EGF coating of titanium surfaces modulates cytokines in oral mucosal primary cells exposed to TNF-α.

OBJECTIVE: This study assessed the metabolism of oral mucosal cells cultured on titanium discs (Ti) coated (or not) with epidermal growth factor (EGF) and exposed to tumor necrosis factor alpha (TNF-α). METHODS: Fibroblasts or keratinocytes were seeded on Ti coated or not with EGF, and then exposed to 100 ng/mL of TNF-α for 24 h. Groups were established: G1: Ti (control); G2: Ti + TNF-α; G3: Ti + EGF; and G4: Ti + EGF + TNF-α. Both cell lines were evaluated for: viability (AlamarBlue®, n = 8); interleukin 6 and 8 (IL-6, IL-8) gene expression (qPCR, n = 5), and protein synthesis (ELISA, n = 6). For keratinocytes cells, the matrix metalloproteinase type 3 (MMP-3) was evaluated by qPCR (n = 5) and ELISA (n = 6). A 3-D culture of fibroblasts was analyzed by confocal microscopy. The data were subjected to ANOVA analysis, α = 5%. RESULTS: Increased cell viability was observed in all groups compared with G1. Enhanced gene expression and synthesis of IL-6 and IL-8 by fibroblasts and keratinocytes in G2 and modulation of hIL-6 gene expression in G4 was noted. Modulation of IL-8 synthesis occurred in keratinocytes in G3 and G4. Keratinocytes in G2 showed enhanced gene expression of hMMP-3. A 3-D culture showed more cells in G3. Fibroblasts in G2 exhibited disrupted cytoplasmic membrane. Cells in G4 showed elongated morphology with intact cytoplasm. CONCLUSIONS: EGF coating increases cell viability and modulates the response of oral cells exposed to an inflammatory stimulus.

Regulation of interleukin-6 and matrix metalloproteinases syntheses by bioflavonoids and photobiomodulation in human gingival fibroblasts.

This study aimed to evaluate the separately effects of bioflavonoids proanthocyanidins, from grape seed extract (GSE) and synthetic naringenin (NA), as well as photobiomodulation (PBM) by low-level laser therapy on interleukin (IL)-6 and matrix metalloproteinases (MMPs) syntheses by human gingival fibroblasts (HGF). For this purpose, a connective tissue exposure (ulceration) model of HGF, stimulated with tumor necrosis factor-alpha (TNF-α), was used. Initially, the highest non-cytotoxic and non-genotoxic concentrations of bioflavonoids were determined by cell viability and micronuclei formation assays. Then, HGF were exposed to different stimuli: culture medium (negative control), dimethyl sulfoxide (DMSO), TNF-α, NA, GSE, TNF-α + NA, TNF-α + GSE, PBM (3 J/cm2, 0.025 W, 780 nm), and TNF-α + PBM. Next, IL-6, MMP-2, and MMP-9 syntheses were assessed. The concentration of 10 µg/mL of bioflavonoids increased cell viability at 24 and 48 h and did not present cytotoxic or genotoxic effects on HGF after 24, 48, and 72 h of contact. This concentration was selected for the assessment of bioflavonoids potential in modulating inflammatory mediators. TNF-α exposure enhanced IL-6 (170%), MMP-2 (10%), and MMP-9 (20%) syntheses, while a decrease of MMP-2 by 55% after exposure to TNF-α + GSE and 20% after TNF-α + NA and TNF-α + PBM was observed. MMP-9 synthesis was decreased by 35% after TNF-α + NA, 20% after TNF-α + GSE, and 30% after PBM. IL-6 was down-regulated by GSE in the presence of TNF-α (80%). In conclusion, TNF-α up-regulated IL-6 and MMPs, while bioflavonoids and PBM down-regulated MMP-2 and MMP-9 syntheses; GSE also decreased IL-6 synthesis, demonstrating the individual promising potential of these therapies for ulceration management.

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.

Photobiomodulation using LLLT and LED of cells involved in osseointegration and peri-implant soft tissue healing.

This study evaluated the influence of photobiomodulation (PBM) using low-level laser therapy (PBM/LLLT) or light-emitting diode (PBM/LED) therapy on peri-implant tissue healing. A laboratory model was used to assess the adhesion and metabolism of osteoblasts (SaOs-2), human gingival fibroblasts (HGF), and normal oral keratinocytes (NOK) seeded on a titanium (Ti) surface. After seeding the cells on disks of Ti placed in wells of 24-well plates, three irradiations were performed every 24 h at energy density of 3 J/cm2. For PBM/LLLT, a LaserTABLE device was used with a wavelength of 780 nm and 25 mW, while for PBM/LED irradiation, a LEDTABLE device was used at 810 nm, 20 mW, at a density of 3 J/cm2. After irradiations, the number of cells (NC) attached and spread on the Ti surface, cell viability (CV), total protein (TP), and collagen (Col) synthesis were assessed. Alkaline phosphate activity (ALP) was evaluated only for SaOs-2. Data were submitted to ANOVA complemented by Turkey statistical tests at a 5% significance level. PBM significantly increased adherence of NOK to the Ti surface, while no significant effect was observed for SaOs-2 and HGF. PBM positively affected CV, as well as Col and TP synthesis, in distinct patterns according to the cell line. Increased ALP activity was observed only in those cells exposed to PBM/LLLT. Considering cell specificity, this investigation reports that photobiomodulation with low-power laser and LED at determined parameters enhances cellular functions related to peri-implant tissue healing in a laboratory model.

Effects of EGF-coated titanium surfaces on adhesion and metabolism of bisphosphonate-treated human keratinocytes and gingival fibroblasts.

OBJECTIVE: To assess the effects of epidermal growth factor (EGF)-coated titanium (Ti) discs on the adhesion and metabolism of keratinocytes and gingival fibroblasts exposed to nitrogen-containing bisphosphonates. MATERIALS AND METHODS: Keratinocytes and fibroblasts were seeded (1 × 105 cells/disc) on Ti discs coated with EGF (100 nM). After 24 h, cells were exposed or not to sodium alendronate (SA) or zoledronic acid (ZA) at different concentrations (0 = control, 0.5, 1, or 5 µM) for 48 h. Cell adhesion to the substrates was evaluated by fluorescence microscopy. Cell viability (alamarBlue, n = 6) and synthesis of vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2), and keratinocytes growth factor (KGF) (ELISA, n = 6) were assessed. Data were statistically analyzed by one-way ANOVA and Tukey tests (α = 0.05). RESULTS: Higher cell adhesion rate was observed when keratinocytes and fibroblasts were seeded onto EGF-coated discs in comparison to uncoated discs. ZA treatment hindered the adhesion of both cell lines on the Ti discs as well as reduced the viability and synthesis of VEGF, KGF and MMP-2 by cells (p < 0.05). SA treatment did not affect cell viability, but interfered negatively on the adhesion and synthesis of EGF and KGF by the cells (p < 0.05). EGF-coated surface increased cell viability and synthesis of growth factors as well as downregulated the synthesis of MMP-2 in comparison to control (p < 0.05). CONCLUSION: EGF applied on Ti surface improves the biological responses of oral mucosa cells exposed to SA and ZA. CLINICAL RELEVANCE: EGF-coating on titanium may be a suitable strategy to improve oral mucosa cellular events related to biological sealing, especially for patients under bisphosphonate therapy.

In vitro effects of photobiomodulation applied to gingival fibroblasts cultured on titanium and zirconia surfaces and exposed to LPS from Escherichia coli.

Photobiomodulation (PBM) therapy is used to stimulate cell proliferation and metabolism, as well as reduce inflammatory cytokine synthesis, which plays a main role in the long-term stability of implants. This study assessed the response of gingival fibroblasts cultured on titanium (Ti) and zirconia (ZrO2), submitted to PBM and exposed to lipopolysaccharide (LPS). Cells seeded on Ti and ZrO2 were irradiated (InGaAsP; 780 nm, 25 mW) 3 times, using 0.5, 1.5, and 3.0 J/cm2 doses, and exposed to Escherichia coli LPS (1 µg/mL). After 24 h, cell viability (alamarBlue, n = 8), interleukin 6 (IL-6) and 8 (IL-8) synthesis (ELISA, n = 6), and IL-6 and vascular endothelial growth factor (VEGF) gene expression (qPCR, n = 5) were assessed and statistically analyzed (one-way ANOVA, α = 0.05). Cell morphology was evaluated by fluorescence microscopy. Increased cell viability occurred in all groups cultured on Ti compared with that of the control, except for cells exposed to LPS. Fibroblasts cultured on ZrO2 and LPS-exposed exhibited reduced viability. PBM at 3.0 J/cm2 and 1.5 J/cm2 downregulated the IL-6 synthesis by fibroblasts seeded on Ti and ZrO2, as well as IL-8 synthesis by cells seeded on ZrO2. Fibroblasts seeded on both surfaces and LPS-exposed showed increased IL-6 gene expression; however, this activity was downregulated when fibroblasts were irradiated at 3.0 J/cm2. Enhanced VEGF gene expression by cells seeded on Ti and laser-irradiated (3.0 J/cm2). Distinct patterns of cytoskeleton occurred in laser-irradiated cells exposed to LPS. Specific parameters of PBM can biomodulate the inflammatory response of fibroblasts seeded on Ti or ZrO2 and exposed to LPS.

Photobiomodulation of inflammatory-cytokine-related effects in a 3-D culture model with gingival fibroblasts.

The aim of this study was to assess the effects of IL-6 and IL-8 cytokines on human gingival fibroblasts (HGF) cultured in a 3-D model and the possible photobiomodulation (PBM) of such effects by low-level laser therapy. In complete culture medium (DMEM), HGF from a healthy patient were seeded in a type I collagen matrix inserted into 24-well plates. After 5 days of incubation, the cytokines were added or not to serum-free DMEM, which was applied to the cell-enriched matrices. Then, PBM was performed: three consecutive irradiations using LaserTable diode device (780 nm, 0.025 W) at 0.5 J/cm2 were delivered or not to the cells. Twenty-four hours after the last irradiation, cell viability and morphology, gene expression, and synthesis of inflammatory cytokines and growth factors were assessed. The histological evaluation demonstrated that, for all groups, matrices presented homogeneous distribution of cells with elongated morphology. However, numerous cytokine-exposed cells were rounded. IL-6 and IL-8 decreased cell viability, synthesis of VEGF, and gene expression of collagen type I. PBM enhanced cell density in the matrices and stimulated VEGF expression, even after IL-6 challenge. Reduced TNF-α synthesis occurred in those cells subjected to PBM. In conclusion, PBM can penetrate collagen matrix and stimulate HGF, highlighting the relevance of this research model for further phototherapy studies and in vitro biomodulation of the healing process.

Proteolytic activity, degradation, and dissolution of primary and permanent teeth.

BACKGROUND: Primary and permanent teeth composition may influence dissolution and degradation rates. AIM: To compare the dissolution and degradation of primary and permanent teeth. DESIGN: Enamel and dentin powders were obtained from primary molars and premolars and incubated within different pH buffers. Calcium and inorganic phosphate release was quantified in the buffers by atomic absorption and light spectrophotometry. A colorimetric assay was used to assess the MMP activity of primary dentin (PrD) and permanent dentin (PeD). Collagen degradation was assessed by dry mass loss, change in elastic modulus (E), and ICTP and CTX release. Data were submitted to ANOVA and Tukey's tests (α = 0.05). RESULTS: Similar dissolution was found between PrD and PeD after 256 hours. At pH 4.5, enamel released more minerals than dentin whereas at pH 5.5 the inverse result was observed. MMP activity was similar for both substrates. PrD showed higher dry mass loss after 1 week. In general, greater reduction in E was recorded for PrD. Higher quantities of ICTP and CTX were released from PrD after 1 week. CONCLUSIONS: Primary and permanent teeth presented similar demineralization rates. Collagen degradation, however, was faster and more substantial for PrD.

Positive influence of simvastatin used as adjuvant agent for cavity lining.

OBJECTIVES: To assess the biological, antimicrobial, and mechanical effects of the treatment of deep dentin with simvastatin (SV) before application of a glass-ionomer cement (GIC). MATERIALS AND METHODS: Dentin discs were adapted to artificial pulp chambers and SV (2.5 or 1.0 mg/mL) was applied to the occlusal surface, either previously conditioned or not with EDTA (±EDTA). The extracts (culture medium + SV that diffused through dentin) was obtained and then applied to cultured odontoblast-like MDPC-23 cells. Cell viability, alkaline phosphatase (ALP) activity, and mineralization nodule (MN) deposition were evaluated. Untreated discs were used as control. The antibacterial activity of SV (2.5 or 1.0 mg/mL) against Streptococcus mutans and Lactobacillus acidophilus, as well as the bond strength of GIC to dentin in the presence of SV 2.5 mg/mL (±EDTA) were also assessed. The data were analyzed by ANOVA/Tukey tests (α = 5%). RESULTS: EDTA + SV 2.5 mg/mL significantly enhanced the ALP activity and MN deposition in comparison with the control, without changing in the cell viability (p < 0.05). The association EDTA + SV 2.5 mg/mL + GIC determined the highest ALP and MN values (p < 0.05). SV presented intense antimicrobial activity, and the EDTA dentin conditioning followed by SV application increased bond strength values compared with SV treatment alone (p < 0.05). CONCLUSION: SV presents antimicrobial activity and diffuses across conditioned dentin to biostimulate odontoblast-like pulp cells. CLINICAL SIGNIFICANCE: The use of SV as adjuvant agent for indirect pulp capping may biostimulate pulp cells thus preserving vitality and function of the pulp-dentin complex.

Epithelial cell-enhanced metabolism by low-level laser therapy and epidermal growth factor.

Reepithelialization and wound closure are the desired outcome for several ulcerative conditions. Such resolution reduces the possibility of wound contamination and maintenance of the injury and improves the reestablishment of tissue morphology and functions. Investigators are seeking adjuvant therapies that can accelerate wound healing and are developing new strategies for clinical applications. This study compared the effects of epidermal growth factor (EGF) application and low-level laser therapy (LLLT) on cultured epithelial cells. Cells were seeded in 24-well plates. After a 24-h incubation, the epithelial cells were either treated with EGF (100 µM in serum-free DMEM for 72 h) or subjected to LLLT (780 nm, 25 mW, 0.5, 1.5, and 3 J/cm2) by three applications every 24 h. Seventy-two hours after cells were treated with EGF or LLLT, cell migration, viability, proliferation, and collagen synthesis were assessed. Cells treated with EGF showed increased cell viability, proliferation, and collagen synthesis compared with those cells that received no treatment. LLLT enhanced cell migration; however, no significant effects of laser irradiation on other cell functions were observed. Comparison of both therapies demonstrated that EGF and LLLT enhanced specific epithelial cell activities related to wound healing.

Influence of bisphosphonates on the adherence and metabolism of epithelial cells and gingival fibroblasts to titanium surfaces.

OBJECTIVES: To evaluate the effects of sodium alendronate (SA) and zoledronic acid (ZA), on the adhesion and metabolism of epithelial cells and gingival fibroblasts to titanium surfaces considering cell functions related to an effective mucosal barrier around the implant. MATERIALS AND METHODS: Cells were seeded onto titanium discs and incubated for 24 h. Then, serum-free DMEM containing selected bisphosphonates (0, 0.5, 1, or 5 µM) was added for 24 and 48 h. Factors related to the achievement of an effective mechanical and immunological barrier-cell adhesion, viability, collagen epidermal growth factor, and immunoglobulin synthesis-were evaluated. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests as well as by ANOVA and Tukey's tests, (α = 0.05). RESULTS: The presence of bisphosphonates culminated in lower cell adhesion to the titanium discs, particularly for SA at 5 µM (40%) and ZA at all concentrations (from 30 to 50%, according to increased concentrations). Reduced cell viability occurred after exposing these cells to ZA (40%); however, only 5 µM SA-treated cells had decreased viability (30%). Reduced synthesis of growth factors and collagen was observed when cells were reated with ZA (20 and 40%, respectively), while about 70% of IgG synthesis was enhanced. CONCLUSION: Bisphosphonates negatively affected the adhesion and metabolism of oral mucosal cells, and this effect was related to the type of bisphosphonate as well as to concentration and period of treatment. CLINICAL RELEVANCE: The negative effects of bisphosphonates on oral mucosal cells can hamper the formation of an effective biological seal in osseointegrated implants.

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.

Odontogenic differentiation potential of human dental pulp cells cultured on a calcium-aluminate enriched chitosan-collagen scaffold.

OBJECTIVE: The study aims to evaluate the odontogenic potential of human dental pulp cells (HDPCs) in contact with an experimental porous chitosan-collagen scaffold (CHC) enriched or not with a mineral phase of calcium-aluminate (CHC-CA). MATERIAL AND METHODS: To assess the chemotactic effect of the materials, we placed HDPCs seeded on transwell membranes in intimate contact with the CHC or CHC-CA surface, and the cell migration was monitored for 48 h. Additionally, cells were seeded onto the material surface, and the viability and proliferation were evaluated at several time points. To assess the odontoblastic differentiation, we evaluated ALP activity, DSPP/DMP-1 gene expression, and mineralized matrix deposition. HDPCs cultured onto a polystyrene surface (monolayer) were used as negative control group. RESULTS: The experimental CHC-CA scaffold induced intense migration of HDPCs through transwell membranes, with cells attaching to and spreading on the material surface after 24-h incubation. Also, the HDPCs seeded onto the CHC-CA scaffold were capable of migrating inside it, remaining viable and featuring a proliferative rate more rapid than that of CHC and control groups at 7 and 14 days of cell culture. At long-term culture, cells in the CHC-CA scaffold featured the highest deposition of mineralized matrix and expression of odontoblastic markers (ALP activity and DSPP/DMP-1 gene expression). CONCLUSIONS: According to the results, the CHC-CA scaffold is a bioactive and cytocompatible material capable of increasing the odontogenic potential of human pulp cells. Based on analysis of the positive data obtained in this study, one can suggest that the CHC-CA scaffold is an interesting future candidate for the treatment of exposed pulps. CLINICAL RELEVANCE: The experimental scaffold composed by a chitosan-collagen matrix mineralized with calcium aluminate seems to be an interesting candidate for in vivo application as a cell-free approach to dentin tissue engineering, which may open a new perspective for the treatment of exposed pulp tissue.

Synthesis of dental matrix proteins and viability of odontoblast-like cells irradiated with blue LED.

To evaluate the effect of irradiation with light-emitting diode (LED; 455 nm) on the viability and synthesis of dentin matrix proteins by odontoblast-like cells, MDPC-23 cells were cultivated (10(4) cells/cm(2)) in 24-well culture plates. After 12 h incubation in Dulbecco's modified Eagle's medium (DMEM), the cells were submitted to nutritional restriction by means of reducing the concentration of fetal bovine serum (FBS) for an additional 12 h. Cells were irradiated one single time with one of the following energy densities (EDs): 0.5, 2, 4, 10, or 15 J/cm(2) and irradiance fixed at 20 mW/cm(2). Non-irradiated cells served as control. After 72 h, cells were evaluated with regard to viability (methylthiazol tetrazolium technique (MTT)), mineralization nodule (MN) formation, total protein (TP) production, alkaline phosphatase activity (ALP), and collagen synthesis (Sircol), n = 8. The data were submitted to Kruskal-Wallis and Mann-Whitney tests (p > 0.05). There was no statistical difference between the viability of cells irradiated or not (control), for all the EDs. However, an increase in TP was observed for all the EDs when compared with the control group. A reduced ALP activity was seen in all irradiated groups, except for the ED of 0.5 J/cm(2), which did not differ from the control. There was no difference between the irradiated groups and control regarding collagen synthesis, with the exception of the ED of 10 J/cm(2), which inhibited this cell function. Significant reduction in MN occurred only for the EDs of 0.5 and 2 J/cm(2). The single irradiation with blue LED (455 nm), irradiance of 20 mW/cm(2), and energy densities ranging from 0.5 to 15 J/cm(2) exerted no effective biostimulatory capacity on odontoblast-like cells.

Metabolic activity of odontoblast-like cells irradiated with blue LED (455 nm).

Blue light emitting diodes (LEDs) are frequently used in dentistry for light activation of resin-based materials; however, their photobiostimulatory effects have not yet been fully investigated. This study aimed to investigate the effect of blue LED (455 nm) on the metabolism of odontoblast-like cells MDPC-23. Energy doses of 2 and 4 J/cm(2) were used at 20 mW/cm(2) fixed power density. MDPC-23 cells were seeded at 10,000 cells/cm(2) density in Dulbecco's modified Eagle's medium (DMEM) containing 10 % fetal bovine serum (FBS). After 12 h, the culture medium was replaced with new DMEM supplemented with 0.5 % of FBS, and the cells were incubated for further 12 h. After that, single irradiation was performed to the culture, under selected parameters. Cell viability evaluations (Alamar Blue Assay, n = 12), number of viable cells (Trypan Blue Assay, n = 12), morphological analysis by scanning electron microscopy (SEM, n = 2), gene expression (n = 6) of alkaline phosphatase (Alp), collagen (Col-1a1), and dental matrix protein (Dmp-1) (quantitative polymerase chain reaction (qPCR)) were performed 72 h after irradiation. Data were analyzed by Kruskal-Wallis, ANOVA, and Tukey tests (p < 0.05). Direct light application at 4 J/cm(2) energy dose had no negative effects on cell viability, while irradiation with 2 J/cm(2) reduced cell metabolism. None of doses affected the number of viable cells compared with the control group. The two energy doses downregulated the expression of Alp; however, expression of Col-1a1 and Dmp-1 had no alteration. Cells presented change in the cytoskeleton only when irradiated with 2 J/cm(2). In conclusion, the blue LED (455 nm) irradiation, under the evaluated parameters, had no biostimulatory effects on MDPC-23 cells.

Transdentinal cytotoxicity of resin-based luting cements to pulp cells.

OBJECTIVES: The aim of this study was to evaluate the transdentinal cytotoxicity of components released from different resin-based luting cements to cultured MDPC-23 odontoblast-like cells and human dental pulp cells (HDPCs). MATERIALS AND METHODS: Artificial pulp chamber (APC)/dentin disc sets were distributed into four groups according to the materials tested (n = 10), as follows: G1, control (no treatment); G2, resin-modified glass-ionomer cement (RelyX Luting 2); G3, self-adhesive resin cement (RelyX U200); and G4, conventional resin cement (RelyX ARC). The materials were applied to the occlusal surfaces (facing up) of the dentin discs adapted to the APCs. The pulpal surfaces of the discs were maintained in contact with culture medium. Then, an aliquot of 400 µL from the extract (culture medium + resin-based components that diffused through dentin) of each luting cement was applied for 24 h to HDPCs or MDPC-23 cells previously seeded in wells of 24-well plates. Cell viability analysis was performed by the MTT assay (1-way ANOVA/Tukey test; α = 5 %). RESULTS: For MDPC-23 cells, RelyX ARC (G4) and RelyX Luting 2 (G2) caused greater reduction in cell viability compared with the negative control group (P < 0.05). Only the HDPCs exposed to RelyX ARC (G4) extract showed a tendency toward viability decrease (9.3 %); however, the values were statistically similar to those of the control group (G1) (P > 0.05). CONCLUSIONS: In accordance with the safe limits of ISO 10993-5:1999 (E) recommendations, all resin-based luting cements evaluated in this study can be considered as non-toxic to pulp cells. CLINICAL RELEVANCE: Cytotoxicity of resin-based luting cements is material-dependent, and the different protocols for the application of these dental materials to dentin may interfere with their cytotoxicity.

Biocompatibility of a restorative resin-modified glass ionomer cement applied in very deep cavities prepared in human teeth.

This study evaluated whether a restorative resin-modified glass ionomer cement, Vitremer (VM), would be biocompatible with pulp tissue when used as a liner in very deep cavities prepared in young human permanent teeth. Two dental cements in current use as liner materials, Vitrebond (VB) and Dycal (DY), were compared to VM. Class V cavities were prepared in 36 sound premolars that were scheduled for extraction, and the cavity floor was lined with the restorative cement (VM) or a liner/base control cement (VB or DY). For VM specimens, the cavity floor was pretreated with a primer (polyacrylic acid plus 2-hydroxyethyl methacrylate). Teeth were extracted after 7 or 30 days and processed for microscopic evaluation. In the VM group, inward diffusion of dental material components through dentinal tubules, associated with disruption of the odontoblastic layer, moderate to intense inflammatory response, and resorption of inner dentin, was observed in 2 teeth at 7 days. These histologic features were observed in 1 tooth at 30 days. In the VB group, mild inflammatory reactions and tissue disorganization observed at 7 days were resolved at 30 days. No pulpal damage occurred in the DY specimens. Of the materials tested, only Vitremer was not considered biocompatible, because it caused persistent pulpal damage when applied in very deep cavities (remaining dentin thickness less than 0.3 mm).

Responses of dental pulp cells to a less invasive bleaching technique applied to adhesive-restored teeth.

PURPOSE: To assess the cytotoxicity of 35% hydrogen peroxide (HP) bleaching gel applied for 15 min to sound or restored teeth with two-step self-etching adhesive systems and composite resin. MATERIALS AND METHODS: Sound and restored enamel/dentin disks were stored in water for 24 h or 6 months + thermocycling. The disks were adapted to artificial pulp chambers and placed in compartments containing culture medium. Immediately after bleaching, the culture medium in contact with dentin was applied for 1 h to previously cultured odontoblast-like MDPC-23 cells. Thereafter, cell viability (MTT assay) and morphology (SEM) were assessed. Data were analyzed by two-way ANOVA and Tukey's test (a = 5%). RESULTS: In comparison to the negative control group (no treatment), no significant cell viability reduction occurred in those groups in which sound teeth were bleached. However, a significant decrease in cell viability was observed in the adhesive-restored bleached groups compared to negative control. No significant difference among bleached groups was observed with respect to the presence of restoration and storage time. CONCLUSION: The application of 35% HP bleaching gel to sound teeth for 15 min does not cause toxic effects in pulp cells. When this bleaching protocol was performed in adhesive-restored teeth, a significant toxic effect occurred.

Immediate and late analysis of dental pulp stem cells viability after indirect exposition to alternative in-office bleaching strategies.

OBJECTIVES: To evaluate human dental pulp stem cell viability and capacity to recover from experimental dental bleaching techniques. MATERIAL AND METHODS: Enamel/dentin disks adapted to trans-wells were positioned on previously cultivated dental pulp stem cells. Bleaching gels containing 35, 17.5, 10, and 8 % hydrogen peroxide (H2O2) were applied one or three times (each application lasting 15 min) on enamel. Cell viability (MTT assay) and morphology (SEM) were evaluated immediately (T1) or 72 h (T2) post-bleaching. RESULTS: The 35 % H2O2 gel promoted intense reduction in viability (93-97 %) and morphological alterations of the cells at T1, irrespective of frequency of application, with absence or limited capacity for recovery being observed at T2. The other bleaching gels presented significant lower toxicity when compared with the 35 % H2O2 gel, in a time/concentration fashion. In T1, no significant difference was observed between the negative control (without bleaching) and the 8 and 10 % H2O2 gels applied on enamel for 15 min, in which the cells presented elevated viability and morphology similar to the negative control at T2. CONCLUSIONS: Bleaching gels with 8 and 10 % H2O2 in their composition cause limited immediate toxic effect on pulp stem cells, which recover their viability 3 days after treatment. CLINICAL RELEVANCE: This study presents proposals for in-office dental bleaching to be performed with limited aggressive effect on dental pulp stem cells. Therefore, we are able to offer interesting clinical alternatives for bleaching vital teeth, under professional supervision, maintaining the integrity and reparative capacity of pulp-dentin complex.

Low-level laser therapy for osteonecrotic lesions: effects on osteoblasts treated with zoledronic acid.

PURPOSE: Clinical studies have shown that low-level laser therapy (LLLT) can improve local tissue healing of bisphosphonate-induced osteonecrosis of the jaw. However, the effects of laser irradiation on bisphosphonate-treated osteoblasts have not been completely elucidated. METHODS: Human osteoblasts were cultured in plain culture medium (DMEM). After 48 h, plain DMEM was replaced by DMEM with no fetal bovine serum, for a 24-h incubation followed by addition of zoledronic acid (5 µM) for additional 48 h. Cells were subjected to LLLT (InGaAsP; 780 ± 3 nm; 0.025 W) at 0.5, 1.5, 3, 5, and 7 J/cm(2), three times every 24 h. Cell viability, total protein production, alkaline phosphatase activity (ALP), mineral nodule formation, gene expression of collagen type I and ALP, and cell morphology were evaluated. RESULTS: LLLT at 0.5 J/cm(2) increased cell viability of cultured osteoblasts. ALP activity and gene expression, in addition to mineral nodule formation and Col-I gene expression, were not increased by LLLT. LLLT applied to ZA-treated cells increased Col-I expression at 0.5, 1.5, and 3 J/cm(2) but did not improve any other cell activity assessed. CONCLUSION: LLLT showed limited effects on bisphosphonate-treated osteoblasts.