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Post-radiotherapy bone fragility: investigating osteoclast activity and novel preventative treatments
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Oest, Megan
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Fall 2025
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2025-08-22
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Introduction: Post-radiotherapy bone fragility is a complex disorder that often results in fragility fractures. These fractures are difficult to predict and often result in complications such as nonunion. Currently no preventative treatments are available to prevent fragility and subsequent fractures following irradiation. One of the main drivers that has stalled development of treatments is a lack of understanding of the cellular mechanisms that drive this condition. Murine model data has identified a short-term increase in osteoclast activity followed by a long-term loss of osteoclasts after radiotherapy. However, the mechanisms that underly this osteoclast dysregulation are unknown. We hypothesized that irradiation directly contributes to the short-term increase in osteoclast activation following irradiation. The overall aim of this dissertation is to identify the cellular mechanisms of post-radiotherapy bone fragility to identify druggable targets for the design of preventative treatments.
Methods: Both in vitro and in vivo methods were used in this dissertation. Osteoclast in vitro studies used a combination of primary murine cells isolated from the bone marrow and the well-established pre-osteoclast like murine RAW 264.7 cell line. Techniques such as qRT-PCR, microscopy, and resorption pit assays were used to understand the transcriptional and functional activity of bone cells following radiotherapy. In vivo studies used female Balb/c mice which received 4 consecutive daily doses of 5 Gy irradiation to the right hindlimb only. Histology, μCT and mechanical testing, among other techniques, were utilized to analyze in vivo samples.
Results: Data from in vitro analysis of osteoclasts showed that osteoclast activation following irradiation is dependent on RANK-L. Irradiated osteocytes significantly increased osteoclast differentiation however osteocytes did not increase RANK-L transcription. In vitro, quercetin decreased osteoclastogenesis in a dose-dependent manner. However, quercetin food supplements did little to attenuate markers of bone fragility in our in vivo mouse model. Caloric restriction attenuated some markers of bone fragility following irradiation in our mouse model, however our preliminary caloric restriction protocol induced signs of stress in mice. Our updated caloric restriction protocol using a cage divider system eliminated signs of stress seen in the preliminary protocol. The updated caloric restriction model was most effective in attenuating short-term markers of bone fragility.
Conclusion: The data presented in this study suggest that short-term osteoclast activation is reliant on RANK-L signaling. Based on in vitro data, osteocytes may play a role in osteoclast activation through secretion of factors that activate RANK/RANK-L signaling cascade. Quercetin showed promise in attenuating the short-term increase in osteoclast activation based on in vitro data, however quercetin supplementation in our murine model did not have a large effect on bone fragility. Improved delivery methods of quercetin may improve efficacy in future studies. Caloric restriction is a promising therapeutic option for post-radiotherapy bone fragility. In vivo data demonstrated improvement in short-term fragility markers. More study is needed to better understand the long-term effects of caloric restriction on bone.
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