Trial design
We conducted a controlled randomized trial with a parallel group design between June 2017 and December 2018. The study was double blinded for patients and the statistical analysis. Dental professionals could not be blinded as the commercial presentation and characteristics of the two varnishes were distinguishable. The study was approved by the Ethics Research Committee and the Research Biosecurity Committee of the University of Murcia, Spain (CIS: 1499/2017; CBE 50/2017). The trial was retrospectively registered on May 26, 2020: ISRCTN13681286, http://www.isrctn.com/ISRCTN13681286.
Participants
The study was carried out at the University of Murcia Dental Clinic, Hospital General Universitario Morales Meseguer, Murcia. Inclusion criteria were children aged 4-12 years attending the Integrated Child Dentistry Clinic of the University of Murcia for checkups or dental treatment who presented a high or extreme risk of caries according to the CAMBRA protocol. A high or extreme risk of caries was defined as a patient who presented ≥ 3 or more cavitated or noncavitated (incipient) carious lesions or restorations (visually or radiographically evident) or teeth missing due to caries in last 36 months [23].
Exclusion criteria were:1) children who had received fluoride varnish or other permanent surface treatment containing fluoride in the previous 6 months, e.g. children who had pits and fissures sealant; 2) children fitted with orthodontic apparatus 3) children living in an area with fluoridated drinking water; 4) children with moderate or severe fluorosis or other morphological or anatomical abnormalities of dental development 5) children with systemic diseases causing physical limitations; 6) children with allergy or proven/suspected sensitivity to milk proteins.
Before the study, an informative leaflet was provided to all parents/guardians, who were shown the expected benefits and risks of the study, after which written informed consent was obtained. A questionnaire was administered in parents containing questions on demographic factors, dietary habits, history of disease, fluoride therapy, and use of medication and vitamin supplements (Supplementary file 1).
The sample size (n=13 patients/group) was calculated using the evolution data of lactic acid and the existing index of loss of children treated by our clinic. An alpha risk of 0.05 and a beta risk of 0.20 (power 0.8) in a bilateral contrast was accepted to detect a minimum difference of 2.0 between two groups, assuming that there were 3 groups, and a standard deviation of 3.0. A loss to follow up of 45% was estimated. We randomly allocated participants to intervention groups using a computer-generated randomization list. AJOR generated and concealed the allocation sequence to intervention groups until assignment. APP enrolled and assigned participants to intervention groups.
Clinical evaluation and interventions (Interventions, compliance measure, and clinical visual evaluation time points)
Patient selection
The history and examination were carried out by an experienced pediatric dentist, who underwent three training sessions on written and visual instructions, standardization, and calibration with participants in vivo. Caries lesions were recorded using a mirror and a WHO probe according to International Caries Detection and Assessment System criteria (ICDAS II) [24]. The ICDAS II system has two-digit coding for the detection criteria of primary coronal caries. The first refers to tooth restoration and is coded from 0 to 9. The second digit is used for coding caries, from 0 to 6.
Verbal and written oral hygiene instructions and dietary advice were given to participants and their responsible adults to facilitate and strengthen preventive measures (Supplementary file 2). Each participant received a fluoridated toothpaste with 1450 ppm of fluoride (Lacer Junior, Lacer SA, Barcelona, Spain) and a manual toothbrush (Lacer Junior, Lacer SA, Barcelona, Spain) which was changed every 3 months. Instructions were given to avoid other sources of fluoride during the study period (environmental products, supplements, professional or other dental products). At each check-up we verified that study hygiene guidelines and the use of the study toothpaste were being complied with. There were five check-ups: baseline and 3, 6, 9 and 12 months. Fluoride and the prevalence of caries were recorded at baseline and 12 months.
Saliva samples
At the beginning of each check-up, 3.5 ml of unstimulated saliva was collected for 5 minutes in a sterile polyethylene tube. Children had not ingested water or food for 1 hour before the examination. Saliva samples were stored at –20oC until measurement. The schedule at which appointments were made was restricted to 15:00 to 18:00 hours for maximum avoidance of circadian fluctuations in the variables studied [25]. Saliva collection was always performed in the waiting room to avoid the effect of anxiety on the amount and composition of saliva.
Experimental groups, application of varnishes.
Patients were assigned randomly to the control group (placebo), the Clinpro White Varnish® group, and the MI Varnish® group. The composition of the study materials is shown in table 1.
Table 1. Product composition according to material safety data sheets (MSDS)
Product
|
Manufacturer
|
Composition
|
MI Varnish
|
GC, Leuven, Belgium
|
30-50% polyvinyl acetate, 10-30% hydrogenated MSDS rosin, 20-30% ethanol, 5% sodium fluoride, 1-5% CPP-ACP, 1-5% silicon dioxide
|
Clinpro White Varnish
|
3 M ESPE, Saint Paul, MN, US
|
30-75% pentaerythritol glycerol ester of colophony resin, 10-15% n-hexane, 1-15% ethyl alcohol, 5% sodium fluoride, 1-5% flavour enhancer, 1-5% thickener, 1-5% food grade flavour, <5% fTCP.
|
CPP-ACP: protein casein-phosphopeptide with amorphous calcium phosphate; fTCP: functionalized tricalcium phosphate.
After obtaining saliva samples and before application of the varnishes, the teeth were dried with dry compressed air and isolated with cotton rolls and saliva ejector. Then, 0.25 ml of varnish was applied to the surface of the teeth and allowed to dry for 30 seconds. In the placebo group, distilled water was applied with a brush identical to that used to apply the varnishes. Patients were instructed not to rinse their mouths, not to eat or drink for an hour and not to brush until 4-6 hours after application, in accordance with the manufacturers' instructions. The procedure was repeated every three months for one year (Figure 1 [see Supplementary file 3]). During the 12-month study period, patients received conventional dental treatment (extractions, seals, pulp treatments, etc.). To avoid uncontrolled sources of fluoride, the materials used were always fluoride-free.
Outcome measures
Caries index
The dmfs/DMFS indexes were calculated from the ICDAS II scores (the second digit ranged from 3 to 6) [24], transforming them into the decayed, missing and filling values of the ICDAS.
pH and Latic acid
Saliva samples were thawed and shaken for 10 seconds at 20°C (ClassicVortex Mixer, Velp Scientifica, Italy) and 15 µL of the saliva sample was poured onto a pH test strip (range 4.0–9.0; Code. 1.16996.0001; Reflectoquant™ Merck, Darmstadt, Germany) which was introduced in a RQflex®10 reflectometer (Merck Millipore, Darmstadt, Germany) to provide the pH value. 30 µL of the saliva sample was poured onto a lactic acid test strip (range 1.0-60.0 mg/L; Code. 1.16127.0001; Reflectoquant™ Merck, Darmstadt, Germany) which was introduced in a RQflex® 10 reflectometer (Merck Millipore, Darmstadt, Germany) to provide the lactic acid value.
Fluoride
Fluoride concentrations were measured using an ion-specific fluoride electrode (Orion 9609 BNWP, Thermo Fisher Scientific Inc. Waltham, USA) coupled to an ion analyzer (Orion EA-940 Thermo Fisher Scientific Inc. Waltham, USA). Before each reading, samples were shaken with a vibrator (Classic Vortex Mixer, Velp Scientifica, Italy) to homogenize the sample. The electrode was calibrated beforehand with standard solutions from 0.125 to 2.0 ppm F, mixing 1mL of each standard solution with 1mL of TISAB II (1.0 M pH acetate buffer 5.0; 1.0 M NaCl and 4% CDTA). Once calibrated, the samples were read, for which we mixed 1mL of each saliva sample with 1mL of TISAB II (Hanna Instruments, Woonsocket, Rhode Island, USA). The results in mV were converted into fluoride concentrations (ppm) using the standard calibration curves measured immediately before the analysis.
Trace elements
We analyzed 2 ml of the homogenized sample using mass spectrometry with inductively coupled argon plasma (ICP-MS Agilent 7900; Agilent Technologies Inc.; CA; USA). Ultrapure water (18.2 M Ω) from a water purification system (Milli-Q® Reference A+, Merck Millipore) was used to prepare the standard reagents and solutions: 100 µL of the saliva sample was diluted up to 1 mL with a 2% HNO3 Suprapur solution in ultrapure water. The samples were introduced in a self- sampling spectrometer by the impulse of a peristaltic pump, and were atomized, ionized and the ions generated detected and quantified subsequently by mass spectrometry. The isotopes selected for each element studied were: 23Na, 24Mg, 27Al, 31P, 39K,44Ca, 52Cr, 55Mn, 57Fe, 59Co, 63Cu, 66Zn, 75As, 111Cd, 137Ba and 208Pb.
Statistical analysis
Values are expressed as mean ± standard deviation. The Kokmogorov-Smirnov test was used to determine sample normality and the Levene test for equality of variance. Pearson’s chi-square test was used to determine between-group differences in sex and a one-way ANOVA test for differences in age. To determine within-group differences in age distribution according to sex we used the Mann-Whitney test. To detect between-group differences in DMFS and dmfs values a Kruskal-Wallis test was performed and one-way analysis of variance was used to detect differences in the Turesky et al Modified Quigley Hein Plaque Index.
Differences in concentrations of trace elements, pH and lactic acid between baseline and 3, 6, 9 and 12 months were determined by simple variance analysis of repeated measures. When there were differences between the times, two-by-two comparisons were made using the Holm–Sidak test.
A paired t-test was used to analyze the within-group evolution of fluoride concentrations between baseline and 12 months when there was normality and a Wilcoxon test when there was no normality. One-way ANOVA was used to detect between-group differences in the same period. A value of p<0.05 was considered significant. The analysis was made using the SigmaStat 3.5 statistical software package (Systat Software Inc., Point Richmond, CA, USA).