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Figure 4. MSC-EVs improved macrophage efferocytosis, inflammation activation, and chondrocyte damage in vivo. (A) Schematic timeline of the in vivo experiment designed to assess whether BMSCs modulate macrophage efferocytosis, inflammatory responses, and cartilage injury through MSC-EV-associated mechanisms; (B and C) Representative micro-CT images of mouse knee joints and corresponding quantitative evaluation of osteophyte formation. Osteophytes are highlighted in yellow (n = 5 mice per group); (D) Representative SO/FG-stained sections of knee joints from mice receiving BMSC or GW4869 treatment. Scale bars: 1,500 and 20 μm; (E) Quantitative assessment of OARSI, synovitis, and osteophyte scores based on SO/FG-stained sections (n = 5 mice per group); (F) Representative immunofluorescence images showing iNOS expression in mouse knee synovial tissue. Scale bar: 20 μm; (G) Quantification of iNOS immunofluorescence signals (n = 5 mice per group); (H) Representative TUNEL-stained images of mouse articular cartilage. Scale bar: 20 μm; (I) Quantitative analysis of TUNEL-positive cells in cartilage (n = 5 mice per group); (J) Representative immunofluorescence images showing Aggrecan and Mmp3 expression in articular cartilage. Scale bar: 20 μm; (K and L) Quantitative assessment of Aggrecan and Mmp3 immunofluorescence signals, respectively (n = 5 mice per group); (M) RT-qPCR measurement of efferocytosis- and inflammation-associated genes in mouse knee synovial tissue (n = 3 mice per group). Osteophyte maturity, OARSI, synovitis, and osteophyte scores were analyzed using the Kruskal-Wallis test followed by Dunn’s multiple-comparison test. For (G), (I), (K), (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. MSC-EVs: Mesenchymal stem cell-derived extracellular vesicles; BMSCs: bone marrow mesenchymal stem cells; micro-CT: micro-computed tomography; SO/FG: Safranin O/Fast Green; OARSI: Osteoarthritis Research Society International; iNOS: inducible nitric oxide synthase; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; RT-qPCR: quantitative real-time polymerase chain reaction; ANOVA: analysis of variance.
