Page 65 - Physico-Chemical Niche Conditions for Bone Cells

along the solid white line visible in Figure 3E. Bar = 50 μm. (H) Nesprin 1 gene expression in control and PFF-treated cells. (I) Nesprin 2 gene expression in control and PFF-treated cells. CON, control; PFF, pulsating fluid flow.
Cell Shape and Area
Static control cells were more oval-shaped, while PFF-treated cells seemed more polygonal- shaped (Figure 2A, B). To further investigate cell spreading, cell area, length, and width were measured. PFF significantly decreased cell surface area by 0.3-fold, indicating differences in cell adhesion surface area due to PFF. The ratio of the major axis (length) versus the minor axis (width) was similar in control and PFF-treated cells (Figure 2D).
Cell Morphology
The total vertical fluorescence signal span was higher (i.e., the distance over which green fluorescent signal was visible in the Z direction) in PFF-treated cells than in static control cells (Figure 3), as could be seen from the signal appearance from Z = 2 to 22 μm in the representative PFF-treated cell versus Z = 8 to 22 μm in the representative control cell (Figure 3B, C). Control cells showed green fluorescence at some distance from the nucleus in the cell periphery when viewed in Z-direction (Figure 2A, 3D). In the XZ and YZ direction, little green fluorescence spots/loci were visible near the nucleus (Figure 3D). Importantly, PFF affected F-actin distribution, since green fluorescence tended to surround the entire nucleus, when cells were viewed in the Z direction (Figure 2B, 3E). In the XZ and YZ direction, the results were consistent with the top view (Figure 3E). F-actin fluorescence intensity of the control cell (60 pixels, 4.9 pixels/μm) was different from that of the PFF-treated cell (100 pixels) (Figure 3F, G). Nesprin 1 (1.52-fold, p = 0.61, n = 3) and nesprin 2 (1.50-fold, p = 0.57, n = 3) gene expression seemed slightly (not significant) up-regulated by PFF (Figure 3H, I).
Expression of Paxillin
To explore cytoarchitectural changes in MC3T3-E1 osteoblasts treated with 1 h PFF compared to static controls, paxillin gene and protein expression levels were measured. PFF did not enhance mRNA expression levels of PXNA (p = 0.053, n = 7), or PXNB (p = 0.14, n = 7) (Figure 4A, B). Immunofluorescent paxillin staining revealed clear clusters at the cell boundary of control cells (Figure 4C, D). PFF resulted in more paxillin dots, resembling short rods (Figure 4E, F, G, H, and Appendix A1). This was confirmed by a significant 2.3-fold increase in paxillin fluorescence intensity and a 4.3-fold increase in paxillin area per cell after PFF, while paxillin number and average size remained unchanged (Figure 4).
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