80°C until further analysis. For 3 horses, 20 pellets in each treatment group were snap-frozen in liquid nitrogen and saved at −80°C for RNA isolation. For 6 horses, 2 pellets/treatment group were saved for histologic examination.

GAG synthesis—New GAG synthesis was determined via 35SO4 incorporation into the pellet and subsequent release into the medium during a 24-hour period. Radiolabeled pellets were digested in papain (150 μg/mL)l at 65°C for 24 hours. Radiolabeled medium was digested in papain (150 μg/mL) at 65°C for 4 hours. Aliquots of 25 μL of radiolabeled papain-digested pellets and 25 μL of radiolabeled papain-digested medium were placed in 96-well filtration plates,m precipitated with 0.2% Alcian blue dye solution, and counted for scintillation.26 Radioisotope decay was accounted for in all values, and scintillation counts were normalized for pellet digestion volume.

Total pellet GAG content—Total GAG content in the pellets and in the medium was determined by use of a dimethylmethylene blue binding assay.27 Pellets were digested in papain as described. Aliquots of 25 μL of papain-digested pellets were placed into 96-well microplates, 200 μL of 1,9-dimethylmethylene blue dyen was added, and samples were analyzed for absorbance. All sample values were compared against a standard curve of chondroitin sulfate values to estimate the total GAG content and normalized for pellet digestion volume.

Total pellet DNA content—Total DNA content of each pellet was determined by use of a fluorometric Hoechst assay.28 Pellets were digested in papain as described. Aliquots of 10 μL of papain-digested pellets were placed into 96-well microplates, 200 μL of dye solutiono was added, and samples were analyzed for fluorescence in a microplate reader.p All sample values were compared against a standard curve of calf thymus values to estimate total DNA content of the digested pellets.

Pellet mRNA content—Real-time PCR data were obtained from chondrocyte pellet cultures of 3 horses. The RNA was extracted from 20 pellets/horse in each treatment group by use of the Trizol reagentq according to the manufacturer's suggested protocol. Complementary DNA was obtained by priming the sample with oligo d(T)r and then adding reverse transcriptase.s Realtime quantitative PCR analysis was performed for collagen type II, aggrecan, and COX-2 and normalized to elongation factor-1α mRNA expression. A PCR detection systemt was used to perform the assay.1,2,29

Histologic examination—After 24 hours in 4% paraformaldehyde, pellets were transferred to a 4% agarose gel and stored at 4°C overnight. The pellets were dehydrated in alcohol, embedded in paraffin, sectioned, and stained with toluidine blue.

Statistical analysis—All nonnormally distributed data were logarithmically transformed and presented as mean ± SE log values. A 1-way repeated-measures ANOVA performed with a software programu was used to compare the positive control (IL-1) with the negative control (no IL-1). The hyaluronic acid and triamcinolone acetonide values were evaluated by use of a 2-way repeated-measures ANOVA performed with the same software program. All post hoc tests were conducted when indicated by use of the Holm-Sidak method. Values of P ≤ 0.05 were considered significant.

Results

Pellet GAG synthesis—Pellet GAG synthesis was designated as the amount of newly synthesized (radiolabeled) GAG (CPM) retained in the pellet. Treatment with IL-1 significantly (P = 0.018) decreased GAG synthesis of the positive control (IL-1 only), compared with the negative control (no IL-1; Figure 1). Treatment with hyaluronic acid (2 mg/mL) significantly (P < 0.001) increased GAG synthesis, compared with the IL-1 control group. Treatment with 0.06 and 0.6 mg of triamcinolone acetonide/mL did not have a significant (P = 0.218) effect on GAG synthesis, compared with 0 mg of triamcinolone acetonide/mL. However, there was a significant (P = 0.004) effect of the combined treatment of hyaluronic acid and triamcinolone acetonide (2 mg of hyaluronic acid/mL combined with 0.06 or 0.6 mg of triamcinolone acetonide/mL), which increased newly synthesized GAG; the 0.06 and 0.6 mg/mL groups were not significantly different. Similarly, results in pellets treated with 2 mg of hyaluronic acid/mL combined with 0.6 mg of triamcinolone acetonide/mL were not significantly different from pellets treated with 0.6 mg of triamcinolone acetonide/mL alone.

Figure 1—
Figure 1—

Log mean ± SE values (n = 7 horses) for incorporation of sulfur 35 (35S)–labeled sodium sulfate into GAG of equine articular chondrocyte pellets treated with various concentrations of hyaluronic acid (HA) and triamcinolone acetonide (TA). *Significant (P < 0.05) difference between negative control (no IL-1) and positive control (IL-1 only). †Significant (P < 0.05) effect of HA, compared with no HA, at the same concentrations of TA. ‡Significant (P < 0.05) effect of combination treatment (HA and TA), compared with the IL-1 control.

Citation: American Journal of Veterinary Research 70, 12; 10.2460/ajvr.70.12.1494

Total GAG synthesis—Total GAG synthesis was designated as the amount of newly synthesized GAG retained in the pellet plus that released into the medium. Treatment with IL-1 resulted in no significant (P = 0.097) difference in total GAG synthesis, compared with the negative control (no IL-1; Figure 2). Treatment with hyaluronic acid (2 mg/mL) resulted in a significant (P = 0.026) increase in total GAG synthesis, compared with the IL-1 control group. Treatment with triamcinolone acetonide did not have a significant (P = 0.607) effect on total GAG synthesis. There was no significant (P = 0.48) synergistic effect of hyaluronic acid and triamcinolone acetonide combined.

Figure 2—
Figure 2—

Log mean ± SE values (n = 7 horses) for incorporation of S35-labeled sodium sulfate into GAG of equine articular chondrocyte pellets and the medium following treatment with various concentrations of HA and TA. *Significant (P < 0.05) effect of HA, compared with no HA, at the same concentrations of TA.

Citation: American Journal of Veterinary Research 70, 12; 10.2460/ajvr.70.12.1494

Percentages of GAG retained in pellet and released into the medium—Percentage of GAG retained in the pellet was calculated by dividing the amount of newly synthesized GAG (CPM) in the pellet by the total GAG in the pellet plus the medium. Percentage of GAG in the medium was calculated by dividing the newly synthesized GAG (CPM) in the medium by the total GAG in the pellet plus the medium. Treatment with IL-1 only significantly (P = 0.04) decreased the percentage of GAG retained in the pellet and significantly (P = 0.04) increased the percentage of GAG released into the medium, compared with the negative control (no IL-1; Figures 3 and 4). Treatment with hyaluronic acid did not have a significant (P = 0.26) effect on percentage of GAG retained within the pellets or on the percentage of GAG released into the medium. Treatment with triamcinolone acetonide (0.06 and 0.6 mg of triamcinolone acetonide/mL) resulted in a significant (P = 0.004) increase in percentage of GAG in pellets and decrease in percentage of GAG released into the medium, compared with the 0 mg of triamcinolone acetonide/mL treatment group. There was no significant (P = 0.67) synergistic effect of hyaluronic acid and triamcinolone acetonide combined on percentage of GAG retained in the pellet or percentage of GAG released into the medium.

Figure 3—
Figure 3—

Mean ± SE percentage values (n = 7 horses) for incorporation of S35-labeled sodium sulfate into GAG of equine articular chondrocyte pellets treated with various concentrations of HA and TA. *Significant (P < 0.05) difference between negative control (no IL-1) and positive control (IL-1 only). †Significant (P < 0.05) effect of TA treatment groups, compared with the non–TA treatment group, at various concentrations of HA.

Citation: American Journal of Veterinary Research 70, 12; 10.2460/ajvr.70.12.1494

Figure 4—
Figure 4—

Mean ± SE percentage values (n = 7 horses) for incorporation of S35-labeled sodium sulfate into GAG released into medium following treatment with various concentrations of HA and TA. See Figure 3 for key.

Citation: American Journal of Veterinary Research 70, 12; 10.2460/ajvr.70.12.1494

Total GAG pellet content—Total pellet GAG content was designated as the total GAG content retained in the pellet after treatment, and this included the newly synthesized GAG. Treatment with IL-1 alone did not have a significant (P = 0.101) effect on the total pellet GAG content, compared with the negative control (no IL-1; Figure 5). Treatment with hyaluronic acid (2 mg/mL) significantly (P = 0.002) increased the total GAG content within the pellet, compared with the IL-1 control group. Treatment of 0.6 mg of triamcinolone acetonide/mL significantly (P = 0.036) increased total GAG pellet content, compared with the 0 mg/mL treatment group. There was no significant (P = 0.732) synergistic effect of hyaluronic acid and triamcinolone acetonide combined.

Figure 5—
Figure 5—

Log mean ± SE values (n = 7 horses) for total GAG content in equine articular chondrocyte pellets treated with various concentrations of HA and TA. *Significant (P < 0.05) effect of HA, compared with no HA, at the same concentrations of TA. †Significant (P < 0.05) effect of TA treatment groups, compared with the non–TA treatment group, at various concentrations of HA.

Citation: American Journal of Veterinary Research 70, 12; 10.2460/ajvr.70.12.1494

Total DNA pellet content—Treatment with IL-1 (P = 0.217), hyaluronic acid (P = 0.781), or triamcinolone acetonide (P = 0.982) had no significant effect on the DNA content of pellets.

Pellet mRNA content—Treatment with IL-1 only significantly (P = 0.026) increased collagen type II mRNA, compared with the negative control (no IL-1; Figure 6). Treatment with hyaluronic acid had no significant (P = 0.102) effect on collagen type II mRNA, compared with the IL-1 control group. Treatment with 0.06 and 0.6 mg of triamcinolone acetonide/mL significantly (P = 0.001) decreased collagen type II mRNA, compared with the IL-1 control. There was no significant (P = 0.121) synergistic effect of hyaluronic acid and triamcinolone acetonide combined.

Figure 6—
Figure 6—

Mean ± SE values (n = 3 horses) for collagen type II mRNA in equine articular chondrocyte pellets treated with various concentrations of HA and TA. *Significant (P < 0.05) difference between negative control (no IL-1) and positive control (IL-1 only). †Significant (P < 0.05) effect of TA treatment groups, compared with the non–TA treatment group, at various concentrations of HA.

Citation: American Journal of Veterinary Research 70, 12; 10.2460/ajvr.70.12.1494

Treatment with IL-1 only significantly (P = 0.045) increased aggrecan mRNA, compared with the negative control (no IL-1; Figure 7). Treatment with hyaluronic acid did not have a significant (P = 0.725) effect on aggrecan mRNA, compared with the IL-1 control. Treatment with triamcinolone acetonide had a significant (P = 0.045) effect on aggrecan mRNA. Specifically, the 0.06 and 0.6 mg of triamcinolone acetonide/mL treatment groups had significantly decreased aggrecan mRNA, compared with the IL-1 control. There was no significant (P = 0.11) synergistic effect of hyaluronic acid and triamcinolone acetonide combined.

Figure 7—
Figure 7—

Mean ± SE values (n = 3 horses) for aggrecan mRNA in equine articular chondrocyte pellets treated with various concentrations of HA and TA. See Figure 3 for key.

Citation: American Journal of Veterinary Research 70, 12; 10.2460/ajvr.70.12.1494

Treatment with IL-1 only significantly (P = 0.021) increased COX-2 mRNA, compared with the negative control (no IL-1; Figure 8). Treatment with hyaluronic acid did not have a significant (P = 0.126) effect on COX-2 mRNA, compared with the IL-1 control. Treatment with triamcinolone acetonide had a significant (P = 0.007) effect on COX-2 mRNA. Specifically, the 0.06 and 0.6 mg of triamcinolone acetonide/mL treatment groups had significantly decreased COX-2 mRNA, compared with the IL-1 control group. There was no significant (P = 0.464) synergistic effect of hyaluronic acid and triamcinolone acetonide combined.

Figure 8—
Figure 8—

Mean ± SE values (n = 3 horses) for COX-2 mRNA in equine articular chondrocyte pellets treated with various concentrations of HA and TA. See Figure 3 for key.

Citation: American Journal of Veterinary Research 70, 12; 10.2460/ajvr.70.12.1494

Histologic examination—The chondrocyte pellets varied in size among horses. The size of the pellets was not measured; instead the pellets were only evaluated for proteoglycan production through the use of the toluidine blue stain. Subjectively, pellets treated with hyaluronic acid, triamcinolone acetonide, or the combination had increased proteoglycan staining, compared with the IL-1 treated control, throughout the pellet matrix (Figure 9).

Figure 9—
Figure 9—

Photomicrographs of sections of equine articular chondrocyte pellets obtained 24 hours after administration of IL-1 treatment, 2 mg of HA/mL, 0.6 mg of TA/mL, or 2 mg of HA/mL and 0.6 mg of TA/mL. Toluidine blue stain; bar = 100 μm.

Citation: American Journal of Veterinary Research 70, 12; 10.2460/ajvr.70.12.1494

Discussion

Results of the study reported here indicated the beneficial effects of hyaluronic acid alone and in combination with triamcinolone acetonide on IL-1–treated equine articular chondrocyte pellets. Hyaluronic acid increased both the new GAG synthesis by chondrocytes and the total GAG content retained in the pellets, compared with the IL-1 control. Treatment with triamcinolone acetonide increased the total GAG content retained in the chondrocyte pellet. These results were similar to those of another study8 in which triamcinolone acetonide mitigated IL-1–induced GAG degradation. Treatment with triamcinolone acetonide also reduced COX-2 mRNA expression, compared with the IL-1 control. Most of these benefits were detected at the high concentration of hyaluronic acid (2 mg/mL) and the high concentration of triamcinolone acetonide (0.6 mg/mL). The combination of 2 mg of hyaluronic acid/mL and 0.6 mg of triamcinolone acetonide/mL resulted in the highest concentrations of total pellet GAG following treatment with IL-1, although a significant synergistic effect was not found.

Treatment with IL-1 had several detrimental effects on chondrocyte metabolism. Administration of IL-1 caused a decrease in retention of newly synthesized GAG by the pellet, compared with the negative control (no IL-1). Treatment with IL-1 also increased COX-2 mRNA expression, compared with the negative control. In addition, IL-1 administration caused an increase in aggrecan and collagen type II mRNA production, compared with the negative control. This may have been caused by an increase in chondrocyte metabolism of aggrecan and collagen type II in response to an IL-1–mediated matrix catabolism.30

Treatment with hyaluronic acid increased GAG synthesis and total GAG pellet content, compared with the positive control (IL-1 treatment), but did not have an effect on mRNA of COX-2, aggrecan, or collagen type II. Specifically, addition of hyaluronic acid (2 mg/mL) was beneficial in negating the effects of IL-1 administration and increasing new GAG synthesis, compared with the positive control. Lower concentrations of hyaluronic acid did not have a significant effect on GAG synthesis. Although hyaluronic acid was beneficial for GAG synthesis, treatment with hyaluronic acid had no effect on COX-2 mRNA, suggesting that matrix degradation continued to occur in these treatment groups. Hyaluronic acid also had no effect on collagen type II and aggrecan mRNA, but resulted in maintained expression rates similar to the IL-1–treated control. The disparity between the aggrecan mRNA expression data and new GAG synthesis suggested that hyaluronic acid may have increased synthesis of products other than aggrecan, such as biglycan and decorin. Alternatively, the retention of aggrecan in the pellet after synthesis may have been more effective in the presence of hyaluronic acid. Ultimately, the GAG synthesis, GAG release, and total GAG retained in the pellet matrix are probably the most important data.

Treatment with triamcinolone acetonide increased total pellet GAG content and decreased mRNA of COX-2, aggrecan, and collagen type II, compared with the IL-1–treated control. Specifically, treatment with triamcinolone acetonide increased total GAG pellet content by increasing GAG retention within the pellet while decreasing GAG lost into the medium. In addition, triamcinolone acetonide was beneficial in suppressing COX-2 mRNA that increased with IL-1 treatment. As seen in other studies2,4,6 using a corticosteroid, treatment with triamcinolone acetonide was effective at a cellular level in blocking mediators of inflammation. In the present study, treatment with triamcinolone acetonide suppressed mRNA of aggrecan and collagen type II. However, the mRNA expression associated with triamcinolone acetonide treatment was similar to the baseline expression of treatment groups with no IL-1. Corticosteroid-induced suppression of aggrecan and collagen type II mRNA has been detected before.31 Corticosteroid suppression of aggrecan and collagen type II mRNA may have detrimental effects in a longer-term study. These results suggest triamcinolone acetonide predominantly works by suppressing inflammation to retain GAG in the pellet, not by increasing the synthesis of GAG in the pellet.

Combination treatment with hyaluronic acid and triamcinolone acetonide mitigated the effects of IL-1 administration by increasing new GAG synthesis and retention within the pellet. The high concentrations of both drugs—hyaluronic acid at 2 mg/mL and triamcinolone acetonide at 0.6 mg/mL—in combination had the most profound effect on GAG synthesis. These results suggest that hyaluronic acid and triamcinolone acetonide mitigate the detrimental effects of IL-1 administration through different mechanisms to increase the total GAG content within the pellet. In an inflammatory environment such as that created by IL-1 administration, high concentrations of hyaluronic acid may be beneficial in supporting chondrocyte GAG production, whereas high concentrations of triamcinolone acetonide may act in an additive or synergistic fashion by retaining GAG within the chondrocyte matrix.

Similar to a previous study, IL-1 was administered to create an inflammatory environment.2 Although IL-1 caused expected detrimental effects in this study, other inflammatory mediators are also known to be present in osteoarthritic joints. These proinflammatory mediators include tumor necrosis factor-α, IL-6, IL-8, IL-11, IL-17, and leukemia inhibitory factor.32 Studies32,33 have documented the detrimental effects of these inflammatory cytokines, suggesting that they may act along different pathways or in a synergistic fashion with IL-1. Results of the present study may have been amplified if other proinflammatory mediators had been used with IL-1 administration to create a model of inflammation.

This study used an in vitro model to study the effects of inflammation on cartilage metabolism. Although an in vitro study provided a controlled environment to evaluate the effect of multiple concentrations of hyaluronic acid and triamcinolone acetonide on a standardized cartilage matrix, many important in vivo influences were lost. Specifically, an in vitro study does not allow for clearance of metabolites from the local environment or for systemic metabolism of administered medications. Therefore, results may not accurately represent what would occur in an in vivo situation. Further in vitro studies evaluating the effects of hyaluronic acid and triamcinolone acetonide in osteoarthritic equine joints may provide further insight into the benefits and deficiencies of intra-articular hyaluronic acid and triamcinolone acetonide administration.

The range of hyaluronic acid and triamcinolone acetonide concentrations used in this study was based on estimated joint concentrations following commonly used intra-articular dosages. In a previous study,13 triamcinolone acetonide at 1.2 mg/mL decreased IL-1–induced GAG degradation, but was unable to maintain GAG synthesis; an overall decrease in GAG synthesis was detected. In the present study, the highest dose of triamcinolone acetonide (0.6 mg/mL) had a protective effect against IL-1–induced GAG degradation without any decrease in GAG synthesis. On the basis of these findings, it is possible that triamcinolone acetonide concentrations > 0.6 mg/mL may have some detrimental effects on chondrocytes8 such as those seen with high doses of methylprednisolone acetonide.1,2,8,9

The effects of triamcinolone acetonide in the present study were similar to results from a previous study2 that used a similar model and used methylprednisolone acetonide. The response to corticosteroid administration was similar for pellet GAG synthesis, total pellet GAG content, total pellet DNA content, and mRNA expression. Both methylprednisolone acetonide and triamcinolone acetonide increased pellet GAG synthesis in combination with hyaluronic acid, suggesting a substantial benefit when corticosteroids and hyaluronic acid are used together. Both corticosteroids, methylprednisolone acetonide and triamcinolone acetonide, significantly increased the total pellet GAG content. Both methylprednisolone acetonide and triamcinolone acetonide had no effect on the total pellet DNA content. Methylprednisolone acetonide and triamcinolone acetonide both had a significant effect on reducing mRNA of COX-2 in the presence of IL-1 administration. The findings of these 2 studies illustrate the beneficial effects corticosteroids have on reducing inflammation and retaining GAG content in the matrix. These studies also revealed that the combination of corticosteroids and hyaluronic acid had the most beneficial effect on increasing GAG synthesis of the pellet and retaining the total GAG content within the pellet.

In the present study, a high–molecular-weight (3,000,000 kDa) hyaluronic acid was used. In previous studies,1,2 use of a medium–molecular-weight (500,000 to 730,000 kDa) hyaluronic acid resulted in no significant effects on new GAG synthesis when used alone. In contrast, the present study revealed an increase in new GAG synthesis and an increase in total pellet GAG with only hyaluronic acid administration at 2.0 mg/mL. These results suggest that a high–molecular-weight hyaluronic acid may be more beneficial in mitigating IL-1–induced proteoglycan catabolism. However, the efficacy of different molecular weights of hyaluronic acid still remains a controversial subject that needs further conclusive in vivo evaluation.18,20,34

The high concentration of hyaluronic acid in combination with triamcinolone acetonide had the most beneficial effects on proteoglycan matrix metabolism in the presence of IL-1 administration. This was a result of both an increase in GAG synthesis and an increase in retention of pellet GAG through a decrease in inflammatory mediators. Future studies may be useful to evaluate the in vivo effects of triamcinolone acetonide and a high–molecular-weight hyaluronic acid in osteoarthritic equine joints.

ABBREVIATIONS

COX-2

Cyclooxygenase-2

CPM

Counts per minute

GAG

Glycosaminoglycan

IL

Interleukin

a.

Dulbecco modified Eagle medium, Mediatech Inc, Herndon, Va.

b.

Fetal bovine serum, Gemini Bioproducts, Woodland, Calif.

c.

L-glutamine, 200mM, Invitrogen, Carlsbad, Calif.

d.

Penicillin-streptomycin, BioWhittaker, Cambrex Bio Science, Walkersville, Md.

e.

Ascorbic acid, WAKO, Richmond, Va.

f.

Collagenase type 2, Worthington Biomedical Corp, Lakewood, NJ.

g.

Trypan blue stain, 0.4%, Invitrogen, Carlsbad, Calif.

h.

IL-1, R&D Systems, Minneapolis, Minn.

i.

Hylartin-V, Pfizer Inc, New York, NY.

j.

Vetalog, Fort Dodge Laboratories, Fort Dodge, Iowa.

k.

S-35–labeled sodium sulfate, MP Biochemicals, Irvine, Calif.

l.

Papain, Sigma-Aldrich, St Louis, Mo.

m.

Multiwell punch plates, polyvinylidene fluoride plate, Millipore, Bedford, Mass.

n.

1,9-dimethyl-methylene blue, Sigma-Aldrich, St Louis, Mo.

o.

Hoechst 33258, Sigma-Aldrich, St Louis, Mo.

p.

Microplate reader, FLUOstar Optima, BMG Laboratories, Durham, NC.

q.

Trizol, Invitrogen, Carlsbad, Calif.

r.

Oligo d(T), Invitrogen, Carlsbad, Calif.

s.

Superscript II, Invitrogen, Carlsbad, Calif.

t.

iCycler iQ real-time PCR detection system, Bio-Rad Laboratories, Hercules, Calif.

u.

SigmaStat, version 3.0, Systat Software Inc, San Jose, Calif.

Appendix

Treatments of hyaluronic acid (HA) and triamcinolone acetonide (TA) applied to chondrocytes collected from 10 horses. Each horse's chondrocytes were evaluated as a separate experiment. All experiments had the same 10 treatment groups. Chondrocytes from 7 horses were evaluated for GAG synthesis, pellet GAG content, and pellet DNA content, and chondrocytes from 6 of the 7 horses were evaluated histologically. Chondrocytes from 3 horses were evaluated for mRNA.

TreatmentIL-1 (ng/mL)HA (mg/mL)TA (mg/mL)
1 (negative control)000
2 (positive control)1000
3100.50
41020
51000.06
6100.50.06
71020.06
81000.6
9100.50.6
101020.6

References

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    Doyle AJ, Stewart AA, Constable PD, et al. Effects of sodium hyaluronate and methylprednisolone acetonide on proteoglycan synthesis in equine articular cartilage explants. Am J Vet Res 2005;66:4853.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2.

    Yates AC, Stewart AA, Byron CR, et al. Effects of sodium hyaluronate and methylprednisolone acetonide on proteoglycan metabolism in equine articular chondrocytes treated with interleukin-1. Am J Vet Res 2006;67:19801986.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3.

    Gilron I. Corticosteroids in postoperative pain management: future research directions for a multifaceted therapy. Acta Anaesthesiol Scand 2004;48:12211222.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4.

    Frisbie DD, Kawcak CE, Baxter GM, et al. Effects of 6A-methylpredinisolone acetonide on an equine osteochondral fragment exercise model. Am J Vet Res 1998;59:16191628.

    • Search Google Scholar
    • Export Citation
  • 5.

    Shoemaker RS, Bertone AL, Martin GS, et al. Effects of intra-articular administration of methylprednisolone acetonide on normal articular cartilage and on healing of experimentally induced osteochondral defects in horses. Am J Vet Res 1992;53:14461453.

    • Search Google Scholar
    • Export Citation
  • 6.

    Murphy DJ, Todhunter RJ, Fubini SL, et al. The effects of methylprednisolone on normal and monocyte-conditioned medium-treated articular cartilage from dogs and horses. Vet Surg 2000;29:546557.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7.

    Frisbie DD, Kawcak CE, Trotter GW, et al. Effects of triamcinolone acetonide on an in vivo equine osteochondral fragment exercise model. Equine Vet J 1997;29:349359.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8.

    Sandler EA, Frisbie DD, McIlwraith CW. A dose titration of triamcinolone acetonide on insulin-like growth factor-1 and interleukin-1-conditioned equine cartilage explants. Equine Vet J 2004;36:5863.

    • Search Google Scholar
    • Export Citation
  • 9.

    Dechant JE, Baxter GM, Frisbie DD, et al. Effects of dosage titration of methylprednisolone acetonide and triamcinolone acetonide on interleukin-1-conditioned equine articular cartilage explants in vitro. Equine Vet J 2003;35:444450.

    • Search Google Scholar
    • Export Citation
  • 10.

    Caron JP. Intra-articular injections for joint disease in horses. Vet Clin North Am Equine Pract 2005;21:559573.

  • 11.

    Schmidt TA, Gastelum NS, Nguyen QT, et al. Boundary lubrication of articular cartilage: role of synovial fluid constituents. Arthritis Rheum 2007;56:882891.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12.

    Akmal M, Singh A, Anand A, et al. The effects of hyaluronic acid on articular chondrocytes. J Bone Joint Surg Br 2005;87:11431149.

  • 13.

    Goodrich LR, Nixon AJ. Medical treatment of osteoarthritis in the horse—a review. Vet J 2006;171:5169.

  • 14.

    Rydell NW, Butler J, Balazs EA. Hyaluronic acid in synovial fluid. Acta Vet Scand 1970;11:139155.

  • 15.

    Young AA, McLennan S, Smith MM, et al. Proteoglycan 4 downregulation in a sheep meniscectomy model of early osteoarthritis. Arthritis Res Ther 2006;8:R41.

  • 16.

    Iwata H. Pharmacologic and clinical aspects of intraarticular injection of hyaluronate. Clin Orthop 1993;289:285291.

  • 17.

    Moreland LW. Intra-articular hyaluronan (hyaluronic acid) and hylans for the treatment of osteoarthritis: mechanisms of action. Arthritis Res Ther 2003;5:5467.

    • Search Google Scholar
    • Export Citation
  • 18.

    White GW, Stites T, Hamm J, et al. Evaluation of the efficacy of various preparations of sodium hyaluronate in an induced equine carpitis model. J Equine Vet Sci 1999;19:331337.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19.

    McIlwraith CW. Synovitis and other soft tissue injuries of equine joints, in Proceedings. Dubai Int Equine Symp 1996;287322.

  • 20.

    Phillips MW. Intraarticular sodium hyaluronate in the horse: a clinical trial, in Proceedings. 26th Annu Conv Am Assoc Equine Pract 1980;389394.

    • Search Google Scholar
    • Export Citation
  • 21.

    Cheney M. One practice's approach to joint therapy in the equine athlete, in Proceedings. 42nd Annu Conv Am Assoc Equine Pract 1996;6974.

    • Search Google Scholar
    • Export Citation
  • 22.

    Aviad AD, Houpt JB. The molecular weight of therapeutic hyaluronan (sodium hyaluronate): how significant is it? J Rheumatol 1994;21:297301.

    • Search Google Scholar
    • Export Citation
  • 23.

    Aviad AD, Arthur RM, Brencick VA, et al. Synacid vs Hylartin V in equine joint disease. J Equine Vet Sci 1988;8:112116.

  • 24.

    Chen CL, Sailor JA, Collier J, et al. Synovial and serum levels of triamcinolone following intra-articular administration of triamcinolone acetonide in the horse. J Vet Pharmacol Ther 1992;15:240246.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    Hilbert BJ, Rowley G, Antonas KN, et al. Changes in the synovia after intra-articular injection of sodium hyaluronate into normal horse joints and after arthrotomy and experimental cartilage damage. Aust Vet J 1985;62:182184.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.

    Masuda K, Shirota H, Thonar E. Quantification of 35S-labeled proteoglycans complexed to alcian blue by rapid filtration in multiwell plates. Anal Biochem 1994;217:167175.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27.

    Farndale RW, Sayers CA, Barrett AJ. A direct spectrophotometric microassay for sulfated glycosaminoglycans in cartilage cultures. Connect Tissue Res 1982;9:247248.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28.

    Kim YJ, Sah RL, Doong JY, et al. Fluorometric assay of DNA in cartilage explants using Hoechst 33258. Anal Biochem 1988;174:168176.

  • 29.

    Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT Method. Methods 2001;25:402408.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30.

    MacLeod JN, Fubini SL, Gu DN, et al. Effect of synovitis and corticosteroids on transcription of cartilage matrix proteins. Am J Vet Res 1998;59:10211026.

    • Search Google Scholar
    • Export Citation
  • 31.

    Fubini SL, Todhunter RJ, Burton-Wurster N, et al.Corticosteroids alter the differentiated phenotype of articular chondrocytes. J Orthop Res 2001;19:688695.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32.

    Shi J, Schmitt-Talbot E, DiMattia DA, et al. The differential effects of IL-1 and TNF-A on proinflammatory cytokine and matrix metalloproteinase expression in human chondrosarcoma cells. Inflamm Res 2004;53:377389.

    • Search Google Scholar
    • Export Citation
  • 33.

    Schuerwegh AJ, Dombrecht EJ, Stevens WJ, et al. Influence of pro-inflammatory (IL-1A, IL-6, TNF-A, IFN-G) and anti-inflammatory (IL-4) cytokines on chondrocyte function. Osteoarthritis Cartilage 2003;11:681687.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 34.

    Ghosh P, Guidolin D. Potential mechanism of action of intra-articular hyaluronan therapy in osteoarthritis: are the effects molecular weight dependent? Semin Arthritis Rheum 2002;32:1037.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Figure 1—

    Log mean ± SE values (n = 7 horses) for incorporation of sulfur 35 (35S)–labeled sodium sulfate into GAG of equine articular chondrocyte pellets treated with various concentrations of hyaluronic acid (HA) and triamcinolone acetonide (TA). *Significant (P < 0.05) difference between negative control (no IL-1) and positive control (IL-1 only). †Significant (P < 0.05) effect of HA, compared with no HA, at the same concentrations of TA. ‡Significant (P < 0.05) effect of combination treatment (HA and TA), compared with the IL-1 control.

  • Figure 2—

    Log mean ± SE values (n = 7 horses) for incorporation of S35-labeled sodium sulfate into GAG of equine articular chondrocyte pellets and the medium following treatment with various concentrations of HA and TA. *Significant (P < 0.05) effect of HA, compared with no HA, at the same concentrations of TA.

  • Figure 3—

    Mean ± SE percentage values (n = 7 horses) for incorporation of S35-labeled sodium sulfate into GAG of equine articular chondrocyte pellets treated with various concentrations of HA and TA. *Significant (P < 0.05) difference between negative control (no IL-1) and positive control (IL-1 only). †Significant (P < 0.05) effect of TA treatment groups, compared with the non–TA treatment group, at various concentrations of HA.

  • Figure 4—

    Mean ± SE percentage values (n = 7 horses) for incorporation of S35-labeled sodium sulfate into GAG released into medium following treatment with various concentrations of HA and TA. See Figure 3 for key.

  • Figure 5—

    Log mean ± SE values (n = 7 horses) for total GAG content in equine articular chondrocyte pellets treated with various concentrations of HA and TA. *Significant (P < 0.05) effect of HA, compared with no HA, at the same concentrations of TA. †Significant (P < 0.05) effect of TA treatment groups, compared with the non–TA treatment group, at various concentrations of HA.

  • Figure 6—

    Mean ± SE values (n = 3 horses) for collagen type II mRNA in equine articular chondrocyte pellets treated with various concentrations of HA and TA. *Significant (P < 0.05) difference between negative control (no IL-1) and positive control (IL-1 only). †Significant (P < 0.05) effect of TA treatment groups, compared with the non–TA treatment group, at various concentrations of HA.

  • Figure 7—

    Mean ± SE values (n = 3 horses) for aggrecan mRNA in equine articular chondrocyte pellets treated with various concentrations of HA and TA. See Figure 3 for key.

  • Figure 8—

    Mean ± SE values (n = 3 horses) for COX-2 mRNA in equine articular chondrocyte pellets treated with various concentrations of HA and TA. See Figure 3 for key.

  • Figure 9—

    Photomicrographs of sections of equine articular chondrocyte pellets obtained 24 hours after administration of IL-1 treatment, 2 mg of HA/mL, 0.6 mg of TA/mL, or 2 mg of HA/mL and 0.6 mg of TA/mL. Toluidine blue stain; bar = 100 μm.

  • 1.

    Doyle AJ, Stewart AA, Constable PD, et al. Effects of sodium hyaluronate and methylprednisolone acetonide on proteoglycan synthesis in equine articular cartilage explants. Am J Vet Res 2005;66:4853.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2.

    Yates AC, Stewart AA, Byron CR, et al. Effects of sodium hyaluronate and methylprednisolone acetonide on proteoglycan metabolism in equine articular chondrocytes treated with interleukin-1. Am J Vet Res 2006;67:19801986.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3.

    Gilron I. Corticosteroids in postoperative pain management: future research directions for a multifaceted therapy. Acta Anaesthesiol Scand 2004;48:12211222.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4.

    Frisbie DD, Kawcak CE, Baxter GM, et al. Effects of 6A-methylpredinisolone acetonide on an equine osteochondral fragment exercise model. Am J Vet Res 1998;59:16191628.

    • Search Google Scholar
    • Export Citation
  • 5.

    Shoemaker RS, Bertone AL, Martin GS, et al. Effects of intra-articular administration of methylprednisolone acetonide on normal articular cartilage and on healing of experimentally induced osteochondral defects in horses. Am J Vet Res 1992;53:14461453.

    • Search Google Scholar
    • Export Citation
  • 6.

    Murphy DJ, Todhunter RJ, Fubini SL, et al. The effects of methylprednisolone on normal and monocyte-conditioned medium-treated articular cartilage from dogs and horses. Vet Surg 2000;29:546557.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7.

    Frisbie DD, Kawcak CE, Trotter GW, et al. Effects of triamcinolone acetonide on an in vivo equine osteochondral fragment exercise model. Equine Vet J 1997;29:349359.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8.

    Sandler EA, Frisbie DD, McIlwraith CW. A dose titration of triamcinolone acetonide on insulin-like growth factor-1 and interleukin-1-conditioned equine cartilage explants. Equine Vet J 2004;36:5863.

    • Search Google Scholar
    • Export Citation
  • 9.

    Dechant JE, Baxter GM, Frisbie DD, et al. Effects of dosage titration of methylprednisolone acetonide and triamcinolone acetonide on interleukin-1-conditioned equine articular cartilage explants in vitro. Equine Vet J 2003;35:444450.

    • Search Google Scholar
    • Export Citation
  • 10.

    Caron JP. Intra-articular injections for joint disease in horses. Vet Clin North Am Equine Pract 2005;21:559573.

  • 11.

    Schmidt TA, Gastelum NS, Nguyen QT, et al. Boundary lubrication of articular cartilage: role of synovial fluid constituents. Arthritis Rheum 2007;56:882891.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12.

    Akmal M, Singh A, Anand A, et al. The effects of hyaluronic acid on articular chondrocytes. J Bone Joint Surg Br 2005;87:11431149.

  • 13.

    Goodrich LR, Nixon AJ. Medical treatment of osteoarthritis in the horse—a review. Vet J 2006;171:5169.

  • 14.

    Rydell NW, Butler J, Balazs EA. Hyaluronic acid in synovial fluid. Acta Vet Scand 1970;11:139155.

  • 15.

    Young AA, McLennan S, Smith MM, et al. Proteoglycan 4 downregulation in a sheep meniscectomy model of early osteoarthritis. Arthritis Res Ther 2006;8:R41.

  • 16.

    Iwata H. Pharmacologic and clinical aspects of intraarticular injection of hyaluronate. Clin Orthop 1993;289:285291.

  • 17.

    Moreland LW. Intra-articular hyaluronan (hyaluronic acid) and hylans for the treatment of osteoarthritis: mechanisms of action. Arthritis Res Ther 2003;5:5467.

    • Search Google Scholar
    • Export Citation
  • 18.

    White GW, Stites T, Hamm J, et al. Evaluation of the efficacy of various preparations of sodium hyaluronate in an induced equine carpitis model. J Equine Vet Sci 1999;19:331337.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19.

    McIlwraith CW. Synovitis and other soft tissue injuries of equine joints, in Proceedings. Dubai Int Equine Symp 1996;287322.

  • 20.

    Phillips MW. Intraarticular sodium hyaluronate in the horse: a clinical trial, in Proceedings. 26th Annu Conv Am Assoc Equine Pract 1980;389394.

    • Search Google Scholar
    • Export Citation
  • 21.

    Cheney M. One practice's approach to joint therapy in the equine athlete, in Proceedings. 42nd Annu Conv Am Assoc Equine Pract 1996;6974.

    • Search Google Scholar
    • Export Citation
  • 22.

    Aviad AD, Houpt JB. The molecular weight of therapeutic hyaluronan (sodium hyaluronate): how significant is it? J Rheumatol 1994;21:297301.

    • Search Google Scholar
    • Export Citation
  • 23.

    Aviad AD, Arthur RM, Brencick VA, et al. Synacid vs Hylartin V in equine joint disease. J Equine Vet Sci 1988;8:112116.

  • 24.

    Chen CL, Sailor JA, Collier J, et al. Synovial and serum levels of triamcinolone following intra-articular administration of triamcinolone acetonide in the horse. J Vet Pharmacol Ther 1992;15:240246.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    Hilbert BJ, Rowley G, Antonas KN, et al. Changes in the synovia after intra-articular injection of sodium hyaluronate into normal horse joints and after arthrotomy and experimental cartilage damage. Aust Vet J 1985;62:182184.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.

    Masuda K, Shirota H, Thonar E. Quantification of 35S-labeled proteoglycans complexed to alcian blue by rapid filtration in multiwell plates. Anal Biochem 1994;217:167175.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27.

    Farndale RW, Sayers CA, Barrett AJ. A direct spectrophotometric microassay for sulfated glycosaminoglycans in cartilage cultures. Connect Tissue Res 1982;9:247248.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28.

    Kim YJ, Sah RL, Doong JY, et al. Fluorometric assay of DNA in cartilage explants using Hoechst 33258. Anal Biochem 1988;174:168176.

  • 29.

    Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT Method. Methods 2001;25:402408.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30.

    MacLeod JN, Fubini SL, Gu DN, et al. Effect of synovitis and corticosteroids on transcription of cartilage matrix proteins. Am J Vet Res 1998;59:10211026.

    • Search Google Scholar
    • Export Citation
  • 31.

    Fubini SL, Todhunter RJ, Burton-Wurster N, et al.Corticosteroids alter the differentiated phenotype of articular chondrocytes. J Orthop Res 2001;19:688695.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32.

    Shi J, Schmitt-Talbot E, DiMattia DA, et al. The differential effects of IL-1 and TNF-A on proinflammatory cytokine and matrix metalloproteinase expression in human chondrosarcoma cells. Inflamm Res 2004;53:377389.

    • Search Google Scholar
    • Export Citation
  • 33.

    Schuerwegh AJ, Dombrecht EJ, Stevens WJ, et al. Influence of pro-inflammatory (IL-1A, IL-6, TNF-A, IFN-G) and anti-inflammatory (IL-4) cytokines on chondrocyte function. Osteoarthritis Cartilage 2003;11:681687.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 34.

    Ghosh P, Guidolin D. Potential mechanism of action of intra-articular hyaluronan therapy in osteoarthritis: are the effects molecular weight dependent? Semin Arthritis Rheum 2002;32:1037.

    • Crossref
    • Search Google Scholar
    • Export Citation

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