Differential effect of steady versus oscillating flow on bone cells

Abstract

Loading induced fluid flow has recently been proposed as an important biophysical signal in bone mechanotransduction. Fluid flow resulting from activities which load the skeleton such as standing, locomotion, or postural muscle activity are predicted to be dynamic in nature and include a relatively small static component. However, in vitro fluid flow experiments with bone cells to date have been conducted using steady or pulsing flow profiles only. In this study we exposed osteoblast-like hFOB 1.19 cells (immortalized human fetal osteoblasts) to precisely controlled dynamic fluid flow profiles of saline supplemented with 2% fetal bovine serum while monitoring intracellular calcium concentration with the fluorescent dye fura-2. Applied flows included steady flow resulting in a wall shear stress of 2 N m(-2), oscillating flow (+/-2 Nm(-2)), and pulsing flow (0 to 2 N m(-2)). The dynamic flows were applied with sinusoidal profiles of 0.5, 1.0, and 2.0 Hz. We found that oscillating flow was a much less potent stimulator of bone cells than either steady or pulsing flow. Furthermore, a decrease in responsiveness with increasing frequency was observed for the dynamic flows. In both cases a reduction in responsiveness coincides with a reduction in the net fluid transport of the flow profile. Thus. these findings support the hypothesis that the response of bone cells to fluid flow is dependent on chemotransport effects.

Fig. 1

Schematic of the parallel plate flow chamber consisting of a quartz glass slide with cells attached, silastic rubber gasket, and polycarbonate manifold. The components are held together by vacuum. The inlet and outlet ports communicate with the inlet and outlet slots.

Fig. 2

A vertically oriented streamwise cross-section through the parallel plate flow chamber depicting the laminar fluid velocity profile. Cells are grown on the quartz glass microscope slide that makes up the bottom of the flow chamber. The top surface is formed by the polycar-bonate of the chamber body. Fully developed laminar flow exhibits a parabolic velocity profile with a maximum velocity in the center that drops to zero at the top and bottom surfaces.

Fig. 3

A schematic of the fluid flow circuit. Steady flow is generated by a Harvard syringe pump (upper left). Oscillating flow is supplied with an Interlaken servohydraulic loading machine (upper right). Flow from the two devices are summed with a “Y” connector and input to the flow chamber via an inline pressure transducer. If the Harvard pump alone is activated steady flow is delivered to the chamber. If only the Interlaken is activated oscillating flow is delivered to the chamber. If both are activated pulsatile flow is delivered to the chamber. Finally, the flow chamber outlet is vented to the air with a short length of tubing.

Fig. 4

An example of two cycles of the three flow profiles analyzed in this study, steady, pulsing, and oscillating. Note that the dynamic flows (pulsing and oscillating) were applied at three different frequencies, 0.5, 1.0, and 2 Hz.

Fig. 5

An example of the [Ca²⁺]_i response traces obtained for steady flow. Note the arrow depicts the onset of flow.

Fig. 6

The fraction of cells responding to flow for each of the flow profiles studied. At all frequencies studied oscillating flow was less stimulatory than pulsatile or steady flow. Also note the trend of decreasing responsiveness with increasing frequency. The number of cells analyzed for each flow regime from left to right was; n = 272, 181, 137, 98, 94, 106, 188, 73. Bars represent standard error of a proportion.

Fig. 7

The corresponding average response amplitudes for each flow profile in nM. Only the response amplitude of pulsing flow at 0.5 Hz was statistically significantly different from the steady-flow condition. Since only one cell responded of the no flow controls, comparisons of response amplitude were not possible with this group. The bars represent standard error of the mean.