Dimethyl sulfoxide affects the viability and mineralization activity of apical papilla cells in vitro

Abstract

Dimethyl sulfoxide (DMSO) is widely used as an adjuvant in dissolving insoluble compounds in an aqueous medium; however, it can induce significant molecular changes in cells. The possible damages may occur obeying a tissue-specific profile, and the effect on human apical papilla cells (hAPC) remains unknown. Therefore, this study aimed to evaluate DMSO effects on the viability and mineralization activity in hAPC cultures in vitro and to establish standards of maximum concentrations for its use in laboratory routines. hAPCs were cultured, plated, and maintained in media containing increasing concentrations of Dimethyl sulfoxide (0.1%, 0.5%, 1%, 5%, and 10%) for 24 h, 48 h, 72 h, and 7 days. At each time point, the cells were subjected to the MTT assay. The Alizarin red S staining assay was performed to evaluate the osteo/odontogenic mineralization potential of hAPC DMSO-exposed (0.1%, 0.5%, and 1%) in the 21-day time-point. Statistical analysis was performed using one-way analysis of variance followed by Tukey's post hoc test (p<0.05). In general, the 5% and 10% DMSO concentrations were shown to be cytotoxic for hAPC at all analyzed time points, and the hAPC DMSO-stimulated presented higher osteo/odontogenic mineralization potential. Therefore, the 5% and 10% DMSO concentrations should be avoided, and the mineralization activity assay should be carefully designed in order to avoid biases at in vitro assays using hAPC cultures.

Figure 1. Viability of human Apical Papilla Cells stimulated with Dimethyl sulfoxide. Absorbance/absorbance 0 (0% DMSO - control group) (570nm) data obtained from the MTT assay at experimental times of 24 h (A), 48 h (B), 72 h (C), and 7 days (D) in hAPC exposed to different concentrations of Dimethyl sulfoxide (0%, 0,1%, 0,5%, 1%, 5%, and 10%). The results showed the mean and standard deviation of the experiments performed in septuplicate. Different letters represent statistical differences between groups. (One-Way ANOVA with Tukey's test, p<0.05)

Figure 2. Human Apical Papilla Cells mineralization activity. Alizarin red S staining assay was performed to analyze the calcium deposition of hAPC exposed to different concentrations of Dimethyl sulfoxide (0.1%, 0.5%, and 1%) in the 21-day time-point. Cells under proliferation medium (A and F). Cells under differentiation medium (B and G). Cells under DMSO 0.1% (C and H), 0.5% (D and I), and 1% (E and J). Macroscopic view (A-E). Microscopic view (F-J). Semi-analytical densitometry of calcium standardized by the Alamar Blue (J). The results showed the mean and standard deviation of the experiments performed in triplicate. Different letters represent statistical differences between groups (One-Way ANOVA with Tukey's test, p<0.05).