NANOINDENTATION OF BONE - Nanoindentation with Biological Applications - page 197

unclear what is being tested, and consequently what the data indicates,
on the final loading cycle of repeated indentations at a single site.
In summary, preconditioning or repeated loading at a single indent
site, prior to nanomechanical testing of bone, has a high likelihood of
altering bone's tissue-level structure at a scale that is easily resolved
with nanoindentation measurements. Most bone samples possess a stiff
surface on which nanoindenter systems tend to adequately detect contact.
Therefore bone's reference state for nanoindentation is not in question.
Preconditioning of bone will more likely complicate, rather than
facilitate, the collection of useful and relevant nanoindentation data.
2.1.4. Influence of contact depth
The volume of material that contributes heavily to a material's overall
nanomechanical response depends on factors that include the indenter
contact depth and the size of the tip. The effective volume contributing to
elastic modulus measurement can be described by a paraboloid of
revolution with radius 3 a and depth 5 a (where a = radius of circle of
contact between the tip and the surface). 43 It is a simple matter, therefore,
to optimize the tip selection and experimental parameters to enable
testing of the microstructures or regions of tissue within bone and,
conversely, to avoid testing volumes that extend beyond the regions of
interest.
The length-scale of bone's microstructural features of interest, for
example a lamellae 5 micrometers thick, is critical to selecting the
appropriate tip shape, tip size, maximum indenter penetration depth and
corresponding maximum applied load. A wide range of indentation
depths, from 100 nm 64 to 3000 nm 55 ( Table 7-1 ) , have been used to
extract bone's modulus and hardness. The volume of material deformed
is laterally seven times the depth of the indent, 72 thus the deeper indents
test a larger volume of bone ( Table 7-2 ). Larger testing volumes include
the influence of heterogeneities such as pore spaces of various length-
scales, changes in the degree of mineralization, increased numbers of
lamellae, and multiple types of bone or other structural features. In
addition, the maximum depth on loading may produce an effective
volume that lies within the boundaries of the region of interest, yet