RESUMEN
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.
Asunto(s)
Subunidad alfa 1 del Factor de Unión al Sitio Principal , Materiales de Obturación del Conducto Radicular , Ratones , Animales , Ensayo de Materiales , Ciclooxigenasa 2 , Materiales de Obturación del Conducto Radicular/toxicidad , Resinas Epoxi , Osteoblastos , InflamaciónRESUMEN
BACKGROUND: Leukotriene B4 (LTB4) is a potent lipid mediator that stimulate the immune response. Because dental pulp inflammation and dentin repair are intrinsically related responses, the aim of this research was to investigate the potential of LTB4 in inducing differentiation of dental pulp stem cells. METHODS: Microspheres (MS) loaded with LTB4 were prepared using an oil emulsion solvent extraction evaporation process and sterility, characterization, efficiency of LTB4 encapsulation and in vitro LTB4 release assay were investigated. Mouse dental pulp stem cells (OD-21) were stimulated with soluble LTB4 or MS loaded with LTB4 (0.01 and 0.1 µM). Cytotoxicity and cell viability was determined by lactate dehydrogenase and methylthiazol tetrazolium assays. Gene expression were measured by quantitative reverse transcription polymerase chain reaction after 3, 6, 24, 48 and 72 h. Mineralized nodule formation was assessed after 28 days of OD-21 cell stimulation with LTB4 in mineralized media or not. Groups were compared using one-way ANOVA test followed by Dunnett's post-test (α = 0.05). RESULTS: Treatment with LTB4 or MS loaded with LTB4 (0.01 and 0.1 µm-µM) were not cytotoxic to OD-21 cells. Treatment with LTB4 modulated the expression of the Ibsp (integrin binding sialoprotein) and Runx2 (runt-related transcription factor 2) genes differently depending on the experimental period analyzed. Interestingly LTB4 loaded in microspheres (0.1 µM) allowed long term dental pulp cell differentiation and biomineralization. CONCLUSION: LTB4, soluble or loaded in MS, were not cytotoxic and modulated the expression of the Ibsp and Runx2 genes in cultured OD-21 cells. When LTB4 was incorporated into MS, odontoblast differentiation and mineralization was induced in long term culture.
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Pulpa Dental , Leucotrieno B4 , Animales , Biomineralización , Diferenciación Celular , Células Cultivadas , Proteínas de la Matriz Extracelular/genética , Humanos , Leucotrieno B4/metabolismo , Leucotrieno B4/farmacología , Ratones , Microesferas , Odontoblastos/metabolismo , Células Madre/metabolismoRESUMEN
The aim of the present study was to investigate the effects of caffeic acid in the interface between the antimicrobial and anti-inflammatory function in macrophage response against S. mutans. S. mutans (108 cfu/mL) were incubated with caffeic acid to determinate the half-maximal inhibitory concentration (IC50) and macrophage cells were incubated with caffeic acid to determinate cell viability and toxicity. Anti-inflammatory effects were measured by nitrite accumulation, TNF-α and PGE2 production, and NF-kB phosphorylation, and S. mutans survival following internalization by macrophages was investigated. We found that caffeic acid presented antimicrobial activity against S. mutans (IC50 = 2.938 ± 0.1225 mM) without exerting cytotoxicity. Caffeic acid inhibited nitrite, TNF-α and PGE2 production by the NF-kB dependent pathway, indicating an immunomodulatory property. Caffeic acid also contributed to macrophage bacteria clearance activity. In summary, caffeic acid presented antimicrobial activity against S. mutans and anti-inflammatory effects in macrophages.
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Antiinfecciosos/farmacología , Ácidos Cafeicos/farmacología , Factores Inmunológicos/farmacología , Macrófagos/inmunología , Streptococcus mutans/efectos de los fármacos , Animales , Ratones , Células RAW 264.7RESUMEN
OBJECTIVES: The aim of this study was to investigate the inflammatory infiltrate, osteoclast formation, and expression of MMP-9 during the healing phase following root canal treatment in teeth with apical periodontitis. MATERIALS AND METHODS: Apical periodontitis was induced in dogs teeth, and root canal treatment was performed in a single visit or using calcium hydroxide as intracanal medication. One hundred and eighty days following treatment the presence of inflammation was examined, and the tissues were stained to detect osteoclasts by means of a tartrate resistant alkaline phosphatase (TRAP) assay. Synthesis of MMP-9 was detected using Western blotting and immunohistochemistry. RESULTS: Teeth with apical periodontitis that had root canal therapy performed in a single visit presented a higher synthesis of MMP-9 compared with root canal treatment using calcium hydroxide. Treatment with calcium hydroxide resulted in a reduced amount of inflammatory cells and MMP-9 positive cells. Osteoclast formation, the number of MMP-9 positive osteoclasts and cementocytes, was reduced following root canal treatment, regardless of the root canal treatment protocol used. CONCLUSION: Root canal treatment reduced the amount of inflammatory cells and osteoclasts in periapical area. The use of calcium hydroxide as intracanal medication resulted in a lower synthesis of MMP-9, though the number of osteoclasts and MMP-9 positive osteoclasts were similar between the groups. CLINICAL RELEVANCE: Periapical bone repair following root canal treatment is impacted by therapy performed either in single visit or using calcium hydroxide dressing measured by inflammatory cell recruitment, osteoclast formation, and MMP-9 synthesis.
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Periodontitis Periapical , Materiales de Obturación del Conducto Radicular , Animales , Hidróxido de Calcio/farmacología , Cavidad Pulpar , Perros , Inflamación , Metaloproteinasa 9 de la Matriz , Osteoclastos , Periodontitis Periapical/tratamiento farmacológico , Irrigantes del Conducto Radicular , Tratamiento del Conducto RadicularRESUMEN
Antimicrobial photodynamic therapy (aPDT) has been used as an adjuvant treatment of oral infections as a minimal intervention clinical approach. Its antimicrobial efficacy was demonstrated in several studies; however, there is a lack of evidence on its cytotoxic effect on mouse fibroblasts (NIH/3T3). The aim of this study was to evaluate the cytotoxicity and apoptotic pathways of methylene blue-mediated aPDT on mouse fibroblasts. Cells were treated with 0.1 or 1.0 mg.L-1 methylene blue (MB), and 0.075 or 7.5 J.cm-² LED at 630 nm. Cell viability was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet (CV) assays, while cDNA expression for Bax, Bad, Bcl-2, VDAC-1, cytochrome C and Fas-L was assessed by qRT-PCR (1, 3, 6 and 24 h). The differences between groups were detected by Kruskal-Wallis and post-hoc Dunn's tests for MTT and CV assays, and by ANOVA and post-hoc Tukey test for qPCR (P < 0.05). The combination of 1.0 mg.L-1 MB and 7.5 J.cm-² LED significantly reduced the cellular viability, whereas MB and LED alone were innocuous to fibroblasts. MB-mediated aPDT increased the expression of cytochrome C and Fas-L after 3 h, and Bax/Bcl-2, Bad/Bcl-2, and VDAC-1 after 6 h from treatment. Based on these results, MB-mediated aPDT induced cytotoxicity on mouse fibroblasts, with consequent activation of Bcl-2 apoptosis signaling pathways. Further studies are needed to determine the adequate parameters of aPDT to inactivate microorganisms without damaging fibroblasts.
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Apoptosis/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Azul de Metileno/farmacología , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/farmacología , Animales , Apoptosis/genética , Supervivencia Celular , Genes bcl-2 , Técnicas In Vitro , Azul de Metileno/toxicidad , Ratones , Fármacos Fotosensibilizantes/toxicidadRESUMEN
BACKGROUND: The aim of this study was to determine the influence of distinct pre-irradiation times (PIT) of curcumin on the effectiveness of antimicrobial photodynamic therapy (aPDT) against intact dentin caries biofilms grown for 3 or 5 days. METHODS: The microcosm biofilms grew on non-fluorescent glass blocks immersed in McBain medium with 1% sucrose, using microaerophilic conditions at 37⯰C for 3 or 5 days. The biofilms were treated by the association of 600⯵mol.L-1 curcumin using different pre-irradiation times (1, 2 or 5â¯min) combined with 0 or 75â¯J.cm-2 blue LED. Then, the vitality of biofilms was determined by confocal scanning laser microscopy (CSLM), after being stained with the mixture of ethidium bromide and fluorescein diacetate. Statistical analysis was performed by two-way ANOVA and post-hoc Tukey tests, after arcsine transformation (Pâ¯<â¯0,05). RESULTS: In comparison to control, curcumin alone (PITâ¯=â¯5â¯min) and all combinations of curcumin and LED reduced significantly the vitality of 3-day biofilms. Distinctly, only curcumin plus LED using PITs of 2 or 5â¯min were effective in reducing the vitality of 5-day biofilms. CONCLUSION: Curcumin-mediated aPDT significantly decreased the vitality of intact dentin caries microcosms grown during 3 or 5 days, although successful treatments of 5-day biofilms required longer PITs in comparison to their counterparts.