NANOINDENTATION OF SOFT TISSUES AND OTHER BIOLOGICAL MATERIALS - Nanoindentation with Biological Applications - page 298

However, adhesive forces between the tip and the sample are typically
higher when the samples are tested moist rather than fully submerged,
and the need to account for adhesion can also complicate the indent
process (see Section 4.3.2).
4.1.2 . Surface preparation
Another challenge associated with indentation testing of soft tissues is
surface preparation. Studies have shown that surface roughness can
heavily influence the modulus measured by nanoindentation. 61-63 For
traditional indentation substrates, and even mineralized tissues such as
bone and teeth, samples are typically microtomed or polished to present a
“smooth” surface to the indenter tip. 64-66 Because of the complex,
heterogeneous structure of many biological materials, sectioning of less
stiff biological materials may also result in local damage on the surface,
which has been a particular problem for indentation of wood. 8 To
minimize the influence of surface damage or surface roughness on
mechanical properties measured through indentation, deep indents should
be performed. 67
Researchers must also consider whether sectioning the tissue will
disrupt the natural microstructure and change the properties that are of
interest in the study. For example, sectioning has been avoided when
surface properties are of interest, such as investigations of the waxy
coatings on plants, 1,41 or when integrity of the surface is important in
maintaining the structure and behavior of the tissue, such as
investigations of cartilage. 18-21 Under these conditions, soft tissues are
typically tested in their “native” state rather than undergoing sectioning
and polishing. If surface roughness is a concern, or if the region of
interest for indentation is not on the surface of the tissue, the best option
for soft tissue sectioning is often cryomicrotoming since it does not
require dehydration and paraffin embedding like other microtoming
methods. Embedding samples in agarose or a different hydrogel of
similar compliance to the tissue may also facilitate sectioning of small
biological samples. Polishing is usually not an option for soft tissues and
many other biological materials, so a sharp knife must be used to ensure
a smooth surface.