Fig. 3.

MC3T3E-1 cells alignment and morphology. a Confocal laser scanning microscopy images of F-actin-stained cells, immediately (0 h) and at 24 h after stretch removal. Green: F-actin, blue: nuclei stained with DAPI, double-headed arrows: the stretch direction, scale bars: 20 μm. b Histograms for distribution of the cell alignment angles which were defined as the angles between the major axis of the cell and the stretch direction (double-headed arrows). The angles were divided into the following three groups for evaluation: pa, parallel (from 0° to 30°); md, middle (above 30°, up to 60°); pe, perpendicular (above 60°, up to 90°). Measurement was performed on three fields of view observed with a × 10 objective lens. The numbers of evaluated cells: control, 0 h = 365, 24 h = 320; continuous, 0 h = 487, 24 h = 524; cyclic, 0 h = 424, 24 h = 647. Data are presented as the ratio (%) to the total cell number. c The aspect ratio (major axis/minor axis) of MC3T3-E1 cells in each group, immediately (0 h) and at 24 h after stretching removal. The aspect ratio was higher in the cyclic group than in the other groups, which means that the cell morphology in the cyclic group was more elongated. The numbers of evaluated cells: control, 0 h = 226, 24 h = 243; continuous, 0 h = 201, 24 h = 306; cyclic, 0 h = 214, 24 h = 333. Data are presented as the mean ± SEM. *P < 0.05, One-way ANOVA followed by Bonferroni’s multiple comparison test