Aligned chitosan nanofiber hydrogel grafted with peptides mimicking bioactive brain-derived neurotrophic factor and vascular endothelial growth factor repair long-distance sciatic nerve defects in rats

Abstract

Autologous nerve transplantation, which is the gold standard for clinical treatment of peripheral nerve injury, still has many limitations. In this study, aligned chitosan fiber hydrogel (ACG) grafted with a bioactive peptide mixture consisting of RGI (Ac-RGIDKRHWNSQGG) and KLT (Ac-KLTWQELYQLKYKGIGG), designated as ACG-RGI/KLT, was used as nerve conduit filler to repair sciatic nerve defects in rats. Methods: Chitosan nanofiber hydrogel was prepared by a combination of electrospinning and mechanical stretching methods, and was then grafted with RGI and KLT, which are peptides mimicking brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF), respectively. The physicochemical properties of ACG-RGI/KLT were fully characterized. In vitro, the distribution, proliferation, and secretory activity of Schwann cells were analyzed. Next, the in vivo repair potential for 15-mm rat sciatic nerve defects was examined. The recovery of regenerated nerve, muscle, and motor function was evaluated by neuromuscular histology, electrophysiology, and catwalk gait analysis. Results: We first constructed directionally aligned chitosan nanofiber hydrogel grafted with RGI/KLT peptide mixture (ACG-RGI/KLT). ACG-RGI/KLT oriented the Schwann cells, and promoted the proliferation and secretion of neurotrophic factors by Schwann cells. At an early injury stage, ACG-RGI/KLT not only enhanced nerve regeneration, but also promoted vascular penetration. At 12 weeks, ACG-RGI/KLT facilitated nerve regeneration and functional recovery in rats. Conclusions: Aligned chitosan nanofiber hydrogel grafted with RGI/KLT peptide provides an effective means of repairing sciatic nerve defects and shows great potential for clinical application.

Keywords: Peripheral nerve injury; chitosan nanofibers; electrospinning; mechanical stretching; peptides..

Figure 1

Preparation and characteristics of aligned chitosan fiber hydrogel. (A) Illustration of the electrospinning and mechanical stretching setup. A voltage of 3-5 kV was applied between the spinning solution and collector. Chitosan polymer chains became increasingly aligned as the chitosan hydrogel was extruded from the needle (Box 1) and stretched during entry (Box 2) into a rotating bath. As the chitosan hydrogel entered the rotating bath (Box 3) containing sodium tripolyphosphate (STPP; black dots) under mechanical stretching, chitosan chains were further aligned and crosslinked by STPP in the bath (Box 4), thus forming a stable chitosan nanofiber hydrogel. (B) Proton nuclear magnetic resonance (NMR) spectroscopy of RGI and KLT. (C) Gross view and light microscopic images of an aligned chitosan hydrogel bundle. Polarized light microscopic images showing optical extinction in the crossover region of two chitosan fiber bundles. (D) Scanning electron microscopy (SEM) images of aligned chitosan fiber hydrogel (ACG) showing hierarchically aligned structures under different magnifications. (E) Test of the mechanical properties of aligned chitosan hydrogels. (F) Elastic modulus of aligned chitosan hydrogels.

Figure 2

Gross view of nerve grafts and operative model of all groups. The chitin conduits were light yellow in color and transparent. ACG and ACG-RGI/KLT were white, similar to sciatic nerves. All groups received the 15-mm sciatic nerve defect.

Figure 3

ACG-RGI/KLT regulated Schwann cells in vitro. (A) Images of cultured Schwann cells in the ACG, ACG-RGI/KLT, and control groups. Schwann cells were stained with anti-S100 (red) and DAPI (blue). (B) The expression levels of several markers reflecting the proliferation, adhesion, and secretory function of cells were examined by Western blotting (WB). (C-I) Statistical analysis of brain-derived neurotrophic factor (BNGF), nerve growth factor (NGF), glial cell-derived neurotrophic factor (GDNF), vascular endothelial growth factor (VEGF), neural cell adhesion molecule 1 (NCAM1), proliferating cell nuclear antigen (PCNA), and phosphorylated protein kinase B (p-AKT) protein levels, respectively. (K-N) qRT-PCR results indicated the relative expression levels of BNGF, NGF, GDNF, VEGF, and NCAM1. Data are presented as means ± standard error of the mean, n = 3 for each group. **p<0.01, * p<0.05.

Figure 4

Evaluation of regenerated nerve fibers at 12 weeks after surgery. (A) Hematoxylin and eosin (HE) staining and toluidine blue staining in all groups. Transmission electron microscopy (TEM) images of the regenerated sciatic nerve. Statistical analysis of regenerated axons: calculation of the density of myelinated axons (B), diameters of myelinated axons (C), and thickness of the myelin sheath (D). All data are expressed as the mean ± standard error of the mean. *p<0.05.

Figure 5

Fluoro-gold (FG) retrograde tracing in all groups. (A) Images of FG-labeled motor neurons in the spinal cord and sensory neurons in DRGs in all groups at 12 weeks after surgery. The average number of FG-positive motor neurons and sensory neurons in each group is shown in (B) and (C), respectively. All data are expressed as the mean ± standard error of the mean. * p<0.05, ** p<0.01.

Figure 6

Recovery of motor function and electrical conduction in rats in all groups at 12 weeks after surgery. (A) Representative images of right hind paw (injured) and left hind paw (normal) footprints in all groups at12 weeks after the operation. (B) Statistical analysis of sciatic function index (SFI) values of all groups. All data are expressed as the mean ± standard error of the mean. *p<0.05, (n = 5). (C) Representative images of gastrocnemius muscles and Masson's trichrome staining images of transverse sections of gastrocnemius muscles. (D) Statistical analysis of the wet weight ratios of gastrocnemius muscles (injured leg vs. normal hind leg) (n = 5). (E) Statistical analysis of the mean cross-sectional area of gastrocnemius muscle fibers. All data are expressed as the mean ± standard error of the mean. * p<0.05. (F) Representative images of the complex muscle action potential (CMAP) of each group, as recorded by electrophysiological apparatus. Statistical analysis of CMAP amplitude (H) and latency (I) in each group. All data are expressed as the mean ± standard error of the mean. * p<0.05.

Figure 7

ACG-RGI/KLT promoted angiogenesis and regeneration of the sciatic nerve. (A) NF200 and CD31 immunofluorescence staining of regenerated nerves at 3 weeks after surgery. (B) WB of GAP43, CD31, and VEGF in regenerated nerves at 3 weeks after surgery. (C-E) Statistical analyses of GAP43, CD31, and VEGF, respectively. Data are presented as the mean ± standard error of the mean, n = 3 for each group. * p<0.05, ** p<0.01.

Figure 8

Illustration of the process of neural regeneration in the conduit. (A) ACG-RGI/KLT was used to bridge the nerve stumps. (B) Schwann cells migrated along the biological cables formed by ACG-RGI/KLT from the proximal and distal nerve stumps. (C) Newly regenerated axons sprouted along these cables. (D) Schwann cells wrapped around the axons to form myelin.