Bone collagen network integrity and transverse fracture toughness of human cortical bone

The final publication is available at Elsevier via https://dx.doi.org/10.1016/j.bone.2018.10.024 © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license https://creativecommons.org/licenses/by-nc-nd/4.0/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, Td) 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, Kinit, 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 (R2 = 24–35%), and to a lesser degree with bound water fraction (BW; R2 = 7.9%) and pore water fraction (PW; R2 = 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-R2 = 36.7–49.0%). Also, the best-fit model for J-int (adjusted-R2 = 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-R2 = 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.U.S. Department of Veterans Affairs ["1I01BX001018"]National Institute of Arthritis and Musculoskeletal and Skin Diseases ["AR063157"]National Science Foundation ["1068988"]Canadian Institutes of Health Research ["115089"

Changes in skeletal collagen crosslinks and matrix hydration in high and low turnover chronic kidney disease

Chronic kidney disease (CKD) increases fracture risk. The results of this work point to changes in bone collagen and bone hydration as playing a role in bone fragility associated with CKD. INTRODUCTION: Clinical data have documented a clear increase in fracture risk associated with chronic kidney disease (CKD). Preclinical studies have shown reductions in bone mechanical properties although the tissue-level mechanisms for these differences remain unclear. The goal of this study was to assess collagen cross-links and matrix hydration, two variables known to affect mechanical properties, in animals with either high- or low-turnover CKD. METHODS: At 35 weeks of age (>75 % reduction in kidney function), the femoral diaphysis of male Cy/+ rats with high or low bone turnover rates, along with normal littermate (NL) controls, were assessed for collagen cross-links (pyridinoline (Pyd), deoxypyridinoline (Dpd), and pentosidine (PE)) using a high-performance liquid chromatography (HPLC) assay as well as pore and bound water per volume (pw and bw) using a 1H nuclear magnetic resonance (NMR) technique. Material-level biomechanical properties were calculated based on previously published whole bone mechanical tests. RESULTS: Cortical bone from animals with high-turnover disease had lower Pyd and Dpd cross-link levels (-21 % each), lower bw (-10 %), higher PE (+71 %), and higher pw (+46 %) compared to NL. Animals with low turnover had higher Dpd, PE (+71 %), and bw (+7 %) along with lower pw (-60 %) compared to NL. Both high- and low-turnover animals had reduced material-level bone toughness compared to NL animals as determined by three-point bending. CONCLUSIONS: These data document an increase in skeletal PE with advanced CKD that is independent of bone turnover rate and inversely related to decline in kidney function. Although hydration changes occur in both high- and low-turnover disease, the data suggest that nonenzymatic collagen cross-links may be a key factor in compromised mechanical properties of CKD.This work was supported by National Institutes of Health grants AR58005 (SM), DL100093 (CN), AR063157 (JSN), and the Indiana Clinical Translational Science Institute grant TR000162 (CN). The cross-link analysis is the result of work supported with resources and the use of facilities at the VA Tennessee Valley Healthcare System. All authors were involved in the design, conduct and analyses of the study. The authors would like to thank Drew Brown, Shannon Roy, and Kali O’Neill for technical assistance. We would also like to acknowledge the late Dr. Vincent H. Gattone II (1951-2013), who was instrumental in developing this animal model

Elasticité et porosité de l'os cortical humain : modèles et expériences

A l'échelle millimétrique, l'os cortical est vu comme une matrice minéralisée traversée de pores (canaux de Havers). Nous avons mesuré la porosité et l'élasticité de 21 échantillons (10 donneurs) et nous montrons que la rigidité de la matrice a une influence très faible sur l'élasticité apparente. Ces données permettent pour la première fois une analyse critique des modèles de changement d'échelle (homogénéisation asymptotique, Mori-Tanaka, bornes, etc.). Nous établissons que la densité apparente à l'échelle millimétrique peut être reliée au tenseur d'élasticité anisotrope grâce aux modèles

Raloxifene improves skeletal properties in an animal model of cystic chronic kidney disease

Patients with chronic kidney disease (CKD) have an increased risk of fracture. Raloxifene is a mild antiresorptive agent that reduces fracture risk in the general population. Here we assessed the impact of raloxifene on the skeletal properties of animals with progressive CKD. Male Cy/+ rats that develop autosomal dominant cystic kidney disease were treated with either vehicle or raloxifene for five weeks. They were assessed for changes in mineral metabolism and skeletal parameters (microCT, histology, whole-bone mechanics, and material properties). Their normal littermates served as controls. Animals with CKD had significantly higher parathyroid hormone levels compared with normal controls, as well as inferior structural and mechanical skeletal properties. Raloxifene treatment resulted in lower bone remodeling rates and higher cancellous bone volume in the rats with CKD. Although it had little effect on cortical bone geometry, it resulted in higher energy to fracture and modulus of toughness values than vehicle-treated rats with CKD, achieving levels equivalent to normal controls. Animals treated with raloxifene had superior tissue-level mechanical properties as assessed by nanoindentation, and higher collagen D-periodic spacing as assessed by atomic force microscopy. Thus, raloxifene can positively impact whole-bone mechanical properties in CKD through its impact on skeletal material properties

Change in porosity is the major determinant of the variation of cortical bone elasticity at the millimeter scale in aged women

International audienceAt 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 R2 = [0.16–0.25], p < 0.05). The mesoscopic elasticity was found to be highly correlated to the cortical porosity (adj-R2 = [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

MRI-derived bound and pore water concentrations as predictors of fracture resistance

AbstractAccurately 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

To what extent can cortical bone millimeter-scale elasticity be predicted by a two-phase composite model with variable porosity?

International audienceAn evidence gap exists in fully understanding and reliably modeling the variations in elastic anisotropy that are observed at the millimeter scale in human cortical bone. The porosity (pore volume fraction) is known to account for a large part, but not all, of the elasticity variations. This effect may be modeled by a two-phase micromechanical model consisting of a homogeneous matrix pervaded by cylindrical pores. Although this model has been widely used, it lacks experimental validation. The aim of the present work is to revisit experimental data (elastic coefficients, porosity) previously obtained from 21 cortical bone specimens from the femoral mid-diaphysis of 10 donors and test the validity of the model by proposing a detailed discussion of its hypotheses. This includes investigating to what extent the experimental uncertainties, pore network modeling, and matrix elastic properties influence the model’s predictions. The results support the validity of the two-phase model of cortical bone which assumes that the essential source of variations of elastic properties at the millimeter-scale is the volume fraction of vascular porosity. We propose that the bulk of the remaining discrepancies between predicted stiffness coefficients and experimental data (RMSE between 6% and 9%) is in part due to experimental errors and part due to small variations of the extravascular matrix properties. More significantly, although most of the models that have been proposed for cortical bone were based on several homogenization steps and a large number of variable parameters, we show that a model with a single parameter, namely the volume fraction of vascular porosity, is a suitable representation for cortical bone. The results could provide a guide to build specimen-specific cortical bone models. This will be of interest to analyze the structure–function relationship in bone and to design bone-mimicking materials

Minima and maxima of acoustic impedance (<i>Z</i>) and mineral orientation (<i>I_SAXS</i>) show a positive correlation (linear regression for the pooled data from <i>ost1</i> (▾) and <i>ost2</i> (•).

<p>Minima and maxima of acoustic impedance (<i>Z</i>) and mineral orientation (<i>I_SAXS</i>) show a positive correlation (linear regression for the pooled data from <i>ost1</i> (▾) and <i>ost2</i> (•).</p

SAM image (amplitude of the reflected ultrasound beam coded in gray levels).

<p>The data analysis was performed on an identical site-matched area on each one of the two investigated osteons (namely <i>ost1</i> and <i>ost2</i>).</p