A Combined Conduit-Bioactive Hydrogel Approach for Regeneration of Transected Sciatic Nerves

Abstract

Transected peripheral nerve injury (PNI) affects the quality of life of patients, which leads to socioeconomic burden. Despite the existence of autografts and commercially available nerve guidance conduits (NGCs), the complexity of peripheral nerve regeneration requires further research in bioengineered NGCs to improve surgical outcomes. In this work, we introduce multidomain peptide (MDP) hydrogels, as intraluminal fillers, into electrospun poly(ε-caprolactone) (PCL) conduits to bridge 10 mm rat sciatic nerve defects. The efficacy of treatment groups was evaluated by electromyography and gait analysis to determine their electrical and motor recovery. We then studied the samples' histomorphometry with immunofluorescence staining and automatic axon counting/measurement software. Comparison with negative control group shows that PCL conduits filled with an anionic MDP may improve functional recovery 16 weeks postoperation, displaying higher amplitude of compound muscle action potential, greater gastrocnemius muscle weight retention, and earlier occurrence of flexion contracture. In contrast, PCL conduits filled with a cationic MDP showed the least degree of myelination and poor functional recovery. This phenomenon may be attributed to MDPs' difference in degradation time. Electrospun PCL conduits filled with an anionic MDP may become an attractive tissue engineering strategy for treating transected PNI when supplemented with other bioactive modifications.

Keywords: electromyography; nerve guidance conduit; peptide; peripheral nerve regeneration; sciatic nerve transection; self-assembly.

Figure 1.

Schematic diagram illustrating the overall experimental design. (a) This blinded study includes the autograft, MDP hydrogel, and HBSS treatment groups that are sacrificed at 3-, 6-, and 16-week post operation. (b) Surgical procedure involves exposing sciatic nerves and introducing 10 mm long defects. Prefilled with hydrogels/HBSS, the PCL conduits bridge the nerve gaps. Secondary injection is then performed to replace hydrogels/HBSS lost during the surgery. (c) Two forms of gait analyses, along with the observation of flexion contractures, are done over 16 weeks to assess motor recovery. As the rats reach sacrificial time points, (d) CMAP signals are recorded at the gastrocnemius muscles, followed by (e) immunofluorescence staining of nerve tissues and the weight measurements of gastrocnemius muscles to evaluate electrical and structural recovery.

Figure 2.

Structural characterizations of MDPs. (a) ATR-FTIR spectra and (b) CD spectra of K₂, D₂, and D₂-V peptides. Oscillatory rheology results of K₂, D₂, and D₂-V peptides in (c) amplitude sweep mode and (d) time sweep mode where 200% oscillation strain occurs at t = −1 to 0 min. SEM images of (e) K₂, (f) D₂, and (g) D₂-V peptides taken at 50 000× magnification. Scale bar = 500 nm.

Figure 3.

(a) 14 mm PCL conduit with its (b) SEM image at 1000× magnification. (c) Inner diameter, (d) length, and (e) thickness of the conduits are consistent before treatment (green) and after 2 days of ethanol and 1 (red) or 2 (blue) days of HBSS. Target inner diameter and length are 2 mm and 10 mm, respectively (n = 12 among 3 batches). (f) Gram force vs strain plot from suture pull-out test (suture retention strength = 1830 ± 50 g m/s², ultimate tensile strain = 400 ± 20%).

Figure 4.

(a) Sciatic nerve is exposed with the defect size measured using a ruler. (b) Nerve is cut to create a 10 mm gap. (c) Image of PCL conduits with MDPs or HBSS bridging the gap after 16-week implantation. (d) Image of reverse autograft at day 0 before wound closing.

Figure 5.

Electromyography (EMG). (a) Electrodes’ placement during EMG recording. (b) An example of CMAP amplitude measured in Labchart software. % CMAP amplitude data normalized with left, uninjured CMAP at (c) 6- and (d) 16-week time points. (One-way ANOVA with Tukey’s HSD. *p < 0.05, ***p < 0.001).

Figure 6.

Gastrocnemius muscle measurements. Representative images of left (uninjured) and right (injured) gastrocnemius muscles at (a) 6- and (b) 16-week time points. Gastrocnemius muscle weight data at (c) 6- and (d) 16-week time points (one-way ANOVA with Tukey’s HSD. * = with respect to neg control and a = with respect to reverse autograft. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 7.

Onset of flexion contractures. Example images of injured rat’s feet (a) without and (b) with flexion contracture, marked by yellow dotted lines. (c) % rats with flexion contracture for 16 weeks after sciatic nerve injury/repair.

Figure 8.

Locations of nerve cross-sections processed throughout the extracted nerve tissue and conduit. The dotted blue lines at the proximal and distal ends show the initial positions of transected nerve, while the black dotted lines denote locations 1–6.

Figure 9.

Percent cross-sectional axon area in conduits measurements. (a) Sample nerve and PCL conduit cross-sectional image. (b) Sample area measurement of conduit (1) and axon (2) using ImageJ. Percent axon area in conduit data of treatment groups after 6 weeks at location (c) 3 and (d) 4 (one-way ANOVA with Tukey’s HSD. *p < 0.05, **p < 0.01).

Figure 10.

Automatic axon counting of 16-week cross-sectional images using Axondeepseg software. (a) Percent myelination and (b) number of myelinated axon at location 3–5 in treatment groups (one-way ANOVA with Tukey’s HSD. *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 11.

Representative confocal images of 6-week nerve cross-sections. A yellow arrow indicates the undegraded K₂ hydrogel (green = β-Tubulin III, red = Myelin basic protein, Blue = DAPI; Scale bar = 500 μm).