Resposta imune inata na estomatite protética: interação in vitro entre células epiteliais de palato humano e Candida albicans / Innate imune response in denture stomatitis: in vitro interaction between human palatal epithelial cells and Candida albicans

A presença de Candida albicans nos biofilmes microbianos da superfície interna das próteses totais superiores está relacionada com uma doença inflamatória no palato, a estomatite protética. Constituinte da defesa inata do hospedeiro, o epitélio bucal, por sua vez, tem a capacidade de reconhecer e reagir aos fatores fúngicos a fim de evitar a invasão pelo microrganismo. O objetivo deste trabalho foi avaliar in vitro o efeito direto e indireto de C. albicans viável sobre as células epiteliais de palato humano (CEPH) ao longo do tempo. Objetivamos correlacionar os eventos de agressão, apoptose e invasão das CEPH provocados pelo fungo, com as respostas de defesa epitelial mediante produção de óxido nítrico (NO) e expressão gênica do peptídeo antimicrobiano β-defensina 2 (hBD-2). Material e Métodos: As CEPH foram obtidas, parte pelo método explante e parte pelo método enzimático, e mantidas em co-cultivo sobre uma camada de sustentação feederlayer (fibroblastos gengivais humanos mitoticamente inativados). Após desafios das CEPH com C. albicans ATCC 90028 por contato direto fungo-epitélio (D.D.) e indireto pelo sobrenadante da cultura do fungo hifal (D.I.), proporções de desafio de 0,01/1; 0,025/1 e 0,1/1 levedura/queratinócito (FUN/EPI) e tempos experimentais de 3, 6 e 10 h foram determinados; via ensaios de viabilidade celular por imunofluorescência (LIVE/DEAD), e análise qualitativa da invasão celular pelo fungo por meio do método colorimétrico com laranja de acridina. A apoptose epitelial foi determinada pela marcação nuclear fluorescente com Hoechtst 33258. A produção de óxido nítrico (NO) e a expressão de RNAm de hBD-2 foram avaliados por reação colorimétrica de Griess e RT-qPCR, respectivamente. Os resultados foram expressos como média ± desvio padrão e submetidos aos testes estatísticos ANOVA Fatorial, Teste de Contraste; ou Teste de Mann-Whitney (p<0,05). Resultados: Em 3 h, foi detectado aumento da apoptose das células epiteliais em relação ao...

The presence of the fungus Candida albicans in the microbial biofilm underlying maxillary prosthesis is related to an inflammatory reaction of the palatal mucosa, the denture stomatitis. As a component of the host innate defense, the oral epithelium has the ability to recognize and react to fungal factors in order to prevent the microrganism invasion. The aim of this study was to in vitro evaluate the direct and indirect effect of viable C. albicans on the human palatal epithelial cells (HPEC) over time. The aggressive events, such as apoptosis and HPEC invasion by the fungus, were correlated with epithelial defense responses through the nitric oxide (NO) production and antimicrobial peptides β-defensin (hBD-2) mRNA expression. Methods: The HPEC were obtained by explant and enzymatic methods, and were maintained in co-culture on a feeder-layer support (mitotically inactivated human gingival fibroblasts). After the HPEC challenges with C. albicans ATCC 90028 by direct contact fungus-epithelium (D.D.) and indirect contact by supernatant from hyphal fungus (D.I.), defiance ratios of 0.01/1, 0.025/1 and 0.1/1 yeast/keratinocyte (FUN/EPI) and experimental times of 3, 6 and 10 h were determined. These conditions were standardized by cell viability immunofluorescence assay (LIVE/DEAD), and cell invasion qualitative analysis (colorimetric method with acridine orange). The apoptotic cells were determined by fluorescent nuclear staining with Hoechtst 33258. The nitric oxide (NO) production and hBD-2 gene expression were evaluated by Griess colorimetric reaction and RT-qPCR, respectively. The results were expressed as mean ± standard deviation and were analyzed using the factorial ANOVA, Contrast Test; or Mann-Whitney Test (p<0,05). Results: At 3 h, the apoptotic epithelial cells under 0.1/1 FUN/EPI increased compared to epithelium unchallenged (p<0,05) that remained over time with increasing concentration and independent of D.D. and D.I. The onset...

Manutenção da viabilidade das células mononucleares de sangue periférico humano em extratos e formulações de própolis / Maintenance of human peripheral blood mononuclear cell viability in propolis extracts and formulations

Neste estudo comparou- se a viabilidade das células mononucleares de sangue periférico (PBMCs) humano quando mantidas em diferentes extratos e formulações de própolis. As PBMCs (106 cels mL-1), provenientes de doadores saudáveis (n=5), foram estocadas a 20ºC nas diferentes soluções de própolis, assim como em solução salina balanceada de Hank's (HBSS), utilizada como controle do experimento. A viabilidade celular foi determinada pelo método de exclusão com azul de Tripan. Quando incubadas por 1h, apenas dois extratos, denominados A70D e D70D, apresentaram desempenho satisfatório na manutenção da viabilidade celular, semelhante (p > 0,05) ao controle HBSS e diferindo estatisticamente (p < 0,05) das demais formulações. A70D e D70D foram então testados em cinco diluições em propilenoglicol, ao longo de 24h, com análise nos tempos 0, 30 min., 1, 3, 6, 10 e 24h. As frações mais concentradas apresentaram pior desempenho (p < 0,05) em relação aos seus extratos originais que mantiveram viabilidade próxima a 80% ao longo de 24h. A redução da viabilidade proporcional ao aumento da concentração das frações foi observada. Os resultados sugerem que soluções de própolis, em concentrações adequadas, podem ser utilizadas nos estudos futuros sobre alternativas aos meios de conservação de dentes avulsionados rotineiramente utilizados na prática odontológica.

In this study a comparison was made of human mononuclear cell (PBMCs) viability, when cells were kept in different propolis extracts and formulations. PBMCs (106 cell mL-1), obtained from healthy donors (n=5), were incubated at 20ºC in the different propolis solutions, as well as in Hank's balanced salt solution (HBSS), used as experimental control. The cell viability was analyzed by Trypan blue exclusion assay. When incubated for 1h, only two extracts, denominated A70D and D70D, showed appropriate results for maintaining cell viability. A70D and D70D showed better viability (p < 0.05) than other formulations and no difference (p > 0.05) from the HBSS control. A70D and D70D were tested in five dilutions in propylene glycol, over 24h, with analysis at 0, 30 minutes, 1, 3, 6, 10 and 24h. The most concentrated fractions showed the worst performance (p < 0.05) in comparison with their original extracts, which remained close to 80% viability over 24h. Reduction in viability proportional to increase in concentration of formulations was observed. The results suggest that propolis solutions in appropriate concentrations may be used in future studies on alternatives to mediums routinely used in dental practice for storing avulsed teeth.

Removal efficiency of propolis paste dressing from the root canal

OBJETIVES: The aim of this study was to investigate through scanning electron microscopy (SEM) the cleaning of root canal walls after the use of experimental propolis or calcium hydroxide root canal dressings. MATERIAL AND METHODS: Twenty single-rooted teeth were used. After conventional cleaning and shaping procedures and removal of the smear layer with 17 percent EDTA, the teeth were divided into four groups according to the medication used (N=5): Group I (control) - No drug, Group II - Calcium hydroxide dressing, Group III - Propolis paste A70D and Group IV - Propolis paste D70D. The medications were introduced into the root canals and maintained for 7 days, then removed with a K-file and 5 mL of 1 percent sodium hypochlorite irrigation. Finally, the canals were flushed with 2 mL of 17 percent EDTA for 3 min. For SEM analysis, the roots were cleaved and microphotographs from the middle third of the root canal were taken at 750x. The cleaning of the root canal walls was determined by the number of open dentinal tubules as verified with the software Image Tool 3.1. The statistical analysis was performed by ANOVA and Tukey's test (p<0.05). RESULTS: The results showed no statistically significant difference between the calcium hydroxide and propolis groups. CONCLUSIONS: The experimental propolis pastes presented acceptable physical characteristics to be used as intracanal medicaments.