Mechanical loading by fluid shear is sufficient to alter the cytoskeletal composition of osteoblastic cells.

Many structural modifications have been observed as a part of the cellular response to mechanical loading in a variety of cell types. Although changes in morphology and cytoskeletal rearrangement have been widely reported, few studies have investigated the change in cytoskeletal composition. Measuring how the amounts of specific structural proteins in the cytoskeleton change in response to mechanical loading will help to elucidate cellular mechanisms of functional adaptation to the applied forces. Therefore, the overall hypothesis of this study was that osteoblasts would respond to fluid shear stress by altering the amount of specific cross-linking proteins in the composition of the cytoskeleton. Mouse osteoblast cell line MC3T3-E1 and human fetal osteoblasts (hFOB) were exposed to 2 Pa of steady fluid shear for 2 h in a parallel plate flow chamber, and then the amount of actin, vimentin, alpha-actinin, filamin, and talin in the cytoskeleton was measured using Western blot analyses. After mechanical loading, there was no change in the amount of actin monomers in the cytoskeleton, but the cross-linking proteins alpha-actinin and filamin that cofractionated with the cytoskeleton increased by 29% (P<0.01) and 18% (P<0.02), respectively. Localization of the cross-linking proteins by fluorescent microscopy revealed that they were more widely distributed throughout the cell after exposure to fluid shear. The amount of vimentin in the cytoskeleton also increased by 15% (P<0.01). These results indicate that osteoblasts responded to mechanical loading by altering the cytoskeletal composition, which included an increase in specific proteins that would likely enhance the mechanical resistance of the cytoskeleton.

Adaptation of cellular mechanical behavior to mechanical loading for osteoblastic cells.

Numerous cellular biochemical responses to mechanical loading are transient, indicating a cell's ability to adapt its behavior to a new mechanical environment. Since load-induced cellular deformation can initiate these biochemical responses, the overall goal of this study was to investigate the adaptation of global, or whole-cell, mechanical behavior, i.e., cellular deformability, in response to mechanical loading for osteoblastic cells. Confluent cell cultures were subjected to 1 or 2 Pa flow-induced shear stress for 2 h. Whole-cell mechanical behavior was then measured for individual cells using an atomic force microscope. Compared to cells maintained under static conditions, whole-cell stiffness was 1.36-fold (p=0.006) and 1.70-fold (p<0.001) greater for cells exposed to 1 and 2 Pa shear loading, respectively. The increase in shear stress magnitude from 1 to 2 Pa also caused a statistically significant, 1.25-fold increase in cell stiffness (p=0.02). Increases in cell stiffness were not altered in either flow group for 70 min after flow was terminated (p=0.15). Flow-induced rearrangement of the actin cytoskeleton was also maintained for at least 90 min after flow was terminated. Taken together, these findings support the hypothesis that cells become mechanically adapted to their mechanical environment via cytoskeletal modifications. Accordingly, cellular mechanical adaptation may play a key role in regulation of cellular mechanosensitivity and the related effects on tissue structure and function.