Journal: Bioactive Materials

Article Title: Microenvironment-educated MSC-EVs loaded injectable smart hydrogel for targeting senescent nucleus pulposus cells and inhibiting ferroptosis against intervertebral disc degeneration

doi: 10.1016/j.bioactmat.2026.02.030

Figure Lengend Snippet: Senescent Microenvironment-Educated Mesenchymal Stem Cells Release High-Affinity Senescent NPC Domesticated Extracellular Vesicles. (A) Schematic diagram of the experimental setup for educating MSCs with SASP-CM to generate D-EVs versus N-EVs. (B) Confocal microscopy images showing different EVs internalization by senescent NPCs after 12 h in vitro. (C) Flow cytometry and quantification analysis of different EVs uptake by senescent NPCs. (D) In vivo validation of the senescent niche. Representative fluorescence images following injection of senescence-tracer (Red). (E) In vivo PKH26-labeled D-EVs tracking. (F) Representative SA-β-Gal images and quantification of MSCs treated with SASP-CM or not. (G) Gene Ontology (GO) analysis confirming enrichment of external encapsulating structure organization and cytokine production in Biological Process (BP) categories. (H) Heatmap indicating gene expression associated with EVs biogenesis within D-MSCs and N-MSCs. (I) Heatmap indicating gene expression associated with cytokine production within D-MSCs and N-MSCs. (J and L) Gene Ontology (GO) analysis confirming enrichment of terms related to vesicle organization and transport in the Cellular Component (CC) categories. (K) Western blot analysis confirmed core senescence markers p16 and p21 and DNA damage marker γ-H2AX in N-MSC and D-MSC. (M) Western blot analysis confirmed the expression of CD9, CD63, TSG101, Calnexin, and GM130 in MSC-EVs, N-EVs, or D-EVs. (N) TEM images showing the morphology and size of MSC-derived EVs, N-EVs, and D-EVs. (O) NTA shows size distribution in MSC-EVs, N-EVs, or D-EVs. The data were presented as mean ± SD. n = 3, ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: After blocked with 5% non-fat milk for 2 h at room temperature, the membranes were incubated with primary antibodies against GAPDH (1:5000, 104941-AP, Proteintech), TSG101 (1:1000, DF8427, Affinity), CD9 (1:1000, AF5139, Affinity), CD63 (1:2000, 25682-1-AP, Proteintech), Calnexin (1:5000, 10427-2-AP, Proteintech), GM130 (1:20000, 11308-1-AP, Proteintech), CXCR3 (1:5000, 26756-1-AP, Proteintech), CXCL10 (1:2000, 10937-1-AP, Proteintech), MMP3 (1:2000, 17873-1-AP, Proteintech), ADAMTS5 (DF13268, Affinity), P16 (AF5484, Affinity), P21 (10355-1-AP, Proteintech), GPX4 (1:1000, 381958, Zen-bio), SLC7A11 (1:1000, 26864-1-AP, Proteintech), ACSL4 (1:5000, 22401-1-AP, Proteintech) and Tubulin (1:10000, T40103 , Abmart) overnight at 4 °C.

Techniques: Confocal Microscopy, In Vitro, Flow Cytometry, In Vivo, Biomarker Discovery, Fluorescence, Injection, Labeling, Gene Expression, Western Blot, Marker, Expressing, Derivative Assay

Journal: Bioactive Materials

Article Title: Microenvironment-educated MSC-EVs loaded injectable smart hydrogel for targeting senescent nucleus pulposus cells and inhibiting ferroptosis against intervertebral disc degeneration

doi: 10.1016/j.bioactmat.2026.02.030

Figure Lengend Snippet: Senescent Microenvironment-Educated Mesenchymal Stem Cells Release High-Affinity Senescent NPC Domesticated Extracellular Vesicles. (A) Schematic diagram of the experimental setup for educating MSCs with SASP-CM to generate D-EVs versus N-EVs. (B) Confocal microscopy images showing different EVs internalization by senescent NPCs after 12 h in vitro. (C) Flow cytometry and quantification analysis of different EVs uptake by senescent NPCs. (D) In vivo validation of the senescent niche. Representative fluorescence images following injection of senescence-tracer (Red). (E) In vivo PKH26-labeled D-EVs tracking. (F) Representative SA-β-Gal images and quantification of MSCs treated with SASP-CM or not. (G) Gene Ontology (GO) analysis confirming enrichment of external encapsulating structure organization and cytokine production in Biological Process (BP) categories. (H) Heatmap indicating gene expression associated with EVs biogenesis within D-MSCs and N-MSCs. (I) Heatmap indicating gene expression associated with cytokine production within D-MSCs and N-MSCs. (J and L) Gene Ontology (GO) analysis confirming enrichment of terms related to vesicle organization and transport in the Cellular Component (CC) categories. (K) Western blot analysis confirmed core senescence markers p16 and p21 and DNA damage marker γ-H2AX in N-MSC and D-MSC. (M) Western blot analysis confirmed the expression of CD9, CD63, TSG101, Calnexin, and GM130 in MSC-EVs, N-EVs, or D-EVs. (N) TEM images showing the morphology and size of MSC-derived EVs, N-EVs, and D-EVs. (O) NTA shows size distribution in MSC-EVs, N-EVs, or D-EVs. The data were presented as mean ± SD. n = 3, ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: After blocked with 5% non-fat milk for 2 h at room temperature, the membranes were incubated with primary antibodies against GAPDH (1:5000, 104941-AP, Proteintech), TSG101 (1:1000, DF8427, Affinity), CD9 (1:1000, AF5139, Affinity), CD63 (1:2000, 25682-1-AP, Proteintech), Calnexin (1:5000, 10427-2-AP, Proteintech), GM130 (1:20000, 11308-1-AP, Proteintech), CXCR3 (1:5000, 26756-1-AP, Proteintech), CXCL10 (1:2000, 10937-1-AP, Proteintech), MMP3 (1:2000, 17873-1-AP, Proteintech), ADAMTS5 (DF13268, Affinity), P16 (AF5484, Affinity), P21 (10355-1-AP, Proteintech), GPX4 (1:1000, 381958, Zen-bio), SLC7A11 (1:1000, 26864-1-AP, Proteintech), ACSL4 (1:5000, 22401-1-AP, Proteintech) and Tubulin (1:10000, T40103 , Abmart) overnight at 4 °C.

Techniques: Confocal Microscopy, In Vitro, Flow Cytometry, In Vivo, Biomarker Discovery, Fluorescence, Injection, Labeling, Gene Expression, Western Blot, Marker, Expressing, Derivative Assay

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