Extracellular vesicles from equine mesenchymal stem cells decrease inflammation markers in chondrocytes in vitro

Summary Background Mesenchymal stem cells (MSCs) have been used therapeutically in equine medicine. MSCs release extracellular vesicles (EVs), which affect cell processes by inhibiting cell apoptosis and regulating inflammation. To date, little is known about equine EVs and their regenerative properties. Objectives To characterise equine MSC‐derived extracellular vesicles (EVs) and evaluate their effect on equine chondrocytes treated with pro‐inflammatory cytokines in vitro. Study design In vitro experiments with randomised complete block design. Methods Mesenchymal stem cells from bone marrow, adipose tissue, and synovial fluid were cultured in vitro. The MSC culture medium was centrifuged and filtered. Isolated particles were analysed for size and concentration (total number of particles per mL). Transmission electron microscopy analysis was performed to evaluate the morphology and CD9 expression of the particles. Chondrocytes from healthy equines were treated with the inflammatory cytokines interleukin (IL)‐1β and tumour necrosis factor‐alpha. MSC‐derived EVs from bone marrow and synovial fluid cells were added as co‐treatments in vitro. Gene expression analysis by real‐time PCR was performed to evaluate the effects of EVs. Results The particles isolated from MSCs derived from different tissues did not differ significantly in size and concentration. The particles had a round‐like shape and positively expressed CD9. EVs from bone marrow cells displayed reduced expression of metalloproteinase‐13. Main limitations Sample size and characterisation of the content of EVs. Conclusions EVs isolated from equine bone marrow MSCs reduced metalloproteinase 13 gene expression; this gene encodes an enzyme related to cartilage degradation in inflamed chondrocytes in vitro. EVs derived from MSCs can reduce inflammation and could potentially be used as an adjuvant treatment to improve tissue and cartilage repair in the articular pathologies.


| INTRODUC TI ON
Osteoarthritis (OA) and tendonitis limit motion and performance of race and sport horses. 1,2 Mesenchymal stem cells (MSCs) have been used for treatment in equine OA and tendonitis 1,3 and isolation of MSCs from bone marrow (BM), adipose tissue, umbilical cord, and synovial fluid has been described. 1

,3 BM cells implanted in injured
tendons can improve the morphological orientation of the affected fibres. 1 In addition, MSCs have anti-inflammatory and immunomodulatory properties. 3 MSCs can regulate the production of tumour necrosis factor-alpha (TNFα) to mediate inflammation and suppress T-cell proliferation. 4 Paracrine mechanisms in MSCs are important for the activation of their immunomodulatory potential.
When MSCs are exposed to an inflammatory environment, inflammatory inhibitors are released. 4 MSCs also release vesicles of different sizes, collectively known as extracellular vesicles (EVs). 5 EVs are classified and named according to their size and release mechanisms. 6 In general, exosomes are secreted by exocytosis and range in size from 30 to 120 nm, while shedding vesicles, also known as microvesicles, are released via budding from the plasma membrane. Microvesicles range in size from 80 to 1000 nm. 5,6 EVs have various functions, including tissue regeneration properties by limiting tissue injury, participating in regenerative processes, 5 and enhancing cell proliferation and apoptosis. 7 EVs also carry proteins and genetic information in the form of mRNA and microRNA and participate in cell-to-cell communication 6 by interacting and fusing with the lipid membranes of target cells, allowing EVs to deliver proteins and genetic information. 5 Extracellular vesicles have also been suggested as a potentially potent treatment for tendon tissue repair in equines, 5 and other studies have demonstrated the performance of EVs in OA. Improvement of cell proliferation in chondrocytes and osteogenesis in bone has been reported after administration of EVs derived from MSCs in vivo. 8 EVs have an anti-inflammatory effect in chondrocytes derived from human OA patients in vitro. 9 In addition, EVs participate in the decrease of pro-inflammatory ILs, 9 reduce T and B lymphocyte proliferation in vitro, and improve arthritic mouse inflammation in vivo. 10 Few studies have been performed on EVs derived from equine MSCs. Transmission electron microscopy (TEM) of EVs derived from equine adipose cells showed rounded shapes of different sizes. 11 Another study characterised EVs from adipose cells by their expression of CD9, CD63, and CD81 surface markers. 12 However, little is known about EVs derived from other types of MSCs in horses, and their effect on inflammation remains unclear. Since MSCs from BM, adipose, and synovial fluid (SF) cells have been proposed as a therapeutic option for joints in equines, 1 the objective of our study was to isolate and evaluate EVs derived from different cell types (BM-MSCs, SF-MSCs, and adipose MSCs) and study the effect of BM-and SF-MSC derived EVs in a pro-inflammatory experiment in vitro using equine primary chondrocytes. These findings could indicate the potential therapeutic value for tissue treatment and MSC-derived EVs.

| Culture of mesenchymal stem cells
Mesenchymal stem cells derived from BM, SF, and adipose cells from the mesenteric, tail, and neck fat (NF) were isolated and cultured as described previously. 13

| Isolation of extracellular vesicles
Approximately 5-15 mL of medium was recovered after 24 hours of MSCs culture. The dish was washed with 5 mL of PBS and was added to the collected medium. Samples were centrifuged at 259 g for 5 minutes at 4°C in a refrigerated centrifuge (Eppendorf 5804R equipped with a model A-4-44 rotor, both from Eppendorf). The supernatant was transferred to a clean tube and centrifuged at 1560 g for 5 minutes at 4°C. The collected supernatant was filtered (0.2 µm) and placed in polyallomer conical tubes (Beckman-Coulter). Ultracentrifugation at 100 000 g was performed for 1 hour at 4°C with a no-break deceleration. The resulting pellet was recovered in 500 µL of PBS (Sigma-Aldrich) for further analysis. Frozen samples were stored at −80°C in 1% dimethyl sulfoxide (DMSO, Sigma-Aldrich).

| Nanoparticle analysis
The EV samples were diluted (1:200) before acquisition with PBS.
They were analysed using a NanoSight LM10 particle size analyser To calculate the yield, the number of EVs from the nanoparticle analysis was multiplied by the dilution factor used for sample preparation (200×) and divided by the volume of the MSCs culture medium recovered after 24 hours. Therefore, the yield represents the number of EVs produced by MSCs per ml of culture medium over a 24 hours time period.

| Transmission electron microscopy
Isolated EVs were placed on coated grids (Pioloform ® film 200 copper mesh, Agar Scientific) and fixed with 1.25% glutaraldehyde in 1% paraformaldehyde in 0.1 M Sorensen's phosphate buffer for 15 minutes. Each grid was washed five times for 10 minutes each time using Sorensen's phosphate buffer. Excess liquid was removed using Whatman ® filter paper. The grids were dried at room temperature prior to analysis. Immunogold staining consisted of washing with Tris-buffered saline (TBS), followed by incubation with goat serum (10%) and an immunogold buffer for 1 hour. Subsequently, a two-night incubation with the primary antibody CD9 (1:10, Clone HI9a, anti-mouse, BioLegend) at room temperature was performed, followed by a 1-hour incubation with anti-mouse IgG immunogold secondary antibody (EM.GAM10, BBI Solutions). Three 15 minutes washes with PBS were then performed, followed by fixation in 2.5% glutaraldehyde for 10 minutes. Three more 15-minutes washes were performed on the samples using PBS, followed by overnight drying at room temperature before image acquisition. A JE1-1010 transmission electronic microscope (JEOL) was used to observe the grids at 250 000× magnification. Image acquisition was performed using soft imaging system analysis® (Megaview III soft imaging system).

| Pro-inflammatory assay
Chondrocytes were isolated from the healthy metacarpal/metatarsophalangeal joint of three young horses (5-10 years of age). 15 Chondrocytes used in the assay were from early passages (passages one to three). EVs were previously isolated from MSCs cultured in TNFα or IL-1β was then added to the culture medium at two different concentrations (10 or 50 ng/mL). Three technical replicates were prepared for each treatment. After 24 hours, the culture medium was removed, and cells were washed once with PBS. Fresh PBS supplemented with 1 mg/mL Hoechst 33342 (Thermo Fisher Scientific) was added to each well to stain the nuclei. Images of ten random fields per well were acquired at 40× magnification using a Leica AF6000 LX inverted microscope equipped with a Leica DFC350 FX monochrome digital camera (Leica Microsystem). Nuclei were then automatically counted using ImageJ software (version 1.52s; https:// imagej.nih.gov/ij/). For the pro-inflammatory assay, 300 000 equine chondrocytes were seeded in individual wells of a 6-well plate with proliferation medium (1× DMEM + 10% EV-free FBS, 4.5 g/L Dglucose, 2 mmol/L L-glutamine, 2.5 μg/mL amphotericin B, and 1% penicillin-streptomycin; all from Sigma-Aldrich). Chondrocytes were starved for 24 hours with DMEM containing 2% BSA. Treatments were initiated and consisted of 10 ng/mL of IL-1β, 10 ng/mL of TNFα, and each cytokine with purified EVs (13 333 EVs/cell, adapted from Collino et al 7 ) derived from the different types of MSCs.
Chondrocytes were then processed for RNA extraction and gene expression analysis. Each treatment was performed in two technical replicates using EVs derived from two types of MSCs (three biological replicates for BM-and SF-MSC derived EVs).

| Gene expression
According to the manufacturer's protocol, chondrocytes were lysed, and total RNA was extracted using TRI Reagent (Sigma-Aldrich). The resulting RNA was eluted with 20 µL diethylpyrocarbonate (DEPC; Sigma-Aldrich) purified water and analysed using a NanoDrop 2000 spectrophotometer and NanoDrop software (ThermoFisher Scientific). Stable RNA was used for reverse transcription-polymerase chain reaction (RT-PCR) using an iScript™ kit (Bio-Rad). Samples were processed using Bio-Rad iCycler. Real-time PCR was carried out using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and run in a Bio-Rad CFX real-time system. Real-time PCR products were obtained following these conditions: initial denaturation at 95°C for 3 minutes and 40 amplification cycles (95°C for 10 seconds, then 62°C for 30 seconds, 95°C for 10 seconds), and a melting curve starting at 65°C with an increment of 0.5°C for 5 seconds. Primers used for this study were verified using the nucleotide basic local alignment search tool (BLAST, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, USA) and the Eurofins Oligo Analysis Tool (www.eurof insge nomics. eu) searching for equine-specific sequences (Equus caballus) ( Table 1).

| Data analyses
Data collected using NanoSight were analysed. Differences in the size and concentration of isolated particles were evaluated using the Kruskal-Wallis test. Differences were considered statistically significant at P < .05. Results derived from the real-time PCR analysis were evaluated first using a Shapiro-Wilk test to evaluate normality in the data. Normally distributed samples were evaluated using a two-way ANOVA.
Samples that were not normal were analysed using a nonparametric Kruskal-Wallis test. If the data were statistically significant (P < .05), a post hoc test was used for both tests

| Isolation of extracellular vesicles from different types of mesenchymal stem cells
Collection of media from derived MSCs was performed after 24 hours in culture with EV-free FBS. Ultracentrifugation of this media from derived MSCs cultures served to recover EVs, and nanoparticle tracking analysis was performed. The size of the particles was the expected size for EVs. 6 The concentration of particles derived from each type of isolated MSCs (BM, SF, NF, mesenteric, and tail fat cells) and size distribution was analysed. No significant differences (P > .05) were observed in the concentration of all samples ( Table 2).
NanoSight analysis provided information on particle size. The histogram distribution of obtained data revealed that particles de- to 669 nm ( Figure 1E).

| Transmission electron microscopic assessment of the morphology of extracellular vesicles
To confirm whether the particles isolated from the derived MSC cultures were indeed EVs, the collected particle suspensions were observed by TEM at a 250 000× magnification. Particles described as "cup-shaped" were evident for the EVs from three types of MSC cultures (BM, SF, and NF adipose cells) (Figure 2).
EVs derived from BM-MSCs displayed small, medium, and large particle sizes (Figure 2A). EVs from SF-MSCs were heterogeneous ( Figure 2B), while those from NF-MSCs were more homogeneous ( Figure 2C).

| Evaluation of extracellular vesicles in a proinflammatory assay in chondrocytes in vitro
On confirming the characteristics of the various EVs, their antiinflammatory potential 5 in pre-inflamed chondrocytes was evaluated.
Assays using equine chondrocytes, which are a cell population affected during OA, 2 were performed. Chondrocytes were treated with either pro-inflammatory cytokines alone (IL-1 or TNFα) or with the same cytokines and isolated EVs. The goal for these treatments was to evaluate whether EVs from BM-and SF-MSCs could  Figure 4A). Starved chondrocytes were incubated for 24 hours with IL-1β and TNFα pro-inflammatory cytokines, and nuclei were counted using Hoechst 33342. The comparison of cell counts using a one-way ANOVA revealed no differences among treatments ( Figure S1) (P > .05). Subsequently, starved chondrocytes were treated for 24 hours with pro-inflammatory cytokines alone or alongside EVs. Cell morphology did not differ among the treatments (Figure 4). when EVs were added. The expression of TIMP-1 was increased when cells were treated with TNFα, which reverted to control levels when EVs were added (P = .001) ( Figure 5B). In the case of TIMP3, a similar behaviour was evident when TNFα was added in combination with EVs (P = .005) ( Figure 5C).

| Extracellular vesicles derived from bone marrow mesenchymal stem cell medium
When using BM-MSC derived EVs and IL1β as an inflammatory signal, MMP1 gene expression did not change with the addition of EVs ( Figure 6A). However, when analysing the effect of TNFα, the addition of EVs was able to counteract the effect of the inflammatory cytokine ( Figure 6A). MMP3 and 13 were significantly different in all the comparisons of CTRLs against IL-1β treated cells, with a difference that was particularly marked for MMP3 (7-fold change, P < .001) ( Figure 6B).
Whereas MMP13, when comparing CTRL and IL-1β, displayed intermediate results (3-fold change) ( Figure 6C). Interestingly, a decrease in MMP13 expression to control levels was observed when chondrocytes were treated with EVs along with IL-1β. A similar pattern was observed after TNFα treatment ( Figure 6C).

| Extracellular vesicles derived from synovial fluid mesenchymal stem cell medium
The effects of EVs derived from SF-MSCs were also evaluated.
However, significant differences were observed only between IL-1β treatment and CTRL for IL-6 (P = .014); however, no differences were observed between control and IL-1β and EVs co-treated cells ( Figure 5A).
The expression of MMP1 was significantly different in the case of CTRL compared to the IL-1β cytokine and CTRL versus IL-1β-EVs (both P < .001) ( Figure 6A). For the MMP3 gene, significant differences were observed between CTRL and IL-1β, and CTRL and IL-1β -EVs (both P < .001) ( Figure 6B).

| D ISCUSS I ON
Extracellular vesicles in culture media derived from different equine MSCs were identified. The recovered EVs were added to equine chondrocytes that were previously subjected to a pro-inflammatory treatment, and the effects during inflammation of EVs derived from BM-and SF-MSCs were evaluated.
In this study, the number of particles isolated per mL derived from adipose-derived MSCs was marginally higher than that previously reported for equine adipose cells. 12  and size exclusion chromatography. 12 TEM determination of particle size diameter may also be influential. 7,22 However, in this study, all the particles derived from the five different MSC types of derived EVs displayed a mixed population that can be observed via nanoparticle tracking analysis. Our findings echoed those of a previous study involving MSCs. 7 Our TEM analysis also detected CD9 expression in round-shaped particles, as similarly reported in equines. 12 We also tried to detect CD63 expression in the EVs we isolated. However, we were not able to detect CD63 positive staining in our equine EVs in TEM, much like Klymiuk et al reported. 12 It seems that in equine CD63 expression is observed in cell cultures but not in isolated EVs via immunostaining. CD81 has been reported to be expressed in equine EVs. 12 However, we decided not to evaluate it since our scope was to confirm the presence of EVs from MSCs media, not to produces an inflammatory response. 23 Our results coincide with another in vitro study, wherein upregulation of the IL-6 gene was observed after treatment with IL-1β in equine chondrocytes. 24 The findings indicate a direct relationship between IL-1β and IL-6 expression in chondrocytes, further indicating that there is the production of IL-6 in exposed tissues in the presence of IL-1β. 23 When EVs derived from BM-and SF-MSCs were added as cotreatments in chondrocytes, a tendency of reduced IL-6 expression was observed, although this was not statistically significant. A reduction in cytokines has been reported in stimulated chondrocytes treated with BM-MSC derived EVs in vitro. 9 Chondrocytes treated with TNFα showed a significant increase in TIMP1 gene expression, which reverted when BM-MSC derived EVs were added. This could correspond to the regulation of TIMP1, an inhibitor of metalloproteinases. 25 TIMPs are important modulators of extracellular matrix (ECM) remodelling, and the expression of TIMPs after inflammatory cytokine exposure seems to be generally reduced. Remodelling of the extracellular matrix has also been observed in healthy patients. 25 Therefore, TIMP3 increase in cells has also been related to the stimulation of cell growth and proliferation. 25 MMPs should be upregulated during inflammation. Thus, when cells are exposed to IL-1β and TNFα, upregulation has been reported. 23 When EVs derived from BM-and SF-MSCs were added, MMP1 expression was slightly reduced in the TNFα treatments.
MMP1 expression is usually higher in OA. 23  Such an MMP13 reduction has been previously demonstrated in cartilage degradation in OA using a conditioned medium from MSCs. 10,27 Since EVs are released from cells in culture media, 6 BM-MSC EVs could be responsible for the effects of the conditioned medium.
Synovial fluid in joints provides growth factors and nutrients for chondrogenesis of articular cartilage, 3 and it is known that SF cells synthesise collagen. 28

E TH I C A L A N I M A L R E S E A RCH
Research ethics committee oversight is not currently required by this journal: the study was performed on material obtained from an abattoir.

I N FO R M E D CO N S E NT
Not applicable.

ACK N OWLED G EM ENTS
We thank Prof Giovanni Camussi from the Department of Medical

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.