Our hypotheses were accepted:
- Adding theobromine to bleaching gels does not adversely affect dental surface roughness, microhardness, or colour change.
- Adding chitosan to bleaching gels does not adversely affect dental surface roughness, microhardness, or colour change.
Evaluation of surface roughness
In previous studies where surface roughness and microhardness measurements were evaluated after bleaching treatments, the different gel components used, the concentration of the agent, its pH, and the duration of application affected the surface roughness and microhardness values [20]. However, in these studies, where the measurements of microhardness and surface roughness are evaluated, the results are not compatible with each other.
Miranda et al. conducted a study in which they used bleaching gels containing 10% CP and 10% HP to investigate the effects of different bleaching gel content and application times on the enamel surface. Surface roughness analyses revealed that the gel containing 10% HP affected the surface roughness by disrupting the hydroxyapatite sequence in the enamel structure 4 weeks after the start of treatment, and the gel containing 10% CP affected the surface roughness 8 weeks after the start of treatment; however, it was emphasised that the increase in roughness was not sufficient for biofilm retention [21]. In an in vitro study by Faraini-Romano et al., the effect of agents with CP content of 10% and 22% and HP content of 38%, 18%, and 7.5% on surface roughness values were compared. In the study, there were no significant differences between the groups in terms of surface roughness values after the bleaching processes [22]. In contrast, in an in vitro study by Souza et al., bleaching gels containing 20–45% CP and 9.5–38% HP were compared, and the greatest increase in surface roughness was observed following treatment with a gel containing 38% HP, while the lowest increase was observed following treatment with a gel containing 20% CP [23].
Mohammed et al. evaluated the effect of different remineralising gels on the susceptibility to recolouration and surface roughness after bleaching treatment. Four different remineralising agents were applied to the enamel surface after a bleaching agent containing 10% CP was applied, and the results were compared with a control group. The group without a remineralised adhesive produced the highest surface roughness values, and it was concluded that the increase in surface roughness also increased the susceptibility to recolouring [24].
Pini et al. conducted a study aiming to evaluate the effect of adding chitosan to an HP-containing bleaching gel on tooth properties and bleaching effectiveness. The authors stated that there was no significant difference between the group treated with HP + 2% chitosan-containing gel and the control group. However, there was a significant increase in the surface roughness of the group treated with HP-containing gel [25]. Ozcetin et al. conducted a study comparing the effect of an experimental bleaching gel enriched with chitosan containing 6% HP and bleaching gels containing 25% and 40% HP on enamel surface roughness. There were no significant differences between the groups in terms of surface roughness. It was concluded that chitosan prevented the increase in surface roughness values without affecting the bleaching efficiency of the gel [26].
Wulandari et al. investigated whether tooth brushing with toothpaste containing theobromine prevented tooth discolouration caused by coffee consumption. It was determined that the acidic environment caused by chlorogenic acid and other types of acids in coffee reduces the crystal size and causes mineral loss on the hydroxyapatite surface. The remineralising effect of theobromine reduced the roughness and thus prevented discolouration [16].
According to the results obtained in the present study, ECP (Group 1) and EHP (Group 2) gels reduced surface roughness values via the remineralising effect of theobromine and chitosan, while FGM Whiteness Perfect household bleaching gel (Group 4) reduced the surface roughness values due to its sodium fluoride content. It is believed that the increase in surface roughness observed in the group treated with BioWhiten ProHome household bleaching gel (Group 3) was due to the citric acid content of the gel. Shehri et al., in a study in which they investigated the effect of over-the-counter teeth bleaching products on enamel surface roughness, reported a significant increase in surface roughness in the group treated with 0.25% citric acid [27]. It was believed that the reason why the increase in tooth roughness was meaningless was due to nanohydroxyapatite, a remineralising agent contained in the gel.
Evaluation of microhardness
Fatima et al. conducted a study to evaluate the effect of a household bleaching agent containing 16% CP and an office bleaching agent containing 38% HP on enamel microhardness. They observed a decrease in microhardness values in both groups, but this decrease was meaningless [28]. Elfallah et al., conducted an in vitro study on bleaching gels to investigate the effect of tooth enamel on protein content and mechanical properties. Bleaching agents containing 16% CP and 35% HP were compared, and although there was no significant difference between the two groups in terms of microhardness, the decrease in microhardness was significant in both groups [29]. Maia et al. evaluated the effect of household bleaching gels containing 10% CP and 7.5% HP on enamel microsertiness and reported no significant difference between the groups. However, it has been reported that the gels containing 7.5% HP tend to reduce the enamel microhardness [30].
Attin et al. investigated how the addition of 0.5% fluoride to a household bleach gel containing 10% CP affected enamel microhardness and the time it took for microhardness to return to baseline values. Fluoride gel caused significantly less hardness loss than the fluoride-free gel. It was concluded that the time required to return to gel and basal values was significantly shorter [31]. There was a decrease in microhardness values in the teeth that underwent bleaching treatment; however, this decrease could be reversed with the use of a solution containing 0.05% fluoride [32].
Suryana et al. aimed to compare the effects of toothpaste containing theobromine and hydroxyapatite on enamel microhardness after soaking in a carbonated drink. The microhardness values in the theobromine group were found to be significantly higher than in the hydroxyapatite group [33].
Puangpanboot et al. evaluated the effect of chitosan on the remineralisation and microhardness of enamel and found that the microhardness of enamel increased significantly following chitosan application [34].
In our study, microhardness values increased in all groups, and no significant differences were observed between VHN1, VHN2, or VHN3 values in the comparison between groups. However, in the intra-group comparison, there was a non-significant increase in microhardness values after bleaching in all groups, and the greatest increase was observed following treatment with ECP (Group 1). The increase in microhardness following ECP (Group 1) and EHP (Group 2) treatment was associated with chitosan and theobromine, which are natural remineralising agents found in the content of gels. We believe that this effect was due to nanohydroxyapatite in BIO (Group 3) treated with BioWhiten ProHome household bleaching gel and sodium fluoride in FGM (Group 4) treated with a household bleaching gel containing FGM Whiteness Perfect.
It has been observed that remineralising agents largely compensate for the decrease in microhardness that occurs after bleaching [35].
To observe the effect of application time on the morphological structure of enamel in bleaching treatments, Vilhena et al. applied a household bleaching agent containing 10% CP for 14, 21, and 28 days. They concluded that the application time of the gel did not have a significant effect on surface roughness; however, the changes in microhardness values were inversely proportional to the application times [36]. Dutra et al. applied home bleaching agents containing 10% and 16% CP for 14 days, 8 hours a day, to investigate the effect of gel concentration and application time on the enamel surface. It was concluded that there was a significant difference between the groups in terms of surface roughness and that surface roughness increased with CP concentration [37]. In our study, bleaching gels containing CP were applied for 4 hours a day and bleaching gels containing HP were applied for 1 hour a day for 14 days; however, no significant negative effects were observed on microhardness or surface roughness values.
Monterubbianesi et al. investigated whether the addition of nanohydroxyapatite could be a safe solution for teeth bleaching, without altering the microstructure or microhardness of the teeth. They compared an at-home bleaching gel containing 6% HP with the same gel enriched with nanohydroxyapatite. Although the household bleaching gel containing 6% HP produced slightly lower average microhardness values than the same gel enriched with nanohydroxyapatite, there was no significant difference between the two groups. However, it has been reported that nanohydroxyapatite largely preserves the enamel surface morphology and compensates for the increased surface roughness [38].
Arnaud et al. applied chitosan to the enamel during remineralisation after demineralisation and reported the lowest phosphate loss in the chitosan group. In addition, the groups were evaluated in terms of microhardness, and it was reported that the most favourable results were observed in the chitosan group [39]. In a study by Ruan et al., it was reported that the amelogenin-chitosan complex is effective in inducing enamel remineralisation and restructuring [40]. In a study evaluating the effect of an experimental bleaching gel enriched with chitosan containing 6% HP on enamel microhardness, Ozcetin et al. concluded that chitosan prevented the decrease in microhardness without affecting the bleaching effectiveness of the gel [26].
In an in vitro study investigating the effect of theobromine on surface microhardness, enamel sections taken from premolars were divided into four groups and treated with distilled water, 100 mg/L, 500 mg/L, or 1000 mg/L solutions. The treatments were applied for 15–80 minutes. After the microhardness test, an increase was observed in the microhardness of the samples treated with theobromine solution, and the highest increase was reported in the group treated with 1000 mg/L theobromine solution [41]. Another study by Taneja et al. compared theobromine and commercially available remineralising agents. They reported no significant difference following SEM-EDX analysis and stated that theobromine could be used as an alternative remineralising agent [42]. Farhad et al. initially investigated the effectiveness of theobromine on the remineralisation of enamel caries lesions by comparing it with 0.05% sodium fluoride solution and reported that the remineralisation effect of theobromine was significantly higher than sodium fluoride [43]. In contrast, Danaswari et al. reported that theobromine prevented a decrease in surface microhardness after bleaching application; they determined that theobromine reduces the increasing microhardness values [44].
Colour evaluation
High positive WID values indicate that the whiteness values of the samples are high, while low values indicate that the whiteness levels of the samples are low [45]. In our study, WID values measured during and at the end of the bleaching treatments increased significantly in all groups, and the highest increase was observed following treatment with FGM (Group 4). In our study, colour changes were evaluated according to CIEL*a*b* and ΔWID formulations, and effective changes in colour and whiteness were observed in all groups. A comparison of the ΔELab1 and ΔWID1 values revealed a difference between the values measured before the bleaching treatment and on the seventh day of bleaching. We made separate measurements on the seventh day of bleaching, that is, half of the total bleaching time, and at the end of the treatment to determine between which days the increase in ΔELab and ΔWID values was highest. A noticeable change occurred in all groups within the first 7 days, with ΔELab1 greater than 3.2 and ΔWID1 greater than 2.60.
Colour changes occurred in all groups during the first 7 days of treatment, and there was a significant difference between ΔELab1 and ΔELab2, but not between ΔELab2 and ΔELab3 values, and ΔELab4 values.
In the first 7 days of treatment, the whiteness change was high in all groups, and there was a significant difference in ∆WID1 and ∆WID2 but no significant difference in ∆WID2 and ∆WID3 and ∆WID4 values.
Lilaj et al. compared the effectiveness of home bleaching gels containing 10% and 16% CP and home bleaching gels containing 6% HP and found no significant difference in ∆WID values between treatment groups [46]. Costacurta et al. compared the efficacy of bleaching agents containing 10% and 22% CP and concluded that the ∆E and ∆WID values following treatment with either gel were similar [47].
Although there was a positive correlation between the ΔELab and ∆WID values in which colour change was evaluated in our study, the results were not compatible with each other. We believe that this was observed because the WID values are affected by each of the L*, a*, and b* values at different rates, and the a* value, which has the highest coefficient in the WID formulation, was significantly different between the groups. Consistent with our findings, Bernardi et al. evaluated the effects of bleaching agents containing 4% and 10% HP on colour change and whiteness index and found that the ∆WID values were higher for the gel with 10% HP content. They emphasised that the most important index for evaluating bleaching effectiveness is WID [48]. Likewise, in a study in which Pini et al. aimed to evaluate the effects of adding chitosan to an office bleaching gel containing 35% HP on tooth properties and bleaching effectiveness, they reported no significant difference between the gels with and without chitosan in terms of ΔE00 and ΔWID values [49]. However, the groups with the highest ΔE00 and ΔWID values supported our study results. Ozcetin et al. compared the bleaching efficacy of an experimental bleaching gel enriched with chitosan containing 6% HP and bleaching gels containing 25% and 40% HP. They found no significant differences between the groups. 26 In another study, Ozcetin et al. evaluated the bleaching efficacy and 3-month follow-up results of an experimental bleaching gel containing 6% HP enriched with chitosan and reported that the gel provided effective bleaching, and there was no significant return in tooth colour after 3 months [50].
No study in the literature has reported the use of chitosan and theobromine, which are used in many areas, such as dentistry and medicine, in bleaching applications, and our study is the first to evaluate this subject.
Within the limitations of this study, experimental bleaching gels enriched with chitosan and theobromine provided us with new alternatives to existing bleaching treatments and an opportunity to avoid the unwanted side effects of these treatments.
In our study, we believed that theobromine inhibited the effects of the HP and CP added in the experimental gels on increasing surface roughness and reducing microhardness in superficial enamel layers. Studies in the literature suggest that theobromine prevents the decrease in microhardness and reduces surface roughness via its remineralisation properties, and the results of our study are consistent with this data.
We believe that chitosan, which was used as an alternative to thickener and carrier synthetic polymers in our experimental gels, prevented the negative effects of bleaching treatments on the enamel surface due to its bioadhesive and remineralising properties. Furthermore, we believe that chitosan slows the release of HP and CP from gels to the external environment and increases the bleaching efficiency at low concentrations.
Limitations of this study include our in vitro evaluation of the experimental gel; we did not apply the maximum surface treatment during polishing of the enamel surface before the surface roughness measurements, and we used a low load and short application time for the microhardness measurements. Further in vitro and in vivo studies are needed to confirm the efficacy of experimental bleaching gels enriched with chitosan and theobromine.