Comprising ~20% of the volume, water is a key determinant of the mechanical behavior of cortical bone. It essentially exists in 2 general compartments: within pores and bound to the matrix. The amount of pore water – residing in vascular-lacunar-canalicular space – primarily reflects intracortical porosity (i.e., open spaces within the matrix largely due to Haversian canals and resorption sites), and as such, is inversely proportional to most mechanical properties of bone. Movement of water according to pressure gradients generated during dynamic loading likely confers hydraulic stiffening to the bone as well. Nonetheless, bound water is a primary contributor to mechanical behavior of bone in that it is responsible for giving collagen the ability to confer ductility or plasticity to bone (i.e., allows deformation to continue once permanent damage begins to form in the matrix) and decreases with age along with fracture resistance. Thus, dehydration by air-drying or by solvents with less hydrogen bonding capacity causes bone to become brittle, but interestingly, it also increases stiffness and strength across the hierarchical levels of organization. Despite the importance of matrix hydration to fracture resistance, little is known about why bound water decreases with age in hydrated human bone. Using 1H nuclear magnetic resonance (NMR), both bound and pore water concentrations in bone can be measured ex vivo because the proton relaxation times differ between the two water compartments giving rise to two distinct signals. There are also emerging techniques to measure bound and pore water in vivo with magnetic resonance imaging (MRI). NMR/MRI-derived bound water concentration is positively correlated with both strength and toughness of hydrated bone, and may become a useful clinical marker of fracture risk.

At the mesoscale (i.e. over a few millimeters), cortical bone can be described as two-phase composite material consisting of pores and a dense mineralized matrix. The cortical porosity is known to influence the mesoscopic elasticity. Our objective was to determine whether the variations of porosity are sufficient to predict the variations of bone mesoscopic anisotropic elasticity or if change in bone matrix elasticity is an important factor to consider. We measured 21 cortical bone specimens prepared from the mid-diaphysis of 10 women donors (aged from 66 to 98 years). A 50-MHz scanning acoustic microscope (SAM) was used to evaluate the bone matrix elasticity (reflected in impedance values) and porosity. Porosity evaluation with SAM was validated against Synchrotron Radiation μCT measurements. A standard contact ultrasonic method was applied to determine the mesoscopic elastic coefficients. Only matrix impedance in the direction of the bone axis correlated to mesoscale elasticity (adjusted R(2)=[0.16-0.25], p<0.05). The mesoscopic elasticity was found to be highly correlated to the cortical porosity (adj-R(2)=[0.72-0.84], p<10(-5)). Multivariate analysis including both matrix impedance and porosity did not provide a better statistical model of mesoscopic elasticity variations. Our results indicate that, for the elderly population, the elastic properties of the mineralized matrix do not undergo large variations among different samples, as reflected in the low coefficients of variation of matrix impedance (less than 6%). This work suggests that change in the intracortical porosity accounts for most of the variations of mesoscopic elasticity, at least when the analyzed porosity range is large (3-27% in this study). The trend in the variation of mesoscale elasticity with porosity is consistent with the predictions of a micromechanical model consisting of an anisotropic matrix pervaded by cylindrical pores.

Fracture risk does not solely depend on strength but also on fracture toughness, i.e. the ability of bone material to resist crack initiation and propagation. Because resistance to crack growth largely depends on bone properties at the tissue level including collagen characteristics, current X-ray based assessment tools may not be suitable to identify age-, disease-, or treatment-related changes in fracture toughness. To identify useful clinical surrogates that could improve the assessment of fracture resistance, we investigated the potential of 1H nuclear magnetic resonance spectroscopy (NMR) and reference point indentation (RPI) to explain age-related variance in fracture toughness. Harvested from cadaveric femurs (62 human donors), single-edge notched beam (SENB) specimens of cortical bone underwent fracture toughness testing (R-curve method). NMR-derived bound water showed the strongest correlation with fracture toughness properties (r=0.63 for crack initiation, r=0.35 for crack growth, and r=0.45 for overall fracture toughness; p<0.01). Multivariate analyses indicated that the age-related decrease in different fracture toughness properties were best explained by a combination of NMR properties including pore water and RPI-derived tissue stiffness with age as a significant covariate (adjusted R2 = 53.3%, 23.9%, and 35.2% for crack initiation, crack growth, and overall toughness, respectively; p<0.001). These findings reflect the existence of many contributors to fracture toughness and emphasize the utility of a multimodal assessment of fracture resistance. Exploring the mechanistic origin of fracture toughness, glycation-mediated, non-enzymatic collagen crosslinks and intra-cortical porosity are possible determinants of bone fracture toughness and could explain the sensitivity of NMR to changes in fracture toughness. Assuming fracture toughness is clinically important to the ability of bone to resist fracture, our results suggest that improvements in fracture risk assessment could potentially be achieved by accounting for water distribution (quantitative ultrashort echo-time magnetic resonance imaging) and by a local measure of tissue resistance to indentation (RPI).