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Additional file 2. Informed consent term.

Additional file 1. SPIRIT 2013 Checklist: Recommended items to address in a clinical trial protocol and related documents*.

ispartof: Huisarts Nu vol:50 pages:192-196 status: published

BACKGROUND The recently introduced 2017 World Workshop on the classification of periodontitis, incorporating stages and grades of disease, aims to link disease classification with approaches to prevention and treatment, as it describes not only disease severity and extent but also the degree of complexity and an individual's risk. There is, therefore, a need for evidence-based clinical guidelines providing recommendations to treat periodontitis. AIM The objective of the current project was to develop a S3 Level Clinical Practice Guideline (CPG) for the treatment of Stage I-III periodontitis. MATERIAL AND METHODS This S3 CPG was developed under the auspices of the European Federation of Periodontology (EFP), following the methodological guidance of the Association of Scientific Medical Societies in Germany and the Grading of Recommendations Assessment, Development and Evaluation (GRADE). The rigorous and transparent process included synthesis of relevant research in 15 specifically commissioned systematic reviews, evaluation of the quality and strength of evidence, the formulation of specific recommendations and consensus, on those recommendations, by leading experts and a broad base of stakeholders. RESULTS The S3 CPG approaches the treatment of periodontitis (stages I, II and III) using a pre-established stepwise approach to therapy that, depending on the disease stage, should be incremental, each including different interventions. Consensus was achieved on recommendations covering different interventions, aimed at (a) behavioural changes, supragingival biofilm, gingival inflammation and risk factor control; (b) supra- and sub-gingival instrumentation, with and without adjunctive therapies; (c) different types of periodontal surgical interventions; and (d) the necessary supportive periodontal care to extend benefits over time. CONCLUSION This S3 guideline informs clinical practice, health systems, policymakers and, indirectly, the public on the available and most effective modalities to treat periodontitis and to maintain a healthy dentition for a lifetime, according to the available evidence at the time of publication.

Supplemental Material, Appendix_I_II_III_IV for Can Periodontitis Influence the Progression of Abdominal Aortic Aneurysm? A Systematic Review by Leila Salhi, Eric Rompen, Natzi Sakalihasan, Isabelle Laleman, Wim Teughels, Jean-Baptiste Michel, and France Lambert in Angiology

Bioactive glass is an attractive biomaterial that has shown excellent osteogenic and angiogenic effects for oral bone repairing procedures. However, anti-biofilm potential related to such biomaterial has not been completely validated, mainly against multispecies biofilms involved in early tissue infections. The aim of the present study was to evaluate the anti-biofilm effect of 58S bioactive glass embedding calcium bromide compounds at different concentrations. Bioactive glass containing 0, 5, or 10wt% CaBr2 was synthesized by alkali sol-gel method and then characterized by physco-chemical and scanning electron microscopy (SEM). Then, samples were tested by microbiological assays using optical density, real time q-PCR, and SEM. Bioactive glass particles showed accurate chemical composition and an angular shape with a bimodal size distribution ranging from 0.6 to 110 μm. The mean particle size was around 29 μm. A significant anti-biofilm effect was recorded for 5wt% CaBr2-doped bioactive glass against S. mitis, V. parvula, P. gingivais, S. gordoni, A. viscosus, F, nucleatum, P. gingivais. F. nucleatum and P. gingivalis. Such species are involved in the biofilm structure related to infections on hard and soft tissues in the oral cavity. The incorporation of calcium bromide into bioactive glass can be a strategy to enhance the anti-biofilm potential of bioactive glasses for bone healing and infection treatment. Key words: Bioactive glass, anti-biofilm, bromide, sol-gel synthesis, bone infection, bone healing ispartof: Journal of Biomedical Materials Research A vol:105 issue:7 pages:1994-2003 ispartof: location:United States status: published

OBJECTIVES: To evaluate clinical, radiographic, microbiologic, and biomechanical parameters related to bone remodeling around implants with external hexagon (EH) and Morse-taper (MT) connections.MATERIALS AND METHODS: Twelve totally edentulous patients received four custom-made implants in the interforaminal region of the mandible. Two of those implants had the same macroscopic design, but different prosthetic connections. All patients received an immediate implant-supported prosthesis. Clinical parameters (periimplant probing pocket depth (PPD), modified gingival index (mGI), and mucosal thickness (MTh)) were evaluated at 12 months follow-up. The distance between the top of the implant and the first bone-to-implant contact (IT-FBIC) was evaluated on standardized digital peri-apical radiographs acquired at 1, 3, 6, and 12 months follow-up. Samples of the subgingival microbiota were collected 1, 3, and 6 months after implant loading and used for the quantification of Tanerella forsythia, Porphyromonas gingivalis, Aggragatibacter actinomycetemcomitans, Prevotella intermedia, and Fusobacterium nucleatum. Further, 36 computerized-tomography based finite element (FE) models were accomplished, simulating each patient under three loading conditions.RESULTS: The evaluated clinical parameters were equal for EH and MT implants. Mean IT-FBIC was significantly different between the tested connections (1.17 ± 0.44 mm for EH, and 0.17 ± 0.54 mm for MT, considering all evaluated time periods). No significant microbiological differences could be observed between tested connections. FE analysis showed a significantly higher peak of equivalent (EQV) strain (p = 0.005) for EH (mean 3,438.65 µε) compared to MT (mean 840.98 µε) connection.CONCLUSIONS: Radiographic periimplant bone loss depends on the implant connection type. MT connections showed less periimplant bone loss, compared to EH connections.

During the last decade an increased interest in alternative, preventive, and therapeutic strategies in dentistry has arisen. Probiotics are living microorganisms which, if administered in sufficient amounts, provide a health benefit to the host. Their precise mechanisms of action have not been identified, but they are able to interfere with the imbalance occurring in biofilm-associated infections. In other fields of medicine, mainly in gastroenterology, their usefulness is already proven. Concerning oral threats, probiotic bacteria may reduce the numbers of pathogens associated with dental caries (mutans streptococci). Clinically, results are encouraging, but further research is needed to demonstrate apparent effects of certain probiotic strains on oral health as well as their desired concentration and vehicle. The use of probiotics in prevention and treatment of caries, periodontal diseases, halitosis, and other oral diseases needs to be further investigated. ispartof: Quintessence International vol:46 issue:3 pages:255-264 ispartof: location:Germany status: published

Objective: The impact of the implant position on the restorative outcome could justify guided surgery even for the single implants particularly in the aesthetic zone and especially when a simplified concept is available. Material and methods: Based on a plaster model, on which the soft tissues were mimicked (according to the thickness measured on a Cone-Beam CT), a tooth-supported, surgical template was prepared. The latter guided all drills so that even flapless implant insertion became possible. All implants were placed by students of the master-after-master training program in Periodontology. Results: The prospective cohort included a total of 34 implants, all of AstraTech (Osteospeed(®) ) type, which were successfully inserted in 29 patients, 16 flapless, 32 onestage. The marginal bone along the integrated implants remained stable over time, with 0.13 mm loss during the first year. The aesthetic parameters were reassuring. Conclusions: This simple model-based concept seems to be reliable for the guided placement of single implants and the pre-operative preparation of their restorations. To cite this article: Marcelis K, Vercruyssen M, Naert I, Teughels W, Quirynen M. Model-based guided implant insertion for solitary tooth replacement: a pilot study. Clin. Oral Impl. Res. xx, 2011; 000-000. doi: 10.1111/j.1600-0501.2011.02242.x. ispartof: Clinical oral implants research vol:23 issue:8 pages:999-1003 ispartof: location:Denmark status: published

As in other fields of healthcare, probiotics have been introduced for prevention and treatment of periodontal diseases. ispartof: Journal of Clinical Periodontology vol:38 issue:SUPPL. 11 pages:159-177 ispartof: location:United States status: published

Objective: The inherent deformation and two-dimensional nature of panoramic radiographs jeopardise their interpretation and quantitative measurements. This study aims to estimate the degree of underestimation of available mesio-distal bone in the premolar area (comparing panoramic radiographs with multi-slice/cone-beam computer tomography [CT]) to determine the prevalence, width, length and position of the bony canal [artery] in the lateral sinus wall and to explore the prevalence, width and length of another (newly detected) bony canal at the palatal aspect of the upper canine. Material and methods: The distance between the distal side of the canine/first premolar and the mesial side of the first molar or the anterior wall of the maxillary sinus was measured on panoramic radiographs and corresponding multi-slice/cone-beam CT images (65 patients). Measurements were made at apical, mid-radicular and crestal regions, parallel to the occlusal plane. The presence and dimensions of the two above-mentioned intra-osseous canals were verified on multi-slice CT scans (144 patients) using reformatted cross-sectional images and/or axial slices. Results: For all 65 patients, panoramic radiographs underscored the mesio-distal distance of available bone in the upper premolar region (mean 2.9 mm, range 0.1-7.5 mm). An intra-osseous canal in the lateral maxillary sinus wall was clearly visible in 49.5% of the cases (mean diameter 1.4 mm). In the canine region, a bony canal was obvious in 32.9% of the cases, with a mean diameter of 1.23 mm. For both canals, there was no correlation between diameter and patient's age. Conclusions: Based on the present data, cone-beam CT imaging can be recommended for visualising anatomical structures during planning of sinus augmentation procedures. To cite this article: Temmerman A, Hertelé S, Teughels W, Dekeyser C, Jacobs R, Quirynen M. Are panoramic images reliable in planning sinus augmentation procedures? Clin. Oral Impl. Res. xx, 2010; 000-000. ispartof: Clinical Oral Implants Research vol:22 issue:2 pages:189-194 ispartof: location:Denmark status: published

Recently, the predation of Bdellovibrio bacteriovorus on a periodontal pathogen has been described. The current study explores the potential antimicrobial activity of a range of predatory bacteria against key periodontal pathogens. A number of representatives from the Bdellovibrio, Bacteriovorax and Peredibacter lineages (called 'BALOs') were tested for their activity towards a group of key periodontal pathogens and an optimal multiplicity of infection was established. As the oral cavity contains a wide variety of bacteria that are not preyed upon, it was investigated if they can have an effect on the predation efficiency of BALOs. It was concluded that a number of important variables involved in bacterial predation are found to be compatible with the composition of the oral microbiota. This finding makes the case for continued study of the potential for BALOs to combat periodontal pathogens. ispartof: Molecular Oral Microbiology vol:26 issue:1 pages:52-61 ispartof: location:Denmark status: published

Periodontitis remains a major public health issue and current management approaches have failed to impact upon the most high-risk proportion of the population and those with the most severe disease. The objective of this session was to assess if and how, current understanding of periodontitis provides the opportunity to develop new preventive and therapeutic strategies.

95% van het tandverlies wordt veroorzaakt door cariës en parodontitis. N aast zijn specifieke orale manifestaties, wordt parodontitis ook beschou wd als een risicofactor voor cardiovasculaire ziekten en voor ongunstige zwangerschapsresultaten. Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis en Tannerella forsythensis w orden beschouwd als de belangrijkste paropathogenen. De capaciteit van e en bacterie om aan zijn gastheer te hechten is belangrijk, vooral voor d e inductie van infectieuze ziekten, zoals gingivitis of periodontitis. O rale bacteriën en vooral pathogene bacteriën, zoals P. gingivali s en A. actinomycetemcomitans, hebben een grote batterij v an virulentiefactoren, waarvan één, de capaciteit is om harde intra -orale oppervlakken en/of de orale mucosa te koloniseren. Omdat bacterië n geen passieve toeschouwers zijn, zullen zij interageren met hun omgevi ng tijdens zulke kolonisatie en vice versa. Onze kennis over de invloed van microbiële interacties tijdens bacteriële kolonisatie van gastheerce llen is zeer beperkt, hoewel de processen betrokken bij individuële bact eriële adhesie aan gastheercellen vrij bekend zijn, zelfs voor paropatho genen. Deze worden tegenwoordig zelfs tot op een moleculair niveau onder zocht. De identificatie van factoren die kolonisatie van pathogenen onde rsteunen of onderdrukken kan helpen in het verder ontrafelen van het zie kteproces en in het ontwikkelen van nieuwe behandelingsstrategieën. Daar om onderzocht deze thesis de bacteriële kolonisatie van epitheliale cell en en specifiek de invloed van klinisch relevante modificerende factoren (roken, virale infectie en bacteriële interacties). In hoofdstuk 1 wordt het effect van nicotine, cotinine en sigaretten roo k extract (CSE) op epitheliale kolonisatie onderzocht. Wanneer de epithe liale cellen werden geïnoculeerd met serum concentraties van nicotine en cotinine werden geen veranderingen in bacteriële kolonisatie ontdekt. D oor niet fysiologische nicotine of cotinine concentraties of door CSE te gebruiken werden nochtans beperkte veranderingen uitgelokt. Hieruit bli jkt dat, vanuit in vitro standpunt, roken slechts op een beperkte manier de gevoeligheid van epitheliale cellen aan kolonisatie door paropathoge nen zal beïnvloeden. Het is echter duidelijk dat deze resultaten niet on tkrachten dat rokers in vivo meer door paropathogenen worden gekolo niseerd. Recente studies hebben de aanwezigheid van een brede waaier van virussen aangetoond in de parodontale pocket. Hoewel het niet duidelijk is of zi j betrokken zijn bij de ontwikkeling van parodontitis, toont hoofdstuk 2 in vitro aan dat hCMV interageert met de epitheliale kolonisatie v an A. actinomycetemcomitans. Een duidelijke virale dosis-resp ons en besmettingstijd afhankelijkheid invloed op bacteriële kolonisatie werd getoond. De relatieve verhoging van kolonisatie met een factor van 2 tot 2.5 ligt binnen de waaier van wat voor andere virale veroorzaakte veranderingen in bacteriële kolonisatie van cellen is gemeld. De result aten steunen de hypothese dat adhesie van A. actinomycetemcomita ns in vitro wordt verhoogd door hCMV. De betekenis van deze bevinding en in de ontwikkeling van agressieve parodontitis blijft onduidelijk. Ondanks een snelle toename van de kennis op vlak van paropathogeen-gasth eer interacties, blijft de rol van beneficiële microbiota in deze intera cties duister. Hoofdstukken 3 en 4 concentreerden zich op het vinden van bacteriële stammen die de kolonisatie van paropathogenen verhinderen en die zelfs reeds gekoloniseerd paropathogenen kunnen verwijderen van epi theliale cellen. Na het selecteren van de drie meest efficiënte stammen en het nader toelichten van sommige mogelijke mechanismen van interactie in vitro, werd hun efficiëntie getest in vivo. Het aanbrengen van deze speciës na rootplaning, als een bacteriële vervangingstherapie, vertraag de beduidend de rekolonisatie van parodontale pockets voor verscheidene paropathogenen. Zelfs een beter klinisch resultaat kon worden waargenome n wanneer deze zogenaamde beneficiële bacteriën werden aangebracht. De r esultaten van hoofdstukken 3 en 4 tonen duidelijk dat een gunstig effect van bacteriële vervangingstherapie, en in het bijzonder GPR, kan worden verwacht. Of de grenzen van GPR al zijn bereikt is hoogst onwaarschijnl ijk. Daarom worden de gegevens voorgelegd als een bewijs van het princip e eerder dan als een behandelingsbenadering die volgende week in patiënt en kan worden toegepast. De momenteel gebruikte technieken om bacteriële kolonisatie van cellen t e modelleren hebben geholpen in het ontrafelen van de interacties tussen pathogene bacteriën en hun gastheer. Nochtans, dringt het huidige beeld van parodontits als een polymicrobiële infectie de ontwikkeling van nie uwe technieken op om de complexe microbiële interacties te analyseren. Hoofdstuk 5 legt de nadruk op het labellen van twaalf verschillende A. actinomycetemcomitans stammen met Green Fluorescent Protein. D eze stammen kunnen in een later stadium worden gebruikt om het proces va n bacteriële kolonisatie van cellen in real time te observeren. De metin g van efficiëntie en frequentie van transformatie en electroporatie over leving tijdens transformatie van deze stammen, gaf extra informatie over de invloed van de genetische achtergrond van deze stammen op het laatst genoemde proces. Het overgaan naar meer complexe, multi-species adhesie experimenten maak t de ontwikkeling van nieuwe bacteriële kwantificatie technieken noodzak elijk. RT-PCR geeft de mogelijkheid om een vrijwel onbeperkt aantal spec ies in één adhesie experiment kwantitatief te bepalen. In hoofdstuk 6, werd een dergelijke techniek, gebaseerd op single copy genen, gevali deerd en geoptimaliseerd voor zijn gebruik in adhesie experimenten. Deze thesis heeft het belang van microbiële interacties op de kolonisati e door paropathogenen in vitro getoond. Dergelijke interacties kunnen ce llen meer gevoelig maken aan kolonisatie door paropathogenen hoewel zij ook in de preventie van rekolonisatie na parodontale therapie kunnen wor den gebruikt. status: published

Antibiotics have played a major role in the improvement of life expectancy in the last 50 years and have led many to believe that bacterial infections were about to vanish as a disease entity of any importance. Emerging problems resulting from a widespread use of antibiotics have modified the general perception of the capabilities of antimicrobial agents. Over the years, bacteria have become increasingly resistant to formerly potent antimicrobial agents, including some antiseptics. The use of antimicrobials may also disturb the delicate ecological equilibrium of the body, allowing the proliferation of resistant bacteria or non-bacterial micro-organisms. This shift may initiate new infections that are worse than the ones originally treated. No antimicrobial drug is absolutely non-toxic and the use of an agent carries accompanying risks. This paper discusses the development and occurrence of antimicrobial resistance in the subgingival flora towards antiseptics and local or systemic antibiotics and is focussed on the question: how can the outcome of periodontal therapy with/without antimicrobials be improved? ispartof: Oral diseases vol:9 issue:s1 pages:30-7 ispartof: location:FRANCE, LYON status: published

ispartof: Periodontology 2000 vol:33 issue:1 pages:119-28 ispartof: location:Denmark status: published

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Odontologia, Florianópolis, 2019. Objetivo: Avaliar a citotoxicidade da furanona C-30 e das lactamas U11, U12 e U26 e seu efeito inibitório na formação de biofilme oral mono e multi-espécies. Materiais e métodos: O efeito da furanona C-30 e lactamas na viabilidade celular de queratinócitos orais humanos (HOK-18A), células da polpa dental humana (HDPC's) e fibroblastos L929 foi avaliado por XTT e MTS. O efeito da furanona C-30 na formação de biofilme mono-espécie foi analisado por cristal violeta, enquanto o efeito da furanona C-30 e das lactamas solubilizadas em meio líquido ou incorporadas em discos de sPEEK na formação do biofilme multi-espécies foi avaliado por PCR quantitativo. Análise estatística foi realizada por comparações entre as condições de crescimento e o grupo controle pelo teste de hipóteses múltiplas (Sidak). Resultados: 100% de viabilidade de HOK-18A foi observada quando expostas a concentrações de 0.5 a 10 µg/mL de furanona C-30, 90% de HDPC's a 10 µg/mL em contraste à concentração de 128 µg/mL que apresentou um efeito citotóxico. Somente o grupo sP3LF128 após 7 dias de exposição demonstrou um efeito citotóxico em fibroblastos L929 resultando em 55% de células viáveis. O crescimento bacteriano planctônico e a formação do biofilme pelos periodontopatógenos Aggregatibacter actinomycetemcomitans, Prevotella intermedia e Porphyromonas gingivalis foram inibidos em >20% e >50%, respectivamente pela furanona nas concentrações de 8 a 128 µg/mL nos experimentos mono-espécie. Nos experimentos multi-espécie a furanona C-30 solubilizada em meio líquido nas concentrações de 10 e 128 µg/mL alterou a composição do biofilme em comparação ao controle reduzindo a concentração bacteriana de A. actinomycetemcomitans e P. intermedia e de P. gingivalis na concentração de 128 µg/mL. A associação da furanona C-30 com lactamas na concentração de 128 µg/mL inibiu significativamente a formação do biofilme de F. nucleatum, P. gingivalis e P. intermedia alterando a composição da comunidade multi-espécies para uma condição mais benéfica e menos patogênica. Quando os compostos antibiofilme foram incorporados em sPEEK a sua atividade inibitória diminuiu consistentemente, apresentando um estímulo na formação do biofilme de S. sobrinus e S. gordonii. Conclusão: O efeito da furanona C-30 e das lactamas na viabilidade celular de HOK-18A, HDPC's e L929, assim como na formação do biofilme mono e multi- espécie foi dose dependente, inibindo principalmente periodontopatógenos Gram- negativos, enquanto a maioria das bactérias benéficas praticamente não foram afetadas pelos compostos. Quando os compostos antibiofilme foram incorporados nos discos de sPEEK o seu efeito inibidor na formação de biofilme multi-espécies foi perdido. Abstract: Aim: To evaluate the cytotoxic effect of furanone C-30 and lactams U11, U12 and U26 and the inhibitory activity on single and multi-species oral biofilm formation. Material and methods: The effect of furanone C-30 and lactams on cell viability of Human Oral Keratinocytes (HOK-18A), Human Dental Pulp Cells (HDPC's) and L929 fibroblasts was evaluated by XTT and MTS assays. The effect of furanone C-30 on single-species biofilm formation was analysed by crystal violet assay, while the effect of furanone C-30 and lactams solubilized in liquid medium or incorporated in sPEEK discs on multi-species biofilms formation was evaluated by quantitative PCR. Statistical analysis were conducted by comparisons between growing conditions and the control group by multiple hypothesis testing (Sidak). Results: 100% viability of HOK-18A was observed when were exposed to 0.5 to 10 µg/mL of furanone C-30, 90% of HDPC's to 10 µg/mL in contrast to 128 µg/mL in which a cytotoxic effect was found. Only group sP/3LF128 at the 7th day of exposure showed a cytotoxic effect on L929 fibroblasts resulting in 55% of viable cells. Planktonic bacterial growth and biofilm formation of the periodontopathogens Aggregatibacter actinomycetemcomitans, Prevotella intermedia and Porphyromonas gingivalis was inhibited by >20% and >50%, respectively at furanone C-30 concentrations of 8 to 128 µg/mL in single-species experiments. In multi-species experiments at 10 µg/mL and 128 µg/mL furanone C-30 solubilized in liquid medium changed the biofilm composition in comparison to the control by reducing the bacterial concentration of A. actinomycetemcomitans and P. intermedia and of P. gingivalis at 128 µg/mL. The association of furanone C-30 and lactams at 128 µg/mL significantly inhibited biofilm formation of F. nucleatum, P. gingivalis and P. intermedia shifting the multi-species community composition to a more beneficial and less pathogenic state. When incorporated in sPEEK discs the inhibitory activity of antibiofilm agents on multi-species biofilms formation decreased consistently, showing an increase in the biofilm formation of S. sobrinus and of S. gordonii. Conclusion: The effect of furanone C-30 and lactams on cell viability of HOK-18A, HDPC's and L929, as well as on single e multi-species biofilm formation was dose dependent, inhibiting mainly Gram-negative periodontopathogens, while most beneficial bacteria were relatively unaffected by the compounds. When antibiofilm compounds were incorporated in sPEEK their inhibitory activity on multi-species biofilm formation has been lost.

Dissertação de mestrado em Engenharia Biomédica (área de especialização em Engenharia Clínica), Periodontitis is an infectious oral disease and remains one of the most prevalent chronic diseases worldwide. Although the exact microbial aetiology is unknown, oral biofilms, also known as dental plaque, seem to play an important role in the development of this disease. Studies already shown that bacterial composition of dental biofilms differs between healthy and diseased sites. To date, the occurrence of this disease is associated with three main factors: (1) the presence of pathogenic bacteria, (2) the absence of beneficial bacteria and (3) the susceptibility of the host. This research project focussed on evaluating the interaction with the outgrowth of beneficial bacteria, where selective nutritional stimulation, here referred to as prebiotics, aimed to modulate oral biofilms by stimulating the growth of beneficial bacteria and, thereby, supress the outgrowth of pathogens, shifting a complex microbiota towards a more healthy-associated composition. The effect of the amino acid L-arginine on selective stimulation of beneficial oral bacteria was tested in single-, dual- and multi-species assays. Further, it was investigated if L-arginine may prevent pathogen incorporation into established biofilms. It was demonstrated that L-arginine treatment could selectively trigger the outgrowth of beneficial bacteria throughout the experiments, leading to a shift in multi-species biofilms in vitro. Although a direct preventive effect of L-arginine could not be observed, continuous treatment showed a reduction in the pathogenic proportion in the complex microbial biofilms. Also, it was demonstrated that predictions about multi-species assays cannot be made based on the results obtained with single and dual-species assays. In conclusion, this study showed that L-arginine has a promising potential to be used as a prebiotic compound in oral health, especially to treat periodontitis, as it is able to modulate oral biofilms towards a more beneficial state. However, in vivo studies are necessary to confirm the observed in vitro prebiotic effect., A periodontite é uma doença oral infeciosa e é uma das doenças crónicas mais prevalentes a nível mundial. Apesar da etiologia exata da doença ser desconhecida, os biofilmes orais, também conhecidos como placa bacteriana, parecem desempenhar um papel importante no seu desenvolvimento. Alguns estudos já demonstraram que a composição dos biofilmes orais de pacientes saudáveis são diferentes dos de pacientes doentes. Até hoje a ocorrência desta doença está associada com três fatores principais: (1) a presença de bactérias patogénicas, (2) a ausência de bactérias benéficas e (3) a suscetibilidade do hospedeiro. Este projeto de investigação focou-se em avaliar a interação com o crescimento de bactérias benéficas, onde uma estimulação nutricional seletiva, aqui referido como prebióticos, visou modular os biofilmes orais, estimulando o crescimento das bactérias benéficas e, desse modo, suprimindo o crescimento dos patogénicos, levando a uma mudança do microbioma oral em direção a um estado mais saudável. O efeito da estimulação seletiva das bactérias benéficas foi testado usando o aminoácido L-arginina em ensaios de mono-, dual- e multi-espécies. Foi ainda investigado se a L-arginina é capaz evitar a incorporação de bactérias patogénicas em biofilmes já estabelecidos na cavidade oral. O tratamento com L-arginina revelou que esta consegue estimular o crescimento das bactérias benéficas em detrimento dos patogénicos, levando a uma mudança nos biofilmes multiespécie in vitro. Apesar de não se ter observado um efeito preventivo direto com o uso da Larginina, o tratamento contínuo com este aminoácido demonstrou uma redução na proporção de espécies patogénicas no biofilme. Percebeu-se ainda que os resultados obtidos com ensaios mono- , dual- e multi-espécies diferem entre si, o que significa que previsões sobre o comportamento das espécies em biofilmes complexos não podem ser feitas através de ensaios onde são apenas testadas uma ou duas espécies. Concluindo, este estudo demonstrou que a L-arginina tem um enorme potencial para ser usada como um prebiótico na saúde oral, com um grande potencial para tratar a periodontite, visto que é capaz de modular os biofilmes orais. No entanto, estudos in vivo são necessários para confirmar os efeitos observados in vitro.

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica), Periodontitis is an inflammatory disease that affects the connective tissues supporting the teeth, being the major cause of tooth loss worldwide. It is nowadays known that this inflammatory disease is related with a shift in the oral microflora. The immune response is responsible for keeping the periodontium healthy – the release of IL-8 to recruit neutrophils to the site of infection is usually very well-orchestrated but becomes dysregulated with the microbial shift. So, the main aim was to try to attenuate the release of IL-8 by human oral keratinocytes in response to several periodontopathogens (Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Prevotella intermedia and Porphyromonas gingivalis), by combining these with probiotic bacterial species (streptococci). Streptococcus oralis proved to exert a beneficial effect when combined with three of the four pathogenic species individually (and was even able to reduce the immune response when facing two pathogenic species at the same time), being therefore considered as the best species to be applied as a probiotic. The pretended effect was optimized when each pathogen was combined with a pair of streptococci, an environment that better replicates a healthy periodontium. Moreover, the study also aimed to better understand the mechanisms through which beneficial bacteria affect the pathogenic species, using heat-killed streptococci or bacterial supernatants. A. actinomycetemcomitans seems to be mainly affected by a physical feature of streptococci, while the opposite happens with P. gingivalis. The results obtained for F. nucleatum, on the other hand, indicate that this species can be affected in both ways. In addition, this study also intended to understand if the use of oral antibiotics could have a negative effect, by confirming if the dead bacteria could feed the resistant microorganisms. P. intermedia seems to be able of using its own and also P. gingivalis’ dead cells as sources of nutrients, which could indicate the resurgence of the problem after antibiotic application. This study highlights the relevance of bacterial interactions on the oral health: if, on the one hand, periodontitis – an inflammatory disease related with a microbial shift – can be attenuated by the use of probiotic bacteria; on the other hand, the bacterial interaction after an oral antibiotic application can have a negative effect, impairing the action of the antimicrobial agent., A periodontite consiste numa doença inflamatória dos tecidos conetivos que dão suporte aos dentes, sendo a maior responsável pela perda de dentes em todo o mundo. Hoje sabe-se que esta doença está relacionada com uma mudança na microflora oral. A resposta imune é responsável por manter o periodonto saudável – a libertação de IL-8 para recrutamento de neutrófilos para o local de infeção é normalmente muito bem orquestrada, mas torna-se desregulada com a alteração microbiana. Assim, o principal objetivo do trabalho é a atenuação da libertação de IL-8 por queratinócitos orais humanos, em resposta à presença de diferentes agentes patogénicos orais (Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Prevotella intermedia e Porphyromonas gingivalis), através da combinação destes com espécies bacterianas probióticas. Streptococcus oralis mostrou ser capaz de exercer um efeito benéfico quando combinada individualmente com três dos quatro agentes patogénicos testados (permitiu inclusivamente reduzir a resposta imune da linha celular quando enfrentando uma associação de dois patogénicos), tendo sido considerada a espécie mais promissora enquanto probiótico. O efeito pretendido foi otimizado quando cada agente patogénico foi combinado com um par de espécies benéficas, numa simulação mais próxima de um periodonto saudável. Para além disto, este estudo pretende adquirir uma melhor compreensão dos mecanismos de ação das espécies benéficas na atenuação do efeito produzido pelos agentes patogénicos, através da utilização de bactérias (streptococci) mortas ou dos sobrenadantes bacterianos. A. actinomycetemcomitans aparenta ser afetada principalmente por uma característica física dos streptococci, enquanto o contrário acontece com P. gingivalis. Os resultados obtidos para F. nucleatum, por outro lado, indicam que esta espécie pode ser afetada de ambas as maneiras. Com o presente trabalho pretende também perceber-se se o uso de antibióticos orais pode ter um efeito negativo, no caso de as bactérias mortas poderem alimentar os microrganismos resistentes. P. intermedia parece ser capaz de usar bactérias mortas da própria espécie e também de P. gingivalis como fontes de energia, o que pode indicar o reaparecimento do problema após aplicação de antibióticos. Este estudo destaca a importância das interações bacterianas na saúde oral: se, por um lado, podem estar na base da atenuação da periodontite, através da aplicação de espécies probióticas; por outro, podem estar relacionadas com a redução do efeito da aplicação de antibióticos orais.

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica), The oral cavity is composed of several bacterial species living in a dynamic and complex ecosystem. Periodontitis and dental caries are two of the most prevalent oral diseases, nowadays. However, current treatments are not enough to fight these oral diseases, so alternative ways are required, as the use of prebiotics and probiotics, which are already being used in several fields. The present work represents the study of the effect of a prebiotic compound (C7) on cariogenic bacteria in order to understand whether addition of this compound leads to a decrease of these bacteria. Streptococcus mutans and Streptococcus sobrinus were the cariogenic bacteria used in this study because these are the main cariogenic bacteria. A probiotic strain, Streptococcus salivarius, was also used in this study. Firstly, quantitative Polymerase Chain Reaction (qPCR) was developed for Streptococcus salivarius in order to allow an accurate determination of its presence in microbial communities. The primers were chosen based on a conserved region of the dextranase gene of S. salivarius and the best combination of primers and probe concentration was determined. This quantification of this strain by this molecular technique was compared with microbial culturing presenting a linear relationship. It was also intended, the find a selective medium for each of the species used, so different media were tested and TYCSB medium showed to be a good selective medium for S. mutans and S. sobrinus. However no selective medium was found for S. salivarius. In order to determine the effect of the prebiotic compound, dual species experiment for each cariogenic bacterium and a probiotic species was carried out. Microbial culturing and qPCR were used for bacteria quantification and pH was also measured. For S. mutans, the main reduction was apparently due to the presence of S. salivarius and was not influenced by C7. For S. sobrinus, the verified reduction was the result of presence of S. salivarius with influence of C7, but no clear conclusions can be made about it. In addition a Denaturing Gradient Gel Electrophoresis (DGGE) was performed to understand the effect of the prebiotic compound in saliva microbiota. The results demonstrated an ecological shift between different bacterial species present in saliva. So, S. salivarius seems to be in higher amounts when C7 is present while other species seem to be present in higher concentration when there is no C7., A cavidade oral é um ecossistema dinâmico e complexo no qual diversas espécies vivem e interagem. Hoje em dia, a periodontite e as caries dentárias são duas das doenças orais mais prevalentes no mundo. No entanto, os tratamentos atuais não têm sido suficientes para responder a estas doenças, havendo necessidade de se utilizar outras alternativas. O uso de prebióticos e probióticos pode ser uma hipótese, tendo em conta que já têm vindo a ser utilizados noutras áreas. Este trabalho representa o estudo do efeito de um composto prebiótico (C7) em bactérias cariogénicas, tentando perceber de que forma a sua presença permite levar à diminuição destas. Deste modo, foram utilizadas as principais bactérias cariogénicas (Streptococcus mutans e Streptococcus sobrinus) e também uma espécie probiótica (Streptococcus salivarius). Assim sendo, foi desenvolvida uma metodologia de Polymerase Chain Reaction em tempo real (qPCR) para S. salivarius, de modo a possibilitar a sua correta quantificação em comunidades microbianas. A melhor combinação de concentrações dos primers e probe foi definida e a quantificação por este método foi comparado com a cultura microbiana, apresentando uma relação linear. A pesquisa de meios seletivos para cada uma das espécies usadas foi também realizada neste trabalho, pelo que foram testados vários meios. O meio TYCSB mostrou ser seletivo para ambas as espécies cariogénicas, contudo não foi encontrado nenhum meio seletivo para o S. salivarius. Para determinar o efeito do composto prebiótico foi utilizado um modelo de espécies dual para cada bactéria cariogénica conjugando-a com a espécie probiótica. A quantificação destas estirpes foi feita através de cultura microbiana e qPCR e o pH foi também medido. Para o S. mutans, a principal redução verificada aparentou dever-se à presença do S. salivarius e não devido à influência do C7. Para o S. sobrinus, a redução que se verificou também resultou da presença do S. salivarius com uma ligeira influência do C7, embora estas conclusões não sejam muito claras. Para perceber o efeito do C7 na saliva foi realizado uma DGGE (Denaturing Gradient Gel Electrophoresis), tendo os resultados demonstrado uma alteração ecológica entre as diferentes espécies presentes na saliva. Assim, S. salivarius, pareceu estar mais presente quando o C7 se encontra adicionado, enquanto outras parecem estar em maiores quantidades quando este composto não se encontra presente.

Periodontitis remains a major public health issue and current management approaches have failed to impact upon the most high-risk proportion of the population and those with the most severe disease. The objective of this session was to assess if and how, current understanding of periodontitis provides the opportunity to develop new preventive and therapeutic strategies. ispartof: Journal of Clinical Periodontology vol:38 pages:114-118 ispartof: location:United States status: published