Novel Raman Spectroscopic Biomarkers Indicate That Postyield Damage Denatures Bone's Collagen

Raman spectroscopy has become a powerful tool in the assessment of bone quality. However, the use of Raman spectroscopy to assess collagen quality in bone is less established than mineral quality. Because postyield mechanical properties of bone are mostly determined by collagen rather than the mineral phase, it is essential to identify new spectroscopic biomarkers that help infer the status of collagen quality. Amide I and amide III bands are uniquely useful for collagen conformational analysis. Thus, the first aim of this work was to identify the regions of amide bands that are sensitive to thermally induced denaturation. Collagen sheets and bone were thermally denatured to identify spectral measures that change significantly following denaturation. The second aim was to assess whether mechanical damage denatures the collagen phase of bone, as reflected by the molecular spectroscopic biomarkers identified in the first aim. The third aim was to assess the correlation between these new spectroscopic biomarkers and postyield mechanical properties of cortical bone. Our results revealed five peaks whose intensities were sensitive to thermal and mechanical denaturation: ∼1245, ∼1270, and ∼1320 cm(-1) in the amide III band, and ∼1640 and ∼1670 cm(-1) in the amide I band. Four peak intensity ratios derived from these peaks were found to be sensitive to denaturation: 1670/1640, 1320/1454, 1245/1270, and 1245/1454. Among these four spectral biomarkers, only 1670/1640 displayed significant correlation with all postyield mechanical properties. The overall results showed that these peak intensity ratios can be used as novel spectroscopic biomarkers to assess collagen quality and integrity. The changes in these ratios with denaturation may reflect alterations in the collagen secondary structure, specifically a transition from ordered to less-ordered structure. The overall results clearly demonstrate that this new spectral information, specifically the ratio of 1670/1640, can be used to understand the involvement of collagen quality in the fragility of bone. © 2015 American Society for Bone and Mineral Research.