Fig. 3

Fluid shear stress strengthens the osteogenic effects of rhBMP-2, PD98059 and Phenamil in ASCs cultured on SBG-PLGA composites. mRNA levels of osteoblastic markers after 7-day ASC culture on SBG-PLGA composites in (A) standard osteogenic medium under either static conditions or with fluid shear stress; and (C) osteogenic medium supplemented with 100 ng/ml rhBMP-2, 50 µM PD98059 and 20 µM Phenamil under either static conditions or with fluid shear stress. Results are presented as relative mRNA expression levels compared to mRNA levels in a control, static culture on PLGA (marked as a black line at 1). (B) The method of fluid shear stress application in ASC cultures using a standard laboratory see-saw rocker (7° tilt angle, 6 RPM frequency). (D) F-actin distribution in ASCs (Phalloidin-Atto488, magenta colored) at day 3 of culture in osteogenic medium supplemented with rhBMP-2, PD98059 and Phenamil after continuous static or dynamic culture conditions applied for 3 days. Scale bar represents 100 μm. (E) Western blot (WB) analysis of p-ERK1/2 and p-SMAD1/5/8 in ASCs after 1-h treatment with rhBMP-2 or rhBMP-2, PD98059 and Phenamil in static or dynamic conditions (upper panel) along with densitometric quantifications of WB results normalized to GAPDH levels. Averages ± SD are indicated. Two-way ANOVA test, *p < 0.05, **p < 0.001, ***p < 0.0001 relative to the static PLGA control or between marked groups