α-IRAK-4 Suppresses the Activation of RANK/RANKL Pathway on Macrophages Exposed to Endodontic Microorganisms.

Periapical lesions are common pathologies affecting the alveolar bone, often initiated by intraradicular lesions resulting from microbial exposure to dental pulp. These microorganisms trigger inflammatory and immune responses. When endodontic treatment fails to eliminate the infection, periapical lesions persist, leading to bone loss. The RANK/RANKL/OPG pathway plays a crucial role in both the formation and the destruction of the bone. In this study, the objective was to inhibit the RANK/RANKL pathway in vitro within exposed Thp-1 macrophages to endodontic microorganisms, specifically Enterococcus faecalis, which was isolated from root canals of 20 patients with endodontic secondary/persistent infection, symptomatic and asymptomatic, and utilizing an α-IRAK-4 inhibitor, we introduced endodontic microorganisms and/or lipoteichoic acid from Streptococcus spp. to cellular cultures in a culture plate, containing thp-1 cells and/or PBMC from patients with apical periodontitis. Subsequently, we assessed the percentages of RANK+, RANKL+, and OPG+ cells through flow cytometry and measured the levels of several inflammatory cytokines (IL-1ß, TNF-α, IL-6, IL-8, IL-10, and IL-12p70) in the cellular culture supernatant through a CBA kit and performed analysis by flow cytometry. A significant difference was observed in the percentages of RANK+RANKL+, OPG+ RANKL+ cells in thp-1 cells and PBMCs from patients with apical periodontitis. The findings revealed significant differences in the percentages of the evaluated cells, highlighting the novel role of the IRAK-4 inhibitor in addressing this oral pathology, apical periodontitis, where bone destruction is observed.

Reduced C-reactive protein levels after root canal treatment in clinically healthy young apical periodontitis individuals at cardiovascular risk. A prospective study.

AIM: To determine the systemic inflammatory burden, including hsCRP and its monomeric forms, in patients with apical lesions of endodontic origin treated with root canal treatment (RCT). METHODOLOGY: Prospective pre-/post-study. Apical periodontitis (AP) individuals aged 16-40 were included (N = 29). Individuals received RCT and were followed at 1 and 6 months. Fasting blood samples were obtained. Apical lesions of endodontic origin (ALEO) diameter (mm), and periapical index (PAI), were recorded. The serum concentrations of total hsCRP were determined by turbidimetry. Tumour necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, IL-1ß, and soluble (s) E-selectin were assessed by Multiplex assay. Additionally, mCRP forms were determined in the serum of AP patients with a baseline moderate to high cardiovascular risk based on hsCRP stratification (hsCRP ≥1 mg/L) by immunowestern blot (n = 15). Also, CRP isoforms were explored in ALEOs from AP individuals (n = 4). Data were analysed with StataV16. RESULTS: Periapical index and ALEO sizes were reduced at both follow-up visits after RCT (p < .05). Serum levels of TNF-α, IL-6, IL-10, IL-1ß, and sE-selectin did not show significant differences. CRP was borderline reduced at 1 month (p = .04); however, in AP individuals at cardiovascular risk (hsCRP ≥ 1 mg/L), hsCRP and its monomeric isoform significantly decreased at 1 and 6 months (p < .05). CONCLUSIONS: High-sensitivity CRP and mCRP are reduced after RCT in AP individuals at cardiovascular risk.

Pretreatment of Root Canal with Photodynamic Therapy Facilitates Adhesion, Viability and Differentiation of Stem Cells of the Apical Papilla.

This study was to test the hypothesis that root canal pretreated with photodynamic therapy (PDT) would promote stem cells from the apical papilla (SCAP) adhesion, proliferation and differentiation without affecting smear layer removal and microhardness of root canal. Standardized root canals were randomized into four groups (n = 30/group): (1) sodium hypochlorite(NaOCl) group, (2) NaOCl + ethylene diaminetetraacetic acid (EDTA) group, (3) NaOCl + PDT group, (4) NaOCl + EDTA + PDT group. After treatments, smear layer removal and microhardness of root canal were evaluated. SCAP with hydroxyapatite-based scaffolds were seeded into root canals for 7 days. SCAP adhesion was observed by scanning electron microscope (SEM), and viable cells were calculated by CellTiter-Glo Luminescent kit. Platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) expression of SCAP were evaluated by Quantitative Reverse Transcriptase-Polymerase Chain Reaction. There was no significant difference in the smear layer removal and microhardness of root dentin between the groups with and without PDT treatment (P > 0.05). SCAP with elongated cytoplasmic processes and cell-cell contact were observed on the dentin surfaces treated with PDT. Elevated cell viability, PDGF and VEGF expression were found in root canal treated with PDT (P < 0.05). Under the experimental conditions, PDT could provide positive microenvironment for SCAP growth.

Comparison of apical irrigant solution extrusion among conventional and laser-activated endodontic irrigation.

The aim of this study was to determine the amount of extruded endodontic irrigant among needle-syringe irrigation (NSI) and laser-activated irrigation (LAI) regimens. Twenty extracted maxillary central incisors were prepared utilizing GT professional rotary files (size 40, taper 0.06). Irrigation was performed with two 27 G irrigation needles (notched open ended (ON) and single side vented (SV)) each at two different irrigant volumetric flow rates (VFR)-0.05 ml/s (3 ml/min) and 0.10 ml/s (6 ml/min). LAI was performed with Er:YAG (erbium-doped yttrium aluminum garnet) using different fiber types (X-Pulse-14/400 cylindrical tip, Preciso- 14/300 flat cylindrical tip, PIPS- 14/400 quartz tapered tip). The Er:YAG laser with a wavelength of 2940 nm (Lightwalker AT, Fotona, Ljubljana, Slovenia) was used according to the following protocol: 10 mJ per pulse, 15 Hz, pulse duration 50 µs. Irrigation time was 60 s for all protocols. Precision syringe pump (PSP) maintained constant irrigant volumetric flow rate. Apically extruded irrigant was collected and net weighed for each protocol (N = 10). Data were analyzed by t tests and Kruskal-Wallis. All LAI regimens had statistically significant lower irrigant extrusion compared with NSI except for the SV 27 G needle used with 0.05 ml/s VFR when compared with the Preciso fiber tip (p = 0,230). The largest amount of extruded irrigant was with the ON 27 G needle at the 0.10 ml/s VFR, while the smallest was after LAI with PIPS fiber tip. The lower quantity of apically extruded irrigant during LAI (X-Pulse and PIPS) points out a safer endodontic irrigation method compared with conventional irrigations.

Root canal contamination or exposure to lipopolysaccharide differentially modulate prostaglandin E 2 and leukotriene B 4 signaling in apical periodontitis

Abstract Purpose To evaluate the kinetics of apical periodontitis development in vivo , induced either by contamination of the root canals by microorganisms from the oral cavity or by inoculation of bacterial lipopolysaccharide (LPS) and the regulation of major enzymes and receptors involved in the arachidonic acid metabolism. Methodology Apical periodontitis was induced in C57BL6 mice (n=96), by root canal exposure to oral cavity (n=48 teeth) or inoculation of LPS (10 µL of a suspension of 0.1 µg/µL) from E. coli into the root canals (n= 48 teeth). Healthy teeth were used as control (n=48 teeth). After 7, 14, 21 and 28 days the animals were euthanized and tissues removed for histopathological and qRT-PCR analyses. Histological analysis data were analyzed using two-way ANOVA followed by Sidak's test, and qRT-PCR data using two-way ANOVA followed by Tukey's test (α=0.05). Results Contamination by microorganisms led to the development of apical periodontitis, characterized by the recruitment of inflammatory cells and bone tissue resorption, whereas inoculation of LPS induced inflammatory cells recruitment without bone resorption. Both stimuli induced mRNA expression for cyclooxygenase-2 and 5-lipoxygenase enzymes. Expression of prostaglandin E 2 and leukotriene B 4 cell surface receptors were more stimulated by LPS. Regarding nuclear peroxisome proliferator-activated receptors (PPAR), oral contamination induced the synthesis of mRNA for PPARδ, differently from inoculation of LPS, that induced PPARα and PPARγ expression. Conclusions Contamination of the root canals by microorganisms from oral cavity induced the development of apical periodontitis differently than by inoculation with LPS, characterized by less bone loss than the first model. Regardless of the model used, it was found a local increase in the synthesis of mRNA for the enzymes 5-lipoxygenase and cyclooxygenase-2 of the arachidonic acid metabolism, as well as in the surface and nuclear receptors for the lipid mediators prostaglandin E2 and leukotriene B4.

Periapical bone response to bacterial lipopolysaccharide is shifted upon cyclooxygenase blockage

Abstract Objectives: Infection, inflammation and bone resorption are closely related events in apical periodontitis development. Therefore, we sought to investigate the role of cyclooxygenase (COX) in osteoclastogenesis and bone metabolism signaling in periapical bone tissue after bacterial lipopolysaccharide (LPS) inoculation into root canals. Methodology: Seventy two C57BL/6 mice had the root canals of the first molars inoculated with a solution containing LPS from E. coli (1.0 mg/mL) and received selective (celecoxib) or non-selective (indomethacin) COX-2 inhibitor. After 7, 14, 21 and 28 days the animals were euthanized and the tissues removed for total RNA extraction. Evaluation of gene expression was performed by qRT-PCR. Statistical analysis was performed using analysis of variance (ANOVA) followed by post-tests (α=0.05). Results: LPS induced expression of mRNA for COX-2 (Ptgs2) and PGE2 receptors (Ptger1, Ptger3 and Ptger4), indicating that cyclooxygenase is involved in periapical response to LPS. A signaling that favours bone resorption was observed because Tnfsf11 (RANKL), Vegfa, Ctsk, Mmp9, Cd36, Icam, Vcam1, Nfkb1 and Sox9 were upregulated in response to LPS. Indomethacin and celecoxib differentially modulated expression of osteoclastogenic and other bone metabolism genes: celecoxib downregulated Igf1r, Ctsk, Mmp9, Cd36, Icam1, Nfkb1, Smad3, Sox9, Csf3, Vcam1 and Itga3 whereas indomethacin inhibited Tgfbr1, Igf1r, Ctsk, Mmp9, Sox9, Cd36 and Icam1. Conclusions: We demonstrated that gene expression for COX-2 and PGE2 receptors was upregulated after LPS inoculation into the root canals. Additionally, early administration of indomethacin and celecoxib (NSAIDs) inhibited osteoclastogenic signaling. The relevance of the cyclooxygenase pathway in apical periodontitis was shown by a wide modulation in the expression of genes involved in both bone catabolism and anabolism.

Butyrate Stimulates Histone H3 Acetylation, 8-Isoprostane Production, RANKL Expression, and Regulated Osteoprotegerin Expression/Secretion in MG-63 Osteoblastic Cells.

Butyric acid as a histone deacetylase (HDAC) inhibitor is produced by a number of periodontal and root canal microorganisms (such as Porphyromonas, Fusobacterium, etc.). Butyric acid may affect the biological activities of periodontal/periapical cells such as osteoblasts, periodontal ligament cells, etc., and thus affect periodontal/periapical tissue destruction and healing. The purposes of this study were to study the toxic effects of butyrate on the matrix and mineralization marker expression in MG-63 osteoblasts. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cellular apoptosis and necrosis were analyzed by propidium iodide/annexin V flow cytometry. The protein and mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) were analyzed by Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). OPG, soluble RANKL (sRANKL), 8-isoprostane, pro-collagen I, matrix metalloproteinase-2 (MMP-2), osteonectin (SPARC), osteocalcin and osteopontin (OPN) secretion into culture medium were measured by enzyme-linked immunosorbant assay. Alkaline phosphatase (ALP) activity was checked by ALP staining. Histone H3 acetylation levels were evaluated by immunofluorescent staining (IF) and Western blot. We found that butyrate activated the histone H3 acetylation of MG-63 cells. Exposure of MG-63 cells to butyrate partly decreased cell viability with no marked increase in apoptosis and necrosis. Twenty-four hours of exposure to butyrate stimulated RANKL protein expression, whereas it inhibited OPG protein expression. Butyrate also inhibited the secretion of OPG in MG-63 cells, whereas the sRANKL level was below the detection limit. However, 3 days of exposure to butyrate (1 to 8 mM) or other HDAC inhibitors such as phenylbutyrate, valproic acid and trichostatin stimulated OPG secretion. Butyrate stimulated 8-isoprostane, MMP-2 and OPN secretion, but not procollagen I, or osteocalcin in MG-63 cells. Exposure to butyrate (2⁻4 mM) for 3 days markedly stimulated osteonectin secretion and ALP activity. In conclusion, higher concentrations of butyric acid generated by periodontal and root canal microorganisms may potentially induce bone destruction and impair bone repair by the alteration of OPG/RANKL expression/secretion, 8-isoprostane, MMP-2 and OPN secretion, and affect cell viability. However, lower concentrations of butyrate (1⁻4 mM) may stimulate ALP, osteonectin and OPG. These effects are possibly related to increased histone acetylation. These events are important in the pathogenesis and repair of periodontal and periapical destruction.

Regenerating a monoblock to obturate root canalsvia a mineralising strategy.

To develop a novel strategy for sealing and obturating dental root canals by tooth-like tissue regeneration, premolars with mature root apices were freshly collected, and root canals were prepared by following the clinical protocols in vitro. The teeth were immersed in supersaturated calcium and phosphate solution containing gallic acid and fluoride. At certain intervals, the dental roots were taken out, and their mineral precipitates were characterised by scanning electron microscopy, energy-dispersive spectroscopy mapping, X-ray diffraction and transmission electron microscopy. The cytocompatibility of the mineralizing products were evaluated with rabbit bone-marrow-derived mesenchymal stem cells in vitro. Results showed that the precipitates were mainly composed of fluoridated hydroxyapatite with ahexagonal prism morphology. Fluoridated hydroxyapatite initially nucleated and grew from the root canal dentine surface to the root canal centre. The fluoridated hydroxyapatite precipitate and root canal dentine intergraded together such that the interface became hardly distinguishable. The fluoridated hydroxyapatite precipitate grew into and obturated the dentinal tubules. In the root canal, the regenerated fluoridated hydroxyapatite densely packed and bundled together with a c-axis extension. After 7 days of mineralisation, the root canal was completely obturated, and the apical foramen was sealed. The mineralizing products had good biocompatibility with the cells, and the cells grew well on the mineralized surface. Biomimetic mineralisation strategy provides a novel means to regenerate tooth-like tissue to seal the root canal system permanently other than by passive synthetic material filling.

Association between bacteria occurring in the apical canal system and expression of bone-resorbing mediators and matrix metalloproteinases in apical periodontitis.

AIM: To evaluate the association between the presence of selected bacterial species/groups in the apical root canal and expression of mediators of soft and bone tissue destruction in apical periodontitis lesions. Relationships between bacteria and some other features of apical periodontitis were also investigated. METHODOLOGY: Seventeen freshly extracted teeth with pulp necrosis and apical periodontitis were included. The apical root segment was sectioned and cryopulverized; DNA was extracted and evaluated for the presence of 9 bacterial species/groups using real-time polymerase chain reaction. Lesions were processed for histopathological and immunohistochemical analyses, which targeted matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9), receptor activator of NFκB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG). Associations of the target bacteria with expression of these mediators, presence of symptoms, lesion size and histopathological diagnosis were evaluated. Data were analysed using the chi-square, Fisher's exact, Mann-Whitney and Pearson tests. P values lower than 0.05 were considered significant. RESULTS: All pulverized apical root samples were positive for bacteria. The most prevalent taxa were Actinobacteria (53%), Streptococcus species (35%), Fusobacterium species and Parvimonas micra (18%). The target mediators exhibited a high mean expression in the lesions (MMP-2: 82%; MMP-9: 73%; RANK: 78%; RANKL; 81%; OPG; 83%). Mean RANKL:OPG ratio was significantly higher in granulomas than cysts (P < 0.05, Mann-Whitney test). Actinobacteria were associated with granulomas, higher MMP-2 expression, lower OPG expression, and higher RANKL:OPG ratio (P < 0.05 for all, Fisher's exact test or Mann-Whitney test). No other significant associations were found. CONCLUSION: Actinobacteria may play an important role in the active phase of soft and bone tissue destruction in apical periodontitis.

Evaluation of Various Pretreatments to Fiber Post on the Push-out Bond Strength of Root Canal Dentin.

INTRODUCTION: The aim of this in vitro study was to compare the effect of different pretreatments (fiber post) with the laser-activated irrigation (LAI) technique (for removal of the smear layer) on root canal dentin in terms of push-out bond strength (PBS) in a fiber post. METHODS: Fifty freshly extracted mandibular single-rooted premolars were prepared, and LAI was applied to all roots (17% EDTA was 5 mL for 120 seconds with an erbium, chromium:yttrium-scandium-gallium-garnet laser [0.50 W, 20 Hz, 25 mJ]). In addition, 50 quartz fiber posts were randomly assigned to 5 groups (n = 10) according to the surface treatments as follows: group S (sandblasting), group N1 and group N2 (neodymium:yttrium-aluminum-garnet laser irradiation [2 W, 200 mJ, 10 Hz, with pulse durations of 180 or 320 microseconds), group HF (9.7% hydrofluoric acid etched), and group C (control with no treatment). Dual-cure resin cement was adhered onto the fiber posts after they were covered with a silane agent, and then the posts were placed into the canal space using a Lentulo spiral. The PBS test was performed after all specimens were transversally sectioned (root slices of 1-mm thickness). Data were analyzed with 1-way analysis of variance/Tukey post hoc test (α = 0.05). RESULTS: The highest PBS value was observed in group S (middle part), and the lowest value was observed in group C (apical part). There were no statistical differences among the groups regardless of the part. Furthermore, when the PBS values of the different parts of dentin were compared within treated groups, significant differences were observed in all groups except group N2 (P < .05). CONCLUSIONS: Within the limitations of this study, it can be concluded that the LAI technique when used with 17% EDTA had a significant effect on the amount of smear layer removed from the root canal dentin, which was also detected in the fracture pattern (adhesive failure [resin-post interface]). However, the various treatments of the fiber post did not improve the PBS of the root dentin.

Electrochemical dissolution of fractured nickel-titanium instruments in human extracted teeth.

AIM: To assess the effect of sodium chloride concentration in fluoridated solutions during the electrochemical dissolution of fractured rotary endodontic instruments. METHODOLOGY: Two solutions were assessed (solution 1: NaF 12 g L-1  + NaCl 1 g L-1 , pH = 5.0; and solution 2: NaF 12 g L-1  + NaCl 180 g L-1 , pH = 5.0) using two tests: the ProTaper Universal F1 (PTU F1) instrument polarization test and the polarization test for intracanal PTU F1 fragments fractured in mandibular incisors. In the first test, two sets of five instruments were separately and partially immersed in each solution, and the electrical current was evaluated over 30 min. In the second test, 45 PTU F1 instruments were fractured within the root canals of mandibular incisors and subjected to potentiodynamic polarization for 30 min. The electrical current and the variations in the length of PTU F1 fragments were measured. The data were analysed statistically (anova and Wilcoxon and Mann-Whitney tests, respectively). RESULTS: Solution 2 was associated with more corrosive effects in both tests. In the first test, the PTU F1 instruments immersed in solution 2 had a higher electrical current (P < 0.001) and had a total dissolution time of approximately 540 s. In the second test, a larger difference between the baseline and final lengths of the fragments was noted in solution 2 (P = 0.011). CONCLUSION: Saturation of fluoridated solution with sodium chloride led to an increase in electrical current and microscopic reductions in the length of fractured instrument fragments subjected to electrochemical dissolution.

Entrococcus faecalis Elimination in Root Canals Using Silver Nanoparticles, Photodynamic Therapy, Diode Laser, or Laser-activated Nanoparticles: An In Vitro Study.

INTRODUCTION: The aim of this study was to compare the efficacy of silver nanoparticles (AgNPs), an 810-nm diode laser (DL), conventional photodynamic therapy (PDT) with the use of indocyanine green (ICG) photosensitizer, and modified PDT with the use of AgNPs for the disinfection of root canals inoculated with Enterococcus faecalis. METHODS: The root canals of 65 extracted human single-rooted teeth were prepared, and E. faecalis was incubated in the root canals for 4 weeks. The teeth were then randomly divided into the following 4 experimental groups: the DL group: 810-nm DL irradiation (1 W, 4 times for 10 seconds), the AN group: 5 minutes of irrigation with 5 mL AgNPs (100 ppm), the ICG/DL group: conventional PDT with ICG (1 mg/mL)/810-nm DL (200 mW, 30 seconds), and the AN/ICG/DL group: modified PDT with AgNPs/ICG/810-nm DL (200 mW, 30 seconds). There was also a control group, which consisted of 5 minutes of irrigation with 5 mL 2.5% sodium hypochlorite (n = 9). Samples were obtained from dentin chips before and after the interventions. A reduction in colony count was assessed by counting the colony-forming units. RESULTS: Significant reductions were noted in E. faecalis colony counts in all groups (P < .05). The greatest reduction in colony count (99.12%) was noted in the AN/ICG/DL group (AgNPs/ICG/810-nm diode laser); however, the differences in this respect between the AN/ICG/DL group and the DL (97.41%), AN (94.42%), and control groups (94.61%) were not significant (P > .05). CONCLUSIONS: PDT with ICG, an 810-nm diode laser, and AgNPs has the potential to be used as an adjunct for disinfection of the root canal system.

Murine Experimental Root Canal Infection: Cytokine Expression in Response to F. nucleatum and E. faecalis

Abstract The aim of this study was to evaluate the gene expression of proinflammatory (RANKL, TNF-a and IFN-g) and regulatory (TGF-b and IL-10) cytokines as reaction to experimental infection by mono or bi-association of Fusobacterium nucleatum (ATCC 10953) and Enterococcus faecalis (ATCC 19433). F. nucleatum and E. faecalis, either in mono- or bi-association were inoculated into the root canal system (RCS) of Balb/c mice. Animals were sacrificed at 10 and 20 days after infection and periapical tissues surrounding the root were collected. The mRNA expression of the cytokines RANKL, TNF-a, IFN- g, TGF-b and IL-10 was assessed using real-time PCR. The Kruskal-Wallis test was used for statistical analysis. F. nucleatum mono-infection induced high expression of RANKL and TNF-a, while its modulation was due to IL-10. High expression of IFN-g at day 20 was up-regulated by E. faecalis and RANKL; TNF-a was up-regulated by an independent mechanism via IL-10 and TGF-b. Bi-association (F. nucleatum and E. faecalis) stimulated high expression of RANKL, TNF-a and IFN-g, which seemed to be modulated by TGF-b 20 days later. The gene expression of proinflammatory cytokines was more prominent in the earlier periods of the experimental periapical infection, which concomitantly decreased in the later period. This expression may be regulated by IL-10 and TGF-b in an infection-specific condition

Resumo O objetivo deste trabalho foi avaliar a expressão gênica de citocinas pró-inflamatórias (RANKL, TNF-a e IFN-g) e regulatórias (TGF-b e IL-10) em resposta à infecção experimental por Fusobacterium nucleatum (ATCC 10953) e Enterococcus faecalis (ATCC 19433) como mono-infecção ou em bi-associação. F. nucleatum e E. faecalis foram inoculados no sistema de canais radiculares de camundongos Balb/c, tanto isoladas como em bi-associação. Os animais foram sacrificados em 10 e 20 dias após a infecção, e os tecidos periapicais foram coletados. As expressões do mRNA das citocinas RANKL, TNF-a, IFN-g, TGF-b e IL-10 foram analisadas por meio do real-time PCR. O teste de Kruskal-Wallis foi utilizado para análise estatística. A mono-infecção com F. nucleatum induziu alta expressão de RANKL e TNF-a, enquanto sua modulação ocorreu devido à IL-10. A alta expressão de IFN-g no dia 20 foi regulada positivamente por E. faecalis e RANKL; TNF-a foi regulada positivamente por um mecanismo independente via IL-10 e TGF-b. A bi-associação (F. nucleatum e E. faecalis) estimulou uma alta expressão de RANKL, TNF-a e IFN-g, que parece ser modulada por TGF-b após 20 dias. A expressão gênica de citocinas pró-inflamatórias foi mais proeminente nos estágios iniciais da infecção periapical experimental, com concomitante redução no período tardio. Este fenômeno pode ser regulado por IL-10 e TGF-b em uma condição de infecção específica.

Expression of Mineralization Markers during Pulp Response to Biodentine and Mineral Trioxide Aggregate.

INTRODUCTION: The purpose of this study was to compare the cell viability of dental pulp cells treated with Biodentine (Septodont, Saint-Maur, France) and mineral trioxide aggregate (MTA) and the in vitro and in vivo expression of mineralization markers induced by the 2 materials. METHODS: Human dental pulp cells isolated from 6 permanent teeth were stimulated with Biodentine and MTA extracts. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and quantitative reverse-transcriptase polymerase chain reaction was used to determine the expression of mineralization markers. Specimens of teeth from dogs treated with Biodentine and MTA after pulpotomy were used to determine the presence of osteopontin and alkaline phosphatase by immunohistochemistry and runt-related transcription factor 2 by immunofluorescence. RESULTS: No significant differences in cell viability were found between MTA and Biodentine extracts and controls after 24 and 48 hours (P > .05). After 48 hours, osteopontin (SPP1), alkaline phosphatase (ALP), and runt-related transcription factor 2 (RUNX2) expression was higher in MTA and Biodentine than in controls (P < .05). Osteopontin staining was more intense and spread over a greater number of areas in Biodentine than in MTA samples (P < .0001). Alkaline phosphatase staining of a mineralized tissue bridge was significantly different between materials (P < .0001), but no difference in alkaline phosphatase staining of pulp tissue was found between MTA and Biodentine (P = .2). Also, no significant difference in the number of cells labeled for runt-related transcription factor 2 by immunofluorescence was observed between materials (P > .05). CONCLUSIONS: Biodentine stimulated similar markers as MTA, but staining was more intense and spread over a larger area of the pulp tissue.

ß-lactam substituted polycyclic fused pyrrolidine/pyrrolizidine derivatives eradicate C. albicans in an ex vivo human dentinal tubule model by inhibiting sterol 14-α demethylase and cAMP pathway.

BACKGROUND: Further quest for new anti-fungal compounds with proven mechanisms of action arises due to resistance and dose limiting toxicity of existing agents. Among the human fungal pathogens C. albicans predominate by infecting several sites in the body and in particular oral cavity and root canals of human tooth. METHODS: In the present study, we screened a library of ß-lactam substituted polycyclic fused pyrrolidine/pyrrolizidine compounds against Candida sp. Detailed molecular studies were carried out with the active compound 3 on C. albicans. Morphological damage and antibiofilm activity of compound 3 on C. albicans was studied using scanning electron microscopy (SEM). Biochemical evidence for membrane damage was studied using flow cytometry. In silico docking studies were carried out to elucidate the mechanism of action of compound 3. Further, the antifungal activity of compound 3 was evaluated in an ex vivo dentinal tubule infection model. RESULTS: Screening data showed that several new compounds were active against Candida sp. Among them, Compound 3 was most potent and exerted time kill effect at 4h, post antifungal effect up to 6h. When used in combination with fluconazole or nystatin, compound 3 revealed an minimum inhibitory concentration (MIC) decrease by 4 fold for both drugs used. In-depth molecular studies with compound 3 on C. albicans showed that this compound inhibited yeast to hyphae (Y-H) conversion and this involved the cAMP pathway. Further, SEM images of C. albicans showed that compound 3 caused membrane damage and inhibited biofilm formation. Biochemical evidence for membrane damage was confirmed by increased propidium iodide (PI) uptake in flow cytometry. Further, in silico studies revealed that compound 3 docks with the active site of the key enzyme 14-α-demethylase and this might inhibit ergosterol synthesis. In support of this, ergosterol levels were found to be decreased by 32 fold in compound 3 treated samples as analyzed by high performance liquid chromatography (HPLC). Further, the antifungal activity of compound 3 was evaluated in an ex vivo dentinal tubule infection model, which mimics human tooth root canal infection. Confocal laser scanning microscopy studies showed 83% eradication of C. albicans and a 6 log reduction in colony forming unit (CFU) after 24h treatment in the infected tooth samples in this model. CONCLUSION: Compound 3 was found to be very effective in eradicating C. albicans by inhibiting cAMP pathway and ergosterol biosynthesis. GENERAL SIGNIFICANCE: The results of this study can pave the way for developing new antifungal agents with well deciphered mechanisms of action and can be a promising antifungal agent or medicament against root canal infection.

Immune-Inflammatory Cell Profile and Receptor Activator of Nuclear Factor Kappa B Ligand/Osteoprotegerin Expression in Persistent Apical Periodontitis after Root Canal Retreatment Failure.

INTRODUCTION: This study assessed the immune-inflammatory profile and the expression of bone resorption activators receptor activator of nuclear factor kappa B ligand (RANKL) and inhibitor osteoprotegerin (OPG) in apical periodontitis (n = 20) that persisted after root canal retreatment. METHODS: Immunohistochemistry was used to characterize lymphocyte populations (CD3+, CD45RO+, CD8+, and FoxP3+ cells), macrophages (CD68+), RANKL+ and OPG+ cells in persistent apical periodontitis (PAP) and primary periapical lesions (PPLs). By using quantitative real-time polymerase chain reaction, the mRNA expression of RANKL and OPG in PAP and periodontal ligament from healthy teeth was comparatively analyzed. The data were analyzed by Mann-Whitney, Pearson χ2, and Wilcoxon tests (5% level). RESULTS: PAP showed an elevated number of FoxP3+ cells compared with PPL (P < .001). The number of CD68+ cells was reduced in the PAP samples compared with the PPLs (P < .001). Similar number of other lymphocyte populations was observed in PAP and PPLs (P > .05 for all comparisons). No differences in the RANKL, OPG, and immune-inflammatory cells were demonstrated when comparing PAP microscopically classified as cyst with those classified as granulomas (P > .05 for all comparisons). The assessment of mRNA expression revealed higher levels of RANKL and OPG in PAP compared with the periodontal ligament from healthy teeth (control) samples (P < .001). Also, a greater expression of RANKL in comparison with OPG was observed in PAP (P < .001). CONCLUSIONS: These findings indicate that PAP consists of biologically active lesions that demonstrate potential of bone resorption (higher expression of RANKL) and is characterized by an immune-inflammatory cell profile that suggests a suppressive and regulatory environment (higher number of FoxP3+ cells and lower number of macrophages) favorable to more chronic clinical behavior.

Revascularization and tissue regeneration of an empty root canal space is enhanced by a direct blood supply and stem cells.

BACKGROUND: Regenerative endodontics is an innovative treatment concept aiming to regenerate pulp, dentin and root structures. In the diseased or necrotic tooth, the limitation in vascular supply renders successful tissue regeneration/generation in a whole tooth challenging. The aim of this study is to evaluate the ability of vascularized tissue to develop within a pulpless tooth using tissue engineering techniques. MATERIALS AND METHODS: A pulpless tooth chamber, filled with collagen I gel containing isolated rat dental pulp cells (DPC) and angiogenic growth factors, was placed into a hole created in the femoral cortex or into its own tooth socket, respectively. The gross, histological and biochemical characteristics of the de novo tissue were evaluated at 4 and 8 weeks post-transplantation. RESULTS: Tooth revascularization and tissue generation was observed only in the femur group, confirming the important role of vascular supply in tissue regeneration. The addition of cells and growth factors significantly promoted connective tissue production in the tooth chamber. CONCLUSION: Successful revascularization and tissue regeneration in this model demonstrate the importance of a direct vascular supply and the advantages of a stem cell approach.

[The role of pulp in the root resorption of primary teeth without permanent tooth germs].

OBJECTIVE: To investigate the role of pulp in the root resorption of primary teeth without permanent tooth germs. METHODS: The animal model without permanent tooth germs was established by surgery in Beagle dog. The root resorption was observed by taking periapical radiographs periodically. The samples of mandibular bone and pulp at different resorption stages were collected. The distribution of odontoclasts and the activating factor was analyzed by histological staining and semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). The role of pulp in the root resorption of primary teeth was tested by early pulpectomy. RESULTS: In the root resorption of primary molars without permanent teeth germs, a large number of odontoclasts were present on the pulpal surface of the root canal. Semi-quantification RT-PCR showed that the ratios of the expression of receptor activator of NF-κB ligand (RANKL) mRNA and ß-actin in the pulp of permanent teeth and primary teeth without permanent teeth germ during different periods of root resorption are 0.1314, 0.1901, 0.2111 and 0.6058 (P > 0.05). The root resorption of primary teeth without permanent teeth germs in test groups was about 5 weeks later than that of control group. CONCLUSIONS: The pulp of primary tooth played an important role in the root resorption of primary tooth without permanent tooth germ.

Effect of dentin treatment time with tetraclean on its residual antibacterial activity.

The goal of this study was to evaluate the effect of dentin treatment duration (10 minutes, 24 hours, and seven days) with Tetraclean on its residual antibacterial activity in bovine root dentin. Results showed that the number of colony-forming units in all three experimental groups was zero at the first culture. Furthermore, the 10-minute group and seven-day group demonstrated the highest and the lowest number of colony-forming units, respectively.

Dentin permeability of the apical third in different groups of teeth

This ex vivo study evaluated dentin permeability of the root canal in the apical third of different human groups of teeth. Eighty teeth were used, 8 from each dental group: maxillary and mandibular central incisors, lateral incisors and canines, maxillary first premolars (buccal and palatal roots), mandibular first premolars, and maxillary and mandibular second premolars, totalizing 88 roots that were distributed in 11 groups. The root canals were instrumented, irrigated with 1 percent NaOCl and 15 percent EDTA. Roots were immersed in 10 percent copper sulfate for 30 min and then in 1 percent rubeanic acid alcohol solution for the same period; this chemical reaction reveals dentin permeability by the formation of copper rubeanate, which is a dark-colored compound. Semi-serial 100-µm-thick cross-sections were obtained from the apical third of the roots. Five sections of each apical third were washed, dehydrated, cleared and mounted on glass slides for examination under optical microscopy. The percentage of copper ion infiltration and the amount of tubular dentin were quantified by morphometric analysis. The penetration of copper ions in the apical third ranged from 4.60 to 16.66 percent. The mandibular central and lateral incisors presented the highest dentin permeability (16.66 percent), while the maxillary canines and mandibular second and first premolars presented the lowest dentin permeability (4.60 percent, 4.80 percent and 5.71 percent, respectively; p<0.001). The other teeth presented intermediate permeability. In conclusion, dye penetration into dentin tubules at the apical region is strongly dependent on the group of teeth evaluated.

Este estudo ex vivo avaliou a permeabilidade da dentina do canal radicular do terço apical de diferentes grupos de dentes humanos. Foram utilizados 80 dentes, sendo 8 de cada grupo dental superior e inferior: incisivos centrais, incisivos laterais, caninos, primeiros pré-molares superiores (raízes vestibulares e palatinas), primeiros pré-molares inferiores, segundos pré-molares superiores e inferiores, totalizando 88 raízes, as quais foram distribuídas em 11 grupos. Os canais foram instrumentados, irrigados com NaOCl a 1 por cento e EDTA a 15 por cento. As raízes foram imersas em sulfato de cobre a 10 por cento por 30 min e acido rubeânico a 1 por cento pelo mesmo período. Esta reação química revela a permeabilidade da dentina por meio da formação de um complexo escurecido denominado rubeanato de cobre. Hemi-secções de 100 µm de espessura foram obtidas do terço apical da raiz. Cinco secções do terço apical foram lavadas, desidratadas, diafanizadas e montadas em lâminas para análise em microscopia óptica. A porcentagem de infiltração de íons cobre e a quantidade de dentina tubular foram quantificadas por meio de análise morfométrica. A penetração de íons cobre no terço apical da raiz variou de 4,60 por cento a 16,66 por cento. Os incisivos centrais e laterais apresentaram a maior permeabilidade dentinária (16,66 por cento), e os caninos superiores e segundos e primeiros pré-molares inferiores as menores (4,60 por cento, 4,80 por cento e 5,71 por cento, respectivamente; p<0,001). Os outros dentes apresentaram permeabilidade intermediaria. Conclui-se que a penetração de corante nos túbulos dentinários da região apical é extremamente dependente do grupo de dentes avaliado.