A significant effort continues to be directed towards improvements in the current assessment methods of bone quality, while in parallel, research into understanding of the fracture resistance is accelerating for assessing collagen matrix and water portions of bone since current diagnostic methods do not encompass the crucial contribution of these variables to bone’s fracture resistance. In this perspective, identifying, describing and understanding the involvement of water and collagen matrix to the fracture resistance of bone could be central to both these efforts and would contribute to improvement of current diagnosis techniques as well as to identify new therapeutic targets. Although there are several different methods available, including microscopic, spectroscopic, physical and chemicals methods to be used for characterizing bone composition, Raman spectroscopy is the only method which can provide quantitative and qualitative information of all bone constituents (i.e., mineral, organic matrix and water), simultaneously at micron-scale spatial resolution as well as holds non-invasive potential clinical use. Therefore, over the past two decades, Raman spectroscopy has become a powerful technique to investigate alterations to bone composition associated with aging and diseases. Although a significant number of spectroscopic studies have investigated the influence of mineral quality at fabric level on bone mechanical competence, the use of Raman spectroscopy to assess collagen quality in bone is limited and the correlation between spectroscopic measurement of collagen quality and bone mechanical properties remains poorly understood. On the other hand, to date, water-related Raman bands have not been studied yet, and Raman OH stretch bands have not been related to varying water compartments within bone or other tissues. The potential of Raman spectroscopy to assess hydration status of bone as a measure of bone quality is still unknown. Therefore, there is a clear need for comprehensive studies investigating Raman spectrum of bone to develop new indicators which infer collagen quality and hydration status of bone, and their associations with bone mechanical properties.

In this work, the potential of Raman spectroscopy to assess hydration and collagen denaturation status of bone was investigated as novel measures of bone quality. For the first time in the literature, four peaks in Raman OH-stretch envelope were identified to be sensitive to dehydration:​ 3220 cm^-1 (collagen-water), 3325 cm^-1 (N-H and collagen-water), 3453 cm^-1 (hydroxyproline and collagen-water), and 3584 cm^-1 (mineral and mineral-water). These peaks were differentially sensitive to deuterium treatment such that some water peaks were replaced with deuterium oxide faster than the rest. Specifically, the peaks at 3325 and 3584 cm^-1 were more physically accessible or/and tightly bound to the matrix than the remaining bands. The OH-stretch range of bone was dominated by collagen and water since the spectral profile of dehydrated demineralized bone was similar to that of the mineralized bone. Furthermore, water associates to bone mainly by collagen as findings of experimentally and theoretically spectra. The current work is among the first thorough analysis of the Raman OH stretch band in bone. Building upon these new findings, four newly identified peaks were investigated:​ I3220/I2949, I3325/I2949 and I3453/I2949 reflect status of organic-matrix related water (mostly collagen-related water) compartments and collagen portion of bone while I3584/I2949 reflects status of mineral-related water compartments and mineral portion of bone. These spectroscopic biomarkers were correlated with elastic and post-yield mechanical properties of bone. Collagen-water related biomarkers (I3220/I2949 and I3325/I2949) correlated significantly and positively with toughness and post-yield toughness. Mineral-water related biomarker correlated significantly and negatively with elastic modulus and positively with strength. While MR-based techniques have been useful in measuring unbound and loosely bound water, this was the first study which probed bound-water compartments differentially for collagen and mineral-bound water. For the first time, an evidence for contributions of different bound-water compartments to mechanical properties of wet bone was also revealed.

Furthermore, it is critical to develop a new spectroscopic biomarker which infers collagen quality in bone since there is only one specific spectroscopic indicator of collagen quality whose validation is still controversial. Amide I and amide III bands of bone are uniquely useful for collagen conformational analysis. Thus, in this work, the regions of amide bands of collagen and bone which are sensitive to thermally and mechanically induced denaturation were identified. Our results revealed five peaks whose intensities were sensitive to thermal and mechanical denaturation:​ ~ 1245, ~1270 and ~1320 cm^-1 in the amide III, and ~ 1640 and ~1670 cm^-1 in the amide I band. Four peak intensity ratios derived as new spectroscopic biomarkers from these peaks were found to be sensitive to denaturation:​ 1670/1640, 1320/1454, 1245/1270 and 1245/1454. Moreover, the correlations between these new spectroscopic biomarkers and post-yield mechanical properties of cortical bone were assessed. Among these four spectroscopic biomarkers, only 1670/1640 displayed significant correlation with all post-yield mechanical properties. The overall results showed that these peak intensity ratios can be used as new spectroscopic biomarkers to assess collagen quality and collagen integrity. The changes in these ratios with denaturation may reflect alterations in the collagen secondary structures, specifically a transition from ordered to less-ordered structure. The overall results clearly demonstrate that these new spectral information, specifically the ratio of 1670/1640, can be used to understand the involvement of collagen quality in the fragility of bone.

In conclusion, the new methods developed in this research can be an important tool not only in a laboratory set up to understand the involvements of water and collagen portions of bone in skeletal fragility due to aging and diseases, but also in a clinical environment to assess bone quality. The reported correlations of new spectroscopic biomarkers to mechanical properties herein underline the necessity for enabling approaches to assess these biomarkers noninvasively in vivo to improve the current diagnosis of those who may be at risk of bone fracture due to aging and diseases.