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1. Ex vivo exposure to calcitonin or raloxifene improves mechanical properties of diseased bone through non-cell mediated mechanisms

   https://doi.org/10.1016/j.bone.2023.116805  

   Surowiec, Rachel K.; Saldivar, Rosario; Rai, Ratan K.; Metzger, Corinne E.; Jacobson, Andrea M.; Allen, Matthew R.; Wallace, Joseph M. (August 2023, Bone)

   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. 

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2. Combining raloxifene and mechanical loading improves bone composition and mechanical properties in a murine model of chronic kidney disease (CKD)

   https://doi.org/10.1016/j.bone.2024.117089  

   Surowiec, Rachel K.; Reul, Olivia N.; Chowdhury, Nusaiba N.; Rai, Ratan K.; Segvich, Dyann; Tomaschke, Andrew A.; Damrath, John; Jacobson, Andrea M.; Allen, Matthew R.; Wallace, Joseph M. (June 2024, Bone)

   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. 

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3. Effects of novel raloxifene analogs alone or in combination with mechanical loading in the Col1a2G610c/+ murine model of osteogenesis imperfecta

   https://doi.org/10.1016/j.bone.2023.116970  

   Kohler, Rachel; Creecy, Amy; Williams, David R; Allen, Matthew R; Wallace, Joseph M (February 2024, Bone)

   Osteogenesis imperfecta (OI) is a hereditary bone disease in which gene mutations affect collagen formation, leading to a weak, brittle bone phenotype that can cause severe skeletal deformity and increased fracture risk. OI interventions typically repurpose osteoporosis medications to increase bone mass, but this approach does not address compromised tissue-level material properties. Raloxifene (RAL) is a mild anti-resorptive used to treat osteoporosis that has also been shown to increase bone strength by a-cellularly increasing bone bound water content, but RAL cannot be administered to children due to its hormonal activity. The goal of this study was to test a RAL analog with no estrogen receptor (ER) signaling but maintained ability to reduce fracture risk. The best performing analog from a previous analog characterization project, named RAL-ADM, was tested in an in vivo study. Female wildtype (WT) and Col1a2G610C/+ (G610C) mice were randomly assigned to treated or untreated groups, for a total of 4 groups (n=15). Starting at 10 weeks of age, all mice underwent compressive tibial loading 3×/week to induce an anabolic bone formation response in conjunction with RAL-ADM treatment (0.5 mg/kg; 5×/week) for 6 weeks. Tibiae were scanned via microcomputed tomography then tested to failure in four-point bending. RAL-ADM had reduced ER affinity, and increased post-yield properties, but did not improve bone strength in OI animals, suggesting some properties can be improved by RAL analogs but further development is needed to create an analog with decidedly positive impacts to OI bone. 

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4. In Vivo Assessment of Bone Quality Without X-rays

   https://doi.org/10.1007/s11914-023-00856-w  

   Surowiec, Rachel K.; Does, Mark D.; Nyman, Jeffry S. (February 2024, Current Osteoporosis Reports)

   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. 

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5. Image Data and Analysis Materials for In-situ Fracture Mechanics Experiments on Human Cortical Bone Treated Ex-vivo with Raloxifene

   https://doi.org/10.4231/F557-WQ66  

   Gallaway, Glynn; Pyrak-Nolte, Laura; Allen, Matthew; Siegmund, Thomas; Gallaway, Glynn; Siegmund, Thomas (January 2024, Purdue University Research Repository)

   This publication documents 3D image stacks for two in-situ loaded fracture mechanics specimens observed with 3D X-ray microscopy, 2D image stacks of the crack mouth opening displacement during said loading, and analysis codes which supported the publication. Materials: The diaphysis of a human (75-year-old, male) cadaveric femur was obtained through the Indiana University School of Medicine Anatomical Donation Program. Nominal 4 mm x 4 mm x 24 mm beams were sectioned from the femur diaphysis. Experiments were conducted on 12 beams. Beams were assigned to 2 groups: treated with Raloxifene (RAL), treated with a vehicle (VEH) control. Image data are provided for one beam from the RAL group and one beam from the VEH group. Additional images can be made available upon request to the corresponding author. Imaging: ;For in-situ fracture mechanics experiments: 2D and 3D scans were acquired by Glynn Gallaway using a 4-point bending rig for single edged notched bend specimens in a water bath 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-Wuerttemberg, Germany) at Purdue University. The 4-point bending frame had a span 16 mm with X-ray transparent, glassy carbon supports. To maintain hydration, bending frame was situated in a waterbath filled with DI water. Displacements were applied at 0.1 mm/min. Load cell outputs were monitored and recorded. During loading 2D images were obtained every 1 second. 3D X-ray images were acquired with a resolution of 4.5 um, exposure time 6 sec., 2401 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 (1 pixel) was applied to reduce image artifacts. Image stacks are exported as tiff files and provided for each specimen. Images are provided for one RAL treated sample and one VEH treated sample. 

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