Electrical aligned polyurethane nerve guidance conduit modulates macrophage polarization and facilitates immunoregulatory peripheral nerve regeneration

Abstract

Biomaterials can modulate the local immune microenvironments to promote peripheral nerve regeneration. Inspired by the spatial orderly distribution and endogenous electric field of nerve fibers, we aimed to investigate the synergistic effects of electrical and topological cues on immune microenvironments of peripheral nerve regeneration. Nerve guidance conduits (NGCs) with aligned electrospun nanofibers were fabricated using a polyurethane copolymer containing a conductive aniline trimer and degradable L-lysine (PUAT). In vitro experiments showed that the aligned PUAT (A-PUAT) membranes promoted the recruitment of macrophages and induced their polarization towards the pro-healing M2 phenotype, which subsequently facilitated the migration and myelination of Schwann cells. Furthermore, NGCs fabricated from A-PUAT increased the proportion of pro-healing macrophages and improved peripheral nerve regeneration in a rat model of sciatic nerve injury. In conclusion, this study demonstrated the potential application of NGCs in peripheral nerve regeneration from an immunomodulatory perspective and revealed A-PUAT as a clinically-actionable strategy for peripheral nerve injury.

Keywords: Aligned nanofibers; Macrophage polarization; Nerve guidance conduit; Nerve regeneration; Polyurethane.

Fig. 1

Characterization of PCL, R-PUAT, and A-PUAT membranes. (A) FT-IR spectra of each membrane. (B) SEM images of each membrane. (C) AFM results of each group. (D) Water contact angles of each membrane. (D) Mechanical properties of each fibrous membrane. (E) CV scanning of PUAT polymer in 1 M HCl.

Fig. 2

A-PUAT membranes stimulate macrophage polarization to M2-phenotype. (A) SEM results revealing the morphology of RAW264.7 cells cultured on different membranes. (B) The viability of RAW264.7 cells seeded on each membrane at indicated time points. (C) Relative transcription levels of pro-inflammatory gene and anti-inflammatory gene in each group. (D) The contents of TNF-α and IL-10 secreted by RAW264.7 cells cocultured on different substrates. (E) Representative immunofluorescent images revealing the cytoskeleton of RAW264.7 cells (FITC, green) and M2-phenotype macrophages (CD206, red) after culturing on different membranes for 3 days. (F) Quantitative analysis of orientation of macrophages cultured on different membranes. (G) Quantitative analysis of CD206 positive cells in RAW264.7 cells seeded on different membranes. (H) Flow cytometry showing the expression of CD80 and CD206 in RAW264.7 cells cultured on different substrates.

Fig. 3

Differentially expressed genes in RAW264.7 cells seeded on R-PUAT and A-PUAT. (A, B) Heatmap and volcano plot of differentially expressed genes in RAW264.7 cells at 1 day after seeding on different membranes. (C) GO analysis of the RNA-sequencing results.

Fig. 4

Effects of polarized macrophages induced by different membranes on RSC96 cell behaviors. (A) Schematic illustration of RSCs incubated with conditioned medium (CM) from macrophages which were cultured on different membranes. (B) The viability of RSCs after cocultured with different CM for 1 or 3 days. (C, D) Representative images and quantitative analysis of migrated RSCs after cocultured with different CM for 1 day. (E, F) Representative images and quantitative analysis of the wound healing assay results of RSCs after cocultured with different CM for the indicated time points. (G) Representative immunofluorescent images of cytoskeleton (FITC, green) and S100 expression (red) of RSCs cocultured with different CM. (H) The transcription levels of myelination-related genes in RSCs after cocultured with different CM for 3 days.

Fig. 5

Relevance between macrophages and SCs at 7 and 14 days after grafting. (A) Representative immunofluorescence images of the transverse sections of different NGCs showing the distribution of macrophages (M0, CD68, green) and SCs (S100β, red). (B) Quantitative analyses showed the number of infiltrated macrophages and SCs in each group. (C) Representative immunofluorescence images of M1-phenotype macrophages (iNOS, red), and M2-phenotype macrophages (CD206, red).

Fig. 6

Evaluation of nerve functional recovery at 12 weeks after implantation. (A) NGC implantation in a 10-mm long sciatic nerve defect in rat models. (B) Gross observation of regenerated sciatic nerves of each group at 12 weeks post-operation. (C, D) Representative images of footprints and quantitative analysis of the SFI values. (E, F) Representative CMAP recordings of each group and quantitative analysis of the CMAP amplitude and latency. (G) Gross observation of the gastrocnemius muscle in each group at 12 weeks after implantation. The surgery side was on the left, and the contralateral side was on the right. (H) Quantitative analysis of the wet weight ratio of the gastrocnemius muscles. (I) Representative images of Masson’s staining of the transverse sections of muscles from each group. (J) Quantitative analysis of the diameter of muscle fibers and the percentage of collagen-positive area.

Fig. 7

Morphologic and histologic analyses of sciatic nerve regeneration at 12 weeks after implantation. (A) Representative H&E staining images. (B, C) Representative images of Luxol fast blue staining and quantitative analysis of the percentage of myelin-positive area. (D, E) TEM images of the cross-sections of regenerated nerves and quantitative analysis of the axon diameter and myelin sheath thickness. (F, G) Representative immunofluorescence images and quantitative analysis of the expression levels of the myelination-related markers PMP22 and NCAM in regenerated nerves.

Fig. 8

Schematic illustration of nerve regeneration promoted by A-PUAT nerve guidance conduits via modulating macrophage polarization towards the M2 phenotype.