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: Regeneration of the femoral trochlea in a rabbit model. Representative images of IHC staining for A) COL1, B) COL10.

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: Immunohistochemistry

Journal: Bioactive Materials

Article Title: An intelligent nanoliposome alleviates disc degeneration and discogenic pain by inhibiting neurovascular ingrowth via a “Soil-conditioning, seed-modulating, and weeds-suppressing” strategy

doi: 10.1016/j.bioactmat.2025.11.048

Figure Lengend Snippet: Oxidative stress and aberrant neurovascularization are progressively enhanced during IVDD. (A) Representative T2-weighted MRI images showing disc structure in human intervertebral discs at varying Pfirrmann degeneration grades (II–IV). (B–C) Fluorescent staining and quantitative analysis of ROS levels in primary NPCs from grades II–IV NP tissues, showing a significant increase in oxidative stress with disease severity (n = 5). Scale bar, 20 μm. (D–F) IHC staining and quantitative analyses of CD31 and Tuj1 across different degenerative grades (n = 5). Scale bar, 50 μm. (G) H&E, SOFG, and IF staining of COL2A1 and MMP3 for IVDs (n = 5). Scale bar, 100 μm. (H) Histological score of the IVD tissues (n = 5). (I–J) Quantification of IF staining for COL2A1 and MMP3 (n = 5). (K–M) IF staining and corresponding quantification analyses of CD31 and Tuj1 in disc tissues of sham and AFP-treated mice (n = 5). Scale bar, 100 μm. Data are presented as mean ± SD. The comparisons among groups were performed using one-way ANOVA. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001; ns = not significant.

Article Snippet: For immunofluorescence (IF) staining, antigen retrieval was conducted using citrate buffer (0.1 M, pH 6.0), followed by blocking in 10 % normal goat serum (Solarbio, Cat. No. SL038) at room temperature for 1 h. Sections were incubated overnight at 4 °C with primary antibodies targeting ACAN (Cat. No. DF7561, Affinity), COL2A1 (Cat. No. AF0135, Affinity), MMP3 (Cat. No. AF0217, Affinity), MMP13 (Cat. No. 18165-1-AP, Proteintech), CD31 (Cat. No. AF0077, Affinity), p-LATS1 (Cat. No. AF7170, Affinity), p-YAP (Cat. No. AF3328, Affinity), Tuj1 (Cat. No. 66375, Proteintech), CGRP (Cat. No. DF7386, Affinity), SP (Cat. No. DF7522, Affinity), and PGP9.5 (Cat. No. AF5490, Affinity) followed by incubation with appropriate fluorescent secondary antibodies for 2 h at room temperature and counterstaining with DAPI (Beyotime, Cat. No. C1006).

Techniques: Staining, Immunohistochemistry

Journal: Bioactive Materials

Article Title: An intelligent nanoliposome alleviates disc degeneration and discogenic pain by inhibiting neurovascular ingrowth via a “Soil-conditioning, seed-modulating, and weeds-suppressing” strategy

doi: 10.1016/j.bioactmat.2025.11.048

Figure Lengend Snippet: NM-LP TK /RSV-MnCDs attenuates H 2 O 2 -induced ECM degradation by enhancing anabolic gene expression and suppressing catabolic activity in NPCs. (A–D) RT-qPCR analysis of mRNA expression levels of anabolic markers Acan and Col2a1 , and catabolic enzymes Adamts5 and Mmp3 in NPCs (n = 3). (E–G) Representative IF images and quantitative analyses of ACAN and MMP3 expression in NPCs across treatment groups, showing reduced ECM degradation in NM-LPTK/RSV-MnCDs-treated NPCs (n = 3). Scale bar, 30 μm. Data are presented as mean ± SD. The comparisons among groups were performed using one-way ANOVA. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001; ns = not significant.

Article Snippet: For immunofluorescence (IF) staining, antigen retrieval was conducted using citrate buffer (0.1 M, pH 6.0), followed by blocking in 10 % normal goat serum (Solarbio, Cat. No. SL038) at room temperature for 1 h. Sections were incubated overnight at 4 °C with primary antibodies targeting ACAN (Cat. No. DF7561, Affinity), COL2A1 (Cat. No. AF0135, Affinity), MMP3 (Cat. No. AF0217, Affinity), MMP13 (Cat. No. 18165-1-AP, Proteintech), CD31 (Cat. No. AF0077, Affinity), p-LATS1 (Cat. No. AF7170, Affinity), p-YAP (Cat. No. AF3328, Affinity), Tuj1 (Cat. No. 66375, Proteintech), CGRP (Cat. No. DF7386, Affinity), SP (Cat. No. DF7522, Affinity), and PGP9.5 (Cat. No. AF5490, Affinity) followed by incubation with appropriate fluorescent secondary antibodies for 2 h at room temperature and counterstaining with DAPI (Beyotime, Cat. No. C1006).

Techniques: Gene Expression, Activity Assay, Quantitative RT-PCR, Expressing

Journal: Bioactive Materials

Article Title: An intelligent nanoliposome alleviates disc degeneration and discogenic pain by inhibiting neurovascular ingrowth via a “Soil-conditioning, seed-modulating, and weeds-suppressing” strategy

doi: 10.1016/j.bioactmat.2025.11.048

Figure Lengend Snippet: NM-LP TK /RSV-MnCDs suppresses vascular ingrowth and matrix degradation in AFP-induced disc degeneration through Hippo pathway inactivation. (A–D) Representative IF images and quantitative analyses of CD31 and Tuj1 in IVDs from AFP-induced IVDD mice treated with MCB, TRULI, or their combinations with NM-LP TK /RSV-MnCDs (n = 5). Scale bar, 100 μm. (E–H) IF staining and quantification of COL2A1 and MMP13 expression in NP regions (n = 5). Scale bar, 100 μm. Data are presented as mean ± SD. The comparisons among groups were performed using one-way ANOVA. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001; ns = not significant.

Article Snippet: For immunofluorescence (IF) staining, antigen retrieval was conducted using citrate buffer (0.1 M, pH 6.0), followed by blocking in 10 % normal goat serum (Solarbio, Cat. No. SL038) at room temperature for 1 h. Sections were incubated overnight at 4 °C with primary antibodies targeting ACAN (Cat. No. DF7561, Affinity), COL2A1 (Cat. No. AF0135, Affinity), MMP3 (Cat. No. AF0217, Affinity), MMP13 (Cat. No. 18165-1-AP, Proteintech), CD31 (Cat. No. AF0077, Affinity), p-LATS1 (Cat. No. AF7170, Affinity), p-YAP (Cat. No. AF3328, Affinity), Tuj1 (Cat. No. 66375, Proteintech), CGRP (Cat. No. DF7386, Affinity), SP (Cat. No. DF7522, Affinity), and PGP9.5 (Cat. No. AF5490, Affinity) followed by incubation with appropriate fluorescent secondary antibodies for 2 h at room temperature and counterstaining with DAPI (Beyotime, Cat. No. C1006).

Techniques: Staining, Expressing

Journal: Aging and Disease

Article Title: Enhanced Chondrogenic Potential and Osteoarthritis Treatment Using Cyaonoside A-Induced MSC Delivered via a Hyaluronic Acid-Based Hydrogel System

doi: 10.14336/AD.2024.10016

Figure Lengend Snippet: CyA promotes chondrogenic differentiation of MSC . ( A ) Relative mRNA expression levels of ACAN and COL2A in MSC. ( B ) Alcian blue staining images of MSC cultured with CyA for 21 days. ( C ) Cell viability of MSC over 21 days. ( D ) Relative mRNA expression levels of ACAN, COL2A, and SOX9 in MSC over 21 days. ( E ) Protein expression levels of ACAN, COL2A, and SOX9 in MSC over 21 days. ( F ) Representative immunocytochemistry images of COL2A in MSC from Day 0 to Day 21. Statistical significance was determined using one-way ANOVA for multiple group comparisons, followed by post hoc tests. All data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, and ns = no statistically significant difference between groups.

Article Snippet: The COL2A primary antibody (1:1000, Cat# 28459-1-AP, Proteintech, USA) was applied, and the sections were incubated overnight at 4°C.

Techniques: Expressing, Staining, Cell Culture, Immunocytochemistry

Journal: Aging and Disease

Article Title: Enhanced Chondrogenic Potential and Osteoarthritis Treatment Using Cyaonoside A-Induced MSC Delivered via a Hyaluronic Acid-Based Hydrogel System

doi: 10.14336/AD.2024.10016

Figure Lengend Snippet: Expression of ACAN, COL2A, SOX9, and MMP13 in chondrocytes . ( A ) Western blot analysis of ACAN, COL2A, SOX9, and MMP13 expression in chondrocytes. ( B ) Relative mRNA expression levels of ACAN, COL2A, SOX9, and MMP13 in chondrocytes. Statistical significance was determined using one-way ANOVA for multiple group comparisons, followed by post hoc tests. All data are presented as mean ± SD (n = 3). **P < 0.01, ***P < 0.001, and ns = no statistically significant difference between groups. ( C ) Alcian blue staining images of chondrocytes. ( D ) Immunofluorescence images of COL2A in chondrocytes. ( E ) Immunofluorescence images of MMP13 in chondrocytes.

Article Snippet: The COL2A primary antibody (1:1000, Cat# 28459-1-AP, Proteintech, USA) was applied, and the sections were incubated overnight at 4°C.

Techniques: Expressing, Western Blot, Staining, Immunofluorescence

Journal: Aging and Disease

Article Title: Enhanced Chondrogenic Potential and Osteoarthritis Treatment Using Cyaonoside A-Induced MSC Delivered via a Hyaluronic Acid-Based Hydrogel System

doi: 10.14336/AD.2024.10016

Figure Lengend Snippet: In vivo therapeutic evaluation of hydrogel loaded with CyA-induced MSC . ( A ) Representative 3D reconstruction images of joints in different groups. ( B ) H&E staining of articular cartilage in different groups. ( C ) S&F staining of articular cartilage in different groups. ( D ) Immunohistochemical staining of COL2A in articular cartilage across different groups. ( E ) Immunofluorescence staining of Caspase-3 in articular cartilage across different groups. (F, G) Relative mRNA expression levels of ACAN and COL2A in joint tissues of different groups. Statistical significance was determined using one-way ANOVA for multiple group comparisons, followed by post hoc tests. All data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, and ns = no statistically significant difference between groups.

Article Snippet: The COL2A primary antibody (1:1000, Cat# 28459-1-AP, Proteintech, USA) was applied, and the sections were incubated overnight at 4°C.

Techniques: In Vivo, Staining, Immunohistochemical staining, Immunofluorescence, Expressing

Journal: Biomedical Reports

Article Title: Rabeprazole attenuates fibrosis by modulating SMAD3 linker region phosphorylation

doi: 10.3892/br.2025.2098

Figure Lengend Snippet: Rabeprazole inhibits fibrosis. After treatment with or without rabeprazole stimulation for 24 h at indicated concertations in (A) GES-1 and (B) AGS-1 cells, reverse transcription-quantitative PCR was conducted to analyze gene expression. Data was displayed as the mean ± SD and quantified by one-way ANOVA, followed by the Dunnett's post hoc test for significance against 0 µM. * P<0.05, ** P<0.01 and **** P<0.0001; n=3. (C) GES-1 and (D) AGS cells were treated as previosuly described in (A and B). The total cells were harvested to detect the indicated protein levels. The quantitation of bands was analyzed using one-way ANOVA, followed by the Dunnett's post hoc test for significance against 0 µM. ** P<0.01, *** P<0.001 and **** P<0.0001; n=3. FN, fibronectin; Col1a1, collagen type I alpha 1 chain; α-SMA, α-smooth muscle actin.

Article Snippet: Antibodies including α-SMA specific monoclonal antibody (mAb) (cat. no. 67735-1-Ig), FN mAb (cat. no. 66042-1-Ig), vimentin polyclonal antibody (pAb) (cat. no. 10366-1-AP), collagen type I (Col1a1) mAb (cat. no. 67288-1-Ig), SMAD3 mAb (cat. no. 66516-1-Ig), lamin A/C pAb (cat. no. 10298-1-AP) and α-tubulin mAb (cat. no. 66031-1-Ig) were purchased from Proteintech Group, Inc. TIF1γ mouse mAb (cat. no. YM1108), SMAD3 (phospho Ser204) rabbit pAb (cat. no. YP0363), SMAD3 (phospho Ser213) rabbit pAb (cat. no. YP0364), SMAD3 (phospho Thr179) rabbit pAb (cat. no. YP0745) and SMAD3 (phospho Ser208) rabbit pAb (cat. no. YP0746) were purchased from Immunoway Biotechnology Co., Ltd.; peroxidase affiniPureTM goat anti-rabbit IgG (H+L) (cat. no. 111-035-003) and peroxidase-conjugated affiniPure goat anti-mouse IgG (H+L) (cat. no. 115-035-003) were obtained from Jackson ImmunoResearch Laboratories, Inc.

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Gene Expression, Quantitation Assay

Journal: Biomedical Reports

Article Title: Rabeprazole attenuates fibrosis by modulating SMAD3 linker region phosphorylation

doi: 10.3892/br.2025.2098

Figure Lengend Snippet: TIF1γ is essential for rabeprazole-modulated ECM. (A) GES-1 and (B) AGS cells were treated with or without rabeprazole for 48 h, and the expression of TIF1γ mRNA was analyzed by reverse transcription-quantitative PCR. Data are shown as the mean ± SD and quantified by one sample t-test for significance against 0 µM. *** P<0.001; n=3. (C) Western blotting was used to detect the protein level of TIF1γ in AGS cells in the absence or presence of rabeprazole for 48 h, and the bands were quantified and analyzed using one sample t-test. Data are displayed as the mean ± SD. **** P<0.0001; n=3. (D) TIF1γ promoter plasmid combined with Renilla plasmid were co-transfected into AGS cell for 24 h, followed by treatment with or without rabeprazole for another 24 h. Relative light units were measured using the dual-luciferase reporter assay system according to the manufacturer's instructions. Data are displayed as the mean ± SD and quantified by two sample t-test for significance against 0 µM. ** P<0.01; n=3. (E) Following transfection with pooled shTIF1γ plasmids overnight, AGS cells were treated with or without rabeprazole for another 48 h, and the bands were quantified and analyzed using one sample t-test. ** P<0.01 and **** P<0.0001; n=3. TIF1γ, transcriptional intermediary factor 1γ; FN, fibronectin; Col1a1, collagen type I alpha 1 chain.

Article Snippet: Antibodies including α-SMA specific monoclonal antibody (mAb) (cat. no. 67735-1-Ig), FN mAb (cat. no. 66042-1-Ig), vimentin polyclonal antibody (pAb) (cat. no. 10366-1-AP), collagen type I (Col1a1) mAb (cat. no. 67288-1-Ig), SMAD3 mAb (cat. no. 66516-1-Ig), lamin A/C pAb (cat. no. 10298-1-AP) and α-tubulin mAb (cat. no. 66031-1-Ig) were purchased from Proteintech Group, Inc. TIF1γ mouse mAb (cat. no. YM1108), SMAD3 (phospho Ser204) rabbit pAb (cat. no. YP0363), SMAD3 (phospho Ser213) rabbit pAb (cat. no. YP0364), SMAD3 (phospho Thr179) rabbit pAb (cat. no. YP0745) and SMAD3 (phospho Ser208) rabbit pAb (cat. no. YP0746) were purchased from Immunoway Biotechnology Co., Ltd.; peroxidase affiniPureTM goat anti-rabbit IgG (H+L) (cat. no. 111-035-003) and peroxidase-conjugated affiniPure goat anti-mouse IgG (H+L) (cat. no. 115-035-003) were obtained from Jackson ImmunoResearch Laboratories, Inc.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Transfection, Luciferase, Reporter Assay