Abstract
With the aim of forming bioactive guides for peripheral nerve regeneration, silk fibroin was electrospun to obtain aligned nanofibers. These fibers were functionalized by incorporating Nerve Growth Factor (NGF) and Ciliary NeuroTrophic Factor (CNTF) during electrospinning. PC12 cells grown on the fibers confirmed the bioavailability and bioactivity of the NGF, which was not significantly released from the fibers. Primary neurons from rat dorsal root ganglia (DRGs) were grown on the nanofibers and anchored to the fibers and grew in a directional fashion based on the fiber orientation, and as confirmed by growth cone morphology. These biofunctionalized nanofibers led to a 3-fold increase in neurite length at their contact, which was likely due to the NGF. Glial cell growth, alignment and migration were stimulated by the CNTF in the functionalized nanofibers. Organotypic culture of rat fetal DRGs confirmed the complementary effect of both growth factors in multifunctionalized nanofibers, which allowed glial cell migration, alignment and parallel axonal growth in structures resembling the ‘bands of Bungner’ found in situ. Graftable multi-channel conduits based on biofunctionalized aligned silk nanofibers were developed as an organized 3D scaffold. Our bioactive silk tubes thus represent new options for a biological and biocompatible nerve guidance conduit.
Highlights
When a peripheral nerve is sectioned, the axonal segment distal to the lesion site undergoes Wallerian degeneration [1,2], while the proximal segment is able to regenerate axonal sprouts that can eventually re-establish motor and sensory nerve function
Cells grown on the multifunctionalized Nerve Growth Factor (NGF)/Ciliary NeuroTrophic Factor (CNTF)-fibers displayed greater neurite length and an increased glial cell/neurons ratio compared to the fibroin nanofibers alone (124 mm and 3.2, respectively)
The restoration of nerve pathways is a prerequisite for successful regeneration in the peripheral nervous system (PNS) in which glial cells play a key role during axonal regeneration [32]
Summary
When a peripheral nerve is sectioned, the axonal segment distal to the lesion site undergoes Wallerian degeneration [1,2], while the proximal segment is able to regenerate axonal sprouts that can eventually re-establish motor and sensory nerve function. Nerve Growth Factor (NGF), for example, promotes neuronal survival and regeneration both in vitro and in vivo [19] Another neurotrophic factor, the ciliary neurotrophic factor (CNTF) produced by astrocytes and abundantly present in peripheral nerves, is localized mainly in the cytoplasm of myelinating Schwann cells and its synthesis is decreased during Wallerian degeneration [20,21]. To emulate the preclinical development of this material, the material system was evaluated using: (i) a neurotrophic sensitive cell line, (ii) rat dorsal root ganglia primary neuronal cultures and (iii) an organotypic culture of the entire rat dorsal root ganglia This multiscale strategy supported the demonstration of the full capacities of this new silk-based nanomaterial system, leading to the synthesis of a biological, biodegradable and bioactive 3D nerve guide for surgical implantation
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