Topographically induced morphogenic neuronal behaviors via neuron-material interfaces

Abstract

As a development of the central nervous system occurs, dynamically changing cellular microenvironments provide local chemical and physical cues in the form of neighboring cells and extracellular matrices for neurons. Neurons respond to the surrounding information of neuron-material interfaces via changing their morphological features. This dissertation deals with unanticipated effects of designed micro- and nano-topographically modified substrates on primary hippocampal neurons and the potential of topographical structures in recapitulating in-vivo neuronal behaviors at cellular/subcellular level in the in-vitro culture platforms. A historical overview of reports discussing neuronal movements on environmental gradient chemical and physical cues is introduced. Pitch-dependent migration behavior of primary hippocampal neurons and different modes of migration were found with the silicon microcone arrays presenting various pitches in the micrometer range. Physical interactions between nanostructures of nanopillar arrays and hippocampal neurons promoted the developmental processes, such as an axon branching and an axon elongation. Environmental chemical and physical cues dynamically interact with migrating neurons and sprouting axons, and in particular, the gradients of environmental cues are regarded as one of the factors intimately involved in the neuronal movement. The gradients of physical cues, such as stiffness and topography, which also interact constantly with the neurons and their axons as a component of the extracellular environments, have rarely been noted regarding the guidance of neurons, despite their gradually increasingly reported influences in the case of nonneuronal-cell migration. In this chapter, chemical (i.e., chemo- and hapto-) and physical (i.e., duro-) taxis phenomena on the movement of neurons including axonal elongation were discussed. In addition, a topotaxis, the most recently proposed physical-taxis phenomenon, was suggested as another potential mechanism in the neuronal movement, based on the reports of neuronal recognition of and responses to nanotopography. Neuronal migration is a complicated but fundamental process for proper construction and functioning of neural circuits in the brain. Migratory behaviors of primary hippocampal neurons were investigated when neurons were cultured on a silicon microcone (SiMC) array that presents fourteen different pitch domains. Neuronal migration becomes the maximum at the pitch of around 3 μm, with an upper migration threshold of about 4 μm. Immunocytochemical studies indicate that the speed and direction of migration, as well as its probability of occurrence, are correlated with the morphology of the neuron, which is dictated by the pitch and shape of underlying SiMC structures. In addition to the effects on neuronal migration, the real-time imaging of migrating neurons on the topographical substrate reveals new in-vitro modes of neuronal migration, which have not been observed on the conventional flat culture plate, but been suggested by in-vivo studies. Axon collateral branches, as a key structural motif of neurons, allow the neurons to integrate information from highly interconnected, divergent networks by establishing the terminal boutons. Although physical cues are generally known to have a comprehensive range of effects on neuronal development, their involvement in axonal branching remains elusive. Herein, it was demonstrated that nanopillar arrays—at about 400 nm of pitch and 280 nm of height—significantly increase the number of axon collateral branches and also promote their growth. Immunocytochemical studies and biochemical analyses indicate that the physical interactions between the nanopillars and the neurons give rise to lateral filopodia at the axon shaft via cytoskeletal changes, leading to the formation of axonal branches. This work, as the first demonstration that nanotopography regulates axonal branching, provides a guideline for the design of sophisticated neuron-based devices and scaffolds for neuro-engineering.