Nerve Guidance Channels Research Articles

Conductive nerve conduit with piezoelectric properties for enhanced PC12 differentiation

Restoration of nerve tissue remains highly challenging, mainly due to the limited regeneration capacity of the nervous system and the development of fibrosis. This limitation necessitates designing new nerve guidance channel to promote nerve repairing. In this study, we developed a novel core/shell conduit to induce PC12 differentiation. Co-electrospinning method was utilized to produce a fibrous shell containing polycaprolactone/polyvinylidene fluoride PCL/PVDF, gelatin and polyaniline/graphene (PAG) nanocomposite. The core section of the conduit was filled with chitosan–gelatin hydrogel containing PAG and ZnO nanoparticles. Such conduit shows antibacterial activity, electrical conductivity and piezoelectric property. The effect of such engineered conduit on PC12 differentiation was investigated by analyzing differentiation markers Nestin and microtubule-associated protein 2 (MAP2) through immunocytochemistry and PCR-RT techniques. The result revealed that such conduit could significantly induce Nestin and MAP2 gene expression in the PC12 cells and, thus, it is a viable option for effective cell differentiation and nerve regeneration.

Regeneration of sciatic nerve injury through nanofiber neural guidance channels containing collagen hydrogel and acetyl L carnitine: An in vitro and in vivo study

Unlike the central nervous system (CNS), peripheral nervous system (PNS) injuries are partially repairable. Nerve guidance channels (NGCs) have been shown to improve the level of nerve repair after injury. In the present study, we developed a nanofiber NGC for the delivery of acetyl L carnitine (ALC) in a rat model of sciatic nerve injury. NGCs were produced by electrospinning a polymer blend of polycaprolacton and gelatin. The physicochemical and biological properties of developed scaffolds were investigated using Scanning electron microscopy, surface hydrophilicity measurement, porosity measurement, tensile strength studies, cell viability assay, and cell attachment assay. ALC was included in the collagen hydrogels at three weight ratios of 1%, 3%, and 5%. Cell viability assay showed that the hydrogels containing 5% ALC demonstrated a more favorable effect on PC-12 metabolic activity. Therefore, this concentration was chosen to treat PNS injury. The NGCs were implanted in rats and then their lumen was filled with collagen hydrogel + 5%ALC. The results of histopathological examinations and functional recovery studies showed that NGCs filled with ALC containing hydrogel have significant recovery potential compared to NGCs loaded with collagen hydrogels without ALC. Our results support the potential use of ALC-delivering NGCs in the treatment of peripheral nerve injury in the clinic.

Implantable Biomaterials for Peripheral Nerve Regeneration-Technology Trends and Translational Tribulations.

The use of autografted nerve in surgical repair of peripheral nerve injuries (PNI) is severely limited due to donor site morbidity and restricted tissue availability. As an alternative, synthetic nerve guidance channels (NGCs) are available on the market for surgical nerve repair, but they fail to promote nerve regeneration across larger critical gap nerve injuries. Therefore, such injuries remain unaddressed, result in poor healing outcomes and are a limiting factor in limb reconstruction and transplantation. On the other hand, a myriad of advanced biomaterial strategies to address critical nerve injuries are proposed in preclinical literature but only few of those have found their way into clinical practice. The design of synthetic nerve grafts should follow rational criteria and make use of a combination of bioinstructive cues to actively promote nerve regeneration. To identify the most promising NGC designs for translation into applicable products, thorough mode of action studies, standardized readouts and validation in large animals are needed. We identify design criteria for NGC fabrication according to the current state of research, give a broad overview of bioactive and functionalized biomaterials and highlight emerging composite implant strategies using therapeutic cells, soluble factors, structural features and intrinsically conductive substrates. Finally, we discuss translational progress in bioartificial conduits for nerve repair from the surgeon’s perspective and give an outlook toward future challenges in the field.

Nanophase surface arrays on poly (lactic-co-glycolic acid) upregulate neural cell functions.

Nerve guidance channels (NGCs) promote cell-extracellular matrix (ECM) interactions occurring within the nanoscale. However, studies focusing on the effects of nanophase topography on neural cell functions are limited, and mostly concentrated on the sub-micron level (>100 nm) surface topography. Therefore, the aim of this study was to fabricate <100 nm sized structures on poly lactic-co-glycolic acid (PLGA) films used in NGC applications to assess the effects of nanophase topography on neural cell functions. For this purpose, nanopit surface arrays were fabricated on PLGA surfaces via replica molding method. The results showed that neural cell proliferation increased up to 65% and c-fos protein expression increased up to 76% on PLGA surfaces having nanophase surface arrays compared to the control samples. It was observed that neural cells spread to a greater extend and formed more neurite extensions on the nanoarrayed surfaces compared to the control samples. These results were correlated with increased hydrophilicity and roughness of the nanophase PLGA surfaces, and point toward the promise of using nanoarrayed surfaces in NGC applications.

Evaluation of polymeric aligned NGCs and exosomes in nerve injury models in diabetic peripheral neuropathy condition

Peripheral nerve injuries in diabetic patients are challenging due to the limited regenerative nerve tissue capacity and additional tissue impairment due to diabetes. Here, we have developed a therapeutic approach for treating traumatic peripheral nerve injuries after diabetic neuropathy. We designed and evaluated the potential of nerve guiding scaffold and stem cell-derived exosomes in the repair of the nerve injuries after diabetic peripheral neuropathy (DPN). For that, polyurethane-based nerve conduits were fabricated by electrospinning technique. Further, in its lumen, aligned chitosan-collagen (1.5% w/v) cryogels were synthesized for developing porous nerve guidance channels (NGCs). Bone marrow stem cell-derived exosomes were isolated and characterized by transmission electron microscopy and CD-9 marker analysis. The developed NGCs have highly aligned porous architecture, as illustrated by the scanning electron microscopy and microCT imaging. In-vitro cell culture experiments showed Neuro2a cells growth and alignment along the porous channels on the chitosan-collagen cryomatrix. The in-vivo analysis was carried by developing two different traumatic sciatic nerve injury models in DPN rats, i.e., nerve crush injury and a nerve transection injury model. The exosomes were delivered via nerve conduits and direct transplantation in case of a crush injury model. In the case of nerve transection injury, exosomes loaded NGCs were implanted at the injury site. The regeneration was analyzed for nerve functionality and morphological assessment. The results showed improvement in the electrophysiological parameters such as nerve conduction velocity, compound muscle action potential, and sciatic nerve morphology after the treatment compared to the non-treated control. Also, the gastrocnemius muscle showed recovery in weight and morphology after treatment. The study demonstrated the role of the PUAO conduits, NGCs, and BMSCs exosomes in clinically challenging nerve injuries and paved the way for its application as a potential therapeutic approach.

The effect of lycopene in egg shell membrane guidance channel on sciatic nerve regeneration in rats.

Objective(s):Peripheral nerves are commonly damaged. Although the nerve autograft still subsists the gold standard in surgery of nerve injury gaps, it has severe detriment. The egg shell membrane (ESM) is a natural material, which has a great potential in practice. The aim of this survey was to appraise the effect of lycopene and the ESM nerve guidance channel on sciatic nerve regeneration.Materials and Methods:In this study, 32 male rats were randomized into four groups: sham surgery, autograft, ESM+ dimethyl sulfoxide (DMSO), and ESM+lycopene. One centimeter of sciatic nerve was removed, and the gap was grafted by ESM channel or autograft. The sciatic function index (SFI) was evaluated at days 7, 21, 30, 49, 60 and 90 after surgery. Nerve regeneration and gastroknemius muscle fibers were evaluated at days 30 and 90 after surgery by withdrawal reflex latency (WRL), histology and immunohistology assessments.Results:At 49, 60 and 90 days after surgery, the mean SFI in ESM+lycopene group was significantly greater than ESM+DMSO group (P<0.05). On day 90, the mean muscle fiber diameters and the mean number of myelinated axons in ESM+lycopene and autograft groups were significantly greater than ESM+DMSO group (P<0.05). In addition, the mean WRL was significantly lower in ESM+lycopene group than in the ESM+DMSO group 90 days after surgery (P<0.05).Conclusion:The results of this study show that the affirmative effects of ESM+lycopene may be beneficial for treating peripheral nerve damages.

The effect of propolis-gum arabic as a novel nerve guidance channel on regeneration of sciatic nerve in male rats

To investigate the effect of Propolis-gum Arabic as a nerve guidance channel. Male rats (n=24) were randomly divided into sham surgery, autograft, and Propolis+gum Arabic groups with equal numbers. Under anesthesia, the sciatic nerve was removed, and the gap between the two ending was repaired by Propolis-gum Arabic or nerve autograft. Nerve regeneration was evaluated by sciatic function index (SFI), withdrawal reflex latency (WRL), muscle fiber diameter, number of myelinated axons, myelinated fiber diameter, and immunohistochemical analysis. At 30, 49, 60 and 90 days after surgery, the mean of SFI in Propolis + gum Arabic group was significantly greater than the autograft group (p < 0.03). The mean muscle fiber diameters (30 and 90 days after surgery) and the mean number of myelinated axons (90 days after surgery) in Propolis + gum Arabic group were significantly greater than autograft group (p < 0.05). In addition, 90 days after surgery, the mean myelinated fiber diameter in Propolis + gum Arabic group was significantly higher than autograft group (p < 0.05). The results of this study suggest that as guidance channel, the Propolis + gum Arabic may be useful in peripheral nerve regeneration.

Systematically engineered electrospun conduit based on PGA/collagen/bioglass nanocomposites: The evaluation of morphological, mechanical, and bio‐properties

Peripheral nerve injury can considerably affect the daily life of affected people through reduced function and permanent deformation of the nerve. One of the conventional treatments used for the management of the disease is the application of autograft, which is recognized as a golden standard method; however, the process of gaining access to autograft has posed a significant challenge to its use. Nerve guidance channels (conduits), which are made in different methods, can act as an alternative therapy for patients that have undergone nerve injury; but, achieving these conduits has always been a major dilemma to be applied for patients with nerve injury. In this study, a novel conduit based on polymer blend nanocomposites of polyglycolic acid (PGA), collagen, and nanobioglass (NBG) were prepared by electrospinning technique and then compared with PGA/collagen and PGA conduits that were made in previous studies. Additionally, their various properties were characterized by scanning electron microscopy (SEM), X‐ray diffraction (XRD), contact angle, dynamic mechanical thermal analysis (DMTA), tensile strength, Fourier‐transform infrared (FTIR), and the porosity and degradation. The results showed that the mechanical, chemical, biocompatibility, and biodegradability properties of PGA/collagen/NBG conduits were more favorable in comparison with other materials. According to 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay and 4′,6‐diamidino‐2‐phenylindole (DAPI) staining technique, nanofibrous electrospun PGA/collagen/NBG conduits are more suitable for cell adhesion and proliferation in comparison with either PGA or PGA/collagen conduits and can have potential for nerve regeneration.

Biomimetic Photocurable Three-Dimensional Printed Nerve Guidance Channels with Aligned Cryomatrix Lumen for Peripheral Nerve Regeneration.

Repair and regeneration of critically injured peripheral nerves is one of the most challenging reconstructive surgeries. Currently available and FDA approved nerve guidance channels (NGCs) are suitable for small gap injuries, and their biological performance is inferior to that of autografts. Development of biomimetic NGCs with clinically relevant geometrical and biological characteristics such as topographical, biochemical, and haptotactic cues could offer better regeneration of the long-gap complex nerve injuries. Here, in this study, we present the development and preclinical analysis of three-dimensional (3D) printed aligned cryomatrix-filled NGCs along with nerve growth factor (NGF) (aCG + NGF) for peripheral nerve regeneration. We demonstrated the application of these aCG + NGF NGCs in the enhanced and successful regeneration of a critically injured rat sciatic nerve in comparison to random cryogel-filled NGCs, multichannel and clinically preferred hollow conduits, and the gold standard autografts. Our results indicated similar effect of the aCG + NGF NGCs viz-a-viz that of the autografts, and they not only enhanced the overall regenerated nerve physiology but could also mimic the cellular aspects of regeneration. This study emphasizes the paradigm that these biomimetic 3D printed NGCs will lead to a better functional regenerative outcome under clinical settings.

Aligned Chitosan-Gelatin Cryogel-Filled Polyurethane Nerve Guidance Channel for Neural Tissue Engineering: Fabrication, Characterization, and In Vitro Evaluation

Recent trends in peripheral nerve regeneration are directed toward the development of nerve guidance channels to assist the regeneration of the nerves across critical size defects. Advanced nerve guidance channels (aNGCs) should possess multifunctional properties to direct the axonal regeneration from proximal to distal end, allow the concentration of growth factors secreted by the injured nerve end, and attenuate the ingrowth of scar tissue at the site of injury. The design of the nerve guidance channel (NGC) is critical for providing the necessary topographical, chemotactic, as well as haptotactic cues for efficient nerve regeneration. In this study, we have designed and fabricated clinically relevant aNGCs comprising an antioxidant polyurethane (PUAO) conduit filled with aligned chitosan-gelatin (CG) cryogel filler for peripheral nerve regeneration. The effects of temperature, polymer concentration, and cross-linker concentration on the physicochemical properties of the CG cryogel filler were studied. The synthesized scaffolds were evaluated by scanning electron microscopy (SEM) and compression testing to obtain the matrix best suited to form the aNGC. The nanofibrous PUAO conduit was fabricated by electrospinning with a wall thickness of 114.16 ± 26.91 μm, which was filled with CG (1.2/6.4%)-aligned cryogel matrix to obtain the aNGCs. The aNGCs with 2.01 ± 0.04 mm internal diameter, 15 mm length, and internal CG filler with a pore diameter of 29.60 ± 9.83 μm were fabricated. The aNGCs were evaluated by SEM and in vitro neuronal culture for biocompatibility and cellular alignment. In vitro dorsal root ganglion cultures showed the aligned growth and cellular migration along the aligned pores of aNGCs. With this study, we conclude that this clinically relevant aligned porous aNGC will have a promising effect in repair and regeneration of peripheral nerve injuries.

3D Printed Polymeric Hydrogels for Nerve Regeneration.

The human nervous system lacks an inherent ability to regenerate its components upon damage or diseased conditions. During the last decade, this has motivated the development of a number of strategies for nerve regeneration. However, most of those approaches have not been used in clinical applications till today. For instance, although biomaterial-based scaffolds have been extensively used for nerve reparation, the lack of more customized structures have hampered their use in vivo. This highlight focuses mainly on how 3D bioprinting technology, using polymeric hydrogels as bio-inks, can be used for the development of new nerve guidance channels or devices for peripheral nerve cell regeneration. In this concise contribution, some of the most recent and representative examples are highlighted to discuss the challenges involved in various aspects of 3D bioprinting for nerve cell regeneration, specifically when using polymeric hydrogels.

Combined Influence of Gelatin Fibre Topography and Growth Factors on Cultured Dorsal Root Ganglia Neurons

Nerve guidance channels facilitate nerve regeneration and represent an attractive alternative to nerve graft. Actually, nano- and microstructured biomaterials for nerve reconstruction have gained much attention, thanks to recent discoveries about topography effects on cell behavior and morphology. Electrospun fibres have been proposed as filler or structural component for nerve guidance channels, principally due to their similarity with extracellular matrices which facilitate nerve regeneration. Among several tested biomaterials, gelatin has been used to prepare fibres able to support Schwann cell migration and neurite outgrowth. In this work, the effects of gelatin fibre size on axon elongation and Schwann cell migration have been tested using dorsal root ganglia cultures. Moreover, we analyzed how fibres might affect the expression of specific neuronal subtype markers in sensory neuron cultures and how the combined effect of substrate and biological cues affects neurite growth and gene expression. Data show that fibre topography differentially affects both neurite outgrowth and gene expression and suggest that fibre size and topography associated to specific growth factor exposure might be used to select neuron subpopulations and favor the axonal growth of specific neurons. Anat Rec, 301:1668-1677, 2018. © 2018 Wiley Periodicals, Inc.

3D Manufacture of Gold Nanocomposite Channels Facilitates Neural Differentiation and Regeneration

AbstractConductive nerve guidance channels are promising alternative therapies in peripheral nerve tissue engineering because they have excellent biocompatibility, biodegradation, and electrical conductivity. Gold, a kind of conductive material, is investigated widely concerning its potential roles in promoting peripheral nerve repair. In the present study, a polydopamine‐coated gold/polycaprolactone nanoscaffold is fabricated via a multilayer molding method and its proliferative, adhesive, and neural differentiation potential for bone marrow mesenchymal stem cells (BMSCs) and Schwann cells (SCs) in vitro is evaluated. Functional, electrophysiological evaluation, and morphological assessment all exhibit satisfactory recovery of sciatic nerves with increased thickness and number of myelinated fibers in vivo. In addition, increased microvessels are confirmed in gold nanocomposite channels, indicating their potential benefits in angiogenesis. Functional regeneration is further enhanced by neurotrophic growth factors released from BMSC and SC loading. The gold nanocomposite channel will have great potential in peripheral nerve restoration.

Gellan Gum-based luminal fillers for peripheral nerve regeneration: an in vivo study in the rat sciatic nerve repair model.

Peripheral nerve injuries (PNI) resulting in a gap to be bridged between the transected nerve ends are commonly reconstructed with autologous nerve tissue, but there is a need for valuable alternatives. This experimental work considers the innovative use of the biomaterial Gellan Gum (GG) as a luminal filler for nerve guidance channels made from chitosan with a 5% degree of acetylation. The engineered constructs should remodel the structural support given to regenerating axons by the so-called bands of Büngner. Four different GG formulations were produced by combining varying amounts of High-Acyl GG (HA-GG) and Methacrylated GG (MA-GG). The effective porosity of the freeze-dried networks was analysed by SEM and micro-CT 3D reconstructions, while the degradation and swelling abilities were characterized in vitro for up to 30 days. The metabolic activity and viability of immortalized Schwann cells seeded onto the freeze-dried networks were also evaluated. Finally, the developed hydrogel formulations were freeze-dried within the chitosan nerve guides and implanted in a 10 mm rat sciatic nerve defect. Functional and histomorphological analyses after 3, 6, and 12 weeks in vivo revealed that although it did not result in improved nerve regeneration, the NGC25:75 formulations could provide a basis for further development of GG scaffolds as luminal fillers for hollow nerve guidance channels.

Optimization of nanofiber scaffold properties towards nerve guidance channel design.

Optimization of nanofiber scaffold properties towards nerve guidance channel design.

Genipin-treated chitosan nanofibers as a novel scaffold for nerve guidance channel design

Schwann cell-seeded nerve guidance channels are designed to assist post-traumatic nerve regeneration in the PNS. Chitosan is a natural polymer well suited for tissue engineering as it is biocompatible, non-immunogenic, and biodegradable. Electrospun chitosan nanofibers utilized in nerve guidance channels have the capacity for guiding axonal growth within the channel lumen yet are limited in their capacity to maintain structural integrity within physiological environments. To address this, we attempted genipin crosslinking of chitosan nanofibers. Compared to neat chitosan nanofibers, genipin-treated nanofibers exhibited increased stiffness, resistance to swelling and lysozymal degradation. Furthermore, alignment and proliferation of purified Schwann cell cultures upon genipin-treated substratum was enhanced. When dorsal root ganglion explants were utilized as an in vitro model of peripheral nerve regeneration, emigrating neurons and Schwann cells assumed the uniaxial pattern of aligned electrospun chitosan nanofibers. Neurite growth along the nanofibers led, reaching a frontier more than twice that of the pursuant Schwann cells. Critically, neurite growth rate upon genipin-treated nanofibers demonstrated a 100% increase. Altogether, genipin treatment improves upon the physical and biological properties of chitosan nanofibers towards their utility in nerve guidance channel design.

Fabrication, properties and cytotoxicity evaluation of degradable poly(trimethylene carbonate-co-lactide) for the use as nerve guidance channels

Strategies to improve healing of damaged nerves include the application of specialized nerve guides, which hold the promise for allowing reanastomosis of the severed or damaged fibers. Studies have demonstrated that the use of a slowly degradable polymeric nerve guide can improve the nature and rate of nerve regeneration across a short gap in small nerves. The objective of this study was to characterize a biodegradable nerve guide based on poly(trimethylene carbonate-co-lactide) for peripheral nerve regeneration and to evaluate its cytotoxicity. The obtained copolymer films were incubated in two different media (distilled water and simulated body fluid), and while the degradation process appeared, pH and ion conductivity changes of solutions were monitored as well as mass loss of the samples. Additionally, mechanical tests (tensile strength, elongation at break and Young’s modulus parameters) before and after different time points were carried out. To evaluate cytotoxicity biological test were done on fibroblasts cells (NIH 3T3). Cell metabolic activity was determined using Alamar Blue reagent and their morphology was observed under fluorescence microscopy. The growth of pH in both media were mostly caused by steadily degradation of carbonate units into alkaline diols. The growth of ion conductivity value at the beginning of the incubation process was associated with the releasing of free ions to the solution. The mechanical parameters decreased with the progress of degradation process. Ringer’s fluid, as more aggressive, caused higher decrease in mechanical properties. The measured contact angles showed good surface wettability. Both surfaces, the top and the bottom, had similar hydrophilicity. Moreover, activity of fibroblasts cells were similar on both sides as well as on the reference TCPS. Good adhesion of NIH 3T3 cells to the surface suggests that the hydrophilic polymers promote colonization of fibroblasts cells on their surface. Biological studies have shown that used cells are very sensitive to surface topography which they colonize and cell viability was higher at the bottom surface, which has a slightly higher average roughness Ra. Thus, fibroblasts cell preferred colonizing rougher than smoother surfaces. Fabricated films does not affect negatively, namely, toxic on cell cultures and forms substrate with favourable surface properties. This was confirmed by the Alamar Blue tests and microscopic observations.

Nerve guidance conduits based on bi-layer chitosan membranes for peripheral nerve regeneration

Chitosan (CS), a derivative of the naturally occurring biopolymer chitin, has been widely used in a variety of biomedical applications including peripheral nerve repair because of its excellent biocompatibility, biodegradability, readily availability and antibacterial activity. In this work, bi-layer CS flat membranes were developed to be easily manipulated and rolled to obtain flexible nerve guidance channels (NGCs). These bi-layer membranes were prepared via solvent casting technique and their chemical composition was previously optimized to realize flexible membranes to be enwrapped for NGC formation. Two kinds of CS films cross-linked with different crosslinking agents were combined to produce scaffold structures with both good biocompatibility and the desired mechanical strength imparted by the inner and the outer layer, respectively. A tight connection between the two layers was achieved and the physicochemical and mechanical properties of CS bi-layer membranes were investigated. Additionally, bi-layer membranes were used for bridge implantation across 10-mm long median nerve defects in rats, and the outcome of peripheral nerve repair at 12 weeks post-implantation was evaluated by a combination of immunohistochemical and histological investigations.

3D printed nerve guidance channels: computer-aided control of geometry, physical cues, biological supplements and gradients.

Nerve guidance channels for peripheral nerve injury: Over the past decade, nerve guidance channels (NGCs) have emerged as a promising technology for regenerating gap injuries in peripheral nerves. Nerve gap injuries resulting from neurodegeneration and trauma, such as car accidents and battlefield wounds, affect hundreds of thousands of people annually. Motivated by suboptimal results obtained with the current gold standard of autologous grafting (i.e., autografts), various commercially available NGCs composed of synthetic and biomaterials are now alternatively available (Jia et al., 2014; Jones et al., 2016).

Thermally drawn fibers as nerve guidance scaffolds

Synthetic neural scaffolds hold promise to eventually replace nerve autografts for tissue repair following peripheral nerve injury. Despite substantial evidence for the influence of scaffold geometry and dimensions on the rate of axonal growth, systematic evaluation of these parameters remains a challenge due to limitations in materials processing. We have employed fiber drawing to engineer a wide spectrum of polymer-based neural scaffolds with varied geometries and core sizes. Using isolated whole dorsal root ganglia as an in vitro model system we have identified key features enhancing nerve growth within these fiber scaffolds. Our approach enabled straightforward integration of microscopic topography at the scale of nerve fascicles within the scaffold cores, which led to accelerated Schwann cell migration, as well as neurite growth and alignment. Our findings indicate that fiber drawing provides a scalable and versatile strategy for producing nerve guidance channels capable of controlling direction and accelerating the rate of axonal growth.