Raman and mechanical properties correlate at whole bone- and tissue-levels in a genetic mouse model

The fracture resistance of bone arises from the composition, orientation, and distribution of the primary constituents at each hierarchical level of organization. Therefore, to establish the relevance of Raman spectroscopy (RS) in identifying differences between strong or tough bone and weak or brittle bone, we investigated whether Raman-derived properties could explain the variance in biomechanical properties at both the whole bone and the tissue-level, and do so independently of traditional measurements of mineralization. We harvested femurs from wild-type mice and mice lacking matrix metalloproteinase 2 because the mutant mice have a known reduction in mineralization. Next, RS quantified compositional properties directly from the intact diaphysis followed by micro-computed tomography to quantify mineralization density (Ct.TMD). Correlations were then tested for significance between these properties and the biomechanical properties as determined by the three-point bending test on the same femurs. Harvested tibia were embedded in plastic, sectioned transversely, and polished in order to acquire average Raman properties per specimen that were then correlated with average nanoindentation properties per specimen. Dividing the ν1 phosphate by the proline peak intensity provided the strongest correlation between the mineral-to-collagen ratio and the biomechanical properties (whole bone modulus, strength, and post-yield deflection plus nanoindentation modulus). Moreover, the linear combination of ν1 phosphate/proline and Ct.TMD provided the best explanation of the variance in strength between the genotypes, and it alone was the best explanatory variable for brittleness. Causal relationships between Raman and fracture resistance need to be investigated, but Raman has the potential to assess fracture risk.