Date of Award
Doctor of Philosophy (PhD)
In this work, various methods of controlling cell growth are examined. Cell-cell interaction, apoptotic cues, three dimensional scaffolds, and non-adherent environments are evaluated for their ability to affect the differentiation, morphology, and growth rate of different cell types. Previous work has shown that cell growth and cell morphology can be influenced by patterns of polymers coated on surfaces in two dimensions, designated here as the x and y dimensions of a standard Cartesian coordinate system. Tissue engineering and regenerative medicine studies have shown limited success in modifying growth in the third, z dimension. This work considers not only the x, y, and z dimensions, but also the fourth dimension, time. The time dimension is explored via apoptosis. The x and y dimensions are inspected through surface patterning, and the three physical dimensions are scrutinized together by means of scaffolding-cultured cells and cytophobic surfaces for non-adherent aspects of cell patterning. The project goals accomplished here are: (1) to develop a simple model of apoptosis that can be used to determine visually the temporal progression of apoptosis and to determine whether staurosporine-induced apoptosis can be delayed by pre-treatment of cells with glutamate, (2) to determine the viability of a cellulose/gelatin biologically-derived 3D cellular scaffolding construct as a platform for tissue engineering, (3) to develop a novel cell-adherence blocking strategy that will improve the localization of cell adherence to patterns deposited by the NanoEnabler® system.
Measures of apoptotic activity based on digital images showing changes in cell area, cell shape, nuclear area and nuclear shape were used to develop the Cell Area Factor and Nuclear Area Factor model. Biochemical assays for mitochondrial activity and for caspase 3 (casp3) activity showed a delay in staurosporine induced apoptosis. Digital images of the scaffold materials demonstrated the scaffold's ability to encourage cell invasion, growth and differentiation. The image observations were supported by MTT assays showing increased metabolic activity of the cell indicating proliferative culture. The adherence blocking strategy discussed resulted in 3D growth of cancerous brain tumor cells tracked via digital imaging and tumor area analysis. Calcein vital dye staining supported the evidence for a growing tumor colony.
The three approaches for cell growth modification, apoptotic stimuli, scaffolding directional cues, and negative adhesive cues (cytophobic surfaces), are considered as building blocks that can be combined in a broader tissue engineering strategy to control the adhesion, morphology, and differentiation of cells. The apoptotic modulator chronicled in this work can be used to modify biological pathways in vitro and provide a more biomimetic environment that can be used to engineer tissues and to formulate and test new experimental hypotheses. The success of the biologically derived cellulose/gelatin material indicates that further work is warranted to develop it as a scaffold to support and cultivate 3D engineered, spatially defined tissues. The cytophobic surface has lead not only to a new blocking strategy, but also to the unexpected result of leading to a novel 3D model of cancer progression in vitro that closely resembles the in vivo situation.
McNamara, James N., "" (2011). Dissertation. 417.