Page 72 - Physico-Chemical Niche Conditions for Bone Cells

Shear stress modulates osteoblast cell and nucleus shape
randomly oriented and smaller [41]. Moreover, nesprin 1 and 2 interact with actin filaments and SUN protein, while mechanical stimuli have been shown to be transmitted from the cytoskeleton to the nuclear skeleton via SUN protein [31]. Thus, osteoblasts adapt to external influences, such as mechanical stimuli by changes in F-actin, which affects cell morphology and function, and possibly affected nucleus volume (Figure 7B). The morphological changes did not hold once the force was removed. We observed that cell morphological changes returned to normal in PFF-treated cells at 4 h after removal of PFF, as PFF-treated cells showed similar morphological features as static control cells at this time point (Appendix A3). Mechanical loading-induced changes in cell and nucleus morphology may drive signaling pathways affecting osteogenic differentiation and bone cell function [49]. Future studies should investigate how long the morphological changes of PFF-treated cells return to normal after removal of mechanical loading, and the relationship of morphological changes and cell function.
The osteoblastic stress response to mechanical stimulation likely involves microtubules that contain a-microtubulin and β-microtubulin, which make up the structural core [50]. Microtubule depolymerization and polymerization in intact cells depend on whether cells are in homeostasis or unstable, moving, in the mitotic stage, or whether they are subjected to external stimuli, e.g., mechanical loading [50,51]. Moreover, microtubules switch quickly between being stably growing and rapidly shrinking [52], which allows them to adapt [52]. In this study, immunofluorescence followed by LSCM revealed that PFF affected microtubule distribution and fluorescence intensity. Nesprin 4 gene expression was decreased by PFF. Nesprin 4 is known to interact with microtubules and SUN protein [31]. Mechanical loading of osteoblasts, thus, likely caused rapid depolymerization and repolymerization of microtubules. PFF altered both actin filaments and microtubules, which likely contributed to changes in cell and nucleus volume. The sequence of PFF-induced changes in polymerization and depolymerization of both microtubules and actin filaments, and the exact impact of these cytoskeletal changes on cell morphology remain to be determined (Figure 5B).
Both paxillin and integrin staining intensity was stronger after PFF-treatment. When paxillin expression is up-regulated in embryonic stem cells, the cell shape turns less round [53]. The currently observed changes in nucleus volume may, thus, be related to alterations in paxillin. In our study, the focal adhesion (paxillin) area was increased by PFF. This data suggests that focal adhesions are indeed important for PFF-induced mechanotransduction in osteoblasts, as has been reported by Young and colleagues [54]. Interestingly, soft stiffness substrates promote the appearance of osteocyte-line features in MC3T3-E1 cells seeded at low density [55]. Soft substrates are generally associated with smaller focal adhesion size. On the other hand, substrates of higher stiffness enhance Rho activity, F-actin formation, focal adhesion size, and osteogenic differentiation of mesenchymal stem cells (MSCs) compared to
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