Osteoporosis is most commonly manifested following menopause when estrogen production is deficient and, as a result, bone loss and changes in bone composition occur leading to bone fractures. Estrogen inhibits bone resorption due to its direct effects on osteoclast differentiation and osteoclast activity, and estrogen deficiency studies have shown an increase in RANKL, a bone resorption marker that binds with RANK on osteoclast surfaces to stimulate their activity. Estrogen also plays an important role in the normal biology of bone cells by regulating their biochemical responses to mechanical loading, such as NO and PGE2 release, and also by protecting against cell death by apoptosis in osteocytes and osteoblasts.
Recent studies have revealed that the effects of estrogen deficiency are not restricted to osteoclasts, but that osteoblasts and osteocytes exhibit an impaired response to mechanical stimulation; however, these changes are not yet fully understood. Although it is known that the osteogenic differentiation of MSCs is regulated by Rho-ROCK signalling, governing actin cytoskeletal changes in response to mechanical stimulation, whether the Rho-ROCK pathway plays a role in the defective bone remodelling cascade during estrogen deficiency has never been established. The global objectives of this PhD thesis are to (a) determine how early estrogen deficiency during osteoporosis governs mechano-responsiveness in osteoblasts and osteocytes and, (b) determine whether ROCK-II inhibition can attenuate estrogen deficiency mediated changes in mechano-responsiveness.
The first study sought to determine alterations in the mechanobiological responses of osteoblasts during early estrogen deficiency and investigate whether an inhibitor of the Rho-ROCK signalling can revert these changes. The results of this study showed that mechanobiological responses (PGE2, RUNX-2 and OPN) were significantly altered in estrogen deficient cells when compared to those that received continuous estrogen treatment. This study is the first to demonstrate such altered mechanobiological responses by osteoblasts during early estrogen deficiency. Interestingly, it was seen that these mechanobiological responses were suppressed when estrogen deficient cells were treated with the ROCK-II inhibitor, and thus pointed to a potential for ROCK-II inhibition for reverting these mechanobiological effects.
Given the paracrine role of osteocytes in regulating bone remodelling by osteoclasts, it is likely that changes in osteocyte mechanobiology play an additive role in the bone loss cascade during estrogen deficiency, but this has never been investigated. The second study sought to investigate osteocyte-mediated osteoclastogenesis during early estrogen deficiency, and the effect of ROCK-II inhibition on osteocyte mediated osteoclastogenesis in vitro. A co-culture approach was undertaken to study the influence of paracrine cell-cell communication from mechanically stimulated MLO-Y4 osteocytes on osteoclasts. The results of this study demonstrated that during estrogen deficiency mechanically stimulated osteocytes increase RANKL and M-CSF gene expression, when compared to estrogen-treated controls. Interestingly these effects were suppressed in MLO-Y4 cells upon treatment with ROCK inhibitor, Y27632. Hence, it was proposed that osteocyte mediated osteoclastogenesis is exacerbated by estrogen deficiency and that ROCK-II inhibition has the potential to override these effects through downregulation of pro-osteoclastogenic signalling.
Calcium oscillations (Ca2+) by osteoblasts and osteocytes are amongst the very first mechanobiological responses that take place intracellularly. Recently it has been demonstrated that Ca2+ oscillations by mechanically stimulated osteocytes are altered during estrogen deficiency in vitro. In vivo, osteocytes communicate with osteoblasts through gap junctions, and, as such, it is possible that changes in Ca2+ oscillations in osteocytes also propagate changes in Ca2+ signalling in osteoblasts. However, these changes have not yet been studied. The third study in this PhD sought to examine how altered calcium signalling in mechanically stimulated osteocytes affects calcium signalling in osteoblasts in their vicinity during early estrogen deficiency. A microfluidics system was used to mimic an in vitro multi-cellular model that enables to study real-time osteocyte mediated Ca2+ signalling in osteocytes and osteoblasts. Using the microfluidics approach, it was possible to capture the effects of signal propagation as well as paracrine factors from osteocytes onto osteoblasts. The results of this study revealed that osteocytes exhibited higher Ca2+ peaks in response to mechanical stimulation during estrogen deficiency conditions. Moreover, osteoblasts cultured in their vicinity, which were not mechanically stimulated, exhibited attenuated calcium signalling peaks during estrogen deficiency.
Altogether, these studies provide an advanced understanding of alterations in the mechanobiology of osteoblasts and osteocytes during early estrogen deficiency. It is also demonstrated that inhibition of ROCK-II has the potential to revert these changes in osteocytes and osteoblasts. However, further research studies are required to explore the direct therapeutic potential of ROCK-II inhibition in targeting osteoclasts during estrogen deficiency. This PhD thesis provides a deeper understanding of the affected mechanobiology in osteoporosis at cellular level.