Keratoconus is the most common global reason for severe visual impairment in childhood and adolescence, with a published prevalence between 1:21 and 1:500 children and adolescents.1–5 Ectasia also may occur after corneal laser ablation surgeries, such as laser-assisted in situ keratomileusis (LASIK) and photorefractive keratectomy (PRK). Reasons for postoperative ectasia may include excessive removal of corneal tissue and hormonal influences.6
Epithelial removal corneal cross-linking (epi-off CXL) is the global standard to treat ectatic corneal diseases.7 Although the success rate of epi-off CXL is exceptionally high,8,9 current clinical protocols have a certain complication profile, mostly linked to the surgical removal of the corneal epithelium.10
Transepithelial CXL protocols have been proposed that leave the corneal epithelium intact (epi-on), shortening the postoperative healing period and decreasing the risk for infection. Although limited clinical benefits with transepithelial CXL have been primarily attributed to low stromal riboflavin penetration, other factors such as stromal oxygen concentration and light absorption may also play a relevant role. Recently, iontophoresis-assisted transepithelial CXL (I-CXL) was introduced, where riboflavin is transported through the epithelium using an electric gradient. When compared with standard epi-off CXL, clinical results have been rather disappointing.11–14 Our working hypothesis is that iontophoresis alone is not enough to improve the efficacy of transepithelial CXL because insufficient oxygenation of the stroma might be another, not yet fully considered factor.15 Tissue oxygenation can be modulated, even when the total energy (fluence) remains constant, by delivering this total energy over a longer period of time with lower intensity.16,17
The objective of this study was to assess whether prolonging irradiation time, and thus tissue oxygenation, might improve the biomechanical outcome in I-CXL.
Materials and Methods
One hundred twelve freshly enucleated porcine eyes were obtained from the local slaughterhouse (Zurich, Switzerland) and used within 12 hours. Eyes had not been steamed, showed an intact epithelium, and were divided in 7 different groups. We chose the porcine cornea as the model due to the large number of samples that were required for biomechanical testing. All enucleated eyes were from pigs aged 6 to 8 months.
For standard epithelium-off CXL protocols (S-CXL), the corneas were completely deepithelialized using surgical blades, followed by soaking with 0.1% riboflavin for 30 minutes and UV-A light irradiation at 3 mW/cm2 for 30 minutes (group 1) or at 9 mW/cm2 for 10 minutes (group 2). Controls were deepithelialized and soaked with riboflavin, but were not irradiated with ultraviolet-A (UV-A) (group 3).
In the experimental I-CXL groups, the corneal epithelium was kept intact. Riboflavin penetration through the cornea was performed using a commercially available iontophoresis device (I-ON CXL; Iacer, Veneto, Italy) and a sodium edetate and trometamol–enriched riboflavin phosphate 0.1% hypotonic solution (Ricrolin; Sooft, Montegiorgio, Italy). I-CXL protocols allow for a delivery of riboflavin in the corneal stroma through the assistance of an electric current, which leads to the reduction of soaking time.12,18 A 1.0-mA electric current was applied to the suction ring for 10 minutes (2 repeated cycles of 5 minutes), followed by irradiation with 1.5 mW/cm2 for 60 minutes (group 4), 3 mW/cm2 for 30 minutes (group 5), or 10 mW/cm2 for 9 minutes (group 6). Controls were subjected to iontophoresis with riboflavin but were not irradiated with UV-A (group 7).
For all irradiated groups, an 11-mm spot diameter was used for UV-A irradiation (365 nm) of the cornea (CCL-Vario Crosslinking; Peschke Meditrade GmbH, Zurich, Switzerland). The total energy was 5.4 J/cm2 for all groups. Frequency of riboflavin application was similar in all groups throughout the entire procedure: corneas were instilled with riboflavin during the soaking period every 3 minutes and during UV-A irradiation every 5 minutes. All eyes had their biomechanical analysis performed after a similar time of withdrawal from the refrigerator. In addition, in the 5 minutes preceding the biomechanical measurement, all corneas remained on balanced saline solution to standardize the hydration of all the samples. Details were summarized in Table 1.
All corneas were analyzed using stress-strain extensiometry as described previously.15,19 After removal of the corneoscleral button from the globe, two corneoscleral strips (5-mm width, full thickness) were prepared centrally in the horizontal axis from each eye. Four millimeters of the end of each strip were dedicated to fixation, leaving approximately 11 mm of central corneal strip length.
Tensile strength measurement was performed using a stress-strain extensometer (Z0.5; Zwick GmbH & Co., Ulm, Germany), calibrated with a distance accuracy of 2 mm and a tensile sensor with no more than 0.21% of measurement uncertainty between 0.25 and 50 N. The Z0.5 is a classic extensometer composed of a linear holder extension arm whose speed can be controlled and a Newton meter, which measures the real-time force in Newton exerted by the arm on the held specimen. The conversion from force to stress was calculated from the thickness and width of the specimen. The arm speed was 2 mm per minute in the conditioning cycles and the position was controlled at the point of load application during the test phase. The biomechanical characterization included elastic and viscoelastic testing up to a force of 2 N.
For the current analysis, we considered both the stress strain and the stress relaxation curve. Based on elastic modulus, the stiffening effect was calculated in each of the groups with respect to their own control group. The slope of the stress-strain curve corresponds to the tangent elastic modulus (Young's modulus) and was determined between 1.5% and 4.5% of strain. This range was selected because it showed a linear relation in the stress-strain diagram. Data analysis was performed using the Xpert II-Testing Software (Zwick GmbH & Co.).
Statistical analysis was performed using SPSS statistical software (version 24; IBM Corporation, Zurich, Switzerland) and the Microsoft Excel program (Excel 11 for Mac; Microsoft Corporation, Redmond, WA). Normal distribution was confirmed with both the Shapiro–Wilk and Kolmogorov–Smirnov tests. In the case of normal distribution and analysis of variance (ANOVA) test with statistical significance, the LSD post-hoc test to multiple comparisons was used to determine significant differences between groups. In the case of non-normal distributions, the non-parametric Kruskal–Wallis test was used to verify statistical significance before the final analysis by the Mann–Whitney test. For both the ANOVA and the Kruskal–Wallis tests, the confidence interval of 95% was used to determine significant differences between groups. Statistical significance was considered significant whenever the P value was .05 or less.
Both the ANOVA analysis and the Kruskal–Wallis test showed significant differences for stress (P < .001) and elastic modulus (P < .001), respectively. Table 2 shows the mean values with standard deviations and Table 3 shows the P values found between each condition tested.
Mean ± SD Values of Stress/Elastic Modulus and Stiffening Effect
Differences Between Groups
Stress After Relaxation Analysis
In the groups submitted to standard epi-off CXL, differences were found between controls (group 3) and 3 mW/cm2 for 30 minutes (group 1) and between controls and 9 mW/cm2 for 10 minutes (group 2). No difference was found between groups 1 and 2. Among the I-CXL groups, stress differences at the end of the relaxation curve were observed between 1.5 mW/cm2 for 60 minutes (group 4) and 9 mW/cm2 for 10 minutes (group 6) and between 1.5 mW/cm2 for 60 minutes (group 4) and their controls (group 7). Among all, group 4 reached the highest stress after relaxation.
Analysis of Elastic Modulus
Elastic modulus at the range of 1.5% and 4.5% of strain showed significant differences between 3 mW/cm2 for 30 minutes (group 1) and its control group (group 3). Group 1 also had the highest elastic modulus, with a stiffening effect of 149%. The stiffening effect of each specific group is shown in Table 1, whereas Figure 1 shows the median of the elastic modulus as a function of strain in all groups.
Median of elastic modulus as a function of strain in all groups. Left images compare standard epithelium-off corneal cross-linking protocols (S-CXL) with their control: Figures A (groups 2 vs 3) and C (groups 1 vs 3). Right images compare the experimental iontophoresis-assisted corneal cross-linking groups (I-CXL) with their control: Figures B (groups 6 vs 7), D (groups 5 vs 7), and E (groups 4 vs 7).
The observation of all corneas of the I-CXL groups did not show epithelial disruption macroscopically, either after the soaking phase of riboflavin or after UV-A irradiation.
Our results suggest that I-CXL with a de-accelerated prolonged irradiation of 1.5 mW/cm2 for 60 minutes resulted in an increased biomechanical stiffening when compared to the other I-CXL protocols. Nevertheless, this improved stiffening effect in I-CXL remained inferior to any of the epi-off S-CXL treated groups. Reported advantages provided by I-CXL include less pain and faster recovery, but clinical studies using I-CXL have shown that it is less effective in stabilizing keratoconus progression,13,20,21 which is consistent with our findings.
There is increasing evidence that the oxygenation of the corneal stroma is an essential and limiting factor for the effectiveness of biomechanical stiffening in CXL. First, tissue oxygenation decreases within seconds after the onset of UV-A light and needs minutes to replenish. Second, each layer of the cornea exhibits metabolism with distinct characteristics. It is known that the epithelium consumes 10 times more oxygen than the stroma, and the epithelium also acts as a barrier to oxygen penetration.22,23 In 2013, our group showed that oxygen is an essential element for the biomechanical effect of CXL.16,24 We performed CXL ex vivo in an atmosphere devoid of oxygen and found no measurable increase in biomechanical stiffness, whereas control corneas cross-linked under normal oxygen content showed the usual increase in stiffness.24
Epi-off CXL using the standard protocol is safe and effective for the prevention of keratoconus progression and postoperative ectasia.25 Theoretically, according to the Bunsen–Roscoe law, as long as the total energy dose remains the same, protocols with higher UV-A energy and shorter irradiation times should have a similar effect. However, reduced experimental and clinical efficacy of accelerated epi-off CXL protocols has been reported.15 Decreased oxygen availability in the corneal stroma has been shown to severely limit the formation of cross-linking.15,24 Interestingly, Webb et al.26 suggested that accelerated CXL lacks stiffening in the deeper regions of the cornea due to limited oxygen availability. Similarly, we have shown that thinner corneas show an increased stiffening effect due to higher oxygen availability.16
As demonstrated by Vinciguerra et al.27 in 2014, I-CXL may help to improve riboflavin diffusion into the stroma and thus increase biomechanical stiffening when compared to transepithelial CXL. Nevertheless, iontophoresis does not influence the speed of oxygen diffusion. We hypothesized that the intact epithelium could be responsible for the reduction in the efficacy of I-CXL technique for two related oxygen reasons: insufficient supply due to the mechanical barrier to diffusion and with the increased metabolic consumption. As a boundary cell layer of the cornea, the epithelium has lower diffusivity than the stroma and tight structure with high cell density and activity.28
Using penetration enhancers and extended soaking time of 60 minutes, an ex vivo study reported that the transepithelial approach had similar effect to S-CXL using the standard protocol, but the authors reported that such prolonged pretreatment induced a remarkable epithelial disruption.29 In our study, epithelial loss was not observed after 10 minutes of iontophoresis nor at the end of the UV-A irradiation. Still, in agreement with our data, the authors noted that transepithelial CXL following 15 minutes of pretreatment did not show more resistance than controls.29
To date, controversial results on I-CXL have been reported. As a possible way of evaluating the effectiveness of the procedure,30 the demarcation line has been shown to be shallower whenever accelerated irradiation times or intact epithelium protocols are used.30–32 Based on assessment by optical coherence tomography, confocal microscopy and clinical data of 80 eyes, Jouve et al.13 demonstrated that I-CXL was less efficient in halting the progression of keratoconus than S-CXL. The 2-year outcomes data showed that the demarcation line was superficially visible in only 35% of cases after I-CXL compared with 95% of cases after S-CXL.13 Moreover, the failure rate was 20% for I-CXL and 7.5% for S-CXL. Conversely, based on clinical data of 1-year follow-up from 40 eyes, Vinciguerra et al.33 suggested that I-CXL may be comparable to S-CXL in stabilization of ectasia progression. Both studies used I-CXL with accelerated irradiation of 9 mW/cm2 for 10 minutes.13,33
Although faster treatments are desired, our ex vivo data suggest that the biomechanical effect of I-CXL increases significantly when deaccelerating the standard protocol, using even lower irradiance levels and longer irradiation time settings (group 4), doubling the time of the classic standard protocol from 30 to 60 minutes. It may be worth mentioning that such an increase in stiffening effect might therefore be better biologically but potentially less useful for clinic flow and utility. Tensile strength measurements of the I-CXL groups could not demonstrate a significant increase in corneal stiffening when using lower irradiances of 3 mW/cm2 for 30 minutes (group 5) or 9 mW/cm2 for 10 minutes (group 6).
In an experimental model with rabbits, a subgroup of 8 eyes treated by I-CXL with accelerated irradiation of 10 mW/cm2 for 9 minutes was compared with the same number of eyes treated by S-CXL and standard protocol and with 10 untreated control eyes.34 In this study, Cassagne et al.34 found comparable results of stress at 10% strain and resistance against corneal collagenase between I-CXL and S-CXL groups. However, the comparison of groups was done directly between both groups (ie, without a specific control group for each I-CXL and S-CXL). From our standpoint, such a direct comparison may have been a bias in the analysis. Furthermore, in a different subgroup of eyes from the same study, the authors observed 45% less riboflavin concentration in corneas soaked by iontophoresis than by standard epi-off application. In the current study, we could show that a difference in stromal riboflavin concentration also leads to a difference in the biomechanical response. This observation reinforces the importance of different control groups for both S-CXL and I-CXL.
A recent morphological and histochemical report showed changes in the organization of collagen fibers in healthy and keratoconic corneas following accelerated S-CXL and I-CXL. Although such an approach cannot quantify corneal stiffness, some corneal structural differences were noticeable with the I-CXL protocol. Still, the arrangement and interweaving of fibers were much more similar to healthy corneas when using the S-CXL protocol.35 These findings are in agreement with ours, and place I-CXL as a potential alternative treatment in selected cases.
This study has some limitations. As an ex vivo study, our extensiometry findings may not be fully equivalent to the biomechanical response in vivo. However, the methodology employed is widely established. Moreover, a potential source of error for such studies is related to alteration of corneal hydration and thickness. Corneal thickness was not documented before and after soaking with riboflavin in our study, but all corneas were exposed to riboflavin in a similar and standardized manner. An additional limitation is that no direct measure of oxygen concentration in the corneal stroma was performed. Although desirable, implanting an optical fiber from an oxygen meter into the cornea would affect the corneal biomechanical evaluation. A further limitation concerns the sensitivity of the method. It is possible that subtle cellular and tissue alterations could be detected with a more sensitive biomechanical measurement technique. The recent introduction of Brillouin microscopy may further elucidate these mechanisms.36 To date, it is not known how much increase in corneal stiffness is needed to arrest the progression of corneal ectasias. Therefore, prospective in vivo studies are necessary to confirm the clinical validity of our findings, in both the short and long term.
The biomechanical effect of I-CXL increased significantly only when using a low irradiance and long irradiation time setting. In addition to the barrier effect on diffusion, the epithelium has a high rate of oxygen consumption, further restricting stromal oxygen availability in the I-CXL protocols. Even when the issue of riboflavin penetration is partially addressed in I-CXL, oxygen diffusion may remain as a limiting factor. To increase I-CXL efficiency, longer irradiation times (which are even slower than the standard protocol) might be needed. Still less effective than S-CXL, this modification might be useful in clinics when the risk for postoperative infection due to eye rubbing is judged higher than the reduced efficacy of the cross-links.
- Torres Netto EA, Al-Otaibi WM, Hafezi NL, et al. Prevalence of keratoconus in paediatric patients in Riyadh, Saudi Arabia [published online ahead of print January 3, 2018]. Br J Ophthalmol. doi:10.1136/bjophthalmol-2017-311391 [CrossRef].
- Godefrooij DA, Soeters N, Imhof SM, Wisse RP. Corneal cross-linking for pediatric keratoconus: long-term results. Cornea. 2016;35:954–958. doi:10.1097/ICO.0000000000000819 [CrossRef]
- Gokhale NS. Epidemiology of keratoconus. Indian J Ophthalmol. 2013;61:382–383. doi:10.4103/0301-4738.116054 [CrossRef]
- Grünauer-Kloevekorn C, Duncker GI. Keratoconus: epidemiology, risk factors and diagnosis [article in German]. Klin Monbl Augenheilkd. 2006;223:493–502. doi:10.1055/s-2005-859021 [CrossRef]
- Nielsen K, Hjortdal J, Aagaard Nohr E, Ehlers N. Incidence and prevalence of keratoconus in Denmark. Acta Ophthalmol Scand. 2007;85:890–892. doi:10.1111/j.1600-0420.2007.00981.x [CrossRef]
- Randleman JB, Woodward M, Lynn MJ, Stulting RD. Risk assessment for ectasia after corneal refractive surgery. Ophthalmology. 2008;115:37–50. doi:10.1016/j.ophtha.2007.03.073 [CrossRef]
- Randleman JB, Khandelwal SS, Hafezi F. Corneal cross-linking. Surv Ophthalmol. 2015;60:509–523. doi:10.1016/j.survophthal.2015.04.002 [CrossRef]
- Li J, Ji P, Lin X. Efficacy of corneal collagen cross-linking for treatment of keratoconus: a meta-analysis of randomized controlled trials. PLoS One. 2015;10:e0127079. doi:10.1371/journal.pone.0127079 [CrossRef]
- Hashemi H, Miraftab M, Seyedian MA, et al. Long-term results of an accelerated corneal cross-linking protocol 18mW/cm2 for the treatment of progressive keratoconus. Am J Ophthalmol. 2015;160:1164–1170. doi:10.1016/j.ajo.2015.08.027 [CrossRef]
- Steinwender G, Pertl L, El-Shabrawi Y, Ardjomand N. Complications from corneal cross-linking for keratoconus in pediatric patients. J Refract Surg. 2016;32:68–69. doi:10.3928/1081597X-20151210-03 [CrossRef]
- Lombardo M, Serrao S, Rosati M, Ducoli P, Lombardo G. Bio-mechanical changes in the human cornea after transepithelial corneal crosslinking using iontophoresis. J Cataract Refract Surg. 2014;40:1706–1715. doi:10.1016/j.jcrs.2014.04.024 [CrossRef]
- Bikbova G, Bikbov M. Standard corneal collagen crosslinking versus transepithelial iontophoresis-assisted corneal crosslinking, 24 months follow-up: randomized control trial. Acta Ophthalmol. 2016;94:e600–e606. doi:10.1111/aos.13032 [CrossRef]
- Jouve L, Borderie V, Sandali O, et al. Conventional and iontophoresis corneal cross-linking for keratoconus: efficacy and assessment by optical coherence tomography and confocal microscopy. Cornea. 2017;36:153–162. doi:10.1097/ICO.0000000000001062 [CrossRef]
- Gatzioufas Z, Raiskup F, O'Brart D, Spoerl E, Panos GD, Hafezi F. Transepithelial corneal cross-linking using an enhanced riboflavin solution. J Refract Surg. 2016;32:372–377. doi:10.3928/1081597X-20160428-02 [CrossRef]
- Hammer A, Richoz O, Mosquera S, Tabibian D, Hoogewoud F, Hafezi F. Corneal biomechanical properties at different corneal collagen cross-linking (CXL) irradiances. Invest Ophthalmol Vis Sci. 2014;55:2881–2884. doi:10.1167/iovs.13-13748 [CrossRef]
- Kling S, Richoz O, Hammer A, et al. Increased biomechanical efficacy of corneal cross-linking in thin corneas due to higher oxygen availability. J Refract Surg. 2015;31:840–846. doi:10.3928/1081597X-20151111-08 [CrossRef]
- Kling S, Hafezi F. Biomechanical stiffening: slow low-irradiance corneal crosslinking versus the standard Dresden protocol. J Cataract Refract Surg. 2017;43:975–979. doi:10.1016/j.jcrs.2017.04.041 [CrossRef]
- Vinciguerra P, Randleman JB, Romano V, et al. Transepithelial iontophoresis corneal collagen cross-linking for progressive keratoconus: initial clinical outcomes. J Refract Surg. 2014;30:746–753. doi:10.3928/1081597X-20141021-06 [CrossRef]
- Kling S, Spiru B, Hafezi F, Sekundo W. Biomechanical weakening of different re-treatment options after small incision lenticule extraction (SMILE). J Refract Surg. 2017;33:193–198. doi:10.3928/1081597X-20161221-01 [CrossRef]
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- Freeman RD. Oxygen consumption by the component layers of the cornea. J Physiol. 1972;225:15–32. doi:10.1113/jphysiol.1972.sp009927 [CrossRef]
- Kamaev P, Friedman MD, Sherr E, Muller D. Photochemical kinetics of corneal cross-linking with riboflavin. Invest Ophthalmol Vis Sci. 2012;53:2360–2367. doi:10.1167/iovs.11-9385 [CrossRef]
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- Webb JN, Su JP, Scarcelli G. Mechanical outcome of accelerated corneal crosslinking evaluated by Brillouin microscopy. J Cataract Refract Surg. 2017;43:1458–1463. doi:10.1016/j.jcrs.2017.07.037 [CrossRef]
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|Parameter||Group 1||Group 2||Group 3||Group 4||Group 5||Group 6||Group 7|
|Fluence (total) (mJ/cm2)||5.4||5.4||0||5.4||5.4||5.4||0|
|Soak time (minutes) & interval||30 (q3)||30 (q3)||30 (q3)||10 (iontophoresis)||10 (iontophoresis)||10 (iontophoresis)||10 (iontophoresis)|
|Treatment time (minutes)||30||10||–||60||30||10||–|
|Chromophore||0.1% riboflavin||0.1% riboflavin||0.1% riboflavin||0.1% riboflavin||0.1% riboflavin||0.1% riboflavin||0.1% riboflavin|
|Protocol abbreviation in article||S-CXL / group 1||S-CXL / group 2||S-CXL / group 3||I-CXL / group 4||I-CXL / group 5||I-CXL / group 6||I-CXL / group 6|
Mean ± SD Values of Stress/Elastic Modulus and Stiffening Effect
|Group||Protocol||UV-A Irradiation (Intensity – Time)||Stress (Pascal)||Elastic Modulus (Pascal)||Normalized Stiffening (%)|
|1||S-CXL||3 mW/cm2 – 30 min||3.63E+05 ± 3.91E+04||2.43E+06 ± 1.51E+06||149%|
|2||S-CXL||9 mW/cm2 – 10 min||3.54E+05 ± 5.03E+04||2.17E+06 ± 1.45E+06||133%|
|3||S-CXL (control)||–||3.31E+05 ± 3.09E+04||1.64E+06 ± 8.53E+05||100%|
|4||I-CXL||1.5 mW/cm2 – 60 min||3.84E+05 ± 2.33E+04||1.60E+06 ± 3.10E+05||112%|
|5||I-CXL||3 mW/cm2 – 30 min||3.70E+05 ± 2.44E+04||1.37E+06 ± 6.76E+05||96%|
|6||I-CXL||9 mW/cm2 – 10 min||3.65E+05 ± 2.41E+04||1.38E+06 ± 6.35E+05||96%|
|7||I-CXL (control)||–||3.67E+05 ± 2.35E+04||1.43E+06 ± 5.41E+05||100%|
Differences Between Groups
|Groups||Stress (P)||Elastic Modulus (P)|
|3 vs 2||.01a||.23|
|2 vs 1||.16||.43|
|7 vs 6||.87||.81|
|6 vs 5||.57||.67|
|5 vs 4||.11||.06|
|3 vs 1||.00a||.04a|
|7 vs 5||.69||.53|
|6 vs 4||.04a||.12|
|3 vs 7||.00a||.45|
|7 vs 4||.05a||.18|