Dentin etch chemistry investigated by Raman and infrared spectroscopy

JOURNAL OF RAMAN SPECTROSCOPYJ. Raman Spectrosc. 31, 171–176 (2000)

Dentin etch chemistry investigated by Ramanand infrared spectroscopy

R. Lemor,1 M. B. Kruger, 1 D. M. Wieliczka,1* P. Spencer2 and T. May3

1 Department of Physics, University of Missouri—Kansas City, Kansas City, Missouri 64110, USA2 School of Dentistry, University of Missouri—Kansas City, Kansas City, Missouri 64110, USA3 Synchrotron Radiation Center, University of Wisconsin—Madison, Madison, Wisconsin, USA

Micro-Raman and infrared spectroscopy were used to investigate the influence of surface treatment on thediffusion of a dental adhesive into dentin. The commercial dentin adhesive Scotchbond MultiPurpose Plus(3M) was placed on coronal dentin substrates that were cut from extracted, unerupted third molars. Priorto placement of the adhesive, one surface was treated with a phosphoric acid etch and the other with acitric acid–iron(III) chloride etch. Thin sections, ∼3 µm in thickness, were prepared and mounted on silverchloride disks for infrared spectroscopic studies with the remaining bulk sample being used for the Ramanstudies. The infrared studies were performed at the Aladdin Synchrotron radiation source at the SynchrotronRadiation Center, Stoughton, WI, USA. The Raman studies employed a krypton ion laser in conjunctionwith a microscope equipped with a 100× objective and confocal aperture. In both studies the sample wastranslated in 1 µm steps at the focus of the respective radiation, providing a line scan across the interface. Theinfrared results clearly show the interface but owing to the diffraction limit of ∼15 µm, accurate dimensionalinformation could not be obtained. The Raman results clearly indicate a broad interface associated with thesample etched with phosphoric acid and the presence of a much more abrupt interface with the citric acidetch. Additionally, a thin layer of exposed collagen exists at the dentin–adhesive interface. Copyright2000 John Wiley & Sons, Ltd.

INTRODUCTION

The formation of an adequate dentin–adhesive bonddepends upon diffusion of the adhesive throughout thedemineralized layer and into the unaffected dentin. If theadhesive does not penetrate the demineralized dentin, thenthe naked protein within this layer will be exposed to oralfluids and vulnerable to degradation by bacterial enzymes.Such degradation can ultimately lead to premature failureof the composite restoration.1

Previously, Raman spectroscopy has been shown to bea useful tool for the investigation of the dentin–adhesiveinterface, provided a laser of appropriate wavelength isused to avoid fluorescence and sample damage.2 Theseresults indicated that the Superbond system employing acitric acid–iron(III) chloride etch produced a thin partiallydemineralized layer which was completely penetrated bythe adhesive. In contrast, it was found that the ScotchBondMultipurpose Plus system generated a thick completelydemineralized layer with only a portion of it penetratedby the adhesive.3

This paper presents both Raman and infrared spectro-scopic results for the dentin–adhesive interface where thesame adhesive was used in conjunction with two different

* Correspondence to: D. M. Wieliczka, Department of Physics, Uni-versity of Missouri—Kansas City, 4747 Troost Avenue, Kansas City,Missouri 64110, USA; e-mail: [email protected]/grant sponsor: University of Missouri Research Board.Contract/grant sponsor: Petroleum Research Fund.Contract/grant sponsor: National Institutes of Health; Contract/grantnumber: DE12252.

etchants to determine the impact of the etching chemistryon the diffusion of the adhesive into the dentin. In addi-tion to the Raman results, infrared transmission spectrawere obtained using a specialized beamline. This beamlineemploys the unique characteristics of synchrotron radia-tion to illuminate a diffraction limited spot on the sample.

Previous applications of infrared transmission spec-troscopy to dentin and the dentin–adhesive interface havebeen only partially successful. The major impediments tothe application of infrared spectroscopy are (1) the watercontent of the sample, (2) diffuse scattering due to themicrocrystalline nature of the dentin and (3) the dimen-sionality of the interface. The presence of water and thestructural characteristics of the sample are inherent andcan only be addressed in a limited fashion. Until recently,the micron dimensions of the interface have precluded theuse of transmission infrared spectroscopy. Conventionalinfrared systems employ a globar with a source size ofa few millimeters. The inherent nature of infrared opticsprecludes the capture and focusing of a sufficient amountof infrared radiation to allow transmission experiments tobe performed with a spot size on the micron scale. Thedevelopment of synchrotron radiation sources and opticalsystems lifts this limitation.

EXPERIMENTAL

Samples

Extracted unerupted human third molars stored at 4°Cin 0.9% (w/v) NaCl containing 0.002% sodium azide

Copyright 2000 John Wiley & Sons, Ltd. Received 3 March 1999Accepted 1 July 1999

172 R. LEMORET AL.

were used in this study. The occlusal one-third of thecrown was sectioned perpendicular to the long axis of thetooth by means of a water-cooled low-speed diamond saw(Buehler, Lake Bluff, IL, USA). The surface was abradedwith 400 and 600 grit SiC under water prior to chemicaletching and application of the dental adhesive. The twoetchants used were a 35% phosphoric acid and a 10%citric acid–3% iron(III) chloride mixture. The prepareddentin was treated with Scotchbond Multipurpose Plusaccording to manufacturer’s instructions.

Cross-sectional samples of the dentine–adhesive inter-face, approximately 1 mm thick, were cut from the toothby means of a diamond saw. A Polycut ‘S’ sledge micro-tome (Leica, Deerfield, IL, USA) was used to cut thinsections,¾3 µm in thickness, which were mounted onsilver chloride substrates for the infrared studies. Theremaining bulk sample was mounted on a suitable sub-strate for securing the sample on the Raman microscope.The same sample was used for both the infrared andRaman measurements.

Raman spectroscopy

Micro-Raman spectroscopy was performed with a Dilor-OMARS 89 Raman spectrometer equipped with an Olym-pus BH-2 BHT microscope and a liquid nitrogen-cooledCCD detector (EG&G 1530 C). The spectral resolution ofthe system was 4 cm�1. The optical microscope allowedfor visual identification of the position at which the Ramansignal was obtained, i.e. adhesive, interface or dentin.The samples were mounted on a computer-controlled,high-precision x–y stage with a spatial resolution of¾0.05 µm.4 The excitation source was a 100 mW kryp-ton ion laser operating at 647 nm. After passing throughthe microscope, the illuminated spot size was¾1 µm2 inarea and approximately 6 mW were incident on the sam-ple. Spectra were obtained at 1µm intervals across the

dentin–adhesive interface and in three spectral regions,350–650, 800–1100 and 1475–1700 cm�1. After acquisi-tion the data were filtered for spikes and high-wavenumbernoise using an FFT filter routine. The composite scanswere generated without further modification. Additionalspectra from regions of pure dentin and pure adhesivewere collected for comparison.

Infrared spectroscopy

The infrared spectra were obtained at the SynchrotronRadiation Center, Stoughton, WI, USA, using the Alladinstorage ring operating at an energy of 800 MeV. Theinfrared spectromicroscopy beamline, port 031, is equip-ped with a Nicolet Magna-IR 550 FTIR spectrometer cou-pled with a NIC-Plan microscope. Spectra were acquiredin the range 700–4000 cm�1 with a resolution of 8 cm�1.The microscope is equipped with a motorizedx–y stagewith 1 µm positioning capability and with dual confocalSchwarzchild reflective lenses. The objective lens param-eters are 0.58 numerical aperture (NA), 15ð and thecondenser lens parameters are 0.71 NA, 10ð. Limitingapertures were adjusted to a dimension of 2ð 18 µm(verticalð horizontal) and oriented to keep the long di-mension parallel to the interface. Spectra were obtainedat 1 µm intervals across the interface with backgroundspectra obtained prior to and after completion of the linescan. Background spectra for conversion to absorbancewere linearly interpolated between these two.

RESULTS

The Raman spectra for the three different spectral regionsand from positions in pure dentin, the interface and pureadhesive are shown in Fig. 1. The top plot shows the

Figure 1. Raman spectra from three different spectral ranges are shown and for sample regions of pure dentin (�), the interface (°)and pure adhesive (M).

Copyright 2000 John Wiley & Sons, Ltd. J. Raman Spectrosc. 31, 171–176 (2000)

DENTIN ETCH CHEMISTRY 173

lower wavenumber region with broad features at 440–470and 580 cm�1 attributable to the phosphate group ofthe mineral.5,6 The ScotchBond Multipurpose has intensefeatures located at 380, 430, 480, 600 and 635 cm�1. TheRaman intensity at 635 cm�1 was used to monitor theadhesive concentration as a function of position acrossthe interface. The center plot shows several weak, broadstructures and the dominant structure associated with thesymmetric stretch of the phosphate group at 960 cm�1.5 – 7

The lack of other structure in this spectral region allowedfor accurate monitoring of the apatitic concentration as afunction of position. The last spectral region is shown inthe lower graph of Fig. 1. This region was found to be thebest possible region to monitor the collagen composition.8

The three strong peaks at 1580 (phenyl), 1610 (phenyl)and 1640 cm�1 (C C) are associated with the adhesiveand the broad weak structure from 1625 to 1700 cm�1 isassociated with the amide I region of collagen.9

Plots of position versus wavenumber are shown inPlates 1 and 2 for the two etch chemistries, with colorcorresponding to the intensity of the Raman scatteredlight. Yellow represents high intensity and blue representslow intensity. Plate 1 shows the composite images ofspectra taken at points across the interface formed withthe phosphoric acid etch. The interface is oriented suchthat the adhesive is in the lower part of the image anddentin is in the upper part. The image on the left is fromthe 1475–1700 cm�1 region showing the intense scatteringat 1600 cm�1. This band begins to decrease in intensityat 13 µm and is not detectable at 6µm. The spectralregion from 1650 to 1690 cm�1 corresponds to Ramanscattering from collagen. Note that the intensity increasesslightly when the Raman scattering due to the adhesivebegins to decline and that the collagen intensity againdecreases when the adhesive signal is no longer detectable.The center figure corresponds to 800–1100 cm�1 wherethe scattering from the orthophosphate group is observed.The Raman scattered intensity associated with the apatitebegins to decrease at 1µm and is no longer detectableat 7 µm. The figure on the right shows the compositeat the lower wavenumber region. Owing to the largebackground and weak spectral features in this region,it is difficult to identify the interface zone clearly. Theimage does confirm the decrease in the adhesive at 15µm.These images qualitatively show the gradual decline inthe intensity of features associated with the adhesive anda large gap between this decline and the increase of theRaman scattered intensity from the apatite. Note that in theregion between 7 and 12µm little structure is observed.

Plate 2 is a similar image but for the interface formedwith the citric acid–iron(III) chloride etch. This interfaceis much more abrupt than the previous one with only1 µm separating the decline in intensity of the signalsfrom the adhesive and apatite. Again the image on theleft shows the intense Raman scattering from the adhesivewith a decrease in intensity occurring at 13µm. Below10 µm an overall increase in intensity is observed, owingto an increasing background. The center image showsthat the Raman scattering from the phosphate extends to10 µm but decreases abruptly after that point. Finally, thelower spectral region shows a reduced background andthe Raman scattered intensity associated with the adhesiveand apatite is clearly observed. The Raman intensity fromthe adhesive begins to decrease near 11µm and the apatitebands begin to increase at 9µm. In the intervening region

the intensity of spectral features associated with bothcomponents gradually decreases.

The infrared absorption spectra for three different pointson the sample are shown in Fig. 2. The top figure showsthe entire spectral range from 650 to 4000 cm�1 and thelower figure shows the range from 750 to 2000 cm�1. Thebackground due to scattering is minimal in the adhesivespectrum, increases in the dentin and is a maximum inthe spectrum taken at the interface. The influence ofdiffuse scattering from the sample has minimal impactbelow 2000 cm�1 and is most evident in the spectralrange 2000–4000 cm�1. A close examination of the lowerwavenumber region shows the characteristic absorptionbands associated with a carbonated apatite. The strongband located from 1000 to 1100 cm�1 is attributable tothe asymmetric stretch of the phosphate group associatedwith the apatite.10 The C–O vibrations associated withthe carbonate appear at 873, 1413 and 1452 cm�1. Thefeatures at 1245, 1545 and 1660 cm�1 are attributedto the amide III, II and I vibrations, respectively.11,12

The transmission spectrum for the adhesive shows strongfeatures at 1157, 1184, 1249, 1455, 1510 and 1730 cm�1.These features are attributed to vibrational modes ofC–O–C, CH2, C6H4 and the C O ester groups.13

A composite image of the infrared absorption spectrafor the limited spectral region is shown in plate 3 forthe phosphoric acid-etched sample. The color scheme isthe same as that used for the Raman composite imagewith yellow representing high and blue representing lowabsorbance. The figure shows a broad interface, greaterthan 30µm, with a decrease in the apatite and collagen

Figure 2. Infrared spectra from the dentin adhesive interfaceand for sample regions of pure dentin (�), the interface (°) andpure adhesive (M). The upper graph shows the entire spectralrange and the lower graph is for the spectral range from 750 to2000 cm�1.

Copyright 2000JohnWiley & Sons,Ltd. J. RamanSpectrosc. 31, 171–176 (2000)

174 R. LEMORET AL.

signatures accompanied by an increase of the absorptiondue to the adhesive. Note that the orthophosphate bandbegins to decrease in intensity at¾12 µm prior to thereduction of the spectral intensity associated with the col-lagen at¾18 µm. The dominant adhesive lines, at 1184and 1730 cm�1, begin to decrease at¾20 µm. Addition-ally, none of the absorption lines shifts or broadens as afunction of distance from the interface. These results arequalitatively the same as those observed with the Ramanspectra.

Plate 4 is the composite infrared image for the citricacid-etched interface. The interface is seen to be abrupt,similar to that structure observed with the Raman spec-troscopy. In this sample the collagen features are morereadily identified. The reductions in the apatite, collagenand adhesive signals occur over a much shorter distancethan with the phosphoric acid-etched interface. Both theapatite and collagen signals begin to decrease in inten-sity at¾20 µm. The features associated with the adhesivebegin to decrease at¾25 µm and easily overlap with theapatite and collagen features. Additionally, the absorptiondue to the collagen is enhanced in the interface zone.

DISCUSSION

Diffusion length

Raman intensity or absorbance data were extracted fromthe line scans at a given wavenumber to provide plots ofintensity versus position. These datasets were fitted to asimple diffusion model as described.3 The concentrationat the position X given by this model is:

c D c0

2

[1� erf

(X

2pDt

)]where erf is the error function and the quantity

pDt is

a characteristic length associated with the diffusion at thegiven interface. The position where the concentration is50% is defined as the interface position. The interfacewidth, defined as the distance between the positions of10% and 90% intensity, is given byX D 3.62

pDt

with the positions given byX D š1.812pDt. Apply-

ing this model to both the dentin–protein and the pro-tein–adhesive interfaces provides a complete descriptionof the region. The fits to the Raman and infrared dataare presented graphically in Figs 3 and 4 and in tabularform in Table 1. The Raman scattered features at 635, 960

Table 1. Characteristics of the dentin–adhesive interfacebased on fits to the Raman and infrared spectra

Parameter Infrared spectroscopy Raman spectroscopyPhosphoric Citric Phosphoric Citric

Sample acid acid acid acid

Overall interface width/µm 19.9 15.4 14.8 7.6Collagen layer width/µm 3.7 2 8.5 2.8(apatite/adhesive <50%)Apatite gradient zone/µm 12.9 16.4 5.6 6.7(apatite 90 10%)Adhesive gradient zone/µm 20.4 10.8 9 1.8(adhesive 90 10%)

Figure 3. Fit to the diffusion equation for the Raman spectra.The solid lines represent the best fit to the data. Wavenumbersat which the data were taken from the spectrum: N, 635; ž, 960;�, 1600 cm�1.

and¾1600cm�1 correspondingto featuresassociatedwithadhesive,apatiteandadhesive,respectively,wereusedtoanalyzethe interfacecomposition.Thecollagencomposi-tion was monitoredwith the broad line at ¾1650 cm�1

but was not fitted to the diffusion equation owing toits weak natureand the dual interface,collagen–dentinand collagen–adhesive.The infrared absorptionfeaturesused to characterizethe interface were at 1038, 1662and 1724 cm�1, correspondingto apatite,collagen andadhesivefeatures,respectively.The fits to the diffusionequationare fairly good and provide a meansof quanti-tatively describingthe interface.

Phosphoric acid etch

The top graph in Fig. 3 shows the fit to the Ramanscatteredintensity for the apatite and adhesivesignalsfrom the phosphoricacid-etchedsample.Two featuresattributableto Ramanscatteringfrom the adhesivewereusedto verify the fitting procedure.The overall interfacewidth as determinedby the 90% points from the fit toscatteredintensityfor theapatiteandadhesiveis 14.8 µm.Of this totalwidth, a regionof 6 µm is partiallydecalcifiedand a 9 µm region is 100% decalcified.The zone overwhich the Ramanscatteredintensity from the adhesivevaries from 10% to 90% of its maximum is 9 µm andhasno directoverlapwith thezoneoverwhich theapatitegradientexists.Finally, the collagenlayer is definedas


Copyright ©1999 John Wiley & Sons, Ltd. J. Raman Spectrosc. 30 (1999)

Plate 2. Composite image of the Raman spectra obtained across the dentin-adhesive interface for the sample etched with citric acid-iron (III). Blue represents low intrensity and yellow high intensity.

Plate 1. Composite image of the Raman spectra obtained across the dentin-adhesive interface for the sample etched with phosphoric acid. Blue represents low intrensity and yellow high intensity.

JRS 470 Pages 14/4/00 2:04 pm Page 1

Plate 3. Composite image of the infrared spectra obtained across the dentin-adhesive interface for the sample etched with phosphoric acid.Blue represents low intrensity and yellow high intensity.

Plate 4. Composite image of the infrared spectra obtained across the dentin-adhesive interface for the sample etched with citric acid-iron (III). Blue represents low intrensity and yellow high intensity.


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Plate 5. Schematic representation of the two interfaces based on the Raman results

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DENTIN ETCH CHEMISTRY 175

Figure 4. Fit to the diffusion equation for the infrared spectra.The solid lines represent the best fit to the data. Wavenumbersat which the data were taken from the spectrum: �, 1038; ž,1662; N, 1724 cm�1.

the positionswhere the Ramanscatteredintensity fromthe adhesiveand apatitebecomeless than 50%, givinga layer of 8.5 µm. This correspondsto the changeinintensityobservedin theRamanspectra.Within this layera regionof ¾0.5 µm hasnomeasurableapatiteor adhesiveconcentrationand the remainingportion of the layer hasminimal incorporationof thesecomponents.

Thefit to theinfraredabsorptiondatafor thephosphoricacid-etchedsampleis shownin the top portion of Fig. 4.The interface width as determinedby this data set is20.9 µm with a region of 12.9 µm partially decalcifiedand 8 µm 100% decalcified.The zone over which theabsorptiondueto theadhesivedecreasesfrom 90%to 10%is 20.4 µm, indicating a sizableoverlapof the adhesiveandapatitegradients.The collagenlayer is determinedtobe 3.7 µm.

Citric acid–ir on(III) chloride etch

The Ramanscatteredintensity from the sampleetchedwith thecitric acid–iron(III) chloridesolutionis presentedin the lower graphof Fig. 3. The overall interfacewidthis 7.6 µm with a regionof 6.7 µm partially decalcifiedand¾1 µm 100%decalcified.Thezoneoverwhich theRamanscatteredintensity from the adhesivevaries is 1.8 µmand overlapswith the similar zone for the apatite.Thecollagen layer has a width of 2.8 µm with the entirelayer having a partial compositionof both apatite andadhesive.Comparedwith the interfacecreatedwith the

phosphoric acid, no region exists which is composedof only collagen and in fact the zones in which theadhesiveandapatitescatteredintensitiesvary overlapby¾1 µm. The correspondinginfraredabsorptionresultsforcitric acid give an interfacewidth of 15.4 µm with theentire region only partially decalcified.The zone overwhich the adhesiveabsorptionvariesis 10.8 µm and thecorrespondingcollagenlayer is 2 µm.

Infrar ed–Raman comparison

The quantitativedifferencesin the resultsderived fromthe Ramanandinfraredspectroscopicanalysesreflectthedissimilar wavelengthsof light used in eachtechnique.The infrared absorptionbandsused to characterizetheinterfaceare all in the 1000 cm�1 wavenumberregion,correspondingto a wavelengthof ¾10 µm. The diffrac-tion limit for this wavelengthis ¾15 µm; hence,basedon the infraredspectroscopicresults,the 20 µm interfaceoverestimatesthe distancewherethereis a changein thechemicalcompositionof thesample.Basedon thediffrac-tion limit, absorptiondatatakenfrom agivenpoint includeinformation from a much larger region. For example,atthe extremesof the map, spectrawere acquiredfrom aregion within 15 µm of the interface;hencethe spectrainclude information from the pure adhesiveas well asthe interface.Similarly at the interface,the spectrumisa compositeof contributionsfrom pure protein in addi-tion to dentinandadhesiveon eithersideof the interface.Deconvolutiontechniquescould improvethespatialreso-lution but the instrumentfunction mustbe known beforethesetechniquescan be applied accurately.Becauseofthediffraction limit, the infraredspectroscopicresultscanonly be usedin a qualitativesense.

In contrast, the Raman spectra were obtained at awavelengthof approximately647 nm, correspondingto adiffractionlimit of 1 µm.Sincetheinterfacedimensionsaregreaterthan1 µm, this limitation doesnot adverselyaffecttheresults.Forexample,theinterfacewidth for thesamplepreparedwith thephosphoricacidetch,asevaluatedat thepositionsof 90%Ramanscatteredintensityassociatedwiththe mineralandadhesive,is approximately15 µm. Usingthe positionswherethe Ramanintensity is 50 and 25%,this width reducesto 8.5 and4 µm, respectively.

CONCLUSION

Plate5 showsa schematicdiagramof the two interfacesbasedon theRamanspectra.Thedrawingon theleft is forthephosphoricacid-etchedsampleandthaton theright isfor the sampletreatedwith citric acid–iron(III) chloride.The striking differencebetweenthesetwo interfacesisthe dramatic increasein overall width associatedwiththephosphoricacidetchant.Theaggressivenatureof thisetchantproducesa wide interfacezonewith the regionsover which the apatiteand adhesiveintensity gradientsexist being much larger than those observedwith thecitric acid.Additionally, a narrowregionof pureprotein,which is not protectedby either the apatiteor adhesive,is found with the phosphoricacid etch.With the currentstatusof understandingfor the Ramanscatteringfromthesematerials,no evidenceis observedfor anychemicalinteraction betweenthe ScotchbondMultipurpose Plusadhesiveandthe collagenor dentin.


176 R. LEMORET AL.

Acknowledgments

This work was funded in part by the University of Missouri ResearchBoard, the Petroleum Research Fund and the National Institutes ofHealth under NIDR Area grant DE12252. The Synchrotron Radiation

Center is part of the Graduate School of the University of Wiscon-sin—Madison, and is funded by the Division of Materials Research ofthe National Science Foundation as a National Facility under AwardNo. DMR-9531009. The authors gratefully acknowledge 3M Corpora-tion for donating the dentin adhesive material used in this project. Theauthors also acknowledge the help of Jim Swafford.

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Dentin etch chemistry investigated by Raman and infrared spectroscopy