Cardiac tissue engineering: designing innovative approaches combining bioreactor-based technologies and biological strategies

Heart disease is the leading cause of morbidity and mortality in the Western World, claiming 7.25 million deaths every year, with an increasing trend destined to rise up to about 23.6 million in 2030. This scenario greatly motivates research into effective therapeutic interventions, since complete native regeneration is unlikely for fully differentiated, loadbearing cardiac tissues. However, current therapeutic cardiac strategies, e.g., transplantation of autologous and donor grafts, and implantation of artificial prostheses or devices as left ventricular assist devices, suffer from limitations. The principal contraindications in using autologous grafts are identified in donor site morbidity, limited availability, risk of infection, and secondary surgical wounds. In case of donor grafts, the disadvantages are the shortage of available donors, the risk of pathogen transfer and rejection, and mandatory lifelong immunosuppressive therapies. Finally, the use of artificial prostheses or devices implies limited durability, inability to completely restore natural functions, and often leads to the establishment of unphysiological conditions with the need of lifelong anticoagulation therapies. Therefore, the innovative field of cardiac tissue engineering (TE) could represent an effective alternative to overcome the limitations of the current clinical therapies. This strategy, in fact, has shown the potential to generate both functional cardiac tissue substitutes for use in the failing heart, and biological in vitro model systems to investigate the cardiac tissue-specific development and diseases, allowing to perform accurate and controlled in vitro tests for cell and tissue-based therapies, drug screening, predictive toxicology and target validation. In the last decade, a great deal of progress was made in this field due to new advances in interdisciplinary areas such as biology, genetic engineering, biomaterials, polymer science, bioreactor engineering, and stem cell biology. Each of these areas has contributed to the development of the three main components of the cardiac TE, i.e., cells, scaffolds and culture environment, that can be used individually or in combination: (1) cells synthesize the new tissue; (2) scaffolds provide physical support to cells and a structural and biochemical cue tailored to promote cell adhesion, migration, proliferation and differentiation; (3) biomimetic in vitro culture environments, designed to replicate the in vivo milieu by using biologically inspired requirements, influence and drive cells to differentiate towards the desired phenotype and to express their functions, promoting extracellular matrix formation and tissue maturation. The objectives of this work concern two complementary aspects of the cardiac TE field, combining innovative bioreactor-based technologies and advanced biological strategies. In particular, during the PhD as engineering approach two different bioreactor prototypes for cardiac TE have been developed (Chapter II and III), while as biological and applicative approach the work dealt with the generation of a contractile cardiac patch by using a perfusion-based bioreactor culture system (Chapter IV). Bioreactors, technological devices designed for monitoring and controlling the culture environment and physiologically stimulating the construct, have become essential support tools in cardiac TE research. Therefore, the first part of the research activity was dedicated to the development of two different bioreactor prototypes in the effort to replicate the native physical microenvironment of the cardiac tissue. Starting from a wide and in-depth review of the state of the art of bioreactors for cardiac TE (Chapter I), two bioreactor prototypes were developed (Chapter II and III), firstly, as model systems to investigate in vitro the effects of biophysical stimuli on cardiac cell culture and cardiac tissue formation and maturation. Secondly, once optimal culture conditions have been identified, bioreactors can be used as production systems for in vitro generation of functional engineered cardiac tissues. In detail, they will be illustrated: (1) a bioreactor for culturing cell-seeded cardiac patches, designed for generating a physiological biochemical and physical environment that mimics the native stimuli of the cardiac tissue, by providing uniaxial cyclic stretching and electrical stimulations (Chapter II); and (2) an innovative and low-cost bioreactor that, with peculiar geometric features, assures the possibility for buoyant vortices to be generate without using electromechanical rotating systems, allowing the establishment of suspension and low-shear conditions (Chapter III). For both the prototypes, preliminary cellular tests demonstrated the suitability of the devices for cardiac TE applications. The second part of the research activity (Chapter IV) concerned a biological approach, with the aim to provide proof-of-principle for engineering reproducible thickrelevant skeletal myoblast-based contractile three-dimensional patches by using perfusionbased bioreactor culture system and type I collagen scaffold. The research activities carried out in this thesis confirm the importance of combining complementary fields such as engineering and biology. A comprehensive approach that joins innovative bioreactor-based technologies with advanced biological strategies could provide an useful model system for cardiac development in the next future, while, in the late future, such approach could represent an effective therapeutic strategy to the reestablishment of the structure and function of injured cardiac tissues in the clinical practice, strongly contributing to the improvement of quality of life