Loading history changes the morphology and compressive force-induced expression of receptor activator of nuclear factor kappa B ligand/osteoprotegerin in MLO-Y4 osteocytes

Published: 19 October 2020| Version 1 | DOI: 10.17632/2yfd2w8jfp.1


Background: 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. Methods: 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 (FRAP) technique was also applied to test the differences of gap-junctional intercellular communications (GJIC) among MLO-Y4 cells. Results: 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. Conclusion: 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.


Steps to reproduce

MLO-Y4 cells were seeded onto the type I collagen-coated 24-well plate, 60-mm culture dish, or 35-mm glass-bottom (glass diameter: 14 mm) plastic dish at 2.63×104 cells/cm2. Three hours after seeding the MLO-Y4 cells, a round micro-cover glass (MATSUNAMI, Japan; area, 78.54 mm2; height, 0.21 mm; diameter,10 mm; volume, 16.49 mm3; weight, 0.041 g) or an assembly of two pieces of the normal cover glass (MATSUNAMI, Japan; area, 960 mm2; height, 0.14 mm; volume, 202.16 mm3; weight, 0.505 g) was placed onto the MLO-Y4 cells as the background continuous compressive force (CCF) of 31 dynes/cm2. As additional CCF, a modified 200-µl pipette tip (density, 0.92 g/cm3; height, 16 mm; volume, 28.26 mm3; weight, 0.211 g) was placed onto the micro-cover glass. The modified 200-µl pipette tip itself generated 32 dynes/cm2 CCF when was immersed into the well of the 24-well plate with 2.1 ml culture media. Therefore, the MLO-Y4 cells in the loading history group were grown under a total of 63 dynes/cm2 CCF, while the cells in the control group were grown under only the background CCF (31 dynes/cm2). After maintaining the MLO-Y4 cells under additional CCF for 36 h, the additional CCF was removed for 10 h (recovery period) before the same additional CCF was applied again for another hour. One hour before applying the additional CCF again for an extra hour, 3.0 µM 18α-GA or 0.1% DMSO was added and kept in the medium until the end of this experiment.


Okayama Daigaku - Shikata Campus


Confocal Microscopy, Western Blot, Morphological Analysis, Image Normalization, Cellular Response to Mechanical Stress, Real-Time Polymerase Chain Reaction, Fluorescence Assay, Cellular Imaging