Composite-derived monomers affect cell viability and cytokine expression in human leukocytes stimulated with Porphyromonas gingivalis

Abstract Objectives: Dental composites release unreacted resin monomers into the oral environment, even after polymerization. Periodontal cells are, therefore, exposed to substances that potentially elicit the immune inflammatory response. The underlying molecular mechanisms associated with the interaction between resin monomers and human immune cells found in the gingival crevicular fluid are not fully understood yet. This study investigated the ability of bisphenol A-glycidyl methacrylate (BISGMA), urethane dimethacrylate (UDMA) and triethylene glycol dimethacrylate (TEGDMA) to induce apoptosis and cytokine release by human leukocytes stimulated with a periodontal pathogen. Methodology: Peripheral blood mononuclear cells (PBMC) from 16 healthy individuals were included in this study. To determine the toxicity, the PBMC were incubated for 20 hours, with monomers, for the analysis of cell viability using MTT assay. To evaluate cell death in the populations of monocytes and lymphocytes, they were exposed to sub-lethal doses of each monomer and of heat-inactivated Porphyromonas gingivalis (P. gingivalis) for 5 hours. Secretions of IL-1β, IL-6, IL-10 and TNF-α were determined by ELISA after 20 hours. Results: UDMA and TEGDMA induced apoptosis after a short-time exposure. Bacterial challenge induced significant production of IL-1β and TNF-α (p<0.05). TEGDMA reduced the bacterial-induced release of IL-1β and TNF-α, whereas UDMA reduced IL-1β release (p<0.05). These monomers did not affect IL-10 and IL-6 secretion. BISGMA did not significantly interfere in cytokine release. Conclusions: These results show that resin monomers are toxic to PBMC in a dose-dependent manner, and may influence the local immune inflammatory response and tissue damage mechanisms via regulation of bacterial-induced IL-1β and TNF-α secretion by PBMC.

Dental pulp fibroblasts response after stimulation with hema and adhesive system

Abstract This study evaluated in vitro cell viability and metabolism, nitric oxide release and production of chemokines by cultured human dental pulp fibroblasts (DPF) under contact with HEMA and Single Bond. Cultures of DPF were established by means of an explant technique. Once plated, cells were kept under contact with increasing concentrations of HEMA (10, 100 and 1000 nM) or Single Bond (SB) [10-fold serially diluted in culture medium (10-4, 10-3 and 10-2 v/v)] and also with polymerized SB components. Cytotoxicity was assessed by Trypan Blue exclusion method and MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Nitric oxide release on cell supernatant was detected by Griess Method whereas chemokines (CXCL12 and CXCL8) were detected by ELISA. RT-qPCR was employed for chemokines gene expression analysis. Cytotoxic tests showed significant differences for SB 10-2. None of the tested materials significantly altered NO levels. Protein levels of CXCL12 were significantly decreased only by HEMA. On the other hand, while CXCL12 mRNA remained unaltered, gene expression of CXCL8 had significant decrease with all materials, except for polymerized SB. In conclusion, Single Bond and HEMA at various concentrations, decreased expression and production of molecules involved in inflammatory processes and, therefore, the use of adhesive systems such as pulp capping materials must be viewed with caution due to its large cytotoxic effect when in close contact with the pulp.

Resumo Este estudo avaliou in vitro a viabilidade e metabolismo celular, liberação de óxido nítrico e produção de quimiocinas em cultura de fibroblastos de polpa dental humana (DPF) em contato com HEMA e Single Bond. Culturas de DPF foram estabelecidas por meio de uma técnica de explante. Uma vez plaqueadas, as células foram mantidas em contato com concentrações crescentes de HEMA (10, 100 e 1000 nM) ou Single Bond (SB) [10 vezes diluídas em série em meio de cultura (10-4, 10-3 e 10-2 v/v)] e também com SB polimerizado. A citotoxicidade foi avaliada pelo método de exclusão de Trypan Blue e pelo ensaio de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólio brometo (MTT). A liberação de óxido nítrico no sobrenadante celular foi detectada pelo método de Griess, enquanto as quimiocinas (CXCL12 e CXCL8) foram detectadas por ELISA. RT-qPCR foi empregada para análise de expressão gênica de quimiocinas. Testes citotóxicos mostraram diferenças significativas para SB 10-2. Nenhum dos materiais testados alterou significativamente os níveis de NO. Os níveis de proteína de CXCL12 foram significativamente diminuídos apenas pelo HEMA. Por outro lado, enquanto o RNAm de CXCL12 permaneceu inalterado, a expressão gênica de CXCL8 teve redução significativa com todos os materiais, com exceção do SB polimerizado. Em conclusão, Single Bond e HEMA, em várias concentrações, diminuíram a expressão e produção de moléculas envolvidas em processos inflamatórios e, portanto, o uso de sistemas adesivos, como o material protetor da polpa, deve ser visto com cautela devido ao seu grande efeito citotóxico quando em contato com a polpa.

Evaluation of physical-mechanical properties, antibacterial effect, and cytotoxicity of temporary restorative materials

Abstract The objective of this study was to compare selective physical-mechanical properties, antibacterial effects and cytotoxicity of seven temporary restorative materials (TRM): five resin-based materials [Bioplic (B), Fill Magic Tempo (FM), Fermit inlay (F), Luxatemp LC (L) and Revotek LC (R)], and zinc oxide-eugenol cement (IRM) and glass ionomer cement (GIC) as the controls. Material and methods The physical-mechanical properties were evaluated by determining microleakage (ML), ultimate tensile strength (UTS) and Shore D hardness (SDH). In addition, the polymerization rate (Pr-1), depth of cure (DC), water sorption and solubility (WS/SL) were evaluated. The antimicrobial effects of the materials were assessed by biofilm accumulation of Streptococcus mutans (BT) and the direct contact test (DCT) by exposure to Enterococcus faecalis for 1 and 24 h, and cytotoxicity by MTT assay. The data were analyzed by ANOVA or Kruskall-Wallis tests, and a complementary post-hoc method (p<0.05). Results Group B, followed by FM and GIC had significantly lower percentages of microleakage in comparison with the other groups; Groups FM and L showed the highest WS, while Groups R and FM showed the significantly lowest SL values (p<0.05). Group R showed the statistically highest UTS mean and the lowest DC mean among all groups. Group F showed the lowest S. mutans biofilm accumulation (p=0.023). Only the Group L showed continued effect against E. faecalis after 1 h and 24 h in DCT. The L showed statistically lower viability cell when compared to the other groups. Conclusions These findings suggest the antibacterial effect of the temporary materials Fill Magic and Bioplic against S. mutans, while Luxatemp showed in vitro inhibition of S. mutans biofilm accumulation and E. faecalis growth. Regarding the cell viability test, Luxatemp was the most cytotoxic and Fill Magic was shown to be the least cytotoxic.

Model resin composites incorporating ZnO-NP: activity against S. mutans and physicochemical properties characterization

Abstract Although resin composites are widely used in the clinical practice, the development of recurrent caries at composite-tooth interface still remains as one of the principal shortcomings to be overcome in this field. Objectives To evaluate the activity against S. mutans biofilm of model resin composites incorporating different concentrations of ZnO-nanoparticles (ZnO-NP) and characterize their physicochemical properties. Materials and Methods Different concentrations of ZnO-NP (wt.%): E1=0, E2=0.5, E3=1, E4=2, E5=5 and E6=10 were incorporated into a model resin composite consisting of Bis-GMA-TEGDMA and barium borosilicate particles. The activity against S. mutans biofilm was evaluated by metabolic activity and lactic acid production. The following physicochemical properties were characterized: degree of conversion (DC%), flexural strength (FS), elastic modulus (EM), hardness (KHN), water sorption (Wsp), water solubility (Wsl) and translucency (TP). Results E3, E4, E5 and E6 decreased the biofilm metabolic activity and E5 and E6 decreased the lactic acid production (p<0.05). E6 presented the lowest DC% (p<0.05). No significant difference in FS and EM was found for all resin composites (p>0.05). E5 and E6 presented the lowest values of KHN (p<0.05). E6 presented a higher Wsp than E1 (p<0.05) and the highest Wsl (p<0.05). The translucency significantly decreased as the ZnO- NP concentration increased (p<0.05). Conclusions The incorporation of 2 - 5 wt.% of ZnO-NP could endow antibacterial activity to resin composites, without jeopardizing their physicochemical properties.

Influence of addition of [2-(methacryloyloxy)ethyl]trimethylammonium chloride to an experimental adhesive

Abstract The aim of this study was to develop an experimental adhesive with addition of [2-(methacryloyloxy)ethyl]trimethylammonium chloride (METAC) and to evaluate its mechanical and biological properties and its in vitro antibacterial activity. An experimental adhesive resin was formulated with Bis-GMA, TEGDMA, and HEMA. The antibacterial monomer was added at concentrations of 1%, 2.5%, and 5% (METAC groups). A group without METAC addition was used as control. The experimental adhesives were evaluated as to their antibacterial potential against Streptococcus mutans, degree of conversion, and softening in ethanol for 2 hours. The data were analyzed by one-way ANOVA, Tukey’s post-hoc test, and the paired Student’s t-test (significance level of 0.05). METAC showed antibacterial activity against S. mutans at all concentrations (p < 0.05). There was no statistical difference across METAC groups (p > 0.05). The 1%, 2.5%, and 5% groups yielded the highest mean values for degree of conversion (p < 0.05). The 1% group did not differ from the control group (p > 0.05). There was no statistical difference in baseline microhardness values (p > 0.05) and microhardness values after immersion in ethanol were lower than at baseline for all groups (p < 0.05). There was no statistical difference in the reduction of Knoop hardness number (KHN) after immersion in ethanol for any of the groups (p > 0.05). The results of the present study indicate that METAC is a promising antibacterial agent when added to an adhesive system.

Catechin-incorporated dental copolymers inhibit growth of Streptococcus mutans

Objective: To test the inhibitory growth activity of green tea catechin incorporated into dental resins compared to resins containing the broad-spectrum antimicrobial compound chlorhexidine against Streptococcus mutans in vitro. Material and Methods: The minimum inhibitory concentrations (MICs) of epigallocatechin-gallate (EGCg) and chlorhexidine (CHX) were determined according to the microdilution method. Resin discs (5 mm × 3 mm) were prepared from Bis-GMA/TEGDMA (R1) and Bis-GMA/CH3Bis-GMA (R2) comonomers (n=9) containing: a) no drug, b) EGCg, c) CHX. Two concentrations of each drug (0.5× MIC and 1× MIC) were incorporated into the resin discs. Samples were individually immersed in a bacterial culture and incubated for 24 h at 37°C under constant agitation. Cell viability was assessed by counting the number of colonies on replica agar plates. Statistical analysis was performed using one-way ANOVA, Tukey and Student t-tests (α=0.05). Results: Both resins containing EGCg and CHX showed a significant inhibition of bacterial growth at both concentrations tested (p<0.05). A significantly higher inhibition was observed in response to resins containing CHX at 0.5× MIC and 1× MIC, and EGCg at 1× MIC when compared to EGCg at 0.5× MIC. Also, EGCg at 0.5× MIC in R1 had a significantly higher growth inhibition than in R2. Conclusions: Both EGCg and CHX retained their antibacterial activity when incorporated into the resin matrix. EGCg at 1× MIC in R1 and R2 resins significantly reduced S. mutans survival at a level similar to CHX. The data generated from this study will provide advances in the field of bioactive dental materials with the potential of improving the lifespan of resin-based restorations.

Pulp capping materials exert an effect on the secretion of IL-1β and IL-8 by migrating human neutrophils

Pulp repair is a complex process whose mechanisms are not yet fully understood. The first immune cells to reach the damaged pulp are neutrophils that play an important role in releasing cytokines and in phagocytosis. The objective of this study was to analyze the effect of different pulp-capping materials on the secretion of interleukin-1 beta (IL-1β) and interleukin-8 (IL-8) by migrating human neutrophils. Neutrophils were obtained from the blood of three healthy donors. The experimental groups were calcium hydroxide [Ca(OH)2], an adhesive system (Single Bond), and mineral trioxide aggregate (MTA). Untreated cells were used as control. Transwell chambers were used in performing the assays to mimic an in vivo situation of neutrophil chemotaxis. The pulp-capping materials were placed in the lower chamber and the human neutrophils, in the upper chamber. The cells were counted and the culture medium was assayed using ELISA kits for detecting and quantifying IL-1β and IL8. The data were compared by ANOVA followed by Tukey's test (p < 0.05). The secretion of IL-8 was significantly higher in all groups in comparison to the control group (p < 0.05). The adhesive system group showed higher IL-8 than the MTA group (p < 0.05). The secretion of IL-1β was significantly greater only in the MTA group (p < 0.001). It was concluded that only MTA is able to improve the secretion of IL-1β, and all materials tested increased IL-8 secretion. These results combined with all the other biological advantages of MTA indicate that it could be considered the material of choice for dental pulp capping.

Effects of a glass-ionomer cement on the remineralization of occlusal caries: an in situ study

Esta pesquisa avaliou a remineralização do esmalte de fóssulas e fissuras de terceiros molares humanos previamente desmineralizados e selados com um cimento de ionômero de vidro (Fuji IX, GC Corporation - Japão) ou com um selante de Bis-GMA (Delton-Dentsply). Dez voluntários participaram deste estudo in situ que consistiu de dois períodos de 30 dias com intervalo de 1 semana entre eles, usando dispositivos intra-orais. Quatro procedimentos de tratamento experimental e um controle foram aleatoriamente distribuídos entre os espécimes que foram usados pelos voluntários: Grupo I, sem qualquer tratamento, controle; Grupo II, processo de cárie artificial; Grupo III, igual ao Grupo II, porém selados com Delton (Dentsply); Grupo IV, igual ao Grupo II, porém selados com Fuji IX (GC Corporation - Japão); Grupo V, igual ao Grupo II, mas sem selamento. Os Grupos I e II não foram submetidos ao meio bucal e serviram de controle. Após um período de trinta dias no meio bucal, os espécimes foram removidos dos dispositivos, montados em resina acrílica, planificados e polidos. Realizaram-se então testes de Dureza Knoop com carga estática de 25 g por 15 segundos. As medidas foram realizadas desde a base da fissura até uma abertura de 600 µm, preestabelecida entre os planos inclinados das cúspides. A cada 100 µm, três penetrações foram realizadas: a primeira a 25 µm da superfície da fissura, a segunda a 75 µm, e a terceira a 125 µm (Micromet 2003). Os dados obtidos foram analisados estatisticamente pelos testes "F" de Brieger e de Bonferroni. Concluiu-se que o selamento com o cimento de ionômero de vidro Fuji IX foi capaz de tornar o esmalte de sulcos e fissuras mais resistente pelo aumento do valor de dureza Knoop.