Biomechanical Weakening of Different Re-treatment Options After Small Incision Lenticule Extraction (SMILE)

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1 Biomechanical Weakening of Different Re-treatment Options After Small Incision Lenticule Extraction (SMILE) Sabine Kling, PhD; Bogdan Spiru, MD; Farhad Hafezi, MD, PhD; Walter Sekundo, MD, PhD ABSTRACT PURPOSE: To determine the corneal weakening induced by different re-treatment options after small incision lenticule extraction (SMILE) and investigate the potential of corneal cross-linking (CXL) to reestablish the original corneal stress resistance. METHODS: A total of 96 freshly enucleated porcine corneas were used. The initial refractive correction was defined to be diopters (D) and the required enhancement to be D. Three different re-treatment options were analyzed: -3D Re-SMILE, -3D photorefractive keratectomy (PRK) on top of the SMILE cap, and cap-to-flap conversion and -3D excimer ablation on the stromal bed (LASIK). The control condition did not receive any treatment. Subsequently, accelerated CXL (9 mw/cm 2, 10 min) was performed in two groups with currently common enhancement techniques: following cap-to-flap conversion (-3D LASIK enhancement) and in controls. Biomechanical properties were measured with stress-strain extensometry ranging from 1.27 to 12.5 N. RESULTS: The Re-SMILE and PRK enhancement did not significantly reduce the overall elastic modulus of the cornea compared to controls (24.7 ± 2.23 and 22.7 ± 2.61 versus 23.8 ± 3.35 MPa, P.176), whereas LASIK enhancement did (22.2 ± 3.37 MPa, P =.048). CXL treatment significantly increased the elastic modulus compared to all non cross-linked conditions (P.001). Refractive surgery decreased the overall elastic modulus by 7%, whereas CXL increased it by 20%. CONCLUSIONS: In enhancement, the corneal biomechanical integrity is less affected with both Re-SMILE and PRK enhancement. Corneal weakening through laser refractive surgery is small compared to the stiffening effect after CXL. [J Refract Surg. 2017;33(3): ] Journal of Refractive Surgery T he small incision lenticule extraction (SMILE) surgical procedure was proposed in and offers an alternative to LASIK and photorefractive keratometry (PRK). The two latter procedures have in common that Bowman s layer, which is a determinant for the biomechanical properties of the cornea, is either severed or ablated. Although iatrogenic ectasia has been reported after both LASIK 2 and PRK 3 procedures, the incidence rate is higher after LASIK because the remaining stromal bed resisting the intraocular pressure (IOP) is thinner. In contrast, SMILE preserves Bowman s layer and therefore has the advantage that the stromal stress increases less than after LASIK or PRK. 4 Although the biomechanical integrity of the cornea is better preserved with SMILE, neither the risk of keratectasia 5 nor the need of enhancements can be totally excluded. Indeed, 0.8% to 4% 6,7 of patients require re-treatment after SMILE. Although after LASIK the flap can be relifted and additional tissue ablated for enhancement, 8 the question arises as to which is the best technique for enhancement after SMILE. The first option is cutting a second layer from the inferior side of the previously generated pocket and extracting it through the same aperture. This technique is known as cap-less SMILE and has been described by Donate and Thaëron. 9 The problem with From the Laboratory of Ocular Cell Biology, Center for Applied Biotechnology and Molecular Medicine, University of Zurich, Zurich, Switzerland (SK, FH); the Department of Ophthalmology, Philipps University of Marburg, Marburg, Germany (BS, WS); ELZA Institute, Dietikon/Zurich, Switzerland (FH); and the Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, California (FH). Submitted: July 20, 2016; Accepted: November 16, 2016 Supported by the Verein zur Förderung der wissenschaftlichen Augenheilkunde in Marburg e.v. (Society to promote Scientific Ophthalmology in Marburg, Germany). The authors have no financial or proprietary interest in the materials presented herein. Drs. Kling and Spiru contributed equally to this work and should be considered as equal first authors. Correspondence: Sabine Kling, PhD, Laboratory of Ocular Cell Biology, CABMM Y13-L86, Winterthurerstrasse 190, 8057 Zurich, Switzerland. kling.sabine@gmail.com doi: / x

2 this Re-SMILE technique is that detaching the lenticule is challenging. It can be difficult to find the edge, especially for small corrections where the new lenticule is thin. The second option is performing an excimer laser ablation on top of the previously generated cap. 10 The problem with this technique is that Bowman s membrane is disrupted 11 and the postoperative pain is greater. Moreover, additional off-label use of mitomycin C is recommended to prevent haze formation. The third option is opening the previously generated cap and converting it into a LASIK flap, 12 then ablating with an excimer laser for enhancement. The disadvantage of this technique is that the biomechanical stress resistance of the cornea is reduced, 4,13 increasing the risk of iatrogenic ectasia. Corneal cross-linking (CXL) was introduced in 2003 as a new treatment modality to stop the progression of keratoconus. 14 CXL is based on the photodynamic induction of additional cross-links in the cornea by means of riboflavin (vitamin B2) instillation and ultraviolet-a (UV-A) irradiation 15,16 to increase the corneal elastic modulus and hence increase the biomechanical stress resistance. With a failure rate of approximately 7.6% and a complication rate of 2.9%, 17 CXL can be considered a safe and effective treatment. More recently, CXL has been applied in combination with LASIK 18 (named LASIK Xtra) to prevent iatrogenic ectasia. Although there is evidence 19 that LASIK Xtra is effective, it is unknown to what extent CXL can compensate for the LASIK-induced weakening of the cornea. Given the higher number of re-treatment options to select from, several factors should be considered: longterm stability, patient comfort, and visual outcome. Long-term stability is determined by the extent of the biomechanical weakening of the cornea; patient comfort is determined by the postoperative pain and the duration of the surgery; and the visual outcome depends on the invasiveness and difficulty of the surgical procedure, as well as the initiated wound healing processes. This study investigated the induced biomechanical weakening of different re-treatment options after SMILE. Moreover, we investigated whether the loss of biomechanical stress resistance after refractive surgery can be reestablished with CXL treatment. MATERIALS AND METHODS SPECIMENS A total of 96 freshly enucleated eyes from 7- to 9-month-old pigs were obtained from a local slaughterhouse (Marburg, Germany) and used within 24 hours. The eyes had not been steamed and showed an intact epithelium. After dividing the eyes into different groups, they were stored in water at 6 C until the refractive surgery or control treatment was performed. We chose the porcine cornea as the model for the current study due to the large number of samples that were required for the biomechanical tests. Also, in contrast to human donor eyes, porcine corneas could be obtained of the same age and at the same post-mortem time, which reduced the variance due to increasing corneal stiffness with age 20 and tissue degradation after death. REFRACTIVE SURGERIES Different re-treatment options for SMILE were analyzed. We assumed the myopic correction of the first surgery to be diopters (D) and the overall desired myopic correction to be D in all treatment conditions. These high dioptric corrections were chosen to remove a similar ratio of the approximately 800-µm postmortem porcine cornea as for a D correction in the 530-µm living human cornea, within an optical zone of 7 mm. Six different groups were studied. The group (15 eyes) corresponds to a -3D SMILE re-treatment procedure for an D SMILE procedure. The femtosecond lenticule extraction (FLEx) -14D group (28 eyes) represents a cap-to-flap conversion and -3D LASIK retreatment procedure for a D SMILE procedure. The SMILE -11D group (19 eyes) represents a -3D PRK re-treatment procedure on top of the cap of an D SMILE procedure. The group (13 eyes) represents the same cap-to-flap conversion re-treatment as the group but with additional corneal reinforcement by CXL treatment. The control group (34 eyes) was not treated at all and the control+cxl group (13 eyes) received only CXL treatment and no refractive correction. For simplicity, the conditions and were created in one step instead of two. Because we were exclusively interested in biomechanical effects and not in the surgical technique as such, this simplification did not affect the outcome. For standardization purposes, all corneas were deepithelialized and mounted on a customized holder, where the intraocular pressure was maintained at 20 mm Hg. The VisuMax 500-kHz femtosecond laser (Carl Zeiss Meditec AG, Jena, Germany) with a pulse energy of 160 nj and a track/spot distance of 4.5 µm was used in combination with the medium contact glass to create a 160-µm, 8.6-mm cap for the SMILE group or a 160- µm, 8.8-mm flap for the FLEx group, respectively. The lenticule diameter was set to 7.6 mm ( optical zone ). The MEL 80 excimer laser (Carl Zeiss Meditec AG) with a 250-Hz repetition rate was used to perform the -3D ablation on top of the cap in the SMILE -11D group using a 7-mm ablation zone. For CXL treatment, a drop of 0.1% riboflavin, dissolved in PBS (Dulbecco s phosphate buffered saline; 194 Copyright SLACK Incorporated

3 Sigma-Aldrich, Buchs, Switzerland), was instilled onto the anterior cornea in control corneas and onto the stromal bed in FLEx corneas every 3 minutes for a total duration of 30 minutes. Then UV irradiation of 365-nm wavelength and 9 mw/cm 2 irradiance was applied for 10 minutes. BIOMECHANICAL CHARACTERIZATION The biomechanical characterization was performed as described before. 21 Briefly, corneoscleral buttons were mounted on a customized 2D holder and placed within a stress-strain extensometer (Z0.5; Zwick GmbH & Co., Ulm, Germany) as depicted in Figure A (available in the online version of this article). Two stress-strain cycles between 1.27 and 12.5 N were performed. The overall tangent elastic modulus was calculated from the stress-strain curves. With overall we refer to the fact that a constant corneal thickness of 800 µm was assumed, and changes in corneal thickness induced by laser ablation were not considered. The elastic modulus of the cornea is an inherent material property and does not change with laser ablation. Therefore, by not considering the changes in corneal thickness we could use the elastic modulus as a measure to express the actual corneal stress resistance after surgery. STATISTICAL ANALYSIS Normal distribution was confirmed with the Shapiro Wilk test. A univariate analysis of variance was performed in IBM SPSS Statistics (version (IBM Corporation, Zurich, Switzerland), individually for stress and elastic modulus. The LSD post-hoc test with a confidence interval of 95% was used to determine significant differences between groups. RESULTS The analysis of variance showed a significant difference between treatment groups, for both stress (P <.001) and the elastic modulus (P <.001). Journal of Refractive Surgery Figure 1. Corneal (top) stress and (bottom) elastic moduli at 0.8% of strain for the different conditions. Error bars represent the standard deviation. FLEx = femtosecond lenticule extraction; D = diopters; SMILE = small incision lenticule extraction; PRK = photorefractive keratectomy; CXL = corneal cross-linking BIOMECHANICAL WEAKENING WITH DIFFERENT RE- TREATMENT OPTIONS Laser refractive surgery reduced the overall corneal elastic modulus maximally by factor 0.93 (-7%). and SMILE -11DPRK-3D did not induce a significant decrease of the elastic modulus at 0.8% of strain compared to the controls (24.7 ± 2.23 and 22.7 ± 2.61 MPa versus 23.8 ± 3.35 MPa, P.264). In contrast, (22.2 ± 3.37 MPa) significantly reduced the elastic modulus compared to (P =.027) and borderline to controls (P =.066). The measurements of stress confirmed the observation with the elastic moduli for versus control (P =.044), but the difference between and did not reach significance (P =.119). BIOMECHANICAL STIFFENING OF FS-LASIK RE-TREATED CORNEAS CXL treatment significantly increased the elastic modulus compared to all non cross-linked conditions (P.008), on average by factor 1.2 (+20%). When comparing between CXL conditions, the control+cxl condition could resist to a significantly higher stress at 0.8% of strain than the condition (458 ± 17.3 vs 378 ± 31.0 kpa, P =.012), whereas no significant differences were observed for the elastic modulus (29.1 ± 2.85 vs 22.2 ± 3.37 MPa, P =.377) (Figure 1). More detailed results are presented in the tables. Table 1 shows individual mean values for stress and elastic moduli and Table 2 the corresponding P values. DISCUSSION Recent studies report that up to 4% of SMILE surgeries require re-treatment. 6,7 In this study, we have analyzed different re-treatment options and showed that creating a LASIK flap significantly reduced the overall corneal stress resistance, whereas other techniques such as cap-less SMILE or SMILE+PRK did not. However, subsequent CXL treatment could easily compensate for the refractive surgery-induced corneal weakening and even further increase the corneal stiffness compared to the non-ablated control. Looking at our experimental results, SMILE enhancement and PRK enhancement were equivalent and did not induce a significant corneal weakening. From a theoretical point of view, 13 we would expect the SMILE enhancement with another SMILE proce- 195

4 TABLE 1 Mean ± SD Values of Stress and Elastic Moduli Group Stress (Pa) emodulus (Pa) Control 3.92E+05 ± 2.75E E+07 ± 3.35E E+05 ± 3.10E E+07 ± 3.37E+06 SMILE -11D 3.85E+05 ± 2.90E E+07 ± 2.61E E+05 ± 1.93E E+07 ± 2.23E+06 Control+ 4.58E+05 ± 1.73E E+07 ± 2.85E E+05 ± 3.19E E+07 ± 3.48E+06 SD = standard deviation; Pa = pascals; control = no treatment; = femtosecond lenticule extraction cap-to-flap conversion and -3D LASIK re-treatment procedure for a diopter (D) small incision lenticule extraction (SMILE) procedure; SMILE -11D = -3D photorefractive keratecomty (PRK) re-treatment procedure on top of the cap of an D SMILE procedure; = -3D SMILE re-treatment procedure for an D SMILE procedure; Control+CXL = only corneal cross-linking (CXL) treatment and no refractive correction; = same cap-to-flap conversion re-treatment as the group but with additional corneal reinforcement by CXL treatment TABLE 2 P Values Using LSD Post hoc Test Group Stress Elastic Modulus Control vs Control vs.044 a.066 Control vs SMILE -11D Control vs Control+ Control vs <.001 a <.008 a vs Control.044 a.066 vs vs SMILE -11D vs vs Control+ vs Control vs a vs SMILE -11D vs <.001 a. 097 vs Control+ <.001 a. 011a + CXL vs Control+CXL.012 a CXL vs LSD = least significant difference; control = no treatment; = femtosecond lenticule extraction cap-to-flap conversion and -3D LASIK re-treatment procedure for an diopter (D) small incision lenticule extraction (SMILE) procedure; SMILE -11D = -3D photorefractive keratecomty (PRK) re-treatment procedure on top of the cap of an D SMILE procedure; = -3D SMILE re-treatment procedure for an D SMILE procedure; Control+CXL = only corneal cross-linking (CXL) treatment and no refractive correction; = same cap-to-flap conversion re-treatment as the group but with additional corneal reinforcement by CXL treatment a Indicates statistical significance. dure to induce the least biomechanical weakening because both Bowman s membrane and the anterior corneal cap remain intact. Accordingly, PRK enhancement should induce the second least biomechanical weakening because Bowman s membrane is damaged but the corneal cap remains intact. However, one factor we could not fully consider within this study is the biomechanical contribution of Bowman s membrane. In fact, Bowman s membrane in porcine corneas is not as highly developed as in humans or primates Therefore, the results of the current study may be interpreted as the sole effect of the different refractive procedures in a homogenous tissue. The absolute biomechanical weakening of PRK and FLEx are likely to not be directly transferrable to humans. Using femtosecond laser-assisted LASIK for en- 196 Copyright SLACK Incorporated

5 Journal of Refractive Surgery hancement, which requires the conversion to a flapbased procedure, is expected to induce the greatest biomechanical weakening because both the corneal cap and Bowman s membrane cannot further contribute to the resistance to the intraocular pressure. This expectation could be confirmed experimentally: LASIK-style enhancement significantly reduced the overall corneal stiffness by -7% and therefore presents an increased risk for iatrogenic ectasia compared to the other re-treatment alternatives. Nevertheless, we could also show that subsequent accelerated CXL treatment could stiffen the corneal tissue to an even higher extent than it gets weakened through the laser ablation. Nevertheless, CXL has been reported to induce corneal flattening in most keratoconic eyes, 25 which brings in an uncertainty factor regarding the refractive outcome. Also, what needs to be considered further is that if CXL treatment is already applied in the first session, the laser ablation rate during re-treatment is different 26 and needs to be adjusted to guarantee an accurate refractive correction. Because CXL efficacy varies locally and many different protocols are being used, adjusting the laser power is not straightforward and currently not possible. The stiffening effect of CXL in porcine corneas ( 1.8) is lower than in human corneas ( 4.5). 27 Therefore, even if the presence of a better developed Bowman s membrane in humans may lead to a stronger weakening of PRK or LASIK style enhancement, we still can expect that subsequent CXL treatment increases the biomechanical stability of the cornea more than the surgically induced weakening. Although from a clinical point of view, conversion of cap into flap using Circle software offers an uncomplicated and almost painless re-treatment option (LASIK-type enhancement), the Re-SMILE enhancement technique is challenging for the surgeon and has limitations in small corrections. However, our experiments suggest that from a biomechanical point of view, LASIK enhancement is not recommended after SMILE. In contrast, PRK enhancement appears to have a low impact on corneal stability (at least in a porcine eye model), but the management of postoperative pain and delayed visual rehabilitation need to be discussed with the patient prior to this intervention. The biomechanical integrity of the post-mortem porcine cornea is best preserved with both SMILE and PRK enhancements. CXL is a potent technique not only to reestablish, but also to additionally strengthen the corneal tissue after laser refractive surgery. A limitation of this study is that we did not evaluate the refractive changes resulting from subsequent CXL treatment. Therefore, we do not recommend this strategy clinically, particularly because the CXL-related corneal flattening is not yet predictable. The results presented here only demonstrate the extent of possible biomechanical weakening and strengthening in refractive surgery. AUTHOR CONTRIBUTIONS Study concept and design (SK, BS, WS); data collection (SK); analysis and interpretation of data (SK, FH, WS); writing the manuscript (SK, BS); critical revision of the manuscript (FH, WS); statistical expertise (SK); administrative, technical, or material support (BS, FH, WS) REFERENCES 1. Sekundo W, Kunert KS, Blum M. Small incision corneal refractive surgery using the small incision lenticule extraction (SMILE) procedure for the correction of myopia and myopic astigmatism: results of a 6 month prospective study. Br J Ophthalmol. 2011;95: McLeod SD, Kisla TA, Caro NC, McMahon TT. 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7 Figure A. Measurement set-up for the biomechanical characterization. (A) 2D-holder for corneal buttons. (B) Corneal button fixed within the 2D-holder during measurement.