Page 91 - Physico-Chemical Niche Conditions for Bone Cells

mechanical loading, and cell morphology, fluorescence intensity, orientation, and metabolic activity were determined, as well as osteogenic, proliferation, and angiogenic gene expression in cell cultures that were lysed in TRIzol® reagent (InVitrogen, Carlsbad, CA, USA) for RNA isolation and quantitative real-time PCR (RT-PCR; Fig. 1A). Some cultures were further incubated in osteogenic induction medium (α-MEM with 10% FBS, 300 μg/ml penicillin, 250 μg/ml streptomycin, 1.25 μg/ml fungizone, 50 μg/ml ascorbic acid (Sigma), and 10 mM β- glycerophosphate (Sigma) for 3 or 4 weeks to determine ALP protein (at 3 weeks), collagen production (at 3 weeks) and ECM mineralization (at 4 weeks) (Fig. 1A).
Nitric oxide production
NO production was measured in medium samples collected at 0, 10, 30, and 60 min of PFF treatment or static culture. NO production was measured as nitrite (NO2) accumulation in conditioned medium using Griess reagent containing 2.5 mol/L H3PO4, 0.1% naphtylethelene- diamine-dihydrochloride, and 1% sulfanilamide. Serial dilutions of NaNO2 in α-MEM containing 2% FBS were used as a standard curve. The absorbance was monitored at 540 nm with a Synergy HTÒ spectrophotometer (BioTek Instruments). Four independent experiments with 8 glass slides (n=4) were performed.
MC3T3-E1 pre-osteoblast metabolic activity
Cell activity was assessed by using AlamarBlueÒ Cell Viability Reagent (Invitrogen, Frederick, MD, USA) at 0, 1, 3, and 6 h of post-incubation without mechanical loading after 1 h static control or PFF treatment as described above. The cells were incubated with AlamarBlueÒ reagent in culture medium (1:100) in a humidified incubator with 5% CO2 in air at 37°C for 4 h. After incubation, the supernatants (100 μl/well) were transferred into a 96-well plate. The absorbance was measured at 450 nm with a Synergy HTÒ spectrophotometer (BioTek Instruments, Winooski, VT, USA). Four independent experiments with 28 glass slides (n=4) were performed.
Cell orientation/morphology
To quantify F-actin fluorescence intensity and cell orientation (angle) using laser scanning confocal microscopy (LSCM; Nikon, A1R/A1, Japan), the cells were fixed in 4% paraformaldehyde (Merck) in PHEM buffer containing 60 mM Pipes (Sigma, St. Louis, MO, USA), 25 Mm Hepes (Sigma), 5 mM EGTA (Sigma), 1 mM MgCl2 (Merck), 3% sucrose, and 0.1% Triton-X100 (Serva, Heidelberg, Germany) for 15 min in the dark at 37°C. After washing for 5 min with PBS, samples were blocked in blocking buffer (PBS containing 5% bovine serum albumin (BSA), 5% glycine, and 0.1% Triton-X100) for 30 min in the dark. Then, the F-actin
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