Culture in vector-averaged gravity under clinostat rotation results in apoptosis of osteoblastic ROS 17/2.8 cells

Space flight experiments and studies carried out in altered gravity environ ments have revealed that exposure to altered gravity conditions results in (mal)adaptation of cellular function. In the present study, we used a clino stat to generate a vector-averaged gravity environment. We then evaluated t he responses of osteoblast-like ROS 17/2.8 cells subsequent to rotation at 50 revolutions per minute (rpm) for 6-24 h, We found that the cells started to detach from the substrate between 12 h and 24 h of rotation in clinosta t but not in stationary cultures or after horizontal rotation (the latter s erving as a motion control for turbulence, shear forces, and vibrations). A t 24 h, 35% of clinorotated cells had detached and the cells underwent apop totic death as evidenced by DNA fragmentation analysis, terminal deoxynucle otidyl transferase-mediated deoxyuridine triphosphate-biotin nick end label ing (TUNEL) staining, and flow cytometry with Annexin V staining. The apopt otic death was associated with perinuclear distribution of cell-surface int egrin beta 1 and disorganization of actin cytoskeleton, These results sugge st that vector-averaged gravity causes apoptosis of osteoblasts by altering the organization of the cytoskeleton, We hypothesize that apoptotic death of osteoblasts might play an important role in the pathogenesis of osteopor otic bone loss as observed in actual space flights.