Dissertations / Theses on the topic 'Gingival Mucosa'

Motivado pela crescente importância que os animais domésticos vêem causando na sociedade humana, tanto em relação à busca de melhor qualidade de vida, quanto em relação ao seu valor epidemiológico, visto que são poucos os estudos sobre este assunto, este trabalho objetivou-se em identificar e traçar o perfil de sensibilidade frente aos antífúngicos das espécies de leveduras potencialmente patogênicas isoladas da mucosa oral de cães sem raça definida, da cidade de Campinas, São Paulo. Por motivos de segurança, os animais selecionados foram anestesiados para a realização de exame clínico da cavidade oral e coleta de amostras da região de mucosa oral, seguida de inoculação em ágar Sabouraund dextrose com cloranfenicol. A partir do crescimento em placa, foram isoladas as colônias de fungos leveduriformes, submetidas a exames macromorfológicos e micromorfológicos, meio cromogênico, provas bioquímicas (urease e método API 20C AUX) e teste de sensibilidade aos antifúngicos. Dos 50 animais participantes do estudo, os cães com idade superior a 4 anos e os que apresentavam doença periodontal tiveram maior percentual de isolamento. Foram identificadas 43 leveduras das 45 amostras isoladas, sendo elas 86% (37) correspondentes ao gênero Candida spp, 11,6% (5) pertencentes ao gênero Trichosporon spp e 2,3% (1) do gênero Malassezia pachydermatis. O perfil de sensibilidade pelo método \"Etest\" identificou importante resistência de algumas amostras à antifúngicos rotineiramente utilizados na clínica médica veterinária, o que ressalta a importância da continuidade deste trabalho para o melhoramento da conduta clínica e para a explicação dos inúmeros tratamentos recidivos tanto em animais como em humanos.<br>Regarding the increasing impact that that the pets have been causing to the human society as its relation to the search for a better quality of life, and its relation with the epidemiological value, this study aimed to identify and draw the profile of the susceptibility to the antifungal drugs of the potentially pathogenic species of yeasts isolated from the oral canine mucosa of animals from indefinite breed of the city of Campinas, São Paulo. For their safety, the selected animals were anesthetized to have a short clinic exam of the oral cavity performed and to have samples from the oral mucosa collected. Later an inoculation in Sabouraud Dextrose Agar with chloramphenicol was performed. After the growth on a dish, colonies of fungi with yeast shape were isolated and submitted to macro morphological and micro morphological exams, chromogenous medium, biochemical proofs (urease and API 20C AUX method), and the test of the susceptibility to the antifungal drugs. Among the 50 animals taken part in the study, the dogs over 4 years old and those which presented periodontal diseases had a higher isolation percentage. Yeasts were identified 43 of the 45 isolates, being that 86%(37) were from the genus Candida spp, 11,6% (5) belonged to the genus Trichosporon spp., and 2,3% (1) belonged to the genus Malasseziapachydermatis.The susceptibility profile through the \"Etest®\" method identified a relevant resistance of some strains to the antifungal drugs commonly used in the veterinary medical clinic. This found highlights the relevance of the continuity of this study to improve the clinical conduct and to explain many relapse treatments in both animals and humans.

O objetivo do presente trabalho é analisar os componentes celulares e de fibras do tecido conjuntivo nas hiperplasias inflamatórias (HI), nos fibromas (F) e na fibromatose gengival hereditária (FGH), além de investigar a imunocompetência e efetuar análises moleculares de pacientes com FGH. Para atingir os objetivos foram desenvolvidos 4 artigos, com diferentes metodologias e universos amostrais. No 1º artigo, pretendeu-se estabelecer critérios microscópicos válidos para diferenciar F e HI. Foram avaliadas em microscópio óptico 136 lesões coradas pela Hematoxilina-eosina (HE) e pelo Tricrômico de Masson quanto às características microscópicas. Os resultados mostraram que uma área central de fibras colágenas dispostas de forma enovelada e mais densa, circundada por uma camada de fibras dispostas de forma paralela são características dos F, enquanto a presença de hiperplasia epitelial, infiltrado inflamatório e fibras colágenas organizadas de forma paralela são características das HI. Tais resultados motivaram o 2º artigo, no qual estudamos 18 lesões de F e 13 de HI, que foram preparadas histologicamente e coradas pelo picrosírius red e pelo direct blue para avaliação quantitativa das fibras colágenas e de fibras do sistema elástico, respectivamente, em microscopia a laser confocal. Os resultados confirmaram a disposição estrutural das fibras colágenas observada no 1º artigo, além de apontarem diferenças nas áreas ocupadas pelas fibras colágenas em todas as regiões estudadas. A fim de proceder a uma avaliação dos componentes fibroso e celular das 3 lesões fibrosas, foi desenvolvido o 3º artigo. Espécimes das 3 lesões foram estudados em microscopia ótica, a fim de avaliar suas populações de fibroblastos e de células inflamatórias e os seguintes componentes fibrosos do tecido conjuntivo: fibras colágenas, sistema de fibras elásticas, fibras reticulares e fibras oxitalânicas. Os resultados mostraram disposição e concentração diferente das fibras colágenas nas 3 lesões e uma maior concentração de fibras reticulares na FGH. A análise dos componentes celulares mostrou um maior número de fibroblastos no F e uma maior contagem de células inflamatórias na HI. A partir do encaminhamento de uma família com FGH, optouse por inclui-la no estudo, tendo em vista serem lesões do mesmo grupo. Com isso, foi desenvolvido um 4º estudo, que utilizou uma avaliação morfológica semelhante à dos 2 artigos anteriormente descritos. Dos pacientes com FGH foi obtido sangue periférico para avaliação da proliferação celular de linfócitos através do teste do MTT e para o sequenciamento do gene SOS-1. Os resultados mostraram hiperplasia epitelial na porção externa da gengiva dos pacientes com FGH, maior concentração de fibras colágenas e poucas células inflamatórias. Os 3 pacientes com FGH não mostraram diferenças no seu índice de proliferação de linfócitos em relação aos controles e não apresentaram a mutação descrita no gene SOS-1 de outras famílias com FGH. Pode se concluir que as 3 lesões apresentam estrutura conjuntiva diferente tanto no aspecto quantitativo quanto na disposição estrutural de seus componentes.<br>The objective of this study was to analyze the cellular and fibrous components of connective tissue in inflammatory hyperplasia (IH), oral fibroma (OF) and hereditary gingival fibromatosis (HGF), and to investigate the immunocompetence and to perform molecular analysis in HGF patients. To achieve the goals were developed 4 articles, with different methodologies and sample universes. In the 1st article, we intended to establish microscopic criteria to differentiate F and IH. The microscopic characteristics of the lesions (n=136) stained by hematoxylin-eosin (HE) and Masson trichrome were evaluated in an optical microscope. The results showed that a central area of wound collagen fibers and arranged in a higher density, surrounded by a layer of parallel fibers are characteristic of F, while the presence of epithelial hyperplasia, inflammatory infiltrate and parallel collagen fibers are characteristics of HI. These results led the 2nd article, which studied 18 F and 13 and IH, histologically prepared and stained by picrosírius red and direct blue for the direct quantitative assessment of collagen fibers and elastic fibers of the system, respectively, in the confocal laser microscope. The results confirmed the structural arrangement of collagen fibers found in Article 1, and indicate differences in the areas of collagen fibers in all regions studied. In order to evaluate the cellular and fibrous components of the 3 fibrous lesions, was developed the 3rd article. Specimens of the 3 lesions were studied in optical microscopy, to assess their populations of fibroblasts and inflammatory cells and the following components of fibrous connective tissue: collagen fibers, elastic fiber system, reticular fibers and oxytalan fibers. The results showed different arrangement and concentration of collagen fibers in the 3 lesions and a higher concentration of reticular fibers in HGF. The analysis of cellular components showed a greater number of fibroblasts in F and a higher count of inflammatory cells in IH. With the identification of a family with HGF, we chose to include it in the study because the lesions belong to the group of benign fibrous lesions. With that, it developed a 4th study, which used a similar morphologic evaluation of the 2 articles described above. Periferic blood was extracted from the HGF patients in order to determine the proliferative capacity of the peripheral lymphocytes, by the MTT test, and in order to sequence the SOS1 gene. The 3 HGF affected patients did not present the described mutation for the SOS1 gene, and the lymphocyte proliferative capacity in HGF patients was similar to those on controls. The results showed epithelial hyperplasia in the outer portion of the gingiva of patients with HGF, greater concentration of collagen fibers and few inflammatory cells. We can conclude that the 3 lesions present a different connective structure, considering both the quantitative aspect and the architectural disposition of their components.

Made available in DSpace on 2014-12-17T15:30:55Z (GMT). No. of bitstreams: 1 Eudivar Correia de Farias Neto_DISSERT.pdf: 1661843 bytes, checksum: 00073311cd74dc1565ed066097489ecc (MD5) Previous issue date: 2010-07-27<br>This study clinically evaluated the relationship of gingival recessions with the periodontal index of gingival and plaque, dental alignment, keratinized mocous, type of periodontal, and occlusal disorders. Study participants were individuals aged between 19 and 33 years. The evaluations were performed by using questionnaires and clinical examinations. In subjects examined, the teeth were assessed and divided into groups (Molars, premolars, canines and incisors). The gingival recession were measured in the central region of the teeth and individuals were subject to disclosure to the plate and observing the poll of plaque and gingival index, respectively. 558 teeth were examined, with 24.1%, 135 had gingival recession greater than or equal to 1mm. Through the combination of tests used to evaluate the average of the recession and its relationship with the variables studied, we observed that the degree of recession of the elements assessed dental showed, almost for the most part, when higher values associated with the index plaque (p = 0.101), Gingival Index (p = 0.053), dental alignment (p = 0.962), width of keratinized mocous (p = 0.004) and type of periodontium (p = 0.033), however statistically significant difference could only be considered when related the recessions in the keratinized mocous and the type of the periodontium. Although we identify, when we evaluate the whole set of teeth that occlusal disturbances (p = 0.002) were more strongly associated with cases of gum recession that the gingival index (p = 0.006), however, these two conditions were correlated with the cases of recession, contributing to its occurrence<br>Este trabalho avaliou clinicamente a rela??o das recess?es gengivais com o ?ndice de placa, ?ndice gengival, alinhamento dental, mucosa ceratinizada, tipo de periodonto, e dist?rbios oclusais. Participaram do estudo indiv?duos com idades variando entre 19 e 33 anos. As avalia??es foram realizadas utilizando-se question?rios e exames cl?nicos. Nos indiv?duos examinados, os dentes foram avaliados e divididos por grupos (Molares, pr?molares, caninos e incisivos). As recess?es gengivais foram mensuradas na regi?o central dos dentes e os indiv?duos foram submetidos ? evidencia??o de placa e sondagem para observa??o dos ?ndices de placa e gengival, respectivamente. Foram examinados 558 dentes, sendo que 24,1%, isto ?, 135 apresentavam recess?o gengival maior ou igual a 1mm. Por meio dos testes de associa??o utilizados para avalia??o da m?dia das recess?es e sua rela??o com as vari?veis pesquisadas, observou-se que o grau de recess?o dos elementos dent?rios avaliados apresentaram, quase que em sua maioria, valores m?dios maiores quando associados ao ?ndice de Placa (p=0,101), ?ndice de Gengival (p=0,053), alinhamento dent?rio (p=0,962), largura da mucosa ceratinizada (p=0,004) e tipo de periodonto (p=0,033), no entanto diferen?a estatisticamente significante s? p?de ser considerada quando relacionamos as recess?es com a mucosa ceratinizada e com o tipo do periodonto. Ainda foi poss?vel identificar, quando avaliamos todo o conjunto dos dentes que os dist?rbios oclusais (p=0,002) estiveram mais fortemente associados aos casos de recess?o gengival que o ?ndice gengival (p=0,006), no entanto, essas duas condi??es se mostraram correlacionadas com os casos de recess?o, contribuindo na sua ocorr?ncia

Enxertos gengivais livres são importantes para garantir condições necessárias para o estabelecimento da homeostasia do periodonto de proteção. O processo de inflamação não ocorre por igual em todos os tecidos conjuntivos do organismo e os fibroblastos têm a capacidade de reagir a estímulos agressivos por meio de liberação de diversas citocinas, que desempenham importante função na formação do infiltrado inflamatório. Até o presente trabalho, não há relatos na literatura acerca da comparação do comportamento dos fibroblastos que compõem a mucosa palatina não marginal e dos fibroblastos provenientes de enxerto gengival livre (EGL) marginal em resistir aos estímulos agressores que ocorrem na doença periodontal. Dessa forma, a proposta do presente trabalho foi investigar se os fibroblastos da mucosa palatina não marginal mudariam seu perfil de secreção de citocinas quando enxertados na margem gengival. Foram coletadas biópsias da mucosa palatina no momento da cirurgia de EGL (período inicial) e após 4 meses (período final) no momento da cirurgia para recobrimento radicular. Os fibroblastos foram cultivados e estimulados com LPS de Porphyromonas gingivalis (Pg) e de Escherichia coli (Ec) por 24h e 48h para avaliação comparativa da expressão de citocinas e mediadores do reparo tecidual, como: IL-6, IL-8/CXCL8, MIP-1&#x3B1;/CCL3, TGF-&#x3B2;, VEGF e CXCL16. As citocinas foram quantificadas no sobrenadante das células por meio de ensaio imunoenzimático (ELISA). Para a citocina IL-6, os fibroblastos da mucosa palatina não marginal mantiveram o mesmo perfil de secreção quando enxertados na área gengival marginal; para MIP-1&#x3B1; a secreção se mostrou aumentada de forma estatisticamente significativa pelos fibroblastos obtidos do enxerto gengival marginal após 48h de estímulo por Pg em comparação com os fibroblastos da área palatina não marginal; a secreção de IL-8 pelos fibroblastos da mucosa palatina não marginal foi maior em resposta ao desafio por LPS de Pg e os fibroblastos obtidos do enxerto gengival marginal exibiram secreção até mesmo sem o estímulo de LPS; apenas os fibroblastos do enxerto gengival marginal apresentaram secreção de TGF-&#x3B2;, mesmo na ausência de estímulo por LPS; a secreção de VEGF e CXCL16 não foi detectada pelos fibroblastos analisados. Conclui-se que os fibroblastos provenientes de uma mucosa palatina não marginal parecem se adaptar às condições locais quando enxertados na área gengival marginal, oferecendo evidência de sua participação efetiva na produção de mediadores inflamatórios importantes para o processo de homeostasia do periodonto marginal.<br>Free gingival grafts are important to ensure conditions for the establishment of homeostasis of the periodontal soft tissues. The process of inflammation does not occur the same way in all connective tissues and fibroblasts have the ability to respond to aggressive stimuli through the release of various cytokines, which play an important role in the inflammatory infiltrate formation. In literature, there are no studies comparing the behavior of fibroblasts from palatal mucosa (not marginal) and fibroblasts from marginal free gingival graft (FGG) regarding their resistance towards periodontal disease aggressive stimuli. Thus, the purpose of this study was to investigate whether fibroblasts from the palatal mucosa behave differently when grafted to the gingival margin considering their mechanism of cytokine secretion. Biopsies from the palatal mucosa were collected at the time of FGG surgery (initial period) and after 4 months (final period) when surgery for root coverage was performed. The fibroblasts were cultured and stimulated with LPS of Porphyromonas gingivalis (Pg) and Escherichia coli (Ec) for 24 and 48 hours in order to make a comparative evaluation of cytokines and mediators of tissue repair expression, such as IL-6, IL-8/CXCL8, MIP-1&#x3B1;/CCL3, TGF-&#x3B2;, VEGF and CXCL16. Cytokines were measured in the cell supernatant by enzyme immunoassay (ELISA). For cytokine IL- 6, fibroblasts from palatal mucosa maintained the same secretion pattern when grafted to the gingival margin; for MIP-1&#x3B1; the secretion was significantly increased by fibroblasts from the marginal gingival graft after 48 hours of stimulation with Pg when compared to palatal mucosa fibroblasts; IL-8 secretion by palatal mucosa fibroblasts did not increase in response to Pg LPS challenge and fibroblasts from marginal gingival graft showed secretion even without the stimulus of LPS; only fibroblasts from marginal gingival graft showed secretion of TGF-&#x3B2;, even in the absence of LPS stimulation; VEGF and CXCL16 secretion by fibroblasts was not detected. It was concluded that fibroblasts from palatal mucosa seem to adapt to local conditions when grafted to the gingival margin area, providing evidence of its effective participation in the homeostasis of marginal periodontium through the production of important inflammatory mediators.

Uma faixa adequada de mucosa ceratinizada (MC) é importante para garantir condições mínimas necessárias para o estabelecimento da homeostasia do periodonto de proteção. Frente à infecção bacteriana, os tecidos periodontais e peri-implantares desenvolvem uma resposta imune inflamatória local, resultando na produção e liberação de diversos mediadores inflamatórios que podem ser encontrados no fluido do sulco gengival e peri-implantar. Entretanto, é escassa a literatura acerca dos níveis desses mediadores em sítios peri-implantares considerando a faixa de MC. Assim, o objetivo deste trabalho foi avaliar a associação entre a quantidade e qualidade da MC peri-implantar e parâmetros clínicos e a qualidade da resposta imune através da análise da concentração de mediadores inflamatórios (IL-1, IL-4, IL-6, IL-8, MIP-1, TNF- e VEGF) presentes no fluido peri-implantar humano antes (T1) e depois (T2) da raspagem subgengival, através de imunoensaio. Parâmetros clínicos avaliaram índice de placa (IP), supuração a sondagem (S), profundidade de sondagem (mesial-PSM, centro-PSC e distal-PSD), índice de sangramento (mesial-ISM, centro-ISC e distal-ISD), nível de inserção relativo (NIR), largura (LMC) e espessura (EMC) da MC na face vestibular. Amostras de fluido sulcular foram coletadas e analisadas. Os implantes foram divididos em grupos de acordo com a faixa de MC (G12mm e G2>2mm) e espessura de MC (GA11mm e GB1>1mm; GA21,5mm e GB2>1,5mm). Foram avaliados 20 pacientes (11 homens e 9 mulheres) com idade entre 40 e 80 anos (53,45±10,32), que apresentaram 42 implantes (G1=25 e G2=17). Os resultados clínicos demonstraram diferença estatística significativa apenas entre T1 e T2 dentro do G1 para IP (T1=56% e T2=16%) e ISM (T1=68% e T2=40%). Foi observada diferença estatística entre G1 e G2 apenas para IL-1 em T2 (G1=9,77pg/ml±12,44 e G2=30,13pg/ml±32,29). Intra-grupos, todas as citocinas aumentaram significativamente, mas apenas no G2, demonstrando diferença de reatividade entre grupos. Quanto à espessura da MC (GA1=6 e GB1=36), resultados clínicos revelaram diferença inter-grupos para ISC em T2 (GA1=16,67% e GB1=61,11%) e intra-grupos para IP no GB1 (T1=52,78% e T2=27,78%). Houve aumento significativo no GB1 para todas as citocinas, exceto VEGF, assim como para IL-1 no GA1. Quando a amostra foi redistribuída em GA2=24 e GB2=18, os resultados clínicos indicaram diferença estatística inter-grupos para PSC em T2 (GA2=2,58mm±1,06 e GB2=3,11mm±1,02) e intra-grupos para IP (T1=62,5% e T2=20,83%) e PSC (T1=2,92mm±1,18 e T2=2,58mm±1,06) no GA2 e para ISM (T1=55,56% e T2=27,78%) no GB2. Intra-grupos observou-se aumento significativo para todas as citocinas no GA2 exceto VEGF, assim como IL-8 no GB2. Conclui-se que as diferenças clínicas apresentadas tenderam a evidenciar a importância da MC principalmente após o preparo inicial e, além disso, uma faixa de MC maior que 2mm influenciou os níveis dos mediadores inflamatórios avaliados após a raspagem subgengival. Adicionalmente, a falta de diferença estatística significativa na comparação entre grupos com diferentes espessuras de MC, bem como tal diferença ora no grupo espesso ora no grupo fino quando se adotam diferentes valores de corte (1mm ou 1,5mm respectivamente), demonstra resultados inconclusivos, ressaltando a importância de novas pesquisas para responder esta questão.<br>An adequate keratinized mucosa (KM) width is important to ensure minimal conditions necessary to establish protect periodontium homeostasis. When a bacterial infection occurs, periodontal and peri-implant tissues develop a local inflammatory immune response that results in production and release of several inflammatory mediators that may be found in gingival crevicular and in peri-implant fluids. However, there is a lack of literature concerning about the levels of these mediators in peri-implant sites considering KM width. The aim of this study was to evaluate the association between KM peri-implant quantity and quality and clinical parameters and immune response quality by analyzing the inflammatory mediators concentration (IL-1, IL-4, IL-6, IL-8, MIP-1, TNF- and VEGF) present in human peri-implant fluid before (T1) and after (T2) subgingival scaling, by immunoassay. Clinical parameters evaluated plate index (PI), probing suppuration (S), probing depth (mesial-PDM, center-PDC and distal-PDD), bleeding index (mesial-BIM, center-BIC and distal-BID), relative attachment level (RAL), keratinized mucosa width (KMW) and thickness (KMT) on the buccal face. Sulcular fluid samples were collected and analyzed. The implants were divided in groups according KM width (G12mm and G2>2mm) and KM thickness (GA11mm and GB1>1mm, GA21,5mm and GB2>1,5mm). Twenty patients (11 men and 9 women) aged 40 to 80 years (53,45±10,32) were evaluated, with 42 implants (G1=25 and G2=17). Clinical results showed a significant statistical difference only between T1 and T2 within G1 for PI (T1=56% and T2=16%) and BIM (T1=68% and T2=40%). Statistical difference was observed between G1 and G2 only for IL-1 in T2 (G1=9,77pg/ml±12,44 and G2=30,13pg/ml±32,29). Intra-groups, all cytokines increased significantly, but only in G2, showing reactivity difference between groups. As to KM thickness (GA1=6 and GB1=36), clinical results revealed intergroup differences for BIC in T2 (GA1=16,67% and GB1=61,11%) and intra-groups for PI in GB1 (T1=52,78% and T2=27,78%). There was a significant increase in GB1 for all cytokines except VEGF, as well as for IL-1 in GA1. When the sample was redistributed in GA2=24 and GB2=18, clinical results indicated statistical inter-group differences for PDC in T2 (GA2=2,58mm±1,06 and GB2=3,11mm±1,02) and intra-groups for PI (T1=62,5% and T2=20,83%) and PDC (T1=2,92mm±1,18 and T2=2,58mm±1,06) in GA2 and for BIM (T1=55,56% and T2=27,78%) in GB2. Intra-groups were observed significantly increase for all cytokines in GA2 except VEGF, as well as IL-8 in GB2. Concluded that clinical differences presented tended to show the KM importance principally after the initial preparation and, in addition, KM width greater than 2mm influenced the inflammatory mediators levels evaluated after subgingival scaling. Additionally, the absence of significant statistic difference between groups when comparing the keratinized mucosa thickness, as well as this difference sometimes in the thick group or in the thin group when different court values was adopted (1mm or 1,5mm respectively), show inconclusive results, emphasizing the importance of new research that may answer this question.

Submitted by (edna.saturno@ufrpe.br) on 2016-10-20T12:27:19Z No. of bitstreams: 1 Mariana Ramos da Silva.pdf: 1888775 bytes, checksum: 19fbece906315d41a063fe0b9d99ddb2 (MD5)<br>Made available in DSpace on 2016-10-20T12:27:19Z (GMT). No. of bitstreams: 1 Mariana Ramos da Silva.pdf: 1888775 bytes, checksum: 19fbece906315d41a063fe0b9d99ddb2 (MD5) Previous issue date: 2006-02-13<br>Periodontal disease is clinical and histological characterized by the degradation of extracellular matrix components associated with a gingival infiltration of inflammatory cell populations. The purpose of the present study was to characterize the inflammatory stages in gingival epithelium of the first upper molar according to the number of cells (mono and polimorfonuclear), concentration of collagen fibers, vascularization and gingival thickness. Samples of free gingival of 21 dogs with different degrees of periodontal disease were collected, one fragment for each animal and compared in order to characterize the different stages of each sample through histological analysis of biopsy from marginal gingival epithelium. For a precise study and to compare each fragment and each portion of the fragment they were subdivided in three portions: I – coronal, II – medium, III – apical. The cuts were evaluated histologically by hemaxoxilin and eosin coloration and tricromic of gomori. Our results showed significant differences in the number of inflammatory cells of groups I, II and III according to severity of periodontal disease and suggests that its progression can be directly related with loss of collagen fibers and decrease in epithelium thickness. Observed in different stages of this disease. Finally quantitative evaluation of the fraction containing gingival collagen fibers may reflect severity in periodontal clinical disease.<br>A doença periodontal é caracterizada clínica e histologicamente por alterações na consistência e perda de inserção clínica em virtude da degradação dos componentes da matriz extracelular associada ao infiltrado de células inflamatórias no tecido gengival. Nosso trabalho tem como objetivo caracterizar e correlacionar aspectos clínicos e histológicos da inflamação, em seus diferentes graus, no epitélio gengival dos quartos pré-molares superiores de cães, de acordo com a quantidade de células (mono ou polimorfonucleares), concentração de fibras colágenas, vascularização e espessura do epitélio. No presente estudo, após avaliação clínica pormenorizada, foram obtidas amostras da gengiva livre de 21 cães portadores de doença periodontal em diferentes graus, coletado um fragmento por animal, e comparados a fim de caracterizar o estágio em que se encontra cada amostra através de análise histológica de biópsia do epitélio gengival marginal. Cada fragmento foi subdividido em três porções: I - coronal, II - média e III - apical, para um estudo mais preciso e os resultados confrontados entre si e entre os fragmentos, totalizando 63 segmentos analizados. Os cortes foram avaliados histologicamente utilizando-se a técnica de coloração hematoxilina e eosina e tricromo de gomori. Os resultados revelaram diferenças significativas entre número de células inflamatórias dos grupos I, II e III de uma mesma amostra de acordo com a severidade da doença periodontal sugerindo que sua progressão pode estar diretamente relacionada com a perda das fibras de colágeno e diminuição na espessura do epitélio observadas durante os estágios da doença. Finalmente, a avaliação quantitativa da fração da área ocupada por fibras de colágeno gengival pode refletir a severidade clínica da doença periodontal.

Periodontitis is a leading cause of tooth loss worldwide. The Gram-negative anaerobe, Porphyromonas gingivalis, has been implicated in the initiation and cyclical progression of this inflammatory disease, which may be associated with its ability to invade oral epithelial cells. The majority of studies investigating P. gingivalis invasion have utilised monolayer cultures of epithelial cells. However, these do not represent the oral mucosa due to the lack of a multi-layered epithelium and fibroblast-embedded connective tissue. Therefore, a fibroblast-containing, connective-tissue collagen scaffold was used to create three-dimensional oral mucosal models (OMM). These were constructed using oral fibroblasts and either the oral keratinocyte cell line (H357) or normal oral keratinocytes (NOK) isolated from healthy patients. OMM were raised to the air-to-liquid interface allowing keratinocyte stratification and differentiation (gingival/buccal OMM) or completely submerged resulting in epithelium consisting of 2-3 cell layers (junctional epithelial OMM). Both models resembled normal oral tissue in terms of immunohistochemical staining for several cytokeratin markers, laminin 5 and E-cadherin. A standard antibiotic protection assay was optimised for OMM and percentage invasion was shown to be similar to that of monolayer cultures. The optimal method was an incubation period of 3-6 hours of OMM with P. gingivalis in an aerobic atmosphere and release of intracellular P. gingivalis by homogenisation. Using these optimised conditions, a range of parameters of P. gingivalis invasion were investigated. At diseased periodontal sites there is an increase in the level of haemin and pocket temperature due to inflammation. The culture of P. gingivalis in both a haemin-rich and high temperature environment resulted in an increase in invasion, suggesting that active periodontal sites may preferentially support bacterial internalisation. Additionally, it was shown that following invasion, P. gingivalis can leave epithelial cells after as little as three hours, which may contribute to the periods of progression and remission commonly observed with this disease. Furthermore, the concentration of environmental haemin has previously been shown to influence the expression of P. gingivalis gingipains and it was thought that this may also influence invasion. Indeed, percentage invasion was shown to increase with loss of gingipain activity, particularly Arg-gingipain. This suggested that the degradation of epithelial cell receptors by gingipains may contribute to a decrease in the ability of this bacterium to invade. Candidate host receptors were the complement receptor CD46, tetraspanin family members and the integrin α5β1. These receptors were blocked using antibodies or cells transfected with siRNA to inhibit their function. A small effect on invasion was seen using anti-α5β1 but the antibodies to other molecules did not influence the invasion of P. gingivalis suggesting that there may be some redundancy in the uptake system exploited by the bacteria. Finally, the response of epithelial cells to invasion by P. gingivalis in terms of cytokine release and expression was determined. Using a semi-quantitative cytokine array, there was a decrease in the majority of cytokines tested in the presence of P. gingivalis when compared with TNFa-stimulated control cells which was assumed to be due to the proteolytic action of P. gingivalis gingipains. Due to the conflicting nature of the literature regarding the modulation of CXCL8 by P. gingivalis, this chemokine was selected for further quantification using monolayer cultures. ELISA and quantitative PCR indicated that, in the presence of P. gingivalis, CXCL8 protein concentration decreased in a gingipain-dependent manner, whereas mRNA expression of CXCL8 increased following stimulation by P. gingivalis, suggesting post-transcriptional and/or post-translational modification of CXCL8 by gingipains. No change in protein concentration or mRNA expression was observed following stimulation of OMM which may reflect the multi-layered nature of this model. Differences between monolayer and OMM indicate a role for OMM to investigate bacterial invasion and resultant cytokine release due to its comparability with the oral mucosa. The work presented in this thesis has described the development, characterisation and optimisation of OMM to investigate invasion by P. gingivalis. Invasion was shown to be influenced by environmental changes and P. gingivalis protease expression. Although P. gingivalis degrades key surface molecules including CD46, tetraspanins and α5β1, blocking experiments with antibodies could not explain the protease-dependent effects on invasion. Modulation of cytokine production, particularly CXCL8, by P. gingivalis gingipains, may contribute to a disruption in leukocyte recruitment resulting in a dysregulated inflammatory response. Future development of OMM in terms of including an immune cell element and endothelial component to extend the study of P. gingivalis-host cell interactions will add value to this model. The data presented here indicate that P. gingivalis invasion of the epithelium is likely to be an important contributor to periodontal disease progression.

Periodontal disease is a chronic inflammation of the gingiva and periodontium that leads to progressive destruction and irreversible damage to the supportive structures of the teeth. It affects nearly half of the United States population and is a particular risk factor in adults older than 65 years of age. Oral microorganisms assemble in plaque as a polymicrobial biofilm and Porphyromonas gingivalis, an important secondary colonizer in oral biofilms, has been implicated in periodontal disease. Although the protective functions of various salivary molecules such as antimicrobial proteins have been delineated, lipids present in saliva and on the oral mucosa have been largely ignored and there is growing evidence that the role of lipids in innate immunity is more important than previously realized. In fact, recent studies suggest that sphingoid bases and fatty acids, which exhibit potent broad spectrum antimicrobial activity against a variety of bacteria and fungi, are likely important innate immune molecules involved in the defense against oral bacterial and fungal infections. However little is known about their spectrum of activity or mechanisms of action. In addition, the effects of these lipids that are endogenous to the oral cavity have not been explored against oral bacteria. In this study I hypothesized that oral mucosal and salivary lipids exhibit dose-dependent antimicrobial activity against P. gingivalis and alter cell morphology and metabolic events. To test this hypothesis, I first examined the effects of two fatty acids: sapienic acid and lauric acid, and three sphingoid bases: sphingosine, dihydrosphingosine, and phytosphingosine, against a variety of gram-positive and gram-negative bacteria including P. gingivalis. Using broth microdilution assays to determine minimum inhibitory and minimum bactericidal concentrations, I show that antimicrobial activity against bacteria is dose-dependent, lipid specific, and microorganism specific. Kill kinetics were also variable across each bacteria-lipid combination. Upon examination of select bacteria-lipid combinations via scanning and transmission electron microscopy, different morphologies were evident across all treatments, demonstrating differential activity of each lipid for a particular bacterium as well as for each bacterium across different lipids. In addition, all sphingoid bases and fatty acids were taken up and retained in association with P. gingivalis cells and could be extracted along with bacterial lipids and separated using thin layer chromatography. Using a combination of two-dimensional in-gel electrophoresis and Western blots followed by mass spectroscopy and n-terminus degradation sequencing, I show that sapienic-acid treatment induces a unique stress response in P. gingivalis, as evidenced by the ability of P. gingivalis to upregulate a set of proteins involved in fatty acid biosynthesis metabolism and energy production, protein processing, cell adhesion, and virulence. Finally, utilizing flow cytometry and confocal microscopy, I assessed the effects of oral antimicrobial lipids against a representative host cell and describe oral lipid concentrations that are both antimicrobial to P. gingivalis cells and non-cytotoxic to the representative host cells tested. Combined, these data strongly suggest that sphingoid bases and fatty acids found within the saliva and on oral mucosa likely do contribute to the innate antimicrobial activity of saliva, mucosal surfaces, and skin and this dose-dependent activity is both lipid specific and bacteria specific. This information adds to current knowledge of the innate functions of endogenous lipids in the oral cavity. With bacterial resistance to current antibiotics increasing, the exploration of new antimicrobial agents is important and these lipid treatments may be beneficial for prophylactic treatments or therapeutic intervention of infection by supplementing the natural immune function of endogenous lipids on skin and other mucosal membranes.

Introdução: Gengivite descamativa (GD) se refere a uma manifestação clínica associada com diversas doenças mucocutâneas. Suas causas mais comuns são penfigóide das membranas mucosas (PMM), pênfigo vulgar (PV) e líquen plano oral (LP). A diagnose específica é melhor estabelecida através de avaliação histopatológica e de imunofluorescência. Objetivos: Examinar casos de gengivite descamativa utilizando microscopia confocal a laser e comparar os achados com aqueles encontrados na gengiva normal. Além disso, comparar os achados de microscopia confocal da gengivite descamativa com os da histopatologia convencional das lesões biopsiadas a fim de estabelecer critérios para este método diagnóstico não invasivo. Método: Doentes com manifestações clínicas de gengivite descamativa foram incluídos, totalizando quarenta e três casos. A microscopia confocal foi realizada na gengiva de um indivíduo saudável e nas lesões gengivais. Todas as lesões sem exame histopatológico prévio foram biopsiadas a fim de permitir uma correlação entre a microscopia confocal e a histopatologia. Resultados: O exame de microscopia confocal das lesões suspeitas de penfigóide das membranas mucosas revelou uma separação ao nível da junção dermo-epidérmica, preenchida por pequenas estruturas brilhantes, interpretadas como hemáceas. Os aspectos histopatológicos e de imunofluorescência confirmaram o diagnose. Para os casos de pênfigo vulgar, os achados da microscopia confocal foram de fenda intraepitelial com células arredondadas interpretadas como queratinócitos acantolíticos. Hiperqueratose e espongiose, associadas com infiltrado inflamatório, caracterizado por células pequenas e brilhantes permeando a estrutura intraepitelial de queratinócitos conhecida como favo de mel foram vistos no líquen plano. Estruturas arredondadas pouco brilhantes, interpretadas como queratinócitos necróticos, e estruturas estelares também pouco brilhantes, interpretadas como melanófagos, foram encontrados na derme. Conclusões: Propõe-se o uso da microscopia confocal como uma ferramenta adicional no diagnose e avaliação da gengivite descamativa<br>Background: Desquamative gingivitis refers to a clinical manifestation associated with several mucocutaneous disorders. The most common are mucous membrane pemphigoid, pemphigus vulgaris and lichen planus. Their specific diagnosis is better established by histopathological and immunofluorescence evaluation. Objective: To examine cases of desquamative gingivitis using reflectance confocal microscopy and compare the findings with those of normal gingiva. Moreover, confocal microscopy findings in desquamative gingivitis were compared to conventional histopathology of the biopsied lesions, in order to establish criteria for this non-invasive diagnostic technique. Methods: Patients with clinical manifestations of desquamative gingivitis were included, totalizing forty-three cases. Reflectance confocal microscopy was performed the gingival of a healthy person and on gingival lesions. All lesions were biopsied in order to perform a reflectance confocal microscopy- histopathologic correlation. Results: Reflectance confocal microscopy exam of the gingival lesions suspected of mucous membrane pemphigoid revealed a separation at the level of dermal-epidermal junction, filled with small bright structures interpreted as blood cells. Histopathological and immunofluorescence aspects confirmed the diagnosis. For pemphigus vulgaris, reflectance confocal microscopy aspects were of intraepithelial cleft with round detached cells interpreted as acantholytic keratinocytes, similar to the histopathological features. Hyperkeratosis and spongiosis associated with infiltration of inflammatory cells, recognized as small bright cells intermingling the honeycomb keratinocyte epithelial structure, were seen in lichen planus. Mild bright round structures interpreted as necrotic keratinocytes and mild bright stellate structures, interpreted as melanophages in the dermis were also seen. These features were present in histopathology, confirming the diagnosis of lichen planus. Conclusion: We propose the use of reflectance confocal microscopy as an additional tool in diagnosis and evaluation of desquamative gingivitis

碩士<br>高雄醫學大學<br>牙醫學研究所<br>82<br>Drug-induced gingival overgrowth (DIGO) is an adverse side effect in patients treated with phenytoin, cyclosporine, and nifedipine. Gingival overgrowth induced by these three drugs is almost identical in clinical and histological features. Nifedipine (NF) is a calcium channel blocker used in the treatment of cardiac angina and arrhythmia. Many observations in cases of drug-induced gingival overgrowth are, however, controversial. The pathogenesis of DIGO remains still obscure. The biochemical natures of NF-induced gingival overgrowth were not studied well enough at present. Interleukin-1β (IL-1β) can modulate a number of biological activities of fibroblasts such as : promote fibroblasts proliferation, increase collagen production, stimulate PGE2 production, and increase proteoglycans & glycosaminoglycans production. These factors may contribute to DIGO. The purpose of this study is to analyze the effects of NF on cellular proliferation and the biosynthesis & release of IL-1β of healthy & inflamed human gingival fibroblasts in vitro. Fibroblast cell lines were cultured with NF at various concentrations (50, 100, 200, 400 ng/ml). Cellular proliferation and viability were assayed by MTT & DNA synthesis, and the cell-free supernatants were measured for IL-1β concentration by ELISA. The results showed that : (1) NF enhanced gingival fibroblasts proliferation at 100∼200 ng/ml, but caused a decrease in cell number at the highest concentration tested (400ng/ml). (2) NF generally increased the production of IL-1β, but there were marked variations among cell lines with regard to the magnitude of stimulation. (3) higher concentrtion of NF (400 ng/ml) generally caused inhibition of IL-1β production. Betel nut chewing habits are commonly seen in the people of Taiwan and the prevalence of betel nut chewing is increasing gradually. Many investigators suggested that betel nut chewing has a untoward effects on the tissue of the oral cavity, including teeth, periodontal tissue and oral mucosa. In recent years, chewing of betel quid has been postulated as the most important etiologic factors in the pathogenesis of oral submucous fibrosis (OSF). Although there is clear evidence that the betel-nut alkaloids (arecoline & arecaidine) can stimulate mucosal fibroblasts proliferation and collagen synthesis, the cause of OSF is still unclear. Fibroblasts are capable of producing a wide variety of cytokines, including IL-1, IL-6, IL-8, and TGF-β etc., which have multiple biological activities. There are seldom reports regarding the modulation of fibroblasts by betel nut in Taiwan. So, this study is designed to investigate the effects of betel nut alkaloids (arecoline & arecaidine) on the biochemical reactions and the biosynthesis & release of cytokine (IL-1β) of oral fibroblasts obtained from healthy buccal mucosa and OSF mucosa. The results revealed that : (1) arecoline and arecaidine can stimulate mucosal fibroblasts proliferation at 1∼10 μg/ml, but arecoline caused a significant decrease in cell number at the highest concentration (100μg/ml) (2) arecoline and arecaidine generally increased the production of IL-1β, but there were variations among cell lines with regard to the magnitude of stimulation. (3) higher concentration of arecoline (100 μg/ml) generally caused inhibition of IL-1β production. In conclusion, there were marked variations in IL-1β production among cell lines, the roles of the production of IL-1β by NF in the DIGO or by arecoline/ arecaidine in OSF need further laboratory studies and clinical investigations.

Objetivo: Avaliar a importância do fenótipo gengival e da presença de mucosa queratinizada na manutenção da saúde e da estética nas regiões peri-implantares. Materiais e métodos: Realizou-se a pesquisa, sem restrição de data ou de idioma, nas bases de dados MEDLINE/PUBMED, Bireme e Scielo. Utilizaram-se como palavras-chaves “Keratinized mucosa”, “peri-implantitis”, “gingival biotypes”, “implants”, “keratinized gingiva” e “gingival phenotype”. 90 artigos foram identificados. Após leitura dos títulos e respetivos resumos, selecionaram-se 27. Pela análise dos textos completos, 22 artigos foram incluídos. Resultados: A presença de mucosa queratinizada parece ser importante para o estabelecimento e a manutenção da saúde e estética periimplantar, ainda que não interfira com a osteointegração. Relativamente ao fenótipo, um fenótipo gengival fino parece ser um fator de risco para o desenvolvimento de peri-implantite. Discussão/Conclusão: Mais estudos clínicos randomizados e controlados são necessários para confirmar e clarificar as informações apresentadas.<br>Objective: To evaluate the importance of gingival phenotype and the influence of keratinized mucosa in maintaining health and aesthetics in the peri-implant regions. Materials and methods: A search was carried out in the MEDLINE / PUBMED, Bireme and Scielo databases, without restriction of date or language. "Keratinized mucosa", "peri-implantitis", "gingival biotypes", "implants", "keratinized gingiva" and "gingival phenotype" were used as keywords. 90 articles were identified. After reading the titles and respective summaries, 27 were selected. After the analysis of the complete texts, 22 articles were included. Results: The presence of keratinized mucosa appears to be important for the establishment and maintenance of peri-implant health and aesthetics, although it does not interfere with osseointegration. Regarding the phenotype, a fine gingival phenotype appears to be a risk factor for the development of peri-implantitis. Discussion / Conclusion: More randomized and controlled clinical trials are needed to confirm and clarify the information presented.

Wound healing is a complex process orchestrated by a variety of known and unknown factors. Oral wound healing presents peculiar characteristics as accelerated wound closure and reduced scar formation when compared with cutaneous wound repair, and it has been recognized the key role of fibroblasts in this concern. In fact, oral-derived fibroblasts represent a resource of great interest for regenerative approaches due to their self-renewal capacity and plasticity. The biomolecular basis of the differences between oral and skin repair have been described by several studies, showing the main differences in transcriptional changes between the first 12-24 hours after injury. Nevertheless, variations in wound healing also exists inside the oral environment. The evidence we have nowadays is that wounding in the alveolar mucosa leads to scarring, while in the gingival tissue does not, but the literature lacks clear evidence concerning comprehensive in vivo biomolecular data on the differences between alveolar mucosa, buccal attached gingiva and palatal tissue-derived fibroblasts behaviour. The course of wound repair process can be modified by different factors. It is well know how the presence of biofilm jeopardizes the healing process. To counteract this during the post-surgical period, chlorhexidine digluconate (CHX) mouthrinse is commonly indicated. Although several in vitro studies have reported cytotoxicity effects on fibroblasts, , in vitro assays cannot fully represent the oral environment as a whole and this could be a limitation. Nevertheless, there are no in vivo data on the effect of post-surgical CHX use in oral cells behaviour in the early phases of wound healing. Improving wound healing through the use of bioactive substances that can influence cells behaviour, supporting tissue repair/regeneration, is of major clinical interest. Hyaluronic acid (HA) is the major endogenous component of ECM, involved in cell proliferation, migration and tissue remodeling. In vitro and animal studies have demonstrated the ability of exogenous HA to improve wound healing, enhancing proliferative and migratory ability of oral-derived fibroblasts. However, in vivo human studies evaluating its specific mechanisms on cellular activation and gene expression modulation in the early phases after oral surgical wounding are lacking. The present thesis was organized and divided in the following sections: Chapter I The first chapter is intended to give evidence on the peculiar characteristics of oral soft tissues in the early phases of wound healing process from a clinical and biomolecular point of view. In addition, evidence available concerning the role of post-surgical chlorhexidine digluconate (CHX) mouthrinse and exogenous hyaluronic acid (HA) in oral wound repair is presented. The aim of this chapter is to provide current knowledge on these concerns, pointing out the missing evidences that lead the research topic during the course of the doctorate and presenting the rationale of the topic that allowed to conclude in the three main branches of this research: - Differences in the gene expression profiles between human alveolar mucosa, buccal attached gingiva and palatal tissue-derived fibroblasts in the early phases of oral wound repair; - Effect of post-surgical CHX mouthrinse on gingival tissue features and oral gingival-derived cells behaviour in the early phases of oral wound repair; - Effect of intra-surgical HA application on gingival tissue features and oral gingival-derived cells behaviour in the early phases of oral wound repair. Chapter II The second chapter is intended to provide evidence on the behaviour of fibroblasts derived from different oral soft tissues in the early phases of the wound healing process. The main aim was to analyse and compare the gene expression profiling of fibroblasts from human alveolar mucosa (M), gingival (G) and palatal (P) tissues in the early phases following surgical wounding, correlating it with the clinical response, autophagy activation and fibrotic markers expression. M, G and P biopsies were harvested from six patients at baseline and twenty-four hours after surgical procedure. Clinical response was evaluated through Early wound Healing Score (EHS). Fibrotic markers expression and autophagy activation were assessed on fibroblasts isolated from those tissues by Western blot and quantitative real time PCR analysis (qRT-PCR). Fibroblasts from two patients were subjected to RT2 profiler array, followed by network analysis of the differentially expressed genes. The expression of key genes was validated with qRT-PCR on all patients. At twenty-four hours after surgery, EHS was higher in P and G than in M. In line with the clinical results, no autophagy and myofibroblast differentiation were observed in G and P. Significant variations in mRNA expression of key genes were observed: RAC1, SERPINE1 and TIMP1, involved in scar formation; CDH1, ITGA4 and ITGB5, contributing to myofibroblast differentiation; and IL6 and CXCL1, involved in inflammation. Some genes involved in the oral soft tissue differential clinical wound healing outcome were identified, providing novel insights into the molecular mechanisms of oral repair and allowing to develop new approaches of essential impact in periodontal surgery. Chapter III The present chapter focuses on the study of the in vivo effect of post-surgical chlorhexidine digluconate (CHX) mouthrinse on the gingival tissue features and oral gingival-derived cells behaviour in the early phases of wound repair. G biopsies were obtained in three patients twenty-four hours after surgery with the indication of post-surgical 0.12% CHX use and were compared with those obtained from the same patients without any antiseptic use. Each gingival biopsy was divided in two parts: one for histological-immunohistochemical (IHC) analysis and one for gene expression analysis in order to carry out a morphological and molecular analysis. For the first one, epithelial tissue/chorion features and collagen fibers organization/content were evaluated through Hematoxylin-Eosin and Masson trichrome staining, respectively. The expressions of proteins related to cell proliferation (ki67) and apoptosis (p53) were examined by IHC analysis. Fibrotic markers expression (Vimetin, Col1a1 and αSMA) were also analysed by IHC in order to evaluate collagen deposition and myofibroblasts differentiation. For the molecular analysis, qRT-PCR was carried out: fibrotic markers expression (Col1a1 and αSMA) and proapoptotic protein (BAX) were analysed in all the patients, and to evaluate the re-epithelialization and collagen turnover, RAC1, SERPINE1 and TIMP1 gene expression were analysing in two patients. Twenty-four hours after surgery, CHX was able to reduce cellular proliferation and to increase collagen deposition, proapoptotic protein and fibrotic markers expression, and myofibroblast differentiation. In addition, a reduction in the expression of RAC1 and triggering in the expression of SERPINE1 and TIMP1 were observed, showing a “scar wound healing response” pattern. The demonstration of CHX-induced fibrotic transformation, leading to scar repair, could support the need for new post-surgical clinical protocols based on a strategic and personalised use of CHX. Chapter IV The present chapter focuses on the study of the in vivo effect of exogenous hyaluronic acid (HA) on the gingival tissue features and oral gingival-derived cells behaviour in the early phases of wound repair. G biopsies were obtained in eight patients twenty-four hours after surgery with intra-surgical application of HA and were compared with those obtained from the same patients without HA application (no treatment group - NT). Clinical response was evaluated through EHS. Each gingival biopsy was divided in three parts: one for histological-IHC analysis, one for protein analysis and one for gene expression analysis in order to carry out a morphological and molecular analysis. For the first one, tissue structure and inflammatory infiltrate, extracellular matrix (ECM) organization and microvascular density (MVD), and collagen fibers organization/content were evaluated through Hematoxylin-Eosin, Sinus red and Masson trichrome staining, respectively; whereas cellular proliferation was evaluated by immunohistochemical detection of Ki67. For the molecular analysis, collagen turnover was evaluated through MMP-1, MMP-2 and MMP-9 protein analysis by Western Blot and LOX, MMP-1, TIMP-1, TGF-β1 genes expression by real time PCR. Since ECM remodeling is also influenced by mechanical stimuli, and fibroblasts are mechanoresponsive cells, the expression of key mechanosensors paxillin (PAX), focal adhesion kinase (FAK) and vinculin (VNC) were also analysed. Twenty-four hours after surgery, EHS was significantly higher in HA than in NT group. In line with the clinical results, gene expression analysis showed that mRNA levels of LOX-involved in collagen maturation-, resulted significantly up-regulated in HA-treated gingiva compared to NT group but this was independent of TGF-β1 since no difference was found its expression. MMP-1/TIMP-1 balance was modified: significant increase of MMP-1 protein and TIMP-1 gene expression in HA compared to NT group were revealed. No significant differences were observed in MVD, key mechanosensors expression, collagen content and cell proliferation. Intra-surgical HA application enhance wound healing properties such as ECM remodeling and collagen maturation, and improve the clinical repair response in human in vivo gingival wounds 24 hrs after injury. HA might be an important component in future regimens aiming to accelerate and improve the wound healing after periodontal surgery. Chapter V The last chapter aims to provide general conclusions concerning the three branches of the research performed during the course of doctorate, integrating the results obtained and pointing out some key points and recommendations. In addition, future perspectives in the wound healing research field are mentioned.

Após a realização de uma exodontia, ocorrem alterações dimensionais no osso alveolar, relacionadas com a cicatrização alveolar que conduz, inevitavelmente, à reabsorção óssea. Estas alterações devem ser conhecidas e correctamente avaliadas antes do procedimento cirúrgico e sempre que se inicia qualquer reabilitação protética. Um grupo de 12 pacientes foi submetido a avaliação pós-exodontia na Consulta de Cirurgia Oral da Clínica Dentária Universitária da Universidade Católica Portuguesa – Viseu. A cada paciente foi extraído, por técnica fechada, um dente maxilar da região compreendida entre o dente 15 e 25, com indicação de exodontia, com a condição de presença de dentes adjacentes. Foram definidos critérios de inclusão e exclusão, sendo que todos os pacientes que apresentavam doença sistémica ou factores locais que potenciassem a reabsorção óssea foram excluídos. As alterações dimensionais ósseas verticais e horizontais foram avaliadas para cada grupo gengival, previamente definido, em três tempos distintos, no momento da exodontia (Baseline), 1 mês após a exodontia (T1) e 3 meses após a exodontia (T2). Os resultados foram previsíveis, de acordo com a bibliografia consultada, verificando-se existiu perda óssea vertical e horizontal, ao longo dos três meses, sendo mais acentuda no grupo gengival G1 e menos acentuada no G3. Verificou-se a existência de correlação forte entre a perda de volume ósseo vertical e a espessura de gengiva aderida.<br>After tooth extraction, dimensional changes occur in the alveolar bone, related to cellular healing which inevitably leads to bone resorption. These changes must be known and properly evaluated before surgery and prosthetic rehabilitation. A group of 12 patients underwent post-extraction evaluation in Oral Surgery appointment at the University Dental Clinic of the Catholic University of Portugal in Viseu. In each patient was extracted, by flapless technique, a single maxillary tooth, with the requirement of the presence of adjacent teeth, located in region between the tooth 15 and 25, with indication for extraction. Inclusion and exclusion criteria were defined, and all patients with a systemic disease or local factors that enhance bone resorption were excluded. The vertical and horizontal bone dimensional changes were evaluated for each gingival group, previously set, in three different times, at the time of extraction (Baseline), 1 month after extraction (T1) and 3 months after extraction (T2). The results were predictable, according to the bibliography, checking whether there has vertical and horizontal bone loss, over the three months, were severe in the gingival group G1 and less pronounced in G3. It has been found that there is a strong correlation between loss of vertical bone volume and thickness of attached gingiva.