**4. 3D bioprinting and neurodegeneration**

ability to maintain a three-dimensional structure once printed and maintained in culture [65]. The main feature of the hydrogel, biocompatible material used as a three-dimensional support for cell growth, is the ability to be extremely hydrophilic, making it an excellent candidate in terms of biocompatibility for its use in bioprinting. It was initially used in TE because it was able to simulate the extracellular matrix, guarantying cell growth and communication [75]. Biomaterials are divided, According to their derivation, biomaterials are divided in natural or synthetic compunds. There are naturally derived polymers such as sodium alginate, gelatin, collagen, chitosan, fibrin, and GelMA [76–78] and synthetic polymers such as Pluronic®, polyethylene glycol (PEG), and polyurethanes [79, 80]. Over time, it has been seen how the natural compounds are more performing when placed in contact with the cells than the synthetic ones. Several cell types associated with different biomaterials to compose the bioink have already been used in several research areas, where cellular viability and motility have been demonstrated, as well as a spatial organization similar to in vivo tissue [81]. To create a new biomaterial, we must consider different physical, mechanical, and biological characteristics that are close to the tissue we want to recreate. Thus, researchers tend to create a combination of biomaterials for each cell type, and with well-defined printability specifications, so as to make the process as standardized and reproducible as possible, despite being a very open field and full of new developments. New-generation bioinks are now able to maintain each of these characteristics, thus improving the success in terms of bioprinting. All this is possible if particular attention is paid to the following chemical, physical, and biological properties: rheological studies (viscosity, thinning, viscoelasticity), biofunctional analysis, biodegradation, and polymerization (cytocompatibility, cell adhesion, migration, proliferation, and differentiation) [82]. One of the most important features that has different biomaterials is the ability to cross-link once the bioink has been printed, reticulating the bioink in which cells are encapsulated, without affecting the viability, the differentiation, and the capacity of migration [83]. The ability to polymerize depends on the material used; for example, collagen needs chemical cross-link, through covalent bonds that bind free amines or carboxylic groups of collagen that is able to reticulate, also through a biological process, and through the interaction with transglutaminase. Other compounds, such as sodium alginate, use an ionic cross-link: divalent cations such as Ca2+ bind to two sodium alginate residues, cross-linking the structure. UV radiation is a very promising cross-link technique given its reaction speed but with many questions regarding the possible damage induced to the cells included in the material. Other materials, such as gelatin and agarose, are heat-sensitive, so they are used during the printing process at the melting temperature and then stiffened with cooling [84–87]. One of the most common types of bioink used in bioprinting techniques is the so-called cell-laden hydrogel, which includes natural hydrogels such as agarose, sodium alginate, chitosan, collagen, gelatin, fibrin, and hyaluronic acid and synthetic hydrogels such as Pluronic® and PEG. Hydrogels can be used with the most common bioplotter that mount different printing techniques, allowing the creation of bioinks that combine the advantages of the natural material with the advantages of synthetic materials [88]. Recent findings have shown also the possibility to transfect cells with target DNA or plasmid, directly during the bioprinting process [80]. A new promising method to develop bioinks is the base on decellularized extracellular matrix. This kind of bioink consists in eliminating cells from a tissue of interest, keeping intact

34 Recent Advances in Neurodegeneration

In the last decade, the possibility of replacing dead cells in degenerative processes affecting the central nervous system opened the way for a more intense and accurate study of stem cells and their possibility of replacing damaged tissue [92]. It was also thought to exploit the ability of stem cells to secrete cytokines and growth factors, offering benefits such as anti-inflammatory effects, protection of neural cells, and endogenous recovery systems. Transplanting these cells into damaged sites presents various problems such as low cell survival and limited engraftment [93]. To minimize these problems, it was decided to use three-dimensional scaffold printing that mimics the complexity both from the biological and functional points of view of the tissue to be replaced [94].

The manufacture of three-dimensional prefabricated scaffolds has already given positive results in the treatment and repair of spinal and nerve damage but with a great limitation in terms of control of the external shape of the scaffold and of its internal architecture [95, 96]. These problems have been overcome with the 3D bioprinting, which leaves the operator complete freedom regarding the shape, the material, and its internal architecture. The recent developments in the field of 3D bioprinting are mostly aimed to the field of regenerative medicine, to respond to the growing demand for tissues and organs for transplants, arriving only later for this technology to be applied to basic scientific research. Until now only few studies have focused on using 3D printing applied to the creation of neural tissue compared to other widely studied tissues such as the skin, bones, heart tissue, and cartilaginous structures [97]. The few studies published so far, in which they use nerve cells in 3D printing processes [98, 99], show a poor characterization of bioinks to be used for nerve cells, due to the delicacy of the tissue to be recreated and of the characteristics necessary for the optimal growth of the nervous tissue [94]. Recently, researchers also think that the nervous tissue printed in 3D may be used for the neural regeneration, a huge possibility in the field of neurodegeneration to replace degenerated neural tissue [78, 80].

The creation of nerve tissue by bioprinting is also used for pharmacological studies, for toxicological screening, and for basic research. It is necessary to underline how this field is still in its infancy and how it is necessary to validate this model for the applications described up to now, to be sure that the model completely recapitulates the pathophysiology that we want to investigate with this tool [94] in particular with regard to neurodegenerative diseases.
