Restor Dent Endod.  2023 Feb;48(1):e6. 10.5395/rde.2023.48.e6.

High-plasticity mineral trioxide aggregate and its effects on M1 and M2 macrophage viability and adherence, phagocyte activity, production of reactive oxygen species, and cytokines

• Vasconcellos BC
• Tavares LCT
• Silva DCd
• Fonseca FO
• Benetti F
• Sobrinho APR
• Tavares WLF

• KMID: 2548174
• DOI: http://doi.org/10.5395/rde.2023.48.e6

Abstract

Objectives
This study evaluated the effects of high-plasticity mineral trioxide aggregate (MTA-HP) on the activity of M1 and M2 macrophages, compared to white MTA (Angelus).
Materials and Methods
Peritoneal inflammatory M1 (from C57BL/6 mice) and M2 (from BALB/c mice) macrophages were cultured in the presence of the tested materials. Cell viability (MTT and trypan blue assays), adhesion, phagocytosis, reactive oxygen species (ROS) production, and tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β production were evaluated. Parametric analysis of variance and the non-parametric Kruskal-Wallis test were used. Results were considered significant when p < 0.05.
Results
The MTT assay revealed a significant decrease in M1 metabolism with MTA-HP at 24 hours, and with MTA and MTA-HP later. The trypan blue assay showed significantly fewer live M1 at 48 hours and live M2 at 48 and 72 hours with MTA-HP, compared to MTA. M1 and M2 adherence and phagocytosis showed no significant differences compared to control for both materials. Zymosan A stimulated ROS production by macrophages. In the absence of interferon-γ, TNF-α production by M1 did not significantly differ between groups. For M2, both materials showed higher TNF-α production in the presence of the stimulus, but without significant between-group differences. Likewise, TGF-β production by M1 and M2 macrophages was not significantly different between the groups.
Conclusions
M1 and M2 macrophages presented different viability in response to MTA and MTA-HP at different time points. Introducing a plasticizer into the MTA vehicle did not interfere with the activity of M1 and M2 macrophages.

Keyword

Cytokines; Macrophages; Mineral trioxide aggregate

Figure

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  Figure 1 Percentage of living M1 (A) and M2 (B) macrophages after incubation in 96-well culture plates with capillaries containing MTA and MTA-HP by the MTT assay. Controls were cultured with empty capillaries. Cultures were maintained for 24, 48, and 72 hours, as described in the Materials and Methods. Bars represent the mean of 2 experiments; lines denote the standard error of the mean.MTA, mineral trioxide aggregate; HP, high-plasticity.*A statistically significant difference in cell viability between the 2 macrophage culture conditions and cell viability between both macrophage cultures stimulated by MTA and MTA-HP compared to control (p < 0.05), by analysis of variance, and ★ indicates difference between the two materials compared to control group (P<0.05).

• 

  Figure 2 Percentage of living M1 (A) and M2 (B) macrophages after incubation in 24-well culture plates with capillaries containing MTA and MTA-HP by the trypan blue exclusion assay. The controls were cultured with empty capillaries. Cultures were maintained for 24, 48, and 72 hours as described in the Materials and Methods. Bars represent the mean of 2 experiments; lines denote the standard error of the mean.MTA, mineral trioxide aggregate; HP, high-plasticity.*A statistically significant difference in cell viability between the 2 macrophage culture conditions and cell viability between both macrophage cultures stimulated by MTA and MTA-HP compared to control (p < 0.05), by analysis of variance.

• 

  Figure 3 Percentage of adherent M1 (A) and M2 (B) macrophages after incubation in culture plates with capillaries containing MTA and MTA-HP. The control were cultures with empty capillaries. Cultures were performed as described in the Materials and Methods. Bars denote the mean of results of 3 experiments performed in duplicate. Lines indicate the standard error of the mean (p < 0.05).MTA, mineral trioxide aggregate; HP, high-plasticity.

• 

  Figure 4 Percentages of M1 (A) and M2 (B) macrophages displaying phagocytosed yeast cells after incubation in culture plates with capillaries containing MTA and MTA-HP. Controls were cultured with empty capillaries. Cultures were performed as described in the Materials and Methods. Bars denote the mean results of 3 experiments performed in duplicate. Lines indicate the standard error of the mean. No statistically significant difference between M1 and M2 cells under the same conditions was observed (p > 0.05).MTA, mineral trioxide aggregate; HP, high-plasticity.

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  Figure 5 Kinetic of ROS production by M1 (A) and M2 (B) macrophages. Cells were cultured with capillaries containing MTA and MTA-HP and stimulated with zymosan A, as described in the Materials and Methods. Bars denote the mean results of 3 experiments performed in duplicate. Lines indicate the standard error of the mean. Different symbols indicate a statistically significant difference (p < 0.05) for ROS production compared with M1 or M2 macrophages in medium with or without stimulation, by analysis of variance.M, macrophage culture containing the materials or not (control); M + Z, macrophage cultures stimulated with zymosan A in the presence or absence of materials (control); ROS, reactive oxygen species; MTA, mineral trioxide aggregate; HP, high-plasticity.

• 

  Figure 6 Mean production of TNF-α by M1 (A) and M2 (B) macrophages cultured in the absence (control) or presence of MTA and MTA-HP. The cells were cultured in the medium alone or in the presence of IFN-γ. Bars denote the mean results of 3 experiments performed in duplicate. Lines indicate the standard error of the mean.M, the presence of macrophages; M + IFN, the presence of macrophages and interferon-γ in the mineral trioxide aggregate, high-plasticity mineral trioxide aggregate, and control groups; TNF, tumor necrosis factor; IFN, interferon; MTA, mineral trioxide aggregate; HP, high-plasticity.* indicates a statistically significant difference (p < 0.05) in TNF-α mean production when specific cultured M2 macrophages were compared with other M1 or M2 macrophages in medium with or without stimulation, by analysis of variance. ★ indicates difference between the two materials compared to control group (P<0.05).

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  Figure 7 Mean production of TGF-β by M1 (A) and M2 (B) macrophages cultured in the absence (control) or presence of MTA and MTA-HP. The cells were cultured in the medium alone or in the presence of LPS. Bars denote the mean results of 3 experiments performed in duplicate. Lines indicate the standard error of the mean. No statistically significant differences were observed (p > 0.05) by analysis of variance.M, the presence of macrophages; M + LPS, the presence of macrophages and lipopolysaccharide; TGF, transforming growth factor; MTA, mineral trioxide aggregate; HP, high-plasticity; LPS, lipopolysaccharide.

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  Figure 8 Manipulation and consistency of MTA-HP.MTA, mineral trioxide aggregate; HP, high-plasticity.

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