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. 2022 Nov;54(6):1133-1143.
doi: 10.1111/evj.13537. Epub 2021 Nov 24.

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

Magdalena Arévalo-Turrubiarte  1 , Mario Baratta  1   2 , Giovanna Ponti  1 , Elisabetta Chiaradia  3 , Eugenio Martignani  1
Affiliations

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

Magdalena Arévalo-Turrubiarte et al. Equine Vet J. 2022 Nov.

Abstract

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.

Keywords: chondrocytes; extracellular vesicles; horse; mesenchymal stem cells; pro-inflammation.

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Conflict of interest statement

No competing interests have been declared.

Figures

FIGURE 1
FIGURE 1
Histogram data from nanoparticle tracking analysis of media from MSCs. Particle sizes are expressed in nm. Particles from adipose cells (C, D, and E) seemed to vary more in size. Mesenteric fat (D) seemed to be more dispersed in comparison with the sizes of other adipose fat cells
FIGURE 2
FIGURE 2
TEM images of isolated particles (EVs)‐derived from BM, SF, and NF cell media. Particles displayed a round‐like shape with a visible membrane. (A) A larger extracellular vesicle observed among the other small extracellular vesicles. (B) and (C) Rounded extracellular vesicles and different particle sizes were observed. Images were acquired at 250 000× magnification. Bar = 0.125 µm
FIGURE 3
FIGURE 3
TEM images show immunogold staining with CD9. Arrows in panels A, B, and C indicate darker dots that represent the staining of marker CD9 surrounding the particles (EVs). Scale bar = 0.1 µm
FIGURE 4
FIGURE 4
Equine chondrocytes after treatment for 24 h with pro‐inflammatory cytokines. (A) Chondrocytes with control medium, (B) and (C) chondrocytes treated with cytokines, (D) chondrocytes treated with control medium supplemented with EVs, (E) and (F) chondrocytes treated with cytokines and EVs. Chondrocytes display rounded morphology. Images are representative of chondrocytes treated with BM‐MSCs cell‐derived EVs. Images were acquired at 10× magnification. Scale bar = 250 μm
FIGURE 5
FIGURE 5
Changes in expressions of genes for IL‐6 and TIMP family members according to treatments. Fold increase or decrease compared to the control (CTRL) from each respective treatment. Gene expression of IL‐6 (A), TIMP 1 (B), and TIMP3 (C). For each panel, the average (n = 3) of each treatment is shown. Differences in letters show statistical significance at P < .05
FIGURE 6
FIGURE 6
Changes in gene expressions for MMP family members according to treatments. Fold increase or decrease compared to the control (CTRL) from each respective treatment. A, B and C show graphs of the gene expression of MMP1, MMP3, and MMP13 respectively. For each panel, the average (n = 3) of each treatment is shown. Differences in letters show significance at P < .05
See this image and copyright information in PMC

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