ultraviolet-A Crosslinking Research Articles

Evaluation of the Safety and Long-term Scleral Biomechanical Stability of UVA Cross-linking on Scleral Collagen in Rhesus Monkeys.

To investigate the changes of retinal and choroidal parameters, scleral biomechanical strength, and ocular histopathology after scleral ultraviolet-A (UVA) cross-linking (CXL) in rhesus monkeys eyes, and to evaluate the safety and long-term biomechanical stability of scleral CXL for preventing myopia from progressing further in clinic. Six 3-year-old male rhesus monkeys (12 eyes) were randomized to receive UVA-CXL procedures applied on the superotemporal equatorial sclera. Optical coherence tomography (OCT) and optical coherence tomography angiography (OCTA) were used for examination before and 1 week and 1, 3, 6, and 12 months after CXL. The stress-strain behaviors of equatorial scleral strips were analyzed 12 months postoperatively by a biomaterial tester. Hematoxylin-eosin and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining were performed 12 months postoperatively. For central retinal thickness, choroidal thickness, and flow density of central retinal superficial vascular networks, no statistical difference was noted between CXL eyes and control eyes at 12 months postoperatively (P > .05). The biomechanical stability of sclera was increased. The scleral stress and Young modulus at 8% strain corresponded to 184% and 183%, respectively, of the control values at 12 months (each P < .001). No retinal damage was detected on histology in scleral CXL eyes. There was no obvious difference between scleral CXL eyes and control eyes by hematoxylin-eosin and TUNEL staining (P > .05). Scleral CXL with riboflavin/UVA in rhesus monkey eyes could strengthen the biomechanical properties of scleral tissues and maintain the stability for 12 months postoperatively. The UVA-CXL on the sclera of rhesus monkey eyes seems to be effective and safe. [J Refract Surg. 2020;36(10):696-702.].

Results of microinvasive cross‐linking of rabbit posterior eye pole sclera

PurposeTo evaluate the results of scleral crosslinking (SXL) with ultraviolet‐A (UVA) radiation and riboflavin in the equator and the posterior pole of the eye using a minimally invasive techniqueMethodsThe tool includes a UV‐LED source (370 nm, 3 mW/cm²) guided through optic fiber located in one of the two channels of a detachable tip. The other channel is used to deliver riboflavin to the scleral surface through a small conjunctival incision, sutured after SXL. It was performed in vivo on 8 rabbit eyes. Intact fellow eyes served as control. Scleral echodensity was measured in vivo with Voluson 730Pro prior to, 2 days and 1 month after the procedure. The biomechanical characteristics of the sclera of enucleated eyes were measured 3 days and 1 month after the procedure with Autograph (Shimadzu). Collagen crosslinking level was measured at the same times using Differential Scanning Calorimetry with Phoenix DSC20144 (Netzsch).ResultsAs a result of the procedure, scleral echodensity (median) increased from 86.7 to 98 dB after 2 days, and to 103 dB after 1 month. The control group revealed no change in the corresponding values. Elasticity modulus (E) of the treated zone was 25.4 MPa after 3 days, while in the control group E was 16.7 MPa. 1 month after, the value of E showed no significant change. Collagen cross‐linking level in the experimental group exceeded that of the control group by 15–18%.ConclusionsThe proposed tool ensures minimally invasive UVA crosslinking of the equatorial and the posterior parts of the sclera and ensures an increase of its biomechanical stability. It opens good prospects for sclera reinforcement treatment of progressive myopia.This work was supported by the Russian Foundation of Basic Research (grant No 15‐29‐03874).

Protective Effects of Soluble Collagen during Ultraviolet-A Crosslinking on Enzyme-Mediated Corneal Ectatic Models.

Collagen crosslinking is a relatively new treatment for structural disorders of corneal ectasia, such as keratoconus. However, there is a lack of animal models of keratoconus, which has been an obstacle for carefully analyzing the mechanisms of crosslinking and evaluating new therapies. In this study, we treated rabbit eyes with collagenase and chondroitinase enzymes to generate ex vivo corneal ectatic models that simulate the structural disorder of keratoconus. The models were then used to evaluate the protective effect of soluble collagen in the UVA crosslinking system. After enzyme treatment, the eyes were exposed to riboflavin/UVA crosslinking with and without soluble type I collagen. Corneal morphology, collagen ultrastructure, and thermal stability were evaluated before and after crosslinking. Enzyme treatments resulted in corneal curvature changes, collagen ultrastructural damage, decreased swelling resistance and thermal stability, which are similar to what is observed in keratoconus eyes. UVA crosslinking restored swelling resistance and thermal stability, but ultrastructural damage were found in the crosslinked ectatic corneas. Adding soluble collagen during crosslinking provided ultrastructural protection and further enhanced the swelling resistance. Therefore, UVA crosslinking on the ectatic model mimicked typical clinical treatment for keratoconus, suggesting that this model replicates aspects of human keratoconus and could be used for investigating experimental therapies and treatments prior to translation.

Spatially heterogeneous corneal mechanical responses before and after riboflavin–ultraviolet-A crosslinking

To determine the heterogeneous through-thickness strains in the cornea at physiologic intraocular pressures before and after corneal collagen crosslinking (CXL) using noninvasive ultrasound. Department of Biomedical Engineering, Ohio State University, Columbus, Ohio, USA. Experimental study. Sixteen paired canine corneoscleral shells were divided into 2 groups. The CXL group completed a standard CXL protocol using riboflavin-ultraviolet-A (UVA) irradiation. The control group was given an identical treatment except UVA irradiation. Ultrasound scans (at 55 MHz) of the cornea were obtained before and after treatment as the corneoscleral shell was inflated from 5 mm Hg to 45 mm Hg to calculate the distributive through-thickness strains in the cornea. The mean radial and tangential strains of the whole cornea layer, as well as those of the anterior, middle, and posterior thirds of the cornea, were compared before and after treatment in the control group and CXL group using linear mixed models with repeated measures. Significant reductions in tangential and radial strains occurred in the CXL group (P=.003 and P=.0025, respectively) but not the control group (P=.08 and P=.63, respectively). The anterior third had the smallest strains in all pretreated corneas (P<.001) and posttreated corneas (CXL group, P=.023; control group, P=.01). Ultrasound speckle tracking showed heterogeneous strain distributions through the cornea and confirmed that CXL results in a stiffer corneal response (ie, smaller strains during physiologic loadings). This technique may provide a clinical tool to quantify the biomechanical effects of CXL. No author has a financial or proprietary interest in any material or method mentioned.

Optimization Model for UV-Riboflavin Corneal Cross-linking

To develop a theoretical model for riboflavin ultraviolet-A cross-linking treatment that can predict the increase in stiffness of the corneal tissue as a function of the ultraviolet intensity and riboflavin concentration distribution, as well as the treatment time. A theoretical model for calculating the increase in corneal cross-linking (polymerization rate) was derived using Fick's second law of diffusion, Lambert-Beer's law of light absorption, and a photopolymerization rate equation. Stress-strain experiments to determine Young's modulus at 5% strain were performed on 43 sets of paired porcine corneal strips at different intensities (3-7 mW/cm²) and different riboflavin concentrations (0.0%-0.5%). The experimental results for Young's modulus increase were correlated with the simulated polymerization increase to determine a relationship between the model and the experimental data. This model allows the calculation of the one-dimensional spatial and temporal intensity and concentration distribution. The total absorbed radiant exposure, defined by intensity, concentration distribution, and treatment time, shows a linear correlation with the measured stiffness increase from which a threshold value of 1.7 J/cm² can be determined. The relative stiffness increase shows a linear correlation with the theoretical polymer increase per depth of tissue, as calculated by the model. This theoretical model predicts the spatial distribution of increased stiffness by corneal cross-linking and, as such, can be used to customize treatment, according to the patient's corneal thickness and medical indication.

Riboflavin/UVA Collagen Cross-Linking-Induced Changes in Normal and Keratoconus Corneal Stroma

PurposeTo determine the effect of Ultraviolet-A collagen cross-linking with hypo-osmolar and iso-osmolar riboflavin solutions on stromal collagen ultrastructure in normal and keratoconus ex vivo human corneas.MethodsUsing small-angle X-ray scattering, measurements of collagen D-periodicity, fibril diameter and interfibrillar spacing were made at 1 mm intervals across six normal post-mortem corneas (two above physiological hydration (swollen) and four below (unswollen)) and two post-transplant keratoconus corneal buttons (one swollen; one unswollen), before and after hypo-osmolar cross-linking. The same parameters were measured in three other unswollen normal corneas before and after iso-osmolar cross-linking and in three pairs of swollen normal corneas, in which only the left was cross-linked (with iso-osmolar riboflavin).ResultsHypo-osmolar cross-linking resulted in an increase in corneal hydration in all corneas. In the keratoconus corneas and unswollen normal corneas, this was accompanied by an increase in collagen interfibrillar spacing (p<0.001); an increase in fibril diameter was also seen in two out of four unswollen normal corneas and one unswollen keratoconus cornea (p<0.001). Iso-osmolar cross-linking resulted in a decrease in tissue hydration in the swollen normal corneas only. Although there was no consistent treatment-induced change in hydration in the unswollen normal samples, iso-osmolar cross-linking of these corneas did result in a compaction of collagen fibrils and a reduced fibril diameter (p<0.001); these changes were not seen in the swollen normal corneas. Collagen D-periodicity was not affected by either treatment.ConclusionThe observed structural changes following Ultraviolet-A cross-linking with hypo-osmolar or iso-osmolar riboflavin solutions are more likely a consequence of treatment-induced changes in tissue hydration rather than cross-linking.

The Absorption Characteristics of the Human Cornea in Ultraviolet-A Crosslinking

With respect to the safety of ultraviolet-A (UVA) crosslinking for the corneal endothelium, an absorption coefficient is used that has been calculated in riboflavin soaked porcine corneas. We aim to validate this value for clinical use by measuring the absorption coefficient for UVA 365 nm in postmortem human corneas after instilling riboflavin on the corneal surface. Corneal thickness was measured in nine pairs of human donor eyes of which one eye was subjected to manual removal of the epithelium, whereas the epithelium of the fellow eye was left intact. Both eyes were instilled with riboflavin 0.1% in dextran 20% on the intact globe. After 20 min, the corneas were rinsed, and a corneoscleral button was trephined. The transmission of the cornea for UVA 365 nm was measured by transillumination, which allows calculation of the absorption coefficient. Measurement of average corneal thickness was 658.5 +/- 51.5 microm when the epithelium was removed, and 758.3 +/- 98.8 microm without epithelial removal. The average transmittance for UVA 365 nm was 12.89 +/- 4.10% with epithelial debridement and 28.52 +/- 4.39% without (P<0.05). The resultant average absorption coefficient is 32 +/- 5 cm when the epithelium is removed and 17 +/- 2 cm when it is left intact (P<0.05). Our results show an absorption coefficient for human corneas that is much lower than the values reported in the literature. This finding may be relevant when considering endothelial safety of the clinical crosslinking treatment.

Staged Intrastromal Delivery of Riboflavin With UVA Cross-linking in Advanced Bullous Keratopathy: Laboratory Investigation and First Clinical Case

To evaluate the safety and efficacy of staged ultraviolet A (UVA) cross-linking following intrastromal 0.1% riboflavin administration in eyes with advanced corneal edema. Ten eye bank corneas divided in two groups (n = 5) were placed on a pressurized artificial anterior chamber following Descemet's membrane stripping. Two consecutive corneal pockets (350- and 150-microm depth) were sequentially created using a femtosecond laser. Sequential intrastromal injections of 0.1% riboflavin (0.2 mL) followed by either UVA irradiation (15 mW/cm2) for 7 minutes or exposure to air were performed for each pocket. Corneal clarity and central thickness were measured before and after the two UVA cross-linking steps. The same steps were clinically applied in an 84-year-old woman with bullous keratopathy prior to corneal transplantation and followed for 6 months. The corneal clarity improved in the treated but not the control eyes. The mean central corneal thickness was significantly reduced by 256 microm (ultrasound, P = .0002) and 273 miccrom (Scheimpflug, P = .0004) in treated eyes, but only 100 microm (ultrasound, P = .048) and 107 microm (Scheimpflug, P = .075) in the control eyes. The clinical treatment of corneal edema showed improved clarity and reduced central corneal thickness from 675 to 550 microm (ultrasound) and 696 to 571 microm (Scheimpflug) at 1 month. Best spectacle-corrected visual acuity improved from finger counting to 20/80 at 1 week and beyond, postponing corneal transplantation for > 6 months. Staged UVA cross-linking (15 mW/cm2) with femtosecond laser facilitated intrastromal 0.1% riboflavin administration may be a safe (no corneal scarring) and effective (marked reduction of edema) temporizing alternative method for managing bullous keratopathy.

Riboflavin and ultraviolet A collagen crosslinking: In vivo thermographic analysis of the corneal surface

To assess the possible thermal damage to the cornea during combined riboflavin and ultraviolet-A crosslinking using in vivo surface thermographic analysis. Department of Oto-Neuro-Ophthalmological Surgical Sciences, Eye Clinic, University of Florence, Florence, Italy. Collagen crosslinking was performed on a day-surgery basis under aseptic conditions with traditional techniques. During the procedure, temperature measurements on the corneal surface were taken using an infrared thermocamera. The temperature values were detected in the area directly exposed to light irradiation, selecting it in the acquired thermographic image. Because the aim of the study was to consider the thermal damage that may be induced during the procedure, the maximum temperature value detected in the area studied was recorded and considered for successive analysis. Infrared thermocamera measurements of the corneal surface during crosslinking treatment showed that the temperature was constant during the entire procedure and remains under the threshold of thermal injury to corneal collagen. Corneal collagen crosslinking was a safe procedure that did not cause thermal injury to the corneal surface.