Riboflavin Imbibitions

After approval by the Review Board of the Department of Medicine and Ageing Sciences (University of Chieti, Italy), 10 human eye bank corneal buttons with scleral rims were included in the study. The mean donor age was 66.8 years (range 56-72 years). The average death-to-enucleation time was 6 hours (range 4 hours and 30 minutes to 11 hours and 15 minutes). The mean storage time (between eye bank procedures and cross-linking imbibitions) was 29.3 hours (range 20-48 hours).

For CXL imbibition, the corneoscleral buttons were mounted on an anterior chamber maintainer (Barron artificial anterior chamber; Katena Products Inc, Denville, New Jersey, USA), filled with Eusol-C storage media (Alchimia s.r.l., Ponte S. Nicolò, Italy) to obtain adequate pressure and stability of the corneal tissue.

Three corneas were treated according to a standard protocol, which consisted of removal of the central 10 mm of corneal epithelium and soaking the stroma with 0.1% riboflavin (Ricrolin; SOOFT Italia S.p.A., Montegiorgio, Italy) for 20 minutes within a dedicated silicon ring (Epi-off group).

Three corneas received riboflavin imbibition via a transepithelial approach. Ricrolin TE (0.1% riboflavin plus enhancers [including destrane T500, edetate sodium, tromethamine, bihydrate sodium phosphate monobasic, and bihydrate sodium phosphate bibasic]; SOOFT Italia S.p.A.) was applied with the same procedure described for the Epi-off group, for 30 minutes, using the same silicon ring imbibition system (Epi-on group). The enhancers in Ricrolin TE facilitate riboflavin penetration into the corneal stroma in the presence of an intact epithelium. In addition, penetration was increased by a longer duration of pretreatment with Ricrolin TE, as per reported protocols.

Three corneas were soaked with riboflavin using an iontophoresis system ( Figure 1 ), composed of a power supply (I-ON CXL; SOOFT Italia S.p.A.), 2 electrodes (the return electrode in the artificial system was a stainless steel wire inserted into the saline circuit and the negative electrode was a steel grid contained in a corneal applicator; Iontofor CXL, SOOFT Italia S.p.A.), and a connection cable. Adherence of the corneal applicator to the eye was maintained with a vacuum system.

Experimental set-up for the iontophoretic imbibition phase in corneal cross-linking of the corneal samples and preparation of the stromal slices using the femtosecond laser system. (First row, left) The cornea is mounted onto the artificial anterior chamber and the corneal applicator with a vacuum suction system is placed on the cornea. The positive electrode is a stainless steel wire inserted into the saline circuit; the negative one is the steel grid contained in the corneal applicator. (First row, right) After the imbibition and epithelial removal, the cornea is docked with the femtosecond laser plastic curved interface. (Second row, left) Frame of the video-recording system internal to the laser that illustrates the first lamellar deep stromal cut performed at 300 μm of depth. Note the green fluorescent color of the corneal tissue, indicating the presence of riboflavin. (Second row, right) Image illustrating the mid phase of second lamellar dissection, performed by the laser on top of the first, at a depth of 150 μm. (Third row, left) The anterior 8.0-mm stromal slice is removed with the aid of surgical forceps. Note the edge of the intermediate slice that has been intentionally wrinkled in order to make it visible (arrows). (Third row, right) The intermediate 8.0-mm slice is gently removed with the aid of forceps. The circular borders of the trephination (arrows) and the stromal bed of the lamellar dissection appear regular and smooth. (Bottom row, left) After 8.0-mm mechanical punching, the posterior stromal slice is isolated. (Bottom row, right) Schematic diagram illustrating characteristics and thicknesses of the 3 stromal slices prepared using the described double-cut femtosecond laser dissections.

After connecting the vacuum syringe to the Luer lock connector and verifying that the stop clamp was in the “open” position, the corneal applicator was put onto the cornea to be treated. Light suction was applied with the syringe (at least 1 mL) and the stop clamp was closed. The operator verified that the applicator was secured to the cornea.

The steel grid (negative electrode) was covered with Ricrolin+ (0.1% riboflavin solution specifically formulated to allow quick passage into the corneal stroma, through intact epithelium with corneal iontophoresis; SOOFT Italia S.p.A.).

The electrical generator was in the “OFF” position up to this point. After connecting the 2 electrodes to the power generator (the female red plug to the connector on the Iontofor CXL and the white male plug to the return electrode), the power generator was turned on (acoustic confirmation). The operator selected 1 mA and pressed START again. The procedure took 5 minutes (total current intensity 5 mA). The power supply software indicated the continuity of the procedure (the generator sounds an alarm if the current is interrupted).

The operator controlled that the steel grid was covered with riboflavin solution for the entire duration of the procedure, thus maintaining a regular flow of the electrical current.

After the iontophoretic procedure, the remaining riboflavin was removed through the specific tube and the stop clamp was opened to allow the air to enter the vacuum ring. The applicator was removed from the cornea and the power generator disconnected from the applicator and from the return electrode. The cornea was rinsed with saline solution.

One cornea was used as a control and was washed with a saline solution.

Femtosecond Laser Stromal Dissections

Three stromal slices of the corneal samples were prepared with a 500-kHz VisuMax femtosecond laser (FSL) (Carl Zeiss Meditec, Jena, Germany) immediately after the imbibition or control, maintaining the corneoscleral rims mounted in the artificial anterior chamber with controlled pressure. Before FSL stromal dissections of the corneas that underwent transepithelial riboflavin imbibition (belonging to Epi-on and Iontophoresis group) and in the control cornea, the epithelium was removed using a blunt spatula for a 10-mm diameter. Therefore, all FSL cuts were performed on denuded stroma in order to standardize the thickness of the 2 superficial slices and to ensure that high-performance liquid chromatography (HPLC) was conducted only on stroma.

The central corneal thickness was measured using an ultrasound pachymeter (Optikon 2000 S.p.A; Altair, Rome, Italy).

Each cornea was divided into 3 8.00-mm circular slices by performing 2 sequential lamellar dissections first at 300 and then at 150 μm from the corneal surface. A 90-degree (perpendicular to the corneal surface) side cut was performed. The FSL parameters were set as follows: spot-line separation between 2.0 and 2.5 μm for the lamellar cuts and 3.0 μm for the side cuts. The spot energy was between 135 and 150 nJ.

The 2 anterior stromal slices were removed using corneal graft toothed forceps and blunt spatula. The residual deep corneal slice was freed using an 8.00-mm cornea punch. Stromal slices were placed in histologic screw-capped containers, shielded from the light with aluminum foil, and stored at −20 C for subsequent HPLC analysis.

High-Performance Liquid Chromatography Analysis

HPLC analysis was subsequently performed to quantify the concentration of riboflavin in each volume of tissue.

Corneal samples were vortexed 1 minute in 500 μL of methanol and centrifuged at 13 200 rpm at room temperature. The supernatants were evaporated under a nitrogen stream at 40 C. The dry residue was dissolved in 50 μL of ultrapure water/methanol 70/30 (vol/vol) and vortexed for 1 minute. Tubes were centrifuged for 2 minutes at 13 200 rpm and 5 μL of the supernatant was injected into an HPLC/MS 1100 series system equipped with a simple Quadrupole LC/MSD 1100 (both Agilent Technologies, Santa Clara, California, USA). The HPLC analysis of riboflavin was performed with a Zorbax SB C18 column (2.1 mm, 50 mm, 3.5 μm; Agilent Technologies). The mobile phase (90% 0.1% formic acid and 10% acetonitrile) was used under isocratic conditions at a flow rate of 0.2 mL/min. Mass spectrometer parameters were: ionization mode: API-ES positive; drying gas flow: 11 l/min; nebulizer pressure: 20 psig; drying gas temperature: 350 C; capillary voltage: 4000 V; and mass-to-charge ratio (M/Z): 377.20. The lower limit for riboflavin quantification was 200 ng/mL. Calibration curves were obtained by plotting riboflavin peak area as a function of concentration.

Statistical Analysis

Statistical analysis was performed using SPSS 20.0 (IBM, Armonk, New York, USA). General linear model for repeated measures was used to compare the mean difference of riboflavin concentration over slices in the 3 treatment groups. A priori–specified contrast analysis was used for within–treatment group comparisons of riboflavin concentration.

Data were expressed as median and interquartile range (IQR) and graphed as a box-whisker plot. Statistical significance was assigned at P ≤ .05.