RESEARCH ARTICLE |
https://doi.org/10.5005/jp-journals-10005-2423 |
Comparative Evaluation of Cariostatic and Remineralizing Potential of Two Commercial Silver Diamine Fluoride Preparations Using Confocal Laser Microscopy and EDX-SEM Spectroscopy: An In Vitro Study
1-5Department of Pediatric and Preventive Dentistry, Maharashtra Institute of Dental Sciences & Research, Latur, Maharashtra, India
6Private Practitioner, Mumbai, Maharashtra, India
Corresponding Author: Sagar Misal, Department of Pediatric and Preventive Dentistry, Maharashtra Institute of Dental Sciences & Research, Latur, Maharashtra, India, Phone: +91 8983879000, e-mail: sagarmisal096@gmail.com
ABSTRACT
Aim: To investigate the cariostatic and remineralizing effect of two commercial silver diamine fluoride (SDF) preparations on enamel and dentinal caries using a plaque bacterial model.
Materials and methods: Thirty-two extracted primary molars were divided into two groups (n = 16) as group I (Advantage Arrest) and group II (e-SDF). Plaque bacterial model was used to induce caries on enamel and dentin. Preoperative evaluation of samples was done using confocal laser scanning microscopy (CLSM) and energy-dispersive X-ray spectroscopy-scanning electron microscope (EDX-SEM). All samples were treated with test materials and evaluated for postoperative remineralization quantification.
Results: EDX revealed that mean preoperative levels (in weight%) of silver (Ag) and fluoride (F) in enamel carious lesions were 0.0 and 0.0, which increased postoperatively to 11.40 and 31.05 for Advantage Arrest and 13.61 and 31.87 for e-SDF, respectively. For dentinal caries, EDX revealed mean preoperative levels (in weight%) of Ag and F were 0.0 and 0.0, which increased to 11.47 and 48.71 for Advantage Arrest and 10.16 and 47.82 for e-SDF, respectively postoperatively. Both groups showed evident demineralization with exposed collagen under SEM. The mean values of enamel lesion depth for the group I and II were 38.64 and 39.30 µm, that reduced to 28.02 and 28.70 µm, while for dentinal caries, the mean depth from 38.05 to 38.29 µm that reduced significantly to 28.96 and 30.10 µm, respectively (p < 0.001). Caries depth declined significantly after the application of both Advantage Arrest and e-SDF (p < 0.001).
Conclusion: Advantage arrest and e-SDF show similar cariostatic and remineralization potential for dental caries. The plaque bacterial model used in this study is an efficient method to induce artificial carious lesions in teeth.
How to cite this article: Kale YJ, Misal S, Dadpe M, et al. Comparative Evaluation of Cariostatic and Remineralizing Potential of Two Commercial Silver Diamine Fluoride Preparations Using Confocal Laser Microscopy and EDX-SEM Spectroscopy: An In Vitro Study. Int J Clin Pediatr Dent 2022;15(4):442-449.
Source of support: Nil
Conflict of interest: None
Keywords: Confocal laser scanning microscopy, Dental caries, Fluoride, Silver diamine fluoride, X-ray emission spectroscopy.
INTRODUCTION
Dental caries continues to remain a global health problem, despite various advancements in dental care. Many chemical agents have been added to stop caries process.1 Ag++ (ionic silver) is known for its bactericidal effect and hence it was used for the first time in japan during late 1960’s and early 1970’s to arrest caries based on its antimicrobial property.2 The initial study done by Suzuki et al. (1974), demonstrated that after 3 minutes of application of SDF (silver diamine fluoride) the fluoride from the composition penetrates into enamel at approximately 25 µm depth, while silver at 20 µm depth.2 Though SDF is available in many concentrations, but 38% of SDF is most commonly used. Many studies reported that an economical, simple, and noninvasive method to arrest dental caries is the application of the SDF.3,4 Antibacterial effect of SDF5 also helps to inhibit matrix metalloproteinase.6 An increase in elemental content of the carious enamel lesions7 and the microhardness of the carious dentin lesions has been shown by the action of SDF.8,9 This cariogenic microflora produces acid, which causes demineralization of dentin. As a result, to prevent dentin demineralization and collagen degradation and to promote remineralization of dentin, the application of SDF is recommended.10,11
Silver diamine fluoride acts on caries by forming Ag phosphate crystals by reacting with the enamel of the tooth surface. Whenever SDF is applied to established carious lesions, it enters dentinal tubules by blocking the lumen of dentinal tubules. Compared to other Ag halides, silver fluoride (AgF2) is highly soluble in water. In addition to AgF2, SDF also has ammonium [NH4]+ in it. Silver diamine ion [Ag (NH3)2]+ is formed by combining [NH4]+ ions with Ag one. This [Ag (NH3)2]+ is reversible and highly stable compared to AgF2, remaining at high intensity for an extended time. Regardless of its advantages, SDF is not preferred by many clinicians in routine practice.12
This study aims to define the outcome of two commercial solutions of SDF on the enamel and dentinal caries using a plaque bacterial model and compare it with CLSM and EDX-SEM.
MATERIALS AND METHODS
We carried out this in vitro experimental study at the Department of Pediatric and Preventive Dentistry and Department of Microbiology, MIMSR Medical College, Latur, after gaining clearance from the Institutional Ethical Committee, letter no. MIDSR/STU/560/714/2019.
Inclusion Criteria
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Primary maxillary and mandibular molar, with an intact and healthy crown.
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Teeth extracted close to exfoliation (age 9–12 years male/female), showing physiological root resorption.
Exclusion Criteria
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Teeth showing evidence of caries, fracture, or other pathological defects.
Collection of Tooth Samples
The external surface of selected teeth was cleaned with curettes to remove soft tissue and debris. All specimens were stored in 5% sodium hypochlorite for 10 minutes to achieve removal of debris, soft tissues, and disinfection, and finally were rinsed with double deionized water. All the samples were kept in normal saline air and a light-protected jar at room temperature until the beginning of the experiment.
Grouping of Samples
Thirty-two extracted primary molars were divided into two experimental groups (n = 16) as follows:
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Group I: (Treated with Advantage Arrest) eight teeth for enamel caries and eight teeth for dentinal caries.
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Group II: (Treated with e-SDF) eight teeth for enamel caries and eight teeth for dentinal caries.
Preparation of Tooth Surface for Caries Induction
Induction of enamel and dentinal caries was done using the plaque bacteria model as follows: a small rounded window of 5 × 5 mm was prepared till the dentinoenamel junction on every specimen exposed the dentin of the tooth. Similarly, a rim of 5 mm intact enamel was left around the dentin well to induce enamel caries. All the remaining surfaces of the tooth except the window were coated with two layers of nail varnish, rendering them resistant to caries induction medium.
Preparation of Biological Caries Induction Media
Plaque from buccal surfaces of the maxillary and mandibular posterior and facial surface of anterior teeth, as well as debris from the carious lesion on teeth, were collected from a patient having high caries index. The plaque and carious dentin were mixed in 20 mL of brain heart infusion (BHI) broth followed by vortexing to gain homogeneous bacterial suspension to get the biological caries induction model. The BHI broth was incubated in a microaerophilic environment using a candle jar for 24 hours at 37°C in the incubator to enhance the growth of cariogenic bacteria. A total of 1 mL of biological caries induction media was added to 16 test tubes containing 5 mL nutrient and BHI broth, respectively, into which tooth samples were immersed completely. All the test tubes were screw-capped tightly to prevent contamination and incubated at 37°C in an aerobic as well as a microaerophilic environment. After every 5 days, broth from test tubes was replenished with freshly prepared broth to maintain the viability and cariogenic potential of the bacteria. This process was repeated for 3013 days for enamel carries induction and 90 days for dentinal caries induction.
Grouping of Samples
After 30 days, all the specimens were evaluated under a stereomicroscope to determine the depth of demineralization and the establishment of enamel and dentinal caries lesion. All the samples were equally separated into two groups, that is, group I (treatment with Advantage Arrest) and group II (treatment with e-SDF) (n = 16). Eight teeth from each group were evaluated for enamel caries and another eight teeth for dentinal caries.
Application of Test Materials
All the specimens from group I were treated with Advantage Arrest, while group II will be treated with e-SDF as recommended by the manufacturer, respectively.
Application of Advantage Arrest
All specimens were removed from the broth and rinsed with double deionized distilled water. The caries lesion was dried using tissue paper, and excess debris was removed using a micro brush. The applicator tip was immersed in a drop of Advantage Arrest (Elevate Oral Care, USA) and applied to the lesion gently for 2 minutes. Two minutes after the application, the teeth were washed with water spray, and these specimens were kept in neutral broth for 1 hour.
Application of e-SDF
All specimens were removed from the broth and rinsed with double deionized distilled water. The caries lesion was dried using tissue paper, and excess debris was removed using a micro brush. The applicator tip was dipped in a drop of e-SDF (Kids-e-Dental LLP, India) and applied to the lesion gently for 2 minutes. Two minutes after the application, the teeth were washed with water spray, and these specimens were kept in neutral broth for 1 hour.
Thermocycling of Samples
All the specimens were subjected to thermocycling at a temperature of 5–55°C for 500 cycles with a dwell time of 30 seconds.14,15 All the samples were checked for loss of acid-resistant varnish from their surface during this procedure.
Microscopic Evaluation of Samples
Surface Morphology
All samples from both groups were washed with distilled water ultrasonically three times, dehydrated in a desiccator, and finally sputter-coated with gold. The surface morphologies of the samples were then observed using SEM.
Elemental Analysis
Elemental analysis was carried out on the surface of all the specimens from each group. The levels of F, calcium (Ca), phosphorus (P), and Ag ions were estimated by EDX under SEM. The elemental analysis was done on the enamel and dentinal surface of each sample, and the mean weight percentage of F, Ca, P, and Ag was calculated for quantification of demineralization and remineralization.
Lesion Depth
All samples from both groups were scanned by a confocal laser microscope using Z-resolution sectioning. The three-dimensional (3D) photos were observed and managed using FLUOVIEW data analyzing software (FV10-ASW 4.2 Viewer). From the reconstructed 3D model of each sample, Z-sectional photos were traced, and the lesion depth was measured.
Statistical Analysis
The data obtained will be subjected to statistical analysis with Statistical Package for the Social Sciences version 22.0 statistical package for M3 Window (SPSS Inc. Chicago, IL, USA). Intragroup comparison in Advantage Arrest (group I) and e-SDF (group II) in cariostatic and remineralization potential in enamel caries was made. Intragroup mean and standard deviation were analyzed using paired “t” test.
RESULTS
Using EDX spectroscopy, it was found that there was an increase in Ca, F, and Ag levels (in weight %) after the application of Advantage Arrest (group I) and e-SDF (group II). In contrast, the decline in P levels (in weight %) was seen in the active carious lesions (Table 1 and Fig. 1).
| Enamel caries | ||||
|---|---|---|---|---|
| Advantage Arrest (group I) | Before application Mean (SD) |
After application Mean (SD) |
Paired t-test | p-value, significance |
| Ca | 16.66 (8.63) | 31.60 (8.13) | t = –3.561 | p = 0.003 |
| P | 12.55 (3.83) | 7.91 (2.06) | t = 3.01 | p = 0.009 |
| F | 0.0 (0.0) | 11.40 (3.17) | t = –10.14 | p < 0.001** |
| Ag | 0.0 (0.0) | 31.05 (8.36) | t = –10.50 | p < 0.001** |
| e-SDF (group II) |
Before application Mean (SD) |
After application Mean (SD) |
Paired t-test | p-value, significance |
| Ca | 22.08 (8.31) | 28.09 (5.87) | t = –1.670 | p = 0.117 |
| P | 12.09 (6.10) | 10.83 (2.11) | t = 0.553 | p = 0.589 |
| F | 0.0 (0.0) | 13.61 (3.77) | t = –10.205 | p < 0.001** |
| Ag | 0.0 (0.0) | 31.87 (5.99) | t = –15.07 | p < 0.001** |
*p < 0.05 - significant difference; **p < 0.001 - highly significant difference; SD, standard deviation
Fig. 1: Graph showing intragroup comparison in Advantage Arrest (group I) and SDF (group II) in cariostatic and remineralization potential in enamel caries
Elemental Analysis
The EDX results were constant with the characteristic mineral concentration outline. In enamel caries, an increase in F and Ag levels (in weight%) postapplication was higher in both groups, and the difference was found to be statistically significant. In both groups increase in Ca levels and a decrease in P levels (in weight%) were seen, but the difference was not found to be statistically significant (Table 1 and Figs 3A to D).
Whereas in dentinal caries, EDX analysis showed that an increase in F-Ag levels (in weight%) postapplication was higher in both groups, and the difference was found to be statistically significant. There was an increase in the level of P in both groups, but the increased level of P in group II was found to be statistically significant as compared to group I (Table 2, Figs 2 and 3E to H).
| Dentinal caries | ||||
|---|---|---|---|---|
| Advantage Arrest (group I) | Before application Mean (SD) |
After application Mean (SD) |
Paired t-test | p-value, significance |
| Ca | 16.66 (8.63) | 16.80 (4.93) | t = –0.042 | p = 0.967 |
| P | 9.11 (5.29) | 5.93 (3.21) | t = 1.452 | p = 0.169 |
| F | 0.0 (0.0) | 11.47 (2.73) | t = –11.86 | p < 0.001** |
| Ag | 0.0 (0.0) | 48.71 (9.49) | t = –14.51 | p < 0.001** |
| e-SDF (group II) |
Before application Mean (SD) |
After application Mean (SD) |
Paired t-test | p-value, significance |
| Ca | 22.08 (8.31) | 15.32 (4.15) | t = 2.059 | p = 0.059 |
| P | 12.90 (3.70) | 4.55 (3.04) | t = 4.918 | p < 0.001** |
| F | 0.0 (0.0) | 10.16 (2.02) | t = –14.17 | p < 0.001** |
| Ag | 0.0 (0.0) | 47.82 (4.19) | t = –32.24 | p < 0.001** |
*p < 0.05 - significant difference; **p < 0.001 - highly significant difference; SD, standard deviation
Fig. 2: Graph showing intragroup comparison in Advantage Arrest (group I) and SDF (group II) in cariostatic and remineralization potential in dentinal caries
Figs 3A to H: Shows elemental analysis (in weight %) using EDX- energy dispersive X-ray spectroscopy. (A) Enamel specimen before application of Advantage Arrest; (B) Enamel specimen after application of Advantage Arrest; (C) Enamel specimen before application of e-SDF; (D) Enamel specimen after application of e-SDF; (E) Dentin specimen before application of Advantage Arrest; (F) Dentin specimen after application of Advantage Arrest; (G) Dentin specimen before application of e-SDF; (H) Dentin specimen after application of e-SDF
Evaluation of Surface Morphology
The surface morphology of the samples was evaluated using an SEM. In arrested dentinal lesions, a smooth surface with exposure to dentine collagen fibers was seen (Figs 4A to D). When viewed under high magnification, disorganized and sparse distribution of collagens and dense structure of spherical granules in the intertubular area was found. The dentinal tubules were blocked (Figs 4E to H).
Figs 4A to H: Representative SEM images of the enamel and dentin surface morphology before and after application of Advantage Arrest and e-SDF. (A) Enamel specimen before application of Advantage Arrest; (B) Enamel specimen after application of Advantage Arrest; (C) Enamel specimen before application of e-SDF; (D) Enamel specimen after application of e-SDF; (E) Dentin specimen before application of Advantage Arrest; (F) Dentin specimen after application of Advantage Arrest; (G) Dentin specimen before application of e-SDF; (H) Dentin specimen after application of e-SDF
Lesion Depth
Confocal imaging showed that there was a decline or decrease in depth (in µm) of enamel and dentinal lesion; that is, increases in cariostatic and remineralization property was found using both interventions (Table 3). The decline in the depth of enamel and the dentinal lesion was seen in both groups, Advantage Arrest (group I) and e-SDF (group II), and the difference was found to be statistically significant (p < 0.05).
| Enamel caries | ||||
|---|---|---|---|---|
| Before application Mean (SD) |
After application Mean (SD) |
Paired t-test | p-value, significance | |
| Advantage Arrest (group I) | 38.64 (1.61) | 28.02 (1.63) | t = 13.089 | p < 0.001** |
| e-SDF | 39.30 (1.53) | 28.70 (1.75) | t = 12.872 | p < 0.001** |
| Dentinal caries | ||||
| Before application Mean (SD) |
After application Mean (SD) |
Paired t-test | p-value, significance | |
| Advantage Arrest (group I) | 38.05 (1.98) | 28.96 (1.77) | t = 9.676 | p < 0.001** |
| e-SDF | 12.90 (3.70) | 30.10 (0.88) | t = 9.409 | p < 0.001** |
*p < 0.05 - significant difference; **p < 0.001 - highly significant difference; SD, standard deviation
DISCUSSION
This was the first research to examine the cariostatic and remineralization potential of two commercial preparation of SDF solutions, that is, Advantage Arrest (Elevate Oral Care, USA) and e-SDF (Kids-e-Dental LLP, India). It showed that the application of both the commercial SDF preparations had a comparable cariostatic and remineralization potential on enamel and dentinal caries. Many types of research have been carried out to examine the demineralization and remineralization process in vitro. In this research, we used a bacterial plaque model followed by pH cycling to induce artificial enamel and dentinal caries to mimic the early caries formation.
The development of caries is an active natural procedure. It comprises demineralization and remineralization of the surface of the tooth,16 which is induced by the acids produced by bacteria that settle on the surface of the tooth. The surface of the enamel contains these oral bacteria, which are part of the dental plaque. The demineralization and remineralization process is affected by the constituents of the dental plaque, which leads to the dissemination of carbohydrates, acids, and ions to and from the surface of the tooth.17 Acid attack leads to the dissolution of enamel crystallites, and caries formation begins.16 It has been debated that successive demineralization of surface and precipitation of mineral content of enamel surface build up the early carious lesions.18 3D models of the investigational caries-like lesions permit inspecting the extension of the lesion into the enamel and defining morphological characteristics of the lesion. Carious lesion induced by this bacterial plaque model was uniform in size and development when the SDF was applied topically. The elemental content and the depth of the lesion were uniforms among all the samples, the analysis was qualitative, and the result of the research could be widespread. Moreover, it is likely to define the volume of the lesion quantitatively with 3D reconstruction models.19
There is a low level of F on the surface layer of a normal, carious lesion, as described by earlier studies.20 Food, saliva, or drinking water are some of the sources of this lower concentration of F in the mouth. As soon as the pH of the oral cavity becomes less than the critical pH of saliva, this F reacts with the hydroxyapatite, and low solvable fluorapatite crystals are formed, which helps in decreasing the value of critical pH and also reduces the enamel dissolution.21 This action of F helps in the inhibition of demineralization. All the clinical situations of the early carious lesion were replicated in the model used for caries induction. The observations of the study showed significant changes in absorption of F before and after the application of both SDF solutions via EDX assessment. The results obtained from the present study were similar to the research led in the past by Mei et al.22 showed that the application of SDF on the arrested caries of primary teeth has a highly mineralized area. There is induction of unbalanced F, which is present at the tooth-saliva boundary with the application of topical F. This unbalanced F occurs in the form of Ca-F deposits, and its suspension is reliant on the pH of the oral cavity. As soon as the rate of pH descends below critical pH, there is a release of labile F, which is bound firmly. Hence, the crystals of F hydroxyapatite are the end result.23 Another reason for the increase in F intake may be the liquidity of the SDF preparations. The fluidity of SDF helps in the full contact of the solution with the carious surface within a very short period of time.
The exposure of collagens in active carious lesions in evasion of hydroxyapatite structure suggests that there was demineralization of the dentine. This collagen is a strength of the dentine, which helps in holding the apatite crystallites together.24 The cross-section of the arrested carious lesion showed compact granules of spherical grains under SEM. This compact granule suggests the formation of extra-fibrillar minerals, which inhibited the collagens from being exposed. Also, the action of SDF helps in the inhibition of action of matrix metalloproteinases,6 cysteine cathepsins,25 and bacterial collagenase.10 This action of SDF might be responsible for the protection of the collagens from being destructed by the endopeptidases and collagenase. Also besides, the increased resistance of collagen and enhanced chemical and physical property of collagen might be due to the higher levels of Ag ion in SDF solution. Similar results were found in the study conducted by Mei et al.,10 which showed inhibition of demineralization and preservation of collagens from degradation in demineralized dentine after the application of 38% SDF.
There was also a significant difference in the depth of the lesion before and after applying both products measured by CLSM. Lesion depth reveals the degree of mineralization and cariostatic effect of both solutions. The result shows that there was a similar degree of remineralization in both groups, that is, Advantage Arrest and e-SDF. The concentration of F in both 38% SDF preparation is 44,800 ppm. There is no commercial product in dentistry having this high level of F. Stable results were obtained with the data from the EDX test, which showed that there was comparable F uptake in both groups, which decreased the depth of lesion measured by confocal microscopy.
Both products showed a more substantial remineralization effect and decreased the depth of the lesion, that is, the cariostatic effect. However, the exact action of SDF on carious lesions was not implicit. The presence of higher levels of F, Ag, and alkaline properties of SDF might be responsible for the cariostatic effect on carious lesions.26 The remineralization of hydroxyapatite is stimulated by F. The alkaline nature of SDF helps to encourage the remineralization and inactivation of dentinal collagenases.27 The earlier studies clarified that the insolubility of the formed Ag composite and the antibacterial effect of Ag is responsible for the action of Ag in SDF on the enamel carious lesion.26,27 In the current research, the increase in Ag was confirmed by EDX before and after the application of both SDF products. The presence of Ag nanoparticles on the hydroxyapatite crystals of both groups, which were treated with SDF, was confirmed using EDX. Hence, there is suppression of the bacterial adhesion by the Ag-containing hydroxyapatite, which helps in the arrest of the caries process.
CONCLUSION
Based on the results of the study, it can be concluded that Advantage Arrest and e-SDF show similar cariostatic and remineralization potential for dental caries. The bacterial model used in this study is an efficient method to induce artificial carious lesions in teeth.
ACKNOWLEDGMENTS
The authors are thankful to Dr Siddiqui, Professor at the Department of Microbiology and Biotechnology, Dayanand Science College, Latur, for her assistance in our study.
ORCID
Yogesh J Kale https://orcid.org/0000-0003-0612-0014
Sagar Misal https://orcid.org/0000-0003-2523-3946
Mahesh Dadpe https://orcid.org/0000-0003-4315-4432
Prasanna T Dahake https://orcid.org/0000-0003-0295-5751
Shrikant B Kendre https://orcid.org/0000-0003-3070-0238
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