Author's School

School of Engineering & Applied Science

Author's Department/Program

Mechanical Engineering and Materials Science

Language

English (en)

Date of Award

January 2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Guy Genin

Abstract

Myofibroblasts play important roles in wound healing and pathological organ remodeling, such as hypertensive cardiac fibrosis and promotion of metastasis. Differentiated myofibroblasts are characterized by increased production of extracellular matrix: ECM) proteins and by the development of α-smooth muscle actin: α-SMA) positive stress fibers that are connected to the ECM through focal adhesion assemblies. Moreover, mounting evidence suggests that development of myofibroblasts is profoundly influenced by the mechanical microenvironment, especially, by the structure, organization or stiffness of the ECM: Hinz and Gabbiani, 2003a). Myofibroblasts are likely signaled by mechanical changes in their environment transduced through their cytoskeletons, and the fundamental goal of this dissertation is to develop our understanding of this transduction, with the long term goal of guiding treatments that modulate the responses of myofibroblasts to their mechanical environment. Previous studies provide a tremendous amount of data for how cells adapt to mechanical stress in two dimensions: 2D). Many techniques exist for probing cyoskeletal responses in 2D. These range from pushing cells with a fluid to poking cells with an AFM probe to pulling cells using micropipette aspiration. However, myofibroblasts in a realistic tissue environment cannot be tested using these approaches. The challenge is that cells adopt very different dynamic morphologies in the network of fibers presented by a natural tissue, and cannot be pushed, poked, or pulled using these techniques inside a tissue. The only way to probe the cytoskeleton of such a cell naturally in a tissue is through its natural adhesions to the ECM. In this work, we developed a 3D tissue culture system to quantify the responses of contractile fibroblasts in an engineered tissue construct: ETC) to external mehanical stimuli. We try to answer the following questions. How does the actin cytoskeleton respond and adapt to mechanical stretch? How does this relate to whole-cell kinematics? How do cells interact with the extracellular environment in 3D? To address the first question, a suite of mechanical tests were performed on transfected myofibroblasts in ETCs. The transfection enabled the visualization of stress fiber dynamics in 3D, and the work showed for the first time that stress fibers can form in response to mechanical stretch. The work also quantified the diversity of cellular responses into three classes, as described in Chapter 2. To address the second question, we studied the nature of the retraction of filopodium-like processes of stained myofiboblasts in response to mechanical stretch. Results, described in Chapter 3, showed that retraction of these processes was independent of the magnitude of stretch and the length of the process. The time constants associated with process retractions were very slow compared to membrane retraction in other cells. The study of the last question confirmed that large focal adhesions existed in a 3D environment, early in remodeling, and that focal adhesion complexes evolve during ECM remodeling. The work in this dissertation helps establish a platform to study cellular mechanics and function in a 3D environment through both real-time imaging and force measurement simultaneously. While the work is at the level of basic biophysics, it provides a quantitative window into the mechanisms underlying myofibroblast-based pathologies.

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Permanent URL: http://dx.doi.org/10.7936/K7416V47

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