Abstract
Mechanical loading induces positive changes in the skeleton due to direct effects on bone cells, which may include regulation of transcription factors that support osteoblast differentiation and function. Flow effects on osteoblast transcription factors have generally been evaluated after short exposures. In this work, we assayed flow effects on osteogenic genes at early and late time points in a preosteoblast (CIMC-4) cell line and evaluated both steady and oscillatory flows. Four hours of steady unidirectional flow decreased the level of RANKL mRNA 53 ± 7% below that of nonflowed cells, but increases in Runx2 and osterix mRNA (44 ± 22% and 129 ± 12%, respectively) were significant only after 12-19 h of continuous flow. Late flow effects on RANKL and osterix were also induced by an intermittent flow-rest protocol (four cycles of 1 h on/1 h off + overnight rest). Four hours of oscillatory flow decreased RANKL mRNA at this early time point (63 ± 2%) but did not alter either osterix or Runx2. When oscillatory flow was delivered using the intermittent flow-rest protocol, Runx2 and osterix mRNA increased significantly (85 ± 19% and 161 ± 22%, respectively). Both β-catenin and ERK1/2, known to be involved in RANKL regulation, were rapidly activated by steady flow. Inhibition of flow-activated ERK1/2 prevented the increase in osterix mRNA but not Runx2; Runx2 phosphorylation was increased by flow, an effect which likely contributes to osterix induction. This work shows that both steady and oscillatory fluid flows can support enhancement of an osteogenic phenotype.