Electromechanical 3D-printed bioreactor for muscle tissue engineering: optimization, prototyping, and characterization

In vivo, physical forces are essential regulators of stem cell behavior within their native microenvironments and contribute significantly to the structural and functional maturation of developing tissues. Cardiac and skeletal muscles, in particular, are constantly exposed to intense physical stimulation, including continuous electrical impulses that induce contraction. In this context, traditional two-dimensional (2D) static cultures, such as Petri dishes and flasks, fail to replicate the three-dimensional (3D) architecture and dynamic nature of the in vivo environment, often leading to non-predictive outcomes. To address these limitations, dynamic culture devices, namely bioreactors, have emerged as a pivotal investigation tool in cardiac and skeletal muscle tissue engineering. These devices are designed for replicating native-like conditions such as mechanical and electrical stimulations, which are crucial for promoting proper tissue development and functional maturation. This thesis focuses on the optimization, prototyping and characterization of a bioreactor designed to deliver combinable mechanical and electrical stimulations to 3D hydrogel-based in vitro models of cardiac and skeletal muscle tissues. The culture chamber (CC) was designed to conform to a standard 12-well plate. The main component of the CC is the dynaframe, a patented support, consisting of two rigid, parallel bars, one constrained and one movable, connected by springs, which can hold up to 3 constructs coupled to flexible pillars. In the CC, up to 4 dynaframes can be housed, allowing the parallel stimulation of up to 12 constructs. Mechanical stimulation is achieved via controlled translation of the dynaframe movable element, while electrical stimulation is provided through pairs of electrodes surrounding each construct. Optimization was conducted adopting an iterative process involving CAD modeling, finite element analysis (FEA), and 3D-printed prototyping. In particular, through mechanical tests, the elasticity and durability of the dynaframe springs were assessed. Simulations confirmed that the materials selected for manufacturing the pillars by 3D-printing allow the contraction of the constructs. Moreover, preliminary biological tests, performed using fibrin-based hydrogels and HS-27A stromal cells, demonstrated that the selected geometry and materials support the formation and retention of fibrin-based constructs. Thus, the dynaframe was refined in response to FEA results and end-user feedbacks and also the CC closure mechanism was updated. In conclusion, the characterization of the developed bioreactor confirmed its mechanical performances, reliability and user-friendliness. This electromechanical 3D-printed bioreactor could represent a powerful in vitro platform for fundamental research, tissue engineering, and pharmacological screening, also contributing to the reduction of animal use in preclinical studies.