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. Author manuscript; available in PMC: 2022 Feb 1.
Published in final edited form as: Plast Reconstr Surg. 2021 Sep 1;148(3):561–570. doi: 10.1097/PRS.0000000000008291

Surgical angiogenesis of decellularized nerve allografts improves early functional recovery in a rat sciatic nerve defect model

Tiam M Saffari 1,2, Femke Mathot 1,3, Patricia F Friedrich 1, Allen T Bishop 1, Alexander Y Shin 1
PMCID: PMC8805151  NIHMSID: NIHMS1772176  PMID: 34292916

Abstract

Background:

Surgical angiogenesis applied to nerve grafts has been suggested to enhance nerve regeneration after nerve injury. We hypothesized that surgical angiogenesis to decellularized nerve allografts would improve functional recovery in a rat sciatic nerve defect model.

Methods:

Sixty Lewis rats were divided in three groups of 20 animals each. Unilateral sciatic nerve defects were repaired with (i) autografts, (ii) decellularized allografts and (iii) decellularized allografts wrapped with a superficial inferior epigastric fascial (SIEF) flap to add surgical angiogenesis. Twelve and 16 weeks after surgery, nerve regeneration was assessed using functional, electrophysiological, histological and immunofluorescence analyses. Ultrasonography was used during the survival period to noninvasively evaluate muscle atrophy and reinnervation by measuring cross-sectional muscle area.

Results:

Surgical angiogenesis of allografts demonstrated significantly improved isometric tetanic force recovery at 12 weeks, compared to allograft alone, which normalized between groups at 16 weeks. Cross-sectional muscle areas showed no differences between groups. Electrophysiology showed superiority of autografts at both time points. No differences were found in histological analysis, besides a significantly inferior N-ratio in allografts at 12 weeks. Immunofluorescent expression of CD34, indicating vascularity, was significantly enhanced in the SIEF group compared to allografts at 12 weeks, with highest expression at 16 weeks compared to all groups.

Conclusions:

Surgical angiogenesis with an adipofascial flap to the nerve allograft increases vascularity in the nerve graft, with subsequent improvement of early muscle force recovery, comparable to autografts.

Introduction

Traumatic nerve injuries are a common cause of severe disability and result in loss of sensory- and motor function.1 The gold standard remains end-to-end tension-free neurorrhaphy.2,3 When this is not possible, autograft interposition is needed, requiring harvest of a functional autologous nerve with the associated donor site morbidity.4 Experimental models have investigated allogenic nerves, processed allografts, conduits and vein grafts for the reconstruction of peripheral nerve injuries.5–7 While these substitutes have demonstrated successful recovery, outcomes have yet to exceed the results of autografts. It is widely accepted that functional results are poor when nerve grafts are transplanted into scarred recipient beds,8 and that independent blood supply of nerve grafts could improve outcomes.9,10

Processed nerve allografts are devoid of cellular materials, have no vascular supply and undergo revascularization from the surrounding tissue bed via centripetal revascularization.11 Surgical addition of vascularization (surgical angiogenesis) has the advantage of ensured blood supply, potentially minimizing the period of ischemia and diminishing fibrosis and central necrosis. Immediate revascularization promotes axonal regeneration12 and faster reinnervation, which could reduce denervation-induced muscle atrophy.13 Furthermore, the addition of cellular-based therapy such as adipose derived mesenchymal stem cells (MSCs), could influence vascularization.13 Adipose tissue itself is a rich source of MSCs, with proven beneficial effects on nerve regeneration.13–18 The superficial inferior epigastric fascial (SIEF) flap is an easily harvested flap that provides surgical angiogenesis while improving nutrition and circulating pluripotent cells to the wound site, as well as a layer of adipose tissue to the nerve injury bed, a potential source of stem cells.19 Application of either vascularized nerve grafts (VNG) or vascularized flaps around nerve grafts has been suggested to improve outcomes, however, there are conflicting clinical and basic science reports on its efficacy.15,20–23 In the present study, we investigated the effect of surgical angiogenesis of decellularized nerve allografts on functional motor recovery after segmental nerve repair in rats by comparing allografts placed in a vascularized bed to allografts alone and autografts.

Materials and Methods

Animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC A3348–18). A total of 60 male Lewis rats, weighing 200–300 grams (Envigo, USA), were randomly divided into three groups. All rats underwent excision of a 10-mm portion of the sciatic nerve. In group I (nerve autograft, gold standard), this nerve defect was repaired with an ipsilateral reversed autologous graft. In group II (optimized processed allograft, OPA), the same gap was reconstructed with a 10-mm OPA. In group III, the OPA was wrapped with a SIEF flap19. All groups had survival periods of 12 and 16 weeks (N=10 per time point/group) for evaluation of outcomes. Time points were chosen based on previous research and the neuroregenerative capacity of rats5. Muscle atrophy and reinnervation was noninvasively measured over time using ultrasonography.

Nerve allograft harvest and processing

Twenty male Sprague-Dawley rats, weighing 250–300 grams, (Envigo, USA) served as donors for harvesting 15-mm segments of the sciatic nerve bilaterally. This species of rats were specifically chosen to create a histocompatibility mismatch to the Lewis rats.24,25 The sciatic nerves were cleaned from external debris and processed into acellular allografts (OPA) using a nerve decellularization protocol.26 Nerves were harvested under sterile conditions and underwent a series of washing steps, were sterilized using γ-irradiation and stored at 4°C until use.26

Surgical procedure

Rats were anesthetized in an isoflurane chamber, shaved and positioned in the nose cone to maintain anesthesia throughout the procedure. Preoperatively Enrofloxacin (Infection prophylaxis, Baytril, Germany, 10mg/kg), 5 mL of NaCl 0.9%, and buprenorphine SR (pain control, Buprenorphine SR-LAB, ZooPharm pharmacy, 0.6mg/kg) were injected subcutaneously. Body temperature was maintained at 37°C with a heating pad.

The sciatic nerve was fully exposed proximally from the inferior margin of the piriformis muscle to approximately 5-mm distal to the bifurcation, under an operating microscope (Zeiss OpMi 6, Carl Zeiss Surgica, Oberkochen, Germany). A 10-mm segment of the sciatic nerve was excised by sharp transection with microsurgical scissors. In group I, the nerve segment was reversed and placed as an interposition autograft with six 10–0 nylon (10–0 Ethilon, Ethicon Inc., NJ, USA) epineural interrupted sutures. In group II, the nerve gap was bridged with a 10-mm OPA using a similar surgical technique and in group III this OPA was wrapped with a pedicled SIEF flap as previously described19. In short, the femoral artery was identified in the groin and the superficial inferior epigastric (SIE) vessels were exposed. The SIEF flap was tunneled subcutaneously toward the nerve reconstruction and wrapped around the nerve to surround both nerve anastomoses (see figure, Supplemental Digital Content 1).

Wounds were closed in layers, approximating muscle with two 5–0 absorbable interrupted sutures (5–0 Vicryl Rapide, Ethicon Inc., NJ, USA) and the skin was closed subcutaneously. After surgery, all animals were individually housed with ad libitum access to food and water.

Survival period evaluation

During the survival period, ultrasonography was used to measure cross-sectional scans of the tibial anterior muscle in the 16-week survival group and these were compared to the contralateral side, as previously described27.

Non-survival evaluation

At 12 and 16 weeks, the rats underwent a non-survival surgical procedure. Anesthesia was induced by an intraperitoneal injection of ketamine (Ketaset, 80 mg/kg; Fort Dodge Animal Health, Iowa, USA) and xylazine (10mg/kg) and maintained by ketamine (40mg/kg) throughout the procedure. Rats were kept warm at 37°C on the heating pad during this experiment.

Ankle contracture angle: The ankle contracture angle was determined by measuring the angle between the anterior side of the tibia and the dorsal aspect of the paw with the ankle in maximal passive plantar flexion.5,28

Electrophysiology (compound muscle action potential, CMAP): The main sciatic nerve proximal to the graft was exposed and CMAP was measured using a miniature bipolar electrode connected to VikingQuest portable electromyelogram (Nicolet Biomedical, Madison, WI). A non-recurrent single stimulation was used with duration of 0.02milliseconds at an intensity level of 2.7mA. The maximal amplitude of the depolarization curve was recorded.5

Maximum isometric tetanic force (ITF): Maximum ITF measurements were performed as previously described.29 Quantification of the ITF provides reproducible evaluation of functional recovery.

After rats were euthanized with Pentobarbital Sodium (Fatal Plus, 390 mg/mL, Vortech, MI, USA), tissues were collected. Tibial muscles were carefully dissected and weighed to evaluate muscle mass. Sciatic nerves and peroneal branches were harvested bilaterally and immediately stored in fixative.

Histology: Peroneal nerve samples were processed and subsequently infiltrated in 50%, 75% and finally 100% epoxy resin and polymerized at 65°C for 12–18 hours to allow cutting 1-μm transverse sections. Samples were stained on a warming plate with toluidine blue (Fisher Scientific, Pennsylvania, USA) for 2–2.5 minutes to evaluate histological outcomes. Nerve area, myelin thickness, axon count, total axon area were determined using NIS-Elements software (NIS-Elements BR 4.51.01), and the N-ratio (ratio between the myelinated fiber area and tissue cable area), indicating the number of axonal sprouting and maturation of the regenerating nerve,30–32 was calculated.

Immunofluorescence: Sciatic nerves were embedded in paraffin, sectioned transversely to a thickness of 5-μm and reacted with immunofluorescent markers CD34, staining vascular endothelial cells and extensively expressed on blood vessels,33 and protein gene product 9.5 (PGP 9.5), a pan-neuronal marker, staining both myelinated and unmyelinated nerve fibers,34 to quantify vascularity and axons, respectively. The CD34 primary antibody (1:2000, rabbit monoclonal, Abcam, UK) was diluted in background reducing diluent (Dako, Agilent Technologies Inc., CA, USA) and incubated for 60 minutes. PGP9.5-Alexa 568 conjugate (1:50, rabbit polyclonal, Dako), using the Zenon-Alexa 568-Rabbit IgG kit (Fisher Scientific) was incubated for 60 minutes, prior to staining with the appropriate secondary antibody (Alexa Goat-Anti-Rabbit 488, 1:200, Fisher Scientific). Counterstain was performed using Hoechst 33342 (Fisher Scientific) for 10 minutes, to stain cell nuclei. Slides were cover slipped and images were obtained using confocal microscope (Zeiss LSM 780, Carl Zeiss Surgical GmbH, Oberkochen, Germany). The total nerve area was defined by the combined area of tissues positively stained for any of PGP 9.5, CD34 and Hoechst. Density of axons and vascularity were normalized to the total nerve area and expressed in percentages.

A detailed description of evaluation measurements is provided (see appendix, Supplemental Digital Content 2).

Statistical analysis

Testing was performed on both the experimental left side and on the contralateral right side, and results were expressed as a percentage of the values of the left to the right side (L/R ratio in %), to diminish the effect of normal biologic variability between animals. Two-way analysis of variance (two-way ANOVA) for ultrasonography analysis and one-way ANOVA for all other outcomes, corrected by Bonferroni post-hoc testing was used for statistical investigation. Sample size (N=10) was calculated by our statistician based on power analysis to find significant differences in functional recovery between groups. All results were reported as the mean and standard error of the mean (SEM), with level of significance set at α≤0.05.

Results

Animal weight

Differences in sacrifice weight among groups at 12 weeks (P=0.33) and 16 weeks (P=0.23) were not significant.

SIEF group

All arteries were patent at both sacrifice time points and the flap was actively bleeding during dissection.

Wet muscle mass

At 12 weeks, the autograft muscle mass (66.6 ± 2.1% of the contralateral tibial muscle) was superior in comparison to allografts (57.6 ± 2.5%, P=0.01) and the SIEF group (56.0 ± 1.4%, P<0.01). At 16 weeks, autografts measured 70.8 ± 1.8%, allografts 65.9 ± 3.4% and SIEF 61.8 ± 1.5%. The autograft was only significantly superior to the SIEF group (P<0.05) (see figure, Supplemental Digital Content 3A).

Ankle contracture angle

At 12 weeks, the ankle contracture angle measured 81.6 ± 3.1% in the autograft group, 72.7 ± 1.2% in allografts and 75.8 ± 2.1% in SIEF. Autografts had a significant larger angle compared to allografts (P<0.05). At 16 weeks, all groups normalized to similar results with around 80% recovery (see figure, Supplemental Digital Content 3B).

Electrophysiology

Recovery of the CMAP at 12 weeks showed that autograft (57.2 ± 2.9%) was superior to allograft (35.9 ± 3.6%, P<0.001) and SIEF (46.5 ± 2.6%, P<0.05). This same pattern was seen at 16 weeks; the autograft (66.0 ± 3.3%) was superior to the allograft (45.9 ± 4.8%, P<0.01) and the SIEF (53.1 ± 3.1%, P=0.03, Figure 1A).

Figure 1. Compound muscle action potential (CMAP, A) and isometric tetanic force testing (ITF, B) at 12 and 16 weeks for the autograft, allograft and the allograft wrapped with a superficial inferior epigastric fascial (SIEF) flap.

Figure 1.

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Results are expressed as a percentage of the experimental left side to the unoperated right side (L/R Ratio) and are given as the mean ± SEM. *Indicates significance at P<0.05, ** P<0.01, ***P<0.001). N=10 per group.

Isometric tetanic force

At 12 weeks, the recovery of the tibial muscle ITF measurements was 54.7 ± 4.7% for the autograft, 33.3 ± 2.7% for allografts, and 52.4 ± 4.9% for SIEF (Figure 1B). In both autografts and the SIEF group, ITF recovered significantly better compared to allografts alone (P<0.01 and P=0.01 respectively). SIEF and autograft ITF outcomes were not different (P=0.58). At 16 weeks, no significant differences were found between groups (autograft 77.3 ± 7.1%, allograft 74.0 ± 5.5%, SIEF 70.2 ± 3.5%, P=0.64).

Muscle atrophy over time

A marked loss of tibial muscle cross-sectional area of 60% was found in week four in all groups, followed by an increase up to 16 weeks. At 16 weeks, recovery muscle loss was averaged at 64.8 ± 5.3% for autograft, 66.4 ± 7.3% for allograft and 72.0 ± 4.9% for SIEF (Figure 2). No significant differences were found among groups at the different time points.

Figure 2. Cross-sectional scans of tibial anterior muscle determined by ultrasonography.

Figure 2.

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Nerve defects repaired with autograft, allograft and the allograft wrapped with a superficial inferior epigastric fascial (SIEF) flap are followed up in time until sacrifice. Results are expressed as a percentage of the experimental left side to the unoperated right side (L/R Ratio) and are given as the mean ± SEM. N=10 per group.

Histological outcomes

No significant differences between groups were found with regard to axon area, axon count and myelin area (see figure, Supplemental Digital Content 4A). The nerve surface area was comparable between groups at 12 weeks. At 16 weeks, nerve area was significantly larger in autografts compared to allografts (P<0.01) and SIEF (P<0.001). Also, nerve area in allograft was larger compared to SIEF (P<0.05). At 12 weeks, the N-ratio showed inferiority of the allograft compared to the autograft and the SIEF group (P<0.05). At 16 weeks, no significant differences were found between groups (see figure Supplemental Digital Content 4B). An overview of representative nerve sections of the different groups has been provided (see figure Supplemental Digital Content 4C).

Evidence of nerve tissue and vascularity

Representative mid-distal graft nerve sections of the experimental groups including control are shown in Figure 3A–D. PGP.9.5 expression showed a consistent distribution throughout the nerve tissue in all groups, as seen in the micrographs. Quantification measured a similar expression between groups for both time points (Figure 3E). Vascularity, determined by CD34 staining, showed consistent distribution throughout the nerve tissue in control and autograft (Figure 3A,B). In allografts, CD34 was mostly expressed in the outer surface of the nerve sample and only little present in the core of the nerve. Wrapping with the SIEF flap resulted in an increased expression in the core of the allograft with consistent distribution throughout the nerve tissue (Figure 3C,D). Quantification measured significant inferiority in allograft compared to other groups at 12 weeks (3F, P<0.001). At 16 weeks, vascularity was significantly higher in the SIEF group compared to other groups (P<0.01 compared to control, P<0.05 compared to autograft, P<0.001 compared to allograft only).

Figure 3. Evidence of nerve tissue and vascularity, determined by immunofluorescent staining of protein gene product 9.5 (PGP9.5) and CD34, respectively.

Figure 3.

Figure 3.

Figure 3.

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Micrographs showing density in mid-distal graft sections from control nerve samples (A), autograft (B), allograft (C) and allograft wrapped with a superficial inferior epigastric fascial flap (SIEF, D) at 12 weeks. PGP.9.5 expression, staining both myelinated and unmyelinated nerve fibers showed a consistent distribution throughout the nerve tissue in all groups. Vascularity, determined by CD34 staining, showed consistent distribution throughout the nerve tissue in control and autograft (A,B). In allografts, CD34 was mostly expressed in the outer surface of the nerve sample and only little present in the core of the nerve. Wrapping with the SIEF flap resulted in an increased expression in the core of the allograft with consistent distribution throughout the nerve tissue (C,D). Density of axons and vascularity (normalized to the combined area of tissues positively stained for any of PGP9.5, CD34 and Hoechst) is shown in E and F, respectively, expressed in percentages. Error bars denote mean ± SEM. *Indicates significance at P<0.05, ** P<0.01, *** P<0.001). Scale bars are set at 100 μm.

Discussion

Interest in the role of vascularity in nerve regeneration has been longstanding.35 In the past, experimental animal models have investigated the effects of vascular growth factors such as vascular endothelial growth factor (VEGF),36 or sought to measure the effect of microsurgical repair of nerve graft with vascular pedicles (e.g. VNG) on nerve regeneration.20,22,23,37,38 VEGF contributes to angiogenesis via several mechanisms and has also been suggested to influence nerve regeneration36,39 by stimulating outgrowth of Schwann cells, the original facilitators of nerve regeneration.40,41 These cited studies have reported conflicting results and are difficult to compare. One previous study evaluated functional recovery in rats and found improved sciatic functional index in VNGs at eight and 12 weeks.20 This is in line with our study, which showed increased muscle force when allografts were wrapped within an adipofascial flap when compared to allografts alone at 12 weeks. Because of its reproducibility, ITF has become a widely used standardized technique to assess mechanical function and the contractile properties of muscles.42,43 This study and others have demonstrated that ITF is a reliable technique in determining the degree of functional recovery of a reinnervated muscle as a direct measure of reinnervation.5,44–46

Electrophysiological outcomes trended to be superior in the allograft wrapped within the SIEF, compared to nerve allograft alone. As CMAP is greatly affected by factors such as changes in temperature and electrode location, there is greater variability than ITF data.31 Although others have reported VNGs to have significantly higher nerve conduction velocity than non-VNGs,20 this could not be corroborated in our study.

Denervated tibial muscles were characterized by differences in muscle mass and ultrasound measurements. Serial cross-sectional muscle scans in vivo provided insights into muscle atrophy and reinnervation over time, but was not sufficiently sensitive enough to determine small differences between groups in our rat model. Although our results did not show a significant difference in muscle mass between allograft and SIEF group, wrapping with the SIEF flap provided a significant, large improvement in muscle force at 12 weeks. Muscle mass includes enlarging but non-contractile muscle fibers, resulting in possible indistinguishable differences in innervated and non-innervated muscles.32,47,48 It is easy to interpret, however, doubtful to be useful as an evaluation tool of functional recovery. Muscle force may therefore be a parameter that expresses functional recovery better than muscle mass or cross-sectional area.32

Histological parameters did not result in differences between groups with respect to axon count, axon area and myelin area. A low N-ratio, seen in allografts at 12 weeks, could be indicative of a relatively larger amount of fibrous tissue,49 compared to SIEF and autografts. This indicates that provision of vascularization to nerve allograft wound bed may decrease fibrosis of the nerve. This is clearly visualized with the expression of CD34 that indicated vascularity in allografts predominantly in the outer layer, while the SIEF flap enhanced vascularity with abundant distribution throughout the nerve graft, reaching the core of the nerve. As few experimental studies have evaluated VNGs or addition of adipofascial flaps,15,20 it remains difficult to compare findings. A previous study has found that at three weeks post-operatively, fat graft wrapping around the nerve coaptation increased lymphocyte infiltration rates and macrophage migration; processes that are involved in the early neural regeneration period.15 This previous study did not find improved neovascularization after fat graft wrapping15, possibly due to the chosen end-point and limitations in evaluating vascularity. Despite this study, little has been reported on the effect of adipose tissue placed around nerve reconstructions.

It is assumed that a pedicled adipofascial flap containing adipose tissue and a vascular bundle improves revascularization of the nerve allografts through excreted angiogenic factors and nutrition, provided by the vascular bundle and stem cells in the adipose tissue, while preventing fibrosis and core necrosis.10,50,51 Blood vessels have been found to precede neural regeneration and stimulate injured axons and non-neuronal cells to produce a supportive microenvironment after nerve injury.35,52,53 Moreover, capillary changes as well as Schwann cell migration suggest axon growth,54 explaining improved early functional recovery. Although surgical angiogenesis to the allograft continued increasing vascularity in the graft at 16 weeks, functional outcomes did not improve at this time point. This raises the possibility of a ceiling effect of vascularity on nerve regeneration. Addition of other factors combined with surgical angiogenesis, such as stem cells, may be another potential strategy to further improve regeneration. The importance of stem cells relies on their ability to secrete various growth factors, such as neurotrophic factors to stimulate myelin formation, and its potency is emphasized to be influenced by its microenvironment (i.e. paracrine properties).55 Adipose tissue, rich in growth factors and stem cells, may be combined with stem cells delivered to the nerve allograft to achieve a higher state of therapeutic potential,56,57 potentially resulting in synergistic mechanisms to enhance nerve regeneration.

In this study, surgical angiogenesis to nerve allograft produced statistical improvement in a number of measured variables at 12 weeks. These differences were less apparent at 16 weeks, consistent with the well-known superlative nerve regenerative capacity of the rat. It could be speculated that earlier time points prior to 12 weeks, would be ideal in future studies as interventions that result in improved and more rapid nerve regeneration in the logistical growth phase are potentially clinically relevant. This can be ascertained from the recent study by Tang and colleagues, whose ITF outcomes at 16 weeks are in line with the results of this study for all groups.46 The effect of surgical angiogenesis to nerve allograft could be evaluated at earlier time points or in a larger nerve gap model to overcome these limitations58. Additionally, future research may further investigate the impact of fibrosis in the nerve graft to elucidate the suppression of fibrosis during axonal regeneration or the combined effect of surgical angiogenesis and stem cells to improve regeneration.

Conclusions

Wrapping of processed nerve allografts with a pedicled adipofascial flap to provide surgical angiogenesis increases vascularity of the allograft, as well as improves early muscle force recovery when compared to allografts alone. Although we still do not have a nerve graft substitute that performs as well as autograft nerve, these data support the use of surgical angiogenesis as part of the equation required to improve processed nerve allograft outcomes.

Supplementary Material

SDC figure 3. Supplemental Digital Content 3, See Figure. Wet muscle mass of the tibial anterior muscle (A) and ankle contracture angle (B) at 12 and 16 weeks for the autograft, allograft and the allograft wrapped with a superficial inferior epigastric fascial (SIEF) flap.

Results are expressed as a percentage of the experimental left side to the unoperated right side (L/R Ratio) and are given as the mean ± SEM. *Indicates significance at P<0.05, ** P<0.01). N=10 per group.

Supplemental Content. Supplemental Digital Content 2, See Appendix Methods. A detailed description of evaluation measurements.

Attached is a supplemental description regarding the methodology of the ultrasonography measurements, isometric tetanic force (ITF) measurements, histological and immunofluorescent outcomes.

SDC Figure 1. Supplemental Digital Content 1, See Figure. Schematic drawing of the superficial inferior epigastric fascia (SIEF) flap harvest.

Depicted is the elevation of the flap from distal to proximal (A), providing a 4 × 3 cm adipofascial flap (B) based on the lateral branch of the superficial inferior (SIE) vessels. The SIEF flap was tunneled subcutaneously toward the nerve without vascular twisting of the epigastric trunk (C) and wrapped around the nerve graft reaching both anastomoses (D). The flap edges were trimmed if needed and two 10–0 nylon sutures were placed to secure the position of the SIEF flap (E). (Copyrighted and used with permission of the Mayo Foundation 2019 for Medical Education and Research. All rights reserved.)

SDC figure 4. Supplemental Digital Content 4, See Figure. Histological outcomes at 12 and 16 weeks for the autograft, allograft and the allograft wrapped with a superficial inferior epigastric fascial (SIEF) flap.

Comparisons are made for axon area, axon count, myelin area and nerve area (A). Results are expressed as a percentage of the experimental left side to the unoperated right side (L/R Ratio) and are given as the mean ± SEM. The N-ratio (B) at 12 weeks showed allograft to be inferior to autograft and allograft wrapped with the SIEF flap. At 16 weeks no differences were found between groups. *Indicates significance at P<0.05, ** P<0.01, *** P<0.001). N=10 per group. Micrographs of toluidine blue staining of different groups are shown (C). Black scale bar is set at 100 nm for upper level photographs. The lower level photographs are taken at a higher magnification; this scale is set at 10 nm.

SDC figure 2

Acknowledgments

We would like to thank Jim Postier for the artwork of Supplemental Digital Content 1

“Research reported in this publication was supported by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health under Award Number RO1 NS 102360. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health”

Footnotes

Disclosure: The authors have no financial interests to disclose.

Poster Presentation at the American Society for Surgery of the Hand (ASSH) 2019 in Las Vegas, Nevada

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

SDC figure 3. Supplemental Digital Content 3, See Figure. Wet muscle mass of the tibial anterior muscle (A) and ankle contracture angle (B) at 12 and 16 weeks for the autograft, allograft and the allograft wrapped with a superficial inferior epigastric fascial (SIEF) flap.

Results are expressed as a percentage of the experimental left side to the unoperated right side (L/R Ratio) and are given as the mean ± SEM. *Indicates significance at P<0.05, ** P<0.01). N=10 per group.

NIHMS1772176-supplement-SDC_figure_3.tif (278.1KB, tif)
Supplemental Content. Supplemental Digital Content 2, See Appendix Methods. A detailed description of evaluation measurements.

Attached is a supplemental description regarding the methodology of the ultrasonography measurements, isometric tetanic force (ITF) measurements, histological and immunofluorescent outcomes.

NIHMS1772176-supplement-Supplemental_Content.docx (19.5KB, docx)
SDC Figure 1. Supplemental Digital Content 1, See Figure. Schematic drawing of the superficial inferior epigastric fascia (SIEF) flap harvest.

Depicted is the elevation of the flap from distal to proximal (A), providing a 4 × 3 cm adipofascial flap (B) based on the lateral branch of the superficial inferior (SIE) vessels. The SIEF flap was tunneled subcutaneously toward the nerve without vascular twisting of the epigastric trunk (C) and wrapped around the nerve graft reaching both anastomoses (D). The flap edges were trimmed if needed and two 10–0 nylon sutures were placed to secure the position of the SIEF flap (E). (Copyrighted and used with permission of the Mayo Foundation 2019 for Medical Education and Research. All rights reserved.)

NIHMS1772176-supplement-SDC_Figure_1.tif (1.8MB, tif)
SDC figure 4. Supplemental Digital Content 4, See Figure. Histological outcomes at 12 and 16 weeks for the autograft, allograft and the allograft wrapped with a superficial inferior epigastric fascial (SIEF) flap.

Comparisons are made for axon area, axon count, myelin area and nerve area (A). Results are expressed as a percentage of the experimental left side to the unoperated right side (L/R Ratio) and are given as the mean ± SEM. The N-ratio (B) at 12 weeks showed allograft to be inferior to autograft and allograft wrapped with the SIEF flap. At 16 weeks no differences were found between groups. *Indicates significance at P<0.05, ** P<0.01, *** P<0.001). N=10 per group. Micrographs of toluidine blue staining of different groups are shown (C). Black scale bar is set at 100 nm for upper level photographs. The lower level photographs are taken at a higher magnification; this scale is set at 10 nm.

NIHMS1772176-supplement-SDC_figure_4.tif (9.9MB, tif)
SDC figure 2
NIHMS1772176-supplement-SDC_figure_2.tif (103.5KB, tif)

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