Nanoindentation under retrocorneal pressure to determine the biomechanical response of the human cornea to accelerated UVA crosslinking and riboflavin osmolarity ex vivo

Riboflavin-mediated UVA corneal crosslinking (CXL) is an established treatment to halt the progression of keratoconus. However, the biomechanical effects of accelerated protocols and varying riboflavin osmolarities under physiological conditions remain poorly understood. Traditional biomechanical assessments using nanoindentation typically analyze tissue flatmounts, disregarding the natural tissue strain exerted by intraocular pressure. We developed a novel experimental setup combining nanoindentation with adjustable retrocorneal fluid pressure (RCP) to simulate physiological conditions during measurements. Using this approach, we first examined five corneas under incrementally increasing RCP levels to quantify the effect of tissue pre-tension on biomechanical properties. In a subsequent series, we investigated 70 human corneas to evaluate seven different protocols, including accelerated treatment protocols and different riboflavin osmolarities, by analyzing the Hertz-elastic modulus (E_HZ) and creep behavior (C_IT). Our results demonstrate that all CXL protocols significantly enhanced corneal stiffness and reduced tissue creep. Compared to the standard Dresden protocol, the biomechanical effect was enhanced with hyperosmolar riboflavin but diminished when high radiation intensity was used for shorter duration. Furthermore, increasing RCP led to non-linear changes in corneal biomechanics, manifesting as increased E_HZ and decreased C_IT. These findings emphasize the importance of considering physiological tissue pre-tension in biomechanical assessments and provide new insights for optimizing CXL treatment protocols.