Design, Prototyping, and Preliminary Validation of Modular Single- and Dual-Chamber Bioreactors for Cartilage and Osteochondral Tissue Engineering

Osteochondral (OC) diseases are a growing medical and socioeconomic burden, yet current treatments remain largely ineffective in restoring the structural and functional complexity of native OC tissue, leaving patients with persistent pain, functional limitations, and long-term morbidity. Tissue engineering (TE) has emerged as a promising strategy, aiming to develop functional in vitro tissues capable of replacing damaged counterparts. However, state-of-the-art TE approaches still face significant limitations, particularly in replicating the in vivo dynamic physicochemical microenvironment that regulates tissue development and cellular responses. In this context, bioreactors represent key enabling technologies, providing controlled culture conditions and multiphysical stimulation, namely shear stress (SS) and hydrostatic pressure (HP), to better mimic physiological environments. Building upon previous developments in bone TE, this thesis focused on the design, prototyping, and preliminary validation of two novel bioreactor systems: a single-chamber bioreactor for cartilage and a dual-chamber bioreactor for OC constructs. The systems were developed through an iterative methodology, combining CAD modeling, computational fluid dynamics (CFD) simulations, rapid prototyping via 3D printing, and experimental testing. Both systems were conceived with a modular architecture, allowing easy assembly, disassembly, sterilization, and reusability, while maintaining compactness and compatibility with sterile operation. CFD simulations guided the optimization of the internal chamber geometries, enabling uniform flow distribution across constructs while minimizing bubble entrapment, stagnation, and recirculation zones. Furthermore, simulations provided quantitative insights into SS and HP stimuli within the chambers, supporting the definition of appropriate stimulation parameters. Prototypes were then experimentally validated through in-house tests assessing pressure resistance, sealing, and, for the dual-chamber system, the absence of cross-contamination between compartments. The tests demonstrated that the cartilage bioreactor can withstand elevated HP without leakage or component failure, confirming its readiness for dynamic cell culture experiments. Future studies will explore long-term resistance under dynamic culture conditions and assess the effects of different stimulations on cell behavior. Although still at an earlier stage, the OC bioreactor, has already achieved fundamental milestones: validation of its dual-chamber separation, demonstration of uniform perfusion across the constructs, and the possibility of scaffold visualization under closed-chamber conditions. The main challenges yet to be addressed concern scaffold permeability, refinement of disassembly procedures, and long-term stability under sustained HP. In conclusion, the developed systems represent promising in vitro platforms for advancing cartilage and OCTE. By integrating modular design, CFD-guided optimization, and 3D-printed prototyping, this work provides innovative tools for investigating fundamental aspects of OC mechanobiology. Although not yet enabling direct clinical translation, these bioreactors establish a solid foundation for future biological validation and experimental exploration, thereby contributing to the progressive development of clinically relevant models for disease research, drug screening, and, in the long term, regenerative therapies.