Page 60 - Physico-Chemical Niche Conditions for Bone Cells

Shear stress modulates osteoblast cell and nucleus shape
μm in a whole cell. Fluorescence intensity (the value of Integrated Density. Because osteoblasts are flat, raw Integrated Density might have a similar level of fluorescence calculated by CTCF (Corrected total cell fluorescence = Integrated Density - (Area of selected cell × Mean fluorescence of background readings)) as measured by Image J software (https://imagej.net/Downloads). For F-actin-stained cells, cell boundaries were drawn using the toolbar, and cell area, length, and width were determined. F-actin fluorescence in the Z direction was visualized via Image J software. Paxillin and integrin-a5 number, area, and average size were also determined using Image J software (image type: 8-bit; threshold: 40/255; size: 5-infinity; circularity: 0.00−1.00). Fifty-two (control) and 47 (PFF) cells from six glass slides from 3 independent experiments (n = 3) were measured.
To observe the live cell morphology before and after PFF, MC3T3-E1 osteoblasts seeded on poly-L-lysine coated glass slides were stained by SiR-actin (Spirochrome, Stein-am-Rhein, Switzerland) for F-actin in a culture medium with 10% FBS for 4 h before the start of PFF. Live cell images were captured by SP8 confocal microscopy (Leica, Solms, Germany) at 0, 10, 20, 30, 40, 50, and 60 min after the start of PFF. Four cells were imaged per glass slide. In total, six glass slides from six independent experiments were analyzed.
Cell and Nucleus Volume
LSCM images were used to select the target cell by utilizing Image J software. Cell threshold data were set to clearly display the cell boundary to reconstruct cell and nucleus shape and to calculate their volume. Afterward, cell and nucleus volume were quantified by MeasureStack software (http://www.optinav.info/MeasureStack.htm). MITK software was utilized to reconstruct the 3D-shape of the cell and nucleus (http://www.mitk.net/download_mitk_ch1.html). The fold-change in cell and nuclear volume was quantified by this equation (mean volume CON - mean volume PFF)/mean volume CON).
RNA Isolation and Quantitative Real Time Polymerase Chain Reaction (RT-PCR)
Total RNA was isolated from MC3T3-E1 osteoblasts using 700 μl Trizol (Invitrogen, Carlsbad, CA, USA). Complementary DNA synthesis was performed using 750 ng of total RNA according to the First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Vilnius, Lithuania) in a thermocycler GeneAmp® PCR System 9700 PE (Applied Biosystems, Foster City, CA, USA). cDNA was diluted to a final concentration of 2 ng/μL for gene expression analysis. RT- PCR reactions were performed using 4 μl cDNA per reaction, 0.5 μl forward-primer (1 μM), 0.5 μl reverse-primer (1 μM), and 5 μl LightCycler® 480 SYBR® Green I Mastermix (Roche Diagnostics, Mannheim, Germany) in a LightCycler® 480 (Roche Diagnostics, Mannheim, Germany). RT-PCR conditions for all genes were as follows: 10 min pre-incubation at 95°C,
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