Changes of the Extracellular Physical Environment of Osteoclast Precursors

Objectives: Osteoclasts (OCs) are differentiated from monocyte/macrophage-lineage hematopoietic precursor cells which are termed OC precursors (OCPs) in the bone microenvironment. OCPs are drawn from the bloodstream to bone by various factors released at sites undergoing resorption in the bone microenvironment, and subsequently differentiate into OCs following stimulation with the RANK ligand (RANKL). The purpose of this study is to investigate the effects of the change of mechanical environment as from the blood to bone on the differentiation of OCPs in the bone microenvironment.
Methods: RAW264.7 cells were used as osteoclast precursors, and cultured on either plastic cell culture dishes (solid condition as bone) or ultra-low attachment surface dishes (fluid condition as blood). We performed cell proliferation assay, and determined TRAP staining and activity. TRAP activity was determined by TRAP solution assay. mTOR, RANK, c-Fos and NFATc1 were detected by Western blot analysis.
Results: The cell proliferation of RAW264.7 cells increased under fluid condition compared with under solid condition. mTOR protein expression level under fluid condition were higher than under solid condition. On day 7, RANKL induced the formation of large multinucleated OC-like cells under solid condition, but failed to differentiate into OC-like cells under fluid condition. TRAP activity was significantly decreased under fluid condition, compared with under solid condition. RANK, c-Fos and NFATc1 protein expression levels under fluid condition were lower than under solid condition.
Conclusions: The results of this study indicate that the change of the extracellular physical environment of OCPs may induce OCPs differentiate into OCs via up-regulated RANK.