corneal collagen cross - linking ( cxl ) is a low - invasive treatment aimed to improve biomechanical stability in eyes with keratectasia [ 13 ] . a photodynamic reaction induced by photosensitizing riboflavin and ultraviolet a ( uva ) light causes an increase of the number of intra- and interfibrillar covalent bonds and the corneal collagen resistance against enzymatic degradation [ 46 ] . increased stromal biomechanical strength and lamellar compaction lead to stabilized corneal shape and better corneal symmetry , potentially causing an improvement in visual function [ 2 , 79 ] .   in cxl , riboflavin has a dual function acting both as a photosensitizer inducing the physical collagen cross - linking and as an absorber of the uva irradiation , preventing damage to deeper ocular structures [ 10 , 11 ] . proper corneal stromal saturation with riboflavin is therefore essential in cxl , and without its presence the uva radiation may cause the collagen fibers to degrade rather than to facilitate cross - linking .   the  standard cxl protocol described by wollensak and colleagues includes removal of the corneal epithelium in a diameter of 9  mm , followed by saturation of the corneal stroma using 0.1% isotonic riboflavin solution in 20% dextran . this procedure is proved to be effective in increasing corneal stiffness , stabilization of keratoconus , and in some cases in improving the refractive and topographic features [ 14 , 15 ] . even so , the epithelial removal may lead to serious complications that include infection [ 16 , 17 ] , stromal haze , and corneal melting   in addition to severe pain and decrease in vision occurring during the first days after the treatment . to avoid such complications , boxer wachler et al . suggested a modification of the technique by keeping the epithelium intact ( epithelium - on or transepithelial cxl ) . however , finding appropriate means of increasing corneal epithelial permeability prior to riboflavin application was warranted as riboflavin has a molecular weight of 338  da , whereas the corneal epithelium is impermeable to compounds with a molecular weight greater than 100  da . accordingly , various approaches have been tried clinically and in the laboratory to enhance the epithelial permeability before the riboflavin application . chemical enhancers such as benzalkonium chloride ( bac ) , ethylenediaminetetraacetic acid ( edta ) , gentamycin , tetracaine , and 20% ethanol [ 2225 ] were used , as well as partial grid - like pattern deepithelialization , excimer laser superficial epithelial removal , and the replacement of the isotonic by hypotonic riboflavin solution [ 24 , 27 ] . the results varied between the studies , but the majority of the aforementioned methods lead to increased epithelial permeability for riboflavin .    in the current study , a multifactorial approach was utilized to enhance the riboflavin penetration by employing : ( 1 ) bac - containing local medication ; ( 2 ) hypotonic riboflavin solution without dextran ; ( 3 ) increased riboflavin solution concentration ; ( 4 ) mechanical disruption of the superficial epithelium ( microabrasions ) ; ( 5 ) prolongation of the riboflavin - induction time until objective verification of the stromal saturation is confirmed . by such an approach , this nonrandomized retrospective study aimed to evaluate the efficacy and safety of the epithelium - on cxl in treatment of progressive keratoconus . in this retrospective , interventional case series , we reviewed the medical records of all patients with advanced progressive keratoconus who had 12-month observation time after the epithelium - on cxl treatment using our multifactorial approach . norway , troms , norway , between september 15 , 2009 and september 15 , 2010 . this study was approved by the regional ethics committee and adhered to the official ethical regulations for clinical research and the tenets of the declaration of helsinki . inclusion criteria included ( 1 ) documented progression of keratoconus during the last 12 months before treatment ( increase of astigmatism or myopia by 1.00 d or increase in average simk by 1.50 d ) ; ( 2 ) minimum corneal thickness of no less than 400  m at the thinnest point measured by ultrasound pachymetry ; ( 3 ) age ranging from 18 to 45 years ; ( 4 ) amsler - krumeich keratoconus classification stage ii to iii . exclusion criteria were : ( 1 ) history of herpes virus keratitis ; ( 2 ) severe dry eye ; ( 3 ) concurrent corneal infections ; ( 4 ) previous ocular surgery ; ( 5 ) hard contact lens wear 4 weeks before the baseline examination . pre- and postoperative assessments consisted of slit lamp biomicroscopy , scheimpflug - based corneal topo-/tomography ( precisio , ivis technology , taranto , italy ) , placido disk - based topography and wavefront aberrometry ( opd - scan ii , nidek . aichi , japan ) , uncorrected ( udva ) and corrected ( cdva ) distance visual acuities ( nidek rt 2100 system , nidek co. ltd . , aichi , japan ) , ultrasound pachymetry ( cornea - gage plus , sonogage inc . , cleveland , ohio ) , tonometry ( icare tonometer , revenio group corporation , helsinki , finland ) , and patients subjective evaluation of postoperative pain . the patients were examined at 1 , 3 , 6 , and 12 months postoperative . to reduce the risk for uv exposure of retroiridal eye structures , miosis was induced by applying two drops of pilocarpine 2% ( pilokarpin , ophtha as , norway ) . it was followed by the application of two drops of local anesthetic proparacaine 0.5% ( alcaine , alcon norway as ) , two drops of local antibiotic gentamycin 0.3% ( garamycin , schering - plough as , norway ) followed by proparacaine again , one drop every minute for five minutes . all the drops were preserved by bac ( 0.001% for pilokarpin , 0.005% for garamycin and 0.01% for alcaine ) , aiming to increase the epithelial permeability by chemically disrupting the tight junction proteins . a round merocel sponge ( medtronic , inc . , minneapolis , mn ) of 5  mm in diameter was inserted into the conjunctival sac to provide a depot of riboflavin , and to produce microabrasions of the superficial epithelial layers caused by friction upon patient 's blinking . thereafter , two drops of proparacaine and two drops of 0.5% aqueous riboflavin solution without dextran ( vitamin b2 ; streuli , uznach , switzerland ) were applied alternating every 30 seconds , until the riboflavin saturation was verified by the slit - lamp inspection of the cornea and by the determination of the presence of riboflavin flare in the anterior chamber ( figure 1 ) . under the same examination the staining of the epithelial microabrasions the initial slit - lamp saturation evaluation was performed 25 minutes after the first application of riboflavin and repeated every five minutes until the saturation was confirmed . during the premedication and riboflavin induction time the patient was instructed to blink normally between eye drop instillation and to remain in a comfortable sitting position . the merocel sponge was then removed and corneal thickness measured with ultrasound pachymetry , at which point the patient was placed in supine position . irrigation with isotonic balanced salt solution ( bss ) was performed before the uva irradiation in order to avoid the shielding effect of riboflavin covering the epithelium . an eyelid speculum was then inserted , and a ring - shaped merocel shield k20 - 5021 ( katena products , inc . denville , nj ) was applied to protect the limbal region and its stem cells from uva radiation . the cornea was subjected to uva radiation for 30 minutes with a wavelength of 365  nm at a working distance of 5  cm . the uv - x lamp ( iroc ag , zrich , switzerland ) provided an irradiance of 3  mw / cm within a circular diameter of 9  mm . during the irradiation , bss was applied every three minutes , and proparacaine drops were added as needed . after the uva irradiation , two drops of atropine 1% ( atropin minims , chauvin , england ) and 2 drops of gentamycin were applied . instructions were given to apply a mixture of 0.1% dexamethasone and 0.5% chloromycetin ( spersadex med kloramfenikol , novartis , norway ) eye drops four times daily for seven days , as well as to use artificial tears as needed . all visual acuity values were recorded as snellen values , converted to logmar for statistical analyses and then changed back to snellen values for presentation purposes . pre- and postoperative topography was analyzed using precisio 's irregularity index ( iri ) as well as by measuring the central 5  mm using opd indices . statistical analysis was performed to compare the post - operative data with the preoperative data using the paired t - test with ibm spss statistics v19.0 ( ibm , armonk , ny ) . the mean age of the patients was 32  10 years ( range , 1552 years ) . figures 2 , 3 , 4 , and 5 and table 1 show the visual acuity and refraction measurements pre- and postoperatively . the udva and cdva improved significantly ( p < 0.05 ) . at 12-month followup , none of the eyes lost lines of cdva , while 27.4% of the eyes gained 2 or more lines and the safety index was 1.14 . at the same time point , mean spherical equivalent refraction decreased by 0.74 d ( less myopic , p = 0.05 ) , while mean cylinder decreased by 1.15 d ( p = 0.00 ) . 
 table 2 shows the postoperative changes of topography and aberrometry . data from the precisio showed reduction in posterior elevation ( p = 0.01 ) , irregularity index ( p = 0.01 ) , and asymmetry ( p = 0.01 ) . the k - value from opd did not significantly alter regarding mean - k , while the max - k showed a significant decrease ( p = 0.02 ) at the location of the cone . figure 6 shows an example of the topographic changes in one of the treated eyes . aberrometry data showed a reduction in odd - order- s 3,5,7 ( p = 0.04 ) and total higher - order - aberrations ( p = 0.05 ) . precisio - measured pachymetry in table 1 shows decrease in thickness at 1-month followup ( p = 0.00 ) and thereafter a gradual increase to preoperative level at 12 months after the treatment ( p = 0.15 ) . patients reported moderate to severe postoperative pain during the first 412 hours , peeking at 46 hours after surgery . discreet superficial epithelial layer damage could be observed on slit - lamp examination upon the verification of riboflavin saturation and after the cxl treatment . previous studies report conflicting results on the effects of epithelium - on cxl . while pinelli and colleagues reported no significant difference in the analyzed parameters between epithelium - on and standard cxl , wollensak and iomdina found that the corneal biomechanical stiffening after epithelium - on cxl was about one - fifth compared to the epithelium - off cxl in an animal model . other clinical and laboratory studies have reported weaker or no effect of cxl using the epithelium - on method [ 22 , 23 , 25 , 2931 ] . collectively , the studies suggested that the significantly weaker biomechanical effect of epithelium - on cxl was due to the insufficient and inhomogeneous transepithelial riboflavin diffusion into the corneal stroma . however , a limitation of most of the studies that procured the low cross - linking effect or low stromal saturation of riboflavin with the epithelium - on cxl includes the use of the standard  or only slightly modified  wollensak / seiler protocol on nondeepithelialized eyes . moreover , the authors did not attempt to enhance the riboflavin penetration , effectively only showing that the epithelium - on cxl does not work with the standard epithelium - off protocol . intriguingly , epithelial permeability can be enhanced by application of several tensioactive substances including bac and gentamicin at concentrations normally used in industrial preparations . such pharmacological enhancements , which are commonly used in epithelium - on cxl [ 20 , 33 ] , were included in the current protocol . on the basis of previous studies reporting increased epithelial riboflavin permeability using hypotonic solution compared to isotonic solution [ 24 , 27 , 34 ] , hypotonic solution was applied in the current protocol . furthermore , we avoided the use of riboflavin solution with dextran due to its high viscosity , which inhibits the penetration through the epithelium . hypotonic riboflavin was originally used with epithelium - off cxl protocol to induce significant edema in corneas thinner than 400  m . however , the swelling of the corneas with intact epithelium seems to be of a considerably lower degree . in the present study , only around 10 microns of swelling were recorded in a subgroup of 29 eyes , presenting respective mean pachymetry of 450.50  42.90 and 471.65  41.43  m before and after the corneal saturation with the 0.5% hypotonic riboflavin solution . even though the clinical safety of cxl with hypotonic riboflavin solution has been documented , issues of the corneal endothelial cell toxicity were considered because of the decreased uv - protective effect with hypotonic riboflavin due to the decrease of uv absorption coefficient from 53  cm for 0.1% isotonic riboflavin solution to 42  cm for 0.1% hypotonic riboflavin solution . to compensate for this , increasing the concentration of riboflavin in the hypoosmolar solution may enhance uva absorption   and hence decrease the uv - radiation at the endothelial level . the current study addresses the endothelial safety as hypotonic riboflavin concentration of 0.5% was applied . a secondary benefit of the increased concentration is the presumably increased availability of the riboflavin molecules to penetrate the epithelium and saturate the stroma . in a subgroup analysis of 21 eyes performing pre- and postoperative specular microscopy using konan cellcheck xl ( konan medical , irvine , ca ) , the endothelial count decreased insignificantly ( p = 0.09 ) from 2738  188  cells / mm to 2608  311  cells / mm . the current protocol also employed mechanical scarification of the epithelial surface by creation of microabrasions caused by movement of a merocel sponge over the corneal surface with patient 's blinking . the amount of such micro - abrasions could obviously not be standardized , and it varied between cases . this may be the reason for a relatively large variation in saturation time ( mentioned in the next paragraph ) . finally , in addition to the chemical and mechanical enhancements , the current protocol demanded a slit - lamp verification of the stromal saturation before the uva radiation ( figure 1 ) . our clinical observations showed that riboflavin saturation was achieved after 2545 minutes , so that a commonly used set induction time of , for example , 30 minutes would in many cases lead to insufficient riboflavin concentration in the stroma . our visual and refractive outcomes were comparable to other published cxl studies . in the current study there were no cases with a loss of 2 lines of cdva , endothelial cell count did not change significantly , and there were no infections or other types of keratitis . most of the patients reported pain peaking 46 hours after the treatment despite the mostly preserved epithelium . this may be explained by actinic - keratoconjunctivitis - like reaction caused by the uv exposure during the treatment and by the microabrasions caused on purpose , to enhance riboflavin penetration . corneal topography change in curvature has often been used to evaluate the effect of cxl [ 23 , 38 , 39 ] . in our study , the maximum - k value and dsi ( differential sector index ) decreased significantly on the opd ii , placido - based topography , as did the posterior elevation , irregularity index and asymmetry on the precisio , scheimpflug - based topography . however , our mean - k did not decrease in contrast to most other studies . in most of our cases , in addition to the max - k decrease , a moderate increase in steepness on the opposite side of the cone occurred ( figure 6 ) , resulting in only minor decrease of the mean - k but more symmetrical corneal optics , decreased higher - order - aberrations , and improved vision . we hypothesize that this may be a consequence of a possibly heavier riboflavin load in the inferior corneal stroma due to the sitting position and blinking during the riboflavin induction , leading to a locally increased cross - linking effect . this theory warrants a further study and may be a small step in the direction of  customized  cxl . detection of the demarcation line   after cxl has been considered the proof of the efficacy and the measure of the depth of the corneal cross - linking . although the precise nature and significance of the demarcation line ( increased optical density ) in relation to the cross - linking process are uncertain , it may be consequent to the keratocyte apoptosis and their subsequent repopulation . epithelium - on cxl with 0.1% isotonic riboflavin solution showed that the demarcation line two weeks postoperatively was located approximately 100  m from the corneal epithelium . in 24 eyes treated with the current protocol that could be evaluated by rtvue ( optovue inc . , fremont , ca ) as - oct ( anterior segment optical coherence tomography ) , the mean demarcation line was located at the depth of 316.92  49.16  m ( range 260 to 367 ) from the surface ( figure 7 ) , which is close to the observations after epithelium - off cxl . epithelial absorption / filtering of the uva light that could potentially lead to lesser energy delivered to riboflavin - saturated stroma has also been stated as an argument against the use of epithelium - on cxl . different studies offer a variety of evaluation backgrounds of this matter . a study performed by baiocci et al .   claimed that human corneal epithelium and the underlying basement membrane naturally absorb 30% to 33% of uva radiation ( 400 to 350  nm ) , while other studies showed that the epithelial uv absorption occurs only with wavelengths lower than 310  nm [ 4244 ] . we may assume that the uv - absorption of the riboflavin within the epithelium is probably low ( since the epithelial cells are hydrophobic and do not absorb riboflavin ) and that the epithelial interstitial space is of negligible volume . the current protocol includes washing off the riboflavin from the corneal surface before the uva - radiation in order to minimize the uv - energy loss due to its possible absorption by the riboflavin . finally , even if the epithelium - on cxl leads to a shallower cross - linking compared to the epithelium - off , the density of collagen fibers in corneal stroma is much higher in the anterior portion where most of the collagen cross - links occur [ 40 , 45 ] . this retrospective study showed that epithelium - on cxl using our novel protocol appeared to be effective and safe in treating progressive keratoconus . the improvements in the visual , refractive , and topographic parameters in our patients indicate that epithelium - on cxl had sufficient effect to halt the progression of keratoconus and improve the corneal shape . a randomized controlled trial is warranted to verify that the effect of the current approach is comparable with the standard epithelium - off cxl . furthermore , the combination of different enhancers , osmolality , and concentration of riboflavin should be explored to further improve the procedure .
purpose . to evaluate the efficacy and safety of epithelium - on corneal collagen cross - linking ( cxl ) using a multifactorial approach to achieve proper stromal riboflavin saturation 
 . 
 methods . 
 this non - randomized retrospective study comprised 61 eyes with progressive keratoconus treated with epithelium - on cxl . 
 chemical epithelial penetration enhancement ( benzalkonium chloride - containing local medication and hypotonic riboflavin solution ) , mechanical disruption of the superficial epithelium , and prolongation of the riboflavin - induction time until verification of stromal saturation were used before the uva irradiation . 
 uncorrected and corrected distance visual acuity ( udva , cdva ) , refraction , corneal topography , and aberrometry were evaluated at baseline and at 1 , 3 , 6 , and 12 months postoperative . 
 results . at 12-month , udva and cdva 
 improved significantly . 
 none of the eyes lost lines of cdva , while 27.4% of the eyes gained 2 or more lines . 
 mean spherical equivalent decreased by 0.74  d , and mean cylindrical reduction was 1.15  d. irregularity index and asymmetry from scheimpflug - based topography and max - k at the location of cone from placido - based topography showed a significant decrease . 
 higher - order - aberration data demonstrated a slight reduction in odd - order aberrations s 3 , 5,7 ( p = 0.04 ) . 
 postoperative pain without other complications was recorded . 
 conclusion . 
 epithelium - on cxl with our novel protocol appeared to be safe and effective in the treatment of progressive keratoconus .