Raloxifene treatment has been shown previously to positively affect bone mechanical properties following 1 year of treatment in skeletally mature dogs. Reference point indentation (RPI) can be used for in vivo assessment of mechanical properties and has been shown to produce values that are highly correlated with properties derived from traditional mechanical testing. The goal of this study was to use RPI to determine if raloxifene-induced alterations in mechanical properties occurred after 6 months of treatment. Twelve skeletally mature female beagle dogs were treated for 6 months with oral doses of saline vehicle (VEH, 1 ml/kg/day) or a clinically relevant dose of raloxifene (RAL, 0.5 mg/kg/day). At 6 months, all animals underwent in vivo RPI (10 N force, 10 cycles) of the anterior tibial midshaft. RPI data were analyzed using a custom MATLAB program, designed to provide cycle-by-cycle data from the RPI test and validated against the manufacturer-provided software. Indentation distance increase (IDI), a parameter that is inversely related to bone toughness, was significantly lower in RAL-treated animals compared to VEH (− 16.5%), suggesting increased bone toughness. Energy absorption within the first cycle was significantly lower with RAL compared to VEH (− 21%). These data build on previous work that has documented positive effects of raloxifene on material properties by showing that these changes exist after 6 months.
Young, skeletally mature mice lacking Cx43 in osteocytes exhibit increased osteocyte apoptosis and decreased bone strength, resembling the phenotype of old mice. Further, the expression of Cx43 in bone decreases with age, suggesting a contribution of reduced Cx43 levels to the age-related changes in the skeleton. We report herein that Cx43 overexpression in osteocytes achieved by using the DMP1-8kb promoter (Cx43OT mice) attenuates the skeletal cortical, but not trabecular bone phenotype of aged, 14-month-old mice. The percentage of Cx43-expressing osteocytes was higher in Cx43OT mice, whereas the percentage of Cx43 positive osteoblasts remained similar to wild type (WT) littermate control mice. The percentage of apoptotic osteocytes and osteoblasts was increased in aged WT mice compared to skeletally mature, 6-month-old WT mice, and the percentage of apoptotic osteocytes, but not osteoblasts, was decreased in age-matched Cx43OT mice. Aged WT mice exhibited decreased bone formation and increased bone resorption as quantified by histomorphometric analysis and circulating markers, compared to skeletally mature mice. Further, aged WT mice exhibited the expected decrease in bone biomechanical structural and material properties compared to young mice. Cx43 overexpression prevented the increase in osteoclasts and decrease in bone formation on the endocortical surfaces, and the changes in circulating markers in the aged mice. Moreover, the ability of bone to resist damage was preserved in aged Cx43OT mice both at the structural and material level. All together, these findings suggest that increased Cx43 expression in osteocytes ameliorates age-induced cortical bone changes by preserving osteocyte viability and maintaining bone formation, leading to improved bone strength.
Introduction Bisphosphonates are highly effective in reducing fracture risk yet some individuals treated with these agents still experience fracture. The goal of this study was to test the hypothesis that genotype influences the effect of zoledronate on bone mechanical properties. Methods Skeletally mature male mice from genetic backgrounds known to have distinct baseline post-yield properties (C57/B6, high post-yield displacement; A/J, low post-yield displacement) were treated for 8 weeks with saline (VEH) or zoledronate (ZOL, 0.06 mg/kg subcutaneously once every four weeks) in a 2×2 study design. Ex vivo µCT and mechanical testing (4-pt bending) were conducted on the femur to assess morphological and mechanical differences. Results Significant drug and/or genotype effects were found for several mechanical properties and significant drug × genotype interactions were found for measures of strength (ultimate force) and brittleness (total displacement, strain to failure). Treatment with ZOL affected bone biomechanical measures of brittleness (total displacement (−25%) and strain to failure (−23%)) in B6 mice significantly differently than in A/J mice. This was driven by unique drug × genotype effects on bone geometry in B6 animals yet likely also reflected changes to the tissue properties. Conclusion These data may support the concept that properties of the bone geometry and/or tissue at the time of treatment initiation play a role in determining the bone’s mechanical response to zoledronate treatment.
Loss of bone and muscle mass are two major clinical complications among the growing list of chronic diseases that primarily affect elderly individuals. Persistent low-grade inflammation, one of the major drivers of aging, is also associated with both bone and muscle dysfunction in aging. Particularly, chronic activation of the receptor for advanced glycation end products (RAGE) and elevated levels of its ligands high mobility group box 1 (HMGB1), AGEs, S100 proteins and Aβ fibrils have been linked to bone and muscle loss in various pathologies. Further, genetic or pharmacologic RAGE inhibition has been shown to preserve both bone and muscle mass. However, whether short-term pharmacologic RAGE inhibition can prevent bone and muscle early loss in aging is unknown. To address this question, we treated young (4-mo) and middle-aged (15mo) C57BL/6 female mice with vehicle or Azeliragon, a small-molecule RAGE inhibitor initially developed to treat Alzheimer's disease. Azeliragon did not prevent the aging-induced alterations in bone geometry or mechanics, likely due to its differential effects [direct vs. indirect] on bone cell viability/function. On the other hand, Azeliragon attenuated the aging-related body composition changes [fat and lean mass] and reversed the skeletal muscle alterations induced with aging. Interestingly, while Azeliragon induced similar metabolic changes in bone and skeletal muscle, aging differentially altered the expression of genes associated with glucose uptake/metabolism in these two tissues, highlighting a potential explanation for the differential effects of Azeliragon on bone and skeletal muscle in middle-aged mice. Overall, our findings suggest that while short-term pharmacologic RAGE inhibition did not protect against early aging-induced bone alterations, it prevented against the early effects of aging in skeletal muscle.
Reference point indentation (RPI) was developed as a novel method to assess mechanical properties of bone in vivo, yet it remains unclear what aspects of bone dictate changes/differences in RPI-based parameters. The main RPI parameter, indentation distance increase (IDI), has been proposed to be inversely related to the ability of bone to form/tolerate damage. The goal of this work was to explore the relationship between RPI parameters and traditional mechanical properties under varying experimental conditions (drying and ashing bones to increase brittleness, demineralizing bones and soaking in raloxifene to decrease brittleness). Beams were machined from cadaveric bone, pre-tested with RPI, subjected to experimental manipulation, post-tested with RPI, and then subjected to four-point bending to failure. Drying and ashing significantly reduced RPI’s IDI, as well as ultimate load (UL), and energy absorption measured from bending tests. Demineralization increased IDI with minimal change to bending properties. Ex vivo soaking in raloxifene had no effect on IDI but tended to enhance post-yield behavior at the structural level. These data challenge the paradigm of an inverse relationship between IDI and bone toughness, both through correlation analyses and in the individual experiments where divergent patterns of altered IDI and mechanical properties were noted. Based on these results, we conclude that RPI measurements alone, as compared to bending tests, are insufficient to reach conclusions regarding mechanical properties of bone. This proves problematic for the potential clinical use of RPI measurements in determining fracture risk for a single patient, as it is not currently clear that there is an IDI, or even a trend of IDI, that can determine clinically relevant changes in tissue properties that may contribute to whole bone fracture resistance.
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