(758d) Biodegradable Scaffolds for Large Peripheral Nerve Defect Regeneration: Electrical and Chemical Stimulation

Every year, more than half a million Americans suffer from peripheral nerve injuries (PNI) that require surgical treatments totaling close to $2 billion in healthcare costs, but many continue to experience pain and/or poor functionality ¹. These severe PNI are typically characterized by large gaps >5 mm that are unable to regenerate on their own without surgical intervention. The main treatments for PNI involve surgical implantation of autografts or allografts, but these are frequently limited in availability and potentially cause immunosuppression, donor site morbidity, scarring, neuroma formation, and sensory loss. Tissue engineering (TE) approaches for nerve repair and regeneration, which use scaffolds, cells, and/or growth factors, can be effective alternatives to biological grafts; however, due to limits on the size of the nerve gap that can be repaired, biocompatibility, and inefficient stimulation of nerve regeneration, they are often not used or only used in specific cases. Ineffective PNI repair – a potential outcome for all current treatments can cause severe loss of sensory and/or motor function, and more severe damage is associated with slower recovery, highlighting an unmet need for better treatments. Chemical and electrical stimulation have the ability to enhance endogenous nerve regeneration in less severe PNI injuries, and may have utility in severe PNI repair ².

Electrical stimulation (ES) as a physical therapy modality has shown benefits in the functional rehabilitation of muscle, bone, skin, tendons, ligament, and nerve injuries (2-4). Specifically, ES enhances cell proliferation, extracellular matrix synthesis, cytokine production, and vasculature development to promote overall wound healing. However, the underlying mechanisms driving these functions are poorly understood. ES of injured peripheral nerves can accelerate axonal regeneration and functional recovery in laboratory animals and human clinical trials; and accelerated axonal regeneration is correlated to higher endogenous expression of BDNF neurotrophin family growth factors and its high-affinity receptor TrkB. Furthermore, although a few clinical trials and animal studies involving ES at low to moderate treatment intensity demonstrated reductions in inflammatory and immunological responses through altered cytokine or chemokine release and/or cell activation, survival, proliferation, or death, ES effects on implant immune responses have not been studied. Additionally, the full potential of ES-mediated tissue regeneration in large-area tissue defects including large-gap PNI is not known, and challenges exist in the application of reliable stimulation to achieve consistent healing outcomes for nerve crush injuries. One challenge with applying ES in vivo is that ES signal transduction from dermis nerves to skin-surface electrodes is not reliable for stimulation or measurement in the absence of suitable connecting materials. Nonetheless, a recent expert article describes the use of ion-conducting hydrogels as an electrode interface that improves signal transduction to deliver reliable ES to nerves, which may be less painful for long-term application. Therefore, more comprehensive and in-depth studies are needed to enhance regenerative capacity and functional outcomes in large-gap PNI.

Chemical stimulation with the potassium-channel blocker 4-aminopyridine (4-AP) can restore the myelination of demyelinated nerves in multiple sclerosis – most likely through its ability to prolong action potentials and amplify neurotransmitter release; myelin ensheathes axons and acts as an electrical insulator, greatly speeding up action potential conduction ³. Application of 4-AP to crush nerve injuries – where axons are intact but the myelin sheath is damaged – can also increase remyelination and nerve conduction velocity, leading to functional recovery. Therefore, 4-AP has potential to enhance large-gap PNI repair, but local application in large-gap PNI and its effects on regeneration and functional recovery have not been studied.

Delivering growth factors or cells with scaffolds can stimulate nerve regeneration processes, but chemical and electrical stimulation may have similar and additional benefits with fewer limitations ¹. Neurotrophins such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) can improve nerve regeneration, but proteins can have high dose requirements, short half-lives, high costs, and undesired side effects. Schwann cells and other neuronal support cells can accelerate nerve regeneration as well, but delivering viable cells can be cumbersome and costly. No efforts have been made to encourage infiltration of endogenous Schwann cells using a drug or any other external cue to improve large-gap PNI. ES and 4-AP may accomplish this goal but will require innovative design of new polymers and delivery methods.

Ionically conductive (IC) polymers and sulfonated chitosan especially, confer high and sustained conductivity, overcoming limitations of other materials in ES applications ^4,5. IC polymers conduct electrical charges via the flow of counter ions in the physiological environment, which is different from electronically conducting polymers, and can be synthesized to carry positive (cation) or negative (anion) charges from well-studied biodegradable polymers, such as chitosan, silk, and poly(lactic acid). Degradation mechanisms and rates vary by polymer type, and include enhanced surface erosion via ionic group modification-conferred hydrophilicity of the polymer. We have developed sulfonated IC polymers with excellent tunable electrical conductivity for neural tissue engineering, and found that sulfonated chitosan has higher initial and sustained conductivity than other sulfonated and non-sulfonated polymers. The non-degradable sulfonated polymers poly(ether ether ketone) (S-PEEK) and poly(aniline) (S-PANI) also showed sustained conductivity.

Results and Discussion: Our scaffold system is based on cross-linked sulfonated (S) chitosan, a natural sugar polymer found in crustaceans and fungi, and designed with: high-sustained conductivity critical for long-term ES; strength and flexibility for kink resistance and suturability; aligned pores for cell guidance; and nanotubes for controllable small molecule release. We developed a freeze-drying technique to create cross-linked S-chitosan IC scaffolds with longitudinally aligned pores optimal for maximum axon penetration and minimum axon misdirection ³. These also accommodated and guided Schwann cells needed for myelination of axons (data not shown). In our preliminary scaffold formulation, 600–900 nm halloysite (alumosilicate clay) nanotubes (HNT) were filled with 4-AP and uniformly dispersed in IC chitosan solution to fabricate porous scaffolds. These IC scaffolds are kink-resistant and suturable, with suture retention strength ranging 18–20 N. Drug-filled HNTs are sealed with chitosan at both ends before combining with chitosan solution. By varying HNT content and cross-link density, we can tune the architecture to match native peripheral nerve mechanical modulus and control drug release. The ability to control localized physical and chemical signals with scaffolds will allow extended treatment periods to achieve motor function not typically seen with short-term treatments.

4-AP induces dose-dependent increases of neurotrophins in human Schwann cells in vitro. Human Schwann cells cultured for 14 days with varying doses of 4-AP induced expression of NGF, P0, and BDNF neurotrophins. These neurotrophins promote axon regeneration, remyelination, and motor function recovery. IC scaffolds support human Schwann cell adhesion, proliferation, and neurotrophin secretion in response to 4-AP and ES in vitro. We published that IC scaffolds can support Schwann cell adhesion, proliferation, and migration along aligned pores, as well as secretion of neurotrophins in response to 4-AP. Intriguingly, our new preliminary data suggest that compared with ES or 4-AP alone, combined 4-AP+ES treatment increases expression of neurotrophins BDNF, NGF, and S100 in Schwann cells grown on IC scaffolds in vitro at day 14 ³.

Combined ES+4-AP treatment improves foreign body responses compared to IC scaffolds alone in vivo. To test whether ES, 4-AP, and/or 4-AP+ES elicit an immune response compared to scaffolds alone, we implanted scaffolds subcutaneously in rats, injected IC scaffold formulations as injectable electrodes to deliver ES treatment groups, and applied ES every other day. Blood samples from tail veins were collected several times over 14 days to analyze IL-6 and IL-10 cytokines by ELISA. Levels of IL-6 and IL-10 fluctuated over time (not shown), and the IL-6/IL-10 ratio – a measure of immune response – dropped initially and stabilized thereafter indicating no strong immune response to implanted scaffolds over 14 days. Fiber capsules can form around implanted foreign material – another measure of immune response – therefore, we measured scaffold fiber capsule thickness using H&E-stained sections. Reduced fiber capsule thickness is an indicator of reduced foreign body response and enhanced host-tissue integration.

IC scaffolds+4-AP and autografts confer similar early-stage sciatic nerve defect repair. Our preliminary data using 4-AP-loaded chitosan-HNT (scaffolds+4-AP) in this same 15 mm nerve defect model showed repair responses (nerve histology, myelin production) and functional recovery (sciatic functional index; SFI) comparable to autografts over 8 weeks⁶. Notably, scaffolds+4-AP appeared better histologically, but similar functionally, than scaffolds alone at this early stage of nerve repair. Thus, investigating long-term 4-AP effects, safety, and full functionality (beyond 8 weeks) is critical. Autografts had the thickest myelin (G-ratio) followed by scaffolds+4-AP, and scaffolds alone. Myelin content in autografts, scaffolds, and scaffolds+4-AP were lower than sham (surgery) controls, however, there were no differences between test groups. Neurofilament-H (NF-H) and S100 staining was observed for all groups, with sham control exhibiting highest and scaffolds alone exhibiting lowest content. In addition, 4-AP application appeared safe and bierosion products didn’t indicate any toxicity in liver histology⁶.

Conclusions: Delivering growth factors or cells with scaffolds can stimulate nerve regeneration processes, but chemical and electrical stimulation may have similar and additional benefits with fewer limitations. This facilitates more efficient and efficacious peripheral nerve regeneration via a drug delivery system that is feasible for clinical application.