Silk-based nerve guidance conduits with macroscopic holes modulate the vascularization of regenerating rat sciatic nerve

Abstract

Peripheral nerve injuries induce a severe motor and sensory deficit. Since the availability of autologous nerve transplants for nerve repair is very limited, alternative treatment strategies are sought, including the use of tubular nerve guidance conduits (tNGCs). However, the use of tNGCs results in poor functional recovery and central necrosis of the regenerating tissue, which limits their application to short nerve lesion defects (typically shorter than 3 cm). Given the importance of vascularization in nerve regeneration, we hypothesized that enabling the growth of blood vessels from the surrounding tissue into the regenerating nerve within the tNGC would help eliminate necrotic processes and lead to improved regeneration. In this study, we reported the application of macroscopic holes into the tubular walls of silk-based tNGCs and compared the various features of these improved silk⁺ tNGCs with the tubes without holes (silk^– tNGCs) and autologous nerve transplants in an 8-mm sciatic nerve defect in rats. Using a combination of micro-computed tomography and histological analyses, we were able to prove that the use of silk⁺ tNGCs induced the growth of blood vessels from the adjacent tissue to the intraluminal neovascular formation. A significantly higher number of blood vessels in the silk⁺ group was found compared with autologous nerve transplants and silk^–, accompanied by improved axon regeneration at the distal coaptation point compared with the silk^– tNGCs at 7 weeks postoperatively. In the 15-mm (critical size) sciatic nerve defect model, we again observed a distinct ingrowth of blood vessels through the tubular walls of silk⁺ tNGCs, but without improved functional recovery at 12 weeks postoperatively. Our data proves that macroporous tNGCs increase the vascular supply of regenerating nerves and facilitate improved axonal regeneration in a short-defect model but not in a critical-size defect model. This study suggests that further optimization of the macroscopic holes silk⁺ tNGC approach containing macroscopic holes might result in improved grafting technology suitable for future clinical use.

Introduction

Peripheral nerve injuries carry a serious global concern and often have a devastating impact on patients’ quality of life, including chronic pain, persisting loss of sensory or motor function, and inability to work due to poor functional recovery. Direct end-to-end repair is the treatment of choice for small nerve lesions and refers to the coaptation of correctly aligned nerve fascicles of the proximal and distal stump without tension and with a minimal number of sutures (Parker et al., 2021). For more severe nerve lesions that cannot be directly sutured without tension, sensory nerve autografts remain the current gold standard treatment, although their use is associated with several problems, including donor site morbidity or limited availability of donor nerves (Daly et al., 2012; Hercher et al., 2019; Vijayavenkataraman, 2020). Consequently, the use of alternative surgical strategies, including that of tubular nerve guidance conduits (tNGCs), has been intensively investigated. However, their use is currently limited to short-distance nerve lesion defects of only up to 3 cm with mostly unsatisfactory functional recovery as Schwann cells fail to form defect-bridging guidance structures (bands of Büngner) indispensable for regenerating axons (Chen et al., 2019; Stocco et al., 2023).

Considerable research efforts have been made to enhance regeneration through longer defects by optimizing tNGC design, such as the addition of intraluminal guidance structures to support directed Schwann cell and axon migration or improving the porosity of the tubular walls to provide sufficient oxygen and nutrient supply (Kokai et al., 2009; Daly et al., 2012; Nectow et al., 2012; Meena et al., 2021). Additionally, several studies highlighted the importance of neovascularization for peripheral nerve regeneration (Muangsanit et al., 2018; Saffari et al., 2020; Prahm et al., 2021). Weddell (1943) has already reported as early as in 1943 on the improved rate of axonal regeneration in areas of larger blood vessels. Others, like Hobson et al. (1997) reported on a morphological interaction between blood vessels and nerve regeneration, showing that the development of longitudinal blood vessels preceded Schwann cell and axon migration. More recently, Cattin et al. (2015) provided further evidence that newly formed blood vessels guide migrating Schwann cells, being crucial for the nerve regeneration process. These findings suggest that inducing neovascularization inside tNGCs could greatly improve functional recovery of peripheral nerve injury by providing (i) oxygen and nutrients to avoid central necrosis, and (ii) a migratory pathway for regenerating Schwann cells and axons.

In our previous study, we developed a custom-made silk fibroin-based tNGC (silk tNGC), which induced neovascularization not only inside the lumen, but also promoted the formation of a finely branched vascular network in the connective tissue surrounding the conduit (Teuschl et al., 2015). Hence, we hypothesized that vascularization was at least partly induced by the regenerating tissue inside the tNGC, signaling the need for additional nutrient supply. This led us to modify our silk tNGCs by adding macroscopic holes to allow vascular ingrowth. In this study, we aimed to investigate two main hypotheses: (i) the generation of periodically arranged macroscopic holes in the tubular walls provides entrance points for blood vessels, which promotes nerve bridge formation inside the lumen and thereby leads to improved nerve regeneration, and (ii) the modified silk⁺ tNGCs lead to improved functional recovery in a critical-size defect model compared with tubes without holes (silk^– tNGCs).

Methods

Preparation of silk tNGCs

tNGCs were fabricated from raw Bombyx mori silk fibers as described previously by our laboratory (Teuschl et al., 2015). Briefly, silk fibers were braided into a tubular structure and the resulting conduits were degummed by boiling in 0.2 M boric acid in 0.05 M sodium borate buffer (pH 9.0) for 45 minutes twice to remove sericin (Teuschl et al., 2014). Afterwards, silk tNGCs were washed in double-distilled water, pulled onto acrylonitrile butadiene styrene rods of 2 mm diameter, and dipped into a boiling solution of the ternary solvent CaCl₂/H₂O/ethanol at a molar ratio of 1:2:8 for 20 seconds. Subsequently, silk tNGCs were treated in 100% formic acid for 20 seconds followed by methanol for 20 minutes, both at room temperature. These serial steps finally resulted in a homogenous crystalline-like outer silk layer giving the silk tNGC its elastic behavior. Processed conduits were rinsed in double-distilled water and air-dried.

To create holes in the wall of silk tNGCs, pores with a diameter of 200–300 µm were laser-ablated into the conduit walls using a CO₂ laser cutter (Speedy 300, Trotec Laser GmbH, Marchtrenk, Austria) applying the following parameters: 10.6 µm wavelength, 60 W laser power and 355 cm/s laser speed. The laser-ablated holes were carried out in lines with 1.5 mm distance between the holes. A total of four of these lines were laser-cut at 90° intervals with a displacement of 750 µm ensuring that only two holes are present at the same longitudinal position. Final silk +/– tNGCs with an inner diameter of 2 mm and length of either 10 mm or 19 mm (for sub-critical 8-mm and critical-size 15-mm nerve defects, respectively) were packed individually and autoclaved.

Scanning electron microscopy

The produced tNGCs were immersed in 2.5% glutaraldehyde solution at room temperature overnight. Subsequently, samples were dehydrated using an ascending ethanol series, followed by hexamethyldisilazane treatment. tNGCs were left to desiccate in a fume hood and subsequently coated with Pd-Au using a Polaron SC7620 sputter coater (Quorum Technologies Ltd., East Grinstead, UK). The samples were examined in a JEOLJSM-6510 scanning electron microscope (JEOL Ltd., Tokyo, Japan) operating at 3 kV.

Ethics statement

Experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health as well as the Convention for the Protection of Vertebrate Animals used for scientific purposes ETS No. 123 and approved by the Animal Protocol Review Board of the City Government of Vienna on July 23, 2015 (MA 58, GZ: 406382/2015/12).

Animals and surgery

Sixty male healthy, naïve Sprague-Dawley rats (n = 36 for the 8-mm sub-critical, n = 24 for the 15-mm critical size defect model; Charles River, Sulzfeld, Germany, weighing 325–415 g, aged 8–10 weeks, bred in SPF grade environment) were housed in groups of 2–3 animals/cage with access to water and food ad libitum and allowed to acclimatize for 7 days prior to experimental procedures. Animals were housed at approximately 22°C and 55% humidity with a day/night cycle of 12 hours. Analgesic treatment was administered subcutaneously using 0.75 mg/kg bodyweight meloxicam (Metacam®; Boehringer Ingelheim, Ingelheim, Germany, once daily) and 1.25 mg/kg butorphanol (Butomidor®; Richter Pharma AG, Wels, Austria, twice daily) immediately prior to surgical procedure and for 2 days thereafter.

8-mm sub-critical size sciatic nerve defect

Thirty-six rats were randomly assigned to three treatment groups (n = 12 in each group, of which n = 8 were used for subsequent histological analysis, and n = 4 for barium sulfate perfusion and Lugol submersion for contrast-enhanced micro-computed tomography [microCT]): autologous nerve transplant (ANT), silk tNGCs without holes (silk^–) and silk tNGCs with holes (silk⁺). Animals were anesthetized by inhalation of isoflurane (Forane®, Abbott, Chicago, IL, USA), and the surgery was carried out under an operating microscope (Leica M651; Leica Microsystems, Vienna, Austria). Induction of anesthesia was performed using 4% isoflurane, continuation under approximately 1.5%–2.5% isoflurane. An 8-mm long segment was excised from the right sciatic nerve, and the proximal and distal nerve stumps were either coapted with the reversed 8-mm sciatic nerve segment (ANT group) or inserted 1 mm deep into 10-mm silk tNGCs, resulting in an 8-mm defect between nerve stumps, and coapted with 10/0 Ethilon® to the conduit by two epineurial sutures. Afterwards, the wound was closed in anatomical layers with intracutane stitches using 4/0 vicryl suture material.

Critical-size (15 mm) sciatic nerve defect

Twenty-four rats were randomly assigned to the ANT, silk^– or silk⁺ treatment groups (n = 8 per group, for contrast-enhanced microCT and subsequent compatible histology). Surgical procedures were performed as described above, except for the length of the tNGC (19 mm), 2 mm insertion of the nerve stumps on either side and resulting defect length (15 mm) (Figure 1). The postoperative observation period lasted 12 weeks.

Figure 1.

Overview of different nerve grafts used.

(A) Based on the observation of finely branched blood vessels (arrows) within a thin layer of connective tissue adjacent to the silk-based tNGC in a previous study (Teuschl et al., 2015), we developed the idea of (B) a silk⁺ tNGC enabling the migration of blood vessels through the tubular wall to the regenerating nerve. (C) Scanning electron micrograph of a line of the laser cut holes in a silk⁺ tNGC. Scale bar: 500 µm. (D–F) Microscopic photographs of an ANT, a silk^– tNGC and a silk⁺ tNGC, respectively, immediately after coaptation. ANT: Autologous nerve transplants; silk^– tNGC: silk-based tNGC without holes; silk⁺ tNGC: silk-based tNGC with macroscopic holes; tNGC: tubular nerve guidance conduit.

Tissue preparation for immunofluorescence stainings

At the end of the observation periods, animals were deeply anesthetized with an intracardial injection of sodium thiopental (Medicamentum pharma, Allerheiligen, Austria) overdose (0.3 g/kg) and sacrificed. Animals were then perfused transcardially with a 4% buffered formaldehyde solution (pH = 7.4 in 0.01M PBS), and ANTs or tNGCs were harvested with the maximal possible length of proximal and distal nerve stumps.

For neurofilament (NF) and Griffonia Simplicifolia lectin B4 (GSA-B4) staining of the 8-mm defect model, dissected nerve grafts and conduits (ANT: n = 7, silk⁺ and silk^–: n = 8 each) were immersion-fixed in 4% buffered formaldehyde for 24 hours, rinsed in distilled water and cryoprotected in 30% sucrose in phosphate-buffered saline (PBS). Nerve samples were cut into 20-µm thick longitudinal sections using a cryostat (Leica 1850, Leica Microsystems) and mounted on gelatine-coated glass slides. After blocking in 5% fat-free milk for 1 hours, sections were incubated in anti-NF heavy polypeptide rabbit anti-rat antibody (1:400, Abcam, Cambridge, UK, Cat# ab8135, RRID: AB_306298) or in biotinylated GSA-B4 (1:200, Vector Laboratories, Burlingame, CA, USA, Cat# B-1205-.5) at 4°C overnight. After washing with PBS, sections were treated with a goat anti-rabbit secondary antibody conjugated with AlexaFluor 488 (1:400, Molecular Probes, Cat# A-11008, RRID: AB_143165) or with streptavidin AlexaFluor 594 (1:400, Thermo Fisher Scientific, Waltham, MA, USA, Cat# S-32356) at room temperature for 1.5 hours to visualize NF or GSA-B4, respectively. The sections were coverslipped with PBS-glycerine for fluorescence microscopic evaluation using a BX-41 epifluorescent microscope (Olympus, Tokyo, Japan).

Tissue processing for microCT and immunohistochemistry

Since contrast-enhanced microCT analysis is not compatible with immunofluorescence stainings, nerve grafts that were analyzed by microCT were subsequently processed for immunohistochemical stainings.

MicroCT analysis

MicroCT analysis was performed as described earlier (Heimelet al., 2019). Briefly, rats underwent terminal anesthesia (1 hour prior terminal anesthesia: 0.75 mg/kg body weight meloxicam (Metacam®; Boehringer Ingelheim, Ingelheim, Germany); intraperitoneal application of 110 mg/kg ketamin (Richter Pharma AG, Wels, Austria) + 12 mg/kg xylazine (Bayer AG, Leverkusen, Germany), and subsequent opening of the auriculum dextrum in deep anesthesia) and were perfused with a barium sulfate contrast agent (Guerbet GmbH, Villepinte, France) to enable visualization of blood vessels within the silk tNGCs (8-mm defect only). Subsequently, whole sciatic nerves including the tNGC were dissected and fixed in formalin for 24 hours.

The nerve grafts were either stained in 50 mL of 0.33% (w/v) Lugol’s Iodine at 4°C for 24–48 hours (8-mm model, n = 4 per group), or in 15 mL Accupaque-350 (GE Healthcare, Munich, Germany, diluted 1:2 in PBS) for 24 hours (15-mm model, n = 8 per group). MicroCT scanning was performed prior to and after the staining using a SCANCO µCT50 (SCANCO Medical AG, Brüttisellen, Switzerland). Samples were placed in 1.5 mL tubes and scanned with 70 kVp (8-mm model) or 90 kVp (15-mm model), 200 µA filtered with 0.5 mm Aluminium (Al). For scans of the 8-mm defect model, 1000 projections/180° were integrated for 500 ms and reconstructed to an isotropic resolution of 7.4 µm, while for scans of the 15-mm model, 512 projections/180° were integrated for 300 ms and reconstructed to anisotropic resolution of 10 µm.

Image processing

Reconstructed images were exported as DICOM slices and evaluated using Fiji, an open-source platform for biological image analysis (Schindelin et al., 2012; Fiji.sc). The images were rotated to align the long axis of the tNGC with the Z axis of the scan to correct for slight twists and bends of the specimens through the use of TransformJ (Meijering et al., 2001). To create a longitudinal view of the conduits, a slice was centered through the middle of the conduit, and re-slicing was performed in 5° increments with a depth of 10–100 slices. Slices were converted to a 2D image by either maximum, minimum, or average intensity projection. Individual projections were combined into a stack, where each slice was the radially re-sliced projection around 360° of the tNGC. Maximum, minimum, and average intensity projections were cross-referenced with the aligned stack to identify vessels and axonal regeneration relative to the conduit.

Axons of Lugol’s iodine-stained samples appeared brighter than the surrounding tissue and were best visible in average and maximum intensity projections, whereas axons of Accupaque-stained samples were darker and best visible in minimum and average intensity projections. To find the regeneration front, the aligned stacks were filtered with a 3D Gauss filter (0.5 × 0.5 × 10 voxel for Lugol; 1 × 1 × 10 voxel for Accupaque samples) and starting at the slice position of the stump of the original nerve, following the region of the regenerating axons deeper into the conduit until they were either no longer visible or they grew across the conduit and formed a full-length bridge. With barium sulfate perfusion (8-mm defect model), the vessels appeared bright and were most visible in maximum intensity projections. As a substantial number of vessels was not successfully perfused in the 8-mm defect model, maximum and minimum intensity projections had to be superimposed to visualize both the bright, perfused and dark, empty vessels in the same image. To achieve this, a maximum intensity projection was windowed to only show contrast agent perfused blood vessels, colorized red and multiplicatively blended with the minimum intensity projection. In case of the non-perfused 15-mm defect model we applied maximum- (for blood-filled vessels with a strong contrast) and minimum-intensity projections (for empty vessels with weak contrast).

Immunohistochemistry

To validate microCT imaging with histological data, we also performed Martius-Scarlet-Blue (MSB) staining of the 8-mm defect model (n = 4). In short, after contrast-enhanced microCT imaging (Lugol’s iodine staining), the nerve segments were transected at the mid-level of the graft/conduit. Samples were fixed in 4% buffered formaldehyde at room temperature for 24 hours and afterwards rinsed in tap water for 1 hour. After dehydration of nerve tissue in 50% ethanol followed by 70% ethanol for 1 hour each, samples were further dehydrated in a vacuum infiltration processor (Sakura Tissue-Tek VIP, Sakura Finetek Germany GmbH, Umkirch, Germany) and subsequently infiltrated with paraffin via the intermedium of xylene. Subsequently, the proximal half was cut into 4-µm thin longitudinal sections, whereas the distal part of the nerve samples was cut into 4-µm thin cross-sections using a Microm HM355S microtome (Thermo Fisher Scientific) and allowed to dry overnight at 37°C. After deparaffinization and rehydration, sections were stained with MSB. To ensure compatibility with contrast-enhanced microCT and automated quantification of axonal regrowth through the 15-mm nerve defect with the IKOSA platform (KML Vision, Graz, Austria), we also opted for immunohistochemical processing of the 15-mm defect model (n = 6). Briefly, following contrast-enhanced microCT, the dissected nerves were fixed and processed as for MSB staining except that no longitudinal sectioning was performed. Instead, 4-µm-thin cross-sections were made proximal and distal of the graft/tNGC as well as at the mid-section of the graft/tNGC. After deparaffinization and rehydration, sections were processed for NF staining. Sections were steamed in sodium citrate buffer (pH 6.0) for 20 minutes, blocked with Bloxall (VectorLaboratories Inc., Burlingame, CA, USA) and afterwards incubated with primary mouse NF antibody (1:100; RRID: AB_2314899 (Dako (Clone2F11), Santa Clara, CA, USA)) overnight at 4°C. Incubation with goat anti-mouse HRP-conjugated secondary antibodies (ImmunoLogic, Arnhem, The Netherlands, DPVR-HRP) was performed for 30 minutes at room temperature, followed by incubation in peroxidase substrate ImmPACT NovaRED (VectorLaboratories Inc.). Nuclei were counterstained using Weigert’s Iron Hematoxylin, and sections were dehydrated and embedded with Shandon Consul-Mount (Thermo Fisher Scientific). Sections were scanned using a Vectra Polaris Automated Quantitative Pathology Imaging System (NF-stained 15-mm defect) (Akoya Biosciences, Marlborough, MA, USA) or a TissueFAXS tissue cytometer (MSB stained 8-mm defect) (TissueFAXS version 7.1.6245.112, TissueGnostics GmbH, Vienna, Austria).

Automated quantification of axons and blood vessels

We employed automated deep learning-based image analysis to quantify the axon counts in whole-slide scans of histological cross-sections (n = 6 per group). A previously established and published deep learning network “Axon Quantification 2.1.0” on the IKOSA platform (KML Vision, Heinzel et al., 2022) was applied to our data set. The validation performance at the axon instance count was 96.3% recall and 94.3% precision. Blood vessel counts of Luxol Fast Blue-stained cross-sections (n = 4 per group) are given as manual counts.

Functional analysis

Muscle electrophysiology

Muscle electrophysiology examination was conducted using a Neuromax EMG device (Natus, Middleton, WI, USA) prior to animal sacrifice (n = 8 for ANT, n = 7 for silk tNGCs; Hercher et al., 2019). Sciatic nerves were carefully set free from the surrounding tissue, the recording electrode was inserted into the tibialis anterior muscle, the bipolar stimulation electrode was placed proximal to the repaired nerve segment using a micromanipulator, whereas the grounding electrode was placed in the surrounding tissue. The contralateral healthy sciatic nerve and tibialis anterior muscle served as internal controls. The core temperature of the animal was measured rectally and used for normalization. The following parameters were assessed after electrical stimulation of the proximal part of the graft: compound muscle action potential and amplitude during supramaximal stimulation.

Automated gait analysis – CatWalk analysis

To analyze postoperative gait changes, an automated gait analysis was performed once a week until the end of the postoperative observation periods (7 and 12 weeks, respectively) by using the CatWalk XT system (Noldus Information Technology, Wageningen, The Netherlands) as previously described (Heinzel et al., 2020). Briefly, rats were moving along a glass plate serving as a walkway. Images were acquired by a camera installed under the plate and processed by the CatWalk XT system software (version 10.6). To ensure sufficient habituation of rats to the system, animals were trained daily for a 7-day long preoperative training period prior to surgery. The following parameters were evaluated: print length, print width and print area, swing time, swing speed, and duty cycle. To account for weight gain over the observation period, gait parameters were calculated as ratios of injured (right) hind limb to contralateral healthy (left) hindlimb.

Static Sciatic Index

Derived from the sciatic functional index measured by CatWalk evaluation, the Static Sciatic Index (SSI) was developed, which measures static paw functional parameters and allows a more reliable statement regarding the functional regeneration than the sciatic functional index (Bozkurt et al., 2008, 2011). Functional recovery in rats with the 15-mm critical-size sciatic nerve defect was evaluated using the SSI by placing the rats in a plexiglass container and static images of the rats were taken from below using a webcam and a customized Fiji plugin. Between 3–5 series of 10 pictures each were taken from each animal at each timepoint, and SSI is calculated after manual measurement of toe spread as well as intermediate toe spread of both hind paws (Bozkurt et al., 2008).

Wet muscle weight

Immediately after sacrificing the rats, the tibialis anterior muscle (8- and 15-mm defect) and the extensor digitorum muscle (15-mm defect) from the injured and intact sides were dissected and immediately weighed. To determine the wet muscle weight following regeneration, the weight of the ipsilateral muscle was normalized to the contralateral one.

Statistical analysis

All statistical analyses were performed using GraphPad Prism (Version 9.4.0, GraphPad Software, Boston, MA, USA, www.graphpad.com). For each parameter, normal distribution was tested using the Anderson-Darling test and the Shapiro-Wilk test. Normally distributed data were compared with parametric tests, i.e. one-way analysis of variance, two-way analysis of variance, or mixed-effects model (REML) (in case of missing values) followed by Tukey’s post hoc test as indicated in the respective figure legends. If data were not normally distributed (only in the case of tibialis anterior muscle weight), the Kruskal–Wallis test followed by Dunn’s multiple comparison test was used.

Results

Macroscopic holes promote axon regeneration in a sub-critical sciatic nerve defect

To investigate whether the generation of periodically arranged macroscopic holes in the tubular walls of silk tNGCs enables vascular ingrowth from the surrounding tissue into the lumen, thereby leading to improved nerve regeneration, we first reconstructed an 8-mm sub-critical sciatic nerve defect with either an ANT (Figure 1D), a silk^– tNGC (silk^–; Figure 1E), or with a silk⁺ tNGC (silk⁺; Figure 1F). We compared axon regeneration as well as blood vessel formation 7 weeks after grafting. Regenerated axons at the distal site of the conduit were present in all animals treated with ANT as well as silk⁺ tNGC, but only in half of the animals with silk^– tNGC (Figure 2A). Immunohistochemical evaluation of longitudinal sections of the whole regenerated nerve segments revealed dense axon bundles spanning the entire nerve segments in the ANT and silk⁺ groups, which were superior to the silk^– groups (Figure 2B). Additionally, although vascularization was observed in all groups, blood vessels appeared to grow from and/or to the conduit walls of silk⁺ grafts (Figure 2B).

Figure 2.

Axon regeneration and vascularization 7 weeks after surgery using ANT, silk⁺ tNGCs or silk^– tNGCs to treat an 8-mm sciatic nerve defect.

(A) Axon regeneration at the distal coaptation site was observed in all animals treated with ANT and silk⁺, but only in 4 out of 8 animals treated with silk^– tNGCs. (B) Regenerating axons (green) were observed across the whole defect size in the ANT and silk⁺ groups, while axon regeneration in silk^– tNGCs was less consistent and absent in the distal part of the graft in 4 out of 8 animals. Vascular ingrowth (red) was observed in all treatment groups. Immunostainings for NF to visualize axons and Griffonia Simplicifolia lectin B4 (GSA-B4) to stain blood vessels are shown for two representative animals per treatment group. Scale bars: 1 mm. ANT: Autologous nerve transplants; NF: neurofilament; silk^– tNGC: silk-based tubular nerve guidance conduit without holes; silk⁺ tNGC: silk-based tubular nerve guidance conduit with macroscopic holes.

Blood vessel migration through macroscopic holes leads to enhanced intraluminal vascularization in a sub-critical as well as critical sciatic nerve defect

Since we could observe at the time of excision of the constructs that blood vessels grew through the holes in the conduit wall (Figure 3A), we also aimed to visualize the blood vessels inside silk tNGCs. Therefore, we perfused the conduits with Lugol’s iodine and Micropaque and performed a microCT scan 7 weeks postoperatively. Blood vessels from the surrounding tissue were seen to have migrated through the tubular wall and anastomosed with blood vessels present in the lumen of the silk⁺ tNGC (Figure 3B–E). Furthermore, to assess whether the silk⁺ tNGCs promote vascularization of the regenerating nerve tissue, we quantified the number of vessels present in ANTs, silk^– and silk⁺ tNGCs (Figure 3F and G). Vessel quantification revealed a significantly higher number of blood vessels in the silk⁺ group compared with ANTs (P = 0.0057) and unmodified silk^– tNGCs (P = 0.0292; Figure 3G).

Figure 3.

Vascularization using silk⁺ tNGCs.

(A) Photograph of the silk⁺ tNGC of the 8-mm defect group perfused with barium sulfate contrast agent at 7 weeks postoperatively. The black arrows indicate blood vessels that migrated through the holes in the tubular wall. (B) MicroCT-scan of a silk⁺ tNGC, previously perfused with barium sulfate and Lugol´s iodine. Blood vessels originating from the surrounding tissue in close proximity to or in the tubular wall as well as luminal vessels are visualized in orange and grey, respectively. Top and side views are shown. Orange and grey structures are connected, indicating anastomosis of luminal blood vessels and vessels from surrounding tissue. Scale bar: 1.0 mm. An animated video of this 3D microCT reconstruction can be found as Additional Video 1. (C, D) Top view of a segment of the blood vessel system along and inside the silk⁺ tNGC identified with barium sulfate (C) and Lugol´s iodine staining (D). The yellow arrows indicate blood vessels migrating through the tubular wall. Scale bars: 1.0 mm. (E) 3D microCT reconstruction of vessel ingrowth. Pink: Vessels growing through holes of the silk⁺ tNGC. Yellow: Anastomosis with vessels in the lumen of the tNGC. Yellow arrow indicates vessel growing through a macroscopic hole. (F) Example of a histological cross section of a silk⁺ tNGC with blood vessels artificially colored in red, that were used to evaluate vessel number in the different treatment groups. Scale bar: 500 µm. (G) Quantification of blood vessels in the different treatment groups at 7 weeks postoperatively. n = 4, one-way analysis of variance with Tukey’s multiple comparison test. ANT: Autologous nerve transplants; microCT: micro-computed tomography; silk^– tNGC: silk-based tubular nerve guidance conduit without holes; silk⁺ tNGC: silk-based tubular nerve guidance conduit with macroscopic holes.

For a more detailed analysis of these newly formed blood vessels, we compared microCT scans of Lugol’s iodine–stained whole regenerated nerve fragments inside the silk tNGCs with histological stainings (Figure 4). Blood vessels grew from the nerve stumps into the center of the lumen of the silk⁺ as well as unmodified silk^– tNGCs (Figure 4A–D). Vascularization seemed to be enhanced within the silk⁺ tNGCs with blood vessels growing through the holes, laser-ablated into the tubular walls (Figure 4H). In contrast to our initial hypothesis, blood vessels did not seem to grow from the surrounding tissue into the tNGCs, but rather grew from the central blood vessel inside the lumen to the outside (Figure 4C and D). Corresponding blood vessels in microCT slices and histological longitudinal and cross-sections are highlighted by color-coded circles (Figure 4E–H).

Figure 4.

microCT scans of Lugol’s iodine-stained silk–based tNGCs in comparison with histology.

(A–D) Empty vessels are visualized by minimum intensity projection through 100 slices, while barium sulfate-filled vessels are visualized by maximum intensity projection and the addition of red coloration. Scale bar: 1 mm. (D) Vessels intersected by histology are highlighted in color. The yellow dashed line indicates the cutting plane for the histological cross section in F. (E) Longitudinal histological section stained with Martius Scarlet Blue (MSB) (E2) and corresponding slice in microCT (E1). A barium sulfate-filled blood vessel is highlighted in yellow and an empty vessel crosses through a hole in the silk tNGC in red. (F) Cross-sectional histology (F2) and corresponding microCT slice (F1) of vessels highlighted in D are circled with color coding. The dashed green line indicates the cutting plane for longitudinal histology shown in E. Scale bar: 1 mm. (G) Magnified highlight from the longitudinal section. The dotted green line demarcates the axonal regeneration front. The dotted orange line surrounds a piece of silk and the surrounding multicellular giant cells. The yellow arrows show barium sulfate-filled blood vessels. Scale bar: 0.25 mm. Red arrow indicates corresponding vessel that is highlighted with a red circle in E1/E2. G1 and H1 are histological magnified highlights, G2 and H2 are corresponding magnified highlights of the respective microCT scans. (H) Magnified highlight from longitudinal section showing blood vessels crossing through the hole in the silk conduit. Scale bar: 0.25 mm. ANT: Autologous nerve transplants; microCT: micro-computed tomography; silk^– tNGC: silk-based tNGC without holes; silk⁺ tNGC: silk-based tNGC with macroscopic holes; tNGCs: tubular nerve guidance conduits.

Based on the promising results observed in the 8-mm subcritical-size sciatic nerve defect in terms of superior axon regeneration and enriched anastomotic network between outside and inside vessels in the silk⁺ tNGCs, we aimed to assess the regeneration-promoting capacity of these conduits in 15-mm critical-size nerve lesions. To account for the longer defect size, we also prolonged the observation period to 12 weeks. To analyze the vascular supply inside silk^– and silk⁺ tNGCs, we studied the regenerated nerves using microCT analysis. Analogous with the short defect model, blood vessels grew from the nerve stumps through the center of both types of silk tNGCs (Figure 5). While very fine vascular branches sprouted towards the luminal wall of unmodified silk^– tNGCs (Figure 5B), growth of blood vessels of various diameters was evident through the laser-edged holes in the tubular wall (Figure 5C and D). We observed ingrowth of larger diameter vessels in the proximal segment but not the distal segment of the silk⁺ tNGCs, where small diameter vessels seemed to grow out of the lumen of the silk⁺ tNGC in the more distal segment, connecting it with the surrounding blood supply.

Figure 5.

MicroCT scans of Accupaque-stained nerve regenerates inside silk-based tNGCs grafted into a 15-mm sciatic nerve defect at 12 weeks postoperatively.

Blood vasculature growing from the nerve stumps into the center of silk^– tNGCs (A, B) and silk⁺ tNGCs (C, D). White asterisks indicate finely branched blood vessels from the central main blood vessel to the tubular walls. The yellow arrows indicate the migration of blood vessels through the conduit wall. Scale bar: 2.5 mm. ANT: Autologous nerve transplants; microCT: micro-computed tomography; silk^– tNGC: silk-based tNGC without holes; silk⁺ tNGC: silk-based tNGC with macroscopic holes; tNGCs: tubular nerve guidance conduits.

Macroscopic holes do not improve axon regeneration in a critical-size sciatic nerve defect

To assess whether the improved vascular supply inside silk⁺ tNGCs, which connected the regenerating tissue with the surrounding tissue, promoted axon regeneration, we quantified axonal outgrowth from the proximal to the distal nerve stumps at 12 weeks postoperatively using neurofilament histological staining. At the most proximal position of the graft, quantified axon numbers were similar in the ANT, silk⁺ and silk^– groups (Figure 6A). However, in the middle and distal positions of the graft, axon counts were significantly lower for both, the silk⁺ and the silk^– groups compared with the ANT groups (mid graft: P < 0.0001, distal: P = 0.0069 for silk⁺ and P = 0.0095 for silk^–; Figure 6B). Surprisingly, no difference in axon numbers was observed between the silk^– and silk⁺ groups, indicating that in this case, blood vessel migration did not lead to improved axon regeneration in the chosen defect model.

Figure 6.

Axon quantification of neurofilament-stained histological sections at different graft positions.

(A) Quantification of axon numbers at proximal, start graft, mid graft, and distal positions. The dotted line represents average axon numbers in healthy sciatic nerves (n = 7). (B) Axon counts were significantly higher at mid-graft and distal positions in the ANT group, but similar in silk⁺ and silk^– tNGCs. n = 6, one-way analysis of variance with Tukey’s multiple comparison test. ANT: Autologous nerve transplants; ns: not significant; silk^– tNGC: silk-based tNGC without holes; silk⁺ tNGC: silk-based tNGC with macroscopic holes; tNGCs: tubular nerve guidance conduits.

Macroscopic holes do not enhance functional recovery of critical-size nerve lesions

To assess functional recovery following the 15-mm sciatic nerve lesion, electrophysiological measurements were performed 12 weeks postoperatively. We observed significantly improved compound muscle action potential (Figure 7A) and amplitude (Figure 7B) in animals treated with ANT compared with both silk tNGC groups. Similarly, muscle weight ratios of the operated and contralateral extensor digitorum longus (Figure 7C) and tibialis anterior (Figure 7D) muscles were significantly higher in the ANT group. No statistically significant differences in any of these parameters were observed between unmodified silk conduits and holey silk conduits (Figure 7A–D).

Figure 7.

Functional analysis of nerve regeneration at 12 weeks postoperatively following a 15-mm critical-size sciatic nerve defect.

Electrophysiological recordings of CMAP (A) and amplitude (B). Comparison of the wet muscle weight of right and left extensor digitorum longus (C) and tibialis anterior muscle (D). (E) Functional recovery was assessed over a 12-week observational period using static sciatic index analysis. (A–D) n = 8 for ANT, n = 7 for silk-based tNGCs, one-way analysis of variance with Tukey’s multiple comparison test (A–C), Kruskal–Wallis test with Dunn’s multiple comparison test (D). (E) n = 7–8, mixed-effects model with Tukey’s multiple comparison test. ANT: Autologous nerve transplants; CMAP: compound muscle action potential; silk^– tNGC: silk-based tNGC without holes; silk⁺ tNGC: silk-based tNGC with macroscopic holes; tNGCs: tubular nerve guidance conduits.

Furthermore, we performed Catwalk automated gait analysis following 8-mm as well as 15-mm sciatic nerve defects with observation times of 7 and 12 weeks, respectively. However, due to a general lack of recovery, no conclusive data could be gathered using this method (Additional Figure 1 ^{(809.9KB, tif)} ).

Additionally, we performed SSI analysis to determine functional regeneration according to static paw functional parameters. Up until postoperative 7 weeks there were no statistically significant differences between the three groups. Interestingly, 7 weeks after surgery, the SSI significantly improved in the ANT group only compared with the silk- tNGC group (P = 0.0057), followed by an improvement in functionality at 9 weeks postoperatively in the ANT group compared with both silk tNGCs. Thereafter, at 11 and 12 weeks postoperatively, the SSI values in the silk⁺ animals were not significantly different compared with the ANT group, while animals of the silk^– group still showed significantly impaired recovery compared with ANT (P = 0.0024 and P = 0.0050 at 11 and 12 weeks postoperatively, respectively; Figure 7E).

Discussion

Peripheral nerve injuries are associated with a dramatic decrease in the patients’ quality of life. Donor site morbidity limits the use of autologous tissue. Therefore, other strategies including the use of hollow tNGCs have been intensively investigated in the past.

There is a wide consensus in the scientific community that the porosity of conduits needs to be fine-tuned to on the one hand allow oxygen and nutrient supply to the regenerating nerve tissue, while on the other hand preventing infiltration of fibrous/scar tissue (Jenq and Coggeshall, 1985, 1987; Brunelli et al., 1994; Oh et al., 2008; Kokai et al., 2009). Therefore, the porosity of tNGCs does generally not exceed holes in the size of nanometers to a few micrometers so that cellular migration through the tubular walls is blocked. Historically, however, it has been considered that critical-size nerve lesions may require tNGCs with bigger pore sizes to enable vascular connection to the adjacent tissue to ensure sufficient oxygen and nutrient supply. Remarkably, Tarlov and Epstein (1945) highlighted the importance of vascular supply for peripheral nerve regeneration. Interestingly, they reported that regeneration of the mid-graft section requires vascularization by the surrounding tissue, concluding that the use of impermeable animal tissue or metal-based membranes is to be condemned because they would prevent ingrowth of blood vessels from surrounding tissues, leading to massive necrosis of the mid-graft section (Tarlov and Epstein, 1945). To the best of our knowledge, Jenq and Coggeshall (1987) for the first time investigated the effect of macroscopic holes in the tubular walls of tNGCs on peripheral nerve regeneration, leading to a significantly increased distance regenerating axons can span compared with impermeable, non-holey tubes. The authors speculated that these microporous holes allowed access for factors from the surrounding tissue, including the “migration of non-neural connective tissue cells” (Jenq and Coggeshall, 1987).

In a sophisticated approach, Oh et al. (2008) showed improved nerve regeneration using tNGCs with asymmetric porosity of 50 µm pores on the outside to allow vascular ingrowth into the tubular walls but not into the lumen, since a layer with 50 nm pores on the inside of their tNGC, intended to prevent scar tissue infiltration, also blocks direct migration of blood vessels to the regenerating nerve tissue. Nevertheless, Oh et al. (2008) speculate that the close approximation of the blood vessel to the tNGC lumen might be sufficient to effectively supply nutrients to the tNGC. Similarly, Vleggeert-Lankamp et al. (2007) showed enhanced regeneration inside porous tNGCs compared with non-porous tNGCs, yielding superior regeneration using micropores between 1–10 µm compared with non-porous tNGCs or macropores of 10–230 µm. In contrast to the pores in our graft, it must be highlighted that the described pores are not single continuous pores but the given range of 10–230 µm rather describes the porosity of single fiber layers the tNGC is fabricated of. There are several overlapping layers forming the tubular walls. Consequently, the reported pore size is rather a description of the tubular wall porosity to assess diffusion properties rather than characterizing entry points for the migration of organized tissue structures including blood vessels.

Despite these reported observations in the past, there is no further graft described in the literature or even on the clinical market showing macroscopic holes to either enhance nerve regeneration or avoid unwanted necrosis in larger nerve defects by enabling the migration of blood vessels from surrounding tissues to the regenerating nerve tissue. To the best of our knowledge, there is no study available investigating the possibly beneficial effects of migrating blood vessels through the tubular walls of tNGCs.

In our previous study (Teuschl et al., 2015), we observed neovascularization within and on the outside of a silk tNGC, which led to this study, in which we test the modification of these tNGCs creating holes of 200–300 µm in the tubular walls. Thereby, we envisioned facilitating the connection of vascular networks from the regenerating tissue inside the tNGC to the surrounding tissue. In a first 8-mm sciatic defect model with 7 weeks of regeneration time post-surgery, we observed superior spanning of the defect area with distally regenerated axons using silk⁺ tNGCs compared with silk^– tNGCs. Using a combination of histological stainings and microCT scans, the formed blood vessels in the regenerated tissues were analyzed. In accordance with literature (Hobson et al., 1997; Cattin et al., 2015; Fornasari et al., 2022), a distinct growth of blood vessels from the nerve stumps is obvious, forming a main blood vessel in the center of the regenerating tissue bridging the created defect. Surprisingly, and in contradiction to our initial idea that blood vessels from the surrounding tissue will enter the regenerating tissue area, the majority of blood vessels that span through the tubular wall grow from the main central blood vessel to the outside. These blood vessels seem to connect to existing adjacent surrounding blood vasculature, thereby most likely improving blood circulation, which in turn leads to an overall enhanced supply of nutrients and oxygen and/or transfer of metabolites. Besides the provision of nutrients, the interlinkage between vascularization and nerve regeneration is well established as it has been shown that endothelial cells secrete factors that favor neurogenesis (Jin et al., 2002) and nerve regeneration (Hobson et al., 2000; Hobson, 2002), and that blood vessels serve as tracks for Schwann cells to grow along and thus guide axon growth (Hobson et al., 1997; Cattin et al., 2015). This is in accordance with the findings of our study, which shows that the regenerating axons from the nerve stumps grow next to aligned blood vessels. Therefore, we propose tNGCs with macroscopic holes to be a potent graft type to further enhance nerve regeneration using tNGCs as they (i) increase the vessel density inside the regenerating nerve tissue, and (ii) might avoid central necrosis observed in nerve grafts for large and long nerve defects (Muangsanit et al., 2018). By providing macroscopic holes into the tubular walls of silk-based tNGCs, we enabled in situ blood vessel growth through our silk tNGC. In addition to successfully enhanced vascularization, we also observed improved axonal bridging in a short rat sciatic nerve defect model. Electrophysiological measurements (Additional Figure 2 ^{(600.9KB, tif)} ) did not show significant differences between autologous grafts and treatment with silk-based tNGCs, neither for silk⁺ nor for silk^– tNGCs, which is most likely due to the limited regeneration time of only 7 weeks for an 8-mm defect. More time might be required to investigate functional target innervation.

Due to the promising results seen in the short defect model, we conducted a follow-up study in a 15-mm critical-size sciatic nerve defect model followed for up to 12 weeks post-surgery. Similar to the short defect model, we were able to see the growth of blood vessels through the silk⁺ tNGCs. Axon counting revealed a similar number of axons for the start graft position for all three treatment groups (ANT, silk⁺, and silk^– tNGCs), followed by a decline in axon count for both silk-based tNGCs compared with ANT. We could not observe a difference between the tNGCs with and without holes despite enabling vascular ingrowth. Functional analyses using SSI revealed an improvement of functionality at 7 weeks post-surgery for ANTs compared with both silk-based tNGCs. Interestingly, beginning with week 11 there was no significant difference between ANTs and the silk⁺ tNGCs. Similar results for functional recovery have been obtained via Catwalk analysis (Additional Figure 1 ^{(809.9KB, tif)} ). It has to be pointed out that the number of regenerated axons in the distal part is sparse compared with ANTs. This is presumably caused by insufficient proliferation, migration, and alignment of endogenous Schwann cells, which consequently fail to form bands of Büngner that are essential guidance structures for regenerating axons. In contrast, ANTs not only provide essential topographical guidance, but also trophic support due to the presence of Schwann cells that secrete neurotrophic factors. Improved functional recovery in the ANT group despite lower numbers of blood vessels suggests that promoting vascularization alone is not sufficient to enhance regeneration of severe critical-size nerve injuries, but that the addition of support cells, such as Schwann cells that are implanted together with silk tNGCs, is required to improve axon regeneration and functional recovery. Hence, in future studies, we aim to investigate the addition of Schwann cell fillers into the lumen of silk⁺ tNGCs on functional recovery to promote axon migration across the defect. Alternatively, loading of the lumen of tNGCs with e.g. neurotrophic factors could boost Schwann cell migration and proliferation to overcome the absence of initial supporting cells. Furthermore, the question arises whether a longer post-operative observation period would have enabled further increased axon regeneration. In general, it is known that the sciatic nerve defect model is a suboptimal model for evaluating functional nerve recovery (Heinzel et al., 2020). Therefore, evaluation of our silk⁺ tNGC in another nerve defect model in the rat, which is more suitable for the evaluation of functional recovery, such as the median nerve model, or in larger animals where it is possible to create defects larger than 2 cm would be of interest to examine the effect of macroscopic holes on the functional outcome or the prevention of necrosis, respectively.

This study has some limitations that should be noted. First, the prepared silk-based tNGCs containing macroscopic holes had a rather uniform distribution of holes with 200–300 µm, which might have not been ideal for enhancing nerve regeneration. Importantly, the size, orientation, and layout of these laser-ablated holes can be easily adjusted to maximize their beneficial effects on nerve regeneration. Additionally, we chose a rather short post-operative observational period of only 7 weeks for the 8-mm sciatic nerve defect model, which might explain the absence of improved electrophysiological measurements in animals treated with silk⁺ tNGCs despite enhanced intraluminal vascularization as well as axon regeneration. Consequently, a longer postoperative observational period might be required to allow for functional target innervation. Similarly, prolonging the postoperative observation period for the critical-size sciatic nerve defect might also improve axon regeneration for the long defect model. Lastly, we here investigated the use of hollow tNGCs for the repair of sciatic nerve lesions, which did not result in improved axon regeneration at the distal site in a 15-mm critical-size sciatic nerve defect. Since it has been previously reported that axon regeneration is impaired by the lack of adequate bands of Büngner formation due to senescence as well as insufficient proliferation, migration, and alignment of endogenous Schwann cells, we aim to investigate the addition of Schwann cell fillers into the lumen of silk⁺ tNGCs on functional recovery to promote axon migration across the defect.

In conclusion, in this study, the introduction of macroscopic holes into the tubular walls of a silk-based tNGC led to a connection of blood vessels from the adjacent tissue to the formed intraluminal vasculature. Contrast-enhanced microCT revealed that this connection was facilitated by both, the ingrowth of vessels into the silk⁺ tNGC containing macroscopic holes as well as outgrowth of blood vessels from the lumen of the tNGC towards the surrounding tissue. In a short rat sciatic nerve defect model, we could see that the thereby created migration holes for blood vessels led to an enhanced rate of nerve defect bridging at the observed time point. However, in a critical-size nerve defect model this effect was not observed, as axonal regeneration did not occur beyond the first millimeters of the tNGCs. Even though ANTs had the lowest number of bloodvessels at the end of the postoperative observation period, axonal regrowth as well as functional recovery was superior in this treatment group. This is presumably caused by the structural guidance of nerves as well as the presence of native Schwann cells, which provide essential trophic support for regenerating axons. Therefore, this finding suggests that promoting vascularization alone is not sufficient to enhance regeneration of severe nerve injuries resulting in a large nerve gap, but that the addition of support cells, such as Schwann cells that are implanted together with silk tNGCs, is required to improve axon regeneration and functional recovery. Despite not being able to see functional improvements with the used nerve defect models and applied functional tests, results of the short defect model make us believe that further optimization of this holey tNGC approach with guidance structures or cellular components will lead to improved grafts for future clinical use.

Additional files:

Additional Figure 1 ^{(809.9KB, tif)} : Functional analysis of nerve regeneration in a rat sciatic defect model was observed via Catwalk.

Additional Figure 1

Functional analysis of nerve regeneration in a rat sciatic defect model was observed via Catwalk.

(A, B) Functional analysis was performed in the 8 mm defect group at 7 weeks postoperatively (n = 10-12) (A) and in the 15-mm critical size defect model at 12 weeks postoperatively (n = 5-6) (B). Mixed-effects model with Tukeys multiple comparison test was used. ANT: Autologous nerve transplants; LH: left hindlimb; RH: right hindlimb; silk- tNGC: silk-based tNGC without holes; silk+ tNGC: silk-based tNGC with macroporous structures; tNGCs: tubular nerve guidance conduits.

Additional Figure 2 ^{(600.9KB, tif)} : Electrophysiology results and relative muscle weight of the tibialis anterior in the 8 mm defect group at 7 weeks postoperatively.

Additional Figure 2

Electrophysiology results and relative muscle weight of the tibialis anterior in the 8 mm defect group at 7 weeks postoperatively.

Electrophysiology results (n=5-8) and relative muscle weight (n=11-12). One-way analysis of variance with Tukeys multiple comparison test. ANT: Autologous nerve transplants; CMAP: compound muscle action potential; L: left; LH: left hindlimb; R: right; RH: right hindlimb; silk- tNGC: silk-based tNGC without holes; silk+ tNGC: silk-based tNGC with macroporous structures; tNGCs: tubular nerve guidance conduits.

Additional Video 1: A rendering of the 3D microCT scan the silk⁺ tNGC depicted in Figure 3B.

Funding Statement

Funding: This work was supported by the Lorenz Böhler Fonds, #2/19 (obtained by the Neuroregeneration Group, Ludwig Boltzmann Institute for Traumatology) and the City of Vienna project ImmunTissue, MA23#30-11 (obtained by the Department Life Science Engineering, University of Applied Sciences Technikum Wien).

Data availability statement:

All relevant data are within the paper and its Additional files.