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Techniques for mechanical

stimulation of cells

We want to know what effect mechanical stimulation has on the biology of the resident

cells and, by extension, on the biology of the whole tissue.

Because of the complexity of cellular

biomechanics, it is not possible to predict this information from theoretical models or measurements of the properties of single

cells.

Therefore, we culture the tissue (or its resident cells) and mechanically stimulate the

cells, observing their ensuing behavior

A wide variety of devices have been developed to

apply mechanical stimuli to cells (and tissues) in

culture.

The choice of device and the mechanical stimulus it applies is dependent on which cells are being studied.

For instance, chondrocytes, the cells in cartilage, are typically compressed within their ECM because cartilage tissue is primarily subjected to compressive loads in vivo.

Smooth muscle cells are usually stretched, since these cells normally experience tensile forces in vivo,

Endothelial cells, which line the inner surface of blood vessels, are typically subjected to fluid flow.

These three loading modes compression, stretching, and fluid flow are the most common and are reviewed

Compressive loading

Hydrostatic compression

One method to compress cultured cells is to increase the gas pressure in the culture system

This results in a hydrostatic pressure being applied to cells within the liquid medium below

the gas phase.

Unfortunately, according to Henry’s law, the solubility of a gas in liquid increases as its

pressure increases, and since cells are sensitive to

the concentrations of dissolved gases in the culture medium (particularly O2 and CO2), it is difficult to determine whether the effects observed result from the mechanical or chemical

stimulus.

It is important to understand that if cells are being cultured on a rigid substrate they will not experience a net deformation from such a hydrostatic pressure increase, since this

increased isotropic stress will presumably be

transmitted into the cytoplasm, resulting in zero net force change on molecular components.

It is therefore difficult to see how hydrostatic pressure variations alone (in the absence of associated deformation, e.g., of a flexible

substrate) can be sensed by the cell.

Platen compression

A common alternative for compressive loading is direct compression by a platform or platen

This method is normally used with tissue specimens or

with cells that have been seeded into a natural or synthetic ECM, yielding a three-dimensional specimen

that can be compressed

Platen compression • A common alternative for compressive loading is direct compression by a platform or

Although this method is conceptually simple, the resulting tissue (and therefore cellular) strains

can be quite complex because of Poisson’s effect,

viscoelasticity of the matrix, the internal architecture of the matrix, and fluid flow that

results from compression (much like how fluid

exudes from a sponge when it is squeezed)

Nonetheless, the similarity of this mode of

loading with that which occurs in vivo for cartilage make it a useful method to study the

response of chondrocytes to compressive forces.

Stretching

The most common approach to stretch cells is to grow them on a flexible surface and to deform the surface once the cells are adhered to it.

This technique has been used in various configurations to apply uniaxial and biaxial strain to cells in culture

Both static and cyclic straining can be applied using these devices.

Of course, care must be taken in selecting a surface that is biocompatible with the cells and allows them

to attach and adhere firmly.

The ability to select the surface for adhesion can be an advantage, however, allowing for

greater experimental control

For instance, by coating the surface with defined matrix molecules (e.g., collagen or

fibronectin), one can investigate whether

specific cellmatrix interactions are important for mechanotransduction in a particular type

of cell

Uniaxial stretch

Uniaxial stretch

One method to stretch cells uniaxially (or longitudinally) is to grow them on a flexible membrane, grip the membrane at either end,

and elongate the membrane.

Another approach is to grow the cells on a substrate that is then bent or flexed in a four-

point bending configuration

The latter method results in a tensile strain on the convex surface.

The longitudinal strain, ε, in this case is:

where F is the applied force, a is the distance between the support and point of force application, E is the elastic modulus of the substrate, and b and h are the width and thickness of the substrate, respectively.

In both the membrane elongation and substrate flexion cases, there is not only

longitudinal deformation of the substrate but

also deformation in the lateral direction owing

to Poisson’s effects.

Biaxial stretch

Rather than pull the membrane in one direction only, the outer edges of a circular membrane can be fixed and the membrane can

be deformed to produce a biaxial deformation,

meaning a circular membrane is strained in both the radial and circumferential directions

This mode of loading has been implemented with several devices that either push the membrane up from the bottom (using a piston

or fluid) or pull the membrane down using

vacuum pressure.

Biaxial stretch

Biaxial stretch

From the strain profiles, it is apparent that the strain experienced by cells depends strongly upon their

location on the membrane.

In some cases, this can be an advantage since a range of strains can be studied in a single experiment

In practice, however, the strain input is often not well

characterized, biological assays are difficult to perform on cells from an isolated area of the membrane, and communication between cells might mask any differences resulting from strain variations from one

area to another

Therefore, the inhomogeneity of the strain stimulus can make interpretation of experimental results

difficult and is a fundamental limitation of these

devices.

EQUI-BIAXIAL STRAIN

To address the strain inhomogeneity, devices that apply uniform biaxial strain have been developed

For these cases, in which the membrane is confined on its periphery and is stretched so that the membrane remains “in- plane,” the theoretical strain profile can be computed by first

realizing that the stresses will be two dimensional or planar for a thin membrane.

EQUI-BIAXIAL STRAIN • To address the strain inhomogeneity, devices that apply uniform biaxial strain have been

For an isotropic linear elastic material in a state of plane stress, the strains (ε) and stresses (σ) are related by:

• For an isotropic linear elastic material in a state of plane stress, the strains (

where r and θ refer to the radial and circumferential components, respectively, and

E and ν are the Young’s modulus and Poisson’s ratio of the membrane material, respectively

Poisson's ratio is the ratio of transverse contraction strain

to

longitudinal

stretching force

extension

strain

in

the

direction

of

Young's modulus,

also known as the tensile modulus or elastic modulus, is a measure of the stiffness of an elastic material and is a quantity used to characterize materials. It is defined as the ratio of the stress (force per unit area) along an axis to the strain (ratio of deformation over initial length) along

that axis in the range of stress in which Hooke's law holds

Hooke's law is a principle of physics that states that the force needed to extend or compress a spring by some

distance is proportional to that distance.

For a circular membrane such as those used in the devices shown in the stress distribution is symmetrical about an axis that passes through the center of the membrane (i.e., axisymmetric) and it can be shown that

σ r = σθ Rearranging Equations equating the two stress components gives εr = εθ In other words, the majority of the membrane

(the part that stays “in-plane”), and presumably the cells attached to it, experience a strain that is spatially constant (not a function of position) and

isotropic (equal in all directions).

Although the primary stimulus applied to the cells in any stretch device is deformation of the substrate, it is

important to realize that movement of the membrane

can cause motion of the liquid medium within the culture dish.

Therefore, the cells might not only respond to the

stretch but also to the shear and pressure forces

generated by the moving liquid

The same is true for platen compression devices:

during compression of a three-dimensional specimen

containing cells, fluid can be expelled from the matrix

and the fluid flows that are generated by this process might affect the cells within the matrix.

Fluid flow

Because there are a number of circumstances in which cells are subjected to shear stresses

in vivo (e.g., endothelial cells in blood vessels

and osteocytes in bone tissue), mechanical stimulation of cells using fluid flow is an

important approach and one that is used

frequently

The devices that have been developed to apply well characterized shear stresses to

cultured cells fall into two categories:

Viscometers and flow chambers

Viscometers

Viscometer systems for mechanical stimulation of cells were adapted from systems originally

used to study the rheological properties of

fluids.

Two configurations have been used: the cone- and-plate viscometer and the parallel disk viscometer.

Cone and plate viscometers

Cone and plate viscometers

In the cone-and-plate viscometer, cells are either attached to a stationary plate or suspended in the medium between the plate and a rotating disc

A second plate, the cone, rotates causing the fluid between the two plates to move in the circumferential direction with a velocity v θ

The cone is not parallel to the stationary plate, but instead makes a small angle, α, with the stationary plate;

Therefore, the distance between the plates, h, varies as a function of the radial distance from the center, r, according to h = r sin α.

For laminar flow in a thin gap, the fluid velocity in the gap depends on the angular velocity of the rotating plate, ω, the separation of the plates, h, the distance from the axis of rotation, r, and the distance from the stationary plate, z, according to:

• For laminar flow in a thin gap, the fluid velocity in the gap depends on

sin α = α, which is valid for small angles when α is expressed in radians.

Because the circumferential velocity is

independent of radial position, the shear stress in a cone-and-plate viscometer is

independent of position:

• sin α = α, which is valid for small angles when α is expressed in

where μ is the viscosity of the fluid. This calculation of the shear stress assumes the fluid is an incompressible Newtonian fluid, which is valid for many fluids (including cell culture medium) but not entirely accurate for

some biological fluids, such as blood

Parallel plate viscometer

• Parallel plate viscometer The parallel disk viscometer differs from the cone- and-plate viscometer in that

The parallel disk viscometer differs from the cone-

and-plate viscometer in that the two plates are parallel to one another, separated by a constant distance, h The circumferential velocity in this case is given by:

• Parallel plate viscometer The parallel disk viscometer differs from the cone- and-plate viscometer in that

shear stress, τ , exerted by the fluid is a function of the

radial position, r:

shear stress, τ , exerted by the fluid is a function of the radial position, r:

From Equation, it is apparent that the shear stress varies from

zero at the center of the plate to a maximum at the edge.

As with the stretching devices that have inhomogeneous strain fields, the inhomogeneity in shear stresses with parallel disk

viscometers can complicate interpretation of experimental

results in many instances.

In some cases, however, such as investigations of the effect of

shear stress on cell shape, a range of shear stresses might be

desirable.

Flow chambers

The other class of fluid shear devices, flow chambers, differs from the viscometers in that

fluid motion is caused not by movement of one surface of the device but by imposing a

pressure gradient.

Flow chambers come in two configurations:

parallel plate and radial

Flow chambers • The other class of fluid shear devices, flow chambers, differs from the viscometers

Parallel plate flow chamber

The most commonly used device is the parallel plate flow

chamber, in which fluid is driven by a pump or hydrostatic head through a rectangular channel

The chamber is designed such that the flow is fully

developed and laminar (approximating plane Poiseuille

flow).

The shear stress on cells adhered to the bottom plate can be approximated by:

Parallel plate flow chamber • The most commonly used device is the parallel plate flow chamber,

where Q is the volumetric flow rate through the chamber, b and h are the width and height of the chamber, respectively, and μ is the viscosity of the fluid.

This approximation is valid if b>> h. Therefore, the

shear stress is constant for a given flow rate. The constant shear stimulus, plus the ease of use of

this system the equipment is relatively simple and the cells can be attached to a microscope slide and

viewed while being sheared make the parallel plate flow chamber an attractive option for flow studies

Radial flow chamber

An alternative configuration for a flow chamber is the radial flow chamber, in which fluid flows from an inlet in the center and moves out between two plates in the radial direction, exiting at the edge.

In this case, the shear stress on cells adhered to the bottom plate is not constant but is a function of the radial position, r:

Radial flow chamber • An alternative configuration for a flow chamber is the radial flow chamber,
Radial flow chamber • An alternative configuration for a flow chamber is the radial flow chamber,

Again, the inhomogeneous stimulus must be

considered carefully when designing an experiment and interpreting results from a radial flow chamber.

In all these devices, the flow is steady, fully developed

and laminar.

In the body, however, fluid flow is often unsteady and in some cases, such as at branch points in large arteries, the flow pattern will be complicated, with

flow reversals and separation.

Investigators have therefore introduced modifications to the devices described above, such as pulsatile flow in parallel plate flow chambers and flow chamber

geometries that cause flow disturbances