Atomic force microscopy (AFM) for measurement of cell elastic properties
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[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] This thesis presents data on the use of atomic force microscopy (AFM) for manipulating, detecting, and resolving the mechanical characteristics of individual cells. In this thesis research, AFM was applied to measure the elasticity and adhesive characteristics of a vascular smooth muscle cells (VSMC). The advantage of high-sensitivity (sub-nano-Newton) and high-resolution (sub-micron) makes AFM to be a powerful tool for cell studies. In these studies an AFM nano-indentation protocol was used with extracellular matrix (ECM) bio-functionalized AFM probes to evaluate both cell stiffness and ECM adhesion simultaneously under physiological conditions in a liquid environment. To characterize the cell's mechanical resistance to compression forces applied with AFM probe the Young's modulus (in cellular mechanical properties' research also called elasticity modulus) that is based on Hertz theory (classical infinitesimal strain theory) was used to calculate elastic properties/stiffness of individual VSMC. Measurement of forces required to rupture bonds between the VSMC and ECM coated AFM probe were measured to evaluate cell adhesive properties. VSMC are the major cellular component of the vascular wall and bears external mechanical forces that are caused by blood flow and pressure. The research here described investigates the individual vascular smooth muscle cell (VSMC) cellular and molecular mechanisms to determine how the mechanical and adhesive properties of the VSMC change in aging and hypertension. The hypothesis tested was that both aging and hypertension would increase the stiffness and adhesion of VSMC. The research reports the novel findings that VSMC stiffness and adhesion to the ECM is significantly increased in cells from older monkeys compared to their younger counterparts. An important feature of this research is the use of non-human primate model (monkeys) because of their genetic proximity to humans and its relatively long life-span in comparison to rats or mice. Hypertension is a common age-related vascular disease, in this research a novel spectral analysis approach that revealed oscillation in the underlying cellular and molecular mechanisms of controlling cell stiffness and adhesion, was undertaken to real-timely and dynamically examine and develop the mechanical behavior and action of single VSMC. Moreover, the molecular mechanisms of this dynamic behavior was explored (e.g. Ca[superscript2+]) to attempt to understand the underlying signaling driving these oscillatory events. In summary, the research demonstrates that age-increased vascular stiffness or hypertension involves not only to alternations in vascular wall ECM, as commonly believed, but also to intrinsic changes in the mechanical stiffness and adhesion of VSMC. This research extends our knowledge of vascular stiffness and contributes some novel findings that may lead to new treatments of vascular diseases associated with vascular stiffening
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