Novel Spiral Structured Nerve Guidance Conduits with Multi-channels and Inner Longitudinally Aligned Nanofibers for Peripheral Nerve Regeneration

Abstract

Nerve guidance conduits (NGCs) are artificial substitutes for autografts, which serve as the gold standard in treating peripheral nerve injury. A recurring challenge in tissue engineered NGCs is optimizing the cross-sectional surface area to achieve a balance between allowing nerve infiltration while supporting maximum axonal extension from the proximal to distal stump. In this study we address this issue by investigating the efficacy of an NGC with a higher cross-sectional surface composed of spiral structures and multi-channels, coupled with inner longitudinally aligned nanofibers and protein on aiding nerve repair in critical sized nerve defect. The NGCs were implanted into 15-mm long rat sciatic nerve injury gaps for 4 weeks. Nerve regeneration was assessed using an established set of assays, including the walking track analysis, electrophysiological testing, pinch reflex assessment, gastrocnemius muscle measurement, and histological assessment. The results indicated that the novel NGC design yielded promising data in encouraging nerve regeneration within a relatively short recovery time. The performance of the novel NGC for nerve regeneration was superior to that of the control nerve conduits with tubular structures.

Introduction

A nerve injury severe enough to be classified as neurotmesis, under the Seddon classification, in one in which there is a disruption in the nerve, axon, and myelin sheath [1]. Peripheral nerve injury (PNI) is damage to the nerves or ganglia of the peripheral nervous system and can be caused by trauma or be a byproduct of surgery [2–4]. PNI can lead to paralysis, the complete loss of voluntary function of the effected limb. There are over 200,000 nerve repair procedures annually performed, often with an autograft, the current gold standard [5–9]. Autografts have haptotactic and chemotactic cues like native nerves, but they are scarce, and can lead to a loss of function at the donor site [10, 11]. Nerve guidance conduits (NGCs) have been studied extensively and numerous efforts have been made to optimize their characteristics to serve as viable alternatives to autografts [12, 13]. Commercial NGCs have a tubular cross section, which limits cell migration.

The process of regeneration of the transected nerve within a nerve guidance conduit is classified under five phases: the fluid phase, matrix phase, cellular migration phase, axonal phase, and myelination phase. Since the nerve guidance conduit serves as a bridge from the proximal stump to the distal stump of a transected nerve all activities occur within a secure environment without disruption from scar tissue or surrounding physiological intervention. The fluid phase consists of the proximal and distal stumps releasing growth factors and extracellular matrix molecules, followed by the formation of a fibrin cable, then Schwann cells, fibroblasts, and endothelial cells migrate along the cable, the Schwann cells then proliferate and align to create a cable known as the bands of Büngner. This cable serves as the structure on which axonal sprouts travel from the proximal stump to the distal stump. The Schwann cells then wrap around the extending axonal sprouts to create the myelin sheath, which completes the myelination phase [14].

It has been noted that this mechanism of nerve regeneration only occurs along surfaces and a tubular structure is noted being hollow, which limits the mechanism of nerve regeneration to occur only along the circular structure of the nerve guidance conduit. In contrast to the tubular structure, nerve guidance conduits with higher cross-sectional surface areas offer a greater area for the mechanism of nerve regeneration to take place [15, 16].

Investigators have fabricated NGCs with higher cross-sectional surface areas, incorporated chemotactic cues in tubular NGCs, or coupled both strategies [16–22]. While full recovery takes 52 to 104 weeks, short term investigations provide insight into the efficacy of an NGC when assessed on critical sized injuries [21–27]. These studies have yielded regenerated nerve fibers with chemotactic factors incorporated in tubular NGCs [28–31]. Previously studied NGCs have not had sufficient surface area, inner longitudinally aligned nanofibers, interconnected pores throughout the NGC to allow for nutrient and waste exchange, and the mechanical integrity to support axonal elongation [18–22, 31]. In circumstances with the absence of such factors dispersion of elongating axons has been observed, especially in larger gaps. [15] The presence of a greater cross-sectional surface area limits dispersion by directing axonal extension, and the presence of aligned nanofibers within the inner surface of an NGC further enables this process [17, 32, 33].

In this paper, we detail the design of a novel multichannel spiral NGC and its efficacy in treating a 15-mm nerve defect in a rat sciatic nerve model, when coupled with haptotactic cues. The NGC is optimized from a previous design of our group, which yielded favorable results [34].

Materials and Methods

Preparation of Spiral NGC with Aligned Nanofiber and outer nanofibrous tube

A solvent evaporation and salt leaching method was used to fabricate a PCL sheet from 8% (w/v) poly-caprolactone (PCL) in dichloromethane (DCM) [35–38]. The sheet was cut into 17-mm by 10-mm rectangles and two smaller rectangles were cut to create chambers at the ends of the NGC to secure the injured nerve stumps with sutures. Inner longitudinally aligned PCL nanofibers (AF) were electrospun on the PCL sheets using a conducting rotary disk. The sheets were then rolled up into a spiral structure. Finally, random nanofibers were electrospun on the outer layer of spiral NGCs to form the outer tube, to prevent scar tissue infiltration and enhance their mechanical strength. All NGCs were disinfected with 70% isopropyl alcohol for 2 hours and rinsed with sterile Phosphate Buffered Saline (PBS) 3 times prior to implantation [36].

Preparation of Multichannel Spiral NGC with Aligned Nanofiber and outer nanofibrous tube

A solvent leaching method was utilized to fabricate the multichannel spiral NGC as aforementioned [35–38]. As the sheet dried, needles that measured 120 mm in length and 160 µm in diameter were placed on the sheet to form the multiple channels on the multichannel spiral structure. The rectangular sheets were then rolled to create a spiral structure in preparation for electrospinning to coat the inner longitudinally aligned nanofibers. Lastly, random nanofibers were electrospun on the outer layer of spiral NGCs to form the outer tube, to prevent scar tissue infiltration and enhance their mechanical strength.

Preparation of the Tubular Structure

The fabrication of the tubular structure was solely through electrospinning. A rod was wrapped in aluminum foil, then attached to a rotating motor and rotated at 150 revolutions per minute. The parameters for electrospinning were kept constant: flow rate of 0.25 ml/hr, distance between the syringe and collector of 5.5 cm, voltage of 12 kilovolts, and a needle size of 20G. The fabricated tubular structure also served as the tubular NGC used in this research study.

Incorporation of Collagen into Multichannel Spiral NGC

Collagen is a major extracellular matrix structural protein and is abundantly available [39, 40]. This is the material of choice and has yielded numerous commercial NGCs: NeuraGen, Neuroflex, NeuroMatrix, NeuraWrap, and NeuroMend [41]. There are 44 different known Collagen genes which develop 28 different kinds of Collagen fibrils of which Type I, II, and III are most abundant [42]. Type I is the most abundant and makes up 90% of the collagen in the human body. Purified collagen has antigenic properties which can be addressed by removing the telopeptide regions of the molecule or cross linking. Cross linking allows for manipulation of its degradation and mechanical strength but can also impact its toxicity levels [43]. The adhesive property of Collagen makes it the material of choice to improve cell attachment, survival, and proliferation for an array of cell types. The potential and capabilities of collagen have allowed it to receive FDA approval and is currently being investigated for CNS injury treatment [41]. Collagen and PCL (50:50) were dissolved in HFIP to create a 16% solution and then electrospun as aligned nanofibers on the multichannel spiral structure [42].

Animal Study

The total number of animal needed for this study was determined using a 1-way analysis of variance. A total of seven groups (n = 5) were evaluated in this study, which are listed in Table I below. An autograft and a tubular NGC were used as the positive control and negative control, respectively. In the study outlined, the autograft was the sciatic nerve severed at the site of surgery, which was then reversed 180º and re-sutured to the nerve. [44 –47]

TABLE I.

Designation of Groups for In Vivo Study

Group	Duration of Study	Designation
Autograft	4 weeks	Positive Control
Multichannel Spiral + AF (PCL/Collagen) + Outer Tube (MC+S+AF(PCL/CG)+T)	4 weeks	Experimental
Multichannel Spiral + AF + Outer Tube (MC+S+AF+T)	4 weeks	Experimental
Multichannel Spiral + Outer Tube (MC+S+T)	4 weeks	Experimental
Spiral + AF + Outer Tube (S+AF+T)	4 weeks	Experimental
Spiral + Outer Tube (S+T)	4 weeks	Experimental
Tubular (T)	4 weeks	Negative Control

A 15-mm gap length was studied for a 4-week period. The animals were maintained according to regulations and standards approved by the Institutional Animal Care and Use Committee at the Stevens Institute of Technology and the National Institutes of Health.

Surgical Procedures

The animal model of choice for this study was the adult male Sprague Dawley rat (250–275 grams) [36]. The rats were anesthetized with isoflurane (2.5%) in oxygen (0.8 L/min) then the right hind legs were shaved with animal clippers and Prepodyne (West Chemicals Inc., Long Island, NY) was applied. A 20-mm incision was made along the femoral axis to separate the thigh muscles and dissect the sciatic nerve. A 15-mm piece of the sciatic nerve was explanted and a 17-mm long NGC was secured with 10–0 nylon monofilament sutures (Fine Scientific Tools, Foster City, CA) maintaining the proximal and distal stumps 15 mm apart. At the 4-week endpoint the rats were anesthetized again and euthanized via CO₂ asphyxiation [36].

Walking track analysis

Walking track analysis is a method to assess functional recovery. Sciatic Functional Index (SFI) is a quantified assessment based on the print length, toe spread, and the intermediate toe spread of the paws. As SFI approaches zero, the corresponding functional recovery improves [37]. Three parameters were derived from the paw prints: print length (PL), toe spread (TS; distance from toe 1 to toe 5), and intermediate toe spread (IT; distance from toe 2 to toe 4). This assay was performed preoperatively, then at week 2 and week 4. The parameters from paw prints taken before and after surgery were normal and experimental respectively. The SFI was calculated using: SFI = −38.3 [(EPL-NPL)/NPL] + 109.5 [(ETS-NTS)/NTS] + 13.3 [(EIT-NIT)/NIT] - 8.8.

Electrophysiological Testing

Electrophysiological studies are performed to assess the restoration of functionality of the regenerated nerve through the implants at 4 weeks after implantation. This assay is based on the presence of myelinated axons and is influenced by the diameter and number of myelinated axons [35–37]. Each rat was placed under anesthesia again during data collection. A recording needle electrode was placed in the gastrocnemius muscle and stimulation electrodes were placed directly posterior to the tibia; the sciatic nerve was stimulated with two stainless steel wire electrodes connected to an electrical stimulator. A ground electrode was placed in the surrounding muscle tissues to remove conduction of stimulation through muscle tissues. The amplitude and nerve conduction velocity (NCV) of the evoked compound muscle action potentials (CMAPs) were recorded.

Pinch Reflex Assessment

Four weeks after implantation, the rats remained under anaesthesia following electrophysiological assessment. The nerve trunk distal to the conduit was pinched with a pair of forceps. Contraction of the muscle on the back or movement of the leg indicated the presence of regenerated nerve inside the conduit. This assessment was critical in evaluating the presence and functionality of the myelinated axons in the regenerated nerve [48].

Gastrocnemius muscle measurement

The mass of the gastrocnemius muscle is proportional to the degree of sciatic nerve innervation and is an indicator of the functional activity of sciatic nerves [39]. The relative gastrocnemius muscle weight (RGMW) is the ratio of the gastrocnemius muscle weight from the experimental (right) side to that of the normal (left) side. RGMW is used as a parameter to represent the “functional” consequences of sciatic nerve regeneration [19, 20].

Histological Assessment

At 4 weeks, the rats were euthanized. The surgical site was reopened to explant the NGCs for a histological analysis. Nerve regeneration was assessed at the midpoint of the NGCs. The explanted autograft and NGCs were embedded in epoxy, sectioned at 900 nm, stained with 1% toluidine blue, and then imaged with a light microscope. Nerve regeneration was evaluated based on the number of myelinated axons at the midpoint of the explanted NGC [21, 22].

Statistical Considerations

The data was expressed as mean values ± standard deviation (SD). Statistical analysis was performed with a single factor analysis of variance (ANOVA). When a significant variance between groups was noted, a Tukey’s test was used to make pairwise comparisons. A value of p <0.05 was statistically significant. A preliminary power calculation showed n=5 defects, type I error (α) of 0.05, and an estimated variance between 10% and 15%, the power of the test (one-way ANOVA) was calculated to be 0.89/1.0000. A value of at n=5 was sufficient for this study.

Results

The data presented was analyzed using a 2-tailed t-test with unequal variance.

The cross section of the implanted NGCs is shown in Fig 1. The inner diameter of all NGCs was 1.8 mm and were 17 mm in length. The tubular cross section (1A) is intended the surface area on the inner circumference for cellular migration and axonal elongation. The spiral NGC (1B) provides a higher cross-sectional surface area for cellular migration and axonal elongation. The gap between the spiral layers in the spiral structure was fixed at 100–150 µm. The multichannel spiral NGC (1C) provides a focused cross-sectional surface area, as evident by the channels along the spiral structure to aid cellular alignment and migration. The channels along the spiral structure in the multichannel spiral NGC may limit axonal dispersion and may aid axonal extension with the presence of aligned nanofibers. The gap was similarly fixed in the multichannel spiral structure. While all NGCs in this study were porous, the spiral and multichannel spiral NGCs contained pores developed using a solvent evaporation and salt leaching approach.

Figure 1:

The NGC types implanted. Tubular (A), Spiral (B), and Multichannel Spiral (C).

The focus of this study was to optimize the space available for nerve infiltration, ensure nutrient exchange through the NGC, and aid cellular migration. A direct comparison of the spiral NGC and multichannel spiral NGC with and without coated nanofibers is shown in Figure 2 above. The channels along the spiral layers of Figure 2G. and Figure 2H. were approximately 170 µm in width and measured 15 mm in length, identical to the length of spiral layers and the multichannel spiral layers. These channels provided additional intraluminal guidance cues for nerve regeneration. The NGCs were 17 mm in length as 1 mm at each end of the NGC was reserved as a chamber to secure the proximal and distal nerve stumps to the NGCs with sutures. The dimensions of the spiral layers, pores, and nanofibers characteristics were optimized based on a review of peer reviewed articles and previous in vitro and in vivo studies conducted by our group [49]. The process of nerve regeneration was expected to occur from the proximal to distal end of the conduit, as this is the direction of cellular migration. Previous studies have shown too many spiral layers inhibit nerve infiltration. The absence of pores has yielded inhibited healthy nerve regeneration, and a pore size of 50+10.5 µm ensured nutrient exchange without the infiltration of scar tissue from previous studies. Similarly, a nanofiber diameter of 850+220 nm for the nanofibers allowed for cell attachment and cell migration from our previous experience.

Figure 2:

The inner morphology of the NGCs implanted. Spiral (A), Spiral with Inner Aligned Nanofibers (B), Multichannel Spiral (C), and Multichannel Spiral with Inner Aligned Nanofibers (D).

The spiral and multichannel spiral NGCs were tested in a 15-mm long SD rat sciatic nerve defect to assess its efficacy relative to an autograft and a tubular structure for peripheral nerve regeneration (PNR). The autograft and NGCs at the implantation using 10–0 sutures are shown in Fig. 3A and Fig. 3B, respectively. The NGCs were all identical in appearance.

Figure 3:

Autograft (A) and a nerve guidance conduit (B) at the time of implant.

Post-implantation

At the 4-week endpoint, the surgical site was reopened to determine the degree of peripheral nerve regeneration as well as any adverse effects caused by the implants. Systemic or regional inflammation and surgical complications after implantation were not observed. The purpose of Fig. 4 is to show that all NGCs part of this study were still intact at the endpoint, without any inflammation around the surgical site. The degree of reinnervation varied for each NGC, and the variation in recovery is discussed in the manuscript through the various assessment used to measure recovery [14]. The images of the autograft and NGCs at the endpoint of 4 weeks are shown in Figure 4. The degree of PNR was measured throughout the 4-week recovery time using an array of functional assays, the results of which are shown and discussed below.

Figure 4:

NGCs and the surrounding environment at the 4 week endpoint. Autograft (A), MC+S+AF(PCL/CG)+T (B), MC+S+AF+T (C), MC+S+T (D), S+AF+T (E), S+T (F), and T (G).

Functional Recovery Assessments

SFI was assessed using a walking track analysis to measure PL, TS, and IT (Fig. 5). The pre-operative SFI value was −5.5±1.3. The SFI value ranged from −86.3 to −91.9 at week 2 post-surgery. Improvement in SFI was observed at week 4. The autograft performed better than the other groups. The SFI values at week 4 ranged from −75.7 to −89.6. The following are the SFI values for week 4 for each group: autograft (−74.9±3.1), MC+S+AF(PCL/CG)+T (−80.2±1.5), MC+S+AF+T (−81.1±2.6), MC+S+T (−83.9±1.6), S+AF+T (−83.5±2.6), S+T (−86.2±3.2), and T (−89.6±3.2).

Figure 5:

Results of the SFI based on a walking track analysis. # indicates p value > .05 compared to autograft.

The MC+S+AF(PCL/CG)+T and MC+S+AF+T were statistically comparable to the autograft.

Electrophysiological Assessment

At the four-week endpoint, CMAPs were measured by stimulation at the surgical limbs and recorded from the gastrocnemius muscle. No signals were detectable in any of the groups except for the autograft, which is consistent with other reports [50].

Pinch Reflex Test

At four weeks post-surgery, the distal nerve of each group was pinched with a pair of forceps. Movement of the leg indicated functional recovery of the regenerated nerve within the NGC. The autograft and MC+S+AF(PCL/CG) +T groups were the only ones that had detectable results, as shown in Table II.

TABLE II.

Results of Pinch Test

Autograft	3/5
MC+S+AF(PCL/CG)+T	1/5
MC+S+AF+T	0/5
MC+S+T	0/5
S+AF+T	0/5
S+T	0/5
T	0/5

Nerve Histology

The results of the histology are shown Fig. 6 and Fig. 7. The toluidine blue stain was present throughout the cross sections of the all groups.

Figure 6:

Histology Images of the Cross Sections of the NGCs from the 4 week study. Autograft (A), MC+S+AF(PCL/CG)+T (B), MC+S+AF+T (C), MC+S+T (D), S+AF+T (E), S+T (F), and T (G).

Figure 7:

Cross Sections of the NGCs from the 4 week study at low magnification at 20x. Autograft (A), MC+S+AF(PCL/CG)+T (B), MS+AF+T (C), MC+S+T (D), S+AF+T (E), S+T (F), and T (G). Arrows indicate blood vessels and arrow heads indicate myelinated axons.

Toluidine blue stain was used as it stains the nucleic acids of tissue and myelin sheath. Figures 4 and 7 represent the regenerated tissue that was present at the midpoint of the autograft and NGCs. The variation in the degree of stain in for each NGC provides an insight into the efficacy of that NGC type in providing a favorable environment for regeneration.

A more detailed set of images from the histology showed the presence of myelinated axons and blood vessels in the center of the regenerated nerve. As evident from the images, the number of myelinated axons varied depending on NGC structure and the presence of aligned nanofibers as well as collagen. The autograft had the highest number of myelinated axons, while the tubular structure had the least.

Using the detailed images of the histology, a quantitative assessment of the neural tissue was performed for all the groups used in this study. The following were the results of the total myelinated axons at the midpoint of the grafts: autograft (3015±584.5), MS+AF(PCL/CG)+T (1198±200.9), MS+AF+T (916.7±208.2), MS+T (783.3±125.8), S+AF+T (583.3±160.7), S+T (523.3±175), and T (475±109) (Fig. 8). While the autograft performed significantly better than the other groups, the MC+S+AF(PCL/CG) +T yielded the second highest number of myelinated axons. The MC+S+AF(PCL/CG) +T was statistically better than the S+T and T groups, while the S+AF+T, S+T, and T groups were statistically comparable to each other.

Figure 8:

Myelinated Axon count at the midpoints of all groups. Paired groups indicate p value > 0.05. # indicates p value < 0.05 compared to all groups.

Gastrocnemius Muscle Weight Ratio

The gastrocnemius muscle shares a proportional relationship with the nerve, as the nerve begins to heal following injury the muscle undergoes reinnervation and regrowth. A value of 1 indicates full recovery. The following are the values of the gastrocnemius muscle weight ratios (GMWRs): autograft (0.35±0.095), MC+S+AF(PCL/CG)+T (0.3±.061), MC+S+AF+T (0.27±.038), MC+S+T (0.256±0.068), S+AF+T (0.22±0.013), ST (0.21±0.036), and T (0.18±0.07) (Fig. 9). The MC+S+AF(PCL/CG)+T group was significantly than the S+AF+T, S+T, and T groups.

Figure 9:

Relative Gastrocnemius Muscle Weight from the 4-week study. # indicates p < .05 than S+AF+T, S+T, and T.

Discussion

An ideal NGC can restore functional recovery for an injured nerve like an autograft which can be achieved by mimicking the architecture of the natural nerve [17, 14]. A constant challenge has been to address large peripheral nerve repair gaps using nerve guidance conduits. While investigators have addressed this challenge in various ways, the approach presented here explored providing additional intraluminal support with channels along the spiral layer, incorporating haptotactic and chemotactic cues in an NGC, while permitting nutrient exchange throughout the environment designed for regeneration.

The addition of haptotactic cues can aid nerve regeneration especially in addressing large nerve gaps. The significance of utilizing intraluminal guidance cues has been denoted by Ngo et al. [51]. The attention to detail at a micro and nano-scale level in this study is shown in Figure 2. While the tubular and autograft were significantly different in architecture, the spiral and multichannel spiral groups only differed in the presence of channels along the spiral layer. The focus was to optimize the spiral structure based on observations from our previous experience, which yielded the multichannel spiral shown in Figure 2C and Figure 2D [49]. As documented in our previous experience the spiral NGC was designed based on an analysis of the studies conducted in the field of peripheral nerve repair, however a challenge addressed was how to further enable cellular migration for a nerve gap of 15mm, as this has been defined as critical gap [52]. The same number of spiral layers as before were retained, however to create channels along these layers to create a micro environment for cellular migration. The structure of the spiral and multichannel spiral NGCs allowed for distribution of aligned nanofibers. The NGCs were wrapped with outer nanofibrous tube to reduce disruption of the process of nerve regeneration occurring in the NGC.

The functional assays highlighted the efficacy of the tubular, spiral, multichannel spiral, and autograft group in the process of nerve regeneration. The differences in quantitative and qualitative assessments provided an insight into the topographical cues of each NGC, especially in the data collected from the tubular group which did not contain any intra luminal cues, and only had a single lumen for the regenerating nerve. While full recovery requires 52 to 104 weeks, a 4-week endpoint for a critical gap of 15 mm was selected to gain an insight into the efficacy of the spiral and multichannel spiral relative to the tubular and autograft, as well as each other.

The initial measure of motor function was SFI, which is intended to illustrate the degree of reinnervation between time of surgery and the end final endpoint of the study. The results of the SFI for this study, shown in Fig. 5, yield comparable results for the MC+S+AF(PCL/CG)+T and MC+S+AF+T to the autograft while being statistically incomparable to the tubular group. It is also interesting to note that while the MC+S+AF(PCL/CG)+T and MC+S+AF+T were comparable to each other, the MC+S+AF(PCL/CG)+T was different from all other groups. This suggests that the presence of collagen may have enhanced motor function. This impact of collagen has been observed in other studies as well [53, 54].

An electrophysiological assessment is useful in evaluating the degree to which sensory and motor fibers have reinnervated. This assay is dependent on the number, diameter, and myelination of axons within a regenerated nerve. Electrophysiological signals were detected in 3 of the 5 autografts, however no signals were detected in any of the other groups. Similar observations have been made in Meek et. Al [55]. The team did not observe electrophysiological data until 7 weeks post-surgery. While the SFI data yielded a different outcome, there have been instances in which there was no correlation between SFI and electrophysiology in such a short period of time [45, 55]. This may be attributed to the degree of maturity that is required of the axons for the strength of the electrophysiological signal to be detected, the maturity of which can be different in conducting a walking track analysis [56].

The pinch test is a measure of nerve regeneration. This assay is conducted on the distal end of the regenerating nerve as the growing tips of the regenerating axons are more sensitive than in the proximal stump. This assay is conducted to measure sensory recovery and particularly useful in short term studies [57]. In the autograft group 3 of the 5 animals responded positively to the pinch test, and 1 of the 5 animals responded positively in the MC+S+AF(PCL/CG) +T group. This may be attributed to the presence of collagen as it was the only distinguishing factor from the MC+S+AF+T. This data suggests that the MC+S+AF(PCL/CG) +T may have the potential to aid full functional recovery in peripheral nerve repair with a longer recovery time.

The nerve histological assessment provides a direct insight into the efficacy of each NGC, specifically allowing an insight of whether certain components are beneficial for regeneration. Since the histology was conducted at the midpoint of the NGC the presence of regenerated tissue and axons indicate that the pores were beneficial in providing an environment for healthy regeneration throughout an NGC. It has been noted that NGCs without pores often have poor regeneration at the midpoint, as that is the maximum distance from the proximal and distal stumps where nutrient exchange can take place [58]. The presence of regenerated tissue at the midpoint of the NGC indicates the success of the pores in allowing nutrient exchange for healthier nerve regeneration. The variation in the amount of nucleic acids and number of axons, based on the presence of toluidine blue, provides an insight into the impact of each NGC type. While the MC+S+AF(PCL/CG)+T, MC+S+AF+T, MC+S+T, and S+AT+T were comparable to each other, a greater number of axons in the multichannel spiral structures indicates the impact of the channels along the spiral structure. The presence of intraluminal guidance cues can improve cellular alignment and migration, which can improve overall recovery [14, 59]. The aim of incorporating channels along the spiral structure was to provide enhanced intraluminal guidance. The spiral structure provided results comparable to an autograft in our previous study, however this optimization was successful as shown in Fig. 9 [49]. The MC+S+T group had more myelinated axons than the S+AT+T, and when aligned nanofibers were coated on the channels of the multichannel spiral it yielded more myelinated axons. This finding suggests that the multichannel spiral coated with aligned nanofibers retains the cell-cell interaction from our previous study, but also provides the intraluminal support needed for Schwann cell migration, proliferation, and formation of the bands of Büngner. The S+AF+T, S+T, and S+T yielded comparable results, which indicates that the spiral structure may be comparable in performance to a tubular structure in a 15mm gap without the channels along the spiral structure. The outer nanofibrous structure provided structural support for the multichannel and spiral structures since both structures types were sustained throughout the recovery time. Finally, the tubular group yielded the lowest number of myelinated axons which may be due to the absence of intraluminal guidance cues and absence of chemotactic factors.

It has been well documented that the gastrocnemius muscle undergoes denervation once peripheral nerve injury occurs. The denervation of the gastrocnemius muscle causes reduction in muscle weight, hyperplasia of connective tissues, and loss of the enzyme creatine kinase. Creatine kinase is essential for the reformation of adenosine triphosphate (ATP), as it allows for muscle contraction and relaxation [60]. The MC+S+AF(PCL/CG) +T, MC+S+AF+T, and MC+S+T were comparable to the autograft. The S+AT+T, S+T, and T were comparable to each other. Since there was data collected using walking track analysis and pinch reflex analysis, there is indication that there was reinnervation of the gastrocnemius muscle. While all groups did not yield functional recovery data, the recovery of the gastrocnemius muscle indicates that the structures were able to aid nerve regeneration.

Fundamentally, all assays are based on presence of the regenerated axons and while the threshold of detection for each assay may differ some assays provide an insight into if parameters are varied then full recovery may be detected. In the case of the gastrocnemius muscle weight ratio, since there is a varied level of recovery in the groups, with increased recovery time other assays may be yield more positive data.

Conclusion

This study focused on understanding the impact of varying the cross-sectional surface area of NGCs applied for a critical-sized rat sciatic nerve defect at 4-week time point. The design of NGCs aimed to mimic the architecture of the natural nerve, with the presence of inner longitudinally aligned nanofibers, to achieve a comparable outcome. As such, a novel multichannel spiral NGC was fabricated and investigated in this study along with a spiral NGC, both coated with and without aligned nanofibers. While the autograft achieved superior performance to the NGCs, the impact and potential of the multichannel spiral NGC was superior to that of the spiral and tubular structures. This study allowed a further understanding of the impact of cross-sectional geometry on nerve regeneration. While improving cross-sectional surface is desirable, especially for severe injuries, guidance cues and surface area must be coupled in an optimal way to ensure a favorable environment for nerve regeneration. In future work the results of the multichannel spiral NGC will be used to optimize the design of NGCs with multiple channels to further enhance their performance.