An in Vitro and in Vivo Study of Fluoride Uptake by Dentine Following Application of Various Topical Fluoride Regimens

AN IN VITRO AND IN VIVO STUDY OF FLUORIDE UPTAKE BY DENTINE FOLLOWING APPLICATION OF VARIOUS TOPICAL FLUORIDE REGIMENS

Nicola Jane Woodley

A thesis submitted for the degree of Doctor of Philosophy in the Faculty of Dentistry University of London

Departments of Biomaterials and Prosthetic Dentistry Eastman Dental Institute for Oral Health Care Sciences

-1999- ProQuest Number: U641832

INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted.

In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest.

ProQuest U641832

Published by ProQuest LLC(2015). Copyright of the Dissertation is held by the Author.

ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT

Restoration of severely worn dentitions frequently involves the use of overlay dentures. Such treatment can lead to the rapid development of caries. Topical fluoride regimens, including sodium fluoride, amine fluoride or stannous fluoride, have been used to reduce this risk. Sodium fluoride is regarded as effective but the other two compounds to be evaluated have benefits such as deposition of acid insoluble salts on the tooth surface. However stannous fluoride can be unstable and it has been suggested that amine fluoride/stannous fluoride combinations may be more effective.

This study investigated the three fluoride containing compounds both alone and in combination to measure the effects on the fluoride content of dentine both in vitro and in vivo. The effect in vivo on caries incidence and on bacteria flora in plaque and saliva (Appendix 7) in patients wearing overlay dentures was also evaluated.

Techniques for biopsy of dentine were assessed and a novel method was developed and evaluated for harvesting dentine in vitro and in vivo for fluoride analysis. The in vitro study examined the uptake of the three materials in paste and rinse, gel or solution.

The results showed that all the regimens significantly increased the fluoride content of dentine except an amine fluoride/stannous fluoride (AmF/SnF:) gel. SEM analysis of the treated dentine surface showed deposition of solid material except following the

AmF/SnF2 gel. The latter behaved inconsistently. This suggested that complexing of the fluoride ion might occur.

The in vitro study also found that demineralised dentine took up fluoride ions but in this instance the AmF/SnF: gel produced a greater fluoride uptake.

The in vivo investigation showed that in a double blind trial using patients wearing overlay dentures, biopsied dentine samples exhibited significantly increased fluoride levels following sodium fluoride and Amf/SnF 2 paste and rinse regimens. No statistical difference was apparent between the treatments.

The use of the products in vivo resulted in a significant increase in new cavitated carious lesions in patients using the AmF/SnF2 regimen compared to the sodium fluoride regimen. The three monthly evaluation of plaque and saliva samples indicated no statistical difference in bacterial counts between the treatments. DECLARATION

I hereby declare that the work embodied in this thesis is the result of my own investigations except where otherwise stated.

The plating of plaque and saliva samples and counting of colony forming units that formed part of the investigation in Appendix 7 were carried out by Dr Tracy Burns and Mr Paul Bhuvanenthiran of the Department of Microbiology, Eastman Dental Institute for Oral Health Care Sciences, 256 Gray’s Inn Road, London. ACKNOWLEDGEMENTS

I would like to thank Professor Gavin Pearson and Dr Brigitte Griffiths for their support, guidance and encouragement not only during the supervision of this thesis but over the last decade. You have been true mentors.

Thank you also to Dr Bieri and his colleagues at GABA International Ltd. both for their financial sponsorship of this project and for their willing and rapid supply of information and materials.

My gratitude for their generous advice and support goes also to:

Professor Hubert Newman Professor Mike Wilson Dr Tracy Burns and Mr Paul Bhuvanenthiran - thank you for your patience and all your hard work My colleagues in the Biomaterials Department - especially Mr Ted Davies and Mr Graham Palmer My colleagues in the Prosthetics Department

Finally, many, many thanks to Pamela, Jim and Caroline Woodley, Joanne Hill, Claire Price, Ruth, William and Alison Hooper and especially to David, Thomas, Robert and Sophie Hooper. I am truly grateful for your help and forbearance. INDEX OF CONTENTS

ABSTRACT...... 2 DECLARATION...... 3 ACKNOWLEDGEMENTS...... 4 INDEX OF CONTENTS...... 5 LIST OF FIGURES...... 9 LIST OF TABLES...... 11 STRUCTURE OF THESIS...... 13

CHAPTER 1 LITERATURE REVIEW...... 14 1. The Management of Tooth Wear with Removable Partial Dentures ...... 15 2. The Incidence of Caries in Patients Wearing Dentures to Restore Tooth WearlS 3. The Development of Topical Fluorides ...... 29 4. The Development of Amine Fluoride, Stabilised Stannous Fluoride and Amino- Stannous Fluoride Preparations...... 38 4.1 The Development of Amine Fluorides Preparations ...... 38 4.2 The Development of Stabilised Stannous Fluoride Preparations ...... 42 4.3 The Development of Preparations Combining Amine and Stannous Fluorides...... 44 5. The Anti-Caries Activity of Stannous Fluorides, Amine Fluorides and Amino- Stannous Fluoride Regimens ...... 46 5.1 Inhibition of Demineralisation ...... 46 5.2 Promotion of Remineralisation ...... 48 5.3 The Formation of Calcium Fluoride ...... 49 5.4 Promotion of Fluoride Uptake by Dental Mineralised Tissues ...... 54 5.5 Surfactant Activity of Amine Fluorides ...... 59 5.6 Anti-Microbial Properties...... 60 5.7 Caries Inhibition...... 72 6. The Measurement of Fluoride Content of Dental Mineralised Tissues ...... 78 6.1 Techniques for Biopsy of Dental Mineralised Tissues ...... 78 6.2 Techniques for the Analysis of the Fluoride Content of Dental Mineralised Tissues...... 89 6.3 The Analysis of the Fluoride Content of Dentine ...... 96 6.4 Altemative Approaches to the Analysis of the Fluoride Content of Dental Mineralised Tissues...... 96 7. Statement of the Problem ...... 97 8. Aims and Objectives ...... 9^ CHAPTER 2 MEASUREMENT OF THE FLUORIDE CONTENT OF DENTINE DEVELOPMENT OF METHOD...... 100 1. Preparation and Storage of Teeth for In Vitro Investigations...... 101 2. Biopsy Techniques...... 103 2.1. An Evaluation of the Use of Trephines to Biopsy Dentine ...... 104 2.2. Evaluation of the Use of a Diamond Bur to Biopsy Dentine In vitro...... 105 3. Methods of Fluoride Analysis...... 109 3.1 Dissolution of Dentine in One Molar Hydrochloric Acid ...... 109 3.2 Analysis of Fluoride...... 112 4. Topical Fluoride Preparations ...... 116 5. Statistical Analysis...... 122

CHAPTER 3 MEASUREMENT OF FLUORIDE UPTAKE BY DENTINE FOLLOWING TOPICAL FLUORIDE TREATMENTS IN VITRO...... 123 1. Assessment of the Effect of Variation in Specimen Thickness on the Fluoride Content of Dentine ...... 124 2. Measurement of Fluoride Uptake by Dentine In Vitro...... 128 3. Weight Loss on Demineralisation of Carious Dentine ...... 135 4. Measurement of the Fluoride Content of the Organic Dentine Remaining after Mineral Dissolution in Hydrochloric Acid...... 138 5. The Effect of Storage in Water on the Fluoride Content of Dentine Biopsies. 142

CHAPTER 4 FLUORIDE UPTAKE BY DENTINE IN VIVO FOLLOWING UNSUPERVISED USE OF TOOTHPASTE AND DAILY MOUTHRINSE FOR ONE MONTH 146 1. Preliminary Investigation of the Measurement of Fluoride Uptake by Dentine In Vivo...... 147 2. Fluoride Uptake by Dentine In Vivo Following Unsupervised Use of Toothpaste and Daily Mouthrinse for One Month ...... 154 CHAPTER 5 THE EFFECT OF UNSUPERVISED USE OF TOOTHPASTE AND DAILY MOUTHRINSE FOR ONE YEAR ON CARIES INCIDENCE...... 160

CHAPTER 6 THE USE OF FLUORIDE GELS TO PROMOTE FLUORIDE UPTAKE 176

CHAPTER 7 INVESTIGATIONS INTO THE MODE OF ACTION OF AMINO-STANNOUS FLUORIDE REGIMENS...... 184 1. Assessment of Fluoride Uptake by Dentine Following Application of Sodium Fluoride, Amine Fluoride 297 and Stannous Fluoride Solutions ...... 185 2. Fluoride Uptake from Amine Fluoride Compounds in the Form of Solutions and Gels...... 190 3. An SEM Study into the Effect of Fluoride Gels and Solutions on the Dentine Surface Morphology ...... 195

CHAPTER 8 INVESTIGATIONS INTO UPTAKE OF FLUORIDE BY DEMINERALISED DENTINE...... 208 1. Following Application of Fluoride Gels ...... 209 2. Following Application of Fluoride Solutions...... 215

CHAPTER 9 A SUMMARY OF THE FINDINGS OF THESE INVESTIGATIONS...... 219

BIBLIOGRAPHY...... 226 Appendix 1. The Prevalence and Incidence of Caries Developing in Patients Wearing Overdentures ...... 257 Appendix 2. Fluoride Uptake by Dental Mineralised Tissues following Application of Amine Fluoride, Stannous Fluoride and Meridol Regimens ...... 267 Appendix 3. Clinical Trials on Caries Incidence following Topical Application of Amine Fluoride, Stabilised Stannous Fluoride and Meridol Regimens...... 292 Appendix 4. Statistical Analyses...... 305 Appendix 5. Results from Chapters 3 to 8 ...... 321 Appendix 6. Patient Information and Consent Forms ...... 340 Appendix 7. The Effect of Unsupervised Use of Toothpaste and Daily Mouthrinse for One Year on the Oral Microflora ...... 349 LIST OF FIGURES

Figure 1. Chemical Formulae of Amine Fluoride Compounds in Common Use ...... 41 Figure 2. Diagram of Sectioning Procedure to Yield Four Sections of Contiguous Occlusal Dentine ...... 102 Figure 3. Diagram showing dissection of an occlusal dentine specimen from the inner right- angled comer of a tooth section ...... 107 Figure 4. Disposable template constructed from two pieces of 0.5mm wide stainless steel rectangular arch wire ...... 107 Figure 5. Frequency distributions of the surface area and depth of the dentine specimens when dissected with a diamond bur...... 108 Figure 6. Weight loss on demineralisation of samples of dentine in 1M Hydrochloric Acid. .111 Figure 7. The differential fluoride electrode cell used for fluoride analysis ...... 113 Figure 8. The microprocessor ionanalyser used for fluoride analysis ...... 113 Figure 9. Dissection of dentine specimens of varying depth ...... 125 Figure 10. Fluoride Content of Dentine Specimens of Varying Thickness Following Application of 1.25% Fluoride Solutions...... 126 Figure 11. Fluoride uptake by dentine following topical application of toothpaste and mouthrinse in vitro...... 131 Figure 12. Percentage weight loss on déminéralisation of samples of dentine in 5 ml 1 M hydrochloric acid...... 136 Figure 13. Fluoride content of the organic portion of dentine following dissolution in 1M hydrochloric acid after topical fluoride treatments with Meridol and sodium fluoride toothpaste and mouth rinse...... 141 Figure 14. Fluoride loss from dentine specimens into storage water at one day, one week and one month after topical application of toothpaste and mouthrinse in vitro 145 Figure 15. Fluoride uptake by dentine foilowing topical application of Meridol and sodium fluoride toothpaste and mouthrinse in vivo. Uptake by abutment and by patient is evaluated ...... 158 Figure 16. Examples of abutments where changes to the dentine were scored as developing new “colour change" lesions ...... 167 Figure 17. Percentage caries incidence following unsupervised use of Meridol and sodium fluoride toothpaste and mouthrinse for 12 months ...... 171 Figure 18. Fluoride uptake of dentine following exposure to fluoride gels (1.25% F) in vitro. 180 Figure 19. Fluoride uptake of dentine following exposure to fluoride solutions in vitro...... 188 Figure 20. Fluoride content of dentine foiiowing exposure to amine fluoride compounds in vitro...... 193 Figure 21. Diagram of identification mark and groove to aid fracture cut into the dentine specimen prior to fluoride treatment and SEM analysis ...... 196 Figure 22. SEM of the surface of untreated dentine ...... 199 Figure 23. SEM of the surface of dentine treated with Meridol gel (1.25% F I ...... 199 Figure 24. SEM of the surface of dentine treated with NaF gel (1.25% F ) ...... 200 10

Figure 25. SEM of the surface of dentine treated with Elmex gel (1.25% F I ...... 200 Figure 26. SEM of the fractured surface of dentine treated with Meridol gel (1.25% F-) ...... 201 Figure 27. SEM of the fractured surface of dentine treated with NaF Gel (1.25% F ) ...... 201 Figure 28. SEM of the fractured surface of dentine treated with Elmex Gel (1.25% F ) ...... 202 Figure 29. SEM of the fractured surface of dentine treated with Elmex Gel (1.25% F ) ...... 202 Figure 30. SEM of the surface of dentine treated with AmF 297 solution (1.25% F*) ...... 204 Figure 31. SEM of the surface of dentine treated with SnFz solution (1.25% F ) ...... 204 Figure 32. SEM of the surface of dentine treated with NaF solution (1.25% F I ...... 204 Figure 33. SEM of the fractured surface of dentine treated with AmF297 solution (1.25%F). 205 Figure 34. SEM of the fractured surface of dentine treated with SnF: solution (1.25% F ) ....205 Figure 35. SEM of the fractured surface of dentine treated with SnFz solution (1.25% F ) .... 205 Figure 36. Fluoride content of the demineralised dentine foiiowing application of fluoride gels (1.25% F ] in vitro. Fluoride released by potassium hydroxide ...... 212 Figure 37. Fluoride content of the demineralised dentine following application of fluoride gels (1.25% F*) in vitro. Fluoride released by hydrochloric acid ...... 213 Figure 38. Fluoride content of the demineralised dentine following application of fluoride solutionsin vitro...... 217 Figure 39. Summary of the fluoride uptake by dentine following the topical regimens evaluated in these investigations ...... 225 11

LIST OF TABLES

Table 1. The fluoride compounds currently in Elmex products (Gaba Intemational Ltd., Switzerland) ...... 42 Table 2. Methods of dissecting enamel by abrasion ...... 79 Table 3. Methods of dissecting enamei by acid dissolution...... 83 Table 4. Altemative approaches available for the determination of fluoride in biological materials (adapted form Venkateswariu, 1977,1990) ...... 90 Table 5. Fluoride Preparations Applied in the Fluoride UptakeInvestigations ...... 117 Table 6. Ingredients of toothpaste and mouthwash (as provided by GABAIntemational) used the in vitro and in vivo investigations...... 118 Table 7. Ingredients of the topical fluoride gels and solutions (as provided by GABA Intemational) used in the in vitro investigations...... 119 Table 8. Tests used for statistical analysis of data...... 122 Table 9. Summary of specimen dimensions, fluoride content and fluoride uptake following application of toothpaste and mouthrinse in vitro to "non-carious" and "carious" dentine ...... 130 Table 10. The weight of the dentine samples prior to and following demineralisation in 1M hydrochloric acid...... 136 Table 11. Fluoride content of Inorganic and organic solutions of dentine following topical fluoride treatments with Meridol and sodium fluoride toothpaste and mouth rinse. 140 Table 12. Fluoride loss from dentine specimens into storage water at 1 day, 1 week and 1 month after topical application of toothpaste and mouthrinse in vitro...... 144 Table 13. Patient details, specimen dimensions, fluoride content and fluoride uptake following topical application of Meridol and sodium fluoride toothpaste and mouthrinse 150 Table 14. Number and description of comments made by patients conceming the flavour of the toothpaste and mouthrinse...... 155 Table 15. Summary of patient age, time regimen used, specimen dimensions, fluoride content and fluoride uptake following topical application of Meridol and sodium fluoride toothpaste and mouthrinse...... 156 Table 16. Age and sex of the patients and the fluoride regimen provided ...... 164 Table 17. Summary of the number of patients, number of teeth, number of abutments considered, caries score at baseline and new lesions recorded over 12 months following unsupervised use of Meridol and sodium fluoride toothpaste and mouthrinse...... 170 Table 18. The sample dimensions, fluoride content and fluoride uptake of dentine samples following exposure to fluoride gels (1.25% F I in vitro...... 179 Table 19. The sample dimensions, fluoride content and fluoride uptake of dentine samples following exposure to fluoride solutions (1.25% F*) in vitro...... 187 Table 20. The sample dimensions and fluoride content of dentine samples following exposure to fluoride solutions or gels (1.25% F") in vitro...... 191 12

Table 21. T opical fluoride preparations applied to the dentine prior to SEM examination 196 Table 22. Groups established for the investigation into fluoride uptake by demineralised dentine following exposure to fluoride gels ...... 210 Table 23. Fluoride uptake by demineralised dentine following application of fluoride gels (1.25% F ) in vitro (fluoride released by exposure to potassium hydroxide) 212 Table 24. Fluoride uptake by demineralised dentine following application of fluoride gels (1.25% F*) in vitro (fluoride released by exposure to hydrochloric acid, remaining fluoride released by potassium hydroxide) ...... 213 Table 25. Fluoride uptake by demineralised dentine following application of fluoride solutions in vitro...... 217 13

STRUCTURE OF THESIS

The thesis is structured so that the first chapter is concerned with the literature, the second chapter outlines the materials and methods common to the studies of the thesis and the third develops the method for use particular to these studies. Investigations described in Chapters 4 to 8 refer to these general methods but otherwise stand alone, each with their own introduction, method, results, discussion and conclusion. Chapter 9 gives a summary of the findings for the whole thesis and proposes areas for future study. 14

CHAPTER 1.

LITERATURE REVIEW. 15

1. The Management of Tooth Wear with Removable Partial Dentures.

The restoration and maintenance of aesthetics and function in patients with severely worn dentitions presents a challenge and a number of treatment options are available.

1. Extraction of Teeth and Replacement with Dentures. This option exists for the patient who does not wish to receive more complex treatment or whose teeth are beyond restoration (Williams, 1987; Smith, 1989). The main disadvantage of this treatment is that the patient is likely to expose the denture-bearing tissues to excessive and damaging forces (Barsby, 1988). Many patients in this situation have a poor tolerance of dentures (Hemmings et a/., 1995).

2. Restoration with Complete or Partial Onlay, Overlay or Overdentures. Onlay dentures have been described as covering the occlusal or incisal surfaces of the teeth, overlay dentures as covering the teeth with a full labial veneer facing and overdentures as covering the teeth with a prosthetic teeth and an acrylic flange (Hemmings et al., 1995). A denture restoring advanced tooth wear may contain one or a combination of these designs depending on:

. the amount of tooth tissue lost • the degree of tooth or alveolar tissue undercut and hence the path of insertion of the denture • the aesthetic demands on the denture • the suitability of teeth remaining for retention of the denture.

The techniques have been comprehensively described (Winstanley 1984; Badr and Unger, 1986; Gankaseer, 1987; Barsby, 1988; Ralph and Basker, 1989; 16

Hemmings et a/., 1995) although evidence based data on the management of tooth wear with removable partial dentures is not available.

3. Restoration of Anterior Teeth using Crowns or Veneers, a Removable Partial Denture Providing Posterior Occlusal Support. (Mack and Allan, 1968; Winstanley, 1984). The main disadvantage of this treatment option becomes apparent if the patient does not wear the denture for a period of time when the anterior teeth are subjected to all of the occlusal loads. Even if the partial denture is worn continuously regular refurbishing of fitting and occlusal surfaces are required for it to remain effective (Hemmings at a/., 1995)

4. Restoration with Conventional Crowns. This is the option most requested by patients (Hemmings at a/., 1995) but requires demanding clinical and laboratory skills (Ibbetson and Setchell, 1989a; Johannson and Omar, 1994). Factors influencing the decision to opt for fixed rather than removable restorations include the amount of tooth structure that remains, the distribution, number of teeth and amount and site of tooth structure remaining, the cost and technical support available (Barsby, 1988; Dahl at a/., 1993; Hemmings at a/., 1995). It may also be dependant on the patient motivation and the skill and experience of the operator.

5. Restoration with Adhesive Restorations. The use of gold, nickel/chrome or composite palatal backings have been described for anterior teeth (Foreman, 1988; Bishop at a/., 1994) and may be used to gain interocclusal space (Darbar and Hemmings, 1997). Adhesive onlays constructed from porcelain, nickel/chrome and gold (Rawlinson and Winstanley, 1988; Foreman, 1988; Harley and Ibbetsson, 1993) have been used for posterior teeth. The long term success of these restorations is yet to be confirmed (Bishop et al., 1994; Darbar and Hemmings, 1997).

Regardless of the treatment modality, the identification and elimination of causative factors of tooth wear is universally agreed to be the first step in management (Barsby, 1988; Johannson and Omar, 1994; Bishop at al., 1994; Hemmings at a/., 1995). This may not be easy. Smith and Knight (1984) found 17

that 31 out of 100 patients could not be confidently allocated to an aetiological group and Kidd and Smith (1993) found that some causes of tooth wear are difficult and embarrassing to discuss. However, medical referral for treatment for acid regurgitation and preventive measures including dietary advice, oral hygiene instruction, topical fluoride application, use of alkaline solutions, sugar free gum or bicarbonate containing toothpaste to raise intra-oral pH and provision of a nocturnal hard acrylic splint might be instigated (Kidd and Smith, 1993; Bishop ef a/., 1994; Imfeld, 1996; Smith ef a/., 1997; Milosevic, 1998).

Before the definitive management of tooth wear is considered, ideally a diagnostic or provisional appliance should be provided (Turner and Missirian, 1984; Barsby, 1988; Ibbetson and Setchell, 1989b; Hemmings et al., 1995). This reduces the effect of bruxing on further tooth wear and allows patient and clinician to assess:

• the appearance of prosthetic teeth • lip support • the occlusal vertical dimension, antero-posterior relationship and occlusal scheme • patient tolerance and motivation to denture wearing • the durability of denture design

A variety of provisional appliances may be used ranging from occlusal splints to removable partial dentures that cover the worn teeth (Barsby, 1988). It is considered unwise during provisional treatment to undertake extensive and irreversible tooth preparation, especially if the patient has no history of successful denture wearing (Hemmings ef a/., 1995).

Following successful provisional therapy, restoration of the severely worn dentition with definitive partial dentures may be considered. Denture construction is carried out conventionally but with reference to parameters established by the provisional appliances. However, in addition to satisfying the patient's aesthetic and functional demands, the definitive treatment needs to provide a durable denture (Hemmings at a/., 1995). To facilitate this tooth preparation may be necessary to make space for prosthetic materials and to 18

remove unfavourable survey lines. If successful denture wearing has been achieved at the provisional stage, tooth preparation can be carried out with greater confidence.

Aesthetically pleasing dental materials do not perform well in thin section and a minimum thickness of acrylic of 2 to 3 mm is required to give reasonable service for patients with tooth wear (Hemmings et al., 1995). Tooth preparation may therefore be needed to avoid excessive bulk of minor connectors and occlusal interference. Sharp changes in contour also have a detrimental effect on these materials and preparation of the tooth to remove sharp or thin edges and to provide smooth contours for denture components might be considered. The use of acrylic veneer facings, which give a more pleasing appearance than onlays that butt fit to the natural teeth, may not be possible unless tooth preparation allows a path of insertion to the gingival margin (Hemmings et al. 1995).

The necessity for tooth preparation in patients with advanced tooth wear for whom partial dentures are constructed gives scope for biopsy of enamel and particularly dentine. This allows examination of these mineralised tissues following clinical trials of therapeutic agents.

Following provision of dentures, it is recognised that the outcome of treatment is dependent on a high level of maintenance (Woodley et al., 1996) but there is little in the dental literature on the sequela following the management of tooth wear with removable partial dentures. However, worn teeth with exposed dentine when covered by a denture might be supposed to be susceptible to caries. 19

2. The Incidence of Caries in Patients Wearing Dentures to Restore Tooth Wear

Hussey and Linden, 1986 reviewed caries incidence following overdenture construction to restore tooth wear (Appendix 1,Table 1). The authors surveyed 40 patients, aged 15 to 72 (average 45 years) and a ratio of males to females of 3 to 1. 36 partial and 9 complete overdentures were worn that covered 165 abutments. 25% of the abutments had been root-filled, 50% of the root surfaces had not been restored and 13% were covered with castings. The dentures had been constructed over 6 years, but while 27% of the abutments had been covered for more than 3 years when surveyed 33% had been covered for less than 1 year. All the patients had been instructed to use supplemental fluoride but only 25% had continued to do so. The fluoride regime recommended to the patients was not recorded but the authors recommended the sucking of a 1 % fluoride tablet last thing at night as a method of topical application of fluoride likely to yield high compliance.

On examination 22% of the abutments were affected by caries (recorded if a sharp probe detected softening of any surface of the abutment) and the incidence increased with the duration of overdenture wear; from 13% in those wearing their dentures for less than 1 year to 27% in those wearing them for more than 2 years. The incidence was found to decrease with the use of fluoride; 15% of the abutments were carious in those who used supplemental fluoride compared with 23% of abutments in patients using fluoride toothpaste only and 40% of abutments in those using no fluoride at all. The patients in this latter group were using desensitising toothpaste and so were presumably similarly motivated to those forming the middle group.

The authors suggest that regular recall is essential to ensure that patient's home care is adequate and caries activity is controlled. But the treatment philosophy of tooth wear patients treated with overdentures should not be based on a single longitudinal study. Gomes and Renner (1990) state that it is logical to speculate that the sequelae to the wearing of removable partial dentures covering worn tooth surfaces would be similar to those observed in patients treated with complete overdentures with regard to abutment teeth and 20 roots. Several studies have investigated the incidence of caries affecting abutment teeth overlaid with complete dentures. A summary of the findings is presented in Appendix 1, Table 2. It is difficult to directly compare these studies due to differing populations and experimental protocols.

The majority of cross sectional studies concern abutments that have been restored with gold copings or precision attachments and reported caries prevalence ranging from 10 to 39% of abutments. In longitudinal investigations the incidence of new carious abutments varied widely with a range of annual caries incidence in abutments of 0 to 40%. A zero incidence was achieved in the fifth to tenth year of overdenture wearing in patients using a 1% sodium fluoride gel daily in their dentures (Toolson and Taylor 1989). It could be argued that these patients must have been highly motivated to continue with ten years of recalls. But the highest annual incidence of carious lesions reported (Laudello and Ciancio 1985) was found amongst 25 patients wearing their overdentures for the sixth year despite daily brushing with 0.4% stannous fluoride gel. These patients could be considered to be similarly motivated.

New caries can develop in a very short period of time; Toolson and Smith (1978) found 29.2% of 233 abutments in 89 patients developed lesions within 8 months and Ralph and Murray (1967) found 2 of their 6 patients developed caries within 15 months.

Most of the authors did not report on tooth loss, but those who did (Appendix 1, Table 3) found the number of abutments extracted due to caries, expressed as a percentage of the total number of abutments utilised by the dentures varied, from 0.8% (Reitz et al., 1977) to 15% (Waas et a/., 1996). So although caries can develop rapidly and attain high incidence the number of teeth extracted due to caries generally has been reported to be low.

Ettinger and Jakobsen (1990) tested a regression model for the 254 individuals in their 12-year longitudinal study in an attempt to determine which factors or covariates measured at each recall visit might relate to caries. Several independent variables were identified as the best predictors of caries, including reduced frequency of brushing, non-use of home fluorides, reduced severity of 21

periodontal problems in the mandibular arch, drugs which may cause neural side-effects and the presence of cardiovascular disease. The authors did not provide an adequate explanation for the finding that the fewer periodontal problems the patients had, the more likely they were to be at risk for caries. But they do point out that the organisms responsible for root caries are very different from those causing chronic periodontitis. Further, although only two of the medicines and diseases evaluated showed a relationship with the development of caries, root surface caries has been found to be associated with a variety of diseases (Beck et a/., 1987). The authors speculated that the aetiology of root caries might involve changes in the general health including depression of local specific immune responses, which may manifest as changes in oral flora related to an increased risk of root caries. Although the specificity of the analysis was very high (98.5%) the sensitivity was very low (7.9%) therefore the model was unable to predict those persons who were in the risk category, although they could identify those who would not get caries. Furthermore, factors known to have a potential effect on root caries, such as rate of salivary flow and snacking habits, were not collected.

Several preventive measures are highlighted by these cross-sectional and longitudinal studies and by clinical trials on this group of patients. These include:

• mechanical plaque control . chemical plaque control . denture wearing habits • topical fluoride regimens . frequent recalls.

Mechanical Plague Control

Many studies have investigated possible relationships between enamel caries and oral cleanliness and a concensus of prevalence and retrospective studies (Sutcliff, 1983) suggests a weak positive association between plaque and caries. Investigations into this relationship, where other preventive agents such as fluoride are absent, have not been performed for root caries or for caries of 22 overdenture abutments. However, it is likely that denture wearing may increase plaque pathogenicity by enhancing anaerobic conditions and by cutting off free access of saliva with its buffering capacities and antibacterial systems including antibodies (Budtz-Jorgensen, 1991).

The composition of plaque beneath dentures has been found to be similar to plaque located at the dento-gingival areas and therefore associated with caries and gingivitis (Theilade et a/., 1982, 1983). Ten-fold higher bacterial counts have been observed in samples from the denture surface compared with underlying oral mucosa (Gusberti et a/., 1985). This suggests good oral (and denture) hygiene may be of more importance in the overdenture wearer. Indeed, several links between plaque control and caries of the abutment teeth are noted in these studies: Renner et al. (1984) noted a high incidence of new caries lesions, 35.7% of abutments in the second to fourth year of overdenture wear despite use of 1 % sodium fluoride gel in the denture thrice weekly. They also found that at no time during the study were any of the abutments clinically free of dental plaque. Hussey and Linden (1986) found that 58% of abutments displayed obvious plaque on at least one surface, and plaque increased with duration of denture wear and that this was mirrored by an increase in caries incidence. Furthermore Toolson et al. (1982) found that the overdenture abutments that developed caries between the initial recall examination and the 1 -year recall had elevated plaque scores when compared to the teeth free of caries. Toolson and Smith (1983) also found that at five years the plaque control of patients motivated to continue with the fluoride regime was superior compared to the group of patients who elected not to use the fluoride solution. The incidence of carious abutments was found to be respectively 2.8% and 21.3%, but the relative importance of plaque control and the use of fluoride could not be separated. Fenton and Hahn (1978) concluded from their study that while patients demonstrated large variations in their ability to maintain low plaque levels, only those who did not apply fluoride to retained tooth root surfaces developed a high incidence of caries.

Ettinger and Jakobsen (1990) did find a reduced frequency of brushing to be a predictor of caries. However, the quality of brushing is not necessarily reflected by its frequency and it may be another factor, such as an increased frequency 23

in the use of fluoride toothpaste that influenced the result.

So, while there is no unequivocal evidence that good oral hygiene reduces caries experience neither is there evidence to dismiss its value. Furthermore good oral hygiene can result in the regular and frequent use of fluoride toothpaste which is known to be capable of reducing the incidence of dental caries.

Chemical Plague Control

Keltjens et al (1990) studied the effect of fluoride and chlorhexidine-fluoride regimens on caries incidence in a group of overdenture wearers. 30 patients (19 women, 11 men) mean age 55 years (34 to 75 years) entered and 26 patients completed the 18-month study. The patients were randomly distributed into 3 groups; The control group applied a daily placebo gel and had a twice yearly professional application of 0.4% NaF gel. The first test group applied 0.1% NaF gel daily putting a drop of the gel in the prosthesis at every abutment site and inserting the denture for 30 minutes once a day. The second test group had a 5 minute professional application of 5% chlorhexidine gel applied 1 week after insertion of the denture and then applied a daily gel containing 1 % chlorhexidine and 0.1% sodium fluoride in the manner described above. All the patients received the same dental treatment and oral hygiene instructions. The gel treatments started one week after insertion. The root caries index according to Katz (1980) was used to score the teeth at baseline and at control visits.

In the control and fluoride groups the caries incidence was found to be 34 and 36% respectively and was not statistically significantly different. But no caries was observed in the chlorhexidine-fluoride group and this was statistically significant compared to the other groups. Survival curves giving the percentage of patients with caries-free abutments during the evaluation period showed no statistically significant difference between the control and fluoride groups but significantly more patients in the chlorhexidine-fluoride group. A chlorhexidine- fluoride gel was therefore found to be very effective in preventing the development of caries in overdenture abutments. Furthermore compliance was high suggesting patients found the gel pleasant and easy to use. But further 24

Study with larger numbers of patients and of a greater length are required to confirm these findings.

Based on the longitudinal studies quoted earlier one would not have expected the fluoride gel to be without effect on caries incidence in comparison with the control group. But this may be due to the low concentration of fluoride in the gel used which was one tenth the strength of that used in other studies.

It should be noted that Barkvoll et al. (1988) have shown that chlorhexidine digluconate and sodium monofluorophosphate are not compatible in equal concentrations, as chlorhexidine difluorate salt forms which is insoluble. The authors state that the gel formulation used for the investigation had, even after complete binding of fluoride, a high concentration of free chlorhexidine remaining, but this could mean that fluoride is not available, and that only the effect of chlorhexidine is being study.

Denture Wearing Habits

Budtz-Jorgensen (1991) conducted a 3-year survey of 35 patients (22 men and 13 women), mean age 68.5 + 10.7 years (range 51 to 81 years) provided with 38 overdentures covering 80 abutments (1 to 4 abutments in each jaw). The patients were instructed to leave their dentures out at night but only 15 (43%) who completed the 3 year survey followed this advice. The patients were recalled every 6 months for oral hygiene instruction and topical fluoride application (regime not given). Active and inactive root caries was recorded (Fejerskov at a/., 1991).

Annually new lesions were seen in approximately 30% of the abutments of patients wearing their dentures day and night, whereas no caries developed in patients wearing their dentures in the day only. This difference was significant. 31 of the patients who retained 72 abutments were followed up at 5 years (Budtz-Jorgensen 1994). Over this period 40 lesions were found in the group of day and night wearers against 3 in the day wearers.

Day and night wearing of dentures then appears to be a major caries risk factor 25

in complete overdenture wearers with controlled oral hygiene. It is likely that continuous denture wearing may further increase the pathogenicity of the plaque by continuance of anaerobic conditions and by eliminating free access of saliva. Leaving overdentures out at night is then to be encouraged, although provision of a nocturnal hard acrylic splint, preferably in the opposite arch to the overdenture, may be required to protect the teeth of the nocturnal bruxer from further wear.

Topical Fluoride Regimens.

Differing experimental protocols make it impossible to compare individual studies, although trends may be established. The early cross-sectional studies in which a fluoride regime was not established certainly showed a high prevalence of caries at recall, ranging from 10% of two year restorations (Dolder 1961) to 39% of abutments (Ratenen et a/., 1971). Ralph and Murray (1967) found 2 of their 6 patients developed caries despite a one off application of fluoride to the abutments following contouring.

Longitudinal studies show wide variation in findings. For example, in the study by Davis at a/., (1981) without a fluoride regime the 2 year caries incidence was 20%, whereas in those studies where fluoride was encouraged the caries rate varies from 0% in the fifth to tenth year (Toolson and Taylor 1989) to 40% in the sixth year of overdenture wearing (Lauciello and Ciancio 1985).

More information can be gained from studies including the "partial" use of fluoride. Toolson and Smith (1978) introduced the partial use of a 1% sodium fluoride solution placed in the denture daily one year into the study. The caries rate then dropped from 34.8% at one year to 19% at two years. When groups using the fluoride rinse and those using a placebo rinse were compared the effect of the topical application daily of a 1 % neutral sodium fluoride solution on the prevention of caries was found to be statistically significant. The fluoride regime was then established for all the patients and at the 5 and 10 year recalls those patients continuing to use the fluoride continued to have statistically significantly fewer lesions. It could, however, be argued that those patients who had continued using the rinse were the better motivated patients and were 26 therefore also more diligent at following oral hygiene or dietary instructions.

All the patients surveyed by Fenton and Hahn (1978) were instructed in the daily use of a fluoride gel. Unfortunately compliance was low, but 21.6% of the abutments not exposed to fluoride developed caries compared to 5.0% of the abutments exposed to fluoride. Again the patients continuing to use the fluoride gel were presumably more motivated and other factors may therefore also have been at work. Interestingly none of the control teeth developed caries. The control teeth were found in only 10 of the 17 patients which may have biased the results, but it does suggest that the patient wearing an overdenture is at high risk of caries.

In contrast, Reitz et al. (1977) found 2 of 9 patients prescribed a daily 2% sodium fluoride mouthrinse developed caries compared with 6 of the 41 patients not so prescribed. The small and disparate sample sizes together with the likelihood that the prescription of the rinse to just some of the patients was presumably done with good reason, suggests that the difference in caries rate cannot safely be attributed to the use of fluoride alone.

Renner at ai. (1984) found a higher caries rate (35.7%) at the fourth year of overdenture wearing following the use of a 1% sodium fluoride gel In the denture thrice weekly, compared to caries incidence (20%) in the second year of denture wearing without fluoride. Patient compliance was not recorded. Also the small sample size means that the loss of 3 of the 11 patients to the study at 4 years makes comparison of the results rather meaningless.

Keltjens et al. (1990) found caries inhibition by daily denture application of 0.1% sodium fluoride gel to be insignificant compared to a control group although the concentration of fluoride in the gel was low (by a factor of 10) compared to that used in other studies. However, Ettinger and Jakobsen (1990) found that use of a daily fluoride gel resulted in remineralistion of a carious lesion within 2 weeks.

Given the strong evidence of caries prevention by fluoride it is unlikely that fluoride is ineffective in patients wearing overdentures. However controlled clinical trials are needed, not only to establish the effectiveness of fluoride in 27 this group but also the most effective regime.

An alternative approach to delivery of topical fluoride was investigated by Yamaga and Nokubi (1997). Overdentures were made from a trial resin incorporating a tannin-fluoride resin. These were found to have a caries preventive effect over an 8 year period although the number of abutments investigated was small, particularly in the control group. This approach has a number of advantages, not least in eliminating the need for patient co-operation.

Frequent Recalls

The early appearance of caries in some studies (Ralph and Murray, 1967; Toolson and Smith, 1976) and the continuing high annual incidence in others despite the use of fluorides (Ettinger and Jakobsen, 1990; Lauciello and Ciancio, 1985) suggests frequent recall examinations are important. Regular recall of patients is essential to ensure the patient's home care is adequate, to encourage the use of topical fluorides and to control caries activity. Indeed Rantanen et al. (1971) states that, since only some patients are capable of maintaining adequate oral hygiene, overdentures should be confined to those who may reasonably be expected to clean the dentures properly and who promise to make regular follow-up visits.

There is a danger, however, in translating data from patients who are candidates for complete dentures to those who are wearing overdentures to restore tooth wear. Those wearing complete overdentures have very few of their remaining teeth left suggesting they have predisposition to dental disease (Rantanen at a/., 1971). Presumably they have not excelled in their plaque control and denture hygiene (Prieskel, 1985). This is not necessarily the case for those candidates for partial tooth coverage due to tooth wear. Indeed clinical impression is of a group with good bone levels and relatively few restorations beyond those restored for tooth wear (Woodley at a/., 1996). This suggests translation of the data from one group to the other must be done cautiously and that longitudinal studies are required based solely on patients wearing removable partial dentures to restore their worn dentition. However, despite difficulties in interpreting this data and the risk of applying it to patients wearing 28 overdentures to restore tooth wear, the risk factors and preventative measures for the development of caries are arguably the same for both populations.

Patients with advanced tooth wear restored with partial dentures that cover the worn teeth therefore form a valuable group of patients. These patients are likely to be at risk of developing caries, they are relatively dentate and a large number of teeth may be covered by the denture (Woodley et al., 1996). This gives the possibility of testing the efficacy of topical fluoride regimens on patients at risk of caries with the unique advantage of being able to obtain samples of dentine for analysis during routine treatment. 29

3. The Development of Topical Fluorides.

Introduction.

The ability of fluoride to affect the teeth was first documented in 1916 when McKay observed characteristic white flecks and yellow/brown spots on the tooth surfaces of children resident in Colorado Springs, USA. in 1942 Dean et al. conclusively linked fluoride levels in drinking water to the prevalence of dental fluorosis and to the reduced prevalence of dental caries. These observations led to artificial fluoridation of the domestic water supply which was first introduced in Grand Rapids, USA in 1945 with Muskagon as a control city (Arnold eta!., 1962). Clinical trials report a reduction in caries (modal values) of 40 to 49% for deciduous teeth and 50 to 59% for permanent teeth (Murray, 1996). The WHO Expert Committee on Oral Health Status and Fluoride Use (1994) concluded that "community water fluoridation is safe and cost effective and that it should be introduced and maintained wherever socially acceptable and feasible”.

The fluoridation of water results in both systemic and topical administration of fluoride to the mineralised dental tissues. Groeneveld at a i (1990) estimated that, at age 15 years, half the maximum DMFS reduction in a fluoridated region is due to pre-eruptive effects and half to post-eruptive effects. However, dental caries has been found to be the predominant cause of tooth loss even up to the age of 60 years in populations from Netherlands and USA (Bouma et a/., 1985; Chauncey et ai, 1989) and is considered to be an increasing problem after this age (Hand ef a/., 1991; Steele et ai, 1996, Fure and Zichert, 1997). In the U.K., in dentate adults over 55, the mean number of carious root surfaces has been estimated to be 0.7 to 0.9 (Hellyer et ai, 1990; Todd and Lader, 1991; Steele et ai, 1996).

Stamm et a i (1990) demonstrated that life long consumption of fluoridated water significantly reduced the prevalence of root caries when compared to similar individuals living in a non-fluoridated community. Burt et a i (1986) found the prevalence to be substantially further reduced in life long residents of a community with naturally fluoridated water at 3.5mg/L (five times the 30 recommended level for it’s climatic zone). Furthermore, Hunt etal. (1989) found a lower root caries incidence over 18 months in adults living throughout their lives in optimally fluoridated areas.

Geographical difficulties or social objections have meant that it has not been possible to fluoridate water supplies to many communities. The use of other vehicles for systemic delivery of fluoride such as orange juice, salt, milk, drops or tablets have also not been adopted globally. Consequently, topical methods of applying fluoride to the teeth have to be considered. These tend to be either self applied agents such as toothpaste and mouthrinse of low fluoride concentration frequently applied or professionally applied agents of high concentration infrequently applied. This thesis seeks to evaluate the former method of delivery.

Fluoridated toothpastes are the most commonly used products that deliver fluoride to the oral cavity. By 1977 over 95% of toothpaste sold in the United Kingdom had been fluoridated (Anderson et al., 1982), approximately 500 million of the world’s population use fluoride toothpaste (Davies at a/., 1995) and 9 out of 10 individuals in industrial countries claim to brush their teeth more or less regularly (Aamdal-Scheie, 1992). When questioned on factors contributing to the decline of caries in industrial countries, 55 international researchers in cariology agreed that the use of fluoridated toothpaste was important (Bratthall at a/., 1996). It has also been cautiously speculated that a link might be made between a resurgence in caries levels in 5 year olds in the United Kingdom and a 12% reduction in the volume of toothpaste sold since 1989 (Downer, 1994).

The Development of Fluoride Toothpaste.

The first reported clinical trial on the addition of fluoride to toothpaste was carried out by Bibby in 1945. However, there was no significant reduction in caries increment over 2 years with a paste containing 0.1% sodium fluoride compared to a placebo paste. The nature of the abrasive system used was not published but calcium carbonate was conventionally used at the time. Subsequent studies found sodium fluoride when combined with a variety of 31 calcium carbonate and phosphate abrasive systems did not have a caries- preventive effect (Winkler et a/., 1953; Muhler et a/., 1955; Kyes et a/., 1961; Brudevold and Chilton, 1966). Ericsson (1961) reported that such abrasive systems inactivate the sodium fluoride due to the formation of insoluble calcium fluoride. Subsequently over 20 clinical trials of toothpastes containing sodium fluoride with abrasive systems of insoluble sodium metaphosphate, silica or acrylic have reported reductions in carious surface increments of 2 to 48% when compared with a placebo (Murray et al., 1991, Clarkson et al., 1993). Mellberg (1991) estimated the mean caries inhibition in such clinical trials to be 21% and Richards and Banting (1996) estimated a reduction in mean DMFS of 32%.

Muhler et al. (1955) also tested a stannous fluoride toothpaste which significantly decreased caries incidence. Succeeding trials with modified formulations led to the first commercially available dentifrice marketed as Crest® (Procter and Gamble, USA). This was recognised in 1960 by the Council on Dental Therapeutics of the American Dental Association as “provisionally therapeutic”, and 4 years later accepted as having definite therapeutic value.

Over 40 clinical trials have been reported (Murray et al., 1991, Clarkson et al., 1993) on the original stannous fluoride-calcium pyrophosphate Crest formulation reporting a reduction in caries incidence of 0 to 49%. Mellberg (1991) estimated the mean caries inhibition in clinical trials comparing stannous fluoride toothpaste with a placebo to be 22% and Richards and Banting (1996) estimated a reduction in DMFS of 25% respectively.

However, the use of stannous fluoride has several disadvantages including a propensity for the development of stain on the enamel (Naylor and Emslie, 1967; Fanning et al., 1968). In addition, stannous fluoride has a short shelf life. It is subject to hydrolysis and oxidation resulting in the formation of complex stannous ions and the reduction of available fluoride. This may be the reason for the variable results achieved in clinical trials (Richards and Banting, 1996). In 1981 the original Crest formulation was replaced by Crest Plus®, a sodium fluoride-silica formulation. The new formulation was shown to be significantly superior (Beiswanger et al., 1981; Zacherl, 1981). However, a resurgence of 32 interest has developed in stannous fluoride products following the commercial development of stabilised compounds (Groat 1983,1985).

Meanwhile, the search for a more potent fluoride agent for caries prevention led to the development of an acidulated phosphate fluoride toothpaste based on good results when this material was applied as a solution (Wellock and Brudevold, 1963). A 2 year clinical investigation by Brudevold and Chilton (1966) of 0.22% sodium fluoride acidulated with 1.5% soluble orthophosphate and the abrasive insoluble sodium metaphosphate did result in significantly lower caries increments than a fluoride free dentifrice. However, clinical investigations on the same combination (Peterson and Williamson, 1968; Slack et al., 1971) and of a 0.22% acidulated phosphate fluoride and calcium pyrophosphate (Zacherl, 1972), did not conclusively establish the efficacy of acidulated sodium fluoride.

Ericsson (1961), in his investigation, also found that sodium monofiuorophosphate (SMFP) was compatible with calcium containing abrasive agents. This combination had several advantages over stannous fluoride, causing less exogenous stain (Naylor and Emslie, 1967; Fanning at a/., 1968), being relatively stable at physiological pH and having a neutral taste. Following clinical trials by Torell and Ericsson (1965) and Naylor and Emslie (1967), a toothpaste containing SMFP and dicalcium phosphate, manufactured by Colgate Palmolive Company, was accepted by The Council on Dental Therapeutics in 1969. Over 40 clinical trials have since been documented (Schmid at al 1984a, Murray at a/., 1991, Clarkson at ai. 1993) with DMFS reductions ranging from 0 to 46% compared to a placebo. Mellberg (1991) estimated the mean caries inhibition in such clinical trials to be 22% and Richards and Banting (1996) estimated a reduction in DMFS of 21%.

Two fluoride formulations have continued to dominate the toothpaste market, sodium fluoride and SMFP. Recent research has attempted to address which of these is more efficacious. Meta-analysis on caries clinical trails by Johnson (1993) and Stamm (1995) found that toothpaste containing sodium fluoride provided significantly lower caries increments (6% and 7% respectively) than those containing SMFP. However, controversy has reigned over the selection of 33 trials forming these analyses (Holloway and Worthington, 1993; Proskin, 1993; Voipe et al., 1993) such that the "International Scientific Assembly on the Comparative Anti-caries Efficacy of Sodium Fluoride and Sodium Monofluorphosphate Dentifrices” (1993) issued a concensus position statement that: "published clinical studies available to date support the conclusion that sodium fluoride and sodium monofluorophosphate at similar concentrations (ranging from 1000 to 1500 ppm fluoride) in commercially available dentifrices across the world provide equivalent anticaries effectiveness”.

However, the "International Scientific Panel Examining the Relative Efficacy of Sodium Fluoride and Sodium Monofluorphosphate as Anti-Caries Agents in Dentifrices” (1995) stated that: "the overwhelming majority of scientific evidence now available supports the statement that sodium fluoride is a more effective caries-preventive agent than sodium monofluorophosphate when delivered in a correctly formulated silica based dentifrice at equivalent fluoride concentration”.

The clinical significance of these statistical differences continues to be disputed (Proskin and VoIpe, 1995; VoIpe etal., 1995).

It has been suggested that sodium fluoride is more effective as fluoride ions (F ) bind more efficiently to oral retention sites than monofluorophosphate ions (Duckworth, 1993). However, SMFP is the world's most widely used fluoride for toothpaste. It is compatible with a wider range of abrasives than other fluorides allowing manufacturers greater freedom in formulating an effective product with desired flavour and texture as well as stability (Mellberg, 1991). Furthermore, the manufacture of effective sodium fluoride toothpaste requires a high level of technology that may not be achieved in developing countries.

Interest has been shown in the use of toothpaste combining more than one fluoride agent, namely sodium fluoride and SMFP. Investigations have been inconclusive as to the superiority of the combination. However, Johnson (1993) noted that, in 5 investigations, there was a trend to combination toothpaste providing an advantage over SMFP alone. Conversely combination products tended not to be aj/effective compared to sodium fluoride alone. A recent investigation (Glass and Naylor, 1997) confirms this, however Richards and Banting (1996) state that "provided the total fluoride concentration is the same. 34 there is no additional benefit from using a combination of the two salts instead of one salt alone”.

A second combination of fluoride agents, amine fluoride and stannous fluoride has recently been developed and marketed as Meridol® (GABA International Ltd., Switzerland) (Page 45).

Research has also been directed at changing the fluoride concentration of toothpaste. It has been found that increasing the fluoride concentration in toothpaste above 1000 ppm improves Its effectiveness although statistical difference has not always been demonstrated (eg. Triol et a/., 1987; Conti et al. 1988; Diodati et a/., 1986; Fogels et a/., 1988; Marks et al., 1994). Indeed, Stephen et al. (1988) suggest a cumulative 6% reduction in caries may be achieved for every additional 500 ppm F" in the range of 1000 to 2500 ppm F'. However, European Directive 78-768 (Dupuis, 1993) suggests an upper limit of 1500 ppm F" for toothpaste sold “over the counter” without prescription.

Conversely, "children’s” toothpaste, with low levels of fluoride, have been developed to negate the increased prevalence in fluorosis reported to be due to children using fluoridated toothpaste from an early age (O’Mullane, 1994). A review of clinical trials by Holt and Murray (1997) suggests caries inhibition decreases with decreasing fluoride content. It is possible that the riskibenefit ratio of therapeutic measures must be considered; the risk of dental fluorosis being weighed against increased cariostatic activity (Stookey, 1990).

The most appropriate fluoride concentration, or indeed preparation, for preventing root surface caries has not been established, indeed most commercial fluoride products were developed for the protection of enamel in children. However, it has been suggested that the aetiology and pathogenesis of enamel and dentinal caries are similar and therefore parallel approaches can be rationally employed (Nyvad and Fejerskov, 1986). Indeed these authors found that the progression of active root surface caries could be controlled with meticulous oral hygiene using an unspecified 0.1% fluoride toothpaste. Nemes et al. (1992) also found that an amino-stannous fluoride toothpaste could convert active lesions to inactive ones. Furthermore, Jensen and Kohout (1988) 35 demonstrated a 67% reduction in root surface DFS scores in those using sodium fluoride (1100ppm F") compared with those using a placebo in areas with unfluoridated water. This reduction was greater than that achieved with coronal caries (41%). Holt and Murray (1997) suggest that there may be a case for developing a toothpaste aimed specifically at the elderly with an increased fluoride content and a low abrasivity. Brands of toothpaste for "sensitive" teeth which do not contain fluoride may be less effective (Hussey and Linden, 1986).

The Development of Fluoride Mouthrinse.

Following the discovery by Dean (1942) that fluoride in drinking water and reduction in caries were related, it has been appreciated that enamel could take up fluoride ions from water solutions. The earliest clinical trials using acidified sodium fluoride solutions (Bibbyet al., 1946, Roberts at a/., 1948) did not give significant results. Neutral sodium fluoride became the most frequently tested compound. Murray at al. (1991) reported 23 studies resulting in caries reductions of 2 to 69%. Since then a 0.05% NaF daily rinse and a 0.2% NaF weekly or fortnightly rinse have been adopted as standard (Wei and Yui, 1993). The clinical trials on these mouthrinses have been sufficiently favourable for Dental Public Health officials to adopt fluoride mouthrinsing in schools as the main alternative to water fluoridation in community prevention in Sweden, Norway, USA, Eire and Cuba.

Trials on rinses containing acidulated phosphate fluoride (APF) began with Brudevold at al. (1963). Two trials reported direct comparison with sodium fluoride; Aasenden at al. (1972a) reported similar effectiveness, whereas Heifetz at al. (1973) found a small but insignificance difference in favour of sodium fluoride. Heifetz at al. (1973) also found APF difficult to manufacture and to have an unacceptable taste.

Investigation into stannous fluoride rinses (Radike at al., 1973; McConchie at al., 1977) report similar reductions in caries increments to sodium fluoride, but a direct comparison has not been reported. Rinses of ammonium fluoride (DePaola at al., 1977) and amine fluoride (Ringelberg at al., 1979) were found to result in similar caries reduction as positive sodium fluoride controls. An 36

amino-stannous fluoride rinse and paste combination compared favourably with a sodium fluoride regime (Banoczy and Nemes, 1991) but other direct comparison has not been reported. These rinses will be discussed further in Section 5.

The scope to increase the concentration of fluoride in a mouthrinse is limited; fluoride concentration in a mouthrinse is usually inversely related to rinsing frequency to avoid frequent ingestion of concentrated solutions. Murray et al. (1991) analysed the percentage caries reduction obtained in 31 clinical trials of fluoride mouthrinsing and found that there was a moderate trend towards increased effectiveness with increased fluoride concentration especially within the range of 0.02 to 0.1% fluoride. The analysis also suggested that rinsing once per week or more frequently is more effective than rinsing every two weeks or less frequently. Studies directly comparing the efficacy of rinsing with 0.2% sodium fluoride weekly with rinsing daily with 0.05% sodium fluoride found the latter to be more effective, but not significantly so (Driscoll at al 1982, Heifetz ef a/., 1982).

Mouthrinsing when performed frequently has been found to be at least as effective in caries reduction as the use of toothpaste (Torell and Ericsson, 1965; Ashley at al., 1977; Ringelberg at al., 1979; Blinkhom at al., 1983). A small additional effect on the percentage caries inhibition has been observed in children when both a fluoride toothpaste and mouthrinse were used (Ashley at al., 1977; Ringelberg at al., 1979; Triol at al., 1980; Boyd, 1993; Karjalainen at al., 1994), although Mathiesen at a/.(1996) only observed the additional effect in those patients with good oral hygiene. However, the caries preventive effect may not continue on cessation of rinsing; Holland at al. (1995) showed the loss of caries inhibition four years after the termination of a school based fortnightly mouth rinsing programme at age 12. Conversely, McConchie at al. (1977), Leske at al. (1986) and Haugejorden at al. (1985) found the effect of rinsing was maintained for up to 12 months, 30 months and 7 years respectively when similar rinsing programmes ceased at the age of 13 years. But an eleven year post-treatment follow up by Haugejorden at al. (1990) found that caries was delayed rather than prevented and suggested that the benefits of a fluoride 37 mouthrinsing programme decreased as the period after discontinuation increased.

Very little information is available on the ability of mouthrinse programmes to prevent root caries. Ripa et al. (1987) reported a 25.1% reduction in new root caries or fillings in adults after 3 years unsupervised use of 0.05% sodium fluoride mouthrinse compared with a placebo. However, the only significant difference in caries increments was found for the mesio-distal root surfaces of 45 to 65 years old participants. Ravald and Birkhead (1992) investigating patients treated for periodontal disease age 33 to 76 years old, found a daily 0.05% sodium fluoride rinse tended to be more effective than professional applications of 5% sodium fluoride varnish or 0.4% stannous fluoride gel carried out 3 to 4 times per year. However, even at 3 years, the results were not statistically significant. These results are complicated by the findings of Wallace et al. (1993) who found in a 2 year study of adults over 60 both twice yearly applications of 1.2% APF gel and daily mouthrinsing with 0.05% sodium fluoride resulted in significantly reduced incremental DMFS compared with placebo daily mouthrinsing. The gel was significantly more effective at reducing the number of new lesions, whereas the mouthrinse significantly increased the number of reversed lesions.

It is interesting to note that Stamm (1993) reported that in the United States, for every $2 spent on toothpaste, $1 is spent on mouthrinse. However, only 2% of the mouthrinse market are made up of fluoride containing products. This suggests that mouthrinse has substantial potential as a caries preventive that is not being exploited to a significant effect.

In summary, both fluoridated toothpaste and mouthrinse have the ability to reduce caries incidence and when used together a small additional reduction might be expected. While the superiority of one fluoride preparation over another has not been conclusively proved, it seems reasonable to adopt sodium fluoride preparations (with a compatible abrasive in toothpaste) as the standard against which the effectiveness of other preparations are measured. 38

4. The Development of Amine Fluoride, Stabilised Stannous Fluoride and Amino-Stannous Fluoride Preparations.

4.1 The Development of Amine Fluorides Preparations.

The possibility of protecting tooth surfaces using fats or oils developed from reports of caries inhibition when rats were fed diets with high fat contents (Rosebury and Kranshaw, 1939; Schweigert et al., 1946). Box (1942) suggested that fat formed a “water proof protective coating preventing acid coming into contact with the tooth surface and showed that both whole teeth and powdered enamel could be protected by oleic acid against décalcification in vitro.

Walsh and Green (1950) examined a number of oily and surface active materials to establish their effect against décalcification of enamel by acidified human saliva in vitro. Long chain aliphatic amines were found to give measurable protection. A 1 % solution of hexadecylamine in paraffin oil at pH 4 gave 80% protection. The authors suggested that the NH 2 group of the amine absorbed onto the enamel surface and the hydrocarbon radicles of hexadecylamine then acted as a bridge between the enamel and the paraffin oil resulting in a stable film which protected the tooth against décalcification.

Green and Walsh (1951) showed that in a homologous series of primary aliphatic amines (carbon lengths 8 to 18), the protective effect passed through a sharp maximum for tetradecylamine, C 1 4 H2 9 NH2 , when a 1% solution in paraffin oil at pH 4 gave 78% protection. The effect virtually vanished when the chain was shortened to 10 carbon atoms. The amines were equally effective on powdered solutions of calcium phosphate as they were on intact enamel, suggesting that amines protect enamel by virtue of their affinity for the inorganic constituents. It was concluded that the protective effect resided in the formation of a mono-molecular layer on the surface of the tooth.

Irwin at al. (1957) found primary amines, in aqueous solutions at pH 4, were very much more effective than secondary and tertiary amines. Quaternary amine gave no protective effect against acid dissolution of calcium phosphate. 39

Pentadecylamine afforded more protection that hexadecylamine but the difference was small. Furthermore, the synthesis of pentadecylamine is costly whereas hexadecylamine is widely available.

Roseman et al. (1969) carried out an investigation into the relation between the absorption ability of surfactants and the solubility of hydroxyapatite and enamel. Absorption behaviour was affected by ionic strength and surfactant type. Retardation of dissolution rates was observed with a long chain homologous series of protenated primary amines (decyl-, dodecyl-, tetradecyl- and hexadecylammonium chlorides). These compounds were found to adsorb more strongly and were considered to form a molecular film at the enamel-liquid or hydroxyapatite-liquid interface. The authors suggested that, in order for a dissolved ion to reach the bulk solution, it must pass through the interfacial film and the diffusion layer regions and the rate determining step was controlled by this film. A linear relationship was demonstrated showing a threefold increase in rate retardation per increase in carbon number reflecting the greater adsorbing tendency of the higher homologues due to the greater number of methylene groups.

Borggreven and Driessens (1986) tested the hypothesis further, measuring coefficients of diffusion of radioisotopes across a bovine enamel membrane

treated with aliphatic amines. C 12 to Cis amines reduced the rate of diffusion by

20 to 30% whereas the Ce and C 2 amines had smaller effects. The authors suggested that the absorption behaviour of surface-active compounds is related

to their critical micelle concentration and that for the Ce and C 2 amines the applied concentration lay below this value, whereas for the other amines it was higher. This accounts for the high diffusion effects, but with little variation,

observed for the C 1 2 to Cie amines.

Nevin at al. (1953) were the first to report on a clinical trial in teenagers using an amine preparation. Brushing with 1.6% tetradecylamine paste with a calcium carbonate abrasive resulted in caries reduction of 37% over 14 months. Ludwig and Taylor (1957) found, in an unsupervised clinical trial also in teenagers that, tetradecylamine emulsion toothpaste resulted in caries reduction in 1 year of 44%. However, Ludwig (1963) reported that tetradecylamine toothpaste with AO calcium carbonate abrasive resulted in only a 10% reduction in a 2 year clinical trial on over 300 children aged 11 years. It was suggested that gradual conversion of the primary amine to a secondary amine, which is less effective, occurred catalysed by the presence in the toothpaste of finely divided calcium carbonate and Sorbitol (Leaver 1971). Interest in amines alone as anti-caries agents then diminished.

However, the physico-chemical protection of tooth surfaces by organic compounds excited interest in the possibility of combining this with chemo- protection afforded by inorganic fluorides. Mühlemann et al. (1957) investigated the effect of pre-treatment with inorganic and organic fluorides on the solubility of intact enamel surfaces of extracted teeth. Fluoridation improved the solubility reduction of the free aliphatic amines. Amino acid fluorides (lysine.KF, leucine.HF, lysine.HF, and cysteine.HF) and particularly the hydrofluorides of aliphatic monoamines (laurylamine.HF and cetylamine.HF) were found to give greater protection than the inorganic fluorides. The authors assumed that the physico-chemical behaviour of the fluoridated aliphatic amines on the enamel surface allowed for improved fluoride chemo-reaction.

This led to the commercial development and production of amine fluoride compounds principally by GABA International Limited (Basel, Switzerland). Figure 1 illustrates the chemical formulae of the amine fluoride compounds commonly used and the various names by which each is known.

Amine fluoride products, under the trade name Elmex®, are widely available in Korea, South Africa and Saudi Arabia and Europe, although not Britain in the form of a fluid, mouthrinse, toothpastes and gel. The fluoride content of these products is made up of various combinations of AmF 242, AmF 335, AmF 297 and sodium fluoride (Table 1). A second amine fluoride solution, Aminfluorid Otopina® (Belupo), has been used in Croatia (Lincir and Rosin-Grget, 1993). 41

Figure 1. Chemical Formulae of Amine Fluoride Compounds in Common Use.

AmF 297

N’-Octadecyl trimethylene diamine-N.N.N'-tris (2-ethanoi)-dihydrofluoride or N,N',N'-Tri (hydroxyaethyl)-N-octadecyl-1,3-propylen-diaminum dihydrofluoricum or N,N',N'-Tri (polyoxyethylene)-N-hexadecyl-propylenediamine-dihydrofluoride or Ethoduomeen dihydrofluoride or Olaflur

CH2 -CH 2 OH CH j -(CH2) i 6-CH2-N|H-CH2-CH2CH2-N*H^ 2F I • - - '^c H2-CH20H CH2 - CH2 0 H

AmF 242 Hexadecylamine hydrofluoride or Cetylamine hydrofluoride or Hetaflur

C H 3 — ( C H 2) i4 — C H 2 — N H3 F

AmF 335 1 -Octadeceneamine hydrofluoride or Oleylamine hydrofluoride or Dectaflur

C H 3 (CH2)7 — C H — CH — (CH2)7 — C H 2 — N H3 42

Table 1. The Fluoride Compounds Currently in Elmex® Products (GABA International Ltd., Switzerland).

Elmex® Products Constituents (according to manufacturer data) Fluid AmF 297/335 (1%F ) Mouthrinse AmF 297 (0.01% F ) and NaF (0.015% F ) Toothpaste Standard AmF 297/242 (0.125% P) Toothpaste Children's AmF 297 (0.025% F ) Toothpaste Sensitive Plus AmF 297 (0.14% P) Gel AmF 297/335 (0.25% P) and NaF (1% P)

4.2 The Development of Stabilised Stannous Fluoride Preparations.

The active ingredients in topical oral preparations must be maintained in a stable, water soluble and bio-available form (Miller et a/., 1994). Stannous fluoride although highly water soluble is susceptible to hydrolysis and oxidation. This can reduce its stability and water solubility. Stannous ion (Sn^*) readily hydrolyses on exposure to water to form stannous hydroxides which precipitate and can lose water to form stannous oxides. This reaction is pH dependant being slowed in acidic conditions of pH 4 and less. (Hefferen, 1963).

The stannous ion also oxidises to the stannic ion (Sn^*) forming stannic fluoride

(Snp4 ) and stannic oxides or hydroxides, the latter having low water solubility. This reaction is not pH dependant but the transition kinetics are slow, so that the two valent state predominates in freshly prepared solutions (Miller et al., 1994).

Once precipitates are formed they "fall out" of solution causing loss of bio- available ions. The loss of titratable Sn^* in aqueous solutions over 15 days has been found to be lower in solutions with relatively high fluoride concentrations (0.5 to 1.0 M). It has also been shown to be a function of temperature and is greater in solutions exhibiting large deviation in pH from their native values 43

(Lim, 1970). The fluoride content has also been shown to gradually decrease in aqueous Snp 2 solutions (Lim and Hsieh, 1971).

This may explain why topical fluoride treatments with freshly prepared, acidic solutions of stannous fluoride have successfully reduced the incidence of caries in children (Howell et al., 1955; Radike et ai., 1973; McConchie et al. 1977) whereas variable results have been achieved in clinical trials of stannous fluoride toothpaste (Richards and Banting, 1996). Means of stabilising stannous fluoride have therefore been investigated.

The incorporation of glycerol and some highly water soluble compounds (sugar or gums) into aqueous solutions of Snp2 have produced a stabilising effect. It was assumed that the activity of the free stannous ions has been reduced consequently reducing the rate of hydrolysis (Hefferren 1963). Lim (1970) showed that sodium fluoride, glycerol and tartaric acid kept stannous fluoride solutions precipitate free. Lim and Hsieh (1971) found that the fluoride content remained constant for these mixed mediums, however, despite the lack of precipitation in the mixed solutions, loss of titratable Sn^* occurred in all solutions such that it was not detectable at 21 days. This suggested that soluble products were formed. It was postulated that such water-soluble complexes would rapidly break down on contact with the teeth and release free and reactive ions. Indeed, significant protection (greater than 50%) against enamel

dissolution was found with solutions of Snp2 both in water and in combination with sodium fluoride, glycerol and tartaric acid in various concentrations even at 21 days (Lim and Hsieh, 1971). It has also been suggested that very small amounts of Sn^* are required for the anti-solubility effect on enamel (Lim and Hsieh, 1971).

Meanwhile, Shannon (1969) reported on an anhydrous 0.4% stannous fluoride dental gel prepared by forcing stannous fluoride into anhydrous glycerine at 150°C. This was found to have excellent stability. Anhydrous preparations, containing no free hydroxide ions (needed for hydrolysis), have since been patented using manufacturing processes to minimise the presence of dissolved oxygen (Groat, 1983, 1985). Such products are further protected from loss of activity by eliminating mineral abrasives that could react with stannous fluoride 44

(Shannon, 1969). Although these gels were generally stable they did not gain widespread consumer acceptance because of an "unpleasant feeling in the mouth", difficulty of use with a toothbrush and the absence of abrasives (Tinanoff, 1995).

Stannous fluoride in aqueous preparations can also be protected from hydrolysis and oxidation by the formation of complexes with the stannous ion.

Aqueous solutions of 0.4% Snp2 have been stabilised with a copolymer of vinyl- methylether and malic anhydride and with polyvinyl alcohol (PVA) (Miller et al., 1994). Similarly toothpaste manufacturers have developed ways of stabilising stannous fluoride for long periods in aqueous toothpaste formulations. These include employment of chelating agents which bind stannous fluoride protecting them from hydrolysis and oxidation. Alternatively stannous reservoirs are included that act both as a supply of stannous ions and as an anti-oxidants, reacting with available oxygen leaving stannous fluoride protected. However, these approaches have the potential of over-stabilising the product making stannous fluoride biologically inactive in the mouth (Tinanoff, 1995).

Miller at al. (1994) investigated the solution and rinse described above and stannous fluoride stabilised with tripolyphosphates to form a toothpaste. Investigation into antibacterial and anti-plaque efficacy showed that some stabilisers can indeed reduce efficacy; only the PVA rinse provided bacterial activity over an extended period of time (hours). The bioavailability of stannous ions appeared to be reduced in preparations when highly stable stannous complexes are formed. The authors concluded that anhydrous glycerine preparations were ideal because they stabilise stannous fluoride without strong complexation.

4.3 The Development of Preparations Combining Amine and Stannous Fluorides.

Mühlemann et al., (1981) found that aged solutions of Snp 2 and AmF 297, equimolar for fluoride, remained precipitate free suggesting that the stannous ions are stabilised and oxidation and hydrolysis reactions have been prevented. 45

The stannous ions were thought to complex with the amine portions of AmF 297 (GABA Product Literature, 1993). This was patented by GABA International Ltd. (Basel, Switzerland) and the product, known as Meridol®, is commercially available as a toothpaste in Switzerland and as a mouthrinse in Switzerland, Israel, Finland, Germany and Luxembourg. 46

5. The Anti-Caries Activity of Stannous Fiuorides, Amine Fiuorides and Amino-Stannous Fiuoride Regimens.

5.1 Inhibition of Déminéralisation.

Stannous fluoride solutions have been found to be more effective in reducing acidic solubility of enamel than sodium fluoride (Muhler and Van Huysen, 1947; Van Huysen and Muhler, 1948; Phillips and Swartz, 1948; Ericsson, 1950; Shannon, 1980). However, Mühlemann et al. (1957) found AmF 297 to be significantly superior to SnF2 . In contrast, Duschner and Uchtmann (1988a) on investigating the inhibition of demineralisation of bovine enamel found acidic

Snp2 , NaF and AmF 335, in order of effectiveness, reduced the etching rate. The depth of the acid resistant zone was measured to be 0.5 pm for the acidic

NaF and 0.7 pm for AmF 335 but was not measurable for SnF 2 .

Groneveld and Arends (1974) using quantitative microradiography found application of solutions (1% F ) combining AmF 297 and 335 were more effective at preventing demineralisation of sound enamel than 4% Snp 2 solutions; the demineralised area had a significantly higher mineral content and a thicker surface layer. However, for white spot enamel the surface layer was statistically more mineralised following treatment with SnF 2 although there was no significant difference in the effect of the solutions over the lesion as a whole.

The increased resistance of SnF 2 treated enamel may be due to formation of a more mineralised surface layer (Groneveld and Arends, 1974) or due to the formation of an acid resistant surface precipitate (Page 50).

Snp2 has also been found to be effective in vitro in reducing the acid solubility of root surfaces (Gordon and Shannon, 1970) and to be more effective than NaF and SMFP on dentine and root surfaces (Shannon at al., 1976; Shannon, 1980). However, these solutions have not been tested at equivalent pH.

Supernatants of centrifuged 50:50 commercial toothpaste-water slurries have also been evaluated for ability to reduce the acid solubility of root surfaces (Stevens etal., 1976). While, stannous fluoride toothpaste provided measurable 47 reduction of solubility, SMFP toothpaste was twice as effective, but no dentifrice provided a statistically significant solubility reduction.

A series of in vitro investigations into the effects of combinations of Snp 2 and AmF 297 on acid dissolution of enamel were carried out by Barbakow et al. (1985, 1986a, 1987). Significantly less acid dissolution occurred on treatment with AmF 297/SnF2 combinations and with Snp2 alone compared with NaF, AmF 297 and water. However, a synergistically decreased enamel dissolution rate was not found. Indeed, the enamel dissolution rate appeared to be inversely proportional to the quantity of Snp 2 in the SnF 2/AmF 297 solution. However, the enamel dissolution rate may also have been a function of pH change resulting from combining the solutions.

The authors suggested that, as the AmF 297/SnF2 mixture produced similar effects on enamel as SnF 2 alone, enamel reacts with the stannous ions of the

SnF2 before it does with AmF 297. However, “over-stabilisation" of stannous fluoride might be occurring as has been documented for stannous fluoride stabilised with a copolymer of vinyl-methylether and malic anhydride or with tripolyphosphates (Miller etal., 1994).

However, AmF 297/SnF2 gel was found to produce a decreased enamel dissolution rate compared to enamel specimens treated with water, sodium fluoride gel (Act®), or Elmex® gel. (Barbakow et al., 1989). This was despite flossing the specimens with waxed floss prior to fluoride treatment. The effects on dentine have not been reported.

The protective effect on enamel solubility increased by increasing the time of exposure to fluoride solutions (Gordon and Shannon, 1970) but brushing specimens following fluoride treatment, increasing the washing time and washing with potassium hydroxide reduced the protective effect (Barbakow 1986a). It was suggested that this indicated the “protection" induced by the solutions affected only the outer most enamel layers. Similar investigations have not been reported for dentine. 48

5.2 Promotion of Remineralisation. inhibition of demineralisation is a useful anti-caries effect, but it is well established that an initial caries lesion is reversible following remineralisation provided cavitation of the lesion has not occurred (ten Cate and Arends 1977; Koulourides and Cameron, 1980; Groeneveld, 1985). If a potential anti-caries agent interferes with the remineralising capacity of a lesion a net decreased inhibition of caries lesion formation might occur. From a preventive point of view it is therefore important to know the effect of fluoride compounds on remineralisation and fluoride uptake of a lesion (Borggreven, 1991).

The ability of amine fluoride, stabilised stannous fluoride and amino-stannous fluoride preparations to effect remineralisation have been reported.

In a pH cycling lesion progression model, stabilised stannous fluoride (1100 ppm F") toothpaste (Crest Gum Care®) and a sodium fluoride toothpaste (Crest®), both with silica abrasive, resulted in similar remineralisation and/or inhibition of demineralisation of coronal enamel, cervical enamel and dentine on evaluation by microhardness and microradiography (Faller et al., 1995; Faller et ai. 1997). These products were found to protect enamel significantly better than the commercially available Chinese SMFP and NaF/SMFP pastes tested in this study.

Arends etal. (1983a) and Goorhuis and Purdell-Lewis (1986) investigated the in vivo ability of amine toothpastes (0.12% F") used twice daily for 21 and 27 days to remineralise previously demineralised enamel specimens embedded into partial dentures. Microradigraphy and microhardness data demonstrated that remineralisation did occur. However, placebo treatment also improved remineralisation, presumably due to the remineralising effects of saliva. Only when amino-sodium fluoride gel once weekly, was additionally used did a significantly improvement in remineralisation occur compared to the placebo group (Goorhuis and Purdell-Lewis, 1986). In a similar investigation, single use of AmF 297/SnF2 mouthrinse for 1 minute significantly increased the microhardness of softened enamel compared to no rinse although hardness was not increased to pre-demineralisation values (Gedalia et al., 1992, 1996). 49

Unfortunately none of these investigations compared the test compound with a positive control.

The effect of these compounds on remineralisation of dentine has not been reported. Furthermore, while in-situ investigations are suggested to be the most meaningful pre-clinical models for predicting clinical efficacy (Stookey et al. 1992), they do not replace assessment by controlled clinical trials.

However, one clinical trial has been reported concerning remineralisation of dentine. The remineralising effect of twice daily use of AmF 297/SnF2 or NaF toothpaste (0.15% F") and mouthrinse (0.025% F’) on exposed root surfaces was assessedin vivo by Nemes at al. (1992). 20 and 24 patients respectively entered the study groups. While root caries index scores decreased by 47% in the AmF 297/SnF2 group and 10% in the NaF group, the difference between these scores was not statistically significant. A statistically significant improvement in the number of softened surfaces between the baseline and examinations was found in the AmF 297/SnF2 group but not in the NaF group. It was suggested that this indicated a remineralising effect of topically applied AmF 297/SnF2 compounds. But the treatment philosophy of patients susceptible to root caries should not be based on one trial, particularly one limited to small populations.

5.3 The Formation of Calcium Fluoride.

The formation of calcium fluoride on the tooth surface is believed to play a role in reducing enamel solubility. Calcium fluoride (CaF 2 ) is stable at neutral pH owing to surface adsorption of phosphate ions onto the crystal surface and formation of a solubility-limiting phase (Lagerlof at a/., 1988). Extended exposure of saliva causes formation of a fluoroapatite layer on the CaF 2 crystals, restricting dissolution further.

It has been suggested that the calcium fluoride crystals serve as pH-controlled reservoirs of fluoride ions on the enamel or in the plaque and release fluoride during caries challenges (Arends at al., 1983b; Rolla, 1988). A low pH, of less than 5, causes loss of the solubility-limiting adsorbed phosphate ions and a 50 slow dissolution of calcium fluoride. Fluoride thereby released from calcium fluoride during caries challenge may subsequently be built into hydroxyfluoroapatite through dissolution/re-precipitation reactions.

Morphology of Precipitates

Scanning electron micrograph studies of polished enamel surfaces reveal the formation of macro- and microglobuli of calcium fluoride after treatment with amine and stannous fluorides.

The precipitates formed by amine fluoride varied in diameter from 0.08 (Gwinnett et a/., 1972) to 30 pm (Barbakow at a/., 1983) whereas Lutz (1983) found they formed a homogenous layer over the tooth surface. The larger globuli appeared to form on top of the layer of small calcium fluoride globules (Barbakow at a/., 1984a). The effect of treatment on dentine has not been reported.

Micrographs of precipitates formed by freshly prepared aqueous solutions of stannous fluoride on enamel showed the presence of small globular particles similar to those formed by stannous chloride and more infrequent larger globules (Dijkman at a/., 1982; Ellingsen, 1986). The dimensions of these globules are not recorded. However, similar globular precipitates with approximate diameters of 0.05 to 5 pm were found on dentine treated with freshly prepared aqueous solutions or anhydrous gels of stannous fluoride in vitro (Ellingsen and Rolla, 1987; Addy and Mostafa, 1988; Miller at a/., 1994) and in vivo (Miller at a/., 1994). The precipitates partially occluded the dentine tubules. Increasing the exposure time results in a thicker globular layer and more variation in globule size.

However, a copolymer stabilised stannous fluoride (Miller at a/., 1994) and a water-based dentifrice (Addy and Mostafa 1989) did not form a deposit on dentine. This may be explained by the overstability of the stannous ion in the former leading to reduction in bioavailabiltiy and the instability of the stannous ion in the latter. 51

The precipitates formed by amine fluorides on enamel demonstrated better retention than sodium fluoride precipitates following ultrasonic energy and washing with water and potassium hydroxide for up to 24 hours (Mühlemann et al., 1968; Barbakow et al., 1984a,b). The larger globuli appeared to be removed sooner than the smaller globuli (Barbakow et al., 1984a,b). The layer was also more resistant to exposure of lactic acid for 60 minutes. Whereas no acid resistance was demonstrated by enamel similarly exposed to sodium fluoride or sodium monofluorophophate (Imfeld, 1996). These solutions did however, exhibit a higher pH and the pH of a fluoride solution is known to be inversely related to calcium fluoride deposition (Rolla and Saxegaard, 1990).

The larger globules precipitated by stannous fluoride on dentine were present after immersion in water for 15 hours (Ellingsen and Relia, 1987) but were removed by immersion for 6 days (Ellingsen, 1985). Both large and small globules were soluble in 1 M KOH alkali. Furthermore, immersion in KOH caused control and Snp2 treated enamel to have similar dissolution rates irrespective of immersion time in SnF: indicating that KOH dissolves the “acid protective" layer (Dijkman etal., 1982).

Chemical Composition of Precipitates.

The composition of precipitates formed on bovine enamel by amine fluoride and sodium fluoride were investigated by Duschner and Uchtmann (1985, 1988b). Fluoride to calcium atomic ratios in both investigations suggested that at the immediate surface pure calcium fluoride was deposited for amine fluoride and sodium fluoride solutions at pH 4.0 and 5.0. Accumulation behaviour was nearly identical in the outermost zone for both preparations but in deeper zones uptake was more effective by the amine fluoride. However, natural values of fluoride concentration were approached at a maximum depth of 3.0 micrometers.

Octadecylamine cation to fluoride ratios in the amine fluoride treated enamel gave evidence of an extremely thin surface coating of octadecylamine. The layer was 0.05 pm deep and therefore in the range of a multimolecular layer. The authors suggested this was a surfactant layer. 52

The depth of zone of acid resistance was found to be almost identical to the extension of the calcium fluoride layer but the retarding effect on surface removal was more pronounced for octadecylamine hydrofluoride (Duschner and Uchtmann, 1988a). It was suggested that the surface activity of the amine fluoride affected the micromorphology of the calcium fluoride precipitate. This assumption was supported by Mühlemann (1983) who found calcium fluoride leachability to be considerably lower when the precipitate was formed from amine hydrofluoride rather than from sodium fluoride solutions.

Analysis of precipitates formed by stannous fluoride has resulted in controversy. Electron Microprobe studies have confirmed that enamel and dentine treated with stannous fluoride absorb both tin and fluoride (Wei and Forbes, 1972; Ellingsen, 1986; Ellingsen and Relia, 1987; Miller et a/., 1994). Other ligands known to readily complex with stannous ions in solution, specifically oxygen, hydroxide and phosphates have been identified in the surface precipitate (Miller at a/., 1994). Tin and phosphorous are present at high levels throughout the deposit, indeed tin has been detected penetrating into dentine tubules to depths of 100 pm (Miller at a/., 1994), whereas calcium and fluoride are found at gradually increasing levels deeper in the deposit.

The large fluoride containing globules are considered to consist of calcium fluoride as this substance is known to form spherical particles on enamel, to be slightly soluble in water and soluble in alkali (Ellingsen, 1986; Relia and Saxegaard, 1990).

The smaller particles have been reported to consist of Sn 2 (P0 4 )0 H (Collins,

1961; Tveit at a/., 1983; Miller at a/., 1994) or Sn 3 p3 P0 4 (Jordan at a/., 1971; Krutchkoff at a/., 1972; Brendt, 1972; Wei and Forbes, 1974; Babcock at a/.,

1978). The presence of Snpz (Miller at a/., 1994) and Ca(SnF 3 ) 2 (Wei, 1974) have also been reported. Miller at al. (1994) also suggested the formation of a consistent level of tin complexation by a phosphate species with increasing

levels of fluoride complexation suggesting the formulation Sn 2 Fx 0 Hi.xP 0 4 . They also suggested that tin is oxidised to Sn*^ on the outer surface while the portion of the tin involved in bonding to fluoride in the interior of the deposit is in the Sn*^ oxidation state. 53

It has been suggested that the tin products are formed under specific conditions of pH (Myers, 1968) and fluoride concentration (Babcock et al. 1978). The detection of SnsFsPO# followed prolonged treatments with high fluoride concentrations (10%) of stannous fluoride (Krutchkoff at al., 1972). Lower concentrations of stannous fluoride applied "short term" probably result in the formation of stannous phosphate, which is also formed by stannous chloride and is alkali soluble (Babcock atal., 1978).

The tin phosphate layer is not, however, thought to be associated with caries protection. This is attributed to the formation of calcium fluoride, which is formed in most conditions, aided by the low pH of Snpz (Miller at al., 1994)

However, Duschner and Uchtmann (1988b) found a 0.1% F" solution of stannous fluoride to be distinctly less effective at depositing fluoride than sodium fluoride and AmF 335 on bovine enamel; the zone of accumulation was limited to 0.2 pm compared with 3.0 pm and the highest level of accumulation was one quarter that of sodium or amine fluoride. Fluoride to calcium atomic ratios suggested that stannous fluoride resulted in no measurable quantities of calcium fluoride formation. It was suggested that this might be due to the simultaneous deposition of stannous or stannic ions. Nevertheless an acid protective layer was formed.

Increased protection from acid dissolution of enamel and root surfaces has been found by combining topical treatments of SnF 2 and APF both sequentially and in combination. The protective layer was significantly more resistant, even to removal by rapidly flowing water, than either compound alone (Shannon and Wightman, 1970; Shannon, 1971; Williams atal., 1974). It was proposed that an intermixture of stannous phoshate superimposed on Cap 2 provides a surface layer that is remarkably resistant to acid dissolution (Shannon, 1971).

Barbakow at al., (1986b) found surface deposition on enamel treated with solutions of Snp2 /AmF 297 combinations. The morphology was not described but the surface products appear to be more resistant to etching than enamel itself, the former producing overhanging edges over the etched enamel. 54

It could be assumed that any of the products found on amine fluoride or stannous fluoride treated enamel might be formed. Whatever the nature of the precipitate more was removed by increasing the water-washing period (Barbakow ef a/. 1987).

Barbakow et al. (1986b) suggested that the chemical nature of the products formed on the enamel after immersion in the precipitate free SnFz/AmF 297 must be analysed to optimise the molar concentration of SnF 2 to AmF 297. They also suggested that this optimal concentration might not be the same as that necessary to optimise the anti-plaque effects.

5.4 Promotion of Fluoride Uptake by Dental Mineralised Tissues.

Amine Fiuoride Regimens

The homogenous adherent layer of calcium fluoride formed by amine fluorides, as well as their mildly acidic pH of between 4.5 and 5.0, could be expected to encourage fluoride uptake by dental mineralised tissues.

Although a large number of investigations have looked at fluoride uptake by mineralised tissues, comparison between them is difficult. Regimens have been applied for different times and a variety of washing procedures have then been carried out. The biopsy technique has not been standardised and fluoride content is usually reported in parts per million which is meaningless unless the volume of solvent used and the volume of tissue biopsied is reported.

However, despite these variations, uptake of fluoride by sound enamel following treatment with amine fluoride solution, gel or toothpaste have been reported (Rinderer et a/., 1965; Antilla and Pohto, 1973; Strübig and Güizow, 1986) (Appendix 3, Table 1). In comparative studies a greater uptake of fluoride by sound enamel in vitro has been found following treatment by amine fluorides compared with treatment by: . Sodium fluoride (Kirkegaard, 1977a,b; Strübig, 1980; Klimek et a/., 1982; M okef a/., 1990, Chan etal., 1991), • APF (Kirkegaard, 1977a), 55

. NaMFP (Kirkegaard, 1977a,b; Klimek, 1981; Klimek et a/.,1982) and . Stannous fluoride (Kirkegaard, 1977a; Barbakow et a/., 1985) (Appendix 3, Table 2).

Mühlemann et al. (1968), however, found uptake by AmF 297 solution to be similar to that by sodium fluoride although treatment with AmF 242 solution led to greater uptake. In contrast, Arends and Schuthof (1975) found uptake by enamel following treatment with Elmex® fluid to be significantly less than that by APF and by ammonium fluoride. These solutions, however, had decreased pH. The influence of pH is confirmed by StObig and Güizow (1986) who found that the pH of fluoride gels applied to enamel had a greater influence on fluoride uptake than the composition of the gel.

Fluoride uptake by plaque covered enamel was also found to be significantly greater following application of amine fluoride solution than sodium fluoride and MFP solution (Klimek etal., 1982).

Fluoride uptake by enamel with artificial caries In vitro has also been reported (Klimek, 1981; Chan etal., 1991). Klimek (1981) found that the uptake by amine fluoride was significantly greater than that by sodium fluoride only at the surface layer whereas Chan et al. (1991) found uptake to be greater throughout the lesion.

In vivo measurements also document fluoride uptake from amine fluoride solution and toothpaste by enamel (Belser et al., 1975; Mushanoff et al., 1981; ten Cate etal., 1988) (Appendix 3, Table 3). In comparative studies use of AmF 297 toothpaste resulted in a similar fluoride uptake by human enamel to the use of MFP over 30 days (Candeli, 1967) in vivo (Appendix 3, Table 4). However, a greater uptake by enamel has been found following use of Elmex® toothpaste compared with MFP toothpaste (Barbakow et al., 1983) and by Elmex® Gel but not Elmex® Fluid when compared with APF gels and MFP gels (Baijot- Stroobants and Vreven, 1980). Amine fluoride toothpastes have also resulted in a greater fluoride uptake over 23 days by rodent enamel in pooled data (Schmid et al. 1984b) and when applied daily or every 5*^ day for 20 days (Schmid etal. 1984c). 56

In contrast, Hotz (1972) however, found no statistical difference in fluoride uptake by enamel in vivo between an amine fluoride and APF gel. The APF gel was, however, considerably more acidic.

Retention of fluoride by enamel has also been noted following treatment with amine fluoride regimens. An in vitro investigation reported that following application of AmF 297/AmF 335 solution more fluoride (65%) was retained in artifically carious enamel than following application of sodium fluoride solution (37%) after storage for 1 week in saline (Chan et al. 1991). Treatment of sound enamel with AmF solution also resulted in retention of more fluoride than treatment with NaF gel or fluorosilicate solution after storage in saline (Strübig, 1980). At 1 week the loss was 5%, 78% and 75% respectively. At 4 weeks fluoride was still present in enamel treated with amine fluoride solution, although significantly lower than at 1 week, and was significantly higher than the fluoride content of enamel treated with the other 2 regimens.

In vivo no difference was found in fluoride retained in deciduous enamel 15 days after withdrawal of amine fluoride or SMFP toothpaste (Candeli et al. 1967). Similarly, there was no difference in fluoride retained by enamel 14 days following treatment with AmF297/AmF335 solution or APF solution (Rinderer et al. 1965). However, the fluoride content of enamel following application of amine fluoride gel was found to be retained for up to 6 weeks compared to 2 weeks following treatment with APF gel (Baijot-Stroobants and Vreven, 1980). Hotz (1972) found both gels to result in retained fluoride for 60 days after treatment.

Only one investigation reports on fluoride uptake by dentine following application of amine fluorides. Hellwig (1992) found that fluoride was taken up by dentine following one application of Elmex® solution for 3 minutes and fluoride was retained when the sample was worn in situ embedded in a partial denture for 5 days. However, comparison with a placebo or a positive control was not documented. 57

Stannous Fluoride Regimens.

The uptake of fluoride by mineralised dental tissues from stannous fluoride has not been consistently proven. Treatment with SnF: has been found to result in a lesser uptake than sodium fluoride or APF for • Sound enamel in vitro (Brudevold et al., 1956a; Cooley, 1961; Mellberg et a!., 1966; Brudevold et a!., 1967; Kirkgaard, 1977a; Dijkman et al., 1982; Sakkab etal., 1984) • Sound enamel in vivo (Heifetz et al., 1970; Mobley, 1981; Reintsema et al., 1985)and • White spot enamel (Gilley and Haberman, 1981 ; Sakkab et al., 1984) (Appendix 3, Tables 6 and 7).

However a similar increase in sound enamel fluoride concentration has been reported in vitro following application of stannous fluoride (Kirkgaard, 1977a) to that following application of SMFP (Kirkgaard, 1977a), AmF 297 (Barbakow et al., 1985) and sodium fluoride (Gilley and Haberman, 1981; Sakkab et al., 1984; Barbakow et al., 1985) and in vivo to that following APF (Brudevold et al., 1967) and MFP (Reintsema, 1985). Brudevold etal., (1956) found a similar increase in enamel fluoride concentration following application of stannous fluoride or sodium fluoride solution. A stannous fluoride dentifrice slurry had no effect.

Faller et al. (1997) found demineralised enamel exposed to a toothpaste slurry in a pH cycling model demonstrated similar fluoride uptake following exposure to a silica based sodium fluoride (1100 ppm F") and stabilised stannous fluoride. These performed significantly better than SMFP and NaF/SMFP pastes commercially available in China, although, as might be expected, exposure to silica based sodium fluoride with increased fluoride content (1450 ppm F ) resulted in greater uptake. It was noted that the sodium fluoride and stannous fluoride products had a lower pH which can enhance fluoride uptake.

Variable enamel fluoride concentrations after applying APF and SnF: sequentially or mixed have been reported but have resulted in less fluoride uptake than that by APF alone (Grail et a/., 1982; Grail and Bjerga, 1984).

However, sequential treatment of APF followed by SnF 2 resulted in retention of 58 fluoride in enamel in vitro even following washing in an inorganic wash solution with KOH for 24 hours. Treatment with stannous fluoride has been reported to result in an increase in fluoride uptake following compared with sodium fluoride for dentine powder (Dowell and Addy, 1984) and demineralised dentine (Dérand et al., 1989) has been reported (Appendix 3, Table 8). Addy and Mostafa (1988) found that pre treatment with saliva to increase uptake by dentine from stannous fluoride such that it was greater than that by sodium fluoride. Other comparative studies have not been reported.

The variability in results may be due to differences in the stability of the stannous fluoride preparations used or due to the composition of the surface products forming. Indeed, Faller et ai. (1995) investigated fluoride uptake by enamel following application of stabilised stannous fluoride toothpaste which resulted in a slight but significant decrease in fluoride uptake compared to sodium fluoride toothpaste but a significantly increased uptake compared with un-stabilised stannous fluoride toothpaste.

Amino-Stannous Fluoride Regimens.

Barbakow et ai. 1985 evaluated the enamel fluoride concentration after topical applications of NaF, AmF 297, SnF& SnFz/AmF 297(1:1) and water. Different depths of etch make assessment of uptake difficult however, all preparations resulted in uptake of fluoride (Appendix 3, Table 9).

Other investigations into the uptake of fluoride following treatment with amino- stannous fluoride regimens have not been published.

Fluoride Uptake and Caries Incidence.

It should be noted that, while a relationship between caries incidence and fluoride concentration in surface enamel has been reported (Brudevold et a/., 1956a), it has not been universally found (Gedalia etal., 1961; Aasenden et al., 1972a; Mellberg, 1977; Schamschula etal., 1979; Haberman etal., 1980; Gilley and Haberman 1981). An inverse relationship has, however, been found 59 between caries Incidence and the fluoride content of white spot enamel (Cllley and Haberman, 1981; Sakkab etal., 1984). Fejerskov at al. (1981) states that different amounts of surface fluoride can produce the same amount of carles Inhibition, this apparent discrepancy suggests that carles reduction by fluorides Is a complex process, which cannot be explained by simple reaction mechanisms. Gron (1977) suggests that that the mode of Incorporation of fluoride and Its distribution In the enamel microenvironment could be Important In determining anti-carles activity of topically applied fluorides. Furthermore, calcium fluoride deposition on the surface enamel may act as a reservoir of fluoride for remlnerallsatlon (Arends at a/., 1983b; Rolla, 1988) or the formation of surface coatings, formed for example by stannous fluoride and amine fluoride regimens, may play a role In reducing the carles susceptibility of the enamel (Crall and Bjerga 1984). This suggests that the anti-carles activity of a fluoride regimen cannot be determined by solubility or fluoride uptake studies alone but requires confirmation by clinical trial.

5.5 Surfactant Activity of Amine Fluorides.

The amine fluorides have fluoride Ions bonded to an organic fatty acid amine and so have a hydrophobic hydrocarbon chain with a hydrophilic head. This Is typical of surfactants, which are distinguished by their surface activity suggesting that amine fluorides might cover the tooth surface with a homogenous molecular layer.

Busschner at al. (1988) found a monolayer absorbed onto enamel following application of Amf 297 and AmF 335. However, Sefton at al. (1996) found absorption onto hydroxyapatlte to continue beyond the critical micelle concentration suggesting formation of a bllayer or even greater build up of surfactant molecules. Increased absorption of AmF 335 was found compared to AmF 297. It was suggested that the single charge on this molecule allowed closer packing of the molecules on the surface and Its greater hydrophoblclty would allow a greater build up of other surfactant molecules. Amine fluoride 335 was not desorbed by water or the salts tested confirming Its strong bond to hydroxyapatlte, whereas AmF 297 was desorbed by calcium chloride (at 20% of 60 its concentration in saliva) among other chlorides, by increases and decreases in pH and by EDTA (Sefton etal., 1996). The formation of such surfactant layers would encourage the formation of a homogenous layer of calcium fluoride on the tooth surface enhancing fluoride uptake at the tooth surface, as was found in Section 3.4. A prolonged oral clearance time would also be expected due to adherence of amine fluoride to the teeth and oral mucosa.

Prolonged salivary fluoride levels have been reported following the use of amine fluoride toothpaste, mouthrinse and gels compared with sodium fluoride (Hassell etal. 1971, Silvera etal. 1974; Mühlemann and Rudolf, 1975; Fritzche and Saxer, 1989; Attin and Hellwig 1996). An increased fluoride content and retention time in plaque has also been reported following application of amine fluoride compared with sodium fluoride. (Flessa and Güizow, 1970; Klimek et al. 1982).

Although similar claims have not been made for stannous fluoride, Bonesvoll and Relia (1978) found tin could be detected in saliva for up to 12 hours following a 1 minute rinse with 22.2 mM SnF 2 solution.

5.6 Anti-Microbial Properties.

Fluoride itself has been found to have anti-microbial activity, affecting a range of metabolic processes (Hamilton, 1990) but these anti-caries effects are usually regarded as of little importance compared to the interactions with the mineralised tissues (Clarkson et al. 1996). Both amine fluorides and stannous fluoride, however, have been reported to have considerable anti-microbial activity.

Amine Fluoride Regimens.

The following anti-plaque and anti-bacterial properties have been described for amine fluorides. 61

1. Reduction of Adhesion to the Tooth Surface.

Aliphatic amine hydrofluorides have been found to reduce the absorption of cariogenic organisms to whale dentine, hydroxyapatite and enamel by 10 to 80% (Olsson and Krasse, 1976; Olsson and Odham, 1978; Borggreven et a/., 1980; Adams and Salem, 1983).

It has been suggested that this reduction in adhesion may be due to the amine moiety lowering the surface energy of enamel by forming a hydrophobic layer on the tooth surface (Luscher et a/., 1974, Warner et a/., 1976, Busscher et a/., 1988). This layer inhibits plaque formation (Loesche, 1977) and forms a physical barrier that prevents the adhesion of the micro-organisms (Shern et al., 1970).

2. Inhibition of Lactic Acid Production.

Strong inhibition of salivary glycolytic activity for up to 6 hours has been found following rinsing with amine fluorides 297, 242 and 335 and their amine chloride analogues (Mühlemann, 1968; Harding et a/., 1974; Breitenmoser, 1975; Schneider and Mühlemann, 1974; Gehring, 1983). The inhibitory effects of sodium fluoride disappeared within one hour.

The inhibition has been attributed to a selective block of the glycolytic enzymes enolase, phosphoglyceromutase and glucosyltransferase, enhanced by the ability of amine fluorides to combine with lipoprotein elements of the bacterial cell wall (Capozzi etal., 1967; Harding etal., 1974; Schneider and Mühlemann, 1974; Ciardi etal., 1978).

3. Inhibition of Extracellular Polysaccharide Formation.

The effect on glycolytic bacterial enzymes may also inhibit the formation of extracellular polysaccharides. The in vitro studies of Shern et al. (1970), Swing and Crawford (1971), Dolan et al. (1972), Balmelli et al. (1974) and Lüscher et al. (1974) variously showed that treatment of stainless steel wires, ni-chrome wires and rat molars with amine hydrofluorides resulted in marked inhibition of 62 bacterial deposits and at inhibitory concentrations similar to those achieved by the equivalent chloride salts. AmF 297 was found to be most effective (Lüscher etal., 1974).

4. Bacteriostatic or Bactericidal Effects.

Amine fluorides have been found to have potent bacteriocidal activity in vitro against cariogenic organisms (Kay and Wilson, 1988; Shani et ai., 1996), mixed bacterial populations from plaque (Shern et ai., 1974; Gehring, 1983), saliva (Salem et ai., 1987) and subgingival plaque (Bullock et ai., 1989; Bansal et ai., 1990; Oosterwaal et ai., 1989,1991a,b).

The bactericidal activity of amine fluorides has not been explained but Bramstedt and Bandilla (1966) postulate that the inhibitory action is potentiated by the ability of hydrofluoride to penetrate the cell uncharged and by the surfactant property of the organic moiety disturbing the integrity of the cell wall. Breitenmoser (1975) suggests that this ability to act as cationic detergents also disturbs the potassium flux of the cell. In addition, aliphatic amines interfere with protein and RNA synthesis (Mills and Dubin 1966, Kabara et ai. 1972). The antibacterial effect also appears to depend upon the surfactant property, enabling gradual release from the tooth surface, a mode of action that has also been suggested in connection with chlorhexidine (Turesky et ai. 1972).

5. Other Anti-Bacterial Effects.

Salivary secretion has been found to be significantly greater following brushing with amine fluoride toothpaste compared with monofluorophosphate toothpaste or a water rinse (Engel-Brill et ai., 1996).

These anti-bacterial properties have been confirmed by investigation into anti plaque activity - in vivo.

Twice daily rinsing with AmF 242/335 (250 ppm F") and twice daily use of an amine fluoride toothpaste significantly reduced the plaque accumulation (Shern et ai., 1974; Lobene and Soparkar, 1974). A combination of amine fluoride - 63 zinc fluoride (200 ppm F') rinse and Elmex® toothpaste was equally as effective in inhibiting established plaque as chlorhexidine rinsing (Renggli, 1983).

Together these investigations suggested that amine fluorides can reduce viable organisms and plaque accumulation in vivo although more comparative and long term investigations need to be implemented as suggested by Overholser (1988) for the assessment of medical oral regimens.

Stannous Fluoride Regimens.

The anti-plaque activity of stannous fluoride has been established since 1959 when Konig found a daily intra-oral spray of 0.4% stannous fluoride to inhibit plaque formation in rats. The following properties have been investigated in association with this anti-plaque activity:

1. Inhibition of Lactic Acid Production.

An acid inhibition effect has been found by stannous fluoride solutions (Lilienthal, 1956; Yankell et a/., 1978a; Svatun and Attramadal, 1978; Opperman and Johansen, 1980) and stabilised stannous fluoride toothpaste (Kasturi at al. 1995; White at a/., 1995; Liang at a/., 1995; Bacca at a/., 1997). Collectively, these investigations showed that the inhibitory effect was related to

Snp2 concentration but that the stannous ion was responsible, the fluoride ion acting solely as a buffer.

2. Inhibition of Adhesion.

Glantz (1969) found stannous fluoride reduced the weight of dental plaque capable of adhering to enamel and dentine while Shern at al. (1970), Doland at al. (1972) and Miller at al. (1994) showed that treatment of steel wires with Snp 2 resulted in marked inhibition of bacterial deposits. However, "over-stabilisation” of the stannous ion with a copolymer of vinyl-methylether and malic anhydride resulted in reduced anti-bacterial activity (Miller atal., 1994). 64

Tinanoff et al. (1976) and R 0 lla (1977), using electron micrography of plaque formation, considered stannous fluoride to cause an alteration in the adhesive properties of bacteria to enamel, as well as reducing cohesive forces within bacterial colonies, thereby inhibiting plaque formation. This was confirmed by the finding that stannous fluoride rendered enamel surfaces more hydrophobic altering the absorption properties of various proteins to the surface (Kambara and Norde, 1995).

3. Bacteriostatic or Bactericidal Effects.

Considerable bacteriocidal activity has been demonstrated in vitro against cariogenic organisms by stannous fluoride solutions (Ferretti and Tinanoff, 1980; Caufield et a/., 1987), gels (Oosterwaal et a!., 1989; Tseng ef a/., 1992; Wade et a/., 1997) and stabilised toothpaste (Weber et a/., 1995; Wade et a/., 1997). Stannous fluoride preparations were more effective than equivalent sodium fluoride preparations.

Tseng et al. (1992) found bacterial inhibition by an anhydrous glycerine gel decreased with stannous fluoride concentration, a 0.4% concentration behaved similarly to 0.12% chlorhexidine.

The use of stannous fluoride rinses have also been found to have bacteriocidal activity against mixed microbial populations in saliva (Andres et a/., 1974; Svanberg and Relia, 1982; McHugh et a/., 1988) and plaque (Yankell et al., 1978b; Tinanoff et al. 1976; Gross and Tinanoff, 1977; Svanberg and Rolla, 1982).

Rinsing with stannous fluoride was found to be more effective than sodium fluoride rinsing (Svanberg and Relia, 1982; McHugh et al., 1988). Miller et al. (1994) found the salivary antibacterial activity of stannous fluoride to increase with increasing concentration, a 0.4% solution resulting in greater than 99% reduction of total salivary bacteria and streptococci. High concentration stannous fluoride gels (15% and 8% respectively) have demonstrated anti-bacterial activity in vivo against plaque and saliva mutans streptococci (Keene et al., 1977; Zickert et al., 1987). Brushing twice daily with 65

0.4% Snp2 gel was also found to result In decreases in salivary and supra gingival plaque levels of mutans streptococci compared to placebos (Potter et a/., 1984; Tinanoff and Zameck, 1985) and to sodium fluoride gels (Vierrou at a/.,1986). A reduction in actinomyces in marginal plaque has also been reported (Yoon and Berry, 1979).

One long term clinical investigation (Keene and Fleming, 1987) also found daily brushing with 0.4% SnF: anhydrous glycerine gel in head and neck cancer patients for up to 3 years resulted in a significant reduction in salivary mutans streptococci compared to use of 1.0% NaF gel. No effect was noted on levels of lactobaccillus.

These anti-bacterial properties have been confirmed by investigation into anti plaque activity - in vivo.

Rinsing with stannous fluoride has been found to reduce plaque indices compared to placebos (Yankell at al., 1978c, Tinanoff at a/., 1980; Frankel at al. 1985; Chikte at al., 1991; Cianco at al., 1992), sodium fluoride and the chloride salts of sodium, tin, aluminum, zinc and magnesium (Skjorlandat al. 1978). Furthermore, the effect of the stannous fluoride rinse was significantly greater than a placebo rinse on both developing and existing plaque (Cianco at al., 1992). Plaque scores decreased with increasing concentration of stannous fluoride (Yankell at al., 1978c). However, rinsing with chlorhexidine has been found to be more effective (Svantun atal., 1977; Chikte atal., 1991; Miller atal., 1994).

Tinanoff at al. (1989) found brushing with 0.4% SnF: gel to be highly effective in plaque inhibition, as it was in combination with 0.1% stannous fluoride mouth rinsing (White and Taylor, 1979) and when used following brushing with sodium fluoride (Boyd and Chan, 1994). However, Wolff at al. (1989) found that daily brushing with the gel resulted in no significant difference in plaque indices over 18 months compared to brushing with 0.22% NaF or a fluoride free placebo gel in addition to sodium MFP paste. These subjects however exhibited poor compliance, although reasons for lack of use were similar for the Snp 2 and placebo gels. Svatun (1978), Bay and Relia (1980), Miller et al. (1994), Kasturi et al. (1995) Weber et al. (1995), Wade et al. (1995), Addy et al. (1997), Bosma et al. (1997) and Mankodi et al. (1997) investigated the ability of various stannous fluoride (0.4%) toothpastes to inhibit plaque formation. The investigations confirmed that

SnF2 functions effectively as a toothpaste in plaque inhibition particularly when combined with stannous pyrophosphate. Although, a stabilised stannous gluconate paste (Bosma et al. 1997) had no significant effect on plaque. The differences in effect may be due to differences in the available dose of stannous ions in these preparations. Furthermore, stannous fluoride could suppress the anti-microbial activity of anionic detergents found in many toothpastes, sufficient stannous ions therefore need to be available to reinstate the lost activity of the detergent and then provide their own action (Addy et al. 1997).

However, while stabilised stannous fluoride toothpaste was found to be significantly better in inhibiting plaque forrnation than sodium fluoride toothpaste by Bacca et al. (1997) other investigations have revealed no significant difference (Perliche et al., 1995; Beiswanger et al., 1997; Owens et al., 1997). Similarly, a significant reduction compared to Triclosan pastes has not been found (Binney etal., 1997; Owens etal., 1997).

In summary while stannous fluoride preparations have been shown to possess good antimicrobial activity in vitro, they have not been universally observed to be as effective in reducing plaque in vivo. This could be due to the low substantivity, for example, when compared to chlorhexidine (Bonesvoll and Rolla, 1978; Elworthy etal., 1996) or due to insufficient bioavalibility of stannous ions in regimens for an anti-bacterial effect (Addy et al. 1997).

Amino-Stannous Fluoride Regimens.

The discovery that a combination of stannous fluoride with AmF 297 retained the anti-plaque activity associated with SnF: led to the development of amino- stannous fluoride regimens. Mühlemann et al. (1981) found AmF 297/SnF2 (in an equimolar ratio for fluoride content) to be significantly more effective than

AmF 297 and SnF 2 alone in decreasing the plaque index in a non-brushing 67 investigation on 16 patients rinsing twice daily. AmF 297 alone, however, was more effective at reducing plaque pH than the other preparations. Investigation into the anti-plaque properties of this combination has continued.

1. Inhibition of Metabolism.

AmF297/Snp2 gel has been found to reduce aerobic metabolism by salivary mixed oral micro-organisms (Bley and Güizow, 1991).

2. Inhibition of Adhesion.

AmF 297/SnF2 gel and rinse has been found to reduce adhesion of mutans streptococci but not lactobacillus to plastic strips (Ostelaet al., 1991; Soderling at a/., 1991). Toothpaste slurry was found to have no effect (Soderling at a/., 1991). Chlorhexidine products were found to result in greater inhibition and the effects were longer lasting.

Weiger at al. (1998) investigated total bacterial counts on protected and unprotected tooth surfaces following application of AmF 297/SnF2 solution. Similar bacterial counts were recored in both sites suggesting that the rinse had anti-bacterial rather that anti-adhesive action.

3. Bacteriostatic or Bactericidal Effects.

AmF 297/SnF2 solution (Raul and Netuschil, 1988) and gel (Ostela and Tenovuo, 1990) have been found to have bacteriocidal effect on cariogenic organisms in vitro. A gel containing 1.2% F was more effective than chlorhexidine (1%) containing gels, but 0.4% F" amino-stannous fluoride gel was less effective and had a similar effect to AmF 297 alone.

The use of AmF 297/SnF2 mouthrinse in vivo was found to reduce plaque vitality as shown by vital fluorescence (Raul and Netuschil, 1988, Weiger at al., 1998). In clinical trials ranging from 3 days to 3 months, use of the rinse resulted in significant reduction of plaque vitality compared to rinsing with a phenolic compound (Listerine) or a placebo (0.02% quinine hydrochloride) 68

(Brecx et al., 1990, 1992, 1993). One investigation found AmF 297/Snp2 combined with Acesulfam also performed better than chlorhexidine (Brecx at al., 1992) but otherwise no statistically significant difference between AmF 297/SnF2 and chlorhexidine have been found (Brecx at al., 1990, 1993; Netuschil ef a/., 1995).

Rinsing with AmF 297/SnF2 (0.025% F") was however found to have a non significant effect on salivary mutans streptococci counts (Hefti and Huber, 1987; Etemadzadeh at al. 1989) unlike 0.12% chlorohexidine gluconate. Neither solution had a significant effect on total colony forming units (Hefti and Huber, 1987) or lactobacilli counts (Etemadzadeh at al. 1989). Weiger at al. (1998) however found that rinsing with AmF 297/SnF2 reduced salivary levels of vital micro-organisms for 24 hours.

Investigations in lymphoma patients receiving chemotherapy (Meurman at al., 1991; Laine at al., 1993) also revealed significant reductions in mutans streptococci on twice daily rinsing with either AmF 297/SnF2 (0.025% F"). Sodium fluoride (0.05% F") rinsing also had an initial effect (two weeks). However, it was suggested that secretion of the cytostatic drugs into saliva might be having an effect, as sodium fluoride would not be expected to be bacteriocidal at that concentration.

Ostela at al. (1991) investigated an AmF 297/SnF2 gel. Professional tooth cleaning three times in 1 week with 1.2% F gel reduced numbers of colony forming units salivary mutans streptococci but had no effect on lactobaccilli or the total aerobic flora. Chlorhexidine similarly affected only salivary mutans streptococci but resulted in greater inhibition of longer duration (11 against 7 weeks). This investigation highlights the potential benefits of brushing with AmF 297/SnF2 gel but professional cleaning three times weekly is not economically or practically feasible, even in long term research programmes.

Clinical investigations on the use of toothpaste and mouthrinse regimens have also been carried out. AmF 297/SnF2 paste (0.15% F ) and rinse (0.025% F ), used over 5 months, had a tendancy to reduce salivary counts of mutans streptococci and lactobaccilli, although not significantly so, compared to a 69 sodium fluoride paste and rinse regimen of identical fluoride content (Herczegh etal., 1991).

In contrast, Mengel et al. (1996) using dark field microscopy found use of the same regimens or AmF 297/Snp2 paste combined with NaF rinse, all significantly increased cocci and decreased rods in supra gingival plaque harvested from patients over 9 months. The changes were significantly greater in the group using the AmF 297/SnF2 paste and rinse regimens. These results were similar to those found on rinsing alone with AmF 297/SnF2 (Zimmerman et al., 1993).

Together these investigations suggest that AmF 297/SnF2 regimens do have a bacteriocidal effect. This is more profound than that of phenolic compounds and of sodium fluoride but less than chlorhexidine gluconate.

4. Other Anti-bacterial Effects.

Effect on Saliva.

Salivary secretion following “one off rinsing with AmF 297/SnF2 was found to be greater than following rinsing with AmF 297 alone but similar to a water rinse (Engel-Brill etal., 1996). However, AmF 297/SnF2 (0.025% F ) and NaF (0.05% F ) rinsing over 1 year were found to have no discernible effect on salivary flow or on buffer capacity in lymphoma patients receiving chemotherapy (Laine et al. 1993).

Effect on Human Neutrophil Functions.

Amine fluoride (type unknown), stannous fluoride or AmF/SnF 2 solutions have been found to enhance by 2 to 3 fold the superoxide release of activated neutrophils and therefore the oxygen-dependent antibacterial activity of neutrophils (Shapira et al. 1997). Furthermore the effective concentration of the

AmF/SnF2 solution was several fold lower than AmF or SnF 2 alone. 70

These anti-bacterial properties have been confirmed by investigation into anti plaque activity - in vivo.

Clinical investigation has revealed rinsing with AmF 297 / Snp 2 (0.025% F") to prevent plaque accumulation compared to rinsing with tap water and Darjeeling tea (Attin et al., 1995), placebo (quinine) solutions (Hefti and Huber, 1987, Brecx et ai. 1990; Zimmerman et al., 1993; Netuschil et al., 1995), sodium fluoride (Meurmann et al. 1991; Laine et al., 1993) and phenolic solutions (Brecx etal. 1990, 1992; Netuschil etal., 1995). However, the amino-stannous fluoride rinse was less effective at preventing plaque accumulation than 0.12% chlorohexidine gluconate and a hexetidine (750 ppm) - zinc (750 ppm) combination. These findings were investigated by plaque weight (Hefti and Huber, 1987; Etemadzadeh et al. 1989), clinical plaque indices (Etemadzadeh et al. 1989; Brecx ef a/. 1990, 1992; Meurmann et al., 1991; Zimmerman et al., 1993; Laine et al., 1993; Netuschil et al., 1995) and planimetrical assessment (calculation of plaque area) (Hefti and Huber, 1987; Attin et al., 1995).

AmF 297/SnF2 rinse regimens appeared to perform better when combined with habitual oral hygiene methods than in the non-brushing investigations. This may be due to low substantivity of the regimen. If plaque is able to accumulate the regimen may not able to kill bacteria in its deepest layers (Brecx et al. 1990). Brushing may keep plaque at a level where the regimen can have an effect.

AmF 297/SnF2 gel applied daily in a custom made tray was compared with a placebo gel (Raab, 1990). Both gels resulted in a decrease in plaque indices but only the AmF 297/SnF2 gel produced a statistically significant fall. Reduction in plaque indices has also been noted following subgingival irrigation of AmF 297/SnF2 gel (Friedman etal. 1992).

Clinical investigations on the use of toothpaste and mouthrinse regimens have also been carried out. A regimen of AmF 297/SnF2 paste (0.15% F") and rinse (0.025% F"), and a sodium fluoride paste and rinse regimen with identical fluoride content, used over 5 and 9 months by adults, were both found to cause a significant reduction in plaque indices scores with a more pronounced reduction in the AmF 297/SnF2 group (Nemes et al., 1991; Mengel et al. 1996). 71

The latter study found a combination of the AmF 297/Snp2 paste and NaF rinse to have an identical effect to the all NaF regimen suggesting use of AmF 297/SnF2 paste alone is not sufficient to render these changes. This was confirmed by Sgan-Cohen et al. 1996 who investigated use of the same toothpastes alone over 6 months by teenagers. Neither regimen resulted In a significant decrease in plaque indices compared to baseline nor was any difference noted between them.

Together these investigations suggest that AmF 297/SnF2 regimens do have a anti-plaque effect which is more profound than that of phenolic compounds and sodium fluoride but less so than chlorhexidine gluconate. However, long term studies are needed.

Summary.

These studies suggest that amine fluoride, stannous fluoride and combinations of the two have potential as chemical adjuncts to oral hygiene. These preparations have been found to have greater anti-bacterial activity than sodium fluoride, but have not been extensively compared with other fluoride regimens or against each other. They are, however, less potent than chlorhexidine gluconate. Long term use of chlorhexidine is associated with unacceptable side- effects such as staining and interference with taste. The fluoride products have not been found to effect taste and amine fluoride regimens have not been reported to cause staining. Stannous fluoride regimens have been associated with staining and this appears to be proportional to the bioavailability of stannous ions (Wade at a/., 1997). However, staining has not caused unacceptable patient loss from clinical trials of these products and therefore might be assumed to be insufficient to cause concern to the general population. Discolouration has also been reported for rinsing with amino-stannous fluoride regimens but less so than chlorhexidine (Brecx at al. 1993). 72

5.7 Caries inhibition.

Amine Fiuoride Regimens.

Animal studies on caries inhibition are usually short term and involve too few subjects for meaningful analysis, however, Schmid et al. (1984b) carried out a pooled statistical analysis on 56 independant caries studies on Osbourne- Mendel rats, resulting in analysis based on 1938 rats. The analysis showed that toothpaste containing amine fluoride was significantly superior to MFP containing preparations with respect to caries prevention on smooth surfaces. In the case of initial and advanced fissure caries there was no statistically significant difference between amine fluoride and MFP preparations although both were statistically superior to the water and fluoride free toothpaste controls.

Human clinical trials on the influence of amine fluoride toothpaste on caries incidence are few (Appendix 2, Table 1) and the early trials did not meet the requirements of controlled clinical trials of caries preventive agents as adopted by the FDI in 1981 (Ainamo, 1982). Although later studies fulfilled these requirements the trials varied in design, methodology and data presentation. Results of individual trials are therefore not comparable. This is frustrating as trials have usually compared the amine fluoride regimen with fluoride free placebo. Variations in design mean that they cannot be used to evaluate the relative efficiency of various formulations against trials carried out with other fluoride regimens.

Nevertheless, clinical trials in children and adolescents using various amine fluoride toothpastes unsupervised (Marthaler, 1965, 1968, 1974; Patz and Naujoks, 1970) found a reduction in caries incidence of 7 to 30% compared to the use of non-fluoridated paste.

The effect of amine fluoride toothpaste used twice daily (with supervised brushing in school) was also investigated in a community preventive programme (Pakhomov at al. 1997). The toothpaste was provided to 12,500 children aged 3 to 12 years for 3 years. Random samples of children were selected from the programme and from a reference community at the start of 73 the study and after 3 years. A statistically significant reduction in mean DMFT scores was noted in 6 and 12 year olds in the programme (86% and 25% respectively) whereas 9 year olds and all three age groups in the reference community had non-significant differences in mean DMFT. Unfortunately the use of toothpaste by children In the reference community is not recorded making it difficult to draw conclusions other than that a community based fluoride toothpaste implemented programme successfully improved the caries status of children.

Two trials have been conducted against positive control toothpaste (Appendix 2, Table 1C). A 36 months clinical trial comparing amine fluoride and monoflurophosphate toothpaste (Cahen et a/., 1982) in 6 to 8 year olds showed a highly significant reduction in DMFS scores for both products compared to a placebo paste. The amine fluoride group performed notably better, but the statistical difference between the products is not reported. However Ringelberg at al. (1979) reporting on an eighteen month clinical study on the use of amine fluoride and stannous fluoride toothpastes by school children found neither dentifrice to be significantly superior to a fluoride free control. The composition of pastes used in this trial is not reported.

It may be concluded that the use of amine fluoride toothpaste in children and adolescents, while proving beneficial compared to fluoride free pastes in preventing caries development, have not yet been established as beneficial compared with positive controls.

The use of higher concentrations of amine fluorides at less frequent intervals have also reported to have an effect on caries incidence in a wider age range (Appendix2, Table 3). Obersztyn etal. (1979, 1984) investigated military cadets aged 19 and 20 and found 6 monthly prophylaxis with a stannous fluoride paste followed by application of a stannous fluoride solution or weekly brushing with an amine fluoride gel to be highly statistically significant in reducing DMFS scores compared with no prophylaxis. However, despite the difference in frequency of the fluoride regimens the difference in effectiveness of the two was non-significant. Denes and Gabris (1991) found, in teenagers, that weekly brushing with Elmex® gel over 3 years to be significantly more effective than 74 use of fluoride free toothpaste alone unlike 2 to 3 weekly professional application of Elmex® fluid. However, the fluoride concentration or composition of the respective treatments was not recorded.

Ran et al., (1991) examined 112 children aged 13 and found a group brushing with Elmex® gel (1.25% F") had a significant decrease in DMFS compared with a group brushing with Elmex® toothpaste (0.125% F"). There was no significant difference between the reduction in DMFS scores between children using a placebo gel, a 0.4% F" gel and the 1.25%F" gel. Only the latter, however, resulted in no increase in DMFS score. The differences however were lost 6 months following cessation of the gel, suggesting that long term use is required for continuation of the protective effect.

Kunzel et al. (1977) reported on a 7 year study, on children aged 6 and 7, receiving professional applications of amine fluoride solutions and using amine fluoride toothpaste which resulted in a reduction of caries incidence by 42% compared to use of a placebo paste. Lincir and Rosin-Grgret (1993) and Rosin- Grgret and Lincir (1995) studied children aged 3 to 4 and 9 to 10 receiving professional applications of amine fluoride solutions. Those treated with 0.5%F' solutions monthly or 1.0%F" solutions once every two months had significant reductions in DMFS compared to those treated with solutions of lower fluoride concentration, applied less frequently. Brambilla et al. (1997) carried out an investigation on 6 year olds using a 1% fluoride toothpaste and receiving professional application of either Elmex® fluid or a non-fluoride placebo fluid twice yearly. A highly significant reduction in DMFT (23%) in first permanent molars was found in the children using the Elmex® fluid.

These investigations highlight the increased anti-caries activity obtainable by increasing the concentration or frequency of fluoride applications. But, additional benefit from the use of such topical applications in conjunction with amine fluoride toothpaste has been established (Marthaler 1970); although this was significant only for the first year, in the three year trial, when applications of amine fluoride gel were applied weekly. Ringelberg et al. (1979) also found the use of amine fluoride mouthrinse in conjunction with amine fluoride toothpaste to be more effective than either used alone (Appendix 2, Table 2). In addition 75 this regimen was more effective than a stannous fluoride toothpaste and sodium fluoride mouthwash combination. This is the only investigation that has shown an amine fluoride regimen to be superior to a positive control regimen but a sodium fluoride mouthrinse and placebo toothpaste regimen was equally effective.

Stannous Fluoride Regimens.

The use of stannous fluoride toothpastes in over 40 clinical trials has been estimated to result in mean caries reduction of 22% (Mellberg, 1991) and 25% (Richards and Banting, 1996) compared to placebo preparations. The variability in results has been suggested to be due to instability of the stannous ion (Richards and Banting, 1996). A few in vivo investigations on the anti-caries activity of stabilised stannous fluoride preparations have been reported (Appendix 2, Table 2B)

Klock et al. (1985) found twice daily rinsing with 0.1% Snp 2 over 2 years resulted in a 33% reduction in caries compared to NaF rinsing.

Faller at si. (1995) reported on a rodent caries incidence trial. A stabilised stannous fluoride toothpaste resulted in a significantly greater reduction in caries than an unstabilised stannous fluoride toothpaste and placebo toothpaste but sodium fluoride toothpaste resulted in a similar reduction.

Human clinical trials have not yet been reported for the toothpaste. However, stabilised 0.4% stannous fluoride gel was more effective at preventing décalcification in adolescents wearing fixed orthodontic appliances, especially when brushed daily, than use of toothpaste alone (Stratemann and Shannon, 1974) and than daily mouthrinsing with 0.05% sodium fluoride mouthrinse (Boyd, 1993). Furthermore the gel, when applied 3 to 4 monthly, resulted in a trend towards decreased root caries indices following periodontal surgery compared to 5% sodium fluoride varnish (Ravald and Birkhead, 1992). 76

Amino-Stannous Fluoride Regimens.

Amino-stannous fluoride preparations have not been tested in clinical caries trials except for two studies on root caries (Appendix 2, Table 3). Ueberschar and Gunay (1991) found root caries incidence to be reduced post-periodontal surgery in patients using AmF 297/Snp2 rinse compared to a control group. This was close to significance (p = 0.06). Bânôczy and Names (1991) and Names et al. (1992) also found a AmF 297/Snp2 toothpaste/mouthrinse regimen to produce a reduction in root caries indices compared to a sodium fluoride toothpaste/mouthrinse regimen but the reduction was not statistically significant.

Summary

These products have not been fully investigated in controlled clinical caries trials against positive controls. Furthermore the regimens have been investigated for their effect on enamel caries, largely in children. The effect on root caries in older populations has not been investigated yet this is recognised as a high risk group and one that is expanding (Section 3).

While, the amine fluoride regimens have been established as effective against enamel caries this has largely been in comparison to fluoride free placebos. When comparing anti-microbial properties of toothpaste (Addy at a/., 1992) and using in situ models to test fluoride containing systems (Faller at si. 1995), it has been suggested that new preparations should be tested against a toothpaste commonly available to the general public. This has been termed a “benchmark control” (Addy at a/., 1992) or “gold standard” (Duckworth, 1995) and is required to assess the effect of the test product over and above that achieved by such a product. Furthermore, if the control could be standardised then comparison of agents would be simplified (Binney at a/.,1996).

It would seem rational to apply this to clinical caries trials. However, it is possible that different active systems exhibit their anti-caries effects in different ways, the choice of this control is therefore important if the assessment of a new product is to be meaningful. An alternative is to compare the test product with a control identical to the test product except for substitution of the active 77 ingredient. The control active ingredient needs to be carefully selected so that it is, not only compatible with the other ingredients in the product, but is also recognised as the current accepted standard regimen. As discussed previously (Section 3) this would arguably be sodium fluoride with a suitably compatible abrasive. This product has indeed been recognised as a reference standard by the American Dental Association to which other toothpastes in the United States are compared (Faller etal. 1997). 78

6. The Measurement of Fluoride Content of Dental Mineralised Tissues.

One method of assessing the relative merits of fluoride regimens is to measure the fluoride uptake by the mineralised dental tissues. But to do this the tissues need to be biopsied and the resultant sample analysed for fluoride.

6.1 Techniques for Biopsy of Dental Mineralised Tissues.

Methods of biopsying mineralised hard tissue have been extensively reported but all present with problems and most are suitable for sampling only fully mineralised dental enamel.

The following techniques are available:

abrasion micro-abrasion acid dissolution micro-drilling sectioning.

Methods of Dissecting Dental Mineralised Tissues by Abrasion.

An abrasion technique was first described by Brudevold et a/. (1956b) and the technique has been modified and used for sampling enamel both in vitro and in vivo (Table 2).

But, despite increasing sophistication such that elaborate and expensive apparatus is required, problems with the technique have not been overcome. Mellberg at a/. (1976) found that the uniformity of abrasion of enamel in the windowed area was poor and on the rounded surfaces of canines and premolars entire portions of the enamel were left unsampled. 79

Table 2. Methods of dissecting enamel by abrasion

INVESTIGATION METHOD OF SAMPUNG

Brudevold etal. (1956b) Grinding layers of enamel from extracted tooth with diamond stone.

Gedalia etal. (1961) As above using a rotating diamond wheel. Depth assessed by measuring opposing tooth surfaces with a micrometer.

Brudevold etal. (1968) Silicon carbide impregnated cones to polish tooth surface in vivo enamel collected In glycerine.

Aasenden etal. (1972b) As above plus area to be polished isolated and depth calculated from specimen weight.

Mellberg etal. (1973) As above plus pressure applied to handpiece controlled and speed of rotating cone limited.

Wei (1973) As above plus improved speed control under varying loads.

Wei and Wefel (1975) As above plus number of revolutions of handpiece pre selected.

Mellberg etal. (1976) As above but foam laminate applicator used to polish surface.

It is possible then that reported differences in surface fluoride concentration may be accounted for by variation in the amount of enamel in the window area that was abraded. Furthermore, the surface of enamel has many irregularities including perikymata, brochs and pits (Newman and Poole, 1974) and any abrasion system is unlikely to remove a perfectly uniform layer of enamel measuring only a few millimetres or less in thickness. The crests of the perikymata and other raised sites will be abraded more heavily than adjacent troughs or depressions. 80

Another problem with this technique lies in the method of calculating the depth of the abraded layer. This is estimated from the following formula;

Depth = ______Weight of the Specimen ______Density of Enamel x Surface Area of the Specimen

Generally the weight of the specimen is obtained by determination of the calcium content and by assuming an average enamel density of 2.95 gcm*^ (Weidmann et al., 1967) and an average calcium content of 37.4 % by weight (Soremark and Samsahl,1961; Robinson ef a/.,1971). But the non-homogenous nature of enamel means some variation from these average values is to be expected. For example, Robinson et el. (1971) found calcium content ranged from 30 to 40 % by weight depending on the region of the tooth sampled and Weidmann et el. (1967) found the surface enamel density of incisors alone ranged from 2.84 to 3.01 gcm"^. If the sample should have a calcium content and density at the extremes of these ranges the calculated depth of the specimen may vary as much as 20% from the actual depth.

These methods were developed to sample enamel, they could, however, be applied to the biopsy of dentine or other mineralised tissues. However, dentine is very much more heterogeneous than enamel, partly due to variation in peritubular dentine formation.

Hence, any reported differences in surface fluoride content of mineralised tissues when biopsied in this manner may in fact be due to variation in the amount abraded, in any event, when abrading partial loss of the sample is unavoidable.

Iyer et el. (1983) described an acid abrasion technique in which 0.5M perchloric acid was added to the slurry produced by a foam cylinder and silicon carbide paste after abrading an exposed area of a tooth and then continuing abrasion. The authors found that the acid-abrasion procedure produced relatively uniform abrasion depths compared with abrasion alone and found it to be a less tedious method of biopsying enamel when sampling to depths of 50 pm or more were required. It also reduced the problems of the non-uniform surface layer of 81 enamel as outlined above. However the problems inherent in the acid etch techniques described below will then influence the data.

Methods of Dissecting Dental Mineralised Tissues by Micro-Abrasion.

This technique, developed by Weatherell etal. (1985) employed abrasion and a Mikrokator (CE Johansson, UK) to measure the thickness of the material removed. The specimen was fixed by adhesive to the end piece of the Mikrokator so that when lowered on to the baseplate a reading could be taken. An abrasive strip was then placed under the specimen and moved backwards and forwards across the face of the specimen. By removing the strip and bringing the specimen back into contact with the baseplate the thickness of specimen removed was indicated on the Mikrokator dial. The authors found that the resolution of the Mikrokator was in the order of 1pm. But, care was needed to ensure that particles of sample or abrasive were not trapped between the ground surface of the specimen and the baseplate.

The sample could be recovered from the abrasive by dissolving it in a small quantity of acid pipetted on to the abrasive surface and then collected by suction, but the authors encountered some difficulty with fluoride recovery from the abrasive by this method. An alternative would be to place both abrasive and sample into the acid, or the abrasive and sample could be ashed prior to sample dissolution. Care was needed to ensure collection of all the abraded dust, most collected on the abrasive but some stuck to the specimen.

This potential loss of material from the specimen made the technique less than ideal for determining the total of any component within a sample or for determining concentration on a volume basis. Furthermore, any abrasion system is unlikely to remove a uniform layer of enamel measuring only a few microns in thickness. Thirdly, this is very much anin vitro technique. However, it can be used to biopsy any of the mineralised tissues. 82

Methods of Dissecting Dental Mineralised Tissues by Acid Dissolution.

Removal of more uniform layers of a desired thickness was made possible by an in vitro technique described by MCihlemann et al. (1964). Extracted premolars were exposed to 2M perchloric acid and the acid was then analysed for fluoride content. An indication of the depth of etch was given by measuring the diameter of the crown using a matrix to ensure exact repositioning of the tooth. But the depth of etch Is unlikely to be even over the surface of the tooth and was shown in the study to vary with differences in the size of the teeth. The method also assumes that mineral concentrations are uniform over the whole surface of the tooth but this is not the case (Robinson etal., 1971).

Similar in vitro techniques have been devised and modified (Table 3) such that small, known areas of tooth surface are etched, the “micro-etch” techniques.

Hotz et al. (1970) and Brudevold et al. (1974) both suggested that an etch method of biopsy might be used in vivo. The former found the technique easy to perform on the buccal surfaces of anterior and premolar teeth and also showed that with polishing and topical fluoridation after treatment no aesthetic deficiencies remained. Munksgaard and Bruun (1973) first reported the use of an acid etch biopsy technique in vivo. The area to be sampled was limited by a piece of sticking plaster, covered with aluminium adhesive tape, punched with a circular perforation. Etching was carried out with 0.5 M perchloric acid. Variations of the technique have been used routinely for sampling sound enamel in vivo.

Patterson et al. (1984) stated that a similar acid etch technique could be used to sample hard tissues other than enamel if it could be guaranteed that a discrete sample of material was removed, that is the etching acid does not diffuse beyond the sampling site. If diffusion does occur material may be leached from adjacent parts of the tissue or dissolved material may be flushed from the etching site into the adjacent porous tissue. 83

Table 3. Methods of dissecting enamel by acid dissolution.

INVESTIGATION METHOD OF SAMPUNG

Mühlemann etal. (1964) Extracted tooth exposed to 2M perchloric acid, depth of etch measured from diameter of tooth.

Cooley (1961) Enamel section immersed in 2M perchloric acid.

Ohmohl etal. (1964) Surface of enamel section exposed to 0.5M perchloric acid. Depth of etched layer calculated from surface area and weight of sample (calculated from calcium content).

Weatherell and 60% perchloric acid used to etch enamel; regions excluded from Hargreaves (1964) etch by nail vamish; depth of etch confinned by measurement from ground sections under LM and by microradiography.

Hotz etal. (1970) 2M perchloric acid used to etch area of enamel, area limited by copalite vamish.

Weatherell etal. (1973) 1M perchloric acid used to etch area of enamel, area limited by nail vamish.

Wei etal. (1974) 0.25M perchloric acid used to etch area of enamel, area limited by pre-punched hole in adhesive tape.

Brudevold etal. (1974) 1.6M hydrochloric acid plus 70% glycerol used to etch enamel, "strong" acid to avoid re-precipitation, glycerol to retard dissolution. 84

To investigate this the authors added a saturated solution of acid fuchsin dye in equal volume to a 2M solution of perchloric acid to indicate any tendency for the acid to spread. They found that even in apparently sound enamel, for example in young teeth or teeth showing mild fluorosis, dye could spread beyond the intentional sampling site. The validity of a sample obtained by acid etching in such cases is doubtful. On sampling dentine the authors found that the dye did not usually spread beyond the sampling site and they stated, therefore, that this tissue could be sampled satisfactorily by acid etching. But in the case of cementum only occasionally was the etching solution confined to the sampling site and sampling of bone was never satisfactory.

This would suggest that acid etch techniques cannot be used for biopsying bone and cementum, must be used with caution in enamel and can be used in dentine. However, the tubular structure of dentine would seem to prohibit the use of acid etching and etch techniques have not found general acceptance for sampling this tissue. Pattersonet al. (1984) did suggest that if the tissue was embedded in methyl methacrylate (first coating the surface required for etching with gelatine) to prevent spreading of the etching acid and dissolved sample, biopsy by etching could be carried out. A series of layers could not be sequentially etched by this method. The authors also point out that a dye technique cannot be used in vivo as the tooth would be permanently stained if organic debris or exposed dentine was present.

In summary enamel sampling by acid etching is practical, has a much greater resolution than most of the mechanical approaches, is convenient and has been used routinely as a sampling method in studies of sound enamel. Unfortunately different conditions, acid strengths and etching periods have been published although most authors use perchloric acid of 0.5 to 2 M concentration. The technique does, however, have several disadvantages:

I. It can only be used on sound, mature enamel. If the enamel is demineralised, carious or immature, the acid diffuses away from the sampling site or is blocked by an insoluble organic component. Enamel may appear sound and yet spread of the acid can occur (Patterson et a/., 1984). For similar reasons, the technique cannot be used to sample other biological material such 85 as bone, dentine, cementum, calculus or acid-insoluble non-biological material.

II. The depth of the etched layer is estimated from the formula:

Depth = ______Weight of the Specimen ______Density of Enamel x Surface Area of the Specimen

The same problems then occur as described for the abrasion method of biopsy.

Furthermore, Dijkman and Arends (1982) found that the resulting distances between the control and the acid etched surfaces determined by chemical and optical depth determination, in most cases showed a statistically significant difference, the optical depth being greater than the chemical depth. The difference was as much as 50% for longer exposure times. The reason for this discrepancy was thought to relate to the prism structure of enamel.

III. Weight estimation is based on the assumption that the fluoride, calcium and phosphate found in the solution are derived from hydroxyapatite and fluorapatite. Steams (1972) states this assumption is generally valid but fails if some non-apatite material is formed such as when the tooth has been treated with stannous fluoride. Similarly the assumption must be invalid if an adherent layer of calcium fluoride forms as when the tooth has been treated with an amine fluoride (Lutz, 1983).

Wei et al. (1976) suggest that both calcium and phosphate determinations be used to calculate the depth of etch in case calcium or phosphate-coatings have occurred.

IV. These estimations are based on the assumption that etching does not occur beyond the designated surface area. This was not found to be the case (Weatherell et a/., 1973, Patterson et a/., 1984). Furthermore Van der Merwe (1974) noted that etched areas ranged from 96 to 105% of the designated surface area.

V. It is not possible to standardise the depth of etch as the rate of acid dissolution varies depending on the region of the tooth sampled as well as 86 varying from tooth to tooth. This makes comparisons of fluoride content difficult. So that meaningful comparisons of the fluoride contents of different groups of teeth can be made at a standardised depth, a number of regression based techniques have been reported (Aasenden and Moreno, 1971; Bookstein etal., 1976). These techniques however are only applicable when the number of samples is large, because they assume that the enamel fluoride contents of all the samples within a given experimental group fall on one fluoride-depth profile. Alternatively Chow at ai. (1985) describe a polynomial function for fluoride gradients to enable calculation of fluoride content at standardised depths. However, due to the inherent variations in biological materials it must be more appropriate to use a biopsy technique which enables accurate and reproducible measurement of depth of specimen.

Methods of Dissecting Dental Mineralised Tissues by Micro-Drilling.

A micro-drill technique was developed by Haberman at a/. (1980) for biopsy of white spot lesions. White spot enamel was biopsied by excision using a specially constructed micro-drill with which depth control of 11 pm in 100 pm was demonstrated. Sakkab at a!. (1984) describe the technique in more detail; a microscope stand and air turbine drill with an inverted-cone carbide bur was used. Calibrated fine focus adjustment which translates the microscope stage was used to control the depth of sampling. Static electric charge was minimised by enclosing the entire micro-drill in a clear plastic tent and purging it with water-saturated nitrogen. The enamel powder was collected, weighed and analysed for fluoride. A metallographic microscope was used to determine the width and depth of the sample pit.

The bulk of the apparatus means this is very much an in vitro technique but its use has been described for in situ studies where samples of enamel were mounted in removable prostheses (Mobely, 1981). The technique has also been used to sample dentine (lijima at a/., 1993)

The micro-drill biopsy technique has several advantages over acid etch biopsy. It can be used to biopsy any of the mineralised tissues. If required, rather than relying on average values, density can be estimated from sample weighing and 87 the calcium content from atomic absorption of analysis of calcium, eliminating error due to biological variation. Variability in sampling depth is negated by defining the depth of drilling, and the depth and diameter of the sample volume can be confirmed by measurement. Furthermore, the method reflects total fluoride uptake rather than calculation of fluoride content per gram. But this technique cannot be adapted for in vivo sampling and there is a possibility of sample loss. The dust may be lost to the atmosphere or adhere to the apparatus itself.

Methods of Dissecting Dental Mineralised Tissues by Sectioning.

A technique of section analysis would overcome the problems of possible sample loss inherent in drilling and abrasion techniques, while enabling surface area, weight and depth of the sample to be measured.

Such a technique was used in vivo by Candeli et al. (1967) on deciduous teeth. Incisions were made with a fissure bur at high speed to delimit the enamel section, which was then biopsied with a scalpel blade. Dentine was then removed from the sample. One or more additional sections could be prepared from the same tooth. The resultant cavity was restored with amalgam. This technique solved problems inherent in other in vivo biopsying techniques, but may present ethical problems.

Weatherell and Hargreaves (1965) developed an etch sectioning technique to enable the removal of small particles from predetermined sites of enamel. Sections were cut through the tooth and ground to approximately 100 pm. The sections were thinly coated on all sides with clear, diluted nail varnish. Coincident cuts were then made through the skin of the nail varnish with a scalpel blade on both sides of the specimen. The section was placed in 15% perchloric acid for 10 to 15 seconds so that the acid penetrated the scalpel cuts and etched into the underlying enamel, separating the areas delineated by the scalpel marks but stopping just before the particles were completely separated from the surrounding tissue. The specimens could then be removed one at a time with a pointed scalpel blade for analysis. But, this technique is limited to sectioning of sound enamel in vitro. 88

Woltgens etal. (1980) described an in vitro micro-sectioning technique. A rod of enamel 1 mm^ in cross section was prepared by cutting perpendicular to the smooth surface of the tooth. This was embedded in methyl methacrylate and a microtome used to obtain slices parallel to the surface with a thickness of 10.0 ± 0.5 pm. The volume of each slice was known within ±7% (Woltgens et al., 1981).

Talib et al. (1993) also described an in vitro sectioning technique. Two adjacent enamel sections 4 mm^ in cross section were biopsied from the middle third of the buccal aspect of an extracted tooth using a band saw. The surface enamel was then biopsied at a depth of 0.7 mm using a diamond bur in the air turbine. One section acted as a control and the surface of the test section (isolated by varnish) was subjected to various fluoride regimes. The whole specimens were then dissolved in 1M hydrochloric acid and subjected to fluoride analysis. The technique utilised the fact that fluoride uptake by enamel following topical fluoride application is a surface phenomenon, involving the outermost 100 pm (Arends et al., 1983b). Below this depth enamel has a relatively low and stable fluoride content. By the use of a sample depth of 700 pm the total fluoride in this fluoride rich layer can be assessed although differences in volume between the specimens will result in variation in the calculated fluoride uptake. Furthermore this technique cannot be used for profiling of fluoride content, for example, from the surface to the interior of a specimen.

The sectioning techniques, however, have much to recommend them. They allow a sample of tooth tissue of known surface area, depth and weight to be obtained without loss of part of the specimen and without relying on properties of the tooth tissue to be sampled (for example requiring it to be non-porous). The techniques can be used for analysis of specimens subjected to regimes in vitro and of extracted teeth following a clinical trial. In situ techniques are also practical whereby specimens are attached, for example, to removable prostheses and subjected to clinical trial before removal and analysis (Meckel and Francis, 1964). Biopsyin vivo is not however possible due to the size of the sample that must be removed for ease of handling, except on those occasions when removal of tooth tissue is scheduled to occur, for example, the tooth is to be re-contoured or prepared for a restoration. 89

In summary, the ideal biopsying technique would allow a sample of tooth tissue to be obtained of known surface area, depth and weight without loss of part of the specimen and without relying on properties of the tooth tissue to be sampled. Ideally it would also be suitable for in vitro and in vivo sampling.

6.2 Techniques for Analysis of the Fluoride Content of Dental Mineralised Tissues.

Following biopsy of the dental mineralised tissue a number of techniques are available for analysis of the fluoride content of the sample. Table 4 lists the alternative approaches available for the determination of fluoride in biological materials (adapted form Venkateswarlu, 1977,1990). Prior to the seventies the common method in use involved a gaseous diffusion procedure (of hydrogen fluoride from perchloric acid) to separate the fluoride from interfering substances with subsequent estimation by a colorimetric procedure (Wharton, 1962). Up to 4 \ig of fluoride could be separated by this method with a reproducibility of 0.05 pg at the Ipg level. However, the method was tedious and required adjustment of sample size to the narrow range of fluoride necessary for an accurate determination. PRETREATMENT OF SAMPLES SEPARATION & CONCENTRATION OF FLUORINE FINAL MEASUREMENT OF FLUORINE

None Distillation (SiF^, HzSiFe) Titrimetry Unmasking the fluoride ion Diffusion (HF, Trimethylfluorosilicane) Spectrophotometry Open ashing Pyrohydrolysis (HF) Fluorimetry

Fusion Pyrolysis (NH4F) Electrochemical methods: Preliminary distilliation Solvent extraction: Spontaneous electrolysis Confined combustion: Complexes of Sb, Ta, La Polarography Oxygen flask Fluorosilicates Potentiometry (Fluoride electrode) Oxygen bomb Organic fluorine Molecular / atomic absorption Tubular furnace Separation as fluoride ion: Gas chromatography Oxyhydrogen flame Ion exchange Ion chromatography Digestion with acid Chromatography Mass spectrometry Digestion with alkaline Electrodialysis Helium microwave plasma detector Fusion with alkali metals Adsorption Polarography Reduction with alkali metals in organic solvents Reverse Extraction Catalytic methods Wet digestion with Teflon decomposition bomb Enzymatic methods Microwave acid digestion bomb Radioanalytical methods

Table 4. Alternative approaches available for the determination of fluoride in biological materials (adapted form Venkateswarlu, 1977,1990 and Grobler and Louw, 1998). (Each phase may be used in combination with the different methods in the other phases)

S 91

In 1966 F rant and Ross reported on the development of an electrode sensor. The electrode consisted of a membrane constructed from a section of single crystal rare earth fluoride, usually lanthanum fluoride, sealed to the end of a rigid polyvinyl chloride tube. The tube was filled with a solution containing fluoride and chloride ions (typically 0.1M sodium fluoride and 0.1M potassium chloride) and the electrode contact was made with a silver chloride wire. The electrode was calibrated on known sodium or potassium fluoride solutions and used with an external reference electrode such as the saturated potassium chloride calomel type. Since the crystal was permeable only to fluoride ions the cell potential was given by the equation;

Cell potential, E = g - R T ______F In (Fluoride ion activity in sample)

R = Gas Constant T = Absolute Temperature F = Faradays Constant The value of a depends on the choice of internal and external electrodes and the fluoride activity in the internal electrode solution and, except for a small liquid-liquid junction potential, a is constant and independent of composition of the sample.

The electrode was found to have a linear response when electrode potential was plotted against the logarithm of fluoride concentration (a Nernstian response) for a concentration of 10"® to 1 M (0.2 to 20,000 ppm) fluoride ions. Below this a non linear response was obtained, requiring a number of standards to be measured but that is still usable to approximately 10*^ M (McCann, 1968; Gron etal. 1969).

The electrode was highly specific, the only significant interference coming from hydroxyl ions. In pure solutions, significant hydroxide interference occurred when the concentration of the hydroxyl ion equalled the concentration of fluoride and a tenfold excess of hydroxide doubled the apparent fluoride content. The interference became more serious in dilute solutions of fluoride. 92

An increased deviation from linearity was also observed in the presence of phosphate ions at neutral pH and fluoride concentrations below 10~^ M. This has been attributed to the ability of phosphate to form strong complexes with lanthanum. But, when measurements were made at lower pH (4.75) this interference disappeared and the response became linear to less than 10*^M (Gron et al. 1968). However, decrease in electrode activity occurred at pH 4 to 5 due to the fact that the pK of HF is 3.14. (Frant and Ross, 1966)

Aluminium also interfered significantly with electrode measurements due to complex formation. This was prevented by the use of buffers containing citrate ions (McCann, 1968) or use of total ionic strength adjustment buffer (TISAB) (Zober and Schellmann, 1975).

The single fluoride electrode has been used for direct determination of fluoride in drinking water (Frant and Ross 1966); in saliva, serum and blood (Cron etal., 1968; Singer and Armstrong, 1969; Ekstrand 1977) and in bone ash and mineralised dental tissues digested with perchloric acid (Singer and Armstrong, 1968, McCann, 1968). Measurement of fluoride by serial etching of enamel has also been described (Kirkgaard etal. 1976).

The use of the single electrode therefore had the advantage of permitting direct measurement of fluoride concentration, requiring no separation procedure or adjustment in sample size (McCann, 1968). The electrode stabilised quickly, solutions with a fluoride concentration greater than 10^ M generally equilibriated in less than 10 minutes, although solutions with lower concentrations took 30 minutes or more to reach a steady state (Raby and Sunderland, 1967). When a series of readings were taken of similar fluoride concentrations equilibrium was approached more rapidly (McCann, 1968). Conditioning the electrode in a fluoride free buffered solution was also found to improve its accuracy (Levine, 1973).

Moreover, comparisons of analysis by the single fluoride electrode and by diffusion and colorimetric methods gave concordant results for enamel, dentine, bone and ashed human sera but with lower standard deviations occurring with the electrode method (MaCann, 1968; Singer and Armstrong, 1969). The 93

accuracy of measurement has been found to be such that when measuring saliva observations could be made with a standard deviation of 2% at concentrations of fluoride down to 0.01 ppm (Yao and Gron, 1970).

The main disadvantages of this technique were the relatively slow response of the electrode and that electrode sensitivity was limited unless volumes exceeding 0.1 ml were used (Yao and Gren, 1970). Methods were developed for use with small sample volumes for example, inverting the electrodes (Weatherell etal. 1973), the hanging drop electrode assembly (Venkateswarlu, 1974) or construction of microelectrodes (Vogel et al. 1983). However, evaporation of the test solution and dilution from the electrode solution could effect accuracy of measurement when small sample volumes were used and prolonged electrode equilibrium times were required (Tyler and Comer, 1985).

Diggens and Ross (1981) showed that these disadvantages could be overcome if the electrochemical analysis of fluoride was carried out using a differential cell consisting of a combination glass pH electrode and a single fluoride electrode at pH values below 3.2, the pK of hydrofluoric acid. Under these conditions hydrofluoric acid would be the predominating species but F" ions at low concentrations would also be present and it was to these that the F" electrode responded. The behaviour of the differential cell conformed to a full Nernstian response where the potential difference established between the electrodes was a logarithmic function of the total fluoride (hydrofluoric acid + F") in solution.

Tyler and Comer (1985) investigated the behaviour of the differential electrode for the direct determination of fluoride in acid solutions of fluoroapatite of stoichiometric composition. They found that it was essential to immerse the electrode membranes completely into the test solutions and to use a magnetic stirrer with an isolated electric motor to avoid changes in Nernstian response due to temperature variation.

While Diggens and Ross (1981) recommended pH values be adjusted to below 2.5 for fluoride analyses, Tyler and Comer (1985) found a Nernstian response was attained for pH values between 1 and 4 for fluoride concentrations between 0.001 to 0.1 ppm at 20°C. Linear deviation for a fluoride concentration of less 94 than 0.005 ppm occurred. The authors suggested that this was due to nanogram amounts of fluoride in de-ionised water and hydrochloric acid used in the experiments. No response was observed for this system using TISAB at pH 5.5 for fluoride concentrations of less than 0.1 ppm. Fluoride kinetics were also impaired at pH 0.1 as very few fluoride ions were present in solution.

It is not known why the differential electrode shows a lower limit to the Nernstian response under experimental conditions of low pH compared to the usually observed limit of 10*^ M fluoride in solution above pH 4 with the single electrode. It is generally held that the lower limit of Nernstian response is determined by the finite solubility of the membrane used in an ion-selective electrode. The fluoride electrode responds to fluoride released by its own dissolution. However the authors speculated that the lower limit was also determined by other factors, such as reduced interference from the hydroxyl ion in acid solution.

Tyler and Comer (1985) found that the mV recorder reading showed a 95% response rate within 30 seconds for a 0.005 ppmF" addition to a vigorously stirred 1 ml sample of 0.1 M HOI, complete equilibrium being obtained within 2 minutes. The differential fluoride cell functioning at a low pH showed a Nernstian type response extended by one decade towards the lower limit of detection compared with the TISAB method for fluoride determination. Furthermore, although it is well known that hydrogen fluoride readily reacts with glass, no deterioration or loss in response of the glass membrane of the pH electrode was observed during 12 months of continuous use. The authors suggested that the fast response of the fluoride differential cell and the low concentrations of fluoride analysed were not conducive to fluoride-glass reactions.

Tyler and Poole (1989) compared the differential electrode with the single electrode in measuring F" in saliva. The differential electrode was found to be as accurate and as consistent, with the marked advantages of requiring a small sample volume (0.05 versus 1.0ml), achieving electrode equilibrium in a short time (2 versus 20 minutes) and having greater sensitivity of the differential cell to low levels of fluoride. 95

6.3 The Analysis of the Fluoride Content of Dentine.

The analysis of the fluoride content of samples of enamel is now well established. The technique involves acid digestion of the sample and analysis with a differential fluoride electrode cell. This was initially carried out by Tyler and Comer (1985).

The single fluoride electrode has been extensively used to analyse the fluoride content of samples of dentine (McCann, 1968; Hellwig and Klimek, 1991; Hellwig, 1992; Tsanidis and Koulourides, 1992; Hellwig and Attin, 1994) but the use of the differential cell has not been described. There is no reason to suppose that the advantages of this technique (described above) will be any different whether measuring fluoride from enamel, dentine or other mineralised tissue.

However, the problems associated with use of acid digestion to separate the fluoride content of dentine have not been fully addressed. McCann (1968) found when comparing fluoride analysis by diffusion and colorimetry with the fluoride ion electrode, that the fluoride content of un-ashed dentine and ashed dentine were identical once the latter was corrected for percentage of ash. But the fluoride content of the organic portion of dentine remaining after acid dissolution has not been directly measured establishing whether fluoride release is complete.

6.4 Alternative Approaches to the Analysis of Fluoride Content of Dental Mineralised Tissues.

Another method of analysis has found use in the analysis of the fluoride content of dental mineralised tissues. The electron probe micro-analyser is capable of elemental analysis of small volumes of tissue in situ. The analysis is based on the emission of characteristic x-ray radiation of elements within a small volume of the sample excited by the impact of a focused beam of electrons. Early studies utilising the technique experienced difficulties in establishing the optimal conditions for determination of fluoride content (Wei and Forbes, 1974; Berkovitz and Heap, 1976; Tveit and Totdal, 1981). This was such that the 96 technique was described as "semi-quantitative" (Tveit and Tôtdal, 1981) giving an estimate of the relative weight percents of the elements analysed rather that a precise determination of true values (Wei and Forbes, 1974). But Chu et al. (1989) found consistency between results obtained by this method and those obtained by an acid abrasion technique.

Nelson et el. (1989) found the technique to have the advantages of being non destructive, simple as regards sample preparation even for large samples and to be highly sensitive to fluorine. But, as the authors state, the proton induced gamma emission is difficult to control and can occur relatively deep within samples, at depths of approximately 20 to 30 pm. As fluoride uptake following topical application is essentially a surface phenomenon (Duschner and Uchtmann 1985, 1988b), this can have a profound effect on the fluoride concentrations calculated within a sample. Figures et el. (1990) also found an inherent variation occurred between individual readings which added to the natural heterogeneity of the sample, in this case dentine.

The greatest disadvantages however are the cost, complexity and accessibility of the apparatus. This is also essentially anin vitro technique although it has been used to measure the fluoride content of enamel in vivo (Baijot-Stroobants and Vreven, 1980). 97

7. Statement of the Problem.

The management of patients with tooth wear has been recognised as an increasing problem (Consultants Restorative Dentistry Group, 1983) with referrals to Restorative Departments of Dental Hospitals ranging from 8 to 22% (Callis et a/., 1993, Shaw et al., 1996). The restoration of advanced tooth wear using removable partial dentures to restore appearance and function is well- established (Hemmings et a/., 1995). The susceptibility of these patients to caries has not been established. Such treatment might, however, be presumed to place the patient at risk of rapid development of caries based on the investigation by Hussey and Linden (1986) and on data from patients wearing conventional overdentures,.

Topical fluoride regimens are proven to reduce the development of root caries (Jensen and Kahout, 1988; Ripa et al., 1987; Wallace et al., 1993) and have been shown to aid prevention of caries in patients wearing conventional overdentures (Toolson and Smith, 1978; Fenton and Hahn, 1978). The most suitable fluoride regimen for prevention of dentinal caries has not been established in clinical trials although sodium fluoride regimens are becoming accepted as standard for prevention of enamel caries. However, amine fluorides are reputed to have additional anti-caries activity including surfactant activity (Busschner et al., 1988; Sefton et al., 1996), increased deposition of calcium fluoride (Lutz, 1983) and anti-bacterial activity (Gehring et al., 1983; Kay and Wilson, 1988). Stannous fluoride additionally forms an acid insoluble surface deposit on the tooth surface (Dusschner and Uchtmann, 1988b) and has anti bacterial activity (Caufield, 1987; Tseng etal., 1992; Wade etal., 1997).

Stannous fluoride preparations can be unstable due to hydrolysis and oxidation of the Sn^* ion, however, a combination of stannous fluoride and Amine Fluoride 297 has been shown to prevent this (Mühlemann et al., 1981). Assuming that the two fluoride compounds will act synergistically, this combination has the potential for improved anti-caries efficacy. The product Is yet to be comprehensively tested in clinical trials. 98

Fluoride uptake - in-situ - studies have been suggested to be a good indicator of anti-caries activity (Faller, 1995). Current methods of mineralised tooth tissue biopsy, especially of dentine, are problematic and a simple yet reproducible method of biopsy in vivo and subsequent fluoride analysis have yet to be established.

Fluoride uptake by mineralised tissues, however, has not been universally associated with caries inhibition (Brudevold et a/., 1956a; Gedalia at a/., 1961; Aasenden et al., 1972) and anti-caries activity needs to be confirmed by controlled double blind clinical trials against accepted standard control regimen. Clinical trials are expensive both in terms of resources and time (O’Mullane, 1976) and methods of increasing efficacy might be considered - the use patients at high risk of developing caries is one such method that is well established (Banting, 1993).

The use of patients who require partial dentures to restore advanced tooth wear as study subjects has several advantages in trials of these types;

1. This population is increasing and sufficient patients numbers for the conduction of a trial are not difficult to obtain. 2. Recontouring of abutments prior to definitive denture construction is a planned procedure for these patients and enables biopsy of dentine to be planned in association with trials on anti-caries activity of topical regimens. 3. The current literature indicates that this group is at high risk of caries following the provision of dentures. The identification of a high risk group enables improvement in the efficacy of a caries trial, the number of patients required and the length of time they are observed can both be decreased. 99

8. Aims and Objectives

This thesis aimed to assess the anti-caries efficacy of an amino-stannous fluoride regimen with a view to prevention of dentinal caries in patients wearing partial dentures overlaying teeth to restore tooth wear. Efficacy was examined by double blind trials to assess the ability of the regimen to promote dentinal fluoride uptake and to reduce caries incidence. These were compared with the effects of a sodium fluoride programme which has become an accepted standard.

Investigations were therefore conducted with the aims of:

1. Developing a sectioning method of dentine biopsy suitable for use In vitro and in vivo 2. Developing a method of analysis of the fluoride content of dentine following topical fluoride application in vitro and in vivo 3. Assessment of fluoride uptake by dentine in vivo following unsupervised use of an amino-stannous fluoride toothpaste and mouthrinse 4. Conducting a double blind clinical caries trial of an amino-stannous fluoride toothpaste and mouthrinse compared to a sodium fluoride programme 5. Researching further the mode of action of the amino-stannous fluoride regimen.

The investigations to achieve these aims are described in Chapters 2 to 8, the objectives of each being described in the introductory passage.

Hypothesis

The hypothesis for these investigations was that a combination of stannous fluoride and Amine Fluoride 297 as a toothpaste and daily mouthrinse regimen would promote fluoride uptake by dentine in vitro and in vivo and would inhibit development of dentinal caries compared to a positive control sodium fluoride regimen. 100

CHAPTER 2.

MEASUREMENT OF THE FLUORIDE CONTENT OF DENTINE. DEVELOPMENT OF METHOD. 101

Introduction

The aim of these initial investigations was to develop methods to measure fluoride uptake by dentine in order to compare the efficacy of different topical fluoride regimens applied in vitro and in vivo.

The objectives were;

1. To develop a method of biopsying dentine suitable for use in vitro and in vivo 2. To develop a method of analysis of the fluoride content of the dentine samples.

These methods are common to the investigations described in Chapters 3,4,6 and 7 and are Included here for ease of reference, along with the fluoride preparations used and statistical analyses performed.

1. Preparation and Storage of Teeth for In Vitro Investigations.

Freshly extracted third molar teeth were used in the in vitro investigations. Following extraction, soft tissue was removed from the root surface using a curette (Gracey, Ash Instruments, Dentsply, U.K.) and the teeth were rinsed in distilled, deionised water. They were stored in distilled, deionised water at 4^0 and used within 1 month of extraction. The patient of origin was identified and their medical history checked before the teeth were prepared. Ethics committee approval was obtained prior to commencement of the studies.

The teeth were sectioned to give access to a standard occlusal dentinal surface by sectioning horizontally through the greatest convexity of the crown of the tooth with a rotary diamond saw (Testboume, Basingstoke U.K.) cooled with distilled, deionised water. When 4 sections of contiguous occlusal dentine were required, the teeth were also sectioned in a longitudinal plane mesio-distally and bucco-lingually providing sufficient dentine for several test and control samples (Figure 2). 102

Figure 2. Diagram of Sectioning Procedure to Yield Four Sections of Contiguous Occlusal Dentine.

Section

•• Section 103

2. Biopsy Techniques.

Introduction

Harvesting samples of dentine to assess fluoride uptake is difficult especially in vivo. As discussed in the literature review, biopsying dentine by dissection allows a sample of tooth tissue of known surface area, depth and weight to be obtained. Unlike abrasion and micro-drilling techniques, loss of part of the specimen is less likely and, unlike etching techniques, biopsy does not rely on the properties of the tooth tissue to be sampled.

Consideration needed to be given to the surface area and depth of biopsy samples. The surface area needed to be large enough for ease of handling but small enough to allow two or more biopsies to be taken from even the smallest (lower incisor) tooth. A larger surface area also limits the effect of local variations across the sample.

Substantial amounts of fluoride have been found from the superficial layers of dentine following exposure to both high concentration fluoride solutions (Hals et al., 1981; Tveit et al., 1985; Figures et al., 1990), fluoride releasing device (Corpron et al. 1991) and a fluoride containing re-mineralisation solution (lijima et al., 1993). This suggested a minimum thickness of biopsy is necessary for assessment of total fluoride uptake.

Biopsy In vivo however, is not always possible due to the size of the sample that must be removed for ease of handling. However, patients with tooth wear often require recontouring of the abutment teeth before a definitive denture can be constructed thereby offering an ideal opportunity to obtain samples of dentine and to assess fluoride uptake in patients at risk of developing caries.

The objective of these investigations was to develop a technique for use in vitro and in vivo for harvesting a sample of dentine of known surface area and similar depth. 104

2.1. An Evaluation of the Use of Trephines to Biopsy Dentine.

Method

Occlusal dentine was exposed on four extracted, unerupted third molars as described above. Three adjacent specimens of dentine were taken from each tooth using a trephine, external diameter of 1.5 mm (Trephan bur, Masseran Kit, Micromega, U.K.), running at slow speed with water-cooling. The core was then removed by fracturing the dentine using an excavator (Ash number 62, Ash instruments, Dentsply, U.K.) inserted along the side of the core as near to the base as possible.

The trephine was then used to biopsy dentine from a patient who required substantial re-contouring of worn teeth before the construction of a definitive overlay denture. The hospital research and ethics committee approved the study design and the patient gave his voluntary, informed, written consent.

Sample dimensions were measured using a micrometer attached to a binocular stereo-microscope (x32 magnification) (Wild 400, Heerbrugg, Switzerland). Each dimension was measured three times to check reproducibility.

Results

In vitro 12 specimens were obtained from 4 teeth. The specimens had a mean surface area of 0.97 mm^ (sd 0.07) and a mean depth of 1.11 mm (sd 0.51). Repetition of measurements showed that the dimensions could be measured to an accuracy of + 0. 02 mm.

In vivo the trephine was unable to make the initial cut into dentine and tended to “skid” across the dentine surface.

Discussion

The surface area of the biopsied specimen was selected to be 1 mm^ as this was considered to be large enough to allow easy handling but small enough to 105 enable biopsy of two specimens from one abutment. A depth of 0.5 mm was selected as substantial levels of fluoride have previously been recorded up to 0.4 mm deep from the dentinal surface following topical fluoride application (Figures et al., 1990).

In vitro dissection by this method resulted in large variation in the depth of the specimens, particularly due to a considerable variation in the length of the fractured surface. This was of concern, as a given specimen of dentine will have a small amount of fluoride throughout its depth depending on systemic fluoride intake of the donor. This fluoride level will influence the apparent amount of fluoride uptake measured. Any difference in depth of specimens dissected, therefore, may be expected to lead to variation in the measured fluoride uptake.

Consideration was given to reducing all the specimens to a standard depth of 0.5 mm by abrasion of the fractured surface using a jig to hold the specimen. However, manipulation of the specimens was found to be difficult and loss of a large percentage occurred.

On using the trephine in vivo, it was not possible to apply sufficient pressure to achieve the initial cut into dentine. The trephines are designed to cut radicular dentine. While their use in vitro on the “young" dentine with patent tubules found in unerupted teeth was possible, the risk of damage to the surface of the dentine caused by the “skidding" of the trephine led to concern that the assessment of the fluoride content in this fluoride rich zone would be impaired. The sclerosed, occlusal dentine found in patients with tooth wear is substantially different such that the use of trephines for dissection was not feasible. An alternative method of dentine biopsy was therefore considered.

2.2. Evaluation of the Use of a Diamond Bur to Biopsy Dentine In vitro.

Method

Ten third molar teeth were sectioned occlusally and longitudinally as described previously. Dentine samples 1 mm x 1 mm wide and 0.5 mm deep were dissected from the inner right-angled corner of each section at the intersection 106 of the bucco-lingual and mesio-distal cuts (Figure 3). Dissection was performed using a fine, long, cylindrical diamond bur (Hopf, RingleS, GMBH, Germany) in an air turbine under water spray. Following preliminary evaluation of the technique a disposable template was constructed from two pieces of 0.5 mm wide stainless steel rectangular arch wire (Figure 4). This was used as a visual guide to specimen dimensions. The width of the template gave a guide to specimen length and the half section was a guide to specimen depth.

Forty dentine samples were dissected. Sample dimensions were measured as before.

Results

The specimens had a mean surface area of 1.34 mm^ (sd 0.19) and a mean depth of 0.53 mm (sd 0.09). The distribution of the specimen dimensions is illustrated in Figure 5.

Discussion

Sectioning of dentine using fine diamond burs enabled biopsies to be obtained such that the surface area and depth of the specimens could be measured directly. The template proved useful as a visual reminder of sample dimensions.

Small variation in surface area of the samples was apparent but this was not regarded as significant as fluoride content was to be calculated per unit surface area. The use of a sample thickness of 0.5 mm yielded samples that were of a size that was easy to handle and with small variation in thickness compared to the mean value. However, investigation was required to ensure that all fluoride uptake was assessed when limiting the depth of dissection to this thickness (Page 124).

This method of biopsy was adopted for all in vitro investigations and was easily adapted for use in vivo (Chapter 4). 107

Figure 3. Diagram showing dissection of an occlusal dentine specimen from the inner right-angled corner of a tooth section.

Figure 4. Disposable template constructed from two pieces of 0.5mm wide stainless steel rectangular arch wire. 108

Figure 5. Frequency distributions of the surface area and depth of the dentine specimens when dissected with a diamond bur.

Frequency Distribution of Surface Area of Dentine Specimens

1.00- 1. 10- 1.20- 1.30- 1.40- 1.50- 1.60- 1.70- 1.09 1.19 1.29 1.39 1.49 1.59 1.69 1.79

Surface Area (mm )

Frequency Distribution of Depth of Dentine Specimens

0.30- 0.70- 0.34 0.74 Depth (mm) 109

3. Methods of Fluoride Analysis.

Introduction

The aim of this study was to develop the method of Tyler and Poole (1989) to produce a method of analysis of the fluoride content of dentine specimens. The objectives of these investigations were:

1 To determine the volume of 1 M hydrochloric acid required for dissolution of the dentine samples and the time of immersion to achieve this. 2 To develop the method of measuring fluoride uptake by dentine.

3.1 Dissolution of Dentine in 1 Molar Hydrochloric Acid.

Method

Twenty-seven cuboidal samples of dentine were taken from the occlusal dentine of 6 teeth (4 to 5 samples per tooth). All the samples were initially stored in 250 ml distilled, deionised water at 4°C for 7 days. Subsequently 3 samples, randomly selected to act as controls, were stored respectively in either 2 ml, 5 ml or 10 ml distilled, deionised water. The remaining 24 samples were randomly distributed such that eight samples were immersed in 2 ml, eight in 5 ml and eight in 10 ml 1 M hydrochloric acid. The hydrochloric acid was prepared by dilution with distilled, deionised water from a 37% hydrochloric acid solution (BDH Laboratory Supplies, U.K.).

The samples were weighed prior to immersion in either the test or control fluids and then after 1,3,6 and 24 hours, 1 and 2 weeks.

The sample was weighed 1 minute after its removal from solution on an electric pan balance (Oertling, Model R51, U.K.), having first removed the surface liquid by blot drying for 15 seconds using tissue paper (Lotus Professional, Jamont, U.K.) and air drying for 15 seconds. 110

Results

The weight of the 27 samples before demineralisation ranged from 1.3- 3.6 mg, mean 2.2mg (sd 0.6). Statistical analysis Is detailed in Appendix 4, Table 1. There was no significant difference in initial weight between the three test groups (p=0.21). Plots of weight of the dentine samples against immersion time are illustrated for the different volumes of acid in Figure 6. Maximum weight loss was found to occur within 1 hour in all specimens except for sample 4 in 2 ml 1 molar hydrochloric acid. This specimen was initially not fully submerged in the acid. The percentage weight loss ranged from 56 to 89% but the mean weight loss for each group of specimens did not differ significantly (p=0.58).

Discussion

The dentine samples were blot dried, air dried and weighed after set times in order to standardise water loss from each sample at the point of weighing. Weight measurements of the control samples showed this method to be accurate to + 0. 2 mg.

On exposure to 1M hydrochloric acid an average 65% of the weight of the dentine was lost. On a weight basis the mineral phase of dentine constitutes approximately 70% of the composition of dentine (Linde, 1988; Berkovitz et al. 1992). This suggests that a weight loss of 70% would be equivalent to complete demineralisation. It was, therefore, considered that a weight loss of 65%, equivalent to approximately 93% demineralisation, was likely to release fluoride ions associated with the mineralised portion of the dentine given that fluoride uptake is a surface reaction.

The weight loss occurred within 1 hour and there was no significant difference between the different volumes of hydrochloric acid. However, 2 ml of acid was not a sufficient volume to guarantee that the sample was fully submerged. For convenience, a volume of 5 ml acid was selected for subsequent investigations to yield sufficient fluid for multiple fluoride measurements and to avoid the problem of incomplete immersion of the sample. Again for convenience, an immersion time of 24 hours was selected for future experiments. 111

Figure 6. Weight loss on déminéralisation of samples of dentine in 1M Hydrochloric Acid.

In 2 ml 1 M HCI

3 3

Period in HCI

In 5 m il M HCI Control

Test 1

3 3 Test 2

Test 3

Test 4

Test 5

Test 6

Test?

Period in HCI Test 8

In 10 ml 1 M HCI

O) 3 E 2 .g (D 1

Period in HCI 112

3.2 Analysis of Fluoride.

Introduction

Following dissolution of dentine in 1 M hydrochloric acid, analysis of fluoride in the subsequent solution may be carried out using the differential electrode cell method. However, a small uptake of fluoride by dentine was expected from the low concentration fluoride compounds employed in this programme. A method of measuring low concentrations of fluoride needed to be established.

The following method was utilised for fluoride measurement throughout the investigations.

Method

Analytical Equipment.

Measurements of solution fluoride concentration were made with a differential fluoride electrode cell according to Tyler and Poole (1989). The differential cell was formed by a combination pH electrode (Model 91-35, Orion Research, USA) and a single fluoride selective electrode (Model 94-09, Orion Research USA) (Figure 7). The potential generated between them was measured with a microprocessor ionanalyser (Model 901, Orion Research, USA) (Figure 8).

As the potential generated by a fluoride solution varies linearly with the log of fluoride concentration (within certain constraints of pH and concentration) a chart can be plotted using calibration solutions and the concentration of test solutions calculated from this. Alternatively, the microprocessor had the facility to convert the potential measured in millivolts so that concentration values in parts per million of fluoride (ppm F ) could be read directly. This was achieved by recording the potentials generated from 2 calibration solutions that differed by a decade in fluoride concentration, selected to ensure that the fluoride concentrations of the solutions to be analysed fell within their range. These values were used to calibrate the machine. 113

Figure 7. The differential fluoride electrode cell used for fluoride analysis.

Figure 8. The microprocessor ionanalyser used for fluoride analysis 114

Solutions to be measured were placed on a polytetrafluoroethylene (PTFE) slide containing a well which housed a PTFE coated magnetic stirring bar. The slide was place on a magnetic stirrer hotplate (HI 200M, Hanna Instruments, U.K.). The electrodes were adjusted to make contact with the solution as it was stirred. A stable reading was taken after 2 minutes. Following each measurement electrodes, slide and stirrer were thoroughly rinsed with distilled, deionised water twice and blot dried with tissue paper. Plasticware was used throughout to avoid fluoride contamination from glass surfaces.

Preparation of the Dentine Samples for Fluoride Analysis.

Following exposure to the topical fluoride regimen, dentine samples were dissected and their dimensions measured as before. The samples were immediately dissolved in 5 ml 1 M hydrochloric acid (sourced as before) at room temperature in a sealed plastic container for 24 hours. Care was taken to keep the sample solution sealed at all times to minimise hydrofluoric acid loss by evaporation.

The test solutions were diluted ten fold with distilled, deionised water to give a pH of approximately 1.5. This ensured that the solution was at the optimum pH value for fluoride analysis by the differential fluoride electrode cell. The fluoride concentrations to be measured in these investigations were expected to be below those limits at which reliable detection could be achieved. To overcome this a standard solution of 0.5 ml 0.4 ppm F" (aqueous sodium fluoride) was added to the diluted dentine solution. The use of a standard blank ensured that the fluoride concentrations to be measured fell within the linear portion of the calibration curve. Thus a 0.1 ppm F" concentration was read as a baseline concentration by the ionanalyser and any additional fluoride was considered to be contributed by the diluted sample solution.

Electrode Calibration.

The electrode was calibrated with standard solutions containing 0.1 and 1 ppm F". These comprised a 1.5 ml aliquot of 0.1 M hydrochloric acid and 0.5 ml of either a 0.4 ppm F" or a 4 ppm F' aqueous (sodium fluoride) standard solution 115 respectively. The 0.1M hydrochloric acid solution was freshly prepared for each calibration by dilution of the 1M solution used to dissolve the dentine samples. The fluoride standard solutions were prepared from analar grade sodium fluoride salt (BDH Chemicals, U.K.) diluted with distilled, deionised water. To ensure accurate calibration, measurements of the calibration fluoride solutions were repeated until a reading stable to ± 0.2 mV was obtained. Re-calibration of the cell with standard samples was performed hourly to overcome the sensitivity to humidity and temperature changes of the differential cell and the Orion Microprocessor.

Analysis of the Prepared Solutions of Dentine.

Duplicate diluted and fluoridated solutions from the dentine solution were made. This allowed each measurement to be repeated to ensure consistency of the readings. The average value was used except where variation greater than ±0.02 ppm F" occurred. Then a third solution was analysed and the outlier discarded.

Fluoride content per unit area of dentine was calculated for each sample by the following formula:

Fluoride Content Of Specimen = 5 x 10 x (IR -0.1) Per Unit Area of Dentine (pgcm'^) A

Where: IR = Fluoride concentration reading by the ionanalyser and A = Surface area (cm^)

(The value of the blank fluoride addition of 0.1 ppm F' concentration was deducted from the ionanalyser reading. The result thus obtained was multiplied by ten to yield the concentration in the undiluted solution and then multiplied by five, since it was dissolved in 5 ml acid. The fluoride content per unit area of the dentine specimen was then calculated by dividing this by the surface area of the specimen) 116

4. Topical Fluoride Preparations

A variety of amine fluoride preparations were examined in these investigations. The type of fluoride preparation used in each investigation is presented in Table 5 and the ingredients of the products are detailed in Table 6 and 7.

Control Preparations.

Sodium fluoride preparations were selected as controls. A fluoride free or low fluoride comparison group were not investigated as recommended by Faller (1995) (for in situ models for evaluating new fluoride containing systems) as these would be unacceptable for use in vivo in patients who are at high risk of developing caries.

The sodium fluoride paste, rinse and gel had formulations identical to those of the amine fluoride/stannous fluoride (Meridol®) products, except that the source of fluoride was sodium fluoride (Table 7). They contained molar equivalents of available fluoride to a tolerance of ±5% , this being the production limit achievable. 117

Table 5. Fluoride Preparations Applied in the Fluoride Uptake Investigations.

INVESTIGATIONSPREPARATIONS

Effect of Depth of Specimen on Dentine Solution (12500ppm F ) Sodium Fluoride Fluoride Content Amine Fluoride 297 (Section 3.1) Stannous Fluoride

Fluoride Uptake by Dentine In vitro Toothpaste (1400ppm F ) Meridol (Section 3.2) Sodium Fluoride

Mouthrinse (250ppm F ) Meridol Sodium Fluoride

Fluoride Uptake by Dentine In vivo Toothpaste (1400ppm F I Meridol (Chapter 4) Sodium Fluoride

Mouthrinse (250ppm F I Meridol Sodium Fluoride

The use of fluoride gels to promotion Gel (12500ppm F ) Meridol fluoride uptake Elmex (Chapter 6) Sodium Fluoride

Investigations into the mode of action of Gel (12500ppm F ) Meridol amino-stannous fluoride regimens Elmex (Chapter 7) Sodium Fluoride

Solution (12500ppm F ) Amine Fluoride 297 Stannous Fluoride Sodium Fluoride “Elmex" “Meridol"

Investigations into uptake of fluoride by Gel (12500ppm F ) Meridol demineralised dentine Elmex (Chapter 8) Sodium Fluoride

Solution (12500ppm F ) Amine Fluoride 297 Stannous Fluoride Sodium Fluoride TOOTHPASTE MOUTHRINSE Active Ingredients: Active Ingredients: MERIDOL 0.46% amine fluoride 297 (350 ppm F*) and MERIDOL 0.164% amine fluoride 297 (125 ppm F ) and 0.44% stannous fluoride (1050 ppm F*) 0.056% stannous fluoride (125 ppm F )

SODIUM FLUORIDE 0.31% sodium fluoride (1400 ppm F) SODIUM FLUORIDE 0.055% sodium fluoride (250ppm F )

Abrasive / Cleaning agent Silica (Sident 8) Other Ingredients: Other Ingredients: Solvent Water Solvent Water and Ethanol (5%) Humectant / Sweetener Sorbitol and glycerin Sweetner Xylitol and acesulfam potassium Foaming agent Cocamidopropyl betain Emulsifier Hydrogenated castor oil Thickener - hydrophilic Silica (Sorbisil AC-33) Flavouring Thickener - hydrophobic Silica (Aerosil R-972) Colouring Gel fonmer Hydroxycellulose Emulsifier of flavour oils Hydrogenated castor oil pH buffering Sodium gluconate and potassium hydroxide Sweetener Saccharin Flavouring Colouring

Table 6. Ingredients of toothpaste and mouthwash (as provided by GABA International) used the in vitro and in vivo investigations. Preparation Source Of Fluoride' Other Constituents' pH Presentation Manufacturer Sodium 2.76% sodium fluoride (12500 ppm F ) As Meridol Gel 5.5 to S O'' Opaque colourless gel GABA International Ltd Gels Fluoride (1.25% F) Elmex 3.03% Amine fluoride 297 (2100 ppm F ) Water, Hydroxyethylcellulose, S.O'' Clear yellow/white gel GABA International Ltd 0.29% Amine fluoride 335 (400 ppm F') Saccharin, Methylparaben, 2.21% sodium fluoride (10000 ppm F ) Flavouring, Colouring Meridol 8.90% Amine fluoride 297 (6250 ppm F ) Glycerin, Water, Propylenglycol, 5.5to6.0'* Clear blue gel GABA International Ltd 2.62% stannous fluoride (6250 ppm F) Liporamnosan, Na-D- Gluconate,Tego-Betain L7, Base 297, Acesulfam, Flavouring, Colouring Sodium 2.76% sodium fluoride (12500 ppm F ) Water 7.9* Clear colourless solution Merck/D Solutions Fluoride (1.25% F) Stannous 5.21% stannous fluoride(12500 ppm F ) Water 2 T Clear colourless solution Riede-de-Haen Fluoride Amine 17.81% Amine fluoride 297(12500 ppm F ) Water 5.2* Clear yellow "oily” solution GABAAG/CH Fluoride 297 "Elmexl" 3.56 % Amine fluoride 297 (2500 ppm F) Water 6.0* Clear yellow solution 2.21% sodium fluoride (10000 ppm F) "Meridol" 8.90% Amine fluoride 297 (6250 ppm F) Water 5.5* Clear yellow solution 2.62% stannous fluoride (6250 ppm F )

^ As provided by GABA International ^ Measured using pH indicator papers (Whatman Paper, U.K.) ^ Measured using pH meter (Jenway 3045 ion analyser, Jenway, U.K.)

Table 7. Ingredients of the topical fluoride gels and solutions (as provided by GABA International) used in the in vitro investigations. 120

Presentation.

All toothpastes and gels were provided in sealed white plastic tubes containing 75 ml. The mouthrinse was provided in sealed white plastic bottles containing 400 ml. The containers provided by GABA International Ltd. (Basel, Switzerland) were labelled with the batch number, the date of manufacture the form of the preparation (eg. "gel") and the percentage of fluoride. No identification was given of the composition therein allowing investigations to be carried out blind. The toothpaste was in the form of a blue gel and the mouthrinse as a blue liquid. These products could not be identified by colour, smell or taste. The presentation of the gels and solutions is described in Table 7.

The solutions were made up by dilution of salts provided by GABA International Ltd. Each solution was freshly made, with distilled, deionised water, immediately prior to application to the dentine except for Amine Fluoride 297. This waxy material was found to require 24 hours to dissolve at room temperature. It was therefore prepared 24 hours prior to application, in a sealed plastic container that was shaken periodically.

The products were designated to have a "shelf life" of 30 months based on stability data from the manufacturer; after this date they were discarded.

Mode of application in vitro.

The teeth were exposed to the topical fluoride regimen in batches of five. Teeth were collected, prepared and sectioned to provide four adjacent sections of occlusal dentine as previously described. The longitudinally cut surfaces were coated with varnish so that only the occlusal dentine was exposed to the topical fluoride regimen. To allow identification of the tooth of origin, each section was marked by engraving "domino” graphics on to the root surface using a round diamond bur (Hi-Di 519, Ash Instruments, Dentsply, U.K.) in an air turbine under water cooling. 121

Exposure to fluoride toothpaste, gel or solution was achieved by placing the sections in 50 ml of the fluoride preparation in a sealed plastic container (Azion specimen bottles, Bibby Sterilin, U.K.) for 1 hour at room temperature. Care was taken to ensure that the occlusal dentine did not come into contact with the side or bottom of the container. The tooth sections were then removed, blot dried with tissue paper and washed three times for 5 minutes in 50 ml distilled, deionised water, placed on a vibrator (Vibro 18, Quayle Dental, U.K.), at room temperature, using fresh water for each wash.

Following exposure to toothpaste the sections were Immediately immersed in 50ml mouthrinse in a sealed plastic container for 30 minutes at room temperature. They were then dried, vibrated in distilled, deionised water as above and immediately biopsied.

In the case of exposure to gels and solutions, dentine samples were immediately dissected from the tooth sections as described above. 122

5. Statistical Analysis.

Unless otherwise stated, the data throughout these investigations was subjected to the non-parametric tests detailed in Table 8. In each case the null hypothesis was that the samples were drawn from the same population. The analysis was adjusted for ties. A 95% confidence interval was selected to indicate a significant difference. When Friedman or Kruskal-Wallis analysis indicated a significant difference, methods of multiple comparisons (Siegel and Castellan, 1988) were used to determine which samples displayed significant differences. 95% confidence intervals for median values of the data were calculated according to Campbell and Gardener (1989).

Table 8. Tests used for statistical analysis of data.

Test Level of Measurement Mann-Whitney Test 2 independent samples Friedman two way analysis of variance by ranks 3 or more related samples Kruskal-Wallis one way analysis of variance. 3 or more independent samples 123

CHAPTER 3.

MEASUREMENT OF FLUORIDE UPTAKE BY DENTINE FOLLOWING TOPICAL FLUORIDE TREATMENTS IN VITRO. 124

Introduction

The objective of these investigations was to develop the method of fluoride analysis to evaluate fluoride uptake by dentine following application of toothpaste and mouthrinse in vitro. The following investigations were carried out to establish the method of analysis.

1. The effect of variation in specimen thickness on fluoride content analysis. 2. The measurement of fluoride uptake by dentine in vitro. 3. The measurement of fluoride content of the organic dentine remaining after dissolution in hydrochloric acid. 4. The effect of storage in water on fluoride content of dentine biopsies.

1. Assessment of the Effect of Variation in Specimen Thickness on the Fluoride Content of Dentine.

introduction

The investigation aimed to verify whether a specimen thickness of 0.5 mm would be sufficient to record the uptake of fluoride by dentine when exposed to a high concentration fluoride solution.

Method

The occlusal dentine was exposed on 30 extracted third molars. The teeth were halved longitudinally to provide 60 sections and the longitudinally sectioned surfaces were varnished as before. One section from each tooth was left untreated while the other was exposed to sodium fluoride, Amine Fluoride 297 or stannous fluoride solutions (equivalent to 12500 ppm F") as previously described. Using the biopsy technique described earlier, three adjacent specimens of dentine were dissected from each section with approximate depths of 0.25 mm (narrow), 0.5 mm (standard) and 1 mm (wide) respectively (Figure 9). The specimens were analysed for fluoride content as described previously. 125

Figure 9. Dissection of dentine specimens of varying depth.

CONTROL 1 TEST Varnish SECTION SECTION

Distilled deionised Fluoride water at 4°C regimen

Specimen Dissection

Narrow Standard Wide 126

Results

180 specimens of dentine were dissected. Plots of fluoride content against depth of specimen are illustrated in Figure 10 (raw data is given in Appendix 5, Table 1). The plot of fluoride content against depth of specimen for the specimens treated with Amine Fluoride 297 revealed a slope that appeared to have a uniform gradient over the range of specimen depth investigated. The specimens treated with stannous fluoride and sodium fluoride revealed curves with an initial steep gradient, which became constant at a depth of approximately 0.40 mm and 0.50 mm respectively. The constant gradient was similar to that plotted for the untreated specimens.

Figure 10. Fluoride Content of Dentine Specimens of Varying Thickness Following Application of 1.25% Fluoride Solutions.

Amine Fluoride 297 • Sodium Fluoride Stannous Fluoride Untreated

300

250

200 * — ?

I 150 o Ü 0) ■O 100 o3 u.

0.2 0.4 0.6 0.8 1 1.2 1.4 Thickness of specimen (mm) 127

Discussion

Following application to dentine of a high concentration sodium and stannous fluoride solution, the fluoride content of the dentine varied with specimen depth, within a range of 0.25 to 1 mm. However, below a depth of approximately 0.5 mm the increase in fluoride content was similar to that found in untreated specimens of dentine. This suggests that a minimum depth of dissection exists that will allow assessment of total fluoride uptake by dentine following exposure to topical fluoride regimen and that a specimen depth of 0.5 mm will accomplish this. These findings are similar to those reported for fluoride taken up by dentine on exposure to topical fluoride application (Hals et al., 1981; Tveit et al., 1985; Figures et al., 1990; Copron et al., 1991; lijima et al., 1993).

However, the influence of the intrinsic fluoride content of dentine should be taken into account. Preferably this should be done by dissection and analysis of untreated samples of dentine; subtraction of the fluoride content of an equivalent volume of untreated dentine from the fluoride content of the treated dentine permits calculation of fluoride uptake per unit area of dentine. The following formula may be used;-

Fluoride Uptake of Specimen per Unit Area of Dentine (pgcm'^) =

(Entire) Fluoride Content - Fluoride Content of Untreated x Volume of of Test Specimen (pg) ^pecimen per Unit Volume (ngcm*®) Test Specimen (cm^)^ Surface Area of Test Specimen (cmi

Conclusions

Selecting a specimen thickness of 0.5mm will enable all the fluoride uptake by dentine to be measured following topical application of high concentration fluoride solutions of sodium fluoride, Amine Fluoride 297 and stannous fluoride. Untreated specimens of dentine were also found to contain significant levels of fluoride. It was determined that the fluoride content of these should be assessed and subtracted from the fluoride content of an equivalent volume of treated dentine. 128

2. Measurement of Fluoride Uptake by Dentine In Vitro.

Introduction

The objective of this study was to investigate the effect of fluoride toothpaste and mouthrinse on fluoride uptake by dentine using the method established in Chapter 2. The effect of caries on uptake was also examined.

Method

Twenty-two freshly extracted third molars were collected, stored and sectioned occlusally and longitudinally to produce four samples of contiguous occlusal dentine as before. The longitudinally cut surfaces were coated with varnish and the sections marked to identify the tooth of origin as above.

One section from each tooth was randomly allocated to a group that was untreated and placed in distilled, deionised water. Two sections from each tooth were randomly allocated to one of two test groups and exposed to either the sodium fluoride or AmF 297/Snp2 (Meridol) toothpaste and mouthrinse regimen described previously. Dentine was biopsied from each section. Samples of carious dentine were taken from eight of the teeth; the other teeth appeared clinically free from caries. The presence or absence of caries was assessed clinically by the presence or absence of colour change and softening of the dentine on probing (Ash number 6, Ash Instruments, Dentsply, U.K). The sample dimensions were measured and the fluoride content analysed as previously described.

The dentine biopsies were exposed to 1M hydrochloric acid for 24 hours. The organic remains of each dentine specimen were then removed from the acid and retained for analysis of fluoride remaining in the organic portion of the dentine (Page 138). The fluoride content of the acid was analysed as previously described. 129

Results

66 dentine specimens were obtained. The specimen dimensions, fluoride content and fluoride uptake are illustrated in Table 9 (raw data in Appendix 5, Table 2). Statistical analysis is detailed in Appendix 4, Table 2.

The specimens were found to have a mean surface area of 1.36 mm (sd 0.30) and a mean depth of 0.52 mm (sd 0.11). The specimen depth (p = 0.19) and surface area (p= 0.37) did not differ significantly between the six groups. Fluoride content of the untreated “non carious" and untreated “carious" specimens also did not differ significantly (p = 0.82).

The fluoride uptake by the specimens is illustrated in Figure 11. Following the Meridol regimen the “non-carious" specimens had a median fluoride uptake of 24 pgcm'^ and the “carious" specimens had a median fluoride uptake of 9 pgcm’^. Following the sodium fluoride regimen the “non-carious" specimens had a median fluoride uptake of 17 pgcm'^ and the “carious" specimens had a median fluoride uptake of 21 pgcm’^.

There was no significant difference in fluoride uptake between the groups of specimens except between fluoride uptake following the Meridol regimen in the “non-carious" specimens which was significantly higher than that in the “carious” specimens (p = 0.035). Depth Surface Area Fluoride content Fluoride uptake

mm mm^ pgcm’^ pgcm’^

Toothpaste and Un-treated Meridol NaF Un-treated Meridol NaF Un-treated Meridol NaF Meridol NaF Mouthrinse NON-CARIOUS DENTINE

Mean 0.54 0.57 0.49 1.41 1.43 1.22 13 59 36 44 24

SD 0.18 0.07 0.09 0.26 0.26 0.18 6 44 17 43 15

Median 0.52 0.57 0.49 1.47 1.47 1.22 13 42 26 24 17

95% Cl 0.46 to 0.61 0.51 to 0.64 0.43 to 0.57 1.13to 1.54 1.29 toi.68 1.03to1.31 9 to 18 31 to 71 21 to 53 21 to 55 12 to 38

CARIOUS DENTINE

Mean 0.50 0.53 0.49 1.40 1.43 1.30 14 31 35 15 22

SD 0.10 0.13 0.07 0.37 0.45 0.21 6 16 18 20 18

Median 0.48 0.52 0.47 1.39 1.37 1.34 14 27 35 9 21

95% Cl 0.39 to 0.63 0.42 to 0.66 0.41 to 0.59 1.07 to 1.79 0.91 to 1.92 1.03 to 1.44 8 to 20 16 to 44 14 to 54 -2 to 33 3 to 42

Table 9. Summary of specimen dimensions, fluoride content and fluoride uptake following application of toothpaste and mouthrinse in vitro to “non-carious" and “carious" dentine. 131

Figure 11. Fluoride uptake by dentine following topical application of toothpaste and mouthrinse in vitro.

Scatter Plot of Fluoride Uptake

180 160 140 ? 120 100 t 80 D 60 40 Io 3 20 0

-20 I I I I I ' ...... I " I I I I I I I I 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Tooth

Meridol Regimen, Non-Carious Dentine Meridol Regimen, Carious Dentine NaF Regimen, Non-Carious Dentine NaF Regimen, Carious Dentine

Median Values and 95% Confidence Intervals for Fluoride Uptake.

50 u ™ 40

30 I20 I 10 o 0

-10 Meridol Regimen, Meridol Regimen, NaF Regimen, NaF regimen. Non-Carious Carious Dentine Non-Carious Carious Dentine Dentine Dentine 132

Discussion

The concentration of fluoride selected for the toothpaste was influenced by the highest concentration of fluoride that can be used within the limits imposed by European Cosmetic Directives (Dupuis, 1993). For the mouthrinse, a concentration was chosen comparable with a 0.05% sodium fluoride mouthrinse (225 ppm F") which has been adopted as standard for daily rinsing (Wei and Yiu, 1993). The periods of exposure to toothpaste and rinse were chosen so as to be equivalent to a cumulative one-month application. This does not reflect exactly the manner of clinical exposure. Such a situation would be better mimicked by cycling periods of exposure with periods in saliva and demineralising solutions (Faller et. a/., 1995) and might be considered if an in vivo investigation is not feasible. However, the laboratory study will give a good indication of fluoride uptake by a method that is quick and easily reproducible for later comparison with other regimens.

There were large variations in fluoride uptake from tooth to tooth. This may be due to the heterogeneous nature of dentine. The structure of dentine may be disturbed by systemic factors during tooth formation, either because the basic components of the dentine are lacking, or because of interference with secretion or maturation. High levels of fluoride ingestion and systemic disorders such as vitamin D dependent rickets, hypophosphataemia, hypophosphatasia and juvenile hypoparathyroidism may affect the degree of mineralisation (Jones and Boyde, 1984; Jones, 1990; Soames and Southam, 1998). On exposure to the oral environment further changes may occur, topical fluoride and cariogenic challenges alter the degree and chemistry of mineralisation (Featherstone at a/., 1987, Shu at a/., 1998). Dentine is also susceptible to ageing and traumatic stimuli when sclerosis can occur. (Frank and Nalbandian, 1989)

Such variation may be reduced by limiting in vitro investigations to extracted, unerupted teeth and highlights the importance of comparative studies being conducted on specimens from adjacent areas of dentine and including untreated specimens of dentine from the same tooth. 133

Uptake of fluoride by dentine following the Meridol regimen was found to be at least as good as that by sodium fluoride in this investigation. Fluoride uptake by dental mineralised tissues follo\A/ing amino-stannous fluoride regimens has not been reported. An increased uptake might have been expected; in comparative studies amine fluorides generally produce higher uptake in enamelin vitro than other regimens (Kirkegaard 1977a,b; StrCibig, 1980; Klimek et al., 1982; Barbakowef a/. 1985; Mok ef a/., 1990; Chan eta!., 1991;) although comparative investigations of uptake by dentine have not been reported. The fluoride uptake by dental mineralised tissues following stannous fluoride regimens in vitro has not been consistently proven (Brudevold et a/., 1956; Cooley, 1961; Mellberg, 1966; Brudevold, 1967; Kirkgaard, 1977a; Dijkmanet a/., 1982; Dowell and Addy, 1984; Sakkab et a/., 1984; Barbakow et al., 1985; Dérand et al., 1989; Faller 1997). Indeed the anti-caries activity has been attributed to the formation of acid insoluble deposits (Duschner and Uchtmann, 1988a; Imfield, 1996) and anti-bacterial properties (Rolla and Ellingsen, 1994) rather than to the formation of fluoride rich surface deposits. Meridol was manufactured with the intention of developing the anti-bacterial effect of both stannous fluoride and amine fluorides. The amine forms a complex with the tin ion either at the nitrogen ion (N*) (Manufacturers Information) or hydroxyl group (-0H) (Personal Communication, ITRI, 1998) thus stabilising the stannous fluoride.

^ C H 2 - C H 2 O H

CH3 — (CH2) i 6 “ CH2 — N j H — CH2 — CH2CH2 — N H ^ 2F CH2 - CH2OH CH2 - CH2OH

However, this complexing of the amine with the tin ion may itself result in a reduction of available fluoride for uptake (Personal Communication, ITRI, 1998). Over-stabilisation of the stannous ion has been documented for other stannous fluoride products (Miller ef a/., 1994; Tinanoff, 1995).

However, such a phenomena can not be deduced from the results in this study, indeed fluoride uptake did occur following the Meridol regimen and this was equivalent to uptake by an accepted standard regimen (sodium fluoride). 134

The presence of clinically diagnosed caries was not found to increase fluoride uptake, indeed a significantly reduced uptake was found following the Meridol regimen. Such a finding was contrary to expectation, as the higher porosity of carious lesions in dentine would suggest that the acquisition of fluoride would be greater. Substantial acquisition of fluoride has been reported in artificial lesions in dentine under oral conditions (Sanchez and Mellberg, 1988), but comparative data is not available. It is possible that the hydroxyapatite available in carious dentine for reaction with fluoride is reduced which may influence fluoride uptake. This is an area for further investigation.

Few studies of fluoride uptake by dentine have been reported and it is difficult to compare the fluoride uptake levels found in this investigation with those of other studies due to differences in fluoride regimens, biopsy and analysis techniques and units of measurement quoted. However from those studies where the similar units of measurement were quoted, (Hellwig and Klimek, 1991 ; Hellwig, 1992; Hellwig and Attin, 1994; Takagi et al., 1997/ the fluoride content of the dentine samples were found to be similar to this investigation.

Conclusions

Application of fluoride rinses and toothpaste increased the fluoride content of the dentine but uptake was not significantly influenced by the type of fluoride compound and not increased by the presence of clinically diagnosed caries. 135

3. Weight Loss on Déminéralisation of Carious Dentine.

Introduction

Treatment of carious dentine was expected to result in increased fluoride uptake following application of topical fluoride regimens. This was not found to be the case in the in vitro study above. It is possible that the increased porosity found in carious dentine that would encourage fluoride uptake is tempered by a reduction in available hydroxyapatite for reaction with fluoride. This investigation was therefore carried out to compare weight loss on demineralisation of specimens of carious dentine with non-carious dentine specimens that were dissolved under similar conditions in Chapter 2, Section 3.1.

Method

Ten cuboidal samples of dentine, determined to be carious using clinical assessments of colour change and softening, were taken from the occlusal dentine of 10 teeth. The samples were initially stored in 250ml distilled, deionised water at 4°C for 7 days. The samples were then immersed in 5 ml 1 M hydrochloric acid.

The samples were weighed prior to immersion and again at 24 hours. Each sample was weighed at 1 minute after its removal from solution on an electric pan balance (Oertling, Model R51, London, U.K.). First, however, the surface liquid was removed by blotting the sample dry for 15 seconds using tissue paper and air drying for 15 seconds.

The weight loss of the specimens were compared with the specimens of non- carious dentine immersed in 5 ml 1M HOI for 24 hours in the previous investigation into weight loss (Page 109).

Results

The weight of the dentine samples prior to and following demineralisation is illustrated in Table 10. Non-Carious Dentine Carious Dentine Pre- Post- Percentage Pre- Post- Percentage Weight (mg) Demineralisation Demineralisation Weight Loss Demineralisation Demineralisation Weight Loss Mean 2.04 0.80 60% 1.04 0.58 43% SD 0.58 0.18 4% 0.19 0.11 11% Median 1.84 0.72 62% 1.05 0.58 44% 95% Cl 1.61 to 3.26 0.66 to 1.19 52% to 64% 0.82 to 1.15 0.48 to 0.58 31% to 54%

Table 10. The weight of the dentine samples prior to and following demineralisation in 1M hydrochloric acid.

90 7} 80 70 60 I f 50 ■ I «► 40 30 20 10 1 0 ------1 "Non Carious" "Carious"

Figure 12. Percentage weight loss on demineralisation of samples of dentine in 5 ml 1 M hydrochloric acid. 137

The weight of the non-carious samples before demineralisation had a median value of 1.84 mg and for the carious samples of 1.05 mg. The weight of the non-carious samples following demineralisation had a median value of 0.72 mg and for the carious samples of 0.58 mg. The percentage weight loss on demineralisation is illustrated in Figure 12. Percentage weight loss for the non- carious samples had a median value of 62% and for the carious samples of 44%. Statistical analysis (Appendix 4, Table 3) revealed a highly significant decrease in the carious dentine samples in percentage weight loss on demineralisation (p = 0.001).

Discussion

The highly significant decrease in percentage weight loss on demineralisation obtained in the carious dentine samples compared to non-carious dentine samples suggests that there was a reduction in the inorganic component of dentine available for reaction with fluoride under the conditions of these investigations. This may explain the lower fluoride uptake achieved by carious dentine following application of Meridol and sodium fluoride toothpaste and mouthrinse in the previous investigation; the reduction in mineral content off setting factors such as increased porosity that would increase fluoride acquisition.

These findings suggest that samples of dentine with similar pathology should be selected for in'vitro fluoride uptake investigations. This is most easily achieved by using dentine taken from extracted unerupted teeth.

Conclusion

When assessed by weight loss on acid dissolution, dentine samples clinically diagnosed as being carious had a reduced inorganic phase compared to samples judged to be non-carious. This suggests that fluoride uptake studies should be conducted on samples of dentine with standardised characteristics. 138

4. Measurement of the Fluoride Content of the Organic Dentine Remaining After Mineral Dissolution In Hydrochloric Acid.

Introduction

Measurement of fluoride uptake by dentine analysed by the method described left an area of potential variability, it was not certain that all fluoride uptake by dentine was released on exposure of the dentine to 5 ml 1 M hydrochloric acid. The surfactant activity of amine fluorides may encourage adsorption of fluoride to the organic matrix of dentine; it may therefore not be released on demineralisation. This investigation aimed to establish the fluoride content remaining in the organic portion of dentine.

Method

Dentine specimens collected from sixteen of the teeth in the previous investigation into measurement of fluoride uptake by dentine in vitro were used in this study. Following exposure to 1M hydrochloric acid for 24 hours, the organic remains of each specimen were retained for further analysis whilst the acid-dentine solution was analysed for fluoride content (detailed in Section 2, Page 128). Each organic dentine sample was blot dried with tissue, washed in 20ml distilled, deionised water, blot dried and then placed in 10 ml 1 M potassium hydroxide (KOH) for 1 week in a sealed container. The KOH solution was prepared by dilution with distilled, deionised water of 85.0% analar KOH (BDH Chemicals, U.K.).

The technique for analysis of the fluoride content of KOH solutions was modified compared to the method previously described for acid solutions. When solutions of high pH (greater than 9.5) are being measured the electrode responds to hydroxide ion as well as to the fluoride ion. The potential reading is then lower than it would be if fluoride alone were present. This was overcome by diluting each calibration and test solution with 4M buffered potassium acetate solution thereby adjusting the pH to between 5 and 6, eliminating hydroxide error and raising the total ionic strength of both samples and standards to the same value. 139

Calibration solutions were prepared by serial dilution, with 1 M KOH, of a 100 ppm F" aqueous (sodium fluoride) solution. A calibration curve was then plotted for the cell with solutions ranging from 10"^ to 10"^ ppm F". The fluoride content of the test solutions was then read from the calibration curve.

Results

The amount of fluoride remaining in the organic portion of untreated and treated dentine specimens is illustrated in Table 11 and Figure 13 (raw data in Appendix 5, Table 3).

The fluoride remaining ranged from 0 to 4 pg (0 to 1.6% of the total fluoride content), median 0.4 pg with a 95% confidence interval of 0.1 to 0.5 pg. There were no statistically significant differences between the topical fluoride treatments (p = 0.72) and ranking of the data indicated that the percentage of fluoride remaining was unrelated to the initial fluoride content of the specimen (details in Appendix 4, Table 4).

Discussion

The technique enabled analysis of the fluoride content of the organic remnants.

Dissolution of dentine in 1M hydrochloric acid resulted in 99.8% (sd 0.2%) of fluoride being released from the specimen for measurement by the established method. The amount of fluoride remaining in the organic residue of the dentine therefore has a minimal effect on the perceived fluoride content when this method is used.

Conclusion

The amount of fluoride remaining in the organic residue of the dentine has a minimal effect on the perceived fluoride content when dentine is dissolved in 5 ml 1 M hydrochloric acid for 24 hours. FLUORIDE REGIMEN

UNTREATED SODIUM FLUORIDE TOOTHPASTE AND MERIDOL MOUTHRINSE TOOTHPASTE AND MOUTHRINSE

Fluoride Fluoride Fluoride Fluoride Fluoride Fluoride content in HOI content In KOH content in HOI content in KOH content in HOI content in KOH

(M9) (no) W) W (xlO^ng) W) Mean 220 0 .6 0.3% 430 0 .6 0.2% 730 0.8 0.1% SD 130 0 .7 0.3% 210 1.1 0.4% 590 1.3 0.2% Median 200 0 .4 0.3% 380 0.3 0.05% 680 0 .3 0.03% 95% 01 150 to 250 0.10 to 1.0 0.1%to0.5% 250 to 650 0.03 to 0.5 0% to 0.2% 400 to 950 0.05 to 0.7 0% to 0.2%

Table 11. Fluoride content of inorganic and organic solutions of dentine following topical fluoride treatments with Meridol and sodium fluoride toothpaste and mouth rinse. 141

Figure 13. Fluoride content of the organic portion of dentine following dissolution in 1M hydrochloric acid after topical fluoride treatments with Meridol and sodium fluoride toothpaste and mouth rinse.

Fluoride Content In Organic Portion of Dentine (Scatter Plot)

4.5

4.0

3.5

3.0 I 2.5 0o 0) 2.0 •O 1.5 1 1.0 0.5 • . )

0.0 r - f ------T-- 1------T------f ------1------* 1 8 9 10 11 12 13 14 15 16 Tooth

Untreated • Sodium Fluoride • Meridol

Percentage of Fluoride Content in Organic Portion of Dentine Compared to Total Fluoride Content. (Median Values and 95% Confidence Intervals )

0.6

0.5

0.4 o (U 3 0.3 o 3 0.2

0.1

0.0 Untreated Sodium Fluoride Meridol 142

5. THE EFFECT OF STORAGE IN WATER ON THE FLUORIDE CONTENT OF DENTINE BIOPSIES

Introduction

To assess fluoride uptake in vivo, untreated and test samples of dentine need to be harvested some time apart. However, on measuring fluoride uptake small variation can occur during the dissolution, dilution and calibration stages of fluoride measurement. These can influence the values obtained even with careful laboratory technique. To reduce the effect of these variables it would be convenient if dentine samples could be stored to allow all measurements to be made together. This investigation aimed to assess the effect of storage in water on the fluoride content of the dentine samples.

Method

Five freshly extracted unerupted third molars were collected, stored, sectioned, prepared and exposed to the toothpaste and mouthrinse regimen as previously described. Dentine was biopsied from each section and the specimens were each stored in 5 ml distilled, deionised water at 4°C. A 0.5 ml aliquot of water was taken from the storage water at one day, one week and one month. Eacg aliquot was placed in a sealed container and stored at 4°C. The fluoride concentration in the storage water for each storage period and in the dentine specimen at the end of the storage period was measured.

The fluoride content of the dentine specimens was analysed as described previously.

The technique for analysis of the fluoride content of the storage water had to be modified because when low concentrations of fluoride are being measured linearity of a plot of potential measured versus the logarithm of fluoride concentration cannot be assumed. A calibration curve for the cell was plotted using calibration solutions prepared from aqueous sodium fluoride solutions ranging from 0.005 ppm F" to 0.5 ppm F". Solutions analysed were composed of a 0.5 ml aliquot of the calibration or test solution plus 1.5 ml of 0.1M 143 hydrochloric acid. The fluoride content of the test solutions was read from the calibration curve. The total amount of fluoride released into the storage water could then be calculated. From this and the fluoride content of the dentine sample, the initial fluoride content of the dentine was deduced and the percentage loss of fluoride into the storage water at each period could then be calculated.

Results

Ten dentine samples were harvested with a mean surface area of 0.93 mm^ (sd 0.07); there was no significant difference (p = 0.07) in surface area between the two groups.

The fluoride content of the samples and the storage water is presented in Table 12 (raw data in Appendix 5, Table 4). Statistical analysis performed is detailed in Appendix 4, Table 5.

Following the Meridol regimen the median fluoride content of the storage water was 0.10 pg at 1 day, 0.08 pg at 1 week and 0.02 pg at 1 month. Following the sodium fluoride regimen the median fluoride content of the storage water was 0.11 pg at Iday, 0.09 pg at 1 week and 0.02 pg at 1 month. There was no significant difference in loss of fluoride between the fluoride regimens at the same storage period or between the fluoride regimens at different storage periods except at 1 month. Here the loss of fluoride following both the sodium fluoride (p = 0.039) and Meridol regimen (p = 0.05) were significantly lower than that following the Meridol regimen at 1 day.

The percentage of fluoride released into the storage water is illustrated in Table 12 and Figure 14 (raw data in Appendix 5, Table 4). Following the Meridol regimen the median percentage of fluoride lost into the water was 8% at 1 day, 6% at 1 week and 1 % at 1 month. Following the sodium fluoride regimen the median percentage of fluoride lost into the water was 7% at 1 day, 7% at 1 week and 2% at 1 month. Specimen Surface Area Fluoride Content Fluoride Released Into Storage Water (pg) Total Fluoride Percentage of Total Fluoride (mm^) Of Dentine (Dentine and Water) Released Into Storage Water (pgcm*^) 1 Day (In 5ml) I 1 Week (In 4.5ml) 1 Month (In 4ml) (us) 1 Day 1 Week 1 Month MERIDOL TOOTHPASTE Ah D MOUTHRINSE REGIMEN Mean 0.89 175 0.12 0.08 0.02 1.51 11% 8% 2% SD 0.08 102 0.04 0.04 0.01 0.81 8% 7% 2% Median 0.87 181 0.10 0.08 0.02 1.54 8% 6% 1% Range 0.80 to 0.97 58 to 280 0.09 to 0.15 0.02 to 0.12 0.01 to 0.03 0.56 to 2.44 4 to 25% 3 to 21% 1to5% SODIUM FLUORIDE TOOTHPAS1FE AND MOUTHRI SISE REGIMEN Mean 0.96 130 0.12 0.09 0.03 1.27 10% 7% 2% SD 0.05 51 0.11 0.05 0.03 0.52 6% 2% 1% Median 0.99 165 0.11 0.09 0.02 1.62 7% 7% 2% Range 0.87 to 1.00 60 to 167 0.03 to 0.30 0.04 to 0.17 0.01 to 0.07 0.52 to 1.65 3 to 19% 6 to 10% 1to4%

^ Total fluoride = Total fluoride content of dentine specimen + Fluoride content in the 0.5ml of water removed at 1 day + Fluoride content in the 0.5ml of water removed at 1 week + Fluoride In the 4 ml of water remaining at 1 month.

Table 12. Fluoride loss from dentine specimens Into storage water at 1 day, 1 week and 1 month after topical application of toothpaste and mouthrinse in vitro. i 145

Figure 14. Fluoride loss from dentine specimens into storage water at 1 day, 1 week and 1 month after topical application of toothpaste and mouthrinse in vitro.

Median values and 95% Confidence Intervals

30%

25%

20%

I 15%

_ 10% (D Û. 5%

0% Meridol NaF Meridol NaF Meridol NaF 1 Day 1 Day 1 Week 1 Week 1 Month 1 Month

Discussion

Storage of the samples in distilled, deionised water demonstrated that even at one day the percentage of fluoride lost into the water was considerable and was still substantial at one week. This suggests that fluoride on the surface of the specimen will diffuse into the water. The reduction at one month suggests that such fluoride may be available for re-uptake by the dentine. Alternatively, insoluble complexes may be forming that are then not detected by the fluoride meter.

Conclusions

The variations noted here imply that dentine specimens exposed to topical fluoride regimens should be analysed immediately on collection. If storage is necessary, the fluoride content in the storage medium should also be taken into account. 146

CHAPTER 4.

FLUORIDE UPTAKE BY DENTINE IN VIVO FOLLOWING UNSUPERVISED USE OF TOOTHPASTE AND DAILY MOUTHRINSE FOR ONE MONTH. 147

1. PRELIMINARY INVESTIGATION OF THE MEASUREMENT OF FLUORIDE UPTAKE BY DENTINE IN VIVO.

Introduction

A preliminary investigation was conducted to evaluate the method of analysis of fluoride uptake by dentine established in vitro for use in vivo.

Method

Six patients with advanced tooth wear restored with provisional overlay dentures took part in the investigation. The study design was approved by the hospital research and ethics committee.

Patient Selection.

The first six patients wearing provisional dentures and requiring re-contouring of one or more denture abutments before the construction of definitive dentures were selected from the waiting list. Three patients had abutments that were sound and unrestored, two patients (4 and 5) had abutments with arrested caries while patient 6 had an abutment to be prepared after removal of a crown that had been cemented with a glass ionomer cement. The patients were included in the study to ascertain the effect of these clinical conditions on the fluoride content of the dentine and to determine whether additional fluoride following the use of a topical fluoride regimen was detectable using this method.

Patient Preparation.

All patients gave their voluntary, informed, written consent (Appendix 6). Prior to the investigation the patients were asked to continue with their normal toothpaste but to refrain from mouthrinsing for 1 month prior to start. The patients were given a patient number based on chronological entry into the trial. 148

Fluoride Regimen.

Each patient was assigned to one of the toothpaste and mouthrinse regimens described previously (Table 6). The allocation of fluoride regimen to patient number was carried out by random number tables generated by GABA International Limited and the codes were retained by the company. Both patient and operator were therefore blind to the nature of the regimen. The products were presented as previously described but the containers were labelled with the patient number rather than the batch number. The patients were also provided with a medium textured toothbrush (Elmex, GABA International, Switzerland).

Written instructions (Appendix 6) were given on the use of the topical fluoride regimen and reinforced verbally. A contact telephone number was given should further supplies be required. They were asked to brush using approximately 1ml of toothpaste (0.14% fluoride) twice daily (morning and night) and to use approximately 10ml of mouthrinse (0.025% fluoride), holding the solution in the mouth for one minute, last thing at night. The toothpaste was measured by delivering a strip of toothpaste from the tube along the full length of the brush. The mouthrinse was measured using a measuring cup provided with the rinse.

At completion, after 1 month, the patients were asked to return any remaining toothpaste and mouthrinse and compliance was assessed by patient comments and by measuring the residue in the containers.

Dentine Biopsy.

Prior to biopsy of the pre-treatment dentine sample, the area planned for dissection of the control and test biopsies was abraded with a white stone (Shofu Dura-White Stone FL2, Shofu, Japan) in a contra-angled handpiece under water-cooling. This gave a consistent surface from which to biopsy and removed the surface rich layer of fluoride found in dentine. The control sample (1 X 1 X 0.5 mm) was then dissected using the method described previously. The provisional denture was adjusted to fit the new abutment contours by a 149 local reline with a light activated denture resin (Triad VLC Provisional Material, Dentsply Trubyte, USA).

After one month a second post-treatment (test) sample was dissected from the abutment from an area adjacent to the first, the abutment was then fully re contoured and polished and the provisional denture adjusted.

Fluoride Analysis.

The dimensions of the specimens were measured immediately after dissection and fluoride analysis was carried out as described previously. Fluoride uptake per unit area by each overdenture abutment was calculated as the fluoride content per unit area of the sample biopsied at one month minus that of the sample biopsied at baseline.

Results

6 patients (3 male, 3 female, aged 58 to 74, mean 70 years) provided 12 dentine specimens.

Table 13 illustrates patient details, the fluoride regimen allocated, the specimen dimensions, the fluoride content of the pre-treatment and treated dentine samples and the mean fluoride uptake for each abutment.

The specimens had a mean surface area of 1.06 mm^ (sd 0.22) and a mean depth of 0.62 mm (sd 0.30). The samples from abutments with carious dentine contained more fluoride than those from sound abutments. The fluoride content of the samples from an abutment exposed to glass ionomer cement were up to ten times higher (330 and 447pgcm"^) than the samples obtained from the sound, unrestored abutments. The fluoride uptake by the sound abutments ranged from 2 to 57 pgcm"^.

Compliance in the use of the fluoride regimen was judged to be 100% from comments by the patients and by the emptied containers. 150

Table 13. Patient details, specimen dimensions, fluoride content and fluoride uptake following topical application of Meridol and sodium fluoride toothpaste and mouthrinse.

PATIENT SEX AGE TOOTH CONDITION REGIME SPECIMEN DEPTH AREA F F (mm) (mm^ CONTENT UPTAKE’ (ligcm'*) (rtjcm*^

1 Male 74.0 13 Meridol Control 0.44 1.24 31 Test 0.52 1.20 88 51 Clinically Sound 2 Female 63.3 35 Sodium Control 1.36 0.96 27 Fluoride Test 0.79 1.14 47 27

3 Female 71.8 33 Meridol Control 0.67 1.41 40 Test 0.52 1.15 42 11

4 Female 58.9 31 Sodium Control 0.43 0.64 185 Arrested Fluoride Test 0.44 0.71 98 -91 Caries

5 Male 80.4 35 Meridol Control 0.73 1.07 115 Test 0.41 0.97 27 -38

6 Male 70.5 25 QIC Sodium Control 0.54 1.24 330 Fiuoiide Test 0.59 1.03 447 87

^ For formula see page 127

Discussion

The technique proved sufficiently sensitive to detect differences in fluoride concentration, both on application of a fluoride regimen and resulting from varying clinical conditions. 151

The carious dentine samples acquired high levels of fluoride compared to sound dentine. It is possible that the fluoride content was influenced by the proximity of the sample to the lesion, hence the negative results obtained for uptake for patients 4 and 5. As discussed in Chapter 3, increased acquisition of fluoride might be expected due to the high porosity of carious lesions and favours reversal of the lesions. The increase in fluoride taken up clinically in comparison to the in vitro investigation could be explained by the déminéralisation- remineralisation cycle that typifies caries development (Silverstone et al. 1981). Calcium fluoride on the tooth surface releases fluoride ions in acidic conditions which is then available for uptake (Rolla and Saxegaard, 1990).

Furthermore, different behaviour might be expected in vitro and in vivo. The properties of dentine could be expected to alter on extraction (with loss of vitality) and on storage. Different solution conditions also exist in vitro and in vivo, including levels of uncomplexing reacting ligands, buffer capacity of solutions, rate of clearance of reactants and presence of adherent salivary proteins (Miller et a/., 1994).

The fluoride content of the samples from an abutment exposed to glass ionomer cement (patient 6) were greatly increased. The acquisition of high levels of fluoride by dentine from glass ionomer cements has been reported in vivo (Mukai et al., 1993). This may be attributed to the ability of the dentine surface to absorb very large amounts of fluoride when given sufficient time to incorporate fluoride by exchange (Robinson eta!., 1996).

From these samples it is apparent that dentine can take up large amounts of fluoride and ways of promoting this need to be investigated.

The wide range in fluoride content between the samples, arising from the various clinical conditions, underlines the importance of standardising or specifying the characteristics of dentine sampled in topical fluoride trials that examine fluoride uptake. Site specific investigations have also been suggested when enamel is sampled (Mok eta!., 1990). 152

As discussed previously studies of the reporting of fluoride uptake by dentine has been limited and comparison between them is difficult. In situ investigations (dentine carried in an intra-oral device) have been reported but a full in vivo study has not been conducted. However, in these In situ studies, where comparison was possible, (Hellwig and Klimek, 1991; Hellwig, 1992; Hellwig and Attin, 1994) the fluoride content of the dentine samples was similar to the current investigation. 153

Conclusions

Dissecting dentine using a fine diamond bur in an air turbine with water cooling, guided by a template, proved an accurate and reproducible technique in vivo, for obtaining samples for fluoride analysis. Analysis of such samples with a differential electrode cell enabled fluoride uptake following a topical regimen to be assessed. The wide range of fluoride found in sound, carious and restored dentine emphasised the need to standardise dentine characteristics when evaluating fluoride uptake. 154

2. FLUORIDE UPTAKE BY DENTINE IN VIVO FOLLOWING UNSUPERVISED USE OF TOOTHPASTE AND DAILY MOUTHRINSE FOR ONE MONTH.

Introduction

Following establishment of the method in the preliminary investigations, the aim of this investigation was to compare the fluoride uptake by dentine in vivo from two fluoride regimens consisting of sodium fluoride or a combination of stannous fluoride and Amine Fluoride 297. The regimens were applied by unsupervised use of a toothpaste and daily fluoride mouthrinse for one month.

Method

Twenty-eight patients with advanced tooth wear restored with provisional overlay dentures took part in the investigation. The patients were selected as before with additional requirement that they presented with one or more abutments for re-contouring that were unrestored and judged clinically to be caries free.

The study design was approved by the hospital research and ethics committee and all patients gave their voluntary, informed, written consent and received instructions on the use of the regimen (Appendix 6) as before.

Preparation and biopsy were carried out as described previously and the fluoride regimen was as described above. The patients were additionally asked to comment on the acceptability of the regimen.

Results

28 patients (20 male, 8 female, aged 36 to 78, mean 62 years) provided 88 samples of dentine.

Compliance was judged to be 100% by the return of emptied containers. Table 14 illustrates the comments made by the patients about the fluoride regimens. 155

Table 14. Number and description of comments made by patients concerning the flavour of the toothpaste and mouthrinse. Total number of patients = 28.

COMMENTMERIDOL SODIUM FLUORIDE FAVOURABLE Nice / Pleasant / Liked Taste 2 6

Very refreshing 1 -

Felt clean - 1 UNFAVOURABLE

Unpleasant taste - 1

Mouth wash unpleasant / awful 2 -

Red wine tasted horrible - 1

Paste gritty 1 -

Very frothy - 1 Didn't feel clean 1 1

Didn’t clean denture - 1

Made mouth sting 1 -

Gave dry mouth 1 -

A summary of the age and sex of the patients, the duration of the fluoride regimen, the dentine specimens dimensions, the fluoride content in the control and test samples and the fluoride uptake by the abutments is illustrated in Table 15 (raw data is presented in Appendix 5, Table 5). A mean value for fluoride uptake for each patient was also calculated when two or more abutments were available for biopsy. The fluoride uptake by the patients is therefore also presented. Statistical analysis is detailed in Appendix 4, Table 6.

The two groups did not exhibit statistical difference for the age (p = 0.19) or sex of the patients. The fluoride regimen was used for an average of 31 (sd 6) days and there was no statistical difference in this between the two group (p = 0.79). AGE TIME DEPTH (mm) SURFACE AREA(mm^) F CONTENT (pgcm^ F UPTAKE F‘ UPTAKE (years) (days) (MOcm^) (ngcm^) Pre-treatment Treated Pre-treatment Treated Pre-treatment Treated BY BY ABUTMENT PATIENT

VIERIDOL REGIMEN Mean 59 0 31 0.53 0.43 1.22 1.26 12 33 23 21 SD 11.1 6 0.12 0.10 0.52 0.48 10 21 16 14 Median 59.6 28 0.55 0.43 1.11 1.24 7 33 21 17 95% 01 48.9 to 71.8 28 to 35 0.44 to 0.61 0.37 to 0.52 0.94 to 1.25 1.06 to 1.47 5 to 15 17 to 43 10 to 33 9 to 33 SODIUM FLUORIDE REGIMEN Mean 65.0 32 0.53 0.52 1.23 1.17 15 28 14 14 SD 8.8 7 0.21 0.14 0.57 0.39 14 19 14 13 Median 65.1 28 0.50 0.50 1.10 1.10 8 21 12 12 95% 01 56.9 to 73.7 28 to 42 0.45 to 0.56 0.4 to 0.6 0.85 to 1.39 0.95 to 1.36 7 to 21 16 to 38 6to19 1to31

Table 15. Summary of patient age, time regimen used, specimen dimensions, fluoride content and fluoride uptake following topical application of Meridol and sodium fluoride toothpaste and mouthrinse.

8 157

The specimens had a mean surface area of 1.22 mm^ (sd 0.49) and a mean depth of 0.51 mm (sd 0.15). Significant difference between the groups was not exhibited for surface area (p = 0.89) or depth (p = 0.07) of the specimens, or for the fluoride content of the control specimens (p = 0.39).

Fluoride uptake by dentine following the regimens is illustrated in Table 15 and Figure 15. When the fluoride uptake by each abutment is considered, the Meridol regimen resulted in a median fluoride uptake of 21 pgcm'^ and the

sodium fluoride regimen in a median fluoride uptake of 12 pgcm'^. Again, these results were not statistically significant (p = 0.09)

When the mean fluoride uptake by each patient is considered, the Meridol regimen resulted in a median fluoride uptake of 17 pgcm'^ and the sodium fluoride regimen in a median fluoride uptake of 12 pgcm'^. These results were not statistically significant (p = 0.29).

Discussion

Despite the negative comments received from 11 of the patients about the regimens all patients completed the investigation. Furthermore, all participants in this study expressed willingness to participate in further studies involving the regimens. The negative comments were evenly split between the regimens. This is in contrast to the findings of Laine et al. (1993) who compared twice daily rinsing of AmF 297/SnF2 (0.025% F") and sodium fluoride (0.05% F ) in lymphoma patients receiving chemotherapy. 52% of subjects recorded unpleasant effects from the AmF 297/SnF2 rinse whereas unfavourable comments on the NaF regimen were made by only 6%. However, a high proportion of the complaints concerned stinging of the mucosa (64%). Chemotherapy can cause mucositis making these patients extra sensitive to such side effects. This study also, however, did not lose participants because of the complaints.

Dimensions in the dentine specimens dissected were not more variable than those dissected in vitro. Variations were taken into account in the calculation of fluoride uptake. 158

Figure 15. Fluoride uptake by dentine following topical application of Meridol and sodium fluoride toothpaste and mouthrinse in vivo. Uptake by abutment and by patient is evaluated.

Scatter Chart

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Patient

• Meridol • Sodium Fluoride

Fluoride Uptake by Abutment Median and 95% Confidence Intervals

i 40 Ï 30 i 20 3 ® 10

Meridol Sodium Fluoride

Fluoride Uptake by Patient Median and 95% Confidence Intervals

I 30 n 20 3

- f -

r? 0 Meridol Sodium Fluoride 159

Fluoride uptake by individual abutments as well as the mean value from abutments sampled from the same patient were compared. The latter gives a more accurate comparison of the regimens. It is well known that observations obtained from multiple specimens in the same patient cannot be treated as independent observations, (Stookey et al., 1992). While there is considerable variation in the local environment surrounding a tooth, individual teeth in any mouth will not behave independently and will respond to the same host factors (Banting, 1993). Such host factors as saliva flow, composition and distribution, the indigenous microflora and oral hygiene practices may affect fluoride uptake.

The superior fluoride uptake claimed for amine fluoride regimens was again not apparent, perhaps due to over-stabilisation on combining it with stannous fluoride. However the Meridol regimen performed as well as the selected accepted standard regimen of sodium fluoride.

Conclusions

The unsupervised use of sodium fluoride and Meridol toothpaste and daily mouthrinse increased the fluoride content of the dentine in vivo but uptake was not significantly influenced by the type of fluoride regimen. 160

CHAPTER 5.

THE EFFECT OF UNSUPERVISED USE OF TOOTHPASTE AND DAILY MOUTHRINSE FOR ONE YEAR ON CARIES INCIDENCE. 161

Introduction

Fluoride uptake on application of a regimen is a useful indicator of its efficacy but amine fluoride, stannous fluoride and amino-stannous fluoride have been reported to effect a range of anti-caries activity (Pages 46 to 77). A very limited number of studies have been carried out to investigate the effect of amino- stannous fluoride regimens on dentinal caries. A medium term study was therefore carried out to establish the effect of the Meridol and sodium fluoride regimens on caries incidence.

Method

40 patients (31 male, 9 female, aged 46.3 to 83.7, mean 64.4 years) with advanced tooth wear restored with definitive overlay partial dentures took part in this investigation. The study design was approved by the hospital research and ethics committee and all patients gave voluntary, informed, written consent (Appendix 6).

Patients were selected from the top of the waiting list and dentures were constructed in the Prosthetic Department of the Eastman Dental Hospital, London to departmental guidelines (Hammings et al. 1995). To participate, patients were required to have two or more sound, unrestored teeth covered by their dentures. Patients on long term antibiotics or requiring antibiotic cover were prohibited from the investigation. The patients had received oral hygiene instruction from a hygienist prior to construction of their dentures and this was reviewed and advice on denture hygiene was given on insertion of the denture.

At the baseline appointment unrestored teeth that were covered by the denture were dried with gauze, photographed and scored for caries using the index described below. Oral hygiene instruction was reinforced and the dentures checked and adjusted as necessary.

Each patient was assigned to one of the toothpaste and mouthrinse regimens described in Table 6. The allocation of fluoride regimen and instructions for its use proceeded as described in Chapter 4. The patients were also provided with 162

a medium textured toothbrush (Elmex, GABA International, Switzerland), a single tufted brush (Super Interspace Brush, CTS, U K ) and a denture brush (Denture-Creme Denture Cleansing Toothbrush, Stafford-Miller, U.K). To reduce the effect of denture cleansers on the oral microflora, the patients were asked to clean their dentures (and nocturnal splints when worn) using the brush and toothpaste provided and to keep the dentures soaking in water overnight. Written instructions were given on the oral and denture hygiene procedures (Appendix 6).

At each visit the patients were requested to return remaining toothpaste and mouthrinse and compliance was assessed by measuring the residue in the containers.

The patients were reviewed 3 monthly for one year. At each review the abutments were photographed and caries scores recorded with reference to previous photographs.

The codes for the fluoride regimens used by each patient were broken when all patients had completed 12 months in the investigation.

CARIES INDEX

A qualitative score was given to the unrestored abutments covered by the denture:

Sound No discoloration or cavitation of the dentine was evident Colour change A colour change of the dentine was apparent. Cavitation Cavitation of the dentine was manifest.

The dentine assessment was made on clinical examination. Colour transparencies were used to confirm assessment of change to the appearance of the dentine. The author carried out all clinical examinations. 163

Intra-examiner calibration was conducted by repeat assessment of colour transparencies of 55 abutments from 10 patients. Assessments were made at baseline, 2 weeks later and at completion of the study.

Cavitated lesions were restored or reduced and polished as appropriate. Restored abutments were exited from the study whereas polished abutments were re-entered as sound abutments from that appointment.

PHOTOGRAPHY

Photographs were taken of the unrestored abutments covered by the denture at the initial appointment and at each review appointment to enable visual changes in the dentine to be identified. Each tooth was wiped dry with gauze and photographed (Camera - F3, Nikon, Japan; Lens - AF Micro Mikkon, Nikon, Japan; Ring flash - AS12, Nikon, Japan; f11; 1/125) in an occlusal mirror at 1:1 magnification using Kodakachrome 64 Daylight Professional Colour Reversal Film (PKP-36PDX). This film was refrigerated at 4°C before use and immediately sent for processing on completion of the film. The films were sent to the same processing laboratory throughout (Kodak Processing Laboratories, London, U.K.).

Images of the transparencies were projected onto a white screen viewed in a darkened room. Care was taken to ensure that on each viewing session, the conditions and equipment arrangements remained the same.

STATISTICAL ANALYSIS

Statistical analysis of the data was carried out using the non parametric tests of Mann Whitney or Kruskal Wallis were appropriate with a confidence limit set at 95%. 164

Results

PATIENT DATA

36 patients completed the investigation. One (no. 23) exited the study at 3 months because she was not wearing her denture and another (no.28) exited the study at 6 months because his denture was irreparably damaged, data was not obtained for these patients. A third patient (no.1) lost his denture and exited the study at 9 months. The fourth (no. 35) exited the study at 12 months. She felt her breathe smelt and her teeth were stained and could not be persuaded from using her customary toothpaste. She was using the sodium fluoride regimen.

The age and sex of the patients and the fluoride regimen provided is illustrated in Table 16 (raw data in Appendix 5, Table 6). The two groups did not exhibit statistical difference for age (p=0.26) or sex of the patients.

Table 16. Age and sex of the patients and the fluoride regimen provided.

Fluoride Regimen Meridol Sodium Fluoride Sex Ratio Male: Female 3:1 4:1 Age Mean 66.2 62.6 Standard Deviation 10.4 10.7 Median 68.0 61.8 95% Confidence Interval 60 to 74 57 to 71

CARIES INCIDENCE

Kappa scoring of the calibration scores (Appendix 4, Table 7) illustrated a high level of conformity (Kappa 0.78 to 0.90). 165

Table 17 records a summary of the number of teeth present and the number of abutments covered by a denture considered for each patient, the caries score at baseline and the number of new lesions recorded over 12 months (raw data in Appendix 5, Table 7 and 8). Statistical analysis performed is illustrated in Appendix 4, Table 8.

The patients were partially dentate with a mean of 18 teeth present (range 10 to 23). 162 unrestored teeth, covered by a denture, were considered. The number of abutments (p = 0.99) and the initial caries score (p =0.53) did not differ significantly between the two groups.

Examples of abutments where changes to the dentine were scored as developing new "colour change” are illustrated in Figure 16. The data on new carious lesions that developed are summarised in Table 17. The percentage carious incidence, both for when the number of abutments and when the number of patients are considered, is illustrated in Figure 17.

In patients following the Meridol regimen 5 abutments (6%) developed a new “colour change” lesion (a mean of 0.28 per patient), and 9 (11%) abutments developed a cavitated lesion (a mean of 0.50 per patient). In patients following the sodium fluoride regimen 4 abutments (5%) developed a new "colour change” lesion (a mean of 0.22 per patient) and 2 abutments (2%) developed a cavitated lesion (a mean of 0.11 per patient). The new "colour change” lesions developed in 4 (22%) of the patients following the Meridol regimen and 3 (17%) of the patients following the sodium fluoride regimen. The cavitated lesions developed in 6 (33%) of the patients following the Meridol regimen and 2 (11%) of the patients following the sodium fluoride regimen

Carious lesions developed early, the first new "colour changes” and cavitations were seen at 3 months

A significant difference was not apparent for new "colour change” lesions (p= 0.38) or for cavitations (p= 0.09) in patients following the Meridol regimen compared to those following the sodium fluoride regimen. Figure 16. Clinical photographs demonstrating examples of abutments where changes to the dentine were scored as developing new "colour change" lesions.

16a. A minor colour change to \1_affecting the junction between primary and secondary dentine.

16b. Colour change to \1_affecting secondary dentine. 167

CD JCl CÛ CD Figure 16. Clinical photographs demonstrating examples of abutments where changes to the dentine were scored as developing new “colour change” lesions.

16c. Further colour change to \1_in areas of the dentine already affected plus development of new areas.

16d. Extensive colour change to the dentine of H I. 16c.

16d. REGIMEN MERIDOL SODIUM FLUORIDE

(18 PATIENTS) (18 PATIENTS)

No. of No. of Colour Change Cavitation No. of No. of Colour Change Cavitation Teeth Abutments^ Initial New Initial New Teeth Abutments^ Initial New Initial New Total 326 81 10 5 0 9 372 81 5 4 0 2

PER PATIENT:

Mean 16 5 0.56 0.28 0 0.50 19 5 0.28 0.22 0 0.11

SO 3 2 0.92 0.57 0 0.79 4 2 0.46 0.55 0 0.32

Median 16 3 0 0 0 0 19 4 0 0 0 0

95% Cl 15to18 3 to 7 0to1 0 0 Otol 17 to 22 2 to 6 Otol 0 0 0

1 _= Unrestored abutments covered by the denture.

Table 17. Summary of the number of patients, number of teeth, number of abutments considered, caries score at baseline and new lesions recorded over 12 months following unsupervised use of Meridol and sodium fluoride toothpaste and mouthrinse.

3 171

Figure 17. Percentage caries incidence following unsupervised use of Meridol and sodium fluoride toothpaste and mouthrinse for 12 months.

Percentage of abutments developing new carious lesions

3? 10

0 Meridol Sodium Fluoride Meridol Sodium Fluoride Colour Change Cavitation

Percentage of patients developing new carious lesions

Meridol Sodium Fluoride Meridol Sodium Fluoride Colour Change Cavitation 172

Discussion

PATIENT DATA

The age group and ratio of sexes of the patients participating in this investigation were similar to those found in a retrospective audit of patients with advance toothwear restored with removable partial dentures (Woodley et al., 1996). One of the patients who exited the study did so following fracture of his denture such that it was irreparable. Fracture is a problem in this group of patients, Woodley et al. (1996) finding that fractures accounted for 47% of denture failures recorded in the audit above.

Another patient exited the study reporting that her teeth were stained. This was not confirmed as she could not be persuaded to attend for a review, however the finding was unexpected as she had been using the sodium fluoride regimen. No other patient complained of staining.

CARIES ASSESSMENT

It was decided that only unrestored denture abutments would be assessed in this investigation. This eliminated the influence that the restorative materials themselves might have of the carious process as well as risk factors such as inadequate design and old leaking margins but had the disadvantage of greatly reducing the number of abutments available for the study.

The caries index used for this investigation varied from those conventionally used to detect dentinal caries of the root surface (Nyvad and Fejerskov, 1982; Katz, 1984) as the dentine was not assessed by probing for softness.

The probing of enamel carious lesions has long been discouraged. This is considered to convert subsurface demineralised lesions to frank cavities as determined by histological examination of teeth (Bergman and Linden, 1969; Ekstrand et al., 1987). The loss of sensitivity caused by use of a visual method of diagnosis is argued to be acceptable given the low prevalence and slow progression of caries (Downer, 1989). Furthermore, probing has not been found 173

to improve the accuracy of diagnosis of fissure caries over visual examination alone (Howat et al., 1981; Lussi, 1991).

Newbrun (1993) suggested that since there was no data concerning remineralisation of subsurface demineralised dentine, probing damage was not an issue. However, as in enamel lesions, progression of mineral loss in dentine occurs deep to a surface zone which varies in thickness and mineral content (Nyvad and Fejerskov, 1987; Nyvad et al. 1989; Schüpbach et al., 1989). Indeed, Nyvad and Fejerskov (1987) found that extensive mineral might be lost from dentine, reflected by extensive softening on probing and yet a surface zone was present. Thylstup and Fejerskov (1994) stated that this zone was vulnerable to damage and therefore probing should be almost totally avoided. Furthermore the root caries lesion is infiltrated by micro-organisms at a very early stage in development (Nyvad and Fejerskov, 1982; Frank et al., 1989; Schupack et al., 1989,1990). Probing a lesion might therefore carry the risk of transferring these organisms deeper into the sterile but softened deeper part of the lesion.

However, it was considered that a diagnosis of colour change could not be used alone. Many substances have been found to stain the dentinal surface extrinsically (substances in the diet, habitual chewing of betel nut and/or tobacco, tobacco smoking, chlorhexidine, stannous fluoride, and chromogenic bacteria) and intrinsically (extrinsic stains, endodontic materials and products of pulp necrosis) (Soames and Southam, 1998). Thylstrup and Fejerskov (1994) felt that from a differential diagnostic point of view, a root surface carious lesion was easy to distinguish from other root surface discolourations as the latter are widespread and ill defined, this should also apply to denture abutments. However, colour change of the dentine is not necessarily a sign that caries is active. Active carious lesions have been described as yellow/orange, tan or light brown in colour and arrested caries as tending to be dark, sometimes almost black (Todd and Lader, 1991). But Beighton et al. (1993) and Lynch and Beighton (1994) found lesions classified as yellow and black respectively harboured the greatest number of microorganisms as compared with brown and dark brown lesions. The authors suggested that that it would therefore be 174 unwise to use colour in the absence of other clinical indices to decide on the disease status and treatment needs of lesions.

The decision not to use colour change alone for diagnosing dentinal caries was confirmed by the findings of this investigation. Six cavitated lesions occurred without initial colour change to the dentine whereas 5 followed colour change. Twelve abutments exhibiting colour change as the beginning of the study remained uncavitated at the end of the 12 month period (Appendix 5, Table 7).

However, the use of cavitation as a criterion to diagnose caries has also been problematical. Cavitation associated with caries needs to be distinguished from that caused by abrasion, erosion or that caused by dental treatment (Lynch and Beighton, 1994). Furthermore, Nyvad and Fejerskov (1986) found inactive lesions to exhibit varying degrees of surface destruction, such that it was speculated that conversion of an inactive to an active lesion was associated with removal of part of the outer softened root surface. The dentine surface of the teeth forming this investigation should have been protected from the other mechanisms of surface removal by the dentures. However, using visual assessment of cavitation carried the risk on leaving early lesions undiagnosed but as patients were being reviewed three monthly this was considered acceptable.

Caries development over 12 months by individual abutments as well as by each patient was reported in this study. The latter gives a more accurate comparison of the regimens. It is well known that observations obtained from multiple sites in the same patient cannot be treated independently (Stookey et al., 1992). While there is considerable variation in the local environment surrounding a tooth, individual teeth in any mouth will not behave independently. They respond to the same host factors such as immune response, diet and nutrition, saliva flow, composition and distribution, the indigenous microflora, oral hygiene practices and concomitant disease (Imrey, 1986; Fleiss ef a/.,1987). 175

CARIES SCORES

Despite the decision to record caries development only in abutments which were sound at baseline, this group of patients developed caries rapidly and with a high incidence. They therefore form a useful group to use in clinical dentinal caries trials. However, while the use of patients at high risk of caries to improves the efficiency of a clinical trial (O’Mullane 1976), the results cannot be reliably extrapolated to the general population (Banting, 1993).

Scores for colour change and cavitation were greater in patients following the Meridol regimen however this difference was not significant at the 95% level and the number of patients in the investigation was small. The findings were at variance to the result of Banoczy and Nemes (1991,1992) who investigated the effect of identical regimens on root caries but their investigation also failed to find a significant difference and had small patient numbers. A larger study should be therefore be considered before the regimen is dismissed. Furthermore, amine fluoride and stannous fluoride regimens themselves have not been proven against accepted standard fluoride regimens in caries trials and the effect of combining the compounds has not proven beneficial for anti caries properties such as acid solubility (Barbakowet al., 1985, 1986a, 1987) or fluoride uptake (Barbakow eta!., 1985).

Conclusions

Unsupervised use of Meridol or sodium fluoride toothpaste and mouthrinse regimens over 12 months in patients wearing partial dentures overlaying teeth with advanced wear resulted in a trend towards reduced new cavitated lesions in the patients using the sodium fluoride regimen. However, no statistically significant differences were observed in this parameter or in colour change to the dentine. 176

CHAPTER 6.

THE USE OF FLUORIDE GELS TO PROMOTE FLUORIDE UPTAKE. 177

Introduction

The high values achieved for fluoride uptake in the previous investigations by carious dentine and from glass ionomer cement in vivo indicate that dentine can take up substantial amounts of fluoride. Ways of promoting this include the application of a higher concentration of fluoride for prolonged periods (Rolla and Saxegaard, 1990). The topical application of fluoride in the form of gels offers an alternative means of exposing tooth tissue for a prolonged period of time.

In the patient wearing an overdenture these gels can be placed in contact with the abutment teeth both professionally and at home. The latter method provides frequent applications at a low fluoride concentration. This may be more effective than infrequent treatments with high concentration fluoride preparations (Ahrens, 1983; Margolis and Moreno, 1990; Arends and Christoffersen, 1990)

Fluoride gel application increases the contact time with the dentine before clearance. Indeed, Billings et al. (1988) and Zero et al. (1992) showed that gels applied in trays provide higher levels and more prolonged retention in whole saliva and dental plaque when compared with fluoride rinses or toothpastes. A prolonged contact time of a high concentration fluoride against dentine will promote not only fluoride uptake but also its anti-microbial action on both dental and denture plaque. The use of daily topical fluoride gels on overdenture abutments has been found to decrease caries incidence (Toolson and Smith, 1978; Fenton and Hahn, 1978) and to remineralise carious lesions (Ettinger and Jakobsen, 1990).

The objective of this study was to investigate the effect of topical fluoride gel application on fluoride uptake by dentine In vitro.

Method

Ten freshly extracted, unerupted third molars were collected, stored, sectioned and prepared as described previously in Chapter 2. One section from each tooth was randomly allocated to a group that was untreated and placed in distilled, deionised water. Three sections from each tooth were randomly 178 allocated to one of three test groups and exposed to either a sodium fluoride gel, an amino-stannous fluoride gel (Meridol) or an amino-sodium fluoride gel (Elmex). These gels are described in Table 7 (Page 119). The gels were applied as previously described (Page 120), for 1 hour and then washed as before.

Dentine samples were immediately dissected, measured and analysed for fluoride as discussed earlier.

Statistical analysis of the data was carried out as before.

Results

The specimen dimensions, fluoride content and fluoride uptake are illustrated in Table 18 (raw data in Appendix 5, Table 9). Statistical analysis carried out on the data is detailed in Appendix 4, Table 9.

40 dentine specimens were obtained and were found to have a mean surface area of 1.05 mm (sd 0.19) and a mean depth of 0.53 mm (sd 0.09). There was no significant difference in specimen depth (p = 0.63) or surface area (p = 0.097).

The untreated samples had a fluoride content with a median of 12 pgcm"^. The fluoride uptake of the samples is illustrated in Figure 18. The median fluoride uptake by samples exposed to sodium fluoride gel was 208 pgcm'^ to Elmex gel was 206 pgcm’^ and to Meridol gel was 16 pgcm'^.

The fluoride uptake exhibited a high statistically significant increase following exposure to sodium fluoride (p = 0.005) and Elmex gel (p=0.001) compared to Meridol gel. There was no statistical difference between the sodium fluoride gel and the Elmex gel. Depth Surface Area Fluoride Content Fluoride Uptake

(mm) (mm^) (pgcm^) (pgcm^)

Fluoride Un NaF Elmex Meridol Un NaF Elmex Meridol Un NaF Elmex Meridol NaF Elmex Meridol Regimen treated Gel Gel Gel treated Gel Gel Gel treated Gel Gel Gel Gel Gel Gel Mean 0.49 0.54 0.54 0.54 0.99 0.98 1.14 1.16 30 241 244 45 207 212 14

SD 0.07 0.09 0.10 0.09 0.24 0.12 0.18 0.14 36 104 106 18 122 125 39

Median 0.50 0.55 0.53 0.52 1.04 1.01 1.12 1.14 12 236 230 43 208 206 16

95% Cl .42 to .55 .46 to .59 .44 to .63 .47 to .64 .66 to 1.17 .81 to 1.17 95 to 1.33 1.00 to 1.26 5 to 51 145 to 342 124 to 372 26 to 65 88 to 330 91 to 362 -20 to 58

Table 18. The sample dimensions, fluoride content and fluoride uptake of dentine samples following exposure to fluoride gels (1.25% F") in vitro.

3 180

Figure 18. Fluoride uptake of dentine following exposure to fluoride gels (1.25% F)my/fro.

Scatter Plot of Fluoride Uptake

500

(7^400

?300 0) 1.200 3 0) ^MOO o = 0

-100 10 Tooth

• NaF ♦ Elmex Merdiol

Median Fluoride Uptake with 95% Confidence Intervals 500

400 - -

§.200 i i.

2 100

-100 NaF Elmex Merdiol 181

Discussion

In order to compare the fluoride uptake by dentine when using topical gels containing different fluoride compounds, gels with the same total fluoride content were prepared by one manufacturer. This enabled a gel containing sodium fluoride and amine fluorides (Elmex, which Is commercially available in Europe) to be compared both with an experimental gel (Meridol) containing stannous fluoride and amine fluoride. The more commonly available sodium fluoride preparation was used as a positive comparison.

The samples were exposed to fluoride gel for 1 hour in vitro. This was based on the finding that when fluoride gels are applied in a tray significant levels of fluoride are found up to 30 minutes after application (Heintze and Petersson, 1979; Seppâ et a/., 1997). Patients wearing overdentures are likely to have an increased clearance time. This was confirmed by the findings of Nârhi et al. (1997) who found significant levels of salivary fluoride concentration 60 minutes after application of 0.1ml of APF gel (5,000 ppm F ) in each abutment depression of an overdenture. A measurable amount of fluoride remained In the dentures at that time (mean 7.5 pg) despite the dentures being on average 8.5 years old and presumably therefore not of the best fit.

Fluoride applied in this manner will be swallowed and absorbed into the systemic circulation. Whitford (1992) estimated that the dose of fluoride necessary to cause systemic toxicity is approximately 5 mg/kg. In overdenture patients a maximum dose of 250 mg may be assumed based on the weight of a small (50 kg) adult. The gels used in this investigation had a fluoride concentration of 1.25% (12500ppm F") which is the equivalent of 12.5 mg/ml. 20 ml of gel therefore needs to be swallowed to achieve the toxic dose. This is unlikely to occur on daily use of the gel but such a small therapeutic ratio suggests that a measured system of delivery is required to ensure this dose is not exceeded along with precise instructions and patient awareness of the risk.

The investigation confirmed that fluoride uptake by dentine can be increased after topical application of fluoride gels in vitro. The greatest uptake of fluoride was achieved after the application of the Elmex and the sodium fluoride gels. 182 the least after the use of Meridol gel. The fluoride uptake after the treatment with sodium fluoride gel was indistinguishable from that after treatment with Elmex gel. The amine fluoride content of Elmex gel was small contributing just 20% of the fluoride content.

Previous investigations with applications of Meridol paste and rinse resulted in fluoride uptakes that were not significantly different from the sodium fluoride preparations. This discrepancy may be due to other components in the gel affecting fluoride uptake. The pH of the gels were similar (Table 7 - within the limits of accurate measurement of the pH of gels) and the components of the sodium fluoride and Meridol gel were identical. However, it has been suggested that components of the gel such as glycerine and propyleneglycol provide an abundance of hydroxyl groups. These might lead to overstabilisation of the amine fluoride - stannous fluoride combination (Personal communication, ITRI, 1998) and a reduction in available fluoride. This may be more apparent in this investigation as other uptake promoting actions may have an opportunity to come into play in the paste/rinse investigation. For example, the surfactant effect of the amine fluoride may have promoted greater fluoride retention during the washing procedures in the in vitro investigations and throughout the in vivo investigation. Another explanation may be that while increasing the concentration of sodium fluoride increases fluoride uptake, increasing the concentration of AmF297/Snp2 has no effect on fluoride uptake. It is possible that a surface reaction occurs preventing further uptake.

Both the individual effects of sodium fluoride, amine fluoride and stannous fluoride need to be assessed in the absence of the other components of the gels. The effects of the compounds on the surface of dentine should also be investigated.

Conclusions

The fluoride content of dentine was increased by application of topical fluoride gels in vitro. The fluoride compound influenced the uptake. The fluoride uptake of the dentine specimens was ranked as Elmex gel = sodium fluoride gel > Meridol gel. There was a highly statistically significant difference in fluoride 183 content of treated compared to untreated dentine after all fluoride treatments except for Meridol gel. There was also a highly statistically significant difference in fluoride uptake between the groups after treatment except between sodium fluoride and Elmex fluoride gels. 184

CHAPTER 7.

INVESTIGATIONS INTO THE MODE OF ACTION OF AMINO-STANNOUS FLUORIDE REGIMENS. 185

Introduction

Following the very low fluoride uptake achieved by specimens of dentine after application of an amino-stannous fluoride gel in vitro compared to those following the application of sodium fluoride and amino-sodium fluoride gels, investigations were conducted to:

1. Assess fluoride uptake following application of sodium fluoride, Amine Fluoride 297 and stannous fluoride in the absence of the other components of the gels 2. Compare uptake of these solutions with uptake by the gel formulations 3. Ascertain the effects of the compounds on the surface morphology of dentine.

1. ASSESSMENT OF FLUORIDE UPTAKE BY DENTINE FOLLOWING APPLICATION OF SODIUM FLUORIDE, AMINE FLUORIDE 297 AND STANNOUS FLUORIDE SOLUTIONS.

Introduction

This study was conducted to assess fluoride uptake following application of sodium fluoride. Amine Fluoride 297 and stannous fluoride solutions in the absence of the other components of the gels investigated in Chapter 6 by use of solutions of these compounds.

Method

Ten freshly extracted, unerupted third molars were collected, stored, sectioned and prepared as described previously in Chapter 2. One section from each tooth was randomly allocated to a group that was untreated and placed in distilled, deionised water. Three sections from each tooth were randomly allocated to one of three test groups and exposed to a 1.25% fluoride solution of sodium fluoride, stannous fluoride or Amine Fluoride 297, as described in Table 7, for 1 hour and then washed as before. 186

Dentine samples were Immediately dissected, measured and analysed for fluoride as previously described.

Statistical analysis of the data was carried out as before.

Results

The specimen dimensions, fluoride content and fluoride uptake are illustrated in Table 19 (raw data in Appendix 5, Table 11). Statistical analysis performed is detailed in Appendix 4, Table 10.

40 dentine specimens were obtained and were found to have a mean surface area of 1.17 mm (sd 0.21) and a mean depth of 0.46 mm (sd 0.09). The specimens exhibited no significant difference for surface area (p = 0.44) or depth (p = 0.27).

The untreated samples had a fluoride content with a median of 50 pgcm'^. The fluoride uptake of the samples is illustrated in Figure 19. The median fluoride uptake by samples exposed to a 1.25% fluoride solution of sodium fluoride was 103 pgcm"^, to stannous fluoride was 107 pgcm"^ and to Amine Fluoride 297 was 318 pgcm'^.

There was a highly statistically significant increase in the fluoride uptake after exposure to the Amine Fluoride 297 solution compared to the sodium fluoride solution (p = 0.022) and the stannous fluoride solution (p = 0.022) but no difference between the sodium fluoride and the stannous fluoride solutions. Depth Surface Area Fluoride Content Fluoride Uptake

(mm) (mm^) (pgcm^) (pgcm^)

Fluoride Un NaF SnF2 AmF297 Un NaF SnF2 AmF 297 Un NaF SnF2 AmF NaF SnF2 AmF 297 Regimen treated treated treated 297 Mean 0.47 0.43 0.44 0.51 1.10 1.18 1.13 1.25 51 169 190 367 121 140 309

SD 0.10 0.08 0.07 0.09 0.19 0.18 0.18 0.26 19 99 109 80 105 96 96

Median 0.49 0.43 0.41 0.53 1.09 1.18 1.08 1.22 50 135 131 388 103 107 318

95% Cl .36 to .54 .34 to .53 .39 to .53 .38 to .60 .89 to 1.34 .99 to 1.38 98 to 1.40 1.05 to 1.43 29 to 77 110to243 97 to 365 311 to 435 22 to 186 55 to 269 185 to 404

Table 19. The sample dimensions, fluoride content and fluoride uptake of dentine samples following exposure to fluoride solutions (^.25%F) in vitro.

25 188

Figure 19. Fluoride uptake of dentine following exposure to fluoride solutions in vitro.

Scatter Plot of Fluoride Uptake

500

400 I § 300 c

Io 200 0 (D T3 100 13

-100 10 Tooth

NaF SnF2 AmF 297

Median Fluoride Uptake with 95% Confidence intervals

500 “T—

E 400 Ü s I 3 0 0 -

Î 200 - I I 100-

NaF SnF2 AmF 297 189

Discussion

From the results, assuming that these compounds work synergistically, one would expect that a preparation consisting of a combination of stannous fluoride and amine fluoride would produce a higher uptake of fluoride than a combination of sodium fluoride and amine fluoride. Sodium fluoride alone would be expected to effect the lowest fluoride uptake. However, the previous investigation showed that an amine fluoride-stannous fluoride gel combination (Meridol) show the least uptake when compared to a sodium fluoride gel and a amine fluoride-sodium fluoride (Elmex) gel. It Is possible that stannous fluoride and amine fluoride compounds are more effective when used alone rather than in combination. This may be due to differences in their mode of action or complexing of the amine and stannous ions. Alternatively another component of the gel may be interfering with fluoride uptake. However, when the results for fluoride uptake by sodium fluoride gel (Page 178) are compared with fluoride uptake by the sodium fluoride solution in this investigation, the gel resulted in a significantly higher fluoride uptake. The pH of the sodium fluoride solution was considerably higher (7.9) than that of the gel (5.5 to 6.0) and an acidic pH is known to encourage fluoride uptake (MCihlemann et al, 1966; Strübig and Güizow, 1986). It was, therefore, decided to repeat the experiment with solution and gel combinations of stannous fluoride/amine fluoride and sodium fluoride/amine fluoride to ascertain this for the amine fluoride compounds.

Conclusions

The fluoride content of dentine was increased by application of topical fluoride solutions in vitro. The fluoride compound influenced the uptake. The fluoride uptake of the dentine specimens was ranked as Amine Fluoride 297 > stannous fluoride = sodium fluoride. There was a highly statistically significant difference between fluoride content of dentine after all fluoride treatments compared to untreated dentine specimens. There was a highly statistically significant difference between fluoride uptake by dentine after exposure to the Amine Fluoride 297 solution compared to the sodium fluoride and the stannous fluoride solution but no difference between the sodium fluoride and the stannous fluoride solutions. 190

2. FLUORIDE UPTAKE FROM AMINE FLUORIDE COMPOUNDS IN THE FORM OF SOLUTIONS AND GELS.

Introduction

The variations in results for fluoride uptake by the amine fluoride gels and their component solutions may be due to a component of the gel interfering with fluoride uptake. This appears not to be the case for sodium fluoride but to test this for the amine fluoride compounds, solutions were made with similar proportions of fluoride components as the Elmex and Meridol gels and fluoride uptake by solution and gel was compared.

Method

Ten freshly extracted, unerupted third molars were collected, stored, sectioned and prepared as described previously. From each tooth, one section was allocated randomly to one of four test groups. Sufficient dentine was not available for the additional dissection of an untreated specimen. The sections were exposed to either a 1.25% fluoride solution of Amine Fluoride 297-sodium fluoride (“Elmex Solution”), a 1.25% fluoride solution of Amine Fluoride 297- stannous fluoride (“Meridol Solution”), Elmex Gel (1.25% F ) or Meridol Gel (1.25% F"), for 1 hour and then washed as described previously.

Dentine samples were immediately dissected, measured and analysed for fluoride as previously described.

Statistical analysis of the data was carried out as before.

Results

The specimen dimensions and fluoride content are illustrated in Table 20 (raw data in Appendix 5, Table 14). Statistical analysis performed is detailed in Appendix 4, Table 11. Depth Surface Area Fluoride Content

(mm) (mm^) (pgcm^)

Fluoride Meridol Meridol Elmex Eimex Meridol Meridol Elmex Eimex Meridol Meridol Elmex Elmex Regimen Solution Gel Solution Gel Solution Gel Solution Gel Solution Gel Solution Gel Mean 0.59 0.48 0.57 0.56 1.37 1.15 1.32 1.47 92 46 207 189

SD 0.06 0.07 0.07 0.08 0.31 0.10 0.21 0.28 34 14 71 60

Median 0.60 0.49 0.56 0.61 1.38 1.14 1.33 1.43 88 48 198 194

95% Cl .47 to .66 .35 to .57 .44 to .68 .43 to .64 .92 to 1.85 1.01 to 1.33 1.04 to 1.63 1.13 to 1.95 58 to 129 30 to 60 138 to 301 114to261

Table 20. The sample dimensions and fluoride content of dentine samples following exposure to fluoride solutions or gels (1.25% F") in vitro. 192

40 dentine specimens were obtained and were found to have a mean surface area of 1.32 mm (sd 0.26) and a mean depth of 0.55 mm (sd 0.08). The specimens treated with Elmex Gel had a significantly greater surface area than those treated with Meridol Gel (p = 0.025). The depth of the specimens treated with “Meridol Solution" had a significantly greater depth than those treated with Meridol Gel (p = 0.023).

The fluoride content of the samples is illustrated in Figure 20. The median fluoride content of the samples exposed to “Meridol Solution” was 88 pgcm'^ to

Meridol Gel was 48 pgcm'^, to “Elmex Solution* was 198 pgcm"^ and to Elmex Gel was 194 pgcm'^.

The fluoride content of the dentine specimens treated with “Elmex Solution” were highly significantly greater compared to those treated with Meridol Gel (p = 0.00036) and significantly greater compared to those treated with “Meridol Solution” (p = 0.023). The fluoride content of the dentine specimens treated with Elmex Gel was also highly significantly greater compared to those treated with Meridol Gel (p = 0.00036). Other differences were not significant.

Discussion

The significant difference in surface area between the specimens treated with Elmex and Meridol Gels is taken account of by calculating fluoride content per surface area of the specimen. However, the significant difference in depth between the specimens treated with “Meridol Solution” and Meridol Gel will affect the perceived fluoride content of the specimens. However the median values for depth differed by only 0.1mm. With reference to the fluoride content plotted against depth for untreated specimens in Chapter 3, Section 1, this would account for a difference of approximately 5 pgcm’^ and is therefore unlikely to affect the difference in fluoride content computed for these compounds. 193

Figure 20. Fluoride content of dentine following exposure to amine fluoride compounds in vitro.

Scatter plot of fluoride content

350

300 tr 1 1 • 1 * • 1 I 250 1 3 4 1 1 ■ ■ c 200 1 1 ■ 1 o 1 « o 1 # ■ 1 0) 150 1 ' • 1 2 ■ # o 1 ♦ 1 3 1 ■ ■ 1 + 1 50 j ^ •* ; ”T ♦ • 1 1 « 1 0 1 2 3 4 5 6 7 8 9 10 Tooth

Meridol Solution • Meridol Gel ■ Elmex Solution ♦ Elmex Gel

Median Fluoride Content with 95% Confidence Intervals

350 -t

300 -

' e o 250 - ?

1 200 - c o U 150 - 0) 2 o 100 - 3 LL 50 - t

Meridol Solution Meridol Gel Eimex Solution Elmex Gel 194

The fluoride content of dentine was not significantly different when exposed to either the gel or solution form of the amine fluoride/sodium fluoride combination (Elmex). However, the fluoride content following exposure to the solution form of the amine fluoride-stannous fluoride combination (Meridol) was greater than that following exposure to the gel form and this difference was highly significant. The pH of the gel and solution were similar suggesting that a component of the gel may be reducing the fluoride uptake by dentine. It is possible that overstabilisation of the amine fluoride-stannous fluoride complex is due to reaction with hydroxyl groups from other components of the gel.

Nevertheless, the fluoride content of dentine following exposure to the “Meridol” preparations was lower than when exposed to the “Elmex” preparations and, referring back to the previous sections, when exposed to the sodium fluoride preparations. This suggests that stannous fluoride and amine fluoride compounds are more effective at promoting fluoride uptake when used on their own rather then in combination.

Conclusions

The uptake of fluoride was influenced by the fluoride preparation. The fluoride content of the dentine specimens was ranked as “Elmex solution”> Elmex Gel > “Meridol solution” > Meridol Gel. There was a statistically significant difference between fluoride content of dentine after all fluoride treatments except between “Elmex solution” and Elmex Gel and between “Meridol solution” and Meridol Gel and “Meridol solution” and Elmex Gel. It can be concluded that other constituents of the Meridol Gel may have interfered with fluoride uptake. This preparation performed less well than the solution and consistently performed less well than Elmex preparations and sodium fluoride alone. 195

3. AN SEM STUDY INTO THE EFFECT OF FLUORIDE GELS AND SOLUTIONS ON THE DENTINE SURFACE MORPHOLOGY

Introduction

Fluoride uptake by dentine following a short exposure to a topical fluoride regimen will mainly be occasioned by the formation of calcium fluoride (Saxegaard et a/., 1987). Distinct patterns of calcium fluoride formation have been described on dentine (Saxegaard ef a/., 1987; Ellingsen etal., 1987, Rolla at a/., 1993). The formation of calcium fluoride on the dentine surface following treatment with the fluoride preparations previously assessed for fluoride uptake may give information as to how these compounds achieve their anti-caries effect. This investigation therefore aimed to analyse, qualitatively, the appearance of deposits on the surface of cut dentine treated with the test fluoride regimens.

Method

Eight extracted, unerupted third molars were collected and randomly distributed into two groups. The occlusal enamel was removed and the teeth sectioned longitudinally to give four sections as before. A sample of approximately 2 mm thick was dissected in a horizontal plane from the occlusal surface of each section using the diamond band saw. Each sample was given an identification mark (as described previously) as to tooth of origin and surface to be observed and then grooved on the opposing surface to aid fracture (Figure 21). The samples were exposed to one of the fluoride regimens detailed in Table 21 (sourced as described previously). Each sample was then fractured, by hand, taking care to touch only the edge of the specimens. 196

Table 21. Topical fluoride preparations applied to the dentine prior to SEM examination.

UNTREATED GELS SODUIM FLUORIDE 1.25% Fluoride ELMEX MERIDOL UNTREATED SOLUTIONS SODIUM FLUORIDE 1.25% Fluoride AMINE FLUORIDE 297 STANNOUS FLUORIDE

Figure 21. Diagram of identification mark and groove to aid fracture cut into the dentine specimen prior to fluoride treatment and SEM analysis.

Identification Mark

Groove 197

The samples were placed separately in labelled plastic containers and stored in a desiccator for 24 hours. They were then mounted on a labelled 0.5" aluminium specimen stub (G301, Agar Scientific, U.K.) with a silver dag (Electrodag 1415M High conductivity paint, Acheson, Colloids, U.K.). The glue was allowed to dry for 30 minutes before sputter coating with gold-palladium (Au-Pd) particles using a sputter coater (81504, Edwards, U.K.). Half the specimens were mounted so that the occlusal surface was examined and half so that the fractured surface was examined. The latter was to allow the thickness of any surface deposits to be assessed.

Specimens were then mounted in a scanning electron microscope (SEM) (Stereoscan 90, Cambridge Instruments, U.K.) from which the surface morphology was observed and photographed. Photographs were taken of the flat surface at approximately 4000x magnification and from the fractured surface at approximately lOOOx and 4000x magnification, allowing dimensions of deposits to be estimated where relevant.

Results

The SEM analysis of the dentine surfaces of the untreated and test groups demonstrated that after exposure of the dentine to topical fluoride, the surface of the dentine was usually coated with a layer of deposits (Figures 22 to 41 ).

Figure 22 shows the surface of the untreated specimens with the dentinal tubules in cross-section and the presence of a smear layer. The openings of the dentinal tubules appeared to be partially plugged with debris. The sectioned dentine surface also showed striations (ridges and troughs) formed by the saw during sectioning.

The specimens treated with fluoride gels showed the inconsistent presence of an amorphous deposit. In the specimens treated with Meridol gel (Figures 23) this was not distinguishable from the smear layer present of the untreated dentine. The specimens treated with NaF gel (Figures 24) showed the amorphous deposit to be more uniformly present although the tubule orifices were not entirely obliterated and differences in morphology compared to the 198 untreated dentine could not be consistently demonstrated. In contrast, the specimens treated with Elmex gel were completely covered with the deposit such that the tubules were not visible (Figure 25). Deposit was not distinguishable on the fractured surfaces of dentine treated with Meridol and NaF gels (Figures 26, 27) but was apparent following treatment with and Elmex gel and had an approximate depth of 5 pm (Figures 28,29).

The specimens treated with fluoride solutions showed the presence of deposit of a different morphology. The treated dentine surface was covered by a series of randomly distributed bead like particles. The particles coalesced in areas to form amorphous deposits (Figures 30,31 ) and flakes (Figure 32). The dentinal tubules were partially obliterated by the deposit in all of these specimens. The fractured surfaces show the presence of a layer of deposit for the AmF 297 and

SnF2 solution treated specimens (Figure 33,34,35). The dentinal deposit following treatment with SnF: solution had a depth of approximately 8 pm. 199

Figure 22. SEM of the surface of untreated dentine.

Field Width = 28 |im. Note the presence of a smear layer and that the openings of the dentinal tubules appear to be partially plugged with debris. Note also the striations formed by the saw during sectioning.

Figure 23. SEM of the surface of dentine treated with Meridol gel (1.25% F )

Field Width = 27 ^im. Note the inconsistent presence of an amorphous deposit that is not distinguishable from the smear layer present of the untreated dentine. 200

Figure 24. SEM of the surface of dentine treated with NaF gel (1.25% F ).

Field Width = 27 urn. Note the amorphous deposit is more uniformly present but differences in morphology compared to untreated dentine cannot be consistently demonstrated.

Figure 25. SEM of the surface of dentine treated with Elmex gel (1.25% F )

Field Width = 27 ^m. Note the surface of the dentine is completely covered with the deposit such that the tubules are not visible. 201

mâlâiim m Figure 26. SEM of the fractured surface of dentine treated with Meridol gel (1.25% F-). Field Width = 74 mm. Note the deposit is not distinguishable on the fractured surface of dentine.

Figure 27. SEM of the fractured surface of dentine treated with NaF Gel (1.25% F ).

Field Width = 76 mm. Note the deposit is not distinguishable on the fractured surface of dentine. 202

Figure 28. SEM of the fractured surface of dentine treated with Elmex Gel

(1.25% F ). Field Width 76 ^m.

Note deposit is apparent on the fractured surface of the dentine.

Figure 29. SEM of the fractured surface of dentine treated with Elmex Gel (1.25% F).

Field Width 19 |.im Note the deposit has an approximate depth of 5 fmn. Figure 30. SEM of the surface of dentine treated with AmF 297 solution (1.25% F ).

Both Field Widths = 16 ^m. Note the presence of deposit in the shape of randomly distributed bead like particles (30a). The particles coalesce in areas to form amorphous deposits (30b).

Figure 31. SEM of the surface of dentine treated with SnF2 solution (1.25% F ).

Both Field Widths = 1 6 ^m. Note the presence of deposit in the shape of randomly distributed bead like particles (31a). The particles coalesce in areas to form amorphous deposits (31b).

Figure 32. SEM of the surface of dentine treated with NaF solution (1.25% F).

Both Field Widths = 1 6 pm. Note the presence of deposit in the shape of randomly distributed bead like particles (32a). The particles coalesce in areas to fomi flakes (32b). 2 04

30a 30b

31a 31b

- 4

32a 32b

W## 2 05

Figure 33. SEM of the fractured surface of dentine treated with AmF297 solution (1.25%F-).

Field Width =76|xm Note deposit is apparent on the fractured surface of the dentine.

Figure 34. SEM of the fractured surface of XT dentine treated with SnF 2 solution (1.25% P).

Field Width = 76 ^irn. Note deposit is apparent on the fractured surface of the dentine.

Figure 35. SEM of the fractured surface of dentine treated with SnF 2 solution (1.25% P). Field Width = 20 f.im. Note the dentinal deposit has a depth of approximately 8 p,m. 206

Discussion

The Investigation showed that there was deposition of particulate matter after exposure of dentine to topical treatment with fluoride gels and solutions. However, in dentine treated with NaF and Meridol gels, it was difficult to distinguish between a smear layer and possible deposition of an amorphous layer as a result of the fluoride treatment. Further investigation is needed by application of the gels to dentine specimens following smear layer removal.

The deposits after treatment with the fluoride solutions were usually spherical particles, which resembled those previously observed on dentine (Saxegaard et al., 1987; Ellingsen et al., 1987, Relia et al., 1993). Application of Elmex gel and appeared to result in deposition of an amorphous layer similar to that reported on enamel (Barbakow et al., 1984; Cruz et al., 1992) and assumed to be due to aggregates of the spherical particles. The deposits are presumed to consist of calcium fluoride (Saxegaard et al., 1987) excepting after treatment with stannous fluoride when the particles could be calcium fluoride, stannous fluoride, stannous phosphate or stannous fluorophosphate (Ellingsen et al., 1987; Miller et al, 1994). Alternatively other constituents from the gels might be deposited on the surface. Abrasives from toothpaste slurries have been reported to form crystalline deposits (calcium carbonate) and granular layers (dicalcium phosphate, alumina and silica) on the dentinal surface (Addy and Mostafa, 1989). These compounds were not present in the gels tested but the effect of the gel constituents alone on the dentinal morphology is an area for further investigation.

The finding of a surface deposition following fracture of the treated dentine specimens was variable and may have been due to loss on fracture. Protection of this layer by coating with resin prior to fracture might be considered In future investigations.

The variable production of a deposit on the surface of dentine following treatment with Meridol gel is consistent with the results on fluoride uptake by dentine, which found Meridol gel to result in a fluoride content that was not statistically different to the untreated specimens. It is, therefore, again 207 reasonable to assume that stannous fluoride and AmF 297 compounds are more effective when used on their own rather than in combination.

Conclusions

Exposure of dentine to topical treatment with fluoride gels and solutions resulted in variable deposition of particulate matter. 208

CHAPTER 8.

INVESTIGATIONS INTO UPTAKE OF FLUORIDE BY DEMINERALISED DENTINE 209

Introduction

The results of the previous investigations have led to some contradictory findings. The application of amino-stannous fluoride gels and solutions to dentine in vitro have resulted in lower fluoride uptake compared to the individual preparations, whereas Meridol paste and rinse used in vitro and in vivo have been at least as effective as sodium fluoride. The dentine in these investigations however had been harvested from erupted teeth and it was postulated that, if demineralisation of the dentine had occurred, the exposed organic matrix of dentine may be attractive to components of the Meridol regimen, particularly the surfactant activity of amine fluorides.

1. Uptake of Fluoride by Demineralised Dentine Following Application of Fluoride Gels.

Introduction

The method previously devised to measure the fluoride content of organic dentine (Page 138) was adapted for this investigation to enable analysis of fluoride adsorbed by the collagen matrix. The study aimed to investigate the effect of topical fluoride gel application on fluoride uptake by the organic dentine matrix in vitro and to assess whether release of the fluoride from the collagen matrix occurred on exposure to acid.

Method

Sixty samples of dentine, 1mm x 1mm x 0.5mm, were prepared from extracted, unerupted third molars and the biopsy dimensions were measured by the established method. However, in this investigation, the whole sample was exposed to the fluoride regimen and the calculations were adjusted accordingly. The samples were demineralised by exposure to 2ml 1M hydrochloric acid for 24 hours to expose the collagen matrix. The samples were then randomly distributed into six groups (Table 22) and exposed to the relevant gel for 1 hour, washed and blot dried as above. 210

Table 22. Groups established for the investigation into fluoride uptake by demineralised dentine following exposure to fluoride gels.

Group 1 and IV Sodium Fluoride Gel Group II and V Meridol Gel Group III and VI Elmex Gel

Fluoride Analysis by Dissolution with Potassium Hydroxide.

Samples in groups I, II and III were exposed to 10 ml 1M potassium hydroxide for 7 days and the resultant solution analysed for fluoride by the method described on Page 138.

Assessment of Fluoride Release by Hydrochloric Acid.

Samples in groups IV, V and VI were exposed to 5ml 1M hydrochloric acid, removed, washed in three 5 minute vibrating washes in 50ml distilled, deionised water, then exposed to 10 ml 1M potassium hydroxide for 7 days. The hydrochloric acid and potassium hydroxide were then analysed for fluoride.

Results

The specimen dimensions and fluoride released by potassium hydroxide Is illustrated in Table 23 (raw data in Appendix 5, Table 12a). The specimen dimensions and fluoride released by hydrochloric acid and then potassium hydroxide is illustrated in Table 24 (raw data in Appendix 5, Table 12b). Statistical analysis performed is detailed in Appendix 4, Table 12.

The surface area of the dentine samples had a mean of 6.4 mm^ (sd 2.3) and did not differ significantly between the six groups (p = 0.97).The fluoride uptake by dentine released by potassium hydroxide (Groups I, II and III) is illustrated in Figure 36. The median fluoride uptake following exposure to Meridol gel was 21 211

ligcrn"^, to sodium fluoride gel was 6 ^gcm"^ and to Elmex gel was 14 ^igcm’^. The uptake was highly significantly greater for specimens treated with Meridol Gel compared with those treated with sodium fluoride gel (p = 0.0002), other differences were not significant.

The fluoride uptake by dentine released by hydrochloric acid (Groups IV, V and VI) is illustrated in Figure 37. The median fluoride uptake following exposure to Meridol gel was 15 pgcm’^ to sodium fluoride gel was 3 pgcm'^ and to Elmex

gel was 4 pgcm'^. The uptake was highly significantly greater for specimens treated with Meridol gel compared with those treated with sodium fluoride gel (p = 0.0002) and Elmex gel (p = 0.001), the difference between Elmex gel and sodium fluoride gel was not significant.

The median fluoride content remaining in the dentine after immersion in 2ml 1M hydrochloric acid for 24 hours was O.lpgcm'^ for the Meridol, sodium fluoride and Elmex samples. The difference between the fluoride contents was not significant (p = 0.62). 212

Table 23. Fluoride uptake by demineralised dentine following application of fluoride gels (1.25% F )in vitro. Fluoride released by exposure to potassium hydroxide. Surface Area (mm^) Fluoride Content (pgcm'^) MERIDOL NaF ELMEX MERIDOL NaF ELMEX Mean 6.3 6.6 6.5 24 7 14 SD 2.5 2.9 2.0 13 3 7 Median 4.7 5.8 6.2 21 6 14 95% Cl 4.3 to 9.2 3.7 to 10.1 4.6 to 8.6 14 to 43 4 to 10 7 to 18

Figure 36. Fluoride content of the demineralised dentine following application of fluoride gels (1.25% F )in vitro. Fluoride released by potassium hydroxide.

Scatter plot of fluoride content

60 i 50 1 ,, Î- I 30 ...... o « • # # • ® 20 • • • ♦ •c • • * • J 10 * • i ♦ « u_ • « • ------1— T ■ ------1------1------1 1------1------J 5 6 10 Tooth

♦ Meridol ♦ Sodium Fluoride • Elmex

Median Fluoride Content with 95% Confidence Intervals

Ü 20

c 10

Meridol Sodium Fluoride Elmex 213

Table 24. Fluoride uptake by demineralised dentine following application of fluoride gels (1.25% F) in vitro. Fluoride released by exposure to hydrochloric acid, remaining fluoride released by potassium hydroxide.

Surface Area (mm'^) Fluoride Content (pgcm'^) Fluoride Content (pgcm'^) Released by HCI Released by KOH Meridol NaF Elmex Meridol NaF Elmex Meridol NaF Elmex Gel Gel Gel Gel Gel Gel Gel Gel Gel Mean 7.0 6.0 6.1 15 4 5 0.4 0.2 0.2 SD 2.8 1.7 2.3 4 2 3 0.4 0.2 0.3 Median 6.6 6.0 6.7 15 3 4 0.1 0.1 0.1 95% Cl 4.4 to 4.1 to 4.1 to 12 to 2 to 3 to 0.06 to 0.06 to 0.01 to 10.1 7.9 9.6 19 7 9 1.00 0.20 0.20

Figure 37. Fluoride content of the demineralised dentine following application of fluoride gels (1.25% F )in vitro. Fluoride released by hydrochloric acid.

Scatter plot of fluoride content

I 50 s c 40 0) E 0 30 u •S 20 •c 1 10 LL 0 5 6 10 Tooth

• Meridol Sodium Fluoride ♦ Elmex

Median Fluoride Content with 95% Confidence Intervals

10 —

Meridol Sodium Fluoride Elmex 214

Discussion

The fluoride content of the demineralised dentine specimens was ranked as Meridol gel > Elmex gel > sodium fluoride gel for both fluoride released by 1 molar potassium hydrochloride and by 1 molar hydrochloric acid. This is very different to the fluoride content found in dentine that had not been demineralised following the same regimens when fluoride content was ranked as Elmex gel = sodium fluoride gel > Meridol gel (Chapter 6). This suggests that components of the Meridol gel have an affinity for the organic component of dentine exposed by demineralisation with hydrochloric acid. This required investigating further with the individual fluoride compounds found in Elmex and Meridol gels.

The two methods of releasing the fluoride content found in the demineralised dentine in this investigation, that is by 1 molar potassium hydrochloride and 1 molar hydrochloric acid, resulted in identical rankings of the three fluoride gels although the significance varied. Furthermore, following immersion in hydrochloric acid, the amount of fluoride released from the dentine samples by immersion in KOH was very small. This suggests that immersion in 1 molar hydrochloric acid alone can be used to release fluoride that is adsorbed to demineralised dentine.

Conclusions

The fluoride uptake by demineralised dentine was influenced by application of topical fluoride gels in vitro. The fluoride content of the demineralised dentine specimens was ranked as Meridol gel > Elmex gel > sodium fluoride gel. There was a statistically significant difference between fluoride content of dentine after all fluoride treatments except the Elmex and sodium fluoride groups in both hydrochloric acid and potassium hydroxide and between the Elmex and Meridol groups in potassium hydroxide. Thus confirming the hypothesis that the nature of the fluoride preparation may influence the uptake of fluoride by demineralised dentine. The majority of the fluoride content of the demineralised dentine was released by 1 molar hydrochloric acid. 215

2. Uptake of Fluoride by Demineralised Dentine Following Application of Fiuoride Soiutions.

Introduction

This investigation aimed to assess the effect of topical fluoride solutions on fluoride uptake by the organic dentine matrix in vitro.

Method

Thirty samples of dentine, 1 mm x 1 mm x 0.5 mm, were prepared from extracted, unerupted third molars and the biopsy dimensions were measured by the established method. The samples were demineralised by exposure to 2ml 1M hydrochloric acid for 24 hours. The samples were then randomly distributed into three groups and exposed to 1.25% fluoride solutions of either sodium fluoride, stannous fluoride or amine fluoride 297 as described previously.

Samples were then exposed to 5ml 1M hydrochloric acid for 24 hours and the hydrochloric acid was analysed for fluoride by the established method.

Results

The specimen dimensions and fluoride content is illustrated in Table 25 (raw data in Appendix 5, Table 13). Statistical analysis performed is detailed in Appendix 4, Table 13.

The surface area of the dentine samples had a mean of 4.1mm^ (sd 0.9). The surface area of the specimens did not differ significantly between the groups except for the sodium fluoride and stannous fluoride groups (p = 0.02).

The fluoride uptake by dentine released by hydrochloric acid is illustrated in Figure 38. The median fluoride uptake following exposure to Amine Fluoride 297 solution was 8 pgcm'^, to sodium fluoride solution was 1 pgcm'^ and to 216 stannous fluoride solution was 33 ^igcm2 . A highly significant difference was exhibited between all three groups. 217

Table 25. Fluoride uptake by demineralised dentine following application of fluoride solutions in vitro. Surface Area (mm^) Fluoride Content (pgcm'^) Fluoride AmF 297 NaF SnFz AmF 297 NaF Snpz Regimen Solution Solution Solution Solution Solution Solution Mean 4.4 4.5 4.0 8 1.5 31 SD 0.5 0.5 0.3 2 0.5 7 Median 4.5 4.6 3.8 8 1 33 95% Cl 3.8 to 5.1 3.8 to 5.1 3.7 to 4.3 6 to 10 1 to 2 21 to 37

Figure 38. Fluoride content of the demineralised dentine following application of fluoride solutions in vitro.

Scatter plot of fluoride content

I 50 ? ^ 40 I i 30 4 Ü 0 20

1 10 4 5 6 10 Tooth

» Amine Fluoride 297 • Sodium Fluoride ■ Stannous Fluoride

Median Fluoride Content with 95% Confidence Intervals

5 30 - -

Ü 2 0 - -

Amine Fluoride 297 Sodium Fluoride Stannous Fluoride 218

Discussion

The significant difference in surface area between two of the groups of specimens will not influence results as the fluoride content is expressed as a function of the surface area. The fluoride content of the demineralised dentine specimens was ranked as stannous fluoride > Amine Fluoride 297 > sodium fluoride in that order. This is different to the fluoride uptake by dentine that has not been demineralised following application of the same regimens where fluoride content was ranked as amine fluoride 297 > stannous fluoride > sodium fluoride (Section 2). Both Amine Fluoride 297 and stannous fluoride appear to have an affinity for the organic component of dentine. This would explain the affinity of Meridol gel for the organic component of dentine seen in the previous section.

This portion of the tooth may facilitate the surfactant activity of the amine component of the amine fluoride. Stannous ions also have an affinity for oxygen and phosphate groups, both of which are found on collagen and so might encourage transport of fluoride ions to the tooth surface.

Conclusions

The fluoride uptake by demineralised dentine was influenced by application of topical fluoride solutions in vitro. The fluoride content of the demineralised dentine specimens was ranked as stannous fluoride > Amine Fluoride 297 > sodium fluoride. There was a highly statistically significant difference between fluoride content of dentine after all fluoride treatments.

The affinity of demineralised dentine for amine fluoride and particularly stannous fluoride might therefore help to explain the conundrum of application of Meridol toothpaste and rinse regimens resulting in fluoride uptake by erupted dentine (exposed to demineralisation-remineralisation cycles) unlike application of gels and solutions of a higher fluoride concentration to unerupted dentine. 219

CHAPTER 9.

A SUMMARY OF THE FINDINGS OF THESE INVESTIGATIONS. 220

This Investigation developed a method for analysis of fluoride uptake by dentine in vitro and in vivo. Biopsy of dentine was carried out using a fine diamond bur in an air turbine under water cooling with a guide to specimen dimensions in the form of a stainless steel rectangular wire template. The technique enabled biopsies with a surface area of 1 mm^ and a depth of 0.5 mm to be consistently obtained. Moreover, in vitro, 4 adjacent sections of dentine from each tooth could be treated and biopsied allowing each tooth to act as its own control. Immediate dissolution of the biopsies in 5 ml 1 M hydrochloric acid for 24 hours allowed the total fluoride content of the specimens to be analysed with a differential electrode cell consisting of a single fluoride selective electrode and a combination pH electrode. The variations in fluoride content of untreated specimens of dentine suggested that, when comparative studies of topical fluoride regimens are conducted, samples should be biopsied from dentine with standardised characteristics. Furthermore the fluoride content of an untreated specimen, preferably from an adjacent area of dentine, should be determined. The fluoride content of treated dentine should then be adjusted to take account of this, thereby recording fluoride uptake by the dentine, rather than fluoride content, following application of each regimen.

Toothpaste and mouthrinse regimens consisting of either a combination of Amine Fluoride 297/stannous fluoride (Meridol) or sodium fluoride were investigated for their affects on fluoride uptake by dentine in vitro and in vivo. The sodium fluoride regimen was selected as an accepted standard against which to compare the Meridol regimen. The in vitro investigation was conducted on contiguous areas of occlusal dentine biopsied from extracted, unerupted third molars. The in vivo investigation involved the exposed dentine of teeth with severe tooth wear. The patients selected had removable partial dentures which covered the worn dentine. Patients used à toothpaste and daily fluoride mouth rinse regimen unsupervised for 1 month. In both investigations the regimens resulted in a statistically significant increase in fluoride content of the dentine and there was no significant difference between the Meridol regimens and the accepted standard - sodium fluoride regimen.

The caries incidence was recorded in a similar group of patients using the regimens for 1 year. Patients using the Meridol regimen developed a greater 221

number of new “colour changes” of the dentine and of cavitated carious lesions compared to the group using the sodium fluoride regimen but, the differences were not statistically significant and the number of patients participating in the trial were small. However, the patients developed caries rapidly and with a high incidence and therefore formed a useful group in which to study the prevention of dentinal caries.

Topical fluoride gels may be used by the overdenture wearer to supplement toothpaste and mouthrinse regimens. An in vitro study was therefore performed on extracted unerupted third molars to ascertain fluoride uptake by dentine following application of gels (1.25% F") containing amine, stannous and sodium fluoride alone and in combination. A highly statistically significant increase in fluoride uptake following application of sodium fluoride or an amino-sodium fluoride (Elmex) combination was found compared to application of amino- stannous (Meridol) gel. Indeed fluoride content of the dentine specimens following application of Meridol gel was not statistically significantly different from that of untreated specimens.

In order to investigate this surprising finding, the fluoride uptake by dentine following application of solutions of the individual fluoride compounds (1.25% F ) and in combinations to match the gels was then analysed (Figure 39). A highly statistically significant increase in fluoride uptake occurred following exposure to Amine Fluoride 297 compared to sodium fluoride or stannous fluoride solutions, the latter two were not significantly different. The amino-sodium fluoride gel and solution were found to result in similar fluoride uptake and both had a highly statistically significantly greater fluoride uptake than the amine-stannous fluoride gel and solution. The amino-stannous fluoride solution however, resulted in a highly statistically significant greater fluoride uptake than the Meridol gel. It was concluded that a combination of AmF 297 and stannous fluoride, rather than acting synergistically, resulted in stabilisation of the combination such that there was a reduction in biovailability of fluoride ions. It was postulated that components of Meridol gel caused further stabilisation. This was probably caused by an abundance of hydroxyl ions from the glycerine and propyleneglycol components of the gel. 222

Scanning electron micrographs of the dentine surface following application of sodium fluoride, Meridol and Elmex gels and solutions of the individual component fluoride compounds revealed the dentine surface to be variably coated with a layer of deposits following all fluoride treatments. However, following application of Meridol gel, the deposit was not distinguishable from the smear layer deposit found on untreated dentine. This tends to confirm the results above.

In the light of these results it was puzzling that the Meridol toothpaste and mouthrinse regimens had performed similarly to the sodium fluoride regimens, both in vitro and in vivo. The dentine in these investigations however was harvested from erupted teeth and it was postulated that, if demineralisation of the dentine had occurred, the exposed organic matrix of dentine may be attractive to components of the Meridol regimen. The fluoride uptake by demineralised dentine was therefore investigated following application of the fluoride gels and solutions. Meridol gel resulted in a highly statistically significant increase in fluoride uptake compared to Elmex or sodium fluoride gel and no difference was observed between the latter two. A highly statistically significant increase in fluoride uptake by the stannous fluoride solution was found compared to the Amine Fluoride 297 whereas the sodium fluoride solution resulted in negligible fluoride uptake. The affinity of demineralised dentine for amine fluoride and particularly stannous fluoride might therefore help to explain the conundrum outlined above.

In summary, fluoride uptake by dentine on application of combinations of amine fluoride and stannous fluoride were less effective than the individual compounds alone. Furthermore, a gel preparation was less effective than a solution of similar fluoride content. Over-stabilisation of the compounds appears to be occurring such that there is a reduction in bio-availability of the fluoride ions. But, a toothpaste and mouthrinse regimen performed at least as well as a sodium fluoride regimen in vivo, perhaps partially due to the affinity of stannous and amine compounds to demineralised dentine.

Unexpectedly, the regimens did not perform significantly differently in prevention of dentinal caries or in reduction of plaque and salivary bacterial 223 counts (Appendix 7). Again, the components of the Meridol regimen may not be acting synergistically as expected but be over-stabilised with reduction therefore in their anti-caries activity. Further investigation is required, therefore, into methods of combining these compounds such that a high bio-availability of the active component remains. 224

Areas for Further Investigation.

Areas of future study therefore need to be considered. These include:

1. Extending the clinical caries trial to include more patients and having a longer period of review

2. Further investigation into the effects of amine, stannous and amino-stannous regimens on the surface morphology of dentine by: • using dentine samples with the smear layer removed • protecting surface deposits by use of resins such as Araldite before fracture of the dentine sample • using solutions with identical pH • using gels with identical components except for the fluoride compound . examination of the effect of the other components of the gel by investigation of non-fluoride containing gels

3. Investigation into the possibility of an over-stabilising effect of combining amine and stannous fluorides and gel components by investigating fluoride uptake and surface deposition (by SEM) following: . application of various proportions of the two compounds, for example in proportions of 0:100, 25:75, 50:50, 75:25 and 100:0, applied as solutions with identical pH . application of gels with identical fluoride compounds but both with and without the other gel components that contain hydroxyl- groups. 225

Am F 297 / Sodium Fluoride Solution (1 2596F) (1) o. (/) w •o S AmF 297 / TO O) Stannous Fluoride I •â Solution (1.25%F) Ü I ( 1) 0 (/) 0 Sodium Fluoride Solution ■D a 0 3 0 Stannous Fluoride > 0 Solution (1.25%F) 0 C 0 E O) 2 AmF 297 Solution (1 2596F) 8 t 0 Sodium Fluoride Gel(1.25%F) Ç I AmF 297&335 / 0 c Sodium Fluoride W-) c (Elmex) Gel 0 (1 2596F) •D È AmF 297 / S Stannous Fluoride 0 (Meridol) Gel Q. 3 (1 2596F) 0 ■D "C 8 Sodium Fluoride y— Toothpaste 0 (0.14%F) and x: Rinse (0 0596) o

0 Meridol Toothpaste E (0.14%F)and E 3 Rinse (0.05%F) co o o O O O) IT ) O S s g O in 8 8 g co co co CM CM ■«- (2-LU06ri) 9)|B%dn apuonid 2 3 CD 226

BIBLIOGRAPHY

Aamdal-Scheie, A. (1992) Dentifrices in the control of dental caries. In: Embery, G. and R^lla, G. (Eds.) Clinical and Biological Aspects of Dentrifces, pp. 29-40. Oxford: Oxford University Press. Aasenden, R. and Moreno, E.G. (1971) Evaluation of biopsy data in human enamel fluoride studies. Arch. Oral Biol. 16,1413-1426. Aasenden, R., de Paulo, P.P. and Brudevold, F. (1972a) Effects of daily rinsing and ingestion of fluoride solutions upon dental caries and enamel fluoride. Arch. Oral Biol. 17,1705-1714. Aasenden, R., Moreno, E.G. and Brudevold, F. (1972b) Gontrol of sampling areas for tooth enamel biopsies. Arch. Oral Biol. 17, 355-358. Adams, D., and Salem, A M. (1983) The use of surfactants to prevent bacterial adhesion to enamel. In: ten Gate, J.M., Leach, S.A. and Arends, J. (Eds.) Bacterial Adhesion and Preventive Dentistry, pp 179-191. Oxford: IRL Press. Addy, M. and Mostafa, P. (1988) Dentine hypersensitivity. I. Effects produced by the uptake in vitro of metal ions, fluoride and formaldehyde onto dentine. J. Oral Rehabil. 15, 575-585. Addy, M. and Mostafa, P. (1989) Dentine hypersensitivity. II. Effects produced by uptake in vitro of tootpastes onto dentine. J. Oral Rehabil. 16, 35-48. Addy, M., Moran, J., Wade, W. and Jenkins, S. (1992) The evaluation of toothpaste products in promoting gingival health. In: Embery, G. and R^lla, G. (Eds.) Clinical and Biological Aspects of Dentrifces, pp. 249-262. Oxford: Oxford United Press. Addy, M., Greenman, J., Renton Harper, P.. Newcombe, R. and Doherty, F. (1997) Studies on stannous fluoride toothpaste and gel (2). Effects on salivary bacterial counts and plaque regrowth in vivo. J. Clin. Periodontol. 24, 86-91. Ahrens, G. (1983) Effizienz der anwendungsformen von fluoriden: Losungen und gelees. Dtsch. ZahnSrztl. Z. 38 (Supp11), S65-S69. Ainamo, J. (1982) Principal requirements for controlled clinical trials of caries preventive agents and procedures. Technical Report No. 1, Third Edition, Gommission on Oral Health, Research and Epidemiology. Int. Dent. J. 32,292-310. Anderson, R.J., Bradnock, G., Beal, J.F. and James, P.M.G. (1982) The reduction of dental caries prevalence in English schoolchildren. J. Dent. Res. 61,1311-1316. Andres, G.J., Shaeffer, J.G. and Windeler, A.S., Jr. (1974) Gomparison of antibacterial properties of stannous fluoride and sodium fluoride mouthwashes. J. Dent. Res. 53, 457- 460. Antila, M.K. and Pohto, P. (1973) Fluoride uptake and retention in dental enamel treated with amine fluoride solution. Proc. Finn. Dent. See. 69, 202-207. Arends, J. and Schuthof, J. (1975) Fluoride content in human enamel after fluoride application and washing - an in vitro study. Canes Res. 9, 363-372. Arends, J., Gelhard, T., Schuthof, J. and Jongebloed, W. (1983a) Enamel remineralization using an amine-fluoride containing tooth paste-a pilot study in vivo. Dtsch. Zahnârztl. Z. 38 (Supp11), 827-830. 227

Arends, J., Nelson, D.G.A., Dijkman, A.G. and Jonegebold, W .L (1983b) Effect of various fluorides on enamel structure and chemistry. In: Guggenheim, B. (Ed.) Cariology Today. International Congress. Zurich 1983, pp. 245-258. Basel: Karger Arends, J. and Christoffersen, J. (1990) Nature and role of loosely bound fluoride in dental caries. J. Dent. Res. 69 (Spec No), 601-605. Arnold, F.A.J., Likins, R.C., Russell, A.L. and Scott, D.B. (1962) Fifteeth year of the Grand Rapids fluoridation study.J. Am. Dent. Assoc. 65, 781-785. Ashiey, F.P., Mainwaring, P.J., Emslie, R.D. and Naylor, M.N. (1977) Clinical testing of a mouthrinse and a dentifrice containing fluoride. A two-year supervised study in school children. Br. Dent. J. 143, 333-338. Attin, T., Hilgers, R.D., Zimmermann, C., Kielbassa, A.M. and Hellwig, E. (1995) Plaque surface area after rinsing with a low-level fluoride-containing Darjeeling tea. Eur. J. Oral Sd. 103, 416-418. Attin, T. and Hellwig, E. (1996) Salivary fluoride content after toothbrushing with a sodium fluoride and an amine fluoride dentifrice followed by different mouthrinsing procedures. J. Clin. Dent. 7, 6-8. Babcock, F.D., King, J.G. and Jordan, T.H. (1978) The reaction of stannous fluoride and hydroxyapatite. J. Dent. Res. 57, 933-938. Bacca, L.A., Leusch, M., Lanzalaco, AC., Macksood, D., Bouwsma, O.J., Shaffer, J.B., Howard Nordan, K.S., Knippenberg, S.H., Kreutzjans, M.K., Miller, J.M., Poore, C.L., Sunberg, R.J., Vastola, K.A., Becus, M., Bartizek, R.D., Block, R.P., Briner, W.W. and White, D.J. (1997) A comparison of intraorai antimicrobial effects of stabilized stannous fluoride dentifrice, baking soda/peroxide dentifrice, conventional NaF dentifrice and essentiai oil mouthrinse. J. Clin. Dent. 8, 54-61. Badr, S.E. and Unger, J.W. (1986) Reconstruction of a severely abraded dentition using an overdenture. Quintessence, int. 17, 293-297. Baijot-Stroobants, J. and Vreven, J. (1980) In vivo uptake of topicaiiy applied fluoride by human dental enamel. Arch. Oral Biol. 25, 617-621. Balmelli, O.P., Regolati, B. and Mühlemann, H.R. (1974) Inhibition of streptococcai deposits on rat moiars by amine fluoride. Heiv. Odontol. Acta 18 (SuppI 8), 45-53. Bânôczy, J. and Nemes, J. (1991) Effect of amine fluoride (AmF)/stannous fluoride (SnF2) toothpaste and mouthwashes on dental plaque accumulation, gingivitis and root-surface caries. Proc. Finn. Dent. Soc. 87, 555-559. Bansai, G.S., Newman, H.N. and Wilson, M. (1990) The survival of subgingival plaque bacteria in an amine fluoride-containing gel. J. din. Periodontol. 17,414-418. Banting, D.W. (1993) Aitematives to conventional clinical caries trials. In: Bowen, W.H. and Tabak, L A. (Eds) Cariology for the Nineties, pp. 273-283. New York: University of Rochester Press Barbakow, F., Comec, S., Rozencweig, D. and Vadot, J. (1983) Enamel fluoride content after using amine fluoride- or monofluorophosphate-sodium fluoride-dentifrices. ASDC J. Dent. Child. SO, 186-191. 228

Barbakow, F., Scherle, W. and Mühlemann, H.R. (1984a) SEM observations of the effects of KOH- and water-washing on amine- and sodium fiuoride-lnduced precipitates on ground human enamel. J. Dent Assoc. S. Afr. 39, 593-600. Barbakow, P., Sener, B. and imfeid, T. (1984b) in vitro fluoride uptake by human enamel after brushing with an amine fluoride gel and subsequent loss of fluoride by water-washing. Schweiz. Monatsschr. Zahnmed. 94,1275-1283. Barbakow, P., Lutz, P. and Sener, B. (1985) In vitro dissolution of human enamel after application of a mixture of stannous fluoride and amine fluoride 297: a pilot study. ASDC J. Dent. CNId. 52, 444-448. Barbakow, P., Lutz, P., Sener. B. and Ransberger, M. (1986a) Dissolution rates of enamel immersed in precipitate-free, aged solutions of SnP: and amine fluoride 297. Schweiz. Monatsschr. Zahnmed. 96,1494-1503. Barbakow, P., Lutz, P., Scherle, W. and Ransberger, M. (1986b) Solubility changes of human

enamel treated with precipitate-free, aged Snp 2/amine fluoride 297 solutions. ASDC J. Dent. ChM 53, 415-419 Barbakow, P., Sener, B. and Lutz, P. (1987) Dissolution of phosphorus from human enamel

pretreated in vitro using Snp 2 stabilized with amine fluoride 297. din. Prev. Dent. 9, 3-6. Barbakow, P., Imfeid, T. and Sener, B. (1989) No effect of waxed dental floss on enamel fluoride uptake or on enamel dissolution changes. Schweiz. Monatsschr. Zahnmed. 99, 40- 43. Barkvoll, P., Rclla, G. and Bellagamba, S. (1988) Interaction between chlorhexidine digluconate and sodium monofluorophosphate in vitro. Scand J. Dent. Res. 96, 30-33. Barsby, M.J. (1988) Partial dentures in the management of severe tooth wear. Dent. Update 15,143-148. Bay, I. and Rglla, G. (1980) Plaque inhibition and improved gingival condition by use of a stannous fluoride toothpaste. Scand. J. Dent. Res. 88, 313-315. Beck, J.D., Kohout, P.J., Hunt, R.J. and Heckert, D.A. (1987) Root caries: physical, medical and psychosocial correlates in an elderly population. Gerodontics 3, 242-247. Beighton, D., Lynch, E. and Heath, M.R. (1993) A microbiological study of primary root-caries lesions with different treatment needs. J. Dent. Res. 72, 623-629. Beiswanger, B.B., Gish, C.W. and Maliatt, M.E. (1981) A three-year study of the effect of a sodium fluoride-silica abrasive dentifrice. Pharmacoi. Ther. Dent. 6, 9-16. Beiswanger, B.B., Doyle, P.M., Jackson, R.D., Maliatt, M.E., Mau Ms, Bollmer, B.W., Crisanti, M.M., Guay, C.B., Lanzalaco, A.C., Lukacovic, M.P. (1995) The clinical effect of dentifrices containing stabilized stannous fluoride on plaque formation and gingivitis - a six-month study with ad libitum brushing.J. din . Dent. 6 (Spec No), 46-53. Beiswanger, B.B., McClanahan, S.P., Bartizek. R.D., Lanzalaco, A.C., Bacca, L A. and White, D.J. (1997) The comparative efficacy of stabilized stannous fluoride dentifrice, peroxide/baking soda dentifrice and essential oil mouthrinse for the prevention of gingivitis. J. d in . Dent. 8, 46-53. 229

Belser, U., Sporri, S. and Mühlemann, H.R. (1975) Uptake and retention of fluoride by Intact and etched enamel. Helv. Odontol. Acta 19, 69-71. Bergman, G. and Linden, L.A. (1969) The action of the explorer on incipient caries. Sven. Tandlak. Tidskr 62,629-634. Berkovitz, B.K.B. and Heap, P.P. (1976) The effect of fluoride on iron, calcium and phosphorous distribution in the rat incisor.Caries Res. 10, 337-351. Bemdt, A.F. (1972) Reaction of stannous fluoride with hydroxyapatite: the crystal structure of SnsPO^Fs.J. Dent. Res. 51, 53-57. Bibby, B.C. (1945) Test of the effect of fluoride containing dentifrices on dental caries. J. Dent. Res. 24, 297-303. Bibby, B.C., Zander, H.G., McKelleget, M. and Labunsky, B. (1946) Preliminary reports on the effect on dental caries of the use of sodium fluoride in a prophylactic cleaning mixture and in a mouthrinse. J. Dent. Res. 25, 207-211. Billings, R.J., Meyerowitz, C., Featherstone, J.D., Espeland, M.A., Fu, J., Cooper, L.F. and Proskin, H.M. (1988) Retention of topical fluoride in the mouths of xérostomie subjects. Caries Res. 22, 306-310. Binney, A., Addy, M., McKeown, S. and Everatt, L. (1996) The choice of controls in toothpaste studies. The effect of a number of commercially available toothpastes compared to water on 4-day plaque regrowth. J. Clin. Periodontol. 23,456-459. Binney, A., Addy, M., Owens, J. and Faulkner, J. (1997) A comparison of triclosan and stannous fluoride toothpastes for inhibition of plaque regrowth. A crossover study designed to assess carry over. J.Clin. Periodontol. 24,166-170. Bishop, K., Briggs, P. and Keileher, M. (1994) The aetiology and management of localized anterior tooth wear in the young adult. Dent. Update 21,153-160. Bley, A. and Güizow, H.J. (1991) Einfluss eines amin-zinnfluoridhaltigen gels auf den stoffwechsel der mundhohlenmikroflora. Dtsch. Stomatol. 41,455-456. Blinkhom, A.S., Holloway, P.J. and Davies, T.G.H. (1983) Combined effects of a fluoride dentifrice and mouthrinse on the incidence of dental caries. Community Dent. Oral Epidemiol. 11,7-11. Bonesvoll, P. and Relia, G. (1978) Retention of tin after mouthrinses. Caries Res. 12, 112(Abstract) Bookstein, F., de Paulo, P.P. and Warram, J. (1976) Regression based techniques for estimating fluoride in surface enamel in large study groups. J. Dent. Res. 55, 97-106. Borggreven, J.M.P.M., Hoppenbrouwers, P., van der Hoeven, J. and Driessens, F. (1980) Influence of hydrophobic amines on diffusion and bacterial adsorption. J. Dent. Res. 59, 919(Abstract) Borggreven, J.M.P.M. and Driessens, F.C.M. (1986) Effect of various aliphatic amines on the permabiiity of bovine dental enamel. Arch. Oral. Biol. 31, 341-344. Borggreven, J.M., Lammers, P.C., Hoeks, T., Zwanenburg, B. and Driessens, F.C. (1991) In vitro remineralization of caries lesions treated with surface-active phosphates. Caries Res. 25, 34-38. 230

Bosma, M.L, Belso, R.W. and Wally, D. (1997) Clinical efficacy of an optimized stannous fluoride dentifrice. Part 5; 3 month study of extrinsic tooth staining. Comp. Con. Ed. 18, 24- 27. Bouma, J., Schaub, R.M.H. and van der Poel, A.C.M. (1985) Periodontal status and total tooth extraction in a medium sized city in Netherlands. Community Dent. Oral Epidemiol. 13, 323- 327. Bouwsma, O.J. (1996) The status, future, and problems of oral antiseptics. Curr. Opin. Periodontol. 3, 78-84. Box, H.K. (1942) The formation of subgingival calculus. J. Canad. Dent. Assoc. 8,415-426. Boyd, R.L. (1993) Comparison of three self-applied topical fluoride preparations for control of décalcification. Angie Orthod. 63,25-30. Boyd, R.L and Chun, Y.S. (1994) Eighteen-month evaluation of the effects of a 0.4% stannous fluoride gel on gingivitis in orthodontic patients. Am. J. Orthod. Dentofaciai Orthop. 105, 35- 41. Brambilla, E., Toselii, A., Felloni, A., Gagliani, M., Malerba, A. and Strohmenger, L. (1997) The effect of biannual applications of amine fluoride solution on caries incidence in permanent first molars; a 5-year study,int. J. Paediatr. Dent. 7, 9-14. Bramstedt, F. and Bandilla, J. (1966) Uber den einfluss organischer fluorverbindungen auf saurebildung und polysaccharidsynthese von plaques-streptokokken. Dtsch. Zahnârztl. Z. 21,1390-1396. Bratthall, D., Hansel Petersson, G. and Sundberg, H. (1996) Reasons for the caries decline: what do the experts believe. Eur. J. Oral Sd. 104,416-422. Brecx, M., Netuschil, L , Reichert, B. and Schreil, G. (1990) Efficacy of Listerine, Meridol and chlorhexidine mouthrinses on plaque, gingivitis and plaque bacteria vitality. J. Clin. Periodontol. 17, 292-297. Brecx, M., Brownstone, E., MacDonald, L , Gelskey, S. and Cheang, M. (1992) Efficacy of Listerine, Meridol and chlorhexidine mouthrinses as supplements to regular tooth cleaning measures. J. Clin. Periodontol. 19, 202-207. Brecx, M., Macdonald, L.L., Legary, K., Cheang, M. and Forgay, M.G. (1993) Long-term effects of Meridol and chlorhexidine mouthrinses on plaque, gingivitis, staining, and bacterial vitality. J. Dent. Res. 72,1194-1197. Breitenmoser, T.H. (1975) The antiglycolytic action on dental plaque of amine chlorides. Helv.Odontol. Acta 19,13-17. Brudevold, F., Steadman, L.T., Gardner, D.E., Rowley, J. and Little, M.F. (1956a) Uptake of tin and fluoride by intact enamel. J. Am. Dent. Assoc. 53,159-164. Brudevold, F., Gardner, D.E. and Smith, F A. (1956b) The distribution of fiuoride in human enamel. J. Dent. Res. 35,420-429. Brudevold, F., Savory, A., Gardner, D.E., Spinelli, M. and Speirs, R. (1963) A study of acidulated fluoride solutions - "In vitro" effects on enamel. Arch. Oral Biol. 8,167-177. 231

Brudevold, F. and Chilton, N.W. (1966) Comparative study of a fiuoride dentifrice containing soiubie phosphate and a caicium-free abrasive: second year report. J. Am. Dent Assoc. 72, 889-894.

Brudevoid, P., McCann, H.G. and Gr 0 n, P. (1968) An enamei biopsy method for determination of fiuoride in human teeth. Arch. Oral Biol. 13, 877-885. Brudevoid, P., Reda, A. and Aasenden, R. (1974) An enamel biopsy method for determination of trace eiements. J. Dent. Res. 54, 581 (Abstract) Bruun, C., Givskov, H. and Thylstrup, A. (1984) Whole saliva fluoride after toothbrushing with NaP and MPP dentifrices with different P concentrations. Caries Res. 18, 282-288.

Budtz-J0 rgensen, E. (1991) Effect of controiied oral hygiene in overdenture wearers: a 3-year study. Int. J. Prosthodont. 4,226-231.

Budtz-J0 rgensen, E. (1994) Effects of denture-wearing habits on periodontal health of abutment teeth in patients with overdentures. J. din. Periodontol 21,265-269. Buiiock, S., Newman, H.N. and Wiison, M. (1989) The in-vitro effect of an amine fluoride gei on subgingivai piaque bacteria. J. Antimicrob. Chemother. 23, 59-67. Burt, B.A., ismaii, A.i. and Ekiund, S.A. (1986) Root caries in an optimaiiy fluoridated and a high-fluoride community. J. Dent. Res. 65,1154-1158. Busscher, N.J., Uyen, N.M., De Jong, H.P., Arends, J. and Kip, G.A. (1988) Adsorption of aminefluorides on human enamel. J. Dent. 16,166-171. Cahen, P.M., Prank, R.M., Turlot, J.C. and Jung, M.T. (1982) Comparative unsupervised ciinicai triai on caries inhibition effect of monofluorophosphate and amine fluoride dentifrices after 3 years in Strasbourg, Prance. Community Derït. Orai Epidemiol 10,238-241. Caiiis, P.O., Chariton, G. and Ciyde, J.S. (1993) A survey of patients seen in Consuitant Clinics in Conservative Dentistry at Edinburgh Dentai Hospital in 1990. Br. Dent. J. 174,106-110. Campbeii, M.J. and Gardener, M.J. (1989) Caicuiating confidence intervais for some nonparametric anaiyses. in: Gardener, M.J. and Aitman, D.G. (Eds.) Statistics with Confidence - Confidence intervais and Statisticai Guideiines, pp. 71-79. London: BMJ Candeii, A., Capozzi, L., Marci, P. and Marchini, G. (1967) The determination of fluoride within the teeth by means of biopsy on the enamel. Caries Res. 1,153-161. Capozzi, L., Brunetti, P.L. and Migiiorini, E. (1967) Enzymatic mechanisms of action of some fluorine compounds. Caries Res. 1, 69-77. Caufield, P.W., Allen, D.N. and Childers, N.K. (1987) In vitro susceptibiiities of suspected periodontopathic anaerobes as determined by membrane transfer assay. Antimicrob. Agents Chemother. 31,1989-1993. Chan, J.C., Hiii, P.J. and Newman, H.N. (1991) Uptake of fluoride by sound and artificiaiiy carious enamei in vitro foiiowing appiication of topical sodium and amine fluorides. J. Dent. 19,110-115. Chauncey, H.H., Giass, R.L. and Aiman, J.E. (1989) Dental Caries. Principie cause of tooth extraction in a sampie of US maie aduits. Caries Res 23, 200-205. 232

Chikte, U.M., Pochee, E., Rudolph, M.J. and Reinach, S.G. (1991) Evaluation of stannous fluoride and chlorhexidine sprays on plaque and gingivitis in handicapped children. J. Clin. Periodontol. 18, 281-286. Chow, L.C., Beaudreau, G.M. and Brown, W.E. (1985) Enamel fluoride profile construction from biopsy data. Caries Res. 19,103-112. Chu, J.S., Fox, J.L., Higuchi, W.l. and Nash, W.P. (1989) Electron probe micro-analysis for subsurface demineralisation and remineralisation of dental enamel. J. Dent. Res. 68,26-31. Giancio, S.G., Shibly, O., Mather, M .L, Bessinger, M.A., Severe, N.C. and Slivka, J. (1992) Clinical effects of a stannous fluoride mouthrinse on plaque. Clin. Prev. Dent. 14, 27-30. Ciardi, J.E., Bowen, W.H. and Relia, G. (1978) The effect of antibacterial compounds on glucosyltransferase activity from Streptococcus mutans. Arch. Oral Biol. 23, 301-305. Ciiley, W.A. and Haberman, J.P. (1981) Fluoride in enamel and correlation to caries. J. Dent. Res. 60, 577(Abstract) Clarkson, J.E., Eilwood, R.P. and Chandler, R E. (1993) Comprehensive summary of fluoride dentifrice caries ciinicai trials. Am. J. Dent. 6, 859-8106. Clarkson, B.H., Fejerskov, O., Ekstrand, J. and Burt, B.A. (1996) Rational use of fluorides in caries control. In: Fejerskov, O., Ekstrand, J. and Burt, B.A. (Eds.)Fluoride in dentistry, 2nd edn. pp. 347-357. Copenhagen: Munksgaard Collins, R., Nebergail, W. and Langer, H. (1961) A study of the reactions of various tin (II) compounds with calcium hydroxyapatite. J. Am. Chem. Soc. 83, 3724-3725. Consultant Restorative Dentistry Group (1983) Roie of the Consultant Restorative Dentist in the General Hospital 8ervice. Br. Dent. J. 153,181-182. Conti, A.J., Lotzkar, 8., Daley, R., Cancro, L., Marks, R.G. and McNeal, D R. (1988) A 3-year clinical trial to compare efficacy of dentifrices containing 1.14% and 0.76% sodium monofluorophosphate. Community Dent. Oral Epidemiol. 16,135-138. Cooley, W.E. (1961) Reactions of tin (II) and fluoride ions with etched enamel. J. Dent. Res. 40,1199-1210. Corpron, R.E., More, F.G., Beltran, E.D., Clark, J.W. and Kowalski, C.J. (1991) In vivo fluoride uptake of human root lesions using a fluoride-reieasing device (short communication). Caries Res. 25,158-160. Crall, J.J., 8ilverstone, L.M., Clarkson, B.H., Wefel, J.8. and Wei, 8.H.Y. (1982) Fluoride uptake and in vitro caries like lesion formation in enamel after two step topical fluoride applications. Caries Res. 16,162-169. Crall, J.J. and BJerga, J.M. (1984) Fluoride uptake and retention following combined applications of APF and stannous fluoride in vitro. Pediatr. Dent. 6, 226-229. Cruz, R., Ogaard, B. and Reila, G. (1992) Acquisition of alkali-soluble fluoride by enamel through treatment with NaF-containing toothpastes in vitro. Scand. J. Dent. Res. 100, 81-87. Cruz, R. and Reiia, G. (1992) The effect of time of exposure on fluoride uptake by human enamel from acidulated fluoride solutions in vitro. Acta Odontol. Scand. 50, 51-56. Dahl, B.L., Carisson, G.E. and Ekfeldt, A. (1993) Occlusal wear of teeth and restorative materials. Acta Odontol. Scand. 51, 299-311. 233

Darbar, U.R. and Hemmings, K.W. (1997) Treatment of localised anterior toothwear with composite restorations at an increased occlusal vertical dimension. Dent. Update 24, 72-75. Davies, R.M., Holloway, P.J. and Eilwood, R.P. (1995) The role of fluoride dentifrices in a national strategy for the oral health of children. Br. Dent. J. 179, 84-87. Davis, R.K., Renner, R.P., Antes, E.W., Jr., Schlissel, E.R. and Baer, P.M. (1981) A two-year longitudinal study of the periodontal health status of overdenture patients. J. Prosthet. Dent. 45, 358-363. Dean, H.T., Amold, FA.J. and Elvove, E. (1942) Domestic water and dental caries. V. Additional studies of the relation of fluoride domestic waters to dental caries experience in 4425 white children aged 12-14 years of 13 cities in 4 states. Publ. Health Rep. 57, 1155- 1179. Denes, J. and Gabris, K. (1991) Results of a 3-year oral hygiene programme, including amine fiuoride products, in patients treated with fixed orthodontic appiiances. Eur. J. Orthod. 13, 129-133. Dérand, T., Lodding, A. and Petersson, LG. (1989) Effect of topical F solutions on caries like lesions in root surfaces. Caries Res. 23,135-140. Diggens, A.A. and Ross, J.W. (1981) Determining ionic species electrochemically. UK Patent GB2604 131A. Dijkman, A.G. and Arends, J. (1982) Thickness of enamel layers removed by HCIO4 etching. Caries Res. 16,129-137. Dijkman, A.G., Tak, J., Smith, M.L, Jongebloed, W.L. and Arends, J. (1982) Fluoride deposited by topicai applications with stannous fluoride in human enamel. J. Biol. Buccale. 10, 63-71. Diodati, R.R., Triol, C.W., Kranz, S.M., Korn, L.R., Voipe, A.R. and Goldman H.M. (1986) Clinical anti-caries efficacy of various fluoride dentifrices. J. Dent. Res. 65,198 (Abstract) Dolan, M.M., Kavanagh, B.J. and Yankell, S.L. (1972) Artificial plaque prevention with organic fluorides. J. Periodontol. 43, 561-563. Dolder, E.J. (1961) The bar joint mandibular denture. J. Prosthet. Dent 11, 689-707. Dowell, P. and Addy, M. (1984) Dentine hypersensitivity. A quantitative comparison of the uptake of metal salts and fluoride by dentine and hydroxyapatite. J. Periodont. Res. 19, 530- 539. Downer, M.G. (1989) Validation of methods used in dental caries diagnosis. Int. Dent. J. 39, 241-246. Downer, M.G. (1994) Caries prevalence in the United Kingdom. Int. Dent J. 44, 365-370. Driscoll, W.C., Swango, P.A., Horowitz, A.M. and Kingman, A. (1982) Caries-preventive effects of daily and weekly fluoride mouthrinsing in a fluoridated community: final results after 30 months. J. Am. Dent. Assoc. 105,1010-1013. Duckworth, R.M. (1993) The science behind caries prevention. Int. Dent. J. 43, 529-539. Duckworth, R.M. (1995) Models for evaluating new fluoride-containing systems: reaction paper. Adv. Dent Res. 9, 300-303. 234

Dupuis, J.(1993) The EEC Cosmetics Directive updated version - incorporating all amendments until 31 August 1993. COLIPA Secretariat. Duschner, H. and Uchtmann, H. (1985) Reaktionen von aminhydrofluoriden mit oberflachenschmeiz. II. Vergleich der wachsel wirkungen von aminhydrofluorid und natriumfluorid bel identischen pH-werten. Dtsch. Zahnârztl. Z. 40,1031-1035. Duschner, H. and Uchtmann, H. (1988a) Effect of sodium fluoride, stannous fluoride, amine hydrofluoride and sodium monofluorophosphate on the formation of precipitates adhering to bovine enamel. Caries Res. 22,65-71. Duschner, H. and Uchtmann, H. (1988b) Etching behaviour of bovine enamel after the formation of precipitates adhering to the surface. Caries Res. 22, 72-75. Ekstrand, J. (1977) A micromethod for the determination of fluoride in blood plasma and saliva. Caldf. Tissue Res. 23. 225-228. Ekstrand, K., Qvist, V. and Thylstrup, A. (1987) Light microscope study of the effect of probing in occlusal surfaces.Caries Res. 21, 368-374. Ellingsen, J.E. (1986) Scanning electron microscope and electron microprobe study of reactions of stannous fluoride and stannous chloride with dental enamel. Scand. J. Dent. Res. 94, 299-305. Ellingsen, J.E., Rolla, G. and Afsth, J. (1987) Exposure of dentin surfaces to metal fluorides. In: Thylstrup, A., Leach, S.A. and Relia, G. (Eds.) Dentine and Dentine Reactions, pp. 189- 198. Oxford: IRL Press Ltd. Ellingsen, J.E. and Relia, G. (1987) Treatment of dentin with stannous fluoride - SEM and electron microprobe study. Scand. J. Dent. Res. 95, 281-286. Elworthy, A., Greenman, J., Doherty, P.M., Newcombe, R.G. and Addy, M. (1996) The substantivity of a number of oral hygiene products determined by the duration of effects on salivary bacteria. J. Periodontoi. 67, 572-576. Engel-Brill, N., Gedalia, I., Raxn, P., Priedwald, E., Rotmann, M. and Rosen, L. (1996) The effect of topical fluoride agents on saliva secretion. J. Orai Rehabii. 23, 501-504. Ericsson, Y. (1950) Reduction of the solubility of enamel surfaces. Acta Odont. Scand. 9, 60- 83. Ericsson, Y. (1961) Pluorides in dentifrices, investigations using radioactive fluorine. Acta Odontol Scand 19, 41-77. Etemadzadeh, H., Meurman, J.H., Murtomaa, H., Torkko, H., Lappi, L. and Roos, M. (1989) Effect on plaque growth and salivary micro-organisms of amine fluoride-stannous fluoride and chlorhexidine-containing mouthrinses. J. Clin. Periodontol. 16,175-178. Ettinger, R.L. and Jakobsen, J. (1990) Caries: a problem in an overdenture population. Community. Dent. Oral Epidemioi. 18,42-45. Palier, R.V. (1995) The application of in situ models for evaluation of new fluoride-containing systems.Adv. Dent. Res. 9, 290-299. Palier, R.V., Best, J.M., Peatherstone, J.D. and Barrett Vespone, N.A. (1995) Anticaries efficacy of an improved stannous fluoride toothpaste. J. Clin. Dent. 6 (Spec No), 89-96. 235

Fallen, R.V., Pfarrer, A.M., Eversole, S.L, Cox, E.R., Landrigan, W.P. and Wang, Q. (1997) The comparative anticaries efficacy of Crest toothpaste relative to some marketed Chinese toothpastes - results of in vitro pH cycling testing. Int. Dent J. 47, 313-320. Fanning, E.A., Gotjamanos, I . and Vowies, N.J. (1968) The use of fiuoride dentifrices in the control of dental caries: methadoiogy and results of a clinical trial. Aust Dent J 13, 201-206. Featherstone, J.D.B., McIntyre, J.M. and Fu, J. (1987) Physico-chemical aspects of root caries progression. In: Thylstrup, A., Leach, S.A. and Qviest, V. (Eds.) Dentine and Dentine Reactions in the Oral Cavity, Oxford: IRL Press Ltd. Fejerskov, O., Thylstrup, A. and Larsen, M.J. (1981) Rational use of fluoride in caries prevention. Acta Odontol. Scand. 3,241-249. Fenton, A.H. and Hahn, N. (1978) Tissue response to overdenture therapy. J. Prosthet. Dent. 40,492-498. Ferretti, G A. and Tinanoff, N. (1980) Bacteriostatic and bacteriocidal effects of different fluoride compounds on S.mutans. J. Dent. Res. 59, 518(Abstract) Figures, K.H., Ellis, B. and Lamb, D.J. (1990a) Fluoride penetration into dentine abutments in vitro. Caries Res. 24, 301-305. Fleiss, J.L., Park, M.H. and Chilton, N.W. (1987) Within-mouth correlation and reliabilities for probing depth and attatchment levels. J. Periodontol. 58,460-463. Flessa, H.J. and Güizow, H.J. (1970) Uber die verweidauer von natriunfluorid und aminfluoriden in den plaques. Dtsch. Zahnârztl. Z. 25, 252-259. Fogels, H.R., Meade, J.J., Griffith, J., Miragliuolo, R. and Cancro, L.P. (1988) A clinical investigation of a high-level fluoride. ASDC J. Dent. Child. 55, 210-215. Foreman, P.C. (1988) Resin-bonded acid etched oniays in two cases of gross attrition. Dent. Update 15,150-153. Frank, R.M. and Nalbandian, J. (1989) Structure and ultrastructure of dentine. In: Oksche, A. and Vollrath, L. (Eds.) Teeth, pp. 173-247. Berlin: Springer-Veriag. Frank, R.M., Steuer, P. and Hemmerie, J. (1989) Uitrastructural study on human root caries. Canes Res. 23,209-217. Frankel, L.S., Bissada, N.F., Maybury, J.E. and Occhino, J.C. (1985) The effect of stannous fluoride mouthrinses on the accumulation of plaque and gingival inflammation following periodontal surgery. Periodont. Case. Rep. 7, 40-42. Frant, M.S. and Ross, J.W. (1966) Electrode for sensing fluoride ion activity in solution. Science 154,1553-1555. Friedman, I., Shapira, L., Gedalia, I., Goultschin, J. and Sela, M. (1992) Effect of subgingival irrigation with AmF and SnF2 in gel form on periodontally diseased sites. J. Dent. Res. 71, 1031 (Abstract) Fritzsche, T. and Saxer, U.P. (1989) Fiuorid retention und ciearence nach spunlungen mit fluoridierten mund wassem. Schweiz. Monatsschr. Zahnmed. 99, 299-306. Fure, S. and Zickert, I. (1997) incidence of tooth loss and dental caries in 60-, 70- and 80-year old Swedish individuals. Community Dent Oral Epidemiol. 25,137-142. 236

Galan, D., Brecx, M. and Heath, M.R. (1995) Oral health status of a population of community- dwelling older Canadians. Gerodontology. 12, 41-48. Gedalia, I., Rosenzweig, K.A. and Sadeh, A. (1961) Fluorine content of superficial enamel layer and its correlation with the fluorine content of saliva, tooth age and DMFT count. J. Dent Res. 40, 865-869. Gedalia, I., Davidov, I., Lewinstein, I. and Shapira, L. (1992) Effect of hard cheese exposure, with and without fluoride prerinse, on the rehardening of softened human enamel. Caries. Res. 26, 290-292. Gedalia, I., Braustein, E., Lewinstein, I., Shapira. L.. Ever Hadani, P. and Sela, M. (1996) Fluoride and hard cheese exposure on etched enamel in neck-irradiated patients in situ. J. Dent. 24, 365-368. Gehring, F. (1983) Wirkung von aminfluorid und natriumfluorid auf keime der plaqueflora. Dtsch. Zahnârztl. Z. 38 (Supp11), S36-40. Glanz, P. (1969) On wettability and adhesiveness. A study of enamel, dentin, some restorative dental materials and dental plaque. Odont. Revy 2 01-132. , Glass, R.L. and Naylor, M.N. (1997) A clinical trial of two fluoride dentifrices in an area of low caries prevalence. Community Dent. Health 14, 74-78. Gomes, B.C. and Renner, R.P. (1990) Periodontal considerations of the removable partial overdenture. Dent. Clin. North Am. 34,653-668. Goorhuis, J. and Purdell Lewis, D.J. (1986) .25% and 0.4% amine fluoride gel for weekly topical application. An in vivo study on human dental enamel. Caries Res. 20,458-464. Gordon, G.E. and Shannon, I.L. (1970) Treatment of root surfaces with stannous fluoride. Bull. N. Z. Soc. Periodontol. 29, 24-28. Green, R.W. and Walsh, J.P. (1951) The protection of the wet enamel surface by adsorbed films. J. Dent. Res. 30, 218-224. Groat, D.E., Sell, R.W. and Kalish, R.J. (1983) Method of forming stable dental gel of stannous fluoride. United States Patent. 4 418 057. Groat, D.E., Sell, R.W. and Kalish, R.J. (1985) Method of forming stable dental gel of stannous fluoride. United States Patent. 4 533 544. Grobler, SR. and Louw, AJ. (1998) A new microwave acid digestion bomb method for the determination of total fluorine. Caries Res. 32, 378-384.

Groeneveld, A. and Arends, J. (1974) The effect of Snp 2 and cetylaminohyrofluoride solutions on the acid demineralisation of enamel. Caries Res. 8, 275-282. Groeneveld, A. (1985) Longitudinal study of prevalence and of enamel lesions in a fluoridated and non-fluoridated area. Community Dent. Oral Epidemiol. 13,159-163. Groeneveld, A., van Eck, A.A.M.J. and Backer Dirks, O. (1990) Fluoride in caries prevention: is the effect pre- or post-eruptive. J. Dent. Res. 69, 751-755.

Gr0 n, P., McCann, H.G. and Brudevold, F. (1968) The direct determination of fluoride in human saliva by a fluoride electrode. Fluoride levels in parotid saliva after ingestion of single doses of sodium fluoride. Arch. Oral Biol. 13, 203-213. G nan, P. (1977) Chemistry of topical fluorides. Caries Res. 11,172-191. 237

Gross, A. and Tinanoff, N. (1977) Effect of Snp2 mouthrinse on Initial bacterial colonization of tooth enamel. J. Dent. Res. 56,1179-1183. Gusberti, F.A., Gada, T.G., Lang, N.P. and Gehring, A H. (1985) Cultivable microflora of plaque from full denture bases and adjacent palatal mucosa. J. Biologie Buccale 13, 227- 236. Gwinnett, A.J., Buonocore, M.G. and Sheykholeslam, Z. (1972) Effect of fluoride on etched human and bovine tooth enamel surfaces as demonstrated by scanning electron microscopy.Arch. Oral Biol. 17,271-278. Haberman, J.P., Ciiley, W.A. and Sakkab, N.Y. (1980) Microanalysis for fluoride in sound enamel and in incipient carious lesions. J. Dent. Res. 59, 918(Abstract) Hals, E., Tveit, A.B., TOtdal, B. and Isrenn, R. (1981) Effect of NaF, TiF^ and APF solutions on root surfaces in vitro, with special reference to uptake of fluoride. Caries Res. 15,468-476. Hals, E. (1991) Coiumn-like structures of human dentin in carious and artificial lesions. Scand. J. Dent. Res. 99, 365-371. Hamilton, I.R. (1990) Biochemical effects of fluoride on oral bacteria. J. Dent. Res. 69 (Spec Iss), 660-667. Hand, J.S., Hunt, R.J. and Kohout, F.J. (1991) Five year incidence of tooth loss in lowans aged 65 and older. Community Dent. Oral Epidemiol. 19,48-51. Harding, E., Dolan, M.M. and Yankell, S.L. (1974) Prolonged effect of an amine fluoride mouthrinse on salivary glycolysis.J. Dent. Res. 53, 267(Abstract) Harley, K.E. and Ibbetson, R.J. (1993) Dental anomolies - are adhesive castings the solution. Br. Dent. J. 174,15-22. Hassell, T.M. (1971) A radio telemetric study of oral fluoride clearance. Helv.Odontol. Acta 15, 29-35. Haugejorden, O., Lervik, T. and Riordan, P.J. (1985) Comparison of caries prevalence 7 years after discontinuation of school-based fluoride rinsing or toothbrushing in Norway.Community Dent. Oral Epidemiol. 13,2-6. Haugejorden, O., Lervik, T., Birkeland, J.M. and Jorkjend, L. (1990) An 11-year foilow-up study of dental caries after discontinuation of school-based fluoride programs. Acta Odontol Scand AS, 257-263. Hefferren, J.J. (1963) Qualitative and quantitative tests for stannous fluoride. J. Pharm. Scl. 52, 1090-1096. Hefti, A.F. and Huber, B. (1987) The effect on early plaque formation, gingivitis and salivary bacterial counts of mouthwashes containing hexetidine/zinc, aminefluoride/tin or chlorhexidine. J. Clin. Periodontol. 14, 515-518. Heifetz, S.B., Meliberg, J R., Winter, S.J. and Doyle, J. (1970) In vivo fluoride uptake by enamel of teeth of human adults from various topical fluoride procedures. Arch. Oral Biol. 15,1171-1181. Heifetz, S.B., Driscoll, W.C. and Creighton, W.E. (1973) The effect on dental caries of weekly rinsing with a neutral sodium fluoride or an acidulated phsphate-fluoride mouthwash. J. Am. Dent. Assoc. 87, 364-368. 238

Heifetz, S.B., Meyers, R.J. and Kingman, A. (1982) A comparison of the anticaries effectiveness of daily and weekly rinsing with sodium fiuoride solutions: results after three years. Pediatr. Dent 4, 300-303. Heintze, U. and Petersson, LG. (1979) Accumulation and clearance of fluoride in human mixed saliva after different topical fluoride treatments. Swed. Dent J. 3,141-148. Hellwig, E. and Klimek, J. (1991) Reaction of NaMFP with plaque-covered and clean dentine. J. Dent Res. 70, 369(Abstract) Hellwig, E. (1992) Fluoride retention in dentin after topical application of aminefluoride. J. Dent Res. 71,1558-1560. Hellwig, E. and Attin, T. (1994) Fluoride retention in dentine after topical application of fluoride vamishes. Caries Res. 2 8 ,199(At>stract) Hellyer, P.H., Beighton, D., Heath, M.R. and Lynch, E.J.R. (1990) Root caries in older people attending a general dental practice in East Sussex. Br. D ent J. 169, 201-206. Hemmings, K.W., Hewlett, J.A., Woodley, N.J. and Griffiths, B.M. (1995) Partial dentures for patients with advanced tooth wear. Dent Update 22, 52-59. Holland, T.J., Whelton, H., O'Mullane, D.M. and Creedon, P. (1995) Evaluation of a fortnightly school-based sodium fluoride mouthrinse 4 years following its cessation. Caries Res. 29, 431-434. Holloway, P.J. and Worthington, H.V. (1993) Sodium fluoride or sodium monofluorophosphate? A critical veiw of meta-analysis on their relative effectiveness in dentifrices. Am. J. Dent. 6, S55-S58. Holt, R.D. and Murray, J.J. (1997) Developments in fiuoride toothpastes - an overview. Community Dent. Oral Epidemiol. 14, 4-10. Hotz, P., Mühlemann, H.R. and Schait, A. (1970) A new method of enamel biopsy for fluoride determination. Heiv. Odontol. Acta 14, 26-29. Hotz, P. (1972) Fluoride levels in surface enamel after application of fluoride gels. Heiv. Odontol. Acta 16, 32-34. Howat, A.P., Holloway, P.J. and Brandt, R.S. (1981) The effect of diagnostic criteria on the sensitivity of dental epidemiological data. Caries Res. 15,117-123. Howell, C.L., Gish, C.W., Smiley, R.D. and Muhler, J.C. (1955) Effect of topically applied stannous fluoride on dental caries experience in children. J. Am. Dent Assoc. 50,14-17. Hunt, R.J., Eldredge, J.B. and Beck, J.D. (1989) Effect of residence in a fluoridated community on the incidence of coronal and root caries in an older adult population. J. Public Health Dent. 49,138-141. Hussey, D.L. and Linden, G.J. (1986) The efficacy of overdentures in clinical practice. Br. Dent. J. 161,104-107. Ibbetson, R.J. and Setchell, D.J. (1989a) Treatment of the wom dentition; 1. Dent. Update 16, 247-253. Ibbetson, R.J. and Setchell, D.J. (1989b) Treatment of the wom dentition:2. Dent. Update 16, 300-305. 239

lijima, Y., Ruben, J.L, Zuidgeest, T.G. and Arends, J. (1993) Fluoride and mineral content in hyper-remineralized coronal bovine dentine in vitro after an acid challenge. Caries Res. 27, 106-110. Imfeid, T. (1996a) Dental erosion. Definition, classification and links. Eur. J. Oral Scl. 104,151- 155. Imfeid, T. (1996b) Micromorphological differences on dental enamel surfaces after exposure to acid with and without previous fluoridation, pp.4.26-4.27. GABA International Report. Imrey, P.B. (1986) Considerations in the statistical analysis of clinical trials in periodontics.J. Clin. Periodont. 13, 517-528. International Scientific Assembly on the Comparative Anticaries Efficacy of Sodium Fluoride and Sodium Monofluorophosphate Dentifrices (1993) Concensus Position Statement. Am. J. Dent. 6, S3 International Scientific Panel Examining the Relative Efficacy of Sodium Fluoride and Sodium Monofluorophosphate as Anti-caries Agents in Dentifrices (1995) Conclusions. In; Bowen, W.H. (Ed.) Relative Efficacy of Sodium Fluoride and Sodium Monofluoroptiosphate as Anti- Caries Agents In Dentifrices, London: Royal Society of Medicine Press Ltd. Irwin, M., Leaver, A.G. and Walsh, J.P. (1957) Further studies on the influence of surface active agents on décalcification of the enamel surface. J. Dent. Res. 36,166-172. ITRI (1998) Complexing and stabilisation of tin. Personal Communication to Woodley, N.J. and Griffiths, B.M. Iyer, S.V., Fox, J.L., Higuchi, W.I., Hefferren, J.J. and Vishnupad, K.S. (1983) A new acid abrasion procedure for studying fluoride profiles in demineralised/remineralised bovine tooth enamel. Caries Res. 17,297-303. Jensen, M.E. and Kohout, F.J. (1988) The effect of a fluoridated dentifrice on root and coronal caries in an older adult population. J. Am. Dent. Assoc. 117, 829-832. Johannson, A. and Omar, R. (1994) Identification and management of tooth wear. Int. J. Prosthodont. 7, 506-516. Johnson, G.K. and Silvers, J.E. (1987) Attrition,abrasion and erosion: diagnosis and therapy. Clin. Prev. Dent. 9,12-16. Johnson, M.F. (1993) Comparative efficacy of NaF and SMFP dentifrices in caries prevention: a meta-analytic overview. Caries Res. 27, 328-336. Jones, S.J. and Boyde, A. (1984) Ultrastructure of dentine and dentinogenesis. In: Linde, A. (Ed.) Dentine and Dentinogenesis, pp. 81-134. Florida: CRS Press Inc. Jones, S.J. (1990) The pulp-dentine complex. In: Elderton, R.J. (Ed.) The Dentition and Dental Care, pp. 63-64. Oxford: Heinemann Medical Books. Jordan, T.H., Wei, S.H., Bromberger, S.H. and King, J.C. (1971) SnsFsPO^: the product of the reaction between stannous fluoride and hydroxyapatite. Arch. Oral Biol. 16, 241-246. Kabara, J.J., Conley, A.J. and Truant, J.P. (1972) Relationship of chemical structure and antimicrobial activity of alkyl amides and amines. Antimicrob. Agents Chemother. 2, 492- 498. 240

Kambara, M. and Norde, W. (1995) Influence of fiuoride applications on some physicochemical surface properties of synthetic hydroxyapatite and human dentai enamei and its consequences for protein adsorption. Caries Res. 29, 210-217. Katjaiainen, S., Eriksson. A.-L, Ruokoia, M. and Toivonen, A. (1994) Caries deveiopment after sut)stitution of supervised fiuoride rinses and toothbrushings by unsupervised use of fiuoride toothpaste. Community Dent Oral Epidemiol. 22,421-424. Kasturi, R., White, D.J., Lanzalaco, A C., Macksood, D., Cox, E.R., Bacca, L., Liang, N. and Baker, R. (1995) Effects of nine weeks' use of a new stabilized stannous fluoride dentifrice on intrinsic piaque virulence expressed as acidogenicity and regrowth: a modified PGRM study. J. Clin. Dent. 6 (Spec No), 71-79. Katz, R.V. (1980) Assessing root caries in populations: The evoiution of the root caries index. J Public health Dent 40, 7-16. Katz, R.V. (1984) Development of an index for the prevalence of root caries. J. Dent. Res. 63 (Spec No), 814-819. Kay, H.M. and Wilson, M. (1988) The in vitro effects of amine fluorides on plaque bacteria. J. Periodontol. 59, 266-269. Keene, H.J. and Fleming, T.J. (1987) Prevalence of caries associated microflora after radiotherapy in patients with cancer of the head and neck. Oral Surg. Oral Med. Oral Pathol. 64, 421-426. Keitjens, H.M., Schaeken, M.J., van der Hoeven, J.S. and Hendriks, J.C. (1990) Caries control in overdenture patients: 18-month evaiuation on fluoride and chlorhexidine therapies. Caries Res. 24, 371-375. Kidd, E.A. and Smith, B.C. (1993) Toothwear histories: a sensitive issue. Dent. Update 20,174- 178. Kirkegaard, E., Müller, I.J. and Skov Jensen, E. (1976) A method for in vitro studies on fluoride uptake in enamei using single tooth surfaces. Caries Res. 10, 370-378. Kirkegaard, E. (1977a) in vitro fluoride uptake in human dental enamel from various fluoride solutions.Caries Res. 11,16-23. Kirkegaard, E. (1977b) in vitro fluoride uptake in human dentai enamel from four different dentifrices. Caries Res. 11,24-29. Kiimek, J. (1981) Fluoridaufnahme kunstlicher karioser initialiasionen nach behandlung mit verschiedenen fluoridverbindungen. Dtsch. Zahnârztl. Z. 36, 520-524. Kiimek, J., Hellwig, E. and Ahrens, G. (1982) Fiuoride taken up by piaque, by the underiying enamel and by clean enamel from three fluoride compounds in vitro. Caries Res. 16, 156- 161. Kiock, B., Seriing, J., Kinder, S., Manwell, M.A. and Tinanoff, N. (1985) Comparison of effect of

SnF2 and NaF mouthrinses on caries incidence, salivary S. mutans and gingivitis in high caries prevalent adults. Scand. J. Dent. Res. 93,213-217. KOnig, K.G. (1959) Dental caries and plaque accumulation in rats treated with stannous fluoride and penicillin. Helv. Odontol. Acta 3, 39-44. 241

Kouiourides, T. and Cameron, B. (1980) Enamel remineralization as a factor in the pathogenesis of dental caries. J. Oral Pathol. 9, 255-269. Krutchkoff, D.J., Jordan, T.H., Wei, S.H. and Nordquist, W.D. (1972) Surface characterization of the stannous fluoride-enamel interaction. Arch. Oral Biol. 17, 923-930. Kunzel, W., Franke, W. and Treide, A. (1977) Klinisch-rontgenologische paralleluberwachung einer langsschnittstudie zum nachweis der karieshemmenden effektivitat 7 jahre lokal angewandten aminfluorids im doppelbindtest. Zahn. Mund. Kieferheilkd. Zentralbl. 65, 626- 637. Kyes, P.M., Overton, N.J. and McKean, T.W. (1961) Clinical trials of a caries inhibitory dentifrices. J. Am. Dent. Assoc. 63,189-193.

Lageriof, P., Ekstrand, J. and R0 lla, G. (1988) Effect of fluoride addition on ionized calcium in saiivary sediment and in saliva. Scand. J. Dent. Res. 96, 399-404. Laine, P., Meurman, J.H., Murtomaa, H., Lindqvist, C., Torkko, H., Pyrhonen, S. and Teerenhovi, L. (1993) One-year trial of the effect of rinsing with an amine fluoride-stannous- fluoride-containing mouthwash on gingival index scores and salivary microbial counts in lymphoma patients receiving cytostatic drugs. J. Clin. Periodontol. 20, 628-634. Lauciello, P.R. and Ciancio, S.G. (1985) Overdenture therapy: a longitudinal report. Int. J. Periodont. Restor. Dent. 4, 63-71. Leaver, A.G. (1971) Long chain aliphatic amines as potential caries-preventing agents. New Zealand Dent. J. 67, 99-106. Leske, G.S., Ripa, L.W. and Green, E. (1986) Post-treatment benefits in a school-based fluoride mouthrinsing program: final results after 7 years of rinsing by all participants. Clin. Prev. Dent. 8 , 19-23. Levine, R.S. (1973) Simple nanogram method for estimation of fluoride in mineralised specimens. J. Dent. Res. 52,1249-1252. Liang, N., White, D.J., Cox, E. and Busemeyer, B.A. (1995) Antimicrobial effects of a stabilized stannous fluoride dentifrice in reducing plaque acid production-a singie-brushing PGRM study. J. Clin. Dent. 6 (Spec No), 80-83. Lilienthal, B. (1956) The effect of a stannous fluoride mouthwash on acid formation in the mouth and some observations on the mechanisms of inhibition. Aust. Dent. J. 1, 221-227. Lim, J.K. (1970) Precipitate-free, dilute aqueous solutions of stannous fluoride for topical application: I. Simpie and mixed mediums. J. Dent. Res. 49, 760-767. Lim, J.K. and Hsieh, J.B. (1971) Solubility of surface enamel treated with precipitate-free aqueous solutions of stannous fluoride.J. Dent. Res. 50, 531-535. Lincir, I. and Rosin Grget, K. (1993a) Caries-preventive effect of two different topical fluoride concentrations with two different frequencies of application in preschool children. Caries Res. 27, 484-487. Linde, A. (1987) Dentin: structure, chemistry and formation. In: Thylstrup, A. and Leach, S.A. (Eds.) Dentine and Dentine Reactions in the Oral Cavity, pp. 17-26. Oxford: IRL Press Ltd. Lobene, R.R. and Soparker, P.M. (1974) The effect of amine fluorides on human plaque and gingivitis. J. Dent. Res. 5 3 ,147(Abstract) 242

Loesche, W.J. (1977) Topical fluorides as an antibacterial agent. J. Prev. Dent 4, 21-26. Ludwig, T.G. and Taylor, W.B. (1957) The effectiveness of a tetradecyiamine dental cream in reducing dental caries. New Zealand D ent J. 53,63-67. Ludwig, T.G. (1963) A ciinicai trial of a dentifrice containing tetradecyiamine. New Zealand D ent J. 59, 220-223. Luscher, B., Regolati, B. and Mühlemann, H.R. (1974) Effect of amine fluorides on plaque and caries (in vitro and animal investigations). Helv. Odontol. Acta 18, SuppI 8 : 71-78 Lussi, A. (1991) Validity of diagnostic and treatment decisions of fissure caries. Caries Res. 25, 296-303. Lutz, F. (1983) Mechanismus der protrahierten Aminfluoridwirkung. Dtsch. Zahnârztl. Z. 38 (Supp11), S31-S35. Lynch, E. and Beighton, D. (1994) A comparison of primary root caries lesions classified according to colour. Caries Res. 28, 233-239. Mack, A.O. and Allan, D.N. (1968) Reconstruction of a severe case of attrition and abrasion. Br. Dent. J. 124,17-19. Mankodi, S., Petrone, M., Devizio, W. and VoIpe, A.R. (1997) Clinical efficacy of an optimized stannous fluoride dentifrice. Part 2: 6-month piaque/gingivitis clinical study. Clin. Con. Ed. 17,10-15. Margolis, H.G. and Moreno, E.G. (1990) Physicochemical perspectives on the cariostatic mechanisms of systemic and topical fluorides.J. Dent. Res. 69 (Spec Iss), 606-613. Marks, R.G., Gonti, A.J., Moorhead, J.E., Gancro, L. and D'Agostino, R.B. (1994) Results from a three-year caries clinical trial comparing NaF and SMFP fluoride formulations. Int. Dent. J. 44, 275-285. Marthaier, T.M. (1965) The caries-inhibiting effect of amine fiuoride dentifrices in children during three years of unsupervised use. Br. Dent. J. 119,153-163. Marthaier, T.M. (1968) Garies-inhibition after seven years of unsupervised use of an amine fluoride dentifrice. Br. Dent. J. 124, 510-515. Marthaier, T.M., KOnig, K.G. and Mühlemann, H.R. (1970) The effect of a fluoride gel used for supervising toothbrushing 15 or 30 times per year. Helv.Odontol. Acta 14,67-77. Marthaier, T.M. (1974) Garies-inhibition by an amine fluoride dentifrice results after 6 years in children with low caries activity. Helv. Odontol. Acta 18, Suppi 8: 35-44. Mathiesen, A.T., 0gaard, B. and R0lia, G. (1996) Oral hygiene as a variable in dental caries experience in 14-year-oids exposed to fluoride. Caries Res 30, 29-33. Mazza, J.E., Newman, M.G. and Sims, T.N. (1981) Giinical and antimicrobial effect of stannous fluoride on periodontitis. J. Clin. Periodont. 8,203-212. McGonchie, J.M., Richardson, A S., Hole, L.W., McGombie, F. and Koltammer, J. (1977) Garies preventive effect of two concentrations of stannous fluoride mouthrinse. Community Dent. Oral Epidemiol. 5, 278-283. McHugh, W.D., Eisenberg, A.D., Leverett, D.H. and Jensen, G.E. (1988) The long-term effects of daily rinsing with stannous fluoride or sodium fluoride on bacteria in dental plaque. Pediatr. Dent. 10, 10-12. 243

McKay, F.S. (1916) An investigation of mottled teeth (1). Dent Cosmos 58, 477-484. Meckel, A H. and Francis, M.D. (1964) intraorai transfer of topically applied stannous fluoride. J. D ent Res. 43, 75-85. Meliberg, J.R., Laakso, P.V. and Nicholson, C.R. (1966) The acquisition and loss of fiuoride by topically fluoridated human tooth enamel. Arch. Oral Biol. 11,1213-1220. Meliberg, J R., Nicholson, C.R., Englander, H.R. and Keene, H.J. (1973) improvement in abrasive biopsy sampling of tooth enamel. J. Dent Res. 52,472-475. Meliberg, J R., Hefferren, J.J., Nicholson, C.R., Zimmerman, M. and Rubin, M. (1976) A new abrasive applicator for human enamel biopsy procedures. J. D ent Res. 55, 964-969. Meliberg, J R. (1977) Enamel fluoride and its anti-caries effects. J. Prevent Dent 4, 8-20. Meliberg, J R. (1991) Fluoride dentifrices: current status and prospects. Int. Dent J. 41, 9-16. Mengel, R., Wissing, E., Schmitz Habben, A. and Flores de Jacoby, L. (1996) Comparative

study of plaque and gingivitis prevention by AmF/SnF 2 and NaF. A clinical and microbiological 9-month study. J. Clin. Periodontol. 23, 372-378. Meurman, J.H., Laine, P., Murtomaa, H., Lindqvist, C., Torkko, H., Teerenhovi, L. and Pyrhonen, S. (1991) Effect of antiseptic mouthwashes on some ciinicai and microbiological findings in the mouths of lymphoma patients receiving cytostatic drugs. J. Clin. Periodontol. 18, 587-591. Miller, S., Truong, T., Heu, R., Stranick, M., Bouchard, D. and Gaffar, A. (1994) Recent advances in stannous fluoride technology: antibacterial efficacy and mechanism of action towards hypersensitivity. Int. Dent. J. 44, 83-98. Mills, J. and Dubin, D.T. (1966) Some effects of spermine on E.coli. Molec. Pharmacol. 2, 311- 318. Milosevic, A. (1998) Toothwear: Management. Dent. Update 25, 50-55.

Mobley, M.J. (1981) Fluoride uptake from in situ brushing with a SnF 2 and a NaF dentifrice. J. Dent. Res. 60,1943-1948. Mok, Y., Hill, F.J. and Newman, H.N. (1990) Enamel fluoride uptake affected by site of application: comparing sodium and amine fluorides. Caries Res. 24,11-17. Mühlemann, H.R., Schmid, H. and KOnig, K.G. (1957) Enamel solubility reduction studies with inorganic and organic fluorides. Helv.Odontol. Acta. 1,23-33. Mühlemann, H.R., Schait, A. and KOnig, K.G. (1964) A chemical method for the removal of enamel surface layers and its suitability for fluoride analysis. Helv.Odontol. Acta 8,147-153. Mühlemann, H.R., Rossinsky,K., Schait, A. and KOnig, K.G. (1966) Microhardness, fluoride content and electro-microscopic appearance of enamei after topicai fluoridation. Helv. Odontol. Acta 10,28-37. Mühlemann, H R., Schait, A. and KOnig, K.G. (1968) Fluorine uptake of enamel and animal caries inhibition of topicai sodium fluoride and aminefluorides at low fluoride concentrations. Helv. Odontol. Acta 12, 61-66. Mühlemann, H.R. and Rudolf, E.R. (1975) Fluoride retention after rinsing with sodium fluoride and amine fluoride. Helv. Odontol. Acta 19, 81-84. 244

Mühlemann, H.R., Saxer, U.P. and Mürmann, W. (1981) Reduction of plaque and gingivitis by stannous fluoride stabilised with amine fluoride. Caries Res. 1 5 ,186(Abstract) Mühlemann, H.R. (1983) Entwicklung der aminfluoride und ihre anwendung in der kariesprophylaxe. Dtsch. Zahnârztl. Z. 38 (Suppi 1), S3-5. Muhler, J.C. and van Huysen, G. (1947) Solubility of enamel protected by sodium fluoride and other compounds. J. Dent. Res. 26,119-127. Muhler, J.C., Radike, A.W., Nebergail, W.H. and Day, H.G. (1955) A comparison between the anticariogenic effects of dentifrices containing stannous fluoride and sodium fluoride. J. Am. Dent. Assoc. 51, 556-559. Mukai, M., Ikeda, M., Yanagihara, T., Hara, G., Kato, K., Nakagaki, H. and Robinson, C. (1993) Fluoride uptake in human dentine from glass-ionomer cement in vivo. Arch. Oral Biol. 38, 1093-1098. Munksgaard, E.G. and Bruun, C. (1973) Determination of fluoride in superficial enamel biopsies from human teeth by means of gas chromatography. Arch. Oral Biol. 18, 735-744. Murray, J.J., Rugg-Gunn, A.J. and Jenkins, G.N. (1991)Fluoride in caries prevention, 3rd edn. Oxford: Butterworth, Heinmann. Mushanoff, O., Gedalia, I. and Daphni, L. (1981) Fluoride acquisition by surface enamel of human teeth in vivo following toothbrushing with an amine-fiuoride toothpaste. J. Dent. 9, 144-149. Narhi, T O., Ettinger, R.L, Heilman, J R. and Wefel, J.S. (1997) Salivary fluoride levels in overdenture wearers after topical fluoride gel application. Int. J. Prosthodont. 10, 553-561. Naylor, M.N. and Emslie, R.D. (1967) Ciinicai testing of stannous fluoride and sodium monofiuorophosphate dentifrices in London school children. Br. Dent. J. 123,17-23. Nelson, D.G.A., Coote, G.E., Vickridge, I.C. and Suckling, G. (1989) Proton micro-probe determination of fluorine profiles in the enamel and dentine of erupting incisors from sheep given low and high daily doses of fluoride. Arch. Oral Biol. 34,419-429. Nemes, J., Bânôczy, J., Wierzbicka, M. and Rost, M. (1991) Aminfluorid es onfluorid tartalmu fogpaszta es szajoblito hatasa felnotteken a plakk-kepzodesre es az inygyulladasra. Fogorv. Sz. 84, 233-236. Nemes, J., Bânôczy, J., Wierzbicka, M. and Rost, M. (1992) Clinical study on the effect of amine fluoride/stannous fluoride on exposed root surfaces. J. Clin. Dent. 3, 51-53. Netuschil, L., Weiger, R., Preisler, R. and Brecx, M. (1995) Plaque bacteria counts and vitality during chlorhexidine, meridol and listerine mouthrinses. Eur. J. Oral Scl. 103, 355-361. Nevin, R.B., Walsh, J.P. and King, R.M. (1953) Clinical trial of a toothpaste containing tetradecyiamine. New Zealand Dent. J. 49,134-138. Newbrun, E. (1993) Problems in caries diagnosis. Int. Dent. J. 43,133-142. Newman, H.N. and Poole, D.F.G. (1974) Observations with scanning and transmission electron microscope on the structure of human surface enamel. Arch. Oral Biol. 19,1135-1143. Nyvad, B. and Fejerskov, O. (1982) Root surface caries: clinical, histopathological and microbiological features and clinical implications. Int. Dent. J. 32, 312-326. 245

Nyvad, B. and Fejerskov, O. (1986) Active root surface caries converted into inactive caries as a response to oral hygiene. Scand J. Dent Res. 94,281-284. Nyvad, B. and Fejerskov, O. (1987) Active and inactive root surface caries - structural entities. In: Thylstrup, A., Leach, S.A. and Qvist, V. (Eds.)Dentine and Dentine Reactions in the Oral Cavity, pp. 165-179. Oxford: IRL Press Ltd. Nyvad, B., ten Cate, J.M. and Fejerskov, O. (1989) Microradiography of experimental root surface caries in man. Caries Res. 23,218-224. O'Mullane, D.M. (1976) Efficiency In clinical trials of caries preventive agents and methods. Community Dent. Oral Epidemiol. 4,190-194. O'Mullane, D.M. (1994) Introduction and rationale for the use of fluoride for caries prevention. Int. Dent. J. 44, 257-261. Obersztyn, A., Kolwinski, K., Trykowski, J. and Starosciak, S. (1979) Effects of stannous fluoride and amine fluorides on caries incidence and enamel solubility in adults. Aust. Dent. J. 24, 395-397. Obersztyn, A. and Kolwinski, K. (1984) Amine fluoride gel in a caries prophylaxis program for soldiers in Poland. Community Dent. Oral Epidemiol. 12, 288-291. Ohmori, I., Brudevold, F. and Gren, P. (1964) Acquisition of fluoride by intact surface enamel from acid fluoride phosphate solutions. J.Dent. Res. 43, 869(Abstract) Olsson, J. and Krasse, B. (1976) A method of studying adherence of oral streptococci to solid surfaces. Scand. J. Dent. Res. 86,108-117. Olsson, J. and Odham, G. (1978) Effect of inorganic ions and surface active organic compounds on the adherence of oral streptococci. Scand. J. Dent. Res. 84,20-28. Oosterwaal, P.J., Mikx, F.H., van den Brink, M.E. and Renggli, H.H. (1989) Bactericidal concentrations of chlorhexidine-digluconate, amine fluoride gel and stannous fluoride gel for sut)gingival bacteria tested in serum at short contact times. J. Periodontal. Res. 24, 155- 160. Oosterwaal, P.J., Mikx, F.H., van t Hof, M.A. and Renggli, H.H. (1991a) Comparison of the antimicrobial effect of the application of chlorhexidine gel, amine fluoride gel and stannous fluoride gel in debrided periodontal pockets. J. Clin. Periodontol. 18, 245-251. Oosterwaal, P.J., Mikx, F.H., van t Hof, M.A. and Renggli, H.H. (1991b) Short-term bactericidal activity of chlorhexidine gel, stannous fluoride gel and amine fluoride gel tested in periodontal pockets. J. Clin. Periodontol. 18, 97-100. Oppermann, R.V. and Johansen, J R. (1980) Effect of fluoride and non-fluoride salts of copper, silver and tin on the acidogenicity of dental plaque in vivo. Scand. J. Dent. Res. 88, 476- 480. Ostela, I. and Tenovuo, J. (1990) Antibacterial activity of dental gels containing combinations of amine fluoride, stannous fluoride, and chlorhexidine against cariogenic bacteria. Scand. J. Dent. Res. 98,1-7. Ostela, I., Karhuvaara, L. and Tenovuo, J. (1991) Comparative antibacterial effects of chlorhexidine and stannous fluoride-amine fluoride containing dental gels against salivary mutans streptococci. Scand. J. Dent. Res. 99, 378-383. 246

Overholser, C D., Jr. (1988) Longitudinal clinical studies with antimicrobial mouthrinses. J. Clin. Periodontol. 15, 517-519. Owens, J., Addy, M. and Faulkner, J. (1997) An 18-week home-use study comparing the orai hygiene and gingival health benefits of triclosan and fluoride toothpastes. J. Clin. Periodontoi. 24, 626-631. Pakhomov, G.N., Mdller, I.J., Atanassov, N.P., Kabackchieva, R.I. and Sharkov, N.I. (1997) Effect of an amine fluoride dentifrice on dental caries used in a community-based oral health education program. J. Pubiic Health Dent. 57,181-183. Patterson, C.M., Weatherell, J.A. and Robinson, 0. (1984) Sampling of porous tissues in vitro by acid etching. Caries Res. 18,231-236. Patz, J. and Naujoks, R. (1970) Die kariesprophylaktische wirkung einer aminfluoridhaltigen zahnpaste bei jugendlichen nach dreijâhrigem unüberwachten gebrauch. Dtsch Zahnarztl Z 25, 617-625. Perlich, M.A., Bacca, L.A., Bollmer, B.W., Lanzalaco, A C., McClanahan, S.F., Sewak, L.K., Beiswanger, B.B., Eichold, W.A., Hull, J R., Jackson, R.D. and et al. (1995) The clinical effect of a stabilised stannous fluoride dentifrice on plaque formation, gingivitis and gingival bleeding; a six month study. J. d in . Dent. 6 (Spec No), 54-58. Peterson, J.K. and Williamson, L. (1968) Three-year caries inhibition of a sodium fluoride acid orthophosphate dentifrice compared with a stannous fluoride and a non-fluoride dentifrice. iADR Abstracts 101 Phillips, R.W. and Swartz, M.L. (1948) Effect of fluorides on hardness of tooth enamel. J. Am. Dent. Assoc. 37,1-13. Potter, D.E., Manwell, M.A., Dess, R., Levine, E. and Tinanoff, N. (1984) SnF: as an adjunct to toothbrushing in an elderly institutionalized population. Spec. Care Dentist. 4,216-218. Prieskel, H.W. (1985) The restoration of the mutilated dentition. J. Dent. 13,173-183. Proskin, H.M. (1993) Statistical considerations related to a meta-analytic evaluation of published caries clinical studies comparing the anti-caries efficacy of dentifrices containing sodium fluoride and sodium monofluorophsphate. Am. J. Dent. 6, S43-S49. Proskin, H.M. and Voipe, A.R. (1995) Comparison of the anticaries efficacy of dentifrices containing fluoride as sodium fluoride or sodium monofluorophosphate. Am. J. Dent. 8, 51- 58. Raab,W.H.M. (1990) Die auswirkungen einer aminfluorid/zinnfluorid-kombination in gelform auf die plaque- und gingivitisentwicklung. In: Flores-de-Jacoby, L. (Ed.) Mogiichkeiten der Piaque- und Ginigivitispravention. pp27-41. Berlin: Quintessenz Veriag GmbH. Raby, B.A. and Sunderland, W.E. (1967) Direct determination of fluoride In tungsten using the fluoride Ion activity electrode. Anai. Chem. 39,1304-1305. Radike, A.W., Gish, C.W., Peterson, J.K., King, J.D. and Segreto, V.A. (1973) Clinical evaluation of stannous fluoride as an anticaries mouthrinse. J. Am. Dent. Assoc. 86, 404- 408. Ralph, J.P. and Murray, F.D. (1967) The use of root abutments in the support of complete dentures. J. Orai Rehabii. 3, 293-297. 247

Ralph, J.P. and Basker, R.M. (1989) The role of overdentures In gerodontics. Dent Update 16, 353-359. Ran, F., Gedalia, i.. Fried, M., Hadani, P. and Tved, A. (1991) Effectiveness of fortnightly tooth brushing with amine fluorides in caries-prone subjects. J. Oral Retiabil. 18, 311-316. Ratanen, T., Makila, E., Antti, Y. and Hannu, S. (1971) investigations of the therapeutic success with dentures retained by precision attatchments. i. Root anchored complete overiay dentures. Suom Hammaslaak Toim 67,356-366. Raul, T. and Netuschil, L. (1988) In vitro and in vivo antibacterial effect of amine fluoride/stannous fluoride. J. Dent. Res. 67, 703(Abstract) Ravald, N. and Birkhead, D. (1992) Prediction of root caries in periodontally treated patients maintained with different fluoride programmes. Caries Res. 26,450-458. Rawlinson, A. and Winstanley, R.B. (1988) The management of severe dental erosion using posterior occlusal porcelain veneers and an anterior overdenture. Restor. Dent 4,10-16. Reintsema, H., Schuthof, J. and Arends, J. (1985) An in vivo investigation of the fluoride uptake in partially demineralised human enamel from several different dentifrices. J. Dent. Res. 64,19-23. Reitz, P.V., Weiner, M.G. and Levin, B. (1977) An overdenture survey: preliminary report. J. Prosthet Dent 37, 246-258. Renggli, H.H. (1983) Plaquehemmung durch aminfluorid. Dtsch. Zahnârztl. Z. 38 (Suppi 1), S45-S49. Renner, R.P., Gomes, B.C., Shakun, M.L., Baer, P.M., Davis, R.K. and Camp, P. (1984) Four- year longitudinal study of the periodontal health status of overdenture patients. J. Prosthet. D ent 51, 593-598. Richards, A. and Banting, D.W. (1996) Fluoride toothpastes, in: Fejerskov, O., Ekstrand, J. and Burt, B. (Eds.) Fluoride in Dentistry, 2nd edn. pp. 328-346. Copenhagen: Munksgaard. Rinderer, L., Schait, A. and Mühlemann, H.R. (1965) Loss of fluoride from dental enamel after topical fluoridation (preliminary report). Helv. Odontol. Acta 9,148-150. Ringelberg, M.L., Webster, D.B., Dixon, D.O. and LeZotte, D C. (1979) The caries-preventive effect of amine fluorides and inorganic fluorides in a mouthrinse or dentifrice after 30 months of use. J. Am. D ent Assoc. 98, 202-208. Ripa, L.W., Leske, G.S., Forte, F. and Varma, A. (1987) Effect of a 0.05% neutral NaF mouthrinse on coronal and root caries of adults. Gerodontology 6,131-136. Roberts, J.F., Bibby, B.G. and Wellock, W.D. (1948) The effect of an acidulated fluoride mouthwash on dental caries. J. D ent Res. 27, 497-500. Robinson, C., Weatherell, J.A. and Hallsworth, A S. (1971) Variation in composition of dentai enamel within thin ground tooth sections. Carles Res. 5, 44-57. Robinson, C., Kirkham, J. and Weatherell, J.A. (1996) Fluoride in teeth and bone. In: Fejerskov, O. Ekstrand, J., and Burt, B.A. (Eds.) Fluoride In Dentistry, Copenhagen: Munksgaard. Rolla, G. (1977) Effects of fluoride on initiation of plaque formation. Caries Res. 11 (Suppi 1), 243-261. 248

R0 lla, G. (1988) On the role of calcium fluoride in the cariostatic mechanism of fluoride. Acta Odontol. Scand. 46, 341-345. Relia, G. and Saxegaard, E. (1990) Critical evaluation of the composition and use of topical fluorides, with emphasis on the roie of calcium fluoride in caries inhibition. J. Dent. Res. 69 (Spec No), 780-785. Relia, G., Ogaard, B. and Cruz, R.D.A. (1993) Topical application of fiuorides on teeth. New concepts of mechanisms of interaction. J. Clin. Periodontoi. 20,105-108. Rosebury, T. and Kranshaw, M. (1939) Susceptibility to dental caries in the cotton rat. J. Dent. Res. 18,189-202. Roseman, T.J., Higuchi, W.I., Modes, B. and Hefferren, J.J. (1969) The retardation of enamel dissolution rates by adsorbed long-chain ammonium chlorides.J. Dent. Res. 48, 509-520 Rosin-Grget, K. and Lincir, I. (1995) Anticaries effect of different amine fluoride concentrations in schoolchiidren. Caries Res. 29,168-171. Sakkab, N.Y., Ciiley, W.A. and Haberman, J.P. (1984) Fluoride in deciduous teeth from an anti-caries clinical study. J.Dent. Res. 63,1201-1205. Salem, A.M., Adams, D., Newman, H.N. and Rawle, L.W. (1987) Antimicrobial properties of 2 aiiphatic amines and chiorhexidine in vitro and in saliva. J. Clin. Periodontol. 14, 44-47. Sanchez, M. and Meiiberg, J R. (1988) Fiuoride uptake in vivo by artificial caries lesions in root surface dentin from sodium monofluorophosphate dentifrices. Caries Res. 22,150-152. Saxegaard, E., Valderhaug, J. and Reila, G. (1987) Deposition of fluoride on dentin and cementum after topical application of 2% NaF. In: Thylstrup, A., Leach, S.A. and Qvist, V. (Eds.) Dentine and Dentine Reactions in the Oral Cavity, pp. 199-206. Oxford: IRL Press Ltd. Schamschula, R.G., Agus, H., Chariton, G., Duppenthaler, J.L. and Un, P. (1979) Associations between fluoride concentration in successive layers of human enamel and individual caries experience. Arch. Oral Biol. 24, 847-852. Schmid, R., Barbakow, F., Mühiemann, H. and De Vecchi, P. (1984a) Amine fluoride and monofluorophosphate: I. Historical review of fiuoride dentifrices. ASDC J. Dent. Child. 51, 99-103. Schmid, R., Barbakow, F., Mühlemann, H. and De Vecchi, P. (1984b) Amine fiuoride and monofluorophosphate: II. Pooled results of 56 independent rat caries tests. ASDC J. Dent. Child. 51,104-106. Schmid, R., Barbakow, F., Mühlemann, H. and De Vecchi, P. (1984c) Amine fluoride and monofluorophosphate: III. Rat caries inhibition by topical applications daily or every 5th day. ASDC J. Dent. Child. 51,107-115. Schneider, P. and Mühiemann, H.R. (1974) The antigiycoiytic action of amine fluorides on dental plaque. Helv.Odontol. Acta 18, 63-70. Schupbach, P., Guggenheim, B. and Lutz, F. (1989) Human root caries: histopathology of initial lesions in cementum and dentin. J. Oral Pathol. Med. 18,146-156. Schupbach, P., Guggenheim, B. and Lutz, F. (1990) Histopathology of root surface caries. J. Dent. Res. 69,1195-1204. 249

Schweigert, B.S., Shaw, J.H., Zepplin, M. and Elvehjem, C.A. (1946) Dental caries in the cotton rat. J. Nutrition 31, 439-447. Sefton, J., Lambert, M., Wilson, M. and Newman, H.N. (1996) Adsorption/desorption of amine fluorides to hydroxyapatite. Biomaterials 17, 37-46. Seppa, L., Salmenkivi, S. and Hausen, H. (1997) Salivary fluoride concentration in adults after different fluoride procedures. Acta Odontol. Scand. 55, 84-87. Sgan Cohen, H.D., Gat, E. and Schwartz, Z. (1996) The effectiveness of an amine fluoride/stannous fluoride dentifrice on the gingivai health of teenagers: results after six months. Int. Dent. J. 46, 340-345. Shani, S., Friedman, M. and Steinberg, D. (1996) Reiation between surface activity and antibacterial activity of amine fluorides. Int. J. Pharm. 131, 33-39. Shannon, I.L. (1969) Water-free solutions of stannous fluoride and their incorporation into a gel for topical application. Caries Res. 3, 339-347. Shannon, I.L. (1971) Antisolubility effects of acidulated phosphofluoride and stannous fluoride in the treatment of crown and root surfaces. Aust. Dent. J. 16, 240-242. Shannon, I.L., Buchanan, W.E., Jr. and Mahan, C.J. (1976) In vitro treatment of human root surfaces with fluorides. J. Public Health Dent. 36, 201-206. Shannon, I.L. (1980) Responses of enamel, dentin, and root surfaces to mouthrinse concentrations of sodium fluoride and stannous fluoride. ASDC J. Dent. Child. 47,17-20. Shapira, L., Schatzker, Y., Gedalia, I., Borinski, R. and Sela, M.N. (1997) Effect of amine and stannous fluoride on human neutrophil functions in vitro. J. Dent. Res. 76,1381-1386. Shaw, J.M., Smith, B.G.N. and Hutton, J.W.A. (1996) National audit of new patient referral to Consultants in Restorative Dentistry. 1, pp.2-3. HMSO Scotland. Shem, R., Swing, K.W. and Crawford, J.J. (1970) Prevention of plaque formation by organic fluorides. J. Oral Med. 25, 93-97. Shem, R.J., Rundell, B.B. and Defever, C.J. (1974) Effects of an amine fluoride mouthrinse on the formation and microbial content of plaque. Helv.Odontol. Acta 18, 57-62. Shu, M., Pearce, EIF., Sissons, CH., Coote, GE. And Miller, JH. (1998) Fluoride distribution in sound and carious root human teeth. Caries Res. 32,239-246. Siegel, S. and Castellan, N.J. (1988) Siegel, S. and Castallan, N.J. (Eds.) Nonparametric Statistics for the Behavioural Sciences, 2nd edn. New York: McGraw-Hill Book Company. Silvera, R.S., Gabathuler, H. and Mühlemann, H.R. (1974) Salivary fluoride levels after mouthrinsing with inorganic fluoride and amine fluoride. Helv. Odontol. Acta 18, Suppi 8: 79- 81. Silverstone, L.M., Wefel, J.S., Zimmerman, B.F., Clarkson, B.H. and Featherstone, M.J. (1981b) Remineralisation of natural and artifical lesions in human dental enamel in vitro. Caries Res. 15,138-157. Singer, L. and Armstrong, W.D. (1969) Total fluoride content of human saliva. Arch. Oral Biol. 14,1343-1347. Skjoriand, K., Gjermo, P. and Rglla, G. (1978) Effect of some polyvalent cations on plaque formation in vivo. Scand. J. Dent. Res. 86,103-107. 250

Slack, G .L, Bulman, J.S. and Osbom, J.F. (1971) Clinical testing of fluoride and non-fluoride containing dentifrices in Hounslow school children, fir. Dent. J. 130,154-158. Smith, B.G. and Knight, J.K. (1984) A comparison of pattems of tooth wear with aetiological factors, fir. D ent J. 157,16-19. Smith, B.G. (1989) Toothwean aetiology and diagnosis. Dent. Update 16, 204-212. Smith, B.G., Bartlett, D.W. and Robb, N.D. (1997) The prevalence, etiology and management of tooth wear in the United Kingdom. J. Prosthet. Dent. 78, 367-372. Soames, J.V. and Southam, J.C. (1998a) Disorders of development of teeth. In: Soames, J.V. and Southam, J.C. (Eds.) Oral Pathology, 3rd edn. pp. 13-16. Oxford: Oxford University Press. Soames, J.V. and Southam, J.C. (1998b) Other disorders of teeth. In: Soames, J.V. and Southam, J.C. (Eds.)Oral Pathology, 3rd edn. pp. 44-46. Oxford: Oxford University Press. Sodetling, E., Ketola, T. and Parviainen, T. (1991) Interference of oral hygiene products with an adhesion-based assay of salivary mutans streptococci.Scand. J. Dent. Res. 99,113-116. SOremark, R. and Samsahl, K. (1961) Gamma ray spectrometric analysis of elements in normal human enamel. Arch. Oral Biol. 6, 275-283. Stamm, J.W., Banting, D.W. and Imrey, P.B. (1990) Adult root caries survey of two similar communities with contrasting natural water fluoride levels. J. Am. Dent. Assoc. 120, 143- 149. Stamm, J.W. (1993) The value of dentifrices and mouthrinses in caries prevention. Int. Dent. J. 43, 517-527. Stamm, J.W. (1995) Clinical studies of neutral sodium fluoride and sodium monofluorophosphate dentifrices. In: Bowen, W.H. (Ed.) Relative efficacy of sodium fluoride and sodium monofluorophosphate as anti-caries agents in dentifrices, pp. 43-55. London: Royal Society of Medicine Press Ltd. Steams, R.l. (1972) Potential errors in analysing enamel for fluoride concentration and rates of acid dissolution subsequent to stannous fluoride treatments. J. Dent. Res. 51, 747-755. Steele, J.G., Walls, A.W.G., Ayatollahi, S.M.T. and Murray, J.J. (1996) Major clinical findings from a dental survey of elderly people in three different English communities, fir. Dent. J. 180,17-23. Stephen, K.W., Creanor, S.L., Russell, J.I., Burchell, C.K., Huntingdon, E. and Downie, C.F.A. (1988) A 3-year oral health dose response study of sodium monofiuorophosphate dentifrices with and without zinc citrate; anti-caries results. Community Dent. Oral Epidemiol. 16, 321- 325. Stevens, F.D., Shannon, I.L. and Williams, H.J. (1976) Effects on fluoride-containing commercial dentifrices on solubility of intact root surfaces. Dent. Hyg. Chic. 50,160-163. Stookey, G.K. (1990) Critical evaluation of the composition and use of topical fluorides. J. Dent. Res. 69, 805-812. Stookey, G.K., Katz, B.P., Beiswanger, B.B. and Dunipace, A.J. (1992) Sample size considerations in designing studies with intra-oral models. J. Dent. Res. 71 (Spec No), 819- 821. 251

Stratemann, M.W. and Shannon, I.L. (1974) Control of décalcification in orthodontic patients by daily self-administered application of a water-free 0.4 per cent stannous fluoride gel. Am. J. Orthod. 66, 273-279. Strubig, W. (1980) Die wirksamkeit lokaler fiuoridiemngsmittel. Dtsch. Zahnârztl. Z. 35, 1067- 1069. Strubig, W. and Güizow, H.J. (1986) Untersuchungen zur lokalen wirksamkeit von gelees mit unterschiedlichem fiuoridgehalt und unterschiedlichem pH-wert. Dtsch. Zahnârztl. Z. 41, 832-835. Sutcliffe, P. (1983) Oral cleanliness and dental caries. In: Murray, J.J. (Ed.) The Prevention of Dental Disease, pp. 159-174. Oxford: Oxford University Press.

Svanberg, M. and R 0 lla, G. (1982) Streptococcus mutans In plaque and saliva after mouthrinsing with SnF:. Scand. J. Dent. Res. 90,292-298. Svatun, B., Gjermo, P., Eriksen, H.M. and Rglla, G. (1977) A comparison of the plaque- inhibiting effect of stannous fluoride and chlorhexidine. Acta Odont. Scand. 35, 247-250. Svatun, 8. and Attramadal, A. (1978) The effect of stannous fluoride on human plaque acidogenicity in situ (Stephan curve). Acta Odontol. Scand. 36, 211-218. Svatun, B. (1978) Plaque-inhibiting effect of dentifrices containing stannous fluoride. Acta Odontol. Scand. 36, 205-210. Swing, W.K. and Crawford, J.J. (1971) Inhibition of plaque-forming streptococci and diptheroids by organic compounds. J. Dent. Res. 5 0 ,104(Abstract) Takagi, S., Chow, L.C., Shih, S. and Sieck, B.A. (1997) Effect of a two solution fluoride mouthrinse on deposition of loosely bound fluoride on sound root tissue and remineralisation of root lesions in vitro. Caries Res. 31, 206-211. Talib, J.M.K., Davies, E.H. and Williams, B. (1993) Fluoride uptake by sound and demineralised enamel from three topical agents. J. Dent. Res. 72, 723(Abstract) ten Cate, J.M. and Arends, J. (1977) Remineralisation of artifical enamel lesions in vitro. Caries Res 11, 277-286. ten Cate, J.M., Exterkate, R.A.M. and Rempt, H E. (1988) Intra-oral retention of fluoride by bovine enamel from amine fluoride toothpaste and 0.4% amine fluoride liquid application. J. Dent. Res. 67, 491-495. Theilade, E., Theilade, J. and Mikkelsen, L. (1982) Microbiological studies on early dento- gingival plaque on teeth and Mylar strips in humans. J. Periodont. Res. 17,12-25. Theilade, E., Budtz-Jgrgensen, E. and Theilade, J. (1983) Predominant cultivable microflora of plaque on removable dentures in patients with healthy oral mucosa. Arch. Oral. Biol. 28, 675-680. Thylstrup, A. and Fejerskov, O. (1994) Clinical and pathological features of dental caries. In: Thylstrup, A. and Fejerskov, O. (Eds.) Textbook of Clinical Cariology, 2nd edn. pp. 145-151. Copenhagen: Munksgaard Tinanoff, N., Brady, J.M. and Gross, A. (1976) The effect of NaF and SnFz mouthrinses on bacterial colonisation of tooth enamel: TEM and SEM studies. Caries Res. 10,415-426. 252

Tinanoff, N., Hock, J., Camosci, D. and Hellden, L (1980) Effect of stannous fluoride mouthrinse on dental plaque formation. J. Clin. Periodontol. 7,232-241. Tinanoff, N. and Zameck, R. (1985) Alteration In salivary and plaque S.mutans In adults

brushing with 0.4% Snp2 gel once or twice a day. Pediatr. Dent 7,180-184. Tinanoff, N., Manwell, M.A., Zameck, R .L and Grasso, J.E. (1989) Clinical and microbiological

effects of dally brushing with either NaF or Snp 2 gels In subjects with fixed or removable dental prostheses. J. Clin. Periodontol. 16,284-290. Tinanoff, N. (1995) Progress regarding the use of stannous fluoride In clinical dentistry. J. Clin. Dent. 6 (Spec No), 37-40. Todd, J.E. and Lader, D. (1991) Adult Dental Health Survey of the United Kingdom, 1988. pp.113-121. London: HMSO. Toolson, L.B., Smith, D.E. and Phillips, 0. (1982a) A 2-year longitudinal study of overdenture patients. Part II: Assessment of the periodontal health of overdenture abutments. J. Prosthet. Dent. 47,4-11. Toolson, L.B. and Smith, D.E. (1978) A 2-year longitudinal study of overdenture patients. Part I: Incidence and control of caries on overdenture abutments. J. Prosthet. Dent. 40,486-491. Toolson, L.B. and Smith, D.E. (1983) A five year longitudinal study of patients treated with overdentures. J. Prosthet. Dent. 49, 749-756. Toolson, L.B. and Taylor, T.D. (1989) A 10-year report of a longitudinal recall of overdenture patients. J. Prosthet. Dent. 62,179-181. Torrell, P. and Ericsson, Y. (1965) Two-year clinical tests with different methods of local caries- preventive fluorine application In Swedish school-children. Acta Odontol. Scand. 23, 287- 322. Triol, C.W., Kranz, S.M., Voipe, A.R., PrankI, S.N., Alman, J.E. and Allard, R.L. (1980) Anticaries effect of a sodium fluoride rinse and an MPP dentifrice in a non-fluoridated water area: a thirty month study. J. Clin. Prev. Dent. 2,13-15, Triol, C.W., Graves, R.C., Webster, D.B. and Clark, B.J. (1987) Anticaries effect of 1450 and 2000ppm P dentifrices. J. Dent. Res. 66, 216 Tseng, C.C., Wolff, L.P. and Aeppll, D.M. (1992) Effect of gels containing stannous fluoride on oral bacteria -an In vitro study. Aust. Dent. J. 37, 368-373. Turesky, S.S., Glickman, 1. and Sandberg, R. (1972) In vitro chemical Inhibition of plaque formation. J. Periodontol. 43, 263-269. Tumer, K.A. and MIsslrilan, D.M. (1984) Restoration of the extremely wom dentition. J. Prosthet. Dent. 54,467-474.

Tvelt, A.B., Hals, E., Isrenn, R. and Tfitdal, B. (1983) Highly add Snp 2 and TIP4 solutions. Effect on and chemical reaction with root dentin in vitro. Caries Res. 17, 412-418. Tveit, A.B., Tôtdal, B., Klinge, B., Nilveus, R. and Selvig, K.A. (1985) Fluoride uptake by dentin surfaces following topical application of TiP^, NaP and fluoride vamlshes In vivo. Caries Res. 19, 240-247. Tvelt, A.B. and TOtdai, B. (1981) Fluoride uptake by cavity walls from a fluoride containing amalgam In vitro. An electron microprobe analysis. Acta. Odont. Scand. 39,107-113. 253

Tyler, J.E. and Comer, J.E. (1985) Novel ion-selective electrode system for the simultaneous analysis of fluoride and calcium in acid solution.Analyst MO, 15-18. Tyler, J.E. and Poole, D.F.G. (1989) The rapid measurement of fluoride concentrations in stored human saliva by means of a differential electrode cell. Arch. Oral Biol. 34, 995-998. Ueberschar, M. and Gunay, H. (1991) Wurzelkaries-lnzidenz unter AmF/SnFrmundspulung. Dtsch. Zahnârztl. Z. 46, 566-568. van der Merwe, E.H., Retief, D.H., Barbakow, F.H. and Friedman, M. (1974) An evaluation of an in vivo enamel acid etch biopsy technique for fluoride determination. J. Dent. Assoc. S. Aft. 29, 81-87. van Huysen, G. and Muhler, J.C. (1948) Enamel solubility reducing effect of flavored low concentration stannous fluoride solution. J.Dent. Res. 27,46-51. Venkateswarlu, P. (1974) Reverse extraction technique for the determination of fluoride In biological materials. Anal. Chem. 46, 878-882. Venkateswarlu, P. (1977) Determination of fluoride in biological materials. In: Glick, D. (Ed.) Methods of Biochemical Analysis, pp. 93-201. New York: Wiley-lnterscience. Venkateswarlu, P. (1990) Evaluation of analytical methods for fluorine in biological and related materials. J. Dent. Res. 69 (Spec No), 514-521. Vierrou, A.M., Manwell, M.A., Zamek, R.L., Sachdeva, R.C. and Tinanoff, N. (1986) Control of Streptococcus mutans with topical fluoride in patients undergoing orthodontic treatment. J. Am. Dent. Assoc. 113, 644-646. Vogel, G .L, Chow, L.C. and Brown, W.E. (1983) A microanalytical procedure for the determination of calcium, phosphate and fluoride in enamel biopsy samples. Carles Res. 17,23-31. VoIpe, A.R., Petrone, M.E. and Davies, R.M. (1993) A critical review of the 10 piviotal caries clinical studies used in a recent meta-analysis comparing the anti-caries efficacy of sodium fluoride and sodium monoflurophosphate dentifrices. Am. J. Dent. 6, 813-842. Voipe, A.R., Petrone, M.E., Davies, R. and Proskin, H.M. (1995) Clinical anti-caries efficacy of NaF and 8MFP dentifrices: overview and resolution of the scientific controversy. J Clin Dent 6,1-28. Waas, MAJ., Kalk, W., van Zetten, B.L. van Os, J.M. (1996) Treatment results with immediate overdentures: An evaluation of 4.5 years. J Prosthet Dent. 76,153-157. Wade, W., Addy, M., Hughes, J., Milsom, 8. and Doherty, F. (1997) 8tudies on stannous fluoride toothpaste and gel (1). Antimicrobial properties and staining potential in vitro. J. Clin. Periodontol. 24, 81-85. Wallace, M.C., Retief, D.H. and Bradley, E.L (1993) The 48 month increment of root caries in an urban population of older adults participating in a preventive dental program. J. Public Health Dent. S 3,133-137. Walsh, J.P. and Green, R.W. (1950) The influence of some surface active substances on décalcification of the enamel surface. J. Dent. Res. 29, 270-277. 254

Warner, V.D., Warner, A.M., Mirth, D.B., Sane, J.N., Turesky, S.S. and Soloway, B. (1976) A physiochemlcal approach to the study of amines as antiplaque agents. J. Dent. Res. 55, 130-134. Weatherell, J.A. and Hargreaves, J.A. (1964) Determination of fluoride In layers of enamel from different surfaces of Individual teeth. J. Dent. Res. 43, 958(Abstract) Weatherell, J.A. and Hargreaves, J.A. (1965) The micro-sampling of enamel In thin layers by means of strong acids.Arch. Oral Biol. 10,139-142. Weatherell, J.A., Robinson, C., Strong, M. and NakagakI, H. (1985) Micro-sampling by abrasion. Caries Res. 19, 97-102. Weber, D.A., Howard Nordan, K., Buckner, B.A., Helslnger, S.A., Lueders, R.A., Court, L.K., Bollmer, B.W., Peiilch, M.A. and Sewak, L.K. (1995) Microbiological assessment of an Improved stannous fluoride dentifrice. J. Clin. Dent. 6 (Spec No), 97-104. Wei, S.H. and Forbes, W.C. (1972) Effect of particle size on reactions of powdered sound human enamel and dentine with stannous fluoride solution. Arch. Oral Biol. 17,1467-1472. Wei, S.H. (1973) Enamel microsampiing technique for assessing fluoride uptake from topical fluoride treatments In vitro. J. Dent. Res. 52,1268-1272. Wei, S.H. (1974) Scanning electron microscope study of stannous fluoride-treated enamel surfaces. J. Dent. Res. 53, 57-63. Wei, S.H. and Forbes, W.C. (1974) Electron microprobe investigations of stannous fluoride reactions with enamel surfaces. J. Dent. Res. 53, 51-56. Wei, S.H., Wefel, J.S., Henderson, W.G. and Tinanoff, N. (1974) Fluoride content of enamel using an acid etch method. J. Dent. Res. 5 3 ,287(Abstract) Wei, S.H. and Wefel, J.S. (1975) An Improved abrasive enamel microsampiing device. J. Dent. Res. 54, 201-205. Wei, S.H., Soboroff, D.M. and Wefel, J.S. (1976) Effects of titanium tetrafluoride on human enamel. J. Dent. Res. 55,426-431. Wei, S.H. and Ylu, C.K. (1993) Mouthrinses: recent clinical findings and implications for use. Int. Dent. J. 43, 541-547. Weidmann, S.M., Weatherell, J.A. and Hamm, S.M. (1967) Variations of enamel density In sections of human teeth. Arch. Oral Biol. 12, 85-97. Weiger, R., Netuschil, L , Wester-Ebbinghaus, T., Brecx, M. (1998) An approach to differentiate between antibacterial and antiadhesive effects of mouthrinses in vivo. Arch. Oral Biol. A2, 559-565. Wellock, W.D. and Brudevoid, F. (1963) A study of acidulated fluoride solutions-ll. The caries inhibiting effect of single annual topical applications of an acidic fluoride and phophate solution. A two year experience. Arch. Oral Biol. 8,179-182. Wharton, H.W. (1962) Isolation and determination of microgram amounts of fluoride In materials containing calcium and orthophosphate. Anal. Chem. 34,1296-1298. White, D.J., Cox, E.R. and Gwynn, A.V. (1995) Effect of a stabilized stannous fluoride dentifrice on plaque acid (toxin) production. J. Clin. Dent. 6 (Spec No), 84-88. 255

White, S T . and Taylor, P.P. (1979) The effect of stannous fluoride on plaque scores. J. Dent. Res. 58,1850-1852. Whitford, G.M. (1992) Acute and chronic fluoride toxicity. J. Dent. Res. 71,1249-1254. Williams, D.R. (1987) A rationale for the management of advanced tooth wear (ATW). J. Oral Rehabii. 14, 77-89. Williams, H.J., Shannon, I.L. and Stevens, F.D. (1974) The treatment of intact root surfaces with combinations of fluoride. J. Am. Soc. Prev. Dent. 4,40-44. Wilson, M.A. and Grant, A.A. (1989) A two-part resin-bonded partial overdenture. Restorative Dent. 5,12-13. Winkler, K.C., Backer Dirks, O. and van Amerongen, J. (1953) A reproducible method for caries evaluation. Test in a therapeutic experiment with a fluoridated dentifrice, fir. Dent. J. 95,119-124. Winstaniey, R.B. (1984) The treatment of severe attrition. Dent. Update 11, 629-642. Wolff, L.F., Pihistrom, B.L., Bakdash, M.B., Aeppii, D.M. and Bandt, C.L. (1989) Effect of toothbrushing with 0.4% stannous fluoride and 0.22% sodium fluoride gel on gingivitis for 18 months. J. Am. Dent. Assoc. 119, 283-289. Woitgens, J.H.M., Vinderiing, P.A. and Witjes, F. (1980) Chemical evidence of two seperate apatite phases in human enamel. Arch. Oral Biol. 25, 435-436. Woitgens, J.H.M., Bervoets, T.J.M., Witjes, F. and Driessens, F.C.M. (1981) Changes in the comosition of the enamel of human preomolar teeth shortly after eruption. Arch. Oral Biol. 26, 717-719. Woodley, N.J., Griffiths, B.M. and Hammings, K.W. (1996) Retrospective audit of patients with advanced toothwear restored with removable partial dentures. Eur. J. Prosthodont. Restor. Denf. 4,185-191. World Health Organisation (1994) Fluorides and oral health. 846, p.19 Geneva: WHO Technical Report Services. Yankeli, S.L., Laster, L.L., Green, P.A. and Shem, R.J. (1978a) Effects of stannous fluoride on salivary glycolysis.J. Dent. Res. 57, 337(Abstract)

Yankeli, S.L., Paskow, E.W. and Shem, R.J. (1978b) Effects of Snp 2 mouthrinses on plaque microbiology. J. Dent. Res. 57, 352(Abstract) Yankeli, S.L., Stoiler, N.H., Tawii, G., Green, P.A. and Shem, R.J. (1978c) Clinical effects of stannous fluoride mouthrinses on plaque accumulation and gingivitis. J. Dent. Res. 57, 353(Abstract) Yao, K. and Gren, P. (1970) Fluoride concentrations in duct saliva and in whole saliva. Caries Res A, 321-331. Yoon, N.A. and Berry, C.W. (1979) The antimicrobial effect of fluorides (acidulated phosphate, sodium and stannous) on Actinomyces viscosus.J. Dent. Res. 58,1824-1829. Zacheri, W.A. (1972) Clinical evaluation of neutral sodium fluoride, stannous fluoride, sodium monofluorophosphate and acid fluoride-phosphate dentifrice. J. Can. Dent. Assoc. 38, 35- 38. 256

Zacheri, W.A. (1981) A three year clinical caries evaluation of the effect of a sodium fluoride- silica abrasive dentifrice. Pharmacol ther dent S, 1-7. Zero, D.T., Raubertas, R.F., Pedersen, A.M., Hayes, A.L and Featherstone, J.D.B. (1992) Studies of fluoride retention by oral soft tissues after the appiication of home use topical fluorides. J. Dent. Res. 71,1546-1552. Zickert, I., Emilson, C.G., Ekblom, K. and Krasse, B. (1987) Prolonged oral reduction of Streptococcus mutans in humans after chlorhexidine disinfection followed by fluoride treatment. Scand. J. Dent. Res. 95, 315-319. Zimmermann, A., Flores de Jacoby, L and Pan, P. (1993) Gingivitis, plaque accumulation and plaque composition under long-term use of Meridol. J. Clin. Periodontol. 20, 346-351. Zober, A. and Schellmann, B. (1975) Determination of fluoride in bone samples from human iliac crest with an ion-specific electrode. Z. Klin. Chem. Klin. Blochem. 13,197-201. 257

APPENDIX 1.

THE PREVALENCE AND INCIDENCE OF CARIES DEVELOPING IN PATIENTS WEARING OVERDENTURES. 258

INDEX OF CONTENTS

Table 1. The incidence of caries developing in patients wearing partial overdentures restoring tooth wear ...... 259 Table 2. The prevalence and incidence of caries developing in patients wearing overdentures ...... 260 Table 3. Extractions due to caries in patients wearing overdentures 264 BIBLIOGRAPY...... 267 INVESTIGATION LENGTH OF NUMBER NUMBER TREATMENT NUMBER OF PERCENTAGE DENTURE OF OF CARIOUS OF CARIOUS SERVICE PATIENTS ABUTMENTS LESIONS ABUTMENTS

Hussey and 1 to 6 years 40 165 Partial use of fluoride 22% Linden 33% < 1 year

1986 40% 1 to 3 years Supplemental fluoride (NR) - 15%

27% > 3 years F‘ toothpaste only - 23%

No fluoride - 40%

Table 1. The incidence of caries developing in patients wearing partial overdentures restoring tooth wear. INVESTIGATION LENGTH OF NUMBER NUMBER TREATMENT NUMBER OF PERCENTAGE DENTURE OF OF CARIOUS OF CARIOUS SERVICE PATIENTS ABUTMENTS LESIONS ABUTMENTS

Dolder1961 Mean 39 months 110 250 No Fluoride 10% of 2 year 18 months minimum restorations Ragnarson & 1 to 5 years 16 43 NR 16 37% Astrad 1963

Ralph & Murray 3 to 15 months 6 NR Topical fluoride after recontouring 2 Patients 33% of patients 1967 Ratenen et ai 7 to 4 9 months 31 52 No Fluoride 20 38.5% 1971 Fenton & Hahn 4 to 30 months 17 57 Partial use of fluoride 9 15.8% 1978 Mean 16 months 20 F gel (NR) in denture dally : 1 6.0% 37 No Fluoride 8-, : 21.6%

Table 2. The prevalence and incidence of caries developing in patients wearing overdentures. A. Cross sectional investigations. INVESTIGATION LENGTH OF NUMBER NUMBER TREATMENT NUMBER OF PERCENTAGE DENTURE OF OF CARIOUS OF CARIOUS SERVICEPATIENTS ABUTMENTS LESIONS ABUTMENTS

Reitz ef a l . 6 to 47 months 50 131 Partial use of fluoride 8 Patients 16.0% of patients 1977 Mean 19 months 2% NaF mouthrinse daily 2 Patients 22.2% of patients No fluoride 6 Patients 14.6% of patients

Reitz ef a/.. 38 to 80 months 35 95 NR 3 Patients 8 ,6 % of patients 1980 Mean 60 months

Toolson and Baseline 89 233 No Fluoride 6 8 29.2% Smith 2 to 8 months

1978 At 1 year 79 2 1 0 No Fluoride 73 34.8% At 2 years 74 190 Partial use of F and AgNOs 36 19.0%

Toolson + Smith At 5 years 16 36 1% NaF gel in denture daily 1 2 .8 %

1983 38 94 No fluoride 2 0 21.3%

Toolson + Taylor At 10 years 28 6 6 1% NaF gel in denture daily 0 0 %

1989 No fluoride 8 1 2 .1 %

Table 2. The prevalence and incidence of caries developing in patients wearing overdentures. B. Longitudinal Investigations. INVESTIGATION LENGTH OF NUMBER NUMBER TREATMENT NUMBER OF PERCENTAGE DENTURE OF OF CARIOUS OF CARIOUS SERVICE PATIENTS ABUTMENTS LESIONS ABUTMENTS

Davis et al. 2 years 1 1 2 0 No fluoride 4 2 0 .0 % 1981

Renner et ai 2 to 4 years 8 14 1 % NaF gel in denture ttirice weekly 5 35.7% 1984 Ettinger et al. Up to 5 years 44 135 0.5% AFP gel in denture daily 13.6% mean annual incidence 1984 Range 2.7% (yearl) to 20.6% (year 3)

Ettinger & Up to 12 years 254 679 0-5% APF gel in denture daily 6 .5 % mean annual incidence Jakobsen Range 1.2% (yearl) 1990 to 11.1% (year 6 )

Lauciello & 2 to 6 years 25 70 0.4% SnF gel in denture daily 2 1 .2 % mean annual incidence Ciancio Range 8 .6% (yearl) 1985 to 40% (year 6) Waas et ai 4.5 years 56 78 Not recorded 14 18% 1996

Table 2. The prevalence and incidence of caries developing in patients vA^earing overdentures. C. Longitudinal Investigations - continued. INVESTIGATION LENGTH OF NUMBER NUMBER TREATMENT NUMBER OF PERCENTAGE DENTURE OF OF CARIOUS OF CARIOUS SERVICE PATIENTS ABUTMENTS LESIONS ABUTMENTS Keltjens et al. 18 months 30

1990 1 0 Control - 34%

1 1 0.1% NaF gel in denture daily - 36%

9 0.1% NaF and 5% chlorhexidine gel - 0 % in denture daiiy Budtz-Jorgresen 3 years 35 80 15.5% mean annual incidence 1991 Range 14.6% to 16.5%

15 36 Day wear only 0 %

2 0 44 Day and night wear 29.0% mean annual incidence Range 27.5% to 30.2%

Budtz-Jorgrensen 5 years 31 72 Professional topical F 6 monthly 43 Year 1 14% 1994,1995 Year 2 15% Year 3 15% Year 5 20% 38 Day wear only 3 34 Day and night wear 40

Yamaga and 8 years 31 80 Denture resin

Nokubi incorperating tannin-fluoride 6 1 0 1997 Conventional resin 3 15 Table 2. The prevalence and incidence of caries developing in patients wearing overdentures. C. Clinical Trials. STUDY LENGTH OF NUMBER NUMBER OF TREATMENT NO. OF PERCENTAGE OF DENTURE OF ABUTMENTS ABUTMENTS ABUTMENTS SERVICE PATIENTS EXTRACTED

Franz 6 to 42 months 1 1 2 268 Stannous fluoride after recontouring 3 1 .1 % 1975 Dentures Ratenen et al. 7 to 49 months 31 52 No fluoride 5 9.6% 1971

Reitz et al. 6 to 47 months 50 131 Partial use of 1 0 .8 % 1977 Mean 19 months 2% NaF mouthrinse daily

Toolson and At 5 years 54 133 Partial use of 1 0 7.5% Smith 1983 1% NaF gel in denture daily

Toolson and At 10 years 28 6 6 Partial use of 7 1 0 .6 % Taylor 1989 1% NaF gel in denture daily

Renner ef a/. 2 to 4 years 8 14 1% NaF gel in denture thrice weekly 2 14.3% 1984

Ettinger 1988 0 to 1 2 years 254 679 0.5% APF gel in denture daily 14 2 .1 %

Lauciello and 0 to 6 years 25 70 0.4% SnF gel in denture daily 2 2.9% Ciancio 1985

Waas et al. 4.5 years 56 78 Not recorded 1 2 15% 1996

Table 3. Extractions due to caries in patients wearing overdentures. 265

BIBLIOGRAPHY

Budtz Jôrgensen, E. (1991) Effect of controlled oral hygiene In overdenture wearers: a 3-year study. Int. J. Prosthodont 4,226-231. Budtz-Jôrgensen, E. (1994) Effects of denture-wearing habits on periodontal health of abutment teeth in patients with overdentures. J. Clin. Periodontol. 21,265-269. Budtz-Jbrgensen, E. (1995) Prognosis of overdenture abutments in elderiy patients with controlled oral hygiene, a 5 year study. J. Oral Rehabii. 22, 3-8. Davis, R.K., Renner, R.P., Antes, E.W., Jr., Schlissel, E.R. and Baer, P.M. (1981) A two-year longitudinal study of the periodontal health status of overdenture patients. J. Prosthet. Derrt. 45, 358-363. □older, E.J. (1961) The bar joint mandibular denture. J. Prosthet. Dent. 11,689-707. Ettinger, R.L., Tayior, T.D. and Scandrett, F.R. (1984) Treatment needs of overdenture patients in a longitudinal study: five-year results. J. Prosthet. Dent. 52, 532-537. Ettinger, R.L. (1988) Tooth loss in an overdenture population. J. Prosthet. Dent. 60, 459-462. Ettinger, R.L. and Jakobsen, J. (1990) Caries: a problem in an overdenture population. Community. Dent. Oral Epidemiol. 18, 42-45. Fenton, A.H. and Hahn, N. (1978) Tissue response to overdenture therapy. J. Prosthet. Dent. 40, 492-498. Franz, W.R. (1975) The use of natural teeth in overlay dentures. J. Prosthet. Dent. 34,135-140. Hussey, D.L. and Linden, G.J. (1986) The efficacy of overdentures in clinical practice. Br. Dent. J. 161,104-107. Keltjens, H.M., Schaeken, M.J., Van der Hoeven, J.S. and Hendriks, J.C. (1990) Caries control in overdenture patients: 18-month evaluation on fluoride and chlorhexidine therapies. Caries Res. 24, 371-375. Lauciello, F.R. and Ciancio, S.G. (1985) Overdenture therapy: a longitudinal report. Int. J. Periodont. Restorative Dent. 5,62-71. Ragnarson, N. and Astrad, P. (1963) Nâgra fall av protesforanking vkJ smâ restbett. En efterkontdl. Svensk Tandl. Tidskr. 56, 335-345. Ralph, J.P. and Murray, F.D. (1967) The use of root abutments in the support of complete dentures. J. Oral Rehabii. 3,293-297. Ratanen, T., Makila, E., Antti, Y. and Hannu, S. (1971) Investigations of the therapeutic success with dentures retained by precision attatchments. I. Root anchored compiete overlay dentures. Suom. Hammasiââk. Toim. 67, 356-366. Reitz, P.V., Weiner, M.G. and Levin, B. (1977) An overdenture survey: preiiminary report. J. Prosthet. Dent. 37, 246-258. Reitz, P.V., Weiner, M.G. and Levin, B. (1980) An overdenture survey: second report. J. Prosthet. Dent. 43, 457-462. Renner, R.P., Gomes, B.C., Shakun, M.L., Baer, P.M., Davis, R.K. and Camp, P. (1984) Four- year longitudinal study of the periodontal health status of overdenture patients. J. Prosthet. Dent. 51, 593-598. 266

Toolson, L.B. and Smith, D.E. (1978) A 2-year longitudinal study of overdenture patients. Part I: Incidence and control of caries on overdenture abutments. J. Prosthet. Dent. 40,486-491. Toolson, L.B. and Smith, D.E. (1983) A five year longitudinal study of patients treated with overdentures. J. Prosthet. Dent. 49, 749-756. Toolson, L.B. and Taylor, T.D. (1989) A 10-year report of a longitudinal recall of overdenture patients. J. Prosthet. Dent. 62,179-181. Waas, MAJ., Kalk, W., van Zetten,B.L van Os, J.H. (1996) Treatment results with immediate overdentures: An evaluation of 4.5 years. J Prosthet Dent. 76,153-157. Yamaga, T. and Nokubi,T. (1997) Clinical observations of noncoping abutments protected by tannin-fluoride preparation. J Prosf/ief Denf. 78, 315-319. 267

APPENDIX 2.

FLUORIDE UPTAKE BY DENTAL MINERALISED TISSUES FOLLOWING APPLICATION OF AMINE FLUORIDE, STANNOUS FLUORIDE AND MERIDOL REGIMENS.

Data given as mean and standard deviation unless otherwise stated. Biopsy techniques described in Chapter 1, Section 4. 268

INDEX OF CONTENTS

Table 1. Fluoride Uptake by Enamel In Vitro Following Application of Amine Fluoride

Regimens ...... 269

Table 2. Comparative Investigations of Fluoride Uptake by Enamel In Vitro Following

Application of Amine Fluoride Regimens ...... 271

Table 3. Fluoride Uptake by Enamel In Vivo Following Application of Amine Fluoride

Regimens ...... 275

Table 4. Comparative Investigations of Fluoride Uptake by Enamel In Vivo Following

Application of Amine Fluoride Regimens ...... 276

Table 5. Fluoride Uptake by Dentine In Vivo Following Application of Amine Fluoride

Regimens ...... 278

Table 6 . Comparative Investigations of Fluoride Uptake by Enamel In Vitro Following

Application of Stannous Fluoride Regimens ...... 279

Table 7. Comparative Investigations of Fluoride Uptake by Enamel In Vivo Following

Application of Stannous Fluoride Regimens ...... 284

Table 8 . Comparative Investigations of Fluoride Uptake by Dentine In Vitro Following

Application of Stannous Fluoride Regimens ...... 286

Table 9. Comparative Investigations of Fluoride Uptake by Enamel In Vitro Following

Application of Amine Fluoride/Stannous Fluoride Regimens ...... 288

BIBLIOGRAPHY ...... 289 INVESTIGATIONMINERALISED BIOPSY FLUORIDE REGIMEN FLUORIDE CONTENT TISSUE TECHNIQUE (ppmF) Rinderer et ai. Human premolar Etch SOLUTION 1965 enamel AmF297/AmF335 {^.25%F') or APF (1.23%F‘) 1385 to 2800 Applied for 3 min. washed 10 min. In tap water

Antila and Pohto Human enamel Etch SOLUTION followed by wash In CaP0 4 : (read from figure) 1973 AmF 297 (0.9% F) for 0 min for 24h 450 At: 59.1 (1.3) pm

“ for 1 min 1900 45.3 (2.8) “ for 5 min « 3800 36.4 (2.7)

“ for 10 min " 7020 (580) 21.9(1.0) for 15 min " 6800 22.9 (1.4) for 0 min for 24 h 450 (42) (Imln etch) “ for 10 min for 15 min 35600(5200)

“ for 10 min for 24 h 7600 (1200)

At 50pm At 75pm At 100pm " for 0 min 440 330 280

« for 10 min 1450 420 2 2 0 AmF 297 (0.9% F ) for 10 min « then coated in Dental varnish 1380 560 270 Elmex Varnish 780 560 380

Table 1. Fluoride Uptake by Enamel In Vitro Following Application of Amine Fluoride Regimens. INVESTIGATION MINERALISED BIOPSY FLUORIDE REGIMEN FLUORIDE CONTENT TISSUE TECHNIQUE (ppmF') Strübig and Human molar Micro- etch GEL Applied for 3 min. Washed 1 min. in water (Read from figure) (Depth not known) Güizow enamel 0.25% (AmF 297 and AmF335) and 1% NaF pH 6.1 2800 1986 0.25% AmF 297and 1% NaF pH 6.1 2800 0.25% AmF 297and 0.15% NaF pH 5.2 4800

0.25% AmF pH 6.2 2 0 0

0.25% AmF pH 5.0 1 2 0 0 0.25% AmF pH 4.7 6600

0.15% AmF pH 6.0 1 0 0 0.15% AmF pH 5.0 500 0.15% AmF pH 4.5 7000

Fluoride Uptake by Enamel In Vitro following Application of Amine Fluoride Regimens - Continued. INVESTIGATION MINERALISED BIOPSY FLUORIDE REGIMEN FLUORIDE CONTENT TISSUE TECHNIQUE (ppmF) Mühlemann et al. Human premolar Etch SOLUTION followed by wash for 30 min. in tap water At; 1968 enamel Water 1313(230) 3.1 (0.6) pm NaF 100 ppmF for 3 min. 1344(188) 2.7 (0.4) AmF 297 100 ppmF' for 3 min. 1399(195) 2.8 (0.5) AmF 242 100 ppmF' for 3 min. 1854 (318) 2.4 (0.3) NaF 200 ppmF' for 6 min. 1300 (293) 2.8 (0.7) AmF 297 200 ppmF' for 6 min. 1376(175) 3.0 (0.5) AmF 242 200 ppmF' for 6 min. 2053 (320) 2.7 (0.4) Arends and Human incisor Etch COMPOUNDS Applied for 3 min. At layer depth of; Washed 1 min In human saliva 8 p.m 18 pm 39 pm 87 pm 120 pm ocnuinOT and premolar Untreated 1270 582 240 147 84 1975 enamel (106) (73) (34) (21) (9) APF Solution (1.2% NaF) 12243 3570 1680 1030 277 (1100) (200)(112) (112) (30) Elmex Fluid (12% AmF297 & 1% AmF355) 2800 2380 910 850 294 (180) (314) (36) (35) (12) NH4F Solution (2%) 8 8 8 8 2038 568 252 2 0 2 (890) (165) (49) (30) (25) Fluoroprotector Laquer (1% difluoro sllane) for 3 min. 3005 1900 910 525 235 (220) (112) (52) (60) (25) Fluoroprotector Laquer (1% difluoro sllane) for 24 h. 2905 1852 671 515 245 (315) (106) (105) (65) (29)

Table 2. Comparative Investigations of Fluoride Uptake by Enamel In Vitro Following Application of Amine Fluoride Regimens. INVESTIGATION MINERALISED BIOPSY FLUORIDE REGIMEN FLUORIDE CONTENT TISSUE TECHNIQUE (ppmF) Kirkegaard Human premolar Etch SOLUTION At layer depth of: 1977a enamel Applied for 5 min. washed 30s. in distilled water 3p.m 6pm 10pm 15pm

NaF (0.9%F1 2073 (299) 810 (108) 485 (67) 247 (37)

APF (1.2%F1 3541 (235) 1498 (115) 752 (112) 307 (62)

NaMFP (1.0%F1 471 (114) 193 (107) 127 (81) 82 (49)

SnFz (1.94%F1 810 (190) 286 (107) 213(66) 79 (67)

NazSnFe (0.6%F') 1150 (94) 603 (68) 272 (53) 112 (32)

AmF 242 (1.0%F) 3906 (569) 3601 (440) 3148 (490) 2364 (616) Kirkegaard Human premolar Etch TOOTHPASTE Applied for 64 min. At layer depth of: 1977b enamel washed in distilled water. 2 ^m 5 pm 10 pm NaF (0.2%) 389(67) 5(126) -48 (41) NaMFP (0.7%) Carbonate Abrasive 410(85) 96(95) 64 (23) NaMFP (0.7%) Silicon Abrasive 5(116) -16(36) 7(36) AmF 242 (1.3%) 606(388) 355(216) 46 (44) Strübig Human molar Micro- etch Applied for 3 min. Washed 1 min. in water At: 27.6 (6.9^m) (Read from figure) 1980 enamel NaF Gel(1.25%F) 1 SOOppm F‘ Elmex Fluid ( 1%F) 2200ppm F Fiuorosllicate Solution 1600ppm F'

mouth Dentine 5 pm 10 pm 20 pm 31 pm 42 pm Untreated 600 (50) 400 (50) 310(50) 300 (50) 250 (50) Sound and NaF Carious 2250(100) 1600(150) 1300(100) 1250(100) 950(100) artifical carious Sound 1950 (200) 1250(150) 850(100) 700(100) 600(100) 1400(150) 1250(100) 12 0 0 (100) lesions Elmex Carious 3100(300) 2450 (300) Sound 2100 (200) 1350(100) 850(100) 650 (50) 600(100) NaMFP Carious 1100(150) 1300(150) 1200(50) 1100(50) 900(80) Sound 700(50) 450(50) 420 (50) 300 (30) 250 (300 Klimek et at. Bovine incisor Etch SOLUTION 10 min. 2xdaily Sdays (Read from figure) At layer depth of: 1982 enamel in artificial SOUND ENAMEL 7 pm 14 pm 27 pm mouth Untreated 500 (100) 400(1000 300(100) NaMFP (200 ppmF") 600 (100) 500(100) 500(100) NaF (200 ppmF) 1680(200) 1100(100) 700(100)

AmF (NK) (200 ppmF) 1820(200) 11 0 0 (100) 800(100) PLAQUE COVERED ENAMEL Untreated 500 (100) 400(100) 350(100) NaMFP (200 ppmF) 1300 (100) 1350(200) 1000(100)

NaF (200 ppmF) 1200 (100) 1 0 0 0 (100) 800(100) AmF (unknown) (200 ppmF) 2200(200) 1600(100) 1 2 0 0 (100)

Comparative Investigations of Fluoride Uptake by EnamelIn Vitro Following Application of Amine Fluoride Regimens - Continued. INVESTIGATION MINERALISED BIOPSY FLUORIDE REGIMEN FLUORIDE CONTENT TISSUE TECHNIQUE (ppmF) Barbakow et ai. Human third Micro etch SOLUTION (250 ppmF^ Applied At ayer depth of: 1985 molar enamel for 2 min. then washed 10s in distilled water, 2x daily, 10days 2 to 3 ^im 5 toi 0 nm 20 to 30pm 40 to60 pm 72 to 85 pm Distilled Water 676 (302) 427 (191) 303 (164) 213 (97) AmF 297 1268 (266) 792 (196) 498 (98) 367 (69)

AmF 297 and SnF 2 (1:1) 1447 (1215) 698 (368) 390 (173) 263 (118) SnF2 1211 (745) 802 (531) 622 (429) 457 (313) NaF 1024 (357) 590 (192) 371 (126) 272 (100) Mok et al. Human pre-molar Micro- etch FLUID (2%F') Applied for 3 min. At 5 pm At 10 pm 1990 enamel Washed 5 min. then 24 h with saline. NaF 360 (309) 224 (253) Elmex (AmF 297/ AmF 335) 1305(778) 796(609) Chan et al. Human pre-molar Micro-etch FLUID (2%F') Applied for 3 min. At 5 pm At 10 pm 1991 enamel Washed 5 min. then 24 h with saline

Sound Enamel Sound and NaF 344(255) 209(149) artifical carious Elmex (AmF 297/ AmF 335) 622(288) 372(215) lesions Carious Enamel NaF 1096(601) 1016(469) Elmex (AmF 297/ AmF 335) 4068(1889) 8698(3821) Comparative Investigations of Fluoride Uptake by Enamel In Vitro Following Application of Amine Fluoride Regimens - Continued. INVESTIGATION MINERALISED BIOPSY FLUORIDE REGIMEN FLUORIDE CONTENT TISSUE TECHNIQUE (ppmF) Belser et al. Human enamel Micro etch TREATMENT 1975 from anterior Elmex fluid = AmF297/AmF335,1%F', for 3 min. at 8.4 (0.7) |im teeth Control, No etch 532 (168) to 763 (374) Control, Etched enamel surface 765 (374) to 954 (464) No etch, Elmex fluid 1142 (509) to 1267 (458) Etched with pyruvic acid then Elmex fluid 3444 (1269)

Etched with phosporic acid then Elmex fluid 2774 (688)

Mushanoffet al. Human enamel Micro etch At layer d( îpth of: 1981 from anterior 0.5 pm 1.0 pm 2.5 pm 5.0 pm teeth Untreated 3821 (1450) 2621 (1300) 846 (325) 128 (27) Elmex Toothpaste One application 7084 (2750) 3329 (1290) 1227 (420) 577 (205) (0.125% F I One week (2x daily) 6993 (2820) 3449 (1250) 1355 (580) 668 (225) ten Cate et al. Bovine enamel Micro-etch COMPOUND FLUORIDE CONTENT (pgcm ' ) In: - 1988 embedded in Single application of liquid for 4 min., rinsed 5 s, Sound Enamel De-mineralised Demineralised partial dentures. then paste used for 7 weeks. Enamel Enamel & Plaque Elmex Paste (0.1% F ) only 16.4 (11.6) Non F' Paste & Elmex Liquid (0.4% F") 9.5 (4.6) 20.6 (8.7) Elmex Paste & Elmex Liquid 9.1 (4.1) 34.0 (12.0) 39.5 (16.8) Elmex Paste (Carry over from group 3) 8.5 (3.8) 17.1 (11.0) 25.7 (15.3) Control (in vitro) 5.1 (2.9) 6.1 (3.2) 8.5 (2.2)

Table 3. Fluoride Uptake by Enamel In Vivo Following Application of Amine Fluoride Regimens. INVESTIGATION MINERALISED BIOPSY FLUORIDE REGIMEN FLUORIDE CONTENT TISSUE TECHNIQUE (ppmF) Candeli et al Human deciduous Section TOOTHPASTE Pre treatment 30 days of brushing 1967 molar enamel AmF 297 (0.101% F") 165 290 NaaPOaF 123 211 Placebo 105 103 Hotz Human premolar Micro-etch TREATMENT At 6.1 to 8.9nm 1972 enamel Applied daily for 5 days For; Untreated T reated AmF297/AmF335/NaF Gel 1.23% F 3 min 687 1179 10 min 817 2195 APFGel 1.23% F 3 min 666 1847 10 min 509 2545

Baijot-Stroobants Human incisor Proton COMPOUND Applied for 4 min. (Depth not known) and Vreven enamel Bombardment Ion Gel (APF, 1.23%F0 1774 (465) 1980 Rafluor Gel (APF, 1.23%F ) 3277 (683) Elmex Gelée (AmF, 1.02% F") 4460 (875) Fluocaril Gel (MFP, 2.0% F) 105 (32) Elmex Fluid (AmF 1 % F') 228 (50)

Table 4. Comparative Investigations of Fluoride Uptake by Enamel In Vivo Following Application of Amine Fluoride Regimens. INVESTIGATION MINERALISED BIOPSY FLUORIDE REGIMEN FLUORIDE CONTENT TISSUE TECHNIQUE (ppmF) Barba ko w et al. Human enamel Micro etch TOOTHPASTE 1983 from anterior 2x daily for 2 min. for 15 days teeth Elmex Paste (AmF297/AmF242, 0.125%FO 959(264) At depth 6.2(1.1) ^m Fluocaril Paste (NaF/NaMFP 0.096%F') 330(119) At depth 9.6(1.6) ^m Schmid et al. Pooled data form Etch TOOTHPASTE (details not known) 1984a 56 independent 2-3x daily for 23 days tests - rodent Water 59 molar enamel Fluoride free 52 Monofluorophosphate 135 Amine Fluoride 408 Schmid et al. Rodent molar Etch TOOTHPASTE (details not known) 1984b enamel Daily applications for 20 days Water 110 Anionic Placebo 100 Anionic MFP (3 types) 140 Cationic AmF 531 Applied once every day for 20 days Water 98 Anionic Placebo 110 Anionic MFP (3 types) 132 Cationic AmF 241 Comparative Investigations of Fluoride Uptake by Enamel In Vivo Following Application o Amine Fluoride Regimens - Continued. INVESTIGATION MINERALISED BIOPSY FLUORIDE REGIMEN FLUORIDE CONTENT TISSUE TECHNIQUE Hellwig Human dentine Micro abrasion ELMEX SOLUTION KOH-Soluble (ngcm'^) Structurally Bound(^gcm'^ 1992 embedded in For 3 min. At layer depth of; partial dentures (AmF297/AmF335, 1.23% F") 20p.m 40|im 60|o.m 48.7 (16.5) 1200(200) 1250(70) 1050(200)

Table 5. Fluoride Uptake by DentineIn Vivo Following Application of Amine Fluoride Regimens. INVESTIGATION MINERALISED BIOPSY FLUORIDE REGIMEN FLUORIDE CONTENT TISSUE TECHNIQUE (ppmFl Brudevold et al. Human molar Abrasion SOLUTION Shaken for 20 min. Layer 1 Layer 2 1956 enamel Distilled water 924 400 1% NaF 1440 561 1.86 %SnF2 1200 580 4.12% SnCIF 1031 570 Exposed as before then washed in saliva 1h., distilled water 30 min. then brushed. Distilled water 777 445 0.0214% NaF 966 508 0.04% SnF] 883 432

Slurry SnF2 dentifrice (0.04% SnF^ 850 467 Cooley 1961 Humal incisor Etch SOLUTION FLUORIDE CONTENT (^cm") Applied with continual brushing for: At layer depth of: enamel 0.01 cm 0.02 cm 0.03 cm 0.04 cm TOTAL (Separate investigation) NaF 0.2M 15 min. 9 30 min. 10 1 h 11 4 0 0 14 4h 26 6 0 0 22 16 h 40 39 8 0 82 SnFz 0.1 M 1 h 7 0 5 2h 6 4h 7 0 6 16 h 20

Table 6. Comparative Investigations of Fluoride Uptake by Enamel In Vitro Following Application of Stannous Fluoride Regimens. INVESTIGATION MINERALISED BIOPSY FLUORIDE REGIMEN FLUORIDE CONTENT TISSUE TECHNIQUE (ppmP") Mellberg et al. Human enamel Etch F SOLUTION SOAKING At layer depth of: 1966 from permanent Applied for 4 min. SOLUTION for 24h 6 to 11 |j.m 14 to 23 ^im 29 to 44 )j,m 59 to 88 |xm teeth APF Water 2322 (167) & 1638 (193) & 717 (80) & 318 (55) & 2405 (257) 1443 (209) 695 (125) 355 (68)

Saliva 2296 (362) & 1240 (280) & 813 (210) & 520 (150) & 2908 (253) 1506 (166) 1147(218) 715(153)

C a P 0 4 2064 (189) 1064 (191) 798 (279) 369 (109) Snp2 Water 1564 (137) 1027 (145) 507 (101) 255 (45)

C a P 0 4 1660 (219) & 2105 (278) & 1639 (156) & 438 (80) & 1404 (186) 874 (138) 460 (83) 240 (44) Brudevold et al. Human enamel Etch SOLUTION FLUORIDE CONTENT (^gcm’"') 1967 Applied for 1 min. then brushed 1 min. under running water

8 % 5n p 2 0.85(0.64)

8% Snp 2 and 8% NaH 2 P0 4 fresh 3.94(1.13)

8% SnP 2 and 8% NaH 2P0 4 aged 7.83 (3.26)

NaPIn H3P0 4 (2 % P ) 16.88 (7.11)

Comparative Investigations of Fluoride Uptake by Enamel In Vitro Following Application of Stannous Fluoride Regimens - Continued. INVESTIGATION MINERALISED BIOPSY FLUORIDE REGIMEN FLUORIDE CONTENT TISSUE TECHNIQUE (ppmF") Kirkegaard Human premolar Etch SOLUTION Applied for 5 min. /Ki layer depth of: 1977a enamel washed 30s. in distilled water 3 |xm 6 ^im 10 nm 15 pm NaF (0.9%F1 2073 (299) 810(108) 485 (67) 247 (37) APF (1.2%F") 3541 (235) 1498 (115) 752 (112) 307 (62) NaMFP (1.0%F-) 471 (114) 193 (107) 127 (81) 82 (49) SnFz (1.94%F-) 810(190) 286 (107) 213 (66) 79 (67) Na2SnF6(0.6%F-) 1150 (94) 603 (68) 272 (53) 112(32) AmF 242 (1.0%FT 3906 (569) 3601 (440) 3148 (490) 2364 (616) Dijkman etal. Human molar Etch TREATMENT followed by FLUORIDE CON TENT (jigcm'^) 1982 enamel 2x 30s rinse In tap water in 24,1 to 28.7>im For: KOH SOLUABLE ACQUIRED Untreated 2.1 (0.9) 6,4 (1.0) SnFz solution (4%) 5 min. 6.2 (4.1) 7 8(2.3 ) 30 min. 7.9 (3.3) 6 7 (1.6) APF gel (unknown) 5 min. 24.7(5.9) 10.1(2.4)

Comparative Investigations of Fluoride Uptake by Enamel In Vitro Following Application of Stannous Fluoride Regimens - Continued. INVESTIGATION MINERALISEDBIOPSY FLUORIDE REGIMEN FLUORIDE CONTENT TISSUETECHNIQUE Crall etal. 1982 Human premolar Micro etch SOLUTIONS followed by inorganic wash 13h. RESULTS Untreated. All treated enamel had significantly greater fluoride enamel content than untreated enamel. APF (1.23%F)for4 min. APF/SnF2 resulted in a lesser fluoride content than APF APF for 2 min. followed by SnF 2 (0.125%F') for 2 min. alone, difference significant to 15 ^im

Dicalcium phosphate dihydrate for 2 min. followed by TiCIVAPF and DCPD/BPF resulted in a greater fluoride basic phosphate fluoride (0.5%F ) for 2 min. content than APF alone, difference significant to 50 pm.

TiCU for 30s followed by APF for 3.5 min. (Details not given) Sakkab et al. Human deciduous Micro drill and TOOTHPASTE FLUORIDE CONTENT (pg/g) 1984 enamel, sound micro etch used for an average of 16 months White Spot Sound Enamel At layer depth of: and white spot At 100 pm 5.5 pm 17,4 pm 30.9 pm Placebo 731 (345) 690 (521) 364(205) 230(144) 4% SnFz- Calcium pyrophosphate 876(572) 717(494) 373(243) 214(147) 0.243 % NaF - Silica 1148(654) 670 (408) 379 (241) 294(406) Crall and Bjerga Human molar Micro etch SOLUTION FLUORIDE CONTENT (ppmF") 1984 enamel At layer depth of: 5 pm 10 pm 15 pm Water 1481 977 740 APF (1.23%F) 2861 1704 1400 APF (1.23%F) then SnF 2 (0.12%F) 2510 1687 1329 APF and SnFz (0.9%F) 2601 1714 1347 Omni APF (0.31%F) then Omni SnF 2 (0.40%F) 2506 1615 1293 Omni APF and Omni SnF2 (0.33%F ) 1744 1167 895 Comparative Investigations of Fluoride Uptake by Enamel In Vitro Following Application of Stannous Fluoride Regimens - Continued. INVESTIGATION MINERALISED BIOPSY FLUORIDE REGIMEN FLUORIDE CONTENT

TISSUE t e c h n iq u e Barbakow et al. Human third Micro etch SOLUTION (250 ppmF*) Applied for (ppmF') 1985 molar enamel 2 min. then washed 10s in At layer depth of: distilled water, 2x daily, lOdays 2 to 3 pm 5 toi 0 pm 20 to 30pm 40 to60 pm 72 to 85 pm Distilled Water 676 (302) 427(191) 303(164) 213(97) AmF 297 1268 (266) 792(196) 498 (98) 367(69) AmF 297 and SnFz (1:1) 1447(1215) 698 (368) 390(173) 263(118) SnFz 1211 (745) 802 (531) 622 (429) 457 (313) NaF 1024(357) 590(192) 371(126) 272(100) Faller et at, Human enamel Micro drill TOOTHPASTE SLURRY (^igcm'O 1995 (1:3, Tocrthpaste:Pooled Human Saliva) At depth of 100 + 10 pm 1100 ppmF' NaF Silica abrasive 28.3(2.2) 1100 ppmF' Stabilised SnFg Silica 22.4(2 5) abrasive 1000 ppmF' SnFz Calcium 16.3(1,8) pyrophosphate abrasive 250 ppmF' NaF 5.8(1 1) Placebo 1.9 (0.7)

Applied 4 X Imin. daily for 6 days. Each application separated by a minimum of 1 hour in pooled human saliva. Also exposed for 3 hours to a demineralisation solution daily

Comparative Investigations of Fluoride Uptake by Enamel In Vitro Following Application of Stannous Fluoride Regimens - Continued. INVESTIGATION MINERALISED BIOPSY FLUORIDE REGIMEN FLUORIDE CONTENT

TISSUE TECHNIQUE (ppmFO Brudevold et al. Human premolar Etch TREATMENT At layer depth of: 1967 enamel Teeth extracted 5/ 6 days after single application 10 pm 20 pm 30 pm Patient C FCFCF Control 800 1400 300 800 200 400 1.2% F , 0.1M H3PO4 pH 3.2 1300 2000 500 1000 300 400 1.2% F , 0.1M H3PO4 pH 5.2 700 1600 300 800 200 400 8% SnF2 pH 2.8 1000 1600 400 1000 300 500 in vitro 1.2% F , 0.1M H3PO4 pH 3.2 Applied 2.5 min., in Calcium phosphate 30 min. 2000 3400 700 1700 500 900 Heifetz etal. 1970 Human enamel Etch TREATMENT At layer depth of: 5 pm 15 pm 30 pm 60 pm

APF Solution (1.23%F) 1085(144) 456 ( 86 ) 218(55) 130 (32) Untreated 894(124) 410(76) 225(52) 127(29) SnFa Solution(2.42%F) 1276 (70) 596(54) 278 (25) 169(18) Untreated 1246 (95) 570 (53) 273(32) 155 (23) APF Prophylaxis Paste(1.2%F) 802 (89) 339 (28) 159(15) 91 (14) Untreated 712(110) 308 (33) 145(12) 97(13) Snp2 Prophylaxsis Paste(4.8%F ) 796(100) 363 (69) 181 (38) 106 (21) Untreated 800(95) 370(59) 198 (41) 100(19)

Table 7. Comparative Investigations of Fluoride Uptake by EnamelIn Vivo Following Application of Stannous Fluoride Regimens. INVESTIGATION MINERALISED BIOPSY FLUORIDE REGIMEN FLUORIDE CONTENT TISSUE TECHNIQUE Gilley and Human deciduous Micro drill and TOOTHPASTE used for 2 years RESULTS (No details given) Haberman enamel, sound micro etch Placebo No difference in fluoride content of sound enamel. 1981 and white spot (exfoliated 4% SnFz Significantly more fluoride in white spot enamel in users (Abstract) teeth) 0.243 % NaF of 0.243% NaF dentifrice.

Mobley Demineralised Micro drill TOOTHPASTE 2x daily for 3 weeks, overnight (^gcm'V At 120 nm 1981 human enamel soak in calcium phosphate solution

embedded in Baseline 1.3 partial derrtures Non F' - Silica abrasive 4.6 SnFa (0.40%) - Calcium pyrophosphate abrasive 8.4 NaF (0.234%) -Silica abrasive 16.0 Reintsema et al. Demineralised Micro drill TOOTHPASTE 2x daily for 2 weeks, (pigcm^) 1985 human incisal and left in overnight. At 120 p.m canine enamel MFP (1000 ppmF ) and NaF (450 ppmF ) (95% Cl) embedded in Dicalcium phosphate-dihydrate abrasive 8.9 (7.8-10.1) partial dentures NaF (1100 ppmF ) Silica abrasive 11.4 (10.0-12.9) MFP (1000 ppmF) Calcium carbonate abrasive 7.4 (6 .5 -8 5 ) Snp2 (1000 ppm F ) Calcium pyrophosphate abrasive 7.4 (6 .5 -8 4 ) MFP (1500 ppmF ) Alumina abrasive 7.9 (6.9-9.0) Non F' - Silica abrasive 4.3 (3.8-4.9)

Comparative Investigations of Fluoride Uptake by Enamel In Vivo Following Application of Stannous Fluoride Regimens - Continued. INVESTIGATION MINERALISED BIOPSY FLUORIDE REGIMEN FLUORIDE CONTENT TISSUETECHNIQUE (ppmF') Dowell and Addy Dentine powder Fluoride probe SOLUTION Mixed for 30 min. Dentine Hydroxyapatite 1984 and analysis Sodium Fluoride 100 ppmF 98 (0) 97 (0) hydroxyapatite 500 ppmF 485 (0.6) 352 (7) 1000 ppmF' 924 (1) 586 (3) 2000 ppmF' 1589(6) 932 (28)

5000 ppmF 2680 (0) 1610(99)

Stannous Fluoride 100 ppmF 98 (0) 98 (0) 500 ppmF 485 (1) 482 (2) 1000 ppmF 929 (7) 898 (1) 2000 ppmF' 1675 (5) 1535 (65)

5000 ppmF' 3722 (65) 3302 (35) Dentine Pre-treatment with water Pre-treatment with saliva Sodium Fluoride 1000 ppmF' 960 (72) 1120 (360) Stannous Fluoride 1000 ppmF 6246 (64) 6426 (179) Addy and Mostafa Human Molar Etch SOLUTION (1000 ppmFl FLUORIDE UPTAKE (ppmF") 1988 Dentine No Pre-treatment Pre-treatment with saliva Applied for 1 h, Unwashed Washed Unwashed Washed Sodium Fluoride 115(3) 37(6) 44 (31) 48 (18)

Stannous Fluoride 70 (48) 70 (37) 104 (50) 66 (23)

Table 8. Comparative investigations of Fluoride Uptake by DentineIn Vitro Following Application of Stannous Fluoride Regimens. INVESTIGATION MINERALISED BIOPSY FLUORIDE REGIMEN FLUORIDE CONTENT TISSUE TECHNIQUE Dérand et al. Human premolar Secondary ion SOLUTION RESULTS 1989 dentine mass Applied for 10 min., washed in distilled water NaF induced high concentrations of F and F spectrometry then in acidified gel for 20 days associated with Ca.

SnF2 induced higher concentrations of fluoride that 2% NaF penetrated the lesion and a depot formed in 3.6% SnFz cementum. 1.5% TiF4 Highest uptake after TiF#, over half the lesion depth. 2% NaF with 0.3% KCI, 0.27% FeCb 0.6% Total F concentration considerably lower with Fe-AI-F

AICI3 solution.

Comparative investigations of Fluoride Uptake by Dentine In Vitro Following Application of Stannous Fluoride Regimens - Continued. INVESTIGATION MINERALISED BIOPSY FLUORIDE REGIMEN FLUORIDE CONTENT TISSUE TECHNIQUE (ppmFl Barbakow et al. Human third Micro etch SOLUTION (250 ppmFl 1985 molar enamel Applied for 2 min. then washed 10s At layer depth 3f: in distilled water, 2x daily, lOdays 2 to 3 nm 5 tolO pm 20 to 30pm 40 to60 pm 72 to 85 pm Distilled Water 676 (302) 427(191) 303 (164) 213(97) AmF 297 1268 (266) 792(196) 498 (98) 367(69) AmF 297 and SnF 2 ( 1:1 ) 1447(1215) 698 (368) 390(173) 263(118) 8nF2 1211(745) 802(531) 622 (429) 457(313) NaF 1024 (357) 590(192) 371 (126) 272(100)

Table 9. Comparative Investigations of Fluoride Uptake by EnamelIn Vitro Following Application of Amine Fluoride/Stannous Fluoride Regimens 289

BIBLIOGRAPHY

Addy, M. and Mostafa, P. (1988) Dentine hypersensitivity. I. Effects produced by the uptake in vitro of metal ions, fluoride and fomnaidehyde onto dentine. J. Oral Rehabil. 15, 575-585. Antiia, M.K. and Pohto, P. (1973) Fluoride uptake and retention in dental enamel treated with amine fluoride solution. Proc. Finn. Dent. Soc. 69,202-207. Arends, J. and Schuthof, J. (1975) Fluoride content in human enamel after fluoride application and washing - an in vitro study. Caries Res. 9,363-372. Baijot-Stroobants, J. and Vreven, J. (1980) in vivo uptake of topically applied fluoride by human dental enamel. Arcti. Oral Biol. 25,617-621. Barbakow, F., Comec, S., Rozencweig, D. and Vadot, J. (1983) Enamel fluoride content after using amine fluoride- or monofluorophosphate-sodium fluoride-dentifrices. J. Dent. Child. 50, 186-191. Barbakow, F., Lutz, F. and Sener, B. (1985) In vitro dissolution of human enamel after application of a mixture of stannous fluoride and amine fluoride 297: a pilot study. J. Dent Child. 52, 444-448. Beiser, U., Sporri, S. and Muhiemann, H R. (1975) Uptake and retention of fluoride by intact and etched enamel. Helv. Odontol. Acta 19,69-71. Brudevold, F., Steadman, L.T., Gardner, D.E., Rowley, J. and Little, M.F. (1956b) Uptake of tin and fluoride by intact enamel. J. Am. Dent. Assoc. 53,159-164. Brudevold, F., McCann, H.G., Nilsson, R., Richardson, B. and Cokiica, V. (1967) The chemistry of caries inhibition. Problems and challenges in topical treatment. J. Dent. Res. 46, 37-45. Candeli, A., Capozzi, L, Marci, F. and Marchini, G. (1967) The determination of fluoride within the teeth by means of biopsy on the enamel. Caries Res. 1,153-161. Chan, J.C., Hill, F.J. and Newman, H.N. (1991) Uptake of fluoride by sound and artificially carious enamel in vitro following application of topical sodium and amine fluorides. J. Dent. 19,110-115. Ciiiey, W.A. and Haberman, J.P. (1981) Fluoride in enamel and correlation to caries. J. Dent. Res. 60, 577 (Abstract No. 1069). Cooley, W.E. (1961) Reactions of tin (11) and fluoride ions with etched enamel. J. Dent. Res. 40, 1199-1210. Crall, J.J., Siiverstone, L.M., Clarkson, B.H., Wefei, J.S. and Wei, S.H.Y. (1982) Fluoride uptake and in vitro caries like lesion formation in enamel after two step topical fluoride applications. Caries Res. 16,162-169. Crall, J.J. and Bjerga, J.M. (1984) Fluoride uptake and retention following combined applications of APF and stannous fluoride in vitro. Pediatr. Dent. 6,226-229. Dérand, T., Lodding, A. and Petersson, L.G. (1989) Effect of topical F solutions on caries like lesions in root surfaces. Caries Res. 23,135-140. 290

Dijkman, A.G., Tak, J., Smith, M.L, Jongebloed, W .L and Arends, J. (1982b) Fluoride deposited by topical applications with stannous fluoride in human enamel. J. Biol. Buccale 10, 63-71. Dowell, P. and Addy, M. (1984) Dentine hypersensitivity. A quantitative comparison of the uptake of metal salts and fluoride by dentine and hydroxyapatite. J. Periodont Res. 19, 530- 539. Faller, R.V., Best, J.M., Featherstone, J.D. and Barrett Vespone, N.A. (1995) Anticaries efficacy of an improved stannous fluoride toothpaste. J. Clin. Dent. 6 (Spec No), 89-96. Heifetz, S B., Mellberg, J.R., Winter, S.J. and Doyle, J. (1970) In vivo fluoride uptake by enamel of teeth of human adults from various topical fluoride procedures. Arch. Oral Biol. 15,1171- 1181. Hellwig, E. (1992) Fluoride retention in dentin after topical application of aminefluoride. J. Dent. Res. 71,1558-1560. Hotz, P. (1972) Fluoride levels in surface enamel after application of fluoride gels. Helv. Odont. Acta 16, 32-34. Kirkegaard, E. (1977a) In vitro fluoride uptake in human dental enamel from various fluoride solutions.Caries Res. 11,16-23. Kirkegaard, E. (1977b) In vitro fluoride uptake in human dental enamel from four different dentifrices. Caries Res. 11, 24-29. Klimek, J. (1981) Fluoridaufnahme kunstlicher karioser initiallasionen nach behandlung mit verschiedenen fluoridverbindungen. Dtsch. Zahnârztl. Z. 36, 520-524. Klimek, J., Hellwig, E. and Ahrens, G. (1982) Fluoride taken up by plaque, by the underlying enamel and by clean enamel from three fluoride compounds in vitro. Caries Res. 16, 156- 161. Mellberg, J R., Laakso, P.V. and Nicholson, C.R. (1966) The acquisition and loss of fluoride by topically fluoridated human tooth enamel. Arch. Oral Biol. 11,1213-1220. Mobley, M.J. (1981) Fluoride uptake from in situ brushing with stannous fluoride and sodium fluoride dentifrices. J. Dent. Res. 60,1943-1948. Mok, Y., Hill, F.J. and Newman, H.N. (1990) Enamel fluoride uptake affected by site of application: comparing sodium and amine fluorides. Caries Res. 24,11-17. Muhiemann, H., Schait, A. and Konig, K.G. (1968) Fluorine uptake of enamel and animal caries Inhibition by topical sodium fluoride and amine fluorides at low fluoride concentrations. Helv. Odont. Acta 12, 61-66. Mushanoff, O., Gedalia, I. and Daphni, L. (1981) Fluoride acquisition by surface enamel of human teeth in vivo following toothbrushing with an amine-fluoride toothpaste. J. Dent. 9, 144-149. Reintsema, H., Schuthof, J. and Arends, J. (1985) An in vivo investigation of the fluoride uptake in partially demineralised human enamel from several different dentifrices. J. Dent. Res. 64, 19-23. Rinderer, L, Schait, A. and Muhiemann, H.R. (1965) Loss of fluoride from dental enamel after topical fluoridation (preliminary report). Helv. Odontol. Acta 9,148-150. 291

Sakkab, N.Y., Cilley, W.A. and Haberman, J.P. (1984) Fluoride in deciduous teeth from an anti caries clinical study. J. Dent Res. 63,1201-1205. Schmid, R., Barbakow, P., Muhiemann, H. and De VecchI, P. (1984a) Amine fluoride and monofluorophosphate: II. Pooled results of 56 independent rat caries tests. J. Dent. Child. 61,104-106. Schmid, R., Barbakow, P., Muhiemann, H. and De Vecchi, P. (1984b) Amine fluoride and monofluorophosphate: III. Rat caries inhibition by topical applications daily or every 5th day. J. Dent. Child. 51,107-115. Strubig, W. (1980) Die wirksamkeit lokaler fluoridierungsmittel. Dtsch. Zahnârztl. Z. 35, 1067- 1069. Strubig, W. and Güizow, N.J. (1986) Untersuchungen zur lokalen wirksamkeit von gelees mit unterschiedlichem fluoridgehalt und unterschiedlichem pH-wert. Dfsch. Zahnârztl. Z. 41, 832-

835. ten Cate, J.M., Exterkate, R.A.M. and Rempt, H.E. (1988) Intra-oral retention of fluoride by bovine enamel from amine fluoride toothpaste and 0.4% amine fluoride liquid application. J. Dent. Res. 67, 491-495. 292

APPENDIX 3.

CLINICAL TRIALS ON CARIES INCIDENCE FOLLOWING TOPICAL APPLICATION OF AMINE FLUORIDE, STABILISED STANNOUS FLUORIDE AND MERIDOL REGIMENS.

Data given as mean and standard deviation unless othen/vise stated

Key:

NR = Not Recorded PpmF’ = Parts per million of fluoride DMFS = Decayed, filled and missing tooth surfaces RCI = Root Caries Index CaCOs — Calcium carbonate CPP = Calcium pyrophosphate BaSÛ4 = Barium Sulphate IMP = Insoluble metaphosphate NaMP = Sodium metaphosphate Stat Sig. = Statistical significance NS = Non significant (p ^ 0.05) BS = Bordering on significance (0.06 >p ^ 0.05) S = Significant (0.05 >p ^ 0.01) HS = Highly significant (p < 0.01 ) 293

INDEX OF CONTENTS

Table 1. Clinical Trials on Caries Incidence Foiiowing Topical Application of Amine Fluoride Toothpaste...... 294 Table 2. Clinical Trials on Caries Incidence Foiiowing Topical Application of Amine Fluoride Toothpaste and Amine Fluoride Mouthrinse ...... 297 Table 3. Clinical Trials on Caries Incidence Following Topical Application of High Concentration Amine Fluoride Regimens ...... 298 Tabie 4. Clinical Trials on Décalcification and Root Caries Incidence Following Topical Application of Stabilised Stannous Fluoride Regimens ...... 301 Tabie 5. Ciinical Trials on Root Caries Incidence Foiiowing Topical Application of Amine Fluoride / Stannous Fluoride Regimens ...... 302 BiBLIOGRAPHY...... 305 INVESTIGATION DESIGN TOOTHPASTE PREPARATION RESULTS Duration Age of Number Other source Active Ingredient PpmF Abrasive DMFS at Increase in Reduction Stat. sig. of trial subjects at of of fluoride Baseline DMFS in DMFS v. versus months start (years) subjects placebo placebo Marthaler 36 6 to 9 145 NR AmF 297/335 1250 IMP 6.6 8.3 30% MS 1965 (mean 7.6) 58 AmF 297 1250 BaS04 NR 10.0 15% NR 124 Placebo 0 IMP 6.8 11.7 --

11 to 14 42 AmF 297/335 1250 IMP 28.9 12.7 24% S (mean 12.7) 32 Placebo 0 IMP 29.5 16.8 - - Marthaler 84 6 to 8 114 NR AmF 297/242 1250 IMP 7.5 29.3(12.1) 22% HS 1968 45 AmF 297 1250 IMP 7.3 34.9(13.2) 7% NS 99 Placebo 0 IMP 6.8 37.5(13.5) - - Patz and Naujoks 36 15 to 18 209 NR AmF NR IMP 23.3 9.99 7% S. 1970 220 Placebo 0 “ 23.7 10.75 - - Marthaler 72 Mean 7.5 50 Domestic salt AmF297/242 1250 IMP 7.2 13.9(10.0) 24% s & 1974 (3 to 42%) NaF solution 59 (0.5-1.0%) Placebo 0 7.3 18.3(10.5) - 6x /year.

44 children received F' tablets for >1 year

Table 1. Clinical Trials on Caries Incidence Following Topical Application of Amine Fluoride Toothpaste.

A. Compared to Fluoride Free Placebo Toothpaste. INVESTIGATION DESIGN TOOTHPASTE PREPARATION RESULTS Duration Age of Number Other source Active Ingredient PpmF' Abrasive DMFT Reduction in Stat. sig.

of trial subjects of of fluoride DMFT V. Baseline versus rrtonths subjects baseline Pakhomov et al. 36 NR 1997 6 476 Baseline NR NR 0.7 (1.3) - 354 AmF (NR) 0.1 (0.4) 86 % HS 100 Baseline 0.6 (1.0) - - 100 Proprietay Brand NR NR 0.7 (0.7) -17% NS

9 503 Baseline NR NR 1.8 (1.6) -- 367 AmF (NR) 1.7 (1.4) 6% NS 100 Baseline 1.4 (2.0) - - 100 Proprietary Brand NR NR 20(1.7) -43% NS

12 500 Baseline NR NR 3.2 (2.7) -- 372 AmF (NR) 24(1.9) 25% HS 101 Baseline 3.5(24) - - 100 Proprietary Brand NR NR 3.2 (2.4) 9% NS

Table 1. Clinical Trials on Caries Incidence Following Topical Application of Amine Fluoride Toothpaste.

B. Supervised Brushing. INVESTIGATION DESIGN TOOTHPASTE PREPARATION RESULTS Duration Age of Number Other source Active Ingredient PpmF Abrasive DMFS at Increase In Reduction Stat. sig.

of trial subjects at of of fluoride Baseline DMFS In DMFS V, versus months start (years) subjects placebo placebo Ringelberg et at. 18 NR 186 None AmF (NR) NR NR NR NR 16% NS 1976/1979 184 SnP2 NR NR NR NR 14% NS 186 Placebo 0 NR NR NR - - Cahen et ai 36 6 to 8 668 NR AmF 297 1500 NaMP NR 3.3 (0.1) 21% HS 1982 632 MFP CaCOs/SIIIca NR 3.9 (0.1) 5% HS 708 Placebo 0 NaMP NR 4.1 (0.1) - -

Table 1. Clinical Trials on Caries Incidence Following Topical Application of Amine Fluoride Toothpaste.

C. Compared to Positive Control Toothpaste. INVESTIGATION DESIGN FLUORIDE PREPARATIONS RESULTS Duration Age of Number DMFS at Increase in Reduction Stat. sig. V of trial subjects at of Baseline DMFS in DMFS V placebo months start (years) subjects placebo Ringelberg etal. 30 Mean 11 TOOTHPASTE at home Mouthrinse at 1979 school daily Fluoride PpmF Abrasive 250 ppmF

92 Placebo-Organic 0 NR Placebo 5.0 6.0 -- 94 Placebo-Inorganic 0 NR Placebo 4.3 6.5 - - 162 Placebo 0 NR AmF (NR) 4.4 4.9 22% NS 179 Placebo 0 NR NaF 3.9 4.8 23% S 186 AmF (NR) 1250 NR Placebo 3.7 5.1 18% NS 184 SnFs 1000 GPP Placebo 4.2 5.1 18% NS 174 AmF (NR) 1250 NR AmF (NR) 4.5 4.4 29% S 174 SnFs 1000 GPP NaF 4.8 5.3 15% NS

Table 2. Clinical Trials on Caries Incidence Following Topical Application of Amine Fluoride Toothpaste and Amine Fluoride Mouthrinse. DESIGN FLUORIDE PREPARATIONS RESULTS INVESTIGATION Duration Age of Number DMFS at Increase in Reduction in Stat. sig. of trial subjects at of Baseline DMFS DMFS V months start (years) subjects placebo Marthaler et al. Domestic salt & 36% children received F' tablets for >1 year 1970 36 6 to 7 TOOTHPASTE at home GEL at school for 6 min. 1x/week for 1 year then 1x/ fortnight for 2 years 57 Non F" IMP Non F' 4.26 9.0 (4.70) F in gel 43 AmF 297/335 0.125% Non F' 4.07 8.1 (4.3) 34% HS 63 Non F AmF 297/335 1.25% 3.19 6.0 (4.3) F' in toothpaste 38 AmF 297/335 0.125% AmF 297/335 1.25% 3.21 5.2 (4.1) 9% NS 48 7 to 9 TOOTHPASTE at home GEL at school for 6 min. 1x/fortnight 20 Non F IMP Non F 9.10 14.3(7.7) F in gel 23 AmF 297/335 0.125% Non F' 7.39 10.1 (5.0) 16% NS 21 Non F AmF 297/335 1.25% 7.86 10.2(6.7) F' in toothpaste 26 AmF 297/335 0.125% AmF 297/335 1.25% 7.54 10.2 (5.7) 15% NS

Table 3. Clinical Trials on Caries Incidence Following Topical Application of High Concentration Amine Fluoride Regimens.

A. Amine Fluoride Gels. INVESTIGATION DESIGN FLUORIDE PREPARATIONS RESULTS Duration Age of Number DMFS at Increase in Reduction Stat, sig. of trial subjects at of Baseline DMFS in DMFS v months start (years) subjects placebo Obersztyn et al. 36 19 to 20 85 No Prophylaxis 20.1 (1.2) 30.6(1.4) -- 1979 79 6 monthly - Prophylaxis with 30%Snp2 paste 18.8(1.1) 24.9(1.2) 42% HS then application of 10%Snp2 solution 84 1x / week supervised brushing with AmP 297/335 (P‘ NR) 21.2(1.2) 25.6(1.3) 48% HS gel for 5 min. Obersztyn and 18 19 to 20 787 AmP 297/335 / NaP Gel (1.25%P‘ ) for 5 min. 1x / week & Kolwinski 1984 F' free dentifrice 2x / daily 21.9(0.5) 24.8(0.5) 41% NR 499 P‘ free dentifrice 2x / daily 23.2 (0.6) 28.1(0.6) -- Denes and Gabris 36 Mean 13.9 Toothbrushing daily with fluoride free paste &: Change In DMFS 1991 63 2-3 weekly - prophylaxis and Elmex fluid (NR) in clinic 1.89 (0.38) NS 53 OHI and home brushing 1x / weekly with Elmex Gelee (NR) 0.49(0.16) S 64 OHI 2.38(0.47) - Ran et al. 18 13 Toothbrushing daily with paste (NR) & fortnightly with: 1991 29 Placebo g el 7.1 (6.1) 7,3 only 5.8 (6.0) 26 AmP paste (0.125%) 8,1 1.25% V 0,125% 30 AmP gel (0.4%) 6.0 (6.5) 6,7 significant 27 AmP gel (1.25%) 6.1 (6.3) 6,1 6 months after cessation of gels Placebo gei 7.7 All groups NS AmF paste (0.125%) 9.6 AmP get (0.4%) 8.2 AmF gel (1.25%) 7.0 Clinical Trials on Caries Incidence Following Topical Application of High Concentration Amine Fluoride Regimens. A. Amine Fluoride Gels - Continued. INVESTIGATION DESIGN FLUORIDE PREPARATIONS RESULTS Duration Age of Number DMFSat Increase in Reduction Stat sig. v

of trial subjects at of Baseline DMFS In DMFS V placebo rrranths start (years) subjects placebo Kunzel et al. 1977 84 6 to 7 Am F Am F 297/335 Solution 43% 297/335 Toothpaste Lincir and Rosin- 24 3 to 4 Tooth Professionally applied for 2 min. ; Grget 55 paste Am F (NR) Solution 5x / year 1%F 2.6 (4.4) 9.0(89) 31.3% S 1993 53 used NR AmF (NR) Solution 5x / year 0.5%F 3.4 (7.7) 9.8 (7.4) 25.2% NS 61 Am F (NR) Solution 10x/year 0.5%F 3.4 (5.5) 86(8.5) 34.4% S 30 Placebo solution 0%F 3.0(45) 13.1(11.5) - - Ro$in-Grget and 36 9 to 10 Tooth Professionally applied for 2 min. Lincir 94 paste A AmF (NR) Solution 5x / year 1%F 4.6 (3.3) 8.5 (5.6) A vB 0.1% NS 1995 85 used NR B AmF (NR) Solution 5x / year 0.5%F 4.6 (3.3) 8.4 (5.7) A vC 1.5% S 94 C AmF (NR) Sdution 5x / year 0.25%F 51.(1) 10.0(5.5) B vC 1.6% S Brambllla et al. 60 6 Tooth Professionally applied for 4 min. : DMFT of permanent first molars

1997 128 paste Elmex Fluid 2x/year 1% F 0.56 1.14 23% HS (1%F) 120 Placebo Solution 2x/year 0 % F 0.23 2.07 Source NR

Table 3. Clinical Trials on Caries Incidence Following Topical Application of High Concentration Amine Fluoride Regimens. C. Amine Fluoride Solutions INVESTIGATION DESIGN FLUORIDE PREPARATION RESULTS Duration Age of Number DMFS at Increase in Stat. sig. of trial subjects at of Baseline DMFS months start (years) subjects Stratemann and Décalcification Shannon 18 to 24 11 to 15+ 110 Toothbrushing 3x daily (Paste NR) NR 58% NR 1974 99 Toothbrushing 3x daily (Paste NR) &. (29) 4% Snp 2 gel brushed at home Total NR 27% (19) 1 / week 66% (51) 2-3x / week 26% Daily (as directed) 2% Klock et al. 1985 24 12 0.1% SnP2 rinse 2x daily 33% Boyd Décalcification 1993 (read from figure) 29.2 12.9 32 OHl & NaF (1100 ppmF) toothpaste 1.7 (2.7) 14.4(10.1) A vB S 29.7 13.4 24 B, OHl & NaF (1100 ppmF) toothpaste & 4.9 (4.9) 4.1 (7.3) A v C S Snp2 gel brushed 2x daily at home or B vC BS 27.3 13.2 26 C. OHl & NaF (1100 ppmF) toothpaste & 3.5 (6.7) 10.1(14.8) 0.05% NaF mouth rinse daily at home. Ravald and 24 51 (11) Every 3 to 4 months: Birkhead Prophylaxis with F" containing paste (NR) &: Root DMFS All groups NS 1992 34 5 % NaF varnish professionally applied or NR 3.1 (0.75) 33 0.4% Snp2 gel professionally applied or NR 2.3 (0.82) 32 Used 0.05% NaF mouthrinse daily at home. NR 2.0 (0.60)

Table 4. Clinical Trials on Décalcification and Root Caries Incidence Following Topical Application of Stabilised Stannous Fluoride Regimens.

8 INVESTIGATION DESIGN FLUORIDE PREPARATION RESULTS Duration Age of Number DMFS at DMFSat Reduction Stat. of trial subjects at of Baseline Finish in DMFS sig. months start (years) subjects Caries incidence following topical application of Meridol preparations Ueberschâr and 16 RCI Günay 1991 44.5 (8.4) 19 AmF/SnFz Mouthrinse 250 ppmF 80s 1x daily 18.1% 24.2% BS 49.5 (8.6) 30 Control Group 24.4% 40.1% Bânôczy and 5 RCI Nemes 1991 45.7 20 AmF/SnF2 (1500 ppmF) Toothpaste & 19.3(24.1) 10.7(13.5) -47% NS & AmF/SnF2 Mouthrinse 250 ppmF 1 min. 2x daily Nemes et al. 48.8 24 NaF (1500 ppmF ) Toothpaste & 10.2(9.5) 9.2(11.3) -10% 1992 NaF Mouthrinse 250 ppmF' 1 min. 2x daily

Table 5. Clinical Trials on Root Caries Incidence Following Topical Application of Amine Fluoride / Stannous Fluoride Regimens 303

BIBLIOGRAPHY

Bânôczy, J. and Nemes, J. (1991) Effect of amine fluoride (AmF)/stannous fluoride (SnFji toothpaste and mouthwashes on dental plaque accumulation, gingivitis and root-surface caries. Proc. Finn. Dent Soc. 87, 555-559. Boyd, R.L. (1993) Comparison of three self-applied topical fluoride preparations for control of décalcification. Angle. Orthod. 63, 25-30. Brambllla, E., Toselll, A., FellonI, A., GagllanI, M., Malerba, A. and Strohmenger, L. (1997) The effect of biannual applications of amine fluoride solution on caries Incidence In permanent first molars: a 5-year study.Int. J. Paedlatr. Dent. 7, 9-14. Cahen, P.M., Frank, R.M., Turiot, J.C. and Jung, M.T. (1982) Comparative unsupervised clinical trial on caries Inhibition effect of monofluorophosphate and amine fluoride dentifrices after 3 years In Strasbourg, France. Community Dent. Oral Epidemiol. 10,238-241. Denes, J. and Gabris, K. (1991) Results of a 3-year oral hygiene programme. Including amine fluoride products. In patients treated with fixed orthodontic appliances. Ear. J. Orthod. 13, 129-133. Klock, B., Selling, J., Kinder, S., Manwell, M.A. and TInanoff, N. (1985) Comparison of effect of SnF2 and NaF mouthrinses on carles Incidence, salivary S. mutans and gingivitis In high caries prevalent adults. Scand. J. Dent. Res. 93,213-217. KOnzel, W., Franke, W. and Trelde, A. (1977) Kllnlsch-rontgenologlsche paralleluberwachung elner langsschnlttstudle zum nachwels der karieshemmenden effektlvltat 7 jahre lokal angewandten amlnfluorids Im doppelblndtest. Zahn. Mund. Kieferheilkd. Zentralbi. 65, 626- 637. LInclr, I. and Rosln-Grget, K. (1993) Caries-preventive effect of two different topical fluoride concentrations with two different frequencies of application In preschool children. Caries Res. 27, 484-487. Marthaler, T.M. (1965) The caries-lnhlblting effect of amine fluoride dentifrices In children during three years of unsupervised use. fir. Dent. J. 119,153-163. Marthaler, T.M. (1968) Caries-lnhlbltlon after seven years of unsupervised use of an amine fluoride dentifrice, fir. Dent. J. 124, 510-515. Marthaler, T.M., Konig, K.G. and Mühlemann, H.R. (1970) The effect od a fluoride gel used for supervising toothbrushing 15 or 30 times per year. Helv. Odont. Acta 14,67-77. Marthaler, T.M. (1974) Caries-lnhlbltlon by an amine fluoride dentifrice results after 6 years In children with low caries activity. Helv. Odontol. Acta 18, Suppl-44. Nemes, J., Bânôczy,J., Wlerblcka,M. and Rost,M. (1992) Clinical study on the effect of amine fluoride/stannous fluoride on exposed root surfaces. J. Clin. Dent. 3, 51-53. Obersztyn, A., KolwlnskI, K., TrykowskI, J. and Starosclak, S. (1979) Effects of stannous fluoride and amine fluorides on caries Incidence and enamel solubility In adults. Aust. Dent. J. 24, 395-397. Obersztyn, A. and KolwlnskI, K. (1984) Amine fluoride gel In a caries prophylaxis program for soldiers In Poland. Community. Dent. Oral Epidemiol. 12, 288-291. 304

Pakhomov, G.N., Moller, I.J., Atanassov, N.P., Kabackchieva, R.l. and Sharkov, N.I. (1997) Effect of an amine fluoride dentifrice on dental caries used in a community-based oral health education program. J. Public Health Dent 57,181-183. Patz, J. and Naujoks, R. (1970) Die kariesprophylaktische wirkung einer aminfluoridhaltigen zahnpaste bei jugendlichen nach dreijâhrigem unüberwachten gebrauch. Dtsch Zahnârztl Z. 25, 617-625. Ran, P., Gedalia, I., Fried, M., Hadani, P. and Tved, A. (1991) Effectiveness of fortnightly tooth brushing with amine fluorides in caries-prone subjects. J. Oral Rehabil. 18, 311-316. Ravald, N. and Birkhed, D. (1992) Prediction of root caries in periodontally treated patients maintained with different fluoride programmes. Caries Res. 26,450-458. Ringelberg, M.L, Webster, D.B. and Dbcon, D.O. (1976) Effect of amine fluoride on dental caries after eighteen months. J. Dent. Res. 55, B159 (Abstract) Ringeiberg, M.L, Webster, D.B., Jr. and Dixon, D.O. (1978) Effects of an amine fluoride dentifrice and mouthrinse on the dental caries of school children after 18 months. J. Prev. Dent. 5, 26-30. Ringelberg, M.L, Webster, D.B., Dixon, D.O. and LeZotte, D.C. (1979) The caries-preventive effect of amine fluorides and inorganic fluorides in a mouthrinse or dentifrice after 30 months of use. J. Am. Dent. Assoc. 98,202-208. Rosin-Grget, K. and Lincir, I. (1995) Anticaries effect of different amine fluoride concentrations in schoolchildren. Caries. Res. 29,168-171. Stratemann, M.W. and Shannon, I.L. (1974) Control of décalcification in orthodontic patients by daily self-administered application of a water-free 0.4 per cent stannous fluoride gel. Am. J. Orthod. 66, 273-279. Ueberschâr, M. and Günay, H. (1991) Wurzelkaries-inzidenz unter AmF/SnFrmundspulung. Dtsch. Zahnârztl. Z. 46, 566-568. 305

APPENDIX 4.

STATISTICAL ANALYSES 306

INDEX OF CONTENTS

Table 1. Statistical analysis performed in the investigation of weight loss of dentine specimens on dissolution in 1 molar hydrochloric acid 308 Table 2. Statistical analysis performed in the investigation into fluoride uptake by carious and non-carious dentine following application of Meridol and sodium fluoride toothpaste and mouthrinse in vitro ...... 309 Table 3. Statistical analysis performed in the investigation into weight loss on dissolution of specimens of "carious" dentine in 1M hydrochloric acid...... 310 Table 4. Statistical analysis performed in the investigation into fluoride content of the organic portion of dentine following demineralisation in IM hydrochloric acid after topical application of toothpaste and mouth rinse in vitro...... 310 Table 5. Statistical analysis performed in the investigation into fluoride loss from dentine specimens into storage water after 1 day, 1 week and 1 month after topical application of toothpaste and mouth rinse in vitro...... :...... 311 Table 6. Statistical difference in patient age, time regimen used, specimen dimensions and initial fluoride content of dentine samples and in fluoride uptake by dentine by abutment and by patient following application of Meridol and sodium fluoride toothpaste and mouthrinse in vivo...... 312 Table 7. Kappa calculations of calibration scoring of colour change of the dentine ...... 313 Table 8. Statistical difference calculated for patient age, number of teeth present, number of abutments considered, initial caries scores and caries scores at 1 year by abutment and by patient following use of Meridol and sodium fluoride toothpaste and mouthrinse...... 314 Table 9. Statistical Analysis performed in the investigation into fluoride uptake by dentine following exposure to fluoride (1.25% F-) gels ...... 315 Table 10. Statistical Analysis performed in the investigation into fluoride uptake by dentine following exposure to fluoride (1.25% F") solutions 316 307

Table 11. Statistical Analysis performed in the investigation into fluoride uptake by dentine following exposure to amine fluoride (1.25% F") gels and solutions...... 317 Table 12. Statistical analysis performed in the investigation into fluoride uptake by demineralised denitine following exposure to fluoride (1.25% F") gels...... 319 Table 13. Statistical analysis performed in the investigation into fluoride uptake by demineralised denitine following exposure to fluoride (1.25% F ) solutions...... 320 308

Table 1. Statistical analysis performed in the investigation of weight loss of

dentine specimens on dissolution in 1 molar hydrochloric acid, i. Initial weight of specimens dissolved in 2ml, 5ml and 10ml 1 M HCI.

Value obtained for Value below which Ho is true. Significance

Kruskal Wallis Analysis (Ho ^ p = 0.05)

KW=3.09 5.99 NS(p=0.21)

Ho = Null hypothesis p = The probabiiity of rejecting Hq when Ho is a true fact NS = Non significant (p ^ 0.05) ii. Weight of specimens at 1 hour, 3 hours, 6 hours, 1 day, 6 days, 1 week and 2 weeks after dissoiution in IM HCI

Value obtained for Value below which Ho is true. Significance

Friedman Analysis (Ho ^ p = 0.05)

Fr= 13.65 12.59 S (p = 0.035)

Ho = Null hypothesis p = The probability of rejecting Ho when Ho is a true fact S = Significant (0.05 > p ^ 0.01) Weight Loss at: Sum of Ranks = 1 Hour 102.5 Critical Difference (p = 0.05) = 45.6 3 Hours 83.5 6 Hours 110.5 Significant difference exists between weight of 1 Day 91.5 specimens at 1 week and at 2 weeks only. 6 Days 96.5 1 Week 70.5 Weight loss at 1 hour can therefore be 2 Weeks 117.0 assumed. ill. Percentage Weight Loss of specimens dissolved in 2ml, 5ml and 10ml IM HCI

Value obtained for Value below which Ho is true. Significance

Kruskal Wallis Analysis (Ho ^ p = 0.05)

KW = 1.09 5.99 NS (p = 0.58)

Ho = Null hypothesis p = The probabiiity of rejecting Ho when Ho is a true fact

NS = Non significant (a ^ 0.05) 309

Table 2. Statistical analysis performed in the investigation Into fluoride

uptake by carious and non-carious dentine following application of

Meridol and sodium fluoride toothpaste and mouthrinse in vitro, a. Analysis of statistical difference within sample populations

Parameter Analysis Significance

Initial Fluoride Mann Whitney NS(p = 0.82) Content

Value obtained for Value below which Ho is true Kruskal Wallis Analysis (Value when Ho^ p = 0.05)

Depth KW=7.50 11.07 NS(p = 0.19)

Surface Area KW=5.38 11.07 NS (p = 0.37)

Fluoride Uptake KW=8.42 7.82 S(p= 0.039)

b. Multiple comparisons between samples

Difference between average rankings and significance where:

Critical Difference (p = 0.05) = 15.2 (n =10 and 10) = 14.5 (n = 10 and 12) = 13.8 (n = 12 and 12) FLUORIDE Meridol Meridol NaF UPTAKE Non-Carious Carious Non-Carious Meridol 15.22 Carious S (p=0.G348) NaF 7.17 4.2 Non-Carious NS NS NaF 11.02 8.05 3.85 Carious NS NS NS

Ho = Null hypothesis p = The probability of rejecting Ho when Ho is a true fact NS = Non significant (p ^ 0.05) S = Significant (0.05 > p ^ 0.01) 310

Table 3. Statistical analysis performed in the investigation into weight loss on dissolution of specimens of “carious” dentine in 1M hydrochloric acid.

Mann Whitney Analysis NS(p = 0.0013) p = The probability of rejecting Ho when Ho is a true fact NS = Non significant (p ^ 0.05)

Table 4. Statistical analysis performed in the investigation into fluoride content of the organic portion of dentine following demineralisation in 1M hydrochloric acid after topical application of toothpaste and mouth rinse in vitro.

Friedman analysis comparing percentage remaining in each treatment group. Value obtained for Value below which Ho is true. Significance Friedman Analysis (Ho ^ p= 0.05)

Fr=0.66 5.99 NS(p=0.72)

Ho = Null hypothesis p = The probability of rejecting Ho when Ho is a true fact NS = Non significant (p ^ 0.05)

Ranking of values for initial fluoride content of dentine against percentage of fluoride remaining for each specimen. Initial Percentage Initial Percentage Initial Percentage Initial Percentage Content Remaining Content Remaining Content Remaining Content Remaining 1 45 9 32 22 1 34 18 2 37 9 1 25 30 37 28 2 14 14 11 25 30 37 43 2 45 15 37 27 1 40 36 5 44 15 37 27 12 40 27 5 42 15 47 27 32 42 1 5 20 15 35 27 8 43 6 5 25 15 48 31 29 44 41 9 23 15 37 32 20 45 22 9 1 15 9 33 19 46 16 9 14 22 34 34 24 47 7 9 14 22 10 34 25 48 13 311

Table 5. Statistical analysis performed in the investigation into fluoride loss

from dentine specimens into storage water after 1 day, 1 week and

1 month after topical application of toothpaste and mouth rinse in

vitro. a. Analysis of statistical difference within sample populations.

Value obtained for Value below which H q is true. Significance Kruskal Wallis Analysis (Ho^p = 0.05)

KW = 16.52 11.07 HS(p= 0.006)

b. Multiple comparisons between samples. Difference between average rankings and significance where: Critical Difference (p = 0.05) = 16.3 Fluoride Meridol NaF Meridol NaF Meridol

Loss 1 day 1 day 1 week 1 week 1 month

NaF 3.1

1 day NS

Meridol 5.5 2.4

1 week NS NS

NaF 4.0 0.9 1.5

1 week NS NS NS

Meridol 16.3 13.2 10.8 12.3

1 month S (p » 0.05) NS NS NS

NaF 16.7 13.6 . 1.2 12.7 0.4

1 month 3 (p ■ 0.039) NS NS NS NS

Ho = Null hypothesis p = The probability of rejecting H q when H q is a true fact NS = Non significant (p ^ 0.05) 8 = Significant (0.05 >p ^ 0.01) HS = Highly significant (p < 0.01) 312

Table 6. Statistical difference in patient age, time regimen used, specimen

dimensions and initial fluoride content of dentine samples and in

fluoride uptake by dentine by abutment and by patient following

application of Meridol and sodium fluoride toothpaste and

mouthrinse in vivo, a. Analysis of statistical difference within sample populations

Parameter Analysis Significance

Mann Whitney

Patient Age NS(p = 0.19)

Time Regimen NS (p = 0.79) Used

initial Fluoride NS (p = 0.31) Content

Fluoride Uptake NS (p = 0.093) by Abutment

Fluoride Uptake NS (p = 0.29) by Patient

Value obtained for Value below which Ho Is true Kruskal Wallis Analysis (Value when Ho ^ p = 0.05)

Depth KW = 6.96 7.82 NS (p = 0.07)

Surface Area KW=0.81 7.82 NS (p = 0.89)

Ho = Null hypothesis p = The probability of rejecting H q when H q is a true fact NS = Non significant (p ^ 0.05) 313

Table 7. Kappa calculations of calibration scoring of colour change of the

dentine.

i. Caliibration scoring of colour change of the dentine.

Patient Initial Scores Calibration Start Calibration End

Sound Colour Sound Colour Sound Colour Change Change Change 1 7 0 7 0 7 0 2 7 0 7 0 7 0 3 5 2 6 1 5 2 4 6 0 6 0 6 0 5 6 0 6 0 6 0 6 5 0 5 0 5 0 7 3 0 3 0 3 0 8 1 1 1 1 1 1 9 6 2 6 2 5 3 10 4 0 4 0 4 0 Total 50 5 51 4 49 6

II. Kappa Scores

Initial to Initial to Calibration Start

Calibration Start Calibration End to Calibration End

Po 0.9818 0.9818 0.9636

Pe 0.8496 0.8198 0.8341

Kappa 0.88 0.90 0.78 314

Table 8. Statistical difference calculated for patient age, number of teeth

present, number of abutments considered, initial caries scores and

caries scores at 1 year by abutment and by patient following use

of Meridol and sodium fluoride toothpaste and mouthrinse.

Mann Whitney Analysis

Patient Age NS(p = 0.26)

Number of Abutments Considered NS (p = 0.99)

Initial Caries Score Colour Change NS (p=0.53)

Final Caries Score Colour Change NS(p = 0.71)

Final Caries Score Cavitations NS(p = 0.09)

NS = Non Significant (p k 0.05) 315

Table 9. Statistical Analysis performed in the investigation into fluoride

uptake by dentine following exposure to fluoride (1.25% F-) gels. a. Analysis of statistical difference within sample populations.

Parameter Analysis Significance

Value obtained for Value below which H q Is true

Kruskal Wallis Analysis (Value when H q ^ p = 0.05)

Depth KW=6.33 7.82 NS (p = 0.097)

Surface Area KW=1.73 7.82 NS (p = 0.63)

Value obtained for Value below which H q Is true

Friedman Analysis (Value when H q ^ p = 0.05)

Fluoride Uptake Fr= 15.2 5.99 HS (p = 0.001

b. Multiple comparisons between samples for fluoride uptake. Difference between average rankings and significance where; Critical Difference (p = 0.05) = 10.7 Fluoride Regimen NaF Gel Elmex Gel

Elmex Gel 2

Meridol Gel 14 16

HS HS

(p = 0.0054) (p = 0.00096)

Ho = Null hypothesis p = The probability of rejecting H q when H q is a true fact NS = Non significant (p ^ 0.05) HS = Highly significant (p < 0.01) 316

Table 10. Statistical Analysis performed in the investigation into fluoride

uptake by dentine following exposure to fluoride (1.25% F")

solutions. a. Analysis of statistical difference within sample populations.

Parameter Analysis Significance

Value obtained for Value below which Ho is true Kruskal Wallis Analysis (Value when Ho ^ p = 0.05)

Depth KW = 2.70 7.82 NS (p = 0.44)

Surface Area KW=3.97 7.82 NS (p = 0.27)

Value obtained for Value t)elow which Ho is true Friedman Analysis (Value when Ho ^ p = 0.05)

Fluoride Uptake Fr = 9.60 5.99 HS(p = 0.008)

b. Multiple comparisons between samples for fluoride uptake. Difference between average rankings and significance where: Critical Difference (p = 0.05) = 10.7 Fluoride Regimen NaF Solution Elmex Solution

Elmex Solution 0

Meridol Solution 12 12

HS HS

(p = 0.022) (p = 0.022)

Ho = Null hypothesis p = The probability of rejecting H q when Ho is a true fact NS = Non significant (p ^ 0.05) HS = Highly significant (p < 0.01 ) 317

Table 11. Statistical Analysis performed in the investigation into fluoride

uptake by dentine following exposure to amine fluoride (1.25% F )

gels and solutions.

a. Analysis of statistical difference within sample populations.

Parameter Analysis Significance

Value obtained for Value below which Ho is true

Kruskal Wallis Analysis (Value whenH q ^ p = 0.05)

Depth KW=9.06 7.82 S(p= 0.029)

Surface Area KW=8.88 7.82 S (p= 0.031)

Fluoride Uptake KW= 29.77 7.82 HS (p = 0)

b. Multiple comparisons between samples for surface area of specimens

Difference between average rankings and significance where: Critical Difference (p = 0.05) = 13.79 Fluoride Regimen Meridol Solution Meridol Gel Elmex Solution

Meridol Gel 9.70 NS Elmex Solution 15.10 24.60 S HS (p = 0.023) (p = 0.00036) Meridol Gel 13.00 22.70 2.10 NS HS NS (p = 0.022) 318

C. Multiple comparisons between samples for depth of specimens Difference between average rankings and significance where: Critical Difference (p = 0.05) = 10.7 Fluoride Regimen Meridol Solution Meridol Gel Elmex Solution

Meridol Gel 15.10 S (p = 0.023) Elmex Solution 4.70 10.40 NS NS Meridol Gel 4.10 11.00 0.60 NS NS NS

d. Multiple comparisons between samples for fluoride content of specimens Difference between average rankings and significance where: Critical Difference (p = 0.05) = 10.7

Fluoride Regimen Meridol Solution Meridol Gel Elmex Solution

Meridol Gel 11.30 NS Elmex Solution 2.00 9.30 NS NS Meridol Gel 3.70 15.00 5.70 NS S NS (p = 0.025)

Ho = Null hypothesis p = The probability of rejecting H q when H q is a true fact NS = Non significant (p ^ 0.05) 8 = Significant (0.05 >p ^ 0.01) HS = Highly significant (p < 0.01) 319

Table 12. Statistical analysis performed in the investigation into fluoride

uptake by demineralised denitine following exposure to fluoride

(1.25% F ) gels. a. Analysis of statistical difference within sample populations.

Parameter Analysis Significance

Value obtained for Value below which Ho is true Kruskal Wallis Analysis (Value when Ho ^ p = 0.05)

Surface Area KW =0.87 11.07 NS (p = 0.97)

Fluoride released KW = 16.96 5.99 HS (p = 0.000) by KOH

Fluoride released KW = 21.15 5.99 HS (p = 0.000) by HCI

Fluoride remaining KW =0.96 5.99 HS (p = 0.62) following HCI

b. Multiple comparisons between samples for fluoride released by KOH. Difference between average rankings and significance where critical difference (p = 0.05) = 9.43 Fluoride Regimen Meridol Gel NaF Gel NaF Gel 16 HS (p = 0.00018) Elmex Gel 6.8 9.3 NS NS c. Multiple comparisons between samples for fluoride released by HCI Difference between average rankings and significance where critical difference (p = 0.05) = 9.43 Fluoride Regimen Meridol Gel NaF Gel NaF Gel 17.6 HS(p = 0.00018) Elmex Gel 12.4 . 5.2 HS(p = 0.0014) NS

Ho = Null hypothesis p = The probability of rejecting H q when Ho is a true fact NS = Non significant (p ^ 0.05) HS = Highly significant (p < 0.01) 320

Table 13. Statistical analysis performed in the investigation into fluoride

uptake by demineralised denitine following exposure to fluoride

(1.25% F") solutions. a. Analysis of statistical difference within sample populations.

Parameter Analysis Significance

Value obtained for Value below which H q is true Kruskal Wallis Analysis (Value whenH q ^ p = 0.05)

Surface Area KW =8.22 5.99 NS (p = 0.017)

Fluoride released KW = 26.14 5.99 H S (p = 0.000) by HCI

b. Multiple comparisons between samples for surface area. Difference between average rankings and significance where: Critical Difference (p = 0.05) = 9.43 Fluoride Regimen Amine Fluoride 297 Solution NaF Solution

NaF Solution 3 NS Stannous Fluoride 7.9 10.9 Solution NS S (p = 0.017) c. Multiple comparisons between samples for fluoride uptake. Difference between average rankings and significance where: Critical Difference (p = 0.05) = 9.43 Fluoride Regimen Amine Fluoride 297 Solution NaF Solution

NaF Solution 10 S (p = 0.033) Stannous Fluoride 10 20 Solution S (p = 0.033) HS (p = 0.00000) Ho = Null hypothesis p = The probability of rejecting H q when H q is a true fact NS = Non significant (p ^ 0.05) S = Significant (0.05 >p ^ 0.01) HS = Highly significant (p < 0.01) 321

APPENDIX 5.

DATA FROM CHAPTERS 3 TO 8. 322

INDEX OF CONTENTS

Table 1. Depth and fluoride content of dentine specimens dissected to assess the effect of variation in specimen depth on the fluoride content of dentine following application of fluoride (1.25% F ) solutions...... 323 Table 2. Specimen dimensions, fluoride content and fluoride uptake following application of Meridol and sodium fluoride toothpaste and mouthrinse in vitro to "non-carious" and "carious” dentine ...... 326 Table 3. Fluoride content of inorganic and organic solutions of dentine following topical fluoride treatments with Meridol and sodium fluoride toothpaste and mouth rinse in vitro...... 327 Table 4. Fluoride loss into storage water at 1 day, 1 week and 1 month from specimens of dentine following application of Meridol and sodium fluoride toothpaste and mouthrinse in vitro...... 328 Table 5. Patient details, specimen dimensions, fluoride content and fluoride uptake following topical application of Meridol and sodium fluoride toothpaste and mouthrinse in vivo...... '...... 330 Table 6. Age and sex of the patients and the fluoride regime provided in patients participating in the investigation into caries incidence and changes to the oral microflora following unsupervised use of Meridol and sodium fluoride toothpaste and mouthrinse 331 Table 7. The number of teeth, number of abutments monitored, caries score at t)aseline and new lesions recorded over 12 months in patients participating in the investigation into caries incidence following unsupervised use of Meridol and sodium fluoride toothpaste and mouthrinse...... 332 Table 8. Summary of the number of teeth, number of abutments monitored, caries score at baseline and new lesions recorded over 12 months in patients participating in the investigation into caries incidence following unsupervised use of Meridol and sodium fluoride toothpaste and mouthrinse ...... 334 Table 9. The sample dimensions, fluoride content and fluoride uptake of dentine samples following exposure to ...... 335 Table 10. The sample dimensions, fluoride content and fluoride uptake of dentine samples following exposure to ...... 336 Table 11. The Sample dimensions and fluoride content of dentine samples following exposure to amine fluoride compounds ...... 337 Table 12. Fluoride uptake by demineralised dentine following application of fluoride gels (1.25% FI/nv/fro ...... 338 Table 13. Fluoride uptake by demineralised dentine following application of fluoride solutions (1.25% F)/nv«ro...... 339 Fluoride Sodium Fluoride Solution (1.25% F) Untreated Regimen

Narrow Specimens Standard Specimens Wide Specimens Narrow Specimens Standard Specimens Wide Specimens

Tooth Depth F Content Depth F Content Depth F Content Depth F Content Depth F Content Depth F* Content (mm) (pgcm'^) (mm) (pgcm'^) (mm) (pgcm'^) (mm) (pgcm'^) (mm) (pgcm*^) (mm) (pgcm'^)

1 0.32 74 0.54 121 0.99 170 0.37 8 0.49 12 0.70 24 2 0.37 86 0.66 137 1.01 143 0.34 38 0.55 33 0.94 58 3 0.22 51 0.49 112 0.91 161 0.20 7 0.51 0 0.98 25 4 0.20 48 0.60 97 1.07 137 0.40 23 0.63 19 1.03 18 5 0.36 92 0.51 75 0.81 163 0.27 4 0.54 47 1.16 55 6 0.27 84 0.49 127 1.00 144 0.24 6 0.47 11 1.10 30 7 0.30 88 0.58 303 0.99 243 0.39 12 0.47 19 0.85 40 8 0.22 51 0.42 113 0.96 130 0.31 9 0.47 20 1.08 40 9 0.30 48 0.53 118 1.02 126 0.30 6 0.46 11 0.99 13 10 0.34 120 0.53 243 0.92 265 0.35 31 0.43 77 1.06 65

a. Sodium fluoride solution (1.25% F ) Fluoride Stannous Fluoride Solution (1.25% F*) Untreated Regimen

Narrow Specimens Standard Specimens Wide Specimens Narrow Specimens Standard Specimens Wide Specimens

Tooth Depth (mm) F Content Depth (mm) F Content Depth (mm) F Content Depth (mm) F Content Depth (mm) F Content Depth (mm) F Content (pgcm^) (pgcm'^) (pgcm'^) (pgcm^) (pgcm*^) (pgcm^)

1 0.32 54 0.51 81 1.08 174 0.35 31 0.46 14 1.08 40 2 0.33 53 0.56 83 1.13 125 0.26 4 0.45 12 0.84 32 3 0.44 73 0.65 55 0.88 164 0.35 3 0.48 16 0.78 19 4 0.34 110 0.80 97 0.85 128 0.37 0 0.64 0 0.99 13 5 0.26 64 0.74 37 1.31 55 0.35 9 0.52 8 0.94 15 6 0.26 88 0.46 119 0.96 108 0.45 6 0.75 16 1.06 26 7 0.44 75 0.86 78 1.23 208 0.31 9 0.63 27 0.89 23 8 0.37 89 0.68 25 1.06 35 0.31 6 0.40 6 0.74 29 9 0.35 82 0.69 39 1.13 105 0.35 2 0.57 8 1.10 15 10 0.36 152 0.58 98 1.00 185 0.30 5 0.59 9 1.21 54

Table 1. Depth and fluoride content of dentine specimens dissected to assess the effect of variation in specimen depth on the fluoride content of dentine following application of fluoride (1.25% F") solutions, b. Stannous fluoride solution (1.25% F‘) Fluoride Amine Fluoride 297 Solution (1.25% F") Untreated Regimen

Narrow Specimens Standard Specimens Wide Specimens Narrow Specimens Standard Specimens Wide Specimens

Tooth Depth (mm) F Content Depth (mm) F* Content Depth (mm) F Content Depth (mm) F' Content Depth (mm) F Content Depth (mm) F Content (pgcm^) (pgcm^) (pgcm^) (pgcm^) (pgcm*^) (pgcm'^)

1 0.39 259 0.40 128 0.97 206 0.21 0 0.43 9 0.75 14 2 0.34 268 0.74 200 0.94 243 0.35 0 0.45 8 0.68 8 3 0.42 223 0.47 220 1.09 194 0.34 14 0.56 24 1.16 69 4 0.39 229 0.48 268 1.13 272 0.50 10 0.59 27 0.95 21 5 0.39 215 0.68 166 0.96 250 0.28 5 0.59 38 1.29 56 6 0.43 198 0.74 226 1.17 177 0.34 3 0.55 8 1.32 31 7 0.31 198 0.73 237 1.11 280 0.25 7 0.42 4 1.03 6 8 0.45 135 0.73 104 1.00 115 0.43 32 0.57 24 0.85 46 9 0.53 136 0.74 138 1.15 89 0.30 30 0.65 25 1.25 38 10 0.39 216 0.74 213 1.28 285 0.50 15 0.62 18 1.14 61

Table 2. Specimen dimensions, fluoride content and fluoride uptake following application of Meridol and sodium fluoride toothpaste and mouthrinse in vitro to “non-carious" and “carious" dentine.

TOOTH UNTREATED MERIDOL SODIUM FLUORIDE Depth Surface F Depth Surface F F Depth Surface F F Area content Area content uptake^ Area content uptake^ mm mm^ ngcm*^ mm mm^ ngcm*^ HQcm'^ mm mm^ ngcm'^ pgcm*^ NON-CARIOUS 1 0.46 1.32 11 0.66 1.63 71 55 0.57 1.00 70 56 2 0.58 1.03 15 0.64 1.01 40 23 0.36 1.03 24 15 3 0.70 1.51 24 0.50 1.41 184 167 0.47 1.18 25 9 4 020 1.46 7 0.58 1.57 31 11 0.43 1.23 53 38 5 0.40 1.61 9 0.51 1.00 35 24 0.50 1.39 43 32 6 0.51 1.13 4 0.52 1.77 54 50 0.43 0.96 21 18 7 0.57 1.50 16 0.51 1.32 66 52 0.54 1.31 27 12 8 0.52 1.48 17 0.64 1.52 43 22 0.32 1.28 51 41 9 0.47 1.36 11 0.66 1.70 35 20 0.45 1.21 21 10 10 0.61 1.05 10 0.57 1.29 31 22 0.55 1.18 21 12 11 0.47 1.54 19 0.56 1.30 90 67 0.63 1.26 56 31 12 0.94 1.96 18 0.47 1.68 30 21 0.59 1.62 25 14 CARIOUS 1 0.47 1.79 14 0.71 2.16 21 0 0.63 1.30 31 15

2 0.59 1.69 15 0.54 0.99 23 9 0.45 1.38 54 42

3 0.53 1.61 12 0.49 1.69 6 -5 0.49 1.44 14 1

4 0.64 1.56 13 0.66 1.46 21 8 0.40 1.60 13 3

5 0.39 0.85 12 0.60 0.91 16 -2 0.48 1.28 16 3

6 0.63 1.97 8 0.42 1.12 62 57 0.51 1.44 45 32

7 0.42 1.10 5 0.47 1.83 38 33 0.59 1.17 51 36

8 0.48 1.15 17 0.63 1.29 31 8 0.41 0.91 22 11

9 0.37 1.07 23 0.48 1.92 42 12 0.45 1.43 38 27

10 0.47 1.22 20 0.25 0.91 44 33 0.44 1.03 63 52

’ Forfomiula see Page 127 327

Table 3. Fluoride content of inorganic and organic solutions of dentine following topical fluoride treatments with Meridol and sodium fluoride toothpaste and mouth rinse in vitro.

FLUORIDE REGIMEN

UNTREATED SODIUM FLUORIDE MERIDOL

TOOTH Fluoride Fluoride Fluoride Fluoride Fluoride Fluoride content content content content content content in MCI InKOH in MCI InKOH in HOI InKOH

W W (^g) (f9) (f9) (no) 1 150 1.00 0.7% 700 0.50 0.1% 1150 0.35 0.0%

2 150 0.80 0.5% 250 4.00 1.6% 400 0.70 0.2%

3 600 0.35 0.1% 300 0.06 0.0% 2600 0.70 0.0%

4 100 0.40 0.4% 650 0.50 0.1% 800 0.04 0.0%

5 150 0.06 0.0% 600 0.40 0.1% 550 0.20 0.0%

6 50 0.40 0.8% 200 0.06 0.0% 950 0.40 0.0%

7 250 1.00 0.4% 350 0.35 0.1% 880 4.00 0.5%

8 250 1.00 0.4% 650 0.20 0.0% 650 4.00 0.6%

9 150 0.10 0.1% 250 1.00 0.4% 600 0.20 0.0%

10 100 0.03 0.0% 250 0.03 0.0% 400 0.04 0.0%

11 300 0.70 0.2% 700 2.50 0.4% 1180 0.10 0.0%

12 350 0.35 0.1% 400 0.00 0.0% 500 0.20 0.0%

13 250 3.00 1.2% 400 0.10 0.0% 450 0.40 0.1%

14 250 0.80 0.3% 750 0.00 0.0% 230 0.05 0.0%

15 200 0.10 0.1% 200 0.35 0.2% 100 0.80 0.8%

16 200 0.00 0.0% 200 0.00 0.0% 300 0.00 0.0% Specimen Surface Area Fluoride Content Fluoride Released into Storage Water (pg) Total Fluoride^ Percentage of Total Fluoride (mm^) Of Dentine (Dentine and Water) Released into Storage Water

(pgcm'^) 1 Day (In 5ml) 1 Week (In 4.5ml) I 1 Month (In 4ml) (F9) 1 Day 1 Week 1 Month Meridol Regimen 1 0.85 181 0.09 0.06 0.02 1.54 6% 4% 1% 2 0.87 280 0.10 0.08 0.02 2.44 4% 3% 1% 3 0.80 270 0.18 0.12 0.03 2.16 8% 6% 1% 4 0.97 58 0.15 0.12 0.03 0.56 25% 21% 5% 5 0.97 86 0.10 0.02 0.01 0.83 12% 7% 1% Sodium Fluoride Regimen 1 0.99 165 0.11 0.09 0.01 1.63 7% 6% 1% 2 0.97 167 0.30 0.17 0.07 1.62 19% 10% 4% 3 0.99 167 0.11 0.11 0.02 1.65 7% 7% 1% 4 1.00 93 0.03 0.06 0.02 0.93 3% 6% 2% 5 0.87 60 0.06 0.04 0.01 0.52 12% 8% 2%

^ Total fluoride = Total fluoride content of dentine specimen + Fluoride content in the 0.5ml of water removed at 1 day + Fluoride content in the 0.5 ml of water removed at 1 week + Fluoride in the 4 ml of water remaining at 1 month.

Table 4. Fluoride loss into storage water at 1 day, 1 week and 1 month from specimens of dentine following application of Meridol and sodium fluoride toothpaste and mouthrinse in vitro. Table 5. Patient details, specimen dimensions, fluoride content and fluoride uptake following topical application of Meridol and sodium fluoride toothpaste and mouthrinse in vivo.

PATIENT SEX AGE REGIME TIME TOOTH SPECIMEN DEPTH AREA F CONTENT F UPTAKE (years) (days) (mm) (mm') (Mflcm') (l*flcm') 1 Male 74.0 Meridol 28 13 Control 0.44 1.24 31 Test 0.52 1.20 88 51 2 Female 63.3 Sodium 42 35 Control 1.36 0.96 27 Fluoride Test 0.79 1.14 47 31 3 Female 71.8 Meridol 28 33 Control 0.67 1.41 40 Test 0.52 1.15 42 11 4 Male 73.5 Sodium 28 13 Control 0.78 1.57 62 Fluoride Test 0.49 1.05 79 40 5 Male 57.9 Meridol 28 43 Control 0.55 1.02 29 Test 0.41 0.82 46 24 6 Male 48.9 Meridol 30 11 Control 0.39 1.32 6 Test 0.57 1.13 33 24 12 Control 0.41 1.22 7 Test 0.35 0.72 52 46 21 Control 0.61 0.44 4 Test 0.43 1.35 38 35 22 Control 0.61 1.11 5 Test 0.37 1.20 24 21 7 Male 56.9 Sodium 28 11 Control 0.51 1.66 29 Fluoride Test 0.37 1.22 40 19 13 Control 0.59 1.39 14 Test 0.76 1.46 31 13 23 Control 0.49 1.14 23 Test 0.37 1.75 34 17 8 Male 61.3 Meridol 28 13 Control 0.58 1.25 6 Test 0.42 0.78 8 4 9 Male 56.9 Sodium 28 11 Control 0.47 1.36 15 Fluoride Test 0.33 0.80 24 13 12 Control 0.35 0.68 15 Test 0.55 1.11 10 -14 10 Female 57.9 Sodium 26 11 Control 0.61 1.77 8 Fluoride Test 0.49 0.93 11 5 23 Control 0.53 2.05 7 Test 0.76 1.25 41 31 24 Control 0.56 1.96 8 Test 0.40 1.33 38 32 11 Male 68.4 Meridol 35 11 Control 0.77 2.20 16 Test 0.40 1.64 28 20 12 Female 62 Meridol 28 11 Control 0.37 0.95 7 Test 0.38 0.72 17 10 13 Male 78.2 Sodium 28 22 Control 0.53 1.07 8 Fluoride Test 0.40 1.96 7 1 14 Male 72.7 Meridol 49 14 Control 0.69 2.87 12 Test 0.47 2.25 41 33 330

Table 5. Patient details, specimen dimensions, fluoride content and fluoride uptake following topical application of Meridol and sodium fluoride toothpaste and mouthrinse in vivo - continued.

PATIENT SEX AGE REGIME TIME TOOTH SPECIMEN DEPTH AREA F CONTENT F UPTAKE’ (years) (days) (mm) (mm^ (Mflcn'*) (Mflcm^ 15 Female 55.1 Sodium 28 21 CorArol 0.53 1.34 5 Fluoride Test 0.52 1.06 16 11 16 Male 46.0 Meridol 35 43 Control 0.54 1.70 7 Test 0.5 1.34 15 9 17 Female 73.7 Sodium 42 43 Control 0.39 0.54 5 Fluoride Test 0.6 1.64 16 8 18 Male 57.9 Sodium 28 31 Control 0.49 0.93 4 Fluoride Test 0.41 0.94 7 4 42 Control 0.45 0.99 4 Test 0.58 1.36 11 6 19 Male 66.9 Sodium 28 33 Control 0.40 0.98 2 Fluoride Test 0.44 1.69 21 19 43 Control 0.57 3.01 7 Test 0.40 1.13 14 9 20 Male 51.5 Meridol 34 22 Control 0.61 1.18 13 Test 0.31 1.06 20 13 21 Male 56.1 Meridol 28 11 Control 0.50 0.76 15 Test 0.46 1.24 43 29 22 Male 67.7 Sodium 28 13 Control 0.56 1.13 24 Fluoride Test 0.65 1.24 62 34 23 Female 35.8 Meridol 28 13 Control 0.55 1.08 3 Test 0.25 2.16 4 3 31 Control 0.29 0.84 0 Test 0.31 1.25 2 2 24 Male 71.4 Sodium 28 31 Control 0.35 0.65 33 Fluoride Test 0.34 0.40 27 -5 41 Control 0.46 0.80 21 Test 0.68 0.39 57 26 42 Control 0.38 0.76 7 Test 0.48 0.95 19 10 25 Male 63.7 Meridol 28 12 Control 0.40 0.84 11 Test 0.46 1.48 62 49 13 Control 0.51 1.19 8 Test 0.27 0.31 48 44 22 Control 0.69 1.09 20 Test 0.57 1.25 42 25 26 Male 77.3 Sodium 42 12 Control 0.40 0.78 17 Fluoride Test 0.47 0.81 18 -2 27 Male 55.7 Meridol 28 13 Control 0.44 0.87 3 Test 0.55 1.47 12 9 11 Control 0.57 0.94 3 Test 0.60 1.96 19 16 28 Female 52.9 Sodium 42 11 Control 0.50 0.85 6 Fluoride Test 0.70 1.40 20 12 ^ Forfoirnula see Page 127 331

Table 6. Age and sex of the patients and the fluoride regime provided in patients participating in the investigation into caries incidence and changes to the oral microflora following unsupervised use of Meridol and sodium fluoride toothpaste and mouthrinse.

Meridol NaF Patient Sex Age Patient Sex Age Number Number

1 M 74 1 2 F 71.8 3 M 68.7 4 M 46.3 7 M 50.5 5 M 59.2 9 M 50.7 6 M 57.9 11 M 62.5 8 M 57.0 13 M 74.1 10 M 65.4 15 F 72.7 12 M 70.2 17 M 74.8 14 F 64.0 20 F 65.3 16 M 61.8 21 M 72.8 18 M 81.1 23 F 60.1 19 M 71.3 26 M 68.8 22 M 49.5 27 F 62.3 24 M 78.3 29 M 76.2 25 M 61.8 33 M 46.7 28 M 46.9 34 F 80.1 30 M 70.9 37 M 55.7 31 F 55.3 38 M 67.3 32 M 78.0 39 M 56.3 35 F 46.8 40 M 83.7 36 M 58.3 Patient Number of Initial Scores New Lesions Abutments 3 Months 6 Months 9 Months 1 Year CC CV S —► CC C C -»C V s-^cv s->cc cc-»cv s->cv S —► CC cc-»cv s-^cv S —► CC cc-»cv s-*cv

1 7 0 0 0 0 0 0 0 0 out 2 7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 7 2 0 0 0 2 0 0 0 0 0 0 0 0 0 4 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 6 5 0 0 0 0 1 0 0 0 2 0 0 0 0 0 7 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 8 2 1 0 0 0 0 0 0 0 0 0 0 0 0 0 9 8 2 0 2 0 0 0 0 0 0 0 0 0 1 0 10 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 11 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 12 2 1 0 0 0 0 0 0 0 0 0 0 0 0 0 13 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 14 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 15 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 16 2 0 0 0 0 0 0 0 0 0 0 0 1 0 0 17 8 0 0 0 0 0 2 0 0 0 0 0 0 2 0 18 2 0 0 0 1 0 0 0 0 0 0 0 0 0 0 19 7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 20 3 1 0 0 1 0 0 0 0 0 0 0 0 0 1

Table 7. The number of teeth, number of abutments monitored, caries score at baseline and new lesions recorded over 12 months in patients participating in the investigation into caries incidence following unsupervised use of Meridol and sodium fluoride toothpaste and mouthrinse. g Patient Number of Initial Scores New Lesions Abutments 3 Months 6 Months 9 Months 1 Year CC CV S-^C C cc-»cv s->cv S-^C C cc-»cv s-^cv S —► CC c c -» c v 8 ->C V 3 -> C C cc-»cv s->cv

21 3 0 0 0 0 1 0 0 0 0 0 0 0 0 0 22 9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 23 4 0 0 out 24 6 1 0 0 0 0 0 0 0 0 0 0 0 0 0 25 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 26 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 27 3 1 0 0 0 0 1 0 0 0 0 0 0 0 1 28 2 1 0 0 0 0 out 29 5 0 0 0 0 0 1 0 0 0 0 0 0 0 0 30 2 0 0 0 0 0 0 0 0 1 0 0 0 0 0 31 5 1 0 0 0 0 0 0 0 0 0 0 0 0 0 32 4 0 0 0 0 0 0 0 0 0 0 0 33 2 0 0 0 0 0 0 0 0 34 7 1 0 0 0 0 0 0 0 0 0 0 35 8 0 0 0 0 0 0 0 0 0 0 0 out 36 4 1 0 0 0 0 0 0 0 0 0 0 0 0 0 37 3 3 0 0 0 0 0 0 0 0 0 0 0 0 0 38 5 0 0 0 0 0 0 0 0 0 0 0 39 3 0 0 0 0 0 0 0 0 1 0 0 0 0 0 40 2 0 0 0 0 0 0 0 0 0 0 0 Table 7. The number of teeth, number of abutments monitored, caries score at baseline and new lesions recorded over 12 months in patients participating in the investigation into caries incidence following unsupervised use of Meridol and sodium fluoride toothpaste and mouthrinse. Regime Meridol Toothpaste and Mouthrinse Sodium Fluoride Toothpaste and Mouthrinse Patient No. of No. of Colour Change Cavitation Patient No. of No. of Colour Change Cavitation Teeth Abutments initial New Initial New Teeth Abutments Initial New Initial New 1 22 7 0 out 0 out 2 19 7 0 0 0 0 3 16 7 2 0 0 2 4 18 6 0 0 0 0 7 19 3 0 0 0 0 5 23 6 0 0 0 0 9 21 8 2 2 0 1 6 22 5 0 2 0 1 11 16 6 0 0 0 0 8 16 2 1 0 0 0 13 18 2 0 0 0 0 10 19 4 0 0 0 0 15 16 8 0 0 0 0 12 22 2 1 0 0 0 17 15 8 0 0 0 2 14 10 4 0 0 0 0 20 14 3 1 0 0 2 16 18 2 0 1 0 0 21 10 3 0 0 0 1 18 12 2 0 0 0 1 23 19 4 0 out 0 out 19 17 7 0 0 0 0 26 14 3 0 0 0 0 22 17 9 0 0 0 0 27 14 3 1 1 0 1 24 20 6 1 0 0 0 29 16 5 0 1 0 0 25 17 4 0 0 0 p 33 15 2 0 0 0 0 28 23 2 1 out 0 out 34 19 7 1 0 0 0 30 23 2 0 1 0 0 37 17 3 3 0 0 0 31 13 5 1 0 0 0 38 16 5 0 0 0 0 32 22 4 0 0 0 0 39 17 3 0 1 0 0 35 21 8 0 out 0 out 40 12 2 0 0 0 0 36 20 4 1 0 0 0

Table 8. Summary of the number of teeth, number of abutments monitored, caries score at baseline and new lesions recorded over 12 months in patients participating in the investigation into caries incidence following unsupervised use of Meridol and sodium fluoride toothpaste and mouthrinse. UNTREATED SODIUM FLUORIDE GEL ELMEX GEL MERIDOL GEL Depth Area F Depth Area F F Depth Area F F Depth Area F F Content Content Uptake^ Content Uptake^ Content Uptake^ mm mm^ pgcm^ mm mm^ pgcm^ pgcm^ mm mm^ pgcm^ pgcm^ mm mm^ pgcm^ pgcm^ 1 0.37 0.60 8 0.57 0.92 342 330 0.45 1.45 372 362 0.47 1.13 40 30

2 0.59 0.66 121 0.74 1.06 175 23 0.63 1.23 163 34 0.53 1.05 52 -57

3 0.49 0.81 12 0.51 0.76 145 133 0.73 0.89 230 212 0.47 1.14 48 36

4 0.50 1.02 10 0.56 0.81 99 88 0.53 1.01 124 113 0.64 1.11 18 5

5 0.45 1.17 47 0.46 0.96 318 270 0.48 1.18 229 179 0.45 1.26 32 -15

6 0 53 1.05 5 0.49 1.13 221 216 0.56 1.06 458 453 0.53 1.04 80 75

7 0.55 0.98 51 0.55 1.02 250 199 0.53 0.95 258 209 0.71 0.97 46 -20

8 0.55 1.07 33 0.55 1.02 157 124 0.44 1.33 117 91 0.50 1.16 26 -4

9 0.49 1.39 11 0.59 1.00 265 252 0.43 1.04 212 202 0.48 1.30 38 27

10 0.42 1.10 5 0.42 1.07 439 434 0.63 1.23 276 269 0.63 1.00 65 58

^ For formula see Page 127

Table 9. The sample dimensions, fluoride content and fluoride uptake of dentine samples following exposure to

fluoride gels (1.25% F") in vitro. UNTREATED SODIUM FLUORIDE SOLUTION STANNOUS FLUORIDE SOLUTION AMINE FLUORIDE 297 SOLUTION Depth Area F Depth Area F F' Depth Area F F' Depth Area F F Content Content Uptake^ Content Uptake^ Content Uptake^ mm mm^ pgcm^ mm mm^ pgcm^ pgcm^ mm mm^ pgcm^ pgcm^ mm mm^ pgcm^ pgcm^ 1 0.54 1.37 29 0.42 0.83 193 170 0.32 1.15 113 96 0.52 1.4 414 386

2 0.67 1.11 58 0.30 0.99 48 22 0.48 1.18 97 55 0.6 1.32 375 323

3 0.34 1.18 38 0.34 1.21 120 82 0.41 1.08 93 47 0.38 0.88 426 384

4 0.40 0.86 23 0.44 1.24 149 124 0.41 0.99 141 117 0.5 1.08 435 406

5 0.54 0.96 47 0.47 1.13 403 362 0.39 0.97 113 79 0.36 1.39 435 404

6 0.36 1.07 84 0.38 1.36 110 21 0.41 0.98 365 269 0.54 1.06 311 185

7 0.49 0.89 56 0.40 1.41 117 71 0.53 1.07 121 60 0.53 1.78 177 116

8 0.49 1.31 53 0.53 1.38 243 186 0.53 1.50 230 173 0.56 1.11 374 313

9 0.43 0.91 77 0.53 1.14 118 23 0.40 1.40 250 178 0.65 1.05 400 284

10 0.48 1.34 41 0.51 1.15 187 143 0.54 0.99 374 328 0.42 1.43 325 289

^ For formula see Page 127

Table 10. The sample dimensions, fluoride content and fluoride uptake of dentine samples following exposure to

fluoride solutions (1.25% F") in vitro. MERIDOL SOLUTION MERIDOL GEL ELMEX SOLUTION ELMEX GEL Depth Area F' Content Depth Area F Content Depth Area F Content Depth Area F Content mm mm^ pgcm^ mm mm^ pgcm^ mm mm^ pgcm^ mm mm^ pgcm^

1 0.66 1.40 129 0.46 1.09 60 0.49 1.63 267 0.60 1.13 235 2 0.57 1.37 99 0.39 1.04 48 0.59 1.30 138 0.44 1.95 279 3 0.61 1.38 58 0.45 1.10 30 0.60 1.07 145 0.61 1.30 114 4 0.59 1.20 72 0.51 1.17 23 0.67 1.48 117 0.61 1.18 158 5 0.66 1.85 89 0.54 1.21 70 0.56 1.36 301 0.64 1.48 213 6 0.60 0.92 158 0.54 1.07 47 0.68 1.41 214 0.61 1.87 128 7 0.51 1.33 113 0.46 1.20 38 0.56 1.04 327 0.63 1.50 113 8 0.47 1.81 52 0.57 1.33 41 0.53 1.57 182 0.51 1.63 261 9 0.59 1.53 62 0.35 1.01 54 0.44 1.22 213 0.43 1.25 176 10 0.65 0.93 86 0.56 1.23 53 0.53 1.07 170 0.51 1.37 212

Table 11. The Sample dimensions and fluoride content of dentine samples following exposure to amine fluoride compounds

(1.25% F") in vitro. 338

Table 12. Fluoride uptake by demineralised dentine following application of fluoride gels (1.25% F ) in vitro. a. Group I, II and III - Fluoride released by exposure to potassium hydroxide.

MERIDOL GEL SODIUM FLUORIDE GEL ELMEX GEL Area (mm^ F Content Area (mm^) F Content Area (mm^) F Content

(FQcm*^) 1 9.2 43 10.3 9 7.1 14 2 4.5 21 8.8 5 8.4 18 3 8.5 24 9.9 5 7.4 14 4 9.2 22 7 7 8.6 17 5 9.6 52 10.1 10 10.1 25 6 4.8 21 3.7 10 5.2 16 7 4.4 18 3.6 9 4.6 14 8 4.3 14 3.8 2 4.3 10 9 4.1 12 4.6 4 4.8 7 10 4.3 14 4.3 4 4.9 1

b. Group IV, V and VI - Fluoride released by exposure to hydrochloric acid, remaining fluoride released by potassium hydroxide.

MERIDOL GEL SODIUM FLUORIDE GEL ELMEX GEL Area F Content Area F Content Area F Content (mm^) (mm^) (ngcm'^) (mm^) HCI KOH HCI KOH HCI KOH 1 7.7 17 0.06 7.4 3 0.07 6.2 7 0.08 2 11.2 19 0.1 6.3 7 0.08 9.6 9 0.05 3 8.8 17 0.06 6.5 2 0.08 6.2 5 0.01 4 7.8 12 0.06 9.1 4 0.05 7.1 10 0.07 5 10.1 22 0.05 7.9 8 0.06 10.4 4 0.01 6 4.4 15 1 4.5 2 0.1 5.2 4 0.1 7 4.5 14 1 4.1 2 0.1 4.1 3 0.1 8 3.8 13 0.1 3.8 3 0.1 3.6 3 0.1 9 4.5 10 0.9 5.1 3 0.2 4.6 4 0.2 10 5.5 15 0.5 5.6 2 0.7 4.4 3 0.9 339

Table 13. Fluoride uptake by demineralised dentine following application of fluoride solutions (1.25% F") in vitro.

AMINE FLUORIDE 297 SODIUM FLUORIDE STANNOUS FLUORIDE SOLUTION SOLUTION SOLUTION Area F" Content Area F" Content Area F" Content

(mm^) (pgcm'^) (mm^) (pgcm'2) (mm^) (pgcm'2) 1 4.6 8 3.8 2 3.7 33 2 4.2 8 4.7 1 3.5 40 3 3.8 6 3.8 1 3.8 24 4 5.1 6 4.5 1 4.4 23 5 5.1 11 5.2 1 4.3 33 6 4.5 9 4.4 1 4.1 21 7 4.5 10 4.9 1 4.1 37 8 3.4 9 4.3 2 3.7 37 9 4.4 8 5.1 2 4.3 36 10 3.9 6 4.6 2 3.7 21 340

APPENDIX 6.

PATIENT INFORMATION AND CONSENT FORMS. 341

INDEX OF CONTENTS

Fluoride Uptake by Dentine In Vivo Following Unsupervised Use of Toothpaste and

Daily Mouthrinse for One Month.

Explanatory Statement ...... 342

Consent Form ...... 343

Instructions on Use of the Toothpaste and Mouthrinse...... 344

The Effect of Unsupervised Use of Toothpaste and Daily Mouthrinse for One Year on

Caries Incidence and the Oral Microflora.

Explanatory Statement ...... 345

Consent Form ...... 346

Instructions For the First Appointment...... 347

Instructions on Use of the Toothpaste and Mouthrinse...... 348 342

Fluoride Uptake by Dentine In Vivo Following Unsupervised Use of

Toothpaste and Daily Mouthrinse for One Month.

Explanatory Statement

In an effort to reduce the risk of patients such as yourself developing decay in worn teeth covered by dentures we are carrying out a short clinical trial to evaluate the use of a fluoride toothpaste and mouthwash. Examination of your mouth has revealed that you have worh teeth which will require reshaping or extraction as part of your treatment. Tooth reshaping will be carried out in two stages and the small amount of tooth substance removed will be collected to help us with our studies. Between these two stages you will be asked to use a fluoride toothpaste or amine fluoride toothpaste and mouthwash.

You should understand that the study will in no way interfere with your treatment and that the procedures form part of your treatment. At the end of the study, you will continue your treatment in the hospital as necessary. You may withdraw from the study at any stage in the investigation without affecting the provision of normal care or treatment.

We thank you for your help in our attempts to improve the treatment we provide.

The details of this study have been explained by:

Signature: 343

Fluoride Uptake by Dentine In Vivo Following Unsupervised Use of

Toothpaste and Daily Mouthrinse for One Month.

Consent Form

I,. of

Hereby fully and freely consent to undergo the procedures involved in the clinical research investigation outlined in the explanatory statement, a copy of which is attached to this form. I understand that the investigation is a contribution to medical knowledge and that I may withdraw my consent at any stage in the investigation, without this affecting the provision of my treatment.

I confirm that the nature and purpose of the study have been explained to me, and I have read the written explanation.

(signed):...... date:

Information explained b y : ...... 344

Fluoride Uptake by Dentine In Vivo Following Unsupervised Use of

Toothpaste and Daily Mouthrinse for One Month.

HOW TO USE THIS TOOTHPASTE AND MOUTHWASH!

TOOTH BRUSHING

Please use this toothpaste instead of your usual toothpaste. It is Important that this is the only toothpaste you use and that only you use the toothpaste. When you clean your teeth please squeeze on to your brush a smear of toothpaste so that it covers the length of your brush. Please clean your teeth twice a day in the way you were shown by your dentist or by the hygienist.

MOUTH RINSING

Please use the mouthwash provided once a day last thing at night (after you have brushed your teeth). Please fill the measuring cup to the line and use this amount of mouthwash every time you rinse. Gently rinse, holding the mouthwash in your mouth, for 1 minute. Then spit out all the mouthwash. Please try not to rinse your mouth out, eat or drink for at least half an hour after you have used the mouthwash. This prolongs its effect. It is important that this is the only mouthwash you use and that only you use the mouthwash.

If you think you are going to run out of toothpaste or mouthwash before your next appointment please let us know and we will send you some more.

Thank you very much for following these instructionsi 345

The Effect of Unsupervised Use of Toothpaste and Daily Mouthrinse for One

Year on Caries Incidence and the Oral Microflora.

Explanatory Statement

In an effort to reduce the risk of patients such as yourself developing decay in worn teeth covered by dentures we are carrying out a short clinical trial to evaluate the use of a fluoride toothpaste and mouthwash. If you agree to take part in this project you will be given a supply of conventional fluoride toothpaste and amine fluoride toothpaste and mouthwash to use during the next year. We will need to collect a sample of sahva and plaque from your front teeth once every three months. This will form part of a normal denture review visit.

Although amine fluoride toothpaste has been newly released in this country, they have been widely used in Switzerland and other European countries for almost 30 years. Research to date indicates that these products may be more beneficial in preventing decay than conventional toothpastes.

We thank you for your help in our attempts to improve the treatment we provide.

The details of this study have been explained by:

Signature: 346

The Effect of Unsupervised Use of Toothpaste and Daily Mouthrinse for One

Year on Caries Incidence and the Oral Microflora.

Consent Form

; ...... o f......

I confirm that the nature and purpose of the study have been explained to me, and I have read the written explanation.

(signed):...... date:......

Information explained b y : ...... date: ...... 347

The Effect of Unsupervised Use of Toothpaste and Daily Mouthrinse for One

Year on Caries Incidence and the Oral Microflora.

INSTRUCTIONS FOR YOUR FIRST APPOINTMENT

Please would you;

1. Not clean your teeth or denture after the meal before your appointment

so that I can collect some plaque. I will give you the opportunity to do so

before your mouth is examined.

2. Not use a mouthwash as this can affect the bacteria in your mouth.

3. Keep a note of any antibiotics you are required to take. 348

The Effect of Unsupervised Use of Toothpaste and Daily Mouthrinse for One

Year on Caries Incidence and the Oral Microflora.

HOWTO USE THIS TOOTHPASTE AND MOUTHWASH!

TOOTH BRUSHING

Please use this toothpaste instead of your usual toothpaste. It is important that this is the only toothpaste you use and that only you use the toothpaste. When you clean your teeth please squeeze on to your brush a smear of toothpaste so that it covers the length of your brush. Please clean your teeth twice a day in the way you were shown by your dentist or by the hygienist.

MOUTH RINSING

DENTURE CLEANSING

Instead of your usual denture cleanser please soak your dentures overnight in plain tap water and clean your denture by gently brushing it with the toothpaste and brush provided. When you clean your dentures please squeeze on to your brush a strip of the toothpaste so that it covers the length of your brush. It is important that this is the only way you clean your dentures.

If you think you are going to run out of toothpaste or mouthwash before your next appointment please let us know and we will send you some more.

Please keep a note of any pills, drugs or medicines that you are required to take.

Thank you very much for foiiowing these instructionsi 349

APPENDIX 7.

THE EFFECT OF UNSUPERVISED USE OF TOOTHPASTE AND DAILY MOUTHRINSE FOR ONE YEAR ON THE ORAL MICROFLORA. 350

INDEX OF CONTENTS

Acknowledgement ...... 351 Introduction...... 351 Method ...... 351 Results ...... 352 Table 1. The median and 95% confidence intervals calculated for the plaque total aerobic count, actinomyces count, streptococci count and lactobacilli count, expressed as a percentage of total anaerobic count, following the use of sodium fluoride or Meridol toothpaste and mouthrinse at 0 to 12 months...... 354 Table 2. The median and 95% confidence intervals calculated for the salivary total aerobic count, actinomyces count, streptococci count and lactobacilli count, expressed as a percentage of total anaerobic count, following the use of sodium fluoride or Meridol toothpaste and mouthrinse at 0 to 12 months...... 355 Figure 1. The median and 95% confidence interval calculated for the plaque and salivary total aerobic count, actinomyces count, streptococci count and lactobacilli count, expressed as a percentage of total anaerobic count, following the use of a sodium fluoride or Meridol topical fluoride regimens at 0 to 12 months...... 356 Discussion...... 357 Conclusions...... 358 Bibliography...... 359 351

Acknowledgement

The plating of plaque and saliva samples and counting of colony forming units that formed part of this investigation were carried out by Dr Tracy Burns and Mr Paul Bhuvanenthiran of the Department of Microbiology, Eastman Dental Institute for Oral Health Care Sciences, 256 Gray’s Inn Road, London.

Introduction

A limited number of studies have been carried out to investigate the effect of amino-stannous fluoride toothpaste and mouthrinse regimens on the microflora associated with dentinal caries (Page 68 to 71). The requirement of long term observation for the assessment of medical oral regimens has not been met (Overholser, 1988). A medium term study was therefore carried out to establish the effect of unsupervised application of toothpaste and a daily fluoride mouthrinse regimen on the oral microflora.

Method

Patients participating in the investigation into caries incidence (Chapter 5) were invited to also participate in this investigation. The study design was approved by the Hospital Research and Ethics Committee. All patients gave voluntary, informed, written consent (Appendix 6).

Only patients who had not received antibiotics within 3 months of the start of the study were included. Patients requiring antibiotic cover were also excluded. The patients were asked to keep a diary of medication taken during the course of the study and this was checked at each appointment.

At the time of consent (1 month prior to the baseline appointment) patients were asked to refrain from using a mouth rinse and were asked not to clean their teeth or their dentures after the meal preceding their appointment.

At the baseline appointment 2 ml of unstimulated, whole saliva was collected by expectoration into a sterile plastic universal container. Plaque was then taken 352 with a paper point (Colour free, Size 70, Produits Dentaire, Switzerland) wiped 5 times over the two most mesial sound, un-restored denture abutments and a second point wiped 5 times over the adjacent fitted denture surfaces. The paper points were placed into 1 ml of transport solution^ Both samples were processed immediately as described below.

The patients were reviewed 3 monthly for one year. At each review further plaque and saliva samples were collected.

Microbiological Processes

The samples were vortexed thoroughly for 30 seconds and then 1/10 serial dilutions were prepared in pre-reduced Wilkins Chalgren broth^. These dilutions were then plated in duplicate on to:

1. Wilkins Chalgren blood agar incubated anaerobically for a total anaerobic count^ 2. Wilkins Chalgren blood agar incubated aerobically for a total aerobic count 3. Mitis salivarius agar incubated anaerobically to give streptococci count^ 4. Rogosa agar incubated anaerobically to give lactobacillus count^ 5. Cadmiun fluoride acriflavin tellurite (CFAT) agar incubated anaerobically to give actinomyces count®.

After 5 to 7 days incubation the average colony forming units per millilitre for each media and a percentage of the total anaerobic count were calculated.

Statistical Analysis

Statistical analysis of the data was carried out using the non parametric tests of Mann Whitney with a confidence limit set at 95%.

Results

With reference to drug diaries kept by the patients, plaque and saliva samples were discarded if antibiotics had been taken within 3 months prior to collection. 353

A summary of the median value and 95% confidence interval calculated for the plaque and salivary total aerobic count, actinomyces count, streptococci count and lactobacilli count, expressed as a percentage of total anaerobic count, following the use of a sodium fluoride or Meridol topical fluoride regimens at 0 to 12 months is provided in Table 1 and 2 and Figure 1.

A downward trend on use of the Meridol regimen was noted for plaque percentage total aerobic and salivary percentage actinomyces counts but these differences were not statistically significant except for a significantly lower (p= 0.046) salivary actinomyces count at 9 months in patients using the Meridol regimen. All other counts did not differ significantly between the two regimens at any period. COUNT %TOTAL AEROBIC % ACTINOMYCES % STREPTOCOCCI % LACTOBACILLI

REGIMEN Meridol NaF Meridol NaF Meridol NaF Meridol NaF

BASELINE 84% 64% 45% 44% 45% 56% 0.05% 0.13% (59 to 98%) (46 to 83%) (23 to 73%) (28 to 78%) (25 to 86%) (40 to 78%) (0.01 to 4.44%) (0.01 to 0.92%) 3 MONTHS 65% 68% 51% 35% 73% 49% 0.67% 0.14% (20 to 99%) (37 to 86%) (16 to 73%) (17 to 69%) (18 to 100%) (34 to 66%) (0.10 to 7.33%) (0.02 to 7.20%) 6 MONTHS 63% 85% 43% 83% 61% 58% 1.38% 0.35% (44 to 91%) (61 to 96%) (18 to 100%) (39 to 94%) (44 to 76%) (34 to 66%) (0.00 to 7.00%) (0.01 to 1.63%) 9 MONTHS 65% 69% 31% 19% 37% 44% 0.10% 0.06% (46 to 87%) (40 to 89%) (7 to 63%) (15 to 43%) (17 to 70%) (9 to 53%) (0.00 to 8.44%) (0.01 to 7.04%) 12 MONTHS 63% 65% 35% 42% 51% 58% 0.16% 0.26% (52 to 100%) (44 to 76%) (18 to 61%) 16 to 98%) (33 to 85%) (27 to 77%) (0.11 to 1.51%) (0.00 to 15.89%)

Table 1. The median and 95% confidence intervals calculated for the plaque total aerobic count, actinomyces count, streptococci count and lactobacilli count, expressed as a percentage of total anaerobic count, following the use of sodium fluoride or Meridol toothpaste and mouthrinse at 0 to 12 months. COUNT %TOTAL AEROBIC % ACTINOMYCES % STREPTOCOCCI % LACTOBACILLI

REGIMEN Meridol NaF Meridol NaF Meridol NaF Meridol NaF

BASELINE 71% 72% 56% 52% 71% 69% 0.05% 0.06% (58 to 76%) (50 to 89%) (52 to 64%) (34 to 74%) (57 to 79%) (61 to 84%) (0.02 to 0.39%) (0.01 to 0.3%) 3 MONTHS 62% 54% 48% 48% 60% 56% 0.09% 0.21% (51 to 86%) (39 to 76%) (33 to 69%) (31 to 65%) (33 to 81%) (46 to 61%) (0.01 to 0.18%) (0.02 to 0.7%) 6 MONTHS 81% 70% 48% 40% 70% 57% 0.15% 0.20% (68 to 87%) (53 to 86%) (36 to 61%) (22 to 68%) (27 to 87%) (41 to 79%) (0.02 to 0.59%) (0.01 to 2%) 9 MONTHS 65% 68% 38% 53% 55% 69% 0.03% 0.13% (56 to 73%) (57 to 85%) (22 to 50%) (43 to 76%) (33 to 78%) (52 to 88%) (0.01 to 0.30%) (0.01 to 3%) 12 MONTHS 65% 64% 24% 50% 59% 54% 0.05% 0.09% (40 to 83%) (60 to 81%) (5 to 58%) (31 to 64%) (33 to 77%) (43 to 69%) (0.00 to 0.27%) (0.01 to 1%)

Table 2. The median and 95% confidence intervals calculated for the salivary total aerobic count, actinomyces count, streptococci count and lactobacilli count, expressed as a percentage of total anaerobic count, following the use of sodium fluoride or Meridol toothpaste and mouthrinse at 0 to 12 months. g 356

Figure 1. The median and 95% confidence interval calculated for the plaque and salivary total aerobic count, actinomyces count, streptococci count and lactobacilli count, expressed as a percentage of total anaerobic count, following the use of a sodium fluoride or Meridol topical fluoride regimens at 0 to 12 months.

Total Aerobic Count Actinomyces Count

100% 1 0 0 % -r -

80% -i 80%

.. 40% 4 20% I 3 6 9 12 Months Months

Streptococci Count Lactobacilli Count

100% 20% -r

80% + 15% - 60% -

40% T

20% 1

0% Months 3 6 9 12 Months

1 Plaque Meridol B Plaque NaF

i Saliva Meridol □ Saliva NaF 357

Discussion

The investigation was designed to observe the affect that the topical toothpaste and mouthrinse fluoride regimens had on mutans streptococci, lactobacilli and actinomyces which are the bacteria that have been associated with active root caries (Beighton et al., 1993).

Pooled samples of plaque were taken from the denture and denture abutments to minimise the effect of variation in plaque composition that occurs throughout mouth. However sampling was limited to two abutments to reduce disparity in the amount of plaque collected. Sampling was, therefore, not necessarily from the abutments that developed caries. It is possible that general levels of bacteria in the mouth may be a poor indication of the type or level of infection at the abutment surface. While pooled counts give an indication of the anti bacterial properties of the regimens, samples from each abutment would give a better indication of bacterial levels associated with the risk of developing caries but this would increase the work-load enormously.

There was no significant difference between the Meridol and sodium fluoride regimens on their effects on the oral microflora. This was different to the findings of Hercezegh et al., (1991) who, on investigating identical regimens to this study, found Meridol to result in decreasing salivary mutans streptoccocci and lactobaciili counts although the differences were not significant. Furthermore, anti-bacterial effects have been found following the use of: • Amine fluoride only regimens (Shern et a/., 1974; Gehring, 1983; Salem et a/., 1987) Bullock et a/., 1989; Bansal et a/., 1990; Oosterwaal et a/., 1989, 1991a,b) • Stannous fluoride only regimens (Andres et a/., 1974; Yankell et a/., 1978; Tinanoff et al. 1976; Gross and Tinanoff, 1977; Svanberg and Relia, 1982; McHugh etal., 1988; Miller ef a/., 1994) . Amino-stannous fluoride rinsing regimens (Raul and Netuschil, 1988; Brecx etal., 1990, 1992, 1993; Meurman etal., 1991; Laine etal., 1993)

Microbiological counts in this investigation were presented as percentages of the total anaerobic count. This was because variables that cannot be controlled 358 such as the amount of plaque collected, the dilution of the saliva collected and variations in plating techniques might make total counts meaningless. The results therefore report on differences in the proportions of the oral microflora and it is possible that a reduction in the total bacterial load may have occurred for both or either regimen that was not detectable.

Alternatively, the amine fluoride-stannous fluoride formulations used in this study might be "over-stabilised" resulting in reduced bioavailability of the amine or stannous ions that are thought responsible for the anti-bacterial effects.

Conclusions

Unsupervised use of Meridol or sodium fluoride toothpaste and mouthrinsing regimens over 12 months in patients wearing partial dentures overlaying toothwear resulted in no significant differences in plaque and salivary percentage total aerobic, streptococci, actinomyces and lactobacillus counts. 359

BIBLIOGRAPHY

Andres, C.J., Shaeffer, J.C. and Windeler, A.S., Jr. (1974) Comparison of antibacterial properties of stannous fiuoride and sodium fiuoride mouthwashes. J. Dent Res. 53, 457- 460. Bansal, G.S., Newman, H.N. and Wilson, M. (1990) The survival of subgingival plaque bacteria in an amine fluoride-containing gel. J. Clin. Periodontol. 17,414-418. Beighton, D., Lynch, E. and Heath, M.R. (1993) A microbiological study of primary root-caries lesions with different treatment needs. J. Dent. Res. 72,623-629. Brecx, M., Netuschil, L , Reichert, B. and Schreil, G. (1990) Efficacy of Usterine, Meridol and chlorhexidine mouthrinses on plaque, gingivitis and plaque bacteria vitality. J. Clin. Periodontol. 17, 292-297. Brecx, M., Brownstone, E., MacDonaid, L , Gelskey, S. and Cheang, M. (1992) Efficacy of Usterine, Meridol and chlorhexidine mouthrinses as supplements to regular tooth cleaning measures. J. Clin. Periodontol. 19, 202-207. Brecx, M., Macdonaid, L.L., Legary, K., Cheang, M. and Forgay, M.G. (1993) Long-term effects of Meridol and chlorhexidine mouthrinses on plaque, gingivitis, staining, and bacterial vitality. J. Dent. Res. 72, 1194-1197. Bullock, S., Newman, H.N. and Wilson, M. (1989) The in-vitro effect of an amine fiuoride gel on subgingival plaque bacteria. J. Antimicrob. Chemother. 23, 59-67. Gehring, F. (1983) Wirkung von aminfluorid und natriumfluorid auf keime der piaqueflora. Dtsch. Zahnârztl. Z. 38 (Suppi 1), 836-40.

Gross, A. and Tinanoff, N. (1977) Effect of SnF2 mouthrinse on initial bacterial colonization of tooth enamel. J. Dent. Res. 56,1179-1183. Herczegh, A., Gombik, A., Rost, M., Wierzbicka, M. and Banoczy, J. (1991) Aminfluorid es onfluorid tartalmu fogkrem es szajoblito mikrobiologiai hatasossaganak vizsgalata. Fogorvosi Sezmie 84,181-184. Laine, P., Meurman, J.H., Murtomaa, H., Lindqvist, C., Torkko, H., Pyrhonen, S. and Teerenhovi, L. (1993) One-year trial of the effect of rinsing with an amine fiuoride- stannous-fiuoride-containing mouthwash on gingival index scores and salivary microbial counts in lymphoma patients receiving cytostatic drugs. J.Clin. Periodontol. 20, 628-634. McHugh, W.D., Eisenberg, A.D., Leverett, D.H. and Jensen, G.E. (1988) The long-term effects of daily rinsing with stannous fiuoride or sodium fluoride on bacteria in dental plaque. Pediatr. Dent. 10,10-12. Meurman, J.H., Laine, P., Murtomaa, H., Lindqvist, C., Torkko, H., Teerenhovi, L. and Pyrhonen, S. (1991) Effect of antiseptic mouthwashes on some clinical and microbiological findings in the mouths of lymphoma patients receiving cytostatic drugs. J. Clin. Periodontol. 18, 587-591. Miller, S., Truong, T., Heu, R., Stranick, M., Bouchard, D. and Gaffar, A. (1994) Recent advances in stannous fluoride technology: antibacterial efficacy and mechanism of action towards hypersensitivity. Int. Dent J. 44, 83-98. 360

Oosterwaal, P.J., Mikx, F.H., van den Brink, M.E. and Renggii, H.H. (1989) Bactericidal concentrations of chlorhexidine-digluconate, amine fluoride gel and stannous fluoride gel for subgingival bacteria tested in serum at short contact times. J. Periodontal. Res. 24, 155-160. Oosten/vaal, P.J., Mikx, F.H., van 1 Hof, M.A. and Renggii, H.H. (1991a) Comparison of the antimicrobial effect of the application of chlorhexidine gel, amine fluoride gel and stannous fluoride gel in debrided periodontal pockets. J. Clin. Periodontol. 18,245-251. Oosterwaal, P.J., Mikx, F.H., van t Hof, M.A. and Renggii, H.H. (1991b) Short-term bactericidal activity of chlorhexidine gel, stannous fluoride gel and amine fluoride gel tested in periodontal pockets. J. Clin. Periodontol. 18, 97-100. Raul, T. and Netuschil, L. (1988) In vitro and in vivo antibacterial effect of amine fluoride/stannous fluoride. J. Dent. Res. 67, 703(Abstract) Salem, A.M., Adams, D., Newman, H.N. and Rawle, L.W. (1987) Antimicrobial properties of 2 aliphatic amines and chlorhexidine in vitro and in saliva. J. Clin. Periodontol. 14, 44-47. Shem, R.J., Rundell, B.B. and Defever, C.J. (1974) Effects of an amine fluoride mouthrinse on the formation and microbial content of plaque. Helv.Odontol. Acta 18, 57-62.

Svanberg. M. and R 0 lla, G. (1982) Streptococcus mutans In plaque and saliva after

mouthrinsing with SnF2 . Scand. J. Dent. Res. 90,292-298.

Tinanoff, N., Brady, J.M. and Gross, A. (1976) The effect of NaF and SnF 2 mouthrinses on bacteriai colonisation of tooth enamel: TEM and SEM studies. Caries Res. 10,415-426.

Yankell, S.L., Paskow, E.W. and Shem, R.J. (1978b) Effects of Snp 2 mouthrinses on plaque microbiology. J. Dent. Res. 57, 352(Abstract) 361

^ Formula for Reduced Transport Fluid.

7.5 ml 0.6% dibasic potassium phosphate solution 7.5 ml Solution of 1.2g potassium chloride, 1.2g ammonium sulphate, 0.6g monobasic potassium phosphate, 1ml 2.5% magnesium sulphate solution 0.5 ml 0.8% sodium carbone solution 81.5 ml Distilled water Autoclave, cool, add: Filtered sterilised solution of 0.02g dithiothreitol in 5 ml distilled water. (All chemicals from BDH, U.K.)

^ Formula for Wilkins Chaigren Anerobe Broth. 33g Wiikins Chaigren agar powder (Oxoid, U.K.) 1 litre distiiied water

^ Fomnula for Wilkins Chalgren Blood Agar. 43g Wilkins Chaigren agar powder (Oxoid, U.K.) 1 litre distiiied water Cooi, add: 50 ml horse blood (Oxoid, U.K.)

^ Fonmuia for Mitis Saiivarius Agar 90g Mitis Saiivarius Agar powder (Difico, U.K.) 1 litre distiiied water Cool, add: 1 ml Chapman Tellurite solution (Difico,U.K.)

® Fonmuia For Rogosa Agar 82g Rogosa agar powder (Oxoid, U.K.) 1 litre distiiied water Boil, add: 1.32 ml glacial acetic acid (Sigma, U.K.) Boil 3 minutes, cover, cool. 362

^ Formula For Cadmiun Fluoride Acriflavin Tellurite Agar 30g tryptone soya broth powder (Oxoid, U.K.) 15g agar technical powder (Oxoid, U.K.) 5g glucose (Sigma, U.K.) 1 litre distilled water Cool, add 50 ml sheep blood (Oxoid, U.K.) filtered, sterilised cadium sulphate (Sigma, U.K.) 13 mg in 1 ml distilled water filtered, sterilised sodium fluoride (Sigma, U.K.) 80 mg in 1 ml distilled water filtered, sterilised neutral acriflavin (Sigma, U.K.) 1.2 mg in 1 ml distilled water filtered, sterilised potassium tellurite (Difco, U.K.) 2.5 mg in 1 ml distilled water filtered, sterilised basic fuschin (Sigma, U.K.) 0.25 mg in 1 ml distilled water Corrigenda - Text

Page 15 Line 4 Replace “Consultants Restorative Dentistry" with “Consultants In Restorative Dentistry” Line 23 Delete “a” from “with a prosthetic teeth” Line 34 Replace “Gankaseer" with “Gankerseer" Page 16 Line 24&27 Replace “nickel/chrome" with “nickel/chromium" Page 20 Line 21 Replace “(1967)” with “(1976)" Page 25 Line 15 Replace “(1967)” with “(1976)” Page 24 Line 26 Replace “(Fejerskov et al., 1991)” with “according to Fejerskov et a!., 1981” Page 26 Line 31 Replace “remlneralistlon” with “remlneraiisation” Page 27 Line 12 Replace “(1967)” with “(1976)” Page 29 Line 13 Replace “(Murray, 1996)” with “(Murray and Naylor, 1996)” Line 28 Replace “1991” with “1991a” Page 32 Line 2 Replace “Groat” with “Groat et al.” Page 33 Line 36 Replace “an” with “as” Page 43 Line 13 Replace “has” with “had” Line 34 Replace “Groat” with “Groat et al.” Page 49 Line 29 Replace “exposure of fluoride” with “exposure to fluoride" Pp 54-59 Section 5.4 Replace “Appendix 3” with “Appendix 2" Page 54 Line 29 Replace “Antllia” with “Antlla” Page 55 Line 10 Replace “Stiibig" with “Strübig” Page 58 Line 4 Delete “following” Lines 5-6 Delete “has been reported’ Line 7 Replace “to increase” with “increased” Page 59 Line 6 Delete “that" Page 66 Line 2 Replace “Wade et al. (1995)” with “Wade et al. (1997)" Line 25 Replace “bioavalibility” with bioavaiiabllity” Page 67 Line 20 Replace “recored” with “recorded” Pp 72-77 Section 5.7 Replace “Appendix 2” with “Appendix 3” Page 73 Line 23 Replace “have” with “has” Page 74 Line 17&18 Replace “Rosln-Grgret" with “Rosin-Grget” Page 75 Line 15 Replace “Appendix 2, Table 2B" with Appendix 3, Table 4 Page 76 Line 4 Replace “Appendix 2, Table 3” with “Appendix 3, Table 5” Page 89 Line 12 Replace “form” with “from” Page 91 Line 28 Replace “1969” with “1968” Page 98 Line 12 Replace “use" with “use of Page 99 Line 9 Add reference “(Faiier et al., 1997)” Page 115 Line 16 Replace “0.02 ppm F"” with “0.002 ppm F" Page 116 Line 17 Replace “(Table 7)” with “(Table 6 and 7)" Page 117 Line 26 Replace “promotion” with “promote” Page 127 Line 3 Replace “sodium and stannous fiuoride” with “sodium fluoride. Amine Fiuoride 297 and stannous fluoride” Page 142 Line 13 Replace “Eacg” with “Each” Page 149 Line 15 Replace “at baseline.” With “at baseline (page 127).” Page 157 Line 4 Replace “(p = 0.39)” with “(p = 0.31)" Page 163 Line 29 Replace “were” with “where” Page 165 Line 33 Replace “(p = 0.38)” with “(p = 0.71)” Page 173 Line 31 Replace “(Todd and Lader,1991)” with “(Todd and Lader,1991b)” Page 178 Line 20 Replace “(p = 0.63)” with “(p = 0.097)" Line 21 Replace “(p = 0.097)" with “(p = 0.63)” Page 181 Line 9 Replace “comparison” with “control" Page 182 Line 3 Delete “highly” Page 186 Line 9 Replace “Table 11” with “Table 10” Line 23 Delete “highly” Page 189 Line 27 Delete “highly” Page 190 Line 32 Replace “Table 14" with “Table 11” Page 192 Line 18 Replace “(p = 0.00036)” with “(p = 0.022)” Page 194 Line 3 Replace “However,” with “The” Line 5 Replace “and this difference was highly significant” with “but this difference was also not significant” Page 197 Line 22 Replace “Figures 22 to 41” with “Figures 22 to 35” Page 199 Line 33 Replace “of with “on” Page 210 Line 18 Replace “hydrochloric acid, removed" with “hydrochloric acid for 24 hours, removed” Page 211 Line 15 Replace “24 hours was” with “24 hours for Groups IV, V and VI released by 1M KOH was’ Page 216 Line 1 Delete “highly” Page 221 Line 31 Replace “biovailability” with “bioavailability” Corrigenda - References

Insert References; Berkovitz, B.K.B., Holland, G.R. and Moxham, BJ. (1992) A colour atlas and textbook of oral anatomy, histology and embryology, 2nd edn. pp. 130. London: Wolfe Publishing Ltd. Brudevold, F., McCann, H.G., Nilsson, R., Richardson, B. and Coklica, V. (1967) The chemistry of caries inhibition. Problems and challenges in topical treatment. J. Dent. Res. 46, 37-45.” DePaola, P.P., Soparkar, P., Foley, S., Bookstein, F. and Bakhos, Y. (1977) Effect of high concentration ammonium and sodium fluoride rinses on dental caries in schoolchildren. Community Dent Oral Epidemiol 5, 7-14. Gankerseer, E.J. (1987) A new technique for the treatment of the severely worn dentition. RestorDentZ, 13-22. Herczegh, A., Gombik, A., Rost, M., Wierzbicka, M. and Banoczy, J. (1991) Aminfluorid es onfluorid tartalmu fogkrem es szajoblito mikrobiologiai hatasossaganak vizsgalata. Fofiro/vos/Sezm/e 84,181-184.

Keene, H.J., Shklair, I.L. and Mickel, G.J. (1977) Effect of multiple dental floss-Snp 2 treatment on Streptococcus mutans in interproximal plaque. J. Dent Res. 56,21-27. McCann, H.G. (1968) Determination of fluoride in mineralised tissues using the fluoride ion electrode. Archs Oral Biol 13,475-477. Murray, J.J. and Naylor, M.N. (1996) Fluorides and dental caries. In: Murray, J.J. (Ed.) The prevention of oral disease, 3rd edn. pp. 34-67. Oxford: Oxford University Press Myers, H.M. (1968) A hypothesis conceming the caries-preventive mechanism of tin. J. Am. Dent. Assoc. 77,1308-1315. Rolla, G. and Ellingsen, J.E. (1994) Clinical effects and possible mechanisms of action of stannous fluoride. Int. Dent. J. 44,99-105. Shannon, I.L. and Wightman, J R. (1970) Treatment of root surfaces with a combination of acidulated phosphofluoride and stannous fluoride. J. La. Dent. Assoc. 28,14-17. Singer, L and Armstrong, W.D. (1968) Determination of fluoride in bone with the fluoride electrode. Anal. Chem. 40,613-614. Todd, J.E. and Lader, D. (1991b) Adult Dental Health Survey of the United Kingdom, 1988. pp. 334. London: HMSO. Tsanidis, V. and Koulourides, T. (1992) An in vitro assessment of fluoride uptake from glass ionomer cements by dentine and its effects on acid resistance. J Dent Res 71, 7-12. Weatherell, J.A., Hallsworth, A.S. and Robinson, C. (1973) The effect of tooth wear on the distribution of fluoride in the enamel surface of human teeth. Archs Oral Biol 18,1175- 1189. Yamaga, T. and Nokubi, T. (1997) Clinical observations of noncoping overdenture abutments protected by tannin-fluoride preparation. J. Prosthet. Dent 78,315-319. Delete References: Page 228 Lines 33-36 Beiswanger ef a/. (1995) Page 230 Lines 7-8 Bouwsma (1996) Page 231 Lines 8-9 Bruun etal. (1984) Page 232 Lines 38-39 Cruz and Relia (1992) Page 236 Lines 1-2 Galan etal. (1995) Page 237 Lines 13-14 Hals (1991) Page 239 Lines 28-29 Johnson and Silvers (1987) Page 242 Lines 34-35 Mazza etal. (1981) Page 255 Lines 8-9 Wilson and Grant (1989) Page 226 Line 10 Replace “for tooth" with “for human tooth” Line 18 Replace “tootpastes" with “toothpastes" Line 21 Replace “dentrifces” with “dentifrices" Page 227 Line 6 Replace “fifteeth" with “fifteenth" Page 229 Line 11 Replace “Zander,H.G.," with “Zander,HA,” Page 231 Line 38 Replace “Principle" with “Principal” Page 232 Line 22 Replace ”153” with “154” Page 234 Line 6-8 Replace “1988a" with “1988b” Line 9-10 Replace “1988b” with “1988a” Line 18 Replace “Afsth” with “Afseth" Line 19 Replace “Rolla,G.” with “Qvist,V.” Page 235 Line 5 Replace “methadology” with “methodology” Line 7 Replace “Qviest" with “Qvist” Line 8 Insert “pp. 127-137.” Line 14 Replace “518” with “397" Line 15 Replace “(1990a)" with “(1990)" Page 237 Line 23 Replace “Hassell, T.M.” with “Hassell, T.M, Gabathuler, H. and Miihlemann, H.R." Line 39 Replace “Driscoll, W.C." with “Driscoll, W.S.” Line 40 Replace “phsphate” with “phosphate” Page 238 Line 23 Replace “Oral Epidemiol.” with “Health” Page 239 Line 26 Replace “Johannson” with “Johansson" Page 241 Line 35 Replace “(1993a)" with “(1993)" Line 36 Replace “and Leach, S.A.” with “Leach, S.A. and Qvist, V. Page 243 Line 3 Replace “75-85” with “78-85" Page 245 Line 20 Replace “86,108-117.” with “84,20-28.” ' Line 22 Replace “84,20-28." with “86,108-117.” Page 246 Line 40 Replace “(1967)” with “(1976)" Page 247 Line 5 Replace “Ratanen" with “Rantanen” Line 38 Insert “2nd edn. pp. 69-83.” Page 249 Line 36 Replace “(1981b)” with “(1981)” Line 38 Replace “saliva” with “serum” Page 250 Line 31 Replace “Huntingdon” with “Huntington" Page 251 Lines 13,15,17 Replace “Svatun” with “Svantun" Page 252 Line 10 Replace “(1991)" with “(1991a)” Line 12 Replace “(1982a)" with “(1982)" Line 21 Replace “Torrell” with “Torell” Line 34 Replace “54" with “52” Page 255 Line 20 Replace “comosition” with “composition" Page 256 Line 3 Replace “Pedersen, A.M., Hayes, A.L.” with “Pedersen, A.M., Fu, J., Hayes, A.L” Line 9 Replace “Flores de Jacoby, L. and Pan, P." with “Flores-de-Jacoby, L., Pan, P. and Pan, P.” Corrigenda - Appendices

Page 257 Insert footnote Key; NR = Not Recorded Pp260-265 Replace “Astrad" with “Astrand” Replace “Ratenen" with “Rantanen" Replace “Franz" with “Frantz” Page 262 Title Replace “C. Longitudinal” with “B. Longitudinal” Page 265 Line 27 Replace “efterkontrll" with “efterkontroll" Page 289 Line 17 Replace “(1956b)" with “(1956)" Page 290 Line 1 Replace “(1982b)” with “(1982)” Page 296 Row 2 Replace “1979" with “1978” Page 300 Title Replace “C. Amine” with “B. Amine" Page 304 Line 9 Replace “Birkhed” with “Birkhead" Line 12 Replace “B159" with “B195" Page 307 Line 5 & 8 Replace “denitine” with “dentine" Page 311 Table 5a Parameter Analysis Significance Fluoride Kruskal Wallià HS (p = 0.006) Loss (Ho true if KW i 11.07; KW =16.52) Surface Mann Whitney NS (p = 0.07) Area

Page 312 Table 6a Insert at end Row 2 Fluoride Content: Meridol Untreated v Treated HS (p = 0.0004) NaF Untreated v Treated HS (p = 0.0044)

Page 315 Table 9a Insert at end Row 5 Fluoride Content Fr= 24.12 7.82 HS (p = 0.000) Insert Table 9c c. Multiple comparisons between samples for fluoride content. Difference between average rankings and significance where: Critical Difference (p = 0.05) = 15.24 NaF Gel Elmex Gel Meridol Gel Control 21.0 21.0 2.0 S(p = 0.019) 8 (p = 0.019) NS

Page 316 Table 10 a Insert at end Row 5 Fluoride Content Fr = 22.20 7.82 HS (p= 0.000)

Table 10b Replace “Elmex Solution" with “SnF 2 Solution" Replace “Meridol Solution" with “AmF 297 Solution" InsertTablelOc c. Multiple comparisons between samples for fluoride content. Difference between average rankings and significance where: Critical Difference (p = 0.05) = 15.24 NaF Solution SnFz Solution AmF 297 Solution Control 16.0 18.0 27.0 S (p = 0.034) S(p = 0.011) HS (p = 0.00036) Page 317 Table 10b Title: Replace “surface area" with “fluoride content" Row 4: Replace “Meridol Gel" with “Elmex Gel” Replace “(p = 0.022)" with “(p = 0.00036) Page 318 Table 10c Title: Replace “Critical Difference = 10.7* with “Critical Difference = 13.79" Row 4: Replace “Meridol Gel" with “Elmex Gel" Table lOd Title: Replace “fluoride content” with “surface area” Replace “Critical Difference=10.7“ with “Critical DifferencezlS.Ty Row 4: Replace “Meridol Gel" with “Elmex Gel" Page 319 Table 12a Title: Replace “denitine" with “dentine" Row 6: Replace “HS" with “NS" Page 320 Table 13a Title: Replace “denitine" with “dentine” Row 3: Replace “NS" with “S” Page 322 Line 30 Title: Add “fluoride gels (1.25% F ) in vitro” Line 32 Title: Add “fluoride solutions (1.25% F") in vitro” Line 34 Title: Add “(1.25% F") in vitro” Page 325 Title Replace “c. Sodium fluoride” with “c. Amine Fluoride 297" Page 342 Line 8 Replace “worh” with “worn” Page 357 Line 21 Replace “Hercezegh" with “Herczegh” Page 360 End Line 10 Insert reference: “Overholser, C.D., Jr. (1988) Longitudinal clinical studies with antimicrobial mouthrinses. J.Clin. Periodontol. 15, 517-519.”