A novel extracellular vesicles production system harnessing matrix homeostasis and macrophage reprogramming mitigates osteoarthritis

Abstract

Osteoarthritis (OA) is a degenerative disease that significantly impairs quality of life. There is a pressing need for innovative OA therapies. While small extracellular vesicles (sEVs) show promising therapeutic effects against OA, their limited yield restricts clinical translation. Here, we devised a novel production system for sEVs that enhances both their yield and therapeutic properties. By stimulating mesenchymal stem cells (MSCs) using electromagnetic field (EMF) combined with ultrasmall superparamagnetic iron oxide (USPIO) particles, we procured an augmented yield of EMF-USPIO-sEVs. These vesicles not only activate anabolic pathways but also inhibit catabolic activities, and crucially, they promote M2 macrophage polarization, aiding cartilage regeneration. In an OA mouse model triggered by anterior cruciate ligament transection surgery, EMF-USPIO-sEVs reduced OA severity, and augmented matrix synthesis. Moreover, they decelerated OA progression through the microRNA-99b/MFG-E8/NF-κB signaling axis. Consequently, EMF-USPIO-sEVs present a potential therapeutic option for OA, acting by modulating matrix homeostasis and macrophage polarization.

Keywords: Electromagnetic field; Extracellular vesicles; Macrophage; Matrix homeostasis; Osteoarthritis; Reprogramming.

Scheme 1

Summary of a novel small extracellular vesicles production system for osteoarthritis treatment

Fig. 1

Production of four distinct sEVs: NC-sEVs, USPIO-sEVs, EMF-sEVs, and EMF-USPIO-sEVs. (A) Schematic representation of an innovative sEVs production system employing EMF and USPIO. B-D: Evaluations of the USPIO include zeta potential (B), diameter measurements (C), and morphological analysis (D). (E) Results from the Cell Counting Kit-8 assay for BMSCs subjected to various conditions, with USPIO concentrations at 0, 10, 25, 50, and 100 µg/mL and EMF intensities of 0, 1, 2, or 5 mT. (F) TEM images of BMSCs without USPIO treatment. The right image is an enlargement of the marked box in the left image. (G) TEM images of BMSCs with USPIO treatment. The black dotted box indicates USPIO internalization by BMSCs, and the red arrows point to the USPIO

Fig. 2

Characterization of the four sEVs. (A) Schematic representation of the sEVs isolation process using differential ultracentrifugation. (B) Western blot analysis for sEV-specific marker proteins, namely CD63, CD81, and TSG101, as well as the negative marker, Calnexin. (C) Morphology of the four sEVs types observed under TEM. (D) NTA analysis of the four sEVs types revealed similar size ranges

Fig. 3

EMF-USPIO-sEVs restored chondrocyte homeostasis. A-E: Western Blotting bands (A) of MMP13, ADAMTS5, Aggrecan, and COL 2, and corresponding semi-quantifications of the expression levels of MMP13 (B), ADAMTS5 (C), Aggrecan (D) and COL 2 (E). F-I: Relative mRNA expression levels of MMP13 (F), ADAMTS5 (G), Aggrecan (H), and COL 2 (I). J-K: Immunofluorescence images of COL 2 (J) and MMP13 (K) following various treatments (PBS, IL-1, NC-sEVs, USPIO-sEVs, EMF-sEVs, and EMF-USPIO-sEVs). COL2/MMP13 were labeled with FITC (green), and nuclei were labeled with DAPI (blue). *p < 0.05, **p < 0.01, ***p < 0.001 compared to PBS group; #p < 0.05, ##p < 0.01 compared to EMF-sEVs group

Fig. 4

EMF-USPIO-sEVs promoted M2 macrophage polarization. (A) EMF-USPIO-sEVs downregulated the proportion of CD206 + macrophages. (B) EMF-USPIO-sEVs upregulated the proportion of CD86 + macrophages. C-D: Immunofluorescence images of CD86 (C) and CD206 (D) following various treatments (PBS, IL-1, NC-sEVs, USPIO-sEVs, EMF-sEVs, and EMF-USPIO-sEVs). CD86 was stained with Cy3, exhibiting a red fluorescence; CD206 was stained with FITC, producing a green fluorescence; and nuclei were stained with DAPI, resulting in a blue fluorescence

Fig. 5

micro-CT assessment of cartilage repair. (A) Schematic illustration of intra-articular injection of EMF-USPIO-sEVs in an ACLT mouse model. (B) Three-dimensional reconstructions generated from micro-CT, illustrating the state of cartilage repair following various treatments (Sham, ACLT, NC-sEVs, USPIO-sEVs, EMF-sEVs, and EMF-USPIO-sEVs)

Fig. 6

Histological verifications (HE, SO-FG, TB staining, and immunohistochemical staining) of cartilage repair. A-C: HE (A), SO-FG (B), and TB (C) staining images detecting the cartilage repair condition. D-E: Immunohistochemical staining of the anti-catabolic marker (Aggrecan) (D) and catabolic marker (MMP13) (E)

Fig. 7

miRNA sequencing and bioinformatics analysis. (A) Heatmap illustrating differentially expressed miRNAs between NC-sEVs and EMF-USPIO-sEVs. (B) Scatter plot depicting the expression profile and global distribution of 28 differentially expressed miRNAs between NC-sEVs and EMF-USPIO-sEVs. (C) KEGG pathway enrichment analysis reveals several signaling pathways associated with OA progression. (D) Volcano plot highlights mRNAs exhibiting a ≥ 2-fold differential expression between NC-sEVs and EMF-USPIO-sEVs. Green represents downregulated mRNAs, while red denotes upregulated ones. (E) GO enrichment analysis of differentially expressed miRNAs

Fig. 8

miR-99b-5p derived from EMF-USPIO-sEVs regulated MFG-E8 expression. (A) Venn diagram displaying the potential target genes of miR-99b-5p, as predicted by miRWalk, RNA22, and miRDB databases. (B) Comparison of the top ten differentially expressed miRNAs (miR-374c-5p, miR-503-5p, miR-28a-5p, miR-146a-5p, miR-744-5p, miR-99b-5p, miR-205-5p, miR-324-5p, miR-221-3p, and miR-542-3p) between NC-sEVs and EMF-USPIO-sEVs using qRT-PCR. (C) miR-99b-5p binding sequence in the 3′-UTR of MFG-E8. D, H: Protein expression of MFG-E8 after cell transfection with miR-99b-5p mimics, miR-99b-5p inhibitor, and their respective NCs and semi-quantification. E-F: Transfection efficiency confirmation of miR-99b-5p in chondrocytes (E) and RAW246.7 (F). (G) Luciferase readings were obtained from either wt or mut MFG-E8 3′-UTR when co-transfected with control mimics or miR-99b-5p mimics. Transfection with miR-99b-5p mimics led to a reduction in luciferase activity relative to the control mimics, substantiating that MFG-E8 is a direct target of miR-99b-5p

Fig. 9

miR-99b-5p exacerbated OA through the MFG-E8/NF- κB pathway. A-D, H-K: Immunofluorescence images of COL 2 (A), MMP13 (B), CD86 (C), and CD206 (D) following various treatments (miR-NC + pcDNA-NC, miR-99b-5p + pcDNA-NC, miR-NC + pcDNA-MFG-E8, and miR-99b-5p + pcDNA-MFG-E8); corresponding semi-quantifications of fluorescence intensity of COL 2 (H), MMP13 (I), CD86 (J) and CD206 (K). COL 2, MMP13, and CD206 were labeled with FITC (green), CD86 was labeled with Cy3 (red), and nuclei were labeled with DAPI (blue). E-G: Western Blotting bands (E) of p-P65 and p-IkB and corresponding semi-quantifications of the expression levels of p-P65 (F) and p-IkB (G). *p < 0.05, **p < 0.01, ***p < 0.001