Bone Microdamage and Its Contributions to Fracture - Skeletal Aging and Osteoporosis: Biomechanics and Mechanobiology - page 87

Both linear microcracks and diffuse damage are typically identified using
histology methods. Along with identification of microdamage types, the rela-
tionship between microdamage and aging has been established, and the role of
microdamage in fracture toughness has also been investigated. This chapter will
review the literature on microdamage detection, changes in microdamage with
aging and/or disease or due to changes in bone quality, differences found in
microdamage between genders and between cancellous and cortical bone types,
and the role of microdamage in bone fragility.
2 Detection of Microdamage
Histology methods are typically used to identify in vivo microdamage. One
commonly used technique was originally developed by Frost [ 16 ] and later
modified by Burr and Stafford [ 12 ]. This technique involves en bloc staining of
bone tissue sections in a 1% basic fuchsin solution based in increasing concen-
trations of ethanol (70, 80, 90 and 100%) in vacuum at room temperature for a
period of five days. The en bloc stained cross sections are then embedded in
poly(methyl methacrylate), serially sectioned to 200 lm thickness, and ground to
100 lm thickness. Thus, only microdamage present in bone at the time of donor
death (i.e. before sectioning) is marked with basic fuchsin. The stained sections
can then be analyzed under a transmitted light microscope to determine the nature
of induced microdamage. Numerous groups have used the above staining method
with traditional fluorescence microscopy or laser confocal microscopy to observe
both linear microcracks and diffuse damage [ 1 , 6 , 15 - 18 ]. Under bright transmitted
light, linear microcracks appear as sharply defined lines with edges that are more
intensely stained than the surrounding space [ 10 , 12 , 19 ]. In contrast, diffuse
damage appears as a focal yet diffused area of pooled staining [ 15 ].
Another technique for marking microdamage incorporates staining with a single
chelating agent (e.g. alizarin complexone, calcein, calcein blue, xylenol orange,
oxytetracylcine). This method involves immersion of the specimen in a 1% stain
solution for a period of 20 h with a solution change after the first 4 h. Specimens
are then rinsed in distilled water to remove excess stain before sectioning and
analysis via microscopy. This procedure was found to be equally effective as the
basic fuchsin technique in identification of in vivo microdamage although the
labeling mechanisms between the two approaches differ [ 19 , 20 ]. Basic fuchsin
fills in void spaces [ 12 , 16 , 21 ] while chelating agents bind to free floating calcium
ions within the damaged areas [ 22 ]. Although any of these methods can be used to
identify native microdamage, multi-labeling with several dyes allows for differ-
entiation between microdamage produced at different time points of the failure
process. One example of a multi-labeling procedure involves staining in 1%
oxytetracycline for 16 h under vacuum to first stain in vivo microdamage. Spec-
imens are subsequently immersed in calcein blue after the first 75% of the fatigue
test and then immersed in xylenol orange after the last 25% of the test (Fig. 2 ).