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2 0 1 5 년
2 월
석 사 학 위 논 문
이
2015년 2월 석사학위 논문
Eval uat i onofe t c he de name l us i ngQuant i t at i vel i ght -i nduc e d
f l uor e s c e nc e( QLF)
조선대학교 대학원
치 의 학 과
이 아 름
Eval uat i onofe t c he de name l us i ngQuant i t at i vel i ght -i nduc e d
f l uor e s c e nc e( QLF)
QLF를 이용한 법랑질 산부식 평가
2 01 5 년 2 월 2 5일
조선대학교 대학원
치 의 학 과
이 아 름
Eval uat i onofe t c he de name l us i ngQuant i t at i vel i ght -i nduc e d
f l uor e s c e nc e( QLF)
지도교수 민 정 범
이 논문을 치의학 석사학위신청 논문으로 제출함.
2 0 1 4 년 1 2 월
조선대학교 대학원
치 의 학 과
이 아 름
이아름의 석사학위논문을 인준함.
위원장 조선대학교 교수 김 병 훈 인 위 원 조선대학교 교수 황 호 길 인 위 원 조선대학교 교수 민 정 범 인
2 0 1 4 년 1 2 월
조선대학교 대학원
CONTENTS
Tablelegends···ii
Figurelegends···iii
Abstract···iv
Ⅰ.Introduction···1
Ⅱ.Materialsandmethods···2
Ⅲ.Results···5
Ⅳ.Discussion···8
Ⅴ.Conclusion···11
References···12
- ii -
TABLE LEGENDS
Table1.ΔF valuesforetchedenamel···5
FI GURE LEGENDS
Figure1.QA2analysisimageoftheetchedenamel···3 Figure 2.Bar diagram showing mean values for fluorescence loss of
etchedenamel···6 Figure3.FE-SEM imagesoftheetchedenamelsurface···7
- iv -
국 문 초 록
QLF를 이용한 법랑질 산부식 효과 평가
이 아 름
지도교수 민정범 조선대학교 대학원 치의학과
본 연구의 목적은 우상치에서 법랑질의 산부식 효과를 Quantitative light-inducedfluorescence(QLF)를 사용하여 정량적으로 평가하는 것이다.
30개의 발거된 소의 절치를 사용하였다.치근을 잘라낸 치관부 치아만을 1000ml 생리식염수 병뚜꼉에 매몰하였다. 치관의 절반을 #400-Grit SiC paper로 삭제하고.나머지 절반은 삭제하지 않았다.각 표면에 투명한 네일바 니쉬를 도포하여 3×3mm 크기의 enamelwindow를 형성하였다.총 120개의 window를 형성하여 무작위로 20개씩 6개 그룹으로 나누어 15,30,60초간 32% 인산을 적용한 후 수세,건조하여 QLF와 FE-SEM분석을 시행하였다.
QLF 분석결과,15초,30초간 산부식한 그룹에서,법랑질 표면을 삭제한 그 룹이 삭제하지 않은 그룹보다 유의적으로 높은 형광소실도를 보였다.60초간 산부식한 그룹에서는,표면삭제유무에 따른 유의적인 차이는 존재하지 않았 다.법랑질 표면을 삭제한 그룹과 삭제하지 않은 그룹 모두에서,15초 산부식 은 60초 산부식보다 유의적으로 낮은 형광소실도를 나타내었다.그러나 15초 와 30초,30초와 60초 산부식 그룹간에 유의적인 차이는 존재하지 않았다.
FE-SEM 분석결과,산부식시간이 증가할수록 법랑소주의 노출이 증가하였으 며,표면삭제한 군은 삭제하지 않은군보다 더 균일한 표면을 보였다.
본 연구에서 다양한 산부식 시간과 삭제유무에 따른 법랑질 산부식 효과를 QLF를 이용해서 정량적으로 평가할 수 있었다.
I .I nt r oduct i on
Sinceacid-etching wasintroduced in 1955,phosphoricacid etching has been used asa standard and predictableprocedure in enamelbonding,1, which enablesthemicromechanicalinterlocking between compositeresins and the enamelsurface.2,3 Acid conditioning removes a few microns of enamelexposingporousprismaticstructureandrougheningthesurface.4
Numerousstudieshavebeen conductedontheeffectofacidetching on enamelregarding severalparameters such as acid concentration,etching time and enamel grinding.5,6 However, Quantitative information on demineralizationafteretchingwithrespecttoenamelbondingislacking.
Quantitative light-induced fluorescence (QLF) is a diagnostic toolfor thequantificationofdemineralizationinanon-destructiveway.7,8,9Whena sound tooth surface is illuminated by blue-green light, it emits fluorescence with a wavelength of 540 nm.However,in demineralized area,increase in the scattering and diffusion of fluorescence reflection causes enamellesions to be visible as a dark spotin the fluorescence image.By means of this noticeable difference in fluorescence intensity betweensoundanddemineralizedenamel,QLF hasbeenwidelyusedasa quantification system for assessing early demineralization or remineralization of enamel by thoroughly investigating the correlation undervarioustreatments.10,11
The objective ofthis study was to quantify the effectofetching to bovine enamelusing QLF.The nullhypothesis was thatthere are no differencesin fluorescencelossbetween groundandungroundenameland thattherearedifferencesinfluorescencelosswithvariousetchingtime.
- 2 -
I I .Mat e r i al sandMe t hods
1.Specimen preparation
Thirty extracted bovine incisors withoutcracks or white spots were used forthisstudy.Theteeth werecleaned and stored frozen.Theroot was removed using low-speed diamond disk.The teeth were embedded intoplasticcapsof1000mlbottleswithutilitywax.One-halfofthelabial surface ofeach tooth was ground with #400-gritsilicon carbide paper. The otherhalfwas leftintact.Ground and intactsurfaces were coated with aclearnailvarnish,leaving rectangularwindowsof3mm × 3mm enamelexposed.A totalofsix groups of120 windows were tested.A 32% phosphoric acid (Uni-Etch®, Bisco, Schaumburg, IL, USA) was applied to the enamelwindow for15,30,or60 seconds.The acid was rinsedoffwithdistilledwaterfor20sandthesurfaceswereairdriedfor 20s.
2.QLF analysis
QLF-D (QLF-D biluminatorTM, Inspektor Research systems BV, Amsterdam,Netherlands)wasusedinthisstudy.Fluorscenceimageifall specimenswerecaptured with a 'liveview'- enabled digitalfull-sensor SLR camera(model550D,Canon,Tokyo,Japan)atthefollowing setting : shutterspeed of1/45 s,aperture value of3.2,and ISO speed of1600.
Measurementheightwas15cm.Proprietary software(C3v1.18,Inspektor Research systems BV)was used to capture and storealldigitalimages on a PC automatically.Allfluorscence images were analyzed using a software (QA2 v1.18, Inspektor Research Systems BV) by a single examiner(Figure1).
Demineralization quantity was calculated before and after etching.A
region ofinterestwasdefinedby manually outlining thesurfaceusing an interface within the capture software. Delta F values (defined as a percentageoffluorscenceloss)wasusedatthe5% thresholdlevel.
Figure1.QA2analysisimageoftheetched enamel.Imageshowed the ΔF values.
- 4 - 3.FE-SEM analysis
AfterQLF analysis,the specimens were mounted on aluminum stubs, sputter-coated with gold-palladium and examined under the FE-SEM(S-4800, Hitachi, Tokyo, Japan). Photographs of the most expressiveregionsweretakeat×2,500magnification.
4.Statisticalanalysis
Theinfluenceofdifferentetching timeon ΔF valueswereanalyzedby one-way ANOVA.Posthoc multiple comparisons were performed using DunnettT3andTukey'stest.Theindependentt-testwasusedtoassess differencesbetweenungroundandgroundenamel.
All statistical procedures were performed using the SPSS 12.0 for windows(IBM corp.,Armonk,NY,USA).Thesignificancelevelwasset atp=0.05
Mean±SD 15s 30s 60s Unground -5.385±0.19A,a -5.485±0.1A,a,b -5.695±0.42A,b,c
Ground -5.46±0.36B,a -5.68±0.19B,a,b -5.885±0.38A,b,c
I I I .Re s ul t s
1.QLF analysis
TheΔF valuesforthesix groupsareshown in Table1and Figure2.
Grindingtheenamelbeforeetchingfor15s,30shadasignificanteffecton enamelfluorescence(valuesas ΔF :averagefluoresceneloss).Butthere wasno singinificantdifferencebetween ground and unground enamelfor 60setching.
ΔF valuesdecreased with increased etching time.TheANOVA results showed that etching time had a significant effect on ΔF values only between15sand60s.Therewasnosignificantdifferencebetween15sand 30s,30sand60sinbothgroundandungroundenamel.
Table1ΔF valuesforetchedenamel
Within a column,significantly differentvalues are followed by different uppercaseletters(p<0.05).Within arow,significantly differentvaluesare followedbydifferentlowercaseletters(p<0.05).
- 6 -
Figure 2.Bar diagram showing mean values for fluorescence loss of etchedenamel.
2.FE-SEM analysis
FE-SEM images showed that there were morphological differences between ground and unground enamel (Figure 3).In ground enamel, micro-irregular etch patterns exposing individualenamelcrystals were clearly observed in the whole surface (Figure 3B,D,F) In unground enamel,porous and numerousenamelcrystallitescould beobserved.The quantity ofgood-quality etch produced by phosphoric acid at37% was time specific,with 15 s being less effective than 30 or 60 seconds. However,60swasnotbetterthan30s(Figure3A,C,E).
Figure 3.FE-SEM images ofthe etched enamelsurface.A,Unground enameletched for 15s;B,Ground enameletched for 15s;C.Unground enameletched for30s;D,Ground enameletched for 30s;E.Unground enamel etched for 60s; F. Ground enamel etched for 60s. ×2,500 magnification.
- 8 -
I V.Di s c us s i on
Adhesion to enamelis achieved through acid etching of this highly mineralized substrate,which substantially enlarges its surface area for bonding.12-14 There had been many attempts to improve the adhesion procedureforsuccessfulbonding andalsominimizing unnecessary mineral loss. Previousresearch on thetopicofetchedenamelwasperformedfor qualitative surface analysis using FE-SEM orcomparing the shearbond strength.1-3 Thus, our study tried to quantitatively evaluate the deminearalizationofetchedenamelusingQLF.
Thepresentstudy revealed thatgrinding theenamelbeforeetching for 15s and 30s had a significant effect on demineralization.This led to rejecting the firstnullhypothesis.These findings could be due to the structuraldifferencebetween twoenameltypes.Itcan beinterpreted that subsurface enamelis more soluble than surface enamel.Previous studies also demonstrated thatthe surface of intactenamelis composed ofa dense layer of hydroxyapatite crystals without any intercrystallite spaces.15,16 In 60s etching group, there was no significant difference between ground and unground enamel.This mighthave resulted from using 32% phosphoric acid.This acid has high acidic capacity (pH<1), and clearly causesenough mineraldissolution to permittheformation of macro- and microretentiveresin tagsbetween and within enamelprisms regard-lessofgrinding.17,18
Etching time had a significanteffecton demineralization between 15s and60inbothgroundandungroundenamel.Thisledtorejectthesecond nullhypothesis.Theresultsindicatethatthequantity ofdemineralization produced by phosphoric acid at32% was time specific,with 15 seconds being significantly less effective than 60 seconds. However,
demineralization ofetched enamelfor 60 seconds was notsignificantly betterthanthatof30seconds.
FE-SEM analysiswasused to exmainethesurfacemorphology ofthe etched enamel.The presentresults showed thata longer etching time resultinamoredissolutionandremovaloftheenamelmineralphase,but there was no morphological difference in the ground enamel surface. There was structuraldifference beteween ground and unground enamel. These findings agree with previous study.19 which reported structural differencebetween twoenameltype.When theprismlesslayerofenamel is removed, the typical prism patterns are obtained from underlying enamelwhenetched.
Thetraditionalvisualmethodsofdetecting etchedenamelisconfirming the "white chalky" surface.But the QLF-D system allows immediate visualeffectand offers quantitative comparison ofcurrentimages with pastimagesfrom thesamepatient.Ithasbeen developed todetectearly caries and monitor its progression or regression longitudinally without destruction of the specimen in the clinical situation.20 Principle of measuring minerallossisbasedon theincreasein fluorescencescattering due to caries formation.21 When the teeth are illuminated with high intensity blue light,green fluorescence is induced from DEJ.The light scatters significantly more within demineralized enamelcompared with sound enamel.Asaresult,demineralized areaappearsdarkerthan sound areaon t,heQLF image.Therefore,theQLF-D system can beusedasa quantification system for assessing the degree ofdemineralization after etchingonenamel.
ΔF valuewasanalyzedtoevaluateminerallossinthisstudy.Sincethe enamelwindow was identicalin sizeforallspecimens,only averageΔF
- 10 -
The enamel window on unground and ground enamel was made adjacent to sound enamel, thus making comparsion the effect of demineralizationbetweenetchedareaandsoundenamel.
It is noteworthy that severalfactors affect the fluorescence loss of QLF-D system. To standardize the measurement conditions when conducting QLF measurements, camera geometry, focal distance, environmentalconditionswasfixed.
Bovine enamelwas used as a substitute forhuman enamel.However, therearesomestructuraldifferencesbetweenbovineandhuman enamel.22 Itwasreported thatbovineenamelismoreporousthan thoseofhuman enamel,thuslessresistanttoaciddiffusion.Suchadifferencemightaffect todisparitiesinΔF valuesbetweenbovineandhumanenamel.
Also,ΔF values is affected by differentsite ofenamelwindow.For example,enamelwindow on cervicalarea ismoreacid-resistantbecause ofaprismaticenamel.
This in vitro study showed thatthe QLF-D system was capable of detecting and monitoring mineral loss in the etched enamel. The fluorescence loss measure by QLF-D system reflected degree ofmineral loss.Itissuggestedthatthenon-destructiveQLF-D system isconsidered tobeusefulnotonlyforclinicalbutalsoinvitroresearch.Futherstudies should beconducted todemonstratethebond strength ofcompositeresin to enamelaccording to theQLF valueofdemineralization.Ifthere isa significant relationship between bond strength and QLF value, QLF method mightbe used to evaluate the prognosis ofenamelbonding in clinicalsituation.
V.Concl usi on
This in vitro study demonstrated the demineralization ofenamelwith regard to varying etching times and surface treatmentis quantitatively analyzed by QLF.Within the limits ofthis study,QLF method may be usedtoevaluatethedemineralizationofetchedenamelconservatively.
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