Animals
This study’s surgical procedures, protocols, and guidelines for animal care were independently IACUC reviewed and monitored to ensure the ethical treatment of animals. The Test Facility is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, International (AAALAC) and registered with the United States Department of Agriculture (USDA) to conduct research in laboratory animals. The veterinary care of the animals were in accordance with the protocol, Test Facility’s SOPs, and regulations outlined in the applicable sections of the Final Rules of the Animal Welfare Act regulations (9 CFR Parts 1, 2, and 3), the Public Health Service Policy on Humane Care and Use of Laboratory Animals, the Guide for the Care and Use of Laboratory Animals, the U.S. Food and Drug Administration (FDA) Good Laboratory Practice (GLP) regulations, standards, and guidelines (US-FDA 21 CFR Part 58.351 and GFI 197), in accordance with ARRIVE guidelines, and the Biomere, Policy on Humane Care. The protocol and any amendments or procedures which involved the care or use of animals in this study were reviewed and approved by the Test Facility’s Institutional Animal Care and Use Committee (IACUC) before the initiation of such procedures.
All xenogeneic nerve transplants used in this study were sourced from one genetically engineered α-1,3-galactosyltransferase knock-out (GalT-KO), designated pathogen free (DPF) porcine donor [70]. Five male and five female na¨ıve Rhesus Macaques (Macaca mulatta) served as xenogeneic nerve transplant recipients.
Cryopreservation
Samples were packaged in cryovials (Simport, T310-1A, Beloeil, QC). Cryoprotective media (5 mL, CryoStor CS5 media, BioLife Solutions, Bothwell, WA) was added to each vial before it was sealed and cryopreserved using a controlled rate freezer at 1°C per minute. The frozen cryopreserved nerve xenotransplants were stored at -80°C, and the ones stored in media were maintained at 4°C until use (24-48 hours).
Surgical Procedures
The porcine donor was euthanized and prepared for surgery as previously described [70]. To isolate the sciatic nerve prior to harvesting, a linear incision was made midway between the sacrum and the ischium and extended ventrally along the posterior aspect of the femur, longitudinally dissecting the gluteus medius, gluteus maximus, piriformis, and biceps femoris muscles, to the proximal tibiofibular joint (Fig. 1a, b). The sciatic nerve was visualized and harvested by radial transections distal to the nerve origin and proximal to the bifurcation into the tibial and common peroneal nerves. This process was repeated on the bilateral side.
One unmodified sciatic nerve segment was stored in RPMI media and maintained at 4°C until surgical use 48 hours later, which was segmented into five xenogeniec nerve transplants, used as the source of the donor nerve to repair the radial nerve defect in 5 Rhesus Macaque recipients. The bilateral porcine sciatic nerve was cryopreserved per protocol, and stored at -80°C for a period of 7 days, after which it was thawed per protocol [52], and used as the source of the donor nerve to repair the radial nerve defects in the remaining 5 Rhesus Macaque recipients. Cryopreservation of the sciatic nerve was necessary to limit variability of surgical personnel, techniques, and conditions, as the number of surgeries and availability of surgical team required more than one series of surgical operations. Preservation of the nerve material at 4°C for this extended timeframe was deemed likely to impair the quality of the nerve material, whereas cryopreservation is a well characterized process that preserves cell viability, basal laminae, and endoneurial structure [11, 14, 63].
Prior to transplantation, xenogeneic nerves were trimmed to 4 cm to fit the defect size. Large-gap ( 4 cm) peripheral nerve defects were surgically introduced bilaterally in all ten Rhesus Macaque recipients. Recipients, under anesthesia [73], were positioned in lateral recumbency with the shoulder at 90º flexion, full internal rotation, and neutral abduction. The subcutaneous tissue and deep fascia were dissected for anatomical orientation. A 6-8 cm skin incision was made along the posterolateral margin of the proximal arm towards the antecubital fossa. This procedure exposed the long and lateral heads of the triceps, which converge to form the triceps aponeurosis [74]. The intramuscular plane between the long and lateral head of the triceps was developed approximately 2.5 cm proximal to the apex of the aponeurosis where the radial nerve and accompanying vessels were observed against the humerus in the radial groove. The surgical plane was extended proximally and distally to minimize unintended injury. The radial nerve was distally transected approximately 1 cm proximal to the origin of the deep branch. A 4 cm segment was removed to create the defect and saved for reattachment or subsequent analysis.
Nerve transplants were attached proximally and distally with four to eight equidistant 8-0 nylon monofilament sutures at each neurorrhaphy site. The incision was then closed in layers using subcuticular, absorbable sutures. This process was performed bilaterally per each of the ten recipients; both xenogeneic and autologous nerves were transplanted in the same surgical procedure. Limb designation (right/left) for xenogeneic or autologous transplants was randomly assigned and blinded from observers for analysis. The ten recipients were randomly, evenly divided between two surgical series, one week apart. Five fresh xenogeneic transplants were used in the first series, and five thawed, previously frozen viable porcine xenogeneic transplants were used in the second.
Porcine sciatic nerve was selected as the source of the xenogeneic transplant due to its superstructural similarity to human and primate nerve [1]. The radial nerve was selected as the transplantation recipient site because there are minimal neighboring nerves. Those in close proximity may reinnervate downstream muscle fibers and complicate electrophysiology and functional analysis of the extensor digitalis muscles. Transplantation at the radial nerve also allowed for ethical loss of function and clearly articulated return of function in an observable and isolated movement. The maximum practical gap size possible was 4 cm based on the measured lengths of the recipients’ limbs. The mean distance from the recipients’ proximal neurorrhaphy site to the site of innervation of the extensor carpi radialis longus and extensor carpi radialis brevis muscles [35] measured 15.7 ± 0.17 cm. Bilateral, 4 cm complete transections of radial nerves were surgically introduced in a total of ten Rhesus Macaque recipients. Two porcine sciatic nerves were harvested from one donor (Fig. 1a), trimmed into ten 4 cm segments (Fig. 1b) and transplanted into one of the radial nerve gaps in each recipient (Fig. 1a), randomized to the left or right limb. In the contralateral arm, excised Rhesus Macaque radial nerve segments were rotated 180°and reimplanted as a surgical control (Fig. 1a). Surgeries were performed synchronously, and the surgical personnel, sterile field, surgical technique, and uniformity of the transplant procedure were independently assessed for quality control at each step.
Due to the large number of surgeries, five sciatic nerve segments were transplanted fresh one to two days after donor harvesting while the remaining five were cryopreserved and subsequently thawed for transplantation over two days the week following based on Holzer et. al., 2020 [70]. Cryopreservation of xenogeneic skin transplants has been shown to be safe and effective for up to seven years [75], demonstrating no significant or meaningful differences between fresh and frozen transplants.
Upon recovery, nerve tissues were immersed in antibiotic solution for decontamination and underwent longitudinal microbiological testing during processing: if a microbiological culture yielded microbial growth, the tissue was discarded. Before the freezing, tissues were immersed in a storage solution composed of RPMI1640 with L-glutamine (Sigma-Aldrich) containing 5% dimethyl sulfoxide as cryoprotectant (Bio-Life Solutions CRYOSTOR-5). Each tissue was preserved in cryoprotective vial and placed in a liquid nitrogen computer-controlled freezer that facilitates a controlled decrease in temperature (1°C/minute) to –80°C. After the freezing, the tissues were stored at –80°C until being thawed in a 37C water bath prior to use.
Toxicology
Oral administration of tacrolimus, at a dosage of 0.15 mg/kg/day, began 14 days before surgery and was continued until 6-months for subjects in Group 2, and 8-months for Group 1.
Pathology
At the designated necropsy time point, the animals will be sedated with Ketamine (10 - 15 mg/kg, IM). An IV catheter will be placed and euthanasia will be performed by administration of Euthasol (≥ 50 mg/kg or to effect, IV). Explants of the entire autologous or xenogeneic transplant, including proximal and distal nerve, as well as samples of spleen, liver, kidney, lung, and heart were collected, fixed in 10% neutral buffered formalin, and transferred to 70% ethanol after approximately 72 hours. Nerve explants were trimmed longitudinally, routinely processed and embedded in paraffin blocks. Resulting blocks were sectioned and stained with either hematoxylin and eosin (H&E), Luxol Fast Blue (LFB), or immunohistochemically stained for neurofilament H (NF-H). Spleen, liver, kidney and heart were trimmed, processed, embedded in paraffin, sectioned and stained with H&E. All tissues were evaluated in a manner blinded to treatment. Nerve explants were evaluated for morphologic changes and underwent semi-quantitative scoring according to the criteria in Table 1. All measurements of axon diameter were made by the pathologist using an ocular micrometer.
At necropsy samples of the spleen, liver, kidney, and other organs were collected. PERV copy number and expression were analyzed by Q-PCR to assess the presence of PERV DNA and mixed chimerism. Samples analyzed included xenogeneic and autologous nerve tissues harvested at 8- and 12-months postoperative, sera and PBMCs from the nine subjects obtained at various time points over the 12-month study, and spleen, kidney, liver, heart, and lung samples obtained at necropsy.
Biodistribution
To confirm whether porcine or primate tissue was present, qualitative PCR using a primate specific target gene demonstrated the presence of primate cells in both autologous and xenogeneic transplants (Fig. 6a). All samples were positive for either the internal positive control (sera) or the control reference genes indicating the validity of the analysis (data not shown).
Histopathology
Explanted tissues were stained by immunohistochemistry for expression of Neurofilament H to demonstrate axons (Fig. 5a). Luxol Fast Blue staining was used to demonstrate myelination levels of the various regions of the explant (Fig. 5c).
Electrophysiology
Sensory and motor nerve conduction was evaluated for all nine recipients in both arms at baseline and postoperatively at 5-, 8-, and 12-months [76] using Natus UltraPro with Electrodiagnostic software [35]. Motor nerve function was assessed across the length of the radial nerve by eliciting orthodromic compound muscle action potentials from the extensor digitorum communis muscle (EDC) via stimulation at four locations proximal and distal to the transplant site. Motor conduction velocity (NCV), compound muscle action potential (CMAP) amplitude and CMAP duration were calculated for each location following the last supramaximal stimulation and then averaged across the nerve sites. Sensory nerve conduction was determined by eliciting sensory antidromic nerve action potentials (SNAPs) directly from the distal branches of the radial nerve as it passes over the extensor pollicis longus tendon. Approximately ten supramaximal stimuli were averaged for each sensory nerve conduction velocity calculation.
Evaluations [76] and analysis were performed using a Natus Neurology System. All motor and sensory responses were elicited with a pediatric stimulator and subcutaneous needle electrodes were used for recordings. Recording procedures were adapted from routinely used neurological clinical protocols. Motor nerve recordings were performed orthodromically at the extensor digitorum communis (EDC) muscle, with successive orthodromic stimulations in the antecubital fossa, at the spiral groove, across the transplant, and at the axilla between the coracobrachialis and the long head of the biceps. Sensory nerve action potentials were recorded antidromically from the radial sensory branch over the extensor pollicis longus tendon with stimulation over the radial side of the forearm, 5 cm proximal. The motor NCV for each segment was calculated using the differences in onset latency and distance between each two points of stimulation along the radial nerve following supramaximal stimulation. Values were averaged to provide an overall mean of each recipient’s motor NCV per arm. CMAP amplitude was determined at the peak of the response following supramaximal stimulation of the associated nerve and the CMAP duration marked at the repolarization. Segmental conduction velocities across the radial nerve were averaged. Sensory nerve responses from approximately ten supramaximal stimuli were averaged at each time point. The conduction velocity was calculated using the onset latency of the response and the distance (for sensory NCV) or the distance difference (for motor NCV) from the stimulation cathode to the recording site.
Functional Evaluation
A previously reported radial nerve injury mode [77] was adapted to assess the functional recovery of xenogeneic and autologous nerve transplant recipients. Radial nerve injury proximal to the elbow results in a loss of wrist extension function, or “wrist drop,” loss of forearm muscle tonality, and digital extension due to motor denervation of the extensor carpi radialis longus and extensor carpi radialis brevis muscles [78, 79]. Radial nerve functional assessments were performed monthly for each recipient and included chair and cage-side observations of active and passive wrist angle flexion during the recipient’s retrieval of objects requiring wrist angle extension to obtain them. A series of wrist extension and gripping attempts by each recipient were video recorded, for each isolated arm, for each month. Observations were performed using food treats or mechanical stimulation to encourage wrist extension and gripping. This resulted in 16:13:33 hours of data, for 18 limbs of nine Rhesus Macaque transplant recipients. Over the entire study period a combined 2,057 total events were recorded. Results were analyzed by two independent investigators in a blinded manner with respect to the transplant type and location.
Qualitative regain of radial nerve functionality was monitored according to the following categorical scale: no observable impairment (Fig. 1d), mild impairment, moderate impairment, and severe impairment (Fig. 1e).
Immunogenicity
Total serum IgM and IgG (Fig. 4d) were measured using a commercial ELISA and overall median and IQR were found all nine animals included in the study. Binding of xenoreactive antibody to pig cells was measured as previously described [80]. Cryopreserved genetically modified α-1,3-galactosyltransferase knockout (GalT-KO) porcine PBMCs were thawed, and cell concentration was determined using Coulter MD II (Coulter Corporation, Miami, FL). Cells were diluted to a concentration of 1.5 x 106 cells/mL in FACS buffer (1X Hanks’ Balanced Salt Solution (HBSS) with calcium and magnesium, 0.1% BSA, and 0.1% sodium azide). Decomplementation of the serum samples was carried out by heat inactivation for 30 min at 56 ºC and diluted at 1:2, 10, 100, 1,000, and 10,000 ratios using FACS buffer. 100 µL of the cells were added into each well in 96-well u-bottom plate with 10 µL of the diluted serum samples and incubated for 30 min at 4 ºC. Cells were washed one time using 200 µL FACS buffer. To prevent nonspecific binding, cells were incubated in 100 µL 10% goat serum for 10 min at room temperature followed by one more additional washing. Cells were stained with goat anti-human IgG PE and goat anti-human IgM FITC (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) for 30 min at 4 ºC. Cells were washed two times using FACS buffer and resuspended in 200 µL 0.5% PFA in MACS/1X PBS buffer. Flow cytometric analysis completed on Novocyte flow cytometer (ACEA Biosciences, Inc. San Diego, CA). Flow cytometry data was analyzed using NovoExpress 1.3.0 (ACEA Biosciences, Inc.).
Binding of IgM and IgG was assessed using relative mean fluorescence intensity (MFI): Relative Actual MFI value/MFI obtained using secondary antibody in the absence of serum. IgG and IgM ELISA kits from Life Diagnostics were used for the quantification of total circulating IgG and IgM in Rhesus serum by following the manufacturer’s instructions.
Recipients were iteratively assessed for anti-porcine IgM and IgG antibodies to evaluate their response to peripheral blood mononuclear cells (PBMCs) from GalT-KO porcine donors, and for total circulating IgM and IgG levels to assess changes in systemic immunogenicity over the course of the study (Figs. 4c, d). These are measurements of circulating IgM and IgG levels in each recipient, therefore each data point in Figures 5a-d represents one recipient Rhesus Macaque, not the xenogeneic or autologous limb. Importantly, tacrolimus withdrawal at 6-months did not increase Group 2 total or anti-porcine IgM or IgG.
Binding of anti-porcine IgM and IgG was assessed using Median Fluorescence Intensity (MFI) and relative MFI obtained as follows: Relative MFI = Actual MFI value / Limit of Blank (MFI obtained using secondary antibody only in the absence of serum).
PCR
Twenty milligrams of the xenogeneic porcine tissue samples and 7 mg of autologous primate tissue samples were treated with the DNeasy Blood and Tissue Kit (Qiagen, Crawley, UK) as described by the manufacturer that included the RNase A-treatment step. The isolated DNA was quantified by UV spectrophotometry. Quantitative PCR amplification of 18S (Eurogentec, Seraing, Belgium) was carried out to assess the DNA homogeneity across samples. Serum samples were processed using the Viral RNA mini kit (Qiagen, Crawley, UK) as described by the manufacturer incorporating the DNA digestion step using DNase I to isolate viral RNA. Samples were then processed using the RNeasy MinElute Cleanup kit (Qiagen, Crawley, UK). All Serum samples shown to have an IPC CT <32 progressed to PERV transcription analysis. PMBC samples were processed for DNA isolation using a modified version of the manufacturers “Whole Blood” protocol for the Gentra Puregene Blood kit (Qiagen, Crawley, UK). The modified protocol involved homogenizing the PBMC samples prior to RNase A treatment, protein precipitation and finally isopropanol and 70% ethanol washes were added before DNA hydration. The DNA product was quantified using UV spectrophotometry and 18S amplification carried out to assess DNA homogeneity between samples while using 200 ng/reaction. PBMC samples shown to have an 18S CT <27 progressed to PERV copy number and mixed chimerism analysis. At necropsy, tissue samples of Kidney, Liver, Lung and Spleen were harvested. RNA isolation was conducted on 35 mg of the tissue samples using the RNeasy mini kit (Qiagen, Crawley, UK) with homogenization using a Fast-Prep 24 (MP Biomedicals, Eschwege, Germany). The RNA product was quantified using UV spectrophotometry and 18S amplification carried out to assess DNA homogeneity between samples while using 200 ng/reaction. Tissue samples shown to have an 18S CT <13 progressed to amplification, reverse transcription, and PERV copy number and mixed chimerism analysis.
Amplification was carried out using an Applied Biosystems ViiA 7 Real-Time PCR System with a polymerase activation step (10 min at 95 °C) and 40 amplification cycles of 15 sec at 95 °C, 30 sec at 53 °C, and 30 sec at 60 °C. All primate and porcine transplantation site samples shown to have an 18S CT <29 progressed to PERV copy number and mixed chimerism analysis. PERV genome copy number quantification and mixed chimerism was assessed by quantitative PCR (Q-PCR) using the QuantiTect virus kit (Qiagen, Crawley, UK), with identical cycling conditions described above. PERV was assessed using TaqMan primers specific to the PERV-pol gene as previously described [81]. PERV quantification was carried out by comparison to standards of known PERV copy number. The limit of quantification (LOQ) for PERV using this assay is ten copies per reaction. Mixed chimerism was assessed using TaqMan primers for porcine centromeric DNA that were also used in the study described [81]. Detection of porcine cells was quantified by comparison to standards of known porcine cell content. The LOQ for porcine cells using this assay is 0.026 cells per reaction.
Qualitative PCR for the reference gene RPL13A (ribosomal protein gene) was carried out to confirm DNA was suitable for amplification. Primers used were 5′-CCT GGA GGA GAA GAG GAA AGA GA-3′ and 5′-TTG AGG ACC TCT GTG TAT TTG TCA AG-3′ giving an amplicon of 126 bp. Fifty nanograms of DNA and 0.5 µM primers in a total volume of 25 µl was cycled under the following conditions; 5 min at 94 °C, 50x (10 sec at 94 °C; 15 sec at 58 °C; 15 sec at 72 °C) 10 min at 72 °C. This PCR will detect both primate and porcine material.
Detection of primate specific DNA was carried out using qualitative PCR as described previously [82] with modifications. In brief, primers used were P5 5′-ATC TGG ACC AGA AAT CCC GAC GAT ATT ACT AAT GAG GAG-3′ and P6 5′-CTT GTA GTT CTC TTT ATC TTC CGC CAG TTC AGT AAA GAG-3′ giving an amplicon of 450 bp. Using the Taq PCR core kit (Qiagen, Surrey, UK) 75 ng of DNA and 0.2 µM primers in a total volume of 50 µl was cycled under the following conditions; 94 °C for 5 min, 40x(30 sec at 94 °C, 30 sec at 5 °C, and 60 sec at 72 °C), 10 min at 72 °C. PCR analysis was preferred for specificity which is not seen by the use of antibodies to nerve components.
Hematology and Clinical Chemistry
Tacrolimus administration was initiated 5-14 days before surgical procedures via intramuscular injection in the ten Rhesus monkey recipients. Postoperatively, all recipients received tacrolimus for at least six months [76] and trough levels were maintained below 30 ng/mL.
Serum chemistry blood samples were processed for serum or transferred to the Biomere Testing Facility laboratory and processed. Whole blood hematology samples were transferred to the Testing Facility laboratory without processing. Whole blood was analyzed on an IDEXX Procyte analyzer for erythrocyte count, hemoglobin, hematocrit, platelet count, leukocyte count, reticulocyte count, and mean corpuscular volume, hemoglobin and hemoglobin concentration, Serum samples were analyzed using an IDEXX Catalyst analyzer (Chem15, Lyte4, Trig, and AST slides for A/G ratio, alanine aminotransferase, albumin, alkaline phosphatase, aspartate aminotransferase, calcium, chloride, cholesterol, creatinine, gamma glutamyl transferase, globulin (by calculation), glucose, inorganic phosphate, potassium, sodium, total bilirubin, total protein, triglycerides, and urea nitrogen.
Statistical Analysis
Data comparisons between autologous and xenogeneic nerve transplant sites, unless otherwise stated, are expressed as medians with interquartile ranges per transplant type. Statistical comparisons were performed as one-way analysis of variance tests with the Student-Newman-Keuls multiple comparisons method. Immunoglobulin analysis was performed as an unpaired t-test using Prism Graph Pad version 9.1.0 software (Prism, San Diego, CA USA). P values less than 0.05 were considered statistically significant for all analysis.