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The objective of this study was to create injectable photo-crosslinkable biomaterials, using gelatin methacryloyl (GelMA) hydrogel, combined with a decellularized bone matrix (BMdc) and a deproteinized (BMdp) bovine bone matrix. These were intended to serve as bioactive scaffolds for dentin regeneration. The parameters for GelMA hydrogel fabrication were initially selected, followed by the incorporation of BMdc and BMdp at a 1% (w/v) ratio. Nano-hydroxyapatite (nHA) was also included as a control. A physicochemical characterization was conducted, with FTIR analysis indicating that the mineral phase was complexed with GelMA, and BMdc was chemically bonded to the amide groups of gelatin. The porous structure was preserved post-BMdc incorporation, with bone particles incorporated alongside the pores. Conversely, the mineral phase was situated inside the pore opening, affecting the degree of porosity. The mineral phase did not modify the degradability of GelMA, even under conditions of type I collagenase-mediated enzymatic challenge, allowing hydrogel injection and increased mechanical strength. Subsequently, human dental pulp cells (HDPCs) were seeded onto the hydrogels. The cells remained viable and proliferative, irrespective of the GelMA composition. All mineral phases resulted in a significant increase in alkaline phosphatase activity and mineralized matrix deposition. However, GelMA-BMdc exhibited higher cell expression values, significantly surpassing those of all other formulations. In conclusion, our results showed that GelMA-BMdc produced a porous and stable hydrogel, capable of enhancing odontoblastic differentiation and mineral deposition when in contact with HDPCs, thereby showing potential for dentin regeneration., (© 2024 Wiley Periodicals LLC.)

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., (Copyright © 2022 Editorial Council for The Journal of Prosthetic Dentistry. Published by Elsevier Inc. All rights reserved.)

Objective: To evaluate whether coating enamel with a polymeric primer (PPol) containing titanium tetrafluoride (TiF 4 ) before applying a bleaching gel with 35% H 2 O 2 (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 2TiF 4 , 6TiF 4 , and 10TiF 4 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, ΔE 00 , 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 H 2 O 2 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 ΔE 00 , no difference was observed among groups 2TiF 4 , 6TiF 4 , and PC (p > 0.05). The ΔWI was similar between groups 2TiF 4 and PC (p > 0.05). The ΔWI of group 6TiF 4 was superior to PC (p < 0.05), and group 10TiF 4 achieved the highest ΔE 00 and ΔWI values (p < 0.05). Besides limiting enamel microstructural changes compared to PC, group 10TiF 4 significantly increased the hardness of this mineralized dental tissue. The highest cellular viability occurred in 10TiF 4 compared to the other bleached groups (p < 0.05). Trans-amelodentinal H 2 O 2 diffusion decreased in groups 2TiF 4 , 6TiF 4 , and 10TiF 4 in comparison with PC (p < 0.05)., Conclusion: Coating enamel with a PPol containing TiF 4 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 TiF 4 promoted the best outcomes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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 H 2 O 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., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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.

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., Competing Interests: Conflict of Interest: No potential conflict of interest relevant to this article was reported., (Copyright © 2023. The Korean Academy of Conservative Dentistry.)

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.

Objective: The study aims to assess the effects of a 10% H 2 O 2 bleaching gel with different MnO 2 concentrations on the bleaching efficacy (BE), degradation kinetics (DK) of H 2 O 2 , 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% H 2 O 2 (positive control/PC); (G3) 10% H 2 O 2 ; (G4) 10% H 2 O 2  + 2 mg/mL MnO 2 ; (G5) 10% H 2 O 2  + 6 mg/mL MnO 2 ; and (G6) 10% H 2 O 2  + 10 mg/mL MnO 2 . After analyzing bleaching efficacy (ΔE 00 and ΔWI), the degradation kinetics of H 2 O 2 and trans-amelodentinal cytotoxicity were determined (n = 8, ANOVA/Tukey; p < 0.05)., Results: G6 presented BE (ΔE 00 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 H 2 O 2 occurred in G3, G4, G5, and G6 compared to G2 (p < 0.0001). The highest DK of H 2 O 2 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 MnO 2 in a 10% H 2 O 2 bleaching gel potentiates the degradation of this reactive molecule, which increases the BE of the product and decreases TC., Clinical Significance: Replacing a 35% H 2 O 2 gel commonly used for conventional in-office dental bleaching by a 10% H 2 O 2 gel containing 10 mg/mL of MnO 2 reduces the cytotoxicity of this professional therapy, maintaining its excellent esthetic efficacy., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., (© 2022 British Endodontic Society. Published by John Wiley & Sons Ltd.)

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., Competing Interests: Disclosure The authors declare no Competing Financial or Non-Financial Interests. This work has been supported by grant from the National University Health System, Singapore (NUHSRO/2020/016/RO5+5/Seed-Aug/14)., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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 β-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βTCP 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Ca SBF , 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Ca SBF 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Ca SBF , CHnHA SBF , and CHβTCP SBF 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., (© 2022 Wiley Periodicals LLC.)

Objective: This study aimed to evaluate the esthetic efficacy, cytotoxicity, and kinetics of decomposition of hydrogen peroxide (H2O2) present in a commercial bleaching gel with 35% H2O2 (BG35%) chemically activated with manganese oxide (MnO2)., Methods and Materials: After incorporating 2 mg/mL, 6 mg/mL, and 10 mg/mL of MnO2 into BG35%, the stability of pH and temperature of the products were analyzed. To assess the esthetic efficacy (ΔE and ΔWI), the BG35%s with MnO2 were applied for 45 minutes on enamel/dentin discs (DiE/D). BG35% or no treatment were used as positive (PC) and negative (NC) controls, respectively. To analyze the cell viability (CV) and oxidative stress (OXS), the same bleaching protocols were performed on DiE/D adapted to artificial pulp chambers. The extracts (culture medium + gel components that diffused through the discs) were applied to pulp cells and submitted to H2O2 quantification. BG35% with MnO2 that showed the best results was evaluated relative to kinetic decomposition of H2O2, with consequent generation of free radicals (FR) and hydroxyl radicals (OH•). The data were submitted to the one-way analysis of variance complemented by Tukey post-test (α=0.05). Data on kinetics of H2O2 decomposition were submitted to the Student's-t test (α=0.05)., Results: All the BG35%s with MnO2 showed stability of pH and temperature, and the gel with 10 mg/mL of this activator had an esthetic efficacy 31% higher than that of the PC (p<0.05). Reduction in OXS and trans-amelodentinal diffusion of H2O2 occurred when all the BG35%s with MnO2 were used. The addition of 6 and 10 mg/mL of MnO2 to BG35% increased the CV in comparison with PC, confirmed by the cell morphology analysis. An increase in FR and OH• formation was observed when 10 mg/mL of MnO2 was added to BG35%., Conclusion: Catalysis of BG35% with MnO2 minimized the trans-amelodentinal diffusion of H2O2 and toxicity of the product to pulp cells. BG35% containing 10 mg/mL of MnO2 potentiated the decomposition of H2O2, enhancing the generation of FR and OH•, as well as the efficacy of the in-office tooth therapy., (©Operative Dentistry, 2022.)

Objectives: Evaluate in vitro the esthetic efficacy and cytotoxicity of a bleaching gel containing 35% hydrogen peroxide (BG-35%H 2 O 2 ), 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%H 2 O 2 , according to the following groups: G1-negative control (NC, without treatment); G2, G3, and G4-BG-35%H 2 O 2 applied for 3 × 15, 2 × 15, and 15 min; G5, G6, and G7-BG-35%H 2 O 2 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 H 2 O 2 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 H 2 O 2 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%H 2 O 2 ., Clinical Significance: Coating the dental enamel with polymeric biomaterials allows safer and faster BG-35%H 2 O 2 application., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Objective: In this paper we propose the association of β-glycerophosphate (βGP) and calcium-hydroxide with chitosan (CH) to formulate a porous bioactive scaffold suitable as a cell-homing platform for dentin regeneration., Methods: Calcium hydroxide and βGP solutions were incorporated into chitosan to modulate scaffold architecture and composition by a phase separation technique. Architecture, chemical composition, and degradability were evaluated, and biological characterizations were performed by the seeding of dental pulp cells (DPCs) onto scaffolds, or by cultivating them in contact with leachable components (extracts), to determine cytocompatibility and odontoblastic differentiation. Cell-free scaffolds were then positioned in intimate contact with a 3D culture of DPCs in a pulp-in-a-chip platform under simulated pulp pressure. Cell mobilization and odontoblastic marker expression were evaluated. Deposition of mineralized matrix was assessed in direct contact with dentin, in the absence of osteogenic factors., Results: Incorporation of calcium hydroxide and βGP generated a stable porous chitosan scaffold containing Ca-P nanoglobule topography (CH-Ca-βGP), which favored cell viability, alkaline phosphatase activity, and mineralized matrix deposition by cells seeded onto the scaffold structure and at a distance. The pulp-in-a-chip assay denoted its chemotactic and bioactive potential, since dentin sialoprotein-positive DPCs from 3D culture adhered to CH-Ca-βGP more than to plain chitosan. The higher deposition of mineralized matrix onto the scaffold and surrounding dentin was also observed., Significance: A CH-Ca-βGP scaffold creates a microenvironment capable of mobilizing DPC migration toward its structure, harnessing the odontogenic potential and culminating in the expression of a highly mineralizing phenotype, key factors for a cell-homing strategy., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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.

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.

Evaluate the kinetics of hydrogen peroxide (H 2 O 2 ) degradation, esthetic efficacy and cytotoxicity of a bleaching gel with 35%H 2 O 2 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%H 2 O 2 (positive control); G3: SNan; G4: PPol; G5: SNan + PPol; G6: SNan + 35%H 2 O 2 ; G7: PPol + 35%H 2 O 2 ; G8: SNan + PPol + 35%H 2 O 2 . The kinetics of H 2 O 2 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 H 2 O 2 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 H 2 O 2 , oxidative stress, and toxicity to the MDPC-23 cells (p < 0.05). Polymeric biomaterials increased the kinetics of H 2 O 2 decomposition, as well as maintained the esthetic efficacy and minimized the cytotoxicity caused by a bleaching gel with 35%H 2 O 2 . CLINICAL SIGNIFICANCE: Application of a bleaching gel with 35%H 2 O 2 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., (© 2021 Wiley Periodicals LLC.)

Engineering multifunctional hydrogel systems capable of amplifying the regenerative capacity of endogenous progenitor cells via localized presentation of therapeutics under tissue inflammation is central to the translation of effective strategies for hard tissue regeneration. Here, we loaded dexamethasone (DEX), a pleotropic drug with anti-inflammatory and mineralizing abilities, into aluminosilicate clay nanotubes (halloysite clay nanotubes (HNTs)) to engineer an injectable multifunctional drug delivery system based on photo-cross-linkable gelatin methacryloyl (GelMA) hydrogel. In detail, a series of hydrogels based on GelMA formulations containing distinct amounts of DEX-loaded nanotubes was analyzed for physicochemical and mechanical properties and kinetics of DEX release as well as compatibility with mesenchymal stem cells from human exfoliated deciduous teeth (SHEDs). The anti-inflammatory response and mineralization potential of the engineered hydrogels were determined in vitro and in vivo . DEX conjugation with HNTs was confirmed by FTIR analysis. The incorporation of DEX-loaded nanotubes enhanced the mechanical strength of GelMA with no effect on its degradation and swelling ratio. Scanning electron microscopy (SEM) images demonstrated the porous architecture of GelMA, which was not significantly altered by DEX-loaded nanotubes' (HNTs/DEX) incorporation. All GelMA formulations showed cytocompatibility with SHEDs ( p < 0.05) regardless of the presence of HNTs or HNTs/DEX. However, the highest osteogenic cell differentiation was noticed with the addition of HNT/DEX 10% in GelMA formulations ( p < 0.01). The controlled release of DEX over 7 days restored the expression of alkaline phosphatase and mineralization ( p < 0.0001) in lipopolysaccharide (LPS)-stimulated SHEDs in vitro . Importantly, in vivo data revealed that DEX-loaded nanotube-modified GelMA (5.0% HNT/DEX 10%) led to enhanced bone formation after 6 weeks ( p < 0.0001) compared to DEX-free formulations with a minimum localized inflammatory response after 7 days. Altogether, our findings show that the engineered DEX-loaded nanotube-modified hydrogel may possess great potential to trigger in situ mineralized tissue regeneration under inflammatory conditions.

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.

Fibronectin (FN)-loaded nanofiber scaffolds were developed and assessed concerning their bioactive potential on human apical papilla cells (hAPCs). First, random (NR) and aligned (NA) nanofiber scaffolds of polycaprolactone (PCL) were obtained by electrospinning technique and their biological properties were evaluated. The best formulations of NR and NA were loaded with 0, 5, or 10 μg/ml of FN and their bioactivity was assessed. Finally, FN-loaded NR and NA tubular scaffolds were prepared and their chemotactic potential was analyzed using an in vitro model to mimic the pulp regeneration of teeth with incomplete root formation. All scaffolds tested were cytocompatible. However, NR and NA based on 10% PCL promoted the highest hAPCs proliferation, adhesion and spreading. Polygonal and elongated cells were observed on NR and NA, respectively. The higher the concentration of FN added to the scaffolds, greater cell migration, viability, proliferation, adhesion and spreading, as well as collagen synthesis and gene expression (ITGA5, ITGAV, COL1A1, COL3A1). In addition, tubular scaffolds with NA loaded with FN (10 μg/ml) showed the highest chemotactic potential on hAPCs. It was concluded that FN-loaded NA scaffolds may be an interesting biomaterial to promote hAPCs-mediated pulp regeneration of endodontically compromised teeth with incomplete root formation., (© 2020 Wiley Periodicals LLC.)

The development of biomaterials based on the combination of biopolymers with bioactive compounds to develop delivery systems capable of modulating dentin regeneration mediated by resident cells is the goal of current biology-based strategies for regenerative dentistry. In this article, the bioactive potential of a simvastatin (SV)-releasing chitosan-calcium-hydroxide (CH-Ca) scaffold was assessed. After the incorporation of SV into CH-Ca, characterization of the scaffold was performed. Dental pulp cells (DPCs) were seeded onto scaffolds for the assessment of cytocompatibility, and odontoblastic differentiation was evaluated in a microenvironment surrounded by dentin. Thereafter, the cell-free scaffold was adapted to dentin discs positioned in artificial pulp chambers in direct contact with a 3-dimensional (3D) culture of DPCs, and the system was sealed to simulate internal pressure at 20 cm/H 2 O. In vivo experiments with cell-free scaffolds were performed in rats' calvaria defects. Fourier-transform infrared spectroscopy spectra proved incorporation of Ca and SV into the scaffold structure. Ca and SV were released upon immersion in a neutral environment. Viable DPCs were able to spread and proliferate on the scaffold over 14 d. Odontoblastic differentiation occurred in the DPC/scaffold constructs in contact with dentin, in which SV supplementation promoted odontoblastic marker overexpression and enhanced mineralized matrix deposition. The chemoattractant potential of the CH-Ca scaffold was improved by SV, with numerous viable and dentin sialoprotein-positive cells from the 3D culture being observed on its surface. Cells at 3D culture featured increased gene expression of odontoblastic markers in contact with the SV-enriched CH-Ca scaffold. CH-Ca-SV led to intense mineralization in vivo, presenting mineralization foci inside its structure. In conclusion, the CH-Ca-SV scaffold induces differentiation of DPCs into a highly mineralizing phenotype in the presence of dentin, creating a microenvironment capable of attracting pulp cells to its surface and inducing the overexpression of odontoblastic markers in a cell-homing strategy.

Objectives: The aim of this review is to highlight recent progress in the field of biomaterials-mediated dental pulp tissue engineering. Specifically, we aim to underscore the critical design criteria of biomaterial platforms that are advantageous for pulp tissue engineering, discuss models for preclinical evaluation, and present new and innovative multifunctional strategies that hold promise for clinical translation., Materials and Methods: The current article is a comprehensive overview of recent progress over the last 5 years. In detail, we surveyed the literature in regenerative pulp biology, including novel biologic and biomaterials approaches, and those that combined multiple strategies, towards more clinically relevant models. PubMed searches were performed using the keywords: "regenerative dentistry," "dental pulp regeneration," "regenerative endodontics," and "dental pulp therapy.", Results: Significant contributions to the field of regenerative dentistry have been made in the last 5 years, as evidenced by a significant body of publications. We chose exemplary studies that we believe are progressive towards clinically translatable solutions. We close this review with an outlook towards the future of pulp regeneration strategies and their clinical translation., Conclusions: Current clinical treatments lack functional and predictable pulp regeneration and are more focused on the treatment of the consequences of pulp exposure, rather than the restoration of healthy dental pulp., Clinical Relevance: Clinically, there is great demand for bioinspired biomaterial strategies that are safe, efficacious, and easy to use, and clinicians are eager for their clinical translation. In particular, we place emphasis on strategies that combine favorable angiogenesis, mineralization, and functional tissue formation, while limiting immune reaction, risk of microbial infection, and pulp necrosis., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., (Copyright © 2021 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2021 The Academy of Dental Materials. Published by Elsevier Inc. All rights reserved.)

Background: A 2011 RCVS report published following review of veterinary referral and specialisation suggested that the general public may have a lack of understanding of what constitutes specialisation. Our questionnaire study evaluated client understanding of the term 'Specialist' and the information obtained prior to referral., Methods: Questionnaires were completed prior to consultation. Client age, medical knowledge, how referral was initiated, options discussed by the primary care practice for treatment, expectations of the referral appointment, understanding of the term Specialist and what constitutes specialist training were assessed., Results: A total of 241 questionnaires were completed: 6.2% (15/241) of clients were working in the veterinary/medical industry; 52% (104/200) of clients assumed they were seeing a Specialist for consultation but only 49% (51/104) saw a Specialist; 0.4% (1/232) of owners correctly identified a Specialist as a clinician with a diploma, and incorrect definitions included having experience with a procedure or a postgraduate certificate; 13.6% (31/228) of clients identified that an internship and residency constituted Specialist training; and 66.8% (155/232) of clients had not researched the referral process., Conclusions: From the population sampled, a minority of clients understand the term Specialist and the postgraduate training this entails. Clients may erroneously assume they are receiving Specialist consultation., (© 2021 British Veterinary Association.)

Purpose: Toilet training (TT) is an important marker in a child's physical and psychosocial development. The present study aimed to evaluate aspects associated to delayed TT., Material and Methods: We interviewed 372 parents of children who had completed TT up to 48 months before the interview. The questionnaires were applied at school exits when parents went to pick their children up and at public parks. Questions included demographics, aspects related to TT, dysfunction voiding symptom score and evaluation of constipation., Results: The interviews were performed at a mean of 15.3±10.4 (0 to 47) months after the end of TT. Girls accounted for 53% of the sample. The mean age at finishing TT was 31.6±9.3 months and similar in both genders (p=0.77). TT occurred before school entry in 45.7% of the children and medical advice for TT was sought only by 4.8% of the parents. No association was observed of age at completing TT and presence of lower urinary tract symptoms (LUTS) (p=0.57) and/or constipation (p=0.98). In the univariate analysis, prematurity (OR=2.7 [95% CI 2.3-3.1], p <0.0001) and mothers who work outside their household (OR=1.8 [95% CI 1.4-2.3], p <0.0001) were associated to delayed TT., Conclusion: Children completed TT at a mean of 2 years and 7 months of age. The age of completing TT was not related to LUTS and/or constipation. Premature children and those whose mothers work outside the home finish TT later., Competing Interests: None declared., (Copyright® by the International Brazilian Journal of Urology.)

Clinical Relevance: Pigments in tooth structures affect the diffusion of H2O2 through enamel and dentin. The bleaching methodology can be impacted., Summary: Objective: The aim of this study was to evaluate the influence of the presence of pigments in tooth structures on the trans-enamel and trans-dentin diffusion of hydrogen peroxide (H2O2) and its cytotoxicity after carrying out an in-office bleaching therapy.Methods and Materials: A bleaching gel with 35% H2O2 was applied for 45 minutes (three times for 15 minutes) on enamel and dentin discs (n=6), either previously submitted to the intrinsic pigmentation protocol with a concentrated solution of black tea, or not, defining the following groups: G1, unbleached untreated discs (control 1); G2, unbleached pigmented discs (control 2); G3, bleached untreated discs; G4, bleached pigmented discs. The discs were adapted to artificial pulp chambers, which were placed in wells of 24-well plates containing 1 mL culture medium (Dulbecco's modified Eagle's medium [DMEM]). After applying the bleaching gel on enamel, the extracts (DMEM + components of bleaching gel that diffused through the discs) were collected and then applied on the cultured MDPC-23 odontoblast-like cells. Cell viability (methyl tetrazolium assay and Live & Dead, Calcein AM, and ethidium homodimer-1 [EthD-1] probes), the amount of H2O2 that diffused through enamel and dentin (leuco-crystal violet product), and the H2O2-mediated oxidative cell stress (SOx) and components of degradation were assessed (analysis of variance/Tukey; α=0.05).Results: There was no significant difference between the groups G1 and G2 for all the parameters tested (p>0.05). Reduction in the trans-enamel and trans-dentin diffusion of H2O2 occurred for G4 in comparison with G3. Significantly lower cell viability associated with greater oxidative stress was observed for G3 (p<0.05). Therefore, in-office tooth bleaching therapy performed in pigmented samples caused lower cytotoxic effects compared with untreated samples submitted to the same esthetic procedure (p<0.05).Conclusion: According to the methodology used in this investigation, the authors concluded that the presence of pigments in hard tooth structures decreases the trans-enamel and trans-dentin diffusion of H2O2 and the toxicity to pulp cells of an in-office bleaching gel with 35% H2O2., (©Operative Dentistry, 2020.)

The present study evaluated the odontogenic potential of human dental pulp cells (HDPCs) exposed to chitosan scaffolds containing calcium aluminate (CHAlCa) associated or not with low doses of simvastatin (SV). Chitosan scaffolds received a suspension of calcium aluminate (AlCa) and were then immersed into solutions containing SV. The following groups were established: chitosan-calcium-aluminate scaffolds (CHAlCa - Control), chitosan calcium-aluminate with 0.5 µM SV (CHAlCa-SV0.5), and chitosan calcium-aluminate with 1.0 µM SV (CHAlCa-SV1.0). The morphology and composition of the scaffolds were evaluated by SEM and EDS, respectively. After 14 days of HDPCs culture on scaffolds, cell viability, adhesion and spread, mineralized matrix deposition as well as gene expression of odontogenic markers were assessed. Calcium aluminate particles were incorporated into the chitosan matrix, which exhibited regular pores homogeneously distributed throughout its structure. The selected SV dosages were biocompatible with HDPCs. Chitosan-calcium-aluminate scaffolds with 1 µM SV induced the odontoblastic phenotype in the HDPCs, which showed enhanced mineralized matrix deposition and up-regulated ALP, Col1A1, and DMP-1 expression. Therefore, one can conclude that the incorporation of calcium aluminate and simvastatin in chitosan scaffolds had a synergistic effect on HDPCs, favoring odontogenic cell differentiation and mineralized matrix deposition.

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., (© 2020 BSPD, IAPD and John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The aim of this study was to develop a highly porous calcium-containing chitosan scaffold suitable for dentin regeneration. A calcium hydroxide (Ca[OH] 2 ) suspension was used to modulate the degree of porosity and chemical composition of chitosan scaffolds. The chitosan solution concentration and freezing protocol were adjusted to optimize the porous architecture using the phase-separation technique. Scanning electron microscopy/energy-dispersive spectroscopy demonstrated the fabrication of a highly porous calcium-linked chitosan scaffold (CH-Ca), with a well-organized and interconnected porous network. Scaffolds were cross-linked on glutaraldehyde (GA) vapor. Following a 28-day incubation in water, cross-linked CH scaffold had no changes on humid mass, and CH-Ca featured a controlled degradability profile since the significant humid mass loss was observed only after 21 (26.0%) and 28 days (42.2%). Fourier-transform infrared spectroscopy indicated the establishment of Schiff base on cross-linked scaffolds, along with calcium complexation for CH-Ca. Cross-linked CH-Ca scaffold featured a sustained Ca 2+ release up to 21 days in a humid environment. This porous and stable architecture allowed for human dental pulp cells (HDPCs) to spread throughout the scaffold, with cells exhibiting a widely stretched cytoplasm; whereas, the cells seeded onto CH scaffold were organized in clusters. HDPCs seeded onto CH-Ca featured significantly higher ALP activity, and gene expressions for ALP, Col1, DMP-1, and DSPP in comparison to CH, leading to a significant 3.5 times increase in calcium-rich matrix deposition. In sum, our findings suggest that CH-Ca scaffolds are attractive candidates for creating a highly porous and bioactive substrate for dentin tissue engineering., (© 2020 Wiley Periodicals, Inc.)

Objectives: This study assessed the human pulp response after adhesive restoration of cavities by indirect pulp capping with a conventional or a resin-modified glass ionomer cement., Materials and Methods: Deep cavities prepared in 26 human premolars were lined with Riva Light Cure (Riva LC), Riva Self Cure (Riva SC), or Dycal, and then restored with composite resin. Four teeth were used as intact control. After time intervals of 7 or 30 days, the teeth were extracted, processed for histological evaluation of the pulp, and the remaining dentin thickness (RDT) between the cavity floor and the pulp was measured., Results: At 7 days, a slight pulp inflammation associated with discrete tissue disorganization was observed in most of t the teeth lined with Riva LC and Riva SC. Moderate pulp inflammation occurred in one tooth lined with Riva LC. Bacteria were identified in one specimen of the same group that exhibited no pulp damage. At 30 days, slight pulp inflammation and discrete tissue disorganization persisted in two specimens treated with Riva LC, in which a thin layer of tertiary dentin was deposited. Mean RDTs ranged from 383.0 to 447.8 μm., Conclusions: Riva LC produced more damage to the pulp than Riva SC. However, the initial pulp damage decreased over time and after 30 days both GICs were labeled as biocompatible., Clinical Relevance: In this study conducted with human teeth, the conventional and the resin-modified glass ionomer cements investigated were shown not to cause post-operative sensitivity or persistent pulp damage when applied as liners in very deep cavities, thereby indicating their biocompatibility.

Objectives: This study aimed to develop a porous chitosan-calcium-aluminate scaffold (CH-AlCa) in combination with a bioactive dosage of 1α,25-dihydroxyvitamin D3 (1α,25VD), to be used as a bioactive substrate capable to increase the odontogenic potential of human dental pulp cells (HDPCs)., Materials and Methods: The porous CH-AlCa was developed by the incorporation of an AlCa suspension into a CH solution under vigorous agitation, followed by phase separation at low temperature. Scaffold architecture, porosity, and calcium release were evaluated. Thereafter, the synergistic potential of CH-AlCa and 1 nM 1α,25VD, selected by a dose-response assay, for HDPCs seeded onto the materials was assessed., Results: The CH-AlCa featured an organized and interconnected pore network, with increased porosity in comparison with that of plain chitosan scaffolds (CH). Increased odontoblastic phenotype expression on the human dental pulp cell (HDPC)/CH and HDPC/CH-AlCa constructs in the presence of 1 nM 1α,25VD was detected, since alkaline phosphatase activity, mineralized matrix deposition, dentin sialophosphoprotein/dentin matrix acidic phosphoprotein 1 mRNA expression, and cell migration were overstimulated. This drug featured a synergistic effect with CH-AlCa, since the highest values of cell migration and odontoblastic markers expression were observed in this experimental condition., Conclusions: The experimental CH-AlCa scaffold increases the chemotaxis and regenerative potential of HDPCs, and the addition of low-dosage 1α,25VD to this scaffold enhances the potential of these cells to express an odontoblastic phenotype., Clinical Relevance: Chitosan scaffolds enriched with calcium-aluminate in association with low dosages of 1α,25-dihydroxyvitamin D3 provide a highly bioactive microenvironment for dental pulp cells prone to dentin regeneration, thus providing potential as a cell-free tissue engineering system for direct pulp capping.

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.

Objective: This study was designed for the chemical activation of a 35% hydrogen peroxide (H2O2) bleaching gel to increase its whitening effectiveness and reduce its toxicity., Methodology: First, the bleaching gel - associated or not with ferrous sulfate (FS), manganese chloride (MC), peroxidase (PR), or catalase (CT) - was applied (3x 15 min) to enamel/dentin discs adapted to artificial pulp chambers. Then, odontoblast-like MDPC-23 cells were exposed for 1 h to the extracts (culture medium + components released from the product), for the assessment of viability (MTT assay) and oxidative stress (H2DCFDA). Residual H2O2 and bleaching effectiveness (DE) were also evaluated. Data were analyzed with one-way ANOVA complemented with Tukey's test (n=8. p<0.05)., Results: All chemically activated groups minimized MDPC-23 oxidative stress generation; however, significantly higher cell viability was detected for MC, PR, and CT than for plain 35% H2O2 gel. Nevertheless, FS, MC, PR, and CT reduced the amount of residual H2O2 and increased bleaching effectiveness., Conclusion: Chemical activation of 35% H2O2 gel with MC, PR, and CT minimized residual H2O2 and pulp cell toxicity; but PR duplicated the whitening potential of the bleaching gel after a single 45-minute session.

The objective of this study was the development of a bioactive glass coating on zirconia (Zr) to modulate the gingival fibroblast phenotype. For this purpose, Biosilicate® (BS) particles in a water/isopropyl alcohol (1:1) vehicle (6 mg/mL) were applied to zirconia discs followed by thermal treatment at 1100 °C for 20 min. The surface topography (SEM), chemical composition (EDX), surface roughness (Ra; confocal microscopy), surface free energy (goniometry), and color alteration (UV-vis spectrophotometry) were assessed (n=6). Thereafter, L929 fibroblasts were seeded onto Zr and Zr+BS discs, and cell proliferation (Alamar Blue; n=6), morphology (SEM; n=2), migration (wound healing; n=4), and collagen synthesis (Sirius Red; n=6) were evaluated up to 7 days. Data were analyzed by ANOVA/Tukey tests (a=5%). A homogeneous coating consisting of Si, Na, O, and Ca was detected on the Zr surface after thermal treatment with BS, which led to a significant increase in surface roughness and free energy (p<0.05). No change in color parameters was observed (p>0.05). Cells seeded on the Zr+BS surface featured increased proliferation, collagen expression, and migration capability in comparison with those cultured on plain Zr (p<0.05). SEM images revealed that cell spreading occurred faster in the presence of BS. Therefore, it was concluded that thermal treatment of the Zr surface with BS led to the deposition of a bioactive coating, which induced gingival fibroblast spread, proliferation, migration, and collagen expression in vitro.

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

Introduction: The improvement of biomaterials capable of driving the regeneration of the pulp-dentin complex mediated by resident cells is the goal of regenerative dentistry. In the present investigation, a chitosan scaffold (CHSC) that released bioactive concentrations of simvastatin (SIM) was tested, aimed at the development of a cell-free tissue engineering system., Methods: First, we performed a dose-response assay to select the bioactive dose of SIM capable of inducing an odontoblastic phenotype in dental pulp cells (DPCs); after which we evaluated the synergistic effect of this dosage with the CHSC/DPC construct. SIM at 1.0 μmol/L (CHSC-SIM1.0) and 0.5 μmol/L were incorporated into the CHSC, and cell viability, adhesion, and calcium deposition were evaluated. Finally, we assessed the biomaterials in an artificial pulp chamber/3-dimensional culture model to simulate the cell-free approach in vitro., Results: SIM at 0.1 μmol/L was selected as the bioactive dose. This drug was capable of strongly inducing an odontoblastic phenotype on the DPC/CHSC construct. The incorporation of SIM into CHSC had no deleterious effect on cell viability and adhesion to the scaffold structure. CHSC-SIM1.0 led to significantly higher calcium-rich matrix deposition on scaffold/dentin disc assay compared with the control (CHSC). This biomaterial induced the migration of DPCs from a 3-dimensional culture to its surface as well as stimulated significantly higher expressions of alkaline phosphatase, collagen type 1 alpha 1, dentin matrix acidic phosphoprotein 1, and dentin sialophosphoprotein on 3-dimensional-cultured DPCs than on those in contact with CHSC., Conclusions: CHSC-SIM1.0 scaffold was capable of increasing the chemotaxis and regenerative potential of DPCs., (Copyright © 2018 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

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., (© 2017 The American Society of Photobiology.)

In this study, we investigated the anti-inflammatory, odontogenic and pro-angiogenic effects of integrating simvastatin and nanofibrous poly(l-lactic acid) (NF-PLLA) scaffolds on dental pulp cells (DPCs). Highly porous NF-PLLA scaffolds that mimic the nanofibrous architecture of extracellular matrix were first fabricated, then seeded with human DPCs and cultured with 0.1 μM simvastatin and/or 10 μg/mL pro-inflammatory stimulator lipopolysaccharide (LPS). The gene expression of pro-inflammatory mediators (TNF-α, IL-1β and MMP-9 mRNA) and odontoblastic markers (ALP activity, calcium content, DSPP, DMP-1 and BMP-2 mRNA) were quantified after long-term culture in vitro. In addition, we evaluated the scaffold's pro-angiogenic potential after 24 h of in vitro co-culture with endothelial cells. Finally, we assessed the combined effects of simvastatin and NF-PLLA scaffolds in vivo using a subcutaneous implantation mouse model. The in vitro studies demonstrated that, compared with the DPC/NF-PLLA scaffold constructs cultured only with pro-inflammatory stimulator LPS, adding simvastatin significantly repress the expression of pro-inflammatory mediators. Treating LPS+ DPC/NF-PLLA constructs with simvastatin also reverted the negative effects of LPS on expression of odontoblastic markers in vitro and in vivo. Western blot analysis demonstrated that these effects were related to a reduction in NFkBp65 phosphorylation and up-regulation of PPARγ expression, as well as to increased phosphorylation of pERK1/2 and pSmad1, mediated by simvastatin on LPS-stimulated DPCs. The DPC/NF-PLLA constructs treated with LPS/simvastatin also led to an increase in vessel-like structures, correlated with increased VEGF expression in both DPSCs and endothelial cells. Therefore, the combination of low dosage simvastatin and NF-PLLA scaffolds appears to be a promising strategy for dentin regeneration with inflamed dental pulp tissue, by minimizing the inflammatory reaction and increasing the regenerative potential of resident stem cells., Statement of Significance: The regeneration potential of stem cells is dependent on their microenvironment. In this study, we investigated the effect of the microenvironment of dental pulp stem cells (DPSCs), including 3D structure of a macroporous and nanofibrous scaffold, the inflammatory stimulus lipopolysaccharide (LPS) and a biological molecule simvastatin, on their regenerative potential of mineralized dentin tissue. The results demonstrated that LPS upregulated inflammatory mediators and suppressed the odontogenic potential of DPSCs. Known as a lipid-lowing agent, simvastatin was excitingly found to repress the expression of pro-inflammatory mediators, up-regulate odontoblastic markers, and exert a pro-angiogenic effect on endothelial cells, resulting in enhanced vascularization and mineralized dentin tissue regeneration in a biomimetic 3D tissue engineering scaffold. This novel finding is significant for the fields of stem cells, inflammation and dental tissue regeneration., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

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.

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/cm 2 ) 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.

Background: Male breast cancer is a rare and less known disease. Therapeutic modalities affect survival. In Burkina Faso, male breast cancers are diagnosed in everyday practice, but the prognosis at short-, middle-, and long-term remains unknown. The objective of this study is to study the diagnosis stages, therapeutic modalities, and 5-year survival in male breast cancer at the General Surgery Unit of Yalgado Ouedraogo University Hospital from 1990 to 2009., Methods: A cohort longitudinal study concerning cases of breast cancer diagnosed in man. Survival was assessed using the Kaplan-Meier method and survival curves were compared through the LogRank test., Results: Fifty-one cases of male breast cancer were followed-up, i.e., 2.6% of all breast cancers. Stages III and IV represented 88% of cases. Eleven patients (21.6%) were at metastatic stage. Patients were operated in 60.8% of cases. The surgery included axillary dissection in 25 (80.6%) out of 31 cases. Lumpectomy was performed on 6.5% of patients (2 cases). Fifteen (29.4%) and 11 (21.6%) patients underwent chemotherapy and hormonal therapy, respectively. The FAC protocol was mostly used. Radiation therapy was possible in two cases. The median deadline for follow-up was 14.8 months. A local recurrence was noticed in 3.2% of cases. The overall 5-year survival rate was 49.9%. The median survival was over 5 years for stages I and II. It was 54 down to 36 months for stages III and IV., Conclusion: Diagnosis is late. The lack of immunohistochemistry makes it difficult to define the proportion of their hormonal dependence. Surgery is the basic treatment. Five-year survival is slow and the median survival depends on the diagnosis stage. It can be improved through awareness-raising campaigns and the conduct of individual screening.

This study evaluated application protocol (etch-and-rinse/ER and self-etching/SE) and dentin wettability (wet and dry) on microtensile bond strength (μTBS) and transdentinal cytotoxicity of ScotchbondTM Universal (SU) adhesive system. The μTBS values and fracture mode were registered 24 h after adhesive system application and resin composite block build-up (n=5). For analysis of transdentinal cytotoxicity, odontoblast-like MDPC-23 cells were seeded on pulpal surface of dentin discs (0.4 mm thick) adapted to artificial pulp chambers (n=8). The adhesive system was applied to occlusal surface, followed by 24-h incubation time. Cell viability (Alamar Blue) and morphology (SEM) were assessed. Adper Single Bond 2 and Clearfil SE Bond were used as positive controls of the ER and SE application protocols, respectively. No treatment was performed on negative control (NC) group. Data were analyzed by ANOVA and Tukey's tests (α=5%). Higher μTBS values were found for ER mode in comparison with SE protocol (p<0.05). Dentin wettability had no effect on bond strength of SU in both the ER and SE techniques (p>0.05). Most fractures involved hybrid layer and/or adhesive layer. Neither variable prevented the intense toxic effects of adhesive systems on MDPC-23 cultured cells, since intense reduction in cell viability (±88%) and severe alterations in cell morphology were observed for all groups compared to NC, with no differences among them (p>0.05). Therefore, it was concluded that application of SU following the ER protocol had better adhesive performance. However, this adhesive system featured intense transdentinal cytotoxicity to pulp cells, regardless of application protocol and dentin wettability.

Several in vitro studies evaluated the cellular and molecular events related to interactions between phototherapy and target tissues, including oral keratinocytes and fibroblasts, providing elucidative data about phototherapy-induced healing. However, these interactions were limited to the application of a bidimensional cell culture model of oral mucosal cells. Thus, thisstudy evaluated the use of an organotypic oral epithelium model to elucidate the morphological and phenotypic responses of cells subjected to low-level laser therapy (LLLT). Oral keratinocytes were seeded in the ex vivo-produced oral mucosal equivalent (EVPOME) model, with a porcine acellular dermal matrix. LLLT was applied by means of the LaserTABLE device (780 nm, 25 mW) at 0.5, 1.5 and 3 J cm -2 . After three irradiations, morphology, proliferation and gene expression of growth factors were assessed. LLLT and control groups presented similar morphological features, characterized by the formation of a stratified, differentiated and keratinized epithelium. LLLT enhanced the cell proliferation and gene expression of keratinocytes (hKGF) as well as epidermal (hEGF) growth factors. In general, analysis of these data shows that the three-dimensional cell culture model can be applied for phototherapy studies and that the positive effects of LLLT were confirmed by the use of an organotypic model., (© 2017 The American Society of Photobiology.)

The aim of this study was to evaluate the bioactivity and cytocompatibility of simvastatin (SV) applied to MDPC-23 odontoblast-like cells. For this purpose, MDPC-23 cells were seeded in 96-well plates and submitted to treatments with 0.01 or 0.1 μM of SV for 24 h, 72 h or continuously throughout the experimental protocol. The negative control group (NC) was maintained in DMEM. Cell viability (MTT), ALP activity (thymolphthalein monophosphate), and mineralized matrix deposition (alizarin red) were analyzed at several time points. The data were submitted to ANOVA and Tukey's test (α = 0.05). Although cell viability was observed in the groups treated with SV, these groups did not differ from the NC up to 7 days. There was a reduction in cell viability for the groups treated with 0.1 μM of SV for 72 h, and submitted to continuous mode after 14 days. A significant increase in ALP activity occurred in the group treated with 0.01 μM of SV for 24 h, compared with the NC; however, only the group treated with 0.1 μM of SV in continuous mode reduced the ALP activity, in comparison with the NC. After 14 days, only continuous treatment with 0.1 μM of SV did not differ from NC, whereas the other experimental groups showed increased mineralized matrix deposition. Thus, it was concluded that low concentrations of simvastatin were bioactive and cytocompatible when applied for short periods to cultured MDPC-23 odontoblast-like cells.

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.

Objectives: This paper aims to assess the whitening effectiveness and toxicity of tooth-bleaching protocols applied to enamel/dentin disks simulating mandibular incisors (ICs) and premolars (PMs)., Materials and Methods: A 10% hydrogen peroxide (H 2 O 2 ) gel was applied for 3 × 15, 1 × 15, or 1 × 5 min to enamel/dentin disks simulating mandibular ICs and PMs, and the trans-enamel and trans-dentinal diffusion products were applied to human dental pulp cells (1 h). Professional therapy (35% H 2 O 2 -3 × 15 min) was used as positive control, and non-bleached samples were used as negative control. Cell viability and morphology, oxidative stress generation, and odontoblastic marker expression were assessed. The H 2 O 2 diffusion and enamel color change (ΔE) were also analyzed., Results: The 10% H 2 O 2 gel induced significant cell viability reduction only when applied 3 × 15 min, with the intensity of oxidative stress and down-regulation of odontoblastic markers being higher in the IC group. The other experimental bleaching protocols caused slight alterations regarding the cell parameters evaluated, with intensity being related to enamel/dentin thickness. These effects were also correlated with higher H 2 O 2 diffusion in the IC group. ΔE values similar as positive control were found for the 10% 3 × 15 and 1 × 15 protocols on IC group, after 4 and 6 sessions., Conclusion: Application of a 10% H 2 O 2 bleaching gel for 15 or 45 min to thin dental substrate significantly minimizes cell toxicity in comparison with highly concentrated gels associated with similar esthetic outcomes by increasing the number of bleaching sessions., Clinical Relevance: Bleaching gels with 10% H 2 O 2 applied in small teeth for short periods may be an interesting alternative to obtain whitening effectiveness without causing toxicity to pulp cells, which may be able to reduce the tooth hypersensitivity claimed by patients.

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/cm 2 ) 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.

Background and Objectives: Increased expression of inflammatory cytokines in the oral cavity has been related to the etiopathogenesis of oral mucositis and to delayed oral mucosal repair. Low-level laser therapy (LLLT) stimulates proliferation and migration of gingival fibroblasts, but the effects of specific inflammatory cytokines on oral mucosal cells and the modulation of these effects by LLLT have not been fully investigated. Therefore, this study investigated the effects of LLLT on oral fibroblasts after being challenged by oral-mucositis-related inflammatory cytokines., Methods: Human gingival fibroblasts were seeded in plain culture medium (DMEM) containing 10% fetal bovine serum (FBS) for 24 hours. Then, cells were kept in contact with inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-8) in serum-free DMEM for 24 hours. After this period, cells were subjected to LLLT with a diode laser device (LaserTABLE, InGaAsP, 780 nm, 25 mW) delivering energy doses from 0.5 to 3 J/cm 2 . Irradiation was repeated for 3 consecutive days. Twenty-four hours after the last irradiation, cell migration (wound-healing and transwell migration assays), cell proliferation (BrdU), gene expression of COL-I and growth factors (real-time PCR), and synthesis of COL-I (Sirius Red assay) and VEGF (ELISA) were assessed. Data were subjected to two-way ANOVA and Tukey's tests or Kruskall-Walis and Mann-Whitney tests (P < 0.05)., Results: The inflammatory cytokines decreased the migration capacity of gingival fibroblasts. However, a statistically significant difference was observed only for IL-6, detected by transwell assay, where 30% less cells migrated through the pores (P < 0.05) and IL-8, with an increased wound area (116%; P < 0.05), detected by the wound healing method. Cell proliferation was not affected by contact with cytokines, while growth factors and COL-I expression (approximately 80%; P < 0.05), as well as VEGF synthesis (approximately 20%; P < 0.05), were decreased after contact to all tested cytokines. The opposite was seen for total collagen synthesis. LLLT promoted an acceleration of fibroblast migration (30%; P < 0.05) and proliferation (112%; P < 0.05) when delivering 0.5 J/cm 2 to the cells previously in contact with the inflammatory cytokines. Gene expression of VEGF (approximately 30%; P < 0.05), and EGF (17%; P < 0.05), was stimulated by LLLT after contact with TNF-α and IL-6., Conclusion: LLLT can counteract the negative effects of high concentrations of inflammatory cytokines, especially IL-6 and IL-8 on gingival fibroblast functions directly related to the wound-healing process. Lasers Surg. Med. 48:1006-1014, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)