Miniaturized system of neurotrophin patterning for guided regeneration

Understanding the fundamentals of cell behaviour is imperative for designing and improving engineering strategies for regenerative medicine. By combining the precision of confocal microscopy with photochemistry, nerve growth factor (NGF) was chemically immobilized on chitosan films either in distinct areas or as concentration gradients. Using rhodamine as a proxy for NGF, a series of immobilized concentration gradients were created, using the number of rastering scans within a defined area and the distance between each area as a way to control the resulting gradient. The same photochemistry was applied to create NGF patterns on chitosan films which were visualized by immunostaining, and the immobilized NGF remained bioactive as demonstrated with a neuron survival assay. Neuron survival was 73.2 ± 1.3% after 3 days of culture on chitosan films with 30 ng/cm 2 of homogenously immobilized NGF, which was comparable to 74.8 ± 3.4% neuron survival on chitosan with 50 ng/ml of soluble NGF present. Interestingly, when neurons were plated on a chitosan film that had distinct immobilized NGF-patterned areas surrounded by unmodified chitosan, the neurons remained predominantly as single cells in the NGF-patterned regions, but formed aggregates outside of these patterns on the plain chitosan film. Thus, the immobilized NGF pattern influenced neuron behaviour and can be used to further probe mechanisms of other neuron behaviour such as axon guidance. Importantly, the versatility of the confocal laser patterning technique reported here can be extended to other factors to elucidate fundamental cell functions, and hence design strategies in regenerative medicine.