Electroconductive polymeric nanowire templates facilitates in vitro C17.2 neural stem cell line adhesion, proliferation and differentiation

Abstract

Stem cells still remain one of the most exciting and lucrative options for treatment of a variety of nervous system disorders and diseases. Although there are neural stem cells present in adults, the ability of both the peripheral and central nervous system for self-repair is limited at best. As such, there is a great need for a tissue engineering approach to solve nervous system disorders and diseases. In this study, we have developed electrically conductive surfaces with controlled arrays of high aspect ratio nanowires for the growth and maintenance of neural stem cells. The nanowire surfaces were fabricated from polycaprolactone using a novel nanotemplating technique, and were coated with an electrically conductive polymer, polypyrrole. The polypyrrole-coated nanowire surfaces were characterized using scanning electron microscopy and X-ray photoelectron spectroscopy. Additionally, the surface resistance of polypyrrole-coated nanowire surfaces was measured. C17.2 neural stem cells were used to evaluate the efficacy of the polypyrrole-coated nanowire surfaces to promote cell adhesion, proliferation and differentiation. The results presented here indicate significantly higher cellular adhesion and proliferation on polypyrrole-coated nanowire surfaces as compared to control surfaces. The differentiation potential of polypyrrole nanowire surfaces was also evaluated by immunostaining key neuronal markers that are expressed when NSCs differentiate into their respective neural lineages.

Figure 1

Schematic of PCL nanowire fabrication: A. A PCL pellet is placed on top of the alumina nanoporous membrane; B,C. The polymer is extruded through the nanoporous membrane in a vacuum oven; D. The alumina nanoporous membrane is dissolved in NaOH to release the nanowires; E. PCL nanowire surfaces.

Figure 2

Representative SEM images of NW and PPy-NW; the high magnification SEM images show an altered and rougher surface architecture on individual nanowires that were coated with PPy.

Figure 3

High-resolution C1s and N1s scan for NW and PPy-NW surfaces indicating precise changes in surface carbon and nitrogen concentrations after coating the surfaces with PPy.

Figure 4

Similar surface morphology of nanowires after 7 days in physiological environment as that of as-coated surfaces (Figure 2, PPy-NW) indicating that the coatings are robust and will not rapidly degrade.

Figure 5

Representative fluorescence microscopy images of NSCs stained with calcien-AM on NW and PPy-NW surfaces after 1, 2, and 7 days of culture; the dotted circle represents cell spreading and contact, and “neuronal network” formation. The figure at the bottom shows cell coverage on NW and PPy-NW surfaces calculated using ImageJ software.

Figure 6

Representative SEM images NSCson NW and PPy-NW surfaces after 1, 2 and 7 days of culture. Significanly higher “neural network” formation is observed on PPy-NW surfaces. The high magnification images (for the dotted circle) confirm that the cells interacting the nanowire architecture.

Figure 7

Representative immunofluorescence images showing expression of Nestin, NF-H, GFAP, MAP2, and APC on PPy-NW surfaces confirming that the NSCs have differentiated into all neural lineages.