Mechanical Overload Regulates Osteoblast Proliferation, Differentiation And Mineralization Through Wnt/Β-Catenin Signaling Pathway

Abstract Objectives: To study the effect of mechanical overload stimulation on proliferation, differentiation and mineralization of osteoblast and the underlying mechanisms.Methods: MC3T3-E1 cells were divided into overload group and control group. Four-point bending loading device was used to exert mechanical overload stimulation on MC3T3-E1 cells for a certain time. The proliferation of osteoblasts was detected by MTT colorimetric assay. Real-time PCR and Western Blot were used to detect the transcription and expression of osteoblast marker genes and proteins. The specific fluorescent dyes were used to label the actin filament and the nucleus, and the changes of cytoskeleton were observed under laser scanning confocal microscope. The mineralization of osteoblasts was evaluated by the number of calcium nodules formed by alizarin red staining. Results: Compared with the control group, the mechanical overload group significantly inhibited the proliferation of osteoblasts (p <0.05). Real-time PCR and Western Blot showed that the expression of osteoblast differentiation marker gene and protein was inhibited by mechanical overload stimulation. Under laser confocal microscopy, the overload group cell shrinkage deformation was observed, also the microfilament arrangement disorder, the skeleton arrangement loose, the direction difference and the skeleton breakage, but the nucleus does not have obvious change. Alizarin red staining showed that mechanical overload inhibited the formation of calcium nodules in osteoblasts. The expression of β-catenin protein in Wnt signaling pathway was inhibited by overload mechanical stimulation under immunofluorescence microscopy.Conclusion: Mechanical overload stimulation reduces the expression of Runx 2 by affecting the classical Wnt/β-catenin signaling pathway, thus it was inhibited osteoblast proliferation, differentiation and mineralization.