Effect of Potassium Iodide and Glutathione on Color Change and Remineralization Potential Induced by Silver Diamine Fluoride Application

BACKGROUND: To evaluate the eﬀect of applying potassium-iodide (KI) and glutathione (GSH) on silver-diamine ﬂuoride (SDF) induced demineralized den:n discolora:on as well as their eﬀect on its remineraliza:on poten:al. MATERIALS AND METHODS : To examine color change, cervical den:nal demineralized cavi:es were performed mesially and distally in 16 human premolars. Glass-ionomer restora:on (GIC) was applied and allocated to four groups I-IV according to the following pre-treatments: No pre-treatment, SDF, SDF+KI, SDF+20% (wt) GSH. Spectrophotometric evalua:on of samples at :me intervals: 1,7 and 14 days of GIC applica:on. To examine the eﬀect of remineraliza:on, 21 bovine den:n blocks were divided into groups I-III: SDF, SDF + KI, and SDF + GSH. Vickers microhardness was measured, before and aZer demineraliza:on and aZer 7 days of treatment. RESULTS: Spectrophotometric results aZer 14 days for groups I-IV were: 1.29±0.18, 12.24±0.19, 2.19±0.32 and 4.76±0.19 respec:vely. Groups III and IV showed signiﬁcant reduc:on in ΔE compared with Group II, although they showed signiﬁcant increase in ΔE compared with group I (p<0.001). KI showed beber management of color changes than GSH. The microhardness test results aZer treatment applica:on to demineralized den:n for Groups I-III were: 30.81±20.87, 30.59±16.42, 24.69±13.21, respec:vely. All groups showed signiﬁcantly increased microhardness of demineralized den:n (P≤0.05), which was comparable to that of Group I. CONCLUSION: Applica:on of KI and GSH aZer SDF signiﬁcantly minimized color changes without aﬀec:ng the remineralizing eﬀect of SDF.


Introduction
Dental caries is one of the most prevalent health conditions worldwide, despite efforts to control it [1].Untreated dental caries lead to general health problems, including pain, poor quality of life, and reduced productivity [1,2].Cariology research currently focuses on the effectiveness of medical treatments for caries arrest and remineralization rather than the conventional invasive drilland-fill approach for caries management [3].
Atraumatic Restorative Technique (ART) is considered a minimal intervention method for caries management using hand instruments for carious tissue removal and restoring decayed tooth structure by high viscosity glass ionomer cement h#p://den*stry3000.pi#.edurestoration (HVGIC) [4].This technique showed less dental anxiety than the traditional drill-and-fill approach [5].As a modification to this technique for a better prognosis, it was suggested to pre-treat the remaining carious tooth structure with SDF prior to HVGIC restoration application.This modification was named Silver Modified Atraumatic Restorative Technique (SMART) [6,7].As lately, SDF gained attention for arresting and remineralizing carious dental lesions [8].In 2014, the FDA approved SDF for caries arrest in both dentitions [9].Furthermore, no need to remove carious dental tissues before SDF application [10], which made its use simple, economical and patient-friendly [11].Previous studies have shown a promising effect of SDF in the control of carious dentin demineralization [12,13].
Despite the advantages of SDF, yet it has limited desirability owing to its dark-staining effect [14].Even application of glass ionomer restoration couldn't effectively mask this dark staining effect [15].The purpose of this study was to reduce this discoloration without affecting the remineralization potential of the SDF.Potassium iodide (KI) application after SDF was suggested, which resulted in a creamy yellowish-white silver iodide (AgI) precipitate by binding to free silver ions from the SDF [16].However, it was shown that the demineralized dentin microhardness was significantly decreased by reducing the silver ion count as a result of KI addition to SDF [17,18].Therefore, in this in vitro study, we tested glutathione (GSH), a relatively unpopular new material for discoloration management.Glutathione (GSH) is an intracellular non-protein thiol (NPSH) antioxidant that acts as a metal chelator, which may decrease free silver ion aggregation by coating its particles without affecting its count [19].
Accordingly, this study aimed to evaluate the effect of applying KI and GSH on SDF induced tooth discoloration as well as their effect on SDF remineralization potential.

Color change spectrophotometric evaluation
Sixteen sound-extracted permanent human premolars were used in this study.Premolars were collected from the Oral and Maxillofacial Surgery Department of the Faculty of Dentistry at Ain Shams University.The Ain-Shams University Faculty of Dentistry Ethical Committee approved the protocol for teeth collection and storage (Approval No. RECEM 011910).The teeth were rinsed with deionized water and cleaned with a periodontal curette to remove any soft tissue debris.Teeth with stains and extreme visual discoloration were excluded, such that teeth were visually with homogenous color.The teeth were then polished using a polishing cup and a pumice.Standardized 2 mm deep square cavities of size 4 × 4 mm were made on the mesial and distal sides of each premolar at the cemento-enamel junction using tungsten carbide bur flat fissure (FG 557-Prima dental-UK) and NSK handpiece high speed under copious amount of water.Each premolar was completely covered with a clear nail varnish (YOLO, A.R.E), except for the mesial and distal cavity windows, which were kept unprotected.Additionally, the apical foramen was occluded externally with the varnish for no contamination.This resulted in 32 specimen, which were then distributed randomly into four experimental groups (n= 8) according to the treatment protocol (Figure 1).Each group had 8 samples (which means 4 teeth), such that the same treatment is applied to both sides of the tooth, avoiding contamination of variable treatments during storage.
h#p://den*stry3000.pi#.eduEach tooth, including two specimens (one on each side), was separately immersed in an acidic buffer solution for 72 h in a separate container at room temperature.The pH was adjusted by the addition of 1 M KOH to maintain a pH of 4. The demineralizing solution was changed every 24 hours [20].As according to the pilot study done, the demineralization depth was average 150 ± 15 µm measured at Oral Pathology Department, faculty of dentistry, Ain-Shams University with polarized light microscope with magnification power X10 using a digital camera (EOS 650D, canon, Japan) which was mounted on a light microscope (BX60, Olympus, Japan).
Thirty-two specimens were allocated randomly to four treatment groups (n=8).In Group I, the cavities of demineralized dentin were filled with GIC restorations without pre-treatment.While in Group II 38% SDF solution was applied for 1 minute with a micro-brush according to the manufacturer's instructions, Group III was treated with the SDF solution as mentioned previously followed by immediate application of saturated KI solution until the creamy white precipitate became clear and in Group IV demineralized dentin was treated by a mixture done by mixing 38% SDF solution with 20% (wt) GSH until the mixture became clear with no precipitates and then applied by a microbrush for one minute.Then all cavities in Groups II, III, IV were then washed with copious amounts of distilled water for 30s and dried with oil-free compressed air and filled with GIC restoration (Table 1).All previous work was done by a single experienced practitioner for standardization.Then all the samples were stored individually in a deionized solution.
Color assessments of the specimens (n=32) were recorded at three-time intervals: baseline after 24 h of GIC restoration application, 7 days, and then 14 days.Color changes were recorded using a spectrophotometer (Agilent Cary 5000-USA) on the GIC restoration surface at the National Institute of Standards (NIS-Egypt-Giza).It was calibrated and used in the standard mode in the visible wavelength region (380nm to 780nm) for all samples.Computed Cie Lab values were measured under Illuminate D65 with a 10 degrees standard observer.The Cie Lab coordinates L* a* b*, hue and chroma of each sample at variable time interval was evaluated.Then the ΔL, Δa, Δb between different time intervals of each sample was calculated and hence the ΔE color change between time intervals was measured.Finally, the mean and standard deviation SD of each group was statistically evaluated.
Teeth coverage with varnish except for mesial & distal window.

Cavities demineralization by immersion in acidic buffer pH.=4 for 72
hrs.

Figure 1. Flow chart for Experimental design of color change assessment
h#p://den*stry3000.pi#.edu

Remineralization and microhardness evaluation
Bovine teeth were collected from cattle slaughtered in the foodmanufacturing industry.None of the animals were harmed to participate in this study.Twenty-one bovine dentin samples (6×6×2 mm) were prepared from the labial and lingual surfaces of the cervical portion of incisor roots.The samples were then cut using a low-speed disc for each dentine disc was used for statistical analysis.

Statistical analysis
Numerical data are presented as both, the mean and standard deviation (SD).Kolmogorov-Smirnov and Shapiro-Wilk tests were used to check the data distribution to assess the normality.All data presented a normal parametric distribution, such that they were analyzed using oneway ANOVA followed by Tukey's post hoc test for intergroup comparisons.While repeated measures were analyzed using ANOVA followed by Bonferroni post hoc test for intragroup comparisons.The significance level was set at p ≤0.05.Statistical analysis was done using the R statistical analysis software, version 4.1.3[21].Cohen's d was calculated and interpreted based on Lakens' formula and thresholds using the online resource available at https://effect-sizecalculator.herokuapp.com/[22].The R package MBESS was used to calculate the confidence intervals (CI) for Cohen's d using the noncentral t method as in Table 3&4, 6&7 [23].

Spectrophotometric results
The results showed that the highest value of color change ΔE was found in SDF group II, followed by SDF + GSH group IV, and SDF + KI group III, while the lowest value was found in Group I at all time intervals.There was a significant interaction between the time and group.Post-hoc pairwise comparisons showed that the values of the different groups were significantly different from each other (p<0.001),as shown in

Discussion
The silver-modified atraumatic restorative technique (SMART) offers an alternative minimal interventional treatment for caries management by arresting caries progression using SDF and restoring tooth form by HVGIC [7,24,25].However, one of the major drawbacks of the SDF treatment is its black staining effect on the carious tooth structure, which has limited its acceptance by aesthetically concerned patients and accordingly its general use [26,27].This discoloration is due to the reaction of SDF with carious dentin resulting in the production of silver phosphate (Ag3PO4), silver chloride, and free metallic silver.Ag3PO4 is yellow when it is first formed but readily turns black under the influence of reducing agents or sunlight.This black impermeable layer prevents the progression of caries [12,13,28,29].Finding a solution for the dark staining without affecting SDF's biological effect on carious dentin would provide a great opportunity for the universal use of SDF.Potassium iodide was introduced commercially as a solution to reverse this discoloration but unfortunately controversies regarding its effect on the antibacterial and remineralization potential of the SDF [30][31][32][33].
Introducing an alternative option for discoloration management was in need.For that reason, glutathione biomolecule was suggested as an alternative to reduce teeth discoloration without affecting the |d| < 0.2 "negligible", |d| < 0.5 "small", |d| < 0.8 "medium", otherwise "large".
There is limited evidence regarding efficiency of GSH compared to commercially available KI.
Accordingly, this study aimed to evaluate the efficiency of GSH compared to KI on remineralization potential and teeth discoloration induced by SDF treatment of demineralized dentine.
High viscosity Glass-ionomer restoration was used as simulation for clinical application of SMART technique as it offers chemical bonding to dentine with good cavity seal, remineralizing effect, biocompatibility, ease of application, and low cost [34].For standardization of tested variables, packable self-cure glass ionomer was applied to avoid any further discoloration that may be caused by the light curing; as the interaction products of both SDF and KI (Ag3PO4 and AgI) are photosensitive [14,15].Cavities were done at the cemento-enamel junction for location standardization and for supporting SDF use in root caries arrest, as previously proposed in other studies due to its effectiveness and ease of application [35][36][37][38][39].
Spectrophotometer was used for evaluation of the teeth discoloration as one of the most commonly used tools to assess the color change [40].
In Group II, the change with time was the highest among all groups as shown in table 2. GIC restoration had limited discoloration masking effect [15].While Group III & IV showed significant reduction in color change relative to Group II at all time intervals (Table 2).Still, they had significant change of color with time.
As in Group III, upon application of KI immediately after SDF, a bright yellow silver iodide (AgI) compound was formed [31,32,41].However, this yellow color soon turned dark and became darker with time.This could be justified by the amount of KI solution that wasn't enough to interact with all the free silver ions, or may be attributed to the high photosensitivity of AgI that readily dissociates into black metallic silver again by light exposure [27].On the other hand, in Group IV glutathione biomolecules reduced discoloration owing to its high adsorption affinity onto metallic surfaces which in turn might have decreased silver particle aggregation.Additionally, GSH has a haemostatic effect which might have controlled the rate of silver ion release [42].Application of a mix of 20%(wt) GSH with SDF prior GIC restoration in SMART for color management was new [19].In Group I color change was visually undetectable, but it was significant along with time.This may be attributed to the degradation of the metal-polyacrylate bonds as an inherent property in GIC restorations, causing lack of color stability [43].
Accordingly, it was assumed that the percentage of GSH was not sufficient to completely coat all the excess silver particles to efficiently reduce the color change.Pilot trials to dissolve more percentage of glutathione powder in 38% SDF solution resulted in mixture with precipitates.However, GSH ability to decrease the color changes of dentin may encourage further studies to better understand simpler ways of increasing the GSH concentration in SDF solutions.According to the results of the current study, KI is more effective than 20%(wt) GSH in reducing the color changes of demineralized dentin.This finding disagree with a pilot study that was done after aging and found no significant difference in color change between application of SDF only and adding KI to it prior to GIC restoration [30].
Regarding the microhardness results, bovine teeth were reported to resemble human teeth in multiple features including dentin surface properties [44].It offered more homogeneity in respect to mineral composition compared with human teeth which is beneficial for invitro studies [45].The base line of the demineralized dentine was standardized without any significant difference between the randomly distributed dentine discs.The different groups showed a significant increase in the microhardness of demineralized dentin, with no significant difference between the different treatments (Table 5).This can be explained by an increase in the mineral content of dentin by extrafibrillar and intrafibrillar mineral formation by the deposition of spherical grains, which were h#p://den*stry3000.pi#.educonfirmed by SEM investigations in a previous study [46].Our results supported the assumptions in previous studies that silver has no role in the SDF remineralizing effect [18].This justifies that addition of KI to the SDF did not affect its remineralization properties.The increase in dentin surface hardness after the application previous treatments was due to the recovery of nanomechanical properties of the demineralized dentin surface by crystal precipitation [47].Meanwhile, our results contrast those of other study that showed that KI application significantly reduced the hardness effect of the SDF compared with the unaccompanied SDF.In this latter study, micro-computed tomography (micro-CT) and Fourier transform infrared (FTIR) spectroscopy were used for indirect measurement of remineralization potential [31].
As an in-vitro study, there are some accompanied limitations, as the artificial demineralization, which was created by an acidic solution rather than combining the bacterial caries model as in real.This method is extensively used in research due to its simplicity and reproducibility [20].In addition, we were focused mainly on the anti-demineralizing effects of SDF.Moreover, the usage of deionized water as a storage medium between variable time intervals after treatment application, was another limitation as ionic concentrations were kept at unrealistic low levels than that of viable dentinal fluid [48], which may affect discoloration intensity by further interacting with SDF.In vitro studies are different from the more complex clinical situation.Such that the results cannot be directly extrapolated to the in-vivo condition.Another limitation was the inability to dissolve more than 20%(wt) of glutathione powder in SDF solution, whether using a magnetic stirrer or glass slab and spatula, since exceeding the 20%(wt) caused white precipitations in mixture.
Accordingly, in this in-vitro study we were concerned with managing the SDF induced discoloration, in a trial to motivate SDF application as a liner beneath final restoration, in deep carious lesions.

Conclusion
Within the limitations of this in-vitro study, 20%(wt) GSH is a promising solution, but with less efficiency than KI, for management of SDF induced teeth discoloration without compromising the remineralization potential.Further studies with higher concentrations of GSH are recommended.

Table 1 :
List of materials used, chemical composition, and manufacturer.

Table 2 :
Mean, Standard deviation (SD) values of color change (ΔE) for different groups.

Table 6 :
Effect sizes (95% confidence intervals) of the intergroup comparisons of microhardness.