ASGSB 1999 Annual Meeting Abstracts

INITIAL RESULTS OF EXPERIMENTS WITH OSTEOBLASTS CULTURED UNDER VARYING DIRECTIONS WITH RESPECT TO THE GRAVITY VECTOR. M.A. Kacena^1,2, L.C. Gerstenfeld1, P. Todd³, and W.J. Landis⁴. ¹Dept. of Musculoskeletal Research, Boston Univ School of Med, Boston, MA, ²Depts. of Aerospace Engineering and ³Chemical Engineering, Univ of Colorado and BioServe Space Technologies, Boulder, CO, and ⁴Dept. of Biochemistry and Molecular Pathology, Northeastern Ohio Univ. College of Med, Rootstown, OH.

Substrate attachment (a) is crucial for normal growth and differentiation of many cell types. To explore 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 the gravity vector, defined as g. Osteoblasts, grown in MEM supplemented with 10% FBS and attached to type I collagen-coated coverslips, were loaded into Fluid Processing Apparatus (FPA) units, placed in incubators (37єC, 5% CO₂), and oriented so that cells were right-side up (ag) or inverted (ag). Other coverslips with attached osteoblasts were placed in clinostats and continuously changed (2 rpm) in orientation with respect to g. Cells in these three different configurations relative to g were collected daily for 6 days and cell viability (attachment) was assessed using a live/dead assay (Molecular Probes, Eugene, OR). Compiled data indicate that viability was significantly decreased (~50%) in inverted and clinostat cultures (compared to ag cultures) for the first 3 days of sampling, but from days 4-6 no significant differences were found in viable cell number for the three conditions. Decreases in viable cell number within the first days of the experiments could result from cell death followed by detachment, detachment followed by death, or differences in cell proliferation rate. To help distinguish among these possibilities, BrdU labeling for 2 or 24 hr is currently being used to measure proliferation rate of cells. In summary, changing the cell-substrate attachment direction with respect to the gravity vector (as ag) causes an immediate response in diminishing viable osteoblast number, but the effect disappears after this initial time period. Implications of these results for cell growth and differentiation are being examined.

(Supported by NASA grants: NGT-51421 and NAG5-7789.)

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