Extrusion-Based Bioprinting of customized polyurethanes for cardiac 3D in vitro model design

Cardiovascular diseases (CVDs) are the leading cause of illness and death worldwide, with heart failure resulting from myocardial infarction being the most fatal among all CVDs. All available approaches for CVD treatment, including medical device implantation, pharmaceutical therapy, and transplants, have significant limitations. In this context, there is a growing demand for advanced and reliable in vitro cardiac tissue models able to provide (i) a deeper understanding of the biophysical and biomolecular pathways of cardiac muscle and CVDs, and (ii) promising new strategies for repairing and regenerating damaged cardiac tissue and discovering potential therapies that could significantly improve patient survival. To address these challenges, traditional Cardiac Tissue Engineering (CTE) approaches are currently translated towards the design of in vitro 3D cardiac models. The aim of this thesis was to develop a new cardiac 3D in vitro model by integrating extrusion-based bioprinting and human induced pluripotent stem cells-derived cardiomyocytes (iPSC-CMs). To achieve this purpose, biological constructs were developed from a custom-made thermo- and photo-sensitive hydrogel. Thermo-sensitivity was achieved by an amphiphilic poly(ether urethane) (PEU) based on Poloxamer® 407, 1,6-hexanediisocyanate, and N-Boc serinol. After the synthesis, the PEU underwent acid deprotection to remove Boc groups, thus exposing primary amino groups along the polymer chains. The photo-sensitivity was introduced by using poly(ethylene glycol) diacrylate (PEGDA), synthesized by reacting poly(ethylene glycol) with acryloyl chloride. Synthesis success was confirmed by spectroscopic and chromatographic techniques. For the bioink cellular component, iPSCs were differentiated into CMs in a 2D culture, mimicking the stages of embryonic cardiogenesis. The first spontaneous contractions were observed on day 12 of the differentiation protocol and the correct differentiation was verified through RT-qPCR by quantifying the expression of cardiac genes. A preliminary biocompatibility test was performed on gel droplets (12.5%w/v SHP407, 10%w/v PEGDA, 0.1%w/v Lithium phenyl-2,4,6-trimethylbenzoylphosphinate as photo-initiator) showing a high percentage of cell death, probably due to the synthetic nature of the gel. Indeed, the addition of 2%v/v Matrigel to the formulation, induced a significant increase in cell viability, with cells continuing to express cardiac genes after one week of culture. Then, the printing process was optimized using an extrusion-based printer. The optimal parameters were defined to be: printing speed of 25±5 mm/s, pressure of 65±5 kPa, and 45 seconds of UV light exposure with 60% brightness for each layer. Five-and seven-layers bioprinted constructs were printed, and their uniformity ratio was quantified demonstrating a good shape fidelity. The bioprinted scaffolds exhibited significant swelling and stability in culture medium for up to 21 days. Lastly, the bioink containing iPSCs-derived CMs was bioprinted using the optimized parameters and the biological behaviour of the printed constructs was investigated, focusing on cell viability and metabolic activity post-printing. Despite the need for further characterizations, this work represents a significant advancement in CTE. This specific combination of polymers was tested for the first time for bioprinting with this type of cardiac cells, paving the way for future innovations in this field and potentially revolutionizing the treatment of CVDs.