The effect of nano silver fluoride, self-assembling peptide and sodium fluoride varnish on salivary cariogenic bacteria: a randomized controlled clinical trial

This study was conducted to assess the secondary aim of a parallel randomized, three arm, clinical trial comparing the effect of NSF, P11-4 and NaF varnish on remineralizing early white spot caries lesions in young adults. The study was conducted from December 2020 to August 2022 in the Pediatric Dentistry clinic of the Faculty of Dentistry, Alexandria University. Microbiological analysis was conducted in the microbiology lab, Faculty of Medicine, Alexandria University. Ethical approval was obtained from the Research Ethics Committee, Faculty of Dentistry, Alexandria University (#0086 − 11/2019). The objectives, risks and benefits of the study were explained to patients and signed informed consent was obtained from participants older than 18 years of age and from guardians of participants aged 10 to 18 years before participating in the study. Also, participants younger than 18 years of age assented to participate in the study. The study was conducted in full accordance with the Helsinki declaration. The trial was registered at ClinicalTrials.gov NCT04929509. Patients were given oral hygiene instructions about brushing twice a day especially before bedtime. The importance of proper diet in preventing caries was explained in detail. Six tubes of fluoridated toothpaste (Signal 2 with 1450 PPm sodium fluoride) and a brush were provided to each participant on the day of the intervention to be used throughout the whole study period. Thus, the 6 tubes were given once/ at the same time and they were meant to last the whole duration of the study as part of the standard basic oral hygiene procedure and this was for all groups. Patients were referred for treatment in the faculty clinics to provide care beyond the study scope.

Participants were enrolled if they were 10–24 years old which is the age of young adolescents and youth as described by the WHO [20], when there is high prevalence of non-cavitated caries lesions [21]. Participants were included if they had at least one visible white spot lesion (WSL) in the buccal surface of permanent teeth with ICDAS II score of 1, or 2 since bacteria colonize the demineralized areas of the tooth [22] and after they or their parents signed an informed consent form to participate in the study [23]. Patients were excluded if they received antibiotics within 1 month before saliva sample collection because it is a cause of dysbiosis and may affect bacterial growth in culture and if they had multiple cavitated carious lesions since salivary SM and LB counts are positively correlated with the decayed, missing and filled teeth (DMFT) score [24, 25].

This study addressed the secondary outcome of a trial where the primary outcome was remineralization of early white spot caries lesions in young adults after P11-4, NSF and NaF varnishes [26]. The sample size of the original study was based on remineralization of white spot lesions after applying P11-4 and NaF varnish and 22 were needed per group. To ensure that the present study was adequately powered, we also calculated the sample size required to assess the antibacterial effect based on 95% confidence level and 80% study power. During the study planning, no estimates of the agents’ salivary antibacterial effects could be found from in vivo studies. Therefore, we used estimates from in vitro studies represented by the mean (SD) percentages inhibition of bacterial adsorption to enamel surface by NSF and NaF (88.94 (31.32) and 41.06 (50.76)) [27], respectively, because bacterial counts are correlated with bacterial adsorption to enamel [28, 29]. Sample size was calculated to be 14 participants per group. Thus, the number of participants required for the primary outcome (n = 20 per group, increased to 22 patients to make up for loss to follow up) would ensure adequate power to asses the secondary outcome addressed in the present study.

Participants were randomly and equally assigned using a computer generated list of random numbers to one of the three arms receiving P11-4 (Curodont Repair), NSF varnish and NaF varnish (Duraflor) (See Fig 1). Allocation was performed by a trial independent individual. Allocation was done in blocks of 3 to insure balanced groups [30]. The group to which each patient was allocated was written on identical sheets of paper, folded and placed inside opaque envelopes carrying the patient’s name. A trial independent person kept the envelopes secure and opened them only at the time of giving each patient the designated regimen. Participants were blinded to the agent they received by removing the labels from the bottles. The investigator could not be blinded since the method of application differed in the three agents.

NSF varnish was prepared in the Faculty of Pharmacy labs, Alexandria University, according to Wei et al’s. method [31]. The synthesis of aqueous solution of AgNPs was carried out via the chemical reduction of silver nitrate (1 mL, 0.11 M) with sodium borohydride (0.3 mL, 0.8 M) and chitosan biopolymer (28.7 mL, 2.5 mg/mL) as a stabilizing agent. Sodium fluoride (10,147 ppm of fluorine) was incorporated at the end of the experiment.

After partial isolation with cotton rolls and saliva ejector, each tooth with WSL in the NSF group received two drops of NSF applied with a micro brush, equivalent to 10 mg of the solution [31]. The NSF solution was left in contact with the tooth surface for 2 min.

The P11-4 was applied on the WSL following the manufacturers’ instructions. The tooth was isolated using rubber dam then cleaned using sodium hypochlorite 2% for 20 s. The WSL was etched with phosphoric acid gel 35–37% for 20 s, then rinsed with water and dried. Then the Curodont Repair was applied on the tooth surface [32].

The third group received 5% NaF varnish (Duraflor^®). After partial isolation, the teeth with WSL were dried before the application of a very thin coat of the varnish, which was then allowed to dry for 10 s. The varnish hardens on contact with saliva or moisture [25]. Each agent was applied once at baseline.

Saliva samples were collected at baseline before the agents were applied, and after one, three and six months.

Saliva collection was done in the morning (9–11 am) and patients were asked to rinse their mouth with water five minutes before sampling. This was done in all study groups and repeated before each saliva sample was taken. No prophylaxis was done at home except brushing with the fluoridated toothpaste distributed to them. The following procedure was followed to collect whole saliva according to Fejerskov and Thylstrup [33]: The patient was instructed not eat or drink (except water) one hour before saliva collection, samples were collected at the same time of day for all participants, and the patient was seated in a relaxed position in an ordinary chair.

Unstimulated whole saliva samples [34, 35] were collected continuously for 5 min in sterile containers which were sealed immediately. The patient was asked to drool out saliva in a sterile container and 2- 3 ml of unstimulated whole saliva were collected. The containers were labelled and stored in an ice box set below 4^oC. Microbiological analyses were done within 45 min after sample collection [12]. Mitis Salivarius Agar (MSA) supplemented with potassium tellurite and Rogosa SL were used as culture media for isolation of salivary S. mutans and Lactobacilli, respectively. Saliva (1 mL) was diluted to 1:10 dilution by adding 9 mL of sterile saline solution and tenfold serial dilutions of saliva were made up to 10^− 6 before plating. Ten microliters of each dilution were spread on the surface of the plates, placed in an anaerobic jar with AnaeroGas Pack system and incubated at 37 °C for 4–5 days [30]. Counting of colonies was done automatically using TotalLab™ 1D software. Then, the count of the colonies was calculated by multiplying the number of colonies on the plate by the dilution factor expressed as the number of colony forming units per milliliter (CFU/mL) of saliva [30, 31]. The outcome variables were the log counts of S. mutans and Lactobacilli at baseline, 1, 3 and 6 months. Participants’ oral health practices such as use of fluoridated toothpaste and sugar consumption was calculated as quantitative variable based on the frequency of daily consumption of any sugar containing food or drink at least once per day and their socioeconomic background (age, sex) were recorded as confounders [36].

Median and Inter Quartile Range (IQR) were used for descriptive statistics. Log counts were calculated. Intention to treat analysis was used. The groups were compared regarding absolute and log bacterial count at each follow-up period using Kruskal Wallis test. Differences across time within each group with post-hoc comparison were assessed using Friedman test. Two multivariate linear regression models were developed to assess the effect of P11-4 and NSF compared to NaF on salivary S. mutans and Lactobacilli log count at baseline, 1, 3 and 6 months adjusting for age, sex, sugar intake frequency, using fluoridated toothpaste, and the number of white spot lesions. We also calculated the adjusted mean salivary S. mutans and Lactobacilli log counts in each group at each follow-up period. Significance level was set at P < 0.05. All tests were two tailed. Data were analyzed using SPSS for Macintosh version 28 [37]. (See Fig. 1).