Three-dimensional directional nerve guide conduits fabricated by dopamine-functionalized conductive carbon nanofibre-based nanocomposite ink printing

Abstract

A potential issue in current nerve guides is that they do not transmit electrical nerve impulses between the distal and proximal end of an injured nerve, i.e. a synapse. Conductivity is a desirable property of an ideal nerve guide that is being considered for peripheral nerve regeneration. Most conductive polymers reported for the fabrication of tissue engineering scaffolds, such as polypyrrole and polyaniline, are non-biodegradable and possess weak mechanical properties, and thus cannot be fabricated into 3D structures. Herein, we have designed a new nanocomposite material composed of dopamine, carbon nanofibers (CNF) and polycaprolactone (PCL) for the fabrication of nerve conduits, which facilitates the growth and migration of neurons toward the targeted end of an injured nerve. This support and navigation of the scaffold leads to better sensory and motor function. The results showed that the mechanical properties of the printed PCL increased by 30% in comparison with the pure PCL film, which is comparable with human nerves. The in vitro cell study of human glioma cells showed that the printed lines provided support for neural cell attachment, migration and differentiation toward the targeted end. In contrast, in the absence of printed lines in the scaffold, the cells attach and grow in random directions, forming a flower shape (cell cluster) on the surface of PCL. Thus, the proposed scaffold is a promising candidate for nerve guide application based on its signal transmission and navigating neurons in a correct pathway towards the targeted end.

Fig. 1. Schematic diagram of navigating directional growth using the functionalized nanocomposite ink.

Fig. 2. (a) FTIR spectra of pure PCL and PCL printed with CNF and DA (40 and 100 μg mL⁻¹), where circles emphasize the OH peak (3700 cm⁻¹) of the carboxylated CNF and NH peak (1565 cm⁻¹) of dopamine. (b) Shear stress of the CNF and CNF + DA nanocomposite inks versus shear rate. (c) Viscosity versus shear rate of the prepared nanocomposite inks.

Fig. 3. (a) Electrical resistance of pure PCL and printed PCL with CNF only and CNF with DA (40 and 100 μg mL⁻¹) nanocomposite ink in the media. (b) Tensile strength of the developed materials. (c) Storage modulus and (d) loss modulus of pure PCL film and printed PCL film with CNF and DA (40 and 100 μg mL⁻¹) nanocomposite ink.

Fig. 4. Confocal images of U87MG cells seeded on pure PCL and printed PCL after 7 and 14 d incubation.

Fig. 5. Number of cells on the scaffold after 7 and 14 d incubation.

Fig. 6. (a) Cellular cluster formed (indicated by arrows) on the pure PCL or pure PCL part of the printed film. (b) Fragmented parts of the printed ink after 14 d incubation (indicated by arrows). (c) SEM images of the pure PCL and printed PCL with ink seeded with U87MG cells after 20 d incubation (arrows and line show the interface between the ink and PCL film).

Fig. 7. Growth and proliferation of neural cell networks on pure PCL film with no directional preference: (a1) SEM image, (a2) possible non-directional growth of nerves cells, and (a3) non-directional growth of glioma cells under fluorescence confocal microscopy (merged image – blue nucleus: DAPI and red actin: rhodamine phalloidin) and directional growth of neural cells on the CNF-based ink line drawn on the PCL film: (b1) SEM image of functionalized nanocomposite ink printed line on PCL film, (b2) possible directional growth of nerves cells, migration and movement to the printed lines then extending axons and dendrites, and (b3) induced directional growth of glioma cells under fluorescence confocal microscopy (merged image – blue nucleus: DAPI and red actin: rhodamine phalloidin); magnified images are shown as inserts.