col2  (Affinity Biosciences)

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: Bone Research

Article Title: Akkermansia muciniphila attenuates intervertebral disc degeneration via extracellular vesicle-mediated delivery of the effector protein B2UKX5

doi: 10.1038/s41413-026-00541-5

Figure Lengend Snippet: Supplementation of Akk and Akk -EVs attenuates IVDD in multiple in vivo models. a Representative ex vivo fluorescence images of L2-6 lumbar intervertebral discs 24 h after intravenous injection of DiR-labeled Akk -EVs in mice. Scale bar: 3 mm. b Quantitative analysis of mean fluorescence intensity across L2-6 in intervertebral discs. n = 3 per group. c Schematic of the experimental protocol for Akk and Akk -EVs treatment in needle puncture mice. d Representative T2-weighted MRI images of the C8-9 caudal spine. Scale bar: 1 mm. e Statistical analysis of Pfirrmann grading for the C8-9 caudal segment. n = 8 per group. Representative DR images ( f ) and micro-CT 3D reconstruction images ( g ) of the caudal spine. Scale bar: 1 mm. h DHI after 12 weeks of needle puncture at the C8-9 caudal segment. n = 8 per group. Representative H&E ( i ) and SO/FG ( j ) stained images showing the macroscopic morphology of the disc, NP, and AF in the C8-9 segment. Scale bars: 200 μm (Disc) or 50 μm (NP, AF). k Histological scores for the C8-9 segment. n = 8 per group. l Schematic of the experimental protocol for Akk and Akk -EVs treatment in naturally aging mice. m Representative T2-weighted MRI images of the lumbar spine in mice. Scale bar: 1 mm. n Statistical analysis of the average Pfirrmann grade in the L1-S1 lumbar disc segments. n = 9–14 per group. o Representative micro-CT 3D reconstruction images of the L5-6 lumbar disc segments. Scale bar: 1 mm. p Statistical analysis of the mean DHI in the L2-6 lumbar disc segments. n = 8–10 per group. q , r Representative images of H&E (q) and SO/FG (r) staining showing the macroscopic morphology of intervertebral disc (Disc), nucleus pulposus (NP), annulus fibrosus (AF), and endplate (EP) in the L5-6 lumbar disc segment. Scale bars: 200 μm (Disc) or 50 μm (NP, AF, EP). s Statistical analysis of histological scores for each lumbar segment (L2-6). n = 8–10 per group. t Representative IF images showing the expression of COL2, ACAN, and MMP13 in the L5-6 lumbar intervertebral discs. Scale bar: 200 μm. u Quantitative analysis of COL2, ACAN, and MMP13 expression. n = 8–10 per group. Data are presented as mean ± SD. Statistical significance was determined by unpaired Student’s t -test ( b ) or one-way ANOVA followed by Bonferroni post hoc test ( e , h , k , n , p , s , u )

Article Snippet: Sections were blocked with 10% goat serum before incubation with primary antibodies: COL2 (1:300; GB11021-100, Servicebio), ACAN (1:300; GB11373-100, Servicebio), MMP13 (1:300; GB11247-100, Servicebio), p16 Ink4a (1:200; GB111143-100, Servicebio), IL-6 (1:300; GB11117-100, Servicebio), Akk -EVs-specific antibody (1:50, custom-made, produced by Wuhan FriendBio Technology Co) and B2UKX5-specific antibody (1:50, custom-made, produced by Nanjing GenScript Biotechnology Co).

Techniques: In Vivo, Ex Vivo, Fluorescence, Injection, Labeling, Micro-CT, Staining, Expressing

Journal: Bone Research

Article Title: Akkermansia muciniphila attenuates intervertebral disc degeneration via extracellular vesicle-mediated delivery of the effector protein B2UKX5

doi: 10.1038/s41413-026-00541-5

Figure Lengend Snippet: B2UKX5 as a potential functional protein in Akk -EVs against IVDD. Hierarchical clustering heatmap ( a ) and count ( b ) of differentially enriched proteins between Akk and Akk -EVs. c Functional annotation for the highly enriched 388 proteins using the COG database. d Relative enrichment levels of the five selected candidate proteins in Akk -EVs compared to Akk , displayed as Akk -EVs/ Akk ratios. e Heatmap showing qPCR-based expression levels of MMP13 , ADAMTS5 , TNF , and CDKN1A in TNF-α-treated human NP cells following treatment with the five candidate proteins. n = 3 per group. f Cytotoxicity assessment of B2UKX5 in human NP cells using Cell Counting Kit-8 assay after 24 h treatment under indicated concentrations. Cell viability is expressed relative to vehicle control. n = 3 per group. g Representative IF staining of COL2 (green) in TNF-α-treated NP cells after B2UKX5 intervention; Nuclei are counterstained with DAPI (blue). Scale bar: 100 μm. h Quantification of COL2 mean fluorescence intensity in human NP cells. n = 3 per group. i Representative IF staining of MMP13 (green) in TNF-α-treated NP cells after B2UKX5 intervention; Nuclei are counterstained with DAPI (blue). Scale bar: 100 μm. j Quantification of MMP13 mean fluorescence intensity in human NP cells. n = 3 per group. k Correlation analysis between age, IVDD grading (Pfirrmann), and serum B2UKX5 levels in human subjects. n = 79. l Quantification of B2UKX5 levels in serum from mice of different age groups. n = 15–16 per group. m Representative IF staining of B2UKX5 (green) in human NP tissue from patients with varying degrees of IVDD; Nuclei are counterstained with DAPI (blue). Scale bar: 50 μm. n Quantification of B2UKX5-positive area (%) in human NP tissue; n = 9–11 per group. o Representative IF staining of B2UKX5 (green) in intervertebral disc sections from young versus aged mice; Nuclei are counterstained with DAPI (blue). Scale bar: 100 μm. p Quantification of B2UKX5-positive area (%) in mouse discs. n = 9–10 per group. Data are presented as mean ± SD. Statistical significance was assessed using one-way ANOVA followed by Bonferroni post hoc test, no significant differences were observed in panel f , and Pearson correlation was used for panel k

Article Snippet: Sections were blocked with 10% goat serum before incubation with primary antibodies: COL2 (1:300; GB11021-100, Servicebio), ACAN (1:300; GB11373-100, Servicebio), MMP13 (1:300; GB11247-100, Servicebio), p16 Ink4a (1:200; GB111143-100, Servicebio), IL-6 (1:300; GB11117-100, Servicebio), Akk -EVs-specific antibody (1:50, custom-made, produced by Wuhan FriendBio Technology Co) and B2UKX5-specific antibody (1:50, custom-made, produced by Nanjing GenScript Biotechnology Co).

Techniques: Functional Assay, Expressing, Cell Counting, Control, Staining, Fluorescence

Journal: Bone Research

Article Title: Akkermansia muciniphila attenuates intervertebral disc degeneration via extracellular vesicle-mediated delivery of the effector protein B2UKX5

doi: 10.1038/s41413-026-00541-5

Figure Lengend Snippet: B2UKX5 Supplementation Alleviates IVDD. a Schematic of the experimental protocol for B2UKX5 treatment in tail needle puncture mice. b Representative T2-weighted MRI images of the C8-9 caudal spine. Scale bar: 1 mm. c Statistical analysis of Pfirrmann grading for the C8-9 caudal segment. n = 8 per group. Representative DR images ( d ) and micro-CT 3D reconstruction images ( e ) of the caudal spine. Scale bar: 1 mm. f DHI after 8 weeks of needle puncture at the C8-9 caudal segment. n = 8 per group. Representative H&E ( g ) and SO/FG ( h ) stained images showing the macroscopic morphology of the disc, NP, and AF in the C8-9 segment. Scale bars: 200 μm (Disc) or 50 μm (NP, AF). i Histological scores for the C8-9 segment. n = 8 per group. j Schematic of the experimental protocol for B2UKX5 treatment in naturally aging mice. k Representative T2-weighted MRI images of the lumbar spine in mice following B2UKX5 treatment. Scale bar: 1 mm. l Statistical analysis of the average Pfirrmann grade in the L1-S1 lumbar disc segments. n = 10–16 per group. m Representative micro-CT 3D reconstruction images of the L5-6 lumbar disc segments. Scale bar: 1 mm. n Statistical analysis of the mean DHI in the L2-6 lumbar disc segments. n = 8–9 per group. Representative images of H&E staining ( o ) and SO/FG staining ( p ) showing the macroscopic morphology of intervertebral discs (Disc), nucleus pulposus (NP), annulus fibrosus (AF), and endplate (EP) in the L4-5 lumbar disc segment. Scale bars: 200 μm (Disc) or 50 μm (NP, AF, EP). q Histological scores for each lumbar segment (L2-6). n = 8–9 per group. r Representative IF images showing expression of COL2, ACAN, and MMP13 in the L5-6 lumbar intervertebral disc segment. Scale bar: 200 μm. s Quantitative analysis of IF staining for COL2, ACAN, and MMP13. n = 8–9 per group. Data are presented as mean ± SD. Statistical significance was assessed by one-way ANOVA followed by Bonferroni post hoc test

Article Snippet: Sections were blocked with 10% goat serum before incubation with primary antibodies: COL2 (1:300; GB11021-100, Servicebio), ACAN (1:300; GB11373-100, Servicebio), MMP13 (1:300; GB11247-100, Servicebio), p16 Ink4a (1:200; GB111143-100, Servicebio), IL-6 (1:300; GB11117-100, Servicebio), Akk -EVs-specific antibody (1:50, custom-made, produced by Wuhan FriendBio Technology Co) and B2UKX5-specific antibody (1:50, custom-made, produced by Nanjing GenScript Biotechnology Co).

Techniques: Micro-CT, Staining, Expressing

Journal: Bioactive Materials

Article Title: Bioinspired scaffold recapitulating chondrogenic ontogeny and microenvironment for functional cartilage regeneration

doi: 10.1016/j.bioactmat.2025.11.041

Figure Lengend Snippet: Biological functional analysis of tri-layer scaffold systems. A) Representative images of migrated cells stained with crystal violet at 48 h. B) Number of migrating cells in control and CE-SKP. C) Glycosaminoglycan concentrations in control and CE-SKP at 7 and 14 days. D) qPCR analysis for COL2, SOX9, and ACAN. E) Western blotting of COL2 and SOX9. F) Alizarin red staining of control and CPH. G) ALP activity of control and CPH. H) qPCR analysis for COL1, COL10, OCN, and RUNX2. I) Western blotting of COL10, COL1, and RUNX2. J) Representative images of migrated cells stained with crystal violet at 48 h. K) Number of migrating cells in control and P2G3. L) Scanning electron microscope micrographs of P2G3. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: After blocking the membranes for an hour at room temperature using fast blocking solution, the membranes were incubated with primary antibodies specific for SOX9 (1:2000, 67439-1-Ig, Proteintech), COL2 (1:200, NBP1-91056, Novus Biologicals), COL10 (1:500, 26984-1-AP, Proteintech), COL1 (1:2000, 67288-1-Ig, Proteintech), RUNX2 (1:200, 20700-1-AP, Proteintech), FGF2 (1:500, 11234-1-AP, Proteintech), FGF18 (1:500, 60341-1-Ig, Proteintech) and GAPDH (1:50000, 60004-1-Ig, Proteintech) for an entire night at 4 °C.

Techniques: Functional Assay, Staining, Control, Western Blot, Activity Assay, Microscopy

Journal: Bioactive Materials

Article Title: Bioinspired scaffold recapitulating chondrogenic ontogeny and microenvironment for functional cartilage regeneration

doi: 10.1016/j.bioactmat.2025.11.041

Figure Lengend Snippet: Repair of rabbit femoral trochlear defects. A) Representative images of Masson's Trichrome staining. B) Representative images of IHC staining for COL2.

Article Snippet: After blocking the membranes for an hour at room temperature using fast blocking solution, the membranes were incubated with primary antibodies specific for SOX9 (1:2000, 67439-1-Ig, Proteintech), COL2 (1:200, NBP1-91056, Novus Biologicals), COL10 (1:500, 26984-1-AP, Proteintech), COL1 (1:2000, 67288-1-Ig, Proteintech), RUNX2 (1:200, 20700-1-AP, Proteintech), FGF2 (1:500, 11234-1-AP, Proteintech), FGF18 (1:500, 60341-1-Ig, Proteintech) and GAPDH (1:50000, 60004-1-Ig, Proteintech) for an entire night at 4 °C.

Techniques: Staining, Immunohistochemistry