FSS induces osteogenic differentiation MC3T3-E1 cells.

(A) FSS induced rearrangement of stress fibers. F-actin microfilaments were visualized with rhodamine phalloidin. Relative means and standard deviations of fluorescence intensity were given upon the images (P<0.01; Scale bar: 20 µm). Transcriptional levels of ALP (B), RUNX2 (C) and SP7 (D) in MC3T3-E1 cells were determined by qRT-PCR in a series of time points after FSS. Data were shown as fold change relative to control. (E) ALP activities were detected by using nitrophenyl phosphate (PNPP) method at 24 h pf.. (F) Extracellular type I collagen was determined by immunostaining at 24 h pf.. Relative integrated optical density (IOD) of immunostaining was calculated and the relative means and standard deviations were shown under each picture. (P<0.001; Scale bar: 50 µm) (G) Microscopic view of extracellular matrix (ECM) mineralization. Cells treated with FSS and stained with Alizarin Red S at day 12 pf. Quantification of Alizarin Red S (ARS) staining was determined via extraction with cetyl-pyridinium chloride. Absorbance was read at 560 nm. Relative means and standard deviations were shown underneath (P<0.001; Scale bar: 50 µm). Transcriptional levels of ALP (H), RUNX2 (I) and SP7 (J), ALP activities (K) and ECM mineralization (L) in primary isolated mesenchymal stem cells from mouse bone marrow were determined after FSS treatment. (pf., post-FSS treatment. Scale bar: 50 µm. Data were shown as mean ± SD. n = 3; **, P<0.001; ***, P<0.001.)