A chitosan/acellular matrix-based neural graft carrying mesenchymal stem cells to promote peripheral nerve repair

Abstract

Background: Treatment of peripheral nerve defects is a major concern in regenerative medicine. This study therefore aimed to explore the efficacy of a neural graft constructed using adipose mesenchymal stem cells (ADSC), acellular microtissues (MTs), and chitosan in the treatment of peripheral nerve defects.

Methods: Stem cell therapy with acellular MTs provided a suitable microenvironment for axonal regeneration, and compensated for the lack of repair cells in the neural ducts of male 8-week-old Sprague Dawley rats.

Results: In vitro, acellular MTs retained the intrinsic extracellular matrix and improved the narrow microstructure of acellular nerves, thereby enhancing cell functionality. In vivo neuroelectrophysiological studies, gait analysis, and sciatic nerve histology demonstrated the regenerative effects of active acellular MT. The Chitosan + Acellular-MT + ADSC group exhibited superior myelin sheath quality and improved neurological and motor function recovery.

Conclusions: Active acellular-MTs precellularized with ADSC hold promise as a safe and effective clinical treatment method for peripheral nerve defects.

Keywords: Acellular nerve; Biomaterials; Chitosan; Mesenchymal stem cells; Peripheral nerve injury; Tissue repair.

Fig. 1

Identification of ADSC. A–D Morphology of ADSC. ADSC exhibited multi-potential differentiation capacity for osteogenesis and adipogenesis. Scale bar: 50 μm, 100 μm, 200 μm. E–H Flow cytometric analysis of cell surface markers of ADSC

Fig. 2

Morphological observation of normal nerve, acellular nerve and chitosan. A–B SEM morphology of normal and acellular nerve. Normal nerves have a compact and ordered structure, while acellular nerves maintain a complete three-dimensional structure. C SEM morphology of chitosan. D HE staining and immunofluorescence staining of the laminin and fibronectin in normal nerve and acellular nerve. Scale bar: 100 μm

Fig. 3

Dead/alive staining of acellular nerve microtissue. FDA/PI bichromatic fuorescence staining was performed at 3d, 5d and 7d of ADSC coculture with acellular-MT. Green fluorescence denotes living cells and red fluorescence denotes dead cells. Scale bar: 200 μm

Fig. 4

Evaluation of neurological function recovery in each group. A–B Gait analysis and footprint strength analysis of 12W groups. C Electrophysiological recovery was assessed in each group at 12W, and CAMP on the operating side was recorded in each group. D–E SFI and Stand/Swing Time Ratio in 4W, 8W, 12W groups (*P < 0.05, **P < 0.01, ***P < 0.001,n = 5), vs. Chitosan group(^##P < 0.01, ^###P < 0.001, n = 5), vs. Autograft groups. Data are represented as mean ± SD

Fig. 5

The distal nerve graft was evaluated by SEM at 12W and analyzed statistically. A Representative images of the transverse section of myelinated nerve fibers in each group. B Calculate the mean diameter of myelinated nerve fibers. C Calculate myelin sheath thickness. (**P < 0.01, ***P < 0.001, ****P < 0.0001, n = 5). Data are represented as mean ± SD

Fig. 6

HE staining was performed on the longitudinal section of nerve graft in each group at 2W. Axons are seen regenerating and growing into the nerve graft. Scale bar: 1 mm

Fig. 7

The longitudinal sections of nerve grafts were stained with immunofluorescence at 4d and 6d. The regenerated myelin sheath was stained with anti-S100 antibody (green), and the regenerated vascular endothelium was stained with anti-endothelial Cell-1 antibody (red), the nucleus was stained with DAPI (blue). Scale bar: 100 μm, 200 μm