See-saw rocking: an in vitro model for mechanotransduction research

Abstract

In vitro mechanotransduction studies, uncovering the basic science of the response of cells to mechanical forces, are essential for progress in tissue engineering and its clinical application. Many varying investigations have described a multitude of cell responses; however, as the precise nature and magnitude of the stresses applied are infrequently reported and rarely validated, the experiments are often not comparable, limiting research progress. This paper provides physical and biological validation of a widely available fluid stimulation device, a see-saw rocker, as an in vitro model for cyclic fluid shear stress mechanotransduction. This allows linkage between precisely characterized stimuli and cell monolayer response in a convenient six-well plate format. Models of one well were discretized and analysed extensively using computational fluid dynamics to generate convergent, stable and consistent predictions of the cyclic fluid velocity vectors at a rocking frequency of 0.5 Hz, accounting for the free surface. Validation was provided by comparison with flow velocities measured experimentally using particle image velocimetry. Qualitative flow behaviour was matched and quantitative analysis showed agreement at representative locations and time points. Maximum shear stress of 0.22 Pa was estimated near the well edge, and time-average shear stress ranged between 0.029 and 0.068 Pa. Human tenocytes stimulated using the system showed significant increases in collagen and GAG secretion at 2 and 7 day time points. This in vitro model for mechanotransduction provides a versatile, flexible and inexpensive method for the fluid shear stress impact on biological cells to be studied.

Keywords: computational fluid dynamics; cyclic fluid flow; particle image velocimetry; rocking; shear stress; tenocytes.

Figure 1.

(a) Graphical representation of a butterfly mesh; (b) vertical extrusion of butterfly mesh by 6.21 mm creates the grid used for CFD modelling; (c) schematic showing model dimensions and the x-coordinate system used for analysis. The plane defined by the dashed circumference is the initial free surface boundary, as specified in the volume of fluid method [14].

Figure 2.

PIV experimental apparatus. A 2 mm thick laser sheet highlights a central plane within the well geometry. A high-speed camera captures the motion of fluorescent microspheres in this plane. Well side-profile: the overlap of camera capture and laser sheet covers 70% of the well base, ensuring that 50% of the well is captured during a complete cycle. C denotes the well centre.

Figure 3.

(a) CFD velocity magnitude. (b) PIV velocity magnitude subjected to a low-pass filter (m s⁻¹). Velocity magnitudes are presented along the centre line of the well for one-half of the diameter owing to phase-symmetric flow behaviour. One complete cycle is shown. (For further information, see electronic supplementary material, ‘CFD velocity profile’ and ‘PIV velocity profile’.)

Figure 4.

Quantitative comparison of PIV raw and filtered velocity magnitudes with CFD velocity magnitudes at 0.1 and 0.4 mm above the base of the well.

Figure 5.

Maximum, time-averaged and minimum shear stress along the well centre line. Standard deviation bars are shown on the time-averaged profile, indicating relatively minimal variance at each location. (For further information, see electronic supplementary material, ‘CFD shear stress profile’.)

Figure 6.

Shear stress maps showing the shear stress at the cell layer every 0.25 s of a 2 s cycle at 30 OPM, starting at 0° (a).

Figure 7.

Velocity magnitude vectors showing the magnitude and direction of velocity 0.1 mm above the base of the well every 0.25 s of a 2 s cycle at 30 OPM, starting at 0° (a).

Figure 8.

Normalized secretion of collagen and GAG: collagen at the cell layer (ECM) and within the medium increases following both SI and constant stimulation. A similar pattern is identified with GAG secreted at the cell layer. All data have been normalized to dsDNA content. (For further information, see electronic supplementary material, ‘Protein secretion data’.)