NANOINDENTATION OF BONE - Nanoindentation with Biological Applications - page 207

Soaking thin bone sections in distilled water for only 6 minutes has
been shown to cause demineralization. 107 Boothroyd suggested using
water saturated with calcium and phosphate ions, 107 however these ions
might precipitate onto the bone structure. An anhydrous preparation
using ethylene glycol as a lubricant and ultrasonic cleaning solution has
been shown to produce excellent smooth surfaces with no
demineralization or precipitation. 74 Alternatively, microtome prepared 60
or micromilled 50,108-111 bone surfaces are optimal in that they possess
significantly less surface roughness than polished samples.
When comparing small structural features ( e.g. , lamellae) at low
depths the surface roughness of the sample can have significant effect on
the measured mechanical properties. Mechanical polishing of bone
selectively removes thin lamellae more quickly and creates a
topographical profile of high and low regions which have been observed
to vary by about 200 nm. 60 In the valleys created by the thin lamellae the
contact area function A c is underestimated, thus the modulus is over
estimated. 60 When comparing the difference in modulus of thick and thin
lamellae, a greater difference was seen with samples prepared via
microtome due the superiorly flat surface preparation. 112
Nanoindentation studies of bone have been shown to generate
standard deviation values upward of 50%. 45,64 This large variability is
partially a reflection of bone's natural tissue structure and heterogeneous
nature, yet some sample preparation and experimental variation must
also contribute to high standard deviation values. In order to probe
individual bone microstructures ( e.g. , individual lamellae) indentations
must be sufficiently shallow to only probe the microstructure of interest,
yet sufficiently deep as to avoid influence of surface roughness.
2.2.5. Nanoindentation of wet bone
Significant challenges are posed by small-scale mechanical testing of
mineralized tissues under physiological conditions: e.g. , hydrated with a
physiological buffered saline solution at 37°C. However the alternative
of testing dry tissue in epoxy or histologically prepared, ethanol
dehydrated and embedded, tissues are apparent from the preceding
discussions in this chapter. A further complication enters in that testing