Electrospun Materials and additives for Nerve Regeneration

The primary objective in the selection of materials for peripheral nerve regeneration is to encourage neurite growth. Materials that favor cell adhesion and proliferation or are found in natural extracellular matrix of the nerve are natural good choices for this purpose. Additives such as growth factors may also be added to promote neurite extension. Nerves are also known to be influenced by stimulants such as electrical signals and materials that can enable this stimulation may also be beneficial.

Bioactive materials and additives

Various bioactive materials and additives have been incorporated into electrospun fibers to improve neurite growth. A common protein used in neural regeneration is laminins which form part of the extracellular matrix that is essential for Schwann cells proliferation and differentiation [Yu et al 2007]. Laminins have been introduced to electrospun fibers by physical adsorption onto the fiber surface, blended into the polymer fiber, core-shell electrospun or covalently bonded [Leach 2013]. IKVAV peptide fragment which represents the primary functional site for laminin protein has also been covalently bonded to electrospun poly(L-lactide) fibers using plasma treatment and amine-modifications [Leach 2013]. Comparison of extension in neurites of motor neurons between aligned fibers of poly(L-lactide), laminin surface adsorbed poly(L-lactide), blended laminin and covalently bonded IKVAV on fiber showed significantly longer neurite growth from bonded IKVAV on fiber. All other samples showed no difference in neurite length [Leach 2013]. Neurite length is also significantly longer on aligned nanofibers than cast films of the same material [Leach 2013].

Hyaluronic acid, a common extracellular matrix glycosaminoglycan used in wound repair has been tested in electrospun fibers for nerve regeneration. RT4-D6P2T rat Schwann cells cultured on electrospun gelatin with hyaluronic acid blended into it showed better organized F-actin stress fibers and significantly higher secretion of neuregulin-1 (Nrg1), glial fibrillary acidic protein (GFAP), and myelin protein zero (P0) on day 6 compared to pure gelatin fibers.

Laminin forms part of nerve extracellular matrix (ECM) and is known to be an essential protein for Schwann cells proliferation and differentiation. Laminin has been incorporated into electrospun fibers by various means such as blending into the solution for electrospinning, physical surface absorption and by covalent bonding on the surface of the nanofibers. In blending and physical adsorption, laminin may leach out from the nanofibers. However, covalently bonded laminin is secured on the surface of the nanofibers. With laminin added to the nanofibers, PC-12 neurite extension has been shown to be much better compared to nanofibers without the protein. However, there is not much difference in neurite extension between physical adsorption and covalently bonded laminin on the nanofiber [Zander et al 2010]. Similar work by Koh (2009) also showed little difference between physically adsorbed and covalently bonded laminin on poly(L-lactide) (PLLA) fibers. However, blended laminin with PLLA showed the longest neurite extension. This may be due to higher amount of laminin that is incorporated into the fiber compared to covalent and physical adsorption as determined by protein assay [Koh et al 2009].

Another additive of interest is collagen which is a common extracellular matrix protein. This has also been physically blended into nanofibers and covalently bonded. When tested with PC-12 cells, neurite extension is better than nanofibers without additives. Collagen that is physically adsorbed into the nanofibers was also found to perform slightly better than covalently bonded collagen. However, the presence of laminin in the nanofibers still outperforms those with collagen [Zander et al 2010]. In the study by Koh et al (2009), covalently bonded collagen to PLLA showed the best neurite extension followed by blending and the last being physically adsorbed collagen. However, covalently bonded collagen to PLLA showed better neurite extension than covalently bonded and physically adsorbed laminin to PLLA although laminin blended PLLA fares better covalently bonded collagen. The differences in the results may be due to different concentration of collagen and laminin that is incorporated to the fiber.

Growth factors of various types are also commonly tested for its influence on peripheral nerve regeneration. Dinis et al (2014) blended a combination of nerve growth factor (NGF) and ciliary neurotrophic factor (CNTF) into silk fibroin/polyethylene oxide (4:1) before electrospinning to form highly aligned nanofibers (diameter of 854 nm). A summary of the PC12 cells and Glial cell response to the various scaffolds are shown in Table 1. Burst release of the growth factors were not observed over 4 days period and this has been attributed to strong adhesion between the negatively charged silk and positively charged growth factors. Core-shell electrospinning has also been used to incorporate growth factors in nanofibers. Using a rat sciatic nerve model, Wang et al (2012) implanted a conduit comprising of poly(DL-lactide-co-glycolide) (PLGA) shell and a poly(ethylene glycol)/β-nerve growth factor core fibers. Comparing electrophysiological response, gastronemius muscle weight ratio and morphological analysis of regenerated nerves between conduit with the nerve growth factor and autograft showed no significant differences except for the number of nerve fibers where autograft is better. All the results showed significantly better result than pure PLGA fiber conduit. A separate in vivostudy based on core-shell fibers with nerve growth factor in the core and poly(lactic acid-caprolactone) (P(LLA-CL)) as the shell also showed comparable results to autograft and this study includes the sciatic function index (SFI) [Liu et al 2011]. This showed the potential benefit of incorporating of nerve growth factor for in vivo reinnervation.

Table 1. Average number of neurites per cell, average neurites length and glia/neurons ratio after 3 days of culture on control fibroin nanofibers, NGF-, CNTF- and NGF/CNTF- functionalized nanofibers; * and # indicate statistically significant difference from fibroin condition for neurites length and glial cells/neuron ratio respectively. [Dinis et al PLoS ONE 2014; 9: e109770. doi:10.1371/journal.pone.0109770. This work is licensed under a Creative Commons Attribution 4.0 International.]

Dorsal Root Ganglia (DRG) neurons adhesion and growth on electrospun fibroin nanofibers. SEM observation of DRG cells adhering on nanofibers (A) and establishing tight contact (B and C). BIII tubulin staining of neurons growing on random (D) and aligned (E) nanofibers. BIII tubulin (red) and actin (green) of neuron growth cones on random (F) and aligned (G) nanofibers. Image acquisition performed after 5 days of culture. Scale bars: A 10µm, B 5µm, C 2µm, D, E, F and G 10µm. Images A, B and C have been recolored for sake of clarity. [Dinis et al PLoS ONE 2014; 9: e109770. doi:10.1371/journal.pone.0109770. This work is licensed under a Creative Commons Attribution 4.0 International.]

Bioactive substance	Base material	Reference
Ile-Lys-Val-Ala-Val (IKVAV) peptide fragment	poly-L-lactide	Leach 2013
Hyaluronic acid	Gelatin	Liou et al 2013
Nerve Growth Factor and ciliary neurotrophic factor	Silk fibroin/ polyethylene oxide (4:1)	Dinis et al 2014

Increased local exudation and inflammatory reactions in peripheral nerve injury may have a negative impact on nerve regeneration and repair. Therefore, inhibiting overactive inflammatory response at the injury site may facilitate peripheral nerve repair. Xu et al (2022) loaded electrospun poly (lactic-co-glycolic acid) (PLGA) nanofibrous scaffold with Tacrolimus (FK506), an FDA-approved immunosuppressant, to improve treatment outcome. The electrospun PLGA/FK506 membrane was rolled into a conduit and tested in a rat sciatic nerve model with a 15 mm nerve segment resected. PLGA scaffold without FK506 loaded was used as a negative control. After 12 weeks, gait analysis, electrophysiology, and neuromuscular histology results showed that PLGA only scaffold performed significantly worse than PLGA/FK506 scaffold. There was no significant difference in the performance between PLGA/FK506 scaffold and autologous graft. The addition of FK506 into electrospun PLGA scaffold is a promising method for repairing peripheral nerve injury.

The addition of biomolecules, peptides and other functional groups may not always lead to better cell response. Schaub et al (2015) tested the neurite extension of chick dorsal root ganglia on electrospun PLLA with GRGDS (cell adhesion peptide) bonded on its surface against unmodified PLLA electrospun fibers and found that there is no significant improvement in the total neurite extension. Similarly, surface functionalization of PLLA electrospun fibers with diethylenetriamine (DTA, for amine functionalization), 2-(2-aminoethoxy)ethanol (AEO, for alcohol functionalization) also showed poorer neurite extension compared to unmodified PLLA.

Conductive Materials

Electrical stimulation is known to promote axonal regeneration following nerve injury [Xu et al 2014] and has been used clinically after carpal tunnel release surgery [Gordon et al 2009]. This has prompted researchers to incorporate electrical conductivity function in electrospun fibers and test for its effect nerve cells behavior. Ghasemi-Mobarakeh et al (2009) construct a nonwoven mesh of nanofibers made out of a blend of polyaniline, polycaprolactone and gelatin solution using electrospinning. The electrospun mesh was found to have a conductance of 0.02x10^-6S. Applying a direct current with potential of 1.5V to the scaffold (100 mV/mm) after 24 h of seeding nerve stem cells on it, significant neurite extension was observed in the scaffold that has been stimulated for 1 h compared to the control while no significant difference was observed when the stimulation duration was 15 or 30 minutes. Another method of electrospinning conductive polymer is by core-shell electrospinning. This has been used for fabrication of core-shell fibers with one part of electrospinnable polymer and with conductive polyaniline [Zhang et al 2014] or polypyrrole [Xie et al 2009] which are generally not electrospinnable on their own.

Conductive material such as carbon nanotube may be added to the solution for electrospinning to produce conductive fibers. In vitro studies have shown that multi-walled carbon nanotube (MWCNT)/collagen/polycaprolactone was able to support Schwann cell adhesion and elongation and has also been demonstrated to promote reinnervation of sciatic nerve defect in rats [Yu et al 2004]. Seonwoo et al (2018) demonstrated the addition of reduced graphene oxide (RGO) in electrospun polycaprolactone (PCL) for dental pulp stem cells (DPSCs) neurogenic differentiation. RGO belongs to the graphene family but has higher electroconductivity than graphene oxide. With 0.1% and 1% RGO in electrospun PCL fibers, there is a high expression of Tuj1, the early marker of neurogenesis, and NeuN, the late marker of neurogenesis. However, with 1% RGO, the cells showed shorter axon-like legs which may be a sign of neurodegeneration of DPSCs at such high concentration of RGO. The study did not report any significant difference in DPSCs neurogenic differentiation between random and aligned RGO/PCL electrospun fibers. They suggested that differentiated neurites exhibited bipolar structures and connects to other cells in a linear direction on aligned fibers which may be more suitable for peripheral nerve regeneration.

Piezoelectric Materials

Piezoelectric materials have the ability to generate electric charge when it experiences mechanical stress. Several piezoelectric materials have been electrospun and tested for biocompatibility in nerve regeneration. Dorsal root ganglion has been cultured on electrospun aligned and random polyvinylidene fluoride-trifluoroethylene (PVDF-TrFE) nanofibers. Aligned PVDF-TrFE fibers were shown to be capable of directing neurite outgrowth for all fiber dimensions from nano to micro-meter diameter [Lee et al 2011]. Electrospub poly(3-hydroxybuty rate-co-3-hydroxyvalerate) (PHBV) is an interesting material for nerve regeneration as it demonstrates piezoelectric property and is biodegradable. A conduit made out of this material has been tested in an in vivo rat sciatic nerve defect model with 30 mm long gap [Biazar et al 2013]. The tests showed good functional recovery of the rats with the implant which is much better than the negative control group although autograft is generally superior to it [Biazar et al 2013].

Charge-storage materials

Materials that is able to store charges which can be subsequently released after implantation may be used to provide low intensity electrical stimulation to encourage neural regeneration. β- tricalcium phosphate (β-TCP) particles were found to have the ability to store electric charge through electrical polarization[Wang et al 2010]. Electrospun chitosan blended with polarized or non-polarized β-TCP was tested using an in vivo rat model. The rats implanted with the fibers containing polarized β-TCP particle were found to exhibit much better recovery compared to non-polarized β-TCP fibers. The electrically polarized composite graft at 12 weeks post implantation in a rat sciatic nerve model also showed no significant difference to autograft in electrophysiological tests and histological tests [Wang et al 2010].

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Content [Biomedical (Peripheral Nerve)]