Root canal contamination or exposure to lipopolysaccharide differentially modulate prostaglandin E 2 and leukotriene B 4 signaling in apical periodontitis

Abstract Purpose To evaluate the kinetics of apical periodontitis development in vivo , induced either by contamination of the root canals by microorganisms from the oral cavity or by inoculation of bacterial lipopolysaccharide (LPS) and the regulation of major enzymes and receptors involved in the arachidonic acid metabolism. Methodology Apical periodontitis was induced in C57BL6 mice (n=96), by root canal exposure to oral cavity (n=48 teeth) or inoculation of LPS (10 µL of a suspension of 0.1 µg/µL) from E. coli into the root canals (n= 48 teeth). Healthy teeth were used as control (n=48 teeth). After 7, 14, 21 and 28 days the animals were euthanized and tissues removed for histopathological and qRT-PCR analyses. Histological analysis data were analyzed using two-way ANOVA followed by Sidak's test, and qRT-PCR data using two-way ANOVA followed by Tukey's test (α=0.05). Results Contamination by microorganisms led to the development of apical periodontitis, characterized by the recruitment of inflammatory cells and bone tissue resorption, whereas inoculation of LPS induced inflammatory cells recruitment without bone resorption. Both stimuli induced mRNA expression for cyclooxygenase-2 and 5-lipoxygenase enzymes. Expression of prostaglandin E 2 and leukotriene B 4 cell surface receptors were more stimulated by LPS. Regarding nuclear peroxisome proliferator-activated receptors (PPAR), oral contamination induced the synthesis of mRNA for PPARδ, differently from inoculation of LPS, that induced PPARα and PPARγ expression. Conclusions Contamination of the root canals by microorganisms from oral cavity induced the development of apical periodontitis differently than by inoculation with LPS, characterized by less bone loss than the first model. Regardless of the model used, it was found a local increase in the synthesis of mRNA for the enzymes 5-lipoxygenase and cyclooxygenase-2 of the arachidonic acid metabolism, as well as in the surface and nuclear receptors for the lipid mediators prostaglandin E2 and leukotriene B4.

Comparison of rRNA-based reverse transcription PCR and rDNA-based PCR for the detection of streptococci in root canal infections

Abstract Objective The rDNA-based method is unable to distinguish between alive and dead cells. Alternatively, bacterial viability can be assessed by molecular methods based on ribosomal RNA (rRNA). Therefore, this study aimed to detect viable streptococci in root canal samples using rRNA-based reverse transcription polymerase chain reaction (RT-PCR), compared to an rDNA-based PCR assay. Methodology Microbiological root canal samples were obtained from 32 teeth with primary endodontic infections before (S1) and after chemomechanical preparation (S2), and after removal of intracanal medication (S3). RNA and DNA were extracted, and complementary DNA (cDNA) was synthesized from RNA using RT reaction. cDNA and genomic DNA were subjected to PCR with primers complementary to the 16S rRNA sequences of Streptococcus spp. McNemar's test was used to compare the detection rate of both assays (P<0.05). Results Streptococci were detected in 28.12% (9/32) and 37.5% (12/32) of S1 samples using rRNA- and rDNA-based PCR assays, respectively. In contrast, they were detected in only 6.25% (2/32) of S2 samples using rRNA-based RT-PCR, compared to 15.62% (5/32) using rDNA-based PCR. Finally, in S3 samples, streptococci were not detected by rRNA, whereas rDNA-based PCR still detected the bacteria in 12.5% (4/32) of the samples. The total number of PCR-positive reactions in the rDNA-based PCR was higher than in the rRNA-based assay (P<0.05). Conclusions The rRNA-based RT-PCR showed a lower detection rate of streptococci when compared to the rDNA-based PCR, suggesting that the latter may have detected dead cells of streptococci in root canal samples.

Periapical bone response to bacterial lipopolysaccharide is shifted upon cyclooxygenase blockage

Abstract Objectives: Infection, inflammation and bone resorption are closely related events in apical periodontitis development. Therefore, we sought to investigate the role of cyclooxygenase (COX) in osteoclastogenesis and bone metabolism signaling in periapical bone tissue after bacterial lipopolysaccharide (LPS) inoculation into root canals. Methodology: Seventy two C57BL/6 mice had the root canals of the first molars inoculated with a solution containing LPS from E. coli (1.0 mg/mL) and received selective (celecoxib) or non-selective (indomethacin) COX-2 inhibitor. After 7, 14, 21 and 28 days the animals were euthanized and the tissues removed for total RNA extraction. Evaluation of gene expression was performed by qRT-PCR. Statistical analysis was performed using analysis of variance (ANOVA) followed by post-tests (α=0.05). Results: LPS induced expression of mRNA for COX-2 (Ptgs2) and PGE2 receptors (Ptger1, Ptger3 and Ptger4), indicating that cyclooxygenase is involved in periapical response to LPS. A signaling that favours bone resorption was observed because Tnfsf11 (RANKL), Vegfa, Ctsk, Mmp9, Cd36, Icam, Vcam1, Nfkb1 and Sox9 were upregulated in response to LPS. Indomethacin and celecoxib differentially modulated expression of osteoclastogenic and other bone metabolism genes: celecoxib downregulated Igf1r, Ctsk, Mmp9, Cd36, Icam1, Nfkb1, Smad3, Sox9, Csf3, Vcam1 and Itga3 whereas indomethacin inhibited Tgfbr1, Igf1r, Ctsk, Mmp9, Sox9, Cd36 and Icam1. Conclusions: We demonstrated that gene expression for COX-2 and PGE2 receptors was upregulated after LPS inoculation into the root canals. Additionally, early administration of indomethacin and celecoxib (NSAIDs) inhibited osteoclastogenic signaling. The relevance of the cyclooxygenase pathway in apical periodontitis was shown by a wide modulation in the expression of genes involved in both bone catabolism and anabolism.

Gliclazide reduced oxidative stress, inflammation, and bone loss in an experimental periodontal disease model

Abstract Objective The aim of this study was to evaluate the effects of gliclazide on oxidative stress, inflammation, and bone loss in an experimental periodontal disease model. Material and Methods Male albino Wistar rats were divided into no ligature, ligature, and ligature with 1, 5, and 10 mg/kg gliclazide groups. Maxillae were fixed and scanned using micro-computed tomography to quantify linear and bone volume/tissue volume (BV/TV) and volumetric bone loss. Histopathological, immunohistochemical and immunofluorescence analyses were conducted to examine matrix metalloproteinase-2 (MMP-2), cyclooxygenase 2 (COX-2), cathepsin K, members of the receptor activator of the nuclear factor kappa-Β ligand (RANKL), receptor activator of nuclear factor kappa-Β (RANK), osteoprotegerin (OPG) pathway, macrophage migration inhibitory factor (MIF), superoxide dismutase-1 (SOD-1), glutathione peroxidase-1 (GPx-1), NFKB p 50 (Cytoplasm), NFKB p50 NLS (nuclear localization signal), PI3 kinase and AKT staining. Myeloperoxidase activity, malondialdehyde and glutathione levels, while interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) levels were evaluated by spectroscopic ultraviolet-visible analysis. A quantitative reverse transcription polymerase chain reaction was used to quantify the gene expression of the nuclear factor kappa B p50 subunit (NF-κB p50), phosphoinositide 3-kinase (PI3k), protein kinase B (AKT), and F4/80. Results Micro-computed tomography showed that the 1 mg/kg gliclazide treatment reduced linear bone loss compared to the ligature, 5 mg/kg gliclazide, and 10 mg/kg gliclazide treatments. All concentrations of gliclazide increased bone volume/tissue volume (BV/TV) compared to the ligature group. Treatment with 1 mg/kg gliclazide reduced myeloperoxidase activity, malondialdehyde, IL-1β, and TNF-α levels (p≤0.05), and resulted in weak staining for COX-2, cathepsin k, MMP-2, RANK, RANKL, SOD-1, GPx-1,MIF and PI3k. In addition, down-regulation of NF-κB p50, PI3k, AKT, and F4/80 were observed, and OPG staining was strong after the 1 mg/kg gliclazide treatment. Conclusions This treatment decreased neutrophil and macrophage migration, decreased the inflammatory response, and decreased bone loss in rats with ligature-induced periodontitis.

Effects of mineral trioxide aggregate, BiodentineTM and calcium hydroxide on viability, proliferation, migration and differentiation of stem cells from human exfoliated deciduous teeth

Abstract Objective: The aim of the study was to evaluate the effects of the capping materials mineral trioxide aggregate (MTA), calcium hydroxide (CH) and BiodentineTM (BD) on stem cells from human exfoliated deciduous teeth (SHED) in vitro. Material and Methods: SHED were cultured for 1 - 7 days in medium conditioned by incubation with MTA, BD or CH (1 mg/mL), and tested for viability (MTT assay) and proliferation (SRB assay). Also, the migration of serum-starved SHED towards conditioned media was assayed in companion plates, with 8 μm-pore-sized membranes, for 24 h. Gene expression of dentin matrix protein-1 (DMP-1) was evaluated by reverse-transcription polymerase chain reaction. Regular culture medium with 10% FBS (without conditioning) and culture medium supplemented with 20% FBS were used as controls. Results: MTA, CH and BD conditioned media maintained cell viability and allowed continuous SHED proliferation, with CH conditioned medium causing the highest positive effect on proliferation at the end of the treatment period (compared with BD and MTA) (p<0.05). In contrast, we observed increased SHED migration towards BD and MTA conditioned media (compared with CH) (p<0.05). A greater amount of DMP-1 gene was expressed in MTA group compared with the other groups from day 7 up to day 21. Conclusion: Our results show that the three capping materials are biocompatible, maintain viability and stimulate proliferation, migration and differentiation in a key dental stem cell population.

Alveolar bone healing in rats: micro-CT, immunohistochemical and molecular analysis

Abstract Alveolar bone healing after upper incisor extraction in rats is a classical model of preclinical studies. The underlying morphometric, cellular and molecular mechanism, however, remains imprecise in a unique study. Objectives The aim of this study was therefore to characterize the alveolar bone healing after upper incisor extraction in rats by micro computed tomographic (Micro-CT), immunohistochemical and real-time polymerase chain reaction (RT-PCR) analysis. Material and Methods Thirty animals (Rattus norvegicus, Albinus Wistar) were divided into three groups after upper incisors extraction at 7, 14, and 28 days. Micro-CT was evaluated based on the morphometric parameters. Subsequently, the histological analyses and immunostaining of osteoprotegerin (OPG), receptor activator of nuclear kappa B ligand (RANKL) and tartrate resistant acid phosphate (TRAP) was performed. In addition, RT-PCR analyses of OPG, RANKL, the runt-related transcription factor 2 (RUNX2), osteocalcin (OC), osteopontin (OPN), osterix (OST) and receptor activator of nuclear kappa B (RANK) were performed to determine the expression of these proteins in the alveolar bone healing. Results Micro-CT: The morphometric parameters of bone volume and trabecular thickness progressively increased over time. Consequently, a gradual decrease in trabecular separation, trabecular space and total bone porosity was observed. Immunohistochemical: There were no differences statistically significant between the positive labeling for OPG, RANKL and TRAP in the different periods. RT-PCR: At 28 days, there was a significant increase in OPG expression, while RANKL expression and the RANKL/OPG ratio both decreased over time. Conclusion Micro-CT showed the newly formed bone had favorable morphometric characteristics of quality and quantity. Beyond the RUNX2, OC, OPN, OST, and RANK proteins expressed in the alveolar bone healing, OPG and RANKL activity showed to be essential for activation of basic multicellular units during the alveolar bone healing.

Immunohistochemical and mRNA expression of RANK, RANKL, OPG, TLR2 and MyD88 during apical periodontitis progression in mice

Abstract Objective To evaluate and correlate, in the same research, the mRNA expression and the staining of RANK, RANKL, OPG, TLR2 and MyD88 by immunohistochemistry in the apical periodontitis (AP) progression in mice. Material and Methods AP was induced in the lower first molars of thirty-five C57BL/6 mice. They were assigned to four groups according to their euthanasia periods (G0, G7, G21 and G42). The jaws were removed and subjected to histotechnical processing, immunohistochemistry and real-time reverse transcription-PCR (qRT-PCR). Data were analyzed with parametric and nonparametric tests (α=0.05). Results An increase of positive immunoreactivity for RANK, RANKL, OPG, TLR2 and MyD88 was observed over time (p<0.05). The RANKL expression was different between the groups G0 and G42, G21 and G42 (p=0.006), with G42 presenting the higher expression in both comparations. The OPG expression was statistically different between the groups G0 and G7, G7 and G21 and G7 and G42 (p<0.001), with G7 presenting higher expression in all the time points. The TLR2 expression was different between the groups G0 and G42 (p=0.03), with G42 showing the higher expression. The MyD88 expression presented a statistical significant difference between groups G7, G21 and G42 compared with G0 (p=0.01), with G0 presenting the smallest expression in all the comparisons. The Tnfrsf11/Tnfrsf11b (RANKL/OPG) ratio increased with the AP progression (p=0.002). A moderate positive correlation between MyD88 and RANKL (r=0.42; p=0.03) and between MyD88 and TLR2 (r=0.48; p<0.0001) was observed. Conclusion The expression of the RANK, RANKL, OPG, MyD88 and TLR2 proteins as well as the ratio Tnfrsf11/Tnfrsf11b (RANKL/OPG) increased with AP progression. There was also a moderate positive correlation between the expression Myd88-Tnfrsf11 and Tlr2-Myd88, suggesting the relevance of Tlr2-Myd88 in bone loss due to bacterial infection.

Human plasma fibronectin promotes proliferation and differentiation of odontoblast

Abstract Objective To assess the effect of fibronectin (Fn) and porcine type I collagen (PCOL) on odontoblast-like cells in vitro. Material and Methods Rat odontoblast-like cells (MDPC-23 cells) were inoculated and cultured on Fn-coated or type I collagen-coated substrates. Proliferation assay, alkaline phosphatase activity (ALP activity), mRNA expression of hard tissue-forming markers, and Alizarin red staining were investigated over a period of 10 days. Results Cells maintained a high proliferation activity on Fn and PCOL even at a low seeding concentration (0.5×104/mL) as demonstrated by CCK-8 assay. The proliferation activity of cells on Fn increases in a concentration-dependent manner while it reached a plateau after 10 µg/mL. Cells adopted long, thin and spindle shape on Fn(10-50) and PCOL. Parallel actin filaments were observed in MDPC-23 cells cultured on Fn and PCOL. ALP activity was markedly up-regulated on Fn and PCOL-coated surfaces. Importantly, gene expression of BSP (Fn10: 2.44±0.32; Fn20: 3.05±0.01; Fn30: 2.90±0.21; Fn40: 2.74±0.30; Fn50: 2.64±0.12; PCOL: 2.20±0.03) and OCN (Fn10: 2.52±0.23; Fn20: 2.28±0.24; Fn30: 2.34±0.21; Fn40: 2.34±0.25; Fn50: 2.20±0.22; PCOL: 1.56±0.16) was significantly enhanced on Fn and PCOL substrates as compared with control; moreover, expression of integrin beta 1 (ITGB1), an ubiquitous cell surface receptor was augmented in Fn(10-50) and PCOL groups simultaneously. In accordance with the ALP activity and gene expression data, calcific deposition in cells grown on Fn(10-50) and PCOL was observed as well. Conclusion Despite the limitation of this study, the findings indicate that a surface coating of Fn enhances the proliferation, differentiation and mineralization of odontoblast-like cells by activation of integrin beta 1 (ITG B1). The promoting effects of Fn on MDPC-23 cells were achieved at a comparatively lower coating concentration than type I collagen (300 µg/mL). Specifically, it is suggested that the optimum coating concentration of Fn to be 10 µg/mL.

Avaliação das citocinas TNF-α, RANKL e OPG e do número de osteoclastos no reparo de defeito ósseo em calvária de ratos diabéticos tratados com matriz óssea desmineralizada / Evaluation of cytokines TNF-α, RANKL and OPG and the number of osteoclasts on repair of bone defects in skulls of diabetic rats treated with demineralized bone matrix

Neste trabalho, foi avaliado a participação dos osteoclastos bem como a ação das citocinas RANKL, OPG e TNF-α durante a formação e remodelação óssea em defeitos ósseos de tamanho crítico em ratos normoglicêmicos e diabéticos tratados ou não com a MAOD. Para isso, foram utilizados 250 ratos machos Wistar. Trinta ratos foram utilizados para coleta dos fêmures e tíbias, os quais foram processados para obtenção da MAOD. Os demais 220 ratos foram divididos em Grupo Não Diabétido (CTL, n=110) e Grupo Diabético (DIAB, n= 110) induzido pela aplicação de uma dose única de 47 mg/Kg de massa corporal de estreptozotocina. Um defeito transósseo de 8 mm de diâmetro foi realizado nos ossos parietais dos ratos, sendo que, nos subgrupos CTL MAOD e DIAB MAOD, os defeitos foram preenchidos com MAOD e nos grupos CTL COAG e DIAB COAG apenas com coágulo sanguíneo. Após 0, 7, 14, 21 e 42 dias, as calotas cranianas foram coletadas para determinação da densidade de volume, número de osteoclastos/mm2 na área do defeito, quantificação por imunoistoquimica e expressão do RNAm para as proteínas RANKL, OPG e TNF-α. Os resultados para volume do tecido ósseo neoformado foi maior nos grupos CTL COAG e CTL MAOD, bem como no grupo DIAB MAOD quando comparado com DIAB COAG (CTL MAOD > CTL COAG e DIAB MAOD > DIAB COAG). O número de osteoclastos nos grupos CTL aumentaram significantemente (3,69 osteoclasto/mm2), enquanto que nos grupos MAOD aumentaram gradualmente até os 42 dias (2,8 osteoclasto/mm2). Os resultados para imunomarcação mostraram que a MAOD promove 1,28 vezes maior expressão de OPG, bem como de TNF-α tanto no grupo CTL (1,59 vezes) como no DIAB (1,76 vezes). Os resultados para expressão do RNAm para OPG mostrou que a média dos valores do grupo COAG comparado com a do grupo MAOD foi 1,91 vezes maior no grupo COAG. Já os valores para expressão de RANKL permaneceram constantes no grupo DIAB MAOD, com aumento significativo de 2,57 vezes aos 42 dias, sendo 4,3 vezes maior, quando comparado com a média dos outros grupos no mesmo período. Conclui-se que nos animais normoglicemicos, o tratamento com a MAOD aumenta a expressão de OPG, RANKL e TNF-α, assim como a atividade osteoclástica, promovendo reabsorção da MAOD e formação de tecido ósseo, enquanto que nos animais diabéticos, a atividade osteoclástica foi reduzida, sem alteração nos níveis de OPG e RANKL, reduzindo a reabsorção da MAOD e consequentemente da formação óssea.(AU)

Participation of osteoclasts was evaluated in reabsorption process of demineralized allogenic bone matrix (DABM) as well as the activity of cytokines RANKL, OPG and TNF- α during formation and bone remodeling in critial size defect of normoglycemic and diabetic rats treated or not with DABM. Therefore, 250 male Wistar rats were used. Thirty rats had femurs and tibias collected and processed to obtain DABM. 220 rats were divided into control group (CTL, n=110) and diabetic group (DIAB, n= 110) injected by a single dose of 47 mg/Kg of body weight streptozotocin. Were made 8mm bone defect on skulls of rats, in subgroups CTL DABM and DIAB DABM, defects were filled with DABM and subgroups CTL CLOT and DIAB CLOT were filled with blood clot. After 0, 7, 14, 21 and 42 days, the skulls were collected to determine the volume density, number of osteoclasts/mm2 into defects area, quantification by immunohistochemistry and RNAm expression of RANKL, OPG and TNF-α cytokines. The results of volume density of newly formed bone was higher in CTL CLOT and CTL DABM, as well as in DIAB DABM compared to DIAB CLOT (CTL DABM > CTL CLOT and DIAB DABM > DIAB CLOT). The number of osteoclasts in CTL groups increased to 3,69 osteoclasts/mm2, while in subgroups treated with DABM gradually increased up until 42 days (2,8 osteoclasts/mm2). Immunohistochemistry showed that DABM promotes an increase of 1.28-fold of OPG expression, as well as TNF-a expression in CTL group (1.59-fold) and DIAB group (1.76-fold). The results of RNAm expression of OPG showed that the average values of the CLOT subgroup compared to the average values of DABM subgroup was 1.91- fold higher in CLOT subgroup. The values of RANKL RNAm expression increase 2.57-fold at 42 days, being 4.3-fold higher than the average os the other groups in the same period. In conclusion, in the normoglicemic animals (CTL group), the treatment with DABM increase the expression of OPG, RANKL and TNF-α as the activity of osteoclasts, leading to DABM resorption and bone tissue formation, while in diabetic animals, the osteoclast activity was reduced, without changes in the leves of OPG and RANKL, decreasing DABM resorption and bone formation.(AU)

Estudo comparativo entre as ações do nimesulida, arnica montana homeopática e arnica montana fitoterápica: possíveis aplicações na terapêutica da doença periodontal e na rotina do consultório odontológico / Comparative study of the actions of nimesulide, homeopathic arnica montana and arnica montana phytotherapic: possible applications in the treatment of periodontal disease and routine dental office

Trata-se de estudo comparativo para avaliar a atividade dos medicamentos nimesulida, Arnica TM, Arnica 6CH comparados com H2O no tratamento da Doença Periodontal experimental induzida por ligadura em ratos. Esta doença se caracteriza por processo inflamatório que pode evoluir até ao ligamento periodontal e tecido ósseo provocando perda óssea. Mediadores produzidos pelo hospedeiro e substâncias do biofilme são responsáveis pelos danos que podem levar até a perda do elemento dentário. O objetivo deste trabalho foi desenvolver estudo exploratório descritivo para analisar, aprofundar e comparar os efeitos das medicações citadas, investigando possíveis inovações no tratamento da doença periodontal. A metodologia consistiu em: 1) colocação, sob anestesia, da ligadura no primeiro molar inferior esquerdo. 2) administração diária por gavagem de H2O (controle) (1μl/g), nimesulida (1μl/g, de uma suspensão com 5mg/ml), Arnica TM (8μl/300g) ou Arnica 6CH (3,21μl/300g) durante 14, 21 e 28 dias a quatro grupos de ratos Wistar (n=30/ grupo); 3) eutanásia e coleta de material para análises histológica, da perda óssea e RT-PCR (alvos: TNF-α, COX-2, IL-6, OPG e RANKL); 4) análise estatística paramétrica realizada por ANOVA a um ou dois critérios, seguida do teste de Tukey, ou por análise não paramétrica por Kruskal-Wallis, seguido do método de Dunns, nível de significância de 5%. Quanto à perda óssea, não houve diferença estatística entre os grupos e nenhum dos tratamentos foi capaz de contê-la. Os escores para o infiltrado inflamatório não mostraram diferença significativa entre os grupos nos períodos estudados. Quanto ao escore geral, que envolve intensidade do infiltrado inflamatório, migração do epitélio juncional, integridade do cemento e da crista óssea alveolar, não houve diferença significativa entre os grupos nos períodos. Na expressão do RNAm para as citocinas vimos para o TNF-α não haver diferença significativa entre os grupos, quando se analisou isoladamente...

This is a comparative study to evaluate the activity of nimesulide medicines, Arnica TM, Arnica 6CH compared with H2O in the treatment of experimental periodontal disease induced by ligation in rats. This disease is characterized by an inflammatory process that may progress to the periodontal ligament and bone tissue leading to bone loss. Mediators produced by the host and biofilm substances are responsible for the damage that can lead to tooth loss. The aim of this exploratory and descriptive study was to analyze, deepen and compare the effects of the mentioned medications, investigating possible innovations in the treatment of periodontal disease. The methodology consisted of: 1) placing under anesthesia ligature in the first lower left molar. 2) daily administration by gavage H2O (control) (1μl/g), nimesulide (1μl/g, a suspension of 5 mg / ml), Arnica TM (8μl/300g) or Arnica 6CH (3,21μl/300g) for 14, 21 and 28 days to four groups of Wistar rats (n = 30 / group); 3) euthanasia and collection of material for histological, bone loss and RTPCRanalysis (targets: TNF-α, COX-2, IL-6, OPG and RANKL); 4) parametric statistical analysis performed by ANOVA to one or two criteria, followed by Tukey test, or nonparametric analysis by Kruskal-Wallis, followed by Dunns method, 5% significance level. Regarding the bone loss, there was no statistical difference between the groups and none of the treatments was able to contain it. The scores for the inflammatory infiltrate showed no significant difference between the groups in the study periods. Regarding the general score, which involves intensity of the inflammatory infiltrate, migration of the junctional epithelium integrity of the cement and the alveolar bone crest, there was no significant difference between groups in the periods. At the mRNA expression for the cytokines we saw for TNF-α no significant difference between groups when analyzed separately periods of 14 and 28 days. However, in the 21day...

Modulation of cell proliferation, survival and gene expression by RAGE and TLR signaling in cells of the innate and adaptive immune response: role of p38 MAPK and NF-KB

Objective: The aim of this study was to evaluate a possible synergism between AGE-RAGE and TLR4 signaling and the role of p38 MAPK and NF-kB signaling pathways on the modulation of the expression of inflammatory cytokines and proliferation of cells from the innate and adaptive immune response. Material and Methods: T lymphocyte (JM) and monocyte (U937) cell lines were stimulated with LPS and AGE-BSA independently and associated, both in the presence and absence of p38 MAPK and NF-kB inhibitors. Proliferation was assessed by direct counting and viability was assessed by a biochemical assay of mitochondrial function. Cytokine gene expression for RAGe, CCL3, CCR5, IL-6 and TNF-α was studied by RT-PCR and RT-qPCR. Results: RAGE mRNA expression was detected in both cell lines. LPS and AGE-BSA did not influence cell proliferation and viability of either cell line up to 72 hours. LPS and LPS associated with AGE induced expression of IL-6 and TNF-α in monocytes and T cells, respectively. Conclusions: There is no synergistic effect between RAGE and TLR signaling on the expression of IL-6, TNF-α , RAGE, CCR5 and CCL3 by monocytes and lymphocytes. Activation of RAGE associated or not with TLR signaling also had no effect on cell proliferation and survival of these cell types. .

Brazilian minipig as a large-animal model for basic research and stem cell-based tissue engineering. Characterization and in vitro differentiation of bone marrow-derived mesenchymal stem cells

Stem cell-based regenerative medicine is one of the most intensively researched medical issues. Pre-clinical studies in a large-animal model, especially in swine or miniature pigs, are highly relevant to human applications. Mesenchymal stem cells (MSCs) have been isolated and expanded from different sources. Objective: This study aimed at isolating and characterizing, for the first time, bone marrow-derived MSCs (BM-MSCs) from a Brazilian minipig (BR1). Also, this aimed to validate a new large-animal model for stem cell-based tissue engineering. Material and Methods: Bone marrow (BM) was aspirated from the posterior iliac crest of twelve adult male BR1 under general anesthesia. MSCs were selected by plastic-adherence as originally described by Friedenstein. Cell morphology, surface marker expression, and cellular differentiation were examined. The immunophenotypic profile was determined by flow cytometry. The differentiation potential was assessed by cytological staining and by RT-PCR. Results: MSCs were present in all minipig BM samples. These cells showed fibroblastic morphology and were positive for the surface markers CD90 (88.6%), CD29 (89.8%), CD44 (86.9%) and negative for CD34 (1.61%), CD45 (1.83%), CD14 (1.77%) and MHC-II (2.69%). MSCs were differentiated into adipocytes, osteoblasts, and chondroblasts as demonstrated by the presence of lipidic-rich vacuoles, the mineralized extracellular matrix, and the great presence of glycosaminoglycans, respectively. The higher gene expression of adipocyte fatty-acid binding protein (AP2), alkaline phosphatase (ALP) and collagen type 2 (COLII) also confirmed the trilineage differentiation (p<0.001, p<0.001, p=0.031; respectively). Conclusions: The isolation, cultivation, and differentiation of BM-MSCs from BR1 makes this animal eligible as a useful large-animal model for stem cell-based studies in Brazil. .

Efeito do extrato de aroeira no processo de proliferação e mineralização de osteoblastos in vitro / Effect of aroira extract in the process of proliferation and mineralization of osteoblasts in vitro

Em virtude do uso indiscriminado de antimicrobianos, a resistência de microrganismos patogênicos à diversas drogas tem aumentado nos últimos anos. Essa situação tem forçado pesquisadores a procurarem por novas drogas, tais como os medicamentos fitoterápicos que são preparações farmacêuticas (extratos, tinturas, pomadas e cápsulas) de ervas medicinais, utilizadas para o tratamento de inúmeras doenças. Uma das espécies brasileiras do cerrado que tem despertado o interesse de várias áreas é a aroeira (Myracrodruon urundeuva), por apresentar ação antiinflamatória, antiproliferativa, antioxidante e antimicrobiana. Dessa maneira o objetivo deste trabalho foi de avaliar o efeito do extrato hidroalcoólico de aroeira na viabilidade celular dos osteoblastos humanos e analisar a influência do extrato hidroalcoólico de aroeira na expressão de metaloproteinase de matriz 2 (MMP-2) e anquilose progressiva humana (ANKH). Nesse estudo foram utilizados osteoblastos humanos cultivados em Meio de Eagle modificado por Dulbecco (DMEM) adicionado de 10% de soro fetal bovino (SFB). Após atingirem a subconfluência as células foram plaqueadas e tratadas com diferentes concentrações do extrato hidroalcoólico de aroeira (Extrato Bruto; 1:10; 1:100; 1:1.000; 1:10.000). Após 24, 48, 72, 96 horas foi realizada a análise da viabilidade celular por meio da redução do MTT (brometo de 3-(4,5-dimetiltiazol-2-yl)-2,5- difeniltetrazólio), captação do vermelho neutro e cristal violeta. Também foi feita a redução do MTT após 7, 14, 21 e 28 dias de tratamento com o extrato, para verificar se as células permaneciam viáveis nesses períodos. A análise da expressão gênica de MMP-2 e ANKH foi realizada pelo RT-PCR em tempo real nos períodos de 24, 48, 72 horas, 7, 14, 21 e 28 dias. Os ensaio de viabilidade celular (redução do MTT, captação do vermelho e cristal violeta) mostraram que houve o aumento da viabilidade celular nos grupos tratados com o extrato a 1:10.000; e a diminuição da viabilidade...

Due to the indiscriminate use of antimicrobial, the resistance of pathogenic microorganisms to various drugs has increased in recent years. This situation has forced researchers to search for new drugs, such as herbal medicines that are pharmaceutical preparations (extracts, tinctures, ointments and capsules) medicinal herb, used for the treatment of numerous diseases. One species of the Brazil that has aroused interest in many areas is the aroeira (Myracrodruon urundeuva) because of its anti-inflammatory, antiproliferative, antioxidant and antimicrobial. Thus the aim of this work was to evaluate the effect of hydroalcoholic extract of aroeira on cell viability of human osteoblasts and to analyze extract on the expression of matrix metalloproteinase 2 (MMP-2) and human progressive ankylosis (ANKH) . In this study, we used human osteoblasts cultured in Eagle's medium modified by Dulbecco (DMEM) supplemented with 10% fetal bovine serum (FBS). After reaching subconfluence cells were plated and treated with different concentrations of the aroeira extract hydroalcoholic (brute extract , 1:10, 1:100, 1:1.000, 1:10:000). After 24, 48, 72 and 96 hours analysis was performed of cell viability by the reduction of MTT (3 - (4,5-dimethylthiazol-2-yl) -2,5 - diphenyltetrazolium bromide) uptake of neutral red and crystal violet. It was also made MTT reduction after 7, 14, 21 and 28 days of treatment with the extract, to check if the cells remained viable during these periods. The analysis of the gene expression of MMP-2 and ANKH was performed by Real Time RT-PCR in periods of 24, 48 hours, 72 hours, 7, 14, 21 and 28 days. The cell viability assay (MTT reduction, uptake of neutral red and crystal violet) showed that there was a tendency of increased cell viability in the groups treated with the extract 1:10.000, and decreased viability of human osteoblast cells in groups with brute extract and 1:10. With respect to expression of MMP-2 and ANKH, the results showed...

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...

Avaliação do papel dos receptores TLR2 e TLR4 na produção de citocinas por fibroblastos humanos periodontais deficientes desses receptores / The role of TLR2 and TLR4 in cytokines production by human periodontal fibroblasts knocked down for these receptors

Os fibroblastos são atualmente considerados componentes ativos da resposta imune porque estas células expressam receptores do tipo Toll (TLRs), são capazes de reconhecer padrões moleculares associados a patógenos e mediar a produção de citocinas e quimiocinas durante a inflamação. A resposta imune inata do hospedeiro a lipopolissacarídeos (LPS) de Porphyromonas gingivalis é incomum, já que diferentes estudos relataram que este LPS pode ser um agonista para TLR2 e um antagonista ou agonista para TLR4. A sinalização por TLRs envolve proteínas adaptadoras, como MyD88 e TRAM, que são necessárias para a transdução do sinal até o núcleo para que ocorra a transcrição de RNAm para os mediadores da inflamação. O objetivo deste estudo foi investigar e comparar se a sinalização por meio de TLR2 ou TLR4 poderia afetar a produção de Interleucina (IL)-6, IL-8 e CXCL12 em fibroblastos humanos gengivais (HGF) e fibroblastos humanos de ligamento periodontal (HPLF). Objetivamos também comparar a participação das moléculas adaptadoras MyD88 e TRAM na expressão do RNAm dos mesmos alvos. Material e Métodos: Após silenciamento mediado por RNA de interferência de TLR2, TLR4, MyD88 ou TRAM, confirmado por RT-qPCR, HGF e HPLF, provenientes de três dadores voluntários, foram estimulados com LPS de Porphyromonas gingivalis ou com dois agonistas sintéticos de TLR2, Pam2CSK4 e Pam3CSK4, por 6 horas. A expressão do RNAm e das proteínas IL-6, IL-8, e CXCL12 foram avaliados por qRT-PCR e ELISA, respectivamente. Resultados: A expressão do RNAm de TLR2 foi regulada em HGF, mas não em HPLF por todos os estímulos. O silenciamento de TLR2 diminuiu IL-6 e IL-8 em resposta ao LPS de P. gingivalis, Pam2CSK4 e Pam3CSK4 de maneira semelhante, em ambas as subpopulações de fibroblastos (p<0,05). Por outro lado, a produção de CXCL12 permaneceu inalterada pelo silenciamento de TLR2 ou TLR4. No caso do silenciamento de MyD88 e TRAM, em ambos os subtipos de fibroblastos, o RNAm para os mesmos alvos também...

Fibroblasts are now seen as active components of the immune response because these cells express Toll-like receptors (TLRs), recognize pathogen associated molecular patterns and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual in that different studies have reported that it can be an agonist for TLR2 and an antagonist or agonist for TLR4. TLRs signaling pathway involves adaptor proteins, like MyD88 and TRAM, which are crucial for signal transduction to the nucleus and mRNA expression of inflammatory mediators. This study investigated and compared whether signaling through TLR2 or TLR4 could affect the production of IL-6, IL-8 and CXCL12 in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPLF). The role of MyD88 and TRAM on the mRNA expression of the same targets were also evaluated. Methods: After small interfering RNA-mediated silencing of TLR2, TLR4, MyD88 or TRAM, confirmed by RT-qPCR, HGF and HPLF from three volunteer donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL-6, IL-8, and CXCL12 mRNA expression and protein production were evaluated by RT-qPCR and ELISA, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPLF by all the stimuli applied. Knockdown of TLR2 decreased IL-6 and IL-8 in response to P. gingivalis LPS, Pam2CSK4 and Pam3CSK4 in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. For MyD88 or TRAM silencing, IL-6 and IL-8 mRNA were also decreased, in both fibroblasts subtypes. However CXCL12 mRNA constitutive expression was increased by siMyD88 or siTRAM. Conclusion: These results suggest that signaling through TLR2 by fibroblasts, the most numerous resident cells in gingiva and periodontal...

Avaliação do papel dos receptores TLR2 e TLR4 na produção de citocinas por fibroblastos humanos periodontais deficientes desses receptores / The role of TLR2 and TLR4 in cytokines production by human periodontal fibroblasts knocked down for these receptors

Os fibroblastos são atualmente considerados componentes ativos da resposta imune porque estas células expressam receptores do tipo Toll (TLRs), são capazes de reconhecer padrões moleculares associados a patógenos e mediar a produção de citocinas e quimiocinas durante a inflamação. A resposta imune inata do hospedeiro a lipopolissacarídeos (LPS) de Porphyromonas gingivalis é incomum, já que diferentes estudos relataram que este LPS pode ser um agonista para TLR2 e um antagonista ou agonista para TLR4. A sinalização por TLRs envolve proteínas adaptadoras, como MyD88 e TRAM, que são necessárias para a transdução do sinal até o núcleo para que ocorra a transcrição de RNAm para os mediadores da inflamação. O objetivo deste estudo foi investigar e comparar se a sinalização por meio de TLR2 ou TLR4 poderia afetar a produção de Interleucina (IL)-6, IL-8 e CXCL12 em fibroblastos humanos gengivais (HGF) e fibroblastos humanos de ligamento periodontal (HPLF). Objetivamos também comparar a participação das moléculas adaptadoras MyD88 e TRAM na expressão do RNAm dos mesmos alvos. Material e Métodos: Após silenciamento mediado por RNA de interferência de TLR2, TLR4, MyD88 ou TRAM, confirmado por RT-qPCR, HGF e HPLF, provenientes de três dadores voluntários, foram estimulados com LPS de Porphyromonas gingivalis ou com dois agonistas sintéticos de TLR2, Pam2CSK4 e Pam3CSK4, por 6 horas. A expressão do RNAm e das proteínas IL-6, IL-8, e CXCL12 foram avaliados por qRT-PCR e ELISA, respectivamente. Resultados: A expressão do RNAm de TLR2 foi regulada em HGF, mas não em HPLF por todos os estímulos. O silenciamento de TLR2 diminuiu IL-6 e IL-8 em resposta ao LPS de P. gingivalis, Pam2CSK4 e Pam3CSK4 de maneira semelhante, em ambas as subpopulações de fibroblastos (p<0,05). Por outro lado, a produção de CXCL12 permaneceu inalterada pelo silenciamento de TLR2 ou TLR4. No caso do silenciamento de MyD88 e TRAM, em ambos os subtipos de fibroblastos, o RNAm para os mesmos alvos também...

Fibroblasts are now seen as active components of the immune response because these cells express Toll-like receptors (TLRs), recognize pathogen associated molecular patterns and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual in that different studies have reported that it can be an agonist for TLR2 and an antagonist or agonist for TLR4. TLRs signaling pathway involves adaptor proteins, like MyD88 and TRAM, which are crucial for signal transduction to the nucleus and mRNA expression of inflammatory mediators. This study investigated and compared whether signaling through TLR2 or TLR4 could affect the production of IL-6, IL-8 and CXCL12 in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPLF). The role of MyD88 and TRAM on the mRNA expression of the same targets were also evaluated. Methods: After small interfering RNA-mediated silencing of TLR2, TLR4, MyD88 or TRAM, confirmed by RT-qPCR, HGF and HPLF from three volunteer donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL-6, IL-8, and CXCL12 mRNA expression and protein production were evaluated by RT-qPCR and ELISA, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPLF by all the stimuli applied. Knockdown of TLR2 decreased IL-6 and IL-8 in response to P. gingivalis LPS, Pam2CSK4 and Pam3CSK4 in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. For MyD88 or TRAM silencing, IL-6 and IL-8 mRNA were also decreased, in both fibroblasts subtypes. However CXCL12 mRNA constitutive expression was increased by siMyD88 or siTRAM. Conclusion: These results suggest that signaling through TLR2 by fibroblasts, the most numerous resident cells in gingiva and periodontal...

Modulação da expressão de VEGF-C por mediador relacionado ao estresse em culturas de carcinomas espinocelulares de boca / Modulation of VEGF-C expression by stress related mediator in oral squamous cell carcinoma cell lines

Os mediadores do estresse, epinefrina e norepinefrina, participam da modulação de diversos processos celulares como a proliferação, a migração celular e a apoptose durante a tumorigênese, influenciando assim, o crescimento e a progressão tumoral. A presença de receptores beta-adrenérgicos e sua expressiva resposta ao estímulo do neurohormônio norepinefrina foram identificadas em várias linhagens de células tumorais, incluindo o carcinoma espinocelular de boca. O objetivo deste estudo foi investigar a influência da norepinefrina na expressão do fator de crescimento endotelial vascular do tipo C (VEGF-C) em cultura de células de carcinoma espinocelular de boca que continham receptores beta adrenérgicos. As linhagens celulares (SCC-9 e SCC-25) foram estimuladas com norepinefrina em diferentes concentrações (0,1; 1 e 10 M) e com 1M de propranolol, sendo analisadas após 1, 6 e 24 horas. A expressão gênica e proteica de VEGF-C foram avaliadas, respectivamente, por RT-PCR em tempo real e por ELISA. A produção de RNAm para VEGF-C teve um comportamento irregular, com tendências a variações da expressão gênica (aumento e inibição). A dosagem proteica nos sobrenadantes das culturas de células malignas não refletiu a expressão gênica de VEGF-C. Somente na linhagem SCC-25 ocorreu uma inibição significativa da produção de VEGF-C (p<0,001) pelas células neoplásicas no ensaio de 24 horas após o estímulo com 10M de norepinefrina. Estes resultados sugerem que a expressão de VEGF-C nas linhagens de carcinomas espinocelulares humanos de boca, parece não ser mediado pela norepinefrina, via receptores beta-adrenérgicos.

The mediators of stress, epinephrine and norepinephrine, are involved in modulation of many cellular processes such as proliferation, migration and apoptosis influencing the tumor growth and progression. The presence of beta-adrenergic receptors and their significant response to stimulation of neurohormone norepinephrine has been identified in various tumor cell lines, including oral squamous cell carcinoma. The aim of this study was to investigate the influence of norepinephrine on the expression of vascular endothelial growth factor type C (VEGF-C) in oral squamous carcinomas cell lines that contained beta-adrenergic receptors. Cell lines (SCC-9 and SCC-25) were stimulated with different concentrations of norepinephrine (0.1, 1 and 10 mM) and 1 M of propranolol, and analyzed after 1, 6 and 24 hours. Gene and protein expressions of VEGF-C were evaluated, respectively, by real time PCR and by ELISA. The results showed an irregular behavior of the oral squamous carcinoma cell lines, with trends to increase or to inhibit the VEGF-C gene expression. Dosage protein in supernatant cultures of malignant cells did not reflect the gene expression of VEGF-C. Only in the SCC-25 cell line was detected a significant inhibition of VEGF-C production by neoplastic cells, twenty-four hours after stimulation with 10M norepinephrine (p<0,001). These results suggest that VEGF-C expression in oral squamous carcinomas cell lines seems not to be mediated by norepinephrine through the beta adrenergic receptor pathway.

Detecção da expressão dos genes HWP1, ALS1 e ALS3 de C. albicans, por meio de RT-PCR em tempo real, após desinfecção química e por microonda, de resina acrílica para confecção de dentaduras / HWP1, ALS1 and ALS3 genes expression in Candida albicans after chemical or microwave disinfection of acrylic resin for denture prosthesis using real time PCR

A desinfecção de dentaduras promove o controle do biofilme microbiano e previne doenças, como a estomatite por uso de dentadura, associada à presença de Candida albicans. A expressão dos genes HWP1, ALS1, ALS3 deste fungo está relacionada à adesão das células fúngicas às do hospedeiro. O objetivo deste estudo foi detectar e quantificar a expressão desses genes em células planctônicas e biofilme de Candida albicans, desenvolvidos sobre superfícies de resina acrílica, após tratamento com dois métodos de desinfecção. Corpos de prova de resina acrílica, previamente tratados por hipoclorito de sódio 1%, microondas, e um grupo não tratado, foram inoculados com Candida albicans para desenvolvimento de biofilme. O biofilme e as células planctônicas foram coletados em três tempos distintos, correspondentes às etapas de desenvolvimento do biofilme: inicial (6h), intermediária (12h) e madura (48h) e a expressão gênica foi quantificada pelo ensaio de RT-PCR em tempo real. Os dados obtidos foram submetidos ao teste estatístico ANOVA two-way e pós-teste de Bonferroni; valores de p<0,05, p<0,01 e p<0,001 foram considerados significantes. Os três genes avaliados foram detectados e quantificados por RT-PCR em tempo real, em biofilmes e células planctônicas, independente do grupo de tratamento ou tempo de desenvolvimento do biofilme. A expressão dos genes ALS1 e ALS3 variou de acordo com o tempo de desenvolvimento do biofilme e, e com o tratamento da superfície. Ocorreu diferença significativa (p<0,001) entre a expressão desses genes nas superfícies tratadas por hipoclorito de sódio e microondas, além de diferenças significativas (p<0,001) na expressão gênica entre células planctônicas e biofilme, para os tratamentos avaliados. Concluiu-se que os tratamentos de desinfecção alteram o padrão da expressão dos genes ALS1 e ALS3 em biofilmes desenvolvidos sobre superfície de resina acrílica e em células planctônicas e este padrão foi diferente entre os tratamentos de desinfecção.

Complete dentures disinfection promotes biofilm control and prevents diseases such as denture stomatitis associated with the presence of Candida albicans infection. The expression of the genes HWP1, ALS1, ALS3 in this fungus is related to the adhesion of fungal cells to the host tissues. Therefore, the aim of this study was to detect and quantify the expression of these genes in planktonic cells and biofilms of Candida albicans, developed on acrylic resin surfaces, after treatment with two disinfection methods. Specimens of acrylic resin, previously treated by 1% sodium hypochlorite or microwave, and an untreated group were inoculated with Candida albicans for biofilm development. Biofilm and planktonic cells were collected at three different time points corresponding to biofilm development stages: initial (6 h), intermediate (12h) and mature (48h). The total RNA of these samples was obtained and the gene expression for mRNA of HWP1, ALS1 and ALS3 were quantified by Real-Time RT-PCR assay. The data obtained were assessed throught two-way ANOVA and Bonferroni post-test. Significant levels were determined for p values at <0.05. The three genes were detected and quantified in both biofilms and planktonic cells regardless of treatment condition or time of biofilm development. ALS1 and ALS3 expression varied according to the time point, and surface treatment. Significant differences (p <0.001) were showed between gene expression on surfaces treated with sodium hypochlorite and microwave, and significant differences (p <0.001) were showed in gene expression between planktonic and biofilm cells. It can be concluded that the disinfection procedures affect the ALS1 and ALS3 expression patterns in Candida albicans denture biofilms and planktonic cells. Additionaly, differential gene expression patterns were observed among the disinfection treatments.