Elsevier

Biomaterials

Volume 29, Issue 21, July 2008, Pages 3117-3127
Biomaterials

The role of aligned polymer fiber-based constructs in the bridging of long peripheral nerve gaps

https://doi.org/10.1016/j.biomaterials.2008.03.042Get rights and content

Abstract

Peripheral nerve regeneration across long nerve gaps is clinically challenging. Autografts, the standard of therapy, are limited by availability and other complications. Here, using rigorous anatomical and functional measures, we report that aligned polymer fiber-based constructs present topographical cues that facilitate the regeneration of peripheral nerves across long nerve gaps. Significantly, aligned but not randomly oriented fibers elicit regeneration, establishing that topographical cues can influence endogenous nerve repair mechanisms in the absence of exogenous growth promoting proteins. Axons regenerated across a 17 mm nerve gap, reinnervated muscles, and reformed neuromuscular junctions. Electrophysiological and behavioral analyses revealed that aligned but not randomly oriented constructs facilitated both sensory and motor nerve regeneration, significantly improved functional outcomes. Additionally, a quantitative comparison of DRG outgrowth in vitro and nerve regeneration in vivo on aligned and randomly oriented fiber films clearly demonstrated the significant role of sub-micron scale topographical cues in stimulating endogenous nerve repair mechanisms.

Introduction

Severe traumatic injury or invasive surgical procedures on a peripheral nerve can result in a gap between two nerve stumps. The clinical “gold standard” for bridging peripheral nerve gaps is the use of autografts (typically, the sensory sural nerve). However, the use of autografts is limited by the following issues: (1) limited availability of nerves to use in the autograft [1], (2) secondary surgery, (3) lack of coaptation between the injured nerve and the nerve graft due to size/length/modality mismatch [2], and (4) functional loss, such as numbness at the donor sites [3]. Moreover, complications at the donor site such as hyperesthesia or formation of painful neuromas also have to be addressed [4], [5]. Therefore, it is imperative that alternative approaches that are ready-to-use, pre-customized for reducing the mismatch, and suitable for both sensory and motor nerve regeneration are developed.

Polymeric tubular nerve constructs have been used clinically for repairing peripheral nerve injury [6]. These nerve constructs, made of either non-porous silicone or porous natural/synthetic polymers, bridge the injured nerve stumps when the gaps are <10–12 mm in rats by enabling the formation of a fibrin cable that provides a substrate for the migration of Schwann cells. The migrated Schwann cells from both nerve stumps reorganize to create longitudinally oriented bands of Bungner, which serve as guiding substrates and a source of neurotrophic factors to foster axonal regrowth [7], [8]. However, polymeric conduits alone are limited in their ability to enable regeneration across long nerve gaps (>15 mm in rats). Failure of nerve regeneration across long gaps seems to be the result of a lack of the formation of an initial fibrin cable, which is necessary for Schwann cell migration into the constructs and the formation of the bands of Bungner which are aligned columns of Schwann cells and laminin [9].

In this study, we used an electrospinning process to create 10–20 μm thin polymer films made of either aligned or randomly oriented sub-micron scale polymeric fibers (∼400–600 nm in diameter). To examine the effects of sub-micron topography on neurite outgrowth/Schwann cell migration, dorsal root ganglia (DRG) were seeded on the films in vitro, and the extent of neurite outgrowth/Schwann cell migration was quantified. These two-dimensional (2D) fiber films were stacked in a 3D configuration to build implantable polymeric constructs with inherent topographical cues that were either aligned or randomly oriented. Polymer fiber-based constructs were implanted across 17 mm tibial nerve gaps in adult rats to determine if topographical cues were sufficient to stimulate endogenous regeneration of nerves even in the absence of biochemical cues such as neurotrophic factors or extracellular matrix proteins. Saline filled polymeric constructs and autografts were used as controls. Our results indicate that robust regeneration across the tibial nerve gap occurred in the aligned fiber and autograft implants, but not in the randomly oriented or saline filled constructs. These results clearly illustrate the significance of using fibers to create sub-micron scale, oriented topographical features in stimulating endogenous repair mechanisms, presumably by enhancing the efficiency of Schwann cell migration and axon outgrowth across the nerve gap. These conclusions are strengthened by a quantitative analysis of regeneration that included evaluation of reinnervation of distal targets (i.e., muscle) by immunohistological, histomorphometrical, electrophysiological, and behavioral analysis.

Section snippets

Fabrication of aligned and random fiber films

Uniaxially aligned fiber films were fabricated by electrospinning poly(acrylonitrile-co-methylacrylate) (PAN-MA), random copolymer, 4 mol% of methylacrylate, on a high-speed rotating metal drum [10]. Briefly, 18% PAN-MA (w/v) solution was prepared by dissolving PAN-MA in the organic solvent N,N,-dimethyl formamide (DMF, Acros) at 60 °C. The polymer solution was loaded into a 10 ml syringe and delivered at a constant flow rate (1 ml/h) through a metal needle (19 gauge) connected to a high-voltage

Fabrication and morphological characterization of fiber films

Either uniaxially aligned or random fiber films were fabricated by an electrospinning process (see Section 2) (Fig. 1E). The diameter of the individual fibers ranged between 400 nm and 600 nm (Fig. 1F and H) and the thickness of the films was approximately 10–20 μm (Supplementary Fig. 1D). This thickness was chosen to enable manual handling (e.g., peeling off from the aluminum foil), which was required for stacking them into the polymeric constructs. Quantification of individual fiber alignment

Discussion

The results of this study demonstrate that aligned polymer fiber-based constructs (aligned constructs), but not randomly oriented polymer fiber-based constructs (random constructs), significantly enhance peripheral nerve regeneration in a challenging 17 mm nerve gaps in rats, as evaluated by anatomical as well as functional measures. The significance of these results is that sub-micron scale topographical cues alone, with no exogenous neurotrophic or extracellular matrix proteins, enabled

Conclusion

Topographical cues can influence endogenous peripheral nerve repair mechanisms even in the absence of exogenous growth promoting proteins. Aligned, but not randomly oriented, polymer fiber constructs successfully promoted regeneration of axons across a 17 mm nerve gap, reinnervating muscles, and reforming new neuromuscular junctions, as verified through histological, electrophysiological, and behavioral analysis. Thus, aligned polymer fiber-based constructs are potentially a significant

Acknowledgements

Funding for this research project was provided by NIH NS44409 (RVB) and GTEC (NSF EEC-9731643). The authors thank Isaac Clements, Dr. Robert H. Lee, Dr. Mahesh Dodla, George McConnell, Tong Wang, and Hong Shen for technical assistance toward this work.

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