Acellular Nerve Allografts Research Articles

Acellular Cauda Equina Allograft as Main Material Combined with Biodegradable Chitin Conduit for Regeneration of Long-Distance Sciatic Nerve Defect in Rats.

Autologous nerve grafting (ANG), the gold standard treatment for peripheral nerve defects, still has many restrictions. In this study, the acellular cauda equina allograft (ACEA), which consists of biodegradable chitin conduit and acellular cauda equina, is developed. The cauda equina is able to complete decellularization more quickly and efficiently than sciatic nerves under the same conditions, and it is able to reserve more basal lamina tube. In vitro, ACEA shows superior guidance capacity for the regeneration of axons and migration of Schwann cells compared to acellular sciatic nerve allograft (ASNA) in dorsal root ganglion culture. In vivo, ACEA is used to bridge 15 mm long-distance defects in rat sciatic nerves. On day 21 after transplantation, the regenerative distance of neurofilaments in the grafting segment is not significantly different between the ACEA and ANG groups. At week 12, ACEA group shows better sciatic nerve repair than chitin conduit only and ASNA groups, and the effect is similar to that in the ANG group as determined by gait analysis, neural electrophysiological, and histological analyses. The above results suggest that the ACEA has the potential to become a new biological material as a replacement for autografting in the treatment of long-distance nerve defects.

Microarray analysis of the expression profile of lncRNAs reveals the key role of lncRNA BC088327 as an agonist to heregulin‑1β‑induced cell proliferation in peripheral nerve injury.

Heregulin‑1β is capable of promoting the nerve regeneration of acellular nerve allografts with skin‑derived precursor differentiated Schwann cell(SC) therapy in peripheral nerve injury. Long non‑coding RNAs(lncRNAs) serve important roles in the regulation of gene transcription and trans-lation in multiple biological processes, but its association with the repair of peripheral nerve injury is unexplored. Therefore, in the present study, the aim was to identify novel indicators for peripheral nerve injury, and to detect whether there is an association between lncRNA expression and the treatment effect of heregulin‑1β on this disorder. The expression status of lncRNAs and mRNAs in a well‑built rat model with sciatic nerve injury was investigated using microarray assays. Based on the results of the microarray assays and quantitative polymerase chain reaction validation, it was inferred that lncRNABC088327 was upregulated to the largest extent among all the lncRNAs. According to these findings, the role of BC088327 in peripheral nerve injury was further assessed by detecting the cell viability, cell cycle and apoptosis in a hypoxic SC cell model after suppressing the expression of BC088327 using specific small interfering RNA. Based on the results of the lncRNA microarray assay, 805lncRNAs were significantly differentially expressed, among which, 323lncRNAs were upregulated and 482lncRNAs were downregulated. Based on the results of the mRNA microarray assay, 1,293lncRNAs were significantly differentially expressed, including 603upregulated and 690downregulated lncRNAs. Moreover, knockdown of lncRNA BC088327 suppressed cell viability and induced cell apoptosis and S-phase cell cycle arrest in the SCs. In conclusion, expression profile changes of lncRNAs in peripheral nerve injuries were closely associated with treatment with heregulin‑1β. lncRNA BC088327 may play a synergistic role with heregulin‑1β in repairing peripheral injury, which has the potential be a biomarker for the detection of peripheral injury and a medical target for the development of therapeutic modalities.

Inhibition of KLF7-Targeting MicroRNA 146b Promotes Sciatic Nerve Regeneration.

A previous study has indicated that Krüppel-like factor 7 (KLF7), a transcription factor that stimulates Schwann cell (SC) proliferation and axonal regeneration after peripheral nerve injury, is a promising therapeutic transcription factor in nerve injury. We aimed to identify whether inhibition of microRNA-146b (miR-146b) affected SC proliferation, migration, and myelinated axon regeneration following sciatic nerve injury by regulating its direct target KLF7. SCs were transfected with miRNA lentivirus, miRNA inhibitor lentivirus, or KLF7 siRNA lentivirus in vitro. The expression of miR146b and KLF7, as well as SC proliferation and migration, were subsequently evaluated. In vivo, an acellular nerve allograft (ANA) followed by injection of GFP control vector or a lentiviral vector encoding an miR-146b inhibitor was used to assess the repair potential in a model of sciatic nerve gap. miR-146b directly targeted KLF7 by binding to the 3'-UTR, suppressing KLF7. Up-regulation of miR-146b and KLF7 knockdown significantly reduced the proliferation and migration of SCs, whereas silencing miR-146b resulted in increased proliferation and migration. KLF7 protein was localized in SCs in which miR-146b was expressed in vivo. Similarly, 4weeks after the ANA, anti-miR-146b increased KLF7 and its target gene nerve growth factor cascade, promoting axonal outgrowth. Closer analysis revealed improved nerve conduction and sciatic function index score, and enhanced expression of neurofilaments, P0 (anti-peripheral myelin), and myelinated axon regeneration. Our findings provide new insight into the regulation of KLF7 by miR-146b during peripheral nerve regeneration and suggest a potential therapeutic strategy for peripheral nerve injury.

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.

The vascularization pattern of acellular nerve allografts after nerve repair in Sprague-Dawley rats

Objective: We have demonstrated that angiogenesis in acellular nerve allografts (ANAs) can promote neuroregeneration. The present study aimed to investigate the microvascular regeneration pattern of ANAs in Sprague-Dawley (SD) rats. Methods: Sixty male SD rats were randomly divided into an autologous group and a rat acellular nerve allograft group (rANA), and 10-mm sciatic nerve defects were induced in these rats. On the 7th, 14th and 21st days after surgery, systemic perfusion with Evans Blue (EB) or lead oxide was performed on the rats through carotid intubation. Samples were then collected for gross observation, and the microvessels in the nerves were reconstructed through microscopic CT scans using MIMICS software. The vascular volume fraction (VF, %) and microvessel growth rate (V, mm/d) in both groups were then measured, and 1 month after surgery, NF-200 staining was performed to observe and compare the growth condition of the axons. Results: Early post-operative perfusion with gelatin/EB showed EB permeation around the acellular nerve. Perfusion with gelatin/lead oxide showed that the blood vessels had grown into the allograft from both ends 7 days after the operation. Fourteen days after the operation, the microvessel growth rate of the autologous group was faster than that of the rANA group (0.39 ± 0.17 mm/d vs. 0.26 ± 0.14 mm/d, p < 0.05), and the vascular VF was also higher than that of the rANA group (8.92% ± 1.54% vs. 6.31% ± 1.21%, p < 0.05). Twenty-one days after the operation, the blood vessels at both ends of the allograft had connected to form a microvessel network. The growth rate was not significantly different between the two groups; however, the vascular VF of the autologous group was higher than that of the rANA group (12.18% ± 2.27% vs. 9.92% ± 0.84%, p < 0.05). One month after the operation, the NF-200 fluorescence (IOD) in the autologous group significantly increased compared with that of the rANA group (540,278 ± 17,424 vs. 473,310 ± 14,636, respectively, p < 0.05), suggesting that the results of the repair after nerve injury were significantly better in the autologous group than in the rANA group. Conclusion: Both the autologous nerve and ANAs rely on the permeation of tissue fluids to supply nutrients during the early stage, and microvessel growth mainly starts at both ends of the graft and enters the graft along the long axis. Compared to ANAs, the growth speed of revascularization in autologous nerve grafts was faster, leading to a better outcome in the autologous nerve group.

Micropuncture and pressure assisted Schwann cell seeding of nerve allograft

BackgroundTissue processing to create immunotolerant nerve allograft removes neurosupportive cells. Few strategies have been described for implanting new cells into the graft to support axonal regeneration. New methodMicropuncture of the nerve allograft surface combined with immersion into a pressurized cell-rich solution to potentiate the introduction of viable Schwann cells (SC) into processed nerve allograft. Allografts were used to repair rodent sciatic nerve defects. At 3, 7, and 21days, grafts were harvested, stained for SCs, and analyzed using total cross sectional area (CSA) occupied by SCs to quantify SC presence. ResultsAt days 3 and 7, SC CSA was significantly greater for the injection group compared to all other groups. At day 21, SC CSA for the injection group (0.2252%±0.2730) was significantly greater compared to following groups: pressurized-punctured (0.0653%±0.0934), nonpressurized-nonpunctured (0.0607%±0.0709), punctured-control (0.0246%±0.0398), and nonpunctured-control (0.0126%±0.0151). A significant decrease in percent CSA occupied by SCs from day 3 to day 21 was noted in nonpressurized-punctured group (p=0.0106), pressurized-nonpunctured group (p=0.0477), and injection group (p=0.0010). Comparison with existing method(s)Most studies have used small caliber hypodermic needles to inject the cells into grafts. ConclusionsDespite a presumed decrease in cell viability over the three weeks of the study, the large initial inoculum achieved by injection technique results in higher levels of final SC seeding in acellular nerve allograft compared with bathing techniques with or without micropuncture or pressurization.

Nerve stepping stone has minimal impact in aiding regeneration across long acellular nerve allografts.

Acellular nerve allografts (ANAs) yield less consistent favorable outcomes compared with autografts for long gap reconstructions. We evaluated whether a hybrid ANA can improve 6-cm gap reconstruction. Rat sciatic nerve was transected and repaired with either 6-cm hybrid or control ANAs. Hybrid ANAs were generated using a 1-cm cellular isograft between 2.5-cm ANAs, whereas control ANAs had no isograft. Outcomes were assessed by graft gene and marker expression (n = 4; at 4 weeks) and motor recovery and nerve histology (n = 10; at 20 weeks). Hybrid ANAs modified graft gene and marker expression and promoted modest axon regeneration across the 6-cm defect compared with control ANA (P < 0.05), but yielded no muscle recovery. Control ANAs had no appreciable axon regeneration across the 6-cm defect. A hybrid ANA confers minimal motor recovery benefits for regeneration across long gaps. Clinically, the authors will continue to reconstruct long nerve gaps with autografts. Muscle Nerve 57: 260-267, 2018.

Transplantation of bone marrow stromal stem cells overexpressing tropomyosin receptor kinase A for peripheral nerve repair

Background aimsPreviously we reported that overexpression of tropomyosin receptor kinase A (TrkA) could improve the survival and Schwann-like cell differentiation of bone marrow stromal stem cells (BMSCs) in nerve grafts for bridging rat sciatic nerve defects. The aim of this study was to investigate how TrkA affects the efficacy of BMSCs transplantation on peripheral nerve regeneration and functional recovery. MethodsRat BMSCs were infected with recombinant lentiviruses to construct TrkA-overexpressing BMSCs and TrkA-shRNA-expressing BMSCs, which were then seeded in acellular nerve allografts for bridging 10-mm rat sciatic nerve defects. ResultsAt 8 weeks post-transplantation, compared with Vector and Control BMSCs-laden groups, TrkA-overexpressing BMSCs-laden group demonstrated obviously improved axon growth, such as significantly higher expression of myelin basic protein and superior results of myelinated fiber density, axon diameter and myelin sheaths thickness. In accordance with this increased nerve regeneration, the animals of TrkA-overexpressing BMSCs-laden group showed significantly better restoration of sciatic nerve function, manifested as greater sciatic function index value and superior electrophysiological parameters including shorter onset latency and higher peak amplitude of compound motor action potentials and faster nerve conduction velocity. However, these beneficial effects could be reversed in TrkA-shRNA-expressing BMSCs-laden group, which showed much fewer and smaller axons with thinner myelin sheaths and correspondingly poor functional recovery. ConclusionsThese results demonstrated that TrkA may regulate the regenerative potential of BMSCs in nerve grafts, and TrkA overexpression can enhance the efficacy of BMSCs on peripheral nerve regeneration and functional recovery, which may help establish novel strategies for repairing peripheral nerve injuries.

Transgenic SCs expressing GDNF-IRES-DsRed impair nerve regeneration within acellular nerve allografts.

Providing temporally regulated glial cell line-derived neurotrophic factor (GDNF) to injured nerve can promote robust axon regeneration. However, it is poorly understood why providing highly elevated levels of GDNF to nerve can lead to axon entrapment in the zone containing elevated GDNF. This limited understanding represents an obstacle to the translation of GDNF therapies to treat nerve injuries clinically. Here, we investigated how transgenic Schwann cells (SCs) overexpressing GDNF-IRES-DsRed impact nerve regeneration. Cultured primary SCs were transduced with lentiviruses (GDNF-overexpressing transgenic SCs), one of which provides the capability to express high levels of GDNF and regulate temporal GDNF expression. These SC groups were transplanted into acellular nerve allografts (ANAs) bridging a 14 mm rat sciatic nerve defect. GDNF-overexpressing transgenic SCs expressing GDNF for as little as 1 week decreased axon regeneration across ANAs and caused extensive extracellular matrix (ECM) remodeling. To determine whether additional gene expression changes beyond GDNF transgene expression occurred in GDNF-overexpressing transgenic SCs, microarray analysis of GDNF-overexpressing transgenic SCs compared to untreated SCs was performed. Microarray analysis revealed a set of common genes regulated in transgenic SC groups expressing high levels of GDNF compared to untreated SCs. A co-culture model of GDNF-overexpressing transgenic SCs with fibroblasts (FBs) revealed differential FB ECM-related gene expression compared to untreated SCs. These data suggest a component of axon entrapment is independent of GDNF's impact on axons. Biotechnol. Bioeng. 2017;114: 2121-2130. © 2017 Wiley Periodicals, Inc.

A novel technique for decellularization of allogenic nerves and in vivo study of their use for peripheral nerve reconstruction.

Autografts represent the gold standard for peripheral nerve reconstruction but their limited availability, the discrepancy of nerve caliber, and long surgical times are drawbacks. Allografts have therefore become a valid alternative option. In particular, acellular nerve allografts (ANAs) rather than fresh allografts do not need immunosuppression and appear to be safe and effective based on recent studies. An innovative method was conceived to obtain ANAs, so as to speed up nerve decellularization, without compromising nerve architecture, and without breaking the asepsis chain. Several detergent-based techniques, integrated with sonication and mechanical stirring, were tested in vitro on rabbit nerves, to identify, by microscopy and immunohistochemistry, the most effective protocol in terms of cell lysis and cellular debris clearance, while maintaining nerve architecture. Furthermore, a pilot in vivo study was performed: ANAs were implanted into tibial nerve defects of three rabbits, and autografts, representing the gold standard, in other three animals. Twelve weeks postoperatively, rabbits were clinically evaluated and euthanasized; grafts were harvested and microscopically and histomorphometrically analyzed. The method proved to be effective in vitro: the treatment removed axons, myelin and cells, without altering nerve architecture. The in vivo study did not reveal any adverse effect: animals maintained normal weight and function of posterior limb during the entire experimental time. A mild fibrotic reaction was observed, macrophages and leukocytes were rare or absent; ANAs regenerated fascicles and bundles were comparable versus autografts. Based on these results, this decellularization protocol is encouraging and deserves deeper investigations with further preclinical and clinical studies. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2228-2240, 2017.

Abstract 51: Acellularized Nerve Allografts Guide Axon Regeneration to a Controlled Termination in a Rat Model

PURPOSE: The myriad of existing management approaches to neuroma highlight the difficulty in treating this clinical entity, and no satisfactory approach has been developed. Evaluation of “off-the-shelf” acellular nerve allografts (ANAs) as a means to bridge nerve gaps has shown an unintentional, controlled termination of axonal regrowth within long (>3cm) ANAs. We hypothesized that long ANAs can be beneficially utilized to “cap” injured nerve and guide regenerating axons to a gradual termination effectively neutralizing neuroma formation. METHODS: Thy1-GFP and Lewis rats were randomized to eight groups which received: 1) nerve transection alone, 2) traction neurectomy, 3) transection and 0.5 cm closed end silicone conduit, 4) transection and 0.5 cm ANA, 5) transection and 2.5 cm ANA, 6) transection and 5.0 cm ANA, 7) transection and proximal nerve crush, or 8) transection, proximal nerve crush and 5.0 cm ANA. In all groups, the distal nerve stump was ligated and the distal nerve turned from the proximal end to remove any trophic influence. The Thy1-GFP rat nerves were serially imaged at 4, 8, and 20 weeks to provide a visual history of regeneration. Lewis rats were sacrificed at 5 and 20 weeks for quantitative nerve histology and IHC. ANOVA with post hoc analysis were performed to evaluate significance (p<0.05). RESULTS: GFP animals that received transection alone, traction neurectomy, or transection and crush showed signs of neuroma with chaotic nerve regeneration (multidirectional axonal regrowth confirmed by histology) extending from the proximal stump as early as 4 weeks. At 5 weeks, axons grew through the entirety of the 0.5 cm ANAs, with neuroma formation extending beyond the grafts. In the 2.5 and 5.0 cm ANAs, robust axonal regeneration was demonstrated in the proximal portions of the grafts with a gradual tapering of regeneration as it moved distally, and axons failed to grow beyond the grafts. At 20 weeks, gross visualization of Thy1-GFP labeled axons demonstrates that regeneration dwindles and terminates within 5.0 cm ANAs without neuroma formation. Further histological analysis is ongoing, as are additional 20 week experiments to evaluate controlled termination with histology and IHC. CONCLUSION: Following nerve transection, long ANA “caps” can be used to control disorganized axonal regrowth, and therefore prevent the formation of a neuroma. As such, the “capping” of a transected nerve with a long ANA is a potential surgical tool in the future of neuroma management. Based upon these results, further studies are underway in a swine model to evaluate the use of ANAs in neuroma prevention in a neuroma model more similar to the human.

Efficacy of Acellular Nerve Allografts in Trigeminal Nerve Reconstruction

During trigeminal nerve repair, a gap is sometimes encountered that prevents the tension-free apposition of nerve endings. The use of a processed acellular nerve allograft is a novel technique that shows promise in overcoming this problem. The goal of the present study was to support the slowly evolving body of evidence that acellular processed nerve allografts (Avance; Axogen, Alachua, FL) are a viable alternative to autogenous nerve grafting and the use of conduits for reconstructing defects of the trigeminal nerve. The study design consisted of a retrospective review of the medical records of patients referred to Rutgers School of Dental Medicine for management of trigeminal nerve injuries from July 2008 to August 2014. Sixteen patients met the inclusion criteria for the present study. All patients underwent nerve grafting using a processed nerve allograft. All operations were performed by the same surgeon (V.Z.). Serial neurosensory testing was performed by 1 clinician (V.Z.) in a standardized fashion. The primary outcome variable was the interval to functional sensory recovery as defined by the Medical Research Council Scale. The participants ranged in age from 16 to 62years (mean 32). Of the 16 patients, 12 were female (75%) and 4 were male (25%), and 3 were smokers (18.75%) and 13 were nonsmokers (81.25%). One half of the patients (n= 8; 50%) underwent surgery on the inferior alveolar nerve, and 8 (50%) underwent surgery on the lingual nerve. The most common mechanism of injury was impacted third molar removal (n= 9; 56.25%) Of the 16 patients, 15 (93.75%) achieved functional sensory recovery during the study period. The results of the present study support the hypothesis that processed nerve allografts are effective in reconstructing small (<2-cm) trigeminal nerve defects.

Introduction of neurosupportive cells into processed acellular nerve allografts results in greater number and more even distribution when injected compared to soaking techniques

Objectives: Processing necessary to remove immunogenic components of nerve allograft renders it acellular. Seeding with supportive cells may improve axon regeneration. We aim to identify the method associated with implantation of the greatest volume and most even distribution of cells.Methods: Hypodermic needle injection was compared to soaking in solution under both normal and pressurized conditions after micropuncture of the allograft. Distribution within the allograft was measured using an in vitro model of fluorescent beads, as well as cultured Schwann cells.Results: Injection treatment resulted in larger volumes and a more uniform cross-sectional distribution of implanted cells. Beads and cells behaved similarly relative to the measured outcomes.Conclusions: Injection instills more cells in a more uniform distribution. In vivo testing may evaluate whether these techniques vary relative to cell survival, cell migration, and clinical outcomes. Size- and concentration-matched fluorescent beads may represent a viable model for analyzing cell implantation.

Healing Effects of Platelet-Rich Plasma on Peripheral Nerve Injuries.

Regeneration of peripheral nerve injuries (PNIs) has been a major challenging issue in regenerative medicine and tissue engineering. Inferior alveolar nerve and lingual nerve injuries are the major difficulties and complications of oral surgeries following dental implant placement, etc. The aim of this study was to systematically review the effects of platelet-rich plasma (PRP) on the regeneration of PNIs. Medline NCBI databases were searched for related articles up to and including May 2016. Being published in English papers, use of PRP in peripheral nerve regeneration, in vivo studies, and having histological evaluations was the inclusion criteria. Seventeen papers were selected according to the inclusion and exclusion criteria, and categorized regarding PNIs types including cut or crushed injuries. The effects of using PRP only or in combination with cells on the functional recovery and histological assessments are discussed and compared with the other treatments such as autologous nerve graft, acellular nerve allograft, and synthetic nerve conduits. The authors also clarified the perspectives in the current field. Consequently, PRP demonstrated positive effects on healing of the nerve function as well histological improvements in cut nerve PNIs model; however, more studies in the field of crushed nerves are needed to arrive at an evidence-based conclusion.

End-to-Side Neurorrhaphy as Schwann Cells Provider to Acellular Nerve Allograft and Its Suitable Application.

Axonal regeneration relies on support from proliferating host Schwann cells (SCs), and previous studies on acellular nerve allografts (ANGs) suggest that axons can regenerate into ANGs within a limited distance. Numerous studies have demonstrated that the supplementation of ANGs with exogenous factors, such as cultured SCs, stem cells, and growth factors, promote nerve regeneration in ANGs. However, there are several problems associated with their utilization. In this study, we investigated whether end-to-side (ETS) neurorrhaphy, which is an axonal provider, could be useful as an SC provider to support axonal elongation in ANGs. We found that ETS neurorrhaphy effectively promoted SC migration into ANGs when an epineurium window combined with partial neurectomy was performed, and the effectiveness increased when it was applied bilaterally. When we transplanted ANGs containing migrated SCs via ETS neurorrhaphy (hybrid ANGs) to the nerve gap, hybrid ANGs increased the number of regenerated axons and facilitated rapid axonal elongation, particularly when ETS neurorrhaphy was applied to both edges of the graft. This approach may represent a novel application of ETS neurorrhaphy and lead to the development of hybrid ANGs, making ANGs more practical in a clinical setting.

Progress of nerve bridges in the treatment of peripheral nerve disruptions

Progress of nerve bridges in the treatment of peripheral nerve disruptions Qiang Ao Department of Tissue Engineering, School of Fundamental Science, China Medical University, Shenyang, Liaoning, Peoples’ Republic of China Abstract: Clinical repair of a nerve defect is one of the most challenging surgical problems. Autologous nerve grafting remains the gold standard treatment in addressing peripheral nerve injuries that cannot be bridged by direct epineural suturing. However, the autologous nerve graft is not readily available, and the process of harvesting autologous nerve graft results in several complications. Thus, it is necessary to explore an alternative to autologous nerve graft. In the last few decades, with significant advances in the life sciences and biotechnology, a lot of artificial nerve grafts have been developed to aim at the treatment of peripheral nerve disruptions. Artificial nerve grafts range from biological tubes to synthetic tubes and from nondegradable tubes to degradable tubes. Among them, acellular nerve allografts and artificial nerve repair conduits are two kinds of the most promising substitutes for nerve autografts. The history, research status, and prospect of acellular nerve allografts and artificial nerve repair conduits are described briefly in this review. Keywords: peripheral nerve injury, repair, acellular nerve graft, nerve conduit

Can Processed Nerve Allografts be Used to Repair Nerve Injuries Greater than 4cm for the Return of Critical Function in the Upper Extremity?: Level 3 Evidence

Consulting Fee: AxoGen, Inc. (Safa)Speakers Bureau: AxoGen, Inc. (Safa, Buncke)Contracted Research: Lead site for Axogen-sponsored RANGER study (Buncke) Recent animal studies suggest acellular nerve allografts may provide inadequate regeneration compared to autografts for gaps greater than 40mm in rodents. To examine whether this observation translates to clinical practice, we queried a national nerve registry on processed nerve allograft (PNA) with autograft controls for injuries between 40-70mm. Based on clinical evidence and historical controls, we hypothesized that PNA would perform similar to nerve autograft in long gap upper extremity nerve injuries in humans. The RANGER registry is an IRB-approved, active database designed to collect injury, repair, safety and outcomes data for processed nerve allografts (Avance® Nerve Graft, AxoGen, Inc). Recently active control groups for autograft and conduit repair were added to RANGER. The registry database was queried for >40mm upper extremity nerve repairs with at least 9 months of quantitative follow-up data. Subject demographics and injury information were also evaluated for observable trends. Outcomes data were incorporated into the Medical Research Council Classification (MRCC) for sensory and motor function with meaningful recovery defined as S3/M3 or higher on the MRCC scale. Outcomes were compared to registry matched controls and historical data for nerve autograft. Twenty-seven subjects with 35 injuries were included. The groups consisted of PNA (n = 23) and nerve autograft (n = 12). Subject demographics, medical history, and concomitant injuries were comparable. PNA repairs were single stranded, caliber matched epineural repairs, while autografts were multi-strand cabled repairs. The PNA group contained a higher incidence of neuroma resections of digital nerves repaired in a delayed fashion. The average gap was 46±6mm and 48±8 mm for PNA and nerve autograft respectively. Meaningful recovery was comparable between the groups at 74% for the PNA and 67% for nerve autograft. In mixed nerve repairs reporting motor function, outcomes were also comparable with 5 of 6 and 6 of 7 repairs reporting return of motor function in the PNA and autograft groups respectively. There were no reported adverse events related to the treatment groups. See Table 1 for summary. •Clinically reported levels of meaningful recovery for >40mm processed nerve allografts are comparable to nerve autografts with both sensory and motor function outcomes.•No adverse events or revisions were reported.•Outcomes compare favorably to historical literature for nerve autograft.•The registry remains ongoing and continues to collect outcomes data on processed nerve allografts in long gap nerve reconstructions.

Molecular examination of bone marrow stromal cells and chondroitinase ABC-assisted acellular nerve allograft for peripheral nerve regeneration.

The present study aimed to evaluate the molecular mechanisms underlying combinatorial bone marrow stromal cell (BMSC) transplantation and chondroitinase ABC (Ch-ABC) therapy in a model of acellular nerve allograft (ANA) repair of the sciatic nerve gap in rats. Sprague Dawley rats (n=24) were used as nerve donors and Wistar rats (n=48) were randomly divided into the following groups: Group I, Dulbecco's modified Eagle's medium (DMEM) control group (ANA treated with DMEM only); Group II, Ch-ABC group (ANA treated with Ch-ABC only); Group III, BMSC group (ANA seeded with BMSCs only); Group IV, Ch-ABC + BMSCs group (Ch-ABC treated ANA then seeded with BMSCs). After 8 weeks, the expression of nerve growth factor, brain-derived neurotrophic factor and vascular endothelial growth factor in the regenerated tissues were detected by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry. Axonal regeneration, motor neuron protection and functional recovery were examined by immunohistochemistry, horseradish peroxidase retrograde neural tracing and electrophysiological and tibialis anterior muscle recovery analyses. It was observed that combination therapy enhances the growth response of the donor nerve locally as well as distally, at the level of the spinal cord motoneuron and the target muscle organ. This phenomenon is likely due to the propagation of retrograde and anterograde transport of growth signals sourced from the graft site. Collectively, growth improvement on the donor nerve, target muscle and motoneuron ultimately contribute to efficacious axonal regeneration and functional recovery. Thorough investigation of molecular peripheral nerve injury combinatorial strategies are required for the optimization of efficacious therapy and full functional recovery following ANA.

Three-dimensional Reconstruction of the Microstructure of Human Acellular Nerve Allograft

The exact inner 3D microstructure of the human peripheral nerve has been a mystery for decades. Therefore, it has been difficult to solve several problems regarding peripheral nerve injury and repair. We used high-resolution X-ray computed microtomography (microCT) to scan a freeze-dried human acellular nerve allograft (hANA). The microCT images were then used to reconstruct a 3D digital model, which was used to print a 3D resin model of the nerve graft. The 3D digital model of the hANA allowed visualization of all planes. The magnified 3D resin model clearly showed the nerve bundles and basement membrane tubes of the hANA. Scanning electron microscopy (SEM) was used to analyse the microstructure of the hANA. Compared to the SEM images, the microCT image clearly demonstrated the microstructure of the hANA cross section at a resolution of up to 1.2 μm. The 3D digital model of the hANA facilitates a clear and easy understanding of peripheral nerve microstructure. Furthermore, the enlarged 3D resin model duplicates the unique inner structure of each individual hANA. This is a crucial step towards achieving 3D printing of a hANA or nerve that can be used as a nerve graft.

Erratum: Light-Activated Sealing of Acellular Nerve Allografts following Nerve Gap Injury.

Erratum: Light-Activated Sealing of Acellular Nerve Allografts following Nerve Gap Injury.