The anti‐inflammatory effects of equine bone marrow stem cell‐derived extracellular vesicles on autologous chondrocytes

Abstract

Background: Osteoarthritis (OA) in the horse is an economic and welfare issue and there are no current disease modifying drugs available. Stem cells have been suggested as a therapeutic intervention for OA, originally on the basis of their regenerative capacity. However, it is hypothesised that mesenchymal stem cells (MSC) exert their effects via paracrine factors including the production of extracellular vesicles that can themselves recapitulate the MSC effects in the joint. Objectives: To isolate extracellular vesicles from bone marrow MSC and investigate their anti-inflammatory effects on chondrocytes. Study design: An in vitro assessment of the effect of direct culturing extracellular vesicles on artificially inflamed chondrocytes. Methods: Extracellular vesicles were isolated from bone marrow MSC using differential sequential ultracentrifugation. Vesicles were characterised using electron microscopy, nanoparticle tracing analysis and protein analysis. Vesicle internalisation was carried out via vesicles being pre-stained and co-cultured with equine chondrocytes before analysis using confocal microscopy. The effects of vesicles on artificially inflamed chondrocytes was examined using quantitative PCR. Results: To the best of the authors' knowledge, this is the first study to isolate and characterise extracellular vesicles from equine bone MSC. Vesicles were taken up by autologous chondrocytes and had anti-inflammatory effects on gene expression following chondrocyte exposure to tumour necrosis factor α and Interleukin 1β. Main limitations: Only three independent biological repeats were performed and the work was done in vitro. Conclusion: Extracellular vesicles can be isolated from equine bone marrow MSC; they may be taken up by chondrocytes and have an anti-inflammatory action.

Temporal extracellular vesicle protein changes following intraarticular treatment with integrin α10β1-selected mesenchymal stem cells in equine osteoarthritis

“…In in vitro equine models of OA, it has been found that chondrocytes stimulated with inflammatory cytokines had a reduced inflammatory phenotype following treatment with MSC -EVs. MSC-EVs also have an anti-catabolic effect evidenced by decreased expression of matrix metalloproteinase 13 (MMP-13) ( 22 , 23 ). These observations were conserved across different species.…”

Human mesenchymal stem cells and derived extracellular vesicles reduce sensory neuron hyperexcitability and pain-related behaviors in a mouse model of osteoarthritis

“…1A). Human MSCs were purchased commercially (Lonza, UK) and derived MSC-EVs were harvested and characterized as previously described (Fig.S1) ( 36 ). To exclude the regenerative effects of MSCs and MSC-EVs in OA that can be observed when administered at week 4 post DMM surgery ( 37 ), we started MSC/MSC-EV treatment from 12 weeks post-DMM surgery at which point OA is well established (Fig.…”

“…Extracellular vesicles were harvested based on previous description ( 36 ). MSCs were cultured in standard cell culture media α-MEM (Thermo, UK) supplemented with 10% v/v fetal calf serum (thermo, UK), 1% (v/v) Glutamax (100×) (Gibco, UK), 1% (v/v) P/S (Gibco, UK), and incubated at 37 °C, 5% CO2.…”

Isolation of Extracellular Vesicles From the Bronchoalveolar Lavage Fluid of Healthy and Asthmatic Horses

“…Nanoparticle tracking analysis (NTA) is a widely used method to determine the size and concentration of small particles, and transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) can be used to directly visualize EVs (23)(24)(25). In horses, EVs have been successfully isolated from plasma, synovial fluid, uterus, bone marrow, and adipose tissue-derived cells (26)(27)(28)(29)(30)(31). However, EVs have not been previously isolated from equine BALF, and the types and content of EVs produced in equine lungs remain unknown.…”