There are several particular problems that must be considered when testing soft tis-sues that do not arise when carrying out conventional tests on engineering materials.
For engineering materials the conventional test methods have evolved over many years in order to address these problems, but the same approaches and solutions do not typically apply when testing different materials and there are many things that are taken for granted in testing conventional engineering materials but which must be considered properly when testing soft tissues.
A fundamental problem in testing any biological material is that they are much more variable than engineering materials (where a great deal of effort goes into making them as consistent as possible) and so much more care is needed in selecting samples to make sure they are representative and carrying out multiple measurements and statistical analysis to assess their variability and its implications for the conclu-sions that can be drawn. This is of course true for all biological measurements and biologists have evolved methods to address these problems. It is highly informative
for any engineer to spend time with biologists and see their approach to experimental work. They take great care in selecting their subjects, often in terms of parameters such as age, sex, body mass, activity levels, diet, and other factors that might be expected to influence the results, and they will document these parameters for future comparison as well as trying to make sure that groups to be compared are similar or even matched between groups. It would not be expected that the same material would have the same properties at different anatomical sites. Multiple samples are always needed and it would be usual also to repeat the entire experiment at least once and make sure that the results are consistent. Standards for testing metals typically require a minimum of five samples and this is about the smallest number that can give a reasonable sense of the variability of the material and hence the uncertainty in the results. To obtain meaningful comparisons between biological samples much larger numbers are needed, often tens or even hundreds of measurements to detect small differences in variable populations, and an appropriate statistical analysis is absolutely essential. Every factor that could conceivably have an influence is kept constant between experiments, and the accuracy and repeatability of measurements is checked thoroughly and regularly. This is also a useful exercise when using engi-neering testing equipment, as even apparently accurate devices such as load cells can frequently produce significant errors and other measurements such as the displace-ment of a testing machine are often not calibrated and may be quite different from the actual relative displacement of the grips or the ends of the sample.
Preparing samples of biological materials presents significant difficulties as it is not easy to cut them accurately to a particular shape. Cutting forces cause significant distortion and it is often unclear what is the unloaded free shape of the material anyway. Methods such as indentation tests which do not require such preparation can have significant advantages in this respect. Cutting materials which have large scale structures such as fibres often significantly alters their properties and this also is an argument for testing materials intact rather than cutting specimens from them.
A further issue to consider in preparing specimens is that the material is often inho-mogenous and anisotropic and so the position and orientation of the specimen within the sample must be carefully considered and defined.
A very important question to consider is the environment, both during the trans-port, preparation, and storage of the specimens and during the actual testing. Soft tissues are likely to be sensitive to temperature and hydration and also to factors such as the concentration of electrolytes which may cause swelling or dehydration.
Preservation of samples is difficult as freezing or chemical fixation are likely to cause significant changes in mechanical properties. For these reasons it is advantageous to test tissuesin situin living subjects although this introduces other complications.
The choice of test configuration is also important. In metals, a tensile test will effectively predict behaviour in compression, shear or other situations, since the mathematical models that are used correctly describe what happens under different types of loading. For soft tissues this is generally not the case; data from tensile tests will not necessarily correctly predict behaviour in compression or vice versa.
Similarly the models that are currently used may not correctly describe what will
happen under multiaxial loading. It is therefore a good idea wherever possible to test in a similar loading configuration to the actual application (this is also the case for some engineering materials such as polymers).
For some simple tests such as uniaxial tensile tests, it may be possible to calculate the material properties directly from the load-displacement data, though this comes at the cost of greater difficulties in preparing accurate, parallel specimens. For more complicated geometries it will probably be necessary to use a computational model to derive the material properties from the experimental data. Note though that even for such apparently simple tests there may be complications. For example, Legerlotz et al. [1] showed that in tensile tests on tendons the interior of the specimen slides relative to the outside where it is gripped and it is difficult to obtain uniform tension except in very long, thin specimens.
Tensile tests are widely used for engineering materials but have some limitations for soft tissues. Gripping the specimens is difficult and many researchers resort to measures such as freezing the specimens to the grips or using adhesives. The fundamental problem is that the specimens are not only slippery but have a high Poisson’s ratio and undergo large strains, so that as they are stretched they become much thinner and slip out of the grips. Using wedge or pneumatic grips that will tighten as the specimen contracts is much more effective than using screw clamps.
Clamping the specimen for a time before testing to squeeze out water can also be helpful. Whichever of these measures is used, the grips will have an effect on the properties and may cause a stress concentration and premature failure. This can be alleviated by making specimens with wider ends but this may not be effective where the material is very anisotropic.
Compression tests are challenging because the specimens must be short to avoid buckling, but this leads to significant effects of friction between the specimen and the platens. This constrains the ends of the specimen so they do not expand as much as they would in pure compression, causing barrelling and inaccurate results. It is possible to compensate for this by modelling the test but this requires assumptions about the friction. Another type of compression test, often used for cartilage, confines the specimen in a cylinder with porous platens that allow fluid to flow through them.
Theoretically this leads to 1D deformation and fluid flow and an analytical solution is possible, although in practice there are again issues with unknown friction between the specimen and the cylinder.
A more satisfactory alternative may be to use an indentation test, which is much less sensitive to friction. For homogenous materials analytical solutions are available [2]; for more complex materials it may be possible to estimate the properties at different depths using different sizes or shapes of indenters [3]. Fundamentally only a limited amount of information is available from the load-displacement curve and this limits what can be learned from the test; using additional strain measurements may allow more information to be extracted [4]. The test is sensitive to the stiffness (for example the Young’s modulus) but insensitive to the Poisson’s ratio, which is useful for measuring the stiffness but unhelpful if the Poisson’s ratio is also needed, and it clearly has serious limitations for anisotropic materials. A major advantage of indentation tests is that they require minimal specimen preparation and so they are
often used forin situtests on living tissue. A suction test where the tissue is drawn into an aperture is similar to an inverted indentation test and shares many of the same advantages and disadvantages [5].
An interesting method for testing tissues that are commonly under biaxial tension, for example aneurysms, is to clamp a circular specimen around its edges and inflate it with a fluid pressure applied to one side [6]. In its most sophisticated form, the curvature and strain distribution can be measured using optical methods and the curvature can be used to calculate the stress, allowing the full stress strain curve to be mapped out locally across the tissue.