In vitro phenotypically stable cartilage regeneration with mechanically adaptable microenvironment using a hydrostatic pressure bioreactor

The regenerative repair of diverse cartilage injuries is a remarkable clinical challenge. Recently, hydrogel-based tissue engineering strategy offers an alternative treatment for cartilage defect repair. However, in vitro cartilage pre-culture period is often confronted with predicaments of insufficient nutrition transport and cartilage phenotypic instability. Inspired by natural mechanical microenvironment of articular cavity, our group has pioneered a hydrostatic pressure (HP) bioreactor for cartilage regeneration. Herein, we present an in vitro hydrostatic bioreactor method using porous hydrogel scaffolds for accelerating phenotypically stable cartilage regeneration. In this study, 3D-printed porous gelatin/chondroitin sulfate composite hydrogel scaffolds exhibit favorable biomechanical properties, biocompatibility, and controllable porous structure. Importantly, it is demonstrated that HP stimulation transcriptionally upregulates the key mechanosensitive channels encoded by TRPV4 and PIEZO1 targets, which initiates the Ca 2+ -dependent TRPV4/PIEZO1-Ca 2+ -SOX9 mechanical transduction. Meanwhile, the mechanical signals effectively promote chondrogenesis while suppress hypertrophic and ossification. In vivo goat models further confirm that in vitro HP-preconditioned cartilage-like constructs show satisfactory long-term regenerative outcomes, particularly in an in situ auricular microenvironment. This study therefore deeply explores the mechanisms of mechanically adaptable microenvironment mediated by HP stimulation in regulating phenotypically stable cartilage regeneration, suggesting a promising clinical treatment by in vitro engineered cartilage. • Successfully promoting cartilage regeneration in vitro through hydrostatic pressure. • Mediating cartilage regeneration through the TRPV4/PIEZO1-Ca 2+ -SOX9 signaling pathway. • Successfully verified the impact of the in situ microenvironment on the maintenance of cartilage phenotype.