Protective effect of various toothpastes and mouthwashes against erosive and abrasive challenge on eroded dentin: an in vitro study


Study planning

A total of 143 sound premolars were extracted for orthodontic reasons from individuals aged 18–20 years according to a protocol approved by the Ethical Review Board of Shiraz University of Medical Sciences (IR.SUMS.DENTAL.REC.1400.146). Thoroughly remove any remaining tissue and debris from the extracted tooth using a preventive brush and disinfect by soaking in 0.1% Chloramine T solution for four weeks. Samples were stored in deionized water at 25°C and replaced weekly until use. Before the start of the study, the specimens were examined under a stereomicroscope (Motic K, Wetzlar, Germany) at 40× magnification to exclude teeth with defects or fracture lines. A total of 130 teeth met the inclusion criteria. Prepared samples were challenged with erosion and abrasion as well as various combinations of toothpastes and mouthwashes. Surface microhardness, surface roughness values, and dentin morphology were evaluated in three stages: sound dentin (baseline), initial eroded dentin, and remineralized dentin.

Sample Preparation

The root was removed approximately 2 mm below the cemento-enamel junction and the crown was inserted into self-curing acrylic resin with the buccal surface parallel to the mold. Enamel was precisely removed using a high-speed handpiece, and exposed dentin was polished using 320, 600, 1200, and 2400 grit waterproof silicone carbide papers (Buehler, Lake Bluff, IL, USA). The polished samples were then immersed in an ultrasonic bath and cleaned for 5 minutes. Complete removal of enamel from the tooth surface was confirmed using a stereomicroscope with 10x magnification. Two layers of nail polish covered the dentin surface and exposed a 2 × 5 mm window to standardize the intervention surface. Then, the samples were randomly assigned to five experimental groups. Each group included 26 teeth, of which 12 teeth were used for surface microhardness testing, 12 teeth were used for surface roughness measurements, and two teeth were used for atomic force microscopy (AFM) evaluation.

Development of initial erosive lesions and film formation

To induce initial erosive lesions, each sample was exposed to 20 mL of 0.1% citric acid solution (pH 2.5) on a stirrer (Alfa D500, Iran) at 60 rpm for 30 min at 25 °C. Refreshed every 5 minutes. Next, rinse the sample with deionized water for 30 seconds.

To better simulate oral conditions, samples were stored in artificial saliva (20 mL per portion) at 25°C for one hour to allow the formation of a salivary film on the dentin surface.The artificial saliva composition consists of 0.2 mM glucose, 0.1 mM C8H15Na2O, 9.9 mM sodium chloride, 1.5 mM calcium chloride2·H2O, 3mMNH4Cl, 17 mM KCl, 2 mM NaSCN, 2.4 mM K2Hydroxyapatite43.3 mM urea, 2.4 mM NaH2back4and 11 µM ascorbic acid (pH 6.8)twenty one. Artificial saliva required for film formation, erosion challenge, and toothpaste slurry preparation was prepared weekly and stored at 25°C.

group allocation

The five experimental groups are as follows:

Group 1 (negative control):

During erosion and abrasion challenges, samples were immersed in artificial saliva.

Group 2 (positive control) (toothpaste and mouthwash containing AmF (Olafur), NaF and SnCl2:

Samples were exposed to Elmex anti-corrosion toothpaste containing 1400 ppm F and 3500 ppm tin2+ (GABA International AG, Switzerland), followed by Elmex Anti-Corrosion Mouthwash, containing 500 ppm F and 800 ppm tin2+.

Group 3 (toothpastes and mouthwashes containing sodium monofluorophosphate (SMFP) and hydroxyapatite):

Sample treated with Vitis anti-caries biorestorative toothpaste containing 1450 ppm F- and 4500 ppm hydroxyapatite (VITIS Oral Health C/O Dent-O-Care, UK), followed by rinsing with Vitis Anti-Caries Biorestorative Mouthwash containing 226 ppm F- and 125 ppm hydroxyapatite.

Group 4 (Toothpaste containing NaF and SnF2 and using NaF mouthwash):

Oral B Pro Toothpaste (1450 ppm F and 3230 ppm tin2+) (Procter and Gamble, USA), then use Oral B fluoride mouthwash (226 ppm F).

Group 5 (toothpaste containing NaF and CPP-ACP and mouthwash containing supersaturated Ca and P ions):

Use MI Paste ONE Toothpaste (1100 ppm F) (GC America) and then rinsed with Caphosol mouthwash (EU Pharma, USA), containing two bottles of A and B, mixed in equal proportions.

The toothpastes and mouthwashes used in this study and their ingredients are shown in Table 1 .

Table 1 Ingredients of toothpastes and mouthwashes used in this study.

Erosion Challenge Protocol

The corrosive challenge protocol consisted of exposure to a 0.1% citric acid solution at pH 2.5 for 90 seconds four times daily (10 am, 12 pm, 2 pm, and 4 pm) for five days. After each challenge, samples were rinsed with deionized water for 10 s and stored in 20 mL of artificial saliva while incubating at 25 °C until the next challenge. Additionally, samples were stored overnight in artificial saliva. Toothpaste and mouthwash from each group were applied to the dentin surface 30 minutes after the first and last acid exposure. Citric acid and artificial saliva were replaced daily.

Remineralization of eroded dentin surfaces

Toothpaste slurries for Groups 2 to 5 were prepared daily by mixing 100 mg of toothpaste with 300 mg of artificial saliva using a mixer device at 60 rpm. The grinding challenge was performed using a two-room automatic toothbrush device (Shiraz Electric, Shiraz, Iran). After placing the sample into the chamber containing the toothpaste slurry for 1 minute, brush the dentin surface with a soft toothbrush (Oral-B Healthy Clean Manual Toothbrush, Procter and Gamble, USA) at a constant force of 2.5 N at 120 times per minute for 15 seconds. . Each tooth was then rinsed with deionized water for 10 seconds and soaked in mouthwash for 30 seconds at 60 rpm to simulate mouthwash rinses in the mouth. The samples in Group 1 (negative control) brushed their teeth with artificial saliva without using toothpaste or mouthwash. At the end of the fifth day, laboratory tests were performed to evaluate changes in surface microhardness, roughness, and dentin morphology.

Surface microhardness test

The microhardness values ​​of each group (n = 12) were measured using a Vickers diamond indenter apparatus (ZwickRoell, Fürstenfeld, Austria) at a force of 50 g for 15 s at five points at a distance of 50 µm for each sample. Measurements were repeated in three stages, including sound dentin (baseline), initially eroded dentin, and remineralized dentin. The percent recovery of dentin microhardness (%RDMH) was calculated using the average Vickers hardness number (VHN) of each group as follows:

$$\mathrm{\%RDMH}=\frac{\mathrm{ VHN\,of\,remineralization\,dentin}-\mathrm{VHN\,of\,erosion\,dentin}}{\mathrm {VHN\,sound\,dentin}-\mathrm{VHN\,eroded\,dentin}}\multiplied by 100$$

Surface roughness test

To evaluate the surface roughness (Ra) of each set of samples (n = 12), 5 points with a distance of 250 µm were scanned using a contact profilometer (TESA RUGOSURF 20, Switzerland). A silicone putty jig was constructed on each mold to provide equal areas of exposed dentin to allow for multiple measurements. The Ra values ​​corresponding to sound dentin (baseline), initial eroded dentin and remineralized dentin were recorded and analyzed using RUGOSOFT software.

Dentin roughness recovery percentage (%RDR) is calculated as follows:

$$\mathrm{\%RDR}=\frac{\mathrm{Ra\,of\,remineralization\,dentin}-\mathrm{Ra\,of\,erosion\,dentin}}{\mathrm {Ra\,sound\,dentin}-\mathrm{Ra\,erosion\,dentin}}\multiplied by 100$$

Additionally, the percent surface loss of remineralized dentin compared to eroded and sound dentin (baseline) was calculated using the following formula:

$${\mathrm{\% surface\,loss}}_{{\text{Remineralization}}-{\text{Erosion}}}=\frac{\mathrm{ Ra\,of\,remineralization \,dentin} -\mathrm{Ra\,of\,erosion\,dentin}}{\mathrm{Ra\,of\,erosion\,dentin}}\times 100$$

$${\mathrm{\%Surface\,Loss}}_{{\text{Remineralization}}-{\text{Baseline}}}=\frac{\mathrm{ Ra\,of\,Remineralization \,dentin} -\mathrm{Ra\,sound\,dentin}}{\mathrm{Ra\,sound\,dentin}}\multiplied by 100$$

Surface topography assessment

Two samples from each group were randomly selected for AFM evaluation (n = 10). Surface characteristics of dentin samples were examined using an AFM device (AFM, JPK Nanowizard II device, JPK Instruments, Berlin, Germany) combined with a non-conductive silicon nitrite cantilever (Acta-Probe, APPNano, CA) and a piezoelectric scanner. .

The measurement area of ​​each sample was 5 µm × 5 µm.

Statistical Analysis

Data analysis was performed using SPSS version 22 software (IBM, New York, USA). Surface microhardness and roughness values ​​of five different groups were compared using one-way ANOVA and Tukey HSD post hoc test. % surface loss remineralization baseline One-way variance analysis and Tukey HSD post hoc test were used to compare differences among the five groups. Remineralization column, Δ value (Δ32), %RDMH, %RDR and % surface lossRemineralization-erosion One-way analysis of variance and Tamhane's T2 post hoc test were used to compare differences among the five groups. Furthermore, repeated measures ANOVA and Sidak's post hoc test were used for intragroup comparison of microhardness and roughness values ​​of sound dentin, eroded dentin, and remineralized dentin. All statistical tests considered a significance level of 0.05.

ethical approval

The study was approved by the Ethical Review Board of the Faculty of Dentistry, Shiraz University of Medical Sciences (IR.SUMS.DENTAL.REC.1400.146).

agree to participate

All methods were performed in accordance with relevant guidelines and regulations (Declaration of Helsinki). Written informed consent for dental use was obtained from participants.



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