Simulated Microgravity Environments Enhance Preosteoblast Mineralization Rates

Previous work has described the use of simulated microgravity as a means to study cell and molecular mechanisms associated with three-dimensional (3D) tissue growth. Our lab has shown that when grown in normal two-dimensional (2D) micromass conditions, human embryonic palatal mesenchymal (HEPM) preosteoblasts supplemented with beta-glycerophosphate (beta-GP) and ascorbic acid (AA) mineralize in approximately 4 weeks. Objective: The objective of this study was to investigate the hypothesis that microgravity environments enhance the mineralization rate of HEPM preosteoblasts. Methods: HEPM cells were cultured in either a rotary vessel to simulate microgravity, or on tissue culture plastic as the normal control for 7 days in either treated (5mM beta-GP and 50ug/ml ascorbate) or untreated media. Alizarin Red S histological staining and EDAX elemental analysis were used to analyze calcium levels. SEM and TEM methods were used to analyze microstructural changes. Results: As compared to normal monolayer cultures, microgravity environments promoted aggregate formations by 2 days. Increased calcium and phosphorous deposition was significantly enhanced 3-18 fold (P<0.001) in microgravity cultures as compared to tissue culture plastic. Microgravity cultures mineralized in less than 1 week as compared to normal monolayer cultures which took nearly 4 weeks. This represented a decrease in mineralization rate of nearly 75%. Although morphologically different, cellular aggregates formed in both treated and nontreated microgravity conditions, but increased calcium deposition was only seen in treated aggregates. When aggregates grown in microgravity environments were re-introduced into normal nonmicrogravity conditions, cell migration from the aggregates was noted demonstrating cell viability if reintroduced into normal monolayer conditions. Conclusions: These results suggest that the microgravity environments promote three dimensional aggregate formations and enhance the rate of mineralization of preosteoblasts. This may translate into the use of microgravity environments for novel osseous tissue engineering strategies. Supported by R03-DE014269 (GS) and P60DE13076 (GS).