published_or_final_version / abstract / Dentistry / Doctoral / Doctor of Philosophy

<p>In the past two decades studies have indicated that oral health might be associated with the prevalence for cardiovascular disease (CVD), although the biological link still remains unknown. Bacteria and inflammatory mediators causing periodontal disease have also been suggested to influence the progression of atherosclerosis.</p><p>The aims of this thesis were to study oral inflammation and associations between different oral health parameters and CVD. </p><p>Inflammatory mediators such as interleukin-1 (IL-1) as well as bone resorption activity (BRA) were significantly higher in gingival crevicular fluid (GCF) from sites with periodontal disease compared to healthy sites. Treatment resulted in a reduction of BRA as well as the levels of IL-1 for most of the diseased pockets. The levels of IL-1 were not correlated to the amount of BRA.</p><p>Number of teeth (NT) was consistently associated to CVD and was the only oral health parameter that related to all-cause mortality and mortality in CVD in a dose-dependent manner. Subjects with <10 teeth had a 7-fold increase risk for mortality in coronary heart disease compared to those with >25 teeth. Furthermore, NT was also significantly associated to the levels of leukocytes and to the metabolic syndrome, which consists of a combination of cardiovascular risk factors.</p><p>Other investigated oral health parameters, such as severity of periodontal disease, number of deepened pockets, and bleeding on probing, were not related to CVD in a consistent way.</p><p> Oral health parameters as well as myocardial infarction (MI) were related to serum antibody levels against Porphyromonas gingivalis (Pg), indicating that Pg might be a link between oral health and MI.</p><p>In conclusion, treatment reduced the increased levels of IL-1 and BRA in GCF from sites with periodontal disease. Oral health was associated to CVD with number of teeth being the only oral health parameter that consistently was associated to CVD. Serum antibody levels against P. gingivalis were related to myocardial infarction (MI) as well as to oral health parameters, suggesting that this bacteria could be a link between oral health and CVD.</p>

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The patient population consisted of a maximum of 18,570 subjects who completed the NHANES III questionnaire and examination from 1988 - 1994. The physical examination included such things as body mass index and serum samples, social and medical history. The periodontal examination recorded probing depth, attachment loss and gingival bleeding. Serum samples were analyzed for CRP levels, cholesterol levels etc. Demographic, cardiovascular and oral health variables were compared in subjects with a history of heart attack. Result showed that history of heart attack is associated with increased odds ratio for elevated CRP, diabetes, hypertension, cholesterol, male gender, non-white race and smoking. Of the periodontal indicators of disease, only gingival bleeding had an increased odds ratio for association with heart attack history. The unadjusted odds ratio was 1.25 with 95% CI[0.84-1.87]. The adjusted odds ratio increase to 1.93 with 95% CI [1.02-3.71]. These findings are consistent with previous research indicating that elevated CRP is associated with increased risk of heart attack. The interesting finding of this study is that only gingival bleeding, not probing depth or attachment loss, had an increased odds ratio for an associated with self-reported history of heart attack.

Porphyromonas gingivalis (P. gingivalis) is a gram-negative, anaerobic bacterium recognized as a major player in progression of periodontal disease. P. gingivalis survives in the oral cavity while being exposed to dynamic environmental conditions such as pH, temperature, nutrient availability and host immune responses such as oxygen tension and nitrosative stress. Survival and pathogenesis of P. gingivalis in the oral cavity require mechanisms to regulate gene expression in response to the extracellular signals. Little is known about the regulatory mechanisms of P. gingivalis in the oral cavity, so it is important to investigate and characterize these regulatory mechanisms. Adaptation to environmental cues using riboregulation is a significant mechanism for post-transcriptional regulation in bacteria. Using bioinformatics, we have identified a putative RNA-binding protein in P. gingivalis: RBP. Bioinformatic studies have led to the selection of HUβ and HUα nucleoid associated proteins as controls for RNA binding. I hypothesize that the candidate proteins RBP, HUβ and HUα bind RNA in P. gingivalis. The first aim is to show that RBP, HUβ and HUα bind RNA. Using electrophoretic mobility shift assays with IRE RNA and synthesized RNA motifs, I have confirmed that the proteins do bind RNA. The second aim is to isolate and sequence the P. gingivalis RNA that bind to RBP, HUβ and HUα. I have isolated the RNAs that bound the proteins and determined identity of the RNA using high throughput sequencing. Finally, I have identified an antibody that specifically binds RBP to use for in vivo immunoprecipitation of RNA-protein complexes from P. gingivalis. In conclusion RBP, HUβ and HUα are novel RNA binding proteins in P. gingivalis, and further investigation of these proteins is necessary to understand the mechanisms of gene regulation in P. gingivalis.

Porphorymonas gingivalis is one of the major bacterial pathogens responsible for the initiation and progression of periodontal disease. The bacterium requires hemin uptake for its growth and has developed sophisticated mechanisms to extract hemin from hemin containing proteins in the oral cavity. Hemin first binds to receptors on the surface of P. gingivalis and is then taken up in an energy dependent manner. TonB is an inner membrane bound protein that spans the periplasm and is believed to be involved in the passage of hemin through the double membrane of P. gingivalis. However, the TonB protein in P. gingivalis is yet to be identified. We identified PG0785 as a possible P. gingivalis TonB based on its bioinformatics data showing similarity to other known TonB proteins. We generated a P. gingivalis mutant lacking a functional PG0785 and then characterized the mutant to determine the role of PG0785. We performed metal content and protease assays, virulence studies and transcriptional and translational analysis of our mutant and wild type P. gingivalis strains. Phenotypic studies showed that the mutant cannot accumulate hemin on its surface. The mutant has significantly lower levels of iron compared to wild type based on metal content assays. The mutant also has significantly lower protease activity compared to the wild type. Virulence studies showed that the mutant interacted and invaded eukaryotic cells at much lower levels than the wild type. These results allowed us to speculate that PG0785 is very important in binding of hemin to surface of P. gingivalis. PG0785 also plays an important role in iron uptake, protease activity and virulence of P. gingivalis. Transcriptional and translational analyses have shown that numerous TonB related genes, metal uptake genes, hemin uptake genes and genes related to virulence have been differentially regulated in the mutant lacking a functional PG0785 gene compared to the wild type strain. In conclusion we believe that based on our results PG0785 is a putative P. gingivalis TonB protein that plays a significant role in the biology of P. gingivalis.

Existem poucos estudos na área de periodontia envolvendo indivíduos portadores de fissura labiopalatina; pesquisas que avaliem as condições periodontais destes pacientes durante tratamento ortodôntico são ainda mais escassas. O objetivo deste trabalho foi fazer um estudo da condição periodontal, durante o tratamento ortodôntico, dos pacientes com fissura labiopalatina com comprometimento de rebordo alveolar e avaliar se a região da fissura apresenta maior incidência e severidade de alterações periodontais. O grupo estudado foi selecionado no Ambulatório da Disciplina de Prótese Bucomaxilofacial da Faculdade de Odontologia da Universidade de São Paulo (FO-USP), sendo constituído por 43 pacientes na faixa etária de 11 a 35 anos, portadores de fissuras labiopalatinas, não sindrômicos, já submetidos às cirurgias reparadoras primárias e em tratamento com ortodontia fixa há mais de 6 meses. Durante o exame clínico periodontal da maxila realizamos medidas de profundidade clínica de sondagem, nível clínico de inserção, sangramento à sondagem e índice de placa. Todos os exames foram feitos por um único pesquisador, especialista em periodontia, com calibração prévia. A avaliação periodontal mostrou que a região da maxila afetada pela fissura apresentou maior índice de sangramento à sondagem o que está associado a maior ocorrência de gengivite. Os dentes da região da fissura apresentaram menores profundidades clínicas de sondagem que a média da maxila, indicando que não há maior prevalência de bolsas periodontais na região fissurada. Não encontramos diferenças estatisticamente significantes no nível clínico de inserção dos dentes adjacentes à fissura, sendo assim a presença da fissura não esteve associada a perda óssea. Os pacientes da amostra apresentaram uma profundidade de sondagem rasa e pouca perda de inserção. A doença periodontal em indivíduos portadores de fissura, neste estudo, demonstrou não ter maior incidência ou severidade na região da fissura. / There is a shortage of studies in the periodontics field about individuals with cleft lip and palate, studies that evaluate the periodontal conditions of these patients during orthodontic treatment are even scarcer. The aim of this research was to analyze the periodontal condition during orthodontic treatment, of patients with cleft lip and palate with compromised alveolar bone and evaluate if the cleft region on maxilla presented a higher incidence and severity of periodontal disease. The study group was selected from the Clinical Division of Bucomaxilofacial Prosthodontics subject, from School of Dentistry of University of São Paulo, which consisted of 43 patients between 11 and 35 years old with cleft lip and palate, non-syndromic, who had already undergone primary reconstructive surgeries, and who have been in treatment with fixed orthodontics for more than 6 months. During a periodontal clinical exam of the maxilla, the measurements of depth probing, clinical level of insertion, bleeding on probing and plaque index were carried out. The exams were performed by an experienced periodontal examiner who was calibrated previously. The periodontal evaluation showed that the region of the maxilla affected by the cleft had a higher index of bleeding on probing, which is associated with a higher occurrence of gingivitis. The teeth in the cleft region showed smaller clinical depths of probing than the maxilla average, indicating that there is no greater prevalence of periodontal pockets in the region of the cleft. We did not find any significant statistical differences in the clinical level of insertion of the teeth adjacent to the cleft, so the presence of cleft was not associated with a higher bone loss. Patients in the sample had a shallow probing depth and low insertion loss. This study showed that periodontal disease in individuals with cleft lip and palate occurs in a similar way at all the maxilla.

Existe a hipótese, dentro da medicina periodontal, de uma possível relação entre doença periodontal e doenças cardíacas. Talvez a doença periodontal possa agir como um fator desencadeante para o desenvolvimento da doença cardiovascular. Vinte e oito pacientes portadores de aterosclerose e periodontite crônica, submetidos à intervenção cirúrgica de revascularização cardíaca e endarterectomia coronariana, participaram do estudo, cujo objetivo foi avaliar nas placas de ateroma de artérias coronárias a presença dos periodontopatógenos do complexo vermelho: Porphyromonas gingivalis, Tannerella forsythia e Treponema denticola. O DNA genômico foi extraídos das amostras de ateromas e se constatou a presença das bactérias através da reação em cadeia da polimerase (PCR). Detectou-se nas 28 amostras de ateroma: P. gingivalis em 50%, T. forsythia em 7,1% e T. denticola em 3,6% respectivamente. Estes resultados ajudam a defender a hipótese de que a doença periodontal pode ser um dos muitos fatores envolvidos com o desenvolvimento da doença cardiovascular, sendo mais um motivo para ser precocemente diagnosticada, prevenida e tratada. / In the field of periodontal medicine there is the hypothesis of a possible connection between periodontal diseases and heart diseases. Maybe the periodontal disease may work as a triggering factor in the development of periodontal of cardiovascular diseases. Twenty-eight patients of atherosclerosis and cronical periodontitis patients were subjected to surgical intervention for cardiac revascularization and coronary endarterectomy, have made part of the study, whose objective was to evaluate the presence of periodontopathogens of the red complex in the atheroma platelets: Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola. Genomic DNA was extracted from atheroma samples, and the presence of bacteria was stated through polymerase chain reaction (PCR). In the 28 samples of atheroma were detected: P. gingivalis at 50%, T. forsythia at 7.1% and T. denticola at 3.6% respectively. These results help to sponsor the hypothesis that the periodontal disease can be one of several factors involved in the development of cardiovascular disease, being this another reason for its early diagnosis, prevention and treatment.

O crescente interesse na utilização de biomateriais para reparação óssea levou a inúmeros estudos envolvendo formação de diversos produtos, neste sentido, os compósitos obtidos utilizando-se gelatina (G), quitosana (QS) e hidroxiapatita (HA) destacam-se por serem bioativos e bioabsorvíveis, além de apresentarem elevada similaridade com o tecido a ser reparado. Barreiras físicas formadas por tais materiais são extremamente importantes para a odontologia, e o seguinte trabalho tem como objetivo a preparação de membranas de G/QS/HA a partir de um método de co-precipitação in situ da HA na matriz polimérica para sua utilização em regeneração tecidual guiada (RTG). As membranas contendo razões de 20/80, 50/50 e 80/20 de G/QS com 0,3M de HA foram preparadas com pequena adição de glicerina (0,5% v/v) e suas características morfológicas e físico-químicas foram analisadas por meio de técnicas como espectroscopia no infravermelho (IV), difração de raios X (DRX), microscopia eletrônica de varredura (MEV), espectroscopia por energia dispersiva (EDS) e análise termogravimétrica (ATG). Além disso, foram realizados ensaios de intumescimento e ensaios de citotoxicidade in vitro com cultura de células para verificar a possibilidade de utilização das membranas confeccionadas. O processo de precipitação in situ foi eficaz para formação das mambranas. Além disso, as amostras reticuladas por GTA apresentaram melhores resultados de intumescimento, ao passo que as membranas reticuladas por TPP não se mostraram citotóxicas. / The increasing growth in the usage of bio-composites in bone repair has led to several studies involving the formation of several products. In this sense, composites obtained with gelatin (G), chitosan (QS) and hydroxyapatite (HA) stand out since they are bioactive and biodegradable, besides showing high similarity with the tissue to be repaired. Physical barriers made by such materials are extremely important in orthopedics, and the present work aims to craft G/QS/HA membranes by an in situ co-precipitation method of HA in the polymeric matrix to use them with a treatment called guided bone regeneration (RTG). The membranes containing 20/80, 50/50 and 80/20 weigh percent of G/QS with 0,3M of HA were prepared with a small amount of glycerin (0,5% v/v) and its morphological and physicochemical properties were analyzed by infrared spectroscopy (IV), X-ray diffraction (DRX), scanning electron microscopy (MEV), electron energy loss spectroscopy (EDS) and thermo gravimetric analysis (ATG). On top of that, swelling essays and in vitro cytotoxicity with cell culture were be carried out to check the availability and possible uses of the crafted membranes. The in situ precipitation method was effective to form the membranes. The samples crosslinked with GTA showed better swelling results, however the TPP method yielded non-cytotoxic membranes.

A doença periodontal (DP) corresponde a um grupo de doenças inflamatórias que acomete as estruturas periodontais de proteção e de suporte e pode levar à perda dentária. A etiologia está relacionada à placa dentobacteriana que leva à produção de grande quantidade de citocinas pró-inflamatórias importantes na destruição tecidual. A angiotensina (Ang) II também pode contribuir para a inflamação e destruição tecidual no periodonto agindo como mediador chave. A utilização de drogas que atuem na cascata do sistema renina-angiotensina (SRA) poderia interferir no estado de saúde ou inflamação do tecido mole, na perda óssea alveolar e na expressão gênica dos componentes do SRA e mediadores inflamatórios. Portanto, o objetivo do presente trabalho foi investigar se o ramipril, um inibidor da enzima conversora de angiotensina (ECA), altera a progressão da DP induzida experimentalmente em ratos. Foi utilizado o modelo de indução da DP por colocação de ligadura ao redor do primeiro molar inferior direito de ratos. Os grupos com 10 animais cada, foram divididos em tratados com ramipril (via gavagem 10 mg/kg/dia) ou água (veículo) durante 14 e 21 dias e o grupo Sham submetido à indução fictícia da DP. Outros quatro grupos foram submetidos ao pré-tratamento com ramipril durante os períodos de 7 e 14 dias e após a indução da DP e tratados por 14 ou 21 dias. As metodologias de avaliação foram: extração de RNA total, transcrição reversa seguida de reação em cadeia da polimerase quantitativa (RTqPCR), análises histológica e da perda óssea alveolar. Os dados foram analisados por meio de gráficos e os resultados foram submetidos à análise unidirecional de variância (ANOVA) e representaram médias e respectivos desvios-padrão. Diferenças entre os grupos foram consideradas estatisticamente significativas quando p < 0,05. Com base nos resultados obtidos pode-se concluir que o ramipril foi capaz de reduzir a progressão da perda óssea no grupo tratado por 21 dias (DP-21d-Rami), entretanto houve aumento do processo inflamatório, além de alteração da expressão de RNAm de ECA-2 e do receptor Mas, alguns mediadores do processo inflamatório, como COX2 e VEGF, e os receptores VEGF-R1 e VEGF-R2. / Periodontitis (PD) consists of a group of inflammatory diseases that affect the protecting and supporting periodontal structures, and may lead to tooth loss. The etiology is related to the dentobacterial plaque that produces a large amount of proinflammatory cytokines with an important action on tissue destruction. Angiotensin (Ang) II may also contribute to the inflammation and periodontal tissue destruction by acting as a key mediator. The use of drugs that affect the cascade of the reninangiotensin system (RAS) might interfere with the healthy or inflammatory status of the soft tissue, alveolar bone loss and gene expression of RAS components and inflammatory mediators. Therefore, the aim of this work was to evaluate whether ramipril, an angiotensin-converting enzyme (ACE) inhibitor, alters the progression of experimentally-induced PD in rats. The model of PD induction by placement of a silk ligature around the right lower first molar was used. Groups with 10 animals each were divided into ramipril-treated (10 mg/kg/day, via gavage), water (vehicle) and the sham surgery group (sham) for 7 or 14 days previously to PD induction and after this period the drug was administered for 14 and 21 days. The techniques employed were: total RNA extraction, reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR) as well as histological and alveolar bone loss analyses. Data were analyzed by means of graphs and the results submitted to unidirectional analysis of variance (ANOVA) and represent the means with respective standard deviations. Differences between groups were considered statistically significant when p < 0.05. Based on the results obtained in this work, it was concluded that ramipril was able to reduce the progression of alveolar bone loss in the group treated for 21 days (DP-21d-Rami), however, there was an increase in the inflammatory process, besides altering the expression of ACE-2, Mas receptor and some inflammatory mediators such as COX2, VEGF and the receptors VEGF-R1 and VEGF-R2.