Assessment of spatially offset Raman spectroscopy to detect differences in bone matrix quality

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 °C and again at 120 °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, ν₁PO₄^3-/Amide III and ν₁PO₄^3-/(proline + OH-proline) 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 ν₃CO₃/ν₁PO₄^3- 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.