Enzymatic Digestion of Fresh-Frozen Human Cornea After Riboflavin/Ultraviolet-A Collagen Crosslinking
Enzymatic Digestion of Fresh-Frozen Human Cornea After Riboflavin/Ultraviolet-A Collagen Crosslinking
Objective: Crosslinking (CXL) of the cornea by using riboflavin (RBF)/ ultraviolet-A (UVA) has been developed for stiffening the collagenous matrix of the cornea. Collagenases and other metalloproteinases are known to play an important role in the pathogenesis of corneal ulceration. Our aim in this study was to show and compare the morphological and histopathological changes of the CXL human corneas against enzymatic digestion by collagenase, trypsin and pepsin solutions. Materials and Methods: Eighteen corneas from nine fresh frozen cadavers were included in this study. The crosslinked group (n=12) was treated with the combined riboflavin (RBF) and ultraviolet-A (UVA) and the control group (n=6) was only treated with RBF. After treatment corneas were exposed to collagenase, trypsin and pepsin enzymes. For each enzyme group one cornea was evaluated morphologically and the other one was evaluated microscopically. Results: The collagenase treated control group, digestion of the cornea was noted in the first days and completed on the 5th day. The control cornea treated with pepsin was fully digested after 14 days. In the trypsin treated CXL cornea complete digestion was noted after 18 days. In both groups, enzyme activity was observed to be parallel to each other morphologically, but CXL cornea was evaluated to be more resistant to enzymatic digestion. Conclusion: In conclusion, CXL increases resistance of the cornea against enzymatic digestion. In addition to the biomechanical support, this study was also showing the histopathological changes of CXL procedure and the results of enzymatic digestion, supporting new treatment options in the corneal diseases.
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- [1] Wollensak G, Wilsch M, Spoerl E, et al. Collagen fiber diameter in the rabbit cornea after collagen crosslinking by riboflavin/UVA. Cornea 2004;23(5):503-507.
- [2] Spoerl E, Wollensak G, Seiler T. Increased resistance of crosslinked cornea against enzymatic digestion. Curr Eye Res 2004;29(1):35-40.
- [3] Wollensak G, Redl B. Gel electrophoretic analysis of corneal collagen after photodynamic cross-linking treatment. Cornea 2008;27(3):353-356.
- [4] Arafat SN, Robert MC, Shukla AN, et al. UV cross-linking of donor corneas confers resistance to keratolysis. Cornea 2014;33(9):955-959.
- [5] Smith VA, Hoh HB, Easty DL. Role of ocular matrix metalloproteinases in peripheral ulcerative keratitis. Br J Ophthalmol 1999;83(12):1376-1383.
- [6] Sternberg SS, editor. Histology for Pathologists. Philadelphia, USA: Lippincott Williams&Wilkins, 1997. [7] Zhu Y, Reinach PS, Zhu H, et al. High-intensity corneal collagen crosslinking with riboflavin and UVA in rat cornea. PLoS One 2017;12(6):e0179580.
- [8] Fini ME, Girard MT, Matsubara M. Collagenolytic/ gelatinolytic enzymes in corneal wound healing. Acta Ophthalmol Suppl 1992;202:26-33.
- [9] Riley GP, Harrall RL, Watson PG, et al. Collagenase (MMP-1) and TIMP-1 in destructive corneal disease associated with rheumatoid arthritis. Eye (Lond) 1995;9(Pt 6):703-718.
- [10] Schilde T, Kohlhaas M, Spoerl E, et al. Enzymatic evidence of the depth dependence of stiffening on riboflavin/UVA treated corneas. Ophthalmologe 2008;105(2):165-169.
- [11] Kanellopoulos AJ, Loukas YL, Asimellis G. Cross-Linking Biomechanical Effect in Human Corneas by Same Energy, Different UV-A Fluence: An Enzymatic Digestion Comparative Evaluation. Cornea 2016;35(4):557-561.
- [12] Kohlhaas M. Complications and postoperative therapeutic strategies in cross-linking. Ophthalmologe 2017;114(8):693-696.
- [13] O’Brart NAL, O’Brart DPS, Aldahlawi NH, et al. An Investigation of the Effects of Riboflavin Concentration on the Efficacy of Corneal Cross-Linking Using an Enzymatic Resistance Model in Porcine Corneas. Invest Ophthalmol Vis Sci 2018;59(2):1058-1065.
- [14] Knyazer B, Krakauer Y, Baumfeld Y, et al. Accelerated Corneal Cross-Linking With Photoactivated Chromophore for Moderate Therapy-Resistant Infectious Keratitis. Cornea 2018;37(4):528-531.
- [15] Laggner M, Pollreisz A, Schmidinger G, et al. Correlation Between Multimodal Microscopy, Tissue Morphology, and Enzymatic Resistance in Riboflavin-UVA Cross- Linked Human Corneas. Invest Ophthalmol Vis Sci 2015;56(6):3584-3592.
- [16] Brekelmans J, Veugen J, Rieff K, et al. Enzymatic Digestion of Porcine Corneas Cross-linked by Hypo- and Hyperosmolar Formulations of Riboflavin/ultraviolet A or WST11/Near- Infrared Light. Transl Vis Sci Technol 2020;9(10):4.
- [17] Tan HY, Chang YL, Lo W, et al. Characterizing the morphologic changes in collagen crosslinked-treated corneas by Fourier transform-second harmonic generation imaging. J Cataract Refract Surg 2013;39(5):779-788.
- [18] McCall AS, Kraft S, Edelhauser HF, et al. Mechanisms of corneal tissue cross-linking in response to treatment with topical riboflavin and long-wavelength ultraviolet radiation (UVA). Invest. Ophthalmol Vis Sci 2010;51(1):129- 138.
- [19] Spoerl E, Huhle M, Seiler T. Induction of cross-links in corneal tissue. Exp Eye Res 1998;66(1):97-103.
- [20] Wollensak G, Sporl E, Reber F, et al. Corneal endothelial cytotoxicity of riboflavin/UVA treatment in vitro. Ophthalmic Res 2003;35(6):324-328.
- [21] Wollensak G, Sporl E, Mazzotta C, et al. Interlamellar cohesion after corneal crosslinking using riboflavin and ultraviolet A light. Br J Ophthalmol 2011;95(6):876-880.
- [22] Kanellopoulos AJ, Asimellis G, Salvador-Culla B, et al. Highirradiance CXL combined with myopic LASIK: flap and residual stroma biomechanical properties studied ex-vivo. Br J Ophthalmol 2015;99(6):870-874.
- [23] Alageel SA, Arafat SN, Salvador-Culla B, et al. Corneal Cross-Linking With Verteporfin and Nonthermal Laser Therapy. Cornea 2018;37(3):362-368.
- [24] Hayes S, Kamma-Lorger CS, Boote C, et al. The effect of riboflavin/UVA collagen cross-linking therapy on the structure and hydrodynamic behaviour of the ungulate and rabbit corneal stroma. PLoS One 2013;8(1):e52860.
- [25] Michael H. Ross LJR, Gordon I. Kaye, editors. Histology, A Text and Atlas. Maryland, USA: Wiliams&Wilkins, 1995.
- [26] Akhtar S, Almubrad T, Paladini I, et al. Keratoconus corneal architecture after riboflavin/ultraviolet A cross-linking: ultrastructural studies. Mol Vis 2013;19:1526-1537.
- [27] Carlson EC, Waring GO, 3rd. Ultrastructural analyses of enzyme-treated microfibrils in rabbit corneal stroma. Invest Ophthalmol Vis Sci 1988;29(4):578-585.
- [28] Meek KM, Tuft SJ, Huang Y, et al. Changes in collagen orientation and distribution in keratoconus corneas. Invest Ophthalmol Vis Sci 2005;46(6):1948-1956.