**Purpose.**:
Ultraviolet (UV) corneal cross-linking is an accepted method for treating corneal ecstatic disorders. The authors evaluated whether a rapid treatment protocol (higher intensity and shorter irradiation time) could achieve the same increase in corneal stiffness as the currently used standard protocol.

**Methods.**:
Stress–strain measurements were performed on porcine corneal strips. The corneas (*n* = 72) were cut into three strips, each randomly receiving a different treatment: rapid (10 mW/cm^{2}, 9 minutes), standard (3 mW/cm^{2}, 30 minutes), or no (control, 0 mW/cm^{2}) irradiation. After irradiation, the Young's modulus of each strip was determined. The results of the stress–strain measurements were analyzed statistically.

**Results.**:
Statistical analysis showed that, after irradiation, the median value of Young's modulus from both active treatment groups (rapid, 3.83 N/mm^{2}; standard, 3.88 N/mm^{2}) was significantly higher (*P* < 0.05) than that of the control group (2.91 N/mm^{2}). Treatment increased Young's modulus by a factor of 1.3. However, there was no significant difference (*P* = 0.43) between the rapid and standard groups in the median of Young's modulus.

**Conclusions.**:
Rapid UV cross-linking treatment can be regarded as equivalent to the standard procedure in terms of increase in corneal stiffness. The new rapid protocol shortens the treatment duration by more than two thirds, from 30 to 9 minutes. The safety of the higher intensities must be addressed in further clinical studies.

^{ 1 –7 }Since its first introduction by Seiler and Spoerl

^{ 8 }in 1997, a standardized protocol has been developed

^{ 9 }: An abrasion of the corneal epithelium is performed, followed by riboflavin application. During the treatment phase, a recommended illumination intensity of 3 mW/cm

^{2}is applied to a 9-mm zone in the cornea for 30 minutes. This intensity corresponds to a total energy dose of 3.4 J or a radiant exposure of 5.4 J/cm

^{2}. The photochemical process that induces the additional cross-links between the corneal fibers is thereby dependent on the applied radiant exposure of UV light.

^{ 8 –10 }

^{2}and 30 minutes) and a rapid cross-linking procedure that uses higher intensities and shorter treatment times. The aim of this work was therefore to experimentally investigate the equivalence of the biomechanical stability of porcine corneas by increasing the intensity to 10 mW/cm

^{2}and simultaneously decreasing the treatment time to 9 minutes (i.e., to less than one third), achieving the same radiant exposure of 5.4 J/cm

^{2}.

^{2}, 30 minutes), the rapid group was treated with the new parameters using higher irradiation intensity and shorter times (10 mW/cm

^{2}, 9 minutes), and the control group was not irradiated with UV light at all, but still underwent the same procedure. The experiment involved three steps: sample preparation, sample treatment with UV irradiation, and stress–strain measurements.

_{2}-riboflavin-5-phosphate 0.5% (G. Streuli & Co AG, Uznach, Switzerland) and four parts 20% dextran T-500 (Carl Roth GmbH & Co. KG, Karlsruhe, Germany). The samples were kept cool overnight in 0.1% riboflavin solution, to ensure a homogenous distribution of riboflavin.

^{2}. Table 1 shows the treatment parameters of the standard and rapid cross-linking treatment.

Treatment Method | Intensity (mW/cm^{2}) | Treatment Time (min) | Dose (J/cm^{2}) |
---|---|---|---|

Standard | 3 | 30 | 5.4 |

Rapid | 10 | 9 | 5.4 |

^{2}, respectively. For the treatment, the UV lamp was placed over the corneal strips, according to the manufacturer's instructions, and the maximum internal aperture of 9 mm ensured full irradiation of the sample area.

^{2}for 9 minutes and then clamped for biomechanical evaluation. The stress–strain measurement was performed on this sample and next on the control group (∼20 minutes). Finally, after 30 minutes of irradiation time, the standard group sample, which had been irradiated with 3 mW/cm

^{2}, was measured.

*a*and

*b*are the fitting parameters. The stiffness (Young's modulus),

*E*, is the first derivative of this function: For the following statistical analysis, Young's modulus was consistently evaluated at 4% strain.

*t*-test): where

*z*is the quantile of the normal distribution, σ

_{SD}is the standard deviation, and

*L*is the maximum allowed difference between the means of the two groups. Our calculation was based on an α = 0.05 level of significance and a statistical power of 1 − β = 0.9. The standard deviation, σ

_{SD}= 31%, was taken from an older study of corneal cross-linking on porcine eyes for Young's modulus at 4% strain.

^{ 11 }Because of the large variability found in biological tissues, a difference of

*L*= 20% was chosen, thereby resulting in a minimum of

*n*= 52 samples per group.

^{ 12,13 }The margin of clinical equivalence, Δ, was set at 1.5 times the standard deviation obtained from the published literature,

^{ 11 }as no other equivalence value has yet been established. The equivalence parameter for Young's modulus was thus defined as Δ = 0.66 N/mm

^{2}.

^{2}or where a procedural error systematically occurred (e.g., slip of the probe in the probe holder). Thus, only 60 of the 72 sample triplets were used for data analysis.

_{untreated}= 2.91 N/mm

^{2}is lower than the medians of the treated groups, which are M

_{rapid}= 3.83 N/mm

^{2}for the rapid and M

_{standard}= 3.88 N/mm

^{2}for the standard group. Consequently, the rapid group's median increased by a factor of 1.32, and the standard group's median rose by a factor of 1.33, compared with that of the untreated group. Figure 4 shows the average increase in Young's modulus of both treated groups compared with that of the untreated control. The medians of the average increase and the interquartile ranges are of comparable size.

*P*< 0.05, Friedman test). In contrast, no statistical significance was found when the two treatment groups were compared (

*P*= 0.43, Friedmann test). The test for equivalence of the difference between the two treatment methods showed also that the methods are equivalent in terms of an increase in stiffness. The average difference in Young's modulus of the two methods is 0.16 ± 1.70 N/mm

^{2}, with a 95% confidence interval of (−0.28 to 0.60 N/mm

^{2}). Thus, the lower bound of the confidence interval is larger than the negative of the equivalence parameter (−0.28 N/mm

^{2}> −Δ = −0.66 N/mm

^{2}) and the upper boundary of the confidence interval is smaller than the equivalence parameter (0.60 N/mm

^{2}< Δ = 0.66 N/mm

^{2}). Therefore, the performed study shows, in a statistically significant manner, the equivalence of both the rapid and standard UV corneal cross-linking procedure.

^{2}and three times shorter illumination time of 9 minutes, demonstrated an increase in Young's modulus by a factor of 1.3. This increase was statistically equivalent to that in the standard treatment group, having an intensity of 3 mW/cm

^{2}and a required illumination time of 30 minutes.

^{ 11 }who measured a 1.8× increase in stability.

^{ 11 }This difference can be attributed to the experimental procedure. The measured stress in the material-testing machine is highly dependent on experimental conditions, such as the load on the fixation clamp, the prestress force of the machine, and the condition of the tissue samples. It should be noted that, in our method, we used tissue samples that had much narrower widths than those used in the work published by Wollensak et al., and this may directly affect the total energy absorbed by the tissue samples. Other possible explanations for the differences between the literature data and the increase in stability reported here may be due to the nonlinear stress–strain behavior of the cornea.

^{ 14 }used a supersonic shear-imaging technique to measure corneal stiffness and showed an increase by a factor of 4.6. He et al.

^{ 15 }obtained a factor of 1.04 by using a quantitative ultrasound method. Kling et al.

^{ 16 }found an average 1.6× increase in Young's modulus in porcine eyes by comparing the corneal geometry changes when the intraocular pressure was changed.

^{2}instead of Δ = 0.66 N/mm

^{2}for Young's modulus, might lead to the conclusion that the rapid and the standard group differ from each other. However, to derive these possible differences statistically, a substantially higher number of eyes is needed. In addition, it is questionable whether the experimental setup used in this study would be capable of detecting these further differences because the handling of the tissue sample also causes large standard deviations.

^{ 17 }that human and porcine corneas show similar behavior in terms of change in biomechanical stability due to cross-linking treatment. To show a statistically significant equivalence in human cornea, more than 50 human corneas would be needed, which seems unjustified in ethical terms.

^{ 10 }the effect of a photochemical or photobiological reaction is directly proportional to the total irradiation dose, irrespective of the time span over which the dose is administered. This law may be valid within a certain dose range for pure photochemical reactions; however, the response of cells and tissue to electromagnetic radiation is more complex. Thus, a linear dose–time relationship is less likely. Currently, only very few studies analyze different tissues within a range in which this law can be applied. Moreover, the few published studies show diverging results. On the one hand, they support the direct application of the Bunsen-Roscoe law but, on the other hand, they also show that biological tissues possess a protective mechanism that can be damaged by electromagnetic radiation when the threshold intensity is exceeded for a prolonged period.

^{ 18 }

^{2}. We assume that the possible protective mechanism of the tissue depends not only on irradiation time, but also on the rate of radical formation, and thus that the period during which the tissue is protected is dependent on the radiation intensity. At lower irradiation intensities, the tissue is protected for a longer time than at higher intensities. The results of this study show that, at the intensity and time regimen used, the possible protective mechanism had an equal influence on both treatment methods as both study groups showed similar increases in stiffness. It may even be possible to further increase the intensity to reach treatment times below 9 minutes.

^{2}and reduced the illumination time from 30 to 9 minutes, thus ensuring an equal energy dose of 5.4 J/cm

^{2}for both cases. Therefore, the expected photoinduced chemical effects on the endothelial cells are assumed to be the same in the rapid and the standard procedures. Since the endothelial cells are not damaged during the standard procedure, no damage is expected to arise as a result of the rapid procedure. This is currently under investigation by our group in human corneas.

^{ 8 }