Immobilized nerve growth factor and microtopography have distinct effects on polarization versus axon elongation in hippocampal cells in culture

Cell interfacing with biomaterial surfaces dictates important aspects of cell behavior. In particular, axon extension in neurons is effectively influenced by surface properties, both for the initial formation of an axon as well as for the maintenance of axon growth. Here, we investigated how neurons behaved on poly(dimethyl siloxane) (PDMS) surfaces decorated with biochemical and physical cues presented individually or in combination. In particular, nerve growth factor (NGF) was covalently tethered to PDMS to create a bioactive surface, and microtopography was introduced to the material in the form of microchannels. Embryonic hippocampal neurons were used to investigate the impact of these surface cues on polarization (i.e., axon initiation or axogenesis) and overall axon length. We found that topography had a more pronounced effect on polarization (68% increase over controls) compared to immobilized NGF (0.1 ng/mm2) (27% increase). However, the effect of NGF was negligible when both types of stimuli were simultaneously presented on the biomaterial surface. In addition to axon formation, chemical and physical cues are also involved in axon growth following the initiation process. Interestingly, for the same studies described above, the effects of microchannels and NGF were opposite from the effects on polarization; the most evident effect was for the immobilized growth factor (10% increase in axon length with respect to controls) whereas there was no effect in general for the microtopography. More importantly, when the two surface stimuli were presented in combination, a synergistic increase in axon length was detected (25% increase with respect to controls), which could be a result of faster polarization triggered by topography plus enhanced growth from NGF. Additionally, axon orientation was also analyzed and we found the well-known tendency of perpendicular or parallel axonal alignment to be dependent on the width and depth of the channels. This investigation thoroughly compared and distinguished the individual and combined impact of material surface properties (chemical and physical) on axogenesis from the effects on axon length. Overall, topography dominated polarization mechanisms, whereas NGF, and particularly a synergy of immobilized NGF plus topography, dominated axon length. These results could be potentially applied for the design of biomaterials in applications were axon growth is critical.