대한안과학회지 2014 년제 55 권제 5 호 J Korean Ophthalmol Soc 2014;55(5):646-655 pissn: 0378-6471 eissn: 2092-9374 http://dx.doi.org/10.3341/jkos.2014.55.5.646 Original Article 미세표면분석기법을이용한미용콘택트렌즈와일반콘택트렌즈의표면거칠기와세균부착성비교 Comparison of Surface Roughness and Bacterial Adhesion between Cosmetic Contact Lenses and Conventional Contact Lenses 지용우 1 홍순호 2 정동룡 3 김응권 1,4 이형근 1,4 Yong Woo Ji, MD 1, Soon Ho Hong 2, Dong Yong Chung 3, Eung Kweon Kim, MD 1,4, Hyung Keun Lee, MD 1,4 연세대학교의과대학안과학교실시기능개발연구소 1, 연세대학교의과대학강남세브란스병원진단검사의학미생물학과 2, 연세의생명연구원형태학연구실 3, 연세대학교의과대학안과학교실각막이상증연구소 4 Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine 1, Seoul, Korea Division of Microbiology, Department of Laboratory Medicine, Gangnam Severance Hospital, Yonsei University College of Medicine 2, Seoul, Korea Morphology Lab., Yonsei Biomedical Research Institute 3, Seoul, Korea Institute of Corneal Dystrophy Research, Department of Ophthalmology, Yonsei University College of Medicine 4, Seoul, Korea Purpose: To determine the surface roughness of cosmetic and conventional contact lenses (CLs) and their susceptibility to bacterial adhesion. Methods: Concave surface roughness of cosmetic and conventional hydrogel (Etafilcon A) CLs was measured by atomic force microscopy (AFM) and scanning electron microscopy (SEM). In particular, the surface of the color tinted area of cosmetic CLs was measured. CLs were immersed into a bacterial solution of Pseudomonas aeruginosa for 1, 12, or 24 hours and culture of P. aeruginosa that had adhered to the CLs was performed. Results: Concave surface roughness of cosmetic CLs significantly increased compared with conventional CLs by AFM (p < 0.05). Bacterial colony formation of P. aeruginosa adhering to cosmetic CLs within one hour significantly increased compared with conventional CLs (p = 0.047). Adhesions of P. aeruginosa to CLs within one hour was found to correlate significantly with the surface roughness of CL (r > 0.9, p < 0.05). By SEM, P. aeruginosa had adhered to the color-tinted area more than to the non-color-tinted area of cosmetic CLs. Conclusions: Surface of cosmetic CLs was significantly rougher and initial adhesion of bacteria was higher to cosmetic CLs than to conventional CLs. In particular, an increased number of bacteria was found to be adhered to the color-tinted area of cosmetic CLs. Initial bacterial adhesion is important because it is the first stage of bacterial attachment process to any surface. After then, the adherent bacteria can progress to form a biofilm. Increased surface roughness of CLs contributes to opportunities for the CL to come into contact with bacteria, and thus, initial bacterial adhesion increases. In this study, it is clear that cosmetic CLs are more vulnerable to bacterial adhesion. To avoid serious complications, such as bacterial keratitis, the manufacturing process for smoothing and treating the surface in order to inhibit bacterial adhesion should be developed in the future. J Korean Ophthalmol Soc 2014;55(5):646-655 Key Words: Atomic force microscope, Bacterial adhesion, Cosmetic contact lens, Pseudomonas aeruginosa, Surface roughness Received: 2013. 9. 13. Revised: 2013. 10. 5. Accepted: 2014. 4. 1. Address reprint requests to Hyung Keun Lee, MD Department of Ophthalmology, Gangnam Severance Hospital, #211 Eonju-ro, Gangnam-gu, Seoul 135-720, Korea Tel: 82-2-2019-3440, Fax: 82-2-3463-1049 E-mail: shadik@yuhs.ac * This study was presented as a narration at the 110th Annual Meeting of the Korean Ophthalmological Society 2013. * This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI13C0055). 미용콘택트렌즈는단순한시력의개선뿐만아니라홍채의색깔을변화시키고강조하여환자의외양을바꿀수있다는점에서최근그사용이증가하고있다. 보고에의하면, 중국, 일본및국내를포함한동아시아에서 2012년도전체콘택트렌즈사용자의 13% 가미용목적으로컬러콘택트렌즈를사용하고있다고한다. 1-4 미용콘택트렌즈역시일반연성콘택트렌즈의사용에서가능한합병증이모두발생할수있는데, 각막미란, 상피결손, 점상각막염, 각막저산소증, c2014 The Korean Ophthalmological Society This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 646
- 지용우외 : 세균부착과관련된미용렌즈표면의특성 - 각막신생혈관등은물론이거니와각막부종, 각막융해, 각막뒤틀림, 데스메막류, 각막혼탁등도보고되고있다. 5-8 특히시력상실의치명적인결과를초래할수있는세균성각막염및궤양등의심각한합병증또한종종접하게되는데, 환자들의소견은일반연성콘택트렌즈사용자의그것보다세균감염에더욱취약한양상이다. 9-13 콘택트렌즈와연관된세균감염의기전에대해서는기존의연구들에서이미렌즈표면의소수성과거칠기가세균부착에매우중요함을제시한바있으나, 이는그대상이모두일반적인무색의연성콘택트렌즈에만국한되어있다. 14-18 미용콘택트렌즈사용으로인한세균성각막염은심각한임상양상및사회적문제인식에비해병인에대한과학적인연구가부족한실정으로, 렌즈의부적절한위생관리및소독등의외부적인요인에만집중되어있을뿐미용콘택트렌즈의직접적인성상과관련해서는논의된바가없다. 19-24 따라서본연구는미용콘택트렌즈사용이세균감염과어떠한직접적관련이있는지알아보기위하여, 미세표면분석기법을이용하여미용콘택트렌즈표면의거칠기와세균부착의연관성을과학적이고객관적으로연구하였다. 대상과방법 국내에서유통되고있는하이드로겔 (Etafilcon A, FDA Group IV, high water & ionic poymers) 재질의일일착용및연속착용콘택트렌즈중, 미용목적의도색처리가동반된컬러콘택트렌즈와일반무색연성콘택트렌즈를대상으로하였다 (1-DAY ACUVUE MOIST /ACUVUE 2 /1-DAY ACUVUE DEFINE / ACUVUE 2 DEFINE, Johnson & Johnson, Vision Care, Inc., NJ, USA). 원자력현미경 (atomic force microscopy, AFM) 촬영을위하여, 형태유지를위한최소의수분만을남겨둔하이드로겔콘택트렌즈샘플을레이저블레이드를이용하여 1/4 크기로잘라서슬라이드위에양면테이프로고정후각각의내측표면 (concave surface) 을촬영하였고, 특히미용콘택트렌즈의경우는도색처리가되어있는부분을직접촬영하였다. 2D XY Flexure 스캐너와 High-Force Z 스캐너가분리되어장착되어있고 Super-Luminescent Diode (SLD) 광원을이용하는 Park Systems (XE-Bio) 의원자력현미경을이용하여비접촉식모드에서진행하였다. 접촉면에알루미늄이코팅된 42 N/m force constant의실리콘캔틸레버 (Si 3N 4 Cantilever) 를사용하여, 50 50 μm 2 의단위면적에대한 Average Roughness (Ra), Root Mean Square Roughness (Rq), Ten Points Mean Height Roughness (Rz) 의거칠기지표를측정하였다. 하이드로겔성분의콘택트렌즈샘플이 탈수되어변형될수있으므로모든검사는 20분이내로최대한빠르게수행하였다. 거칠기지표 1. Average Roughness (Ra): arithmetic average height (z) : 평균 height 를먼저구하여이를기준으로요철부위 (peak) 즉 (+) 값과, 패인부위 (valley) 즉 (-) 값을있는그대로계산한, 단순히평균으로부터벗어나있는정도만을계산한것이다. (+), (-) 값이상쇄될수있으므로실제로는다른값의데이터가같은결과값을보일수있다. 2. Root Mean Square (RMS) Roughness (Rq): 수식에서보듯이평균과의편차에제곱을하여모두양의차이값으로만든후이것을제곱근한, 표면높이에대한표준편차이다. Ra값이비슷할때, 이것을통해서더분별력을가질수있다. 계산시측정값의제곱이되므로심한요철부위나패인부위가있다면이값은더욱커지게된다. Artifact에의해영향을많이받을수도있겠으나, 반대로특정부분의좋지않은거칠기값이더분명하게반영될수있다는장점이있다. 3. Ten Points Mean Height Roughness (Rz): 10개의점을찍어 peak의평균과 valley의평균을더한값으로, 표면의도장두께또는접점계면의발생정도를간접적으로알수있어, 불규칙한표면에미생물을비롯한미세물질의포획을비교할수있다. 25-27 콘택트렌즈표면에세균을부착하기위하여, 렌즈에의한세균성각막염에서흔한균주인 Pseudomonas aeruginosa의대표균주 (P. aeruginosa ATCC No. 29853) 를사용하였다. 탁도계 (DensiCHEK TM Plus, BioMérieux, Inc.) 를이용하여 1 McFarland, 즉 3.0 10 8 CFU/mL의세균용액을준비한후그중 500 μl만취하여, 2.5 10 7 CFU/mL의 3 cc 세균용액을 12-well plate에준비하였다. 먼저상온 2 5 에서세균용액을 24시간이상방치해도세균증식으로인하여탁도가변하지않음을확인하였다. 준비된세균용액에각각의콘택트렌즈를충분히잠기도록담근후, Parafilm으로밀봉하여상온 25 에서각각 1시간, 12시간, 647
- 대한안과학회지 2014 년제 55 권제 5 호 - 24시간동안렌즈표면에세균이부착되도록유도하였다. 해당시간이경과하면렌즈를꺼내어부드럽게 2-3회가량 A 물기를털어내고천천히흐르는생리식염수 20 cc로천천히 3회세척하여콘택트렌즈표면에부착력없이존재하는세균을제거하였다. 이후생리식염수 5 cc가담겨있는멸균 tube에오염되지않도록조심히렌즈를옮겨담은후 Laboratory Vortex Mixer (VORTEX-GENIE 2, Scientific Industries, Inc., USA) 를이용하여, 3000 rpm로 1분씩렌즈표면에부착된세균을생리식염수에탈착및분리시켰다. 분리된세균이담겨있는용액에서 1:1000 백금이를이용하여 Right angle streaking method for urine culture 방법으로혈액한천배지 (BAP) 에선상도말하여 C 세균을순수분리하였다. 도말한배지는 37, 5% CO 2 incubator 안에서 18 시간동안뒤집어배양하였고, 사진을촬영하여발생한세균군집수를계산, 비교하였다. ( 단, 24시간동안세균을부착하는경우세균군집수가육안으로세기힘들정도로증가하여 1시간, 12시간세균부착시보다세균용액의농도를 1/4로희석하여시행한후, 발생한세균군집수에 4배를하여계산하였다.) 미용콘택트렌즈의표면상태및세균부착양상을직접관찰하기위하여주사전자현미경 (Scanning electron microscopy, SEM) 을이용하였다. 멸균상태및 24시간동안세균을부착한상태의콘택트렌즈를촬영대상으로, 0.1 M PBS 완충액 (ph 7.4) 으로조정한 Karnovsky 고정액 (2% Glutaraldehyde, 2% Paraformaldehyde, 0.5% CaCl 2) 으로 6 시간이상전고정하였다. 0.1 M PBS로 2시간세척하여 1% OsO 4 (0.1 M PBS buffer) 로 2시간고정하였다. 고정된샘플은 0.1 M PBS buffer로 10분간세척후저농도 Alcohol 에서무수알코올까지탈수 (50%, 60%, 70%, 80%, 90%, 95%, 100% 순서 ) 한후에 Isoamyl acetate로치환후약 30분-1시간후에임계점건조 (CPD-critical point dry: Hitachi HCP-2) 한다음 Ion coater (Eiko IB-3형 - Japan) 를이용하여 30 nm 두께로 Gold coating하여 FE-SEM (Japan, Hitachi S-800) 으로콘택트렌즈내측표면의도색처리된부분과그렇지않은부분및세균이부착된모습을촬영하였다. 통계학적분석은통계프로그램 SPSS (version 21.0, SPSS Inc., Chicago, IL, USA) 를사용하여독립표본 t 검정과 Pearson 상관분석을이용하여비교분석하였다. 통계학적유의성의 기준은 p<0.05 로정의하였다. Daily wear Extended wear B 결 Conventional 과 실험에사용된하이드로겔콘택트렌즈의성분은모두 Etafilcon A (FDA Group IV) 로함수율 (Water Content 58%) 과산소투과율 (Dk/t 25.5 10-9 ) 은차이가없었다. 동일사용형태 (Daily wear, Extended wear) 의콘택트렌즈는도색처리와상관없이모든기초성상이동일하였으며, 연속착용렌즈 (Extended wear) 의경우는일일착용렌즈 (Daily wear) 에비하여직경과곡률반경에서약간의차이를보였다 (Table 1). D연구에사용된미용콘택트렌즈는렌즈전면과후면의사이에염료를넣고접합시키는 Sandwich method (ACUVUE Beauty-Wrapped-In-Comfort TM ) 공법을사용하여실제노출된표면의재질은일반무색의콘택트렌즈와동일한성분으로간주할수있었으며, 그무늬및형상은 Fig. 1과같았다. 원자력현미경을이용한콘택트렌즈표면의거칠기평가각콘택트렌즈의내측표면에대하여 2500 μm 2 단위면적의 3-dimensional image를구현한후거칠기의지표인 Average Roughness (Ra), Root Mean Square Roughness (Rq), Ten Points Mean Height Roughness (Rz) 를각각측정 Cosmetic Figure 1. Shape and tinted pattern of four hydrogel contact lenses (CL) used in the study. Table 1. Contact lens (CL) used in the study Contact lens type Recommended Water Dk/t value BC (mm) Center thickness Material replacement content (%) (@-3.00D) /Dia (mm) (@-3.00D) (mm) Conventional Daily wear 1 day Etafilcon A 58 25.5 10-9 8.5/14.2 0.084 Extended wear 2 weeks Etafilcon A 58 25.5 10-9 8.3/14.0 0.084 Cosmetic Daily wear 1 day Etafilcon A 58 25.5 10-9 8.5/14.2 0.084 Extended wear 2 weeks Etafilcon A 58 25.5 10-9 8.3/14.0 0.084 648
- 지용우외 : 세균부착과관련된미용렌즈표면의특성 - 50 um Conventional 50 um Cosmetic nm 160 Daily wear 120 80 40 0 0 50 um 0 50 um 50 um 50 um Extended wear 0 50 um 0 50 um Figure 2. Atomic force micrographs of surface of different contact lenses (CLs) on the concave side. The surface topography of four different CLs was obtained through atomic force microscopy (AFM) analysis over a 2500 μm 2 surface area on the concave side. Especially measuring cosmetic CLs, color tinted area was contained. (A) Conventional CL daily wear. (B) Cosmetic CL daily wear. (C) Conventional CL extended wear. (D) Cosmetic CL extended wear. Table 2. Surface roughness of contact lenses (CLs) measured by atomic force microscope (AFM) on concave side (nm at 2500 μm 2 ) Average roughness (Ra) Root mean square roughness (Rq) Ten points mean height roughness (Rz) Conventional Cosmetic Conventional Cosmetic Conventional Cosmetic Daily wear 9.68 34.03 12.39 54.86 182.12 809.08 Extended wear 10.89 51.69 14.27 73.14 203.50 819.35 하였다 (Fig. 2). 그결과, 일반콘택트렌즈와일일착용렌즈에비하여미용콘택트렌즈와연속착용렌즈표면의거칠기측정치가더증가한양상을보였다 (Table 2). 일반콘택트렌즈의거칠기값은 Ra=10.29 ± 0.85 nm, Rq=13.33 ± 1.33 nm, Rz=192.81 ± 15.11 nm인반면, 미용콘택트렌즈는 Ra=42.86 ± 12.49 nm, Rq=64.00 ± 12.93 nm, Rz=814.21 ± 7.26 nm이었으며, 특히 Rq와 Rz는독립표본 t 검정상통계학적으로의미있는차이를보였다 (Ra; p=0.067, Rq; p=0.031, Rz; p=0.002). 일일착용콘택트렌즈의거칠기값은 Ra=21.86 ± 17.22 nm, Rq=33.62 ± 30.03 nm, Rz=495.60 ± 443.33 nm 인반면, 연속착용렌즈는 Ra=31.29 ± 28.85 nm, Rq=43.71 ± 41.63 nm, Rz=511.42 ± 435.47 nm로증가된양상을보였으나, 이는모두통계학적으로의미있는차이를보이지는않았다 (Ra; p=0.730, Rq; p=0.807, Rz; p=0.975) (Table 3). 콘택트렌즈의종류에따른 P. aeruginosa 부착비교각콘택트렌즈의표면에 P. aeruginosa 균이부착되도록유도시간을달리하여 (1시간, 12시간, 24시간 ) 시행한후, 배양된세균군집수를측정하였다 (Fig. 3). 그결과, 일반콘택트렌즈와일일착용렌즈에비하여미용콘택트렌즈와연속착용렌즈표면의 P. aeruginosa 부착으로인한세균군집수가더증가하였다 (Table 4). 일반렌즈에부착배양된세균군집수는 1 시간부착시 4.53 ± 0.04 10 5 CFU/mL, 12 시간부착시 12.68 ± 0.88 10 5 CFU/mL, 24시간부착시 94.50 ± 7.50 10 5 CFU/mL인반면, 미용콘택트렌즈는 1 시간부착시 7.98 ± 1.10 10 5 CFU/mL, 12시간부착시 17.55 ± 3.11 10 5 CFU/mL, 24시간부착시 117.50 ± 21.92 10 5 CFU/mL이었으며, 특히초기 1시간동안세균을부착하여배양된세균군집수는독립표본 t 검정상통계학적으 649
- 대한안과학회지 2014 년제 55 권제 5 호 - Table 3. Comparison of concave surface roughness between different contact lenses (CLs) (a) Conventional vs Cosmetic (nm at 2500 μm 2 ) Average roughness (Ra) Root mean square roughness (Rq) Ten points mean height roughness (Rz) Conventional Cosmetic Conventional Cosmetic Conventional Cosmetic 10.29 ± 0.85 42.86 ± 12.49 0.067 13.33 ± 1.33 64.00 ± 12.93 0.031 * 192.81 ± 15.11 814.21 ± 7.26 0.002 Values are presented as mean ± SD. * p < 0.05 by independent t-test; p <0.01 by independent t-test. (b) Daily wear (DW) vs Extended wear (EW) (nm at 2500 μm 2 ) Average roughness (Ra) Root mean square roughness (Rq) Ten points mean height roughness (Rz) DW EW DW EW DW EW 21.86 ± 17.22 31.29 ± 28.85 0.730 33.62 ± 30.03 43.71 ± 41.63 0.807 495.60 ± 443.33 511.42 ± 435.47 0.975 Values are presented as mean ± SD. Contact lens type Pseudomonas aeruginosa: Bacterial culture 1 hour 12 hours 24 hours 10 5 CFU/mL로증가된양상을보였으나이는모두통계학적으로의미있는차이를보이지는않았다 (1 hour; p=0.779, 12 hours; p=0.517, 24 hours; p=0.363) (Table 5). Conventional Daily wear Cosmetic Conventional Extended wear Cosmetic 콘택트렌즈내측표면의거칠기와 P. aeruginosa 부착과의연관성콘택트렌즈표면의거칠기가세균부착과관련이있는지를, 특히초기 1시간동안표면의세균부착과의연관성을통계학적으로분석하였다. Pearson 상관분석상렌즈내측표면의거칠기 (Ra, Rq, Rz) 는초기 1시간동안 P. aeruginosa 부착에따른세균군집수와유의한양의상관관계를보였으며, 12시간및 24시간동안세균부착을시행한경우에서는통계학적으로유의한차이를보이지않았다 (Ra-1 hour; r=0.998, p=0.002, Rq-1 hour; r=0.998, p=0.002, Rz-1 hour; r=0.956, p=0.044) (Fig. 4). Figure 3. Bacterial culture of Pseudomonas aeruginosa adhering to contact lenses after immersing the contact lenses (CLs) into bacterial solutions for 1 hour, 12 hours, or 24 hours, using right angle streaking method. (The bacterial solution for P. aeruginosa adhesion to CLs during 24 hours was diluted to 1:4, compared to others for 1 and 12 hours.) 로의미있는차이를보였다 (1 hour; p=0.047, 12 hours; p=0.167, 24 hours; p=0.295). 일일착용렌즈에부착배양된세균군집수는 1시간부착시 5.85 ± 1.91 10 5 CFU/mL, 12시간부착시 13.70 ± 2.33 10 5 CFU/mL, 24시간부착시 95.60 ± 9.05 10 5 CFU/mL인반면, 연속착용렌즈는 1 시간부착시 6.65 ± 2.97 10 5 CFU/mL, 12시간부착시 16.53 ± 4.56 10 5 CFU/mL, 24시간부착시 116.40 ± 23.48 주사전자현미경으로관찰한미용콘택트렌즈의표면및 P. aeruginosa의부착양상미용콘택트렌즈의표면을주사전자현미경을이용하여관찰하였다. 특히초기 1시간동안 P. aeruginosa를부착한렌즈의표면을살펴본결과 Fig. 5와같았다. 색깔이없는부분 (N) 은표면이매끈한반면, 도색처리된부분 (C) 은거칠고불규칙하였다 (Fig. 5A; 100배율, Fig. 5B; 1000배율, B 사진은 A의검은색사각형부분을확대하였음 ). 미용콘택트렌즈의표면내에서도색깔이없는부분에비하여도색처리된거칠고불규칙한표면에 P. aeruginosa가더부착되어있는모습 ( 화살표머리 ) 을확인할수있었고 (Fig. 5C; 2000 배율 ), 이를고배율로촬영하였다 (Fig. 5D; 10000 배율 ). 650
- 지용우외 : 세균부착과관련된미용렌즈표면의특성 - Table 4. Bacterial culture of Pseudomonas aeruginosa adhering to different contact lenses (CLs), after immersing the CLs into bacterial solutions for 1 hour, 12 hours, or 24 hours ( 10 5 CFU/mL) Immersing time - 1 hour Immersing time - 12 hours Immersing time - 24 hours Conventional Cosmetic Conventional Cosmetic Conventional Cosmetic Daily wear 4.50 7.20 12.05 15.35 89.20 102.00 Extended wear 4.55 8.75 13.30 19.75 99.80 133.00 Table 5. Comparison of Pseudomonas aeruginosa adhering to different contact lenses (CLs), after immersing the CLs into bacterial solutions for 1 hour, 12 hours, or 24 hours (a) Conventional vs Cosmetic ( 10 5 CFU/mL) Immersing time - 1 hour Immersing time - 12 hours Immersing time - 24 hours Conventional Cosmetic Conventional Cosmetic Conventional Cosmetic 4.53 ± 0.04 7.98 ± 1.10 0.047 * 12.68 ± 0.88 17.55 ± 3.11 0.167 94.50 ± 7.50 117.50 ± 21.92 0.295 Values are presented as mean ± SD. * p < 0.05 by independent t-test. (b) Daily wear (DW) vs Extended wear (EW) ( 10 5 CFU/mL) Immersing time - 1 hour Immersing time - 12 hours Immersing time - 24 hours DW EW DW EW DW EW 5.85 ± 1.91 6.65 ± 2.97 0.779 13.70 ± 2.33 16.53 ± 4.56 0.517 95.60 ± 9.05 116.40 ± 23.48 0.363 Values are presented as mean ± SD. 10 r = 0.998, p = 0.002 r = 0.998, p = 0.002 r = 0.956, p = 0.044 10 10 x10 CFU/mL 5 8 6 4 2 x10 CFU/mL 5 8 6 4 2 x10 CFU/mL 5 8 6 4 2 0 0 0 0 10 20 30 40 50 60 0 20 40 60 80 0 200 400 600 800 nm at 2500 µ m 2 nm at 2500 µ m 2 nm at 2500 µ m 2 (Ra-concave) (Rq-concave) (Rz-concave) Figure 4. Correlation of surface roughness and initial (1 hour) number of Pseudomonas aeruginosa adhering to contact lenses (CLs) (r: Pearson correlation coefficient -1 r 1 by the Pearson correlation test). 고찰 본연구는동일한제조사, 동일한공정및동일성분의콘택트렌즈를대상으로연구한결과, 일반적인무색콘택트렌즈에비하여미용콘택트렌즈의내측표면의거칠기가통계학적으로유의하게증가하였음을확인하였다. 또한미용콘택트렌즈에서초기 1시간동안의렌즈표면의 P. aeruginosa 균의부착이증가하였고, 이는일반렌즈에비하여통계학적으로유의한차이를보였다. 이를바탕으로렌즈표면의거칠기와초기세균의표면부착정도가통계학적으로유의한양의상관관계를갖음을알수있었다. 세균의감염에있어초기세균의부착과정은매우중요하다. 혈관도관, 도뇨관, 심혈관이식재등의인체내삽입 용의료기기에서많이연구된바와같이, 일반적으로세균의부착과정은크게두단계로나눌수있다. 28 첫단계는브라운운동을하던세균세포들이반데르발스힘, 이온차등에의해불규칙하게표면에붙게되는것이다. 이것은초기약 1시간내에일어나는현상으로, 가역적이고우연적인결합이나특히편모나섬모등의물리적능력이없는세균의경우는더더욱감염의시작이라는점에서중요하다. 부착후입자간의소수성, 전위차등으로인하여결합이좀더고착화되면서두번째단계인비가역적인부착과정으로넘어가게된다. 이것은시간종속적인과정으로, 세균과의접촉후약 24시간정도지나서나타나게된다. 초기부착된세균들이점액성다당류 (Exopolymer) 를분비하여세균-표면뿐만아니라세균-세균간의비가역적인생물막 651
- 대한안과학회지 2014년 제 55 권 제 5 호 - A B C D Figure 5. Scanning electron microscopic photographs of cosmetic contact lens surface and its Pseudomonas aeruginosa adhesions. Cosmetic contact lenses (CLs) were immersed into P. aeruginosa for 24 hours. After the CLs were washed and processed for scanning electron microscopy (SEM ). Then, the microscopic photographs of concave side of CLs were taken from SEM. (A) Color tinted area of cosmetic CL was rougher than non-color tinted ( 100). (B) Black lined square of Fig. A was observed at higher magnification ( 1000). (C) P. aeruginosa (white arrow head) was adhered to irregular surface of cosmetic CL. Especially, more bacterial adhesions were found to color tinted area ( 2000). (D) P. aeruginosa adhesion to surface of CL was observed at high magnification ( 10000). (N: non-color tinted area, C: color tinted area, white arrow head: P. aeruginosa adhesions). (biofilm)을 형성하게 되므로, 일반적인 세척 및 소독으로도 23,29-32 파괴되지 않을 수 있다. 는 일일 또는 2주 착용 후 교체하도록 되어 있어 세균 감염 이처럼 초기 세균의 표면 부 의 기회가 비교적 적으며, 도색 처리의 방식도 염료가 각막 착 과정은 생물막 형성으로 진행할 수 있으므로 중요하다. 에 직접적으로 닿지 않게 하는 sandwich 방식(ACUVUE 본 연구에 사용된 미용 콘택트렌즈는 시중에 유통되는 BWICTM)이라 좀더 안정적이라고 알려졌다. 그러나 본 연 것들 중에서도 비교적 고가이면서 안정성이 높은 제품으로, 구에서 원자력 현미경으로 미용 콘택트렌즈의 내측 표면 함수율이 높고 이온성을 갖는 FDA Group IV의 하이드로 중 도색 처리된 부분의 거칠기를 측정한 결과, 일반 렌즈보 겔(Etafilcon A) 중합체로 이루어져 있는 것을 대상으로 하 다 통계학적으로 유의하게 증가되어 있었으며, 주사 전자 였다. 함수율이 낮고 비이온성의 polyhema만으로 구성된 현미경을 통한 직접적인 관찰을 통해서도 도색 처리된 부 (FDA Group I) 재질이 아닌, Etafilcon A 하이드로겔 렌즈 분이 무색의 부분에 비하여 더 거칠고 불규칙한 표면을 갖 652
- 지용우외 : 세균부착과관련된미용렌즈표면의특성 - 음을확인하였다. 원자력현미경으로측정한거칠기지표중 Average Roughness (Ra) 는평균 height를먼저구하여이를기준으로 peak 즉 (+) 값과 valley 즉 (-) 값을있는그대로계산한, 단순히평균으로부터벗어나있는편차만을계산한것이므로, (+), (-) 값이상쇄되어실제거친정도에비하여작은값을갖을수있다. 따라서 Ra 값으로분별력이떨어질때에는 Root Mean Square Roughness (Rq) 를사용하게되는데, 계산시제곱이되므로매우심한요철부위나패인부위가있다면이값은더욱커지게된다. Artifact에의해영향을많이받을수도있겠으나, 반대로특정좋지않은부분의거칠기가더욱많이반영이될수있다는장점이있다. Ten Points Mean Height Roughness (Rz) 는 10 개의점을찍어 peak의평균과 valley의평균을더한값으로, 지엽적이기는하나도료의두께나이물질이포착될수있는공간을알아볼수있는지표이다. 본연구는수분이있어야형태를유지하는하이드로겔연성콘택트렌즈의오목한내측표면을대상으로하였는데, 2500 μm 2 의넓은면적을원자력현미경으로촬영하는데에는수십분이걸리게되어최소의수분으로렌즈의형태를유지시키는것이쉽지않았다. 측정한시료가전체적으로편평하지못하여평균 height가달라지게되면서 Ra 값이유의한차이를보이지않았던것으로생각되나, Rq에서는불규칙한부분이더반영되므로미용콘택트렌즈의값이더증가하였고이는통계학적으로유의한결과를보였던것으로해석된다. Rz 역시미용콘택트렌즈에서더증가하는유의한결과를보였는데, 용매속에서브라운운동을하던세균의포착이미용콘택트렌즈의거친표면에서더일어나게되어초기세균부착이증가할수있음을예상할수있다. 미용콘택트렌즈사용자의많은수가청소년들로, 부족한관리교육및위생관념으로인하여비위생적인렌즈보관함으로부터세균오염이시작되는경우가대부분이다. 본연구는이러한임상행태와유사한조건으로실험하기위하여, 탁도계를이용하여일정한농도의세균용액을준비하여콘택트렌즈를충분히잠기도록담근후일정시간을경과하도록하였다. 콘택트렌즈와연관된세균성각막염의흔한균주중, 보다소수성을갖는 P. aeruginosa를이용하여콘택트렌즈의표면에균이부착되도록유도하였는데, 거칠고불규칙한표면을갖고있는미용콘택트렌즈의세균부착이초기 1시간에서유의하게증가하였다. 이는렌즈표면의불규칙함으로인하여세균과렌즈의접촉면적및기회가증가하게되어실제세균-표면간의결합력이극대화됨으로써나타난결과로생각할수있다. 미용콘택트렌즈내에서도무색부분보다도색처리된부분에서세균의 부착이더많이일어나있음을주사전자현미경을통하여확인할수있었다. 반면 24시간동안세균용액에보관하였던콘택트렌즈의표면에부착한세균의군집수는매우많이증가하였으나, 예상과는달리생물막을형성한모습을관찰하지는못하였다. 이를바탕으로, 동일한성분의콘택트렌즈들을대상으로측정된거칠기값과초기 1시간동안의세균부착의정도를분석한결과, 통계학적으로매우유의한양의상관관계를갖음을밝혔다. 예상했던대로렌즈표면의거칠기가초기의세균부착에중요한영향을미친다는결론을얻었다. 12시간및 24시간동안세균용액에담근후배양한세균군집수는콘택트렌즈표면의거칠기와통계학적으로유의한상관관계를갖지않았는데, 이는초기에불규칙한면적에세균의부착이완료되면그이후에는세균-표면간의관계가아닌세균- 세균간의결합이더중요하게작용하여그러할것으로생각한다. 본문의결과에명시하지는않았으나, 초기의세균부착은 12시간및 24시간후의부착된세균수와당연하게도통계학적으로유의한양의상관관계를보였다 (Pearson coefficient r>0.9, p<0.05). 연구에사용된콘택트렌즈중연속착용렌즈는장기사용의편의를위해일일착용렌즈에비하여직경과곡률반경이약간더작았으나, 일반무색콘택트렌즈와미용콘택트렌즈에상관없이일일착용과연속착용렌즈간의거칠기, 세균의부착정도등의모든데이터는통계학적으로유의한차이를보이지않았다. 본연구는 Etafilcon A를중합체로사용한하이드로겔연성콘택트렌즈만을대상으로하였다는한계가있다. 현재시중에유통되는제품으로는하이드로겔재질뿐만아니라, Enfilcon A, Narafilcon A, Galyfilcon A, Comfilcon A, Senofilcon A, Lotrafilcon A&B, Balafilcon A 등의다양한실리콘하이드로겔재질또한사용되고있고, 재질성분에따라서도모두다른성상을띠고있다. 특히청소년들에게문제가되는저가의미용콘택트렌즈는단순 polyhema만으로구성된렌즈인경우가많아, 본연구의결과가이러한모든렌즈의경우를대표하기에는한계가있을수있다. 그러나기존의연구에서실리콘하이드로겔콘택트렌즈는하이드로겔렌즈에비하여더소수성을띠고있어, 소수성을보이는세균세포와의부착이더용이하다고알려졌다. 17,18,33 또한 Etafilcon A는하이드로겔중에서도 FDA Group IV에속하는보다친수성의우수한재질로, 본재질만의일반무색의콘택트렌즈와미용콘택트렌즈를비교함과동시에실제임상에서세균성각막염의가장흔한균주를사용함으로써, 보편적이고의미있는결과로받아들이기에무리가없다. 단, 시간, 비용등의한계로많은수의렌즈를연구할수없었으 653
- 대한안과학회지 2014 년제 55 권제 5 호 - 며, 콘택트렌즈의내측표면중일부분 (2500 μm 2 ) 의거칠기를측정하였다는점이추후보완해야할부분이겠다. 주사전자현미경으로외측표면을먼저촬영해본결과, 내측과차이를보이지않아각막과직접적으로접촉하는내측표면만을대상으로하였다. 물론, 세균의부착정도를비교하는실험을할때에도콘택트렌즈의오목한내측에만세균용액을넣어서시행할수도있었으나, 연성콘택트렌즈의조작이매우어려울뿐만아니라이는실제렌즈의보관행태와도다르므로렌즈전체를세균에노출시키는방법을사용하였다. 본연구는실험적연구에한하였므로, 실제생물연구또는임상연구에적용할직접적인데이터를제시하지못한다는점도향후에추가연구가필요한부분이다. 미용목적의컬러콘택트렌즈는우리나라를비롯한많은나라에서의사의처방없이유통되고있다. 일반무색의콘택트렌즈에비하여, 미용콘택트렌즈의표면은더거칠고불규칙하여초기세균의부착이용이하기때문에비위생적인관리를하는경우감염에더욱취약할수있는데, 이는비단시중에유통되는저가제품에서만발생가능한문제가아니라정상적인제조공정을거친고가제품조차도갖을수있는문제점이다. 그러나이제까지미용콘택트렌즈의성상에대한연구는전무한실정으로, 향후기존의미용콘택트렌즈제품에대한후향적연구뿐만아니라, 제작공정에서부터의료인과렌즈제조산업체가협력하여모든변수들을통제하는체계적이고전향적인연구가필요하겠다. 특히세균성각막염과같은시력예후에치명적인합병증을예방하기위해서미용콘택트렌즈의도색처리과정에서표면거칠기를감소시킬수있는공정기술과적정밀도인식신호차단 (Anti-Quorum Sensing) 혹은입체장벽 (Steric Barrier) 과같이세균의부착을줄일수있는처치및신소재의개발이필요하다. REFERENCES 1) Efron N, Morgan PB, Woods CA. International survey of contact lens prescribing for extended wear. Optom Vis Sci 2012;89:122-9. 2) Efron N, Morgan PB, Woods CA. Survey of contact lens prescribing to infants, children, and teenagers. Optom Vis Sci 2011;88: 461-8. 3) Morgan PB, Efron N, Woods CA. Determinants of the frequency of contact lens wear. Eye Contact Lens 2013;39:200-4. 4) Efron N, Morgan PB, Woods CA. An international survey of daily disposable contact lens prescribing. Clin Exp Optom 2013;96: 58-64. 5) Park YM, Hahn TW, Choi SH, et al. Acanthamoeba Keratitis Related to Cosmetic Contact Lenses. J Korean Ophthalmol Soc 2007;48:991-4. 6) Song JS, Lee H, Kim JW, et al. The effects of cheap tinted contact lenses on corneal swelling and ocular surface inflammation. J Korean Ophthalmol Soc 2008;49:1888-93. 7) Lim TH, Lee JR, Choi KY, et al. Corneal melting And descemetocele resulting from noninfectious keratitis related to the cosmetic contact lenses. J Korean Ophthalmol Soc 2009;50:774-8. 8) Park SJ, Lee SM, Kim MK, et al. Cosmetic contact lens-related complications: 9 cases. J Korean Ophthalmol Soc 2009;50:927-35. 9) Sauer A, Bourcier T. Microbial keratitis as a foreseeable complication of cosmetic contact lenses: a prospective study. Acta Ophthalmol 2011;89:e439-42. 10) Steinemann TL, Fletcher M, Bonny AE, et al. Over-the-counter decorative contact lenses: Cosmetic or Medical Devices? A Case Series. Eye Contact Lens 2005;31:194-200. 11) Steinemann TL, Pinninti U, Szczotka LB, et al. Ocular complications associated with the use of cosmetic contact lenses from unlicensed vendors. Eye Contact Lens 2003;29:196-200. 12) McKelvie J, Patel D, McGhee C. Cosmetic contact lens-related Acanthamoeba keratitis. Clin Experiment Ophthalmol 2009;37: 419-20. 13) Williams D. Great expectations and the grapes of wrath: contamination of contact lenses. Med Device Technol 1999;10:10-3. 14) Giraldez MJ, Resua CG, Lira M, et al. Contact lens hydrophobicity and roughness effects on bacterial adhesion. Optom Vis Sci 2010; 87:E426-31. 15) Burnham GW, Cavanagh HD, Robertson DM. The impact of cellular debris on Pseudomonas aeruginosa adherence to silicone hydrogel contact lenses and contact lens storage cases. Eye Contact Lens 2012;38:7-15. 16) Dutta D, Cole N, Willcox M. Factors influencing bacterial adhesion to contact lenses. Mol Vis 2012;18:14-21. 17) Vijay AK, Zhu H, Ozkan J, et al. Bacterial adhesion to unworn and worn silicone hydrogel lenses. Optom Vis Sci 2012;89:1095-106. 18) Willcox MD. Microbial adhesion to silicone hydrogel lenses: a review. Eye Contact Lens 2013;39:61-6. 19) Willcox MD, Holden BA. Contact lens related corneal infections. Biosci Rep 2001;21:445-61. 20) Robertson DM. The effects of silicone hydrogel lens wear on the corneal epithelium and risk for microbial keratitis. Eye Contact Lens 2013;39:67-72. 21) Bailey CS. A review of relative risks associated with four types of contact lenses. Cornea 1990;9 Suppl 1:S59-61; discussion S62-3. 22) Willcox MD, Harmis N, Cowell, et al. Bacterial interactions with contact lenses; effects of lens material, lens wear and microbial physiology. Biomaterials 2001;22:3235-47. 23) Bruinsma GM, van der Mei HC, Busscher HJ. Bacterial adhesion to surface hydrophilic and hydrophobic contact lenses. Biomaterials 2001;22:3217-24. 24) McGlinchey SM, McCoy CP, Gorman SP, Jones DS. Key biological issues in contact lens development. Expert Rev Med Devices 2008;5:581-90. 25) Loyola-Rodriguez JP, Zavala-Alonso V, Reyes-Vela E, et al. Atomic force microscopy observation of the enamel roughness and depth profile after phosphoric acid etching. J Electron Microsc (Tokyo) 2010;59:119-25. 26) Lira M, Santos L, Azeredo J, et al. Comparative study of silicone-hydrogel contact lenses surfaces before and after wear using atomic force microscopy. J Biomed Mater Res B Appl Biomater 2008;85:361-7. 654
- 지용우외 : 세균부착과관련된미용렌즈표면의특성 - 27) Giraldez MJ, Serra C, Lira M, et al. Soft contact lens surface profile by atomic force microscopy. Optom Vis Sci 2010;87:E475-81. 28) Desrousseaux C, Sautou V, Descamps S, Traore O. Modification of the surfaces of medical devices to prevent microbial adhesion and biofilm formation. J Hosp Infect 2013;85:87-93. 29) Mueller RF, Characklis WG, Jones WL, Sears JT. Characterization of initial events in bacterial surface colonization by two Pseudomonas species using image analysis. Biotechnol Bioeng 1992;39: 1161-70. 30) Chen L, Wen YM. The role of bacterial biofilm in persistent infections and control strategies. Int J Oral Sci 2011;3:66-73. 31) Fletcher EL, Weissman BA, Efron N, et al. The role of pili in the attachment of Pseudomonas aeruginosa to unworn hydrogel contact lenses. Curr Eye Res 1993;12:1067-71. 32) Evans DJ, Fleiszig SM. Microbial keratitis: could contact lens material affect disease pathogenesis? Eye Contact Lens 2013;39:73-8. 33) Vermeltfoort PB, Rustema-Abbing M, de Vries J, et al. Influence of day and night wear on surface properties of silicone hydrogel contact lenses and bacterial adhesion. Cornea 2006;25:516-23. = 국문초록 = 미세표면분석기법을이용한미용콘택트렌즈와일반콘택트렌즈의표면거칠기와세균부착성비교 목적 : 미용콘택트렌즈사용에따른세균감염에대한문제인식에비해객관적이고과학적인연구는전무한실정으로, 일반연성콘택트렌즈에비하여미용콘택트렌즈표면의특성이세균부착에직접적으로미치는영향에대해체계적으로연구하였다. 대상과방법 : 하이드로겔 (Etafilcon A) 재질의일일착용및연속착용콘택트렌즈중미용목적의컬러렌즈와일반투명렌즈를대상으로하였다. 각렌즈의내측표면거칠기를원자력현미경으로분석하였고, Pseudomonas aeruginosa 를 1, 12, 24 시간동안렌즈에접촉시킨후표면에부착된세균을분리 배양하여군집수를계산하였다. 또한부착된세균의양상을주사전자현미경으로관찰하였다. 특히미용콘택트렌즈는도색처리된부분을집중검사하였다. 결과 : 원자력현미경으로렌즈의내측표면을분석한결과, 일반렌즈에비하여미용콘택트렌즈의거칠기가유의하게증가하였다 (p <0.05). 1 시간동안의 P. aeruginosa 의렌즈표면부착은통계학적으로유의하게미용콘택트렌즈에서증가하였다 (p=0.047). 또한표면거칠기는 1 시간동안렌즈에부착된세균수와유의한상관관계를보였다 (r>0.9, p<0.05). 주사전자현미경을통하여도색처리된표면에세균이더부착됨을확인하였다. 결론 : 본연구는미용콘택트렌즈표면의거칠기와세균부착의연관성을객관적으로밝힘으로써, 세균성각막염등의합병증을예방하기위하여렌즈표면의거칠기를감소시키고세균부착을억제할수있는콘택트렌즈의개발및도색처리공정의개선이필요함을제시하였다. < 대한안과학회지 2014;55(5):646-655> 655