Serra, Elisa (2018)A hydrogel in combination with essential oils for oral ther- apy. Doctoral thesis (PhD), Manchester Metropolitan University. Downloaded from: http://e-space.mmu.ac.uk/622355/ Usage rights: Creative Commons: Attribution-Noncommercial-No Deriva- tive Works 4.0 Please cite the published version https://e-space.mmu.ac.uk A HYDROGEL IN COMBINATION WITH ESSENTIAL OILS FOR ORAL THERAPY A thesis submitted in partial fulfilment of the requirements of the Manchester Metropolitan University for the degree of Doctor of Philosophy School of Healthcare Science Faculty of Science and Engineering Elisa Serra December 2018 DECLARATION AND STATEMENTS Declaration This work has not previously been accepted in substance for any degree and is not concurrently submitted in candidature for any degree. Signed…………………………………………(candidate) Date…………. Statement 1 This thesis is being submitted in partial fulfilment of the requirements for the degree of PhD. Signed…………………………………………(candidate) Date…………. Statement 2 This thesis is the result of my own independent work/investigation, except where otherwise stated. Other sources are acknowledged by explicit references. Signed…………………………………………(candidate) Date…………. Statement 3 I hereby give consent for my thesis, if accepted, to be available for photocopying and for interlibrary loan, and for the title and summary to be made available to outside organisations. Signed…………………………………………(candidate) Date…………. i ACKNOWLEDGEMENTS Firstly, I would like to express my gratitude to Dr Sladjana Malic for her constant support. Without her advice and encouragement, I would have never completed the thesis on time. My sincere thanks for reading through the dissertation when she was on annual leave. I would like to extend my appreciation to the other two members of the team, Prof Dr Joanna Verran and Dr Araida Hidalgo-Bastida. My sincere gratitude goes to Prof Alastair Sloan, who gave me the possibility to spend four months at Cardiff University, and to Prof David Williams. I am grateful to them for their supervision and scientific advice. A special thank goes to Dr Amr Alraies and Dr Wayne Nishio Ayre for their constant and essential help with the ex vivo model. I would like to express my gratefulness to Prof Thomas Wilkinson for giving me the possibility to work in his lab at Swansea University. Even if I stayed there for a short time, I learned a lot. I would like to extend my gratitude to all the people that helped me in the lab. Among all of them, I would like to give my sincere thanks to the lab technicians Paul, Glenn and Lee that taught an engineer how to work in a microbiology, cell, and chemistry lab, respectively. In addition, I am deeply grateful to Rhiannon Parkhouse for proofreading the thesis. I would like to thank all my friends in Manchester, Zurich, and Milan. My sincere gratitude goes to Robertinho for always being there and for letting me feel at home. Thanks to the Moss Side volleyball team, for both the trainings and the nights out. My sincere gratitude goes to Zalessio, Marco, and Monica. Despite the distance, their support was essential. Last and the most important, thanks to my family and brother. Even if I do not live anymore with them, they are always on my mind and in my heart. ii TABLE OF CONTENTS DECLARATION AND STATEMENTS i ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iii LIST OF FIGURES ix LIST OF TABLES xiii LIST OF ABBREVIATIONS xv LIST OF UNITS xviii LIST OF SYMBOLS xix PREFACE xx ABSTRACT xxi OVERVIEW OF THE THESIS STRUCTURE xxii CHAPTER 1: LITERATURE REVIEW 1.1 Biofilms 2 1.1.1 Dental plaque 3 1.1.2 Mechanisms of dental plaque formation 4 1.1.3 Biofilm resistance to antimicrobials 7 1.2 Oral diseases 8 1.2.1 Dental caries 10 1.2.1.1 Pathogenic mechanisms in dental caries 12 1.2.2 Periodontal diseases 13 1.2.2.1 Pathogenic mechanisms in periodontal 16 diseases 1.2.3 Oral candidiasis 19 1.2.3.1 Pseudomembranous candidiasis 19 1.2.3.2 Acute atrophic candidiasis 20 1.2.3.3 Chronic hyperplastic candidiasis 20 1.2.3.4 Chronic atrophic candidiasis 21 1.2.3.5 C. albicans virulence factors 22 1.3 Risk factors 24 1.3.1 Dental caries 24 1.3.2 Periodontal diseases 25 1.3.3 Oral candidiasis 26 1.4 Current therapies 28 1.4.1 Prevention and treatment for dental caries 28 1.4.2 Prevention and treatment for periodontal diseases 29 1.4.3 Prevention and treatment for oral candidiasis 33 1.4.4 Challenges: antibiotic and antifungal resistance 34 iii 1.5 Biocides 36 1.5.1 Chlorhexidine 36 1.5.2 Triclosan 38 1.6 Essential oils 40 1.6.1 Basil 41 1.6.2 Bergamot 42 1.6.3 Cinnamon 42 1.6.4 Citronella 43 1.6.5 Geranium 43 1.6.6 Lavender 44 1.6.7 Melissa 44 1.6.8 Myrtle 45 1.6.9 Peppermint 46 1.6.10 Sage 46 1.6.11 Spearmint 47 1.6.12 Tea tree oil 47 1.6.13 E-cinnamaldehyde 48 1.6.14 Linaool 49 1.7 Hydrogels 51 1.7.1 Synthetic polymers 52 1.7.1.1 Poly(lactic acid) and poly(glycolic acid) and 53 their copolymers 1.7.1.2 Poly(ethylene oxide) and poly(ethylene glycol) 54 1.7.1.3 Poly(2-hydroxyethyl methacrylate) 55 1.7.1.4 Poly(vinyl alcohol) 56 1.7.2 Natural polymers 57 1.7.2.1 Collagen 58 1.7.2.2 Hyaluronate 58 1.7.2.3 Fibrin 59 1.7.2.4 Alginate 60 1.7.2.5 Chitosan 60 1.7.2.6 Methylcellulose 61 1.7.3 Hydrogel characterisation 62 1.7.3.1 Morphology 62 1.7.3.2 Molecular characterisation 62 1.7.3.3 Gelation 62 1.7.3.4 Swelling 63 1.7.3.5 Degradation 63 1.7.3.6 Porosity and pore size 64 1.7.3.7 In vitro drug release 65 1.8 Ex vivo models 65 1.9 Aims and objectives 66 iv CHAPTER 2: ANTIMICROBIAL PROPERTIES OF ESSENTIAL OILS AND BIOCIDES 2.1 Oral candidiasis 69 2.1.1 Candida 69 2.1.1.1 Pseudomembranous candidiasis 69 2.1.1.2 Acute atrophic candidiasis 70 2.1.1.3 Chronic hyperplastic candidiasis 70 2.1.1.4 Chronic atrophic candidiasis 71 2.1.1.5 Candida virulence factors 71 2.2 Periodontal diseases 73 2.2.1 Microbial complexes in the subgingival plaque 73 2.2.1.1 Porphyromonas gingivalis 75 2.2.1.1.1 Porphyromonas gingivalis 76 virulence factors 2.2.1.2 Fusobacterium nucleatum 78 2.2.1.2.1 Fusobacterium nucleatum 79 virulence factors 2.2.1.3 Prevotella intermedia 80 2.2.1.3.1 Prevotella intermedia virulence 80 factor 2.3 Essential oils: mode of action 81 2.4 Biocides: mode of action 84 2.5 Synergistic activity 85 2.6 Aims and objectives 88 2.7 Materials and methods 89 2.7.1 Essential oils and biocides preparation 89 2.7.2 Microorganisms 90 2.7.3 Minimum inhibitory concentration (MIC) and minimal 91 lethal concentration (MLC) 2.7.4 Minimal biofilm eradication concentration (MBEC) 92 2.7.5 Checkerboard method: essential oils and biocides 93 synergy 2.7.6 Statistical analysis 94 2.8 Results 95 2.8.1 Minimum inhibitory concentration (MIC) and minimal 95 lethal concentration (MLC) for Candida albicans 2.8.2 Minimum inhibitory concentration (MIC) for periodontal 100 pathogens 2.8.3 Minimal biofilm eradication concentration (MBEC) 102 2.8.4 Essential oils and biocides synergy 104 2.9 Discussion 107 2.10 Conclusions 113 v CHAPTER 3: EVALUATION OF THE CYTOTOXICITY AND IMMUNE RESPONSE TO ANTIMICROBIALS 3.1 Introduction 115 3.1.1 Evaluation of the cytotoxicity 115 3.1.1.1 Dye exclusion method 115 3.1.1.2 Metabolic assay 116 3.1.2 Immune response to Candida infection 119 3.2 Aims and objectives 124 3.3 Materials and methods 125 3.3.1 Half maximal inhibitory concentration on mouse 125 fibroblasts 3.3.2 Inflammatory response to Melissa officinalis essential 126 oil 3.3.2.1 Preparation of Melissa officinalis essential oil, 126 chlorhexidine and zymosan 3.3.2.2 Candida albicans 126 3.3.2.3 Ex vivo model of whole blood 126 3.3.2.4 ELISA 128 3.3.3 Statistical analysis 128 3.4 Results 129 3.4.1 Half maximal inhibitory concentration 129 3.4.2 Inflammatory response of whole blood 138 3.5 Discussion 142 3.6 Conclusion 146 CHAPTER 4: DEVELOPMENT OF A METHYLCELLULOSE HYDROGEL WITH MELISSA OFFICINALIS ESSENTIAL OIL 4.1 Introduction 148 4.1.1 Hydrogels 148 4.1.1.1 Methylcellulose hydrogel 149 4.1.1.2 Applications and antifungal activity of 150 methylcellulose hydrogels 4.1.2 Rheology 152 4.1.2.1 Rheological analysis 154 4.2 Aims and objectives 157 4.3 Material and methods 158 4.3.1 Hydrogel preparation 158 4.3.2 Rheology 159 4.3.3 Melissa officinalis composition 160 4.3.4 Melissa officinalis release 161 4.3.5 Disc diffusion method 162 4.3.6 Time-kill assay 162 vi 4.3.7 Statistical analyses 163 4.4 Results 164 4.4.1 Rheology 164 4.4.1.1 Strain sweep 164 4.4.1.2 Frequency sweep 166 4.4.1.3 Time sweep - Gelation time 168 4.4.2 Melissa officinalis composition 171 4.4.3 Melissa officinalis release 171 4.4.4 Disk diffusion method 176 4.4.5 Time-kill assay 177 4.5 Discussion 178 4.6 Conclusions 183 CHAPTER 5: DEVELOPMENT OF AN EX VIVO MANDIBLE RODENT MODEL TO MIMIC ORAL CANDIDIASIS 5.1 Introduction 185 5.1.1 In vitro, in vivo and ex vivo models 185 5.1.2 In vitro and ex vivo models for oral candidiasis 185 5.1.3 Ex vivo rodent model for oral diseases 187 5.2 Aims and objectives 189 5.3 Materials and methods 190 5.3.1 Cell culture medium 190 5.3.2 Microorganisms 190 5.3.3 Diffusion of Melissa officinalis essential oil into agar 191 5.3.4 Culture of rodent mandibles 192 5.3.5 Gingiva infection 194 5.3.6 Melissa officinalis essential oil treatment 196 5.3.7 Histological examination 197 5.3.8 Colony forming units (CFUs) from C.