Investigating the coupled effects of stiffness and stretch on the trabecular meshwork cells using a hydrogel-integrated microfluidic system

Glaucoma is characterized by progressive stiffening of the trabecular meshwork (TM), which elevates intraocular pressure and contributes to tissue dysfunction. Although substrate stiffness and mechanical stimulation both regulate TM homeostasis, their combined effects remain poorly understood. Here, a hydrogel-integrated microfluidic platform is presented that enables simultaneous control of substrate stiffness via tunable gelatin methacryloyl (GelMA) hydrogels and equi-biaxial quasi-static stretch via hydraulic actuation. Finite element analysis validates the applied strain field, and optimized crosslinking ensures structural stability. Primary normal TM (nTM) and glaucomatous TM (gTM) cells cultured under coupled conditions exhibit selective mechanotransduction dysregulation rather than global mechanosensory impairment. While nTM cells dynamically regulate -smooth muscle actin (-SMA), myocilin (MYOC), matrix metalloproteinase-2 (MMP2), and collagen type I (COL1), gTM cells display constitutively elevated -SMA, loss of mechanical regulation of MMP2, and impaired stretch-mediated COL1 suppression, while retaining stiffness-dependent focal adhesion kinase and MYOC sensitivity. Key differences between normal and glaucomatous cells emerge only under combined stiff and stretched conditions, underscoring the importance of coupled mechanical cues in revealing disease-relevant phenotypes. These findings implicate tissue stiffening in selective pathway dysregulation and highlight mechanotransduction-targeted therapeutic strategies.