Aligned Fingolimod-Releasing Electrospun Fibers Increase Dorsal Root Ganglia Neurite Extension and Decrease Schwann Cell Expression of Promyelinating Factors

Abstract

Researchers are investigating the use of biomaterials with aligned guidance cues, like those provided by aligned electrospun fibers, to facilitate axonal growth across critical-length peripheral nerve defects. To enhance the regenerative outcomes further, these aligned fibers can be designed to provide local, sustained release of therapeutics. The drug fingolimod improved peripheral nerve regeneration in preclinical rodent models by stimulating a pro-regenerative Schwann cell phenotype and axonal growth. However, the systemic delivery of fingolimod for nerve repair can lead to adverse effects, so it is necessary to develop a means of providing sustained delivery of fingolimod local to the injury. Here we created aligned fingolimod-releasing electrospun fibers that provide directional guidance cues in combination with the local, sustained release of fingolimod to enhance neurite outgrowth and stimulate a pro-regenerative Schwann cell phenotype. Electrospun fiber scaffolds were created by blending fingolimod into poly(lactic-co-glycolic acid) (PLGA) at a w/w% (drug/polymer) of 0.0004, 0.02, or 0.04%. We examined the effectiveness of these scaffolds to stimulate neurite extension in vitro by measuring neurite outgrowth from whole and dissociated dorsal root ganglia (DRG). Subsequently, we characterized Schwann cell migration and gene expression in vitro. The results show that drug-loaded PLGA fibers released fingolimod for 28 days, which is the longest reported release of fingolimod from electrospun fibers. Furthermore, the 0.02% fingolimod-loaded fibers enhanced neurite outgrowth from whole and dissociated DRG neurons, increased Schwann cell migration, and reduced the Schwann cell expression of promyelinating factors. The in vitro findings show the potential of the aligned fingolimod-releasing electrospun fibers to enhance peripheral nerve regeneration and serve as a basis for future in vivo studies.

Keywords: Schwann cells; biomaterial; dorsal root ganglia; drug delivery; electrospun fibers; fingolimod hydrochloride; neurons; peripheral nervous system injury.

FIGURE 1

An overview of aligned fingolimod-releasing electrospun fiber fabrication and in vitro assessment. (A) Fingolimod hydrochloride was incorporated into aligned poly(lactic-co-glycolic acid) fibers via blend electrospinning. Aligned fingolimod-releasing fibers (B) increased whole dorsal root ganglia (DRG) neurite (green) extension and Schwann cell (red) migration, (C) increased individual DRG neuron (green) neurite outgrowth in dissociated DRG cultures, and (D) decreased Schwann cell (red) expression levels of promyelinating factors in purified Schwann cell cultures.

FIGURE 2

Electrospun fibers containing various loading concentrations of fingolimod share similar morphological features. SEM images of (A) 0.0%, (B) 0.0004%, (C) 0.02%, or (D) 0.04% w/w fingolimod/poly(lactic-co-glycolic acid) electrospun fibers with inlaid images of water droplets used to measure static water contact angle for each (scale bar = 20 μm). (E) Fiber alignment data for each loading concentration are represented by histograms displaying the percentage of fibers with a deviation from the mean fiber angle in degrees. (F) Fiber diameter data are represented as mean diameter (in μm) ± standard deviation. (G) Fiber surface coverage data are represented by the mean percentage of the coverslip covered by fibers ± standard deviation. (H) Static water contact angle data (in degrees) are represented by the mean contact angle ± standard deviation.

FIGURE 3

Fingolimod-loaded fibers continuously release fingolimod for at least 28 days. (A) Cumulative release (in ng) of fingolimod from 350 mg of 0.0004% (green), 0.02% (pink), and 0.04% (blue) fingolimod-loaded electrospun fibers over 28 days. (B) Predicted cumulative release of fingolimod (in ng) from a single 0.0004% (green), 0.02% (pink), and 0.04% (blue) fingolimod-loaded electrospun fiber scaffold over 28 days. An individual fingolimod-loaded electrospun fiber scaffold contains 3 mg of fibers. Data are represented by the mean mass of drug released (in ng) ± standard deviation.

FIGURE 4

Control poly(lactic-co-glycolic acid) (PLGA) electrospun fibers show significant degradation over 6 weeks. SEM images of control PLGA fibers incubated in cell culture media at 37°C for (A) 7, (B) 14, (C) 21, (D) 28, (E) 35, or (F) 42 days illustrate the gradual degradation of the fiber scaffolds over 6 weeks. The scale bar of these images represents 20 μm. The scale bar of the inlaid image represents 4 μm.

FIGURE 5

The 0.0004 and 0.02% fingolimod-loaded electrospun fiber groups significantly increased neurite extension from whole dorsal root ganglia (DRG) explants. Confocal images of whole DRG stained against neurofilament (green) and cultured for 4 days on (A) 0.0%, (B) 0.0004%, (C) 0.02%, or (D) 0.04% fingolimod-loaded poly(lactic-co-glycolic acid) electrospun fibers (scale bar = 500 μm). (E) Whole DRG total percent adhesion data show the percentage of the total number of whole DRG that remained adhered to each fiber type after 4 days in culture. (F) Neurite length data are represented by the mean length (in μm) ± standard error of the mean. Statistical significance compared with the 0.0% control fibers was assessed using one-way ANOVA and post hoc Dunnett’s test (**p < 0.001).

FIGURE 6

The addition of fingolimod to poly(lactic-co-glycolic acid) (PLGA) electrospun fibers affects neurite outgrowth from individual dorsal root ganglia (DRG) neurons. Confocal images of individual DRG neurons stained against neurofilament (green) and cultured for 12 h on (A) 0.0%, (B) 0.0004%, (C) 0.02%, or (D) 0.04% fingolimod-loaded PLGA electrospun fibers (scale bar = 100 μm). (E) Total neurite length (in μm), (F) longest neurite length (in μm), (G) the number of branch points, and (H) the number of primary neurites data are represented by the mean ± standard error of the mean. Statistical significance compared with the 0.0% control fibers was assessed for total neurite length, longest neurite length, and the number of branch points data using Welch’s ANOVA and post hoc Games–Howell test and for the number of primary neurites using one-way ANOVA and post hoc Dunnett’s test (**p < 0.001, *p < 0.05).

FIGURE 7

All fingolimod-loaded electrospun fiber groups significantly increased Schwann cell migration outward from the dorsal root ganglia (DRG) body. Confocal images of whole DRG stained with 4′,6-diamidino-2-phenylindole and cultured on (A) 0.0%, (B) 0.0004%, (C) 0.02%, or (D) 0.04% fingolimod-loaded poly(lactic-co-glycolic acid) electrospun fibers (scale bar = 500 μm). (E) The migration distance data for each fiber group are represented by the mean distance traveled from the DRG body ± the standard error of the mean. Statistical significance compared with the 0.0% control fibers was assessed for Schwann cell migration distance using one-way ANOVA and post hoc Dunnett’s test (**p < 0.001).

FIGURE 8

The 0.02% fingolimod-loaded electrospun fibers significantly decreased Schwann cell expression levels of several promyelinating factors. qPCR was conducted to determine Schwann cell mRNA expression levels of the (A) regenerative factors BDNF, cJun, GAP43, NCAM1, and PDGF-BB and the (B) myelinating factors Cx32, Krox20, MBP, Oct6, and PMP2. Schwann cell gene expression data are represented by the mean RNA fold change ± standard deviation. Statistical significance was assessed using general linear regression (*p < 0.05).