Page 93 - Physico-Chemical Niche Conditions for Bone Cells

control: 3, PFF: 3) were used for quantification. Five pseudopodia were quantified in every cell.
Alkaline phosphatase activity
ALP activity (short-term) was measured to assess the osteoblastic phenotype of MC3T3-E1 pre-osteoblasts treated with or without 1 h PFF after 0, 1, 3, and 6 h of post-incubation without mechanical loading (short-term down-stream impact). Cells were lysed with 1.5 ml milli-Q water, and stored at -20°C until use. 4-Nitrophenyl phosphate disodium salt (Merck, Darmstadt, Germany) at pH 10.3 was used as a substrate for ALP, according to the method as described by Lowry [19]. The absorbance was read at 405 nm with a Synerg HT® spectrophotometer (BioTek Instruments). ALP activity (short-term) was expressed as μmol/μg cell protein. BCA Protein Assay Reagent Kit (PierceTM, Rockford, III, USA) was utilized to measure the amount of protein. The absorbance was read at 540 nm with a Synergy HTÒ spectrophotometer (BioTek Instruments). Four independent experiments with 32 glass slides (n=4) were performed.
ALP protein (long-term) was determined after 21 days of culture 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, and 10 mM β-glycerophosphate). The cells were washed 3 times with PBS, and fixed with 4% paraformaldehyde in PBS for 15 min at 37°C. The BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) phosphatase color development kit (Roche Diagnostics, Mannheim, Germany) was used for the colorimetric detection of ALP intensity (long-term) by incubation for 30 min at 37°C. Quantification of ALP intensity was performed using Image-Pro Plus software. Note that the images had to be converted to gray scale 8. Three independent experiments providing 72 images from 24 glass slides (n=3) were performed.
Collagen production
Total collagen production by MC3T3-E1 pre-osteoblasts attached to the glass slides was visualized and quantified by using picrosirius red stain kit (Chondrex, Inc., Redmond, WA, USA). Cells were cultured for 21 days in osteogenic induction medium following 1 h static control or PFF treatment, and post-incubation (both static control and PFF treatment) in normal culture medium without mechanical loading. Then cells were washed with PBS thrice, and fixed with 4% paraformaldehyde for 15 min at 37°C. Samples were stained for 1 h with picrosirius (0.1 wt%) at room temperature. Then, samples were washed twice with acidified water (5 ml acetic acid/L distilled water) to remove unbound stain, and visualized under a stereo and inverted microscope. For semiquantitative collagen analysis, picrosirius red stain
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