GNB2023, June 21st-23rd 2023, Padova, Italy 1 Abstract—Humans are continuously exposed to a wide variety of chemicals. Animal and bi-dimensional (2D) in vitro models currently represent the gold standard for chemical toxicity assessment, although they do not adequately reproduce the real human scenario, thus resulting in poorly predictive results. In this context, New Approach Methodologies are needed for a proper assessment of chemical toxicity. For instance, three- dimensional (3D) bioengineered in vitro models have the potential to closely represent the human physio-pathological milieu, enabling a more reliable evaluation of chemical toxicity. Among human tissues/organs, the myocardium is a target organ for chemical- and drug-induced toxicity. In this work, a custom- made poly(ester urethane) was synthesized and used as raw material to fabricate a platform of multi-layered structures with different geometrical features through melt extrusion additive manufacturing. The designed matrices were also surface functionalized with proteins to improve their biomimesis of the native cardiac extracellular matrix. The physico-chemical properties of the constructs proved their suitability for the development of in vitro bioengineered cardiac tissue models, enabling the investigation of physio-pathological processes and cardiotoxicity testing. Keywords—poly(urethane)s, in vitro cardiac tissue models, additive manufacturing, surface functionalization. I. INTRODUCTION Drug development, chemical toxicity assessment, and disease modeling heavily rely on the use of animal models and two- dimensional in vitro models [1]. However, these models exhibit several drawbacks that make their outcomes poorly predictive of the real human scenario. Two-dimensional models are extremely simple and do not allow to replicate the complexity of human tissue structure [2]. Conversely, animal models present high management costs and ethical issues. Furthermore, due to interspecies differences, animal models are unlikely to faithfully replicate the human physio- pathological behavior. The concordance between toxicity levels evaluated in humans and laboratory animals is around 71% when both rodent (mice and rats) and non-rodent (dogs and monkeys) models are used. This concordance is reduced to 43% using only rodents as animal models [3]. The consequences of these issues are diverse and heterogeneous. The selection effectiveness of preclinical trials is reduced: only 11.8% of drugs entering clinical trials reaches approval. In addition, there are considerable cases of approved drugs showing undesirable toxic effects. For instance, during the last decade eight non-cardiovascular drugs have been reported to induce ventricular arrhythmia and sudden death, and thus they have been withdrawn from the market [4]. Furthermore, the proper and reliable assessment of the toxicity of chemicals and chemical mixtures is another key target to achieve, with important health, social and economic repercussions. Until 2008, only 5,000 chemicals out of 100,000 present on the western market had been subjected to an approved analysis process due to the limitations of two- dimensional and animal models [3]. In this complex scenario, the development of reliable three- dimensional (3D) bioengineered in vitro models is of central importance since they can be very useful tools for overcoming the limitations of previously adopted approaches [5] [6]. In particular, the design of reliable 3D in vitro models of the cardiac tissue in gaining increasing interest in the research community. On the PubMed database the number of publications related to “cardiac tissue models” is exponentially increasing over time, with around 4000 publications in 2022. This trend can be correlated to the very high social and economic impact of heart diseases, that are responsible for around 17.5 million deaths worldwide each year [7]. Secondly, the high complexity of the cardiac tissue and the very limited possibilit