Page 129 - Physico-Chemical Niche Conditions for Bone Cells

proteins, and fluorescently labeled antibodies have become indispensable for the observation of organelles using high contrast and resolution, which results in advanced understanding of organelle dynamics [49]. To assess how mitochondria change their morphology and function, we chose MitoTracker and SiR-actin to stain live cell mitochondria and actin filaments, respectively. In our study, we studied live cells, that still might be spreading, proliferating, or differentiating. This means that we studied the cells in different stages with different morphology, and likely also with different distribution of mitochondrial network structure. In addition, earlier we showed using finite element modeling that the distribution of fluid velocity, fluid pressure, and fluid shear stress on the bone cell membrane and cytoplasm was affected during the initial stage of mechanical loading, especially at the first cycle (Jin et al. accepted by Physiological Reports). Therefore, a movie was made during 2 min showing the effect of the first, second, and third cycle of PFF within the cell. During the 2 min PFF treatment, mitochondrial track speed, displacement, footprint and mean network branch were affected, which might change the function of mitochondria, e.g. reactive oxygen production and respiratory property of the electron transport chain [50].
To support mitochondrial diverse functions, including migration, contraction, cell division, and response to physical and chemical cues from the environment, mitochondria form an extensive and complicated network with small clusters, which move along the cytoskeletal filaments with the aid of motor proteins [51]. Furthermore, the mitochondrial network is highly dynamic where the network is constantly undergoing alteration including fission and fusion [52]. These processes are important for the integrity of the cell [15]. Therefore, we investigated the movement of mitochondria including track speed and displacement in cells treated by PFF. The values of track speed or track displacement were different between pre and post PFF (mitochondrial track speed ranged from 0-14.9 μm/s, and displacement from 0-62.7 μm). This may be explained by the fact that many mitochondria within the cell formed a complex network, e.g. spots, rods, and branches. The size of those structures was different in cells with and without PFF treatment, as well as the density of mitochondria. In pre and post PFF, there were clearly more mitochondria surrounding the nucleus compared to the number of mitochondria in the area of the cell membrane. We found differences in footprint and mean network branch in 3 cells pre and post PFF, i.e. PFF increased mitochondrial footprint as well as mitochondrial network branch by 1.1-fold (post PFF/pre PFF). These differences might be explained by superoxide production within the cell, as these are stimulated by external force applied to cells [53]. This could have resulted in cell shrinkage to decrease the cell volume, as well as the mitochondrial size. After adaptation to the mechanical loading, the mitochondria moved in the direction of PFF, especially away from the nucleus. The extend of movement of mitochondria is likely restricted by cytoskeletal
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