Coil-reinforced hydrogel tubes promote nerve regeneration equivalent to that of nerve autografts

Abstract

Despite spontaneous sprouting of peripheral axons after transection injury, peripheral regeneration is incomplete and limited to short gaps, even with the use of autograft tissue, which is considered to be the “gold” standard. In an attempt to obviate some of the problems associated with autografts, including limited donor tissue and donor site morbidity, we aimed to synthesize a synthetic nerve guidance channel that would perform as well as the nerve autograft. Given that the patency of the nerve guidance channel is critical for repair, we investigated a series of nerve guidance channel designs where patency and the resulting regenerative capacity were compared in a transected rat sciatic nerve injury model. Three tube designs were compared to autograft tissue: plain, corrugated and coil-reinforced tubes of poly(2-hydroxyethyl methacrylate-co-methyl methacrylate). Of the three designs, the coil-reinforced tubes demonstrated superior performance in terms of patency. By electrophysiology and histomorphometry, the coil-reinforced tubes demonstrated outcomes that were comparable to autografts after both 8 and 16 weeks of implantation. The nerve action potential (NAP) velocity and muscle action potential (MAP) velocity for the coil-reinforced PHEMA-MMA tube was 54.6±10.1 and 10.9±1.3 m/s, respectively at 16 weeks, which was statistically equivalent to those of the autograft at 37.5±7.9 and 11.3±2.0 m/s. The axon density in the coil-reinforced tube was 2.16±0.61×10⁴ axons/mm², which was statistically similar to that of the autograft of 2.41±0.62×10⁴ axons/mm² at 16 weeks. These coil-reinforced tubes demonstrated equivalence to autografts for nerve regeneration, demonstrating the importance of channel design to regenerative capacity and more specifically the impact of patency to regeneration.

Introduction

Spinal cord injury results from either compression of the cord by, for example, vertebrate fracture, or cord transection due to a stabbing or gunshot wound. Severed spinal tissue lacks the capacity to regenerate due to the secretion of inhibitory and neurodegenerative molecules after injury, the presence of a glial scar, induction of apoptosis, lack of neuroregenerative molecules and the lack of a pathway along which regeneration could be stimulated [1]. The use of entubulation or guidance channels has been investigated by us using biostable PHEMA-MMA tubes [2] and others using biostable PAN/PVC [3], [4], [5] to promote regeneration of the transected spinal cord. Interestingly, most spinal cord entubulation repair strategies have studied non-degradable tubes, such as PHEMA-MMA and PAN/PVC, described above; however, some studies have investigated degradable tubes [6], yet with limited success, possibly due to the instability of the tube during regeneration or the swelling of the tube during degradation. Although the regenerative capacity of the central nervous system is not as profound as that of the peripheral nervous system, both systems require chemotactic cues and appropriate guidance for axonal growth. Thus the peripheral nervous system can be thought of as a model system for spinal cord repair. While repair in the PNS is spontaneous (and it is not in the CNS), repair of the peripheral nerve presents a simpler surgical site and allows many of the design criteria to be more rapidly tested than would be possible in the spinal cord. Thus in this study, where our focus is tube design and determining the optimal design for long-term patency, we investigated regeneration of the rat peripheral sciatic nerve.

After peripheral nerve injury, Wallerian degeneration is observed in the distal end of the injured axon, followed by spontaneous sprouting of fibres from the proximal stump to reinnervate the distal nerve [7], [8], [9]. However, depending on the size of the injury gap and the formation of neuroma and scar tissue, spontaneous reinnervation fails, requiring surgical intervention to bridge the gap. Short gaps, less than 1 cm in humans, can be repaired by suturing the peripheral nerve ends together whereas longer gaps require an autograft [10], [11], [12]. While the autograft is considered the gold standard for nerve repair, it is plagued by numerous deficiencies, including inconsistent results, scar and neuroma pain, scarce availability of donor tissue and donor site morbidity, not to mention the additional surgery required to harvest the donor tissue. To overcome the limitations associated with autografts, several researchers, including ourselves, have investigated synthetic nerve guidance channel alternatives to autografts [13], [14], [15], [16], [17], [18], [19], [20], [21], [22]; however, none of these, including commercially available guidance channels can effectively bridge gaps longer than 3 cm in humans, limiting their use to short gaps. Moreover, the in vivo performance is often sub-optimal as compared to the nerve autograft.

In recent studies we found that poly(2-hydroxyethyl methacrylate-co-methyl methacrylate) (PHEMA-MMA) nerve guidance channels demonstrated equivalent regenerative capacity to nerve autografts for 8 and 16 weeks, but in the 16-week samples, a minority of the channels were sub-optimal, giving rise to a bimodal population in regenerative capacity [23]. PHEMA-MMA has been used in medicine for over 25 years because of its biocompatibility. We have been able to tune the transport and mechanical properties of the hollow fibre PHEMA-MMA membrane used as the nerve guidance channel through control of the formulation [24], [25]. However, it was these tubes that resulted in the bimodal regenerative capacity observed after 16 weeks when used to repair a severed rat sciatic nerve injury gap. We hypothesized that the mechanical integrity of our previous PHEMA-MMA tubes was insufficient to withstand long-term (i.e. 16 weeks) in vivo forces [17]. To overcome this limitation, we investigated a series of designs for the nerve guidance channel and specifically examined the regenerative capacity in corrugated wall tubes and coil-reinforced composite tubes vs. plain tubes and autografts in the transected rat sciatic nerve injury model. Both corrugation and coils are common designs used to reinforce tubes in industrial applications [26], [27], [28], [29], [30], [31], [32] and based on this precedence we chose to investigate these designs for improved patency in nerve repair. Moreover, we were already using a high monomer concentration (of 33 wt%) and were limited by monomer solubility from using greater percent solids and thus could not simply increase wall thickness to achieve sufficient modulus for patency. All tubes were “enhanced” with fibroblast growth factor-1 (FGF-1), dispersed with heparin in a dilute collagen gel because we previously demonstrated enhanced regeneration with this growth factor/matrix mixture [33].