# Patent

## Title
Implantable nerve conduit having a polymer fiber spiral guidance channel

## Abstract

A nerve guidance conduit includes a spiral structured porous sheet decorated with channels on its surface and electrospun nanofibers in a parallel alignment with the channels and an outer tubular structure including randomly-oriented nanofibers. Such a structure provides augmented surface areas for providing directional guidance and augmented surfaces for enhancing and peripheral nerve regeneration. The structure also has the mechanical and nutrient transport requirements required over long regeneration periods. To prepare a nerve guidance conduit, porous polymer sheet is prepared by a solvent casting method while using a template of thin rods to form parallel channels on a surface of the sheet. Aligned nanofibers are deposited on the sheet parallel to the channels. The polymer sheet is then wound to form a spiral structure. A dense layer of randomly-oriented nanofibers may be deposited on the outside of the spiral.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims the benefit of U.S. Provisional Patent Application No. 61/838,553, which was filed on Jun. 24, 2013, and which is incorporated by reference herein.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
The present invention was supported in part by funds from the U.S. government (i.e., NIH Grant No. RO3NS058595, NIH Grant No. R15 NS074404, and the Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Orthopaedic Research Program under Award No. W81XWH-13-02301), and the U.S. government may therefore have certain rights in the invention.


FIELD OF THE INVENTION OR TECHNICAL FIELD
The present invention relates to the field of nerve regeneration, in particular to nerve conduits for the regeneration of peripheral nerves.
BACKGROUND OF THE INVENTION
In the United States, each year more than 700,000 people suffer from peripheral nerve injuries (PNI) that can lead to a lifelong disability, such as paralysis. The most frequent causes include motor vehicle accidents, gunshot wounds, stabbings, and birth trauma.
Currently, there are two gold standard treatments for nerve repair, which are end-to-end suturing and application of autograft or allograft biological tissue. However, each strategy suffers from a number of limitations. For example, end-to-end suturing cannot be performed when the nerve gap is larger than 1 cm. The use of autograft results in potential donor site morbidity for the patient and can potentially exacerbate the condition. The use of allograft tissue has an associated risk of immunogenicity.
Recent advances in tissue engineering and biomaterials suggest that there may be other approaches to nerve repair and regeneration that may overcome the limitations associated with harvesting natural tissues. One such approach would be the use of biomaterials to produce natural or synthetic nerve guidance conduits (NGCs). These NGCs may overcome some of the limitations of nerve autograft and allograft methods. The NGCs act as an essential precursor for nerve repair, since they can reduce tension at the suture line, can protect the regenerating axons from the infiltrating scar tissue, and can exhibit a low immune response. Although FDA-approved tissue engineered nerve devices have been available in the market for several years, these implant devices do not possess the proper physical topography or chemical cues for nerve repair and regeneration. Also, most of them are currently limited to a critical nerve gap of approximately 4 cm. To design an optimal NGC for enhancing PNR still remains a challenge.
Current laboratorial NGCs developed using haptotactic strategies alone are not yet comparable to autograft. For example, multichannel NGCs may have an insufficient cross sectional area and or inhibit cell-cell interaction between each of the individual channels. This may lead to functional mismatches and an insufficient level of regeneration. Controlling the position of inner filament bundles within NGCs has yet to be achieved, despite the fact that the presence of microfilaments has been demonstrated to enhance axonal regeneration and provide contact guidance for the regenerating axons in rats. Alternatively, microfilaments can mislead cell migration which can result in uneven distribution of cells within the NGC. These failures in NGCs may be attributed to the inadequate design of intra-luminal guidance channels/filament, forming incomplete fibrin cables during the initial stages of regeneration. Without the formation of this aligned bridge of extracellular material (ECM), further mechanisms for nerve repair are limited. Therefore, it still remains a challenge to design an optimal NGC for enhancing PNR, when compared to the use of autografts.
SUMMARY OF THE INVENTION
An embodiment of the present invention provides a fabricated implantable NGC. In some embodiments, the NGC comprises an inner spiral structured porous sheet. Such conduits have the potential to serve as medical devices to treat PNI and restore function to the site of the injury. This may be achieved by the spiral structure's ability to facilitate regeneration of nerve tissues.
In another embodiment of the present invention, the NGC has an integrated spiral structured porous sheet decorated with surface channels. Such a structure increases the surface area available for cell migration and attachment, and may reduce the length of time needed for recovery. Additionally, such a structure can reduce the wear and tear that is often observed with single lumen tubular NGCs. A highly-aligned set of electrospun fibers are present within the surface channels and on the backs thereof. The presence of aligned fibers in such areas ensures that the regenerating nerve will come into contact with aligned fibers. In order to place and suture the nerve tissue without tension, there are two reserved chambers at the proximal and distal end of the conduit. The chambers allow for nerve stumps to be sutured without tension due to the fact that the chambers provide space to house the nerve in place with an optimal grip. A dense layer of randomly-oriented fibers on the outside of the spiral structure can greatly improve the mechanical properties of the NGC and provides integrated structural support for nerve regeneration. The spiral conduit is tunable such that its length and diameter can be varied controllably depending on how it is to be used. The length and the outer diameter of the conduit depend on the size of its intermediate sheet, which is the spiral structured porous layer of the NGC. The method of fabricating the conduit does not limit its length, thus enabling the application for longer gap repair/regeneration for PNI.



BRIEF DESCRIPTION OF FIGURES
FIG. 1 is a schematic illustration in cutaway view of a nerve guidance conduit (NGC) according to an embodiment of the present invention bridging the stumps of a damaged nerve;
FIG. 2 is a schematic end-on cross-sectional view of the NGC of FIG. 1;
FIG. 3 is a scanning electomicrograph (SEM) image of a first side of a portion of a porous polymeric sheet of a type used to fabricate NGCs according to an embodiment of the present invention;
FIG. 4 is an SEM image of the side opposite the first side of the porous polymeric sheet of FIG. 3;
FIG. 5 is an SEM image of a porous polymeric sheet having aligned nanofibers thereupon according to an embodiment of the present invention;
FIG. 6 is an SEM image of a porous polymeric sheet having randomly-distributed nanofibers thereupon;
FIG. 7 is a stereomicroscopic image of the exterior of a second NGC;
FIG. 8 is stereomicroscopic image of the NGC of FIG. 7 after being sectioned longitudinally;
FIG. 9 is a stereomicroscopic image of an end-on view of the NGC of FIG. 7;
FIG. 10 is an SEM image of surface channels on a polymer sheet of a type used to fabricate an NGC according to an embodiment of the present invention;
FIG. 11 is a schematic diagram of a polymer sheet of the type shown in FIG. 10;
FIG. 12 is a group of stress-strain plots generated from tests performed on various NGCs which are embodiments of the present invention;
FIG. 13 is a bar chart comparing cell proliferation on various NGCs which are embodiments of the present invention;
FIG. 14 is a plot showing changes in sciatic functional index (SFI) over time for rats having implanted NGCs according to embodiments of the present invention;
FIG. 15 is a bar chart of compound muscle action potentials (CMAP) for rats having implanted NGCs according to embodiments of the present invention;
FIG. 16 is a bar chart of nerve conduction velocities (NCV) for rats having implanted NGCs according to embodiments of the present invention;
FIG. 17 is a bar chart of percent of neural tissue regenerated in sciatic nerves bridged by NGCs according to embodiments of the present invention;
FIG. 18 is a bar chart comparing muscle weight ratios for the gastrocnemius muscle of rats for which the sciatic nerve was bridged by NGCs according to embodiments of the present invention;
FIG. 19 is a bar chart comparing muscle fiber diameter for the gastrocnemius muscle of rats for which the sciatic nerve was bridged by NGCs according to embodiments of the present invention; and
FIG. 20 is a bar chart comparing muscle fiber coverage for the gastrocnemius muscle of rats for which the sciatic nerve was bridged by NGCs according to embodiments of the present invention.



DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the present invention provide NGCs with integrated spiral structured porous sheets decorated with surface channels and electrospun fibers. Such NGCs provide superior mechanical strength compared to NGCs in the prior art, along with integrated multiple channels, stable aligned fibrous layers, good inter-cell communication, and high surface/volume ratios within the NGCs. Chambers at the distal and proximal ends of the NGC provide additional space for fitting nerve stumps in order to reduce the tension at the suture line between the NGC and the nerve stump. A dense outer fibrous tube on the outside of the spiral structured porous sheet can prevent the infiltration of scar tissue while the regeneration process takes place. One embodiment of the NGC of the present invention comprises a three-dimensional (3-D) spiral structured porous sheet having two chambers at the ends thereof. The spiral structure includes a highly porous polycaprolactone (PCL) sheet, which may be formed as a spiral-wound sheet using known methods and decorated with surface channels on a surface of the spiral wound sheet, coated with a thin layer of aligned electrospun fibers on the surface channels, and a dense randomly-oriented fibrous tube on the outside of the NGC. Other bioresorbable materials known for use in the biomedical arts may be used in place of PCL for the sheet and fibers (e.g., collagen/PCL blends for the fibers).
Other embodiments of the present invention provide a process for fabricating an implantable NGC, such as the embodiment of an NGC described above, which can be used as a medical device for facilitating the repair and regeneration of nerve tissues.
Several features of NGCs according to embodiments of the present invention are discussed herein below.
1. Three-Dimensional (3-D) Integrated Spiral Structured Porous Sheet with Proximal and Distal Reserved Chambers
Collagen tubes, which have been approved by the FDA, lack sufficient mechanical strength to support nerve regeneration. As for multi-channel NGCs, the major drawback is that cells/axons in each channel do not interact well with those in the other channels, which adversely affects nerve regeneration and would affect nerve function recovery even if the nerve gap were bridged. In comparison, the integrated spiral structure makes the NGC of the present invention superior to those in the prior art in that mechanical properties are greatly improved and favorable for inter-cellular interaction and neural myelination. This is important for nerve regeneration because of the time required for nerve regeneration to bridge large nerve gaps. Further, a NGC should have enough mechanical strength to provide structural support to the nerve during regeneration. Also, the proximal and distal chambers in the ends of the NGC provide an optimal initial environment for nerve ingrowth. These chambers can prevent stress from accruing when the nerve tissue is sutured with the conduit in an end-to-end fashion. Moreover, the increased surface/volume ratio and the highly porous intermediate layers of the PCL sheet are preferred for cell attachment and nutrient transportation during nerve regeneration.
2. Decorated Surface Channels on the Spiral Porous Sheet with Additional Electrospun Aligned Fibers and an Outer Fibrous Tube
Electrospinning is an approach for polymer biomaterial processing that provides an opportunity to control morphology, porosity and composition of an NGC using relatively unsophisticated equipment. Unlike conventional fiber spinning processes that produce fibers with diameters in the micrometer range, electrospinning is capable of producing fibers in the nanometer diameter range, which are typically deposited in the form of nonwoven fabrics. Nanofibers provide a connection between the nanoscale and the macroscale world, since, although their diameters are in the nanometer range, the fibers are very long, sometimes having lengths of the order of kilometers. A major problem of all hollow tubes is misdirection of cellular migration: since transected axons produce axon sprouts proceeding in a distal direction, a neuroma is always formed which consists of minifascicles proceeding in an abnormal manner, proliferating Schwann cells (SCs), fibroblasts and capillaries. If there is a directional factor of any kind (e.g., an artificial nerve tube which usually provides no endoneurial structure), the neuroma proceeds in the desired direction. This phenomenon has been called “neuromateous neurotization”. In consequence, only a few dispersed axons are able to enter the right fascicle and endoneurial tube in the distal nerve stump once they have reached the end of the conduit in the interior of the NGC.
One successful tissue engineering strategy for nerve repair is to create aligned features on the conduit to provide guidance for cell migration and directional axonal regeneration across the glial scar and lesion site in both central nervous system and peripheral nervous system injuries. Such features may include aligned surface channels and electrospun fiber-based conduits for nerve repair, according to embodiments of the present invention.
Consequently, the construction of a spiral structure conduit with highly aligned surface channels and nano-fibers is very helpful for nerve proliferation and neurite extension. Meanwhile, the intricate aligned structure can also influence the growth and distribution of seeded SCs, which further directs the longitudinal extension of the neural axons. Further, there is a wide range of polymers available that are suitable for deposition on the spiral sheet to meet the individualized specifications for the NGC (e.g., collagen/PCL copolymer nanofibers, rather than pure PCL sheets).
Fibers spun along the outside of the NGC not only assist in stabilizing the spiral structure, but also inhibit infiltration of scar tissue through the inter-connective pores. By increasing the mechanical strength of the NGC, the risk of structural failure can be minimized, promoting more uniform and natural regeneration of nerve tissue.
Tunable Features of the NGC
In order to solve the conflict between optimizing the mechanical properties of the NGC and maximizing its length, many techniques may be used to reinforce the NGC. In a method according to an embodiment of the present invention, a spiral conduit (e.g., a spiral structured porous sheet) is placed onto a rotator and a nanofiber is spun in random orientations along the spiral structure to form an outer fibrous tube. The thickness of the outer fibrous tube can be controlled. This dense layer of randomly-oriented fibers deposited on the outside of the spiral conduit can improve the mechanical properties of the entire structure, and meanwhile provide a stable structural support during nerve regeneration. In a method according to an embodiment of the present invention, depositing the outside layer of fibers on the spiral conduit is the final and separate step of fabricating the NGC, so it is practical to modify the polymers used to form the fibers before the electrospinning step. The outer fibrous tube can be made from polymers that are different from that of the spiral sheet or the aligned fibers.
In another aspect, the process of the present invention is tunable in that the sizes of the spiral conduit are controllable, and both the length and the outside diameter are dependent on the size of the spiral-wound sheet. Therefore, in order to fabricate a spiral conduit with a particular size, (e.g., a length larger than 15 mm, which is the maximum length of nerve regeneration achieved with silicone tubes in rats), it is only necessary to cut a polymer sheet to the appropriate size.
Embodiments of the Present Invention
FIG. 1 is a schematic illustration in cutaway view of a nerve guidance conduit (NGC) 10 according to an embodiment of the present invention bridging the stumps 12, 14 of damaged nerve 16. The stumps 12, 14 are received in reserved chambers 18, 20 at the proximal and distal ends 22, 24 of the NGC 10, and held in place with sutures 26, 28, or by other means known in the art. The reserved chambers 18, 20 allow the nerve stumps 12, 14 to be placed in the NGC 10 and sutured without tension by housing the nerve stumps 12, 14 in place with an optimal grip.
FIG. 2 is a schematic cross-sectional view of the NGC 10 showing that the NGC 10 includes an outer fibrous tube 30 surrounding one or more spiral wound sheets 32 The fibrous tube 30 includes a dense structure of randomly oriented polymer fibers (not shown). The spiral wound sheets 32 define a lumen 34 inside the NGC 10. The lumen 34 is bounded by an inner surface 36 of the spiral wound sheets 32. The NGC 10 further includes an integrated guidance spiral 38 having a plurality of surface channels 40. The guidance spiral 38 is are composed of multiple layers (e.g., layers 42, 44), and together define a spiral guidance channel 46 within the lumen 34. In some embodiments of the present invention, the surface channels 40 are arranged such that they are substantially parallel to each other and to a longitudinal axis (not shown) of the NGC 10. The layers 42, 44 may be extensions of the spiral-wound sheets 32, or may be formed separately therefrom, then integrated with the spiral-wound sheets 32. The plurality of surface channels 40 increases the surface area of the guidance spiral 38 that is available for cell migration and may reduce the length of time needed for nerve regeneration. Additionally, the integrated layers 42, 44 may reduce the wear and tear that can occur in NGCs known in the art. Such wear and tear is often observed with single lumen tubular NGCs.
In some embodiments of the present invention, a highly aligned orientation of electrospun nanofibers (not shown) are provided as coats on the surface channels 40, and on both layers 42, 44 of the spiral sheet 38, and dense randomly-oriented fibers are provided on an outer surface 48 of the NGC 10, which greatly improves the mechanical properties of the NGC 10, as discussed above. In some embodiments, the aligned fibers are substantially parallel to each other. In some embodiments, the aligned fibers are substantially parallel to a longitudinal axis of the NGC 10. The presence of aligned fibers ensures that all areas of the regenerating axon will come into contact with aligned fibers.
The NGC 10 is tunable such that its size can be varied in a controlled fashion depending on how it is to be used. The length and the outer diameter of the NGC 10 are dependent on the size of guidance spiral 38. An NGC 10 according to the present invention may have any length, thus enabling it to be used to repair long gaps in the axon for the repair or regeneration of peripheral nerves.
FIGS. 3 and 4 are scanning electromicrograph (SEM) images a first side and a second side opposite the first side of a portion of a porous polymeric sheet 50 of a type that may be used to fabricate the spiral-wound sheets 32 or guidance spiral 38 of an NGC of the same type as NGC 10, before the application of electrospun nanofibers. Interconnected pores (e.g., pores 52) are present throughout the polymeric sheet 50. FIG. 5 is an SEM image of a porous polymeric sheet 54 of the same type as polymeric sheet 50, showing aligned nanofibers 56 that have been deposited on the polymeric sheet 54 by electrospinning. FIG. 6 is an SEM image of a porous polymeric sheet 58 of the same type as polymeric sheets 50, 54 showing randomly-distributed nanofibers 60 that have been deposited on the polymeric sheet 58 by electrospinning.
FIGS. 7-9 are stereomicroscopic images of an NGC 62 according to an embodiment of the present invention. NGC 62 is of the same general type as the NGC 10 discussed with respect to FIGS. 1 and 2. FIG. 7 is an image of the intact NGC 62 showing its outer fibrous tube 64. FIG. 8 is an image of the interior of the NGC 62 after it has been cut lengthwise, showing an interior surface 66 of the outer fibrous tube 64, the guidance spiral 66, and the reserved chambers 68, 70. FIG. 9 is an end view of the NGC 62 showing the outer spiral wall 64, the guidance spiral 66 and the channels 72 of the guidance spiral 66. FIG. 10 is a SEM image of a portion of polymer sheet 74, which is of a type for making an NGC according to an embodiment of the present invention, showing the substantially parallel alignment of channels 76, which are separated by ridges 78.
Exemplary Fabrication Method
In a method of fabricating an NGC according to an embodiment of the present invention, a polycaprolactone (PCL) sheet was fabricated using a combination of the solvent evaporation method and the salt-leaching method. An 8% (w/v) PCL solution was poured onto a glass petri dish, and acupuncture needles having a diameter of 150 μm were placed on top of the PCL solution to form multi-channels having widths of about 180 μm. The dish was moved to a hood to let it air dry. After an hour, the resulting PCL sheet was immersed into deionized water so that the salt was dissolved, producing pores in the PCL sheet. The needles were also removed, having formed multi-channels on the PCL sheet with widths of about 180 μm. After 30 minutes, the PCL sheet was taken out and dried on a paper towel. Subsequently, 2 hours later, the fully dried PCL sheet was cut into a rectangular shape having dimensions of about 12 mm by 10.5 mm to bridge a 10 mm nerve gap in an animal study.
Referring to FIG. 11, in an exemplary embodiment of the method, the cut PCL sheet 80 had opposite longer edges 82, 84 (i.e., the 12 mm edges), and opposite shorter edges 86, 88 (i.e., the 10.5 mm edges). It may be noted that the channels 90 are substantially parallel to the longer edges 82, 84. Two rectangular areas 92, 94 were cut out from the opposite corners 96, 98 of the edge 82, such that edge 82 was then shorter than edge 84.
PCL aligned nanofibers were spun on the cut PCL sheet 80 using a conductible rotation disk method known in the art. A 16% (w/v) solution of PCL in 1,1,1,3,3,3 Hexafluoroisopropanol (HFIP) (Oakwood Products, Inc) was prepared for electrospinning. Aligned fibers were deposited on the 12 mm×10.5 mm PCL sheet longitudinally on the edge of the rotating disk such that the fibers were substantially parallel to channels 90. The fibers were deposited such that they would be substantially longer than the cut PCL sheet 80. The sheet was carefully removed from the disk to ensure the fibers deposited remained aligned. The excess lengths of fiber (i.e., the portions of the fibers that extended beyond the edges of the cut PCL sheet 80 were collected and folded onto the back of the cut PCL sheet 80.
Turning back to FIG. 11, the cut PCL sheet 80 with the aligned nanofibers thereon was then wound in a spiral fashion from the edge 82 to the edge 84, such that the edge 82 was in the interior of the resulting spiral NGC and the channels 90 were substantially parallel to a longitudinal axis of the spiral NGC. In the spiral NGC, the cutaway areas 92, 94 become reserved chambers (e.g. reserved chambers 68, 70 of spiral NGC 64 of FIGS. 7-9, or reserved chambers 18, 20 of spiral NGC 10 of FIG. 1).
Random nanofibers were then spun onto the outside of the spiral NGC to form an outer fibrous tube on the spiral NGC. The thickness of the outer fibrous tube was approximately 150 μm. The outer fibrous tube is intended to secure the entire spiral structure, enhance the mechanical strength, and prevent tissue infiltration during nerve regeneration. The resulting spiral NGC with its outer fibrous tube was 1.8 mm in diameter and 12 mm in length, suitable for bridging a 10 mm nerve gap.
Tensile Properties of the NGCs of the Present Invention
FIG. 12 is a plot of stress versus strain for several NGCs fabricated according to a method of the present invention: an outer fiber tube comprising a dense layer of randomly-oriented nanofibers; the outer fiber tube with a spiral sheet therein, and the outer fiber tube with the spiral sheet and aligned nanofibers (“AF”). The following tensile properties were measured: Young's Modulus, percent elongation to failure, and tensile strength of the different NGCs. The Young's Modulus, calculated through the stress-strain curve shown FIG. 12, ranged between 0.262-0.7625 Mpa. All three of the NGCs yielded a Young's Modulus that can stand force stretching and be applicable for in vivo use. The values reported for the outer fibrous tube and the other NGCs all in a useful range for use in nerve regeneration and repair. High tensile strength will provide a mechanically strong NGC that can be sutured well during coaptation of the nerve stump and NGC, and preserve the suture after surgery. The measured physical properties of the NGCs of FIG. 11 are summarized in Table 1, below.







TABLE 1







Tensile Properties of Nerve Guidance Conduits











Young's

Tensile



Modulus (MPa)
% Elongation
Strength (MPa)














Outer Fibrous Tube
0.7625
296.4
8.98


Outer Fibrous Tube +
0.33766
171
2.08


Spiral


Outer Fibrous Tube +
0.32766
301
1.78


Spiral + AF









Porosities of the NGCs

The measured porosity values for the outer fibrous tube (hereinafter, NGC-T), outer fibrous tube+spiral (hereinafter, NGC-T-S), and outer fibrous tube+spiral+AF (hereinafter, NGC-T-S-AF) were respectively 71.98±1.22%, 75.01±2.69%, and 78.41±3.64%. The differences in porosities for these three types of NGCs are not statistically significant (p<0.05).
Cell Proliferation
Schwann cells were adopted as the model for evaluation of cellular response on the fiber-based spiral NGCs. At day 4, NGC-T-S-AF showed significantly greater cell proliferation than NGC-T and NGC-T-S. The cell numbers for each type of NGC are shown in FIG. 13. The degrees of cell proliferation for the NGC-T and NGC-T-S are significantly lower (p<0.05) than for the NGC-T-S-AF.
Implantation of NGCs
The NGCs were tested in a 10 mm Sprague Dawley (SD) rat sciatic nerve defect to evaluate the effect of nanofibers on peripheral nerve regeneration through porous spiral NGCs. The sciatic nerve of each rat was cut, then bridged with one of the NGCs. One group received an autograft rather than a NGC. One group received no grafts. All rats were in good condition during the survival weeks. There were no obvious signs of systemic or regional inflammation and surgical complications after implantation
The recovery of motor function was assessed based on the walking track evaluation Referring to FIG. 14, normal sciatic functional index (SFI) value of −9.4±1.4 was measured from all healthy rats (n=30) before surgery. All experimental animals had decreased SFI of values between −85.6 and −94.5 (n=30) by week 2 after surgery. During the initial 4 weeks, there was no significant improvement in any of the groups. At 6 weeks after surgery, the overall SFI reached the levels between −72.2 and −91.7, which was equivalent to an improvement of 2.8-13.4 index points from week 2. Each group's 6-week SFI value was recorded as follows: autograft (−72.2±6.6), T-S-AF (−81.5±3.2), T-S (−88.4±4.9), and T (−91.7±4.2). The autograft SFI revealed a significant difference (p<0.05) as compared to the T-S and T groups. The SFI in the T-S-AF group was significantly higher than for the T groups (p<0.05).
Functional recovery was further evaluated with electrophysiological assessment to determine whether functional recovery occurred through the NGCs. Six weeks post-surgery, compound muscle action potentials (CMAP) were evoked by stimulation at the surgical limbs and recorded from gastrocnemius muscle following by measurements of amplitude and nerve conduction velocity (NCV). Signals were absent and no muscle contractions were observed in the non-grafted group. Referring to FIG. 15, for the amplitude measurements, each group's value was recorded as follows: autograft (5.25±1.51 mV), T-S-AF (4.96±1.58 mV), T-S (3.6±1.39 mV), and T (2.0±0.64 my). Significant differences in amplitude were observed in the T group as compared to the autograft and T-S-AF groups (p<0.05). However, the difference between the autograft, T-S-AF, and T-S groups (p>0.05) was not statistically significant. Similar results were found in NCV measurement: autograft (31.57±4.13 m/s), T-S-AF (26.47±6.87 m/s), T-S (18.28±4.16 m/s), and T (13.3±5.65 m/s) (See FIG. 16). Significant differences in NCV were observed in the autograft group as compared to the T-S and T groups (p<0.05). The NCV result in the T group also showed a significant difference as compared to autograft and T-S-AF groups (p<0.05). However, there were no significant differences when the NCV values of the autograft group were compared to those of the T-S-AF group, which may indicate that nanofibers can accelerate the level of muscle reinnervation as well as autograft.
After 6 weeks post-surgery, the distal nerve segment from each group was explored and carefully isolated from the surrounding tissues. A pinch reflex test was performed distally. A reflex movement of the back muscles indicates that the sensory fibers are positively regenerated through the NGCs, while no movement was considered as lack of sensory fibers in the NGCs. The results are presented in Table 2, below.







TABLE 2







Pinch Test Results









Number of rats



responding to pinch test



(n = 5)














Autograft
5/5



T-S-AF
5/5



T-S
4/5



T
3/5










Further histological evaluations of nerve regeneration behavior with NGCs were investigated under a light microscope. The results clearly demonstrated the potential of the NGCs of the present invention to house a large number of supportive cells, both with and without nanofibers to enhance the surface area of the channel. The NGCs possessed durable mechanical strength to support the entire regeneration process. Low magnifications of micrographs showed that neural tissues, including myelinated axons and myelin sheath, were all successfully presented among the groups. Angiogenesis occurred through which new blood vessels were formed during the nerve regeneration process. Normal axons were nearly all surrounded by uniform thicknesses of myelin sheaths and presented large fiber diameters. Nevertheless, the studied groups presented premature morphologies (i.e., diverse nerve fiber sizes and thinner myelin sheaths).
Quantitative analysis of the total occupied neural tissue coverage in the NGCs compared to those of normal rat nerves (70.57±3.81%) further confirmed the above findings. Referring to FIG. 17, each group's value was recorded as follow: autograft (29.29±4.61%), T-S-AF (26.52±3.77%), T-S (17.37±2.97%), and T (5.88±1.43%). No significant differences were found among autograft and T-S-AF groups. However, the area occupied by neural tissue in T-S group showed significantly lower values than the autograft, and T-S-AF groups. High significance was observed in the T group as compared to the other groups (p<0.01). Finally, it should be noted that the cross-sectional micrograph of T group was covered with a large white area. That implied the single lumen repair limited the nerve regeneration.
When severe nerve injury occurs, the muscle is denervated and the balance of muscle metabolism could be shifted from protein synthesis toward protein degradation. As a consequence, the target muscle presents a decreased muscle cell size, muscle weight loss, hyperplasia of connective tissues, and new blood vessel formation. To evaluate the reinnervation of the gastrocnemius muscle, Masson trichrome staining was applied to the section followed by measurements of muscle weight ratio, diameter of muscle fibers, and muscle fiber coverage per cross section. Referring to FIG. 18, for comparisons of muscle weight ratio, each group's value was recorded as follows: autograft (39.73±4.19%), T-S-AF (25.64±3.01%), T-S (22.31±2.18%), and T (19.2±2.03%). The muscle weight ratio of the autograft group was greater than that of the other groups by a statistically significant amount (p<0.05). However, there were no significant differences between the T-S-AF and T-S groups (p>0.05). The T group revealed a significant lower ratio than the T-S-AF group.
Referring to FIG. 19, for comparisons of muscle fiber diameter, each group's value was recorded as follows: autograft (34.62±1.05 μm), T-S-AF (31.81±2.18 μm), T-S (25.5±6 μm), and T (21.56±2.98 μm). Although the autograft group showed a significant difference from the T-S and T groups, it was not significantly higher than the T-S-AF group. Also, there were no significant differences between the T-S and T groups (p>0.05). Further findings showed that the value for the T group was significantly lower than that for the autograft, and T-S-AF groups.
Referring to FIG. 20, for comparisons of muscle fiber coverage, each group's value was recorded as follows: autograft (96.84±4.1%), T-S-AF (93.72±4.63%), T-S (86.99±10.31%), and T (58.42±4.69%). There were no significant differences between the values for the autograft, T-S-AF, and T-S groups (p>0.05); however, they were all significantly greater than the value for the T group (p<0.05).
From qualitative analyses and histological observations discussed above, spiral NGCs of the present invention, with or without nanofibers, revealed the potential to prevent muscle atrophy as well as the effect of autograft. Both the surface channels and the aligned fibers provide good topographical cues for nerve regeneration, and thus allow muscle reinnervation faster than single lumen NGCs, thus suggesting that the surface channels and nanofibers further assisted NGC structures in promoting nerve regeneration.
It should be understood that the embodiments described herein are merely exemplary in nature and that a person skilled in the art may make many variations and modifications thereto without departing from the scope of the present invention. All such variations and modifications, including those discussed above, are intended to be included within the scope of the invention, as defined by the appended claims.

## Claims

1. A nerve guidance conduit for promoting regeneration of a severed nerve, comprising:
a spiral structured porous sheet wound in a first spiral geometry defining a lumen extending from a proximal end of said spiral structured porous sheet to a distal end of said spiral structured porous sheet opposite said proximal end, said proximal and distal ends of said spiral structured porous sheet defining a longitudinal axis of said nerve guidance conduit said lumen, said proximal chamber being arranged to receive a first stump of a nerve and said; and
a guidance spiral within said lumen and aligned with said longitudinal axis, said guidance spiral integrated with said spiral structured porous sheet, said guidance spiral being porous and having at least two layers, wound in a second spiral geometry, said guidance spiral including at least one surface channel that is on a surface of said guidance spiral, said at least one surface channel extending from said first end of said guidance spiral to said second end of said guidance spiral and providing guidance for cell migration and directional axonal regeneration, said spiral structured porous sheet and said first end of said guidance channel defining a proximal chamber within said lumen that is arranged to receive, house, and grip a first stump of a severed nerve at said proximal end of said spiral structured porous sheet, said spiral structured porous sheet and said second end of said guidance channel defining a distal chamber within said lumen that is arranged to receive, house, and grip a second stump of the severed nerve at said distal end of said spiral structured porous sheet, whereby said nerve guidance conduit bridges a gap between the first and second stumps of the severed nerve and prevents stress from accruing in the first and second stumps when the first and second stumps are sutured to the nerve guidance conduit.

2. The nerve guidance conduit of claim 1, further comprising a plurality of aligned nanofibers on said plurality of surface channels, said plurality of aligned nanofibers being substantially parallel to said plurality of surface channels.

3. The nerve guidance conduit of claim 1, further comprising an outer fibrous tube around said spiral structured porous sheet, said outer fibrous tube including a plurality of nanofibers randomly distributed on said spiral structured porous sheet.

4. The nerve guidance conduit of claim 1, wherein said at least one surface channel includes a plurality of surface channels which are substantially parallel to each other.

5. The nerve guidance conduit of claim 1, wherein at least one of said layers of said guidance spiral is continuous with said spiral structured porous sheet.

6. The nerve guidance conduit of claim 1, wherein said plurality of surface channels are defined by at least one of said at least two layers.

7. A nerve guidance conduit for promoting regeneration of a severed nerve, comprising:
a spiral structured porous sheet wound in a first spiral geometry defining a lumen extending from a proximal end of said spiral structured porous sheet to a distal end of said spiral structured porous sheet opposite said proximal end, said proximal and distal ends of said spiral structured porous sheet defining a longitudinal axis of said nerve guidance conduit, said porous sheet further defining at least one layer of a guidance spiral that is wound in a second spiral geometry and aligned with said longitudinal axis, said guidance spiral including a plurality of surface channels that are substantially parallel to each other on a surface of said guidance spiral, said plurality of surface channels extending from said first end of said guidance spiral to said second end of said guidance spiral and providing guidance for cell migration and directional axonal regeneration;
a plurality of aligned nanofibers on at least one of said plurality of surface channels; and
an outer fibrous tube around said spiral structured porous sheet, said outer fibrous tube including a plurality of nanofibers randomly distributed on said spiral structured porous sheet,
wherein said spiral structured porous sheet and said first end of said guidance channel define a proximal chamber within said lumen that is arranged to receive, house, and grip a first stump of a severed nerve at said proximal end of said spiral structured porous sheet, said spiral structured porous sheet and said second end of said guidance channel define a distal chamber within said lumen that is arranged to receive, house, and grip a second stump of the severed nerve at said distal end of said spiral structured porous sheet, whereby said nerve guidance conduit bridges a gap between the first and second stumps of the severed nerve and prevent stress from accruing in the first and second stumps when the first and second stumps are sutured to the nerve guidance conduit.

8. The nerve guidance conduit of claim 7, wherein said guidance spiral includes at least another porous sheet arranged such that said guidance spiral has at least two layers.