Journal: Cartilage

Article Title: Regenerative Variability of Human Juvenile Chondrocyte Sheets From Different Cell Donors in an Athymic Rat Knee Chondral Defect Model

doi: 10.1177/19476035241277946

Figure Lengend Snippet: Analysis of image stained with anti-type 2 collagen antibody using Image J. The area of the entire implanted cell sheet regenerative area ( A ); and the positive cell area ( B ). % COL2 area is shown as the percentage of ( B ) to ( A ). Bars: 500 μm.

Article Snippet: Polyclonal goat anti-COL1 (1:200, SouthernBiotech, Birmingham, USA), monoclonal mouse anti-COL2 (1:200, 2B1.5, ThermoFisher, USA), and monoclonal rabbit anti-hVIM (1:200, SP20, Abcam) were used as primary antibodies.

Techniques: Staining

Journal: Cartilage

Article Title: Regenerative Variability of Human Juvenile Chondrocyte Sheets From Different Cell Donors in an Athymic Rat Knee Chondral Defect Model

doi: 10.1177/19476035241277946

Figure Lengend Snippet: Scores of each system in all samples. The scores were arranged in order of modified O’Driscoll score. Bars sharing the same pattern represent samples from the same donor. Sample numbered 1, 3, and 5 represent the defect groups. ( A ) modified O’Driscoll score, ( B ) %COL1 area, ( C ) %COL2 area, and ( D ) COL1/2 ratios.

Article Snippet: Polyclonal goat anti-COL1 (1:200, SouthernBiotech, Birmingham, USA), monoclonal mouse anti-COL2 (1:200, 2B1.5, ThermoFisher, USA), and monoclonal rabbit anti-hVIM (1:200, SP20, Abcam) were used as primary antibodies.

Techniques: Modification

Journal: Cartilage

Article Title: Regenerative Variability of Human Juvenile Chondrocyte Sheets From Different Cell Donors in an Athymic Rat Knee Chondral Defect Model

doi: 10.1177/19476035241277946

Figure Lengend Snippet: Scatter plot of the correlation between modified O’Driscoll score and %COL 1 area ( A ), %COL 2 area ( B ), and COL2/1 ratio ( C ). Modified O’Driscoll score correlated significantly with each score (P<0.01).

Article Snippet: Polyclonal goat anti-COL1 (1:200, SouthernBiotech, Birmingham, USA), monoclonal mouse anti-COL2 (1:200, 2B1.5, ThermoFisher, USA), and monoclonal rabbit anti-hVIM (1:200, SP20, Abcam) were used as primary antibodies.

Techniques: Modification

Journal: Cartilage

Article Title: Regenerative Variability of Human Juvenile Chondrocyte Sheets From Different Cell Donors in an Athymic Rat Knee Chondral Defect Model

doi: 10.1177/19476035241277946

Figure Lengend Snippet: Comparison of each score between groups. ( A ) modified O’Driscoll score, ( B ) %COL1 area, ( C ) %COL2 area, ( D ) COL2/1 ratio. *<0.01, **<0.05.

Article Snippet: Polyclonal goat anti-COL1 (1:200, SouthernBiotech, Birmingham, USA), monoclonal mouse anti-COL2 (1:200, 2B1.5, ThermoFisher, USA), and monoclonal rabbit anti-hVIM (1:200, SP20, Abcam) were used as primary antibodies.

Techniques: Comparison, Modification

Journal: Science advances

Article Title: Bioenergetic-active exosomes for cartilage regeneration and homeostasis maintenance.

doi: 10.1126/sciadv.adp7872

Figure Lengend Snippet: Fig. 4. Bioenergetic-active exosomes promotes BMSC chondrogenic differentiation and chondrocyte’s homeostasis maintenance through the P2X7-mediated path- way. (A) Hematoxylin and eosin (H&E) (top)–, Toluidine blue (middle)–, Alcian blue (bottom)–stained sections of BMSC microspheres cocultured with N-EXO and Suc-EXO for 28 days. (B) Relative mRNA level of chondrogenic differentiation gene after stimulated by N-EXO and Suc-EXO for 7 days (bottom), 14 days (middle), and 21 days (top) in chondro- genic differentiation medium. (C) Relative mRNA level of Col2, ACAN, MMP9, and MMP13 of BMSCs in common medium. (D) The protein levels of MMP13, MMP9, and Col2 in BMSCs quantified by Western blot. (E) Relative mRNA level of PI3K (top), AKT (middle), and mTOR (down) after incubation with N-EXO, N-EXO, and KN62, Suc-EXO or Suc-EXO and KN62. (F) The protein levels of P2X7, P-mTOR, mTOR, P-S6K, S6K, P-AKT, and AKT in BMSCs quantified by Western blot. (G to I) Relative mRNA level of Col2 (G), ACAN (H), and MMP13 (I) in chondrocytes. (J) The protein levels of MMP13 and Col2 in chondrocytes quantified by Western blot. (K and L) Relative mRNA level of SIRT3 (K) and AMPK (L) in chondrocytes. (M) The protein levels of P2X7, SIRT3, p-AMPK, and AMPK in chondrocytes quantified by Western blot. Results in (B), (C), (E), (G) to (I), and (K) and (L) represent the mean ± SD (n = 3 biological repeats). Statistical analysis: *P < 0.05, **P < 0.01, and ***P < 0.001, analyzed using ANOVA with Tukey’s multiple comparisons test.

Article Snippet: 10, eadp7872 (2024) 18 October 2024 16 of 18 Proteintech, China), anti- MMP13 (1:1000; rabbit polyclonal, Proteintech, China), anti- Col2 (1:1000; rabbit polyclonal, Proteintech, China), anti–phospho- AMPK (1:1000; rabbit polyclonal, Proteintech, China), anti- SIRT3 (1:1000; rabbit polyclonal, ABclonal, China), anti- AMPK (1:1000; rabbit polyclonal, Proteintech, China), anti- P2RX7 (1:1000, rabbit polyclonal, ABclonal, China), anti- AKT(1:1000; rabbit monoclonal, Cell Signaling Technology (CST), USA), anti–phospho- AKT (Ser473) (1:2000; rabbit monoclonal, CST, USA), anti- calnexin (1:1000; rabbit polyclonal, Proteintech, China), anti- S6K (1:2000; rabbit monoclonal, Proteintech, China), anti–phospho- S6K (Thr389) (1:1000; rabbit monoclonal, CST, USA), anti–β- ACTIN (1:20,000; mouse monoclonal, Proteintech, China), and anti- GAPDH (1:50,000, mouse monoclonal, Proteintech, China).

Techniques: Staining, Western Blot, Incubation

Journal: Science advances

Article Title: Bioenergetic-active exosomes for cartilage regeneration and homeostasis maintenance.

doi: 10.1126/sciadv.adp7872

Figure Lengend Snippet: Fig. 7. Immunohistochemical and immunofluorescence assessment of cartilage regeneration. (A) Immunohistochemical staining for Col2, ACAN, MMP9, and MMP13. Relative staining intensity of ACAN (B), Col2 (C), MMP9 (D), and MMP13 (E) in cartilage samples at 6 and 12 weeks. (F) Immunofluorescence staining for SIRT3 and Col2 at 12 weeks. Results in (B) to (E) represent the mean ± SD (n = 3 technical repeats). Statistical analysis: **P < 0.01 and ***P < 0.001, analyzed using ANOVA with Tukey’s multiple comparisons test.

Article Snippet: 10, eadp7872 (2024) 18 October 2024 16 of 18 Proteintech, China), anti- MMP13 (1:1000; rabbit polyclonal, Proteintech, China), anti- Col2 (1:1000; rabbit polyclonal, Proteintech, China), anti–phospho- AMPK (1:1000; rabbit polyclonal, Proteintech, China), anti- SIRT3 (1:1000; rabbit polyclonal, ABclonal, China), anti- AMPK (1:1000; rabbit polyclonal, Proteintech, China), anti- P2RX7 (1:1000, rabbit polyclonal, ABclonal, China), anti- AKT(1:1000; rabbit monoclonal, Cell Signaling Technology (CST), USA), anti–phospho- AKT (Ser473) (1:2000; rabbit monoclonal, CST, USA), anti- calnexin (1:1000; rabbit polyclonal, Proteintech, China), anti- S6K (1:2000; rabbit monoclonal, Proteintech, China), anti–phospho- S6K (Thr389) (1:1000; rabbit monoclonal, CST, USA), anti–β- ACTIN (1:20,000; mouse monoclonal, Proteintech, China), and anti- GAPDH (1:50,000, mouse monoclonal, Proteintech, China).

Techniques: Immunohistochemical staining, Immunofluorescence, Staining

Journal: Tissue Engineering and Regenerative Medicine

Article Title: Towards Clinical Translation of In Situ Cartilage Engineering Strategies: Optimizing the Critical Facets of a Cell-Laden Hydrogel Therapy

doi: 10.1007/s13770-022-00487-9

Figure Lengend Snippet: Minimum expansion time frame to obtain chondrogenic hADSCs from the isolation phase. A Representative brightfield images of cryosections from pellet culture stained with SafraninO (in pink) and Haematoxylin (in purple) to identify cell’s nuclei. B Representative confocal images of cryosections from pellet immunostained with Phalloidin-RFP (Actin, in red), Collagen type 2 (Col 2, in cyan) and counterstained to detect cells nuclei (DAPI, in white). Superimposed channels are shown in the last row of panels (MERGE). For all the stainings, the cryosections were obtained from cells pelleted after 5 and 7 days of non-passaged proliferation, pushed into 3 weeks of chondrogenic differentiation. The calculated Collagen type 2 intensity from day 0 to day 21 in reported as Fold Increase (F.I.) in the day 21 panels of their corresponding groups

Article Snippet: After washing, samples were dropped in blocking solution (10% goat serum diluted in PBT) for 60 min and then incubated overnight at 4 °C with mouse anti-human Collagen type 2 (Col2) (#II6B3, DSHB) and goat anti-human Collagen type 1 (Col1) (# sc8784, Santa Cruz Biotechnology).

Techniques: Isolation, Staining

Journal: Tissue Engineering and Regenerative Medicine

Article Title: Towards Clinical Translation of In Situ Cartilage Engineering Strategies: Optimizing the Critical Facets of a Cell-Laden Hydrogel Therapy

doi: 10.1007/s13770-022-00487-9

Figure Lengend Snippet: Assessment of minimal hADSCs concentration required for chondrogenesis in cell-laden hydrogel bioscaffolds. A Representative confocal images of cryosections from bioscaffolds from the 3 different hADSCs/ml groups, assessed using immunostaining for DAPI, Actin and Collagen type 2 (Col 2). The cryosections has been obtained by cutting the samples along the z axis to provide spatial information from the top to the bottom of the bioscaffolds. B Superimposed high magnification of the selected white square areas (I and II) in ( A ). C The graphs show the quantification expressed as fold change relative to 1.25 hADCSs/ml group at day 21 post chondrogenesis. The Collagen type 2 (Col 2) intensity signal was calculated and averaged from 16 different ROI from the Collagen II stained cryosections (see Figure S7). D The graphs show the quantification of Glycosaminoglycan (GAG) content measured via the normalisation of GAG over total DNA present in the processed bioscaffolds and expressed as fold change relative to 1.25 hADSCs/ml group at day 21 post chondrogenesis. E Chondrogenic gene expression analysis: the bar graphs represent the fold changes calculated with 2 ΔΔCΤ method of Collagen type 2A1 (COL2A1), Aggrecan (ACAN), Sox9 (SOX9) and Collagen type 1A2 (COL1A2) markers in RT-qPCR assay, relative to 1.25 hADSCs/ml group at day 21 post chondrogenesis. GAPDH was used as the housekeeping gene. Graph bars represent standard error margin between three biological replicates. Statistical analysis was performed using an unpaired t-test

Article Snippet: After washing, samples were dropped in blocking solution (10% goat serum diluted in PBT) for 60 min and then incubated overnight at 4 °C with mouse anti-human Collagen type 2 (Col2) (#II6B3, DSHB) and goat anti-human Collagen type 1 (Col1) (# sc8784, Santa Cruz Biotechnology).

Techniques: Concentration Assay, Immunostaining, Staining, Expressing, Quantitative RT-PCR

Journal: Tissue Engineering and Regenerative Medicine

Article Title: Towards Clinical Translation of In Situ Cartilage Engineering Strategies: Optimizing the Critical Facets of a Cell-Laden Hydrogel Therapy

doi: 10.1007/s13770-022-00487-9

Figure Lengend Snippet: In situ stem cells-laden hydrogel therapy in a rabbit in vivo cartilage repair model. A Representative macroscopic pictures (Macro) and images from Haematoxylin and Eosin (H&E) stained paraffin sections from explanted samples of the indicated groups. Representative confocal images of paraffin sections from explanted samples of the indicated groups assessed using immunostaining for Collagen type 2 (Col 2, in cyan) and Collagen type 1 (Col 1, in red). Overimposed images of the two channels are shown in the Merge raw. B The graph shows the macroscopic score using the International Cartilage Repair Society (ICRS) system for the indicated groups, calculated at the end of the 8 weeks study on the explants. C The graph shows the microscopic score calculated at the end of the 8 weeks study on the HandE and Col1 and 2 stained paraffin sections. D The graph shows the percentage of the Collagen 1 (Col 1) and Collagen 2 (Col 2) positive areas. Graph bars represents the mean with standard deviation of 4 different regions along the entire diameter of the defect for each sample analysed calculated at the end of the 8 weeks study on the immunostained paraffin sections. E The graph shows the biomechanical evaluation using atomic force microscopy calculated at the end of the 8 weeks study on the explants and expressed as Young Modulus (kPa kilopascals). Statistical analysis was performed using unpaired t-test

Article Snippet: After washing, samples were dropped in blocking solution (10% goat serum diluted in PBT) for 60 min and then incubated overnight at 4 °C with mouse anti-human Collagen type 2 (Col2) (#II6B3, DSHB) and goat anti-human Collagen type 1 (Col1) (# sc8784, Santa Cruz Biotechnology).

Techniques: In Situ, In Vivo, Staining, Immunostaining, Standard Deviation, Microscopy