Correlation of cell strain in single osteocytes with intracellular calcium, but not intracellular nitric oxide, in response to fluid flow

Abstract

Osteocytes compose 90-95% of all bone cells and are the mechanosensors of bone. In this study, the strain experienced by individual osteocytes resulting from an applied fluid flow shear stress was quantified and correlated to two biological responses measured in real-time within the same individual osteocytes: (1) the upregulation of intracellular calcium and (2) changes in intracellular nitric oxide. Osteocyte-like MLO-Y4 cells were loaded with Fluo-4 AM and DAR-4M and exposed to uniform laminar fluid flow shear stresses of 2, 8, or 16 dyn/cm(2). Intracellular calcium and nitric oxide changes were determined by measuring the difference in fluorescence intensity from the cell's basal level prior to fluid flow and the level immediately following exposure. Individual cell strains were calculated using digital image correlation. MLO-Y4 cells showed a linear increase in cell strain, intracellular calcium concentration, and nitric oxide concentration with an increase in applied fluid flow rate. The increase in intracellular calcium was well correlated to the strain that each cell experienced. This study shows that osteocytes exposed to the same fluid flow experienced a range of individual strains and changes in intracellular calcium and nitric oxide concentrations, and the changes in intracellular calcium were correlated with cell strain. These results are among the first to establish a relationship between the strain experienced by osteocytes in response to fluid flow shear and a biological response at the single cell level. Mechanosensing and chemical signaling in osteocytes has been hypothesized to occur at the single cell level, making it imperative to understand the biological response of the individual cell.

Figure 1

For the application of fluid flow to the cells, a closed system, parallel plate, live-cell micro-observation chamber (Focht Chamber System 2, Bioptechs Inc., Butler, PA) was utilized. (A) Exploded view of the chamber: 1-heater, 2-upper half of the chamber, 3-perfusion tubes, 4-upper gasket, 5-microaqueduct slide, 6-lower gasket, 7-coverslip, 8-lower half of the chamber which locks to the microscope stage. (B) Illustration of the microaqueduct slide perfusion technique with the laminar flow region designated by arrows.

Figure 2

Utilizing fluorescent microscopy, (A) intracellular calcium and (B) nitric oxide levels were imaged in MLO-Y4 cells prior to and then immediately following the initiation of fluid flow over the cells. (C) ROIs were chosen for each cell, and the changes in calcium and nitric oxide levels relative to basal levels were determined. (D) DIC images were also captured before and immediately after the application of fluid flow, and average cell body strains were calculated from strain vectors using DISMAP.

Figure 3

Increasing imposed shear stress results in an increase in osteocyte intracellular calcium levels (**p < 0.05, error bars show the standard deviation).

Figure 4

Increasing imposed shear stress results in an increase in osteocyte intracellular nitric oxide levels (**p < 0.05, error bars show the standard deviation).

Figure 5

Increasing imposed shear stress results in an increase in the average osteocyte cell body strain (**p < 0.05, error bars show the standard deviation).

Figure 6

The osteocytes showed an increase in intracellular calcium concentration with an increase in cell strain in response to fluid flow regardless of the induced shear stress.

Figure 7

The osteocytes showed a slight increase in intracellular nitric oxide concentration with increasing cell strain, however there was not a significant correlation between the two.