Substrate Stiffness Regulated Regulatory Volume Decrease (RVD) and Calcium Signaling of Chondrocyte

Substrate stiffness is an important physical cue of the microenvironment and plays a critical role in transducing biochemical and biomechanical signals for chondrocytes. But how substrate stiffness modulates the chondrocyte volume and calcium signaling remains unknown. This study aims to recapitulate the physiologically relevant stiffness and to investigate the effects of substrate stiffness on the chondrocyte regulatory volume decrease (RVD) and calcium signaling. The mechanical properties of chondrocytes on varying substrate stiffness in both iso-osmotic and hypo-osmotic medium were measured by using atomic force microscopy (AFM). The cell diameter rate during the RVD response was measured. TRPV4-mediated calcium signaling of chondrocytes was captured by calcium dye solution. TRPV4 protein and mRNA expression levels were quantified by Western Blot and Semi-quantitative reverse transcription polymerase chain reaction, respectively. Soft substrate induced faster cell swelling but slower cell recovering during chondrocyte RVD response. Stiff substrate enhanced the cytosolic Ca2+ oscillation of chondrocytes in iso-osmotic medium. Moreover, chondrocytes exhibited a completely distinctive cytosolic Ca2+ oscillation during the RVD response. Soft substrate significantly improved the Ca2+ oscillation during the cell swelling whereas stiff substrate enhanced cytosolic Ca2+ oscillation during the cell recovering. More importantly, TRPV4 channel is involved in the chondrocyte sensing substrate stiffness and RVD response by mediating Ca2+ signaling in a stiffness-dependent manner. Our work demonstrates that varying substrate stiffness induces completely different swelling and recovering processes during chondrocyte RVD response. Moreover, the TRPV4 channel is involved in chondrocyte sensing substrate stiffness and RVD response by mediating calcium signaling.