| During the tissue development and morphogenesis,cells located in specific parts of embryo with specific mechanical microenvironment can developed into different organs and tissues.Recent evidence shows that mechanical factores play crucial roles of tissue morphogenesis.For instance,a growing body of evidence shows that the geometric constraints and substrate rigidity can influence the adhesion-based cell behaviors,such as focal adhesion distribution,cytoskeleton structure,cell migration,differentiation and apoptosis.In this thesis,we experimentally studied the substrate togography and rigidity induced cell arrangement,polarization and fusion process;and the flow induced migration of osteoclast and its signaling pathways.The main results are summarized as follows:(1)Osteoblastic cells(MC3T3-E1)were seeded on the micropatterned substrate of different rigidity,and the cell arrangement and polarization behaviors were analyzed.The traction force and the in-plane stress in cell layer were measured.We showed that cells exhibit position-dependent arrangement and polarization which are regulated by geometry and stiffness of the patterned substrate.We found that the driving force of the collective behaviors is the in-plane maximum shear stress in the cell layer.The larger the maximum shear stresses,the more the cells prefer to align along the direction of the maximum principal stress,and the higher the degree that cells to polarize.The biphasic dependence of degree of cell alignment and cell polarization on substrate stiffness was consistent with the corresponding biphasic behaviors of the maximum shear stress.We further showed that when the contractility of cytoskeleton,cell-cell interaction or cell-matrix interaction was inhibited,the collective cell mechanosensing behaviors then diminished.(2)Osteoclast precursors(RAW264.7)were seeded on the micropatterned substrate,the pattern dependent fusion of multinucleate osteoclasts in presence of induction medium was studied.The effect of density,induction time,and cell-cell interation on the fusion of osteoclasts was analyzed.We showed that the fusion of multinucleated osteoclasts occurred mostly in the inner region of the ring pattern.The actin staining results suggested that the actin preferred to align along the maximum principal stress at the edge of pattern,while the actin align along the cell periphery in the inner region.Comparing with the FEM calculation,we showed that high shear stress promoted the polarization of actin while inhibited the fusion process.Blocking the cell-cell interaction inhibited the fusion of osteoclasts,and the pattern dependent behavior vanished.(3)MC3T3-E1 cells were seeded on the half-cylinder like 3-dimensional curved substrate,and the effect of curvature on cell arrangement and polarization was analyzed.The cellular arrangement and polarization were estimated by the arrangement and polarization of actin cytoskeleton and nucleus.Combined with the finint element modeling,we found that on the substrate of high curvature,both the nucleus and actin cytoskeleton more preferred to polarize and aligne along the axis of the half-cylinder.The FEM calculation suggested that the curvature of substrate regulated cell arrangement and polarization behavior because it could influence the in-plane stress in cell layer.(4)RAW264.7 monocytes were exposed to 1 and 10 dyne/cm2 FSS for 30 min,and the cell migration were analyzed using the home-made program.We found that the RAW264.7 cells tended to migrate along the flow direction and the migration speed and orientation increased with FSS level.Calcium pathways(i.e.,MSCC,ER,PLC,or extracellular calcium)had significant impact on their migration.When one of these pathways is blocked,the migration speed along flow direction significantly reduced.The induction time had profound effect on the calcium pathways regulated migration.We showed that the osteoclasts induced for 4 or 8 d also preferentially migrated along the direction of fluid flow,but calcium pathways did not influence their migration. |
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