Repair of peripheral nerve defects with chemically extracted acellular nerve allografts loaded with neurotrophic factors-transfected bone marrow mesenchymal stem cells

Chemically extracted acellular nerve allografts loaded with brain-derived neurotrophic fac- tor-transfected or ciliary neurotrophic factor-transfected bone marrow mesenchymal stem cells have been shown to repair sciatic nerve injury better than chemically extracted acellular nerve allografts alone, or chemically extracted acellular nerve allografts loaded with bone marrow mesenchymal stem cells. We hypothesized that these allografts compounded with both brain-derived neurotrophic factor- and ciliary neurotrophic factor-transfected bone marrow mesenchymal stem cells may demonstrate even better effects in the repair of peripheral nerve injury. We cultured bone marrow mesenchymal stem cells expressing brain-derived neuro- trophic factor and/or ciliary neurotrophic factor and used them to treat sciatic nerve injury in rats. We observed an increase in sciatic functional index, triceps wet weight recovery rate, myelin thickness, number of myelinated nerve fibers, amplitude of motor-evoked potentials and nerve conduction velocity, and a shortened latency of motor-evoked potentials when al- lografts loaded with both neurotrophic factors were used, compared with allografts loaded with just one factor. Thus, the combination of both brain-derived neurotrophic factor and cili- ary neurotrophic factor-transfected bone marrow mesenchymal stem cells can greatly improve nerve injury.

Combining acellular nerve allografts with brainderived neurotrophic factor transfected bone marrow mesenchymal stem cells restores sciatic nerve injury better than either intervention alone

In this study, we chemically extracted acellular nerve allografts from bilateral sciatic nerves, and repaired 10-mm sciatic nerve defects in rats using these grafts and brain-derived neurotrophic factor transfected bone marrow mesenchymal stem cells. Experiments were performed in three groups： the acellular nerve allograft bridging group, acellular nerve allograft ＋ bone marrow mesenchymal stem cells group, and the acellular nerve allograft ＋ brain-derived neurotrophic factor transfected bone marrow mesenchyrnal stem cells group. Results showed that at 8 weeks after bridging, sciatic functional index, triceps wet weight recovery rate, myelin thickness, and number of myelinated nerve fibers were significantly changed in the three groups. Variations were the largest in the acellular nerve allograft ＋ brain-derived neurotrophic factor transfected bone marrow mesenchymal stem cells group compared with the other two groups. Experimental findings suggest that chemically extracted acellular nerve allograft combined nerve factor and mesenchymal stem cells can promote the restoration of sciatic nerve defects. The repair effect seen is better than the single application of acellular nerve allograft or acellular nerve allograft combined mesenchymal stem cell transplantation.

Acellular nerve allograft promotes selective regeneration

Acellular nerve ailograft preserves the basilar membrane tube and extracellular matrix, which promotes selective regeneration of neural defects via bridging. In the present study, a Sprague Dawley rat sciatic nerve was utilized to prepare acellular nerve allografts through the use of the chemical extraction method. Subsequently, the allograft was transplanted into a 10-mm sciatic nerve defect in Wistar rats, while autologous nerve grafts from Wistar rats served as controls. Compared with autologous nerve grafts, the acellular nerve allografts induced a greater number of degenerated nerve fibers from sural nerves, as well as a reduced misconnect rate in motor fibers, fewer acetylcholine esterase-positive sural nerves, and a greater number of carbonic anhydrase-positive sensory nerve fibers. Results demonstrated that the acellular nerve allograft exhibited significant neural selective regeneration in the process of bridging nerve defects.

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.

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.

Various changes in cryopreserved acellular nerve allografts at-80°C

The experimental design evaluated histological,mechanical,and biological properties of allogeneic decellularized nerves after cryopreservation in a multi-angle,multi-directional manner to provide evidence for long-term preservation.Acellular nerve allografts from human and rats were cryopreserved in a cryoprotectant（10% fetal bovine serum,10% dimethyl sulfoxide,and 5% sucrose in RPMI1640 medium） at-80°C for 1 year,followed by thawing at 40°C or 37°C for 8 minutes.The breaking force of acellular nerve allografts was measured using a tensile test.Cell survival was determined using L-929 cell suspensions.Acellular nerve allografts were transplanted into a rat model with loss of a 15-mm segment of the left sciatic nerve.Immunohistochemistry staining was used to measure neurofilament 200 expression.Hematoxylin-eosin staining was utilized to detect relative muscle area in gastrocnemius muscle.Electron microscopy was applied to observe changes in allograft ultrastructure.There was no obvious change in morphological appearance or ultrastructure,breaking force,or cytotoxicity of human acellular nerve allografts after cryopreservation at-80°C.Moreover,there was no remarkable change in neurofilament 200 expression,myelin sheath thickness,or muscle atrophy when fresh or cryopreserved rat acellular nerve allografts were applied to repair nerve injury in rats.These results suggest that cryopreservation can greatly extend the storage duration of acellular nerve tissue allografts without concomitant alteration of the physiochemical and biological properties of the engineered tissue to be used for transplantation.

Cartilage oligomeric matrix protein enhances the vascularization of acellular nerves

Vascularization of acellular nerves has been shown to contribute to nerve bridging.In this study,we used a 10-mm sciatic nerve defect model in rats to determine whether cartilage oligomeric matrix protein enhances the vascularization of injured acellular nerves.The rat nerve defects were treated with acellular nerve grafting（control group） alone or acellular nerve grafting combined with intraperitoneal injection of cartilage oligomeric matrix protein（experimental group）.As shown through two-dimensional imaging,the vessels began to invade into the acellular nerve graft from both anastomotic ends at day 7 post-operation,and gradually covered the entire graft at day 21.The vascular density,vascular area,and the velocity of revascularization in the experimental group were all higher than those in the control group.These results indicate that cartilage oligomeric matrix protein enhances the vascularization of acellular nerves.

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.

Limited recovery following acellular nerve allograft reconstruction in major peripheral nerve injuries

Recent studies suggest that acellular nerve allografts(ANA)have similar efficacy as nerve autografts in certain applications of nerve surgery.However,multiple studies also demonstrate the limitations of nerve allografts,resulting in poor patient outcomes.This submission discusses a recent case series of patients who failed allograft use with subsequent histologic analyses of these allografts.Recommendations on the treatment of nerve gaps are presented,drawing from our current understanding of allograft and autograft utility in reconstruction.Factors taken into account include recipient critical nerve function,existent nerve gap,and nerve diameter.

Biomechanical properties of acellular sciatic nerves treated with a modified chemical method

Nerve grafts are able to adapt to surrounding biomechanical environments if the nerve graft itself exhibits appropriate biomechanical properties （load, elastic modulus, etc.）. The present study was designed to determine the differences in biomechanical properties between fresh and chemically acellularized sciatic nerve grafts. Two different chemical methods were used to establish acellular nerve grafts. The nerve was chemically extracted in the Sondell method with a combination of Triton X-100 （nonionic detergent） and sodium deoxycholate （anionic detergent）, and in the modified method with a combination of Triton X-200 （anionic detergent）, sulfobetaine-10 （SB-10, amphoteric detergents）, and sulfobetaine-16 （SB-16, amphoteric detergents）. Following acellularization, hematoxylin-eosin staining and scanning electron microscopy demonstrated that the effect of acellularization via the modified method was similar to the traditional Sondell method. However, effects of demyelination and nerve fiber tube integrity were superior to the traditional Sondell method. Biomechanical testing showed that peripheral nerve graft treated using the chemical method resulted in decreased biomechanical properties （ultimate load, ultimate stress, ultimate strain, and mechanical work to fracture） compared with fresh nerves, but the differences had no statistical significance （P 〉 0.05）. These results demonstrated no significant effect on biomechanical properties of nerves treated using the chemical method. In conclusion, nerve grafts treated via the modified method removed Schwann cells, preserved neural structures, and ensured biomechanical properties of the nerve graft, which could be more appropriate for implantation studies.

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.

Acellular nerve xenografts based on supercritical extraction technology for repairing long-distance sciatic nerve defects in rats

Compared to conventional artificial nerve guide conduits (NGCs) prepared using natural polymers or synthetic polymers, acellular nerve grafts (ACNGs) derived from natural nerves with eliminated immune components have natural bionic advantages in composition and structure that polymer materials do not have. To further optimize the repair effect of ACNGs, in this study, we used a composite technology based on supercritical carbon dioxide (scCO_(2)) extraction to process the peripheral nerve of a large mammal, the Yorkshire pig, and obtained an innovative Acellular nerve xenografts (ANXs, namely, CD + scCO_(2) NG). After scCO_(2) extraction, the fat and DNA content in CD + scCO_(2) NG has been removed to the greatest extent, which can better supported cell adhesion and proliferation, inducing an extremely weak inflammatory response. Interestingly, the protein in the CD + scCO_(2) NG was primarily involved in signaling pathways related to axon guidance. Moreover, compared with the pure chemical decellularized nerve graft (CD NG), the DRG axons grew naturally on the CD + scCO_(2) NG membrane and extended long distances. In vivo studies further revealed that the regenerated nerve axons had basically crossed the CD + scCO_(2) NG 3 weeks after surgery. 12 weeks after surgery, CD + scCO_(2) NG was similar to autologous nerves in improving the quality of nerve regeneration, target muscle morphology and motor function recovery and was significantly better than hollow NGCs and CD NG. Therefore, we believe that the fully decellularized and fat-free porcine ACNGs may be the most promising “bridge” for repairing human nerve defects at this stage and for some time to come.

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.

Chemically extracted acellular allogeneic nerve graft combined with ciliary neurotrophic factor promotes sciatic nerve repair

A chemically extracted acellular allogeneic nerve graft can reduce postoperative immune rejection, similar to an autologous nerve graft, and can guide neural regeneration. However, it remains poorly understood whether a chemically extracted acellular allogeneic nerve graft combined with neurotrophic factors provides a good local environment for neural regeneration. This study investigated the repair of injured rat sciatic nerve using a chemically extracted acellular allogeneic nerve graft combined with ciliary neurotrophic factor. An autologous nerve anastomosis group and a chemical acellular allogeneic nerve bridging group were prepared as controls. At 8 weeks after repair, sciatic functional index, evoked potential amplitude of the soleus muscle, triceps wet weight recovery rate, total number of myelinated nerve fibers and myelin sheath thickness were measured. For these indices, values in the three groups showed the autologous nerve anastomosis group 〉 chemically extracted acellular nerve graft ＋ ciliary neurotrophic factor group 〉 chemical acellular allogeneic nerve bridging group. These results suggest that chemically extracted acellular nerve grafts combined with ciliary neurotrophic factor can repair sciatic nerve defects, and that this repair is inferior to autologous nerve anastomosis, but superior to chemically extracted acellular allogeneic nerve bridging alone.

Biomechanical properties of peripheral nerve after acellular ：reatment

Peripheral nerve injury causes a high rate of disability and a huge economic burden, and is currently one of the serious health problems in the world. The use of nerve grafts plays a vital role in repairing nerve defects. Acellular nerve grafts have been widely used in many experimental models as a peripheral nerve substitute. The purpose of this study was to test the biomechanical properties of acellular nerve grafts. Methods Thirty-four fresh sciatic nerves were obtained from 17 adult male Wistar rats （age of 3 months） and randomly assigned to 3 groups： normal control group, nerve segments underwent no treatment and were put in phosphate buffered saline （pH 7.4） and stored at 4℃ until further use; physical method group, nerve segments were frozen at -196℃ and then thawed at 37℃; and chemical method group, nerve segments were chemically extracted with the detergents Triton X-200, sulfobetaine-10 （SB-10） and sulfobetaine-16 （SB-16）. After the acellularization process was completed, the structural changes of in the sciatic nerves in each group were observed by hematoxylin-eosin staining and field emission scanning electron microscopy, then biomechanical properties were tested using a mechanical apparatus （Endura TEC ELF 3200, Bose, Boston, USA）. Results Hematoxylin-eosin staining and field emission scanning electron microscopy demonstrated that the effects of acellularization, demyelination, and integrity of nerve fiber tube of the chemical method were better than that of the physical method. Biomechanical testing showed that peripheral nerve grafts treated with the chemical method resulted in some decreased biomechanical properties （ultimate load, ultimate stress, ultimate strain, and mechanical work to fracture） compared with normal control nerves, but the differences were not statistically significant （P 〉0.05）. Conclusion Nerve treated with the chemical method may be more appropriate for use in implantation than nerve treated with the physical method.