Figure 4.

Effects of PACAP and/or oxidative stress and/or mechanical stress (MS) on metachromatic cartilage formation. Metachromatic cartilage areas in six-day-old cultures were visualized with DMMB dissolved in 3% acetic acid. Metachromatic (purple) structures represent cartilaginous nodules formed by many cells and a cartilage matrix rich in polyanionic GAG chains. Original magnification was 4×. Scale bar, 500 µm. Optical density (OD₆₂₅) was determined in samples containing toluidine blue (TB) extracted with 8% HCl dissolved in absolute ethanol. Absorbance and means of three independent experiments (±standard error of the mean) are shown in the figures. Asterisks indicate significant differences compared with the six-day cultures (p < 0.05, one-way ANOVA followed by Tukey’s HSD test). * shows significant differences between Control cultures and treated samples; ** shows significant differences between single-H₂O₂ treatment and combined treatments.