Suppression of osteoblast activity by disuse is prevented by low magnitude mechanical loading through a bone morphogenic protein-dependent Mechanism
Patel, Mamta Jashvantlal
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Musculoskeletal pathologies associated with decreased bone mass, including osteoporosis and disuse-induced bone loss, affect millions of Americans annually. Many pharmaceutical treatments have slowed osteoporosis, but there is still no countermeasure for bone loss observed in astronauts. Additionally, high magnitude and low frequency impact has been recognized to increase bone and muscle mass under normal but not microgravity conditions. However, a low magnitude and high frequency (LMHF) mechanical load experienced in activities such as postural control has also been shown to be anabolic to bone. While several clinical trials have demonstrated that the LMHF mechanical loading normalizes bone loss in vivo, the target tissues and cells of the mechanical load and underlying mechanisms mediating the responses are unknown. As such, the objectives of this project are to analyze cellular and molecular changes induced in osteoblasts by LMHF loading and to investigate the utility of a LMHF mechanical load in mitigating microgravity-induced bone loss. The central hypothesis of the project is that simulated microgravity or disuse conditions induce bone loss by inhibiting expression of genes critical in regulating bone formation, osteoblast differentiation, and subsequent mineralization while a LMHF mechanical load prevents these effects. To test this hypothesis, we developed an in vitro disuse system using the Random Positioning Machine (RPM). For the first time, we reported systemic gene expression studies in 2T3 preosteoblasts using the RPM disuse system showing that 140 genes were altered by RPM exposure with over two-fold statistically significant changes. Moreover, we also utilized an independent simulator called the Rotating Wall Vessel (RWV) to partially validate the in vitro disuse systems and to confine the list of genes to those most critical in regulating bone formation. After comparative studies, we constricted the list to 15 commonly changed genes, three of which were not only decreased with disuse but also increased with mechanical loading in vivo. Furthermore, we employed the RPM disuse system to evaluate the mechanism by which a LMHF load mitigates bone loss. Exposure of osteoblasts to the RPM decreased both ALP activity and mineralization even in the presence of bone morphogenic protein 4 (BMP4), and the LMHF mechanical loading prevented the RPM-induced decrease in both markers. Mineralization induced by LMHF mechanical loading was enhanced by treatment with BMP4 and blocked by the BMP antagonist noggin, suggesting a role for BMPs in this response. In addition, LMHF mechanical loading rescued the RPM-induced decrease in gene expression of ALP, runx2, osteomodulin, parathyroid hormone receptor 1, and osteoglycin. These findings show that osteoblasts directly respond to LMHF mechanical loading, potentially leading to normalization or prevention of bone loss caused by disuse or microgravity conditions. The mechanosensitive genes identified here provide potential targets for pharmaceutical treatments that may be used in combination with LMHF mechanical loading to better treat osteoporosis, disuse-induced bone loss, or microgravity-induced bone loss.