NANOINDENTATION OF BONE - Nanoindentation with Biological Applications - page 206

A commonly used alternative to including dry bone in epoxy resin is
to, instead, embed bone samples in a low viscosity, infiltrating resin or
polymer ( e.g. , PMMA). Embedding facilitates complete infiltration of
the embedding medium into pore spaces to preserve cells, maintains
tissue morphology, and permits excellent surface preparation. High-
quality tissue embedding is time-consuming and typically requires that
the bone samples are dehydrated in a series of ethanol, 'cleared' with a
solvent such as acetone or xylene that is miscible with the monomer or
epoxy, and then slowly infiltrated with the embedding medium
(unpolymerized monomer). As rapid curing or polymerization often
results in incomplete infiltration into smaller pore spaces, the infiltration
process may be prolonged over many days or weeks prior to encouraging
polymerization with the addition of a chemical initiator ( e.g. , benzoyl
peroxide) and/or low heat (37-40°C).
Tissue embedding is useful for stabilizing the porous 3-D architecture
of bone. Additionally, PMMA can stabilize delicate bone samples such
as fossilized, demineralized, or deproteinated specimens. PMMA, with a
low modulus of 5 GPa, is thought to minimally contribute to the

measured mechanical properties of PMMA-embedded bone. 106 This is
demonstrated by the similarity between measured values between dry
bone and PMMA-embedded bone. 59

2.2.4. Surface preparation

Surface preparation for nanoindentation is generally achieved by
mechanical polishing or with microtome sectioning. Preparation of dried,
included, or embedded samples may follow conventional polishing
methods whereby the surface is smoothed by progressively finer grit
silicon carbide paper and then polished with diamond suspension paste. 58
Between each step, samples are ultrasonically cleaned in an appropriate
solution. Preparation of wet samples is possible, yet the fine polish
afforded by pastes or slurries finer then 1200 grit paper appears to add

little benefit. A simple approach to preparing surfaces of wet bone tissue
is cryopolishing via placing polishing paper over a frozen metal
surface. 43