Influence of Manganese Oxide on the Esthetic Efficacy and Toxicity Caused by Conventional In-office Tooth Bleaching Therapy.

OBJECTIVE: This study aimed to evaluate the esthetic efficacy, cytotoxicity, and kinetics of decomposition of hydrogen peroxide (H2O2) present in a commercial bleaching gel with 35% H2O2 (BG35%) chemically activated with manganese oxide (MnO2). METHODS AND MATERIALS: After incorporating 2 mg/mL, 6 mg/mL, and 10 mg/mL of MnO2 into BG35%, the stability of pH and temperature of the products were analyzed. To assess the esthetic efficacy (ΔE and ΔWI), the BG35%s with MnO2 were applied for 45 minutes on enamel/dentin discs (DiE/D). BG35% or no treatment were used as positive (PC) and negative (NC) controls, respectively. To analyze the cell viability (CV) and oxidative stress (OXS), the same bleaching protocols were performed on DiE/D adapted to artificial pulp chambers. The extracts (culture medium + gel components that diffused through the discs) were applied to pulp cells and submitted to H2O2 quantification. BG35% with MnO2 that showed the best results was evaluated relative to kinetic decomposition of H2O2, with consequent generation of free radicals (FR) and hydroxyl radicals (OH•). The data were submitted to the one-way analysis of variance complemented by Tukey post-test (α=0.05). Data on kinetics of H2O2 decomposition were submitted to the Student's-t test (α=0.05). RESULTS: All the BG35%s with MnO2 showed stability of pH and temperature, and the gel with 10 mg/mL of this activator had an esthetic efficacy 31% higher than that of the PC (p<0.05). Reduction in OXS and trans-amelodentinal diffusion of H2O2 occurred when all the BG35%s with MnO2 were used. The addition of 6 and 10 mg/mL of MnO2 to BG35% increased the CV in comparison with PC, confirmed by the cell morphology analysis. An increase in FR and OH• formation was observed when 10 mg/mL of MnO2 was added to BG35%. CONCLUSION: Catalysis of BG35% with MnO2 minimized the trans-amelodentinal diffusion of H2O2 and toxicity of the product to pulp cells. BG35% containing 10 mg/mL of MnO2 potentiated the decomposition of H2O2, enhancing the generation of FR and OH•, as well as the efficacy of the in-office tooth therapy.

Chitosan in association with osteogenic factors as a cell-homing platform for dentin regeneration: Analysis in a pulp-in-a-chip model.

OBJECTIVE: In this paper we propose the association of ß-glycerophosphate (ßGP) and calcium-hydroxide with chitosan (CH) to formulate a porous bioactive scaffold suitable as a cell-homing platform for dentin regeneration. METHODS: Calcium hydroxide and ßGP solutions were incorporated into chitosan to modulate scaffold architecture and composition by a phase separation technique. Architecture, chemical composition, and degradability were evaluated, and biological characterizations were performed by the seeding of dental pulp cells (DPCs) onto scaffolds, or by cultivating them in contact with leachable components (extracts), to determine cytocompatibility and odontoblastic differentiation. Cell-free scaffolds were then positioned in intimate contact with a 3D culture of DPCs in a pulp-in-a-chip platform under simulated pulp pressure. Cell mobilization and odontoblastic marker expression were evaluated. Deposition of mineralized matrix was assessed in direct contact with dentin, in the absence of osteogenic factors. RESULTS: Incorporation of calcium hydroxide and ßGP generated a stable porous chitosan scaffold containing Ca-P nanoglobule topography (CH-Ca-ßGP), which favored cell viability, alkaline phosphatase activity, and mineralized matrix deposition by cells seeded onto the scaffold structure and at a distance. The pulp-in-a-chip assay denoted its chemotactic and bioactive potential, since dentin sialoprotein-positive DPCs from 3D culture adhered to CH-Ca-ßGP more than to plain chitosan. The higher deposition of mineralized matrix onto the scaffold and surrounding dentin was also observed. SIGNIFICANCE: A CH-Ca-ßGP scaffold creates a microenvironment capable of mobilizing DPC migration toward its structure, harnessing the odontogenic potential and culminating in the expression of a highly mineralizing phenotype, key factors for a cell-homing strategy.

Effect of Time and Temperature of Air Jet on the Mechanical and Biological Behavior of a Universal Adhesive System.

OBJECTIVES: To evaluate the influence of heat application on the degree of conversion (DC) of the 3M Single Bond Universal Adhesive System, as well as its transdentinal cytotoxicity and microtensile bond strength to dentin. METHODS: Experimental groups were established according to the time and temperature of the air jet: G1: 5 seconds-25°C; G2: 10 seconds-25°C; G3: 20 seconds-25°C; G4: 5 seconds-50°C; G5: 10 seconds-50°C; G6: 20 seconds-50°C. In control group (G7), no treatment was performed. The DC was assessed using the Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR) technique. For the transdentinal cytotoxicity test, dentin discs fitted in artificial pulp chambers (APC) received the application of the adhesive system and the air jets. For the microtensile bond strength, healthy molars were restored and submitted to the microtensile test after 24 hours and 6 months, respectively. RESULTS: Significant reduction in viability of Mouse Dental Papilla Cell-23 (MDPC-23), which exhibited morphological changes, was observed in all experimental groups compared to control (p<0.05). Although all tested protocols resulted in transdentinal diffusion of 2-hydroxyethyl methacrylate (HEMA), the group G6 presented the highest degree of monomeric conversion and the lowest cytotoxic effect, with higher dentin bond strength values in comparison to group G1 (p<0.05). CONCLUSIONS: Applying an air blast at 50°C for 20 seconds increases the DC and microtensile bond strength of the 3M Single Bond Universal Adhesive System to dentin, as well as reduces the transdentinal cytotoxicity of the material to pulp cells.

Chitosan-Calcium-Simvastatin Scaffold as an Inductive Cell-Free Platform.

The development of biomaterials based on the combination of biopolymers with bioactive compounds to develop delivery systems capable of modulating dentin regeneration mediated by resident cells is the goal of current biology-based strategies for regenerative dentistry. In this article, the bioactive potential of a simvastatin (SV)-releasing chitosan-calcium-hydroxide (CH-Ca) scaffold was assessed. After the incorporation of SV into CH-Ca, characterization of the scaffold was performed. Dental pulp cells (DPCs) were seeded onto scaffolds for the assessment of cytocompatibility, and odontoblastic differentiation was evaluated in a microenvironment surrounded by dentin. Thereafter, the cell-free scaffold was adapted to dentin discs positioned in artificial pulp chambers in direct contact with a 3-dimensional (3D) culture of DPCs, and the system was sealed to simulate internal pressure at 20 cm/H2O. In vivo experiments with cell-free scaffolds were performed in rats' calvaria defects. Fourier-transform infrared spectroscopy spectra proved incorporation of Ca and SV into the scaffold structure. Ca and SV were released upon immersion in a neutral environment. Viable DPCs were able to spread and proliferate on the scaffold over 14 d. Odontoblastic differentiation occurred in the DPC/scaffold constructs in contact with dentin, in which SV supplementation promoted odontoblastic marker overexpression and enhanced mineralized matrix deposition. The chemoattractant potential of the CH-Ca scaffold was improved by SV, with numerous viable and dentin sialoprotein-positive cells from the 3D culture being observed on its surface. Cells at 3D culture featured increased gene expression of odontoblastic markers in contact with the SV-enriched CH-Ca scaffold. CH-Ca-SV led to intense mineralization in vivo, presenting mineralization foci inside its structure. In conclusion, the CH-Ca-SV scaffold induces differentiation of DPCs into a highly mineralizing phenotype in the presence of dentin, creating a microenvironment capable of attracting pulp cells to its surface and inducing the overexpression of odontoblastic markers in a cell-homing strategy.

Influence of Tooth Pigmentation on H2O2 Diffusion and Its Cytotoxicity After In-office Tooth Bleaching.

CLINICAL RELEVANCE: Pigments in tooth structures affect the diffusion of H2O2 through enamel and dentin. The bleaching methodology can be impacted. SUMMARY: Objective: The aim of this study was to evaluate the influence of the presence of pigments in tooth structures on the trans-enamel and trans-dentin diffusion of hydrogen peroxide (H2O2) and its cytotoxicity after carrying out an in-office bleaching therapy.Methods and Materials: A bleaching gel with 35% H2O2 was applied for 45 minutes (three times for 15 minutes) on enamel and dentin discs (n=6), either previously submitted to the intrinsic pigmentation protocol with a concentrated solution of black tea, or not, defining the following groups: G1, unbleached untreated discs (control 1); G2, unbleached pigmented discs (control 2); G3, bleached untreated discs; G4, bleached pigmented discs. The discs were adapted to artificial pulp chambers, which were placed in wells of 24-well plates containing 1 mL culture medium (Dulbecco's modified Eagle's medium [DMEM]). After applying the bleaching gel on enamel, the extracts (DMEM + components of bleaching gel that diffused through the discs) were collected and then applied on the cultured MDPC-23 odontoblast-like cells. Cell viability (methyl tetrazolium assay and Live & Dead, Calcein AM, and ethidium homodimer-1 [EthD-1] probes), the amount of H2O2 that diffused through enamel and dentin (leuco-crystal violet product), and the H2O2-mediated oxidative cell stress (SOx) and components of degradation were assessed (analysis of variance/Tukey; α=0.05).Results: There was no significant difference between the groups G1 and G2 for all the parameters tested (p>0.05). Reduction in the trans-enamel and trans-dentin diffusion of H2O2 occurred for G4 in comparison with G3. Significantly lower cell viability associated with greater oxidative stress was observed for G3 (p<0.05). Therefore, in-office tooth bleaching therapy performed in pigmented samples caused lower cytotoxic effects compared with untreated samples submitted to the same esthetic procedure (p<0.05).Conclusion: According to the methodology used in this investigation, the authors concluded that the presence of pigments in hard tooth structures decreases the trans-enamel and trans-dentin diffusion of H2O2 and the toxicity to pulp cells of an in-office bleaching gel with 35% H2O2.

Effects of Enzymatic Activation of Bleaching Gels on Hydrogen Peroxide Degradation Rates, Bleaching Effectiveness, and Cytotoxicity.

OBJECTIVES: The aim of this study was to evaluate the effect of horseradish peroxidase (HRP) on the release of free radicals, bleaching effectiveness, and indirect cytotoxicity of a 35% hydrogen peroxide (HP) bleaching gel. METHODS AND MATERIALS: First, HP degradation rates and free radical release were evaluated for 35% HP in contact or not with HRP (10 mg/mL). The bleaching gel associated or not with HRP was then applied (3 × 15 minutes) to enamel/dentin discs adapted to artificial pulp chambers, and the culture medium in contact with dentin surfaces (extract) was collected and exposed to cultured odontoblast-like cells. Membrane damage and viability of cells as well as oxidative stress were evaluated. Residual HP/free radical diffusion was quantified, and bleaching effectiveness (ΔE) was assessed. Unbleached discs served as negative controls. RESULTS: The addition of HRP to the 35% HP bleaching gel enhanced the release of free radicals in comparison with plain HP gel. The 35% HP-mediated cytotoxicity significantly decreased with HRP in the bleaching gel and was associated with reduced HP/free radical diffusion through the enamel/dentin discs. ΔE values increased every bleaching session for HRP-containing gel relative to positive control, accelerating the whitening outcome. CONCLUSION: The enzymatic activation of a 35% HP bleaching gel with HRP accelerated HP degradation mediated by intensification of free radical release. This effect optimized whitening outcome as well as minimized residual HP and free radical diffusion through enamel and dentin, decreasing the harmful effects on odontoblast-like cells.

PAR-1 and PAR-2 Expression Is Enhanced in Inflamed Odontoblast Cells.

Protease-activated receptors (PARs) are G protein-coupled receptors, which are activated by proteolytical cleavage of the amino-terminus and act as sensors for extracellular proteases. We hypothesized that PAR-1 and PAR-2 can be modulated by inflammatory stimulus in human dental pulp cells. PAR-1 and PAR-2 gene expression in human pulp tissue and MDPC-23 cells were analyzed by quantitative polymerase chain reaction. Monoclonal PAR-1 and PAR-2 antibodies were used to investigate the cellular expression of these receptors using Western blot, flow cytometry, and confocal microscopy in MDPC-23 cells. Immunofluorescence assays of human intact and carious teeth were performed to assess the presence of PAR-1 and PAR-2 in the dentin-pulp complex. The results show for the first time that human odontoblasts and MDPC-23 cells constitutively express PAR-1 and PAR-2. PAR-2 activation increased significantly the messenger RNA expression of matrix metalloproteinase (MMP)-2, MMP-9, MMP-13, and MMP-14 in MDPC-23 cells ( P < 0.05), while the expression of these enzymes decreased significantly in the PAR-1 agonist group ( P < 0.05). The high-performance liquid chromatography and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analysis showed the presence of MMP-13 activity cleaving PAR-1 at specific, noncanonical site TLDPRS42↓F43LL in human dental pulp tissues. Also, we detected a presence of a trypsin-like activity cleaving PAR-2 at canonical site SKGR20↓S21LIGRL in pulp tissues. Confocal microscopy analysis of human dentin-pulp complex showed intense positive staining of PAR-1 and PAR-2 in the odontoblast processes in dentinal tubules of carious teeth compared to intact ones. The present results support the hypothesis of activation of the upregulated PAR-1 and PAR-2 by endogenous proteases abundant during the inflammatory response in dentin-pulp complex.

Correction: In vivo evaluation of photodynamic inactivation using Photodithazine® against Candida albicans.

Correction for 'In vivo evaluation of photodynamic inactivation using Photodithazine® against Candida albicans' by J. C. Carmello, et al., Photochem. Photobiol. Sci., 2015, 14, 1319-1328.

Influence of enamel/dentin thickness on the toxic and esthetic effects of experimental in-office bleaching protocols.

OBJECTIVES: This paper aims to assess the whitening effectiveness and toxicity of tooth-bleaching protocols applied to enamel/dentin disks simulating mandibular incisors (ICs) and premolars (PMs). MATERIALS AND METHODS: A 10% hydrogen peroxide (H2O2) gel was applied for 3 × 15, 1 × 15, or 1 × 5 min to enamel/dentin disks simulating mandibular ICs and PMs, and the trans-enamel and trans-dentinal diffusion products were applied to human dental pulp cells (1 h). Professional therapy (35% H2O2-3 × 15 min) was used as positive control, and non-bleached samples were used as negative control. Cell viability and morphology, oxidative stress generation, and odontoblastic marker expression were assessed. The H2O2 diffusion and enamel color change (ΔE) were also analyzed. RESULTS: The 10% H2O2 gel induced significant cell viability reduction only when applied 3 × 15 min, with the intensity of oxidative stress and down-regulation of odontoblastic markers being higher in the IC group. The other experimental bleaching protocols caused slight alterations regarding the cell parameters evaluated, with intensity being related to enamel/dentin thickness. These effects were also correlated with higher H2O2 diffusion in the IC group. ΔE values similar as positive control were found for the 10% 3 × 15 and 1 × 15 protocols on IC group, after 4 and 6 sessions. CONCLUSION: Application of a 10% H2O2 bleaching gel for 15 or 45 min to thin dental substrate significantly minimizes cell toxicity in comparison with highly concentrated gels associated with similar esthetic outcomes by increasing the number of bleaching sessions. CLINICAL RELEVANCE: Bleaching gels with 10% H2O2 applied in small teeth for short periods may be an interesting alternative to obtain whitening effectiveness without causing toxicity to pulp cells, which may be able to reduce the tooth hypersensitivity claimed by patients.

Transdentinal photobiostimulation of stem cells from human exfoliated primary teeth.

AIM: To evaluate the effects of infrared light-emitting diode (LED) irradiation on stem cells from human exfoliated deciduous teeth (SHEDs). METHODOLOGY: Exfoliated primary teeth were obtained (n = 3), and SHEDs obtained from the teeth were seeded on the pulpal surface of 0.2-mm-thick dentine discs produced from permanent molars. The cells were incubated for 24 h by placing the discs in plain Dulbecco's modified Eagle's medium (DMEM). The DMEM was then replaced with new culture medium formulated for odontoblast differentiation. After 12 h in the second medium, SHEDs were irradiated through the dentine discs using an infrared LED (850 nm) with a power density of 80 mW cm-2 . Energy doses (EDs) delivered to the occlusal surface of the dentine discs were 0 (control), 2 and 4 J cm-2 (n = 6). Subsequent tests were performed 72 h after irradiation. These tests included cell viability (MTT), alkaline phosphatase activity (ALP), total protein production (TP), scanning electron microscopy (SEM), as well as gene expression for ALP, Col I, DSPP and DMP-1. Data were analysed using Kruskal-Wallis and Mann-Whitney t-tests (α = 0.05). RESULTS: Both EDs (2 and 4 J cm-2 ) significantly increased cell viability and ALP activity. For TP, ALP and Col I gene expression, only the 4 J cm-2 group had significantly higher values compared to the control group. Cell morphology was not affected by irradiation. CONCLUSION: Infrared LED irradiation was capable of biostimulating SHEDs through a 0.2 mm thickness of dentine, especially at the 4 J cm-2 level.

Development of an oral mucosa equivalent using a porcine dermal matrix.

We evaluated the suitability of a porcine acellular dermal matrix for the development of a 3-dimensional oral mucosal equivalent using an ex vivo-produced oral mucosal equivalent (EVPOME). Oral keratinocytes were seeded in a submerged model, and then in an air-liquid interphase model, using Transwell® inserts. EVPOME showed good cell viability and increased glucose consumption over time. Histological evaluation showed that stratified differentiated epithelium had formed in all matrices.

Nutritional deprivation and LPS exposure as feasible methods for induction of cellular - A methodology to validate for vitro photobiomodulation studies.

Previous studies have demonstrated that high biostimulation takes place when cells under stress are subjected to phototherapy by laser or light-emitting-diode (LED) devices. Several studies selected nutritional deprivation by reducing the concentration of fetal bovine serum (FBS) in the culture medium or the exposure of cultured cells to lipopolysaccharide (LPS) as an in vitro cellular stress condition. However, there are no data certifying that these stimuli cause stressful conditions for cultured cells. This investigation assessed the induction of cellular stress by decreasing the concentration of FBS or adding LPS to culture medium. Odontoblast-like cells (MDPC-23) were cultured in complete culture medium (DMEM) containing 10% FBS. After a 12-hour incubation period, the DMEM was replaced by fresh medium containing 10% FBS (control), low concentrations of FBS (0, 0.2, 0.5, 2, or 5%) or LPS from Escherichia coli (10µg/ml). After an additional 12-hour incubation, cell viability, total cell-counting, total protein production, and gene expression of heat shock protein 70 (HSP70) were assessed. Data were statistically analyzed by ANOVA complemented by the Tukey test, with 5% considered significant. Cell viability was negatively affected only for 0% FBS, while reduced viable cell numbers and total protein production were detected for FBS concentrations lower than 2%. Higher HSP70 gene expression was also observed for FBS concentrations lower than 2% and for cells exposed to LPS. The nutritional deprivation model with culture medium lower than 2% of FBS can be safely used to induce cellular stress for in vitro photobiomodulation studies.

In vivo photodynamic inactivation of Candida albicans using chloro-aluminum phthalocyanine.

This study evaluated the photoinactivation of Candida albicans in a murine model of oral candidiasis using chloro-aluminum phthalocyanine (ClAlP) encapsulated in cationic nanoemulsions (NE) and chloro-aluminum phthalocyanine (ClAlP) diluted in DMSO (DMSO) as photosensitizer (PS). Seventy-five 6-week-old female Swiss mice were immunosuppressed and inoculated with C. albicans to induce oral candidiasis. PDT was performed on the tongue by the application of the photosensitizers and LED light (100 J cm(-2) -660 nm). Twenty-four hours and 7 days after treatments, microbiological evaluation was carried out by recovering C. albicans from the tongue of animals (CFU ml(-1) ). Then, mice were sacrificed and the tongues were surgically removed for histological and biomolecular analysis of pro- and anti-inflammatory cytokines. Data were analyzed by ANOVA followed by Tukey's post hoc test. ClAlP-NE-mediated PDT reduced 2.26 log10 of C. albicans recovered from the tongue when compared with the control group (P-L-) (P < 0.05). PDT did not promote adverse effects on the tongue tissue. Seven days after treatment, all animals were completely healthy. In summary, PDT mediated by chloro-aluminum phthalocyanine entrapped in cationic nanoemulsions was effective in reducing C. albicans recovered from the oral lesions of immunocompromised mice.

Indirect cytocompatibility of a low-concentration hydrogen peroxide bleaching gel to odontoblast-like cells.

AIM: To assess the initial cytotoxicity and the late phenotype marker expression of odontoblast-like cells (MDPC-23) subjected to less aggressive in-office bleaching therapies. METHODOLOGY: A 17.5% hydrogen peroxide (H2O2) gel was applied for 45, 15 or 5 min to enamel/dentine discs adapted to trans-wells positioned over cultured MDPC-23 cells. No treatment was performed on the negative control. Immediately after bleaching, the cell viability, gene expression of inflammatory mediators and quantification of H2O2 diffusion were evaluated. The ALP activity, DSPP and DMP-1 gene expression and mineralized nodule deposition (MND) were assessed at 7, 14 or 21 days post-bleaching and analysed statistically with Mann-Whitney U-tests (α = 5%). RESULTS: H2O2 diffusion, proportional to treatment time, was observed in all bleached groups. Reductions of approximately 31%, 21% and 13% in cell viability were observed for the 45-, 15- and 5-min groups, respectively. This reduction was significant (P < 0.05) for the 45- and 15-min groups, which also presented significant (P < 0.05) over-expression of inflammatory mediators. The 45-min group was associated with significant (P < 0.05) reductions in DMP-1/DSPP expression at all periods, relative to control. The ALP activity and MND were reduced only in initial periods. The 15-min group had less intense reduction of all markers, with no difference to control at 21 days. CONCLUSIONS: The 17.5% H2O2 applied to tooth specimens for 5 min caused no alteration in the odontoblast-like cells. When this gel was applied for 45 or 15 min, a slight cytotoxicity, associated with alterations in phenotypic markers, was observed. However, cells were able to recover their functions up to 21 days post-bleaching.

Cytocompatibility of HEMA-free resin-based luting cements according to application protocols on dentine surfaces.

AIM: To evaluate the transdentinal cytotoxicity of resin-based luting cements (RBLCs), with no HEMA in their composition, to odontoblast-like cells. METHODOLOGY: Human dentine discs 0.3 mm thick were adapted to artificial pulp chambers (APCs) and placed in wells of 24-well plates containing 1 mL of culture medium (DMEM). Two categories of HEMA-free RBLCs were evaluated: group 1, self-adhesive Rely X Unicem (RU; 3M ESPE), applied directly to the dentine substrate; and group 2, Rely X ARC (RARC; 3M ESPE), applied to dentine previously acid-etched and treated with a bonding agent. In group 3 (control), considered as representing 100% cell metabolic activity, no treatment was performed on dentine. The APC/disc sets were incubated for 24 h or 7 days at 37 °C and 5% CO2 . Then, the extracts (DMEM + dental materials components that diffused through dentine) were applied to cultured odontoblast-like MDPC-23 cells for 24 h. After that, the cell viability (MTT assay), cell morphology (SEM), total protein production (TP) and alkaline phosphatase (ALP) activity were assessed. Data from MTT assay and TP production were analysed by Kruskal-Wallis and Mann-Whitney tests (α = 5%). Data from ALP activity were analysed by one-way anova and Tukey's test (α = 5%). RESULTS: In group 1, a slight reduction in cell viability (11.6% and 16.8% for 24-h and 7-day periods, respectively) and ALP activity (13.5% and 17.9% for 24-h and 7-day periods, respectively) was observed, with no significant difference from group 3 (control) (P > 0.05). In group 2, a significant reduction in cell viability, TP production and ALP activity compared with group 3 (control) occurred (P < 0.05), regardless of incubation time. Alteration in MDPC-23 cell morphology was observed only in group 2. CONCLUSIONS: HEMA-free Rely X ARC cement caused greater toxicity to odontoblast-like MDPC-23 cells than did Rely X Unicem cement when both resin-based luting materials were applied to dentine as recommended by the manufacturer.

Influence of Restoration Type on the Cytotoxicity of a 35% Hydrogen Peroxide Bleaching Gel.

OBJECTIVES: The tooth/restoration interface may act as a pathway for hydrogen peroxide (H2O2) diffusion into the pulp chamber. Therefore, the influence of resin-modified glass ionomer cement (RMGIC) and resin composite simulated restorations on the cytotoxicity of an in-office bleaching gel was assessed in vitro. MATERIALS AND METHODS: Cavities in enamel/dentin discs restored with RMGIC Vitremer (3M ESPE) or Single Bond/Filtek Z350 (3M ESPE) resin composite (RC) were subjected or not subjected to hydrolytic degradation (HD). A 35%-H2O2 bleaching gel was applied to simulated restored and nonrestored enamel surfaces, and culture medium in contact with the dentin substrate (extract) was collected and applied to MDPC-23 cells. Nonrestored discs subjected or not subjected to bleaching were used as positive and negative controls, respectively. Cell viability, oxidative stress, interleukin (IL)-1ß expression, alkaline phosphatase (ALP) activity, and mineralized nodule deposition were evaluated. The H2O2 in the extracts was quantified. Data were subjected to statistical analysis. RESULTS: Higher oxidative stress associated with reduced cell viability, ALP activity, and mineralized nodule deposition was observed for all bleached groups compared with the negative control group. The RMGIC/HD group, which presented the highest H2O2 diffusion, had the lowest values of cell viability, ALP activity, and mineralized nodule deposition, as well as significantly increased IL-1ß expression. CONCLUSIONS: Dental cavities restored with the RMGIC subjected to hydrolytic degradation allowed for more intense diffusion of H2O2 into the pulp chamber, intensifying the toxicity of a 35%-H2O2 bleaching gel to pulp cells.

Effect of LPS treatment on the viability and chemokine synthesis by epithelial cells and gingival fibroblasts.

OBJECTIVE: Several local factors can affect the wound-healing process, delaying its progression and postponing tissue homeostasis. It is known that local inflammation is related to wound healing; however, the maintenance of the inflammatory reaction can impair the proliferation and migration of oral mucosal cells. The aim of this study was to evaluate the viability and chemokine expression of epithelial cells and gingival fibroblasts exposed to long-term lipopolysaccharide (LPS) treatment. DESIGN: Epithelial cells (HaCaT, Cell Lines Service, 300493) and human gingival fibroblasts (HGFs) were seeded (1×10(5) cells/well) in 24-well plates and incubated for 24h. To simulate the responses of cells to a local chronic oral mucosal inflammation, we added LPS of Escherichia coli (10 µg/ml) to Dulbecco's modified Eagle's medium (DMEM), kept in contact with fibroblasts and epithelial cells for 24, 48, and 72h. Then the cells were assessed for viability (alamarBlue assay), number (trypan blue assay), and expression of CCL2 and CCL5 inflammatory chemokines (enzyme-linked immunosorbent assay (ELISA)). Data were statistically analyzed by nonparametric Kruskal-Wallis and Mann-Whitney tests at a significance level of 5%. RESULTS: Cell treatment with LPS caused significant decrease in viability for both cell lines. No time-dependent effect was observed for epithelial cells. However, reduction in fibroblast viability was greater at 48 and 72 h. CCL2 and CCL5 synthesis was significantly increased for both LPS-treated cells, and this expression decreased with time. CONCLUSION: The maintenance of an inflammatory cell stimulus by LPS decreases the number and viability of cultured oral mucosal cells, which may be related to delayed wound healing.

In vivo evaluation of photodynamic inactivation using Photodithazine® against Candida albicans.

This study describes the photoinactivation of Candida albicans in a murine model of oral candidosis, mediated by Photodithazine® (PDZ). Six-week-old female Swiss mice were immunosuppressed, and inoculated with C. albicans to induce oral candidosis. After five days, photodynamic inactivation (PDI) mediated by PDZ at concentrations of 75, 100, 125 and 150 mg L(-1) was applied on the tongue of mice. Next, microbiological evaluation was performed by recovering C. albicans from the tongue via colony forming units (CFU mL(-1)). After 24 h of treatment, the animals were killed and the tongues were surgically removed for histological analysis. PDI was effective in reducing C. albicans on the tongue of mice using 100 mg L(-1) of PDZ, when compared to the positive control group (without treatment). No adverse effect on the tongue tissue was verified after PDI. Therefore, PDI was effective for inactivation of C. albicans without causing any harmful effects on host tissues, which is promising for future clinical trials.

Increased Durability of Resin-Dentin Bonds Following Cross-Linking Treatment.

OBJECTIVES: This study evaluated the long-term effect of carbodiimide treatments of acid-etched dentin on resin-dentin bond strength of a simplified etch-and-rinse adhesive system. METHODS: Forty-eight sound third molars were divided into three groups (n=16) according to the dentin treatment: G1: deionized water; G2: 0.5 mol/L 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) applied for 30 seconds; and G3: 0.5 mol/L EDC applied for 60 seconds. Flat dentin surfaces were produced, etched with 37% phosphoric acid for 15 seconds, and then treated with deionized water for 60 seconds or with 0.5 mol/L EDC for 30 or 60 seconds prior to the application of Single Bond 2. Crowns were restored with resin composite, and beam specimens were prepared for microtensile testing. The beams from each group were tested 24 hours or 6 or 12 months after the adhesive procedures. One slab from each tooth was prepared and analyzed for nanoleakage. Bond strength (MPa) data were submitted to analysis of variance and Tukey test (α=0.05). RESULTS: The treatment of dentin with 0.5 mol/L EDC for 30 seconds (24.1±6.2 MPa) and 60 seconds (25.5±5.1 MPa) did not negatively affect the immediate bond strength of Single Bond 2 when compared to the control group (24.6±7.3 MPa). Additionally, EDC prevented resin-dentin bond degradation after 12 months in artificial saliva for both periods of treatment. An increased accumulation of silver ions was seen for the control group over time, while a much lower amount of silver grains was observed for the EDC-treated groups. CONCLUSIONS: 0.5 mol/L EDC was able to prevent resin-dentin bond degradation after 12 months, especially when applied for 60 seconds.

Cytotoxicity of dimethyl sulfoxide (DMSO) in direct contact with odontoblast-like cells.

OBJECTIVES: To evaluate the cytotoxicity of dimethyl sulfoxide (DMSO) on the repair-related activity of cultured odontoblast-like MDPC-23 cells. METHODS: Solutions with different concentrations of DMSO (0.05, 0.1, 0.3, 0.5 and 1.0 mM), diluted in culture medium (DMEM), were placed in contact with MDPC-23 cells (5 × 104 cells/cm(2)) for 24 h. Eight replicates (n = 8) were prepared for each solutions for the following methods of analysis: violet crystal dye for cell adhesion (CA), quantification of total protein (TP), alizarin red for mineralization nodules formation (MN) and cell death by necrosis (flow cytometry); while twelve replicates (n = 12) were prepared for viable cell number (Trypan Blue) and cell viability (MTT assay). Data were analyzed by ANOVA and Tukey or Kruskal-Wallis and Mann-Whitney's tests (p < 0.05). RESULTS: Cell viability, adhesion and percentage of cell death by necrosis were not affected by DMSO at any concentration, with no statistical significant difference among the groups. A significant reduction in total protein production was observed for 0.5 and 1.0 mM of DMSO compared to the control while increased mineralized nodules formation was seen only for 1.0 mM DMSO. SIGNIFICANCE: DMSO caused no or minor cytotoxic effects on the pulp tissue repair-related activity of odontoblast-like cells.