Eberhard Spoerl

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.

Collagen crosslinking with riboflavin and ultraviolet-A light in keratoconus: long-term results.

PURPOSE: To prove the long‐term dampening effect of riboflavin‐ and ultraviolet‐A‐induced collagen crosslinking on progressive keratoconus. SETTING: Department of Ophthalmology, C.G. Carus University Hospital, Dresden, Germany. METHODS: Four hundred eighty eyes of 272 patients with progressive keratoconus were included in this long‐term retrospective study. The maximum follow‐up was 6 years. At the first and all follow‐up examinations, refraction, best corrected visual acuity (BCVA), corneal topography, corneal thickness, and intraocular pressure were recorded. RESULTS: The analysis included 241 eyes with a minimum follow‐up of 6 months. The steepening decreased significantly by 2.68 diopters (D) in the first year, 2.21 D in the second year, and 4.84 D in the third year. The BCVA improved significantly (≥1 line) in 53% of 142 eyes in the first year, 57% of 66 eyes in the second year, and 58% of 33 eyes in the first year or remained stable (no lines lost) in 20%, 24%, and 29%, respectively. Two patients had continuous progression of keratoconus and had repeat crosslinking procedures. CONCLUSIONS: Despite the low number of patients with a follow‐up longer than 3 years, results indicate long‐term stabilization and improvement after collagen crosslinking. Thus, collagen crosslinking is an effective therapeutical option for progressive keratoconus.

Safety of UVA-riboflavin cross-linking of the cornea.

Purpose: To study potential damage to ocular tissue during corneal collagen cross-linking (X-linking) by means of the riboflavin/UVA (370 nm) approach. Methods: Comparison of the currently used technique with officially accepted guidelines regarding direct UV damage and the damage created by the induced free radicals (photochemical damage). Results: The currently used UVA radiant exposure of 5.4 mJ/cm2 and the corresponding irradiance of 3 mW/cm2 is below the known damage thresholds of UVA for the corneal endothelium, lens, and retina. Regarding the photochemical damage caused by the free radicals, the damage thresholds for keratocytes and endothelial cells are 0.45 and 0.35 mW/cm2, respectively. In a 400-μm-thick cornea saturated with riboflavin, the irradiance at the endothelial level was 0.18 mW/cm2, which is a factor of 2 smaller than the damage threshold. Conclusions: After corneal X-linking, the stroma is depopulated of keratocytes ∼300 μm deep. Repopulation of this area takes up to 6 months. As long as the cornea treated has a minimum thickness of 400 μm (as recommended), the corneal endothelium will not experience damage, nor will deeper structures such as lens and retina. The light source should provide a homogenous irradiance, avoiding hot spots.

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.

Biomechanical evidence of the distribution of cross-links in corneas treated with riboflavin and ultraviolet A light.

PURPOSE: To examine to which depth of the cornea the stiffening effect is biomechanically detectable. SETTING: Department of Ophthalmology, University of Dresden, Dresden, Germany. METHODS: Of 40 enucleated porcine eyes, 20 eyes were treated with the photosensitizer riboflavin (0.1%) and ultraviolet A (UVA) light (370 nm, 3 mW/cm2, 30 minutes); the other 20 eyes served as control. From each eye, 2 flaps of 200 μm thickness were cut with a microkeratome, and strips of 5 mm width and 7 mm length were prepared. Stress–strain behavior was measured with a material tester to characterize the stiffening effect. Five pairs of human donor eyes were tested in the same way. RESULTS: In porcine corneas, the stiffening effect was stronger in the anterior‐treated flaps than in the posterior‐treated flaps and the control flaps (P = .001). A 5% strain was achieved at a stress of 261.7 ± 133.2 × 103 N/m2 in the anterior‐treated flaps and 104.1 ± 52.7 × 103 N/m2 in the anterior control flaps. The posterior‐treated flaps (105.0 ± 55.8 × 103 N/m2) and the posterior control flaps (103.7 ± 61.8 × 103 N/m2) showed no difference (P = .95). A similar stiffening effect was observed in human eyes, but contrary to findings in porcine corneas, in human corneas the anterior control flaps were stiffer than the posterior control flaps (P = .027). CONCLUSIONS: Treatment of the cornea with riboflavin and UVA significantly stiffened the cornea only in the anterior 200 μm. This depth‐dependent stiffening effect may be explained by the absorption behavior for UVA in the riboflavin‐treated cornea. Sixty‐five percent to 70% of UVA irradiation was absorbed within the anterior 200 μm and only 20% in the next 200 μm. Therefore, deeper structures and even the endothelium are not affected.

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.

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.

Techniques for stiffening the cornea.

In keratoconus, corneal stiffness is decreased. The purposes of this study were to search for techniques to stiffen the cornea and to determine a dose-response relation. The stiffness of collagenous tissues can be increased by cross-linking. Cross-linking techniques--riboflavin and ultraviolet irradiation (365 nm, 2 mW/cm2, 45 min), glutaraldehyde of low concentration, and several aldehyde sugars--were tested on corneas of enucleated porcine eyes. The stress-strain relation was measured and compared to untreated corneas. Aldehyde sugars produced cross-links by nonenzymatic glycation only after a prolonged time (realized in diabetics). Riboflavin and UV seems to be a promising technique to stabilize the cornea by artificial cross-linking.