Synthetic Mechanogenetic Gene Circuit Response in Human Induced Pluripotent Stem Cell‐Derived Chondrocytes Is Altered by Matrix Stiffness

Transient receptor potential vanilloid 4 (TRPV4) is a mechanosensitive ion channel found in both excitatory and non-excitatory cells in the body. In chondrocytes, TRPV4 plays a crucial role in transducing physiologic levels of mechanical loading and osmotic stimuli. One of the earliest tissue-level events that occur in early stages of osteoarthritis is the degradation of the cartilage extracellular and pericellular matrices. In this technical note, we highlight the development of an in vitro model system for studying the relationship between TRPV4-mediated mechanotransduction and matrix stiffness. We modeled the effects of varying degrees of degradation on cartilage mechanical properties using 3D agarose hydrogel constructs with varying concentrations, which were used to encapsulate human induced pluripotent stem cell (iPSC)-derived chondrocytes. Hydrogel stiffness significantly altered the volume of encapsulated chondrocytes, with cells in soft constructs demonstrating significantly larger volume compared to those in medium and stiff constructs. Next, we used a synthetic mechanogenetic gene circuit as a tool to investigate the stiffness-dependence of TRPV4-mediated mechanoresponsive transcription. Interestingly, chemical activation of TRPV4 (using GSK101) and hypotonic stimuli resulted in opposite trends: chondrocytes in stiffer constructs demonstrated stronger circuit response to GSK101 compared to those in softer constructs, and vice versa in response to hypotonic stimuli. These findings suggest that while increased matrix stiffness can modulate TRPV4 activity, the increased physical swelling that occurs in a softer matrix may counterbalance these effects. These results provide an initial understanding of the interplay between matrix stiffness and TRPV4-mediated mechanotransduction in chondrocytes.