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Effects of Hydrogen Peroxide on Human Dentin Structure
1 Key Laboratory for Oral Biomedical Engineering, Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, PR China; Correspondence: * corresponding authors, yiningwang{at}whuss.com and jmhu{at}whu.edu.cn
It has been hypothesized that hydrogen peroxide (H2O2) bleaching may cause destruction of dentin by a mechanism of protein oxidation. However, to our knowledge, there has been no direct chemical evidence to validate this viewpoint. To investigate the effects of H2O2 on the structure of human dentin, we used Fourier transform infrared spectroscopy (FTIR) and attenuated total reflection (ATR) spectroscopy. Human intact dentin specimens were treated either with 30% H2O2 or Hanks’ balanced salt solution (HBSS). Significant differences were observed in ATR spectra parameters. Additionally, demineralized dentin specimens were also tested. They were completely dissolved in the H2O2, but remained intact in the 0.1 N HCl and HBSS. The results suggested that H2O2 attacked both the organic and mineral components of dentin. Destruction of the organic components was mainly because of the oxidizing ability of H2O2, while changes in the mineral components were probably due to its acidity.
Key Words: dentin • hydrogen peroxide • FTIR • ATR
Intracoronal bleaching is a conservative alternative to the more invasive esthetic treatment of discolored, endodontically treated teeth (Dahl and Pallesen, 2003). The most commonly used bleaching agents are hydrogen peroxide (H2O2) and sodium perborate. Although there is little question about their efficacy, concerns have been expressed regarding the safety of bleaching agents, especially H2O2. The associated undesirable complications included changes in the surface morphology of dentin (Kawamoto and Tsujimoto, 2004), alterations in the structure of dentin (Rotstein et al., 1996), loss of mechanical integrity (Chng et al., 2002, 2005), increased dentin permeability (Heling et al., 1995), and external cervical root resorption (Harrington and Natkin, 1979; Madison and Walton, 1990). It has been hypothesized that H2O2 bleaching may cause destruction of dentin by a mechanism of protein oxidation (Rotstein et al., 1992; Kawamoto and Tsujimoto, 2004; Chng et al., 2005). However, to our knowledge, there has been no direct chemical evidence to validate this viewpoint. The purpose of the present study was to investigate the effects of 30% H2O2 on the structure of human dentin, and to explore the possible mechanism by which the H2O2 affects dentin.
The study protocol was reviewed and approved by the Ethics Committee of the School and Hospital of Stomatology, Wuhan University. Patients from whom teeth were being extracted were asked to read and sign a consent form prior to the extraction.
Specimen Preparation
Twenty intact dentin specimens were prepared. The crown was sectioned at 4 mm apical to the buccal cusp tip, with the slicing plane parallel to the occlusal surface, by means of a low-speed diamond saw (Isomet, Buehler Ltd., Evanston, IL, USA). A flat dentin surface on the remaining tooth specimen was exposed. This surface was polished with silicon carbide papers to 1000-grit size and 0.5-µm aluminum oxide slurry under continuous water cooling. The tooth was then sectioned at 2 mm from the exposed dentin surface and fabricated into slabs, approximately 4 x 6 x 2 mm. The specimens were ultrasonicated for 5 min with distilled water to remove smear layer before treatment. Twenty specimens were randomly assigned to 2 groups treated either with 30% H2O2 (pH
Fifteen demineralized dentin specimens were also prepared, first as above, then demineralized for 7 days at 25°C in 0.5 M EDTA (pH Each specimen was placed in 1 well of a 24-well tissue culture plate containing 1 mL treatment solution for 24 hrs at 37°C. The plates were sealed, and the solutions were not refreshed during the test.
FTIR and ATR Spectroscopy During the FTIR measurement, the dentin was gently scraped off with a fresh scalpel blade before and after treatment. We prepared KBr pellets by mixing 1 mg of dentin powder with 100 mg of KBr. FTIR spectra were recorded in the range from 400 to 4000 cm–1 at 4 cm–1 resolution. The sample was scanned 128 times in each FTIR measurement, and the spectrum acquired is the average of all these scans. Each specimen was measured 3 times before and after treatment. For the ATR testing, the unpolished surface of each specimen was marked in 3 different places by means of a water-cooled high-speed handpiece with a fine bur. The specimens were then put onto the face of the Ge crystal of the smart OMNI-sampler accessory, with the unpolished surface up. They were carefully adjusted so that the pointed tip of the standard pressure tower would be just pressed onto the center of the mark. This procedure kept the specimens measured at the same place before and after treatment. Spectra were collected in the range from 675 to 4000 cm–1 at 4-cm–1 resolution, with 128 scans co-added. Each specimen was measured at 3 different places before and after treatment. FTIR and ATR spectra of water were obtained (Nara et al., 2006) and subtracted from the sample by OMNIC 7 software (Nicolet, Madison, WI, USA). After baseline correction and normalization, second derivative spectra and difference spectra were calculated by use of the software. In addition, parameters of FTIR and ATR spectra of the Dentin+H2O2 group and the Denitn+HBSS group were examined: (1) mineral:matrix ratio (the ratio of the integrated areas of the phosphate v1, v3 contour to the amide I peak); (2) carbonate:mineral ratio (the ratio of the integrated areas of carbonate v2 contour to the phosphate v1, v3 contour); and (3) crystallinity determined as the 1030/1020 cm–1 intensity ratios (only for the FTIR spectra).
Statistical Analyses
The typical ATR spectrum of intact dentin was not identical to that from FTIR in peak shapes and locations (Fig. 1A  ). The second derivative spectra of phosphate v1, v3 also demonstrated obvious differences (Fig. 1B), especially in the spectra area 990–1050 cm–1.
FTIR of Intact Dentin In both groups, the spectra after treatment showed slight changes compared with those before treatment (data not shown). The mineral:matrix ratios and the crystallinity increased, and the carbonate:mineral ratios decreased after treatment in both groups (Table ). The two-way RMANOVA revealed significant main effects (p < 0.001, p = 0.003, and p = 0.017) for time and no significant main effects for treatment (p = 0.106, p = 0.334, and p = 0.134) in all 3 parameters. The time x treatment interaction effects were not significant (p = 0.051, p = 0.514, and p = 0.444).
ATR of Intact Dentin Amide peaks are representative of protein conformation. In the Dentin+H2O2 group, the relative intensity of amide I and amide II decreased dramatically, and amide III almost disappeared after treatment (Fig. 2A ). In the Dentin+HBSS group, only slight variations were observed in these regions (Fig. 2A). The difference spectra showed the same results (Fig. 2B).
Mineral investigations of crystallinity were focused essentially on the phosphate v1, v3 region. The contour in the region became sharpened in the Dentin+H2O2 group, but slightly widened in the Dentin+HBSS group (Fig. 2A  ). The difference spectra of the Dentin+H2O2 group showed a positive peak at 1028 cm–1 and negative peaks at 995, 1076, and 1107 cm–1 (Fig. 2B), while the spectra of the Dentin+HBSS group showed a positive peak at 999 cm–1 and a negative peak at 1028 cm–1 (Fig. 2B). Second derivative spectra were also used. In the –1 became Dentin+H2O2 group, the peaks at 1102 and 1072 cm weak, but the peak at 1091 cm–1 and shoulder at 1028 cm–1 appeared more intense after treatment (Fig. 2C). In the Dentin+HBSS group, the shoulder at 1028 cm–1 was less pronounced after treatment (Fig. 2C).
The mineral:matrix ratio increased after treatment in both groups (Table The carbonate:mineral ratio decreased after treatment in the Dentin+H2O2 group, but increased in the Dentin+HBSS group. The main effect for time was not significant (p = 0.058). There were significant time x treatment interaction (p < 0.001) and significant main effects for treatment (p < 0.001).
ATR of Demineralized Dentin
In this study, we confirmed that H2O2 attacked both the organic and mineral components of dentin. The results not only provide direct chemical evidence for the hypothesis that H2O2 may cause destruction of dentin by a mechanism of protein oxidation (Rotstein et al., 1992, 1996; Kawamoto and Tsujimoto, 2004; Chng et al., 2005), but also offer new insights into the changes in mineral components of dentin. Unlike ATR, FTIR did not detect differences between the Dentin+H2O2 and Dentin+HBSS groups. The low sensitivity of FTIR was probably because of the sample preparation. For measurement, the sample need to be scraped off, ground to a fine powder, desiccated, and dispersed in KBr. Hence, the small surface changes may have been masked by the less-affected subsurface region. In contrast, ATR was ideal for surface measurement. Specimens could be repeatedly measured at the same place without sample preparation, which ensured high comparability between spectra before and after treatment. Because of the fundamental differences between these 2 techniques (Grdadolnik, 2002), the spectrum obtained by ATR was not identical to that obtained by FTIR. ATR revealed relative shifts in peak intensity and absolute shifts in frequency. Therefore, the crystallinity parameter for FTIR was not used in ATR. One important finding was that the mineral:matrix ratio increased dramatically in the Dentin+H2O2 group. The change indicated that the organic component of surface dentin was removed by H2O2. This was confirmed by the changes of amide II and amide III in the spectra. Another important observation was that all demineralized dentin specimens were completely dissolved in H2O2. It is well-known that H2O2 is a strong oxidant and is also acidic. The dissolution of demineralized dentin should mainly be attributed to the strong oxidizing ability, but not the acidity, of H2O2, because the specimens in the Demin+HCl group remained intact after treatment. It should be pointed out that the behaviors of intact dentin and demineralized dentin subjected to H2O2 were quite different, probably because intact dentin contains mineral components, but demineralized dentin does not. We propose that the surface organic components are quickly removed by H2O2; meanwhile, the surface mineral components collapse and form a protective layer for the underlying dentin. This layer lessens the direct contact of H2O2 with underlying dentin and greatly slows the attack. The results of ATR also suggested that the mineral components were affected by H2O2. In the Dentin+H2O2 group, the carbonate:mineral ratios decreased, and the contour for phosphate v1, v3 became sharpened after treatment. The sharpened contour for phosphate v1, v3 indicated that the crystallinity (Pleshko et al., 1991) of dentin increased after H2O2 treatment. Both difference spectra and second-derivative spectra confirmed the change in crystallinity. The 1102 and 1072 cm–1 peaks have been attributed to HPO4 and PO4 groups in poorly crystalline apatite (Rey et al., 1991; Gadaleta et al., 1996; Magne et al., 2001). The 1091 and 1028 cm–1 are due to PO4 in stoichiometric apatite (Rey et al., 1991; Gadaleta et al., 1996; Magne et al., 2001). Changes in these peaks indicated increased crystallinity after treatment. Similar phenomena were also found in an earlier study (LeGeros and Tung, 1983), in which the carbonate-containing hydroxyapatite had decreased carbonate and increased crystallinity after exposure to acid buffer. The changes in the mineral components can be explained by the dissolution of the hydrated layer of dentin mineral. It has been suggested that one of the most important characteristics of biologically poorly crystalline apatites (dentin and bone) is the presence of labile non-apatitic environments of phosphate and carbonate ions (Cazalbou et al., 2004; Rey et al., 2007). These environments are believed to be mainly located in a hydrated layer at the surfaces of the crystals, while the core of the crystals may contain the relatively ordered hydroxyapatite phase (Wu et al., 2002; Cazalbou et al., 2004). The hydrated layer seems to be involved in homeostasis and other interactions of mineral crystals with their surrounding media (Cazalbou et al., 2005). It might also play a role in the mechanical properties of mineralized tissues, related to the hydration level, and possibly in biological regulatory processes (Cazalbou et al., 2005). It is likely that the surface layer preferentially dissolves because of its high reactivity and solubility and the acidity of H2O2. The mineral:matrix ratios and carbonate:mineral ratios increased slightly in the Dentin+HBSS group. This is probably due to the remineralization of dentin in the HBSS. The results of the Demin+HBSS group also confirmed that some mineral deposited on the demineralized dentin. It has been proven that demineralized dentin with phosphoproteins has the ability to induce apatite formation (Saito et al., 1998). The HBSS is rich in Ca2+ and PO4 3–. When the specimens were put into the solution, it is likely that remineralization was initiated. The present study was designed to explore the mechanism by which H2O2 affects dentin. H2O2 was used in a manner similar to that described in previous studies (Rotstein et al., 1992; Kawamoto and Tsujimoto, 2004; Chng et al., 2005). However, it does not represent a common clinical application. In the ’walking bleach’ technique, the bleaching agents are used in paste form or gel. H2O2 may be mixed with sodium perborate and sealed in the pulp chamber for 3–7 days. The procedure is repeated several times until a satisfactory tooth color is achieved. Further studies are planned to test these materials under more clinically typical conditions.
This work was supported by grant No. 30400507 from the National Natural Science Foundation of China. We thank Ms. Fei Niu, the Center of Analysis and Testing, Wuhan University, for her technical assistance. A preliminary report was presented at the IADR/AADR/CADR 85th General Session & Exhibition in New Orleans, LA, USA. in 2007 (Abstract #1772). Received for publication October 17, 2006. Revision received July 16, 2007. Accepted for publication July 31, 2007.
Journal of Dental Research, Vol. 86, No. 11,
1040-1045 (2007)
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ABSTRACT
INTRODUCTION
3.0, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) (Dentin+ H2O2 group) or Hanks’ balanced salt solution (HBSS) (
