Effects of Swelling on Nanomechanical Properties of Corneal Lamellae: Assessed by Atomic Force Microscopy

The cornea exhibits significant swelling behavior during mechanical characterization in a liquid environment. This technical brief quantified the impact of swelling on nanomechanical characterization during an atomic force microscopy (AFM) test. Corneal sections from the African green monkey were tested in this study. Results showed that lamellar stiffness in swollen corneal section was slightly lower than that in controls, particularly in the anterior and posterior regions (172.4 ± 107.8 kPa and 107.6 ± 69.3 kPa versus 221.7 ± 62.5 kPa and 124.3 ± 42.2 kPa, respectively), while the middle region remained nearly unchanged (105.9 ± 30.1 kPa versus 116.2 ± 44.3 kPa); however, these differences were not statistically significant (p > 0.05). These results indicate that the increased interlamellar spacing leads to a larger thickness, which likely contributes to reduced bulk stiffness measured by uniaxial testing. During AFM testing in liquid, adhesion between the tissue section and the glass slide mechanically constrained free swelling. In addition, 14 days of frozen storage at -20 °C significantly increased lamellar stiffness in both control and swollen corneal sections. After storage, control corneal section exhibited stiffness values of 307.7 ± 103.4 kPa, 161.4 ± 30.6 kPa, and 171.9 ± 52.0 kPa at the anterior, middle, and posterior regions, respectively, and 268.8 ± 154.0 kPa, 163.1 ± 101.5 kPa, and 163.2 ± 72.0 kPa in the swollen corneal section. These results indicate that immediate testing following cryosectioning is necessary to better preserve the nanomechanical integrity of the tissue.