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The study of nervous system regeneration and axonal outgrowth control are relevant in several research areas, like neurophysiology or biomedical engineering. Among the elements that control neuron dynamics, host substrate topography is a key parameter in driving cell differentiation [Sapelkin et al, Biomaterials 27, 842 (2006)]. Many research efforts were directed to control axon outgrowth direction [Recknor et al, Biomaterials 25, 2703 (2004)], but only recently a few experiments reported succesful topographical guidance. Johansson et al. demonstrated forced axon alignment to nano-ridges fabricated on silicon substrates [Johansson et al., Biomaterials 27, 1251 (2005)], Foley et al. observed increased response to nerve growth factor (NGF) in PC12 cells growing along SiO2 nano-gratings [Foley et al., Biomaterials 26, 3639 (2005)].
We present time-lapse experiments and analyze the differentiation dynamics of PC12 cells on nano-patterned bio-compatible substrates. Particular attention was paid to the implementation of a full physiological environment. To this end we chose tissue culture polystyrene (TCPS) dishes (without any chemical functionalization) as substrates and induced the desired topography by nanoimprint lithography (NIL). This configuration is optimal for in vivo studies since it allows cell development for very long time without any interference due to the interaction with toxic materials or chemically active molecules. Gratings with 200 nm depth and different line-widths and pitches were studied down to 500 nm and 1000 nm, respectively. PC12 cells were cultured on the patterned substrates and, after NGF addiction to the medium, body morphology, cell-movement and neurito-genesis were monitored over 24-48 hour periods. In addition to demonstrating guided differentiation (our analysis was supported by statistical analysis over a 100-cell sample), our studies show strong non-linear change in body-morphology and axon length, and guided cell-movement. We followed unstable synaptic connections and cell-body polarization, and we shall show strong evidence of competition between topographical guidance and cell-to-cell interaction. |