The experimental methods for studying the mechanotransduction in osteoblasts in two dimensional culture in vitro are based on controlled propagation of external force upon the culture plate via induced intra- and extracellular fluid flow in cells which are adherent to plastic surface of the culture plate. The ability of ostoblast to adhere to plastic surfaces (Fig. 26.1), which is also common in other cells of mesenchumal origin, is indespensible characteristic that enables to induce cellular deformation with subsequential activation of the cytoskeleton indirectly by controlling the fluid flow by external mechanical means [3, 4]. Accordingly many of the experimental methods which are related to mechanotransduction in osteoblasts utilize two dimensional cultures with cells adherent to plastic surfaces and exposed to external mechanical force. This type of experimental model is easily reproducible and relatively easy for maintenance (Fig. 26.2).

Osteoblasts in the two dimensional cultures adhere to the plastic surface of the culture container by protein extracellular extensions of the cytoskeleton. These extensions through the cell membrane are proteins from the groups of cadhedrins, integrins and selectines. The magnitude of anchorage energy that they generate with the plastic surface is in the scale of 10⁻¹⁶ J, which is two orders higher than the total cellular membrane bending energy of 10⁻¹⁸ J [5]. Therefore a significant deformation of the whole cell can occur without disrupting the cellular membrane integrity and without disconnecting of the cellular adhesion to the container surface. This difference in the strength of anchorage and of the membrane integrity allows a safe 4–5 % strain application to a pure vesicle bound by a lipid bilayer membrane when it is adherent to a surface [5]. Since the mechanical structure of osteoblast is reinforced by a cytoskeleton, much higher mechanical deformation is possible without its disconnection from the surface and without distraction of the cellular membrane.

Accordingly in most of the methods of evaluation of mechanotransduction in adherent osteoblast in two dimensional culture, a similar concept of mechanical movements of the surface and/or of the culture media over the adherent cells are utilized. By these methods a deformation of the cellular membrane on its free, not adherent, edge is generated following flow of the surrounding media and cyclic movement of the adherent to the surface cellular part (Fig. 26.2).

Although the basic principle of mechanical stimulation of osteoblasts in monolayer culture is similar in different studies there is a high range of mechanical parameters that was applied by the different authors, e.g., cellular displacement between 0.0003 and 0.025 mm (estimated from strains applied to cells and assuming that the diameters of the osteoblasts are in the range of 20–40 μm), acceleration of mechanical force in the range of 0.0009–500 mm/s², if a close to the sine shaped alternating force was used at frequencies between 0.05 and 20 Hz [6–10]. Currently there is no uniform experimental setup for mechanotransduction studies of the cells in a monolayer culture and therefore a comparison between different studies is sometimes difficult or even impossible.

The most popular research model for the in vitro study of mechanotransduction in osteoblasts utilizes a controlled cyclic stretching of an artificial silicon membrane, when the cells are adherent to its surface [11]. The membrane is stretched radially or bended with subsequent change in its surface. This process generates a mechanical strain on the adherent cells. This method does not allow generating uniform stretching forces on all the adherent cells because the strain magnitude changes radially when the highest strain is generated on the bending axis or on the periphery of two dimensional stretchable membrane, while on the center of the membrane the strain is almost negligible. By using this experimental setup strains of cells up to 20 % can be generated [11, 12]. The generated strains on cells are also dependent on the material properties of the membrane surface or on the geometrics of the bending curvature.

Additional widely used experimental method for mechanotransuction research is by exposure of a monolayer of cultured osteblasts, which are adherent to a plastic surface, to a controlled flow of culture media. By this method an alternating or continuous cell deformation can be achieved by controlled inflow of media from an external pump into the culture container in a closed circuit [13]. The flow can be continuous, pulsatile or oscillatory.