A new model of in vitro dentin intratubular contamination for Fusobacterium nucleatum: Validation by confocal laser scanning microscopy.

Objectives: To test and validate a new protocol for in vitro contamination of dentinal tubules using Fusobacterium nucleatum (F. nucleatum) by confocal laser scanning microscopy (CLSM), in addition to evaluating the effectiveness of conventional endodontic irrigants such as sodium hypochlorite (NaOCl) and chlorhexidine (CLX) on this biofilm. Material and methods: Thirty lower premolars were contaminated with F. nucleatum (ATCC 51190) for 7 days under anaerobic conditions using the proposed new model. The specimens were divided into a control group and experimental groups, according to the irrigants: NaOCl 2.5% and CLX 2%. Then, the samples were submitted for analysis by CLSM and the LIVE/DEAD technique to quantify bacterial viability. Data normality was verified by the Shapiro-Wilk test. Intragroup and intergroup comparisons were performed using the Kruskal-Wallis test, followed by Dunn's post-test. Results: The CLSM images obtained demonstrated the effectiveness of the proposed new contamination protocol, with a high percentage of viable bacteria in relation to the treated groups (p < 0.05). Lower viability values were observed for the 2.5% NaOCl group. Conclusion: The new contamination protocol resulted in a high and homogeneous percentage of viable bacteria in the dentinal tubules in all specimens evaluated. Both irrigating solutions proved to be effective in reducing the intratubular microbiota, especially 2.5% NaOCl.

Osteoblastic response to biomaterials surfaces: Extracellular matrix proteomic analysis.

The cellular response to surfaces is mediated, among other factors, by the extracellular matrix (ECM). However, little is known about the ECM proteome during mineralization. Our objective was to compare the ECM composition formed by osteoblast on different materials surfaces with proteomic analysis. Three types of biomaterial surfaces (pure titanium, anodized titanium, and zirconia) were used. Osteoblasts (MC3T3 linage) cells were cultivated on the biomaterials for 7, 14, and 21 days with the osteogenic medium. For the proteomic analysis, the specimens were washed, decellularized, and the ECM was collected. The majority of the typical ECM proteins, out of a total of 24 proteins identified, was expressed and regulated equally on the three biomaterials tested. Alpha-1,4 glucan phosphorylase was found to be down-regulated on zirconia on the seventh day, while at the same time, glycogen phosphorylase brain form was up-regulated on anodized titanium, both when compared with pure titanium (ratio: 1.06 and 0.97, respectively). And after 14 days of culture, glycogen phosphorylase brain form was downregulated on zirconia when compared with pure titanium (ratio: 0.90), suggesting the influence of material surface roughness and chemical composition on energy metabolism. Proteins related to bone development like Transforming growth factor beta-3 and Fibroblast growth factor 8 were found exclusively on pure titanium on the 21st day. Altogether, our results show a possible influence of material surfaces on the composition of ECM.

Effects of Qualea grandiflora Extract on the Expression of MMP-14 and HIF-1α in Cultured Fibroblasts and Preosteoblasts

Abstract Background: Qualea grandiflora (QG) (Vochysiaceae), also known as "pau-ferro", "pau-terra" or "pau-de-tucano", is a very common deciduous tree in the Brazilian Cerrado used in traditional medicine to treat inflammations, ulcers, diarrhea, and infections. There are reports in the scientific literature that demonstrate the medicinal effects of the bark and leaf of the QG. However, studies involving this plant are rather imited. Aim of the study: To perform the phytochemical analysis of the QG hydroalcoholic extract (HAE) of leaves, and to investigate it effects on fibroblast and preosteoblasts. Methods: Phytochemical analysis was done by HPLC-DAD. Murine NIH/3T3 fibroblasts and MC3T3-E1 preosteoblasts cell lines (ATCC) were used for the experiments. Cell viability was assessed by the MTT colorimetric assay and the expression of MMP-14 and HIF-1α by immunofluorescence. Results and conclusion: The following compounds were identified by HPLC-DAD, such as quinic acid, ethyl galate, ellagic acid derivatives as O-methylellagic acid O-galloyl, O-methylellagic acid O-deoxyhexoside, galloyl derivatives, flavonol glycoside as kaempferol-O-deoxyhexoside, quercetin-O-deoxyhexoside, myricetin-O-deoxyhexoside and the pentacyclic triterpene arjunglucoside. Cell viability results demonstrated no cytotoxic effects in the studied concentrations. We found in QG HAE some compounds with therapeutic properties that can increase the expression of MMP-14 and HIF-1α, in fibroblasts and preosteoblasts. These data suggest that QG HAE has an action on these two molecules widely involved in physiological conditions, such as collagen remodeling, bone development and growth and pathological processes as HIF signaling in cancer metastasis.

Intratubular decontamination ability and physicochemical properties of calcium hydroxide pastes.

OBJECTIVE: This in vitro study compared the penetration, pH, calcium ion release, solubility, and intradentinal decontamination capacity of calcium hydroxide (CH) pastes with different vehicles and additives. MATERIALS AND METHODS: Infected standard bovine dentine contaminated with Enterococcus faecalis were treated with propolis extract, chlorhexidine, and camphorated paramonochlorophenol (CPMC) loaded in CH paste for the bacterial viability evaluation made by confocal laser scanning microscopy (CLSM) and microbiological culture. Beside this, 50 acrylic teeth were filled with the previously mentioned pastes to evaluate the pH and calcium ion release (pHmeter and atomic absorption spectrophotometer at time intervals of 7, 15, and 30 days) and solubility (micro-computed tomographic imaging before and after 15 days). RESULTS: After treatment, all samples decreased intra-dentinal contamination, specially, the CH paste with CPMC. There was no statistically significant difference between the groups when evaluating the intra-canal paste penetration. In the pH measurements, CH with distilled water showed the smallest pH values. Regardless the solubility percentage of the pastes, the paste of CH + PG presented the highest values. CONCLUSION: The vehicles and additives tested may increase CH antimicrobial effect, but with small differences. In general, all CH pastes tested here were effective in reducing Enterococcus faecalis and were similar in the penetration, pH, calcium ion release, and solubility of calcium hydroxide when compared to distilled water. CLINICAL RELEVANCE: The use of calcium hydroxide pastes as intracanal medication with an aqueous or viscous vehicle, as propylene glycol, can be useful, since all formulations of the tested pastes resulted in great bacterial reduction inside root canals.

Effect of ethanol-dissolved rhodamine B marker on mechanical properties of non-simplified adhesives.

OBJECTIVES: Rhodamine B (RB) is commonly used to evaluate dental polymers, including dental bonding systems (DBS). For reliability assessments, its effect should not only allow visualization of the dentin-polymer interface but also must not interfere with the bonding of the DBS to dentin as measured by the microtensile bond strength and hardness tests. MATERIAL AND METHODS: Flat human dentin surfaces were prepared and randomly distributed (n = 10) into six groups: Adper Scotchbond Multi-Purpose (MP) or Clearfil SE Bond (SE) in concentrations of none/control, 0.02 or 0.1 mg/mL. These combinations were prepared through ethanol dissolution to improve their penetration into the dentin. All specimens were fabricated with Filtek Z250 (n = 10) and prepared for a microtensile bond test (µTBS) (0.5 mm/min) after 7 days and 6 months. The failure modes were determined using a stereomicroscope (×40). For the hardness test, flat human dentin blocks were prepared and treated as previously described (n = 6). The specimens were stored at 37 °C/48 h and were tested (Knoop indenter - 25 gF/10 s). Data were analyzed with two-way ANOVA and Tukey tests for multiple comparisons (α = 0.05). The effect of time was evaluated using the Student t-test. RESULTS: For 7-day µTBS, both the DBS and RB concentrations were significant factors (p < 0.01). After 6 months, only the RB concentration was significantly different. Adhesive failures were prevalent for all groups. Regarding hardness, the DBS differed only with the use of 0.10 mg/mL of RB. CONCLUSIONS: Ethanol-dissolved rhodamine B in concentrations of 0.02 and 0.10 mg/mL in non-simplified adhesives can affect the physical-mechanical properties of functional monomer-based systems rather more than those of BisGMA systems.

Intratubular disinfection with tri-antibiotic and calcium hydroxide pastes.

OBJECTIVE: The aim of this study was to compare the in vitro intradentinal antimicrobial ability of the calcium hydroxide and tri-antibiotic pastes. MATERIALS AND METHODS: Standard bovine dentin tubes were sterilized and then infected with Enterococcus faecalis by a new contamination protocol of great depths of dentin. The specimens were filled with the medications, divided into two test-groups: calcium hydroxide (Group 1) and tri-antibiotic (Group 2) pastes. After 15 days, the teeth were evaluated by microbiological culture and confocal laser scanning microscopy (CLSM) with viability dye assay LIVE/DEAD inside dentinal tubules. In experiment of culture, the bacterial collection of the dentin fragments was done for counting the colony-forming units. RESULTS: The tri-antibiotic paste had a slightly greater antimicrobial effect; however, there was no statistical difference between the groups. CONCLUSIONS: It was concluded that the tri-antibiotic paste and the calcium hydroxide paste exercise the same effect on intra-tubular decontamination against E. faecalis. So, due the multiples advantages, the calcium hydroxide paste can be the choice for dentinal decontamination in regenerative procedures.

Effect of ultrasonic streaming on intra-dentinal disinfection and penetration of calcium hydroxide paste in endodontic treatment

ABSTRACT Objective The antimicrobial effect of ultrasonic agitation of calcium hydroxide (CH) pastes in infected bovine dentin and their penetrability were evaluated using confocal laser scanning microscopy (CLSM) and microbiological culture. Material and Methods Fifty-two bovine teeth were infected with Enterococcus faecalis using a new contamination protocol; then they received CH paste and were divided into groups with or without ultrasound. Ultrasonic agitation was conducted for 1 min with a plain point insert. After 15 d, the CLSM analyzed the viable and dead bacteria with Live and Dead assay. The dentinal wall debris was collected by burs, and the colony forming units (CFU/mL) were counted. The penetrability of the paste inside dentinal tubules was tested using the B-rodamine dye. Results The calcium hydroxide paste showed better results with the use of ultrasonic agitation (p<0.05). Conclusion The ultrasonic agitation of CH paste increased its antimicrobial action and was responsible for intradentinal penetration with the fulfilment of the tubules.

Effect of ultrasonic streaming on intra-dentinal disinfection and penetration of calcium hydroxide paste in endodontic treatment.

OBJECTIVE: The antimicrobial effect of ultrasonic agitation of calcium hydroxide (CH) pastes in infected bovine dentin and their penetrability were evaluated using confocal laser scanning microscopy (CLSM) and microbiological culture. MATERIAL AND METHODS: Fifty-two bovine teeth were infected with Enterococcus faecalis using a new contamination protocol; then they received CH paste and were divided into groups with or without ultrasound. Ultrasonic agitation was conducted for 1 min with a plain point insert. After 15 d, the CLSM analyzed the viable and dead bacteria with Live and Dead assay. The dentinal wall debris was collected by burs, and the colony forming units (CFU/mL) were counted. The penetrability of the paste inside dentinal tubules was tested using the B-rodamine dye. RESULTS: The calcium hydroxide paste showed better results with the use of ultrasonic agitation (p<0.05). CONCLUSION: The ultrasonic agitation of CH paste increased its antimicrobial action and was responsible for intradentinal penetration with the fulfilment of the tubules.

A new improved protocol for in vitro intratubular dentinal bacterial contamination for antimicrobial endodontic tests: standardization and validation by confocal laser scanning microscopy

Objectives To compare three methods of intratubular contamination that simulate endodontic infections using confocal laser scanning microscopy (CLSM). Material and Methods Two pre-existing models of dentinal contamination were used to induce intratubular infection (groups A and B). These methods were modified in an attempt to improve the model (group C). Among the modifications it may be included: specimen contamination for five days, ultrasonic bath with BHI broth after specimen sterilization, use of E. faecalisduring the exponential growth phase, greater concentration of inoculum, and two cycles of centrifugation on alternate days with changes of culture media. All specimens were longitudinally sectioned and stained with of LIVE/DEAD® for 20 min. Specimens were assessed using CLSM, which provided images of the depth of viable bacterial proliferation inside the dentinal tubules. Additionally, three examiners used scores to classify the CLSM images according to the following parameters: homogeneity, density, and depth of the bacterial contamination inside the dentinal tubules. Kruskal-Wallis and Dunn’s tests were used to evaluate the live and dead cells rates, and the scores obtained. Results The contamination scores revealed higher contamination levels in group C when compared with groups A and B (p<0.05). No differences were observed between group A and B (p>0.05). The volume of live cells in group C was higher than in groups A and B (p<0.05). Conclusion The new protocol for intratubular infection resulted in high and uniform patterns of bacterial contamination and higher cell viability in all specimens when compared with the current methods.

Influência do hidróxido de cálcio na adaptação e na penetração às paredes do canal de dentes obturados com um cimento resinoso à base de metacrilato / The influence of calcium hydroxide on adaptation and root canal penetration in teeth filled with methacrylate-based resin sealer

Introdução: o objetivo deste trabalho foi avaliar, por meio da microscopia confocal de varredura a laser (MCVL), o efeito da medicação intracanal com pasta de hidróxido de cálcio sobre a penetração e a porcentagem de adaptação — nos terços cervical, médio e apical — de canais obturados com o sistema obturador à base de metacrilato (Sistema Epiphany). Métodos: trinta incisivos inferiores humanos foram instrumentados até a lima 40.04 do sistema ProFile e a solução irrigadora usada foi o hipoclorito de sódio. Os dentes foram aleatoriamente divididos em três grupos (n = 10): Grupo I = sem hidróxido de cálcio (Ca(OH)2) (grupo controle); Grupo II = com Ca(OH)2 por 14 dias e remoção com solução salina + lima K #40; e Grupo III = similar ao Grupo II, mas utilizando o EDTA a 17% para remoção da medicação. O cimento Epiphany foi corado com rodamina B, e todos os canais foram obturados com o sistema Epiphany. Três secções de cada dente foram avaliadas sob magnificações de cinco e de quarenta vezes. Resultados: os testes estatísticos de ANOVA e Tukey indicaram significância estatística na redução dos valores de penetração do cimento no terço apical, comparado aos outros terços (p < 0,05). A maior profundidade de penetração foi observada nos terços coronal e médio do Grupo II. Os Grupos II (93%) e III (86%) tiveram as maiores porcentagens de adaptação, comparados ao Grupo I (78%) (p < 0,05). Conclusões: o Ca(OH)2 favoreceu a profundidade de penetração do cimento e a porcentagem de adaptação na interface dentina/cimento em dentes obturados com o cimento à base de metacrilato.

A new improved protocol for in vitro intratubular dentinal bacterial contamination for antimicrobial endodontic tests: standardization and validation by confocal laser scanning microscopy.

OBJECTIVES: To compare three methods of intratubular contamination that simulate endodontic infections using confocal laser scanning microscopy (CLSM). MATERIAL AND METHODS: Two pre-existing models of dentinal contamination were used to induce intratubular infection (groups A and B). These methods were modified in an attempt to improve the model (group C). Among the modifications it may be included: specimen contamination for five days, ultrasonic bath with BHI broth after specimen sterilization, use of E. faecalisduring the exponential growth phase, greater concentration of inoculum, and two cycles of centrifugation on alternate days with changes of culture media. All specimens were longitudinally sectioned and stained with of LIVE/DEAD for 20 min. Specimens were assessed using CLSM, which provided images of the depth of viable bacterial proliferation inside the dentinal tubules. Additionally, three examiners used scores to classify the CLSM images according to the following parameters: homogeneity, density, and depth of the bacterial contamination inside the dentinal tubules. Kruskal-Wallis and Dunn's tests were used to evaluate the live and dead cells rates, and the scores obtained. RESULTS: The contamination scores revealed higher contamination levels in group C when compared with groups A and B (p<0.05). No differences were observed between group A and B (p>0.05). The volume of live cells in group C was higher than in groups A and B (p<0.05). CONCLUSION: The new protocol for intratubular infection resulted in high and uniform patterns of bacterial contamination and higher cell viability in all specimens when compared with the current methods.

Erosive cola-based drinks affect the bonding to enamel surface: an in vitro study

Objective: This study aimed to assess the impact of in vitro erosion provoked by different cola-based drinks (Coke types), associated or not with toothbrushing, to bonding to enamel. Material and methods: Forty-six bovine enamel specimens were prepared and randomly assigned into seven groups (N=8): C- Control (neither eroded nor abraded), ERO-RC: 3x/1-minute immersion in Regular Coke (RC), ERO-LC: 3x/1-minute immersion in Light Coke (LC), ERO-ZC: 3x/1-minute immersion in Zero Coke (ZC) and three other eroded groups, subsequently abraded for 1-minute toothbrushing (EROAB-RC, EROAB-LC and EROAB-ZC, respectively). After challenges, they were stored overnight in artificial saliva for a total of 24 hours and restored with Adper Single Bond 2/Filtek Z350. Buildup coronal surfaces were cut in 1 mm2 -specimens and subjected to a microtensile test. Data were statistically analyzed by two-way ANOVA/Bonferroni tests (α=0.05). Failure modes were assessed by optical microscopy (X40). The Interface of the restorations were observed using Confocal Laser Scanning Microscopy (CLSM). Results: All tested cola-based drinks significantly reduced the bond strength, which was also observed in the analyses of interfaces. Toothbrushing did not have any impact on the bond strength. CLSM showed that except for Zero Coke, all eroded specimens resulted in irregular hybrid layer formation. Conclusions: All cola-based drinks reduced the bond strength. Different patterns of hybrid layers were obtained revealing their impact, except for ZC. .

Erosive cola-based drinks affect the bonding to enamel surface: an in vitro study.

OBJECTIVE: This study aimed to assess the impact of in vitro erosion provoked by different cola-based drinks (Coke types), associated or not with toothbrushing, to bonding to enamel. MATERIAL AND METHODS: Fifty-six [Corrected] bovine enamel specimens were prepared and randomly assigned into seven groups (N=8): C- Control (neither eroded nor abraded), ERO-RC: 3x/1-minute immersion in Regular Coke (RC), ERO-LC: 3x/1-minute immersion in Light Coke (LC), ERO-ZC: 3x/1-minute immersion in Zero Coke (ZC) and three other eroded groups, subsequently abraded for 1-minute toothbrushing (EROAB-RC, EROAB-LC and EROAB-ZC, respectively). After challenges, they were stored overnight in artificial saliva for a total of 24 hours and restored with Adper Single Bond 2/Filtek Z350. Buildup coronal surfaces were cut in 1 mm2 -specimens and subjected to a microtensile test. Data were statistically analyzed by two-way ANOVA/Bonferroni tests (α=0.05). Failure modes were assessed by optical microscopy (X40). The Interface of the restorations were observed using Confocal Laser Scanning Microscopy (CLSM). RESULTS: All tested cola-based drinks significantly reduced the bond strength, which was also observed in the analyses of interfaces. Toothbrushing did not have any impact on the bond strength. CLSM showed that except for Zero Coke, all eroded specimens resulted in irregular hybrid layer formation. CONCLUSIONS: All cola-based drinks reduced the bond strength. Different patterns of hybrid layers were obtained revealing their impact, except for ZC.

Comparative analysis of Enterococcus faecalis biofilm formation on different substrates.

INTRODUCTION: The aim of this study was to compare Enterococcus faecalis biofilm formation on different substrates. METHODS: Cell culture plates containing growth medium and E. faecalis (ATCC 29212) were used to grow biofilm on bovine dentin, gutta-percha, hydroxyapatite, or bovine bone. Substrates were incubated at 37°C for 14 or 21 days, and the medium was changed every 48 hours. After the growth induction periods, specimens (n = 5 per group and per induction period) were stained by using Live/Dead, and the images were analyzed under a confocal microscope. The total biovolume (µm(3)), live bacteria biovolume (µm(3)), and substrate coverage (%) were quantified by using the BioImage_L software. Results obtained were analyzed by nonparametric tests (P = .05). RESULTS: Biofilm formation was observed in all groups. Gutta-percha had the lowest total biovolume at 14 days (P < .05) and hydroxyapatite the highest at 21 days (P < .05). No significant difference was observed in green biovolume at 14 days. At 21 days, however, hydroxyapatite had the highest volume (P < .05). The percentages of coverage were similar among all substrates at 21 days (P > .05), but at 14 days, bovine bone presented the highest coverage (P < .05). CONCLUSIONS: E. faecalis was capable of forming biofilm on all substrates during both growth periods; hydroxyapatite presented the highest rates of biofilm formation. The type of substrate influenced the biofilm characteristics, according to the parameters evaluated.

The effects of occlusal loading on the margins of cervical restorations.

BACKGROUND: Researchers have proposed the restoration of abfraction lesions, but limited information is available about the effects of occlusal loading on the margins of such restorations. Because abfraction is a well-recognized problem, the authors conducted a study to assess the effects of occlusal loading on the margins of cervical restorations. METHODS: The authors prepared 40 wedge-shaped cavities in extracted premolars and restored them with a resin-based composite. They subjected specimens to occlusal loading (150 newtons, 10(6) cycles) on the buccal cusp, on the central fossa or on the lingual cusp, and they stored the control group specimens in deionized water. The authors used fluorescein to delimit marginal defects and evaluated the defects by using laser scanning confocal microscopy. RESULTS: Results of chi2 and Kruskal-Wallis tests (P < .05) showed that specimens subjected to occlusal loading had a higher percentage of marginal gaps (53.3 percent) than did the control specimens (10.0 percent). There were no differences between groups in marginal defect formation or in defect location, length or width. CONCLUSIONS: Occlusal loading led to a significant increase in gap formation at the margins of cervical resin-based composite restorations. CLINICAL IMPLICATIONS: The clinician cannot underestimate the effects of occlusal loading when restoring teeth with cervical wedge-shaped lesions. If occlusal loading is the main factor contributing to lesion formation, the clinician should identify and treat it before placing the restoration or otherwise run the risk that the restorative treatment will fail because of marginal gap formation.