Conditioning of MLO-Y4 Osteocyte like Cells to Fluid Shear Stress

Objective: To determine the functional adaptation of MLO-Y4 osteocyte like cells to shear stress stimulation over multiple days.  

Method: MLO-Y4 cells were cultured on 100-mm plates to 70-80% confluency. Oscillatory shear stress of 1 Pa was applied at 0.5 Hz for 1, 2, 4, and 5 d.  Additional cultures were stressed for 1 and 4 d then allowed to rest for a period of 24 h.  Thus, repeated cycles of 1-h stimulation followed by 1-d rest then 1-h re-stimulation were applied over 5 d in order to determine the extent to which conditioning stabilized cellular activity.  Real-time PCR was used to evaluate the effects of stimulation duration on genes representing osteogenic, osteoclastogenic, second messenger, and cell attachment modulators.  ANOVA and t-tests were applied to determine between-group and within-group differences, respectively (alpha = 0.05).

Result: In the first two days, rest increased the regulatory pattern observed in the first hour of testing and re-stimulation had no effect.  Osteoprotegrin, SOST, and DMP-1 increased significantly.  In the last two days, RANKL, osteopontin, and E11 were significantly downregulated, while iNOS increased.

Conclusion: Durations of loading and resting affect gene expression.  Steady-state is not readily achievable in vitro within 5 days of conditioning.  Longer test periods are needed to approach steady-state response in osteocytes and thus clarify the impact of loading.