Combined use of decellularized allogeneic artery conduits with autologous transdifferentiated adipose-derived stem cells for facial nerve regeneration in rats
Introduction
Inflammation, trauma, tumor resection and surgical manipulation can all cause peripheral facial nerve injury accompanied with facial paralysis which is a common but devastating condition producing functional, esthetic, mental and psychological deficits in patients [1]. Although numerous modern microsurgical techniques are available for facial nerve repair, the resulting facial function sometimes remains unsatisfactory, together with inevitable post-paralytics syndrome (paresis, synkinesis and dysreflexia) [2]. Autologous nerve grafting is the gold standard technique used to coaptate long nerve gaps without tension, where it achieves optimal nerve regeneration and functional restoration by providing an ideal nerve scaffold containing Schwann cells (SCs) and their basal lamina, neurotrophic factors (NTFs) and cell adhesion molecules (CAMs) [3]. However, disadvantages associated with this approach have inspired the search for alternative strategies that are more effective for peripheral nerve reconstruction.
To date, tubulization techniques using nerve guidance conduits (NGCs) to bridge long nerve defects has drawn increasing attention in the fields of tissue engineering and reconstructive surgery. To improve regenerative outcomes, tissue-engineered nerve grafts combined with seed cells, NTFs, and extracellular matrix (ECM) components that mimic the biological features of nerve autografts, represent an effective solution to facilitate regenerating axonal sprouts in finding distal pathways and reinnervating target organs [4], [5], [6], [7]. Artificial NGCs have been fabricated using a variety of natural and synthetic, biodegradable and nonbiodegradable materials [4], [5]. Indeed, some natural biological conduits such as veins, arteries and decellularized grafts have been widely used to bridge peripheral nerve gaps [8], [9], [10], [11], [12], [13].
Since SCs are the most important supportive and myelinating cells and play a critical role in peripheral nerve regeneration [14]. Recent work has found that nerve conduits supplemented with viable SCs represent an efficient strategy for the treatment of nerve injuries [8], [12], [15]. However, it is difficult to obtain a sufficient number of autologous SCs exhibiting high purity and viability within a short time, therefore their clinical application is greatly limited [8]. Mesenchymal stromal cells (MSCs), including adipose-derived stem cells (ADSCs) and bone marrow mesenchymal stem cells (BMSCs), are multipotent adult stem cells derived from adipose tissue and bone marrow. They are considered as an alternative cell source in tissue regeneration because of their sufficient availability, easy accessibility, rapid proliferation, multipotent differentiation properties, and successful integration into host tissue with immunological tolerance [16], [17], [18]. Of the two MSCs, ADSCs are superior candidate cells for autologous cell transplantation and promoting peripheral nerve regeneration, as they can be harvested by less-invasive procedures and cultured with higher proliferation rates [18], [19]. Importantly, their ability to transdifferentiate into SC-like cells and their trophic effects, as well as myelin-forming ability make them an excellent candidate which can overcome the limited application of SCs in nerve regeneration [20], [21], [22]. Therefore, dADSCs or transdifferentiated BMSCs (dBMSCs) induced by a cocktail of growth factors and subsequently seeded into different guidance conduits is effective in promoting nerve regeneration and functional recovery [12], [23], [24], [25], [26], [27], [28].
In this study, we aimed to investigate the effects of a decellularized allogeneic artery conduit containing autologous dADSCs on the lesioned buccal branch of the facial nerve in a rat model.
Section snippets
Animals
Young female Sprague Dawley rats (provided by the Laboratory Animal Center of the Fourth Military Medical University, Xi’an, China) were used in the present study, since testosterone has proved to beneficially promote nerve regeneration [29], [30]. Sixty animals (weighing 100–120 g) were randomly divided into six groups (n = 10 in each group): Artery conduit group (1, negative control); Artery-ADSCs group (2); Artery-dADSCs group (3); Artery-SCs group (4); Nerve autograft group (5); and
Characterization of rat autologous cells
ADSCs within 3–5 passages appeared to form an adherent layer containing abundant dispersed spindle-like cells, continuously proliferated and formed a confluent layer comprised of fibroblastic-like and spindle-like cells (Fig. 2A). Whereas, SCs were typically spindle-shaped and were either bipolar or occasionally multipolar, often aligning in fascicles (Fig. 2F). Fifteen days after transdifferentiation, the ADSC-derived SC-like cells (dADSCs) exhibited spindle-shaped morphology and were bi- or
Discussion
In this current study, we fabricated a decellularized allogeneic artery conduit, and then combined it with autologous seed cells including dADSCs to bridge an 8-mm gap in the rat buccal branch of the facial nerve. Given an 8 week recovery period, we demonstrated that the decellularized artery conduit served as a scaffold without any immunogenicity to support cells and guide regenerating axons across the nerve gap towards functional reinnervation of the target whisker pad muscles, while the
Conclusion
In the current study, decellularized allogeneic artery conduits supplemented with autologous dADSCs were used to bridge an 8-mm gap in the buccal branch of the facial nerve. Subsequently, this method achieved satisfying regenerative outcomes approaching those achieved with SC-seeded artery conduits at week 8 postoperatively. Meanwhile, the transplanted dADSCs maintained their acquired SC-phenotype and myelin sheath-forming capacity inside decellularized artery conduits, and were involved in the
Acknowledgments
This research was supported by grant from Natural Science Foundation of China, No. 30930098.
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These authors contributed equally to this work.