Biomedical Engineering Reference
In-Depth Information
16.3
NANOINDENTATION OF ORAL MINERALIZED TISSUES
16.3.1
Sample Preparation
One of the concerns when working with oral mineralized tissues is their small sizes. To overcome this
problem, the tissues are sometimes embedded in resins such as polymethyl methacrylate to facilitate
handling during processing, as well as to provide mechanical support during indentation. Although
infiltration of the embedding material could be prevented by a careful choice of the resin, nanoinden-
tation should only be conducted in areas of the tissues away from the resin-tissue interfaces, since the
presence of the resin may alter the mechanical properties of the tissues
[9]
. In addition, the mechani-
cal properties of the mineralized tissues could be affected, to varying extents, when embedded in dif-
ferent resins. This makes the choice of the embedding material crucial if valid comparisons are to be
made between different studies
[10]
.
Nanoindentation requires a flat surface with surface roughness
10% of the indentation depth
[11]
. Since the surfaces exposed by most cutting techniques do not satisfy this requirement, the speci-
men surfaces are normally further smoothened with mechanical polishing or microtome section-
ing. In mechanical polishing, the specimens are first polished with silicon carbide paper to achieve
a relatively flat surface before they are fine polished to fulfill the stringent surface requirement. Fine
polishing is normally conducted by lapping with a slurry containing diamond or aluminum oxide
particles ranging from 0.05 to 6 μm in size. Alternatively, in microtome sectioning, the specimen is
first sectioned using a glass knife until a relatively flat surface is achieved. The specimen surface is
then further trimmed down with a diamond knife to decrease the surface roughness in preparation
for nanoindentation. These ultra-sectioned surfaces are reputed to possess smaller surface roughness
and less subsurface damages in comparison to mechanical polishing. These characteristics are valu-
able for investigating localized variations in mechanical properties of the mineralized tissues since the
natural properties are better preserved, and some of the finer details in the hierarchical structure of the
tissues may be more readily revealed
[12,13]
.
16.3.2
Hydration
As with most other biological tissues, the mineralized tissues in the oral cavity are hydrated in their
natural state. Thus, water is an important constituent of the mineralized tissues, and it is well proven
that major alterations in their mechanical properties can result from dehydration
[14-17]
. Therefore,
at any stage during the storage, preparation, and experiment, the specimens have to remain hydrated
by a suitable fluid. When an inappropriate fluid is used, problems such as demineralization may arise
which may induce changes in the mechanical properties of the tissues. Some of the common fluids
used for such a purpose include Hank's balanced salt solution (HBSS), calcium chloride (CaCl
2
)
solution, and distilled water
[18,19]
. Relative to CaCl
2
and deionized water, HBSS has been demon-
strated to be able to prevent surface demineralization in both dentin and enamel for at least 14 days
[19]
, so it is frequently used in nanoindentation investigations
[20-24]
.
Several strategies have been devised to keep the specimens hydrated during nanoindentation.
The simplest method would be to perform indentation within a short time frame after removal of
the specimen from the storage medium, such that no significant dehydration could occur during the
experiments
[25]
. Similar techniques include hydrating the specimens from the sides during inden-
tation
[26]
or applying a fluid to the surface just prior to indentation
[7,23,24]
. Some of the more
