Surveillance of salivary properties of pre-orthodontic patients in relation to age and sex

Saliva plays an important role in masticatory function and protection from dental caries. Although studies have been conducted on saliva properties, their results vary widely depending on population settings. Hence, this study was performed to evaluate the results of saliva properties in individuals who attended their first visit for orthodontic treatment. A total of 619 participants were included (387 females and 232 males; mean age: 14.6 years). We conducted oral examinations and saliva (stimulated) tests and evaluated the saliva flow rate, pH, and buffering capacity, along with bacterial culture. Saliva flow rate, pH, and buffering capacity were significantly higher in males than in females. However, the Streptococcus mutans score was significantly higher in females than in males even though oral hygiene was better in females. Significant positive correlations were found between age and saliva flow rate and S. mutans score. On the contrary, significant negative correlations were found between age and pH and buffering capacity. These results were similar to other studies where the target population was children or teenagers. Saliva properties of patients starting orthodontic treatment were almost the same as in populations of similar ages.

www.nature.com/scientificreports/ orthodontic appliances [20][21][22][23][24] . Presently, orthodontists try to address these problems through specific guidance on prevention and oral care protocols for each patient and by further adjusting their visit intervals 25 . Despite the availability of numerous studies about the properties of saliva, the data are scarce with respect to the population of patients starting orthodontic treatment. The goal of our research was to predict iatrogenic disorders that could be caused by bacterial infections during orthodontic treatment by performing specific tests at the first visit.

Materials and methods
Study design and ethics statement. This study used a cross-sectional design and was approved by the Independent Ethics Committee of Hiroshima University Hospital, Hiroshima, Japan (No. E-1039). Written informed consent was obtained from all the subjects following provision of a full explanation of the study. This study included subjects under the age of 18, in which case informed consent was obtained from the parent or legal guardian. All the methods were performed in accordance with the relevant guidelines and regulations. Study population. A total of 1,321 patients visited the Department of Orthodontics, Hiroshima University Hospital from April 2015 to December 2019. All patients received dental check-ups including plaque control record (PCR) performed by oral hygienists and underwent orthodontic examinations such as dental models, oral photos, lateral cephalometric records, and salivary tests at the first visit. We excluded patients from the final sample who lacked even one type of examination result, those who were on medicines such as antibiotics within 14 days leading up to the saliva test and who had congenital diseases including oligodontia. Thus, a final sample of 619 subjects (387 females, 232 males) was included. The subjects were classified by tooth alignment and malocclusion traits, such as arch length discrepancy (ALD), and antero-posterior, vertical, or horizontal aspects of malocclusion. Three orthodontists performed the analysis of the dental models and intraoral photos, and classified the malocclusions. With regard to ALD, subjects were classified according to the following criteria based on the amount of discrepancy; crowding < − 2 mm, − 2 mm < normal < 2 mm, and 2 mm < space arch. In the antero-posterior aspect of the malocclusion, subjects were classified as per the following criteria: with overjet; mandibular protrusion < 0 mm, 0 mm < normal < 4 mm, and 4 mm < maxillary protrusion. In the vertical aspect of the malocclusion, subjects were classified as per the following criteria: with overbite; open bite < 0 mm, 0 mm < normal < 4 mm, and 4 mm < deep bite. In the horizontal aspect of the malocclusion, subjects were classified according to premolar and molar crossbites. Saliva collection. Before sampling, subjects were asked to refrain from eating, drinking, smoking, and brushing their teeth for at least 2 h and to avoid using mouthwash and dentifrices. We collected the stimulated saliva samples while the subjects were seated on a dental chair in a relaxed state. Subjects were instructed to chew a tasteless gum contained in Checkbuf (Horiba, Kyoto, Japan). The saliva secreted in the first minute was spat out into a dental spittoon and stimulated saliva was collected in conical centrifuge tubes for 5 min by continued chewing. If the amount of saliva was less than 2 mL, the subjects continued to chew the tasteless gum until the total volume reached at least 2 mL. The collected saliva was immediately sent to a laboratory for subsequent examinations.
Saliva examinations. Saliva flow rate, the volume of the saliva secreted per min (mL/min), was calculated.
Saliva pH was measured with a digital pH metre. The buffering capability of saliva was estimated by the CAT-21Buf Risk Test of Saliva (Morita Co., Osaka, Japan) 26 . The mixture of saliva and acid load liquid was vigorously shaken, and the final pH was measured. S. mutans and Lactobacillus spp. were cultured for 48 h at 37 °C using the CRT caries risk test (Ivoclar Vivadent, Tokyo, Japan) and semi-quantitative evaluation was performed in four stages (score 1, 2 < 10 5 colony-forming units [CFU], score 3, 4 > 10 5 CFU). Candida albicans was cultured in CHROMagar Candida medium (CHROMagar, Paris, France) for 48 h at 37 °C and semi-quantitative evaluation was performed in four stages (score 0: colony not found, score 1: colonies were found [at least single colony]).

Statistical analysis.
All data were processed with BellCurve for Excel (Society Survey Research Information Co., Ltd, Tokyo, Japan). Descriptive statistics were used for all variables including standard, means, and frequency. In the comparison between females and males, the data were compared using the Mann-Whitney U test and Fisher's exact test. To consider the influence of age in each oral condition, the data were analysed using the Spearman's rank correlation coefficient and Kruskal-Wallis test. The regression line in the regression analysis was drawn using the least-squares method.

Results
The study subjects comprised 619 patients (387 females and 232 males; mean age 14.6 ± 9.9 years). Details of the distribution of the subjects in terms of age, sex, and malocclusions or dentofacial deformities are shown in Table 1.
Oral hygiene status was better in females than males and the variation was large in the younger generation. The average of PCR score was significantly higher (p = 0.0129) in males (50.7 ± 20.3%) than in females (47.1 ± 19.5%) (Fig. 1a). The overall mean PCR value was 48.44 ± 19.91% and ranged from 2 to 100% with large individual differences, especially among the younger generation. Spearman's rank correlation coefficient revealed that PCR had a statistically significant negative correlation with age (p < 0.001) and the value of the correlation coefficient (ρ) was -0.3033 (Fig. 2a). In other words, the older the subjects, the lower the PCR value.  (Fig. 1b). The overall mean saliva flow (mL/min) rate was 1.28 ± 0.66, ranging from 0.1 www.nature.com/scientificreports/ to 4.4. There was significant positive correlation with age (ρ = 0.1553, p < 0.001) (Fig. 2b). Salivary pH was significantly higher in male subjects (7.61 ± 0.29) than in females (7.50 ± 0.32) (p < 0.001) (Fig. 1c). In addition, salivary buffering capability was significantly higher in male subjects (6.32 ± 0.47) than in females (6.15 ± 0.58) (p = 0.0021) (Fig. 1d). There were large individual differences among the younger generations in salivary pH and buffering capability. Both salivary pH and buffering capability had significant negative correlations with age and the correlation coefficients were − 0.2586 (Fig. 2c) and − 0.0984, respectively (Fig. 2d).
Gender difference in salivary S. mutans. Bacterial culture tests revealed that the S. mutans score was significantly higher in the female (2.57 ± 1.32) oral cavity than in the male (2.29 ± 1.32) oral cavity (p = 0.0152) (Fig. 1e). In relation to age, the S. mutans score showed significant positive correlation (ρ = 0.1314, p = 0.0011) (Fig. 2e). The average age for each S. mutans score was compared using the Kruskal-Wallis test, and the average age for score 1 was significantly lower than that for scores 2, 3, and 4 (Fig. 3a). Although Lactobacillus spp. showed the same tendency as S. mutans, there were no statistically significant differences based on sex. The average Lactobacillus spp. score for males was 2.51 ± 1.27 and for females was 2.67 ± 1.18 (Fig. 1f). The Lactobacillus spp. score showed a positive correlation with age but there was no significant difference (ρ = 0.0736) (Fig. 2f). The average age for each Lactobacillus spp. score was compared using the Kruskal-Wallis test, and the average age for score 1 was significantly lower (p = 0.0184) than that for score 3 (Fig. 3b). In terms of the relationship between the two genera of bacteria, a positive correlation was found (ρ = 0.3383). The results of the Candida albicans culture test showed no significant differences based on sex and age (ρ = − 0.0108). Positive correlations were found between Candida albicans and S. mutans (ρ = 0.2211) and Lactobacillus spp. (ρ = 0.1470) but the results were not significant.

Discussion
Overall, males had poorer oral hygiene than females. However, their caries risk was lower based on the saliva flow rate, pH, buffering capability, and S. mutans count. We found that oral hygiene improved, and the saliva flow rate increased with age. In contrast, we found that pH, buffering capability, and bacterial counts were indicative of an increased caries risk.   27 . There are a few reports that found no significant difference between females and males for oral hygiene during orthodontic treatment 28 . However, several investigations, using full fixed appliances, have proposed that females have better oral hygiene conditions than males during orthodontic treatment 29,30 . Kudirkaite et al. reported that the 16-to 18-year-old patient-group had better oral hygiene than a younger group 30 . In the present study, based on the PCR index, the results of oral hygiene conditions reflected previous reports in terms of sex and age differences (Figs. 1a, 2a). This could be because the age groups of the evaluated population were similar.
Salivary properties. Saliva contains various antibacterial substances, namely lysozyme, lactoperoxidase, lactoferrin, immunoglobulin A, and mucin, in addition to water, electrolytes, and enzymes 31 . Thus, a reduced salivary flow might predispose to dental caries 32 . The specific timepoints were not uniform and we did not analyze the diurnal effects. Unstimulated salivary flow rate tends to increase from morning to night, much like diurnal variations in body temperature under control of the autonomic nervous system 33 . It has been reported that in females, unstimulated salivary flow rate and pH are higher during the ovulation period than during the menstrual period 33 . It was reported that saliva microbiome differed more between individuals than the diurnal variation 34 , and the pH differed greatly between day and night but does not fluctuate significantly among different timepoints during daytime 35 . Hence, we utilised a relatively stable stimulated saliva. Yeh et al. studied more than 1000 subjects (35 to 75 years) and reported a significant age-related decrease for both stimulated and unstimulated saliva. However, the trend was stronger in unstimulated saliva 4 . In elderly people over the age of 60, there were no sex differences in stimulated saliva flow rate 36 . In adults, the secretion of unstimulated saliva, as well as stimulated saliva, was significantly lower for females than for males. In females, the resting secretion rate negatively correlated with age. The buffer effect was also significantly lower in females and had a positive correlation with age. Therefore, with advancing age, females tend to catch up with males 6 . In a study conducted on various age groups, the secretion volume of both stimulated and unstimulated saliva secreted from the parotid gland was irrelevant regarding age 37 . Parvinen et al. reported that there was no significant influence of age on the stimulated saliva flow rate and the pH, and females had lower output than males 17 . On the contrary, other investigations on persons ranging in age from 21 to 93 years have insisted that the secretion rate of stimulated saliva increases with age 38 . Percival et al. have reported that the secretion rate of unstimulated saliva decreases with age, whereas stimulated saliva has no correlation with age 7 . In addition, they reported a tendency towards lower secretion rate for both unstimulated and stimulated saliva in females than in males, and found a negative correlation between the DMF index and the flow rates of unstimulated saliva, but no relationship to stimulated parotid saliva 7 . Evaluation of salivation volume of young children under 5 or 12 years reported that the flow rate of both unstimulated saliva and stimulated saliva showed no age-or sex-related differences 39,40 . Mazengo et al. reported that the mean salivary flow rate and buffer effect were slightly lower in females than males, and salivary flow rate was significantly lower in the 12-year-old group than the older age groups 10 . A study focusing on Japanese children aged from 5 to 12 years reported that at all ages, unstimulated saliva flow rate was more than double that of adults, and girls tended to have lower saliva volume than boys 5 . Taking into account that the average age of the population in this study was 14.6 years, the results that salivary flow rate, pH, and buffering capability were significantly higher in males than in females (Fig. 1b-d) are in agreement with the other studies. In addition, the significant positive correlation between saliva flow rate and ageing (Fig. 2b) was consistent with previous reports on populations similar to our study 41,42 . www.nature.com/scientificreports/ Bacteria cultural tests. The earlier S. mutans is detected in children, the higher the occurrence of dental caries 43 . In children between the ages of 2 and 5 years, S. mutans were detected in 43% while the detection frequency of Lactobacillus spp. was 11.5%. Despite the low correlations between the number of S. mutans or Lactobacillus spp. and diet in terms of sugar intake, there were highly significant correlations between caries incidence and the amount of bacteria in saliva 44 . The essential requirements, such as low pH, anaerobic environment, and access to carbohydrates, were necessary for sustained colonisation of Lactobacillus spp. in the human oral cavity 45 . Lactobacillus spp. are known to downregulate several virulence genes including acid tolerance genes such as aguD, and suppress growth and biofilm formation of S. mutans 46,47 . The ability of Lactobacillus spp. to inhibit S. mutans was more pronounced in clinical isolates from caries-free subjects 48 . The detection rate of S. mutans in saliva by the PCR method was higher in subjects aged 12 years old than in those aged 15 years old 49 . In our study, the significant increase in the number of S. mutans with age (Figs. 2e, 3a) seems to be due to the increase in the number of teeth associated with the eruption of permanent molars. Therefore, it cannot be concluded that caries risk is increasing just because of the increase in the number of cariogenic bacteria. Caries occurrence may be affected by diet rather than the prevalence of S. mutans 50 . Candida albicans correlated with advancing age by analysis of its protein in a study population with a mean age of 53.4 years 51 . A previous study focusing on children found no significant difference in the detection rate of Candida according to age or sex 52 . Likewise, in other saliva tests, Candida cultural tests show a concordance of the results due to population approximation.
In this study, we found that the salivary properties of pre-orthodontic patients had a very similar tendency to that of other studies that included healthy subjects. However, this study had the limitation of being a single time point cohort study. Despite this, our findings suggest that future studies of orthodontic patients may be applicable to other age-matched populations. In the future, we aim to build a preventive system to predict infectious diseases during orthodontic treatment and by expanding the population, adding other examinations such as microbiome analysis, and longer follow up.

Conclusions
In conclusion, among pre-orthodontic patients, males had worse oral hygiene than females; however, males had a lower risk of caries. With ageing, improved oral hygiene and an increased saliva flow rate were noted.