Investigation of neuronal pathfinding and construction of artificial neuronal networks on 3D-arranged porous fibrillar scaffolds with controlled geometry

Dongyoon Kim,Myung-Han Yoon,Seyeong Lee,Seong-Min Kim

doi:10.1038/s41598-017-08231-3

Abstract

Herein, we investigated the neurite pathfinding on electrospun microfibers with various fiber densities, diameters, and microbead islands, and demonstrated the development of 3D connected artificial neuronal network within a nanofiber-microbead-based porous scaffold. The primary culture of rat hippocampal embryonic neurons was deposited on geometry-controlled polystyrene (PS) fiber scaffolds while growth cone morphology, neurite outgrowth patterns, and focal adhesion protein expression were cautiously examined by microscopic imaging of immunostained and live neuronal cells derived from actin-GFP transgenic mice. It was demonstrated that the neurite outgrowth was guided by the overall microfiber orientation, but the increase in fiber density induced the neurite path alteration, thus, the reduction in neurite linearity. Indeed, we experimentally confirmed that growth cone could migrate to a neighboring, but, spatially disconnected microfiber by spontaneous filopodium extrusion, which is possibly responsible for the observed neurite steering. Furthermore, thinner microfiber scaffolds showed more pronounced expression of focal adhesion proteins than thicker ones, suggesting that the neuron-microfiber interaction can be delicately modulated by the underlying microfiber geometry. Finally, 3D connected functional neuronal networks were successfully constructed using PS nanofiber-microbead scaffolds where enhanced porosity and vertical fiber orientation permitted cell body inclusion within the scaffold and substantial neurite outgrowth in a vertical direction, respectively.

Highlights

Electrospinning is a simple but versatile technique for fabricating micro/nanofibers, and both single fibers and entangled fibrillar scaffolds have been extensively exploited for energy storage devices[1, 2], sensors[3, 4], and biomedical applications[5,6,7,8,9]
Electrospun fibers were visualized by confocal reflection microscopy (CRM), which permits the overlay of neuronal processes and micro/nanofibers, the microscopic examination of neuron-fiber interaction in terms of growth cone morphology and focal adhesion expression using fibrillar scaffolds with distinct fiber density and diameter
3D connected artificial neuronal networks were constructed within a nanofiber-microbead-based porous scaffold where the enhanced porosity and vertical fiber orientation enabled cell body inclusion within the scaffold and substantial neurite outgrowth in a vertical direction, respectively

Summary

Introduction

Electrospinning is a simple but versatile technique for fabricating micro/nanofibers, and both single fibers and entangled fibrillar scaffolds have been extensively exploited for energy storage devices[1, 2], sensors[3, 4], and biomedical applications[5,6,7,8,9]. It was reported that axonal guidance could occur in the direction perpendicular to aligned fibers[27] These apparently contradictory results imply that there exists a certain possibility that neurite pathfinding and outgrowth may be guided by ‘static’ environmental factors, i.e., the underlying micro/nanofiber directionality, and by ‘dynamic’ cellular factors, i.e., growth cone movement and its interaction with extracellular structures. There exist only few studies on CNS-derived neurons on electrospun micro/nanofibers such a fibrillary network can be employed to define 3D artificial neuronal networks which could be useful in the field of fundamental neuroscience and/or developmental neurobiology It has not been clarified how neurite outgrowth of CNS neurons can be modulated by structural parameters of underlying fibrillar scaffolds, for instance, fiber diameter, density, porosity, etc. 3D connected artificial neuronal networks were constructed within a nanofiber-microbead-based porous scaffold where the enhanced porosity and vertical fiber orientation enabled cell body inclusion within the scaffold and substantial neurite outgrowth in a vertical direction, respectively

Methods

Results

Discussion

Conclusion