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.

Raman spectroscopy (RS) is used to analyze the physiochemical properties of bone because it is non-destructive and requires minimal sample preparation. With over two decades of research involving measurements of mineral-to-matrix ratio, type-B carbonate substitution, crystallinity, and other compositional characteristics of the bone matrix by RS, there are multiple methods to acquire Raman signals from bone, to process those signals, and to determine peak ratios including sub-peak ratios as well as the full-width at half maximum of the most prominent Raman peak, which is nu1 phosphate (ν₁PO₄). Selecting which methods to use is not always clear. Herein, we describe the components of RS instruments and how they influence the quality of Raman spectra acquired from bone because signal-to-noise of the acquisition and the accompanying background fluorescence dictate the pre-processing of the Raman spectra. We also describe common methods and challenges in preparing acquired spectra for the determination of matrix properties of bone. This article also serves to provide guidance for the analysis of bone by RS with examples of how methods for pre-processing the Raman signals and for determining properties of bone composition affect RS sensitivity to potential differences between experimental groups. Attention is also given to deconvolution methods that are used to ascertain sub-peak ratios of the amide I band as a way to assess characteristics of collagen type I. We provide suggestions and recommendations on the application of RS to bone with the goal of improving reproducibility across studies and solidify RS as a valuable technique in the field of bone research.

Developing clinical tools that assess bone matrix quality could improve the assessment of a person’s fracture risk. To determine whether Raman spectroscopy (RS) has such potential, we acquired Raman spectra from human cortical bone using microscope- and fiber optic probe-based Raman systems and tested whether correlations between RS and fracture toughness properties were statistically significant. Calculated directly from intensities at wavenumbers identified by second derivative analysis, Amide I sub-peak ratio I₁₆₇₀/I₁₆₄₀, not I₁₆₇₀/I₁₆₉₀, was negatively correlated with K_init (N = 58; R² = 32.4%) and J-integral (R² = 47.4%) when assessed by Raman micro-spectroscopy. Area ratios (A₁₆₇₀/A₁₆₉₀) determined from sub-band fitting did not correlate with fracture toughness. There were fewer correlations between RS and fracture toughness when spectra were acquired by probe RS. Nonetheless, the I₁₆₇₀/I₁₆₄₀ sub-peak ratio again negatively correlated with K_init (N = 56; R² = 25.6%) and J-integral (R² = 39.0%). In best-fit general linear models, I₁₆₇₀/I_1640, age, and volumetric bone mineral density explained 50.2% (microscope) and 49.4% (probe) of the variance in K_init. I₁₆₇₀/I₁₆₄₀ and v₁PO₄/Amide I (microscope) or just I₁₆₇₀/I₁₆₄₀ (probe) were negative predictors of J-integral (adjusted-R² = 54.9% or 37.9%, respectively). While Raman-derived matrix properties appear useful to the assessment of fracture resistance of bone, the acquisition strategy to resolve the Amide I band needs to be identified.