ISSN 1229-5418 Implantology 2016; 20(3): 124~131 임플란트지대주재료에따른구강세균부착 이성훈 1, 신현승 2, 박정철 2, 조인우 2, 송영균 3 단국대학교치과대학 1 구강미생물학교실, 2 치주과학교실, 3 치과보철학교실 The Adhesion of Oral Bacteria on Saliva-Coated Abutments Sung-Hoon Lee 1, Hyun-Seung Shin 2, Jung-Chul Park 2, In-Woo Cho 2, Young-Gyun Song 3 Departments of 1 Oral Microbiology and Immunology, 2 Periodontology, and 3 Prosthodontics, College of Dentistry, Dankook University, Cheonan, Korea Abstract Purpose: The aim of the present in vitro study was to compare bacterial adhesion on four different implant abutment materials with diverse surface roughness. Materials and Methods: The specimens of gold and zirconia were prepared and modified from the commercially available titanium rods (Ti) and TiN coating on machined titanium (TiN). Fusobacterium nucleatum, Porphyromonas gingivalis, and Treponema denticola in the saliva-coated specimens were analyzed by real-time polymerase chain reaction using the extracted DNA. Results: F. nucleatum adhered well with an exception of gold. P. gingivalis adhered well to zirconia and T. denticola adhered more to zirconia, Ti than gold and TiN. In conclusion, bacterial adhesion in the saliva-coated specimens had less effect on surface roughness. Conclusion: In stages of early adhesion, zirconia exhibited higher adhesion rate, but Ti coated TiN and gold materials show a low adhesion tendency. Key Words: adhesion, implant abutment, oral bacteria Reprint requests: Young-Gyun Song Department of Prostodontics, College of Dentistry, Dankook University, 119 Dandae-ro, Dongnam-gu, Cheonan 31116, Korea Tel: 82-41-550-1932, Fax: 82-41-550-1859 E-mail: ygsong@dankook.ac.kr Received for publication: August 16, 2016 Revised for publication: August 22, 2016 Accepted for publication: August 23, 2016 교신저자 : 송영균 (31116) 천안시동남구단대로 119 단국대학교치과대학치과보철학교실 Tel: 82-41-550-1932, Fax: 82-41-550-1859 E-mail: ygsong@dankook.ac.kr 원고접수일 : 2016 년 8 월 16 일원고수정일 : 2016 년 8 월 22 일게재확정일 : 2016 년 8 월 23 일 Copyright 2016. The Korean Academy of Oral & Maxillofacial Implantology This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 124 Implantology Vol. 20 No. 3, 2016
I 서론 치과임플란트술식의보편화와더불어구강내임플란트주위감염과연관된세균총에대한연구는비교적활발히진행되어왔다. 임플란트와관련한치주조직의미생물에대한기존의연구에따르면건강한상태의치주조직및치유단계에있는치주조직에서는치주질환에이환된조직과비교하여적은숫자의세균이치은열구내에존재하며분포하는세균군에있어서다른양상을보인다 1,2. 치주염은치은주변에서기인하는경우가대부분이며, 임플란트의경우치은과접하고있는임플란트의지대주부근에서임플란트주위염이기인한다. 기존의임플란트지대주는금주조합금이대부분을차지하였으나, 최근에는재료및표면처리도매우다양해지고있다. 초기에는임플란트치료가완전무치악자를위한치료에서시작되었기때문에임플란트매식체와마찬가지로기계적으로가공된순수티타늄이임플란트지대주의주된재료였다. 그러나티타늄지대주는다양한임상적상황에유연하게적용되는데에는한계가있었다. UCLA 지대주는금합금을주조하여각각의임상적상황에맞도록제작할수있도록고안된지대주이다 3,4. 이지대주의형태나각도는임의로조절될수있기때문에식립된매식체의각도나위치가원래치아의위치에서많이벗어난경우, 또는전치부에서임플란트보철물의 emergence profile을자연치와유사하게모방할수있기때문에심미적인보철물의제작을위해서도이용되었다. 최근기성지대주에사용되는 titanium nitride (TiN) 와 zirconium nitride (ZrN) 와같은질화물코팅기술을적용하고있다. 질화물코팅기술은우선금속구조물을금빛 이띠게함으로써심미적개선은물론기계적인성질역시증가시킬수있다 4-7. 또한고온에서티타늄의산화를어렵게하여도재와의결합을증진시킬수있으며 5, 뿐만아니라항세균성효과를얻을수있기때문에임플란트대주에이미널리사용되고있는실정이다 8. 표면경도의증가는임플란트의유지관리시, 초음파기구나금속으로된큐렛에의한표면의손상을감소시킬수있기때문에유리하다 9,10. 지대주표면의손상은표면거칠기를증가시키고세균의부착을용이하게하기때문에 11, 표면의견고함은장기적인예후에유리할것으로예상된다. TiN 코팅은치은을통해서투과되어보일때, 기존의티타늄지대주에비하여치은의색을보다정상적으로보이게하므로심미적으로도유리하다. 최근많은임플란트제조사들이지대주를 TiN으로코팅하고있다. 비슷한이유로 ZrN 코팅도지대주의기계적성질을개선시키는방법으로연구되고있다. 임플란트표면에대한세균부착에관해서는연구가많지않지만, 세균의부착은세균의종류, 임플란트표면의물리, 화학적특징, 그리고구강분비액의도말여부등에좌우되는것으로알려지고있다 12-15. 상이한임플란트재료에대한구강세균부착에대한연구에따르면, 재료의표면거칠기는표면의자유에너지보다더중요하며, 이는재료에따라세균의부착정도가차이가있다는것을의미하는것이다 13. 본연구에서는이러한여러가지지대주재료에치주세균이부착하는정도를비교분석해보고자하였다. 대한구강악안면임프란트학회지 20 권 3 호, 2016 125
Original Article II 연구재료및방법 1. 시편의준비 Gold, Ti, Ti-TiN, zirconia 4가지시편을직경 4 mm, 높이 1 mm의디스크형태로제작하였다. 1) Ti: 임플란트가공을위해상업적으로유통되는직경 4 mm의원통형티타늄봉을절삭가공하여제작하였다. 2) Gold: 직경 4.2 mm, 높이 1.2 mm의왁스패턴을이용한금주조를시행하여연마후직경 4 mm, 높이 1 mm 에가장근접한시편을제작하였다. 3) Ti-TiN: 위의 Ti 시편위에 TiN을증착시켰다. 4) Zirconia: 치과용 computer aided design/computer aided manufacturing (CAD/CAM) 을이용하여직경 4 mm 높이 1 mm의시편을제작하였다. 2. 세균의준비 Fusobacterium nucleatum ATCC 25586과 Porphyromonas gingivalis W50은 5 mg/ml의 hemin과 0.1 μg/μl 의 vitamin K가첨가된 brain heart infusion 배 지를이용하여배양하였다. Treponema denticola ATCC 35321은 OMIZ-Pat 배지를이용하였다. 모든세균은 37 o C의혐기성환경 (5% H 2, 10% CO 2 및 85% N 2 ) 에서배양하여준비하였다. 3. 프라이머의설계각세균의 16S ribosomal DNA 염기서열을이용하여프라이머 (primer) 를설계하였다. Melting temperature는 60 o C로설정하였고중합연쇄반응의 product size는 200~300 bp로설계하였다 (Table 1). 4. 정량분석을위한표준 DNA 농도의생성세균양에대한염색체의표준곡선 (standard curve) 을얻기위하여배양된각세균의수를 Petroff-Hausser bacterial counting chamber (Hausser Scientific, Horsham, PA, USA) 를이용하여 10 9 에서 10 3 범위로 10 배단위로측정하고, G-spin TM Genomic DNA extraction kit (intron Biotech, Seongnam, Korea) 를사용하여 genomic DNA 를추출하였다. 5. 시편에의세균부착건강한성인의비자극성타액을냉장보관후 6,500 Table 1. The primer sequences for real-time PCR Name Fusobacterium nucleatum Porphyromonas gingivalis Treponema denticola PCR: polymerase chain reaction. Forward Reverse Forward Reverse Forward Reverse Primer sequence 5 -CGC AGA AGG TGA AAG TCC TGT AT-3 5 -TGG TCC TCA CTG ATT CAC ACA GA-3 5 -TGC AAC TTG CCT TAC AGA GGG-3 5 -TAC TCG TAT CGC CCG TTA TTC C-3 5 -GAT AAT ACC GAA TGT GCT CAT TTA CAT A-3 5 -TCA AAG AAG CAT TCC CTC TTC TTC TTA-3 Sung-Hoon Lee et al. : The Adhesion of Oral Bacteria on Saliva-Coated Abutments. Implantology 2016 126 Implantology Vol. 20 No. 3, 2016
g에서 30분간원심분리하여상청액을취하여준비된시편을넣고 37 o C에서 1시간타액도말하였다. 이후시편을직경이 35 mm 접시로옮긴후, 1 μci/ml의 methyl- [ 3 H] thymidine을함유한각배지 5 ml를첨가하고, 준비한각세균 100 μl ( 파장 660 nm에서 OD=0.6) 를식균하였다. 혐기상태에서 T. denticola 는 48시간, 그외의세균은 24시간배양하였다. 시편을인산완충액으로세척하고세균을시편에서분리하기위해서파쇄완충액 (lysis buffer; 0.5 M Tris ph 9.0, 10 mm NaCl, 20 mm ethylene diamine tetraacetic acid, 1% sodium dodecyl sulfate) 을넣고 vortexing하였다. 6. 정량실시간중합효소연쇄반응추출된구강내미생물 DNA 의측정을위해정량실시간중합효소연쇄반응이실시되었다. 표준곡선의형성을위해제작되었던표준용액과추출된구강내미생물 DNA를각각 SYBR Premix Ex Taq (Takara, Kyoto, Japan), forward primer, reverse primer, ROX Reference Dye II, 증류수와혼합하였다. 혼합된용액은 ABI PRISM 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) 으로 94 o C에서 15초, 60 o C에서 15초, 72 o C에서 33초간 40회반응되었다. 단일증폭생성물을확인하기위해서온도를올려서증폭산물이단일가닥으로될때의온도 ( 형광소멸온도 ) 를측정하여단일증폭생성물임을확인하는 dissociation curve 단계를프로그램상으로추가하였다. 표준곡선을이용하여얻어진중합연쇄반응생산물에서세균양을측정하였다. 각 primer 에대하여 3회씩실시하였으며평균값을통계분석에이용하였다. 7. 통계적분석통계적분석을위해 IBM SPSS Statistics version 23 (IBM Co., Armonk, NY, USA) 프로그램을사용하였다. 통계적인유의성은 one-way ANOVA 와 multiple comparison Scheffé test를시행하여유의수준 5% 에서검정하였다. Rn 10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 2 Amplification plot 1E+10 1E+09 100,000,000 10,000,000 1,000,000 100,000 10,000 1000 100 10 1 4 6 8101214161820222426 28 30 3234 36 38 40 0 Cycle Bacterial count 10 20 30 Log concentration of sample 40 Fig. 1. Standard curve of Fusobacterium nucleatum. Sung-Hoon Lee et al. : The Adhesion of Oral Bacteria on Saliva-Coated Abutments. Implantology 2016 대한구강악안면임프란트학회지 20 권 3 호, 2016 127
Original Article III 연구결과 구강내미생물의정량분석을위해실시간중합효소연 Bacterial count 30,000 * 25,000 20,000 15,000 10,000 5,000 0 Gold Zirconia TiN coating Ti 쇄반응을이용하였다. 특정 primer 를이용하여증폭시킬때효과적인증폭이이루어지는지알아보고세균수와임계값주기의상관관계가있는지알아보기위하여표준곡선을확인하였다. 그결과세균수의 log 값과임계값수치가함수관계를가짐을확인할수있었다 (Fig. 1). F. nucleatum은 gold, TiN, zirconia, Ti 순으로부착이증가하였다. Gold 는다른시편에비해세균부착수가적게나타났다 (p<0.05; Fig. 2). P. gingivalis는 gold, TiN, Ti, zirconia 순으로부착이증가하였다. Zirconia 시편은다른시편에비해통계적으로유의하게많은부착을보였다 (p<0.05; Fig. 3). T. denticola 는 gold, TiN, zirconia, Ti 순으로부착이증가하였다. Gold 와 TiN 코팅은 zirconia와 Ti에비해적은부착을보였다 (p<0.05; Fig. 4). Fig. 2. Mean bacterial count of Fusobacterium nucleatum on specimens asterisks indicate significant difference (p<0.05). TiN: titanium nitride, Ti: titanium. Sung-Hoon Lee et al. : The Adhesion of Oral Bacteria on Saliva-Coated Abutments. Implantology 2016 35,000 140,000 30,000 * 120,000 b Bacterial count 25,000 20,000 15,000 10,000 Bacterial count 100,000 80,000 60,000 40,000 a b a 5,000 20,000 0 Gold Zirconia TiN coating Ti 0 Gold Zirconia TiN coating Ti Fig. 3. Mean bacterial count of Porphyromonas gingivalis on specimens asterisks indicate significant difference (p<0.05). TiN: titanium nitride, Ti: titanium. Sung-Hoon Lee et al. : The Adhesion of Oral Bacteria on Saliva-Coated Abutments. Implantology 2016 Fig. 4. Mean bacterial count of Treponema denticola on specimens. Means with different letters are significantly different (p<0.05). TiN: titanium nitride, Ti: titanium. Sung-Hoon Lee et al. : The Adhesion of Oral Bacteria on Saliva-Coated Abutments. Implantology 2016 128 Implantology Vol. 20 No. 3, 2016
IV 총괄및고찰 본연구의실험에서추출된구강내미생물 DNA 측정을위해실시된정량실시간중합효소연쇄반응은 fluorescence signal을사용하여미생물의종류와양을확인하는정확성과민감성이높은방법으로효과적이고신속한장점이있어치주병원성미생물의검출에적절히활용될수있다 16,17. 임플란트지대주는점막을관통하는특성상, 이곳에치태가형성되면결과적으로임플란트주위염을유발하여, 하부의임플란트매식체의건강한골유착에위해한영향을줄수있다. 매식체의표면이너무거칠면세균의부착이쉽게일어나고, 염증이발생하는경우, 일상적인구강위생행위에의해염증이조절되지않기때문에예후가불리해진다 18,19. 임플란트지대주주변의세균조성에대한연구에서, 표면거칠기가증가함에따라치태형성이증가하는것이관찰되었으나, 지대주의표면거칠기가 0.2 μm 이하인경우에는, 치은연하또는치은연상의세균조성에는거칠기에따른차이가거의없는것으로보고되었다 15,20. 그러나타액의여부에따른결과는일반실험실논문의결과와는다른결과를보였다. 타액여부에따른구강세균부착실험에서타액도말하지않은재료들에대한 Streptococcus mutans의부착은재료의표면자유에너지의변화와높은상관성이있었다. 그러나사용한재료들을타액또는혈청으로도말한경우에는 S. mutans의부착과표면자유에너지의변화와는상관성이없었다 12. 이번연구에서는기존에알려진것과다르게 zirconia 에서세균의부착이높게나타났다. 이것은단시간에이루어진실험때문으로생각된다. 재료에따라타액이개 재된상태에서구강내에서나타나는초기획득피막형성과정의차이가발생하는데, 티타늄은표면에일정한방식으로부착되지만, zirconia의경우세균 cluster를형성하며집락화된다 21. 이러한치면세균막형성기전의차이때문에지르코니아의세균부착이높게나타났을것으로생각된다. 장기간의실험에서는지르코니아의세균부착수가티타늄보다월등히적은것으로보고되고있다 22. Kohavi 등 23 은구강에장착된티타늄지대주에타액의아밀라제와혈청알부민이주로흡착함을보고하였다. 여러연구보고에따르면, 세균은피막의일정한단백질에선택적으로부착한다. Okte 등 24 은전자현미경에의한관찰을통하여, 0.14~1.00 μm 의표면거칠기와약 35 mn/ m의표면에너지를보이는티타늄시편들에대한세균의부착을비교하였다. 타액비도말시편으로행한실험에서는, 연마된티타늄시편에비하여 Ti-ZrN, Ti-TiN 또는열산화처리 (thermal oxidation) 한티타늄 (TiO 2 ) 에세균이유의하게적게부착하였다. 한편이시편들을타액으로도말하여실험하면, S. mutans는 TiO 2, Ti polished, Ti-ZrN, Ti-TiN, 그리고레이저방사선으로변모시킨티타늄 (Ti-laser) 의순으로많이부착하였다. T. denticola는가장거친표면의시료인 Ti에더많이부착하고, 여타의시료에도그람음성세균가운데서제일많이부착하였다. 구강에서는치아와임플란트지대주재료들이타액및치은열구액과같은체액으로젖어있고, 이런체액에는여러물질들이들어있어세균의부착에영향을준다 23. In vivo 에서치태형성은피막으로덮힌치아와티타늄표면에연쇄구균과여타의조기정착자들이군락하면서시작된다 24,25. 치태가성숙함에따라치주낭에혐기성환경이형성되면서영양요구가까다로운그람음성혐기성세균들이자라게된다 26. 그리고이혐기성세균들이치은연하치태에서탈락될때어떤세균은표피에부착 대한구강악안면임프란트학회지 20 권 3 호, 2016 129
Original Article 하거나또는치은조직을침입할수도있고, 또는치과기구를사용하여청결이한티타늄표면에재정착할수도있는것이다. 본실험의결과는혐기성세균들이피막으로덮힌티타늄표면에직접부착할수있음을시사한다. 또한 gold 와 TiN 코팅재료에서세균의낮은부착률을보여줌을알수있다. 이것은표면거칠기외에타액이개재된상태에서는타액성분등다른요소가작용함을알수있었다. 일반적으로임플란트지대주재료를일정한수준까지부드럽게하면세균의부착과정착을감소시킬수있다 19,27. 그러나세균의 in vitro에서행동은 in vivo 에서의행동과상이할수있다는것을주지해야할것이다. 앞으로단순타액이아닌타액의성분에따른세균부착경향에대한연구가추가적으로이루어져야할것이다. V 결론 타액에도말된지대주재료는구강내세균부착양상이표면거칠기와는다른양상으로나타난다. 초기부착의경우 zirconia가다른재료들에비해더높은경향을보이며, 기성지대주의재료인 TiN과 gold 의경우전반적으로낮은세균부착률을보인다. References 1. Fürst MM, Salvi GE, Lang NP, et al. Bacterial colonization immediately after installation on oral titanium implants. Clin Oral Implants Res. 2007; 18: 501-508. 2. De Boever AL, De Boever JA. Early colonization of non-submerged dental implants in patients with a history of advanced aggressive periodontitis. Clin Oral Implants Res. 2006; 17: 8-17. 3. Lewis S, Beumer J 3rd, Hornburg W, et al. The "UCLA" abutment. Int J Oral Maxillofac Implants. 1988; 3: 183-189. 4. Lewis S, Avera S, Engleman M, et al. The restoration of improperly inclined osseointegrated implants. Int J Oral Maxillofac Implants. 1989; 4: 147-152. 5. Habig KH. Friction and wear of sliding couples coated with TiC, TiN or TiB2. Surf Coat Technol. 1990; 42: 133-147. 6. Al Jabbari YS. Physico-mechanical properties and prosthodontic applications of Co-Cr dental alloys: a review of the literature. J Adv Prosthodont. 2014; 6: 138-145. 7. Endo K, Sachdeva R, Araki Y, et al. Effects of titanium nitride coatings on surface and corrosion characteristics of Ni-Ti alloy. Dent Mater J. 1994; 13: 228-239. 8. Lai CH, Chang YY, Huang HL, et al. Characterization and antibacterial performance of ZrCN/amorphous carbon coatings deposited on titanium implants. Thin Solid Films. 2011; 520: 1525-1531. 9. Tamura Y, Yokoyama A, Watari F, et al. Surface properties and biocompatibility of nitrided titanium for abrasion resistant implant materials. Dent Mater J. 2002; 21: 355-372. 10. Mengel R, Meer C, Flores-de-Jacoby L. The treatment of uncoated and titanium nitride-coated abutments with different instruments. Int J Oral Maxillofac Implants. 2004; 19: 232-238. 11. Speelman JA, Collaert B, Klinge B. Evaluation of different methods to clean titanium abutments. A scanning electron microscopic study. Clin Oral Implants Res. 1992; 3: 120-127. 12. Fujioka-Hirai Y, Akagawa Y, Minagi S, et al. Adherence of streptococcus mutans to implant materials. J Biomed Mater Res. 1987; 21: 913-920. 13. Nakazato G, Tsuchiya H, Sato M, et al. In vivo plaque formation on implant materials. Int J Oral Maxillofac Implants. 1989; 4: 321-326. 14. Wolinsky LE, de Camargo PM, Erard JC, et al. A study of in vitro attachment of Streptococcus sanguis and Actinomyces viscosus to saliva-treated titanium. Int J Oral Maxillofac Implants. 1989; 4: 27-31. 15. Quirynen M, Marechal M, Busscher HJ, et al. The influence of surface free energy and surface roughness on early plaque formation. An in vivo study in man. J Clin Periodontol. 1990; 17: 138-144. 16. Edgerton M, Lo SE, Scannapieco FA. Experimental salivary pellicles formed on titanium surfaces mediate adhesion of streptococci. Int J Oral Maxillofac Implants. 1996; 11: 443-449. 17. Smith BA, Shanbour GS, Caffesse RG, et al. In vitro polishing 130 Implantology Vol. 20 No. 3, 2016
effectiveness of interdental aids on root surfaces. J Clin Periodontol. 1986; 13: 597-603. 18. Quirynen M, Bollen CM, Papaioannou W, et al. The influence of titanium abutment surface roughness on plaque accumulation and gingivitis: short-term observations. Int J Oral Maxillofac Implants. 1996; 11: 169-178. 19. Bollen CM, Papaioanno W, Van Eldere J, et al. The influence of abutment surface roughness on plaque accumulation and peri-implant mucositis. Clin Oral Implants Res. 1996; 7: 201-211. 20. Astrand P, Anzén B, Karlsson U, et al. Nonsubmerged implants in the treatment of the edentulous upper jaw: a prospective clinical and radiographic study of ITI implants--results after 1 year. Clin Implant Dent Relat Res. 2000; 2: 166-174. 21. Rimondini L, Cerroni L, Carrassi A, et al. Bacterial colonization of zirconia ceramic surfaces: an in vitro and in vivo study. Int J Oral Maxillofac Implants. 2002; 17: 793-798. 22. Nakamura K, Kanno T, Milleding P, et al. Zirconia as a dental implant abutment material: a systematic review. Int J Prosthodont. 2010; 23: 299-309. 23. Kohavi D, Klinger A, Steinberg D, et al. Adsorption of salivary proteins onto prosthetic titanium components. J Prosthet Dent. 1995; 74: 531-534. 24. Okte E, Sultan N, Doğan B, et al. Bacterial adhesion of Actinobacillus actinomycetemcomitans serotypes to titanium implants: SEM evaluation. A preliminary report. J Periodontol. 1999; 70: 1376-1382. 25. Ahn SJ, Kho HS, Lee SW, et al. Roles of salivary proteins in the adherence of oral streptococci to various orthodontic brackets. J Dent Res. 2002; 81: 411-415. 26. Socransky SS, Haffajee AD, Cugini MA, et al. Microbial complexes in subgingival plaque. J Clin Periodontol. 1998; 25: 134-144. 27. Quirynen M, Teughels W. Microbiologically compromised patients and impact on oral implants. Periodontol 2000. 2003; 33: 119-128. 대한구강악안면임프란트학회지 20 권 3 호, 2016 131