Impairment of 7F2 osteoblast function by simulated partial gravity in a Random Positioning Machine

Abstract

The multifaceted adverse effects of reduced gravity pose a significant challenge to human spaceflight. Previous studies have shown that bone formation by osteoblasts decreases under microgravity conditions, both real and simulated. However, the effects of partial gravity on osteoblasts' function are less well understood. Utilizing the software-driven newer version of the Random Positioning Machine (RPM^SW), we simulated levels of partial gravity relevant to future manned space missions: Mars (0.38 G), Moon (0.16 G), and microgravity (Micro, ~10^-3 G). 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. Our data suggest 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. Our study is of relevance, given the dearth of research into the effects of Lunar and Martian gravity for forthcoming space exploration.

Fig. 1. Inhibition of 7F2 cell proliferation in simulated partial gravity.

a Increase in cell numbers on days 2 and 4 of culture in 1 G (Earth) and under the various altered simulated partial gravity conditions (Mars, Moon, Micro) (N = 3). b Specific partial population doubling times between days 2 and 4 and taking into account the mean absolute error (MAE). The average population doubling times were calculated as 0.96 ± 0.14 days for Earth (1 G), 1.4 ± 0.08 days for Mars, 1.5 ± 0.30 days for Moon, and 2.95 ± 0.17 days for simulated microgravity (Micro). c Semilogarithmic plot of the specific population doubling times vs. simulated partial gravity (R² = 0.9919) for that 48-h window of time. Data are presented as means ± standard deviation. Asterisk (*) shows p < 0.05, (**) shows p < 0.01, (***) p < 0.001 as determined by Tukey’s post hoc analysis (panels (a) and (c)) or mean absolute error (MAE) (panel (b)).

Fig. 2. Simulated partial gravity inhibits osteogenic differentiation in 7F2 osteoblasts.

a ALP activity as a result of exposure to different simulated partial-gravity conditions at different time points (N = 3). b ALP activity as a function of time between day 2 and day 6. c ALP activity as a function of altered simulated partial gravities at day 6. Data are presented as means ± standard deviation. Asterisk (*) shows p < 0.05, (**) shows p < 0.01, (***) p < 0.001 as determined by Tukey’s post hoc analysis.

Fig. 3. Effects of simulated partial gravities on osteogenic marker gene expression.

a Time course of ALPL gene expression for multiple simulated gravity conditions, following exposure of confluent 7F2 osteoblasts to osteogenic media. Relative expression was analyzed by real-time PCR and normalized to the levels of a housekeeping gene (GAPDH), with 1 G confluent cell culture (prior to osteogenic induction) as control. The relative expression of each gene to control is presented as a fold-change expression for each transcript. b Inhibition by simulated partial gravity of the three osteogenic marker genes is best discernable at the peak of their expression at day-6 Alkaline phosphatase (ALPL), Runt-related Transcription Factor 2 (RUN), Sparc/osteonectin (ON). Data are presented as means ± standard deviation. Values are means ± SD of three independent cultures. Asterisk (*) shows p < 0.05, (**) shows p < 0.01, (***) p < 0.001 as determined by Tukey’s post hoc analysis.

Fig. 4. Effect of simulated partial gravity on long-term mineralization.

a Alizarin red-stained mineralized nodules after 18 days on Earth (1 G) and in the RPM exposed to simulated partial -gravities of Moon and Mars. Images are representative micrographs of 1 × 1 cm² areas from the bottom of T-12.5 Falcon™ Tissue Culture Treated Flasks. b Long-term mineralization under variable simulated gravity conditions, quantified as micrograms of calcium per square centimeter (µg/cm²). c Percentage inhibition of mineralization normalized versus Earth controls with modeled trendlines for various days: 14 days (R² = 0.8708), 16 days (R² = 0.92), 18 days (R² = 0.9735), 21 days (R² = 0.9308). d Three-dimensional surface plot and heatmap of the interplay between simulated gravity levels and mineralization over time. Data are presented as means ± standard deviation (N = 3). Asterisk (*) shows p < 0.05, (**) shows p < 0.01, (***) p < 0.001 as determined by Tukey’s post hoc analysis.