RESEARCH ARTICLE Image J 프로그램을사용한모공측정및노화도의새로운정량분석법개발과화장품인체적용시험에의활용 차화준 1, 권승빈 2, 김지현 2,3, 김수경 2, 안인숙 2, 안성관 1,3 * 1 건국대학교생물공학과, 2 한국피부임상과학연구소, 3 건국대학교대학원향장학과 Development of a Novel Quantitative Evaluation Method for Skin Pore Areas and Aging Degrees Using the Image J and It Application for Clinical Studies Hwa Jun Cha 1, Seungbin Kwon 2, Jihyun Kim 2,3, Sookyung Kim 2, In-Sook An 2, Sungkwan An 1,3 * 1 Department of Biological Engineering, Konkuk University 2 KISCS Incorporated 3 Department of Cosmetology, Graduate School of Engineering, Konkuk University Abstract Sebum is emitted by skin pores, the openings of sebaceous glands. Skin pore size increases due to excessive sebum emission and decreased skin elasticity caused by aging. These larger skin pores result in cosmetic problems. Therefore, methods to regulate skin pore size were examined. Additionally, effective methods for measurement of skin pores are also needed. In the present study, we developed a novel quantitative method for evaluation of skin pore area and degree of aging using the Image J software. First, we evaluated the pixel size of skin pores following the application of pore-tightening cream to the skin using the Cosmetic Full-Face Photography System, which is used to measure skin pore size, as well as a novel system that combines both microscopy and Image J analysis. In both the Cosmetic Full-Face Photography System and the novel system, skin pore size decreased following application of pore-tightening cream; however, the novel skin pore detection system effectively determined the decreases in skin pore size. Additionally, the degree of aging of skin pores was determined using Feret s diameter. Thus, we suggest that this novel skin pore-detection system can be utilized as a substitute for the existing skin pore-detection method. Keywords: Image J, Skin pore, Aging, Quantitative evaluation, Clinical study 모공은피지샘또는아포크린샘의개구부가확대되어육안 으로확인하였을경우를말한다 (Uhoda et al., 2005). 실제 모공은정상적인피부에존재하는구조물이기때문에모공자 체가문제가되는것은아니지만성별, 유전적영향, 나이, 피 *Corresponding author: Sungkwan An, Department of Biological Engineering, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea Tel.: +82 2 450 4054, Fax: +82 502 770 2278 Email: ansfgrc@konkuk.ac.kr Received July 2, 2015; Revised August 8, 2015; Accepted August 17, 2015; Published August 30, 2015 지분비량, 여드름등여러가지요인들에의해서모공이확장되게되면육안으로식별가능해지기때문에미적으로문제가되고있다 (Roh et al., 2006; Kim et al., 2013; Zoublulis & Boschanakow, 2001). 때문에모공을조절하는화장품소재가활발하게개발되고있고, 이에대한임상적측정이필수적으로필요하기때문에동시에모공측정에대한임상시험법또한여러방법이개발되어있는실정이다 (Suk et al., 2013). 하지만개발된측정법은특정장비를사용해야하는단점이있고, 피부의미백, 주름등과함께다른임상시험항목들과동시에측정하기때문에상대적으로감도가떨어질수밖에없다 (Lee, 2014; Jo et al., 2007). 따라서본논문에서는모공을확대하여촬영하고, 이를 Image J로분석하여높은감도로모공을분석하고자하였다. www.kosac.or.kr 477
모공의확장은기존에는과도한피지의분비와피지샘의개구부가막혀서주로생성이되는것으로알려져, 모공확장을치료하기위해서각질층의필링과피지분비억제를통해서모공의확장을억제시키려는연구가최근까지도계속되어왔다 (Kim et al., 2015; Lee et al., 2015). 하지만최근연구에서는모공이노화에의해서피부표면탄력이저하되면서형성되며, 모공이확장될때점차중력방향인아래방향으로늘어지기때문에세로로긴형태의모공이생성되는것으로알려져있어 (Suk et al., 2013), 현재피부의탄력을회복하여모공을축소시키는방법에대한연구가활발하게진행중이다. 또한노화와모공간에상관관계가있기때문에노화에의한모공을측정하는방법역시개발되고있다 (Masuda et al., 2014). 노화성모공을측정하는경우, 모공의세로축의길이를이용하여측정하는것이일반적이다 ( 김신한등, 2010; Kim et al., 2004). 일반적으로이러한모공의측정은상업용으로개발된전용프로그램을이용해서측정하는경우가대부분이다. 그러나본연구에서는미국 National Institute of Health (NIH) 에서개발하여무료배포및사용되고있는 Image J를이용하여모공의개수, 면적및노화성모공측정파라미터인세포축의길이를측정하는기법을개발하고자하였다. Image J는 JAVA를이용하여각종다른프로그램과효율적으로상호조작이가능하고, 거의모든학술적자료의화상분석에이용이가능하다는특징을가지고있다 (Pettiaux et al., 1997). 또한간단한사용법때문에프로그램에대한간단한정보만으로쉽게사용할수있어많은연구자들에의해서사용되고있다. 이러한 image J는무료배포기반으로되어있기때문에다양한플러그인이개발되어계속적으로사용범위또는분석방법들이생겨나고있다 (Rittweger et al., 2004; Weitkamp et al., 2011). Image J를이용한측정의대표적인예로써, western blotting 기술을통해단백질 band의 density를디지털화면에서 pixel 대조를통해값을수치화하고, 이를화면상의수치 (arbitrary unit) 로변환하여간단하게화상의결과를수치데이터로변환할수있다 (Tovée et al., 2003; Rocha- Martins et al., 2012). 때문에본연구에서는피부의모공을 pixel 단위로분석하여전체모공의개수및면적의수치데이터를측정하고, 세로축의 pixel값을구하여노인성모공에대한상대적수치를측정하는화장품임상평가기법을개발하고자하였다. 1. 연구대상및모공촬영모든임상시험은한국피부임상과학연구소기관윤리심의위원 회 (Institutional Review Board) 를통해연구계획을심의및승인받은후실시되었으며, 헬싱키선언에따라실험을실시하였다. 모공의촬영은본시험에참여한 20-30대여성피험자들중, 육안으로평가시모공확장으로인해모공이보이는피험자 20명을대상으로실시하였다. 이들피험자에게는시험목적및내용에대해충분히설명을하고동의를구한후시험을개시하였다. 이중시험참가시점 3개월이내면역억제제치료를받은경우, 시험참가시점 1개월이내에전신스테로이드또는광선치료를받은경우, 시험부위에병변이있어측정이곤란한경우, 심한여드름, 아토피또는감염성피부질환이있는자, 화장품, 의약품또는일상적인광노출에대한반응이심하거나알러지가있는경우, 6개월이내에본시험과유사한인체시험에참여한적이있는경우, 임신또는수유중인여성, 기타인체시험수행이곤란하다고판단되는경우등의제외기준을두었다. 피험자는세안후실내온도 20~25, 습도 40~60% 의항온항습실에서 30 min 간안정을취하여피부표면온도와습도를측정공간의환경에적응하게하였으며, 안정을취하는동안의수분섭취는제한하였다. 모공변화는공인기관을통해모공수축효과가입증된 C사의모공수축크림을사용하였고, 사용전과사용후의모공면적변화를측정하였다. 측정은피부에서모공분포가많은볼부위에현미경카메라 (Kong Camera; Domax Care, Seoul, Korea) 를이용하여서근접촬영을하였고, 동일한조명과위치에서측정하였다. 또한추가적으로기존의모공측정장비인전안촬영시스템 (Cosmetic Full-Face Photography System; Facial Stage DM-3; MORITEX Corp., Saitama, Japan) 을적용하여측정하였다. 2. 모공촬영사진에서 Image J를이용한모공분석 Image J는이미지를 pixel 단위로분석하는프로그램이다 (Jung et al., 2013). 때문에촬영된모공사진을동일한조건으로 BMP ( 비트맵 ) 파일로변환하였고, Image J에서각각의이미지에 Gaussian blur를이용하여영상이미지를최적화하고, 지역적최대값 (FInd Maxima) 을찾아모공의위치및영역을검출하였다. 검출된모공을기반으로 pixel 단위모공영역과장축의길이를측정하였다. 또한이를 arbitrary unit로변환하여수치화된값을분석하였다. 3. 전안촬영시스템을이용한모공분석전안촬영시스템 Facial Stage는촬영부터분석까지일체화된장비이며, 얼굴측정은얼굴을얼굴틀 (cage) 아래에있는툴바 (tool bar) 의상단에고정시킨채얼굴정면을촬영하였다 (Baek, 2013; Choi et al., 2013; Lee et al., 2008). 측정의재현성을위하여시험물질사용전안면촬영이미지와사용후 478 www.kosac.or.kr
Image J 프로그램을사용한새로운모공분석방법 원본이미지영상최적화모공위치찾기 모공영역추출모공영역분석결과오버레이 Figure 1. Process of skin pore detection method using Image J. 이미지를오버랩시켜동일부위를측정하여모공면적을분석하였고, 측정단위는 mm 2 이다. 3. 수치분석및통계적분석모든수치분석과통계적수치의차이는 Microsoft Office Excel 2010을사용하여분석하였으며, 수치분석은사용전과비교하여시용후의상대적인개선률을측정하였다. 통계분석법으로는student paired t-test를통하여유의성을나타내는 p value가.05 이하의범위에있을때통계적차이가유의한것으로간주하였다. 1. Microscope camera와 image J를통한모공분석법개발 Image J에서 microscope camera를이용하여측정한모공사진을불려오고, 불러온영상을분석에최적화하기위해 서 Gaussian blur를적용하였다. 이후각각의모공의위치를찾기위해서 noise tolerance 기능을적용하여모공위치를추출한다. 추출된모공의위치를분석하기위해서 analyze particles 기능을적용하여모공영역을분석한다. 분석한결과중 Area 값과 Feret 값을분석하여모공의넓이변화와장축의길이변화를측정하여각각의결과를도출하였다. 또한측정후 from ROI manager를적용하여원본이미지와분석이미지의모공부위가동일하게오버레이되는결과를도출하였다 (Figure 1). 2. 전안촬영시스템과비교분석기존에공인기관을통해모공수축효과를입증된 C사의모공수축크림을이용하여임상적용후기존의전안촬영시스템과본연구에서개발된 microscope camera와 Image J를이용한모공분석결과를비교분석하였다. 모공면적을분석한결과, 사용전 359.90 mm 2, 사용후 328.50 mm 2 로유의적으로모공이감소한것을확인하였고, 8.72% 개선율을보였다 (Figure 2, Table 1. Comparative analysis between cosmetic full-face photography system and microscope camera and Image J system Cosmetic Full-Face Photography System Microscope camera +Image J Before After Before After Mean (mm 2 and AU) 359.90 328.50 613.417 436.13 Improvement rate (%) 8.72 28.90 www.kosac.or.kr 479
Figure 2. Elevation of skin pore size using cosmetic full-face photography system. Figure 3. Elevation of skin pore size using Image J. Figure 4. Elevation of aging degree using Image J. (A) Quantitative evaluation of aging degree, (B) Comparative analysis between morphology of before and after skin pore tightening cream applied skin Table 1). 동일한조건에서 microscope camera와 image J를이용하여분석한결과사용전 613.417 AU, 사용후 436.13 AU로감소하였고, 28.90% 개선율을나타냈다 (Figure 3, Table 1). 분석결과전안촬영시스템과 microscope camera 와 image J를이용한시스템에서결과적인경향성은유사하게나왔지만 microscope camera와 image J를이용한방법이좀더경향성이명확하게나타났다. 이는전안촬영시스템의경우얼굴전면을모공감소효능측정에사용하였고, microscope camera와 image J를이용한방법은국소부위를확대하여촬영하기때문에위와같은차이가생기는것으로판단된다. 따라서본연구에서개발된 microscope camera와 Image J을이용한모공분석법을통해서좀더감도높은모공측정법이개발된것으로사료된다. 3. Image J를이용한노인성모공측정본논문에서개발된 microscope camera와 Image J를이용한시스템에서모공의장축의길이는 Suk et al. (2013) 의연구결과처럼노인성모공에서나타나는현상을측정한것이다. 때문에 microscope camera와 Image J를이용한시스템에서모공의장축의길이를분석한결과장축의길이를의미하는 Feret s diameter가 4.792 AU에서 3.265 AU로감소하였고, 개선율은 32% 로나타났다. 따라서모공의감소에따라모공의크기가감소하고 (Figure 4A), 육안상으로사용전과사용후를비교하였을때모공의형태가동그랗게일정해지는것을확인할수가있다 (Figure 4B). 따라서모공수축크림을통해피부의탄력성이증가되면노인성모공이감소하는데이를본논문에서개발된방법을통하여확인하였다. 480 www.kosac.or.kr
Image J 프로그램을사용한새로운모공분석방법 모공은피지를배출하는개구부로써피지의배출을목적으로하고있으나피지의과도한분출이나노화에의해서모양과크기가커지기때문에미용적인문제가발생되게된다. 따라서이러한모공을감소시키려는연구가계속되고있고, 이와같이모공의감소를평가하는분석방법의개발역시많은연구가진행중이다. 미적으로문제가되는모공은육안상으로도관찰되기때문에이미지분석장비를이용하여분석을한다. 하지만분석장비마다차이가많고공인된평가방법이없기때문에공인된이미지분석장비인 Image J를이용하여정확하게모공을분석하는것이본연구의목적이다. 또한본연구에사용된방법을통해서좀더쉽고간단하게모공의변화를정확하게분석해낼수있을것으로사료된다. 연구결과본연구를통해개발된모공측정방법을통해서더감도가좋은모공측정법을제안하였고, 추가로동일한방법을이용하여모공의노화도를측정할수있다. 때문에기존의방법보다효과적이고고가의장비없이측정가능한모공측정방법을개발하였다고사료된다. 감사의글본논문은보건복지부보건의료연구개발사업의지원 ( 과제번호 : HN13C0075) 에의한것이며, 이에감사드립니다. 김신한, 고재완, 김영구, 강진문, 이상주, 정원순, 이정은, 조한경. 확장된모공에대한임상적고찰. 대한피부과학회지, 48: 212, 2010. Baek SW. A Clinical study on effects of the face pack containing Kaolin, Adenosine, and Plant Extracts on the Skin Improvement. Master s Thesis, Konkuk Univ., 2013. Choi SJ, Baeck SW, Choi MH, Jo A, Jang HH, Oh CR, Ahn KJ, An IS, An S, Oh JS. Effects of the Face Pack Containing Kaolin, Adenosine and Plant Extracts on the Skin Improvement. 11: 993-1000, 2013. Jung HM, Won DH, JunG JE. Quantitative Analysis Methods for Adapting Image J programs on Mouse Calvarial defected Model. J. Digit. Policy Management, 11: 365-370, 2013. Jo HY, Yu DS, Oh CH. Quantitative research on skin pore widening using a stereoimage optical topometer and Sebutape. Skin Res. Technol., 13: 162-168, 2007. Kim BR, Jung SW, Ryoo HC, Noh YK, Ahn BK, Ajn SK, Lee SH. Measuring technique for skin pore size. J. Skin Barrier Res., 6: 97-104, 2004. Kim BY, Choi JW, Park KC, Youn SW. Sebum, acne, skin elasticity, and gender difference which is the major influencing factor for facial pores? Skin Res. Technol., 19: 45-53, 2013. Kim SJ, Baek JH, Koh JS, Bae MI, Lee SJ, Shin MK. The effect of physically applied alpha hydroxyl acids on the skin pore and comedone. Int. J. Cosmet. Sci., Epub ahead of print, 2015. Lee BM, An S, Kim SY, Han HJ, Jeong YJ, Lee KR, Roh NK, Ahn KJ, An IS, Cha HJ. Topical application of a cleanser containing extracts of Diospyros kaki folium, Polygonum cuspidatum and Castanea crenata var. dulcis reduces skin oil content and pore size in human skin. Biomed. Rep., 3: 343-346, 2015. Lee JY. Pore Extraction Method using Commodity Cameras. Master s Thesis, Korea Univ., 2014. Lee SR, Lee YK, Park SY, Ko KM, Lee YK, Kim JS, Lee BH, Lim SC, Jung TY, Lee KM. Reproducibility assessment of skin furrows measurement using facial skin photographing system. J. Kor. Acupuncture Moxibustion Soc., 25: 187-197, 2008. Masuda Y, Oguri M, Morinaga T, Hirao T. Threedimensional morphological characterization of the skin surface micro-topography using a skin replica and changes with age. Skin Res. Technol., 20: 299-306, 2014. Rittweger J, Michaelis I, Giehl M, Wüsecke P, Felsenberg D. Adjusting for the partial volume effect in cortical bone analyses of pqct images. J. Musculoskelet Neuronal Interact, 4: 436-441, 2004. Roh M, Han M, Kim D, Chung K. Sebum output as a factor contributing to the size of facial Pores. Br. J. Dermatol., 155: 890-894, 2006. Rocha-Martins M, Njaine B, Silveira MS. Avoiding pitfalls of internal controls: validation of reference genes for analysis by qrt-pcr and Western blot throughout rat retinal development. PLoS. One, 7: e43028, 2012. Suk JM, Park SY, Choi MR, An SY, Kim BJ, Park OJ, Jung SW. A Method of Facial Pore Size Evaluation. J. www.kosac.or.kr 481
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