INHIBITED MINERALIZATION IN OSTEOBLASTS CULTURED UNDER VARIED SIMULATED PARTIAL GRAVITY CONDITIONS AND THE USE OF PHYTONUTRIENTS FOR MITIGATING THE EFFECTS OF REDUCED GRAVITY

Abstract

The multifaceted adverse effects of reduced gravity on the skeletal system pose a significant challenge to human spaceflight. There is an interest in investigating any hypothetical differences between partial gravity and microgravity, and in the unmet need to identify countermeasures to both. A hypothesis to be tested is that reduced gravity impairs a variety of osteogenic cell functions, such as proliferation and differentiation, and that these inhibitory effects can be mitigated by nutritional countermeasures or by interrupting signaling pathways that drive undesired osteogenic remodeling. Utilizing the Random Positioning Machine, it is possible to simulate a variety of reduced gravity levels relevant to future manned space missions: Mars, Moon, and Microgravity of the Low Earth Orbit (LEO) environment. In this study, the effects of altered gravity on the physiology and morphology of cultured osteoblasts were investigated, specifically on their proliferation, osteogenic differentiation, and matrix mineralization. In assessing the role of mechanotransduction in microgravity-induced cytoskeletal dysfunction, this thesis also explored whether selective inhibition of specific signaling steps within the Rho-ROCK pathway can be used to modulate the effects of microgravity on osteoblast differentiation and function. Finally, in developing new countermeasures, an investigation was made into the effectiveness of curcumin and carnosic acid, two nutritional antioxidants with pro-osteogenic properties, contrasted with the trace element zinc, as potential alimentary supplements that may mitigate or alleviate the deleterious effects of microgravity. Results showed that short-term (6 days) culture yielded a dose-dependent reduction in proliferation and the enzymatic activity of alkaline phosphatase (ALP), while long-term studies (21 days) showed a distinct dose-dependent inhibition of mineralization. By contrast, expression levels of key osteogenic genes (Alkaline phosphatase, Runt-related Transcription Factor 2, Sparc/osteonectin) exhibited a threshold behavior: gene expression was significantly inhibited when the cells were exposed to Mars-simulating partial gravity, and this was not reduced further when the cells were cultured under simulated Moon or microgravity conditions. My data suggests that impairment of cell function with decreasing simulated gravity levels is graded and that the threshold profile observed for reduced gene expression is distinct from the dose dependence observed for cell proliferation, ALP activity, and mineral deposition. My studies into the gravity-induced re-organization of the cytoskeleton indicate that selective interruption of the Rho-ROCK pathway at ROCK can prevent morphological changes that result in impaired differentiation and mineralization. Further, I found that nutraceuticals partially reversed the inhibitory effects of SMG on ALP activity and promoted osteoblast proliferation and differentiation in the absence of traditional osteogenic media. I further observed a synergistic effect of the intermix of the phytonutrients on ALP activity. Intermixes of phytonutrients may serve as convenient and effective nutritional countermeasures against bone loss in space.