room temperature  (Proteintech)

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: D-EVs Alleviate Cellular Senescence and Restore ECM anabolic/catabolic metabolism in Senescent NPCs. (A) The CCK8 assay was used to determine D-EVs concentrations on cell viability. (B) Flow cytometry analysis of proliferative capacity in the above group, and (C) quantitative analysis. (D) Representative ROS images of senescent NPCs treated with N-EVs, D-EVs, or D-EVs + GW4869. (E) Representative SA-β-Gal images of senescent NPCs treated with N-EVs, D-EVs, or D-EVs + GW4869, and (F) quantitative analysis. (G) Confocal analysis of γ-H2A with IF staining depicting DNA damage in the control, TBHP, N-Evs, or D-EVs group. (H) WB analysis of ECM metabolism–related and aging-related proteins in NPCs following treatment with Control, TBHP, N-Evs, or D-EVs. (I) Western blot analysis of p53, p21, and p16 in senescent NPCs treated with D-EVs, D-CM, or D-CM EV-dep . (J) Confocal analysis of COL2 with IF staining in the control, TBHP, N-EVs, or D-EVs group. The data were presented as mean ± SD. n = 3, ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: For immunohistochemistry, the tissue sections were incubated overnight at 4 °C with primary antibodies against COL2 (1:1000, 28459-1-AP, Proteintech) and GPX4 (1:500, 381958, Zen-bio).

Techniques: CCK-8 Assay, Flow Cytometry, Staining, Control, Western Blot

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: Histological evaluation of the D-EVs@Gel ROS in a rat IDD model. (A) Representative histological staining of intervertebral disc tissues (H&E, Safranin O, and Masson) at 4 and 8 weeks post-treatment. Scale bars: 1 mm. (B, E) Immunohistochemical staining and quantification for GPX4, a key inhibitor of ferroptosis, at 4 and 8 weeks post-treatment. (C, F) Immunohistochemical staining and quantification for COL2 at 4 and 8 weeks post-treatment. (D) Quantitative analyses of histological score at 4 and 8 weeks post-treatment. Scale bars: 500 μm. The data were presented as mean ± SD. n = 6, ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: For immunohistochemistry, the tissue sections were incubated overnight at 4 °C with primary antibodies against COL2 (1:1000, 28459-1-AP, Proteintech) and GPX4 (1:500, 381958, Zen-bio).

Techniques: Staining, Immunohistochemical staining

Journal: Bioactive Materials

Article Title: A dual-functional hydrogel integrating adhesive and lubricating interfaces for mitochondrial protection–Driven cartilage regeneration

doi: 10.1016/j.bioactmat.2026.02.051

Figure Lengend Snippet: Regulation of extracellular matrix metabolism by AdHy@Pae in LPS-induced chondrocytes. (A) Representative immunofluorescence images showing the expression of COL2A1, SOX9, MMP9, and ADAMTS5 in different treatment groups and corresponding (B) quantitative analysis. Data are shown as mean ± SD (n = 3, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001).

Article Snippet: Rat chondrocytes (P1) were seeded in 24-well plates (5 × 10 4 cells per well) and cultured to ∼70–80% confluence, stimulated with LPS (10 μg mL −1 , 12 h) to induce an inflammatory phenotype, and then treated with hydrogel extracts (Hy, AdHy, AdHy@Pae) for 24 h. Cells were washed three times with PBS, fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 (Aladdin, T434386) for 10 min, and blocked with 5% BSA (Solarbio, SW3015) at room temperature for 1 h. Primary antibodies were applied overnight at 4 °C: COL2A1 (Proteintech, 28459-1-AP, 1:200), SOX9 (HUABIO, HA723548, 1:1000), MMP9 (HUABIO, ET1704-69, 1:200), and ADAMTS5 (HUABIO, HA722011, 1:100).

Techniques: Immunofluorescence, Expressing

Journal: Bioactive Materials

Article Title: A dual-functional hydrogel integrating adhesive and lubricating interfaces for mitochondrial protection–Driven cartilage regeneration

doi: 10.1016/j.bioactmat.2026.02.051

Figure Lengend Snippet: Expression analysis of cartilage-related proteins after treatment with AdHy@Pae. (A) Representative Western blot images showing ECM synthesis–related proteins (COL2A1, ACAN, COMP, and SOX9) and antioxidant markers (NRF2 and HO-1) in different groups. (B) Quantitative analysis of matrix-degrading enzymes (MMP13, ADAMTS5, ADAMTS1) and apoptosis-related proteins (BCL-2, Bax, and Caspase-3). Data are presented as mean ± SD (n = 3; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001).

Article Snippet: Rat chondrocytes (P1) were seeded in 24-well plates (5 × 10 4 cells per well) and cultured to ∼70–80% confluence, stimulated with LPS (10 μg mL −1 , 12 h) to induce an inflammatory phenotype, and then treated with hydrogel extracts (Hy, AdHy, AdHy@Pae) for 24 h. Cells were washed three times with PBS, fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 (Aladdin, T434386) for 10 min, and blocked with 5% BSA (Solarbio, SW3015) at room temperature for 1 h. Primary antibodies were applied overnight at 4 °C: COL2A1 (Proteintech, 28459-1-AP, 1:200), SOX9 (HUABIO, HA723548, 1:1000), MMP9 (HUABIO, ET1704-69, 1:200), and ADAMTS5 (HUABIO, HA722011, 1:100).

Techniques: Expressing, Western Blot

Journal: Bioactive Materials

Article Title: A dual-functional hydrogel integrating adhesive and lubricating interfaces for mitochondrial protection–Driven cartilage regeneration

doi: 10.1016/j.bioactmat.2026.02.051

Figure Lengend Snippet: In vivo validation of the chondroprotective and mitochondrial regulatory effects of AdHy@Pae after therapy for 4 weeks. (A) Representative immunohistochemical staining of cartilage sections for COL2A1, SOX9, MMP3, and ADAMTS1 in different groups (Sham, PBS, Hy, AdHy, and AdHy@Pae). (B) Quantitative analysis of the immunohistochemical staining intensity showing relative expression levels of anabolic (COL2A1, SOX9) and catabolic (MMP3, ADAMTS1) markers. (C) TEM images of chondrocytes showing mitochondrial ultrastructure in each group, with high-magnification views (bottom panels) indicating cristae integrity and membrane morphology. Data are presented as mean ± SD (n = 3); ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗P < 0.0001.

Article Snippet: Rat chondrocytes (P1) were seeded in 24-well plates (5 × 10 4 cells per well) and cultured to ∼70–80% confluence, stimulated with LPS (10 μg mL −1 , 12 h) to induce an inflammatory phenotype, and then treated with hydrogel extracts (Hy, AdHy, AdHy@Pae) for 24 h. Cells were washed three times with PBS, fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 (Aladdin, T434386) for 10 min, and blocked with 5% BSA (Solarbio, SW3015) at room temperature for 1 h. Primary antibodies were applied overnight at 4 °C: COL2A1 (Proteintech, 28459-1-AP, 1:200), SOX9 (HUABIO, HA723548, 1:1000), MMP9 (HUABIO, ET1704-69, 1:200), and ADAMTS5 (HUABIO, HA722011, 1:100).

Techniques: In Vivo, Biomarker Discovery, Immunohistochemical staining, Staining, Expressing, Membrane