Aligned microchannel polymer-nanotube composites for peripheral nerve regeneration: Small molecule drug delivery

Abstract

Peripheral nerve injury accounts for roughly 2.8% of all trauma patients with an annual cost of 7 billion USD in the U.S. alone. Current treatment options rely on surgical intervention with the use of an autograft, despite associated shortcomings. Engineered nerve guidance conduits, stem cell therapies, and transient electrical stimulation have reported to increase speeds of functional recovery. As an alternative to the conduction effects of electrical stimulation, we have designed and optimized a nerve guidance conduit with aligned microchannels for the sustained release of a small molecule drug that promotes nerve impulse conduction. A biodegradable chitosan structure reinforced with drug-loaded halloysite nanotubes (HNT) was formed into a foam-like conduit with interconnected, longitudinally-aligned pores with an average pore size of 59.3 ± 14.2 μm. The aligned composite with HNTs produced anisotropic mechanical behavior with a Young's modulus of 0.33 ± 0.1 MPa, very similar to that of native peripheral nerve. This manuscript reports on the sustained delivery of 4-Aminopyridine (4AP, molecular weight 94.1146 g/mol), a potassium-channel blocker as a growth factor alternative to enhance the rate of nerve regeneration. The conduit formulation released a total of 30 ± 2% of the encapsulated 4AP in the first 7 days. Human Schwann cells showed elevated expression of key proteins such as nerve growth factor, myelin protein zero, and brain derived neurotrophic factor in a 4AP dose dependent manner. Preliminary in vivo studies in a critical-sized sciatic nerve defect in Wistar rats confirmed conduit suturability and strength to withstand ambulatory forces over 4 weeks of their implantation. Histological evaluations suggest conduit biocompatibility and Schwann cell infiltration and organization within the conduit and lumen. These nerve guidance conduits and 4AP sustained delivery may serve as an attractive strategy for nerve repair and regeneration.

Keywords: Halloysite nanotube; Nerve guidance conduit; Peripheral nerve regeneration; Polymer composite; Sciatic nerve defect; Small molecule drug delivery; Sustained release.

Figure 1.

(a) Unidirectional freezing of chitosan solution was achieved by creating a uni-axial thermal gradient by exposing the bottom surface of chitosan solution in molds (insulated in Styrofoam, not shown) to a stainless-steel plate submersed in liquid nitrogen. Upon exposure, the uni-axial thermal gradient results in linear formation of ice crystals. (b) A simplified schematic illustrating the process of incorporating 4AP drug into Halloysite Nanotubes (HNT) and subsequently mixing with chitosan in a custom mold to produce drug-loaded conduits. A photograph of the prototype is shown along with representative SEM images of the aligned, porous microstructure.

Figure 2.

SEM images showing highly porous channel microstructure of (a) conduit cross sectioned, SB = 1 mm; (b) conduit sectioned longitudinally, SB = 100 µm; (c) conduit cross sectioned, SB = 100 µm. A highly linear pore configuration can be seen throughout the construct. (d) FITC-loaded HNTs were imaged within the chitosan matrix, showing an even distribution throughout the construct. (e) The directionality of HNTs was calculated, showing a highly aligned distribution of HNTs resulting from the alignment of the polymer matrix. (f) Human Schwann cells seeded on conduit showed alignment and proliferation in the direction of the aligned polymer matrix. (g) The directionality of seeded Schwann cells confirmed an aligned Distribution of cells on the aligned microchannel conduit.

Figure 3.

Tensile mechanical testing of fabricated nerve guidance conduits, both with aligned pores and random porosity, and with and without halloysite reinforcement (labelled 5% HNT and 0% HNT, respectively). Young’s modulus is shown to increase in composite samples with 5% HNT as compared to samples with 0% HNT. Alignment of pores was shown to have anisotropic mechanical properties, increasing the modulus, particularly for the aligned composite with 5% HNT which showed the greatest moduli. Where native peripheral nerve is generally considered to have a Young’s modulus of 0.50 MPa, aligned composite conduits showed very similar moduli. *=p<0.05, **=p<0.01, and ***=p<0.001.

Figure 4.

TGA spectra (a) before and (b) after loading of 4AP into HNT. The modification of HNT loaded with 4AP (b) is shown as the TGA curve shows decomposition peaks for both 4AP and halloysite. The amount of 4AP loaded into HNT was quantified as mass lost over the given temperature range corresponding to the decomposition of 4AP and is expressed as the percent of total mass of HNT-4AP. (c) Pristine chitosan and composite Cht/HNT-4AP.

Figure 5.

(a) Degradation of crosslinked composite Cht/HNT-4AP samples in PBS pH 7.4 (black) and PBS with 4mg/mL lysozyme (red) (n=3 samples/timepoint, mean±s.d.). Degradation represented by the weight (%) of polymer remaining. *=p<0.05 vs PBS, **=p<0.01 vs PBS, and ***=p<0.001 vs PBS. (b) Representative SEM images showing crosslinked composite samples at various timepoints during degradation in PBS+lysozyme.

Figure 6.

(a) Schematic of 4AP drug loading into HNT, followed by sustained release of drug from lumen. (b) Cumulative release of 4AP from halloysite nanotubes; (c) cumulative release of 4AP from Cht (black diamond), crosslinked Cht (blue square), composite Cht/HNT-4AP (red circle), and crosslinked composite Cht/HNT-4AP (orange triangle) (n=5 samples/group, mean±s.d.). (d) Burst release of 4AP from samples, where burst release is quantified as percent cumulative drug release within the first 1 h (n=5 samples/group, mean±s.d.). **=p<0.01, ****=p<0.0001.

Figure 7.

(a) Lactase dehydrogenase (LDH) leakage cytotoxicity assay testing various doses of 4AP treatment on human Schwann cells in culture. ns=no significance. (b) MTS assay of human Schwann cells cultured on TCPS with no drug (control) or 1, 5, and 10 µg/mL of 4AP for up to 21 days. *=p<0.05 vs control, ***=p<0.001 vs control, and ****=p<0.0001 vs control. (c) MTS assay of human Schwann cells cultured on composite conduits for 21 days. **=p<0.01 vs day 1. (d) Human Schwann cells cultured on composite conduits and stained using live/dead cell staining. A Z-stack projection of 10 confocal microscopy images, each 10µm deep, is shown (i) and the same image is shown pseudo-colored for cell z-position using a color-depth gradient (ii).

Figure 8.

(a) In vitro immunofluorescent staining images showing human Schwann cells stained for NGF, P0, and BDNF (all stained red, respectively by row) in a dose response study to 4AP drug treatment. Control groups, which received media without drug, were compared to 1, 5, and 10 µg/mL drug media solutions, respectively by column. SB=10µm. (b) Quantification of immunofluorescent staining of NGF, P0, and BDNF (left to right, respectively). The intensity of staining obtained with each antibody was measured and displayed as box-plots with 5 to 95% confidence intervals (n=5 images/group). *=p<0.05 vs control, ***=p<0.001 vs control, and ****=p<0.0001 vs control.

Figure 9.

(a) Native rat sciatic nerve prior to transection. (b) 15 mm sciatic nerve defect surgically repaired with drug-loaded composite conduit. (c) H&E stain of a longitudinal section of native sciatic nerve shows well-organized myelin sheaths and round axons in wave formation, typical of axons and Schwann cells. (d) H&E stain of a longitudinal section of drug-loaded conduit repaired nerve 4-weeks post-operative. (e) H&E stain of a cross-section of native sciatic nerve shows well-organized axons and Schwann cells, surrounded by the connective tissue perineurium. (f) H&E stain of a cross-section of drug-loaded conduit repaired nerve 4-weeks post-operative. Partial infiltration and organization of Schwann cells has begun through the conduit lumen and into the conduit scaffold matrix (stained dark red). The composite conduit scaffold shows robust suturability, sufficient ability to withstand ambulatory forces, and strong biocompatibility – with promising results that it is favorable to facilitate nerve repair and regeneration.