Experiments With Osteoblasts Cultured Under Hypergravity Conditions
bone; cells; differentiation; Engineering; expression; human dermal fibroblasts; in-vitro; Mechanics; microgravity; rats; space-flight; system; Thermodynamics
To understand further the role of gravity in osteoblast attachment, osteoblasts were subjected to hypergravity conditions in vitro. Scanning electron microscopy of all confluent coverslips from FPA units show that the number of attached osteoblasts was similar among gravitational levels and growth durations (similar to90 cells/microscopic field). Specifically. confluent 1.0G control cultures contained an average of 91+/-8 cells/field, 3.3G samples had 88+/-8 cells/field, and 4.0G cultures averaged 90+/-7 cells/field. The sparsely plated cultures assessed by immunohistochemistry also had similar numbers of cells at each time point (1.0G was similar to 3.3 and 4.0G), but cell number changed from one time point to the next as those cells proliferated Immunohistochemistry of centrifuged samples showed an increase in number (up to 160% increase) and thickness (tip to 49% increase) of actin fibers, a decrease in intensity of fibro-nectin fluorescence (18-23% decrease) and an increase in number of vinculin bulbs (202-374% increase in number of vinculin bulbs/area). While hypergravity exposure did not alter the number of attached osteoblasts, it did result in altered actin, fibronectin, and vinculin elements, changing some aspects of osteoblast-substrate adhesion.
Kacena M A; Todd P; Gerstenfeld L C; Landis W J
Microgravity Science and Technology
2004
2004
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1007/bf02870949" target="_blank" rel="noreferrer noopener">10.1007/bf02870949</a>
Experiments With Osteoblasts Cultured Under Varying Orientations With Respect To The Gravity Vector
attachment; averaged gravity; Biotechnology & Applied Microbiology; bone; Cell Biology; cell-culture; clinostat; gene-expression; growth; growth rate; in-vitro; inversion; microgravity; osteoblasts; proliferation; space; substrate
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 (similar to2 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 (similar to50%) 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.
Kacena M A; Todd P; Gerstenfeld L C; Landis W J
Cytotechnology
2002
2002
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1023/a:1023936503105" target="_blank" rel="noreferrer noopener">10.1023/a:1023936503105</a>
Osteoblasts Subjected To Spaceflight And Simulated Space Shuttle Launch Conditions
acceleration; bone cells; bone-formation; Cell Biology; cells; Developmental Biology; differentiation; flight; gene-expression; growing rats; in-vitro; messenger-rna expression; microgravity; vibration; weightlessness
To understand further the effects of spaceflight on osteoblast-enriched cultures, normal chicken calvarial osteoblasts were flown aboard shuttle flight STS-77, and the total number of attached cells was determined. Spaceflight and control cultures were chemically fixed 3 h and 3 d after launch. These fixed cultures were processed for scanning electron microscopy (SEM). The SEM analysis showed that with just 3 d of exposure to spaceflight, coverslip cultures contained 300 +/- 100 cells/mm(2), whereas 1G control samples contained a confluent monolayer of cells (2400 +/- 200 cells/mm(2)). Although the cultures flown in space experienced a drastic decline in cell number in just 3 d, without further experimentation it was impossible to determine whether the decline was a result of microgravity, the harsh launch environment, or some combination of these factors. Therefore, this research attempted to address the effect of launch by subjecting osteoblasts to conditions simulating shuttle launch accelerations, noise, and vibrations. No differences, compared with controls, were seen in the number of total or viable cells after exposure to these various launch conditions. Taken together, these data indicate that the magnitude of gravitational loading (3G maximum) and vibration (7.83G rms maximum) resulting from launch does not adversely affect osteoblasts in terms of total or viable cell number immediately, but launch conditions, or the microgravity environment itself, may start a cascade of events that over several d contributes to cell loss.
Kacena M A; Todd P; Landis W J
In Vitro Cellular & Developmental Biology-Animal
2003
2003-11
Journal Article or Conference Abstract Publication
n/a