Analysis of the cytotoxicity and bioactivity of CeraSeal, BioRoot™ and AH Plus® sealers in pre-osteoblast lineage cells.

BACKGROUND: The objective of the present study was to evaluate in vitro the cytotoxicity and bioactivity of various endodontic sealers (CeraSeal, BioRoot™ and AH Plus®) in pre-osteoblast mouse cells (MC3T3 cells). METHODS: MC3T3 cells (ATCC CRL-2594) were plated in 1 × 104 cells/well in 96-well plates in contact with endodontic sealers at concentrations of 1:10 and 1:100. Cell viability was evaluated by MTT assay after 24 and 48 h. In addition, sealer bioactivity was measured by RT-PCR for mediator of inflammation (Tnf, Ptgs2) and mineralization (Runx2, Msx1, Ssp1 and Dmp1) after 24 h and by Alizarin Red S Assay of mineralization after 28 days. Data were analyzed using one-way ANOVA followed by the Tukey's post-test at a significance level of 5%. RESULTS: BioRoot™ presented 24-hour cytotoxicity (p < 0.05) at 1:10 concentration. In the period of 48 h, no endodontic cement was cytotoxic to the cells compared to the control (p > 0.05). TNF-α gene expression was induced by AH Plus® (p < 0.05), while Ptgs2 was induced by the CeraSeal and BioRoot™ (p < 0.05). The expression of Runx2 was stimulated by BioRoot™ and AH Plus® (p < 0.05). In contrast, the expression of Dmp-1 Dmp1 was higher for the CeraSeal and BioRoot™ (p < 0.05). Nonetheless, the sealers did not impact the formation of mineralization nodules (p > 0.05). CONCLUSION: CeraSeal, BioRoot™ and AH Plus® sealers were not cytotoxic to MC3T3 cells within 48 h, but differentially induced the expression of genes related to inflammation and mineralization without impacting biomineralization by the cells.

Leukotriene B4 Loaded in Microspheres Inhibits Osteoclast Differentiation and Activation.

To investigate osteoclast formation in vivo and if leukotriene B4 (LTB4) loaded in microspheres (MS) could be used as a therapeutical strategy to promote a sustained delivery of the mediator and prevent osteoclast differentiation. Methods: In vivo, apical periodontitis was induced in mice to investigate osteoclast differentiation and signaling in absence of 5-lipoxygenase (5-LO). In vitro, LTB4-MS were prepared using an oil-in-water emulsion solvent extraction-evaporation process. Characterization and efficiency of LTB4 encapsulation were investigated. J774A.1 macrophages were cultured in the presence of monocyte colony-stimulating factor (M-CSF) and ligand for receptor activator of nuclear factor kappa B (RANKL) and then stimulated with LTB4-MS. Cytotoxicity, in vitro MS-LTB4 uptake, osteoclast formation and gene expression were measured. Results: We found that 5-LO negatively regulates osteoclastic formation in vivo during apical periodontitis development. In vitro, LTB4-MS were up-taken by macrophages and were not cytotoxic to the cells. LTB4-MS inhibited osteoclast formation and the synthesis of osteoclastogenic genes Acp5, Mmp9, Calcr and Ctsk. LTB4-MS inhibited differentiation of macrophages into an osteoclastic phenotype and cell activation under M-CSF and RANKL stimulus.

Leukotriene B4 Loaded in Microspheres Inhibits Osteoclast Differentiation and Activation

Abstract To investigate osteoclast formation in vivo and if leukotriene B4 (LTB4) loaded in microspheres (MS) could be used as a therapeutical strategy to promote a sustained delivery of the mediator and prevent osteoclast differentiation. Methods: In vivo, apical periodontitis was induced in mice to investigate osteoclast differentiation and signaling in absence of 5-lipoxygenase (5-LO). In vitro, LTB4-MS were prepared using an oil-in-water emulsion solvent extraction-evaporation process. Characterization and efficiency of LTB4 encapsulation were investigated. J774A.1 macrophages were cultured in the presence of monocyte colony-stimulating factor (M-CSF) and ligand for receptor activator of nuclear factor kappa B (RANKL) and then stimulated with LTB4-MS. Cytotoxicity, in vitro MS-LTB4 uptake, osteoclast formation and gene expression were measured. Results: We found that 5-LO negatively regulates osteoclastic formation in vivo during apical periodontitis development. In vitro, LTB4-MS were up-taken by macrophages and were not cytotoxic to the cells. LTB4-MS inhibited osteoclast formation and the synthesis of osteoclastogenic genes Acp5, Mmp9, Calcr and Ctsk. LTB4-MS inhibited differentiation of macrophages into an osteoclastic phenotype and cell activation under M-CSF and RANKL stimulus.

Resumo O objetivo deste trabalho foi Investigar a formação de osteoclastos in vivo e se o leucotrieno B4 (LTB4) incorporado em microesferas (MS) poderia ser usado como estratégia terapêutica para promover uma entrega sustentada do mediador e prevenir a diferenciação dos osteoclastos. Métodos: In vivo, a periodontite apical foi induzida em camundongos para investigar a diferenciação e sinalização de osteoclastos na ausência de 5-lipoxigenase (5-LO). In vitro, LTB4-MS foi preparado usando um processo de evaporação e extração de solvente de emulsão de óleo em água. A caracterização e a eficiência do encapsulamento do LTB4 foram investigadas. Macrófagos J774A.1 foram cultivados na presença de fator estimulador de colônia de monócitos (M-CSF) e ligante para o receptor ativador do fator nuclear kappa B (RANKL) e, então, estimulados com LTB4-MS. Citotoxicidade, captação in vitro de MS-LTB4, formação de osteoclastos e expressão gênica foram avaliadas. Resultados: A via 5-LO regula negativamente a formação de osteoclastos in vivo durante o desenvolvimento da periodontite apical. In vitro, LTB4-MS foram fagocitadas pelos macrófagos e não foram citotóxicos para as células. LTB4-MS inibiu a formação de osteoclastos e a síntese dos genes pró-osteoclastogênicos Acp5, Mmp9, Calcr e Ctsk. Conclusões: LTB4-MS inibiu a diferenciação de macrófagos em um fenótipo osteoclástico e a ativação celular sob estímulo de M-CSF e RANKL.

Prostaglandin E2 Induces Expression of Mineralization Genes by Undifferentiated Dental Pulp Cells.

Prostaglandin E2 (PGE2) is a lipid mediator usually released during inflammation. This study aimed to investigate the potential of soluble or microsphere-loaded PGE2 on inducing differentiation of dental pulp stem cells. PGE2-loaded microspheres (MS) were prepared using an oil-in-water emulsion solvent extraction-evaporation process and were characterized. Mouse dental pulp stem cells (OD-21) were stimulated with soluble or PGE2-loaded MS (0.01 and 0.1 µM). Cell viability was determined by MTT colorimetric assay. Ibsp, Bmp2 and Runx2 expression was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) after 3, 6, and 24 h. The results showed that the soluble PGE2 reduced dental pulp stem cells viability after 24 h of stimulation whereas PGE2-loaded MS did not. Soluble PGE2 up-regulated Ibsp and Bmp2 at 3 h, differently from PGE2-loaded MS. On the other hand, PGE2-MS induced Bmp2 and Runx2 at 6 h and Ibsp at 24 h. In conclusion, our in vitro results show that PGE2, soluble or loaded in MS are not cytotoxic and modulateIbsp,Bmp2, andRunx2gene expression in cultured OD-21 cells.

Human teeth biobank: Microbiological analysis of the teeth storage solution.

The cross-infections may occur during handling of dental elements, affecting the health of dental practitioners and researchers. This study aimed to analyze the influence of the storage medium temperature on the bacterial contingent of the human teeth used for research purposes. Thirty human teeth were donated to the Human Teeth Biobank immediately after extraction. The teeth were cleaned with tap water and neutral soap. The teeth were randomly distributed according to the temperature of the storage solution (deionized water): at 4 °C (refrigerator) or at -10 °C (freezer) and were stored individually in sterile vials during 60 days. After this period, a microbiological analysis (CFU/mL) of the storage solutions was performed and teeth were submitted to SEM analysis. Data were analyzed by Kruskal-Wallis test followed by Dunn's post-test (p ≤ .05). Total aerobic bacteria ranged from 5.8 to 8.4 log10 CFU/mL for refrigerated solution and from 1.9 to 8.5 log10 CFU/mL for frozen solution. No statistical differences were found between the storage solutions (p > .05). The counts of Streptococcus spp., Lactobacillus spp., and Staphylococcus spp. were similar for both storage solutions (p > .05). SEM analysis showed spiral- and rod-shaped bacteria attached on teeth stored under 4 °C, which may suggest the presence of Treponema spp. and Lactobacillus spp. Similar morphological forms were found on teeth stored under -10 °C. A biofilm organized in honeycomb-like form was found in the frozen teeth. Cocci were eventually found in all the samples. It was concluded that bacterial growth and survival were not influenced by the temperature of the teeth storage solution.