Animal models of vascularized nerve grafts:a systematic review

The aim of this review is to present and compare the various animal models of vascularized nerve grafts described in the literature as well as to summarize preclinical evidence for superior functional results compared to non-vascularized free nerve grafts. We also will present the state of the art on prefabricated vascularized nerve grafts. A systematic literature review on vascularized nerve graft models was conducted via the retrieval with the Pub Med database on March 30, 2019. Data on the animal, nerve, and vascularization model, the recipient bed, the evaluation time points and methods, and the results of the study results were extracted and analyzed from selected articles. The rat sciatic nerve was the most popular model for vascularized nerve grafts, followed by the rabbit;however, rabbit models allow for longer nerve grafts, which are suitable for translational evaluation, and produced more cautious results on the superiority of vascularized nerve grafts. Compared to free nerve grafts, vascularized nerve grafts have better early but similar long-term results, especially in an avascular bed. There are few studies on avascular receiving beds and prefabricated nerve grafts. The clinical translation potential of available animal models is limited, and current experimental knowledge cannot fully support that the differences between vascularized nerve grafts and free nerve grafts yield a clinical advantage that justifies the complexity of the procedure.

Artificial nerve graft constructed by coculture of activated Schwann cells and human hair keratin for repair of peripheral nerve defects

Studies have shown that human hair keratin(HHK) has no antigenicity and excellent mechanical properties. Schwann cells, as unique glial cells in the peripheral nervous system, can be induced by interleukin-1β to secrete nerve growth factor, which promotes neural regeneration. Therefore, HHK with Schwann cells may be a more effective approach to repair nerve defects than HHK without Schwann cells. In this study, we established an artificial nerve graft by loading an HHK skeleton with activated Schwann cells. We found that the longitudinal HHK microfilament structure provided adhesion medium, space and direction for Schwann cells, and promoted Schwann cell growth and nerve fiber regeneration. In addition, interleukin-1β not only activates Schwann cells, but also strengthens their activity and increases the expression of nerve growth factors. Activated Schwann cells activate macrophages, and activated macrophages secrete interleukin-1β, which maintains the activity of Schwann cells. Thus, a beneficial cycle forms and promotes nerve repair. Furthermore, our studies have found that the newly constructed artificial nerve graft promotes the improvements in nerve conduction function and motor function in rats with sciatic nerve injury, and increases the expression of nerve injury repair factors fibroblast growth factor 2 and human transforming growth factor B receptor 2. These findings suggest that this artificial nerve graft effectively repairs peripheral nerve injury.

Hypoxic pre-conditioned adipose-derived stem/progenitor cells embedded in fibrin conduits promote peripheral nerve regeneration in a sciatic nerve graft model

Recent results emphasize the supportive effects of adipose-derived multipotent stem/progenitor cells(ADSPCs)in peripheral nerve recovery.Cultivation under hypoxia is considered to enhance the release of the regenerative potential of ADSPCs.This study aimed to examine whether peripheral nerve regeneration in a rat model of autologous sciatic nerve graft benefits from an additional custom-made fibrin conduit seeded with hypoxic pre-conditioned(2%oxygen for 72 hours)autologous ADSPCs(n=9).This treatment mode was compared with three others:fibrin conduit seeded with ADSPCs cultivated under normoxic conditions(n=9);non-cell-carrying conduit(n=9);and nerve autograft only(n=9).A 16-week follow-up included functional testing(sciatic functional index and static sciatic index)as well as postmortem muscle mass analyses and morphometric nerve evaluations(histology,g-ratio,axon density,and diameter).At 8 weeks,the hypoxic pre-conditioned group achieved significantly higher sciatic functional index/static sciatic index scores than the other three groups,indicating faster functional regeneration.Furthermore,histologic evaluation showed significantly increased axon outgrowth/branching,axon density,remyelination,and a reduced relative connective tissue area.Hypoxic pre-conditioned ADSPCs seeded in fibrin conduits are a promising adjunct to current nerve autografts.Further studies are needed to understand the underlying cellular mechanism and to investigate a potential application in clinical practice.

The role of vascularization in nerve regeneration of nerve graft

Vascularization is an important factor in nerve graft survival and function. The specific molecular regulations and patterns of angiogenesis following peripheral nerve injury are in a broad complex of pathways. This review aims to summarize current knowledge on the role of vascularization in nerve regeneration, including the key regulation molecules, and mechanisms and patterns of revascularization after nerve injury. Angiogenesis, the maturation of pre-existing vessels into new areas, is stimulated through angiogenic factors such as vascular endothelial growth factor and precedes the repair of damaged nerves. Vascular endothelial growth factor administration to nerves has demonstrated to increase revascularization after injury in basic science research. In the clinical setting, vascularized nerve grafts could be used in the reconstruction of large segmental peripheral nerve injuries. Vascularized nerve grafts are postulated to accelerate revascularization and enhance nerve regeneration by providing an optimal nutritional environment, especially in scarred beds, and decrease fibroblast infiltration. This could improve functional recovery after nerve grafting, however, conclusive evidence of the superiority of vascularized nerve grafts is lacking in human studies. A well-designed randomized controlled trial comparing vascularized nerve grafts to non-vascularized nerve grafts involving patients with similar injuries, nerve graft repair and follow-up times is necessary to demonstrate the efficacy of vascularized nerve grafts. Due to technical challenges, composite transfer of a nerve graft along with its adipose tissue has been proposed to provide a healthy tissue bed. Basic science research has shown that a vascularized fascial flap containing adipose tissue and a vascular bundle improves revascularization through excreted angiogenic factors, provided by the stem cells in the adipose tissue as well as by the blood supply and environmental support. While it was previously believed that revascularization occurred from both nerve ends, recent studies propose that revascularization occurs primarily from the proximal nerve coaptation. Fascial flaps or vascularized nerve grafts have limited applicability and future directions could lead towards off-the-shelf alternatives to autografting, such as biodegradable nerve scaffolds which include capillary-like networks to enable vascularization and avoid graft necrosis and ischemia.

An update–tissue engineered nerve grafts for the repair of peripheral nerve injuries

Peripheral nerve injuries（PNI） are caused by a range of etiologies and result in a broad spectrum of disability. While nerve autografts are the current gold standard for the reconstruction of extensive nerve damage, the limited supply of autologous nerve and complications associated with harvesting nerve from a second surgical site has driven groups from multiple disciplines, including biomedical engineering, neurosurgery, plastic surgery, and orthopedic surgery, to develop a suitable or superior alternative to autografting. Over the last couple of decades, various types of scaffolds, such as acellular nerve grafts（ANGs）, nerve guidance conduits, and non-nervous tissues, have been filled with Schwann cells, stem cells, and/or neurotrophic factors to develop tissue engineered nerve grafts（TENGs）. Although these have shown promising effects on peripheral nerve regeneration in experimental models, the autograft has remained the gold standard for large nerve gaps. This review provides a discussion of recent advances in the development of TENGs and their efficacy in experimental models. Specifically, TENGs have been enhanced via incorporation of genetically engineered cells, methods to improve stem cell survival and differentiation, optimized delivery of neurotrophic factors via drug delivery systems（DDS）, co-administration of platelet-rich plasma（PRP）, and pretreatment with chondroitinase ABC（Ch-ABC）. Other notable advancements include conduits that have been bioengineered to mimic native nerve structure via cell-derived extracellular matrix（ECM） deposition, and the development of transplantable living nervous tissue constructs from rat and human dorsal root ganglia（DRG） neurons. Grafts composed of non-nervous tissues, such as vein, artery, and muscle, will be briefly discussed.

Acellular allogeneic nerve grafting combined with bone marrow mesenchymal stem cell transplantation for the repair of long-segment sciatic nerve defects:biomechanics and validation of mathematical models

We hypothesized that a chemically extracted acellular allogeneic nerve graft used in combination with bone marrow mesenchymal stem cell transplantation would be an effective treatment for long-segment sciatic nerve defects.To test this,we established rabbit models of 30 mm sciatic nerve defects,and treated them using either an autograft or a chemically decellularized allogeneic nerve graft with or without simultaneous transplantation of bone marrow mesenchymal stem cells.We compared the tensile properties,electrophysiological function and morphology of the damaged nerve in each group.Sciatic nerves repaired by the allogeneic nerve graft combined with stem cell transplantation showed better recovery than those repaired by the acellular allogeneic nerve graft alone,and produced similar results to those observed with the autograft.These findings confirm that a chemically extracted acellular allogeneic nerve graft combined with transplantation of bone marrow mesenchymal stem cells is an effective method of repairing long-segment sciatic nerve defects.

A novel artificial nerve graft for repairing longdistance sciatic nerve defects:a self-assembling peptide nanofiber scaffold-containing poly (lactic-co-glycolic acid) conduit

In this study, we developed a novel artificial nerve graft termed self-assembling peptide nanofiber scaffold （SAPNS）-containing poly（lactic-co-glycolic acid） （PLGA） conduit （SPC） and used it to bridge a 10-mm-long sciatic nerve defect in the rat. Retrograde tracing, behavioral testing and histomorphometric analyses showed that compared with the empty PLGA conduit implantation group, the SPC implantation group had a larger number of growing and extending axons, a markedly increased diameter of regenerated axons and a greater thickness of the myelin sheath in the conduit. Furthermore, there was an increase in the size of the neuromuscular junction and myofiber diameter in the target muscle. These findings suggest that the novel artificial SPC nerve graft can promote axonal regeneration and remyelination in the transected peripheral nerve and can be used for repairing peripheral nerve injury.

Facial reanimation with interposition nerve graft or masseter nerve transfer:a comparative retrospective study

Both interposition nerve grafts and masseter nerve transfers have been successfully used for facial reanimation after irreversible injuries to the cranial portion of the facial nerve.However,no comparative study of these two procedures has yet been reported.In this two-site,twoarm,retrospective case review study,32 patients were included.Of these,17 patients(eight men and nine women,mean age 42.1 years)underwent interposition nerve graft after tumor extirpation or trauma between 2003 and 2006 in the Ear Institute,School of Medicine,Shanghai Jiao Tong University,China,and 15 patients(six men and nine women,mean age 40.6 years)underwent masseter-to-facial nerve transfer after tumor extirpation or trauma between November 2010 and February 2016 in Shanghai Ninth People's Hospital,China.More patients achieved House-Brackmann III recovery after masseter nerve repair than interposition nerve graft repair(15/15 vs.12/17).The mean oral commissure excursion ratio was also higher in patients who underwent masseter nerve transfer than in patients subjected to an interposition nerve graft.These findings suggest that masseter nerve transfer results in strong oral commissure excursion,avoiding obvious synkinesis,while an interposition nerve graft provides better resting symmetry.This study was approved by the Institutional Ethics Committee,Shanghai Ninth People's Hospital,China(approval No.SH9 H-2019-T332-1)on December 12,2019.

Brain-derived neurotrophic factor expression in dorsal root ganglion neurons in response to reanastomosis of the distal stoma after nerve grafting

Studies have shown that retreatment of the distal stoma after nerve grafting can stimulate nerve regeneration. The present study attempted to verify the effects of reanastomosis of the distal stoma, after nerve grafting, on nerve regeneration by assessing brain-derived neurotrophic factor expression in 2-month-old rats. Results showed that brain-derived neurotrophic factor expression in L2-4 dorsal root ganglia began to increase 3 days after autologous nerve grafting post sciatic nerve injury, peaked at 14 days, decreased at 28 days, and reached similar levels to the sham-surgery group at 56 days. Brain-derived neurotrophic factor expression in L2-4 dorsal root ganglia began to increase 3 days after reanastomosis of the distal stoma, 59 days after autologous nerve grafting post sciatic nerve injury, significantly increased at 63 days, peaked at 70 days, and gradually decreased thereafter, but remained higher compared with the sham-surgery group up to 112 days. The results of this study indicate that reanastomosis of the distal stoma after orthotopic nerve grafting stimulated brain-derived neurotrophic factor expression in L2.4 dorsal root ganglia.

Preparation of acellular nerve grafts with triton X-100

BACKGROUND: The source of nerve allograft enriches. We may choose expediently nerve allograft to repair injured nerve and the structure of choice nerve homology or similar with the injured nerve, but the immunological rejection limits the clinical application of nerve allograft. The ideal substitute of autograft never is researching. OBJECTIVE: In this experiment, Triton X-100 was used to extract the Schwann cells and myelin sheaths of allograft nerve and obtain the inartificial and eliminated antigenicity nerve-transplanter (nerve grafts). DESIGN: Controlled experiment. SETTING: Department of Hand Surgery, the Third Affiliated Hospital of Hebei Medical University; Second Department of Orthopedics, Fourth Center Hospital of Tianjin. MATERIALS: Thirty health New Zealand big ear white rabbit, of either sex (gender), weighing 2000-3000 g, were provided by the Center of Experimental Animal of Hebei Medical University. TritonX-100 was offered by SIGMA Company. METHODS: The experiment was carried out at the Central Laboratory of the Third Affiliated Hospital of Hebei Medical University from December 2003 to December 2004. Sixty pieces of sciatic nerves, 10-mm-long nerve segment, which were taken from 30 rabbits, were incised. They were randomly divided into chemical extraction group (n =50) and control group (n =10). In the chemical extraction groups, the nerves were put into 3% Triton X-100 solution. They were treated with Triton X-100 for 12 hours, 24 hours, 48 hours, 96 hours and 1 week, respectively. They were examined in every period. The control groups did not treated with anything. ① Respectively two segments of nerve by 2 mm length were taken from each nerve in the every periods. ② The laminin immunohistochemical stained sections were performed with image acquisition and analyzed with multicolor pathological image analysis system. Measured the laminin antibody reaction part of each section and computed laminin average gray degrees of the unit area. All dates were analyzed by SPSS 10.0 software. MAIN OUTCOME MEASURES: ① General observation and histological observation in two groups; ② Compared with laminin average gray degrees of the unit area in each section. RESULTS: ① General observation: In the control groups, fresh nerve was polish, rigidity and elasticity. After the nerves were chemical extracted, the floccules was seen at two ends and around of the nerves. The nerves being extraction presented ivory and lackluster. Its diameter and length compared reduced, tenderness and tenacity with the fresh nerve. Observed by light microscope, Schwann cells, myelin sheaths and basement membrane distribute uniformly in control groups. After the nerves were extracted, Schwann cells and myelin sheaths disappeared. Basement membrane presented barrier array in longitudinal sections. Between the membranes was the basement membrane tube. Observed with scanning electron microscope, the basement membrane tubes composed by collagen fibers were remained and collagen fibers maintained their former position, form and structure. Further, the structure of membrane was seen in the tubes. It was Schwann cells basement membrane. ② In chemical extraction groups, laminin average gray degrees of the unit area were 140.1±3.41 (12 hours), 142.1±3.14 (24 hours), 142.1±3.14 (48 hours), 140.4±4.03 (96 hours), 141.7±2.62 (1 week). In the control groups, laminin average gray degree of the unit area was 142.7±7.24. There were not significant differences among the groups (P > 0.05). CONCLUSION: The method of chemical extraction by using of Triton X-100 may be an ideal measure for preparing tissue-engineered nerve-transplanter and reserved the live of laminin in the basement membrane.

Autologous nerve graft repair of different degrees of sciatic nerve defect:stress and displacement at the anastomosis in a three-dimensional finite element simulation model

In the repair of peripheral nerve injury using autologous or synthetic nerve grafting, the mag- nitude of tensile forces at the anastomosis affects its response to physiological stress and the ultimate success of the treatment. One-dimensional stretching is commonly used to measure changes in tensile stress and strain; however, the accuracy of this simple method is limited. There- fore, in the present study, we established three-dimensional finite element models of sciatic nerve defects repaired by autologous nerve grafts. Using PRO E 5.0 finite element simulation software, we calculated the maximum stress and displacement of an anastomosis under a 5 N load in 10-, 20-, 30-, 40-mm long autologous nerve grafts. We found that maximum displacement increased with graft length, consistent with specimen force. These findings indicate that three-dimensional finite element simulation is a feasible method for analyzing stress and displacement at the anas- tomosis after autologous nerve grafting.

Repairing whole facial nerve defects with xenogeneic acellular nerve grafts in rhesus monkeys

Acellular nerve allografts conducted via chemical extraction have achieved satisfactory results in bridging whole facial nerve defects clinically,both in terms of branching a single trunk and in connecting multiple branches of an extratemporal segment.However,in the clinical treatment of facial nerve defects,allogeneic donors are limited.In this experiment,we exposed the left trunk and multiple branches of the extratemporal segment in six rhesus monkeys and dissected a gap of 25 mm to construct a monkey model of a whole left nerve defect.Six monkeys were randomly assigned to an autograft group or a xenogeneic acellular nerve graft group.In the autograft group,the 25-mm whole facial nerve defect was immediately bridged using an autogenous ipsilateral great auricular nerve,and in the xenogeneic acellular nerve graft group,this was done using a xenogeneic acellular nerve graft with trunk-branches.Examinations of facial symmetry,nerve-muscle electrophysiology,retrograde transport of labeled neuronal tracers,and morphology of the regenerated nerve and target muscle at 8 months postoperatively showed that the faces of the monkey appeared to be symmetrical in the static state and slightly asymmetrical during facial movement,and that they could actively close their eyelids completely.The degree of recovery from facial paralysis reached House-Brackmann grade II in both groups.Compound muscle action potentials were recorded and orbicularis oris muscles responded to electro-stimuli on the surgical side in each monkey.Fluoro Gold-labeled neurons could be detected in the facial nuclei on the injured side.Immunohistochemical staining showed abundant neurofilament-200-positive axons and soluble protein-100-positive Schwann cells in the regenerated nerves.A large number of mid-graft myelinated axons were observed via methylene blue staining and a transmission electron microscope.Taken together,our data indicate that xenogeneic acellular nerve grafts from minipigs are safe and effective for repairing whole facial nerve defects in rhesus monkeys,with an effect similar to that of autologous nerve transplantation.Thus,a xenogeneic acellular nerve graft may be a suitable choice for bridging a whole facial nerve defect if no other method is available.The study was approved by the Laboratory Animal Management Committee and the Ethics Review Committee of the Affiliated Wuxi No.2 People's Hospital of Nanjing Medical University,China(approval No.2018-D-1)on March 15,2018.

Histological observation on acellular nerve grafts co-cultured with Schwann cells for repairing defects of the sciatic nerve

BACKGROUND： Animal experiments and clinical studies about tissue engineering method applied to repair nerve injury mainly focus on seeking ideal artificial nerve grafts, nerve conduit and seed cells. Autologous nerve, allogeneic nerve and xenogeneic nerve are used to bridge nerve defects, it is one of the methods to promote the repair of nerve injury by culturing and growing Schwann cells, which can secrete various neurotrophic factor activities, in the grafts. OBJECTIVE ： To observe the effect of acellular nerve grafts co-cultured with Schwann cells in repairing defects of sciatic nerve. DESIGN： An observational comparative study.SETTING： Tissue Engineering Laboratory of China Medical University.MATERIALS： The experiment was carried out in the Tissue Engineering Laboratory of China Medical University between April 2004 and April 2005. Forty neonatal Sprague-Dawley rats of 5-8 days （either males or females） and 24 male Wistar rats of 180-220 g were provided by the experimental animal center of China Medical University. METHODS： ① Culture of Schwann cells： The bilateral sciatic nerves and branchial plexus were isolated from the 40 neonatal SD rats. The sciatic nerves were enzymatically digested with collagenase and dispase, isolatd, purified and cultured with the method of speed-difference adhersion, and identified with the SABC immunohistochemical method. ② Model establishment： In vitro Schwann cells were microinjected into 10-mm long acellular nerve grafts repairing a surgically created gap in the rat sciatic nerve. According to the different grafted methods, the animals were randomly divided into three groups： autografts （n=8）, acellular nerve grafts （n=8）, or acellular nerve grafts with Schwann cells （n=8）. ③ The regenerated nerve fiber number and average diameter of myeline sheath after culture were statistically anlayzed. MAIN OUTCOME MEASURES： ① The regenerated nerve ultrastructure, total number and density of myelinated nerve fibers, and the thickness of myeline sheath were observed under electron microscope. ② The images were processed with the Mias-1000 imaging analytical system to calculate the number of myelinated nerve fibers, and the thickness of myeline sheath. RESULTS： All the 24 Wistar rats were involved in the analysis of results. ① Results observed under transmission electron microscope： The regenerated myelinated nerve fibers in the group of acellular nerve grafts with Schwann cells were more even than those in the group of acellular nerve grafts, the number of myelinated nerve fibers and thickness of myelin sheath were close to those in the allografts group （P 〉 0.05）, but significantly different from those in the group of acellular nerve grafts （P 〈 0.05）. ② Results observed under scanning electron microscope： A great amount of Schwann cells with two polars were observed in the group of grafts with Schwann cells, the feature of cultured Schwann cells showed shoulder by shoulder, head to head. ③ The number of myelinated nerve fibers and thickness of myelin sheath analyzed by Mias-1000 imaging system in the group of acellular nerve grafts with Schwann cells were close to those in the autografts group （P 〉 0.05）, but significantly different from those in the group of acellular nerve grafts （P 〈 0.05）.CONCLUSION： Host axonal regeneration is significantly increased after implant of acellular nerve grafts. Acellular nerve grafts with Schwann cells offers a novel approach for repairing the gap of nerve defect.

Effect of a Nerve Graft Substitute Single-walled Carbon Nanotubes on Rat Pheochromocytoma Cells

Carbon nanotubes （CNTs） were extensively explored for their beneficial use in nervous system tissue engineering. However, an important concern regarding the use of CNTs is their toxicity during the interaction between cells and the nano particles. The rat pheochromocytoma cell line （PC12） was co-cultured with three types of single-walled carbon nanotubes （SWNTs）, purified raw SWNTs （C）, hydroxyl purified SWNTs （C-OH） and carboxyl purified SWNTs （C-COOH） at 25 μg/mL and 100 μg/ml. The experimental results revealed that SWNTs at the concentration below 100 μg/mL did not affect the cell viability. Notably, powerful antioxidant system in nerous system tissue is able to counteract with the toxicity of CNTs, which is characterized by the prominently enhanced expression of main antioxidant enzymes （catalase （CAT）, superoxide dismutase （SOD）, glutathione peroxidase （GPx） and glutathione-S-transferase （GST））. Therefore, we believe that CNTs can be good candidates for the fabrication of biomedical scaffolds for the nerve tissue repair.

Functional recovery and muscle atrophy in pre-clinical models of peripheral nerve transection and gap-grafting in mice:effects of 4-aminopyridine

We recently demonstrated a repurposing beneficial effect of 4-aminopyridine(4-AP),a potassium channel blocker,on functional recove ry and muscle atrophy after sciatic nerve crush injury in rodents.However,this effect of 4-AP is unknown in nerve transection,gap,and grafting models.To evaluate and compare the functional recovery,nerve morphology,and muscle atrophy,we used a novel stepwise nerve transection with gluing(STG),as well as 7-mm irreparable nerve gap(G-7/0)and 7-mm isografting in 5-mm gap(G-5/7)models in the absence and presence of 4-AP treatment.Following surgery,sciatic functional index was determined wee kly to evaluate the direct in vivo global motor functional recovery.After 12 weeks,nerves were processed for whole-mount immunofluorescence imaging,and tibialis anterior muscles were harvested for wet weight and quantitative histomorphological analyses for muscle fiber crosssectional area and minimal Feret's diameter.Average post-injury sciatic functional index values in STG and G-5/7 models were significantly greater than those in the G-7/0 model.4-AP did not affect the sciatic functional index recovery in any model.Compared to STG,nerve imaging revealed more misdirected axons and distorted nerve architecture with isografting.While muscle weight,cross-sectional area,and minimal Feret's diameter were significantly smaller in G-7/0 model compared with STG and G-5/7,4-AP treatment significantly increased right TA muscle mass,cross-sectional area,and minimal Feret's diameter in G-7/0 model.These findings demonstrate that functional recovery and muscle atrophy after peripheral nerve injury are directly related to the intervening nerve gap,and 4-AP exerts diffe rential effects on functional recove ry and muscle atrophy.

Ganglioside promotes the bridging of sciatic nerve defects in cryopreserved peripheral nerve allografts

Previous studies have shown that exogenous gangliosides promote nervous system regeneration and synapse formation. In this study, 10 mm sciatic nerve segments from New Zealand rabbits were thawed from cryopreservation and were used for the repair of left sciatic nerve defects through allograft bridging. Three days later, 1 mL ganglioside solution （1 g/L） was sub- cutaneously iniected into the right hind leg of rabbits. Compared with non-injected rats, muscle wet weight ratio was increased at 2-12 weeks after modeling. The quantity of myelinated fibers in regenerated sciatic nerve, myelin thickness and fiber diameter were elevated at 4-12 weeks after modeling. Sciatic nerve potential amplitude and conduction velocity were raised at 8 and 12 weeks, while conduction latencies were decreased at 12 weeks. Experimental findings indicate that ganglioside can promote the regeneration of sciatic nerve defects after repair with cryopre- served peripheral nerve allografts.

Acellular nerve grafts supplemented with induced pluripotent stem cell-derived exosomes promote peripheral nerve reconstruction and motor function recovery

Peripheral nerve injury is a great challenge in clinical work due to the restricted repair gap and weak regrowth ability.Herein,we selected induced pluripotent stem cells(iPSCs)derived exosomes to supplement acellular nerve grafts(ANGs)with the aim of restoring long-distance peripheral nerve defects.Human fibroblasts were reprogrammed into iPSCs through non-integrating transduction of Oct3/4,Sox2,Klf4,and c-Myc.The obtained iPSCs had highly active alkaline phosphatase expression and expressed Oct4,SSEA4,Nanog,Sox2,which also differentiated into all three germ layers in vivo and differentiated into mature peripheral neurons and Schwann cells(SCs)in vitro.After isolation and biological characteristics of iPSCs-derived exosomes,we found that numerous PKH26-labeled exosomes were internalized inside SCs through endocytotic pathway and exhibited a proliferative effect on SCs that were involved in the process of axonal regeneration and remyelination.After that,we prepared ANGs via optimized chemical extracted process to bridge 15 mm long-distance peripheral nerve gaps in rats.Owing to the promotion of iPSCs-derived exosomes,satisfactory regenerative outcomes were achieved including gait behavior analysis,electrophysiological assessment,and morphological analysis of regenerated nerves.Especially,motor function was restored with comparable to those achieved with nerve autografts and there were no significant differences in the fiber diameter and area of reinnervated muscle fibers.Taken together,our combined use of iPSCs-derived exosomes with ANGs demonstrates good promise to restore long-distance peripheral nerve defects,and thus represents a cell-free strategy for future clinical applications.

In vivo application of poly-3-hydroxyoctanoate as peripheral nerve graft

Objective:This study aims to investigate the degree of biocompatibility and neuroregeneration of a polymer tube,poly-3-hydroxyoctanoate(PHO)in nerve gap repair.Methods:Forty Wistar Albino male rats were randomized into two groups:autologous nerve gap repair group and PHO tube repair group.In each group,a 10-mm right sciatic nerve defect was created and reconstructed accordingly.Neuroregeneration was studied by sciatic function index(SFI),electromyography,and immunohistochemical studies on Days 7,21,45 and 60 of implantation.Biocompatibility was analyzed by the capsule formation around the conduit.Biodegradation was analyzed by the molecular weight loss in vivo.Results:Electrophysiological and histomorphometric assessments demonstrated neuroregeneration in both groups over time.In the experimental group,a straight alignment of the Schwann cells parallel to the axons was detected.However,autologous nerve graft seems to have a superior neuroregeneration compared to PHO grafts.Minor biodegradation was observed in PHO conduit at the end of 60 d.Conclusions:Although neuroregeneration is detected in PHO grafts with minor degradation in 60 d,autologous nerve graft is found to be superior in axonal regeneration compared to PHO nerve tube grafts.PHO conduits were found to create minor inflammatory reaction in vivo,resulting in good soft tissue response.

Gene expression profiles of human adipose-derived mesenchymal stem cells dynamically seeded on clinically available processed nerve allografts and collagen nerve guides

It was hypothesized that mesenchymal stem cells(MSCs) could provide necessary trophic factors when seeded onto the surfaces of commonly used nerve graft substitutes. We aimed to determine the gene expression of MSCs when influenced by Avance■ Nerve Grafts or Neura Gen■ Nerve Guides. Human adipose-derived MSCs were cultured and dynamically seeded onto 30 Avance■ Nerve Grafts and 30 Neura Gen■ Nerve Guides for 12 hours. At six time points after seeding, quantitative polymerase chain reaction analyses were performed for five samples per group. Neurotrophic [nerve growth factor(NGF), glial cell line-derived neurotrophic factor(GDNF), pleiotrophin(PTN), growth associated protein 43(GAP43) and brain-derived neurotrophic factor(BDNF)], myelination [peripheral myelin protein 22(PMP22) and myelin protein zero(MPZ)], angiogenic [platelet endothelial cell adhesion molecule 1(PECAM1/CD31) and vascular endothelial cell growth factor alpha(VEGFA)], extracellular matrix(ECM) [collagen type alpha I(COL1A1), collagen type alpha III(COL3A1), Fibulin 1(FBLN1) and laminin subunit beta 2(LAMB2)] and cell surface marker cluster of differentiation 96(CD96) gene expression was quantified. Unseeded Avance■ Nerve Grafts and Neura Gen■ Nerve Guides were used to evaluate the baseline gene expression, and unseeded MSCs provided the baseline gene expression of MSCs. The interaction of MSCs with the Avance■ Nerve Grafts led to a short-term upregulation of neurotrophic(NGF, GDNF and BDNF), myelination(PMP22 and MPZ) and angiogenic genes(CD31 and VEGFA) and a long-term upregulation of BDNF, VEGFA and COL1A1. The interaction between MSCs and the Neura Gen■ Nerve Guide led to short term upregulation of neurotrophic(NGF, GDNF and BDNF) myelination(PMP22 and MPZ), angiogenic(CD31 and VEGFA), ECM(COL1A1) and cell surface(CD96) genes and long-term upregulation of neurotrophic(GDNF and BDNF), angiogenic(CD31 and VEGFA), ECM genes(COL1A1, COL3A1, and FBLN1) and cell surface(CD96) genes. Analysis demonstrated MSCs seeded onto Neura Gen■ Nerve Guides expressed significantly higher levels of neurotrophic(PTN), angiogenic(VEGFA) and ECM(COL3A1, FBLN1) genes in the long term period compared to MSCs seeded onto Avance■ Nerve Grafts. Overall, the interaction between human MSCs and both nerve graft substitutes resulted in a significant upregulation of the expression of numerous genes important for nerve regeneration over time. The in vitro interaction of MSCs with the Neura Gen■ Nerve Guide was more pronounced, particularly in the long term period(> 14 days after seeding). These results suggest that MSC-seeding has potential to be applied in a clinical setting, which needs to be confirmed in future in vitro and in vivo research.

Potential application of let-7a antagomir in injured peripheral nerve regeneration

Neurotrophic factors,particularly nerve growth factor,enhance neuronal regeneration.However,the in vivo applications of nerve growth factor are largely limited by its intrinsic disadvantages,such as its short biological half-life,its contribution to pain response,and its inability to cross the blood-brain barrier.Considering that let-7(human miRNA)targets and regulates nerve growth factor,and that let-7 is a core regulator in peripheral nerve regeneration,we evaluated the possibilities of let-7 application in nerve repair.In this study,anti-let-7a was identified as the most suitable let-7 family molecule by analyses of endogenous expression and regulatory relationship,and functional screening.Let-7a antagomir demonstrated biosafety based on the results of in vivo safety assessments and it entered into the main cell types of the sciatic nerve,including Schwann cells,fibroblasts and macrophages.Use of hydrogel effectively achieved controlled,localized,and sustained delivery of let-7a antagomir.Finally,let-7a antagomir was integrated into chitosan conduit to construct a chitosan-hydrogel scaffold tissue-engineered nerve graft,which promoted nerve regeneration and functional recovery in a rat model of sciatic nerve transection.Our study provides an experimental basis for potential in vivo application of let-7a.