Optimizing the formation of the acquired enamel pellicle in vitro for proteomic analysis

Abstract Saliva is the major contributor for the protein composition of the acquired enamel pellicle (AEP), a bacteria-free organic layer formed by the selective adsorption of salivary proteins on the surface of the enamel. However, the amount of proteins that can be recovered is even smaller under in vitro condition, due to the absence of continuous salivary flow. Objective This study developed an in vitro AEP protocol for proteomics analysis using a new formation technique with different collection solutions. Methodology 432 bovine enamel specimens were prepared (4x4 mm) and divided into four groups (n=108). Unstimulated saliva was provided by nine subjects. The new AEP formation technique was based on saliva resupply by a new one every 30 min within 120 minutes at 37ºC under agitation. AEP was collected using an electrode filter paper soaked in the collection solutions according with the group: 1) 3% citric acid (CA); 2) 0.5% sodium dodecyl sulfate (SDS); 3) CA followed by SDS (CA+SDS); 4) SDS followed by CA (SDS+CA). The pellicles collected were processed for analysis through LC-ESI-MS/MS technique. Results A total of 55 proteins were identified. The total numbers of proteins identified in each group were 40, 21, 28 and 41 for the groups CA, SDS, CA+SDS and SDS+CA, respectively. Twenty-three typical AEP proteins were identified in all groups, but Mucin was only found in CA and CA+SDS, while three types of PRP were not found in the SDS group. Moreover, a typical enamel protein, Enamelin, was identified in the CA+SDS group only. Conclusion The new technique of the in vitro AEP formation through saliva replacement was essential for a higher number of the proteins identified. In addition, considering practicality, quantity and quality of identified proteins, citric acid seems to be the best solution to be used for collection of AEP proteins.

Influence of salivary conditioning and sucrose concentration on biofilm-mediated enamel demineralization

Abstract The acquired pellicle formation is the first step in dental biofilm formation. It distinguishes dental biofilms from other biofilm types. Objective To explore the influence of salivary pellicle formation before biofilm formation on enamel demineralization. Methodology Saliva collection was approved by Indiana University IRB. Three donors provided wax-stimulated saliva as the microcosm bacterial inoculum source. Acquired pellicle was formed on bovine enamel samples. Two groups (0.5% and 1% sucrose-supplemented growth media) with three subgroups (surface conditioning using filtered/pasteurized saliva; filtered saliva; and deionized water (DIW)) were included (n=9/subgroup). Biofilm was then allowed to grow for 48 h using Brain Heart Infusion media supplemented with 5 g/l yeast extract, 1 mM CaCl2.2H2O, 5% vitamin K and hemin (v/v), and sucrose. Enamel samples were analyzed for Vickers surface microhardness change (VHNchange), and transverse microradiography measuring lesion depth (L) and mineral loss (∆Z). Data were analyzed using two-way ANOVA. Results The two-way interaction of sucrose concentration × surface conditioning was not significant for VHNchange (p=0.872), ∆Z (p=0.662) or L (p=0.436). Surface conditioning affected VHNchange (p=0.0079), while sucrose concentration impacted ∆Z (p<0.0001) and L (p<0.0001). Surface conditioning with filtered/pasteurized saliva resulted in the lowest VHNchange values for both sucrose concentrations. The differences between filtered/pasteurized subgroups and the two other surface conditionings were significant (filtered saliva p=0.006; DIW p=0.0075). Growing the biofilm in 1% sucrose resulted in lesions with higher ∆Z and L values when compared with 0.5% sucrose. The differences in ∆Z and L between sucrose concentration subgroups was significant, regardless of surface conditioning (both p<0.0001). Conclusion Within the study limitations, surface conditioning using human saliva does not influence biofilm-mediated enamel caries lesion formation as measured by transverse microradiography, while differences were observed using surface microhardness, indicating a complex interaction between pellicle proteins and biofilm-mediated demineralization of the enamel surface.

Comparison of protein profiles of the pellicle, gingival crevicular fluid, and saliva: possible origin of pellicle proteins

BACKGROUND: The pellicle, the acellular organic material deposited on the surface of tooth enamel, has been thought to be derived from saliva. In this study, protein compositions of the pellicle, gingival crevicular fluid, and saliva collected from healthy adults were compared to elucidate the origin of pellicle proteins. RESULTS: The pellicle, gingival crevicular fluid, and saliva from the parotid gland or mixed gland were collected; subsequently, protein expression in samples from the respective individual was compared by SDS-PAGE and mass spectrometry. Following SDS-PAGE, proteins in the major bands were identified by mass spectrometry. The band pattern of pellicle proteins appeared different from those of gingival crevicular fluid, or saliva samples. Using mass spectrometry, 13 proteins in these samples were identified. The relative abundance of the proteins was quantitatively analyzed using mass spectrometry coupled with stable isotope labeling and by western blot. Cystatin S and α-amylase detected in pellicle were enriched in saliva samples, but not in gingival crevicular fluid, by western blot, and their abundance ratios were high in saliva and low in gingival crevicular fluid when analyzed by stable isotope labeling. Serotransferrin, however, was found only in the pellicle and gingival crevicular fluid by western blot and its abundance ratio was low in saliva. CONCLUSIONS: Our study revealed that the gingival crevicular fluid appears to contribute to pellicle formation in addition to saliva.

Decontamination methods to restore the biocompatibility of contaminated titanium surfaces

PURPOSE: The reaction of cells to a titanium implant depends on the surface characteristics of the implant which are affected by decontamination. The aim of this study was to evaluate the cytocompatibility of titanium disks treated with various decontamination methods, using salivary bacterial contamination with dental pellicle formation as an in vitro model. METHODS: Sand-blasted and acid-etched (SA) titanium disks were used. Three control groups (pristine SA disks [SA group]; salivary pellicle-coated SA disks [pellicle group]; and biofilm-coated, untreated SA disks [NT group]) were not subjected to any decontamination treatments. Decontamination of the biofilm-coated disks was performed by 14 methods, including ultrasonic instruments, rotating instruments, an air-powder abrasive system, a laser, and chemical agents. MG63 cells were cultured in the presence of the treated disks. Cell proliferation assays were performed on days 2 and 5 of cell culture, and cell morphology was analyzed by immunofluorescence and scanning electron microscopy (SEM). A vascular endothelial growth factor (VEGF) assay was performed on day 5 of culture. RESULTS: The cell proliferation assay revealed that all decontaminated disks, except for the 2 groups treated using a plastic tip, showed significantly less cell proliferation than the SA group. The immunofluorescence and SEM analyses revealed that most groups showed comparable cell density, with the exception of the NT group, in which the cell density was lower and bacterial residue was observed. Furthermore, the cells grown with tetracycline-treated titanium disks showed significantly lower VEGF production than those in the SA group. CONCLUSIONS: None of the decontamination methods resulted in cytocompatibility similar to that of pristine SA titanium. However, many methods caused improvement in the biocompatibility of the titanium disks in comparison with the biofilm-coated, untreated titanium disks. This suggests that decontamination is indispensable for the treatment of peri-implantitis, even if the original biocompatibility cannot be restored.

Proteomic analysis of the acquired enamel pellicle formed on human and bovine tooth: a study using the Bauru in situ pellicle model (BISPM)

Abstract The acquired enamel pellicle (AEP) is an organic film, bacteria-free, formed in vivo as a result of the selective adsorption of salivary proteins and glycoproteins to the solid surfaces exposed to the oral environment. Objective: This study aimed to compare the proteomic profile of AEP formed in situ on human and bovine enamel using a new intraoral device (Bauru in situ pellicle model - BISPM). Material and Methods: One hundred and eight samples of human and bovine enamel were prepared (4×4 mm). Nine subjects with good oral conditions wore a removable jaw appliance (BISPM) with 6 slabs of each substrate randomly allocated. The AEP was formed during the morning, for 120 minutes, and collected with an electrode filter paper soaked in 3% citric acid. This procedure was conducted in triplicate and the pellicle collected was processed for analysis by LC-ESI-MS/MS. The obtained mass spectrometry MS/MS spectra were searched against human protein database (SWISS-PROT). Results: The use of BISPM allowed the collection of enough proteins amount for proper analysis. A total of 51 proteins were found in the AEP collected from the substrates. Among them, 15 were common to both groups, 14 were exclusive of the bovine enamel, and 22 were exclusive of the human enamel. Proteins typically found in the AEP were identified, such as Histatin-1, Ig alpha-1, Ig alpha 2, Lysozyme C, Statherin and Submaxillary gland androgen-regulated protein 3B. Proteins not previously described in the AEP, such as metabolism, cell signaling, cell adhesion, cell division, transport, protein synthesis and degradation were also identified. Conclusion: These results demonstrate that the proteins typically found in the AEP appeared in both groups, regardless the substrate. The BISPM revealed to be a good device to be used in studies involving proteomic analysis of the AEP.

Effect of vegetable oils applied over acquired enamel pellicle on initial erosion

Abstract Objective The prevalence of dental erosion has been recently increasing, requiring new preventive and therapeutic approaches. Vegetable oils have been studied in preventive dentistry because they come from a natural, edible, low-cost, and worldwide accessible source. This study aimed to evaluate the protective effect of different vegetable oils, applied in two concentrations, on initial enamel erosion. Material and Methods Initially, the acquired pellicle was formed in situ for 2 hours. Subsequently, the enamel blocks were treated in vitro according to the study group (n=12/per group): GP5 and GP100 - 5% and pure palm oil, respectively; GC5 and GC100 - 5% and pure coconut oil; GSa5 and GSa100 - 5% and pure safflower oil; GSu5 and GSu100 - 5% and pure sunflower oil; GO5 and GO100 - 5% and pure olive oil; CON− - Deionized Water (negative control) and CON+ - Commercial Mouthwash (Elmex® Erosion Protection Dental Rinse, GABA/positive control). Then, the enamel blocks were immersed in artificial saliva for 2 minutes and subjected to short-term acid exposure in 0.5% citric acid, pH 2.4, for 30 seconds, to promote enamel surface softening. The response variable was the percentage of surface hardness loss [((SHi - SHf) / SHf )×100]. Data were analyzed by one-way ANOVA and Tukey's test (p<0.05). Results Enamel blocks of GP100 presented similar hardness loss to GSu100 (p>0.05) and less than the other groups (p<0.05). There was no difference between GP5, GC5, GC100, GSa5, GSu100, GSa100, GSu5, GO5, GO100, CON− and CON+. Conclusion Palm oil seems to be a promising alternative for preventing enamel erosion. However, further studies are necessary to evaluate a long-term erosive cycling.

Evaluación de la microdureza del esmalte afectado por caries incipiente y tratados por dos tipos de barnices fluorados: Estudio in vitro / Avaliação da microdureza do esmalte afetado por cárie incipiente e tratada por dois tipos de vernizes fluoretados: Estudo in vitro / Evaluation of the microhardness of enamel affected by incipient caries and treated by two types of fluoride varnishes: In vitro study

El flúor ha demostrado su efectividad en la terapia de lesiones iniciales de caries. En la actualidad, existen nuevos barni-ces de flúor con agentes remineralizantes adicionales, con la finalidad de tratar las lesiones de caries no cavitadas de una forma no invasiva, promoviendo la remineralización del esmalte. Objetivo: Determinar la microdureza del esmalte con lesiones de caries incipientes tratados con dos barnices fluorados. Materiales y métodos: Estudio experimental in vitro. La muestra estuvo constituida por 35 bloques de esmalte, que fueron divididos de manera aleatoria en 3 grupos. G1(n=5): control negativo; G2 (n=15): Barniz de Flúor (Flúor protector- Ivoclar Vivadent) y G3(n=15): Barniz de flúor con Fosfato Tricálcico (Clinpro White Varnish- 3M ESPE). Se realizaron 3 indentaciones mediante un microdurómetro, con el fin de obtener la dureza inicial, luego se desmineralizaron las muestras con la finalidad de producir lesiones incipientes, donde se realizaron nuevamente 3 indentaciones para obtener la microdureza del esmalte desmineralizado, concluida esta fase se colocó el remineralizante en cada muestra y se siguió un régimen de pH cíclico durante 7 días para simular las condiciones bucales, transcurrido este periodo se realizaron 3 indentaciones en cada muestra para obtener la dureza final del esmalte remineralizado, llegando a un total de 9 indentaciones por muestra. Los datos se analizaron mediante el test de Scheffé y la prueba T de Student con un nivel de significancia del 5%. Resultados: Existió diferencia significativa entre el barniz fluorado con Fosfato Tricálcico (TCP) con el grupo control (p=0,03). No se encontraron diferencias significativas entre los diferentes barnices fluorados (p=0,09). Fue observada una mejora significativa de la microdureza postratamiento de ambos barnices de flúor (p<0.001). Conclusión: Los dos tipos de barnices de flúor incrementaron de manera eficiente la microdureza del esmalte con lesión de caries incipiente, sin existir diferencia significativa entre estos dos.

Fluoride has shown its effectiveness in the therapy of initial lesions of caries. Currently, there are new fluoride varnishes with additional remineralizing agents, to treat lesions of non-cavitated caries in a non-invasive way, promoting the remineralization of the enamel. Objective: To determine the microhardness of enamel with incipient caries lesions treated with two fluoride varnishes. Materials and methods: In vitro experimental study. The sample consisted of 35 enamel blocks, which were randomly divided into 3 groups. G1 (n=5): negative control; G2 (n=15): Fluorine varnish (Flúor protector - Ivoclar Vivadent) and G3 (n=15): Fluorine varnish with Tricalcium Phosphate (Clinpro White Varnish - 3M ESPE). Three indentations were made by means of a microhardness meter, in order to obtain the initial hardness, then the samples were demineralized with the purpose of producing incipient lesions, where 3 indentations were made again to obtain the microhardness of the demineralized enamel, after this phase the remineralizing solution in each sample and a cyclical pH regime was followed during 7 days to simulate the oral conditions, after this period 3 indentations were made in each sample to obtain the final hardness of the remineralized enamel, reaching a total of 9 indentations per sample. The data were analyzed using the Scheffé test and the Student's T test with a level of significance of 5%. Results: There was a significant difference between the fluoride varnish with Tricalcium Phosphate (TCP) and the control group (p=0.03). No significant differences were found between the different fluorinated varnishes (p=0.09). A significant improvement of the microhardness after treatment of both fluoride varnishes was observed (p<0.001). Conclusion: The two types of fluoride varnishes efficiently increased the microhardness of enamel with incipient caries lesion, without significant difference between these two.

O flúor tem mostrado sua efetividade na terapia das lesões iniciais de cárie. Atualmente, existem novos vernizes de flúor com agentes remineralizantes adicionais, a fim de tratar lesões de cárie não cavitadas de forma não invasiva, promovendo a remineralização do esmalte. Objetivo: Determinar a microdureza do esmalte com lesões de cárie incipientes tratadas com dois vernizes de flúor. Materiais e métodos: Estudo experimental in vitro. A amostra consistiu em 35 blocos de esmalte, que foram divididos aleatoriamente em 3 grupos. G1 (n=5): controle negativo; G2 (n=15): Verniz de flúor (Fluor protector - Ivoclar Vivadent) e G3 (n=15): Verniz de flúor com fosfato tricálcico (Clinpro White Varnish -ESPE 3M). Três indentações foram feitas por meio de um microdurômetro para obter a dureza inicial, depois as amostras foram desmineralizadas com o objetivo de produzir lesões incipientes, onde foram feitas novamente três indentações para obter a microdureza do esmalte desmineralizado, após essa fase foi colocado a solução remineralizante em cada amostra e um regime de pH cíclico foi fei-to durante 7 dias para simular as condições bucais, após esse período foram feitas 3 indentações em cada amostra para obter a dureza final do esmalte remineralizado, atingindo um total de 9 indentações por amostra. Os dados foram analisados uti-lizando o teste Scheffé e o teste T de Student com um nível de significância de 5%. Resultados: Houve diferença significa-tiva entre o verniz fluoretado com fosfato tricálcico (TCP) e o grupo controle (p=0,03). Não foram encontradas diferenças significativas entre os diferentes vernizes fluoretados (p=0,09). Observou-se uma melhoria significativa da microdureza após o tratamento de ambos os vernizes de flúor (p<0,001). Conclusão: Os dois tipos de vernizes de flúor aumentaram de forma eficiente a microdureza do esmalte com lesão de cárie incipiente, sem existir diferença significativa entre estes dois.

Estudio de la microdureza de dientes permanentes con fluorosis incipiente, tratados con resina infiltrante / Micro-hardness of permanent teeth with incipient fluorosis treated with infiltrant resin / Estudo da microdureza de dentes permanentes com fluorose incipiente tratados com resina infiltrante

Objetivo: Evaluar la microdureza del esmalte afectado con fluorosis sometido a tratamiento con resina infiltrante, compa-rándolo con dientes sanos y dientes con fluorosis incipiente. Materiales y métodos: La muestra estuvo constituida por 15 dientes permanentes humanos recolectados del Banco de Dientes de la Facultad de Odontología de la Universidad Central del Ecuador. Para la selección de los dientes se tuvieron en consideración los criterios diagnósticos de fluorosis dental según Thylstrup y Ferjeskov (1978), teniendo 5 dientes con score 0 y 10 dientes con fluorosis incipiente (score 1-3). Todos los dientes considerados en el estudio no presentaron lesiones de caries, grietas ni fracturas. La muestra fue dividida en 3 grupos, G1: dientes sanos (control negativo), G2: dientes con fluorosis incipiente y G3: dientes con fluorosis incipiente tratado con resina infiltrante Icon®. A cada grupo (n=5) se le realizó una profilaxis y posteriormente al G3 se le aplicó la resina infiltrante. La microdureza knoop fue obtenida mediante tres indentaciones con microdurometro (Wilson Tukon Microhardness Tester). Los datos fueron analizados a través del método de Rho de Spearman con significancia de 5%. Resultados: La media de la microdureza knoop de los grupos y sus desviaciones estándar fueron de: G1=284,8 ± 56,2 G2=325,7 ± 95,1 G3=226,2 ± 67,4. no se encontraron diferencias estadísticamente significativas entre los grupos estudia-dos (p>0,05). Conclusión: La microdureza del esmalte afectado por fluorosis incipiente sometida a tratamiento con resina infiltrante, esmalte de dientes sanos y esmalte de dientes con fluorosis incipiente no mostró diferencia estadística.

Objective: To evaluate the microhardness of the enamel affected by fluorosis subjected to treatment with infiltrating resin, comparing it with sound teeth and teeth with incipient fluorosis. Materials and methods: The sample consisted of 15 permanent human teeth collected from the Teeth Bank of the Faculty of Odontology of the Central University of Ecuador. For the selection of the teeth, diagnostic criteria of dental fluorosis were considered according to Thylstrup and Ferjeskov (1978), having 5 teeth with score 0 and 10 teeth with incipient fluorosis (score 1-3). None of the teeth that were examined in the study had caries, cracks or fractures. The sample was divided into 3 groups. G1: sound teeth (negative control), G2: teeth with incipient fluorosis and G3: teeth with incipient fluorosis treated with Icon® infiltrating resin. To each group (n=5), dental prophylaxis was performed and afterwards to G3, infiltrating resin was applied. The Knoop micro-hard-ness was obtained through 3 indentations with microhardness tester (Wilson Tukon Microhardness Tester). The data were analyzed through the Spearman's Rho method with 5% significance. Results: The median of Knoop microhardness of the groups and their standard deviations were: G1=284.8 ± 56.2 G2=325.7 ± 95.1 G3=226.2 ± 67.4, no statistically significant differences were found between the examined groups (p>0.05). Conclusion: The microhardness of the enamel affected by incipient fluorosis subjected to treatment with infiltrating resin, enamel of sound teeth, and enamel of teeth with incipient fluorosis did not demonstrate statistical difference.

RESUMOObjetivo: Avaliar a microdureza do esmalte afetado com fluorose incipiente submetido ao tratamento com resina infiltrada, fazendo uma comparação com dentes higidos dentes com fluorose incipiente. Materiais e métodos: A amostra estava constituída por 15 dentes permanentes humanos coletados do Banco de Dientes de la Facultad de Odontología de la Uni-versidad Central del Ecuador (Banco de dentes da Faculdade de Odontologia da Universidade Central do Equador). Para a seleção dos dentes se considerou os critérios de diagnóstico de fluorose dental segundo Thylstrup y Ferjeskov (1978), tendo 5 dentes com pontuação de 0 e 10 dentes com fluorose incipiente (pontuação de 1 a 3). Todos os dentes considerados no estudo não apresentaram lesões de cárie, rachaduras nem fraturas. A amostragem foi dividida em 3 grupos: G1: dentes higidos (controle negativo); G2: dentes com fluorose incipiente e G3: dentes com fluorose incipiente tratados com resina infiltrante Icon®. Para cada grupo (n=5) uma profilaxia foi realizada, e posteriormente ao G3 se aplicou a resina infiltrante. A microdureza knoop foi obtida mediante três entalhes com microdurómetro (Wilson Tukon Microhardness Tester). Os dados foram analisados através do método de Rho de Spearman com um nivel de 5%. Resultados: A média da microdu-reza knoop dos grupos e seus desvios padrões foram de G1=284,8 ± 56,2; G2=325,7 ± 95,1; G3=226,2 ± 67,4, não foram estatisticamente encontradas diferenças significativas entre os grupos estudados (p>0,05). Conclusão: A microdureza do esmalte afetado com fluorose incipiente submetido ao tratamento com resina infiltrante, esmalte de dentes hígidos e de dentes com fluorose incipiente demostrou não ter diferença estatisticamente significativa.

Influence of substratum position and acquired pellicle on Candida albicans biofilm

The purpose of this study was to evaluate the influence of the substratum position and the saliva acquired pellicle (AP) on Candida albicans biofilm development. Poly(methylmethacrylate) (PMMA) disks were fabricated and randomly allocated to experimental groups: HNP (disks placed in a horizontal position and uncoated by pellicle), VNP (disks placed in a vertical position and uncoated by pellicle), HCP (disks placed in a horizontal position and coated by pellicle), and VCP (disks placed in a vertical position and coated by pellicle). Disks were placed in a 24-well plate and a suspension of 107 cells/mL of Candida albicans was added to each well for biofilm development. The plates were aerobically incubated at 35°C. The biofilms were evaluated at 1.5 (adhesion time point), 24, 48, 72, and 96 hours. The number of viable cells was quantified in terms of the colony-forming units per milliliter (CFU/mL). Metabolic activity was measured by the XTT assay. The biofilm structure was analyzed by scanning electron microscopy. The data were analyzed by three-way ANOVA followed by Tukey's test, with significance set at 5%. The vertical groups showed less biofilm formation and lower metabolic activity than the horizontal groups (p< 0.05). Significant differences in cell viability and metabolic activity were observed between the adhesion and other time points (p< 0.05), but these variables were not affected by the presence of the pellicle (p > 0.05). It can be concluded that the substratum position influenced biofilm development.

Saliva and dental erosion

Dental erosion is a multifactorial condition. The consideration of chemical, biological and behavioral factors is fundamental for its prevention and therapy. Among the biological factors, saliva is one of the most important parameters in the protection against erosive wear. Objective: This review discusses the role of salivary factors on the development of dental erosion. Material and Methods: A search was undertaken on MeDLINe website for papers from 1969 to 2010. The keywords used in the research were "saliva", "acquired pellicle", "salivary flow", "salivary buffering capacity" and "dental erosion". Inclusion of studies, data extraction and quality assessment were undertaken independently and in duplicate by two members of the review team. Disagreements were solved by discussion and consensus or by a third party. Results: Several characteristics and properties of saliva play an important role in dental erosion. Salivary clearance gradually eliminates the acids through swallowing and saliva presents buffering capacity causing neutralization and buffering of dietary acids. Salivary flow allows dilution of the acids. In addition, saliva is supersaturated with respect to tooth mineral, providing calcium, phosphate and fluoride necessary for remineralization after an erosive challenge. Furthermore, many proteins present in saliva and acquired pellicle play an important role in dental erosion. Conclusions: Saliva is the most important biological factor affecting the progression of dental erosion. Knowledge of its components and properties involved in this protective role can drive the development of preventive measures targeting to enhance its known beneficial effects.

Película adquirida do esmalte - a importância de seus componentes para a proteção contra a desmineralização do esmalte dentário / Enamel pellicle (AEP) formed by different components against the in vitro dental enamel

O presente estudo teve como objetivo avaliar o efeito protetor da película adquirida do esmalte (PAE), formada por diferentes componentes, contra a desmineralização in vitro do esmalte dentário. Na primeira etapa, uma revisão sistemática da literatura buscou evidências sobre a relação entre proteínas salivares e a doença cárie. Foram selecionados estudos observacionais controlados comparando a presença e/ou concentração de proteínas salivares entre indivíduos cárie-resistentes (CPO=0) e cárie-susceptíveis (CPO>0). A busca eletrônica pelos estudos foi feita nas bases de dados PubMed, Ovid Medline, ISI Web of Science, Medline, Cochrane, Lilacs, Scielo, BBO, Paho e Wholis, usando os descritores “dental caries” OU “tooth demineralization” OU “dental caries susceptibility” OU “dental enamel solubility” E“salivary proteins and peptides” OU “saliva” E “proteins”. De 188 estudos identificados, somente nove foram incluídos na revisão sistemática e cinco apresentaram evidência de relação entre proteínas salivares e cárie dental. Contudo, apesar de alta evidência encontrada, mais estudos com metodologia bem definida são necessários para que se possa identificar proteínas salivares como biomarcadores para a doença cárie. Na segunda etapa, um estudo in vitro avaliou o efeito protetor da PAE, formada por diferentes amostras salivares, contra a desmineralização do esmalte. Espécimes dentários foram expostos, por duas horas, à saliva total (ST), saliva da parótida (SP), ST dializada e SP dializada, permitindo a adsorção de proteínas sobre o esmalte dentário e, conseqüentemente, a formação de PAE. A diálise das amostras salivares foi realizada com membrana de peso molecular de corte igual a 1 kDa, o que permitiu a remoção de íons, principalmente, o cálcio e fosfato, da amostras salivares. Para o grupo controle foi empregada água deionizada (sem a presença de íons ou proteínas), não havendo assim a formação de PAE sobre os espécimes dentários deste grupo...

Influência da formação de película adquirida e aplicação tópica de flúor na microdureza do esmalte tratado com peróxido de hidrogênio a 35 por cento / Influence of acquired pellicle formation and topical fluoride application on microhardness of bleached enamel with 35 per cent hydrogen peroxide

O objetivo deste estudo foi avaliar a influência da formação da película adquirida (PEA) e da aplicação tópica de flúor (ATF) após o tratamento com peróxido de hidrogênio a 35% na microdureza Knoop do esmalte. Foram obtidas 120 amostras de esmalte (4x4x4 mm), a partir das superfícies vestibulares de 60 incisivos bovinos. As amostras foram preparadas para a leitura de microdureza de superfície (inicial) e aleatoriamente divididas em quatro grupos (n=30): (1) Esmalte sem formação de PEA e sem ATF pós-tratamento clareador (controle); (2) Esmalte sem formação de PEA e com ATF pós-tratamento clareador; (3) Esmalte com formação de PEA e sem ATF pós-tratamento clareador; (4) Esmalte com formação de PEA e com ATF pós-tratamento clareador. Os dentes foram submetidos a 12 dias de ciclagem de pH, concomitante com o clareamento (Pola Office, SDI, Bayswater, Victoria, Austrália), que foi realizado no 1º, 6º e 12º dia de ciclagem. Após a ciclagem de pH, foi realizada a leitura da microdureza superficial final e da microdureza longitudinal do esmalte tratado. Todos os grupos experimentais mostraram redução da microdureza superficial do esmalte após os tratamentos realizados. Os valores médios (iniciais e finais) foram semelhantes entre os grupos experimentais. Com relação à microdureza longitudinal, somente na primeira profundidade (10 μm) observou-se redução significativa da microdureza, com relação às demais profundidades analisadas. Esses valores médios, em 10 μm, não diferiram entre os grupos experimentais, assim como as outras profundidades analisadas também não diferiram entre os grupos. A microdureza do esmalte não foi afetada pela formação de PEA, tampouco pela ATF

The objective of this study was to evaluate the influence of acquired salivary pellicle formation (SPF) and the topical application of fluoride (TAF) post-bleaching with 35% hydrogen peroxide on the Knoop microhardness of enamel. Sixty bovine incisors were used. One hundred twenty enamel blocks (4x4x4 mm) were obtained from the buccal surfaces of these teeth. The enamel surfaces were prepared for microhardness measurements (baseline) and randomly divided into 4 groups (n=30): 1) enamel without SPF and TAF (control); 2) enamel without SPF and treated with ATF; 3) enamel with PEA and without ATF; and 4) enamel with PEA and treated with TAF. The enamel blocks were submitted to a 12-day pH-cycling, and the bleaching agent (Pola Office, SDI, Bayswater, Victoria, Australia) was applied at the 1st, 6th and 12th days of cycling. After the treatments, the surface microhardness and the internal microhardness were measured. All the groups showed a reduction in the enamel surface microhardness after the treatments. The mean values (baseline and final) were similar among the groups. The internal microhardness was lower at the first depth (10 μm). The mean values of microhardness at 10 μm were similar among the groups, and the other means did not differ among them. SPF and TAF did not affect enamel microhardness

Oral streptococci growth on aging and non-aging esthetic restorations after radiotherapy

The aim of this study was to examine Streptococcus mutans biofilm growth on both aged and non-aged restorative dental resins, which were submitted to therapeutic irradiation. Sixty-four disks of an esthetic restorative material (Filtek Supreme) were divided into 2 groups: aged group (AG) and a non-aged group (NAG). Each group was subdivided into 4 subgroups: non-irradiated and irradiated with 10Gy, 35Gy, and 70Gy. The biofilms were produced by Streptococcus mutans UA159 growing on both AG and NAG surfaces. The colony-forming units per mL (CFU/mL) were evaluated by the ANOVA and the Tukey LSD tests (α=0.05). AG presented smaller amounts of CFU/mL than the NAG before irradiation and after 10Gy of irradiation (p<0.05). AG irradiated with 35 and 70Gy showed increased amount of bacterial biofilm when compared to non-irradiated and 10Gy-irradiated disks (p<0.05). The exposure to ionizing radiation at therapeutic doses promoted changes in bacterial adherence of aged dental restorative material.

O objetivo deste estudo foi avaliar a formação do biofilme de Streptococcus mutans crescido em resina restauradora envelhecida e não-envelhecida, submetidas à radiação terapêutica. Sessenta e quatro discos do material restaurador Filtek Supreme foram divididos em 2 grupos: grupo envelhecido (AG) e grupo não-envelhecido (NAG) e cada grupo foi dividido em 4 sub-grupos: não-irradiado e irradiado com 10Gy, 35Gy e 70Gy. O biofilme de S. mutans UA159 foi produzido na superfície de ambos os discos AG e NAG. As unidades formadoras de colônia/mL (UFC/mL) foram avaliadas por ANOVA e teste de Tukey (α=0,05). O grupo AG demonstrou menores quantidades de UFC/mL que o grupo NAG antes da radiação e após a radiação de 10Gy (p<0,05). Os sub-grupos AG irradiados com 35 e 70Gy demonstraram aumento na quantidade de biofilme quando comparado aos não irradiados e irradiados com 10Gy (p<0,05). A exposição à radiação ionizante nas doses terapêuticas promoveu mudanças na aderência bacteriana no material restaurador.

The cariogenic dental biofilm: good, bad or just something to control?

This paper discusses the role of dental biofilm and adjunctive therapies in the management of dental caries. Dental biofilm is a site of bacterial proliferation and growth, in addition to being a location of acid production. It also serves as a reservoir for calcium exchange between the tooth and saliva. The salivary pellicle, a protein-rich biofilm layer, regulates the reaction between tooth surface, saliva and erosive acids. The protective effects of this pellicle on enamel are well established. However, understanding the effects of the pellicle/biofilm interaction in protecting dentin from erosive conditions requires further research. Saliva interacts with the biofilm, and is important in reducing the cariogenic effects of dental plaque as acidogenic bacteria consume fermentable carbohydrates producing acids that may result in tooth demineralization. Adequate supplies of healthy saliva can provide ingredients for successful remineralization. Strategies for managing the cariogenic biofilm are discussed with emphasis on the effectiveness of over-the-counter (OTC) products. However, since many toothpaste components have been altered recently, new clinical trials may be required for true validation of product effectiveness. A new generation of calcium-based remineralizing technologies may offer the ability to reverse the effects of demineralization. Nevertheless, remineralization is a microscopic subsurface phenomenon, and it will not macroscopically replace tooth structure lost in a cavitated lesion. Optimal management of cavitations requires early detection. This, coupled with advances in adhesive restorative materials and microsurgical technique, will allow the tooth to be restored with minimal destruction to nearby healthy tissue.

Bond strength of a pit-and-fissure sealant associated to etch-and-rinse and self-etching adhesive systems to saliva-contaminated enamel: individual vs. simultaneous light curing

This study evaluated in vitro the shear bond strength (SBS) of a resin-based pit-and-fissure sealant [Fluroshield (F), Dentsply/Caulk] associated with either an etch-and-rinse [Adper Single Bond 2 (SB), 3M/ESPE] or a self-etching adhesive system [Clearfil S3 Bond (S3), Kuraray Co., Ltd.] to saliva-contaminated enamel, comparing two curing protocols: individual light curing of the adhesive system and the sealant or simultaneous curing of both materials. Mesial and distal enamel surfaces from 45 sound third molars were randomly assigned to 6 groups (n=15), according to the bonding technique: I - F was applied to 37 percent phosphoric acid etched enamel. The other groups were contaminated with fresh human saliva (0.01 mL; 10 s) after acid etching: II - SB and F were light cured separately; III - SB and F were light cured together; IV - S3 and F were light cured separately; V - S3 and F were light cured simultaneously; VI - F was applied to saliva-contaminated, acid-etched enamel without an intermediate bonding agent layer. SBS was tested to failure in a universal testing machine at 0.5 mm/min. Data were analyzed by one-way ANOVA and Fisher's test (α=0.05).The debonded specimens were examined with a stereomicroscope to assess the failure modes. Three representative specimens from each group were observed under scanning electron microscopy for a qualitative analysis. Mean SBS in MPa were: I-12.28 (±4.29); II-8.57 (±3.19); III-7.97 (±2.16); IV-12.56 (±3.11); V-11.45 (±3.77); and VI-7.47 (±1.99). In conclusion, individual or simultaneous curing of the intermediate bonding agent layer and the resin sealant did not seem to affect bond strength to saliva-contaminated enamel. S3/F presented significantly higher SBS than the that of the groups treated with SB etch-and-rinse adhesive system and similar SBS to that of the control group, in which the sealant was applied under ideal dry, noncontaminated conditions.

Este estudo avaliou in vitro a resistência ao cisalhamento (RC) de um selante resinoso [Fluroshield (F), Dentsply/Caulk] em associação com um sistema adesivo de condicionamento total [Adper Single Bond 2 (SB), 3M/ESPE] ou auto-condicionante [Clearfil S3 Bond (S3), Kuraray Co., Ltd.] após contaminação salivar do esmalte, comparando dois protocolos: fotopolimerização individual do sistema adesivo e do selante ou simultânea de ambos os materiais. Superfícies mesiais e distais de esmalte de 45 terceiros molares hígidos foram aleatoriamente alocadas em 6 grupos (n=15), de acordo com a técnica adesiva empregada: I - F foi aplicado sobre o esmalte condicionado com ácido fosfórico a 37 por cento. Os demais grupos foram contaminados com saliva (0,01 mL por 10 s) após o condicionamento ácido. II - SB e F foram fotopolimerizados separadamente; III - SB e F foram fotopolimerizados simultaneamente; IV - S3 e F foram fotopolimerizados separadamente; V - S3 e F foram fotopolimerizados simultaneamente; VI - F foi aplicado sobre o esmalte condicionado e contaminado sem sistema adesivo. RC foi testada em uma máquina universal de ensaios (0,5 mm/min; 50 kgf) e os dados analisados por ANOVA a 1 fator e teste exato de Fisher (α=0,05). As interfaces adesivas foram analisadas quanto ao padrão de fraturas em estereomicroscópio. Três espécimes de cada grupo foram analisados qualitativamente em microscópio eletrônico de varredura. As médias de RC em MPa foram: I-12,28 (±4,29); II-8,57 (±3,19); III-7,97 (±2,16); IV-12,56 (±3,11); V-11,45 (±3,77); e VI-7,47 (±1,99). Conclui-se que a fotopolimerização individual ou simultânea do sistema adesivo e do selante não afetou os valores de RC ao esmalte contaminado. S3/F apresentou RC estatisticamente maior do que os grupos tratados com o sistema adesivo etch-and-rinse SB e estatisticamente semelhante ao grupo controle, no qual o selante foi aplicado em condições ideais, na ausência de contaminação salivar.

Película adquirida salival: revisión de la literatura / Acquired salivary pellicle: a review of the literature

En este artículo de revisión se presenta y analiza la información actualizada disponible sobre la composición química, mecanismo de formación y factores que afectan la producción de la película adquirida salival. Asimismo se discuten aspectos vinculados con la función que cumple dicho integumento, en especial la relacionada con su desempeño como antecesor de la placa bacteriana de la cual dependen las afecciones de mayor prevalencia e incidencia en odontología, como son la caries dental y la enfermedad periodontal.

In this article is presented and analyzes the information brought up to date on the chemical composition, mechanism of formation and factors that affect the production of salivary acquired pellicle. Likewise aspects related to the function are discussed that complies said integument, especially it related to its performance as the ancestor of the bacterial plaque of which the affections of grater prevalence and incidence in dentistry they depend, like are the dental decay and the periodontal illness.

Effect of dental film on adhesive bond strength to enamel using different adhesive systems

The purpose of this study was to evaluate the effect of the dental film, forme in vitro, on the adhesive bond strength of resins to enamel. Fragments of previously planed bovine enamel were submitted to different periods of time in contact with human saliva, to form dental film (1, 12 and 24 hours). Next, the following adhesive systems were used: Scotchbond Multi-Purpose (3M ESPE, Brazil), and Clearfil SE Bond (Kuraray, Osaka, Japan) and over them an approximately 4 mm thick layer of composite resin Z100 (3M ESPE, Brazil)was constructed. After 24 hours of storage, the specimens were sectioned to obtain sticks with a cross sectional area of approximately 1 mm², which were submitted to microtensile tests in an Instron testing machine. Statistical analysis of the results, by means of the ANOVA and Tukey tests showed similarities between salivary contact periods using the Clearfil SE Bond (p=0.108) adhesive system. For the Scotchbond Multi-Purpose adhesive system, the ANOVA and Dunnett tests detected statistically significant differences between the 1 and 12 hour groups (p=0.010), between 1 and 24 hour groups (p=0.003), and between the 1 hour and control groups (p=0.021). Comparing the two adhesives used, the Student-t test detected similarity between adhesive bond strength values obtained at different periods of salivary contact and the two systems tested. Adhesive failure was the most frequent type of fracture observed in both systems used, in the 12 hours and 24 hours groups. Cleaning procedures prior to the application of the adhesive systems to enamel are indicated.

Adhesion of Streptococcus mutans to salivary proteins in caries-free and caries susceptible individuals

La adhesión de los microorganismos a las superficies dentales, es el paso inicial en la formación de la placa dentobacteriana, Streptococcus mutans (S. mutans) es uno de los encontrados en ésta y está asociado como el principal agente causal de una delas enfermedades más comunes en los humanos, la caries dental. La adherencia de esta bacteria se da por la interacción de adhesinas que la constituyen con los componentes salivales, específicamente con los que están formando parte de la película adquirida. La complejidad de esta interacción ha sido motivo deestudio durante los últimos años, hasta el punto de identificar algunos componentes salivales relacionados con la unión a las superficies del esmalte, tales como Proteínas ricas en prolina(PRPs), staterinas,Histatinas,Cistatinas, etc. En el presente trabajo se buscó determinar la capacidad de adhesión de S. mutans a hidroxilapatita sintética incubada con muestras de salivade personas con y sin experiencia de caries. Para estos ensayos se utilizó tanto la muestra de saliva completa como extractos de proteínas salivales, obtenidos por medio de la separaciónde las proteínas contenidas en la muestra por SDS-PAGE, en tres rangos de peso molecular seleccionados de acuerdo con el perfil electroforético que fue comúnmente encontrado. Losresultados indican que la adhesión de este microorganismo es mayor en las personas sin experiencia de caries cuando se ensayó con saliva completa y con las proteínas separadas en el rangode peso molecular de 120-159 kDa. Sugiriendo que la adhesión por sí sola no tiene un efecto determinante en los mecanismos que producen la enfermedad en algunas personas. Sin embargo estos hallazgos son interesantes ya que pueden contribuir en el diseño de estrategias para intervenir en la adhesión de S. mutanssobre las superficies dentales

Adhesion of microorganisms to dental surfaces is the initial step in the formation of dental bacterial plaque. Streptococcus mutans (S. mutans) is considered the main causal agent of one of the most common diseases in humans: dental caries. Adherence of these bacteria results from the interaction of adhesins that form part of their structure with salivary components, specifically those that compose the acquired pellicle. The complexity of this interaction has been the subject of studies in past years, to the extent of identifying certain salivary components related to adhesion to enamel surfaces, such as proline-rich proteins (PRSs), Staherins, Histatins, Cystatins, etc. One of the objectives of this study was to determine the adhesion capacity of S. mutans to synthetic hydroxyapatite incubated with saliva samples of caries-active and caries-inactive individuals. For the purpose of these assays, both the whole saliva samples and the salivary protein extracts were used. They were obtained by separating the proteins contained in the simple SDS-PAGE, in three ranges of molecular weight, selected in accordance with the electrophoresis profile that was usually found. The results indicated that the adhesion of this microorganism was greater in caries-inactive patients, when tested with whole saliva and proteins in the 120-159 kDa molecular weight range. This suggests that adhesion, per se, does not have a definite effect on the mechanisms that cause the disease in some individuals. However, these are interesting findings that may contribute to the design of strategies to control the adhesion of S. mutans to the tooth’s surface.

Adhesion of salivary components to Streptococcus mutans peptides

Streptococcus mutans es el principal microorganismo asociadoa la caries dental, esta bacteria se une al esmalte a través de su interacción con las proteínas de la película adquirida yla proteína de superficie celular comúnmente denominada PAc. Por lo menos dos sitios de PAc interactúan in vitro con los receptores salivales, uno está dentro de la región más conservadade esta proteína que comprende los residuos de 816-1213 y el otro dentro de la secuencia rica en Alanina, residuos186-469. El objetivo del presente trabajo fue establecer similitudes o diferencias en la interacción de péptidos de PAc con los componentes salivales de individuos con y sin experienciade caries, para lo cual se tomaron muestras de saliva por salivación espontánea de 20 individuos con caries y 20 sin caries. A partir de las muestras de saliva se extrajeron las proteínas de la película adquirida (PA) utilizando hidroxilapatita sintética y fueron sometidas a la interacción con trespéptidos sintéticos de los segmentos de unión de PAc con los componentes salivales: PAc (301-319), PAc (365-377) y PAc (1024-1044). Los resultados muestran una baja interacciónentre los componentes de la PA y los péptidos en todos los individuos, sugiriendo que con base en las similitudes entre los individuos sanos y los individuos con la enfermedad lospéptidos de PAc estudiados no son relevantes en la adhesión

Streptococcus mutans is the main microorganism associated to dental caries; it adheres to the dental enamel by interacting with the acquired film’s proteins and the cell surface adhesin, called variously antigen PAc. At least two distinct sites in PAc interact with salivary receptors in vitro, these are within residues 816-1213, the most conserved portion of PAc, and within residues 186-469, the alanine-rich sequence. Our purpose was to establish differences or similarities in PAc’s peptides interactions with the salivary components of individuals with and without previous caries experience. 40 saliva samples were obtained from patients with (n=20) and without (n=20) caries. The acquired film’s proteins were extracted using hydroxyapatite, and subjected to interaction with three synthetic PAc peptides (PAc (301-319), PAc (365-377), and PAc (1025-1044)) synthesized from PAc’s bonding sites to the salivary components. The results show low interaction between the acquired pellicle components and the peptides in all patients. This suggests that the examined PAc’s are not relevant as far as the initial adhesion of Streptococcus mutans to the tooth’s surface is concerned, as defined by the similarities in the results for healthy and affected individuals.