Gregor Wollensak

Stress-strain measurements of human and porcine corneas after riboflavin–ultraviolet-A-induced cross-linking

Purpose: To evaluate the biomechanical effect of combined riboflavin–ultraviolet A (UVA) treatment on porcine and human corneas. Setting: Department of Ophthalmology, Technical University of Dresden, Dresden, Germany. Methods: Corneal strips from 5 human enucleated eyes and 20 porcine cadaver corneas were treated with the photosensitizer riboflavin and irradiated with 2 double UVA diodes (370 nm, irradiance = 3 mW/cm2) for 30 minutes. After cross‐linking, static stress‐strain measurements of the treated and untreated corneas were performed using a microcomputer‐controlled biomaterial tester with a prestress of 5 × 103 Pa. Results: There was a significant increase in corneal rigidity after cross‐linking, indicated by a rise in stress in treated porcine corneas (by 71.9%) and human corneas (by 328.9%) and in Youngs modulus by the factor 1.8 in porcine corneas and 4.5 in human corneas. The mean central corneal thickness was 850 &mgr;m ± 70 (SD) in porcine corneas and 550 ± 40 &mgr;m in human corneas. Conclusions: Riboflavin−UVA‐induced collagen cross‐linking led to an increase in mechanical rigidity in porcine corneas and an even greater increase in human corneas. As collagen cross‐linking is maximal in the anterior 300 &mgr;m of the cornea, the greater stiffening effect in human corneas can be explained by the relatively larger portion of the cornea being cross‐linked in the overall thinner human cornea.

Increased resistance of crosslinked cornea against enzymatic digestion

Purpose. Collagen-crosslinking using combined riboflavin/UVA treatment has been developed by us as a new treatment for keratoconus by stiffening the collagenous matrix. Recently, we have started to use the same method for the treatment of corneal ulcers. The aim of the present study was to evaluate the influence of the crosslinking treatment on the resistance of the cornea against enzymatic degradation. Methods. 60 enucleated porcine eyes were treated with the photosensitizer riboflavin and UVA-irradiation (370 nm; irradiance of 1, 2 or 3 mW/cm2) for 30 minutes and compared with 20 untreated control eyes. After crosslinking treatment, the corneal buttons were trephined and exposed to pepsin, trypsin and collagenase solutions. The extent of the corneal digestion was monitored daily. Selected cases were examined by light microscopy. Results. The corneal buttons crosslinked with riboflavin/UVA at 3 mW/cm2 were dissolved only by day 13 following pepsin digestion and by day 14 following collagenase treatment versus 6 days in the untreated control corneas. Digestion by trypsin was observed on day 5 in buttons crosslinked at 3 mW/cm2 compared to day 2 in the control corneas. Microscopically, a prolonged preservation especially of the anterior portion of the crosslinked corneas could be demonstrated. Conclusions. Photochemical crosslinking of the cornea using riboflavin and UVA results in a markedly increased resistance versus collagen digesting enzymes. The findings support the use of the new method in the treatment of corneal ulcers.

Keratocyte apoptosis after corneal collagen cross-linking using riboflavin/UVA treatment.

Purpose Combined riboflavin/UVA treatment inducing collagen cross-links in the cornea has been shown to increase the biomechanical rigidity of the cornea and has been used successfully in the treatment of progressive keratoconus. The current study was undertaken to investigate the possible cytotoxic effect of combined riboflavin/UVA treatment on corneal keratocytes in vivo. Methods Thirty-four New Zealand white rabbits were treated with 0.1% riboflavin solution and surface UVA irradiances ranging from 0.75 to 4 mW/cm2 (1.35– 7.2 J/cm2) for 30 minutes. The animals were euthanized either 4 (n = 6) or 24 (n = 28) hours postoperatively. Four additional control eyes underwent epithelial debridement alone. The corneas of the enucleated eyes were evaluated in routine histologic sections. In addition, the TUNEL technique and transmission electron microscopy were used for the detection of keratocyte apoptosis. Results In the control eyes with corneal epithelial debridement only, apoptotic keratocytes were found in the anterior 50 &mgr;m of the corneal stroma 4 hours postoperatively. However, riboflavin/UVA-induced apoptosis was only visible in the rabbit eyes enucleated 24 hours postoperatively. In these eyes, we found apoptosis of keratocytes down to a variable stromal depth depending on the applied UVA irradiance. A cytotoxic UVA irradiance for keratocytes in the range of 0.5–0.7 mW/cm2 could be deduced. Conclusions Riboflavin/UVA treatment leads to a dose-dependent keratocyte damage that can be expected in human corneas down to a depth of 300 &mgr;m using a surface UVA dose of 5.4 J/cm2. Future studies should be done to examine the keratocyte repopulation and exclude possible adverse sequelae of keratocyte loss like stromal scarring or thinning.

Endothelial cell damage after riboflavin- ultraviolet-A treatment in the rabbit

Purpose: To evaluate the possible cytotoxic effect of combined riboflavin–ultraviolet‐A (UVA) treatment on the corneal endothelium. Setting: Department of Ophthalmology, Technical University of Dresden, Dresden, Germany. Methods: The right eyes of 34 New Zealand White rabbits were treated with riboflavin and various endothelial UVA doses ranging from 0.16 to 0.9 J/cm2 (0.09 to 0.5 mW/cm2, 370 nm) and postoperative enucleation times of 4 hours and 24 hours. The endothelial cells were evaluated in histological sections. The terminal deoxynulceotidyl transferase deoxy‐UTP‐nick‐end labeling (TUNEL) technique and transmission electron microscopy were used to detect apoptosis. Results: There was no endothelial damage in the 6 rabbit eyes enucleated at 4 hours. In those enucleated at 24 hours, there was significant necrosis and apoptosis of endothelial cells in the corneas treated with an endothelial dose of ≥0.65 J/cm2 (0.36 mW/cm2), which is about twice the endothelial UVA dose used in the treatment of keratoconus patients. Conclusions: In rabbit corneas with a corneal thickness less than 400 &mgr;m, the endothelial UVA dose reached a cytotoxic level of ≥0.65 J/cm2 (0.36 mW/cm2) using the standard surface UVA dose of 5.4 J/cm2 (3 mW/cm2). Pachymetry should be routinely performed before riboflavin–UVA treatment; in thinner corneas, irradiation should not be done because of the cytotoxic risk to the endothelium.

COLLAGEN FIBER DIAMETER IN THE RABBIT CORNEA AFTER COLLAGEN CROSSLINKING BY RIBOFLAVIN/UVA

Objective: Collagen crosslinking of the cornea has been developed recently as a quasiconservative treatment of keratoconus. Biomechanical in vitro measurements have demonstrated a significant increase in biomechanical stiffness of the crosslinked cornea. The aim of the present study was to evaluate the effect of this new procedure on the collagen fiber diameter of the rabbit cornea. Methods: The corneas of the right eyes of 10 New Zealand White albino rabbits were crosslinked by application of the photosensitizer riboflavin and exposure to UVA light (370 nm, 3 mW/cm2) for 30 minutes. The left fellow control eyes were either left untreated (rabbits 1–4), deepithelialized (rabbits 5–7), or deepithelialized and treated with riboflavin/dextran solution (rabbits 8–10) to exclude an influence of epithelial debridement or hydration changes on the fiber diameter. On ultrathin sections of samples from the anterior and posterior cornea, the collagen fiber diameter was measured semiautomatically with the help of morphometric computer software. Results: In the anterior stroma, the collagen fiber diameter in the treated corneas was significantly increased by 12.2% (3.96 nm), and in the posterior stroma by 4.6% (1.63 nm), compared with the control fellow eyes. In the crosslinked eyes, the collagen fiber diameter was also significantly increased by, on average, 9.3% (3.1 nm) in the anterior compared with the posterior stroma within the same eye. Conclusions: Collagen crosslinking using riboflavin and UVA leads to a significant increase in corneal collagen diameter. This alteration is the morphologic correlate of the crosslinking process leading to an increase in biomechanical stability. The crosslinking effect is strongest in the anterior half of the stroma because of the rapid decrease in UVA irradiance across the corneal stroma as a result of riboflavin-enhanced UVA absorption.

Biomechanical and histological changes after corneal crosslinking with and without epithelial debridement

PURPOSE: To test the biomechanical efficiency of corneal crosslinking with riboflavin without epithelial debridement (C3‐R). SETTING: Moscow Helmholtz Research Institute of Eye Diseases, Moscow, Russia. METHODS: The left eyes of rabbits were crosslinked using standard crosslinking including epithelial removal (Group 1), using benzalkonium chloride–containing proxymetacaine eyedrops without epithelial removal (Group 2), or using preservative‐free oxybuprocaine eyedrops without epithelial removal (Group 3). All left eyes received riboflavin solution and were irradiated with an ultraviolet‐A double diode for 30 minutes (irradiance 3 mW/cm2). The animals were killed 1 day after crosslinking. Biomechanical and histological analyses were performed. RESULTS: Fourteen eyes were evaluated. There was a statistically significant increase in Youngs modulus in Group 1 (102.45%) and in Group 2 (21.30%). In Group 3, no biomechanical changes were measured. Histology showed complete cell loss of keratocytes and endothelium in Group 1 and inhomogeneous keratocyte loss down to 200.0 μm in Group 2. No changes were observed in Group 3. CONCLUSIONS: Corneal crosslinking without epithelial debridement reduced the biomechanical effect by approximately one fifth compared with standard crosslinking, probably because of restricted and inhomogeneous stromal distribution of riboflavin. The cytotoxic damage was restricted to 200.0 μm stromal depth, which is an advantage over the standard method. Therefore, C3‐R is not recommended for the routine treatment of keratoconus but primarily for cases with a corneal thickness less than 400.0 μm in which standard crosslinking cannot be used without serious risk to the endothelium.

Corneal Endothelial Cytotoxicity of Riboflavin/UVA Treatment in vitro

Recently, we have developed collagen crosslinking induced by combined riboflavin/UVA treatment, thus increasing the biomechanical rigidity of the cornea to treat progressive keratoconus. The present safety study was performed to evaluate possible cytotoxic effects of combined riboflavin/UVA treatment on the corneal endothelium in vitro. Endothelial cell cultures from porcine corneas were treated with 500 µM riboflavin solution, exposed to various endothelial UVA irradiances (370 nm) ranging from 0.1 to 1.6 mW/cm2 for 30 min and evaluated 24 h later using trypan blue staining and Yopro fluorescence staining. The effect of either treatment alone (UVA irradiation ranging from 0.2 to 6 mW/cm2) was also tested. An abrupt cytotoxic threshold irradiance level was found at 0.35 mW/cm2 after combined treatment with riboflavin plus UVA irradiation and at 4 mW/cm2 with UVA irradiation alone. Riboflavin alone was not toxic. A cytotoxic effect of the combined riboflavin/UVA treatment on corneal endothelial cells is to be expected with a corneal thickness of less than 400 µm. Therefore, pachymetry should be routinely performed before riboflavin/UVA treatment to exclude patients at risk.

Keratocyte cytotoxicity of riboflavin/UVA-treatment in vitro

AbstractPurpose 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/cm2 and with UVA alone between 2 and 9 mW/cm2 for 30 min. The cell cultures were evaluated for cell death 24 h after irradiation using trypan-blue and Yopro-fluorescence staining.Results An abrupt cytotoxic irradiance level was found at 0.5 mW/cm2 for keratocytes after UVA-irradiation combined with the photosensitizer riboflavin, which is 10-fold lower than the cytotoxic irradiance of 5 mW/cm2 after UVA-irradiation alone.Conclusions A cytotoxic effect of combined riboflavin/UVA-treatment on keratocytes is to be expected at 0.5 mW/cm2, which is reached in the clinical setting in human corneas down to a depth of 300 μm using the standard surface UVA-irradiance of 3 mW/cm2.

Hydration behavior of porcine cornea crosslinked with riboflavin and ultraviolet A

PURPOSE: To examine the influence of a new crosslinking treatment on corneal swelling properties that correlate with the degree of crosslinking. SETTING: Department of Ophthalmology, Vivantes‐Klinikum Neukölln, Berlin, Germany. METHODS: Twenty freshly enucleated porcine eyes were crosslinked by applying the photosensitizer riboflavin and ultraviolet‐A (UVA) light (370 nm, 3 mW/cm2) for 30 minutes. After the eyes were treated and incubated for 24 hours in a moist chamber, 15 eyes were examined by biomicroscopy and optical coherence tomography (OCT); 5 eyes were examined by light microscopy. Five control eyes were included. RESULTS: Using light microscopy, a characteristic swelling pattern with 3 zones was identified in the crosslinked porcine cornea: an anterior intensely crosslinked zone of 242 μm, an intermediate partially crosslinked zone of 238 μm (hydration factor 2.2), and a noncrosslinked posterior zone of 1355 μm (hydration factor 2.7). A condensed OCT signal was demonstrated in the treated portion of the anterior stroma to a depth of 520 μm with a pronounced line at 540 μm, correlating with the combined anterior and intermediate layers after hydration in the histological analysis. In the nonhydrated state of the crosslinked cornea, the anterior zone was deduced to be 242 μm; the intermediate zone, 109 μm; and the posterior zone, 501 μm. Therefore, the maximum depth of the crosslinking effect was 351 μm. CONCLUSIONS: Collagen crosslinking using riboflavin and UVA led to a significant change in the swelling behavior of the anterior stroma, confirming prior findings that the crosslinking effect is strongest in the anterior half of the stroma. Crosslinked cornea did not induce a specific signal on OCT, and OCT is therefore not suited for clinical controls of the crosslinking effect.

COLLAGEN CROSSLINKING OF HUMAN AND PORCINE SCLERA

Purpose: To develop methods of collagen crosslinking the sclera to increase its biomechanical strength for the treatment of progressive myopia. Setting: Department of Ophthalmology, Technical University of Dresden, Dresden, Germany. Methods: Sagitally oriented scleral strips of 4.0 mm × 8.0 mm were prepared from 5 human postmortem eyes and 50 porcine cadaver eyes and treated with various crosslinking methods including physical crosslinking by combined riboflavin–ultraviolet A (UVA) or rose bengal/white‐light irradiation and chemical crosslinking by incubation with glucose, ribose, glyceraldehyde, and glutaraldehyde solutions. Parallel scleral strips from the same eye were used as untreated controls. After crosslinking, stress‐strain measurements of the treated and control scleras were performed using a microcomputer‐controlled biomaterial tester. Results: A statistically significant increase in scleral rigidity was found after crosslinking with riboflavin–UVA, with a rise in stress in treated porcine (157%) and human (29%) sclera, and after treatment with glyceraldehyde, with a rise in stress in treated porcine (487%) and human (34%) sclera, and with glutaraldehyde, with a rise in stress in treated porcine (817%) and human sclera (122%) at 8% strain. The other crosslinking methods proved ineffective. The untreated human sclera had a 4‐fold higher stiffness than porcine sclera. Conclusions: Collagen crosslinking induced by riboflavin–UVA, glyceraldehyde, and glutaraldehyde led to a significant increase in biomechanical strength in human and porcine sclera. Using these methods, collagen crosslinking might become a treatment possibility for progressive myopia. Future animal and clinical studies must determine the best application methods and the long‐term effects of increased crosslinking on scleral rigidity and prevent potential toxicity or serious side effects.