Corneal riboflavin gradients and UV-absorption characteristics after topical application of riboflavin in concentrations ranging from 0.1 to 0.5.

Avoiding damage of the endothelial cells, especially in thin corneas, remains a challenge in corneal collagen crosslinking (CXL). Knowledge of the riboflavin gradients and the UV absorption characteristics after topical application of riboflavin in concentrations ranging from 0.1% to 0.5% could optimize the treatment. In this study, we present a model to calculate the UV-intensity depending on the corneal thickness. Ten groups of de-epithelialized porcine corneas were divided into 2 subgroups. Five groups received an imbibition of 10 min and the other five groups for 30 min. The applied riboflavin concentrations were 0.1%, 0.2%, 0.3%, 0.4% and 0.5% diluted in a 15% dextran solution for each subgroup. After the imbibition process, two-photon fluorescence microscopy was used to determine fluorescence intensity, which was compared to samples after saturation, yielding the absolute riboflavin concentration gradient of the cornea. The extinction coefficient of riboflavin solutions was measured using a spectrophotometer. Combining the obtained riboflavin concentrations and the extinction coefficients, a depth-dependent UV-intensity profile was calculated for each group. With increasing corneal depth, the riboflavin concentration decreased for all imbibition solutions and application times. The diffusion coefficients of 10 min imbibition time were higher than for 30 min. A higher RF concentration and a longer imbibition time resulted in higher UV-absorption and a lower UV-intensity in the depth of the cornea. Calculated UV-transmission was 6 percentage points lower compared to the measured transmission. By increasing the riboflavin concentration of the imbibition solution, a substantially higher UV-absorption inside the cornea is achieved. This offers a simple treatment option to control the depth of crosslinking e.g. in thin corneas, resulting in a lower risk of endothelial damage.

Corneal Cross-Linking: An Example of Photoinduced Polymerization as a Treatment Modality in Keratoconus.

The cornea is one of the principal refractive elements in the human eye and plays a crucial role in the process of vision. Keratoconus is the most common corneal dystrophy, found mostly among young adults. It is characterized by a reduced number of collagen cross-links in the corneal stroma, resulting in reduced biomechanical stability and an abnormal shape of the cornea. These changes lead to progressive myopia, corneal thinning, central scarring and irregular astigmatism, causing severely impaired vision. Hard contact lenses, photorefractive keratectomy or intracorneal rings are the most common treatment options for refractive error caused by keratoconus. However, these techniques do not treat the underlying cause of the corneal ectasia and therefore are not able to stop the progression of the disease. Riboflavin photoinduced polymerization of corneal collagen, also known as corneal cross-linking (CXL), has been introduced as the first therapy which, by stabilizing the structure of the cornea, prevents the progression of keratoconus. It stiffens the cornea using the photo-sensitizer riboflavin in combination with ultraviolet irradiation. This is a current review of the CXL procedure as a therapy for keratoconus, which relies on photoinduced polymerization of human tissue. We have focused on its biomechanical and physiological influences on the human cornea and have reviewed the previous and current biochemical theories behind cross-linking reactions in the cornea.

Ultraviolet A: Visible spectral absorbance of the human cornea after transepithelial soaking with dextran-enriched and dextran-free riboflavin 0.1% ophthalmic solutions.

PURPOSE: To evaluate the stromal concentration of 2 commercially available transepithelial riboflavin 0.1% solutions in human donor corneas with the use of spectrophotometry. SETTING: University of Calabria, Rende, Italy. DESIGN: Experimental study. METHODS: The absorbance spectra of 12 corneal tissues were measured in the 330 to 700 nm wavelength range using a purpose-designed spectrophotometry setup before and after transepithelial corneal soaking with a 15% dextran-enriched riboflavin 0.1% solution (n = 6) or a hypotonic dextran-free riboflavin 0.1% solution (n = 6). Both ophthalmic solutions contained ethylenediaminetetraacetic acid and trometamol as enhancers. In addition, 4 deepithelialized corneal tissues underwent stromal soaking with a 20% dextran-enriched riboflavin 0.1% solution and were used as controls. All the riboflavin solutions were applied topically for 30 minutes. The stromal concentration of riboflavin was quantified by analysis of absorbance spectra of the cornea collected before and after application of each solution. RESULTS: The mean stromal riboflavin concentration was 0.012% ± 0.003% (SD), 0.0005% ± 0.0003% (P < .001), and 0.004% ± 0.001% (P < .01) in tissues soaked with 20% dextran-enriched, 15% dextran-enriched, and hypotonic dextran-free solutions, respectively. The difference of stromal riboflavin concentration between the 2 transepithelial solutions was statistically significant (P < .01). CONCLUSIONS: Dextran-enriched solutions required complete corneal deepithelialization to permit effective stromal soaking with riboflavin. Nevertheless, riboflavin in hypotonic dextran-free solution with enhancers permeates across stroma through an intact epithelium. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Measuring the Refractive Index of Bovine Corneal Stromal Cells Using Quantitative Phase Imaging.

The cornea is the primary refractive lens in the eye and transmits >90% of incident visible light. It has been suggested that the development of postoperative corneal haze could be due to an increase in light scattering from activated corneal stromal cells. Quiescent keratocytes are thought to produce crystallins that match the refractive index of their cytoplasm to the surrounding extracellular material, reducing the amount of light scattering. To test this, we measured the refractive index (RI) of bovine corneal stromal cells, using quantitative phase imaging of live cells in vitro, together with confocal microscopy. The RI of quiescent keratocytes (RI = 1.381 ± 0.004) matched the surrounding matrix, thus supporting the hypothesis that keratocyte cytoplasm does not scatter light in the normal cornea. We also observed that the RI drops after keratocyte activation (RI = 1.365 ± 0.003), leading to a mismatch with the surrounding intercellular matrix. Theoretical scattering models showed that this mismatch would reduce light transmission in the cornea. We conclude that corneal transparency depends on the matching of refractive indices between quiescent keratocytes and the surrounding tissue, and that after surgery or wounding, the resulting RI mismatch between the activated cells and their surrounds significantly contributes to light scattering.

Deep anterior lamellar keratoplasty using irradiated acellular cornea with amniotic membrane transplantation for intractable ocular surface diseases.

PURPOSE: To report the clinical outcomes of deep anterior lamellar keratoplasty (DALK) when sterile gamma-irradiated acellular corneal tissues (VisionGraft) are used in combination with amniotic membrane transplantation (AMT) for intractable ocular surface diseases. METHODS: The medical records of fifteen patients who had DALK with AMT were retrospectively reviewed. Indications for surgery included ocular burn, bacterial keratitis, herpes simplex virus keratitis, corneal opacity with Stevens-Johnson syndrome, Mooren's ulcer, idiopathic myxoid degeneration of corneal stroma, and recurrent band keratopathy. DALK was performed using partial-thickness acellular corneal tissue and a temporary amniotic membrane patch was added at the end of the operation. RESULTS: All cases that underwent DALK with AMT became epithelialized within 2 postoperative weeks. Twelve patients showed favorable outcomes without graft rejection, corneal opacification, or neovascularization. The other three grafts developed corneal opacification and neovascularization, and required additional penetrating keratoplasty (PK). Unlike the results of previous PKs, there were no graft rejections and the graft clarity was well-maintained in these three cases for at least 8 months after PK. CONCLUSIONS: DALK using sterile acellular corneal tissues in combination with AMT may be a good therapeutic strategy for treating intractable ocular surface diseases because of lowered immune rejection, fibroblast activation, and facilitation of epithelialization. Furthermore, DALK can help stabilize the ocular surface, prolong graft survival, and may allow better outcomes when combined with subsequent PK.

Epithelium-off photochemical corneal collagen cross-linkage using riboflavin and ultraviolet a for keratoconus and keratectasia: a systematic review and meta-analysis.

This report presents the results of a systematic review and meta-analyses of studies on epithelium-off photochemical corneal collagen cross-linkage for the management of keratoconus and secondary ectasia. The literature search identified 3,400 records of which 49 were considered for inclusion in the meta-analyses. Eight papers reported 4 unique randomized controlled trials, 29 studies were prospective, and 12 were retrospective studies. The majority of the studies (39/49) were graded as very low quality evidence. Twenty-six studies described adverse events and were included in the safety analysis. Meta-analyses are presented for changes in four outcomes: visual acuity, topography, refraction and astigmatism, and central corneal thickness. Statistically significant improvements were found in all efficacy outcomes at 12 months after the operation. Common side effects were pain, corneal edema, and corneal haze, which resolved usually within a few days after the procedure. The remaining uncertainty is duration of benefit to establish the procedure's potential benefit in avoiding or delaying disease progression and possibly reducing the need for corneal transplantation.

Intrastromal application of riboflavin for corneal crosslinking.

PURPOSE: To experimentally evaluate the efficacy of corneal crosslinking (CXL) by injecting the photomediator riboflavin into the corneal stroma via intrastromal channels. METHODS: Five groups of pig corneas, nine each, were compared regarding stress-strain relationship and UV-absorption. Group 1 had intrastromal channels floated with riboflavin 0.5%-solution followed by UVA-irradiation (3 mW/cm(2) for 30 minutes); group 2 was handled like group 1, but were irradiated with 9 mW/cm(2) for 10 minutes; group 3 was treated according to the Dresden protocol (epi-off, 9 mW/cm(2) for 10 minutes); group 4 had the identical channel system, no riboflavin but identical irradiation; group 5 with native corneas served as a control group. The intrastromal channels were created with a femtosecond laser. The stress-strain relations were measured in corneal strips using a uniaxial material tester at strains up to 12%. The UV-transmission of the corneas was measured in groups 1, 3, and 5. RESULTS: The stress needed for a 10% strain was significantly increased by 82% in the corneas treated with the Dresden protocol compared with native cornea (P = 0.0005). With intrastromal application of riboflavin the significant increase was 87% (P = 0.0005) in group 1 and 64% (P = 0.007) in group 2. The channel formation alone did not alter biomechanics (P = 0.923). The corneal UVA-transmission was 2.4% after intrastromal riboflavin application, 8.9% after the treatment according to the Dresden protocol, and 57.9% in native corneas. CONCLUSIONS: The experiments demonstrate the intrastromal application of riboflavin by means of intrastromal channels a feasible "epi-on" approach for CXL. More experimental data are needed before clinical testing.

Intraoperative corneal thickness measurements during corneal collagen cross-linking with isotonic riboflavin solution without dextran in corneal ectasia.

UNLABELLED: Abstract Objective: To monitor the changes in corneal thickness during the corneal collagen cross-linking procedure by using isotonic riboflavin solution without dextran in ectatic corneal diseases. MATERIALS AND METHODS: The corneal thickness measurements were obtained before epithelial removal, after epithelial removal, following the instillation of isotonic riboflavin solution without dextran for 30 min, and after 10 min of ultraviolet A irradiation. RESULTS: Eleven eyes of eleven patients with progressive keratoconus (n = 10) and iatrogenic corneal ectasia (n = 1) were included in this study. The mean thinnest pachymetric measurements were 391.82 ± 30.34 µm (320-434 µm) after de-epithelialization of the cornea, 435 ± 21.17 µm (402-472 µm) following 30 min instillation of isotonic riboflavin solution without dextran and 431.73 ± 20.64 µm (387-461 µm) following 10 min of ultraviolet A irradiation to the cornea. CONCLUSION: Performing corneal cross-linking procedure with isotonic riboflavin solution without dextran might not induce corneal thinning but a little swelling throughout the procedure.

Corneal thickness changes during corneal collagen cross-linking with UV-A irradiation and hypo-osmolar riboflavin in thin corneas / Alterações na espessura da córnea durante "cross-linking" do colágeno com irradiação UV-A e riboflavina hipo-osmolar em córneas finas

PURPOSE: To evaluate the thinnest corneal thickness changes during and after corneal collagen cross-linking treatment with ultraviolet-A irradiation, using hypo-osmolar riboflavin solution in thin corneas. METHODS: Eighteen eyes of 18 patients were included in this study. After epithelium removal, iso-osmolar 0.1% riboflavin solution was instilled to the cornea every 3 minutes for 30 minutes. Hypo-osmolar 0.1% riboflavin solution was then applied every 20 seconds for 5 minutes or until the thinnest corneal thickness reached 400 µm. Ultraviolet-A irradiation was performed for 30 minutes. During irradiation, iso-osmolar 0.1% riboflavin drops were applied every 5 minutes. Ultrasound pachymetry was performed at approximately the thinnest point of the cornea preoperatively, after epithelial removal, after iso-osmolar riboflavin instillation, after hypo-osmolar riboflavin instillation, after ultraviolet-A irradiation, and at 1, 6 and 12 months after treatment. RESULTS: Mean preoperative thinnest corneal thickness was 380 ± 11 µm. After epithelial removal it decreased to 341 ± 11 µm, and after 30 minutes of iso-osmolar 0.1% riboflavin drops, to 330 ± 7.6 µm. After hypo-osmolar 0.1% riboflavin drops, mean thinnest corneal thickness increased to 418 ± 11 µm. After UVA irradiation, it was 384 ± 10 µm. At 1, 6 and 12 months after treatment, it was 372 ± 10 µm, 381 ± 12.7, and 379 ± 15 µm, respectively. No intraoperative, early postoperative, or late postoperative complications were noted. CONCLUSIONS: Hypo-osmolar 0.1% riboflavin solution seems to be effective for swelling thin corneas. The swelling effect is transient and short acting. Corneal thickness should be monitored throughout the procedure. Larger sample sizes and longer follow-up are required in order to make meaningful conclusions regarding safety.

OBJETIVO: Avaliar as alterações da espessura mínima da córnea durante e após o cross-linking do colágeno corneano com radiação ultravioleta A e solução hipo-osmolar de riboflavina em córneas finas. MÉTODOS: Dezoito olhos de 18 pacientes foram incluídos neste estudo. Após a remoção do epitélio, solução iso-osmolar de riboflavina 0,1% foi instilada a cada 3 minutos por 30 minutos. Solução hipo-osmolar de riboflavina 0,1% foi então aplicada a cada 20 segundos por 5 minutos ou até que a espessura mínima da córnea atingisse 400 µm. Irradiação UVA foi feita durante 30 minutos. Durante a irradiação, riboflavina iso-osmolar 0,1% foi aplicada a cada 5 minutos. Paquimetria ultrassônica foi realizada no ponto mais fino da córnea antes da cirurgia, após a remoção do epitélio, após a instilação de riboflavina iso-osmolar, após a instilação de riboflavina hipo-osmolar, após a irradiação com UVA e após 1, 6 e 12 meses do tratamento. RESULTADOS: Antes da cirurgia, a espessura mínima da córnea era de 380 ± 11 µm. Após a remoção do epitélio, este valor foi reduzido para 341 ± 11 µm e após 30 minutos de riboflavina iso-osmolar, caiu para 330 ± 7,6 µm. Após a riboflavina hipo-osmolar, a espessura mínima da córnea aumentou para 418 ± 11 µm. Após a irradiação com UVA, era de 384 ± 10 µm. Após 1, 6 e 12 meses do tratamento este valor era de 372 ± 10, 381 ± 12,7 e 379 ± 15 µm, respectivamente. Não foram observadas complicações no intra ou no pós-operatório precoce ou tardio. CONCLUSÕES: A solução de riboflavina hipo-osmolar 0,1% parece ser eficaz para edemaciar córnea finas. Este efeito é transitório e de curta duração. A espessura da córnea deveria ser monitorada durante todo o procedimento. Maior número de casos e seguimento prolongado são necessários para tirarmos conclusões quanto à segurança.

Inverse computational analysis of in vivo corneal elastic modulus change after collagen crosslinking for keratoconus.

Corneal collagen crosslinking with riboflavin photosensitization and ultraviolet irradiation is a novel approach to limiting the progression of keratoconus in patients by increasing the elastic modulus of the degenerate cornea. Beneficial reductions in corneal steepness and aberrations after crosslinking also frequently occur. In a previous study, we described a computational modeling approach to simulating topographic progression in keratoconus and regression of disease with corneal collagen crosslinking. In the current study, this model has been expanded and applied to the inverse problem of estimating longitudinal time-dependent changes in the corneal elastic modulus after crosslinking using in vivo measurements from 16 human eyes. Topography measured before crosslinking was used to construct a patient-specific finite element model with assumed hyperelastic properties. Then the properties of the cornea were altered using an inverse optimization method to minimize the difference between the model-predicted and in vivo corneal shape after crosslinking. Effects of assumptions regarding sclera-to-cornea elastic modulus ratio and spatial attenuation of treatment effect due to ultraviolet beam characteristics on the predicted change in elastic modulus were also investigated. Corneal property changes computed by inverse finite element analysis provided excellent geometric agreement with clinical topography measurements in patient eyes post-crosslinking. Over all post-treatment time points, the estimated increase in corneal elastic modulus was 110.8 ± 48.1%, and slightly less stiffening was required to produce the same amount of corneal topographic regression of disease when the sclera-to-cornea modulus ratio was increased. Including the effect of beam attenuation resulted in greater estimates of stiffening in the anterior cornea. Corneal shape responses to crosslinking varied considerably and emphasize the importance of a patient-specific approach.

Early confocal microscopy findings after cross-linking treatment.

OBJECTIVE: To determine the effects of in vivo cross-linking treatment of the cornea. METHODS: Eighteen eyes of eighteen keratoconus patients underwent cross-linking treatment using a 0.1% riboflavin solution and ultraviolet A radiation at 370 nm at 3 mW/cm² for 30 minutes. In vivo confocal microscopy was performed before, and at 1 week and 1 month after treatment. RESULTS: At 1 week after treatment, keratocyte activation and collagen fiber organization showed as hyper-reflective structures and were observed from the first sub-epithelial image to a corneal stromal depth of 275.1 ± 85.9 µm. At 1 month after treatment, activated keratocytes and fiber organization were also observed from the first sub-epithelial image to a corneal stromal depth of 324.9 ± 66.0 µm. The deepest hyper-reflective structures at 1 month showed as thick, linear-shaped hyper-reflective structures. CONCLUSION: In vivo confocal microscopy in humans showed corneal stromal changes at 1 week and 1 month after cross-linking treatment, in some cases at depths in excess of 300 µm.

Wavelength-dependent ultraviolet induction of cyclobutane pyrimidine dimers in the human cornea.

Exposition to ultraviolet (UV) light is involved in the initiation and the progression of skin cancer. The genotoxicity of UV light is mainly attributed to the induction of cyclobutane pyrimidine dimers (CPDs), the most abundant DNA damage generated by all UV types (UVA, B and C). The human cornea is also exposed to the harmful UV radiations, but no UV-related neoplasm has been reported in this ocular structure. The probability that a specific DNA damage leads to a mutation and eventually to cellular transformation is influenced by its formation frequency. To shed light on the genotoxic effect of sunlight in the human eye, we have analyzed CPD induction in the cornea and the iris following irradiation of ex vivo human eyes with UVA, B or C. The extent of CPD induction was used to establish the penetrance of the different UV types in the human cornea. We show that UVB- and UVC-induced CPDs are concentrated in the corneal epithelium and do not penetrate deeply beyond this corneal layer. On the other hand, UVA wavelengths penetrate deeper and induce CPDs in the entire cornea and in the first layers of the iris. Taken together, our results are undoubtedly an important step towards better understanding the consequences of UV exposure to the human eye.

The effect of riboflavin/UVA collagen cross-linking therapy on the structure and hydrodynamic behaviour of the ungulate and rabbit corneal stroma.

PURPOSE: To examine the effect of riboflavin/UVA corneal crosslinking on stromal ultrastructure and hydrodynamic behaviour. METHODS: One hundred and seventeen enucleated ungulate eyes (112 pig and 5 sheep) and 3 pairs of rabbit eyes, with corneal epithelium removed, were divided into four treatment groups: Group 1 (28 pig, 2 sheep and 3 rabbits) were untreated; Group 2 (24 pig) were exposed to UVA light (3.04 mW/cm(2)) for 30 minutes and Group 3 (29 pig) and Group 4 (31 pig, 3 sheep and 3 rabbits) had riboflavin eye drops applied to the corneal surface every 5 minutes for 35 minutes. Five minutes after the initial riboflavin instillation, the corneas in Group 4 experienced a 30 minute exposure to UVA light (3.04 mW/cm(2)). X-ray scattering was used to obtain measurements of collagen interfibrillar spacing, spatial order, fibril diameter, D-periodicity and intermolecular spacing throughout the whole tissue thickness and as a function of tissue depth in the treated and untreated corneas. The effect of each treatment on the hydrodynamic behaviour of the cornea (its ability to swell in saline solution) and its resistance to enzymatic digestion were assessed using in vitro laboratory techniques. RESULTS: Corneal thickness decreased significantly following riboflavin application (p<0.01) and also to a lesser extent after UVA exposure (p<0.05). With the exception of the spatial order factor, which was higher in Group 4 than Group 1 (p<0.01), all other measured collagen parameters were unaltered by cross-linking, even within the most anterior 300 microns of the cornea. The cross-linking treatment had no effect on the hydrodynamic behaviour of the cornea but did cause a significant increase in its resistance to enzymatic digestion. CONCLUSIONS: It seems likely that cross-links formed during riboflavin/UVA therapy occur predominantly at the collagen fibril surface and in the protein network surrounding the collagen.

Comparison of central corneal thickness using ultrasound pachymetry during corneal collagen cross-linking.

PURPOSE: To study variation in central corneal thickness (CCT) during corneal collagen cross-linking(CXL) using ultrasound pachymetry. METHODS: Twenty patients (26 eyes) with progressing keratoconus undergoing riboflavin-UVA-induced CXL were involved in this study. Intraoperative CCT measurement using ultrasonic pachymetry was performed during the procedure. Measurements were obtained before operation, after epithelial removal, after riboflavin drop instillation, and after UVA irradiation. RESULTS: Mean CCT was 495 +/- 56 and 450 +/- 52 microm before and after epithelial removal, respectively. Mean CCT was 443 +/- 42 and 411 +/- 39 microm after riboflavin drop instillation and after UVA irradiation, respectively. Statistically significant decreases in CCT occurred between the preoperative period and after epithelial removal, after riboflavin drop instillation and after UVA irradiation. Twenty-six eyes from 20 patients undergoing CXL were divided into 2 groups (I with CCT > or = 400 microm after UVA irradiation and II with CCT < 400 microm after UVA irradiation). No statistically significant difference was noted between I and II in preoperative endothelial cell count, but a statistically greater postoperative endothelial cell count was noted in I compared to II. A statistically significant difference was evident between preoperative and postoperative endothelial cell counts in Group II (P < 0.05). CONCLUSION: Performing CXL with the use of riboflavin and UVA irradiation resulted in a statistically significant decrease in CCT, to a level where the corneal endothelium may be damaged.

Stiffening of rabbit corneas by the bacteriochlorophyll derivative WST11 using near infrared light.

PURPOSE: We evaluated the efficacy and safety of photochemical corneal stiffening by palladium bacteriochlorin 13'-(2-sulfoethyl)amide dipotassium salt (WST11) and near infrared (NIR) illumination, using ex vivo and in vivo rabbit eye models. METHODS: Corneas of post mortem rabbits and living rabbits were pretreated topically with 2.5 mg/mL WST11 in saline or in 20% dextran T-500 (WST-D), washed and illuminated with an NIR diode laser (755 nm, 10 mW/cm(2). Studies with corneas of untreated fellow eyes served as controls. Tensile strength measurements, histopathology, electron spin resonance, and optical spectroscopy and fluorescence microscopy were used to assess treatment effects. Comparative studies were performed with standard riboflavin/ultraviolet-A light (UVA) treatment. RESULTS: WST11/NIR treatment significantly increased corneal stiffness following ex vivo or in vivo treatment, compared to untreated contralateral eyes. The incremental ultimate stress and Young's modulus of treated corneas increased by 45, 113, 115%, and 10, 79, and 174% following 10, 20, and 30 minutes of incubation with WST11, respectively. WST-D/NIR had a similar stiffening effect, but markedly reduced post-treatment edema and shorter time of epithelial healing. WST11/NIR and WST-D/NIR generate hydroxyl and superoxide radicals, but no singlet oxygen in the cornea. Histology demonstrated a reduction in the keratocyte population in the anterior half of the corneal stroma, without damage to the endothelium. CONCLUSIONS: Treatment of rabbit corneas, with either WST11/NIR or WST-D/NIR, increases their biomechanical strength through a mechanism that does not involve singlet oxygen. The WST-D/NIR treatment showed less adverse effects, demonstrating a new potential for clinical use in keratoconus and corneal ectasia after refractive surgery.

Synergistic effects of ultraviolet A/riboflavin and glucose on corneal collagen cross-linking.

PURPOSE: To investigate the possible synergistic effect of ultraviolet A (UVA)/riboflavin and glucose on corneal collagen cross-linking (CXL) by means of the mechanical properties and thermal shrinkage temperatures. METHODS: Forty rabbits were divided into four groups: control group; UVA/glucose group; UVA/riboflavin group; and UVA/riboflavin/glucose group. After the treatments, the mechanical properties and thermal shrinkage temperatures of the corneal strips were analyzed to determine the CXL effects of corneal collagen induced by different treatments. RESULTS: Compared with the control group and other experimental groups, the UVA/riboflavin/glucose group showed significant alterations of the biomechanical properties. The ultimate stress value for the UVA/riboflavin/glucose group was increased by 120%, ultimate strain decreased by 80%, and Young's modulus increased by 70% relative to the control group. An increase in ultimate stress by 24.5%, a decrease in ultimate strain by 34%, and an increase in Young's modulus by 17.4% were found in the UVA/riboflavin/glucose group compared to the UVA/riboflavin group. A significant increase in thermal shrinkage temperatures was also noted after corneal collagen CXL induced by UVA/riboflavin/glucose relative to other groups. CONCLUSIONS: The combination of glucose and UVA/riboflavin may synergistically induce corneal collagen CXL, resulting in increased mechanical strength of corneal collagen.

Measurement of corneal changes after collagen crosslinking using a noninvasive ultrasound system.

PURPOSE: To determine the changes in corneal properties induced by riboflavin-ultraviolet A (UVA) irradiation using a noninvasive ultrasound (US) method in a porcine eye model. SETTING: Department of Biomedical Engineering, Ohio State University, Columbus, Ohio, USA. METHODS: Fresh porcine globes acquired within 24 hours postmortem were divided into equal groups. In the collagen crosslinking (CXL) group, the globes were irradiated with UVA in combination with riboflavin application following the standard protocol. The control group had the same procedures as the CXL group but with no UVA irradiation. Corneal properties were measured before and after treatments in both groups using a quantitative US method. Changes in corneal thickness, density, and stiffness were examined and the difference between the changes in the CXL group and the control group was analyzed. RESULTS: Each group comprised 14 porcine globes. After riboflavin-UVA treatment, there was a significant increase in corneal stiffness (mean 2.59 +/- 0.08 GPa before and 2.70 +/- 0.08 GPa after CXL) (P<.001) and in the speed of sound in corneal tissue (1557 +/- 23 m/s versus 1588 +/- 22 m/s) (P<.001). There was no significant change in corneal stiffness or in the speed of sound in the control group. Corneal thickness increased significantly after the treatment in both groups. No significant change in corneal density was observed in either group. CONCLUSION: Corneal stiffness increased after riboflavin-UVA CXL, a finding detected using a noninvasive US technique. FINANCIAL DISCLOSURE: Dr. Liu has filed a patent application for the general ultrasound technique used in the study. No other author has a financial or proprietary interest in any material or method mentioned.