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Femtosecond laser-assisted conjunctival autograft preparation for pterygium surgery
Accepted Manuscript Femtosecond laser-assisted conjunctival autograft preparation for pterygium surgery Matthias Fuest, Yu-Chi Liu, Gary Hin-Fai Yam, Ericia Pei Wen Teo, Hla Myint Htoon, Minas T. Coroneo, Jodhbir S. Mehta PII:
S1542-0124(16)30112-4
DOI:
10.1016/j.jtos.2016.12.001
Reference:
JTOS 210
To appear in:
Ocular Surface
Received Date: 1 August 2016 Revised Date:
2 December 2016
Accepted Date: 2 December 2016
Please cite this article as: Fuest M, Liu Y-C, Yam GH-F, Teo EPW, Htoon HM, Coroneo MT, Mehta JS, Femtosecond laser-assisted conjunctival autograft preparation for pterygium surgery, Ocular Surface (2017), doi: 10.1016/j.jtos.2016.12.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT SECTION: Original Research, Ali Djalilian, MD, Editor TITLE: Femtosecond Laser-Assisted Conjunctival Autograft Preparation for Pterygium Surgery AUTHORS: Matthias Fuest, MD,1,2 Yu-Chi Liu, MD, MCI,1,3 Gary Hin-Fai Yam, PhD,1,4 Ericia Pei Wen Teo, BSc (Hons II),1 Hla Myint Htoon, PhD,1,4 Minas T Coroneo, MBBS,
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FRANZCO,5 Jodhbir S Mehta, MBBS1,3,4,6 Running Head: Fs-assisted CAG preparation /Fuest et al
Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore
2
Department of Ophthalmology, RWTH Aachen University, Aachen, Germany
3
Singapore National Eye Centre, Singapore
4
Eye-ACP, Duke-NUS Graduate Medical School, Singapore
5
Faculty of Medicine, University of New South Wales, Australia
6
School of Materials Science and Engineering, Nanyang Technological University,
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1
Singapore FOOTNOTES
Accepted for publication November 2016.
in this article.
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The authors have no commercial or proprietary interest in any product or concept discussed
This study was supported by the Singapore National Research Foundation Grant NMRC TCR 1021.
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Corresponding author: Assoc. Prof. Jodhbir S. Mehta, Singapore National Eye Centre, 11 Third Hospital Avenue, Singapore 168751. E-mail: [email protected]. Telephone: +65
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62277266. Fax: +65 62261884.
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ACCEPTED MANUSCRIPT Abstract Purpose: Pterygium is a common ocular surface disorder. Conjunctival autografting (CAG) following pterygium resection is the gold standard treatment. CAGs without Tenon’s tissue provide better results but are more technically difficult to achieve. In this study, we evaluated the feasibility and reproducibility of femtosecond laser (FSL)-assisted
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CAG preparation. Methods: Fifteen porcine globes were fixed in a suction holder and CAGs of different
diameters were created by 1) an experienced consultant and 2) a less experienced fellow
using the Ziemer LDV Z8. The CAG´s dimension was measured and thickness analyzed by
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optical coherence tomography (OCT) and histology (HE). Statistical analysis was performed by Mann-Whitney-U, Wilcoxon and Spearman-test.
Results: FSL-assisted CAGs prepared at 100 µm (146.4 ±45.7µm) showed a significantly
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higher deviation from desired depth (p=.04) and a higher variability (p=.03) in thickness than those prepared at 60 µm (71.4 ±12.7 µm). The experienced (68.3 ±14.3 µm) and inexperienced surgeon (73.9 ±11.9 µm) produced 60 µm grafts of comparable thickness (p=.6) and variability (p=.7). The CAG area measured after dissection (37.5 ±12.1mm2) did not differ significantly from the FSL settings (40.6 ±12.7mm2, p=.3). FSL cutting time
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at 60 µm took 18.1 ±2.2s, at 100 µm 20.7 ±2.4s. Graft separation time was not significantly influenced by depth or surgeon. No buttonholes or CAG tags occurred during surgery. Conclusions: The FSL allowed the accurate and reliable preparation of very thin CAGs,
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independent of surgeon experience and may represent a valuable tool in pterygium surgery.
Outline
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KEY WORDS conjunctiva, femtosecond laser, graft, pterygium
I.
Introduction
II.
Methods A.
Porcine eyes
B.
Graft preparation
C.
Optical Coherence Tomography (OCT)
D.
Histology and Hematoxylin-Eosin histochemistry (HE)
E.
Immunohistochemistry
F.
Statistical analysis
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ACCEPTED MANUSCRIPT Results A.
Creation of autografts
B.
Optical Coherence Tomography measurements
C.
Histology measurements
D.
Immunohistochemistry
Discussion
V.
Conclusion
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IV.
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III.
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I.
Introduction
A pterygium is a wing-shaped corneal incursion of an aberrant conjunctival wound healing response, characterized by the centripetal growth of a squamous metaplastic
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epithelium with goblet cell hyperplasia and an underlying stroma of activated proliferating fibroblasts, neovascularization, inflammatory cells, and extracellular matrix.1, 2 Although
seen worldwide, it is commonly found in the peri-equatorial latitudes, at high altitude and in highly reflective environments, due to elevated levels of ultraviolet (UV) radiation, a
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known risk factor.3 The proliferation of pterygium tissue can cause changes in corneal
topography and refraction, occlusion of the visual axis, as well as visual field loss, corneal scarring and consequently reduction in visual acuity.1, 4 In advanced cases, scarring of the
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ocular surface can also lead to symblepharon formation, reduction of motility, and diplopia.5 In addition, pterygium can cause chronic inflammation, a dry eye state, and discomfort for the patient.6
Currently the management of pterygium is surgical. Ophthalmic surgeons have been treating this disease since 1000 BC.7 For many years a bare sclera technique, in which
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the pterygium was simply excised from the cornea, leaving only bare sclera exposed, was the standard approach.8 However, there were high recurrence rates, up to 88%, 9 months after surgery.9 Hence, alternative surgical procedures were sought, including the use of amniotic membrane (AM) or a conjunctival autograft (CAG) onto the bare sclera. This led
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to a significant reduction in recurrence rates.10, 11 A recent Cochrane meta-analysis of 1947 eyes of 1866 patients revealed superiority of CAG over AM, with risk ratio for recurrence
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of pterygium (primary and recurrent) of 0.87 and 0.53 at 3 and 6 months follow-up intervals and recurrence rates from 3.33% to 16.7% in the CAG and 2.6% to 42.3% in the AM groups at 6 months.11
It has been shown that the outcome of pterygium surgery is dependent on several factors, including thorough Tenon´s tissue removal and adequate management of the ocular surface,8 as well as on the surgical experience in harvesting the CAG, with greater experience leading to lower recurrence rates and fewer complications.12-14 An important factor for success in pterygium surgery is the ability to dissect a thin and adequately sized graft to cover the conjunctival defect with minimal inclusion of
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ACCEPTED MANUSCRIPT Tenon’s tissue.13 The resulting thin, tension-free grafts have been shown to not subsequently retract after surgery, providing a good cosmetic outcome.13 Femtosecond lasers (FSLs) have been employed in ophthalmic surgery since 2001, and more recently in cataract surgery,15, 16 but the most widespread application remains in corneal refractive surgery.17 FSLs have shown greater precision in corneal flap diameter
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and thickness creation and a more uniform flap thickness in comparison to mechanical microkeratomes.18, 19 In an attempt to transfer these favorable characteristics of FSLs to
pterygium surgery, we aimed to study and evaluate the feasibility of the creation of thin and
new technique.
A.
Porcine eyes
Methods
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II.
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uniform CAGs by FSLs and to investigate possible effects of surgeon experience on this
Fresh, whole cadaveric eyes from 24-week old pigs were obtained from an abattoir with a permit from Agri-Food & Veterinary Authority of Singapore. Eyes were kept in chilled balanced salt solution (BSS, Amo Endosol, Abbott Laboratories, Abbott Park,
enucleation.
Graft preparation
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B.
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USA) during both transit and dissection. Eyes were processed within 8 hours after
Porcine eyes were fixed in a suction holder (Figure 1A). Bulbi were pressurized by
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injecting (21G hypodermic needle, B. Braun, Melsungen, Germany) BSS from an infusion height of 27 cm above the eye (20 mmHg), to allow for easier tissue manipulation. The FSL-assisted CAG creation process is shown in Figure 1B and Figure 2. Briefly, peripheral Tenon´s tissue and conjunctiva were fixed to the sclera by 5-0 nylon sutures (Ethicon Inc, Somerville, NJ, USA), the designated CAG center was marked with a marker, and 10 ellipsoid CAGs of different diameters (6 x 5 cm – 9 x 8 cm) were created by an experienced consultant (JSM), using the Ziemer LDV Z8 (5 at 100 µm, 5 at 60 µm depth) without suction.20 The Z8 dissected the CAGs with adjunctive 1 µm laser spots, a frequency of 10 MHz, 100 % energy for the horizontal stroma and 130% for the side cuts.20
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ACCEPTED MANUSCRIPT The CAG´s length and width were measured immediately after dissection using a surgical calliper, and the CAG´s area was calculated (area ellipse: π x length/2·x height/2). The corner of the CAG was then slightly elevated by forceps, and closed scissors entered into the CAG plane to separate the flap form the underlying Tenon´s tissue; cutting by scissors was not permitted. The CAG was then lifted by 90% of extension, maintaining a small
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conjunctival bridge. After marking of the underlying Tenon´s tissue for histology purposes, the conjunctival flap was resutured (nylon 10-0, Ethicon) to the surrounding conjunctiva. The procedure was repeated by a less experienced fellow (YCL, 5 at 60 µm depth). FSL
were recorded.
Optical Coherence Tomography
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dissection times and time required to manually separate the CAG from underlying tissue
Directly after FSL-CAG preparation, a viscoelastic gel (Viscoat, Alcon, Fort Worth, USA) was injected underneath the CAG to cleave the FSL plane (Figure 3). Three optical coherence tomography (OCT) measurements were taken for the central and the peripheral (2 mm to each side) thickness of the CAG by anterior segment spectral-domain OCT
D.
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(Figure 3B, RTVue, Optovue, Fremont, USA).
Histology and Hematoxylin-Eosin histochemistry (HE)
The CAGs and underlying Tenon´s tissue and sclera were excised with a 1-3 mm
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margin, fixed in 10% buffered formalin (Sigma-Aldrich, St. Louis, USA) for 6 hours, and stored in 70% ethanol (Merck, Darmstadt, Germany) overnight. Tissue samples were then dehydrated through a series of ascending concentrations until absolute ethanol, with each
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change of 30 min, cleared in xylene (Thermo Fisher Scientific, Massachusetts, USA) and infiltrated and embedded in paraffin (Thermo Fisher Scientific). Serial sections of 5 µm in thickness were obtained by microtome (Leica RM 2135; Leica Micro Systems, Wetzlar, Germany). After discarding of the sections adjacent to the cut edge (to avoid crush artefacts), the sections were mounted on glass slides, processed for hematoxylin and eosin histochemistry (HE, Sigma-Aldrich). Images were scanned and captured using the Eclipse Ti-E inverted microscope (Nikon Instruments Inc., Melville, USA). The HE-CAG thickness was measured (central and 250 µm to each side) using NIS Elements software (Nikon Instruments Inc.) (Figure 3A). All OCT and HE thickness measurements were taken by an investigator (MF) masked for designated flap thickness and surgeon. 6
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E.
Immunohistochemistry
Sections were deparaffinized and rehydrated. After rinses in 0.01 M PBS (First Base, Singapore), sections were treated with 0.025% trypsin-EDTA (Life Technologies, Carlsbad, USA) at 37°C for 5 minutes for antigen retrieval. After further PBS washes, the
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samples were blocked with 5% bovine serum albumin (Sigma-Aldrich) and 0.1% Triton X100 (Sigma-Aldrich) in PBS, followed by incubation in AlexaFluor 568 Griffonia
simplicifolia isolectin 4 (GSI-B4) conjugate (Life Technologies) for 30 minutes at room temperature, which stains N-acetyl-D-galactosamine end groups and terminal α-D-
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galactosyl residues,21 commonly found in porcine perivascular and endothelial vessel
cells.22 After PBS washes, the sections were mounted with UltraCruz Mounting Medium
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containing 4',6-diamidino-2-phenylindole (DAPI ,Santa Cruz Biotech, Dallas, USA), examined and imaged under fluorescence microscopy (Figure 5B and C, AxioImager Z1; Carl Zeiss, Oberkochen, Germany). Porcine limbal tissue with its´ inherent vasculature served as a positive control (Figure 5A).
Statistical Analysis
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F.
All statistical analysis was performed by the Statistical Package for the Social Sciences version 22.0 (SPSS, Inc., Chicago, USA). Values were expressed as mean ± standard deviation. Intergroup comparison was performed using the Mann-Whitney-U test.
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Central and peripheral measurements, FSL dissection times, as well as OCT and HE values, were compared with the Wilcoxon signed-rank test. The correlation between OCT and HE thickness measurements, as well as between FSL dissection/separation times and
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CAG area, were calculated by the Spearman test. A p-value of less than .05 was considered statistically significant.
A.
III.
Results
Creation of Autografts
The CAG area measured after dissection (37.5 ±12.1 mm2) did not significantly differ from the FSL settings (40.6 ±12.7 mm2, p=.3). No buttonholes or CAG tags occurred during surgery. Calculating the FSL dissection times for 7 ellipsoid CAGs (mean area 43.6 ±10.3 mm2) measuring from 6 x 5 mm (23.6 mm2) to 9 x 8 mm (56.5 mm2) the preparation
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ACCEPTED MANUSCRIPT of a 60 µm deep CAG took on average 2.6 seconds shorter than the preparation of a same sized CAG at 100 µm (mean time 60 µm 18.1 ±2.2 s; mean time 100 µm 20.7 ±2.4 s, p<.001). Dissection time was highly correlated to CAG area (60 and 100 µm r=.96, p<.001). There was no significant difference in graft separation time following FSL dissection between the consultant´s 100 (mean 59.6 ±8.3 s) and 60 µm CAGs (mean 42.4
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±6.9 s, p=.17), nor between consultant and fellow (mean 39.4 ±6.4 s) at 60 µm dissection depth (p=.75). Separation time and CAG area were not significantly correlated (r=.66, p=.16).
Optical Coherence Tomography Measurements
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B.
Optical coherence tomography measurements of FSL-assisted CAGs prepared at
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100 µm (mean 146.4 ±45.7 µm) showed a significantly higher deviation from the designated cutting depth (p=.04) and a higher variability (p=.03) in thickness than those prepared at 60 µm (mean 71.4 ±12.7 µm, Figure 4). Measurements of the periphery did not differ significantly from central values at 60 µm (mean periphery 78.6 ±9.4 µm; p=.06) or 100 µm cutting depth (mean periphery 156.0 ±46.0 µm; p=.5). The resulting central/peripheral ratios (CP) of 0.91 ±0.13 for 60 µm and 0.94 ±0.18 for 100 µm grafts did
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not differ between the two groups either (p=.52). Experienced (mean 68.3 ±14.3 µm) and less experienced surgeon (mean 73.9 ±11.9 µm) produced 60 µm grafts of comparable
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thickness (p=.6) and variability (p=.7, Figure 4).
Histology Measurements
Central histology and OCT thickness measurements were highly correlated (r=.89,
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p=.007), yet HE (after ethanol dehydration) measured in general 16.0 ±16.5 µm thinner than OCT (p=.04), 28.8 ±18.5 µm for 100 µm and 10.9 ±4.9 µm for 60 µm grafts.
D.
Immunohistochemistry
The positive control, using limbal tissue, revealed a specific labelling of porcine vasculature by GSI-B4 (red, Figure 5A). The porcine conjunctival epithelium also stained GSI-B4 positive (Figure 5A and B). CAGs dissected with the FSL at 60 µm thickness showed maintenance of conjunctival stromal vasculature with both large (Figure 5B) and small caliber vessels (Figure 5C).
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IV.
Discussion
In this study we demonstrated the successful preparation of CAGs by FSL in a
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porcine whole globe model. Accuracy and reliability proved higher for dissection depths of 60 µm than 100 µm with no significant differences between experienced and inexperienced surgeons. The CAG area measured after dissection did not significantly differ from the FSL settings. FSL cutting times at 60 µm depth took on average 2.6 seconds less than the
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cutting of a same-sized CAG at 100 µm. Mean graft separation time took less than one
minute for all cases and did not differ between the consultant´s 100 µm and 60 µm CAGs or between consultant and fellow, nor was it correlated to the CAG area.
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The ultra-short near-infrared pulses of the FSL pass through transparent tissues at low power densities without exerting significant collateral damage. However, at higher power densities, the structures absorb light energy, leading to plasma generation and tissue disruption, vaporizing small volumes of tissue with the formation of cavitation gas bubbles consisting of carbon dioxide and water (Figures 1B and 2B).23 To a limited degree, the FSL
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is also capable of passing through optically hazy media, such as edematous cornea and even the relatively translucent perilimbal sclera, because its infrared-wavelength energy undergoes much less attenuation than visible light.17 The conjunctiva is considered a translucent tissue. Gardiner found that age, sex, and nutrition influenced the grade of
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conjunctival transparency, which gradually increased with age as a sign of progressive atrophy of subepithelial layers.24 Hence we hypothesized, that it may be possible to make a
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conjunctival graft using an FSL.
Manual CAG creation represents a difficult and time-consuming part of the conventional pterygium surgical procedure.14 Several studies have shown a correlation of surgeon experience with lower recurrence and intraoperative complication rates.12, 13 Farrah et al showed that the recurrence rate produced by a trainee ophthalmologist (19.4%) was more than double that of an experienced ophthalmologist (6.8%). In addition, trainees caused 3.5 times more surgical complications.12 Ti et al retrospectively analyzed 139 cases with primary and 64 cases with recurrent pterygia and found the recurrence rates to vary widely among surgeons, from 5% to 82%. The recurrence rates were found to be inversely related to previous experience of the surgeon in performing conjunctival grafting. The
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ACCEPTED MANUSCRIPT author inferred that an adequately sized graft with minimum Tenon’s tissue (i.e., a thin graft) required considerable surgical skill and was associated with a substantial learning curve.13 This conclusion was also recently supported by Kuo et al, who demonstrated in a porcine cadaveric eye teaching model that the conjunctival graft thickness diminished significantly with the experience of the trainee, who needed approximately 50 attempts to
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reach CAG thicknesses of 87 ±23 µm.14 Using the same model, we were able to show that the FSL allowed the accurate cutting of ultra-thin CAGs with no significant influence of
surgeon experience and higher accuracy and reliability for dissection depths of 60 µm than 100 µm.
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There are no previous published studies that have correlated intraoperative CAG thickness with recurrence rates and complications. However, two recent studies investigated the postoperative CAG thickness after pterygium surgery using OCT.
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Kheirkhah et al found that the average thickness of the grafts decreased from 458 ±171 µm at 1 week after surgery to 291 ±124 µm at 3 months.25 Ozgurhan et al described similar results in 20 patients with a mean graft thickness of 430 ±127 µm in primary and 461 ±178 µm in recurrent pterygium at 1 week after surgery, which decreased to 109 ±15 µm and 107 ±18 µm at 3 months after surgery.26 The results suggested that the CAG undergoes
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significant swelling after surgery that requires approximately 3 months to deterge. The values at 3 months showed a wide range between 107 - 291 µm, but might give an idea of the actual CAG thickness transplanted. FSL assistance in our study allowed the reproducible production of CAGs with little variability in dissection depth, particularly at
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60 µm.
We were first able to demonstrate that FSL allowed the accurate and reliable
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preparation of very thin (60 µm) CAGs. The accuracy was higher and variability lower at a dissection depth of 60 µm in comparison to 100 µm, which we attribute to an increasing light scattering and absorption of the laser with conjunctival cutting depth.27 Tse et al showed that cutting depths up to 1 mm are possible in a porcine skin model. They also found that the smaller the numerical aperture (NA), the lower the precision of the cut.27 Hence, not all current commercially available FSLs may be able to achieve the results obtained from this study. The Ziemer LDV Z8 provides a large NA and a low energy level, permitting accurate cuts. The device also proved suitable for the preparation of FSLassisted CAGs as the handheld laser (Figure 1) allowed free positioning on the ocular surface and the conjunctival laser photodisruption can be performed without the use of suction.28 In order to ensure appropriate centration, the conjunctiva was marked in the 10
ACCEPTED MANUSCRIPT center of the intended graft before the laser head was docked. The laser head was held steady by the surgeons during the photodisruption. Kuo et al demonstrated in a porcine model that a trainee could reduce manual CAG preparation time from 191 to 126 seconds over 58 (5 x 5 mm) grafts.14 The inexperienced surgeon in our study, having never used the FSL before, needed on average only 57.5
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seconds for laser and preparation of the first 5 CAGs at 60 µm performed. Even though the area of the dissected CAG correlated with the laser dissection time, the actual surgical
times are clinically less meaningful due to speed of graft creation (e.g., 21 seconds for 9 x 8 mm and 14 seconds for 6 x 5 mm graft). Manual removal of the graft, following FSL
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dissection, and graft area were not significantly correlated. The conjunctival graft was
simply peeled from the underlying Tenon’s tissue and did not require meticulous surgical dissection as in manual CAG preparation. Certainly the use of an FSL involves additional
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preparation time and programming, which we did not record in this study, but this is no longer than for a standard refractive procedure and can be done in advance. The FSLassisted CAG procedure with the LDV Z8 did not demand a high level of experience in the use of FSL or pterygium surgery, as the CAGs prepared by a fellow at 60 µm did not differ significantly in thickness or variability from the experienced surgeon´s cases. The FSL consequently did not require a difficult and time-consuming learning curve as shown by
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Kuo et al.14 In addition, with the FSL, the dissection can be performed at any programmed depth.14 In an attempt to increase precision, maneuverability and ergonomics, Bourcier et al investigated the possibility of robot-assisted pterygium surgery relying on the DaVinci
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surgical system in a porcine model29 and recently in the first patient.30 While they found the procedure to be feasible and without complications, the surgery time (porcine model 36
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minutes, patient 60.5 minutes) still limits its application in routine clinical cases. In particular, the CAG preparation time in the porcine model (7 minutes) took substantially longer than our FSL-assisted grafts. The human conjunctiva consists of a nonkeratinized epithelium and a vascularized stroma.31 OCT measurements revealed variability in overall conjunctival thickness of 197.7 ±32.5 µm32 to 270 ±90 µm,31 increasing from limbus to periphery and being thicker in the nasal than temporal region.31 The conjunctival epithelium was measured at 42.4 ±7.4 µm32 to 61 ±12 µm.25 However, appreciating the exact transition from epithelium to stroma in OCT images can be difficult, and confocal microscopy found thinner epithelial thicknesses of 32.9 ± 1.1µm.33 In our study, setting the FSL to a cutting depth of 60 µm, histology showed that the dissection plane was in the conjunctival stroma in all cases (Figure 3A). 11
ACCEPTED MANUSCRIPT However, the preparation of even thinner CAGs could bear the danger of intraepithelial spiting. While thinner CAGs with less adjacent Tenon´s tissue are associated with lower recurrence rates,13 remaining vascular tissue in a free skin graft has been shown to promote an early vascularization of the new graft.34 Assuming similarities between skin and
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conjunctival grafts, excessively thin CAG´s with little and/or damaged vasculature
consequently might incur higher complication rates, such as graft necrosis or scarring. In
our ultra-thin 60 µm CAGs, we were able to demonstrate the persistence of small and midsize stromal vessels by immunohistochemistry (Figure 5). While the isolectin-B4
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glycoprotein originally isolated from the seeds of the tropical African legume Griffonia simplicifolia has proven high affinity to porcine perivascular and vascular endothelial
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cells,22 it has also been shown to interact with other cell types such as neurons or epithelial cells,35 which also occurred in our sections.
Older generation low frequency (15kHz) high energy FSLs have previously been reported to cause more keratocyte cell necrosis and consequently inflammation in corneal incisions,36, 37 which could raise concerns of elevated scarring and recurrence rates in FSLassisted pterygium surgery (FLAPS). However, the use of lower energy level FSL has
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significantly attenuated the inflammation and apoptosis adjacent to the photo-disruptive site.38 The Ziemer Z8 is a low energy (50 – 2500 nJ) high frequency (0.1 - 10 MHz) laser and previous studies have shown minimal signs of inflammation in animal, corneal incision models.20 Nevertheless, human scarring responses and recurrence rates in FLAPS can only
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be evaluated in clinical trials.
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Our study is limited by the utilization of a porcine model with possible anatomical and mechanical differences to human conjunctiva, which, to date, have not been investigated in detail. However, at least for porcine sclera, a high similarity in histology and collagen bundle organization to human eyes has been shown.39 In addition, pig eyes have proven to be realistic and valuable surgical models and allowed us to do a direct comparison to previous reports using conventional manual CAGs.14, 29, 40 We also evaluated the CAG thickness by 2 methods, as the conjunctival tissue was subjected to significant shrinking due to alcohol dehydration during tissue fixation with standard histology preparation.41 Consequently the OCT measurements obtained provided a more realistic estimation of intraoperative graft thicknesses.
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ACCEPTED MANUSCRIPT While FLAPS is currently experimental, given the growing use of lasers in ophthalmic surgery, the quick, reliable and automated preparation of CAGs by FSL might assist in further standardizing the surgical procedure and reducing intraoperative and postoperative complications.
Conclusion
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We have shown that CAGs could be prepared with the Ziemer LDV Z8 FSL.
Accuracy, reliability and speed of graft preparation were higher for dissection depths of 60 rather than 100 µm irrespective of surgeon experience. Clinical trials are currently
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underway (NCT02866968), to show whether these ultra-thin FSL-assisted CAGs do result in good clinical cosmetic outcomes and low recurrence rates.
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References
1. Kim KW, Park SH, Kim JC. Fibroblast biology in pterygia. Exp Eye Res 2016;142:32-39 2. Di Girolamo N, Chui J, Coroneo MT, Wakefield D. Pathogenesis of pterygia: role of cytokines, growth factors, and matrix metalloproteinases. Prog Retin Eye Res 2004;23:195-228 3. Saw SM, Tan D. Pterygium: prevalence, demography and risk factors. Ophthalmic Epidemiol 1999;6:219-228
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4. Chui J, Coroneo MT, Tat LT, et al. Ophthalmic pterygium: a stem cell disorder with premalignant features. Am J Pathol 2011;178:817-827 5. Shimazaki J, Shinozaki N, Tsubota K. Transplantation of amniotic membrane and limbal autograft for patients with recurrent pterygium associated with symblepharon. Br J Ophthalmol
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1998;82:235-240
6. Coroneo MT, Chui JJY. Pterygium, in: Holland EJ, Mannis MJ, Lee WB (eds). Ocular surface disease: cornea, conjunctiva and tear film: Elsevier Health Sciences; 2013:125-144.
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7. Rosenthal JW. Chronology of pterygium therapy. Am J Ophthalmol 1953;36:1601-1616 8. Hirst LW. The treatment of pterygium. Surv Ophthalmol 2003;48:145-180 9. Chen PP, Ariyasu RG, Kaza V, et al. A randomized trial comparing mitomycin C and conjunctival autograft after excision of primary pterygium. Am J Ophthalmol 1995;120:151-160 10. Kaufman SC, Jacobs DS, Lee WB, et al. Options and adjuvants in surgery for pterygium: a report by the American Academy of Ophthalmology. Ophthalmology 2013;120:201-208 11. Clearfield E, Muthappan V, Wang X, Kuo IC. Conjunctival autograft for pterygium. Cochrane Database Syst Rev 2016;2:CD011349 12. Farrah JJ, Lee GA, Greenrod E, Vieira J. Outcomes of autoconjunctival grafting for primary pterygia when performed by consultant compared with trainee ophthalmologists. Clin Experiment Ophthalmol 2006;34:857-860 13
ACCEPTED MANUSCRIPT 13. Ti SE, Chee SP, Dear KB, Tan DT. Analysis of variation in success rates in conjunctival autografting for primary and recurrent pterygium. Br J Ophthalmol 2000;84:385-389 14. Kuo MX, Sarris M, Coroneo MT. Cadaveric porcine model for teaching and practicing conjunctival autograft creation. Cornea 2015;34:824-828 15. Trikha S, Turnbull AM, Morris RJ, et al. The journey to femtosecond laser-assisted cataract surgery: new beginnings or a false dawn? Eye (Lond) 2013;27:461-473
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16. Pahlitzsch M, Torun N, Pahlitzsch ML, et al. Impact of the femtosecond laser in line with the femtosecond laser-assisted cataract surgery (FLACS) on the anterior chamber characteristics in
comparison to the manual phacoemulsification. Semin Ophthalmol 2016; Apr 19:0. [Epub ahead of print] 17. Soong HK, Malta JB. Femtosecond lasers in ophthalmology. Am J Ophthalmol
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2009;147:189-197 e182
18. Zhou Y, Zhang J, Tian L, Zhai C. Comparison of the Ziemer FEMTO LDV femtosecond laser and Moria M2 mechanical microkeratome. J Refract Surg 2012;28:189-194
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19. Santhiago MR, Kara-Junior N, Waring GO. Microkeratome versus femtosecond flaps: accuracy and complications. Curr Opin Ophthalmol 2014;25:270-274
20. Riau AK, Liu YC, Lwin NC, et al. Comparative study of nJ- and muJ-energy level femtosecond lasers: evaluation of flap adhesion strength, stromal bed quality, and tissue responses. Invest Ophthalmol Vis Sci 2014;55:3186-3194
21. Murphy LA, Goldstein IJ. Five alpha-D-galactopyranosyl-binding isolectins from Bandeiraea
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simplicifolia seeds. J Biol Chem 1977;252:4739-4742
22. Kirkeby S, Moe D. Binding of Griffonia simplicifolia 1 isolectin B4 (GS1 B4) to alphagalactose antigens. Immunol Cell Biol 2001;79:121-127 23. Juhasz T, Loesel FH, Kurtz RM, et al. Corneal refractive surgery with femtosecond lasers. IEEE
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J Sel Top Quantum Electron 1999;5:902-910
24. Gardiner PA. Observations on the transparency of the conjunctiva. Br J Ophthalmol 1944;28:538-554
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25. Kheirkhah A, Adelpour M, Nikdel M, et al. Evaluation of conjunctival graft thickness after pterygium surgery by anterior segment optical coherence tomography. Curr Eye Res 2011;36:782786
26. Ozgurhan EB, Kara N, Bozkurt E, et al. Comparison of conjunctival graft thickness after primary and recurrent pterygium surgery: Anterior segment optical coherence tomography study. Indian J Ophthalmol 2014;62:675-679 27. Tse C, Zohdy MJ, Ye JY, O'Donnell M. Penetration and precision of subsurface photodisruption in porcine skin tissue with infrared femtosecond laser pulses. IEEE Trans Biomed Eng 2008;55:1211-1218 28. Pepose J, Lubatschowski H. Comparing femtosecond lasers. Cataract Refract Surg Today 2008;10:45-52
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ACCEPTED MANUSCRIPT 29. Bourcier T, Nardin M, Sauer A, et al. Robot-assisted pterygium surgery: feasibility study in a nonliving porcine model. Transl Vis Sci Technol 2015;4:9 30. Bourcier T, Chammas J, Becmeur PH, et al. Robotically assisted pterygium surgery: first human case. Cornea 2015;34:1329-1330 31. Read SA, Alonso-Caneiro D, Vincent SJ, et al. Anterior eye tissue morphology: Scleral and conjunctival thickness in children and young adults. Sci Rep 2016;6:33796
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32. Zhang X, Li Q, Xiang M, et al. Bulbar conjunctival thickness measurements with optical
coherence tomography in healthy chinese subjects. Invest Ophthalmol Vis Sci 2013;54:4705-4709 33. Efron N, Al-Dossari M, Pritchard N. Confocal microscopy of the bulbar conjunctiva in contact lens wear. Cornea 2010;29:43-52
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34. Calcagni M, Althaus MK, Knapik AD, et al. In vivo visualization of the origination of skin graft vasculature in a wild-type/GFP crossover model. Microvasc Res 2011;82:237-245
35. Shimizu T, Nettesheim P, Mahler JF, Randell SH. Cell type-specific lectin staining of the
B4. J Histochem Cytochem 1991;39:7-14
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tracheobronchial epithelium of the rat: quantitative studies with Griffonia simplicifolia I isolectin
36. Netto MV, Mohan RR, Medeiros FW, et al. Femtosecond laser and microkeratome corneal flaps: comparison of stromal wound healing and inflammation. J Refract Surg 2007;23:667-676 37. Angunawela RI, Riau A, Chaurasia SS,et al. Manual suction trephination versus femtosecond laser corneal trephination: intraocular pressure, endothelial cell loss and wound healing responses.
22427557
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Invest Ophthalmol Vis Sci. 2012 May 4;53(6):2571-9. doi: 10.1167/iovs.11-8403. PMID:
38. de Medeiros FW, Kaur H, Agrawal V, et al. Effect of femtosecond laser energy level on corneal stromal cell death and inflammation. J Refract Surg 2009;25:869-874
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39. Nicoli S, Ferrari G, Quarta M, et al. Porcine sclera as a model of human sclera for in vitro transport experiments: histology, SEM, and comparative permeability. Mol Vis 2009;15:259-266 40. Lee GA, Chiang MY, Shah P. Pig eye trabeculectomy-a wet-lab teaching model. Eye (Lond)
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2006;20:32-37
41. Bahr GF, Bloom G, Friberg U. Volume changes of tissues in physiological fluids during fixation in osmium tetroxide or formaldehyde and during subsequent treatment. Exp Cell Res 1957;12:342-355
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ACCEPTED MANUSCRIPT Figure legends Figure 1. A. Porcine bulbi were fixed in a suction holder. Conjunctival autografts were then dissected with the Ziemer LDV Z8. B. Close-up of the surgery site after femtosecond laser preparation. The black arrow indicates typical cavitation bubbles, marking the corner of the
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graft.
Figure 2. Steps of the femtosecond laser-assisted conjunctival autograft (CAG) creation. A. Peripheral Tenon´s tissue and conjunctiva was fixed to the sclera by 5-0 nylon sutures. B.
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The designated CAG center was marked with a marker and ellipsoid CAGs of different
diameters created using the Ziemer LDV Z8, the black arrows show the corner of the CAG with characteristic cavitation bubbles. C. The corner of the CAG was slightly elevated by
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forceps and closed scissors entered into the CAG plane, cutting by scissors was not permitted. D. 90% of the CAG was then lifted, leaving a small conjunctival bridge. (E. The underlying Tenon´s tissue was marked. F. The conjunctival flap re-sutured (nylon 10-0) to the surrounding conjunctiva.
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Figure 3. Directly after the preparation of the porcine conjunctival autograft (CAG) and resuturing it to the surrounding conjunctiva, a viscoelastic gel was injected in the dissection plane to improve optical coherence tomography (OCT) visualization (B). Three measurements (central and 250 µm to each side) were then taken of the central and the
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peripheral (2 mm to each side) thickness of the CAG. The CAGs were then fixed and dissected for histology (A). The CAG dissection plane was in the conjunctival stroma in all
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cases. Three central measurements of the histology samples were taken (central and 250 µm to each side).
Figure 4. Box-plots of the mean central conjunctival autograft (CAG) thickness for 100 and 60 µm grafts prepared by consultant or fellow (A) and the resulting deviation from the desired dissection depth (B). FS-assisted CAGs prepared at 100 µm showed a significantly higher deviation from the designated cutting depth (p=.04) and a higher variability (p=.03) in thickness than those prepared at 60 µm. Experienced and less experienced surgeon produced 60 µm grafts of comparable thickness (p=.6) and variability (p=.7).
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ACCEPTED MANUSCRIPT Figure 5. Immunohistochemistry with Griffonia simplicifolia isolectin 4 conjugate (GSIB4, red) and 4',6-diamidino-2-phenylindole (DAPI, blue). A. Staining of the limbal area revealed a high specificity of GSI-B4 for porcine vessels. The porcine conjunctival epithelium also stained GSI-B4 positive (e.g., in B). Conjunctival autografts dissected with the FS at 60 µm thickness showed maintenance of conjunctival stromal vasculature with
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Figure 1
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Figures
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both large (B) and small calibre vessels (C).
Figure 2
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S1542-0124(16)30112-4
DOI:
10.1016/j.jtos.2016.12.001
Reference:
JTOS 210
To appear in:
Ocular Surface
Received Date: 1 August 2016 Revised Date:
2 December 2016
Accepted Date: 2 December 2016
Please cite this article as: Fuest M, Liu Y-C, Yam GH-F, Teo EPW, Htoon HM, Coroneo MT, Mehta JS, Femtosecond laser-assisted conjunctival autograft preparation for pterygium surgery, Ocular Surface (2017), doi: 10.1016/j.jtos.2016.12.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT SECTION: Original Research, Ali Djalilian, MD, Editor TITLE: Femtosecond Laser-Assisted Conjunctival Autograft Preparation for Pterygium Surgery AUTHORS: Matthias Fuest, MD,1,2 Yu-Chi Liu, MD, MCI,1,3 Gary Hin-Fai Yam, PhD,1,4 Ericia Pei Wen Teo, BSc (Hons II),1 Hla Myint Htoon, PhD,1,4 Minas T Coroneo, MBBS,
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FRANZCO,5 Jodhbir S Mehta, MBBS1,3,4,6 Running Head: Fs-assisted CAG preparation /Fuest et al
Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore
2
Department of Ophthalmology, RWTH Aachen University, Aachen, Germany
3
Singapore National Eye Centre, Singapore
4
Eye-ACP, Duke-NUS Graduate Medical School, Singapore
5
Faculty of Medicine, University of New South Wales, Australia
6
School of Materials Science and Engineering, Nanyang Technological University,
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1
Singapore FOOTNOTES
Accepted for publication November 2016.
in this article.
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The authors have no commercial or proprietary interest in any product or concept discussed
This study was supported by the Singapore National Research Foundation Grant NMRC TCR 1021.
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Corresponding author: Assoc. Prof. Jodhbir S. Mehta, Singapore National Eye Centre, 11 Third Hospital Avenue, Singapore 168751. E-mail: [email protected]. Telephone: +65
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62277266. Fax: +65 62261884.
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ACCEPTED MANUSCRIPT Abstract Purpose: Pterygium is a common ocular surface disorder. Conjunctival autografting (CAG) following pterygium resection is the gold standard treatment. CAGs without Tenon’s tissue provide better results but are more technically difficult to achieve. In this study, we evaluated the feasibility and reproducibility of femtosecond laser (FSL)-assisted
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CAG preparation. Methods: Fifteen porcine globes were fixed in a suction holder and CAGs of different
diameters were created by 1) an experienced consultant and 2) a less experienced fellow
using the Ziemer LDV Z8. The CAG´s dimension was measured and thickness analyzed by
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optical coherence tomography (OCT) and histology (HE). Statistical analysis was performed by Mann-Whitney-U, Wilcoxon and Spearman-test.
Results: FSL-assisted CAGs prepared at 100 µm (146.4 ±45.7µm) showed a significantly
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higher deviation from desired depth (p=.04) and a higher variability (p=.03) in thickness than those prepared at 60 µm (71.4 ±12.7 µm). The experienced (68.3 ±14.3 µm) and inexperienced surgeon (73.9 ±11.9 µm) produced 60 µm grafts of comparable thickness (p=.6) and variability (p=.7). The CAG area measured after dissection (37.5 ±12.1mm2) did not differ significantly from the FSL settings (40.6 ±12.7mm2, p=.3). FSL cutting time
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at 60 µm took 18.1 ±2.2s, at 100 µm 20.7 ±2.4s. Graft separation time was not significantly influenced by depth or surgeon. No buttonholes or CAG tags occurred during surgery. Conclusions: The FSL allowed the accurate and reliable preparation of very thin CAGs,
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independent of surgeon experience and may represent a valuable tool in pterygium surgery.
Outline
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KEY WORDS conjunctiva, femtosecond laser, graft, pterygium
I.
Introduction
II.
Methods A.
Porcine eyes
B.
Graft preparation
C.
Optical Coherence Tomography (OCT)
D.
Histology and Hematoxylin-Eosin histochemistry (HE)
E.
Immunohistochemistry
F.
Statistical analysis
2
ACCEPTED MANUSCRIPT Results A.
Creation of autografts
B.
Optical Coherence Tomography measurements
C.
Histology measurements
D.
Immunohistochemistry
Discussion
V.
Conclusion
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IV.
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III.
3
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I.
Introduction
A pterygium is a wing-shaped corneal incursion of an aberrant conjunctival wound healing response, characterized by the centripetal growth of a squamous metaplastic
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epithelium with goblet cell hyperplasia and an underlying stroma of activated proliferating fibroblasts, neovascularization, inflammatory cells, and extracellular matrix.1, 2 Although
seen worldwide, it is commonly found in the peri-equatorial latitudes, at high altitude and in highly reflective environments, due to elevated levels of ultraviolet (UV) radiation, a
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known risk factor.3 The proliferation of pterygium tissue can cause changes in corneal
topography and refraction, occlusion of the visual axis, as well as visual field loss, corneal scarring and consequently reduction in visual acuity.1, 4 In advanced cases, scarring of the
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ocular surface can also lead to symblepharon formation, reduction of motility, and diplopia.5 In addition, pterygium can cause chronic inflammation, a dry eye state, and discomfort for the patient.6
Currently the management of pterygium is surgical. Ophthalmic surgeons have been treating this disease since 1000 BC.7 For many years a bare sclera technique, in which
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the pterygium was simply excised from the cornea, leaving only bare sclera exposed, was the standard approach.8 However, there were high recurrence rates, up to 88%, 9 months after surgery.9 Hence, alternative surgical procedures were sought, including the use of amniotic membrane (AM) or a conjunctival autograft (CAG) onto the bare sclera. This led
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to a significant reduction in recurrence rates.10, 11 A recent Cochrane meta-analysis of 1947 eyes of 1866 patients revealed superiority of CAG over AM, with risk ratio for recurrence
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of pterygium (primary and recurrent) of 0.87 and 0.53 at 3 and 6 months follow-up intervals and recurrence rates from 3.33% to 16.7% in the CAG and 2.6% to 42.3% in the AM groups at 6 months.11
It has been shown that the outcome of pterygium surgery is dependent on several factors, including thorough Tenon´s tissue removal and adequate management of the ocular surface,8 as well as on the surgical experience in harvesting the CAG, with greater experience leading to lower recurrence rates and fewer complications.12-14 An important factor for success in pterygium surgery is the ability to dissect a thin and adequately sized graft to cover the conjunctival defect with minimal inclusion of
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ACCEPTED MANUSCRIPT Tenon’s tissue.13 The resulting thin, tension-free grafts have been shown to not subsequently retract after surgery, providing a good cosmetic outcome.13 Femtosecond lasers (FSLs) have been employed in ophthalmic surgery since 2001, and more recently in cataract surgery,15, 16 but the most widespread application remains in corneal refractive surgery.17 FSLs have shown greater precision in corneal flap diameter
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and thickness creation and a more uniform flap thickness in comparison to mechanical microkeratomes.18, 19 In an attempt to transfer these favorable characteristics of FSLs to
pterygium surgery, we aimed to study and evaluate the feasibility of the creation of thin and
new technique.
A.
Porcine eyes
Methods
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uniform CAGs by FSLs and to investigate possible effects of surgeon experience on this
Fresh, whole cadaveric eyes from 24-week old pigs were obtained from an abattoir with a permit from Agri-Food & Veterinary Authority of Singapore. Eyes were kept in chilled balanced salt solution (BSS, Amo Endosol, Abbott Laboratories, Abbott Park,
enucleation.
Graft preparation
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B.
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USA) during both transit and dissection. Eyes were processed within 8 hours after
Porcine eyes were fixed in a suction holder (Figure 1A). Bulbi were pressurized by
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injecting (21G hypodermic needle, B. Braun, Melsungen, Germany) BSS from an infusion height of 27 cm above the eye (20 mmHg), to allow for easier tissue manipulation. The FSL-assisted CAG creation process is shown in Figure 1B and Figure 2. Briefly, peripheral Tenon´s tissue and conjunctiva were fixed to the sclera by 5-0 nylon sutures (Ethicon Inc, Somerville, NJ, USA), the designated CAG center was marked with a marker, and 10 ellipsoid CAGs of different diameters (6 x 5 cm – 9 x 8 cm) were created by an experienced consultant (JSM), using the Ziemer LDV Z8 (5 at 100 µm, 5 at 60 µm depth) without suction.20 The Z8 dissected the CAGs with adjunctive 1 µm laser spots, a frequency of 10 MHz, 100 % energy for the horizontal stroma and 130% for the side cuts.20
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ACCEPTED MANUSCRIPT The CAG´s length and width were measured immediately after dissection using a surgical calliper, and the CAG´s area was calculated (area ellipse: π x length/2·x height/2). The corner of the CAG was then slightly elevated by forceps, and closed scissors entered into the CAG plane to separate the flap form the underlying Tenon´s tissue; cutting by scissors was not permitted. The CAG was then lifted by 90% of extension, maintaining a small
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conjunctival bridge. After marking of the underlying Tenon´s tissue for histology purposes, the conjunctival flap was resutured (nylon 10-0, Ethicon) to the surrounding conjunctiva. The procedure was repeated by a less experienced fellow (YCL, 5 at 60 µm depth). FSL
were recorded.
Optical Coherence Tomography
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dissection times and time required to manually separate the CAG from underlying tissue
Directly after FSL-CAG preparation, a viscoelastic gel (Viscoat, Alcon, Fort Worth, USA) was injected underneath the CAG to cleave the FSL plane (Figure 3). Three optical coherence tomography (OCT) measurements were taken for the central and the peripheral (2 mm to each side) thickness of the CAG by anterior segment spectral-domain OCT
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(Figure 3B, RTVue, Optovue, Fremont, USA).
Histology and Hematoxylin-Eosin histochemistry (HE)
The CAGs and underlying Tenon´s tissue and sclera were excised with a 1-3 mm
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margin, fixed in 10% buffered formalin (Sigma-Aldrich, St. Louis, USA) for 6 hours, and stored in 70% ethanol (Merck, Darmstadt, Germany) overnight. Tissue samples were then dehydrated through a series of ascending concentrations until absolute ethanol, with each
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change of 30 min, cleared in xylene (Thermo Fisher Scientific, Massachusetts, USA) and infiltrated and embedded in paraffin (Thermo Fisher Scientific). Serial sections of 5 µm in thickness were obtained by microtome (Leica RM 2135; Leica Micro Systems, Wetzlar, Germany). After discarding of the sections adjacent to the cut edge (to avoid crush artefacts), the sections were mounted on glass slides, processed for hematoxylin and eosin histochemistry (HE, Sigma-Aldrich). Images were scanned and captured using the Eclipse Ti-E inverted microscope (Nikon Instruments Inc., Melville, USA). The HE-CAG thickness was measured (central and 250 µm to each side) using NIS Elements software (Nikon Instruments Inc.) (Figure 3A). All OCT and HE thickness measurements were taken by an investigator (MF) masked for designated flap thickness and surgeon. 6
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E.
Immunohistochemistry
Sections were deparaffinized and rehydrated. After rinses in 0.01 M PBS (First Base, Singapore), sections were treated with 0.025% trypsin-EDTA (Life Technologies, Carlsbad, USA) at 37°C for 5 minutes for antigen retrieval. After further PBS washes, the
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samples were blocked with 5% bovine serum albumin (Sigma-Aldrich) and 0.1% Triton X100 (Sigma-Aldrich) in PBS, followed by incubation in AlexaFluor 568 Griffonia
simplicifolia isolectin 4 (GSI-B4) conjugate (Life Technologies) for 30 minutes at room temperature, which stains N-acetyl-D-galactosamine end groups and terminal α-D-
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galactosyl residues,21 commonly found in porcine perivascular and endothelial vessel
cells.22 After PBS washes, the sections were mounted with UltraCruz Mounting Medium
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containing 4',6-diamidino-2-phenylindole (DAPI ,Santa Cruz Biotech, Dallas, USA), examined and imaged under fluorescence microscopy (Figure 5B and C, AxioImager Z1; Carl Zeiss, Oberkochen, Germany). Porcine limbal tissue with its´ inherent vasculature served as a positive control (Figure 5A).
Statistical Analysis
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F.
All statistical analysis was performed by the Statistical Package for the Social Sciences version 22.0 (SPSS, Inc., Chicago, USA). Values were expressed as mean ± standard deviation. Intergroup comparison was performed using the Mann-Whitney-U test.
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Central and peripheral measurements, FSL dissection times, as well as OCT and HE values, were compared with the Wilcoxon signed-rank test. The correlation between OCT and HE thickness measurements, as well as between FSL dissection/separation times and
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CAG area, were calculated by the Spearman test. A p-value of less than .05 was considered statistically significant.
A.
III.
Results
Creation of Autografts
The CAG area measured after dissection (37.5 ±12.1 mm2) did not significantly differ from the FSL settings (40.6 ±12.7 mm2, p=.3). No buttonholes or CAG tags occurred during surgery. Calculating the FSL dissection times for 7 ellipsoid CAGs (mean area 43.6 ±10.3 mm2) measuring from 6 x 5 mm (23.6 mm2) to 9 x 8 mm (56.5 mm2) the preparation
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ACCEPTED MANUSCRIPT of a 60 µm deep CAG took on average 2.6 seconds shorter than the preparation of a same sized CAG at 100 µm (mean time 60 µm 18.1 ±2.2 s; mean time 100 µm 20.7 ±2.4 s, p<.001). Dissection time was highly correlated to CAG area (60 and 100 µm r=.96, p<.001). There was no significant difference in graft separation time following FSL dissection between the consultant´s 100 (mean 59.6 ±8.3 s) and 60 µm CAGs (mean 42.4
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±6.9 s, p=.17), nor between consultant and fellow (mean 39.4 ±6.4 s) at 60 µm dissection depth (p=.75). Separation time and CAG area were not significantly correlated (r=.66, p=.16).
Optical Coherence Tomography Measurements
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B.
Optical coherence tomography measurements of FSL-assisted CAGs prepared at
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100 µm (mean 146.4 ±45.7 µm) showed a significantly higher deviation from the designated cutting depth (p=.04) and a higher variability (p=.03) in thickness than those prepared at 60 µm (mean 71.4 ±12.7 µm, Figure 4). Measurements of the periphery did not differ significantly from central values at 60 µm (mean periphery 78.6 ±9.4 µm; p=.06) or 100 µm cutting depth (mean periphery 156.0 ±46.0 µm; p=.5). The resulting central/peripheral ratios (CP) of 0.91 ±0.13 for 60 µm and 0.94 ±0.18 for 100 µm grafts did
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not differ between the two groups either (p=.52). Experienced (mean 68.3 ±14.3 µm) and less experienced surgeon (mean 73.9 ±11.9 µm) produced 60 µm grafts of comparable
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thickness (p=.6) and variability (p=.7, Figure 4).
Histology Measurements
Central histology and OCT thickness measurements were highly correlated (r=.89,
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p=.007), yet HE (after ethanol dehydration) measured in general 16.0 ±16.5 µm thinner than OCT (p=.04), 28.8 ±18.5 µm for 100 µm and 10.9 ±4.9 µm for 60 µm grafts.
D.
Immunohistochemistry
The positive control, using limbal tissue, revealed a specific labelling of porcine vasculature by GSI-B4 (red, Figure 5A). The porcine conjunctival epithelium also stained GSI-B4 positive (Figure 5A and B). CAGs dissected with the FSL at 60 µm thickness showed maintenance of conjunctival stromal vasculature with both large (Figure 5B) and small caliber vessels (Figure 5C).
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IV.
Discussion
In this study we demonstrated the successful preparation of CAGs by FSL in a
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porcine whole globe model. Accuracy and reliability proved higher for dissection depths of 60 µm than 100 µm with no significant differences between experienced and inexperienced surgeons. The CAG area measured after dissection did not significantly differ from the FSL settings. FSL cutting times at 60 µm depth took on average 2.6 seconds less than the
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cutting of a same-sized CAG at 100 µm. Mean graft separation time took less than one
minute for all cases and did not differ between the consultant´s 100 µm and 60 µm CAGs or between consultant and fellow, nor was it correlated to the CAG area.
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The ultra-short near-infrared pulses of the FSL pass through transparent tissues at low power densities without exerting significant collateral damage. However, at higher power densities, the structures absorb light energy, leading to plasma generation and tissue disruption, vaporizing small volumes of tissue with the formation of cavitation gas bubbles consisting of carbon dioxide and water (Figures 1B and 2B).23 To a limited degree, the FSL
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is also capable of passing through optically hazy media, such as edematous cornea and even the relatively translucent perilimbal sclera, because its infrared-wavelength energy undergoes much less attenuation than visible light.17 The conjunctiva is considered a translucent tissue. Gardiner found that age, sex, and nutrition influenced the grade of
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conjunctival transparency, which gradually increased with age as a sign of progressive atrophy of subepithelial layers.24 Hence we hypothesized, that it may be possible to make a
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conjunctival graft using an FSL.
Manual CAG creation represents a difficult and time-consuming part of the conventional pterygium surgical procedure.14 Several studies have shown a correlation of surgeon experience with lower recurrence and intraoperative complication rates.12, 13 Farrah et al showed that the recurrence rate produced by a trainee ophthalmologist (19.4%) was more than double that of an experienced ophthalmologist (6.8%). In addition, trainees caused 3.5 times more surgical complications.12 Ti et al retrospectively analyzed 139 cases with primary and 64 cases with recurrent pterygia and found the recurrence rates to vary widely among surgeons, from 5% to 82%. The recurrence rates were found to be inversely related to previous experience of the surgeon in performing conjunctival grafting. The
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ACCEPTED MANUSCRIPT author inferred that an adequately sized graft with minimum Tenon’s tissue (i.e., a thin graft) required considerable surgical skill and was associated with a substantial learning curve.13 This conclusion was also recently supported by Kuo et al, who demonstrated in a porcine cadaveric eye teaching model that the conjunctival graft thickness diminished significantly with the experience of the trainee, who needed approximately 50 attempts to
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reach CAG thicknesses of 87 ±23 µm.14 Using the same model, we were able to show that the FSL allowed the accurate cutting of ultra-thin CAGs with no significant influence of
surgeon experience and higher accuracy and reliability for dissection depths of 60 µm than 100 µm.
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There are no previous published studies that have correlated intraoperative CAG thickness with recurrence rates and complications. However, two recent studies investigated the postoperative CAG thickness after pterygium surgery using OCT.
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Kheirkhah et al found that the average thickness of the grafts decreased from 458 ±171 µm at 1 week after surgery to 291 ±124 µm at 3 months.25 Ozgurhan et al described similar results in 20 patients with a mean graft thickness of 430 ±127 µm in primary and 461 ±178 µm in recurrent pterygium at 1 week after surgery, which decreased to 109 ±15 µm and 107 ±18 µm at 3 months after surgery.26 The results suggested that the CAG undergoes
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significant swelling after surgery that requires approximately 3 months to deterge. The values at 3 months showed a wide range between 107 - 291 µm, but might give an idea of the actual CAG thickness transplanted. FSL assistance in our study allowed the reproducible production of CAGs with little variability in dissection depth, particularly at
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60 µm.
We were first able to demonstrate that FSL allowed the accurate and reliable
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preparation of very thin (60 µm) CAGs. The accuracy was higher and variability lower at a dissection depth of 60 µm in comparison to 100 µm, which we attribute to an increasing light scattering and absorption of the laser with conjunctival cutting depth.27 Tse et al showed that cutting depths up to 1 mm are possible in a porcine skin model. They also found that the smaller the numerical aperture (NA), the lower the precision of the cut.27 Hence, not all current commercially available FSLs may be able to achieve the results obtained from this study. The Ziemer LDV Z8 provides a large NA and a low energy level, permitting accurate cuts. The device also proved suitable for the preparation of FSLassisted CAGs as the handheld laser (Figure 1) allowed free positioning on the ocular surface and the conjunctival laser photodisruption can be performed without the use of suction.28 In order to ensure appropriate centration, the conjunctiva was marked in the 10
ACCEPTED MANUSCRIPT center of the intended graft before the laser head was docked. The laser head was held steady by the surgeons during the photodisruption. Kuo et al demonstrated in a porcine model that a trainee could reduce manual CAG preparation time from 191 to 126 seconds over 58 (5 x 5 mm) grafts.14 The inexperienced surgeon in our study, having never used the FSL before, needed on average only 57.5
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seconds for laser and preparation of the first 5 CAGs at 60 µm performed. Even though the area of the dissected CAG correlated with the laser dissection time, the actual surgical
times are clinically less meaningful due to speed of graft creation (e.g., 21 seconds for 9 x 8 mm and 14 seconds for 6 x 5 mm graft). Manual removal of the graft, following FSL
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dissection, and graft area were not significantly correlated. The conjunctival graft was
simply peeled from the underlying Tenon’s tissue and did not require meticulous surgical dissection as in manual CAG preparation. Certainly the use of an FSL involves additional
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preparation time and programming, which we did not record in this study, but this is no longer than for a standard refractive procedure and can be done in advance. The FSLassisted CAG procedure with the LDV Z8 did not demand a high level of experience in the use of FSL or pterygium surgery, as the CAGs prepared by a fellow at 60 µm did not differ significantly in thickness or variability from the experienced surgeon´s cases. The FSL consequently did not require a difficult and time-consuming learning curve as shown by
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Kuo et al.14 In addition, with the FSL, the dissection can be performed at any programmed depth.14 In an attempt to increase precision, maneuverability and ergonomics, Bourcier et al investigated the possibility of robot-assisted pterygium surgery relying on the DaVinci
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surgical system in a porcine model29 and recently in the first patient.30 While they found the procedure to be feasible and without complications, the surgery time (porcine model 36
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minutes, patient 60.5 minutes) still limits its application in routine clinical cases. In particular, the CAG preparation time in the porcine model (7 minutes) took substantially longer than our FSL-assisted grafts. The human conjunctiva consists of a nonkeratinized epithelium and a vascularized stroma.31 OCT measurements revealed variability in overall conjunctival thickness of 197.7 ±32.5 µm32 to 270 ±90 µm,31 increasing from limbus to periphery and being thicker in the nasal than temporal region.31 The conjunctival epithelium was measured at 42.4 ±7.4 µm32 to 61 ±12 µm.25 However, appreciating the exact transition from epithelium to stroma in OCT images can be difficult, and confocal microscopy found thinner epithelial thicknesses of 32.9 ± 1.1µm.33 In our study, setting the FSL to a cutting depth of 60 µm, histology showed that the dissection plane was in the conjunctival stroma in all cases (Figure 3A). 11
ACCEPTED MANUSCRIPT However, the preparation of even thinner CAGs could bear the danger of intraepithelial spiting. While thinner CAGs with less adjacent Tenon´s tissue are associated with lower recurrence rates,13 remaining vascular tissue in a free skin graft has been shown to promote an early vascularization of the new graft.34 Assuming similarities between skin and
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conjunctival grafts, excessively thin CAG´s with little and/or damaged vasculature
consequently might incur higher complication rates, such as graft necrosis or scarring. In
our ultra-thin 60 µm CAGs, we were able to demonstrate the persistence of small and midsize stromal vessels by immunohistochemistry (Figure 5). While the isolectin-B4
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glycoprotein originally isolated from the seeds of the tropical African legume Griffonia simplicifolia has proven high affinity to porcine perivascular and vascular endothelial
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cells,22 it has also been shown to interact with other cell types such as neurons or epithelial cells,35 which also occurred in our sections.
Older generation low frequency (15kHz) high energy FSLs have previously been reported to cause more keratocyte cell necrosis and consequently inflammation in corneal incisions,36, 37 which could raise concerns of elevated scarring and recurrence rates in FSLassisted pterygium surgery (FLAPS). However, the use of lower energy level FSL has
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significantly attenuated the inflammation and apoptosis adjacent to the photo-disruptive site.38 The Ziemer Z8 is a low energy (50 – 2500 nJ) high frequency (0.1 - 10 MHz) laser and previous studies have shown minimal signs of inflammation in animal, corneal incision models.20 Nevertheless, human scarring responses and recurrence rates in FLAPS can only
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be evaluated in clinical trials.
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Our study is limited by the utilization of a porcine model with possible anatomical and mechanical differences to human conjunctiva, which, to date, have not been investigated in detail. However, at least for porcine sclera, a high similarity in histology and collagen bundle organization to human eyes has been shown.39 In addition, pig eyes have proven to be realistic and valuable surgical models and allowed us to do a direct comparison to previous reports using conventional manual CAGs.14, 29, 40 We also evaluated the CAG thickness by 2 methods, as the conjunctival tissue was subjected to significant shrinking due to alcohol dehydration during tissue fixation with standard histology preparation.41 Consequently the OCT measurements obtained provided a more realistic estimation of intraoperative graft thicknesses.
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ACCEPTED MANUSCRIPT While FLAPS is currently experimental, given the growing use of lasers in ophthalmic surgery, the quick, reliable and automated preparation of CAGs by FSL might assist in further standardizing the surgical procedure and reducing intraoperative and postoperative complications.
Conclusion
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We have shown that CAGs could be prepared with the Ziemer LDV Z8 FSL.
Accuracy, reliability and speed of graft preparation were higher for dissection depths of 60 rather than 100 µm irrespective of surgeon experience. Clinical trials are currently
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underway (NCT02866968), to show whether these ultra-thin FSL-assisted CAGs do result in good clinical cosmetic outcomes and low recurrence rates.
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References
1. Kim KW, Park SH, Kim JC. Fibroblast biology in pterygia. Exp Eye Res 2016;142:32-39 2. Di Girolamo N, Chui J, Coroneo MT, Wakefield D. Pathogenesis of pterygia: role of cytokines, growth factors, and matrix metalloproteinases. Prog Retin Eye Res 2004;23:195-228 3. Saw SM, Tan D. Pterygium: prevalence, demography and risk factors. Ophthalmic Epidemiol 1999;6:219-228
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4. Chui J, Coroneo MT, Tat LT, et al. Ophthalmic pterygium: a stem cell disorder with premalignant features. Am J Pathol 2011;178:817-827 5. Shimazaki J, Shinozaki N, Tsubota K. Transplantation of amniotic membrane and limbal autograft for patients with recurrent pterygium associated with symblepharon. Br J Ophthalmol
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1998;82:235-240
6. Coroneo MT, Chui JJY. Pterygium, in: Holland EJ, Mannis MJ, Lee WB (eds). Ocular surface disease: cornea, conjunctiva and tear film: Elsevier Health Sciences; 2013:125-144.
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7. Rosenthal JW. Chronology of pterygium therapy. Am J Ophthalmol 1953;36:1601-1616 8. Hirst LW. The treatment of pterygium. Surv Ophthalmol 2003;48:145-180 9. Chen PP, Ariyasu RG, Kaza V, et al. A randomized trial comparing mitomycin C and conjunctival autograft after excision of primary pterygium. Am J Ophthalmol 1995;120:151-160 10. Kaufman SC, Jacobs DS, Lee WB, et al. Options and adjuvants in surgery for pterygium: a report by the American Academy of Ophthalmology. Ophthalmology 2013;120:201-208 11. Clearfield E, Muthappan V, Wang X, Kuo IC. Conjunctival autograft for pterygium. Cochrane Database Syst Rev 2016;2:CD011349 12. Farrah JJ, Lee GA, Greenrod E, Vieira J. Outcomes of autoconjunctival grafting for primary pterygia when performed by consultant compared with trainee ophthalmologists. Clin Experiment Ophthalmol 2006;34:857-860 13
ACCEPTED MANUSCRIPT 13. Ti SE, Chee SP, Dear KB, Tan DT. Analysis of variation in success rates in conjunctival autografting for primary and recurrent pterygium. Br J Ophthalmol 2000;84:385-389 14. Kuo MX, Sarris M, Coroneo MT. Cadaveric porcine model for teaching and practicing conjunctival autograft creation. Cornea 2015;34:824-828 15. Trikha S, Turnbull AM, Morris RJ, et al. The journey to femtosecond laser-assisted cataract surgery: new beginnings or a false dawn? Eye (Lond) 2013;27:461-473
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16. Pahlitzsch M, Torun N, Pahlitzsch ML, et al. Impact of the femtosecond laser in line with the femtosecond laser-assisted cataract surgery (FLACS) on the anterior chamber characteristics in
comparison to the manual phacoemulsification. Semin Ophthalmol 2016; Apr 19:0. [Epub ahead of print] 17. Soong HK, Malta JB. Femtosecond lasers in ophthalmology. Am J Ophthalmol
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2009;147:189-197 e182
18. Zhou Y, Zhang J, Tian L, Zhai C. Comparison of the Ziemer FEMTO LDV femtosecond laser and Moria M2 mechanical microkeratome. J Refract Surg 2012;28:189-194
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19. Santhiago MR, Kara-Junior N, Waring GO. Microkeratome versus femtosecond flaps: accuracy and complications. Curr Opin Ophthalmol 2014;25:270-274
20. Riau AK, Liu YC, Lwin NC, et al. Comparative study of nJ- and muJ-energy level femtosecond lasers: evaluation of flap adhesion strength, stromal bed quality, and tissue responses. Invest Ophthalmol Vis Sci 2014;55:3186-3194
21. Murphy LA, Goldstein IJ. Five alpha-D-galactopyranosyl-binding isolectins from Bandeiraea
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simplicifolia seeds. J Biol Chem 1977;252:4739-4742
22. Kirkeby S, Moe D. Binding of Griffonia simplicifolia 1 isolectin B4 (GS1 B4) to alphagalactose antigens. Immunol Cell Biol 2001;79:121-127 23. Juhasz T, Loesel FH, Kurtz RM, et al. Corneal refractive surgery with femtosecond lasers. IEEE
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J Sel Top Quantum Electron 1999;5:902-910
24. Gardiner PA. Observations on the transparency of the conjunctiva. Br J Ophthalmol 1944;28:538-554
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25. Kheirkhah A, Adelpour M, Nikdel M, et al. Evaluation of conjunctival graft thickness after pterygium surgery by anterior segment optical coherence tomography. Curr Eye Res 2011;36:782786
26. Ozgurhan EB, Kara N, Bozkurt E, et al. Comparison of conjunctival graft thickness after primary and recurrent pterygium surgery: Anterior segment optical coherence tomography study. Indian J Ophthalmol 2014;62:675-679 27. Tse C, Zohdy MJ, Ye JY, O'Donnell M. Penetration and precision of subsurface photodisruption in porcine skin tissue with infrared femtosecond laser pulses. IEEE Trans Biomed Eng 2008;55:1211-1218 28. Pepose J, Lubatschowski H. Comparing femtosecond lasers. Cataract Refract Surg Today 2008;10:45-52
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ACCEPTED MANUSCRIPT 29. Bourcier T, Nardin M, Sauer A, et al. Robot-assisted pterygium surgery: feasibility study in a nonliving porcine model. Transl Vis Sci Technol 2015;4:9 30. Bourcier T, Chammas J, Becmeur PH, et al. Robotically assisted pterygium surgery: first human case. Cornea 2015;34:1329-1330 31. Read SA, Alonso-Caneiro D, Vincent SJ, et al. Anterior eye tissue morphology: Scleral and conjunctival thickness in children and young adults. Sci Rep 2016;6:33796
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32. Zhang X, Li Q, Xiang M, et al. Bulbar conjunctival thickness measurements with optical
coherence tomography in healthy chinese subjects. Invest Ophthalmol Vis Sci 2013;54:4705-4709 33. Efron N, Al-Dossari M, Pritchard N. Confocal microscopy of the bulbar conjunctiva in contact lens wear. Cornea 2010;29:43-52
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34. Calcagni M, Althaus MK, Knapik AD, et al. In vivo visualization of the origination of skin graft vasculature in a wild-type/GFP crossover model. Microvasc Res 2011;82:237-245
35. Shimizu T, Nettesheim P, Mahler JF, Randell SH. Cell type-specific lectin staining of the
B4. J Histochem Cytochem 1991;39:7-14
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tracheobronchial epithelium of the rat: quantitative studies with Griffonia simplicifolia I isolectin
36. Netto MV, Mohan RR, Medeiros FW, et al. Femtosecond laser and microkeratome corneal flaps: comparison of stromal wound healing and inflammation. J Refract Surg 2007;23:667-676 37. Angunawela RI, Riau A, Chaurasia SS,et al. Manual suction trephination versus femtosecond laser corneal trephination: intraocular pressure, endothelial cell loss and wound healing responses.
22427557
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Invest Ophthalmol Vis Sci. 2012 May 4;53(6):2571-9. doi: 10.1167/iovs.11-8403. PMID:
38. de Medeiros FW, Kaur H, Agrawal V, et al. Effect of femtosecond laser energy level on corneal stromal cell death and inflammation. J Refract Surg 2009;25:869-874
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39. Nicoli S, Ferrari G, Quarta M, et al. Porcine sclera as a model of human sclera for in vitro transport experiments: histology, SEM, and comparative permeability. Mol Vis 2009;15:259-266 40. Lee GA, Chiang MY, Shah P. Pig eye trabeculectomy-a wet-lab teaching model. Eye (Lond)
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2006;20:32-37
41. Bahr GF, Bloom G, Friberg U. Volume changes of tissues in physiological fluids during fixation in osmium tetroxide or formaldehyde and during subsequent treatment. Exp Cell Res 1957;12:342-355
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ACCEPTED MANUSCRIPT Figure legends Figure 1. A. Porcine bulbi were fixed in a suction holder. Conjunctival autografts were then dissected with the Ziemer LDV Z8. B. Close-up of the surgery site after femtosecond laser preparation. The black arrow indicates typical cavitation bubbles, marking the corner of the
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Figure 2. Steps of the femtosecond laser-assisted conjunctival autograft (CAG) creation. A. Peripheral Tenon´s tissue and conjunctiva was fixed to the sclera by 5-0 nylon sutures. B.
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diameters created using the Ziemer LDV Z8, the black arrows show the corner of the CAG with characteristic cavitation bubbles. C. The corner of the CAG was slightly elevated by
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forceps and closed scissors entered into the CAG plane, cutting by scissors was not permitted. D. 90% of the CAG was then lifted, leaving a small conjunctival bridge. (E. The underlying Tenon´s tissue was marked. F. The conjunctival flap re-sutured (nylon 10-0) to the surrounding conjunctiva.
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Figure 3. Directly after the preparation of the porcine conjunctival autograft (CAG) and resuturing it to the surrounding conjunctiva, a viscoelastic gel was injected in the dissection plane to improve optical coherence tomography (OCT) visualization (B). Three measurements (central and 250 µm to each side) were then taken of the central and the
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peripheral (2 mm to each side) thickness of the CAG. The CAGs were then fixed and dissected for histology (A). The CAG dissection plane was in the conjunctival stroma in all
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cases. Three central measurements of the histology samples were taken (central and 250 µm to each side).
Figure 4. Box-plots of the mean central conjunctival autograft (CAG) thickness for 100 and 60 µm grafts prepared by consultant or fellow (A) and the resulting deviation from the desired dissection depth (B). FS-assisted CAGs prepared at 100 µm showed a significantly higher deviation from the designated cutting depth (p=.04) and a higher variability (p=.03) in thickness than those prepared at 60 µm. Experienced and less experienced surgeon produced 60 µm grafts of comparable thickness (p=.6) and variability (p=.7).
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ACCEPTED MANUSCRIPT Figure 5. Immunohistochemistry with Griffonia simplicifolia isolectin 4 conjugate (GSIB4, red) and 4',6-diamidino-2-phenylindole (DAPI, blue). A. Staining of the limbal area revealed a high specificity of GSI-B4 for porcine vessels. The porcine conjunctival epithelium also stained GSI-B4 positive (e.g., in B). Conjunctival autografts dissected with the FS at 60 µm thickness showed maintenance of conjunctival stromal vasculature with
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