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Development of an in Vitro Gingivitis Model Development of an in vitro gingivitis model Thesis submitted by Joanna Maria Wiecek For the degree of DOCTOR OF PHILOSOPHY in the Faculty of Medical Sciences University College London Division of Microbial Diseases UCL Eastman Dental Institute 256 Gray’s Inn Road London WC1X 8LD UK Declaration I hereby certify that the work embodied in this thesis is the result of my own investigation. Where the information was derived from other sources, I confirm that this has been indicated in the thesis. The contents have not been submitted for any other qualification. Signed ................................................... Date ...................................................... Acknowledgements My sincere appreciation goes to Dr David Spratt whom I would like to thank for being a great supervisor and a great friend. I would like to express my appreciation for his invaluable guidance, assistance, and support throughout the period of this project, and for proofreading this thesis. The knowledge I have gained from our discussions over the past years has been instrumental in the success of this research project, it would have been truly impossible without him. I would like to express my deepest gratitude to Don White for funding this project and his continuous support. I am most appreciative for the countless brainstorming sessions and the knowledge he has shared. The positive attitude shown by him throughout the entirety of this project has truly inspired me to persevere and endure throughout this PhD programme. I would like to acknowledge the financial, academic and technical support provided by University College London and Procter & Gamble (Impact Scholarship). Stay hungry Stay foolish 6 Abstract Gingivitis a gum disease, affects 50-90% of the adult population worldwide, if left untreated gingivitis can lead to periodontitis. However, even though gingivitis is highly prevalent, the pathogenicity of the disease is still poorly understood; highlighting the need for a reliable and reproducible in vitro gingivitis model which could help elucidate clinically important questions and provide a better understanding of gingivitis aetiology. In this study the original Constant Depth Film Fermentor (CDFF) was modified to create a Triple-CDFF (T- CDFF) allowing concurrent growth of oral biofilms that can be treated separately in a controlled, flexible environment. Several mechanical changes were implemented to increase the model’s reliability, reproducibility and manoeuvrability; including improving air- tightness of the model by applying better seals, re-shaping model parts to increase portability. A standardised experimental methodology was devised; this included sampling procedures to allow reliable and reproducible growth of oral biofilms across the individual units of this system. Biofilms obtained from T-CDFF under health and disease conditions were screened for eight bacteria; using qPCR primers for S. sanguinis, V. dispar, N. subflava, S. mutans, L.casei, F. nucleatum, P. intermedia, and A. naeslundii. To investigate the presence of other bacteria associated with gingivitis such as T. denticola, P. gingivalis, a trypsin-like-protease assay was applied. Next generation sequencing combined with metabolomics gave a better understanding of the bacterial changes occurring during simulated disease progression. In conclusion, this study has successfully (i) developed and validated a new complex in vitro T-CDFF system and also (ii) showed great potential for modelling gingivitis in vitro; although further verification needed. The system was considered reliable and shows a great potential for being used as a standardised model for testing dentifrices and antimicrobials. 7 8 Table of content Abstract.........................................................................................................................7 List of Figures...............................................................................................................17 List of Tables................................................................................................................27 1 CHAPTER ..................................................................................................................................... 33 Introduction ........................................................................................................................................ 33 1.1 Oral cavity ................................................................................................................ 35 1.2 Dental plaque formation .......................................................................................... 36 1.2.1.1 Enamel Pellicle Formation .......................................................................... 37 1.2.1.2 Initial colonization ...................................................................................... 37 1.2.1.3 Biofilm maturation...................................................................................... 38 1.3 Periodontal diseases and the oral microbiota ......................................................... 39 1.3.1 Gingivitis ............................................................................................................. 39 1.3.2 Periodontitis ....................................................................................................... 41 1.3.3 Plaque hypotheses of periodontal diseases ...................................................... 43 1.3.4 Oral microbiome ................................................................................................ 46 1.3.4.1 Gingivitis microbiome ................................................................................. 47 1.3.5 Reductionist approach ....................................................................................... 49 1.3.6 Cultivation methods ........................................................................................... 50 1.3.7 Culture independent methods........................................................................... 51 1.3.7.1 PCR .............................................................................................................. 51 1.3.7.2 Quantitative PCR (qPCR) ............................................................................. 52 1.3.7.3 Fluorescence In Situ Hybridisation (FISH) ................................................... 52 1.3.7.4 DNA – DNA hybridisation checkerboard .................................................... 53 1.4 Holistic approaches .................................................................................................. 54 9 1.4.1 Proteomics ......................................................................................................... 54 1.4.2 Transriptomics ................................................................................................... 55 1.4.3 Genomics ........................................................................................................... 56 1.4.4 Next generation sequencing .............................................................................. 56 1.4.4.1 454 platform using pyrosequencing ........................................................... 57 1.4.4.2 SOLiD platform based on Ligation .............................................................. 58 1.4.4.3 Illumina platform based on sequencing by synthesis ................................ 59 1.4.5 Metabolomics .................................................................................................... 61 1.4.5.1 1H NMR spectroscopy ................................................................................. 62 1.4.5.2 Mass spectrometry based techniques........................................................ 63 1.4.5.2.1 Liquid chromatography – mass spectrometry ....................................... 63 1.4.5.2.2 Gas chromatography – mass spectrometry (GC - MS) ........................... 64 1.5 Complex in vitro models .......................................................................................... 65 1.5.1 The Chemostat ................................................................................................... 66 1.5.2 The Robbins device ............................................................................................ 67 1.5.3 Calgary Biofilm Device (CBD) ............................................................................. 68 1.5.4 Multiple Sorbarod Device .................................................................................. 68 1.5.5 CDC Biofilm reactor ............................................................................................ 70 1.5.6 Constant Depth Film Fermentor (CDFF) ............................................................ 72 1.6 Aims of the project .................................................................................................. 74 2 CHAPTER ..................................................................................................................................... 75 2.1 Triple-Constant Depth Film Fermentor model (T-CDFF) ......................................... 77 2.2 Model description .................................................................................................... 78 2.2.1 T-CDFF construction ........................................................................................... 78 2.2.2 T-CDFF set-up ..................................................................................................... 81 10 2.2.3 T-CDFF calibration process................................................................................
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