Deuterium nuclear magnetic resonance unambiguously quantifies pore and collagen-bound water in cortical bone

Bone water (BW) plays a pivotal role in nutrient transport and conferring bone with its viscoelastic mechanical properties. BW is partitioned between the pore spaces of the Haversian and lacuno-canalicular system, and water predominantly bound to the matrix proteins (essentially collagen). The general model of BW is that the former predominantly experiences fast isotropic molecular reorientation, whereas water in the bone matrix undergoes slower anisotropic rotational diffusion. Here, we provide direct evidence for the correctness of this model and show that unambiguous quantification in situ of these two functionally and dynamically different BW fractions is possible. The approach chosen relies on nuclear magnetic resonance (NMR) of deuterium ((2) H) that unambiguously separates and quantifies the two fractions on the basis of their distinguishing microdynamic properties. Twenty-four specimens of the human tibial cortex from 6 donors (3 male, 3 female, ages 27-83 years) were cored and (2) H spectra recorded at 62 MHz (9.4 Tesla) on a Bruker Instruments DMX 400 spectrometer after exchange of native BW with (2) H(2) O. Spectra consisted of a doublet signal resulting from quadrupole interaction of water bound to collagen. Doublet splittings were found to depend on the orientation of the osteonal axis with respect to the magnetic field direction (8.2 and 4.3 kHz for parallel and perpendicular orientation, respectively). In contrast, the isotropically reorienting pore-resident water yielded a single resonance line superimposed on the doublet. Nulling of the singlet resonance allowed separation of the two fractions. The results indicate that in human cortical bone 60% to 80% of detectable BW is collagen-bound. Porosity determined as the difference between total BW and collagen bound water fraction was found to strongly parallel micro-computed tomography (µCT)-based measurements (R(2)  = 0.91). Our method provides means for direct validation of emerging relaxation-based measurements of cortical bone porosity by proton MRI.