Introduction
Navicular disease is a major cause of forelimb lameness in horses.1,2,3,4,5,6 Although pathological changes in multiple structures of the foot are common,1,3,5 primary deep digital flexor tendinitis at the level of and just proximal to the NB and pathological changes in the DDFT accompanying degenerative changes of the NB contribute substantially to lameness in horses with navicular disease.1,4,7,8 Tendon healing is a prolonged process, and fibrous adhesions between the healing tendon and synovial sheath can negatively influence the long-term functional outcome of affected horses.1,3,4,5,7,8 Corticosteroids are commonly administered intrasynovially as part of the management of horses with navicular disease.6,9 Despite their potent anti-inflammatory benefits, the potential toxic effects of corticosteroids on DDFT and navicular fibrocartilage cells are largely unknown.
Lesions or erosions of the NB fibrocartilage and degeneration of the opposing surface of the DDFT may develop concurrently or be present as independent conditions.3 The DDFT at the level of and just proximal to the NB is located within the navicular bursa. This segment of the DDFT is subject to compressive forces; from a structural standpoint, it is composed of fibrocartilage tissue that is located dorsally.4,7,8 The flexor surface of the NB consists of superficial uncalcified and deeper calcified fibrocartilage superficial to the subchondral bone plate.3 Fibrocartilage on the flexor surface of the NB and the dorsal surface of the opposing DDFT contains chondrocyte-like cells within a proteoglycan-rich extracellular matrix.3,4,7 Several in vitro and in vivo studies10,11,12,13 of horses and other species have revealed that corticosteroids impact chondrocyte viability and alter matrix homeostasis in articular cartilage; however, there is minimal information regarding their effects on tendons. Results of an ex vivo study14 involving canine supraspinatus tendons indicate that betamethasone and methylprednisolone significantly reduce the cell viability of tendon tissue. Because intrabursal or intrasynovial administration of corticosteroids in horses with navicular disease is common practice, identification of the drugs' potential deleterious effects is critical. The objective of the ex vivo study of the present report was to investigate the effects of TA and MPA on the viability of resident cells in FC-DDFT and FC-NB explants from cadaveric horses. Live-dead cell staining and metabolic activity assays were used as 2 measures of explant cell viability.
Materials and Methods
Explants of FC-DDFT and FC-NB were harvested from both forelimbs of 5 horses euthanatized for reasons unrelated to the study. All horses were Quarter Horses (4 mares and 1 gelding) between 9 and 15 years of age. All horses had body condition scores > 6/9 and were free of forelimb lameness when trotted in hand on a smooth, hard surface. Horses appeared free of musculoskeletal abnormalities on the basis of a complete physical examination. The DDFT opposing the NB and the FC-NB were determined to be normal on the basis of gross assessment at the time of explant harvest.
Harvest and incubation of tissue explants
Immediately following euthanasia of each horse with an IV injection of pentobarbital sodium (150 mg/kg), both forelimb digits were disarticulated at the metacarpophalangeal joint. Each hoof was cleaned, the hair was clipped, and the solar surface was pared with a hoof knife. Each foot was rinsed with water to remove gross debris, and the entire digit was scrubbed with disinfectant solution.a Each foot was disarticulated at the level of the distal inter-phalangeal joint to expose the proximal aspect of the NB without severing the DDFT. With an aseptic technique, the NB was dissected from the foot en bloc by transecting the surrounding soft tissues. The DDFT segment that was 1 cm proximal to and directly opposing the NB was harvested with aseptic technique. Two-millimeter-thick tissue explants from the FC-DDFT and FC-NB were harvested with a 4-mm dermal biopsy punchb (Figure 1). Twelve to 14 explants of the FC-DDFT and of the FC-NB were collected from each forelimb.
Photograph depicting FC-DDFT explants harvested with a 4-mm biopsy punch. In this image, forelimb DDFT within the hoof capsule has been collected for explant harvesting.
Citation: American Journal of Veterinary Research 82, 2; 10.2460/ajvr.82.2.125
Explants were cultured in 24-well platesc (1 ex-plant/well) in Dulbecco modified Eagle mediumd containing glucose (4.5 g/L) and l-glutamine (300 μg/mL) with supplements of 2% fetal bovine serum,d 1% insulin-transferrin-selenium,e sodium penicillinf (100 U/mL), streptomycin sulfatef (100 μg/mL), and l-ascorbic acide (50 μg/mL). Explants were placed in the wells with 1 mL of medium so that the opposing surfaces of the FC-DDFT and FC-NB faced upwards throughout the experiment. Prior to start of the experiments, all explants were first allowed to equilibrate to culture conditions (37°C with 5% CO2 at 95% humidity) for 24 to 36 hours in the aforementioned medium.
Following adaptation to culture conditions, the FC-DDFT and FC-NB explants were placed in an equivalent volume of fresh medium (control) or medium containing TAg (0.6 or 6 mg/mL) or MPAh (0.5 or 5 mg/mL) with 3 replicates for each incubation period (6 or 24 hours). The concentrations of TA and MPA used in treatment groups were adopted from other studies that assessed corticosteroid-induced cytotoxicosis,14,15 corticosteroid dose divided by the typical estimated volume of equine navicular bursa–distal interphalangeal joint,14 and preliminary data collected in our laboratory. The explants were maintained in 24-well plates (1 explant/well) with 1 mL of control or drug-supplemented medium and incubated at 37°C with 5% CO2 at 95% humidity for either 6 or 24 hours.
Cell metabolic activity assay
The metabolic activity of cells present in individual FC-DDFT and FC-NB explants (3 replicate explants/treatment or control group/horse) was assessed with a resazurin-based assay.12,14,15,e This fluorescent metabolic assay detects the conversion of resazurin to a fluorescent compound, resorufin, by metabolically active cells. Once the incubation period with TA or MPA was complete, the explants were rinsed in PBS solution and transferred to new 24-well plates in 1 mL of fresh culture medium without corticosteroids to avoid interference of corticosteroid with resazurin. Resazurin (100 μL) was added to each well and incubated at 37°C in the dark for 4 hours. A 200-μL aliquot of the medium from each well was transferred in duplicate to 96-well fluoro-microtiterd plates, and fluorescence was measured at 570 nm (excitation) and 585 nm (emission) with a plate reader.i The optical density (mean fluorescence) from each explant incubated for 6 or 24 hours with TA or MPA at either of the 2 concentrations was expressed as the percentage change in mean fluorescence from findings for the respective control FC-DDFT or FC-NB explant group at the corresponding time point. The metabolic activity of freshly harvested explants prior to equilibrating to ex vivo culture was also measured with a similar protocol and expressed as an absolute mean fluorescence value.
Assessment of live-dead cell staining by confocal microscopy and quantitative image analysis
By use of live-dead cell staining,14 the numbers of live or dead cells were determined in FC-DDFT and FC-NB explants (2 replicates/treatment or control group/horse) after 6 and 24 hours in control medium or medium containing TA or MPA at either of the 2 concentrations. The explants were sectioned in half longitudinally prior to staining to view cells through the thickness of the explant. Cell status (live or dead) was assessed by means of fluorescent microscopy with fluorescent stains, namely calceind (15μM) for live cells (excitation, 495 nm; emission, 515 nm; green staining) and a high-affinity nucleic acid staind (50nM) for dead cells (excitation, 633 nm; emission, 658 nm; red staining). The explants were incubated in both stains simultaneously for 30 minutes at 37°C, according to the manufacturer's instructions. The ex-plants were secured so that the dorsal surface of the DDFT and the flexor surface of the FC-NB faced toward the microscope objective in a 35-mm glass-bottom cell culture dishd and observed with a 20X objective lens of an inverted, confocal microscope.j Digital images of FC-DDFT and FC-NB explants were acquired in double-channel sequential scans (green fluorescence: excitation, 488 nm and emission, 498 to 544 nm; red fluorescence: excitation, 514 nm and emission, 563 to 663 nm). A minimum of 3 image stacks/explant was obtained for a thickness of 1 mm, and imaging was performed with the same settings throughout the study.
Quantitative image analysis was conducted on the image stacks to determine the percentage of dead cells in the control and treated FC-DDFT and FC-NB explants. The number of live and dead cells and total number of cells in each image were determined with software.k The data were thresholded on the basis of pixel intensity; individual cells were then identified and counted on the basis of cell volume. In each image, cell death was calculated as the number of dead cells (red channel) divided by the total number of cells (live and dead) detected in both the green and red channels. The final reported values for cell death were the mean of at least 3 image stacks from each explant. Cell death in TA- or MPA-treated explants was expressed as the change in percentage of dead cells relative to findings for the respective control FC-DDFT or FC-NB explant group at the 6- and 24-hour time points.
Statistical analysis
Normal distribution of data was assessed with the Shapiro-Wilk test. Within each explant type (FC-DDFT or FC-NB), metabolic activity and cell viability data (expressed as percentage change in metabolic activity and cell viability from control values) were analyzed with a mixed-model ANOVA,l with random effects of horse and replicate, and fixed effects of corticosteroid (TA and MPA) at each concentration and time point (6 and 24 hours after treatment). All possible 2- and 3-way interactions among the independent variables and a random effect of horse to account for the use of multiple tissue samples from each horse were assessed. When multiple pairwise comparisons were performed, P values were adjusted with the Tukey post hoc test. No comparisons were made between FC-DDFT and FC-NB data. Differences were considered significant at values of P ≤ 0.05.
Results
Metabolic activity
The ex vivo culture system did not affect the metabolic activity of FC-DDFT and FC-NB explants. The mean ± SD fluorescence of freshly harvested and untreated FC-DDFT explants was 45,481 ± 1,274, and that of freshly harvested and untreated FC-NB explants was 41,632 ± 2,234. After an incubation period of 6 hours, mean ± SD fluorescence of control FC-DDFT and FC-NB explants was 43,481 ± 1,112 and 40,331 ± 1,734, respectively. After an incubation period of 24 hours, mean ± SD fluorescence of control FC-DDFT and FC-NB explants was 46,580 ± 2,215 and 39,631 ± 1,450, respectively. Within each explant type, these values were not significantly different (P = 0.1) from each other.
Treatment with TA did not significantly affect the metabolic activity of FC-DDFT explants, except when explants were exposed to 6 mg of TA/mL for 24 hours (decrease in mean fluorescence, compared with the respective control explant value, 21 ± 3.5%; P = 0.04; Figure 2). In contrast, MPA treatments significantly (P < 0.01) reduced the metabolic activity of FC-DDFT explants. After an incubation period of 6 hours, FC-DDFT explants exposed to MPA at a concentration of 5 mg/mL had a decrease in mean fluorescence of 23 ± 6%, compared with the respective control explant value. After an incubation period of 24 hours, FC-DDFT explants exposed to MPA at a concentration of 0.5 or 5 mg/mL had a decrease in mean fluorescence, compared with the respective control explant value, of 23 ± 5% and 54 ± 7%, respectively. Similar to the FC-DDFT explants, exposure of FC-NB explants to MPA at a concentration of 5 mg/mL significantly (P < 0.01) reduced their metabolic activity at the 6- (36 ± 6%) and 24-hour (61 ± 9%) time points.
Metabolic activity of FC-DDFT explants (A) and FC-NB explants (B) maintained in medium containing TA at a concentration of 0.6 mg/mL (TA 0.6) or 6 mg/mL (TA 6) or containing MPA at a concentration of 0.5 mg/mL (MPA 0.5) or 5 mg/mL (MPA 5) for 6 or 24 hours. There were 5 of each type of explant in each treatment group; for each treatment group, control explants were incubated in medium without drug supplementation (data not shown). The metabolic activity of cells present in individual FC-DDFT and FC-NB explants (3 replicate explants/treatment or control group/horse) was assessed with a resazurin-based assay. Fluorescence was measured at 570 nm (excitation) and 585 nm (emission) with a plate reader. The optical density (mean fluorescence) from each explant incubated with TA or MPA at either of the 2 concentrations was expressed as the percentage change (decrease) in mean fluorescence from findings for the respective control FC-DDFT or FC-NB explant group at each time point. *Value differs significantly (P ≤ 0.05) from that of the respective control explant group.
Citation: American Journal of Veterinary Research 82, 2; 10.2460/ajvr.82.2.125
Live-dead cell staining
No dead cells were observed in the FC-DDFT and FC-NB explants maintained in control medium at the 6- and 24-hour time points. Subjectively, live-dead cell staining revealed that cell death was higher in the FC-DDFT and FC-NB explants maintained in MPA-supplemented medium, compared with explants maintained in TA-supplemented medium, at both the 6-hour (Figure 3) and 24-hour (Supplementary Figure S1, available at: avmajournals.avma.org/doi/suppl/10.2460/ajvr.82.2.125) time points.
Representative confocal microscopic images of FC-DDFT and FC-NB explants at 6 hours in control medium and medium containing TA at a concentration of 0.6 mg/mL (TA 0.6) or 6 mg/mL (TA 6) or containing MPA at a concentration of 0.5 mg/mL (MPA 0.5) or 5 mg/mL (MPA 5). Cell status was assessed by means of fluorescent microscopy with calcein to detect live cells (excitation, 495 nm; emission, 515 nm; green staining) and a high-affinity nucleic acid stain to detect dead cells (excitation, 633 nm; emission, 658 nm; red staining). The explants were incubated in both stains simultaneously for 30 minutes at 37°C and observed with an inverted, confocal microscope. In each panel, scale bar = 100 μm.
Citation: American Journal of Veterinary Research 82, 2; 10.2460/ajvr.82.2.125
These subjective findings were consistent with dead cell counts determined by means of quantitative image analysis. At the 6-hour time point, TA treatments did not significantly (P = 0.7) affect cell death in FC-DDFT explants, compared with findings for the respective control explants (Figure 4). However, both MPA treatments significantly (P < 0.01) increased the percentage of dead cells in FC-DDFT explants after 6 or 24 hours of incubation, compared with findings for the respective control explants. These results were similar for the FC-NB explants, although when explants were exposed to 6 mg of TA/mL for 6 hours, there was also a significant (P = 0.04) increase in the percentage of dead cells (21 ± 3%). For the FC-NB explants, the live-dead cell staining (Supplementary Figure S1) and percentage of dead cells (Figure 3) at 24 hours were consistent with findings at the 6-hour time point.
Results of quantitative image analysis of live-dead cell staining in FC-DDFT explants (A) and FC-NB explants (B) maintained in medium containing TA at a concentration of 0.6 mg/mL (TA 0.6) or 6 mg/mL (TA 6) or containing MPA at a concentration of 0.5 mg/mL (MPA 0.5) or 5 mg/mL (MPA 5) for 6 or 24 hours. There were 5 of each type of explant in each treatment group; for each treatment group, control explants were incubated in medium without drug supplementation (data not shown). Digital images of FC-DDFT and FC-NB explants were acquired in double-channel sequential scans (green fluorescence: excitation, 488 nm and emission, 498 to 544 nm; red fluorescence: excitation, 514 nm and emission, 563 to 663 nm). A minimum of 3 image stacks/explant was obtained, and quantitative image analysis was conducted on the image stacks to determine the percentage of dead cells in the control and treated FC-DDFT and FC-NB explants. Cell death in TA- or MPA-treated explants was expressed as the change in percentage of dead cells relative to findings for the respective control FC-DDFT or FC-NB explant group at the 6- and 24-hour time points. See Figures 2 and 3 for remainder of key.
Citation: American Journal of Veterinary Research 82, 2; 10.2460/ajvr.82.2.125
Discussion
The effects of 2 commonly used corticosteroids, TA and MPA, on FC-DDFT and FC-NB explant cell viability were investigated. Live-dead cell staining and metabolic activity assays were used as 2 measures of explant cell viability. Use of explants enabled the preservation of the extracellular matrix and cell heterogeneity in the tissues. Results of the present study indicated that, at the concentrations used, TA was less toxic to FC-DDFT explants than MPA, although after exposure at a concentration of 6 mg/mL for 24 hours, TA did decrease metabolic activity of the FC-DDFT explants. Findings for the FC-NB explant groups were similar to those of the FC-DDFT explant groups; however, the higher concentration of TA resulted in increased cell death. The levels of metabolic activity and cell death in the explants maintained in control medium were unaffected by the ex vivo culture system used in this study.
The results of the present study corroborated the findings of Nuelle et al,14 who determined that TA is less toxic than MPA to canine supraspinatus tendon explants. Also, a recent study by Edmonds et al15 revealed that although both TA and MPA were toxic to equine tendon–derived cell suspensions, cell death was less rapid with TA exposure. The in vitro and in vivo toxic effects of commonly used corticosteroids on chondrocytes and articular tissues have been investigated.16,17,18 The greater chondrotoxicity of MPA, compared with that of TA, in those studies was reflected in the findings of the present study involving equine FC-DDFT and FC-NB explants. Despite the differences in navicular fibrocartilage and articular cartilage,19,20 the reduced toxicity of TA, compared with that of MPA, is consistent across equine tendon and cartilage tissues.12,13,14,16,17,18,21,22
The concentrations of corticosteroids to which DDFT and navicular fibrocartilage cells are exposed in vivo after intrasynovial (distal interphalangeal joint or navicular bursa) injection are likely to be lower than those used in the present study.23,24,25 Equine experimental studies26,27,28,29 of tarsocrural or carpal joints have been performed to determine synovial fluid disposition of commonly used corticosteroids following intra-articular administration (80 to 100 mg of MPA and 10 to 18 mg of TA). Intra-articular dosages used in those studies correspond to synovial fluid molar concentrations in the range of 10−3M to 10−8M and are therefore commonly used for in vitro studies with articular tissue.10,17,30,31 Higher concentrations of TA and MPA (in the range of 10−3M and 10−4M) were used in the present study because assessment of cytotoxicity was the major objective; however, it is important to determine whether such differential cytotoxicity of the drugs exists at lower concentrations. Given that high concentrations of corticosteroids are cytotoxic to articular chondrocytes, the results of the present study must be interpreted with that limitation in mind.
The mechanisms of corticosteroid-mediated chondrocyte apoptosis, such as direct toxic effects of preservatives in medications, autophagy, matrix metalloproteinase release, oxidative stress, and reactive oxygen species release, are likely also active in tendon fibrocartilaginous tissues.21,31 In addition to cytotoxic effects, the influence of corticosteroids on transcriptional and translational activity of DDFT cells warrants further investigation. With regard to horses, most published reports are focused on the metabolic effects of corticosteroids on articular cartilage. Given the disparate results regarding the anabolic and catabolic effects of various corticosteroids in articular tissues, investigation of tendon tissues for similar differences in cytotoxicity is warranted. Evaluation of the anabolic and catabolic effects of corticosteroids in tenocytes in addition to the drugs' tenotoxicity will be useful for determining the optimal corticosteroid in clinical settings, especially because tenocytes are responsible for maintaining tendon extracellular matrix.
The ex vivo study of the present report had several limitations. In addition to the high doses of corticosteroids used, the study design did not account for the diffusion of corticosteroids from the intrasyno-vial space that occurs in vivo.23,24,25,26,27 Consequently, such diffusion could increase cytotoxic effects of a drug. Although the DDFT and navicular fibrocartilage tissues used in these experiments were determined to be normal on the basis of gross assessment, nascent navicular pathological changes could have influenced responses to corticosteroids. Further, it is not known whether navicular pathological changes, which likely exist in most clinical cases, alter tissue susceptibility to corticosteroid-mediated sequelae. Lastly, cell viability was determined after relatively short exposure times (6 or 24 hours); therefore, delayed toxic effects of either corticosteroid were not evaluated.
The results of the present study suggested that a high concentration (in the range of 10−3M) of TA was less cytotoxic to DDFT and navicular fibrocartilage, compared with a high concentration of MPA. Further studies to determine whether this differential tenotoxicity is evident at lower concentrations of TA and MPA are warranted. Regardless of the inherent limitations of ex vivo experiments, findings of the present study have supported testing for the cytotoxic effects of commonly used corticosteroids on equine DDFT tissue. Results of the present study may serve as a foundation for further in vitro and in vivo evaluations that could lead to clinical treatment recommendations.
Acknowledgments
Funding obtained from The Ohio State University Equine Research Fund by the Ohio State Racing Commission.
Presented in part in abstract form at the American College of Veterinary Surgeons Surgical Summit, Las Vegas, Nev, October 2019.
Abbreviations
| DDFT | Deep digital flexor tendon |
| FC-DDFT | Fibrocartilage on the dorsal surface of the deep digital flexor tendon |
| FC-NB | Fibrocartilage on the flexor surface of the navicular bone |
| MPA | Methylprednisolone acetate |
| NB | Navicular bone |
| TA | Triamcinolone acetonide |
Footnotes
Decon Laboratories Inc, King of Prussia, Pa.
Uni-Punch, Premier Medical Products, Pa.
Corning Inc, Corning, NY.
ThermoFisher Scientific, Waltham, Mass.
Sigma-Aldrich Corp, St Louis, Mo.
BioWhittaker, Cambrex Bio Science, Walkersville, Md.
Kenalog-40 (TA suspension), Bristol-Myers Squibb Co, Princeton, NJ.
Depo-Medrol (MPA suspension), Zoetis, Parsippany, NJ.
Infinite M1000PRO, Tecan Group Ltd, Morrisville, NC.
Nikon A1+ (NA 0.75), Nikon Instruments Inc, Melville, NY.
Imaris imaging software, verson 9.2, Bitplane Inc, Concord, Mass.
Sigmastat 4, Systat Software Inc, San Jose, Calif.
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