Using acoustic waves to modulate stem cell growth and differentiation

During spaceflight, the loss of mechanical loads due to microgravity leads to rapid bone loss, where bone deteriorates at a rate of 1-2% per month, where some astronauts can lose as much as 20% of their skeletal mass in a single expedition [NASA, 2001]. In order to prevent muscle and bone loss, longterm space flight exercise regimes are strictly implemented [Shackleford, 2004]. Current research has demonstrated that mechanical vibrations can help to maintain or improve bone mass [Chan, 2013] and reduce adiposity [Chen, 2015, Sen, 2011] when signals are applied at the appropriate frequency and amplitude. We have developed an acoustic sound chamber that can apply sound waves to stem cells grown in vitro. Characterization of the culture conditions inside the vibration chamber showed considerable variance across the culture plates where an applied acceleration of 0.6g varied at different spots in a 12-well tissue culture plate from as low as 0.47g to 0.78g. We believe the variance is caused by differences in the rigidity of the culture plates that makes the waves transmit inconsistently through the plastic. We hypothesized acoustic waves would induce osteogenic differentiation when applied to stem cells. We utilized pre-osteoblastic stem cells (MC3T3-E1-Subclone 4) to observe the effects of acoustic waves when applied at 0.3g and 0.6g, compared to non-vibrated controls. Cells were vibrated for 30 minutes a day for either 6 days (n=24/group) or 12 days (n=12/group). Cellular changes were characterized by assessing well-by-well cell number by a manual cell count and mineral content by Alizarin Red S staining. Differences between groups were determined using One-Way ANOVA with a post hoc test: Student's t-test. To assess the effects of the variance across the culture plates, correlative analysis was conducted for well-by-well variation using Regression Analysis. Acoustically vibrated wells had 10x more cells after 6 days and showed more mineralization than non-vibrated wells at both 6 and 12 days. Acoustic waves have the ability to increase cell proliferation and can drive stem cell differentiation towards an osteoblastic lineage, this could lead to therapies that prevent bone loss during spaceflight.