The physiological environment of neurons in vivo is composed of complex, physical structures in the form of support cells, other neurons, and the extracellular matrix. However, conventional substrates for neuron culture in vitro (flat, glass coverslips or tissue culture plates) fail to embody this biological topography. This dissertation deals with the design of physical surfaces that better represent the structural complexity of the in vivo environment, and characterizes the interaction and behavior of neurons with such platforms. A brief overview of recent research involving neurons on different topographies, and nanotopography in particular, is introduced. Neurons were cultured on anisotropic pillar arrays for the directional guidance of neurite outgrowth. Vertically-grown silicon nanowires were used to induce an alternate pathway of neuron development. Bioinert replicas of real neurons were developed as scaffolds by silicifying live neuronal networks.
Control over neurite directionality has been extensively researched through the use of aligned nanofibers or grooved substrates. However, most studies have relied on the use of continuous, line-based topographies to guide neurite outgrowth, and the use of interrupted topographies is rare, despite their unique effects on primary hippocampal neuron behavior. Anisotropic micropillar arrays were designed to successfully control the directionality of neurite outgrowth in primary hippocampal neurons. The degree of anisotropy in the micropillar arrays was found to be correlated to the fidelity of neurite alignment, and the rate of neurite elongation was affected as well. The three-dimensional interactions between neurites and micropillars was also explored. This topographical platform can contribute to both studies in fundamental neuron behavior, as well as the practical development of neuroregenerative scaffolds.
In neuron-material interfaces, recent findings that nanometric features accelerate the neurite outgrowth in vitro have important biological implications in mechanobiology, considering neurons and other cells encounter the hierarchical nano/microstructures of extracellular matrices in vivo. High-density, vertically grown silicon nanowires (vg-SiNWs) directed a new in vitro developmental pathway of primary hippocampal neurons. Neurons on vg-SiNWs formed a single, extremely elongated major neurite earlier than minor neurites, which led to accelerated polarization. Additionally, the development of lamellipodia, which generally occurs on 2D culture coverslips, was absent on vg-SiNWs.
Developing neurons in vivo rely on several physiological cues for neurites to find their intended synaptic target. Included are other ‘pioneer neurons’ which act as physical guideposts to facilitate the efficient and accurate axon pathfinding of developing neurons. Previous works in topography, which have utilized structures such as nanowires, gratings, nanofibers and micropillars, have endeavored to more faithfully represent the complex physical environment of neurons in vivo in order to better understand the mechanism behind neurite outgrowth phenomenon. However, the majority of these structures have been singular in form and scale, and are considered simplified representations of a true neuronal network. Additionally, while the importance of hierarchical topographies, which feature both nano- and micrometric structures, has been increasingly recognized for other cell types, their effects on primary neurons have yet to be seen. In response to these needs, the use of neurotemplated scaffolds as a neuron culture platform, which are not only hierarchical in nature, but also act as accurate replicas of a real neuronal network, is presented. Neurons cultured on neurotemplated scaffolds displayed the ability to recognize templated somas amidst the ‘nanotopographical noise’ of templated neurites, and used the micrometric, rounded structures as guideposts throughout neurite outgrowth. Additionally, neurite complexity could be modulated by varying the nanotopographical density of the neurotemplated scaffolds, and that nano- and micrometric features on hierarchical topographies govern different aspects of neurite development. A potential evolution of this work involving a novel patterning technique using lipids is also discussed.