Page 76 - Physico-Chemical Niche Conditions for Bone Cells

Shear stress modulates osteoblast cell and nucleus shape
Appendix A1
Figure A1. Focal adhesions visualized by staining of paxillin in MC3T3-E1 cells cultured without or with 1 h PFF. Line tool (Image J) was used to draw the target area. The “Reslice” function was used, and the following parameters applied: Output spacing (microns): 0.125, Slice count: 1, Avoid interpolation (use 1 pixel spacing). (A) Paxillin morphology in control cells without PFF. Bar: 20 μm. (B) Paxillin morphology in PFF-treated cell. Bar: 20 μm. (C) Side view of the paxillin staining based on the white dotted line in (A). Bar = 10 μm. (D) Side view of paxillin based on the white dotted line in (B). Bar: 10 μm. (E) Side view of the paxillin staining based on the red dotted line in (A). Bar: 5 μm. (F) Side view of the paxillin staining based on the red dotted line in (B). Bar: 5 μm. CON, control; PFF, pulsating fluid flow.
Appendix A2
A potential cytotoxic effect of different culture media before and after PFF on MC3T3-E1 osteoblasts was assessed by measuring lactate dehydrogenase (LDH; Roche, Mannheim, Germany). Prior to 1 h PFF treatment, the medium of MC3T3-E1 osteoblast cultures was refreshed by a-MEM containing 2% FBS, 300 μg/mL penicillin, 250 μg/mL streptomycin, and 1.25 μg/mL fungizone. After 1 h of culture, the medium was collected and replaced by a-MEM containing 10% FBS, 0.1% penicillin, 0.1% streptomycin, and 0.5% fungizone. After 1 h with or without PFF treatment, the medium was also collected. The medium of all samples (100 μl) were pipetted into 96-well plates and incubated with 100 μl reaction mixture for 20 min at room
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