Abstract

Keratoconus (KC) is a prevalent ectatic corneal disease influenced by corneal biomechanics, though its pathogenesis remains unclear. Constructing an in vitro corneal model with adjustable biomechanics is essential for studying keratoconus. In this study, a KC disease group (L group) was created using a low matrix stiffness collagen hydrogel containing human corneal stromal cells (hCSCs), while a high matrix stiffness group (H group) was established using a plastic compression method. Additionally, a cross-linking treatment model was applied to the L group using corneal collagen cross-linking (CXL). Results showed that the matrix stiffness of the L group was significantly lower compared to the H group. Both the L group and clinical KC samples exhibited lower collagen fibrils density. Transcriptomic and proteomic analyses revealed decreased antioxidant capacity and increased levels of inflammatory factors and reactive oxygen species (ROS) in the L group. The NRF2 pathway activity was downregulated. In the L group cells, inflammation-related genes were upregulated, antioxidantrelated genes were downregulated, and ROS levels were elevated, accompanied by a reduction in mitochondrial membrane potential. The concentrations of IL-6 and TNF-α in the culture medium were increased. Histological results indicated that the L group, similar to clinical KC samples, exhibited high expression of inflammatory and oxidative stress factors, while signals of antioxidant-related factors were decreased. After cross-linking treatment, the L group demonstrated increased matrix stiffness, higher collagen fibrils density, upregulated expression of antioxidant factors, and decreased expression of inflammatory and immune factors. This study successfully established an in vitro corneal stromal model with adjustable matrix stiffness, providing a platform for investigating the pathogenesis of KC.