Experiments with osteoblasts cultured under varying orientations with respect to the gravity vector

Abstract

Substrate attachment is crucial for normal growth and differentiation of many cell types. To better understand the role of gravity in osteoblast attachment and growth in vitro, 17-day-old embryonic chick calvarial osteoblasts were subjected to directional variations with respect to gravity. Osteoblasts, grown in MEM or DME supplemented with 10% FBS and attached to type I collagen-coated coverslips, were loaded into cylindrical containers completely filled with medium and oriented so that cells were either atop or beneath, or coverslips continuously rotated (∼2 rpm) in a clinostat, thereby continuously changing their orientation with respect to gravity. Cells in these three conditions were collected daily for up to 6 days, and cell viability, two osteoblast functions, and proliferation were assessed. Data suggest the number and function of attached osteoblasts is unaltered by inversion or clino-rotation in initially confluent cultures. In sparsely plated cultures, however, osteoblast viability was significantly decreased (∼50%) in inverted and rotated cultures during the first 3 days of sampling, but from days 4–6 no significant difference was found in viable cell number for the three conditions. Decreases in viable cell number within the first days of the experiments could result from death followed by detachment, detachment followed by death, differences in proliferation rate, or lag-phase duration. To help distinguish among these, BrdU labeling for 2 or 24 hr was used to assess cell proliferation rate. Log-phase growth rates were calculated and were unchanged among the three conditions tested. These results point to an increase in lag-phase duration in inverted and rotated cultures. In summary, changing the cell-substrate attachment direction with respect to gravity causes an immediate response in the form of diminished viable osteoblast number in sparse, early cultures, but the effect disappears after 3–4 days and does not occur in mature, confluent cultures.