The present study indicated that tooth bleaching efficacy is partly positively correlated with the treatment time, prolonging the treatment time may lead to color regression, which is contrary to our hypothesis. There are essentially two factors that are of concern: (1) the concentration of H2O2 in the dental gel, (2) the total time of the procedure. Previous studies showed that with the assistance of cold plasma, even low concentrations of H2O2 (down to 6%) can result in an excellent tooth bleaching efficacy [11] without an unacceptable damage on tooth enamel [17]. Thus, this study focused on the operating time because opening mouth for long time is not only an additional load for the patients.

During the bleaching process, the appearance of the teeth is due to the complex interplay of the breaking of the long molecular chain of pigment stains, change of enamel surface roughness and enamel demineralization. A 5-min treatment results in 65% of the maximum color change. A 10-min treatment leads to approximately 90% of the maximum color change. A saturation of color change is likely achieved with a 15-min treatment. Further increase of treatment time may lead to color regression. This is in particular clear in the 35% H2O2 and plasma group. This finding is consistent with the so-called “saturation point” in teeth bleaching as reported in reference [18]. Beyond this “saturation point”, the porosity and brittleness of the enamel may increase (i.e. enamel damage) and the bleaching process must be stopped. The third 5-min treatment contributes little to the overall color change and may even be omitted in clinics.

In present study, 55% showed visible color change (ΔE* > 3.0) and 40% showed significant color change (ΔE* > 3.7) after a 5-min treatment. These percentages increase to 85% and 80%, respectively, after a 10 min treatment. There are, however, 10% of the teeth do not have visible color change even after 20 min treatment. This means combining H2O2 with cold plasma works majority of the time but is not effective for very single sample, as the bleaching efficacy depends on enamel and dentin structure of individual tooth and baseline of the tooth color to a certain extent.

The enamel microhardness in general decreases with the increase of treatment time, including in the naturally drying group and the negative control group. No significant differences on microhardness were observed within different treatment groups in each 5 min treatment period. On the other hand, enamel surface roughness in general increases with the increase of treatment time, with the first 5 min treatment contributing the most to the changes. These changes are attributed to the demineralization of enamel during the treatment process. The demineralization (erosion) patterns are related to the distribution of various enamel crystals (e.g. carbonated apatite, hydroxyapatite and fluorapatite) in the enamel rods (enamel prisms)[19]. The actual arrangement of the crystals in each enamel rod is rather complex. However, near the head of the enamel rod, the crystals are arranged parallel to the long axis of the rods. The central regions of the enamel rods are richer in carbonated apatite, which is more susceptible to acid demineralization than other crystallites found in enamel rods. Therefore, demineralization occurs preferentially in the central regions at the head of the enamel rods, leading to erosion patterns and thus rougher surface. The erosion then progresses along the central core, smoothing out the surface gradually, leading to the smoother surface at 20 min. This rough-smooth process repeat once the active agents been used.

No significant differences on surface roughness were observed within different treatment groups in each five-min treatment period, indicating the treatment time probably plays a more important role to demineralization than does the concentration of H2O2. The surface roughness results are further supported by the SEM data of teeth from the 35% H2O2 and plasma group: 5–15 min treatments result in surface roughness increase, while prolonged treatment of 20 min led to a smoother surface, which is similar to that of an untreated tooth surface. We believe this is actually caused by the extensive melt down of the enamel rods, which is completely different from the state of the teeth before the treatments.

Enamel is the hardest and most resilient tissue in the human body, which mainly composed of a hard mineral, carbonated hydroxyapatite (HA, Ca10 (PO4)6 (OH)2), packed at high density (95 wt% in mature enamel) [20, 21]. The main chemical elements are Ca, P and O. It is reported that HA is in contact with water, the following formula occurs [22]:

$$} \rightleftarrows }$$

(1)

$$}_ \left( }_ } \right)_ \left( }} \right)_ }_ \rightleftarrows 6}_}^}} + \, 2} -$$

(2)

$$} \rightleftarrows }$$

(3)

A small amount of HA dissolves, releasing calcium, phosphate and hydroxyl ions. As Fig. 8 shows, although there is no significant difference between 0 min, 5 min, 10 min and 20 min (p > 0.05), the atom amounts of Ca and P decreased within 10 min, this is in accordance with formula. This indicated that demineralization occurs, which is also in accordance with SEM results. The O increased within 10 min mainly because that there are large amounts of ROS generated after cold plasma treatment. As treatment prolong to 15 min, the hydrogen ions has been removed: H+ + OH− ⇄ H2O, so the O element decreased. However, the significant increase of Ca and P at 15 min are contrary to the formula. We speculate that enamel is mineralized layer by layer during growth and development, the new enamel surface explored after 15 min treatment.

Despite the similarity of the surface morphology, surface roughness and the atom amount of Ca, P and O between the teeth treated for 20 min and the untreated teeth, the microhardness is indeed different. This infers that teeth over-bleached may have a good appearance but the extensive melting of the enamel rods will eventually affect the strength of the teeth.

The tolerance of the temperature increase of pulp and bone tissue is limited. Zach and Cohen were the first to observe that a temperature increase of higher than 5.5 °C irreversibly damage the pulp tissue [18]. Other reports showed that the maximum pulp temperature increase is about 6 °C, and the acceptable value of bone temperature is below 42 °C [23, 24]. Several studies have proposed the use of thermocouples to evaluate tooth temperature directly during light irradiation for bleaching purpose [25,26,27,28]. K-type thermocouple was used in this study. The temperature increase was below 6 °C during a 20-min bleaching assisted by the plasma. This value is blow what was reported in our previous work [12], due to the fact that we measured the temperature change directly on teeth without applying dental gel. This value is also lower than that measured in LED bleaching for 30 s [24]. Other than mediating the reactive species between the plasma and the teeth surface, the dental gel may absorb the light to the tooth surface, and meanwhile serve as a good heat sink as it was replenished every 30 s during the treatment process, therefore reducing the internal pulp chamber temperature [29]. Actually, H2O2 gel was used to maintain humidness of tooth surface to avoid white plaque caused by dry and covered bleaching effect. The gel of a certain thickness can also buffer the directional heat irradiation from the plasma, averting local heating of the teeth.