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, <i>in vitro</i> 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 <i>in vitro</i> 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 <i>TRPV4</i> and <i>PIEZO1</i> targets, which initiates the Ca²⁺-dependent <i>TRPV4/PIEZO1-Ca</i> ^<i>2+</i> <i>-SOX9</i> mechanical transduction. Meanwhile, the mechanical signals effectively promote chondrogenesis while suppress hypertrophic and ossification. <i>In vivo</i> goat models further confirm that <i>in vitro</i> HP-preconditioned cartilage-like constructs show satisfactory long-term regenerative outcomes, particularly in an <i>in situ</i> 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 <i>in vitro</i> engineered cartilage.