Electrostimulation via a 3D-Printed, Biomimetic, Neurotrophic, Electroconductive Scaffold for the Promotion of Axonal Regrowth After Spinal Cord Injury

Spinal cord injury (SCI) is a devastating neurotrauma, affecting up to 500,000 people annually, and typically results in lifelong paralysis. Electrostimulation can promote neuronal growth, but the formation of a lesion cavity post-SCI inhibits regrowth, limiting its efficacy. Bridging the lesion with a structured, electroactive substrate to direct electrostimulation to growing axons could support and drive regrowth to enable functional recovery. Through mimicking the architecture of the tissue, regrowing neurons could also be guided to meet their distal targets. However, to date, no such platform exists. This study describes the application of electrostimulation via an electroconductive 3D-printed scaffold, comprising an electroconductive polypyrrole/polycaprolactone framework filled with biomimetic & neurotrophic extracellular matrix, to induce significantly longer process extension in neurons.<br/><br/>Biomimetic polycaprolactone (PCL) scaffolds were 3D-printed (Allevi 2) to produce uniaxially aligned, interlocking cylindrical architectures, utilising several channel sizes to match the diameters of human lateral corticospinal cord axonal tracts. Polypyrrole (PPy) was polymerised <i>in situ </i>to form an electroconductive nanoparticle coating on the scaffolds, verified using FTIR and SEM imaging. Electroconductivity was measured via the 4-point probe method. A hyaluronic acid-based extracellular matrix (ECM) was directionally freeze-dried within scaffold channels to provide a neurotrophic environment, and a physical guide to direct neuronal growth. Biocompatibility was assessed by growing neurons and astrocytes on 2D PPy/PCL films and measuring metabolic activity, cellular DNA and morphology. Neurons were electrically stimulated for 7 days on ECM-functionalised PPy/PCL and PCL scaffolds using an Ionoptix bioreactor and imaged to assess the effect of electrostimulation-induced neurite extension.<br/><br/>PCL scaffolds with anatomically relevant channel sizes were 3D-printed and functionalised with PPy and ECM. The conductivity of the PPy coating was measured at 15±5 S/m, 30 times higher than native spinal cord tissue. PPy/PCL substrates displayed excellent biocompatibility – with both neurons and astrocytes colonising the surface with typical morphologies and no difference in metabolic activity or cellular DNA on PPy/PCL in comparison to PCL controls. The freeze-dried neurotrophic ECM formed aligned pore structures within scaffold channels, to guide neuronal neurite outgrowth. Scaffolds of varying channel sizes exhibited no difference in electrical properties or cell activity, showing scaffolds can be scaled to match native cord tracts without losing functionality. Electrostimulation induced significantly longer neurite outgrowth and average neurite length was significantly increased on stimulated PPy/PCL scaffolds compared to all other groups.<br/><br/>This work represents the development of a novel, biphasic electroconductive scaffold, which can direct electrostimulation to promote neurite growth through its longitudinal, ECM-filled channels. The data indicates that PPy/PCL is an excellent substrate for neural tissue engineering and can be developed into ordered structures that can be tailored to match the native tracts to direct neurite growth. Applying electrical stimulation via this electroconductive scaffold results in significantly longer neurite length through the ECM phase of the scaffold, demonstrating the potential of this system to direct and guide axonal growth post-SCI to restore connection and promote functional recovery.<br/><br/><b>FUNDING</b><br/>Irish Rugby Football Union Charitable Trust and SFI Advanced Materials and Bioengineering Research (AMBER) Centre (SFI/12/RC/2278_P2).