Biomechanical changes after repeated collagen cross-linking on human corneas assessed in vitro using scanning acoustic microscopy

PURPOSE. To explore the biomechanical changes induced by repeated cross-linking using scanning acoustic microscopy (SAM). METHODS. Thirty human corneas were divided into three groups. In group A, five corneas were cross-linked once. In group B, five corneas were cross-linked twice, 24 hours apart. In group C, five corneas were cross-linked three times, 24 hours apart. The contralateral controls in all groups had similar treatment but without UV-A. The speed of sound, which is directly proportional to the square root of the tissue's elastic modulus, was assessed using SAM. RESULTS. In group A, the speed of sound of the treated corneas was 1677.38 ± 10.70 ms^-1 anteriorly and 1603.90 ± 9.82 ms^-1 posteriorly, while it was 1595.23 ± 9.66 ms^-1 anteriorly and 1577.13 ± 8.16 ms^-1 posteriorly in the controls. In group B, the speed of sound of the treated corneas was 1746.33 ± 23.37 ms^-1 anteriorly and 1631.60 ± 18.92 ms^-1 posteriorly, while it was 1637.57 ± 22.15 ms^-1 anteriorly and 1612.30 ± 22.23 ms^-1 posteriorly in the controls. In group C, the speed of sound of the treated corneas was 1717.97 ± 18.92 ms^-1 anteriorly and 1616.62 ± 17.58 ms^-1 posteriorly, while it was 1628.69 ± 9.37 ms^-1 anteriorly and 1597.68 ± 11.97 ms^-1 posteriorly in the controls. The speed of sound in the anterior (200 × 200 μm) region between the cross-linked and control corneas in groups A, B, and C was increased by a factor of 1.051 (P = 0.005), 1.066 (P = 0.010), and 1.055 (P = 0.005) respectively. However, there was no significant difference among the cross-linked corneas in all groups (P = 0.067). CONCLUSIONS. A significant increase in speed of sound was found in all treated groups compared with the control group; however, the difference among the treated groups is not significant, suggesting no further cross-links are induced when collagen cross-linking treatment is repeated.