Weakly and strongly associated nonfreezable water bound in bones

Water bound in bone of rat tail vertebrae was investigated by ¹H NMR spectroscopy at 210–300 K and by the thermally stimulated depolarization current (TSDC) method at 190–265 K. The ¹H NMR spectra of water clusters were calculated by the GIAO method with the B3LYP/6-31G(d,p) basis set, and the solvent effects were analyzed by the HF/SM5.45/6-31G(d) method. The ¹H NMR spectra of water in bone tissue include two signals that can be assigned to typical water (chemical shift of proton resonance δ_H = 4–5 ppm) and unusual water (δ_H = 1.2–1.7 ppm). According to the quantum chemical calculations, the latter can be attributed to water molecules without the hydrogen bonds through the hydrogen atoms, e.g., interacting with hydrophobic environment. An increase in the amount of water in bone leads to an increase in the amount of typical water, which is characterized by higher associativity (i.e., a larger average number of hydrogen bonds per molecule) and fills larger pores, cavities and pockets in bone tissue.