Raman Spectroscopy of Bone and Cartilage - Emerging Raman Applications and Techniques in Biomedical and Pharmaceutical Fields - page 350

rather than the higher frequency component found in the fracture study, a re-
sult that is explainable by an increase rather than a decrease in lattice strain.
The addition of either a higher or lower frequency component is consistent
with high-pressure studies on calcium hydroxyapatite, where the frequency
of the phosphate ν 1 band initially increases and then decreases with greater

pressure [21]. Importantly, in both the indent and control areas, only one ma-
trix factor was found. A second matrix factor was found at the edge of the
indent where shear forces were great. The spectral features of this factor were
characteristic of the absence or at least reduced number of mature Pyr cross-
links [5] and were interpreted as rupture of these cross-links. This conclusion
was strengthened by the scanning electron microscopy, which showed shear
bands at the edges of the indents. No collagen damage was spectroscopically
observable in the center of indents, consistent with the known weakness of
bone in shear mode, relative to compression.
Other studies have produced apparently conflicting results on the relation-
ship between local composition and microcrack formation [50, 51]. In 2005,
Akkus and coworkers used Raman microspectroscopy and human male femurs
to examine mineralization in the vicinity of microdamage. Mineralization was
evaluated by calculating the intensity ratio of the phosphate

ν 1 band to the

1450 cm − 1 ). Raman maps of this mineral/matrix ratio
showed that the mineralization near microcracks was greater than elsewhere,
and that the mineralization was uniform in regions of microcracks and did not
vary with distance from the crack. Statistical analysis showed average miner-
alization of areas near microcracks was consistently and significantly greater
than overall mineralization, suggesting that the formation of some microcracks
may be composition dependent.
Pezzotti and coworkers monitored the spectral shift of Raman bands with
mechanical loading to examine the in situ response of femoral and cortical
bone to external stress [47, 48]. It has been proposed that collagen operates
by a crack-bridging mechanism during fracture initiation and propagation
[52]. Tensile microstresses were quantitatively assessed for the first time by
constructing microscopic stress maps from shifts of the phosphate

CH 2 -scissoring band ( ∼

ν 1 band.
Figure 14.5 shows the stress maps in bovine femoral bone near a fracture. An
area of high stress exists ahead of the crack tip, and subsequent micrographs
showed that microcracks were present in this area of high stress [47]. Con-
sistent with the crack-bridging mechanism, the stress field contained areas of
stress relaxation and stress intensification, seen as the striped pattern in the
maps.
A follow-up study showed the relation between the composition and the
redistribution of stress [47]. Cortical bone was loaded with external compres-
sion and tension, and the stress stored within the apatite crystals was as-
sessed via the shift in phosphate

ν 1 band in two regions: a collagen-rich area
and an apatite-rich area. In the collagen-rich areas, stress was released under
external tension, but localized stress intensification occurred under external.
In apatite-rich areas, both tensile and compressive stresses were observed.