Quantification of Axonal Outgrowth on a Surface with Asymmetric Topography
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Quantification of Axonal Outgrowth on a Surface with Asymmetric Topography Elise Spedden1 and Cristian Staii1 Tufts University Department of Physics and Astronomy, 4 Colby St, Medford, MA 02155
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ABSTRACT Topographical features are known to influence the axonal outgrowth of neurons. Understanding what kinds of topographical features are most effective at growth cone guidance and how outgrowth responds to these structures is of great importance to the study of nerve regeneration. To this end we analyze axonal outgrowth on tilted nanorod substrates which have been shown to impart directional bias to neuron growth. We utilize the Atomic Force Microscope to characterize the surface features present on these substrates and how such features are influencing the axonal outgrowth. Additionally, using a model which considers the neuronal growth cone as an object influenced by an effective potential we determine an effective force imparted on the growth cone by the surface topography. INTRODUCTION The study of neuronal outgrowth on patterned surfaces is important for understanding nervous system development and repair 1, 2. Such studies focus both on understanding the underlying sensory mechanisms behind directed neuronal outgrowth, and on optimizing biocompatible surfaces for directed growth in the field of neural tissue engineering. Directed axonal outgrowth has been studied on a variety of surfaces. Particular focus has been paid to the sensing of surface bound proteins 3, signaling molecules 4, and topographical features such as ridges 5, 6 or indentations 7. Axons have been shown to respond to topographical patterns 5-9. The preferred growth orientation with respect to repeating patterns is cell-type dependent 5, 6, 10, and neurons have been shown to consistently respond to variations in surface topography and micropatterning 5-7, 9-11. Many studies of axonal outgrowth on patterned substrates rely on symmetric patterns to impart outgrowth along one or more pattern axes 5-7, 9. The neurons in these studies typically prefer alignments which can be characterized as parallel or perpendicular to the repeating topographical surface patterns. Similar results have been achieved on symmetric patterns of chemical rather than topographical signals 3. Such symmetric surfaces can bias outgrowth along an axis, but cannot be used to direct neuronal growth towards a single direction, as can be achieved through electrical stimulation 12. Asymmetric tilted nanorod surfaces, however, have been shown to impart axonal outgrowth bias towards a single dominant direction 11. Here we utilize similar tilted nanorod substrates. We make use of the high spatial resolution of the Atomic Force Microscope (AFM) to characterize the features of these surfaces, and analyze the directed outgrowth on these surfaces of embryonic rat cortical neurons after 5 days. Additionally, we model the growth cone as an object under the influence of an effective force to determine the relative strengths of axonal outgrowth bias towards perpendicular aligned axon growth
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