cells Article Repair of Long Nerve Defects with a New Decellularized Nerve Graft in Rats and in Sheep Estefanía Contreras 1,2 , Sara Traserra 1,2 , Sara Bolívar 1,3 , Joaquim Forés 1,4 , Eduard Jose-Cunilleras 5 , Felix García 5 , Ignacio Delgado-Martínez 1,3 , Sandra Holmgren 6 , Raimund Strehl 6 , Esther Udina 1,3 and Xavier Navarro 1,3, * 1 Department of Cell Biology, Physiology and Immunology, Institute of Neuroscience, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain 2 Integral Service for Laboratory Animals (SIAL), Faculty of Veterinary, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain 3 Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), 28031 Madrid, Spain 4 Hand and Peripheral Nerve Unit, Hospital Clínic i Provincial, Universitat de Barcelona, 08036 Barcelona, Spain 5 Department of Animal Medicine and Surgery, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain 6 VERIGRAFT AB, Arvid Wallgrens Backe 20, 41346 Göteborg, Sweden * Correspondence: xavier.navarro@uab.cat; Tel.: +34-935811966; Fax: +34-935812986 Abstract: Decellularized nerve allografts (DC) are an alternative to autografts (AG) for repairing severe peripheral nerve injuries. We have assessed a new DC provided by VERIGRAFT. The de- cellularization procedure completely removed cellularity while preserving the extracellular matrix. We first assessed the DC in a 15 mm gap in the sciatic nerve of rats, showing slightly delayed but effective regeneration. Then, we assayed the DC in a 70 mm gap in the peroneal nerve of sheep Citation: Contreras, E.; Traserra, S.; compared with AG. Evaluation of nerve regeneration and functional recovery was performed by Bolívar, S.; Forés, J.; Jose-Cunilleras, clinical, electrophysiology and ultrasound tests. No significant differences were found in functional E.; García, F.; Delgado-Martínez, I.; recovery between groups of sheep. Histology showed a preserved fascicular structure in the AG Holmgren, S.; Strehl, R.; Udina, E.; while in the DC grafts regenerated axons were grouped in small units. In conclusion, the DC was et al. Repair of Long Nerve Defects permissive for axonal regeneration and allowed to repair a 70 mm long gap in the sheep nerve. with a New Decellularized Nerve Graft in Rats and in Sheep. Cells 2022, Keywords: nerve regeneration; autograft; allograft; decellularization; sheep 11, 4074. https://doi.org/10.3390/ cells11244074 Academic Editor: Stefano Geuna Received: 21 October 2022 1. Introduction Accepted: 9 December 2022 Peripheral nerve injuries results in partial or total loss of the sensory, autonomic and Published: 16 December 2022 motor functions dependent on the injured nerve, with important consequences for the Publisher’s Note: MDPI stays neutral quality of life of affected patients [1]. It is estimated that more than 300,000 persons are with regard to jurisdictional claims in affected by traumatic nerve injury every year in North America and Europe [2,3]. Although published maps and institutional affil- peripheral neurons are able to regenerate after axotomy, and eventually reinnervate target iations. organs, functional recovery is often unsatisfactory, especially following severe injuries [4,5]. Poor functional outcomes generally stem from long regenerative distances coupled with a relatively slow rate of axonal regeneration (~1–2 mm/day), creating prolonged periods of denervation that ultimately limit the regenerative capacity of the distal nerve structure [6,7]. Copyright: © 2022 by the authors. Therefore, a special challenge regarding recovery after peripheral nerve injury are long Licensee MDPI, Basel, Switzerland. distance nerve defects and proximal injuries in long limbs. This article is an open access article When the length of the gap created by a nerve injury is too long to allow apposi- distributed under the terms and tion and direct suture without tension, nerve grafts are usually employed for repairing. conditions of the Creative Commons The graft between the stumps of a transected nerve offers mechanical guidance, and the Attribution (CC BY) license (https:// Schwann cells of the graft and their basal lamina play an essential role in promoting axonal creativecommons.org/licenses/by/ growth [8]. The autologous nerve graft represents the “gold standard” surgical treatment 4.0/). Cells 2022, 11, 4074. https://doi.org/10.3390/cells11244074 https://www.mdpi.com/journal/cells Cells 2022, 11, 4074 2 of 19 for complex peripheral nerve injuries in the clinic; however, autografts present a number of limitations. Apart from the paucity of expendable nerve tissue, harvesting autologous nerves results in significant donor site morbidity, increased risk of infection, and longer intraoperative times [9]. The alternative use of allografts has been discouraging, since the immune rejection directed against Schwann cells and myelin sheaths of the graft impedes axonal regeneration [10]. Processed acellular nerve allograft has been promoted as a po- tential replacement [9,11]. If adequately decellularized, such grafts do not induce immune response, whereas the internal nerve structure, including endoneurial tubules, basal lamina and extracellular matrix (ECM) components, remains supporting axonal regeneration, despite the absence of the activated Schwann cells [8]. Allograft and autograft repair of nerve defect injury in small animals have indicated that regeneration is possible on critical length nerve gaps [12,13]. Even though the anatomy of rodent nerves has been well studied and is similar to that of humans [14], the important difference in size between these species compared to humans limits the translation of the results obtained, since axonal regeneration is faster in smaller animals and relatively short gaps can be produced in them [15,16]. Indeed, it is not clear if the mechanisms involved in peripheral nerve trauma, repair and regeneration are closely conserved across species [6,16,17]. In order to achieve results that may be applicable to humans, a translational large animal model for peripheral nerve surgery is important [6,17,18]. A valid animal model for nerve regeneration studies should have similar anatomy and length of the nerves to humans and follow a similar regeneration rate after nerve injury. Sheep have emerged as one of the most adequate large animals for pre-clinical studies on a variety of peripheral nerves [18–23]. Thus, experimental studies in small and large animal models are fundamental to optimize the decellularization process and to evaluate the regenerative capacity of novel allografts. In this study, we assessed a newly prepared decellularized nerve graft used for repairing a critical gap of 15 mm of the sciatic nerve in rats, and given the obtained success, we extended to a comparative assessment of nerve regeneration along a nerve gap of 70 mm length in the peroneal nerve of adult sheep repaired with the new decellularized nerve graft, provided by VERIGRAFT AB, or with an autograft as the gold standard method of repair. 2. Materials and Methods 2.1. Nerve Decellularization Rat sciatic nerves were harvested from five euthanized Sprague Dawley rats under aseptic conditions. Specimens of sheep nerves for the decellularized allografts were ob- tained from four donor sheep from the colony of Servei de Granges i Camps Experimentals (SGiCE) of the Universitat Autònoma de Barcelona (UAB). Peroneal nerves were harvested from euthanized sheep under aseptic conditions. The harvested nerves were stored in phosphate buffered saline (PBS) plus antibiotic-antimycotic agents, maintained at 4 ◦ C and shipped to VERIGRAFT AB. For decellularization, the nerves were thawed and agitated in NaCl (Fisher Scientific, Göteborg, Sweden) for 24 h, PBS (Medicago, Uppsala, Sweden) for 6 h and Deoxyribonucle- ase (DNase; VWR, Stockholm, Sweden) overnight. The nerves were washed 3 × 5 min in H2 O after NaCl and DNase. This process was repeated three times. Then the nerves were washed in PBS for 24 h. After decellularization, samples were taken for DNA quantification and histology before peracetic acid sterilization and final washes in PBS under sterile conditions. The sterilized decellularized nerves were shipped in PBS at 2–4 ◦ C. One nerve segment was decellularized for each animal to be transplanted. DNA was extracted from 10–25 mg wet tissue with the DNeasy Blood & Tissue kit (Qiagen, Mettmann, Germany) and quantified with the Qubit dsDNA HS assay kit (Invitrogen, Waltham, MA, USA), according to the manufacturer’s instructions. DNA concentration was calculated using the DNA standard supplied with the Qubit dsDNA HS assay kit. After decellularization, a small segment of each decellularized nerve was fixed in paraformaldehyde 4% for 4 h at room temperature (RT) and then transferred to PBS with sucrose 30% for 24 h. Samples were cryo-embedded with OCT and serially cut in 15 µm Cells 2022, 11, 4074 3 of 19 thick transverse sections with a cryotome. The sections were blocked and permeabilized with 0.3% Triton-X100 and 10% normal donkey serum and/or goat donkey serum for 1 h. Then, slides were incubated overnight at 4 ◦ C with primary antibodies against anti-rabbit S-100 protein (S100; to label Schwann cells; 1:50; 22520-DiaSorin), anti-chicken neurofil- ament (NF200; myelinated axons; 1:400; AB5539-Millipore), anti-rabbit non-collagenous connective tissue glycoprotein (laminin; 1:500; AHP420-Biorad), and with myelin stain (Fluoromyelin; 1:300; F34651-Invitrogen), all diluted in 0.1% Triton-PBS. Following three washes in PBS, the sections were incubated with secondary antibodies conjugated to Alexa Fluor 488 goat anti-chicken (1:200; A11039-Invitrogen) and Alexa Fluor 594 goat anti-rabbit (1:200; A21207-Invitrogen) for 1 h. After two washes with PBS, samples were incubated with DAPI stain for nuclei (1:100; D9564-10MG-Sigma) diluted in PBS for 2 min. After three more washes in PBS, immunolabeled sections were cover-slipped with Fluoromount (Sigma-Aldrich, St. Louis, MI, USA) and viewed under epifluorescence microscopy (Olym- pus BX51). All the procedures were approved by the Ethics Committees of the UAB and Generali- tat de Catalunya, and followed the European Community Council Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. 2.2. Rat Study For the implantation, ten female Sprague Dawley rats (12 weeks of age) were randomly assigned into two different experimental groups: autograft (AG) (n = 5) or decellularized rat allograft (DRA) (n = 5). Animals were anesthetized with ketamine (75 mg/kg) and medetomidine (0.01 mg/kg) intraperitoneally. A skin incision was made on the right hindlimb and the biceps femoris muscle was incised to expose the sciatic nerve. A 15 mm in length was removed and then, in the AG group, the same nerve was sutured again maintaining the same orientation. In the DRA group, the nerve removed was substituted by a 15 mm length decellularized rat allograft. In both cases, the proximal and distal nerve stumps were sutured by 10-0 nylon epineural sutures. Postoperative care included amitriptyline (20 mL/L) in drinking water to prevent autotomy [24] and buprenorphine (0.03 mg/kg subcutaneously) to treat postoperative pain. Reinnervation of target muscles were assessed by monthly noninvasive nerve conduc- tion tests until the end time, set at 120 days post injury (dpi). Under general anesthesia, the rat was placed on a warm plate, and the sciatic nerve was stimulated with transcutaneous needle electrodes placed at the sciatic notch using a Sapphire 4M electromyograph (EMG) (Medelec, Vickers, Surrey, U.K.), by delivering single pulses of 0.1 ms of increasing intensity. The compound muscle action potential (CMAP) was recorded from the tibialis anterior (TA), gastrocnemius (GM) and plantar interosseus (PL) muscles with small needle elec- trodes transcutaneously placed on the active muscle belly and the reference in the fourth toe [25,26]. The ground electrode was placed at the knee. The contralateral hindlimb was used as a control since there are not significant changes in electrophysiological properties of the contralateral limb after a unilateral nerve lesion [25]. At the end of the study, animals were euthanized by an overdose of pentobarbital (200 mg/kg i.p.) and samples from the sciatic nerve were harvested. Samples were fixed in paraformaldehyde 4% and then, divided in three different parts. The proximal and distal segments, including suture levels were post-fixed in 3% glutaraldehyde-3% paraformaldehyde in cacodylate-buffer solution (0.1 M, pH 7.4) at 4 ◦ C for 48 h. To evaluate the microstructure of the nerve, samples were embedded in epon resin. Semithin 0.5 µm sections were stained with 1% toluidine blue and visualized with a light microscope (Olympus BX40). The cross-sectional area of the whole nerve was measured at 40X. Images were obtained at 1000X from fields chosen by systematic sampling of squares, representing at least 30% of the transverse area of the nerve [27,28]. Counts of the number of myelinated nerve fibers were conducted using ImageJ software (version 1.52d). Only myelinated fibers whose contour was completely within each field photograph or cut at two borders of the square were counted. The total number of myelinated fibers per nerve was obtained Cells 2022, 11, 4074 4 of 19 from the fiber density of the sampled fields and the total transverse endoneurial area. The middle segment of the sciatic nerve was processed for immunolabeling as explained above for axons (NF200), Schwann cells (S100), laminin and macrophages (anti-goat iba 1; ionized calcium-binding adapter molecule 1; 1:500; 19-19741-Rafer). Samples were washed and incubated with secondary antibodies Alexa Fluor 488 goat anti-chicken (1:200; A11039-Invitrogen), Alexa Fluor 488 donkey anti-goat (1:200; A11055-Invitrogen) and Alexa Fluor 594 goat anti-rabbit (1:200; A21207-Invitrogen) diluted in PBS-Triton 0.3%. Finally, sections were cover-slipped with Fluoromount containing DAPI (1:10,000; Sigma- Aldrich). Sections were visualized with an epifluorescence microscope (Olympus BX51) for qualitative assessment of the regeneration process. Observations were focused on the relationship between axons and Schwann cells and the number of infiltrating macrophages. 2.3. Sheep Study Ten female ripollesa sheep (Ovis aries), weighing 55–75 Kg, from the animal facility of SGiCE of the UAB were used. The sheep were divided in two experimental groups of 5 animals each, according to the repair procedure performed: autograft and decellularized allograft. A blood sample was taken before the surgery and at the end of the study to perform hematological and biochemical standard analyses, to ensure that the sheep were in good health state. A general clinical assessment was performed once a week until the end of the study. 2.4. Surgical Procedure The animals were fasted 16 h prior to the surgery to reduce the ruminal content and prevent deviant swallowing. Animals were sedated by midazolam (0.2 mg/Kg) and morphine (0.4 mg/Kg i.m.). Anesthesia was induced by propofol (4 mg/Kg i.v.) and maintained by isofluorane 2% in 2 L/min oxygen administered with a pressure-controlled ventilator. Analgesia was provided by diazepam (0.5 mg/Kg i.v.), and an antibiotic dose of cefazoline (20 mg/Kg i.v.). Fluid therapy was applied with Ringer solution infusion (10 mL/Kg/h). The operative procedure was conducted using a sterile technique with the sheep in a left lateral decubitus position on an operating table. On the right hindlimb, the peroneal nerve was exposed using a longitudinal lateral skin incision along the thigh followed by splitting of the semitendinous and biceps femoris muscles. Under the operating microscope, the common peroneal nerve was resected 1 cm above the iliac vein to create a 7 cm gap. The repair was conducted by bridging the two nerve stumps with a decellularized nerve allograft or with an autograft (Figure 1). The resected nerve segment was used as autograft maintaining the original orientation. The decellularized nerve graft was easy to handle and suture, without notable difference compared with the nerve autograft. The nerve stumps were sutured to each end of the graft by means of 8/0 epineural sutures. The suture resistance was tested against light stretching. The incision was closed in layers and disinfected with chlorhexidine and Aluspray® . After the surgery, the animals were allowed to recover in couples in the animal’s facility. During the post-operative period, analgesia was provided twice a day with buprenorphine (0.01 mg/Kg s.c.) for 2 days and once a day with meloxicam (0.2 mg/Kg s.c.) for 3 days. Cells Cells 2022, 2022, 11, 11, x4074 FOR PEER REVIEW 5 5ofof 20 19 1. Decellularized Figure 1. Figure Decellularizednerve nerveallograft (A) allograft was (A) wastrimmed trimmedintointo 7-cm7-cm nerve long long nerve and then anditthen was it placed was into the gap created in the common peroneal nerve (B). The decellularized nerve allograft placed into the gap created in the common peroneal nerve (B). The decellularized nerve allograft(C) or the same (C) or nerve segment the same nerveresected segmentused as autograft resected used as (D) were sutured autograft (D) werebridging suturedthe gap. the gap. bridging 2.5. Functional Tests 2.5. Functional Tests At monthly intervals, animals were tested for evidence of hindlimb functional recovery. At monthly intervals, animals were tested for evidence of hindlimb functional recov- Each parameter was assessed in a semiquantitative arbitrary scale from 0 (no deficit), −1 ery. Each parameter was assessed in a semiquantitative arbitrary scale from 0 (no deficit), (partial deficit or functional loss) to −2 (complete loss of response in the maneuver tested). −1 (partial deficit or functional loss) to −2 (complete loss of response in the maneuver Locomotion was assessed qualitatively while freely walking in the stable, paying particular tested). Locomotion was assessed qualitatively while freely walking in the stable, paying attention to the foot-drop position of the operated hindlimb (0 = normal walking; −1 = a particular attention to the foot-drop position of the operated hindlimb (0 = normal walk- few failures; −2 = foot drop in mots steps). The TA muscle mass was assessed by manual ing; −1 = a comparing palpation few failures; −2 =the both foot drop in mots denervated and steps). controlThesideTA muscle (0 = similarmass mass;was −1assessed = slight by reduction; −2 = marked reduction). The proprioceptive response was tested by themass; manual palpation comparing both the denervated and control side (0 = similar capa- −1 = slight bility reduction; of replacing the−2 = marked hoof from a reduction). plantarflexion Theposition proprioceptive forced byresponse was testedtobya the experimenter the capability plantar support ofposition replacing (0 the hoof from = consistent a plantarflexion response; −1 = one position failure; −forced 2 = twobyorthe experi- 3 failures). menter The withdrawal reflex of the limb was evaluated by pinching with a hemostat the two to a plantar support position (0 = consistent response; −1 = one failure; −2 = skin or of 3the failures). dorsumThe withdrawal of the paw at three reflex of between sites the limb the wasankle evaluated by hoof and the pinching with (Figure a hemostat S1), proximal, the skinand middle of the dorsum distal. Eachof the site paw was at three tested twicesites between in the the ankle same session andand the hoof separately (Figure scored as: S1), proximal, middle and distal. Each site was tested twice in the same 0 = fast and strong withdrawal response of the limb; −1 = weak response or not consistent session and sepa- rately in the scored 2 trials;as: −20== nofastresponse and strongin 2withdrawal trials. response of the limb; −1 = weak response or not consistent in the 2 trials; −2 = no response in 2 trials. 2.6. Electrophysiological Tests 2.6. Electrophysiological Motor nerve conductionTests tests were performed at 5, 6.5 and 9 months after the operation under general Motor anesthesia nerve (diazepam conduction 0.25 tests were mg/Kg and performed at 5,ketamine 6.5 and 9 5months mg/Kgafter i.v.).the Anopera- EMG apparatus tion under (Sapphire 4ME, Vickers, general anesthesia Surrey, (diazepam U.K.) 0.25 was and mg/Kg usedketamine for these5tests. mg/Kg Thei.v.). sciatic An nerve EMG was stimulated, apparatus at supramaximal (Sapphire 4ME, Vickers,intensity, at thewas Surrey, U.K.) sciatic usednotch with tests. for these a pairThe of percutaneous sciatic nerve was stimulated, at supramaximal intensity, at the sciatic notch with a pair of percutaneous Cells 2022, 11, 4074 6 of 19 needle electrodes, and the compound muscle action potential (CMAP) was recorded from the TA muscle with monopolar needle electrodes, the active electrode on the muscle belly, and the reference at the distal tendon. Moreover, free-running EMG recordings were made to detect fibrillation potentials, as a sign of muscle denervation. The contralateral hindlimb was tested at the same session and its values used as control. 2.7. Ecographic Evaluation of Hindlimb Muscles Ultrasound examinations were performed at 6.5 and 9 months after the electrophysio- logical tests, while animals were anesthetized, using a MyLab® Gamma (Esaote, Genova, Italy) device and a linear ultrasound probe. The cross-sectional area and perimeter of the TA muscle was determined using a B-mode ultrasound, employing a 15 MHz linear transducer, and optimizing the image at a depth of 3 cm and focal point at 1.5 cm. To optimize image acquisition and good skin contact, the hair over the area of interest was clipped and the skin cleaned with water and mild soap, and acoustic gel was used. In preliminary studies, we established that the point of interest to standardize image acquisition was the cranial aspect of the crus at a midpoint between the tibial crest and the tuber calcanei (Figure S2). 2.8. Histological Evaluation After performing the functional test 9 months after injury and when still anesthetized, animals were euthanized using an intravenous injection of Euthasol (400 mg/Kg i.v.). Nerve segments extending at least 1 cm from the proximal suture and 2 cm from the distal suture of the graft were harvested and fixed in paraformaldehyde 4% for 7 days at 4 ◦ C. TA muscles were taken and weighed, and samples of TA muscles and of the skin of the dorsum of the paw were obtained and fixed in paraformaldehyde 4% for 7 days at room temperature. After fixation, the peroneal nerves were divided into 5 different sections (see Sec- tion 3.3.4), and each section was further divided in two halves. The first half of Section 2 from the middle of the graft and Section 4 from the distal end were embedded in paraffin and 5 µm thick cross sections were obtained with a microtome. Samples from the midpoint of the graft were deparaffinized and stained with hematoxylin at 10% in absolute ethanol (Hematoxylin cryst. 1.04302.0100, Sigma Aldrich) and eosin at 0.5% in distilled water (Eosin Y, 1.15935.0100, Sigma Aldrich) to visualize the general structure of the nerve. Briefly, the samples were hydrated in H2 O for 10 min, then, stained for 5 min in hematoxylin. After two washes in H2 O, samples were introduced in 70% ethanol with 1% of hydrochloric acid for 30 s. Samples were washed again and stained with eosin for 5 min. Finally, samples were dehydrated, and mounted in gelatinized glass slides. Other sections were dewaxed and processed for immunohistochemistry against S-100 protein to label Schwann cells, and neurofilament NF200 to label myelinated axons, as explained above. Following washes, the sections were incubated with secondary antibodies bound to Alexa Fluor 488 and Alexa Fluor 594, mounted on gelatinized slides and viewed under epifluorescence microscopy. Quantitative analysis was performed with sets of images obtained at 1000X by measuring the cross-section area of the nerves and grafts, and then counting the number of NF200 labeled axons in systematically selected fields, with at least 8 representative fields compris- ing more than 30% of the transverse area, under epifluorescence microscopy (Olympus BX51) using ImageJ software. The second half of Section 2 from the middle of the graft and Section 4 from the distal nerve were postfixed in 3% paraformaldehyde and 3% glutaraldehyde in phosphate- buffered saline. The nerve segments were postfixed in osmium tetroxide, dehydrated in a series of ethanol, and embedded in epon resin. Semithin 0.5 µm thick sections were stained with toluidine blue, and representative images were taken by light microscopy. TA muscle and skin samples were also processed for paraffin embedding. Samples were stained with hematoxylin and eosin, as explained above for the nerve samples, to visualize the general structure and to evaluate qualitatively the areas showing denerva- tion atrophy. 22, 11, x FOR PEER REVIEW 7 of 20 visualize the general structure and to evaluate qualitatively the areas showing denerva- Cells 2022, 11, 4074 7 of 19 tion atrophy. 2.9. Data Analysis 2.9. Data Analysis Data are expressed as mean ± standard error of the mean (SEM). The results of func- Data are tional tests and histology expressed were analyzedasby mean ± standard the Student’s error t test andofa two-way the meanANOVA (SEM). The results of after checking functional for normaltests and histology distribution, were using analyzed GraphPad by the Prism 8 software. t test Student’s A p < and 0.05awas two-way ANOVA after checking considered as significant. for normal distribution, using GraphPad Prism 8 software. A p < 0.05 was considered as significant. 3. Results 3. Results 3.1. Effectiveness of Effectiveness 3.1. the Decellularization Procedure of the Decellularization Procedure The decellularization procedure developed The decellularization removed procedure all donor developed cells all removed and DNAcells donor fromand DNA from the nerve segments, while the ECM as well as structural layers of the graft remained the nerve segments, while the ECM as well as structural layers of the graft remained intact. In this way, theintact. nerveIn grafts this can way,provide the nerveguidance to the grafts can regenerating provide guidance axons upon to the implan- axons upon regenerating tation. DNA content implantation. DNA content averaged 159.7 ± 18.7 ng/mg tissue in and averaged 159.7 ± 18.7 ng/mg tissue in the native nerves of sheep, the native nerves 0.35 ± 0.02 ng/mg tissue in of sheep, the0.35 and decellularized ± 0.02 ng/mg nerves. Cross tissue in sections of decellularized the decellularized nerves.nerve Cross sections of grafts were processed for immunohistochemistry before implantation. Immunolabeling decellularized nerve grafts were processed for immunohistochemistry before implantation. against laminin showed that theagainst Immunolabeling structure of the laminin ECM was showed that well preserved. the structure Faint of the ECMstaining was well preserved. against NF200,Faint S100staining and Fluoromyelin in the decellularized nerve grafts, of against NF200, S100 and Fluoromyelin in the decellularized both rats andnerve grafts, of sheep, was compatible with lack of axons (NF200) and Schwann cells (S100), and good both rats and sheep, was compatible with lack of axons (NF200) and Schwann cells (S100), decellularization and(absence of DAPI, general good decellularization markerofofDAPI, (absence nucleus), thus general indicating marker that cellthus indicating of nucleus), contents were that completely removed cell contents were(Figure 2). removed (Figure 2). completely Figure 2. Representative Figure 2. Representative micrographsmicrographs showing immunofluorescence showing immunofluorescence of myelinated of myelinated axonsaxons (A,F,K,G), Schwann (A,F,K,G), Schwann cells (B,G,L,Q), cells (B,G,L,Q), nucleinuclei (C,H,M,R), (C,H,M,R), extracellular extracellular matrix matrix proteinsproteins (D,I,N,S) (D,I,N,S) and and myelin myelin (E,J,O,T), in a control (E,J,O,T), in arat sciaticrat control nerve (A,B,C,D,E), sciatic in a decellularized nerve (A,B,C,D,E), rat nerve (F,G,H,I,J), in a decellularized rat nerve (F,G,H,I,J), in a in a control sheep peroneal control sheepnerve (K,L,M,N,O) peroneal and in a and nerve (K,L,M,N,O) decellularized sheep nerve in a decellularized sheep(P,Q,R,S,T). nerve (P,Q,R,S,T). Scale bar Scale bar 150 µm. 150 µm. 3.2. Rat Study 3.2. Rat Study Denervation ofDenervation the GM, TA of the andGM, PL TA and PLwas muscles muscles was observed observed in both in both experimental groups experimental at 30At groups at 30 dpi. dpi. 60 At 60all dpi, dpi, all animals animals fromfrom the AG the AG andand DRA DRA groups groups showed showed electrophysiological electro- evidence of starting reinnervation in the GM and TA muscles. Three physiological evidence of starting reinnervation in the GM and TA muscles. Three of the of the animals of animals of the AG group had recorded CMAPs of small amplitude evoked by electrical stimulation, the AG group had recorded CMAPs of small amplitude evoked by electrical whereas none of the animals of the DRA group showed evidence of reinnervation in the PL muscle. At 90 dpi, the amplitude of the CMAPs increased for TA and GM muscles in both groups. All the rats of the AG group but only two thirds of the rats of the DRA group showed positive values for PL muscle. At the end of the follow-up, set up at 120 dpi, group Cells 2022, 11, x FOR PEER REVIEW 8 of 20 stimulation, whereas none of the animals of the DRA group showed evidence of reinner- Cells 2022, 11, 4074 vation in the PL muscle. At 90 dpi, the amplitude of the CMAPs increased for TA and8GM of 19 muscles in both groups. All the rats of the AG group but only two thirds of the rats of the DRA group showed positive values for PL muscle. At the end of the follow-up, set up at 120 dpi, group AG had significantly higher mean CMAP amplitude values than the DRA AG had significantly higher mean CMAP amplitude values than the DRA group in TA group in TA (28.8 ± 1.8 mV vs. 19.3 ± 2.8 mV; p < 0.05), GM (41.3 ± 3.9 mV vs. 23.7 ± 1.5 (28.8 ± 1.8 mV vs. 19.3 ± 2.8 mV; p < 0.05), GM (41.3 ± 3.9 mV vs. 23.7 ± 1.5 mV; p < 0.05) mV; p < 0.05) and PL (2.3 ± 0.4 mV vs. 0.5 ± 0.1 mV; p < 0.05) muscles (Figure 3A). and PL (2.3 ± 0.4 mV vs. 0.5 ± 0.1 mV; p < 0.05) muscles (Figure 3A). Figure 3. Results of the electrophysiological tests and histological evaluation after repair of a 15 mm long gap Figure in the rat 3. Results sciatic of the nerve. (A) Electrophysiological electrophysiological evaluation tests and histological of nerve evaluation afterregeneration repair of a 15along mm long gap in the rat sciatic nerve. (A) Electrophysiological evaluation of nerve regeneration 120 days follow-up after sciatic nerve section and repair with an autograft (AG, n = 5) or with along 120a days follow-up after sciatic nerve section and repair with an autograft (AG, n = 5) decellularized allograft (DRA, n = 5). Results are presented as mean ± SEM. Statistical analysis was or with a decellu- larized performedallograft using(DRA, 2way nANOVA. = 5). Results Plots are showpresented as mean the amplitude of ±CAMP SEM. ofStatistical plantar (*analysis p < 0.05was vs. per- AG), formed using 2way ANOVA. Plots show the amplitude of CAMP of plantar (* p < 0.05 vs. AG), tibialis anterior (* p < 0.05 vs. AG) and gastrocnemius (* p < 0.05 vs. AG) muscles. (B) Representative tibialis anterior (* p < 0.05 vs. AG) and gastrocnemius (* p < 0.05 vs. AG) muscles. (B) Representative micrographs showing immunofluorescence of myelinated axons labeled against Neurofilament 200, micrographs showing immunofluorescence of myelinated axons labeled against Neurofilament 200, Schwanncells Schwann cellslabeled labeledagainst againstS100, S100,nuclei nucleilabeled labeledwith withDAPI, DAPI,macrophages macrophageslabeled labeledwith withIBA1 IBA1and and extracellularmatrix extracellular matrixlabeled labeledwith withlaminin laminininincross crosssections sectionsofofnerve nervegraft graftin incontrol, control,AGAGand andDRA DRA groups;scale groups; scalebar bar200 200µm. (C)Representative µm.(C) Representativetransverse transversesemithin semithinsections sectionsofofthe themid midgraft graftandanddistal distal tothe to thegraft graftinin AG AG andand DRA DRA groups, groups, stained stained with with toluidine toluidine blue. blue. Scale Scalebar bar1010µm. Plotsshow µm. Plots showthe the density densityandandthethenumber numberof ofmyelinated myelinatedaxonsaxonsininthe thesciatic sciaticnerve nerveatatmid midgraft graftand anddistal distaltotothe thegraft. graft. Normal Normalvalues valuesin inour ourlaboratory laboratoryforforthe the sciatic sciatic nerve nerve inin rats rats average average 8076 8076 ±±215 215myelinated myelinatedaxons,axons, with a density of 11,336 ± 698 per with a density of 11,336 ± 698 per mm . mm 2. 2 The histological observations of transverse nerve sections showed the typical ap- pearance of regenerated nerves, with numerous small size myelinated and unmyelinated axons inside the endoneurial compartments of the nerve graft and in the distal nerve. Immunohistochemical labeling showed numerous axons accompanied by Schwann cells Cells 2022, 11, 4074 9 of 19 along the grafts, but at lower density than in control nerves (Figure 3B). Quantitative analysis of the myelinated axons under light microscopy showed similar density in the middle of the graft in the AG group (26,320 ± 5013 axons/mm2 ) compared to the DRA group (27,858 ± 2257 axons/mm2 ), and similar total number of axons (17,395 ± 1525 axons/nerve) in the AG group and DRA group (18,773 ± 3464 axons/nerve). Distally to the graft, all animals from the AG and DRA groups had regenerated myelinated axons, but the density was higher in the AG group (26,105 ± 2991 axons/mm2 ) than in the DRA group (18,992 ± 3358 axons/mm2 ); similarly, the total number of axons distal to the graft was also higher in the AG group (17,224 ± 1329 axons/nerve) compared to the DRA group (11,983 ± 2664 axons/nerve) (Figure 3C), but without significant differences between the two groups. 3.3. Sheep Study All sheep from both groups recovered well from the surgical procedure without any side effect and survived until the end of the study. No significant clinical signs, except for the right hindlimb dysfunction, were observed in the animals during the experimental follow-up. The sheep were able to stand and walk and have good mobility. Due to the peroneal nerve injury, two animals, one of each group, had a marked foot-drop posture that produced pressure ulcers on the dorsal skin of the paw. A splint was placed 30 days post-operation to prevent further foot lesions, and ulcers were treated with chlorhexidine and antibiotic cream and covered. Only one of the sheep, in group DC, did not recover and continued with the foot-drop posture until the end of the study. 3.3.1. Functional Test Results All the sheep showed close to normal locomotion activity after the surgery. In the resting orthostatic position, all the sheep, except one from the AG group, were able to correctly position the right hind hoof, maintaining the plantar support. When tested during fast walking, a foot-drop gait was evidenced in all sheep, as they failed to make plantar stepping in some steps (scored −1 or −2). A significant improvement (p < 0.05) in dorsiflexion was observed at the end of the follow-up in the AG group with respect to values at 30 days (Figure 4). The right TA muscle showed a reduction in size from one month after the surgery. There was a significative improvement appreciated in the last two months of the follow-up in the AG group. The reposition of the injured hindlimb when displaced to the dorsum, indicating proprioceptive inputs, was partially reduced after the surgery and the score significantly recovered to normal value at the end of the follow-up. The withdrawal reflex response was induced in 3 different sites: proximal, mid and distal in the dorsum of the paw. The response was suppressed after the surgery and slowly recovered with time. At the distal site, the test was more sensitive, without full recovery in all the sheep at 9 months post-operation (Figure 4). The AG group showed significant recovery from 4 months post-operation while the DC group had significant improvement from 5 months post-operation with respect to values at 30 days. Overall, the functional tests showed the expected failure due to a peroneal nerve injury, and partial recovery of sensory-mediated functions, whereas motor recovery and muscle mass remained relatively steady until the last month of follow-up. 3.3.2. Electrophysiological Test Results In the left, control hindlimb, the TA CMAP evoked by stimulation at the sciatic notch appeared at an onset latency of 4.2–4.4 msec and had a maximal amplitude of 20–22 mV, with small variations between sheep (Figure 5A). In the right, operated side, at 5 months none of the animals showed evidence of TA muscle reinnervation. Fibrillation potentials, signs of existing muscle denervation, were detected. At 6.5 months, CMAPs of late latency, small amplitude and disperse shape were recorded in 3 sheep of group AG and in 3 sheep of group DC. Fibrillation potentials were also recorded in TA muscles without CMAP response (Figure S3). At 9 months, CMAPs, recorded in 4 sheep of each group, were of Cells 2022, 11, 4074 10 of 19 slightly higher amplitude and still of long latency, compatible with early reinnervation of Cells 2022, 11, x FOR PEER REVIEW the TA muscle. No significant differences were found between groups AG and DC 10 in of any 20 parameter of the nerve conduction tests, although CMAP amplitude was comparatively higher in group AG than in group DC (Table 1). Plotsofofthe Figure4.4.Plots Figure thefunctional functionalevaluation evaluationalongalongthe the9 9months monthsfollow-up follow-upininthe thetwo twogroups groupsofofsheep. sheep. Similarevolution Similar evolutionwas wasobserved observedforforall allthe thetests testsapplied, applied,without withoutsignificant significantdifferences differencesbetween betweenthethe groupsAG groups AGand andDC.DC.Regarding Regarding the the time evolution, AG AG group group(n(n== 5) 5) showed showedaasignificant significantrecovery recoveryin inall allthe thetests applied(#(#pp<< 0.05) testsapplied 0.05) vs. vs. baseline at 30 30 days days post-operation, post-operation,while whileanimals animalsofofDC DCgroup group 22, 11, x FOR PEER REVIEW (n(n==5)5)only onlyshowed showeda asignificant significantrecovery recoveryininthe thewithdrawal withdrawalresponse-distal response-distal($ ($pof 11 p<<0.05) 200.05)vs. vs.baseline baseline atat3030days dayspost-operation. post-operation. 3.3.2. Electrophysiological Test Results In the left, control hindlimb, the TA CMAP evoked by stimulation at the sciatic notch appeared at an onset latency of 4.2–4.4 msec and had a maximal amplitude of 20–22 mV, with small variations between sheep (Figure 5A). In the right, operated side, at 5 months none of the animals showed evidence of TA muscle reinnervation. Fibrillation potentials, signs of existing muscle denervation, were detected. At 6.5 months, CMAPs of late la- tency, small amplitude and disperse shape were recorded in 3 sheep of group AG and in 3 sheep of group DC. Fibrillation potentials were also recorded in TA muscles without CMAP response (Figure S3). At 9 months, CMAPs, recorded in 4 sheep of each group, were of slightly higher amplitude and still of long latency, compatible with early reinner- vation of the TA muscle. No significant differences were found between groups AG and DC in any parameter of the nerve conduction tests, although CMAP amplitude was com- paratively higher in group AG than in group DC (Table 1). Figure 5. Representative Figure 5.EMG recordings of Representative the CMAP EMG recorded recordings of thein CMAP the TA muscle recorded evoked in theby stim- TA muscle evoked by ulation of the sciatic nerve in stimulation of the intact left the sciatic hindlimb nerve (A), and in the intact in the operated left hindlimb (A), andright hindlimb in the operatedof right a hindlimb of sheep at 6.5 (B) and 9 (C)atmonths a sheep after9operation. 6.5 (B) and (C) months Labels: “1” at the onset, after operation. “2”“1” Labels: at at thethe peak, “4”“2” onset, at the at the peak, “4” at end of the CMAP. Scale in A: amplitude 10 mV/square and time 3 ms per square; in B and C: am- the end of the CMAP. Scale in A: amplitude 10 mV/square and time 3 ms per square; in B and C: plitude 500 µV/square and time 5 ms per square. Representative echographic images of the TA mus- amplitude 500 µV/square and time 5 ms per square. Representative echographic images of the TA cle recorded in the intact left hindlimb (D) and in the operated right hindlimb of a sheep repaired with an autograftmuscle (E) orrecorded in the intact left with a decellularized hindlimb nerve (D)(F). allograft and in the operated right hindlimb of a sheep repaired with an autograft (E) or with a decellularized nerve allograft (F). Table 1. Values of the nerve conduction tests in the peroneal nerve and of the echography tests in the peroneal innervated muscles, in the control left limb and in the operated right limb (n = 5 each) at 6.5 and 9 months postoperation. * p < 0.05 vs. AG; ** p < 0.01 vs. AG. Results were analyzed by unpaired t test using GraphPad Prism 8 software. Cells 2022, 11, 4074 11 of 19 Table 1. Values of the nerve conduction tests in the peroneal nerve and of the echography tests in the peroneal innervated muscles, in the control left limb and in the operated right limb (n = 5 each) at 6.5 and 9 months postoperation. * p < 0.05 vs. AG; ** p < 0.01 vs. AG. Results were analyzed by unpaired t test using GraphPad Prism 8 software. CMAP Nerve Group Latency (msec) Amplitude n + Response Conduction. (mV) Control (L) AG 4.38 ± 0.16 21.26 ± 1.54 5/5 Control (L) DC 4.24 ± 0.09 22.84 ± 1.02 5/5 6.5 mo AG 11.05 ± 1.52 0.97 ± 0.48 3/5 6.5 mo DC 16.87 ± 4.54 0.21 ± 0.11 3/5 9 mo AG 11.58 ± 1.06 1.87 ± 0.72 4/5 9 mo DC 13.16 ± 1.50 0.46 ± 0.16 4/5 TA perimeter Echography TA area (cm2 ) (cm) Control (L) AG 5.30 ± 0.29 10.30 ± 0.24 Control (L) DC 5.31 ± 0.48 10.30 ± 0.44 6.5 mo AG 1.90 ± 0.07 7.34 ± 0.23 6.5 mo DC 1.48 ± 0.03 ** 6.87 ± 0.08 9 mo AG 2.44 ± 0.21 7.95 ± 0.17 9 mo DC 1.85 ± 0.09 * 7.45 ± 0.11 3.3.3. Echographic Evaluation of TA Muscles In denervated muscles, the density of the muscle imaging changed, and the size (measured both as area and perimeter) was decreased, evidencing muscle atrophy and increased connective tissue secondary to denervation (see Figure 5D–F). At 6.5 months post-surgery, the TA muscle area in the DC group was significantly lower than in the AG group (** p < 0.01). At the end of the follow-up, there was a tendency to recover muscle size, although it was still significantly lower in the DC group than in the AG group (* p < 0.05) (Table 1). The size of the TA muscles in the two sheep without electrophysiological re- sponses, one from each experimental group, was the smallest (1.85 cm2 in area and 7.41 cm in perimeter in the AG group and 1.56 cm2 and 7.36 cm in the DC group), pointing to a relationship between muscle size and degree of reinnervation. 3.3.4. Histological Assessment Most of the animals from AG and DC groups presented grafted nerves with a well- preserved structure and neuroma visible at proximal and distal ends corresponding with the suture lines (Figure 6A–C). The control peroneal nerve was composed of multiple small fascicles, usually more than 30, each containing numerous nerve fibers densely packed in the endoneurium. In the AG group, the structure of the fascicles was preserved in the graft, but regenerating axons were detected both inside and outside of the perineurium delimiting the fascicles. In contrast, no clear fascicles were observed in the decellularized grafts, where regenerating axons were seen grouped in small regenerative units, disperse within the graft structure (Figure 6E–M). Semithin sections stained with toluidine blue showed, in the AG group, a preserved structure of the fascicles with many myelinated axons of different sizes. In addition, there were small new regenerative units that contained a small number of myelinated axons. In the DC group, the structure of the fascicles was not conserved, although new regenerative units were observed throughout the nerve with a large number of myelinated axons (Figure 6M; marked with red arrows). disperse within the graft structure (Figure 6E–M). Semithin sections stained with toluidine blue showed, in the AG group, a preserved structure of the fascicles with many myelinated axons of different sizes. In addition, there were small new regenerative units that contained a small number of myelinated axons. In the DC group, the structure of the fascicles was not conserved, although new regenerative Cells 2022, 11, 4074 units were observed throughout the nerve with a large number of myelinated axons12(Fig- of 19 ure 6M; marked with red arrows). 6. Macroscopic Figure 6. Figure Macroscopicrepresentative representativepictures of of pictures a control sheep a control peroneal sheep nervenerve peroneal (A), a(A), peroneal nerve a peroneal autograft nerve (B), and autograft a decellularized (B), nerve allograft and a decellularized (C), both nerve allograft with (C), a neuroma both visible (marked with a neuroma with red visible (marked arrows) at proximal and distal sutures, harvested at the end of the study. (D) Schema of the division in sections of nerves harvested. Section 1 includes the proximal suture and Section 3 includes the distal suture of the graft. Sections 2 and 4 were divided into two different segments (dotted line). Micrographs of cross sections of the proximal segment of the graft of sheep stained with hematoxylin and eosin. (E,H) control nerve, (F,I) autograft and (G,J) decellularized nerve graft; E, F, G scale bar 200 µm, and H, I, J scale bar 100 µm. Representative semithin transverse sections of the graft of sheep in AG and DC groups, stained with toluidine blue. (K) control nerve, (L) autograft and (M) decellularized nerve graft, scale bar 10 µm. Red arrows in M point to the newly formed regenerative small fascicles. Distal to the graft, the multifascicular structure of the peroneal nerve was maintained and most regenerating axons grew within the distal nerve fascicles. Immunohistochemical labeling at the middle part of the grafts (noted with dotted line in Figure 6D) from AG animals showed axons and Schwann cells (Figure 7) present within both fascicular and extrafascicular areas, whereas in the decellularized grafts of the DC group, axons and glial cells were dispersed in small regenerative clusters. Regarding the analysis of NF200 labeled axons, group AG had 1 animal with a very low number of regenerating axons while 2 animals had higher than control values. One animal did not show regenerated axons, likely Distal to the graft, the multifascicular structure of the peroneal nerve was maintained and most regenerating axons grew within the distal nerve fascicles. Immunohistochemical labeling at the middle part of the grafts (noted with dotted line in Figure 6D) from AG animals showed axons and Schwann cells (Figure 7) present within both fascicular and extrafascicular areas, whereas in the decellularized grafts of the DC group, axons and glial Cells 2022, 11, 4074 cells were dispersed in small regenerative clusters. Regarding the analysis of NF200 13 of la-19 beled axons, group AG had 1 animal with a very low number of regenerating axons while 2 animals had higher than control values. One animal did not show regenerated axons, likely attributable attributable to suture to suture dehiscence dehiscence earlythe early after after the operation. operation. GroupGroup DChad DC also also had 1 1 animal animal withoutwithout regenerating regenerating axons. axons. Representativemicrographs Figure7.7.Representative Figure micrographsshowing showingimmunofluorescence immunofluorescenceofofmyelinated myelinatedaxons axonslabeled labeled againstNeurofilament against Neurofilament 200 200 in cross sections sections of of the theproximal proximalgraft graftininAGAGandandDC DCgroups. groups.(A,D) (A,D)control con- trol nerve, nerve, (B,E) (B,E) autograft autograft andand (C,F) (C,F) decellularized decellularized nerve nerve graft.A,A,B,B,CCscale graft. scalebar bar200 200µm, µm,andandD, D,E,E,F Fscale scalebar bar100 100µm. µm. Representative Representative micrographs showing showing immunofluorescence immunofluorescencelabeling labelingof ofSchwann Schwann cells cellswith withan anantibody antibodyagainst againstthe theS100 S100protein proteininincross crosssections sectionsofofthe theproximal proximalgraft graftininAG AGand andDCDC groups. (G,J) control nerve, (H,K) autograft and (I,L) decellularized nerve graft. G, H, I scale bar 100 µm, and J, K, L scale bar 100 µm. The estimated mean number of myelinated axons was 21,676 ± 3716 in the control per- oneal nerve, whereas in the mid segment of the grafts (Figure 6 Section 2 dotted line) there were 26,213 ± 2798 axons in the four regenerated sheep of the AG group, and 18,724 ± 3410 in the four sheep of the DC group at 9 months post-surgery. The corresponding values at the nerve distal to the graft were 20,600 ± 6082 for the AG group and 7780 ± 1871 (p < 0.05) for the DC group (Figure 6 Section 4 dotted line). The higher number of regrowing axons in the graft is indicative of some excessive regenerative sprouting that was remodeled at the distal nerve. 3.3.5. Histological Evaluation of Reinnervated Targets In the operated hindlimb, the TA muscle showed a heterogeneous structure in both groups, AG and DC (Figure 8A–F). Some areas appeared normal, although muscle fibers had a smaller diameter than normal, likely corresponding to reinnervated areas of the muscle. Other areas showed signs of atrophy and inflammatory infiltration. The skin modeled at the distal nerve. 3.3.5. Histological Evaluation of Reinnervated Targets In the operated hindlimb, the TA muscle showed a heterogeneous structure in both Cells 2022, 11, 4074 groups, AG and DC (Figure 8A–F). Some areas appeared normal, although muscle fibers 14 of 19 had a smaller diameter than normal, likely corresponding to reinnervated areas of the muscle. Other areas showed signs of atrophy and inflammatory infiltration. The skin sam- ples, processed samples, in sagittal processed sections, in sagittal showed sections, distinct showed organization distinct in three organization layers: in three epider- layers: epi- mis, dermis, dermis and subcutaneous tissue. The skin of the dorsum of the paw had asimilar dermis and subcutaneous tissue. The skin of the dorsum of the paw had a similar aspect aspecttotothe theskin skinfrom fromthe thecontralateral contralateralside, side,without withoutsigns signsofofcell cellinfiltration infiltrationororatrophy atrophy (Figure (Figure8G–L). 8G–L). Figure 8. Representative micrographs of cross sections of the Tibialis Anterior muscle stained with hematoxylin and eosin from (A,D) the contralateral side (control), (B,E) an animal from AG group, (C,F) an animal from DC group. A, B, C scale bar 200 µm, and D, E, F scale bar 100 µm. Representative micrographs of perpendicular sections of the skin of the dorsum of the foot stained with hematoxylin and eosin from (G,J) the contralateral side (control), (H,K) an animal from AG group, (I,L) an animal from DC group. G, H, I scale bar 200 µm, and J, K, L scale bar 100 µm. 4. Discussion The gold standard repair technique for long gap peripheral nerve injuries is grafting an autologous nerve. However, sources for autologous nerve grafts are limited, so it is not possible to use this method to treat extended or multiple nerve lesions [29]. Acellular nerve allografts appear as a good alternative for autografts, considering that they come from a natural and abundant source, are not overtly immunogenic, and may keep a normal-like ECM structure which helps guiding regenerating axons [8,30]. Moreover, an allograft may offer several technical advantages over the autograft, such as off-the-shelf availability, easy handling, shorter surgical time, and no donor graft site morbidity [31]. Nevertheless, acellular allografts also have some limitations, mainly related to the lack of cell support Cells 2022, 11, 4074 15 of 19 for axonal regeneration, since it is important to eliminate cellularity in order to avoid immune rejection. The results of our studies indicate that the used, novel procedure for tissue decellu- larization is highly efficient, eliminating all cells and DNA from the nerve segment, while preserving a normal-like structure of the perineurium and endoneurial tubules. Conse- quently, the decellularized graft did not induce an immune rejection response after in vivo implantation and proved to be a good substrate for axon growth and migration of host cells into the graft, allowing effective regeneration and functional recovery. The results of the two studies made in rats and in sheep showed promising results in terms of functional reinnervation and axonal regeneration, although the outcomes of the DC allograft were comparatively lower than those achieved with an ideal AG (i.e., the same nerve segment resected and re-sutured in place). The DC allograft allowed effective regeneration across a critical 15 mm long nerve gap in the rat sciatic nerve, and a 7 cm long gap in the sheep peroneal nerve, in both cases a length that cannot be effectively bridged with synthetic nerve conduits and represents a challenge for surgical nerve repair. The limiting gap size depends on the nerve and the animal species. Seminal experimental reports showed that regenerating axons are able to bridge synthetic conduits such as silicone, up to 4 mm in the mouse and 10 mm in the rat [15,32], and less than 30 mm in primates [33,34], but failed through longer gaps. 4.1. Nerve Regeneration in the Sheep Peroneal Nerve Model Small animal models are commonly used to replicate challenging clinical scenarios [6,26,35,36]. In contrast to small-animal models, large-animal models allow investigation of longer and more clinically relevant nerve defects and harvesting long segments of donor nerves. This reflects the actual situation in most human nerve injuries in which damage to the nerve often extends over several centimeters [22]. Hence, standardized models of long nerve gap injury in large animals are necessary to evaluate tolerability and efficacy of new treatment strategies for severe nerve injuries [6]. In this sense, sheep are proposed as an optimal large animal model, since they have similarities in general body and neural structures to humans [37,38], and particularly peripheral nerve dimensions and structure similar to humans [20,39–41]. Moreover, their calm nature, compared to other large animals used as experimental models, such as pigs, enables easier postoperative management and clinical testing [18,42]. In the present study, the regenerative potential of a novel decellularized nerve allo- graft was evaluated and compared to autograft repair of a 70 mm long resection of the peroneal nerve in sheep. This nerve is the most commonly affected in injuries involving the lower limb in humans [41,43]. Moreover, it is adequate because it is similar in size and plurifasciular, as is the human peroneal nerve, and induces a limited motor deficit that still allows the animals to stand and walk [20,44]. Since the common peroneal nerve is a mixed nerve, both motor and sensory functional loss in the denervated targets is expected [45]. Therefore, paralysis of dorsiflexion muscles and lack of sensitivity of the dorsum of the foot was observed, although all the sheep showed close to normal locomotion after recovery from the surgery, and most of them were able to position the hind hoof in a normal resting position. Similar to motor function, proprioceptive sensibility of the denervated muscles was also affected, and complete recovery was achieved with time in all the animals. We hypothesize that this recovery could be accounted by the normal processing of information by the intact tibial and sural nerves, also innervating the hind limb, so that the contri- bution of the peroneal nerve is limited. The response observed as a withdrawal flexion of the hind limb at early times, when no axons of the injured peroneal nerve could have regenerated to the ankle level, can be attributed to collateral reinnervation by sprouts from nearby cutaneous nerve tributaries of tibial and sural nerves, a phenomenon that has been well studied in other smaller mammals [46,47] as well as in human subjects [48]. Therefore, results obtained in the functional sensory tests can be confounded by collateral reinnervation and are not completely useful to report nerve regeneration and cutaneous reinnervation. In contrast, electrophysiological assessment of the recovery of the CMAP of Cells 2022, 11, 4074 16 of 19 the TA muscle was the most sensitive test. Although the electrophysiological parameters rarely return to normal levels after nerve injury, they are frequently used to evaluate nerve regeneration and muscle reinnervation in humans and animal models [6,49–51]. Electro- physiological tests evidenced reinnervation of the TA muscle in a 3/5 sheep of both AG and DC groups at 6.5 months, and in 4/5 at 9 months. The time needed for axonal regeneration to distal targets in the sheep hindlimb is considerably long, taking into consideration that the distance between the proximal section of the peroneal nerve and the entrance of the nerve into the TA muscle is about 34 to 36 cm. Since we found early electrophysiological evidence of reinnervation of the TA muscle at 6.5 months, a regeneration rate between 1.7 and 2 mm/day can be estimated. The lower amplitude of the recorded CMAPs in the DG group than in the AG group, may suggest a slightly slower regeneration along the decellularized graft. At the end of follow up, histological assessment of the graft and the distal regenerated nerve was performed. The extent of axonal regeneration in the grafts was evaluated by using neurofilament immunolabeling [22,52]. Schwann cells were observed within the decellularized nerve graft 9 months after the repair, indicating that the host cells migrated from proximal and distal stumps into the graft. Neurofilament labeling was present in most of the grafts, corroborating successful axonal regeneration in these animals and confirming the functional results. However, the incomplete functional recovery observed, as evidenced by the low amplitude of TA CMAPs recorded at 9 months (about 8% and 2% of control values in AG and DC groups, respectively), compared with the extensive amount of regeneration into the grafts suggests that many regenerating axons need more time to reach target organs and therefore, longer follow up periods are needed in this model to assess maximal functional outcomes, as reported also in humans [53]. 4.2. Regenerative Potential of the Acellular Nerve Graft The only available and FDA approved processed nerve allograft to repair human nerve defects of a length up to 5 cm is the marketed allograft Axogen Avance® . This product provides an off-the-shelf alternative to synthetic conduits while maintaining some proregenerative properties of autologous nerve grafts [54], providing overall 82% mean- ingful recovery in repairs of nerve gaps up to 70 mm [55]. No adverse events have been observed, despite some failures reported pointed to a careful consideration of acellular nerve allografts in long, large diameter injured nerves [56]. In this study, we have evaluated a novel decellularized nerve graft that allowed axonal regeneration across not only a 15 mm long gap in rats, but also a 70 mm gap in sheep, offering good perspectives for marketing applications. Electrophysiological and functional test results indicated a similar course, although with some delay, of muscle reinnervation than with the ideal autograft used. However, the organization of the regenerating axons was different in the decellu- larized graft compared to the autograft. In the decellularized grafts, regenerated axons were found in an extrafascicular location, and only in the distal nerve sections the fascicles were consistently well defined, with very few extrafascicular axons visible, as described in previous studies [22,57]. This indicates that the matrix of the decellularized nerve is not so consistent as in a fresh nerve autograft, that has not been previously manipulated, even when the macroscopic observation indicates a well-preserved structure of the connective tissue, ECM molecules and fascicles in the decellularized grafts before implantation. Al- though further optimization of the decellularization protocol can be performed, there is a delicate equilibrium between preserving nerve architecture and completely decellularizing the graft to avoid immune rejection. Finally, the number of regenerated axons distal to the graft was lower in the DC group compared to the AG group, indicative of a slower regeneration along the decellularized graft. Therefore, although the decellularized graft is permissive for regeneration, the lack of a cellular component causes slower regeneration into the graft compared to the ideal cellular autografts. This is expected, mainly due to the important role of Schwann cells in axonal regeneration [8]. Interestingly, this slower regeneration into the graft implies a Cells 2022, 11, 4074 17 of 19 delayed onset but does not have to translate in a significantly poorer functional recovery, according to previous observations [9]. In fact, electrophysiological recovery was quite limited in the two groups of rats followed for 4 months (see Figure 3A), and in the two groups of sheep evaluated up to 9 months after injury (Table 1), comparing to control values obtained in the contralateral hind limb. These results point out the long gap bridged by the graft and the long distance that axons had to grow to reinnervate target organs as determinants for the functional outcome. In this sense, the optimization of acellular grafts and recellularization methods to improve their regenerative capability offers a window of opportunity to improve recovery in this type of injuries, when the gold standard technique has already limited potential. Supplementary Materials: The following supporting information can be downloaded at https://www. mdpi.com/article/10.3390/cells11244074/s1, Figure S1: Flexor withdrawal reflex test; Figure S2: Perfor- mance of the echography of the Tibialis Anterior muscle; Figure S3: Fibrillation potentials. Author Contributions: Conceptualization, R.S. and E.U.; Data curation, E.C.; Funding acquisition, X.N. and R.S.; Investigation, E.C., S.T., S.B., J.F., E.J.-C., F.G., I.D.-M., S.H., R.S., E.U. and X.N.; Methodology, J.F., E.J.-C., F.G., I.D.-M., S.H., R.S. and X.N.; Resources, S.H., R.S. and X.N.; Supervision, E.U. and X.N.; Writing-original draft, E.C.; Writing-review and editing, E.J.-C., E.U. and X.N. All authors have read and agreed to the published version of the manuscript. Funding: This study was funded by Project E10668 eBIONERVE from EUREKA, and partially supported by project SAF2017-84464R from Ministerio de Ciencia, Innovación y Universidades of Spain, CIBERNED (CB06/05/1105), and TERAV RICORS (RD21/0017/008) funds from the Insti- tuto de Salud Carlos III of Spain, co-funded by European Union (NextGenerationEU, Recovery, Transformation and Resilience Plan). Institutional Review Board Statement: The animal study protocol was approved by the Ethics Committees of Universitat Autònoma de Barcelona and Generalitat de Catalunya. Rat study protocol code 10306 and date of approval 07/09/2018 and sheep study protocol code 10314 and date of approval 20/11/2018). Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on reasonable request from the corresponding author. Acknowledgments: The authors thank Jessica Jaramillo and Mónica Espejo for their excellent techni- cal support, and the technical personnel of the SGICE-UAB for their help in managing the sheep. Conflicts of Interest: E.C., S.T., S.B., J.F., E.JC., I.D.M., E.U. and X.N. declare that they have no competing financial interests. R.S. and S.H. are employees of VERIGRAFT AB. References 1. Navarro, X.; Vivó, M.; Valero-Cabré, A. Neural Plasticity after Peripheral Nerve Injury and Regeneration. Prog. Neurobiol. 2007, 82, 163–201. [CrossRef] [PubMed] 2. Noble, J.; Munro, C.A.; Prasad, V.S.; Midha, R. Analysis of upper and lower extremity peripheral nerve injuries in a population of patients with multiple injuries. J. Trauma 1998, 45, 116–122. [CrossRef] [PubMed] 3. Robinson, L.R. Traumatic Injury to peripheral nerves. Muscle Nerve. 2000, 23, 863–873. [CrossRef] 4. Allodi, I.; Guzmán-Lenis, M.S.; Hernàndez, J.; Navarro, X.; Udina, E. 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