Spinal cord injury (SCI) induces paralysis by severing the long axons of neurons and recovery is inhibited by poor regrowth rates. As neural cells are electroactive, electrical stimulation (ES) may present a promising method of promoting axonal regrowth when applied in conjunction with electroconductive (EC) biomaterials1. To efficiently deliver ES to regrowing motor and sensory axons, it is essential to have precise control of scaffold geometry.
This work focused on producing novel EC scaffolds for spinal cord injury by coating 3D-printed polycaprolactone (PCL) with polypyrrole (PPy) and assessing its suitability as a substrate for neuronal growth.
PCL scaffolds consisting of multiple interlocking ‘axon’ channels were 3D-printed (Allevi 2 printer). PPy was then polymerised in situ to form an EC coating2. Electroconductivity was measured via the 4-point method and surface morphology and coating thickness were assessed using SEM.. Biocompatibility was tested by seeding SH-SY5Y neurons on PPy/PCL films and measuring metabolic activity and total cellular DNA. Neurons were immunostained for beta-III tubulin, counterstained with DAPI for nuclei and imaged using a Nikon 90i fluorescent microscope to determine neurite outgrowth. Images were analysed using ImageJ.
A method was developed to coat complex 3D-printed PCL structures with a biocompatible, EC PPy layer. SEM images of coated films show that PPy forms a network of particles over the PCL surface. Conductivity of the PPy coating was 15 ± 5 S/m, rendering the scaffold suitably electroconductive for biological applications. In cultures of SH-SY5Y neurons on 2D PCL and PPy/PCL films, metabolic rate and total cellular DNA increased significantly (p<0.05) in both groups between day 1 and 3, with no significant difference in either metric between groups, showing the PPy coating is as biocompatible as uncoated PCL, providing a suitable substrate for neural proliferation. Neurons cultured on both film types (7 days) exhibited robust neurite outgrowth and typical morphology with no significant difference in cell number or neurite length between groups, confirming neuronal viability.
Biocompatible, 3D EC scaffolds with complex architectures were produced for SCI repair. Coating 3D-printable PCL with EC PPy allows printing of biocompatible EC structures with precise, controlled geometries and porosities that can match the organisation of grey and white matter tracts in the cord. Conductivity of the PPy coating is over 30 times higher than central nervous system grey matter, and 8 times higher than cerebrospinal fluid, potentially allowing for efficient direction of electrical stimulation3. Taken together, these data indicate that PPy/PCL is biocompatible, supports neuronal growth and is a suitable substrate for growth of spinal cord neurons. Ongoing optimisation work will determine the ability of the EC scaffold to increase efficacy of ES to further promote neurite extension as a prerequisite for SCI applications.
Research was funded by Irish Rugby Football Union Charitable Trust and Science Foundation Ireland Advanced Materials and Bioengineering Research (AMBER) Centre (SFI/12/RC/2278_P2).