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    Assessment of spatially offset Raman spectroscopy to detect differences in bone matrix quality
    (Pergamon-Elsevier Science Ltd, 2023) Gautam, Rekha; Ahmed, Rafay; Haugen, Ezekiel; Unal, Mustafa; Fitzgerald, Sean; Uppuganti, Sasidhar
    Since spatially offset Raman spectroscopy (SORS) can acquire biochemical measurements of tissue quality through light scattering materials, we investigated the feasibility of this technique to acquire Raman bands related to the fracture resistance of bone. Designed to maximize signals at different offsets, a SORS probe was used to acquire spectra from cadaveric bone with and without skin-like tissue phantoms attenuating the light. Autoclaving the lateral side of femur mid-shafts from 5 female and 5 male donors at 100 degrees C and again at 120 degrees C reduced the yield stress of cortical beams subjected to three-point bending. It did not affect the volumetric bone mineral density or porosity. Without tissue phantoms, autoclaving affected more Raman characteristics of the organic matrix when determined by peak intensity ratios, but fewer matrix properties depended on the three offsets (5 mm, 6 mm, and 7 mm) when determined by band area ratios. The cut-off in the thickness of the tissue phantom layers was-4 mm for most properties, irrespective of offset. Matching trends when spectra were acquired without phantom layers between bone and the probe, & nu;1PO43 ? /Amide III and & nu;1PO43%(proline + OHproline) were higher and lower in the non-treated bone than in the autoclaved bone, respectively, when the thickness of tissue phantom layers was 4 mm. The layers, however, caused a loss of sensitivity to autoclaving related changes in & nu;3CO3/& nu;1PO43 ? and crystallinity. Without advanced post-processing of Raman spectra, SORS acquisition through turbid layers can detect changes in Raman properties of bone that accompany a loss in bone strength.
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    Sensitivity of the amide I band to matrix manipulation in bone: a Raman micro-spectroscopy and spatially offset Raman spectroscopy study
    (Royal Soc Chemistry, 2023) Ahmed, Rafay; Unal, Mustafa; Gautam, Rekha; Uppuganti, Sasidhar; Derasari, Shrey; Mahadevan-Jansen, Anita
    The fracture resistance of bone arises from the hierarchical arrangement of minerals, collagen fibrils (i.e., cross-linked triple helices of & alpha;1 and & alpha;2 collagen I chains), non-collagenous proteins, and water. Raman spectroscopy (RS) is not only sensitive to the relative fractions of these constituents, but also to the secondary structure of bone proteins. To assess the ability of RS to detect differences in the protein structure, we quantified the effect of sequentially autoclaving (AC) human cortical bone at 100 & DEG;C (& SIM;34.47 kPa) and then at 120 & DEG;C (& SIM;117.21 kPa) on the amide I band using a commercial Raman micro-spectroscopy (& mu;RS) instrument and custom spatially offset RS (SORS) instrument in which rings of collection fiber optics are offset from the central excitation fiber optics within a hand-held, cylindrical probe. Being clinically viable, measurements by SORS involved collecting Raman spectra of cadaveric femur mid-shafts (5 male & 5 female donors) through layers of a tissue mimic. Otherwise, & mu;RS and SORS measurements were acquired directly from each bone. AC-related changes in the helical status of collagen I were assessed using amide I sub-peak ratios (intensity, I, at & SIM;1670 cm(-1) relative to intensities at & SIM;1610 cm(-1) and & SIM;1640 cm(-1)). The autoclaving manipulation significantly decreased the selected amide I sub-peak ratios as well as shifted peaks at & SIM;1605 cm(-1) (& mu;RS), & SIM;1636 cm(-1) (SORS) and & SIM;1667 cm(-1) in both & mu;RS and SORS. Compared to & mu;RS, SORS detected more significant differences in the amide I sub-peak ratios when the fiber optic probe was directly applied to bone. SORS also detected AC-related decreases in I-1670/I-1610 and I-1670/I-1640 when spectra were acquired through layers of the tissue mimic with a thickness & LE;2 mm by the 7 mm offset ring, but not with the 5 mm or 6 mm offset ring. Overall, the SORS instrument was more sensitive than the conventional & mu;RS instrument to pressure- and temperature-related changes in the organic matrix that affect the fracture resistance of bone, but SORS analysis of the amide I band is limited to an overlying thickness layer of 2 mm.