Comparative Analysis of Two Measurement Modalities for Ex Vivo Analysis of Corneal Stiffness in Porcine Corneas

Uniaxial tensile testing and atomic force microscopy (AFM) nanoindentation experiments are two valuable methods used to quantify changes in stiffness after corneal crosslinking (CXL). Here, we apply these methods by characterizing corneal stiffness ex vivo before and after CXL. Sixty-two fresh porcine corneas were divided into three groups: an untreated control group, a CXL3 group treated with the Dresden protocol, and a CXL9 group treated with the accelerated protocol. Biomechanical testing was then performed using either uniaxial tensile testing or AFM nanoindentation. Uniaxial tensile testing revealed a significant increase in corneal stiffness for the CXL3 group compared to the control group (p < 0.05). At 10% strain, the CXL3 and CXL9 groups exhibited increases in stiffness of 96% and 48%, respectively, compared to the control group. In contrast, AFM analysis revealed no significant differences in stiffness, showing 28% and 16% increases in the CXL3 and CXL9 groups, respectively, compared to the control group. The results suggest that uniaxial tensile testing provides a robust, sample-averaged measure of global stiffening. Interestingly, AFM nanoindentation enables mapping of localized biomechanical changes with high spatial resolution but is less sensitive to overall biomechanical changes induced by CXL.