Improvement of In Vitro Three‐Dimensional Cartilage Regeneration by a Novel Hydrostatic Pressure Bioreactor

: In vitro three-dimensional (3D) cartilage regeneration is a promising strategy for repair of cartilage defects. However, inferior mechanical strength and tissue homogeneity greatly restricted its clinical translation. Simulation of mechanical stress through a bioreactor is an important approach for improving in vitro cartilage regeneration. The current study developed a hydrostatic pressure (HP) bioreactor based on a novel pressure-transmitting mode achieved by slight deformation of a flexible membrane in a completely sealed stainless steel device. The newly developed bioreactor efficiently avoided the potential risks of previously reported pressure-transmitting modes and simultaneously addressed a series of important issues, such as pressure scopes, culture chamber sizes, sealability, contamination control, and CO2 balance. The whole bioreactor system realized stable long-term (8 weeks) culture under high HP (5-10 MPa) without the problems of medium leakage and contamination. Furthermore, the results of in vitro 3D tissue culture based on a cartilage regeneration model revealed that HP provided by the newly developed bioreactor efficiently promoted in vitro 3D cartilage formation by improving its mechanical strength, thickness, and homogeneity. Detailed analysis in cell proliferation, cartilage matrix production, and cross-linking level of collagen macromolecules, as well as density and alignment of collagen fibers, further revealed the possible mechanisms that HP regulated in vitro cartilage regeneration. The current study provided a highly efficient and stable bioreactor system for improving in vitro 3D cartilage regeneration and thus will help to accelerate its clinical translation. SIGNIFICANCE Inferior mechanical strength and tissue homogeneity of in vitro engineered three-dimensional (3D) cartilage greatly restricted its clinical translation. The current study developed a hydrostatic pressure (HP) bioreactor based on a novel pressure-transmitting mode, which efficiently avoided potential risks of the previously reported bioreactors in culture environment and contamination controls. The newly developed bioreactor realized stable long-term culture under high HP and efficiently promoted in vitro 3D cartilage formation by improving its mechanical strength, cartilage regeneration thickness, tissue homogeneity, cell proliferation, extracellular matrix contents, and collagen cross-linking level. This study provided a highly efficient and stable bioreactor system for improving in vitro 3D cartilage regeneration and thus helped to accelerate the clinical translation of this system.