Biopolymeric microbeads as a 3D scaffold for soft tissue engineering

The increase of different types of cell cultures, which can be used for the in vitro studies of physiological and/or pathological processes, has introduced the need to improve culture techniques through the use of materials and culture media that promote growth, recreating a cellular micro-environment that can be asserted in in vivo condition. Therefore, it is important to design and develop new biologically sustainable methods, such as to contribute to the “closer-to-in vivo” condition. In particular, the design of a 3D in vitro model of neuronal culture is an important step to better understand the mechanisms of cell-cell communication, synaptogenesis and neurophysiological circuits. In order to mimic the ECM environment, a granular, porous and soft structure is preferred in the design of an artificial neural network. The granular structure is preferred due to the fact that CNS tissue seems to be organized as a greater proportion of the microscale tissue, that can be thought of as granular. For this reason, the thesis is focused on the production and characterization of bipolymeric microbeads as a 3D scaffold for soft tissue engineering. The biopolymer Chitosan is presented as an alternative adhesion factor and support for 2D and 3D neuronal cell cultures. 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. Chitosan backbone shows positive charges of primary ammines that favor the electrostatic interactions with the negatively charged cell membranes promoting cell adhesion and growth. The standard studies focoused on the development of nervous system, have been performed using traditional monolayer culture onto supports modified by extracellular matrix components or synthetic biopolymers such as poly-ornithine and poly-lysine which are expressed at stages critical for neuronal differentiation in situ and are functional in neurite outgrowth in vitro, acting as adhesion proteins. Morphological and functional characterization of 2D neuronal culture grew up onto chitosan susbtrates are carried out and compared with the gold standard reported in literature, in order to validate the ability of chitosan to support neuronal adhesion, networks development and the differentiation capacity. 3D cultured neurons on chitosan microbeads based-scaffold, showed a structural development of a functional network that are more representative of the in vivo environment. The studies reported in this thesis, successfully demonstrate the alternative use of the polysaccharide chitosan as adhesion factor and as a structural component for 2D/3D neuronal cultures. Definitely, thanks to its low cost and versatility, it could be easily functionalized for the fabrication of personalized of in vitro models. In this thesis, a new technology to converts monodisperse microbead hydrogels to fine powders, is reported. Microengineered emulsion-to-powder (MEtoP) technology generates microgels with all the molecular, colloidal, and bulk characteristics of fresh microbeas upon resuspension in aqueous media. GelMA microbeads are fabricated by microfluidic technique, that is one of the most effective techniques, and allows precise tuning of the compositions and geometrical characteristics of microbeads. Gelatin-methacryloyl (GelMA) is a semi-synthetic hydrogel which consists of gelatin derivatized with methacrylamide and methacrylate groups. These hydrogels provide cells with an optimal biological environment (e.g., RGD motifs for adhesion) and can be quickly photo-crosslinked, which provide shape fidelity and stability at physiological temperature. MEtoP technology is based on protecting the dispersed phase of an emulsion to preserve its physical and chemical cues during harsh freezing and lyophilization procedures. This technology avoids the persistent problems of colloids, including difficulty in sterilization, bacterial and viral contamination, impaired stability, high processing costs, and difficult packaging and transportation.