Page 69 - Physico-Chemical Niche Conditions for Bone Cells

DISCUSSION
This study aimed to investigate whether PFF affected cell and nucleus morphology and volume of osteoblasts, and how these effects relate to the actin cytoskeleton and focal adhesion formation. In 2D and 3D, we showed that PFF decreased cell and nucleus volume and cell surface area. These changes corresponded to increased fluorescence staining intensity for paxillin and integrin-a5, and altered distribution of F-actin and a-tubulin. This suggests that PFF alters osteoblast volume and nuclear volume, and that the changed morphology of osteoblasts after PFF coincides with changes in cytoskeletal and focal adhesion protein expression.
The actin and microtubule cytoskeleton are highly adaptive structures, rapidly adapting to internal and external triggers. Alterations in the osteoblast cytoskeletal structure in response to mechanical stimuli, such as shear stress, take place within minutes [27,28]. Hence, we have chosen this time point as an end point for our investigations. When primary mouse bone cells were exposed in vitro to different shear stresses that have been calculated to occur around osteocytes in situ in response to physiological loading, the production of nitric oxide (NO) and prostaglandin E2 (PGE2) was enhanced in a dose-dependent manner [29]. Increasing the shear stress magnitude and rate beyond physiological ranges leads to a catabolic response of MLO-Y4 osteocytes to fluid shear stress [9], more reminiscent of overloading. Here we have tested the effect of shear stress of a single magnitude within the physiological range to determine bone cell morphological changes. It was not our goal to test shear stresses resembling disuse or overuse, since these regimes are known to cause bone cell apoptosis and cell death [30]. PFF caused live cell deformation at different time points. The decreased live cell shape could result from actual cell shrinkage, but also from fluorescence bleaching. Fluorescence bleaching is affected by excited fluorophores and ambient light. Here, the cells stained for F-actin were exposed to the environment for only 1 h, and cells were kept in the dark between taking images to avoid fluorescence bleaching. In addition, fixed cells were only imaged once, and still showed a clear difference between static and PFF-exposed cells, suggesting that the decrease in cell volume was not an artifact. Cell shrinkage was not a result of cell death either (Appendix A2). Changes in cell and nucleus volume upon mechanical loading have been shown for chondrocytes [17], and is thus, not unprecedented.
Mechanical stimuli can alter gene expression levels, and the physical connection of the cytoskeleton to the nucleus provides an optimal conduit for signal transduction from the exterior of the cell, all the way to the nucleus. Sad1 and UNC-84 (SUN), NESPRIN, and Klarsicht, ANC-1, Syne homology (KASH) proteins form a bridge across the nuclear envelope, often referred to as the linker of nucleoskeleton and cytoskeleton (LINC) complex, connecting the nucleoskeleton to the cytoskeleton [31–35]. In our study, we showed that nucleus volume
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