Figure 1.
Strain is efficiently transmitted to the collagen gel surface.
The longitudinal (A) and lateral (B) strains (mean ± SEM; n = 6) within the region of the silicone rubber chamber used to support the collagen gels were quantified at the surfaces of the silicone rubber sheet (black) and the collagen gel (grey).
Figure 2.
Cyclic stretch-induced SF alignment on soft collagen gels and stiff silicone rubber sheets.
A–D: Representative images and circular histograms depicting SF angular distributions of non-confluent U2OS cells (A, B) (n = 90 for each condition) and hMSCs (C, D) (n = 60 for each condition) subjected to 3 h of 10% cyclic uniaxial stretch at frequency of 1 Hz on collagen-coated rubber sheets (A, C) and collagen gels (B, D). Scale bar, 50 µm. E, F: Representative confocal reflectance images of collagen fibrils (red) in regions containing a U2OS cell (E) and devoid of cells (F) and circular histograms depicting collagen fibril alignment after 3 h of 10% cyclic stretching at 1 Hz.; Scale bar, 5 µm.
Figure 3.
Cyclic stretch-induced cell and SF alignment on soft collagen gels depends on stretch frequency.
Representative images of non-confluent U2OS cells adhered on a soft collagen matrix subjected to 3 h of 10% cyclic stretch at frequencies of 0.01 (A), 0.1 (B) and 1 Hz (C). Order parameters for cells (D) and SFs (E) were computed for each cell to quantify the extents of alignment and the results were summarized (mean ± SEM; n = 90). * indicates significant differences between groups as determined by ANOVA followed by Student-Neuman–Keuls post-hoc multiple comparison testing (P<0.01). Scale bar, 50 µm.
Figure 4.
Effects of pre-stretch and strain rate on steady stretch-induced cell and SF alignment.
Representative images of non-confluent U2OS cells seeded on 10% pre-stretched collagen gel (A) or adhered onto collagen gels (that were not pre-stretched) subjected to 10% stretch at ramp rates of 20%/s (B) and 0.2%/s (C). Representative image of U2OS cells adhered onto collagen-coated silicone rubber sheets subjected to 10% stretch at 20%/s (D). Cell (E) and SF (F) order parameters (n = 90) are summarized. * indicates significant differences between groups as determined by ANOVA followed by Student-Neuman–Keuls post-hoc multiple comparison testing (P<0.01). Scale bar, 50 µm.
Figure 5.
Extent of cell and SF alignment depends on the duration of transient step stretch.
Representative images of non-confluent U2OS cells adhered onto collagen gels subjected to 10% transient step stretch, i.e. a regimen consisting of rapid ramp increase in stretch, a hold, and subsequent release of the stretch. The collagen gels were subjected to 10% stretch and held for 1 s (A), 10 min (B) and 1 h (C). Cell (E) and SF (F) order parameters (n = 90) are summarized. Significant differences between groups were determined by ANOVA followed by Student-Neuman–Keuls post-hoc multiple comparison testing (* = P<0.01, ** = P<0.05). Scale bar (A–C), 50 µm.
Figure 6.
Roles of Rho-kinase and MLCK on cyclic stretch-induced SF alignment in cells on 3-D collagen gels.
Representative images of non-confluent U2OS cells (n = 60) adhered on soft collagen gels subjected to 3 h of 10% cyclic uniaxial stretch at 1 Hz after treatment with 30 µM ML7 (A) or 10 µM Y27632 (B). Scale bar, 50 µm.
Figure 7.
Time-lapse images of a U2OS cell expressing GFP-actin subjected to subjected to 10% stretch at ramp rates of 20%/s. Imaging began immediately after the collagen hydrogel was stretched, with subsequent images captured at 10 min intervals for 2 h. Scale bar, 5 µm.