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Cornea  |   June 2013
Protective Effects of Deswelling on Stromal Collagen Denaturation After a Corneal Femtosecond Laser Cut
Author Affiliations & Notes
  • Marco Rossi
    Ophthalmology Department, Azienda Ospedaliera di Busto Arsizio, Busto Arsizio, Varese, Italy
  • Raffaela Mistò
    Monza Eye Bank, Azienda Ospedaliera San Gerardo, Monza, Monza Brianza, Italy
  • Claudio Gatto
    Research and Development, AL.CHI.MI.A. Srl, Ponte San Nicolò, Padova, Italy
  • Paolo Garimoldi
    Ophthalmology Department, Azienda Ospedaliera di Busto Arsizio, Busto Arsizio, Varese, Italy
  • Marino Campanelli
    Monza Eye Bank, Azienda Ospedaliera San Gerardo, Monza, Monza Brianza, Italy
  • Jana D'Amato Tóthová
    Research and Development, AL.CHI.MI.A. Srl, Ponte San Nicolò, Padova, Italy
  • Correspondence: Jana D'Amato Tóthová, Research and Development AL.CHI.MI.A. Srl, Viale Austria 14, 35020 Ponte San Nicolò, Padova, Italy; jtothova@alchimiasrl.com  
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 4148-4157. doi:10.1167/iovs.12-10818
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      Marco Rossi, Raffaela Mistò, Claudio Gatto, Paolo Garimoldi, Marino Campanelli, Jana D'Amato Tóthová; Protective Effects of Deswelling on Stromal Collagen Denaturation After a Corneal Femtosecond Laser Cut. Invest. Ophthalmol. Vis. Sci. 2013;54(6):4148-4157. doi: 10.1167/iovs.12-10818.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To evaluate the effect of corneal deswelling prior to a microkeratome and femtosecond (fs) laser cut and the impact of the fs laser energy settings on the tissue surface and collagen quality.

Methods.: Porcine and human corneas were incubated in THIN-C (deswelled) or EUSOL-C (control) at 4°C for 4 hours. Porcine corneas were cut using a microkeratome or an Intralase fs laser 150 Hz and 0.75 μJ energy. Human corneas were cut using the laser at different energy settings. The tissue thickness was measured using Visante OCT and the endothelial cell density (ECD) was evaluated by an Eye Bank KeratoAnalyzer or trypan blue staining. The tissue was analyzed using scanning electron microscopy (SEM).

Results.: In porcine corneas, tissue deswelling resulted in a reduction of 14% in the central corneal thickness (CCT), a regular surface, and increased tissue stiffness without affecting ECD. After fs laser cutting, the control corneas showed superficial collagen denaturation that was absent in the deswelled corneas. The deswelled human corneas showed a CCT reduction of 20%, better surface smoothness, preserved collagen, and increased stiffness after fs laser cutting at 1.0 to 1.2 μJ, compared with controls. Surface smoothness decreased and collagen fibers showed a melted-like aspect both in deswelled and control corneas at greater than 1.4 μJ.

Conclusions.: Fs laser cutting parameters can reduce corneal surface smoothness and increase collagen melting damage. The use of a deswelling medium resulted in tissues for Descemet stripping automated endothelial keratoplasty (DSAEK) with increased stiffness, smooth surfaces, and no thermal damage to the collagen matrix.

Introduction
Descemet stripping automated endothelial keratoplasty (DSAEK) is a state of the art procedure for the treatment of visual losses caused by dysfunction of the corneal endothelium. 13 One of the main requisites for successful DSAEK is a thin graft with a regular and predictable geometry. Both microkeratome and femtosecond (fs) laser procedures are used to produce precut corneal tissues with suitable features for DSAEK. 47 Endothelial cell density (ECD) and viability have been shown to be very similar in lamellar tissues prepared using the two methods. 8,9  
Preparation of the lamellar graft using a microkeratome is a worldwide-accepted procedure with known drawbacks, such as limited graft thickness predictability and irregular graft geometry. 10 Recently, a double pass of the microkeratome technique has been proposed to obtain ultrathin tissues and improve optical outcomes. 11,12 However, this challenging technique faces additional drawbacks, including a high rate of tissue perforation and increased endothelial damage. 12 Besides tissue thickness, the graft smoothness is generally considered an important determinant of the DSAEK surgical outcome because it favors the juxtaposition of the donor and recipient surfaces with a regular topography. 13  
On the other hand, fs lasers potentially allow the operator to finely customize the tissue geometry and size with high accuracy and reproducibility. However, currently used fs lasers have been described to show some limitations including limited cutting depth, insufficient surface smoothness, applanation-derived tissue deformation, and generation of scatter at the cutting interface. An inverse cutting approach using an fs laser was proposed with the aim of preparing thin and plane corneal grafts. 14,15 Nevertheless, most of the graft recipients did not get a visual benefit from the operation due to dense interface scatter. 15 Furthermore, it has been reported that the lamellar interface obtained with fs lasers can bear topographic irregularities, which degrade visual acuity. 1619  
The drawbacks of both a microkeratome and fs laser approach are the handling of very thin and flaccid tissue with possible negative effects on the endothelial vitality. 
In the present study, we hypothesized that the reduction of water in donor corneas could lead to tissues with more suitable characteristics for lamellar graft preparation. We examined the hypothesis first in porcine corneas and then in human tissues to validate the suitability of the procedure. Corneas were deswelled prior to microkeratome or fs laser cutting to obtain grafts of reduced thickness and increased stiffness due to water loss. After microkeratome and fs laser cutting, the smoothness of the cutting surface and the collagen ultrastructure were analyzed using scanning electron microscopy (SEM) in porcine corneas and then the effects of the laser cut at different energy levels were investigated in human corneas. The main finding of our study is the stromal collagen denaturation phenomenon observed in fs laser–cut tissues and the possible protective effect of deswelling. 
Methods
Tissue Origin and Handling
Sixteen porcine eye globes were collected from 6-month-old Danish Landrance pigs at a local abattoir (Venegoni Spa, Boffalora Sopra Ticino, Italy), rinsed with sterile 0.9 % NaCl, and transported at 4°C in a moist chamber containing a phosphate buffered saline solution (PSS-L; AL.CHI.MI.A. Srl, Ponte San Nicolò, Italy) to the Research and Development (R&D) Department of AL.CHI.MI.A. Srl. The corneoscleral rims were dissected and stored in EUSOL-C (AL.CHI.MI.A. Srl) at 4°C for 24 hours. 
Eighteen human donor corneas, not suitable for transplantation due to donor serology, were collected and processed by the Eye Bank of Monza, Italy, according to their standard operating procedures. After dissection, the corneas were stored in EUSOL-C at 4°C for 4 days, transferred to TISSUE-C (AL.CHI.MI.A. Srl), and incubated under organ culture conditions at 31°C for 20 days on an average. Before use, the corneas were placed in CARRY-C (AL.CHI.MI.A. Srl) at room temperature for 24 hours and transported either to the Ophthalmology Department of the Busto Arsizio Hospital (ODBAH), Italy, or to the Vista Vision Clinic (VVC), Milan, Italy. All experiments were conducted with the Declaration of Helsinki and the ARVO Statement for the Use of Animals in Ophthalmic Research. 
Tissue Treatment Prior to Cutting
In order to induce tissue deswelling before cutting, porcine (n = 8) and human donor (n = 12) corneas were placed in THIN-C (AL.CHI.MI.A. Srl) which contains a proprietary mixture of high and low molecular weight dextrans, at 4°C for 4 hours. The deswelling phase was skipped for control porcine (n = 8) and human (n = 6) tissues, which were stored in EUSOL-C at 4°C for 4 hours before cutting. 
Central Corneal Thickness Measurements
Central corneal thickness (CCT) measurements of control and THIN-C–treated corneas was performed before deswelling and cutting, using an OCT-Visante (Visante; Carl Zeiss Meditec Inc., Dublin, CA) equipped with a FLEB OCT adaptor (Ophthalmic Biophysics Center, Miami, FL) to carry out measurements through the optically clear vial bottom with the cornea placed in the vial. 20 All CCT measurements of human tissues were performed by the same operator at ODBAH. 
CCT measurements of porcine tissues were performed either at the Eye Bank of Monza or ODBAH by two different investigators using validated instruments and methods and internal control tissues. For each cornea, at least two measurements were performed. 
Endothelial Cell Density Measurements
In porcine corneas, ECD was evaluated using trypan blue staining (TB-S; AL.CHI.MI.A. Srl) according to the method of Stocker using an inverted microscope (Zeiss Axiovert 30; Carl Zeiss, Thornwood, NY) and counting the cell number in at least three different areas of 1 mm2 each. In human corneas, ECD measurements were performed by the Eye Bank of Monza, using an Eye Bank KeratoAnalyzer (Konan Medical Inc., Tokyo, Japan) and the “center method,” which involved counting all visible cells in at least three different areas of 1 mm2 each. For each cornea, the mean ECD was determined. 
Microkeratome and Femtosecond Laser Cutting
Sixteen porcine tissues were cut by an expert surgeon either with a Moria microkeratome (THIN-C, n = 5; control, n = 5) equipped with a 350-μm cutting head (Moria S.A., Antony, France) or with a iFS Advanced Femtosecond Laser (THIN-C, n = 3; control, n = 3; Abbott Medical Optics Inc., Santa Ana, CA), 150 kHz setting raster mode, 330-μm cutting depth, 4-μm spot separation, 0.75 μJ energy, and 10.6 J/cm2 fluence. The fluence was estimated by dividing the pulse energy by the spot area. Microkeratome cuts of the porcine tissue were performed by ODBAH, and VVC performed fs laser cuts. 
All 18 human donor corneas were cut using a iFS Advanced Femtosecond Laser, with 150 kHz setting raster mode, cutting depth of approximately 200 μm from endothelium, 4-μm spot separation, and using low (1.0 μJ), medium (1.2–1.4 μJ), or high (1.8 μJ) energies. Estimated respective fluence was 14.2 J/cm2 (low), 17.0 to 19.8 J/cm2 (medium), and 25.5 J/cm2 (high). Estimated fluence values correspond to the unperturbed beam. The spot size at focus, in the volume of the corneal tissue, might be higher and, thus, result in considerably lower fluence values. For each energy range, corneas treated with THIN-C (n = 4) were compared with control tissues (n = 2). All human tissue cuts were performed by ODBAH. 
Scanning Electron Microscopy
Immediately after microkeratome or fs laser cutting, the corneal lamellar tissue was fixed in 2.5% glutaraldehyde in PBS at room temperature for 2 hours and transported at 4°C to the R&D department of AL.CHI.MI.A. Srl for further analysis. The tissue was dehydrated using gradient ethanol solutions and a critical point dryer (Balzer CPD030; Balzers, Vaduz, Liechtenstein), Au-metalized in a Edwards S150A sputter coater (Edwards Ltd., Crawley, UK), and finally analyzed using a JEOL JSM-6490 Scanning Electron Microscope (JEOL Ltd., Tokyo, Japan) for surface quality. 
Qualitative Analysis of Surface Smoothness and Collagen Structure
For qualitative analysis of the surface smoothness and the presence of folds, SEM images of the whole tissue surface were acquired at low magnifications (×25–30). Image of the corneal surface was reconstructed using Photoshop CS5 software (Adobe Systems Incorporated, San Jose, CA) from low magnification SEM images. Three independent operators evaluated the surface smoothness, in a masked manner, by attributing a score in arbitrary units to the smooth surface on cornea images. For qualitative analysis of the stromal collagen structure, SEM images were acquired at high magnifications (×500, ×1000, and ×5000) in both central and peripheral areas. Three independent operators, in masked fashion, scored the grade of damage on four different areas per cornea. 
Results
Effect of Porcine Cornea Deswelling on the Central Corneal Thickness and Endothelial Cell Density
Figure 1 shows a representative OCT image of a porcine cornea treated with THIN-C, at the beginning (Fig. 1a) and after 4 hours of treatment (Fig. 1b). At the end of the incubation period, all porcine corneas treated with THIN-C showed an average reduction of 159 ± 13 μm (14%), from an initial 1138 ± 12 μm to a final 979 ± 6 μm, in CCT. Control tissues did not show any variation in CCT within 4 hours (Fig. 1c). Deswelling occurred progressively and a significant difference in CCT between control and THIN-C–treated tissues was already detected after 1 hour of treatment (not shown). In addition, surgeons reported an increased stiffness in the THIN-C–treated tissues as compared with that in controls. 
Figure 1
 
Effects of deswelling on porcine corneas. (a) A representative OCT image of porcine cornea at the beginning of the treatment with the deswelling medium THIN-C and (b) the same deswelled porcine cornea after 4 hours of treatment at 4°C. (c) CCT of control porcine corneas (white bars, n = 6) and deswelled cornea (gray bars, n = 6) at the beginning (time 0) and after 4 hours of incubation at 4°C (time 4). (d) ECD of porcine corneas treated with the control medium (white bars) and the deswelling medium (gray bars) before cutting (n = 6), and after microkeratome (n = 3), and fs laser (n = 3) cutting.
Figure 1
 
Effects of deswelling on porcine corneas. (a) A representative OCT image of porcine cornea at the beginning of the treatment with the deswelling medium THIN-C and (b) the same deswelled porcine cornea after 4 hours of treatment at 4°C. (c) CCT of control porcine corneas (white bars, n = 6) and deswelled cornea (gray bars, n = 6) at the beginning (time 0) and after 4 hours of incubation at 4°C (time 4). (d) ECD of porcine corneas treated with the control medium (white bars) and the deswelling medium (gray bars) before cutting (n = 6), and after microkeratome (n = 3), and fs laser (n = 3) cutting.
Trypan blue staining showed an average ECD of 4072 ± 186 cells/mm2 in control porcine corneas. ECD did not vary after 4 hours of deswelling in THIN-C. Tissues cut with a microkeratome or fs laser showed small, not statistically significant, reduction in the ECD (Fig. 1d). 
Scanning Electron Microscopy Analysis of Interface After Porcine Cornea Microkeratome and Femtosecond Laser Cutting
After microkeratome cutting, SEM analysis at low magnification of the porcine corneal flaps, previously treated with THIN-C, showed generally a regular interface without macroscopic folds (Fig. 2a). The control tissues showed reduced surface regularity due to the presence of deep central and peripheral folds. Furthermore, the peripheral areas of the control tissues often exhibited repetitive patterns of crests and valleys, probably produced by the discontinuous movement of the microkeratome blade (Fig. 2c). At higher magnifications, we observed a regular collagen structure with parallel and uniformly spaced collagen fibers in both control and THIN-C–treated tissues (Figs. 2b, 2d). 
Figure 2
 
SEM images of deswelled and control porcine corneal tissue after microkeratome cutting. (a) Porcine cornea deswelled in THIN-C at 4°C for 4 hours and cut using a microkeratome showing regular and smooth stromal surfaces. (b) Higher magnification (×5000) of the same deswelled tissue showing normal morphology and organization of collagen fibers. (c) Porcine cornea incubated in the control medium and cut using a microkeratome showing macro folds (encircled) and peripheral irregularities on the stromal surface (arrows). (d) Higher magnification (×5000) of the same control cornea showing normal morphology and organization of collagen fibers.
Figure 2
 
SEM images of deswelled and control porcine corneal tissue after microkeratome cutting. (a) Porcine cornea deswelled in THIN-C at 4°C for 4 hours and cut using a microkeratome showing regular and smooth stromal surfaces. (b) Higher magnification (×5000) of the same deswelled tissue showing normal morphology and organization of collagen fibers. (c) Porcine cornea incubated in the control medium and cut using a microkeratome showing macro folds (encircled) and peripheral irregularities on the stromal surface (arrows). (d) Higher magnification (×5000) of the same control cornea showing normal morphology and organization of collagen fibers.
After fs laser cutting, SEM analysis of the tissues pretreated with THIN-C showed a smooth and regular surface at low magnifications (Fig. 3a). Similar to microkeratome-cut corneas, the control tissues showed deep, peripheral folds (Fig. 3d). 
Figure 3
 
SEM images of deswelled and control porcine corneal tissue after fs laser cut. (a) Porcine cornea deswelled in THIN-C at 4°C for 4 hours and cut using an fs laser showing regular and smooth stromal surface. (b) Higher magnification (×500) of the same deswelled tissue showing the normal stacked organization of collagen lamellae. (c) Higher magnification (×5000) of the same deswelled tissue showing normal morphology of the collagen fibers. (d) Porcine control cornea incubated in the control medium and cut using an fs laser showing a peripheral fold (encircled). (e) Higher magnification (×500) of the same control tissue showing loss of stacked organization and smoothened aspect of collagen lamellae. (f) High magnification (×5000) of the same control cornea showing damaged, superficially denatured collagen fibers.
Figure 3
 
SEM images of deswelled and control porcine corneal tissue after fs laser cut. (a) Porcine cornea deswelled in THIN-C at 4°C for 4 hours and cut using an fs laser showing regular and smooth stromal surface. (b) Higher magnification (×500) of the same deswelled tissue showing the normal stacked organization of collagen lamellae. (c) Higher magnification (×5000) of the same deswelled tissue showing normal morphology of the collagen fibers. (d) Porcine control cornea incubated in the control medium and cut using an fs laser showing a peripheral fold (encircled). (e) Higher magnification (×500) of the same control tissue showing loss of stacked organization and smoothened aspect of collagen lamellae. (f) High magnification (×5000) of the same control cornea showing damaged, superficially denatured collagen fibers.
Unexpectedly, in fs laser–cut corneas, we observed a clear difference in the collagen aspect of the THIN-C–treated and control tissues, at higher magnifications. THIN-C–treated corneas showed a well preserved, essentially normal array of collagen lamellae and fibers (Figs. 3b, 3c). Strikingly, in control corneas, cut under identical fs laser settings, collagen lamellae appeared tightly packed with a loss of collagen filamentous appearance and regular orientation (Figs. 3e, 3f). This superficially smoothened aspect of collagen fibers (Fig. 3f) is reminiscent of the melting of a heat-sensitive collagen structure. 
Effect of Donor Cornea Deswelling on Increasing Femtosecond Laser Energy
Representative OCT Visante images of human donor corneas show the CCT reduction in corneal thickness at the beginning and after 4 hours of deswelling in THIN-C (Figs. 4a, 4b). At the beginning, the mean donor cornea thickness was 553 ± 33 μm and 598 ± 42 μm for the THIN-C–treated and control tissues, respectively. Deswelling in THIN-C caused a significant reduction of 113 ± 41 μm (20%) in CCT after 4 hours of treatment (Fig. 4c). 
Figure 4
 
Effects of deswelling and an fs laser cut on human corneas. (a) Representative OCT image of human cornea at the beginning of the treatment with the deswelling medium THIN-C and (b) the same deswelled cornea after 4 hours of treatment at 4°C. (c) CCT of control corneas (white bars, n = 6) and deswelled corneas (gray bars, n = 9), at the beginning (time 0) and after 4 hours of incubation at 4°C (time 4). (d) ECD of human corneas treated with the control medium (white bars) and the deswelling medium (gray bars) before cutting (n = 6), and after an fs laser cut (n = 9).
Figure 4
 
Effects of deswelling and an fs laser cut on human corneas. (a) Representative OCT image of human cornea at the beginning of the treatment with the deswelling medium THIN-C and (b) the same deswelled cornea after 4 hours of treatment at 4°C. (c) CCT of control corneas (white bars, n = 6) and deswelled corneas (gray bars, n = 9), at the beginning (time 0) and after 4 hours of incubation at 4°C (time 4). (d) ECD of human corneas treated with the control medium (white bars) and the deswelling medium (gray bars) before cutting (n = 6), and after an fs laser cut (n = 9).
ECD was not modified immediately after the cut, as measured using a KeratoAnalyzer (Fig. 4d). 
The best smoothness results were observed at 1.0 μJ energy for both deswelled and control tissues (Figs. 5a, 5d). At energies ranging from 1.0 μJ to 1.4 μJ, THIN-C–treated tissues showed better surface smoothness and regularity as compared with the equivalent tissues in the control medium, which showed more folds and gross unevenness (Fig. 5). 
Figure 5
 
SEM images at low magnification (×30) of human corneas after fs laser cutting at different energy settings. Left column: The posterior stromal surface of corneas deswelled in THIN-C at 4°C for 4 hours and after fs laser cutting with (a) 1.0 μJ energy showing smooth surface, (b) 1.2 μJ energy showing increased surface irregularities, and (c) 1.4 μJ energy showing increased roughness, irregularities, and a cavitation bubble imprint (arrow). Right column: The posterior stromal surface of control corneas cut with an fs laser using (d) 1.0 μJ energy showing smooth stromal surface, (e) 1.2 μJ energy showing increased surface roughness and macro folds, and (f) 1.4 μJ energy showing irregular and rough surface with numerous macro folds. At same energy settings, deswelled corneas (left column) show better surface smoothness and regularity as compared with control tissues (right column). Scale bars: 500 μm.
Figure 5
 
SEM images at low magnification (×30) of human corneas after fs laser cutting at different energy settings. Left column: The posterior stromal surface of corneas deswelled in THIN-C at 4°C for 4 hours and after fs laser cutting with (a) 1.0 μJ energy showing smooth surface, (b) 1.2 μJ energy showing increased surface irregularities, and (c) 1.4 μJ energy showing increased roughness, irregularities, and a cavitation bubble imprint (arrow). Right column: The posterior stromal surface of control corneas cut with an fs laser using (d) 1.0 μJ energy showing smooth stromal surface, (e) 1.2 μJ energy showing increased surface roughness and macro folds, and (f) 1.4 μJ energy showing irregular and rough surface with numerous macro folds. At same energy settings, deswelled corneas (left column) show better surface smoothness and regularity as compared with control tissues (right column). Scale bars: 500 μm.
For both control and THIN-C treatments, the cut at 1.4 μJ energy setting resulted in increased surface irregularity with extensive formation of cavitation bubbles and longitudinal parallel depressions reflecting the laser beam trajectory and probably corresponding to the laser beam tissue ablation (Figs. 5d, 5f). 
High laser energy setting at 1.8 μJ led to extensive tissue damage visible both on the stromal and endothelial sides of the tissue flap (Fig. 6). Abundant gas bubbling was observed in the liquid contained in the artificial chamber during the whole lamellar laser cutting. 
Figure 6
 
SEM images of a deswelled human cornea after fs laser cutting using high energy (1.8 μJ). (a) Cornea deswelled in THIN-C and cut with an fs laser using high 1.8 μJ energy showing a posterior stromal surface with irregularities and fissures; (b) the same tissue at higher magnification (×300), exhibiting damaged surface with deep superficial valleys and holes; (c) the same tissue at a higher magnification (×1000) showing undistinguishable collagen lamellae and fibers. (d) Transversal section of the same tissue with the upper stromal side showing a damaged white superficial layer estimated to be 8 to 10 μm. (e) The endothelial side of the corneal tissue cut with low energy (1.0 μJ) showing well preserved endothelial morphology. (f) The endothelial side of the corneal tissue cut with high energy (1.8 μJ) showing loss of endothelial cells morphology (g) the same corneal tissue at a higher magnification (×2200) showing damaged endothelial cell depleted of the plasma membrane with visible nucleus (N) and intracellular vesicles (arrows). (h) The same endothelium with numerous micro holes localized along intercellular spaces (arrows).
Figure 6
 
SEM images of a deswelled human cornea after fs laser cutting using high energy (1.8 μJ). (a) Cornea deswelled in THIN-C and cut with an fs laser using high 1.8 μJ energy showing a posterior stromal surface with irregularities and fissures; (b) the same tissue at higher magnification (×300), exhibiting damaged surface with deep superficial valleys and holes; (c) the same tissue at a higher magnification (×1000) showing undistinguishable collagen lamellae and fibers. (d) Transversal section of the same tissue with the upper stromal side showing a damaged white superficial layer estimated to be 8 to 10 μm. (e) The endothelial side of the corneal tissue cut with low energy (1.0 μJ) showing well preserved endothelial morphology. (f) The endothelial side of the corneal tissue cut with high energy (1.8 μJ) showing loss of endothelial cells morphology (g) the same corneal tissue at a higher magnification (×2200) showing damaged endothelial cell depleted of the plasma membrane with visible nucleus (N) and intracellular vesicles (arrows). (h) The same endothelium with numerous micro holes localized along intercellular spaces (arrows).
SEM analysis of the stromal side at high magnifications showed the presence of longitudinal, parallel, and deep valleys with possible deep central fissures (Fig. 6a). At higher magnifications, the collagen fibers were not recognizable; the surface consisted of a superficial amorphous layer with long depressions and holes (Figs. 6b, 6c). The superficial layer close to the stromal surface appeared whiter and tightly packed, indicating that collagen denaturation was limited to the superficial layers up to a depth of approximately 12 μm (Fig. 6d). At the endothelial side of the flap, SEM analysis showed extensive endothelial cell damage (Fig. 6f) compared with the endothelial cell morphology of the tissue cut with a low energy setting of 1.0 μJ (Fig. 6e). Some endothelial cells appeared without a cell membrane, yet preserving intracellular structures such as nuclei and vesicles (Fig. 6g). Holes with diameters ranging from 0.7 to 5 μm, probably formed by the passage of gas bubbles from the cutting interface into the artificial chamber, were noted along the intercellular spaces (Fig. 6h). 
Figure 7 shows the morphology and structure of stromal collagen at a high magnification, and at low (1.0 μJ) and medium (1.2–1.4 μJ) energy settings. 
Figure 7
 
SEM images at low magnification (×30) of human corneas after fs laser cutting at different energy settings. Left column: The collagen fibers on the posterior stromal surface of corneas deswelled in THIN-C at 4°C for 4 hours and after fs laser cutting with (a) 1.0 μJ energy showing a normal collagen organization, (b) 1.2 μJ energy showing well distinguishable collagen fibers, and (c) 1.4 μJ energy showing damaged collagen with undistinguishable fibers. Right column: Collagen morphology on the posterior stromal surface of control corneas cut with an fs laser using (d) 1.0 μJ energy showing the superficial layer of denatured collagen and the well-preserved collagen organization in deeper layers (arrow), (e) 1.2 μJ showing increased collagen denaturation damage, and (f) 1.4 μJ showing a smooth layer of denatured collagen with undistinguishable fibers. At same energy settings deswelled tissues (left column) show better preservation of collagen morphology and organization as compared with control tissues (right column). Scale bars: 5 μm.
Figure 7
 
SEM images at low magnification (×30) of human corneas after fs laser cutting at different energy settings. Left column: The collagen fibers on the posterior stromal surface of corneas deswelled in THIN-C at 4°C for 4 hours and after fs laser cutting with (a) 1.0 μJ energy showing a normal collagen organization, (b) 1.2 μJ energy showing well distinguishable collagen fibers, and (c) 1.4 μJ energy showing damaged collagen with undistinguishable fibers. Right column: Collagen morphology on the posterior stromal surface of control corneas cut with an fs laser using (d) 1.0 μJ energy showing the superficial layer of denatured collagen and the well-preserved collagen organization in deeper layers (arrow), (e) 1.2 μJ showing increased collagen denaturation damage, and (f) 1.4 μJ showing a smooth layer of denatured collagen with undistinguishable fibers. At same energy settings deswelled tissues (left column) show better preservation of collagen morphology and organization as compared with control tissues (right column). Scale bars: 5 μm.
We observed a difference between deswelled and control corneas in terms of collagen organization and morphology. In control tissues, the corneal flaps showed signs of superficial collagen melting and disarray (Fig. 7d), which were absent in THIN-C pretreated corneas (Fig. 7a). The difference in the collagen morphology was also visible after cutting with 1.2 μJ energy (Figs. 7b, 7e). 
Higher laser energy (1.4 μJ) resulted in a dramatic disarray and flattening of superficial collagen fibers in both controls and THIN-C deswelled tissues (Figs. 7c, 7f). 
Discussion
We showed that incubation in the optimized deswelling solution THIN-C is able to reduce tissue thickness significantly before cutting, thus, creating a favorable condition for deeper tissue cut when using the standard one-pass microkeratome technique or fs laser cut with an optimal cutting depth and energy. As a result of deswelling, the increased tissue stiffness facilitates handling, prevents folding, and potentially preserves the endothelium integrity during both microkeratome and fs laser cutting. The tissue folds, mostly observed in control corneas, could be explained as diluted collagen stroma due to a high water content that leads to low structural rigidity and easy tissue deformation and folding. In contrast, the more compact collagen stroma of deswelled corneas may protect the tissue from deformation. 
From the observed deswelling rate, we believe that thicker donor tissues, with a high water content, treated with THIN-C may deswell faster than corneas with physiologic thickness. If so, the deswelling treatment may make the thickness of the donor corneas uniform prior to cutting, allowing standardization and predictability of the cutting procedure. 
The unexpected finding of the collagen denaturation phenomenon, initially observed in porcine corneas after fs laser cutting and not after microkeratome cutting prompted us to perform a more in depth investigation of this phenomenon in human tissues and in relation with laser energy settings. 
The study on human corneas confirmed the stromal collagen denaturation phenomenon observed in fs laser–cut tissues with wide range of energies, which can have potentially dramatic effects on the outcome of corneal grafts after DSAEK surgery. We observed that increasing the laser energy causes a rearrangement of stromal collagen layers that perturbs severely the delicate geometry of the corneal tissue with possible negative consequences on light penetration and scattering. 
The impact of laser energy on corneal tissue has been the object of intensive research that has revealed molecular aspects and given precise quantification of collagen damage. 2123 Due to the high intensities at the focal region, several effects, such as self-focusing, photo dissociation, and UV-light production, were reported during fs laser photodisruption, inducing both mechanical and thermal tissue damage and resulting in streak formation inside the cornea. 24 In our study, using increasing laser energies, we detected the appearance of progressively larger areas of collagen denaturation, where the fine filamentous structure of the stromal bed flattened and disorganized as a result of apparent thermal damage induced by photodisruption. 
Since collagen fibers with a correct spacing and orientation are fundamental for corneal stromal transparency, 2527 any modification of the stromal organization is predicted to have severe consequences on the functionality of the cornea intended for transplantation. 
The novel contribution of our study resides in the observation that deswelling the cornea before fs laser cutting can partially prevent collagen denaturation caused by fs laser pulses. This was seen both in porcine and human cornea, despite the structural and size differences between the two species. If the fs laser energy is kept at low to medium levels (i.e., below 1.2 μJ) the corneas deswelled in THIN-C are structurally intact compared with control corneas, which show clear signs of collagen denaturation. The beneficial effect of the deswelling process is probably due to the improvement of the corneal transparency in terms of arrangement of corneal fibers, which leads to better beam quality of the surgical laser. In the edematous donor cornea, the structural arrangement of the corneal fibrils is perturbed and this results in scattering and optical aberrations of the laser beam that may induce damage of the overlying or surrounding tissues. 26 Peyrot et al. measured the percentage of scattered light as a function of corneal thickness and showed that scattering increased with increased tissue thickness, with the lowest scattering value for the physiologic corneal thickness (∼480 μm without epithelium). 27 In our study, the thickness of human corneas deswelled in THIN-C corresponds approximately to the minimal light scattering found by Peyrot et al. 27 This condition favors the optimal performance of the laser beam and could explain the better cutting of deswelled tissues. 
Miles and Ghelashvili studied the thermal stability of collagen molecules at different hydration levels. 28 They reported that the process of swelling coincides with the loss of thermal stability, and accordingly, the loss of water molecules leads to a thermal stabilization of collagens. This could partially explain why water removal from the collagen stroma in deswelled corneas yields higher resistance to thermal damage induced by fs pulses. Moreover, the presence of dextrans in the deswelling medium THIN-C could specifically contribute to the observed protective effects, as dextran polymers were shown to have a protective effect during thermal treatments by protein stabilization through noncovalent interactions. 29,30  
On the other hand, there is basically no difference between THIN-C– and control-treated tissues at a higher laser energy (> 1.4 μJ), suggesting that a higher degree of collagen damage occurred and no protective effect of THIN-C incubation could be observed compared with that in control. Altogether, our observations indicate that a laser energy higher than 1.4 μJ should be avoided in the preparation of corneal tissue for DSAEK. 
In our study the surface smoothness varied based on fs laser energy settings. At very low energies (<0.9 μJ), irregular and folded surface with collagen bridges and locally extracted collagen lamellae suggest the incomplete tissue cut (not shown). On the other hand, the surface roughness observed at a very high energy (1.8 μJ) showed a different aspect that evokes widely diffused thermal damage with formation of depressions, fissures, and holes in the melted collagen layer. 
According to Cherian and Rau, high energy enhances the photodisruption phenomenon throughout the tissue, yielding smoother cut surfaces. 31 In fact, in our study we observed an improvement of tissue smoothness with increased energy within the range of 0.8 to 1.2 μJ. We also noticed that extremely high smoothness after an fs laser cut could be due to the loss of the collagen fibrous structure and its transformation into an amorphous melted material that formed a superficial flat layer (not shown). Consequently, the excessive smoothness of the cutting surface could be a sign of collagen denaturation. Therefore, appropriate fs laser cutting parameters including energy intensity, cutting depth, spot size, and distance have to be established in order to carefully calibrate the balance between graft smoothness and collagen denaturation. 
The morphologic characteristics of our control tissue are essentially in accord with the data on human cornea by Cheng et al. who reported both relatively smooth stromal bed after fs laser cutting and the presence of a damage zone of few micrometers at the cutting interface, which consisted of irregularly oriented collagen fibril, as observed using transmission electron microscopy. 18  
Conclusions
Our study showed that the reduction of water in donor corneas leads to tissues with more suitable characteristics for DSAEK tissue preparation. Deswelling of donor corneas in THIN-C prior to microkeratome and fs laser cutting to prepare lamellar grafts increased tissue smoothness, stiffness, and fs cutting efficiency, and reduced tissue folding. The main finding is the stromal collagen denaturation phenomenon observed only in fs laser–cut tissues and its increase with fs laser energy increase. We showed that corneal collagen could be preserved from thermal damage by corneal deswelling in THIN-C. Although our study is limited to qualitative evaluation of the stromal bed and it remains to be verified whether the clear benefit at morphologic and structural levels that we highlighted may affect the clinical outcomes of lamellar keratoplasty, our results emphasize the importance of standardizing tissue pretreatment and cutting procedures during DSAEK surgery in order to meet the surgeon's requirements and optimize visual outcome in patients. 
Acknowledgments
Disclosure: M. Rossi, AL.CHI.MI.A. Srl (R); R. Mistò, None; C. Gatto, AL.CHI.MI.A. Srl (E); P. Garimoldi, None; M. Campanelli, None; J. D'Amato Tóthová, AL.CHI.MI.A. Srl (E) 
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Figure 1
 
Effects of deswelling on porcine corneas. (a) A representative OCT image of porcine cornea at the beginning of the treatment with the deswelling medium THIN-C and (b) the same deswelled porcine cornea after 4 hours of treatment at 4°C. (c) CCT of control porcine corneas (white bars, n = 6) and deswelled cornea (gray bars, n = 6) at the beginning (time 0) and after 4 hours of incubation at 4°C (time 4). (d) ECD of porcine corneas treated with the control medium (white bars) and the deswelling medium (gray bars) before cutting (n = 6), and after microkeratome (n = 3), and fs laser (n = 3) cutting.
Figure 1
 
Effects of deswelling on porcine corneas. (a) A representative OCT image of porcine cornea at the beginning of the treatment with the deswelling medium THIN-C and (b) the same deswelled porcine cornea after 4 hours of treatment at 4°C. (c) CCT of control porcine corneas (white bars, n = 6) and deswelled cornea (gray bars, n = 6) at the beginning (time 0) and after 4 hours of incubation at 4°C (time 4). (d) ECD of porcine corneas treated with the control medium (white bars) and the deswelling medium (gray bars) before cutting (n = 6), and after microkeratome (n = 3), and fs laser (n = 3) cutting.
Figure 2
 
SEM images of deswelled and control porcine corneal tissue after microkeratome cutting. (a) Porcine cornea deswelled in THIN-C at 4°C for 4 hours and cut using a microkeratome showing regular and smooth stromal surfaces. (b) Higher magnification (×5000) of the same deswelled tissue showing normal morphology and organization of collagen fibers. (c) Porcine cornea incubated in the control medium and cut using a microkeratome showing macro folds (encircled) and peripheral irregularities on the stromal surface (arrows). (d) Higher magnification (×5000) of the same control cornea showing normal morphology and organization of collagen fibers.
Figure 2
 
SEM images of deswelled and control porcine corneal tissue after microkeratome cutting. (a) Porcine cornea deswelled in THIN-C at 4°C for 4 hours and cut using a microkeratome showing regular and smooth stromal surfaces. (b) Higher magnification (×5000) of the same deswelled tissue showing normal morphology and organization of collagen fibers. (c) Porcine cornea incubated in the control medium and cut using a microkeratome showing macro folds (encircled) and peripheral irregularities on the stromal surface (arrows). (d) Higher magnification (×5000) of the same control cornea showing normal morphology and organization of collagen fibers.
Figure 3
 
SEM images of deswelled and control porcine corneal tissue after fs laser cut. (a) Porcine cornea deswelled in THIN-C at 4°C for 4 hours and cut using an fs laser showing regular and smooth stromal surface. (b) Higher magnification (×500) of the same deswelled tissue showing the normal stacked organization of collagen lamellae. (c) Higher magnification (×5000) of the same deswelled tissue showing normal morphology of the collagen fibers. (d) Porcine control cornea incubated in the control medium and cut using an fs laser showing a peripheral fold (encircled). (e) Higher magnification (×500) of the same control tissue showing loss of stacked organization and smoothened aspect of collagen lamellae. (f) High magnification (×5000) of the same control cornea showing damaged, superficially denatured collagen fibers.
Figure 3
 
SEM images of deswelled and control porcine corneal tissue after fs laser cut. (a) Porcine cornea deswelled in THIN-C at 4°C for 4 hours and cut using an fs laser showing regular and smooth stromal surface. (b) Higher magnification (×500) of the same deswelled tissue showing the normal stacked organization of collagen lamellae. (c) Higher magnification (×5000) of the same deswelled tissue showing normal morphology of the collagen fibers. (d) Porcine control cornea incubated in the control medium and cut using an fs laser showing a peripheral fold (encircled). (e) Higher magnification (×500) of the same control tissue showing loss of stacked organization and smoothened aspect of collagen lamellae. (f) High magnification (×5000) of the same control cornea showing damaged, superficially denatured collagen fibers.
Figure 4
 
Effects of deswelling and an fs laser cut on human corneas. (a) Representative OCT image of human cornea at the beginning of the treatment with the deswelling medium THIN-C and (b) the same deswelled cornea after 4 hours of treatment at 4°C. (c) CCT of control corneas (white bars, n = 6) and deswelled corneas (gray bars, n = 9), at the beginning (time 0) and after 4 hours of incubation at 4°C (time 4). (d) ECD of human corneas treated with the control medium (white bars) and the deswelling medium (gray bars) before cutting (n = 6), and after an fs laser cut (n = 9).
Figure 4
 
Effects of deswelling and an fs laser cut on human corneas. (a) Representative OCT image of human cornea at the beginning of the treatment with the deswelling medium THIN-C and (b) the same deswelled cornea after 4 hours of treatment at 4°C. (c) CCT of control corneas (white bars, n = 6) and deswelled corneas (gray bars, n = 9), at the beginning (time 0) and after 4 hours of incubation at 4°C (time 4). (d) ECD of human corneas treated with the control medium (white bars) and the deswelling medium (gray bars) before cutting (n = 6), and after an fs laser cut (n = 9).
Figure 5
 
SEM images at low magnification (×30) of human corneas after fs laser cutting at different energy settings. Left column: The posterior stromal surface of corneas deswelled in THIN-C at 4°C for 4 hours and after fs laser cutting with (a) 1.0 μJ energy showing smooth surface, (b) 1.2 μJ energy showing increased surface irregularities, and (c) 1.4 μJ energy showing increased roughness, irregularities, and a cavitation bubble imprint (arrow). Right column: The posterior stromal surface of control corneas cut with an fs laser using (d) 1.0 μJ energy showing smooth stromal surface, (e) 1.2 μJ energy showing increased surface roughness and macro folds, and (f) 1.4 μJ energy showing irregular and rough surface with numerous macro folds. At same energy settings, deswelled corneas (left column) show better surface smoothness and regularity as compared with control tissues (right column). Scale bars: 500 μm.
Figure 5
 
SEM images at low magnification (×30) of human corneas after fs laser cutting at different energy settings. Left column: The posterior stromal surface of corneas deswelled in THIN-C at 4°C for 4 hours and after fs laser cutting with (a) 1.0 μJ energy showing smooth surface, (b) 1.2 μJ energy showing increased surface irregularities, and (c) 1.4 μJ energy showing increased roughness, irregularities, and a cavitation bubble imprint (arrow). Right column: The posterior stromal surface of control corneas cut with an fs laser using (d) 1.0 μJ energy showing smooth stromal surface, (e) 1.2 μJ energy showing increased surface roughness and macro folds, and (f) 1.4 μJ energy showing irregular and rough surface with numerous macro folds. At same energy settings, deswelled corneas (left column) show better surface smoothness and regularity as compared with control tissues (right column). Scale bars: 500 μm.
Figure 6
 
SEM images of a deswelled human cornea after fs laser cutting using high energy (1.8 μJ). (a) Cornea deswelled in THIN-C and cut with an fs laser using high 1.8 μJ energy showing a posterior stromal surface with irregularities and fissures; (b) the same tissue at higher magnification (×300), exhibiting damaged surface with deep superficial valleys and holes; (c) the same tissue at a higher magnification (×1000) showing undistinguishable collagen lamellae and fibers. (d) Transversal section of the same tissue with the upper stromal side showing a damaged white superficial layer estimated to be 8 to 10 μm. (e) The endothelial side of the corneal tissue cut with low energy (1.0 μJ) showing well preserved endothelial morphology. (f) The endothelial side of the corneal tissue cut with high energy (1.8 μJ) showing loss of endothelial cells morphology (g) the same corneal tissue at a higher magnification (×2200) showing damaged endothelial cell depleted of the plasma membrane with visible nucleus (N) and intracellular vesicles (arrows). (h) The same endothelium with numerous micro holes localized along intercellular spaces (arrows).
Figure 6
 
SEM images of a deswelled human cornea after fs laser cutting using high energy (1.8 μJ). (a) Cornea deswelled in THIN-C and cut with an fs laser using high 1.8 μJ energy showing a posterior stromal surface with irregularities and fissures; (b) the same tissue at higher magnification (×300), exhibiting damaged surface with deep superficial valleys and holes; (c) the same tissue at a higher magnification (×1000) showing undistinguishable collagen lamellae and fibers. (d) Transversal section of the same tissue with the upper stromal side showing a damaged white superficial layer estimated to be 8 to 10 μm. (e) The endothelial side of the corneal tissue cut with low energy (1.0 μJ) showing well preserved endothelial morphology. (f) The endothelial side of the corneal tissue cut with high energy (1.8 μJ) showing loss of endothelial cells morphology (g) the same corneal tissue at a higher magnification (×2200) showing damaged endothelial cell depleted of the plasma membrane with visible nucleus (N) and intracellular vesicles (arrows). (h) The same endothelium with numerous micro holes localized along intercellular spaces (arrows).
Figure 7
 
SEM images at low magnification (×30) of human corneas after fs laser cutting at different energy settings. Left column: The collagen fibers on the posterior stromal surface of corneas deswelled in THIN-C at 4°C for 4 hours and after fs laser cutting with (a) 1.0 μJ energy showing a normal collagen organization, (b) 1.2 μJ energy showing well distinguishable collagen fibers, and (c) 1.4 μJ energy showing damaged collagen with undistinguishable fibers. Right column: Collagen morphology on the posterior stromal surface of control corneas cut with an fs laser using (d) 1.0 μJ energy showing the superficial layer of denatured collagen and the well-preserved collagen organization in deeper layers (arrow), (e) 1.2 μJ showing increased collagen denaturation damage, and (f) 1.4 μJ showing a smooth layer of denatured collagen with undistinguishable fibers. At same energy settings deswelled tissues (left column) show better preservation of collagen morphology and organization as compared with control tissues (right column). Scale bars: 5 μm.
Figure 7
 
SEM images at low magnification (×30) of human corneas after fs laser cutting at different energy settings. Left column: The collagen fibers on the posterior stromal surface of corneas deswelled in THIN-C at 4°C for 4 hours and after fs laser cutting with (a) 1.0 μJ energy showing a normal collagen organization, (b) 1.2 μJ energy showing well distinguishable collagen fibers, and (c) 1.4 μJ energy showing damaged collagen with undistinguishable fibers. Right column: Collagen morphology on the posterior stromal surface of control corneas cut with an fs laser using (d) 1.0 μJ energy showing the superficial layer of denatured collagen and the well-preserved collagen organization in deeper layers (arrow), (e) 1.2 μJ showing increased collagen denaturation damage, and (f) 1.4 μJ showing a smooth layer of denatured collagen with undistinguishable fibers. At same energy settings deswelled tissues (left column) show better preservation of collagen morphology and organization as compared with control tissues (right column). Scale bars: 5 μm.
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