Experimental Design
The Human Ethical Committee of São Paulo State University (UNESP), School of Dentistry, Araçatuba, Brazil, approved this study (Protocol: 60803722.2.0000.5420), and all participants read and signed informed consent statements before study onset. This in situ, crossover, double-blind study was conducted in five phases of 7 days each [16]. The sample size of 10 volunteers was determined based on a previous study [16, 17], with the primary outcome being surface and cross-sectional hardness analysis, mean difference between groups (30 and 1300, respectively), standard deviation (20 and 900, respectively), an α-error of 5%, and a β-error of 20%. Volunteers (n=10), aged 20–30 years, who were in good general and oral health, were included in the study. The initial exclusion criteria include the use of medication or carriers of alterations that influence salivary flow and those allergic to milk protein (CPP). Exclusion criteria during the experiment were voluntary withdrawal, change in health with consequent alteration in salivary flow, the necessity to use antibiotics and similar demineralizing capacity between Placebo and 1100F. Each participant wore an acrylic palatal appliance with sound bovine enamel blocks (4 mm× 4mm), previously polished and selected according to the initial surface hardness (SHi) (baseline). The specimens were allocated to different treatments: 1) Placebo (No F-TMP-CPP-ACP), 2) 1100 ppm F (1100F), 3) 1100F+3%TMP (1100F-TMP), 4) 1100F+10%CPP-ACP (1100F-CPP-ACP) and 5) 1100F-CPP-ACP-TMP. After each phase, the biofilm was collected for the analysis of F, Ca, P, and insoluble extracellular polysaccharides (EPS). In the enamel blocks, the percentage of surface hardness loss (%SH) and integrated loss of subsurface area (ΔKHN) were determined. F, Ca, and P contents in the enamel were then determined (Figure 1).
Enamel block preparation
Enamel specimens (4 mm× 4 mm, n = 200) obtained from bovine incisors were stored in 2% formaldehyde solution with a pH of 7.0 for 30 days at room temperature [18]. Subsequently, the blocks underwent flattening and polishing using a BETA polisher (Buehler, Lake Bluff, Illinois, USA) with silicon carbide sandpaper in 600, 800, and 1200 grits (Extec Corp, Enfield, CT, USA), with continuous water irrigation. Final polishing was carried with felt disks moistened with 1 μm diamond solution (Extec Corp. Enfield, CT, USA). Next, the blocks were cleaned with deionized water using ultrasound (Unique USC 1400, Indaiatuba, SP, Brazil), operated at 40 Hz and 135 W for 20 min at room temperature [19].
Toothpaste formulation, fluoride and pH assessment
The experimental toothpastes were formulated with the following components: titanium dioxide, carboxymethylcellulose, sodium methyl-p-hydroxybenzoate, saccharin, mint oil, glycerin, abrasive silica, sodium lauryl sulfate, and water. The toothpaste was prepared to contain at concentrations: F (1100 ppm F, in the form of NaF), TMP (3%) and CPP-ACP (10%). A toothpaste without F/CPP-ACP/TMP (Placebo) and with F/CPP-ACP/TMP (1100F-CPP-ACP-TMP) were prepared (Table 1). The toothpastes used in this study were stored at room temperature and kept sealed to prevent any change to the samples. The total fluoride (TF) and ionic fluoride (IF) amounts were determined with a F-specific electrode (Orion 9609-BN; Orion Research Inc., Beverly, USA) connected to an ion analyzer (Orion 720 A+; Orion Research Inc.). The pH levels of the toothpaste slurries were determined via a pH electrode (2A09E, Analyser, São Paulo, Brazil) calibrated with standard pH levels of 7.0 and 4.0 [5].
Palatal appliance preparation and treatments
The palatal appliance was prepared in acrylic resin (Jet, Articles Classic Odontológico, São Paulo, Brazil), and four enamel specimens were fixed with a different device used in each phase of the experiment. In order to allow biofilm accumulation on the enamel blocks, a piece of plastic mesh was fixed to the acrylic appliance, leaving a space of 1mm from the block surface [16, 17]. To provide a cariogenic challenge, the volunteers were instructed to remove the device and drip 30% sucrose solution (Sucrose, Synth, Diadema, Brazil) onto each enamel block 6x/day at predetermined times (8:00 am, 11:00 am, 2:00 pm, 5:00 pm, 7:00 pm, and 9:00 pm) and five minutes later (i.e. and five minutes later, after diffusion of the 30% sucrose solution over the surface of the enamel), the device was reinserted into the mouth [16, 17]. The volunteers were instructed to use the appliances during the entire day (including night time), except when drinking or eating anything, and brushed their natural teeth 3x/day (08:00 am, 1:00 pm, 9:30 pm) for 2 min, with palatal appliance in the oral cavity, allowing the natural saliva/toothpaste slurry to come into contact with the enamel blocks by gently squishing the slurry in their mouths. Subsequently, the devices were removed from the oral cavity and gently rinsed with tap water; volunteers then brushed their natural teeth and rinsed their mouths as usual, returning the devices to the oral cavity immediately afterward. During a seven-day pre-experimental and washout period, the volunteers brushed their teeth with the Placebo toothpaste.
Five experimental phases of 7 days each [16, 17, 20] were performed with the following toothpastes: 1) Placebo (No F-TMP-CPP-ACP), 2) 1100 ppm F (1100F), 3) 1100F+3%TMP (1100F-TMP), 4) 1100F+10%CPP-ACP (1100F-CPP-ACP) and 5) 1100F-CPP-ACP-TMP. After each phase, the biofilm was collected for analysis of F, Ca, P, and insoluble extracellular polysaccharides (EPS). In the enamel blocks, the percentage of surface hardness loss (%SH) and integrated loss of subsurface area (ΔKHN) were assessed again. F, Ca, and P contents in enamel were determined.
Hardness Analysis
The SH was determined before (initial hardness surface, SHi) and after each experimental phase (final hardness surface, SHf), using a Shimadzu HMV-2000 microhardness tester (Shimadzu Corp., Kyoto, Japan) under a 25-g load for 10 s [7]. The percentage of surface hardness loss was calculated as follows: (%SH = [(SHf-SHi)/SHi] × 100). For cross-sectional hardness measurements, the enamel blocks were longitudinally sectioned through their center, embedded in acrylic resin with the cut face exposed and gradually polished. A sequence of 14 indents was created 100 μm apart at different distances (5, 10, 15, 20, 25, 30, 40, 50, 70, 90, 110, 130, 220, and 330 μm) from the outer enamel surface using a Micromet 5114 hardness tester (Buehler, Lake Bluff, USA) and the software Buehler OmniMet (Buehler, Lake Bluff, USA) with a Knoop diamond indenter under a 5-g load for 10 s. Integrated hardness (KHN × μm) for the lesion into sound enamel was calculated using the trapezoidal rule (GraphPad Prism, version 3.02) and subtracted from the integrated hardness for sound enamel to obtain the integrated area of subsurface regions in enamel, referred to as integrated loss of subsurface hardness (ΔKHN; KHN × μm) [16, 17].
Analysis of F, Ca and P in the enamel
The F present in the enamel was determined as described by Weatherell et al. [21] with modifications by Alves et al. [22]. Self-adhesive polishing disks (diameter, 13 mm) and 400-grit silicon carbide (Buehler) were attached to the bottom of polystyrene glass tubes (J-10; Injeplast, São Paulo, SP, Brazil). A layer of enamel (50.9 ± 0.2 µm) was removed from each block, followed by the addition of 0.5 mL of 1.0 mol L-1 HCl; these were kept under constant agitation for 1 hour. F analysis was performed on 0.30 mL of this solution after adding an equal volume of TISAB II, modified with NaOH, using a specific electrode 9409BN (Thermo Scientific, Beverly, MA, USA), and a reference microelectrode (Analyser, São Paulo, Brazil) coupled to an ion analyzer (Orion 720A+, Thermo Scientific, Beverly, MA, USA). The results were expressed in µg/mm3 [13]. Ca analysis was conducted using the Arsenazo III colorimetric method [23]. Absorbance readings were recorded at 650 nm using a plate reader (PowerWave 340, Biotek, Winooski, VT, USA). P was analyzed according to Fiske & Subbarow [24] and absorbance readings were recorded at 660 nm. The results were expressed in µg/mm3.
Analysis of F, Ca, P and EPS in the biofilm
The biofilm formed on the enamel was collected and stored in microcentrifuge tubes. Biofilm samples were vacuum-dried over P2O5 (Vetec Química Fina Ltda., Duque de Caxias, Rio de Janeiro, Brazil) for 12 hours at room temperature. After extraction for 3 hours at room temperature with 0.5 mol L-1 hydrochloric acid (250 μL/mg, wet weight of the biofilm) under constant agitation, an equal volume of NaOH (0.5 mol L-1) was added [13]. The samples were then centrifuged (11,000 × g) for 1 minute, and the supernatant was retained for the determination of F, Ca, and P. The F analysis was conducted using a specific ion electrode (Orion 9409 BN) and a potentiometer (Orion 720 Aplus). Ca concentration evaluation was performed using the Arsenazo III colorimetric method [23, 25, 26]. For calibration, standards containing 40-200 μg Ca/mL were used. Aliquots of 3 μL (duplicate) were placed in 96-well plates (Plate for flat-bottomed cell culture - Model 92096 - TPP, Switzerland) coupled to a plate reader (PowerWave 340, Biotek), using a wavelength of 650 nm. P was measured using a 0.1 mL aliquot using the colorimetric method described by Fiske & Subarrow [24]. Duplicate readings were performed on 96-well plates in the same way as for Ca concentration, using a spectrophotometer (MicroplateSpectrophotometer EONC, Biotek, Winooski, VT, USA). Absorbance readings were recorded at 660 nm and the results were expressed as μg/mm3. EPS was extracted by adding 1.0 mol L-1 NaOH (10 μL/mg dry weight) to the biofilm [27]. The amount of EPS was determined using the phenol-sulfuric acid method [28]. The results were expressed as moles per kilogram (F, Ca, and P) and milligrams per gram (EPS) dry weight.
Statistical analysis
SigmaPlot 12.0 software (version 12.0, Systat Software Inc., San Jose, CA, USA) was used for statistical analysis, and the significance level was set at 5%. The statistical power calculated was 85%, considering all differences between groups for each outcome. Data from the dental biofilm analysis (F, Ca, P and EPS content) and enamel analysis (%SH, ΔKHN and F, Ca, and P content) exhibited normal (Shapiro–Wilk) and homogeneous (Bartlet) distribution and were therefore subjected to one-way ANOVA, repeated measures, followed by the Student–Newman–Keuls’ testing.