Bioactivity effects of extracellular matrix proteins on apical papilla cells

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

Increased whitening efficacy and reduced cytotoxicity are achieved by the chemical activation of a highly concentrated hydrogen peroxide bleaching gel

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

Atividade anti fúngica do α-terpinen sobre Candida albicans

Objetivo: avaliar a atividade antifúngica do α- terpinen sobre culturas planctonicas e biofi lme de Candida albicans. Material e Métodos: Primeiramente, foi determinada a Concentração Inibitória Mínima (CIM) e a Concentração Fungicida Mínima (CFM) do α-terpinen sobre microrganismos planctônicos. A Nistatina foi utilizada como controle positivo. Biofi lme de Candida albicans foi desenvolvido e, após o tratamento com diferentes concentrações de α-terpinen, foi quantifi cado em UFC/mL, além da atividade metabólica das células ser avaliada por XTT. Resultados: a menor concentração capaz de inibir o crescimento (CIM) foi 0,2 % para o α-terpinen e 4 μg/mL para a Nistatina. Na CIM, os resultados mostraram que a partir da concentração 0,05 % de α-terpinen e 2 μg/mL de Nistatina houve diminuição de C.albicans quando comparado ao controle. A CFM foi para α-terpinen 0,2 % e Nistatina 8 μg/mL. Na quantifi cação as concentrações efi cazes foram de α-terpinen (0,1%) e Nistatina (128μg/mL), e no teste do XTT, observou-se que α –terpinen (0,1%) e Nistatina (256μg/mL) diminuem a viabilidade quando comparado com o controle. Conclusão: Assim, pode-se afi rmar que α-terpineol pode ser uma alternativa para tratamento de infecções fúngicas

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.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/155906/1/jbmb34586.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/155906/2/jbmb34586_am.pd

Avaliação dos fatores de virulência, atividade antimicrobiana e viabilidade celular de bactéria cariogênica na presença do Terpinen-4-ol: estudo in vitro

A utilização de óleos essenciais tem sido amplamente difundida no tratamento de doenças infecciosas que acometem a cavidade oral por serem eficazes contra patógenos presentes em biofilme. Dentre estes produtos de origem natural, destaca-se o Terpinen-4-ol um biocida membrana-ativo, com amplo espectro de ação contra bactérias gram-positivas, gram-negativas e bactérias multi-resistentes. Neste estudo, investigou-se o efeito antimicrobiano do Terpinen-4-ol sobre culturas planctônicas e biofilmes de Streptococcus mutans (S. mutans) e a expressão gênica de glucano de ligação de proteína A (gbpA) envolvido na adesão em biofilme. A atividade antimicrobiana do Terpinen-4-ol (0.059 % a 0.95 %) foi avaliada pelo teste de microdiluição em caldo com a determinação da Concentração Inibitória Mínima (CIM) e Concentração Bactericida Mínima (CBM) para o microrganismo na forma planctônica. O biofilme formado em placa de cultura celular foi tratado com diferentes concentrações de Terpinen-4-ol e o metabolismo celular do biofilme resultante foi avaliado por meio de ensaio de hidróxido de 2,3-bis (2-metoxi-4-nitro-5-sulfo-fenil) -2H-tetrazólio-5-caboxanilide (XTT). A análise do biofilme formado sobre blocos de esmalte e dentina tratados com Terpinen-4-ol (0.24 % e 0.95 %) e Clorexidina (CHX 0.12 %) durante 60 segundos foi realizada por meio de ensaio de XTT e razão espectral (verde/vermelho) das imagens obtidas por Microscopia Confocal de Varredura a Laser (MCVL). A expressão gênica de gbpA foi investigada por RT-PCR quantitativo após exposição de S. mutans ao Terpinen-4-ol e CHX por 15 e 30 minutos. Os dados obtidos apresentaram distribuição normal e, portanto, foram utilizados os testes paramétricos de ANOVA One-way e de Tukey. Todos os testes estatísticos foram considerados em nível de significância de 5% (p<0.05). Terpinen-4-ol demonstrou atividade antimicrobiana a partir da concentração de 0.24 % tanto para o microrganismo em cultura planctônica quanto para biofilme formado em placa de cultura celular e esmalte, com redução microbiana próxima a 100 %. Não houve diferença estatisticamente significante entre Terpinen-4-ol e CHX. Para biofilme em dentina, a concentração de Terpinen-4-ol 0.95 % apresentou atividade antimicrobiana tanto pela análise por XTT quanto por MCVL. Houve redução na expressão de gbpA quando em contato com Terpinen-4-ol durante 15 minutos, no entanto a superexpressão gênica ocorreu a partir dos 30 minutos de exposição. CHX levou a superexpressão do gene gbpA em todos os tempos testados. Estes achados demonstram que Terpinen-4-ol apresenta importante atividade antimicrobiana e capacidade de modulação da expressão de genes envolvidos na formação de biofilme cariogênico, podendo ser utilizado no tratamento de doenças infecciosas bucais.The use of essential oils has been widespread in the treatment of infectious diseases that affect the oral cavity to be effective against pathogens in biofilms. Among these natural products, there is the Terpinen-4-ol a biocide membrane-active, with broad spectrum of action against gram-positive bacteria, gram-negative and multi-resistant bacteria. In this study, we investigated the antimicrobial effect of Terpinen-4-ol on planktonic and biofilm cultures of Streptococcus mutans (S. mutans) and gene expression gbpA (glucan binding protein A) involved in the adhesion of biofilm. The antimicrobial activity of Terpinen-4-ol (0.059 % to 0.95 %) was assessed by the broth microdilution test for the determination of MIC and MBC for the microorganism in planktonic form. The biofilm formed in cell culture plate were treated with different concentrations of Terpinen-4-ol and cell metabolism resulting plaque was assessed using the hidróxido de 2,3-bis (2-metoxi-4-nitro-5-sulfo-fenil) -2H-tetrazólio-5-caboxanilide (XTT). The analysis of the biofilm formed on enamel blocks and dentin treated with Terpinen-4-ol (0.24 % and 0.95 %) and chlorhexidine (CHX 0.12 %) for 60 seconds was performed by XTT assay and spectral Ratio (Green/Red) of images obtained by microscopy Laser Scanning Confocal (CLSM). The gbpA gene expression was investigated by quantitative RT-PCR after S. mutans have been exposed to Terpinen-4-ol and CHX for 15 and 30 minutes. The data were normally distributed and therefore parametric tests of ANOVA One-way and Tukey were used. All statistical tests were considered at a significance level of 5 % (p<0.05). Terpinen-4-ol showed antimicrobial activity at concentrations 0.24% for both planktonic microorganisms in culture and to biofilm formation in cell culture plate and enamel with microbial reduction near 100 %. There was no statistically significant difference between Terpinen-4-ol and CHX. For biofilm on dentin, the concentration of Terpinen-4-ol 0.95 % showed antimicrobial activity both by analysis by XTT and by CLSM. There was a reduction in gbpA expression when in contact with Terpinen-4-ol for 15 minutes, however the gene overexpression occurred after 30 minutes of exposure. CHX caused overexpression of the gbpA gene in all tested times. These findings demonstrate that Terpinen-4-ol shows a significant antimicrobial activity and ability to modulate the expression of genes involved in cariogenic biofilm formation, can be used in treating oral infectious diseases.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Antimicrobial therapeutics in regenerative endodontics : a scoping review

Introduction: This review aimed to provide a critical appraisal of alternative antimicrobial strategies in lieu of traditional triple antibiotic paste (TAP). Methods: This review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. The literature search was performed in 8 databases (PubMed/Medline, Embase, LILACS, Web of Science, Scopus, BVS, SciELO, and the Cochrane Library), selecting clinical, in vitro, in vivo, and in situ studies that evaluated antimicrobial alternatives to TAP in regenerative endodontics. Studies lacking an experimental TAP group were excluded. Results: A total of 1705 potentially relevant records were initially identified. From the 38 studies retrieved for full- text reading, 16 fulfilled all selection criteria and were included in the qualitative analysis. According to the study design, 11 studies were solely in vitro, 1 study was both in vitro and in vivo (animal model), 2 studies were solely animal experiments, and 2 studies were clinical trials. The alternative antimicrobial agents to TAP consisted of modified TAP formulations (eg, a combination of TAP with chitosan); TAP-eluting nanofibers; propolis; chlorhexidine (CHX) gels/solutions; double antibiotic pastes composed of distinct combinations of antibiotics; Ca(OH)2-based formulations; and sodium hypochlorite. Overall, most of the alternative agents performed similarly to TAP, although some strategies (eg, Ca(OH)2- and CHX-based formu- lations) seemed to present dubious importance in the control of infection. Conclusions: TAP still remains an excellent option in terms of the complete elimination of microorganisms. This review points to the use of electrospun fibers as a drug delivery system to offer a controlled release of the antimicrobial agent, as well as the use of natural compounds, deserving future investigation