Experiments with osteoblasts cultured under varying orientations with respect to the gravity vector |
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Authors: | Melissa A Kacena Paul Todd Louis C Gerstenfeld William J Landis |
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Institution: | (1) Dept. of Orthopaedics and Rehabilitation, Yale University School of Medicine, 333 Cedar St., TMP 510, New Haven, 06510, USA (e-mail;(2) SHOT Inc., Greenville, USA;(3) Dept. of Musculoskeletal Research, Boston University School of Medicine, Boston, USA;(4) Dept. of Biochemistry and Molecular Pathology, Northeastern Ohio Universities College of Medicine, Rootstown, USA |
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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.
This revised version was published online in September 2006 with corrections to the Cover Date. |
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Keywords: | Clinostat Growth rate Inversion Osteoblasts Proliferation Substrate attachment |
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