Cell-substrate traction force regulates the fusion of osteoclast precursors through cell-cell interaction

The adhesion morphology of a cell monolayer results in a mechanical force inside cells, between cells, or between cells and substrates. The mechanical force regulates the differentiation of stem cells, but its influence on cell fusion is seldom studied. The present study is focused on osteoclast precursors, RAW264.7 monocytes, which can fuse into multinucleated cells (MNCs) responsible for bone resorption. Cells were cultured on circular and ring-like patterned substrates. Then, cell fusion, cell-substrate traction force, and force-sensitive molecules in different regions were measured and analyzed. Results showed that MNCs mainly appeared in the interior of the ring-like pattern and the central zone of the circular pattern, where both cell-substrate traction force and in-plane maximal shear stress were smaller than that at the patterns' edge. The immunostaining results revealed that F-actin, vinculin, β-catenin, and E-cadherin were highly distributed at the edge of patterns. High seeding density of cells promoted mechanical force-dependent fusion. When calcium-dependent cell-cell connections were inhibited by E-cadherin antibody or low-calcium medium, the fusion into MNCs was greatly reduced. Thus, the morphology of cell monolayer decides the mechanical state of cell-substrate interaction and cell-cell connection, ultimately regulating the fusion of osteoclast precursors.