Investigation Into The Role Of Stromal Stiffness In Keratoconus Pathogenesis Using A Tunable Extracellular Matrix Model

The primary aim of this research is to delve into the complexities of Keratoconus (KC), a common ectatic disorder of the cornea that significantly affects vision by altering corneal biomechanics and structure. Despite being widely studied, the exact pathogenesis of KC remains elusive, hindering the development of targeted and effective treatments. Recognizing the pivotal role of stromal stiffness in corneal biomechanics, our study seeks to develop a sophisticated in vitro model that allows for the modulation of stromal stiffness. By employing this model, we aim to closely replicate the conditions of KC, thereby facilitating a deeper understanding of how reductions in stromal stiffness contribute to the disease's progression.

This study was conducted in a modern biomedical lab, aimed at investigating KC by developing an in vitro model to study stromal stiffness effects. It involved human corneal stromal cells and tissue from KC patients and controls undergoing specific corneal surgeries. The controlled lab environment allowed for detailed cellular, molecular, and imaging analyses to understand KC's pathogenesis and the potential of treatment strategies like corneal collagen cross-linking.

To model KC, we utilized a low-stiffness collagen hydrogel with human corneal stromal cells (hCSCs) for the KC mimic group (L group), and a high-stiffness model (H group) via plastic compression. A corneal collagen cross-linking (CXL) treatment was applied to the L group. We assessed microstructure using TEM and matrix stiffness. Gene and protein expressions related to immune, oxidative, and antioxidant processes were analyzed using RNA-seq, qPCR, proteomics, immunofluorescence (IF), and western blot. Reactive oxygen species (ROS) were evaluated with and without H2O2, and cytokine levels (IL-6, TNF-α) were measured using ELISA. Patient-derived corneal stromal tissues and controls from SMILE surgery were also examined via TEM and IF.

TEM revealed that the microstructure of collagen fibers and cell arrangements in the H/L/CXL groups resembled natural corneal stroma. The L group showed significantly lower stromal stiffness compared to H and CXL. RNA-seq, proteomics, qPCR, and western blot analyses indicated increased expression of immune and oxidative stress-related factors in the L group, alongside decreased collagen and antioxidant-related factors. The L group exhibited increased ROS production. ELISA results revealed elevated IL-6 and TNF-α levels in the L group. IF showed significant decreases in YAP and antioxidant factors in the KC patient group, with increased inflammation and oxidative stress-related factors, mirroring the L group's findings compared to H/CXL.

This study established an innovative in vitro model of KC that allows for the precise manipulation of stromal stiffness, providing a significant leap forward in understanding the biomechanical underpinnings of KC pathogenesis. Our findings demonstrate that reduced matrix stiffness closely replicates the pathological landscape of KC, as evidenced by alterations in cellular microstructure, increased expression of immune and oxidative stress markers, and enhanced reactive oxygen species production. These results not only elucidate the critical role of stromal stiffness in KC development but also highlight the therapeutic promise of biomechanical interventions in mitigating the disease's progression.