Effect of Er:YAG laser and diamond drill on hybrid layer morphology obtained with self-etch adhesive: analysis by SEM and confocal laser scanning microscopy (CLSM)

This study aimed to evaluate the effect of Er:YAG (L) and diamond drills (DD) on: 1) the microshear bond strength (MPa); 2) the adhesive interface of two-step (TS) – Adper Scotchbond Multipurpose and one-step (OS) adhesives – Adper EasyOne, both from 3M ESPE. Material and methods: According to the preparation condition and adhesives, the samples were divided into four groups: DD_TS (control); DD_OS; L_TS and L_OS. 60 bovine incisors were randomly divided into experimental and groups: 40 for microshear bond strength (n = 10) and 20 for the adhesive interface morphology [6 to measure the thickness of the hybrid layer (HL) and length of tags (t) by CLSM (n = 3); 12 to the adhesive interface morphology by SEM (n = 3) and 2 to illustrate the effect of the instruments on dentine by SEM (n = 1)]. To conduct the microshear bond strength test, four cylinders (0.7 mm in diameter and 1 mm in height with area of adhesion of 0.38 mm) were constructed with resin composite (Filtek Z350 XT – 3M ESPE) on each dentin surface treated by either L or DD and after adhesives application. Microshear bond strength was performed in universal testing machine (EMIC 2000) with load cell of 500 kgf and a crosshead speed of 0.5 mm / min. Adhesive interface was characterized by thickness of hybrid layer (HL) and length of tags (t) in nm, with the aid of UTHSCSA ImageTool software. Results: Microshear bond strength values were: L_TS 34.10 ± 19.07, DD_TS 24.26 ± 9.35, L_OS 33.18 ± 12.46, DD_OS 21.24 ± 13.96. Two-way ANOVA resulted in statistically significant differences only for instruments (p = 0.047). Mann-Whitney identified the instruments which determined significant differences for HL thickness and tag length (t). Concerning to the adhesive types, these differences were only observed for (t). Conclusion: It can be concluded that 1) laser Er:YAG results in higher microshear bond strength values regardless of the adhesive system (TS and OS); 2) the tags did not significant affect the microshear bond strength; 3) the adhesive interface was affected by both the instruments for cavity preparation and the type of adhesive system used.

The applicability of hematoxylin-eosin staining plus fluorescence or confocal laser scanning microscopy to the study of elastic fibers in cartilages

This study focuses on the use of hemotoxylin-eosin staining plus fluorescence microscopy for the investigation of elastic fibers in some elastic cartilages. We have observed that elastic fibers are consistently imaged by the proposed procedure and the resolution attained is similar to that obtained with the classical Weigert's fuchsin-resorcin. The results also demonstrate that elastin autofluorescence gives little or no contribution to the final fluorescence and that the use of the confocal laser scanning microscope adds to the resolution, permits the use of thicker sections and reveals of minute structural features. We conclude that this is a relevant tool in elastin research.

Fluorescence and confocal laser scanning microscopy imaging of elastic fibers in hematoxylin-eosin stained sections

We have studied the possibility of associating fluorescence microscopy and hematoxylin-eosin staining for the identification of elastic fibers in elastin-rich tissues. Elastic fibers and elastic laminae were consistently identified by the proposed procedure, which revealed itself to be easy and useful for the determination of such structures and their distribution. The fluorescence properties of stained elastic fibers are due to eosin staining as revealed by fluorescence analysis of the dye in solution, with no or only minor contribution by the elastin autofluorescence. The main advantage of this technique resides in the possibility of studying the distribution of elastic fibers in file material without further sectioning and staining. The use of the confocal laser scanning microscope greatly improved the resolution and selectivity of imaging elastic fibers in different tissues. The determination of the three-dimensional distribution and structure of elastic fiber and laminae using the confocal laser scanning microscope was evaluated and also produced excellent results.

Antibiofilm activity of irrigating solutions associated with cetrimide. Confocal laser scanning microscopy

AimTo evaluate the antibiofilm activity of sodium hypochlorite (NaOCl) and chlorhexidine (CHX) solutions associated with cetrimide (CTR), and QMiX using confocal laser scanning microscopy.MethodologyEnterococcus faecalis (ATCC- 29212) biofilms were induced on bovine dentine blocks for 14days. The dentine blocks containing biofilm were immersed for 1min in the following solutions: 2.5% NaOCl; 2.5% NaOCl+0.2% CTR; 2% CHX; 2% CHX+0.2% CTR; 0.2% CTR; QMiX. After contact with the solutions, the dentine blocks were stained with Live/Dead((R)) BacLight for analysis of the remaining biofilm using confocal laser scanning microscope. Images were evaluated using the BioImage_L software to determine the total biovolume (m(3)), the green biovolume (live cells) (m(3)) and the percentage of substrate coverage (%). The data were subjected to nonparametric statistical test using Kruskal-Wallis and Dunn's tests at 5% significance level.ResultsAfter exposure to irrigants, the total biovolume observed for CHX, CHX+CTR, CTR, QMiX was similar to distilled water (P>0.05). NaOCl and NaOCl+CTR had the lowest total and green biovolume. The CTR and QMiX had intermediate green biovolume, with greater antibacterial activity than CHX and CHX+CTR (P<0.05). The NaOCl and NaOCl+CTR solutions were associated with microorganism removal and substrate cleaning ability.ConclusionsNaOCl and NaOCl+CTR solutions were effective on microorganism viability and were able to eliminate biofilm. The addition of cetrimide did not influence antibacterial activity.

Confocal laser scanning microscopy investigation of bond interfaces involved in fiber glass post cementation

Objective: This confocal microscopy study evaluated the cement/dentin and cement/post interfaces along theroot canalwallswhenfiberglasspostswerebonded to dentin using different types of cements. Material & Methods: Thirty endodontically treated premolars were divided into 3 groups according to the adhesive materials used in the bonding procedure: Prime & Bond 2.1/Self Cure + Enforce, RelyX Unicem and RelyX Luting. Rhodamine B dye was incorporated in the luting materials for the cementation of the fiber glass posts (Exacto, Angelus) to dentin. Three transversal slices (apical, middle and coronal) were examined under confocal laser scanning microscopy. Statistical analysis was performed using the Kappa, Kruskal-Wallis and Dunnet tests, in a significance level of 5%. Results: The Prime & Bond 2.1/Self Cure + Enforce presented a uniform formation of tags in the dentin but gaps in the cement/dentin interface. The RelyX Unicem and RelyX Luting presented an adhesive interface with a fewer amount of gaps, but showed shorter tag formation than the Enforce system. All cements presented the same pattern of bubbles inside the cements. The RelyX Luting presented a greater amount of cracks inside the cement in comparison with the other cements in the coronal third, while no difference was observed between RelyX Unicem and Enforce. The RelyX Luting showed the lowest quantity of cement penetration into the post. Conclusion: In general, the quality of bonding interfaces of fiber posts luted to root canals was affected by both location and type of cement.

Effect of bleaching on sound enamel and with early artificial caries lesions using confocal laser microscopy

The aim of this study was to evaluate effect of bleaching agents on sound enamel (SE) and enamel with early artificial caries lesions (CL) using confocal laser scanning microscopy (CLSM). Eighty blocks (4 × 5 × 5 mm) of bovine enamel were used and half of them were submitted to a pH cycling model to induce CL. Eight experimental groups were obtained from the treatments and mineralization level of the enamel (SE or CL) (n=10). SE groups: G1 - unbleached (control); G2 - 4% hydrogen peroxide (4 HP); G3 - 4 HP containing 0.05% Ca (Ca); G4 - 7.5% hydrogen peroxide (7.5 HP) containing amorphous calcium phosphate (ACP). CL groups: G5 - unbleached; G6 - 4 HP; G7 - 4 HP containing Ca; G8 - 7.5 HP ACP. G2, G3, G6, G7 were treated with the bleaching agents for 8 h/day during 14 days, while G4 and G8 were exposed to the bleaching agents for 30 min twice a day during 14 days. The enamel blocks were stained with 0.1 mM rhodamine B solution and the demineralization was quantified using fluorescence intensity detected by CLSM. Data were analyzed using ANOVA and Fisher's tests (α=0.05). For the SE groups, the bleaching treatments increased significantly the demineralization area when compared with the unbleached group. In the CL groups, no statistically significant difference was observed (p>0.05). The addition of ACP or Ca in the composition of the whitening products did not overcome the effects caused by bleaching treatments on SE and neither was able to promote remineralization of CL.

Confocal Laser Microscopic Analysis of Biofilm on Newer Feldspar Ceramic

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Texture and Microstructure of Gelatin/Corn Starch-Based Gummy Confections

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Photodynamic inactivation of planktonic cultures and biofilms of Candida albicans mediated by aluminum-chloride-phthalocyanine entrapped in nanoemulsions

New drug delivery systems, such as nanoemulsions (NE), have been developed to allow the use of hydrophobic drugs on the antimicrobial photodynamic therapy. This study evaluated the photodynamic potential of aluminum-chloride- phthalocyanine (ClAlPc) entrapped in cationic and anionic NE to inactivate Candida albicans planktonic cultures and biofilm compared with free ClAlPc. Fungal suspensions were treated with different delivery systems containing ClAlPc and light emitting diode. For planktonic suspensions, colonies were counted and cell metabolism was evaluated by XTT assay. Flow cytometry evaluated cell membrane damage. For biofilms, the metabolic activity was evaluated by XTT and ClAlPc distribution through biofilms was analyzed by confocal laser scanning microscopy (CLSM). Fungal viability was dependent on the delivery system, superficial charge and light dose. Free ClAlPc caused photokilling of the yeast when combined with 100 J cm-2. Cationic NE-ClAlPc reduced significantly both colony counts and cell metabolism (P < 0.05). In addition, cationic NE-ClAlPc and free ClAlPc caused significant damage to the cell membrane (P < 0.05). For the biofilms, cationic NE-ClAlPc reduced cell metabolism by 70%. Anionic NE-ClAlPc did not present antifungal activity. CLSM showed different accumulation on biofilms between the delivery systems. Although NE system showed a lower activity for planktonic culture, cationic NE-ClAlPc showed better results for Candida biofilms. Candida albicans biofilm overview after 30 min of contact with free ClAlPc. This study presents the photodynamic potential of aluminum-chloride-phthalocyanine (ClAlPc) entrapped in cationic and anionic nanoemulsions (NE) to inactivate C. albicans planktonic cultures and biofilm comparing with free ClAlPc. The photodynamic effect was dependent on the delivery system, superficial charge and light dose. Cationic NE-ClAlPc and free ClAlPc caused significant reduction in colony counts, cell metabolism and damage to the cell membrane (P < 0.05). However, only the free ClAlPc was able to cause photokilling of the yeast. The anionic NE-ClAlPc did not present antifungal activity. Although NE system showed a lower activity for planktonic culture, cationic NE-ClAlPc showed better results for Candida biofilms. © 2012 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2012 The American Society of Photobiology.

Effect of different pre-irradiation times on curcumin-mediated photodynamic therapy against planktonic cultures and biofilms of Candida spp

Objectives: The aim of this study was to evaluate the effects of pre-irradiation time (PIT) on curcumin (Cur)-mediated photodynamic therapy (PDT) against planktonic and biofilm cultures of reference strains of Candida albicans, Candida glabrata and Candida dubliniensis. Materials and methods: Suspensions and biofilms of Candida species were maintained in contact with different concentrations of Cur for time intervals of 1, 5, 10 and 20 min before irradiation and LED (light emitting diode) activation. Additional samples were treated only with Cur, without illumination, or only with light, without Cur. Control samples received neither light nor Cur. After PDT, suspensions were plated on Sabouraud Dextrose Agar, while biofilm results were obtained using the XTT-salt reduction method. Confocal Laser Scanning Microscopy (CLSM) observations were performed to supply a better understanding of Cur penetration through the biofilms after 5 and 20 min of contact with the cultures. Results: Different PITs showed no statistical differences in Cur-mediated PDT of Candida spp. cell suspensions. There was complete inactivation of the three Candida species with the association of 20.0 μM Cur after 5, 10 and 20 min of PIT. Biofilm cultures showed significant reduction in cell viability after PDT. In general, the three Candida species evaluated in this study suffered higher reductions in cell viability with the association of 40.0 μM Cur and 20 min of PIT. Additionally, CLSM observations showed different intensities of fluorescence emissions after 5 and 20 min of incubation. Conclusion: Photoinactivation of planktonic cultures was not PIT-dependent. PIT-dependence of the biofilm cultures differed among the species evaluated. Also, CLSM observations confirmed the need of higher time intervals for the Cur to penetrate biofilm structures. © 2012 Elsevier Ltd. All rights reserved.

Bifunctional silica nanoparticles for the exploration of biofilms of Pseudomonas aeruginosa

Luminescent silica nanoparticles are frequently employed for biotechnology applications mainly because of their easy functionalization, photo-stability, and biocompatibility. Bifunctional silica nanoparticles (BSNPs) are described here as new efficient tools for investigating complex biological systems such as biofilms. Photoluminescence is brought about by the incorporation of a silylated ruthenium(II) complex. The surface properties of the silica particles were designed by reaction with amino-organosilanes, quaternary ammonium-organosilanes, carboxylate-organosilanes and hexamethyldisilazane. BSNPs were characterized extensively by DRIFT, 13C and 29Si solid state NMR, XPS, and photoluminescence. Zeta potential and contact angle measurements exhibited various surface properties (hydrophilic/hydrophobic balance and electric charge) according to the functional groups. Confocal laser scanning microscopy (CLSM) measurements showed that the spatial distribution of these nanoparticles inside a biofilm of Pseudomonas aeruginosa PAO1 depends more on their hydrophilic/hydrophobic characteristics than on their size. CLSM observations using two nanosized particles (25 and 68 nm) suggest that narrow diffusion paths exist through the extracellular polymeric substances matrix. © 2013 Copyright Taylor and Francis Group, LLC.

Eficácia do óleo de Melaleuca alternifolia sobre bactérias cariogênicas e sua citoxicidade sobre cultura de queratinócitos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Eficácia da terapia fotodinâmica antimicrobiana em biofilmes de Staphylococcus Aureus suscetível e resistente á meticilina

Pós-graduação em Reabilitação Oral - FOAR

Avaliação in vitro da dentina remanescente após remoção químico-mecânica do tecido cariado em dentes decíduos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo da eficácia da terapia fotodinâmica, mediada pelos fotossensibilizadores Photodithazine® e Curcumina, sobre biofilmes multi-espécies formados por Streptococcus mutans, Candida albicans e Candida glabrata

Pós-graduação em Reabilitação Oral - FOAR