Journal: Bioactive Materials

Article Title: NsPEFs-enriched ADSCs-EVs alleviate osteoarthritis via RSPO3-mediated dual pro-chondrogenic and pro-M2 macrophage properties

doi: 10.1016/j.bioactmat.2026.01.006

Figure Lengend Snippet: NsPEFs engineering boosts the production of ADSCs-EVs with superior yield and stability A. Schematic illustration of the high-efficiency extraction of extracellular vesicles (EVs) from adipose-derived stem cells (ADSCs) using nanosecond pulsed electric fields (NsPEFs). B. Representative transmission electron microscopy (TEM) images of isolated Ctrl-ADSCs-EVs and NsPEFs-ADSCs-EVs, showing characteristic cup-shaped morphology and bilayer membrane (scale bars: 150 nm and 75 nm). C. Nanoparticle tracking analysis (NTA) showing the particle size distribution of EVs (n = 3). D. Western blot (WB) analysis confirming the positive expression of EV-specific markers (CD81, CD63, TSG101) and the absence of the negative markers (Calnexin, Histone H3, LaminA/C). Quantification is shown on the right (n = 3). E. The particle concentration of EVs. F. NsPEFs stimulation significantly enhanced both yield and protein output compared to Ctrl-ADSCs-EVs. G. Zeta potential measurement indicating colloidal stability (n = 3). H. Purity assessment expressed as the particle-to-protein ratio ( × 10 9 particles/μg). I. Viability of cells post-NsPEFs-ADSCs-EVs treatment assessed by trypan blue exclusion assay (scale bar: 1.7 mm). Data are presented as mean ± SEM from at least three independent experiments. Statistical significance was determined by unpaired two-tailed Student's t-test or one-way ANOVA with Tukey's post-hoc test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001; ns: not significant.

Article Snippet: The antibodies used and the dilution ratios were as follows:Anti-INOS (1:800, Cohesion), Anti-Arginase 1 (1:800, BOSTER), Anti-LRP6 (1:800, BOSTER), Anti-Beta-catenin (1:800, BOSTER), Anti-CD163 (1:800, Abclonal), Anti-CD86 (1:800, BOSTER), Anti-LGR4 (1:800, Abclonal), Anti-IL-1β (1:800, BOSTER), Anti-IL-10 (1:1000, Bioss), Anti-MMP13 (1:800, BOSTER), Anti-COL2A1 (1:800, BOSTER), Anti-Histone H3 (1:1000, Nature Biosciences), Anti-Lamin A/C (1:1000, Nature Biosciences), Anti-Akt (1:1000, Nature Biosciences), Anti-pAkt (1:1000, Nature Biosciences), Anti-RSPO3 (1:1000, Abcam), Anti-CD63(1:800, BOSTER), Anti-CD81(1:800, BOSTER), Anti-TSG101(1:800, BOSTER), Anti-Calnexin(1:800, BOSTER).

Techniques: Extraction, Derivative Assay, Transmission Assay, Electron Microscopy, Isolation, Membrane, Western Blot, Expressing, Concentration Assay, Zeta Potential Analyzer, Trypan Blue Exclusion Assay, Two Tailed Test

Journal: Bioactive Materials

Article Title: NsPEFs-enriched ADSCs-EVs alleviate osteoarthritis via RSPO3-mediated dual pro-chondrogenic and pro-M2 macrophage properties

doi: 10.1016/j.bioactmat.2026.01.006

Figure Lengend Snippet: NsPEFs-ADSCs-EVs induce RSPO3 secretion via an ITGA4/PI3K/Akt-dependent mechanism. A- D.Proteomic profiling identifies ITGA4 as a key mediator linking NsPEFs-ADSCs-EVs to RSPO3. (A). Significantly enriched proteins in NsPEFs-ADSCs-EVs using proteomic analysis (n = 3). (B, C). Gene Ontology and KEGG pathway enrichment analyses of proteins in NsPEFs-ADSCs-EVs, highlight integrin binding, cell adhesion and PI3K-Akt signaling. (D). The protein-protein interaction network integrating RSPO3 with top enriched EV proteins, reveals a potential functional link with ITGA4. E- I.EV-surface ITGA4 is essential for chondrocyte targeting and RSPO3 induction. (E). The schematic hypothesizes the ITGA4-initiated signaling axis related to RSPO3 secretion. (F) qPCR analysis shows that the increased transcriptional level of Rspo3 in chondrocytes treated with NsPEFs-ADSCs-EVs is inhibited by an ITGA4-neutralizing antibody (Trosunilimab) (n = 6). (G). qPCR validation of Itga4 knockdown efficiency in ADSCs (n = 6). (H). Western blot analysis confirms the successful generation of ITGA4-deficient EVs (NsPEFs-EVs-ITGA4-KD) from Itga4 -knockdown ADSCs, while maintaining EV purity (CD63 + /Calnexin − ) (n = 3). (I) Cellular uptake of DiR-labeled NsPEFs-ADSCs-EVs-ITGA4-KD by chondrocyte is significantly impaired compared to that of NsPEFs-ADSCs-EVs-NC. Quantification of fluorescence intensity is shown (scale bar: 36.8 μm; n = 3). J-L.ITGA4 initiates RSPO3 expression through the PI3K/Akt pathway. (J). Western blot analysis of Akt phosphorylation (p-Akt) and RSPO3 in chondrocytes treated with the indicated EVs (n = 3). (K). qPCR analysis of Rspo3 confirms that ITGA4-deficient EVs fail to induce RSPO3 expression (n = 6). (L). Pharmacological inhibition of PI3K (LY294002) or Akt (MK-2206) abolishes NsPEFs-ADSCs-EVs-induced Rspo3 upregulation in chondrocytes (n = 6). Data are presented as mean ± SEM. Statistical significance was determined by unpaired two-tailed Student's t-test or one-way ANOVA with Tukey's post-hoc test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

Article Snippet: The antibodies used and the dilution ratios were as follows:Anti-INOS (1:800, Cohesion), Anti-Arginase 1 (1:800, BOSTER), Anti-LRP6 (1:800, BOSTER), Anti-Beta-catenin (1:800, BOSTER), Anti-CD163 (1:800, Abclonal), Anti-CD86 (1:800, BOSTER), Anti-LGR4 (1:800, Abclonal), Anti-IL-1β (1:800, BOSTER), Anti-IL-10 (1:1000, Bioss), Anti-MMP13 (1:800, BOSTER), Anti-COL2A1 (1:800, BOSTER), Anti-Histone H3 (1:1000, Nature Biosciences), Anti-Lamin A/C (1:1000, Nature Biosciences), Anti-Akt (1:1000, Nature Biosciences), Anti-pAkt (1:1000, Nature Biosciences), Anti-RSPO3 (1:1000, Abcam), Anti-CD63(1:800, BOSTER), Anti-CD81(1:800, BOSTER), Anti-TSG101(1:800, BOSTER), Anti-Calnexin(1:800, BOSTER).

Techniques: Binding Assay, Functional Assay, Biomarker Discovery, Knockdown, Western Blot, Labeling, Fluorescence, Expressing, Phospho-proteomics, Inhibition, Two Tailed Test