NANOINDENTATION OF BONE - Nanoindentation with Biological Applications - page 199

heterogeneities as less material volume contributes to the overall
mechanical response within a single indentation. Surface roughness,
porosity, polishing relief, and other near-surface characteristics may
cause a high degree of variability in measured indentation properties. 74
Deeper indents sample large volumes of material and measured
mechanical properties are averaged properties of the multiple
heterogeneities. Using both experimental results and finite element
models, nanoindentation modulus values have been shown to converge to
a single value as the depth of the indent, and consequently the effective
volume of measurement, increases. 75
2.2. Sample preparation
Small-scale mechanical testing is complicated by the need to prepare
samples for analysis. The mere process of removing tissues from in vivo
physiological conditions triggers a series of events that potentially alter
tissue properties. These include altered pH and ionic environment,
dehydration, temperature, enzymatic degradation, and cell death. Further
processing to prepare samples for analysis may involve storage over
time; handling in the form of cleaning, sectioning, and polishing;
decalcification (in some cases); and mechanically stabilizing the sample
via embedding or through the use of a fixture. Throughout collection,
sample preparation, and testing processes, the maintenance of
physiologically relevant conditions is often desired. However, more
important is the uniform treatment, or control, of variables that may skew
results in ways that are often difficult to recognize or interpret.
While some have attempted to mechanically probe 76,77 or measure
strains 78-80 in vivo , the bulk of mechanical bone measurements are
performed ex vivo . Depth-sensing indentation testing of bone in live
human subjects is under development and may have great clinical
potential 76,77 ; however, the challenges of obtaining high quality and
repeatable data are immense. Testing bone samples ex vivo provides such
advantages as the ability to selectively alter bone properties, test specific
orientations within the 3-D structure of bone, and couple indentation
testing with other analysis methods to enable a multi-factor interpretation
of the nanomechanical results. 50