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Browsing by Author "Sheets, Kevin Tyler"

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    Cell-Fiber Interactions: A New Route to Mechano-Biological Investigations in Developmental and Disease Biology
    Sheets, Kevin Tyler (Virginia Tech, 2014-11-03)
    Cells in the body interact with a predominantly fibrous microenvironment and constantly adapt to changes in their neighboring physiochemical environment, which has implications in developmental and disease biology. A myriad of in vitro platforms including 2D flat and 3D gel substrates with and without anisotropy have demonstrated cellular alterations to subtle changes in topography. Recently, our work using suspended fibers as a new in vitro biological assay has revealed that cells are able to sense and respond to changes in fiber curvature and structural stiffness as evidenced by alterations to cytoskeleton arrangement, including focal adhesion cluster lengths and nucleus shape indices, leading to altered migration speeds. It is hypothesized that these behaviors occur due to modulation of cellular inside-out forces in response to changes in the external fibrous environment (outside-in). Thus, in this study, we investigate the role of fiber curvature and structural stiffness in force modulation of single cells attached to suspended fibers. Using our previously reported non-electrospinning Spinneret based Tunable Engineered Parameters (STEP) fiber manufacturing platform, we present our findings on single cell inside-out and outside-in forces using fibers of three diameters (250 nm, 400 nm and 800 nm) representing a wide range of structural stiffness (3-45 nN/μm). To investigate cellular adaptability to external perturbation, we present the development of a first-of-its-kind force measurement 'nanonet' platform capable of investigating cell adhesion forces in response to symmetric and non-symmetric (injury model) loading. Our combined findings are multi-fold: (i) Cells on suspended fibers are able to form focal adhesion clusters approximately four times longer than those on flat substrates, which gives them potential to double their migration speeds, (ii) Nanonets as force probes show that the contractility-based inside-out forces are nearly equally distributed on both sides of the cell body, and that overall force magnitudes are dependent on fiber structural stiffness, and (iii) External perturbation can evenly (symmetric) or unevenly (non-symmetric) distribute forces within the cell, and the resulting bias causes diameter-dependent outside-in adhesion force response. Finally, we demonstrate the power of the developed force measurement platform by extending our studies to cell-cell junctional forces as well as single-cell disease models including cancer and aortic aneurysm.
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    Investigating Cell Viscoelastic Properties with Nanonet Force Microscopy
    Zhang, Haonan (Virginia Tech, 2022-08-04)
    Determining the mechanical properties of living cells accurately and repeatably is critical to understanding developmental, disease, and repair biology. The cellular environment is composed of fibrous proteins of a mix of diameters organized in random and aligned configurations. In the past two decades, several methods, including modified atomic force microscopy (AFM) and micro-pipette aspiration have been developed to measure cellular viscoelastic properties at single-cell resolution. We inquired if the fibrous environment affected cellular mechanobiology. Using our non-electrospinning Spinneret based Tunable Engineered Parameters (STEP) fiber manufacturing platform, we developed fused nanonets to measure single-cell forces and viscoelasticity. Using computer-controlled probes, we stretched single cells attached to two-fiber and three-fiber systems precisely and recorded the relaxation response of cells. The viscoelastic properties were determined by fitting the data to the standard linear viscoelastic solid model (SLS), which includes a spring (k0) in parallel with a spring (km)-damper (cm) series. In cases in which cells are seeded on two fibers, we tested hMSCs and BJ-5TA cells, and the viscoelastic components measurements k0, km, and cm are 26.16 ± 3.38 nN/µm, 5.81 ± 0.81 nN/µm, and 41.15 ± 5.97 nN-s/µm, respectively for hMSCs, while the k0, km, and cm, measurements of BJ-5TA cells are 20.02 ± 2.89 nN/µm, 4.62 ± 0.75 nN/µm, and 45.46 ± 6.00 nN-s/µm respectively. Transitioning to the three-fiber system resulted in an overall increase in native contractility of the cells while allowing us to understand how the viscoelastic response was distributed with an increasing number of fibers. Viscoelastic experiments were done twice. First, we pulled on the outermost fiber similar to the two-fiber case. The cell was then allowed to rest for two hours, sufficient time to regain its pre-stretching contractility. The cell was then excited by pulling on the middle fiber. The experimental results of cell seeding on three fibers proved that the viscoelastic property measurements depend on the excitation position. Overall, we present new knowledge on the cellular viscoelasticity of cells attached to ECM-mimicking fibers.