Axon-like aligned conductive CNT/GelMA hydrogel fibers combined with electrical stimulation for spinal cord injury recovery

Abstract

Rehabilitation and regenerative medicine are two promising approaches for spinal cord injury (SCI) recovery, but their combination has been limited. Conductive biomaterials could bridge regenerative scaffolds with electrical stimulation by inducing axon regeneration and supporting physiological electrical signal transmission. Here, we developed aligned conductive hydrogel fibers by incorporating carbon nanotubes (CNTs) into methacrylate acylated gelatin (GelMA) hydrogel via rotating liquid bath electrospinning. The electrospun CNT/GelMA hydrogel fibers mimicked the micro-scale aligned structure, conductivity, and soft mechanical properties of neural axons. For in vitro studies, CNT/GelMA hydrogel fibers supported PC12 cell proliferation and aligned adhesion, which was enhanced by electrical stimulation (ES). Similarly, the combination of aligned CNT/GelMA hydrogel fibers and ES promoted neuronal differentiation and axon-like neurite sprouting in neural stem cells (NSCs). Furthermore, CNT/GelMA hydrogel fibers were transplanted into a T9 transection rat spinal cord injury model for in vivo studies. The results showed that the incorporating CNTs could remain at the injury site with the GelMA fibers biodegraded and improve the conductivity of regenerative tissue. The aligned structure of the hydrogel could induce the neural fibers regeneration, and the ES enhanced the remyelination and axonal regeneration. Behavioral assessments and electrophysiological results suggest that the combination of aligned CNT/GelMA hydrogel fibers and ES could significantly restore motor function in rats. This study demonstrates that conductive aligned CNT/GelMA hydrogel fibers can not only induce neural regeneration as a scaffold but also support ESto promote spinal cord injury recovery. The conductive hydrogel fibers enable merging regenerative medicine and rehabilitation, showing great potential for satisfactory locomotor recovery after SCI.

Keywords: CNT/GelMA; Conductive hydrogel fibers; Electrical stimulation; NSCs differentiation; Spinal cord injury.

Graphical abstract

Fig. 1

Characterization of CNT/GelMA directed conductive hydrogel fibers. (A) Schematic representation of CNT/GelMA hydrogel fibers preparation inspired by the spinal cord and axons. (B) SEM images of fibers at the micron level. (C) TEM images of CNTs (red arrows) on the longitudinal section of GelMA. (D–E) Electrical conductivity of hydrogel fibers. (**p < 0.01).

Fig. 2

Proliferation and adhesion characterization of PC12 in vitro. (A) Confocal images of cells cultured in different conditions. Red fluorescence (Rhodamine phalloidin) and blue fluorescence (DAPI) indicate cytoskeletons and nucleus, respectively. (B) Adhesion of PC12 on fibers observed by SEM. (C) Quantitative comparison of PC12 cell length diameter ratio. (D) Characterization of cell proliferation capacity by CCK-8. (*p < 0.05, **p < 0.01).

Fig. 3

Differentiation and adhesion characterization of NSCs in vitro. (A) Confocal images of cells cultured in different conditions. Red fluorescence (Rhodamine phalloidin) and blue fluorescence (DAPI) indicate cytoskeletons and nucleus, respectively. (B) Adhesion of NSCs on fibers with electrical stimulation observed by SEM. (C) Quantitative comparison of NSCs cell length diameter ratio. (*p < 0.05, **p < 0.01).

Fig. 4

2CNT/GelMA + ES promoted neural stem cells maturation and prevented astrocyte formation in vitro. (A–B) GFAP (green) and Nestin (red) immunofluorescence. (C–D) Quantitative analysis of the optical density of Nestin and GFAP. (*p < 0.05, **p < 0.01).

Fig. 5

2CNT/GelMA + ES promoted the differentiation and elongation of dendrites and axons in vitro. (A) NF (green) and MAP2 (red) immunofluorescence. (B–C) Quantitative analysis of the optical density of MAP2 and NF. (D) Quantitative analysis of average neuron length. (*p < 0.05, **p < 0.01).

Fig. 6

Evaluation of biosecurity of directed conductive hydrogel fibers in rats. (A) After 8 weeks, the histology of the heart, kidney, spleen, brain and liver (top to bottom) of rats treated with GelMA, 2CNT/GelMA and 2CNT/GelMA + ES were shown. Magnified images were to the right of the macroscopic image. The histology of the organs showed no pathological changes in the brain, kidney, liver, heart and spleen of the rats treated with materials and ES. Scale bars: 5 mm on macroscopic images and 100 μm on magnified images. (B) Illustration of the tissue was obtained for the TGA test. (C) Middle part of the spinal cord in the 2CNT/GelMA group, (D) rostral and caudal of the spinal cord in the 2CNT/GelMA group.

Fig. 7

Characterization of inflammation after SCI and materials implantation in rats. (A–B) CS-56 (green) and Iba-1 (red) immunofluorescence at 2 and 4 weeks after surgery. (C) Quantitative analysis of the optical density of CS-56 and Iba-1 among the four groups. (*p < 0.05, **p < 0.01).

Fig. 8

Observation of nerves and axons regeneration after 8 weeks surgery in rats. (A) NF (green) and β-tubulin (red) immunofluorescence indicating nerve fibers. (B) TEM images of axon regeneration in the injure after transplantation. (C) The nerve fiber density at different locations of the lesion site. (D) Quantitative analysis of axons number (*p < 0.05, **p < 0.01).

Fig. 9

2CNT/GelMA + ES promoted hindlimb locomotion and pain perception recovery in rats. (A) Electrical conductivity of spinal cords 8 weeks after surgery. (B)The hindlimb locomotion of rats was evaluated by the BBB score. (C) The pain perception function of rats was evaluated by the thermal pain experiment. (D and E) Quantitative analyses of the latency and amplitude of MEP of bilateral hindlimbs of all experimental groups. (*p < 0.05, **p < 0.01).