The null hypothesis was partially proven, since the application of the varnishes did not change either the pH, the concentration of lactic acid or most trace metals studied.
The age of the children included ranged from 4 to 12 years, a population in which caries prevention is common (21). The varnishes used contained 5% sodium fluoride, equivalent to 22,600 ppm or 1.19 M of fluoride, and calcium phosphate in two chemical forms: CPP-ACP and fTCP. In vitro studies have demonstrated their preventive and remineralizing capacity as they generate supersaturated calcium and phosphate solutions in biofilm and saliva (16),(24),(25).
In vulnerable populations, such as infants and children, saliva is the perfect diagnostic medium due to its non-invasive collection, and easy handling and storage (26). A wide range of saliva biomolecules are related to the physiological state and provide useful data on oral and systemic diseases. We selected pH, lactic acid and some trace elements in total unstimulated saliva as biomarkers of the predisposition to caries and the ability of varnishes to modify the risk (2),(10),27. Reports (27) have found differences in saliva composition depending on whether there is caries or not, but found no difference between stimulated and unstimulated saliva in children, or according to sex or the dental status.
The baseline pH value was between 7.6 and 7.7, although the patients were at high or extreme risk of cavities, and the value did not change significantly throughout the study in any group. Although some reports (28) (29) have recorded lower pH values and observed significant differences in pH in unstimulated saliva in children without versus children with early childhood caries (7.20 vs. 6.07) (30), other studies recorded a wider range than ours in salivary pH values in children with active caries (6.20–7.90) and found no correlation between pH and caries activity (30),(31). Therefore, the static measurement of salivary pH is of little use in assessing the risk of caries (32) because, within 20–40 minutes, saliva neutralizes pH variations caused by sugary foods and the activity of microorganisms (2),(33), while it is the acidification of the pH of the biofilm in the area of the lesion that determines the generation of tooth decay and correlated with a high risk of caries (2),(16),(33),(34).
There are few studies of lactic acid concentrations in children’s total saliva. Fidalgo et al. 2013 (35) and Pereira et al. 2019 (27) detected a higher concentration of lactate and other organic acids (acetate and n-butyrate) in the saliva of children with caries. We found higher baseline lactic acid values than did other studies (27), probably because our patients were at are high and extreme risk of caries, lactic acid concentrations were not significantly reduced in any group throughout the study.
The role of trace elements present in saliva on caries remains unclear (36). Of the trace elements measured in our study, Ca, P, F, Mg, Zn and Cu are, in some way, related to tooth mineralization. However, their saliva concentrations do not always reflect the degree of tooth demineralization/remineralization, caries or its risk (29),(37). It might be thought that concentrations of Ca and P, the main components of tooth hydroxyapatite, would be increased in saliva during tooth demineralization of the tooth, although some reports have found an inverse relationship between caries and salivary calcium (31),(38),(39) and phosphate (38) levels, while other studies found no relationship (39).
The application of calcium phosphate varnishes should reflect an increase in the concentrations of these two ions in children's saliva, as has been observed in vitro. Cochrane et al. 2014 (40) and Shen P et al. 2011 (19), determined in vitro release of calcium, phosphate and fluoride ions in five varnishes (MI Varnish Clinpro White Varnish, Emanel Pro, Bifluorid 5, and Duraphat) and found a greater cumulative release of the ions by MI Varnish. The calcium concentrations observed in our study did not increase significantly after varnish application because saliva was collected 3 months after each application and there is no cumulative in vivo effect of in vitro studies, since the varnish remains in situ only for up to 24 hours as it is eliminated by chewing, salivary flow, rubbing by the cheeks and tongue and oral hygiene (41). Phosphorus was significantly reduced at 12 months in the control group but not in the varnish groups.
Baseline fluoride levels in all groups were around 0.05 ppm and there was a non-significant increase, which was higher in the MI group than in the other groups, reaching 0.0920 ± 0.0402 ppm. Initial concentrations were similar to those described by Sekhri et al. 2018 (36) in caries-free groups and higher than those described by Dehailan et al. 2017 (42) and Rechman et al. 2018 (43). As one of the exclusion criteria was consumption of fluoridated running water, the higher levels of baseline salivary fluoride of study children might be due to consumption of external sources of fluoride, such as bottled waters. In fact, a study that analyzed the fluoride content of 20 bottled water brands marketed in the area of origin of the study children found fluoride concentrations between 0.05 ppm and 0.95 ppm (44). Increases in salivary fluoride seem to predict increases in fluoride content of dental plaque fluid, implying that both would be good indicators of intraoral levels of fluoride (45).
There is no consensus on the significance of Cu, Zn and Mg levels in relation to caries. Brookes et al. 2003 (46) suggested Cu2+ might have a direct protective effect on enamel dissolution, although other studies (32),(37),(47),(48),(49) suggest a high Cu level is observed in patients with caries and comes from destroyed hydroxyapatite crystals. We found high salivary Cu levels in our sample with a basal dmfs between 18.33 ± 10.07 and 32.34 ± 19.93 and the levels did not fall in any group throughout the study. We also found high zinc levels. Elevated Cu and Zn values could reflect the action of saliva's antioxidant systems, as both ions act as co-enzymes of superoxide dismutase (49). Sejdini et al. 2017(50) suggested Mg promotes caries resistance, so children with low Mg concentrations would have a high caries index. This might explain the low levels of Mg recorded in our children, which are similar to those recorded by Rajesh et al. 2015 (51). While the application of varnishes kept Mg levels stable during the follow-up, there was a fall in levels in the control group.
Saliva is the main remineralizing agent and generally protects the teeth. Knowledge of its composition may help detect deficiencies in patients at high risk of caries and thus provide individualized treatments that reverse the risk (47). The parameters studied in our work did not serve to define a risk situation or monitor the treatment with fluoride varnishes and calcium phosphate. Continuous salivary flow and the influence of the diet, hormonal status, hydration status and anxiety levels have on salivary composition, and the short half-life of varnishes in the oral environment may have been influencing factors. It is estimated that the half-life of CPP in plaque is 124.8 minutes and that casein is hydrolyzed by salivary bacteria in a similar time (11). Therefore, varnishes must release their ions in a relatively short time and the initial high ion load is diluted over time.
Although there is no difference in the composition of some elements between plaque and saliva (sodium, ammonium, potassium, magnesium and chlorine), the metabolic activity of bacterial plaque can vary the inorganic composition between it and saliva (50). It may be useful to measure the composition of bacterial plaque and not the composition of saliva to determine the risk of caries and their monitoring.
Controlled, randomized clinical trials often have a problem due to patient losses throughout the study. Likewise, children at high or extreme risk of caries often miss appointments due to socioeconomic or other factors.
In conclusion, the quarterly application of two calcium phosphate varnishes, MI Varnish and Clinpro White Varnish, for 12 months, did not change pH, lactic acid concentrations or most trace metals in the saliva of children at high and extreme risk of caries, and therefore were not useful in in detecting a high risk of cavities or monitoring their follow-up.