An injectable, self-healing, electroconductive hydrogel loaded with neural stem cells and donepezil for enhancing local therapy effect of spinal cord injury

Abstract

Background: Spinal cord injury (SCI) is a serious injury with high mortality and disability rates, and there is no effective treatment at present. It has been reported that some treatments, such as drug intervention and stem cell transplantation have positive effects in promoting neurological recovery. Although those treatments are effective for nerve regeneration, many drawbacks, such as low stem cell survival rates and side effects caused by systemic medication, have limited their development. In recent years, injectable hydrogel materials have been widely used in tissue engineering due to their good biocompatibility, biodegradability, controllable properties, and low invasiveness. The treatment strategy of injectable hydrogels combined with stem cells or drugs has made some progress in SCI repair, showing the potential to overcome the drawbacks of traditional drugs and stem cell therapy.

Methods: In this study, a novel injectable electroactive hydrogel (NGP) based on sodium hyaluronate oxide (SAO) and polyaniline-grafted gelatine (NH₂-Gel-PANI) was developed as a material in which to load neural stem cells (NSCs) and donepezil (DPL) to facilitate nerve regeneration after SCI. To evaluate the potential of the prepared NGP hydrogel in SCI repair applications, the surface morphology, self-repairing properties, electrical conductivity and cytocompatibility of the resulting hydrogel were analysed. Meanwhile, we evaluated the neural repair ability of NGP hydrogels loaded with DPL and NSCs using a rat model of spinal cord injury.

Results: The NGP hydrogel has a suitable pore size, good biocompatibility, excellent conductivity, and injectable and self-repairing properties, and its degradation rate matches the repair cycle of spinal cord injury. In addition, DPL could be released continuously and slowly from the NGP hydrogel; thus, the NGP hydrogel could serve as an excellent carrier for drugs and cells. The results of in vitro cell experiments showed that the NGP hydrogel had good cytocompatibility and could significantly promote the neuronal differentiation and axon growth of NSCs, and loading the hydrogel with DPL could significantly enhance this effect. More importantly, the NGP hydrogel loaded with DPL showed a significant inhibitory effect on astrocytic differentiation of NSCs in vitro. Animal experiments showed that the combination of NGP hydrogel, DPL, and NSCs had the best therapeutic effect on the recovery of motor function and nerve conduction function in rats. NGP hydrogel loaded with NSCs and DPL not only significantly increased the myelin sheath area, number of new neurons and axon area but also minimized the area of the cystic cavity and glial scar and promoted neural circuit reconstruction.

Conclusions: The DPL- and NSC-laden electroactive hydrogel developed in this study is an ideal biomaterial for the treatment of traumatic spinal cord injury.

Keywords: Conductivity; Donepezil; Hydrogel; Neural stem cells; Spinal cord injury.

Scheme 1

A brief schematic drawing of the application of NSCs and DPL-loaded NGP hydrogels for SCI repair

Fig. 1

A Images of the SAO and NGP solutions and the NGP hydrogels. B Adhesion of NGP hydrogels to spinal cord tissue. (A) FITR spectra of NH₂-Gelatin, NH2-Gelatin-PANI, SAO and NGP hydrogels. D SEM images of NGP hydrogels, scale bar lengths are 100 μm

Fig. 2

A The display of the self-healing ability of NGP hydrogels. B The electrical conductivity results of NGP hydrogels

Fig. 3

A In vivo degradation images of NGP hydrogels at different time points. B Effect of NGP hydrogel on surrounding skin tissue after subcutaneous implantation, a: Normal mice b: 4 days c: 8 days d: 16 days, scale bar lengths are 200 μm

Fig. 4

In vitro drug (DPL) release of the NGP hydrogel over 8 days; error bars represent the standard deviation for n = 3

Fig. 5

A Morphology of NSCs under a light microscope. Left: ruler: 200 μm; right: ruler: 100 μm. B Nestin immunofluorescence identification of NSCs. C Live and dead cell staining of NSCs on NGP hydrogels; scale bar lengths are 100 μm. D Cell proliferation of NSCs cultured in the control, NGP, DPL and NGP + DPL groups, *P < 0.05, error bars represent the standard deviation for n = 3

Fig. 6

Quantitative real-time PCR analysis of Tuj-1, OSP and GFAP expression in NSCs seeded in different groups. *P < 0.05, error bars represent standard deviation for n = 4

Fig. 7

A Immunofluorescent images for Tuj-1, OSP and GFAP expression by NSCs seeded on different groups. DAPI staining for nuclei (blue) and Cy3-conjugated secondary antibody for protein (red), scale bar lengths are 50 μm. B Axon length of new neurons and the proportion of new neurons, oligodendrocytes and astrocytes in each group, P < 0.05, error bars represent standard deviation for n = 3

Fig. 8

A BBB scores of different treatment groups at ten weeks postinjury. B The results of MEP amplitude measurement in each group. C Electromyography of rats in each experimental group. *P < 0.05, error bars represent standard deviation for n = 6

Fig. 9

A Image of spinal tissue specimen. B H&E staining of spinal cord tissues in different groups; scale bar lengths are 1000 μm. C LFB staining of spinal cord tissue in different groups, Left: scale bars = 500 μm; right: scale bars = 50 μm. D Semiquantitative analysis of the cystic cavity area in different groups, *P < 0.05, error bars represent standard deviation for n = 3. E Semiquantitative analysis of the LFB staining area in different groups, *P < 0.05, error bars represent standard deviation for n = 3

Fig. 10

Immunofluorescent images for Tuj-1 (A-1), NF200 (B-1) and CS56 (C-1) at the lesion sites of the control group and NGP group, Left: scale bars = 500 μm. Right: scale bars = 20 μm. Percentage of Tuj-1 (A-2), NF200 (B-2) and CS56 (C-2) positive area in each group, *P < 0.05, error bars represent standard deviation for n = 3