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 μm) 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 μm 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 μm assessed by the PrestoBlue assay. The Reverse Transcription-Polymerase Chain Reaction (RT-PCR) results involving the three most important genes concerning neural differentiation, namely β3-tubulin, NF-H, and GAP-43, confirm that the scaffolds with a pore size of 125 ± 15 μm 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 β3-tubulin and NF200, showed directional alignment of the neurite growth along the fibre direction in EHD-jet 3D printed scaffolds.

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The incidence of peripheral nerve injuries is on the rise and the current gold standard for treatment of such injuries is nerve autografting. Given the severe limitations of nerve autografts which include donor site morbidity and limited supply, neural guide conduits (NGCs) are considered as an effective alternative treatment. Conductivity is a desired property of an ideal NGC. Reduced graphene oxide (rGO) possesses several advantages in addition to its conductive nature such as high surface area to volume ratio due to its nanostructure and has been explored for its use in tissue engineering. However, most of the works reported are on traditional 2D culture with a layer of rGO coating, while the native tissue microenvironment is three-dimensional. In this study, PCL/rGO scaffolds are fabricated using electrohydrodynamic jet (EHD-jet) 3D printing method as a proof of concept study. Mechanical and material characterization of the printed PCL/rGO scaffolds and PCL scaffolds was done. The addition of rGO results in softer scaffolds which is favorable for neural differentiation. In vitro neural differentiation studies using PC12 cells were also performed. Cell proliferation was higher in the PCL/rGO scaffolds than the PCL scaffolds. Reverse transcription polymerase chain reaction and immunocytochemistry results reveal that PCL/rGO scaffolds support neural differentiation of PC12 cells.