(350g) Effect of Frequency of Pulsatile Fluid Flow on Mechanotransductive Signaling of Osteoblasts

A promising strategy for the development of tissue engineered bone involves culturing osteoprogenitor cells within porous biomaterial scaffolds under conditions that induce the deposition a bioactive extracellular matrix (ECM). One such condition is perfusion culture, which is known to stimulate the expression of numerous growth and differentiation factors, including bone morphogenetic protein (BMP)-2, vascular endothelial growth factor (VEGF)-A, and transforming growth factor (TGF)-β. Our working theory is that these proteins act back on osteoblastic cells to induce the expression of bone-specific ECM proteins, such as osteocalcin (OC) and bone sialoprotein (BSP). However, the molecular signaling mechanisms by which fluid flow induces the expression of these growth and differentiation factors are not understood.

The objective of this study is to probe the mechanism by which fluid flow stimulates osteoblastic differentiation through the induction of growth and differentiation factors. To accomplish this, MC3T3-E1 cells were seeded onto glass slides, loaded into parallel plate flow chambers (PPFCs), and exposed to various regimens of pulsatile flow (square wave form pattern) to modulate mechanotransductive signaling and gene expression. Experimental conditions included continuous flow, 0.074 Hz pulsatile flow, 0.044 Hz pulsatile flow, 0.015 Hz pulsatile flow, and no flow. The time-average shear stress for all flow conditions was 2.8 dyn/cm². To probe the signaling mechanisms we characterized the phosphorylation of extracellular signal-regulated kinase (ERK) by western blot, the activation of transcription factors activator protein (AP)-1 and Osterix by electrophoretic mobility shift assay, and the mRNA expression of BMP-2, BMP-4, BMP-7, and VEGF-A by quantitative real time PCR. Finally, we characterized the induction of OC and BSP mRNA and correlated this to the expression of growth and differentiation factors. Our preliminary data suggests that expression of ECM proteins OC and BSP are correlated to BMP-2 induction and enhanced by dynamic flow regimens.