There are a number of unanswered questions regarding new caries preventive methods such as ozone treatment as well as novel caries detection methods that monitor and evaluate these preventive methods. This thesis seeks answers to some of these questions. Aims: Paper I investigates the in vitro sealing capacity of a novel ozone delivery system and its re-suction capacity. Paper II studies the in vitro antibacterial effect of ozone on cariogenic bacterial species with and without the presence of saliva and its possible effect on the saliva proteins. Paper III assesses the in vitro validity of laser-induced fluorescence (LF) measurements, visual inspection (colour), and tactile examination (surface texture) on root caries lesions and correlates these with histopathological measurements of lesion depths.  In addition, Paper III investigates how inter-device, inter-examiner, and intra-examiner levels influence LF reading agreements and whether lesion colour and surface texture influences LF readings. Paper IV evaluates the in vivo effect of ozone and fluoride varnish treatments on occlusal caries in primary molars. Material and methods: Paper I: Full ozone application cycles, including the re-suction period, and interrupted cycles with displacement of the cup during the delivery cycle were studied using an ozone air analyser. Paper II: Ozone gas was exposed to the bacteria in the solution’s buffer and saliva via a tube connected to the ozone generator for 10, 30, and 60 seconds. Paper III: Calibrated examiners assessed lesion colour and surface texture and performed measurements with two LF devices for three separate one-week intervals.  Sections (300µm thick) of 64 out of 93 teeth were obtained and examined under a microscope. Lesion depth was assessed with two references: from the delineated borderline of the original exposed root surface (ref I) or, if loss of surface continuity, the absolute lesion depth (ref II). Paper IV: The split mouth study was conducted in two parts. The participants in part A were children (mean age 4.8 years) with medium-high caries risk. Inclusion criteria were bilateral matched pairs of cavitated or non-cavitated occlusal lesions in primary molars (Ekstrand index score ≤ 3). Children in part B (mean age 4.5 years) with low-medium caries risk had pairs of non-cavitated lesions only (Ekstrand index score ≤ 2a). The assessments and treatments with ozone and fluoride varnish were performed at baseline and at three, six, and nine months. At the 12-month follow-up, only assessments were performed. Results: Paper I: Ozone leakage levels varied between 5.2 and 9.8 µg/m3. Paper II: In the salt buffer, 92%, 73%, and 64% of the initial numbers of A. naeslundii, S. mutans, and L. casei, respectively, were killed after 10 s ozone exposure and approximately 99.9% of the bacteria were dead after a 60 s exposure. After 10 and 30 s but not after 60 s exposure to ozone, S. mutans and L. casei were less efficiently killed in saliva compared to in the salt buffer. Various saliva proteins were degraded by ozone after the 60 s exposure. Paper III: The correlation between LF readings and histological depth was low with values ranging from 0.22 (p ˃ 0.05) to 0.31 (p < 0.05). The LF devices were significantly correlated with discolouration and with a surface texture denoted as hard. A significant correlation was found between colour and histological depth. No significant correlation was found between surface texture and histological depth. The reliability, evaluated as intra-class correlation coefficient, was 0.99 for intra-examiner, 0.97 for inter-examiner, and 0.98 inter-device level. Large differences were found between two consecutive measurements and high measurement errors indicated considerable deviation of individual measurements.Paper IV: In the first 15 pairs of part A, eight lesions treated with ozone and nine treated with fluoride, including all cavitated lesions, progressed to failure, i.e., required operative treatment during the study time. Due to non-acceptable results, the sample collection was discontinued because of ethical reasons. In part B, of 35 pairs, one of the ozone treated lesions failed at 12 months. A small shift towards increased VI scores was recorded for both ozone and fluoride lesions in this second part. Conclusions: - The ozone delivery system can be considered a safe system with low leakage levels in air, also with accidental displacements. - The cariogenic species S. mutans, L. casei, and A. naeslundii were sensitive to ozone gas treatment. The presence of saliva hampered the antibacterial effect of ozone. A low correlation between the LF readings and the histopathological depth of root caries lesions was shown. The LF device was found not to be appropriate for application to root caries diagnosis. - Neither ozone nor fluoride varnish treatments arrested the progression of cavitated occlusal caries lesions. In low and medium caries risk children non-cavitated occlusal lesions remained mainly unchanged during the study period.  No difference in the effect of ozone and fluoride varnish treatments on occlusal caries in primary molars was seen.

A utilização de enxaguatórios bucais é um recurso importante na manutenção da saúde oral, uma vez que esta pode suprir as limitações da higienização tradicional mecânica, devido ao maior acesso às bactérias do biofilme dental. Em função de sua fácil utilização, palatabilidade e poder de refrescância, os enxaguatórios podem ser considerados um produto de fácil adesão, sendo especialmente importantes na manutenção da saúde bucal de usuários com menor destreza ou impossibilidade de realizar uma escovação adequada. Entre os diversos componentes ativos que podem estar presentes nos enxaguatório bucais estão os constituintes de óleos essenciais como mentol, eucaliptol e timol. Uma grande vantagem da utilização de óleos essenciais em produtos para saúde bucal é a existência de uma gama de propriedades biológicas e organolépticas que os mesmos podem conferir às formulações, uma vez que podem atuar como agentes antimicrobianos, inibidores da produção de ácidos e sulfetos voláteis por bactérias orais, antioxidantes, anti-inflamatórios, aromatizantes e flavorizantes simultaneamente. Dessa forma, formulações contendo óleos essenciais podem ser potenciais agentes na prevenção e tratamento das afecções bucais mais comuns como a cárie dental, placa, tártaro, gengivite, periodontite e halitose. O presente trabalho teve por objetivo inicial realizar um estudo comparativo das atividades antimicrobianas e anticariogênicas dos óleos essenciais de Cymbopogon citratus, Illicium verum, Zingiber officinale, Eucalyptus globulus, Tithonia diversifolia e Aldama arenaria, por meio da determinação de suas concentrações inibitórias mínimas (CIM) e concentrações bactericidas mínimas (CBM) e através da medida potenciométrica do pH de suspensões bacterianas, as quais foram tratadas com concentrações seriadas dos óleos essenciais. Entre os componentes voláteis analisados, o óleo essencial das inflorescências de A. arenaria demonstrou-se o mais promissor, apresentando-se ativo contra S. mutans (CIM = 15,6 ?g/mL e CBM = 62,5 ?g/mL) e S. mitis (CIM = 31,2 ?g/mL e CBM = 31,2 ?g/mL) e moderadamente ativo contra L. casei (CIM = 125?g/mL e CBM = 250 ?g/mL) e S. salivarius (CIM = 250 ?g/mL e CBM = 250 ?g/mL). A concentração capaz de inibir 50% da produção de ácidos orgânicos por S. mutans foi de 26,5 ?g/mL. Este óleo essencial foi caracterizado quimicamente e avaliado quanto à sua segurança para incorporação em produtos de higiene bucal. Os principais componentes majoritários identificados foram os sesquiterpenos carotol (12,67%), falcarinol (6,71%) e espatulenol (5,48%). Quanto à citotoxicidade, não foi observado efeito anti-proliferativo do óleo essencial em fibroblastos gengivais humanos tratados com concentrações iguais ou menores que 250 ?g/mL, após 30 minutos de exposição. Assim, esta concentração de óleo essencial foi escolhida para ser veiculada na microemulsão de enxaguatório bucal. A microemulsão obtida apresentou-se como um sistema translúcido e estável, de densidade relativa 1,22 g/mL, viscosidade de 2,28 cP, tamanho de gotícula 24,96 nm, condutividade 1.246,67 ?S/cm e pH final igual a 6,56. Embora o sistema da microemulsão tenha sido caracterizado como físico-quimicamente estável, por meio de análise cromatográfica foi possível constatar que óleo não se encontrava homogeneamente disperso no sistema. Considerando a não homogeneidade da formulação e, visando determinar o real potencial do óleo essencial, optou-se por avaliar seu efeito isoladamente sobre o biofilme de S. mutans. Em concentrações equivalentes a 250 e ii 1.000 ?g/mL, o óleo essencial não apresentou atividade inibitória sobre o crescimento e a produção de ácidos dos micro-organismos sésseis. No entanto, a atividade significativa contra a proliferação e a produção de ácidos orgânicos de S. mutans planctônico, o qual é considerado um dos principais iniciadores do processo de formação da cárie e do próprio biofilme, abrem perspectivas para a aplicação dos constituintes deste óleo essencial em formulações de enxaguatórios bucais. / The use of mouthwashes are an important resource in maintaining oral health as it can overcome the limitations of traditional mechanical cleaning because of their greater access to bacterial biofilm. Due to their ease of use, palatability and freshness, mouthwashes are considered as products of easy adhesibility, being especially important in maintaining oral health of users with less dexterity or inability to perform a proper teeth brushing. Among the several active components which may compose mouthwashes are the constituents from essential oils such as menthol, eucalyptol and thymol. A major advantage of the use of essential oils in oral health products is the range of biological and organoleptic properties that can be confered to the formulations, once these oils can work as antimicrobials that inhibit the production of acids and volatile sulfides by oral bacteria, also as antioxidants, antiinflammatory and flavoring agents simultaneously. Thus, essential oil containing formulations can work for preventing and treating the most common oral diseases such as dental caries, plaque, tartar, gingivitis, periodontitis and halitosis. The initial goal of the present work was to conduct a comparative study of antimicrobial and anticariogenic activities of essential oils extracted from Cymbopogon citratus, Illicium verum, Zingiber officinale, Eucalyptus globulus, Tithonia diversifolia and Aldama arenaria, by determining their minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) and by the potentiometric measurement of the pH from bacterial suspensions treated with serial essential oils concentrations. Among the volatile compounds analyzed, the essential oil from A. arenaria inflorescences was considered the most promising, for being active against S. mutans (MIC = 15.6 ?g/ml and MBC = 62.5 ?g/ml) and S. mitis (MIC = 31.2 ?g/ml and MBC = 31.2 ?g/ml) and moderately active against L. casei (MIC = 125 ?g/mL and CBM = 250 ?g/ml) and S. salivarius (MIC = 250 ?g/ml and MBC = 250 ?g/ml). The concentration that inhibits 50% of organic acids production by S. mutans was determined as 26.5 ?g/mL. This essential oil was chemically characterized and evaluated about its safety for incorporation in oral hygiene products. The major identified constituents were the sesquiterpenes carotol (12.67%), falcarinol (6.71%) and spathulenol (5.48 %). The essential oil displayed no anti-proliferative effects against human gingival fibroblasts treated during 30 minutes with concentrations up to 250 ?g/mL. Thus, this concentration of essential oil was chosen to be conveyed in the microemulsion mouthwash. The microemulsion was characterized as a translucent and stable system, with 1.22 g/ml relative density, 2.28 cP viscosity, 24.96 nm droplet size, 1246.67 ?S/cm of conductivity and final pH 6.56. Although characterized as a physical-chemically stable system, through a chromatograph analysis it was found that the oil was not homogeneously dispersed in the system. Considering the inhomogeneity of the formulation and, in order to determine the real potential of the essential oil, it was decided to evaluate the isolated effect of the oil against S. mutans biofilm. The essential oil displayed no inhibitory activity on the growth and acid production of sessile microorganisms at concentrations equal to 250 and 1,000 ?g/mL. However, the significant activity against proliferation and organic acid production of planktonic S. mutans, which is considered one of the main initiators of dental caries process and biofilms,indicated good perspectives for its constituents application in mouthwashes formulations.

A utilização de enxaguatórios bucais é um recurso importante na manutenção da saúde oral, uma vez que esta pode suprir as limitações da higienização tradicional mecânica, devido ao maior acesso às bactérias do biofilme dental. Em função de sua fácil utilização, palatabilidade e poder de refrescância, os enxaguatórios podem ser considerados um produto de fácil adesão, sendo especialmente importantes na manutenção da saúde bucal de usuários com menor destreza ou impossibilidade de realizar uma escovação adequada. Entre os diversos componentes ativos que podem estar presentes nos enxaguatório bucais estão os constituintes de óleos essenciais como mentol, eucaliptol e timol. Uma grande vantagem da utilização de óleos essenciais em produtos para saúde bucal é a existência de uma gama de propriedades biológicas e organolépticas que os mesmos podem conferir às formulações, uma vez que podem atuar como agentes antimicrobianos, inibidores da produção de ácidos e sulfetos voláteis por bactérias orais, antioxidantes, anti-inflamatórios, aromatizantes e flavorizantes simultaneamente. Dessa forma, formulações contendo óleos essenciais podem ser potenciais agentes na prevenção e tratamento das afecções bucais mais comuns como a cárie dental, placa, tártaro, gengivite, periodontite e halitose. O presente trabalho teve por objetivo inicial realizar um estudo comparativo das atividades antimicrobianas e anticariogênicas dos óleos essenciais de Cymbopogon citratus, Illicium verum, Zingiber officinale, Eucalyptus globulus, Tithonia diversifolia e Aldama arenaria, por meio da determinação de suas concentrações inibitórias mínimas (CIM) e concentrações bactericidas mínimas (CBM) e através da medida potenciométrica do pH de suspensões bacterianas, as quais foram tratadas com concentrações seriadas dos óleos essenciais. Entre os componentes voláteis analisados, o óleo essencial das inflorescências de A. arenaria demonstrou-se o mais promissor, apresentando-se ativo contra S. mutans (CIM = 15,6 ?g/mL e CBM = 62,5 ?g/mL) e S. mitis (CIM = 31,2 ?g/mL e CBM = 31,2 ?g/mL) e moderadamente ativo contra L. casei (CIM = 125?g/mL e CBM = 250 ?g/mL) e S. salivarius (CIM = 250 ?g/mL e CBM = 250 ?g/mL). A concentração capaz de inibir 50% da produção de ácidos orgânicos por S. mutans foi de 26,5 ?g/mL. Este óleo essencial foi caracterizado quimicamente e avaliado quanto à sua segurança para incorporação em produtos de higiene bucal. Os principais componentes majoritários identificados foram os sesquiterpenos carotol (12,67%), falcarinol (6,71%) e espatulenol (5,48%). Quanto à citotoxicidade, não foi observado efeito anti-proliferativo do óleo essencial em fibroblastos gengivais humanos tratados com concentrações iguais ou menores que 250 ?g/mL, após 30 minutos de exposição. Assim, esta concentração de óleo essencial foi escolhida para ser veiculada na microemulsão de enxaguatório bucal. A microemulsão obtida apresentou-se como um sistema translúcido e estável, de densidade relativa 1,22 g/mL, viscosidade de 2,28 cP, tamanho de gotícula 24,96 nm, condutividade 1.246,67 ?S/cm e pH final igual a 6,56. Embora o sistema da microemulsão tenha sido caracterizado como físico-quimicamente estável, por meio de análise cromatográfica foi possível constatar que óleo não se encontrava homogeneamente disperso no sistema. Considerando a não homogeneidade da formulação e, visando determinar o real potencial do óleo essencial, optou-se por avaliar seu efeito isoladamente sobre o biofilme de S. mutans. Em concentrações equivalentes a 250 e ii 1.000 ?g/mL, o óleo essencial não apresentou atividade inibitória sobre o crescimento e a produção de ácidos dos micro-organismos sésseis. No entanto, a atividade significativa contra a proliferação e a produção de ácidos orgânicos de S. mutans planctônico, o qual é considerado um dos principais iniciadores do processo de formação da cárie e do próprio biofilme, abrem perspectivas para a aplicação dos constituintes deste óleo essencial em formulações de enxaguatórios bucais. / The use of mouthwashes are an important resource in maintaining oral health as it can overcome the limitations of traditional mechanical cleaning because of their greater access to bacterial biofilm. Due to their ease of use, palatability and freshness, mouthwashes are considered as products of easy adhesibility, being especially important in maintaining oral health of users with less dexterity or inability to perform a proper teeth brushing. Among the several active components which may compose mouthwashes are the constituents from essential oils such as menthol, eucalyptol and thymol. A major advantage of the use of essential oils in oral health products is the range of biological and organoleptic properties that can be confered to the formulations, once these oils can work as antimicrobials that inhibit the production of acids and volatile sulfides by oral bacteria, also as antioxidants, antiinflammatory and flavoring agents simultaneously. Thus, essential oil containing formulations can work for preventing and treating the most common oral diseases such as dental caries, plaque, tartar, gingivitis, periodontitis and halitosis. The initial goal of the present work was to conduct a comparative study of antimicrobial and anticariogenic activities of essential oils extracted from Cymbopogon citratus, Illicium verum, Zingiber officinale, Eucalyptus globulus, Tithonia diversifolia and Aldama arenaria, by determining their minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) and by the potentiometric measurement of the pH from bacterial suspensions treated with serial essential oils concentrations. Among the volatile compounds analyzed, the essential oil from A. arenaria inflorescences was considered the most promising, for being active against S. mutans (MIC = 15.6 ?g/ml and MBC = 62.5 ?g/ml) and S. mitis (MIC = 31.2 ?g/ml and MBC = 31.2 ?g/ml) and moderately active against L. casei (MIC = 125 ?g/mL and CBM = 250 ?g/ml) and S. salivarius (MIC = 250 ?g/ml and MBC = 250 ?g/ml). The concentration that inhibits 50% of organic acids production by S. mutans was determined as 26.5 ?g/mL. This essential oil was chemically characterized and evaluated about its safety for incorporation in oral hygiene products. The major identified constituents were the sesquiterpenes carotol (12.67%), falcarinol (6.71%) and spathulenol (5.48 %). The essential oil displayed no anti-proliferative effects against human gingival fibroblasts treated during 30 minutes with concentrations up to 250 ?g/mL. Thus, this concentration of essential oil was chosen to be conveyed in the microemulsion mouthwash. The microemulsion was characterized as a translucent and stable system, with 1.22 g/ml relative density, 2.28 cP viscosity, 24.96 nm droplet size, 1246.67 ?S/cm of conductivity and final pH 6.56. Although characterized as a physical-chemically stable system, through a chromatograph analysis it was found that the oil was not homogeneously dispersed in the system. Considering the inhomogeneity of the formulation and, in order to determine the real potential of the essential oil, it was decided to evaluate the isolated effect of the oil against S. mutans biofilm. The essential oil displayed no inhibitory activity on the growth and acid production of sessile microorganisms at concentrations equal to 250 and 1,000 ?g/mL. However, the significant activity against proliferation and organic acid production of planktonic S. mutans, which is considered one of the main initiators of dental caries process and biofilms,indicated good perspectives for its constituents application in mouthwashes formulations.