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.

Activation of gelatinases in permanent human teeth after different experimental radiotherapy protocols.

The objective of this study was to compare the activation of gelatinases in dentin-enamel junction (DEJ) and underlying dentin of permanent teeth after experimental radiotherapy in conventional and hypofractionated modalities. Newly extracted third molars (n = 15) were divided into three experimental radiotherapy groups: control, conventional (CR), and hypofractionated (HR) (n = 5 per group). After in vitro exposure to ionizing radiation, following standardized protocols for each modality, a gelatinous substrate was incubated on the tooth slices (n = 10 per group). Activation of gelatinases was measured by in situ zymography, expressed in arbitrary fluorescence units (mm2) from three tooth regions: cervical, cuspal, and pit. Fluorescence intensity was compared among radiotherapy protocols and tooth regions in each protocol, considering a significance level of 5%. Considering all tooth regions, the fluorescence intensity of the CR group was higher than the HR and control groups, both in DEJ and underlying dentin (p <0.001). In addition, the fluorescence intensity was higher in underlying dentin when compared to DEJ in all groups (p <0.001). Considering each tooth region, a statistically significant difference between CR and HR was only observed in the pit region of underlying dentin (p <0.001). Significant and positive correlations between fluorescence intensities in DEJ and underlying dentin were also observed (p <0.001). Experimental radiotherapy influenced the activation of gelatinases, as well as exposure to the conventional protocol can trigger a higher activation of gelatinases when compared to hypofractionated, both in DEJ and underlying dentin.

Reparative Dentin Formation Following Dental Pulp Capping is Mediated by TNFR1 In Vivo.

INTRODUCTION: Tumor necrosis factor (TNF)-α is a pro-inflammatory cytokine that promotes biomineralization in vitro in dental pulp cells. However, the role of TNF-α-TNF receptor 1 (TNFR1) signaling in reparative dentin formation and related inflammatory pathways is not known. Therefore, the aim of this study was to evaluate the role of the TNF-α-TNFR1 axis in dental pulp repair following pulp capping in vivo. METHODS: Dental pulp repair response of genetically deficient TNF-α receptor-1 mice (TNFR1-/-; n = 20) was compared with that of C57Bl6 mice (wild type [WT]; n = 20). Pulp capping was performed with mineral trioxide aggregate on the mandibular first molars of mice. After 7 and 70 days, tissues were collected and stained with hematoxylin and eosin for histopathological and histometric evaluation, and assessed by the Brown and Brenn methods for histomicrobiological analysis and by immunohistochemistry to localize TNF-α, Runt-related transcription factor 2, Dentin Sialoprotein (DSP) and Osteopontin (OPN) expression. RESULTS: Compared with WT mice, TNFR1-/- mice showed significantly decreased reparative dentin formation with a lower mineralized tissue area (P < .0001). Unlike WT mice, TNFR1-/- mice also exhibited significant dental pulp necrosis, neutrophil recruitment, and apical periodontitis formation (P < .0001) without bacterial tissue invasion. TNFR1-/- animals further exhibited decreased TNF-α, DSP, and OPN expression (P < .0001), whereas Runt-related transcription factor 2 expression was unchanged (P > .05). CONCLUSION: The TNF-α-TNFR1 axis is involved in reparative dentin formation following dental pulp capping in vivo. Genetic ablation of TNFR1 modified the inflammatory process and inhibited the expression of the DSP and OPN mineralization proteins, which culminated in dental pulp necrosis and development of apical periodontitis.

Detection of Apical Dental Resorption Caused by Endodontic Infection in Mice Using Fluorescence and Bright-Field Microscopy.

The aim of this study was to evaluate the sensitivity, specificity, and predictive values of the fluorescence microscopy method in the detection of apical dental reabsorption after induction of apical periodontitis in animal models. Forty-first molars of mice, aged 6 to 8 weeks, had their root canals exposed to the oral environment or were maintained healthy as controls (n = 20). After 14 and 42 days, mice were euthanized and tissues were collected for histological evaluation by means of bright field and fluorescence microscopy. The accuracy of fluorescence microscopy in identifying apical external dental resorption was investigated using a diagnostic validation test based on the sensitivity (S) and specificity (E) properties. Bright-field microscopy revealed a higher number of specimens with scores of 1 to 3 - absence of apical dental resorption (n = 29; 52%), while fluorescence microscopy revealed a higher number of specimens with scores of 4 to 6 - presence of apical dental resorption (n = 37; 66%). Out of 56 specimens, 26 were TP, 11 were FP, and 19 were TN. No FN result was observed. Fluorescence microscopy presented a sensitivity value of 1, similar to the bright-field method, while specificity was lower (0.633). The accuracy of the fluorescent method to detect apical dental resorption was 0.804. Fluorescence microscopy revealed a higher number of false positive apical dental resorption than bright-field microscopy. The detection of apical dental resorption was not impacted by the sensitivity of the method but by its specificity.

Activation of gelatinases in permanent human teeth after different experimental radiotherapy protocols

Abstract The objective of this study was to compare the activation of gelatinases in dentin-enamel junction (DEJ) and underlying dentin of permanent teeth after experimental radiotherapy in conventional and hypofractionated modalities. Newly extracted third molars (n = 15) were divided into three experimental radiotherapy groups: control, conventional (CR), and hypofractionated (HR) (n = 5 per group). After in vitro exposure to ionizing radiation, following standardized protocols for each modality, a gelatinous substrate was incubated on the tooth slices (n = 10 per group). Activation of gelatinases was measured by in situ zymography, expressed in arbitrary fluorescence units (mm2) from three tooth regions: cervical, cuspal, and pit. Fluorescence intensity was compared among radiotherapy protocols and tooth regions in each protocol, considering a significance level of 5%. Considering all tooth regions, the fluorescence intensity of the CR group was higher than the HR and control groups, both in DEJ and underlying dentin (p <0.001). In addition, the fluorescence intensity was higher in underlying dentin when compared to DEJ in all groups (p <0.001). Considering each tooth region, a statistically significant difference between CR and HR was only observed in the pit region of underlying dentin (p <0.001). Significant and positive correlations between fluorescence intensities in DEJ and underlying dentin were also observed (p <0.001). Experimental radiotherapy influenced the activation of gelatinases, as well as exposure to the conventional protocol can trigger a higher activation of gelatinases when compared to hypofractionated, both in DEJ and underlying dentin.

Resumo O objetivo deste estudo foi comparar a ativação de gelatinases na junção dentina-esmalte (DEJ) e na dentina subjacente de dentes permanentes após a radioterapia experimental nas modalidades convencional e hipofracionada. Os terceiros molares recém-extraídos (n = 15) foram divididos em três grupos de radioterapia experimental: controle, convencional (CR) e hipofracionada (HR) (n = 5 por grupo). Após a exposição in vitro à radiação ionizante, seguindo protocolos padronizados para cada modalidade, um substrato gelatinoso foi incubado nas fatias de dente (n = 10 por grupo). A ativação das gelatinases foi medida por zimografia in situ, expressa em unidades arbitrárias de fluorescência (mm2) de três regiões do dente: cervical, cúspide e fossa. A intensidade da fluorescência foi comparada entre os protocolos de radioterapia e as regiões do dente em cada protocolo, considerando um nível de significância de 5%. Considerando todas as regiões do dente, a intensidade de fluorescência do grupo CR foi maior do que a dos grupos HR e controle, tanto no DEJ quanto na dentina subjacente (p <0,001). Além disso, a intensidade da fluorescência foi maior na dentina subjacente quando comparada à DEJ em todos os grupos (p <0,001). Considerando cada região do dente, uma diferença estatisticamente significativa entre CR e HR foi observada apenas na região da fossa da dentina subjacente (p <0,001). Também foram observadas correlações significativas e positivas entre as intensidades de fluorescência no DEJ e na dentina subjacente (p <0,001). A radioterapia experimental influenciou a ativação das gelatinases, assim como a exposição ao protocolo convencional pode desencadear uma maior ativação das gelatinases quando comparada ao hipofracionamento, tanto no DEJ quanto na dentina subjacente.

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.

Enamel biomineralization under the effects of indomethacin and celecoxib non-steroidal anti-inflammatory drugs.

The aim of this study was to explore the effects of nonsteroidal anti-inflammatory drugs on biomineralization of enamel. Sixty C57Bl6 male mice were used, which were assigned into three groups: celecoxib (n = 20) or indomethacin (n = 20) treatment for a period of 28 days or received no medication (control group, n = 20). Visual inspection and microcomputed tomography were used to analyze enamel morphology. Scanning electron microscopy-Energy dispersive X-ray and Knoop microhardness test were used to quantify chemical element content (Ca, P, C, O) and enamel microhardness, respectively. Tissues were collected to investigate the synthesis, activity or nuclear translocation of metalloproteinase-20, transcription factor Runx2, dentin sialoprotein and cyclooxygenase-2 enzyme by means of immunohistochemistry, in situ zymography and indirect immunofluorescence. Treatment with indomethacin and celecoxib reduced the Ca and P content, microhardness and mineral density in enamel. Treatment with nonsteroidal anti-inflammatory drugs caused an accumulation of metalloproteinase-20 and overall increased enzymatic activity in enamel matrix, while the synthesis of the transcription factor Runx2 was inhibited by these drugs. Interestingly, indomethacin inhibited Runx2 translocation to the nucleus whereas celecoxib did not. Those findings show that non-steroidal anti-inflammatory drugs impact the enamel biomineralization and could be involved in the etiology tooth enamel defects if used during the period of tooth formation and mineralization.

Absence of Tumor Necrosis Factor Receptor 1 Inhibits Osteoclast Activity in Apical Dental Resorption Caused by Endodontic Infection in Mice.

INTRODUCTION: The aim of this study was to evaluate osteoclastogenesis and dental resorption resulting from endodontic infection in wild-type (WT) and tumor necrosis factor receptor 1 genetically deficient (TNFR1 KO) mice. METHODS: After approval by the ethics committee on the use of animals, 40 mice were distributed into 2 experimental groups based on time periods: 14 days (n = 10 WT mice and n = 10 TNFR1 KO mice) and 42 days (n = 10 WT mice and n = 10 TNFR1 KO mice). After these periods, morphometric analysis was performed using bright field and fluorescence microscopy and tartrate-resistant acid phosphatase histoenzymology to identify osteoclasts. One-way analysis of variance followed by the Tukey post hoc test was used for the statistical analysis (α = 0.05). RESULTS: WT mice in the 42-day period had a greater apical dental resorption in the distal root of the first molar than TNFR1 KO mice (P < .05). On the other hand, TNFR1 KO mice showed a smaller number of osteoclasts on the dental surface than WT mice (P < .05). CONCLUSIONS: WT mice with apical periodontitis had more extensive apical dental resorptions and a larger number of osteoclasts on the tooth surface than TNFR1 KO mice.

Influence of digital media in the oral health education of mother-child pairs: study protocol of a parallel double-blind randomized clinical trial.

BACKGROUND: Dental caries is the most common non transmissible chronic disease in childhood and the control of dental biofilm in children is one of the greatest challenges in oral disease prevention. Digital media applications can help patients in improving their oral hygiene performance and reducing the number of appointments due to pain and discomfort reasons. This study aims to investigate the use of an smartphone application (WhatsApp) to deliver oral health education to mother-child pairs, with the ultimate goal of controlling dental biofilm and caries through digital activities focused on oral hygiene. METHODS: This study was designed as a randomized, double-blind, parallel clinical trial involving 100 pairs of mothers and children (6-12 years old). The mothers and children will be randomly allocated to the control group (n = 50 pairs), who will receive a single visit conventional oral health education, or to the experimental group (n = 50 pairs), who will receive both a single visit conventional oral health education and educational videos through WhatsApp Messenger, twice a week. Before randomization of the groups and after the intervention, pairs will be evaluated regarding to plaque index (VPI), gingival bleeding index (GBI), and number of decayed, missing and filled permanent or primary teeth (DMF-T) modified by the inclusion of active non-cavitated carious lesions (Nyvad criteria). Socioeconomic data, dental history, and oral health literacy will obtained using questionnaires (Oral Health Literacy Assessment Task for Paediatric Dentistry; BOHLAT-P). Chi-square, Student's t-test, paired Student's t-test, Mann-Whitney, and Friedman tests will be used with a 5% significance level. DISCUSSION: This intervention proposal is designed to motivate behavioral change in mother-child pairs. We hypothesize that adding digital media to traditional oral health programs will provoke improvements in oral hygiene behavior and health outcomes. To our knowledge, this is the first study evaluating the effect of educational videos communicated by digital media (WhatsApp) on the oral health of mother-child pairs evaluated by long-term dental examinations. In addition, we will assess the maternal level of comprehension of the provided information via a literacy assessment tool. The clinical trial is registered at the Brazilian Registry of Clinical Trials (No. RBR-7s8bw6m).

Impact of cigarette smoke on osteogenic and osteoclast signaling in middle palatal suture.

Considering that smoking is a public health problem that has been growing among adolescents, the aim of this study was to investigate the impact of cigarette smoke on osteogenic and osteoclastogenic signaling in middle palatal suture of rats. Male Wistar rats exposed (n = 30) or not to cigarette smoke (n = 30) were used. Exposure to smoke was carried out for two daily periods of 3 minutes each, with an interval of 12 hours between exposures. After the experimental periods of 3, 7, 14 and 21 days, the animals were euthanized. The collected tissues were analyzed using light microscopy and real-time RT-PCR was performed to investigate gene expression. The data obtained were compared using the Kruskal Wallis and Dunn tests (⍺ = 5%). Morphologically, there were no significant changes in the middle palatal suture of rats exposed or not to cigarette smoke during 3, 7, 14 and 21 days (p> 0.05). On the other hand, osteoclastogenic signaling was increased in animals exposed to smoke and was characterized by a higher production of RANKL at 3 and 14 days (p <0.05), with no change in the synthesis of RANK and osteoprotegerin (p> 0.05). Interestingly, in the exposed animals, an early increase in the synthesis of osteocalcin, bone sialoprotein and osteopontin was also identified at 3 days of exposure (p <0.05), not sustained over time (p> 0.05). Cigarette smoke modulates osteogenic and osteoclastogenic signaling in the middle palatal suture of young rats, although morphological changes have not been evidenced.

Impact of cigarette smoke on osteogenic and osteoclast signaling in middle palatal suture

Abstract Considering that smoking is a public health problem that has been growing among adolescents, the aim of this study was to investigate the impact of cigarette smoke on osteogenic and osteoclastogenic signaling in middle palatal suture of rats. Male Wistar rats exposed (n = 30) or not to cigarette smoke (n = 30) were used. Exposure to smoke was carried out for two daily periods of 3 minutes each, with an interval of 12 hours between exposures. After the experimental periods of 3, 7, 14 and 21 days, the animals were euthanized. The collected tissues were analyzed using light microscopy and real-time RT-PCR was performed to investigate gene expression. The data obtained were compared using the Kruskal Wallis and Dunn tests (⍺ = 5%). Morphologically, there were no significant changes in the middle palatal suture of rats exposed or not to cigarette smoke during 3, 7, 14 and 21 days (p> 0.05). On the other hand, osteoclastogenic signaling was increased in animals exposed to smoke and was characterized by a higher production of RANKL at 3 and 14 days (p <0.05), with no change in the synthesis of RANK and osteoprotegerin (p> 0.05). Interestingly, in the exposed animals, an early increase in the synthesis of osteocalcin, bone sialoprotein and osteopontin was also identified at 3 days of exposure (p <0.05), not sustained over time (p> 0.05). Cigarette smoke modulates osteogenic and osteoclastogenic signaling in the middle palatal suture of young rats, although morphological changes have not been evidenced.

Resumo Considerando que a fumaça de cigarro é um problema de saúde pública que está crescendo entre os adolescentes, o objetivo deste estudo foi investigar o impacto da fumaça de cigarro na sinalização osteogênica e osteoclastogênica da sutura palatina mediana de ratos. Foram utilizados ratos Wistar machos expostos (n=30) e não expostos à fumaça de cigarro (n=30). A exposição à fumaça de cigarro foi realizada duas vezes ao dia por 3 minutos, com um intervalo de 12 horas entre as exposições. Os animais foram mortos após o período experimental de 3, 7, 14 e 21 dias. Os tecidos coletados foram analisados em microscópico de luz e pelo RT-PCR em tempo real foi realizado para investigar a expressão gênica. s dados obtidos foram comparados usando o testes de Kruskal Wallis e Dunn (⍺ = 5%). Morfoligicamente, não houve mudança significativa na sutura palatina mediana nos ratos expostos ou não à fumaça de cigarro durante os tempo de 3, 7, 14 e 21 dias (p> 0.05). Por outro lado, a sinalização osteogênica esta aumentada nos animais expostos à fumaça e foi caracterizado por um aumento da produção de RANKL aos 3 e 14 dias (p <0.05), sem mudança na síntese da produção de RANK e osteoprotegerina (p> 0.05). Curiosamente, nos animais expostos, também foi observado um aumento precoce da síntese de osteocalcina, sialoproteína óssea e de osteopontina aos 3 dias de exposição, o que não foi mantido ao longo do tempo. A fumaça de cigarro modula a sinalização osteogênica e osteoclastogênica na sutura palatina mediana de ratos jovens, apesar de não tenha sido evidenciado alterações morfológicas.

Low-level laser therapy (LLLT) improves alveolar bone healing in rats.

The main objective of the present study was to evaluate the effect of low-level laser therapy (LLLT) in enhancing bone healing in irradiated alveolus post-tooth extraction. Sixty male Wistar rats (180 ± 10 g) were used in the present study. The left maxillary first molars were extracted, and the alveolar region was irradiated by diode laser device (GaAlAs) immediately after extraction and for more 3-day daily applications. The animals were randomly assigned into two groups: control group (n = 30, with left maxillary molar extraction-CG) and experimental group (n = 30, with tooth extraction and low-level laser therapy applied to the dental alveolus for 42 s-EG). These groups were divided into subgroups (five rats per subgroup) according to the observation time point-1, 2, 3, 5, 7, and 10 days-post-tooth extraction. The maxillary bone was separated, and the specimens were stained with hematoxylin and eosin, Masson's trichrome, and picrosirius red and immunohistochemistry for RUNX-2. Parametric and nonparametric tests were used with a significance level of 5%. LLLT accelerated bone healing with mature collagen fiber bundles and early new bone formation. Histomorphometric analysis revealed an increase of osteoblast (RUNX-2) and osteoclast (TRAP) activity and in the area percentage of cancellous bone in the lased alveolus compared to the control group. This increase was statistically significant (p < 0.05). Application of LLLT with a GaAlAs diode laser device enhanced bone healing and mineralization on alveolar region.

In Vivo Detection of External Apical Root Resorption Induced by Apical Periodontitis Using Periapical Radiography and Cone-Beam Computed Tomography

Abstract Objective: To compare the accuracy of periapical radiography (PR) and cone-beam computed tomography (CBCT) for the detection of external apical root resorption (EARR) due to root canal contamination. Material and Methods: Dog's teeth with experimentally induced root resorption due to root canal contamination underwent or not root canal treatment (n=62). True positives (TP), false positives (FP), true negatives (TN), and false negatives (FN) in PR and CBCT diagnoses were determined using histopathologic findings as the gold standard. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy (TP + TN) in the diagnosis of EARR were calculated. Data were compared using chi-squared test (α=0.05). Results: EARR was detected in 35% of roots by PR, in 47% by CBCT, and in 50% of the roots by microscopy (p=0.03 PR versus microscopy; p=0.67 CBCT versus microscopy). Overall, CBCT produced more accurate diagnoses than PR (p=0.008). PR and CBCT allowed the identification of large resorption in 100% of the cases and showed the same accuracy. However, for small resorptions, PR showed an accuracy of 0.83, whereas CBCT showed an accuracy of 0.96 (p=0.003). Conclusion: Cone-beam computed tomography showed higher accuracy in detecting external apical root resorption of endodontic origin.

Effect of non-steroidal anti-inflammatory drugs on pulpal and periapical inflammation induced by lipopolysaccharide.

OBJECTIVES: The objective of this study was to evaluate the effect of non-steroidal anti-inflammatory drugs (NSAIDs) in controlling pulpal and periapical inflammation in vivo as a potential coadjutant systemic therapy for pulpitis. MATERIALS AND METHODS: A suspension containing E. coli lipopolysaccharide (LPS; 1.0 µg/µL) was inoculated into the pulp chamber of the first molars of C57BL/6 mice (n = 72), and the animals were treated daily with indomethacin or celecoxib throughout the experimental periods. After 7, 14, 21, and 28 days, the tissues were removed for histopathological, histoenzymology, histometric, and immunohistochemical evaluation. RESULTS: Inoculation of LPS into the pulp chamber induced the synthesis of the enzyme cyclooxygenase-2 (COX-2) in dental pulp and periapical region. Indomethacin and celecoxib treatment changed the profile of inflammatory cells recruited to dental pulp and to the periapex, which was characterized by a higher mononuclear cell infiltrate, compared to LPS inoculation alone which recruited a higher amount of polymorphonuclear neutrophils. Administration of indomethacin for 28 days resulted in the development of apical periodontitis and increased osteoclast recruitment, unlike celecoxib. CONCLUSIONS: NSAIDs indomethacin and celecoxib changed the recruitment of inflammatory cells to a mononuclear profile upon inoculation of LPS into the pup chamber, but indomethacin enhanced periapical bone loss whereas celecoxib did not. CLINICAL RELEVANCE: Celecoxib, a selective COX-2 inhibitor, can change the profile of inflammatory cells recruited to the dental pulp challenged with LPS and might a be potential systemic coadjutant for treatment of pulpitis.

Cytotoxicity and Inflammatory Mediators Release by Macrophages Exposed to Real Seal XT and Sealapex Xpress.

This study evaluated the cytotoxicity of Sealapex Xpress and Real Seal XT and their effect on macrophage activation. J774.1 macrophages were incubated with Sealapex Xpress and Seal Real XT (0.1, 1.0, and 10 mg/mL) for 24 and 48 h. Cell viability was assessed by the MTT assay and macrophage activation was measured by pro- and anti-inflammatory cytokine production using ELISA. Data were analyzed using one-way ANOVA and Tukey's post-test (a=0.05). Cell viability was not affected with 0.1 or 1.0 mg/mL of extracts of Sealapex Xpress and Real Seal XT at 24 and 48 h (p>0.05), but was significantly lower when cells were exposed to 10 mg/mL of both sealers (p<0.05). Sealapex Xpress inhibited the production of TNF-a, whereas Real Seal XT induced TNF-a secretion at 24 h (p<0.05). IL-6 production was induced by Real Seal XT, but not by Sealapex Xpress (p<0.05). Real Seal XT and Sealapex Xpress induced the secretion of anti-inflammatory IL-10. IL-4 was not detected in any group. In conclusion, both sealers had low toxicity but differentially activated macrophages. Macrophage activation by Sealapex Xpress was characterized by inhibition of TNF-a and induction of IL-10, whereas Real Seal XT induced IL-6 solely.

VEGF and FGF-2 Released In Palatal Suture after Rapid Maxillary Expansion (RME).

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2) have the ability to increase vascular proliferation and permeability. The aim of this study was to quantify the release of two diffusible angiogenic growth factors (VEGF and FGF-2) after rapid maxillary expansion (RME). Thirty animals were randomly assigned to two groups. Control group (5 rats - intact suture) and Experimental groups (25 rats with RME) which were evaluated in different periods of treatment. Five animals were euthanized in different periods of healing at 0, 1, 2, 3, 5 and 7 days after RME. RT-PCR was used to evaluate the gene expression of angiogenic growth factors released on different periods of study. Data were submitted to statistical analysis using ANOVA followed by Tukey test and significance was assumed at a=0.05. RT-PCR showed that mRNAs of VEGF and FGF-2 were expressed in intact palatal suture tissue. mRNAs of VEGF and FGF-2 was upregulated in early periods (24 h) after RME (p<0.001 and p<0.01, respectively). The molecular levels of VEGF never returned to its original baseline values, and FGF-2 expression decreased up to day 5 (p<0.001) and suddenly increased at day 7, returning to its original level. RME increased VEGF secretion, but decreased FGF-2 secretion when compared to intact tissue. The results showed that these angiogenic growth factors are released and regulated in the palatal suture tissue after RME and could make an important contribution to the knowledge of overall reparative response of the suture tissue during the bone remodeling process.

Cytotoxicity and inflammatory mediators release by macrophages exposed to real seal xt and sealapex xpress

Abstract This study evaluated the cytotoxicity of Sealapex Xpress and Real Seal XT and their effect on macrophage activation. J774.1 macrophages were incubated with Sealapex Xpress and Seal Real XT (0.1, 1.0, and 10 mg/mL) for 24 and 48 h. Cell viability was assessed by the MTT assay and macrophage activation was measured by pro- and anti-inflammatory cytokine production using ELISA. Data were analyzed using one-way ANOVA and Tukey's post-test (a=0.05). Cell viability was not affected with 0.1 or 1.0 mg/mL of extracts of Sealapex Xpress and Real Seal XT at 24 and 48 h (p>0.05), but was significantly lower when cells were exposed to 10 mg/mL of both sealers (p<0.05). Sealapex Xpress inhibited the production of TNF-a, whereas Real Seal XT induced TNF-a secretion at 24 h (p<0.05). IL-6 production was induced by Real Seal XT, but not by Sealapex Xpress (p<0.05). Real Seal XT and Sealapex Xpress induced the secretion of anti-inflammatory IL-10. IL-4 was not detected in any group. In conclusion, both sealers had low toxicity but differentially activated macrophages. Macrophage activation by Sealapex Xpress was characterized by inhibition of TNF-a and induction of IL-10, whereas Real Seal XT induced IL-6 solely.

Resumo O objetivo deste estudo foi avaliar in vitro a citotoxicidade dos cimentos endodônticos Sealapex Xpress e Real Seal XT pelo ensaio de MTT e a ativação de macrófagos J774.1. Os cimentos endodônticos Sealapex Xpress e Real Seal XT foram pesados e os extratos foram obtidos a partir da diluição em meio de cultura DMEM por 48 horas (10mg/mL, 1mg/m, e 0,1 mg/mL). A viabilidade celular foi avaliada pelo ensaio MTT e a produção de citocinas (TNF-a, IL-6 e IL-10) foi investigada pelo ensaio imunoenzimático (ELISA) em células de linhagem (macrofagos J774.1). Os dados obtidos foram analisados utilizando-se análise de variância de uma via e pós-teste de Tukey (a=0,05). A viabilidade celular após 24 ou 48 horas não foi afetada nas concentrações de 0,1 ou 1 mg/mL dos dois cimentos estudados (p>0,05). Por outro lado, na concentração 10 mg/mL, a viabilidade celular foi significativamente mais baixa (p <0,05). Observou-se que o Sealapex Xpress inibiu a produção de TNF-a, enquanto o Real Seal XT induziu a secreção de TNF-a às 24 h (p<0,05). A produção de IL-6 foi induzida pelo Real Seal XT, mas não pelo Sealapex Xpress (p<0,05). A secreção da citocina anti-inflamatória IL-10 foi induzida tanto pelo Real Seal XT quanto pelo Sealapex Xpress. IL-4 não foi detectada em nenhum grupo. Em conclusão, os dois cimentos obturadores apresentaram baixa toxicidade, mas ativaram os macrófagos de modo distinto. A ativação pelo Sealapex Xpress foi caracterizada pela inibição do TNF-a e indução da IL-10, enquanto o Real Seal XT induziu somente IL-6.

Osteoclast formation, inflammation, and matrix metalloproteinase-9 are downregulated in bone repair following root canal treatment in dogs teeth.

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.

Metaloproteinases da matriz: papel no desenvolvimento das lesões periapicais e modulação após tratamento endodôntico / Matrix metalloproteinases: role in the development of apical periodontitis and modulation after endodontic treatment

Introduction: Apical periodontitis represents a local immune response directed against the progression of microorganisms from the dental pulp to the apical foramen and periapical tissues, which results in bone and dental resorption. The aim of this review is to describe the expression of this group of proteases in apical periodontitis and its modulation during the periapical healing phase following root canal treatment. Literature review: The pathogenesis of apical periodontitis involves degradation of several extracellular matrix components. Matrix metalloproteinases (MMPs) are expressed in response to specific stimuli by resident cells of connective tissue during tissue remodeling and by inflammatory cells that arrive into the surrounding tissues during inflammatory events. MMPs have been reported in apical periodontitis, either experimentally induced or obtained from humans and there is evidence that these enzymes show diff erent expression patterns in granulomas and periapical cysts. Root canal therapy is important for the reduction of periapical inflammation as well as the synthesis of MMPs, especially when using a calcium hydroxide-based dressing. Conclusion: Apical periodontitis show high expression of matrix metalloproteinases and root canal treatment results in less expression of MMPs when compared to untreated apical periodontitis.

Introdução: A lesão periapical representa a resposta imunoinflamatória devido ao aumento do número e progressão de micro-organismos advindos dos canais radiculares contaminados em direção aos tecidos apicais e periapicais, resultando em reabsorção óssea. O objetivo desta revisão será abordar a importância das metaloproteinases da matriz no desenvolvimento das lesões periapicais e sua modulação durante a fase de reparação tecidual depois de instituído o tratamento endodôntico. Revisão da literatura: A patogênese da lesão periapical envolve a degradação progressiva de diversos componentes da matriz extracelular. Dentre as proteases responsáveis pela degradação destes componentes estão as metaloproteinases da matriz (MMPs). Estas proteinases são expressas em resposta a estímulos específicos pelas células residentes do tecido conjuntivo durante o processo de remodelação tecidual e por células inflamatórias que invadem os tecidos durante eventos inflamatórios. As MMPs foram descritas em lesões periapicais experimentais e em humanos e existem evidências de que estas enzimas apresentam padrões de expressões diferentes em granulomas e cistos periapicais. A terapia endodôntica é importante para a redução da inflamação periapical assim como da síntese das MMPs, principalmente quando utilizado um curativo de demora à base de hidróxido de cálcio. Conclusão: As lesões periapicais apresentam alta expressão de metaloproteinases da matriz e o tratamento endodôntico em dentes com lesão periapical resulta em menor expressão de MMPs quando comparado às lesões periapicais não tratadas.