Reduced graphene oxide-embedded nerve conduits loaded with bone marrow mesenchymal stem cell-derived extracellular vesicles promote peripheral nerve regeneration

We previously combined reduced graphene oxide(rGO)with gelatin-methacryloyl(GelMA)and polycaprolactone(PCL)to create an rGO-GelMA-PCL nerve conduit and found that the conductivity and biocompatibility were improved.However,the rGO-GelMA-PCL nerve conduits differed greatly from autologous nerve transplants in their ability to promote the regeneration of injured peripheral nerves and axonal sprouting.Extracellular vesicles derived from bone marrow mesenchymal stem cells(BMSCs)can be loaded into rGO-GelMA-PCL nerve conduits for repair of rat sciatic nerve injury because they can promote angiogenesis at the injured site.In this study,12 weeks after surgery,sciatic nerve function was measured by electrophysiology and sciatic nerve function index,and myelin sheath and axon regeneration were observed by electron microscopy,immunohistochemistry,and immunofluorescence.The regeneration of microvessel was observed by immunofluorescence.Our results showed that rGO-GelMA-PCL nerve conduits loaded with BMSC-derived extracellular vesicles were superior to rGO-GelMA-PCL conduits alone in their ability to increase the number of newly formed vessels and axonal sprouts at the injury site as well as the recovery of neurological function.These findings indicate that rGO-GelMA-PCL nerve conduits loaded with BMSC-derived extracellular vesicles can promote peripheral nerve regeneration and neurological function recovery,and provide a new direction for the curation of peripheral nerve defect in the clinic.

Ultrasound imaging of chitosan nerve conduits that bridge sciatic nerve defects in rats

The repair of peripheral nerve injuries with autologous nerve remains the gold standard （Wang et al., 2005; Yao et al., 2010; Deal et al., 2012; Kriebel et al., 2014; Liu et al., 2014; Tamaki et al., 2014; Yu et al., 2014; Zhu and Lou, 2014）. With advances in tissue engineering and biomaterials, tissue-engineered nerve conduits with various biomaterials and structures, such as collagen and chitosan nerve conduits, have already been used in the clinic as alternatives to autologous nerve in the repair of peripheral nerve injury （Wang et al., 2012; Svizenska et al., 2013; Eppenberger et al., 2014; Gu et al., 2014; Koudehi et al., 2014; MoyaDiaz et al., 2014; Novajra et al., 2014; Okamoto et al., 2014; Shea et al., 2014; Singh et al., 2014; Tamaki et al., 2014; Yu et al., 2014）. Therefore, new simple and effective methods

Roles of reinforced nerve conduits and low-level laser phototherapy for long gap peripheral nerve repair

Peripheral nerve injuries are common in clinical practice because of traumas such as crushing and sectioning. Lesions of the nerve structure result in lost or diminished sensitivity and/or motor activity in the innervated territory. The degree of lesion depends on the specific nerve involved, the magnitude and type of pres- sure exerted, and the duration of the compression. The results of such injuries commonly include axonal degeneration and retro- grade degeneration of the corresponding neurons in the spinal medulla, followed by very slow regeneration （Rochkind et al., 2001）. The adverse effects on the daily activities of patients with a peripheral nerve injury are a determining factor in establishing the goals of early recovery （Rodriguez et al., 2004）.

Growth Factors and Supporting Cells of Nerve Conduits for Peripheral Nerve Regeneration

Peripheral nerve injury is a common disease that endangers human health.There is a variety of methods to repair peripheral nerve injury,the current"gold standard"approach is autograft,however it still faces many disadvantages.A new choice is the use of artificial nerve conduits,which are tubular structures and are designed to bridge nerve gaps.In order to bridge longer nerve gaps and gain ideal nerve regeneration effects,multiple technologies have been developed to the design of nerve conduits,such as selecting sutible materials,supplementing growth factors,transplanting supporting cells and so on.This review mainly introduce current progess in growth factors supplementation and supporting cells transplantation technology of nerve conduits.

Use of nerve conduits for peripheral nerve injury repair:A Web of Science-based literature analysis

OBJECTIVE： To identify global research trends in the use of nerve conduits for peripheral nerve injury repair. DATA RETRIEVAL： Numerous basic and clinical studies on nerve conduits for peripheral nerve injury repair were performed between 2002-2011. We performed a bibliometric analysis of the institutions, authors, and hot topics in the field, from the Web of Science, using the key words peripheral nerve and conduit or tube. SELECTION CRITERIA： Inclusion criteria： peer-reviewed published articles on nerve conduits for peripheral nerve injury repair, indexed in the Web of Science; original research articles, reviews, meeting abstracts, proceedings papers, book chapters, editorial material, and news items. Exclusion criteria： articles requiring manual searching or telephone access; documents not published in the public domain; and several corrected papers. MAIN OUTCOME MEASURES： （a） Annual publication output; （b） publication type; （c） publication by research field; （d） publication by journal; （e） publication by funding agency; （f） publication by author; （g） publication by country and institution; （h） publications by institution in China; （i） most-cited papers. RESULTS： A total of 793 publications on the use of nerve conduits for peripheral nerve injury repair were retrieved from the Web of Science between 2002-2011. The number of publications gradually increased over the 10-year study period. Articles constituted the main type of publication. The most prolific journals were Biomaterials, Microsurge and Joumal of Biomedical Materials Research PartA. The National Natural Science Foundation of China supported 27 papers, more than any other funding agency. Of the 793 publications, almost half came from American and Chinese authors and institutions. CONCLUSION： Nerve conduits have been studied extensively for peripheral nerve regeneration; however, many problems remain in this field, which are difficult for researchers to reach a consensus.

Silk Nerve Conduits for Peripheral Nerve Regeneration

Silk can promote the penetration of oxygen and water and support the attachment and proliferation of neurons and Schwann cells,and has appropriate biodegradation kinetics and flexible physical strength.It is a high-quality natural tissue engineering material and can be used as an ideal matrix for nerve repair.This paper reviewed the structure and properties of silk and the research progress of silk nerve conduits in peripheral nerve regeneration in recent years,and prospected its clinical application.

Human umbilical cord mesenchymal stem cell-derived exosomes loaded into a composite conduit promote functional recovery after peripheral nerve injury in rats

Complete transverse injury of peripheral nerves is challenging to treat.Exosomes secreted by human umbilical cord mesenchymal stem cells are considered to play an important role in intercellular communication and regulate tissue regeneration.In previous studies,a collagen/hyaluronic acid sponge was shown to provide a suitable regeneration environment for Schwann cell proliferation and to promote axonal regeneration.This three-dimensional(3D)composite conduit contains a collagen/hyaluronic acid inner sponge enclosed in an electrospun hollow poly(lactic-co-glycolic acid)tube.However,whether there is a synergy between the 3D composite conduit and exosomes in the repair of peripheral nerve injury remains unknown.In this study,we tested a comprehensive strategy for repairing long-gap(10 mm)peripheral nerve injury that combined the 3D composite conduit with human umbilical cord mesenchymal stem cell-derived exosomes.Repair effectiveness was evaluated by sciatic functional index,sciatic nerve compound muscle action potential recording,recovery of muscle mass,measuring the cross-sectional area of the muscle fiber,Masson trichrome staining,and transmission electron microscopy of the regenerated nerve in rats.The results showed that transplantation of the 3D composite conduit loaded with human umbilical cord mesenchymal stem cell-derived exosomes promoted peripheral nerve regeneration and restoration of motor function,similar to autograft transplantation.More CD31-positive endothelial cells were observed in the regenerated nerve after transplantation of the loaded conduit than after transplantation of the conduit without exosomes,which may have contributed to the observed increase in axon regeneration and distal nerve reconnection.Therefore,the use of a 3D composite conduit loaded with human umbilical cord mesenchymal stem cell-derived exosomes represents a promising cell-free therapeutic option for the treatment of peripheral nerve injury.

Fabrication and Characterization of Chitosan Nerve Conduits with Microtubular Architectures

Porous multi-channel chitosan conduits were fabricated using a novel phase-separation technique with an axial temperature gradient. First, porous chitosan tubes were made with a mold that was composed of two concentric polytetrafiuoroethylene tubes. Then 1%-3% （w/v） chitosan solution was injected into the chitosan tube while the two ends of the tube were closed with steel rods. Then the outside of the tube was wrapped with a layer of thermal insulating material to reduce the heat transfer through the outside, and the tubes were placed in a freezer. The resulting phase separation then occurred in the presence of an axial temperature gradient. The porosity, microtubule diameter, and orientation were controlled by adjusting the polymer concentration and temperature gradient. After the preparation course, no poisonous substances remained on the conduits. The mechanical properties, swelling, and biodegradability of the chitosan conduits were investigated, and a scanning electron microscope was used to observe the tubular morphology and growth of neuroblastoma cells （N2A, mouse） in the conduits. The results demonstrate that the multi-channel chitosan conduits have suitable mechanical strength, swelling, degradation properties, and nerve cell affinity, so they hold promise for use as neural tissue engineering scaffolds.

Sustained release of exosomes loaded into polydopamine-modified chitin conduits promotes peripheral nerve regeneration in rats

Exosomes derived from mesenchymal stem cells are of therapeutic interest because of their important role in intracellular communication and biological regulation.On the basis of previously studied nerve conduits,we designed a polydopamine-modified chitin conduit loaded with mesenchymal stem cell-derived exosomes that release the exosomes in a sustained and stable manner.In vitro experiments revealed that rat mesenchymal stem cell-derived exosomes enhanced Schwann cell proliferation and secretion of neurotrophic and growth factors,increased the expression of Jun and Sox2 genes,decreased the expression of Mbp and Krox20 genes in Schwann cells,and reprogrammed Schwann cells to a repair phenotype.Furthermore,mesenchymal stem cell-derived exosomes promoted neurite growth of dorsal root ganglia.The polydopamine-modified chitin conduits loaded with mesenchymal stem cell-derived exosomes were used to bridge 2 mm rat sciatic nerve defects.Sustained release of exosomes greatly accelerated nerve healing and improved nerve function.These findings confirm that sustained release of mesenchymal stem cell-derived exosomes loaded into polydopamine-modified chitin conduits promotes the functional recovery of injured peripheral nerves.

PDLLA/β-TCP/HA/CHS/NGF Sustained-release Conduits for Peripheral Nerve Regeneration

Using nerve guide conduits (NGCs) to promote the regeneration of PNI is a feasible alternative to autograft.Compared with NGCs made of single material,composite NGCs have a greater development prospect.Our previous research has confirmed that poly(D,L-lactic acid)/β-tricalcium phosphate/hyaluronic acid/chitosan/nerve growth factor (PDLLA/β-TCP/HA/CHS/NGF) NGCs have excellent physical and chemical properties,which can slowly release NGF and support cell adhesion and proliferation.In this study,PDLLA/β-TCP/HA/CHS/NGF NGCs were prepared and used to bridge a 10 mm sciatic nerve defect in 200-250 g Sprague-Dawley (SD) rat to verify the performance of the NGCs in vivo.Substantial improvements in nerve regeneration were observed after using the PDLLA/β-TCP/HA/CHS/NGF NGCs based on gross post-operation observation,triceps wet weight analysis and nerve histological assessment.In vivo studies illustrate that the PDLLA/β-TCP/HA/CHS/NGF sustained-release NGCs can effectively promote peripheral nerve regeneration,and the effect is similar to that of autograft.

Chitosan conduits combined with nerve growth factor microspheres repair facial nerve defects

Microspheres containing nerve growth factor for sustained release were prepared by a compound method, and implanted into chitosan conduits to repair 10-mm defects on the right buccal branches of the facial nerve in rabbits. In addition, chitosan conduits combined with nerve growth factor or normal saline, as well as autologous nerve, were used as controls. At 90 days post-surgery, the muscular atrophy on the right upper lip was more evident in the nerve growth factor and normal sa- line groups than in the nerve growth factor-microspheres and autologous nerve groups. Electro- physiological analysis revealed that the nerve conduction velocity and amplitude were significantly higher in the nerve growth factor-microspheres and autologous nerve groups than in the nerve growth factor and normal saline groups. Moreover, histological observation illustrated that the di- ameter, number, alignment and myelin sheath thickness of myelinated nerves derived from rabbits were higher in the nerve growth factor-microspheres and autologous nerve groups than in the nerve growth factor and normal saline groups. These findings indicate that chitosan nerve conduits com- bined with microspheres for sustained release of nerve growth factor can significantly improve facial nerve defect repair in rabbits.

Peripheral nerve regeneration with conduits:use of vein tubes

Treatment of peripheral nerve injuries remains a challenge to modern medicine due to the com-plexity of the neurobiological nerve regenerating process. There is a greater challenge when the transected nerve ends are not amenable to primary end-to-end tensionless neurorraphy. When facing a segmental nerve defect, great effort has been made to develop an alternative to the au-tologous nerve graft in order to circumvent morbidity at donor site, such as neuroma formation, scarring and permanent loss of function. Tubolization techniques have been developed to bridge nerve gaps and have been extensively studied in numerous experimental and clinical trials. The use of a conduit intends to act as a vehicle for moderation and modulation of the cellular and molecular ambience for nerve regeneration. Among several conduits, vein tubes were validated for clinical application with improving outcomes over the years. This article aims to address the investigation and treatment of segmental nerve injury and draw the current panorama on the use of vein tubes as an autogenous nerve conduit.

Efficacy of nanofibrous conduits in repair of longsegment sciatic nerve defects

Our previous studies have histomorphologically confirmed that nanofibrous poly（3-hydroxybutyrate- co-3-hydroxyvalerate） conduit can be used to repair 30-mm-long sciatic nerve defects. However, the repair effects on rat behaviors remain poorly understood. In this study, we used nanofibrous poly（3-hydroxybutyrate-co-3-hydroxyvalerate） conduit and autologous sciatic nerve to bridge 30-ram-long rat sciatic nerve gaps. Within 4 months after surgery, rat sciatic nerve functional re- covery was evaluated per month by behavioral analyses, including toe out angle, toe spread anal- ysis, walking track analysis, extensor postural thrust, swimming test, open-field analysis and no- ciceptive function. Results showed that rat sciatic nerve functional recovery was similar after nanofibrous poly（3-hydroxybutyrate-co-3-hydroxyvalerate） conduit and autologous nerve grafting. These findings suggest that nanofibrous poly（3-hydroxybutyrate-co-3-hydroxyvalerate） conduit is suitable in use for repair of long-segment sciatic nerve defects.

Additive-lathe 3D bioprinting of bilayered nerve conduits incorporated with supportive cells

Nerve conduits have been identified as one of the most promising treatments for peripheral nerve injuries,yet it remains unsolved how to develop ideal nerve conduits with both appropriate biological and mechanical properties.Existing nerve conduits must make trade-offs between mechanical strength and biocompatibility.Here,we propose a multi-nozzle additive-lathe 3D bioprinting technology to fabricate a bilayered nerve conduit.The materials for printing consisted of gelatin methacrylate(GelMA)-based inner layer,which was cellularized with bone marrow mesenchymal stem cells(BMSCs)and GelMA/poly(ethylene glycol)diacrylate(PEGDA)-based outer layer.The high viability and extensive morphological spreading of BMSCs encapsulated in the inner layer was achieved by adjusting the degree of methacryloyl substitution and the concentration of GelMA.Strong mechanical performance of the outer layer was obtained by the addition of PEGDA.The performance of the bilayered nerve conduits was assessed using in vitro culture of PC12 cells.The cell density of PC12 cells attached to cellularized bilayered nerve conduits was more than 4 times of that on acellular bilayered nerve conduits.The proliferation rate of PC12 cells attached to cellularized bilayered nerve conduits was over 9 times higher than that on acellular bilayered nerve conduits.These results demonstrate the additive-lathe 3D bioprinting of BMSCs embedded bilayered nerve conduits holds great potential in facilitating peripheral nerve repair.

Electrospun and woven silk fibroin/poly(lactic-coglycolic acid) nerve guidance conduits for repairing peripheral nerve injury

We have designed a novel nerve guidance conduit（NGC） made from silk fibroin and poly（lactic-co-glycolic acid） through electrospinning and weaving（ESP-NGCs）. Several physical and biological properties of the ESP-NGCs were assessed in order to evaluate their biocompatibility. The physical properties, including thickness, tensile stiffness, infrared spectroscopy, porosity, and water absorption were determined in vitro. To assess the biological properties, Schwann cells were cultured in ESP-NGC extracts and were assessed by morphological observation, the MTT assay, and immunohistochemistry. In addition, ESP-NGCs were subcutaneously implanted in the backs of rabbits to evaluate their biocompatibility in vivo. The results showed that ESP-NGCs have high porosity, strong hydrophilicity, and strong tensile stiffness. Schwann cells cultured in the ESP-NGC extract fluids showed no significant differences compared to control cells in their morphology or viability. Histological evaluation of the ESP-NGCs implanted in vivo indicated a mild inflammatory reaction and high biocompatibility. Together, these data suggest that these novel ESP-NGCs are biocompatible, and may thus provide a reliable scaffold for peripheral nerve repair in clinical application.

Combined use of Y-tube conduits with human umbilical cord stem cells for repairing nerve bifurcation defects

Given the anatomic complexity at the bifurcation point of a nerve trunk,enforced suturing between stumps can lead to misdirection of nerve axons,thereby resulting in adverse consequences.We assumed that Y-tube conduits injected with human umbilical cord stem cells could be an effective method to solve such problems,but studies focused on the best type of Y-tube conduit remain controversial.Therefore,the present study evaluated the applicability and efficacy of various types of Y-tube conduits containing human umbilical cord stem cells for treating rat femoral nerve defects on their bifurcation points.At 12 weeks after the bridging surgery that included treatment with different types of Y-tube conduits,there were no differences in quadriceps femoris muscle weight or femoral nerve ultrastructure.However,the Y-tube conduit group with longer branches and a short trunk resulted in a better outcome according to retrograde labeling and electrophysiological analysis.It can be concluded from the study that repairing a mixed nerve defect at its bifurcation point with Y-tube conduits,in particular those with long branches and a short trunk,is effective and results in good outcomes.

A hyaluronic acid granular hydrogel nerve guidance conduit promotes regeneration and functional recovery of injured sciatic nerves in rats

A hyaluronic acid granular hydrogel can promote neuronal and astrocyte colony formation and axonal extension in vitro,suggesting that the hydrogel can simulate an extracellular matrix structure to promote neural regeneration.However,in vivo experiments have not been conducted.In this study,we transplanted a hyaluronic acid granular hydrogel nerve guidance conduit to repair a 10-mm long sciatic nerve gap.The Basso,Beattie,and Bresnahan locomotor rating scale,sciatic nerve compound muscle action potential recording,Fluoro-Gold retrograde tracing,growth related protein 43/S100 immunofluorescence staining,transmission electron microscopy,gastrocnemius muscle dry/wet weight ratio,and Masson’s trichrome staining results showed that the nerve guidance conduit exhibited similar regeneration of sciatic nerve axons and myelin sheath,and recovery of the electrophysiological function and motor function as autologous nerve transplantation.The conduit results were superior to those of a bulk hydrogel or silicone tube transplant.These findings suggest that tissue-engineered nerve conduits containing hyaluronic acid granular hydrogels effectively promote the morphological and functional recovery of the injured sciatic nerve.The nerve conduits have the potential as a material for repairing peripheral nerve defects.

Electrodeposition of chitosan/graphene oxide conduit to enhance peripheral nerve regeneration

Currently available commercial nerve guidance conduits have been applied in the repair of peripheral nerve defects.However,a conduit exhibiting good biocompatibility remains to be developed.In this work,a series of chitosan/graphene oxide(GO)films with concentrations of GO varying from 0-1 wt%(collectively referred to as CHGF-n)were prepared by an electrodeposition technique.The effects of CHGF-n on proliferation and adhesion abilities of Schwann cells were evaluated.The results showed that Schwann cells exhibited elongated spindle shapes and upregulated expression of nerve regeneration-related factors such as Krox20(a key myelination factor),Zeb2(essential for Schwann cell differentiation,myelination,and nerve repair),and transforming growth factorβ(a cytokine with regenerative functions).In addition,a nerve guidance conduit with a GO content of 0.25%(CHGFC-0.25)was implanted to repair a 10-mm sciatic nerve defect in rats.The results indicated improvements in sciatic functional index,electrophysiology,and sciatic nerve and gastrocnemius muscle histology compared with the CHGFC-0 group,and similar outcomes to the autograft group.In conclusion,we provide a candidate method for the repair of peripheral nerve defects using free-standing chitosan/GO nerve conduits produced by electrodeposition.

Evaluating nerve guidance conduits for peripheral nerve injuries:a novel normalization method

The peripheral nervous system （PNS） is composed of the nerves and ganglia outside of the brain and spinal cord whose primary function is to connect the central nervous system to the limbs and organs. A peripheral nerve injury （PNI） is damage to the nerves and/or its surrounding tissue. These injuries can affect up to 5% of patients that are hospitalized for trauma （Taylor et al., 2008） and over 50,000 surgical repair procedures are performed annually in the United States alone （Evans, 2001）.

Nerve transfer with 3D-printed branch nerve conduits

Background:Nerve transfer is an important clinical surgical procedure for nerve repair by the coaptation of a healthy donor nerve to an injured nerve.Usually,nerve transfer is performed in an end-to-end manner,which will lead to functional loss of the donor nerve.In this study,we aimed to evaluate the efficacy of 3D-printed branch nerve conduits in nerve transfer.Methods:Customized branch conduits were constructed using gelatine-methacryloyl by 3D print-ing.The nerve conduits were characterized both in vitro and in vivo.The efficacy of 3D-printed branch nerve conduits in nerve transfer was evaluated in rats through electrophysiology testing and histological evaluation.Results:The results obtained showed that a single nerve stump could form a complex nerve network in the 3D-printed multibranch conduit.A two-branch conduit was 3D printed for transferring the tibial nerve to the peroneal nerve in rats.In this process,the two branches were connected to the distal tibial nerve and peroneal nerve.It was found that the two nerves were successfully repaired with functional recovery.Conclusions:It is implied that the two-branch conduit could not only repair the peroneal nerve but also preserve partial function of the donor tibial nerve.This work demonstrated that 3D-printed branch nerve conduits provide a potential method for nerve transfer.