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대한화상학회지제 14 권제 2 호 77 Journal of Korean Burn Society Vol. 14, No. 2, 77-84, 2011 저친화도와고친화도의 Carboxymethylcellulose 의창상치유에미치는효과 이지원ㆍ지소영ㆍ최재일ㆍ석정훈ㆍ양완석 동강병원성형외과 Effect of Carboxymethylcellulose with Low and High Degree of Substitution on Wound Healing Ji Won Lee, M.D., So Young Ji, M.D., Jae Il Choi, M.D., Jeong Hoon Suhk, M.D. and Wan Suk Yang, M.D. Department of Plastic and Reconstructive Surgery, Dong Kang General Hospital, Ulsan, Korea Purpose: Wound healing is a dynamic and complex process of tissue repair that involves a number of cellular and molecular events, which is characterized by intercalating degradation and re-assembly of connective tissue and epidermal layer. Carboxymethyl cellulose (CMC) is one of the most common hydrofiber dressing. This material have a various degree of substitution. Our goal was to test the efficacy of Carboxymethyl cellulose with low and high degree of substitution on wound healing. Methods: Four round (diameter 2 cm) wounds were made bilaterally on the dorsum of the all rat s trunk and were divided into four groups of dressing material: Group A with gauze dressing as a negative control group, Group B with high gel (DS=0.3), Group C with Aquacel R (ConvaTec, US) as a positive control group, Group D with low gel (DS= 0.15). We compared each group with gross findings by means of percentage of wound contraction, percentage of wound epithelization, and percentage of total wound healed area by tracing the remained wound area on each time periods. Results: Group B and C show statistically higher percentage of wound contraction and higher percentage of wound healed than the other groups until 14th day after wound formation. Group D shows higher percentage of wound epithelization rate in early days, but it was reversed that Group B and C show statistically higher percentage of wound epithelization on 21st day after wound formation. Histologically, Group B and C show less inflammatory cell infiltration, faster and more prominent angiogenesis. On the 21st day after wound formation, collagen fibers was more regularly and densely arranged on Group B and C than the other groups. 책임저자 : 양완석, 울산시중구태화동 123-3 681-320, 동강병원성형외과 Tel: 052-241-1324, Fax: 052-241-1324 E-mail: artpsyang@yahoo.co.kr Conclusion: This study suggest Carboxymethylcellulose with high degree of substitution induces stable reepithelization and collagen synthesis in the wound healing process and have optimal effective results and is expected as more application of a various property of Carboxymethylcellulose for treatment of wound healing. (J Korean Burn Soc 2011; 14:77-84) Key Words: Carboxymethylcellulose, Hydrofiber dressing, Degree of substitution (DS) 서 창상은어떠한원인에의해해부학적으로조직의연속성이파괴된것을의미하며, 다양한방법으로창상을치유하려는노력은지속되고있다. 창상치유과정은손상된조직을복구하기위한수많은요소들의상호작용을통해이루어지고, 이러한환경체제는상피세포, 진피세포, 진피하세포, 세포외기질, 단백분해효소와그억제인자등수없이많은인자들의균형과항상성을유지하기위한신호에의해조절받는다. 창상의치유는염증단계, 상피화단계, 증식단계, 성숙단계로구분할수있으며, 각각의단계는중첩되면서연속적으로진행된다. 이는각과정에서나타나는세포들, 세포들에의해유래되는각종인자들과조직간의복잡한유기적인상호작용으로그과정이이루어지기때문이다 1,2). 임상에서사용되는다양한드레싱제재들은창상치유에서염증반응, 육아조직및섬유아세포의활동, 상피화촉진등에주로그효과를나타낸다. 드레싱재료의효과는이상적인드레싱재료의조건인창상의외부노출차단을통한감염예방, 생체에대한우수한친화성, 생체분해및흡수성, 과다한삼출물의적절한흡수와지나친염증반응의억제등을통해이루어진다. 이러한조건을만족하기위해현재까지다양한드레싱재료들이개발되어왔다 3-5). Carboxymethylcellulose (CMC) 는셀룰로오스의가장잘알려진수용성유도체이며, 알칼리셀룰로오스를모노클로로아세테이트에반응시킴으로얻을수있다. Sodium carboxymethylcellulose (CMC) 는음이온의수용성중합체로 론

78 대한화상학회지 Vol. 14, No. 2, 2011 셀룰로오스로부터유래되고친수성을가지며, 수용액의고점성, 피막형성능이있어기름이나유지그리고유기용매에저항성을가진다. 또한강력한접착력을가진안정화제, 콜로이드보호성을가지며흘러넘침을억제하는물질로쓰인다. 차거나뜨거운물에빠르게녹는성질을가지고있으며여러음식을만드는재료로적합한성질을나타낸다. 친수성기와배합물을만들면중성을띠는특성을가지고있다. 저자들은이러한특징을가지는 CMC를주성분으로하는드레싱재료로서치환도의차이에따라 gel화되는정도및친수성및용매에대한유기도가달라짐에착안하여, 신선창상에있어창상치유과정특히, 창상치유속도및상피화정도교원질침착등에미치는영향과새로운드레싱재료로서의가능성을알아보기위해치환도에차이를둔 sodium carboxymethylcellulose (CMC) 2개의타입과거즈드레싱및 Aquacel R (ConvaTec, US) 을각각사용하여창상치유를비교하는실험을시행하였다. 대상및방법 1. 실험재료체중 250 300 g의흰색백서 (Sprague-Dawley) 20마리를일정한온도및습도의사육사에서사육하여사용하였으며실험에사용된드레싱재료에따라 4개의실험군으로나누었다. A군은음성대조군 (negative control) 으로고식적인거즈드레싱을하였고시중에시판중인 gamma radiation으로살균된제품을구입하여사용하였다. 시험을위해사용된 CMC 드레싱재제는 100% cotton 섬유를 Spunlace 공법으로제작한부직포를 carboxymethylation 하여제조하였다. 부직포의셀룰로오스고분자쇄의 hydroxy 치환정도 (DS= Degree of substitution) 에따라겔화정도가달라지며, 시험에사용한것은 low gel (DS=0.15), high gel (DS=0.3) 두가지로 B군은고친화도의 high gel (DS=0.3) CMC로드레싱하였고, C군은양성대조군으로 Aquacel R (ConvaTec, US) 로드레싱하였다. D군의경우는저친화도의 low gel (DS=0.15) CMC로드레싱하였다 (Fig. 1). ketamine-hcl R, Huons, Korea) 를복강내에주사하여마취를유지하였다. 엎드린자세의백서의털을면도하고 betadine과 alcohol로소독한후 15번수술칼과 metzembaum 을이용하여동일한크기, 동일한깊이로전층피부창상을만들었다 (Fig. 1). 원칙적으로모든군에서술후 7일까지는하루에한번씩, 7일부터 14일까지는이틀에한번씩, 그이후에는 3 4일에한번씩드레싱하였으며드레싱후드레싱이탈락되는것을방지하기위해몸통에탄력붕대를이용하여가볍게고정하였다. 3. 실험평가 1) 육안적평가총 20마리의백서의육안적평가를위해서술후 0, 4, 7, 10, 14, 18, 21일째에백서등의창상면적을 Visitrak grid R (Smith & Nephew, UK) 필름에그린다음이를면적분석장치 Visitrak digital R (Smith & Nephew, UK) 을이용하여면적을측정하였다. 위와같이구한값을토대로창상치유에따른창상수축률, 창상치유률, 창상상피화률을구하였다 (Fig. 2). 측정된값들은시간에따른각실험군간의상호비교로분산분석법 (ANOVA test) 과쉐페법 (Scheffe's test) 을이용하여평가하였다. 2) 조직학적평가총 20마리의백서는조직학적평가를위해술후 4, 7, 10, 14, 18, 21일에창상전체를적출하여 10% 중성포르말린에고정시키고파라핀포매한후조직절편을만들어 Hematoxylin and Eosin과 Masson's trichrome으로염색하여광학 2. 실험방법 20마리의백서를대상으로등에지름 2 cm의원형의전층피부창상을 4군데에만들어각각의창상에 4가지드레싱재료의내용에따라 4개의실험군으로할당하여모든창상이치유될때까지관찰하였다. 수술시 kg당 10 mg의 xylazine-hcl (Rompun R, Bayer Korea, Korea) 과 kg당 100 mg의 ketamine-hcl (Huons Fig. 1. (Left) Full thickness skin defects, 2 cm in diameter, were made on the back of a rat. Four different types of dressing were applied. An gauze (group A) was applied on the upper-left wound, an High gel (group B) on the upper-right, an Aquacel R (ConvaTec, US) (group C) on the lower-left, low gel (group D) on the lower-right. (Right) Immediate post-operative view.

이지원등 :Carboxymethylcellulose 79 Fig. 2. Method for measuring changes in wound size. Fig. 4. The mean percentage of the wound epithelization rate according to time proceeding of each groups in defected wound by the application of four different dressings (P<0.05, values are significantly different from the control by ANOVA & Scheffe test). Fig. 3. The mean percentage of the wound contraction rate according to time proceeding of each groups in defected wound by the application of four different dressings (P<0.05, values are significantly different from the control by ANOVA & Scheffe test). 현미경하에창상부의염증반응, 혈관신생, 교원섬유의형성과재생상피를관찰하였다. Fig. 5. The mean percentage of the wound healing rate according to time proceeding of each groups in defected wound by application of four different dressings (P<0.05, values are significantly different from the control by ANOVA & Scheffe test). 결과 1. 육안적소견창상은시간이경과함에따라창상의수축과상피화가동시에진행되어상피화되지않은육아조직이점점감소하면서창상이치유되는것을관찰할수있었다. 창상생성후 4일째까지모든군에서다량의삼출물이배출되었으며이는 A군 : 거즈군에서상대적으로심하였으며, 드레싱교환과정에서창상표면의손상에의한출혈이관찰되기도하였다. 창상수축율은 14일이전까지의창상치유과정동안 B군 : high gel과 C군 : Aquacel R (ConvaTec, US) 에서통계적으로유의하게높았다 (P<0.05) (Fig. 3). 창상상피화율은 D군 : low gel에서창상생성후 14일째까지높게나타났 으며 14일이후에서는다른군에비해 B군 : high gel과 C군 : Aquacel R (ConvaTec, US) 에서통계적으로유의하게높았으며 (P<0.05) (Fig. 4), 창상치유율역시 14일이전까지의창상치유과정동안 B군 : high gel과 C군 : Aquacel R (ConvaTec, US) 에서높게나타났으나통계적유의성은없었으며, 창상치유 21일군에서는거즈군을제외한다른군에서비슷한결과를나타내었다 (Fig. 5). 2. 조직학적소견창상치유초기에모든군에서전층피부결손부상층에삼출물과염증세포가층을이루어관찰되었다. 초기의조직학적소견으로다핵구및림프구등의염증세포의침윤은술후 7일째에서 high gel 및 Aquacel R (ConvaTec, US)

80 대한화상학회지 Vol. 14, No. 2, 2011 Fig. 6. The degree of infiltration of inflammatory cells on the 7th post-operative day (Hematoxylin-Eosin stain, 100). Group A (above, left), Group B (above, right), Group C (below, left), Group D (below, right). Profuse inflammatory cell infiltration in Group A, moderate infiltration in Group D, and mild infiltration in Group B and C were observed. 군에서다른군들에비해염증세포침윤이조기에감소하는소견을보였다 (Fig. 6). 혈관증식소견은술후 7일째에거즈군을제외한다른군에서증식된혈관이관찰되었고, 술후 14일째에 high gel 및 Aquacel R (ConvaTec, US) 군에서타군들에비해뚜렷하게혈관증식소견을보였다 (Fig. 7). 창상상피화율은 D군 : low gel에서창상생성후 14일째까지높게나타났으며 14일이후에서는다른군에비해 B군 : high gel과 C군 : Aquacel R (ConvaTec, US) 에서교원섬유의배열이규칙적이고밀집된배열로나타났다 (Fig. 8). 고찰창상은염증과세포반응에의해치유가되는데, 이과정에는재상피화, 수축, 육아조직형성및교원질합성등이포함된다. 창연면적의감소는재상피화와수축이라는두가지과정에의해동시에유도되는현상인데창상수축은 섬유아세포의수축력에의해일어나며, 창연의면적감소에우선적으로기여한다고알려져있다 6,7). 재상피화는피부손상후정상표피세포가유사분열하여기저세포를대치해주고기저세포는피부결손부를향해이동하게되는데, 이기저세포는섬유소그물을따라창상경계면에서부터창상중심부로이동하며창상연양쪽에서이동해오던기저세포가서로만나게되면이동을멈추고표피의두께가정상이될때까지유사분열을계속하여표피의밀도를증가시키는과정을뜻한다. BC 400년무렵의히포크라테스시대의의학에서부터습윤한환경조성은창상치유에있어서널리사용되어온중요방법이며 8), Gilje는 adhesive tape 하에서조성된습윤드레싱법을이용한 Unna's boot법을이용하여하지궤양의치료에있어습윤환경의조성이창상치유에있어서중요함을보고하였으며, 1962년에 Winter의실험보고이후습윤환경은창상치유에있어가장중요한방법의하나로

이지원등 :Carboxymethylcellulose 81 Fig. 7. A comparison of the angiogenesis on the 14th post-operative day (Masson's trichrome stain, 100). Group A (above, left), Group B (above, right), Group C (below, left), Group D (below, right). Degree of angiogenesis is most abundant in Group B and Group C. 사용되어오고있다 4). 그러나습윤한환경의지속은창상및주위조직의짓무름을유발하는단점을가지기도하며과도한삼출물이있는상황에서는문제가되기도한다 9). 현재널리상용되는드레싱제재는크게 hydrocolloid dressing, hydrofoam dressing, silver-based dressing 등이있다. Hydrocolloid dressing 및 hydrofoam dressing 제재는습윤환경을제공하고삼출물을적당하게흡수하는작용이있으나, 창상의삼출물이과도하면밀봉 (occlusiveness) 효과가떨어져감염및침연 (maceration) 의우려가있다. 또한재료및제조방식에따라차이가있으나합성고분자소재로인체친화성이떨어져역할을다하면반드시제거되어야한다 9). 또한 silver based dressing material의경우높은항균성을지녀단독또는다른드레싱재료와의조합으로도널리쓰이고있으나, silver의 cytotoxicity로감염이없는창상의사용에는재고해야한다. Carboxymethylcellulose (CMC) 는제1차세계대전후독 일에서 gelatin의대체용으로개발되기시작하였으며, Carboxymethylcellulose (CMC) 염의일반적인형태는 sodium salt로 CMC라불리기보다는정확히표현을하자면 SCMC (Sodium carboxymethylcellulose) 가옳을것이다. CMC는금속이온이나암모늄이온과착염을형성할수있는데, 그용해특성과제조공정, 사용처에따라현재로서는가장적합한것이 sodium salt로산업적으로도중요한 cellulose 유도체이다. CMC는식품분야에서는보통 cellulose gum으로알려지는데, sodium염형태로뜨거운물과차가운물에녹으며, 유기용매에는불용이나, 저급알코올수용액이나아세톤에는녹는게된다 10). 그리고그특성은치환도 (DS) 와점도입자에따라다른성질을가지며, 안정한용액점도를이끌어내는 CMC의용해속도는여러가지의요인에따라달라진다 11). 첫째, 입자크기로적당히분산되었을때, 최종적으로 ground CMC가 coarse CMC보다더빨리녹게되지만후자의 coars CMC는점차로응집하지않고, 훨씬쉽

82 대한화상학회지 Vol. 14, No. 2, 2011 Fig. 8. A comparison of re-epithelization on the 21th post-operative day in the infected wound (Masson's trichrome stain, 100). Group A (above, left), Group B (above, right), Group C (below, left). Group D (below, right). Re-epithelization was still not completed in Group A. The thick layer of re-epithelization was observed in Group B and C. 게분산된다. 용해속도는치환의정도가증가함에따라, 분자량이감소함에따라증가한다. 낮은점도의 CMC가높은점도의 CMC보다안정화하려는성질이있고, 용액의점도는 CMC의농도에따라급격히증가하고, 용액들은의가소성유동 (pseudoplastic flow) 을보여주고있다. 낮은 DS 형태 (0.4 0.7) 의것들은빈번히요변성유동 (thixotrophy flow) 을하게되고, 적용이나전단의제거에관해점도의변화를보여준다. 하지만낮은농도의용액이나높은점도형태의것은거의뉴우튼유동을하고특히낮은전단속도일때더그렇다. CMC 용액의점도는온도의증가에따라감소한다. 이것의영향은고온과높은 ph를제외하고는역으로도가능하다. 용액이정상적인점도이상의광범위한점도를지닐때라도 ph 7과 ph 9사이에서용액의최상적인안정성이나타난다. ph 4 아래에서는 3차원상의네트워크를형성하는덜녹는산성 CMC가우세하여점도가증가한다. 이론적으로무수글루코오스반복단위안에 3개의모든수산기는에테르화에이용이가능하다. 하지만최고치환율인 DS 3은얻을수없으며, 최적용해도와실행에대해필요한것은아니다. 대개폭넓게사용되는 cellulose gum 은치환율 0.65 1.0을갖는다. 낮은치환율의것들은물에잘녹지않는다. 일반적으로 CMC는치환율 0.4에서는불용성 fiber가많은부분존재하게될지모르고, 치환율 0.05 0.25 사이에서는단지알칼리수용액에서만녹는성질이있다. CMC는증점, suspending, stabilzing, binding 등으로서역할이뛰어나고 film-forming 성질이매우우수하다 10). 저자들은이러한특징을가지는 CMC를주성분으로하는드레싱재료의치환도의차이에따라 gel화정도및친수성및용매에대한유기도가달라짐에착안하여, 신선창상에있어창상치유과정특히, 창상치유속도및상피화정도교원질침착등에미치는영향과새로운드레싱재료로서의치환도에차이를둔 sodium carboxymethylcellulose

이지원등 :Carboxymethylcellulose 83 (CMC) 과거즈드레싱및 Aquacel R (ConvaTec, US) 을각각사용하여창상치유정도를비교하는실험을시행하였다. 창상수축율은 14일이전까지의창상치유과정동안 B 군 : high gel과 C군 : Aquacel R (ConvaTec, US) 에서통계적으로유의하게높았고, 창상상피화율은 D군 : low gel에서창상생성후 14일째까지다른군에비해높았으며, 14일이후군에서는 B군 : high gel과 C군 : Aquacel R (ConvaTec, US) 에서통계적으로유의하게높은결과를나타내었다. 술후 14일째조직소견에서 B군 : high gel과 C군 : Aquacel R (ConvaTec, US) 에많은양의혈관신생을관찰할수있었다. 따라서두군에서나타난삼출물의조기감소및조직소견상에서염증세포의침윤감소및보다많은양의신생혈관생성이결국염증단계의빠른종결을의미하는것으로사료된다. 상처치유를매개하는대표적인염증세포는호중성구및대식세포로서 CMC와같은 hydrofiber wound dressing에서는드레싱자체가흡수하는호중성구의숫자가늘어남으로결국상처표면자체에남게되는호중성구의숫자가감소하게되어상처치유를촉진하게되는결과를유도하게되는것이며, 동시에각질세포의성장을유도하여상피재생을유도하게되는것이다 12-14). 현재 Aquacel R (ConvaTec, US) 과같은 hydrofiber wound dressing material은 fibronectin 및 fibrin을통한상처표면과의흡착으로밀봉의드레싱효과및삼출물을충분히흡수하고, 습윤환경을유지해주는제품으로볼수있다. 이러한 CMC 유도체의유변학적성질은용매, 농도, 온도, ph, 염의첨가등에의해다양하게나타날수있다. 현재 CMC 유도체는다양한산업에이용되며, 의료분야에있어서도훌륭한창상치유촉진제로서의기능을하고있다. 본실험을통해서는치환도에따라겔화정도의차이를보이는성질을이용하여창상치유에미치는영향을조사하였으며, 상처수축, 혈관생성, 상피화가전반적으로치환도 0.3의 CMC에서좋은결과를보여주었으나. 14일이전의상피화에서낮은치환도에서초기의빠른변화를보여줌을동시에관찰할수있어, 일률적인치환도의 CMC를사용하기보다는시기에따라좀더다양한치환도를가지는 CMC를적용하고, 다양하게변하는유변학적성질을좀더이용한다면더좋은창상치유효과를나타낼수있으리라사료된다. 결론저자들은치환율의정도에따라점도, 친수성등이달라지며다양한물성을가지는 carboxymethylcellulose (CMC) 를저친화도와고친화도의그룹으로나누어신선창상에있어나타나는창상회복을육안적, 조직학적으로비교하였다. 결과적으로급성창상의창상회복에있어고친화도그룹과양성대조군인 Aquacel R (ConvaTec, US) 에서창상치유초기 2주간수축율, 창상치유율에서높은성과를보였으며, 창상상피화율은창상치유초기에는저친화도그룹에서다른군에비해높은정도를보였으나, 이후에는고친화도그룹과양성대조군인 Aquacel R (ConvaTec, US) 에서좀더높은성과를보여주었다. 치환율의정도에따라점도, 친수성등이달라지는 CMC 의특징에따라시기별적절한드레싱을적용시킬수있음을본실험을통해알수있었으며, 다양한치환율을더적용하여접목한다면, 좀더구체적이고상세한시기별적정농도를파악할수있으리라여겨진다. 또한 CMC의다양한유변학적성질을응용하여좀더향상된창상치유촉진제를개발할수있으리라여겨진다. REFERENCES 1) Broughton G 2nd, Janis JE, Attinger CE, Wound healing: an overview. Plastic Reconstr Surg. 2006;117:1e-s-32e-s. 2) Hardig KG, Morris HL, Patel GK. Science, medicine and the future: healing chronic wounds. BMJ. 2002;324:160-163. 3) Hinnman CD, Maibach H. Effect of air exposure and occlusion on experimental human skin wounds. Nature. 1963;200:377-378. 4)Winter GD. Formation of the scab and the rate of epithelization of superficial wounds in the skin of the young domestic pig. Nature. 1962,193:293-294. 5) Hoffman AS. Hydrogels for biomedical applications. Adv Drug Deliv Rev. 2002;54:3-12. 6) Liu M, Warn JD, Fan Q, Smith PG. Relationships between nerves and myofibroblasts during cutaneous wound healing in the developing rat. Cell Tissue Res. 1999;297:423-433. 7) Baek RM, Song YT, Baek SJ, Lee JH, Im TG, Yoon BH. Effects of the recombinant human epidermal growth factor on full-thickness wound of the rat skin. J Korean Soc Plast Reconstr. 2003;30:201-208. 8) Adams F: The genuine works of Hippocrates. 1st ed. 271-336, London: Sydenham Society; 1939. 9) Phillips T, Stanton B, Provan A, Lew R. A study of the impact of leg ulcers on quality of life, social and psychologic implication. J Am Acad Dermatol. 1994;31:49-53. 10) Otto E, Spurlin H: Cellose and cellulose derivatives. 2nd ed. 882-888, Interscience, NY: Interscience Publishers, Inc.; 1954. 11)Rees DA. Structure, conformation, and mechanism in the formation of polysaccharide gels and networks. Adv Carbohyd Chem Biochem. 1969;24:267-332.

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