Role of enamel and dentin on color changes after internal bleaching associated or not with external bleaching

Abstract Objective To evaluate the effect of the association between external and internal tooth bleaching on color changes in dentin and enamel, individually or recombined, previously stained with triple antibiotic paste (TAP). Methodology Forty enamel-dentin specimens from bovine incisors were separated into ten blocks according to similarity in their whiteness index (WID). Three specimens within each block were stained by dentin exposure to TAP, and the remaining specimen was used as control to estimate color changes. Specimens were sectioned to separate tissues, and dentin and enamel colors were measured individually and after being recombined. Alterations in color (CIEDE2000 - ΔE00) and translucency parameter (TP) resulting from staining were estimated by color difference between stained and control specimens. The contribution of each tissue to the color change (CTCC) was also calculated. Non-sectioned stained specimens were bleached by applying sodium perborate on dentin, associated or not with 35% hydrogen peroxide on enamel. Color changes caused by bleaching procedures were estimated and data were analyzed using the paired t-test or Two-way repeated measures ANOVA. Results TAP caused more pronounced changes in dentin, but enamel color was also affected. Both protocols presented a similar ΔE00, and dentin showed the greater color change. After exposure to TAP, we observed a reduction in WID; WID values were the same for bleached and control specimens regardless of protocol. We found no significant effect of substrate and bleaching technique on TP. Enamel played a more critical role in color changes caused by either staining or bleaching procedures. Conclusion Enamel color played a greater role on tooth color changes than dentin. External and internal bleaching association did not improve bleaching effect on specimens stained with TAP.


Introduction
Tooth color results from the combination between dentin and enamel individual color and optics characteristics. 1 Being more chromatic, dentin is the main dental tissue affecting overall tooth color; 2,3 however, enamel surface characteristics affect light transmission, modifying tooth color. 3 Enamel is a chromatic translucent tissue that modifies the underlying dentin appearance, 1,4,5 so changes within the color or translucency of both of these dental hard tissues may cause tooth discoloration.
Despite the high success of root canal therapy, endodontic interventions may cause tooth discoloration not only due to dental trauma and pulp necrosis, but also due to some filling materials, sealers, and intracanal medications. [6][7][8] Triple antibiotic paste (TAP) was used for a long time as an intracanal medication.
However, this medication was reported to entail significant tooth discoloration 9,10 attributed to the presence of minocycline, 11,12 which lead researchers to recommend its replacement by amoxicillin to avoid discoloration. 13 Internal bleaching is a relatively safe and conservative treatment for discoloring endodontically treated teeth. It consists of applying the bleaching agent closer to dentin, as this tissue is strongly related to color changes. Each tissue (dentin and enamel) contribution to overall tooth color changes in bleaching procedures remains controversial. 1,3,14,15,16 Studies reported that enamel opacity increase due to the action of hydrogen peroxide is the major responsible for bleaching effect, 1,17,18 indicating that associating both internal and external bleaching procedures could optimize bleaching effect. 19 Considering that, this study aimed to evaluate the effect of the association between external and internal tooth bleaching on color changes in dentin and enamel, individually or recombined, previously stained with TAP. Our null hypotheses were that (1) associating external and internal bleaching techniques would not affect bleaching effectiveness and (2) Enamel and dentin contribution to overall tooth color change are not different.

Methodology Specimen preparation
Forty enamel-dentin specimens (6x6x2 mm thickness) were sectioned from bovine incisors using a water-cooled low-speed saw and a double-sided diamond disk (#7020; KG Sorensen, Barueri, SP, Brazil

Baseline measurements
Specimens initial color was measured with a portable spectrophotometer (Easyshade Compact V5, Vita-Zahnfabrik, Bad Säckinge, Germany) positioned on enamel surface. As the spectrophotometer tip diameter (6 mm) was similar to specimens surface dimensions, no index was used. Color was measured in triplicate, with specimens placed over a white background (L* = 94.5; a* = -0.9; and b* = 2.9).
The parameters L* (lightness), a* (red-green axis), and b* (yellow-blue axis), defined by the International Commission on Illumination (Commission Internationale de L'Eclairage -CIE), were recorded and used to calculate whiteness index (WID) according to the following formula: 21 WID=0.551×L -2.324×a-1.1×b Specimens which WID values ranged more than the standard deviation from specimen averages were replaced. Then, they were separated into ten blocks (n=4) according to similarity in their WID values.
Staining procedure Three specimens of each block were randomly allocated to undergo staining procedures; the remaining were used as control to estimate color changes caused by the procedure. The dentin surface opposite to buccal surface was exposed to TAP (ciprofloxacin, metronidazole, and minocycline) J Appl Oral Sci. 2021;29:e20200511 3/9 and the other faces were protected with wax. Before staining procedure, the exposed dentin was acidetched with 35% phosphoric acid for 15 s, rinsed, and air-dried. Then, TAP was mixed, applied onto the exposed dentin, and left undisturbed for 15 days. After this period, wax was removed and specimens were washed in running water and kept in distilled water at 37°C during one week for rehydration. Control specimens were kept in distilled water throughout staining period.
One stained specimen and the control specimen were sectioned to separate enamel from dentin using a diamond saw (Extec; Enfield, CT, USA) in a cutting machine (Isomet low-speed, Buehler; Lake Bluff, IL, USA). The color of each tissue was individually measured as above described, and WID values were calculated by placing the spectrophotometer tip on either enamel or dentin buccal surface (not exposed to TAP). Tissues were also recombined by applying  showed the more pronounced color change, without differences between enamel and combined substrates.  Table 3 shows the contribution of each tissue to  Data analyzed by two-way repeated measured ANOVA.Distinct letters (uppercase for lines, lowercase for columns) indicate significant differences at Tukey`s test (p < .05). WID -whiteness index.     (Figure 3). Figure 4 shows ΔTP results.
After the staining procedure, one-way repeated measure ANOVA found no significant difference among substrates (p=.140). Two-way repeated measures

Discussion
Both bleaching techniques evaluated in this study reached similar ΔE 00 values for enamel, dentin, or combined tissues and changed WID values only in combined tissues, confirming our first null hypothesis.
We found combined specimens to present close ΔE 00 and WID values to those measured in enamel, suggesting that color change within enamel had a greater effect on combined tissues ultimate color. This is confirmed by the fact that enamel showed higher CTCC values than dentin, rejecting our second null hypothesis.
As most studies in this subject assess bleaching effect on teeth without approaching individual effects on enamel and dentin, 24-27 the exact mechanism explaining peroxides action on each tooth substrate bleaching has not yet been fully elucidated. 5,18,23 Although both substrates present similar compositions, differences within their inorganic and organic components ratio modify their interaction with light, such as reflection, transmission, refraction, and scattering. 4 We separated specimens hard dental tissues after staining and bleaching procedures to assess color changes within each substrate both separately and in recombination. A prior study has employed this method 1 and calculated color changes by comparing different specimens. This procedure requires that only specimens with similar colors at baseline are used to calculate color change.
In that study, CTCC was calculated by comparing color differences between specimens from a same tooth (premolar). 1 In our study, specimens were divided into ten blocks (corresponding to n) with four specimens each (corresponding to the number of treatments), all with similar WID values at baseline. Color changes were calculated based on color differences between specimens within the same group. Although this method does not enable an exact color change measurement, it does allow us to estimate these changes and compare each tissue effect on the overall tooth color.
Tooth bleaching is a dynamic process not fully elucidated. Peroxides and their subproducts react with both dentin and enamel to yield whiter teeth.
Recently, peroxides bleaching effect on dentin was proved to be associated with peroxide interaction with phosphoproteins or, more specifically, with benzene ring oxidation on aromatic amino acid complexes. 28,29 Some studies report that enamel increased opacity due to its organic content oxidation contributes to tooth bleaching. 23 However, both internal and external bleaching techniques caused no clinically significant changes in enamel translucency parameter (TP) values, as their averages were lower than the acceptability threshold (TP=4.33). 30 We also verified no significant ΔTP values for dentin and recombined tissues. In fact, data on TP presented high variability and seemed unreliable, which may be explained by the dental spectrophotometer used. The Easyshade was developed to read teeth color, which contains yellow and yellow-red colorations. 31 Although this device has been used to calculate the TP of dental materials, 32-34 placing a thin specimen (≈ 1.0 mm) over a black background results in a grayish color, compromising Easyshade's ability to accurately measure specimens color.
After staining procedure, dentin presented the highest ∆E 00 values and the greatest changes within WID. This may be explained by the fact that triple antibiotic paste (TAP) was only applied on dentin inner surface while specimens surrounding walls and enamel surface were protected with wax, simulating discoloration caused by an endodontic treatment using TAP. Minocycline (a component of TAP) is an antibiotic derived from tetracycline that binds to calcium and form an insoluble complex, resulting in a darker substrate. 11 Before applying TAP, dentin was etched to remove the coarse smear layer from flattening procedures performed with SiC sandpapers.
We expected no significant reduction in dentin calcium content, as the 35% phosphoric acid application for 15 s over dentin only demineralizes approximately 2.0 µm of this tissue. 35 Considering that, we expected more pigment within the dentin matrix and an increased bleaching effect, as a darker substrate tends to achieve better bleaching outcomes. Interestingly, enamel played a greater role on overall color changes than dentin, besides reaching the lowest color change values. This may be justified by the spectrophotometer tip, which was placed on enamel during combined specimens color measurement. Unlike dentin, no overlay substrate modified enamel color, and even a slight change within this tissue modified the combined J Appl Oral Sci. 2021;29:e20200511 8/9 specimen color, which is supported by the fact that enamel color changes values are closer than dentin values to those found in combined specimens.
Although tooth dentin is thicker than enamel, this study used both tissues at the same thickness (1 mm), resulting in specimens with an overall thickness of 2 mm, which may help explain the lack of color change differences between the two bleaching techniques.
Thin tissues might enable peroxide and its subproducts to penetrate throughout the entire specimen, causing peroxide application on enamel to have no significant effect on reactive oxygen specimens availability in either tissue. The 1-mm thickness of bovine incisors dentin was a limitation of this study; using thicker dentin would hinder specimen thickness standardization due to the high variability in pulpal chamber volume of bovine incisors.
Besides overall color change, analyzing modifications within each parameter is crucial to understand staining and bleaching effects on color. As expected, the staining procedure reached lower lightness values than those measured in control specimens. Regardless of the protocol, bleaching procedure increased lightness in dentin, but reached moderate L* values in enamel.
We found no significant ΔTP; yet, the highest L* after bleaching procedures may be related to the white background reduced visualization, indicating an increase in enamel opacity. After staining, we verified that specimens were more red and yellow, especially dentin. These findings confirm TAP ability to increase dentin chromaticity 27 and minocycline ability to reach enamel, modifying its color (increased yellowness).
Dental bleaching reduced yellowness in both dentin and enamel, as expected. In turn, peroxide reduced dentin redness but increased enamel a* values.
Although a* has the greatest effect on WID, the two bleaching protocols reached higher WID values than those of dentin and enamel control.
The methodology employed in this study allows us to estimate individual color changes in each hard dental tissue and dentin and enamel contributions to the overall color change. Besides the aforementioned limitations, our methodology is unable to measure the real color change caused by the staining and bleaching procedures, and causes some tissue loss during specimen sectioning. Another limitation is that applying an optical solution, such as glycerin, between tissues before measuring recombined specimens might fail in reproducing enamel-dentin junction, affecting light interaction with tooth structure.

Conclusions
Our study found that enamel color significantly affects tooth color changes even when discoloration is caused by dentin staining. Additional application of a bleaching agent on enamel plays no role in improving internal bleaching efficacy.

Acknowledgments
This study was financed in part by the Coordination