Hyung Keun Lee, MD, Jin Kook Kim, MD, Sung Soo Kim, MD, Eung Kweon Kim, MD, Kwang One Kim, MD, In Sik Lee, MD, Gong Je Seong, MD

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1 Effect of amniotic membrane after laser-assisted subepithelial keratectomy on epithelial healing Clinical and refractive outcomes Hyung Keun Lee, MD, Jin Kook Kim, MD, Sung Soo Kim, MD, Eung Kweon Kim, MD, Kwang One Kim, MD, In Sik Lee, MD, Gong Je Seong, MD Purpose: To evaluate the effect of an amniotic membrane (AM) on reepithelialization time, corneal haze, and postoperative visual and refractive outcomes after laserassisted subepithelial keratectomy (LASEK) for myopia and myopic astigmatism. Setting: Department of Ophthalmology, Yonsei University College of Medicine, and Balgeunsesang Ophthalmology Clinic, Seoul, Korea. Methods: One hundred fifty-two eyes of 84 patients with myopia or myopic astigmatism were prospectively evaluated for 6 months after LASEK. An AM was placed as a strip on the inferior limbus in 94 eyes of 54 patients after LASEK; 58 eyes of 30 patients served as the control group. Postoperative epithelial healing time, uncorrected visual acuity (UCVA), best corrected visual acuity, remaining refractive error, and corneal haze were examined. Results: The reepithelialization time was shorter in the AM group (2.40 days 0.94 [SD]) than in the control group ( days) (P.001). At 6 months, 86 eyes (91.5%) in the AM group had a UCVA of 20/25 or better and 90 eyes (95.7%) had a UCVA of 20/40 or better; 48 eyes (82.8%) and 53 eyes (91.4%) in the control group had a UCVA of 20/25 or better and 20/40 or better, respectively. The mean spherical equivalent in the AM group was diopter (D) and in the control group, D (P.001). The corneal haze was significantly less in the AM group than in the control group (P.001). Conclusion: Amniotic membrane use after LASEK induced rapid epithelial healing with more favorable visual and refractive outcomes and lower corneal haze scores than conventional LASEK. J Cataract Refract Surg 2004; 30: ASCRS and ESCRS Laser-assisted subepithelial keratectomy (LASEK) ocular pain after surgery or stromal opacity similar to was introduced and popularized with the advan- that after PRK. 3 tages of decreased pain, tearing, irritating symptoms, Amniotic membrane (AM), the innermost memand corneal opacity compared to photorefractive kerabrane lining the placenta facing the fetus, is known to tectomy (PRK). It also enables the correction of refracinduce rapid corneal epithelial healing and is used to tive errors, especially in eyes with thin corneas. 1,2 There is, however, some controversy about the possibility of reconstruct the ocular surface in cases of partial limbal- delayed epithelial healing resulting in complaints of cell deficiency and persistent epithelial defects. 4 7 Recent studies show that AM transplantation results in less stromal infiltration of inflammatory cells and a reduced Accepted for publication June 17, loss of keratocytes in rabbit corneas. 8,9 These results Reprint requests to Eung Kweon Kim, MD, Institute of Vision Research, suggest that AM application to the cornea after LASEK Department of Ophthalmology, College of Medicine, Yonsei University, 134 Shinchon-dong, Sudaemoon-gu, Seoul, Korea. eungkkim@ would affect the epithelial healing pattern and stroyumc.yonsei.ac.kr. mal haze ASCRS and ESCRS /04/$ see front matter Published by Elsevier Inc. doi:s (03)

2 This study prospectively evaluated the effect of AM placement after LASEK on epithelial healing time, postoperative visual outcomes including refractive status and visual acuity, and corneal opacities. Patients and Methods LASEK WITH AMNIOTIC MEMBRANE One hundred fifty-two consecutive eyes of 84 patients were enrolled between September 2000 and December The preoperative ophthalmic examination of all patients included slitlamp biomicroscopy, intraocular pressure, fundus examination, pupil diameter measurements, Schirmer test, manifest refraction, corneal keratometry, corneal topography, corneal pachymetry, and visual field examination. No patient had a history of refractive procedures or cataract surgery, Figure 1. (Lee) One day after LASEK with AM strip transplantation. keratoconus, diabetes, glaucoma, connective tissue disorders, Arrowheads mark the upper margin of the AM. or retinal disease. After the preoperative examinations, the patients were proparacaine hydrochloride 0.5% (Alcaine ) instilled. An told about the AM and its intended use after LASEK. They alcohol solution cone (J2905, Janach) with an 8.5 mm diamewere also told that this might be the first human trial after ter was placed on the eye. Twenty percent of the alcohol photoablation, and written informed consent was obtained solution was instilled inside the cone, left for about 20 seconce the patients agreed to participate. The patients who did onds, and then carefully washed off with a balanced salt not agree to the use of the AM on the LASEK-treated eye solution so the epithelium around the flap was not disturbed. were considered the control group after they provided in- The epithelial flap was gently lifted with an epithelial formed consent. microhoe (J2915A, Janach). If the edges were difficult to lift, Preparation of Preserved Human Amniotic Membrane the alcohol application was repeated for another 10 seconds. The epithelial flap was then peeled back as a sheet toward the The AM was prepared following the methods of Lee 12 o clock position using a spatula (J2910A, Janach). To avoid and Tseng. 10 Human placenta was obtained during an elective tears in the epithelial flap, the basal lamina was carefully Cesarean section in a seronegative (human immunodeficiency separated from Bowman s layer instead of separating the virus, human hepatitis type B and C, and syphilis) woman. epithelium from the basal lamina. If the epithelial flap was Informed consent was obtained from the AM donor after the not applied to the denuded stromal as an intact sheet, the purpose of the AM was explained. Under a laminar flow hood, eyes were excluded from the study. the placenta was cleaned of blood clots with sterile phosphate- Excimer laser treatment was performed in the usual manbuffered saline solution containing penicillin 50 g/ L, ner using the nomogram for PRK with the EC-5000 laser streptomycin 50 g/ L, neomycin 100 g/ L, and ampho- system (Nidek). The flap was washed with a balanced salt tericin B 2.5 g/ L. The amnion was separated from the solution and then repositioned carefully with a spatula. In chorion by blunt dissection and flattened on a nitrocellulose the control group, the treatment was finished with the applipaper, with the epithelium-basement-membrane surface fac- cation of a therapeutic soft contact lens on the operated eye. ing away from the paper. The paper with adherent AM was In the AM group, the AM was washed thoroughly and then cut into 1.5 cm blocks and stored at 80 C until immersed in a balanced salt solution containing gentamicin transplantation in a sterile vial containing Dulbecco s modi- 8 g/ L and cefaxolin 4 g/ L for 30 minutes before surfied Eagle medium (GIBCO Life Technologies, Inc.) and gery. The membrane was then cut into 1.5 cm 0.3 cm glycerol (GIBCO Life Technologies, Inc.) at the ratio of 1:1 rectangular pieces. With the mesenchymal side facing the (vol/vol). Six months after delivery, the AM donors were cornea, the membrane was attached to the stromal bed after retested by the previous serologic examinations for the win- excimer laser ablation. The slender AM strip was placed on dow period of transmittable diseases. Only the AM that the inferior limbus so it would not touch the ablated corneal passed prenatal and postnatal serologic tests as seronegative bed and secured with 2 interrupted 10-0 nylon sutures placed was used. on the limbal conjunctiva. A therapeutic soft contact lens was also placed on the eyes with the AM strip (Figure 1). Laser-Assisted Subepithelial Keratectomy Procedure One drop of ofloxacin 0.3% (Tarivid ) and diclofenac The LASEK procedure was the same in the control and 0.1% (Optanac ) was given to the patients in both groups AM groups. A speculum was applied to the patient s eye and immediately after LASEK. All patients were checked daily J CATARACT REFRACT SURG VOL 30, FEBRUARY

3 Table 1. Preoperative independent varaibles. illumination; 1, for an opacity of minimal density seen with difficulty under direct and diffuse illumination; 2, for an AM Group Control Characteristics (n 98) (n 58) P Value easily visible opacity; 3, for a dense opacity that significantly decreased visualization of intraocular structures such as the Age* NS iris and retina; and 4, for an opaque cornea. Sex (male/female) 35/12 21/9 NS Statistical analysis was performed using Statistical Analysis System (version 6.12, SAS Institute Inc.). A P value less Spherical equivalent* NS than 0.05 was considered statistically significant. Degree of cylinder* NS Corneal thickness* NS Mean SD AM amniotic membrane; NS not significant *Student t test Chi-square test Results The AM group and the control group consisted of 94 eyes of 54 patients and 58 eyes of 30 patients, respectively. Preoperative data are shown in Table 1. There were no statistically significant differences between the 2 groups in the preoperative independent variables. Eyes with an AM had a significantly faster rate of epithelial healing than the control eyes (P.001). Round or oval epithelial defects under the epithelial flap and therapeutic soft lens were observed on the day of treatment; the defects decreased concentrically at each follow-up. The mean epithelial healing time was 2.40 days 0.94 (SD) in the AM group and days in the control group. Three days after surgery, 82 eyes (87.2%) in the AM group and 18 eyes (31.8%) in the control group were completely reepithelialized. Ten eyes (10.6%) in the AM group showed complete Figure 2. (Lee) Completion of epithelial healing in the control and reepithelialization within 36 hours of surgery, and 2 AM groups after LASEK. eyes (3.45%) in the control group showed complete reepithelialization after 5 days (Figure 2). until the epithelial defect completely healed. They were in- At 1 week, 61 eyes (64.9%) in the AM group and structed to apply 1 drop of diclofenac and ofloxacin every 31 eyes (53.5%) in the control group had a UCVA 2 hours and artificial tears (Hyalein 0.1%) every hour until epithelial healing was complete. of 20/25 or better. At 6 months, 48 eyes (82.8%) Complete epithelialization was determined by daily slitlamp observation. Once the epithelium was healed, the thera- eyes (91.4%), of 20/40 or better; in the AM group, in the control group had a UCVA of 20/25 and 53 peutic contact lenses and AM, if used, were removed from 86 eyes (91.5%) had a UCVA of 20/25 or better and the cornea. Then, ofloxacin 0.3% and fluorometholone 0.1% 90 eyes (95.7%), of 20/40 or better (Table 2). (Fluorometholone ) were administered 4 times daily for There were no statistically significant differences in 1 week and 2 times daily for 1 month. Uncorrected visual acuity (UCVA), best corrected visual BCVA between the 2 groups during the follow-up. acuity (BCVA), manifest refraction, tonometry, and slitlamp Most patients in both groups showed no changes in biomicroscopy were performed at each follow-up examina- BCVA. However, 7 eyes (7.5%) in the AM group and tion. Subepithelial corneal haze levels were checked with a 5 eyes (8.6%) in the control group lost 1 or 2 Snellen slitlamp 1, 3, and 6 months after surgery. Two of the authors, lines of BCVA at the final examination. who did not know whether the eye examined was part of Statistically significant differences in the mean refracthe AM group or the control group, observed and graded the corneal opacities from 0 to 4, according to the method tive error were found between 1 month and 6 months of Hanna et al. 11 A grade of 0 was given for totally clear ; (Table 3). At the final examination, 70 eyes (74.5%) 0.5, for a faint corneal opacity seen only by oblique indirect in the AM group and 40 eyes (69.0%) in the control 336 J CATARACT REFRACT SURG VOL 30, FEBRUARY 2004

4 Table 2. Visual acuity results in the control and AM groups over time after LASEK. Month(s) After LASEK 1* 3* 6* UCVA AM Control AM Control AM Control 20/20 74 (78.7) 37 (63.8) 79 (84.0) 38 (65.6) 80 (85.1) 41 (70.7) 20/25 83 (88.3) 46 (79.3) 86 (91.5) 49 (84.5) 86 (91.5) 48 (82.8) 20/30 85 (90.4) 54 (93.1) 90 (95.7) 51 (87.9) 87 (92.6) 50 (86.2) 20/40 94 (100) 57 (98.3) 92 (97.9) 55 (94.8) 90 (95.7) 53 (91.4) 20/50 94 (100) 58 (100) 94 (100) 58 (100) 94 (100) 58 (100) Number of eyes (%) AM amniotic membrane; LASEK laser-assisted subepithelial keratectomy *p.01, chi-square test group had a mean spherical equivalent (SE) within in the control group had a corneal haze score greater 0.50 diopter of the attempted myopic correction, than 2 (P.01). At 6 months, the AM group had a more with a significant between-group difference (P.01). favorable corneal haze score. Forty-five eyes (47.8%) in The cylinder magnitude between the groups was also the AM group and 21 eyes (35.2%) in the control group significantly different from 1 month to 6 months had a haze score below grade 1. Three eyes (5.2%) in (Table 3). the control group and 4 eyes (4.3%) in the AM group Corneal haze was examined and graded under the had grade 3 corneal opacity 6 months after surgery slitlamp according to the previously described grading (Table 4). system. At 1 month, the corneal haze score was less The correlations between duration of the epithelial than grade 1 in all eyes. But the AM group had less defect, corneal opacity, SE, and cylinder magnitude in haze than the control group (P.01). At 3 months, all eyes are shown in Table 4. The strongest correlation 8 eyes (8.5%) in the AM group and 20 eyes (34.7%) was between corneal opacity and SE at 6 months (correlation coefficient 0.711, P.000). The correlation Table 3. Refractive error in the control and AM groups after coefficient showed a positive correlation between the LASEK. duration of the epithelial defect and the stromal opacity Mean D SD (r 0.653, P.000) and negative correlations between Time Preoperative AM Group Control Group P Value* the stromal opacity and the SE (r 0.607, P.000) (Table 5). SE Cyl Discussion Postoperative From our results, we conclude that the epithelialization and the wound-healing process after LASEK was SE * influenced by the AM strip. Both visual and refractive Cyl * outcomes were acceptable in all patients. However, the AM group had a shorter epithelial healing time and SE * more favorable visual and refractive outcomes. At the Cyl * beginning of the study, we used the AM to cover the entire LASEK-treated surface directly. However, in SE * many eyes, the regenerated corneal epithelium was detached during removal of the AM so a longer period Cyl * was needed for reepithelialization. We therefore modified the so-called overlay technique to use the AM as a narrow strip fixed on the limbus. 1mo 3mo 6mo AM amniotic membrane; Cyl cylinder; LASEK laser-assisted subepithelial keratectomy; SE spherical equivalent *p.05, Student t test J CATARACT REFRACT SURG VOL 30, FEBRUARY

5 Table 4. Corneal haze score in the control and AM groups after LASEK. Month(s) After LASEK 1* 3* 6* Score AM Control AM Control AM Control 0 48 (51.1) 20 (34.4) 44 (46.8) 6 (10.2) 10 (10.6) 4 (6.9) (31.9) 23 (39.7) 20 (21.3) 10 (17.2) 35 (37.2) 17 (29.3) 1 16 (17.0) 15 (25.9) 22 (23.4) 22 (37.9) 38 (40.4) 24 (41.4) 2 0 (0) 0 (0) 5 (5.3) 18 (31.3) 7 (7.5) 10 (17.2) 3 0 (0) 0 (0) 3 (3.2) 2 (3.4) 4 (4.3) 3 (5.2) 4 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Number of eyes (%) AM amniotic membrane; LASEK laser-assisted subepithelial keratectomy *p.01, chi-square test The exact mechanism of how AM use after LASEK first is that the AM strip may act as a mechanical barrier is effective in corneal epithelial healing was not demon- against white-blood-cell migration that emerges from strated in this study. The AM was not directly in contact the limbal blood vessels or tear fluid in the conjunctival with the wound bed, so the direct physical and mechani- sac. After excimer laser refractive surgery, the concentracal roles of the AM would hardly affect the wound tion of inflammatory cells and cytokines such as transhealing after LASEK. A bandage soft contact lens forming growth factor- is increased immediately. 12,13 (BSCL) was used in both groups until the epithelial The greatest amount of tear flow occurs in the meniscus healing was complete. Both groups were studied at the adjacent to the lower lid. 14 The AM, which was placed same time by the same surgeon and observers. We on the inferior limbus, might act as a mechanical barrier therefore think the BSCL had the same effect on both so it would decrease inflammatory cell infiltration in groups and did not act as a bias in 1 group. the wound bed and reduce epithelial cell destruction We suggest 2 possible mechanisms to explain how by the white blood cells and the inflammatory factors the AM strip facilitates corneal epithelial healing. The released from the white cell. The second possibility is that the various cytokines Table 5. Correlation coefficients between duration of epithelial de- and epithelial-growth-associated factors within the AM fect and corneal opacity, refractive error, and cylinder magnitude at would help epithelial regeneration and wound healing 6 months. after LASEK. The AM has been shown to express epi- DED SE CM SO dermal growth factor, hepatocyte growth factor, and DED keratinocyte growth factor 15 and suppress the proinflamr matory cytokines during wound healing, just as interleu- Sig (2-tailed) kin does. 16 Although the exact role of these factors in SE AM associated with facilitating cornea wound healing r is not known, the factors may play some role in corneal Sig (2-tailed) epithelial healing. However, further studies of how the CM AM promotes epithelial healing are needed. r The UCVA was better in the AM eyes than in the Sig (2-tailed) control group from 1 week to 6 months after surgery. SO We believe the faster epithelial healing that resulted in r a smoother refractive surface in the AM group provided Sig (2-tailed) better UCVA a week after LASEK. At 6 months, corneal CM cylinder magnitude; DED duration of epithelial defect; SD opacities, which might cause irregular astigmatism and stromal opacity; SE spherical equivalent; Sig significance increase postoperative refractive errors, were fewer in 338 J CATARACT REFRACT SURG VOL 30, FEBRUARY 2004

6 the AM group. We think these could be related to the 2. Dastjerdi MH, Soong HK. LASEK (laser subepithelial better UCVA. keratomileusis). Curr Opin Ophthalmol 2002; 13: We did not find statistically significant differences 3. Litwak S, Zadok D, Garcia-de Quevedo V, et al. Laserin the BCVA between the 2 groups until 6 months assisted subepithelial keratectomy versus photorefractive postoperatively. After PRK or LASEK, the corneal haze keratectomy for the correction of myopia; a prospective is associated with BCVA loss. 17 Up to 6 months postop- comparative study. J Cataract Refract Surg 2002; 28: eratively, most patients showed no changes in BCVA and corneal haze below grade 2. We do not think this 4. Azuara-Blanco A, Pillai CT, Dua HS. Amniotic mem- brane transplantation for ocular surface reconstruction. mild to moderate grade of corneal haze would affect Br J Ophthalmol 1999; 83: BCVA significantly. 5. Tseng SCG, Prabhasawat P, Barton K, et al. Amniotic Although the refractive outcomes in the 2 groups membrane transplantation with or without limbal allografts were relatively comparable, there was a statistically siglimbal for corneal surface reconstruction in patients with stem cell deficiency. Arch Ophthalmol 1998; nificant difference in the remaining refractive errors. 116: Besides the spherical component of the postoperative 6. Tsubota K, Satake Y, Ohyama M, et al. Surgical reconrefractive error, the magnitude of astigmatism was statis- struction of the ocular surface in advanced ocular cicatritically significantly different between the groups. More- cial pemphigoid and Stevens-Johnson syndrome. Am J over, we found correlations among corneal opacity, Ophthalmol 1996; 122:38 52 epithelial healing time, and remaining refractive errors. 7. Chen H-J, Pires RTF, Tseng SCG. Amniotic membrane From these results, we think it is possible that the AM transplantation for severe neurotrophic corneal ulcers. Br J Ophthalmol 2000; 84: affects postoperative wound healing after LASEK. 8. Wang MX, Gray TB, Park WC, et al. Reduction in The excessive synthesis of collagen and glycosami- corneal haze and apoptosis by amniotic membrane manoglycans by keratocytes in the ablation zone can lead to trix in excimer laser photoablation in rabbits. J Cataract a thickening that yields myopic regression, astigmatism, Refract Surg 2001; 27: irregular topography, and optical aberration. 18 Tabin 9. Park WC, Tseng SCG. Modulation of acute inflamma- tion and keratocyte death by suturing, blood, and amniet al. 19 report that surgically induced refractive errors otic membrane in PRK. Invest Ophthalmol Vis Sci including astigmatism may be the result of irregular 2000; 41: epithelial thickening or epithelial hyperplasia. There- 10. Lee S-H, Tseng SCG. Amniotic membrane transplantafore, the postoperative wound healing after excimer laser tion for persistent epithelial defects with ulceration. Am surgery could significantly affect the postoperative visual J Ophthalmol 1997; 123: and refractive outcomes. It is possible that corneal haze 11. Hanna KD, Pouliquen YM, Waring GO III, et al. Cor- neal wound healing in monkeys after repeated excimer and surgically induced refractive errors could be regulaser photorefractive keratectomy. Arch Ophthalmol lated and moreover reduced with antiinflammatory 1992; 110: treatment, including AM use. 12. Tuominen ISJ, Tervo TMT, Teppo A-M, et al. Human In conclusion, a strip of AM fixed on the limbus tear fluid PDGF-BB, TNF- and TGF- 1 vs corneal after LASEK may reduce the duration of epithelial denerve haze and regeneration of corneal epithelium and subbasal plexus after PRK. Exp Eye Res 2001; 72: fects and decrease corneal haze, which improves visual 13. Ramirez-Florez S, Maurice DM. Inflammatory cells, reoutcomes in LASEK eyes. Further studies are needed fractive regression, and haze after excimer laser PRK. to investigate the subcellular mechanisms of the AM J Refract Surg 1996; 12: in facilitating epithelial healing. We also believe that a 14. Ōgūt MS, Bavbek T, Kazokoglu H. Assessment of tear longer follow-up of the patients is needed. drainage by fluorescein dye disappearance test after ex- perimental canalicular obstruction. Acta Ophthalmol References 1993; 71: Koizumi N, Inatomi T, Sotozono C, et al. Growth factor 1. Lee JB, Seong GJ, Lee JH, et al. Comparison of laser mrna and protein in preserved human amniotic memepithelial keratomileusis and photorefractive keratec- brane. Curr Eye Research 2000; 20: tomy for low to moderate myopia. J Cataract Refract 16. Solomon A, Rosenblatt M, Monroy D, et al. Suppression Surg 2001; 27: of interleukin 1 and interleukin 1 in human limbal J CATARACT REFRACT SURG VOL 30, FEBRUARY

7 epithelial cells cultured on the amniotic membrane stro- pic astigmatism; the Melbourne Excimer Laser Group. mal matrix. Br J Ophthalmol 2001; 85: J Cataract Refract Surg 1996; 22: Van Gelder RN, Steger-May K, Yang SH, et al. Comparison of photorefractive keratectomy, astigmatic PRK, From the Institute of Vision Research, Department of Ophthalmology, laser in situ keratomileusis, and astigmatic LASIK in the College of Medicine (H.K. Lee, S.S. Kim, E.K. Kim, Seong) and treatment of myopia. J Cataract Refract Surg 2002; 28: BK21 Project for Medical Science (E.K. Kim); Yonsei University; and Balgeunsesang Ophthalmology Clinic (J.K. Kim, I.S. Lee), Seoul, Korea. 18. Shah S, Chatterjee A, Smith RJ. Predictability and out- Supported by a grant (02-PJ1-PG1-CH ) from the Korea comes of photoastigmatic keratectomy using the Nidek Health 21 R&D Project, Ministry of Health and Welfare, Republic EC-5000 excimer laser. J Cataract Refract Surg 2002; of Korea. 28: Tabin GC, Alpins N, Aldred GF, et al. Astigmatic change None of authors has a financial or proprietary interest in any product 1 year after excimer laser treatment of myopia and myo- mentioned. 340 J CATARACT REFRACT SURG VOL 30, FEBRUARY 2004