The Cytocompatibility of Silver Diamine Fluoride on Mesenchymal Stromal Cells from Human Exfoliated Deciduous Teeth: An In Vitro Study

Abstract

Silver diamine fluoride (SDF) has been used for many years for the treatment of caries, and minimally invasive dentistry concepts have made it popular again. The fact that its application does not require the administration of anesthesia makes its use in children more desirable. The aim of this study was to determine the cytotoxicity of two new commercial SDF products: Riva Star (SDI Dental Limited) and e-SDF (Kids-e-Dental) on mesenchymal stromal cells from human exfoliated deciduous teeth (SHEDs). SHEDs were exposed to SDF products at different concentrations (0.1%, 0.01% and 0.005%). Then different assays were performed to evaluate their cytocompatibility on SHEDs: IC50, MTT, cell migration (wound healing), cell cytoskeleton staining, cell apoptosis, generation of intracellular reactive oxygen species (ROS), and ion chromatography. Statistical analyses were performed using one-way ANOVA and Tukey’s post hoc test (p < 0.05). Riva Star Step 2 showed the same cell metabolic activity when compared to the control condition at any time and concentration. Meanwhile, e-SDF displayed high cytotoxicity at any time and any concentration (*** p < 0.001), whereas Riva Star Step 1 displayed high cytotoxicity at any time at 0.1% and 0.01% (*** p < 0.001). Only e-SDF showed a statistically significant decreased cell migration rate (*** p < 0.001) at all times and in all concentrations. At 0.1%, e-SDF and Riva Star Step 1 only showed 4.37% and 4.47% of viable cells, respectively. These results suggest that Riva Star has better in vitro cytocompatibility on SHEDs than does e-SDF. Riva Star Step 1 was found to be as cytotoxic as e-SDF, but it had better biological properties when mixed with Riva Star Step 2. Our findings suggest that Riva Star is more suitable when used in deciduous teeth due to its lower cytotoxicity compared to e-SDF.

Keywords: SHEDs; cytocompatibility; cytotoxicity; silver diamine fluoride.

Figure 1

SHED mesenchymal stem cell phenotype analysis by flow cytometry with specific antibodies for the mesenchymal surface markers CD73, CD90, and CD105, and the hematopoietic markers CD14, CD20, CD34, and CD45. Control isotype staining is also shown (grey histograms). Numbers inside histograms represent the percent of positive cells for each marker.

Figure 2

Calculation of IC50 values of the various SDF. The percent of concentration of each SDF in the extract was calculated for 50% inhibition of SHED metabolic activity. Data were analyzed by non-linear regression by plotting the percentage of the metabolic activity on the y-axis and the percentage of each varnish on the x-axis. Curves shown are representative from n = 3 separate experiments.

Figure 3

MTT assay. Metabolic activity of SHEDs after exposure to different concentrations of SDF products for 24, 48, and 72 h. Absorbance values at 570 nm were significantly different from the control group (** p < 0.01; *** p < 0.001) according to one-way ANOVA and Tukey’s post hoc test.

Figure 4

Migration of SHEDs after treatment with different SDF concentrations was analyzed by wound healing assays. Confluent SHED monolayers were cultured with complete growth medium (control) or different concentrations (0.1%, 0.01% and 0.005%) of the indicated SDF products for 72 h. Cell migration was expressed as the percentage of open wound area at each time point relative to the same wound area at 0 h (100%). Migration was significantly reduced compared to control (*** p < 0.001) according to one-way ANOVA and Tukey’s post hoc test.

Figure 5

Morphological aspects and cytoskeleton F-actin fibers organization on SHEDs cultures exposed to the indicated SDF by confocal fluorescence microscopy. F-actin fibers were stained with AlexaFluor™594-labeled phalloidin (Thermo Fisher, Carslbad, CA, USA) (red fluorescence); meanwhile, cell nuclei were counterstained with DAPI (blue fluorescence). Images shown are representative from three independent experiments carried on in triplicate for each material. Scale bar: 100 μm.

Figure 6

Flow cytometry analysis of cell apoptosis and necrosis induced by the different SDF concentrations on SHEDs by Annexin-V-FITC and 7-AAD staining. Numbers inside density plots represent percentages of live (Q4), early apoptotic (Q3), and late apoptotic necrotic cells (Q1 and Q2) at different concentrations (0.1%, 0.01% and 0.005%) and are representative from three independent experiments carried on in triplicate for each material.

Figure 7

Analysis of intracellular reactive oxygen production (ROS) after treatment with different dilutions of the indicated SDF products by CM-H2DCFDA staining. Representative histograms obtained in each experimental condition are shown. Bar graphs show the quantification of positive CM-H2DCFDA SHEDs obtained with each resin extract dilution and are represented as mean ± SD from three independent experiments performed in triplicate. Percentages of CM-H2DCFDA-positive cells were significantly increased compared to the control,*** p < 0.001; ns: no significant; respectively, according to one-way ANOVA and Tukey’s post hoc test.