Distribution of Young's modulus in porcine corneas after riboflavin/UVA-induced collagen cross-linking as measured by atomic force microscopy.
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.
Department of Applied Physics and LISA+, Eberhard-Karls-University, Tübingen, Germany.
Jan
J
Seifert
Department of Anatomy II, Friedrich-Alexander-University, Erlangen, Germany.
Christian M
CM
Hammer
Department of Applied Physics and LISA+, Eberhard-Karls-University, Tübingen, Germany.
Johannes
J
Rheinlaender
Department of Ophthalmology, Ruprecht-Karls-University, Heidelberg, Germany.
Saadettin
S
Sel
Department of Anatomy II, Friedrich-Alexander-University, Erlangen, Germany.
Michael
M
Scholz
Department of Anatomy II, Friedrich-Alexander-University, Erlangen, Germany.
Friedrich
F
Paulsen
Department of Applied Physics and LISA+, Eberhard-Karls-University, Tübingen, Germany.
Tilman E
TE
Schäffer
D000818:Animals; D003315:Cornea / Q000737:chemistry / Q000648:ultrastructure; D003432:Cross-Linking Reagents / Q000737:chemistry*; D055119:Elastic Modulus; D018625:Microscopy, Atomic Force; D017319:Photosensitizing Agents / Q000737:chemistry; D012256:Riboflavin / Q000737:chemistry*; D013552:Swine; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D017319:Photosensitizing Agents; D012256:Riboflavin