The main goal of this study was to investigate the tooth-whitening effect of nano-HAP, used as an ingredient in toothpaste. Compared with the negative control group, the nano-HAP groups showed significantly higher △E values due to the increased L* values and the decreased a* and b* values, indicating that the nano-HAP toothpaste could make teeth appear brighter, less red and less yellow. These optical alterations can be explained by the increased diffuse light reflection and reduced light transmission through the tooth caused by the HAP particles adhering to the enamel[19, 27]. After three applications, the △E mean values of the 10 wt% and the 1 wt% nano-HAP groups were 4.47 and 2.55, respectively. Considering that △E > 3.7 can be recognized by human eyes, the color changes of the 10 wt% nano-HAP group were visually perceivable. The whitening performance of the 10 wt% nano-HAP toothpaste seemed better than that of a 44.4 wt% nano-HAP aqueous suspension, of which the △E value was 3.30 after the third use. Several randomized clinical trials reported that the △E values of commercial abrasive toothpastes and peroxide-based toothpastes ranged from 2.25 to 4.46 with a clinical application period of up to 90 days[29, 30]. Therefore, it is considered that the 10 wt% nano-HAP toothpaste in our study had a satisfying postbrushing tooth-whitening effect. It is worth mentioning that the negative control group exhibited a slight whitening effect compared with the water group, with the mean values of △E fluctuating around approximately 1. The abrasive particles adhering to the enamel surface could also contribute to light scattering to some extent.
Understanding the relationship between the nano-HAP concentration and tooth-whitening effect is crucial for optimizing the efficiency of nano-HAP toothpaste. To analyze the role of the concentration more accurately, we did not make any structural modifications to the nano-HAP. To avoid interference from the ingredients of commercial toothpastes, none of them were incorporated into the toothpaste. Nano-HAP has been applied to some commercial toothpastes at concentrations up to 10 wt%, which was considered optimal for remineralization of early enamel caries. Within this concentration, nano-HAP would not have any significant systemic exposure via the oral mucosa or cytotoxicity after a 48-hr exposure. Therefore, a concentration of 10 wt% was also chosen as the upper limit in the present study.
The null hypothesis that the HAP concentration does not influence the whitening effect of HAP toothpaste could be rejected, as we found a significant main effect of the concentration on the △E, △L, △a, and △b values. Compared with the 1 wt% nano-HAP group, the 10 wt% nano-HAP group exhibited significantly higher △E values throughout the observation period. This finding could be explained by the SEM images. After HAP 1–3, more adhered HAP crystallites and agglomerates were observed in the 10 wt% nano-HAP group than in the 1 wt% group. The enamel coverage area was quantitatively calculated and shown to be increased from 10 to 30% with the HAP concentration increasing from 1 to 10 wt%. More coverage may result in an increase in the reflection of light on the HAP layer and thereby lead to the increase in the △L values. This finding appeared to be well substantiated by a previous study, in which the increase in the amount of HAP in toothpaste resulted in the enhancement in the degree and rate of brightness. At the same time, more coverage could decrease the light transmission through enamel and dentin, which could lower the a* and b* values.
A significant main effect of the repeated application on the color changes was found in the current study. For both nano-HAP groups, the △E values increased significantly with the reapplication, which could be caused by the increased enamel coverage and the size change of the adherent particles from nanosized to microsized. These changes in the particle size appeared to be well substantiated by the previous studies, in which it was confirmed that the photoelectric characteristics and maturation time enabled the nano-HAP to be gathered within microsized conglomerates[33, 34]. A previous study reported that there was a maximal adhering load on enamel. The tooth color does not change much when adhering saturation has been achieved. However, we did not find this saturation within three HAP reapplications. In our study, nano-HAP was applied to the enamel by tooth brushing. The loosely attached nano-HAP particles may be brushed away by mechanical friction.
From a clinical view of point, the adherent HAP agglomerates are exposed to mechanical forces in the patient’s mouth after brushing. HSF is often used to create force comparable to the mechanical stress caused by the movement of the lips and checks[35, 36]. For both nano-HAP groups, statistical reductions in the △E values were observed after the HSF application. Nevertheless, the △E mean value of the 10 wt% was still higher than 4, suggesting that a tooth whitening effect of the nano-HAP toothpaste is still to be expected under a loaded condition. After HSF application, most of the microsized agglomerates were removed from the enamel surfaces, while the nanosized agglomerates remained in place, which could be explained by their higher surface charges and stronger electrostatic forces[22, 33].
Our study has some particular strengths. First, we took a further step toward real clinical situations. We applied nano-HAP toothpaste to enamel by tooth brushing. The whitening effect of HAP toothpaste has been confirmed. Second, the interference factors were strictly controlled. For instance, tooth dehydration could decrease enamel translucency and increase luminosity, making the tooth falsely appear whiter[37, 38]. Compared with previous studies[17, 18, 24], in which the samples were air-dried before the color measurement, we measured the tooth color in a liquid environment, which could avoid the interference of dehydration in tooth color assessment. Accurate repositioning is a key factor for color measurement, as the teeth are multilayered, translucent and exhibit color transitions in all directions. However, the previously published studies did not address how the repositioning was achieved in their work[16, 18, 19]. To bridge this information gap, we invented a repositioning system by using 3D printing technology to enable accurate color measurement (Fig. 1).
Within the framework of laboratory tests, one tries to select the conditions in such a way that they can be transferred as well as possible to the clinical situation. Ideally, the tests would be carried out with human teeth. However, due to the success of prevention and modern filling materials, hardly any teeth are extracted in industrialized nations today that can be used for laboratory examinations. Although bovine enamel is somewhat more porous than human enamel, its chemical composition and surface properties are identical to those of human teeth. Therefore, we used bovine teeth in our study.
We used artificial saliva instead of human saliva. The statherin and proline-rich glycoproteins in human saliva could bond strongly to HAP and thereby might influence its tooth whitening effect. Moreover, human saliva has high variability of individual factors and complexity of its components. Therefore, human saliva was not chosen.
The findings of this study have to be seen in light of some limitations. First, although we confirmed for the first time the whitening effect of nano-HAP toothpaste and the significant main effect of its concentration on tooth color changes, the whitening effect of nano-HAP after a prolonged application period needs to be further proven. Second, our in vitro study cannot reflect the complexity of the oral environment. Oral pH fluctuations and temperature changes also influence nano-HAP adhesion behavior.