institutional review board / ethics committee , and animal care and use committee approvals were obtained ( authorization no . the solution used contained 0.1% wt / vol riboflavin-5-phosphate and 15% ( wt / vol ) dextran t-500 ( iros , naples , italy ) , similar to those typically used in the standard cxl procedure described by the dresden protocol . human corneas , not suitable for transplant because of biological contamination of the carrier solution ( optisol gs ; bausch & lomb , rochester , ny ) , were provided by the spin - off consortium fast - linking srl and by the pellegrini hospital regional eye bank . the porcine corneas , harvested from a local slaughterhouse , were preserved with the same type of carrier solution and were tested within 24 hours postmortem . all the samples showed intact epithelium , a normal density of endothelial cells , and absence of epithelial or stromal opacity . before experimentation , each cornea was excised circularly 2 mm from the limbus , and the thickness ( d ) of the sample was measured with a 5-m resolution ultrasound pocket pachymeter ( quantel medical , clermont , france ) . the uv - a source used in this study ( cf  x linker modulated megaride , iros ) uses light - emitting diodes with a 370-nm wavelength and an intensity ( i ) varying from 1 to 10 mw / cm . the uv detector used was a lasermate - q laser ( coherent inc , santa clara , ca ) , set orthogonal to the radiation wave front . the detector has been previously calibrated with a known intensity , so that standard error of the mean was always within 0.01 mw / cm . the uv - a intensity values ( i0 ) were measured placing the microscope glass directly in contact with this device . the intensity of the beam emerging from the posterior surface of every microscope slide ( i0 ) when exposed to uv - a ( 3 mw / cm ) was previously measured ; only slides presenting clear and unscratched glass and i0 values of 2.527  0.01 mw / cm were used in this study . the specific absorbance value  of the riboflavin solution was then measured using a spectrophotometer ( perkinelmer lambda 25 uv / vis ; perkinelmer , waltham , ma ) ; the starting solution was diluted to 1:20 , to obtain a molarity of 2.6  10 the i0 values at the posterior surface of each corneal sample were initially measured , as described above . these values at time 0 ( t0 ) were used as a comparative control for the subsequent readings . the cornea , with a 2-mm ring of sclera ( approximate ) was then mounted on a modified franz - type diffusion cell (  9 mm , 5-ml receptor volume ; ses gmbh- analysesysteme , bechenheim , germany ) , with the endothelium facing the receptor compartment . this compartment was constituted with isotonic phosphate buffer at ph 7.4 and was maintained at 37  1c . when stipulated , epithelium was removed using a surgical scraper and epithelial melting by ethanol as already described.5 before each experiment , corneas were equilibrated with the use of 0.5-ml of balanced salt solution for 10 minutes , after which 1.0 ml of riboflavin solution was applied . after 30 minutes , cells were dismantled , the excess donor solution was washed away with balanced salt solution and the samples were set on the microscope slide to be exposed to uv - a radiation . the first group ( h1 , 6 samples ) was soaked with the riboflavin solution after the removal of the epithelium ( epi - off ) ; the second group ( h2 , 6 samples ) was soaked with intact epithelium ( epi - on ) . all the samples were then placed on glass slides and were irradiated for 30 minutes at 3 mw / cm . the intensity of uv - a light emerging from the posterior surface of each cornea ( i0 ) was measured every 5 minutes , with the last reading at 30 minutes . porcine corneas were divided into 3 groups of 8 samples each . the first group ( p1 ) was not soaked with the riboflavin solution after epithelium removal and was not exposed to uv - a rays ( baseline group ) . the second group ( p2 ) was soaked with the riboflavin solution , in epi - on conditions , and received a 10-minute uv - a exposure at 1.5 mw / cm ( negative control group ) . the third group ( p3 ) was soaked with the riboflavin solution , in epi - off conditions , and received the same 10-minute uv - a exposure at 1.5 mw / cm . after irradiation , 2 strips of each sample underwent static stress test measurements as described below . a previously described experimental model was used for stress - strain measurement.5 after cxl , 2 corneal strips of 5.0  0.2 mm width , 11.0  0.4 mm length ( comprising 1 mm of sclera at both ends ) , and 890  20 m thickness were cut from each sample . static stress - strain measurements were performed using an electronic dynamometer ( ametek lloyd instruments lrx plus , st . young modulus was calculated at 2% , 4% , 6% , and 8% strain . the method used and the force parameters were the same as previously described.4 statistical analyses were conducted using graph - pad prism ( graphpad software inc , san diego , ca ) . the significance of differences between groups was determined by one - way analyses of variance followed by bonferroni post hoc tests for multiple comparisons . the solution used contained 0.1% wt / vol riboflavin-5-phosphate and 15% ( wt / vol ) dextran t-500 ( iros , naples , italy ) , similar to those typically used in the standard cxl procedure described by the dresden protocol . human corneas , not suitable for transplant because of biological contamination of the carrier solution ( optisol gs ; bausch & lomb , rochester , ny ) , were provided by the spin - off consortium fast - linking srl and by the pellegrini hospital regional eye bank . the porcine corneas , harvested from a local slaughterhouse , were preserved with the same type of carrier solution and were tested within 24 hours postmortem . all the samples showed intact epithelium , a normal density of endothelial cells , and absence of epithelial or stromal opacity . before experimentation , each cornea was excised circularly 2 mm from the limbus , and the thickness ( d ) of the sample was measured with a 5-m resolution ultrasound pocket pachymeter ( quantel medical , clermont , france ) . the uv - a source used in this study ( cf  x linker modulated megaride , iros ) uses light - emitting diodes with a 370-nm wavelength and an intensity ( i ) varying from 1 to 10 mw / cm . the uv detector used was a lasermate - q laser ( coherent inc , santa clara , ca ) , set orthogonal to the radiation wave front . the detector has been previously calibrated with a known intensity , so that standard error of the mean was always within 0.01 mw / cm . the uv - a intensity values ( i0 ) were measured placing the microscope glass directly in contact with this device . the intensity of the beam emerging from the posterior surface of every microscope slide ( i0 ) when exposed to uv - a ( 3 mw / cm ) was previously measured ; only slides presenting clear and unscratched glass and i0 values of 2.527  0.01 mw / cm were used in this study . the specific absorbance value  of the riboflavin solution was then measured using a spectrophotometer ( perkinelmer lambda 25 uv / vis ; perkinelmer , waltham , ma ) ; the starting solution was diluted to 1:20 , to obtain a molarity of 2.6  10 mol / l . the i0 values at the posterior surface of each corneal sample were initially measured , as described above . these values at time 0 ( t0 ) were used as a comparative control for the subsequent readings . the cornea , with a 2-mm ring of sclera ( approximate ) was then mounted on a modified franz - type diffusion cell (  9 mm , 5-ml receptor volume ; ses gmbh- analysesysteme , bechenheim , germany ) , with the endothelium facing the receptor compartment . this compartment was constituted with isotonic phosphate buffer at ph 7.4 and was maintained at 37  1c . when stipulated , epithelium was removed using a surgical scraper and epithelial melting by ethanol as already described.5 before each experiment , corneas were equilibrated with the use of 0.5-ml of balanced salt solution for 10 minutes , after which 1.0 ml of riboflavin solution was applied . after 30 minutes , cells were dismantled , the excess donor solution was washed away with balanced salt solution and the samples were set on the microscope slide to be exposed to uv - a radiation . the first group ( h1 , 6 samples ) was soaked with the riboflavin solution after the removal of the epithelium ( epi - off ) ; the second group ( h2 , 6 samples ) was soaked with intact epithelium ( epi - on ) . all the samples were then placed on glass slides and were irradiated for 30 minutes at 3 mw / cm . the intensity of uv - a light emerging from the posterior surface of each cornea ( i0 ) was measured every 5 minutes , with the last reading at 30 minutes . porcine corneas were divided into 3 groups of 8 samples each . the first group ( p1 ) was not soaked with the riboflavin solution after epithelium removal and was not exposed to uv - a rays ( baseline group ) . the second group ( p2 ) was soaked with the riboflavin solution , in epi - on conditions , and received a 10-minute uv - a exposure at 1.5 mw / cm ( negative control group ) . the third group ( p3 ) was soaked with the riboflavin solution , in epi - off conditions , and received the same 10-minute uv - a exposure at 1.5 mw / cm . after irradiation , 2 strips of each sample underwent static stress test measurements as described below . a previously described experimental model was used for stress - strain measurement.5 after cxl , 2 corneal strips of 5.0  0.2 mm width , 11.0  0.4 mm length ( comprising 1 mm of sclera at both ends ) , and 890  20 m thickness were cut from each sample . static stress - strain measurements were performed using an electronic dynamometer ( ametek lloyd instruments lrx plus , st . statistical analyses were conducted using graph - pad prism ( graphpad software inc , san diego , ca ) . the significance of differences between groups was determined by one - way analyses of variance followed by bonferroni post hoc tests for multiple comparisons . differences with p transcorneal uv - a transmission data for the human cadaver eyes are shown in table 1 for the cases of cross - linking with epithelium removed . after riboflavin presoaking for 30 minutes , those corneas with epithelium removed transmitted only 24% of the uv - a light through the cornea ( second column ) , but after 5 minutes of uv - a irradiation fully 75% of the uv - a light was transmitted ( third column ) ; both these were statistically significantly different from baseline ( p < 0.05 ) . in the interval from 10 to 30 minutes after commencing irradiation , 100% uv - a transmission was observed , within the limits of statistical variation . there was no statistically significant difference between transmission from 10 to 30 minutes and baseline ( p > 0.19 in all cases ) . time - related ( minutes ) uv - a intensity readings ( mw / cm ) ( i0 ) in group h1 ( epi - off , i = 3 mw / cm ) in the group where the epithelium was left intact , there was no statistically significant change in uv - a transmission after riboflavin application for 30 minutes or after uv - a exposure throughout the entire 30-minute period ( p > 0.19 in all cases , data not shown ) . table 2 shows the average values of young modulus measured at various levels of strain for the 3 test groups . group p3 demonstrated a statistically significantly higher young modulus at 6% and 8% strain when compared with groups p1 and p2 ( p < 0.05 ) . although the average young modulus seemed slightly higher in group p2 than p1 at each level of strain , the differences were not statistically significant ( p > 0.05 ) . measured young modulus ( e ) at 2% , 4% , 6% , and 8% strain in porcine corneas spectroscopic results obtained in this study show that the uv - a intensities emerging from the posterior surface of the cornea during standard cxl procedures are not constant over time , as suggested by the lambert  beer law . the average intensity of uv - a radiation emerging from the posterior corneal surface in the epithelium off group , calculated immediately after topical riboflavin application , was 0.27  0.041 mw / cm , a value considered safe as it is within the 0.35-mw / cm limit to avoid endothelial damage.4 however , by 10 minutes the apparent uv - a exposure exceeded this safety limit in all tested corneas . these results are consistent with those reported previously ; several authors have noted that during cross - linking , the endothelial uv light dose would exceed safety limits.6,7 this is why the current standard cxl protocol mandates the presence of a riboflavin solution film on the cornea during the cxl procedure , to shield the endothelium from an excess of uv - a radiation . there is a negative consequence to this ; by reducing the penetration of uv - a , the precorneal film allows only the cross - linking of the superficial stroma , thus producing the so - called  demarcation line  effect.810 avoiding the use of the precorneal riboflavin film and lowering uv - a intensities should provide a deeper biomechanical effect while maintaining overall safety . in addition , the results obtained in this study suggest that the riboflavin has been oxidized long before the standard uv - a irradiation period is complete . this implies that the shielding fades long before the standard 30-minute uv - a exposure ends . in the present study , the riboflavin shielding effect completely disappeared in 10 minutes . as such , a shorter irradiation time may be feasible , which would be likely to produce a similar effect to the dresden protocol with lower potential uv - a exposure to the endothelium . the differences between the groups in the second experiment were most notable at 6% and 8% strain , which is consistent with previous observations in the literature.5,11 wollensak et al2 reported that the young modulus of cross - linked porcine corneas increased by a factor of 1.82.0 with 6% and 8% of strain . our results are very similar , with a corresponding increase of 1.7 and 2.2 times ( table 2 ) at those levels of strain . we encountered some variation among the porcine samples in measured values of stress at each strain level . we attribute these variations in young modulus to the variations in porcine corneal thicknesses and to the fact that only the outer stromal layers were cross - linked.5 however , the sds of our measurements were lower than those reported in previous studies.5,11 the dresden protocol for cross - linking has been demonstrated to be effective in stabilizing keratoconus.15 it requires a precorneal riboflavin film and 30 minutes of uv - a exposure , and it hardens the outer portion of the cornea . higher uv - a intensity and short - duration treatment produce an even more superficial effect and have the potential to exceed published safety limits to endothelial irradiation.3,6,12 experimental cross - linking in the present study , using lower uv - a intensity and not adding riboflavin during uv irradiation , provided corneal stiffening similar to that achieved with the dresden protocol . these effects were achieved with a 10-minute exposure time , stopping the treatment once the riboflavin has already oxidized . this suggests that a quicker , safer , and more reproducible cross - linking protocol may be viable . the experimental data reported here , along with additional experimental results , are being used to develop a mathematical model describing the rate of photolysis of riboflavin by unit of volume ( rate equation ) . it is hoped that such a model may allow for a uv - a energy optimization , further reducing unnecessary uv - a exposure . that material will be the subject of a separate article . results in the clinical study demonstrate that stabilization of corneal and refractive parameters can be achieved with the shorter , lower energy protocol.13 the results of 2 laboratory experiments , reported here , indicate that the lambert  beer law must be modified in cross - linking to account for the reduction in uv filtration over time . they also suggest that it may be possible to produce a shorter duration , lower energy , time - variable uv - a intensity cross - linking treatment that will accomplish the goal of adequately increasing corneal stiffness while lowering the risk of endothelial cell damage . such a treatment would not require a precorneal riboflavin film during uv - a exposure . a new cxl protocol based on these findings may yield a safer , equally effective , more reproducible , and more rapid cross - linking treatment . it must be kept in mind that the results presented here are based on in vitro experiments and can not be generalized to in vivo experiments . preliminary results have been obtained with the procedure in a clinical setting , but only cases of mild keratoconus were treated.13 additional clinical research is required to determine if the procedure studied here can be generalized to broader clinical application .
purpose : to determine the cross - linking effect of a riboflavin ultraviolet - a ( uv - a ) corneal cross - linking treatment that is both shorter and has lower energy than the dresden protocol.methods:in a first experiment , 12 human corneas were presoaked with riboflavin and then irradiated with uv - a at 3 mw / cm2 after clearing the surface of riboflavin , with no added riboflavin during irradiation . 
 percent uv - a transmission through the corneas was measured at intervals up to 30 minutes . a second experiment involved 24 porcine corneas . 
 eight were de - epithelialized , presoaked in riboflavin for 30 minutes , and irradiated at 1.5 mw / cm2 for 10 minutes . 
 an additional 8 were riboflavin treated and similarly irradiated , but with epithelium intact and a final 8 corneas were not treated . 
 young modulus was measured in all 24 corneas at the end of the experiment.results:the first experiment showed essentially complete riboflavin oxidation after only 10 minutes . 
 based on these results , a shortened uv - a exposure cross - linking experiment was designed using a reduced uv - a fluence of 1.5 mw / cm2 , an endothelial exposure within safety limits in humans . with this protocol 
 young modulus was the same in the irradiated porcine corneas but with epithelium intact as in the untreated corneas . 
 in contrast , young modulus increased by a factor of 1.99 in the uv - a cross - linked corneas at 1.5 mw / cm2 for 10 minutes with the epithelium removed.conclusions:a shorter , lower energy protocol than the dresden protocol seems to provide a significant increase in young modulus , similar to published results with higher energy , longer exposure protocols .