The Regulation Mechanism Of Cell Monolayer’s Morphology On The Fusion Of Osteoclast Precursors

Bone loss may occur in those aging people, menopausal women, long-term bedridden patients and astronauts. There are two processes during bone remodeling, i.e. bone resorption and bone formation. Bone resorption is performed by osteoclasts, and osteoclast precursors derive from bone marrow hematopoietic stem cells, which are the monocyte-macrophage cells. It has been demonstrated that these monocytes may differentiate into mature multinucleated osteoclasts induced by chemicals, but it is still unclear whether mechanical microenvironment around osteoclast precursors gets involved in the regulation of cell fusion. Previously it had been found in our lab that for the cell monolayer cultured on a ring-like pattern or circular pattern, the maximum in-plane shear stress occurs at the edges. In this thesis, it will be studied whether the adhesion morphology affects the fusion of osteoclast precursors and its mechanism. Firstly the micro-contact printing technology was adopted to build multicellular monolayer with ring-like and circular patterns. Then the distribution of fused mulnucleated osteoclasts in different regions along radial direction was measured and analyzed. In addition, the immunofluorescent staining was used to observe the expression and distribution of F-actin, Vinculin, β-catenin and E-cadherin in different regions. The experimental results show that both RANKL and the conditioned medium from MC3T3-E1 osteoblasts could incduce the fusion osteoclast precursors on the pattern after 4 days or 6 days. However, for the conditioned medium from osteoblasts, a region-dependent fusion phenomenon was found, i.e. the cell fusion was lowest at the outer edge of ring-like and circular patterns. Interestingly, the expression of F-actin, Vinculin, β-catenin and E-cadherin are highest at the outer edge. Therefore, we speculate that the maximum shear stress at the outer edge of cell monolayer enhances intercellular connection, cell-substrate adhesion as well as intracellular tension, which finally leads to the low-level fusion. These results indicate that the mechanical microenvironment within the cell monolayer plays an important role in the fusion of osteoclast precursors and provide insight into the molecular mechanism.