Optimization, characterization and development of a novel electromechanical bioreactor for 3D hydrogel-based cardiac and skeletal muscle constructs

The evaluation of drug efficacy and safety still relies predominantly on two-dimensional (2D) cell cultures and animal models, both of which present significant limitations. Conventional 2D cultures fail to reproduce the complex three-dimensional (3D) architecture and mechanical cues of native tissues, while animal testing remains expensive, ethically debated, and often poorly predictive of human responses. Within this framework, the implementation of bioreactors, systems designed to regulate and monitor the cellular microenvironment, is increasingly crucial to replicate physiological conditions and produce 3D reliable in vitro tissue models for preclinical drug evaluation. Indeed, physical forces within microenvironments are key determinants of stem cell behavior and play a decisive role in tissue growth and maturation. In particular, cardiac and skeletal muscles are continuously subjected to dynamic mechanical and electrical signals that guide their structural organization and functional maturation. In this context, this thesis presents the optimization, multi-level characterization and development of a 3D-printed bioreactor aimed at delivering electromechanical stimulation to hydrogel-based constructs for cardiac and skeletal muscle tissue engineering applications. The system is based on the Dynaframe, a patented mechanical support consisting of fixed and movable elements connected by elastic springs and equipped with flexible pillars that anchor the constructs. The culture chamber, designed to fit a standard 12-well plate, allows parallel stimulation of up to twelve samples through coordinated mechanical and electrical inputs. An iterative process combining computer-aided design (CAD) through SolidWorks, finite element analysis (FEA) using COMSOL Multiphysics, and mechanical characterization guided the optimization of the device. The redesigned culture chamber closure system and refined Dynaframe geometry enhanced usability, robustness, and compatibility with standard biological workflows. The mechanical performance of the Dynaframe springs was evaluated through tensile and fatigue testing, confirming their elasticity and long-term endurance under cyclic loading. The flexural stiffness of the pillars was analyzed through analytical, numerical, and experimental approaches, ultimately revealing the possibility of achieving stiffness values suitable for muscle construct culture. In addition, a dedicated pre-culture platform was developed to standardize hydrogel casting directly around the Dynaframe pillars, ensuring construct integrity during transfer and reducing manual handling. The device was delivered to collaborators at the HEDGe group, Molecular Biotechnology Center (University of Turin), for preliminary biological tests to assess the suitability of the design for laboratory use and to identify the most appropriate printing materials for the Dynaframe component. The obtained results confirmed the suitability of the materials used for the Dynaframe prototyping in allowing cardiac construct formation. Overall, the developed bioreactor and its complementary pre-culture platform provide a robust, modular, and user-friendly system for dynamic 3D culture. Their mechanical reliability, biological compatibility, and reproducibility make them valuable tools for tissue engineering, pharmacological studies, and fundamental research, helping to reduce animal experimentation in preclinical models.