Silver diamine fluoride remineralized artificial incipient caries in permanent teeth after bacterial pH-cycling in-vitro

Abstract

Objectives

To investigate the remineralizing effect of 38% silver diamine fluoride (SDF) application on enamel artificial caries in adjunct to 1000 ppm fluoride toothpaste compared with fluoride toothpaste alone by analyzing the mineral density, depth of remineralization, and remineralization percentage of the lesions.

Methods

Eighteen artificial caries slabs were created from the proximal surfaces of nine chemically demineralized premolars. The slabs were scanned by Micro-Computed Tomography (Micro-CT) to determine the baseline mineral density of the initial lesions and randomly allocated into 2 groups. The test group was applied with 38% SDF in adjunct to fluoride toothpaste and the control group was treated with fluoride toothpaste alone. The specimens underwent bacterial pH-cycling for 5 d and were re-evaluated using Micro-CT. The pre-treatment and post-treatment mineral densities were plotted and the areas under the curves were used to calculate the remineralization percentage of both groups.

Results

Mineral density significantly increased in both groups after pH-cycling (p < 0.05) although to different depths (control group = 260 μm, test group = 300 μm). The test group demonstrated a significantly higher mineral density to a depth of 120 μm and higher remineralization percentage (p < 0.05) compared with the control group.

Conclusion

The adjunctive use of 38% SDF enhances the remineralization of initial carious lesions based on mineral density, depth, and remineralization percentage compared with the use of 1000 ppm fluoride toothpaste alone. SDF might be used as an adjunct to fluoride toothpaste to remineralize incipient caries lesions on smooth tooth surfaces.

Clinical significance

In non-compliant patients, the application of 38% SDF might be used as an adjunct to fluoride toothpaste, to remineralize incipient caries lesions of permanent teeth where esthetics is not a concern.

Introduction

Fluoride toothpaste has been marketed worldwide since the 1990s, and it might be the most important source of fluoride for reducing caries globally. Experts agree that the use of fluoride toothpaste has contributed to the decline of caries .

The prevented fraction of fluoride toothpaste was reported to be 24% . Despite the widespread use of fluoride toothpaste, in high caries risk patients, incipient proximal caries still develop and can be detected in bitewing radiographs.

The current concept in managing an incipient proximal lesion is to remineralize the lesion with the additional use of home-use or professional use fluoride . Twice-daily use of 0.32% NaF mouthrinse in adjunct to brushing with 1500 ppm fluoride dentifrice two times per day yielded less proximal caries increment compared with the use of fluoride toothpaste alone . Fluoride mouthrinse was shown to raise salivary fluoride levels at the proximal area . However, in patients with poor compliance, professional treatment using the high concentration, less frequency concept could be employed by directly applying high fluoride concentration solution to the incipient lesions .

The use of 38% silver diamine fluoride (SDF) can prevent and inhibit the progression of cavitated dentin lesions . Studies have demonstrated SDF’s antibacterial activities against cariogenic microorganisms, such as Streptococcus mutans and lactobacilli . SDF has been used to arrest caries in primary tooth dentin , however, is not yet used to arrest white spot lesions in enamel in permanent tooth proximal caries. Moreover, the depth of remineralization and mineral content change of arrested lesions after SDF treatment have not yet been reported. We hypothesized that SDF can be applied to remineralize early proximal lesions in posterior permanent teeth. Thus, the objective of our study was to compare the remineralizing effect of SDF used as an adjunct to fluoride toothpaste and fluoride toothpaste alone as shown by the depth of mineralization and mineral density gain at every 20 μm depth in artificial white spot lesions. The null hypothesis was there is no difference between mineral density gains after pH-cycling within and between the two groups.

Materials and methods

Subjects

The study protocols were approved by the Ethics Committee of the Faculty of Dentistry, Chulalongkorn University (HREC-DCU 2014-011). A sample size calculation determined that 9 samples were required to demonstrate an absolute difference of 5% in mean mineral density change between the two fluoride products with 90% power (α = 0.05, β = 0.10) . After the experimental protocol had been explained and informed consent was obtained, nine human premolars were collected from private dental clinics in Bangkok. These teeth were from patients who had their premolars extracted for orthodontic reasons and were kept in 0.9% normal saline until used. The inclusion criteria were no white spot lesions or any other defects with a minimum proximal surface width of 6 mm.

Specimen preparation and lesion formation

Specimen preparation was performed as illustrated in Fig. 1 . The enamel slabs were prepared from the proximal surfaces of nine sound extracted human premolars. Each of the proximal surfaces was polished using an automatic polishing machine (DPS3200, IMPETECH, South Africa) at 100 rpm for 45 s. Each tooth was then immersed in 16 ml of polyacrylic acid, 1.76 ml 85% lactic acid, 100 mg hydroxyapatite, 184 ml deionized water, and 6 M sodium hydroxide at pH 4.8 and stored at 37 °C on a vibrating table to create artificial caries that were approximately 250 μm in depth . The demineralizing solution was changed every day for 18 d. Two slabs 2 × 1 × 3 mm 3 in width, length and height, respectively, were prepared from each proximal surface of a tooth, for a total of 18 slabs.

Fig. 1
Schematic of the experimental procedures.

Artificial carious lesion baseline mineral density determination

Each tooth slab was individually fixed in a resin holder. The specimens’ surfaces were covered with nail varnish (Enchanting, Revlon Nail Lacquer, USA), except for a targeted proximal 2 × 1 mm 2 window on each specimen. A reference line was prepared on the lateral surface of the holder, perpendicular to the targeted window, of each specimen ( Fig. 1 ). This reference mark was used in the Micro-Computed Tomography (Micro-CT) evaluations for baseline mineral density (pre-treatment) and subsequent mineral density evaluations of all 18 slabs. The slabs were then sterilized with ethylene dioxide and equally divided into control and test groups. The control group (F-toothpaste group) was applied with deionized water and the test group was applied with SDF. Both treatments were performed by applying 5 μl of each liquid with a micro-brush.

Bacterial pH-cycling model

The 18 slabs, individually fixed on resin holders, were inserted into hard plastic sheet holes made to lock them in place, and then kept in a 24-well plate, where they were immersed in a bacterial pH-cycling solution. The specimens underwent bacterial pH-cycling for 5 d as shown in Fig. 1 .

The demineralizing solution used in the pH-cycling was made as previously described . Briefly, Streptococcus mutans ATCC 25175 from a −80 °C glycerol stock were inoculated on Tryptic Soy Agar and then incubated for 24 h in a 37 °C and 5% CO 2 environment. An isolated colony was transferred to tryptic soy broth containing 0.5% yeast extract, 2% sucrose, and 1% glucose, following by overnight incubation at 37 °C in a 5% CO 2 atmosphere.

The fluoride toothpaste slurry was made at a ratio of 1:3, fluoride toothpaste: deionized water . The specimens were soaked in fluoride toothpaste slurry for 2 min before and after demineralization solution immersion. The specimens were placed in the demineralizing solution and incubated for 4 h in a 37 °C and 5% CO 2 environment. Subsequently, the specimens were immersed in artificial saliva overnight. A new sterilized plate was used at each step of pH-cycling.

Assessment of the Micro-CT scan and data analysis

After pH-cycling for 5 d, the mineral density at each 20 μm depth interval was measured by Micro-CT. The specimens were single-blindly masked before measurement to avoid bias.

The mineral density of the 2 × 1 mm 2 window of each specimen was measured using a Micro-CT system (μCT35, Scanco Medical AG. Switzerland). The specimens were scanned at 70 kVp with a current of 114 mA and evaluated under medium resolution (1024 × 1024). A 0.5-mm-thick aluminum filter was placed in the beam path to reduce beam-hardening effects. To calibrate the Micro-CT, a series of hydroxyapatite phantom standards with a range of densities were scanned.

The mineral density of each specimen (mgHA/cm 3 ) was determined by volumetric measurements, and the grey scale value was calculated from the Micro-CT scan images. Mineral density values were calculated from each specimen every 20 μm through the entire lesion depth. To calculate the lesion depth and mineral gain, a mineral density profile was created by plotting mean mineral density against lesion depth. The mean mineral density gain (MD gain) was calculated by subtracting the pre-treatment area under the curve (AUC) from the post-treatment AUC of each group. The lesion depth was marked at the depth where the mineral density was equivalent to 95% of the maximum density . The percent remineralization (% Remineralization) indicated the proportion of the MD gain and the mineral density of the original lesion, which was obtained from the equation :

% Remineralization = (AUC post − AUC pre /AUC pre ) × 100

The percent increase in mineral density compared between the two groups was calculated from the following equation :

% Increase in mineral density compared between groups = [(ΔZ d − ΔZ r )/ΔZ d ] × 100
ΔZ d = difference in AUC between post-cycling of the control group and median sound enamel.
ΔZ r = difference in AUC between post-cycling of the SDF group and median sound enamel.

Statistical analysis

The data were assessed for a normal distribution using the Shapiro-Wilk test for normality (p > 0.05). The dependent t -test was used to compare the mineral density between baseline and post-cycling values within each group. Mineral density and remineralization percentage between the SDF and control groups were compared by the independent t -test. All of the analyses were conducted using the SPSS program version 16 (SPSS Inc., Chicago, Illinois, USA) with a significance level set at 0.05.

Materials and methods

Subjects

The study protocols were approved by the Ethics Committee of the Faculty of Dentistry, Chulalongkorn University (HREC-DCU 2014-011). A sample size calculation determined that 9 samples were required to demonstrate an absolute difference of 5% in mean mineral density change between the two fluoride products with 90% power (α = 0.05, β = 0.10) . After the experimental protocol had been explained and informed consent was obtained, nine human premolars were collected from private dental clinics in Bangkok. These teeth were from patients who had their premolars extracted for orthodontic reasons and were kept in 0.9% normal saline until used. The inclusion criteria were no white spot lesions or any other defects with a minimum proximal surface width of 6 mm.

Specimen preparation and lesion formation

Specimen preparation was performed as illustrated in Fig. 1 . The enamel slabs were prepared from the proximal surfaces of nine sound extracted human premolars. Each of the proximal surfaces was polished using an automatic polishing machine (DPS3200, IMPETECH, South Africa) at 100 rpm for 45 s. Each tooth was then immersed in 16 ml of polyacrylic acid, 1.76 ml 85% lactic acid, 100 mg hydroxyapatite, 184 ml deionized water, and 6 M sodium hydroxide at pH 4.8 and stored at 37 °C on a vibrating table to create artificial caries that were approximately 250 μm in depth . The demineralizing solution was changed every day for 18 d. Two slabs 2 × 1 × 3 mm 3 in width, length and height, respectively, were prepared from each proximal surface of a tooth, for a total of 18 slabs.

Fig. 1
Schematic of the experimental procedures.

Artificial carious lesion baseline mineral density determination

Each tooth slab was individually fixed in a resin holder. The specimens’ surfaces were covered with nail varnish (Enchanting, Revlon Nail Lacquer, USA), except for a targeted proximal 2 × 1 mm 2 window on each specimen. A reference line was prepared on the lateral surface of the holder, perpendicular to the targeted window, of each specimen ( Fig. 1 ). This reference mark was used in the Micro-Computed Tomography (Micro-CT) evaluations for baseline mineral density (pre-treatment) and subsequent mineral density evaluations of all 18 slabs. The slabs were then sterilized with ethylene dioxide and equally divided into control and test groups. The control group (F-toothpaste group) was applied with deionized water and the test group was applied with SDF. Both treatments were performed by applying 5 μl of each liquid with a micro-brush.

Bacterial pH-cycling model

The 18 slabs, individually fixed on resin holders, were inserted into hard plastic sheet holes made to lock them in place, and then kept in a 24-well plate, where they were immersed in a bacterial pH-cycling solution. The specimens underwent bacterial pH-cycling for 5 d as shown in Fig. 1 .

The demineralizing solution used in the pH-cycling was made as previously described . Briefly, Streptococcus mutans ATCC 25175 from a −80 °C glycerol stock were inoculated on Tryptic Soy Agar and then incubated for 24 h in a 37 °C and 5% CO 2 environment. An isolated colony was transferred to tryptic soy broth containing 0.5% yeast extract, 2% sucrose, and 1% glucose, following by overnight incubation at 37 °C in a 5% CO 2 atmosphere.

The fluoride toothpaste slurry was made at a ratio of 1:3, fluoride toothpaste: deionized water . The specimens were soaked in fluoride toothpaste slurry for 2 min before and after demineralization solution immersion. The specimens were placed in the demineralizing solution and incubated for 4 h in a 37 °C and 5% CO 2 environment. Subsequently, the specimens were immersed in artificial saliva overnight. A new sterilized plate was used at each step of pH-cycling.

Assessment of the Micro-CT scan and data analysis

After pH-cycling for 5 d, the mineral density at each 20 μm depth interval was measured by Micro-CT. The specimens were single-blindly masked before measurement to avoid bias.

The mineral density of the 2 × 1 mm 2 window of each specimen was measured using a Micro-CT system (μCT35, Scanco Medical AG. Switzerland). The specimens were scanned at 70 kVp with a current of 114 mA and evaluated under medium resolution (1024 × 1024). A 0.5-mm-thick aluminum filter was placed in the beam path to reduce beam-hardening effects. To calibrate the Micro-CT, a series of hydroxyapatite phantom standards with a range of densities were scanned.

The mineral density of each specimen (mgHA/cm 3 ) was determined by volumetric measurements, and the grey scale value was calculated from the Micro-CT scan images. Mineral density values were calculated from each specimen every 20 μm through the entire lesion depth. To calculate the lesion depth and mineral gain, a mineral density profile was created by plotting mean mineral density against lesion depth. The mean mineral density gain (MD gain) was calculated by subtracting the pre-treatment area under the curve (AUC) from the post-treatment AUC of each group. The lesion depth was marked at the depth where the mineral density was equivalent to 95% of the maximum density . The percent remineralization (% Remineralization) indicated the proportion of the MD gain and the mineral density of the original lesion, which was obtained from the equation :

% Remineralization = (AUC post − AUC pre /AUC pre ) × 100
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Jun 17, 2018 | Posted by in General Dentistry | Comments Off on Silver diamine fluoride remineralized artificial incipient caries in permanent teeth after bacterial pH-cycling in-vitro
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