Page 59 - Physico-Chemical Niche Conditions for Bone Cells

MATERIALS AND METHODS
Cell Culture
MC3T3-E1 osteoblasts (passage 16–28) were cultured in α-minimal essential medium (a- MEM; Gibco, Paisly, UK) with 10% fetal bovine serum (FBS; Gibco), 300 μg/mL penicillin (Sigma-Aldrich, St. Louis, MO, USA), 250 μg/mL streptomycin (Sigma-Aldrich), and 1.25 μg/mL fungizone (Gibco) in a humidified atmosphere of 5% CO2 in air at 37°C. Upon reaching 80−90% confluence, cells were harvested with 0.25% trypsin and 0.5 mM EDTA for 5 min.
Treatment with Pulsating Fluid Flow
One day before PFF-treatment, MC3T3-E1 osteoblasts were harvested and seeded at 1×103 cells/cm2 (for cell structure analysis), and at 3 × 103 cells/cm2 (for gene and protein expression analysis) 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). Prior to 1 h PFF, the medium was refreshed by a-MEM containing 2% FBS and antibiotics. PFF was generated using a flow apparatus containing a parallel-plate flow chamber [69]. Two different chambers were used, i.e., a “big chamber” (5.8 × 3.2 × 0.03 cm (inner dimension)) for measuring gene and protein expression, and a “small chamber” (1.4 × 1.4 × 0.02 cm (inner dimension)) for measuring cell morphology and volume. 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 (for fixed cell analysis) or at room temperature (for live cell analysis). Static control cultures were kept in a petri-dish under similar conditions as experimental cultures.
Cell Morphology
After 1 h PFF, MC3T3-E1 osteoblasts were fixed with 4% paraformaldehyde solution at 37°C for 15 min, treated with 0.2% TritonX−100 (Sigma-Aldrich) for 15 min, and blocked in 5% bovine serum albumin (BSA) for 30 min. Expression of F-actin, phospho-paxillin (p-paxillin), a-tubulin, and integrin-a5 was analyzed by immunofluorescence staining with respectively rhodamine-phalloidin (Invitrogen, Fisher Scientific, Carlsbad, CA, USA), p-paxillin pTy31 polyclonal rabbit immunoglobulin G (IgG) (ab32084, Abcam, Cambridgeshire, UK), a-tubulin (Abcam), and a5 rat monoclonal IgG−2a (Abcam). The secondary antibody was Alexa Fluor−488 goat anti-rat IgG (Abcam), or Alexa Fluor−555 goat anti-rat IgG (Abcam). Nuclei were stained with 1 μg/mL DAPI (Sigma-Aldrich). After glycerol mounting, cell imaging was performed by laser scanning confocal microscopy (LSCM; Nikon, A1R/A1, Tokyo, Japan). LSCM images were captured randomly at three representative sites/glass slide at 40x magnification. Captured images did not overlap. The LSCM z-stack was executed every 0.15
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