The nervous system is the most complex in the human body, coordinating a detailed network of signals and responses that regulate all of our actions and sensations. Given its complexity, comprehending how the nervous system operates and its associated disorders is exceptionally complicated. Neuroscience has long grappled with unraveling the mysteries of this system, particularly the intricate relationship between its structure and function, which has captivated researchers for decades. In this pursuit, in vitro models have emerged as invaluable tools, offering simplified yet insightful representations of neural processes. However, to further our understanding, there is an urgent need to expand and refine biofabrication techniques, driving the development of more technologically advanced models. Through the integration of engineering technologies, materials science, and nanotechnologies, in this thesis, I focus on biomaterials and their use in biofabrication in order to develop enhanced models where it is possible to control the structural and/or functional aspects of a neural network. Chitosan is a copolymer of glucosamine and N-acetyl-glucosamine, obtained by the deacetylation of chitin; it is well known for its low cost, biocompatibility, biodegradability, muco-adhesiveness, antibacterial activity as well as its bioaffinity. This natural polymer can be used as an adhesion factor since it can anchor the cells thanks to the electrostatic interaction generated between the cell membrane negatively charged and the amine group exposed along the chitosan backbone. On the other side, gold particles synthesized on the nanoscale, known as gold nanoparticles, exhibit unique optical properties due to surface plasmon resonance when interacting with light. Anisotropic shapes, like gold nanorods, are particularly intriguing, with tunable surface plasmon resonance bands in the near-infrared wavelength range. The gold nanorods' plasmonic properties make it possible to convert light energy into heat and luminescence, making gold nanorods particularly useful in numerous biological applications. By merging chitosan's bioadhesive and biomimetic attributes with the plasmonic properties of gold nanorods, I realized a photosensitive ink suitable for printing using a commercial drop-on-demand inkjet printer. This ink facilitates precise control over the geometry of neural networks through a quick and straightforward deposition. Printing onto a microelectrode array allows for the recording of neural activity. Furthermore, the plasmonic properties of the ink enable the modulation of patterned neural network activity via the photothermal effect. As a result, the newly developed engineering system can both record and inhibit neural network activity while also controlling its geometry. Considering the limitations of two-dimensional models in replicating the in vivo environment due to its three-dimensional nature, this thesis introduces the pioneering application of inhibiting neural activity through the thermoplasmonic effect in threedimensional culture. The scaffold is constructed using glass microspheres, previously validated as a supportive structure for the three-dimensional growth of a functional neural network. Utilizing the layer-by-layer technique, I functionalized the glass microspheres with gold nanoparticles, conferring the plasmonic properties to the scaffold. I demonstrated the scaffold's efficiency in supporting the development of a functional three-dimensional network and in modulating its activity through the thermoplasmonic effect. Nonetheless, the mechanical properties of the glass scaffold significantly differ from those of soft tissues, making it incapable of accurately mimicking the brain microenvironment. To create a more biomimetic scaffold, I functionalized a chitosanbased thermosensitive solution with gold nanorods and spatially controlled its crosslinking using the photothermal effect. This process resulted in the fabrication of a photosensitive hydrogel with mechanical properties within the range of the brain. Gold nanorods release heat on the nanoscale, so in comparison to the typical crosslinking process in the incubator, photothermal crosslinking is selective for the area where the laser spot is applied. Finally, the scaffold's efficiency in modulating the activity of the 3D neural network is confirmed. In addition, controlling the microstructure in three dimensions is essential to replicate the physiological organization of anisotropic tissues, such as muscle and nerve fibers. The unidirectional ice-templating technique is commonly employed to fabricate anisotropic scaffolds that guide the alignment of post-seeded neural cells. However, post-seeding imposes limitations due to its low control over cell arrangement within the scaffold. One of the primary challenges associated with using this technique with embedded cells is their compromised viability during the freezing and thawing, where ice crystal formation and osmotic effects damage the cell membrane. Among various cryoprotective agents, disaccharides and trisaccharides have been particularly effective in limiting osmotic damage due to their high molecular weight. Hyaluronic acid is a linear polysaccharide widely found in the nervous system and is one of the most physiologically relevant extracellular matrices discovered. It is possible to synthesize hyaluronic acid methacryloyl, which can be used to fabricate hydrogel scaffolds capable of mimicking the extracellular matrix of the nervous system. In my thesis, I optimized a cryo-bioink formulation based on hyaluronic acid methacryloyl to preserve cell viability during unidirectional freeze casting. Furthermore, I demonstrated the effectiveness of the anisotropic scaffold in aligning both muscle and neural cells, and I showed the printability of the cryo-bioink using a multi-material cryo-bioprinter. In summary, the new engineering approaches presented in this thesis enable control over the structure and neural activity in in vitro models, encompassing both the simplified two-dimensional case and the more complex three-dimensional models that closely resemble the in vivo environment. These advancements are poised to offer novel tools for deepening our comprehension of the nervous system and its associated disorders.

Biofabrication Techniques for Controlling Structure and Function in In Vitro Neural Models

ANDOLFI, ANDREA
2024-05-31

Abstract

The nervous system is the most complex in the human body, coordinating a detailed network of signals and responses that regulate all of our actions and sensations. Given its complexity, comprehending how the nervous system operates and its associated disorders is exceptionally complicated. Neuroscience has long grappled with unraveling the mysteries of this system, particularly the intricate relationship between its structure and function, which has captivated researchers for decades. In this pursuit, in vitro models have emerged as invaluable tools, offering simplified yet insightful representations of neural processes. However, to further our understanding, there is an urgent need to expand and refine biofabrication techniques, driving the development of more technologically advanced models. Through the integration of engineering technologies, materials science, and nanotechnologies, in this thesis, I focus on biomaterials and their use in biofabrication in order to develop enhanced models where it is possible to control the structural and/or functional aspects of a neural network. Chitosan is a copolymer of glucosamine and N-acetyl-glucosamine, obtained by the deacetylation of chitin; it is well known for its low cost, biocompatibility, biodegradability, muco-adhesiveness, antibacterial activity as well as its bioaffinity. This natural polymer can be used as an adhesion factor since it can anchor the cells thanks to the electrostatic interaction generated between the cell membrane negatively charged and the amine group exposed along the chitosan backbone. On the other side, gold particles synthesized on the nanoscale, known as gold nanoparticles, exhibit unique optical properties due to surface plasmon resonance when interacting with light. Anisotropic shapes, like gold nanorods, are particularly intriguing, with tunable surface plasmon resonance bands in the near-infrared wavelength range. The gold nanorods' plasmonic properties make it possible to convert light energy into heat and luminescence, making gold nanorods particularly useful in numerous biological applications. By merging chitosan's bioadhesive and biomimetic attributes with the plasmonic properties of gold nanorods, I realized a photosensitive ink suitable for printing using a commercial drop-on-demand inkjet printer. This ink facilitates precise control over the geometry of neural networks through a quick and straightforward deposition. Printing onto a microelectrode array allows for the recording of neural activity. Furthermore, the plasmonic properties of the ink enable the modulation of patterned neural network activity via the photothermal effect. As a result, the newly developed engineering system can both record and inhibit neural network activity while also controlling its geometry. Considering the limitations of two-dimensional models in replicating the in vivo environment due to its three-dimensional nature, this thesis introduces the pioneering application of inhibiting neural activity through the thermoplasmonic effect in threedimensional culture. The scaffold is constructed using glass microspheres, previously validated as a supportive structure for the three-dimensional growth of a functional neural network. Utilizing the layer-by-layer technique, I functionalized the glass microspheres with gold nanoparticles, conferring the plasmonic properties to the scaffold. I demonstrated the scaffold's efficiency in supporting the development of a functional three-dimensional network and in modulating its activity through the thermoplasmonic effect. Nonetheless, the mechanical properties of the glass scaffold significantly differ from those of soft tissues, making it incapable of accurately mimicking the brain microenvironment. To create a more biomimetic scaffold, I functionalized a chitosanbased thermosensitive solution with gold nanorods and spatially controlled its crosslinking using the photothermal effect. This process resulted in the fabrication of a photosensitive hydrogel with mechanical properties within the range of the brain. Gold nanorods release heat on the nanoscale, so in comparison to the typical crosslinking process in the incubator, photothermal crosslinking is selective for the area where the laser spot is applied. Finally, the scaffold's efficiency in modulating the activity of the 3D neural network is confirmed. In addition, controlling the microstructure in three dimensions is essential to replicate the physiological organization of anisotropic tissues, such as muscle and nerve fibers. The unidirectional ice-templating technique is commonly employed to fabricate anisotropic scaffolds that guide the alignment of post-seeded neural cells. However, post-seeding imposes limitations due to its low control over cell arrangement within the scaffold. One of the primary challenges associated with using this technique with embedded cells is their compromised viability during the freezing and thawing, where ice crystal formation and osmotic effects damage the cell membrane. Among various cryoprotective agents, disaccharides and trisaccharides have been particularly effective in limiting osmotic damage due to their high molecular weight. Hyaluronic acid is a linear polysaccharide widely found in the nervous system and is one of the most physiologically relevant extracellular matrices discovered. It is possible to synthesize hyaluronic acid methacryloyl, which can be used to fabricate hydrogel scaffolds capable of mimicking the extracellular matrix of the nervous system. In my thesis, I optimized a cryo-bioink formulation based on hyaluronic acid methacryloyl to preserve cell viability during unidirectional freeze casting. Furthermore, I demonstrated the effectiveness of the anisotropic scaffold in aligning both muscle and neural cells, and I showed the printability of the cryo-bioink using a multi-material cryo-bioprinter. In summary, the new engineering approaches presented in this thesis enable control over the structure and neural activity in in vitro models, encompassing both the simplified two-dimensional case and the more complex three-dimensional models that closely resemble the in vivo environment. These advancements are poised to offer novel tools for deepening our comprehension of the nervous system and its associated disorders.
31-mag-2024
Biofabrication, Neuromodulation, Gold nanoparticles, Hydrogel, 3D neuronal model, Photothermal effect, Human induced pluripotent stem cells, Electrophysiology, Neural cells alignment
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11567/1176315
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