Journal: Scientific Reports

Article Title: Catabolic mediators from TLR2-mediated proteoglycan aggrecan peptide-stimulated chondrocytes are reduced by Lactobacillus -conditioned media

doi: 10.1038/s41598-024-68404-9

Figure Lengend Snippet: Effects of TLR2-blockade on expression levels of chondrocyte development and cartilage degradation-related genes in PG peptide-stimulated chondrocytes. Knee OA chondrocytes (N = 10) were treated with 50 ng/ml of IFNγ and 10 µg/ml of PG peptides (p16-31, p263-280 or p2379-2394), the aggrecan 32-mer peptide or Col2 peptide in the presence of 20 ng/ml of anti-TLR2 antibodies and expression levels of chondrocyte development and cartilage degradation-related genes were determined by quantitative realtime PCR. Comparison of expression levels of chondrocyte differentiation and development (HOXA11, SOX5 and RUNX2) ( A ), cartilagenous matrix (COL2A1) ( B ), and cartilage degradation (IL-6) ( C ) -related genes from chondrocytes stimulated with IFNγ and PG peptides, aggrecan 32-mer peptide and Col2 peptide with and without anti-TLR2 treated chondrocytes were normalized to GAPDH and interpreted as relative expression levels (fold change) (Y-axis). Data was shown as mean ± SEM and statistical significance was calculated by two-way ANOVA with bonferroni post hoc test (*, p < 0.0500; **, p < 0.0100; ***, p < 0.0010; ****, p < 0.0001).

Article Snippet: Isolated chondrocytes were cultured in a 48-well plate (1 × 10 5 cells/well) and stimulated with 50 ng/ml IFNγ (R&D System) with either 10 μg/ml p16-31, p263-280, p2379-2394, 32-mer or Col2 peptides (GenScript) for 30 min with or without anti-TLR2 antibodies or LCM- LS- B60.

Techniques: Expressing, Comparison

Journal: Scientific Reports

Article Title: Catabolic mediators from TLR2-mediated proteoglycan aggrecan peptide-stimulated chondrocytes are reduced by Lactobacillus -conditioned media

doi: 10.1038/s41598-024-68404-9

Figure Lengend Snippet: Effects of TLR2 blockade on cartilage-degrading enzyme production in PG peptide-stimulated chondrocytes. Knee OA chondrocytes (N = 10) were treated with 50 ng/ml of IFNγ and 10 µg/ml of PG peptides (p16-31, p263-280 or p2379-2394), the aggrecan 32-mer peptide or Col2 peptide in the presence of 20 ng/ml of anti-TLR2 antibodies. Cartilage degrading- enzymes (MMP-1, MMP-9, MMP-13, ADAMTS-4 and ADAMTS-5) and IL-6 production were determined by ELISA. ( A ) Bar graphs showing comparison of MMP-1, MMP-9, MMP-13, ADAMTS-4 and ADAMTS-5 production from IFNγ and PG peptide, aggrecan 32-mer peptide and Col2 peptide stimulation with ( +) and without (-) anti-TLR2 antibodies. Y-axis represents cartilage-degrading enzyme concentrations (pg/ml). ( B ) Bar graphs showing percentage of inhibition of MMP-1, MMP-9, MMP-13, ADAMTS-4 and ADAMTS-5 production after TLR2 blockade in each stimulation condition. ( C ) Bar graph showing comparison of IL-6 production from IFNγ and PG peptide stimulation with ( +) and without (-) anti-TLR2 antibody. Y-axis represents IL-6 concentration (pg/ml). ( D ) Bar graph showing the percentage of inhibition of IL-6 production after TLR2 blocking in IFNγ and PG peptide-stimulated chondrocytes. Data was shown as mean ± SEM and significant difference of cartilage degrading factor production was calculated by two-way ANOVA with bonferroni post hoc test, while significant difference of percentage of inhibition was calculated by one-way ANOVA with post-hoc Turky HSD (*, p < 0.0500; **, p < 0.0100; ***, p < 0.0010; ****, p < 0.0001).

Article Snippet: Isolated chondrocytes were cultured in a 48-well plate (1 × 10 5 cells/well) and stimulated with 50 ng/ml IFNγ (R&D System) with either 10 μg/ml p16-31, p263-280, p2379-2394, 32-mer or Col2 peptides (GenScript) for 30 min with or without anti-TLR2 antibodies or LCM- LS- B60.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Inhibition, Concentration Assay, Blocking Assay

Journal: Scientific Reports

Article Title: Catabolic mediators from TLR2-mediated proteoglycan aggrecan peptide-stimulated chondrocytes are reduced by Lactobacillus -conditioned media

doi: 10.1038/s41598-024-68404-9

Figure Lengend Snippet: Inhibition effect of Lactobacillus- conditioned medium on cartilage degraded mediators released from chondrocytes after IFNγ with PG peptides treatment. Chondrocytes isolated from OA patients (N = 4) were treated with 10% of Lactobacillus -conditioned medium (LCM), L. gasseri- L10 (LG-L10), L. rhamnosus-L34 (LR-L34), L. casei -L39 (LC-L39), L. salivarius -B60 (LS-B60) and L. plantarum -XB7 (LP-XB7) before stimulating with IFNγ and either PG peptides (p16-31, p263-280 and p2379-2394) or control peptides (32-mer and Col2). The cultured medium was collected for cartilage degraded mediators measurement by ELISA. ( A ) Comparison of MMP-1, MMP-9, MMP-13, ADAMTS-4, ADAMTS-5 and IL-6 production from IFNγ and PG peptides stimulated chondrocytes between LCM-treated and untreated conditions ( B ) Bar graphs show the MMP-1 and MMP-13 production in 10% LCM-treated chondrocytes (without IFNγ and PG peptides stimulation) compared with that of chondrocytes treated with 10% of heated LCM. ( C ) Bar graphs show the MMP-1 and MMP-13 production in 10% LCM-treated chondrocytes (without IFNγ and PG peptides stimulation) compared with that of chondrocytes treated with 10% of proteinase K-treated LCM. Data was shown as mean ± SEM and statistical significance was calculated by two-way ANOVA with Bonferroni post hoc test (*, p < 0.0500; **, p < 0.0100; ***, p < 0.0010; ****, p < 0.0001).

Article Snippet: Isolated chondrocytes were cultured in a 48-well plate (1 × 10 5 cells/well) and stimulated with 50 ng/ml IFNγ (R&D System) with either 10 μg/ml p16-31, p263-280, p2379-2394, 32-mer or Col2 peptides (GenScript) for 30 min with or without anti-TLR2 antibodies or LCM- LS- B60.

Techniques: Inhibition, Isolation, Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Scientific Reports

Article Title: Catabolic mediators from TLR2-mediated proteoglycan aggrecan peptide-stimulated chondrocytes are reduced by Lactobacillus -conditioned media

doi: 10.1038/s41598-024-68404-9

Figure Lengend Snippet: Effects of TLR2 blockade and Lactobacillus- conditioned media treatment on intracellular signaling activation in PG peptide-stimulated chondrocytes. Chondrocytes isolated from OA patients (N = 5) were treated with 20 μg/ml of anti-TLR2 antibodies or 10% of LCM, L. salivarius -B60 for 1 h before stimulating with IFNγ and PG peptides (p16-31, p263-280 and p2379-2394) or control peptides (32-mer and Col2 peptides). Intracellular phosphorylated proteins; p-STAT3, p-IkBα, p-ERK1/2, p-p38 and p-MAPK9 (JNK2) were determined by flow cytometry. ( A ) Bar graph showing the fold change of mean fluorescent intensity of phosphorylated proteins of stimulated chondrocyte which was divided by that of unstimulated chondrocytes. Statistical significance was calculated by one-way ANOVA with post-hoc Turky HSD. ( B ) Comparison of mean fluorescence intensity (MFI) of phosphorylated protein expression after stimulating with IFNγ and PG peptides between no blocking and anti-TLR2 antibody treatment (upper panel) or between no blocking and L. salivarius -B60 LCM treatment in each individual. Statistical significance was calculated by paired-T test. ( C ) Bar graphs showing the comparison of percentage of phosphorylated protein- expressing chondrocytes in no blocking, anti-TLR2 antibody treatment and L. salivarius -B60 LCM treatment conditions after stimulation with IFNγ and PG peptides or control peptides. Data was shown as mean ± SEM and statistical significance was calculated by two-way ANOVA with Bonferroni post hoc test (*, p < 0.0500; **, p < 0.0100; ***, p < 0.0010; ****, p < 0.0001).

Article Snippet: Isolated chondrocytes were cultured in a 48-well plate (1 × 10 5 cells/well) and stimulated with 50 ng/ml IFNγ (R&D System) with either 10 μg/ml p16-31, p263-280, p2379-2394, 32-mer or Col2 peptides (GenScript) for 30 min with or without anti-TLR2 antibodies or LCM- LS- B60.

Techniques: Activation Assay, Isolation, Control, Flow Cytometry, Comparison, Fluorescence, Expressing, Blocking Assay

Journal: Bioengineering

Article Title: Electrical Stimulation of Mesenchymal Stem Cells as a Tool for Proliferation and Differentiation in Cartilage Tissue Engineering: A Scaffold-Based Approach

doi: 10.3390/bioengineering11060527

Figure Lengend Snippet: Forward and reverse sequences of primers used for gene analysis.

Article Snippet: Samples were stained with HE and COL2 stainings using anti-collagen type II (rabbit) antibody (Rockland Immunochemicals Inc., Limerick, PA, USA) at a 1:100 dilution according to internal protocols.

Techniques:

Journal: Bioengineering

Article Title: Electrical Stimulation of Mesenchymal Stem Cells as a Tool for Proliferation and Differentiation in Cartilage Tissue Engineering: A Scaffold-Based Approach

doi: 10.3390/bioengineering11060527

Figure Lengend Snippet: ( a – d ) Show photos of histological sections. ( a , b ) HE stainings of D4 at 10×: ( a ) is the control, ( b ) is the ES group. ( c ) An HE staining of the D7 ES group at 40×; ( d ) a COL2 immunohistochemical staining from the D10 ES group at 40× magnification. ( e , f ) Show 3D renderings of stacks from two-photon imaging from D7. ( e ) Displays the ES group; ( f ) the control group. ( g ) Shows a physical image of the scaffolds at D10. The left scaffold is from the ES group, the right scaffold is from the control group.

Article Snippet: Samples were stained with HE and COL2 stainings using anti-collagen type II (rabbit) antibody (Rockland Immunochemicals Inc., Limerick, PA, USA) at a 1:100 dilution according to internal protocols.

Techniques: Control, Staining, Immunohistochemical staining, Imaging

Journal: Bioengineering

Article Title: Electrical Stimulation of Mesenchymal Stem Cells as a Tool for Proliferation and Differentiation in Cartilage Tissue Engineering: A Scaffold-Based Approach

doi: 10.3390/bioengineering11060527

Figure Lengend Snippet: Mean relative gene expression (RGE) of ( a ) ACAN, ( b ) COL1, ( c ) COL2, ( d ) SOX9, and ( e ) MMP13 across the different time points (D4, D7, D10) and conditions (control—C, stimulated—S). Bars represent the mean RGE and error bars show the standard error of the mean. Significance levels are indicated by asterisks (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: Samples were stained with HE and COL2 stainings using anti-collagen type II (rabbit) antibody (Rockland Immunochemicals Inc., Limerick, PA, USA) at a 1:100 dilution according to internal protocols.

Techniques: Gene Expression, Control