Paulo Emílio C. Leite’s research while affiliated with Fluminense Federal University and other places

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Publications (2)

The elemental chemical nature of silica powder (SAS-NM-200) used in the assays was confirmed using EDS, with the majority presence of O and Si (A), while the spherical shape of NPs was visualized using SEM–scale bar: 100 and 20 μm (B). The XRD analysis proves the amorphous structure of the silica powder used in the assays, owing to a wide peak (pointed by an arrow) with a Bragg angle close to 2θ = 20° (C).
Schematic design demonstrating the dispersion process performed using an ultrasonic sonicator, followed by analysis in DLS and MET (A). SiO2 NPs were dispersed in Milli-Q water at a concentration of 1.25 mg ml⁻¹. The analysis of the mean diameter in the nanometer scale and PDI was performed in the DLS at 0 and 24 h after dispersion. The statistical analysis was performed using a paired t-test (*p < 0.05; **p < 0.01; *** p < 0.001) (B). SiO2 NP average size (1.25 mg ml⁻¹) when diluted in water at 0 and 24 h after dispersion and diluted in low-glucose DMEM with 10% FBS in the absence or presence of BSA 24 h after dispersion. The statistical analysis was performed using the unpaired t-test (*p < 0.05; **p < 0.01; *** p < 0.001) (C). SiO2 NP zeta potential (1.25 mg ml⁻¹) when diluted in water and low-glucose DMEM with 10% FBS 24 h after dispersion. SiO2 NP zeta potential in water presented a more negative charge than NPs diluted in DMEM (D). Three independent experiments were conducted. Images of the population of SiO2 NPs diluted in water (1.25 mg ml⁻¹ 24 h after dispersion) obtained using MET show the presence of clusters/aggregates in the sample, with heterogeneous sizes and diffuse distribution (E) and (F), range: 2000 and 200 nm, respectively).
Real-time monitoring assay of A549 cell adhesion and proliferation after exposure to different SiO2 NP concentrations (0.01, 0.1, 1, 5, 10; 25, 50, and 100 μg ml⁻¹). The analysis was performed from plating (initially in the absence of NPs) to the end of the experiment, totaling 96 h. The DMEM 10% FBS condition represents viable cells in the absence of NPs, while TRITON represents cell death through lysis using 0.1% Triton X-100, and the DMEM 10% FBS condition without cells represents only culture medium. It is possible to identify three moments in the graph: 1 (adherence and proliferation in the absence of SiO2 NPs), 2 (proliferation after treatment with NPs), and 3 (plateau).
MTT reduction assay following A549 cell challenge with SiO2 NPs at 0.01, 0.1, 1, 5, 10, 25, 50, and 100 μg ml⁻¹ at 24, 48, and 72 h (A). Cell viability assay using flow cytometry using the Anexin-V/PI kit. The A549 line was challenged with 5, 10, 25, 50, and 100 μg ml⁻¹ SiO2 NPs for 72 h (B) and (C). In the trials presented here, the DMEM condition represents the control of viable cells, only cultivated in low-glucose DMEM with 10% FBS without NPs. Cell death control was performed through lysis using 0.01% Triton X-100. Three independent experiments were conducted. There was no exchange of culture medium during the assay. The statistical analysis was performed using a paired t-test (*p < 0.05; **p < 0.01; *** p < 0.001).
Effects of different SiO2 NP concentrations (0.01, 0.1, 1, 10, and 100 μg ml⁻¹) on the secretion of inflammatory mediators (TNF-α, VEGF, G-CSF, GM-CSF, IL-8, MCP-1, eotaxin, and RANTES) and anti-inflammatory mediators (IL-1ra and IL-10) by A549 cells after 24, 48, and 72 h of exposure. Three independent experiments were conducted. The statistical analysis was performed using a paired t-test (*p < 0.05; **p < 0.01; *** p < 0.001).
In vitro inflammatory and cytotoxic responses of human alveolar cells to amorphous silica nanoparticles exposure
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  • Publisher preview available

March 2025

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25 Reads

Beatriz K A Dal-Cheri

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Wanderson de Souza

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Luis Mauricio T R Lima

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Paulo Emilio C Leite

Silicon dioxide nanoparticles (SiO2 NPs) are widely used to manufacture products for human consumption. However, their large-scale use in many fields poses risks to industrial workers. In this study, we investigated the cytotoxic and inflammatory potential of SiO2 NPs in the human cell line A549, representing the human alveolar epithelium. The NPs were characterized using energy-dispersive x-ray spectroscopy coupled with scanning electron microscopy, x-ray diffraction, transmission electron microscopy, dispersion, and dynamic light scattering. The effects on A549 cells were monitored by cell adhesion and proliferation using electrical impedance, as well as cell viability, apoptosis, necrosis, and secretion of multiple inflammatory mediators. SiO2 NPs did not alter the adhesion and proliferation of A549 cells but led to cell death by apoptosis at the highest concentrations tested. SiO2 NP impacted the secretion of pro-inflammatory (tumor necrosis factor-α, interleukin (IL)-8, monocyte chemoattractant protein-1, eotaxin, regulated upon activation, normal T cell expressed and secreted, vascular growth factor, granulocyte–macrophage colony-stimulating factor, and granulocyte-colony stimulating factor) and anti-inflammatory (IL-1ra and IL-10) mediators. These results indicate that, even with little impact on cell viability, SiO2 NPs can represent a silent danger, owing to their influence on inflammatory mediator secretion and unbalanced local homeostasis.

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Cell viability of three-dimensional culture of MG-63 spheroids exposed to the different sealers by 24 h, as measured through the release of lactate dehydrogenase by cells with compromised membranes (LDH assay). Results are represented as mean and Standard deviation of the percentage relative to the unexposed control group. An asterisk indicates significant difference from the other experimental groups (p < 0.05)
Cell viability as revealed by a fluorescent live/dead assay from the 3D model exposed to the different sealers. Cells were stained with calcein (living cells, green) and ethidium (dead cells, red). Extracts of polystyrene beads were used as negative control, while extracts of latex fragments and 1% Triton X100 were employed as positive, cytotoxic controls. The bar indicates 100 micrometers
Morphological evaluation of the histological structure of spheroids exposed to sealer extracts for 24 h. (A) polystyrene beads (negative control); (B) latex fragments (positive control); (C) 1% Triton X100 (positive control), (D) AH Plus; (E) Bio C Sealer; (F) Sealer Plus (G) Sealer Plus BC; (H) AH Plus BC
Heatmap of the variation of concentrations of different inflammatory mediators secreted by the MG-63 spheroids after 24 h exposure to extracts of epoxy resin-based and calcium silicate-based endodontic sealers, relative to the measured concentration of unexposed cells (experimental control)
Comparison of inflammatory mediators released by MG-63 spheroids after 24 h exposure to extracts of epoxy resin-based and calcium silicate-based endodontic sealers. The results show mean concentration and standar deviation of the pro-inflammatory interleukins (A) IL-5, (B) IL-7, (C) IP-10, (D) RANTES, (E) IL-1beta and (F) IL-15, as well as the anti-inflammatory interleukins (G) IL-1RA and (H) FGF2. An asterisk indicates significant difference between groups
A three-dimensional cell culture approach to investigate cytotoxic effects and production of inflammatory mediators by epoxy resin-based and calcium silicate-based endodontic sealer

May 2024

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91 Reads

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1 Citation

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Sandro J.O. Tavares

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Objectives The aim of the present study was to assess the cytocompatibility of epoxy resin-based AH Plus Jet (Dentsply De Trey, Konstanz, Germany), Sealer Plus (MK Life, Porto Alegre, Brazil), calcium silicate-based Bio-C Sealer (Angelus, Londrina, PR, Brazil), Sealer Plus BC (MK Life) and AH Plus BC (Dentsply) through a tridimensional (3D) culture model of human osteoblast-like cells. Methods Spheroids of MG-63 cells were produced and exposed to fresh root canal sealers extracts by 24 h, and the cytotoxicity was assessed by the Lactate Dehydrogenase assay (LDH). The distribution of dead cells within the microtissue was assessed by fluorescence microscopy, and morphological effects were investigated by histological analysis. The secreted inflammatory mediators were detected in cell supernatants through flow luminometry (XMap Luminex). Results Cells incubated with AH Plus Jet, AH Plus BC, Sealer Plus BC and Bio-C Sealer extracts showed high rates of cell viability, while the Sealer Plus induced a significant reduction of cell viability, causing reduction on the spheroid structure. Sealer Plus and Seaker Plus BC caused alterations on 3D microtissue morphology. The AH Plus BC extract was associated with the downregulation of secretion of pro-inflammatory cytokines IL-5, IL-7, IP-10 and RANTES. Conclusions The new AH Plus BC calcium silicate-based endodontic sealer did not reduce cell viability in vitro, while led to the downregulation of pro-inflammatory cytokines. Clinical significance Choosing the appropriate endodontic sealer is a crucial step. AH Plus BC demonstrated high cell viability and downregulation of pro-inflammatory cytokines, appearing reliable for clinical use, while Sealer Plus presented lower cytocompatibility.

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Citations (1)

... The presence of voids in HC may be influenced by the type of sealer employed 21 . Initial laboratory studies have shown that AH Plus Bioceramic Sealer has adequate physicochemical and biological properties 32,33 , but there is limited information on its clinical performance. Although most CSS manufacturers recommend using a syringe and needle for application, there is no evidence regarding the impact of different delivery methods within the canals 34 . ...

Reference:

Teaching a New Obturation Technique in Preclinical Endodontic Training: Assessment of Student Learning Experience, Performance, and Self-Evaluation
A three-dimensional cell culture approach to investigate cytotoxic effects and production of inflammatory mediators by epoxy resin-based and calcium silicate-based endodontic sealer

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Sandro J.O. Tavares

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