Resistance of corneal RFUVA-cross-linked collagens and small leucine-rich proteoglycans to degradation by matrix metalloproteinases

Purpose. Extracellular matrix metalloproteinases (MMPs) are thought to play a crucial role in corneal degradation associated with the pathological progression of keratoconus. Currently, corneal cross-linking by riboflavin and ultraviolet A (RFUVA) has received significant attention for treatment of keratoconus. However, the extent to which MMPs digest cross-linked collagen and small leucine-rich proteoglycans (SLRPs) remains unknown. In this study, the resistance of RFUVA–cross-linked collagens and SLRPs to MMPs has been investigated. Methods. To investigate the ability of MMPs to digest cross-linked collagen and SLRPs, a model reaction system using purified collagen type I, type IV, and nonglycosylated, commercially available recombinant SLRPs, keratocan, lumican, mimecan, decorin, and biglycan in solution in vitro has been compared using reactions inside an intact bovine cornea, ex vivo. Results. Our data demonstrate that corneal cross-linked collagen type I and type IV are resistant to cleavage by MMP-1, MMP-2, MMP-9, and MMP-13, whereas non–cross–linked collagen I, IV, and natively glycosylated SLRPs are susceptible to degradation by MMPs. In addition, both cross-linked SLRPs themselves and cross-linked polymers of SLRPs and collagen appear able to resist degradation. These results suggest that the interactions between SLRPs and collagen caused by RFUVA protect both SLRPs and collagen fibrils from cleavage by MMPs. Conclusions. A novel approach for understanding the biochemical mechanism whereby RFUVA cross-linking stops keratoconus progression has been achieved

Fibrinogen, riboflavin, and UVA to immobilize a corneal flap - conditions for tissue adhesion

Purpose. Laser-assisted in situ keratomileus (LASIK) creates a permanent flap that remains non-attached to the underlying laser-modified stroma. This lack of permanent adhesion is a liability. To immobilize a corneal flap, a protocol using fibrinogen (FIB), riboflavin (RF), and ultraviolet (UVA) light (FIB+RF+UVA) was devised to re-adhere the flap to the stroma. Methods. A model flap was created using rabbit (Oryctolagus cuniculus) and shark (Squalus acanthias) corneas. Solutions containing FIB and RF were applied between corneal strips as glue. Experimental corneas were irradiated with long wavelength (365 nm) UVA. To quantify adhesive strength between corneal strips, the glue-tissue interface was subjected to a constant force while a digital force gauge recorded peak tension. Results. In the presence of FIB, substantive non-covalent interactions occurred between rabbit corneal strips. Adhesiveness was augmented if RF and UVA also were applied, suggesting formation of covalent bonds. Additionally, exposing both sides of rabbit corneas to UVA generated more adhesion than exposure from one side, suggesting that RF in the FIB solution catalyzes formation of covalent bonds at only the interface between stromal molecules and FIB closest to the UVA. In contrast, in the presence of FIB, shark corneal strips interacted non-covalently more substantively than those of rabbits, and adhesion was not augmented by applying RF+UVA, from either or both sides. Residual RF could be rinsed away within 1 hour. Conclusions. Glue solution containing FIB and RF, together with UVA treatment, may aid immobilization of a corneal flap, potentially reducing risk of flap dislodgement

Fibrinogen, riboflavin, and UVA to immobilize a corneal flap - molecular mechanisms

Purpose. Tissue glue containing fibrinogen (FIB) and riboflavin (RF), upon exposure to long wavelength ultraviolet light (UVA, 365 nM) has been proposed potentially to solve long-standing problems presented by corneal wound and epithelial ingrowth side-effects from laser-assisted in situ keratomileuis (LASIK). Data presented in a previous study demonstrated an ability of FIB + RF + UVA to adhere two stromal surfaces; however, to our knowledge no molecular mechanisms have been proposed to account for interactions occurring between corneal extracellular matrix (ECM) and tissue glue molecules. Here, we document several covalent and noncovalent interactions between these classes of macromolecules. Methods. SDS-PAGE and Western blot techniques were used to identify covalent interactions between tissue glue molecules and corneal ECM molecules in either the presence or absence of RF and UVA, in vitro and ex vivo. Surface plasmon resonance (SPR) was used to characterize noncovalent interactions, and obtain ka, kd, and KD binding affinity values. Results. SDS-PAGE and Western blot analyses indicated that covalent interactions occurred between neighboring FIB molecules, as well as between FIB and collagen type I (Coll-I) proteins (in vitro and ex vivo). These interactions occurred only in the presence of RF and UVA. SPR data demonstrated the ability of FIB to bind noncovalently to corneal stroma molecules, Coll-I, decorin, dermatan sulfate, and corneal basement membrane molecules, laminin and heparan sulfate – only in the presence of Zn2+. Conclusions. Covalent and (zinc-mediated) noncovalent mechanisms involving FIB and stromal ECM molecules contribute to the adhesion created by FIB + RF + UVA

Enzymatic resistance of corneas crosslinked using riboflavin in conjunction with low energy, high energy, and pulsed UVA irradiation modes

Purpose: To investigate the effect of various riboflavin/ultraviolet light (UVA) crosslinking (CXL) protocols on corneal enzymatic resistance. Methods: A total of 66 enucleated porcine eyes, with the corneal epithelium removed, were divided into 6 groups. Group 1 remained untreated. Groups 2 to 6 received riboflavin/dextran for 30 minutes. Group 3 underwent standard CXL (SCXL) with 3 mW/cm2 UVA for 30 minutes (total energy dose 5.4 J/cm2). Groups 4 and 5 underwent high intensity CXL (HCXL) using 30 mW/cm2 UVA for 3 minutes (5.4 J/cm2) and 30 mW/cm2 for 4 minutes (7.2 J/cm2), respectively. Group 6 was exposed to 8 minutes of 30 mW/cm2 UVA in a 10-second on/10-second off pulsed-radiation mode (p-HCXL; 7.2 J/cm2). A central 8-mm disk from each cornea was submerged in pepsin digest solution at 23°C and measured daily. After 13 days, the dry weight was recorded from 5 samples in each group. Results: The CXL-treated corneas took longer to digest than nonirradiated corneas (P < 0.0001). Differences in digestion time also were observed between CXL groups, such that, HCXL (5.4 J/cm2) < SCXL (5.4 J/cm2) < HCXL (7.2 J/cm2) < p-HCXL (7.2 J/cm2; P < 0.0001). The dry weight of the SCXL (5.4 J/cm2) group was higher than the HCXL (5.4 and 7.2 J/cm2; P < 0.001) and p-HCXL 7.2 J/cm2 (P <0.05) groups. No difference was detected between the HCXL and p-HCXL 7.2 J/cm2 groups. Conclusions: The intensity and distribution of the crosslinks formed within the cornea vary with different UVA protocols. The precise location and amount of crosslinking needed to prevent disease progression is unknown

Fibrinogen, Riboflavin, and UVA to Immobilize a Corneal Flap – Molecular Mechanisms

Purpose. Tissue glue containing fibrinogen (FIB) and riboflavin (RF), upon exposure to long wavelength ultraviolet light (UVA, 365 nM) has been proposed potentially to solve long-standing problems presented by corneal wound and epithelial ingrowth side-effects from laser-assisted in situ keratomileuis (LASIK). Data presented in a previous study demonstrated an ability of FIB + RF + UVA to adhere two stromal surfaces; however, to our knowledge no molecular mechanisms have been proposed to account for interactions occurring between corneal extracellular matrix (ECM) and tissue glue molecules. Here, we document several covalent and noncovalent interactions between these classes of macromolecules. Methods. SDS-PAGE and Western blot techniques were used to identify covalent interactions between tissue glue molecules and corneal ECM molecules in either the presence or absence of RF and UVA, in vitro and ex vivo. Surface plasmon resonance (SPR) was used to characterize noncovalent interactions, and obtain ka, kd, and KD binding affinity values. Results. SDS-PAGE and Western blot analyses indicated that covalent interactions occurred between neighboring FIB molecules, as well as between FIB and collagen type I (Coll-I) proteins (in vitro and ex vivo). These interactions occurred only in the presence of RF and UVA. SPR data demonstrated the ability of FIB to bind noncovalently to corneal stroma molecules, Coll-I, decorin, dermatan sulfate, and corneal basement membrane molecules, laminin and heparan sulfate – only in the presence of Zn2+. Conclusions. Covalent and (zinc-mediated) noncovalent mechanisms involving FIB and stromal ECM molecules contribute to the adhesion created by FIB + RF + UVA