Engineering Spatial Patterns of Gene Expression: Fundamental Studies of Guided Cellular Processes and Applications to Tissue RegenerationPublic Deposited
Natural tissues can have complex architectures characterized by the organization of multiple cells into structures, such as branching networks of the vascular or nervous systems. This cellular organization arises, in part, from spatial patterns in the expression of soluble factors, which create concentration gradients that direct cellular processes during morphogenesis. Regenerative strategies for damaged tissue must recreate these cellular architectures to restore function. Biomaterials serve a central role in the engineering of functional tissue replacements, and are designed to present a combination of insoluble and soluble signals that direct tissue formation. Gradients of insoluble signals have been created at biomaterial surfaces; however, generation of gradients of soluble signals has proven more difficult. By combining non-viral gene delivery strategies with soft lithography, I have developed methods to spatially pattern gene expression. Using DNA encoding for soluble growth factors, transfection leads to localized and sustained secretion thereby creating concentration gradients as the factors diffuse. In this thesis, the systems are utilized to investigate fundamental questions in neurite guidance and are applied to the rational design of tissue engineering scaffolds. Spatial patterns of gene expression within a cluster of cells were established and the gradients formed by diffusion were mathematically modeled. Neuronal responses to NGF gradients formed by patterned expression were experimentally determined using an in vitro co-culture model, and the width of the pattern governed neuronal response. Patterns 100-250 m in width confined neuron survival and neurite extension to the region of localized expression. Patterns of 500-1000 m in width guided neurite extension up the NGF gradient, with guidance dependent on the amount of NGF on the surface and the distance a neuron was cultured from the pattern. Spatial patterns of gene expression were next established within single cells, by altering the extent of transgene expression and transfection efficiency. The gene expression patterns were combined with topographically patterned scaffolds to determine the design parameters necessary for directed neurite extension during nerve regeneration strategies. Neurite guidance was governed by the topographical pattern width and the extent of transgene expression by transfected cells. Photopolymerizable hydrogels were developed to extend spatially patterned gene expression to three-dimensional systems. Hydrogels were characterized in terms of mechanical properties, DNA vector release, and in vivo cell migration and transfection. These studies demonstrate the capacity of patterned gene expression to create concentration gradients of soluble factors that can locally organize tissue formation. The systems developed in this thesis provide a platform with which to investigate concentration gradients in tissue formation, and may be applied for the engineering of functional tissue replacements.