Tissue-engineered spiral nerve guidance conduit for peripheral nerve regeneration

Abstract

Recently in peripheral nerve regeneration, preclinical studies have shown that the use of nerve guidance conduits (NGCs) with multiple longitudinally channels and intra-luminal topography enhance the functional outcomes when bridging a nerve gap caused by traumatic injury. These features not only provide guidance cues for regenerating nerve, but also become the essential approaches for developing a novel NGC. In this study, a novel spiral NGC with aligned nanofibers and wrapped with an outer nanofibrous tube was first developed and investigated. Using the common rat sciatic 10-mm nerve defect model, the in vivo study showed that a novel spiral NGC (with and without inner nanofibers) increased the successful rate of nerve regeneration after 6 weeks recovery. Substantial improvements in nerve regeneration were achieved by combining the spiral NGC with inner nanofibers and outer nanofibrous tube, based on the results of walking track analysis, electrophysiology, nerve histological assessment, and gastrocnemius muscle measurement. This demonstrated that the novel spiral NGC with inner aligned nanofibers and wrapped with an outer nanofibrous tube provided a better environment for peripheral nerve regeneration than standard tubular NGCs. Results from this study will benefit for future NGC design to optimize tissue-engineering strategies for peripheral nerve regeneration.

Statement of Significance

We developed a novel spiral nerve guidance conduit (NGC) with coated aligned nanofibers. The spiral structure increases surface area by 4.5 fold relative to a tubular NGC. Furthermore, the aligned nanofibers was coated on the spiral walls, providing cues for guiding neurite extension. Finally, the outside of spiral NGC was wrapped with randomly nanofibers to enhance mechanical strength that can stabilize the spiral NGC. Our nerve histological data have shown that the spiral NGC had 50% more myelinated axons than a tubular structure for nerve regeneration across a 10 mm gap in a rat sciatic nerve. Results from this study can help further optimize tissue engineering strategies for peripheral nerve repair.

Introduction

Peripheral nerve injuries (PNI), often caused by trauma or iatrogenic injury [1], present a serious clinical problem, potentially affecting motor activity or sensation in the affected area. Although FDA-approved nerve guidance conduits (NGCs) have been commercially available for several years, these tubular NGCs provide an insufficient cross sectional surface area to support a large number of cells needed for complete functional regeneration. Additionally, the lack of sufficient mechanical strength to support peripheral nerve regeneration (PNR) may cause further injury and inhibit nerve regeneration. Most tubular NGCs are not able to successfully repair critical sized nerve gaps (2.5 cm in human) [2]. Additionally in current implementations Fibrin cables and Schwann cell tubes are formed in a discontinuous manner without any inner structure support that lead to reduced functional regeneration [3]. Therefore, tubular NGCs often limit the degree of PNR, leading to inappropriate regenerating axons surrounded by extracellular matrix tissue [4], [5].

New strategies may include chemotactic (ex. additives) or combinations of both chemotactic and haptotactic (ex. multi-channel), which can enhance the successful rate of nerve regeneration [6], [7], [8], [9]. However, chemotactic approaches require a length approval process by FDA as they are considered a therapeutic and many of the effects are currently unknown. Moreover, usage of haptotactic alone with NGCs (ex. Multi-channel or intra-luminal fillers) have not yielded any comparable outcomes to autograft [10], [11], [12]. This is due to the absence of interconnected pores in multi-channel NGCs, which inhibit the cell-cell interaction between each individual channel. When aligned fibers/filaments are incorporated within conduit, these fibers/filaments are randomly inserted into the hollow conduit, leading to uneven distribution. Without any well-organized inner support, these aligned fibers/filaments may stick together or precipitate to the bottom of conduit after implantation, which may cause uneven tissue regeneration [13]. Thus, inadequate intra-luminal guidance cues could form incomplete fibrin cables during the initial stages of regeneration, which could limit further processes of nerve reinnervation [3], [14], [15], [16], [17]. The difficulty of creating complex 3D structures with even stereological aligned fibers matrix for novel NGC remains a challenge, this in turn reduces efficacy of the treatment leading to lessened motor function and sensation.

Recent advances in tissue engineering seek to overcome the limitations associated with these aforementioned issues. Our previous in vitro studies for nerve regeneration [18], [19], cartilage regeneration [20], [21], [22], [23], and bone regeneration [24], [25], [26] showed that a novel spiral scaffold with electrospun fibers aided cellular activities and tissue formation significantly higher than tubular or cylindrical scaffold. These results can be attributed to a highly porous 3D spiral channel and electrospun nanofibers on the spiral wall that increases the surface area to volume ratio, which can evenly disperse the spiral channel in stereological fashion. The addition of electrospun fibers enable formation of 3D fibrous matrices providing a guidance cue to improve cellular activities. Several in vivo studies [27], [28], [8], [29] have shown that aligned nanofibers enhance nerve regeneration and are able to bridge large nerve gaps (over 15 mm in rats) by ensuring that the axons would follow the shortest distance, extending toward the distal stumps.

Based on the aforementioned concepts, the novel spiral NGC with inner aligned nanofibers and wrapped outer nanofibrous tube was developed and scaled down the wall thickness and diameter in order to match the optimal conditions for our first animal study. The efficacy of the NGC was investigated based on the results of walking track analysis, electrophysiology, gastrocnemius muscle measurement, and histological assessment. It was hypothesized that a novel spiral NGC with aligned nanofibers and wrapped with an outer nanofibrous tube could enhance the rate of nerve regeneration for a 10 mm rat sciatic nerve gap.