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One possibility for the disproportionate increase in fracture risk with aging relative to the decrease in bone mass is an accumulation of changes to the bone matrix which deleteriously affect fracture resistance. In order to effectively develop new targets for osteoporosis, a preclinical model of the age-related loss in fracture resistance needs to be established beyond known age-related decreases in bone mineral density and bone volume fraction. To that end, we examined long bones of male and female BALB/c mice at 6-mo. and 20-mo. of age and assessed whether material and matrix properties of cortical bone significantly differed between the age groups. The second moment of area of the diaphysis (minimum and maximum principals for femur and radius, respectively) as measured by ex vivo micro-computed tomography (μCT) was higher at 20-mo. than at 6-mo. for both males and females, but ultimate moment as measured by three-point bending tests did not decrease with age. Cortical thickness was lower with age for males, but higher for old females. Partially accounting for differences in structure, material estimates of yield, ultimate stress, and toughness (left femur) were 12.6%, 11.1%, and 40.9% lower, respectively, with age for both sexes. The ability of the cortical bone to resist crack growth (right femur) was also 18.1% less for the old than for the young adult mice. These decreases in material properties were not due to changes in intracortical porosity as pore number decreased with age. Rather, age-related alterations in the matrix were observed for both sexes: enzymatic and non-enzymatic crosslinks by high performance liquid chromatography increased (femur), volume fraction of bound water by 1 H-nuclear magnetic resonance relaxometry decreased (femur), cortical tissue mineral density by μCT increased (femur and radius), and an Amide I sub-peak ratio I 1670 /I 1640 by Raman spectroscopy increased (tibia). Overall, there are multiple matrix changes to potentially target that could prevent the age-related decrease in fracture resistance observed in BALB/c mouse., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Greater understanding of the determinants of skeletal fragility is highly sought due to the great burden that bone affecting diseases and fractures have on economies, societies and health care systems. Being a complex, hierarchical composite of collagen type-I and non-stoichiometric substituted hydroxyapatite, bone derives toughness from its organic phase. In this study, we tested whether early observations that a strong correlation between bone collagen integrity measured by thermomechanical methods and work to fracture exist in a more general and heterogeneous sampling of the population. Neighboring uniform specimens from an established, highly characterized and previously published collection of human cortical bone samples (femur mid-shaft) were decalcified in EDTA. Fifty-four of the original 62 donors were included (26 male and 28 females; ages 21-101 years; aging, osteoporosis, diabetes and cancer). Following decalcification, bone collagen was tested using hydrothermal isometric tension (HIT) testing in order to measure the collagen's thermal stability (denaturation temperature, T d ) and network connectivity (maximum rate of isometric tension generation; Max.Slope). We used linear regression and general linear models (GLMs) with several explanatory variables to determine whether relationships between HIT parameters and generally accepted bone quality factors (e.g., cortical porosity, pentosidine content [pen], pyridinoline content [pyd]), age, and measures of fracture toughness (crack initiation fracture toughness, K init , and total energy release/dissipation rate evaluated at the point of unstable fast fracture, J-int) were significant. Bone collagen connectivity (Max.Slope) correlated well with the measures of fracture toughness (R 2  = 24-35%), and to a lesser degree with bound water fraction (BW; R 2  = 7.9%) and pore water fraction (PW; R 2  = 9.1%). Significant correlations with age, apparent volumetric bone mineral density (vBMD), and mature enzymatic [pyd] and non-enzymatic collagen crosslinks [pen] were not detected. GLMs found that Max.Slope and vBMD (or BW), with or without age as additional covariate, all significantly explained the variance in Kinit (adjusted-R 2  = 36.7-49.0%). Also, the best-fit model for J-int (adjusted-R 2  = 35.7%) included only age and Max.Slope as explanatory variables with Max.Slope contributing twice as much as age. Max.Slope and BW without age were also significant predictors of J-int (adjusted-R 2  = 35.5%). In conclusion, bone collagen integrity as measured by thermomechanical methods is a key factor in cortical bone fracture toughness. This study further demonstrates that greater attention should be paid to degradation of the overall organic phase, rather than a specific biomarker (e.g. [pen]), when seeking to understand elevated fracture rates in aging and disease., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Changes in the distribution of bone mineralization occurring with aging, disease, or treatment have prompted concerns that alterations in mineralization heterogeneity may affect the fracture resistance of bone. Yet, so far, studies assessing bone from hip fracture cases and fracture-free women have not reached a consensus on how heterogeneity in tissue mineralization relates to skeletal fragility. Owing to the multifactorial nature of toughening mechanisms occurring in bone, we assessed the relative contribution of heterogeneity in mineralization to fracture resistance with respect to age, porosity, and area fraction of osteonal tissue. The latter parameters were extracted from quantitative backscattered electron imaging of human cortical bone sections following R-curve tests of single-edge notched beam specimens to determine fracture toughness properties. Microstructural heterogeneity was determined as the width of the mineral distribution (bulk) and as the sill of the variogram (local). In univariate analyses of measures from 62 human donors (21 to 101 years), local but not bulk heterogeneity as well as pore clustering negatively correlated with fracture toughness properties. With age as covariate, heterogeneity was a significant predictor of crack initiation, though local had a stronger negative contribution than bulk. When considering all potential covariates, age, cortical porosity and area fraction of osteons explained up to 50% of the variance in bone׳s crack initiation toughness. However, including heterogeneity in mineralization did not improve upon this prediction. The findings of the present work stress the necessity to account for porosity and microstructure when evaluating the potential of matrix-related features to affect skeletal fragility., Competing Interests: The authors do not have a conflict of interest with present work., (Published by Elsevier Ltd.)

Accurately predicting fracture risk in the clinic is challenging because the determinants are multi-factorial. A common approach to fracture risk assessment is to combine X-ray-based imaging methods such as dual-energy X-ray absorptiometry (DXA) with an online Fracture Risk Assessment Tool (FRAX) that includes additional risk factors such as age, family history, and prior fracture incidents. This approach still does not adequately diagnose many individuals at risk, especially those with certain diseases like type 2 diabetes. As such, this study investigated bound water and pore water concentrations (Cbw and Cpw) from ultra-short echo time (UTE) magnetic resonance imaging (MRI) as new predictors of fracture risk. Ex vivo cadaveric arms were imaged with UTE MRI as well as with DXA and high-resolution micro-computed tomography (μCT), and imaging measures were compared to both whole-bone structural and material properties as determined by three-point bending tests of the distal-third radius. While DXA-derived areal bone mineral density (aBMD) and μCT-derived volumetric BMD correlated well with structural strength, they moderately correlated with the estimate material strength with gender being a significant covariate for aBMD. MRI-derived measures of Cbw and Cpw had a similar predictive ability of material strength as aBMD but did so independently of gender. In addition, Cbw was the only imaging parameter to significantly correlate with toughness, the energy dissipated during fracture. Notably, the strength of the correlations with the material properties of bone tended to be higher when a larger endosteal region was used to determine Cbw and Cpw. These results indicate that MRI measures of Cbw and Cpw have the ability to probe bone material properties independent of bone structure or subject gender. In particular, toughness is a property of fracture resistance that is not explained by X-ray based methods. Thus, these MRI-derived measures of Cbw and Cpw in cortical bone have the potential to be useful in clinical populations for evaluating fracture risk, especially involving diseases that affect material properties of the bone beyond its strength., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

In addition to the loss in bone volume that occurs with age, there is a decline in material properties. To test new therapies or diagnostic tools that target such properties as material strength and toughness, a pre-clinical model of aging would be useful in which changes in bone are similar to those that occur with aging in humans. Toward that end, we hypothesized that similar to human bone, the estimated toughness and material strength of cortical bone at the apparent-level decreases with age in the male Fischer F344 rat. In addition, we tested whether the known decline in trabecular architecture in rats translated to an age-related decrease in vertebra (VB) strength and whether non-X-ray techniques could quantify tissue changes at micron and sub-micron length scales. Bones were harvested from 6-, 12-, and 24-month (mo.) old rats (n=12 per age). Despite a loss in trabecular bone with age, VB compressive strength was similar among the age groups. Similarly, whole-bone strength (peak force) in bending was maintained (femur) or increased (radius) with aging. There was though an age-related decrease in post-yield toughness (radius) and bending strength (femur). The ability to resist crack initiation was actually higher for the 12-mo. and 24-mo. than for 6-mo. rats (notch femur), but the estimated work to propagate the crack was less for the aged bone. For the femur diaphysis region, porosity increased while bound water decreased with age. For the radius diaphysis, there was an age-related increase in non-enzymatic and mature enzymatic collagen crosslinks. Raman spectroscopy analysis of embedded cross-sections of the tibia mid-shaft detected an increase in carbonate subsitution with advanced aging for both inner and outer tissue., (Published by Elsevier Inc.)

Even though age-related changes to bone tissue affecting fracture risk are well characterized, only a few matrix-related factors have been identified as important to maintaining fracture resistance. As a gene critical to osteoblast differentiation, activating transcription factor 4 (ATF4) is possibly one of these important factors. To test the hypothesis that the loss of ATF4 affects the fracture resistance of bone beyond bone mass and structure, we harvested bones from Atf4+/+ and Atf4-/- littermates at 8 and 20 weeks of age (n≥9 per group) for bone assessment across several length scales. From whole bone mechanical tests in bending, femurs from Atf4-/- mice were found to be brittle with reduced toughness and fracture toughness compared to femurs from Atf4+/+ mice. However, there were no differences in material strength and in tissue hardness, as determined by nanoindentation, between the genotypes, irrespective of age. Tissue mineral density of the cortex at the point of loading as determined by micro-computed tomography was also not significantly different. However, by analyzing local composition by Raman Spectroscopy (RS), bone tissue of Atf4-/- mice was found to have higher mineral to collagen ratio compared to wild-type tissue, primarily at 20 weeks of age. From RS analysis of intact femurs at 2 orthogonal orientations relative to the polarization axis of the laser, we also found that the organizational-sensitive peak ratio, ν1Phosphate per Amide I, changed to a greater extent upon bone rotation for Atf4-deficient tissue, implying bone matrix organization may contribute to the brittleness phenotype. Target genes of ATF4 activity are not only important to osteoblast differentiation but also in maintaining bone toughness and fracture toughness., (Published by Elsevier Inc.)

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Ultrasound axial transmission (UAT), a technique using propagation of ultrasound waves along the cortex of cortical bones, has been proposed as a diagnostic technique for the evaluation of fracture healing. Quantitative ultrasound parameters have been reported to be sensitive to callus changes during the regeneration process. The aim of this work was to identify the specific effect of cortical bone mineralization on UAT measurements by means of numerical simulations and experiments using a reverse fracture healing approach. A cortical bovine femur sample was used, in which a 3mm fracture gap was drilled. A 3mm thick cortical bone slice, extracted from another location in the bone sample, was submitted to a progressive demineralization process with EDTA during 12 days. UAT measurements and simulations using a 1MHz probe were performed with the demineralized slice placed into the fracture gap to mimic different stages of mineralization during the healing process. The calcium loss of the slice due to the EDTA treatment was recorded everyday, and its temporal evolution could be modeled by an exponential law. A 50MHz scanning acoustic microscopy was also used to assess the mineralization degree of the bone slice at the end of the intervention. These data were used in the numerical simulations to derive a model of the time evolution of bone slice mechanical properties. From both the experiments and the simulations, a significant and progressive increase in the time of flight (TOF; p<0.001) of the propagating waves measured by UAT was observed during the beginning of the demineralization process (first 4 days). Although the simulated TOF values were slightly larger than the experimental ones, they both exhibited a similar time-dependence, validating the simulation approach. Our results suggest that TOF measured in axial transmission is affected by local changes of speed of sound induced by changes in local mineralization. TOF may be an appropriate indicator to monitor callus maturation., (Copyright © 2011 Elsevier Inc. All rights reserved.)