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.

Synergistic potential of 1α,25-dihydroxyvitamin D3 and calcium-aluminate-chitosan scaffolds with dental pulp cells.

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.

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.

Characterization of novel calcium hydroxide-mediated highly porous chitosan-calcium scaffolds for potential application in dentin tissue engineering.

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 Ca2+ 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.

Yttria-stabilized tetragonal zirconia polycrystal/resin luting agent bond strength: Influence of Titanium dioxide nanotubes addition in both materials.

PURPOSE: To evaluate the shear bond strength (SBS) between Y-TZP and a resin luting agent, after 1 of 2 enhancing strategies with TiO2--nts was applied, either to the resin luting agent or the Y-TZP mass, in different concentrations. METHODS: In the Strategy TiO2-nts on ceramic, the resin luting agent Panavia F2.0™ (Kuraray) and an experimental Y-TZP with added concentrations of TiO2--nts (0%, 1%, 2%, and 5% vol/vol) and a commercial Y-TZP, comprised 5 different groups (n = 10). In the Strategy TiO2-nts on cement, the resin luting agent RelyX U200™ (3 M ESPE) was added with different concentrations of TiO2--nts (0%, 0.3%, 0.6%, 0.9% wt/wt) luted to a commercial Y-TZP, comprising 4 different groups (n = 10). The Y-TZP discs were included in acrylic bases, and a cylinder (3 × 3 mm) of the correspondent luting agent for each respective group was applied over them. After 24 h, specimens were subjected to SBS assessments in a universal testing machine. Field emission scanning electron microscopy and energy dispersive X-ray spectroscopy analyses were also performed on Y-TZP surfaces. Data were analyzed via analysis of variance and Tukey tests (α = 0.05). RESULTS: TiO2-nts on ceramic influenced the bond strength significantly, but not linearly; TiO2-nts on cement did not influence bond strength when analyzed separately, nor in comparison with the first. CONCLUSION: Y-TZP enhancements with TiO2-nts led to a higher SBS with Panavia F2.0, a 5% TiO2--nt concentration presented the highest bond strength. Modified Rely X U200 did not improve SBS.

The effects of TiO2 nanotubes on bond strength and radiopacity of a self-adhesive resin cement in self-curing mode / Os efeitos de nanotubos de TiO2 na resistência de união e radiopacidade de um cimento resinoso auto-adesivo no modo auto-polimerizável

Objective: The aim of this in vitro study w as t o analyze the influence of the titanium dioxide nanotubes i n a self-cure mode polymerization of a dual resin luting agent through push out bond strength and radiopacity tests. Material and Methods: After mixed with a commercial du al self-adhesive resin cement, three concentrations o f tit anium dioxide nanotubes (0.3, 0.6, and 0.9% by weight) we re analyzed in self-curing mode. T he bond strength to bovine root dentin and fi berglass posts was assessed with the push out bond str ength t est and was evaluated in three thirds (cervical, middle and apical) (n=10), followed by failure mode analysis (SEM), and the ISO standard 9917-2 was followed for radiopacity test (n=10). Data were statistically analyzed by one-way ANOVA test, followed by Tukey's test (α=0.05). Results: Reinforced self-adhesive resin cement with 0.6% titanium dioxide nanotubes showed significant difference compared to the control gr oup for push out test (p=0.00158). The modified groups did not s how significant difference among thirds (p=0.782). Radiopacity sh owed higher value for group w ith 0.9% titanium dioxide nanotubes in comparison w ith control group (p<0.001). Conclusion: The addition of titanium dioxide nanotubes to a self-adhesive resin cement increased the bond strength to dentin and radiopacity values in the self-cure polymerization mode (AU)

Objetivo: O objetivo deste estudo in vitro foi analisar a influência de nanotubos de dióxido de titânio na polimerização química de um agente cimentante resinoso dual através de testes de resistência à união e radiopacidade. Material e Métodos: Após misturado com um cimento resinoso auto-adesivo comercial, três concentrações de nanotubos de dióxido de titânio (0,3, 0,6 e 0,9% em peso) foram analisadas. A resistência da união para a dentina da raiz bovina e os pinos de fibra de vidro foi avaliada pelo teste de push-out e avaliada em três terços (cervical, médio e apical) (n = 10), seguido pelo análise de modo de falha (MEV) e a norma ISO 9917-2 foi seguido para teste de radiopacidade (n = 10). Os dados foram analisados estatisticamente pelo teste ANOVA um fator seguido do teste de Tukey (α = 0,05). Resultados: O cimento resinoso auto-adesivo reforçado com nanotubos de dióxido de titânio a 0,6% mostrou diferença significativa em comparação com o grupo controle para teste de push-out (p=0,00158). Os grupos modificados não mostraram diferença significativa entre os terços (p=0,782). A radiopacidade mostrou maior valor para o grupo com nanotubos de dióxido de titânio 0,9% em comparação com o grupo controle (p<0,001). Conclusão: A adição de nanotubos de dióxido de titânio a um cimento resinoso auto-adesivo aumentou a os valores de resistência de união à dentina e radiopacidade no modo de polimerização química do agente cimentante (AU)

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.