THE EFFECT OF HIGH INTENSITY FOCUSED ULTRASOUND ON ENTEROCOCCUS FAECALIS BIOFILM KULSUM IQBAL (BDS) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DISCIPLINE OF PROSTHODONTICS, OPERATIVE DENTISTRY AND ENDODONTICS FACULTY OF DENTISTRY NATIONAL UNIVERSITY OF SINGAPORE 2013 Supervisor: A/P Jennifer Neo Co-Supervisor: Dr Amr Fawzy i DECLARATION I hereby declare that the thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information which have been used in the thesis This thesis has also not been submitted for any degree in any university previously _ Kulsum Iqbal August 2013 ii Acknowledgements With great pleasure in the completion of my project I would like to express my gratitude to all those who provided their kind support and motivation First and foremost I would like to thank my supervisors, Prof Jennifer Neo and Dr.Amr for giving me an opportunity to join as a graduate student at this esteemed university Their leadership, support, motivation and continuous hard work have inspired me a lot I deeply appreciate Dr Amr for his contributions, help and uncountable hours spent in guiding me throughout my project It has been a great odyssey with which I would cherish a lifetime I would like to express my gratitude to Dr Tan Kai Soo for her invaluable support in allowing me to perform my experiments in her lab Furthermore, I would like to thank Prof Khoo and Siew Wan for sharing their transducer for my project They have been helping and guiding me in carrying out my research which would not have been possible without their continuous support I would like to specially thank Mr Chan for his technical support always and Aunty Leena and Ms Han for providing a comfortable working environment The sincere help from my lab members helped me a lot in working as a team I would like to pay my sincere appreciation to my husband and my family who provided me support and encouragement Finally I would like to thank the Faculty of Dentistry for its support throughout my whole Master’s program Kulsum Iqbal iii Table of Contents Chapter I – Introduction Chapter II – Literature Review 2.1 Basic Concepts of Ultrasound Physics 2.2 Properties of Ultrasound Waves 10 2.2.1 Wavelength 10 2.2.2 Frequency 11 2.2.3 Ultrasound Speed 11 2.2.4 Medium Density and Compressibility 11 2.2.5 Absorption 12 2.2.6 Intensity 12 2.2.7 Power 13 2.3 History of Ultrasound in Medicine 14 2.3.1 Therapeutic Ultrasound 17 2.3.2 High Intensity Focused Ultrasound (HIFU) 20 2.3.2.1 Historical Background 21 2.3.2.2 Principle of HIFU 22 2.3.2.3 Thermal effects of HIFU 24 2.3.2.4 Non thermal effects 25 iv 2.3.2.5 Stable cavitation 26 2.3.2.6 Inertial Cavitation 27 2.3.2.7 Radiation force 27 2.3.3 Current Clinical Applications of HIFU 28 2.3.3.1 Liver tumors 28 2.3.3.2 Renal tumors 29 2.3.3.3 Prostate cancer 30 2.3.3.4 Gynecology 31 2.3.3.4.1 Uterine fibroids 31 2.3.3.5 Neurosurgical 31 2.3.3.5.1 Targeted Drug Delivery: 32 2.3.3.5.2 Enhancement of drug delivery 32 2.3.4 HIFU application in dentistry 33 2.3.4.1 Role of biofilms in infections 34 2.3.4.2 Biofilms in endodontics 35 2.3.4.3 Techniques in root canal disinfection 36 2.3.4.3.1 Mechanical debridement 36 2.3.4.3.2 Chemical disinfection 37 2.3.4.3.2.1 Sodium hypochlorite 37 2.3.4.3.2.2 Chlorhexidine digluconate 38 2.3.4.3.2.3 Ethylenediaminetetraacetic acid (EDTA) 38 2.3.4.3.3 Agitation of the irrigants 39 v 2.3.4.3.3.1 Manual agitation techniques 39 2.3.4.3.3.2 Machine assisted irrigation 40 Chapter III: Hypotheses and Objectives 43 3.1 Hypotheses: 43 3.2 Objectives 43 3.2.1 Phase 1: To study the effect of HIFU on E faecalis planktonic suspensions 43 3.2.2 Phase 2: To study the effect of HIFU on E faecalis biofilms on glass-bottom petri dish ……………………………………………………………………………………………….43 3.2.3 Phase 3: To study the effect of HIFU on E faecalis biofilm on root dentin –disc and whole root dentin surface 44 3.2.4 Phase 4: To study the effect of irrigants on HIFU efficacy 44 Chapter IV- Methods and Materials 46 4.1 Experimental set up 46 4.1.1 Water tank 47 4.1.2 Driving circuit 48 4.1.3 Waveform function generator 48 4.1.4 Amplifier 48 4.1.5 Transducer 49 4.1.6 Hydrophone 50 vi 4.2 Measurement of temperature changes 51 4.3 Inoculation of E faecalis 51 4.3.1 Phase 1: HIFU exposure on planktonic suspensions 52 4.4 Biofilm formation 53 4.5 Specimen preparation 53 4.5.1 Root discs 53 4.5.2 Root canal dentin 54 4.5.3 Root disc and root canal contamination 55 4.6 Phase 2: HIFU exposure on biofilms -Petri dish 56 4.7 Phase 3: HIFU Exposure on biofilms- Dentin Disc and Root Canal Dentin 57 4.7.1 Dentin Disc 57 4.7.2 Root canal Dentin 58 4.8 Phase 4: HIFU Exposure on biofilms- Root canal Dentin with Irrigants 59 4.8.1 Root canal Irrigants used: EDTA and sodium hypochlorite (NaOCl) 59 4.8.2 Classification of the Groups 59 4.8.2.1 Control group (no HIFU): 59 4.8.2.2 Conventional syringe irrigation group with NaOCl: 60 4.8.2.3 Conventional syringe irrigation group with EDTA: 60 4.8.2.4 “HIFU NaOCl”: 60 vii 4.8.2.5 “HIFU EDTA”: 60 4.8.2.6 Harvesting the Biofilm 60 4.8.2.6.1 Microbiological analysis for petri dish and root dentin biofilm 60 4.9 Scanning electron microscopy (SEM) 61 4.10 Confocal laser scanning microscopy (CLSM) 62 4.11 Statistical analysis 63 Chapter V: Results 65 5.1 Temperature variation 65 5.2 Phase 1: The effect of HIFU on E faecalis planktonic suspensions 66 5.3 Phase 2: The effect of HIFU on E faecalis biofilms on glass-bottom petri dish 67 5.4 Phase 3: The effect of HIFU on E faecalis biofilm on root dentin disc and root dentin surface ………………………………………………………………………………………………71 5.4.1 Root dentin disc 71 5.4.2 Root canal dentin surface 74 5.5 Phase 4: The effect of different irrigants along with HIFU 76 Chapter VI: Discussion 79 Chapter VII: Conclusions 89 Chapter VIII: Future perspectives 91 Chapter IX: Bibliography 93 viii Summery The foremost aim of endodontic treatment is the complete removal of micro-organisms, their byproducts and pulpal tissue remnants Currently, chemo-mechanical preparation (chemical irrigant combined with mechanical instrumentation) of the root canal system is in use Bacteria are naturally present in oral milieu as biofilm which is known to be a survival mechanism of bacteria Previous literatures have shown that conventional endodontic disinfection techniques are not able to disinfect the root canal completely Therefore, the limitations of the conventional root canal disinfection procedures demand the need of alternative disinfection strategies In this study, High Intensity Focused Ultrasound (HIFU) was used as one of these potential methods To verify our hypothesis, the effect of HIFU on planktonic suspensions and biofilm of Enterococcus faecalis (E faecalis) on different substrates were analysed The main aims of this study were to examine the potential of HIFU as an alternative and efficient method for root canal disinfection To achieve our aims, sequential phase wise studies were conducted The objective of Phase was to study the effect of HIFU on E faecalis planktonic suspensions The results indicated that a bactericidal effect of HIFU was time dependent i.e a significant reduction in Colony forming units (P ≤ 0.05) was found with increasing HIFU exposure Phase was carried out to study the effect of HIFU on two-week old E faecalis biofilms on glassbottom petri dish at different time periods of 30, 60, 120 s The results from these experiments showed significant increase in CFU after HIFU exposure for 30 s compared to the control group (P ≤ 0.001) At 60 s exposure to HIFU, the CFU was significantly higher than the control group (P ≤ 0.05) Furthermore, a significant reduction in biofilm thickness was found with increased exposure time of HIFU ix In Phase 3, the effect of HIFU was observed on biofilm attached to root dentin disc and whole root canal dentin The results were analysed by CFU, SEM and CLSM From 30 s exposure, the 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strategies In this study, High Intensity Focused Ultrasound (HIFU) was used as one of these potential methods To verify our hypothesis, the effect of HIFU on planktonic suspensions and biofilm. .. List of Publications Journal publications: ? ?Effect of High Intensity Focused Ultrasound on Enterococcus faecalis planktonic suspensions and biofilms” Kulsum Iqbal, Siew-Wan Ohl, Boo-Cheong Khoo,... coefficient of the exposed tissue, tissue perfusion, the time period for which the ultrasound is on and the ultrasound intensity From all the developing applications of therapeutic ultrasound, the one