Influence of coating dental enamel with a TiF4-loaded polymeric primer on the adverse effects caused by a bleaching gel with 35% H2O2.

OBJECTIVE: To evaluate whether coating enamel with a polymeric primer (PPol) containing titanium tetrafluoride (TiF4) before applying a bleaching gel with 35% H2O2 (35% BG) increases esthetic efficacy, prevents changes in morphology and hardness of enamel, as well as reduces the cytotoxicity from conventional in-office bleaching. MATERIALS AND METHODS: Standardized enamel/dentin discs were stained and bleached for 45 min (one session) with 35% BG. Groups 2TiF4, 6TiF4, and 10TiF4 received the gel on the enamel previously coated with PPol containing 2 mg/mL, 6 mg/mL, or 10 mg/mL, respectively. No treatment or application of 35% BG directly on enamel were used as negative control (NC), and positive control (PC), respectively. UV-reflectance spectrophotometry (CIE L*a*b* system, ΔE00, and ΔWI, n = 8) determined the bleaching efficacy of treatments. Enamel microhardness (Knoop, n = 8), morphology, and composition (SEM/EDS, n = 4) were also evaluated. Enamel/dentin discs adapted to artificial pulp chambers (n = 8) were used for trans-amelodentinal cytotoxicity tests. Following the treatments, the extracts (culture medium + bleaching gel components diffused through the discs) were collected and applied to odontoblast-like MDPC-23 cells, which were assessed concerning their viability (alamarBlue, n = 8; Live/Dead, n = 4), oxidative stress (n = 8), and morphology (SEM). The amount of H2O2 in the extracts was also determined (leuco crystal violet/peroxidase, n = 8). The numerical data underwent one-criterion variance analysis (one-way ANOVA), followed by Tukey's test, at a 5% significance level. RESULTS: Regarding the ΔE00, no difference was observed among groups 2TiF4, 6TiF4, and PC (p > 0.05). The ΔWI was similar between groups 2TiF4 and PC (p > 0.05). The ΔWI of group 6TiF4 was superior to PC (p < 0.05), and group 10TiF4 achieved the highest ΔE00 and ΔWI values (p < 0.05). Besides limiting enamel microstructural changes compared to PC, group 10TiF4 significantly increased the hardness of this mineralized dental tissue. The highest cellular viability occurred in 10TiF4 compared to the other bleached groups (p < 0.05). Trans-amelodentinal H2O2 diffusion decreased in groups 2TiF4, 6TiF4, and 10TiF4 in comparison with PC (p < 0.05). CONCLUSION: Coating enamel with a PPol containing TiF4 before applying a 35% BG may increase enamel microhardness and esthetic efficacy and reduce the trans-amelodentinal cytotoxicity of conventional in-office tooth bleaching. The PPol containing 10 mg/mL of TiF4 promoted the best outcomes.

Chitosan-Calcium Aluminate as a Cell-homing Scaffold: Its Bioactivity Testing in a Microphysiological Dental Pulp Platform.

In vitro models of the dental pulp microenvironment have been proposed for the assessment of biomaterials, to minimise animal use in operative dentistry. In this study, a scaffold/3-D dental pulp cell culture interface was created in a microchip, under simulated dental pulp pressure, to evaluate the cell-homing potential of a chitosan (CH) scaffold functionalised with calcium aluminate (the 'CHAlCa scaffold'). This microphysiological platform was cultured at a pressure of 15 cm H2O for up to 14 days; cell viability, migration and odontoblastic differentiation were then assessed. The CHAlCa scaffold exhibited intense chemotactic potential, causing cells to migrate from the 3-D culture to its surface, followed by infiltration into the macroporous structure of the scaffold. By contrast, the cells in the presence of the non-functionalised chitosan scaffold showed low cell migration and no cell infiltration. CHAlCa scaffold bioactivity was confirmed in dentin sialophosphoprotein-positive migrating cells, and odontoblastic markers were upregulated in 3-D culture. Finally, in situ mineralised matrix deposition by the cells was confirmed in an Alizarin Red-based assay, in which the CHAlCa and CH scaffolds were adapted to fit within dentin discs. More intense deposition of matrix was observed with the CHAlCa scaffold, as compared to the CH scaffold. In summary, we present an in vitro platform that provides a simple and reproducible model for selecting and developing innovative biomaterials through the assessment of their cell-homing potential. By using this platform, it was shown that the combination of calcium aluminate and chitosan has potential as an inductive biomaterial that can mediate dentin tissue regeneration during cell-homing therapies.

Chronic exposure to lipopolysaccharides as an in vitro model to simulate the impaired odontogenic potential of dental pulp cells under pulpitis conditions.

BACKGROUND: Simulating a bacterial-induced pulpitis environment in vitro may contribute to exploring mechanisms and bioactive molecules to counteract these adverse effects. OBJECTIVE: To investigate the chronic exposure of human dental pulp cells (HDPCs) to lipopolysaccharides (LPS) aiming to establish a cell culture protocol to simulate the impaired odontogenic potential under pulpitis conditions. METHODOLOGY: HDPCs were isolated from four healthy molars of different donors and seeded in culture plates in a growth medium. After 24 h, the medium was changed to an odontogenic differentiation medium (DM) supplemented or not with E. coli LPS (0 - control, 0.1, 1, or 10 µg/mL) (n=8). The medium was renewed every two days for up to seven days, then replaced with LPS-free DM for up to 21 days. The activation of NF-κB and F-actin expression were assessed (immunofluorescence) after one and seven days. On day 7, cells were evaluated for both the gene expression (RT-qPCR) of odontogenic markers (COL1A1, ALPL, DSPP, and DMP1) and cytokines (TNF, IL1B, IL8, and IL6) and the production of reactive nitrogen (Griess) and oxygen species (Carboxy-H2DCFDA). Cell viability (alamarBlue) was evaluated weekly, and mineralization was assessed (Alizarin Red) at 14 and 21 days. Data were analyzed with ANOVA and post-hoc tests (α=5%). RESULTS: After one and seven days of exposure to LPS, NF-κB was activated in a dose-dependent fashion. LPS at 1 and 10 µg/mL concentrations down-regulated the gene expression of odontogenic markers and up-regulated cytokines. LPS at 10 µg/mL increased both the production of reactive nitrogen and oxygen species. LPS decreased cell viability seven days after the end of exposure. LPS at 1 and 10 µg/mL decreased hDPCs mineralization in a dose-dependent fashion. CONCLUSION: The exposure to 10 µg/mL LPS for seven days creates an inflammatory environment that is able to impair by more than half the odontogenic potential of HDPCs in vitro, simulating a pulpitis-like condition.

Effect of medium or high concentrations of in-office dental bleaching gel on the human pulp response in the mandibular incisors.

Objectives: The present study evaluated the pulp response of human mandibular incisors subjected to in-office dental bleaching using gels with medium or high concentrations of hydrogen peroxide (HP). Materials and Methods: The following groups were compared: 35% HP (HP35; n = 5) or 20% HP (HP20; n = 4). In the control group (CONT; n = 2), no dental bleaching was performed. The color change (CC) was registered at baseline and after 2 days using the Vita Classical shade guide. Tooth sensitivity (TS) was also recorded for 2 days post-bleaching. The teeth were extracted 2 days after the clinical procedure and subjected to histological analysis. The CC and overall scores for histological evaluation were evaluated by the Kruskal-Wallis and Mann-Whitney tests. The percentage of patients with TS was evaluated by the Fisher exact test (α = 0.05). Results: The CC and TS of the HP35 group were significantly higher than those of the CONT group (p < 0.05) and the HP20 group showed an intermediate response, without significant differences from either the HP35 or CONT group (p > 0.05). In both experimental groups, the coronal pulp tissue exhibited partial necrosis associated with tertiary dentin deposition. Overall, the subjacent pulp tissue exhibited a mild inflammatory response. Conclusions: In-office bleaching therapies using bleaching gels with 20% or 35% HP caused similar pulp damage to the mandibular incisors, characterized by partial necrosis, tertiary dentin deposition, and mild inflammation.

Assessment of the regenerative potential of macro-porous chitosan-calcium simvastatin scaffolds on bone cells.

This study evaluated the bioactive potential of a macro-porous chitosan scaffold incorporated with calcium hydroxide (CH-Ca) and functionalized with bioactive doses of simvastatin (SV) for bone tissue regeneration. Initially, the bioactive dose of SV in osteoblastic cells (SAOS-2) was determined. For the direct contact experiment, SAOS-2 cells were plated on scaffolds to assess cell viability and osteogenic differentiation. The second assay was performed at a distance using extracts from scaffolds incubated in culture medium to assess the effect of conditioned medium on viability and osteogenic differentiation. The initial screening showed that 1 µM SV presented the best biostimulating effects, and this dose was selected for incorporation into the CH-Ca and pure chitosan (CH) scaffolds. The cells remained viable throughout the direct contact experiment, with the greatest cell density in the CH-Ca and CH-Ca-SV scaffolds because of their higher porosity. The CH-Ca-SV scaffold showed the most intense bio-stimulating effect in assays in the presence and absence of osteogenic medium, leading to an increased deposition of mineralized matrix. There was an increase in the viability of cells exposed to the extracts for CH-Ca, CH-SV, and CH-Ca-SV during the one-day period. There was an increase in ALP activity in the CH-Ca and CH-Ca-SV; however, the CH-Ca-SV scaffold resulted in an intense increase in the deposition of mineralized nodules, approximately 56.4% at 7 days and 117% at 14 days, compared with CH (control). In conclusion, functionalization of the CH-Ca scaffold with SV promoted an increase in bioactivity, presenting a promising option for bone tissue regeneration.

Assessment of the regenerative potential of macro-porous chitosan-calcium simvastatin scaffolds on bone cells

Abstract This study evaluated the bioactive potential of a macro-porous chitosan scaffold incorporated with calcium hydroxide (CH-Ca) and functionalized with bioactive doses of simvastatin (SV) for bone tissue regeneration. Initially, the bioactive dose of SV in osteoblastic cells (SAOS-2) was determined. For the direct contact experiment, SAOS-2 cells were plated on scaffolds to assess cell viability and osteogenic differentiation. The second assay was performed at a distance using extracts from scaffolds incubated in culture medium to assess the effect of conditioned medium on viability and osteogenic differentiation. The initial screening showed that 1 μM SV presented the best biostimulating effects, and this dose was selected for incorporation into the CH-Ca and pure chitosan (CH) scaffolds. The cells remained viable throughout the direct contact experiment, with the greatest cell density in the CH-Ca and CH-Ca-SV scaffolds because of their higher porosity. The CH-Ca-SV scaffold showed the most intense bio-stimulating effect in assays in the presence and absence of osteogenic medium, leading to an increased deposition of mineralized matrix. There was an increase in the viability of cells exposed to the extracts for CH-Ca, CH-SV, and CH-Ca-SV during the one-day period. There was an increase in ALP activity in the CH-Ca and CH-Ca-SV; however, the CH-Ca-SV scaffold resulted in an intense increase in the deposition of mineralized nodules, approximately 56.4% at 7 days and 117% at 14 days, compared with CH (control). In conclusion, functionalization of the CH-Ca scaffold with SV promoted an increase in bioactivity, presenting a promising option for bone tissue regeneration.

Chronic exposure to lipopolysaccharides as an in vitro model to simulate the impaired odontogenic potential of dental pulp cells under pulpitis conditions

Abstract Simulating a bacterial-induced pulpitis environment in vitro may contribute to exploring mechanisms and bioactive molecules to counteract these adverse effects. Objective To investigate the chronic exposure of human dental pulp cells (HDPCs) to lipopolysaccharides (LPS) aiming to establish a cell culture protocol to simulate the impaired odontogenic potential under pulpitis conditions. Methodology HDPCs were isolated from four healthy molars of different donors and seeded in culture plates in a growth medium. After 24 h, the medium was changed to an odontogenic differentiation medium (DM) supplemented or not with E. coli LPS (0 - control, 0.1, 1, or 10 µg/mL) (n=8). The medium was renewed every two days for up to seven days, then replaced with LPS-free DM for up to 21 days. The activation of NF-κB and F-actin expression were assessed (immunofluorescence) after one and seven days. On day 7, cells were evaluated for both the gene expression (RT-qPCR) of odontogenic markers (COL1A1, ALPL, DSPP, and DMP1) and cytokines (TNF, IL1B, IL8, and IL6) and the production of reactive nitrogen (Griess) and oxygen species (Carboxy-H2DCFDA). Cell viability (alamarBlue) was evaluated weekly, and mineralization was assessed (Alizarin Red) at 14 and 21 days. Data were analyzed with ANOVA and post-hoc tests (α=5%). Results After one and seven days of exposure to LPS, NF-κB was activated in a dose-dependent fashion. LPS at 1 and 10 µg/mL concentrations down-regulated the gene expression of odontogenic markers and up-regulated cytokines. LPS at 10 µg/mL increased both the production of reactive nitrogen and oxygen species. LPS decreased cell viability seven days after the end of exposure. LPS at 1 and 10 µg/mL decreased hDPCs mineralization in a dose-dependent fashion. Conclusion The exposure to 10 µg/mL LPS for seven days creates an inflammatory environment that is able to impair by more than half the odontogenic potential of HDPCs in vitro, simulating a pulpitis-like condition.

Regenerating the Dental Pulp-Scaffold Materials and Approaches.

Novel technologies and platforms have allowed significant breakthroughs in dental pulp tissue engineering. The development of injectable scaffolds that can be combined with stem cells, growth factors, or other bioactive compounds has enabled the regeneration of functional dental pulps able to secrete dentin in preclinical and clinical studies. Similarly, cell-homing technologies and scaffold-free strategies aim to modulate dental pulp self-regeneration mediated by resident stem cells and can evade some of the technical challenges related to cell-based tissue engineering strategies. This article will discuss emerging technologies and platforms for the clinical applications of dental pulp tissue engineering.

Manganese oxide increases bleaching efficacy and reduces the cytotoxicity of a 10% hydrogen peroxide bleaching gel.

OBJECTIVE: The study aims to assess the effects of a 10% H2O2 bleaching gel with different MnO2 concentrations on the bleaching efficacy (BE), degradation kinetics (DK) of H2O2, and trans-amelodentinal cytotoxicity (TC). MATERIALS AND METHODS: Standardized bovine enamel/dentin disks (n = 96) were placed in artificial pulp chambers, and the bleaching gels were applied for 45 min. Thus, the following groups were established: (G1) no treatment (negative control/NC); (G2) 35% H2O2 (positive control/PC); (G3) 10% H2O2; (G4) 10% H2O2 + 2 mg/mL MnO2; (G5) 10% H2O2 + 6 mg/mL MnO2; and (G6) 10% H2O2 + 10 mg/mL MnO2. After analyzing bleaching efficacy (ΔE00 and ΔWI), the degradation kinetics of H2O2 and trans-amelodentinal cytotoxicity were determined (n = 8, ANOVA/Tukey; p < 0.05). RESULTS: G6 presented BE (ΔE00 and ΔWI) statistically similar to G2, which represented conventional in-office bleaching (p = 0.6795; p > 0.9999). A significant reduction in the diffusion of H2O2 occurred in G3, G4, G5, and G6 compared to G2 (p < 0.0001). The highest DK of H2O2 occurred in G6 (p < 0.0001), which had the lowest TC in comparison with all other bleached groups (p ≤ 0.0186). CONCLUSION: The addition of 10 mg/mL of MnO2 in a 10% H2O2 bleaching gel potentiates the degradation of this reactive molecule, which increases the BE of the product and decreases TC. CLINICAL SIGNIFICANCE: Replacing a 35% H2O2 gel commonly used for conventional in-office dental bleaching by a 10% H2O2 gel containing 10 mg/mL of MnO2 reduces the cytotoxicity of this professional therapy, maintaining its excellent esthetic efficacy.

Poly(caprolactone)-aligned nanofibers associated with fibronectin-loaded collagen hydrogel as a potent bioactive scaffold for cell-free regenerative endodontics.

AIM: Guided tissue regeneration has been considered a promising strategy to replace conventional endodontic therapy of teeth with incomplete root formation. Therefore, the objective of this study was to develop a tubular scaffold (TB-SC) with poly (caprolactone)-aligned nanofibres associated with a fibronectin (FN)-loaded collagen hydrogel and assess the pulp regeneration potential mediated by human apical papilla cells (hAPCs) using an in vitro model of teeth with incomplete root formation. METHODOLOGY: Aligned nanofibre strips based on 10% poly(caprolactone) (PCL) were synthesized with the electrospinning technique to produce the TB-SCs. These were submitted to different treatments, according to the following groups: TB-SC (negative control): TB-SC without treatment; TB-SC + FN (positive control): TB-SC coated with 10 µg/ml of FN; TB-SC + H: TB-SC associated with collagen hydrogel; TB-SC + HFN: TB-SC associated with FN-loaded collagen hydrogel. Then, the biomaterials were inserted into cylindrical devices to mimic the regenerative therapy of teeth with incomplete root formation. The hAPCs were seeded on the upper surface of the TB-SCs associated or not with any treatment, and cell migration/proliferation and the gene expression of markers related to pulp regeneration (ITGA5, ITGAV, COL1A1 and COL1A3) were evaluated. The data were submitted to anova/Tukey's tests (α = 5%). RESULTS: Higher values of cell migration/proliferation and gene expression of all markers tested were observed in groups TB-SC + FN, TB-SC + H, and TB-SC + HFN compared with the TB-SC group (p < .05). The hAPCs in the TB-SC + HFN group showed the highest values of cell proliferation and gene expression of COL1A1 and COL3A1 (p < .05), as well as superior cell migration results to groups TB-SC and TB-SC + H (p < .05). CONCLUSION: Aligned nanofibre scaffolds associated with the FN-loaded collagen hydrogel enhanced the migration and proliferation of hAPCs and gene expression of pulp regeneration markers. Therefore, the use of these biomaterials may be considered an interesting strategy for regenerative pulp therapy of teeth with incomplete root formation.

An organotypic model of oral mucosa cells for the biological assessment of 3D-printed resins for interim restorations.

STATEMENT OF PROBLEM: Three-dimensionally (3D) printed resins have become popular as a new class of materials for making interim restorations. However, little is known about how the fabrication parameters can influence biological compatibility with oral tissues. PURPOSE: The purpose of this in vitro study was to evaluate the effect of the postpolymerization time on the cytotoxicity of resins for printing interim restorations by using a 3D organotypic model of the oral mucosa. MATERIAL AND METHODS: Cylindrical specimens were prepared with conventional acrylic resin (AR), computer-aided design and computer-aided manufacture (CAD-CAM) resin (CC), composite resin (CR), and 2 resins for 3D printing (3DP) marketed as being biocompatible. The 3DPs were submitted to postpolymerization in an ultraviolet (UV) light chamber for 1, 10, or 20 minutes (90 W, 405 nm). Standard specimens of the materials were incubated for 1, 3, and 7 days in close contact with an organotypic model of keratinocytes (NOK-Si) in coculture with gingival fibroblasts (HGF) in a 3D collagen matrix, or directly with 3D HGF cultures. Then, the viability (Live/Dead n=2) and metabolism (Alamar Blue n=6) of the cells were assessed. Spectral scanning of the culture medium was performed to detect released components (n=6) and assessed statistically with ANOVA and the Tukey post hoc test (α=.05). RESULTS: Severe reduction of metabolism (>70%) and viability of keratinocytes occurred for 3DP resin postpolymerized for 1 minute in all periods of analysis in a time-dependent manner. The decrease in cell metabolism and viability was moderate for the 3D culture of HGFs in both experimental models, correlated with the intense presence of resin components in the culture medium. The resins postpolymerized for 10 and 20 minutes promoted a mild-moderate cytotoxic effect in the period of 1 day, similar to AR. However, recovery of cell viability occurred at the 7-day incubation period. The 3DP resins submitted to postpolymerization for 20 minutes showed a pattern similar to that of CR and CC at the end of the experiment. CONCLUSIONS: The cytotoxic potential of the tested 3DP resins on oral mucosa cells was influenced by postprinting processing, which seemed to have been related with the quantity of residual components leached.

Pro-inflammatory mediators expression by pulp cells following tooth whitening on restored enamel surface.

This paper aimed to assess the influence of adhesive restoration interface on the diffusion of hydrogen peroxide (H2O2), indirect toxicity, and pro-inflammatory mediators expression by odontoblast-like cells, after in-office tooth whitening. Dental cavities prepared in bovine enamel/dentin discs were adhesively restored and subjected or not to hydrolytic degradation (HD). A whitening gel with 35% H2O2 (WG) was applied for 45 min onto restored and non-restored specimens adapted to artificial pulp chambers giving rise to the groups: SD- intact discs (control); SD/HP- whitened intact discs; RT/HP- restored and whitened discs; and RT/HD/HP- restored and whitened discs subjected to HD. The extracts (culture medium + WG components diffused through enamel/dentin/restoration interface) were collected and applied to odontoblast-like MDPC-23 cells. The study evaluated the amount of H2O2 in the extracts, as well as the cell viability (CV), cell morphology (CM), and gene expression of inflammatory mediators (TNF-α and COX-2) by the pulp cells exposed to the extracts (ANOVA and Tukey tests; 5% significance). All whitened groups presented lower CV than SD (control; p<0.05). The highest CV reduction and gene expression of TNF-α and COX-2 was observed in the RT/HD/HP group in comparison with SD/HP and RT/HP (control; p<0.05). CM alterations occurred in all whitened groups. The intensity of these cell side effects was directly related with the amount of H2O2 in the extracts. We concluded that adhesive restoration of dental cavity increases the H2O2 diffusion after in-office whitening, enhancing the indirect toxicity of this therapy and trigger pro-inflammatory overexpression by MDPC-23 cells.

Pro-inflammatory mediators expression by pulp cells following tooth whitening on restored enamel surface

Abstract This paper aimed to assess the influence of adhesive restoration interface on the diffusion of hydrogen peroxide (H2O2), indirect toxicity, and pro-inflammatory mediators expression by odontoblast-like cells, after in-office tooth whitening. Dental cavities prepared in bovine enamel/dentin discs were adhesively restored and subjected or not to hydrolytic degradation (HD). A whitening gel with 35% H2O2 (WG) was applied for 45 min onto restored and non-restored specimens adapted to artificial pulp chambers giving rise to the groups: SD- intact discs (control); SD/HP- whitened intact discs; RT/HP- restored and whitened discs; and RT/HD/HP- restored and whitened discs subjected to HD. The extracts (culture medium + WG components diffused through enamel/dentin/restoration interface) were collected and applied to odontoblast-like MDPC-23 cells. The study evaluated the amount of H2O2 in the extracts, as well as the cell viability (CV), cell morphology (CM), and gene expression of inflammatory mediators (TNF-α and COX-2) by the pulp cells exposed to the extracts (ANOVA and Tukey tests; 5% significance). All whitened groups presented lower CV than SD (control; p<0.05). The highest CV reduction and gene expression of TNF-α and COX-2 was observed in the RT/HD/HP group in comparison with SD/HP and RT/HP (control; p<0.05). CM alterations occurred in all whitened groups. The intensity of these cell side effects was directly related with the amount of H2O2 in the extracts. We concluded that adhesive restoration of dental cavity increases the H2O2 diffusion after in-office whitening, enhancing the indirect toxicity of this therapy and trigger pro-inflammatory overexpression by MDPC-23 cells.

Resumo Este trabalho teve como objetivo avaliar a influência da interface de uma restauração adesiva na difusão do peróxido de hidrogênio (H2O2), toxicidade indireta e expressão de mediadores pró-inflamatórios por células odontoblastóides, após clareamento dental em consultório. Cavidades dentárias preparadas em discos de esmalte / dentina foram restauradas com adesivo e submetidas ou não à degradação hidrolítica (HD). Um gel clareador com 35% H2O2 (WG) foi aplicado por 45 min em discos restaurados e não restaurados adaptados às câmaras pulpares artificiais dando origem aos grupos: SD- discos intactos (controle); SD / HP - Discos intactos clareados; RT / HP - discos restaurados e clareados; e RT / HD / HP - discos restaurados, clareados e submetidos a HD. Os extratos (meio de cultura + componentes WG difundidos através da interface esmalte/dentina/restauração) foram coletados e aplicados em células odontoblastóides MDPC-23. Foi avaliada a quantidade de H2O2 nos extratos, bem como a viabilidade (CV), morfologia (CM) e expressão gênica de mediadores inflamatórios (TNF-α e COX-2) pelas células pulpares expostas aos extratos (ANOVA e testes de Tukey; 5% de significância). Todos os grupos clareados apresentaram menor CV do que SD (controle; p <0,05). A maior redução CV e expressão gênica de TNF-α e COX-2 foi observada no grupo RT / HD / HP em comparação com SD / HP e RT / HP (controle; p <0,05). Alterações na CM ocorreram em todos os grupos clareados. A intensidade desses efeitos celulares teve relação direta com a quantidade de H2O2 nos extratos. Concluímos que a presença de uma cavidade contendo restauração adesiva aumenta a difusão de H2O2 após o clareamento em consultório, o que, por sua vez, aumenta a toxicidade indireta dessa terapia e desencadeia a expressão de mediadores pró-inflamatórios pelas células pulpares MDPC-23.

Mineral-induced bubbling effect and biomineralization as strategies to create highly porous and bioactive scaffolds for dentin tissue engineering.

The objective of the study was to assess the biological and mechanical characteristics of chitosan-based scaffolds enriched by mineral phases and biomineralized in simulated body fluid (SBF) as a possible biomaterial for dentin regeneration. Thus, porous chitosan scaffolds were prepared by the mineral-induced bubbling-effect technique and subjected to biomineralization to create biomimetic scaffolds for dentin tissue engineering. Suspensions containing calcium hydroxide, nanohydroxyapatite, or ß-tricalcium phosphate were added to the chitosan (CH) solution and subjected to gradual freezing and freeze-drying to obtain CHCa, CHnHA, and CHßTCP porous scaffolds, respectively, by the bubbling effect. Then, scaffolds were incubated in SBF for 5 days at 37°C, under constant stirring, to promote calcium-phosphate (CaP) biomineralization. Scanning electron microscopy revealed increased pore size and porosity degree on mineral-containing scaffolds, with CHCa and CHnHA presenting as round, well-distributed, and with an interconnected pore network. Nevertheless, incubation in SBF disrupted the porous architecture, except for CHCaSBF , leading to the deposition of CaP coverage, confirmed by Fourier Transform Infrared Spectroscopy analyses. All mineral-containing and SBF-treated formulations presented controlled degradation profiles and released calcium throughout 28 days. When human dental pulp cells (HDPCs) were seeded onto scaffold structures, the porous and interconnected architecture of CHCa, CHnHA, and CHCaSBF allowed cells to infiltrate and spread throughout the scaffold structure, whereas in other formulations cells were dispersed or agglomerated. It was possible to determine a positive effect on cell proliferation and odontogenic differentiation for mineral-containing formulations, intensely improved by biomineralization. A significant increase in mineralized matrix deposition (by 8.4 to 18.9 times) was observed for CHCaSBF , CHnHASBF , and CHßTCPSBF in comparison with plain CH. The bioactive effect on odontoblastic marker expression (ALP activity and mineralized matrix) was also observed for HDPCs continuously cultivated with conditioned medium obtained from scaffolds. Therefore, biomineralization of chitosan scaffolds containing different mineral phases was responsible for increasing the capacity for mineralized matrix deposition by pulpal cells, with potential for use in dentin tissue engineering.

Strategy for reducing cytotoxicity and obtaining esthetic efficacy with 15 min of in-office dental bleaching.

OBJECTIVES: Evaluate in vitro the esthetic efficacy and cytotoxicity of a bleaching gel containing 35% hydrogen peroxide (BG-35%H2O2), applied for different time intervals, on enamel coated or not with polymeric biomaterials. MATERIALS AND METHODS: Nanofiber scaffolds (NSc) and a primer catalyst (PrCa) were used to coat the bovine enamel/dentin discs before the application of BG-35%H2O2, according to the following groups: G1-negative control (NC, without treatment); G2, G3, and G4-BG-35%H2O2 applied for 3 × 15, 2 × 15, and 15 min; G5, G6, and G7-BG-35%H2O2 applied on enamel coated with NSc and PrCa for 3 × 15; 2 × 15, and 15 min, respectively. The culture medium with components of gel diffused through the discs was applied on MDPC-23 cells, which were evaluated regarding to viability (VB), integrity of the membrane (IM), and oxidative stress (OxS). The quantity of H2O2 diffused and esthetic efficacy (ΔE/ΔWI) of the dental tissues were also analyzed (ANOVA/Tukey; p < 0.05). RESULTS: Only G7 was similar to G1 regarding VB (p > 0.05). The lowest value of H2O2 diffusion occurred in G4 and G7, where the cells exhibited the lowest OxS than G2 (p < 0.05). Despite G5 showing the greatest ΔE regarding other groups (p < 0.05), the esthetic efficacy observed in G7 was similar to G2 (p > 0.05). ΔWI indicated a greater bleaching effect for groups G5, G6, and G7 (p < 0.05). CONCLUSION: Coating the dental enamel with polymeric biomaterials reduced the time and the cytotoxicity of BG-35%H2O2. CLINICAL SIGNIFICANCE: Coating the dental enamel with polymeric biomaterials allows safer and faster BG-35%H2O2 application.

Dose- and time-dependent effects of taxifolin on viability and mineralization markers of osteoblast-like cells.

The current study evaluated the effects of taxifolin treatments on the viability of osteoblast-like cells, and on the expression of early mineralization markers, as part of the ongoing search for new endodontic materials able to induce periapical healing without causing cytotoxicity. Saos-2 osteoblast-like cells were exposed to different concentrations of taxifolin (5 and 10 µM), applied as pretreatments either for 24h and 72h, or continuously throughout the experimental protocol. Cell viability using the methylthiazole tetrazolium (MTT) assay, alkaline phosphatase activity using thymolphthalein monophosphate assays, deposition of mineralized nodules using alizarin red staining, and expression of ALP and COL-1 by qPCR were determined after 6 and 13 days of treatment. The data were analyzed statistically (p<0.05). Taxifolin was not cytotoxic in the concentrations tested. Pretreatments with taxifolin for 24h and 72h at 10 µM stimulated ALP activity, and increased mineralized nodule deposition by Saos-2 cells. Continuous treatment with taxifolin was not effective in stimulating ALP activity and mineralization. ALP and COL-1 gene expression increased with taxifolin pretreatments, since the highest mRNA levels were observed after 72h of pretreatment with taxifolin at 10 µM on day 13. In conclusion, taxifolin was cytocompatible, and induced mineralization markers when applied for short periods in osteoblast-like cell culture.

Bioactivity effects of extracellular matrix proteins on apical papilla cells.

BACKGROUND: Potent signaling agents stimulate and guide pulp tissue regeneration, especially in endodontic treatment of teeth with incomplete root formation. OBJECTIVE: This study evaluated the bioactive properties of low concentrations of extracellular matrix proteins on human apical papilla cells (hAPCs). METHODOLOGY: Different concentrations (1, 5, and 10 µg/mL) of fibronectin (FN), laminin (LM), and type I collagen (COL) were applied to the bottom of non-treated wells of sterilized 96-well plates. Non-treated and pre-treated wells were used as negative (NC) and positive (PC) controls. After seeding the hAPCs (5×103 cells/well) on the different substrates, we assessed the following parameters: adhesion, proliferation, spreading, total collagen/type I collagen synthesis and gene expression (ITGA5, ITGAV, COL1A1, COL3A1) (ANOVA/Tukey; α=0.05). RESULTS: We observed greater attachment potential for cells on the FN substrate, with the effect depending on concentration. Concentrations of 5 and 10 µg/mL of FN yielded the highest cell proliferation, spreading and collagen synthesis values with 10 µg/mL concentration increasing the ITGA5, ITGAV, and COL1A1 expression compared with PC. LM (5 and 10 µg/mL) showed higher bioactivity values than NC, but those were lower than PC, and COL showed no bioactivity at all. CONCLUSION: We conclude that FN at 10 µg/mL concentration exerted the most intense bioactive effects on hAPCs.

Polymeric biomaterials maintained the esthetic efficacy and reduced the cytotoxicity of in-office dental bleaching.

Evaluate the kinetics of hydrogen peroxide (H2 O2 ) degradation, esthetic efficacy and cytotoxicity of a bleaching gel with 35%H2 O2 applied on enamel previously covered or not with polymeric nanofibrillar scaffold (SNan), polymeric primer catalyst (PPol), and both. Standardized enamel/dentin discs (n = 128) obtained from bovine teeth were adapted to pulp chambers. After covering enamel with the polymeric products, the bleaching gel was applied for 45 min, establishing the following groups: G1: no treatment (negative control); G2: 35%H2 O2 (positive control); G3: SNan; G4: PPol; G5: SNan + PPol; G6: SNan + 35%H2 O2 ; G7: PPol + 35%H2 O2 ; G8: SNan + PPol + 35%H2 O2 . The kinetics of H2 O2 degradation (n = 8), bleaching efficacy (ΔE/ΔWI; n = 8), trans-amelodentinal cytotoxicity (n = 8), and cell morphology (n = 4) were assessed (ANOVA/Tukey test; p < 0.05). Greater H2 O2 degradation occurred in G7 and G8. Bleaching efficacy (ΔE) was higher in G6, G7, and G8 in comparison with G2 (p < 0.05). However, no difference was observed for ΔWI (p > 0.05). G8 presented the lower level of trans-amelodentinal diffusion of H2 O2 , oxidative stress, and toxicity to the MDPC-23 cells (p < 0.05). Polymeric biomaterials increased the kinetics of H2 O2 decomposition, as well as maintained the esthetic efficacy and minimized the cytotoxicity caused by a bleaching gel with 35%H2 O2 . CLINICAL SIGNIFICANCE: Application of a bleaching gel with 35%H2 O2 on enamel previously covered by polymeric biomaterials maintains the esthetic efficacy and reduces the cytotoxicity caused by a single session of in-office dental bleaching.

Fibronectin-loaded Collagen/Gelatin Hydrogel Is a Potent Signaling Biomaterial for Dental Pulp Regeneration.

INTRODUCTION: Guided tissue regeneration has been considered a promising biological strategy to replace conventional endodontic therapies of teeth with incomplete root formation. Therefore, in the present study, a collagen/gelatin hydrogel either containing dosages of fibronectin (FN), or not, was developed and assessed concerning their bioactive and chemotactic potential on human apical papilla cells (hAPCs). METHODS: Hydrogels were prepared by varying the ratio of collagen and gelatin (Col/Gel; v/v), and used to establish the following groups: Collagen (positive control); Col/Gel 4:6; Col/Gel 6:4; Col/Gel 8:2. The viability, adhesion, and spreading of cells seeded on the hydrogels were evaluated. Different concentrations of FN (0, 5, or 10 µg/mL) were incorporated into the best formulation of the collagen/gelatin hydrogel selected. Then, the hAPCs seeded on the biomaterials were assessed concerning the cell migration, viability, adhesion and spreading, and gene expression of ITGA5, ITGAV, COL1A1, and COL3A1. RESULTS: The Col/Gel 8:2 group exhibited better cell viability, adhesion and spreading in comparison with Control. Higher values of hAPC migration, viability, adhesion, spreading and gene expression of pulp regeneration markers were found, the higher the concentration was of FN incorporated into the collagen/gelatin hydrogel. CONCLUSION: Collagen/gelatin hydrogel with 10 µg/mL of FN had potent bioactive and chemotactic effects on cultured hAPCs.

Response of pulp cells to resin infiltration of enamel white spot-like lesions.

OBJECTIVES: To investigate the trans-enamel and trans-dentinal biological effects of treating enamel white spot-like lesions (EWSLs) with resin infiltration components (RICs) on odontoblast-like cells (MDPC-23) and human dental pulp cells (HDPCs). METHODS: EWSLs were induced in 60 enamel/dentin discs (4.0 ± 0.2 mm thick) using S. mutans. The discs were adapted into artificial pulp chambers and MDPC-23 were seeded on the dentin surface. The components of a resin infiltration system (Icon) were applied individually or in combination on the enamel surface as following (n = 10/treatment): Etch, Infiltrant, Etch+Infiltrant, or Etch+Dry+Infiltrant. The application of water or hydrogen peroxide served as negative and positive controls, respectively. After 72 h, MDPC-23 viability was evaluated. The extracts were exposed for 72 h to pre-cultured MDPC-23 and HDPCs in 96-well plates to evaluate cell viability, alkaline phosphatase activity (ALP), mineralized nodule formation (MN), and the expression of inflammatory cytokines (ICs) and mineralization-related genes (MRs). Data were analyzed by ANOVA complemented with Tukey or Games-Howell post-hocs (α = 5%). RESULTS: Cell viability, ALP activity, and MN formation were significantly reduced in response to the RICs, presenting intermediate values compared to positive and negative controls. Likewise, ICs were upregulated, whereas MRs were downregulated. Among the RICs, the Etch component caused the most notorious detrimental effects. SIGNIFICANCE: Resin infiltration of EWSLs negatively affected the metabolism of pulp cells in vitro. Therefore, even though resin infiltration is a micro-invasive therapy for non-cavitated caries in enamel, it should be closely followed up seen that components may diffuse and unbalance pulp homeostasis.