Lameness is a common problem for equine athletes with an estimated prevalence of up to 5%.1 Among the most common manifestations of lameness is osteoarthritis of the appendicular skeleton. Osteoarthritis is a complex disease process characterized by articular tissue catabolism with progressive cartilage deterioration. Regardless of the inciting etiology, proinflammatory cytokines (including IL-1β) are released by multiple intra-articular tissues.2 Reduction of inflammatory mediators is 1 aim of therapeutic intervention, and intra-articular administration of corticosteroids is commonly used for that purpose in horses.3 Corticosteroids act by blocking the arachidonic acid cascade, limiting capillary dilation, and interrupting the congregation of inflammatory cells capable of releasing cytokines that contribute to inflammation and catabolism.4
The in vitro effects of commonly used corticosteroids on equine articular tissues in inflammatory environments have been investigated.5–7 Some controversy exists regarding the metabolic effects of corticosteroids on articular cartilage, with both potentially positive (eg, GAG synthesis and abrogation of histopathologic changes)8 and negative (eg, erosion and morphological lesions)9 sequelae reported. However, the disparate results regarding the effects of corticosteroids on articular tissues may be attributable to dose differences rather than the biologic properties of the drugs. Results of other studies suggest that low concentrations of corticosteroids downregulate gene expression of degradative enzymes with no or minimal detrimental effects on equine articular cartilage6 and that equivalent molarities of MPA and TA have similar effects on gene expression.7
Results of a survey3 of equine practitioners suggest that TA and MPA are the corticosteroids most commonly administered intra-articularly for the treatment of osteoarthritis in horses; however, a substantial proportion (> 15%) of respondents reported that they administered IPA intra-articularly for the same purpose. Although the effects of corticosteroids other than IPA on cartilage have been investigated, to our knowledge, the in vitro effects of IPA on equine cartilage have not been documented.
In vitro models used to evaluate the effects of corticosteroids on articular tissues typically include only cartilage explants or chondrocytes.5–7,10–12 As osteoarthritis progresses, articular cartilage becomes irreversibly damaged, which results in a loss of function7; however, cross talk among cartilage, subchondral bone, and synovium is important in disease pathogenesis.13,14 Results of several studies indicate that coculture of cartilage with other articular tissues, such as synovium,15,16 subchondral bone,17 or synovium and subchondral bone,18 has important effects on biologic responses to inflammatory stimuli and treatments. Synovial tissue may be the largest contributor of catabolic cytokines within the articular environment.19–21 Inclusion of subchondral bone with articular cartilage as an osteochondral explant significantly reduces in vitro chondrocyte death.17 Those coculture models17,19–21 indicate cellular interplay may contribute to or mitigate articular degeneration.
The objective of the study reported here was to compare the in vitro effects of 3 equimolar concentrations of MPA, TA, and IPA on equine articular cocultures in an inflammatory environment. Various biomarkers of articular metabolism associated with inflammation (PGE2), degradation of extracellular matrix (MMP-13), cytotoxicosis (LDH), and articular catabolism (GAG) were analyzed. The hypotheses were that all 3 corticosteroids would counteract the negative effects (upregulation of inflammatory biomarkers and dysregulation of cartilage metabolism) of IL-1β on equine articular cocultures consisting of synovium, cartilage, and subchondral bone and that cocultures treated with low concentrations (10−7 and 10−10M) of the corticosteroids would have more beneficial and fewer detrimental effects than those treated with high concentrations (10−4M).
Materials and Methods
Samples
All equine articular tissue specimens were collected in accordance with a protocol approved by the Virginia-Maryland College of Veterinary Medicine Institutional Animal Care and Use Committee (No. 14–259). Horses from which articular tissue specimens were obtained were euthanized by a barbituratea overdose for reasons unrelated to the study. Tissue specimens were harvested immediately after death was confirmed. The horses from which tissue specimens were obtained included 3 mares, 2 geldings, and 1 sexually intact colt with ages that ranged from 2 to 11 years. Breeds included Quarter Horse (n = 2), Thoroughbred (2), Belgian (1), and warmblood (1). None of the horses had a history or clinical signs of musculoskeletal disease, and no gross evidence of degenerative joint disease (cartilage fibrillation, erosion, scoring, or discoloration) was detected during specimen collection. From each horse, osteochondral and synovial explants were aseptically collected from either the right or left femoropatellar joint; the joint (right or left) from which specimens were obtained was determined in a random manner (ie, coin flip). Osteochondral explants (diameter, 7.9 mm; cartilage depth, 2 mm; subchondral bone depth, 4 mm) were collected from the axial surface of the lateral trochlear ridge of the femur by use of a hollow punchb and orthopedic mallet. Synovium without fibrous articular capsule tissue was collected, and 6-mm-diameter explants were prepared. Approximately 25 explants (more than the number of explants necessary for all treatments) were obtained from the selected femoropatellar joint of each horse, and only grossly undamaged and uniform explants were used for the cocultures. Immediately following harvest, explants were placed in PBS solution containing 1% penicillin and streptomycin and stored at room temperature (25°C) for approximately 1 hour prior to tissue culture.
Tissue culture
Tissue specimens were transferred to 12-well polystyrene transwell platesc with inserts lined with polyester porous membranes (diameter, 12 mm; pore size, 3 μm). Synovium specimens were placed in the bottom of the plate wells, and osteochondral specimens were suspended in well inserts. One 7.9-mm-diameter osteochondral specimen and two 6-mm-diameter synovium specimens were included in each well. This yielded a synovium-to-articular cartilage surface area ratio of 1.2:1, which is similar to that in human and mouse joints.22 All articular tissue specimens were fully submerged in Dulbecco modified eagle medium (2.5 mL/well) that contained glucose (1 g/L), l-glutamine (584 mg/L), and sodium pyruvated (110 mg/L). Tissue cultures were incubated at 37°C in an atmosphere with 95% relative humidity and 5% CO2 for 48 hours before initiation of experimental treatments.
All experimental treatments were performed in duplicate. Treatment groups included explant cocultures with no rhIL-1βe (negative control), rhIL-1β alone (10 ng/mL; positive control), and rhIL-1β (10 ng/mL) plus 10−4, 10−7, or 10−10M of MPAf, TA,g or IPA.h The corticosteroid concentrations used were chosen on the basis of results of another study6 and preliminary data collected in our laboratory that indicate marked cell death occurs at corticosteroid concentrations > 10−4M. Tissue cultures were incubated with the assigned treatment at 37°C in an atmosphere with 95% relative humidity and 5% CO2 for 48 hours. Then, the culture medium and assigned treatment was replaced, and tissue cultures were incubated for another 48 hours. A sample of the culture medium was collected from each coculture after 48 and 96 hours of incubation with the assigned treatment. All culture medium samples were stored frozen at −80°C until analysis.
PGE2 assay
For each culture medium sample, PGE2 was quantified in duplicate by use of a commercially available colorimetric assayi in accordance with the manufacturer's instructions. Briefly, each culture medium and standard sample was diluted 25-fold with an assay-supplied buffer and incubated in goat anti-mouse antibody-precoated wells at room temperature for 1 hour on a horizontal plate shaker set at a speed of 400 revolutions/min. Horseradish peroxidase– labeled PGE2 was added to each well, and the plate was incubated for another 2 hours under the same conditions as previously described. The wells were washed with assay-supplied wash buffer, then tetramethylbenzidine was added to each well for luminescence of bound substrate. The plates were covered and incubated at room temperature for 30 minutes. Sulfuric acid (1 mol/L) was added to each well as a stop solution. Light absorbance was determined at 450 nm with a commercial microplate reader.j A standard curve was generated to determine the PGE2 concentration in each well. The mean PGE2 concentration of the paired replicates was calculated for each culture medium sample and used for statistical analyses.
MMP-13 assay
For each culture medium sample, MMP-13 concentration was determined in duplicate by use of a commercially available kitk in accordance with the manufacturer's instructions. Briefly, each culture medium and standard sample was placed in duplicate wells that were precoated with anti–MMP-13 antibody. The assay plate was incubated at room temperature for 2.5 hours and then washed with assay-supplied wash solution. Biotinylated anti-human MMP-13 antibody was added to each well, and the plate was incubated at room temperature for 1 hour. Then, the plate was washed with wash solution to remove any unbound biotinylated antibody and horseradish peroxidase–conjugated streptavidin from the wells. The plate was incubated at room temperature for 45 minutes and washed again. Then, 3,3,5,5′-tetramethylbenzidine was added to each well for colorimetric change of bound substrate. The plate was covered and incubated at room temperature on a benchtop for 30 minutes. Sulfuric acid was added to each well to stop the reaction. The optical density of each well was immediately determined at 450 nm. A standard curve was generated to determine the MMP-13 concentration in culture medium samples. The mean MMP-13 concentration of the paired replicates was calculated for each culture medium sample and used for statistical analyses.
LDH assay
For each culture medium sample, the LDH concentration was determined in duplicate by use of a colorimetric assayl in accordance with the manufacturer's instructions. Each culture medium and standard sample was placed in duplicate wells of a clear flat-bottom 96-well polystyrene plate. Substrate solution that included a tetrazolium salt was added to each well, and the plate was incubated at room temperature for 30 minutes. The coupled enzymatic reaction produced red formazan in an amount proportional to the amount of LDH in the sample. The reaction was halted by the addition of a stop solution to each well, and the light absorbance of each well was measured at 490 nm. A standard curve was generated and used to quantify the LDH concentration in each replicate. The mean LDH concentration of the paired replicates was calculated for each culture medium sample and used for statistical analyses.
GAG assay
The GAG concentration in each culture medium sample was determined in duplicate as described by other investigators.23 Briefly, each replicate for each culture medium sample was diluted 1:3 in formate buffer and placed in a well of a clear flat-bottom 96-well polystyrene plate. Serial dilutions of chondroitin-6-sulfate standards were combined with 1,9-dimethylmethylene blue dye and ethyl alcohol substrate. After gentle mixing, the light absorbance of each well was determined at 525 nm. A standard curve was generated to determine the GAG concentration in culture medium samples. The mean GAG concentration was determined for the paired replicates of each culture medium sample and used for statistical analyses.
Statistical analysis
For each biomarker evaluated (PGE2, MMP-13, LDH, and GAG), the distribution of the concentration data was assessed for normality by means of normal probability plots. Concentration data for GAG and LDH were normally distributed, and results for those 2 biomarkers were reported as the mean ± SD. Concentration data for PGE2 and MMP-13 were not normally distributed, and results for those 2 biomarkers were reported as the median (range). The PGE2 and MMP-13 concentration data were logarithmically transformed to normalize the respective distributions for statistical analyses. For each biomarker, the effect of corticosteroid (MPA, TA, or IPA), concentration (10−4, 10−7, or 10−10M), and duration of treatment exposure (48 or 96 hours; time) on culture medium concentration was assessed by use of a mixed-model ANOVA.m The model included fixed effects for the 3 independent variables (corticosteroid, concentration, and time) and all possible 2- and 3-way interactions among the independent variables and a random effect for horse to account for the use of multiple tissue specimens from each horse. The denominator degrees of freedom for each fixed effect were approximated by use of the Kenward-Roger method. The 3-way interaction of corticosteroid, concentrations, and time was significant for each biomarker evaluated. Therefore, the culture medium biomarker concentration was compared between the 2 times for each corticosteroid and concentration combination, among the 3 concentrations for each corticosteroid and time combination, and among the 3 corticosteroids for each concentration and time combination. When multiple pairwise comparisons were performed, P values were adjusted with the Tukey procedure to prevent type I error inflation. For each biomarker, an additional mixed-model ANOVA was created to compare the culture medium concentrations for the positive and negative control samples with those for each combination of corticosteroid, concentration, and time, and P values were adjusted with the Dunnett procedure when multiple pairwise comparisons were necessary. For all ANOVA models, residuals were plotted and visually inspected to verify that they had a normal distribution and constant variance. Unless otherwise specified, values of P < 0.05 were considered significant.
Results
PGE2
The mean PGE2 concentration in culture medium samples obtained from cocultures after 48 and 96 hours of incubation with each corticosteroid treatment as well as the positive and negative control treatments was summarized (Figure 1). After 48 hours of incubation, the mean PGE2 concentration for the lowest (10−10M) concentration of each corticosteroid was significantly less than that for the positive control (IL-1β–stimulated) treatment, and the mean PGE2 concentration for the lowest (10−7 and 10−10M) concentrations of MPA and TA was significantly less than that for the highest concentration of each of those corticosteroids. After 96 hours of incubation, the mean PGE2 concentration for the 10−7M MPA treatment was significantly less than that for the 10−4 M MPA treatment, and the mean PGE2 concentration for the 10−10M TA treatment was significantly less than that for the 10−4M TA treatment.
Mean PGE2 (A), MMP-13 (B), LDH (C), and GAG (D) concentrations in culture medium samples obtained from cocultures of equine synovial and osteochondral tissue explants after 48 (black bars) and 96 (gray bars) hours of incubation with rhIL-1β (10 ng/mL; positive control), culture medium without rhIL-1β or a corticosteroid (negative control), or rhIL-1β and 1 of 3 corticosteroids (MPA, TA, or IPA) at 1 of 3 concentrations (10−4, 10−7, or 10−10M). Explants were obtained from the femoropatellar joints of 6 horses that were euthanized for reasons other than musculoskeletal disease. A sufficient number of explants was obtained from each horse to create a coculture for each of the 11 experimental treatments in duplicate. Thus, the bars represent the mean for 12 cocultures, and brackets represent the interquartile (25th to 75th percentile) range. Concentration data for PGE2 and MMP-13 underwent a logarithmic transformation to normalize them for analysis, and the mean loge concentration is reported for those 2 biomarkers. *Within an incubation duration, value differs significantly (P < 0.05) from the corresponding value for the positive control treatment.
Citation: American Journal of Veterinary Research 79, 9; 10.2460/ajvr.79.9.933
MMP-13
The mean MMP-13 concentration in culture medium samples obtained from cocultures after 48 and 96 hours of incubation with each treatment was summarized (Figure 1). Mean MMP-13 concentration of all tested corticosteroid treatments was less than positive control after 96 hours. After 48 hours of incubation, the mean MMP-13 concentration for the 10−7M TA treatment was significantly less than that for the 10−4M TA treatment. After 96 hours of incubation, the mean MMP-13 concentration for the 10−7M MPA treatment was significantly less than that for the 10−10M MPA treatment, and the mean MMP-13 concentration for the 10−10M TA treatment was significantly less than that for the 10−4M TA treatment.
LDH
The mean LDH concentration in culture medium samples obtained from cocultures after 48 and 96 hours of incubation with each treatment was summarized (Figure 1). After both 48 and 96 hours of incubation, the mean LDH concentration for the 10−4M IPA treatment was significantly greater than that for each of the other corticosteroid treatments and the positive control treatment. The mean LDH concentrations for the MPA and TA treatment groups did not differ among the 3 doses evaluated or from those for the positive and negative control treatments after both 48 and 96 hours of incubation.
GAG
The mean GAG concentration in culture medium samples obtained from cocultures after 48 and 96 hours of incubation with each treatment was summarized (Figure 1). For each of the 3 corticosteroids, the mean GAG concentration for the lower-concentration treatments (10−10M MPA, 10−7M TA, and 10−10M IPA) was significantly lower than that for the highest concentration treatment after 48 hours of incubation. After 96 hours of incubation, the mean GAG concentration did not differ significantly among any of the 9 corticosteroid treatments evaluated.
Discussion
In the present in vitro study, treatment of equine synovium and osteochondral cocultures with MPA, TA, or IPA at each of the 3 concentrations (10−4, 10−7, or 10−10M) evaluated mitigated the inflammatory and catabolic effects of IL-1β as determined by quantification of PGE2 and MMP-13 concentrations in culture medium samples obtained from cocultures after 48 and 96 hours of incubation with the assigned treatment relative to those in the culture medium samples obtained from the cocultures of the positive control (rhIL-1β–stimulated) treatments. Additionally, the mean culture medium GAG concentration for cocultures incubated for 48 hours with the 10−10M MPA, 10−7M TA, and 10−10M IPA treatments was significantly lower than the mean culture medium GAG concentration for cocultures incubated for 48 hours with the highest concentration (10−4M) of the corresponding corticosteroid. Importantly, none of the 3 corticosteroids evaluated in this study induced cytotoxicosis (as determined on the basis of LDH quantification) in the explant cocultures when administered at the lower concentrations (10−7 and 10−10M).
An abnormally increased intra-articular eicosanoid (eg, PGE2) concentration is a clinically relevant indicator of joint disease,24,25 and for horses with joint disease, a decrease in synovial fluid PGE2 concentration is associated with clinical improvement independent of the treatment administered.26,27 In the present study, stimulation of equine articular tissues with rhIL-1β resulted in a predictable increase in culture medium PGE2 concentration, and treatment of cocultures with low concentrations (10−7 and 10−10M) of MPA, TA, or IPA caused a decrease in culture medium PGE2 concentration relative to the mean PGE2 concentration for positive control cocultures. That finding was expected and corroborates results of other in vitro studies,7,12,28 which indicate that various corticosteroids impede upregulation of eicosanoid production.
Treatment of cocultures with low concentrations of the 3 corticosteroids evaluated likewise caused a significant decrease in the mean culture medium MMP-13 concentration relative to that for the positive control cocultures. Matrix metalloproteinase-13 degrades type II collagen, and abnormally increased concentrations of MMP-13 have been reported in the joints of horses with naturally occurring joint disease19,29 and in in vitro models of short-term30 and long-term31 equine joint disease. Results of other studies21,32 indicate that MMP-13 production in cartilage is upregulated by stimulation with IL-1 and coculture with synovial tissue and downregulated following treatment with MPA. Interestingly, results of another study18 conducted in our laboratory suggest that the inclusion of subchondral bone in the in vitro model might partially mitigate upregulation of MMP-13 in an inflammatory environment. The equine cocultures created for the present study included all major articular tissues, and results indicated that treatment of those cultures with low concentrations of a corticosteroid following stimulation with rhIL-1β (to create an inflammatory environment) caused downregulation of MMP-13 relative to positive control cocultures. Moreover, the mean MMP-13 concentration in culture medium obtained from cocultures treated with low concentrations of a corticosteroid was similar to or lower than that for the negative control (ie, healthy) cocultures. That finding may have important clinical implications because it suggested that low concentrations of corticosteroids can be clinically effective without inducing detrimental effects in cartilage.
In the present study, the mean culture medium GAG concentration for cocultures treated with the 10−7M TA and 10−10M MPA treatments was significantly lower than that for the positive control cocultures after 48 and 96 hours of treatment incubation, respectively. Also, the mean culture medium GAG concentration for the 10–10M MPA, 10–7M TA and 10–10M IPA treatment after 48 hours of incubation was significantly lower than the mean GAG concentration for the corresponding 10–4M treatments of those corticosteroids. The effects of MPA and TA on IL-1–conditioned cartilage explants were investigated in another study.5 Results of that study5 indicate that cartilage homeostasis (as determined by GAG metabolism) did not differ between explants that were treated with low concentrations of a corticosteroid and those treated with a high concentration of a corticosteroid; however, none of the corticosteroid-concentration combinations evaluated were able to completely nullify the detrimental effects of IL-1 stimulation. Interestingly, the investigators of that study5 suggested that the in vitro model used (explants consisting of articular cartilage only) might not be representative of in vivo joint physiology. The use of articular tissue cocultures, such as those used in the present study, might increase the physiologic relevance of the findings to clinical patients, and the discrepancies in the results between that study5 and the present study are likely attributable to in vitro model differences (articular cartilage monocultures vs synovial and osteochondral tissue cocultures).
We used LDH quantification as a nonspecific biomarker of cytotoxicosis and cell death for the cocultures of the present study. The 10−4M IPA treatment was the only treatment evaluated that appeared to have a significant increase in cytotoxicosis relative to the positive control. However, 10−4M exceeds the intra-articular dose of IPA commonly used in horses with naturally occurring osteoarthritis. The tissue or tissues from which LDH originated was not identified. The cocultures included synoviocytes, chondrocytes, and osteocytes, and LDH could have been produced by any or all of those cell types. Aside from the apparent cytotoxicity of the 10−4M concentration of IPA, the effects of the lower concentrations of IPA on the evaluated biomarkers were similar to those for MPA and TA. The apparent discrepancy in the cytotoxicity of the 3 corticosteroids evaluated may reflect differences in the anti-inflammatory potency of those compounds. The glucocorticoid activity of IPA is 5 to 10 times that of MPA and TA,33–35 even though the results of the present study and effects of commonly used doses in clinical patients do not reflect that. It is possible that the cytotoxicity of the 10−4M concentration of IPA was related to its increased potency on an equimolar basis. Preliminary data from our laboratory (unpublished results) indicated marked cell death at a corticosteroid concentration of 10–3M.
The present study was not without limitations. The tissue cocultures were not histologically evaluated; therefore, the treatment effects in the various anatomic tissues could not be characterized and culture medium biomarker concentration results could not be corroborated with pathological lesions. Also, the cocultures were only incubated with the assigned treatments for 96 hours, which represents a short-term simulation of inflammatory joint disease; therefore, the results cannot be extrapolated to a chronic disease process. Recombinant human IL-1β was selected as the inflammatory stimulus on the basis of results of a preliminary investigation conducted in our laboratory as well as to be consistent with procedures used in other in vitro studies.16,18,36 Nevertheless, the concentration (10 ng/mL) of rhIL-1β used as the inflammatory stimulus for the cocultures of the present study in the absence of other inflammatory and degradative stimuli may not accurately mimic naturally occurring disease. The evaluated corticosteroid concentrations used in this study were selected on the basis of results of pilot trials conducted in our laboratory. The 10−4M concentration exceeded doses commonly used in clinical practice, but the 10−7 and 10−10M concentrations were clinically relevant.37,38 The in vitro coculture model used in the present study included all major articular tissue types and may have been more representative of in vivo physiologic processes than monoculture models. However, similar to another study17 that involved the use of osteochondral explants, the cut edges of subchondral bone were exposed to the culture medium. Those edges are not exposed to the joint environment in vivo, and that component of our coculture model is not physiologically accurate and requires further refinement.
Results of the present study suggested that low concentrations (10−7 and 10−10M) of MPA, TA, and IPA mitigate the negative effects of IL-1β in equine tissue cocultures consisting of synovium and osteochondral explants, but a high concentration (10−4M) of those corticosteroids, particularly IPA, was detrimental to equine articular tissues in vitro. The effects of those 3 corticosteroids on articular tissues in an inflammatory environment were similar at clinically relevant equimolar concentrations. Therefore, we propose that the intra-articular dose for those 3 corticosteroids should be approximately 10 mg/joint rather than 100 mg/joint. That suggestion is supported by the results of this study and findings of other studies that indicate intra-articular administration of 12 mg of TA into a middle carpal joint has primarily beneficial effects,8 whereas intra-articular administration of 100 mg of MPA into a joint has detrimental effects.9 Collectively, those results suggest that it may be best to use similar doses of MPA, TA, and IPA for intra-articular administration in horses with osteoarthritis, but in vivo studies are warranted to validate that supposition.
Acknowledgments
Supported by Zoetis Inc.
Presented in abstract form at the American College of Veterinary Surgeons Surgical Summit, Seattle, October 2016.
The authors thank Kristel Fuhrman for technical assistance.
ABBREVIATIONS
| GAG | Glycosaminoglycan |
| IL | Interleukin |
| IPA | Isoflupredone acetate |
| LDH | Lactate dehydrogenase |
| MMP-13 | Matrix metalloproteinase-13 |
| MPA | Methylprednisolone acetate |
| PGE2 | Prostaglandin E2 |
| rhIL-1β | Recombinant human interleukin-1β |
| TA | Triamcinolone acetonide |
Footnotes
Fatal-Plus, Vortech Pharmaceuticals Ltd, Dearborn, Mich.
Tekton Hollow Punch Set, Michigan Industrial Tools, Grand Rapids, Mich.
Transwell, Corning Inc, Corning, NY.
Corning cellgro, Mediatech Inc, Manassas, Va.
rhIL-1β, R&D Systems, Minneapolis, Minn.
Depo-medrol, Zoetis Inc, Kalamazoo, Mich.
Kenalog-40, Bristol-Myers Squibb Co, Princeton, NJ.
Predef 2x, Zoetis Inc, Kalamazoo, Mich.
Parameter Prostaglandin E2, R&D Systems, Minneapolis, Minn.
SpectraMax M5 microplate reader, Molecular Devices, Sunnyvale, Calif.
Human MMP-13 ELISA Kit, RayBiotech Inc, Norcross, Ga.
Pierce LDH Cytotoxicity Assay Kit, Thermo Scientific, Rockford, Ill.
SAS, version 9.4, SAS Institute Inc, Cary, NC.
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