Considerando-se a localização intra-óssea das lesões periapicais, o diagnostico clinico e dificultado e a avaliação radiográfica não fornece informações suficientes para o diagnostico de um periodonto apical sadio pós-tratamento. Dessa maneira, o objetivo deste estudo foi comparar os achados radiográficos e por tomografia computadorizada de feixe cônico com a avaliação microscópica após tratamento de canais radiculares em dentes de cães e avaliar a participação das metaloproteinases da matriz (MMPs) na lesão periapical e nos tecidos em processo de reparação, assim como em cistos e granulomas periapicais obtidos de humanos. A seguir, os mecanismos envolvidos na cementogênese apical foram investigados utilizando células do ligamento periodontal de humanos. Foram induzidas lesões periapicais em dentes de cães e o tratamento endodôntico foi realizado em sessão única ou apos utilização de um curativo de demora a base de hidróxido de cálcio [Ca(OH)2]. Avaliações radiográficas e tomográficas foram realizadas previamente, após a indução das lesões periapicais e 180 dias apos o tratamento endodôntico. Os tecidos periapicais foram avaliados por meio de microscopia de luz, imunofluorescência, imunoistoquímica e RT-PCR em tempo real. In vitro, células do ligamento periodontal foram utilizadas para avaliar os efeitos da estimulação com Ca(OH)2 nos processos de migração, proliferação, diferenciação celular e mineralização. As vias de sinalização envolvidas na diferenciação cementoblastica foram investigadas por meio de inibidores bioquímicos da via das proteínas quinases ativadoras de mitose (MAPK), bloqueadores de canais de cálcio e silenciadores de RNA para proteínas quinases reguladas por sinal extracelular (ERK-1 / ERK-2). De acordo com os resultados obtidos, a tomografia computadorizada permitiu a detecção de lesões periapicais com maior sensibilidade e acuracia que a radiografia periapical convencional, utilizando-se a avaliação microscópica como padrão-ouro. Histologicamente, as lesões periapicais experimentalmente induzidas apresentaram bactérias distribuídas pelo sistema de canais radiculares e lacunas de reabsorção do cemento e estavam associadas a desorganização das fibras colágenas e alta expressão de MMPs. A presença das MMPs em processos inflamatórios periapicais de humanos (cistos e granulomas) foi confirmada. Nos dentes com lesão periapical submetidos ao tratamento endodôntico em sessão única o desfecho do tratamento endodôntico foi caracterizado pela manutenção ou progressão da lesão periapical e alta expressão de MMPs. Por outro lado, nos dentes submetidos ao tratamento endodôntico utilizando o Ca(OH)2 como curativo de demora, maior numero de espécimes apresentaram regressão da lesão periapical e houve modulação da expressão de MMPs pelo tratamento. Neste grupo foi evidenciada neoformação de cemento no forame apical. Células do ligamento periodontal estimuladas com Ca(OH)2 expressaram proteínas especificas de cementoblastos (CEMP-1, CAP) e foram capazes de sintetizar nódulos de mineralização, mediados via ERK MAPK. A ação do Ca(OH)2 ocorreu via canais de cálcio, uma vez que o bloqueio destes canais inibiu a fosforização de ERK-1 e ERK-2 e, portanto, a expressão de CEMP-1 e CAP. CEMP-1 estimulou a migração, proliferação e mineralização mediada por células, exercendo um papel central na cementogenese, uma vez que o bloqueio de CEMP-1 inibiu a migração celular e mineralização. Juntos, estes resultados permitem concluir que a tomografia computadorizada e superior a radiografia periapical convencional para detecção de lesões periapicais refratarias ao tratamento de canais radiculares. Ainda, o tratamento endodôntico realizado utilizando o hidróxido de cálcio como curativo de demora propiciou um reparo apical e periapical mais favorável do que o tratamento endodôntico em sessão única, possivelmente devido a capacidade do Ca(OH)2 induzir a diferenciação de células do ligamento em células com um fenótipo cementoblastico e posterior mineralização. / Clinical diagnosis of apical periodontitis is difficult due to the intraosseous nature of the disease and radiographic evaluation does not provide sufficient information to determine a healthy apical periodontium following root canal therapy. Therefore, the aim of this study was to compare the radiographic and cone beam computed tomographic findings with microscopic evaluation following root canal treatment in dogs teeth. Then, the presence of matrix metalloproteinases (MMPs) in apical periodontitis and during the healing phase following treatment was evaluated and compared to the expression of MMPs in periapical cysts and granulomas obtained from human. Finally, the mechanisms involved in apical cementogenesis were investigated using human periodontal ligament cells. Apical periodontitis was induced in dogs teeth and then root canal treatment was performed in a single visit or using calcium hydroxide [Ca(OH)2] as the root canal dressing. Tomographic and radiographic evaluations were performed prior to and following induction of apical periodontitis, and 180 days following root canal therapy. Periapical tissues were evaluated by conventional light microscopy, immunofluorescence, immunohistochemistry, and real time RT-PCR. In vitro, periodontal ligament cells were used to evaluate the effects of Ca(OH)2 treatment on cell migration, proliferation, differentiation, and mineralization. The signaling pathways triggered by treatment with Ca(OH)2 were investigated using mitogen activated protein kinase (MAPK) biochemical inhibitors, calcium channel blockers, and extracellular regulated protein kinase (ERK-1 / ERK-2) silencing RNAs. Based on the results obtained, apical periodontitis was detected with higher sensitivity and accuracy by means of cone beam computed tomography compared to conventional periapical radiographs, using microscopic evaluation as the gold standard. Histological evaluation revealed that teeth with apical periodontitis presented microorganisms throughout the root canal system and in areas with resorption of cementum. Apical periodontitis was characterized by collagen fiber disorganization and high expression of MMPs. The presence and activity of MMPs was confirmed in periapical inflammatory diseases (cysts and granulomas) obtained from humans. Root canal treatment outcome was characterized by maintenance or progression of apical periodontitis and high expression of MMPs in teeth submitted to root canal treatment in a single visit, whereas root canal treatment outcome in teeth submitted to root canal treatment using Ca(OH)2 as the root canal dressing, higher number of teeth presented reduced apical periodontitis and lower expression of MMPs. In this group, cementum neogenesis was evident in the apical foramina. Periodontal ligament cells stimulated with Ca(OH)2 expressed cementoblastic specific proteins (CEMP-1, CAP) and were able to synthesize mineralized nodules via ERK MAPK. The effects of Ca(OH)2 occurred via calcium channels, since their blockade prevented ERK-1 and ERK-2 phosphorylation and therefore expression of CEMP-1 and CAP. CEMP-1 stimulated cell migration, proliferation, and mineralization and it was central to cementogenesis because blockade of activity of CEMP-1 prevented cell migration and mineralization. Taken together, these findings demonstrate that cone beam computed tomography is superior to conventional periapical radiography for detection of refractory apical periodontitis. Furthermore, the root canal treatment using Ca(OH)2 as the root canal dressing permitted a more favorable outcome than the root canal treatment performed in a single visit, probably due to the ability of Ca(OH)2 induce cementoblastic differentiation of periodontal ligament cells and mineralization.

In der vorliegenden Arbeit wurden vier Studien vorgestellt, die sich mit der Notwendigkeit und Art medikamentöser Einlagen im Rahmen von Wurzelkanalbehandlungen beschäftigen. In Studie 1 wurde in vitro untersucht, in wieweit Kalziumhydroxid in der Lage ist, bakterielle Endotoxine zu neutralisieren. Hierzu wurde die TNFalpha-Ausschüttung menschlicher Monozyten als Indikator für die biologische Aktivität von Escherichia coli LPS bestimmt. Aus der Studie konnte geschlossen werden, E. coli LPS durch Kalziumhydroxid neutralisiert werden kann. In Studie 2 wurde die Keimreduktion durch Kalziumhydroxid und Chlorhexidin (CHX) in einem In-situ-Modell untersucht. Die Wurzeln extrahierter menschlicher Zähne wurden in intraoral zu tragende herausnehmbare Apparaturen einpolymerisiert und von zwei Freiwilligen jeweils für eine Woche getragen. So konnte eine Besiedlung der Wurzelkanäle durch orale Mikroorganismen stattfinden. Vor und nach Applikation von Kalziumhydroxid und CHX in verschiedenen Darreichungsformen wurde die Keimzahl im Wurzelkanal bestimmt. Kalziumhydroxid-Paste und 5%iges CHX-Gel führten zu einer signifikant besseren Keimreduktion als in Guttaperchaspitzen integriertes Kalziumhydroxid oder CHX. In Studie 3 wurde die Re-Infektion des Wurzelkanals nach Füllung in vitro untersucht. Hierbei wurden adhäsive (Clearfil, CoreRestore) und nichtadhäsive provisorische Füllungsmatierialien (Ketac-Fil, IRM) als Deckfüllungen appliziert und die bakterielle Passage über einen Zeitraum von einem Jahr bestimmt. Nach diesem Zeitraum war in allen Gruppen eine ähnliche Anzahl undichter Proben zu finden. Es wurde die Schlußfolgerung gezogen, dass wurzelkanalgefüllte Zähne möglichst innerhalb der ersten drei Monate nach Obturation eine definitive Versorgung erhalten sollten. In Studie 4 wurde die Re-Infektion gefüllter Wurzelkanäle nach vorheriger medikamentöser Einlage mit Kalziumhydroxid, CHX oder Ledermix in vitro untersucht. Ab dem achten Monat zeigten die mit Kalziumhydroxid behandelten Zähne bis zum Ende des Experimentes nach 12 Monaten signifikant weniger undichte Proben als die Zähne aller anderen Gruppen. Kalziumhydroxid ist unter dem in dieser Studie beleuchteten Aspekt als günstigste Zwischeneinlage zu sehen. Unter Berücksichtigung aller vorgestellten Studien darf die Empfehlung ausgesprochen werden, im Falle der Behandlung eines infizierten Wurzelkanals eine mindestens einwöchige medikamentöse Einlage mit Kalziumhydroxid vorzunehmen, da hierdurch bakterielle Endotoxine neutralisiert werden, die Zahl der Mikroorganismen im Wurzelkanal signifikant reduziert und ein prolongierter Schutz vor Re-Infektion des nachfolgend obturierten Wurzelkanals erreicht wird. Nach Wurzelkanalfüllung sollte möglichst schnell eine dichte, adhäsiv befestigte provisorische oder definitive Deckfüllung appliziert werden, um Schutz vor einer Re-Infektion des Wurzelkanalsystems zu gewährleisten. / This work presents four studies dealing with endodontic treatment and microorganisms or microbial cell wall components, respectively. The first study examined the effect of calcium hydroxide on Escherichia coli LPS. TNF-alpha production in human monocytes was used as indicator for the biological activity of LPS. Results showed that calcium hydroxide is able to eliminate the ability of E. coli LPS to stimulate TNF-alpha production in peripheral blood monocytes. The second study determined the antibacterial effectiveness of either chlorhexidine or calcium hydroxide integrated in gutta-percha points or chlorhexidine or calcium hydroxide delivered as gel or paste. Roots from extracted human teeth were carried with open accesses for one week in the oral cavities of two volunteers. The number of CFU's was determined before and after medication of the contaminated root canals. The chlorhexidine-gel and the calcium hydroxide paste resulted in significantly more samples without microbial colonization as compared to gutta-percha point groups. The third study examined whether obturated roots combined with several adhesive and temporary filling materials can be bypassed by bacteria. After one year of observation, there was no significant difference in number of leaking samples among the groups. It could be concluded that root-filled teeth should receive definitive restoration within three months after obturation. The aim of the fourth study was to examine whether intracanal medication prior to root canal obturation has an inhibitory effect on corono-apical penetration of bacteria. After one year of observation, the group pre-treated with calcium hydroxide paste showed significantly less leaking samples as compared to the groups pretreated with chlorhexidine, Ledermix or no medication. Reconsidering the results of all four studies it may be recommended to perform multi-visit treatment in case of an infected root canal. A one week intermediate dressing with calcium hydroxide will not only neutralize bacterial LPS, but also reduce bacteria significantly and provide prolonged protection against bacterial penetration after root canal filling. After obturation of the root canal system, it is recommended to quickly provide the tooth with a tight adhesively inserted temporary filling or a definitve restoration to achieve additional protection against bacterial ingress.