Os poliomavírus compõem uma grande família de vírus que causam infecções primárias geralmente na infância, e se mantem em condições subclínicas. Em situações de imunossupressão podem causar algumas doenças. Os indivíduos com HIV/AIDS frequentemente apresentam deficiência imunológica e por isso podem exibir maior risco de doenças causadas pelos poliomavírus. A utilização da saliva no diagnóstico e acompanhamento de doenças infecciosas tem sido explorado na literatura. As vantagens de usar a saliva para rastreio se pautam especialmente na coleta não invasiva e segurança no manuseio. O presente estudo teve como objetivo, detectar e quantificar o DNA dos poliomavírus BKV, JCV, de células Merkel e TSV, em fluídos orais (saliva, lavado bucal e fluído gengival crevicular) e comparar com a detecção em soro e urina, meios usualmente utilizados para detecção. Foram coletadas 299 amostras de 42 indivíduos, sendo 22 HIV positivos (GE) e 20 pacientes controle (GC). No GE, 63,6% dos pacientes apresentaram positividade para JCV em pelo menos uma amostra analisada, 54,5% foram positivos para BKV, 18,2% para células Merkel e não houve amostra positiva para TSV. No GC, 45% exibiu positividade para o JCV em pelo menos uma amostra analisada, 80% para BKV e nenhuma participante controle exibiu positividade para células Merkel e TSV. Não houve diferença de frequência de detecção viral entre os grupos estudados em relação às amostras coletadas, ou ainda em relação à idade ou sexo. Entretanto, nas amostras de fluídos orais houve maior prevalência de detecção para o BKV e para células Merkel. Concluímos que fluídos orais, especialmente saliva e lavado bucal, podem ser usados para o rastreamento do BK e JC; e que os indivíduos HIV positivos, sob tratamento antirretroviral não exibem frequências maior de poliomavírus, comparativamente a indivíduos controle. / Polyomavirus is one of the large family of viruses that cause primary infections usually in childhood, and can remain subclinical. In immunosuppression may cause some diseases. Individuals with HIV/AIDS often have immune deficiencies and may be at increased risk for diseases caused by polyomaviruses. The use of saliva in the diagnosis and follow-up of infectious diseases has been explored in the literature. The advantages of using saliva for screening are based on non-invasive collection and handling safety. The aim of present study was to detect and quantify the DNA from BKV, JCV, Merkel cell and TSV polyomaviruses in oral fluids (saliva, mouthwash and gingival crevicular fluid) and to compare it with serum and urine detection, the means usually used for detection. A total of 299 samples were collected from 42 individuals, 22 HIV positive (GE) and 20 control patients (GC). In GE, 63,6% of the patients presented positive for JCV in at least one sample analyzed, 54,5% were positive for BKV, 18,2% for Merkel cell and there was no positive sample for TSV. In GC, 45% showed JCV positivity in at least one analyzed sample, 80% in BKV, and no control participant exhibited positivity for Merkel cell and TSV. There was no difference in the frequency of viral detection among the groups studied in relation to the samples collected, or in relation to age or gender. However, in oral fluid samples there was a higher prevalence of detection for BKV and Merkel cell. We conclude that oral fluids, especially saliva and mouthwash, can be used for the screening of BK e JC; and that HIV positive individuals under antiretroviral treatment do not exhibit higher frequencies of polyomavirus compared to healthy control subjects.

Novas abordagens clínicas para o diagnóstico e monitoramento de pessoas com doenças sistêmicas têm sido empregadas, através da utilização de fluidos biológicos orais, como a saliva e o fluido gengival crevicular (FGC). Alguns autores têm avaliado o potencial desses fluidos no diagnóstico e acompanhamento de doenças, por apresentarem vantagens tais como coleta não invasiva e segurança no manuseio. Até o presente momento, poucos trabalhos detectaram os poliomavírus humano BK (BKV) e JC (JCV) em saliva e nenhum trabalho procurou sua presença no FGC. Esses poliomavírus infectam assintomaticamente cerca de 80% da população geral, mantendo-se latente no trato urinário. No caso de imunossupressão mediada por células, pode ocorrer o aumento da replicação e indução de reação inflamatória. Uma das doenças causadas pela replicação do BKV é a nefropatia associada ao poliomavírus (NAP), caracterizada pela disfunção e perda do próprio rim ou do rim transplantado, enquanto a Leucoencefalopatia Multifocal Progressiva (LMP), causada pela replicação do JCV, infecta os oligodendrócitos, causando desmielinização. Métodos não invasivos para o screening dos poliomavírus podem facilitar a detecção de novos casos e a monitoração de casos previamente conhecidos. O objetivo deste estudo foi verificar a possibilidade de detecção e quantificação do BKV e JCV em fluidos orais (saliva, lavado bucal e FGC) de indivíduos com insuficiência renal crônica (IRC), transplantados renais (TR), e controles em relação ao sangue e urina, fluidos frequentemente usados para esse teste. Para tanto, foram incluídos no estudo 38 sujeitos, divididos em 3 grupos, sendo 14 indivíduos no grupo com IRC (GIR), 12 TR no grupo transplantado renal (GTR) e 12 indivíduos saudáveis no grupo controle (GC). No total, coletamos 283 amostras dos participantes, sendo 151 de FGC, 38 amostras de saliva, 38 de lavado bucal, 35 de soro e 21 amostras de urina. No GIR, 100% (14) dos indivíduos apresentaram positividade para BKV em pelo menos uma amostra analisada e 14% (2) foram positivos para JCV. No GTR, 91,7% (11) dos indivíduos foram positivos para BKV e 51,7% (5) foram positivos para JCV. Dentre os sujeitos do GC, 91,7% (11) foram positivos para BKV e 50% (6) para JCV, em pelo menos uma amostra testada. Não houve diferença de frequência de detecção viral entre os 3 grupos de participantes, com relação às amostras coletadas. As amostras de fluidos orais (saliva, lavado e FGC) exibiram alta prevalência de detecção, principalmente do BKV, com muitas amostras com níveis quantificáveis de carga viral. Concluímos que fluidos orais, especialmente saliva e lavado bucal, podem ser usados para o rastreamento do BKV e JCV. / New clinical approaches for diagnosis and monitoring of individuals with systemic diseases have been employed through the use of oral biological fluids such as saliva and gingival crevicular fluid (GCF). Some authors have evaluated the potential of these fluids in the diagnosis and monitoring of diseases, because they have advantages such as noninvasive collection and safe handling. To date, few studies have demonstrated the detection of human polyomavirus BK (BKV) and JC (JCV) in saliva and no study reached for its presence in GCF. These polyomavirus infect asymptomatically around 80% of general population, remaining latent in the urinary tract. In case of immunosuppression mediated by cells, there is increased inflammation and induction of replication. One of the diseases caused by BKV replication is polyomavirus associated to the nephropathy (PVAN), characterized by the dysfunction or loss of the kidney or transplanted kidney, while the progressive multifocal leukoencephalopathy (PML) is caused by replication of JCV, infects oligodendrocytes causing demyelination. Noninvasive screening could facilitate the detection of new cases and monitoring of cases previously known. The objective of this study was to investigate the possibility of BKV and JCV detection and quantification in oral fluids (saliva, mouthwash and GCF) of individuals with chronic kidney failure (CKF), kidney transplantation (KT), and controls compared with blood and urine, often used for this test. Therefore, we included 38 subjects, divided into 3 groups, being 14 individuals with CKF (KFG), 12 individuals with KT (KTG) and 12 healthy control individuals (CG). In a total, we collected 283 samples, being 151 of GCF, 38 of saliva, 38 of mouthwash, 35 of serum and 21 samples of urine. In the KFG, 100% (14) of the individuals were positive for BKV in at least one of the collected sample and 14% (2) were positive for JCV. In the KTG, 91.7% (11) were positive for BKV and 51.7% for JCV. Among the subjects of the CG, 91.7% (11) were positive for BKV and 50% (6) to JCV, in at least one tested sample. There was no difference in viral detection frequency between the 3 studied groups with respect to the collected samples. Oral fluids samples (saliva, mouthwash and GCF) exhibited high prevalence of detection, especially of BKV, and several samples showed detectable viral load. We conclude that oral fluids, especially saliva and mouthwash, can be used for the screening of BKV and JCV.

As metaloproteinases da matriz (MMPs) foram identificadas na saliva, na placa dental, na dentina e no cemento. Este trabalho teve como objetivos: Estudo I (I) - avaliar a expressão de MMP-2 e MMP-9 presentes na saliva total e parotidiana e no fluido gengival crevicular (FGC) de pacientes portadores e não portadores de lesões cervicais não cariosas (LCNC); Estudo II (II) - investigar se a presença de MMP-8 e -9/TIMPs poderia influenciar a remineralização de lesões artificialmente criadas na superfície radicular, com ou sem desgaste por abrasão. Os métodos utilizados foram: (I) Coleta de amostras de saliva e do FGC de 32 pacientes, com (n=16) e sem LCNC (n=16). A atividade gelatinolítica das MMPs foi avaliada através de análise zimográfica e Western Blot. (II): Espécimes de dentina humana radicular foram obtidos. O grupo controle G1(10) não sofreu nenhum tratamento. Os demais segmentos radiculares foram desmineralizados G2(60). O Grupo A não foi submetido à escovação e o Grupo B foi submetido à abrasão por escovação em uma máquina de escovação simulada. G2(10) foi apenas desmineralizado, G3(10) desmineralizado e remineralizado, e os Grupos G4(10), G5(10), G6(10), G7(10) foram desmineralizados e remineralizados em presença de tampão neutro, TIMP, MMP-8 e -9, MMP-8,-9 e TIMP, respectivamente. Para a análise elemental, as concentrações de Ca+2, P, Mg+2 assim como a relação molar Ca/P e Mg/Ca foram determinadas através de uma sonda eletrônica para microanálise (EPMA). A análise qualitativa por retrodispersão (BSE) foi realizada para demonstrar a distribuição global da densidade mineral. Os resultados (I) mostraram que a principal gelatinase presente, tanto na saliva total quanto no FGC, é a proMMP-9. Na saliva secretada pela glândula parótida, não foram detectadas bandas indicando a presença de gelatinases. Os resultados do estudo (II) indicaram que os espécimes escovados apresentaram maior conteúdo de Ca+2 a 20µm e maior conteúdo de Mg+2 a 30 e 50µm. Em presença de TIMPs, ocorreu uma redução do conteúdo de Ca+2 a 20µm. Para os espécimes não escovados, em todas as profundidades, as amostras incubadas com MMPs apresentaram maiores valores de Ca+2. Portanto, pode-se concluir que (I) a comparação entre pacientes com e sem LCNC mostrou não haver diferença estatisticamente significante quanto à atividade gelatinolítica; (II) quando não inibidas pelos TIMPs, as MMPs degradaram o colágeno completamente desmineralizado na superfície radicular, permitindo melhor recalcificação na superfície subjacente. Esse fenômeno foi também facilitado pela abrasão por escovação. / Matrix metalloproteinases (MMPs) have been identified in saliva, plaque, gingival crevicular fluid (GCF), dentin and cementum. Study (I) aimed at evaluating the presence and quantity of gelatinases MMP-2 and MMP-9 in total and parotid saliva and in GCF (GCF) of subjects with and without NCCL. Study (II) aimed at investigating whether the presence of matrix metalloproteinase (MMP)-8 and - 9/TIMPs would influence the remineralization of artificial root lesions with and without mechanical wear. (I) Total stimulated saliva, parotid saliva, and GCF from patients with (n=16) and without NCCL (n=16) were collected and assessed for gelatin zymography and for western immunoblot analysis. (II) Human root segments from Group A (n=35) were not brushed and from Group B (n=35) were subjected to machine-controlled brushing, simulating mechanical wear. Specimens from Group 1 (control, n=10) were left untreated. Group 2 (n=10), was just demineralized; Group 3 (n=10) was demineralized and remineralized. The other samples G4 (n=10), G5 (n=10), G6 (n=10), G7 (n=10) were subjected to remineralization with HEPES buffer, tissue inhibitor of matrix metalloproteinase-2 (TIMP-2), activated MMP-8 and MMP-9 and activated MMP-8, MMP-9 and TIMP-2, respectively. Ca+2, P, Mg+2 concentrations as well as Ca/P and Mg/Ca molar ratios were determined through an Electron Probe Microanalyser (EPMA). (I) Densitometric analysis revealed that the main gelatinase was proMMP-9. No statistically significant difference was observed for MMP-2 and MMP-9 levels, separately. In parotid saliva, gelatinolytic activity was very low or absent. Western immunoblots revealed that, while little immunoreactivity was detected for MMP-2, there was positive immunoreaction for MMP-9, both in total saliva and in GCF. Gelatinases do not seem to originate from parotid gland. (II) The results indicated that the brushed specimens presented higher Ca+2 levels at 20 µm and higher Mg+2 content at 30 and 50 µm. Ca+2 content at 20 µm decreased in the presence of TIMPs. For the non-brushed specimens, in all depths, samples incubated with MMPs showed highest Ca+2 values. It can be concluded that (I) the main gelatinase present in the oral cavity is MMP-9. No significant differences were found in total gelatinolytic activity among NCCL+ and NCCL- patients. (II) When not inhibited by TIMPs, MMPs degraded the completely demineralized collagen in the root surface, allowing for better recalcification in the deeper areas. This phenomenon was also facilitated by the brushing procedure.

The Dissertation work presented herein monitors the significance of oral hygiene in adults, while aiming at preventive measures leading to lowering mouth cavity illness occurrences. Within the theoretical part, I focus on the delineation of important terms relating to preventive care for oral cavity, information concerning anatomy, physiology of oral cavity, influence of foods and other problems within the oral cavity, among which there are tooth cavities and diseases of gingiva. In the practical part of the work I aim on the assessment of the level of oral health, trough the help of applied clinical research methodologies and investigative tooth indices such as API, PBI, and CPITN. Furthermore, educational materials are passed on, for the purpose of dissemination of needed information in the area of dental hygiene, teaching correct teeth brushing techniques, and utilization of other between-the-tooth tools. The following graphic presentation reveals the level of dental health. As well, within my work, I have verified that, within a larger proportion of patients an improved oral hygiene ensued; thanks to educational program together with an improved home oral hygiene care. Familiarity, care of oral cavity and dental hygiene are the most important prerequisites for each individual, when attempting to improve dental health.

Submitted by Suelen Reis (suziy.ellen@gmail.com) on 2013-04-24T13:33:20Z No. of bitstreams: 1 dissertacao- Patríciasec.pdf: 452651 bytes, checksum: 406c286b26c6f4211a97270a3b5b972d (MD5) / Approved for entry into archive by Rodrigo Meirelles(rodrigomei@ufba.br) on 2013-05-08T12:10:20Z (GMT) No. of bitstreams: 1 dissertacao- Patríciasec.pdf: 452651 bytes, checksum: 406c286b26c6f4211a97270a3b5b972d (MD5) / Made available in DSpace on 2013-05-08T12:10:20Z (GMT). No. of bitstreams: 1 dissertacao- Patríciasec.pdf: 452651 bytes, checksum: 406c286b26c6f4211a97270a3b5b972d (MD5) Previous issue date: 2005 / A doença periodontal (DP) é uma doença infecciosa crônica que acomete os tecidos periodontais, sendo a maior causa de perda dentária em adultos acima de 40 anos, precedida apenas pela cárie dentária como importante problema de saúde bucal coletiva no Brasil. Nos últimos anos vários pesquisadores encontraram evidências ligando as doenças periodontais com um risco aumentado para arterosclerose, independente de outros fatores de risco para doenças cardiovascular, o que nos motivou a realizar esta pesquisa, na qual verificou-se a prevalência da doença periodontal nos pacientes atendidos no Ambulatório de Cardiopatia Isquêmica do Hospital Universitário Professor Edgard Santos, da Universidade Federal da Bahia(HUPES). O desenho é de um estudo de corte transversal. A amostra foi composta de 135 indivíduos (78 do sexo masculino e 57 do sexo feminino), com faixa etária de 35 até 99 anos. Os dados da pesquisa foram obtidos por estágios: leitura de prontuário, questionário e exame periodontal. Foram utilizados o Índice Gengival (LÖE e SILNESS, 1963), o Índice de Placa (SILNESS e LÖE, 1964), a Profundidade de Sondagem (PS) e Perda de Inserção Clínica como parâmetros clínicos de avaliação periodontal. A prevalência de doença periodontal em pacientes portadores de Cardiopatia Isquêmica no HUPES foi de 55,7%. A presença de doença periodontal entre aqueles com Insuficiência Cardíaca Congestiva (ICC) foi 1,5 vezes maior (p=0,001). Entre os pacientes diabéticos a presença de doença periodontal foi 1,4 vezes maior (p=0,02). Dessa forma, os achados deste estudo contribuem para fortalecer o conceito de que fatores etiopatogênicos comuns podem existir entre doença periodontal e doença isquêmica do coração. / Salvador

Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2016-01-27T15:07:23Z No. of bitstreams: 2 Tese - Germana Jayme Borges - 2013.pdf: 1472999 bytes, checksum: d3966b343549a12f4a7f7511fdeb6fa7 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-01-28T11:38:50Z (GMT) No. of bitstreams: 2 Tese - Germana Jayme Borges - 2013.pdf: 1472999 bytes, checksum: d3966b343549a12f4a7f7511fdeb6fa7 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-01-28T11:38:50Z (GMT). No. of bitstreams: 2 Tese - Germana Jayme Borges - 2013.pdf: 1472999 bytes, checksum: d3966b343549a12f4a7f7511fdeb6fa7 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-06-19 / This study aimed to compare clinical measurements of gingival thickness (GT) and distance between gingival margin and vestibular (GMBC-V) and interproximal bone crests (GMBC-I) with those using cone-beam computed tomography (CBCT).Material and methods: GT and GMBC-V were measured in 348 teeth and GMBC-I, in 377 tooth regions of 29 patients with gummy smile. GT was assessed using transgingival probing (TP), ultrasound (US) and CBCT, whereas GMBC-V and GMBC-I, by trans-surgical clinical evaluation (TCE) and CBCT. Statistical analyses used independent t-test, Mann-Whitney test, ANOVA Tamhane‟s post hoc test, Pearson correlation coefficient and simple linear regression.Results: Significant difference was observed for GT: between TP, CBCT and US considering all teeth; between TP and CBCT and between TP and US in incisors and canines; between TP and US in premolars and molars. No significant difference was observed between TP and CBCT in premolarsand molars. TP presented the highest means for GT. Significant positive correlation and linear regression were observed (p < 0.05) between TP and CBCT, TP and US and CBCT and US. Significant difference was observed for GMBC-V and GMBC-I using TCE and CBCT, considering all teeth and groups of teeth. Pearson correlation and linear regression were significant for GMBC-V and GMBC-I (p < 0.05), except in molars(r = 0.014; p = 0.915) and region between molars(r = 0.239; p = 0.071).Conclusions:Measurements obtained using CBCT and US presented lower means for GT than TCE. GMBC-V and GMBC-I means were higher using CBCT than TCE. / Avaliar a TCFC como método de diagnóstico da espessura gengival (EG) e da distância da margem gengival à crista óssea vestibular(MGCO-V) e interproximal(MGCO-I), comparando mensurações clínicas com as medidas obtidas em imagens de TCFC.Material e métodos: o estudo foi desenvolvido em 29 pacientes com queixa de sorriso gengival e indicação de cirurgia para aumento de coroa clínica com finalidade estética. A EG e a MGCO-V foram mensuradas em 348 dentes e a MGCO-I em 377 regiões. A EG foi avaliada por meio de sondagem transgengival (ST), com aparelho de ultrassom (US) e nas imagens de TCFC, enquanto que a MGCO-V e MGCO-I foram verificadas por avaliação clínica transcirúrgica (ACT) e nas imagens de TCFC. Para padronizar o local de aferição foi utilizado um guia tomográfico e um guia clínico. A diferença entre as medidas de EG, MGCO-V e MGCO-I obtidas pelos diferentes métodos foi analisada pelo Teste-t independente, Mann-Whitney e ANOVA post hocTamhane, enquanto que a relação entre as mesmas foi avaliada pelo teste de Correlação de Pearson e Regressão Linear Simples. Resultados:para a EG foi observada diferença significante entre ST, TCFC e US quando considerados todos os dentes. No grupo dos incisivos e caninos houve diferença significativa entre ST e TCFC, e entre ST e US. Nos pré-molares e molares (M) não foi observada diferença entre ST e TCFC, mas houve entre ST e US. A ST registrou maiores médias de EG em relação aos demais métodos. Observou-se correlação e regressão linear positivas significativas (p< 0,05) entre ST e TCFC, ST e US e entre TCFC e US. Em relação à MGCO-V e MGCO-I, notou-se diferença significante entre as medidas obtidas por ACT e por TCFC, quando considerados todos os dentes e os grupos dentários. A análise de correlação e regressão linear foram significativas para MGCO-V e MGCO-I (p< 0,05) no grupo dos incisivos, caninos e pré-molares. Conclusão: A TCFC é um método de diagnóstico eficaz para visualização e mensuração da espessura gengival e distância da margem gengival à crista óssea, apresentando medidas correlacionadas às obtidas clinicamente e contribuindo dessa forma, para o planejamento de procedimentos estéticos em periodontia.

As metaloproteinases da matriz (MMPs) foram identificadas na saliva, na placa dental, na dentina e no cemento. Este trabalho teve como objetivos: Estudo I (I) - avaliar a expressão de MMP-2 e MMP-9 presentes na saliva total e parotidiana e no fluido gengival crevicular (FGC) de pacientes portadores e não portadores de lesões cervicais não cariosas (LCNC); Estudo II (II) - investigar se a presença de MMP-8 e -9/TIMPs poderia influenciar a remineralização de lesões artificialmente criadas na superfície radicular, com ou sem desgaste por abrasão. Os métodos utilizados foram: (I) Coleta de amostras de saliva e do FGC de 32 pacientes, com (n=16) e sem LCNC (n=16). A atividade gelatinolítica das MMPs foi avaliada através de análise zimográfica e Western Blot. (II): Espécimes de dentina humana radicular foram obtidos. O grupo controle G1(10) não sofreu nenhum tratamento. Os demais segmentos radiculares foram desmineralizados G2(60). O Grupo A não foi submetido à escovação e o Grupo B foi submetido à abrasão por escovação em uma máquina de escovação simulada. G2(10) foi apenas desmineralizado, G3(10) desmineralizado e remineralizado, e os Grupos G4(10), G5(10), G6(10), G7(10) foram desmineralizados e remineralizados em presença de tampão neutro, TIMP, MMP-8 e -9, MMP-8,-9 e TIMP, respectivamente. Para a análise elemental, as concentrações de Ca+2, P, Mg+2 assim como a relação molar Ca/P e Mg/Ca foram determinadas através de uma sonda eletrônica para microanálise (EPMA). A análise qualitativa por retrodispersão (BSE) foi realizada para demonstrar a distribuição global da densidade mineral. Os resultados (I) mostraram que a principal gelatinase presente, tanto na saliva total quanto no FGC, é a proMMP-9. Na saliva secretada pela glândula parótida, não foram detectadas bandas indicando a presença de gelatinases. Os resultados do estudo (II) indicaram que os espécimes escovados apresentaram maior conteúdo de Ca+2 a 20µm e maior conteúdo de Mg+2 a 30 e 50µm. Em presença de TIMPs, ocorreu uma redução do conteúdo de Ca+2 a 20µm. Para os espécimes não escovados, em todas as profundidades, as amostras incubadas com MMPs apresentaram maiores valores de Ca+2. Portanto, pode-se concluir que (I) a comparação entre pacientes com e sem LCNC mostrou não haver diferença estatisticamente significante quanto à atividade gelatinolítica; (II) quando não inibidas pelos TIMPs, as MMPs degradaram o colágeno completamente desmineralizado na superfície radicular, permitindo melhor recalcificação na superfície subjacente. Esse fenômeno foi também facilitado pela abrasão por escovação. / Matrix metalloproteinases (MMPs) have been identified in saliva, plaque, gingival crevicular fluid (GCF), dentin and cementum. Study (I) aimed at evaluating the presence and quantity of gelatinases MMP-2 and MMP-9 in total and parotid saliva and in GCF (GCF) of subjects with and without NCCL. Study (II) aimed at investigating whether the presence of matrix metalloproteinase (MMP)-8 and - 9/TIMPs would influence the remineralization of artificial root lesions with and without mechanical wear. (I) Total stimulated saliva, parotid saliva, and GCF from patients with (n=16) and without NCCL (n=16) were collected and assessed for gelatin zymography and for western immunoblot analysis. (II) Human root segments from Group A (n=35) were not brushed and from Group B (n=35) were subjected to machine-controlled brushing, simulating mechanical wear. Specimens from Group 1 (control, n=10) were left untreated. Group 2 (n=10), was just demineralized; Group 3 (n=10) was demineralized and remineralized. The other samples G4 (n=10), G5 (n=10), G6 (n=10), G7 (n=10) were subjected to remineralization with HEPES buffer, tissue inhibitor of matrix metalloproteinase-2 (TIMP-2), activated MMP-8 and MMP-9 and activated MMP-8, MMP-9 and TIMP-2, respectively. Ca+2, P, Mg+2 concentrations as well as Ca/P and Mg/Ca molar ratios were determined through an Electron Probe Microanalyser (EPMA). (I) Densitometric analysis revealed that the main gelatinase was proMMP-9. No statistically significant difference was observed for MMP-2 and MMP-9 levels, separately. In parotid saliva, gelatinolytic activity was very low or absent. Western immunoblots revealed that, while little immunoreactivity was detected for MMP-2, there was positive immunoreaction for MMP-9, both in total saliva and in GCF. Gelatinases do not seem to originate from parotid gland. (II) The results indicated that the brushed specimens presented higher Ca+2 levels at 20 µm and higher Mg+2 content at 30 and 50 µm. Ca+2 content at 20 µm decreased in the presence of TIMPs. For the non-brushed specimens, in all depths, samples incubated with MMPs showed highest Ca+2 values. It can be concluded that (I) the main gelatinase present in the oral cavity is MMP-9. No significant differences were found in total gelatinolytic activity among NCCL+ and NCCL- patients. (II) When not inhibited by TIMPs, MMPs degraded the completely demineralized collagen in the root surface, allowing for better recalcification in the deeper areas. This phenomenon was also facilitated by the brushing procedure.

No description available.

Zielsetzung: Verschiedene chirugische Techniken sind für die Deckung entblößter Wurzeloberflächen vorgeschlagen worden. Die vorliegende Studie sollte die Ergebnisse koronaler Verschiebelappen mit bzw. ohne Einsatz von Schmelzmatrixprotein (SMP) bei der Therapie fazialer Rezessionen vergleichen. Material und Methode: Die Studie war als intraindividueller longitudinaler Vergleich über 12 Monate in einem doppelt verblindeten plazebo-kontrollierten randomisierten Design gestaltet. 22 Patienten im Alter von 24-64 Jahren, die 2 paarige faziale Rezessionen von mindestens 3 mm aufwiesen, wurden untersucht. Beide Rezessionen wurden in derselben Sitzung nach der Technik des koronalen Verschiebelappens chirurgisch gedeckt. Eine Rezession wurde dabei zusätzlich mit einem kommerziell erhältlichen SMP (Emdogain) und die jeweils andere mit dem entsprechenden Trägergel (Vehikel: Propylen-glykol-alginat) behandelt. Die Zuweisung der Therapien erfolgte zufällig. Präoperativ sowie 1 und 3 Wochen, 3, 6 und 12 Monate postoperativ wurden durch einen verblindeten Untersucher klinische Parameter (Höhe und Breite der Rezession, Breite der keratinisierten Gingiva, Sondierungstiefe, Attachmentniveau, Knochenniveau) mittels manueller sowie elektronischer Parodontalsonde bzw. Schieblehre auf 0,5 mm genau erhoben. Ergebnisse: 12 Monate postoperativ zeigten beider Therapievarianten signifikante Rezessionsdeckungen und Attachmentgewinne. Die fazialen Rezessionen verringerten sich von 4,5 mm auf 1,5 mm in der SMP- und von 4,4 mm auf 1,5 mm in der Vehikel-Gruppe was einer Rezessionsdeckung von 71,7% bzw. 73,6% entspricht. Der Unterschied zwischen den zwei Gruppen war nicht signifikant. Alle anderen klinische Parameter zeigten keine Unterschiede zwischen den Gruppen. Schlußfolgerungen: Der Einsatz von SMP zusätzlich zum koronalen Verschiebelappen zur chirurgischen Rezessionsdeckung ergab keine wesentliche Unterschiede in den klinischen Resultaten im Vergleich zum koronalen Verschiebelappen in Kombination mit dem Trägergel. / Objectives: Various surgical techniques have been proposed for root coverage of denuded root surfaces. The aim of this study was to evaluate a comparison of coronally advanced flap procedure with or without the use of enamel matrix proteins in the treatment of recession defects. Material and methods: This study was an intra-individual longitudinal test of 12 months duration conducted as a blinded, split-mouth, placebo-controlled and randomised design. 22 patients, aged 24-64 years, with 2 paired buccal recession defects of at least 3 mm participated. Surgical recession coverage was performed as coronally advanced flap technique at both sites in the same session. One site was additionally treated with commercially available enamel matrix proteins (Emdogain) and the other site with placebo (propylene glycol alginate) in accordance with the randomisation list. A blinded examiner assessed pre- and post-surgical measurements. Clinical measurements and photographs were taken pre-surgically and after 1 week, 3 weeks, 3 months, 6 months and 12 months postoperatively. Measurements comprised height and width of the gingival recession, height of keratinized tissue, probing attachment level, probing pocket depth and alveolar bone level by periodontal probe, Florida Probe or caliper to the nearet 0.5 mm. Results: Twelve months after therapy, both treatment modalities showed significant root coverage and probing attachment gain. Gingival recession decreased from 4,5 mm to 1,5 mm for the Emdogain treated sites and from 4,4 mm to 1,5 mm for the control sites, corresponding to mean root coverages of 71,7% and 73,6%, respectively. This difference was not significant. All other clinical variables were not different in the between-group comparison. Conclusions: The use of Emdogain together with coronally advanced flap technique for recession coverage appeared to be equally effective in the overall clinical outcome, there is no clear benefit to combine Emdogain with this surgical technique.

Oral Submucous Fibrosis (OSF) is a chronic inflammatory disease resulting in progressive fibrosis of the oral tissues that can cause difficulty in chewing, swallowing, speaking, and mouth opening. Epidemiological studies have shown that OSF is a precancerous condition and 2-8% of the OSF patients develop squamous cell carcinoma. This disease affects 0.5% of the population in the Indian subcontinent and is now a growing public health issue in many parts of the world. Habit of chewing betel quid has been proposed as an important etiological factor in the development of this disease and is coline, a principle alkaloid of areca nut is considered as major causative factor for OSF development. But the exact molecular mechanism of OSF pathogenesis is not known. Therefore, we set the following objectives for this study: 1) Gene expression profiling of OSF using microarray. 2) Role of areca nut constituents in OSF pathogenesis. 3) Effect of areca nut on epithelial and fibroblast cells. In order to delineate the possible molecular mechanism of OSF pathogenesis, we took microarray approach and identified differentially regulated genes in ten OSF tissues against eight pooled normals using whole human genome oligonucleotide arrays. Microarray results revealed differential expression of 5288 genes (p≤0.05 and Fold change≥1.5), among them 2884 were up-regulated and 2404 were down-regulated. Validation employing quantitative real-time PCR and immunohistochemistry confirmed up-regulation of transforming growth factor-β1 (TGF-β1), TGFBI, THBS1, SPP1, TIG1 and down-regulation of bone morphogenic protein 7 (BMP7), C4orf7 and ALOX12 in OSF tissues. Furthermore, activation of TGF-β pathway was evident in OSF tissues as demonstrated by p-SMAD2 strong immunoreactivity. Analysis of IHC data showed that in all the normal tissues and in 70% of the OSF tissues the expression of TGF-β and BMP7 are inversely correlated. In good correlation, treatment of keratinocytes (HaCaT) by TGF-βdown-regulated BMP7, while BMP7 expression could not be detected in fibroblast cells. Hence, the imbalance between TGF-βand BMP7 signalling, which are positive and negative modulators of extracellular matrix production, respectively may trigger the manifestation of OSF. We also studied the regulation few genes (CTGF, TGM2 and THBS1) identified in OSF microarray in response to TGF-βand arecoline. TGF-βwas able to induce all the above genes in both HaCaT and hGF cells but arecoline could only induce TGM2 in hGF and THBS1 in HaCaT. Therefore TGF-βpathway came out to be the most important pathway in OSF microarray and subsequent validations. But areca nut constituents responsible for TGF-βpathway activation and the source (epithelial or fibroblast cells) through which it activates TGF-βare not known. In an attempt to understand the role of areca nut and its constituents in inducing TGF-βsignalling in epithelial cells, we performed microarray on epithelial cells (HaCaT) treated with areca nut water extract. Surprisingly, 64% of the differentially regulated genes by areca nut water extract matched with TGF-βinduced gene expression profile. To find out areca nut induced genes through TGF-β, epithelial cells were treated with areca nut in presence of ALK5 (TβRI) inhibitor. Out of 64% differentially induced genes, 57% genes induced by areca nut got compromised in presence of ALK5 and 7% were independently induced by areca nut, highlighting the effect of areca nut via TGF-β. Accordingly, areca nut treatment induced both p-SMAD2 and TGF-βdownstream targets TGFBI, TGM2, TMEPAI and THBS1 in HaCaT cells. One possible mechanism of TGF-βsignalling induction by areca nut could be via induced ligand (TGF-β2) and its activator (THBS1). Induction of TGF-β2 ligand by areca nut was shown at both RNA (Real Time) and protein (ELISA) levels. To find out areca nut components responsible for inducing TGF-β signalling, areca nut fractionation was performed which gave three fractions namely, Ethyl acetate (polyphenol), water supernatant (alkaloids) and Dichloromethane (impurity). Out of these; polyphenol and alkaloid fractions were found to be responsible for the induction of TGF-β signalling and its downstream targets. Upon treatment with purified components, catechin and tannin of polyphenol fraction and arecoline, arecaidine and guvacine of alkaloid fraction were found to be responsible for inducing TGF-β signalling, as seen by increased appearance of phopho-SMAD2 in HaCaT cells. Areca nut treatment on human gingival fibroblast cells (hGF) did not induce TGF-β signalling, highlighting that the source of TGF-β induction by areca nut could possibly be the epithelium. Further treatment of areca nut along with TGF-β on hGF cells potentiated TGF-β effect both in terms of TGF-β downstream targets like TGFBI, TGM2, TMEPAI, COL1A1 etc and activation of fibroblast by inducing α-SMA. Increasing concentration of areca nut is cytotoxic on HaCaT cells and pro-proliferative on hGF cells. This could provide a possible explanation for epithelial atrophy and proliferating fibroblast cells in connective tissue of OSF patients. Further exploration on HaCaT cell cytotoxicity by areca nut suggests the involvement of Reactive Oxygen Species (ROS) as a key molecule induced by areca nut. Compromising ROS generation by NAC (N-Acetyl-L-Cysteine) led to reversal of Sub-G1 peak induced by areca nut in HaCaT cells. This highlighted that cell death caused by areca nut could be ROS mediated. Areca nut treatment on hGF cells did not induce ROS generation, leading to no cytotoxicity on these cells. A possible explanation of this differential ROS generation can be due to dose dependent suppression of Catalase activity by areca nut in HaCaT cells but not in hGF cells. We also compared cytotoxicity of areca nut with all the alkaloids and found a good match with arecoline as both of them induce ROS, apoptotic ladder formation, annexin V positivity, suppression of Catalase activity and the cell death induced by them was compromised by NAC. The above results indicated that arecoline could be a mediator of areca nut water extract cytotoxicity on HaCaT cells. Betel nut chewer’s oral epithelium gets regularly exposed to areca nut and hence this exposure could be cytotoxic to oral epithelial cells too. We performed Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) in normal and OSF tissues. Our data showed 62.5% of OSF patients having significant percentage of epithelial cells with TUNEL positivity (Labeling index = 2-60%) compared to all normal tissues that were TUNEL negative. TUNEL positivity was predominantly seen in the upper keratin and supra basal layer of the epithelium. We also studied proliferation status of OSF epithelium and observed that 3-17% (LI) of epithelial cells in all normal tissues showed Ki-67 positivity in the germinal layer of epithelium. However, 65% of the OSF patients showed staining for Ki-67 (LI=.2-58%) in their epithelium. Also analysis of TUNEL positive and Ki-67 positive sections indicated that OSF patients with high TUNEL positivity have high Ki-67 labeling index, but stains in the supra basal or keratin layer (TUNEL) and basal layer (Ki-67) of epithelium respectively. This induced proliferation of epithelial cells could be the result of heavy apoptosis in the outer epithelium. But as these patients are regularly exposed to areca nut, this increased proliferation may not be able to cope up with the heavy apoptosis induced by areca nut, leading to atrophied epithelium. To understand the germinal status of OSF atrophied epithelium we performed staining for OCT4 in OSF tissues. To our surprise there were no OCT4 positive nuclei in the epithelium of 53% of OSF patients but a regular spread of OCT4 positivity has been seen in the epithelium of normal subjects. In conclusion, this thesis highlights the involvement of TGF-β pathway in OSF patho-physiology. In addition, activation of TGF-β pathway by areca nut constituents has been demonstrated. Moreover, the atrophied epithelium of OSF appears to be a consequence of apoptosis and stem cell deprivation. Taken together, areca nut perhaps causes atrophy of the epithelium and activates TGF-β pathway that may lead to manifestation of OSF.