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1 <Abstract> 대한치과재료학회지 43(4) : , 2016 ISSN: (Print); (Online) Available online at 불소방출성수복재의불소유리량, 표면경도및세포독성평가 이명진, 김광만 * 연세대학교치과대학치과생체재료공학교실및연구소, BK21 플러스통합구강생명과학사업단 A Study on the Fluoride Release, Microhardness and Cytotoxicity of Fluoride Releasing Restorative Materials Myung-Jin Lee, Kwang-Mahn Kim * Department and Research Institute of Dental Biomaterials and Bioengineering, College of Dentistry, Yonsei University, 50-1 Yonsei-ro, Seodeamun-gu, Seoul 03722, Republic of Korea, BK21 PLUS Project, Yonsei University College of Dentistry, Seoul, Republic of Korea The objective of this study was to examine fluoride release, microhardness and cytotoxicity of three different types of restorative materials; giomer (Beautifil Ⅱ), composite resin (Filtek TM Z250) and glass ionomer (Fuji Filling LC). Samples were prepared as disk-shaped specimens with diameter of 10 mm and thickness of 1 mm. Fluoride release was measured using ph meter (920A, Orion, Boston, USA) every 24 hours for the first 7 days followed by measurement every week until the 28th day. The Vickers hardness measurement was carried out for the microhardness, while in vitro cytotoxicity test of agar diffusion test was carried out according to ISO and ISO The results showed that microhardness of Filtek TM Z250 was the highest, followed by Beautifil Ⅱ, and Fuji Filling LC, in order. All groups, except Filtek TM Z250 released most amount of fluoride during first 24 hours and the release level was decreased over time. Cytotoxicity test indicated that all experimental materials were mildly toxicity. This study confirmed the variability of fluoride release, microhardness and cytotoxicity in these restorative materials and therefore such features should be considered in clinical application. Key words: Fluoride releasing materials, Fluoride release, Microhardness, Cytotoxicity Ⅰ. 서론 심미수복재에대한관심과수요가증가하고있는가운데여전히지적되고있는문제점중하나는이차우식으로인한수복치료의실패이다. 이에이차우식의발생을억제할수 * Correspondence: 김광만 (ORCID ID: ) (03722) 서울시서대문구연세로 50-1 연세대학교치과대학치과생체재료공학교실및연구소 Tel: , Fax: KMKIM@yuhs.ac Received: Aug. 4, 2016; Revised: Dec. 9, 2016; Accepted: Dec. 9, 2016 있는수복재가필요하게되었고, 이중대표적인것이불소를유리하는수복재이다 (Wiegand 등, 2007). 이는치아의탈회를막고법랑질을재광화시켜우식발생을억제하고초기우식의재석회화를촉진하는작용이있어이차우식을예방하기위해불소유리가가능한수복물들이널리사용되어져왔다 (Valpio, 1993). 불소이온이다양한기전을통해우식발생을억제하고초기우식의재석회화를촉진한다는것은이미많은연구들에서보고되고있다 (Eichmiller 등, 1998; Yaman 등, 2004). 이에, 불소를유리하며일정한강도를가지는수복재를개발하려는노력들이지속되어왔다 (Dionysopoulos 등, 299

2 2003). 불소를유리하는대표적인수복재로글라스아이오노머시멘트가있으며생체친화성이우수하고구강내에서불소를유리하고재충전할수있는장점을지니지만복합레진에비해수분에민감하고조작성, 심미성등이떨어지며초기물성이취약한한계가있다 (Williams 등, 2001). 이와같은단점들을상호보완하기위하여다중산강화복합레진인컴포머가개발되었다 (Andersson-Wenckert 등, 1997). 컴포머는저농도불소유리광중합형복합레진으로서산성화된이중기능성레진단량체와 fluoroalumi-nosilicate glass 입자로구성되어구강내수분을흡수하여이차적인산- 염기반응의결과로불소를유리한다고알려져있다 (Harry FA, 2003). 이는복합레진에버금가는심미성, 조작의용이성, 양호한표면연마성을가지며불소유리능력도가지고있다 (Çehreli, 2000). 하지만, 전통적인글라스아이오노머시멘트에비하여낮은불소유리양상을보이며 (Millen, 1998), 복합레진보다낮은결합강도, 높은중합수축률등이단점으로지적되었다 (Tate, 2000). 따라서불소를유리하면서물성의저하가없는재료를개발하기위하여 Roberts 등은 pre-reacted glasss ionome r(prg) 필러기술을개발하였고, 이를기반으로하여자이오머라는수복재료가소개되었다 (Roberts, 1999). 자이오머는 PRG technology 를이용한불소유리광중합레진제재 Glass ionomer 와 Polymer 의합성어이다 (Ikemura 등, 2003). Fluoro-aluminosilicate glass filler 와폴리아크릴산사이의산- 염기반응을통해만들어진실리카겔을동결건조처리하고가공하여실란처리후 PRG 필러를만들어이를레진기질내에포함시켜제조한다 (Ikemura, 2008). 기존의양대심미수복재료인복합레진과글라스아이오노머의단점을보완할 목적으로자이오머는복합레진의물리적성질과글라스아이오노머의불소유리능력을동시에가지고있는새로운형태의치과용수복재료로개발되어널리사용되고있다 (Itota, 2004). 그러나, 자이오머는임상활용도에비해이를뒷받침할만실제불소방출수준과경도뿐만아니라재료의생물학적친화성에관한연구가아직까지미흡한실정이다. 이에본연구의목적은기존임상에서널리쓰이고있는자이오머의시간경과에따른불소유리량, 표면경도및세포독성을측정하고기존에소개된불소유리수복재료인글라스아이오노머와비교하여새로소개된수복재로서의자이오머의임상적유용성을평가하는데있다. Ⅱ. 재료및방법 1. 연구재료본연구에사용된재료는대표적인심미수복용치과재료 3종을선택하였고, 실험군으로자이오머 Beautifil Ⅱ(Shofu Inc., Japan) 와글라스아이오노머 (GC Co., Japan), 대조군으로는충전용복합레진 Filtek TM Z250(3M ESPE, USA) 을선택하였다 (Table 1). 제조사의지시사항에따라작업하였으며중합을위해광조사기 (Eliper S10, 3M ESPE, Seefeld, Germany) 로 600 mw/cm² 수준의광도를유지한상태에서 40초간광중합을시행하였다. 2. 연구방법 1) 시편제작시편제작을위해내경 10 mm 1 mm의원판형아크릴 Table 1. Test materials used in this study Product Code Type Composition Manufacturer Beautifil Ⅱ BF Giomer BisGMA, TEGDMA, fluoroboroaluminosilicate glass Shofu Inc., Japan Filtek TM Z250 FZ Composite resin BisGMA, UDMA, BisEMA, zirconia/silica filler 3M ESPE, USA Fuji Filling LC FL Glass Ionomer Polyacrylic acid, distilled water, initiator, aluminosilicate glass, HEMA, UDMA BisGMA : Bisphenol-A-glycidyl methacrylate, TEGDMA : Triethylene glycol dimethacrylate, UDMA : Urethane dimethacrylate, BisEMA : Ethoxylated bisophenol-a-glycidyl methacrylate, HEMA : 2-hydroxyl methacrylate GC Co., Japan 300

3 몰드를준비하였다. Slide glass에 polyester film을놓고그위에몰드를올린뒤재료를주입하고기포가생기지않게조금씩충전한후, polyester film과 slide glass를덮어과잉재료를제거하였다. 그후상, 하면에광중합을시행하여각군당 5개씩시편을제작하였다. 2) 불소유리량측정플라스틱용기에각각 2 ml 탈이온증류수를채운후, 시편을넣고 parafilm 으로밀봉한후 37 로고정된항온기에보관하였다. 불소농도측정을위하여 ph/ion meter(920a, Orion, Boston, USA) 에 fluoride ion specific electrode(96-09, Orion, Boston, USA) 를부착시킨다음 0.1 ppm, 1 ppm, 10 ppm, 100 ppm 불소표준용액으로농도보정을시행하고시편으로부터유리된불소이온이담겨있는용액에동량의 TISAB Ⅱ 용액을첨가하여용액을안정화시킨후불소이온농도를측정하였다. 1, 2, 3, 4, 5, 6, 7, 14, 21 및 28일간보관후유리된불소량을측정하였으며, 매측정후용액은새로운증류수로교환하였다. 3) 표면경도측정비커스미세경도기 (DMH-2; Matsuzawa Seiki, Japan) 에시편을장착하고측정부위에 200g 의하중을 20 초간적용하였다. 시편에형성된다이아몬드형압흔의장축길이를측정하여미세경도 (Vicker s Hardness Number, VHN) 를계산하였다. 또한, 수분이표면경도에주는영향을평가하기위하여시편을 37 증류수에서 5 일간침적시킨후, 표면경도를측정하여비교하였다. 각시편당 3부위를측정하여평균치를산정하였다. 4) 세포독성평가 ISO 에따라한천중층법을이용하여세포독성을평가하였다. petri-dish 에 L-929 세포부유액 2.5x10 5 cells/ml 을 10 ml을첨가하여 37, 5% CO 2 incubator에서 24시간동안배양하였다. 배양액의단층배양상태와세포의형태를현미경으로확인한후배양액제거후 2X RPMI 1640배지와 3% 한천용액을동일한비율로섞은 Agar medium을 10 ml 첨가하였다. 고체화된 Agar medium에 neutral red 염색용액 을주입하고빛이차단되게밀폐하여보관후, 염색약을제거하고시편을조심스럽게위치하였다. ISO 의제시에따라양성및음성대조군은라텍스고무와폴리에틸렌필름으로설정하였으며, 각각직경 5 mm로잘라고압멸균소독후시험에사용하였다. 24시간동안 37, 5% CO 2 incubator 에서배양하였다. Table 2, 3에따라탈색지수및용해지수를를평가하였고, 이를 4번씩반복실험하였다. Table 2. Zone index criteria Index Description of zone No detectable decolorization zone around or under 0 specimen 1 Zone limited to area under specimen 2 Zone not > 0.5cm beyond specimen 3 Zone not > 1.0cm beyond specimen Zone > 1.0cm beyond specimen, but not involving 4 entire dish 5 Zone involves entire dish Table 3. Lysis index criteria Index Description of zone 0 No observable lysis 1 Up to 20% of zone lysed % of zone lysed % of zone lysed % of zone lysed 5 over 80% lysed within zone 5) 통계분석 각재료에따른기간별불소유리량및미세경도의측정치를 SPSS 21.0(SPSS Inc., Chicago, IL, U.S.A.) 프로그램을이용하여통계분석하였다. 유의수준은 0.05로설정하였고, One-way ANOVA로유의성을검정하고 Tuckey test로사후검정을시행하였다. 301

Table 4. Results of fluoride release (ppm) 1 to 28 days (Mean±SD) Group 1 day 3 day 7 day 14 day 21 day 28 day BF 0.198±0.040 b 0.082±0.014 b 0.042±0.004 b 0.043±0.014 b 0.033±0.007 b 0.027±0.

4 Table 4. Results of fluoride release (ppm) 1 to 28 days (Mean±SD) Group 1 day 3 day 7 day 14 day 21 day 28 day BF 0.198±0.040 b 0.082±0.014 b 0.042±0.004 b 0.043±0.014 b 0.033±0.007 b 0.027±0.012 b FZ 0.022±0.004 c 0.020±0.004 c 0.017±0.003 c 0.001±0.001 c c c FL 9.081±0.358 a 2.890±0.423 a 1.459±0.151 a 1.446±0.169 a 1.301±0.118 a 1.170±0.180 a *values in columns having the same letter were not significantly different(p>0.05) Ⅲ. 결과 1. 불소유리량측정결과각군의불소유리량측정결과를 Table 4에나타내었다. 복합레진인 FZ군은다른군에비해유의하게낮은불소유리량을나타내었고, 자이오머인 BF군이글라스아이오노머군인 FL군보다낮은측정치를나타내었다. 불소유리량은 FZ을제외한모든실험군에서첫째날가장높게측정되었고, 이후급격히감소하여 7일이후부터는완만하게유지되는양상을보였다. 글라스아이오노머에서가장불소유리량이높게측정되었고복합레진에서는측정기간내내불소의유리를거의관찰할수없었다. 2. 표면경도측정결과증류수에침적하기전표면경도는 FZ군이 79.64로가장높았고, BF군이 71.1로다음으로높았으며 FL군이 35.68로가장낮았다. 증류수침적후의표면경도역시 FZ군이 로가장높았고, BF군이 63.52로다음으로높았으며, FL군이 31.84로가장낮았다 (Table 5). 표면경도는각군간유의한차이를보였다. 전반적으로증류수침적후에표면경도가다소감소되는양상을보였으며통계적로유의한차이를보였다 (p<0.05). Table 5. Results of Vickers microhardness (VHN) test (Mean±SD) Group Dry Aged BF ±3.921 b ±4.143 b FZ ±1.942 a ±2.911 a FL ±2.288 c ±2.765 c *values in columns having the same letter were not significantly different(p>0.05) 3. 세포독성결과 한천중층시험후탈색지수및용해지수를 Table 2, 3에따라평가한결과, Figure 1과같이모든시편에서 0.5cm 이하의탈색을보여탈색지수 2로나타났다. 위상차현미경사진에서는, 양성대조군과같이탈색지수및세포사멸지수가큰재료를적용할때세포가탈색되고원형에가까운모양을나타냈으며, 모든시편에서세포의형태는거의동일했으며, 음성대 Figure 1. Decolorization zones of experimental materials in the agar diffusion test. The empty Teflon mold (NC; Negative Control), the latex sheet from the latex glove (PC; Positive Control), and experimental samples of (A)BF; Giomer, (B)FZ; Composite resin, (C)FL; Glass ionomer. 302

5 Figure 2. Cellular images of experimental materials in the agar diffusion test. The empty Teflon mold (NC; Negative Control), the latex sheet from the latex glove (PC; Positive Control), and experimental samples of (A)BF; Giomer, (B)FZ; Composite resin, (C)FL; Glass ionomer. 조군과유사한세포형태를나타내었다. sample 의탈색부위과 sample 이놓여있던 sample 바로아래부위의용해도를관찰하였을때탈색부위의 20% 이하로용해지수는 1이었다 (Figure 2). 음성대조군과양성대조군과비교한이상의탈색지수와용해지수의결과에따라반응지수를구하였고, 이에따른세포독성도를평가한결과경미 (mild) 한세포독성을나타내는것으로판단되었다. Ⅳ. 고찰이차우식은수복물의빈번한실패의원인으로이를예방하기위하여수복재의개발에대한노력들이이어져왔으며, 1990년대초에컴포머가소개되어사용되고있으나여전히복합레진보다낮은물성과글라스아이오노머보다약한불소유리량으로인해임상적사용에제한이있는실정이다 (Hattab 등, 1989; Ten Cate 등,2004). 이와같은한계점을극복하고자하이브리드레진계수복재로자이오머가개발되었다 (Yoon 등, 2010). 자이오머의 PRG 필러는실란처리되어탈수된 hydrogel layer와레진기질간의 coupling이증진되며, 추가로함유된 4-AET 와 HEMA 는친수성을띠게하여 PRG 필러로부터이온성분의적절한수화를도와이온의이동을돕게된다. 따라서 PRG 필러는불소이온의지속적인유리와재충전이유리한특성을가지면서도재료의물성은저하되지않는장점을갖는다고한다 (Kim 등, 2010). Sunico 등은자이오머 로수복한 1급및 5급와동수복물의 2년후임상검사를시행하여 80% 성공률을보고하였고 (Sunico 등, 2005), Fujimoto 등은 S-PRG 필러는산과접촉하게되면이온을유리하고이러한이온들이산과중화반응을한다고보고하여자이오머의산중화능은수분과접촉시에유리되는이온들이주위 ph를증가시킴으로써발휘된다고보고하였으며 (Fujimoto 등, 2010), Yap 등은자이오머가복합레진이나컴포머등의수복재보다유의하게높은표면경도를보이며열순환후에경도가증가됨을관찰하였다 (Yap 등, 2004). 본연구에서는혼합시간및보관시간의오차를줄이기위하여한사람의술자가제조회사의지시에따라실험하였으며, 그결과로자이오머가글라스아이오노머에비해약 2배정도의높은표면경도를나타내었으며복합레진에비해서는통계적으로낮은수치였지만유사한결과를나타내었다. 37 증류수에 7일간침적한후조사한결과모든군에서다소감소되는양상을보였지만, 마찬가지로자이오머의표면경도가글라스아이오노머의표면경도보다높았다. Roberts 는기존의불소유리수복재의 filler 로주로사용된 NaF 는많은양의불소를유리하지만나트륨이온과불소이온의용출로인해재료자체의구조적붕괴를야기하지만 PRG filler 는 hydrogel 내부에서불소이온과반대양이온의리간드교환을통해지속적으로불소를유리하므로내구성이유지되는것이라보고하였다 (Roberts, 1999). 불소유리량을일주일간매일측정한이유는 1일째가장높은수치를나타낸후급격한감소를보이며 1주일후어느정도일정한유리량을유지하기때문이다 (Cranfield 등, 1982; Swarts 등, 1984). 복합레진을제외한 303

6 수복재에서불소유리가관찰되었고불소유리량은 1일째가장높은수치를나타내고이후급격히감소하여 7일이후부터는완만한상태로일정불소량이유지되는양상을보였다. 여러문헌들에서도이와유사한불소유리양상이보고된바있으며, 이번연구에서도이러한양상을확인하였다 (Itota 등, 2004; Kamijo 등, 2009; Verbeeck 등, 1998). 글라스아이오노머는 burst effect 라불리는초기중합이이루어지고첫날에나타나는높은불소유리에이은이후 3일간의급격한유리량감소와낮은수준으로유지되는불소유리양상을관찰하였다. 이러한 burst effect 는수복직후에는살균효과를기대할수있지만장기간으로는이차우식을예방할수있는효과를기대하기어렵다 (Dijkman 등, 1993). Yap 등의연구에의하면자이오머와같인불소방출성수복재는상당히미미한불소유리를보였으며 2주후부터는불소유리가중지되었다고보고되었으나, 본연구에서자이오머는글라스아이오머보다연구기간동안유의하게낮은불소유리를보였으나낮은수준이지만지속적으로유리되는양상을보였다 (Yap 등, 1999). 이러한현상은각수복재의초기불소유리량의차이에기인하는것으로생각된다. 불소유리량이증가하는만큼수복재주위치질에뛰어난항우식효과를나타내는지는정립되어있지않은상태이다. 실제로치질에대한불소의재석회화효과는높은불소농도보다는낮은농도라도지속적으로유리될때훨씬우수한것으로보고된바있다 (Silverstone, 1985). 따라서, 자이오머는낮은불소유리량이지만글라스아이오노머와유사한양상으로긴시간동안지속적인불소유리량을보이기때문에주변치질에대한불소유리효과를기대할수있는것으로사료된다. 또한, 재료의생물학적특성은중요한성질로서대부분의수복재는구강내에서장기간접촉하므로임상적으로발생할수있는부작용을미리평가하는것이필요하다. 본실험에서사용된한천중층법은한천을통해재료가확산되기때문에재료의독성뿐만아니라독성물질의확산도평가가가능하다. 본연구의세포독성시험결과에서는모든군에서경미 (mild) 한세포독성양상으로같은정도의세포독성도를보였다. 이는이들단량체의상당부분이중합이되어잔류양이적기때문에세포생존도에영향을줄정도가아니기때문이라고사료된다. 이에, 세포독성정도는우려할정도는아니라고생각되며추후연구에서생체내 시험을추가로시행하여그재료의생체적합성을좀더정확하게평가하여야할것이다. 본연구에서는시편의보관매체를증류수로한정하여사용하였다. 그러나실제구강내환경은다양한형태의응력이작용할뿐아니라온도와산도의다양한변화가있으므로 in vitro 와 in vivo 에서의차이의한계를고려하여실험을설계하여야할것으로생각된다. 그리고불소유리관찰기간이 28일로비교적짧아장기간불소유리양상을관찰할수없다는한계가있다. 또한, 불소재충전양상을파악하지못하였다. 일반적으로초기불소유리가높은재료일수록우수한재충전능력을보이는것으로보고되고있으나 (Itota 등, 2004), 이를바탕으로결론내리는것은다소한계가있음을고려해야한다. 따라서임상적으로적용하기위해서는이러한한계점들을보완하여추가적인연구가필요할것으로사료된다. Ⅴ. 결론본연구에서는자이오머의불소유리량, 표면경도및세포독성을기존의복합레진과글라스아이오노머수복재와비교평가해보고자본실험을수행하여다음과같은결과를얻었다. 1. 불소유리량은 FL 군 > BF군 > FZ 군순으로높게나타났다. 2. 표면경도는증류수침적전, 후모두 FZ군 > BF군 > FL군순으로높게나타났다 (p<0.05). 3. 세포독성도를평가한결과세가지재료모두경미 (mild) 한세포독성을나타냈다. 이상의연구결과로자이오머는기존의글라스아이오노머와유사한불소유리량을보일것으로기대하였으나글라스아이오노머보다낮은불소유리량을보이지만지속적인유리량을보이는것을관찰하였고, 복합레진과유사한표면경도를보이는것을확인하였다. 이에, 임상적인활용도를결정하기위해서는보다다양하고발전된방법으로보완연구가필요할것으로사료된다. 304

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J Korean Acad Pediatr Dent 39(4) 2012 광을받고있으며이를뒷받침하는많은연구들이발표되고있다. 저자는새로개발된유동성자이오머의물성이기존유동성복합레진에어느정도근접했는지에초점을맞추고압축강도와표면미세경도를측정한후비교분석하여다소의지견을얻었기에보고하는바이다

J Korean Acad Pediatr Dent 39(4) 2012 광을받고있으며이를뒷받침하는많은연구들이발표되고있다. 저자는새로개발된유동성자이오머의물성이기존유동성복합레진에어느정도근접했는지에초점을맞추고압축강도와표면미세경도를측정한후비교분석하여다소의지견을얻었기에보고하는바이다 http://dx.doi.org/10.5933/jkapd.2012.39.4.383 대한소아치과학회지 39(4) 2012 유동성자이오머와복합레진의압축강도및표면미세경도비교 김종수 단국대학교치과대학소아치과학교실 국문초록본연구의목적은유동성자이오머인 Beautifil flow와유동성복합레진인 Filtek TM Z350, Z350XT의압축강도와표면미세경도를비교평가하는것이었다.