Tesis sobre el tema "In vitro oral tribology"

L’étude de la lubrification orale devient une problématique actuelle pour l’industrie agroalimentaire. Les analyses quantitatives permettent de comprendre et d’anticiper des mécanismes physiologiques, tels que la prédiction des phénomènes d’astringence des produits alimentaires. L’astringence se manifeste par une diminution de la lubrification de la muqueuse orale après la consommation de produits d’origine végétale. Cependant, les recherches actuelles sur la lubrification orale s’appuient sur des matériaux synthétiques qui représentent mal les tissus buccaux. Elles négligent les interactions entre les protéines salivaires sécrétées et les protéines transmembranaires, limitant ainsi la compréhension des mécanismes de lubrification.Cette thèse s’inscrit dans le projet MACARON qui vise à étudier le rôle de la muqueuse orale dans la perception sensorielle. Des modèles in vitro de muqueuse orale qui expriment la protéine transmembranaire MUC1 ont été développés pour simuler les interactions fondamentales entre MUC1 et les protéines salivaires. Ces interactions sont responsables de la lubrification et de l’hydratation des tissus. Par ailleurs, un tribomètre a été conçu pour effectuer des tests tribologiques in vitro sur ces modèles d’épithélium afin de suivre leur état de lubrification.Cette thèse se concentre ainsi sur l’étude des mécanismes moléculaires de la lubrification orale à travers une approche tribologique in vitro, en utilisant des paramètres physiques macro et micrométriques. Ce manuscrit propose en premier lieu une étude sur le rôle crucial de la mucine MUC1 et de sa structure dans la lubrification orale. La présence de MUC1 améliore la lubrification grâce à une meilleure rétention des protéines salivaires à la surface cellulaire. Ensuite, le manuscrit présente une exploration des mécanismes moléculaires de l’astringence. Les essais tribologiques in vitro en présence de composés astringents montrent que ces substances forment des agrégations à la surface épithéliale qui diminuent la lubrification orale. Parallèlement, nos travaux montrent que des mécanismes de protection, notamment la dissociation de MUC1 et l’interaction des protéines riches en proline avec les tanins, atténuent ces effets néfastes sur la lubrification.À travers une étude complémentaire, des corrélations entre la perception sensorielle et nos propriétés physiques mesurées sont établies, démontrant la capacité de notre méthodologie à classer des individus selon leur sensibilité à l’astringence. Enfin, la dernière étude présente le développement d’un nouveau modèle de muqueuse orale visant à reproduire les propriétés mécaniques et physico-chimiques de la muqueuse in vivo.Cette thèse propose une méthodologie innovante pour l’étude de la lubrification orale, en particulier en s’intéressant à des mécanismes responsables de la sensation d’astringence grâce à l’utilisation de modèles de muqueuse toujours plus proches des tissus oraux physiologiques
The study of oral lubrication has become a current concern for the food industry. Quantitative analyses allow for understanding and anticipating physiological mechanisms, such as predicting astringency phenomena in food products. Astringency is characterized by a decrease in oral mucosa lubrication following the consumption of plant-based products. However, current research on oral lubrication relies on synthetic materials that poorly represent oral tissues, neglecting interactions between secreted salivary proteins and transmembrane proteins, thus limiting the understanding of lubrication mechanisms. This thesis is part of the MACARON project, which aims to investigate the role of oral mucosa in sensory perception. In vitro models of oral mucosa expressing the transmembrane protein MUC1 have been developed to simulate fundamental interactions between MUC1 and salivary proteins responsible for tissue lubrication and hydration. Additionally, a tribometer has been designed to perform in vitro tribological tests on these epithelial models to monitor their lubrication state. Thus, this thesis focuses on studying the molecular mechanisms of oral lubrication through an in vitro tribological approach, using macro- and micrometric physical parameters. Firstly, this manuscript provides a study on the crucial role of MUC1 mucin and its structure in oral lubrication. The presence of MUC1 enhances lubrication by improving the retention of salivary proteins on the cell surface. Secondly, the manuscript explores molecular mechanisms of astringency. In vitro tribological tests in the presence of astringent compounds show that these substances form aggregations on the epithelial surface, reducing oral lubrication. Concurrently, our work demonstrates protective mechanisms, including the dissociation of MUC1 and the interaction of proline-rich proteins with tannins, mitigating these adverse effects on lubrication. Through additional study, correlations between sensory perception and our measured physical properties are established, demonstrating the ability of our methodology to classify individuals based on their sensitivity to astringency. Finally, the last study presents the development of a new oral mucosa model aiming to reproduce mechanical and physicochemical properties of in vivo mucosa. This thesis proposes an innovative methodology for studying oral lubrication, particularly focusing on mechanisms responsible for astringency sensation through the use of mucosa models increasingly closer to physiological oral tissues

This thesis studies how to increase the expected satiating ability in dairy products analyzing the effect of the incorporation of certain ingredients. The study of food reformulation with high expected satiating ability contributes to moderate size meal choice and to regulate food intake. In this thesis, two factors influencing the satiating ability perception of semi-solid foods have been mainly studied: a) the increase in oral processing time that produces prolonged orosensory signals, and b) the increase in protein content. The increase in oral processing time is produced by, for example, higher consistencies, mouth-coating or creaminess sensations. These features can be modified by the addition of hydrocolloids. Native starch, modified starch, ¿-carrageenan and guar gum were added to increase the viscosity and consistency of milkshakes. Rheological characterization of the products revealed very different behavior patterns depending on the type of hydrocolloid. The addition of saliva in the studies showed modified sensory perceptions, so its inclusion is desirable and more realistic. The addition of hydroxypropyl methylcellulose (HPMC) and the effect of milk solids and cream in milk-based desserts was also studied. In addition to HPMC dominant effect on the temporality of perceptions, the presence of fat globules or milk solids modulated such perceptions. Yogurt was selected to increase protein content, adding twice the original amount of protein from different milk fractions, and starch. Yogurt with milk powder addition was the most accepted sample and it was closer to the 'ideal' characteristics of a satiating yogurt according to consumers, with a series of sensory attributes that contribute to the feeling of expected satiety. On the other hand, in vitro oral digestion showed that physically modified starch granules remain unchanged after the attack of ¿-amylase, and would be responsible for its consistency and creaminess. In addition, it was found that the sensation of astringency was undesirable; tribological studies allowed to interpret yogurt sensory characteristics. The lubricity of some samples did not reflect the difference in astringency between samples indicating that this was not a purely tactile perception caused by an increase in friction. The addition of physically modified starch significantly reduced friction coefficients values due to soluble starch polymers and starch granules preservation. All the strategies allowed to gain understanding on how to reformulate food increasing its satiating ability perception. Such strategies could be extrapolated to other food categories, what broadens the scope of the results obtained in this thesis.
La presente tesis estudia cómo aumentar las expectativas de la capacidad saciante en productos lácteos analizando el efecto de la incorporación de determinados ingredientes. El estudio de la reformulación de alimentos con alta expectativa sobre su capacidad saciante contribuye a moderar la elección de las raciones de alimentos y a regular la ingesta. En la presente tesis se han estudiado principalmente dos de los factores que más influyen en la percepción de capacidad saciante de alimentos semisólidos: a) el aumento en el tiempo de procesado oral que produce saciedad debido a una prolongación de las señales orosensoriales, y b) el alto contenido en proteínas. El aumento del tiempo de procesado oral lo producen, por ejemplo, mayores consistencias, recubrimiento oral o sensaciones de cremosidad, que son características que se pueden modificar por adición de hidrocoloides. Se emplearon almidón nativo y modificado, goma guar y ¿-carragenato para aumentar la viscosidad y consistencia de batidos lácteos. La caracterización reológica de los productos reveló patrones de comportamiento muy diferentes dependiendo del tipo de hidrocoloide. La adición de la saliva en los estudios demostró modificar las percepciones sensoriales, por lo que su inclusión resulta recomendable y más realista. Se estudió también la adición de hidroxipropilmetilcelulosa (HPMC) y el efecto de dos componentes normales en productos lácteos (sólidos lácteos y nata). Además del efecto dominante del HPMC en la temporalidad de las percepciones, la presencia de glóbulos de grasa o sólidos de la leche modularon dichas percepciones. Para aumentar el contenido en proteína se seleccionó como alimento el yogur, al que se añadió el doble de la cantidad de proteína de distintas fracciones lácteas y almidón. El yogur con el doble de leche en polvo fue globalmente el más aceptado y más cercano a las características "ideales" según los consumidores, con una serie de atributos sensoriales que contribuyen a la sensación de saciedad esperada. Por otro lado, la digestión oral in vitro mostró que los gránulos de almidón procedentes de almidón físicamente modificado permanecen inalterados después del ataque de la ¿-amilasa de la saliva, y serían los responsables de la mayor consistencia y cremosidad. Adicionalmente, se encontró que la sensación de astringencia resultaba negativa; los estudios tribológicos permitieron interpretar las características sensoriales. Las propiedades lubricantes de algunas muestras no reflejaron la diferencia de astringencia sensorial indicando que ésta no era una percepción puramente táctil causada por un aumento en la fricción. La adición de almidón modificado físicamente redujo significativamente los valores de los coeficientes de fricción relacionados con los polímeros de almidón solubles y la conservación de gránulos en el almidón. Todas las estrategias abordadas permitieron comprender y ahondar en los conocimientos que conducen a cómo reformular un alimento para aumentar la percepción de saciedad. Dichas estrategias son extrapolables a otras categorías de alimentos lo que amplía el alcance de los resultados obtenidos en la presente tesis.
La present tesi estudia com augmentar les expectatives de la capacitat saciant en productes lactis analitzant l'efecte de la incorporació de determinats ingredients. L'estudi de la reformulació d'aliments amb alta expectativa sobre la seua capacitat saciant contribueix a moderar l'elecció de les racions d'aliments i a regular la ingesta. En la present tesi s'han estudiat principalment dos dels factors que més influeixen en la percepció de capacitat saciant d'aliments semisòlids: a) l'augment en el temps de processat oral que produeix sacietat a causa d'una prolongació dels senyals orosensorials, i b) l'alt contingut en proteïnes. L'augment del temps de processat oral el produeixen, per exemple, majors consistències, recobriment oral o sensacions de cremositat, que són característiques que es poden modificar per addició d'hidrocol·loïdes. S'emprà midó natiu i modificat, goma guar i ¿-carragenat per augmentar la viscositat i consistència de batuts lactis. La caracterització reològica dels productes revelà patrons de comportament molt diferents depenent del tipus d'hidrocol·loïde. L'addició de la saliva en els estudis demostrà modificar les percepcions sensorials, per la qual cosa la seua inclusió resulta recomanable i més realista. S'estudià també l'addició d'hidroxipropilmetilcel·lulosa (HPMC) i l'efecte de dos components normals en productes lactis (sòlids lactis i nata). A més de l'efecte dominant del HPMC en la temporalitat de les percepcions, la presència de glòbuls de greix o sòlids de la llet van modular les citades percepcions. Per augmentar el contingut en proteïna es va seleccionar com a aliment el iogurt, a què s'afegí el doble de la quantitat de proteïna de diverses fraccions làcties i midó. El iogurt amb el doble de llet en pols fou globalment el més acceptat i més pròxim a les característiques "ideals" segons els consumidors, amb una sèrie d'atributs sensorials que contribueixen a la sensació de sacietat esperada. D'altra banda, la digestió oral in vitro mostrà que els grànuls de midó procedents de midó físicament modificat romanen inalterats després de l'atac de l'¿-amilasa de la saliva, i serien els responsables de la major consistència i cremositat. Addicionalment, es trobà que la sensació d'astringència resultava negativa; els estudis tribològics permeteren interpretar les característiques sensorials. Les propietats lubricants d'algunes mostres no reflectiren la diferència d'astringència sensorial indicant que esta no era una percepció purament tàctil causada per un augment en la fricció. L'addició de midó modificat físicament reduí significativament els valors dels coeficients de fricció relacionats amb els polímers de midó solubles i la conservació de grànuls en el midó. Totes les estratègies abordades permeteren comprendre i aprofundir en els coneixements que condueixen a com reformular un aliment per augmentar la percepció de sacietat. Les citades estratègies són extrapolables a altres categories d'aliments, cosa que amplia l'abast dels resultats obtinguts en la present tesi.
Morell Esteve, P. (2017). Diseño de alimentos lácteos con capacidad saciante. Relación entre estructura, procesamiento oral y percepción [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/89093
TESIS

The canine oral microbiota is poorly understood compared to that of humans. The aim of this work was to improve understanding of the canine oral microbiota. This was achieved by surveying the canine oral microbiota, determining coaggregation interactions between its members, and developing a laboratory microcosm. Bacteria were isolated from the dental plaque and saliva of dogs, and isolates were identified by comparative 16S rRNA gene sequencing. From 339 isolates, 84 phylotypes belonging to 37 genera were identified. Approximately half were identified to species level, and 28 % of these were also members of the human oral microbiota. Thirty eight phylotypes were tentatively identified as candidate new species. The genera most frequently isolated from saliva were Actinomyces, Streptococcus, and Granulicatella. Porphyromonas, Actinomyces, and Neisseria were most frequently isolated from plaque. On average, sequences from this study differed by almost 7 % in the 16S rRNA gene compared to similar organisms from humans. Targeted PCR was used to detect culture resistant bacteria from canine plaque. Successful amplification indicated that Spirochaetes and candidate division TM7 bacteria were present, however the identities of the originating organisms were not determined. The entire cultivable plaque microbiota from a single dog was assessed for coaggregation reactions. Eight (6.7 %) unique interactions were detected from 120 crosses, indicating that the prevalence of coaggregation is similar in the canine and human oral microbiotas. Genera common to both hosts generally exhibited similar coaggregation reactions, however autoaggregation was more common among bacteria isolated from dogs. The constant depth film fermenter was used to grow microcosms from canine plaque and saliva using a mucin containing artificial saliva supplemented with horse serum as the growth medium. The model produced biofilms similar to natural dental plaque, which could be used to investigate the canine oral microbiota further.

Manufacturers are under pressure to reformulate products to make healthier foods, without changing desirability or flavour. A better understanding of product breakdown under oral conditions is essential to structure novel products which are healthier without consumers noticing. In-vitro methods were developed to explore product behaviour when subject to a range of phenomena relevant to those in the mouth, with particular emphasis on lubrication behaviour. Polysaccharides common in food products were mainly used as model systems and salt release was studied in some systems. Three in-vitro systems were developed and used in this study. Firstly, a stirred vessel was developed to gather data of salt release from gelatin, gellan and alginate systems, under quiescent conditions. This was a reliable method of tracking diffusion of sodium ions through the gel structures into a surrounding liquid, showing that diffusion was unaffected by the differing structure of the gels. The second system introduced the effect of compression. Only when pressures are sufficient to rupture the gel samples did compressions affect salt release over that observed in the stirred vessel study. Samples released the majority of their contained salt up to nine times faster, as a result of greater surface areas being exposed. Finally, tribology equipment was developed, which explores the thin film, high shear behaviour of materials. An exploration of available equipment, processing parameters and configurations was carried out to determine optimum surfaces, normal forces and speed ranges which could be related to phenomena occurring in the mouth. The lubricating properties of inhomogeneous polysaccharides with different physical properties were studied. The mixing behaviour of the polysaccharide greatly affected the lubrication response; some mixed quickly so lubricated more efficiently and vice versa. Finally, the developed tribology equipment was used to study the ordering process of a series of fluid gel samples, indicating that lubrication tracks the ordering process, with a decrease in lubrication when structure forms. The pattern of this response is a result of polysaccharide and salt content of the materials, with increasing content enhancing the change in lubrication experienced as more rigid gel particles are produced. The work presented in this thesis shows that the use of in-vitro methods can provide repeatable information on structure behaviour under conditions relevant to the mouth. This information could then be used to develop and assess future food products for their expected performance when consumed.

In vitro models of dental plaque are a valuable tool for understanding the development of plaque-related diseases and assessing potential treatments for these diseases. The main focus of this study was the development of an in vitro model to characterise the changes in bacterial populations from dental plaque associated with health to one associated with gingivitis. By emulating environmental conditions in the oral cavity associated with gingivitis it was possible to see changes in the oral microbiota associated with gingivitis. Using traditional culture techniques the ascendancy of Actinomyces spp. at the expense of Streptococcus spp. was observed with the onset of gingivitis conditions, along with increased proportions of Gram-negative species. To assess the range of cultivable species present isolates, which had previously been cultured in the model, were identified by sequencing of the 16S rRNA gene. After the onset of gingivitis conditions a greater richness of species was identified. Examination of these communities with confocal microscopy and viability staining also revealed structural changes associated with environmental conditions emulating gingivitis. To assess the presence of species which were not frequently identified by culture, but previously shown to be associated with gingivitis, quantitative PCR (qPCR) was used to enumerate Prevotella spp., Fusobacterium spp. and Porphyromonas gingivalis. Prevotella and Fusobacterium spp. were found to be significant members of the microbial communities developed in the CDFF, with Prevotella spp. increasing significantly under conditions emulating gingivitis. Furthermore, the total bacterial counts enumerated by culture were underestimated by approximately 80% compared to the total counts obtained by qPCR. This model was ultimately used to assess the effectiveness of tetracycline, chlorhexidine and silver ion-releasing dental materials against the accumulation of plaque. All of these agents influenced the microbial composition, rather than total microbial numbers, with reduced levels of Actinomyces, Prevotella and Fusobacterium spp. This study has shown that in vitro models of microbial communities associated with health and disease are valuable tools for observing key factors in disease progression. When disease results from changes in the resident microflora the use of such models allows the influence of individual environmental factors to be assessed and also allows the effect of potential treatments on these communities to be examined.

Around half of oral bacteria have yet to be cultured, and their role in disease is therefore unknown. It is hypothesised that bacteria in biofilms have become dependent on growing in multi-species communities. TM7 phylum has no cultured representatives and some oral TM7 phylotypes have been associated with oral diseases such as periodontitis. The aims of this study were therefore to evaluate the ability of two model culture systems: Cooked Meat Medium and the Calgary Biofilm Device (CBD), to support the growth of mixed oral bacterial communities including uncultured bacteria, and to attempt to culture representatives of the TM7 division. The Cooked Meat Medium was used to establish a mixed bacterial community from 3 endodontic samples and their composition was analysed by Sanger sequencing and 454 pyrosequencing. A diverse bacterial community closely related to the original endodontic samples was maintained up to 480 days and included some uncultured bacteria present in the original samples. A mixed oral biofilm was established on the CBD from saliva. The effect of the presence of mucin and glucose in the growth media on community composition was evaluated, but no significant differences were seen. The effect of using propidium monoazide to remove extracellular DNA was assessed and was found to significantly affect the perceived composition of the biofilms. Uncultured taxa detected in culture included representatives of deep branches of Bacteroidetes and Clostridiales, and TM7 and SR1 phyla. TM7 members were detected in both models with specific PCR primers, but their proportion never exceeded 1 %. In an attempt to isolate TM7 division representatives a saliva microcosm was grown on agar. TM7 representatives were detected by colony hybridization and specific PCR and subcultured, producing enrichment. Two simple co-cultures of TM7 HOT352/HOT353 with Slackia exigua or Atopobium parvulum were obtained, but were not maintained.

SyfteSyftet med denna laborativa pilotstudie in vitro var att undersöka hur den mikrobiella sammansättningen och pH i den orala biofilmen påverkades vid upprepad exponering för Stevia (steviosid och rebaudiosid A), sackaros eller en kombination av reb A och sackaros. Ett ytterligare syfte var att studera om isolat av orala bakterier kunde metabolisera reb A i buljongkultur. Material och metodBiofilmer från tre personer blev exponerade för 0,025 % steviosid, 0,025 % reb A eller 10 % Todd Hewitt (TH) under 15 minuter tre gånger dagligen i fyra dagar. Artbestämning gjordes i början och slutet av försöken. Biofilmerna exponerades för 0,025 % reb A och 10 % sackaros eller en kombination av dessa varefter pH-värdet mättes. Vidare studerades 23 bakteriestammar avseende förmåga att metabolisera 0,025% respektive 0,5% reb A. ResultatStreptokocker dominerade i samtliga biofilmer vid försökets slut oavsett om de exponerats för steviosid, reb A eller enbart TH. En pH-sänkning kunde ses hos biofilmerna som enbart blivit exponerade för sackaros eller för en kombination av sackaros och reb A. Däremot kunde endast en marginell pH-sänkning observeras efter exponering för enbart reb A. Ingen av de enskilda bakteriestammar som studerades kunde metabolisera reb A.SlutsatsIngen större skillnad i biofilmens bakteriesammansättning kunde ses efter upprepad exponering för steviosid eller reb A i förhållande till TH.Upprepade exponeringar av reb A i kombination med sackaros gav en liknande pH-sänkning som enbart sackaros, dock sågs ingen pH-sänkning efter exponering för enbart reb A. Enskilda bakteriestammar kunde inte metabolisera reb A.
AimsIn this in vitro laboratory pilot study, the effect on the microbial composition and pH of the oral biofilms after repeated exposure to Stevia (stevioside and rebaudioside A), saccarose or a combination, was investigated. Another aim was to study whether isolate of oral bacteria were able to metabolise reb A in broth bacteria culture.Material and MethodBiofilms from three adult individuals were repeatedly exposed to 0,025% stevioside, 0,025% reb A or 10% Todd Hewitt (TH). Samples were taken at the beginning and at the end of the experiment for cultivation and bacterial identification.Biofilms were exposed to 0,025% reb A and 10% saccharose, either separately or combined, followed by a pH-measurement. Furthermore, 23 bacterial species were studied in regards to ability to metabolize reb A. ResultsAll biofilms were dominated by Streptococcus regardless of whether they were exposed to stevioside, reb A, or TH. A pH-decrease was seen in biofilms exposed to saccharose or a combination of saccharose and reb A. However, only minor changes were noted after exposure to reb A.None of the species studied were able to metabolize reb A.ConclusionNo major differences in the bacterial composition of the biofilms were seen after repeated exposure to stevioside or reb A compared to TH.No pH-decrease after repeated exposure to reb A was noted. Exposure to reb A combined with saccharose resulted in a pH-decrease similar to exposure to saccharose. The bacterial species tested in this study were not able to metabolise reb A.

Dipeptides can be absorbed into cells via the dipeptide transporter (which also transported tripeptides and dipeptide derivatives). The optimum conditions for measuring the inhibition of Gly-Pro uptake in Caco-2 cells were identified. A number of structure-activity relationships were identified. These included the effects of increasing the amino-acid chain-length, and the presence of a thiol or hydroxyl group in the side-chain increased IC50 while the presence of a hydroxyl group did not. The benzyl esters had lower or equal IC50 values compared to the parent dipeptides while the methyl esters had higher values. These results indicated that while molecular properties did affect IC50, the size, charge and composition of three particular groups caused the most significant effects, supporting the structure-activity relationship identified. An assay was developed using calcein-AM to show the inhibition of p-glycoprotein activity. There was no significant change due to the presence of mannitol but there was in the presence of clyclosporin A (p<0.01). Incubating the cells with the test solution for 30 minutes before the addition of the ester resulted in a significant (p<0.001) difference. The assay was specific for p-glycoprotein, as the presence MRP inhibitors had no effect (p>0.05). The modified protocol allowed the identification of p-glycoprotein inhibitors quickly and simply using a cell suspension of unmodified cells. The clinically relevant buffering of grapefruit juice to pH 7 led to a four-fold increase in intracellular calcein and hence significant inhibition of p-glycoprotein. Buffered orange and lemon juices had no effect on the assay. Flavone derivatives had previously been found to be inhibitors of CYP3A4 yet neither naringin nor naringenin had any significant effect at concentrations found in grapefruit juice. Of the other (non-grapefruit) flavone derivatives tested, hesperidin, found in orange juice, had no significant effect, kaempferol and rutin also had no effect while genistein significantly inhibited p-glycoprotein (results that support previous studies).

Abstract Desmoglein 3 (Dsg3) is an adhesion receptor in desmosomes, but relatively little is known about its role in cancer. In this study, the function of Dsg3 was investigated in oral squamous cell carcinoma (SCC) cell lines in vitro using locally established human leiomyoma tumor microenvironment (TME) matrices. Since Dsg3 has been identified as a key regulator in cell adhesion, we hypothesized that it may play a role in oral SCC cells adhesion and motility. Thus, one aim of the study was to explore this hypothesis by both gain and loss of function methods in four human buccal mucosa SCC SqCC/Y1 cell lines: transduction of vector control (Ct), full-length (FL) or two different C-terminally truncated Dsg3 mutants (Δ238 and Δ560). Live cell imaging was performed for 2D migration and 3D sandwich, alongside other assays. In 3D sandwich, we tested the effects of the monoclonal antibody, AK23, targeting the extracellular domain of Dsg3 in SqCC/Y1 cells. Our results showed that loss of Dsg3 disrupted cell adhesion and protein expression. In 2D assays, FL and Dsg3 mutants migrated faster with higher accumulated distances than Ct. In contrast with 2D, mutants showed accelerated invasion over the Ct in 3D models. The AK23 antibody inhibited only the invasion of FL cells. The TME in vivo consists of cellular and matrix elements playing a leading role in carcinoma progression. To study carcinoma cells invasion in vitro, mouse Matrigel® and rat type 1 collagen are the most commonly used matrices in 3D models. Since they are non-human in origin, they do not perfectly mimic human TME. To address this, we have developed a solid organotypic myoma disc model derived from human uterus leiomyoma tumor. Here, we introduce a novel Myogel, prepared from leiomyoma similar to Matrigel®. We validated Myogel for cell-TME interactions in 3D models, using SqCC/Y1 and HSC-3 cell lines. Compared with Matrigel® and type I collagen, oral SCC cell lines invaded more efficiently in Myogel containing matrices. This study describes promising 3D models using human TME mimicking Myogel which is suitable to analyze oral SCC cells both in carcinoma monocultures and in co-cultures, such as with TME fibroblasts. We also introduce a possible novel therapeutic target against Dsg3 to suppress cancer cell invasion
Tiivistelmä Desmogleiini 3 (Dsg3) on desmosomien adheesioreseptori, jonka merkityksestä syövässä tiedetään vähän. Koska Dsg3 on tärkeä epiteelisolujen välisissä liitoksissa, oletimme sillä olevan vaikutusta myös suun karsinoomasolujen tarttumisessa ja niiden liikkuvuudessa. Testasimme hypoteesiamme muuttamalla Dsg3:n toimintaa ihmisen posken karsinoomasolulinjassa SqCC/Y1, josta oli aiemmin valmistettu neljä erilaista muunnosta: tyhjän vektorin sisältävä kontrollisolulinja (Ct), kokopitkää Dsg3 tuottava solulinja (FL), sekä kaksi Dsg3 C-päästä lyhennettyä mutanttisolulinjaa (Δ238 ja Δ560). Immunofluoresenssi-menetelmää käyttäen analysoimme solulinjoissamme solujen välisiä liitoksia. Lisäksi mittasimme solujen liikkeitä 2D-migraatio- ja 3D-sandwich-kokeissa. Testasimme myös Dsg3:n solunulkoista osaa tunnistavan monoklonaalisen vasta-aineen (AK23) vaikutusta solujen invaasioon. Osoitimme, että Dsg3:n rakenteen muuttaminen ja toiminnan estyminen häiritsi solujen tarttumista. 2D-kokeissa sekä FL että mutanttilinjat (Δ238 ja Δ560) migroivat kontrollisoluja nopeammin ja pidemmälle, mutta 3D-kokeissa vain mutanttilinjat invasoituivat kontrollisoluja tehokkaammin. AK23-vasta-aine esti vain FL-solujen invaasiota. Syöpäsolujen 3D-invaasiota mittaavissa kokeissa käytetään yleensä hiiren kasvaimesta valmistettua kaupallista Matrigeeliä® tai rotan kudoksista eristettyä tyypin I kollageenia. Tutkimusryhmämme on jo aiemmin kehittänyt organotyyppisen myoomamallin, jossa valmistamme myoomakudosnapit ihmisen kohdun leiomyoomakasvaimista. Tässä työssä valmistimme leiomyoomasta Myogeelia, vertasimme sitä Matrigeeliin®, sekä tutkimme tarkemmin Myogeeli-valmisteen soveltuvuutta 3D-tutkimuksiin. Totesimme, että kielen (HSC-3) ja posken (SqCC/Y1) karsinoomasolut invasoituivat tehokkaimmin Myogeeli-pitoisissa matrikseissa kuin Matrigeeliä® tai kollageeniä sisältävissä kasvatusalustoissa. Tutkimustulostemme perusteella Myogeeli-pohjaiset 3D-mallit soveltuvat hyvin sekä syöpäsolulinjojen invaasiotutkimuksiin että yhteisviljelmiin, joissa syöpäsoluja viljellään yhdessä syöpäkasvaimen ympärillä olevien solujen, kuten fibroblastien, kanssa

Porcine buccal mucosa has been extensively used as in vitro model to study the permeability of drugs and assess their potential to deliver through buccal route. Porcine buccal mucosa is found to be very similar to human oral mucosa in structure and function. However, the in vitro permeation studies across porcine buccal mucosa show high variability which is mostly due to the various experimental and biological variables that are often overlooked while conducting such studies. The precise nature of the permeability barrier offered by the various tissue layers of buccal mucosa was investigated in this study. It was observed that the permeability of model diffusants decreased significantly with an increase in the connective tissue layer. However, the epithelium offered a stronger barrier to permeation of all diffusants studied at mucosal thickness of up to 500 |tm. The epithelium acted as a stronger barrier for hydrophilic diffusants when compared to lipophilic diffusants. It was also observed that the permeability of model diffusants was significantly higher in the region behind lip when compared to the middle cheek region which is due to lower epithelial thickness in that region. Porcine buccal mucosa retained its integrity in Kreb's bicarbonate Ringer solution at 4 °C for 24 hours and many other storage conditions resulted in loss of epithelial integrity. Separation of epithelium from the underlying connective tissue by heat treatment, did not adversely affect its permeability and integrity characteristics. Influence of experimental temperature on the permeability of model compounds across porcine buccal mucosa was also investigated in vitro. An exponential relationship was observed between the apparent permeability and temperature. It was found that the activation energy of diffusion of the model compounds decreased linearly with increasing distribution coefficients across porcine buccal mucosa. This suggested that the buccal mucosa acted as a stronger barrier for diffusion of hydrophilic diffusants when compared to the lipophilic diffusants.

The prevalence of oral diseases such as dental caries and periodontitis and the universal need for effective control of oral health has stimulated a great deal of interest in oral hygienic formulations both scientifically and commercially driven. Such formulations are normally deployed as complex formulations commonly containing antimicrobial actives together with excipients, where both classes of ingredients may contribute to the bacteriological effect of the oral hygienic product. However, the mode of action and/or the bacteriological and microecological effects of exposure of microorganisms to oral hygiene products are poorly understood. In this context, this doctoral dissertation represents a series of investigations to contribute to knowledge in the area. The impact of selected oral antimicrobial actives (triclosan, sodium lauryl sulphate, stannous fluoride and zinc lactate) on a key aspect of bacterial cellular membrane function was investigated. This involved measuring major cellular respiratory pathways during exposure to the test agents using two types of tetrazolium dyes possessing different redox potentials as respiration pathway indicators. Spectrophotometric analyses indicated that sub-lethal levels of triclosan and sodium lauryl sulphate act as uncoupling agents, an observation not previously been reported. Sub-lethal concentrations of stannous fluoride and zinc lactate however, blocked cellular respiration with resulting shifts towards glycolytic/fermentative pathways. The contribution of a variety of test agents to the overall antimicrobial effect of a complex formulation (Listerine®) was investigated in order to understand the relative efficacy of the actives. This was achieved by testing the essential oils present in the formulation singly and in combination utilising in vitro models. The use of the hydroxyapatite disc model (HDM) to grow salivary microcosms to test the efficacy of the ingredients revealed hitherto unreported synergistic activity between the active ingredients thymol and menthol. Proprietary dentifrices (Colgate Total® and Crest ProHealth®) containing the antimicrobial agents triclosan or stannous fluoride/zinc lactate, respectively, were comparatively evaluated. This was performed by simultaneously establishing salivary microcosms in Sorbarod Biofilm Devices (SBDs). Following the establishment of dynamic steady-states, paired devices were dosed with each of the two proprietary dentifrices. Bacteriological data generated after multiple dosing indicated that both dentifrices were comparably effective in the reduction of all tested bacterial functional groups in the plaque models. However, data generated using HDM models indicated greater reductions in Gram-negative anaerobes after exposure to Colgate total®. The observations presented in this thesis may contribute to the development of oral formulations with optimised antimicrobial efficacies against adventitious pathogens present in the oral cavity and help in reducing the incidence of oral diseases and potentially related systemic interface.

Orala bakterier, såsom de tidiga kolonisatörerna; Streptococcus gordonii, Streptococcus oralis, Streptococcus mitis och Actinomyces naeslundii uttrycker ett brett spektrum av ytadhesiner som möjliggör inbindning till receptorer i tandpellikeln. Saliv, gingivalt exudat (GCF) och kollagen I i cement på blottade rotytor erbjuder möjliga ytor i munhålan där bakterier kan adherera och bilda biofilm. Syftet med denna studie var att undersöka huruvida utvalda bakterier kan förändra genuttryck beroende på innehållet i olika ytor. Laborativa in vitro-försök genomfördes där de fyra bakteriearterna tilläts växa på ytor täckta med de tre olika substrat; saliv, serum och kollagen I. Graden av inducerad proteolytisk aktivitet, yt-associerad såväl som utsöndrad, uppskattades därefter med hjälp av ett FITC-konjugerat substrat, radiell diffusionsteknik samt spektrofotometri. Enligt studiens hypotes skulle bakteriearterna anpassa sig beroende på ytan de fäste till, och därigenom ändra metabol aktivitet såsom proteasuttryck. Baserat på resultaten kunde små förändringar noteras. Dock kunde inga bestämda slutsatser dras vad gäller förändrad proteolytisk förmåga hos de utvalda bakterierna exponerade för de olika orala ytorna i studien.
Oral bacteria, such as the early colonizers; Streptococcus gordonii, Streptococcus oralis, Streptococcus mitis and Actinomyces naeslundii display a wide range of surface adhesins which enable them to bind to receptors in the tooth pellicle. Saliva, gingival crevicular fluid (GCF) and collagen I in cementum on uncovered root surfaces present possible binding sites in the oral cavity onto which microorganisms can adhere and form a biofilm. The aim of this study was to assess whether these selected bacteria can alter their gene expression in response to protein components found on the various surfaces. In vitro laboratory assays were conducted, where the four oral species were added to surfaces coated with three substrates; human saliva, human serum and collagen I. The degree of induced proteolytic activity, surface-associated as well as secreted, was subsequently assessed using a FITC-labelled protease substrate, radial diffusion assays on skim milk agar and spectrophotometry. The hypothesis underlying the study was that bacterial species adapt depending on the surfaces they adhere to, thus altering protease expression. Based on the results, small variations could be detected, although no firm conclusion can be drawn regarding proteolytic abilities of the selected bacteria when exposed to the surfaces tested here.

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Dissertacao (Mestrado Profissionalizante em Lasers em Odontologia)
IPEN/D-MPLO
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP; Faculdade de Odontologia, Universidade de Sao Paulo, Sao Paulo

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The aims of this study was the oral cavity evaluation of females stray dogs from Pelotas-RS, as well as yeast isolation and identification from three different areas of this cavity, comparison of two technique of sample collection (swabs and curettes) and test the in vitro susceptibility of isolated yeasts against common oral antiseptics used in pets. Fifty-nine randomized animals were included in the study. All of them were SRD, without age defined and from Pelotas-RS. They were divided in three groups of ages by the evaluation of dental arc, and were examined to determine the cranial morphology, presence of dental plaque, tooth fractures, malocclusion, halitosis and gingival bleeding. Measure of periodontal groove depth was also done. Samples were collected from three areas, gingival mucosa, dental biofilm and periodontal grooves, with different collection techniques: swab, curettes, millimeter periodontal probe and cellulose polymers membranes. In vitro susceptibility tests by microdilution methods was done with 15 of those yeasts against A product (0,12% chlorhexidine gluconate, 0,12% benzalkonium chloride and 0,10% chlorophila extract) and B product (0,2% benzalkonium chloride, 1% propolis and 0,5% peppermint arome). It was also evaluated chemical principles of products in separated, as chlorhexidine gluconate, benzalkonium chloride and propolis. Tooth fractures was observed in higher proportion at animals with six or more years old (p=0,0030). Dogs younger than 2 years old had lower rate of dental plaques than the others groups (p=0,0000). The association of positive areas of yeast isolation with age, tooth fracture, dental plaque, halitosis, gingival bleeding or malocclusion was not statistically significant. Sixty-one yeasts were isolated, characterized by M. pachydermatis (50,82%), Rhodotorula spp. (13,11%), C. albicans (4,92%), C. catenulata (3,28%), C. famata (1,64%), C. guilliermondii (1,64%), C. parapsilosis (1,64%), C. intermedia (1,64%), T. asahii (13,11%), T. mucoide (1,64%) and C. albidus (6,56%), from 30 (50,85%) animals. Yeasts growing inhibition was observed in all products tested and in all concentrations of them, with exception of propolis that did not show activity against yeasts. In conclusion, at oral cavity exam of female dogs, the mainly alterations found were dental plaques, tooth fractures and malocclusion. Isolated yeasts are normal habitants from the three different areas of oral cavity studied, without resulting in clinical signs. In relation to the techniques, it was not found difference between swab and curettes for oral sample collection. Yeast isolation was higher in female dogs that show halitosis. Oral antiseptics tested, as well as chlorhexidine gluconate and benzalkonium chloride were effective against yeasts isolated from canine oral cavity in all dilution tested. Propolis, at the conditions and dilutions tested, is not recommended as an oral antiseptic against yeast.
Objetivou-se avaliar a cavidade oral de fêmeas caninas errantes de Pelotas-RS, isolar e identificar leveduras de três sítios desta cavidade, comparar duas técnicas de colheita da mucosa gengival (swab e cureta) e testar a suscetibilidade in vitro destas leveduras frente a anti-sépticos orais utilizados na rotina odontológica de pequenos animais e seus princípios ativos. Foram avaliadas 59 fêmeas caninas, errantes, SRD, provenientes de Pelotas/RS. Os animais foram selecionados aleatoriamente, e distribuídos em três grupos de idade conforme avaliação da arcada dentária. Estes foram avaliados quanto à conformidade cranial, presença de cálculo dentário, fraturas dentárias, maloclusão, halitose, sangramento gengival e mensuração da profundidade de sulco peridontal. As amostras foram obtidas da mucosa gengival, biofilme dental e sulco periodontal, com diferentes formas de coletas: swab, cureta, sonda periodontal milimetrada e ponta de membrana em éster de celulose. O teste de suscetibilidade in vitro foi realizado pela técnica de microdiluição em caldo testando o produto A (0,12% de gluconato de clorexidina, 0,12% de cloreto de benzalcônio e 0,10% de extrato de clorofila) e, o produto B (0,2% de cloreto de benzalcônio, 1% de tintura de própolis e 0,5% de aroma hortelãpimenta) frente a 15 leveduras isoladas. Também foram avaliados os princípios ativos gluconato de clorexidina, cloreto de benzalcônio e tintura de própolis isoladamente. As fraturas dentárias foram mais frequentes nos animais com seis anos ou mais (p=0,0030). Nos cães com até dois anos a presença de cálculo dentário aresentou-se com menor frequência (p=0,0000) em relação aos outros dois grupos. Na relação entre o número de locais de isolamento com idade, conformidade cranial, presença de fratura, cálculo dentário, halitose, sangramento gengival e maloclusão não foi encontrada diferença estatisticamente significativa. Foram isoladas 61 leveduras, M. pachydermatis (50,82%), Rhodotorula spp. (13,11%), C. albicans (4,92%), C. catenulata (3,28%), C. famata (1,64%), C. guilliermondii (1,64%), C. parapsilosis (1,64%), C. intermedia (1,64%), T. asahii (13,11%), T. mucoide (1,64%) e C. albidus (6,56%) distribuídas em 30 (50,85%) animais. Foi observada inibição do crescimento de todas as leveduras, em todos os produtos, em todas as concentrações, com exceção da tintura de própolis que não demonstrou ação nenhuma nas concentrações testadas. Sendo assim, na avaliação da cavidade oral das fêmeas caninas predominaram presença de cálculo dentário, fraturas dentárias e maloclusões. As leveduras isoladas fazem parte da microbiota dos diferentes sítios da cavidade oral das fêmeas caninas estudadas, estando presentes sem causar alterações. O isolamento de leveduras foi maior naquelas fêmeas que tinham halitose. Não houve diferença entre as técnicas utilizadas (swab ou cureta) para colheita de material da mucosa gengival. Os anti-sépticos orais e os compostos gluconato de clorexidina e o cloreto de benzalcônio foram eficazes frente às leveduras isoladas da cavidade oral de fêmeas caninas em todas as concentrações testadas, inclusive nas abaixo da recomendada para uso. Nas condições estudadas, a tintura de própolis não é recomendada para utilização como anti-séptico oral frente às leveduras nas concentrações estudadas.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Este estudo objetivou: (1) comparar a topografia superficial e a interface entre a obturação e as paredes do canal de raízes seccionadas com laser, ultra-som ou broca; (2) avaliar o efeito do preparo retrógrado com retropontas ultra-sônicas ou laser na integridade da superfície apical; (3) e avaliar a espessura de dentina remanescente após retropreparos com laser ou ultra-som verificando sua correlação com o surgimento de trincas. Trinta dentes instrumentados e obturados foram usados para cada situação. No experimento 1, foram avaliados três métodos de apicectomias: broca Zekrya, ponta ultra-sônica CVD (9.5107-8) e laser de ErCr:YSGG (Waterlase). Para os experimentos 2 e 3, cavidades retrógradas foram preparadas com retropontas CVD (6.1107-6), retropontas EMS (DT-060/Berutti) ou laser de ErCr:YSGG. O tempo de preparo das apicectomias e das cavidades apicais foi registrado. Réplicas em resina epóxica das superfícies radiculares foram avaliadas por MEV. Todos os dados foram submetidos ao devido tratamento estatístico (Shapiro-Wilk, ANOVA, Tukey, Kruskal-Wallis, Student-Newman-Keuls) com α = 5%. A avaliação das réplicas após as apicectomias não revelou trincas e nem fraturas em nenhum dos grupos. A Zekrya apresentou o menor tempo para concluir as apicectomias (p<0,05) e produziu a superfície mais lisa e plana (p<0,05). A melhor adaptação entre obturação/dentina ocorreu no grupo do laser, mas sem diferença significativa para o ultra-som (p>0,05). A avaliação das réplicas das cavidades retrógradas mostrou que o laser de ErCr:YSGG removeu mais estrutura dental que as retropontas CVD e EMS (p<0,05). O Waterlase teve a maior média de tempo para os retropreparos (p<0,05). Fraturas no ângulo cavo-superficial ocorreram apenas no grupo da retroponta EMS. Os grupos CVD e EMS apresentaram melhores escores relacionados à qualidade do preparo do que o grupo com laser ErCr:YSGG (p<0,05).
This study aimed: (1) to compare the surface topography, and the interface between the obturation and the root canal walls of roots resected with laser, ultrasonic tip, or bur; (2) to investigate the effect of retrograde preparations by using ultrasonic tips or laser on the integrity of root-end surfaces; (3) and to compare the thickness of surrounding dentine after ultra-sonics or laser root-end cavity preparation and its relationship with cracking formation. Thirty instrumented and root filled teeth were select for each situation. For the experiment 1, three root-end resections methods were evaluated: Zekrya bur, CVD (9.5107-8) ultra-sonic tip, and ErCr:YSGG laser (Waterlase). For the experiments 2 and 3, root-end cavities were prepared by CVD (6.1107-6) ultra-sonic tip, EMS (DT060/Berutti) ultra-sonic tip or ErCr:YSGG laser.The time required to cut through the apical portion of the root and to prepare the rootend cavity was recorded. Epoxy resin replicas of the root surfaces were assessed by SEM. Statistical analyses were performed using Shapiro-Wilk, ANOVA, Tukey, Kruskal-Wallis, and Student-Newman-Keuls tets (α = 5%). Assessment of the replicas of apices after apicectomy did not reveal cracks or fractures in any specimen. The Zekrya bur showed the lowest mean time to cut through the root-ends (p<0.05) and produced the smoothest and most uniplanar resected root surfaces (p<0.05). The best adaptation between the obturation and the root canal walls of roots was observed in laser group, but it did not differ from ultra-sonics group (p>0.05). The evaluation of the root-end cavities replicas showed that the ErCr:YSGG laser removed more dental structure than the CVD and EMS tips (p<0,05). The Waterlase showed the highest mean time to prepare the root-end cavity (p<0.05).

A Saúde Bucal está relacionada à exposição ao flúor, hábitos dietéticos e higiênicos menos cariogênicos, além de fatores sócio-econômico-culturais favoráveis. A melhor intervenção farmacêutica constitui-se em formular um agente fluoretado capaz de prevenir e/ou retardar o processo carioso. Procedeu-se a coleta de dentes terceiros molares inclusos que foram fragmentados e adaptados em suportes adequados para serem submetidos aos testes. Determinou-se o protocolo experimental baseado, seqüencialmente, em: avaliação da microdureza das amostras dentárias hígidas; desmineralização das mesmas; avaliação da microdureza das amostras desmineralizadas; remineralização das amostras com diferentes fontes de flúor, em duas concentrações distintas, com três valores de pH e duas posologias diversas; nova avaliação da microdureza no 5 º, 10 º , 15º e 20º dias, seguida de análise por Microscopia Eletrônica de Varredura, com comparação dos resultados quanto à eficácia remineralizante e estabilidade dos enxagüatórios à base de flúor propostos neste experimento.
Oral Health is directly related to the increase of exposure to fluorine, as well as hygienic and dietetic habits that are less cariogenic, and favorable social and economical factors. Consequently, the best pharmaceutical intervention consists in formulating a fluorine agent that is able to prevent and/or retard the carious process. A protocol was executed for the collection of teeth at odontological clinics. After being extracted, those third molar included teeth were, taken to the laboratory so that all the necessary removals of blood deposits, as well as residual tissues, which are unnecessary to the experiment, could be done. The teeth were fragmented and adapted to supports that were adequate to the preparation of the samples that would be tested. The experimental protocol was based on: the evaluation of the hardness of samples of included teeth; demineralization of the samples; the evaluation of the hardness of the samples; the remineralization of the samples with different sources of fluoride, in two different oncentrations, with three pH values and two diverse posologies ; new hardness evaluation on the 5th, 10th, and 15th and 20 th days, followed by an analysis by Electronic Microscopy of Screening with a comparative analysis of the results regarding the efficacy and stability of the mouth rinse propouse in this experiment.

Orientador: Fabio Luiz Camargo Villela Berbert
Banca: Clóvis Monteiro Bramante
Banca: Mário Tanomaru Filho
Resumo: Este estudo objetivou: (1) comparar a topografia superficial e a interface entre a obturação e as paredes do canal de raízes seccionadas com laser, ultra-som ou broca; (2) avaliar o efeito do preparo retrógrado com retropontas ultra-sônicas ou laser na integridade da superfície apical; (3) e avaliar a espessura de dentina remanescente após retropreparos com laser ou ultra-som verificando sua correlação com o surgimento de trincas. Trinta dentes instrumentados e obturados foram usados para cada situação. No experimento 1, foram avaliados três métodos de apicectomias: broca Zekrya, ponta ultra-sônica CVD (9.5107-8) e laser de ErCr:YSGG (Waterlase). Para os experimentos 2 e 3, cavidades retrógradas foram preparadas com retropontas CVD (6.1107-6), retropontas EMS (DT-060/Berutti) ou laser de ErCr:YSGG. O tempo de preparo das apicectomias e das cavidades apicais foi registrado. Réplicas em resina epóxica das superfícies radiculares foram avaliadas por MEV. Todos os dados foram submetidos ao devido tratamento estatístico (Shapiro-Wilk, ANOVA, Tukey, Kruskal-Wallis, Student-Newman-Keuls) com α = 5%. A avaliação das réplicas após as apicectomias não revelou trincas e nem fraturas em nenhum dos grupos. A Zekrya apresentou o menor tempo para concluir as apicectomias (p<0,05) e produziu a superfície mais lisa e plana (p<0,05). A melhor adaptação entre obturação/dentina ocorreu no grupo do laser, mas sem diferença significativa para o ultra-som (p>0,05). A avaliação das réplicas das cavidades retrógradas mostrou que o laser de ErCr:YSGG removeu mais estrutura dental que as retropontas CVD e EMS (p<0,05). O Waterlase teve a maior média de tempo para os retropreparos (p<0,05). Fraturas no ângulo cavo-superficial ocorreram apenas no grupo da retroponta EMS. Os grupos CVD e EMS apresentaram melhores escores relacionados à qualidade do preparo do que o grupo com laser ErCr:YSGG (p<0,05).
Abstract: This study aimed: (1) to compare the surface topography, and the interface between the obturation and the root canal walls of roots resected with laser, ultrasonic tip, or bur; (2) to investigate the effect of retrograde preparations by using ultrasonic tips or laser on the integrity of root-end surfaces; (3) and to compare the thickness of surrounding dentine after ultra-sonics or laser root-end cavity preparation and its relationship with cracking formation. Thirty instrumented and root filled teeth were select for each situation. For the experiment 1, three root-end resections methods were evaluated: Zekrya bur, CVD (9.5107-8) ultra-sonic tip, and ErCr:YSGG laser (Waterlase). For the experiments 2 and 3, root-end cavities were prepared by CVD (6.1107-6) ultra-sonic tip, EMS (DT060/Berutti) ultra-sonic tip or ErCr:YSGG laser.The time required to cut through the apical portion of the root and to prepare the rootend cavity was recorded. Epoxy resin replicas of the root surfaces were assessed by SEM. Statistical analyses were performed using Shapiro-Wilk, ANOVA, Tukey, Kruskal-Wallis, and Student-Newman-Keuls tets (α = 5%). Assessment of the replicas of apices after apicectomy did not reveal cracks or fractures in any specimen. The Zekrya bur showed the lowest mean time to cut through the root-ends (p<0.05) and produced the smoothest and most uniplanar resected root surfaces (p<0.05). The best adaptation between the obturation and the root canal walls of roots was observed in laser group, but it did not differ from ultra-sonics group (p>0.05). The evaluation of the root-end cavities replicas showed that the ErCr:YSGG laser removed more dental structure than the CVD and EMS tips (p<0,05). The Waterlase showed the highest mean time to prepare the root-end cavity (p<0.05).
Mestre

É crescente o interesse dos consumidores por produtos naturais com apelo funcional. Por isso tem se estudado novos veículos de liberação de compostos bioativos, como por exemplo, os filmes de desintegração oral. A própolis é uma fonte natural rica em compostos ativos, como por exemplo, os compostos fenólicos, que lhe confere atividade antimicrobiana e antioxidante. O colágeno hidrolisado pode apresentar diversos benefícios quando ingerido, como aumento da densidade óssea e representa uma boa fonte de peptídeos. Este trabalho teve como objetivo desenvolver e caracterizar os filmes de desintegração oral à base de gelatina aditivados com colágeno hidrolisado (CH) e extrato etanólico de própolis (EEP). Os filmes foram produzidos pela técnica de casting mantendo constante a massa de gelatina(mG) colágeno hidrolisado(mCH) em 2 g de mG+CH (g/100g de solução filmogênica), e a concentração de sorbitol em 30g (g/100g de mG+CH). Foram avaliadas diferentes concentrações de CH (0, 10, 20 e 30 g/100 g de mG+CH) e de EEP (0, 100 e 200 g /100 g de mG+CH). Os filmes foram caracterizados em relação à cor e opacidade, matéria total solúvel, umidade, propriedades mecânicas (tensão na ruptura e elongação), microscopia eletrônica de varredura, mucoadesividade, ângulo de contato, tempo de desintegração, grau de intumescimento, concentração de compostos fenólicos, liberação in vitro, cinética de liberação, calorimetria diferencial de varredura, espectroscopia de infravermelho com transforma de Fourier (FTIR), atividade antimicrobiana e estabilidade dos compostos fenólicos em função do tempo armazenamento. O aumento da concentração de CH provocou aumento significativo da solubilidade. Nas propriedades mecânicas foi observado aumento da elongação para os filmes com adição de colágeno em relação ao filme controle e redução significativa da tensão na ruptura com o aumento da concentração de CH. Não foi observado variação da microestrutura com a adição de colágeno e os filmes com maior concentração de CH foram mais mucoadesivos. O aumento da concentração de CH provocou redução do tempo de desintegração, apresentando uma liberação mais rápida dos compostos fenólicos. Em relação ao aumento da concentração de EEP foram observados alterações nos parâmetros de cor e opacidade devido a cor característica do extrato. Observou-se redução da solubilidade e aumento na tensão de ruptura com o aumento de EEP. Para filmes aditivados com EEP foi observada uma microestrutura menos compacta e desordenada e aumento da mucoadesividade. O aumento da concentração de EEP provocou aumento do tempo de desintegração, com redução do grau de intumescimento, e uma liberação mais lenta dos compostos fenólicos. O processo de produção dos filmes não provocou degradação dos compostos fenólicos e os filmes aditivados apresentaram atividade antimicrobiana contra Staphylococcus aureus. Os filmes de desintegração oral mantiveram a concentração de compostos fenólicos após 84 dias de armazenamento. Desta forma, os filmes de desintegração oral aditivados com extrato etanólico de própolis e colágeno hidrolisado representam uma boa alternativa como veículo de compostos fenólicos na cavidade oral.
There is an increase in the interest of consumers for natural products with functional appeal. Therefore, it has been studied new vehicles for release of bioactive compounds, such as orally disintegrating films. Propolis is a rich natural source of active compounds, such as phenolic compounds, responsible for antimicrobial and antioxidant activity. Hydrolyzed collagen may have several benefits when ingested, such as increased bone density and it is a good source of peptides. This study aimed the development and characterization of orally disintegrating films gelatin-based additives with hydrolyzed collagen (CH) and ethanol extract of propolis (EEP). The films were produced by casting, keeping mass constant of gelatin (mG) and hydrolyzed collagen (mCH) at 2 g of mG+CH/100 g filmogenic solution, and the concentration of sorbitol at 30 g/100 g mG+CH. It was evaluating different concentrations of CH (0, 10, 20 and 30 g/100 g of mG+CH) and EEP (0, 100 and 200 g/100 g of mG+CH). The films were characterized regarding color and opacity, total soluble matter, moisture, mechanical properties (tensile strength and elongation), scanning electron microscopy, mucoadhesion, contact angle, disintegration time, swelling degree, concentration of phenolic compounds, in vitro release, release kinetics, differential scanning calorimetry, Fourier transform infrared spectroscopy (FTIR), antimicrobial activity and stability of phenolic compounds were evaluated during the storage time. Increasing in concentration of CH caused a significant increase of solubility. For mechanical properties was observed increased at elongation for films with collagen in relation to control film and significant reduction of tensile strength with increasing of CH. It was not observed difference in microstructure because of CH addition and the films with higher concentrations of CH showed higher mucoadhesive. The increased of CH concentration promoted a reduction in disintegration time, showed a faster release of phenolic compounds. The increased of EEP concentration influenced color parameters and opacity due to color of the extract. It was observed a reduction of the solubility and an increase in tensile strength with the increase of EEP. The addition of EEP promoted a microstructure less compact and more disordered structure and increased mucoadhesion. Increased in EEP concentration increased disintegration time, reduced swelling degree, and caused a slower release of phenolic compounds. The production process did not cause degradation of phenolic compounds and additives films exhibited antimicrobial activity against Staphylococcus aureus. The orally disintegrating films remained the concentration of phenolic compounds after 84 days of storage. Thus, the orally disintegrating film additives with ethanol extract of propolis and hydrolyzed collagen represent a good alternative as a vehicle of phenolic compounds in the oral cavity.

CAD/CAM technology had evolved extensively from the time when it was first used clinically in the 1980s. Today, CAD/CAM technology can be used to fabricate crowns, design and mill fixed partial denture frameworks, set teeth and fabricate complete dentures as well as to mill interim restorations. An extensive literature review found many research studies on the evaluation of the accuracy of the CAD/CAM ceramic crowns as compared to other ceramic crowns but little research had been done to evaluate the accuracy of interim restorations. This article describes the method in which CAD/CAM as well as conventional interim crowns were fabricated, cemented onto their dies, dye stained, thermocycled as well as sectioned with their marginal discrepancies investigated.

Orientadores: Luiz Andre Freire Pimenta, Lourenço Correr Sobrinho
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: Dentes naturais, bem como restaurações estão sujeitos à degradação inta-oral. Nas resinas compostas, além das interferências químicas, térmicas e mecânicas, a composição e a estrutura pós-cura são mecanismos considerados responsáveis pela durabilidade do procedimento restaurador. Neste estudo, avaliou-se o efeito da formulação do material, métodos de fotoativação (MF) e condições de armazenamento (CA) na resistência à degradação química e/ou mecânica de resinas-compostas. Os objetivos, métodos e resultados deste trabalho, composto por dois artigos científicos foram: CAPÍTULO 1: avaliar como o MF e a CA afetam a degradação química (DQ) de duas resinas-compostas de nanopartículas, um ormocer de 1a e um de 2a geração. Discos de 4 mm de diâmetro e 2 mm de altura foram confeccionados e fotoativados com os métodos convencional (C) ou soft-start (S). Testaram-se quatro CA (n=5): grupo 1: imediato; grupo 2: seco e escuro; grupo 3: etanol e grupo 4: água destilada. A dureza Knoop (KHN) foi mensurada imediatamente após a cura e 24 horas após armazenagem. Aplicaram-se os testes estatísticos two-way ANOVA e Tukey (p=0.05) comparando os valores de KHN entre as CA e entre os MF. O MF não interferiu na DQ de nenhum material testado. Entretanto, diferenças foram observadas entre as CA, e independente do material, o etanol produziu a maior DQ. O ormocer de 2a geração foi o único material que não degradou após imersão em água. Concluiu-se que: o MF não influenciou na resistência à DQ; o etanol causou maior DQ e o ormocer de 2a geração foi o material mais estável em água após 24 h de armazenamento. CAPÍTULO 2: investigar o efeito do tamanho de partículas de carga na rugosidade superficial (RS), retenção de brilho (RB) e estabilidade de cor (C) antes e após abrasão utilizando a escovação mecânica (EM). Avaliaram-se quatro resinas-compostas experimentais e uma comercial com partículas variando de 100 a 1000 nm. Para cada material, seis discos medindo 10 mm de diâmetro e 2 mm de altura foram confeccionados. Após acabamento e polimento, as amostras foram submetidas a 20.000 ciclos de EM. Os parâmetros RS, RB e C foram mensurados antes e após escovação. Aplicaram-se os testes estatísticos de ANOVA e Bonferroni (p=0.05) e o teste de correlação de Pearson para determinar a relação entre tamanho das partículas e os parâmetros avaliados; e a relação entre RS e RB. A EM resultou em superfícies mais rugosas e menos brilhantes para todos materiais testados. Após a EM houve correlação significativa entre o tamanho das partículas e os parâmetros de rugosidade Ra (r = 0.95/ p = 0.013) e Rt (r = 0.93 / p = 0.022). Não houve correlação significativa entre o tamanho das partículas e ?E*. Houve correlação negativa significante entre brilho e rugosidade (r = - 0.97 / p = 0.001). Concluiu-se que a abrasão causou modificações na rugosidade, relacionadas ao tamanho das partículas, e no brilho dos materiais testados. A C não foi alterada pelo tamanho das partículas de carga e nem por modificações na textura superficial
Abstract: Natural teeth, as well as restorations, are composed of materials that are subject to deterioration. Besides chemical, mechanical and thermo degradation, the formulation and post-cure composite¿s network are dominant mechanism to determine the restoration¿s longevity. The purpose of this study was to investigate the effect of material¿s formulation, light-activation mode (LM) and storage condition (SC) on resin-composite¿s resistance to chemical and/or mechanical degradation. The aims, methods and results of this study, composed of two chapters were: CHAPTER 1: to evaluate how the LM and the SC would affect the chemical degradation, through the softening test, of two nanohybrids; a 1st and a 2nd generation ormocer. Disk specimens (4 mm x 2 mm) were prepared from each material and light-activated by full-cure (F) and soft-start (S) modes. Four storage conditions were evaluated (n=5): group 1: immediately after cure, group 2: dry and dark, group 3: absolute ethanol and group 4: distilled water. Knoop hardness values (KHN) were measured immediately after cure and after immersion in the different SC for 24 hours. Two-way ANOVA with Tukey¿s post-hoc test (p = 0.05) was used to compare KHN among each SC and between the two LM. The LM presented no significant effect on the softening test, in all groups. However, significant differences were observed among the SC. Absolute ethanol produced the higher softening effect in all materials tested. The 2nd generation ormocer was the more stable material when stored in water. LM did not affect the final KHN. CHAPTER 2: to investigate the effect of filler-particle size of resin-composites, undergoing toothbrush abrasion (TA), on three surface properties: surface roughness (SR), surface gloss (G) and color stability (CS). Four models and one commercial resin-composite and with varying filler-size from 100 to 1000 nm were examined. Six discs (10 mm x 2 mm) from each - 0.97). Filler size did not affect CS. product were mechanically polished. The samples were then submitted to 20,000 brushing strokes in a TA machine. SR parameters (Ra, Rt and RSm), G and CS (CIE-L*a*b*) were measured before and after TA. Changes in SR and G were analyzed by 2-way ANOVA, with Bonferroni post-hoc test. CS values were submitted to one-way ANOVA and Bonferroni post-hoc test (p=0.05). There was no statistically significant difference in CS. TA resulted in rougher and matte surfaces for all materials tested. Although the individual differences in SR among filler sizes were not always significant, the correlation showed a trend that larger filler sizes result in higher surface roughness after abrasion for Ra and Rt parameters (r = 0.95; r = 0.93, respectively). RSm showed an increase after TA for all resin-composites; however no significant correlation was detected. Initial G values ranged between 73 and 83 gloss units (GU) and were reduced after TA to a range of 8.5 to 64 GU. TA revealed significant modifications in surface roughness and gloss amongst the materials tested, that related to filler sizes. There was a significant correlation between gloss and roughness (Ra) (r = ) - 0.97). Filler size did not affect CS
Doutorado
Dentística
Doutor em Clínica Odontológica

Orientador: Rosangela Gonçalves Peccinini
Banca: Marina Galdino da Rocha Pitta
Banca: Anderson Rodrigo Moaraes de Oliveira
Banca: Marcelo Tadeu Marin
Banca: Marlus Chorilli
Resumo: As tiazolidinodionas (TZDs) são fármacos utilizados no tratamento de Diabetes Mellitus tipo 2 (DM2), seu mecanismo de ação é a ativação dos receptores PPAR-γ do núcleo celular. Além de sua atividade sobre o metabolismo glicídico e lipídico, há um particular interesse em sua atividade sobre a aterosclerose decorrente da síndrome metabólica que acomete estes pacientes. Tendo em vista a ampla aplicação das TZDs e a presença de apenas uma alternativa terapêutica relativamente segura no mercado, a pioglitazona, pesquisadores do Laboratório de Planejamento e Síntese de Fármacos da Universidade Federal de Pernambuco sintetizaram e selecionaram TZDs promissoras em relação à atividade anti-aterosclerose. Dentre elas destacaram-se as GQ-2, GQ-11, GQ-19 e GQ-177. Tendo em vista a maior aceitação da administração oral de medicamentos, prever a absorção destas novas moléculas para esta via empregando métodos de screening farmacocinético pode acelerar o desenvolvimento do produto farmacêutico. Neste estudo utilizou-se o método de screening farmacocinético Cassette Accelerated Rapid Rat Screen (CARRS), o screening físico-químico, principalmente através da avaliação do Log P, e screeningin vitro, utilizando modelo de monocamadas de células Caco-2, para obter informações preditivas acerca da biodisponibilidade de moléculas. Estas técnicas foram utilizadas para selecionar dentre as TZDs - GQ-2, GQ-11, GQ-19 e GQ-177 - o(s) candidato(s) com características mais apropriadas para a continuidade d... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Thiazolidinediones (TZDs) are drugs used to treat type 2 diabetes mellitus (DM2); its mechanism of action is the activation of the cell nucleus receptor PPAR-γ. In addition to its activity on glucose and lipid metabolism, there is a particular interest in its activities on atherosclerosis that is caused by the metabolic syndrome that affects these patients. Given the wide application of TZDs and the presence of a single therapeutic alternative in the market, the pioglitazone, the researchers from the Laboratório de Planejamento e Síntese de Fármacos from Universidade Federal de Pernambuco synthesized and selected promising TZDs in relation to antiatherosclerosis activity. Among them stood out the GQ-2, GQ-11, GQ-19 and GQ177. Taking into account the greater acceptance oral administered drugs, to predict the absorption of these new molecules for this route using pharmacokinetic screening methods can accelerate the development of a pharmaceutical product. In this study we used the screening method CARRS (PK), the physico-chemical screening, particularly by evaluating the Log P, and in vitro screening using Caco-2 cell monolayer model to obtain predictive information regarding the bioavailability of molecules. These techniques were used to select among the TZDs - GQ-2, GQ-11, GQ-19 and GQ-177 - the candidate(s) with the most appropriate characteristics for the continuation of pre-clinical studies. The physic-chemical screening showed that the GQ-19 and GQ-177 as favorable oral absorption, but in the in vitro screening, only the GQ-19 showed permeation in Caco-2 cell model. In in vivo screening, GQ-19 appeared unstable in animal plasma, degraded completely in less than 20 minutes. For other molecules, during the in vivo screening, the expectations arisen from the behavior observed in in vitro... (Complete abstract click electronic access below)
Doutor

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico
O tratamento das doenÃas ocasionadas por biofilmes orais envolve basicamente a remoÃÃo mecÃnica e o uso de antibiÃticos e agentes anti-sÃpticos os quais podem originar cepas resistentes aos antimicrobianos tradicionais. A Terapia FotodinÃmica Antimicrobiana (TFDA) apresenta-se como uma opÃÃo alternativa ao tratamento clÃssico, promovendo a morte bacteriana por meio da fotossensibilizaÃÃo dos componentes microbianos. Este estudo verificou a aÃÃo antimicrobiana da terapia fotodinÃmica sobre biofilmes orais produzidos in vitro e in situ utilizando um diodo emissor de luz (LED) associado ao fotossensibilizador azul de orto-toluidina (TBO). No estudo in vitro, biofilmes de Streptococcus mutans UA159, foram formados sobre discos de hidroxiapatita utilizando um modelo de banhos de cultura e submetidos à TFDA apÃs 5 dias. Para o estudo in situ, vinte e um voluntÃrios foram previamente selecionados para utilizar dispositivos intra-orais palatinos contendo 8 blocos de dentina humana durante 7 dias. SoluÃÃo de sacarose 10% foi gotejada sobre os blocos dentais 8 vezes ao dia. O biofilme formado em um dos lados do dispositivo recebeu tratamento da TFDA, e o lado oposto serviu como grupo controle. O material coletado passou por um processo de disrupÃÃo para a dispersÃo das cÃlulas e diluiÃÃo em sÃrie decimal de 10-1 a 10-4. Em ambos os experimentos, meios de cultura especÃficos para o crescimento de estreptococos totais e estreptococos do grupo mutans foram inoculados e incubados em condiÃÃes ideais para o crescimento desses microrganismos. ReduÃÃes significativas acima de 99,99% (p<0.05) foram observadas na viabilidade das colÃnias de S. mutans UA159 quando expostos ao TBO e LED no estudo in vitro. Entretanto, nos biofilmes formados in situ e submetidos Ãs mesmas condiÃÃes experimentais, nÃo foram verificadas diferenÃas estatisticamente significantes (p≥0.05) da contagem microbiana quando comparadas ao grupo controle. Portanto, podemos concluir que a TFDA foi efetiva na reduÃÃo microbiolÃgica de S. mutans UA159 crescidos em modelo de formaÃÃo de biofilme in vitro, mas, pouco efetiva sobre biofilmes de estreptococos orais formados in situ.
The treatment of diseases caused by oral biofilms involves mechanical removal and use of antibiotics and antiseptics which can lead to problems of bacterial resistance. Photodynamic antimicrobial chemotherapy (PACT) represents an alternative option to conventional treatment, promoting bacterial killing by photo-sensitization of microbial components. This study assessed the antimicrobial action of photodynamic therapy on oral biofilms produced in vitro and in situ using a light emitting diode (LED) associated with the photosensitizer toluidine blue O (TBO). Biofilms of Streptococcus mutans UA159 were grown on hydroxyapatite discs immersed in bathing culture and submitted to PACT after 5 days. For the in situ study, twenty-one volunteers were previously selected to use intra-oral palatal appliances containing eight blocks of human dentin during 7 days. Sucrose solution (10%) was dripped onto the dental blocks 8 times a day. The biofilm formed on one side of the device received treatment (PACT), and the opposite side acted as control. The collected material has gone through a process of disruption to the dispersion of cells and diluted in decimal series (10-1 to 10-4). In both experiments, specific culture media for the growth of total streptococci and mutans streptococci were inoculated and incubated under optimal conditions for growth of these microorganisms. Significant reductions in excess of 99.99% (p<0.05) were observed in the viability of colonies of S. mutans UA159 when exposed to TBO and LED on in vitro study. However, the biofilms formed in situ and subjected to the same experimental conditions showed no statistically significant differences (p≥0.05) in the microbiological counting when compared with control group. Therefore, we conclude that PACT was effective in microbiological reduction of S. mutans UA159 grown in an in vitro biofilm model, but very little effective on oral streptococci biofilms produced in situ.

Publicación a texto completo no autorizada por el autor
Determina el grado de microfiltración marginal de los materiales de obturación temporal en preparaciones cavitarias clase I según la clasificación de Black, a los 3, 5 y 7 días. Se usan 60 terceras molares superiores e inferiores que son extraídas por razones ortodóntica. Se prepara una cavidad de 4x4x4mm; se aislan usando barniz de uñas para sellar toda la pieza. Se dividen en cuatro grupos de 15 dientes según el material de obturación temporal usado (óxido de zinc -eugenol, policarboxilato de zinc, óxido de zinc/sulfato de zinc y óxido de zinc/sulfato de calcio). Se termocicla; se incuban en azul de metileno, después del tiempo señalado se realizan cortes longitudinales y medición en microscopio electrónico. Como resultado se encuentra que a los 3 días, la microfiltración marginal entre los grupos no presentaba diferencia significativa. A los 5 días el óxido de zinc presenta mayor microfiltración marginal. A los 7 días, todos los grupos alcanzan el grado 4 de microfiltración marginal a excepción del óxido de zinc/sulfato de calcio. Se concluye que el grupo óxido de zinc/sulfato de calcio presenta menor microfiltración marginal al término de los 3, 5 y 7 días en comparación con el óxido de zinc-eugenol, policarboxilato de zinc, óxido de zinc/sulfato de zinc.
Tesis

Thesis (MSD) --Boston University, Henry M. Goldman School of Dental Medicine, 2009 (Department of Periodontology and Oral Biology).
Includes bibliographic references: leaves 52-59.
Platelet-rich plasma (PRP) is a concentrated gel of platelets that contains several growth factors. Growth factors have been recognized as the part of PRP that play role in regeneration of the bone. It is not clear how these growth factors in PRP affect the bone regeneration. Resolvin El (RvEl; 5S,12R,18R-trihydroxyeicosapentaenoic acid) is an pro-resolving lipid mediator derived from omega-3 fatty acid eicosapentaenoic acid and shown to have potent effects on the resolution of inflammation. The purpose of this study was to analyze the action of PRP and RVEl on the proliferation and behavior of osteoblasts and osteoclasts in vitro. PRP was prepared from 14 healthy donors. Osteoblast cultures were from a cell line (Saos2) of osteosarcoma cells. Osteoclasts were differentiated from primary human peripheral blood monocytes. Osteoclastic morphology was studied and activity was analyzed via resorption on dentin discs using SEM. PRP and RVE 1 were added at different doses and time-points. Osteoblast function was analyzed by osteocalcin expression and release. Osteoclast activity was assessed by resorption and cathepsin K expression. PRP and RvEl comparably increased the osteoblastic activity and suppressed the osteoclast differentiation and function. These results suggest that multiple tools are available to reverse the inflammation and restore the lost bone architecture as a result of periodontal disease.

Las fluoroquinolonas se han convertido en agentes importantes en quimioterapia, especialmente en pacientes con infecciones graves que requieran fármacos potentes administrados por vía oral. Sin embargo, la aparición de resistencias a las quinolonas ha conducido a la búsqueda de nuevas moléculas, principalmente incluyendo modificaciones en el núcleo base de la benzopiridona, lo que ha llevado al hallazgo y comercialización de nuevas quinolonas, más activas pero, en muchos casos, más tóxicas, hasta el punto de que algunas han dejado de utilizarse o han sido retiradas del mercado tras haberse demostrado su peligrosidad a medio o largo plazo.En este contexto, el ciprofloxacino es una de las quinolonas más clásicas y de uso más extendido por su amplio espectro de acción, su eficacia, su excelente tolerancia y por permitir su administración tanto por vía parenteral como por vía oral, si bien presenta el problema de poseer una biodisponibilidad oral relativamente baja e irregular. Por este motivo y tomando esta quinolona como referencia de actividad, se han sintetizado nuevos derivados homólogos pertenecientes a dos series: 4'N-alquilciprofloxacino y 3'metil,4'N-alquilciprofloxacino que podrían mejorar la biodisponibilidad y la actividad. En esta línea se incluye el Proyecto de Investigación SAF 96-1710 titulado "Predicción de la biodisponibilidad en los estudios de desarrollo de fármacos: nuevas quinolonas" que pretende comprobar si el aumento de biodisponibilidad esperado en los derivados afecta a la actividad antimicrobiana.En resumen, se ha estudiado y comparado la actividad de estos homólogos con la del ciprofloxacino. Para ello, se ha determinado la CMI de los derivados, sobre 160 cepas bacterianas de origen clínico, utilizando el método de diluciones dobles seriadas en MHA según las especificaciones del NCCLS.Se demuestra que la adición del sustituyente alquílico en 4'N no altera el espectro de acción del patrón utilizado. Sin embargo, los compuestos sintetizados presentan diferente actividad frente a los gérmenes ensayados. En1conjunto los más activos son 4'N-metilciprofloxacino, 4'N-etilciprofloxacino, 3'metilciprofloxacino(97-100) y 3'metil,4'N-metilciprofloxacino (97-101).De forma general, se puede afirmar que respecto a la serie 4'N-alquilciprofloxacino, los compuestos con un radical metilo o etilo son los más activos: 4'N-metilciprofloxacino es el más activo frente a los Gram negativos (a excepción de E. coli) y 4'N-etilado el más activo frente a los Gram positivos. No obstante, 4'N-propilciprofloxacino también presenta mejor actividad que el patrón en los microorganismos Gram positivos (a excepción de E. faecalis), y en A. calcoaceticus. 4'N-butilciprofloxacino no presenta mejor actividad que el compuesto de referencia en ningún caso, si bien en los microorganismos ya mencionados la actividad es la misma.Respecto a la serie 3'metil, 4'N-alquilciprofloxacino, se puede colegir que los compuestos 97-100 y 97-101 son los más activos. En resumen, el derivado 97-101 presenta la mayor actividad frente a A. calcoaceticus, M. catarrhalis, S. aureus, SCN y S. pneumoniae. Por otro lado, 97-100 es el más activo frente a Achromobacter spp. No obstante, 97-102 y 97-103 también presentan mejor actividad que el ciprofloxacino en los microorganismos A. calcoaceticus, S. aureus y S. pneumoniae. El derivado 97-104 no presenta mejor actividad que el patrón en ningún caso, si bien en los microorganismos ya mencionados la actividad es la misma.En lo que se refiere a los heterólogos estudiados, en general, el grepafloxacino es el compuesto más activo. El ofloxacino y el sarafloxacino presentan una actividad muy similar a la del ciprofloxacino, y el flumequino es el menos activo.También se ha estudiado la cinética de letalidad de algunos de estos compuestos en Staphylococcus aureus ATCC 25923. Este estudio proporciona una serie de parámetros de gran interés en lo que se refiere al mecanismo de acción. Los resultados obtenidos demuestran que la dinámica de crecimiento y letalidad que presentan los homólogos es la misma que su patrón, siguen la misma cinética bactericida y muestran un efecto paradógico similar. Si se2considera la concentración absoluta, el antibiótico con mayor velocidad de letalidad es el 97-101. Se planteó si el grupo metilo adicional que presenta este derivado en posición 3' con respecto a ciprofloxacino y los compuestos de la otra serie, podría ser el responsable de su mayor letalidad. Dado que la selección de la girasa como primera diana por parte de las quinolonas se asocia con un aumento en la velocidad de letalidad, se determinó la diana de primera elección para este derivado. Se comprobó que la mayor letalidad de este compuesto no se debe a un cambio en la selección de la diana intracelular; pues se demostró, mediante estudios de CMI sobre cepas mutantes, que la topoisomerasa IV es su primera diana en S. aureus, al igual que para el ciprofloxacino.Por otro lado, debido a que los antibióticos estudiados pertenecen a series homólogas, se ha estudiado la relación entre la lipofilia, como parámetro estructural, y la inversa de la media geométrica de la CMI, como índice de actividad. Esta correlación proporciona ajustados bilineales y permite identificar el coeficiente de reparto óptimo para una especie dada, que es en general más bajo para los microorganismos Gram negativos que para los Gram positivos.Además, se ha investigado acerca del mecanismo de acción de estas quinolonas a nivel molecular. En concreto, la investigación se ha centrado en Streptococcus pneumoniae, determinando los efectos de inhibición de las quinolonas ensayadas en la girasa y topoisomerasa IV bacteriana in vitro, con el propósito de conocer la diana específica de los derivados alquilados en comparación con el ciprofloxacino, proporcionar información acerca del modo de acción de estos compuestos y comprobar si ello permite explicar las diferencias en la actividad observadas sobre este microorganismo. Esta parte del trabajo se ha desarrollado en el St. George's Hospital Medical School, University of London, bajo la dirección del Dr LM Fisher.3Se demostró por estudios de CMI sobre cepas mutantes de S. pneumoniae que los homólogos mantienen la diana principal en Topoisomerasa IV, como el patrón. Por otra parte, la determinación de la CC25 mediante electroforesis en gel de agarosa demuestra que los compuestos estudiados no provocan mayor formación del complejo ternario ni con la topoisomerasa IV ni con la girasa. Sólo con el derivado 97-100 éste es igual que ciprofloxacino, lo que indica que la alquilación en 4'N del anillo piperacínico del ciprofloxacino no es favorable, si bien afecta a las interacciones entre fármaco y enzima. El efecto del sustituyente en C7 sobre la bomba de secreción PmrA no se conoce puesto que los resultados de este estudio no fueron concluyentes. No se puede descartar, pues, la penetración intracelular como posible responsable de la diferencia de actividad de los homólogos, debido a que para un microorganismo dado, este parámetro está bajo el control de las propiedades fisicoquímicas de la quinolona.Por último, al considerar el equilibrio actividad-biodisponibilidad oral comparada para la selección de los candidatos, existen tres compuestos que presentan las mejores propiedades en conjunto: 4'N-metilciprofloxacino, compuesto de elección en todos los microorganismos Gram negativos a excepción de E. coli. 4'N-etilciprofloxacino, muy eficaz frente a los microorganismos Gram positivos, entre los que es el derivado de elección en todos los casos, y además presenta buenas propiedades en los microorganismos Gram negativos. 3'-metilciprofloxacino (97-100) presenta una eficacia en general mejor a la del patrón en todas las especies estudiadas, sin embargo sólo resulta el candidato de primera elección en E. coli, en el que la mayoría de compuestos son menos activos que el ciprofloxacino.No obstante, aunque estos tres compuestos son los candidatos de elección, los demás derivados también podrían resultar de interés en algunos de los microorganismos estudiados como A. calcoaceticus, S. aureus y S. pneumoniae.
The thesis entitled "In vitro antimicrobial activity of new 6-fluoroquinolones for oral administration. Structure-activity relationship" is a part of the research Project SAF 96-1710 "Bioavailability prediction in drug development studies: new quinolones". In this study, we have synthesized new ciprofloxacin homolog derivatives belonging to two series: 4'N-alkylciprofloxacin and 3'methyl,4'N-alkylciprofloxacin, whose antimicrobial activity (minimum inhibitory concentration, MIC) we have studied and compared with that of ciprofloxacin against 160 bacterial strains from clinical sources. MICs were determined by the standard NCCLS, M7-A5 method. The results showed that 4'N alkyl substitution does not modify the reference drug antibacterial spectrum. Nevertheless, the synthesized compounds present different activity against the assayed microorganisms.Time-kill kinetics of some homolog compounds against Staphylococcus aureus ATCC-25923 were determined. These derivatives present the same lethality kinetics than ciprofloxacin. The 3'methyl,4'N-methylciprofloxacin derivative shows the quickest lethality, which is not due to a change in the primary target selection, as topoisomerase IV is its primary target for S. aureus, as it is for ciprofloxacin.Furthermore, we have studied the relationship between the lipophilicity, as a structural parameter, and the inverse of the geometric mean MIC, as an activity index. The activity-lipophilicity correlations can be fitted to a bilineal equation and allow the selection of the optimum partition coefficient for each species studied. This optimum tends to be lower for gramnegative microorganisms than for grampositive ones.We have also studied the mechanisms of action of these quinolones by determining the in vitro inhibition effects through Streptococcus pneumoniae gyrase and topoisomerase IV (carried out in St. George's Hospital Medical School, London). Topoisomerase IV is the primary target of ciprofloxacin homologs for S. pneumoniae. Ciprofloxacin C7 substitution affects drug-enzyme interaction. The cleavage through gyrase or topoisomerase IV is low, suggesting that 4'N ciprofloxacin alkylation is not favourable.Finally, the activity-bioavailability balance has been considered, in order to select the best drug candidates. Three compounds are pointed out: 4'N-methylciprofloxacin and 3'methylciprofloxacin against gramnegative microorganisms and 4'N-ethylciprofloxacin against grampositive ones. These finding are consistent with the established activity-lipophilicity correlations and the selected candidates against gramnegatives are more hydrophilic than those against grampositives.

Objective: Oral squamous cell carcinoma (OSCC) is known to aggressively invade bone in a complex process that remains vague and poorly understood in many aspects. It is known that OSCC recruits osteoclasts, bone cells uniquely capable of bone destruction. The goal of this study was to assess the effects of soluble factors produced by OSCC on osteoclasts formation and responsiveness of bisphosphonates.Methods: BHY is the human cell line originally isolated from bone invasive OSCC. BHY OSCC cells were cultured and condition media was collected. Osteoclast formation from mouse murine monocytes RAW 264.7 cells was assessed for both osteoclast number and shape. Bisphosphonate pamidronate was used in concentrations 10-6-10-8 M.Results: BHY CM stimulated osteoclastogenesis from RANKL-primed RAW 264.7 cells in a dose-dependent manner. BHY CM stimulated phosphorylation of Akt in osteoclast precursors this was assessed using immunoblotting. Quantitative RT-PCR demonstrated that BHY CM induced upregulation of MMP-9 in osteoclasts. Pamidronate decreased osteoclast survival in physiological conditions, but was not effective in the presence of BHY-CM.Conclusion: These data suggest that bisphosphonates such as pamidronate may not be effective in treating osteoclast-dependent bone invasion of OSCC. Inhibition of Akt may represent an alternative strategy that deserves further investigation in the context of OSCC treatment and management.
Objectif : Le carcinome épidermoïde oral est un cancer qui a un potentiel d'envahissement osseux; ce processus complexe reste vague ainsi que mal compris dans plusieurs aspects. Il est documenté que le carcinome épidermoïde oral recrute les ostéoclastes, les seules cellules osseuses qui possèdent la capacité de détruire l'os. Le but de cette étude était d'évaluer les effets des facteurs solubles produit par le cancer épidermoïde auprès de la formation des ostéoclastes ainsi que sur la réponse face aux bisphosphonates. Méthodes : BHY est la première lignée de cellule humaine isolée de l'invasion osseuse. Suite à l'inoculation des cellules BHY du carcinome épidermoïde oral, ce dernier a été recueillit et analysé. La formation des ostéoclastes provenant de monocytes RAW 264.7 de souris murines ont été analysés pour leur nombre ainsi que leur forme. La forme pamidronate de biphosphanate a été employée avec une concentration de 10-61-10-8 MRésultats : BHY CM a stimulé la genèse d'ostéoclastes à partir des cellules RANKL-primed RAW 264.7, de manière dépendante à la dose. Avec l'aide du Western blot, il a été démontré que BHY CM a aussi stimulé la phosphorylation de Akt dans les précurseurs d'ostéoclastes. L'utilisation de RT-PCR a établit que BHY CM a induit l'augmentation de l'expression de MMP-9 chez les ostéoclastes. L'utilisation de pamidronate a réduit la survie des ostéoclastes dans des conditions physiologiques, cependant n'a eu aucun effet lors de la présence de BHY CM.Conclusion : Les données obtenues suggèrent que les biphosphanates telle que le pamidronate peuvent être inefficace dans le traitement de l'invasion osseuse ostéoclastique du carcinome épidermoïde oral. L'inhibition de Akt est une alternative de traitement qui mérite d'être analysé plus profondément pour le traitement et la gestion du carcinome épidermoïde oral.

In vitro dissolution, release and permeation testing is a common practice during drug product research and development. These in vitro tests, if predictive, are referred to as biorelevant tests and can play diverse roles to facilitate and expedite product development in a cost effective manner. Oral transmucosal products (OTPs) are currently tested using compendial and modified in vitro tests which may or may not be good predictors of in vivo performance due to a lack of biorelevance. A critical need for a broadly applicable and biorelevant in vitro system for OTPs has been expressed in the literature and the goal of this research was the development and validation of a biorelevant in vitro method that can facilitate accurate prediction of the in vivo behavior of OTPs. A combined strategy of appropriate apparatus design and relevant physiological and in vitro variable adjustment was investigated to incorporate biorelevance into evaluation of OTPs. A novel in vitro device, the bidirectional transmucosal apparatus (BTA), was designed and fabricated which allowed simulation of the oral cavity and its physiological variables to evaluate OTPs in a more realistic fashion. The BTA was tested using snus (a type of smokeless tobacco) as the OTP product. A simple and selective high performance liquid chromatographic (HPLC) method with photodiode array (PDA) detection was developed and validated to assess in vitro nicotine release and permeation (Linearity: 0.5 – 32 μg/mL; calibration curve accuracy (%recovery, n=5 ): 97.98-103.20%; calibration curve precision (%RSD, n=5): 0.15-3.14%). The performance of BTA was compared with the modified USP IV flow through apparatus (USP IV) and a commercially available vertical diffusion cell (VDC). The observed in vitro in vivo relationship (IVIVR) slopes with the USP IV, VDC and BTA were 0.27, 2.01 and 2.11 respectively. The BTA was selected over the VDC and USP IV devices because of better simulation and adjustment of variables to incorporate biorelevance in the test of OTPs. Additionally, the BTA allows study of permeation and release simultaneously unlike VDC and USP IV apparatuses. Further, the different BTA parameters were sequentially screened for their impact on in vitro rate of nicotine permeation that can be employed for the optimization of IVIVR for snus. Based on the results, stimulated saliva swallowing rate (SSSR) and media temperature were considered as significant factors affecting in vitro permeation of nicotine and used to further optimize IVIVR for snus. A 32 multifactorial experimental design integrating SSSR (0.32, 1.66 and 3 mL/min) and media temperature (25, 37 and 45 °C) was employed. Based on the response surface analysis, 0.55 mL/min SSSR and 43 °C media temperature were identified as optimal BTA conditions that would give perfect IVIVR (i.e. IVIVR slope close to one) for snus. The experimental value of IVIVR slope (0.92) at these optimal conditions indicated that the BTA is a valid in vitro system for evaluation of OTPs in a biorelevant manner. The applicability of BTA for predicting nicotine permeation from ‘Stonewall’, a dissolvable compressed tobacco was also evaluated. However, comparable in vitro nicotine permeation and in vivo nicotine absorption profiles were not obtained (ratio of in vitro permeation to in vivo absorption rate ranged from 0.04 to 0.14 at different in vitro conditions) either due to the unavailability of reliable clinical data or due to inherently different in vivo behavior of Stonewall compared to snus that would require further modification in the BTA. In conclusion, this research demonstrated the potential of the novel in vitro device to be a valuable tool for the prediction of in vivo performance of snus. The application of the novel bidirectional transmucosal apparatus for other types of OTPs will be an interesting subject for further investigation.

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O presente estudo teve como objetivo avaliar a atividade antimicrobiana in vitro dos extratos das espécies: Anacardium ocidentalis; Harpagophytum procumbens; Momordica charantia L.; Punica granatum; Psidium guajava L.; Stryphnodendron adstringens e Uncaria tomentosa, frente ao microbioma oral de 12 cães Labradores retriviers. Para isso, utilizaram-se extratos polares das amostras vegetais. Realizouse a análise fitoquímica qualitativa e quantitativa para determinação das principais classes de metabólitos presentes. Em seguida, determinou-se a concentração inibitória mínima dos extratos pelo método de difusão em disco em Brain Heart Infusion Agar, a partir de soluções das amostras vegetais nas concentrações de 250 mg/mL, 125 mg/mL, 62,5 mg/mL, 31,25 mg/mL, 5 mg/mL, aplicadas sobre discos de papel filtro. A seguir, os discos foram transferidos para placas de petri, com meio de cultura agar BHI semeadas com saliva dos 12 cães, contendo microbioma oral dos mesmos. Após 12 horas de incubação a 370C foram avaliados os halos de inibição resultante da ação dos extratos sobre o microbioma e comparados com os halos de inibição de disco de clorexidina 0,12% como controle. Para interpretação dos dados foi empregada a análise de variância seguida do teste de Tukey com nível de significância de 5%. O extrato de Punica granatum demonstrou maior halo de inibição nas concentrações 250 mg/mL e 125 mg/mL, enquanto a Psidium guajava apresentou maior halo de inibição na concentração de 250 mg/mL. Os extratos de Anacardium ocidentalis, Uncaria tomentosa, Stryphnodendron adstringens, Momordica charantia L, tiveram halos semelhantes aos halos do controle clorexidina 0.12%. Pode-se relacionar que a atividade antimicrobiana dos referidos extratos esteja associada à presença de compostos fenólicos, taninos condensados e flavonóides, cujas presenças foram confirmadas nos ensaios fitoquímicos. Os resultados obtidos demonstraram que o microbioma oral dos 12 cães, foram mais sensíveis aos extratos de Punica granatum e Psidium guajava 125mg/mL e 250 mg/mL, quando comparados ao controle clorexidina a 0,12%, reforçando a importância das plantas medicinais como recurso terapêutico e sua maplicabilidade na odontologia veterinária.

Abstract Tumor associated macrophages (TAMs) are linked to the invasion of oral tongue squamous cell carcinoma (OTSCC). We modified THP-1 leukemia cells to M1 (inflammatory), M2 (TAM-like) and R848 (imidazoquinoline-treated) type macrophages in order to examine their interactions with OTSCC-cells (HSC-3) by using different kinds of in vitro migration and invasion models. We observed that interaction of TAM-resembling M2-type macrophages with HSC-3 cells induced invasion and migration, whereas the influence of M1 macrophages reduced them. Patient response to chemoradiotherapy is highly reliant on the characteristics such as the aggressiveness and stage of the cancer. Therefore, new methods for treatment testing are needed in order to design personalized therapies. We tested the applicability and consistency of human TME mimicking tissue methods for analyzing the effects of chemoradiation using commercial OTSCC cell lines. Based on our trials, both our human uterine leiomyoma tissue -based matrix models provide viable platforms for future in vitro chemoradiotherapy testing. Conventionally pro-tumorigenic activities of matrix metalloproteinase (MMP)9 have been linked with oral squamous cell carcinoma, but recently its tumor-suppressor role has also been revealed. Our study provides strong evidence that MMP9 also has an anti-invasive effect in OTSCC and is a potential mediator of the protective effects of arresten in tongue cancer cells
Tiivistelmä Makrofageilla on yhteys kielen levyepiteelikarsinooman invaasioon eli syöpäkasvaimen tunkeutumiseen ympäröivään kudokseen. Tutkimuksessamme muokkasimme ihmisen THP-1 leukemiasoluja kemiallisesti tulehdusreittejä aktivoiviksi M1-makrofageiksi, kasvaimeen liittyvien makrofagien kaltaisiksi M2-makrofageiksi sekä imidatsokinoliini-käsitellyiksi R848-makrofageiksi. Tarkoituksenamme oli tutkia makrofagien ja kielisyöpäsolujen vuorovaikutuksia erilaisilla in vitro migraatio- ja invaasiomalleilla. Anti-inflammatoristen, syövän etenemistä edesauttavien TAM-makrofagien kaltaisiksi erilaistetut M2-tyypin makrofagit lisäsivät HSC-3 kielikarsinoomasolujen invaasiota ja migraatiota, kun taas M1-tyypin makrofagien vaikutus oli päinvastainen. Potilaan vaste kemosädehoitoon riippuu syöpäkasvaimen ominaisuuksista, kuten syöpäsolujen aggressiivisuudesta ja syövän levinneisyysasteesta. Tämän vuoksi on tarve uusille menetelmille, joiden avulla voidaan ottaa huomioon potilaan sekä syöpätyypin yksilölliset ominaisuudet hoitoa suunniteltaessa. Testasimme syöpäkasvaimen mikroympäristöä mallintavien, ihmiskudokseen perustuvien menetelmien käyttökelpoisuutta ja luotettavuutta kemosädehoidon vaikutusten arvioimiseen. Testiemme perusteella myoomakudokseen pohjautuvat menetelmät voivat auttaa kemosädehoidon vaikutusten testauksessa. Matriksin metalloproteinaasi (MMP) 9:n on pitkään uskottu olevan yksinomaan syövän etenemistä edesauttava molekyyli. Viimeaikaisissa tutkimuksissa on myös havaittu, että MMP9:llä voi olla syövältä suojaavia vaikutuksia. Tutkimme MMP9:n vaikutusta kielisyöpäsoluihin ja havaitsimme, että MMP9:llä on myös invaasiota hillitseviä vaikutuksia. Lisäksi MMP9 saattaa toimia verisuonten muodostumista estävän arresten-molekyylin syövältä suojaavien mekanismien välittäjänä

OBJETIVO: A etiologia das LNCs é complexa e não está totalmente esclarecida e parecem influir e interagir na sua formação fatores tão diversos como a concentração de tensões, a presença de ácidos de diversas origens e mecanismos tribológicos variados. O objetivo deste estudo in vitro foi avaliar se há diferenças na influência da tensão de tração, de compressão e da escovação na profundidade da lesão, microdureza e rugosidade do esmalte bovino submetido a desafio ácido. (O trabalho constituiu-se da avaliação dos danos ao esmalte bovino submetido a um desafio ácido erosivo e desafio abrasivo) com carregamento mecânico simultâneo. MÉTODOS: Foram avaliados 48 palitos contendo esmalte e dentina, com dimensões de (2,7 x 2,7 x 16 mm). O esmalte dos palitos foi planificado com lixas de granulação 600 e 800 e depois posteriormente polidos com lixas de granulação 1000, 1200, 2400 e 4000. Foi confeccionado um entalhe transversal de 1,5 mm de diâmetro na dentina, deixando um \"pescoço\" de 1,7 mm de esmalte e dentina. Toda a superfície do palito, exceto uma janela de 4 mm na vestibular do esmalte (na região de maior concentração de tensão), recebeu uma camada que protegia frente ao desafio ácido. Os palitos foram fixados em uma de suas extremidades e divididos em 2 grupos (n=24): submetidos a uma carga contínua (650 gf) à flexão na outra extremidade (sendo que 12 deles com o entalhe voltado para cima compressão e 12 com o entalhe para baixo tração) e outro grupo de palitos descarregados (n=24). Com esta divisão, todos os palitos sofreram o primeiro desafio ácido (2 horas em 400 ml de solução de ácido cítrico tamponado, ajustado ao pH 3,75, a 37° C) e, a seguir, foram mensurados (degrau e rugosidade com o perfilômetro, além de microdureza). Depois, cada grupo foi subdividido em dois com igual número (n=6 para os carregados, e n=12 para os sem carregamento): um grupo recebeu escovação com dentifrício e outro com água, foram novamente protegidos e foram expostos ao segundo desafio ácido (com ou sem carregamento). Após nova medição (degrau e rugosidade com o perfilômetro, além de microdureza) e re-proteção, foram submetidos ao terceiro desafio ácido e novamente mensurados. Um grupo extra de 24 palitos foi destinado à avaliação por microscopia de luz polarizada, dividido em três subgrupos (n=8): sem carga, submetidos à tração e submetidos à compressão e desafio ácido por 2 horas. RESULTADOS: em todos os estágios houve aumento na profundidade da lesão (dependendo do grupo, variou de 1,9 m no primeiro estágio a 7,9 m no último a 4,5 m no primeiro estágio a 19,2 m no último). A tensão de tração produziu profundidade de lesão (segundo estágio: 16,2 m; último estágio: 19,2 m) significativamente maior que a de compressão (segundo estágio: 9,2 m; último estágio: 13,4 m) apenas do lado fixado no grupo de espécimes escovados com dentifrício. A rugosidade foi maior na área exposta (Ra variando de 0,36 a 0,55) que na protegida (Ra variando de 0,13 a 0,28), mas nenhuma das tensões influiu significativamente em nenhuma das duas. Em todos os casos constatou-se diminuição significativa da rugosidade no segundo estágio (da ordem de 0,8 unidades de Ra para áreas protegidas e 0,16 para as expostas), atribuível à escovação, tanto com dentifrício quanto com água. A tensão não provocou diferenças significantes na microdureza em nenhum dos grupos, mas a escovação com dentifrício, no segundo estágio, produziu microdureza significativamente maior, tanto nas áreas protegidas (410 KNH) quanto nas expostas (140 KNH) quando comparadas com os respectivos primeiros estágios (318 KHN e 60 KHN, respectivamente). CONCLUSÃO: o efeito da tensão sobre a profundidade da lesão depende da presença de outros fatores, pois nos espécimes escovados com água, a tensão não provocou efeito estatisticamente significante na profundidade da lesão, nem do lado do entalhe nem do lado fixado. Apenas nos palitos escovados com dentifrício e do lado da fixação foi possível constatar aumento significante da profundidade da lesão (no segundo e terceiro estágios) da tração em relação à compressão, sendo que os sem tensão tiveram profundidade intermediária. A tensão de tração levou ao aparecimento de micro-trincas detectáveis pela microscopia de luz polarizada. Já em relação à microdureza, a tensão não provocou diferenças significantes em nenhum caso, mas apenas a escovação com dentifrício pode ser responsabilizada por aumento da dureza. A rugosidade na área exposta foi bem maior que na protegida, mas a tensão não influiu significativamente. A escovação (com água ou com dentifrício) diminuiu a rugosidade no segundo estágio. No segundo estágio, a rugosidade diminui nas áreas expostas e protegidas e a microdureza aumenta nos grupos escovados com dentifrício nas áreas expostas e protegidas.
OBJECTIVE: The etiology of LNC\'s is complex and not fully understood and seem to influence and interact in their formation diverse factors as stress concentration, the presence of acids of various origins and various tribological mechanisms. The purpose of this in vitro study was to evaluate whether there are differences in the influence of tensile stress, compression stress and brushing on the lesions depth, hardness and roughness of bovine enamel subjected to acid challenge. The work consisted of assessing damage to bovine enamel subjected to an erosive acid challenge (and abrasive) with simultaneous mechanical loading. METHODS: The work consisted of assessing the mineral loss in enamel subjected to an erosive acid challenge and biomechanical loading. We evaluated 48 sticks containing enamel and dentin, with dimensions (2.7 x 2.7 x 16 mm). These sticks were planned with sandpapers with granulation 600 and 800 and after subsequently polished with sandpaper granulation 1000, 1200, 2400 and 4000. A transverse notch of 1,5 mm diameter was made on dentin leaving a neck of 1,7mm of enamel and dentin. The entire surface of the specimen, except for a window of 4 mm on the buccal enamel (the region of highest stress concentration), received a protection against the acid challenge. The specimens were fixed at one end and divided into two groups (n = 24): subjected to a continuous bending load (650 gf) applied to the other end (where 12 of them were fixed with the notch facing up - compression - and 12 with the notch down - tensile) and another group of specimens unloaded (n = 24). With this division, all specimens suffered the first acid challenge (2 hours in 400 ml of buffered citric acid solution, adjusted to pH 3.75 at 37 ° C) and, then, were measured (lesion depth and surface roughness with profilometer, and also micro hardness). Then each group was subdivided into two with the same number (n = 6 for loaded ones and n=12 for unloaded ones): A group received brushing with toothpaste and other just with water, then they were protected again and exposed to the second acid challenge (with load or without load). After remeasurement (lesion depth and roughness with profilometer, and micro hardness) and re-protection, the specimens were subjected to the third acid challenge and again measured. An extra set of 24 sticks was used for the evaluation by polarized light microscopy, divided into three subgroups (n = 8) without load, subjected to tension and under compression. RESULTS: In all stages the lesion depth have increased:(depending of the group, ranged from 1.9 m in the first stage to 7.9 m in the last stage and 4.5 m in the first stage to 19.2 m in the last stage). The tensile stress produced lesion depth (second stage: 16.2 mm; last stage: 19.2 mm) significantly higher than the compression (the second stage: 9.2 mm; last stage: 13.4 mm) only in the fixed side in the group of specimens brushed with toothpaste. The roughness was higher in the exposed area (Ra ranging from 0.36 to 0.55) than in the protected one (Ra varying from 0.13 to 0.28), but the stress was not significantly correlated in either. In all cases it was observed a significant reduction in roughness in the second stage (about 0.8 units of Ra for the protected areas to 0.16 for the exposed), attributable to brushing with toothpaste as well with water. The stress did not cause significant differences in the micro hardness in either group, but brushing with toothpaste, in the second stage, produced significantly higher micro hardness in, both areas, protected areas (410 KNH) and the exposed (140 KNH) when compared with their early stages (318 KHN and 60 KHN, respectively). CONCLUSION: The effect of stress on the lesion depth depends of the presence of other factors; therefore the specimens brushed with water, the stress caused no statistically significant effect on lesion depth neither in the fixed nor in the notch side. Just in the specimens brushed with toothpaste in the side of fixation was possible to observe a significant increase of the lesion depth (in the second and third stages) of the tensile stress in relation to the compression, whereas the specimens not submitted to stress presented intermediate lesion depth values. The tensile stress led to the emergence of micro-cracks detectable by polarized light microscopy. Regarding to the micro hardness, the stress did not cause significant differences in any case but only brushing with toothpaste may be responsible for increased hardness. The roughness in the exposed area was much higher than in the protected, but the stress was not significantly correlated. Brushing with water or toothpaste reduces the roughness in the second stage for the specimens brushed with toothpaste. In the second stage, the roughness decreases in the exposed and protected areas and the micro hardness increases in the groups brushed with toothpaste in the exposed and protected areas.

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Os estÃgios iniciais da formaÃÃo das lesÃes de cÃrie em humanos estÃo relacionados diretamente com a presenÃa de Streptococcus mutans no biofilme dental. O tratamento das doenÃas ocasionadas por biofilmes orais envolve basicamente a remoÃÃo mecÃnica e o uso de antibiÃticos e agentes anti-sÃpticos os quais podem originar cepas resistentes aos antimicrobianos tradicionais. A Terapia FotodinÃmica Antimicrobiana (TFDA) apresenta-se como uma opÃÃo alternativa ao tratamento clÃssico, promovendo a morte bacteriana por meio da fotossenssibilizaÃÃo dos componentes microbianos. O objetivo dessa dissertaÃÃo foi avaliar o potencial antimicrobiano da TFDA e sua capacidade de alterar o padrÃo de expressÃo gÃnica de Streptococcus mutans em biofilmes orais produzidos in vitro utilizando para tanto um diodo emissor de luz (LED) associado ao fotossenssibilizador azul de orto-toluidina (TBO). Para tanto biofilmes de .S. mutans UA159, foram formados sobre discos de hidroxiapatita utilizando um modelo de banhos de cultura e submetidos a TFDA apÃs 5 dias. O material coletado passou por um processo de disrrupÃÃo para a dispersÃo das cÃlulas e diluiÃÃo em sÃrie decimal, apÃs o que foi plaqueado em meio de cultura e incubado em condiÃÃes ideais de crescimento. Adicionalmente, e expressÃo dos genes de virulÃncia gtfB, gtfC e gbpB de S. mutans foram avaliados apÃs a extraÃÃo do RNA total, sÃntese de cDNA e realizaÃÃo da tÃcnica da reaÃÃo da polimerase em cadeia em tempo real utilizando primers especÃficos. O gene ribossomal 16S foi utilizado com gene de referÃncia.. ReduÃÃes significativas (p<0,05) foram observadas na viabilidade das colÃnias de S. mutans quanto exposto ao TBO e LED. A expressÃo gÃnica de gtfC em biofilme de S. mutans foi significantemente reduzida apÃs a realizaÃÃo da terapia fotodinÃmica antimicrobiana enquanto a expressÃo de gtfB parece ter sido menos influenciada pela terapia. NÃo houve alteraÃÃes na expressÃo de gbpB em funÃÃo da terapia fotodinÃmica antimicrobiana. Assim, outros estudos sÃo necessÃrios para avaliar aÃÃo da terapia fotodinÃmica antimicrobiana em condiÃÃes mais semelhantes daquelas encontradas no ambiente clÃnico, bem como para favorecer o entendimento de seus efeitos na expressÃo de importantes genes de virulÃncia de S. mutans.
The early stages of the formation of caries lesions in humans are directly related to the presence of Streptococcus mutans in biofilm. The treatment of diseases caused by oral biofilms basically involves mechanical removal and use of antibiotics and antiseptic agents which may lead to strains resistant to traditional antimicrobials. Antimicrobial Photodynamic Therapy (PACT) presents itself as an alternative option to conventional treatment, promoting bacterial killing by photosensitizing of microbial components. The objective of this study was to evaluate the antimicrobial potential of PACT and its ability to alter the pattern of gene expression of S. mutans in oral biofilms produced in vitro using both a LED light (LED) associated with the photosensitizer ortho-toluidine blue ( TBO). To this end, S. mutans UA159 biofilms were formed on hydroxyapatite discs using a model of bathing culture and were subjected to PACT after 5 days. The collected material has gone through sonication for dispersion and dilution of cells in decimal series. After that, material was plated in rich medium and incubated under optimum growth conditions. Additionally, expression of virulence gtfB, and gtfC gbpB genes of S. mutans were evaluated after extraction of total RNA, cDNA synthesis and realization of the technique of polymerase chain reaction in real time using specific primers. The 16S ribosomal gene was used as the reference gene. Significant reductions (p <0.05) were observed in the viability of colonies of S. mutans when exposed to TBO and LED at the same time. Gene expression of gtfC in S. mutans biofilm was significantly reduced after the completion of antimicrobial photodynamic therapy, whereas expression of gtfB seems to have been less influenced by therapy. No change in the expression of gbpB a function of antimicrobial photodynamic therapy was observed. Therefore, further studies are needed to evaluate the action of antimicrobial photodynamic therapy in conditions more like those found in the clinical environment and to encourage understanding of their effects on the expression of important virulence of S. mutans genes.

A fototerapia laser (FTL) tem sido usada clinicamente para auxiliar na cicatrização de inúmeras doenças bucais, especialmente no tratamento de lesões ulceradas. Os mecanismos celulares através dos quais o laser é capaz de promover a bioestimulação não são completamente compreendidos. Dessa forma, o objetivo do presente estudo foi avaliar in vitro o efeito da FTL no comportamento de queratinócitos bucais no processo de cicatrização. Células epiteliais bucais (NOK-SI) foram cultivadas sob duas condições nutricionais: suplementadas com 10% de soro fetal bovino (FBS) e sob déficit nutricional (2% FBS) seguido de irradiação com laser de diodo InGaAlP (660nm, 40mW, 4 e 20J/cm2, 4 e 20s), através da técnica pontual e em contato. Foram realizados ensaio de viabilidade celular (MTT), migração celular (cicatrização) e análise proteica (Western Blotting e Fluorescência). Os resultados obtidos indicaram que a FTL influencia diretamente a migração epitelial evidenciado pelo fechamento acelerado das feridas irradiadas e polarização do citoesqueleto celular (F-actina). Conclui-se que os efeitos clínicos da FTL estão associados, entre outros fatores, ao aumento da migração epitelial.
Laser phototherapy (LPT) has been used clinically to accelerate wound healing in a variety of oral diseases. The cell mechanisms by which LPT can promote biostimulation have not yet been fully elucidated. Epithelial cells play an important role in the reparative process since it proliferation and migration from the wound margin is crucial for restore epithelial continuity. It is unclear whether LPT has an effect on epithelial cell migration. Based on this, the aim of this study was to investigate the effect of LPT in oral wound healing process using oral keratinocytes. Oral keratinocytes were maintained under two nutritional conditions supplemented with 10% of fetal bovine serum (FBS) and in nutritional deficit (2% FBS). Laser irradiation was delivered with InGaAlP laser (660nm, 40mW, 4 e 20J/cm2, 4 e 20s). Irradiations were performed in contact, using the punctual irradiation mode. The following tests were performed cell viability, cell migration and protein analysis. Results obtained suggest that LPT influences epithelial migration and cytoskeleton polarization. Interestingly, LPT effect under epithelial cell migration occurs independently of cell viability. In conclusion, clinical LPT effects are associated with an increase in epithelial cell migration.

Over the past few decades many researchers have investigated the utility of in vitro - in vivo correlations for the assessment of dosage forms. These investigations are, however, dependent on reproducible dissolution data and well conducted biostudies in order to establish meaningful and robust correlations. Despite the fact that the establishment of such correlations is perhaps idealistic, considerable interest has still been shown in this area of research. Various Controlled/Modified Release Dosage Forms (CMRD's) of theophylline, a weakly basic drug, and indomethacin, a weakly acidic drug, were assessed in order to establish in vitro - in vivo correlations. Dissolution rate studies were carried out using either the USP basket or paddle apparatus. The dissolution rate studies were conducted in a range of dissolution media of varying pH. Bioavailability studies were conducted on the dosage forms used by the Biopharmaceutics Research Institute at Rhodes University. The results of these biostudies were kindly made available for use in this research project. Type A correlations were established using a mathematical simulation process whereby expected in vivo responses are simulated and compared to actual profiles obtained for the dosage forms. In order to perform the simulations the dissolution rate profiles were stripped and using linear regression and the methods of residuals the dissolution rate order and the relevant dissolution rates were obtained. The results of the s imulations indicated that the in vivo serum concentration-time curves could be accurately predicted for the theophylline dosage forms but to a lesser extent, for the indomethacin formulations. The dissolution rate studies indicated that the paddle method is a suitable method for dissolution rate studies of theophylline CMRD's, although it appeared that the optimum pH of the dissolution medium was formulation dependent. Dissolution rate studies conducted on indomethacin formulations indicated that the USP specified basket method for extended-release indomethacin formulations was not able to distinguish between two formulations which exhibited different in vivo profiles. The conversion to the paddle method was, however, able to highlight the differences between these formulations. The use of three dimensional topographs to depict dissolution rate profiles was demonstrated for formulations of both theophylline and indomethacin. The topographs enabled the successful differentiation between bioinequivalent formulations. The dissolution rate profiles were also fitted to the Wei bull equation and the parameters obtained from this were compared to the Weibull parameters obtained from the in vivo absorption plots obtained using the Wagner-Nelson method. The results indicated that the Weibull function was suitable to describe both the in vivo and in vitro data. The following recommendations for the preformulation dissolution studies of weakly acidic and weakly basic drugs are proposed. The dissolution rate studies of weakly acid drugs, such as indomethacin, should be carried out over a range of pH utilising the paddle apparatus. Three dimensional topographs based on the dissolution data should be constructed and used as a comparative tool for different formulations. Based on these comparisons the appropriate formulation can then be selected for a pilot scale in vivo bioavailability study. The dissolution rate studies of weakly basic drugs, such as theophylline, should be carried out over a range of pH utilising the paddle apparatus. The dissolution data should then be used to simulate the expected in vivo profile and on this basis the appropriate formulation selected for a pilot scale bioavailability study. The above approach to the preformulation studies of new CMRO's would allow for the more careful selection of new dosage forms and could thus eliminate costly and unnecessary bioavailability studies performed on inferior formulations.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Anfotericina B (AmB) é o fármaco de escolha para o tratamento das infecções fúngicas invasivas, importante causa de morbidade e mortalidade em pacientes imunodeprimidos. A toxicidade da AmB na forma convencional tem estimulado o desenvolvimento de novas formulações para a administração do fármaco. Neste trabalho foram estudadas microemulsões óleo/água, contendo fosfatidilcolina de soja/tween 20 como agentes tensoativos, CaptexTM 200 como fase oleosa e tampão fosfato 50mM pH 7,2 como fase aquosa, com o objetivo de reduzir a toxicidade e aumentar a absorção oral da AmB. Os sistemas obtidos com diferentes proporções dos componentes foram descritos através de um diagrama de fase pseudo-ternário. As microestruturas foram caracterizadas por espalhamento dinâmico de luz (DLS), reologia, microscopia de luz polarizada e espalhamento de raios X a baixo ângulo (SAXS). Foi desenvolvido e validado um método por CLAE para a determinação de AmB em plasma para aplicação em estudo do perfil farmacocinético do fármaco veiculado na ME em ratos. A nefrotoxicidade da AmB foi avaliada pela determinação da creatinina plasmática dos ratos após administração oral da nova formulação desenvolvida (50 mg/kg), e comparada à administração da formulação convencional na mesma dose. Foi observado que o tamanho das gotículas das microemulsões aumenta quando a AmB é incorporada ao sistema. As amostras apresentaram comportamento Newtoniano e, dependendo da composição do sistema, antitixotropia foi observada. Também foi observado que a viscosidade aumenta com o aumento da fase oleosa assim como a formação de estruturas lamelares ordenadas que são desfavorecidas com a adição do fármaco. A incorporação do fármaco depende das proporções de fase oleosa e tensoativo. As interações da AmB...
Amphotericin B (AmB) is the drug of choice for therapy of invasive fungal infections, an important cause of morbidity and mortality among immunodeficient patients. The high toxicity of AmB in its conventional formulation have induced the development of innovative formulations for the drug administration. In this work, oil-in-water microemulsions containing soya phosphatidylcholine/Tween® 20 (1:1) as surfactant, captexTM 200 as oil phase, and phosphate buffer 50mM, pH 7.2 as aqueous phase were studied in order to reduce AmB toxicity and increase its oral absorption. Systems obtained with different proportions of the components were described by pseudoternary phase diagram. The microstructures of the system were characterized by dynamic light scattering (DLS), rheological behavior, polarized light microscopy and small angle X-ray scattering (SAXS). Was developed and validated a fast and selective HPLC method to determine AmB for application to study of pharmacokinetic profile of drug in rats. The nephrotoxicity of AmB was assessed by determining of rat plasma creatinine after oral administration of the novel formulation (50 mg/kg) and comparing with it a conventional formulation in the same dose. It was observed that the oil droplets size increase when AmB is incorporated into the system. The samples presented Newtonian behavior depending on the system composition. An anti-thixotropic behavior was found, as well, the viscosity increases withthe oil phase. The data showed the formation of ordered structures with lamellar arrangements in the drug unloaded systems and that order decrease with the drug incorporation. The AmB incorporation into the system was dependent on both the oil phase and surfactant. The interactions of AmB with the systems can control both the drug solubility and release... (Complete abstract click electronic access below)