Continuous Chelation in Endodontics with a Focus on the Novel Chelator Clodronate Patricia Paule Wright Bsc, Bdsc (Hons), BA

Home , EGTA (chemical), Pentetic acid

Continuous Chelation in Endodontics with a Focus on the Novel Chelator Clodronate Patricia Paule Wright BSc, BDSc (Hons), BA

A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2020 School of Dentistry

Abstract Background Sodium hypochlorite (NaOCl) is essential to endodontic irrigation because it uniquely dissolves organic matter and disinfects. Chelators remove the smear layer. Sequences of NaOCl and the chelator ethylenediaminetetraacetic acid (EDTA) are in common use because alone neither fulfils all the core functions required of an irrigant. In 2005, the concept of continuous chelation was introduced, whereby a chelator is combined with NaOCl for simultaneous antimicrobial and proteolytic actions and with the capacity to remove smear layer. Not only is this simpler compared to standard irrigation, but some authors report improved antimicrobial effectiveness, enhanced dentinal debris removal, and improved bonding of endodontic materials to dentine. Combinations of alkaline EDTA with NaOCl have been investigated, although the only commercial application of this technique employs the chelator etidronate. The major shortcoming of etidronate-NaOCl mixtures is that a chemical reaction occurs between etidronate and NaOCl, causing reduced NaOCl concentrations over time. Given that continuous chelation may hold advantages, finding a chelator more compatible with NaOCl than etidronate would improve the lifespan of continuous chelation mixtures.

Objectives The initial objective was to examine the effect of heating to intracanal temperature on known continuous chelation mixtures. A number of chelators were then screened, aiming to identify one, which when combined with NaOCl had better stability with NaOCl than etidronate and was capable of removing smear layer. Once identified, the NaOCl stability of such a mixture was examined at root canal temperature and in refrigerated storage. Subsequent objectives focused on comparing the identified mixture to etidronate-hypochlorite mixtures, standard sequences and NaOCl with respect to organic tissue dissolution, antimicrobial activity and tooth fracture resistance.

Methods Free available chlorine (FAC), pH and temperature were used to assess NaOCl stability in known continuous chelation mixtures at root canal temperature versus room temperature. Iodometric titration was used to determine the FAC. This methodology was then employed to identify a stable chelator-NaOCl solution from several novel chelator mixtures at room

i temperature. The chelator inclusion criteria were the calcium ion ligand stability constant and chemical structure. Once a stable mixture was identified, scanning electron microscopy (SEM) in conjunction with image analysis software was employed to assess smear layer removal. The NaOCl stability was then tested for the mixture, as previously described, at root canal temperature and additionally in cold storage. The novel mixture and other irrigant regimens were then compared with respect to important irrigant functions. Tissue dissolution was assessed by measuring the weight loss of porcine mucosa samples at 32oC. The antimicrobial capacity against 7-day Enterococcus faecalis biofilms on hydroxyapatite discs was examined using microbial counts, SEM imaging and the 2,3-bis (2-methoxy-4-nitro-5- sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) metabolic activity assay. Compressive strength testing, using an Instron universal testing machine, evaluated the mechanical properties of teeth treated with continuous chelation mixtures versus standard sequences.

Results 1. At 20 minutes, heating from 23oC to 35oC accelerated FAC falls in etidronate and EDTA mixtures with NaOCl. This reduced the therapeutic window of etidronate mixtures to 20 minutes and rendered EDTA mixtures unsuitable for use. Declines in FAC were accompanied by falls in pH. 2. The chelators EDTA, egtazic acid (EGTA), pentetic acid (DTPA), cyclohexanediaminetetraacetic acid (CDTA), aminotris(methylenephosphonic acid) (ATMP), hydroxyphosphonic acetic acid, clodronate and etidronate were chosen for assessment of their stability with NaOCl. Clodronate was the only chelator that when combined with NaOCl maintained its FAC and was associated with neither a temperature spike nor a pH fall. 3. Clodronate-NaOCl mixtures removed smear layer and did not erode the peritubular dentine. 4. FAC in clodronate mixtures was unaffected by heating to root canal temperature over 3 hours. Clodronate-hypochlorite mixtures lost minimal FAC in cold storage over 3 months. 5. At a clodronate concentration able to remove smear layer, clodronate-NaOCl mixtures dissolved porcine mucosa better than etidronate mixtures and equally as well as the control NaOCl.

6. Colony counting methodology on E. faecalis biofilms on hydroxyapatite discs was invalidated. 7. The resistance to compressive force was the same in teeth irrigated with the sequence NaOCl/EDTA/NaOCl or with etidronate or clodronate mixtures with NaOCl. 8. SEM imaging showed that clodronate-NaOCl, etidronate-NaOCl and NaOCl all removed biofilm on hydroxyapatite discs. The XTT assay showed no differences between continuous chelation mixtures and NaOCl.

Conclusions Clodronate, compared with etidronate, had superior NaOCl stability in continuous chelation mixtures. Clodronate-NaOCl mixtures removed smear layer, dissolved organic material and disinfected. The addition of clodronate to NaOCl neither impaired antimicrobial activity nor the dissolution of organic tissue by NaOCl. The use of clodronate in continuous chelation did not negatively impact the mechanical properties of teeth compared with a standard sequence.

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Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award.

I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School.

I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co- authors for any jointly authored works included in the thesis.

Patricia Paule Wright

Publications included in this thesis 1. Wright PP, Kahler B, Walsh LJ (2019) The effect of heating to intracanal temperature on the stability of sodium hypochlorite admixed with etidronate or EDTA for continuous chelation. Journal of Endodontics 45, 57-61. https://doi.org/10.1016/j.joen.2018.09.014 2. Wright PP, Cooper C, Kahler B, Walsh LJ (2020) From an assessment of multiple chelators clodronate shows potential for use in continuous chelation. International Endodontic Journal 53, 122-134. https://doi.org/10.1111/iej.13213 3. Wright PP, Scott S, Kahler B, Walsh LJ (2020) Organic tissue dissolution in clodronate and etidronate mixtures with sodium hypochlorite. Journal of Endodontics 46, 289-294. https://doi.org/10.1016/j.joen.2019.10.020 4. Wright PP, Kahler B, Walsh LJ (2020) The effect of temperature on the stability of sodium hypochlorite in a continuous chelation mixture containing the chelator clodronate. Australian Endodontic Journal 46, 244-8. https://doi.org/10.1111/aej.12399 5. Wright PP, Scott S, Shetty S, Kahler B, Walsh LJ. (2020) Resistance to compressive force in continuous chelation. Australian Endodontic Journal https://doi.org/10.1111/aej.12440 [Epub ahead of print].

Submitted manuscripts included in this thesis 1. Wright PP, Cooper C, Kahler B, Walsh LJ. The antibiofilm actions of sodium hypochlorite mixed with clodronate or etidronate using multiple biofilm assessment methodologies. Submitted to Microscopy Research and Technique on 12 May 2020.

Other publications during candidature 1. Wright PP, Walsh LJ (2017) Optimizing antimicrobial agents in endodontics. In: Kumavath R, ed. Antibacterial Agents, pp. 87-108. Rijeka, Croatia: InTech. https://doi.org/10.5772/67711 2. PP, Kahler B, Walsh LJ (2017) Alkaline sodium hypochlorite irrigant and its chemical interactions. Materials (Basel) 10: 1147 https://doi.org/10.3390/ma10101147

Poster Presentations 1. Wright PP, Kahler B, Walsh LJ. The effect of heating to intracanal temperature on the stability of sodium hypochlorite admixed with etidronate or EDTA for continuous chelation. 58th Annual Scientific Meeting of the IADR ANZ Division, Perth, Australia, September 2018. 2. Wright PP, Kahler B, Walsh LJ. The effect of temperature on the stability of sodium hypochlorite admixed with clodronate. 4th Meeting of the IADR Asia Pacific Region, Brisbane, Australia, November 2019.

Oral Presentations 1. Wright PP. The art and the science of continuous chelation, an alternative irrigation technique. Australian Society of Endodontology (Qld), October 18, 2019. 2. Wright PP, Kahler B, Walsh LJ. From an assessment of multiple chelators clodronate shows potential for use in continuous chelation. School of Dentistry Research Day, The University of Queensland, August 2019.

Contributions by others to the thesis There are no contributions by others except for those listed in the author contributions of the published and submitted manuscripts. Such contributions appear on the page immediately preceding the relevant thesis chapter.

Statement of parts of the thesis submitted to qualify for the award of another degree No works submitted towards another degree have been included in this thesis. Research Involving Human or Animal Subjects 1. Animal ethics approval for use of tissues from cows and pigs was obtained from The University of Queensland, Animal Ethics Unit, approval number ANRFA/DENT/523/17. 2. Approval for the use of extracted human teeth and plaque was obtained from The University of Queensland, Health and Behavioural Sciences, Low and Negligible Risk Ethics Sub-Committee, approval number, 2018001868. 3. Approval for the use of human teeth was obtained from the Metro South Human Research Ethics Committee, HREC reference number, HREC/17/QPAH/521. Clearance was obtained from The University of Queensland to use this ethics approval, clearance number 2017001377/HREC/QPAH/521. Site approval was obtained from the Royal Brisbane and Women’s Hospital Metro North Hospital and Health Service. 4. Approval for the use of saliva was obtained from the University of Queensland, Health and Behavioural Sciences, Low and Negligible Risk Ethics Sub-Committee, approval number, 2017001492.

Copies of the ethics approval letters are included in the thesis appendix.

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Acknowledgements Foremost, I would like to thank my supervisors, Professor Laurie Walsh and Honorary Associate Professor Bill Kahler. Without them this PhD thesis would not have been possible. Early on in my PhD, Laurie spent considerable time with me ploughing through the endodontic literature. It was this early foundation which engendered in me the importance of reading the literature. Laurie’s door was always open and despite being an incredibly busy and productive person, Laurie addressed concerns as they arose and put ideas on the table. He was also the generous funder of my research. This was a godsend as it ensured that products could be purchased when required. Bill was my moral support when times were tough. He always had time to listen and understand. His knowledge on publication procedures streamlined the submission procedure and gave us the best chance of getting papers into press. As a practicing endodontist, he was able to view research from a clinical perspective.

I would also like to show my appreciation to Suzanne Scott, Crystal Cooper and Sowmya Shetty, co-authors on some of my publications. Their expertise and assistance filled gaps in my own knowledge and experience. I am particularly grateful to Professor Ove Peters who generously spent considerable time with me, both in the lab and off site, to work on a project together. Although the result was disappointing, it was a chance to experience the inspiration, enthusiasm and encyclopaedic knowledge of the endodontic literature that is Ove Peters. I would also like to acknowledge Professor Christine Peters, who at my confirmation, asked if I was going to look at the effect of heat in continuous chelation. What a good idea that turned out to be!

It is important to recognize the spirit of camaraderie that exists in the lab. My fellow PhD students made the journey easier. Specifically, Nebu Philip went out his way to pass on knowledge and share techniques with me. We also enjoyed many a good laugh together. Nebu was the ‘gold standard’ in the lab, no one could possibly ever attain the level of output that he had. Andrea Kazoulis was motivational. Not only did she source many much needed items for communal lab use, but she was a terrific teacher to many and the go to person for many pieces of equipment in the lab, including the notorious autoclave. Thanks also to Srinivas Ramachandra. Sri and I often shared ideas, techniques and problems, not to mention popcorn in the late afternoon. We watched out for one another. Two fellow PhD students, Reuben Staples and Tulio Fernandez, never failed to brighten up my days. They

viii were pleasant, supportive and helpful. They had me in fits of laughter so often that it helped relieve the stress of being a PhD student.

My gratitude also goes to The School of Dentistry at The University of Queensland for providing the staff and facilities necessary to complete a PhD. The level 6 research laboratory at the Oral Health Centre at Herston is well equipped, spacious and modern. For me however, its most precious asset is the lab manager Nigel Bennett. Nigel is a font of practical knowledge and spontaneously gave me and many other lab users tips. The school also provided many other staff, including Associate Professor Ratilal Lalloo, the Postgraduate Co-ordinator, my candidature team of Professor Alex Moule, Dr Sobia Zafar and Dr Unni Krishnan Pillai, all of whom have worked on my behalf. Thank you also to Associate Professor Roy George from Griffith University for being the independent expert. I am indebted to Bruce Rex in the workshop for his assistance. Bruce machined mechanical components for me on several occasions. Thanks to Chris Sexton for taking time out from his busy schedule to discuss statistics and share his knowledge. I would also like to thank Alan White and Jasper Bowman from Griffith University for their willingness and collaboration in trying to help me to look at possible chemical by-products, if any, of my irrigation mixtures.

Finally to my long suffering family and friends, thank you for your understanding in helping me through a difficult period of my life. My cycling friends Karen and Kerryn not only kept me sane by enabling me to distance myself a little from my work but displayed incredible friendship by listening stoically to my regular renditions of the week’s disasters in the lab. To my dear friends Elaine and Jo, thanks for keeping in touch and making the effort without expectation of reciprocation. My children John, Suzanne and Nicole helped me in so many ways, from assistance with IT problems to an unfaltering faith in my ability to complete the work required for thesis completion. My husband Noel was full of practical advice. He not only assured me of the emotional nature of a PhD and advised me on the finer points on endnote, but his insistence that I just take it one day at a time paid off. The process was hard and painful, but the rewards were many.

Financial support The acquisition of materials for this research was primarily funded by Professor Laurence Walsh. Funding was also obtained from The School of Dentistry, The University of Queensland, both in the form of an annual grant and in the provision of laboratory facilities, staff and infrastructure. I also acknowledge that this research was supported by an Australian Government Research Training Program Scholarship.

Keywords Clodronate, continuous chelation, etidronate, disinfection, smear layer, sodium hypochlorite, dissolution. Australian and New Zealand Standard Research Classifications (ANZSRC)

ANZSRC code 110503, Endodontics 50% ANZSRC code 110502, Dental Therapeutics, Pharmacology and Toxicology, 50%

Fields of Research (FoR) Classification

FoR code 1105, Dentistry 100%

Dedication

To Noel, John, Suzanne and Nicole For their love assistance and understanding

Table of Contents

Abstract ...... i

Declaration by author ...... iv

List of Figures ...... xvi

List of Tables ...... xvi

List of Abbreviations ...... xviii

Preface ...... 1

...... 3

Requirements of an ideal endodontic irrigant ...... 4

Biofilms, antimicrobial irrigants and assessment methodologies ...... 6

1.2.1 Biofilms ...... 6

1.2.2 The root canal biofilm ...... 6

1.2.3 Antimicrobial aspects of endodontic irrigants...... 11

1.2.4 Assessment models for endodontic disinfection ...... 15

Dentine structure, smear layer, chelating irrigants and methodologies ...... 26

1.3.1 The Smear Layer ...... 27

1.3.2 Factors influencing chelation in dentine ...... 27

1.3.3 Common chelators in endodontics for smear layer removal ...... 28

1.3.4 Methodologies for assessing smear layer removal ...... 31

Root canal organic tissue ...... 33

1.4.1 Structure of the dental pulp ...... 33

1.4.2 Tissue dissolution capacity of NaOCl ...... 33

1.4.3 Methodologies for assessing organic tissue dissolution ...... 35

Effect of irrigation on dentine structure, function and mechanical properties ...... 36

1.5.1 Dentine structure and related functionality ...... 36

1.5.2 Methodologies for assessing dentine structure and strength ...... 37

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1.5.3 Effects of NaOCl, chelators and irrigation sequences ...... 38

Alternative irrigants, sequences and strategies for irrigation enhancement ...... 42

1.6.1 CHX ...... 43

1.6.2 MTAD and Tetraclean ...... 44

1.6.3 Peracetic acid ...... 45

1.6.4 Photoactivated disinfection (PAD) and other strategies for improving irrigation …… ...... 45

1.6.5 Conclusion to Section 1.6 ...... 46

Continuous chelation in endodontic irrigation ...... 47

1.7.1 Maintenance of FAC ...... 47

1.7.2 Removal of dentine debris and smear layer ...... 52

1.7.3 Organic tissue dissolution ...... 53

1.7.4 Antimicrobial activity ...... 55

1.7.5 Detrimental effects on dentine ...... 59

1.7.6 Safety, bond strength ...... 60

Gaps in the continuous chelation literature ...... 61

Thesis aims ...... 63

Thesis objectives ...... 63

...... 65

Abstract ...... 66

Introduction ...... 67

Materials and Methods ...... 69

Results ...... 70

Discussion ...... 73

Conclusion ...... 75

...... 76

Paper 1 ...... 77 xiii

3.1.1 Abstract ...... 77

3.1.2 Introduction ...... 78

3.1.3 Materials and Methods ...... 81

3.1.4 Results ...... 85

3.1.5 Discussion ...... 93

3.1.6 Conclusion ...... 96

Paper 2 ...... 97

3.2.1 Abstract ...... 97

3.2.2 Introduction ...... 98

3.2.3 Materials and Methods ...... 99

3.2.4 Results ...... 101

3.2.5 Discussion ...... 103

3.2.6 Conclusion ...... 105

...... 106

Abstract ...... 107

Introduction ...... 108

Materials and Methods ...... 109

Results ...... 111

Discussion ...... 114

Conclusion ...... 116

...... 117

Abstract ...... 118

Introduction ...... 119

Materials and methods ...... 120

Results ...... 125

Discussion ...... 130

Conclusion ...... 133 xiv

...... 134

Abstract ...... 135

Introduction ...... 136

Materials and Methods ...... 137

Results ...... 140

Discussion ...... 141

Conclusion ...... 144

7 General Discussion ...... 145

7.1 Experimental outcomes and methodological considerations ...... 147

7.1.1 Free available chlorine and NaOCl compatibility ...... 147

7.1.2 Smear layer removal ...... 149

7.1.3 Organic tissue dissolution ...... 150

7.1.4 Antibiofilm actions ...... 151

7.1.5 Compressive strength testing ...... 152

7.2 Directions for future research ...... 153

7.3 Concluding remarks ...... 154

8 Bibliography ...... 156

9 Appendix ...... 188

9.1 Ethics, site and biosafety approvals ...... 189

9.2 Certificates of analysis ...... 202

9.3 Experimental protocols ...... 212

9.3.1 Protocol 1, pH measurement of chelator-NaOCl mixtures ...... 212

9.3.2 Protocol 2, Determination of FAC with iodometric titration ...... 214

9.3.3 Protocol 3, Smear layer assessment by SEM ...... 217

9.3.4 Protocol 4, Porcine mucosa dissolution ...... 221

9.3.5 Protocol 5, Antimicrobial studies ...... 227

9.3.6 Protocol 6, Fracture resistance ...... 233 xv

9.3.7 Protocol 7, Disinfection of oral biofilm on acrylic tooth root model ...... 238

9.4 Raw data ...... 243

9.4.1 Data from Chapter 2 ...... 243

9.4.2 Data from Chapter 3 ...... 250

9.4.3 Data from Chapter 4 ...... 275

9.4.4 Data from Chapter 5 ...... 278

9.4.5 Data from Chapter 6 ...... 287

List of Figures

Figure 1-1. States of NaOCl at various pH levels ...... 12 Figure 2-1. The effect of temperature on FAC ...... 72 Figure 2-2. Changes in FAC and pH ...... 73 Figure 3-1. pH values for chelator-NaOCl mixtures ...... 87 Figure 3-2. Temperature values for chelator-NaOCl mixtures ...... 88 Figure 3-3. FAC values for chelator-NaOCl mixtures ...... 89 Figure 3-4. Area fraction of open dentinal tubules as a percentage of total area (% AF) 91 Figure 3-5. SEM images of smear layer removal and Image J output file…………………92 Figure 4-1. Percentage change in FAC and weight for plain chelators and mixtures ...... 112 Figure 4-2. Percentage change in weight of clodronate-NaOCl 6 hours from mixing ...... 113 Figure 5-1. Colony forming units (CFU) following biofilm disinfection ...... 126 Figure 5-2. Scanning electron micrographs of control and treated HA discs ...... 128

Figure 5-3. A492 data from the XTT assay ...... 129 Figure 6-1. Sample preparation and load testing ...... 140 Figure 6-2. Load at fracture in Newtons (N) ...... 141 Figure 7-1. Research gaps in continuous chelation, thesis aims and outcomes……….. 146

List of Tables

Table 1-1. Requirements of an ideal irrigant…………………………………………………....5 Table 1-2. Abundance of bacteria in primary endodontic infections.…………………………8

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Table 1-3. Abundance of bacteria in secondary endodontic infections ..………………….. .9 Table 1-4. Summary of EDTA requirements for smear layer removal……………………..30 Table 1-5. Studies in continuous chelation with FAC data from 2005-2018……………… 51 Table 2-1. Changes over time in FAC with solution temperature and pH………………….71 Table 3-1. Chelator common names and Ca2+ stability constants………………………….80 Table 3-2. Changes over 3 hours in pH and temperature at 23 and 34-35oC……………102 Table 3.3. FAC at 2-4oC over 3 months………………………………………………………103

Table 7-1. Chelator structures. Na2 Clodronate, Na4 EDTA and Na4 Etidronate……….. 148

List of Abbreviations

% AF Percent area fraction (of open dentine tubules)

A492 Absorbance at 492 nm

AAA Amsterdam Active Attachment (plate/model)

AO Acridine orange

AMP Antimicrobial peptide

ATC Automated temperature control

ATR-FTIR Attenuated total reflectance - Fourier transform infrared spectroscopy

ANOVA Analysis of variance

ATMP Aminotris(methylene phosphonic acid)

BHI Brain heart infusion

Ca2+ Calcium ion

xvii

CBD Calgary biofilm device

CDTA Cyclohexanediaminetetraacetic acid

CFU Colony forming unit

Cl− Chloride ion

Cl2 Chlorine molecule

CHX Chlorhexidine

CLSM Confocal laser scanning microscopy

EDS Energy dispersive X-ray spectroscopy

EDTA Ethylenediaminetetraacetic acid

EGTA Egtazic acid

EPS Extracellular polymeric substance

ESEM Environmental scanning electron microscopy

EtOH Ethyl alcohol (ethanol)

FAC Free available chlorine

FISH Fluorescence in situ hybridization

FTIR Fourier transform infrared spectroscopy

HA Hydroxyapatite

xviii

HOCl Hypochlorous acid

I− Iodide ion

I2 Iodine

− IO3 Iodate ion

M Moles per litre

Na2 EDTA Disodium EDTA

Na3 EDTA Trisodium EDTA

Na4 EDTA Tetrasodium EDTA

Na4 etidronate Tetrasodium etidronate

NaOCl Sodium hypochlorite

NaOH Sodium hydroxide

OD600 Optical density at 600 nm

OCl− Hypochlorite ion

OH− Hydroxide ion

PAA Peracetic acid

PAD Photoactivated disinfection

PBS Phosphate-buffered saline

xix

3− PO4 Phosphate ion qPCR Quantitative polymerase chain reaction rpm Revolutions per minute

2− S203 Thiosulphate ion

SD Standard deviation

SEM Scanning electron microscopy

TEM Transmission electron microscopy

VBNC Viable but nonculturable

W Watt, unit of power

Wt/v Weight/volume

XTT 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]- 2H-tetrazolium hydroxide

Preface

The body of work presented in this thesis aims to broadly demonstrate that the chelator clodronate admixed with sodium hypochlorite (NaOCl) can function in some of the various roles of an endodontic irrigant. In such demonstrations, it additionally seeks to compare this novel mixture with the only available product for use in continuous chelation, etidronate combined with NaOCl, as well as with NaOCl or standard endodontic sequences. The thesis begins with a literature review which mirrors these aims. The review, which comprises the bulk of Chapter 1, first details the roles of an endodontic irrigant. The following sections expand on specific irrigant requirements, all of which have experimental counterparts in following chapters. Those sections concerning irrigant roles all begin with a description of the environment associated with them. For example, the section on the antimicrobial function of irrigation starts with a description of biofilms. This is followed by details of some of the irrigants associated with that particular role and with the methodologies used for assessing irrigant effectiveness in this area. The review next considers some common irrigation sequences or combinations and identifies associated problems. Finally, the review turns to continuous chelation, detailing the literature in the field, for both issues specific to continuous chelation and from the viewpoint of the functional requirements of irrigation. The review identifies knowledge gaps and problematic areas, leading to the thesis aims and hypotheses. The first experimental chapter, Chapter 2, examines the effect of heating known continuous chelation mixtures to root canal temperature. Changes in NaOCl stability, pH and temperature are measured. Chapter 3 reports on the results from screening a number of chelator mixtures with NaOCl by measuring, at room temperature, the maintenance of NaOCl concentration via assessments of free available chlorine (FAC), pH and temperature. This process identifies that the clodronate-NaOCl mixture maintains FAC over many hours. Thus, the mixture is likely to maintain the core irrigant functions of tissue dissolution and antimicrobial action. Its potential for use then hinges on the ability to remove smear layer. Chapter 3 evaluates this aspect together with the effect that temperature changes have on the clodronate-NaOCl combination. These experiments firmly establish that clodronate is compatible with NaOCl and possibly suitable for use in continuous chelation. Chapters 4 and 5 respectively deal with organic tissue dissolution and antimicrobial activity. Chapter 6 addresses concerns over the possible deleterious effects to tooth structure that prolonged irrigation with a mixture containing both NaOCl and a chelator may 1 have. It does this by comparing the resistance to compressive force following irrigation of teeth with clodronate or etidronate mixtures to a standard endodontic sequence. The final thesis sections are the discussion, bibliography and appendix. The bibliography presents the references as a collated whole and uses a style adapted from The International Endodontic Journal. The thesis concludes with the appendix which lists ethics approvals, certificates of analysis for the chelators, protocols and raw data. The thesis type is “thesis with publications” and journal articles have been included. These texts have been revised to include examiner recommendations and requirements, and they have been formatted to allow for consistency of presentation. Hyperlinks have been provided to the published texts. Because each journal article was a standalone publication, and because the concept of continuous chelation is new to many readers, the opening parts of the introduction for these experimental papers contain similar explanatory material. While this was necessary for the purpose of publication, it has resulted in some repetition at the beginning of chapters.

Literature review

Requirements of an ideal endodontic irrigant

The fundamental role that bacteria play in apical periodontitis was established in a seminal experiment in 1965 when it was demonstrated that germ free rats did not develop apical periodontitis, even when exposed dental pulps were left open to the oral environment (Kakehashi et al.1965). Similarly, experiments in dogs showed that when necrotic pulps were not infected, no periapical inflammation was present (Lin et al. 2006). Bacterial biofilms located predominantly within the root canal, but also in extraradicular sites, are inevitably associated with root canal infections (Ricucci & Siqueira 2010). This is not to deny that secondary factors such as traumatic injury (Clark & Levin 2019) or the host immune response (Kreth & Herzberg 2015) can play a contributing role. Additionally, the outcome of endodontic treatment and the resolution of apical periodontitis is influenced by several factors. These include the absence of periapical pathology at the time of treatment, and the adequacy of both the coronal restoration and the root canal filling (Ng et al. 2011). Accordingly, the primary goals of root canal treatment are biofilm elimination and prevention of recurrence of infection (Gulabivala & Ng 2015). While mechanical instrumentation removes a significant volume of the microbial load (Byström & Sundqvist 1981), it cannot completely debride the root canal system. This is due to the incomplete contact of instrumentation with the canal walls (Peters et al. 2001). Other steps, including irrigation and the use of intracanal medicaments are needed to compliment the mechanical preparation of the root canal (Johnson & Flake 2014). Because bacteria are the cause of root canal infections, the only essential function associated with endodontic irrigation is the removal of biofilm. Thus, the disinfection process would ideally totally eliminate all biofilms both from within the main canal and in anatomically inaccessible sites. The removal of smear layer is another important irrigant function, and the chelator EDTA (ethylenediaminetetraacetic acid) is commonly used in endodontic treatment for this purpose (Zehnder 2006). There is some evidence to support the view that the removal of smear layer in secondary endodontic infections (Ng et al. 2011) and in the deciduous dentition (Barcelos et al. 2012) assists in positive treatment outcomes. However, smear layer removal during the treatment of primary endodontic infections in permanent teeth has not been associated with clinical success. Rather, the use of EDTA or other chelators to remove the smear layer is advocated for other reasons. These include the potential for smear layer to act as a nutritional source for microbial growth, to assist the penetration of endodontic sealers into the dentine tubules, or to facilitate the antimicrobial

4 action of irrigants (Violich & Chandler 2010). Indeed, in vitro research has shown that the antibacterial effectiveness of irrigation is diminished in the presence of smear layer (Wang et al. 2013). Finally, the dissolution of organic matter plays a significant role in endodontic irrigation (Zehnder 2006). Thus, the three main functions of an endodontic irrigant are to disinfect the root canal system, to remove smear layer, and to dissolve organic material. However, the properties of an ideal irrigant extend beyond the effectiveness of these three core irrigant functions. Biofilm-associated endotoxins require inactivation (Torabinejad 2011). Additionally, tooth or root canal factors, safety and cost all require consideration. Irrigants must not stain or weaken teeth (Basrani & Haapasalo 2014). They should leave dentine in a condition compatible with the usage of endodontic sealants (Vilanova et al. 2012) and restorative materials (Torabinejad 2011). Irrigants need to facilitate the chemomechanical preparation of the canal by providing lubrication, instrument and canal cooling, and by flushing out dentine and soft tissue debris (Haapasalo et al. 2014). With respect to safety, irrigants need to be well tolerated by the periapical tissues (Cunha & da Silveira Bueno 2015), non-toxic and non-allergenic (Zehnder 2006). Finally, from a clinician’s perspective, irrigants should be cost effective. This is achieved by a low initial cost, rapid action and good stability over time (Wright & Walsh 2017a). Table 1-1 summarizes these attributes. It is noteworthy that no ideal endodontic irrigant currently exists (Haapasalo et al. 2010).

Table 1-1. Requirements of an ideal irrigant Effectiveness in core functions: Broad spectrum antimicrobial action, including biofilm disinfection and dissolution in areas of complex canal anatomy, removal of smear layer and dissolution of vital and necrotic pulp.

Tooth factors: Non-staining, maintenance of tooth mechanical properties, compatibility with dental materials, lubrication and cooling during instrumentation, flushing of debris.

Safety: Non-toxic, non-allergenic, non-irritant to periapical tissues, inactivation of endotoxins.

Cost: Low purchase price, rapid action, stable in storage. Adapted from Wright & Walsh (2017a).

In assessing how endodontic irrigation fulfils the first of these key roles, antimicrobial action, it is necessary to examine root canal infections and how antimicrobial treatment is assessed.

Biofilms, antimicrobial irrigants and assessment methodologies

1.2.1 Biofilms

Biofilms consist of closely associated microbial communities bound together in a polyanionic matrix called the extracellular polymeric substance (EPS) (Kreth & Herzberg 2015). This consists largely of polysaccharides, but includes other components such as proteins and extracellular DNA (Azeredo et al. 2017). The EPS contains a considerable amount of water in the form of water channels which function in the exchange of nutrients and waste (Sedgley & Dunny 2015). The EPS also acts as a scaffold for microbial growth (Kreth & Herzberg 2015). The biofilm structure imparts several advantages to the microbial flora. These include protection from shear stress (van der Sluis et al. 2014), increased resistance to antimicrobial agents (Neelakantan et al. 2017), and the ability to survive during periods of nutrient depletion (Svensäter & Bergenholtz 2004). The latter is due to the ability of biofilm bacteria to enter into a dormant state, accompanied by a slowed metabolic rate (Chávez de Paz & Marsh 2015). Additional survival mechanisms for biofilm microbes include the transmission of antibiotic resistance due to gene transfer (Sedgley et al. 2008), bacterial communication via the production of signalling molecules (quorum sensing) (Kreth & Herzberg 2015), and the presence of persister cells (Shen et al. 2016).

1.2.2 The root canal biofilm

The first identification of biofilms in the root canal is attributed to Nair (Siqueira et al. 2015), who in 1987, described the aggregation of several bacterial species into organized clusters (Nair 1987). Since then, others have firmly established apical periodontitis as a biofilm disease with the vast majority of endodontic biofilms occurring in intraradicular areas (Ricucci & Siqueira 2010). Root canal anatomy is complex, particularly in molars, where deltas, isthmuses and ramifications abound, acting as a reservoir for debris accumulation (Endal et al. 2011; Paqué et al. 2011). Importantly from a disinfection viewpoint, biofilms are

6 located in these inaccessible sites, particularly in isthmuses, and lateral canals (Ricucci & Siqueira 2010). Biofilms located in dentinal tubules are especially difficult to eradicate (Berutti et al. 1997; Matsuo et al. 2003).

1.2.2.1 Bacterial flora

The understanding of the composition of endodontic biofilms has developed with the advent of new molecular biology techniques such as pyrosequencing, polymerase chain reaction and fluorescence in situ hybridization (FISH) which are all described in more detail in the “Molecular techniques” subsection of Section 1.2.4.2. Just as the introduction of anaerobic culturing vastly expanded our knowledge of the root canal microbiome (Sundqvist 1994), so the evolution of molecular methodology, to include techniques such as the polymerase chain reaction and next generation pyrosequencing, has paved the way for further understanding of endodontic biofilms (Siqueira & Rôças 2014). Although next generation pyrosequencing has its own limitations, such as the inclusion in the analysis of non-biofilm bacteria that may be present in the sampling (Siqueira et al. 2015), it has allowed for the rapid identification of many fastidious, microorganisms that are difficult to culture (Keijser et al. 2008). Pyrosequencing has challenged the view that persistent endodontic infections are less diverse than primary ones (Hong et al. 2013; Tzanetakis et al. 2015). Additionally, pyrosequencing has shown that some endodontic bacteria are much less abundant than previously thought. For example, Enterococcus faecalis, identified from culturing techniques, was considered to be the most common microorganism in persistent endodontic infections (Pinheiro et al. 2003). However, according to pyrosequencing studies, the abundance of the genus Enterococcus in such infections is only 0.7% (Hong et al. 2013), 1.3% (Tzanetakis et al. 2015) or 5.1% (Keskin et al. 2017). FISH has confirmed the low abundance of E. faecalis in secondary endodontic infections (Zehnder et al. 2017). Tables 1-2 and 1-3 collate three pyrosequencing studies of the most abundant bacterial species in respectively, primary, and secondary endodontic infections (Hong et al. 2013; Tzanetakis et al. 2015; Keskin et al. 2017). There are many similarities between these studies as noted in the following discussion. The root canal bacterial flora is dominated by Gram-negative anaerobes. The genera Prevotella, Pyramidobacter, Porphyromonas and Fusobacterium are common in primary infections. In secondary infections, Fusobacterium is the most commonly encountered genus. Prevotella and Porphyromonas are also

7 abundant. The Gram-positive anaerobe Parvimonas and to a lesser extent, the Gram- positive, mostly anaerobic genus Streptococcus, are also prevalent in secondary apical periodontitis. An important common feature of Tables 1-2 and 1-3 is the increase in abundance of Fusobacterium, and to a lesser extent Porphyromonas, in secondary compared to primary infections. This agrees with another pyrosequencing study, examining only apical areas, which found that Fusobacterium and Porphyromonas were more prevalent in secondary compared to primary infections. It was hypothesised in that study that, these, rather than E. faecalis, are the main bacteria that resist chemomechanical preparation and which proliferate after treatment (Chugal et al. 2011). This latter study also highlighted that the bacterial flora in the root canal apex consists predominantly of Gram-negative anaerobes. In the Hong, Tzanetakis and Keskin analyses presented in Tables 1-2 and 1-3, whole teeth, rather than just apical areas, were used, and aerobic or facultative anaerobic genera such as Streptococcus and microaerophilic/facultative genera like Lactobacillus were also well represented. This difference between the two studies can be explained by site specificity due to a higher abundance of anaerobic bacteria in the apical compared to coronal regions of the root canal. (Özok et al. 2012). Table 1-2. Abundance of bacteria in primary endodontic infections (Hong et al. 2013) (Tzanetakis et al. 2015) (Keskin et al. 2017)

Genera/other (%) Genera/other (%) Genera/other (%) taxonomy taxonomy taxonomy Proprionibacterium 16.6 Bacteroidaceae_G1 15.1 Prevotella 23.5 Pyramidobacter 13.4 Pyramidobacter 10.1 Porphyromonas 16.8 Prevotella 9.0 Parvimonas 9.1 Neisseria 14 Fusobacterium 6.7 Prevotella 8.7 Fusobacterium 12.6 Streptococcus 5.7 Actinobacteria 8.6 Parvimonas 12.1 Bacteroidetes[G-1] 5.3 Atopobium 6.8 Lactobacillus 9.6 Porphyromonas 5.0 Eubacterium_XIG6 5.1 Streptococcus 9.4 Neisseria 4.8 Pseudoramibacter 4.5 Campylobacter 2.1 Atopobium 1.9 Porphyromonas 4.3 Enterococcus 2.0 Capnocytophagea 1.9 Fusobacterium 3.6 Actinomyces 1.1 Afipia 1.4 Dialister 2.8 Synergistes 1.1 TM7[G-1] 1.3 Bacteroides 2.6 Granulicatella 0.9

Table 1-3. Abundance of bacteria in secondary endodontic infections (Hong et al. 2013) (Tzanetakis et al. 2015) (Keskin et al. 2017)

Genera/other (%) Genera/other (%) Genera/other (%) taxonomy taxonomy taxonomy Fusobacterium 14.6 Fusobacterium 11.0 Fusobacterium 18.4 Porphyromonas 10.6 Bacteroidaceae_G1 10.1 Porphyromonas 16.4 Prevotella 8.6 Prevotella 9.4 Prevotella 15.7 Peptostreptococcus 5.4 Actinobacteria 7.6 Lactobacillus 13.8 Dialster 4.9 Parvimonas 7.2 Neisseria 12.4 Tannerella 4.8 Tannerella 6.2 Streptococcus 12.0 Bacteroidetes[G-1] 4.2 Porphyromonas 5.2 Parvimonas 10.2 Streptococcus 4.0 Atopobium 5.2 Enterococcus 5.1 Bacilius 2.6 Lactobacillus 5.2 Rothia 1.2 Treponema 2.2 Streptococcus 3.7 Granulicatella 1.1 Solobacterium 1.5 Bacteroidetes_G3 3.6 Selenomonas 0.8 Actinomyces 1.5 Treponema 2.5 Synergistes 0.8

The differences in these pyrosequencing studies, in the order, abundance and genera of bacteria, can be attributed to several methodological factors. An important difference between the studies is that one used cryogenically ground whole teeth (Keskin et al 2017) while the other two employed sampling of the canals using paper points. The use of paper points may fail to accurately represent the bacteria present in the canals as described in the “Substrates” subsection of Section 1.2.4.1. Research in the field has alluded to variations in sample size and DNA extraction techniques as likely reasons for study differences (Özok et al. 2012). Sample numbers did vary between studies with a minimum of 18 (Hong et al. 2103) to a maximum of 48 (Tzanetakis et al. 2015). The DNA extractions kits all varied, and importantly, one study (Keskin et al. 2017) included a more complex lysis buffer. Finally, the primers and analysis software used were unique to each study. Bacteria located in dentine tubules Bacteria are present in the tubules in 70% of endodontic infections and even with root canal instrumentation and disinfection, post treatment, 65% of tubules still remain infected (Matsuo et al. 2003). An immunohistological analysis, involving the identification of bacteria by

9 antibody staining, revealed that that the obligate anaerobes Fusobacterium, Eubacterium and Parvimonas (formerly named Peptostreptococcus micros) and the facultative microbe Lactobacillus are frequently encountered (Matsuo et al. 2003). Streptococci are also commonly found in dentinal tubules (Love et al. 2002).

1.2.2.2 Nonbacterial microbes

According to a recent pyrosequencing study, fungi are found in over 50% of endodontic biofilms from primary infections (Persoon et al. 2017). This agrees with early studies using scanning electron microscopy (SEM) which showed that yeasts are commonly present on the root canal walls (Sen et al. 1995). However, the diversity in fungal infections is limited. Candida is the most abundant genera, but Malassezia is also present (Persoon et al. 2017). The presence of fungi in the root canal positively relates to increased numbers of acidophilic bacterial species (Persoon et al. 2017). Archaea are also associated with apical periodontitis (Siqueira & Rôças 2014).

1.2.2.3 Non-microbial features and current state of knowledge

Similar to biofilms in other locations, in root canal biofilms, microbes are embedded in an amorphous matrix (Schaudinn et al. 2009). The amount of EPS in endodontic biofilms is highly variable. Some biofilms are cell dense, while others are matrix dense (Ricucci & Siqueira 2010). EPS production is species dependant (Siqueira & Rôças 2014), and in view of the extensive bacterial diversity present in apical periodontitis, this variation is not surprising. However, the literature appears devoid of specific detail of the EPS present in actual endodontic infections. Such details would include the type of sugars or glycoside linkages present. Equally, descriptions of other biofilm features, for example, water channels, seem not to have been elucidated for endodontic biofilms. Since endodontic biofilms do not form under shear force from fluid movement, such features may differ from classic biofilms such as supragingival dental plaque. Furthermore, the actual conditions under which biofilms form and propagate in root canals are poorly understood (Svensäter & Bergenholtz 2004). Thus, it appears that considerable research is required to fully elucidate biofilm structures in endodontic infections.

1.2.3 Antimicrobial aspects of endodontic irrigants.

1.2.3.1 NaOCl

It was Alfred Walker who, in 1936, first used NaOCl solutions for treating infected root canals (Walker 1936). NaOCl is now the most popular endodontic irrigant (Zehnder 2006), largely due to its unique and dual capacity for disinfection and tissue dissolution (Haapasalo et al. 2014). From a practical perspective, NaOCl loses stability over time and with temperature increases. Attention must therefore be paid to storage conditions, keeping it in the cool and away from light (Schäfer 2007). An early in vivo study showed no differences in the antimicrobial effectiveness of high and low concentration NaOCl (Byström & Sundqvist 1985). However, the use of paper point canal sampling, a technique that may lack accuracy as described in Section 1.2.4.1 on page 18, irrigation with NaOCl at pH 9 as opposed to current solutions at pH 12, together with a lack of stipulation of irrigation times and volumes, mean this result needs verification. While in vivo studies are more clinically relevant, several in vitro studies have conversely shown that the antimicrobial action of NaOCl increases with concentration (Clegg et al. 2006; Retamozo et al. 2010; Wang et al. 2013). However, it should be borne in mind that tissue toxicity rises as concentration increases. (Shen et al. 2015). NaOCl is also a potent antifungal agent (Radcliffe et al. 2004). Its in vivo effectiveness against a wide range of aerobic and anaerobic bacteria (Ballal et al. 2019b) attests to broad spectrum anti-biofilm actions. This is also borne out by in vitro experiments with polymicrobial biofilms derived from isolates from root canal infections where the use of 2.5.% NaOCl resulted in superior antimicrobial effectiveness compared to other irrigants including 2% chlorhexidine (CHX) and 2% alexidine (Ruiz‐Linares et al. 2017). However, it is noteworthy that in the same experiment, 2.5 % NaOCl was capable of neither total bacterial killing nor complete biofilm eradication, a result which can be explained by the dentine model used. Bacteria located in dentine tubules are difficult to eradicate, as mentioned, and the staining technique used was able to identify them. The ability of NaOCl to kill microorganisms is related to pH. The percentage of live bacteria remaining in biofilms is lower at pH 5 compared to pH 12, but at pH 12 more of the biofilm is removed (del Carpio-Perochena et al. 2015a). This is explained by the fact that NaOCl exists as various states dependent upon pH, as seen in Figure 1-1. Below pH 2, chlorine gas (Cl2) prevails, while at pH 3-6 hypochlorous acid (HOCl) predominates and at

11 pH levels over 9 the hypochlorite ion ( OCl−) is mostly present. HOCl is more antimicrobial in action than OCl−; however, it is OCl− that is responsible for tissue dissolution (Fukuzaki 2006). This rationalizes why NaOCl at pH 5 kills more bacteria and at pH 12 more of the biofilm is removed. However, NaOCl solutions used in endodontics are alkaline, ranging from pH 11.7 for a 1% solution to pH 12.4-12.5 for a 5% solution (Biel et al. 2017), although variations may occur (Clarkson et al. 2006). Thus, NaOCl is present as OCl−. This explains a salient feature of NaOCl disinfection. The antimicrobial mechanism of NaOCl is dual and unique, both dissolving biofilms and killing the bacteria within them (Tawakoli et al. 2017; Busanello et al. 2019). While OCl−, due to its negative charge, cannot enter bacterial cells as the more antimicrobial HOCl does, it is nonetheless an oxidizing agent and it can still exert an antimicrobial effect on the exterior of bacterial cells (Fukuzaki 2006). The OCl− species is also responsible for tissue dissolution (Fukuzaki 2006), and thus it will physically remove the organic components of biofilms. Furthermore, NaOCl breaks glycosidic bonds, and reduces biofilm EPS (Tawakoli et al. 2017), thus potentiating its ability to dissolve biofilm. The antimicrobial effect of NaOCl is weakened in the presence of organic matter and dentine (Haapasalo et al. 2000; Morgental et al. 2013). This is related to the proteolytic ability of NaOCl, and this aspect will be discussed later in Section 1.4.2, on pages 33-34. Suffice to say here that high bacterial loads, reduce the effectiveness of NaOCl (Fukuzaki 2006).

Figure 1-1. States of NaOCl at various pH levels

− Chlorine gas (Cl2), hypochlorous acid (HOCl) and the hypochlorite anion ( OCl), also known as OCl−, may be present. Note the predominance of HOCl at pH 3-6 and OCl− above pH 9. HCl, hydrochloric acid. Curves A and B result from, respectively, the absence or presence of additional 100 mM sodium chloride. Adapted from (Fukuzaki 2006). 12

1.2.3.2 CHX

CHX is a bisbiguanide cationic antimicrobial agent (Russell & Day 1993). Its positive charge explains its mode of action, facilitating its binding to negatively charged bacterial cell membranes (Basrani & Haapasalo 2014). CHX can bind to hydroxyapatite, resuting in substantivity (Rölla et al. 1971), an attribute that is important to its antimicrobial actions (Basrani & Haapasalo 2014). An issue concerning the use of CHX is its reaction with NaOCl is that of precipitate formation, and this aspect is discussed in more detail in Section 1.6.1, on page.44. CHX has been used in endodontics as a final flush (Haapasalo et al. 2014). It has also been employed in retreatment cases (Schäfer 2007). Although many describe the action of CHX as broad spectrum (Glassman 2011; Chiniforush et al. 2015; Plotino et al. 2016), others highlight that it is not as effective against Gram-negative bacteria as it is against Gram-positive bacteria (Russell & Day 1993; Zehnder 2006; Schäfer 2007). Specifically, although CHX possesses antimicrobial actions against Gram-negative Prevotella and Porphyromonas species (Vianna et al. 2004), its overuse, for example as a hand wash, has led to resistance in many other Gram-negative species (Kampf 2016). Additionally, CHX has difficulty penetrating the cell wall of Gram-negative bacteria, and the concentration of CHX needed to kill them is considerably more than for Gram- positive microbes (Russell & Day 1993). This has obvious clinical repercussions because of the predominance of Gram-negative bacteria in apical periodontitis. From a research perspective, endodontic infection modelling either has used or still uses the Gram-positive organism E. faecalis in CHX studies (Portenier et al. 2006; Retamozo et al. 2010; Bukhary & Balto 2017; da Silva et al. 2018). Thus, there is potential to overestimate the effectiveness of CHX. Like NaOCl, the antimicrobial action of CHX is negatively impacted by the presence of dentine (Portenier et al. 2001). An important difference between the antimicrobial actions of CHX and that of NaOCl is the inability of CHX to dissolve biofilms (Tawakoli et al. 2017; Busanello et al. 2019). CHX tends to rearrange the biofilm rather than remove it (Busanello et al. 2019). A further point of variance between NaOCl and CHX is their differing efficacy against biofilms of divergent types. The antimicrobial actions of CHX are most effective on cell rich biofilms, while NaOCl acts preferentially on biofilms with a large EPS content (Busanello et al. 2019).

When used in concentrations from 0.2-2.0%, CHX is considered safe for endodontic use (Schäfer 2007), with less negative impact than NaOCl on the periapical tissues (Haapasalo et al. 2010; Glassman 2011). However, allergic reactions to CHX can occur. These consist mainly of redness, itching and urticaria, although they can potentially progress to anaphylactic reactions (Nagendran et al. 2009).

1.2.3.3 Other endodontic antimicrobial agents

Apart from NaOCl, CHX and the irrigant BioPure™ MTAD, discussed in Section 1.6.2, the use of other antimicrobial agents in endodontic irrigation is rare. In a 2011 survey with a response rate of 28.5% of 3707 endodontist members of the American Association of Endodontists, the use of no other antimicrobial agent was recorded (Dutner et al. 2012). However, historically, the irrigants hydrogen peroxide and iodine potassium iodide have played a part in endodontic irrigation, and it is possible that novel ways to use them may be identified. Hydrogen peroxide will not dissolve biofilm (Tawakoli et al. 2017). Rather, its antimicrobial action derives from the fact that it is a small molecule, similar in size to water, and with no electrical charge (Fukuzaki 2006). In this respect, the action of hydrogen peroxide is like the action of HOCl mentioned in Section 1.2.3.1. These characteristics permit it to diffuse through bacterial cell membranes where oxidation reactions, including the Fenton reaction, cause bacterial destruction (Fukuzaki 2006). In the Fenton reaction, hydrogen peroxide oxidizes a ferrous ion to form a hydroxy radical, a hydroxyl ion and a ferric ion. The hydroxy radical is a reactive oxygen species and it is highly antimicrobial (Fukuzaki 2006). The Iodine potassium iodide is rapidly bactericidal and fungicidal owing to its ability to penetrate microorganisms (Haapasalo et al. 2005). It is also another antimicrobial irrigant which is adversely affected by the presence of dentine (Portenier et al. 2001). Looking to the future, recent additions to the antibacterial armamentarium include nanoparticles and antimicrobial peptides (AMPs). Nanoparticles, due to their small size and large surface area, possess increased chemical reactivity and their nanometre dimensions may allow them to better kill microbes in narrow spaces such as dentinal tubules (Kishen & Shrestha 2015). Chitosan nanoparticles can disrupt biofilm EPS (Kishen & Shrestha 2015), an effect which has been exploited by their incorporation into endodontic sealers (del Carpio- Perochena et al. 2015b).

AMPs can be naturally occurring enzymes such as trypsin (Niazi et al. 2014), or manufactured peptides that have been specifically designed to include cationic amino acids, such as lysine and arginine, or non-naturally occurring amino acids, to help evade degradation via host proteolysis (Winfred et al. 2014). Their intent is to disrupt negatively charged biofilm EPS. Although AMPs can be potentially deactivated by pathogenic proteases (Pathirana et al. 2006), they may have use in endodontic disinfection when combined with other irrigants (Tong et al. 2011; Wang D et al. 2017).

1.2.4 Assessment models for endodontic disinfection

There is no model that that is widely accepted for testing antimicrobial agents in endodontics (Kishen & Haapasalo 2015). A contributing factor to this is likely the lack of detailed knowledge concerning endodontic biofilm structure and propagation. The implications are that results from various studies are either difficult to compare to one another, or may not hold true clinically. However, modelling is a necessary part of irrigant research and as a lead up to preclinical assessments. The use of a specific disinfection model is influenced by the stage of development of the antimicrobial of interest, the resources available and the particular question being examined. The use of planktonic bacteria may be appropriate in the initial stages of antimicrobial testing because the result is more clearly assessed due to the limitation of confounding factors (Stojicic et al. 2012). However, realistically a biofilm model needs to be incorporated to more closely simulate clinical situations (Shen et al. 2012). Biofilm models can be broken down into two broad categories, the growth of the biofilm itself and the assessment methodology used to measure the antimicrobial effects.

1.2.4.1 Biofilm growth

The propagation of biofilms for in vitro and ex vivo endodontic research is a complex interplay of several components: the device used to grow the biofilm, the biofilm substrate, the choice of microorganism, the age of the biofilm and the growth media. Devices Biofilms for the assessment of endodontic irrigants have been grown under static (Yang et al. 2016; Ruiz‐Linares et al. 2017) or dynamic conditions (Seet et al. 2012; Busanello et al. 2019). Dynamic growth may result in more reproducible biofilm formation (Neelakantan et

15 al. 2017). Under dynamic conditions, biofilms continuously receive nutrients due to the circulation of growth medium in the system, while for static devices nutrients must be replenished periodically (Azeredo et al. 2017). The root canal is mostly a closed system and the actual rate of supply of nutrients is unknown. Thus, it seems difficult to create a dynamic model that corresponds to the root canal environment. The choice of a static versus dynamic model may therefore be more a question of addressing a particular research aim. Static devices are suitable for the assessment of large numbers of biofilms, such as might occur in high throughput screening (Azeredo et al. 2017). If however the research question is oriented towards an examination of biofilm formation, then a dynamic growth model is preferable (Kishen & Haapasalo 2015). Increasing the shear forces in dynamic systems can produce thicker biofilms, and alter the microbial composition (Fernandez et al. 2017). This may be of relevance in studies of biofilm removal with lasers or ultrasonic devices which generate such forces in fluids. Additionally, biofilm structures can be tailored, based on the growth method chosen, to fulfil a specific need. For example, research may seek to differentiate the action of irrigants on various biofilm components (Busanello et al. 2019). Static growth promotes biofilms rich in EPS, while dynamic growth, using devices such as constant depth film fermenters with their accompanying compacting action, results in cell dense biofilms (Busanello et al. 2019). Microtiter plates are the most frequently used device for growing static biofilms (Azeredo et al. 2017). A limitation of their use is the incorporation into the biofilm of planktonic cells due to sedimentation at the bottom of the plate where the biofilm forms (Azeredo et al. 2017). Two devices which overcome this problem are the Calgary Biofilm Device (CBD) (Ceri et al. 1999) and the Amsterdam Active Attachment (AAA) plate (Exterkate et al. 2010). In both, biofilm formation occurs in the vertical plane, thus overcoming sedimentation. The vertical orientation of the biofilm substrate also mirrors that found in the root canal. The CBD is based on 96 wells and was devised for measuring the minimal concentration of an antimicrobial agent that will eradicate biofilms. It was designed for growing biofilms on a sole substrate, the pegs of the CBD itself (Ceri et al. 1999); however, it can be adapted for the attachment of dentine blocks (Arias-Moliz et al. 2015). A problem with the CBD is that harvesting biofilm from the pegs is incomplete (Azeredo et al. 2017). The AAA plate can accommodate different substrates. These are suspended from nylon pegs attached to the metallic lid of the device and positioned over the wells of a 24 well plate (Exterkate et al. 2010). The setup of both systems allows for not only the easy changing of

16 growth media, but also for the simultaneous disinfection of multiple biofilms using different irrigants. This seems beneficial to experimental accuracy particularly when disinfection times are short. Substrates Substrates used for growing in vitro endodontic biofilms include: microtiter plates (Duggan & Sedgley 2007), cell culture plates (Upadya et al. 2011), glass cover slips (George & Kishen 2008), hydroxyapatite (HA) discs (Tawakoli et al. 2017; Jing et al. 2019) and resin blocks. Resin blocks attempt to duplicate canal volume and structure and thus more closely resemble teeth. They have been used for assessments where fluid dynamics are important, for example when comparing standard needle irrigation to the use of sonics, ultrasonics or lasers (Mohmmed et al. 2016; Swimberghe et al. 2019a). In vitro substrates have the obvious advantage of uniformity of shape, allowing for better experimental standardization. Additionally, they can be purchased without the need for ethics approval, as is required for material from human sources. Similar to the use of planktonic bacteria, such substrates have a place in the early stages of disinfectant screening (Azeredo et al. 2017). HA discs are used commonly for growing biofilms both in caries research (Exterkate et al. 2010; Koo et al. 2010) and to examine the efficacy of endodontic irrigants (Shen et al. 2011; Wang et al. 2018; Busanello et al. 2019). Despite their widespread use, it has never been shown that all the bacteria can be removed from these discs, particularly using common methods such as vortex mixing. Problems therefore arise if the assessment methodology requires bacterial removal and counting. A case in point is a study that employed HA discs to assess biofilm structure at 3 days as compared to 21 days with various disc coatings (Ali et al. 2020). A colony forming unit (CFU) count was used to determine the number of biofilm cells. A finding of the study was that the CFU count for both ages of biofilms was the same for three of the groups. Yet, although not quantified, the SEM images presented as typical clearly showed that the 21 day biofilms contained many more bacteria than the 3 day biofilms for those coatings. Because complete bacterial removal from the disc was not demonstrated, the discrepancy may be due to residual bacteria in deeper layers remaining on the discs after vortex mixing. This is supported by the observation that the superficial layers of biofilms are more easily removed than deeper layers (Siqueira et al. 2015). This is of concern, if true, because the base layer contains bacteria resistant to antimicrobial agents, and in low metabolic states (Siqueira et al. 2015).

A final detail about the use of HA discs is the materials used to coat the discs. Collagen (Stojicic et al. 2013; Shen et al. 2016) or saliva (Tawakoli et al. 2017; Wang et al. 2018) are commonly employed for surface conditioning. Pre-conditioning influences the biofilm structure compared to when no coating is used. With saliva conditioning, biofilms contain more protein and polysaccharide (Ali et al. 2020). The use of collagen results in thicker biofilms with an increased resistance to the action of disinfectants (Shen et al. 2009). In ex vivo models, substrates utilising extracted teeth facilitate the formation of more clinically relevant biofilms (Swimberghe et al. 2019b). This permits an examination of the effect of chemomechanical preparation in all parts of the main canal and dentinal tubules (Azim et al. 2016). However, the use of extracted teeth suffers from a lack of standardization and hence comparison between studies is difficult. Differences between studies include: methods of canal preparation, tooth anatomy, size, shape and volume, and whether the teeth are of human or animal origin. A compromise is seen in the use of dentine slices (Meire et al. 2012; Shrestha & Kishen 2014). A standardized dentine block model which assesses bacterial killing within dentinal tubules (Ma et al. 2011) has been used widely to evaluate the antimicrobial effect of various irrigants (Wang et al. 2012a; Du et al. 2014; Morago et al. 2016). A limitation on the use of dentine blocks and slices as substrates is that, similar to the situation with HA discs, if bacteria are to be counted, then their removal may be difficult to achieve. Given the dense manner in which bacteria pack into tubules (Taschieri et al. 2014), it is possible that many bacteria cannot be removed from the substrate. Nonetheless, removal of bacteria from dentine is attempted by vortex mixing or ultrasonic agitation (da Silva et al. 2018; Halkai et al. 2018). Other authors have advised caution in the extraction of bacteria from dentine, particularly when using ultrasonic devises which have the capacity to kill bacteria (Kishen & Haapasalo 2015). In the case of whole teeth, bacterial sampling or counting following chemomechanical preparation is a common approach. Sampling may be done with or without some canal filing, by placing sterile fluids into the canal and absorbing them onto paper points (Kishen et al. 2018). Given that endodontic files will not reach all areas of the canal (Peters et al. 2001), bacterial removal from the main canal is difficult to gauge. It is also reasonable to assume that different bacterial species will adhere to dentine with different strengths. Thus, some species may preferentially remain attached to dentine.

Bacterial species Disinfection studies largely employ monospecies biofilms with the vast majority of them composed of E. faecalis (Swimberghe et al. 2019b). In particular, the strain ATCC 29212 is commonly encountered in the literature (Ma et al. 2015; de Freitas et al. 2016; Estrela et al. 2018; Kishen et al. 2018). While this achieves some standardization between studies, it does not relate well to the bacteria present in apical periodontitis. As suggested earlier, the genera Fusobacterium and Porphyromonas are possibly key players in endodontic infections, and thus inclusion of these taxa can be justified. Recently, there does appear to be increased incorporation of F. nucleatum into endodontic biofilms as part of a three species or four species biofilm which also includes E. faecalis (Wang L et al. 2017; AlSaeed et al. 2018; Race et al. 2019). However, there is a need for polymicrobial models to be incorporated into endodontic research (Shen et al. 2012; Chávez de Paz & Marsh 2015). Subgingival dental plaque can be used as a diverse bacterial source for growing endodontic biofilms (Shen et al. 2016), while saliva is commonly used in caries research (Fernandez et al. 2017). The problem that arises with polymicrobial biofilm models in current endodontic research is that the bacterial composition of the biofilms grown are mostly not assessed beyond a basic level, if at all. Hence it is difficult to prove their reproducibility. It is one thing to use a polymicrobial source, but another to demonstrate the breakdown of the bacteria present in the biofilm and its structure. This is particularly relevant given that the bacterial diversity in dental plaque or saliva is far more complex than that of the biofilms derived from these (Tian et al. 2010; Fernandez et al. 2017). The best attempts seem to be SEM imaging to show some morphological variety in the bacteria present (Shen et al. 2016). This is surprising given the rise of next generation pyrosequencing. In other areas of dental research, pyrosequencing and other tools are used to validate the composition of polymicrobial biofilms that are produced (Fernandez et al. 2017). Biofilm age The effect of antimicrobial agents on biofilms is partially determined by biofilm age (Du et al. 2014). The period between 2 and 3 weeks is critical for the development of biofilms with increased resistance to endodontic irrigants (Shen et al. 2011; Stojicic et al. 2013b). This increase in resistance is associated with the appearance of persister cells, and hence it has been suggested that endodontic infections be treated early (Zhao et al. 2016). Without specific proof, it is logical that long standing periapical lesions contain older and hence more resistant biofilms, while recently or rapidly acquired infections of vital pulps would be

19 composed of younger and less resistant biofilms. It may be therefore beneficial to include both types of biofilms in antimicrobial testing. This is particularly pertinent given that, as biofilms age, they increase in thickness and contain higher relative proportions of EPS, which in turn influences the type of antimicrobial agents to which they are susceptible (Busanello et al. 2019). Growth media Nutrition is a key determinant in biofilm formation and structure. One issue that arises is that, as mentioned, little is known regarding the actual nutrition supplied to root canal bacteria in vivo. Thus, in vitro and ex vivo experiments can only address the nutritional aspects of biofilm proliferation from the viewpoint of the growth needed to fulfil a particular experimental need. For example, some assessment methodologies require certain biovolumes for validity (Stiefel et al. 2016). For E. faecalis, biofilms are commonly grown with brain heart infusion (BHI) as the sole nutrient (Liu et al. 2010; Du et al. 2014; Ali et al. 2020), but when a thicker biofilm is required, then this can be achieved by supplementation of the BHI with glucose (Seneviratne et al. 2013). In polymicrobial models, researchers frequently add blood or supplement media with hemin and Vitamin K to encourage the proliferation of fastidious Gram-negative anaerobes (Soukos et al. 2005; Chow et al. 2019). The Shi medium, which can be made in the laboratory, additionally contains, amongst other ingredients, mucin, arginine, urea, yeast extract and sucrose (Tian et al. 2010), is of possible interest in endodontics. Although the Shi medium was formulated to be representative of the general oral microbiome, the bacterial profile in the biofilms formed with salivary inoculums after 24 hours resembles that seen in primary endodontic infections (Table 1-2). Pyrosequencing has revealed the following bacterial distributions: 39% Prevotella, 20% Streptococcus, 16% Veillonella, 10% Fusobacterium, 5% Porphyromonas and minor components of other bacteria including many Gram-negative anaerobes (Tian et al. 2010). It should be recognized though that the composition of older biofilms may differ from this. In 85% of cases, media replenishment of static endodontic biofilms occurs every 24 or 48 hours (Swimberghe et al. 2019b). However, some maintain that for polymicrobial biofilms, such regular media changes favour the proliferation of facultative cocci, and hence recommend changing the media no less than weekly (Stojicic et al. 2013b). Nutrient deprivation is also important because it can lead to increased virulence, where bacteria enter a dormant state with more resistance to antimicrobials (Gulabivala & Ng 2015). However, given the wide-spread use of more frequent media replenishment, there may be a fine line

20 between producing bacterial diversity and resistant biofilms by weekly changes of media, and supplying enough nutrition to either keep the bacteria alive or to grow a biofilm of sufficient volume in order to achieve a useful laboratory result.

1.2.4.2 Biofilm disinfection assessment methods

An assessment of the methodologies used to measure biofilm removal or bacterial killing is essential in order to evaluate the antimicrobial action of irrigants (Kishen et al. 2018). A variety of techniques exist, each with a different focus. Some techniques measure the total biomass (live and dead cells) while others quantify the number of vital bacteria. Still others concentrate on the identification of particular biofilm components such as EPS or protein (Stiefel et al. 2016). The reliance on just one methodology should be avoided as each has its strengths and shortcomings (Azeredo et al. 2017). This common-sense approach is even more relevant in endodontics where different antimicrobial and chelating agents have the potential to react chemically with one another (Wright et al. 2017b), or with assay reagents, thus causing methodological problems. Colony Forming Units A recent review reported that 87% of endodontic biofilm research employs CFUs as an assessment tool (Swimberghe et al. 2019b). It is a simple and low cost technique involving bacterial sampling, plating, cultivation and counting. Yet, as previously mentioned, accurate bacterial sampling cannot be guaranteed. Additionally, direct culturing will not recover many fastidious anaerobic microorganisms (Love & Jenkinson 2002). This is of particular concern in endodontics where the bacterial flora consists predominately of Gram-negative anaerobes. In this methodology, microbes must be capable of forming colonies. In stressful situations, such as during starvation or changes in oxygen tension, temperature or osmolarity, microbes enter a low metabolic state where they are not dead, but cannot be cultured. Some refer to this as a viable but non-culturable state (VBNC) (Oliver 2005). Indeed, in endodontics, colony counting has been shown to overestimate the effectiveness of antimicrobial agents during biofilm nutrient deprivation (Shen et al. 2010). Light microscopy Light microscopy is a basic and long standing technique for observing microorganisms and the histopathology caused by infective processes. Tissue staining and light microscopy has been used to examine microorganisms in the main canal and dentinal tubules as well as in uninstrumented isthmus areas (Nair et al. 2005). In fact, the classification of apical

21 periodontitis as a biofilm disorder was achieved through light microscopy (Ricucci & Siqueira 2010). The technique does have some disadvantages. Fixation steps not only devitalise tissue, but are time consuming and introduce artefacts (Swimberghe et al. 2019b). The use of light microscopy in endodontic research has been superseded by more sophisticated microscopic techniques. Scanning electron microscopy SEM suffers from the same sort of fixation and sample preparation problems that occur in light microscopy. In particular, the EPS is barely visualised because of biofilm dehydration (Peters et al. 2015). However, the increased magnification and greater resolution of SEM make it a commonly utilised tool in endodontic research. It is used frequently to verify biofilm structure and composition (Shen et al. 2011; Shen et al. 2016; Ali et al. 2020). Microbial quantification with SEM is also possible in conjunction with image analysis (Vyas et al. 2016), although it must be remembered that dead bacteria will be included in assessment. In endodontic research, it does not appear to be used to any great extent for this purpose. Confocal laser scanning microscopy and biofilm staining Confocal laser scanning microscopy (CLSM) has been heralded as a ground-breaking noninvasive tool for biofilm research (Lawrence & Neu 1999). Images can be viewed in the x, y and z planes as well as in real time. Hence, 3D images evolving over time can be recorded. Staining procedures cause minimal biofilm disruption and do not result in specimen dehydration (Lawrence & Neu 1999). Major applications include the elucidation of biofilm features such as EPS, and the determination of bacterial viability (Azeredo et al. 2017). The use of this analytical method in endodontic research has mirrored its rising popularity in other fields (Kishen & Haapasalo 2015). Despite the popularity of CLSM, there are caveats and limitations in the applications of its use. Like other microscopic techniques, CLSM has been criticized for the possibility of operator bias when used to quantify results (He et al. 2013). Hence, there is a need for operator blinding of methodical sampling systems such as grids which consistently image the same areas of substrates. Partial attempts have been made to do this, for example, imaging from the central area of a sample (del Carpio-Perochena et al. 2011). A further reported problem is that of incomplete dye penetration of the biofilm (Busanello et al. 2019). There is widespread use in endodontics of CLSM with viability staining to assess dead and live bacteria in endodontic biofilms (Ma et al. 2011; Wang et al. 2012b; Azim et al. 2016; Morago et al. 2019). The technique, often referred to as “live/dead”, involves the staining of all cells, dead and alive, with the green stain Syto9 and the red dye propidium

22 iodide. Syto9 stains live bacteria, while propidium iodide selectively enters cells with damaged or permeable cell membranes and replaces the Syto9. Thus, green bacteria are counted as alive and red bacteria are deemed dead (Invitrogen 2004). The use of this technique to quantify the live/dead percentages can be misleading when the action of the irrigant is to remove the biofilm, as is the case for NaOCl (Tawakoli et al. 2017). In these situations, the biovolume as well is calculated (del Carpio-Perochena et al. 2015a). A further limitation of this technique is dye bleaching, e.g. from quenching of the fluorescence signal. In specific cases, 4-8% of the Syto 9 signal can be lost over 5 minutes (Stiefel et al. 2015). This would have repercussions for experiments involving a large number of samples. Additionally, individual scan times can be prolonged, up to 20 minutes (Azim et al. 2016), and thus there is added potential for dye bleaching. Furthermore, the live/dead technique is not universally applicable. In particular, it should not be applied to the assessment of polymicrobial biofilms (Netuschil et al. 2014). The reason is that in live Gram-negative bacteria, the Syto9 does not easily penetrate the two cell membranes, but it does readily enter these same bacteria when they are dead. Because propidium iodide replacement is incomplete, and because the green signal is much more intense than the red one, this results in a stronger green fluorescent signal from dead Gram-negative bacteria than from live ones (Stiefel et al. 2015). This causes an overestimation of the population of live cells. In biofilms where Gram-negative bacteria predominate, as for endodontic biofilms, this then becomes problematic. Additionally, the manufacturer’s instructions make no mention of how the technique can be applied to biofilms, using the terminology ‘cell suspension’ rather than biofilm. Moreover, the instructions imply that the cell suspensions are from one species and that calibration curves need to be determined for each species (Invitrogen 2004). The impossibility of calibrating a polymicrobial biofilm are obvious. A further complication with the use of polymicrobial biofilms is that the dye concentration depends upon the bacterial species (Netuschil et al. 2014). Yet, in spite of these varied problems, live/dead staining has been a commonly used tool for assessing the efficacy of irrigants in polymicrobial biofilms (del Carpio-Perochena et al. 2015a; Ruiz‐Linares et al. 2017; Yang et al. 2018; Jing et al. 2019). The fact remains that it is important to assess polymicrobial biofilms in endodontics, and alternative solutions need to be sought. An issue specific to the use of live/dead staining in endodontics is the impact of the use of a chelator or detergent. Propidium iodide will enter bacterial cells not only when they are dead, but also when there are changes to the cell membrane as occurs in the presence

23 of surfactants and for cells dividing in the exponential growth phase (Shi et al. 2007). This effect has been observed for the chelator EDTA. In a study which calculated the percentage of red fluorescence, compared to a control, increased red fluorescence was attributed to enhanced membrane permeability caused by the EDTA treatment, and not to dead bacteria. This was verified by plating the same bacteria, which showed no reduction in the microbial numbers (Wang et al. 2013). Others have also reported that EDTA alters cell permeability and increases the amount of red fluorescence in live/dead staining (Chávez de Paz et al. 2010). Thus, the use of live/dead staining in conjunction with a chelator which alters the membrane permeability could give a false result, overestimating the amount of bacterial killing. Molecular techniques: Polymerase chain reaction, pyrosequencing and FISH The quantitative polymerase chain reaction (qPCR) has been used for quantifying biofilm bacteria following irrigant action. This approach matches microbial DNA sequences with primer nucleotide sequences (Rôças et al. 2016; Zhang et al. 2019). The primers may be species specific or universal, with the latter enabling the detection of the total number of surviving bacteria following irrigation (Zhang et al. 2019). A criticism of this technique is that quantification can include extracellular DNA (Swimberghe et al. 2019b), the main source of which is from autolysed (Sedgley & Dunny 2015) and hence dead bacteria .This results in the inclusion of DNA from non-vital microbes in the count of vital bacteria that survived treatment. Thus the number of surviving bacteria is overestimated and the irrigant effect is underestimated. More importantly, whether the biofilm is formed on an artificial substrate or in a root canal, a sample must be obtained for analysis, and the same problems are then encountered as occur for CFUs. Hence, this more sophisticated and sensitive technology is limited by the accurate extraction of bacteria from the substrate. Pyrosequencing is another molecular technique able to provide the order of nucleotide sequences in segments of DNA (Margulies et al. 2005). It has been used in endodontics for the identification of microorganisms in apical periodontitis (Persoon et al. 2017). In FISH, oligonucleotide probes are hybridized with target RNA in bacteria that have undergone fixation and then been permeabilised (Azeredo et al. 2017). The use of different fluorescent labelled probes for various species enables visualization in 3D of the distribution of different bacteria in the biofilm (Thurnheer et al. 2004). Universal probes are available to enable the identification of the biofilm as a whole. Indeed, such a probe, EUB338, has been used with CLSM and SEM, to enable the visualization of remaining biofilm within root canals following failed endodontic treatment (Schaudinn et al. 2009). Fluorescence in situ

24 hybridization has also been applied to the identification of bacterial species recovered from cases of periapical periodontitis (Zehnder et al. 2017). Colorimetric assays Stains such as crystal violet and safranin red stain the total biomass, including live and dead cells, as well as EPS (Stiefel et al. 2016). They have high detection limits and are suited to large biovolumes (Stiefel et al. 2016). Many versions of the crystal violet assay appear in the literature, and this leads the reader to some confusion as to which protocol to use. The differences include the crystal violet concentration, the extraction chemical and the measurement wavelength (Borucki et al. 2003; Burton et al. 2007; Chavant et al. 2007; Duggan & Sedgley 2007; Seneviratne et al. 2013; Bandara et al. 2016). A protocol employing a fixation step with 1% paraformaldehyde containing 1 mM calcium chloride in PBS was reported as being optimized for biofilms of E. faecalis (Seneviratne et al. 2013). Colorimetric assays are also useful for measuring the metabolic activity of bacteria within biofilms. The tetrazolium salt 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5- [(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) (Philip et al. 2019) is converted to a highly coloured formazan product when dehydrogenase enzymes are present in the biofilm cells (Azeredo et al. 2017). This reaction involves the reduction of XTT (Xu et al. 2016) and hence care must be exercised to completely remove any reducing agents used to neutralize disinfectants. Commonly, sodium thiosulphate is employed to neutralize NaOCl (Gomes et al. 2001; Stojicic et al. 2012; Morgental et al. 2013) and hence if the washing steps to remove it are not complete, results from the XTT assay could be inaccurate. A further caveat on the use of the XTT assay is that differential colour changes can occur between various species or strains (Kuhn et al. 2003). Summary and concluding remarks on biofilm methodology The methodology used to assess the in vitro action of disinfectants on endodontic biofilms lacks a consistent model (Kishen & Haapasalo 2015). Variations include: the device used to propagate the biofilm, the substrate, the type of bacteria chosen, biofilm age, the growth medium and the frequency of its replenishment. Even if different researchers adopted the same model, the relevance of that model to actual periapical periodontitis would need to be established (Chávez de Paz & Marsh 2015). This again highlights the urgency for further research into the basic nature of the biofilms present in endodontic infection. Furthermore, many assessment techniques suffer from limitations and inaccuracies, and hence there is often a need to apply more than one of them. It is also important, in choosing a methodology,

25 to tailor it to the irrigant used. Overall, there is a clear need to develop better techniques to assess the antimicrobial action of endodontic irrigants.

Dentine structure, smear layer, chelating irrigants and methodologies

The elucidation of dentine structure is important in understanding another core function of endodontic irrigation, smear layer removal. Additionally, a knowledge of dentine structure is of prime importance when considering the impact that endodontic irrigants may have on the structural integrity and mechanical properties of teeth. Dentine is dominated by the presence of dentinal tubules. These tubules vary in diameter depending upon location and age, being larger closer to the pulp and in youth (Ryou et al. 2015). Surrounding the tubular lumen is a band of highly mineralized, collagen- poor dentine, the peritubular dentine, while between the tubules, the intertubular dentine forms a dense network of mineralised matrix, that is rich in collagen (Ryou et al. 2015). Dentine is a hydrated tissue consisting of, by weight, 20% organic material, 70% inorganic material and 10% water (Goldberg et al. 2011). The organic matrix is comprised predominantly of type I collagen, with noncollagenous proteins such as glycoproteins and proteoglycans accounting respectively for 5% and 3% (Bertassoni 2017). At a molecular level, fibrillar collagen has a helical structure, formed from three intertwined polypeptide chains, where every third amino acid is a glycine residue (Brodsky et al. 2008). The high percentage of glycine, proline and hydroxyproline help stabilize and tighten this structure through hydrogen bonding. (Brodsky et al. 2008). The inorganic component of dentine consists predominantly of calcium HA 2+ 3− − nanocrystals, containing calcium (Ca ), phosphate (PO4 ) and hydroxide (OH ) ions in the − formula Ca10(PO4)6(OH)2 (Wu et al. 2012). Carbonate groups may substitute for OH and 3− PO4 to some extent, giving type A carbonated HA and type B carbonated HA, respectively (Ortiz-Ruiz et al. 2018). Additionally, small amounts of sodium, magnesium and potassium can replace Ca2+ (Teruel Jde et al. 2015). The HA nanocrystals are irregularly shaped, and are located both within and external to the collagen fibrils (Kishen 2006; Bertassoni 2017). The nanocrystals outside the fibrils have a more random orientation, because, intrafibrillarly, the mineral aligns with the collagen (Bertassoni 2017). The water component binds to both the collagen and inorganic components, and is also found unbound in the dentinal tubules (Kishen 2006).

1.3.1 The Smear Layer

A second key role of endodontic irrigation is the removal of smear layer. The smear layer covers the instrumented areas of the canal and is a direct result of the mechanical preparation of the root canal (Haapasalo et al. 2010). It consists of dentine debris, containing the inorganic component, in addition to organic debris which contain collagen remnants and many microorganisms (Violich & Chandler 2010). Whilst historically there has been some debate about whether it should be left or removed (Violich & Chandler 2010), evidence shows that the smear layer impedes the action of antimicrobial agents by blocking dentinal tubules, and thus its removal is recommended (Wang et al. 2013). Because of its bacterial and nutrient content, leaving the smear layer may also encourage further bacterial proliferation (Violich & Chandler 2010). Lastly, the bonding of endodontic materials, such as epoxy resin sealants to the root canal wall, is enhanced by removal of smear layer (Vilanova et al. 2012). While the removal of smear layer is largely a chemical reaction as described below in Section 1.3.2, chelator and irrigant activation and agitation, probably best described as a physical process, can enhance smear layer removal, and this aspect is further discussed in Section 1.6.4.

1.3.2 Factors influencing chelation in dentine

Chelators, also known as ligands, act as Lewis bases, donating electrons to metal ions and binding them (Blackman et al. 2008). In the context of endodontics, chelators assist in the removal of smear layer because of their high affinity for metal ions, particularly for Ca2+ (Cobankara et al. 2011), by pulling dentine debris containing such ions from tooth structure. However, the smear layer also contains organic components, and thus the adjunctive use of NaOCl as a proteolytic agent is also required (Haapasalo et al. 2012). Many factors influence the ability of a chelator to bind Ca2+ ions. In medical applications, including assessments of bone metabolism, ligand stability constants and the pKa are important considerations (Ke et al. 2016). Chelators with a high stability constant for a particular metal ion will bind strongly to it (Blackman et al. 2008). The degree of Ca2+ binding is also dictated by the pH at which the chelator is used and by its pKa (Wen et al. 2004). This is because chelators bind metal ions more strongly when they are fully

27 dissociated, and it is the pKa which determines that pH level for any particular chelator (Smith & Martell 1989; Blackman et al. 2008). Ligands are more dissociated at higher pH levels, and so higher pH levels should favour stronger chelation, with the exact pH that this occurs at being determined by the pKa value for that specific ligand (Nikiforuk & Sreebny 1953; Blackman et al. 2008). However, this statement is valid only when considering chelation of freely available Ca2+ ions. Chelation in teeth however is rather more complex, with low pH values favouring chelation. This is because in dentine, Ca2+ is bound up in HA, and its release and hence availability for chelation is limited by the equilibrium, equation (1) below (Dawes 2003).

2+ 3− − Ca10(PO4)6(OH)2 10 Ca + 6 PO4 + 2 OH (1)

⇌ The solubility constant for this equilibrium is extremely low, and the equilibrium lies predominately to the left, with little Ca2+ released. However, if acid is added to this system, − − 3- 2+ then OH is consumed, and to restore the equilibrium more OH , PO4 and Ca are released. The extent of Ca2+ release from HA is estimated to be 10 times higher for each decrease in pH unit (Dawes 2003). Thus, in teeth, as opposed to when Ca2+ ions are free in solution, acidic conditions favour chelation.

1.3.3 Common chelators in endodontics for smear layer removal

1.3.3.1 EDTA

EDTA is the most commonly used chelator in contemporary endodontics (Dutner et al. 2012). It has no antibacterial actions, although it can weaken bacterial cell walls (Haapasalo et al. 2014). EDTA is a strong chelator, with a Ca2+ ligand stability constant of 10.7 (Kroll & Gordon 1960). The account given in the previous section clarifies the role that pH plays in the ability of EDTA not just to chelate, but also to remove smear layer. It thus explains the apparent dichotomy of why Ca2+ in a solution of calcium carbonate is 40% better chelated by EDTA at pH 7 than at pH 6 (Nikiforuk & Sreebny 1953), yet smear layer is removed to a greater extent by EDTA at pH 8.5 than at pH 11.4 (Deari et al. 2019). EDTA, as employed in standard endodontic treatment, is used at a neutral to slightly alkaline pH (Haapasalo et al. 2014).

Such EDTA solutions can be derived from dissolution of the di-sodium salt (Na2

EDTA) or the tri-sodium salt (Na3 EDTA) (Tartari et al. 2017; Deari et al. 2019). This is an important point because when the tetrasodium EDTA salt (Na4 EDTA) is dissolved, a solution of pH 11.4 results (Deari et al. 2019). EDTA of this alkalinity is not used in standard irrigation, but it has been applied to continuous chelation, and this point will be discussed in a later section. A criticism of the literature on the use of EDTA to remove smear layer is that the majority of authors do not cite the brand or type of salt, nor the pH. More astute authors (Çalt & Serper 2002) show an understanding for the mode of action of EDTA on removing smear layer by citing the pH of their solution in the methods section of their study. There is no consensus on actual clinical protocols involving the use of EDTA to remove layer (Haapasalo et al. 2012). However, clinical recommendations exist in the form of reference books and review articles. EDTA at a concentration of 15% or 17%, at near neutral pH, is typically used as a final flush after the application of NaOCl, with two minutes being effective in most cases (Haapasalo et al. 2014). Even a one minute application of 17% EDTA can be effective after canal instrumentation and this can be followed with an optional 30 second flush with NaOCl (Basrani & Haapasalo 2014). Early ex vivo experiments involving the irrigation of single rooted teeth, while stating irrigant volumes rather than irrigation times, concluded that a final flush with equal volumes of either 8.5% or 17% EDTA solutions removed smear layer almost to the same extent (Yamada et al. 1983). Another experiment, involving the instrumentation and irrigation of standardized roots and using a final flush with 17% EDTA, reported smear layer removal after 3 minutes, although shorter irrigation times were not tested. (Lottanti et al. 2009). When 17% EDTA was used, and with a final rinse of NaOCl, effective removal of the smear layer was achieved in only one minute (Çalt & Serper 2002). Several researchers have used a simpler model involving dentine discs to examine the removal of smear layer by EDTA. In such experiments, and without the prior use of NaOCl, smear layer removal was considered complete after exposure to 17% EDTA at slightly alkaline pH after one minute (De‐Deus et al. 2007; Reis et al. 2008; De‐Deus et al. 2011). The concentration and time of EDTA use for smear layer removal in the dentine disc experiments together with the tooth studies and clinical recommendations are summarised in Table 1-4 .

Table 1-4. Summary of EDTA requirements for smear layer removal.

1.3.3.2 Citric acid

Citric acid has a long history of use in endodontics for the removal of smear layer (Wayman et al. 1979; Baumgartner et al. 1984). Recommended clinical protocols involve the use of citric acid as contained in the BioPure™ MTAD and Tetraclean™ formulations. These are used following instrumentation with NaOCl (Basrani & Haapasalo 2014). A solution of 10% citric acid shows some antimicrobial activity, including actions against Gram- negative anaerobes including Fusobacterium nucleatum (Yamaguchi et al. 1996). Solutions of 10% citric acid are the most commonly used for smear layer removal (Haapasalo et al. 2010), and even 5% citric acid is effective (Haznedaroğlu 2003). A 15-30 second exposure to 10% citric acid is sufficient to remove smear layer (Reis et al. 2008). However, even though the smear layer is fully removed, some debris may remain in isolated tubules (Reis et al. 2008). Citric acid is a weak chelator compared to EDTA with a ligand 2+ stability constant for Ca of only 3.5-3.6 (Smith & Martell 1989) and pKa 1, pKa 2 and pKa 3 values of 3.13, 4.76 and 6.4, respectively. The pH of a 10% solution of citric acid is 1.8 (Machado-Silveiro et al. 2004). The low pH at which citric acid is used explains the effectiveness of this weak chelator in removing smear layer. At pH 7.4, citric acid, which

30 according to the pKa values, exists predominantly as the citrate, is not effective at removing Ca2+ from dentine (Sousa & Silva 2005; Spanó et al. 2009).

1.3.3.3 Other chelating agents

Although other irrigants are not popular in endodontic practice for the removal of smear layer, research exists on a variety of chelators. Of these, in recent times, peracetic acid (PAA) and etidronate have received some attention. Both will be discussed in other sections of the review. The EDTA analogues, EGTA (egtazic acid) and CDTA (cyclohexanediaminetetraacetic acid) are strong chelators, with Ca2+ ligand stability constants of 11.0 (Kroll & Gordon 1960) and 12.7 (Smith & Martell 1989), respectively. CDTA has a similar capacity for smear layer removal compared to EDTAC (a mixture of EDTA plus cetrimide (also known as cetavlon) (Marques et al. 2006). This is possibly explained by its high stability constant. However, EGTA removes less smear layer than either EDTA or EDTAC (Çalt & Serper 2000; Marques et al. 2006), as well as chelating less Ca2+ from dentine (Sayin et al. 2007a).

1.3.4 Methodologies for assessing smear layer removal

A relatively simple measure of chelation ability is to measure Ca2+ concentration using atomic absorption spectroscopy. This approach measures the Ca2+ concentration of root canal elutes without visualization of the smear layer itself (Zehnder et al. 2005). While this allows for a comparison between different irrigants, there is no guarantee that the smear layer has actually been removed. For this reason, this method is often combined with other techniques such as SEM (Zehnder et al. 2005) or laser scanning microscopy (Deari et al. 2019). The latter is a simpler technique than SEM, requiring less complex sample preparation (Deari et al. 2019). A refinement of SEM is the use of environmental SEM (ESEM) which requires no sample metallization (i.e. sputter coating) and maintains tissue hydration (Reis et al. 2008). However, SEM studies are frequently encountered in the literature and in this regard, they act as a standard technique. In the late 1990s, a scoring system was developed to assess the degree of smear layer removal (Hülsmann et al. 1997). SEM images are scored for both remaining smear layer and debris on a scale from 1-5. A score of 1 represents a clean canal wall with open dentinal tubules, and 5 is scored when the canal is predominately covered with smear layer

31 or debris. Because scoring images is somewhat of a subjective procedure, studies using such methodologies have multiple blinded scorers and then calculate the degree of interobserver agreement (Spanó et al. 2009). The advent of image analysis software has allowed for the batch processing of SEM images to assess the effect of chelators on smear layer removal. This removes the subjectivity of observer scoring. The percentage area faction (% AF) of open tubules is calculated as a percentage of the whole image (George et al. 2008a). The same criticism of CLSM image analysis also applies to this technique, that is, taking images is open to operator bias. This is particularly the case for dentine where the distribution of the dentinal tubules is heterogeneous both within and between samples (Deari et al. 2019). The use of a blinded operator to record the images is of assistance in this regard. A further criticism is that image analysis measures the degree of tubular opening and it does not account for canals that have been overly widened by chelator action. In this scenario, what might be deemed as a superior ability to remove smear layer may actually be the result of chelator induced dentinal erosion. High resolution images may be useful in this regard, and could enable visualization of the erosion. Lastly the parameters chosen for the image analysis must be applied universally. Because of minor variations in image quality, it is therefore necessary to ensure that all tubules in all images are counted, and to exclude images that do not conform (Deari et al. 2019). Co-site optical digital microscopy has further refined the assessment of smear layer removal (De‐Deus et al. 2007). In this technique chelators can be applied to a sample at various time intervals and the same set of tubules can be observed for widening at the different time points. Thus, the chelator effect is effectively observed in real time. It also helps remove operator bias because the same tubules are followed throughout. A limitation is that, with the reduced resolution of an optical microscope compared to SEM, high resolution images to check for erosion are not possible. This methodology can be used on a dentine sample which is divided into portions using tape. This allows for the application of different irrigants to the same dentine sample (De-Deus et al. 2008b). However, because of the difficulty in dividing small dentine slices into many compartments, its use is limited to three or four chelator groups.

Root canal organic tissue

A third key function of irrigation is to remove organic components. Irrigants must remove both vital and necrotic pulpal soft tissue (Zehnder 2006). In addition to the organic material contained in the dental pulp, biofilms themselves contain organic material, and removing this by irrigation reduces the mass of the biofilm (del Carpio-Perochena et al. 2015a).

1.4.1 Structure of the dental pulp

The dental pulp is composed of a cellular component embedded in an extracellular matrix. The former consists predominately of fibroblasts, but also contains odontoblasts, a small percentage of pluripotent stem cells as well as cells associated with pulpal innervation, vascularity and the immune response (Goldberg 2014). The odontoblasts line the pulpal lumen, where they are instrumental in the formation of dentine (Kuo et al. 1992). Collagen forms a large part of the proteinaceous extracellular matrix, of which 34% is Type III (van Amerongen et al. 1983), a highly cross-linked form of collagen containing many disulphide bonds (Linde 1985). The remainder is largely Type I collagen (Goldberg 2014). The noncollagenous component of the extracellular matrix consists of proteoglycans and glycoproteins (Linde 1985).

1.4.2 Tissue dissolution capacity of NaOCl

Early landmark research in the area of tissue dissolution by various chemical solutions established the superior ability of NaOCl to dissolve organic material. (Grossman & Meiman 1982). Current clinical usage recognizes that NaOCl is the only irrigant able to significantly dissolve organic tissue, and therefore its use is essential in any endodontic irrigation regimen (Zehnder 2006). Because NaOCl is a neutral molecule containing sodium and oxygen atoms in oxidation states of +1 and -2 respectively, the chlorine carries an effective charge of +1. It is thus an electrophile, seeking out centres of negative charge, such as proteinaceous amino and sulphydryl groups (Fukuzaki 2006). This helps explain organic tissue dissolution, a process which consumes NaOCl and produces chloramines from the reaction of NaOCl with organic nitrogen (Fukuzaki 2006). NaOCl can thus assist in the disinfection process by dissolving organic matter associated with pulpal tissue and

33 biofilms. However, NaOCl also reacts with the collagen component of dentine, which lowers the effective NaOCl concentration, as measured by FAC (Macedo et al. 2010). This explains why the antimicrobial action of NaOCl is reduced in the presence of dentine and organic load. This point has implications for the chemomechanical preparation of the root canal where dentine debris may accumulate. Because OCl− is the species of NaOCl responsible for tissue dissolution, theoretically, as seen in Figure 1-1, at a pH above 9 organic matter will be dissolved. However, dentine has a buffering effect (Wang & Hume 1988), and because additionally NaOCl is consumed when it reacts with organic matter, in practice, NaOCl solutions used in endodontics must have a pH well above 9. This is observed clinically where commercial NaOCl solutions have a pH around 12 (Clarkson et al. 2006). At high pH, by definition, OH− abounds. While some authors have suggested that OH− assists in tissue dissolution by the neutralization of amino acids and digestion of fatty acids (Estrela et al. 2002), the role of OH− itself may not be significant. Experiments comparing the tissue dissolution ability of NaOCl and sodium hydroxide (NaOH) solutions of the same pH level show that NaOH does not dissolve organic matter (Clarkson et al. 2006). Thus, it is not high OH− levels that cause tissue dissolution, but rather that highly alkaline pH conditions allow for NaOCl to be present as OCl−. Factors that increase the ability of NaOCl to dissolve tissue are temperature, time, concentration, refreshment and activation. Increasing the temperature of NaOCl solutions enhances organic tissue dissolution, as shown in in vitro experiments (Sirtes et al. 2005; Dumitriu & Dobre 2015). In such studies, either pulpal tissue or collagen matrix was dissolved directly in NaOCl. The effect of temperature is limited by the prevailing temperature in the root canal. When irrigants at 66oC are applied into the root canal, they equilibrate to 37±0.9oC in 1 minute and to 35.7±0.9oC in 4 minutes (de Hemptinne et al. 2015). Thus, the suggestion of heating NaOCl to temperatures such as 45-60oC, to obtain more rapid tissue dissolution (Sirtes et al. 2005), seems of little practical value. The exposure of organic matter to solutions of higher concentration for longer time frames results in faster tissue dissolution than when lower concentrations are used for shorter times (Clarkson et al. 2006). However, the limiting factor is that prolonged times are impractical and may not show a nett benefit because of NaOCl degradation with depletion of FAC. In this respect, frequent irrigant refreshment becomes essential (Macedo et al. 2014). Lastly, agitating NaOCl solutions in techniques such as pulsed lasers, sonics or ultrasonics assists in their ability to dissolve organic tissue (Guneser et al. 2015.; Conde et al. 2017).

1.4.3 Methodologies for assessing organic tissue dissolution

Methodological considerations encompass the above influencing factors and include: the experimental measure of dissolution, NaOCl concentration, the volume of NaOCl, the weight of tissue, the option to measure the FAC in solutions following tissue dissolution, the tissue type, the experimental temperature, the use of agitation, the presence of dentine, and the use of refreshment. The ability of NaOCl to dissolve organic tissue has been measured most commonly using three methods: weight changes over time (Conde et al. 2017; Ulusoy et al. 2018), time to complete dissolution (Clarkson et al. 2012; Cullen et al. 2015) and the release of the imino acid, hydroxyproline (Koskinen et al. 1980; Nakamura et al. 1985). The first two methodologies are commonly employed, and the choice between them could be of practical nature, such as difficulty in visualizing the endpoint of tissue dissolution. However, measuring hydroxyproline release is inaccurate because NaOCl reacts with it (Koskinen et al. 1980). A fourth methodology, based on a histological approach, has also been reported in the literature (De-Deus et al. 2013). Because the concentration of NaOCl is known to deteriorate in storage (Clarkson et al. 2001), a fundamental aspect of tissue dissolution methodologies is the verification at the outset of the NaOCl concentration that is being used. Similarly, given the importance of pH, the pH level of irrigants needs to be recorded. It is also essential to ensure a balance between the volume of NaOCl used and the amount of tissue to be dissolved. If the amount of tissue is too large for the volume, then the dissolution process will stagnate, and the results will be invalid (Moorer & Wesselink 1982). To counter this, regular irrigant refreshment is employed. This not only ensures the continued ability of NaOCl to dissolve tissues, particularly in the presence of dentine (Cullen et al. 2015), but it also mimics clinical practice. The ratio of irrigant volume and tissue mass is also important when measuring the residual FAC following dissolution. In cases where the NaOCl volume is too high for the amount of tissue, only extremely small differences in FAC are seen (Clarkson et al. 2013). The type of tissue used in dissolution studies reported in the literature varies over a wide range, including human (Cullen et al. 2015), bovine (Almeida et al. 2015) or porcine pulp (Clarkson et al. 2013), porcine (Christensen et al. 2008) or bovine muscle (Tartari et al. 2017; Ulusoy et al. 2018) and porcine palatal mucosa (Naenni et al. 2004). While stating the actual sample weight and standard deviation (SD) would seem a prerequisite for dissolution studies, so that sample standardization can be verified, often this detail is omitted in studies

35 involving pulpal tissue (Clarkson et al. 2006; Jungbluth et al. 2012; Cullen et al. 2015). Part of the reason may be that achieving a consistent sample size for pulp is difficult, because, when it is stated, variations can be over 30% (Clarkson et al. 2013; Almeida et al. 2015). This could also be due to small sample weights or difficulties with handling specific types of tissue. Changing the parameters of size and tissue type may result in more uniformity. For bovine muscle samples of around 60 mg, 4% variations are achievable (Stojicic et al. 2010; Tartari et al. 2017). Uniformity of surface area is also important for standardization (Moorer & Wesselink 1982), and this could be expected to vary according to the source and type of tissue.

Effect of irrigation on dentine structure, function and mechanical properties

1.5.1 Dentine structure and related functionality

Both the inorganic and organic components of dentine play a part in the mechanical strength of dentine. The inorganic component is responsible for dentine stiffness and elasticity, with apatite located intrafibrillarly playing a significant role in this respect (Kinney et al. 2003). The collagen absorbs strain energy and it is important in fracture resistance and toughness (Kishen 2006). The toughness of dentine is also profoundly affected by water content, with dehydrated dentine being considerably more prone to fracture (Kahler et al. 2003). Glycoproteins are thought to play a role in dentine toughness, as they are attached to the collagen and can unfold to absorb energy, thereby shielding the fibrils (Bertassoni 2017). Thus, it can be seen that the various dentine components play an important role in maintaining the mechanical properties and fracture resistance of teeth. However, endodontic irrigation has the potential to impair the functional properties of dentine because it can change the organic and inorganic structure of dentine. One of the attributes of an ideal irrigant is that it does not damage the dentine by impairing its physical properties (Basrani & Haapasalo 2014). The same proteolytic action of NaOCl that dissolves biofilms and kills bacteria can dissolve the dentine collagen (Mai et al. 2010). The ability of chelators to unblock dentine tubules and remove smear layer can cause dentinal erosion by excessive removal of Ca2+ (Reis et al. 2008). There is concern that the irreversible damage to dentine caused by NaOCl may increase the rate of fracture in endodontically treated teeth (Gu et al.

2017). A variety of methodologies exist to examine the potential adverse effects of irrigation on dentine structure and mechanical strength.

1.5.2 Methodologies for assessing dentine structure and strength

Methodologies for evaluating the possible undesirable effects of irrigation on dentine borrow in part from the assessment techniques for the removal of smear layer. SEM and % AF (Qian et al. 2011), ESEM showing high resolution images of the erosion of peritubular dentine (Reis et al. 2008), co-site optical microscopy in conjunction with % AF (Reis et al. 2008) and flame photometry (Sayin et al. 2007a) have all been applied to show excessive Ca2+ removal from dentine following irrigation. Additional techniques that have been used include transmission electron microscopy (TEM) (Gu et al. 2017) to examine altered dentine ultrastructure. Attenuated total reflectance of Fourier transform infrared spectroscopy (ATR- FTIR) or Fourier transform infrared spectroscopy (FITR) can measure the ratios of dentine 3− components such as amide III or amide I and PO4 , where amide I and II are measures of 3− the collagen content, and PO4 reflects the apatite component (Gu et al. 2017; Tartari et al. 2018a). Measurements of dentine hardness with Knoop (Cruz-Filho et al. 2011) or Vicker’s (Eldeniz et al. 2005) indenters, micro elemental analysis (Marending et al. 2007) or elemental analysis with energy dispersive X-ray spectroscopy (EDS) (Wang et al. 2016) and measurements on dentine roughness and wettability (Tartari et al. 2018b) have all been used to show the effects of irrigation on dentine. A criticism of many of the studies which have used these methodologies is that they examine slices, slabs or portions of dentine that have been soaked in irrigants, and hence do not represent the irrigation process. Thus, the implication that certain irrigation protocols cause increased fracture of endodontically treated teeth cannot be made. However, such studies can be useful to compare various irrigants or sequences and to examine ultrastructural changes. Using a variety of methodologies to confirm results adds extra validity. Even studies that are more focused on strength testing cannot prove a cause and effect relationship between the use of irrigation and clinical tooth fracture because of limitations in the models used. Possibly the least clinically relevant of these is the 3-point bend test. This test employs bars of dentine to measure the modulus of elasticity and flexural strength after exposure to various irrigants (Grigoratos et al. 2001; Marending et al. 2007; Cecchin et al. 2017; Gu et al. 2017). The thickness of bars used in these experiments ranges from 0.15 to 1 mm. Regardless of the thickness used, because the bars are exposed to

37 irrigant solutions from every side, the detrimental effects of particular protocols can be overstated. In practice only a band of dentine adjacent to the canal is affected by the irrigant. With thin bars, either the entire dentine volume, or a large proportion of it, is infiltrated by irrigant. Despite this limitation, the 3-point bend test, when used on bars of reasonable thickness has application for comparing the influence of irrigant concentration or exposure time; however the results must be used in conjunction with alternative evidence. Other experimental models can exhibit a higher degree of clinical relevance. Such models involve the chemomechanical preparation of roots with various irrigants and the subsequent application of a compressive force (Souza et al. 2014). A further improvement on this technique is the addition of obturation to the protocol (Uzunoglu et al. 2016; Turk et al. 2017). Nevertheless, increased relevance is accompanied by the confounder of dentine bonding. The ultimate force at fracture is influenced not only by the effect of the irrigant on the mechanical strength of dentine, but also by the differential effects that irrigants have on the dentine surface with respect to bond strength to a variety of sealants (Uzunoglu-Özyürek et al. 2018). A further methodological refinement is the use of heat cycling and fatigue loading prior to the application of a compressive force, to better simulate tooth fracture as it relates to cycles of masticatory force (Ambica et al. 2013). Fatigue loading typically requires 1.2 x 106 cycles at a frequency of 1.3 Hz (Ambica et al. 2013).

1.5.3 Effects of NaOCl, chelators and irrigation sequences

1.5.3.1 Effect of NaOCl alone

Despite the protective layer of apatite crystals surrounding the collagen fibrils, the hypochlorite ion is small and can penetrate into dentine to degrade collagen, even in the absence of mineral binding chelating agents (Gu et al. 2017). SEM imaging shows that this proteolytic action creates a zone of apatite-rich permeable dentine. When dentine is exposed to 5% NaOCl for 1 hour this zone measures approximately 100 µm (Marending et al. 2007). Apatite-rich but collagen-poor dentine has also been demonstrated by FTIR, with 3− increases in the PO4 /amide I ratio (Gu et al. 2017). As could be expected from the relative rise in the inorganic component, these changes are associated with increased stiffness, as is demonstrated by a reduction in both the modulus of elasticity and flexural strength using the 3-point bend test (Grigoratos et al. 2001; Marending et al. 2007). Increases in both exposure time and NaOCl concentration, ranging from 2-8%, augment these effects (Gu et

38 al. 2017). However, when used at a concentration of 1%, NaOCl alters neither dentine carbon content nor flexural strength over 1 hour (Marending et al. 2007). By itself, NaOCl does not affect the surface roughness or wettability of dentine (Tartari et al. 2018b). A factor which could impact the effect of NaOCl irrigation is the distance that this irrigant penetrates into dentine. This penetration distance has been reported as 131µm (Giardino et al. 2017) or 300 µm (Zou et al. 2010). However, both these studies were conducted using dentine samples where there was no infection. In a study where tubular infection was present, NaOCl penetration was limited to 130 µm from the root canal lumen, even with the additional use of EDTA and a detergent (Berutti et al. 1997). Because NaOCl is consumed in its reaction with organic matter, it could be thought that bacterial infection in the tubules would exhaust the effectiveness of NaOCl. However, this effect can be expected to be countered by irrigant refreshment.

1.5.3.2 Effect of chelating agents alone

While chelators are a necessary part of irrigation protocols for the removal of smear layer, by their very nature, they demineralize teeth. Hence, they have the potential to alter dentine structure and mechanical properties, even without the adjunctive use of NaOCl. In the following comparisons, 17% EDTA is assumed to have a pH at or near neutral. Chelators vary in their ability to extract Ca2+ from dentine. When dentine discs are exposed to solutions of 10% citric acid or 17% EDTA, more Ca2+ is removed by the former than the latter (Machado-Silveiro et al. 2004). This agrees with the previously mentioned faster removal of smear layer by 10% citric acid compared to 17% EDTA. It is also in line with the ESEM observation that 5% and 10% citric acid cause erosion of the tubules after 60 seconds, while 17% EDTA shows no erosion at 3 minutes, and the larger % AF seen in co-site optical microscopy when 5% and 10% citric acid are compared to 17% EDTA (Reis 3− et al. 2008). Using ATR-FTIR, 17% EDTA and 0.5% PAA result in a similar amide III/ PO4 . However, for 2.0% PAA, this ratio is significantly increased, due to its heightened removal of the inorganic component (Tartari et al. 2018a). The chelator EGTA has also been compared with EDTA. Not only does 17% EGTA remove less Ca2+ than 17% EDTA as measured by flame photometry (Sayin et al. 2007a) but it causes less reduction in dentine hardness (Sayin et al. 2007b).

1.5.3.3 Effect of irrigation sequences on dentine structure and function

Irrigation usually involves the sequencing of NaOCl and chelators, and in some cases other antimicrobial agents. This is because NaOCl cannot remove smear layer by itself, and chelators cannot dissolve organic matter (Zehnder 2006). This introduces a significant factor in relation to the erosive potential of irrigation protocols. The order in which NaOCl is sequenced with other irrigants influences the extent of the resulting erosion (Qian et al. 2011; Wang et al. 2016). Standard irrigation protocols universally employ NaOCl as the main irrigant throughout chemomechanical preparation (Zehnder 2006; Dutner et al. 2012; Basrani & Haapasalo 2014; Haapasalo et al. 2014). While alternative irrigation solutions and regimens exist (Basrani & Haapasalo 2014), most often chemomechanical preparation with NaOCl is followed by a flush with EDTA or an alternate chelator such as citric acid, and finally with an antimicrobial rinse (Zehnder 2006). The use of NaOCl or CHX for this purpose has been advised, (Zehnder 2006; Schäfer 2007). More recently the recommendation is to limit the final NaOCl rinse following the chelator to 30 seconds, or to eliminate it completely by substituting saline, followed by CHX (Basrani & Haapasalo 2014). The explanation for this lies in the creation of a permeable layer of dentine following the initial application of NaOCl, and the subsequent treatment of this layer with a chelating agent, followed again by NaOCl. The first application of NaOCl removes collagen, as shown 3− by a lowered amide III/ PO4 value (Tartari et al. 2016). When 17% EDTA follows, an 3− experiment involving dentine discs demonstrated that high amide III/ PO4 ratios that considerable collagen is exposed with little accompanying apatite (Tartari et al. 2018b). EDS has shown that the further application of NaOCl after EDTA can cause severe erosion and demineralization, with relatively large amounts of organic material remaining (Wang et al. 2016). In this experiment molar teeth were irrigated and sectioned dentine blocks from the palatal roots were analysed with EDS. The result appears counter intuitive because NaOCl, which is not a chelator, seemingly preferentially removes apatite compared to organic matter. The authors explained that the collagen exposed by EDTA was not protected by apatite and the additional NaOCl then dissolved this rapidly, facilitating more mineral depletion. It is also possible that disintegration of the dentine architecture allowed apatite to fall away, leaving an organic shell. The EDS data also showed that the erosive effect of a final rinse with NaOCl is exaggerated when the application time is 5 minutes rather than 1 minute. This led the authors to recommend that NaOCl should not follow EDTA, or if it does,

40 it should be done with great care, the latter statement possibly meaning to minimize the contact time. SEM experiments measuring % AF confirm this view. The application of NaOCl after a chelator further widens the tubular diameter, an effect which is more pronounced for a 5- minute exposure to NaOCl than a 1 minute exposure (Qian et al. 2011).The type of chelator used is also important. For 10% citric acid, severe erosive effects are seen, but the tubular opening for 17% EGTA is significantly less than for 17% EDTA (Qian et al. 2011). However, using % AF as a measure of erosion can be problematic. Because of dentine heterogeneity, both between and within dentine samples, it seems difficult to define the point where a beneficial result, i.e. smear layer removal, turns into a detrimental one, i.e. dentinal erosion. Dentine hardness studies have also been used to illustrate the differing effect of various chelators in the sequence NaOCl/chelator/NaOCl. A solution of 17% EDTA caused less reduction in dentine hardness than 19% citric acid when Vicker’s testing was employed (Eldeniz et al. 2005). However using the same testing methodology, the use of 17% EDTA reduced dentine hardness compared to 17% EGTA (Sayin et al. 2007b). However another hardness study, employing dentine micro indentation and atomic force microscopy, reported no hardness differences between extracted root filled and extracted controls (Cheron et al. 2011). Mechanical strength testing of whole teeth or roots provides the opportunity to apply force throughout the entire root mass and hence it can be of more clinical relevance. The bite force at maximal clenching in human permanent molars versus anterior teeth has been recorded respectively at 365±160 N and 65±50 N (Kumagai et al. 1999). For first permanent molars in the human adult dentition, the maximum bite force in brachyfacial and dolichofacial types has been measured respectively at 680±120 N and 450±100 N (Abu Alhaija et al. 2010). From the perspective of clinical relevance, experimental fracture loads should be in this range. Additionally, it must be recognized that in reality tooth fracture is associated with fatigue loading and thermal cycling (Torbjörner & Fransson 2004), and the inclusion of such features contributes to the relevance of a fracture study. Of particular clinical relevance are experiments involving strength testing where root filling follows chemomechanical preparation with different irrigant sequences. Because the use of NaOCl as a primary irrigant is universal, it is the final irrigation protocol used that has the potential to influence the fracture resistance of teeth. In such an experiment, which used an irrigation time of 1 minute per irrigant, with the irrigants 2.5 % NaOCl, 5% or 17% EDTA or 2% CHX or water in various combinations, teeth that received a final sequence of EDTA

41 then NaOCl or EDTA followed by NaOCl then CHX, all fractured at a higher compressive load than the teeth where the final irrigant was only water (Turk et al. 2017). The teeth receiving a final rinse with CHX had the best resistance. The use of 5% EDTA rather than 17% EDTA also resulted in improved fracture resistance. A similar study, also with accompanying root canal filling, looked at the effect of EDTA exposure time and concentration on fracture resistance, however 1% NaOCl was used. Again, irrigation with 5% or 17% EDTA, before to the use of NaOCl, resulted in improved fracture resistance compared to when water was used, provided that for 17% EDTA, the exposure time was 1 rather than 5 minutes (Uzunoglu et al. 2012). These results conflict with the SEM, EDS and hardness studies mentioned above. Possibly, this can be explained in terms of the improved bond strength to sealers. When smear layer is removed, bond strengths to dentine are increased (Vilanova et al. 2012). It may also be associated with the larger bulk of tooth structure that was tested. In conclusion, it appears that the results of the studies involving root filling in conjunction with strength testing of whole teeth (Turk et al. 2017; Uzunoglu et al. 2012) contrast with the experiments employing EDS (Wang et al. 2016), SEM (Qian et al. 2011) 3− and III/ PO4 (Tartari et al. 2018b). The former group of studies had higher clinical relevance, and reported no adverse effects of canal irrigation. However, in the later group, where dentine slices or blocks were tested, and therefore arguably less clinically relevant, adverse irrigant effects were reported. Clinical relevance may also help explain the differences noted in the hardness studies. Although the techniques in these studies varied and therefore the results are difficult to compare, the only one reporting no changes in dentine hardness was also the study where the teeth tested had previously undergone in vivo root canal treatment (Cheron et al. 2011).

Alternative irrigants, sequences and strategies for irrigation enhancement

The use of NaOCl is fundamental to endodontic debridement. Although it is used commonly both before and after the chelator EDTA, there is no consensus in the literature concerning the exact details of such irrigation regimens (Qian et al. 2011). Not only can the sequence NaOCl/EDTA/NaOCl be erosive, but EDTA at neutral pH, as is normally used, reacts rapidly with NaOCl, severely reducing its effectiveness (Grawehr et al. 2003). Thus, when NaOCl follows EDTA, additional rinsing steps with water have been advocated

(Clarkson et al. 2011; Haapasalo et al. 2014). The sequence NaOCl/EDTA, while less erosive than its three step counterpart above, lacks a final antimicrobial rinse. Additionally, because NaOCl/EDTA results in the exposure of collagen fibrils, there is some evidence that E. faecalis more readily attaches to the exposed fibrils when compared to three step sequences involving a final rinse with CHX, or to a continuous chelation mixture (Tartari et al. 2018b). Thus, for NaOCl/EDTA there may be an increased potential for reattachment of surviving bacteria that have not been killed in the disinfection process . Furthermore, there is concern that the antibiofilm action of NaOCl is incomplete. In polymicrobial biofilms 2.5 % NaOCl, without a subsequent EDTA rinse, neither removes all the biofilm mass nor kills all the bacteria (Ruiz‐Linares et al. 2017). For biofilms of E. faecalis in dentine tubules, higher concentrations, namely 5.25% rather than 2.5%, and exposure times of 40 minutes are needed for eradication (Retamozo et al. 2010). However, this increased effectiveness at higher concentration comes with greater risks of toxicity from accidental extrusion of irrigant solutions past the apical foramen. Adverse responses to extruded NaOCl, involving severe pain, swelling and ecchymosis, are well documented, particularly for NaOCl solutions at higher concentrations (Hülsmann & Hahn 2000; Glassman 2011; Torabinejad 2011). It is not surprising then that considerable endodontic irrigant research is focused on developing alternative regimens which are both efficacious and with minimal adverse effects. These sequences necessarily involve the use of NaOCl as the main irrigant. They include other three step sequences, often employing CHX, and two step sequences where the final irrigant contains both a chelator and another antimicrobial agent.

1.6.1 CHX

An irrigant sequence involving NaOCl, a chelator and incorporating a final rinse with 2% CHX has been proposed for endodontic irrigation (Haapasalo et al. 2014). This avoids the effects of a final rinse with NaOCl, and indeed, as mentioned, in sequences of CHX, rather than NaOCl, as the final irrigant, teeth exhibit higher resistance to compressive force. However, the disadvantage of this approach is that this final rinse will have a narrower spectrum of action, with studies showing that NaOCl is more effective against polymicrobial biofilms compared with CHX (del Carpio-Perochena et al. 2011; Ruiz‐Linares et al. 2017). CHX is also a component of the formulation QMix™ which additionally contains EDTA and a detergent (Stojicic et al. 2012). QMix is recommended for use following debridement 43 with NaOCl to remove both smear layer, and as final antimicrobial rinse (Basrani & Haapasalo 2014). The action of QMix against polymicrobial biofilms is equivalent to 2% NaOCl (Stojicic et al. 2012), which is less than the concentrations commonly used (Dutner et al. 2012). Furthermore, in the study which reported this result, the concentration of NaOCl was not confirmed, and given the propensity for NaOCl to degrade in storage, the actual NaOCl concentration used may have been less than reported. A caveat on the use of CHX is that it must not come into contact with NaOCl because of a chemical reaction between the two resulting in the formation of an orange-brown precipitate (Basrani et al. 2007). Although there is debate in the literature about the actual composition of this by-product (Wright et al. 2017b), it resembles the toxic substance chloroguanidine (Nowicki & Sem 2011) and it can occlude the dentinal tubules (Bui et al. 2008; Gasic et al. 2012). Before the use of QMix or CHX, an intervening rinse of saline is advised (Basrani & Haapasalo 2014).

1.6.2 MTAD and Tetraclean

The commercial formulations BioPure™ MTAD and Tetraclean™ both combine citric acid, doxycycline and a detergent with the former containing a higher concentration of doxycycline (Basrani & Haapasalo 2014). The detergent component also differs. MTAD contains Tween 80, while Tetraclean contains cetrimide (Pappen et al. 2010). Because of the combination of an antibiotic and a chelator, both were formulated as a final rinse following the use of NaOCl (Haapasalo et al. 2010). The cetrimide component of Tetraclean gives it a superior antimicrobial action compared to MTAD (Pappen et al. 2010). The recommended time for the application of a final rinse with MTAD is 5 minutes, during which time it will remove smear layer with no resulting erosion (Torabinejad et al. 2003). However, the statement that the sequence 5.25% NaOCl/17% EDTA causes more erosion than 5.25% NaOCl/MTAD is unjustified. In the relevant study (Torabinejad et al. 2003), the application time for EDTA was 5 minutes. While this is appropriate for MTAD, it is significantly longer than the recommended treatment time for EDTA. As was the case for citric acid (Reis et al. 2008), after the application of MTAD, SEM images, appearing in the publication concerned (Torabinejad et al. 2003), show that even though the tubules are free of smear layer, debris may remain in some tubules. While the usage of MTAD as a secondary irrigant is somewhat prevalent in the United States (Dutner et al. 2012), there are some problems associated with its usage. A reaction

44 occurs between MTAD and NaOCl. This probably involves oxidation of the doxycycline by NaOCl, and decreases the antimicrobial effectiveness of NaOCl (Tay et al. 2006b). Additionally, when NaOCl and MTAD come into contact, a yellow precipitate forms and purple staining of the dentine occurs over time (Tay et al. 2006a). This has prompted the recommendation that ascorbic acid be used in between irrigation with NaOCl and rinsing with MTAD (Tay et al. 2006a). Additionally of concern is the rise of antibiotic resistance (Clatworthy et al. 2007), and the use of antibiotics in preference to biocides has been discouraged (Zehnder 2006).

1.6.3 Peracetic acid

At 2.0%, PAA acid causes excessive loss of the inorganic component of dentine (Tartari et al. 2018a). However, at the lower concentration of 0.5% there has been some interest in the use of PAA as a final rinse following NaOCl (Fernandes et al. 2015). A solution of 0.5% PAA can chelate Ca2+ from dentine similarly to EDTA. It will dissolve smear layer in 60 seconds (De‐Deus et al. 2011). PAA is also an antimicrobial agent. A final rinse of 1% PAA eradicates E. faecalis to a similar extent as 17% EDTA followed by 2.5% NaOCl (Cord et al. 2014). However, the erosive capacity of a 1% solution has not been explored.

1.6.4 Photoactivated disinfection (PAD) and other strategies for improving irrigation

PAD has been used successfully as an adjunctive form of antimicrobial therapy following NaOCl/EDTA sequences (Rios et al. 2011; Trindade et al. 2015). PAD is a non- thermal photochemical process which uses photosensitive dyes to absorb photons of laser or LED light energy, leading to the production of reactive oxygen species. These reactive species kill bacteria (Singh et al. 2015). Research has focused on improving PAD because, when used alone, it only achieves a 1-2 log reduction of the bacterial load (Trindade et al. 2015). Nanoparticles have been used with PAD, as they can be loaded with photosensitizers for enhancing the antimicrobial actions (Pagonis et al. 2010; Shrestha & Kishen 2014). Because photosensitizer dyes are highly coloured, staining of tooth structure is a potential problem. While it is not the focus of this literature review, the use of agitation techniques to enhance antimicrobial action, smear layer removal and tissue dissolution has received

45 considerable attention in the literature. Fluid dynamic studies show that standard needle irrigation results in the stagnation of irrigants apically (Boutsioukis et al. 2009), with minimal flow directed laterally onto the canal wall (van der Sluis et al. 2014). Several agitation devices have been developed to enhance irrigation by improving mixing, refreshment and delivery. A simple and inexpensive method of agitation is achieved by the manual plunging of gutta-percha points into irrigant solutions in the root canal (Caron et al. 2010). The EndoVac™ is a negative pressure device that was introduced to overcome the problem of apical vapour lock. Its use enhances the removal of smear layer apically (Akyuz Ekim & Erdemir 2015). Sonic devices in use include the EndoActivator™ (Caron et al. 2010) and the Vibringe™ (Glassman 2011) and the EDDY® which has been shown to enhance removal of canal debris (Urban et al. 2017). However, sonic devices do not generate the complex microstreaming and cavitation that occurs with lasers and ultrasonics (van der Sluis et al. 2014), and several studies demonstrate the inferior action of sonic devices (Ordinola‐ Zapata et al. 2014; Guneser et al. 2015; Jiang et al. 2016). GentleWave™ is a multi-sonic device advocated for effective root canal debridement with minimal canal preparation (Molina et al. 2015). Ultrasonic agitation causes complex streaming of irrigants with the formation of jets and cavitation (van der Sluis et al. 2014). Its use in endodontics enhances antimicrobial effects (Ordinola‐Zapata et al. 2014), and the removal of smear layer (Akyuz Ekim & Erdemir 2015) and debris (De Moor et al. 2009; Deleu et al. 2015) compared with standard needle irrigation. Finally, laser activation of irrigants has become popular to augment disinfection (Jaramillo et al. 2012; Olivi et al. 2014), remove smear layer (George et al. 2008b), dissolve organic material (Guneser et al. 2015) and remove debris, particularly in areas of complex anatomy such as isthmuses (Li et al. 2015).

1.6.5 Conclusion to Section 1.6

Although the use of NaOCl is nonnegotiable in contemporary endodontics, protocols involving NaOCl are not standardized and do not fully remove biofilm. This has led to the development of new devices, irrigants and sequencing protocols. One positive aspect of this is the development of new sonic, ultrasonic and laser devices, all of which create irrigant agitation. Such agitation is beneficial for canal disinfection, tissue dissolution and smear layer removal. However, there is now a vast choice of irrigants and irrigant sequences, many

46 with their own associated problems. This adds to the complexity of choice for the clinician. It also indicates the need to simplify irrigation protocols and this has led to the notion of combining a chelator and NaOCl, a topic which is the focus of the next section.

Continuous chelation in endodontic irrigation

In the search for new and improved methodologies and strategies in endodontic irrigation, the concept of continuous chelation was introduced in 2005 (Zehnder et al. 2005), with the actual terminology being used first in 2012 (Neelakantan et al. 2012). Continuous chelation involves mixing a chelator with NaOCl for use as a sole irrigant throughout the chemomechanical preparation of the root canal, to simultaneously perform all three core roles of an endodontic irrigant. A body of literature now exists, consisting of over 50 journal articles. The research scope is wide, covering not only core irrigant functions but also examining other aspects of irrigation such as bond strengths to endodontic materials, instrument lubrication, surface tension, dentine debris, effects on dentine properties, safety and toxicity. The work in this field centres on the mixture etidronate-NaOCl, with a minor contribution from alkaline EDTA mixtures.

1.7.1 Maintenance of FAC

Due to the oxidizing potential of NaOCl, mixing many chemical substances with NaOCl causes chemical reactions (Wright et al. 2017b). Thus, in continuous chelation, the maintenance of NaOCl levels, as measured by FAC, is of prime importance. When FAC levels decline, so does the ability of NaOCl to dissolve tissue (Clarkson et al. 2006) and kill microorganisms (Retamozo et al. 2010; Morgental et al. 2013). The reduction of FAC over time determines the useful lifespan of mixtures. This is otherwise known as the therapeutic window or “use-life” (Wright et al. 2019).

1.7.1.1 Measurement of FAC and iodometry

FAC measurements report chlorine (Cl2) concentrations in g/L (Standards Australia − 2003). The FAC is the amount of Cl2 released when OCl decomposes, in an acidic environment and in the presence of chloride (Cl−), with 1 mole of OCl− producing 1 mole of

Cl2 according to equation (2) (Vogel 2000). Because the molecular weight of Cl2 is 70.9 and 47 that of NaOCl is 74.44 (NCBI), FAC weight/volume (wt/v) can be converted to NaOCl (wt/vol) by multiplying by a factor of 1.05. Thus 47.62 g/L FAC is 50g/L NaOCl or 5% NaOCl. FAC can be measured by a variety of methodologies, including colorimetric assays such as diethyl-p-phenylene diamine where a red colour change occurs with oxidation by 2- Cl2 (Palin 1957), or by standard iodometry involving the use of thiosulphate (S203 ) (Standards Australia 2003). Alternatives include amperometry (Wilde 1991) and thermometric titration, which also measures other ions that are present such as chlorate and chlorite (Clarkson et al. 2013). In continuous chelation, FAC has been measured only by iodometry (Biel et al. 2017; Tartari et al. 2017; Zollinger et al. 2018), and thus direct comparisons between studies are possible. The basis of iodometric titration is the reaction between OCl− with colourless iodide − 2− (I ) to form brown iodine (I2), according to equation (3).The I2 then reacts with S203 to form I−, according to equation (4), at which point the solution becomes colourless again (Vogel − 2− 2000). From equations (3) and (4) it is seen that 1 mole of OCl reacts with 2 moles of S203 . The amount of OCl− consumed is then typically expressed as FAC. Starch can be added for − 2− a more visual discernment of the endpoint (Vogel 2000). Before titration with OCl , S203 − must first be standardized. Iodate (IO3 ) has a purity of 99.9% and is used for this purpose − − (Vogel 2000). IO3 reacts with acidified I as in equation (5) (Vogel 2000), and which − combined with 3x equation (4) results in the overall equation (6). Hence, 1 mole of IO3 2− reacts with 6 moles of S203 .

− − + OCl + Cl +2H = Cl2 + H2O (2) − − + − OCl + 2I + 2H Cl + I2 + H20 (3) 2− 2− − 2S203 + I2 S4O⇌6 + 2I (4) − − + IO3 + 5I +6H⇌ = 3I2 +3 H20 (5) − + 2- − 2− IO3 + 6H +6S203 = I + 3S4O6 + 3H20 (6)

1.7.1.2 FAC measurements in continuous chelation

The factors reported in the literature as influencing FAC in continuous chelation mixtures are the type of chelator, NaOCl and chelator concentrations, and the time from mixing (Zehnder et al. 2005; Biel et al. 2017). A seminal study in 2005 combined various chelators with 1% NaOCl in differing ratios (Zehnder et al. 2005). The FAC was measured at 1 minute, 1 hour, 1 day and 21 days. The chelators used were 17% EDTA (pH = 8.2-8.4), 48

10% citric acid (pH = 2.4-4.3), sodium triphosphate (pH = 9.3-10.6), 15% aminotris(methylene phosphonic acid) (ATMP), referred to in the study as ATMA (pH = 0.6- 1.1), and 7% 1-hydroxyethylidene-1,1-bisphosphonate (pH = 10.6-11.3), also known as etidronate or HEDP (NCBI 2018). While the sodium triphosphate mixtures maintained all their FAC, even at 21 days, they were not able to remove smear layer and were eliminated. However, the etidronate mixtures maintained reasonable FAC at 1 hour and showed some capacity to remove smear layer. All other chelator mixtures lost FAC rapidly. The study above illustrates the importance of the pH of the chelator solution. Only those with high pH, etidronate and sodium triphosphate, in mixtures with NaOCl maintained their FAC over time. Acidic mixtures were not stable. This is explained by equation (2), and indeed, the poor performance of the acidic chelators could have been predicted by this equation. The incompatibility of EDTA at slightly alkaline pH mixed with NaOCl is supported by other work (Grawehr et al. 2003; Clarkson et al. 2011). However, if the tetrasodium salt of EDTA (Na4 EDTA), rather than Na2 EDTA or Na3 EDTA, is dissolved in water, the resulting pH is considerably higher, at around pH 12 (Tartari et al. 2017).

Na4 EDTA-NaOCl mixtures have been examined for use in continuous chelation because of their high pH. There is a clear and marked influence of both the chelator and NaOCl concentration on FAC levels. In one study, mixtures containing concentrations of 3%

Na4 EDTA and 1% NaOCl (3% Na4 EDTA-1% NaOCl) or 18% Na4 EDTA-1% NaOCl, maintained respectively 17% and 6% FAC at 1 hour, and when the NaOCl concentration increased, for 3% Na4 EDTA-5% NaOCl and 18% Na4 EDTA-5% NaOCl, only 5 % and 1% of baseline FAC were maintained at 10 minutes (Biel et al. 2017). Thus, increases in both

NaOCl and Na4 EDTA concentrations reduce FAC. Comparisons between studies can be difficult because of the variations in concentrations used. However, a second study reporting FAC levels of 62% and 49% of baseline after 1 hour for the mixtures 5% Na4 EDTA-2.5% NaOCl and 10% Na4 EDTA-2.5% NaOCl respectively (Tartari et al. 2017), did not concur with the first. In the second study increased NaOCl and chelator concentrations corresponded to a higher FAC, yet a lower FAC result is expected. The pH measurements in the second study are also inconsistent with other research, because when the pH fell from around 12 to 9, FAC was maintained at high levels. In other studies, the decline in FAC is mirrored by a corresponding fall in pH (Arias-Moliz et al. 2016; Biel et al. 2017). Thus, it is possible that there was a methodological error in the FAC data in the second Na4 EDTA-NaOCl study.

The issue of a pH fall is an important one, because under pH 9, little OCl− is present (Figure 1-1), and hence the capacity to dissolve organic material is also reduced. When dentine is present, both the pH and FAC fall more rapidly (Arias-Moliz et al. 2016). In in vitro studies in the absence of dentine, the maintenance of high levels of pH therefore becomes important to counter this effect, and it is important to record pH concurrently with FAC data. Authors have reported a superiority of FAC maintenance in the alternative mixture, tetrasodium etidronate (Na4 etidronate)-NaOCl (Biel et al. 2017; Zehnder et al. 2005). Na4 etidronate is cited in the literature under a variety of common names including Na4 HEDP (NCBI 2018) and tetrasodium 1-hydroxyethane 1,1-diphosphonic acid (Deari et al. 2019).

Dissolving Na4 etidronate also results in a high pH solution, above pH 11 (Deari et al. 2019).

Na4 etidronate has a ligand stability constant of 6.04-6.08 (Smith & Martell 1989), thus it is a weaker chelator than EDTA. There is reasonable agreement between studies on the residual FAC in etidronate mixtures with NaOCl. For 9% etidronate-5% NaOCl, after 1 hour, the effective NaOCl concentration maintained 74% FAC (Zollinger et al. 2018) or 86% in a second study (Biel et al. 2017). As was the case for Na4 EDTA, these declines in FAC are concentration dependant. When the NaOCl concentration is halved to 2.5%, in mixtures with 9% etidronate, after 1 hour, two studies reported a 92-96% maintenance of the original NaOCl concentration (Arias-Moliz et al. 2014; Zollinger et al. 2018). A third study however reported only 80% of the baseline FAC at 10 minutes (Arias-Moliz et al. 2016). The improved stability of etidronate mixtures with 2.5% NaOCl is possibly the reason that a recent clinical trial was performed with etidronate solutions of 9% etidronate-2.5 % NaOCl (Ballal et al. 2019b). A selection of the results from the only FAC studies in continuous chelation in the literature from 2005-2018 involving chelator-NaOCl solutions are listed in Table 1-5. A salient feature of the seven studies is that none were performed at root canal temperature. Only one contains FAC measurements at a temperature warmer than room temperature (Zollinger et al. 2018). In that study, solutions originally containing 1%, 2.5% or 5% NaOCl, after 1 hour at 60oC, were found to contain 0.0% 0.0% and 0.2% NaOCl, respectively. This result is not unexpected because chemical reactions are hastened by heating. At root canal temperature, there may also be increased deterioration of FAC, and this clinically relevant temperature requires investigation.

Table 1-5. Studies in continuous chelation with FAC data from 2005-2018.

Author and date NaOCl Chelator and Min Result and NaOCl-chelator combination. Conc Conc Result expressed as a % of theoretical maximum for FAC or NaOCl conc.

Arias-Moliz et al. 2016 2.5% 9% Na4 etidronate 10 2.5% NaOCl-9% Na4 etidronate = 80

Arias-Moliz et al. 2014 2.5% 9% Na4 etidronate 60 2.5% NaOCl-9% Na4 etidronate = 96

Biel et al. 2017 5.0% 9% Na4etidronate 60 5%NaOCl-9% Na4 etidronate = 86

1.0% 18% Na4 EDTA 60 5%NaOCl-18% Na4 EDTA = 1

3% Na4 EDTA 60 5% NaOCl-3 % Na4 EDTA = 3

10 5% NaOCl-18% Na4 EDTA =1

10 5% NaOCl-3% Na4 EDTA = 5

10 1% NaOCl-18% Na4 EDTA = 10

10 1% NaOCl-3% Na4 EDTA = 93

1 5% NaOCl-18% Na4 EDTA = 5

1 5% NaOCl-3% Na4 EDTA = 96

Girard et al. 2005 0.9% 1.6% 60 0.9% NaOCl-1.6% Na4 etidronate = 96

Na4etidronate

Tartari et al. 2017 2.5% 10% Na4 EDTA 60 2.5% NaOCl-10%EDTA = 49

5% Na4 EDTA 60 2.5% NaOCl-5%EDTA = 62 10 2.5% NaOCl-10%EDTA = 83.5 10 2.5% NaOCl-5%EDTA =90

Zehnder et al. 2005 0.83% 1.17% 60 0.83% NaOCl-1.17%Na4etidronate =

0.5% Na4etidronate 60 100

3.5% 0.5% NaOCl-3.5% Na4 etidronate =80

Na4etidronate

Zollinger et al. 2018 2.5% 9% Na4 etidronate 60 2.5% NaOCl-9% Na4 etidronate = 92

5.0% 60 5.0% NaOCl-9% Na4 etidronate = 74 Conc, concentration. Studies were conducted at room temperature. All concentrations are final concentrations in the mixtures. Only results for selected NaOCl and chelator concentrations and time points are shown. None of the results include experiments where dentine was added.

It should be pointed out that confusion potentially exists in the literature concerning etidronate, because of the purity of the chemicals used. The etidronate in all the FAC experiments is derived from either of two sources, Cublen K8514 GR (Girard et al. 2005; Zehnder et al. 2005; Arias-Moliz et al. 2014; Arias-Moliz et al. 2016; Biel et al. 2017), made by Zschwimmer-Schwarz, or the commercial product Dual Rinse® HEDP (Zollinger et al. 2018). According to company information, from Zschwimmer-Schwarz in the case of the Cublen product (Zschimmer & Schwarz 2018), and from Medcem for Dual Rinse HEDP (Medcem 2016), both products contain only 85% etidronate. It is feasible that authors are unaware of this, and if they are, none stipulate that they have taken this into account. Hence it is possible that the etidronate concentrations are actually less than stated. Because lower concentrations are associated with higher levels in FAC, the FAC data for etidronate mixtures could be overly optimistic.

1.7.2 Removal of dentine debris and smear layer

There is some evidence to suggest that fewer dentine debris accumulate during continuous chelation. Using micro-computed tomography, the accumulation of debris in the isthmuses of the mesial roots of mandibular molars was less when teeth were irrigated with

9% Na4 etidronate-2.5% NaOCl compared to 2.5% NaOCl (Paqué et al 2012). However, the comparison with NaOCl only is inequitable because rinsing with EDTA following the application of NaOCl is known to reduce dentine debris accumulation (Paqué et al 2011). The comparison of etidronate mixtures with standard sequences would have been more conclusive. The smear layer studies using continuous chelation can be divided into two broad categories: those that contain groups where the continuous chelation mix has been used throughout (Zehnder et al. 2005; Lottanti et al. 2009; Tartari et al. 2017), and those with only a final rinse of either a plain chelator solution or a continuous chelation mixture (De-Deus et al. 2008b; Yadav et al. 2015; Ulusoy et al. 2017; Patil et al. 2018; Deari et al. 2019; Erik et al. 2019). The studies employing only a final rinse of a chelator only use etidronate, and serve to illustrate the difference in chelation in teeth between 17% EDTA, at near neutral pH, and etidronate at around pH 11. From the lower stability constant for etidronate and the higher pH of etidronate solutions it can be predicted that etidronate will remove less smear layer than EDTA at neutral pH. This was indeed found to be the case (De-Deus et al. 2008b; Deari et al. 2019).

However another study where root canals were irrigated for only 1 minute reported no significant differences (Ulusoy et al. 2017).The pH of the etidronate mixture was not stated, and hence a possible explanation is that the etidronate used was not highly alkaline. The latter study raises an important feature of some of the smear layer studies in continuous chelation. Some authors (Yadav et al. 2015; Emre Erik et al. 2019) cite neither the pH nor the brand of etidronate that was used. The results of these studies cannot therefore be compared with others and their validity is questionable. Some authors also fail to understand that continuous chelation uses a chelator-NaOCl mixture throughout the entire irrigation process, because they include groups where the mixture is used as a final rinse for short time periods. Because etidronate is a weak chelator and is used at high pH, it requires longer to remove smear layer than chelators which either have a higher stability constant or are acidic. Using etidronate mixtures for 3-5 minutes as a final rinse, following NaOCl, and comparing them to a similar application of solutions known to chelate well in such time frames such as MTAD (Yadav et al. 2015; Patil et al. 2018) or standard 17% EDTA (Emre Erik et al. 2019) is of little avail. The most relevant research used continuous chelation mixtures throughout the whole irrigation process. There is one study which used Na4 EDTA mixtures. It reported no differences between dentine discs treated for 3-5 minutes with 17% EDTA at pH 8 or 5-10%

Na4 EDTA- 2.5% NaOCl mixtures at pH 12 (Tartari et al. 2017). Hence, despite the short time frame, and the higher pH and lower concentration of the continuous chelation mix, it removed smear layer just as well. Possibly a part of the explanation for this result was that the disc exposed to 17% EDTA had no exposure to NaOCl, thus reflecting the necessary adjunctive role of NaOCl in smear layer removal.

For etidronate mixtures, when dentine discs are exposed to 9% Na4 etidronate-1% NaOCl over 15 minutes, smear layer is removed, but less Ca2+ is eluted from the root canal compared to 1% NaOCl for 15 minutes followed by 17 % EDTA (pH 8) for 3 minutes (Lottanti et al. 2009). In a second study (Zehnder et al 2005), 17% EDTA removed smear layer better than 3.5% Na4 etidronate-0.5% NaOCl over 1 minute. This can be explained by both the low etidronate concentration and the short time period.

1.7.3 Organic tissue dissolution

Because FAC is lowered over time in continuous chelation mixtures, some diminution in the ability to dissolve organic tissue may result. There is only one tissue dissolution study

53 using the chelator Na4 EDTA. For mixtures of 5-10% Na4 EDTA-2.5% NaOCl over 15 minutes, compared to a 2.5% NaOCl control, the percentage reduction in bovine muscle was not significantly different (Tartari et al. 2017). This contrasts with the reported results for etidronate mixtures. For 9% Na4 etidronate-2.5% NaOCl, also using bovine muscle, 27% and 41% reductions in tissue mass were seen respectively for the etidronate mixture and the control (Tartari et al. 2015).

The better result for the Na4 EDTA mixture compared to the Na4 etidronate mixture is surprising, given the greater maintenance of FAC in the latter. Part of the reason is the different conditions under which the experiments were conducted. The EDTA experiment was presumably conducted at room temperature, because that experimental detail was not included. It also included irrigant refreshment every 5 minutes. However, it is unclear whether the new irrigant was a freshly mixed batch or more of the original mix. If it was a freshly mixed batch, then the effect of a decaying FAC level in the irrigant was not accounted for. The etidronate experiment was carried out at 32oC without irrigant refreshment. The rise in temperature possibly reduced the FAC, given that heat increases the rate of chemical reactions. In addition, the lack of refreshment meant that the FAC in the irrigant decayed over time. A further etidronate study looked at the effect on the removal of minced bovine muscle, packed into concavities created within human root canals, using passive ultrasonic irrigation and the XP-endo® shaper (Ulusoy et al. 2018). The irrigant groups were the 2.5% NaOCl, the sequence 2.5% NaOCl/17% EDTA, 9% etidronate-2.5% NaOCl and water. In the case of no activation or passive ultrasonic irrigation the sequence resulted in the same amount of tissue removal as the water control, while the effects of the etidronate mixture were similar to plain NaOCl. The result is not surprising given that all irrigants were present in the canal for only 2 minutes. For the sequence, there was a 1 minute application of NaOCl followed by another minute of EDTA. Thus, the samples in the sequence were exposed to NaOCl for half the time compared with either the NaOCl control or the etidronate-NaOCl mixture. Thus, it could not be expected to dissolve as much organic tissue. The similar result for the NaOCl control and the etidronate mixture is also reasonable given the short time frame. Interestingly, this same experiment also reported that agitation did not alter the efficacy of the etidronate-hypochlorite combination. It could be expected that, like heat, agitation might cause an increased likelihood of molecules colliding and reacting. However, over 2 minutes, this effect may be negligible.

The three studies discussed above highlight the importance of differences in experimental details between investigations and the interpretation of their effect on the results. Differing conditions resulted in a mixed result for the etidronate mixture. Whether or not etidronate mixed with NaOCl does in fact impede organic tissue dissolution needs to be clarified.

1.7.4 Antimicrobial activity

This review, as far as can be known, assesses all published papers which included a chelator-NaOCl mixture as an irrigant throughout the disinfection process that were published from 2005-2019. Studies that used plain chelators only were omitted. The research on the antimicrobial effects of continuous chelation, except for a clinical trial (Ballal et al. 2019b), have exclusively used E. faecalis (Zehnder et al. 2005; Arias-Moliz et al. 2014; Arias-Moliz et al. 2015; Neelakantan et al. 2015; Arias-Moliz et al. 2016; Morago et al. 2016; Solana et al. 2017; Tartari et al. 2018b; Morago et al. 2019). One study additionally included Candida albicans (Tartari et al. 2018b). All but two studies involved biofilm disinfection. The in vitro biofilm studies stated that disinfection occurred in 3-10 minutes, and that the chelator and the NaOCl were mixed immediately prior to use. The latter detail is important because, as seen in the FAC studies, continuous chelation mixtures lose their FAC, and hence antimicrobial effectiveness over time. If different studies used varying times between mixing and the start of the experiment, comparisons would be difficult as the actual NaOCl concentration would not be known. However, because mixing occurred immediately before experimentation, the studies do not reflect longer clinical scenarios and the effect of a decaying irrigant. In this respect the FAC data over time becomes crucial.

1.7.4.1 Na4 EDTA

Only one study has assessed alkaline EDTA. It concluded that 5% or 10% EDTA- 2.5% NaOCl was as effective on 3 week biofilms as 2.5% NaOCl (Solana et al. 2017). The methodologies used were colony counting and the adenosine triphosphate assay. When 0.2% cetrimide was added to the mixture, the result was the same. Thus, the addition of a detergent may be unnecessary.

1.7.4.2 Na4 etidronate

The initial antimicrobial evaluation of etidronate in continuous chelation was with a planktonic suspension (Zehnder et al. 2005). In this study 3.5% etidronate-0.5% mixtures together with a 1/10 and a 1/100 dilution were tested. The disinfection time was 15 minutes, and the final bacterial concentration in the disinfection solution was 106/mL. In another planktonic study, not involving continuous chelation, with a final concentration of 0.9% NaOCl and 2.25 x 107/mL E. faecalis, no viable bacteria remained after 5 seconds (Stojicic et al. 2012). Thus, the disinfection time for the continuous chelation mixture was long, and the bacterial challenge used was low. The conclusion that continuous chelation was as effective as 0.5% NaOCl needs verification. However, the study did serve to show that 3.5% etidronate was not antimicrobial when tested over 15 minutes. The only other study not employing a biofilm examined the potential for reinfection following irrigation using a 2 hour growth period for E. faecalis and for C. albicans (Tartari et al. 2018b). The methodology employed was the live/dead technique. A finding of this study was that, compared to the standard sequence 2.5% NaOCl/17% EDTA, fewer viable E. faecalis were attached when 9% etidronate-2.5 % NaOCl was used. The opposite occurred for C. albicans. The result for E. faecalis was attributed to the higher amounts of exposed collagen in the etidronate mixture. Interestingly the mixture had significantly better wettability than any of the other irrigant protocols, but this was not linked to improved antimicrobial activity. Typically, the in vitro studies using etidronate involved the disinfection of 5-7 day biofilms (Arias-Moliz et al. 2014; Arias-Moliz et al. 2015; Arias-Moliz et al. 2016; Morago et al. 2016; Morago et al. 2019), with only one study reporting a 4-week biofilm growth period (Neelakantan et al. 2015). All the in vitro biofilm experiments used live/dead staining as the assessment method to examine the extent of disinfection occurring in the dentinal tubules. However, two studies additionally employed colony counting (Arias-Moliz et al. 2014; Neelakantan et al. 2015). As has already been discussed, the use of CFU methodology can be problematic. The heavy reliance on live/dead staining also needs to be addressed, particularly in view of its application to the tubules only, with no application of the technique to the main canal. Of specific relevance to continuous chelation is that chelators can overestimate the dead bacterial count in the live/dead technique (Wang et al. 2013), as discussed in Section 1.2.4.2.

A case in point is the discrepancy between the live/dead and CFU result seen for a 9% solution of plain etidronate (Arias-Moliz et al. 2014). In this study using live/dead staining, 9% etidronate was as effective an antimicrobial agent as both 2.5% NaOCl and 9% etidronate-2.5% NaOCl. However, the CFU methodology used produced only a 1-log reduction for plain etidronate solutions, yet the biofilms were eradicated for the groups containing NaOCl. The authors explained that the high pH in the etidronate solution was antimicrobial, and that considerably heavier bacterial load for the microbial count experiment, resulted in a lowering of pH in the alkaline etidronate solution. Thus, it was reasoned that in the CFU experiment there was no real antimicrobial effect. This compared to the live/dead result in the tubules with a low bio load and hence the pH was presumably higher. While this is certainly possible, it is also likely that the pH was also lower in in the dentinal tubules. This could occur because alkaline solutions are buffered by dentine (Wang & Hume 1988) Thus, it is feasible that the live/dead staining technique, when used over the 10-minute disinfection period, caused an unduly high red fluorescence signal. In contrast, in an experiment over 3 minutes, for the 9% plain etidronate, the percent of live bacteria was lower, but not significantly different from the control (Arias-Moliz et al. 2016). There are probably various factors at play, such as chelator strength and time of exposure. For a standard 17% EDTA solution, this effect was noticed at both 3 and 10 minutes (Wang et al. 2013), but not for 8.5% EDTA over 1 or 3 minutes (Wang D et al. 2017). Lastly, when considering total biovolume and thus disregarding the potential for error in counting bacteria as dead when they are not, 9% etidronate showed a minor capacity to remove biofilm (Arias-Moliz et al. 2016). This was attributed to its action as a chelator. There is a consensus that 9% etidronate-NaOCl mixtures are at least as antibacterial as NaOCl of the same concentration (Arias-Moliz et al. 2014; Arias-Moliz et al. 2015; Neelakantan et al. 2015; Arias-Moliz et al. 2016; Morago et al. 2016; Ballal et al. 2019b; Morago et al. 2019). Some authors report additional efficacy under certain conditions. In the presence of dentine powder, solutions of 9% etidronate-1% NaOCl recorded significantly lower biofilm biovolume and higher levels of bacterial killing than 1% NaOCl controls (Arias- Moliz et al. 2016). The explanation given was that the etidronate assisted by detaching bacteria from the biofilm. Although the effective NaOCl concentration in the presence of dentine powder had fallen to 0.5%, this was still sufficient to kill the bacteria that were now essentially planktonic. When the NaOCl concentration was at 2.5% there were no differences between NaOCl controls and etidronate mixtures.

The presence of smear layer is another situation reported as increasing the antimicrobial actions of etidronate mixtures relative to NaOCl controls. For mixtures of 9% etidronate and 2.5% NaOCl, there was greater bacterial death than for 2.5% NaOCl alone in the presence of smear layer (Morago et al. 2016). Both the dentine powder and smear layer studies above involved blocks of dentine that were soaked in irrigant for 3 minutes. A further third study that involved the chemomechanical preparation of 12 mm sections of roots with single canals conversely reported no differences between 2.5% NaOCl followed by 17% EDTA and 9% etidronate- 2.5% NaOCl (Morago et al. 2019). This was despite the fact that the canal preparation would have resulted in the production of both dentine debris and smear layer. Some understanding of the difficulty in interpreting the results of the above mentioned three studies can be gained from the following considerations. The root canal experiment is more clinical in nature. The use of root canal instrumentation would have resulted in only partial instrument contact with the root canal (Peters et al. 2001), and hence smear layer coverage of the canal was incomplete. Secondly, irrigation provided the opportunity to flush debris out of the canal. Thus, it is possible that the more simplistic nature of the two dentine block studies produced a heavier smear layer, and the irrigant solutions contained more debris than were present in the clinically orientated root canal experiment. Conversely, it is true that the use of single rooted teeth was not representative of teeth with complex anatomy, such as molar teeth are known to accumulate debris (Paqué et al 2011). If instead, a molar had been used, then the third experiment could have contained enough debris to show the additional effect noticed in the first two experiments. This is because the molar would have accumulated debris in areas of complex anatomy. Finally, a second irrigation study, using 8 mm lengths of the coronal-middle sections of root canals, did report an advantage in a mixture of 9% etidronate-5% NaOCl compared to 5% NaOCl followed by 17% EDTA (Giardino et al. 2019). However, in this second irrigation study, no accompanying chemomechanical preparation occurred. Thus, it was less clinically relevant. Although neither smear layer nor dentine debris were created, the bacterial load in the root canal would have been significantly higher than in the first irrigation experiment because of the lack of canal preparation. This may have provided the opportunity for the constant presence of etidronate to be beneficial. The author suggested that the increased hypertonicity in the etidronate mixture may have been responsible. Alternatively, more bacteria may have been pulled from the canal wall, as previously suggested. Ultimately, it is difficult to know whether or not etidronate-NaOCl mixtures are additionally

58 beneficial compared to NaOCl controls or to standard sequences. Comparing studies conducted under varied conditions is problematic, and further experimentation including molar teeth is required. The most relevant study concerning the antimicrobial effect of continuous chelation is a recent clinical trial which compared irrigation with 9% etidronate-2.5% NaOCl to irrigation with 2.5% NaOCl (Ballal et al. 2019b). The study used paper points for sampling of the canal bacteria, and as previously alluded to in the “Substrates” part of Section 1.2.4.1, this is a limitation of such studies. Both single and multirooted teeth were incorporated, although, in the multirooted teeth, only roots with single canals were included. The reason given for omitting a comparison with a standard sequence was that the trial’s objective was to determine if the addition of etidronate altered the action of NaOCl or caused any untoward effects. While trial numbers were low, canal sampling revealed that the etidronate mixture and plain NaOCl resulted in canals free of bacteria in respectively 50% and 40% of cases. However, the difference between the two protocols was not significant. A comparison with a standard endodontic sequence would seem a logical next step.

1.7.5 Detrimental effects on dentine

Studies examining the possible detrimental effects of chelator-NaOCl mixtures on dentine are limited. Given the proteolytic action of NaOCl and the demineralizing capacity of chelators, assessments in this area are important. For combinations of Na4 EDTA with NaOCl, only one such study exists (Tartari et al 2018a). In that study, after exposure of dentine to 5% Na4 EDTA-2.5% NaOCl over 5 or 10 minutes, the amide III/phosphate ratio remained unaltered. This means that the collagen dissolution was matched by demineralization. However, it does not imply that the dentine thickness was maintained. The same study examined 9% Na4 etidronate-2.5% NaOCl mixtures and found that the ratio was decreased. Thus, demineralization was comparatively less compared to collagen dissolution. This agrees with the fact that, as is seen later in Table 3-1 in Section 3.1.2, the ligand stability for etidronate is less than that for EDTA and hence more demineralization is to be expected with the latter than the former. An SEM study, at low magnification and using dentine discs, examined the differences in the length of demineralization zones caused by mixtures of 9% Na4 etidronate- 1% NaOCl over 18 minutes (Lottanti et al 2009). The study reported no observed erosive

59 effects for the mixture, while erosion was observed in dentine treated with the sequence 1% NaOCl for 15 minutes followed by 17% EDTA ( pH = 8.0) for 3 minutes. In contrast, a more clinically relevant study using roots found that irrigation with the sequence 2.5% NaOCl/17% EDTA was less likely to cause alterations in mechanical strength than irrigation with 9% etidronate-2.5% NaOCl (Domínguez et al. 2018). The fracture resistance to compressive force was lower for the continuous chelation mixture than it was for the sequence.

1.7.6 Safety, bond strength

One in vitro study (Ballal et al. 2019a) and one in vivo clinical trial (Ballal et al. 2019b) report safety data for the most commonly used mixture in continuous chelation, Na4 etidronate-2.5%. The aim of both was to determine if any safety concerns resulted from the addition of etidronate to plain NaOCl solutions. The in vitro study examined cell viability with the use of the tetrazolium dye MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and the clonogenic assay. Additionally, genotoxicity was evaluated by a micronucleus test. For all assays, no additional effects were seen for the etidronate-NaOCl mixture. The clinical trial also evaluated post-operative pain at 24 hours and levels of matrix metallopeptidase 9 immediately post treatment and at 1 week. A reduction in the latter indicated a decline in the inflammatory response (Ballal et al. 2019b). Again, no differences were seen between groups, with both reporting minimal pain at 24 hours and significant decreases in metallopeptidase 9 levels. Thus, the addition of etidronate to NaOCl does not appear to raise safety concerns based on these assessments. Irrigation protocols are known to effect the bond strength of endodontic materials to dentine (Prado et al. 2013). Three studies have evaluated the pushout bond strength following irrigation with Na4 etidronate-NaOCl mixtures compared to the sequence NaOCl/17% EDTA. Whether bonding occurred to Biodentine® (Paulson et al. 2018), AH Plus™ (Neelakantan et al. 2012) or to Resilon™/Epiphany™ (De-Deus et al. 2008a), the pushout bond strengths were higher when irrigation occurred with the etidronate mixture rather than the sequence. While bond strengths can be expected to improve with the removal of smear layer (Villanova et al. 2012), the enhanced result for the etidronate mixture in AH Plus study was attributed to better bonding to collagen and penetration of the sealer

60 in the dentine (Neelakantan et al. 2012). For Biodentine it was thought that the poorer result for the sequence was due to dehydration of the cement caused by EDTA.

Gaps in the continuous chelation literature

1. The effect of temperature on NaOCl stability in continuous chelation mixtures has not been demonstrated.

2. Only two chelators, Na4 etidronate and Na4 EDTA, have been examined in continuous chelation. Both chelators when mixed with NaOCl produce an irrigant mixture that decays over time, resulting in the loss of FAC, and an accompanying reduction in the therapeutic window of use. Testing novel chelator mixtures may result in an irrigant with an improved therapeutic window.

3. Any newly identified chelator-NaOCl mixture would need to be evaluated with respect to the main irrigant functions of smear layer removal, tissue dissolution and disinfection as well as to confirm that the mechanical strength of teeth is not compromised in comparison to a standard irrigation protocol.

4. No smear layer and tissue dissolution studies exist for continuous chelation mixtures containing 5% NaOCl. Only one antimicrobial study incorporates the use of 5% NaOCl.

5. It is uncertain whether etidronate impedes the tissue dissolution properties of NaOCl.

6. The antimicrobial testing of continuous chelation mixtures has relied heavily on live/dead staining. This technique may be unsuitable for mixtures containing a chelator. Different antimicrobial assessment methodologies are needed to confirm the existing antimicrobial result for etidronate mixtures with NaOCl and additionally to test novel mixtures.

7. There are differing results for the antimicrobial actions of plain etidronate solutions when live/dead compared to colony counting is used as the assessment methodology.

8. It is unclear if etidronate mixtures with NaOCl have better antimicrobial actions compared to NaOCl, alone or in sequences.

9. CFU methodologies involving HA discs need to be validated to confirm that bacteria are adequately removed from both from the control and treatment discs.

10. Only one study has examined fracture resistance in continuous chelation.

Thesis aims

1. To evaluate the effect of heating to root canal temperature in continuous chelation.

2. To identify a novel chelator, which when mixed with sodium hypochlorite, results in the maintenance of higher free available chlorine levels compared to mixtures of the existing known chelators in continuous chelation.

3. To ensure that a novel chelator is compatible with sodium hypochlorite at root canal temperature.

4. To ascertain that such a novel chelator, when combined with sodium hypochlorite, can perform the main functions required of an endodontic irrigant, that is, smear layer removal, organic tissue dissolution and disinfection.

5. To use a variety of methodologies in the antimicrobial assessments and to examine the usefulness of the CFU technique.

6. To ensure that the novel chelator-sodium hypochlorite mixture will not adversely affect the fracture resistance of teeth.

Thesis objectives

1. To compare alkaline EDTA and etidronate mixtures with NaOCl at both root canal and room temperature by an examination of FAC, pH and temperature changes.

2. To compile a list of chelators for an evaluation of compatibility with NaOCl. The considerations for inclusion are the ligand stability constant for Ca2+ and chemical structure.

3. To compare, at room temperature, FAC, pH and temperature in NaOCl mixtures of alkaline EDTA or etidronate and novel chelator-NaOCl mixtures with their NaOCl controls and to identify the mixture with the best NaOCl stability.

4. To compare, at root canal temperature, the FAC, pH and temperature a newly identified chelator-NaOCl mixture with NaOCl.

5. To compare the removal of smear layer in novel chelator-NaOCl mixtures, using SEM and dentine discs, with corresponding etidronate solutions and NaOCl/EDTA and NaOCl/EDTA/NaOCl sequences.

6. To compare novel chelator and etidronate mixtures with NaOCl in their capacity to dissolve porcine palatal mucosa by an assessment of both weight loss and FAC decline.

7. To evaluate the antimicrobial effect of etidronate and novel chelator-NaOCl mixtures compared with NaOCl using colony counting, SEM and XTT assessment methodologies.

8. To include final concentrations of 5% NaOCl in objectives 5-7 above.

9. To compare the potential of etidronate and clodronate mixtures to the sequence 5% NaOCl/17% EDTA/5% NaOCl with respect to the resistance to compressive force using a bovine root canal model.

The effect of heating to intracanal temperature on the stability of sodium hypochlorite admixed with etidronate or EDTA in continuous chelation

This chapter examines the effect of heating on existing continuous chelation mixtures of etidronate or alkaline EDTA, to root canal temperature on NaOCl stability as measured by FAC. Accompanying pH and temperature changes in the mixtures are presented also. Previously, the stability of NaOCl in such mixtures was measured at room temperature only. Temperature is an important consideration because it establishes the length of time that mixtures are likely to be useful in a clinical scenario, i.e. their therapeutic window. It sets the benchmark to later assess novel chelator-NaOCl mixtures once they have been identified. This chapter includes the following publication and a link to the published article.

Wright PP, Kahler B, Walsh LJ (2019) The effect of heating to intracanal temperature on the stability of sodium hypochlorite admixed with etidronate or EDTA for continuous chelation. Journal of Endodontics 45, 57-61. https://doi.org/10.1016/j.joen.2018.09.014

Author contribution statement

In Patricia Wright: Design, execution, methods, statistics and writing of the original draft (100%); Analysis (80%); Editing/review (30%).

Bill Kahler: Supervision (25%); Editing/review (10%).

Laurence Walsh: Supervision (75%); Editing/review (60%); Analysis (20%).

Abstract

Introduction: Na4 etidronate and Na4 EDTA are chelators which can combine with sodium hypochlorite NaOCl as a one-mix endodontic irrigant in a process called continuous chelation. The therapeutic window of these mixtures is determined by the chemical reaction between NaOCl and the chelator. At room temperature, this window is 60 minutes for Na4 etidronate and 30 minutes for Na4 EDTA. Because reaction kinetics are influenced by heat, this study assesses the influence of heating to an intracanal temperature of 35oC on the therapeutic window in continuous chelation. Methods: The loss of FAC in NaOCl mixtures o o with Na4 etidronate or Na4 EDTA was determined by iodometric titration at 23 C±0.7 C (23oC) and 34.6oC±0.3oC (35oC) at 1, 20, 40 and 60 minutes after mixing. The pH and o temperature of the mixtures were measured. Results: At 23 C, 18% Na4 etidronate-5% NaOCl solutions at 20, 40 and 60 minutes lost, 4%, 9% and 18% FAC and at 35oC, they lost

20%, 68% and 92% FAC; 5% Na4 EDTA-2.5% NaOCl solutions at 20, 40 and 60 minutes at 23oC lost 88%, 94% and 97% FAC and at 35oC, 96%, 99% and 100%. Decreases in FAC were accompanied by pH declines. Conclusions: The effect of heating to 35 oC from a o room temperature of 23 C on 18% Na4 etidronate-5% NaOCl solutions reduces its therapeutic window to 20 minutes. Solutions of 5% Na4 EDTA-2.5% NaOCl are not useful in the continuous chelation technique. Tracking pH changes could be used to estimate NaOCl degradation.

Introduction

Irrigant solutions of NaOCl are used widely because of their unique capacity to both dissolve organic material and to exert rapid antimicrobial actions (Haapasalo et al. 2014). However, because NaOCl does not remove the smear layer created by instrumentation, a chelating agent is employed to perform this role (Haapasalo et al. 2014). In standard endodontic irrigation protocols, the additional chelation step uses EDTA (Haapasalo et al.

2012). The form commonly used for this is Na2 EDTA (O’Connell et al. 2000). An alternative approach, termed continuous chelation, was introduced in 2005. This involves the concurrent use of a solution containing NaOCl and a chelator during chemomechanical preparation (Zehnder et al. 2005). Na4 etidronate and Na 4EDTA are both alkaline chelators that operate in the pH range from 10.8-12.2 and have been admixed with NaOCl for continuous irrigation (Tartari et al. 2015; Tartari et al. 2017). In contrast, Na2 EDTA works at a neutral pH. Alkaline conditions used with Na4 etidronate and Na4 EDTA should favour their use with alkaline solutions of NaOCl (Biel et al. 2017). Maintaining alkalinity maximizes the concentration of the hypochlorite anion, which is the species involved in organic tissue dissolution (Fukuzaki 2006). Continuous chelation is of interest because it not only simplifies the clinical technique but also improves removal of debris from the root canal (Paqué et al. 2012). Additionally, it enhances the antimicrobial effect of NaOCl in the presence of smear layer (Morago et al. 2016) and shows good compatibility with some dental materials (Paulson et al. 2018). Chelators are thought to promote detachment of biofilms from the walls of the root canal (Zehnder 2006) . Biofilm disruption may be driven by the constant presence of the chelator (Arias-Moliz et al. 2016). Chelators will also remove metal ions that are required for bacterial nutrition (Estrela et al. 2018). Despite the advantages, a major concern is the potential for chemical reactions to occur between NaOCl and the chelator. In endodontics, significant interactions are known to occur between it and other irrigants (Wright et al. 2017b). In continuous chelation, the concomitant presence of a chelator will lower FAC and reduce the pH (Zehnder et al. 2005). As FAC falls, the antimicrobial and tissue dissolving actions are impaired (Zehnder 2006). Previous studies of continuous chelation have shown that the effective use life of solutions may be up to 60 minutes (Biel et al. 2017). The measured FAC reduction for NaOCl solutions is less for Na4 etidronate than for Na4 EDTA when the mixtures are kept for 60 minutes at 67 room temperature (Biel et al. 2017). Under these same conditions, solutions containing 18%

Na4 etidronate and 5% NaOCl, or 3% Na4 EDTA and 1% NaOCl showed reductions in FAC of 16% and 83%, respectively (Biel et al. 2017). In contrast, a reduction in FAC of 38% has been recorded for a mixture with final concentrations of 5% Na4 EDTA and 2.5% NaOCl kept at room temperature for 60 minutes (Tartari et al. 2017). Because chemical reactions occur more readily at elevated temperatures, in continuous chelation, increases in temperature have the capacity to accelerate the depletion of FAC. Reductions in FAC have been studied previously for NaOCl combined with Na4

EDTA at room temperature (Biel et al. 2017; Tartari et al. 2017), for Na4 etidronate mixtures o at 23 C (Biel et al. 2017), and for the commercially available Na4 etidronate, Dual Rinse® HEDP (Medcem, Weinfielden, Switzerland) 5oC, 23oC and 60oC (Zollinger et al. 2018). The effect at 60oC was tested at 1 hour when all the FAC was found to be depleted. This result is congruent with the expectation that a temperature rise causes an increase in the reaction rate between NaOCl and the chelator. However, the temperatures chosen (ie, 5oC, 23oC and 60oC) relate more to storage, rather than intracanal usage. There has been one study o examining the ability of Na4 etidronate mixtures at 32 C to dissolve organic tissue (Tartari et al. 2015). In that investigation, there was no comparison to any other temperature, as the effect of heat was not the focus of the experiment, and conclusions about differences due to temperature changes cannot be made. Clinically relevant temperature studies of FAC at various time points up to 1 hour are lacking. Endodontic irrigants rapidly equilibrate from both high and low temperatures in vivo. In 15 seconds irrigants injected at 19oC reach 31.8±0.9oC and then finally equilibrate to 35±1oC over 4 minutes. Conversely, irrigants injected at 66oC attain 40.5±1.5oC in 15 seconds and 35.7±0.8oC in 4 minutes (de Hemptinne et al. 2015). Knowing this, it is essential that studies of the possible impact of chelators on FAC levels use temperatures that are experienced during canal irrigation. Such data can then be used to give a realistic value of the in vivo lifespan of these mixtures. The present study was designed to track the reduction in FAC and corresponding changes in pH and temperature for NaOCl mixtures with chelators at room temperature (23oC) versus 35oC, at intervals of 1, 20 40 and 60 minutes after mixing. The current study was also devised to test the chelators in mixtures at the highest final NaOCl concentrations examined in previous studies, that is 5% NaOCl for Na4 etidronate (Zollinger et al. 2018) and 2.5% NaOCl for Na4 EDTA (Tartari et al. 2017). Additionally, a final concentration of 18% Cublen etidronate was used because this was the maximum concentration used for

68 etidronate in other research (Biel et al. 2017). Because FAC declines as chelator and NaOCl concentrations rise (Biel et al. 2017), the concentrations chosen would more likely reveal a heat effect should one be present. The null hypothesis was that raising the temperature would not affect the reduction in FAC over time.

Materials and Methods

Solutions tested The following solutions were prepared in closed 15 mL polypropylene Falcon tubes (Corning, Tewksbury, MA, USA) (n=10 replicates of each).

1. 10% Na4 EDTA (Merck 34104-500G, Darmstadt, Germany) (pH = 11.1) mixed equally

with 5% NaOCl (pH = 12.6) for final concentrations of 5% Na4 EDTA and 2.5% NaOCl. 2. Demineralised water mixed in equal volumes with 5% NaOCl (giving 2.5% NaOCl and serving as a control for the EDTA solution in [1]).

3. 36% Na4 etidronate (Cublen K 8514 GR, Zschwimmer & Schwarz, Burgstädt, Germany) (pH = 11.2-11.5) mixed equally with 10% NaOCl (pH = 12.7) for final

concentrations of 18% Na4 etidronate and 5% NaOCl. 4. Demineralised water mixed in equal volumes with 10% NaOCl (giving 5% NaOCl as

a control for the Na4 etidronate solution in [3]).

Experiments for Na4 EDTA with its relevant control and Na4 etidronate with its control were conducted separately. All chelator solutions were freshly prepared with demineralised water. FAC of the NaOCl stock solution (AJA-458-5L, Ajax Finechem, Scoresby, VIC, Australia) was determined by iodometric titration using a burette (Standards Australia 2003). The NaOCl stock solution was diluted to 5% or 10% with demineralised water and stored in the dark at 2-4oC until used.

FAC, pH and temperature recordings Solutions were kept on the bench at room temperature of 23±0.7oC (23oC) or were equilibrated to 34.6±0.3oC (35oC) using a water bath (Ratek Instruments, Boronia, VIC, Australia). At the designated time 0, the chelator solution was added to the NaOCl, to give a total volume of 8 mL. FAC was measured after 1, 20, 40 and 60 minutes. FAC was measured by iodometric titration. In a separate series of experiments, changes in pH and temperature over time were measured, starting from either 23oC or 35oC with the same solutions described previously.

The electrode used (Ionode IJ40CT, Ionode, Brisbane, QLD, Australia) incorporated an internal temperature probe. The pH meter (Orion Star 211, Thermo Scientific, Beverly, MA, USA) used automated temperature correction. The pH readings were adjusted for high sodium concentrations and for high pH (Thermo Scientific 2014).

Statistical analysis R 3.5 (Core Team, Vienna, Austria) was used for the normal Q-Q plot of the FAC data, which revealed a normal distribution. Prism 7.0 (GraphPad Software, La Jolla, CA, USA) was used in the statistical analysis. Means and standard deviations of FAC (normalised), pH and temperature were calculated from the 10 replicates. A three-way ANOVA was used, followed by the Tukey multiple comparison test. Alpha was set at 0.05.

Results

The impact of raising the temperature from 23oC to 35oC is summarized in Table 2- 1, where FAC values are presented as a percentage of the baseline control value. Data for FAC (in g/L) are shown in Figure 2-1. In both solutions, the fall in FAC was more pronounced at the elevated temperature at all time points recorded, except at 1 minute for Na4 etidronate solutions (P < 0.01). Thus, the null hypothesis was discarded. There were significantly lower levels of FAC after mixing chelators with NaOCl for both mixtures compared to their controls for all time periods excluding the 1 minute values (P < 0.01). o For Na4 etidronate-5% NaOCl at 23 C, at 20, 40 and 60 minutes, FAC fell to 96%, 91% and 82% respectively. At the same time points, but at 35oC, the FAC declined to 80%, o 32% and 8% respectively. Likewise, for Na4 EDTA-2.5% NaOCl at 23 C, FAC fell to 12%, 6% and 3% of baseline, while at 35oC the levels were 4%,1% and 0%. As shown in Figure 2-2, changes in measured levels of FAC and in pH followed the same trend over time. Concerning the temperature (Table 2-1), for alkaline EDTA at 23oC the temperature change occurred at 20 minutes, rising to 34.5±1.3oC compared to the control of 23.8±0.4oC. At 35oC for EDTA, at all times, temperatures were comparable to the control. For etidronate, the temperature rise occurred at 60 minutes and was considerably less pronounced. At this time point, at 23oC, the temperature rose to 25.6±0.8oC compared to the control at 22.6±0.3oC, while at 35oC the rise was to 35.5±0.1oC compared to a control of 34.7±0.2oC.

Table 2-1. Changes over time in FAC with solution temperature and pH

Temp, temperature. Data for FAC are expressed as a percentage of the baseline value.

Figure 2-1. The effect of temperature on FAC

Figure 2-2. Changes in FAC and pH (A) 18% Na4 etidronate-5% NaOCl kept at 23oC, (B) 5% Na4 EDTA-2.5% NaOCl kept at 23oC, (C) 18% Na4 etidronate-5% NaOCl kept at 35oC, (D) 5% Na4 EDTA-2.5% NaOCl kept at 35oC.

Discussion

This study provides important insights into the reactions between NaOCl and chelators that can occur when they are combined. Mixtures containing 18% Na4 etidronate and 5% NaOCl or 5% Na4 EDTA and 2.5% NaOCl have been examined in previous studies (Biel et al. 2017; Tartari et al. 2017), but at room temperature rather than at the more clinically relevant temperature of 35oC. This study extends previous work by showing that degradation of NaOCl, with concomitant reductions in FAC and reductions in pH, occur more readily at 35oC than 23oC.

In this study, 35oC was used because NaOCl rapidly equilibrates to this temperature when it is used in root canal irrigation in vivo (de Hemptinne et al. 2015). From the viewpoint of standard sequential irrigation protocols, it is known that warming NaOCl increases the rate of dissolution of soft tissues (Cunningham & Balekjian 1980; Sirtes et al. 2005); however, in continuous chelation mixtures, an increase in temperature causes an increase in the rate of the chemical reaction between the chelator and the NaOCl, causing a decrease in FAC. Once mixed, the effective use life of the solution of NaOCl combined with a chelator at room temperature is in the order of 60 minutes. In the present study, for mixtures o containing 18% Na4 etidronate and 5% NaOCl kept at 23 C, FAC fell to 96 % of the baseline level in 20 minutes, and to 82% at 60 minutes. This agrees with similar experiments by others where the FAC fell to 95% in 20 minutes and 84% in 60 minutes, with the “therapeutic window” estimated to be 60 minutes (Biel et al. 2017).The present investigation broadens this by showing that at 35oC FAC falls to 80% at 20 minutes, 32% at 40 minutes, and 8% at 60 minutes. Based on this, the increase in temperature reduces the therapeutic window from 60 minutes to 20 minutes. Not only does frequent refreshment of irrigants then become necessary, but consideration needs to be given to mixing fresh solutions, to overcome the problems of inactivation of NaOCl. It is also recognised that lowering the concentrations of both NaOCl and Na4 etidronate, would prolong the therapeutic window beyond 20 minutes.

The results of the present study indicate that Na4 EDTA is less compatible with NaOCl than Na4 etidronate. For mixtures containing 5% Na4 EDTA and 2.5% NaOCl, the current study found that the FAC after 20 minutes was 12% at room temperature, and only 6% at 35oC. Hence, the use of these solutions is not recommended. Reducing the concentration of both agents may extend the therapeutic window by slowing the reaction rate. For example, for final concentrations of 1% NaOCl and 3% EDTA, 87% of the FAC remains after 30 minutes at room temperature) (Biel et al. 2017). Such dilute solutions would however have limited therapeutic benefit, so such an approach may not be practicable.

The Na4 etidronate used in the current study and in most previous studies of etidronate is an industrial sequestering agent, Cublen K8514 GR (Paqué et al. 2012; Arias- Moliz et al. 2014; Tartari et al. 2015; Morago et al. 2016; Biel et al. 2017). The purity as stated by the manufacturer is 85% (Zschimmer & Schwarz). This raises two issues. The first of these concerns the effective concentration. A final concentration of 18% Cublen will result in a concentration of Na4 etidronate of 15.3%. The second issue is the possibility that the

74 impurities present may have interacted with the NaOCl. This could be examined by assessing etidronate preparations of higher purity. For the present study, the method applied to assess FAC was iodometric titration. This approach has been used in previous investigations of continuous chelation (Biel et al. 2017; Tartari et al. 2017; Zollinger et al. 2018). Although it is believed that thermometric titration is more accurate than iodometric titration for assessing FAC in NaOCl solutions (Clarkson et al. 2013), its use in continuous chelation is problematic. This is because the thermometric technique requires the conversion of NaOCl to sodium hypobromite (Clarkson et al. 2013), which has a different oxidation-reduction potential compared to NaOCl (Vanysek 2010). Because in continuous chelation the oxidizing capacity of NaOCl dictates its reaction with the chelator, the presence of sodium hypobromite would cause erroneous FAC measurements, which is why thermometric titration was not used. A final point of note is that the current study shows that the reduction in FAC over time is tracked closely by a fall in pH. These follow one another because the concentration of the hypochlorite anion falls as it is consumed by the reaction with the chelator. Tracking changes in pH over time could be a useful means to assess the stability of mixtures of NaOCl with various chelators. Assessing pH can be done in real time and is less complex than measuring FAC. Further work on continuous chelation could exploit the predictive power of pH measurements for real-time assessment of various mixtures.

Conclusion

Raising the temperature from room temperature (23oC) to the intra-canal temperature of o 35 C accelerates the loss of FAC in continuous chelation employing mixtures of either Na4 etidronate or Na4 EDTA with NaOCl. This results in a shortening of the therapeutic window of use, with the effect being more pronounced for Na4 EDTA.

The search for chelators compatible with sodium hypochlorite

This chapter screens NaOCl stability in novel chelator-NaOCl mixtures. Clodronate was identified as being stable with NaOCl at room temperature. This mixture was further examined with respect to the main irrigant function of smear layer removal and maintenance of FAC at root canal temperature. The chapter includes the following publications and links to the published texts. 1. Wright PP, Cooper C, Kahler B, Walsh LJ (2020) From an assessment of multiple chelators clodronate has potential for use in continuous chelation. International Endodontic Journal 53, 122-134 https://doi.org/10.1111/iej.13213

Author contribution statement Patricia Wright: Analysis, conception, ethics, statistics, writing of draft manuscript (100%); Execution (80%); Graphics (75%); Methodology (70%); Editing/review (30%).

Crystal Cooper: Editing/review (25%); Execution and graphics (20%); Methodology (15%).

Bill Kahler: Publishing advice (50%); Supervision (25%); Editing/review (10%).

Laurence Walsh: Supervision (75%); Publishing advice (50%); Editing/review (35%); Methodology (15%); Graphics (5%).

2. Wright PP, Kahler B, Walsh LJ (2020) The effect of temperature on the stability of sodium hypochlorite in a continuous chelation mixture containing the chelator clodronate. Australian Endodontic Journal 46, 244-8. https://doi.org/10.1111/aej.12399

Author contribution statement Patricia Wright: Conception, design, execution, graphics, methods, statistics, writing of draft manuscript (100%); Editing/review (35%). Bill Kahler: Supervision (25%), Editing/review (10%). Laurence Walsh: Supervision (75%); Editing/review (55%).

Paper 1

From an assessment of multiple chelators clodronate has potential for use in continuous chelation

3.1.1 Abstract

Aim To identify chelators which when mixed with NaOCl are stable, exhibiting minimal loss of FAC over 80 minutes and to further investigate potential mixtures by assessing FAC over 18 hours and the capacity to remove smear layer. Methodology 0.25 M EDTA (10%), 0.25 M EGTA (egtazic acid), 0.25 M CDTA (cyclohexanediaminetetraacetic acid), 0.25 M DTPA (pentetic acid), 0.5 M ATMP (aminotris(methylene phosphonic acid)) and 1 M HPAA (hydroxyphosphonoacetic acid), all at alkaline pH, were mixed equally with 5% NaOCl. 0.5 M alkaline clodronate and 0.5 M Na4 etidronate (15%) were mixed equally with 10% NaOCl. For all mixtures, the pH and temperature were measured over 80 minutes and additionally for the clodronate mixture over 18 hours. Iodometric titration was used to measure the FAC of all mixtures except for HPAA. The following were compared with respect to their ability to remove smear layer: 1 M clodronate+10% NaOCl, 0.5 M clodronate+10% NaOCl, 1 M etidronate+10% NaOCl, 0.5 M clodronate+10% NaOCl and the sequences 5% NaOCl/17% EDTA/5% NaOCl and 5% NaOCl/17% EDTA. The area fraction occupied by open dentinal tubules as a percentage of the total area (% AF) from scanning electron microscopy micrographs was calculated using Image J. The results were statistically analysed with alpha set at 0.05. Results Compared to its control, the mixture 0.5 M clodronate+10% NaOCl lost no FAC over 18 hours (p < 0.05). The FAC of 0.25 M CDTA mixed with 5% NaOCl fell to 96%, 92%, 75% and 4.9% at 20, 40, 60 and 80 min, respectively. Temperature rises were observed in all cases except in the etidronate and clodronate mixtures. Only in the clodronate mixture did the pH remain above pH 12 for the whole experiment. Although smear layer was removed, the % AF in 1 M clodronate+10% NaOCl, 0.5 M clodronate+10% NaOCl, 1 M etidronate+10% NaOCl was less than for 0.5 M etidronate + 10% NaOCl and 5% NaOCl/17% EDTA/5% NaOCl and 5% NaOCl/17% EDTA. Conclusion Alkaline 0.5 M clodronate mixed equally with 10% NaOCl, has potential for use in continuous chelation, based on this assessment of stability and smear layer removal. Further research is needed to establish its efficacy and safety. 77

3.1.2 Introduction

Endodontic irrigation involves the use of NaOCl and a chelator. The application of NaOCl both dissolves organic material and exerts antimicrobial actions (Haapasalo et al. 2014). The subsequent use of a chelator removes the instrument-created smear layer. The prior application of NaOCl assists the chelator in removing the inorganic component of the smear layer because it removes associated organic matter (Haapasalo et al. 2014). The removal of smear layer is recommended as it can contain not only bacteria and nutrients for bacterial growth, but it impedes irrigant penetration into the dentinal tubules (Violich & Chandler 2010). EDTA is the most commonly employed chelator, although the use of citric acid is also well documented (Schäfer 2007). Typically, EDTA solutions are either pH neutral or slightly alkaline (Wright et al. 2017b). The sequence NaOCl/EDTA/NaOCl is common, and although this combination gives powerful antimicrobial actions, biofilm removal in the root canal system is incomplete (Neelakantan et al. 2015). Additionally, the risk of eroding peritubular dentine is of concern, and some have advised against the use of a final rinse with NaOCl for this reason (Wang et al. 2016). Also of concern is the inactivation of NaOCl, as it reacts chemically with EDTA. This has led to the recommendation that NaOCl and EDTA should not be used sequentially, without first emptying and drying the canal (Clarkson et al. 2011). The other common sequence, NaOCl/EDTA, lacks a final rinse with an antimicrobial agent, and its use results in less killing of bacteria in biofilms when compared to NaOCl/EDTA/NaOCl (Neelakantan et al. 2015). In 2005, in the search for new improved irrigation regimens to address some of the above issues, the concept of continuous chelation was introduced. This involves the simultaneous use of a chelator and NaOCl in a single solution, thus simplifying the irrigation procedure (Zehnder et al. 2005). Considerable research has focused on the use of the weak chelator etidronate at alkaline pH combined with NaOCl, and from this, a commercial product, Dual Rinse HEDP® has been developed (Zollinger et al. 2018). The form of etidronate used is Na4 etidronate, also known as Na4 HEDP. The reported benefits include the increased removal of dentinal debris (Paqué et al. 2012), enhanced antimicrobial effects (Arias-Moliz et al. 2016; Morago et al. 2016), and good compatibility with dental materials (De-Deus et al. 2008a). Even though these findings are promising, etidronate-hypochlorite mixtures lack chemical stability, and lose their FAC over time. The “use life” of solutions containing 18% 78

Cublen etidronate and 5% NaOCl (Biel et al. 2017), or 9% Dual Rinse HEDP and 2.5% NaOCl (Zollinger et al. 2018) is 1 hour and 2 hours respectively at room temperature.

Alkaline EDTA in the form of Na4 EDTA, as opposed to the pH neutral EDTA used in standard irrigation, has also been tested in continuous chelation. Even at concentrations as low as 5% EDTA and 2.5% NaOCl, the chemical stability is considerably less than the etidronate mixtures, with a use life of only 30 minutes after mixing (Biel et al. 2017). The identification of a suitable chelator with improved stability when combined with NaOCl would extend the therapeutic window of continuous chelation. It would streamline endodontic irrigation, and may give benefits similar to those described for etidronate mixtures. Particular chemical properties may be useful in the search for chelators compatible with NaOCl. These are the ligand stability constant for the formation of Ca2+ complexes, pH, chemical structure and solubility. The stability constant is a measure of how well a chelator binds metal ions, with a higher value indicating stronger binding. EDTA has a higher stability constant than etidronate (Smith & Martell 1989). This explains the better removal of smear layer by the former under the same conditions (Deari et al. 2019). A high stability constant is then a desirable quality for a chelator. The alkalinity of chelating agents is particularly important. Acidic chelators such as citric acid react rapidly with NaOCl, with a concomitant rapid loss of FAC (Zehnder et al. 2005). Alkaline chelator solutions are needed to ensure that when mixed with NaOCl, the pH remains high. Na4 EDTA and Na4 etidronate, when mixed with water, give solutions of over pH 11 (Deari et al. 2019). Solutions of other chelators can be made alkaline by the addition NaOH. Structural differences in chelators, such as elemental variances, where chelators contain phosphorous rather than nitrogen or have altered stereochemistry, can also be expected to contribute to differences in stability. Table 3-1 lists the nomenclature and the Ca2+ stability constant for the chelators evaluated in this study: EGTA, CDTA, DTPA, ATMP, clodronate and HPAA. Na4 EDTA and

Na4 etidronate are included for comparison. Except for ATMP, none of these chelators have previously been tested in continuous chelation. ATMP has been previously examined (Zehnder et al. 2005), but in the current study the pH was alkaline rather than acidic. The EDTA analogues, EGTA, CDTA and DTPA all have high stability constants, with the values for CDTA and EGTA being higher than that of EDTA. HPAA and clodronate were included because they are structurally similar to etidronate, containing phosphorous rather than nitrogen. ATMP is a known chelator containing only one nitrogen atom. All the chelators

79 salts chosen for examination are water soluble at the desired pH and concentration. ATMP and HPAA are both liquids and can be diluted with water to the desired concentration.

Table 3-1. Chelator common names and Ca2+ stability constants

Chelator common names and Ca2+ stability constant IUPAC nomenclature*

Na4 EDTA, ethylenediaminetetraacetic acid tetrasodium salt, 10.65 (Smith & Martell 1989) sodium edetate tetrasodium, tetrasodium; 2- [2[bis(carboxylatomethyl)amino]ethyl- (carboxylatomethyl)amino]acetate* EGTA, ethylene glycol tetraacetic acid, egtazic acid, 2-[2-[2-[2- 11.0 (Owen 1976) [bis(carboxymethyl)amino]ethoxy]ethoxy]ethyl- (carboxymethyl)amino]acetic acid* CDTA, cyclohexanediaminetetraacetic acid, 2-[[-2- 12.5 (Kroll & Gordon 1960) [bis(carboxymethyl)amino]cyclohexyl]- (carboxymethyl)amino]acetic acid* DTPA, diethyltriaminepentaacetic acid, pentetic acid, 2-[bis[2- 10.63 (Kroll & Gordon 1960) [bis(carboxymethyl)amino]ethyl]amino]acetic acid* ATMP, aminotris(methylene phosphonic acid), 6.7 (Smith & Martell 1990) [bis(phosphonomethyl)amino]methylphosphonic acid* HPAA, hydroxyphosphonoacetic acid, Not found

2-[hydroperoxy(hydroxy)phosphoryl]acetic acid*

Na4 Etidronate, 1-hydroxyethane 1,1-diphosphonic acid 6.04-6.08 (Smith & Martell

tetrasodium salt, Na4HEDP, tetrasodium;1- 1989) diphosphonatoethanol*

Na2Clodronate; disodium clodronate, clodronic acid disodium 5.95 (Smith & Martell 1989) sat, disodium;[dichloro-[hydroxy(oxido)phosphoryl]methyl]- hydroxyphosphinate* IUPAC; International Union of Pure and Applied Chemistry.

If a chelator with improved compatibility with NaOCl were identified, its useful application to endodontics would need to be demonstrated. As the main function of a chelator is to remove smear layer, the ability of any new chelator-NaOCl mixtures to clear the dentinal tubules of debris would help establish its clinical relevance. This can be 80 achieved by employing a variety of techniques including SEM (Tartari et al. 2017), co-site optical microscopy (De-Deus et al. 2008b), and laser microscopy (Deari et al. 2019), all of which have assessed smear layer removal in endodontics. Calcium removal from dentine can be determined by atomic absorption spectroscopy (Zehnder et al. 2005; Deari et al. 2019). The aim of this study was to identify chelators that have improved chemical compatibly with NaOCl, as measured by FAC levels, when compared to those currently identified for use in continuous chelation. An additional goal of this research was to demonstrate that such a chelator-NaOCl mixture can remove smear layer. Thus, the hypothesis tested was that a chelator with better stability with NaOCl than either Na4 EDTA or Na4 etidronate can be identified, and that such a chelator when mixed with NaOCl is capable of removing smear layer from dentine.

3.1.3 Materials and Methods

Chemicals The following chelators were used:

• Na4 EDTA ( (34104-500G; Merck, Darmstadt, Germany). • EGTA (egtazic acid) (BePharm, Shanghai, China). • CDTA (cyclohexanediaminetetraacetic acid) (BePharm, Shanghai, China) • DTPA (pentetic acid) (BePharm, Shanghai, China). • ATMP (aminotris(methylene phosphonic acid)) (BePharm, Shanghai, China). • HPAA (hydroxyphosphonoacetic acid) (iChemical Shanghai, China).

• Na4 etidronate (Cublen, Zschwimmer & Schwarz, Burgstädt Germany).

• Na2 clodronate.4H2O (iChemical, Shanghai, China) for the FAC experiment over 80

minutes and Na2 clodronate (Enke Pharma-tech, Cangzhou, China) for all other experiments. Chelator solutions were prepared freshly with demineralised water and the purity of the chemical accounted for. All solid chelators were 98-99% pure except for the Cublen product which contained 85% Na4 etidronate. ATMP and HPAA were aqueous solutions with a concentration of 50%. All pH adjustments were made with NaOH (SA178-500G, Chem- supply, Gillman, SA, Australia). NaOCl (AJA-485-5L; Ajax Finechem, Scoresby, VIC,

Australia) was titrated by iodometric titration, and diluted to either 5% or 10%. The stock and diluted solutions were stored at 2-4 oC in the dark for a maximum of 5 weeks, during which time regular titrations showed that the NaOCl concentration was maintained.

Mixtures tested for FAC and pH assessment over 80 minutes • (i) 0.25 M EDTA (10%) (pH 11.1) mixed equally with 5% NaOCl (pH 12.6). • (ii) 0.25 M EGTA adjusted to pH 11.0, then mixed equally with 5% NaOCl (pH 12.5- 12.6). • (iii) 0.25 M CDTA adjusted to pH 11.4, then mixed equally with 5% NaOCl (pH 12.5- 12.6). • (iv) 0.25 M DTPA adjusted to pH 10.8, then mixed equally with 5% NaOCl (pH 12.7). • (v) 0.5 M ATMP adjusted to pH 10.3, then mixed equally with 5% NaOCl (pH 12.6). • (vi) 1 M HPAA adjusted to pH 11.1, then mixed with 5% NaOCl (pH 12.5). Only the pH-temperature experiment was performed. • (vii) 0.5 M etidronate (15%) (pH 11.6) mixed equally with 10% NaOCl (pH 12.6-12.7). • (vii) 0.5 M clodronate adjusted to pH 11.4, then mixed equally with 10% NaOCl (pH 12.6-12.7). Each of solutions i-vii above were matched with a control of demineralised water mixed with equal volumes of NaOCl. The final concentration of NaOCl was the same as that used in the chelator-NaOCl mixtures.

Mixture tested for FAC and pH assessment over 18 hours 0.5 M clodronate was adjusted to pH 10.7, then mixed equally with 10% NaOCl (pH 12.7) with a control of 10% NaOCl mixed with an equal volume of demineralised water.

Mixtures and sequences tested for smear layer assessment • (i) 1 M clodronate adjusted to pH 10.7, then mixed equally with 10% NaOCl (pH 12.7). • (ii) 0.5 M clodronate adjusted to pH 10.7, then mixed equally with 10% NaOCl (pH 12.7). • (iii) 1 M (30%) etidronate (pH 11.5) mixed equally with 10% NaOCl (pH 12.7). • (iv) 0.5 M (15%) etidronate (pH 11.5) mixed equally with 10% NaOCl (pH 12.7). • (v) 0.14 M PBS (vi). 5% NaOCl (pH 12.5) followed by 17% EDTA (pH 7.2).

• (vii) 5% NaOCl (pH 12.5) followed by 17% EDTA (pH 7.2) and then followed by 5% NaOCl (pH 12.5). pH-temperature and FAC recordings All measurements were performed at a room temperature of 23oC. A total of 10 replicates was used for every group. The pH-temperature experiments were conducted separately to FAC testing. At T=0, 4 mL of either the chelator solution or demineralised water was mixed with 4 mL of NaOCl, and stored in a closed 15 mL Falcon tube (Corning, Tewksbury, MA, USA). pH and temperature recordings were performed at 1, 20, 40, 60 and 80 minutes and additionally for the clodronate solution at 1 minute, 3 and 18 hours after mixing, using a pH electrode (Ionode IJ40CT; Ionode, Brisbane, QLD, Australia) connected to a pH meter (Orion Star 211; Thermo Scientific, Beverly, MA, USA). The pH electrode, which incorporated an automated temperature control (ATC) sensor, was rinsed in demineralised water between recordings and wiped dry. The pH meter was calibrated daily. Under the same conditions, FAC was measured by iodometric titration with a burette. A variation of a standard iodometry technique was employed (Standards Australia 2003). This variation entailed the delivery of a 1 mL sample of the neat solution with a 1000 µL pipette, as opposed to a 10 mL sample of a 1 in 10 dilution. The variation was tested by comparison of the variation with the standard technique.

Smear layer assessment Disc preparation and treatment The study was approved by the ethics committee of both the University of Queensland, Brisbane, QLD, Australia (approval number: 2018001868) and Metro South Health, Brisbane, QLD, Australia (reference number: HREC/17/QPAH/521). Following extraction, the external surface of 71 human third molars was scraped to remove soft tissue and cementum from the coronal aspect of the root, stored in 0.05% thymol in PBS and then sterilised by gamma irradiation at a dose of 25 kGy. A precision saw (IsoMet 1000, Buehler, Lake Bluff, IL, USA) was used to cut a 1.8-mm thick cross sectional slice from immediately below the CEJ. The coronal aspect of each slice was polished with a series of coarse, medium and fine Softlex discs (3M ESPE, St Paul, MN, USA), rinsed in demineralised water and subsequently stored in 0.05% thymol in PBS at 2-4oC until 24 hours before the commencement of the experiment when they were transferred to demineralised water.

The discs were allocated randomly to the seven groups, each group containing 10 discs except for the PBS group which was comprised of 11 discs. All discs were treated in 24-well cell culture plates (Costar 3738, Corning, Kannebunk, ME, USA), at room temperature, without agitation. A total of 7.5 mL of active irrigant over 15 minutes was used for all groups, with additional demineralised water rinses between the NaOCl and EDTA, in groups (vi) and (vii). All discs were finally rinsed in a beaker containing 100 mL of demineralised water. For groups (i)-(v) the dentine discs were moved to a new well containing 1.5 mL of fresh irrigant every 3 minutes. This was also the pattern in groups (vi) and (vii) for the first 9 minutes, where NaOCl served as the irrigant. After this, in group (vi), the discs were immersed in 1.5 mL of NaOCl for 4 minutes and finally 1.5 mL EDTA for 2 minutes and for group (vii), 1.0 mL NaOCl for 3.5 minutes, then 1.5 mL EDTA for 2 minutes and lastly 0.5 mL NaOCl for 30 seconds. These changes for groups (vi) and (vii) ensured that equal irrigant times and volumes for all groups were maintained. Disc imaging and image analysis The discs were air-dried, mounted on aluminium stubs using conductive adhesive carbon tabs and carbon paint before being coated in 4 nm of platinum using a Leica ACE EM600 splutter coater (Leica Microsystems, Wetzlar, Germany.) The slices were imaged at 3 kV using the secondary electron detector on a Tescan Mira3 field emission SEM (Tescan, Brno, Czech Republic). The microscope operator, who was blinded to the experimental details, was instructed to obtain five images/disc at low magnification (3,000x, field of view 92.3 µm), from a region roughly equidistant to the canal and root surface, where the tubules were round and approximately equal in number. Additionally, a representative image from each group was obtained at high magnification (20,000x, field of view 13.8 µm). A total of nine discs in each group were chosen for image analysis using Image J 1.52i (NIH, Bethesda, MD, USA). Discs were excluded for variance in tubule number or shape. The 16-bit images were batch analysed with a sequence of operations which included image thresholding at 11,000/65,535 in order to calculate the area occupied by open tubules as a percentage of the total area, that is, the percentage area fraction (% AF). The output images were checked to ensure that the Image J routine had not excluded any open tubules. Of the 315 images analysed, three were excluded for this reason.

Statistical analysis Prism software version 7.00 (GraphPad Software, La Jolla, CA, USA) was used for the statistical analyses. The D’Agostino and Pearson test was used to assess normality. For

84 the FAC data over 80 minutes, the Mann-Whitney test was used to test each chelator-NaOCl mixture against its control at each time point, after which the Bonferroni correction was applied to adjust for multiple comparisons. Unpaired t- tests with the Bonferroni correction were employed for the 18 hour FAC experiment of the 0.5 M clodronate+10% NaOCl mixture and its control at each time period. The smear layer assessment was performed with a one- way ANOVA. Alpha was set at 0.05 for each experiment. Means of the pH and temperature data were calculated to connect replicate values in the line plots.

3.1.4 Results pH, and FAC measurements over 80 minutes Replicate measurements for the pH and temperature experiments are shown in Figure 3-1 (pH) and Figure 3-2 (temperature). Only the clodronate mixture maintained a pH level of 12 or above for the entire experiment. The etidronate mixture held this pH level for 60 minutes, dropping to pH 11.9 at 80 minutes, whilst the CDTA mixture maintained a pH of 12 or above for 40 minutes, which then fell to pH 11.6 and pH 9.2 at 60 and 80 minutes, respectively. In contrast, the EDTA mixture had a pH of 11.2 at 20 minutes, but by 40 minutes this had fallen to a pH of just 9.0. The EGTA, DTPA, ATMP and HPAA mixtures all showed dramatic falls in pH, with the latter two dropping to pH 8.8 and pH 7.4, respectively, by 1 minute. Large pH falls were accompanied by temperature spikes, the highest of which were for HPAA and ATMP. The temperature in these solutions rose, from the baseline of 23oC, to 42.2oC and 37.1oC, respectively, at 1 minute. The minor variation in the titration method used in this study did not give results that were significantly different from those obtained by using the standard titration technique with larger volumes, and thus the altered method was accepted as valid. Individual FAC data points for the chelator mixtures and corresponding controls at all time points appear in Figure 3-3. The mixture of 0.5 M clodronate+10% NaOCl was the only solution where there was no significant difference in FAC compared to the control at any time period (P > 0.05). FAC values for all other mixtures were significantly different (P < 0.05) to their controls at every time point except for T=1 minute. However, two of the other chelator mixtures showed reasonable maintenance of FAC over time. In the etidronate mixture, FAC fell to 93% of the control value at 80 minutes, while for the CDTA mixture the FAC was 96%, 92% and 75% at 20, 40 and 60 minutes, but by the end of the experiment this had diminished to just 4.9%. 85

The EDTA mixture maintained a reasonable level of FAC at 20 minutes (82%), but with large variations in the FAC values between replicates at this time point. By 40, 60 and 80 minutes, the FAC had fallen to 6.1%, 4.5% and 2.9%, respectively. FAC levels in the remaining mixtures were low. In the ATMP, EGTA and DTPA mixtures, the FAC had fallen to 15%, 7.6% and 1.5%, respectively, at 20 minutes. pH, temperature and FAC measurements of 0.5 M clodronate+10% NaOCl over 18 hours. There were no significant differences in the FAC levels between the test and control solutions at each of the time periods (p > 0.05). At 1 minute, 3 and 18 hours, for 0.5 M clodronate+10% NaOCl, the FAC, respectively, was 47.5±0.1, 47.4±0.1 and 47.4±0.1 g/L, whilst for the 5% NaOCl control at the same time points it was 47.6±0.1, 47.5±0.1 and 47.5±0.1 g/L. All test and control solutions maintained a pH of 12.5. Temperature variations in both the test and control solutions were minor and less than 1oC.

Figure 3-1. pH values for chelator-NaOCl mixtures Replicate values are connected by their mean at 1 minute, then at 20 minute intervals for groups: (a) 0.25 M EDTA+5% NaOCl (b) 0.25 M EGTA+5% NaOCl (c) 0.25 M CDTA+5% NaOCl (d) 0.25 M Pentetic acid (DTPA)+5% NaOCl (e) 0.5 M ATMP+5% NaOCl (f) 1.0 M HPAA+5% NaOCl. (g) 0.5 M Etidronate+10% NaOCl (h) 0.5 M Clodronate+10% NaOCl.

Figure 3-2. Temperature values for chelator-NaOCl mixtures

Replicate values connected by their mean at 1 minute then at 20 minute intervals for groups: (a) 0.25 M EDTA+5% NaOCl (b) 0.25 M EGTA+5% NaOCl (c) 0.25 M CDTA+5% NaOCl (d) 0.25 M Pentetic acid (DTPA)+5% NaOCl (e) 0.5 M ATMP+5% NaOCl (f) 1.0 M HPAA+5% NaOCl. (g) 0.5 M Etidronate+10% NaOCl (h) 0.5 M Clodronate+10% NaOCl. Temperature in degrees Celsius (oC).

Figure 3-3. FAC values for chelator-NaOCl mixtures

Replicate values connected by their medians at 1 minute then at 20 minute intervals for FAC in g/L for groups: (a) 0.25 M EDTA+5% NaOCl (b) 0.25 M EGTA+5% NaOCl (c) 0.25 M CDTA+5% NaOCl (d) 0.25 M Pentetic acid (DTPA)+5% NaOCl (e) 0.5 M ATMP+5% NaOCl (f) The HPAA group was not used in the FAC experiment. (g) 0.5 M Etidronate+10% NaOCl (h) 0.5 M Clodronate+10% NaOCl.

Smear layer removal Figure 3-4 illustrates the % AF results for 1 M clodronate+10% NaOCl, 4.39±1.27; 0.5 M clodronate+10% NaOCl, 4.18±0.72; 1 M etidronate+10% NaOCl, 4.07±1.06; 0.5 M etidronate+10% NaOCl, 5.09±1.21; 5% NaOCl/17% EDTA, 5.39±1.47 and 5% NaOCl/17% EDTA/5% NaOCl, 5.27±1.33. There were no significant differences between the first three groups mentioned (p > 0.05), nor between the second three groups (p > 0.05), while each of the first three groups differed significantly to all of the second three groups (p < 0.05). Every treatment group differed significantly to the PBS control (p < 0.05), for which the % AF was 0.23±0.16. Scanning electron micrographs are shown for each group taken at high magnification as well as, for the 1 M clodronate+10% NaOCl group, an image at low magnification and its corresponding Image J output file, illustrating the identification of open tubules (Figure 3-5). On a qualitative level, the high magnification images (d)-(g) revealed a similar texture in the intertubular dentine for mixtures containing either clodronate or etidronate together with a lack of erosion of the peritubular dentine. Canalicular branches were not obvious in these groups. For sequences containing 17% EDTA, (h) and (i), canalicular branches are seen both within the tubule itself and in the peritubular dentine. The peritubular dentine is eroded in the sequence 5% NaOCl/17% EDTA/5% NaOCl, (i), while in the sequence 5% NaOCl/17% EDTA, (h), the erosion observed was considerably less.

Figure 3-4. Area fraction of open dentinal tubules as a percentage of total area (% AF)

Groups: 1 M clodronate+10% NaOCl, 0.5 M clodronate+10% NaOCl, 1 M etidronate+10% NaOCl, 0.5 M etidronate+10% NaOCl, PBS, 5% NaOCl/17% EDTA/5% NaOCl, 5% NaOCl/17% EDTA. The statistical analysis was performed with a one-way analysis of variance (ANOVA) and Tukey’s multiple comparison tests, with alpha set at 0.05. ns = no significant differences.

Figure 3-5. SEM images of smear layer removal and Image J output file

Low magnification (a) and high magnification (c)-(i) micrographs collected with a secondary electron detector. (a) From the group 1 M clodronate+10% NaOCl and (b) the corresponding Image J analysis. (c) PBS control. (d) From the group 1 M clodronate+10% NaOCl. (e) From the group 0.5 M clodronate+10% NaOCl. (f) From the group 1 M etidronate+10% NaOCl. (g) From the group 0.5 M etidronate+10% NaOCl. (h) From the group 5% NaOCl/17% EDTA/5% NaOCl. (i) From the group 5% NaOCl/17% EDTA. Scale bars: (a)-(b) = 20µm, (c)-(i) =2 µm.

3.1.5 Discussion

This study clearly identified NaOCl mixtures of CDTA or clodronate at high pH as being more compatible with NaOCl than alkaline EDTA or etidronate, respectively. At room temperature, solutions of 0.25 M CDTA mixed equally with 5% NaOCl have a use life of up to 60 minutes and 0.5 M clodronate mixed equally with 10% NaOCl loses no FAC over 18 hours. This latter result compares with a drop in the effective NaOCl concentration from 5% to 3.7% in 1 hour for etidronate mixtures of the same concentration (Zollinger et al. 2018). All other potential chelators when combined with NaOCl lost their FAC rapidly, with a concurrent spike in temperature. In the case of HPAA, the 1 minute fall to pH 7.4 coupled with a temperature rise to 42.2oC precluded its inclusion in the FAC experiment. The reason for the observed chelator compatibility of CDTA and clodronate with NaOCl relates to their chemical structure. Comparing EDTA and its analogues, CDTA is the only chelator that contains a cyclic hexane group, and this would impose stereochemical restrictions, limiting molecular movement. This is likely responsible for its lower reaction rate with NaOCl as compared to EDTA. Clodronate, like etidronate, is a non-nitrogenous chelator containing phosphorous instead. In NaOCl, the chlorine essentially carries a positive charge and will attack electrophilic centres such as nitrogen atoms (Fukuzaki 2006). Phosphorous is less electronegative than nitrogen (Rumble 2017) so is less likely to react with NaOCl. Additionally, as clodronate already contains two chlorine atoms bonded at the central carbon atom, chlorine addition here is unlikely. Indeed, NaOCl is used in the synthesis of clodronate, with the endpoint of the reaction being the formation of clodronate (Vepsäläinen et al. 1991) and this fact helps explain its lack of reaction with NaOCl. The actual concentrations employed in the FAC experiments were based on previous research. All the chelator experiments contained their own controls and were thus standalone experiments, designed to identify a concentration for both chelator and NaOCl that resulted in NaOCl stability. For etidronate, much of the literature has used the Zschwimmer & Schwarz product Cublen K8514 GR, in concentrations of 18% Cublen (0.5 M (15%) etidronate) mixed equally with 5% NaOCl, giving final concentrations of 2.5% NaOCl and 9% Cublen etidronate (Neelakantan et al. 2012; Tartari et al. 2015; Arias-Moliz et al. 2016). However, because 5% NaOCl is a better antimicrobial agent than 2.5% NaOCl (Vianna & Gomes 2009), the former was chosen for the current study. Additionally, etidronate mixtures containing 5% NaOCl had shown reasonable stability (Biel et al. 2017). The clodronate concentration copied that used commonly for etidronate. For all other 93 chelators a final concentration of 2.5% NaOCl was used in anticipation that they would behave in a similar manner to Na4 EDTA mixtures because it has been shown that FAC was maintained for 30 minutes in Na4 EDTA-2.5% NaOCl solutions (Tartari et al. 2017), but that

Na4 EDTA-5% mixtures lost FAC rapidly (Biel et al. 2017). The same chelator concentrations that were previously shown to result in a use life for Na4 EDTA mixtures of 30 minutes were used for the Na4 EDTA, EGTA, CDTA and DTPA solutions. For ATMP and HPAA, 0.5 M (15%) and 1.0 M (15%) solutions were used, respectively, to match those used in a previous experiment using ATMP (Zehnder et al. 2005).The experimental times chosen of 80 minutes and 18 hours allowed for time extensions compared with other studies (Biel et al. 2017; Tartari et al. 2017). The superior stability of the clodronate mixture warranted further examination of its clinical potential and its ability to remove smear layer was assessed. Two concentrations of clodronate were tested and compared with corresponding etidronate solutions and standard endodontic sequences. Smear layer was removed in the clodronate mixtures; however, the % AF was found to be lower when compared with the standard sequences and the mixture 0.5 M etidronate+10% NaOCl, with differences being of the order of 1% AF. Both clodronate mixtures and 1 M etidronate+10% NaOCl showed a similar % AF. In continuous chelation increased chemical reactions occur between the chelator and NaOCl as the concentrations increase (Biel et al. 2017) and the somewhat surprising result of the 1 M etidronate mixture compared to the 0.5 M mixture is possibly explained by this fact. Of concern in using chelators concurrently with a proteolytic agent such as NaOCl is the potential for peritubular erosion because solutions remain in contact with dentine for prolonged periods. Thus, the current study did not use short chelator rinses following the application of NaOCl as some other etidronate smear layer studies, possibly with other focuses, have done (De-Deus et al. 2008b; Deari et al. 2019). Instead, it followed a methodology where the chelator is admixed with NaOCl (Zehnder et al. 2005; Lottanti et al. 2009). Because when chelator pH increases, the chelation ability in dentine is reduced (Deari et al. 2019), the method used has the additional benefit of making smear layer assessments at the pH of the actual mixture, rather than the at lower pH of the chelator. An important finding of the present study was that even though dentine discs were exposed to the chelator-hypochlorite mixtures for 15 minutes, neither the clodronate nor etidronate mixtures caused peritubular erosion. This is consistent with previous research which reported negligible decalcifications, but smear layer free dentine, following irrigation with a mixture of 2% NaOCl and 18% etidronate over 18 minutes (Lottanti et al. 2009). While

94 this in vitro study cannot be expected to correspond to the clinical situation, the time period of 15 minutes was regarded as a minimum time for endodontic treatment and future evaluations should assess longer timer frames. Traditionally, the analysis of smear layer removal has employed a scoring system and a team of scorers using SEM images (Hülsmann et al. 1997). More recently, image analysis software has facilitated and refined this evaluation (George et al. 2008a). Nonetheless, the assessment of smear layer removal from dentine can be problematic due to variations in dentine tubules both from within a single sample and between samples (Deari et al. 2019). Dentine sclerosis can also lead to erroneous results (Lottanti et al. 2009). The technique known as co-site optical microscopy has been successfully employed to automate image analysis and to follow the evolution of the opening of a defined set of tubules over time (De‐Deus et al. 2007). This method was not employed in the current study due to difficulties in adapting the technique to a large number of test groups. Additionally, the SEM analysis used allowed for high magnification micrographs and additional insights. An endeavour was made to address some of the above mentioned problems firstly by choosing coronal slices of dentine, where sclerosis is known to be less common (Lottanti et al. 2009). Next, as much as possible, image acquisition was standardised during microscope operation and although large standard deviations in the data were seen, significant differences were recorded. Clodronate has many desirable attributes, including some antibacterial activity against Pseudomonas aeruginosa (Kruszewska et al. 2002). While the systemic release of clodronate during a procedural mishap would be low, such release may not be without consequence. As a first-generation bisphosphonate, clodronate has been used for the treatment of osteoporosis, Paget’s disease, osteoarthritis and malignant hypercalcaemia for over 30 years (Frediani & Bertoldi 2015). Extensive medical research exists on its safety and effectiveness (Frith et al. 2001; Atula et al. 2003; Frediani & Bertoldi 2015; Rodrigues et al. 2015). Clodronate inhibits osteoclastic activity, and it is anti-inflammatory (Atula et al. 2003) with both these properties being potential advantages for an endodontic irrigant. A final point in its favour is that the use of clodronate is not associated with osteonecrosis of the jaw, a complication that has only been associated with nitrogen containing bisphosphonates (Rodrigues et al. 2015). Nonetheless, areas of concern exist, notably the recommendation for the avoidance of the use of bisphosphonate drugs such as clodronate and etidronate in pregnancy, or in women of childbearing age (Ioannis et al. 2011). This cautionary approach has been advised

95 because of a paucity of human safety studies, but the topic is considered open to debate (Losada et al. 2010). Indeed, even for NaOCl itself, possible adverse effects during pregnancy remain undefined (Agency for Toxic Substances and Disease Registry 2002; UK Teratology Information Service 2017). Other issues include the high cost of disodium clodronate, and the lack of a commercially available tetrasodium salt. However, tetrasodium clodronate can be synthesised, and this has been used as the starting material for the manufacture of clodronate pro-drugs (Kunnas-Hiltunen et al. 2010). If it were necessary to use solutions of clodronate to mix with NaOCl, experimentation would need to establish an appropriate storage temperature and pH in order to achieve long term stability. The hypothesis tested was accepted. A chelator, clodronate, was identified with improved stability in NaOCl mixtures compared to Na4 EDTA and Na4 etidronate and which when mixed with NaOCl was able to remove smear layer. Although clodronate has potential for use in continuous chelation, other irrigant requirements need investigation. Such aspects include: the ability to dissolve organic material and kill microorganisms, penetration into the intricacies of the root canal system, safety, compatibility with dental materials, and the fracture resistance of tooth structure.

3.1.6 Conclusion

In this in vitro experiment, alkaline 0.5 M clodronate mixed equally with 10% NaOCl, resulting in final concentrations of 0.25 M clodronate and 5% NaOCl, has potential for use in continuous chelation. This mixture did not lose FAC over 18 hours. It was capable of removing smear layer, although the opening of dentine tubules was to a lesser extent when compared with standard endodontic sequences and the mixture of 0.5 M etidronate+10% NaOCl.

Paper 2

This paper firstly addressed the potential of clodronate mixtures with NaOCl to maintain their NaOCl concentrations at root canal temperatures for 3 hours. Secondly, it examined NaOCl stability in refrigerated storage over 3 months.

3.2.1 Abstract

This study evaluated the stability of continuous chelation mixtures of clodronate admixed with NaOCl at room temperature (23oC), at root canal temperature (34-35oC), and in refrigerated storage (2-4oC). In continuous chelation, one solution containing a chelator and NaOCl, simultaneously disinfects and removes organic matter and smear layer. This technique is may enhance antimicrobial action and debris removal. However, sodium hypochlorite stability and free available chlorine levels may decline with elevated temperature and through chemical interactions with the chelator, thus reducing the therapeutic window of these mixtures. Employing iodometric titration, the FAC for clodronate-hypochlorite mixtures was measured at 34-35oC, 23oC and 2-4oC. Clodronate- hypochlorite solutions were stable for 180 minutes at 34-35oC. When kept at 2-4oC over 3 months, they maintain 95% FAC compared with baseline. It was concluded that the therapeutic window of clodronate-hypochlorite mixtures is unaffected at root canal temperature.

3.2.2 Introduction

NaOCl is the most popular endodontic irrigant as it performs the dual roles of dissolving organic material and disinfecting the root canal system (Zehnder 2006). Typically, following instrumentation with NaOCl, the canal is flushed with the chelating agent EDTA, at near neutral pH, to remove the smear layer created during canal preparation (Haapasalo et al. 2014). Often, this is followed by a final rinse with NaOCl (Schäfer 2007). Endodontic irrigation can however be simplified with the use of a single irrigant (Zehnder et al. 2005). A commercial product containing 80-90% of the chelator etidronate, also known as HEDP, has recently become available (Medcem 2016). Etidronate mixtures with hypochlorite generate an irrigant solution that concurrently disinfects, removes smear layer and dissolves organic material (Lottanti et al. 2009; Arias-Moliz et al. 2014; Tartari et al. 2015). This technique of using a single multi-functional irrigant solution is known as continuous chelation (Neelakantan et al. 2012). As well as simplicity, it has the additional reported benefits of improved disinfection (Arias-Moliz et al. 2016; Morago et al. 2016), removal of dentine debris (Paqué et al. 2012), and compatibility with dental materials (Neelakantan et al. 2012; Paulson et al. 2018). A mixture containing 0.26 M etidronate and 5% NaOCl (0.26 M etidronate-5% NaOCl) results in a solution with a therapeutic window of one hour at a nominal room temperature of 23oC (Zollinger et al. 2018). Lowering the NaOCl concentration to 2.5% or 1% extends the “use-life” at room temperature to 2 hours and 4 hours respectively (Zollinger et al. 2018). This limitation on use is due to the loss of FAC resulting from a reaction between NaOCl and etidronate. FAC is directly proportional to the NaOCl concentration. Root canal temperature is considered to be 35oC (de Hemptinne et al. 2015). After the application of room temperature irrigants into the root canal, a temperature of .8±0.9oC is reached after 15 seconds and an equilibrium establishes after 4 minutes at 35±0.9oC (de Hemptinne et al. 2015). At root canal temperature, the stability of chelator mixtures in continuous chelation is affected profoundly, because chemical interactions are accelerated by heating (Wright et al. 2019). This shortens their therapeutic window. Conversely, in storage conditions at low temperature, for example using conventional refrigeration, it could be expected that the FAC would be maintained at an adequate level for longer time periods.

The FAC of etidronate admixed with NaOCl has been examined at various temperatures; at 60oC and 5oC (Zollinger et al. 2018), and at 35oC (Wright et al. 2019). Recently, clodronate has been identified as having potential for use in continuous chelation (Wright et al. 2020a). The mixture 0.25 M (7.3%) clodronate-5% NaOCl has the capacity to remove smear layer, with no loss of FAC when kept for 18 hours at room temperature (Wright et al. 2020a). No studies exist however on the effect on the FAC of clodronate and NaOCl mixtures at temperatures other than room temperature. Thus, the use life of clodronate mixtures at a clinically relevant temperature has not been investigated. Time and concentration have also been identified as factors that lower the FAC of continuous chelation mixtures (Biel et al. 2017; Wright et al. 2019). Given the potential for clodronate to be used in continuous chelation, the current study examined the effect on FAC of heating the mixture 0.52 M (15.2%) clodronate-5% NaOCl to the intracanal temperature of 34-35oC, compared with a room temperature of 23oC over 3 hours. This is more than double the previously tested concentration of clodronate. The storage potential of the same mixture when kept for 3 months at 2-4oC was also studied.

3.2.3 Materials and Methods

Materials

The chelator solution used was 1.04 M (30.4%) clodronate (Na2 clodronate, Enke Pharma-tech, Cangzhou, China) adjusted to pH 11.1-11.2, for the 34-35oC and 23oC study, or to pH 10.7 for the 3 month storage experiment at 2-4oC. The chelator solution was mixed equally with 10% NaOCl (pH 12.5-12.7), resulting in final concentrations of 0.52 M (15.2%) and 5% (wt/vol) respectively. NaOH (Chem-supply, Gillman, SA, Australia) was used for the pH adjustments. Clodronate solutions were made freshly with demineralised water. The concentration of a stock solution of NaOCl (Ajax Finechem, Scoresby, VIC, Australia) was determined by iodometric titration (Wright et al. 2020a). The NaOCl stock solution and a 10% dilution were stored in the dark at 2-4oC.

Experimental design All experiments were performed with 10 replicates. Each experiment consisted of a test group of the clodronate-NaOCl mixture and a control of NaOCl. A repeated measures design was used whereby FAC, pH or temperature were measured on the same solutions at set times.

Measurement of FAC, pH and temperature The FAC experiments were conducted at three different temperatures: an intracanal temperature of 34-35oC, a room temperature of 23oC, and a storage temperature of 2-4oC. The FAC was determined with iodometric titration, utilising a burette, as previously described (Wright et al. 2020a). The pH and temperature were recorded at 34-35oC and at 23oC in a separate experiment with a pH electrode (Ionode IJ40CT; Ionode, Brisbane, QLD, Australia), which incorporated automated temperature compensation, using a pH meter (Orion Star 211: Thermo Scientific, Beverly, MA, USA). The pH recordings were adjusted for high sodium levels and high pH. Measurements at 34-35oC and 23oC were performed, in a water bath (Ratek instruments, Boronia, VIC, Australia) or on the bench, respectively. For the FAC study at 2-4oC, solutions were stored in a refrigerator. For the experiments conducted at 34-35oC and 23oC, at T=0, 4 mL of 10% NaOCl was mixed with either 4 mL of a 1.04 M clodronate solution, or with 4 mL of demineralised water as a control. For the cold storage experiment, 4.5 mL of each solution was used. The concentration of the resulting solutions were thus either 0.52 M clodronate and 5% NaOCl, or 5% NaOCl in the case of the control. Volumes were measured with a serological pipette (Costar Stripette, Corning, NY, NY, USA). Solutions were stored in closed polypropylene falcon tubes (Corning, Tewksbury, MA, USA). For experiments conducted at room temperature and at intracanal temperature, measurements were recorded at T=1, 20, 40, 60, 120 and 180 minutes, while for solutions stored at 2-4oC, the time points for measurements were: 1 minute, 1 day, 3 days, 1 week, 2 weeks, 6 weeks and 3 months.

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Statistical analysis Statistical calculations were performed with Prism version 7.0 (GraphPad Software, La Jolla, CA, USA). All FAC measurements were tested for normality with the D’Agostino and Pearson test, then analysed using the Mann-Whitney test, with alpha set at 0.05. The Bonferroni correction was applied to correct for multiple comparisons of baseline data at subsequent times. Means and standard deviations were determined for the pH and temperature data.

3.2.4 Results . At room temperature and at root canal temperature, for the 5% NaOCl control, no significant changes occurred from baseline in terms of FAC at any time point at either temperature (P > 0.05). Concerning 0.52 M clodronate-5% NaOCl, when kept for 180 minutes at both 23oC and at 34-35oC, solutions were found to maintain 99% of their baseline FAC (Figure 3-6). When comparing FAC at 1 minute versus the subsequent time periods, significant differences were seen at 180 minutes only at 23oC, and from 60 minutes onwards at 34-35oC (P < 0.05). The pH of these mixtures remained above 12 (Table 3-2). During this same 180 minute period, no temperature spike occurred after the solutions were mixed (Table 3-2). When stored at 2-4oC over 3 months, the initial FAC of the clodronate-NaOCl mixture differed from subsequent values at 3 days and onwards (P < 0.05). In the 5% NaOCl control, significant reductions in FAC from the baseline value were seen from 6 weeks onwards (P < 0.05). At 6 weeks and 3 months, the clodronate-NaOCl mixture had lost 3% and 5% of its baseline FAC respectively, whereas for 5% NaOCl, at both these time points, the loss of FAC was 1% (Table 3-3). .

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Table 3-2. Changes over 3 hours in pH and temperature at 23 and 34-35oC Mixtures and Mean and Controls SD 1 min 20 min 40 min 60 min 120 min 180 min pH

0.52 M clodronate- 12.73±0.05 12.70±0.01 12.68±0.01 12.66±0.01 12.75±0.03 12.76±0.02 5% NaOCl, 23oC

5% NaOCl, 12.57±0.02 12.55±0.02 12.54±0.01 12.52±0.01 12.56±0.03 12.57±0.02 23oC

0.52 M clodronate- 12.37±0.05 12.27±0.07 12.20±0.06 12.13±0.04 12.28±0.04 12.28±0.04 5% NaOCl, 34-35oC

5% NaOCl, 12.14±0.06 12.12±0.07 12.06±0.04 11.94±0.07 12.08±04 12.08±0.03 34-35oC Temperature, oC

0.52 M clodronate- 23.1±0.1 23.1±0.1 23.2±0.1 23.2±0.1 23.0±0.2 23.0±0.1 5% NaOCl, 23oC

5% NaOCl, 23oC 23.0±0.1 23.1±0.1 23.2±0.2 23.2±0.2 23.0±0.1 23.1±0.1

0.52 M clodronate- 33.8±0.2 34.5±0.3 34.4.2±0.1 34.4±0.2 34.6±0.1 34.5±0.3 5% NaOCl, 34-35oC

5% NaOCl, 34-35oC 33.6±0.4 34.4±0.2 34.4±0.2 34.5±0.2 34.6±0.1 34.6±0.1

SD, standard deviation. Temperature is expressed in degrees Celsius. Values show means and standard deviations for 10 replicates.

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Table 3-3. FAC at 2-4oC over 3 months Time 0.52 M (15.2%) clodronate-5% 5% NaOCl NaOCl FAC g/L FAC % of FAC g/L FAC % of 95% CI Baseline 95% CI Baseline 1 Minute 47.5-47.9 100 47.3-47.5 100 1 Day 47.2-48.1 99 47.3-47.7 100 3 Days 47.1-47.6 99 47.4-47.7 100 1 Week 46.9-47.2 99 47.3-47.8 100 2 Weeks 46.8-47.5 99 47.1-47.5 100 6 Weeks 46.3-46.7 97 47.0-47.1 99 3 Months 45.0-45.6 95 46.4-46.9 99 CI, Confidence interval. FAC values are expressed as both a concentration in g/L, shown with the 95% CI, and as a percentage of the baseline control value, for 10 replicates. Temperature is expressed in degrees Celsius.

3.2.5 Discussion

The importance of this study is the finding that NaOCl concentrations are maintained in clodronate-hypochlorite mixtures not only at room temperature, but also at root canal temperature. Heating to 34-35oC does not lower its use life. This level of stability is unparalleled in continuous chelation. The FAC in etidronate-NaOCl solutions of the same molarity as those used in the current study, falls at root canal temperature to 80% of the baseline at 20 minutes, and to only 8% at 60 minutes (Wright et al. 2019). Moreover, at root canal temperature, alkaline EDTA when used with NaOCl in continuous chelation, causes such a steep and rapid decline in FAC levels that its use is not recommended (Wright et al. 2019). In addition, the pH of clodronate-NaOCl solutions was maintained at over pH 12 during the 180 minutes of the experiment. This is significant because for NaOCl to dissolve organic material, the pH must remain above 9 (Christensen et al. 2008). Additionally, because dentine buffers alkaline solutions (Wang & Hume 1988), when irrigation is performed in teeth, the pH of alkaline mixtures must be high, to counter this effect. Finally, no temperature spikes were recorded, indicating that no exothermic chemical reaction occurred between the two active ingredients. 103

It is advantageous, from a practical and cost saving perspective, to be able to store solutions for long periods of time after mixing the chelator and NaOCl. Under refrigerated storage conditions of 2-4oC over 3 months, the FAC of the 0.52 M clodronate-5% NaOCl mixture fell only 5%, which indicates that this mixture has high stability. This contrasts with the stability of mixtures of etidronate and NaOCl in cold storage. In the case of 0.26 M etidronate-5% NaOCl, once the etidronate and NaOCl are mixed, the concentration of NaOCl falls from 5% to 4.1%, a drop of 18%, within 7 hours of being stored at 5oC (Zollinger et al. 2018). It should be pointed out however that although the clodronate mixtures in the current study retained 95% of their FAC over 3 months, maintenance of the chelation ability of the solution after having been stored for this time was not verified. The temperature at which the FAC of continuous chelation mixtures is measured is an important consideration. Most prior work in this area measured FAC at room temperature (Zehnder et al. 2005; Biel et al. 2017; Tartari et al. 2017). It is more informative to examine the stability of continuous chelation mixtures at the temperature at which they act, namely at root canal temperature, as in the current study. A point of relevance is that because FAC does not decline in clodronate-NaOCl mixtures at root canal temperature, the key irrigant functions of disinfection and tissue dissolution are likely to be performed as well as for NaOCl at the same concentration. When lower NaOCl concentrations are used, organic tissue dissolution is less in terms of weight loss (Jungbluth et al. 2012) and it takes longer for dissolution to occur (Clarkson et al. 2006). Indeed, due to the maintenance of high levels of FAC, continuous chelation mixtures of 0.26 M clodronate-5% NaOCl at 32oC dissolve organic tissue as well as 5% NaOCl (Wright et al. 2020b). The variables of concentration and time are worthy of note. Increases in both augment the loss of FAC in continuous chelation solutions (Biel et al. 2017; Tartari et al. 2017). In the current study, extended times and high concentrations were chosen to test the stability of the clodronate mixtures at the extremes. However, it must be recognized that with irrigant refreshment, the timeframe of three hours was not clinically relevant. With regard to concentration, in the present study, the final concentration of clodronate in the mixture was 0.52 M. This is over twice the concentration examined previously, which was 0.25 M (Wright et al. 2020a). The doubling in chelator concentration in the current experiment explains why changes in FAC occurred at 3 hours at room temperature, compared to 18 hours in the prior study. The present study used 5% NaOCl. This allowed for a comparison with a previous etidronate study which examined the same chelator and NaOCl concentrations, also at root canal temperature (Wright et al. 2019).

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Additionally, several in vitro studies have shown the antimicrobial superiority of 5-6% NaOCl compared with solutions of 1-2%. (Clegg et al. 2006; Retamozo et al. 2010; Wang et al. 2013). However, it should be acknowledged that such concentrations carry a higher risk of tissue toxicity (Hülsmann & Hahn 2000). In some countries, such as Australia, 1% NaOCl is commonly used (Clarkson et al. 2003), and further investigations could include this concentration. A further technical point worthy of explanation is that higher pH values were seen in the test solutions with clodronate compared to the NaOCl controls. This difference is explained by the fact that the clodronate solutions were already basic when mixed with NaOCl, while in the control, NaOCl was mixed with water. The reductions in FAC that occurred over 3 hours at 34-35oC, and over 3 months at 2-4oC in mixtures containing 0.26 M (15.2%) clodronate and 5% NaOCl were small, indicating that these mixtures have good stability. However, further research is required to establish if disinfection capabilities are maintained in such mixtures, and whether they are safe and viable for use in endodontic irrigation.

3.2.6 Conclusion

Mixtures containing 0.52 M (15.2%) clodronate and 5% NaOCl are stable at root canal temperature over 180 minutes, maintaining 99% of their free available chlorine. This same mixture can be kept in storage at 2-4oC for 3 months, and maintains 95% of its baseline FAC.

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Organic Tissue dissolution in continuous chelation

This chapter assesses a second core function of endodontic irrigation, organic tissue dissolution. Continuous chelation mixtures of clodronate or etidronate are compared with sodium hypochlorite controls of the same concentration with respect to tissue weight loss and residual free available chlorine following dissolution. It includes the following publication and link to the original text.

Wright PP, Scott S, Kahler B, Walsh LJ (2020) Organic tissue dissolution in clodronate and etidronate mixtures with sodium hypochlorite. Journal of Endodontics 46, 289-294. https://doi.org/10.1016/j.joen.2019.10.020

Author contribution statement Patricia Wright: Conception, design, ethics, execution, methodology and writing of draft manuscript (100%); statistics (50%); editing/review (25%).

Suzanne Scott: Graphics (100%); statistics (50%); editing/review (25%).

Bill Kahler: Supervision (25%); editing/review (10%).

Laurence Walsh: Supervision (75%); editing/review (40%).

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Abstract

Introduction: Mixtures of clodronate with NaOCl better maintain FAC than etidronate- hypochlorite mixtures. This research aimed to compare organic tissue dissolution and residual FAC between clodronate and etidronate mixtures. Additionally, clodronate- hypochlorite mixtures lose no FAC over several hours. The second aim was to examine how well such mixtures dissolve organic material 6 hours from mixing. Methods: Soon after mixing, porcine palatal mucosa samples were added to 32oC solutions of 0.26 M clodronate- 5% NaOCl, 0.26 M etidronate-5% NaOCl, 5% NaOCl, 0.26 M clodronate, 0.26 M etidronate, or phosphate-buffered saline. Weights and FAC, where applicable, were recorded initially and at 15 minutes. FAC was measured by iodometric titration. Secondly, 6 hours following mixing, mucosa was added to 0.26 M clodronate-2.5% NaOCl, 2.5% NaOCl, 0.52 M clodronate-5% NaOCl, 5% NaOCl or phosphate-buffered saline. Sample weights at 0, 5, 10 and 15 minutes were recorded. Analysis of variance was used for statistical analyses (alpha = 0.05). Results: Soon after mixing, 0.26 M clodronate-5% NaOCl dissolved mucosa as well as 5% NaOCl, and better than 0.26 M etidronate-5% NaOCl, compared to which it retained more FAC. At 6 hours following mixing, 0.26 M clodronate-2.5% NaOCl dissolved organic material as well as 2.5% NaOCl. However, 0.52 M clodronate-5% NaOCl dissolved less mucosa than 5% NaOCl. Conclusions: Soon after mixing, clodronate mixtures better dissolve organic material than etidronate mixtures and have higher residual FAC. At 6 hours from mixing, 0.26 M clodronate-2.5% NaOCl mixtures dissolve organic material similarly to control.

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Introduction

Root canal irrigation aims to remove bacteria, organic matter and smear layer from the root canal (Zehnder 2006). Typically, endodontic irrigation involves the sequential use of NaOCl and EDTA, as in the sequences NaOCl/EDTA, or NaOCl/EDTA/NaOCl. NaOCl disinfects the canal and dissolves organic material, while the chelator, EDTA, removes the smear layer (Torabinejad 2011). However, by combining a suitable chelator with NaOCl, a single irrigant solution can simultaneously perform all three core irrigant functions (Zehnder et al. 2005). This technique is known as continuous chelation (Neelakantan et al. 2012). The only commercial product available for use in this method is Dual Rinse HEDP, a powder containing 80-90% of the chelator HEDP, also known as etidronate (Medcem 2016). It can be mixed with NaOCl solutions of varying concentrations. A 9% solution of Dual Rinse HEDP would correspond to 7.7% (0.26 M etidronate). While mixtures of etidronate with hypochlorite can disinfect (Arias-Moliz et al. 2016; Morago et al. 2016), dissolve organic material and remove smear layer (Lottanti et al. 2009), their therapeutic window is limited by an inherent lack of stability (Biel et al. 2017). After mixing the FAC begins to fall. Depending on chelator and NaOCl concentrations, the “use-life” for mixtures is 1-4 hours (Biel et al. 2017; Zollinger et al. 2018). A reduction in FAC and the corresponding NaOCl concentration over time could be expected to lessen the ability to dissolve soft tissues (Clarkson et al. 2006; Christensen et al. 2008; Dumitriu & Dobre 2015). Etidronate-hypochlorite mixtures do in fact dissolve less organic material than NaOCl controls of the same concentration (Tartari et al. 2015). The corresponding fall in FAC in these mixtures has not been measured. Recording FAC before and after tissue dissolution may help explain differences in weight loss of tissue samples treated with NaOCl alone versus mixtures of NaOCl and chelators. Clodronate at alkaline pH is another chelator of interest for potential use in continuous chelation. At room temperature, 0.25 M clodronate-5% NaOCl maintains FAC over 18 hours and also removes smear layer (Wright et al. 2020a). Such mixtures could be expected to better remove organic material compared to etidronate mixtures. Thus, the first hypothesis was that, soon after combining the chelator and NaOCl, 0.26 M clodronate-5% NaOCl better dissolves organic material with higher residual FAC than 0.26 M etidronate-5% NaOCl. Due to sustained FAC levels over time, clodronate mixtures potentially dissolve organic material for several hours. The second hypothesis was then, that 6 hours from mixing, 0.52 M

108 clodronate-5% NaOCl and 0.26 M etidronate-2.5% NaOCl dissolve organic material as well as 5% NaOCl and 2.5% NaOCl, respectively. The final hypothesis was that the plain chelator solutions, 0.26 M clodronate and 0.26 M etidronate, have no capacity to dissolve organic material.

Materials and Methods

Materials

The materials used were Na2 clodronate (Enke Pharma-tech, Cangzhou, China), 99% pure, adjusted to pH 10.7 with NaOH (SA178-500G, Chem-supply, Gillman, SA,

Australia); Na4 etidronate (Cublen K 8514 GR, Zschwimmer & Schwarz, Burgstädt, Germany), 85% pure; NaOCl (AJA-458-5L, Ajax Finechem, Scoresby, VIC, Australia); PBS; Demineralised water for NaOCl dilutions and chelator solutions; Porcine palatal mucosa.

Experimental design Two experiments were conducted with ten replicates per experimental group. Groups included a positive control of NaOCl and a negative control of PBS. Experiment 1 was based on one independent variable, irrigant group, while experiment 2 incorporated a repeated measures design for irrigant groups over fixed times. The order of testing and allocation of samples was randomized (https://www.random.org/integers).

Experimental procedures Porcine palatal mucosa was used as an organic tissue source (Zehnder et al. 2002; Naenni et al. 2004; López et al. 2018). Animals were butchered for human consumption and not for the purpose of this study. Approval was obtained from the institutional animal ethics committee (ANRFA/DENT/523/17). Mucoperiosteal flaps were excised from pigs within 1-2 hours of slaughter, rinsed in PBS and transported in sterile closed tubes on ice. Mucosal samples were obtained from the rugae crest (Naenni et al. 2004) using a 6-mm diameter punch. Specimens were blotted dry, and trimmed at the base to ensure uniform weights of 55±0.6 mg (mean and standard deviation). After recording individual weights, samples were stored at -30oC and subsequently thawed in PBS at room temperature for 30 minutes before use. The concentration of NaOCl was determined by iodometric titration (Standards Australia 2003) as previously described (Wright et al. 2020a). The stock solution, 5% and

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10% dilutions were stored in the dark at 2-4oC for 6 days. Chelator solutions were made freshly before use. The concentration calculations accounted for chemical purity. Thus 9% Cublen solutions contained 7.7% (0.26 M) etidronate. All experiments were conducted in closed polypropylene tubes. In experiment 1, the groups were as follows:

1. 0.26 M (7.6%) clodronate, pH = 10.7 2. 0.26 M (7.7%) etidronate, pH = 11.6 3. PBS 4. 5% NaOCl, pH = 12.6 5. 0.26 M (7.6%) clodronate-5% NaOCl, pH = 12.7 6. 0.26 M (7.7%) etidronate-5% NaOCl, pH = 12.7

For groups 4-6, 4.5 mL of 10% NaOCl was mixed with either 4.5 mL water (group 4), 0.52 M clodronate (group 5) or 0.52 M etidronate (group 6). A 1.0-mL sample was taken for immediate iodometric titration to determine the initial FAC. In groups 1-3, 8 mL of solution was used. All groups were equilibrated to 32oC (Naenni et al. 2004; Tartari et al. 2015) over 11 minutes, after which a sample of mucosa was added, exactly 15 minutes after the chelator and NaOCl were mixed in groups 5 and 6. The samples of mucosa were treated for 15 minutes, then removed, rinsed with demineralised water for 10 seconds, stored temporarily in 5 mL PBS, blotted dry and weighed. Immediately prior to the removal of the tissue sample in groups 4-6, the tubes were mixed by inversion five times, and a further 1.0- mL sample was taken for immediate titration. Experiment 2 was conducted in two parts, A and B. The groups were as follows:

A1. 2.5% NaOCl, pH = 12.3 A2. 0.26 M (7.6%) clodronate-2.5% NaOCl, pH = 12.4 A3. PBS B1. 5% NaOCl, pH = 12.6 B2. 0.52 M (15.2%) clodronate-5% NaOCl, pH = 12.7 B3. PBS A1 and A2 were obtained by mixing equal volumes of 5% NaOCl with water or 0.52 M clodronate, respectively. For B1 and B2, 10% NaOCl was mixed equally with water or 1.04 M clodronate, respectively. At 6 hours after the clodronate and NaOCl were mixed and

110 stored in their sealed polypropylene tubes at room temperature, a mucosal sample was added to 4 mL of all solutions that had been preheated to 32oC for 10 minutes, and then kept at that temperature. The samples of mucosa were removed at 5 minute intervals over 15 minutes, rinsed, placed in PBS, blotted dry and weighed. At 5 and 10 minutes, the samples were returned to 4 mL of fresh warmed solution that was mixed 6 hours previously.

Statistical analysis Graph Pad Prism 7.00 (GraphPad Software, La Jolla, CA, USA) was used for statistical analyses. R 3.5.1; (running in R Studio [RStudio Team 2018]. RStudio: Integrated Development for R. RStudio, Inc., Boston, MA, USA) was used to confirm the normality of the data with Q-Q plots and to produce the graphs, making use of the tidyverse 1.2.1 packages. Changes in weight and FAC were expressed as a percentage reduction of the initial value. Group-wise comparisons were performed with analysis of variance and Tukey multiple comparison tests. A one-way ANOVA for experiment 1 and a two-way ANOVA for experiment 2 were used. Alpha was set at 0.05.

Results

For each experiment, the initial sample weights, and FAC in the case of experiment 1, were similar between groups (P > 0.05). In experiment 1 (Figure 4-1A and B), considering weight changes, plain clodronate, plain etidronate and PBS all showed no change over time (P > 0.05). Groups that contained NaOCl differed to those without NaOCl. Etidronate- hypochlorite mixtures differed to clodronate-hypochlorite mixtures, and to the NaOCl control (P < 0.0001), however the latter two groups were not significantly different from one another (P > 0.05). The mean percentage weight reductions for 5% NaOCl, 0.26 M clodronate-5% NaOCl and 0.26 M etidronate-5% NaOCl were 60.5%, 56.2% and 46.8%, respectively (Figure 4-1B). With regard to FAC, 5% NaOCl, 0.26 M clodronate-5% NaOCl and 0.26 M etidronate-5% NaOCl all differed to each other (P < 0.05). Mean declines in FAC from baseline were 5.5%, 4.8%, and 10.3%, respectively (Figure 4-1A). In experiment 2 (Figure 2A and B), there were no significant weight changes in samples treated with PBS at any time point (P > 0.05). For all other groups, weight changes occurred between each time point. (P < 0.0001). There were no significant differences between the effects of treatment with 0.26 M clodronate-2.5% NaOCl and the NaOCl control at any time point (P > 0.05) (Figure 4-2A).

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Conversely, the more concentrated solution, 0.52 M clodronate-5% NaOCl, exhibited differences at all time points other than the starting time (P < 0.05)(Figure 4-2B). After treatment for 15 minutes in 2.5% NaOCl, 0.26 M clodronate-2.5% NaOCl, 5% NaOCl, 0.52 M clodronate-5% NaOCl, the mean reductions in weight were 61.1%, 56.6%, 86.2% and 71.9%, respectively.

Figure 4-1. Percentage change in FAC and weight for plain chelators and mixtures

Boxplots of (A) The percentage change in FAC for groups 5% NaOCl, 0.26 M (7.6%) Clodronate-5% NaOCl and 0.26 M (7.7%) Etidronate-5% NaOCl. (B) The percentage weight change for groups: 0.26 M (7.6%) Clodronate, 0.26 M (7.7%) Etidronate, PBS, 5% NaOCl (sodium hypochlorite), 0.26 M (7.6%) Clodronate-5% NaOCl and 0.26 M (7.7%) Etidronate- 5% NaOCl.

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Figure 4-2. Percentage change in weight of clodronate-NaOCl 6 hours from mixing

(A) Groups: 2.5% sodium hypochlorite (NaOCl), 0.26 M (7.6%) Clodronate (Clo)-2.5% NaOCl and PBS. (B) Groups: 5% NaOCl, 0.52 M (15.2%) Clodronate (Clo)-5% NaOCl and PBS. Measurements were made at 5 minute intervals over 15 minutes.

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Discussion

This study provides insights into the tissue dissolution capabilities of mixtures of chelators with NaOCl. All hypotheses tested were accepted, except in experiment 2, part B, where 0.52 M clodronate-5% NaOCl dissolved less organic material than 5% NaOCl. Comparing clodronate and etidronate mixtures of the same concentration soon after mixing, clodronate mixtures better dissolve porcine palatal mucosa. Moreover, the percentage reduction in sample weight in 0.26 M clodronate-5% NaOCl solutions did not differ significantly from 5% NaOCl, although the numerical value was less. The FAC data explains such observations. FAC falls as chlorine is consumed when NaOCl dissolves organic tissue (Clarkson et al. 2013). Additionally, in continuous chelation, chelator-hypochlorite interactions cause a loss in FAC (Wright et al. 2020a). The greater stability of clodronate mixtures results in higher residual FAC, which in turn causes more tissue dissolution. Unexpectedly, 0.26 M clodronate-5% NaOCl mixtures contained significantly higher residual FAC compared with 5% NaOCl. This, coupled with a lesser numerical value for weight change, may be indicative of slight inhibition by clodronate on the ability of NaOCl to dissolve organic material. Neither the plain chelator solutions nor PBS dissolved soft tissues. This result is consistent with previous findings that etidronate solutions do not dissolve organic tissue (Tartari et al. 2015). Although alkaline, the plain chelator solutions contained no OCl−. It is not high pH per se that causes tissue dissolution (Clarkson et al. 2006). Instead, at high pH, NaOCl is present as OCl− and it is these ions which dissolve organic matter (Fukuzaki 2006; del Carpio-Perochena et al. 2015). Furthermore, as predicted from prior stability studies, 0.26 M clodronate-2.5% hypochlorite solutions, 6 hours following mixing, dissolved organic material similarly to 2.5% NaOCl. This equates clinically to clodronate-hypochlorite solutions being mixed in the morning for use in the afternoon. The apparent stability is, however, concentration dependant, since some reduction in performance occurs when chelator and NaOCl concentrations are both doubled. The chelation ability of these mixtures was not however examined, and future research could investigate this aspect. NaOCl dissolves organic tissues at a higher rate with increasing temperature (Stojicic et al. 2010; Dumitriu & Dobre 2015), time (Moorer & Wesselink 1982; Naenni et al. 2004), and concentration (Zehnder et al. 2002; Clarkson et al. 2006; Dumitriu & Dobre 2015). In continuous chelation, these same factors need careful consideration because they have a

114 negative effect. FAC falls more quickly at elevated temperatures (Wright et al. 2019), at longer times following mixing, and at higher NaOCl concentrations (Biel et al. 2017), giving reduced tissue dissolution. Continuous chelation studies conducted at room temperature likely overestimate the ability of chelator mixtures to dissolve organic tissues, because clinically, irrigation is conducted at root canal temperature. One study where 5% alkaline EDTA-2.5% hypochlorite mixtures dissolved as much soft tissue as 2.5% NaOCl (Tartari et al. 2017) was presumably conducted at room temperature. Such mixtures are known to rapidly lose FAC at root canal temperatures (Wright et al. 2019). The surprising result may be explained in full if the experiment was conducted at room temperature. Because most chelator-hypochlorite mixtures can be regarded as decaying irrigants (Wright et al. 2020a), it is important that any time delay should be clearly documented. Differences in such conditions may explain variations in the results of tissue dissolution studies using continuous chelation mixtures. On first look, the present study employing 0.26 M etidronate-5% NaOCl mixtures is consistent with a previous study which used a 0.26 M etidronate-2.5% NaOCl mixture with agitation (Tartari et al. 2015). Both were conducted at 32oC over 15 minutes and weight changes were likewise 14% less than the NaOCl controls. The 0.26 M etidronate-2.5% NaOCl experiment was conducted however at half the NaOCl concentration and at double the irrigant volume. Because fewer chelator-hypochlorite interactions occur at lower concentrations, a better result, closer to the control value, could have been expected. Possibly, FAC was additionally lost in the 32oC solutions because the heating time was unclear. Another possible explanation is that agitation enhanced etidronate-hypochlorite interactions, causing a deterioration of the FAC. However, there was no direct evidence to support this. Some details of the experimental protocol used in the current study are worthy of comment. Time to dissolution (Clarkson et al. 2006; Dumitriu & Dobre 2015), weight loss over a set time (Stojicic et al. 2010; Guneser et al. 2015) and residual protein levels (Koskinen et al. 1980; Nakamura et al. 1985) have been employed to gauge the extent or rate of tissue dissolution. Weight loss was used in the current study because assessing the endpoint of dissolution in samples contained in a water bath is difficult. Moreover, measuring protein levels has proven problematic, since reactions occur between NaOCl and reagents (Koskinen et al. 1980). In the selected methodology, samples must be dried methodically. Although porcine mucosa contains collagen, it cannot replace dental pulp and its use is a limitation. However, its firm texture allows for samples of uniform dimensions. This is

115 important for ensuring consistency of results (Moorer & Wesselink 1982). Agitation was not employed in the present study. Even though agitation accelerates tissue dissolution in techniques such as laser activation and ultrasonic (Guneser et al. 2015; Conde et al. 2017), in standard needle irrigation, fluids stagnate at 1.0-1.5 mm from the needle tip (Boutsioukis et al. 2010). Merely agitating solutions without replicating a specific agitation technique was not considered necessary given the aims of the study.

Conclusion

Several hours following the combination of clodronate solutions and sodium hypochlorite, 0.26 M (7.6%) clodronate-2.5% NaOCl dissolved organic materials as well as 2.5% NaOCl. However, mixtures of double these concentrations dissolved less organic material than the 5% NaOCl control. Clodronate mixtures, compared to etidronate ones of the same concentration, better dissolved organic material with higher residual FAC.

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Antibiofilm actions in continuous chelation using multiple assessment methodologies

This chapter uses colony counting, SEM and the XTT assay to assess the antimicrobial actions of NaOCl mixed with clodronate or etidronate. It also highlights the problems associated with colony counting on HA discs. The chapter includes the following journal article submitted to Microscopy Research and Technique.

Wright PP, Cooper C, Kahler B, Walsh LJ. The antibiofilm actions of sodium hypochlorite mixed with clodronate or etidronate using multiple biofilm assessment methodologies. Submitted to Microscopy Research and Technique on 12 May 2020.

Author contribution statement Patricia Wright: Analysis, conception, ethics, graphics, statistics and writing of draft manuscript (100%); Execution (80%); Methodology (90%); Editing/review (35%). Crystal Cooper: Execution (10%); Editing/review (10%).

Bill Kahler: Supervision (25%); Editing/review (5%), Execution (5%). Laurence Walsh: Supervision (75%); Editing/review (50%); Methodology (10%); Execution (5%).

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Abstract

This study aimed to use multiple methodologies, including scanning electron microscopy (SEM), to compare the antimicrobial actions of sodium hypochlorite (NaOCl) with clodronate or etidronate admixed with NaOCl. A second objective was to validate colony counting as a disinfection methodology, with the use of SEM. Seven day E. faecalis biofilms were grown on HA discs. The discs were disinfected with 0.26 M clodronate-5% NaOCl, 0.26 M etidronate-5% NaOCl, 5% NaOCl, or treated with PBS. Assessments were performed using colony counting, SEM and the XTT reduction assay. The XTT assessment used the same groups but with 2.5% NaOCl. For colony counting, bacteria were removed from the discs, by two minutes of vortex mixing. Evaluators scored the SEM micrographs for remaining bacteria. A weighted Kappa test and Spearman r were used for evaluator comparisons. There were nine replicates/group in the treatment groups. Antibiofilm actions were assessed with the Kruskal-Wallis and Dunn’s multiple comparison tests. SEM micrographs and the XTT assay revealed no differences between the NaOCl controls and the clodronate or etidronate mixtures with NaOCl (P > 0.05). The statistical tests applied showed a good level of agreement between the SEM evaluators. Biofilm removal from HA discs in the PBS controls was incomplete. It was concluded that the chelator mixtures with NaOCl had antibiofilm actions comparable to NaOCl. Furthermore, colony counting, when used with E. faecalis biofilms on HA discs and vortex mixing, is an invalid methodology.

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Introduction

Several authors have written on the importance of choosing appropriate methodologies to assess antibiofilm actions. A common theme is the discouragement of the use of a single methodology (Stiefel et al. 2016; Azeredo et al. 2017). Rather, because of the differing strengths, weaknesses and suitability of particular techniques, the use of a variety of methodologies is stressed. Colony counting is the most commonly encountered tool in endodontic antimicrobial research and the majority of studies use the species E. faecalis (Swimberghe et al. 2019b). In this methodology, E. faecalis biofilms are grown on teeth, blocks of dentine or HA discs, disinfected, and the remaining bacteria are removed by vortex mixing or sonication, subsequent to plating and counting (Deng et al. 2009; Ali et al. 2020). One of the problems associated with colony counting is that the removal of biofilm bacteria from the substrate for both test and control groups is typically not experimentally verified. Thus, calculating percentage reductions and performing intergroup comparisons may not be valid. SEM has been used to qualitatively assess the removal of E. faecalis biofilm from teeth with different irrigation protocols employing NaOCl (Seet et al. 2012). Because NaOCl effectively dissolves biofilm (Zehnder 2006), SEM could also be applied in a quantitative manner to examine antibiofilm effects by counting bacteria remaining on substrates such as HA discs following treatment. Secondly, after vortex mixing or sonication, SEM could demonstrate the effectiveness of removing E. faecalis biofilm from discs, and thus, it would help ascertain the validity of colony counting in this situation. The 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H- tetrazolium hydroxide (XTT) reduction assay is a measure of cell viability (Kuhn et al. 2003). In this assay, XTT undergoes chemical reduction due to the action of bacterial dehydrogenase enzymes with a resulting colour change that is quantified spectrophotometrically (Koban et al. 2012). The use of an additional assessment methodology would provide information on antibiofilm actions from another perspective, helping to consolidate any experimental results from other assays. In continuous chelation, a single irrigant mixture consisting of a chelator combined with NaOCl is used throughout the entire preparation of the root canal of the tooth (Neelakantan et al. 2012). Thus, continuous chelation aims to simultaneously perform the three key functions of an endodontic irrigant, that is, to disinfect, remove smear layer and dissolve organic matter (Zehnder et al. 2005). In contrast, standard irrigation sequences

119 involve an antimicrobial agent and a chelator, because neither one alone will perform all the key roles required of an irrigant. Typically, EDTA at near neutral pH is used as the chelating agent, while NaOCl disinfects and dissolves organic matter (Zehnder 2006). Several studies have assessed the ability of etidronate-NaOCl mixtures to disinfect biofilms. The in vitro studies have employed a model incorporating 5-7 day E. faecalis biofilms, and based predominantly on the use of live/dead staining in conjunction with confocal laser scanning microscopy (Arias-Moliz et al. 2014; Morago et al. 2016). In vitro studies, in addition to the live/dead technique have, on occasion, also incorporated colony counting as the assessment methodology (Arias-Moliz et al. 2014). There is a general consensus among studies that etidronate mixtures with NaOCl are at least as antimicrobial as NaOCl. Recently, another chelator, clodronate at alkaline pH, has been identified for use in continuous chelation. Clodronate-NaOCl mixtures can remove smear layer and maintain their free available chlorine at root canal temperature over clinically relevant time frames (Wright et al. 2020a). Given sustained levels of NaOCl, it is probable that clodronate-NaOCl mixtures will be at least equally as antimicrobial as etidronate-NaOCl mixtures and NaOCl controls. However, to date, there is no published data on the capacity of clodronate mixtures to disinfect biofilms. The principal aim of this research was to assess the antimicrobial effectiveness of clodronate and etidronate mixtures with NaOCl using three assessment methods. The current study tested two hypotheses using colony counting, SEM and the XTT assay as assessment methods. The first was that the mixtures 0.26 M clodronate-NaOCl and 0.26 M etidronate-NaOCl are equally as effective against E. faecalis biofilms as NaOCl of the same concentration. The second hypothesis was that E. faecalis biofilms can be removed from treatment and control HA discs by vortex mixing, and thus colony counting can provide valid information about the antibiofilm actions of antimicrobial agents.

Materials and methods

Solutions

1. 0.26 M (7.6%) clodronate-5% NaOCl, (pH = 12.5), derived from 0.52 M Na2 clodronate, (Enke Pharma-tech, Cangzhou, China) (99% pure), adjusted to pH 10.7 with sodium hydroxide (NaOH) (SA178-500G, Chem-supply, Gillman, SA, Australia) and mixed with equal volumes of 10% NaOCl.

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2. 0.26 M clodronate-2.5% NaOCl (pH = 12.4) derived from 0.52 M Na2 clodronate mixed with equal volumes of 5% NaOCl. 3. 0.26 M clodronate (pH = 10.7).

4. 0.26 M (7.7%) Na4 etidronate (9% Cublen)-5% NaOCl, (pH = 12.5) derived from 0.52

M Na4 etidronate (Cublen K 8514 GR, Zschwimmer & Schwarz, Burgstädt, Germany) (85% pure), mixed with equal volumes of 10% NaOCl.

5. 0.26 M Na4 etidronate-2.5% NaOCl (pH = 12.4), derived from 0.52 M Na4 etidronate mixed with equal volumes of 5% NaOCl.

6. 0.26 M Na4 etidronate (pH = 11.6). 7. 5% NaOCl (pH = 12.5-6). 8. 2.5% NaOCl (pH = 12.2). 9. 5% sodium thiosulphate made from sodium thiosulphate pentahydrate (Fischer Scientific, Longborough, UK).

The stock solution of NaOCl (AJA-458-5L, Ajax Finechem, Scoresby, VIC, Australia) was titrated as described previously (Wright et al. 2020a), diluted to 10% with demineralised water and stored in the dark at 2-4oC for up to 6 weeks. The 5% and 2.5% NaOCl solutions were diluted on the experimental day from 10% NaOCl. The amounts of materials used accounted for the chemical purity. For the 5% sodium thiosulphate solution, a weight adjustment was made to account for the existing degree of hydration.

Biofilm formation Biofilm substrate and conditioning Biofilms were grown on HA discs with a diameter of 9.6 mm and a thickness of 2 mm (Himed, Old Bethpage, NY, USA). The discs were autoclaved at 121oC, then mounted on an Amsterdam Active Attachment (AAA) plate, suitable for high throughput screening of antimicrobial agents (Deng et al. 2009). The AAA plate consists of a metallic lid with 24 nylon pegs affixed on the underside for substrate attachment. Each is positioned over the wells of a 24-well plate. The HA discs were suspended in 1.7 mL of sterile salivary supernatant which covered two thirds of the disc. A salivary pellicle was then allowed to form on the HA discs by incubation for 4 hours (Wang et al. 2015) at 37oC on a rotary shaker at 80 rpm. The saliva for the sterile supernatant was derived from one healthy donor. Institutional ethics approval for saliva collection was obtained from The University of Queensland, approval number

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2017001492. The collected saliva was pooled, centrifuged at 3129 g at 4oC for 20 minutes and the supernatant was filter sterilised with a low binding fast flow PES membrane filter with a 0.22 µm pore size (Merck Millipore, Carrigtwohill, Ireland) and stored at -80oC until required (Philip et al. 2019). Inoculation and biofilm growth E. faecalis, ATCC strain 29212, was obtained directly from a supplier, (In Vitro Technologies, Noble Park, VIC, Australia), propagated according to instructions, and stored in aliquots as glycerol stock at -80oC until required. A previously unused aliquot was steaked onto a BHI agar plate and incubated aerobically at 37oC for 24 hours. A single colony was then dispersed into 20 mL of BHI broth, which was then incubated at 37oC on an orbital shaker at 180 rpm for 18 hours. Following centrifugation and washing steps, the bacterial pellet was suspended in phosphate buffered saline containing PBS to an OD600 of 0.996-0.999. This corresponded to an E. faecalis count of 7.6 x 108, as determined by serial dilution and triplicate plating of two cell suspensions, each on two separate occasions. The inoculum consisted of a 1/50 dilution of the cell suspension in BHI supplemented with 0.2% sucrose for a final bacterial count of 1.5 x 107. The HA discs were lowered into 1.7 mL of inoculum, and incubated anaerobically in a BD GasPak EZ® container system (Becton Dickinson, Sparks, MD, USA) for 7 days, on an orbital shaker at 80 rpm at 37oC . The BHI-sucrose growth medium was changed daily.

Microbial counts Four irrigant groups were tested. (i) 0.26 M clodronate-5% NaOCl, (ii) 0.26 M etidronate-5% NaOCl, (iii) 5% NaOCl, and (iv) PBS. Each AAA plate contained four HA discs/group. Experiments were repeated on three independent occasions. Biofilms were rinsed to remove non-adherent bacteria by moving the AAA plates up and down gently in two plates of PBS, 15 times each. They were then exposed to 1.85 mL of irrigant for 1 minute and subsequently to 2 mL of 5% sodium thiosulphate for 3 minutes. This was followed by two more PBS rinses, without plate movement. The discs were then detached, placed in 2 mL PBS, and kept on ice. Three discs from each group were vortex mixed at 25 W, 50 Hz for 2 minutes, resulting in nine replicates/group over the three experiments. The resulting bacterial suspensions were serially diluted in PBS and hand plated in triplicate onto BHI agar. The plates were incubated aerobically for 48 hours at 37oC, after which colonies were counted with a digital colony counter. A vortex mixed disc from

122 each group, together with the fourth unmixed disc from each group, were processed for SEM analysis.

SEM Further experimentation was conducted using the same protocol as detailed above, except that after treatment none of the discs were vortex mixed nor colonies counted. This experiment, repeated in duplicate, contained three discs/group. Thus, a total of nine treated but unmixed discs/group, from five separate occasions were available for SEM analysis (n=9). The three vortexed discs/group from the colony counting triplicate experiments were included in the SEM assessment to check if the vortex mixing had removed all bacteria from the discs (n=3). Finally, six fresh HA discs, three autoclaved and three non-autoclaved, were included to visualize discs that had been neither infected nor treated. Discs were fixed in 2.5% glutaraldehyde for 1 hour, rinsed in PBS, dehydrated in an ascending ethanol series followed by overnight drying, before splutter coating with 2 nm of platinum using a Leica ACE EM600 sputter coater (Leica Microsystems, Wetzlar, Germany). The discs were imaged by a blinded microscope operator using the secondary electron detector of a Tescan Mira3 field emission SEM (Tescan, Brno, Czech Republic) at 3 kV, with a field width of 70 µm. For each disc, one image was obtained from five random sites located in five different locations, one from the centre and one from each of the four quadrants. The number of bacteria on the surface of the treated vortex mixed and unmixed discs were counted by three independent blinded counters according to protocol (A) below. The PBS controls were scored by two blinded scorers, using the surface percentage coverage scores (B) below by looking at each image and estimating the percentage of bacterial coverage of the HA disc. Counters received methodological instruction which included examples of disc counting and the trial assessment of six discs.

(A). Bacterial counting protocol 1. Enlarge the image and count all bacteria on the surface of the disc. 2. Several features distinguish bacteria from small detached pieces of the HA discs. a. Size: 1 µm b. Marking: There is often a distinguishable fine line in the middle c. Shape: Oval, with a smooth non-angular contour d. Colour: Uniform and usually darker than the HA disc

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e. Position: Located on the surface, and often aligned with a disc grain boundary or depression 3. Bacteria often appear in groups of two, but individual bacteria and larger groups are possible. When in groups, the bacteria are easily identifiable because they appear contiguous with adjacent organisms, and often are aligned in a straight line.

(B). Surface percentage coverage scores Score 1: < 40% Score 2: 40-60% Score 3: 61-80% Score 4: > 80%

XTT reduction assay Two experiments, A and B, were conducted. Experiment A examined continuous chelation mixtures with the following groups: (A1) 0.26 M clodronate-2.5% NaOCl. (A2) 0.26 M etidronate-2.5% NaOCl. (A3) 2.5% NaOCl. (A4) PBS. Experiment B evaluated plain chelator solutions with the following groups: (B1) 0.26 M clodronate. (B2) 0.26 M etidronate. (B3) 2.5% NaOCl. (B4) PBS. Both experiments contained three discs/group and were repeated in triplicate (n=9). Additionally, one disc/group was included on one occasion for both experiments A and B to act as a background blank for the absorbance measurements. Such discs were exposed to uninoculated media over 7 days at 37oC on an orbital shaker at 80 rpm in an anaerobic environment, and were treated in the same manner as the other discs as detailed below. A stock solution of XTT (Sigma Aldrich, St Louis, MO, USA) at 1 mg/mL in PBS was filter sterilized and stored at -80oC. A 0.4 mM menadione solution was prepared immediately before use by dissolving 7 mg menadione (Sigma Aldrich, St Louis, MO, USA) in 1 mL acetone, followed by two 10-fold dilutions in water, with vortex mixing of each diluted solution. Activated XTT was prepared by first mixing 100 µL of the menadione solution with 2 mL of thawed XTT, to which was added 7.9 mL PBS (Philip et al. 2019). XTT solutions were protected from light exposure. All discs were rinsed before disinfection in the same manner as for the microbial counts. In experiment A, the continuous chelation mixtures or controls were disinfected for 30 seconds, while in experiment B the discs were exposed to the plain chelator solutions or controls for 3 minutes. This was followed in all cases by neutralization in 2 mL of 5% sodium

124 thiosulphate for 1 minute, followed by three PBS rinses. The discs were then placed into 600 µL of activated XTT solution and incubated in the dark at 37oC for 4 hours. The last 5 minutes only of this incubation period included agitation shaking at 80 rpm. The absorbance at 492 nm (A492) (Philip et al. 2019) was read on an Infinite 200 PRO plate reader (Tecan, Grödig, Austria).

Statistical analysis Statistical calculations were performed in Prism 7.0 (GraphPad Software, La Jolla, CA, USA), except for the weighted Kappa calculation, which was performed using GraphPad QuickCalcs (GraphPad 2014). Data were tested for normality with the D’Agostino and Pearson test. Bartlett’s test was applied to check for significant differences in standard deviation. The data distribution was not normal and/or there were significant differences in the data standard deviations. Thus, nonparametric tests were used. The median and 95% confidence interval of the log10 transformed colony counts was graphed. For the SEM data, the number of bacteria, and the score of percentage coverage were both averaged over the counters and scorers respectively, and groupwise comparisons were performed with the Kruskal-Wallis and Dunn’s multiple comparisons tests. The number of bacteria recorded by the counters was assessed for agreement with the Spearman r correlation. An interscorer linearly weighted Kappa test was performed for the percentage coverage scores for the PBS controls. The XTT results were also analysed with Kruskal-Wallis and Dunn’s multiple comparison tests.

Results

Microbial counts The SEM results showed that the microbial counts could not be used for intergroup comparisons in a valid manner. Therefore, statistical analysis was not performed. However, the counts are nonetheless presented because they form a basis for a discussion of this methodology. Figure 5-1 shows the logarithmic transformation of data for CFU (colony forming unit)/mL for the median and 95% CI from the average of plating in triplicate for three HA discs/irrigant group in three independent experiments (n=9). Large variations in CFU counts were seen in groups containing NaOCl, while tight ranges were recorded for the PBS group.

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Figure 5-1. Colony forming units (CFU) following biofilm disinfection Data points show the median and 95% confidence intervals for the average of triplicate plating of log10 transformed CFU/mL. The groups were: 0.26 M clodronate (Clo)-5% NaOCl, 0.26 M etidronate (Eti)-5% NaOCl, 5% NaOCl and PBS, (n=9).

SEM analysis The PBS controls for unmixed HA discs showed good coverage with a uniform E. faecalis biofilm (Figure 5-2 (c)). No other bacterial species were observed, illustrating the purity of the monospecies biofilms produced. The median score was 4, with a 95% confidence interval of 4-4, signifying a disc coverage greater than 80%. When the HA discs were vortex mixed for 2 minutes the median score was 2, with a 95% confidence interval of 2-2.5, denoting a disc coverage of 40-60%. Thus, a large number of bacteria remained on the control discs following vortex mixing. The images in Figure 5-2 contrast unmixed (Figure 5-2 (c)) and vortex mixed (Figure 5-2 (d)) PBS controls. The weighted kappa for the two scorers was 0.841, indicating a high level of scorer agreement. 126

The median number of bacteria remaining on the surface of the discs in the unmixed treatment groups, 0.26 M clodronate-5% NaOCl, 0.26 M etidronate-5% NaOCl and 5% NaOCl was respectively 0.00, 0.67 and 0.33 with corresponding 95% Confidence intervals of 0.00 - 1.00, 0.00 - 2.33 and 0.00 - 1.33. There were no statistical differences between groups (P > 0.05). That is, in all groups containing NaOCl, treatment was able to remove biofilm from the surface of the disc. Figure 5-2 (e) shows an unmixed disc following treatment with 5% NaOCl for 1 minute which is free of bacteria. However, in some images, a small number of bacteria remained, as indicated by the arrows in Figure 5-2 (f). When the discs were vortex mixed following treatment, the median number of bacteria remaining for the groups 0.26 M clodronate-5% NaOCl, 0.26 M etidronate-5% NaOCl and 5% NaOCl was respectively, 0.00, 0.00 and 1.67 with corresponding 95% confidence intervals of 0.00 - 0.67, 0.00 - 0.67 and 0.67 - 2.33. There were no significant differences between the two continuous chelation mixtures (P > 0.05). However, both mixtures differed significantly to the 5% NaOCl control (P < 0.01). Thus, in the NaOCl group, vortex mixing resulted in a higher number of bacteria remaining on the disc. Considering the agreement between the three counters, the Spearman r calculation included all groups where bacterial median numbers were reported. The Spearman r correlation was 0.802, 0.735 and 0.836 for the three possible combinations, indicating a good level of agreement. A new disc that was rinsed and autoclaved, but that was not exposed to a bacterial inoculum, is shown in Figure 5-2 (a). Like all such discs, a spotty deposit formed on the disc during autoclaving, and fine particles of hydroxyapatite were evident in scattered clumps. When the discs were not autoclaved (Figure 5-2 (b)), large particles of rubble were present on the disc surface without accompanying areas of staining. Both autoclaved and non- autoclaved discs displayed cavities in the disc surface where the sintering of the powder was incomplete. However, in the latter group, the cavities appeared smaller.

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Figure 5-2. Scanning electron micrographs of control and treated HA discs

Images taken at 3 kV using a secondary electron detector. Biofilms, when present, are from the bacteria E. faecalis. (a) Rinsed and autoclaved, with no biofilm. (b) Unrinsed and not autoclaved, with no biofilm. (c) Biofilm on unmixed PBS control. (d) Remaining biofilm on PBS control following vortex mixing for 2 minutes. (e) Following biofilm treatment with 5% NaOCl with no mixing, with no remaining bacteria. (f) Small numbers of remaining bacteria (arrows) after treatment of biofilm with 5% NaOCl.

XTT assay All absorbance readings were adjusted to account for the background blank of the disc in uninfected media. The A492 of 0.26 M clodronate-2.5% NaOCl and 0.26 M etidronate- 2.5% NaOCl and the 2.5% NaOCl control were all significantly different to the PBS control (P < 0.005), but none were significantly different to each another (P > 0.05) (Figure 5-3 (a)). The median percentage reductions compared to PBS for the clodronate mixture, the etidronate mixture and 2.5% NaOCl were, respectively, 90.4%, 90.0% and 91.4%. The plain chelator solutions, 0.26 M clodronate and 0.26 M etidronate, did not differ from each other, nor from the PBS control (P > 0.05), while the plain chelators and PBS all differed to 2.5% NaOCl (P < 0.05) (Figure 5-3 (b)). The median percentage differences to the PBS control

128 for 0.26 M clodronate, 0.26 M etidronate and 2.5% NaOCl were 4.2%, 10.4% and 93.0% respectively.

Figure 5-3. A492 data from the XTT assay (a) 0.26 M clodronate (Clo)-2.5% NaOCl, 0.26 M etidronate (Eti)-2.5% NaOCl, 2.5% NaOCl and PBS. (b) 0.26 M clodronate, 0.26 M etidronate, 2.5% NaOCl and PBS. ns = (P >0.05), * = (P < 0.05), ** = (P < 0.005),*** = (P = 0.0005), **** = (P < 0.0001). 129

Discussion

The results of this study add to the body of knowledge supporting the view that etidronate mixtures with NaOCl are at least equally as effective against E. faecalis biofilms as NaOCl of the same concentration. The findings further indicate that these effects can be extended to the chelator clodronate, that is that clodronate mixtures with NaOCl were equally effective as NaOCl. The first hypothesis was thus accepted. A second important outcome was the SEM result which demonstrated that, in the colony counting technique, E. faecalis biofilms were incompletely removed from PBS controls by vortex mixing. This resulted in the rejection of the second hypothesis, and raises questions concerning the validity of this technique. While other in vitro studies established the antibiofilm actions of etidronate-NaOCl mixtures predominantly via live/dead staining, the current study reached the same conclusion using alternative techniques. This is important, not only because of the need for diversified methodologies in antimicrobial assessments, but also because of potential problems that exist when a chelator is used in conjunction with live/dead staining. Other research employing this technique reported high proportions of dead cells when biofilms were treated with another chelator, EDTA, as used in standard endodontic irrigation (Chavez de Paz et al. 2010; Wang et al. 2013). In live/dead staining the green dye, Syto9, is used to designate viable cells. The red dye, propidium iodide, identifies dead cells due to its penetration of damaged cell membranes, as occurs when cells die (Invitrogen, 2004). However, propidium iodide can enter viable cells when changes in membrane permeability occur (Shi et al. 2007; Stiefel et al. 2015). Indeed, altered cell permeability was reported as the reason for a false positive result for EDTA using live/dead staining, because when verification was performed with colony counting, cell viability was maintained (Wang et al. 2013). In the current study the antimicrobial effect of plain etidronate was not significantly different to PBS when examined with the XTT assay. However, it did result in a 10.4 % reduction in bacterial survival. The possible role that chelators play in the results of live/dead staining may be a reason why the current result both agrees and conflicts with that of another study (Arias-Moliz et al. 2014). In that study, a 9% Cublen etidronate solution, as in the current study, resulted in the death of 80% of biofilm cells in tubular dentine, when assessed with live/dead staining. However, when the assessment was performed on biofilms grown on the Calgary Biofilm Device (CBD), little effect was detected with colony counting

130 methodology. This was rationalized in terms of the high pH of the etidronate solution and the low bio-load in tubular infection. It was further explained that there is better resistance to the actions of alkaline solutions when bio-load is high as would occur on the CBD. While these comments certainly have a sound foundation, it is also true that the pH in dentinal tubules falls because of the buffering effect of dentine proteins (Wang & Hume 1988), and that less rather than more bacterial death could be expected in the tubules compared to the CBD. Such controversy resulted in the choice of SEM, the XTT assay and colony counting as the assessment methods for examining the antibiofilm effects of continuous chelation mixtures. In the current study, SEM was applied to quantitatively examine the effects of various irrigants by counting the number of remaining bacteria on the HA discs. The use of a monospecies biofilm, where all bacteria were identified using the same morphological criteria, aided in this respect. It was also achievable because the discs were largely devoid of bacteria or only low bacterial numbers remained. Due to sample preparation, SEM cannot differentiate vital from non-vital bacteria (Peters et al. 2015). However, in the present study, the scant number of remaining bacteria in the treatment groups alleviated concerns in this area. When the HA discs were not vortex mixed, the effects of treatment were observed directly by SEM. The results showed that 0.26 M clodronate-5% NaOCl, 0.26 M etidronate- 5% NaOCl and 5% NaOCl were all able to remove biofilm effectively and equally. When the HA discs were vortex mixed, SEM was used to determine if the mixing procedure had removed all the bacteria from the discs in order to validate the colony counting methodology. The results decisively demonstrated that biofilm remained to a large extent on the PBS treated HA discs. Thus, calculating percentage reductions using the control was not possible in the colony counting experiment. Furthermore, although the differences were small, fewer bacteria remained on the discs following disinfection and vortex mixing for the continuous chelation mixtures compared to plain NaOCl. This helps explain why the colony counts were slightly higher for those groups. Importantly, the varying ability of irrigants to remove bacteria following vortex mixing highlights the problem that a low bacterial count may not necessarily be reflective of a better antimicrobial effect. Instead, it could represent a differential ability between irrigants to remove bacteria from substrates. As seen, PBS failed to remove all bacteria. When this methodology is applied to irrigants which do not act by removing biofilm, inaccurate results may be possible. In general, for colony counting to be used as a valid methodology, the removal of biofilm is required. As revealed in the present study, complete removal of bacteria from HA

131 discs from both control and treatment groups does not occur in all cases, and indeed cannot be assumed. The onus should be on authors to demonstrate that for the bacterial species, substrate and biofilm detachment methods used, that extraction of bacteria from the substrate has been achieved. In dentine, where bacteria pack tightly into dentinal tubules (Matsuo et al. 2003), this seems a difficult task. Additionally, extraction methods themselves may in fact cause bacterial death (Kishen & Haapasalo 2015). This will affect CFU counts that are subsequently made. The SEM results revealed that biofilm removal using 5% NaOCl with a time of 1 minute was all but complete. If these same parameters were used for the XTT assay, any differential result between irrigants would not have been discerned. In the XTT assay, some biofilm needs to remain on the substrate following disinfection for colour changes to occur (Koban et al. 2012). Thus, both the NaOCl concentration and disinfection time were halved. The XTT assay further confirmed that clodronate and etidronate mixtures with NaOCl are equally as antimicrobial as NaOCl, and further showed that the plain chelators, 0.26 M etidronate and clodronate, were not statistically different to PBS. Again, the use of a monospecies biofilm improved the validity of these findings. Other work has shown that differences between microbial species and strains can result in varying capacities for XTT to be reduced and for colour changes to occur (Kuhn et al. 2003). While the current study results agreed with many others that etidronate-NaOCl mixtures are as antimicrobial as NaOCl (Arias-Moliz et al. 2014; Ballal et al. 2019b), some studies report additional effectiveness (Morago et al. 2016; Giardino et al. 2019). In the current study, treatment times were short. Experiments using longer irrigation time frames in whole teeth are needed to answer this question more definitively. Those experiments should employ a variety of assessment methodologies. Ideally, polymicrobial biofilms should also be used because endodontic infections are polymicrobial in nature, with a predominance of Gram-negative anaerobic bacteria (Tzanetakis et al. 2015; Keskin et al. 2017). However, this is not an easy task. In endodontics, polymicrobial biofilms are frequently assessed with live/dead staining (Wang et al. 2015; del Carpio-Perochena et al. 2015a). Additional to the specific concerns for continuous chelation, others have advised against the use of this technique on polymicrobial biofilms, due to the inaccurate staining of Gram-negative organisms (Netuschil et al. 2014; Stiefel et al. 2015). However, staining techniques in conjunction with confocal microscopy are advantageous because biofilms can remain relatively undisturbed on the substrate (Lawrence & Neu 1999). Alternative techniques such as fluorescein diacetate and

132 ethidium bromide have been suggested (Netuschil et al. 2014). Methodologies such as qualitative real time polymerase chain reaction can identify a wide variety of vital microorganisms, although dead cells and their DNA can also be detected (Swimberghe et al. 2019b). Ultimately, even though molecular techniques are more sophisticated compared to colony counting, in the root canal, microbial samples still need to be extracted from the substrate. At present, no one methodology can be relied upon, and using not just one but instead a variety of techniques should be the norm.

Conclusion

Clodronate or etidronate mixtures with sodium hypochlorite were as equally effective against E. faecalis biofilms as sodium hypochlorite of the same concentration. However, the mixtures showed no additional benefits. Colony counting should not be used as a biofilm assessment methodology in conjunction with hydroxyapatite discs and E. faecalis biofilms. A variety of methodologies is needed to accurately assess biofilm disinfection.

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Resistance of bovine tooth roots to compressive force after continuous chelation

One of the tenets of endodontic irrigation is the avoidance of damage to tooth structure. This chapter compares irrigation between a standard endodontic sequence and continuous chelation mixtures of clodronate or etidronate from the viewpoint of changes in the mechanical properties of teeth. It includes the following publication.

Wright PP, Scott S, Shetty S, Kahler B, Walsh LJ. (2020) Resistance to compressive force in continuous chelation. Australian Endodontic Journal https://doi.org/10.1111/aej.12440 [Epub ahead of print].

Author contribution statement Patricia Wright: Conception, design, ethics, execution, graphics, and writing of draft manuscript (100%); Methodology (65%); Statistics (40%); Editing/review (30%).

Suzanne Scott: Statistics, (60%), Editing/Review (30%).

Sowmya Shetty: Methodology (25%); Editing/review (5%).

Bill Kahler: Photography (100%); Supervision (25%); Methodology (5%); Editing /review (5%).

Laurence Walsh: Supervision (75%); Editing/review (30%); Methodology (5%).

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Abstract

Continuous chelation involves the simultaneous use of sodium hypochlorite and a chelating agent. Given the combination of a proteolytic agent and a demineralizing chelator, this study aimed to investigate if mixtures containing the weak chelators etidronate or clodronate and sodium hypochlorite could adversely affect the mechanical strength of teeth compared to the sequence sodium hypochlorite/EDTA/sodium hypochlorite. Matching pairs of bovine teeth were tested on a universal testing machine with respect to their resistance to compressive force. One root from each pair was prepared with the sequence, and the matching tooth was prepared with either water, the clodronate mixture or the etidronate mixture. The load at fracture was recorded. A paired t-test was used to compare irrigants in matching pairs. No differences in load at fracture were seen between either mixture and the sequence, indicating that the continuous chelation mixtures did not alter tooth mechanical properties compared to the standard sequence.

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Introduction

Endodontic irrigation often involves the sequential use of sodium hypochlorite (NaOCl) and the chelator EDTA at near neutral pH. NaOCl kills bacteria and dissolves organic material, whereas EDTA removes inorganic components, assisting in the removal of the smear layer (Lottanti et al. 2009). While both the proteolytic capacity of NaOCl and the chelating ability of EDTA are essential for successful irrigation, these same irrigant properties can cause detrimental changes within the dentine (Wang et al. 2016). During irrigation, NaOCl dissolves collagen, causing dentine ultrastructural changes and decreased flexural strength, whereas chelating agents demineralize the dentine (Gu et al. 2017) and reduce microhardness (De‐Deus et al. 2006). It is important then that irrigation mixtures which expose dentine simultaneously to both NaOCl and a chelator be assessed in terms of their effects on the mechanical properties of teeth. In continuous chelation, a chelator is admixed with NaOCl and used as a single irrigant throughout chemo-mechanical preparation (Biel et al. 2017). The only commercially available product for use in this technique contains the chelator etidronate (Zollinger et al. 2018). However, research exists on two other chelators, EDTA and clodronate, both at alkaline pH. Alkaline EDTA is not well suited to continuous chelation because of the higher loss of free available chlorine that occurs when it is mixed with NaOCl (Biel et al. 2017). By comparison, clodronate is stable in mixtures with NaOCl (Wright et al. 2020c). However, to date no studies have examined the effect of clodronate mixtures on the mechanical properties of teeth. Fracture resistance to compressive force is used commonly in endodontic research to examine the effect of various interventions such as irrigation (Uzunoglu et al. 2016; Turk et al. 2017), or instrumentation (Capar et al. 2014), on the mechanical properties of teeth. In this methodology, the force is directed down the long axis of a root, with the indenter placed over the canal orifice. The placement of the indenter in this position simulates root cracking in one functional direction. The aim of this study was to compare the resistance to compressive force in teeth irrigated with etidronate or clodronate mixtures with NaOCl and those irrigated with the sequence 5% NaOCl/ 17% EDTA/5% NaOCl. The hypothesis tested was that there are differences in the resistance to compressive force between irrigation regimens comprised of mixtures containing 0.26 M clodronate or 0.26 M etidronate and 5% NaOCl compared to the clinical sequence of 5% NaOCl/17% EDTA/5% NaOCl.

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Materials and Methods

Bovine teeth Contralateral, non-carious pairs of deciduous bovine mandibular canines, containing one root canal, were obtained within 1-2 hours of slaughter, and stored in 0.05% thymol in phosphate buffered saline containing 0.14 M sodium chloride (PBS). The periodontal ligament was removed, and the teeth were stored in PBS at 2-4oC for up to 6 weeks. The teeth were inspected with a stereo microscope at 10x magnification. Only those without cracks, chips or root resorption were used. The teeth were subsequently gamma irradiated at a dose of 25 kGy. Ethics approval was obtained from the institutional animal ethics committee (ANRFA/DENT/523/17). Animal slaughter was unrelated to the purpose of this study.

Solutions The EDTA solution and chelator mixtures used were: 1. 17% EDTA (disodium EDTA dihydrate, Chem-supply, Gillman, SA, Australia) adjusted to pH 7.3. 2. 0.52 M clodronate (15.2%) (disodium clodronate, Enke Pharma-tech, Cangzhou, China) adjusted to pH 10.7 with sodium hydroxide and mixed equally with 10% NaOCl for final concentrations of 0.26 M (7.6%) clodronate and 5% NaOCl and a pH of 12.6. 3. 0.52 M (15.4% ) etidronate (Cublen K 8514 GR, Zschwimmer & Schwarz, Burgstädt, Germany; 18% Cublen), pH 11.6 mixed equally with 10% NaOCl for final concentrations of 0.26 M (7.7%) etidronate (9% Cublen) and 5% NaOCl and a pH of 12.6. The Cublen formulation contained 85% etidronate. The preparation of all solutions accounted for chemical purity and any hydration already present. The clodronate and etidronate solutions were prepared daily with deionized water, and mixed with NaOCl immediately before use. NaOCl (AJA-458-5L, Ajax Finechem, Scoresby, VIC, Australia) was diluted to 5% and 10%. NaOCl solutions were stored in the dark at 2-4oC for 17 days. The concentration of the stock solution of NaOCl was determined by iodometric titration as previously described (Standards Australia 2003; Wright et al. 2020a). The pH of the 5% NaOCl solution used in the sequence was 12.6.

Experimental design 137

The experimental design was based around the benefits obtained from utilising contralateral matching pairs of teeth from the same animal. Three separate experiments were conducted, with each designed to answer a different question. Question 1. Is the methodology sensitive enough to discriminate between differences where they are likely to occur, in this case, between a standard endodontic sequence and water? Question 2. Are there differences in the resistance to compressive force between irrigation with 0.26 M etidronate-5% NaOCl and the sequence 5% NaOCl/17% EDTA/5% NaOCl? Question 3. Are there differences in the resistance to compressive force between irrigation with 0.26 M clodronate-5% NaOCl and the sequence 5% NaOCl/17% EDTA/5% NaOCl? A total of 45 matching pairs of teeth were divided randomly into three sets (n=15). One tooth from each tooth pair was prepared with the irrigant sequence 5% NaOCl/17% EDTA/5% NaOCl and the matching tooth prepared with either 1. Demineralised water, 2. The 0.26 M clodronate-5% NaOCl mixture or 3. The 0.26 M etidronate-5% NaOCl mixture.

Tooth preparation Teeth were cut 13 mm from the apex (Capar et al. 2014; Uzunoglu et al. 2016) (Figure 6-1A) with a diamond saw (Exakt Advanced Technologies GmbH, Norderstedt, Germany), to give a consistent root length. Dental pulp tissue was removed with nerve broaches and Hedstrom files. The apical foramen and lateral canals were sealed with flowable composite resin (Admira Fusion Flow, Voco GmbH, Cuxhaven, Germany) and the roots were stored as matched pairs in PBS at 2-4oC for up to 1 week. During chemo-mechanical preparation, roots were suspended in wax slices to avoid irrigant contact with the external root surface, and to facilitate instrumentation (Figure 6-1C). The canals were prepared with an endodontic motor (X-Smart, Dentsply Maillefer, Ballaigues, Switzerland) at settings of 3.0 Ncm and 300 rpm with ProTaper Next® instruments from X1 to X5 (Dentsply Sirona, Ballaigues, Switzerland) at a working length of 12 mm. The canals were irrigated over 62.5 minutes, with a total active irrigant volume of 18.5 mL, using ProRinse™ 30 GA needles (Dentsply Maillefer, Ballaigues, Switzerland). The first 60 minutes were comprised of 4 x 15minute cycles, where in each cycle, 4 mL of irrigant was used with the following instruments: cycle 1-X1and X2, cycle 2-X3, cycle 3-X4 and cycle

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4-X5. In every cycle, instrumentation and irrigation occurred during the first 4 minutes at a flow rate of 1 mL/minute. The irrigant was then allowed to remain in the canal for a further 11 minutes. Finally, 2 mL of irrigant was delivered over 2 minutes, then 0.5 mL in 30 seconds. For water and the clodronate and etidronate mixtures, the same irrigant was used throughout. For the sequence NaOCl/EDTA/NaOCl, 5% NaOCl was used for 60 minutes, then 2 mL of 17% EDTA over 2 minutes followed by. 0.5 mL of 5% NaOCl for 30 seconds. In all cases, irrigation was followed immediately by a flush with 5 mL of demineralised water over 2 minutes, and a further 1mL flush 10 minutes later. The roots were stored in pairs in 5 mL of demineralised water at 2-4oC until strength testing.

Sample mounting The roots were dipped in molten wax (Soares et al. 2005) (Investo Bite & Boxing Wax, Ainsworth Dental, Marrickville, NSW, Australia) to cover the apical 11 mm (Figure 6- 1A). The wax layer thickness was 100-150 µm. The roots then were mounted in a cardboard frame with the waxed section on one side (Soares et al. 2005). This assembly was lowered into a stabilized M10 stainless steel dome nut containing investment dye stone (GC Fujirock® EP, GC Europe, Leuven, Belgium) (Figure 6-1B). Upon setting, the roots were removed from the stone by soaking in tepid water to soften and remove the wax. The roots were coated in a soft polyether impression material (Impregum™, 3M ESPE, Neuss, Germany) to simulate a periodontal ligament (Ossareh et al. 2018), and then replaced in the investment stone. Strength testing was conducted immediately after the Impregum had set.

Fracture testing The dome nuts were mounted into the grips of a universal testing machine (Instron 3365, Instron, Norwood, MA, USA), fitted with a 5000 N load cell. An indenter with a spherical tip measuring 5 mm in diameter (Uzunoglu et al. 2016) was positioned over the canal orifice, and moved vertically at a cross head speed of 1 mm/minute (Capar et al. 2014; Ossareh et al. 2018) (Figure 6-1D and E). The compressive load to fracture in Newtons (N) was recorded using Bluehill 3 software, version 3.15 (ToolWorks Inc, Glenview, IL, USA).

Statistical analysis Statistical analysis was performed with Prism 7.00 (GraphPad, La Jolla, CA, USA). The D’Agostino and Pearson test was used to test for normality. The effectiveness of comparing matched teeth from the same animal was assessed with the Pearson correlation

139 coefficient. A corresponding P value was calculated. A paired two-tailed t-test was applied, with alpha set at 0.05. A power calculation with alpha = 0.05, beta = 0.8, and n=15 (one sample, two-sided) revealed that the experiment had sufficient samples to detect an effect size (Cohen’s d) of 0.8 (Cohen 1969), calculated using pwr.t.test from the pwr library (1.2) (Champely 2018) for R (3.5.1) (R-Core-Team 2019).

Figure 6-1. Sample preparation and load testing (A) Specimen marking, cutting and wax placement. (B) Cardboard frame and investment in M10 dome nut. (C) Tooth preparation set up. (D-E) Strength testing.

Results

The D’Agostino and Pearson test revealed that the data sets were normally distributed. The pairing of samples was effective in all experiments, with Pearson r values of 0.859 (P < 0.001), 0.653 (P = 0.004), and 0.573 (P = 0.013) respectively for water, the clodronate mixture and the etidronate mixture. Irrigation with water resulted in higher fracture resistance compared to the sequence 5% NaOCl/17% EDTA/5% NaOCl, with a mean difference in the load between pairs of +66 N. This difference was statistically significant (P < 0.0001). Both chelator mixtures showed less fracture resistance than the sequence. The mean differences in load were -23 N and - 3 N for the etidronate and clodronate mixtures, respectively. These differences were not statistically significant (P > 0.05). The 95% confidence intervals for the differences for water, etidronate and clodronate were, respectively, +40 to +91 N, -76 to +23 N and -53 to +48.

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Cohen’s d for the etidronate and clodronate groups were 0.3 and 0.03, respectively, which were below the detectable threshold of 0.8 for the experimental design. Figure 6-2 shows the mean differences in load at fracture for both teeth in every pair for the three experiments with matching teeth connected by lines.

Figure 6-2. Load at fracture in Newtons (N)

Individual teeth are designated with circles and matching pairs are connected with straight lines. In all cases the sequence is 5% NaOCl/17% EDTA/5%NaOCl. (A) The sequence versus water. (B) The sequence versus 0.26 M (7.6%) Clodronate-5% NaOCl (Clo-NaOCl). (C) The sequence versus 0.26 M (7.7%) Etidronate-5% NaOCl (Eti-NaOCl).

Discussion

In the present study, continuous chelation mixtures containing 0.26 M (7.6%) clodronate or 0.26 M (7.7%) etidronate (9% Cublen) and 5% NaOCl did not cause reduced resistance to compressive force compared with the sequence 5% NaOCl/17% EDTA/5% NaOCl. This was despite an exposure of just over 1 hour to the chelators clodronate and etidronate, compared with a 2 minute exposure to EDTA in the sequence. The hypothesis posed was rejected. This result can be explained by several factors. The capacity of a chelator to bind metal ions is determined by the ligand stability constant, which is expressed as a logarithmic value for a particular ion of interest. Etidronate and clodronate are weak chelators, having lower Ca2+ stability constants of 6.04-6.08 and 5.95 respectively, compared to the strong chelator EDTA with a ligand stability constant of 10.65 (Smith & Martell 1989). In the present study, the clodronate and etidronate concentrations used (0.26 M) was roughly half that of 141

EDTA (17% or 0.5 M). Higher chelator concentrations are associated with lower fracture resistance (Turk et al. 2017). Additionally, the prevailing pH in dentine determines the bioavailability of Ca2+ (Dawes 2003). For hydroxyapatite, there will be a corresponding release of OH− together with Ca2+. In alkaline conditions, the high OH− concentration drives the equilibrium back towards the formation of hydroxyapatite, with the result that little Ca2+ is available for chelation (Dawes 2003). Thus, in standard irrigation, with EDTA close to neutral pH, the bioavailability of Ca2+ is higher than in continuous chelation where the pH of solutions is highly alkaline. These combined effects account for the fact that no differences were seen between the chelator mixtures and the sequence. In the current study the root canals were not root filled. This was done to remove the confounding effect of bonding between dentine and sealer on the mechanical properties of dentine. Following the use of NaOCl or water, the application of EDTA can increase push out bond strengths due to the removal of smear layer by chelating agents (Vilanova et al. 2012). Thus, including obturation in this study would have made it difficult to attribute differences in fracture resistance between groups to variations in bond strength, versus mechanical changes in tooth structure. The present study employed the sequence NaOCl/17% EDTA/NaOCl. The usage of this sequence has been recommended for root canal preparation, although there is no consensus on the NaOCl concentration, and solutions of between 0.5% and 6% are in use (Peters & Arias 2017). A member survey conducted by the American Association of Endodontists revealed that the majority of clinicians use NaOCl solutions with concentrations greater than 5% (Dutner et al. 2012). Thus, 5% NaOCl was employed in the current study. The final NaOCl rinse was limited to 0.5 mL and 30 seconds. Others have advised a final NaOCl flush of 30 seconds (Basrani & Haapasalo 2014). If this last NaOCl rinse is for one minute or longer, erosion of the dentine tubules can occur (Qian et al. 2011). The final experimental variable of note was time. The active irrigation time was just over one hour and this gave the chelator mixtures the opportunity to exert an effect should one be present. Only one other study has examined fracture resistance in continuous chelation (Domínguez et al. 2018). That study used a treatment time of 28 minutes with mixtures containing 9% Cublen etidronate and 2.5% NaOCl, without obturation, and found that the etidronate mixture lowered fracture resistance compared to the sequence NaOCl/EDTA. The contrasting result in the current study may reflect the actions of a higher NaOCl concentration and a different sequence.

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In the present study, irrigation with water as opposed to the sequence 5% NaOCl/17% EDTA/5% NaOCl gave higher fracture resistance, and this confirms the ability of the methodology used to discern differences. Another study, also without obturation of the roots, likewise reported that irrigation with 5-6% NaOCl compared to water lowered the resistance to compressive force (Souza et al. 2014). However, this result does not imply that the use of such sequences causes tooth fracture. Clinically, whole teeth rather than just roots are root filled, and this is a limitation of the study. In studies where root obturation was conducted after irrigation, the sequence NaOCl/EDTA/NaOCl, compared with NaOCl during preparation followed by water irrigation, improved the resistance to compressive force. The concentration of NaOCl used in these particular studies was 2.5% (Uzunoglu et al. 2012; Turk et al. 2017). The present study utilised bovine rather than human teeth. Bovine teeth are sufficiently similar to human teeth for comparable trends to be seen between different irrigant and medicament groups with respect to mechanical properties such as flexural strength, microhardness and modulus of elasticity (Cochrane et al. 2019). Standardised human teeth are difficult to procure, and although many studies attempt to match teeth based on age (Ossareh et al. 2018), weight (Capar et al. 2014) or size (Capar et al. 2014; Uzunoglu et al. 2016), the teeth are inevitably obtained from different individuals. The bovine teeth used in the current study were matched pairs from the same animal which allowed for standardization of canal preparation. The choice of paired teeth also refined the selection process to allow not only for size, age and weight but also biological matching, and thus resulted in a reduction of confounding factors. The model employed in this study included a periodontal ligament, to better distribute stresses applied to the root (Soares et al. 2005), and incorporated design features common in other studies such as the use of roots with 1.5-2.0 mm above the investment material and similar indenter shapes and positioning (Uzunoglu et al. 2012; Domínguez et al. 2018; Ossareh et al. 2018). However, consideration could have been given to different root orientations in the investment material which may have better simulated the direction of clinical forces leading to tooth fracture. The methodology employed many steps, and each contributed to an accumulated experimental error, and similar fracture studies have reported data standard deviations of about ± 20% of the force at fracture (Capar et al. 2014; Cicek et al. 2015). However, the use of contralateral pairs allowed for statistical differences to be examined between pairs within an experiment, rather than between two groups where the teeth were unrelated. This design meant that only the experimental procedures of canal

143 preparation and irrigation, and tooth processing were responsible for differences in the force to fracture between pairs. Future studies could examine fatigue loading and assess a variety of NaOCl concentrations. Alternative sequences such as NaOCl/EDTA and final rinses with other antimicrobial agents should also be evaluated. Furthermore, a 1% solution of NaOCl, as commonly used in Australia, could be examined. Lastly, further research, could incorporate more frequent irrigant refreshment to better align with clinical practice.

6.6 Conclusion

Irrigation performed with continuous chelation mixtures showed no differences in the fracture resistance of bovine roots compared to irrigation utilizing a standard endodontic sequence.

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7 General Discussion

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The concept of continuous chelation was introduced in 2005 when it was demonstrated that a single solution was able to simultaneously perform the main functions of an endodontic irrigant (Zehnder et al. 2005). The clinical assumption is that such a technique would not only save time, but the combination of a chelator and NaOCl would also require a lower volume of total irrigant. Figure 7-1 outlines the body of work conducted in this thesis and which led to the identification of a novel chelator, clodronate, for potential use in the irrigation technique of continuous chelation. NaOCl was more stable in the presence of clodronate compared with the only viable alternative, etidronate, and when clodronate was combined with NaOCl,

Figure 7-1. Research gaps in continuous chelation, thesis aims and outcomes 146

it performed the key functions required of an endodontic irrigant. Clodronate-NaOCl mixtures did not cause dentinal erosion nor negatively impact the mechanical properties of teeth compared to a standard sequence. The gaps identified in the literature lead to the research aims which dictated the experiments performed, the results of which in turn helped make the literature more complete.

7.1 Experimental outcomes and methodological considerations

The discussion which follows not only summarises the experiments performed, but presents additional insights and explanations as well as logical next steps for extension experiments. It is also worth noting that in the experiments described in 7.1.1-7.1.5, the chelator and NaOCl concentrations chosen differed, in accordance with the aims of particular experiments as discussed in the relevant chapters.

7.1.1 Free available chlorine and NaOCl compatibility

Clodronate showed exceptional compatibility with NaOCl, while the chelators Na4

EDTA and Na4 etidronate both lost FAC over time. Differences between these chelators can be attributed to differences in their chemical structure (Table 7-1).

It is important to realise that when Na4 EDTA and Na4 etidronate are dissolved, both contain four deprotonated hydroxyl groups, as depicted in Table 7-1. In order for Na2 clodronate to achieve this same form, NaOH must be added to Na2 clodronate solutions. If NaOH is not added, the solution will be acidic and mixtures with NaOCl will lose FAC rapidly. This phenomenon has been observed for EDTA at neutral pH (Grawehr et al. 2003) and relates to the pH dependant changes in NaOCl species that were seen in Figure 1-1. The 4th pKa for clodronate is 8.3 (Fonong et al. 1983), and because the pH of clodronate solutions in the experimental protocols was adjusted to 10.7, fully deprotonated forms of clodronate were achievable.

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Table 7-1. Chelator structures. Na2 Clodronate, Na4 EDTA and Na4 Etidronate

Chelator Chemical structure (NCBI 2018)

Na2 Clodronate

Na4 EDTA

Na4 Etidronate

EDTA, even at alkaline pH, displayed poor compatibility with NaOCl compared with etidronate and clodronate. This is explained by the fact that EDTA contains two nitrogen atoms. Attached to both are two lone pair electrons, because only three of the five electrons in the outer atomic orbitals are involved in bonding. That is, the nitrogen in EDTA is trivalent. However, in etidronate and clodronate, phosphorous rather than nitrogen is present. Although phosphorous is in the same column of the periodic table as nitrogen, in bisphosphonates nitrogen is pentavalent rather than trivalent, and thus there are no lone pair electrons. Because, as previously mentioned, the chlorine of NaOCl essentially carries a positive charge, it will attack negatively charged centres, such as nitrogen lone pair

148 electrons in EDTA to form chemical bonds. This mechanism is not possible for bisphosphonate compounds. The difference between clodronate and etidronate is that the former has two chlorine atoms bonded at the central carbon, while in the latter, a methyl and hydroxyl group are present. That is, the clodronate is already chlorinated. Intuitively, it could be imagined that this is the reason for the lack of signs of a reaction between clodronate and NaOCl. Indeed, because NaOCl is used to chlorinate the precursor chemical in the manufacture of clodronate (Vepsäläinen et al. 1991), a reaction between NaOCl and clodronate is unlikely. However, this supposition remains to be proved. Prior to the work of this thesis, all FAC studies in continuous chelation were performed at room temperature. The demonstration that FAC decay is accelerated by heating to root canal temperature helped establish the usefulness of chelator mixtures with NaOCl in a clinical scenario. The therapeutic window in mixtures containing Na4 EDTA or Na4 etidronate was significantly reduced compared with previous reported values. For clodronate mixtures, NaOCl stability was maintained over 3 hours, showing that no reduction in the therapeutic window occurs. The enhanced stability of NaOCl in mixtures with clodronate compared to etidronate dictated some of the steps in the experimental protocols. It was important to ensure that time delays were minimized so as to ensure that the same NaOCl concentration was present in both the clodronate and etidronate mixtures. For example, this requirement resulted in the quite complicated timing schedule seen in Protocol 4. In all cases where etidronate was used, the mixing of the chelator and NaOCl occurred as soon as possible before the commencement of the particular experiment.

7.1.2 Smear layer removal

Given that FAC is maintained in clodronate mixtures with NaOCl, it was reasonable to assume that the antimicrobial actions and tissue dissolution properties of NaOCl would also be maintained. This was subsequently shown to be the case. However, the ability of the mixture to remove smear layer was the crucial next step that would ascertain if clodronate mixtures could function as a single solution in endodontic irrigation. The high resolution images presented in the smear layer study, not only demonstrated the removal of smear layer, but they were the first to show the ultrastructural definition of the peritubular dentine following exposure to continuous chelation mixtures. One

149 of the aims of the thesis was to investigate the potential for clodronate-NaOCl mixtures to cause erosion, and the finding that the peritubular dentine remained intact for such mixtures is an important one. Although the % AF for the clodronate groups was less than for the two standard sequences, it was considerably higher than for the PBS control, and smear layer was assessed as having been removed. It is also possible that some degree of peritubular erosion is responsible for the larger tubular opening seen for the sequences. The image J analysis revealed no significant differences between 0.52 M and 0.26 M clodronate-5% NaOCl mixtures. The smear layer study in this thesis, differed to other such studies in endodontics. Typically, chelating agents are applied to dentine for anything from 1 minute (Deari et al. 2019) to 3-5 minutes (De‐Deus et al. 2007; Reis et al. 2008; De‐Deus et al. 2011). While the application of 17% Na2 EDTA respected these time frames, clodronate or etidronate mixtues with NaOCl were applied to the dentine for 15 minutes. This was nececessary to reflect the essentially different nature of the continous chelation technique whereby chelator mixtures are present throughout the entire irrigation period. Additionally, because clodronate and etidronate are weak chelators compared to EDTA, applications for short time frames are unlikely to be efficacious. A 15-minute period could be considered a minimal irrigation time. Further studies should extend this time frame to assess if the observed integrity of peritubular tubular dentine is likewise maintained.

7.1.3 Organic tissue dissolution

The porcine mucosa study recorded a significant difference in tissue dissolution between 0.52 M clodronate-5% NaOCl and the 5% NaOCl control, but no differences between either 0.26 M clodronate-2.5% NaOCl and its control nor between 0.26 M clodronate-5% NaOCl and the corresponding control. Given that smear layer removal was equal when either 0.26 M clodronate-5% NaOCl or 0.52 M clodronate-5% NaOCl was used, it therefore seems reasonable to recommend the use of 0.26 M clodronate, rather than 0.52 M clodronate in mixtures with either 5% or 2.5% NaOCl. Concerning the NaOCl concentration, previous dissolution studies in continuous chelation used only 2.5% NaOCl and this was one on the concentrations used in the current study along with 5% NaOCl. Extensions studies could include also a 1% NaOCl solution. Mixtures containing 0.26 M clodronate and 5% NaOCl removed more organic material compared to the corresponding etidronate mixture. This effect was particularly

150 obvious because the experiment was conducted at 32oC. The superior tissue dissolution displayed by clodronate mixtures at this temperature was a direct result of their better stability with NaOCl at root canal temperature.

7.1.4 Antibiofilm actions

The antimicrobial studies revealed no differences between 5% or 2.5% NaOCl and the corresponding clodronate or etidronate mixtures. Hence, no further benefits were identified that could be attributed to the addition of a chelator to NaOCl. However, three papers, all using live/dead staining as the sole methodology, identified additional benefits for etidronate mixtures (Arias-Moliz et al. 2016; Morago et al. 2016; Giardino et al. 2019). This remains an area for further investigation. It must be recognised that the antimicrobial results reported in this thesis were based on the use of HA discs in conjunction with the AAA system. Ongoing research could incorporate other models accompanied by longer time frames, giving any chelator effect a chance to be apparent. Others have reported that the chelator EDTA can remove metallic ions from biofilms. These ions may be important to bacterial survival (Estrela et al. 2018). Non-biofilm research related how manganese ions play a crucial role in bacterial virulence (Colomer-Winter et al. 2018). Thus, the use of chelators in endodontic irrigation may have benefits as yet undefined. Definitively showing that the constant presence of a chelator such as clodronate or etidronate during canal preparation provides advantages compared to standard irrigation would represent a considerable advance in establishing continuous chelation as a valuable technique. The SEM study demonstrated that considerable amounts of E. faecalis biofilm remained on the PBS treated discs following vortex mixing. Thus, the control in the CFU experiment was not valid for calculating the percentage reductions achieved. Colony counting and the use of the E. faecalis are respectively the most commonly encountered methodology and bacterial species in endodontic research (Swimberghe et al. 2019 b). This raises serious concerns about the accuracy of studies that employ these techniques and more specifically for studies that use HA discs. It reiterates the emphasis expressed in review articles, that antimicrobial assessments be performed using a variety of methods (Azeredo et al. 2017). In this work, SEM and the XTT assay were used as additional evaluation tools.

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SEM was a good methodological choice, given that NaOCl acts by dissolving biofilm (Tawakoli et al. 2017). The few bacteria that remained were easily counted because of the homogenous nature of the biofilm. Therefore, the SEM methods used here are not necessarily transferable to other irrigants and biofilms. The use of a monospecies biofilm was additionally beneficial in the XTT assay. It avoided the problem of differential rates of conversion of XTT to the water soluble formazan dye observed between differing species and strains of microorganisms (Kuhn et al. 2003). During the work of this thesis, other antimicrobial assessments were performed, but not reported. Studies were conducted employing a 3-week polymicrobial biofilm based on a salivary inoculum in conjunction with a 3D printed model representing the mesial root of a lower molar. Initially the FISH technique was trialled and then studies with the dye acridine orange were performed. The sequences NaOCl/EDTA and NaOCl/EDTA/NaOCl were compared with clodronate mixtures. However, the methodology was fundamentally flawed due to dye bleaching and difficulties estimating the remaining biofilm after treatment, and a simpler model was finally adopted in order to be able to report a result in this important area. The protocol for the acridine orange experiment appears in 9.3.7.

7.1.5 Compressive strength testing

The experiments involving the resistance to compressive force revealed no differences between chemomechanical preparation with the sequence 5% NaOCl/17% EDTA/ 5% NaOCl and either 0.26 M clodronate-5% NaOCl or 0.26 M etidronate-5% NaOCl. This positive result, particularly in light of the smear layer result which revealed no peritubular erosion, augers well for the potential of the continuous chelation technique in endodontic irrigation. Further research needs to be performed in this area and this would include the use of alternative sequences employing a variety of NaOCl concentrations and other antimicrobial agents. The limitations of this study must be recognized. While the protocol used was based on several literature studies (Capar et al. 2014 ;Cicek et al. 2015; Uzunoglu et al. 2016), in reality, endodontically treated whole human teeth in a functional dentition are at risk of fracture. Clinically, fracture occurs not under compressive loading, but in conjunction with thermal and fatigue events (Torbjörner et al 2004). Even though some authors incorporate cyclic thermal and fatigue loading (Ossareh et al. 2018), clinical relevance is still uncertain because the events occur experimentally over days compared to months or years in real

152 life. Thus, the methodology chosen could not ultimately answer whether continuous chelation increases the risk of tooth fracture. However, it did allow for a comparison of irrigation protocols. Also of relevance is that the fracture loads recorded in the current study, reported in raw data format in Section 9.4.5, correspond to the bite force at maximal clenching in human molar teeth as described in Section 1.5.3.3. The collection of teeth for this study was conducted in a timely fashion because of the ample availability of bovine teeth. Additionally, the animal ethics approvals were considerably easier to obtain than for human teeth. The use of matching pairs of teeth was beneficial because the two teeth in each pair were almost identical. The statistical results showed that tooth pairing was effective, and thus fewer pairs were required to obtain a particular effect size than would have been needed for unrelated teeth. In the study conducted, the research question asked to compare a standard sequence with either a clodronate or etidronate mixture. This suited the use of matching pairs, whereby one tooth was assigned to the sequence and the other to the chelator mixture. However, many studies seek to perform groupwise comparisons where the number of groups is more than two. In this case, the benefits of using paired teeth would be lost, unless specific measures were taken in the allocation of teeth. For a study with four groups, some advantage could still exist. For example, the 12 teeth from six matching pairs of teeth, A, B, C, D, E and F could be split between four groups as follows: Group 1, A, D and F. Group 2, E, A and C. Group 3, E, B and D. Group 4 B, C, and F. In this manner, if any two groups are compared, they will all contain one tooth from a matched pair. Additional size matching could also be employed. Thus, the use of paired teeth can be beneficial when the group number is not only two, but also four. These sorts of strategies are an improvement in the current methodologies employed in the strength testing of teeth.

7.2 Directions for future research

The preliminary nature of the work conducted in this thesis was necessary to establish the viability of a new irrigant across several major areas in a limited amount of time. However, the fundamental research performed here in key areas of endodontic irrigation must evolve to incorporate more clinically relevant studies. This would include the use of commercially available NaOCl. Some of these products contain surfactants and it would be necessary to establish that no chemical reaction occurred with clodronate.

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Extension studies would also use more clinically relevant models. Ideally, future research would eventually lead to better treatment outcomes. Additionally, considerable work is required in areas not covered by this thesis. Returning to the requirements of an ideal endodontic irrigant, as outlined at the beginning of the literature review, future research needs to address essential tooth and safety factors. For etidronate mixtures, studies in the areas of debris removal (Paqué et al. 2012), and the compatibility of treated dentine with other endodontic materials (Neelakantan et al. 2012; Paulson et al. 2018) established possible benefits of the continuous chelation technique. Similar studies are needed for clodronate mixtures. Research is also required on irrigant penetration into dentine which, to date, has not been conducted for any continuous chelation mixture. Another area not covered in the current literature is the ability of chelator mixtures with NaOCl to neutralize endotoxin. It is essential that safety studies be performed. These would be centred on toxicity, genotoxicity, biocompatibility and allergenicity. Ultimately, a clinical trial should be performed. Thus, the question of product availability for clinical use is premature. Even if further research could demonstrate safety and additional efficacy, obstacles exist in relation to cost effectiveness. Currently, commercial clodronate is available in the disodium form, for use in its various medical applications. Hence, the cost is high. Chemical companies, such as those listed in 9.2 (Certificates of analysis), stock clodronate with a purity of at least 99% at considerably lower prices. Specific negotiations with chemical manufactures would be required to not only provide clodronate at a reasonable price and purity, but also to supply it in the tetrasodium form. These issues would need to be addressed before irrigant delivery systems could even be contemplated. Finally, there is the risk that dentists will not adopt this new methodology. For a potential developer of this technology, the path to commercialisation seems long and difficult, but not unattainable.

7.3 Concluding remarks

The work carried out during this thesis identified that NaOCl is stable in mixtures with clodronate. Such mixtures can function in the core functions of an endodontic irrigant, although considerable further research is required. Nonetheless, the solid foundation laid here establishes that further studies in this area are warranted. Currently, continuous chelation using clodronate has potential as a time saving technique for the clinician.

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However, the best outcome of future investigations would be the demonstration of biological benefits for the endodontic patient.

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9 Appendix

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9.1 Ethics, site and biosafety approvals

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9.3 Experimental protocols

9.3.1 Protocol 1, pH measurement of chelator-NaOCl mixtures

1. NaOCl a. Remove NaOCl from the fridge and store in a closed 50 mL Falcon tube. b. Allow this sufficient time to reach room temperature, approximately 45-60 minutes, depending on volume. c. For experiments at 35oC, heat in the water bath with the thermostat control dial adjusted to around 36-38oC so the solution itself reaches 35oC. Measure and record the temperature of the solution with the thermometer. d. The NaOCl used in this experiment will have been standardised by iodometric titration beforehand (see Iodometric Titration, Protocol 2) and stored in a dark container at 2-4oC. 2. Chelator solution a. Weigh the exact amount of chelator in a glass beaker (for a particular desired

concentration) and dissolve in d-H2O. b. In weighing out and diluting the chemicals, take into account the purity of the chemical as recorded on the COA (certificate of analysis) or the supplier website. Additionally, make allowances for the degree of hydration. c. Adjust with NaOH to the appropriate pH.

d. Use pipette to transfer to a volumetric flask. Rinse the beaker with d-H2O and transfer that also to the flask. Make up the final volume to the flask marking. 3. Calibrate pH meter a. Wash the electrode in distilled water, removing any potassium chloride salt that has been deposited on the electrode sleeve. b. Use a 3-point calibration with the following buffers: 7.00, 10.01 and 12.46. Use fresh buffer solutions. c. Rinse the electrode first in each buffer before actual immersion in the buffer. d. For the Orion Star A211 meter, follow the calibration on-screen prompts. 4. Fill in the recording sheet a. Chelator type, concentration, NaOCl concentration, date, time, temperature.

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5. Set up and run the experiment

a. Using a serological pipette, place 4 mL of d-H2O in each of 5, 15 mL Falcon tubes and 4 mL of chelator solution in another 5, 15 mL tubes. b. If doing the experiment in the water bath, check the temperature of all solutions o (NaOCl, chelator and d-H2O) is 35 C. c. Measure the pH of the chelator and NaOCl and write it on the recording sheet. d. Pipette 4 mL of NaOCl, start the timer and add it to the first tube of chelator. At 2 minute intervals add 4 mL of NaOCl successively to the remaining 4

chelator tubes and the 5 d-H2O tubes. e. After the initial addition of NaOCl, cap the tube and invert contents 5 times. Remove the cap and measure the pH. Record the temperature and the pH at

time 1 minute. Rinse the electrode after each reading with d-H2O and pat it dry with Kim wipes. Rinse the electrode between readings to limit cross contamination. f. Successively record the pH and temperature at the time intervals for the particular experiment. Place the cap back on the tube after the reading is complete. These readings must be started 45 seconds beforehand to allow time for equilibration of the pH meter. g. Need to do 2 runs of 5/ group. The recording sheet layout will ensure that each addition of NaOCl is 2 minutes apart. (n=10).

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9.3.2 Protocol 2, Determination of FAC with iodometric titration

1. Make up a standardised potassium iodate (KIO3) solution. (2.A)

a. Weigh out roughly 3.7 g KIO3, cover with filter paper so air can get out but any oven contaminants cannot get in, and place in oven at 105 oC for 1 hour.

b. Dissolve 3.5667 g of the dried KIO3 in d-H2O in a 1 L volumetric flask. Once dissolved, make up to the 1L mark. 2. Make up a solution of potassium iodide (KI) (2.B)

a. Weigh out 200 g of KI and dissolve it in 1 L of d-H2O. 3. Dilute 10.2 M Hydrochloric acid (HCl) to approximately 2.5 M HCl (2.C) a. Use a fume hood.

b. Add 50 mL 10.2 M HCl cautiously, slowly and with stirring to 150 mL d-H2O.

4. Make up a roughly 0.1 M solution of sodium thiosulphate (Na2S2O3 ) (2.D)

a. Dissolve 25g of sodium thiosulphate pentahydrate (Na2S2O3.5H2O) in 1 L ultrapure water (from the Elga Classic machine).

b. After rinsing the burette 3 times with this solution, fill the burette with Na2S2O3 solution.

5. Standardized Na2S2O3 solution (2.E).

a. Add 50 mL d-H2O to a 250 mL conical flask

b. Using a volumetric pipette, pipette 25 mL of the KIO3 solution (2.A) into the flask. c. Add 10 mL KI solution (2.B) and 10 mL HCl (2.C). A measuring cylinder can be used for measuring KI and HCl. The solution will go brown.

d. Without delay, titrate with the Na2S2O3 solution (2.D). Near the endpoint, the solution becomes less and less yellow. Slow the titration when the colour becomes pale yellow. The titration is complete when one drop distinctly changes the solution to clear. This endpoint is an obvious change.

e. Record the volume of the Na2S2O3 solution used (V1) and calculate and record

the molarity of the Na2S2O3 solution (c1) as follows according to AS 1087-2003 p11-12: c1 = 25 x 0.1/ V1

f. The concentration of the Na2S2O3 solution will be approximately 0.1 M. Repeat in triplicate and average results. Record the exact molarity. 6. Dilute acetic acid (2.F). a. Use a fume hood.

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b. Place 100 mL d-H2O into a beaker and add 10 mL glacial acetic acid cautiously and with stirring. 7. Determine the concentration of the stock solution of NaOCl and dilute to 5%

and 10% with d-H2O. a. The stock solution needs to be kept at 4oC in a light proof container. b. Allow a 10 mL sample of the stock solution to reach room temperature.

c. Place 150 mL d-H2O in a 500 mL conical flask. d. Using a Gilson 1000 µL pipette, pipette 1000 µL of the neat sample into the flask. e. Add KI (2.B) solution. The volume added depends on the concentration of the NaOCl. For unknown concentrations or known concentrations of NaOCl of 10 % or higher, add 50 mL of KI, for 5% NaOCl use 25 mL and for 2.5% or less add 12.5 mL KI. This ensures there is an excess of KI. f. Add 10 mL of acetic acid (2.F). The solution will go brown.

g. Titrate without delay with the standardised Na2S2O3 solution (2.E), until the

yellow colour turns distinctly clear, and record the volume of Na2S2O3. h. The [NaOCl] in % of the original solution is calculated as follows:

[NaOCl] = V Na2S2O3 x [Na2S2O3] x 74.44/20. i. Dilute the stock solution into 5% and 10% NaOCl by the addition of the o appropriate amount of d-H2O, label, and store at 4 C in light proof containers. 8. Prepare the chelator and NaOCl solutions. a. Calculate the number of grams of chelator needed to make up 50 mL of the desired concentration. Take into account any hydration present in the solid and the degree of purity. Calibrate the pH meter as per Protocol 1.

b. For Na4 EDTA and Na4 etidronate, mix the solids with d-H20 in a volumetric flask. These solutions are already alkaline. c. For other solutions, dissolve in a beaker with stirring, and adjust the pH with 10 M and 2 M NaOH (even 0.2 M NaOH may be needed if the pKa is low) to bring the solution to alkaline pH. The final pH should be between 10.8-12, with the pH for each solution reproduced each time the solution is made. Make up final volume in a volumetric flask and transfer washings from the beaker. Record the chelator solution pH. d. Remove 100 mL of either 5% or 10% NaOCl from the refrigerator and warm to room temperature.

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e. For etidronate and clodronate use 10% NaOCl, for all other chelators use either 5% or 10% NaOCl as determined by the particular experiment. 9. Set up the experiment a. Each run of this experiment consists of two test samples and two control samples per run, with 5 runs being performed on each chelator group. A recording sheet is used to record experimental set up details as well as to

record the volume of Na2S2O3 solution used in each titration. b. Fill out a recording sheet for each run. Record the pH and concentration for both the NaOCl and chelator solution, date, time, name of chelator, room temperature. c. Have ready 3 x 10 mL serological pipettes and label them, ‘water’, ‘chelator’ and ‘NaOCl’. d. Label 4 x 15 mL Falcon tubes 1-4. With the appropriate serological pipette, fill tubes 1 and 2 with 4 mL of chelator solution (test) and tubes 3 and 4 with 4 mL

of d-H20 (control). 10. Start the experiment a. At T=0, pipette 4 mL NaOCl into tube 1. b. Cap and invert 5 times.

c. Uncap and titrate with Na2S2O3 solution (2.E) as in steps 7. c-g. d. At T=4:30, T=9:00, and T=13:30, add 4 mL NaOCl into tubes 2,3 and 4 respectively and repeat 10. b-c. This will enable a measurement at T=1 for all tubes. e. Repeat the above titrations such that aliquots are taken from the tubes at starting at 19:00, 39:00, 59:00 and 79:00 after the addition of the initial NaOCl. This means that FAC can be calculated for each tube at T= 20:00, 40:00, 60:00 and 80:00 (for the 80 minute experiments) and involves staggering the taking of aliquots in tubes 1-4 by 4:30 as occurred in 10 d. The recording sheet outlines the exact time that each aliquot is taken.

f. The calculation of FAC can be done: FAC in g/L = V Na2S2O3 x [Na2S2O3] x 70.906/2 (AS 1087-2003).

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9.3.3 Protocol 3, Smear layer assessment by SEM

1. Cutting of dentine slices a. Make sure only third molars teeth in patients of age group 18-30 are collected. After extraction, ask the surgeon to place immediately in 0.05% thymol in PBS. In the lab, scrape off soft tissue and cementum with a curette and store at 2- 4oC in new container of 0.05% thymol in PBS. When approximately 100 teeth are collected, take to Steritech (Potassium St, Narangba), for gamma irradiation at 25 kGy. Store again at 2-4oC. b. Make a mould from polyvinylsiloxane putty using a 1.5 cm diameter plastic storage bottle. Mount the apical 3-4 mm of each tooth in pink boxing wax in the mould. Ensure the tooth is mounted so that the root is exactly vertical. c. Mount the encased apical tooth in the Isomet precision saw. Cut a 1.8 mm of radicular dentine. The blade thickness is 0.6 mm. Decoronate the tooth at the CEJ. Cut at 150 rpm and weight = 150 g. Mark the exposed surface of the tooth with a high-speed bur so as to identify the coronal aspect. Set the Isomet saw at 2.4 mm. This allows for the blade thickness. d. Polish the apical aspect of each slice. This ensures a flat surface for imaging. Use a water trough to immerse the slice as it is polished. Turn the water spray off (to avoid oil contamination) and successively polish with coarse, medium and fine Soflex discs. Polish all slices in a methodical manner: Take 1 minute to polish with each Soflex disc, spending equal amounts of time on each portion, using only light pressure. e. Rinse with water and store with the polished side facing up in petri dishes containing 0.05% thymol in PBS in the fridge at 2-4oC until needed. 2. Experiment preparation

a. 24 hours before experiment, transfer dentine slices to d-H2O and store at 2- 4oC. b. Prepare 250 mL sterile 1x PBS. c. The day before the experiment prepare 5% and 10% NaOCl as per Protocol 2. Record the pH of the solutions. Store NaOCl solutions at 2-4oC.

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d. On the experimental day, place 150 mL 10% NaOCl in 50 mL labelled Falcon tubes. Place 150 mL of 5% NaOCl in in 50 mL labelled Falcon tubes. Seal all tubes, keep in the dark and allow to equilibrate to room temperature. e. Calibrate pH meter.

f. Make up 50 mL 1.0 M Na2 clodronate.

g. Make up 50 mL 0.5 M Na2 clodronate.

h. Make up 50 mL 1.0 M Na4 etidronate.

i. Make up 50 mL 0.5 M Na4 etidronate.

j. Make up 50 mL 17% w/V Na2 EDTA. k. Record pH of chelator solutions. 3. Treatment of discs a. Groups i. 1.0 M clodronate+10% NaOCl, 1:1 ii. 0.5 M clodronate+10% NaOCl, 1:1 iii. 1.0 M etidronate+10% NaOCl, 1:1 iv. 0.5 M etidronate+10% NaOCl, 1:1 v. 1 x PBS vi. 5% NaOCl followed by 17% EDTA vii. 5% NaOCl followed by 17 % EDTA followed by 5% NaOCl b. Randomly allocate slices to groups, 10 slices/group. c. Irrigant volumes and timing for groups. i. For all groups, the total active irrigant volume = 7.5 mL and the time for active irrigants = 15 minutes. ii. For groups i-v this consists of 1.5 mL irrigant refreshed every 3 minutes. iii. For group vi, 1.5 mL NaOCl is refreshed every 3 minutes 9 minutes, followed by 1.5 mL NaOCl for 4 minutes and finally 1.5 mL EDTA for 2 minutes. iv. For group vii, 1.5 mL of NaOCl refreshed every 3 minutes for 9 minutes, followed by 1.0 mL NaOCl for 3.5 minutes, then 1.5 mL EDTA for 2 minutes and lastly 0.5 mL NaOCl for 30 seconds. v. These changes for groups vi and vii are due to the extra steps in these groups and are necessary to maintain equal irrigant times and volumes for all groups.

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vi. Groups vi and vii also receive additional rinses rapidly (3 seconds) in a beaker containing 100 ml water between NaOCl and EDTA and EDTA and NaOCl to avoid mixing of irrigants. vii. Refreshing is achieved by moving slices into new wells containing fresh irrigant by carefully gripping disc with tweezers. d. Combine the chelator and NaOCl just before their use. e. Fill 24-well plates with irrigant mixtures according to irrigant requirements (one group at a time). Each row of the plate will treat one dentine disc. f. At T=0 place the first dentine slice into the solution in well A1 using tweezers. 10 seconds later place the second slice in the group in irrigant and follow this pattern until all 10 slices (groups i-vi) are in the first solution. Move the slices at the allotted time according to 3.c above. g. For timing reasons, in group vii where the final rinse is for 30 seconds only, it will only be possible to do 2 slices at a time.

h. After irrigation is complete rinse in a tub of d-H2O. Finally transfer to a storage 12-well plate (1/group) containing 3 mL PBS. Seal the 12-well plate with parafilm and place at 4oC awaiting SEM imaging. This is not necessary if the imaging is done within a day of the experiment. i. Before imaging rinse all slices in demineralised water and leave to dry in 12- well plates. 4. Sample preparation and Imaging at QUT a. Place plates of samples in oven at 37oC overnight to dry. b. Splutter coat with 2 nm platinum with EM600 Leica Ace. c. Imaging instructions i. 5 images /slice. ii. Try to image midway between the canal and the external root surface. iii. Choose areas where the canals are circular in cross section and where there appears to be an equal density of tubules. iv. The magnification is 3000x, field of view 92.3 µm. v. Additional imaging at 20000x for every group, field of view, 13.8 µm. 5. Image Analysis a. Use Image J 1.52i. b. Need to optimize several parameters. i. Sphericity, pixel range and thresholding.

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ii. Use 15 random images /group and apply same parameters to make sure that all tubules are counted and size of tubule corresponds to image. iii. This process can take several hours. c. Write macro for batch application of same parameters to all the discs. d. Apply macro.

e. Look at all output files and ensure all tubules have been counted on each image. Discard images where all tubules are not counted.

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9.3.4 Protocol 4, Porcine mucosa dissolution

1. Porcine mucosa sample preparation a. Excise porcine palatal mucosa flaps within 2 hours of slaughter. b. Rinse in sterile PBS, place in Falcon tubes, place in esky on ice for transport. c. In the lab use a 6 mm leather punch to obtain specimens of regular surface area from the rugae. Blot samples on Kim wipes so no moisture remains. Weigh each sample and adjust to 55 mg. Place in labelled individual 2 mL centrifuge tubes. Record sample numbers and corresponding exact weights on tubes. Freeze at -30oC. 2. Experiment 1. FAC combined with weight a. Groups i. 0.26 M clodronate ii. 0.26 M etidronate iii. PBS iv. 5% NaOCl v. 0.26 M clodronate-5% NaOCl vi. 0.26 M etidronate-5% NaOCl b. The day before the experiment, titrate stock NaOCl as per Protocol 2 and dilute to 10%. Store in dark at 2-4oC. c. Make up the following i. 0.52 M clodronate

ii. 0.26 M clodronate (1:1 dilution of i with d-H20) iii. PBS iv. 0.52 M etidronate

v. 0.26 M etidronate (1:1 dilution of iv with d-H20) d. Take 10% NaOCl from fridge and allow to equilibrate to room temperature. e. Make up trial solutions as per groups above in a. (except PBS) in order to record pH. Chelator mixtures are derived equal volumes of the 0.52 M chelator solution with 10% NaOCl. f. Use a random number generator to create 2 lists, each 1-30, to determine the order of testing. One list is for plain solutions (P): PBS, 0.26 M etidronate 0.26

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M clodronate, the other is for solutions containing NaOCl (N): 5% NaOCl, 0.26 M clodronate-5% NaOCl, 0.26 M etidronate-5% NaOCl. g. The experiment consists of 10 runs, each with 6 mucosal samples (n=10). The order of solutions within a run is determined from the randomly derived lists. Runs follow the pattern N (1), P (1), N (2), P (2), N (3), P (3), as seen in the run execution section. For example, for Run 1, P (1), P (2) and P (3) would correspond to solutions 1, 2, 3 from the list (P) and N (1), N (2) and N (3) would correspond to solutions 1, 2, 3 from the list (N), and in run 2, P (1) ,P (2) and P (3) would correspond to solutions 4, 5, 6 from the list (P) and N (1) ,N (2) and N (3) would correspond to solutions 4, 5, 6 from the list (N). This pattern is continued for the 10 runs. h. Have available a weight of approximately 1 kg. This will help with blotting samples dry. i. Place 1.5 mL PBS in 60 x 2 mL centrifuge tubes. j. Turn on the water bath, ensure solutions will equilibrate to 32 oC within 11 minutes and stay at that temperature. k. Record weights of 6 mucosal samples to be used for N (1), P (1), N (2), P (2), N (3), P (3). Weights are on tubes. Defrost mucosal samples in PBS for 30 minutes before start. l. During the execution of each run, keep Falcon tubes capped when not adding/removing samples. m. Run execution timing

i. T=0 Add 4.5 mL 10% NaOCl to 4.5 mL either d-H20, 0.52 M etidronate or 0.52 M clodronate (as predetermined by random list). Mix by 5 inversions, ASAP take 1 mL sample, titrate. Record volume thiosulphate. This is N (1) ii. T=3 Add 8 mL either PBS, 0.26 M etidronate or 0.26 M clodronate (as predetermined) to water bath. P (1) iii. T=4 Add N (1) to water bath.

iv. T=4:30 Add 4.5 mL 10% NaOCl to 4.5 mL either d-H20, 0.52 M etidronate or 0.52 M clodronate. Mix by 5 inversions, ASAP take 1 mL sample, titrate. Record volume thiosulphate. N (2) v. T=7:30. Add 8 mL either PBS, 0.26 M etidronate or 0.26 M clodronate to water bath. P (2)

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vi. T=8:30 Add N (2) to water bath.

vii. T=9 Add 4.5 mL 10% NaOCl to 4.5 mL either d-H20, 0.52 M etidronate or 0.52 M clodronate. Mix by 5 inversions, ASAP take 1 mL sample, titrate. Record volume thiosulphate. N (3) viii. T=12 Add 8 mL either PBS, 0.26 M etidronate or 0.26 M clodronate to water bath. P (3) ix. T=13 Add N (3) to water bath. x. T=14 Add mucosa to P (1). xi. T=15 Add mucosa to N (1). xii. T=18:30 Add mucosa to P (2). xiii. T=19:30 Add mucosa to N (2). xiv. T=23 Add mucosa to P (3). xv. T=24 Add mucosa to N (3).

xvi. T=29 Drain (tea strainer), rinse (d-H2O, approximately 5 mL), and place mucosa from P (1) in 1.5 mL PBS. xvii. T=29:40 Inversions x 5, 1 mL sample, N (1). xviii. T=30 Drain, rinse, and place mucosa from N (1) in 1.5 mL PBS, titrate. Record volume thiosulphate N (1). xix. T=33:30 Drain, rinse, and place mucosa from P (2) in 1.5 mL PBS. xx. T=34:10 Inversions x 5, 1 mL sample, N (2). xxi. T=34:30 Drain, rinse, and place mucosa from N (2) in 1.5 mL PBS, titrate. Record volume thiosulphate N (2). xxii. T=38 Drain, rinse, and place mucosa from P (3) in 1.5 mL PBS. xxiii. T=38:40 Inversions x 5, 1 mL sample, N (3). xxiv. T=39 Drain, rinse, and place mucosa from N (3) in 1.5 mL PBS, titrate. Record volume thiosulphate N (3). xxv. T= 43+ Weigh 6 samples. n. Weigh each sample by blotting dry in a systematic fashion on absorbent paper (Kim wipe). Cover the mucosa in the paper and cover with the 1 Kg weight. Hold it there for 5 seconds. Repeat the blotting for another 5 seconds, if necessary, use the weight again to ensure that no moisture will be present on the paper. Record final weights.

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3. Experiment 2, (no FAC, clodronate mixtures 6 hours from mixing) a. Experiment 2 is conducted in 2 separate parts, Part A and Part B. Each part consists of 3 runs, each with 10 samples (n=10). b. Groups Part A i. 2.5% NaOCl ii. 0.26 M clodronate-2.5% NaOCl iii. PBS c. Groups Part B i. 5% NaOCl ii. 0.52 M clodronate-5% iii. PBS d. Take 10% NaOCl solutions out of the fridge. For Part A, dilute to 5% NaOCl. Keep NaOCl solutions in the dark to warm to room temperature. e. Solutions: 0.52 M clodronate (Part A) and 1.04 M (Part B), both at pH 10.7. Final concentrations are achieved by mixing equal volumes of clodronate with 5% NaOCl (Part A) or 10% NaOCl (Part B). Do trial mixtures to record the pH of each solution. f. For the part being performed, set up Run 1, Run 2 and Run 3 with the aid of a random number generator. Each run uses 10 mucosa samples. Because irrigants will be refreshed twice (at 5 minute intervals), 3 lots (X,Y,Z) of 10 labelled tubes (15 mL Falcon) need to be set up . g. Print off a data collection sheet indicating mucosa sample number, initial weight, group replicate designated to each sample. Leave columns for T=5, T=10 and T=15 to be recorded on the experimental sheet at the time of the experiment.

h. Dilute half of NaOCl 1:1 with d-H20, giving the control 2.5% NaOCl (Part A) and 5% NaOCl (Part B) solutions. i. Timing of combination of clodronate 1:1 with 5% NaOCl (Part A) or 10% NaOCl (Part B). i. Run 1 Tubes X (for first 5 minutes), T=0:00:00 ii. Run 1 Tubes Y (for 5-10 minutes), T=0:40:00 iii. Run 1 Tubes Z (for 10-15 minutes), T= 1:20:00 iv. Run 2 Tubes X, T= 2:00:00 v. Run 2 Tubes Y, T= 2:40:00

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vi. Run 2 Tubes Z, T=3:20:00 vii. Run 3 Tubes X, T=4:00:00 viii. Run 3 Tubes Y, T=4:40:00 ix. Run 3 Tubes Z, T=5:20:00 j. Put 4 mL of each group’s solution in labelled tubes corresponding to the designation of samples allotted to group replicates from the random number generator. Seal tube and leave it on the bench at room temperature until required. For each part, Run 1 will be for samples 1-10, Run 2 for 11-20, and Run 3 for 21-30. k. Place 1 mL PBS in 30 x 2 mL centrifuge tubes. l. Have the 1 Kg weight available. m. Turn on the water bath and ensure solutions can reach 32oC in 10 minutes. n. Half an hour before the start of each experimental run, remove the 10 designated porcine mucosa samples from the freezer. Add 1mL PBS to each microtube to allow them to thaw. o. Timing of solution heating and sample placement. i. T=5:50:00, place Run 1 tubes X (10) in the water bath, 30 seconds apart, starting with the first tube. Equilibrate to 32oC over 10 minutes. ii. T=6:00:00, place sample 1 in tube 1 of 10 (X), and at 30 second intervals place subsequent 9 samples in the corresponding tubes. The 10th sample will go in at T=6:04:30

iii. T=6:05:00 tip the first sample into a tea strainer and rinse with d-H2O for 10 seconds. Place in the PBS in the prepared centrifuge tube. Repeat at 30 second intervals for samples 2-10. The 10th sample will come out at 6:09:30. iv. Place samples for next run in PBS to thaw, then weigh current samples as per experiment 1. Record. the 5 minute weight. v. T= 6:30:00, place Run 1 tubes Y (10) in the water bath, 30 second apart, starting with the first tube. Equilibrate to 32oC over 10 minutes. vi. T= 6:40:00, place samples in the order as before, 30 seconds apart. The 10th sample will go in at 6:44:30.

vii. T=6:45:00 tip the first sample into a tea strainer and rinse with d-H2O for 10 seconds. Place in the PBS in the prepared centrifuge tube.

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Repeat at 30 second intervals for samples 2-10. The 10th sample will come out at 6:49:30. viii. Weight current samples and record the 10 minute weight. ix. T=7:10, place Run 1 tubes Z (10) in the water bath, 30 second apart, starting with the first tube. Equilibrate to 32oC over 10 minutes. x. T=7:20:00 place samples in the same order as before, 30 seconds apart. The 10th sample will go in at 7:24:30. xi. T=7:25:00 tip the first sample into a tea strainer and rinse with d-H2O for 10 seconds. Place in the PBS in the prepared centrifuge tube. Repeat at 30 second intervals for samples 2-10. The 10th sample will come out at 7:29:30. xii. Weight current samples and record 15 minute weight. xiii. For Run 2 and Run 3 repeat steps i-xii above, however times are continuously stagged for each set of 10 tubes (Run 2, X, Y, Z and Run 3, X, Y, Z) by 40 minutes.

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9.3.5 Protocol 5, Antimicrobial studies

1. Preparation for biofilm propagation a. Prepare sterile salivary supernatant. i. This can be done months beforehand. ii. Collect 50 mL stimulated (parafilm) saliva from one donor on several days, depending on amount required. iii. Freeze at -80oC between collections. iv. Thaw all collected saliva, centrifuge at 3129 g at 4oC for 20 minutes, remove supernatant and pool. v. Filter sterilised with a low binding fast flow PES membrane filter with a 0.22 µm pore size, Store at -80oC until required.

b. Prepare BHI. Dissolve 37 g/L BHI granules in d-H20. Autoclave. c. Prepare a 10% sucrose solution. Filter sterilize. d. Prepare BHI agar plates.

i. Add 37 g BHI granules to 15 g agar-agar and make up to 1L with d-H20 and autoclave on a liquid cycle at 121oC. ii. Place in a water bath at 55oC. iii. Pour plates in laminar flow cabinet when temperature equilibrates. iv. Just cover the bottom of the plates and ensure there are no bubbles. If bubbles appear shake plate to move bubbles to the edge. v. Cover with lid over approximately 70% of plate while dying over 30 minutes, time it. When dry place lid fully. If moisture then develops, leave ajar again until dry. o e. Wash HA discs under running d-H20. Autoclave them at 121 C, along with the AAA plate. f. Streak E. faecalis from glycerol stock in -80oC freezer onto BHI agar plate. Seal with parafilm. Incubate aerobically at 37oC for 20-24 hours. Place in the micro fridge for up to 1 week. g. Take 1 colony of BHI and place in 20 mL BHI. Incubate at 37oC for 18 hours at 180 rpm. 2. Biofilm propagation a. In the biosafety cabinet (BSC) use sterile gloves to mount HA discs onto AAA Plate.

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b. Expose HA discs to UV light for 2 hours, turning though 180o after 1 hour. c. Suspend discs in 1.7 mL of sterile salivary supernatant in the corresponding wells of a 24-well plate. Seal with parafilm. Incubate at 37oC for 4 hours at 80 rpm. d. Remove overnight E. faecalis culture and centrifuge at 3000g for 8 minutes at 23oC. Remove supernatant and resuspend in 20 mL PBS. Centrifuge again

and suspend in PBS to an OD600 of 0.996-0.999. This corresponds to an E. faecalis density of 7.6 x108. e. The inoculum is a 1/50 dilution of the E. faecalis suspension and a 1/50 dilution of the 10% sucrose solution (0.2 % sucrose) in BHI. For example 20 mL of inoculum consists of: i. 400 µL E. faecalis suspension ii. 400 µL of 10% sucrose iii. 19.2 mL BHI f. Transfer the AAA plate from the sterile salivary supernatant into a new 24-well plate containing 1.7 mL inoculum/well. Seal with parafilm. g. Place in the BD GasPak EZ®, and light candles. Seal GasPak. Incubate at 37oC for 7 days at 80 rpm. h. Change medium every 24 hours with BHI supplemented with 0.2% sucrose by transferral of the AAA plate to a new 24-well plate containing the medium. 3. Disinfection a. Preparation for disinfection. i. Titrate stock NaOCl solution as per Protocol 2. Dilute to 10% and store in the dark at 2-4oC.

ii. Make up a 5% solution of Na2S2O3, taking into account hydration factor. Filter sterilize. iii. Make up 1x PBS. Autoclave. iv. On the morning of each disinfection experiment, make up 0.52 M

clodronate (pH =10.7), 0.52 M Na4 etidronate. b. Execution i. Rinse biofilms twice in 2 mL PBS. With each rinse move AAA plate up and down gently 15 times. ii. Transfer to 1.85 mL disinfection solutions or control for allotted time:1 minute for CFU and SEM studies or 30 seconds for XTT assay.

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iii. Transfer to 5% thiosulphate: 3 minutes for CFU and SEM studies, 1 minute for XTT assay. iv. Rinse x 2 (for CFU and SEM) or x 3 (for XTT) in PBS. 4. CFU counts a. Groups are i. 5% NaOCl ii. 0.26 M clodronate-5% NaOCl iii. 0.26 M etidronate-5% NaOCl iv. PBS

b. Combine 10% NaOCl with either d-H20, 0.52 M clodronate or 0.52 M etidronate immediately before disinfection. c. Disinfect as per 3.b. above. d. Detach discs with sterile tweezers and place in 2 mL PBS contained in a 5 mL centrifuge tube. e. Place tubes on ice. f. Vortex mix discs (reserving 1 disc/group for SEM without vortex mixing) on maximum speed for 2 minutes. g. Keep 1 disc/groups from mixed and unmixed discs for SEM fixation as described in 5.b. h. Serially dilute in PBS. For irrigants containing NaOCl dilute to 10-2, for PBS dilute to 10-4. i. Hand plate 100 µL onto BHI agar. j. For irrigants containing NaOCl, plate neat solution and 10-1 and 10-2 dilutions. For PBS plate 10-3 and 10-4 dilutions. k. Incubate at 37oC aerobically for 48 hours. l. Count colonies with a digital colony counter, with the aid of illumination and magnification. 5. SEM a. Proceed as per 4. a-d. above. b. Fixation and dehydration i. In a fume hood, thaw 25% glutaraldehyde from -30 oC freezer. ii. Dilute to 2.5% in PBS. iii. Fix discs in 2.5% glutaraldehyde for 1 hour, enough to just cover the disc.

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iv. Rinse x 3 in PBS, 10 minutes each. v. Dehydrate x 2 in 50% ethanol, 10 minutes each. vi. Dehydrate x 2 in 70% ethanol, 10 minutes each. vii. Dehydrate x 2 in 90% ethanol, 10 minutes each. viii. Dehydrate x 3 in 100% ethanol, 15 minutes each. ix. Dispose of glutaraldehyde and ethanol in specific waste containers. x. Dehydrate overnight in fume hood. c. Coat in 2 nm platinum at QUT on Leica ACE EM 600 splutter coater. d. Imaging i. 5 images/disc, 1 in centre, and 1 in each of the 4 quadrants. Image in the area at least 3 mm in from disc periphery (inoculum did not cover whole of disc). ii. 1024 x1024 pixels iii. Magnification 3.95 kx iv. 3.0 kV v. Field of view, 70µm vi. Blinded operator. e. Counting criteria of bacteria in treatment groups i. Often in groups of 2-3, joined and in a straight line. Single bacteria and larger groups are possible. ii. Size: 1 µm iii. Marking: There is often a distinguishable fine line in the middle iv. Shape: Oval, with a smooth non-angular contour. v. Colour: Uniform and usually darker than the HA disc vi. Position: Above the plane of the disc and often aligned with grain boundaries or in a depression. f. Percentage surface coverage scores for biofilms in PBS controls i. Score 1: <40% ii. Score 2: 40-60% iii. Score 3: 61-80% iv. Score 4: >80%

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6. XTT assay a. This experiment was conducted in 2 parts. The first part (Part A) was to examine etidronate and clodronate mixtures with NaOCl. The second part (Part B) examined plain chelator solutions b. Groups (Part A) i. 2.5% NaOCl ii. 0.26 M clodronate-2.5% NaOCl iii. 0.26 M etidronate-2.5% NaOCl iv. PBS c. Groups (Part B) i. 0.26 M clodronate ii. 0.26 M etidronate iii. 2.5% NaOCl iv. PBS d. Prepare XTT in advance at 1 mg/mL in PBS, filter sterilize, store in 10 mL aliquots in -80oC freezer. e. Prepare a 4 mM solution of menadione. i. Weight 7 mg menadione. ii. Dissolve in 1 mL acetone (solution A).

iii. Add 100 µL solution A to 900 µL sterile d-H20, vortex mix until cloudy (solution B).

iv. Add 100 µL solution B to 900 µL sterile d-H20, vortex mix (solution C). Keep in dark. f. Take a tube of XTT from the freezer, wrap in alfoil, thaw and vortex mix. g. Prepare activated XTT solution. i. Mix 2 mL of XTT with 100 µL 4 mM menadione, vortex mix. ii. Add 7.9 mL PBS, vortex mix. iii. Store in dark. h. Place 600 µL of activated XTT in wells of a 24-well plate. i. Wrap in alfoil until disinfection is complete. j. Disinfect biofilms on AAA plates as per 3., noting the variances. Included in the disinfection are 1 HA disc/ group that were exposed to sterile media sterile for 7 days. These act as spectrophotometric blanks.

231 k. Detach HA discs with sterile tweezers, place each in the prepared 600 µL of activated XTT. l. Wrap 24-well plate in aluminium foil. Place in aerobic incubator at 37oC for 3:55. After this transfer to orbital shaker for 5 minutes of shaking at 80 rpm at 37oC. m. Place 100 µL from each well in the wells of a 96 well plate. n. Read A492. o. Subtract blank readings from corresponding readings for each group.

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9.3.6 Protocol 6, Fracture resistance

1. Tooth extraction and storage a. Choose the contralateral 4th incisors (also known as canines) from each calf. Calf age is approximately 12 months, canines are deciduous. b. Use a scalpel to incise the thick periodontal ligament, then place a small screwdriver vertically in the space created and move it apically, widening the ligament. Elevate distally. Place each pair in a vial labelled with the animal number and filled with 0.05% thymol in PBS. Place in an esky for transport. c. Scrape periodontal ligament from teeth. This is very time consuming. Store in pairs in 0.05% thymol in PBS fridge in at 2-4oC. d. Change storage medium to PBS and get teeth gamma irradiated at 25 kGy After irradiation, return to 2-4oC. 2. Preparation of the teeth a. Inspect teeth under 10x magnification and discard cracked ones. Also eliminate those with curved roots, resorption or uneven wear. At all times, after any intervention (cutting measuring, extirpation etc), keep teeth in their vials in

d-H20 (from Elga Classic). b. Create 13 mm of root. Using the digital callipers, measure from the apex and mark all around the circumference at 13.5. Section teeth in the middle of the line with the Exakt diamond saw. Check length is 13.0±0.3. Use a permanent marker to number each tooth A or B and the sample number. Store each tooth in PBS in vials labelled with the tooth number and A or B. c. Measure and record the M-D and B-L dimensions of both the tooth at this point with digital callipers. d. Extirpate the pulp with #30 H files or #15 barbed broaches and ensure #10

and #15 H files fit at 12 mm, use d-H20 if needed. 12 mm is the working length. Discard any pairs that are shorter than the WL or have calcified canals. e. Use a random number generator to assign 46 tooth pairs into 3 groups of 15, plus one spare. Each group will be used for a separate experiment. f. In each experiment A of each pair will be the positive control sequence and B

will be either d-H20 (Elga Classic) (negative control) or test chelator-NaOCl.

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g. Experiment 1 i. A-teeth, 5% NaOCl/17% EDTA/5% NaOCl.

ii. B-teeth, d-H20. h. Experiment 2 i. A-teeth, 5% NaOCl/17% EDTA/5% NaOCl. ii. B-teeth, 0.26 M etidronate-5% NaOCl (mix equal volumes of 0.52 M

Na4 etidronate and 10% NaOCl). i. Experiment 3 i. A-teeth, 5% NaOCl/17% EDTA/5% NaOCl. ii. B-teeth, 0.26 M clodronate-5% NaOCl. (mix equal volumes of 0.52 M clodronate and 10% NaOCl). j. Seal each apex with composite resin. i. Use etch for 5 seconds, rinse. ii. Prime and Bond 20s. iii. Admira flowable composite, 20s cure. iv. When sealing the apex, place a plugger in the canal from the coronal aspect to foramen to ensure composite does not block the canal. Check for multiple foramina as the apical delta area of bovine canines can be complex.

v. Fill with d-H20 and check for leakage. If a leak is present, locate it and seal it with composite resin. k. Titrate NaOCl beforehand, and store 10% NaOCl in dark at 2-4oC. l. Solutions i. All clodronate and etidronate solutions are made freshly, and mixed with NaOCl immediately before use.

ii. For 5% NaOCl, dilute 10% NaOCl with d-H20, on the experimental day. m. 17% EDTA at pH 7.2-.4, can be made up beforehand, filter sterilised, and stored in Falcon tubes. 3. Irrigation and rotary instrumentation a. Irrigation with active irrigants. i. Irrigation is over 62.5 minutes. ii. Deliver irrigants with ProRinse™ 30 GA needles. iii. Irrigation is comprised of 4 x 15 minutes cycles with 4 mL of irrigant/cycle.

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iv. Cycle 1, X1 and X2 ProTaper Next. v. Cycle 2, X3 ProTaper Next vi. Cycle 3, X4 ProTaper Next vii. Cycle 4, X5 Protaper. viii. Deliver the 4 mL over 4 minutes. Filing with each instrument occurs with 1 mL of irrigant for 1 minute and the remainder of the irrigation time is without instrumentation. ix. Leave in the canal for 11 minutes until the next cycle. x. Finish with 2 mL of irrigant in 2 minutes, then 0.5 mL in 30 seconds. xi. For clodronate and etidronate groups, use the same irrigant throughout. For NaOCl/EDTA/NaOCl, NaOCl is used for 60 minutes, then 2mL of EDTA over 2 minutes and finally 0.5 ml NaOCl over 30s. b. In all cases, irrigation is followed immediately by a flush with 5 mL of demineralised water over 2 minutes and a further 1mL flush 10 minutes later. o c. Place in pairs in 5.0 mL of sterile d-H2O, store at 2- 4 C. d. This cyclic arrangement allows 3 teeth to be prepared and irrigated at any one time. 4. Mounting of teeth and creation of a PDL a. Mark the circumference of each tooth with a permanent marker 2 mm from the cut surface. b. Dip each tooth in molten wax (low melting point soft red wax) as close as possible to the mark. c. Trim with a scalpel blade to the lower edge of the mark. d. Pare back the wax in any areas where it has pooled. This occurs mostly around the apex.

e. Place each tooth back in its vial and replace with new d-H2O. f. Create a 2 cm x 2 cm rectangle out of cardboard which is plasticised on one side. Make a hole on the plasticised side, roughly the tooth size, in the middle, with a screwdriver. Trim on the plain cardboard side with nail clippers. g. Do 4 teeth at a time. Place each tooth in the hole so that 2 mm of the tooth is above the cardboard and the rest below. The fit must be snug but not too tight as this will push the wax apically. If the hole is the correct size, the wax will hold it at this point.

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h. Set 4 labelled M10 dome nuts on a hollow hex. This is done to level and stabilize the dome nut.

i. Mix Fuji Rock using 25 g powder and 5.1 mL d-H20. Use the vortex to aid in the removal of bubbles. Fill half of each dome nut with Fuji Rock ensuring the sides of the dome nut are coated. Vortex. Fill the remaining half and vibrate manually to ensure no bubbles are trapped. The level must be a little below the rim. j. Slowly lower each tooth with its cardboard into the middle of the dome nut, until the cardboard is level with the nut. k. Allow to set for 60 minutes. Label the Fuji rock. l. Remove each tooth by running under warm water for 10 seconds and by pulling on the root. The tooth should slide out. If it doesn’t, apply the warm water for longer.

m. Replace the tooth in its vial in new d-H2O. n. Soak the dome nut in warm water. Remove the soft wax from the mould with an excavator. Use triplex air to remove any debris from the mould. Allow the Fuji rock to dry. o. Immediately before fracture testing create a simulated PDL. i. Do this for only one pair of teeth at any time. This ensures teeth do not dehydrate. ii. Remove teeth from the water and allow to dry for 1 minute. iii. Mark the tooth and its corresponding position in the mould. iv. Mix Impregum soft and coat the tooth by rolling it in the Impregum. v. Seat the tooth in the mould and wipe away most of the excess Impregum. vi. At 5 minutes from the start of mix, place water in the canal with a needle and remove all the excess Impregum with a scalpel blade. vii. Strength test immediately. viii. The PDL on tooth B is created first and while its Impregum is setting, start tooth A. 5. Strength testing a. Instron settings i. A compression test software setting is used. ii. The load cell is 5000 N.

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iii. Crosshead speed is 1 mm/minute. iv. Stop parameter is a 40% force reduction but the machine can also be stopped manually. The software safety stops are 4900 N and 6 mm extension. Set the manual stops on the Instron frame. v. The indenter is a shaft with a semi-sphere at the tip, measuring 5 mm in diameter. b. Sample positioning i. Set the sample with the root oriented in a vertical position, coronal surface level, in the grips of the base. ii. Position the indenter tip in the cradle of the canal so that it sits in the hollow. iii. Tighten the grips manually, and then with the lever arm hex. Ensure the tooth is not loaded at this stage by more than 2.5 N. A small amount of preloading stops the indenter slipping once the test is started. c. Label each specimen in the software. Zero the load and extension. Start the test in the software. If the tooth obviously cracks before the stop is triggered, stop the test. A noise occurs at the time of cracking followed by a visual crack. d. Ensure that data is transferred to a USB after each test session.

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9.3.7 Protocol 7, Disinfection of oral biofilm on acrylic tooth root model

1. 3-week salivary biofilm

a. Sand blast the canal side of 38 pairs of 3D printed root canal models of the mesial root of a lower molar (the “model”), using 50 µm alumina oxide. Rinse to remove debris. b. Place 36 pairs of models on the metal pegs of 3 modified AAA plates, ensuring that the printed surfaces are all facing the same direction. Separate deep from shallow halves (7 pairs are spares in case biofilm is uneven). Put aside 2 pairs for later use. c. Autoclave the loaded AAA plates. d. Defrost 34 mL saliva. This saliva was previously collected from multiple donors, mixed 1:1 with 60% glycerol then aliquoted and frozen at -80oC. The 60% glycerol was obtained by diluting 100% to 60% with demineralised water and autoclaved. e. Defrost 8.5 mL defibrinated sheep’s blood. f. Mix up 187 mL of inoculum by combing the saliva, sheep’s blood and 144.5 mL BHI. This represents 17x the basic mix of 11 mL which consists of 2 mL defrosted saliva, 0.5 mL sheep’s blood and 8.5 mL BHI. g. Place 2.5 mL of the inoculum in all the wells of 3x 24 well plates. h. Place the AAA plates in the wells, seal with parafilm. i. Light the candles in the anaerobic chamber (BD Gas Pak EZ), place the plates in the chamber and seal it. j. Incubate in an orbital shaker at 37oC, 80 rpm k. Replace the media at 4 days, 1 week and 2 weeks by filling new wells with media and transferring the plate containing the models into those wells. The media is the same as for (g) minus the saliva: 1 mix is 8.5 mL BHI and 0.5 mL sheep’s blood.

2. Preparation

a. Set up 25 sets of files in a sponge and autoclave. Sets consist of: 1x # k file, X1, X2 and X3 Protaper Next Ni-Ti instruments. Stopper all instruments to 9.25 mm. Autoclave. b. Mark 10 model holders S at the top for ‘Shallow’ and D at the base for “deep”,

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c. Use iodometric titration to determine the NaOCl concentration. Dilute to 5% and 10% with demineralised water. Store at 2-4oC in the dark. d. Prepare 17% EDTA pH 7.2-7.4. e. Prepare Acridine orange (AO) Stock solution: Dissolve 50 mg of AO in 10 mL demineralised water and store in the dark at 2-4oC. f. Prepare AO working solution (on the day): To 50 mL demineralised water add 1 mL AO stock solution and 0.5 mL glacial acetic acid. Store in the dark in the fridge. g. Autoclave 1xPBS. h. Prepare 25 mL of 30.4% (1.04 M) clodronate solution (on the day): Dilute half the volume to 15.2% (0.52 M) with demineralised water. i. Mix 30.4% clodronate 1:1 with 10% NaOCl just before filling the irrigation syringe for use in section 6. j. Prepare fixation solution, 2.5% glutaraldehyde, for SEM Images of biofilm: Unfreeze an aliquot of 25% glutaraldehyde form the -30oC freezer. Mix 5 mL of 25% glutaraldehyde with 45 mL PBS. k. Fill 1 mL Luer Lock labelled syringes with the following solutions: 1x PBS, 5% NaOCl, 17%EDTA, 10% NaOCl+30.4% clodronate, 1:1 mix, 15.2% clodronate. Place a 30G Pro Rinse needle on each, and store in separate containers. l. Place wax on the base of the moulds and place them under UV light for 60 minutes.

3. Biofilm rinsing

Place all 3 AAA plates with the 3-week biofilms in 3 x 24 well plates containing PBS, to remove both media and non-adherent biofilm. Keep models in PBS until required as in Sections 4, 5 or 6.

4. Fixation of biofilms for SEM

a. Detach 3 models (6 halves) from one AAA plate. Rinse in PBS. Use 3 halves for fixation and 3 halves in Section 4 (sequencing). b. Fix each half in 2.5% glutaraldehyde for 1 hour at room temperature. Ensure the printed surface is face up. Ensure the orientation of the model is with the canal to the top of the plate for this and subsequent steps. c. Rinse in PBS, 3x10 minutes. 239

d. Place models in the storage plate containing PBS, printed side up. Seal with parafilm. Label. Store in the fridge at 2-4oC for later dehydration and drying. e. After the main experiment and before SEM imaging, dehydrate and dry. This involves: f. 50% EtOH (ethanol) 10:00 x2, 70% EtOH 10:00x2, 90% EtOH 10:00x2, 100% EtOH 15:00x3, final drying is achieved with 2x30:00 hexamethyldisilizine (note this is very toxic and needs correct disposal). Follow by fume cupboard drying 1 hour-over/night. Place in a clean plate and seal with parafilm.

5. Preparation for sequencing

a. Label 6 x 2 mL Eppendorf tubes with the sample codes for the ACE submission. Use tubes from an unopened pack, but do not autoclave them. b. Use the other 3 halves detached in Section 4. c. Defrost an aliquot of frozen saliva. d. Place 500 µl PBS in 3 x 2 mL Eppendorf tubes. Leave another 3 x 2 mL Eppendorf tubes empty. e. Using a sterile excavator, scrape the biofilm from each model half and place into PBS tubes . f. Place 500 µl of unfrozen saliva in each of the empty tubes. g. Deliver the 6 samples to ACE UQ, St Lucia.

6. Root canal preparation

a. The remaining canal models are divided into 5 groups, n=5 for groups 1-6 and n=2 for group 7. The groups are: i. Group 1: The sequence, 5% NaOCl / 17% EDTA / 5% NaOCl ii. Group 2: The sequence, 5% NaOCl / 17% EDTA iii. Group 3: 30.4% clodronate + 10% NaOCl (final concentrations 15.2% clodronate, 5% NaOCl iv. Group 4: 15.2% clodronate v. Group 5: PBS vi. Group 6: Biofilm control, no treatment vii. Group 7: No biofilm, no treatment, for staining only

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b. Remove models from the AAA plate with sterile tweezers. Using 2 moulds / group, place the complementary model halves together in the mould, making sure to orientate the model correctly, shallow at the top, deep at the base. The total time for instrumentation and irrigation per model is 4 minutes. Time in minutes:seconds is apportioned in groups as: i. Group 1: 2:30, 5% NaOCl / 1:00, 17% EDTA / 0:30, 5% NaOCl ii. Group 2: 3:00 5% NaOCl /1:00 17% EDTA iii. Groups 3,4 and 5: 4:00 iv. Groups 6 and 7: no preparation c. The total volume for irrigation is 1 mL. This is apportioned as: i. Group 1: 0.7 mL 5% NaOCl / 0.2 mL 17% EDTA / 0.1 mL 5% NaOCl ii. Group 2: 0.8 mL 5% NaOCl / 0.2 mL 17% EDTA iii. Group 3: 1 mL 30.4% clodronate +10% NaOCl iv. Group 4: 1 mL 15.2% clodronate v. Group 5: 1 mL PBS vi. Groups 6 and 7: no irrigation d. The order of instrumentation of individual model sets is determined at the time of the experiment and models from each set allocated a random order i.e. groups are not filed as a set. File with #10 K file, followed by a sequence of, Protaper X1, Protaper X2, Protaper X3. Irrigate before and after each. Instrumentation is complete at 2:30, irrigation after instrumentation until 4:00. e. After the 4:00 period, irrigate groups 1-5 with 1 mL PBS over 1 minute. f. Place all models in PBS in 3 x24 well plates and then rinse a second time ( for 5:00) in PBS 3x24 well plates. Take models out of wells and dry in a fume hood, making sure to keep deep and shallow halves separated. In all steps ensure the models are oriented coronal to apical going down the wells. Place the shallow half in rows 1 and 4 and the deep halved in rows 2 and 3. 7. Fixation and staining of prepared models with acridine orange

a. All rinsing and fixation steps can be performed in 24 well plates. Ensure that the printed surface is facing up. Maintain the canal orientation b. Fix models in 100% methanol for 2:00. Follow this with 3x PBS rinses, each 5:00. Dry models in the fume cabinet. c. Place 2 mL of acridine orange working solution in 3 x 24 well plates. Cover each plate with alfoil. 241

d. Stain group by group for 2:00 in the acridine orange solution. Keep out of the light. e. Rinse in 3 x (PBS x5:00) in alfoil covered 24 well plates or until no dye stains the liquid. f. Store each model group in a separate glass bottomed 12 well plates containing 1mL PBS/well. Place the printed surface of the model against the glass. Make sure orientation is maintained and the deep and shallow halves are separated (shallow left, deep right). Cover with alfoil and store at 2-4oC until ready for imaging.

8. CLSM Imaging

a. Obtain 5 x Z stacks per model half. Take images of the isthmus area and label as follows: ‘s’ for shallow models and ‘d’ for deep models. For each half take 5 stacks. In the coronal and mid root sections align the model so the canal is just out of view, apically have the canal on the left and right on the image. For the ‘s’ model half the numbers 1 2 3 4 and 5 correspond to L(left) coronal, L mid root, apical, R(right) mid root, R coronal respectively and the symbols used are 1s 2s 3s 4s 5s. For the ‘d’ half the numbers 1 2 3 4 and 5 correspond to R coronal, R mid root, apical, L mid root, L coronal respectively and the symbols used are 1d 2d 3d 4d 5d. b. Scan size - 1024x1024 pixels; biofilm depth 200µm (determined on actual day by assessment of the non-treatment biofilm controls; 21 steps of 10 µm starting at 0 and ending at -200µm; laser 488 parameters are: laser 1.0%, HV 391, Gain 3500 offset 3%. c. Excitation is with the 488 nm laser and the emission filter is set to capture emissions from 530-600 nm. d. Image analysis- fluorescence measurements in Image J.

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9.4 Raw data

9.4.1 Data from Chapter 2

The effect of heating to intracanal temperature on the stability of sodium hypochlorite admixed with etidronate or EDTA for continuous chelation. .

Na4 EDTA o o pH data Na4 EDTA, 23 C and 35 C o pH data for 5% Na4 EDTA-2.5% NaOCl at 23 C, time in minutes vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 12.3 12.3 12.3 12.29 12.28 12.3 12.29 12.29 12.27 12.26 20 9.14 9.09 9.09 9.07 9.06 9.1 9.05 9.03 9.02 9.02 40 8.93 8.91 8.92 8.9 8.9 8.95 8.93 8.92 8.92 8.91 60 8.89 8.88 8.88 8.88 8.88 8.93 8.91 8.9 8.9 8.9 pH data for 2.5% NaOCl at 23 oC (control), time in minutes vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 12.25 12.26 12.25 12.25 12.24 12.24 12.24 12.22 12.21 12.2 20 12.16 12.21 12.21 12.21 12.2 12.16 12.17 12.16 12.15 12.18 40 12.18 12.19 12.19 12.19 12.19 12.16 12.17 12.15 12.14 12.14 60 12.19 12.18 12.18 12.17 12.17 12.19 12.16 12.14 12.12 12.14

o pH data for 5% Na4 EDTA-2.5% NaOCl at 35 C, time in minutes vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 11.94 11.94 11.93 11.93 11.92 11.85 11.84 11.81 11.78 11.76 20 8.82 8.81 8.82 8.8 8.8 8.83 8.81 8.83 8.81 8.81 40 8.73 8.74 8.76 8.77 8.77 8.73 8.76 8.74 8.74 8.73 60 8.74 8.74 8.75 8.75 8.75 8.72 8.74 8.75 8.74 8.73

243 pH data for 2.5% NaOCl at 35 oC (control), time in minutes vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 11.96 11.97 11.96 11.98 11.97 11.82 11.83 11.81 11.93 11.91 20 11.94 11.94 11.94 11.94 11.93 11.86 11.84 11.83 11.83 11.82 40 11.94 11.92 11.92 11.92 11.93 11.82 11.84 11.82 11.8 11.79 60 11.91 11.91 11.91 11.9 11.95 11.84 11.8 11.8 11.78 11.78

o o Temperature data Na4 EDTA, 23 C and 35 C o Temperature data for 5% Na4 EDTA-2.5% NaOCl at 23 C, time in minutes vs repetition number. Temperature in degrees Celsius. Time 1 2 3 4 5 6 7 8 9 10 1 23 23.1 23.1 23.1 23.2 23.3 23.3 23.5 23.6 23.6 20 33 35.4 35.5 35.8 35.5 31.6 34.8 34.6 35 33.8 40 26.9 27.4 27.1 27.3 27 26.2 27 26.9 27.1 27 60 24.4 24.7 24.7 24.7 24.5 24.2 24.5 24.6 24.6 24.5

Temperature data for 2.5% NaOCl (control) at 23 oC, time in minutes vs repetition number. Temperature in degrees Celsius. Time 1 2 3 4 5 6 7 8 9 10 1 23 23 23.1 23.2 23.2 23.6 23.5 23.5 23.6 23.4 20 24.6 23.7 23.5 23.5 23.5 24.4 23.8 23.9 23.7 23.7 40 23.9 23.6 23.5 23.5 23.5 24 23.7 23.6 23.5 23.6 60 23.7 23.5 23.6 23.6 23.5 23.5 23.5 23.5 23.5 23.5

o Temperature data for 5% Na4 EDTA-2.5% NaOCl at 35 C, time in minutes vs repetition number. Temperature in degrees Celsius. Time 1 2 3 4 5 6 7 8 9 10 1 34.2 34.4 34.5 34.4 34.5 34 33.8 34.2 34.3 34.4 20 34.8 34.9 34.5 34.4 34.3 34.6 34.6 34.6 34.8 34.7 40 34.7 34.7 34.6 34.5 34.5 34.7 34.7 34.8 34.8 34.6 60 34.6 34.8 34.8 34.4 34.4 34.8 34.8 34.6 34.7 34.7

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Temperature data for 2.5% NaOCl (control) at 35 oC, time in minutes vs repetition number. Temperature in degrees Celsius. Time 1 2 3 4 5 6 7 8 9 10 1 34.1 34.5 34.2 34.3 34.2 34 34 33.9 34.1 34.3 20 34.7 34.6 34.3 34.7 34.7 34.6 34.6 34.5 34.6 34.5 40 34.4 34.6 34.6 34.5 34.6 34.6 34.8 34.7 34.7 34.6 60 34.8 34.7 34.6 34.6 34.6 34.7 34.7 34.8 34.8 34.6

o o Titration data Na4 EDTA, mL thiosulphate, 23 C and 35 C o Thiosulphate in mL for 5% Na4 EDTA-2.5% NaOCl at 23 C, time in minutes vs repetition number. Thiosulphate concentration = 0.09947 M. Time 1 2 3 4 5 6 7 8 9 10 1 7.02 7.01 7.00 7.00 6.97 7.00 6.98 6.98 7.00 7.00 20 0.90 0.90 0.89 0.82 0.84 0.94 0.82 0.92 0.75 0.81 40 0.40 0.41 0.38 0.40 0.46 0.39 0.33 0.40 0.37 0.34 60 0.21 0.22 0.25 0.23 0.22 0.24 0.21 0.24 0.22 0.21

Thiosulphate in mL for 2.5% NaOCl (control) at 23 oC, time in minutes vs repetition number. Thiosulphate concentration = 0.09947 M. Time 1 2 3 4 5 6 7 8 9 10 1 7.01 7.03 7.08 7.02 7.03 7.02 7.02 7.04 7.00 7.08 20 7.02 7.02 7.09 7.01 7.03 7.04 7.07 7.01 7.01 7.08 40 7.01 7.03 7.09 7.01 7.04 7.01 7.09 7.02 7.02 7.02 60 7.05 7.01 7.05 7.02 7.00 7.01 7.09 7.02 7.00 7.04

o Thiosulphate in mL for 5% Na4 EDTA-2.5% NaOCl at 35 C, time in minutes vs repetition number. Thiosulphate concentration = 0.09973 M. Time 1 2 3 4 5 6 7 8 9 10 1 6.87 6.86 6.81 6.75 6.85 6.90 6.86 6.90 6.90 6.93 20 0.27 0.25 0.29 0.22 0.25 0.22 0.28 0.30 0.25 0.25 40 0.11 0.10 0.10 0.07 0.10 0.10 0.10 0.10 0.11 0.09 60 0.02 0.03 0.03 0.02 0.02 0.04 0.03 0.03 0.02 0.02

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Thiosulphate in mL for 2.5% NaOCl (control) at 35 oC, time in minutes vs repetition number. Thiosulphate concentration = 0.09973 M. Time 1 2 3 4 5 6 7 8 9 10 1 6.99 7.02 7.03 6.91 6.97 7.00 6.96 7.07 7.00 7.00 20 7.00 7.00 6.95 6.91 7.02 6.98 6.94 7.00 7.00 7.00 40 7.00 6.99 6.94 6.97 6.93 7.00 6.98 7.04 6.99 7.00 60 6.99 7.00 6.96 6.94 6.94 7.02 7.00 7.03 6.99 7.00

Na4 etidronate o o pH data Na4 etidronate, 23 C and 35 C o pH data for 18% Cublen Na4 Etidronate-5% NaOCl at 23 C, time in minutes vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 12.51 12.49 12.47 12.45 12.42 12.53 12.47 12.48 12.43 12.41 20 12.07 12.03 12.02 12.02 12 12.1 12.06 12.08 12.02 12.01 40 11.23 11.18 11.19 11.18 11.19 11.4 11.32 11.33 11.29 11.32 60 10.55 10.52 10.51 10.49 10.51 10.66 10.62 10.63 10.58 10.62

pH data for 5% NaOCl (control) at 23 oC, time in minutes vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 12.35 12.33 12.33 12.32 12.32 12.39 12.35 12.33 12.31 12.27 20 12.25 12.3 12.29 12.3 12.29 12.28 12.28 12.33 12.31 12.31 40 12.3 12.33 12.31 12.3 12.27 12.26 12.25 12.3 12.32 12.27 60 12.32 12.28 12.26 12.24 12.23 12.29 12.29 12.22 12.24 12.24

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o pH data for 18% Cublen Na4 Etidronate-5% NaOCl at 35 C, time in minutes vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 12.22 12.26 12.28 12.28 12.28 12.24 12.26 12.25 12.22 12.21 20 10.66 10.69 10.65 10.67 10.65 10.61 10.58 10.61 10.6 10.62 40 9.87 9.84 9.9 9.92 9.9 9.92 9.87 9.92 9.9 9.9 60 9.5 9.46 9.54 9.51 9.53 9.45 9.49 9.53 9.52 9.49 pH data for 5% NaOCl (control) at 35 oC, time in minutes vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 12.17 12.16 12.15 12.13 12.11 12.11 12.09 12.08 12.07 12.09 20 12.08 12.06 12.07 12.04 12.03 12.09 12.08 12.07 12.09 12.05 40 12.06 12.04 12.06 12.04 12.02 12.06 11.99 12.02 11.97 12.01 60 12.07 12.01 12.04 12.03 12.03 12.02 12 11.97 11.98 11.94

o o Temperature data Na4 etidronate, 23 C and 35 C o Temperature data for 18% Cublen Na4 Etidronate-5% NaOCl at 23 C, time in minutes vs repetition number. Temperature in degrees Celsius. Time 1 2 3 4 5 6 7 8 9 10 1 23.2 23 23.1 23 23.2 22.1 22.2 22.1 22.1 22.1 20 23.9 24.5 24.3 24.1 24.1 23.1 23.4 23.3 23.3 23.3 40 24.2 24.6 24.7 24.8 24.7 23.6 23.9 23.7 23.9 24 60 25 26.2 26.3 26.7 26.5 24.4 25.1 25.3 25.4 24.8

Temperature data for 5% NaOCl (control) at 23 oC, time in minutes vs repetition number. Temperature in degrees Celsius. Time 1 2 3 4 5 6 7 8 9 10 1 22.7 22.8 22.5 22.6 22.5 21.9 22.4 22 22 22 20 22.7 23 22.7 22.7 22.6 21.8 21.9 22.1 22.1 22.2 40 22.9 22.7 22.6 22.6 22.6 22.3 22 22.1 22 22.2 60 23.1 22.9 22.9 22.9 22.7 22.3 22.2 22.4 22.3 22.4

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o Temperature data for 18% Cublen Na4 Etidronate-5% NaOCl at 35 C, time in minutes vs repetition number. Temperature in degrees Celsius. Time 1 2 3 4 5 6 7 8 9 10 1 34.7 34.7 34.5 34.5 34.5 34.2 34.3 34.3 34.6 34.6 20 36.5 36.7 36.6 36.4 36.6 35.6 36.6 36.6 36.6 36.5 40 37.2 37.3 37.6 37.1 37.1 37 37.1 36.8 37.3 37.3 60 35.6 35.7 35.4 35.7 35.5 35.6 35.4 35.4 35.3 35.5

Temperature data for 5% NaOCl (control) at 35 oC, time in minutes vs repetition number. Temperature in degrees Celsius. Time 1 2 3 4 5 6 7 8 9 10 1 34.1 34.1 34.1 34.2 34.3 34.2 34.2 34.4 34.2 33.9 20 35.1 34.9 34.9 35 34.9 34.6 34.7 34.7 34.7 34.6 40 35 35 34.8 34.9 34.9 34.8 34.7 34.7 34.7 34.8 60 34.9 34.9 34.9 34.8 34.8 34.6 34.5 34.6 34.6 34.6

o o Titration data Na4 etidronate, mL thiosulphate, 23 C and 35 C o Thiosulphate in mL for 18% Cublen Na4 Etidronate-5% NaOCl at 23 C, time in minutes vs repetition number. Thiosulphate concentration = 0.10052 M. Time 1 2 3 4 5 6 7 8 9 10 1 13.36 13.32 13.42 13.39 13.4 13.54 13.32 13.3 13.52 13.31 20 12.87 12.8 12.89 12.91 12.76 12.94 12.8 12.7 12.84 12.72 40 12.16 12.19 12.21 12.19 12.14 12.27 12.03 11.96 12.11 12.03 60 11.18 11.14 11.05 11.02 10.9 10.99 10.77 10.6 10.9 10.63

Thiosulphate in mL for 5% NaOCl (control) at 23 oC, time in minutes vs repetition number. Thiosulphate concentration = 0.10052 M Time 1 2 3 4 5 6 7 8 9 10 1 13.35 13.34 13.38 13.36 13.4 13.33 13.38 13.32 13.35 13.4 20 13.3 13.33 13.34 13.39 13.35 13.32 13.41 13.35 13.3 13.4 40 13.29 13.39 13.4 13.35 13.38 13.32 13.39 13.34 13.28 13.39 60 13.33 13.36 13.37 13.36 13.39 13.34 13.4 13.35 13.34 13.34

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o Thiosulphate in mL for 18% Cublen Na4 Etidronate-5% NaOCl at 35 C, time in minutes vs repetition number. Thiosulphate concentration = 0.09923 M. Time 1 2 3 4 5 6 7 8 9 10 1 13.45 13.42 13.4 13.27 13.41 13.35 13.45 13.45 13.3 13.34 20 10.85 10.8 10.88 10.95 10.8 11.22 10.95 10.88 10.82 10.84 40 4.3 4.52 4.44 4.62 4.39 3.44 4.61 4.52 4.62 4.46 60 1.09 1.08 1.12 1.14 1.18 1.1 1.17 1.14 1.21 1.18

Thiosulphate in mL for 5% NaOCl (control) at 35 oC, time in minutes vs repetition number. Thiosulphate concentration = 0.09923 M. Time 1 2 3 4 5 6 7 8 9 10 1 13.51 13.51 13.53 13.46 13.52 13.53 13.47 13.4 13.4 13.47 20 13.46 13.46 13.46 13.37 13.48 13.33 13.36 13.32 13.51 13.46 40 13.5 13.5 13.46 13.35 13.46 13.37 13.45 13.43 13.37 13.5 60 13.45 13.57 13.44 13.4 13.43 13.31 13.41 13.37 13.4 13.49

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9.4.2 Data from Chapter 3

Paper 1. “From an assessment of multiple chelators, clodronate shows potential for use in continuous chelation” pH, temperature and FAC data over 80 minutes at room temperature pH and temperature experiments conducted separately to titration experiments.

ATMP

ATMP pH data pH data for 0.25 M ATMP-2.5% NaOCl, time in minutes vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 8.99 8.76 8.78 8.73 8.74 9.09 8.69 8.70 8.74 8.69 20 7.70 7.55 7.57 7.52 7.52 7.93 7.45 7.46 7.48 7.45 40 7.35 7.27 7.30 7.25 7.26 7.41 7.21 7.22 7.23 7.2 60 7.22 7.18 7.18 7.16 7.16 7.29 7.14 7.14 7.15 7.13 80 7.17 7.13 7.13 7.12 7.12 7.21 7.1 7.11 7.11 7.09

pH data for 2.5% NaOCl (control), time in minutes vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 12.3 12.33 12.34 12.35 12.35 12.19 12.21 12.24 12.24 12.22 20 12.29 12.31 12.31 12.31 12.3 12.16 12.18 12.2 12.2 12.19 40 12.27 12.28 12.29 12.27 12.27 12.17 12.17 12.19 12.19 12.18 60 12.25 12.27 12.26 12.26 12.27 12.17 12.17 12.18 12.17 12.17 80 12.23 12.25 12.24 12.24 12.25 12.16 12.16 12.16 12.15 12.16

ATMP temperature data

Temperature data for 0.25 M ATMP-2.5% NaOCl, time in minutes vs repetition number. Temperature in degrees Celsius. Time 1 2 3 4 5 6 7 8 9 10 1 35.6 36.9 37.4 37.4 37.7 35.5 37.2 37.3 37.9 38.1 20 26.8 26.8 27.1 27.2 27.3 26.9 28 28 28.1 28.2 40 23.5 23.8 23.8 24 24 24.3 24.8 24.9 25.1 25 60 23 23.1 23.1 23.1 23.1 23.9 23.8 23.9 23.9 23.8 80 22.8 22.8 22.8 22.8 22.8 23.7 23.7 23.7 23.8 23.8

250

Temperature data for 2.5% NaOCl (control), time in minutes vs repetition number. Temperature in degrees Celsius. Time 1 2 3 4 5 6 7 8 9 10 1 22.3 22.5 22.3 22.4 22.3 22.8 23.2 23.3 23.3 23 20 22.8 22.5 22.5 22.5 22.4 24 23.5 23.4 23.3 23.2 40 22.6 22.5 22.5 22.5 22.5 23.4 23.3 23.3 23.3 23.3 60 22.8 22.6 22.6 22.7 22.7 23.4 23.4 23.4 23.4 23.4 80 22.8 22.7 22.7 22.7 22.7 23.5 23.5 23.5 23.5 23.5

ATMP titration data, mL thiosulphate

Thiosulphate in mL for 0.25 M ATMP-2.5% NaOCl, time in minutes vs repetition number. Thiosulphate concentration = 0.09959 M. Time 1 2 3 4 5 6 7 8 9 10 1 1.2 1.41 1.3 1.4 1.12 1.27 1.12 1.52 1.42 1.46 20 0.59 1.08 0.88 1.05 0.6 0.97 0.59 1.11 1.2 1.1 40 0.3 0.9 0.63 0.89 0.32 0.69 0.32 1.05 0.88 1 60 0.2 0.7 0.33 0.73 0.2 0.5 0.21 1 0.63 0.82 80 0.15 0.45 0.21 0.51 0.13 0.31 0.18 0.93 0.41 0.68

Thiosulphate in mL for 2.5% NaOCl (control), time in minutes vs repetition number. Thiosulphate concentration = 0.09959 M. Time 1 2 3 4 5 6 7 8 9 10 1 6.82 6.77 6.82 6.8 6.81 6.84 6.73 6.79 6.82 6.78 20 6.8 6.8 6.86 6.85 6.85 6.82 6.8 6.83 6.8 6.79 40 6.78 6.77 6.9 6.81 6.83 6.79 6.79 6.64 6.86 6.82 60 6.77 6.78 6.8 6.8 6.8 6.9 6.79 6.89 6.78 6.8 80 6.9 6.76 6.78 6.79 6.79 6.8 6.81 6.81 6.78 6.78

251

CDTA

CDTA pH data pH data for 0.125 M CDTA-2.5% NaOCl, time in minutes vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 12.3 12.31 12.32 12.33 12.36 12.26 12.28 12.28 12.29 12.29 20 12.26 12.27 12.26 12.26 12.26 12.15 12.15 12.18 12.18 12.17 40 12.02 12.06 12.05 12.05 12.05 12.05 12.08 12.09 12.09 12.07 60 11.71 11.65 11.75 11.72 11.78 11.53 11.45 11.5 11.48 11.48 80 9.25 9.25 9.24 9.22 9.22 9.26 9.12 9.15 9.1 9.12

pH data for 2.5% NaOCl (control), time in minutes vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 12.29 12.29 12.26 12.3 12.31 12.27 12.17 12.13 12.16 12.16 20 12.28 12.03 12.05 12.01 12.07 12.18 12.18 12.18 12.19 12.18 40 12.14 12.18 12.17 12.17 12.18 12.18 12.15 12.18 12.15 12.18 60 12.23 12.23 12.23 12.23 12.23 12.14 12.13 12.15 12.15 12.14 80 12.15 12.2 12.21 12.21 12.2 12.09 12.13 12.14 12.16 12.16

CDTA temperature data

Temperature data 0.125 M CDTA-2.5% NaOCl, time in minutes vs repetition number. Temperature in degrees Celsius. Time 1 2 3 4 5 6 7 8 9 10 1 22.9 23.1 23 22.8 22.4 22.9 22.8 22.6 22.9 22.8 20 22.6 22.7 22.7 22.7 22.7 23.3 23.2 23.2 23.3 23.3 40 22.5 22.5 22.6 22.8 22.7 23.4 23.6 23.7 23.6 23.7 60 23.3 23.4 23.3 23.5 23.4 24.5 24.9 24.9 24.8 24.8 80 30.1 30.2 31.2 32.1 32.2 30 30.8 31 31.5 31.4

252

Temperature data 2.5% NaOCl (control), time in minutes vs repetition number. Temperature in degrees Celsius. Time 1 2 3 4 5 6 7 8 9 10 1 22.7 22.7 22.7 22.4 22.2 22.8 22.8 22.9 22.9 22.9 20 22.4 22.4 22.4 22.2 22.2 23.1 23 23 23.1 23.1 40 22.3 22.3 22.3 22.2 22.1 23.3 23.2 23.2 23.2 23.2 60 22.4 22.3 22.3 22.3 22.3 23.5 23.5 23.4 23.4 23.4 80 22.5 22.7 22.6 22.5 22.5 24.4 23.8 23.6 23.5 23.5

CDTA titration data, mL thiosulphate

Thiosulphate in mL for 0.125 M CDTA-2.5% NaOCl, time in minutes vs repetition number. Thiosulphate concentration = 0.09959 M. Time 1 2 3 4 5 6 7 8 9 10 1 6.78 6.78 6.77 6.88 6.8 6.83 6.8 6.73 6.9 6.75 20 6.58 6.57 6.57 6.55 6.59 6.59 6.53 6.57 6.6 6.51 40 6.29 6.27 6.24 6.2 6.3 6.28 6.6 6.24 6.2 6.17 60 5.3 5.3 5.13 4.95 5.35 5.39 5.15 5.02 4.99 4.85 80 0.39 0.32 0.32 0.3 0.36 0.31 0.31 0.35 0.34 0.34

Thiosulphate in mL for 2.5% NaOCl (control), time in minutes vs repetition number. Thiosulphate concentration = 0.09959 M. Time 1 2 3 4 5 6 7 8 9 10 1 6.85 6.81 6.79 6.8 6.82 6.8 6.81 6.8 6.81 6.79 20 6.87 6.83 6.79 6.79 6.8 6.83 6.8 6.82 6.89 6.88 40 6.89 6.82 6.8 6.8 6.83 6.8 6.78 6.81 6.77 6.8 60 6.82 6.87 6.9 6.79 6.82 6.87 6.8 6.89 6.8 6.8 80 6.83 6.8 6.8 6.81 6.85 6.74 6.77 6.8 6.8 6.86

253

Clodronate

Clodronate pH data pH data for 0.25 M Clodronate-5% NaOCl, time in minutes vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 12.49 12.51 12.49 12.49 12.5 12.5 12.5 12.57 12.55 12.54 20 12.46 12.46 12.46 12.46 12.45 12.45 12.45 12.45 12.44 12.43 40 12.44 12.44 12.44 12.43 12.43 12.43 12.43 12.44 12.41 12.38 60 12.44 12.45 12.43 12.43 12.42 12.42 12.42 12.38 12.37 12.38 80 12.43 12.42 12.42 12.42 12.41 12.41 12.41 12.37 12.37 12.35

pH data for 5% NaOCl (control), time in minutes vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 12.43 12.42 12.42 12.41 12.4 12.45 12.46 12.45 12.43 12.41 20 12.38 12.39 12.39 12.37 12.37 12.38 12.38 12.37 12.36 12.35 40 12.36 12.37 12.37 12.36 12.36 12.33 12.33 12.32 12.31 12.32 60 12.36 12.36 12.36 12.36 12.34 12.36 12.34 12.35 12.33 12.32 80 12.36 12.34 12.34 12.34 12.33 12.34 12.32 12.3 12.3 12.3

Clodronate temperature data

Temperature data for 0.25 M Clodronate-5% NaOCl, time in minutes vs repetition number. Temperature in degrees Celsius. Time 1 2 3 4 5 6 7 8 9 10 1 23.4 23.3 23.3 23.3 23.3 22.7 22.8 22.7 22.7 22.8 20 23.3 23.1 23.2 23.2 23.3 23.1 23.1 23.1 23.1 23.1 40 23 23.2 23.1 23.1 23.1 23.2 23.2 23.1 23.1 23.3 60 23 23 23.1 22.9 22.9 23.2 23.3 23.2 23.3 23.3 80 22.9 22.9 22.9 22.9 22.9 23.4 23.3 23.2 23.2 23.3

254

Temperature data for 5% NaOCl (control), time in minutes vs repetition number. Temperature in degrees Celsius. Time 1 2 3 4 5 6 7 8 9 10 1 23.1 23 23 23 23 22.9 22.8 23 23 23 20 23.2 23.1 23.1 23.2 23.1 23.2 23.2 23.2 23.2 23.3 40 23.2 23.1 23.1 23 23 23.3 23.3 23.4 23.3 23.3 60 22.9 22.9 22.9 22.9 22.9 23.4 23.3 23.3 23.3 23.4 80 22.8 22.8 22.9 23 23 23.3 23.3 23.3 23.3 23.2

Clodronate titration data, mL thiosulphate

Thiosulphate in mL for 0.25M Clodronate-5% NaOCl, time in minutes vs repetition number. Thiosulphate concentration = 0.09913 M. Time 1 2 3 4 5 6 7 8 9 10 1 13.6 13.79 13.35 13.42 13.41 13.4 13.3 13.4 13.5 13.4 20 13.48 13.62 13.3 13.37 13.31 13.4 13.37 13.3 13.4 13.45 40 13.52 13.64 13.3 13.47 13.31 13.4 13.32 13.38 13.45 13.43 60 13 13.7 13.32 13.39 13.3 13.42 13.4 13.42 13.47 13.43 80 13.52 13.75 13.33 13.4 13.32 13.4 13.31 13.34 13.48 13.45

Thiosulphate in mL for 5% NaOCl (control), time in minutes vs repetition number. Thiosulphate concentration = 0.09913 M. Time 1 2 3 4 5 6 7 8 9 10 1 13.35 13.42 13.02 13.45 13.4 13.37 13.41 13.4 13.48 13.4 20 13.29 13.55 13.33 13.48 13.53 13.48 13.42 13.4 13.46 13.5 40 13.3 13.4 13.45 13.46 13.41 13.41 13.44 13.39 13.52 13.4 60 13.24 13.38 13.45 13.37 13.49 13.41 13.42 13.38 13.65 13.48 80 13.25 13.4 13.41 13.45 13.5 13.47 13.5 13.48 13.48 13.47

255

EDTA (Na4 EDTA)

Na4 EDTA pH data pH data for 0.125 M Na4 EDTA-2.5% NaOCl, time in minutes vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 12.27 12.28 12.28 12.27 12.31 12.25 12.26 12.26 12.22 12.27 20 9.42 11.65 11.62 11.56 9.58 11.5 11.67 11.54 11.52 11.52 40 9.06 9 9.01 9 9.01 9.02 8.93 8.96 8.96 9 60 9.01 8.95 8.96 8.96 8.93 8.95 8.96 8.99 8.95 8.98 80 8.94 8.91 8.9 8.93 8.92 8.97 8.97 8.97 8.94 8.98

pH data for 2.5% NaOCl (control), time in minutes vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 12.32 12.37 12.39 12.38 12.34 12.23 12.24 12.33 12.31 12.3 20 12.22 12.29 12.31 12.3 12.3 12.2 12.24 12.28 12.27 12.27 40 12.24 12.25 12.25 12.24 12.27 12.2 12.21 12.19 12.22 12.22 60 12.18 12.17 12.23 12.24 12.23 12.23 12.25 12.22 12.27 12.25 80 12.23 12.24 12.24 12.24 12.24 12.31 12.33 12.33 12.33 12.33

Na4 EDTA temperature data

Temperature data 0.125 M Na4 EDTA-2.5% NaOCl time in minutes vs repetition number. Temperature in degrees Celsius. Time 1 2 3 4 5 6 7 8 9 10 1 23 23 23.2 23.3 23.3 22.8 22.9 23 22.4 23 20 31.7 25.8 25.4 24.9 31.3 24.1 24.4 24.8 24.9 30.7 40 27 28.3 28.3 28.2 27.8 27 28.1 27.9 27.3 27.5 60 23.9 24.7 24.6 24.4 24.7 24.3 24.6 24.5 24.8 24.5 80 23.6 23.8 23.6 23.8 23.7 23.4 23.5 23.4 23.6 23.5

256

Temperature data 2.5% NaOCl (control) time in minutes vs repetition number. Temperature in degrees Celsius. Time 1 2 3 4 5 6 7 8 9 10 1 22.9 23 22.7 22.8 22.8 22.9 22.9 22.9 23 22.8 20 24.2 23.5 23.3 23.2 23.4 24.4 23.5 23.3 23.2 23.1 40 23.6 23.6 23.3 23.4 23.3 23.7 23.4 23.2 23.2 23.1 60 23.5 23.4 23.3 23.3 23.2 23.3 23.3 23.2 23.2 23.1 80 23.5 23.4 23.1 23.3 23.2 23.1 23.2 23.1 23.2 23.2

Na4 EDTA titration data, mL thiosulphate

Thiosulphate in mL for 0.125 M Na4 EDTA -2.5% NaOCl, time in minutes vs repetition number. Thiosulphate concentration = 0.09959 M. Time 1 2 3 4 5 6 7 8 9 10 1 6.79 6.78 6.71 6.75 6.75 6.9 6.72 6.69 6.72 6.7 20 5.72 5.7 1.06 2.97 5.71 5.72 5.55 5.6 1 1 40 0.5 0.44 0.4 0.44 0.41 0.42 0.48 0.4 0.41 0.41 60 0.32 0.29 0.31 0.34 0.32 0.3 0.31 0.3 0.35 0.3 80 0.21 0.2 0.2 0.22 0.2 0.24 0.2 0.2 0.2 0.2

Thiosulphate in mL for 2.5% NaOCl (control), time in minutes vs repetition number. Thiosulphate concentration = 0.09959 M. Time 1 2 3 4 5 6 7 8 9 10 1 6.81 6.8 6.79 6.9 6.81 6.82 6.93 6.86 6.8 6.83 20 6.8 6.88 6.82 6.83 6.83 6.83 6.81 6.78 6.85 6.8 40 6.85 6.79 6.78 6.88 6.81 6.84 6.81 6.9 6.78 6.85 60 6.81 6.78 6.85 6.9 6.81 6.81 6.81 6.81 6.75 6.8 80 6.81 6.78 6.81 6.84 6.8 6.8 6.85 6.9 6.81 6.84

257

EGTA

EGTA pH data pH data for 0.125 M EGTA-2.5% NaOCl, time in minutes vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 12.43 12.48 12.46 12.44 12.48 12.47 12.5 12.5 12.48 12.46 20 9.13 9.12 9.08 9.06 9.07 9.13 9.04 9.04 9.03 9.04 40 9.04 9.05 9.05 9.05 9.05 9.04 9.02 9.02 9.02 9.02 60 9.03 9.04 9.04 9.04 9.05 9.03 9.01 9 9 9 80 9.03 9.04 9.04 9.05 9.04 9 8.99 9 8.99 8.98

pH data for 2.5% NaOCl (control), time in minutes vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 12.39 12.39 12.38 12.42 12.41 12.3 12.39 12.4 12.41 12.38 20 12.36 12.38 12.37 12.38 12.38 12.25 12.33 12.34 12.34 12.32 40 12.37 12.4 12.39 12.4 12.4 12.17 12.28 12.29 12.29 12.29 60 12.41 12.41 12.41 12.42 12.42 12.21 12.29 12.31 12.29 12.3 80 12.41 12.42 12.42 12.43 12.43 12.2 12.27 12.25 12.29 12.27

EGTA temperature data

Temperature data for 0.125 M EGTA-2.5% NaOCl, time in minutes vs repetition number. Temperature in degrees Celsius. Time 1 2 3 4 5 6 7 8 9 10 1 23.1 22.7 22.7 22.9 22.7 21.8 22.8 22.9 23.2 23.3 20 27.2 28 28.1 28 28.2 28.2 30 29.7 29.5 29.7 40 22.9 23.3 23.3 23.3 23.2 24.4 25.1 25.2 25.2 25.1 60 21.9 21.9 21.9 21.9 21.8 23.6 23.5 23.7 23.8 23.8 80 21.4 21.5 21.6 21.5 21.5 23.4 25.5 23.4 23.4 23.4

258

Temperature data for 2.5% NaOCl (control), time in minutes vs repetition number. Temperature in degrees Celsius. Time 1 2 3 4 5 6 7 8 9 10 1 23.2 22.2 22.1 22.1 22.1 23.3 23 22.9 22.8 23.2 20 22.7 22.3 22.1 22.1 22 23.8 23.4 23.2 23.1 23.1 40 21.9 21.6 21.6 21.6 21.4 23.5 23.3 23.1 23.1 23.1 60 21.6 21.4 21.4 21.4 21.3 23.3 23.3 23.2 22.9 23 80 21.4 21.4 21.4 21.4 21.5 23.2 23.2 23.2 23.1 23.1

EGTA titration data, mL thiosulphate

Thiosulphate in mL for 0.125 M EGTA -2.5% NaOCl, time in minutes vs repetition number. Thiosulphate concentration = 0.M. 0.09959 Time 1 2 3 4 5 6 7 8 9 10 1 6.69 6.69 6.74 6.73 6.69 6.7 6.71 6.67 6.69 6.69 20 0.52 0.5 0.52 0.53 0.52 0.6 0.57 0.52 0.52 0.52 40 0.4 0.38 0.36 0.37 0.45 0.46 0.38 0.4 0.4 0.39 60 0.3 0.33 0.31 0.32 0.37 0.36 0.3 0.32 0.3 0.3 80 0.25 0.23 0.23 0.29 0.31 0.3 0.27 0.24 0.24 0.26

Thiosulphate in mL for 2.5% NaOCl (control), time in minutes vs repetition number. Thiosulphate concentration = 0.M. 0.09959 Time 1 2 3 4 5 6 7 8 9 10 1 6.82 6.9 6.82 6.88 6.82 6.8 6.79 6.81 6.83 6.82 20 6.84 6.82 6.82 6.89 6.83 6.79 6.83 6.82 6.86 6.87 40 6.82 6.8 6.83 6.9 6.85 6.79 6.81 6.83 6.83 6.84 60 6.8 6.81 6.8 6.79 6.85 6.8 6.8 6.82 6.85 6.8 80 6.78 6.83 6.8 6.8 6.81 6.78 6.8 6.79 6.79 6.81

259

Na4 Etidronate

Na4 Etidronate pH data pH data for 0.25 M Na4 Etidronate-5% NaOCl, time in minutes vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 12.47 12.48 12.48 12.48 12.47 12.52 12.55 12.55 12.53 12.53 20 12.38 12.38 12.38 12.38 12.38 12.4 12.4 12.38 12.38 12.38 40 12.26 12.26 12.26 12.26 12.26 12.24 12.22 12.23 12.21 12.2 60 12.13 12.12 12.12 12.12 12.12 12.16 12.14 12.12 12.12 12.11 80 11.98 11.96 11.95 11.95 11.95 11.97 11.93 11.92 11.91 11.92

pH data for 0.25 M Na4 Etidronate-5% NaOCl, time in minutes vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 12.42 12.44 12.43 12.42 12.42 12.48 12.47 12.47 12.47 12.46 20 12.39 12.4 12.41 12.39 12.39 12.39 12.4 12.4 12.38 12.4 40 12.37 12.38 12.39 12.38 12.37 12.36 12.35 12.35 12.34 12.35 60 12.37 12.36 12.36 12.37 12.35 12.42 12.41 12.38 12.37 12.37 80 12.37 12.37 12.37 12.36 12.36 12.39 12.35 12.35 12.33 12.32

Na4 Etidronate temperature data

Temperature data for 0.25 M Na4 Etidronate-5% NaOCl, time in minutes vs repetition number. Temperature in degrees Celsius. Time 1 2 3 4 5 6 7 8 9 10 1 22.7 22.8 22.8 22.9 22.9 23.1 22.9 22.8 22.9 23 20 23 23.1 23.1 23.1 23.1 23.3 23.3 23.3 23.4 23.4 40 23.4 23.3 23.3 23.3 23.3 23.6 23.5 23.7 23.7 23.7 60 23.3 23.5 23.3 23.4 23.5 23.6 23.8 23.7 23.8 23.7 80 23.3 23.5 23.5 23.6 23.5 23.6 23.7 23.8 23.9 23.7

260

Temperature data for 5% NaOCl (control), time in minutes vs repetition number. Temperature in degrees Celsius. Time 1 2 3 4 5 6 7 8 9 10 1 22.6 22.4 22.3 22.5 22.5 22.9 22.8 22.8 22.8 22.7 20 22.8 22.8 22.8 22.9 22.8 23.2 23.2 23.2 23.2 22.9 40 23 22.9 22.9 23 22.9 23.2 23.2 23.1 23.1 23.1 60 23.1 23.1 23.1 23.1 23.1 23.2 23 23.1 23.1 23.1 80 23.1 23 23.1 23.1 23.1 23.2 23.3 23.3 23.3 23.2

Na4 Etidronate titration data, mL thiosulphate

Thiosulphate in mL for 0.25 M Na4 Etidronate-5% NaOCl, time in minutes vs repetition number. Thiosulphate concentration = 0.09935 M Time 1 2 3 4 5 6 7 8 9 10 1 13.35 13.4 13.3 13.32 13.38 13.42 13.32 13.5 13.4 13.32 20 13.1 13.1 13.1 13.12 13.15 13.1 13.11 13.1 13.1 13.16 40 13 13.05 12.98 12.95 12.9 12.88 12.87 12.9 13.14 12.95 60 12.8 12.88 12.7 12.7 12.7 12.72 12.67 12.68 12.69 12.69 80 12.6 12.6 12.55 12.51 12.5 12.59 12.44 12.49 12.55 12.57

Thiosulphate in mL for 5% NaOCl (control), time in minutes vs repetition number. Thiosulphate concentration = 0.09935 M Time 1 2 3 4 5 6 7 8 9 10 1 13.42 13.35 13.38 13.4 13.55 13.41 13.72 13.5 13.6 13.55 20 13.41 13.39 13.4 13.45 13.43 13.36 13.45 13.42 13.7 13.55 40 13.4 13.4 13.35 13.6 13.43 13.37 13.54 13.48 13.5 13.55 60 13.45 13.35 13.38 13.5 13.45 13.37 13.45 13.5 13.51 13.5 80 13.4 13.38 13.4 13.42 13.49 13.31 13.6 13.42 13.51 13.5

261

HPAA

HPAA pH data pH data for 0.5 M HPAA-2.5% NaOCl, time in minutes vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 7.52 7.5 7.5 7.48 7.48 7 7.41 7.48 7.47 7.5 20 7.51 7.48 7.47 7.47 7.45 7.06 7.45 7.51 7.49 7.5 40 7.46 7.44 7.45 7.48 7.45 7.05 7.45 7.51 7.55 7.5 60 7.5 7.48 7.49 7.47 7.48 7.06 7.48 7.52 7.53 7.52 80 7.49 7.49 7.5 7.49 7.5 7.08 7.48 7.54 7.55 7.55

pH data for 2.5% NaOCl (control), time in minutes vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 12.12 12.17 12.19 12.19 12.2 12.09 12.12 12.15 12.17 12.19 20 12.12 12.13 12.14 12.14 12.14 12.11 12.14 12.13 12.14 12.16 40 12.12 12.13 12.13 12.16 12.14 12.15 12.14 12.15 12.13 12.16 60 12.13 12.12 12.15 12.15 12.14 12.13 12.15 12.13 12.12 12.12 80 12.1 12.13 12.12 12.11 12.12 12.14 12.14 12.14 12.13 12.13

HPAA temperature data

Temperature data for 0.5 M HPAA-2.5% NaOCl, time in minutes vs repetition number. Temperature in degrees Celsius. Time 1 2 3 4 5 6 7 8 9 10 1 41.1 42.9 42.9 43.1 42.5 39.3 40.8 43.1 43.6 42.7 20 25.9 26.8 27.2 27.3 26.8 26.4 27.9 28.2 28.3 28.4 40 23.4 23.2 23.5 23.6 23.8 23.7 24.1 24.2 24.6 24.7 60 22.7 22.6 22.8 22.9 22.9 23.6 23.7 23.7 23.7 23.6 80 22.7 22.6 22.6 22.8 22.7 23.3 23.3 23.4 23.3 23.3

262

Temperature data for 2.5% NaOCl(control), time in minutes vs repetition number. Temperature in degrees Celsius. Time 1 2 3 4 5 6 7 8 9 10 1 22.7 22.8 22.4 22.4 22.3 23.2 23.2 22.8 22.7 22.6 20 22.7 22.4 22.3 22.4 22.3 23.6 23.2 23 22.9 23 40 22.4 22.3 22.3 22.3 22.3 23.3 23.3 23.2 23.2 23.2 60 22.5 22.4 22.4 22.4 22.5 23.3 23.3 23.2 23.2 23.2 80 22.6 22.5 22.5 22.5 22.5 23.1 23.1 23 23.1 23

Pentetic acid (DTPA)

Pentetic acid pH data pH data for 0.125 M Pentetic acid-2.5% NaOCl, time in minutes vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 12.37 12.34 12.37 12.32 12.39 12.36 12.39 12.36 12.34 12.38 20 9.26 9.23 9.21 9.22 9.22 9.24 9.21 9.21 9.2 9.2 40 9.23 9.22 9.22 9.23 9.24 9.23 9.23 9.22 9.22 9.22 60 9.23 9.24 9.24 9.24 9.25 9.23 9.22 9.21 9.22 9.21 80 9.22 9.22 9.22 9.23 9.24 9.21 9.21 9.21 9.21 9.21

pH data for 2.5% NaOCl (control), time in minutes vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 12.28 12.35 12.34 12.35 12.35 12.31 12.34 12.33 12.31 12.33 20 12.25 12.31 12.33 12.32 12.32 12.28 12.3 12.31 12.3 12.31 40 12.24 12.31 12.32 12.32 12.32 12.28 12.29 12.3 12.3 12.31 60 12.25 12.3 12.32 12.31 12.31 12.28 12.28 12.29 12.28 12.3 80 12.24 12.29 12.3 12.31 12.31 12.27 12.27 12.28 12.27 12.27

263

Pentetic acid temperature data

Temperature data for 0.125 M Pentetic acid-2.5% NaOCl, time in minutes vs repetition number. Temperature in degrees Celsius. Time 1 2 3 4 5 6 7 8 9 10 1 24.2 24.5 24.2 24.3 24.1 23.6 23.9 23.9 24.2 24 20 28.4 29.8 29.9 29.9 29.9 28.4 29.7 29.5 29.9 29.7 40 24.9 25.2 25.4 25.4 25.4 24.7 25.2 25.4 25.5 25.3 60 23.9 23.9 24 24.1 24.1 24 24 24.1 24.1 24.1 80 23.6 23.6 23.6 23.5 23.6 23.7 23.8 23.8 23.7 23.8

Temperature data for 2.5% NaOCl Control), time in minutes vs repetition number. Temperature in degrees Celsius. Time 1 2 3 4 5 6 7 8 9 10 1 23.6 23.4 23.4 23.4 23.5 23.8 23.4 23.6 23.6 23.4 20 24.3 23.8 23.8 23.7 23.6 24.3 23.7 23.7 23.6 23.7 40 23.8 23.7 23.7 23.6 23.6 23.9 23.6 23.7 23.7 23.6 60 23.6 23.6 23.5 23.5 23.5 23.7 23.6 23.6 23.6 23.6 80 23.5 23.5 23.5 23.4 23.3 23.8 23.6 23.7 23.6 23.6

Pentetic acid titration data, mL thiosulphate

Thiosulphate in mL for 0.125 M Pentetic acid-2.5% NaOCl, time in minutes vs repetition number. Thiosulphate concentration = 0.09946 M Time 1 2 3 4 5 6 7 8 9 10 1 6.14 6.2 6.37 6.35 6.48 6.5 6.43 6.41 5.89 6.48 20 0.11 0.09 0.13 0.1 0.12 0.12 0.1 0.14 0.1 0.09 40 0.05 0.04 0.05 0.05 0.05 0.04 0.08 0.07 0.07 0.03 60 0.02 0.02 0.03 0.05 0.04 0.03 0.03 0.04 0.02 0.03 80 0.02 0.01 0.01 0.02 0.02 0.01 0.02 0.03 0.02 0.02

264

Thiosulphate in mL for 2.5% NaOCl (control), time in minutes vs repetition number. Thiosulphate concentration = 0.09946 M Time 1 2 3 4 5 6 7 8 9 10 1 6.89 6.86 6.83 6.89 6.83 6.84 6.78 6.81 6.9 6.87 20 6.85 6.82 6.9 6.85 6.84 6.87 6.83 6.82 6.85 6.81 40 6.95 6.83 6.91 6.83 6.82 6.82 6.84 6.8 6.8 6.86 60 6.85 6.86 6.82 6.8 6.88 6.81 6.83 6.82 6.82 6.88 80 6.87 6.85 6.88 6.85 6.88 6.83 6.84 6.9 6.88 6.85

265 pH, temperature and FAC data over 18 hours at room temperature for 0.25 M clodronate-5% NaOCl pH and temperature experiments conducted separately to titration experiments. pH Data pH data for 0.25 M Clodronate-5% NaOCl, time vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 min 12.5 12.49 12.47 12.47 12.5 12.5 12.5 12.46 12.46 12.48 3 h 12.5 12.51 12.54 12.5 12.54 12.51 12.53 12.52 12.52 12.52 18 h 12.5 12.53 12.51 12.5 12.52 12.5 12.51 12.51 12.5 12.51 Min, minute; h, hours

pH data for 5% NaOCl (control), time vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 min 12.46 12.46 12.43 12.47 12.46 12.46 12.44 12.43 12.41 12.48 3 h 12.47 12.48 12.47 12.48 12.49 12.49 12.5 12.47 12.48 12.49 18 h 12.49 12.48 12.47 12.47 12.45 12.48 12.47 12.48 12.46 12.47 Min, minute; h, hours

Temperature data

Temperature data for 0.25 M Clodronate-5% NaOCl, time vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 min 23.9 24.1 24 24.1 23.5 23.5 23.5 23.7 23.9 23.6 3 h 22.9 23 22.9 22.8 22.9 23.1 22.9 23.1 23 22.9 18 h 23.2 23.3 23.4 23.5 23.6 23.6 23.5 23.5 23.5 23.5 Min, minute; h, hours. Temperature in degrees Celsius.

Temperature data for 5% NaOCl (control), time vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 min 23.8 24 23.8 23.5 23.3 23.8 23.9 23.6 23.8 23.4 3 h 23 23.1 23.1 22.9 22.9 23.1 23.1 23.2 23 22.9 18 h 23.3 23.4 23.5 23.5 23.5 23.5 23.7 23.4 23.6 23.6 Min, minute; h, hours. Temperature in degrees Celsius.

266

Titration data, mL thiosulphate

Thiosulphate in mL for 0.25 M Clodronate-5% NaOCl, time vs repetition number. Thiosulphate concentration = 0.10048 M. Time 1 2 3 4 5 6 7 8 9 10 1 min 13.3 13.38 13.21 13.29 13.2 13.25 13.36 13.41 13.36 13.46 3 h 13.31 13.38 13.15 13.3 13.19 13.22 13.37 13.39 13.31 13.4 18 h 13.3 13.4 13.2 13.3 13.19 13.27 13.32 13.31 13.36 13.33 Min, minute; h, hours. Temperature in degrees Celsius.

Thiosulphate in mL for 5% NaOCl (control), time vs repetition number. Thiosulphate concentration =0.10048 M Time 1 2 3 4 5 6 7 8 9 10 1 min 13.4 13.42 13.31 13.42 13.28 13.29 13.31 13.31 13.42 13.31 3 h 13.44 13.3 13.31 13.36 13.28 13.3 13.28 13.31 13.4 13.27 18 h 13.41 13.4 13.31 13.37 13.29 13.29 13.31 13.3 13.42 13.31 Min, minute; h, hours. Temperature in degrees Celsius.

267

Smear Layer removal, percent area fraction of open tubules (% AF). Experiment conducted over 15 minutes.

Group 1. 0.5 M Clodronate-5% NaOCl Group 2. 0.25 M Clodronate-5% NaOCl Group 3. 0.5 M Etidronate-5% NaOCl Group 4. 0.25 M Etidronate-5% NaOCl Group 5. PBS Group 6. Sequence 5% NaOCl/17% EDTA Group 7. Sequence 5% NaOCl/17% EDTA/5% NaOCl

Percent area fraction of open tubules for each group for 45 image/group from 9 dentine slices for all groups except Group 3 (42 images, from 9 slices). Group1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7

2.422 5.314 3.408 3.96 0.012 2.859 3.155 4.499 5.118 3.124 5.861 0.064 4.921 3.288 4.513 5.533 5.48 6.406 0.171 1.819 3.255 5.31 3.442 3.843 6.216 0.125 2.621 4.584 5.296 4.722 3.787 3.302 0.027 3.322 6.288 5.359 5.092 3.821 3.959 0.414 5.159 4.265 4.051 5.224 4.283 3.986 0.616 6.604 4.148 4.174 4.029 2.594 4.358 0.606 7.359 3.776 4.977 4.357 2.576 3.975 0.4 7.604 3.687 4.079 5.34 1.797 4.216 0.115 8.602 3.653 3.759 3.726 2.402 6.772 0.429 5.178 4.875 4.331 3.902 1.72 6.11 0.321 7.335 6.376 4.051 4.11 3.71 5.733 0.35 4.176 5.155 2.943 3.621 3.889 4.965 0.298 5.151 4.861 3.942 3.807 4.002 5.758 0.16 8.852 4.725 5.177 4.681 3.887 5.674 0.39 4.484 5.425 4 3.859 3.877 5.819 0.302 5.376 5.177 3.903 3.953 3.609 5.108 0.378 6.527 5.712 3.352 3.312 3.937 5.929 0.089 5.482 6.468 3.411 3.537 4.107 5.143 0.11 4.789 3.89

268

5.318 3.879 3.3 3.712 0.207 5.58 5.451 2.703 4.081 3.381 5.089 0.075 5.832 6.629 2.029 3.38 4.653 3.032 0.121 5.487 6.787 3.057 3.487 5.109 3.255 0.181 4.909 7.341 2.41 3.082 4.225 3.572 0.267 5.09 8.184 5.095 4.402 4.334 6.029 0.142 4.547 7.211 6.207 4.519 3.546 6.003 0.191 5.627 6.167 5.955 4.393 4.011 7.284 0.248 5.81 5.576 4.186 3.926 3.63 7.504 0.333 6.524 5.809 5.155 4.67 4.199 8.051 0.575 5.269 5.503 2.919 6.015 5 5.366 0.417 6.145 5.82 3.864 3.308 5.087 5.846 0.15 6.014 5.828 2.721 3.629 4.653 5.665 0.35 6.043 5.4 2.826 4.425 4.322 5.191 0.114 6.281 5.161 4.172 4.412 4.43 5.538 0.309 7.123 3.671 4.623 4.87 4.205 4.527 0.244 3.878 5.419 4.57 4.503 3.723 4.604 0.086 3.439 7.233 4.725 4.542 4.532 4.98 0.14 5.449 8.892 3.455 4.347 6.14 5.556 0.405 6.867 5.091 4.72 4.392 6.113 5.984 0.08 6.326 5.115 7.41 2.819 6.798 4.461 0.134 4.215 3.804 6.803 3.965 5.624 3.627 0.07 3.795 3.753

6.457 3.133 3.42 0.078 4.176 5.986

6.401 3.46 3.731 0.05 4.697 4.105

6.372 3.683 3.714 0.09 5.194 4.416

269

Paper 2, Chapter 3. The effect of temperature on the stability of sodium hypochlorite admixed with clodronate. pH data at 23 oC and 35 oC pH data for 0.52 M Clodronate-5% NaOCl at 23 oC, time in minutes vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 12.65 12.67 12.68 12.70 12.70 12.79 12.78 12.78 12.77 12.76 20 12.69 12.70 12.70 12.69 12.72 12.70 12.70 12.70 12.70 12.69 40 12.69 12.69 12.69 12.68 12.69 12.67 12.67 12.68 12.68 12.68 60 12.66 12.67 12.66 12.66 12.67 12.66 12.67 12.67 12.66 12.65 120 12.76 12.76 12.76 12.74 12.75 12.80 12.77 12.75 12.72 12.70 180 12.77 12.78 12.76 12.77 12.77 12.78 12.78 12.77 12.74 12.71

pH data for 5% NaOCl (control) at 23 oC, time in minutes vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 12.56 12.56 12.56 12.56 12.55 12.59 12.59 12.59 12.58 12.56 20 12.56 12.57 12.57 12.56 12.55 12.55 12.56 12.54 12.53 12.53 40 12.54 12.55 12.54 12.54 12.55 12.53 12.54 12.54 12.54 12.53 60 12.53 12.53 12.53 12.52 12.51 12.53 12.52 12.5 12.52 12.51 120 12.59 12.59 12.59 12.57 12.57 12.54 12.54 12.53 12.52 12.53 180 12.61 12.6 12.58 12.58 12.58 12.57 12.57 12.56 12.54 12.55

pH data for 0.52 M Clodronate-5% NaOCl at 35 oC, time in minutes vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 12.42 12.4 12.42 12.39 12.39 12.39 12.38 12.33 12.3 12.27 20 12.35 12.31 12.36 12.3 12.32 12.19 12.17 12.22 12.23 12.24 40 12.24 12.24 12.28 12.23 12.24 12.2 12.18 12.12 12.14 12.08 60 12.17 12.16 12.16 12.16 12.13 12.09 12.12 12.06 12.13 12.07 120 12.32 12.3 12.26 12.23 12.24 12.34 12.31 12.28 12.26 12.24 180 12.35 12.31 12.28 12.25 12.24 12.33 12.31 12.29 12.25 12.23

270 pH data for 5% NaOCl (control) at 35 oC, time in minutes vs repetition number. Time 1 2 3 4 5 6 7 8 9 10 1 12.22 12.2 12.18 12.2 12.17 12.12 12.1 12.09 12.07 12.07 20 12.21 12.19 12.16 12.14 12.16 12.06 12.09 12.1 12.02 12.04 40 12.13 12.09 12.11 12.06 12.04 12.07 12.02 12.01 12.02 12.02 60 11.98 11.93 12.02 12 11.98 12.01 11.91 11.88 11.83 11.84 120 12.09 12.05 12.07 12.05 12.11 12.14 12.11 12.06 12.04 12.04 180 12.1 12.05 12.08 12.05 12.12 12.11 12.1 12.09 12.07 12.05

Temperature data at 23 oC and 35 oC

Temperature data for 0.52 M Clodronate-5% NaOCl at 23 oC, time in minutes vs repetition number. Temperature in degrees Celsius. Time 1 2 3 4 5 6 7 8 9 10 1 23.1 23.1 23.2 23.1 23.3 22.9 23 23 23.1 23.1 20 23 23.1 23.1 23.1 23 23.1 23.2 23.2 23.1 23.2 40 23 23 23.1 23.1 23.1 23.3 23.2 23.2 23.2 23.3 60 23 23.1 23.1 23.1 23 23.4 23.3 23.3 23.2 23.2 120 22.8 22.7 22.9 22.9 22.9 22.8 23 23.1 23.2 23.2 180 22.9 22.9 23 23 23 23 23 23.1 23.1 23.2

Temperature data for 5% NaOCl (control) at 23 oC, time in minutes vs repetition number. Temperature in degrees Celsius. Time 1 2 3 4 5 6 7 8 9 10 1 23 23.1 22.9 23 23.1 23 23 23 23 23.2 20 22.9 23 22.9 23 23 23.2 23.1 23.2 23.2 23.3 40 23 23 23 23 23.1 23.3 23.2 23.2 23.3 23.4 60 22.9 23 23.1 23.1 23 23.2 23.2 23.3 23.4 23.3 120 22.9 22.9 22.9 23 23 23.1 23.1 23 23 23.1 180 23 23 23.1 23.1 23.1 23.1 23 23.1 23.1 23.1

271

Temperature data for 0.52 M Clodronate-5% NaOCl at 35 oC, time in minutes vs repetition number. Temperature in degrees Celsius. Time 1 2 3 4 5 6 7 8 9 10 1 33.6 33.6 34 33.8 34 34.1 34 33.8 33.6 33.9 20 34.6 34.6 33.9 34.6 34.7 34 34.8 34.8 34.7 34.5 40 34.5 34.4 34.6 34.6 34.4 34.5 34.4 34.3 34.4 34.3 60 34.5 34.4 34.6 34.5 34.7 34.5 34.3 34.3 34.3 34.2 120 34.8 34.6 34.5 34.6 34.5 34.8 34.7 34.7 34.7 34.5 180 34.3 34.2 34.1 34.4 34.8 34.9 34.7 34.6 34.5 34.5

Temperature data for 5% NaOCl (control) at 35 oC, time in minutes vs repetition number. Temperature in degrees Celsius. Time 1 2 3 4 5 6 7 8 9 10 1 33.6 33.7 33.9 34.1 34.1 33.7 33.5 33.2 33 33 20 34.4 34.5 34.5 34.6 34.2 34.3 34.4 34.1 34.3 34.5 40 34.1 34.3 34.6 34.7 34.7 34.3 34.3 34.2 34.5 34.6 60 34.5 34.4 34.9 34.6 34.5 34.1 34.7 34.5 34.3 34.2 120 34.4 34.6 34.5 34.6 34.6 34.7 34.7 34.6 34.3 34.6 180 34.6 34.8 34.6 34.6 34.7 34.8 34.7 34.6 34.5 34.5

Titration data, mL thiosulphate, 23 oC and 35 oC over 3 hours

Thiosulphate in mL for 0.52 M Clodronate-5%-5% NaOCl at 23 oC, time in minutes vs repetition number. Thiosulphate concentration = 0.09973 M. Time 1 2 3 4 5 6 7 8 9 10 1 13.32 13.37 13.45 13.28 13.25 13.25 13.3 13.29 13.3 13.43 20 13.28 13.29 14.49 13.27 13.31 13.3 13.18 13.34 13.39 13.44 40 13.33 13.28 13.31 13.2 13.31 13.31 13.18 13.31 13.39 13.41 60 13.21 13.23 13.39 13.3 13.28 13.24 13.19 13.27 13.27 13.35 120 13.27 13.26 13.3 13.18 13.34 13.27 13.18 13.37 13.2 13.32 180 13.29 13.3 13.19 13.2 13.25 13.26 13.12 13.28 13.2 13.2

272

Thiosulphate in mL for 5% NaOCl (control) at 23 oC, time in minutes vs repetition number. Thiosulphate concentration = 0.09973 M. Time 1 2 3 4 5 6 7 8 9 10 1 13.32 13.45 13.4 13.39 13.4 13.39 13.38 13.27 13.39 13.39 20 13.36 13.41 13.39 13.41 13.39 13.4 13.3 13.26 13.43 13.39 40 13.36 13.39 13.4 13.36 13.34 13.42 13.31 13.29 13.47 13.31 60 13.37 13.45 13.48 13.31 13.43 13.39 13.36 13.29 13.44 13.37 120 13.37 13.42 13.34 13.39 13.4 13.41 13.3 13.21 13.39 13.32 180 13.3 13.42 13.36 13.39 13.39 13.38 13.29 13.3 13.4 13.36

Thiosulphate in mL for 0.52 M Clodronate-5%-5% NaOCl at 35 oC, time in minutes vs repetition number. Thiosulphate concentration = 0.09973 M. Time 1 2 3 4 5 6 7 8 9 10 1 13.4 13.38 13.33 13.32 13.24 13.26 13.29 13.32 13.3 13.36 20 13.27 13.32 13.3 13.31 13.27 13.3 13.22 13.34 13.17 13.3 40 13.15 13.23 13.28 13.37 13.29 13.27 13.22 13.29 13.28 13.25 60 13.17 13.21 13.28 13.2 13.21 13.24 13.19 13.31 13.28 13.23 120 13.22 13.16 13.32 13.37 13.2 13.22 13.21 13.23 13.21 13.22 180 13.18 13.22 13.22 13.3 13.28 13.41 13.25 13.2 13.22 13.23

Thiosulphate in mL for 5% NaOCl (control) at 35 oC, time in minutes vs repetition number. Thiosulphate concentration = 0.09973 M. Time 1 2 3 4 5 6 7 8 9 10 1 13.42 13.48 13.41 13.39 13.47 13.5 13.31 13.32 13.3 13.28 20 13.3 13.4 13.36 13.42 13.35 13.43 13.35 13.36 13.3 13.31 40 13.33 13.37 13.4 13.38 13.32 13.32 13.36 13.31 13.25 13.39 60 13.4 13.31 13.41 13.42 13.36 13.31 13.33 13.3 13.31 13.29 120 13.33 13.35 13.43 13.41 13.35 13.35 13.34 13.26 13.3 13.33 180 13.41 13.4 13.5 13.41 13.35 13.4 13.37 13.31 13.25 13.34

273

Titration data, mL thiosulphate, 2-4 oC over 3 months Thiosulphate in mL for 0.52 M Clodronate-5%-5% NaOCl at 2-4 oC, time in minutes, days, weeks and months vs repetition number. Thiosulphate concentration = 0.09984 M for 1 minute, 1 day, 3 days, 1 week and 2 weeks, = 0.09815 M for 6 weeks, = 0.10048 for 3 months. Time 1 2 3 4 5 6 7 8 9 10 1 min 13.5 13.54 13.45 13.41 13.6 13.47 13.52 13.85 13.42 13.5 1 d 13.32 13.6 13.41 13.32 13.52 13.4 13.58 13.72 13.4 13.34 3 d 13.3 13.33 13.3 13.45 13.4 13.35 13.4 13.55 13.32 13.42 1 wk 13.2 13.29 13.27 13.29 13.28 13.34 13.35 13.24 13.29 13.3 2 wk 13.3 13.29 13.28 13.41 13.21 13.32 13.31 13.1 13.41 13.21 6 wk 13.3 13.32 13.39 13.29 13.31 13.42 13.42 13.1 13.4 13.44 3 mth 12.68 12.72 12.74 12.64 12.7 12.9 12.78 12.44 12.8 12.8 min, minute; d, day/s; wk, week/s; mth, months.

Thiosulphate in mL for 5% NaOCl (control) at 2-4 oC, time in minutes, days, weeks and months vs repetition number. Thiosulphate concentration = 0.09984 M for 1 minute, 1 day, 3 days, 1 week and 2 weeks, = 0.09815 M for 6 weeks, = 0.10048 for 3 months. Time 1 2 3 4 5 6 7 8 9 10 1 min 13.4 13.32 13.4 13.4 13.35 13.35 13.42 13.4 13.43 13.42 1 d 13.46 13.37 13.47 13.39 13.41 13.41 13.36 13.42 13.4 13.43 3 d 13.46 13.32 13.5 13.42 13.38 13.4 13.41 13.41 14.44 13.45 1 wk 13.5 13.31 13.41 13.35 13.45 13.37 13.4 13.5 13.4 13.47 2 wk 13.33 13.3 13.42 13.31 13.41 13.32 13.34 13.41 13.36 13.4 6 wk 13.5 13.45 spilt 13.49 13.5 13.51 13.5 13.54 13.53 13.6 3 mth 13.07 13.08 13.15 13.18 13.12 13.11 13.03 13.11 13.13 12.21 min, minute; d, day/s; wk, week/s; mth, months.

274

9.4.3 Data from Chapter 4

Organic tissue dissolution in clodronate and etidronate mixtures with sodium hypochlorite.

Experiment 1. Measurement of FAC (groups 1,2,3) and weight changes (all groups) over 15 minutes. Group 1. 0.26 M clodronate-5% NaOCl Group 2. 0.26 etidronate-5% NaOCl Group 3. 5% NaOCl Group 4. PBS Group 5. 026 M clodronate Group 6. 0.26 M etidronate

Titration data. Thiosulphate in mL. Thiosulphate concentration = 0.09796 M Sample 0.26 M 0.26 M 0.26 M 0.26 M 5% 5% number Clodronate- Clodronate- Etidronate- Etidronate- NaOCl NaOCl 5% NaOCl 5% NaOCl 5% NaOCl 5% NaOCl Initial Final Initial Final Initial Final 1 13.11 12.5 13.11 11.72 13.19 12.53 2 13.11 12.38 13.16 11.8 13.2 12.42 3 13.1 12.52 13.11 11.73 13.18 12.4 4 13.23 12.5 13.1 11.71 13.15 12.42 5 13.11 12.62 13.22 11.85 13.05 12.45 6 13.13 12.49 13.2 11.87 13.2 12.41 7 13.2 12.61 13.13 11.79 13.2 12.46 8 13.2 12.61 13.07 11.9 13.12 12.49 9 13.19 12.53 13.12 11.76 13.2 12.5 10 13.21 12.57 13.16 11.78 13.18 12.4

275

Weight data in mg. Sam, sample number; Gp, group; In, initial; Fin, final. Sam Gp1 Gp1 Gp2 Gp2 Gp3 Gp3 Gp4 Gp4 Gp5 Gp5 Gp6 Gp6 In Fin In Fin In Fin In Fin In Fin In Fin 1 55.3 26.2 55.5 28.3 55.2 24.5 54.7 54.8 55.2 53.5 56.3 56.5 2 55.6 26.9 56 28.4 55.7 25.7 55.1 56.4 54.9 57.6 55.4 57.5 3 55.6 27.1 54.7 32.6 55.9 22 55.5 57.4 55.7 54.1 56 56 4 55.9 25.1 55.8 30.3 56.5 25.6 56.3 55.4 54 53.9 54.7 53.1 5 55.2 26 55.3 29.6 55.6 20.8 56.4 54.5 56.5 56.9 56.1 55.7 6 54.7 19 55.9 35.6 56 20.7 56.3 55.2 56.4 56.8 55.4 54.9 7 55.9 22.7 55.8 29.5 55.3 19.6 55.6 54.3 55.4 55.8 55.6 56.8 8 55.5 21.3 55.5 29.3 55.8 19.1 56 51.5 55.9 57.2 55.3 54 9 55.4 24.1 54.9 26.7 55.6 22.4 55 53.3 55 53.5 56.5 56.6 10 55.9 25 56 25.3 55 19.5 54.9 53.3 56 53.1 55.6 57.1

Experiment 2. Repeated measures data. Measurement of weight changes over 15 minutes. 6 hour time delay from mixing.

Part A

Weight data in mg for 0.26 M Clodronate-2.5% NaOCl, weight measurements at Time = 5, 10 and 15 minutes vs repetition/sample number. Time 1 2 3 4 5 6 7 8 9 10 0 55.1 55.2 55.5 54 53.8 55.8 54.9 54.9 55 Drop 5 43.6 43.3 47.4 43 41.3 47.4 42.4 44.2 39.3 Drop 10 33.8 29.9 38.7 29.4 32.6 35.5 32.2 31.1 28.6 Drop 15 23.6 23.2 30.5 21.2 23.1 28.3 23.6 23 18.3 Drop

Weight data in mg for 2.5% NaOCl (positive control), weight measurements at Time = 5, 10 and 15 minutes vs repetition/sample number. Time 1 2 3 4 5 6 7 8 9 10 0 53.8 55.3 54.4 55.6 55.3 54.6 56 55.6 55 55.7 5 41.3 46.4 41.3 46.6 46 43 46.8 43.2 41.8 45.1 10 30.5 37.2 28.5 33.7 33.7 29 35.2 32.3 27.4 32.8 15 20.9 27.1 15.9 22.5 21.4 18.7 24.9 21.8 18.6 22.8

276

Weight data in mg for PBS (negative control), weight measurements at Time = 5, 10 and 15 minutes vs repetition/sample number. Time 1 2 3 4 5 6 7 8 9 10 0 54.3 53.8 55.2 54.6 54.6 55.8 55.6 55.9 54.8 55 5 56.5 49.4 55 56.2 52.7 52.9 55.6 56.4 56 55.5 10 57.5 47.3 54.1 57 52.4 52.9 55.7 53.3 55.7 53.6 15 58.3 50.4 54.5 54.2 51.7 54.8 56.8 53.3 56.4 54.8

Part B Weight data in mg for 0.52 M clodronate-5% NaOCl, weight measurements at Time = 5, 10 and 15 minutes vs repetition/sample number. Time 1 2 3 4 5 6 7 8 9 10 0 54.7 55.2 55.1 55.1 54.9 55.1 56.2 55.6 55.1 55.7 5 36.8 34.4 38.5 35.5 38 39.4 41.2 39.4 42.3 37.4 10 25.2 20.6 26.3 24.1 22 28.5 28.1 27.9 28.4 24.2 15 16.8 10.2 15.7 16.5 13.4 19.6 16 18.1 16.4 12.4

Weight data in mg for 5% NaOCl (positive control), weight measurements at Time = 5, 10 and 15 minutes vs repetition/sample number. Time 1 2 3 4 5 6 7 8 9 10 0 56 55.8 56.1 55.3 54.5 55.6 55.5 55.5 55.5 55.7 5 33 34 35.7 33.4 33 36.3 36.8 37.7 35.7 39.2 10 13.4 17.7 20 15.3 12.9 18.9 20.4 22.3 20.7 22.8 15 3.8 5.6 9.1 5.3 6.1 8.2 9.6 9.9 8.3 10.6

Weight data in mg for PBS (negative control), weight measurements at Time = 5, 10 and 15 minutes vs repetition/sample number. Time 1 2 3 4 5 6 7 8 9 10 0 55.6 55.3 55.5 55.8 55.4 54 55.9 55.7 55.2 55.2 5 54.8 53.1 53.1 52.7 56.1 53.2 57.9 58.5 61.2 56.3 10 55.6 53.2 51 52.6 53 54.2 57.5 58.3 62.3 56.9 15 57.6 52.8 50.5 50.9 56.2 51 53.8 55.9 63.2 56.2

277

9.4.4 Data from Chapter 5

The antibiofilm actions of sodium hypochlorite mixed with clodronate or etidronate using multiple biofilm assessment methodologies

CFU results, chelator mixtures with NaOCl CFU/mL from 3 experiments for HA discs treated with 0.26 M Clodronate-5% NaOCl. Disc Exp_Disc Replicate 1 Replicate 2 Replicate 3 Average 1_1 5.48 x 104 4.22 x 104 3.72 x 104 4.47 x 104 1_2 3.86 x 104 3.68 x 104 3.54 x 104 3.69 x 104 1_3 1.86 x 105 1.76 x 105 2.42 x 105 2.01 x 105 2_1 6.70 x 103 6.10 x 103 5.86 x 103 6.22 x 103 2_2 9.60 x 104 1.54 x 105 1.74 x 105 1.41 x 105 2_3 6.60 x 104 7.20 x 104 4.80 x 104 6.20 x 104 3_1 2.02 x 105 1.94 x 105 1.80 x 105 1.92 x 105 3_2 1.00 x 104 1.04 x 104 1.88 x 104 1.31 x 104 3_3 1.37 x 106 1.20 x 106 1.27 x 106 1.28 x 106

CFU/mL from 3 experiments for HA discs treated with 0.26 M Etidronate-5% NaOCl. Disc Exp_Disc Replicate 1 Replicate 2 Replicate 3 Average 1_1 5.94 x 104 5.78 x 104 5.84 x 104 5.85 x 104 1_2 4.70 x 104 5.42 x 104 3.56 x 104 4.56 x 104 1_3 3.34 x 103 2.44 x 103 3.36 x 103 3.05 x 103 2_1 6.42 x 104 5.28 x 104 4.38 x 104 5.36 x 104 2_2 3.26 x 104 2.42 x 104 2.18 x 104 2.62 x 104 2_3 1.60 x 105 1.22 x 105 7.00 x 104 1.17 x 105 3_1 1.72 x 105 1.38 x 105 8.60 x 104 1.32 x 105 3_2 2.72 x 104 2.98 x 104 smudgy 2.85 x 104 3_3 9.80 x 105 8.98 x 105 smudgy 9.39 x 105 Exp_Disc, experiment number and disc number.

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CFU/mL from 3 experiments for HA discs treated with 5% NaOCl (positive control). Disc Exp_Disc Replicate 1 Replicate 2 Replicate 3 Average 1_1 2.94 x 104 2.62 x 104 2.82 x 104 2.79 x 104 1_2 4.60 x 103 4.26 x 103 6.14 x 103 5.00 x 103 1_3 2.78 x 104 3.50 x 104 4.06 x 104 3.45 x 104 2_1 1.26 x 104 1.78 x 104 1.70 x 104 1.58 x 104 2_2 1.24 x 104 1.48 x 104 1.54 x 104 1.42 x 104 2_3 5.22 x 103 5.42 x 103 5.32 x 103 5.32 x 103 3_1 1.10 x104 1.14 x 104 8.8 x 103 1.04 x 104 3_2 4.86 x105 4.02 x105 3.76 x105 4.21 x 105 3_3 1.36 x105 8.80 x 104 7.2 x 104 9.87 x 104 Exp_Disc, experiment number and disc number.

CFU/mL from 3 experiments for HA discs treated with PBS (negative control). Disc Exp_Disc Replicate 1 Replicate 2 Replicate 3 Average 1_1 2.74 x 107 2.42 x 107 2.18 x 107 2.45 x 107 1_2 1.58 x 107 2.66 x 107 2.08 x 107 2.11 x 107 1_3 2.94 x 107 3.26 x 107 3.70 x 107 3.30 x 107 2_1 1.78 x 107 3.86 x 107 3.60 x 107 3.08 x 107 2_2 3.52 x 107 1.52 x 107 2.68 x 107 2.57 x 107 2_3 9.40 x 106 6.20 x 106 5.20 x 106 6.93 x106 3_1 2.66 x 107 2.28 x 107 2.28 x 107 2.41 x 107 3_2 3.00 x 107 2.58 x 107 2.46 x 107 2.68 x 107 3_3 2.42 x 107 1.78 x 107 1.98 x 107 2.06 x 107 Exp_Disc, experiment number and disc number.

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SEM analysis

Effects of treatment only, that is not vortex mixed. Number of bacteria left on disc. Clo = Clodronate, Eti = Etidronate, C1 = counter 1, number of bacteria counted, C2 = counter 2, number of bacteria counted, C3 = counter 3, number of bacteria counted.

0.26 M Clo-5%NaOCl 0.26 M Eti-5%NaOCl 5% NaOCl

image C 1 C2 C3 Image C 1 C2 C3 Image C 1 C2 C3 1_1_1 0 0 0 9_1_1 0 0 0 10_1_1 0 0 0 1_1_2 0 0 0 9_1_2 1 0 0 10_1_2 0 0 0 1_1_3 0 0 0 9_1_3 0 0 0 10_1_3 0 0 0 1_1_4 0 0 0 9_1_4 0 2 0 10_1_4 0 0 0 1_1_5 0 0 0 9_1_5 20 20 14 10_1_5 0 0 0 1_2_1 0 1 2 9_2_1 1 1 1 10_2_1 1 1 1 1_2_2 0 0 0 9_2_2 0 0 0 10_2_2 6 7 9 1_2_3 0 0 0 9_2_3 1 2 1 10_2_3 1 2 2 1_2_4 0 2 2 9_2_4 0 0 0 10_2_4 2 0 0 1_2_5 0 0 0 9_2_5 0 0 0 10_2_5 4 3 2 1_3_1 0 0 0 9_3_1 16 15 15 10_3_1 1 1 1 1_3_2 0 0 0 9_3_2 17 13 12 10_3_2 0 0 0 1_3_3 11 11 11 9_3_3 4 6 6 10_3_3 4 0 0 1_3_4 2 1 3 9_3_4 17 16 16 10_3_4 4 3 2 1_3_5 0 0 0 9_3_5 10 10 9 10_3_5 4 3 2 1_4_1 3 7 3 9_4_1 1 3 1 10_4_1 0 0 0 1_4_2 0 5 1 9_4_2 5 9 5 10_4_2 5 4 4 1_4_3 0 2 2 9_4_3 6 4 4 10_4_3 1 1 1 1_4_4 0 0 6 9_4_4 2 1 0 10_4_4 0 0 0 1_4_5 0 0 0 9_4_5 0 0 0 10_4_5 3 4 4 1_5_1 2 2 3 9_5_1 23 16 24 10_5_1 16 19 17 1_5_2 0 0 0 9_5_2 8 9 8 10_5_2 14 12 13 1_5_3 0 4 6 9_5_3 0 2 2 10_5_3 0 0 0 1_5_4 0 0 0 9_5_4 2 2 3 10_5_4 5 5 4 1_5_5 2 3 0 9_5_5 23 17 22 10_5_5 24 27 24 1_6_1 0 0 0 9_6_1 1 3 6 10_6_1 7 6 7

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1_6_2 2 2 2 9_6_2 18 15 20 10_6_2 2 2 2 1_6_3 0 2 0 9_6_3 3 0 4 10_6_3 2 1 0 1_6_4 4 2 2 9_6_4 6 7 7 10_6_4 0 1 0 1_6_5 2 3 2 9_6_5 7 4 4 10_6_5 16 17 16 1_7_1 0 0 0 9_7_1 0 0 0 10_7_1 0 0 0 1_7_2 0 0 0 9_7_2 1 0 0 10_7_2 0 0 0 1_7_3 0 0 0 9_7_3 0 0 0 10_7_3 0 0 0 1_7_4 0 0 0 9_7_4 0 0 0 10_7_4 4 0 0 1_7_5 0 0 0 9_7_5 0 0 0 10_7_5 0 0 0 1_8_1 0 0 0 9_8_1 0 0 0 10_8_1 0 0 0 1_8_2 1 0 1 9_8_2 0 0 0 10_8_2 0 0 0 1_8_3 0 1 0 9_8_3 0 0 0 10_8_3 0 0 0 1_8_4 0 0 1 9_8_4 0 0 0 10_8_4 0 0 0 1_8_5 0 0 0 9_8_5 0 0 0 10_8_5 0 0 0 1_9_1 0 0 0 9_9_1 0 0 0 10_9_1 0 0 0 1_9_2 0 2 1 9_9_2 1 0 0 10_9_2 1 0 0 1_9_3 0 0 0 9_9_3 1 0 0 10_9_3 1 0 1 1_9_4 4 4 4 9_9_4 0 0 0 10_9_4 0 0 0 1_9_5 0 0 0 9_9_5 0 0 0 10_9_5 0 0 0

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Effect of vortex mixing on removing bacteria from disc following treatment. Clo = Clodronate, Eti = Etidronate, C1 = counter 1, number of bacteria counted, C2 = counter 2, number of bacteria counted, C3 = counter 3, number of bacteria counted.

0.26 M Clo-5%NaOCl 0.26 M Eti-5%NaOCl 5% NaOCl

image C 1 C2 C3 Image C 1 C2 C3 Image C 1 C2 C3 3_1_1 0 0 0 2_1_1 0 0 1 8_1_1 4 1 3 3_1_2 1 0 1 2_1_2 1 0 2 8_1_2 2 2 2 3_1_3 0 0 0 2_1_3 0 0 0 8_1_3 2 1 1 3_1_4 0 0 0 2_1_4 1 0 1 8_1_4 5 0 2 3_1_5 0 0 0 2_1_5 0 0 0 8_1_5 5 1 9 3_2_1 0 0 0 2_2_1 0 0 0 8_2_1 1 0 2 3_2_2 0 0 0 2_2_2 0 1 1 8_2_2 1 1 1 3_2_3 0 0 0 2_2_3 0 0 0 8_2_3 0 0 0 3_2_4 0 2 0 2_2_4 2 4 2 8_2_4 0 0 0 3_2_5 0 0 0 2_2_5 2 0 1 8_2_5 0 3 2 3_3_1 1 1 1 2_3_1 0 0 0 8_3_1 0 0 0 3_3_2 1 1 1 2_3_2 0 0 0 8_3_2 2 2 2 3_3_3 1 0 0 2_3_3 0 0 0 8_3_3 1 0 1 3_3_4 1 1 2 2_3_4 0 0 0 8_3_4 3 2 2 3_3_5 0 0 0 2_3_5 0 0 0 8_3_5 4 2 4

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Effect of removing bacteria from PBS control with vortex mixing. S1 = score for scorer 1, S2 = score for scorer 2

PBS control, not vortex mixed PBS control, vortex mixed

Image S1 S2 Image S1 S2

4_1_1 4 4 6_1_1 2 2 4_1_2 4 3 6_1_2 2 2 4_1_3 3 3 6_1_3 2 2 4_1_4 3 2 6_1_4 2 2 4_1_5 1 1 6_1_5 2 2 4_2_1 4 4 6_2_1 2 2 4_2_2 4 4 6_2_2 2 3 4_2_3 4 4 6_2_3 2 3 4_2_4 3 3 6_2_4 3 3 4_2_5 4 4 6_2_5 3 3 4_3_1 4 4 6_3_1 2 2 4_3_2 4 4 6_3_2 2 2 4_3_3 4 4 6_3_3 2 3 4_3_4 4 4 6_3_4 2 2 4_3_5 4 3 6_3_5 1 1

4_4_1 4 4

4_4_2 4 4

4_4_3 4 4

4_4_4 4 3

4_4_5 3 3

4_5_1 4 4

4_5_2 4 4

4_5_3 4 4

4_5_4 4 3

4_5_5 4 4

4_6_1 3 3

4_6_2 4 4 283

4_6_3 2 2

4_6_4 3 2

4_6_5 4 4

4_7_1 3 4

4_7_2 4 4

4_7_3 4 4

4_7_4 2 2

4_7_5 3 3

4_8_1 4 4

4_8_2 4 4

4_8_3 4 4

4_8_4 4 4

4_8_5 4 4

4_9_1 4 4

4_9_2 4 4

4_9_3 3 3

4_9_4 4 4

4_9_5 4 4

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XTT assay

Chelator mixtures with NaOCl

A492, background reading for each group Group 0.26 M 0.26 M Na4 2.5% NaOCl PBS clodronate- etidronate- 2.5% NaOCl 2.5% NaOCl 0.073 0.072 0.0733 0.0724

A492, for each group, blank not subtracted Disc Exp_Disc 0.26 M 0.26 M Na4 2.5% NaOCl PBS clodronate- etidronate- 2.5% NaOCl 2.5% NaOCl 1_1 0.0842 0.0895 0.0888 0.2569 1_2 0.0831 0.0798 0.0868 0.2298 1_3 0.0959 0.0884 0.0846 0.2363 2_1 0.0831 0.0805 0.1022 0.2166 2_2 0.0965 0.0879 0.0861 0.2126 2_3 0.1322 0.096 0.0885 0.239 3_1 0.0924 0.0848 0.0785 0.2273 3_2 0.0881 0.0962 0.0798 0.2416 3_3 0.0822 0.0821 0.0887 0.2064 Exp_Disc, experiment number and disc number.

285

Plain chelators

A492, background reading for each group Group 0.26 M 0.26 M Na4 2.5% NaOCl PBS clodronate etidronate 0.0727 0.071 0.0708 0.07

A492, for each group, blank not subtracted Disc Exp_Disc 0.26 M 0.26 M Na4 2.5% NaOCl PBS clodronate etidronate 1_1 0.2296 0.2169 0.0818 0.2145 1_2 0.2221 0.2367 0.0847 0.2277 1_3 0.2456 0.2014 0.0841 0.2147 2_1 0.2383 0.188 0.0773 0.2178 2_2 0.2436 0.1924 0.0801 0.2111 2_3 0.1982 0.217 0.0872 0.2851 3_1 0.2057 0.2123 0.0948 0.2571 3_2 0.2238 0.178 0.0733 0.2376 3_3 0.1818 0.2296 0.0732 0.2833 Exp_Disc, experiment number and disc number.

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9.4.5 Data from Chapter 6

Experiment 1: Sequence 5% NaOCl/17% EDTA/5% NaOCl and d-H20

Force in Newtons (N) at point of fracture Pair number Sequence d-H20 N N

1 442.8 461.7 2 423.1 432.6 3 385.1 510.3 4 309.4 342.1 5 208.2 271.7 6 356.6 432.7 7 443.1 482.3 8 530.3 550.8 9 339.6 359.5 10 404.8 560.4 11 412.9 446.1 12 310.3 413 13 380.2 485.8 14 547.3 609.1 15 382.8 502

287

Experiment 2: Sequence 5% NaOCl/17% EDTA/5% NaOCl and 0.26 M Clodronate -5% NaOCl

Force in Newtons (N) at point of fracture Pair number Sequence 0.26 M clodronate-5% NaOCl N N

1 435.2 457.7 2 251 319 3 518.9 386.3 4 451.1 501.4 5 551.2 603.2 6 590.7 532.4 7 479.2 387.3 8 362.7 351 9 471.7 566.4 10 408.7 432.9 11 636.8 543 12 251.8 351.5 13 334.3 469.6 14 373.8 341.9 15 403.8 237.6

288

Experiment 3: Sequence 5% NaOCl/17% EDTA/5% NaOCl and 0.26 M Na4 etidronate-5% NaOCl

Force in Newtons (N) at point of fracture Pair number Sequence 0.26 M Na4 etidronate- 5% NaOCl N N

1 494.2 513.3 2 422.7 331 3 264.3 285.5 4 385.2 364.1 5 593.3 491.6 6 525.7 409.8 7 440.3 340.9 8 526.3 653.8 9 360.1 408.6 10 490.9 356 11 315.1 291.6 12 422.9 399.1 13 463.9 376 14 379.6 542.4 15 395.3 314.2

289