In Vivo Evaluation of Antibacterial Toothpaste Efficiency and Patients' Satisfaction: a Double-blind Randomised Controlled Trial
Phase: NA
Conditions: Caries, Gingivitis
Sponsor: University of Bologna

In Vivo Evaluation of Antibacterial Toothpaste E7iciency and Patients' Satisfaction: 
a Double-blind Randomised Controlled Trial  
NCT05569850 
November, 20th 2024 
 
Study Protocol 
 
This was a single-site, double-blind RCT  that was conducted in the Dental Clinic of 
Bologna University (DIBINEM), Bologna, Italy in the period from November 2022 until July 
2023. Before initiating the trial, the study protocol was reviewed and approved by the 
University Ethics Committee (793/2022/SPER/AUSLBO). The trial was in accordance with 
Helsinki Declaration of Human Rights [21] and followed the CONSORT statement. The 
tested dentifrices included a newly introduced ZCT-, HAP- and KCit- 1450 ppm  sodium 
monoﬂuorophosphate containing (Experimental group; Mentadent PROTECT+ Carie, 
Mentadent, Unilever, Milan, Italy) and 1450 ppm NaF containing (Control group; AZ Multi-
Protezione Scudo Protettivo Famiglia) toothpastes.  
 
Sample size, recruitment and eligibility criteria 
The number of patients was determined based on the power analysis performed using 
data from a previously published study in order to assure a power of 80% for ﬁnding the 
statistically signiﬁcant diderences given the standard value of type I errors (0.05). Based 
on the outcomes of the power calculation (edect size f=0.2291667, at the level of α 0.05, 
and 1-β prob 0.80), the determined sample size was 92 patients. Considering the 
probability of loss to follow-up 10% of the minimum size per group was added, leading to 
the ﬁnal sample size of 100 patients.  
  
Potential patients were recruited through local advertisement or on-site, during routine 
dental check-ups. Study details were explained to the potential participants and only 
those who voluntarily signed the informed consent were scheduled for the ﬁrst visit. 
During the screening phase, one dentist evaluated the eligibility of the patients in the 
study based on the following inclusion criteria: male and female patients aged 18-50 with 
minimum of 20 natural teeth in stable occlusion and good oral hygiene level (bleeding on 
probing not exceeding 20%, no advanced periodontal disease, absence of active caries 
lesions, pulpitis), patients with good general health (no systemic diseases reported in the 
medical anamnesis), and subjects with good language comprehension. Exclusion 
criteria were tooth anomalies (i.e. amelogenesis imperfecta, dentinogenesis imperfecta 
etc.), intrinsic stain (i.e. ﬂuorosis, molar incisors hypomineralization [MIH]), active caries 
lesions, advanced periodontal disease, smokers, presence of orthodontic devices, use 
of antibiotics in the last 3 months, use of antibacterial mouthrinses in the last 3 months, 
reported allergies, drug and alcohol addiction.  
 
Study procedures  
One week before the beginning of the trial, all patients received sub- and supra-gingival 
full-mouth prophylaxis. At the same appointment, patients were given detailed oral 
hygiene instructions by the dental hygienist and a brief explanatory video was shared via 

messaging applications (i.e., WhatsApp) to serve as a reminder for good habits consulting 
during the study period. The patients were scheduled to come back after 7 days (baseline 
visit) and were asked not to brush or eat at least 5 hours before the appointment.  
During the baseline visit, all patients completed a custom-made questionnaire 
(supplementary ﬁle) containing questions regarding dental sensitivity (“self-reported” 
sensitivity), their perception of tooth color and overall perception of the toothpaste they 
were using at the time. Subsequently, they were asked to collect 2 ml of unstimulated 
saliva in sterile tubes (Greiner centrifuge tube, Sigma-Aldrich, St Louis, MO, USA). Saliva 
was immediately processed for microbiological analysis as thereafter described. Upon 
saliva collection, a sensitivity test was conducted by applying a tactile and air-stimulus 
(duration: 2 s, distance from the tooth surface: 1 cm) test to all teeth of the four 
quadrants. The subjects’ response was recorded on a 4-point Schid Sensitivity Scale [25] 
and visual-analogue scale, respectively. Additionally, plaque index (Modiﬁed Quigley and 
Hein) was scored on a 6 point scale at 3 buccal and 3 lingual sites per tooth after it had 
been disclosed with a disclosing agent (Bioﬁlm Disclosure, EMS Dental, Nyon, 
Switzerland); gingival index according to Löe-Silness was also recorded, as well as 
bleeding on probing index. Patients’ tooth color was determined under daily light using 
the Vita classical shade guide. Finally, at the end of the baseline visit, the participants 
were provided with the same soft-bristled toothbrush (Mentadent P, Mentadent, Unilever) 
and randomly assigned into one of the two groups (n=50) according to the dentifrice 
used. An online software (https://www.sealedenvelope.com) was utilized for 
randomization purposes by a stad member unaware of the study protocol. In order to 
enable complete blinding of the participants and dental examiner to the group 
assignment, the two dentifrices were distributed in identical plain white tubes provided 
with only letters A or B, with no other visible marks. The patient’s code and dentifrice 
assigned (A or B) were documented in the chart for later reference. From the baseline 
visit, the subjects had to restrain from using any other products or means of oral hygiene 
except those provided and were invited to follow the domiciliary oral hygiene instructions 
as previously described. In case of any adverse events were to occur, the subjects were 
advised to discontinue the use of the toothpaste and were withdrawn from the study. The 
4 weeks follow-up visit was ﬁxed (recall). 
After 4 weeks (recall), the patients returned to the study site for a ﬁnal visit after having 
abstained from brushing or eating at least 5 hours before the visit. During this visit, the 
patients were asked to ﬁll in the questionnaire (supplementary ﬁle), while the saliva 
collection and all clinical procedures described above were repeated by the same 
dentist.  
 
Microbiological procedures 
The saliva collected during the baseline and recall visits was processed for 
microbiological analysis within 3 hours of sample acquisition. Brieﬂy, 0.1 ml of saliva was 
diluted in 9.9 ml of sterile phosphate-budered saline solution until 10-8 was reached. 
Subsequently, the diluted samples were dispersed on Mitis Salivarius Selective Agar 
(MSB, Microbiol Diagnostici, UTA, CA, Italy) and the plates were incubated at 37oC for the 
next 48-72h. The bacterial colonies were identiﬁed by morphology  and counted by one 
trained investigator, blinded to the groups. S. mutans colonies (CFU) were multiplied by 
their respective dilution ratio to calculate the number of colonies per one milliliter 

(CFU/mL) of each subject’s saliva sample. In order to perform statistical analysis, a base-
10 logarithm transformation was applied to the calculated CFU/ml values.  
 
Statistical analysis 
After checking the normality (Kolmogorov–Smirnov) and the homoscedasticity (modiﬁed 
Levene’s test) of the data, the Two Way Repeated Measures ANOVA (One Factor 
Repetition) followed by Bonferroni post-hoc were run to investigate the edect of the 
tested dentifrices on salivary counts of S. mutans, as well as plaque and gingival indexes. 
Since the data retrived from questionnares where not normally distributed (Kolmogorov–
Smirnov, p<0.05), the non-parametric Mann-Whitney U-test was run. All analyses were 
performed by a statistician blinded to the groups using SigmaPlot 14.0 (Systat Software, 
Chicago, IL, USA). In all tests, the signiﬁcance level was set at α=0.05.