Separate contribution of enamel and dentine to overall tooth colour change in tooth bleaching

Separate contribution of enamel and dentine to overall tooth colour change in tooth bleaching

Journal of Dentistry, 2011-11-01, Volume 39, Issue 11, Pages 739-745, Copyright © 2011 Elsevier Ltd

Summary

Objectives

The aim of this study was to evaluate the separate contribution of enamel (E) and dentine (D) to the colour change of tooth which subjected to 10% carbamide peroxide (CP) gels using a novel recombined enamel–dentine (Recombined-ED) study model.

Methods

120 enamel–dentine (ED) samples (four homogeneous premolar ED from each patient; total = 30 × 4 ED) were involved in the present study. Two homogeneous ED samples were bleached with 10% CP and the other two ones were stored in artificial saliva for one, two or four weeks. After treatment, four kinds of layers were prepared from each four homogeneous ED samples by removing enamel or dentine part: bleached-enamel (BE), bleached-dentine (BD), control-enamel (CE) and control-dentine (CD). Initial and final colour records of samples were taken with a spectrophotometer in CIELab system. The contribution of enamel/dentine to the colour change of tooth (CTCC) was calculated by measuring the colour difference Δ E between two different enamel–dentine combinations (Δ E between BE/BD and CE/BD for enamel; Δ E between BE/BD and BE/CD for dentine). Translucency parameter (TP) was obtained by calculating the colour difference between enamel on black and white backings.

Results

ED and recombined-ED were significantly correlated in L * a * b * values both for unbleached samples and bleached samples. Bleaching resulted in a significant colour change (Δ E ) of E, D and ED samples. The TP of BE was significantly lower than that of CE. The CTCC of enamel was significantly higher than that of dentine all through the time points.

Conclusions

Enamel played a more important role than dentine in tooth bleaching due to the changes in translucency and colour.

Introduction

Tooth bleaching has gained popular acceptance in recent years, especially after the introduction of night-guard vital bleaching. Nowadays this technique has been recognized as an efficacious and safe method to treat discoloured teeth.

Numerous scientists and dentists have investigated the efficacy of tooth bleaching in the laboratory and clinic. Although most studies have demonstrated teeth experienced good whitening effects immediately and several months after bleaching treatments, the exact optical mechanism of tooth bleaching has not been fully revealed yet. It has been proved that bleaching agents lighten the tooth by penetrating into enamel and dentine. The optical properties of a tooth are influenced by both enamel and dentine, and tooth colour is the result of diffuse reflectance from the inner dentine through the outer translucent enamel layer.

Although the opinion that both enamel and dentine influenced the colour of teeth has been approved by lots of studies, whether enamel or dentine contributed more to the tooth colour change during tooth bleaching still remains unclear. Some researchers believed that the colour change of the bleached teeth mainly resulted from alterations in the colour of subsurface dentine. However, others argued that the majority of colour change of tooth crowns after bleaching was because of the colour and translucency change in enamel.

Moreover, most of studies compared the contribution of enamel and dentine through calculating respective colour changes. However, the simple comparison of respective colour changes would be not enough to determine the contribution of translucent enamel and underlying dentine for they could influence each other and act together on the tooth colour. Therefore, the optical interactions between enamel and dentine should be mostly retained in the investigation of the function of enamel and dentine.

In the present study, we developed a novel recombined enamel–dentine model to study the separate contribution of enamel and dentine to the tooth colour change in tooth bleaching. The hypothesis was that enamel might contribute more than dentine in tooth bleaching due to the masking effects of enamel on the underlying dentine.

Materials and methods

Sample preparation and bleaching

Thirty pairs of intact premolars were obtained from orthodontic departments and then stored in 0.1% thymol solution at room temperature until required.

Four enamel–dentine (ED) samples were prepared from the labial surface of each pair of tooth by means of a low-speed saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) allowed a homogeneous distribution amongst the experimental groups with respect to baseline colour values of the ED specimens. Make sure the colour difference among every four homogeneous samples did not exceed 0.9, which was reported as the mean colour difference between contralateral natural teeth. The thickness of enamel and dentine was mostly reserved (total ≈ 2 mm × 3 mm × 4 mm, average thickness of enamel = 0.88 ± 0.07 mm), and homogeneous ED samples were trimmed to the same thickness. The labial and pulpal surfaces of each ED specimen were serially ground flat with water-cooled SiC paper 500–2000 grits and then polished with cloth and diamond polishing paste (1–0.5 μm). After preparation, the specimens were stored in artificial saliva.

The flow chart of treatments was shown in Fig. 1 . Two of each four homogeneous ED specimens were firstly bleached with 10% carbamide peroxide (pH ≈ 6.8; Ultradent Products Inc., South Jordan, UT, USA) for 8 h per day, by covering the enamel surfaces with 1 mm thickness of bleaching gel. Then the bleaching gel was removed under running distilled water, and the specimens were individually kept in artificial saliva for the remaining 16 h. The other two were stored in artificial saliva (control) for 24 h per day. This procedure was repeated for 7 days, 14 days and 28 days with each n = 10 × 4 ED. During these cycles, the specimens were kept in a humid atmosphere at 37 °C and the artificial saliva was replaced daily. Baseline L * a * b * values of ED specimens were assessed according to the CIE-Lab system.

The flow chart of study.
Fig. 1
The flow chart of study.

Separation and recombination of enamel and dentine sections

Due to the limited size of the crown, the enamel–dentine unit could not be separated into enamel and dentine slabs perfectly. Therefore, careful removal of the enamel or dentine by grinding and polishing was necessary to obtain pure dentine or enamel slabs.

The above separation of each pair of teeth ended in four kinds of sections: bleached enamel (BE), bleached dentine (BD), control enamel (CE) and control dentine (CD). Recombined enamel–dentine specimens (Recombined-ED) were obtained by recombining different kinds of enamel and dentine sections (Enamel/Dentine: BE/BD, CE/BD, BE/BD and BE/CD).

To obtain recombined-ED samples, glycerol was firstly applied on the contact surfaces of the enamel and dentine sections, then enamel and dentine sections were impacted tightly by a consistent pressure. Make sure the gap between enamel and dentine was filled with glycerol to avoid great changes of refractive index caused by air.

Colour measurement

The colour coordinates ( L * , a * , b * ) of each specimen were measured with a spectrophotometer (Spectrascan PR650, Photo Research, CA, USA) for it was accurate and objective in colour measurement. A D65 illuminant was used with a 45-degree entrance angle and 0-degree observation angle geometry. Before the measurement, the spectrophotometer was calibrated according to the manufacturer’s protocol.

A circular area with 1.0 mm in diameter was measured at the middle third region of the specimen. The measurement was repeated three times for each specimen and the values were averaged to get the final reading. Wet cotton pellets were used to inhibit dehydration of samples.

The colour of D and ED samples were measured directly, and the colour of enamel was measured over an A3 coloured resin background (3M, St. Paul, MN, USA) with the medium of glycerol.

The colour differences were calculated between bleached ones and respective controls by the following expression:

ΔE=(ΔL2+Δa2+Δb2)1/2 Δ E = ( Δ L 2 + Δ a 2 + Δ b 2 ) 1 / 2
where Δ L * , Δ a * , and Δ b * are the respective differences in the L * , a * and b * colour parameters between two colours.

Translucency of a single enamel slab was expressed by translucency parameter (TP). The colour of enamel slab was firstly measured over a black background and a white background separately, and then translucency parameter (TP) was obtained by calculating the colour difference between enamel on the backings. Glycerol was also used between enamel sections and backgrounds.

TP=[(LBLW)2+(aBaW)2+(bBbW)2]1/2 TP = [ ( L B L W ) 2 + ( a B a W ) 2 + ( b B b W ) 2 ] 1 / 2
The subscript B refers to the colour coordinates of enamel slabs over the black background and the subscript W refers to those over the white background.

TP difference (ΔTP) was calculated by the following expression:

ΔTP=TPBleachedTPControl Δ TP = TP Bleached TP Control

Validation of recombined-ED model

To validate the efficacy of recombined-ED model, Pearson correlation test was used to discover the relationships between L * a * b * values of ED specimens and recombined-ED specimens both for bleached and unbleached specimens.

Calculation of contribution of enamel and dentine

After enamel–dentine samples being bleached, either enamel or dentine layers were replaced with non-bleached controls, and then the colour change Δ E of recombined-ED samples were calculated before and after replacement. The Δ E was considered to be able to indirectly reflect the influence of the replaced layers on the colour expression of bleached enamel–dentine samples. Therefore, an index was developed in the present study to describe respective roles of enamel and dentine in tooth bleaching, and it was calculated by measuring the colour difference Δ E between two different enamel–dentine combinations (Δ E between BE/BD and CE/BD for enamel; Δ E between BE/BD and BE/CD for dentine). The index was named the contribution of enamel/dentine to the colour change of tooth (CTCC).

The CTCC of enamel was calculated by measuring the colour difference between bleached enamel and control enamel over bleached dentine.

CTCCofenamel=[ΔL2(BE&BDCE&BD)+Δa2(BE&BDCE&BD)+Δb2(BE&BDCE&BD))]1/2 CTCC of enamel = [ Δ L ( BE & BD CE & BD ) 2 + Δ a ( BE & BD CE & BD ) 2 + Δ b ( BE & BD CE & BD ) 2 ) ] 1 / 2

The CTCC of dentine was calculated by measuring the Δ E between bleached dentine and control dentine under bleached enamel.

CTCCofdentine=[ΔL2(BE&BDBE&CD)+Δa2(BE&BDBE&CD)+Δb2(BE&BDBE&CD))]1/2 CTCC of dentine = [ Δ L ( BE & BD BE & CD ) 2 + Δ a ( BE & BD BE & CD ) 2 + Δ b ( BE & BD BE & CD ) 2 ) ] 1 / 2

Statistical analysis

We conducted a paired T -test to examine the colour and TP differences between bleached E, D and ED specimens and respective controls. T test was also used to compare the CTCC of enamel and dentine. P < 0.05 was considered significantly different.

Results

Before treatment, the L * a * b * values of ED specimens at three time points were detected no significant difference. The correlation test certified that the recombined-ED sample was highly correlated with original ED samples in L * a * b * values both for bleached and control ones ( P ≤ 0.01) ( Table 1 ), although the L * a * b * values were significantly different between them.

Table 1
Correlation test between ED and Recombined-ED.
Group Parameter ED Recombined-ED T -test Correlation test
P P r
Control L * 71.00 ± 3.09 68.17 ± 2.61 <0.01 <0.01 0.84
a * 1.35 ± 1.44 −0.57 ± 0.45 <0.01 <0.01 0.67
b * 19.89 ± 3.95 12.89 ± 3.70 <0.01 <0.01 0.91
Bleached L * 75.61 ± 3.43 71.27 ± 3.29 <0.01 <0.01 0.80
a * 0.49 ± 0.84 −0.70 ± 0.39 <0.01 0.01 0.55
b * 11.50 ± 3.51 6.02 ± 2.72 <0.01 <0.01 0.86
Significantly different at level of p = 0.01.

Bleaching treatment led to a significant colour change of bleached-ED and bleached-E specimens compared to control values ( P < 0.01, Table 2 ). For all bleached ED and E samples, significant increase of L * values and decrease of b * values could be observed, indicating a shift in the direction of more white and less yellow ( Figs. 2 and 3 ). Compared with controls, colour of bleached D samples was not significantly improved by bleaching agents except for the b * values at the second week. Significant overall colour change Δ E of bleached-ED samples were also detected after 7, 14, 28 days bleaching (8.91 ± 0.85, 10.51 ± 1.7 and 12.02 ± 1.54, respectively; P < 0.05). Δ E of ED and E samples were significantly higher than that of D samples ( Fig. 4 ).

Table 2
The L * a * b * values of ED, E and D samples at three time points.
1st week 2nd week 4th week
Bleached Control P Bleached Control P Bleached Control P
ED L * 71.48 ± 1.15 67.83 ± 1.40 <0.001 ** 73.68 ± 1.74 68.22 ± 1.41 <0.001 ** 75.77 ± 1.08 69.89 ± 0.95 <0.001 **
a * 1.61 ± 0.43 2.49 ± 0.35 <0.001 ** −0.03 ± 0.26 1.05 ± 0.28 <0.001 ** 0.90 ± 0.15 2.78 ± 0.66 <0.001 **
b * 12.84 ± 2.43 20.87 ± 2.20 <0.001 ** 12.03 ± 1.97 20.78 ± 3.59 <0.001 ** 11.55 ± 1.14 21.76 ± 0.83 <0.001 **
E L * 70.05 ± 2.82 64.68 ± 1.60 <0.001 ** 70.53 ± 2.10 65.23 ± 1.23 <0.001 ** 69.57 ± 1.23 64.07 ± 0.77 <0.001 **
a * −1.23 ± 0.12 −1.25 ± 0.23 0.749 −0.94 ± 0.29 −1.00 ± 0.30 0.423 −1.03 ± 0.31 −1.29 ± 0.22 0.001
b * 4.02 ± 0.88 10.16 ± 1.24 <0.001 ** 2.91 ± 1.67 9.12 ± 4.00 <0.001 ** 2.92 ± 0.59 10.13 ± 1.80 <0.001 **
D L * 72.82 ± 2.16 73.90 ± 2.45 0.053 77.52 ± 2.78 75.47 ± 2.26 0.153 79.96 ± 1.95 79.62 ± 4.03 0.689
a * −2.08 ± 0.55 −2.21 ± 0.34 0.219 −1.72 ± 0.33 −1.74 ± 0.27 0.679 −2.32 ± 0.59 −2.19 ± 0.41 0.150
b * 13.98 ± 2.31 14.17 ± 2.47 0.747 12.52 ± 3.11 14.79 ± 2.10 0.004 ** 14.27 ± 4.10 15.55 ± 4.07 0.051
** Significant different at level of p = 0.01.
Δ L * of E, D and ED samples with time.
Fig. 2
Δ L * of E, D and ED samples with time.
Δ b * of E, D and ED samples with time.
Fig. 3
Δ b * of E, D and ED samples with time.
Δ E of E, D and ED samples and ΔTP of enamel with time.
Fig. 4
Δ E of E, D and ED samples and ΔTP of enamel with time.

Besides colour change, the TP values of bleached-E samples were sharply decreased in the period 0–14 days and were significantly different from that of controls ( P < 0.05).

The contribution of enamel/dentine to tooth colour change (CTCC) at three time points (day 7, 14, and 28) of the bleaching period were presented in Fig. 5 . The CTCC of E samples were consistently higher than that of dentine all through the treating period ( P < 0.01).

The CTCC of enamel and dentine at three time points.
Fig. 5
The CTCC of enamel and dentine at three time points.

Discussion

To allow for direct measurements of the optical changes of enamel and dentine with bleaching, careful separation was performed on the ED samples. As expected, pronounced changes of L * and b * values were observed in E and D samples, which reflected the increase of lightness and reduction in yellowness by bleaching treatment. This was consistent with the results of former researches, which demonstrated both enamel and dentine could be lightened by bleaching agents in different degrees. Moreover, the results also demonstrated that enamel experienced larger colour improvement than dentine, indicating enamel was easier to be bleached in direct contact with bleaching gel.

As we know, once a light falls on tooth surfaces, a multitude of interactions between enamel and dentine, such as transmission, reflection, scattering and refraction, may occur simultaneously. Separation of single enamel and dentine from a tooth would obviously detach these interactions. Therefore, in the present study we developed a novel enamel–dentine model by recombining enamel layer and dentine layer which were cut from bleached or unbleached tooth specimens. The method of “separation and recombination” has been successfully utilized before in two former studies to evaluate bleaching effects. Wiegand et al. obtained single enamel and dentine samples by grinding and polishing and then recombined them into enamel–dentine samples to measure separate and combined colour parameters. The similar pattern has been also adopted in an in vitro study aiming to bleach tetracycline-stained rat teeth by attaching a rat tooth to the human enamel layer.

The thickness of enamel has been proved to be highly correlated with its translucency. Previous studies revealed that the natural enamel varies greatly in thickness among individuals and types of teeth. In the present study, be respect to original thickness of enamel, we did not create an uniform thickness of enamel but keep original thickness of enamel almost unchanged (despite the thickness of 200 μm by necessary abrasion). The varied thickness of enamel would reflect the real covering effect of enamel on the dentine.

Through recombining enamel and dentine, the model mostly preserved the optical interactions between them, and encouraged us to investigate the optical changes of combined ED samples after separation of enamel and dentine. The optical contact between E samples and D samples was improved by an interfacing layer of glycerol with a refractive index ( n ) of approximately 1.5, which approached that of enamel ( n ≈ 1.65). Obviously this model would not perform like non-separated tooth samples and the results showed that the colour parameters ( L * , a * , b * ) were different between original ED and recombined-ED. However, the correlation tests showed that ED samples and recombined-ED samples were highly correlated although the absolute values of L * a * b * values were different. The results suggested that this model could provide a relatively reliable study model for investigating the mechanism of tooth bleaching.

Vieira and Ma et al. reported that the bleaching procedure significantly changed the enamel translucency, making it more opaque. In the enamel–dentine system, along with the decrease in enamel translucency, more light was reflected within the enamel, leading to more light reflected to the human eye. And more importantly, enamel acted as a light filter for dentine. The present study indicated that, when bleaching resulted in a decrease in enamel translucency (which means “more opaque”), less light would fall on dentine and less light from dentine would be reflected to human eye. Consequently, the influence of dentine colour on tooth colour would be decreased. In other words, the object would seem lighter.

Enamel consisted of large amount of inorganic materials and very small amount of organic phase such as protein and water. The decrease in translucency of enamel sample would be related to partial removal of mineralized tissues and organic matrix, which might be affected by the etching and oxidization of bleaching gels. A study carried out by Li et al. showed the density of enamel was detected significantly decreased after treatment with 30% hydrogen peroxide (HP) using μ-CT. Jiang et al. analysed Raman scattering and laser-induced fluorescence of enamel subjected to 30% HP, and found that HP may have adverse effects on the mineral and the organic matter of human tooth enamel. As the matter was destroyed by HP, the distance among enamel crystals increased and thus the distribution of enamel crystals was less compact than before, which could increase the refractive index of enamel.

The background was also an important aspect to consider. A translucent material placed against two distinct different coloured backgrounds gave two different visual expressions. The light went through the translucent material and fell on the background. Then the light returned to the observer, carrying colour information both of the translucent layer and the background. In the present study, results demonstrated that the colour of dentine was less affected by bleaching agents. This result would be partly owing to the consumption of bleaching agents when penetrating through enamel.

The index “CTCC”, integrating the colour and translucency change of tooth and simplifying the complicated optical interactions between enamel and dentine, successfully assessed the contribution through measuring final colour expression of different combinations of ED samples. The results of the present study showed that the contribution (CTCC) of enamel was higher than that of dentine all through the time points. Compared with dentine, enamel functioned in tooth bleaching not only by the colour changes, but also by the decrease of its translucency. Although tooth colour has been proved to be mainly determined by dentine colour, overall tooth colour change during tooth bleaching was strongly influenced by enamel. Taking all the previous information into consideration, it could be said that enamel and dentine colour as well as enamel translucency determine the colour of teeth.

It has been pointed out that the results were condition dependent. Most of the time enamel and dentine are both treated simultaneously in vivo and the optical changes are both acting together. Additionally, variations in the age of tooth samples and the initial colour changes of teeth caused by grinding and polishing would also influence the final results. Therefore, the results of present study could not fully reflect the real situation of intra-tooth changes in clinical tooth bleaching process. Within the limitation of the present study and based on the results of CTCC, it could be speculated that the teeth with more transparent enamel might be easier to be whitened than the teeth with less transparent enamel.

Conclusions

This finding indicated that both enamel and dentine contributed to the tooth colour change and enamel played a more important role than dentine in tooth bleaching.

Acknowledgements

This work was supported by the Natural Science Foundation of China ( No. 81071190 ), the Youth Chenguang Project of Science and Technology of Wuhan City (No. 200950431186 ), the Fundamental Research Funds for the central Universities ( No. 4103003 ) and the Self-Research Program for Doctoral Candidates of Wuhan University .

a These authors contributed equally to the work.

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Separate contribution of enamel and dentine to overall tooth colour change in tooth bleaching Xiao Ma , Rong Li , Yue Sa , Shanshan Liang , Lili Sun , Tao Jiang and Yining Wang Journal of Dentistry, 2011-11-01, Volume 39, Issue 11, Pages 739-745, Copyright © 2011 Elsevier Ltd Summary Objectives The aim of this study was to evaluate the separate contribution of enamel (E) and dentine (D) to the colour change of tooth which subjected to 10% carbamide peroxide (CP) gels using a novel recombined enamel–dentine (Recombined-ED) study model. Methods 120 enamel–dentine (ED) samples (four homogeneous premolar ED from each patient; total = 30 × 4 ED) were involved in the present study. Two homogeneous ED samples were bleached with 10% CP and the other two ones were stored in artificial saliva for one, two or four weeks. After treatment, four kinds of layers were prepared from each four homogeneous ED samples by removing enamel or dentine part: bleached-enamel (BE), bleached-dentine (BD), control-enamel (CE) and control-dentine (CD). Initial and final colour records of samples were taken with a spectrophotometer in CIELab system. The contribution of enamel/dentine to the colour change of tooth (CTCC) was calculated by measuring the colour difference Δ E between two different enamel–dentine combinations (Δ E between BE/BD and CE/BD for enamel; Δ E between BE/BD and BE/CD for dentine). Translucency parameter (TP) was obtained by calculating the colour difference between enamel on black and white backings. Results ED and recombined-ED were significantly correlated in L * a * b * values both for unbleached samples and bleached samples. Bleaching resulted in a significant colour change (Δ E ) of E, D and ED samples. The TP of BE was significantly lower than that of CE. The CTCC of enamel was significantly higher than that of dentine all through the time points. Conclusions Enamel played a more important role than dentine in tooth bleaching due to the changes in translucency and colour. 1 Introduction Tooth bleaching has gained popular acceptance in recent years, especially after the introduction of night-guard vital bleaching. Nowadays this technique has been recognized as an efficacious and safe method to treat discoloured teeth. Numerous scientists and dentists have investigated the efficacy of tooth bleaching in the laboratory and clinic. Although most studies have demonstrated teeth experienced good whitening effects immediately and several months after bleaching treatments, the exact optical mechanism of tooth bleaching has not been fully revealed yet. It has been proved that bleaching agents lighten the tooth by penetrating into enamel and dentine. The optical properties of a tooth are influenced by both enamel and dentine, and tooth colour is the result of diffuse reflectance from the inner dentine through the outer translucent enamel layer. Although the opinion that both enamel and dentine influenced the colour of teeth has been approved by lots of studies, whether enamel or dentine contributed more to the tooth colour change during tooth bleaching still remains unclear. Some researchers believed that the colour change of the bleached teeth mainly resulted from alterations in the colour of subsurface dentine. However, others argued that the majority of colour change of tooth crowns after bleaching was because of the colour and translucency change in enamel. Moreover, most of studies compared the contribution of enamel and dentine through calculating respective colour changes. However, the simple comparison of respective colour changes would be not enough to determine the contribution of translucent enamel and underlying dentine for they could influence each other and act together on the tooth colour. Therefore, the optical interactions between enamel and dentine should be mostly retained in the investigation of the function of enamel and dentine. In the present study, we developed a novel recombined enamel–dentine model to study the separate contribution of enamel and dentine to the tooth colour change in tooth bleaching. The hypothesis was that enamel might contribute more than dentine in tooth bleaching due to the masking effects of enamel on the underlying dentine. 2 Materials and methods 2.1 Sample preparation and bleaching Thirty pairs of intact premolars were obtained from orthodontic departments and then stored in 0.1% thymol solution at room temperature until required. Four enamel–dentine (ED) samples were prepared from the labial surface of each pair of tooth by means of a low-speed saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) allowed a homogeneous distribution amongst the experimental groups with respect to baseline colour values of the ED specimens. Make sure the colour difference among every four homogeneous samples did not exceed 0.9, which was reported as the mean colour difference between contralateral natural teeth. The thickness of enamel and dentine was mostly reserved (total ≈ 2 mm × 3 mm × 4 mm, average thickness of enamel = 0.88 ± 0.07 mm), and homogeneous ED samples were trimmed to the same thickness. The labial and pulpal surfaces of each ED specimen were serially ground flat with water-cooled SiC paper 500–2000 grits and then polished with cloth and diamond polishing paste (1–0.5 μm). After preparation, the specimens were stored in artificial saliva. The flow chart of treatments was shown in Fig. 1 . Two of each four homogeneous ED specimens were firstly bleached with 10% carbamide peroxide (pH ≈ 6.8; Ultradent Products Inc., South Jordan, UT, USA) for 8 h per day, by covering the enamel surfaces with 1 mm thickness of bleaching gel. Then the bleaching gel was removed under running distilled water, and the specimens were individually kept in artificial saliva for the remaining 16 h. The other two were stored in artificial saliva (control) for 24 h per day. This procedure was repeated for 7 days, 14 days and 28 days with each n = 10 × 4 ED. During these cycles, the specimens were kept in a humid atmosphere at 37 °C and the artificial saliva was replaced daily. Baseline L * a * b * values of ED specimens were assessed according to the CIE-Lab system. Fig. 1 The flow chart of study. 2.2 Separation and recombination of enamel and dentine sections Due to the limited size of the crown, the enamel–dentine unit could not be separated into enamel and dentine slabs perfectly. Therefore, careful removal of the enamel or dentine by grinding and polishing was necessary to obtain pure dentine or enamel slabs. The above separation of each pair of teeth ended in four kinds of sections: bleached enamel (BE), bleached dentine (BD), control enamel (CE) and control dentine (CD). Recombined enamel–dentine specimens (Recombined-ED) were obtained by recombining different kinds of enamel and dentine sections (Enamel/Dentine: BE/BD, CE/BD, BE/BD and BE/CD). To obtain recombined-ED samples, glycerol was firstly applied on the contact surfaces of the enamel and dentine sections, then enamel and dentine sections were impacted tightly by a consistent pressure. Make sure the gap between enamel and dentine was filled with glycerol to avoid great changes of refractive index caused by air. 2.3 Colour measurement The colour coordinates ( L * , a * , b * ) of each specimen were measured with a spectrophotometer (Spectrascan PR650, Photo Research, CA, USA) for it was accurate and objective in colour measurement. A D65 illuminant was used with a 45-degree entrance angle and 0-degree observation angle geometry. Before the measurement, the spectrophotometer was calibrated according to the manufacturer's protocol. A circular area with 1.0 mm in diameter was measured at the middle third region of the specimen. The measurement was repeated three times for each specimen and the values were averaged to get the final reading. Wet cotton pellets were used to inhibit dehydration of samples. The colour of D and ED samples were measured directly, and the colour of enamel was measured over an A3 coloured resin background (3M, St. Paul, MN, USA) with the medium of glycerol. The colour differences were calculated between bleached ones and respective controls by the following expression: ΔE=(ΔL2+Δa2+Δb2)1/2 Δ E = ( Δ L 2 + Δ a 2 + Δ b 2 ) 1 / 2 where Δ L * , Δ a * , and Δ b * are the respective differences in the L * , a * and b * colour parameters between two colours. Translucency of a single enamel slab was expressed by translucency parameter (TP). The colour of enamel slab was firstly measured over a black background and a white background separately, and then translucency parameter (TP) was obtained by calculating the colour difference between enamel on the backings. Glycerol was also used between enamel sections and backgrounds. TP=[(LB−LW)2+(aB−aW)2+(bB−bW)2]1/2 TP = [ ( L B − L W ) 2 + ( a B − a W ) 2 + ( b B − b W ) 2 ] 1 / 2 The subscript B refers to the colour coordinates of enamel slabs over the black background and the subscript W refers to those over the white background. TP difference (ΔTP) was calculated by the following expression: ΔTP=TPBleached−TPControl Δ TP = TP Bleached − TP Control 2.4 Validation of recombined-ED model To validate the efficacy of recombined-ED model, Pearson correlation test was used to discover the relationships between L * a * b * values of ED specimens and recombined-ED specimens both for bleached and unbleached specimens. 2.5 Calculation of contribution of enamel and dentine After enamel–dentine samples being bleached, either enamel or dentine layers were replaced with non-bleached controls, and then the colour change Δ E of recombined-ED samples were calculated before and after replacement. The Δ E was considered to be able to indirectly reflect the influence of the replaced layers on the colour expression of bleached enamel–dentine samples. Therefore, an index was developed in the present study to describe respective roles of enamel and dentine in tooth bleaching, and it was calculated by measuring the colour difference Δ E between two different enamel–dentine combinations (Δ E between BE/BD and CE/BD for enamel; Δ E between BE/BD and BE/CD for dentine). The index was named the contribution of enamel/dentine to the colour change of tooth (CTCC). The CTCC of enamel was calculated by measuring the colour difference between bleached enamel and control enamel over bleached dentine. CTCCofenamel=[ΔL2(BE&BD−CE&BD)+Δa2(BE&BD−CE&BD)+Δb2(BE&BD−CE&BD))]1/2 CTCC of enamel = [ Δ L ( BE & BD − CE & BD ) 2 + Δ a ( BE & BD − CE & BD ) 2 + Δ b ( BE & BD − CE & BD ) 2 ) ] 1 / 2 The CTCC of dentine was calculated by measuring the Δ E between bleached dentine and control dentine under bleached enamel. CTCCofdentine=[ΔL2(BE&BD−BE&CD)+Δa2(BE&BD−BE&CD)+Δb2(BE&BD−BE&CD))]1/2 CTCC of dentine = [ Δ L ( BE & BD − BE & CD ) 2 + Δ a ( BE & BD − BE & CD ) 2 + Δ b ( BE & BD − BE & CD ) 2 ) ] 1 / 2 2.6 Statistical analysis We conducted a paired T -test to examine the colour and TP differences between bleached E, D and ED specimens and respective controls. T test was also used to compare the CTCC of enamel and dentine. P < 0.05 was considered significantly different. 3 Results Before treatment, the L * a * b * values of ED specimens at three time points were detected no significant difference. The correlation test certified that the recombined-ED sample was highly correlated with original ED samples in L * a * b * values both for bleached and control ones ( P ≤ 0.01) ( Table 1 ), although the L * a * b * values were significantly different between them. Table 1 Correlation test between ED and Recombined-ED. Group Parameter ED Recombined-ED T -test Correlation test P P r Control L * 71.00 ± 3.09 68.17 ± 2.61 <0.01 <0.01 0.84 a * 1.35 ± 1.44 −0.57 ± 0.45 <0.01 <0.01 0.67 b * 19.89 ± 3.95 12.89 ± 3.70 <0.01 <0.01 0.91 Bleached L * 75.61 ± 3.43 71.27 ± 3.29 <0.01 <0.01 0.80 a * 0.49 ± 0.84 −0.70 ± 0.39 <0.01 0.01 0.55 b * 11.50 ± 3.51 6.02 ± 2.72 <0.01 <0.01 0.86 Significantly different at level of p = 0.01. Bleaching treatment led to a significant colour change of bleached-ED and bleached-E specimens compared to control values ( P < 0.01, Table 2 ). For all bleached ED and E samples, significant increase of L * values and decrease of b * values could be observed, indicating a shift in the direction of more white and less yellow ( Figs. 2 and 3 ). Compared with controls, colour of bleached D samples was not significantly improved by bleaching agents except for the b * values at the second week. Significant overall colour change Δ E of bleached-ED samples were also detected after 7, 14, 28 days bleaching (8.91 ± 0.85, 10.51 ± 1.7 and 12.02 ± 1.54, respectively; P < 0.05). Δ E of ED and E samples were significantly higher than that of D samples ( Fig. 4 ). Table 2 The L * a * b * values of ED, E and D samples at three time points. 1st week 2nd week 4th week Bleached Control P Bleached Control P Bleached Control P ED L * 71.48 ± 1.15 67.83 ± 1.40 <0.001 ** 73.68 ± 1.74 68.22 ± 1.41 <0.001 ** 75.77 ± 1.08 69.89 ± 0.95 <0.001 ** a * 1.61 ± 0.43 2.49 ± 0.35 <0.001 ** −0.03 ± 0.26 1.05 ± 0.28 <0.001 ** 0.90 ± 0.15 2.78 ± 0.66 <0.001 ** b * 12.84 ± 2.43 20.87 ± 2.20 <0.001 ** 12.03 ± 1.97 20.78 ± 3.59 <0.001 ** 11.55 ± 1.14 21.76 ± 0.83 <0.001 ** E L * 70.05 ± 2.82 64.68 ± 1.60 <0.001 ** 70.53 ± 2.10 65.23 ± 1.23 <0.001 ** 69.57 ± 1.23 64.07 ± 0.77 <0.001 ** a * −1.23 ± 0.12 −1.25 ± 0.23 0.749 −0.94 ± 0.29 −1.00 ± 0.30 0.423 −1.03 ± 0.31 −1.29 ± 0.22 0.001 b * 4.02 ± 0.88 10.16 ± 1.24 <0.001 ** 2.91 ± 1.67 9.12 ± 4.00 <0.001 ** 2.92 ± 0.59 10.13 ± 1.80 <0.001 ** D L * 72.82 ± 2.16 73.90 ± 2.45 0.053 77.52 ± 2.78 75.47 ± 2.26 0.153 79.96 ± 1.95 79.62 ± 4.03 0.689 a * −2.08 ± 0.55 −2.21 ± 0.34 0.219 −1.72 ± 0.33 −1.74 ± 0.27 0.679 −2.32 ± 0.59 −2.19 ± 0.41 0.150 b * 13.98 ± 2.31 14.17 ± 2.47 0.747 12.52 ± 3.11 14.79 ± 2.10 0.004 ** 14.27 ± 4.10 15.55 ± 4.07 0.051 ** Significant different at level of p = 0.01. Fig. 2 Δ L * of E, D and ED samples with time. Fig. 3 Δ b * of E, D and ED samples with time. Fig. 4 Δ E of E, D and ED samples and ΔTP of enamel with time. Besides colour change, the TP values of bleached-E samples were sharply decreased in the period 0–14 days and were significantly different from that of controls ( P < 0.05). The contribution of enamel/dentine to tooth colour change (CTCC) at three time points (day 7, 14, and 28) of the bleaching period were presented in Fig. 5 . The CTCC of E samples were consistently higher than that of dentine all through the treating period ( P < 0.01). Fig. 5 The CTCC of enamel and dentine at three time points. 4 Discussion To allow for direct measurements of the optical changes of enamel and dentine with bleaching, careful separation was performed on the ED samples. As expected, pronounced changes of L * and b * values were observed in E and D samples, which reflected the increase of lightness and reduction in yellowness by bleaching treatment. This was consistent with the results of former researches, which demonstrated both enamel and dentine could be lightened by bleaching agents in different degrees. Moreover, the results also demonstrated that enamel experienced larger colour improvement than dentine, indicating enamel was easier to be bleached in direct contact with bleaching gel. As we know, once a light falls on tooth surfaces, a multitude of interactions between enamel and dentine, such as transmission, reflection, scattering and refraction, may occur simultaneously. Separation of single enamel and dentine from a tooth would obviously detach these interactions. Therefore, in the present study we developed a novel enamel–dentine model by recombining enamel layer and dentine layer which were cut from bleached or unbleached tooth specimens. The method of “separation and recombination” has been successfully utilized before in two former studies to evaluate bleaching effects. Wiegand et al. obtained single enamel and dentine samples by grinding and polishing and then recombined them into enamel–dentine samples to measure separate and combined colour parameters. The similar pattern has been also adopted in an in vitro study aiming to bleach tetracycline-stained rat teeth by attaching a rat tooth to the human enamel layer. The thickness of enamel has been proved to be highly correlated with its translucency. Previous studies revealed that the natural enamel varies greatly in thickness among individuals and types of teeth. In the present study, be respect to original thickness of enamel, we did not create an uniform thickness of enamel but keep original thickness of enamel almost unchanged (despite the thickness of 200 μm by necessary abrasion). The varied thickness of enamel would reflect the real covering effect of enamel on the dentine. Through recombining enamel and dentine, the model mostly preserved the optical interactions between them, and encouraged us to investigate the optical changes of combined ED samples after separation of enamel and dentine. The optical contact between E samples and D samples was improved by an interfacing layer of glycerol with a refractive index ( n ) of approximately 1.5, which approached that of enamel ( n ≈ 1.65). Obviously this model would not perform like non-separated tooth samples and the results showed that the colour parameters ( L * , a * , b * ) were different between original ED and recombined-ED. However, the correlation tests showed that ED samples and recombined-ED samples were highly correlated although the absolute values of L * a * b * values were different. The results suggested that this model could provide a relatively reliable study model for investigating the mechanism of tooth bleaching. Vieira and Ma et al. reported that the bleaching procedure significantly changed the enamel translucency, making it more opaque. In the enamel–dentine system, along with the decrease in enamel translucency, more light was reflected within the enamel, leading to more light reflected to the human eye. And more importantly, enamel acted as a light filter for dentine. The present study indicated that, when bleaching resulted in a decrease in enamel translucency (which means “more opaque”), less light would fall on dentine and less light from dentine would be reflected to human eye. Consequently, the influence of dentine colour on tooth colour would be decreased. In other words, the object would seem lighter. Enamel consisted of large amount of inorganic materials and very small amount of organic phase such as protein and water. The decrease in translucency of enamel sample would be related to partial removal of mineralized tissues and organic matrix, which might be affected by the etching and oxidization of bleaching gels. A study carried out by Li et al. showed the density of enamel was detected significantly decreased after treatment with 30% hydrogen peroxide (HP) using μ-CT. Jiang et al. analysed Raman scattering and laser-induced fluorescence of enamel subjected to 30% HP, and found that HP may have adverse effects on the mineral and the organic matter of human tooth enamel. As the matter was destroyed by HP, the distance among enamel crystals increased and thus the distribution of enamel crystals was less compact than before, which could increase the refractive index of enamel. The background was also an important aspect to consider. A translucent material placed against two distinct different coloured backgrounds gave two different visual expressions. The light went through the translucent material and fell on the background. Then the light returned to the observer, carrying colour information both of the translucent layer and the background. In the present study, results demonstrated that the colour of dentine was less affected by bleaching agents. This result would be partly owing to the consumption of bleaching agents when penetrating through enamel. The index “CTCC”, integrating the colour and translucency change of tooth and simplifying the complicated optical interactions between enamel and dentine, successfully assessed the contribution through measuring final colour expression of different combinations of ED samples. The results of the present study showed that the contribution (CTCC) of enamel was higher than that of dentine all through the time points. Compared with dentine, enamel functioned in tooth bleaching not only by the colour changes, but also by the decrease of its translucency. Although tooth colour has been proved to be mainly determined by dentine colour, overall tooth colour change during tooth bleaching was strongly influenced by enamel. Taking all the previous information into consideration, it could be said that enamel and dentine colour as well as enamel translucency determine the colour of teeth. It has been pointed out that the results were condition dependent. Most of the time enamel and dentine are both treated simultaneously in vivo and the optical changes are both acting together. Additionally, variations in the age of tooth samples and the initial colour changes of teeth caused by grinding and polishing would also influence the final results. Therefore, the results of present study could not fully reflect the real situation of intra-tooth changes in clinical tooth bleaching process. 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