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.

1. Introduction

Peripheral nerve injuries are common. However, owing to the slow regeneration rate of axons, the target muscle after nerve injury faces long-term denervation, causing irreversible damage [1, 2]. Severe nerve deficits are often accompanied by long nerve spaces that do not heal spontaneously; thus, bridging grafts are often required to accelerate axon regeneration [3]. Several advances have been made in nerve graft scaffolds, including the use of abundant natural biomaterials, synthetic materials, and autografts. These scaffolds have good biocompatibility, suitable degradation cycles, and tensile strength, and are suitable for different degrees of peripheral nerve defect repair [4–7].

Acellular nerve allografts (ANA) are an alternative to autografts. Numerous studies have suggested that ANA combined with cell or molecular therapy holds promise for nerve regeneration [8]. After nerve injury, endogenous Schwann cells have limited repair functions, particularly in acellular nerve grafts with long nerve defects (>3 cm) [7, 9]. Stem cell therapy can accelerate axon regeneration and help neurons survive, and has shown significant advantages in the repair of peripheral nerve defects [10]. Adipose-derived mesenchymal stem cells (ADSCs) promote tissue regeneration by releasing active factors. ADSCs have the potential for self-renewal and multidirectional differentiation, and are readily available and expanded in vitro, making them one of the best cells for combination therapy with acellular nerves [11, 12].

Important components of acellular nerves, such as the intrinsic extracellular matrix (ECM) and complex microstructure [13], can recruit Schwann cells to migrate from the stump of the nerve defect to the interior of the nerve graft to induce neurotrophic factors and other biologically active substances [3, 14]. Injured axons regenerate and extend to the distal end of the graft [15]. As such, preserving an intact ECM structure is key to promoting axon regeneration [16]. However, the internal structure of normal acellular nerves is very dense, making it difficult for the regenerated axons to span the graft. In addition, the degree of control of the cell-free chemical processes affects their clinical application. Although existing decellularization methods have been shown to decellularize well, there still have some shortcomings in preserving relatively abundant ECM components, such as laminin and fibronectin [17]. Conversely, preserving more ECM or the dense structure of normal acellular nerves may result in insufficient immunogenic clearance. However, excessive detergent residue can spread to the connection between the graft and nerve endings, which is potentially dangerous for tran