Peripheral nerve injury is a common clinical disorder that commonly results in motor dysfunction and loss of sensory function, resulting in long-term impacts on patient quality-of-life [11]. Prompt and effective treatment after injury is necessary to promote functional recovery [22]. The results of the present study showed that transplantation of allogeneic acellular microtissue pre-cellularized with ADSCs into a chitosan-constructed 3D culture platform to bridge the 10 mm gap in rats could enhance the regenerative repair of peripheral nerves [3, 23].

Selecting an appropriate nerve graft can further improve the efficacy of the surgical repair of nerve defects in clinical settings. Autografts are widely recognized as the “gold standard” for the treatment of severe nerve defects [24, 25]. However, there are many limitations to obtaining autologous nerves, including difficulty in obtaining a suitably sized and functional graft from the patient, intolerable neuroma, and increased operation time [26,27,28]. As such, it is necessary to develop new biomaterials to make up for the defects of autologous nerve transplantation [29, 30]. As an alternative to autografts, ANAs have been widely recognized as suitable materials for bridging nerve defects. Previous studies have revealed that motor nerve-derived grafts have a better effect at promoting axon regeneration and myelin formation than sensory nerve-derived grafts [2, 29, 31]. Therefore, we used rat sciatic nerves to prepare an acellular nerve scaffold. The main components of acellular nerves, the extracellular matrix (ECM), and internal microstructure are key to recruiting Schwann cells, which tend to migrate and induce myelinated nerve regeneration [9, 32]. Cell growth and culture are complex processes that require the co-regulation of several glycoproteins [6, 17]. The composition of the ECM, as an effective “soil” for cell growth, is primarily determined by its abundant glycoproteins [9, 33]. LN, together with collagen, forms the basement membrane; its primary biological function is to promote cell adhesion to the matrix and to regulate cell growth and differentiation [27, 28]. As an important ECM component, FN have various biological functions [34]. The most important of which is to promote adhesion and growth between cells, as effective adhesion of cells can promote injury healing and exert biological functions [35]. The two-step procedural decellularization method developed by Sondell et al. has been proven to maintain relatively rich ECM components and a complete microstructure [7, 10] that is close to the endogenous environment of cell growth [36]. However, the detergent control remains problematic [24]. Excessive chemical processes are used to remove immunogenicity from the pores to a greater extent in the traditional decellularization process, resulting in the destruction of ECM components of ANA, such as laminin, collagen, and bioactive factor [13]. Excessive detergent can also come into contact with the ANA nerve stump, which was not conducive to anastomosis and repair of the graft and nerve stump [2, 30, 37]. However, to retain an abundant ECM, it is necessary to reduce the amount of detergent required [31, 34, 35]. Although the decellularization process can almost completely remove the cellular components that induce autoimmune reactions, some substances will inevitably remain, leading to hidden dangers when using the decellularized nerve as a nerve defect bridging the graft [1, 23, 33, 38].

In addition to the effect of the degree of decellularization, the dense internal structure of the acellular nerve graft limits the growth of regenerated axons at the severed end of the nerve into this segment [13, 21, 22]. Under physiological conditions, normal nerves have low porosity, a small pore size, and a dense structure, while the wrapping of axons and myelin in basal tubes is conducive to maintaining their growth and physiological roles [11, 39,40,41]. During the repair process of injured nerves, Schwann cells migrate into the graft, release neurotrophic factors, and form Büngner bands, which induce the extension of regenerated axons [7, 9, 10, 36, 42, 43]. However, the original structure of traditional acellular nerves is dense, which is not conducive to the growth and extension of regenerative myelinated nerves [8, 18, 44, 45].

It is worth noting that the use of acellular scaffolds could avoid these problems [6]. Indeed, microtissue engineering has been used to develop acellular scaffolds as carriers for regenerative medicine [8]. The overarching goal is to construct an acellular nerve with low immunogenicity and ECM integrity that allows for a large number of cells to survive and perform their repair functions [6]. Acellular scaffolds have a loose structure, which is more conducive to endogenous Schwann cell infiltration during nerve dissection, and promotes nerve regeneration [29, 32]. To achieve this structure, we obtained the motor nerves of allogeneic species and prepared them into 1 mm diameter microparticles [9, 30]. Acellular microparticles were subsequently obtained by traditional decellularization neurochemical processes and decellularization was performed using an appropriate detergent [12, 19, 20]. Owing to the small diameter of the prepared microparticles, full contact with the stain remover resulted in a high degree of decellularization, while the ECM and other components favorable for cell growth were retained to the greatest extent [6, 17].

ADSCs have good regenerative and secretory abilities, and can be targeted to deliver stimulants related to nerve regeneration, such as neurotrophic factors, soluble factors, and cytokines [35, 36]. The levels of many biological factors at the injured site were increased to provide the optimal environmental conditions for tissue regeneration [25, 27, 30]. The recruitment of endogenous Schwann cells to injured sites can accelerate the extension of axons and play a positive role in repairing peripheral nerve defects [30, 32]. In addition, ADSCs can be obtained through a simple in vitro amplification method and are promising nerve regeneration cells for cell therapy [3, 15, 18]. Stem cell therapy can further compensate for the lack of endogenous Schwann cells in acellular MTs [19, 36, 38]. For acellular nerve grafts, sufficient autologous Schwann cells are required to infiltrate the nerve ends and promote the growth of regenerated nerves throughout the defect [32, 40]. The repair process is believed to be slow because of the insufficient driving effect of Schwann cells [4, 5]. This process involves the infiltration, expansion, and arrangement of Schwann cells [32]. The acellular neural bridge strategy also does not apply to the treatment of long-segment defects (> 3 cm), as Schwann cells cannot complete the graft [1, 41]. The failure of repair may result in an insufficient migration driving force and/or a short proliferation cycle of Schwann cells, which makes it difficult to meet the demands of transplanting segments [23, 25]. As such, microtissue engineering scaffolds have been used to promote the proliferation and arrangement of Schwann cells in the graft, induce the formation of Büngner bands, and accelerate the axon regeneration rate [28, 29]. Using acellular nerve particles as neural scaffolds, supporting cells were cultured on neural scaffolds to modify the ECM, while a three-dimensional culture environment suitable for cell growth was constructed [6, 9]. On the one hand, acellular-MT can accommodate more supporting cells, simulate the physical and chemical characteristics of natural ECM in vivo, and exert the positive effects of the ECM on the in situ environment [10, 39]. However, on the other hand, the neurotrophic factors secreted by the supporting cells contribute to the recruitment of endogenous Schwann cells that participate in myelinated nerve regeneration [11, 21]. This acellular MT, with a large pore size and low biotoxicity, is conducive to the extension of regenerative axons to the distal end of the graft, resulting in faster repair and regeneration quality [3, 23, 26].

Chitosan is one of the most popular scaffolds in tissue engineering due to its high biocompatibility, good tensile resistance, and biodegradability [12, 39]. There have been many studies investigating the utility of chitosan as a natural biological scaffold to repair damaged peripheral nerves using combined cell or factor therapy, such as chitosan combined with fibroin filaments to construct a good regenerative peripheral nerve scaffold, which has achieved good curative effects in the treatment of peripheral nerve defects in rats [9, 16, 29, 41]. This prior research helped us to develop the encapsulation vector of Acellular-MT.

In the present study, we filled chitosan with acellular nerve microparticles combined with ADSC to construct MT and bridge a 1 cm nerve defect of peripheral nerves in rats to explore the ability of MT to promote peripheral nerve regeneration and repair. Our results showed that no cellular components were present in the acellular MTs, and that the fibers were loose and porous. Immunofluorescence staining revealed that the main bioactive components of the ECM, glycoproteins including LN and FN, remained intact, which is considered to be key for the acellular-MT to help cells perform the repair function and promote axon regeneration and extension. After 7 days, Schwann cells were co-cultured with acellular-MT, showing good survival and proliferation, indicating that the constructed acellular-MT had good biocompatibility. To evaluate the degree of recovery of nerve function after the repair of peripheral nerve defects with acellular-MT, we analyzed the sciatic nerve SFI value and the stand/swing time ratio of each group, finding that the Chitosan + Acellular-MT + ADSC group showed a recovery advantage second only to the autograft group at 8 W postoperatively, while the degree of recovery showed no significant difference with the autograft group at 12 W postoperatively. The electrophysiological characteristics of the nerves were similar to those of the autograft group, which also reflected a strong ability to promote the regeneration of peripheral nerve defects. SEM analysis of the distal nerve grafts at 12 W after the operation in each group and the effect on sciatic nerve regeneration were evaluated, revealing that the structure of myelinated nerve fibers in the Acellular-MT group showed good continuity of the regenerative myelin sheath, smooth shape, and wider diameter of nerve fibers, similar to that in the autograft group.