Aligned electrospun nanofibers specify the direction of dorsal root ganglia neurite growth

Abstract

Nerve injury, a significant cause of disability, may be treated more effectively using nerve guidance channels containing longitudinally aligned fibers. Aligned, electrospun nanofibers direct the neurite growth of immortalized neural stem cells, demonstrating potential for directing regenerating neurites. However, no study of neurite guidance on these fibers has yet been performed with primary neurons. Here, we examined neurites from dorsal root ganglia explants on electrospun poly-L-lactate nanofibers of high, intermediate, and random alignment. On aligned fibers, neurites grew radially outward from the ganglia and turned to follow the fibers upon contact. Neurite guidance was robust, with neurites never leaving the fibers to grow on the surrounding cover slip. To compare the alignment of neurites to that of the nanofiber substrates, Fourier methods were used to quantify the alignment. Neurite alignment, however striking, was inferior to fiber alignment on all but the randomly aligned fibers. Neurites on highly aligned substrates were 20 and 16% longer than neurites on random and intermediate fibers, respectively. Schwann cells on fibers assumed a very narrow morphology compared to those on the surrounding coverslip. The robust neurite guidance demonstrated here is a significant step toward the use of aligned, electrospun nanofibers for nerve regeneration.

The design of electrospun PLLA nanofiber scaffolds compatible with serum-free growth of primary motor and sensory neurons

“…Alignment of fibers and neurites was quantified as previously described [1]. SEM images of fibers and dark field images of neurites were taken digitally and analyzed using ImageJ (http://rsb.info.nih.gov/ij/).…”

“…Aligned electrospun nanofibers powerfully direct the growth of regenerating neurites in vitro, offering promise as scaffolds for peripheral nerve repair [1][2][3][4]. Additionally, nanofiber scaffolds may be useful in guiding regenerating neurons in the spinal cord and brain, as well as serve to differentiate and guide transplanted neurons and stem cells in nervous system lesions.…”

“…Instead of using primary neurons harvested from the nervous system, most in vitro testing of biomaterials relies on the use of renewable cell lines [1,2,[9][10][11][12][13] or, more rarely, primary cells grown in media supplemented with serum [1,3,14,15]. Both of these approaches have significant drawbacks.…”

“…Neuroblastoma cells and transformed neural stem cells have neurites that extend using contact guidance and thus grow along fibers, but their neurites do not resemble true axons and dendrites as judged by standard morphological and immunocytochemical criteria [16]. Typically, these cells attach and grow readily on materials without the surface coatings required by primary neurons to adhere and survive [1,2]. Therefore, their use to predict behavior of primary neurons in a specific regeneration application is very limited.…”

“…In these experiments, PLLA fibers electrospun on the target wheel were anchored on a glass cover slip with adhesive bandage [1]. When we tried growing dissociated neurons in serum-free conditions using these substrates, the neurons died by 4 days in vitro (DIV).…”