Effects of Mechanical Stimulation on the Expression of Na⁺/Ca²⁺ Exchanger mRNA and Orientation of F-actin Filaments in Cultured Osteoblastic Cells

Abstract

It is well known that the application of physiological mechanical stress to skeletal tissue is important in regulating bone remodeling and modeling. The aim of this study is to examine the expression of Na⁺/Ca²⁺ exchanger (NCX1) mRNA and the activation of stretch-activated (SA) Ca²⁺ channels when three-dimensional strain is applied to osteoblast-like cells (MC3T3-E1 cell). The cells were mixed with collagen solution at a cell density of 6×10⁵ cells/ml and layered on nylon mesh, then pre-incubated in αMEM containing 10% fetal bovine serum. After 24 hours of pre-incubation, cells in the collagen gel were incubated for up to three days with or without intermittent mechanical stimulation (1 Hz, 10% stretch, 15 min, three times a day) in the presence or absence of Gd³⁺, an inhibitor of SA channel. The expression of NCX1 mRNA was assessed using RT-PCR, and cells were examined using a confocal laser microscope after visualizing actin filaments using rhodamine-phalloidin staining. Accelerated NCX1 mRNA expression was observed in the stretched cells. The mechanical strain also caused the cells to change shape and orient perpendicular to the direction of stretching. Consistent with the morphological changes of the cells, strong intensity of the staining for actin filaments was observed in the stretched group, which was abolished by the addition of Gd³⁺. Our results suggest that the mechanical strain induces the Ca²⁺ influx through activation of the SA channel and NCX1 resulting in the morphological changes in osteoblastic cells.