Temporal Exposure of Dermal Fibroblasts to Silver Diamine Fluoride and Fluorine NMR Quantitation

Objective: Silver diamine fluoride has been advocated as a caries arresting material for Early Childhood Caries (ECC) and has received considerable public attention as the “silver bullet”. However, cytotoxicity tests on the current concentrations of Silver Diamine Fluoride (SDF) to soft tissue have not been thoroughly assessed and analyzed at selected time intervals. The level of fluoride that is present within human cells has yet to be quantified. Preliminary SDF toxicity studies in our lab determined exposures of Dermal Fibroblasts to 0.03% SDF for 18 hours resulted in 100% cytotoxicity and complete monolayer loss. Endpoint titration of SDF determined that morphologic cytotoxic effects were ameliorated at input SDF levels lower than 0.002%. Because of the small culture sample volumes, we were unable to effectively assay fluoride concentrations using commercially available assays. In this study we attempted to assess fluoride levels in culture supernatants in a temporal fashion using quantitative Nuclear Magnetic Resonance (NMR).

Study design: Dermal Fibroblast (DF) cells were grown in 24 well cluster plates fitted with 0.4 micron TranswellTM inserts to confluency in 0.9mL of DF culture media. Then the DF cells were challenged with 0.1 mL of SDF in sterile water in the Transwell chamber to achieve a final concentration of 0.03% SDF. Cultures were reincubated for 30 minutes, 1, 2, 4 and 8 hours. At the selected time points Transwell inserts were removed. SDF culture media was removed and replaced with fresh media and allowed to re-incubate up to 8 hours. Harvested SDF culture media was centrifuged at 15,000 x g to remove any resulting SDF precipitates and supernatants were harvested and stored at −70°C for fluoride assay. After 8 hours, media was aspirated from all wells and DF cells were fixed and then stained with methylene blue and assessed for cytotoxicity. Harvested supernatants were assessed for fluoride content. While SDF is soluble in pure water, it precipitates instantly in the presence of other media constituents and 0.85% saline. Transwells inserts capture the precipitate but allow soluble SDF and constituents pass through to the cell monolayer. NMR was used to assess SDF (fluoride) prepared in water, in DF media or in normal saline at the same concentrations used in the DF cell studies. The 19F NMR spectra were acquired at 25 °C on an Agilent DD2 500 MHz spectrometer equipped with a 5mm HFX z gradient probe operating at 470.3 MHz for fluorine. For quantitative measurements, all spectra were collected with 64 scans and a delay of 5 seconds. The spectrum width is 220 ppm with offset at resonance of −110 ppm. The processing and analyzing were done by MNOVA. The dataset consists 45371 complex points and is zero-filled to the size of 128k points after applying 5Hz exponential line broadening. The 19F chemical shift was referenced indirectly based on proton chemical shift, which was referenced with respect to the water proton signal of 4.75 ppm at 25°C.

Results: Visible DF cell morphology changes begin to appear as early as 1 hour exposure to 0.03% SDF in Transwells and continue with degradation of cell morphology through 8 hours exposure at which point 100% of the cell monolayer is lost. The 8 hour image shows complete cell loss which is consistent with earlier studies using 24 hour exposures at 0.03% concentration. Note that the actual concentration of SDF affecting cell viability is shown in this study to be far lower than the 0.03% input because of the aggregate precipitation captured with the Transwell inserts. In this study, our NMR fluoride assessments showed that only 6–12 % of the input SDF fluoride reaches the lower cell chamber.

Conclusions: Considering that the SDF reagent is applied orally at ~40%, these results warrant more refined testing to identify true lower limit of toxicity end points of SDF. SDF should be utilized only by trained professionals and never contact soft tissue. NMR may be utilized to determine relative amounts of fluoride both in cell culture media and within fluoride exposed cells.

SDF; Cytotoxicity; Dermal fibroblasts cells; Transwells

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