Due to the difficulty of electrospinning nerve conduits composed of nanofibers aligned along the conduit's long axis, researchers in previous studies21,22
either rolled a film with aligned nanofibers to form a conduit or filled a silicone tube with aligned nanofiber sheets. Here we developed a one-step electrospinning process to fabricate a novel, seamless, tubular nanofibrous nerve conduit composed of two fully integrated layers: a luminal layer with longitudinally aligned nanofibers and an outer layer with randomly organized nanofibers. To our knowledge, this was the first demonstration that a bi-layer tubular device with longitudinally aligned nanofibers can be directly electrospun as a unified, seamless construct.
Unlike previous attempts, the device and process described here are much more amenable and scalable for manufacturing and clinical use. The bi-layer design is likely to provide better suturability and mechanical integrity than a conduit composed entirely of longitudinally aligned nanofibers. Mechanical testing and in vivo results showed that this bi-layer nerve conduit has adequate mechanical strength for suturing and for supporting nerve growth. The in vitro degradation study demonstrated that the nerve guides maintained structural integrity to support nerve growth under physiological conditions for clinically relevant time periods. Direct electrospinning of bi-layer nanofibrous conduits is a fast process that avoids the tedious and unreliable process of rolling and sealing sheets and easily adapts to larger conduit sizes and longer lengths. The seamless construction of the bi-layer nanofibrous conduit also presents a smooth, even luminal surface for nerve growth and poses no risk of mechanical failure or separation at the seam.
We evaluated the nerve regeneration capacity of bi-layer aligned nanofibrous conduits in a rat sciatic nerve transection model with random nanofibrous conduits and autografts as controls. Nerve regeneration and muscle innervation were assessed at 2-month and 12-month time points by using histomorphometry analysis, electrophysiology measurement, and behavior test.
Electrophysiological analysis demonstrated the superior capability of aligned nanofibrous nerve conduits in nerve regeneration when compared to random nanofibrous nerve conduits. Based on the 2-month results, better functional recovery in terms of CMAP amplitude and conduction velocity was observed in the aligned nanofibrous nerve conduit group than in the random nanofibrous nerve conduit group. Interestingly, the advantage of the autograft was not shown in this early recovery period. One explanation is that that autograft may have to remodel its existing cellular and matrix contents (e.g., degradation and reorganization) to allow the ingrowth of regenerating axons. The 2-month electrosphysiology results suggest that aligned nanofibrous conduits were the most efficient in accelerating nerve functional recovery at the early phase. At 12-month time point, both aligned nanofibrous nerve conduit and autograft performed significantly better than random nanofibrous nerve conduit, and there was no statistical difference between aligned nanofibrous nerve conduits and autografts. These results indicate that nanofiber organization had long-term effects on nerve regeneration and that the in vivo performance of aligned nanofibrous nerve conduits is similar to autografts, which is the current gold standard of treatment for peripheral nerve injuries. The results from behavior tests also demonstrated the same trend.
Histological analysis of explanted nerve samples showed myelinated axons, vasculature, and epineurial sheaths in both random and aligned nanofibrous nerve conduits at 2 months and 12 months, similar to that in autografts. Quantitative analysis revealed a higher frequency of large-diameter axons and thick myelin sheaths for the aligned nanofibrous nerve conduit group compared to random nanofibrous nerve conduit group at both time points. Temporal comparison showed obvious shifts toward larger axons and thicker myelin sheath at 12 months for both the aligned nanofibrous nerve conduit and autograft groups. In contrast, the axon diameter and myelin sheath thickness in the random nanofibrous nerve group at 12 months only showed marginal increase. The axon diameter in aligned nanofibrous conduits and autografts had a similar distribution profile. Interestingly, the myelin sheath was generally thicker in autografts than in aligned nanofibrous conduits. It is possible that pre-existing Schwann cells in autografts played an important role in the myelination of regenerating axons, which may explain the difference in myelination between synthetic grafts and autografts.
The difference in the distribution profiles of axon characteristics between aligned and random nanofibrous nerve conduits suggests that longitudinally aligned nanofibers accelerate growth of large myelinated axons, which are morphological characteristics of motor neurons. The presence of larger axons with thicker myelin sheaths may also account for the higher CMAP amplitude and faster conduction velocity measured in the aligned nanofibrous nerve conduit group. A major limitation of nerve repair in humans is the slower growth of motor nerve fibers and relatively poor re-innervation of target muscle compared to sensory nerve fiber growth.1,15
The potential ability of aligned nanofibrous nerve conduits to improve the regeneration of motor nerve fibers and to match the biological performance of autografts merits further study.
Another interesting finding is the difference in the thickness of the fibrous tissue layer on the luminal surface of random and aligned nanofibrous nerve conduits. At 2 months, a thin continuous epineurial-like layer developed at the nerve–conduit interface in both random and aligned nanofibrous nerve conduit groups. At the 12 month time point, a dense connective tissue stroma formed around the regenerated nerve in the random nanofibrous nerve conduit group, whereas a thin tissue layer resembling a normal epineurium was observed in the aligned nanofibrous conduit group. These results suggest that random and aligned nanofibers not only had different effects in the early phase of nerve regeneration, but also exerted long-term effects during the maturation and remodeling of the regenerated nerve. These results may also suggest another advantage of the seamless nanofibrous conduit over nerve conduits with seams or discontinuous luminal surfaces, which may be more susceptible to fibrous capsule formation. The underlying mechanism of fibrous layer formation on the luminal surface of nerve conduits is not clear. Intriguingly, a recent study demonstrated that aligned poly(caprolactone) nanofibers reduced monocyte adhesion but had thicker fibrous capsule on the surface compared to random nanofibers within 4 weeks,28
which could be explained as acute foreign body responses. However, the growth of fibrous layer in our nerve conduits was only found on the luminal surface but not outer surface, and the growth happened between 2 and 12 months. It is likely caused by long-term tissue remodeling instead of acute inflammatory responses. One possible explanation for the differences in epineurial thickness is the faster cell proliferation rate and matrix synthesis rate on random nanofibers. Indeed, we have shown that aligned smooth muscle cells on micropatterned surfaces have lower proliferation rate.29,30
Whether this is the case in nerve conduits awaits further investigation.