Harnessing three-dimensional porous chitosan microsphere embedded with adipose-derived stem cells to promote nerve regeneration

Abstract

Background Nerve guide conduits are a promising strategy for reconstructing peripheral nerve defects. Improving the survival rate of seed cells in nerve conduits is still a challenge and microcarriers are an excellent three-dimensional (3D) culture scaffold. Here, we investigate the effect of the 3D culture of microcarriers on the biological characteristics of adipose mesenchymal stem cells (ADSCs) and to evaluate the efficacy of chitosan nerve conduits filled with microcarriers loaded with ADSCs in repairing nerve defects. Methods In vitro, we prepared porous chitosan microspheres by a modified emulsion cross-linking method for loading ADSCs and evaluated the growth status and function of ADSCs. In vivo, ADSCs-loaded microcarriers were injected into chitosan nerve conduits to repair a 12 mm sciatic nerve defect in rats. Results Compared to the conventional two-dimensional (2D) culture, the prepared microcarriers were more conducive to the proliferation, migration, and secretion of trophic factors of ADSCs. In addition, gait analysis, neuro-electrophysiology, and histological evaluation of nerves and muscles showed that the ADSC microcarrier-loaded nerve conduits were more effective in improving nerve regeneration. Conclusions The ADSCs-loaded chitosan porous microcarrier prepared in this study has a high cell engraftment rate and good potential for peripheral nerve repair.

1. Introduction

Peripheral nerve injury (PNI) is very common in clinical practice, with more than 50,000 new cases of PNI each year worldwide, mainly caused by traffic accidents, natural disasters, disease, and iatrogenic injury [1]. The peripheral nervous system has a certain regenerative capacity, but when the nerve is severed, or a large gap defect is caused, surgery is usually required to restore its continuity. Autologous nerve grafting is the gold standard for repairing a long-segment nerve defect [2]. Autologous nerve grafts are harvested typically from the sural nerve, the superficial branch of the radial nerve, the medial and lateral anterior cutaneous nerves of the arm, and the intercostal nerves [3]. This approach has some inherent disadvantages, such as limited autologous nerve supply, donor site morbidity (neuroma and pain), mismatch of donor and recipient nerve size or nerve fibre arrangement, and the risk of secondary surgery [4–6].

These issues have led to extensive investigation of alternative repair strategies, including nerve allografts [7], autologous venous catheter grafts [8], and tissue-engineering nerve conduits [9–11]. Of these, tissue-engineering nerve conduits are the most promising, offering several advantages, such as unlimited sources, customizable sizes and configurations, and personalized functionality by setting different loads [12]. The core elements of the tissue engineering nerve conduit are scaffold materials and seed cells. As a natural polymer, chitosan is widely used in gene delivery, cell culture, and tissue engineering due to its low toxicity, good histocompatibility, and biodegradability [13]. The United States Food and Drug Administration has approved nerve catheters made from chitosan materials for clinical use [14]. Seed cells and their derivatives are critical components of tissue-engineering, releasing various bioactive substances, providing a suitable microenvironment for nerve regeneration, guiding and promoting axon regeneration [15]. Schwann cells (SCs) and various types of stem cells have been shown to act as seed cells in tissue engineering nerve conduits, actively promoting nerve regeneration [16, 17]. However, SCs are end-stage cells that are difficult to expand in vitro and have limited clinical application. Among all types of stem cells, adipose-derived mesenchymal stem cells (ADSCs) are not only abundant in source, easy to obtain, and fast in proliferation rate, but also have a robust pro-angiogenesis function and few ethical restrictions.