Development of an in vitro 3D Model of the Spinal Cord

Spinal cord injuries (SCI) are a devastating neurological and pathological condition that often results in severe motor, sensory and autonomic impairments. Its pathophysiology includes acute and chronic phases that begin immediately after the injury and continue for days, or even weeks, after the event. Many treatments have been developed to prevent neurodegenerative processes, reduce secondary neuronal damage, and improve neuronal recovery. Despite the different degrees of success achieved in the past few decades, there is currently no effective therapy for SCI. The complexity of SCI and the numerous biochemical and physiological changes in the damaged spinal cord are primarily responsible for this gradual but slow improvement. In this context, realistic preclinical models are needed, aiming to allow in vitro studies of the neurodegenerative process and enable the creation of new platforms for pharmacological screening, as complementary tools to reduce and refine animal tests. 3D bioprinting technologies, in particular, provide the possibility of creating engineered tissues capable of reproducing the structural complexity of the native tissue, thanks to the ability to integrate multiple cell types within the construct with high spatial control and provide biomaterial-specific stimuli. In this work, a biomimetic bioink was developed by encapsulating neural stem cells (NE-4C cell line) in a gelatin methacrylate (GelMA) hydrogel. In vitro tests have been conducted to evaluate cell viability after the printing process. Results showed similar cell survival rates compared to cells encapsulated in 3D GelMA hydrogels. To induce the alignment of printed neural cells, a polycaprolactone (PCL) scaffold with aligned fibers has been designed. Melt electrowriting (MEW) technique has been exploited to obtain the final construct, considering a fiber diameter and a distance among the fibers suitable for cell culture. Finally, 3D-bioprinting technique has been used to deposit encapsulated cells on the MEW construct. Printing parameters have been adapted in order to promote good adhesion between the bioink and the PCL scaffold. Neural stem cells were differentiated and cultured for up to 21 days. Immunofluorescence staining results revealed the presence of both neurons and astrocytes, highlighting also the scaffold’s potential to induce alignment of printed cells. Overall, the combination of 3D bioprinting and MEW techniques showed good potential to develop a reliable in vitro model of the spinal cord.