Electrohydrodynamic Jet 3D Printed Nerve Guide Conduits (NGCs) for Peripheral Nerve Injury Repair

The prevalence of peripheral nerve injuries resulting in loss of motor function, sensory function, or both, is on the rise. Artificial Nerve Guide Conduits (NGCs) are considered an effective alternative treatment for autologous nerve grafts, which is the current gold-standard for treating peripheral nerve injuries. In this study, Polycaprolactone-based three-dimensional porous NGCs are fabricated using Electrohydrodynamic jet 3D printing (EHD-jetting) for the first time. The main advantage of this technique is that all the scaffold properties, namely fibre diameter, pore size, porosity, and fibre alignment, can be controlled by tuning the process parameters. In addition, EHD-jetting has the advantages of customizability, repeatability, and scalability. Scaffolds with five different pore sizes (125 to 550 mu m) and porosities (65 to 88%) are fabricated and the effect of pore size on the mechanical properties is evaluated. In vitro degradation studies are carried out to investigate the degradation profile of the scaffolds and determine the influence of pore size on the degradation rate and mechanical properties at various degradation time points. Scaffolds with a pore size of 125 +/- 15 mu m meet the requirements of an optimal NGC structure with a porosity greater than 60%, mechanical properties closer to those of the native peripheral nerves, and an optimal degradation rate matching the nerve regeneration rate post-injury. The in vitro neural differentiation studies also corroborate the same results. Cell proliferation was highest in the scaffolds with a pore size of 125 +/- 15 mu m assessed by the PrestoBlue assay. The Reverse Transcription-Polymerase Chain Reaction (RT-PCR) results involving the three most important genes concerning neural differentiation, namely beta 3-tubulin, NF-H, and GAP-43, confirm that the scaffolds with a pore size of 125 +/- 15 mu m have the highest gene expression of all the other pore sizes and also outperform the electrospun Polycaprolactone (PCL) scaffold. The immunocytochemistry results, expressing the two important nerve proteins beta 3-tubulin and NF200, showed directional alignment of the neurite growth along the fibre direction in EHD-jet 3D printed scaffolds.