Keratocyte cytotoxicity of riboflavin/UVA-treatment in vitro

Abstract

Purpose Collagen crosslinking using ultraviolet-A (UVA) -irradiation combined with the photosensitizer riboflavin is a new technique for treating progressive keratoconus. It has been shown to increase effectively the biomechanical strength of the cornea and to stop or even reverse the progression of keratoconus. As part of a safety evaluation, the present study was undertaken to investigate in vitro the possible cytotoxic effect of combined riboflavin/UVA-treatment on corneal keratocytes and to compare it to UVA-irradiation alone. Methods Cell cultures established from porcine keratocytes were treated with 0.025% riboflavin solution and various UVA (370 nm)-irradiances ranging from 0.4 to 1.0 mW/cm 2 and with UVA alone between 2 and 9 mW/cm 2 for 30 min. The cell cultures were evaluated for cell death 24 h after irradiation using trypanblue and Yopro-fluorescence staining. Results An abrupt cytotoxic irradiance level was found at 0.5 mW/cm 2 for keratocytes after UVA-irradiation combined with the photosensitizer riboflavin, which is 10-fold lower than the cytotoxic irradiance of 5 mW/ cm 2 after UVA-irradiation alone. Conclusions A cytotoxic effect of combined riboflavin/UVA-treatment on keratocytes is to be expected at 0.5 mW/cm 2 , which is reached in the clinical setting in human corneas down to a depth of 300 mm using the standard surface UVA-irradiance of 3 mW/cm 2 .

UV light crosslinking regresses mature corneal blood and lymphatic vessels and promotes subsequent high-risk corneal transplant survival

“…Consistent with previous studies,26, 27, 28 our CXL procedure reduced corneal keratocyte numbers in the stroma. After the topical application of the riboflavin solution and UVA irradiation, in addition to the biomechanical stiffening effect of the procedure, the reaction of riboflavin and UVA leads to the release of highly reactive singlet oxygen and other reactive oxygen radicals, which can cause apoptosis of keratocytes and endothelial cells in the cornea 26, 27, 28.…”

“…After the topical application of the riboflavin solution and UVA irradiation, in addition to the biomechanical stiffening effect of the procedure, the reaction of riboflavin and UVA leads to the release of highly reactive singlet oxygen and other reactive oxygen radicals, which can cause apoptosis of keratocytes and endothelial cells in the cornea 26, 27, 28. Because corneal BVs and LVs mainly grow in the upper corneal stromal layer, the reaction of riboflavin and UVA should also induce similar apoptosis of endothelial cells in corneal vessels and thereafter lead to the regression.…”

“…Normally, the human corneal stroma absorbs 32% of the UVA light. After riboflavin application, UVA absorption in the stroma increases to 95% 27, 39. The reason for the high absorption rate is that the wavelength range of UVA llight (315‐400 nm) corresponds to one of the riboflavin absorption maxima (ie, 370 nm) 26.…”

“…Recently, CXL with the interaction of riboflavin and UV irradiation was reported to also be used in corneal infections 23, 24, 25. After the topical application of riboflavin and UVA light, the reaction causes the release of very reactive singlet oxygen and other reactive oxygen radicals, which leads to apoptosis of, for example, keratocytes in the cornea 26, 27, 28. It has been shown that CXL can lead to a reduction in corneal keratocytes 29.…”

Collagen crosslinking with ultraviolet-A and hypoosmolar riboflavin solution in thin corneas

“…[7][8][9][10] Before the first application in humans in 1999, 3 the parameters of the method (riboflavin concentration, intensity and wavelength of UVA light, and duration of treatment) were established in a series of time-and dose-response assays in animal models over several years. [2][3][4][11][12][13][14] To protect the corneal endothelium and deeper ocular structures, the currently used treatment parameters are set so the anterior 250 to 350 mm of corneal stroma are treated. 15 Accordingly, the current inclusion criteria require a minimum stromal thickness (without the corneal epithelium) of 400 mm, including a safety margin.…”