Determining the Relationship Between Corneal Stiffening and Tissue Dehydration After Corneal Cross-Linking.

Purpose: To quantify corneal cross-linking (CXL)-induced stiffening via mechanical testing to estimate the impact of changes in hydration levels (H) and evaluate depth-dependent tissue hydration after CXL. Methods: Eighty-three porcine corneal buttons were divided into three groups: Standard protocol CXL (S-CXL), accelerated CXL (A-CXL), and untreated (nonirradiated riboflavin-only) controls. Samples were hydrated or dehydrated to modulate H and dynamic mechanical analyzer compression tests were performed to measure Young's modulus (E). To extract the solid tissue network modulus, the cornea was modeled as a biphasic material after measuring E at different H. Corneal hydration was correlated with depth-dependent tissue thickness characterized by confocal reflection microscopy (CRM). Results: Young's modulus increased fourfold after S-CXL (0.72 ± 0.1 MPa) and threefold after A-CXL E (0.53 ± 0.12 MPa) versus controls (0.17 ± 0.045 MPa). However, H decreased from 4.07 ± 0.35 in controls to 2.06 ± 0.2 after S-CXL and 2.79 ± 0.12 after A-CXL. After H modulation and biphasic mechanical modeling, Young's modulus for corneal solid tissue network showed only a 1.8-fold increase after S-CXL (2.25 MPa) and 1.5-fold increase after A-CXL (1.85 MPa) versus controls (1.22 MPa). With CRM, the overall thickness of the corneal tissue was found to linearly correlate to hydration H as expected. No appreciable depth dependence of hydration-induced thickness changes throughout the corneal buttons were observed. Conclusions: Corneal tissue hydration changes significantly impact measured corneal stiffness after CXL using mechanical testing. Not considering H leads to major overestimation of the stiffening effect of the CXL procedure. Depth-dependence of corneal thickness because of changing hydration is strongly dependent on the integrity of the tissue.