The molecular basis of bone structure and strength is mineralized collagen fibrils at the submicron scale (∼500 nm). Recent advances in optical photothermal infrared (O-PTIR) spectroscopy allow the investigation of bone composition with unprecedented submicron spatial resolution, which may provide new insights into factors contributing to underlying bone function. Here, we investigated (i) whether O-PTIR-derived spectral parameters correlated to standard attenuated total reflection (ATR) Fourier transform infrared spectroscopy spectral data and (ii) whether O-PTIR-derived spectral parameters, including heterogeneity of tissue, contribute to the prediction of proximal femoral bone stiffness. Analysis of serially demineralized bone powders showed a significant correlation (r = 0.96) between mineral content quantified using ATR and O-PTIR spectroscopy, indicating the validity of this technique in assessing bone mineralization. Using femoral neck sections, the principal component analysis showed that differences between O-PTIR and ATR spectra were primarily attributable to the phosphate ion (PO4) absorbance band, which was typically shifter toward higher wavenumbers in O-PTIR spectra. Additionally, significant correlations were found between hydrogen phosphate (HPO4) content (r = 0.75) and carbonate (CO3) content (r = 0.66) quantified using ATR and O-PTIR spectroscopy, strengthening the validity of this method to assess bone mineral composition. O-PTIR imaging of individual trabeculae at 500 nm pixel resolution illustrated differences in submicron composition in the femoral neck from bones with different stiffness. O-PTIR analysis showed a significant negative correlation (r = -0.71) between bone stiffness and mineral maturity, reflective of newly formed bone being an important contributor to bone function. Finally, partial least squares regression analysis showed that combining multiple O-PTIR parameters (HPO4 content and heterogeneity, collagen integrity, and CO3 content) could significantly predict proximal femoral stiffness (R2 = 0.74, error = 9.7%) more accurately than using ATR parameters. Additionally, we describe new findings in the effects of bone tissue orientation in the O-PTIR spectra. Overall, this study highlights a new application of O-PTIR spectroscopy that may provide new insights into molecular-level factors underlying bone mechanical competence.
Keywords: ATR; Bone composition; O-PTIR; attenuated total reflection; bone stiffness; infrared spectral imaging; optical photothermal infrared; submicron.