Page 90 - Physico-Chemical Niche Conditions for Bone Cells

PFF affects pre-osteoblast behavior
residual algorithm (GMRES)) was used to evaluate the variables fluid velocity, fluid pressure, and fluid shear stress on an adherent pre-osteoblast inside a parallel-plate flow chamber.
Average fluid velocity, fluid pressure, and fluid shear stress calculation
The average values of fluid velocity, fluid pressure, and fluid shear stress were evaluated as described previously [17].
MC3T3-E1 pre-osteoblast culture
MC3T3-E1 pre-osteoblasts were cultured in 75 cm2 flasks (Nunc, Roskilde, Denmark) in α– minimal essential medium (α-MEM, Gibco, Paisly, UK) supplemented with 10% fetal bovine serum (FBS; Gibco, Paisly, UK), 300 μg/ml penicillin (Sigma-Aldrich, St. Louis, MO, USA), 250 μg/ml streptomycin (Sigma-Aldrich), and 1.25 μg/ml fungizone (Gibco, Paisly, UK) in a humidified atmosphere of 5% CO2 in air at 37°C. The medium was exchanged every 72 h. Upon reaching 80-90% confluence, cells were harvested using 0.5 mM ethylenediaminetetraacetic acid (EDTA) and 0.25% trypsin (Gibco, Paisly, UK) for 5 min at 37°C, replated at 1.5×105 cells per 75 cm2 flask (Greiner Bio-One, Kremsmuenster, Austria), and passaged until the cells reached 80-90% confluence again. Cells used for PFF experiments were between passage 20 and 29 (P20-P29).
Pulsating fluid flow
One day before mechanical loading by PFF, MC3T3-E1 pre-osteoblasts were seeded at 1×103 cells/cm2, or 3×103 cells/cm2 on poly-L-lysine-coated (50 μg/mL; poly-L-lysine hydrobromide; Sigma-Aldrich) glass slides (24×24×0.15 mm or 36×76×1 mm). One hour before the start of PFF, the medium was changed by α-MEM with 2% FBS, 300 μg/ml penicillin, 250 μg/ml streptomycin, and 1.25 μg/ml fungizone. PFF was generated using a flow apparatus containing a parallel-plate flow chamber [18]. A “small chamber” (14×14×0.2 mm (inner dimension)) was used for FE modeling, and measuring metabolic activity, cell orientation, cell morphology, F-actin fluorescence intensity, ALP activity (short-term), ALP protein (long-term), collagen production, and ECM mineralization. A “big chamber” (58×32×0.3 mm (inner dimension)) was used for measuring NO production and gene expression. In both chambers, cells were treated with the same intensity of PFF (amplitude: 1.0 Pa, peak shear stress rate: 6.5 Pa/s, frequency: 1 Hz) for 1 h at 37°C. Static control cultures were kept in a petri-dish under similar conditions as experimental cultures, i.e. α-MEM with 2% FBS, 300 μg/ml penicillin, 250 μg/ml streptomycin, and 1.25 μg/ml fungizone, as well as 1 h incubation at 37°C.
Medium samples of 500 μl were taken at 10, 30, and 60 min of static or PFF treatment, and assayed for NO production. After 60 min of static or PFF treatment, cells were post- incubated in fresh α-MEM containing 10% FBS and antibiotics for 1, 3, or 6 h without
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