Ex vivo effects of corticosteroids on equine deep digital flexor and navicular fibrocartilage explant cell viability

Abstract

OBJECTIVE To investigate the effects of triamcinolone acetonide (TA) and methylpredniso-lone acetate (MPA) on the viability of resident cells within the fibrocartilage on the dorsal surface of the deep digital flexor tendon (FC-DDFT) and fibrocartilage on the flexor surface of the navicular bone (FC-NB) of horses. SAMPLE 12 to 14 explants of FC-DDFT and of FC-NB from grossly normal forelimbs of 5 cadavers of horses aged 9 to 15 years without evidence of musculo-skeletal disease. PROCEDURES Explants were incubated with culture medium (control) or TA-supplemented (0.6 or 6 mg/mL) or MPA-supplemented (0.5 or 5 mg/mL) medium for 6 or 24 hours. Explant metabolic activity and percentage of dead cells were assessed with a resazurin-based assay and live-dead cell staining, respectively, at each time point. Drug effects were assessed relative to findings for the respective control group. RESULTS Application of TA (at both concentrations) did not significantly change the cell viability of FC-DDFT explants. For FC-NB explants, TA at 6 mg/mL significantly reduced the metabolic activity and increased the percentage of dead cells at both time points. With either MPA concentration, FC-DDFT and FC-NB explants had reduced metabolic activity and an increased percentage of dead cells at 24 hours, whereas only MPA at 5 mg/mL was cytotoxic at the 6-hour time point. CONCLUSIONS AND CLINICAL RELEVANCE In ex vivo explants, TA was less cytotoxic to equine FC-DDFT and FC-NB cells, compared with MPA. Further work is warranted to characterize the drugs' transcriptional and translational effects as well as investigate their cytotoxicity at lower concentrations.

Review of glucocorticoid therapy in horses: Intra‐articular corticosteroids

“…The reduction in the viability of chondrocytes and synoviocytes following treatment with 40 mg MPA suggests that it is somewhat cytotoxic to synovial cells (Sherman et al, 2015). Similar findings were observed in deep digital flexor tendon and navicular bone fibrocartilage explants treated with MPA (0.5 mg and 5 mg/ml, 6‐ and 12‐h exposure time), suggesting that use in synovial structures other than joints may also be questionable (Sullivan et al, 2021).…”

Autologous platelet-rich plasma effects on Staphylococcus aureus–induced chondrocyte death in an in vitro bovine septic arthritis model

“…Prior to experimental setup, all cartilage explants were allowed to equilibrate to in vitro culture conditions (37 °C with 5% CO 2 at 95% humidity) for 36 to 48 hours in the aforementioned medium. 32,33 Prior to experimental setup, the explants were grossly evaluated to ensure that they were of uniform size and thickness.…”

“…The metabolic activity of cells in individual cartilage explants (3 randomly allocated replicate explants/treatment group/cow) was assessed with a resazurin-based assay as described. 33,34 This is a fluorescent metabolic assay that detects the conversion of resazurin to the fluorescent compound resorufin by metabolically active cells. After the 24hour incubation was complete, cartilage explants were rinsed twice with PBS solution and transferred to new wells in 24-well plates in 1 mL of DMEM containing glucose (4.5 g/L) and l-glutamine (300 µg/ mL) and supplemented with 2% fetal bovine serum, 1% insulin-transferrin-selenium, sodium penicillin (100 U/mL), streptomycin sulfate (100 µg/mL), and l-ascorbic acid (50 µg/mL).…”

“…The numbers of live and dead cells were determined among cartilage explants (2 randomly allocated replicates/treatment group/cow) after 24 hours of incubation was determined by use of live-dead cell staining as described. 27,28,33 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 calcein (15 µM) for live cells (excitation, 495 nm; emission, 515 nm; green staining) and a high-affinity nucleic acid stain (50 nm) for dead cells (excitation, 633 nm; emission, 658 nm; red staining).…”

“…Quantitative image analysis was conducted on the image stacks to determine the percentage of dead cells in cartilage explants groups. 33 The numbers of live and dead cells and total number of cells in each image were determined with a software program (Imaris version 9.5; Bitplane Inc). The data were thresholded on the basis of pixel intensity; individual cells were then identified and counted on the basis of cell volume.…”