Stepwise increasing and decreasing fluid shear stresses differentially regulate the functions of osteoblasts.

It is well accepted that osteoblasts respond to fluid shear stress (FSS) depending on the loading magnitude, rate, and temporal profiles. Although in vivo observations demonstrated that bone mineral density changes as the training intensity gradually increases/decreases, whether osteoblasts perceive such slow temporal changes in the strength of stimulation remains unclear. In this study, we hypothesized that osteoblasts can detect and respond differentially to the temporal gradients of FSS. In specific, we hypothesized that when the temporal FSS gradient is high enough, i) the increasing FSS inhibits the osteoblastic potential in supporting osteoclastogenesis and enhances the osteoblastic anabolic responses; ii) on the other hand, the deceasing FSS would have opposite effects on osteoclastogenesis and anabolic responses. To test the hypotheses, stepwise varying FSS was applied on primary osteoblasts and osteogenic and resorption markers were analyzed. The cells were subjected to FSS increasing from 5, 10, to 15 or decreasing from 15, 10, to 5 dyn/cm(2) at a step of 5 dyn/cm(2) for either 6 or 12 hours. In a subset experiment, the cells were stimulated with stepwise increasing or decreasing FSS at a higher step (10 dyn/cm(2)) for 12 hours. Our results showed that, with the step of 5 dyn/cm(2), the stepwise increasing FSS inhibited the osteoclastogenesis with a 3- to 4-fold decrease in RANKL/OPG gene expression versus static controls, while the stepwise decreasing FSS increased RANKL/OPG ratio by 2- to 2.5-fold versus static controls. Both increasing and decreasing FSS enhanced alkaline phosphatase expression and calcium deposition by 1.0- to 1.8 fold versus static controls. For a higher FSS temporal gradient (three steps of 10 dyn/cm(2) over 12 hour stimulation), the increasing FSS enhanced the expression of alkaline phosphatase expression and calcium deposition by 1.3 fold, while the decreasing FSS slightly inhibited them by -10% compared with static controls. Taken together, our results suggested that osteoblasts can detect the slow temporal gradients of FSS and respond differentially in a dose-dependent manner, which may account for the observed bone mineral density changes in response to the gradual increasing/decreasing exercise in vivo. The stepwise FSS can be a useful model to study bone cell responses to long-term mechanical usage or disuse. These studies will complement the short-term studies and provide additional clinically relevant insights on bone adaptation.