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Figure 2. BMSC-derived EVs enhance macrophage efferocytosis and promote a pro-resolving phenotype in vitro. (A) Scheme of MSC-EVs isolation; (B) Representative microscopic image showing the characteristic spindle-shaped appearance of BMSCs; (C) Flow cytometry was performed to assess BMSC surface-marker expression. Cells were positive for CD44, CD29, and CD90 and negative for CD11b and CD45; (D) Representative TEM image showing the morphology of MSC-EVs. Scale bar: 100 nm; (E) Western blot analysis of characteristic MSC-EV protein markers; (F) Size distribution of MSC-EVs determined by NTA; (G) Schematic overview of the experimental design used to evaluate the effects of MSC-EVs on macrophages exposed to LPS (100 ng/mL); (H) Expression of efferocytosis-associated genes in macrophages under the indicated experimental conditions was determined by RT-qPCR (n = 3 independent biological experiments); (I and J) Representative immunofluorescence images and corresponding quantification of efferocytic activity in macrophages following a 2-h co-culture with CFSE-labeled ACs (n = 5 independent biological experiments). Scale bar: 20 µm; (K and L) Representative immunofluorescence staining and quantitative assessment of Arg1 and iNOS expression in macrophages (n = 3 independent biological experiments); (M) RT-qPCR measurement of inflammation-associated genes in macrophages exposed to LPS (100 ng/mL) for 24 h in the presence or absence of MSC-EV treatment (n = 3 independent biological experiments). For (H), (J), (L), and (M), comparisons among multiple groups were performed using one-way ANOVA followed by Tukey’s multiple-comparison test. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. (A) and (G) were created with BioRender.com. BMSCs: Bone marrow mesenchymal stem cells; EVs: extracellular vesicles; MSC-EVs: mesenchymal stem cell-derived extracellular vesicles; TEM: transmission electron microscopy; NTA: nanoparticle tracking analysis; LPS: lipopolysaccharide; RT-qPCR: quantitative real-time polymerase chain reaction; CFSE: carboxyfluorescein succinimidyl ester; ACs: apoptotic chondrocytes; iNOS: inducible nitric oxide synthase; ANOVA: analysis of variance.