D. M. WIELICZKA,* P. SPENCER, and M. B. K R U G E R Department of Physics, University of Missouri, Kansas City, Missouri 64110 (D.M.W., M.B.K.) and School of Dentistry, University of Missouri, Kansas Ci~, Missouri 64110 (P.S)
The degree of adhesive penetration into dentin has been studied through micro-Raman spectroscopic examination of the dentin/ad- hesive interface. In contrast to previous studies, for the specimen examined in this work the adhesive penetrates less than 2 p~m into the acid-etched and, thus, decalcified dentin. There is strong spec- troscopic evidence that, upon acid etching of the dentin surface, which is typically performed immediately before the adhesive is ap- plied, the collagen matrix collapses upon itself.
Index Headings: Dentin/adhesive interface; Collagen; Micro-Raman spectroscopy.
I N T R O D U C T I O N
In an effort to understand and define the adhesive forc- es that contribute to bonding between dental adhesives and the dentin substrate, numerous morphologic and structural characterization studies have been completed on the dentin/adhesive interface. 1-5 On the basis of the results of these studies, it is generally accepted that two factors are critical in determining adhesion at this inter- face: wetting of the dentin substrate by components of the adhesive system 6 and micromechanical interlocking via resin penetration and entanglement of exposed col- lagen fibrils in the decalcified dentin, zT,s A third factor which has been considered, but to date remains unre- solved, is the nature of the chemical bond between the dentin and adhesive.
It has been suggested that dental adhesives potentially adhere to the dentin surface via chemical bonding to the organic or inorganic phase. Such bonding could be dem- onstrated spectroscopically by a shift in the absorption bands attributable to the dentin substrate. 9 No substantial binding energy shifts that would indicate pr imary chem- ical bonding between the resin molecule and the dentin substrate were reported in a previous study that used X-ray photoelectron spectroscopy to examine the dentin/ adhesive interface. 1° Comparable results were reported f rom Fourier transform infrared (FT-IR) spectroscopic analyses of the resin/dentin interface. 4,1~ The results of micro-Raman spectroscopic investigations also indicated there is no chemical reaction between the resin and den- tin. 3,12 In contrast, FT-IR photoacoustic phase analysis suggests that 2-hydroxyethyl methacrylate is capable of chemical bonding to the organic fraction of dentin. ~3 In view of these conflicting results, further chemical char- acterization of the dentin/adhesive interface is required.
A critical tool in surface spectroscopic analysis of the dentin/adhesive interface is micro-Raman spectrosco- py.12.14 Using this technique, an investigator can detect compound-specif ic molecules from a sample without spectral interference f rom water at a lateral resolution of
Received 11 January 1996; accepted 28 August 1996. * Author to whom correspondence should be sent.
approximately 1 txm. One factor that traditionally limits the application of micro-Raman spectroscopy in the study of many biological samples, including the dentin/adhe- sive interface, is interference f rom fluorescence. The flu- orescence is due to electronic excitation of the organic component by the 488-nm or 514-nm lines of the mr ÷ laser that are the standard excitation wavelengths for these studies. The background from fluorescence is often so strong that the noise in the fluorescence is larger than the Raman signal.
Previous authors have attempted to reduce the back- ground fluorescence by removing the organic component, i.e., the collagen, This requires either exposure of the sample to hydrazine or prolonged exposure of the sample to a high-power laser beam. 15-~9 Such destructive sample preparation techniques alter the sample, thereby prohib- iting any further accurate analysis. Another little used approach to overcome the problem of high background due to fluorescence is to use red or near-infrared excita- tion, thus avoiding the electronic absorption process that leads to fluorescence5 ° The advantage of this technique is that minimal sample preparation is required-- i .e . , the collagen is left intact.
The purpose of this paper is to study the dentin/adhe- sive interface using micro-Raman spectroscopy in con- junction with laser light that is optimized to reduce the background fluorescence. Proper selection of the wave- length of the laser light also allows for identification of the protein which has been eliminated in the samples studied by previous investigators. This provides for a complete characterization of the dentin/adhesive interface since the protein, the mineral, and the adhesive lines can be monitored at all points on the interface.
EXPERIMENTAL
Extracted noncarious, unerupted human molars, which were stored upon extraction at 3 °C in buffered saline, were randomly selected for treatment with ScotchBond multipurpose dentin adhesive (3M Company, St. Paul, MN). The occlusal one-third of the crown was sectioned perpendicular to the long axis of the tooth by means of a water-cooled low-speed diamond saw. The surface was abraded with 400 and 600 grit silicon carbide under wa- ter, etched with 35% phosphoric acid for 15 s, then washed and dried. The pr imer agent and dentin adhesive were applied according to the manufacturer 's instructions. The adhesive was polymerized by exposure to visible light for 60 s. Cross-sectional samples of the dentin/ad- hesive interface, approximately 1 m m thick, were cut f rom the tooth. The surface of the cross section was cleaned with sodium hypochlorite to remove the remnant smear layer created by the diamond cutting.
Raman spectra were recorded with a Di lor-OMARS 89
Raman spectrum of ScotchBond mult ipurpose adhesive.
Raman spectrometer, equipped with an Olympus micro- scope and a liquid nitrogen-cooled CCD detector. The excitation source was a Kr + laser operating at 647 nm with a power of 50 roW. After passing through the band- pass filter and condensing optics, approximately 4 m W was incident upon the sample. The spectral resolution was 1.5 cm ~. The sample was placed at the focus of a 100× microscope Objective and translated under the mi- croscope using a high-precision x - y stage with a spatial resolution of 0.1 ~xm. The focus of the laser beam in conjunction with a confocal aperture provided a spatial resolution of - 1 txm.
Spectra were obtained at positions corresponding to 2-p~m intervals across the dentin/adhesive interface. In the collection of these spectra, the zero position was set at the optical interface of the hybrid layer and unmodified dentin. The resulting linescans provide a chemical and morphological map across the interface. Additional spec- tra from regions of pure dentin and pure adhesive were collected for comparison. No post-processing of the data was performed, and a linear interpolation of the data was used to generate the contour plots.
RESULTS
The Raman spectrum of dentin is shown in Fig. 1. The spectrum reveals vibrations of both the organic and inor- ganic components with the more intense lines being due to the inorganic components. The strength of the inorganic contribution with respect to the organic contribution is due in part to the relative composition of the dentin ( - 6 5 % inorganic and 35% organic). The most intense peaks that are associated with the inorganic component occur at 431 cm -1 ( O - P - O bending mode), 591 cm -~ ( O - P - O bending mode), and 961 cm ' (P-O symmetric stretch) and the major peaks associated with the organic component appear at 1236 cm -I (amide 1II), 1272 cm -~ (amide III), 1447 cm -1 (CH2), and 1661 cm ~ (amide I). The Raman spec- trum of ScotchBond multipurpose adhesive is shown in Fig. 2. The spectrum reveals vibrations of both the ali- phatic and aromatic groups. The more intense lines occur at 640 cm -~, 1111 cm -~ (C-O-C) , 1187 cm ~, 1457 cm -I (CH2), and 1607 cm -l (phenyl C=C) .
As shown in the photomicrograph of the dentin/adhesive
interface (Fig. 3), there is sufficient contrast between the adhesive, the hybrid layer, and the dentin to allow for easy identification of the position at which the Raman spectra are obtained. The following two contour plots (Figs. 4 and 5) are composites of the accumulated line scans. The zero position was visually set at the hybrid layer/unmodified dentin interface. The peaks at - 1 1 0 0 cm -~ and 1175 cm -~ are due to scattering from the adhesive. These features clearly disappear from the spectra as the sample is translated and data are acquired from the hybrid layer. The lines lo- cated at 1082 cm -~, 1243 cm -1, and 1269 cm -~ are due to collagen 2~ within the hybrid layer. Further translation of the sample moves the point o1' acquisition into the unmodified dentin, resulting in a decrease in the spectral intensity as- sociated with the collagen.
Figure 5 is a similar contour plot of Raman scattering over the same spatial region as Fig. 4, but a different spectral region. The intense peak at --960 cm -1 (red) is associated with the P - O stretching vibration in the min- eral component of the dentin. The position at which its intensity vanishes spectroscopically defines the dentin/ hybrid interface. The numerous peaks in the hybrid layer are due to scattering f rom the collagen. The three weak features that appear at approximately 10 txm from the dentin/hybrid interface are due to scattering f rom the ad- hesive. As in the previous figure, the peaks due to scat- tering f rom the adhesive :ire not observed within the hy- brid layer.
DISCUSSION
A limitation of the Raman technique as reported by previous investigators is the presence of fluorescence from the organic component of the dentin. The two tech- niques that have commonly been used to reduce the flu- orescence are incinerating the collagen by exposing the sample to intense laser it:radiation or removing the col- lagen by exposing the sample to hydrazine. 15-~9 Frequent- ly, this technique did not eliminate the fluorescence in the spectra, and the investigators were forced to subtract an arbitrary background from the spectra. Such manage- ment of the data can complicate the identification of spec- tral band positions and intensities.
One additional distinct disadvantage of these sample
APPLIED SPECTROSCOPY 1501
FIG. 3. Photomicrograph of the dentin/adhesive interface, taken at a magnification of 1000×. The picture represents a region 51 x 64 l.Lm. The darker region at top is the adhesive, with the lighter region at the bottom the dentin. The narrow band across the center of the picture represents the hybrid layer.
1502 Volume 50, Number 12, 1996
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preparation techniques is the removal of collagen from the interface and the obvious alteration of the sample. Fur- thermore, with the removal of the collagen, the technique must be limited to mapping of the mineral or adhesive lines. Little or no information can be collected on the structure or distribution of the collagen at the interface.
Micro-Raman spectroscopy is well suited as a non- destructive tool for the detection of spatial inhomogene- ities that are on the order of 1 txm in samples with Ra- man-act ive modes. With the use of the advantages of this technique, in combination with a Kr + laser, chemical composit ional changes in the region of the dentin/adhe- sive interface have been analyzed. Figure 5 shows the three chemically distinct regions studied. At distances of 1 txm and below, the 960-cm-1 line of the P - O stretching vibration f rom the mineral component of the dentin is observed. For all distances above 1 ~xm this peak is ab- sent, indicating the absence of the apatitic or mineral por- tion of the dentin. Indeed, this is known to be the case because the region f rom 1 to - 7 p~m is the hybrid layer in which the apatite has been etched away, leaving only the collagen matrix of the dentin. The interface between the etched portion (hybrid layer) and the unetched portion is clearly visible in the photomicrograph (Fig. 3), and the spectroscopic differences at this interface have been re- corded in this investigation.
The relative increase in intensity of the lines associated with the collagen within the hybrid layer would indicate
that the density of the collagen has increased as compared to that in dentin. For example, if the etchant simply re- moved the apatite, leaving voids in the hybrid layer, the intensity of the collagen lines should not be greatly af- fected. However, the approximate threefold increase in intensity of the collagen lines, within the hybrid layer versus the unmodified dentin, would suggest a threefold increase in the collagen density. Thus, it appears the col- lagen fills the voids created by the removal of the min- eral, increasing the density of the collagen. On the basis of the increase in observed intensity, we would speculate that the dentin was initially etched to a depth of - 1 5 - 1 8 txm and that the collagen layer collapsed to - 5 - 6 ixm before application of the adhesive.
A comparison of Figs. 4 and 5 allows one to map the chemical variation across the interfaces from 800 to 1300 cm ~. It is apparent that the hybrid layer extends f rom - 1 to - 7 txm. The weakly scattering peaks due to the adhesive (Fig. 5) do not extend below --7 txm, suggesting that the adhesive does not penetrate into the hybrid layer. The spatial dependence of the intensity of the two more intense lines from the adhesive (Fig. 4) similarly do not extend below - 7 txm. These peaks, while prominent in the adhesive region, are not detectable within the hybrid layer. Thus, spectroscopic examination of the adhesive/ hybrid interface suggests two possible interpretations for this line scan. The adhesive penetration is limited to < 2 txm, or if the penetration is greater than 2 pom, the amount
APPLIED SPECTROSCOPY 1503
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of adhesive that penetrates into the hybrid layer must be less than the detectable limit.
C O N C L U S I O N
Through the use of micro-Raman spectroscopy, prelim- inary evidence for the collapse of the collagen upon re- moval of the mineral component of the dentin has been established. A line scan across the dentin/adhesive inter- face has demonstrated the usefulness of micro-Raman scattering in defining the chemical variation across this boundary. The data from this line scan suggest the fol- lowing: adhesive penetration is limited to less than 2 ~m or the amount of adhesive which penetrates is below the present detection limit. Future work in which 2-dimen- sional regions of different dentin/adhesive surfaces are mapped with both optical and micro-Raman spectroscopy would be desirable to support these conclusions.
ACKNOWLEDGMENTS
This work was supported by a grant from NIH/NIDR (#DE09696), UMRB(Spencer 94)UMFG, and the Research Corporation.
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