Development of innovative 3-D neuronal scaffolds and related additive manufacturing technology for multi-material / multi-functional printing.

Neurodegenerative states pose a significant challenge in modern medicine, affecting over 55 million individuals, and with an estimated global cost of 2$ trillion by 2030. Despite scientific advancements, the intricate interactions between neuronal cells and their environment are not yet fully understood. Stem cell-based in vitro research holds promise in overcoming ethical and physical limitations of in vivo studies. However, the reproduction of the complex 3D soft biochemical neural niche is still an intricate ongoing area of research. Advanced microchips, known as microelectrode array (MEA), have been optimized for neural monitoring and stimulation since late 1970s, however they lack in reproducing the 3D in vivo neural environment, being conventionally planar and rigid interfaces. Their customization is also limited due to high prototyping costs. Twenty years later, neural tissue engineering has emerged in the 1990s as a multidisciplinary field aimed at replicating this complex niche, by means of 3D bioengineered and/or cell-driven platforms. Complex brain-on-chip systems were also developed, but all solutions still have limitations in terms of suitable biomaterials, technical resolution, complexity, and unit integration. In this context, the field of additive manufacturing (AM) can be exploited for the fabrication of multi-material and multi-functional customisable and personalised prototypes, offering high flexibility in design and sensing integration. This thesis aims to the development of innovative proof-of-concept 3D in vitro neural bioelectrical devices through AM technologies and compatible with MEA systems. It particularly explores 3D printing of bioelectronics in combination with MEA and advanced bioprinting as key bioengineered technologies towards the development of an in vitro neural enhanced platform. The ideal platform should integrate a 3D soft bioprinted neural construct on top of a 3D bioconductive hybrid printed MEA. The manufacturing strategy involves Aerosol Jet® Printing for the manufacturing of 3D bioconductive microelectrodes printed on a MEA chip and syringe extrusion 3D FRESH bioprinting for the fabrication of the 3D soft construct. Specifically, Aerosol Jet® Printing, conventionally employed for 2D printed electronics, is here investigated for the development and validation of innovative polymeric and metal-based 3D bioconductive micropillars. Syringe extrusion printing is then explored for the fabrication of early-stage 3D soft neural tissues, starting from low-viscous free-standing hydrogels to 3D FRESH bioinks printed as a 3D soft construct containing mesenchymal or neural lineages. Materials, both commercial and novel formulations, are thoroughly investigated and critically analysed based on their functionality. Most importantly, their biocompatibility is tested with human induced pluripotent stem cells (hiPSC)-derived neural stem cells. In summary, the technological advancements of thesis are believed to hold potential for future research in the development of 3D integrated biomimetic systems, applicable in regenerative and personalized medicine, disease modelling, and drug testing. The findings also extend beyond the main goal of this thesis, with potential applications in lab-on-chips, energy harvesting devices, batteries, and wearable devices.