Migration and differentiation of osteoclast precursors under gradient fluid shear stress

The skeleton can adapt to mechanical loading through bone remodeling, and osteoclasts close to microdamages are believed to initiate bone resorption. However, whether local mechanical loading, such as fluid flow, regulates recruitment and differentiation of osteoclast precursors at the site of bone resorption has yet to be investigated. In the present study, finite element analysis first revealed the existence of a low-fluid shear stress (FSS) field inside microdamage. Based on a custom-made device of cone-and-plate fluid chamber, finite element analysis and particle image velocimetry measurement were performed to verify the formation of gradient FSS flow field. Furthermore, the effects of gradient FSS on the migration, aggregation, and fusion of osteoclast precursors were observed. Osteoclast precursor RAW264.7 cells migrated along a radial direction toward the region with decreased FSS during exposure to gradient FSS stimulation for 40 min, thereby deviating from the direction of actual fluid flow indicated by fluorescent particles. When calcium signaling pathway was inhibited by gadolinium and thapsigargin, cell migration toward a low-FSS region was significantly reduced. For the other cell lines MC3T3-E1, PDLF, rat mesenchymal stem cells, and Madin-Darby canine kidney epithelial cells, gradient FSS stimulation did not lead to low-FSS inclined migration. After being cultured under gradient FSS stimulation for 6 days, RAW264.7 cells showed significantly higher density and ratio of TRAP-positive multinucleated osteoclasts in the low-FSS region to those in the high-FSS region. Therefore, osteoclast precursor cells may exhibit the special ability to sense FSS gradient and tend to actively migrate toward low-FSS regions, which are regulated by calcium signaling pathway.