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.