Bone Microdamage and Its Contributions to Fracture - Skeletal Aging and Osteoporosis: Biomechanics and Mechanobiology - page 89

evaluation have been described. One method is based on a novel automated fluo-
rescent imaging method that was recently developed by Kazakia et al. [ 24 ]. Bone
specimens are stained using fluorescent dyes and are embedded in a resin. Using a
computer numeric controlled (CNC) mill, the specimen surface is serially sectioned
in small increments. Fluorochromes that are exposed after the milling of each
section are excited and hence, fluorescence imaging allows for image aquisition of
the evenly spaced two-dimensional sections. The two-dimensional images are
reconstructed into a three-dimensional model in which stained components can be
visualized. This technique is an improvement over available histological micro-
damage quantification techniques. However, it is currently limited to overall
microdamage quantification and cannot be used to distinguish between morpholo-
gies of microdamage [ 25 ]. We have described a second method, a non-invasive
microcomputed tomography based technique using a heavy metal stain to charac-
terize microdamage quantity and morphology [ 26 - 28 ]. The staining procedure,
modified from a previous protocol [ 29 ], involves a 14 day immersion of specimens
in an equal mixture of 8% uranyl acetate in 70% acetone and 20% lead acetate in
70% acetone, followed by a 7 day immersion in 1% ammonium sulfide in acetone
[ 27 , 28 ]. The central cubic region of stained specimens is scanned by microcom-
puted tomography. Scanning electron microscopy studies have confirmed that
regions of heavy metal staining correspond to areas of microdamage in bone (Fig. 3 )
[ 27 ]. From the regions stained with heavy metal, the ratios of damaged volume to
bone volume (DV/BV) and damaged surface area to damaged bone volume (DS/
DV) can be calculated. Here, an increase in DV/BV represents an increase in mi-
crodamage quantity. The surface-to-volume ratio of the microdamage, DS/DV,
illustrates microdamage morphology where higher DS/DV represents linear mi-
crocracks and lower DS/DV represents diffuse damage (Fig. 4 )[ 26 , 28 ]. A DS/DV
based numerical index may help to eliminate the observer bias in describing
microdamage as a linear microcrack or diffuse damage. Although the lead-uranyl
acetate based microcomputed tomography technique may not be able to fully resolve
a linear microcrack that is smaller than imaging resolution, pure linear microcracks
are rarely found in bone. A microdamage zone develops around linear microcracks
during growth. The zone allows the lead uranyl acetate to permeate into a wider area
(Fig. 3 )[ 29 ] and makes it possible to detect microdamage using microcomputed
tomography.
3 The Effect of Aging and Disease on Microdamage
in Cortical and Cancellous Bone
The formation of microdamage is observed in both cancellous and cortical bone.
Previous work on the detection of linear microcracks and diffuse damage in human
vertebral cancellous bone [ 18 , 30 , 31 ] and recent observations of these damage
morphologies in human cortical bone [ 8 ] indicate that both cortical and cancellous
tissues form linear microcracks and diffuse damage in vivo.