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Not AvailaMineral imbalances in the body from chronic kidney disease can impact bone turnover and cause cortical bone loss. Synthetic salmon calcitonin is an FDA-approved treatment for bone fragility observed in diseases such as osteoporosis, and clinical trials have demonstrated a reduction in fractures compared to untreated individuals. This study documents the effects of calcitonin on cortical bone using an in vivo mouse model of chronic kidney disease. Serum BUN and PTH are reported. Calcitonin was found to impact at a dose of 50/IU/kg/day five times a week for five weeks. MicroCT was used to evaluate bone quantity measures, such as cortical porosity, thickness, bone area, and long bone structural geometric parameters. It was found that porosity, thickness, and bone geometry are affected by disease, but not by treatment at the specified regime. Small and wide-angle x-ray scattering (SAXS and WAXS) was used to obtain the nanostructure of the mineral-collagen-water composite, including mineral dimensions, -periodicity and collagen spacing. Data from thermogravimetric analysis (TgA) were used to quantify wt.% of the mineral, collagen, and bound water of each sample. Chronic kidney disease was found to decrease collagen spacing to increase mineral weight fractions, and to reduce loosely bound water. There were no changes from chronic kidney disease on the -Periodicity. Treatment increased the weight percent of collagen, with no effect on other bone quality parameters. 

Understanding bone strength is important when assessing bone diseases and their treatment. Bending experiments are often used to determine strength. Then, flexural stresses are calculated from elastic bending theory. With a brittle failure criterion, the maximum flexural tensile stress is equated to (nominal) strength. However, bone is not a perfectly brittle material. A quasi-brittle failure criterion is more appropriate. Such an approach allows for material failure to occur before full fracture. The extent of the subcritical damage domain then introduces a length scale. The intrinsic strength of the bone is calculated from the critical load at fracture and the failure process zone dimensions relative to the specimen size. We apply this approach to human cortical bone specimens extracted from a femur. We determine strength measures in the untreated reference state and after treatment with the selective estrogen receptor modulator raloxifene. We find that the common nominal strength measure does not distinguish between treatments. However, the dimensions of the failure process zone differ between treatments. Intrinsic strength measures then are demonstrated as descriptors of bone strength sensitive to treatment. An extrapolation of laboratory data to whole bone is demonstrated. 

This publication documents measurement data for two in-situ loaded fracture mechanics specimens observed with 3D X-ray microscopy. Materials The diaphysis of a human (92-year-old, male) cadaveric femur was obtained through the Indiana University School of Medicine Anatomical Donation Program. Bars (nominally 4.0 mm x 4.0 mm cross section) were extracted from the diaphysis as demonstrated in Figure-samplelocation. Two single Edge Notch Bend, SEN(B), specimens for a load span s=20 mm were machined for a three-point bend fixture for crack growth in the transverse direction. SEN(B) specimens had the following dimensions (height d, depth b, initial crack length a0): beam 1 d=4.0 mm, b=4.0 mm, a0=1.8 mm, beam 2 d=4.1 mm, b=3.9 mm, a0=1.7 mm). Osteon diameter was measured was measured on polished sections by using backscatter SEM images following Britz (2009), Figure samplelocation.jpg. Using ImageJ, a grid is imposed on the images and On.Dm is determined as the Feret Diameter for at least 40 On.Dm measures. For beam 1 mean On.Dm is 242 micrometer and for beam 2 284 micrometer. Experiments and Data Fracture experiments were conducted with a Deben 5000 load rig in a Zeiss XRADIA 3D microscope. For system details see https://www.physics.purdue.edu/xrm/about-our-instruments/index.html. Data for these experiments is given in the two csv files of this project data set. In these experiments force F (load cell) data and image frame data are obtained as machine output. Crack mouth opening displacement (CMOD) is obtained from 3D X-ray images at frame numbers synchronized to force readings. Fracture process zone (FPZ) length L. FPZ length data is obtained from 3D image data in Gallaway, G. E.; Allen, M. R.; Surowiec, R. K.; Siegmund, T. H. (2025). 3D Image Data from In-situ X-ray Imaging Transverse Crack Growth Experiments in Human Cortical Bone. Purdue University Research Repository. doi:10.4231/94PZ-AB06 Code Code (Analysis_Main.m, Analysis_Func.m) takes data from the .csv files and determines the linear elastic fracture mechanics quantities (LEFM toughness), the quasi-brittle fracture mechanics quantities (QBFM toughness), and the tissue intrinsic (size-independent) fracture properties (tissue toughness, tissue strength, tissue lengthscale). Output is depicted as force-CMOD and fracture process zone length - CMOD records, and as crack growth resistance curves (quasibrittle energy release rate vs. fracture process zone length). In addition, the microstructure constant eta is obtained as the ratio between the tissue intrinsic lengthscale and the mean osteon diameter. Code (P_star.m) is provided to determine maximum sustainable load of a femoral shaft in three-point bending. It is assumed that the beam is a pipe with a surface crack of depth equal to the mean osteon diameter. This code can be used for sensitivity studies of the dependence of whole bone maximum sustainable load on cortical thickness, tissue intrinsic strength and microstructure constant eta. Example calculations are depicted in two relevant figures. 

This publication documents 3D image stacks from HR-pQCT imaging of a femur diaphysis, as well as image stacks for two in-situ loaded fracture mechanics specimens observed with 3D X-ray microscopy. Imaging For HR-qQCT: HR-pQCT scans were acquired by Rachel Surowiec using an XtremeCT II scanner (SCANCO Medical AG, Bruttisellen, Switzerland) within the Musculoskeletal Function, Imaging and Tissue (MSK-FIT) Resource Core of the Indiana Center for Musculoskeletal Health’s Clinical Research Center (Indiana University, Indianapolis, IN). Scans are performed at 60.7 um resolution, a 68 kV, 1467 uA, 43 ms integration time, 1 frame averaging. Raw scans are ‘.RSQ’ file types. The ISQ file type were read into ImageJ using the Import-KHKs Scanco uCT ISQ file reader plug-in, and exported as bmp image stacks, image stacks are provided in two parts. Reconstructed images are rotated in dataviewer so that all bones are in the same orientation (prox/distal/anterior/posterior for the Femur). For in-situ fracture mechanics experiments: 3D scans were acquired by Glynn Gallaway using a 3-point bending rig for single edged notched bend specimens with a Deben CT5000N load cell (Deben, Bury St. Edmunds, UK) in a Zeiss XRADIA 510 Versa 3D X-Ray microscope (Carl Zeiss AG, Baden-Württemberg, Germany) at Purdue University. The 3-point bending frame had a span 20 mm with X-ray transparent, glassy carbon supports. To maintain hydration, the beam was wrapped in a plastic film slit at the notch. Displacements were applied at 0.1 mm/min. Load cell outputs were monitored and recorded. Displacements are held constant during image acquisitions. The first 3D image was obtained at the onset of non-linearity. Subsequently, the displacement was increased until a load increase of 10 N was observed, and another image was obtained. This sequence was repeated 6-times until peak load. 3D X-ray images were acquired with a resolution of 4.5 um, exposure time 5 sec., 801 projections, 120 kV, 10 W, 4 x objective, and a LE2 filter. X-ray projections were processed through XRADIA Scout-and-Scan Reconstructor. A recursive Gaussian smoothing filter (s=1 pixel) was applied to reduce image artifacts. Image stacks are exported as tiff files and provided individually for each load step and specimen. Two experiments are documented (beam 1 and beam 2). MaterialstThe diaphysis of a human (92-year-old, male) cadaveric femur was obtained through the Indiana University School of Medicine Anatomical Donation Program. 

Quasi-brittle fracture mechanics is used to evaluate fracture of human cortical bone in aging. The approach is demonstrated using cortical bone bars extracted from one 92-year-old human male cadaver. In-situ fracture mechanics experiments in a 3D X-ray microscope are conducted. The evolution of the fracture process zone is documented. Fully developed fracture process zone lengths at peak load are found to span about three osteon diameters. Crack deflection and arrest at cement lines is a key process to build extrinsic toughness. Strength and toughness are found as size-dependent, not only for laboratory-scale experimental specimens but also for the whole femur. A scaling law for the length fracture process zone is used. Then, size-independent, tissue fracture properties are calculated. Linear elastic fracture mechanics applied to laboratory beam specimens underestimates the tissue toughness by 60%. Tissue fracture properties are used to predict the load capacity of the femur in bending within the range of documented data. The quasi-brittle fracture mechanics approach allows for the assessment of the combined effect of bone quantity and bone quality on fracture risk. However, further work is needed considering a larger range of subjects and in the model validation at the organ length scale. 

Introduction: Patients with chronic kidney disease (CKD) are at an alarming risk of fracture compared to age and sex-matched non-CKD individuals. Clinical and preclinical data highlight two key factors in CKD-induced skeletal fragility: cortical porosity and reduced matrix-level properties including bone hydration. Thus, strategies are needed to address these concerns to improve mechanical properties and ultimately lower fracture risk in CKD. We sought to evaluate the singular and combined effects of mechanical and pharmacological interventions on modulating porosity, bone hydration, and mechanical properties in CKD. Methods: Sixteen-week-old male C57BL/6J mice underwent a 10-week CKD induction period via a 0.2 % adenine-laced casein-based diet (n = 48) or remained as non-CKD littermate controls (Con, n = 48). Following disease induction (26 weeks of age), n = 7 CKD and n = 7 Con were sacrificed (baseline cohort) to confirm a steady-state CKD state was achieved prior to the initiation of treatment. At 27 weeks of age, all remaining mice underwent right tibial loading to a maximum tensile strain of 2050 μƐ 3× a week for five weeks with the contralateral limb as a non-loaded control. Half of the mice (equal number CKD and Con) received subcutaneous injections of 0.5 mg/kg raloxifene (RAL) 5× a week, and the other half remained untreated (UN). Mice were sacrificed at 31 weeks of age. Serum biochemistries were performed, and bi-lateral tibiae were assessed for microarchitecture, whole bone and tissue level mechanical properties, and composition including bone hydration. Results: Regardless of intervention, BUN and PTH were higher in CKD animals throughout the study. In CKD, the combined effects of loading and RAL were quantified as lower cortical porosity and improved mechanical, material, and compositional properties, including higher matrix-bound water. Loading was generally responsible for positive impacts in cortical geometry and structural mechanical properties, while RAL treatment improved some trabecular outcomes and material-level mechanical properties and was responsible for improvements in several compositional parameters. While control animals responded positively to loading, their bones were less impacted by the RAL treatment, showing no deformation, toughness, or bound water improvements which were all evident in CKD. Serum PTH levels were negatively correlated with matrix-bound water. Discussion: An effective treatment program to improve fracture risk in CKD ideally focuses on the cortical bone and considers both cortical porosity and matrix properties. Loading-induced bone formation and mechanical improvements were observed across groups, and in the CKD cohort, this included lower cortical porosity. This study highlights that RAL treatment superimposed on active bone formation may be ideal for reducing skeletal complications in CKD by forming new bone with enhanced matrix properties. 

A 2D plane strain extended finite element method (XFEM) model was developed to simulate three-point bending fracture toughness tests for human bone conducted in hydrated and dehydrated conditions. Bone microstructures and crack paths observed by micro-CT imaging were simulated using an XFEM damage model. Critical damage strains for the osteons, matrix, and cement lines were deduced for both hydrated and dehydrated conditions and it was found that dehydration decreases the critical damage strains by about 50%. Subsequent parametric studies using the various microstructural models were performed to understand the impact of individual critical damage strain variations on the fracture behavior. The study revealed the significant impact of the cement line critical damage strains on the crack paths and fracture toughness during the early stages of crack growth. Furthermore, a significant sensitivity of crack growth resistance and crack paths on critical strain values of the cement lines was found to exist for the hydrated environments where a small change in critical strain values of the cement lines can alter the crack path to give a significant reduction in fracture resistance. In contrast, in the dehydrated state where toughness is low, the sensitivity to changes in critical strain values of the cement lines is low. Overall, our XFEM model was able to provide new insights into how dehydration affects the micromechanisms of fracture in bone and this approach could be further extended to study the effects of aging, disease, and medical therapies on bone fracture. 

Raloxifene (RAL) reduces clinical fracture risk despite modest effects on bone mass and density. This reduction in fracture risk may be due to improved material level-mechanical properties through a non-cell mediated increase in bone hydration. Synthetic salmon calcitonin (CAL) has also demonstrated efficacy in reducing fracture risk with only modest bone mass and density improvements. This study aimed to determine if CAL could modify healthy and diseased bone through cell-independent mechanisms that alter hydration similar to RAL. 26-week-old male C57BL/6 mice induced with chronic kidney disease (CKD) beginning at 16 weeks of age via 0.2 % adenine-laced casein-based (0.9 % P, 0.6 % C) chow, and their non-CKD control littermates (Con), were utilized. Upon sacrifice, right femora were randomly assigned to the following ex vivo experimental groups: RAL (2 μM, n = 10 CKD, n = 10 Con), CAL (100 nM, n = 10 CKD, n = 10 Con), or Vehicle (VEH; n = 9 CKD, n = 9 Con). Bones were incubated in PBS + drug solution at 37 ◦C for 14 days using an established ex vivo soaking methodology. Cortical geometry (μCT) was used to confirm a CKD bone phenotype, including porosity and cortical thinning, at sacrifice. Femora were assessed for mechanical properties (3-point bending) and bone hydration (via solid state nuclear magnetic resonance spectroscopy with magic angle spinning (ssNMR)). Data were analyzed by two-tailed t-tests (μCT) or 2-way ANOVA for main effects of disease, treatment, and their interaction. Tukey's post hoc analyses followed a significant main effect of treatment to determine the source of the effect. Imaging confirmed a cortical phenotype reflective of CKD, including lower cortical thickness (p < 0.0001) and increased cortical porosity (p = 0.02) compared to Con. In addition, CKD resulted in weaker, less deformable bones. In CKD bones, ex vivo exposure to RAL or CAL improved total work (+120 % and +107 %, respectively; p < 0.05), post-yield work (+143 % and +133 %), total displacement (+197 % and +229 %), total strain (+225 % and +243 %), and toughness (+158 % and +119 %) vs. CKD VEH soaked bones. Ex vivo exposure to RAL or CAL did not impact any mechanical properties in Con bone. Matrix-bound water by ssNMR showed CAL treated bones had significantly higher bound water compared to VEH treated bones in both CKD and Con cohorts (p = 0.001 and p = 0.01, respectively). RAL positively modulated bound water in CKD bone compared to VEH (p = 0.002) but not in Con bone. There were no significant differences between bones soaked with CAL vs. RAL for any outcomes measured. RAL and CAL improve important post-yield properties and toughness in a non-cell mediated manner in CKD bone but not in Con bones. While RAL treated CKD bones had higher matrix-bound water content in line with previous reports, both Con and CKD bones exposed to CAL had higher matrix-bound water. Therapeutic modulation of water, specifically the bound water fraction, represents a novel approach to improving mechanical properties and potentially reducing fracture risk. 

Purpose of Review This review summarizes recent advances in the assessment of bone quality using non-X-ray techniques. Recent Findings Quantitative ultrasound (QUS) provides multiple measurements of bone characteristics based on the propagation of sound through bone, the attenuation of that sound, and different processing techniques. QUS parameters and model predictions based on backscattered signals can discriminate non-fracture from fracture cases with accuracy comparable to standard bone mineral density (BMD). With advances in magnetic resonance imaging (MRI), bound water and pore water, or a porosity index, can be quantified in several long bones in vivo. Since such imaging-derived measurements correlate with the fracture resistance of bone, they potentially provide new BMD-independent predictors of fracture risk. While numerous measurements of mineral, organic matrix, and bound water by Raman spectroscopy correlate with the strength and toughness of cortical bone, the clinical assessment of person’s bone quality using spatially offset Raman spectroscopy (SORS) requires advanced spectral processing techniques that minimize contaminating signals from fat, skin, and blood. Summary Limiting exposure of patients to ionizing radiation, QUS, MRI, and SORS has the potential to improve the assessment of fracture risk and track changes of new therapies that target bone matrix and micro-structure.