Page 128 - Physico-Chemical Niche Conditions for Bone Cells

Regulation of bone cell mitochondrial structure and dynamics
DISCUSSION
This study aimed to investigate whether mitochondrial network structure and dynamics were affected by PFF in single live osteoblast. We found that mitochondria movement followed the direction of PFF, and that mitochondrial track speed and displacement were affected by PFF. Mitochondrial footprint and mean network branch were also altered by PFF, especially after 50 sec of PFF. F-actin did not move in the direction of PFF. This suggests that in bone cells, PFF alters mitochondrial network structure and dynamics possibly independent from F-actin filaments.
Mechanical loading of bone is an important physical stimulus for bone tissue remodeling and adaptation, and plays a vital role in fracture repair, bone growth, and treatment of bone diseases [38]. Bone is a composite and dynamic structure made up of hydroxyapatite- collagen matrix, and a lacuno-canalicular network occupied by osteocytes, which are surrounded by interstitial fluid [39]. In vitro studies show that shear stress induced by fluid flow is a regulator of osteocyte as well as osteoblast fate and function [39,40]. Fluid flow not only changes bone cell morphology, but also alters bone cell behavior, e.g. nitric oxide production and gene expression [41,42]. Here we employed PFF to subject osteoblasts in vitro to mechanical loading. We treated cells with PFF of 6.5 Pa/s (peak shear stress rate), since we have shown previously that the osteoblast response to PFF linearly depends on the fluid shear stress rate, which depends on the stress amplitude and frequency [43,44]. Moreover, mouse osteocytes in situ have been estimated to experience shear stress up to 5 Pa [45]. In vitro studies have confirmed that this range of fluid shear stress can stimulate bone cells [35,46]. Osteocytes as well as osteoblasts respond to loading-induced fluid flow, but osteoblasts are less responsive [35]. Since cultured osteoblasts respond to fluid flow-induced shear stress, they provide a practical model to analyze the response of bone cells to shear stress. In addition, alterations in osteoblast cytoskeletal structure in response to shear stress occur within minutes [45,47]. Therefore, we have chosen 2 minutes PFF as an end point for our investigations. Primary mouse bone cells subjected to different physiological loading-induced shear stresses showed a dose-dependent increase in NO and PGE2 production [42]. Enhancing the shear stress magnitude and rate beyond physiological ranges results in a catabolic response of osteocytes to fluid shear stress [46]. In the current study, we have tested the effect of shear stress of a single magnitude within the physiological range, but not shear stresses resembling disuse or overuse, since this will cause bone cell apoptosis and cell death [48].
Dynamic observation and testing of cells, organelles, molecules, and cell collections are best accomplished by microscopy and confocal microscopy. The direct observation of organelles and molecules in the cell is even more challenging. Fluorescent molecules,
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