Engineering of 3D biphasic constructs based on a custom-made poly(ester urethane) and gelatin methacryloyl hydrogels for the development of in vitro 3D bioengineered cardiac tissue models

As chemicals can strongly influence heart proper functioning, the study of their biological effects on the cardiac tissue is highly relevant in the scientific community. Currently, chemicals are tested in animal and bi-dimensional (2D) in vitro models. Despite their widespread use, animal models have limitations due to anatomical and physiological differences from humans and ethical concerns. Conversely, 2D models only provide indicative results, lacking complexity and three-dimensionality (3D). These issues have recently opened the way towards the design of in vitro bioengineered 3D cardiac tissue models. This thesis aimed to contribute to this emerging field by designing 3D scaffolds and hydrogels to be used as 3D framework and cell carrier, respectively, to assemble in vitro cardiac models for cardiotoxicity testing. To this aim, the first part of the thesis project focused on the fabrication of poly(ester urethane) (PUR)-based 3D scaffolds by melt extrusion additive manufacturing (AM). First, a polycaprolactone (PCL)-based PUR was synthesized, showing number average molecular weight (Mn) of ca. 36 kDa and polydispersity index (D) of 1.37. Differential scanning calorimetry was conducted for PUR thermal characterization, evidencing melting temperature of 60 °C and crystallinity of 62%, both attributed to the PCL block. These data coupled with rheological characterization were used to optimize the printing process that was performed at 120 °C cartridge temperature, 30 °C bed temperature, 400 kPa pressure and 8 mm/s printing speed. The PUR also showed excellent thermal stability during printing: the extruded PUR showed Mn of 32 kDa and D of 1.43 after 180 minutes of continuous heating. 3D anisotropic scaffolds were then fabricated and characterized by optical and scanning electron microscopies (average strand diameter 0.41 mm). Surface functionalization was realized by plasma treatment for acrylic acid surface grafting/polymerization, followed by fibronectin (FN) grafting through carbodiimide chemistry. The bicinchoninic acid assay (BCA) proved scaffold correct functionalization. In parallel, Gelatin Methacryloyl (GelMA) hydrogels (5% and 10% w/v concentration) were prepared and used as scaffold fillers. The resulting biphasic 3D structs were characterized by compression tests in longitudinal and transverse directions. The expected anisotropic mechanical behaviour was not clearly visible, most likely because of the struct external polymeric shell significantly contributing to the definition of sample mechanical performances. Similarly, GelMA gels did not significantly affect struct macroscopic mechanical behaviour. Scanning Electron Microscopy images evidenced an interconnected porosity useful for nutrient and oxygen diffusion. Lastly, biphasic structures stability was studied in a watery medium at 37 °C. After a burst dissolution on the first day, GelMA gels remained relatively stable for up to 14 days, irrespective of their starting concentration (dried weight loss of ca. 38%). Overall, the synthesized PUR showed optimal characteristics for processing by melt extrusion AM and the surface functionalization protocol was effective. Furthermore, the PUR scaffolds and GelMA hydrogels showed excellent interconnection. Based on these premises, the developed biphasic constructs hold promise towards the design of in vitro bioengineered cardiac tissue models, potentially replacing animal and 2D models in the future.