Development of an in vitro model of peri-implantitis Thesis submitted by Elham Hazeim Abdulkareem BDS, MSc Oral & Maxillofacial Surgery (Baghdad) For the degree of DOCTOR OF PHILOSOPHY In the Faculty of Medicine, University College London Department of Microbial Diseases, UCL Eastman Dental Institute, 256 Gray’s Inn Road London WC1X 8LD -2013- 1 Abstract Abstract Introduction Peri-implantitis is a bacterially induced inflammatory reaction surrounding dental implants leading to loss of supporting bone. Microbial biofilm development seems to play an important role in altering the biocompatibility of the implant surface leading to loss of the implant. Aims The aim of this study was to develop an in vitro model of peri-implantitis. Material and Methods Microcosm biofilms were grown on titanium discs in a constant depth film fermentor (CDFF). Artificial saliva and peri-implant sulcular fluid (PISF) were delivered to simulate three communities associated with dental implants (health, peri-implant mucositis and peri-implantitis). The intact biofilms were visualised by confocal laser scanning microscope (CLSM) at different time points. Biofilms were cultured on non-selective and selective media. PCR-cloning and comparative sequencing of the 16S rRNA gene was carried out to determine bacterial richness. To quantify bacterial species, multiplex qPCR was used for a range of important oral species; qPCR probes were designed, tested and used to quantify Capnocytophaga in the communities. Evaluation of current and novel treatment modalities, to eliminate the microcosm biofilm, on commercial titanium surfaces (polished, SLA and SLActive surfaces) was carried out. Furthermore, novel antimicrobial surfaces were prepared and initial biofilm formation assessed by cultural analysis and CLSM. Results The biofilms shifted from coccid dense communities, to those dominated by rods and long filaments. The clones shifted from healthy to mixed pathogens. The qPCR revealed significant differences between a healthy and disease conditions. The combined treatment (mechanical and chemical) revealed the greatest reduction of biofilm on all surfaces tested. The antibacterial surfaces showed promotion towards a healthy community on old mature biofilm. Discussion/Conclusion The CDFF allowed successful growth of microbial communities and the ability to monitor the bacterial shifts between three communities associated with dental implants. In addition, it has allowed the testing of a range of titanium surfaces and treatment modalities. This model will allow further understanding of the 2 Abstract microbiology of peri-implantitis and provide appropriate biofilm communities for testing surfaces infection and treatments. 3 Declaration Declaration I hereby certify that the work embodied in this thesis is entirely the result of my own investigations. This research project has not been submitted either in part or in full for a degree or diploma to this or any other University or examination board elsewhere. Elham Hazeim Abdulkareem 2013 4 Dedication Dedication To my Mama & Daddy, Sisters, Brothers & their respective families 5 Acknowledgement Acknowledgement It is indeed a great honour and privilege to have been supervised by Dr David Spratt and Dr Jonathan Pratten. They have been stood by me through thick and thin, generously giving of their time, support and encouragement. Their most humane and unstinting approach in guiding me through this period of research from the initial to the final level enabled me to develop an understanding of the subject. I am heartily thankful to them and owe them this PhD. I fully acknowledge the guidance and support of Professor Nikos Donos, Head of Periodontology department, Eastman Dental Institute, University College London. I fully acknowledge the support, help and collaboration of Professor Rob Allaker University of Bart’s and the London, Queen Mary's School of Medicine. I would like to thank all Doctors in the microbiology unit, in particular Dr Anna Tymon, Dr Lena Ciric, Dr Derren Ready and Dr Haitham Hussain for their time and patience especially during the early days of the project. I would like to thank Dr Nicky Mordan for her time and help during CLSM and SEM imaging techniques, Biomaterials & Engineering, Eastman Dental Institute, University College London. I would like to thank Dr Jie Huang, Kaveh Memarzadeh and Dr Megul Vergus for their help during antibacterial samples preparation, Department of Mechanical Engineering, University College London. I would like to thank Dr Aviva Petrie for her time and help during statistical analysis, Statistical analysis department, Eastman Dental Institute, University College London. I would like to thank Dr Daniel Ciantar of the Anatomy department, University College London. 6 Acknowledgement I fully acknowledge the guidance and support of Dr Nickos Mardas during periodontology clinic sessions, Periodontology department, Eastman Dental Institute, University College London. I thank Dr Mohammed Parker of the department of Biomaterials & Engineering for his time and help in the laboratory. I thank all my colleagues in the Microbial Diseases department, Eastman Dental Institute, University College London. I fully acknowledge the funding provided for this project by the Iraqi Ministry of Higher Education and Scientific Research. I appreciate the period of study sponsorship by Iraq, which made it is possible for me to undertake research at the Eastman Dental Institute. The greatest gratitude is due to my parents, for their love, support (both psychological and financial), guidance and encouragement, throughout my life in general and throughout this PhD in particular. I would not have come this far without you. This is your PhD as much as it is mine. If all parents were like you the world would be such a better place. Considerable credit is also due to my sisters Noor, Marwa, Ola and my brothers Omar, Ahmed and Ali. Despite us being in different parts of the world, their support flowed through e-mail and telephone calls. Above all I thank Allah for giving me the possibility to undertake this adventurous journey in science which gave me some insight into His Creation. 7 Index of contents Index of contents Abstract ................................................................................................................................... 2 Declaration .............................................................................................................................. 4 Dedication ............................................................................................................................... 5 Acknowledgement ................................................................................................................... 6 Index of contents ..................................................................................................................... 8 List of figures ......................................................................................................................... 18 List of tables .......................................................................................................................... 30 List of presentations pertaining to this research ................................................................... 32 Chapter 1 ............................................................................................................................. 34 Literature review ................................................................................................................. 34 Overview ............................................................................................................................... 35 1.1 History of osseointegration ............................................................................................. 35 1.2 Peri-implantitis (PI) .......................................................................................................... 36 1.2.1 Definition .................................................................................................................. 36 1.2.2 Classification ............................................................................................................ 37 1.2.3 Diagnosis .................................................................................................................. 37 1.2.4 Clinical signs and symptoms .................................................................................... 38 1.2.5 Prevalence of peri-implantitis ................................................................................... 39 1.2.6 Etiopathogenesis ...................................................................................................... 41 1.2.7 Peri-implant microbiota ............................................................................................. 43 1.2.7.1 Microbiota associated with healthy peri-implant tissues .................................... 43 1.2.7.2 Microbiota associated to peri-implant infection .................................................. 43 1.3 Importance of saliva in oral hygiene ............................................................................... 45 1.4 Peri-implant sulcular fluid (PISF) .................................................................................... 46 1.5 Biofilms ........................................................................................................................... 47 1.5.1 Oral biofilm formation ..............................................................................................