Analysis of neuronal growth on micropatterned substrates.

摘要

Currently, nerve injuries can result in conditions ranging from full functional recovery to partial loss of function to paralysis. In vivo, growth cones, sensory-motile structures on the tips of regenerating axons, are guided along specific innervation pathways by spatially oriented cues in the extracellular environment. To understand the mechanisms of axon-pathway interactions, embryonic day 7 chick dorsal root ganglia (DRG) were cultured on micropatterned substrates of the extracellular matrix protein laminin (20 or 30 mum-wide stripes). The micropatterned substrates were created using photolithography, a technology used in the manufacture of electronics circuit boards.;Chick DRG response to the substrates was evaluated using both low and high magnification microscopy. The low magnification studies showed that the micropatterned substrates were capable of modulating both the density and pattern of neurite outgrowth around the explant. The dynamic behavior of the growth cones was investigated using high magnification, phase-contrast videomicroscopy. Growth cone morphology was evaluated both in response to substrate type ( e.g. uniform versus patterned laminin) and to the type of interactions the growth cone has with other growth cones or neurites. Isolated growth cones extending along uniform substrates tended to be smaller, less complex, and more circular in shape compared to both those on micropatterned substrates and those interacting with other cells. A comparison of the morphology data by culture age revealed that there were time-dependent variations in parameters such as growth cone area and number of filopodia extended. Several methods were used to model growth cone migration dynamics. A geometric analysis showed directed movement on the micropatterned substrate. Probability distribution modeling showed there was no one single distribution model that represented all parameters. Last, stochastic modeling yielded both random and deterministic components in migration.;In summary, the patterned substrates were capable of regulating the pattern of neurite outgrowth, growth cone morphology, and migration trajectories. However, some refinement of the experimental system will be required in order to develop a more consistent picture of regeneration dynamics. In the future, these results can potentially be applied to the design of in vitro neural networks or nerve regeneration devices.