NANOINDENTATION OF BONE - Nanoindentation with Biological Applications - page 202

may also serve to plasticize bone tissue. 43,47,48,93 Subsequent mechanical
testing performed on rehydrated samples demonstrated little measureable
effect at the scale of the whole bone. 94 However, the effects of
dehydration in air followed by rehydration on nanomechanical behavior
at the tissue-level are unknown.
Air-dried surfaces are readily polished and easy to test, yet substantial
microstructural damage occurs through shrinking of the high water-
containing organic phase. This is readily seen in tooth tissues, where
large cracks typically separate the dental enamel from the underlying
dentin in air-dried samples. Volumetric shrinkage measured by
dimensional changes has been reported to be 6-7%. 95,96 However true

volumetric changes may be significantly larger as shrinkage measured by
helium pycnometer and the Archimedes principle decrease by 15-16%. 97
Water is a main constituent of bone, thus the hydration state of
the sample plays a vital role in the measured mechanical properties.
Dehydration can cause increased modulus of elasticity, tensile and
bending strengths along with reduced values of fracture toughness. 59,94,98
Nanoindentation experiments have shown that dehydration increased
modulus values up to 50%, yet most studies found an average increase of
15-25%. 43,64,99 While the magnitude of mechanical properties is increased
with dehydration, the inherent material relationships ( e.g. , variations in
type of bone) are maintained.
Chemical Fixation: Samples can also be dehydrated via chemical
means through submersion in solvents, such as ethyl alcohol (ethanol,
C 2 H 5 OH), which are commonly used to dehydrate tissues in preparation
for histology. 47 Ethanol is pH neutral and avoids mineral dissolution,
and may be most effective at storage temperatures of 4 to 7°C. 100 Such
solvents simultaneously displace the water molecules and preserve the
tissue from bacterial invasion and significant enzymatic degradation. It is
thought that recovery from this form of dehydration is possible, 101 after
rehydrating in an isotonic saline solution. However microcracks often
form with such histological preservation methods. The extent of
permanent alterations within the bone tissue that may occur following
chemical dehydration and that affect nanomechanical behavior remain
unknown.