<sec>
<title>Background</title>
In this study, we investigated the effect of the mechanical loading history on the expression of receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) in MLO-Y4 osteocyte-like cells.


</sec>
<sec>
<title>Methods</title>
Three hours after MLO-Y4 osteocytes were seeded, a continuous compressive force (CCF) of 31 dynes/cm² with or without additional CCF (32 dynes/cm²) was loaded onto the osteocytes. After 36 h, the additional CCF (loading history) was removed for a recovery period of 10 h. The expression of RANKL, OPG, RANKL/OPG ratio, cell numbers, viability and morphology were time-dependently examined at 0, 3, 6 and 10 h. Then, the same additional CCF was applied again for 1 h to all osteocytes with or without the gap junction inhibitor to examine the expression of RANKL, OPG, the RANKL/OPG ratio and other genes that essential to characterize the phenotype of MLO-Y4 cells. Fluorescence recovery after photobleaching technique was also applied to test the differences of gap-junctional intercellular communications (GJIC) among MLO-Y4 cells.


</sec>
<sec>
<title>Results</title>
The expression of RANKL and OPG by MLO-Y4 osteocytes without a loading history was dramatically decreased and increased, respectively, in response to the 1-h loading of additional weight. However, the expression of RANKL, OPG and the RANKL/OPG ratio were maintained at the same level as in the control group in the MLO-Y4 osteocytes with a loading history but without gap junction inhibitor treatment. Treatment of loading history significantly changed the capacity of GJIC and protein expression of connexin 43 (Cx43) but not the mRNA expression of Cx43. No significant difference was observed in the cell number or viability between the MLO-Y4 osteocyte-like cells with and without a loading history or among different time checkpoints during the recovery period. The cell morphology showed significant changes and was correlated with the expression of OPG, Gja1 and Dmp1 during the recovery period.


</sec>
<sec>
<title>Conclusion</title>
Our findings indicated that the compressive force-induced changes in the RANKL/OPG expression could be habituated within at least 11 h by 36-h CCF exposure. GJIC and cell morphology may play roles in response to loading history in MLO-Y4 osteocyte-like cells.


</sec>