NANOINDENTATION OF BONE - Nanoindentation with Biological Applications - page 201

cleaning ( i.e. , removal of non-osseous tissue), and short-term storage (on
the order of hours) to prevent significant degradation and dehydration.
2.2.2. Storage and preservation methods
While storage and preservation of bone samples beyond the day of
collection is often necessary, it is critical to consider the benefits and
limitations of each storage method on tissue-level properties. Other more
comprehensive reviews of storage and preservation methods exist. 83 A
brief overview here examines preservation methods from the perspective
of tissue-level alterations that may influence nanomechanical testing
results.
Storage in isotonic, calcium-based solutions is often used to maintain
hydration and conditions that are similar to those experienced in vivo .
Significant decreases in bending stiffness with storage in isotonic saline
for ten days were prevented by buffering with CaCl 2 . 84 However, storage
of machined equine cortical bone samples in a CaCl 2 buffered solution
has been observed to result in the deposition of amorphous calcium
phosphate crystals onto the sample surface. 85,86 Interactions between the
mineral phase of bone and the solution in which samples are stored are
also demonstrated by the energetically favorable replacement of
hydroxyl groups with fluorine to form fluoroapatite. 87 Mineral deposits
have been shown to form on samples following storage in phosphate-
buffered fixative solutions. 88 In dental tissues, elastic modulus and
hardness decreased significantly following storage in deionized water 89
but showed little change after storage in a balanced salt solution 89,90 or in
70% ethanol. 90 The mineral composition, and possibly the nanomechanical
behavior, of a bone sample may therefore be significantly affected by the
ionic environment of its storage solution.
Dehydration is a cheap and simple approach that can be employed to
store bone samples over long durations. Following removal of non-
osseous (non-bony) tissue, bone can be dried in ambient air or under an
applied vacuum and stored for extended durations. Mechanical properties
of dry bone are significantly different than for hydrated samples due to
the formation of microfractures, decreased elasticity of the collagen, and
possibly increased packing density of the mineral platelets. 91,92 Water