|Year : 2016 | Volume
| Issue : 2 | Page : 49-55
Surface quality of human corneal SMILE lenticules in comparison with microkeratome free caps
Ihab M Osman MD, FRCS (Glasgow) 1, Amira Y Madwar2
1 Department of Ophthalmology, Faculty of Medicine, Alexandria University, Alexandria, Egypt
2 Department of Histology, Faculty of Medicine, Alexandria University, Alexandria, Egypt
|Date of Submission||06-Mar-2016|
|Date of Acceptance||12-May-2016|
|Date of Web Publication||30-Aug-2016|
Ihab M Osman
Department of Ophthalmology, Faculty of Medicine, Alexandria University, Alexandria 2667
Source of Support: None, Conflict of Interest: None
The aim of this study was to compare electron microscopic morphological images of the posterior surface of corneal lenticules removed during femtosecond laser small-incision lenticule extraction (SMILE) with free corneal caps created using a mechanical microkeratome.
An in-vitro comparative study was carried out including 30 human corneal lenticules from 15 patients who underwent bilateral SMILE refractive procedure (SMILE group) using the 500 kHz VisuMax femtosecond laser and 20 cadaveric donor corneas that were cut creating free caps using the Moria M2 microkeratome.
The present study was carried out at Roayah Vision Correction Center and Faculty of Science, Alexandria University.
Participants and methods
A total of 15 SMILE lenticules and 15 microkeratome free caps were examined by scanning electron microscopy (SEM), and another 15 SMILE lenticules and five microkeratome free caps were examined by transmission electron microscopy. The JEOL JSM-5300LV SEM was used for observations. A new scoring system was used for comparison.
The surface and edge of SMILE lenticules showed more roughness in the form of tissue bridges, dome-shaped elevations, more cavitational changes, and tiny tissue fragments compared with the undersurface of free corneal microkeratome caps, but the difference was not statistically significant on SEM (P=0.40654).
SMILE induces corneal surface roughness and structural changes that are slightly more pronounced than those induced by the microkeratome. The observed changes may cause surgically induced optical aberrations. Energy settings still need further adjustment to be able to create reproducible, predictable smooth surfaces in lenticules of SMILE cases.
Keywords: electron microscopy, microkeratome, small-incision lenticule extraction
|How to cite this article:|
Osman IM, Madwar AY. Surface quality of human corneal SMILE lenticules in comparison with microkeratome free caps. Delta J Ophthalmol 2016;17:49-55
|How to cite this URL:|
Osman IM, Madwar AY. Surface quality of human corneal SMILE lenticules in comparison with microkeratome free caps. Delta J Ophthalmol [serial online] 2016 [cited 2022 Aug 18];17:49-55. Available from: http://www.djo.eg.net/text.asp?2016/17/2/49/189471
| Introduction|| |
The femtosecond laser uses ultra-short pulses of infrared laser at high frequency to elicit a cleavage of tissue planes by creating small cavitation bubbles, thus making highly accurate incisions with minimal tissue damage .
At first, the femtosecond laser was used as an initial step in laser in-situ keratomileusis surgeries to replace the use of mechanical microkeratomes in creating corneal flaps ,,.
The refractive lenticule extraction is the first all-in-one femtosecond laser refractive procedure aiming for the correction of myopia/myopic astigmatism ,. This procedure encompasses two different techniques − (a) femtosecond lenticule extraction and (b) small-incision lenticule extraction (SMILE), which is a flapless technique creating a small, arcuate incision through which a corneal lenticule of predetermined thickness is extracted ,.
The main and theoretical benefits of the absence of flap creation are elimination of flap-associated complications with microkeratome flap cuts, reduced risk of induced astigmatism, fewer high-order aberrations ,,, and reduced postoperative inflammation following SMILE .
There is a significant number of published studies evaluating the safety and efficacy of the SMILE technique at the clinical level, whereas very few studies address the ultrastructural changes induced by this technique. Most published studies were performed on eye bank in-vitro human corneas; however, more precise data can be obtained from in-vivo human corneas.
| Participants and methods|| |
A total of 30 human corneal lenticules were included in this study. They were extracted from 30 eyes that underwent SMILE refractive procedure for correction of myopia or myopic astigmatism (SMILE group): half of these specimens (15) were examined by scanning electron microscopy (SEM) and the other half (15) by transmission electron microscopy (TEM). In addition, 20 free corneal caps (microkeratome group) were included. They were obtained from 20 donor eyes from human cadavers that did not meet criteria for keratoplasty because of an endothelial cell count of less than 1800 cells. The time between collecting the donor corneas and the time of inclusion to the present study was less than 6 days for all specimens. Of these, 15 specimens were examined by SEM and five by TEM.
Small-incision lenticule extraction procedure
The VisuMax 500 kHz laser system (Carl Zeiss Meditec, Jena, Germany) platform was used for the SMILE surgeries. In all eyes, the spot distance was 3 µm for lamellar cuts and 2 µm for side cuts. The spot energy was set to 130 nJ for all patients. The minimum lenticule side-cut thickness was set to 10 µm. The lenticule side-cut angle was 130°, the incision side-cut angle was 70°, and the optical zone was 6.5 mm, as SMILE is not performed in patients with a mesopic pupillary diameter greater than 6.5 mm. A small-sized cone was used in all patients; the cap diameter was set to 7.8 mm. The attempted depth of the lenticule was set to 130 µm to compare with the attempted free-cap depth in the second group. The scan mode was single with scan direction spiral-in for the lenticule and spiral-out for the cap, allowing the patient to fix at the flickering fixation target as long as possible.
The donor corneas and surrounding scleral rims were mounted on the Barron artificial anterior chamber (Katena, Denville, New Jersey, USA) with the endothelial side facing downward, and then the tissue retainer was carefully placed over the unit and was advanced to the bottom of the base. The artificial anterior chamber was then secured by placing the locking ring over the unit. Moria Surgical M2 microkeratome (Moria, Antony, France) with disposable heads was used to create free caps with thickness of 130 µm. This was performed using suction rings of size zero and without locking the stop. The resulting free cap was taken and processed in the same way as the SMILE lenticules to examine the posterior surface of the free cap.
The specimens designated for SEM were fixed by immediate immersion in formaldehyde gluteraldehyde in phosphate buffer solution (pH 7.2) at 4°C for 3 h. The specimens were then postfixed in 2% osmic oxide in the same buffer at 4°C for 2 h. The specimens were washed in the buffer and dehydrated at 4°C using a graded series of ethanol. The specimens were dried using the critical point method and then mounted using carbon paste on an Al-stub and coated with gold up to a thickness of 400 Å in a sputter-coating unit (JFC-1100E). Observation of the specimens was performed using a JEOL JSM-5300 SEM (JEOL USA Inc., Peabody, Massachusetts, USA) operated between 15 and 20 KeV.
The lenticules for TEM were dehydrated in the same way as specimens for scanning microscopy and then embedded in prelabeled plastic capsules to be polymerized for 48 h. The sections were trimmed and semithin sections of 1 µm were cut using LKB ultra-microtome to be mounted on copper grids (mesh size 200). The grids were stained with urinyl acetate for 20 min, followed by lead citrate for 10 min. Subsequently, the grids were examined and photographed by JEOL 100 CX TEM.
The size of the lenticules allowed the examination of the complete lenticular surface in all SMILE cases, whereas the free caps of the corneas had to be cut into halves by a scalpel to allow their mounting on the SEM because of their larger diameter.
Analysis of the specimens
A qualified clinical histologist (A.Y.M.) examined the resulting samples. The examiner was blinded to the groups. The examiner used the same histological evaluation protocol with special attention to standardizing the angle of SEM tilt in all specimen evaluations.
A scoring system for evaluating the electron microscopic quality of the specimens was used, based on the edge serrations and surface tissue fragments. A point was assigned for every quadrant with edge serrations − best edge quality was assigned 0 points and the worst edge quality was assigned 4 points. As for the surface quality, it was assessed by the height and presence of tissue fragments at the lenticular surface. The highest tissue fragment in each quadrant was measured. A point was assigned for each quadrant of surface tissue fragments less than 5 μm, 4 points were assigned for each quadrant with fragments of 5–10 μm, and 3 points were assigned for each quadrant with tissue fragments greater than 10 μm; fewer points thus suggest a better surface quality. The total points assigned for both the edge and the surface quality were given for each specimen.
The Mann–Whitney U-test was used to assess the score difference in SEM between the two groups. A cut-off P value of 0.05 was used to declare statistical significance. No statistical analysis was performed for TEM between the groups because of the very small size of the TEM microkeratome group.
| Results|| |
Scanning electron microscopy
On low maginification (×10, ×1000, and ×2000), in the SMILE group, the edges of the lenticules showed infoldings in some areas due to the thinness of the edges, and minimal serrations were noted at several points along the lenticule circumference [Figure 1]. Moreover, the SMILE group showed tissue fragmentation, with cavitational changes along the edge with less preservation of the lamellar collagen layer arrangment compared with the microkeratome group [Figure 2].
|Figure 1 Left: ×50 magnifiation of the SMILE lenticule showing the complete lenticule. Right: free microkeratome cap being cut into halves with the epithelial side facing upward. SMILE, small-incision lenticule extraction.|
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|Figure 2 (a–d) Variable magnifications of SMILE and microkeratome corneal specimens. (a, c) Show infoldings of the edge with more tissue fragmentations in the SMILE lenticule. (b, d) In the microkeratome group, more preservation of the lamellar collagen layers is observed. SMILE, small-incision lenticule extraction.|
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With higher magnification (×2000 and ×7500), the plasma bubbles of the femtosecond laser were seen in the lenticular edges. These bubbles were of variable sizes, with confluence of some bubbles forming bigger cavities with tissue bridges in between, mimicking a marine coral reef [Figure 3].
|Figure 3 ×7500 magnification of the SMILE lenticule edge showing cavitational bubbles with a tissue bridge in a big cavity. SMILE, small-incision lenticule extraction.|
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The surface of the lenticules in the SMILE group showed roughness and elevations corresponding to tissue bridges and depressions corresponding to the concavities of cavitational bubbles that were all more prominent compared with the microkeratome group. These changes were more apparent in some samples than in others and in some areas in the same sample than in other areas. However, in the microkeratome group, classic chatter lines were seen more evenly in most samples in a more consistent way. Collagen bundles were seen in both groups but were more prominent in the microkeratome group [Figure 4].
|Figure 4 (a–d) Different magnifications of the corneal specimens. (a, c) Show surface depressions and tissue debris less than 5 μm in the SMILE group (white arrows). (b, d) Show chatter lines and more prominent collagen bundles in the microkeratome group with no tissue debris. SMILE, small-incision lenticule extraction.|
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In the SMILE group, with higher magnifications (×2000 and ×3500), the surface roughness was identified more easily [Figure 5]. The shape of the rough areas differed from one place on the surface of the lenticule to another with no specific pattern. This roughness appeared in the form of tissue bridges, dome-shaped elevations, and tiny tissue fragments. The average height of these bridges was 4.3±2.1 μm with a range of 1.6–12.4 μm. The larger tissue bridges were mostly apparent in areas where the femtosecond laser was not seen by the surgeon, at the end of the laser application, denoting absence of its effect, and the dissection made at these points were pure shearing forces by the spatula [Figure 6].
|Figure 5 (a) ×2000 magnification of the lenticular surface with tissue bridges (black arrow) more than 10 μm in height, with cavities (white arrow). (b) Higher magnification at ×3500 and dome-shaped elevation.|
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|Figure 6 Pattern of the femtosecond laser at the end of the laser application, showing an area of absent laser effect due to meibomian secretion (white arrow).|
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The scoring system showed a difference between the two groups with regard to surface roughness and edge serrations: in total, 93 points could be assigned to the SMILE group and 73 points to the microkeratome group (P=0.40654), a statistically insignificant difference.
Transmission electron microscopy
TEM showed more uniform arrangement of the corneal collagen fibers in the microkeratome group than in the SMILE group, with preservation of the longitudinal and transverse cross-sectional cuts of the collagen fibers. The bundles appeared thread-like in the longitudinal sections and appeared rounded in the transverse sections, whereas the femto SMILE group showed separation of the collagen bundles and cavity formations within the collagen bundles [Figure 7].
|Figure 7 (a) Transmission electron microscopy of lenticules in the SMILE group showing cavities with collagen fragments in it (arrows). (b) Note the more regular arrangement of the collagen bundles without cavities in the microkeratome group. SMILE, small-incision lenticule extraction.|
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| Discussion|| |
SMILE is a novel and expanding technique in refractive surgery. It offers great advantages with regard to both safety and efficacy compared with microkeratome flap creation in laser in-situ keratomileusis procedures . Several studies have investigated the optical and refractive outcomes of this technique. However, only a few studies have investigated the anatomical and morphological outcomes . The anatomical analysis of the surface of these lenticules is very important, as they are the only available specimens obtained from living humans undergoing refractive procedures.
To date, we are unable to directly examine corneas that have undergone SMILE from the anatomical point of view. The surface changes seen on the undersurface of the cap of extracted lenticules are supposed to be an exact replica of the changes seen on the upper surface of the residual stroma.
The relationship between the spot size, spot distance, laser frequency, as well as the laser energy plays a vital role in the outcome of the femtosecond laser cutting properties . Higher pulse energy creates larger bubbles and more cavitational changes, whereas larger spot distances increase tissue bridges between the cavitational bubbles. The slower frequency and longer duration of the procedure increase the chances of suction loss. Better cuts are obtained with higher frequency, smaller spot distances, and lower pulse energy. The ultimate settings of these variable parameters are yet to be determined ,,.
The earliest micromorphological studies were on porcine eyes using a 60 kHz femtosecond laser system (IntraLase FS60) . Another study was perfomed using a 200 kHz laser (WaveLight UltraFlap − Femtosecond laser) by applying 500 nJ pulse energy and 5 µm spot distance. The resulting non-quantitative assessment showed smooth cuts as seen by SEM .
Heichel and colleagues, worked on an ex-vivo pig model using the VisuMax laser with 200 kHz repetition rate, 185 nJ pulse energy, and 3×3 µm spot separation.
They found the lenticules to be of lower surface quality than those obtained with a mechanical microkeratome .
Kunert and colleagues analyzed the quality of lenticules extracted from patients using the 200 kHz VisuMax system with 3×3 µm spot separation while using three different pulse energies (150, 180, and 195 nJ). The best quality resections were generated with the lowest pulse energy used in the study (150 nJ), suggesting that lower energy levels produced smaller gas bubbles that resulted in smoother surfaces .
Ang et al.  used the new higher-frequency VisuMax femtosecond laser with 500 kHz repetition rate, 130 nJ pulse energy, as in the current study, and 3×3 µm spot separation to create refractive lenticules in 12 cadaveric eyes. Although they found improvement in surface quality compared with earlier studies, however, there were still cavitation bubbles and rough patches seen in their samples.
Ziebarth et al.  in a recent study evaluated the surface of eight lenticules extracted using the 500 kHz VisuMax femtosecond laser, with 130 nJ pulse energy, as in the present study, and a spot separation of 2.5×2.5 µm. They used environmental SEM, which was not used in the present study. They found smoother lenticule surfaces with easier lenticule extraction, attributing these results to the closer spot placement that likely leaves fewer collagen fibers intact. Another factor for the smoother surface was attributed to the use of environmental SEM that enables imaging of hydrated, uncoated biological samples, thereby eliminating artifacts solely caused by tissue preparation .
The present study of 30 SMILE lenticules is the largest published study on the electron microscopic changes in femtosecond laser performed on living human corneas. It assessed both the SEM and TEM changes in SMILE lenticules and compared the SEM changes in SMILE with the microkeratome cuts in donor human corneas.
In contrast with the study of Ziebarth et al. , and more in accordance with the study of Ang et al. , the present study showed that SMILE lenticules still harbor both in surface and edges more surface roughness, tissue bridges, and cavitational changes, with both SEM and TEM, even though the difference did not reach statistical significance. Larger studies are needed to confirm these results. Nevertheless, these observations are of importance if one considers their potential impact on the visual results of the SMILE procedure. Indeed, the described effect of the laser could produce optical aberrations that may affect the visual outcome as well.
In the present study, the surface of the lenticules showed fine tissue fragments in all quadrants of the SMILE lenticules in almost all samples, whereas larger tissue bridges were seen in only three samples. These larger bridges were attributed to the absence of laser marks due to meibomian secretions obscuring a clear optical pathway to the desired corneal depth in two cases and to difficult lenticule dissection in another sample. Other factors such as repeated docking and fine saccadic eye movements were also noted as factors affecting the smoothness of the lenticule surface and edge. As for the microkeratome surface cuts, they were more homogenous with fine tissue fragments in some quadrants, and larger bridges were either related to blade serrations or rough tissue handling. Factors such as donor age, time of acquisition of the cornea from the cadaver, tissue processing of the graft, storage, and transportation issues could have played a role in the quality of the electron microscopic evaluations in the second group.In contrast to all previous studies, the present study used higher magnifications − ×1000, ×2000, and ×3500–to demonstrate the femtosecond laser effect on the corneal lenticules. This may explain the exaggerated morphological picture obtained in the present study compared with the previous studies of Ziebarth et al.  who used ×100, ×200, and ×500 magnifications.
Nevertheless, the exact comparison between the present study and the two previous published studies on SMILE lenticules performed with the 500 kHz VisuMax femtosecond laser and 130 nJ pulse energy is not possible, as the present study used different spot sizes ,.
The question as to whether this surface roughness provokes stronger healing and increases the corneal biomechanical strength or adds to the optical aberrations remains open ,. Indeed, these changes may be an explanation for some of the delayed visual recovery observed in some of the SMILE patients.
| Conclusion|| |
The SMILE procedure induced more corneal surface roughness and structural changes that may be even more obvious than those induced by a microkeratome manual cutting. Nevertheless, this more significant surface roughness may not translate to poorer visual outcomes. Energy settings need further adjustment to be able to create reproducible, predictable, and smooth surfaces in SMILE cases. Further and comparative studies using novel machines with higher frequency should answer questions about the optimal energy, spot size, and spot distance to be used to reach the best surface quality for the SMILE procedure.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]