A proteomic view on the differential phenotype of Schwann cells derived from mouse sensory and motor nerves.

Schwann cells (SCs) are myelin-forming glial cells of the peripheral nervous system. Recent studies suggested that SCs comprise two phenotypes: sensory SCs and motor SCs, which are associated with the modality-specific promotion of sensory and motor axon growth during peripheral neuronal regeneration. However, the molecular basis of the two phenotypic SCs is unclear. We established a workflow to obtain highly purified SCs derived from sensory nerve (SNdSCs) and motor nerve (MNdSCs) from B6; D2-Tg(s100B-EGFP)1Wjt/J mice. Subsequently, a quantitative proteomic analysis based on iTRAQ labeling was performed to compare the proteome of SNdSCs and MNdSCs. A total of 6,567 proteins were identified, of which 63 and 11 proteins were overexpressed in SNdSCs and MNdSCs, respectively. Three of the overexpressed proteins were further validated by western blot and immunocytochemistry: GMFB and CNPase, which were overexpressed in sensory SNdSCs, and histone H4, which was overexpressed in MNdSCs. The expression pattern of the three proteins was also validated in the dorsal roots and ventral roots. Bioinformatics analysis indicated that proteins highly expressed in SNdSCs are mainly involved in RNA processing and protein synthesis, while those overexpressed in MNdSCs are related to cell proliferation. Real-time cell analysis confirmed that the proliferation activity of MNdSCs is higher than that of SNdSCs. This study is the first to provide a proteomic view of the differential phenotype of mouse SNdSCs and MNdSCs. The data may serve as a valuable source for the study of the biological characteristics of these two SC phenotypes and their roles in nerve-specific regeneration.

Axonal extension from dorsal root ganglia on fibrillar and highly aligned poly(lactic acid)-polypyrrole substrates obtained by two different techniques: Electrospun nanofibres and extruded microfibres.

The biological behaviour of Schwann cells (SCs) and dorsal root ganglia (DRG) on fibrillar, highly aligned and electroconductive substrates obtained by two different techniques is studied. Mats formed by nanometer-sized fibres of poly(lactic acid) (PLA) are obtained by the electrospinning technique, while bundles formed by micrometer-sized extruded PLA fibres are obtained by grouping microfibres together. Both types of substrates are coated with the electrically conductive polymer polypyrrole (PPy) and their morphological, physical and electrical characterization is carried out. SCs on micrometer-sized substrates show a higher motility and cell-cell interaction, while a higher cell-material interaction with a lower cell motility is observed for nanometer-sized substrates. This higher motility and cell-cell interaction of SCs on the micrometer-sized substrates entails a higher axonal growth from DRG, since the migration of SCs from the DRG body is accelerated and, therefore, the SCs tapestry needed for the axonal growth is formed earlier on the substrate. A higher length and area of the axons is observed for these micrometer-sized substrates, as well as a higher level of axonal sprouting when compared with the nanometer-sized ones. These substrates offer the possibility of being electrically stimulated in different tissue engineering applications of the nervous system.

Glial cells influence cardiac permittivity as evidenced through in vitro and in silico models.

Excitation-contraction (EC) coupling in the heart has, until recently, been solely accredited to cardiomyocytes. The inherent complexities of the heart make it difficult to examine non-muscle contributions to contraction in vivo, and conventional in vitro models fail to capture multiple features and cellular heterogeneity of the myocardium. Here, we report on the development of a 3D cardiac µTissue to investigate changes in the cellular composition of native myocardium in vitro. Cells are encapsulated within micropatterned gelatin-based hydrogels formed via visible light photocrosslinking. This system enables spatial control of the microarchitecture, perturbation of the cellular composition, and functional measures of EC coupling via video microscopy and a custom algorithm to quantify beat frequency and degree of coordination. To demonstrate the robustness of these tools and evaluate the impact of altered cell population densities on cardiac µTissues, contractility and cell morphology were assessed with the inclusion of exogenous non-myelinating Schwann cells (SCs). Results demonstrate that the addition of exogenous SCs alter cardiomyocyte EC, profoundly inhibiting the response to electrical pacing. Computational modeling of connexin-mediated coupling suggests that SCs impact cardiomyocyte resting potential and rectification following depolarization. Cardiac µTissues hold potential for examining the role of cellular heterogeneity in heart health, pathologies, and cellular therapies.

Dedifferentiated Schwann cells secrete progranulin that enhances the survival and axon growth of motor neurons.

Schwann cells (SCs), the primary glia in the peripheral nervous system (PNS), display remarkable plasticity in that fully mature SCs undergo dedifferentiation and convert to repair SCs upon nerve injury. Dedifferentiated SCs provide essential support for PNS regeneration by producing signals that enhance the survival and axon regrowth of damaged neurons, but the identities of neurotrophic factors remain incompletely understood. Here we show that SCs express and secrete progranulin (PGRN), depending on the differentiation status of SCs. PGRN expression and secretion markedly increased as primary SCs underwent dedifferentiation, while PGRN secretion was prevented by administration of cAMP, which induced SC differentiation. We also found that sciatic nerve injury, a physiological trigger of SC dedifferentiation, induced PGRN expression in SCs in vivo. These results suggest that dedifferentiated SCs express and secrete PGRN that functions as a paracrine factor to support the survival and axon growth of neighboring neurons after injury.

A Subpopulation of Foxj1-Expressing, Nonmyelinating Schwann Cells of the Peripheral Nervous System Contribute to Schwann Cell Remyelination in the Central Nervous System.

New myelin sheaths can be restored to demyelinated axons in a spontaneous regenerative process called remyelination. In general, new myelin sheaths are made by oligodendrocytes newly generated from a widespread population of adult CNS progenitors called oligodendrocyte progenitor cells (OPCs). New myelin in CNS remyelination in both experimental models and clinical diseases can also be generated by Schwann cells (SCs), the myelin-forming cells of the PNS. Fate-mapping studies have shown that SCs contributing to remyelination in the CNS are often derived from OPCs and appear not to be derived from myelinating SCs from the PNS. In this study, we address whether CNS remyelinating SCs can also be generated from PNS-derived cells other than myelinating SCs. Using a genetic fate-mapping approach, we have found that a subpopulation of nonmyelinating SCs identified by the expression of the transcription factor Foxj1 also contribute to CNS SC remyelination, as well as to remyelination in the PNS. We also find that the ependymal cells lining the central canal of the spinal cord, which also express Foxj1, do not generate cells that contribute to CNS remyelination. These findings therefore identify a previously unrecognized population of PNS glia that can participate in the regeneration of new myelin sheaths following CNS demyelination.SIGNIFICANCE STATEMENT Remyelination failure in chronic demyelinating diseases such as multiple sclerosis drives the current quest for developing means by which remyelination in CNS can be enhanced therapeutically. Critical to this endeavor is the need to understand the mechanisms of remyelination, including the nature and identity of the cells capable of generating new myelin sheath-forming cells. Here, we report a previously unrecognized subpopulation of nonmyelinating Schwann cells (SCs) in the PNS, identified by the expression of the transcription factor Foxj1, which can give rise to SCs that are capable of remyelinating both PNS and CNS axons. These cells therefore represent a new cellular target for myelin regenerative strategies for the treatment of CNS disorders characterized by persistent demyelination.

Fabrication, characterization and biocompatibility of collagen/oxidized regenerated cellulose-Ca composite scaffold for carrying Schwann cells.

Schwann cell (SC) is the primary structural and functional part of the peripheral nervous system, and it plays a key role in the repair and regeneration of peripheral nerve. In order to develop a suitable scaffold for SC nerve tissue engineering, three kinds of scaffolds, including pristine collagen, pure oxidized regenerated cellulose-Ca (ORCCa) and collagen/ORC-Ca composite scaffolds, have been fabricated for carrying SC in this study. SC is then seeded on the scaffolds to form SC-scaffold nerve tissue engineering composites and evaluate their biocompatibility. The chemical and physical structure of the scaffolds are investigated by FTIR, NMR and SEM. The wettability of the collagen/ORC-Ca composite scaffold is close to that of pristine collagen, and the tensile strength of the composite scaffold (0.58 MPa) is better than that of pristine collagen (0.36 MPa). Cytotoxicity, cell proliferation, cell adhesion and western blotting assays are conducted to evaluate the biocompatibility and properties of different scaffolds. The results show that the three scaffolds exhibit no toxicity, and the proliferation rate of SC on the collagen/ORC-Ca composite scaffold is significantly higher than that of the other scaffolds (p < 0.05). The number of the adhesion cells on the composite scaffold (244.67 ±â€¯13.02) is much more than that in the pure ORC-Ca group (p < 0.01). Furthermore, the expression of N-Cadheri and PMP22 proteins in the collagen/ORC-Ca composite scaffold is significantly superior to the other two scaffolds (both p < 0.01). Therefore, it could be concluded that the collagen/ORC-Ca composite is a promising candidate as a scaffold for carrying SC to form nerve tissue engineering composites in order to assist the peripheral nervous in the repair and regeneration.

Effects of processing on structural, mechanical and biological properties of collagen-based substrates for regenerative medicine.

The aim of this work was to investigate the structural features of type I collagen isoforms and collagen-based films at atomic and molecular scales, in order to evaluate whether and to what extent different protocols of slurry synthesis may change the protein structure and the final properties of the developed scaffolds. Wide Angle X-ray Scattering data on raw materials demonstrated the preferential orientation of collagen molecules in equine tendon-derived collagens, while randomly oriented molecules were found in bovine skin collagens, together with a lower crystalline degree, analyzed by the assessment of FWHM (Full Width at Half Maximum), and a certain degree of salt contamination. WAXS and FT-IR (Fourier Transform Infrared) analyses on bovine collagen-based films, showed that mechanical homogenization of slurry in acidic solution was the treatment ensuring a high content of super-organization of collagen into triple helices and a high crystalline domain into the material. In vitro tests on rat Schwannoma cells showed that Schwann cell differentiation into myelinating cells was dependent on the specific collagen film being used, and was found to be stimulated in case of homogenization-treated samples. Finally DHT/EDC crosslinking treatment was shown to affect mechanical stiffness of films depending on collagen source and processing conditions.

A new electrospun graphene-silk fibroin composite scaffolds for guiding Schwann cells.

Graphene (Gr) has been made of various forms used for repairing peripheral nerve injury with favorable electroactivity, however, graphene-based scaffolds in peripheral nerve regeneration are still rarely reported due to the difficulty of realizing uniform dispersion of graphene and electroactive materials at nanoscale as well as lacking biocompatibility. In this paper, graphene-silk fibroin (SF) composite nanofiber membranes with different mass ratios were prepared via electrospinning. Microscopic observation revealed that electrospun Gr/SF membranes had a nanofibrous structure. Electrochemical analysis provided electroactivity characterization of the Gr/SF membranes. The physiochemical results showed that the physiochemical properties of electrospun Gr/SF membranes could be changed by varying Gr concentration. Swelling ratio and contact angle measurements confirmed that electrospun Gr/SF membranes possessed large absorption capacity and hydrophilic surface, and the mechanical property was improved with increasing Gr concentration. Additionally, in-vitro cytotoxicity with L929 revealed that all the electrospun Gr/SF membranes are biocompatible. Moreover, the morphology and quantity showed that the membranes supported the survival and growth of the cultured Schwann cells. Collectively, all of the results suggest that the electrospun Gr/SF membranes combine the excellent electrically conductivity and mechanical strength of the graphene with biocompatibility property of silk to mimic the natural neural cell micro-environment for nerve development.

Proteomic analysis of mesenchymal to Schwann cell transdifferentiation.

While transplantation of Schwann cells facilitates axon regeneration, remyelination and repair after peripheral nerve injury clinical use is limited by cell bioavailability. We posit that such limitation in cell access can be overcome by the use of autologous bone-marrow derived mesenchymal stem cells (MSCs). As MSCs can transdifferentiate to Schwann cell-phenotypes and accelerate nerve regeneration we undertook proteomic evaluation of the cells to uncover the protein contents that affects Schwann cell formulation. Transdifferentiated MSCs secrete significant amounts of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in cell-conditioned media that facilitated neurite outgrowth. MSC proteins significantly regulated during Schwann cell transdifferentiation included, but were not limited to, GNAI2, MYL9, ACTN4, ACTN1, ACTB, CAV-1, HSPB1, PHB2, TBB4B, CTGF, TGFI1, ARF6, EZR, GELS, VIM, WNT5A, RTN4, EFNB1. These support axonal guidance, myelination, neural development and neural growth and differentiation. The results unravel the molecular events that underlie cell transdifferentiation that ultimately serve to facilitate nerve regeneration and repair in support of cell transplantation. SIGNIFICANCE STATEMENT: While Schwann cells facilitate axon regeneration, remyelination and repair after peripheral nerve injury clinical use is limited by cell bioavailability. We posit that such limitation in cell access can be overcome by the use of bone-marrow derived mesenchymal stem cells (MSCs) transdifferentiated to Schwann cell-phenotypes. In the present study, we undertook the first proteomic evaluation of these transdifferentiated cells to uncover the protein contents that affects Schwann cell formulation. Furthermore, these transdifferentiated MSCs secrete significant amounts of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in cell-conditioned media that facilitated neurite outgrowth. Our results demonstrate that a number of MSC proteins were significantly regulated following transdifferentiation of the MSCs supporting roles in axonal guidance, myelination, neural development and differentiation. The conclusions of the present work unravel the molecular events that underlie cell transdifferentiation that ultimately serve to facilitate nerve regeneration and repair in support of cell transplantation. Our study was the first proteomic comparison demonstrating the transdifferentiation of MSCs and these reported results can affect a wide field of stem cell biology, tissue engineering, and proteomics.

Human eyelid adipose tissue-derived Schwann cells promote regeneration of a transected sciatic nerve.

Schwann cells (SCs) can promote the regeneration of injured peripheral nerves while the clinical application is limited by donor site complications and the inability to generate an ample amount of cells. In this study, we have isolated human eyelid adipose-derived Schwann cells (hE-SCs) from human eyelid adipose tissue and identified the cell phenotype and function. Using immunofluorescence and H &E staining, we detected subtle nerve fibers and SCs in human eyelid adipose tissue. Immunofluorescence staining indicated that hE-SCs expressed glial markers, such as S100, p75NTR GFAP, Sox10 and Krox20. To explore whether hE-SCs promote the regeneration of injured peripheral nerves in vivo, a Balb/c-nu mice model was used in the study, and mice were randomly assigned to five groups: Matrigel; hE-SCs/P0; hE-SCs/P2; hE-FLCs/P2; and Autograft. After 12 weeks, functional and histological assessments of the regenerated nerves showed that sciatic nerve defect was more effectively repaired in the hE-SCs/P2 group which achieved 66.1 ± 6.5% purity, than the other three groups and recovered to similar level to the Autograft group. These results indicated that hE-SCs can promote the regeneration of injured peripheral nerve and the abundant, easily accessible supply of adipose tissue might be a promising source of SCs for peripheral nerve repair.

The Transcription Factors EBF1 and EBF2 Are Positive Regulators of Myelination in Schwann Cells.

Myelin formation by Schwann cells is tightly controlled by multiple pathways and regulatory molecules. The Ebf2 gene, belonging to the Ebf family of transcription factors regulating cell development and differentiation, is expressed in Schwann cells, and Ebf2 knockout mice show peripheral nerve defects. We also found that Ebf1 is expressed in Schwann cells. To investigate Ebf function in myelination, we silenced Ebf genes in myelinating dorsal root ganglia cultures. Combined downregulation of Ebf genes leads to a severe impairment of myelin formation that is completely rescued by their specific overexpression, suggesting that the expression level of Ebf genes strongly influences axon myelination. In addition, by profiling Ebf target genes, we found several transcripts belonging to pathways actively involved in peripheral myelination, including Gliomedin, a gene with a role in the formation of the nodes of Ranvier and recently implicated in the pathogenesis of the nodo-paranodopathies. Our results suggest that Ebf genes act as positive regulators of myelination and directly regulate the promoter of Gliomedin.

Low-grade Schwann cell neoplasms with leptomeningeal dissemination: clinicopathologic and autopsy findings.

Leptomeningeal dissemination of low-grade Schwann cell neoplasms is an exceptionally rare occurrence and has not been well documented in the literature. We encountered 2 cases of leptomeningeal dissemination of low-grade Schwann cell neoplasms. Patient 1 was a 63-year-old woman with neurofibromatosis type 1 and a progressive low-grade malignant peripheral nerve sheath tumor developing from a diffuse/plexiform orbital neurofibroma that arose in childhood. The neoplasm demonstrated local and leptomeningeal dissemination intracranially leading to the patient's death. There was partial loss of H3K27 tri-methylation, p16 and collagen IV. Patient 2 was a 60-year-old man without neurofibromatosis type 1 who presented with cranial nerve symptoms and a disseminated neoplasm with a Schwann cell phenotype. The neoplasm stabilized after irradiation and chemotherapy, but the patient died of medical complications. Autopsy findings documented disseminated leptomeningeal disease in the intracranial and spinal compartment. H3K27M tri-methylation was preserved. The clinicopathologic and autopsy findings are studied and presented, and the literature is reviewed.

An innovative protocol for schwann cells extracellular matrix proteins extraction.

The evidence that extracellular matrix (ECM) components could represent new targets for drugs designed to approach degenerative disease, requires their analysis. Before the analysis, proteins should be extracted from ECM and solubilized. Currently, few protocols for ECM proteins extraction and solubilization are available in literature, and most of them are based mainly on the use of proteolytic enzymes, such as trypsin, which often lead to proteins damage. Moreover, no methods have been so far proposed to solubilize Schwann Cell ECM, which may represent an important target for the therapy of neurodegenerative disorders. In our study, we propose to solubilize SC ECM through the use of surfactants and urea. We compared our method of solubilization, with one of that proposed in literature for a general ECM, mainly based on the use of enzymes. We want to highlight the benefit of solubilizing SC ECM, avoiding the use of proteolytic enzymes. To compare the amount of proteins extracted with both methods, MicroBCA assay was used, while the quality of the proteins extracted was observed through the SDS-PAGE. The results obtained confirm a better solubilization of SC ECM proteins with the proposed protocol, both quantitatively and qualitatively, showing a higher concentration of proteins extracted and a better enrichment of protein fractions, if compared to the enzyme-based protocol. Our results show that SC ECM could be efficiently solubilized through the use of surfactant and urea, avoiding the use of enzyme-base methods. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3175-3180, 2016.

Mechanoreceptors in the Anterior Horn of the Equine Medial Meniscus: an Immunohistochemical Approach.

Lameness due to stifle and especially meniscal lesions is frequent in equine species. In humans, mechanoreceptors involved in proprioceptive function are well studied. Given the high incidence of meniscal injuries in horses, and the lack of information concerning them in equine menisci, our objective was to study these corpuscles in six healthy anterior horns of the equine medial meniscus, which is the most common localisation reported for equine meniscal injuries. Immunohistochemical stainings were performed using antibodies against high molecular weight neurofilaments and glial fibrillary acidic proteins. From a purely fundamental point of view, our work highlights for the first time the presence of Ruffini, Pacini and Golgi corpuscles in equine meniscus. They were found, isolated or in clusters and always located at the vicinity of blood vessels, at the level of the anterior horn of the equine medial meniscus. This morphological approach could serve as a basis for clinical studies, to evaluate the impact of these corpuscles on the poor sportive prognosis in equine meniscal tears.

[Sciatic nerve intraneural perineurioma]. / Périneuriome intraneural du nerf sciatique.

Intraneural perineurioma is a benign tumor developed from the perineurium and responsible for localized nerve hypertrophy. This uncommon tumor is characterized by a proliferation of perineural cells with a "pseudo-onion bulb" pattern. We report a sciatic nerve intraneural perineurioma in a 39-year-old patient.

Tactile Corpuscle-like Bodies in Gastrointestinal-type Mucosa: A Case Series.

Tactile corpuscle-like bodies (TCLB) are microscopic Schwannian structures that simulate the superficial mechanoreceptors of the peripheral nervous system (Wagner-Meissner corpuscles). They have been described nearly exclusively in peripheral nerve sheath tumors, namely diffuse neurofibromas, and schwannomas but also in cellular nevi. There are rare reports of these structures in the gastrointestinal tract (predominantly the lower tract), with the presumption that they are incidental reactive neural proliferations. We compiled 9 cases showing this rare phenomenon in gastrointestinal-type mucosa in nonsyndromic patients to further characterize its features. There were 6 men and 3 women (age range, 39 to 79 y, mean 56 y) with lesions involving esophagus/gastro-esophageal junction (n=7), sigmoid colon (n=1), and gastric heterotopia of the cricopharynx (n=1). Endoscopic examination was abnormal in 6 of the 7 cases (including changes consistent with Barrett esophagus and polypoid/nodular mucosa) and normal in 1 of 7 cases for which this information was available. The histologic features were similar in all cases, with unencapsulated clusters of lamellated and concentrically arranged spindle cells in the lamina propria. The foci of TCLB ranged in size from <0.1 to 1.5 mm in the greatest dimension. Abnormal histopathologic findings were identified in the background mucosa in 6 of 9 cases (including Barrett esophagus, active and inactive chronic gastritis, enterochromaffin-like cell hyperplasia, and gastric intestinal metaplasia). None of the patients showed signs of neurofibromatosis type 1, multiple endocrine neoplasia type 2B, Cowden syndrome, or other inherited syndrome. No morbidity related to TCLB was reported for the patients with available follow-up.

Chronic inhibitory effect of riluzole on trophic factor production.

Riluzole is the only FDA approved drug for the treatment of amyotrophic lateral sclerosis (ALS). However, the drug affords moderate protection to ALS patients, extending life for a few months by a mechanism that remains controversial. In the presence of riluzole, astrocytes increase the production of factors protective to motor neurons. The stimulation of trophic factor production by motor neuron associated cells may contribute to riluzole's protective effect in ALS. Here, we investigated the effects of media conditioned by astrocytes and Schwann cells acutely or chronically incubated with riluzole on trophic factor-deprived motor neuron survival. While acute riluzole incubation induced CT-1 secretion by astrocytes and Schwann cells, chronic treatment stimulated a significant decrease in trophic factor production compared to untreated cultures. Accordingly, conditioned media from astrocytes and Schwann cells acutely treated with riluzole protected motor neurons from trophic factor deprivation-induced cell death. Motor neuron protection was prevented by incubation with CT-1 neutralizing antibodies. In contrast, conditioned media from astrocytes and Schwann cells chronically treated with riluzole was not protective. Acute and chronic treatment of mice with riluzole showed opposite effects on trophic factor production in spinal cord, sciatic nerve and brain. There was an increase in the production of CT-1 and GDNF in the spinal cord and CT-1 in the sciatic nerve during the first days of treatment with riluzole, but the levels dropped significantly after chronic treatment with the drug. Similar results were observed in brain for CT-1 and BDNF while there was no change in GDNF levels after riluzole treatment. Our results reveal that riluzole regulates long-lasting processes involving protein synthesis, which may be relevant for riluzole therapeutic effects. Changing the regimen of riluzole administration to favor the acute effect of the drug on trophic factor production by discontinuous long-term treatment may improve the outcome of ALS patient therapy.

Fingolimod induces the transition to a nerve regeneration promoting Schwann cell phenotype.

Successful regeneration of injured peripheral nerves is mainly attributed to the plastic behavior of Schwann cells. Upon loss of axons, these cells trans-differentiate into regeneration promoting repair cells which provide trophic support to regrowing axons. Among others, activation of cJun was revealed to be involved in this process, initiating the stereotypic pattern of Schwann cell phenotype alterations during Wallerian degeneration. Nevertheless, the ability of Schwann cells to adapt and therefore the nerve's potential to regenerate can be limited in particular after long term denervation or in neuropathies leading to incomplete regeneration only and thus emphasizing the need for novel therapeutic approaches. Here we stimulated primary neonatal and adult rat Schwann cells with Fingolimod/FTY720P and investigated its impact on the regeneration promoting phenotype. FTY720P activated a number of de-differentiation markers including cJun and interfered with maturation marker and myelin expression. Functionally, FTY720P treated Schwann cells upregulated growth factor expression and these cells enhanced dorsal root ganglion neurite outgrowth on inhibitory substrates. Our results therefore provide strong evidence that FTY720P application supports the generation of a repair promoting cellular phenotype and suggest that Fingolimod could be used as treatment for peripheral nerve injuries and diseases.

Nerve guidance conduits based on double-layered scaffolds of electrospun nanofibers for repairing the peripheral nervous system.

Compared to the nerve guidance conduits (NGCs) constructed from a single layer of aligned nanofibers, bilayer NGCs with random and aligned nanofibers in the outer and inner layers are more robust and tear-resistant during surgical procedures thanks to an isotropic mechanical property provided by the random nanofibers. However, it remains unclear whether the random nanofibers will interfere with the aligned nanofibers to alter the extension pattern of the neurites and impede regeneration. To answer this question, we seeded dorsal root ganglia (DRG) on a double-layered scaffold, with aligned and random nanofibers on the top and bottom layers, respectively, and evaluated the outgrowth of neurites. The random nanofibers in the bottom layer exerted a negative impact on the extension of neurites projecting from the DRG, giving neurites a less ordered structure compared to those cultured on a single layer of aligned nanofibers. The negative impact of the random nanofibers could be effectively mitigated by preseeding the double-layered scaffold with Schwann cells. DRG cultured on top of such a scaffold exhibited a neurite outgrowth pattern similar to that for DRG cultured on a single layer of aligned nanofibers. We further fabricated bilayer NGCs from the double-layered scaffolds and tested their ability to facilitate nerve regeneration in a rat sciatic nerve injury model. Both histomorphometric analysis and functional characterization demonstrated that bilayer NGCs with an inner surface that was preseeded with Schwann cells could reach 54%, 64.2%, and 74.9% of the performance of isografts in terms of nerve fiber number, maximum isometric tetanic force, and mass of the extensor digitorum longus muscle, respectively. It can be concluded that the bilayer NGCs hold great potential in facilitating motor axon regeneration and functional motor recovery.