THE INFLUENCE OF INTERMITTENT COMPRESSIVE FORCE ON OSTEOGENIC DIFFERENTIATION OF HUMAN MANDIBULAR-DERIVED OSTEOBLAST-LIKE CELLS

Abstract

Mechanical force can regulate osteoblast differentiation in osteoblasts. Different types of force have different effects on osteoblasts. These effects are associated with the activation of purinergic receptors, especially purinergic 2X7 receptor (P2X7R). However, the molecular response of human osteoblasts to intermittent compressive force (ICF) associated with P2X7 receptors has not been clarified. ​In this study, the influence of ICF on human mandibular-derived osteoblast-like cells (hMOBs) was investigated. The role of ICF-induced ATP through P2X7R was examined. The hMOBs were obtained from non-pathologic mandibular bone. Cells were subjected to ICF for 20 h. The hMOBs were cultured in osteogenic medium in the absence or presence of 0.5-5 µM 2’(3’)-O-(4-benzoyl) benzoyl-ATP (BzATP), a selective P2X7R agonist. The mRNA expression was investigated by quantitative real time polymerase chain reaction. In vitro mineral deposition was investigated by Alizarin Red S staining. Transfection of small interfering RNA was performed to confirm the effect of P2X7R activation. WNT/β-catenin signaling was detected by immunofluorescence staining for β-catenin. The results demonstrated that ICF increased the OSX and ALP mRNA expression and enhanced in vitro mineralization. In addition, WNT3A mRNA expression and β-catenin nuclear translocation were also increased. These effects were related to ICF-induced ATP. However, BzATP significantly attenuated in vitro mineralization and RUNX2 and OSX mRNA expression in osteogenic medium-induced hMOBs. Knockdown P2X7R expression by siRNA rescued BzATP-inhibited RUNX2 and OSX expression. Interestingly, a reduction of WNT3A mRNA expression and blockage of osteogenic medium-induced β-catenin nuclear translocation were also found in BzATP treated group. The addition of recombinant human WNT3A abolished the effect of BzATP-reduced osteogenic marker gene expression and in vitro mineralization. In conclusion, our results demonstrated that ICF-induced ATP enhanced osteoblast differentiation and WNT/β-catenin signaling pathway. P2X7R pathway participates in BzATP-inhibited osteoblast differentiation of hMOBs. This inhibitory effect was associated with inhibition of the WNT/β-catenin signaling pathway.