Viability and oxidative stress of dental pulp cells after indirect application of chemomechanical agents: An in vitro study.

AIM: This study evaluated the transdentinal cytotoxic effects of enzymatic agents (EA) for chemomechanical carious tissue removal on human dental pulp cells. METHODOLOGY: The groups were based on the performed dentine treatments (n = 8): G1: Positive Control (PC - no treatment); G2: Negative Control (NC - 35% H2 O2 for 2 min); G3: Brix 3000™ (BX) for 30 s; G4: BX for 2 min; G5: Papacarie Duo™ (PD) for 30 s; G6: PD for 2 min. The cells were evaluated for viability (VB; MTT assay) and production of reactive oxygen species (ROS; DCFH-DA assay) and nitric oxide (NO; Griess reagent). A scanning electron microscope provided morphological chemical analyses and energy-dispersive X-ray spectroscopy. The data were submitted to the one-way anova statistical test complemented by Tukey (p < .05). RESULTS: Cell viability decreased by 21.1% and 58.4% in G5 and G6, respectively. ROS production in G3 and G4 maintained basal levels but increased by 171.2% and 75.1% in G5 and G6, respectively. CONCLUSIONS: The Brix3000™ enzymatic agent did not cause indirect cytotoxic effects on pulp cells, regardless of the application time. Conversely, Papacarie Duo™ reduced viability and increased ROS production by pulp cells.

Roughness assessment of colored compomers: Results after an erosive-abrasive in vitro cycling test.

Background: The present in vitro study aimed to evaluate and compare the surface roughness of a colored compomer and a composite resin, after 15 days of erosive-abrasive cycling. Material and Methods: The sample included ninety circular specimens, randomly divided (n = 10): G1 Berry, G2 Gold, G3 Pink, G4 Lemon, G5 Blue, G6 Silver, G7 Orange and G8 Green, referring to the different colors of compomer (Twinky Star®, VOCO, Germany) and G9 for composite resin (Z250®, 3M ESPE). The specimens were submerged in artificial saliva and stored at 37°C for 24 hours. After polishing and finishing, the specimens were submitted to initial roughness (R1). Then, the specimens were submerged in an acidic cola-based drink for 1 minute and then exposed to electric toothbrushing for 2 minutes for 15 days. After this period, the final roughness (R2) and the ΔRa were performed. Data were submitted to ANOVA and Tukey's test for intergroup comparison and paired T-test for intragroup comparison (p<0.05). Results: Among compomers, the green color presented the higher/lower initial and final roughness values (0.94 ± 0.44, 1.35 ± 0.55) with lemon color presenting the most prominent real roughness increase (ΔRa = 0,74) whereas composite resin showed the lower values (0,17 ± 0.06, 0,31 ± 0.15; ΔRa = 0,14). Conclusions: All compomers, after the erosive-abrasive challenge, presented an increase in roughness values when compared to composite resin with a highlight to green tones. Key words:Compomers, composite resins, surface properties.

Antibacterial activity of Brazilian red propolis and in vitro evaluation of free radical production.

OBJECTIVE: This study aimed to evaluate the antibacterial activity of crude Brazilian red propolis (BRP) extract against anaerobic bacteria involved in primary endodontic infection. Additionally, we evaluate the cell viability and free radical production of human dental pulp fibroblasts (HDPF) in direct contact with mineral trioxide aggregate (MTA) and BRP. DESIGN: The Minimum Inhibitory Concentration, Minimum Bactericidal Concentration (MIC, MBC) and Minimum Inhibitory Concentration of Biofilm (MICB50) of BRP against anaerobic endodontic pathogens were determined. HDPF were exposed to BRP10 (10 µg/mL), BRP50 (50 µg/mL), MTA extract (1:1, 1:2, 1:4 e 1:8), dimethyl sulfoxide 0.5% (DMSO), and cell culture medium (DMEM). The groups were tested for cell viability (MTT assay), and free radical production (reactive oxygen species - ROS, DCFH-DA probe and nitric oxide - NO, Griess reagent). The one-way ANOVA and Tukey's tests were employed at a significance level of 5%. RESULTS: MIC/MBC values of BRP performed antibacterial activity for Parvimonas micra (6.25/6.25 µg/mL), Fusobacterium nucleatum (25/25 µg/mL), Prevotella melaninogenica (50/100 µg/mL), Prevotella nigrescens (50/100 µg/mL), Prevotella intermedia (50/100 µg/mL), and Porphyromonas gingivalis (50/200 µg/mL). The MICB50 values ranged from 1.56 to 50 µg/mL. BRP and MTA stimulated cell viability, emphasizing BRP10 (p = 0.007). Furthermore, it was observed that MTA 1:1, MTA 1:2, and BRP50 slightly increased ROS (p < 0.001) and NO production (p = 0.008, p = 0.007, and p < 0.001 respectively) compared to DMEM group. CONCLUSIONS: BRP exhibits good antibacterial activity against endodontic pathogens, and both BRP and MTA promote the viability of HDPF without increasing NO and ROS production.

Photobiomodulation effect of red LED (630 nm) on the free radical levels produced by pulp cells under stress conditions.

The aim of this study was to assess the ability of red light emitting diodes (LED) to modulate oxidative stress in human dental pulp fibroblasts (HDPFs) when different irradiation parameters are employed. Cells from primary teeth were seeded (100,000 cells/well) in 24-well plates in culture medium (DMEM). At 24 h after incubation, the culture medium was replaced with DMEM containing 10 µg/mL lipopolysaccharide (LPS). Thereafter, the cells were irradiated (LED 630 nm, 0.04 W/cm2 and 0.08 W/cm2) at 0 J/cm2 (control group), 4 J/cm2, 15 J/cm2, and 30 J/cm2; and their viability (MTT assay), number (Trypan Blue), synthesis of nitric oxide (NO) (Griess reagent), and reactive oxygen species (ROS) (fluorescence probe, DCFH-DA) were assessed. The Kruskal-Wallis and Mann-Whitney statistical tests using Bonferroni correction were employed (significance level of 5%). Compared to that in control fibroblasts, increased viability was observed in HDPFs exposed to LPS and irradiated with 15 J/cm2 and 30 J/cm2 at 0.04 W/cm2 and 4 J/cm2 and 15 J/cm2 at 0.08 W/cm2 (p < 0.05). Exposure to 4 J/cm2 at 0.04 W/cm2 and 15 J/cm2 and 30 J/cm2 at 0.08 W/cm2 modulated the oxidative stress in cells relative to that observed in non-irradiated LPS-treated pulp cells (p < 0.05). It was concluded that the irradiation strategies of using red LED with radiant exposures of 15 J/cm2 and 30 J/cm2 at 0.04 W/cm2 and 15 J/cm2 at 0.08 W/cm2 were the best parameters to decrease NO and ROS concentration and to stimulate viability of HDPFs exposed to LPS challenge.

Proliferation rate and expression of stem cells markers during expansion in primary culture of pulp cells.

The aim of the present study was to evaluate the proliferation rate and the expression of stem cells markers during expansion in primary culture of dental pulp stem cells (DPSCs), comparing different techniques (explant and enzymatic digestion), subject ages (up to 40 and over 40) and cell passages (#2, #5 and #8). DPSCs were isolated using either the enzymatic digestion (ED) or explant (EX) technique. The number of days needed for the cells to reach confluence was determined. Immunophenotyping was performed by immunofluorescence and flow cytometry analysis using antibodies specific for nestin, vimentin, CD44, CD146, Oct3/4 and CD34. Data were subjected to three-way analysis of variance (n = 6/group). The ANOVA tests were complemented by Tukey's or t-tests (p < 0.05). The variables "donor age" and "technique" were analyzed to define the optimal desirability value using a response optimization. DPSCs presented a high proliferation rate from passages 2 to 5 while cells from passage 8 proliferated at a slower rate. For all markers, no significant difference was observed among passages, irrespective of the technique used or the donor's age. The mean fraction of specific antibodies was 73.7% (± 11.5), 49.0% (± 18.7), 80.1% (± 8.0), 45.2% (± 13.7), 64.7% (± 5.3) and 2.0% (± 1.5) for CD44, OCT, vimentin, nestin, CD146 and CD34, respectively. The highest optimal desirability value was obtained using the ED technique and cells from younger patients (d = 0.92). However, it was concluded that neither the isolation technique nor the donor age or cell passage significantly interfered with the stem cell phenotype and proliferation rate during cell expansion.

Specific parameters of infrared LED irradiation promote the inhibition of oxidative stress in dental pulp cells.

OBJECTIVES: The present study aimed to assess the oxidative stress and the viability of dental pulp cells stimulated by lipopolysaccharide (LPS) and submitted to photobiomodulation (PBM) with infrared light-emitting diode (LED, 850 nm). DESIGN: Three healthy primary teeth (n = 3) were collected and seeded in 24-well plates with 10 µg/mL of LPS to induce inflammatory mediator formation. The cells were irradiated (850 nm, 40 mW/cm2 and 80 mW/cm2) at the proposed radiant exposures of 0 (control), 4, 15, and 30 J/cm2 shortly after LPS supplementation. The tests were performed 24 h after irradiation to assess mitochondrial activity (MTT assay), the number of viable cells (Trypan Blue), cell morphology (Scanning Electron Microscopy - SEM), and the quantification of Nitric Oxide (NO) and Reactive Oxygen Species (ROS). The data were analyzed using Kruskal-Wallis and Dunn's tests (p < 0.05). RESULTS: The irradiated groups showed larger viable cells number than the non-irradiated group with LPS (p < 0.0001). All irradiation parameters decreased ROS concentrations after LPS application compared to the non-irradiated group (p < 0.05). All irradiation parameters enhanced the NO values compared to those of the control group (p < 0.05). The SEM images showed cells with regular morphology that adhered to the substrate. CONCLUSIONS: According to the parameters used in this study, the radiant exposure of 15 J/cm2 and irradiance of 40 mW/cm2 were the most effective irradiation parameters to stimulate and modulate oxidative stress in the primary teeth-derived dental pulp cells.

Cytotoxicity and dentin composition alterations promoted by different chemomechanical caries removal agents: A preliminary in vitro study.

BACKGROUND: The use of chemomechanical agents for caries removal has been indicated as a non-invasive treatment option; however, their possible deleterious effects on the dental-pulp complex have been insufficiently studied. This study assessed the direct cytotoxicity of two chemomechanical caries removal agents (Brix 3000™ - BX and Papacarie Duo™ - PD) on pulp cells from deciduous teeth, as well as to assess the morphology and chemical compositions of the dentin surface after the application of these materials. MATERIAL AND METHODS: The cells were seeded (50,000 cells/cm²) in a culture medium (DMEM with 10% fetal bovine serum - FBS). After 24 hours, the BX and PD materials were added to 1:20, 1:100, and 1:1000 dilutions. Non-exposed cells were considered as the control group. The viability test (MTT), Trypan Blue assay (TB), and cell morphology (Scanning Electron Microscopy - SEM) were performed after 24 hours of agent application. For the SEM and chemical (energy-dispersive X-ray spectrometry - EDS) dentin evaluation, 0.3-mm-thick dentin discs were obtained and divided into control group (no treatment) and surfaces covered with 37% phosphoric acid, BX, or PD. Data were compared by one-way ANOVA and Tukey's test (p<0.05). RESULTS: Decreases in cell viability and numbers of viable cells were observed for both materials, at all dilutions, when compared with the control group (p<0.05). The BX and PD materials did not cause visually perceptible changes, according to SEM, on the surfaces of dentin discs. The EDS analysis did not indicate a statistically significant difference in the levels of calcium (Ca) and phosphorus (P) between the materials and the control group (p>0.05). CONCLUSIONS: Both materials showed cytotoxicity when in direct contact with the pulp cells from deciduous teeth, and the BX material presented lower cytotoxicity than the PD material. Moreover, both materials did not significantly change the dentin composition. Key words:Cell culture, cytotoxicity, dental pulp, papacarie, primary teeth.

Biological parameters, discolouration and radiopacity of calcium silicate-based materials in a simulated model of partial pulpotomy.

AIM: To analyse the discolouration, radiopacity, pH and calcium ion release of Biodentine (BD), Bio-C repair (BCR) and Bio-C temp (BCT), as well as their biological effects on human dental pulp cells (hDPCs). METHODOLOGY: Sixty-four extracted bovine incisors were prepared to simulate crown fractures with pulp exposure and open root apex. The roots were filled using a mixture of agar and blood (control), and BD, BCR or BCT were placed over this mixture. Colour assessment analyses of the samples were performed before and immediately after material insertion and repeated at 30 and 90 days, using a spectrophotometer. The colour change of each specimen was evaluated at the crown and calculated based on the CIELab colour space. Digital radiographs were acquired for radiopacity analysis. hDPCs were placed in contact with different dilutions of culture media previously exposed to such materials and tested for cell viability using the MTT assay. The pH and calcium ion release of all materials were measured after 24 h; the data were assessed using one-way analysis of variance (ANOVA). Cell viability was analysed by two-way ANOVA. Differences in colour parameters and wound-healing data were assessed by two-way repeated measures ANOVA (α = 0.05). Tukey's and Dunnett's tests were used to compare the experimental groups with the control group. RESULTS: BCR had grater radiopacity and smaller colour alteration (ΔEab/ΔE00) than the other materials tested (p < .005; p < .001). No significant differences in pH were found amongst the tested materials (p > .05). BCT was associated with the largest release of calcium ions (p < .0001). BD had cell viability similar to that of the control at the lowest dilutions, and BCR was similar to that of the control, regardless of the dilution tested (p > .05). BCT had a lower percentage of viability than that of the control at all tested dilutions (p < .0001). Cell migration rates in BD and BCR were similar to those in the control group after 24 h and 48 h (p > .05), whilst BCT had larger voids than the control in both periods (p < .0001). CONCLUSIONS: BCR, BCT and BD were associated with tooth discolouration. BCR had the lowest staining values, the highest radiopacity and viability greater than 80% hDPCs.

Oral Health Status of Children Who Require In-Home Medical Care.

Purpose: To evaluate the oral health status of children who require in-home medical care, their oral hygiene and eating habits, and the association between oral health status and medical conditions.
Methods: Legal guardians of children who need in-home medical care were interviewed regarding their socioeconomic level and their children's medical and dental history, as well as their oral hygiene habits. An oral exam assessed the children's plaque level, caries experience, and periodontal disease. Descriptive and bivariate analyses were performed.
Results: Fifty-six children participated. Almost 61 percent had never received dental care and 58.9 percent had fair or poor oral hygiene. The most observed oral problems were gingival hyperplasia (46.4 percent), calculus (46.4 percent), and gingivitis (30.3 percent). The use of anticonvulsants and type of food were factors that correlated to calculus, gingivitis, or hyperplasia (P <0.05).
Conclusion: A significant number of children who require in-home medical care presented with deficient oral hygiene and periodontal problems, which were correlated with the use of anticonvulsants and gastrostomy feeding.

Proliferation rate and expression of stem cells markers during expansion in primary culture of pulp cells

Abstract: The aim of the present study was to evaluate the proliferation rate and the expression of stem cells markers during expansion in primary culture of dental pulp stem cells (DPSCs), comparing different techniques (explant and enzymatic digestion), subject ages (up to 40 and over 40) and cell passages (#2, #5 and #8). DPSCs were isolated using either the enzymatic digestion (ED) or explant (EX) technique. The number of days needed for the cells to reach confluence was determined. Immunophenotyping was performed by immunofluorescence and flow cytometry analysis using antibodies specific for nestin, vimentin, CD44, CD146, Oct3/4 and CD34. Data were subjected to three-way analysis of variance (n = 6/group). The ANOVA tests were complemented by Tukey's or t-tests (p < 0.05). The variables "donor age" and "technique" were analyzed to define the optimal desirability value using a response optimization. DPSCs presented a high proliferation rate from passages 2 to 5 while cells from passage 8 proliferated at a slower rate. For all markers, no significant difference was observed among passages, irrespective of the technique used or the donor's age. The mean fraction of specific antibodies was 73.7% (± 11.5), 49.0% (± 18.7), 80.1% (± 8.0), 45.2% (± 13.7), 64.7% (± 5.3) and 2.0% (± 1.5) for CD44, OCT, vimentin, nestin, CD146 and CD34, respectively. The highest optimal desirability value was obtained using the ED technique and cells from younger patients (d = 0.92). However, it was concluded that neither the isolation technique nor the donor age or cell passage significantly interfered with the stem cell phenotype and proliferation rate during cell expansion.

Effects of zinc oxide and calcium-doped zinc oxide nanocrystals on cytotoxicity and reactive oxygen species production in different cell culture models.

OBJECTIVES: This study aimed to synthesize nanocrystals (NCs) of zinc oxide (ZnO) and calcium ion (Ca2+)-doped ZnO with different percentages of calcium oxide (CaO), to evaluate cytotoxicity and to assess the effects of the most promising NCs on cytotoxicity depending on lipopolysaccharide (LPS) stimulation. MATERIALS AND METHODS: Nanomaterials were synthesized (ZnO and ZnO:xCa, x = 0.7; 1.0; 5.0; 9.0) and characterized using X-ray diffractometry, scanning electron microscopy, and methylene blue degradation. SAOS-2 and RAW 264.7 were treated with NCs, and evaluated for viability using the MTT assay. NCs with lower cytotoxicity were maintained in contact with LPS-stimulated (+LPS) and nonstimulated (-LPS) human dental pulp cells (hDPCs). Cell viability, nitric oxide (NO), and reactive oxygen species (ROS) production were evaluated. Cells kept in culture medium or LPS served as negative and positive controls, respectively. One-way analysis of variance and the Dunnett test (α = 0.05) were used for statistical testing. RESULTS: ZnO:0.7Ca and ZnO:1.0Ca at 10 µg/mL were not cytotoxic to SAOS-2 and RAW 264.7. +LPS and -LPS hDPCs treated with ZnO, ZnO:0.7Ca, and ZnO:1.0Ca presented similar NO production to negative control (p > 0.05) and lower production compared to positive control (p < 0.05). All NCs showed reduced ROS production compared with the positive control group both in +LPS and -LPS cells (p < 0.05). CONCLUSIONS: NCs were successfully synthesized. ZnO, ZnO:0.7Ca and ZnO:1.0Ca presented the highest percentages of cell viability, decreased ROS and NO production in +LPS cells, and maintenance of NO production at basal levels.

Cytotoxicity and Effects of a New Cacium Hydroxide Nanoparticle Material on Production of Reactive Oxygen Species by LPS-Stimulated Dental Pulp Cells.

Introduction: The aim of this study was to synthesize and characterize calcium hydroxide (CH) nanoparticles [CH-NP] and compare the cytotoxicity of these materials with that of mineral trioxide aggregate (White MTA) in human dental pulp mesenchymal cells (hDPMCs) stimulated by lipopolysaccharide (LPS). Methods and Materials: The CH-NP were synthesized by the co-precipitation method, and the physical properties were investigated through X-ray diffraction, scanning electron microscopy (SEM) and energy dispersive x-ray spectrometry (EDS). LPS-stimulated hDPMCs were placed in contact with different dilutions of culture media previously exposed to CH-NP and white MTA for 24 h. The groups were tested for cell viability by MTT formazan and Alamar Blue assays, the production of nitric oxide (NO) by Griess method and the production of reactive oxygen species (ROS) by means of the fluorescent oxidant-sensing probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Control groups for viability test were maintained in DMEM (not LPS-stimulated). For NO and ROS production, negative control group was cells in DMEM, and positive control was cells stimulated by LPS. The results were statistically analyzed by two-way ANOVA, Tukey's test and Dunnett's test (ɑ=0.05). Results: The results showed that the cell viability remained above 50% in all materials, independent of the dilution in MTT formazan and Alamar Blue tests. MTA showed a reduction in NO production at dilutions of 1:4 to 1:32 compared with the positive control group (P<0.05). The tested materials exhibited lower ROS production by DPMCs than that by cells in the positive control group (P<0.05), and similar ROS production to the negative control group (P>0.05). Conclusion: The outcomes of present in vitro study showed that MTA and [CH-NP] were not cytotoxic materials, with MTA closer to the results of control group (DMEM). MTA and [CH-NP] reduced ROS production at basal levels, with MTA inhibiting NO production at higher dilutions.

Talon cusp in the temporary dentition of a patient with Kabuki syndrome: Case report with a two-year follow-up.

The presence of dental abnormalities in the same individual may be related to syndromic cases and occur through associated systemic changes. Kabuki syndrome presents well-defined systemic changes, but its clinical characteristics related to the oral cavity have not been fully explained. This study aimed to report the dental changes in a child diagnosed with Kabuki syndrome. A male brown patient aged 2 years and 7 months, accompanied by his mother to the dental visit, they main complaint was the presented of an additional tooth behind upper right central incisor. Anamnesis, intra- and extraoral examinations, and dental X-rays were performed, revealing a talon cusp. Considering the dental clinical findings, the patient was referred to a medical geneticist who additionally requested cardiological and genetic examinations, which established the Kabuki syndrome. The caregivers were advised that the talon cusp would not cause any injury to the natural exfoliation of the tooth and that oral hygiene should be performed carefully. Abnormalities in the oral cavity and developmental delay may be associated with a potential undiagnosed syndrome. The medical evaluation becomes decisive for investigation, diagnosis, and final conduct of the case.

Photobiomodulation in the Metabolism of Lipopolysaccharides-exposed Epithelial Cells and Gingival Fibroblasts.

This study assessed the effects of photobiomodulation (PBM) to cells previously exposed to lipopolysaccharides (LPS). Human gingival fibroblasts (HGF) and epithelial cells (HaCaT) were seeded in wells of 24-well plates containing complete culture medium (DMEM). After 24 h, the DMEM was replaced by serum-free DMEM, and cells were exposed to LPS of Escherichia coli (E. coli) (10 µg mL-1 ) for 24, 48, and 72 h. The cells were subjected to specific parameters of phototherapy (PT) (LASERTable-InGaAsP-780 ± 3 nm, 25 mW, 3 J cm-2 ). Cell proliferation (alamarBlue® ), viability (Trypan Blue) and synthesis of CCL2 (ELISA) were evaluated. Data were statistically analyzed by the Kruskal-Wallis and Mann-Whitney test (α = 5%). Proliferation and viability of both cell lines decreased after LPS treatment at 48 and 72 h. Enhanced synthesis of CCL2 by gingival fibroblasts occurred at 24 h, while epithelial cells increased synthesis of this chemokine at 48 and 72 h. PBM enhanced cell proliferation and viability in a time-dependent manner for both cell lines exposed or not to LPS, while synthesis of CCL2 by cells exposed to PT decreased over time. PBM caused biomodulatory effects on gingival fibroblasts and epithelial cells previously treated with LPS. These effects may decrease tissue inflammatory response and accelerate wound healing of oral mucosal tissue.

"Metabolism of Odontoblast-like cells submitted to transdentinal irradiation with blue and red LED".

OBJECTIVES: The present study evaluated the trans-dentinal effect of light emitting diodes (LEDs) irradiation on the metabolism of odontoblast-like cells. METHODS: Seventy-two dentin discs (0.2mm thick) were obtained from human molar teeth. MDPC-23 cells (20,000 cells/disc) were seeded on the pulpal side of the discs using DMEM, supplemented with 10% fetal bovine serum (FBS). After 12h, the culture medium was replaced with DMEM containing 0.5% FBS. After additional 12h, blue (455±10nm) or red (630±10nm) LEDs were used at irradiances of 80 and 40mW/cm2, respectively, to irradiate the occlusal side of the discs. The energy doses were fixed at 2 or 4J/cm2. Cell viability, alkaline phosphatase activity (ALP), total protein production and collagen synthesis were evaluated 72h after irradiation. Data were submitted to Kruskal-Wallis and Mann-Whitney tests (α=0.05). RESULTS: Red light promoted proliferative effects at the energy dose of 4J/cm2. Conversely, cell cultures irradiated with 2J/cm2 emitted by the blue light showed reduced viability. ALP production was stimulated by red light in comparison with blue light at 4J/cm2. Total protein production was reduced after exposure to blue light at 4J/cm2, while no effect was observed on collagen production. CONCLUSIONS: Irradiation with red LED at 4J/cm2 bio-stimulated the viability of odontoblast-like cells, whilst blue light had unfavorable effects on the cellular metabolism.

Effects of low-level laser therapy and epidermal growth factor on the activities of gingival fibroblasts obtained from young or elderly individuals.

This study evaluated the effects of low-level laser therapy (LLLT) and epidermal growth factor (EGF) on fibroblasts obtained from young and elderly individuals. Gingival fibroblasts from young (Y) and elderly (E) individuals were seeded in wells of 24-well plates with Dulbecco's modified Eagle's medium (DMEM) containing 10 % of fetal bovine serum (FBS). After 24 h, the cells were irradiated (LASERTable-InGaAsP-780 ± 3 nm, 25 mW, 3 J/cm2) or exposed to EGF (100 µM). After 72 h, cells were evaluated for viability, migration, collagen and vascular endothelial growth factor (VEGF) synthesis, and gene expression of growth factors. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests (α = 5 %). Y and E fibroblasts irradiated with laser or exposed to EGF showed increased viability and collagen synthesis. Enhanced cell migration was observed for Y fibroblasts after both treatments, whereas only the LLLT stimulated migration of E cells. VEGF synthesis was higher for Y and E cells exposed to EGF, while this synthesis was reduced when E fibroblasts were irradiated. Increased gene expression of VEGF was observed only for Y and E fibroblasts treated with LLLT. Regardless of a patient's age, the LLLT and EGF applications can biostimulate gingival fibroblast functions involved in tissue repair.

Response of a co-culture model of epithelial cells and gingival fibroblasts to zoledronic acid.

Osteonecrosis of the jaw is an adverse effect of bisphosphonates. While the etiopathogenesis of this condition has been investigated, the interactions and effects of bisphosphonates on oral mucosa cells remain unclear. It is hypothesized that cell culture models, such as co-culture or three-dimensional cell culture models, can provide valuable insight. Therefore, the aim of this study was to evaluate the effects of zoledronic acid (ZA) on epithelial cells and gingival fibroblasts in a co-culture model. Briefly, epithelial cells were seeded on transwell inserts and gingival fibroblasts were seeded in the lower well of 24-well plates. The latter were treated with ZA (5 µM) for 24 or 48 h. Cell viability and synthesis of the inflammatory chemokine, CCL2, were subsequently assessed. Data were subjected to statistical analysis with a 5% significance level. In the presence of ZA, the epithelial cells exhibited significant toxicity in both cell culture models and at both time points. However, greater cytotoxicity was observed in the co-culture model. Greater viability for the gingival fibroblasts was also associated with the co-culture model, and ZA-mediated toxicity was observed for the 48 h time point. ZA promoted a significant increase in CCL2 synthesis in both sets of cells, with greater CCL2 synthesis detected in the gingival fibroblasts. However, this effect was diminished in the co-culture model. Taken together, these results confirm the specific response patterns of the cells seeded in the co-culture model and also demonstrate the protective mechanism that is mediated by epithelial/mesenchymal cell interactions upon exposure to ZA.

Red LED Photobiomodulates the Metabolic Activity of Odontoblast-Like Cells.

Phototherapy has been indicated as an adjunctive treatment for tissue repair, including the pulp tissue. However, there are no defined irradiation parameters, which is a great challenge to the clinical use of phototherapy. The aim of this study was to evaluate the effect of phototherapy with red LED on odontoblast-like MDPC-23 cells, using different parameter settings. Cells were seeded (104 cells/cm²), incubated for 12 h in complete DMEM and then the culture medium was replaced by DMEM supplemented with 0.5% FBS. After 12 h incubation, irradiations were performed (630±10 nm) using a LEDTable device with a 20 or 40 mW/cm² power density and 2 J/cm² energy dose. The cells were irradiated 1 or 3 times, at 1 min intervals. Non-irradiated cells served as control. The cells were evaluated for viability (MTT assay), total protein dosage (Lowry method) and number of viable cells (Trypan blue). The data (n=12 per group) were submitted to Kruskal-Wallis and Mann-Whitney tests (p=0.05). A single irradiation with 20 or 40 mW/cm² enhanced cell viability, which was negatively affected after 3 consecutive irradiations. Cells irradiated only once with 20 mW/cm² produced more proteins compared with those irradiated with 40 mW/cm². Reduction in the number of viable cells occurred only after 3 consecutive irradiations with 40 mW/cm². In conclusion, red LED was capable of biomodulating the metabolic activities of cultured MDPC-23 odontoblast-like cells. The best cell biostimulation was obtained when a single irradiation with 2 J/cm2 energy dose and 20 mW/cm2 power density was delivered to the pulp cells.

Red LED Photobiomodulates the Metabolic Activity of Odontoblast-Like Cells

Abstract Phototherapy has been indicated as an adjunctive treatment for tissue repair, including the pulp tissue. However, there are no defined irradiation parameters, which is a great challenge to the clinical use of phototherapy. The aim of this study was to evaluate the effect of phototherapy with red LED on odontoblast-like MDPC-23 cells, using different parameter settings. Cells were seeded (104 cells/cm²), incubated for 12 h in complete DMEM and then the culture medium was replaced by DMEM supplemented with 0.5% FBS. After 12 h incubation, irradiations were performed (630±10 nm) using a LEDTable device with a 20 or 40 mW/cm² power density and 2 J/cm² energy dose. The cells were irradiated 1 or 3 times, at 1 min intervals. Non-irradiated cells served as control. The cells were evaluated for viability (MTT assay), total protein dosage (Lowry method) and number of viable cells (Trypan blue). The data (n=12 per group) were submitted to Kruskal-Wallis and Mann-Whitney tests (p=0.05). A single irradiation with 20 or 40 mW/cm² enhanced cell viability, which was negatively affected after 3 consecutive irradiations. Cells irradiated only once with 20 mW/cm² produced more proteins compared with those irradiated with 40 mW/cm². Reduction in the number of viable cells occurred only after 3 consecutive irradiations with 40 mW/cm². In conclusion, red LED was capable of biomodulating the metabolic activities of cultured MDPC-23 odontoblast-like cells. The best cell biostimulation was obtained when a single irradiation with 2 J/cm2 energy dose and 20 mW/cm2 power density was delivered to the pulp cells.

Resumo Fototerapia tem sido indicada como um tratamento adjuvante para o reparo de tecidos, incluindo o tecido pulpar. Entretanto, não há parâmetros de irradiação definidos, o que representa um grande desafio para o uso clínico da fototerapia. O objetivo deste estudo foi avaliar o efeito da fototerapia com LED vermelho em células MDPC-23 com fenótipo odontoblastóide, usando vários parâmetros. As células foram semeadas (104 células/cm2), incubadas por 12 h em DMEM completo e então o meio de cultura foi trocado por DMEM com 0,5% SFB. Após 12 h de incubação, as irradiações foram realizadas (630±10 nm) usando um dispositivo com densidade de potência de 20 ou 40 mW/cm2 e dose de energia de 2 J/cm2. As células foram irradiadas 1 ou 3 vezes, com intervalos de 1 min. Células não irradiadas serviram como controle. Foram avaliadas a viabilidade (ensaio de MTT), dosagem de proteína total (método de Lowry) e número de células viáveis (ensaio de Trypan blue). Os dados (n=12 por grupo) foram submetidos aos testes de Kruskal-Wallis e Mann-Whitney (p=0,05). Uma única irradiação com 20 ou 40 mW/cm2 aumentou a viabilidade celular, a qual foi negativamente afetada após 3 irradiações. Células irradiadas apenas uma vez com 20 mW/cm2 produziram mais proteínas comparadas com aquelas irradiadas com 40 mW/cm2. Redução no número de células viáveis ocorreu apenas após 3 irradiações com 40 mw/cm2. Em conclusão, o LED vermelho foi capaz de biomodular a atividade metabólica de células MDPC-23. A melhor bioestimulação celular foi obtida quando uma única irradiação com dose de energia de 2 J/cm2 e densidade de potência de 20 mW/cm2 foi administrada às células pulpares.

Tumor Necrosis Factor-α and Interleukin (IL)-1ß, IL-6, and IL-8 Impair In Vitro Migration and Induce Apoptosis of Gingival Fibroblasts and Epithelial Cells, Delaying Wound Healing.

BACKGROUND: Multiple factors affect oral mucosal healing, such as the persistence of an inflammatory reaction. The present study evaluates effects of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß, IL-6, and IL-8 on epithelial cells (ECs) and human gingival fibroblasts (GFs) in vitro. METHODS: GFs and ECs were seeded in 96-well plates (1 × 10(4) cells/well) in plain culture medium (Dulbecco's modified Eagle's medium [DMEM]) containing 1% antibiotic/antimycotic solution and 10% fetal bovine serum, and incubated for 24 hours. Both cell lines were exposed for 24 hours to the following cytokines: 1) TNF-α (100 ng/mL); 2) IL-1ß (1 ng/mL); 3) IL-6 (10 ng/mL); and 4) IL-8 (10 ng/mL). All cytokines were diluted in serum-free DMEM. Control cultures were exposed only to serum-free DMEM. Effects of exposure to inflammatory cytokines were determined by means of: 1) apoptosis (anexin V); 2) cell migration (wound healing assay); 3) inflammatory cytokine synthesis (enzyme-linked immunosorbent assay). Data were analyzed by Kruskal-Wallis and Mann-Whitney U tests (α = 0.05). RESULTS: Increased apoptosis rates were noted when cells were exposed to inflammatory cytokines, except ECs exposed to IL-1ß. Cell migration was negatively affected by all inflammatory cytokines for both cell lines. ECs and GFs exposed to IL-6 and IL-8 significantly increased synthesis of TNF-α and IL-1ß. CONCLUSIONS: Demonstrated results indicate negative effects of tested inflammatory cytokines on ECs and GFs, inducing apoptosis and impairing cell migration. These results can justify delayed oral mucosa healing in the presence of inflammatory reaction.