Experimental And Numerical Approach For Cell Mechanics Of Osteocytes

Osteocytes are mature bone cells encased in mineralized extracellular matrix.Abundant evidence has shown that the osteocytes are the key mechanonsensor cells thatdirectly regulate bone-forming osteoblast and bone-removing osteoclast activities.Thus, osteocyte functions are critical to the etiology and new treatments forosteoporosis, which is one of the most challenging health issues of human. It has beenhypothesized that the dominant loading mechanism of the mechanosensing osteocytes isoscillating fluid shear resulting from the deformation of bone. The relationship betweenoscillatory loading and cellular mechanotransduction events is still poorly understood,especially at the initial events of biochemical signal activation and transduction.For adherent cells under fluid flow, the interactions among the flow field, the cell,and the substrate are dynamic and complex, which cannot be visualized via theconventional top-to-bottom view microscopy. However, the mechanotransduction of theadhered cells under flow depends on the fluid shear stress on their apical surface and theadherence with the substrate on the basal surface, which is a truly3D dynamics.Traditional techniques to obtain this3D information of a cell-body, such as by confocaland deconvolution microscopy, are inherently limited by the timescale under which thedeformational information can be visualized due to the necessity of scanning a z-stack.Given the dynamic nature of oscillating flow at physiological frequencies (i.e.,1Hz)and the timescale of Src/FAK activation (<0.3seconds), any imaging technique musthave a temporal resolution capable of capturing cell deformation at these timescales.Here, we propose using a real-time “quasi-3D” technique to greater capture the spatialdynamics of cytoskeletal deformation and Src/FAK activations. In this project, we willstudy the real-time Quasi-3D deformation dynamics of the cytoskeleton, cell properties(via computation), and Src/FAK activation in osteocytes under oscillatory fluid flow.Several in vitro techniques have been developed to determine the mechanicalproperties of a single cell, including micropipette aspiration, cell compression within agel matrix, unconfined cell compression, and various forms of cellular indentation,including atomic force microscopy. However, none of these techniques can be used to determine the viscoelastic properties of individual osteocytes under fluid flow,non-invasively. In this study, we determine the viscoelastic properties of osteocytesunder flow using a combined finite element modeling and experimental approach. Thisis a novel technique to determine cell mechanical properties under flow. The osteocyteis modeled as an incompressible viscoelastic standard linear solid with finitestrain. The instantaneous and equilibrium modulus of an osteocyte is obtained byminimizing the deviation between model predicted and experimental deformation ofwhole cell at different time, respectively. The apparent viscosity is determined by fittingexperimental data of creep using theoretical formula. This fluid-structure cell modelbased on individual cell geometry provides a novel technique to measure theviscoelastic properties of individual cells and determine intracellular deformation.We can obtain whole-cell and regional deformation of the cytoskeleton byquantitative analysis of side-view and bottom-view cellular image using a quasi-3Dmicroscopy technique. In this study, we aim to study the accuracy of whole-cell andregional deformation of the cytoskeleton measured experimentally. A numerical modelof a virtual fluorescently dyed cell is created in3D space, and the cell is subjected tounidirectional fluid shear flow via finite element analysis. Therefore, the intracellulardeformation in the simulated cells is exactly prescribed. Two-dimensional fluorescentimages simulating the quasi-3D technique are created from the virtual cell and itsdeformed states in3D space using a point-spread function (PSF) and a convolutionoperation. These simulated original and deformed images are processed by a digitalimage correlation technique to calculate quasi-3D-based intracellular strains. Thecalculated strains are compared to the prescribed strains, thus providing a theoreticalbasis for the measurement of the accuracy of quasi-3D intracellular strain measurementsagainst the true3D strains. Our computational study demonstrates that quasi-3D strainmeasurements closely represente the true3D strains in uniform and complex strainstates with high precision.In future study, we have confidence that our quantitative research will not onlyhelp to elucidate the mechanisms of mechanotransduction in osteocytes, but also make abreakthrough in cancer cell migration/motility and endothelial cell mechanobiology.