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Marcos Garza-Madrid, Jennifer Elisseeff; Soluble collagen protects the corneal fibrils during riboflavin crosslinking. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4074.
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Cornea crosslinking with riboflavin and UV-A is a novel procedure able to stop the progression of keratoconus. Our aim is to characterize and prevent the fibrillar damage associated with this procedure.
Fresh bovine corneas were obtained from a local slaughterhouse. Six mm biopsies, devoid of epithelium and endothelium were treated with a Riboflavin isotonic solution with or without soluble collagen. The tissue was then crosslinked using UV-A light. Hydration was maintained by adding crosslinking solution periodically. Samples were then either processed for TEM or analyzed through DSC to identify the temperature at which the molecular structure breaks down. Fibril counting was performed manually on a minimum of 180 fibrils per group.
After 30 minutes of crosslinking the denaturation temperature of the corneas increased from 66.77±0.92°C to 70.94±1.72°C (p<0.01). After 45 minutes, however, the temperature dropped to 67.99±1.15°C (p<0.01). When compared to an untreated control (Figure 1: A), cornea crosslinked for one hour (Figure 1: B) shows an accumulation of material in the space between fibers. This accumulation bears remarkable similarity to those formed when collagen is added to the cornea tissue without exposure to UV light (Figure 1: C). There was a significant increase in fibril width after crosslinking (25.91±4.1 nm vs 28.41±5.5 nm, p<0.01). The addition of collagen was able to prevent the change in fibril width (28.41±5.5 nm without collagen vs 24.66±4.2 nm with collagen, p<0.01). Adding collagen to the solution, without exposing the tissue to UV light, had no effect on fibril width (25.91±4.1 nm without collagen vs 25.79±4.1 nm with collagen, p=0.78).
Cornea collagen fibrils are damaged during riboflavin-UV crosslinking. This damage can be seen through fibril thickening, collagen release and decreased denaturation temperatures. Adding collagen to the crosslinking solution can prevent the damage to the cornea nanostructure during crosslinking.
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