Figure 4. Extracellular ATP signaling via P2X₄ receptors mediates ES-driven ATP oscillations, prechondrogenic condensation, and chondrogenesis.

(a) Real-time gene expression analyses of P2X₄ receptors in micromass cultures of MSCs after culture for 3 days in maintenance medium (control) or maintenance medium under ES. Data are presented as means ± S.D. and differences were identified using Students t-test, *p < 0.05 (b) Effects of 5-BDBD on ES-driven ATP oscillations; MSCs in micromass culture were treated with 5-BDBD after induction of P_ACTIN-Luc oscillations by ES. Bioluminescence monitoring was performed after application of ES to MSCs (time = 0 h). (c) Effects of apyrase and 5-BDBD on ES-driven MSC condensation; MSCs in micromass culture were examined using phase contrast images after 3 days culture in maintenance medium (control), with ES (ES), with ES plus apyrase (ES + apyrase), or with ES plus 5-BDBD (ES + 5-BDBD); Scale bars, 500 μm. (d) Suppressive effects of apyrase and 5-BDBD on ES-induced type II collagen (COL2A1), aggrecan (AGC), and Sox9 expression. After 3 days culture in maintenance medium (control), with ES (ES), with ES plus apyrase (ES + apyrase), or with ES plus 5-BDBD (ES + 5-BDBD), gene expression was analyzed in MSCs using real-time PCR. Data are presented as means ± S.D. and differences were identified using ANOVA; **p < 0.01, *p < 0.05.