Effect of clinostat rotation on differentiation of embryonic bone in vitro.

We have investigated the effect of changes in the gravity vector on osteoblast behaviour, using the clinostat set at 8 rpm. Two sources of osteoblasts were used: secondary cultures of fetal rat bone cells, and the rat osteosarcoma line 17/2.8 (ROS). Cell number was determined by incubation with 3-(4,dimethyl-2yl)-2,3 diphenyl) tetrazolium bromide (MTT) and measurement of optical density at 570 nm (OD). Alkaline phosphatase activity was detected by standard cytochemical methods. Dividing cells were localised by labelling dividing nuclei with Bromodeoxyuridine (BrdU), detected by immunofluorescence. Cell culture was initiated at densities between 1-4x10(4) cells ml-1. Growth rates in all cultures during the first 48 hours exposure to clinostat rotation were less than in stationary controls. After 3 days, ROS cell numbers were 35% lower, and calvarial cells 39% lower than their respective controls. Alkaline phosphatase activity in calvarial control cultures was uniformly present in characteristically polygonal cells, but after culture in the clinostat the enzyme was present sporadically, and the cells were cuboid. There was also no BrdU uptake in nuclei, but it was present in cell cytoplasms. We conclude that the clinostat decreases cell numbers and cell division. Both cell shape and the distribution of alkaline phosphatase activity in calvarial cell cultures were also affected. This implies that changes in the gravity vector can affect osteoblasts directly, without interaction with other cell types.