Investigation of synergic effects of nanogroove topography and polyaniline-chitosan nanocomposites on PC12 cell differentiation and axonogenesis

Abstract

Axonal damage is the main characteristic of neurodegenerative diseases. This research was focused on remodeling cell morphology and developing a semi-tissue nanoenvironment via mechanobiological stimuli. The combination of nanogroove topography and polyaniline-chitosan enabled the manipulation of the cells by changing the morphology of PC12 cells to spindle shape and inducing the early stage of signal transduction, which is vital for differentiation. The nanosubstarte embedded with nanogooves induced PC12 cells to elongate their morphology and increase their size by 51% as compared with controls. In addition, the use of an electroconductive nanocomposite alongside nanogrooves resulted in the differentiation of PC12 cells into neurons with an average length of 193 $\pm 7$ μm for each axon and an average number of seven axons for each neurite. Our results represent a combined tool to initiate a promising future for cell reprogramming by inducing cell differentiation and specific cellular morphology in many cases, including neurodegenerative diseases.

Keywords: Bioengineering; Biotechnology; Nanoscience; Nanotechnology; Tissue engineering.

Graphical abstract

Figure 1

The FTIR spectra of PANI, chitosan, and PANI-C

Figure 2

Characterization of PANI, chitosan, and PANI-C using X-ray diffraction and UV-visible spectroscopy (A) X-ray diffraction of PANI. (B) X-ray diffraction of chitosan. (C) X-ray diffraction of PANI-C nanocomposites. (D) UV-visible spectra of chitosan, PANI, and PANI-C.

Figure 3

FESEM images of PANI-C molecules (A) Single PANI-C molecules. (B) PANI-C populations accumulated as nanoclusters.

Figure 4

The MTT viability assay of cultured PC12 cells incubated with different concentrations of PANI and PANI-C (A) Various concentrations of PANI. (B) Various concentrations of PANI-C.

Figure 5

The microscopic images of PC12 cell line morphology before and after the alignment in the direction of the nanogrooves (A) phase contrast. (B) Cytoplasm stained by Calcein AM. (C) nucleus stained by Hoechst. (D) The merging of phase contrast and fluorescent images. (E) The phase contrast of the control group. (F) Cytoplasm of the control group stained by Calcein AM. (G) Nucleus of the control group stained by Hoechst. (H) The merged phase contrast and fluorescent images of the control group.

Figure 6

FESEM images of cells cultured on nanogroove topography (A) The morphology of the cells beside filopodia and cell-cell interactions. (B) The early formation of axons in cells. (C) Single cells cultured on nanogroove topography. (D) The early formation of axons in a single cell. (E) Random direction of a positive control group of cells in bulk level. (F) SEM image of bare nanosubstrate.

Figure 7

The fluorescent and confocal microscopy images of neurons differentiated from PC12 cells incubated with a primary antibody against neurofilaments and Alexa Fluor 488 as the secondary antibody (A and D) The images were captured in phase contrast. (B and E) Fluorescent microscopy images. (C and F) Merged images of phase contrast and fluorescent microscopy images. (G) The confocal light images of the positive control (PC12 cells treated with NGF). (H) Fluorescent confocal image of the positive control. (I) Merged images of light and fluorescent confocal microscopy images.

Figure 8

The mechanical properties of bare PDMS and PDMS coated with PANI and PANI-C nanosubstrates (A) Hardness. (B) Stiffness. (C) Young’s modulus.

Figure 9

AFM topographic images (A) 3D image of a flat PDMS substrate. (B) 2D image of a flat PDMS substrate. (C) 3D image of a nanogrooved PDMS substrate with a ridge width of 125 ± 94 nm. (D) 2D image of nanogrooved PDMS substrate. (E) 3D image of nanogrooved PDMS substrate coated with PANI-C. (F) 2D image of nanogrooved PDMS substrate coated with PANI-C.