Front Vet Sci 7: 486

PMC: PMC7419577

<em>In vitro</em> Effects of Methylprednisolone Acetate on Equine Deep Digital Flexor Tendon-Derived Cells

DDFT Harvesting and Cell Isolation

Immediately following euthanasia with sodium pentobarbital (150 mg/kg i.v.), both forelimb digits were disarticulated at the metacarpophalangeal joint. The hoof was cleaned, hair was clipped and the solar surface pared with a hoof knife. The feet were rinsed with water to remove gross debris and the entire digit was scrubbed with disinfectant solution. Each foot was disarticulated at the level of the distal interphalangeal joint to expose the proximal aspect of the navicular bone, without severing the DDFT. Using aseptic technique, the navicular bone was dissected en-bloc from the foot by transecting the surrounding soft tissues and set aside. The DDFT segment directly opposing the navicular bone and 0.5-cm proximal (within the navicular bursa) was harvested using aseptic technique. The dorsal fibrocartilaginous region was dissected from the palmar portion, diced into 0.25 cm^{segments and digested in 0.15% collagenase II (Worthington, Lakewood, NJ) in DMEM supplemented with 2% fetal bovine serum (Gemini Biomedicals, Calabasas, CA) at 37°C overnight. Following digestion, the isolated cells were filtered through a 40-um filter (Thermo-Fisher, Waltham, MA). The cells were collected by centrifuging at 300 × g for 5 min. The supernatant was removed, and the cell pellet was resuspended in culture medium containing Dulbecco's modified Eagle's medium containing 4.5 g/l glucose and 300 μg/mL L-glutamine, supplemented with 10% fetal bovine serum, 100 U sodium penicillin/mL and 100 μg streptomycin sulfate/mL (basal expansion medium). Cell yield was determined by use of a hemocytometer, and viability was estimated via trypan blue dye exclusion (Sigma, St. Louis, MO) (}25).

DDFT-Derived Cell Culture

DDFT-derived cells were seeded at 5,000 cells/cm^{in monolayer cultures in basal expansion medium and maintained at 37°C, 95% air/5% CO}₂ in a humidified incubator. Non-adherent cells were removed at day 2, the resulting colony forming units were trypsinized at day 7–9 and expanded in monolayers for two passages. Third passage DDFT-derived cells were used in subsequent experiments as described below. DDFT-derived cells were maintained as aggregate cultures to support their chondrogenic phenotype on the basis of results of another study and preliminary data collected in our laboratory (26).

Aggregate Culture

Aggregate cultures were established from third passage DDFT-derived cells isolated from each individual horse (Figure 1A) by resuspending the cells in reduced serum medium (OptiMEM, Thermo-Fisher, Waltham, MA) supplemented with 50 μg/mL ascorbic acid, 100 U sodium penicillin/mL, 100 μg streptomycin sulfate/mL, 1% insulin-transferrin-selenium and 2% fetal bovine serum (27). Cell suspensions containing a total of 3 × 10^{cells and 0.5 × 10}^{cells were aliquoted into each well of a 6- and 24-well hydrogel-coated, Ultra Low Attachment plates, respectively (Corning Inc, Corning, NY) (}Figure 1B). Cultures were maintained for 72–96 h while the floating cells formed gross and microscopically visible aggregates (Figure 1C) (27). Fresh medium was supplemented every 36–48 h. Just prior to adding treatments, the medium was collected and frozen at −80°C for later analysis of total soluble collagen and matrix metalloproteinase (MMP) contents. Treatment groups consisted of medium alone (control), medium + 0.05 mg/mL methylprednisolone acetate (0.05 MPA), or medium + 0.5 mg/mL MPA (0.5 MPA). After 24 h of incubation with the aforementioned treatment groups, the media and aggregates were collected separately, snap frozen in liquid nitrogen and stored at −80°C for further analyses. The concentrations of MPA evaluated in this study were based on previous studies measuring corticosteroid-induced biosynthesis in equine chondrocytes (28, 29), corticosteroid dose divided by the average estimated volume of navicular bursa/distal interphalangeal joint (8, 30), and preliminary data collected in our laboratory.

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Figure 1

(A) Photomicrograph of second passage DDFT-derived cell monolayer culture prior to trypsinization for aggregate culture. (B) Dispersed third passage DDFT-derived cells seeded in hydrogel-coated, Ultra Low attachment plates at the start of aggregate culture. (C) DDFT-derived cell aggregates formed in 72–96 h of non-adherent culture prior to MPA treatment. Scale bar: 100 μm.

RNA Isolation and Quantitative RT-PCR

Total RNA was isolated using a previously described protocol (31, 32). Cells from respective aggregate samples in 6-well plates were homogenized in a guanidinium thiocyanate-phenol-chloroform solution reagent (TRIzol, ThermoFisher, Waltham, MA) according to manufacturer's suggested protocol. RNA purity and quantity were assessed spectrophotometrically (Nanodrop, ThermoFisher, Waltham, MA) on the basis of A_260/280 measurement. Total RNA of 1 μg was reverse-transcribed to cDNA which was used to measure the expression of cardinal cartilage ECM related markers (Sox9, collagen type II, aggrecan) and tendon ECM related genes (collagen type I, collagen type III, COMP) by SYBR green fluorescence-based RT-qPCR; all primer pairs were previously optimized for the mRNAs of interest (Table 1). A total of 10 ηg of cDNA template was utilized for each reaction. Each treatment group from individual horses were run as triplicates and threshold values from the triplicates were averaged prior to calculations. Expression of these genes in DDFT-derived cells treated with control, 0.05 and 0.5 MPA were compared with the expression levels in third passage DDFT-derived cells. Relative gene expression was quantified using the 2^{method, normalized to expression of the reference gene, elongation factor-1α (EF1α) (}33).

Table 1

Primers for SYBRgreen RT-qPCR.

Gene	Accession Number		Sequence	Amplicon (bp)
Col I	{"type":"entrez-nucleotide","attrs":{"text":"NC_009154","term_id":"1325362985","term_text":"NC_009154"}}NC_009154	S A	5′ GAA AAC ATC CCA GCC AAG AA 5′ GAT TGC CAG TCT CCT CAT CC	231
Col III	{"type":"entrez-nucleotide","attrs":{"text":"AW261123","term_id":"6636833","term_text":"AW261123"}}AW261123	S A	5′ AGG GGA CCT GGT TAC TGC TT 5′ TCT CTG GGT TGG GAC AGT CT	215
COMP	{"type":"entrez-nucleotide","attrs":{"text":"NM_001081856","term_id":"126352423","term_text":"NM_001081856"}}NM_001081856	S A	5′ TCA TGT GGA AGC AGA TGG AG 5′ TAG GAA CCA GCG GTA GGA TG	223
Sox9	{"type":"entrez-nucleotide","attrs":{"text":"XM_023452130","term_id":"1321330000","term_text":"XM_023452130"}}XM_023452130	S A	5′ GAA CGC ACA TCA AGA CGG AG 5′ CTG GTG GTC TGT GTA GTC GT	304
Col II	{"type":"entrez-nucleotide","attrs":{"text":"NM_001081764.1","term_id":"126352360","term_text":"NM_001081764.1"}}NM_001081764.1	S A	5′ AGC AGG AAT TTG GTG TGG AC 5′ TCT GCC CAG TTC AGG TCT CT	223
Aggrecan	{"type":"entrez-nucleotide","attrs":{"text":"XM_023650277.1","term_id":"1333549081","term_text":"XM_023650277.1"}}XM_023650277.1	S A	5′ GAC GCC GAG AGC AGG TGT 5′ AAG AAG TTG TCG GGC TGG TT	202
EF1-alpha	{"type":"entrez-nucleotide","attrs":{"text":"NM_001081781.1","term_id":"126352303","term_text":"NM_001081781.1"}}NM_001081781.1	S A	5′ CCC GGA CAC AGA GAC TTC AT 5′ AGC ATG TTG TCA CCA TTC CA	328

Total Glycosaminoglycan (GAG) Content

The cell aggregates from 0.5 × 10^{DDFT-derived cells in control, 0.05 and 0.5 MPA treatment groups were individually digested in papain. Three replicates per treatment group per horse were used for this measurement. Total GAG content of representative aggregates and the culture treatment medium was determined through the use of a 1,9-dimethylmethylene blue binding assay (}31, 34). All sample values were compared against a standard curve of chondroitin sulfate values to estimate the total GAG content in paired replicates.

Total DNA Content

Total DNA content was determined by fluorometric measurement of Hoechst 33258 (Sigma, St. Louis, MO) dye incorporation in DDFT-derived cell aggregates digested with papain that was utilized for GAG assay (31). All measurements (ug) were from 0.5 × 10^{DDFT-derived cells in 0.5 mL of the respective culture medium.}

Total Soluble Collagen Content

Total soluble collagen content in each treatment culture medium of DDFT-derived cell aggregates (three replicates per treatment per horse) was measured using a commercially available kit (Biocolor, Carrickfergus, County Antrim, UK) according to manufacturer's recommendations (35). All measurements (ng) were from 0.5 × 10^{DDFT-derived cells in 0.5 mL of the respective culture medium.}

Medium MMP-3 and MMP-13 Activity

Matrix metalloproteinase-3 and−13 activity in treatment culture medium (three replicates per treatment per horse) were determined with commercially available ELISA kits (Quantikine, R&D Systems, Minneapolis, MN) in accordance with the manufacturer's protocol. Briefly, the collected control, 0.05 or 0.5 MPA medium samples was combined with MMP-3 or MMP-13 conjugates. The samples were washed and incubated with a substrate solution, and optical density was measured with a microplate reader set at 450 nm.

Statistical Analysis

Normal distribution of data was assessed by Shapiro-Wilks test. Data were compared with one-way ANOVA (GAG) or the non-parametric equivalent, Kruskal-Wallis test on ranks (chondrocytic gene expression, tendon ECM gene expression, total soluble collagen, MMP-3 and−13). Post hoc comparisons for the detection of statistically significant differences between the control, 0.05 and 0.5 MPA treatment groups were conducted with Tukey's method. Differences were considered statistically significant at P < 0.05.

Tendon ECM Related mRNA Expression

Mean fold changes in mRNA expression with MPA treatments are depicted in Figure 2. 0.5 MPA treatment significantly decreased collagen type I and collagen type III mRNA compared to 0.05 MPA (5-fold, P = 0.013; 4.5-fold, P = 0.03) and control (6.25-fold, P = 0.013; 5-fold; P = 0.036) treatments, respectively. Cartilage oligomeric matrix protein (COMP) mRNA was significantly decreased with 0.5 MPA (8.3-fold, P = 0.02) treatment alone. Overall, 0.05 MPA treatment did not significantly affect the tendon related ECM genes of DDFT-derived cells compared to control.

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Figure 2

Tendon extracellular matrix (ECM) mRNA expression: Median (and range) fold change collagen type I (Col I), collagen type III (Col III) and cartilage oligomeric matrix protein (COMP) mRNA levels (normalized to EF1a) in DDFT-derived cells after 24 h of treatment with culture media containing 0 mg/mL MPA (Control), 0.05 mg/mL MPA (0.05 MPA) and 0.5 mg/mL MPA (0.5 MPA). *Represents significant difference (P < 0.05) between treatment groups.

Cartilage ECM Related mRNA Expression

0.5 MPA treatment significantly decreased Sox-9, collagen type II and aggrecan mRNA expressions compared to 0.05 MPA (5-, 5.6-, 4.6-fold, P = 0.01, 0.003, 0.04) and control (4.5-, 10-, 7.6-fold, P = 0.013, 0.005, 0.002) treatments, respectively (Figure 3). In contrast to tendon ECM mRNAs, 0.05 MPA significantly decreased collagen type II and aggrecan mRNAs (2.1-, 1.8-fold, P = 0.015, 0.02) compared to control. 0.05 MPA treatment did not significantly (P = 0.6) affect Sox-9 mRNA expression of DDFT-derived cells.

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Figure 3

Chondrocytic mRNA expression: Mean (± standard deviation) fold change SRY-box transcription factor-9 (Sox-9), collagen type II (Col II) and aggrecan mRNA levels (normalized to EF1a) in DDFT-derived cells after 24 h of treatment with culture media containing 0 mg/mL MPA (Control), 0.05 mg/mL MPA (0.05 MPA) and 0.5 mg/mL MPA (0.5 MPA). *Represents significant difference (P < 0.05) between treatment groups.

GAG

Control and MPA-treated DDFT-derived cell aggregates secreted small amounts of GAG (ug range) during in-vitro culture, and was predominantly retained within the aggregates (Figure 4). There was no significant difference (P = 0.182) in the total GAG contents of control, 0.05 and 0.5 MPA treated DDFT-derived cell aggregates. This finding remained consistent (P = 0.283) when the total GAG content was normalized to cell number (total DNA content). There was no significant difference (P = 0.182) in the GAG content released into culture medium of control, 0.05 and 0.5 MPA DDFT-derived cell aggregates.

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Figure 4

Mean (± standard deviation) glycosaminoglycan (GAG) content (ug) in DDFT-derived cell aggregates, aggregate normalized to total DNA and culture media after 24 h of treatment with 0 mg/mL MPA (Control), 0.05 mg/mL MPA (0.05 MPA) and 0.5 mg/mL MPA (0.5 MPA).

Total Soluble Collagen

There was no significant difference (P = 0.6) in the total soluble collagen concentrations of culture medium from control, 0.05 and 0.5 MPA treated DDFT-derived cells (Table 2).

Table 2

Median (and range) soluble collagen, MMP-3 and MMP-13 concentrations in DDFT-derived cell culture media following treatment with 0 mg/mL MPA (Control), 0.05 mg/mL MPA (0.05 MPA) and 0.5 mg/mL MPA (0.5 MPA).

Median; range	Control	0.05 MPA	0.5 MPA	P-value
Soluble collagen (nanograms/mL)	244 (235–343)	240 (236–308)	254 (237–348)	0.9
MMP-3 (nanograms/mL)	0.62 (0.62–0.65)	0.63 (0.62–0.64)	0.63 (0.62–0.65)	0.6
MMP-13 (picograms/mL)	3.32 (3.31–3.32)	3.34 (3.33–3.36)	3.36 (3.35–3.38)	0.6

MMP-3 and MMP-13

There were no significant differences in the culture medium MMP-3 and MMP-13 concentrations of control, 0.05 and 0.5 MPA treated DDFT-derived cell aggregates (Table 2).

Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, United States

Edited by: Micaela Sgorbini, University of Pisa, Italy

Reviewed by: Vincenzo Miragliotta, University of Pisa, Italy; Gerald Fritz Schusser, Leipzig University, Germany

*Correspondence: Sushmitha S. Durgam ude.uso@3.magrud

This article was submitted to Comparative and Clinical Medicine, a section of the journal Frontiers in Veterinary Science

Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, United States

Edited by: Micaela Sgorbini, University of Pisa, Italy

Reviewed by: Vincenzo Miragliotta, University of Pisa, Italy; Gerald Fritz Schusser, Leipzig University, Germany

Received 2020 May 12; Accepted 2020 Jun 29.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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.

Keywords: navicular disease, deep digital flexor tendon, methylprednisolone acetate, ECM mRNA expression, collagen, GAG, MMP-3/-13, horse

Abstract

Footnotes

Funding. Funding was obtained from the Ohio State University Equine Research Fund by the Ohio State Racing Commission.

Footnotes

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