In-vitro-cytotoxicity of cariostatic agents based on fluorides and lanthanide salts in L-929 fibroblasts

Abstract

Objectives: Fluoride-based cariostatic agents are commonly used in dental products and are generally considered safe. However, ongoing discussions about potential adverse effects are driving research into alternative agents, such as lanthanide salts. This study aims to evaluate the cytotoxic effects of different cariostatic agents, including fluoride compounds (NaF, Na₂PO₃F, NH₄F) and lanthanide salts (Ce(NO₃)₃, CeCl₃, Sm(NO₃)₃, SmCl₃).

Materials and methods: Mouse fibroblasts (L-929) were cultured in Eagle's Minimum Essential Medium supplemented with 5% fetal bovine serum and 1% penicillin-streptomycin. Cell viability was assessed via MTT assay after 24 h of exposure to test compounds at concentrations of 0.0128, 0.064, 0.32, 1.6, 8, 40, 200 and 1000 mM, and lactate dehydrogenase (LDH) release was quantified to assess membrane integrity. The accumulation of reactive oxygen species (ROS) was determined after 24 h. Data were analyzed by non-parametric methods at a significance level of α = 0.05 (Mann-Whitney U and Kruskal-Wallis tests).

Results: Cell viability decreased significantly for NaCl and NaNO₃ at 200 mM, and for NaF, NH₄F, Ce(NO₃)₃, CeCl₃, Sm(NO₃)₃ and SmCl₃ at 1.6 mM, falling below 70% of control (P ≤ 0.0178). Similarly, LDH assays indicated a significant incline in cytotoxicity at a concentration of 200 mM for NaCl, NaNO₃ and Na₂PO₃F, and at 8 mM for NaF, NH₄F and all lanthanide compounds (P ≤ 0.0016). ROS quantification showed that NaF, NH₄F, CeCl₃, Sm(NO₃)₃ and SmCl₃ induced oxidative stress at 1.6 mM with statistical significance (P ≤ 0.0065).

Conclusions: Fluoride and lanthanide compounds exhibited similar in vitro biocompatibility, comparable to that of table salt.

Clinical relevance: Both fluoride- and lanthanide-based cariostatic agents appear to pose a low biological risk to surrounding oral tissues when used at appropriate doses in dental products.

Keywords: Cariostatic agents; Cell death; Cell survival; Fluorides; Lanthanoid series elements; Toothpastes.

Fig. 1

Cell viability (green, left y-axis) and cytotoxicity (orange, right y-axis) assessed by MTT and LDH assays, respectively. Viability is expressed relative to the untreated control, toxicity relative to the lysis control (median: 1.16785; interquartile range: 0.9471–1.32915). Data are presented as medians with interquartile range (25th-75th percentiles). Asterisks denote significant differences compared to the untreated control (P ≤ 0.05). Concentrations marked with downward arrows were excluded due to visible precipitation

Fig. 2

Light microscopy images of L-929 cells cultured for 24 h without (a) or with (b-j) 8 mM test compounds. While untreated cells (a), NaCl (b), NaNO₃ (c) and Na₂PO₃F (d) showed normal morphology, cytotoxic changes were observed for NaF (e), NH₄F (f), SmCl₃ (g), CeCl₃ (h), Sm(NO₃)₃ (i) and Ce(NO₃)₃ (j). Scale bar: 50 μm

Fig. 3

Cell number (a) and reactive oxygen species production (b) in L-929 cells after 24 h exposure to test compounds. The dotted line indicates the median for the untreated control. Data are presented as medians with interquartile range (25-75th percentiles). Asterisks denote significant differences compared to the untreated control (P ≤ 0.05)