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Compressive Force Induces Osteoblast Apoptosis via Caspase-8

¹ Division of Orthodontics and Dentofacial Orthopedics and
² Division of Oral Dysfunction Science, Department of Oral Health and Development Sciences, School of Dentistry, Tohoku University, 4-1 Seiryo-cho, Aoba-ku, Sendai 980-8575, Japan; and
³ Shimizu Orthodontic Clinic, 6-1, 2F ESTA-Build., Sakae-cho, Fukushima 960-8031, Japan

View larger version (75K): [in a new window]   Figure 1. Morphology and viability were changed by compressive force. Phase-contrast microscopic appearance of MG-63 cells under compressive force for 24 hrs. Bar = 100 µm. The compressive force resulted in a change of the morphology of MG-63 cells. The arrangement of the cells became irregular, and several spaces were observed between cells, as compared with control cells. As the compressive force became greater, some atrophic cells began to appear. (A) Control; (B) 1.02 x 10–4 N/cm2; (C) 2.04 x 10–4 N/cm2; (D) 4.08 x 10–4 N/cm2; and (E) 6.12 x 10–4 N/cm2. (F) Viability of MG-63 cells under compressive force. Values represent the mean ± SD of the relative absorbance of 3 experimental samples (exp.) per control sample (cont.). Cell viability was decreased at all levels of compressive force applied for 12 hrs, and there was no significant difference between cell viability at 12 and that at 24 hrs. Greater compressive force tended to inhibit cell viability to a greater extent. Significantly different from controls: * p < 0.05 (Bonferroni/Dunn method); a, vs. control; b, vs. 6.12 x 10–4 N/cm2 (n = 3).

View larger version (47K): [in a new window]   Figure 2. Apoptosis detection by the TUNEL method with compressive force application for 24 hrs in MG-63 cells. The apoptotic cells among MG-63 cells under compressive force were detected by the TUNEL method. Stained nuclei were observed in some cells under compressive force, but no stained nuclei were seen in control cells. (A) Control, magnification, x200; (B) cells under 2.04 x 10–4 N/cm2 of compressive force, x200; (C) cells under 2.04 x 10–4 N/cm2 of compressive force, x400. Bar = 100 µm. (D) Percentage of apoptotic cells induced by compressive force in MG-63 cells. Values represent the mean ± SD of the relative absorbance of 3 experimental samples (exp.) per control sample (cont.). Percentages were significantly different from controls at all forces except 1.02 x 10–4 N/cm2. At 2.04 x 10–4 and 4.08 x 10–4 N/cm2 of compressive force, the percentage of apoptotic cells increased between 12 and 24 hrs. The percentage of apoptotic cells increased in a force-dependent manner. Significantly different from controls: * p < 0.05 (Bonferroni/Dunn method); a, vs. control; b, vs. 1.02 x 10–4 N/cm2; c, between 12 hrs and 24 hrs (n = 3).

View larger version (15K): [in a new window]   Figure 3. Caspase-3 activity increased via the caspase-8 signaling cascade. (A) Force-dependent caspase-3 induction. Relative absorbance of caspase-3 is shown following the application of a compressive force of 2.04 x 10–4 or 4.08 x 10–4 N/cm2 for 24 hrs. Values represent the mean ± SD of the relative absorbance of 3 experimental samples (exp.) per control sample (cont.). Caspase-3 activity in the cells increased markedly under continuous compressive forces of 2.04 x 10–4 and 4.08 x 10–4 N/cm2. Caspase-3 activity increased with greater compressive force. Significantly different from controls at each weight: ** p < 0.01 (Bonferroni/Dunn method) (n = 3). (B) Effects of caspase-8 and -9 inhibitors on caspase-3 activity. Relative absorbance of caspase-3 activity is shown following the application of a compressive force of 4.08 x 10–4 N/cm2 for 24 hrs. To examine the signal pathway of the compressive-force-induced caspase-3 activation, we treated the cells with an inhibitor specific for caspase-8 (LETD-CHO) or caspase-9 (LEHD-CHO) before applying the compressive force, and then measured caspase-3 activity in the cells. The caspase-8 inhibitor significantly reduced the compressive-force-induced caspase-3 activation, while the caspase-9 inhibitor did not. Values represent the mean ± SD of the relative absorbance of 3 experimental samples (exp.) per control sample (cont.). ** p < 0.01 (Student’s t test). White columns, non-compressive force; black columns, the application of a compressive force of 4.08 x 10–4 N/cm2 (n = 3).