Cora-Cruz, José J.

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    Expression of focal adhesions in response to inducing cellular orientation through mechanical transduction
    (2014) Cora-Cruz, José J.; Sundaram, Paul A.; College of Engineering; Díaz, Rubén; Diffoot, Nanette; Department of Mechanical Engineering; Castillo, Paul
    Loading frequency and the stiffness of the substrate are associated with changes in the expression of focal adhesions in adherent cells. In turn, any change in the characteristics of the focal adhesions generates a corresponding change in the orientation, morphology, motility and mechanical properties of the cell. This research aims to understand the manner in which substrate stiffness in combination with mechanically transduced stimuli influences the cell’s expression of its focal adhesions and orientation. To accomplish this a small cyclical tensile force was transmitted to mouse pre-osteoblast MC3T3-E1 cells through PDMS substrates of different stiffness. Cells were seeded on the substrates and incubated for 12 hours under static (no load) conditions, followed by 4 days of cyclic tensile loading for 5 minutes intervals interspersed with a 5 minute no-load phase. After the mechanical stimulus was applied, immunofluorescence labeling was performed on the cells to identify vinculin at the focal adhesion points, the actin stress fibers and nucleus. In conjunction with image processing through ImageJ®, the images were carefully analyzed to quantify the changes in cell behavior. The substrate’s modulus of elasticity, “E”, and surface roughness were measured and taken into account during analysis of the results. Proliferation, number of focal adhesion points, area of each focal adhesion and amount of inactive vinculin on each sample was influenced by the substrate’s stiffness and loading condition (cyclic/static). Proliferation and amount of inactive vinculin was higher on substrates subjected to cyclic stimulation when compared with the results of the static case, whereas the number and area of focal adhesion points underwent a reduction. The images obtained through confocal microscopy, did not reveal any significant orientation of the cells with respect to the applied strain axis as a result of the applied cyclic load. Thus a small strain of 1% at 0.05 Hz is probably not enough to align the cells either parallel or perpendicular to the principal strain axis. Nevertheless a significant difference was observed in cell distribution after analyzing the axis parallel and perpendicular to the direction of strain up to ±45°. Cells grown on the stiffer materials had a higher percentage of cells aligned parallel to the direction of strain when compared to those grown on the less rigid PDMS substrates. This remains true on the statically held substrates but to a lesser degree. Cells showed an increase in elongation with respect the substrate’s stiffness. Hence the cyclic stimulus to MC3T3-E1 cells appears to be important since it plays a role in the proliferation rate, characteristics of focal adhesions, vinculin expression, cell orientation and elongation.