Distribution of Young's Modulus in Porcine Corneas after Riboflavin/UVA-Induced Collagen Cross-Linking as Measured by Atomic Force Microscopy

Abstract

Riboflavin/UVA-induced corneal collagen cross-linking has become an effective clinical application to treat keratoconus and other ectatic disorders of the cornea. Its beneficial effects are attributed to a marked stiffening of the unphysiologically weak stroma. Previous studies located the stiffening effect predominantly within the anterior cornea. In this study, we present an atomic force microscopy-derived analysis of the depth-dependent distribution of the Young's modulus with a depth resolution of 5 µm in 8 cross-linked porcine corneas and 8 contralateral controls. Sagittal cryosections were fabricated from every specimen and subjected to force mapping. The mean stromal depth of the zone with effective cross-linking was found to be 219±67 µm. Within this cross-linked zone, the mean Young's modulus declined from 49±18 kPa at the corneal surface to 46±17 kPa, 33±11 kPa, 17±5 kPa, 10±4 kPa and 10±4 kPa at stromal depth intervals of 0–50 µm, 50–100 µm, 100–150 µm, 150–200 µm and 200–250 µm, respectively. This corresponded to a stiffening by a factor of 8.1 (corneal surface), 7.6 (0–50 µm), 5.4 (50–100 µm), 3.0 (100–150 µm), 1.6 (150–200 µm), and 1.5 (200–250 µm), when compared to the Young's modulus of the posterior 100 µm. The mean Young's modulus within the cross-linked zone was 20±8 kPa (2.9-fold stiffening), while it was 11±4 kPa (1.7-fold stiffening) for the entire stroma. Both values were significantly distinct from the mean Young's modulus obtained from the posterior 100 µm of the cross-linked corneas and from the contralateral controls. In conclusion, we were able to specify the depth-dependent distribution of the stiffening effect elicited by standard collagen cross-linking in porcine corneas. Apart from determining the depth of the zone with effective corneal cross-linking, we also developed a method that allows for atomic force microscopy-based measurements of gradients of Young's modulus in soft tissues in general.

Quantitative assessment of corneal viscoelasticity using optical coherence elastography and a modified Rayleigh–Lamb equation

“…Figure 2(b) shows the estimated Young's modulus (E ¼ 60 kPa) and shear viscosity (η ¼ 0.33 Pa · s) of three porcine corneas at an artificially controlled IOP of 20 mmHg, which were estimated by fitting the phase velocities from the OCE measurements with the modified RLFE numerical results. The estimated Young's modulus is of the same order as measured by atomic force microscopy, 17 which suggests that the proposed method could provide accurate quantitative viscoelastic characterization of the cornea under the proper assumptions.…”

Noncontact depth-resolved micro-scale optical coherence elastography of the cornea

“…Also, real-time in vivo measurement of the spatial elasticity distribution with microscopic scale in the cornea could lead to adaptive mechanical modeling of the individual corneal structure [5], which is extremely important to prevent overcorrections, under-corrections and ectasia from refractive surgeries, such as LASIK, and to further optimize the laser ablation procedures [6,7]. More importantly, collagen cross-linking with riboflavin has been extensively used for corneal therapy [8], and the monitoring of the depth-dependent elastic properties of the cornea could help to achieve a better management of this treatment [9]. Thus, noninvasive corneal elastographic techniques with depth-resolving capability are sorely needed for various clinical ophthalmological applications.…”

Current and future applications of corneal cross-linking

“…Evidence of effectiveness in laboratory studies shows objective data that confirm that CXL is in fact effective with increase in collagen fiber diameter on electron microscopy [62], resistance to enzymatic digestion, increase in biomechanical stiffness indicated by increase in Young modulus [2] and other imagining modalities such as atomic force microscopy [63] and second harmonic generation microscopy. Second harmonic generation microscopy offers an opportunity to image collagen fibrils in a high-contrast manner, generates a three-dimensional reconstruction and detects differences in patterns of lamellae between treated and untreated corneas [64].…”