Identification of water compartment in bone using ATR-FTIR spectroscopy

Water bound to the matrix in bone contributes significantly to its mechanical integrity, particularly to the ductility of the organic matrix. Attenuated Total Reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy has been widely used to evaluate bone compositional quality and is sensitive to the detection of water. However, no study has used this technique to quantify bone hydration. Therefore, we sought to use ATR-FTIR spectroscopy to identify and validate the water compartment in bone (including both free and bound water). We conducted a series of dehydration, hydrogen/deuterium (H/D) exchange, and glycation experiments on human bone tissue and investigated changes in signal intensity in the OH region of ATR-FTIR spectra. We identified four sub-peaks (∼3200 cm^-1, ∼3313 cm^-1, ∼3415 cm^-1, ∼3508 cm^-1) that decreased in intensity with each dehydration process in the OH range. In particular, the sub-peak at ∼3200 cm^-1 was no longer present with H/D exchange over time, indicating that this sub-peak is solely related to the water compartment. The reduction in the sub-peak at ∼3200 cm^-1 was also significantly correlated with the corresponding weight loss during serial dehydration (R² = 0.92, p = 0.04). Compared to the untreated samples, the intensities at the sub-peak of ∼3200 cm^-1 for glycated samples were significantly lower (p = 0.02). This study is the first to apply ATR-FTIR spectroscopy on bone to quantify the hydration status of bone. With one hour of air drying, the sub-peak at ∼3200 cm^-1 can be used to indicate water bound to the bone matrix. This fast and simple methodology provides an additional key angle to evaluate bone quality ex vivo.