In vitro Effects of Methylprednisolone Acetate on Equine Deep Digital Flexor Tendon-Derived Cells

Abstract

Primary deep digital flexor tendon (DDFT) pathologies and those accompanying degenerative changes of navicular bone fibrocartilage are major causes of lameness associated with navicular disease. Intrasynovial corticosteroids are mainstay in the treatment due to the anti-inflammatory effects, but their effect on DDFT cell biosynthesis are unknown. The objective of this in-vitro study was to investigate the effects of methylprednisolone acetate (MPA) on cells isolated from the dorsal fibrocartilaginous region of forelimb DDFTs (DDFT-derived cells) of 5 horses (aged 11-17 years). Non-adherent aggregate cultures were established from third passage cells over a 72 to 96-h duration prior to treating with medium containing 0 (control), 0.05 and 0.5 mg/mL MPA for 24 h. Tendon and cartilage extracellular matrix (ECM) related gene expression, cell aggregate and culture medium GAG contents, culture medium collagen and MMP-3 and−13 concentrations were measured. After 24 h of treatment, only the higher MPA concentration (0.5 mg/mL) significantly down-regulated tendon ECM related genes; whereas, both MPA doses significantly down-regulated cartilage ECM related genes. MPA treatment did not affect the total GAG content of DDFT-derived cells or total GAG, soluble collagen and MMP-3 and−13 contents in culture medium compared to untreated controls. Future studies to determine the response of DDFT-derived cells with longer exposure times to corticosteroids and in the presence of inflammatory cytokines are necessary. These results are a first step in assessing the effects of intrasynovial medications on equine DDFT, for which currently no information exists.

Zonal characterization and differential trilineage potentials of equine intrasynovial deep digital flexor tendon-derived cells

“…DDFT tissue was harvested from the forelimbs of five equine cadavers euthanized for reasons unrelated to musculoskeletal disorders (age range 6 to 12 years). Intrasynovial DDFT tissue within the proximal aspect of the podotrochlear bursa and adjacent to the distal sesamoid bone was used for TDC isolation and was determined free of gross pathology at the time of harvesting [ 11 , 38 ]. The dorsal fibrocartilaginous and underlying tendinous zones of DDFT were dissected by gross assessment (Fig.…”

Interleukin-1β and methylprednisolone acetate demonstrate differential effects on equine deep digital flexor tendon and navicular bone fibrocartilage cells in vitro

“…The dorsal 2 mm thickness of DDFT tissue extending from distal to the T-ligament attachment up to the distal margin of the navicular bone was dissected. 18,19 NBF and DDFT tissues were diced into 0.25 cm 3 pieces and digested in 0.15% collagenase II (ThermoFisher Scientific) in Dulbecco's modified Eagle's medium (DMEM; Gemini Biomedicals) supplemented with 2% fetal bovine serum (ThermoFisher Scientific) in a humidified shaker incubator at 37 °C overnight. After digestion, the isolated cells were filtered through a 40-um filter (ThermoFisher Scientific).…”

“…DDFT and NBF cells were seeded at 5,000 cells/ cm 2 in monolayer cultures in basal expansion medium and maintained at 37 °C, 95% air/5% CO 2 in a humidified incubator. 18,21 Non-adherent cells were removed on day 2 and the resulting colonyforming units trypsinized (0.05% Trypsin-EDTA, Gibco) on days 7-9. The cells were subsequently expanded in monolayers until 80% confluence was reached and passaged twice.…”

“…DDFT and NBF cells were maintained as aggregate cultures to support their chondrogenic phenotype. 18,21…”

“…When DDFT cells maintained under noninflammatory conditions were treated with 0.05 mg/ mL and 0.5 mg/mL MPA, both concentrations of MPA downregulated the cartilage ECM gene expression, whereas only 0.5 mg/mL MPA downregulated the tendon-related ECM gene expression. 18 To our knowledge, the effects of IL-1β and IL-1β combined with MPA treatments on DDFT or NBF cells have not been investigated.…”