Abstract

Objective. Neural tissue engineering holds incredible potential to restore functional capabilities to damaged neural tissue. It was hypothesized that patterned and functionalized nanofiber scaffolds could control neurite direction and enhance neurite outgrowth. Approach. A method of creating aligned electrospun nanofibers was implemented and fiber characteristics were analyzed using environmental scanning electron microscopy. Nanofibers were composed of polycaprolactone (PCL) polymer, PCL mixed with gelatin, or PCL with a laminin coating. Three-dimensional hydrogels were then integrated with embedded aligned nanofibers to support neuronal cell cultures. Microscopic images were captured at high-resolution in single and multi-focal planes with eGFP-expressing neuronal SH-SY5Y cells in a fluorescent channel and nanofiber scaffolding in another channel. Neuronal morphology and neurite tracking of nanofibers were then analyzed in detail. Main results. Aligned nanofibers were shown to enable significant control over the direction of neurite outgrowth in both two-dimensional (2D) and three-dimensional (3D) neuronal cultures. Laminin-functionalized nanofibers in 3D hyaluronic acid (HA) hydrogels enabled significant alignment of neurites with nanofibers, enabled significant neurite tracking of nanofibers, and significantly increased the distance over which neurites could extend. Specifically, the average length of neurites per cell in 3D HA constructs with laminin-functionalized nanofibers increased by 66% compared to the same laminin fibers on 2D laminin surfaces, increased by 59% compared to 2D laminin-coated surface without fibers, and increased by 1052% compared to HA constructs without fibers. Laminin functionalization of fibers also doubled average neurite length over plain PCL fibers in the same 3D HA constructs. In addition, neurites also demonstrated tracking directly along the fibers, with 66% of neurite lengths directly tracking laminin-coated fibers in 3D HA constructs, which was a 65% relative increase in neurite tracking compared to plain PCL fibers in the same 3D HA constructs and a 213% relative increase over laminin-coated fibers on 2D laminin-coated surfaces. Significance. This work demonstrates the ability to create unique 3D neural tissue constructs using a combined system of hydrogel and nanofiber scaffolding. Importantly, patterned and biofunctionalized nanofiber scaffolds that can control direction and increase length of neurite outgrowth in three-dimensions hold much potential for neural tissue engineering. This approach offers advancements in the development of implantable neural tissue constructs that enable control of neural development and reproduction of neuroanatomical pathways, with the ultimate goal being the achievement of functional neural regeneration.

Highlights

  • Damage to neural tissue is one of the leading causes of death and permanent disability in the world and presents one of the greatest challenges in current medical care

  • It was found that aligned nanofibers enabled significant directional control of neurite outgrowth both on 2D surfaces and in 3D mediums, and that the use of lamininfunctionalized nanofibers in 3D hyaluronic acid (HA) hydrogel significantly enhanced neurite length and neurite tracking along the nanofibers

  • Low neurite outgrowth was expected in the 3D HA hydrogels without fibers since hyaluronan hydrogels do not possess functional groups to which cells can attach, but the addition of nanofibers through a hydrogel with weak cell attachment points significantly enhanced neurite outgrowth along the fiber

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Summary

Introduction

Damage to neural tissue is one of the leading causes of death and permanent disability in the world and presents one of the greatest challenges in current medical care. The regeneration and restoration of neural tissue requires many important cellular and extracellular components, which interact with each other in several important ways. Extracellular materials serve as scaffolding for cellular architecture and can provide many biochemical signals that influence stem cell differentiation and cell behavior, and cells themselves interact with each other to facilitate neural function, provide trophic support, and direct differentiation during development [1]. Guiding cells to achieve the intended goal of survival, proliferation, differentiation, and network formation is a difficult challenge, but integration of multiple components will provide the best approach to engineering functional neural tissue

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