State Of The Art And Future Perspectives

Cariology in the 21st Century State of the Art and Future Perspectives Proceedings of a Symposium held at the 50th Anniversary ORCA Congress, July 2–6, 2003, Konstanz, Germany

Guest Editors

B. Nyvad, Aarhus J.M. ten Cate, Amsterdam C. Robinson, Leeds

28 figures, 4 in color, and 21 tables, 2004

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Vol. 38, No. 3, 2004

Contents

167 Introduction Nyvad, B. (Aarhus); ten Cate, J.M. (Amsterdam); Robinson, C. (Leeds)

254 Fluorides in Caries Prevention and Control:

168 Clinical Manifestations and Treatment of Caries

258 Systemic versus Topical Fluoride Hellwig, E.; Lennon, Á.M. (Freiburg/Göttingen)

from 1953 to Global Changes in the 20th Century König, K.G. (Nijmegen) 173 Changes in Dental Caries 1953–2003 Marthaler, T.M. (Zurich) 182 Changing Paradigms in Concepts on Dental Caries:

Consequences for Oral Health Care Fejerskov, O. (Aarhus)

Empiricism or Science ten Cate, J.M. (Amsterdam)

263 How to Improve the Effectiveness of Caries-

Preventive Programs Based on Fluoride Hausen, H. (Oulu) 268 The Effect of Fluoride on the Developing Tooth Robinson, C.; Connell, S.; Kirkham, J.; Brookes, S.J.; Shore, R.C.; Smith, A.M. (Leeds)

192 Diagnosis versus Detection of Caries Nyvad, B. (Aarhus)

277 Sugars – The Arch Criminal? Zero, D.T. (Indianapolis, Ind.)

199 Diagnostic Levels in Dental Public Health Planning Ismail, A. (Ann Arbor, Mich.)

286 Sugar Alcohols: What Is the Evidence for Caries-

204 Dental Plaque as a Microbial Biofilm Marsh, P.D. (Salisbury) 212 Application of the Zürich Biofilm Model to Problems

of Cariology Guggenheim, B.; Guggenheim, M.; Gmür, R. (Zürich); Giertsen, E. (Bergen); Thurnheer, T. (Zürich) 223 Antimicrobials in Future Caries Control? A Review

with Special Reference to Chlorhexidine Treatment Twetman, S. (Umeå) 230 A Caries Vaccine? The State of the Science of

Immunization against Dental Caries Russell, M.W. (Buffalo, N.Y.); Childers, N.K.; Michalek, S.M. (Birmingham, Ala.); Smith, D.J.; Taubman, M.A. (Boston, Mass.) 236 How Much Saliva Is Enough for Avoidance of

Xerostomia?

Preventive and Caries-Therapeutic Effects? van Loveren, C. (Amsterdam) 294 Are We Ready to Move from Operative to

Non-Operative/Preventive Treatment of Dental Caries in Clinical Practice? Pitts, N.B. (Dundee) 305 How ‘Clean’ Must a Cavity Be before Restoration? Kidd, E.A.M. (London) 314 The Future Role of a Molecular Approach to Pulp-

Dentinal Regeneration Tziafas, D. (Thessaloniki) 321 Getting Research into Clinical Practice – Barriers and

Solutions Clarkson, J.E. (Dundee) 325 Summaries of Discussions at 50th Anniversary

ORCA Symposium

Dawes, C. (Winnipeg) 241 Salivary Enhancement Therapies Fox, P.C. (Charlotte, N.C.)

330 Announcement

247 Salivary Proteins: Protective and Diagnostic Value in

331 Author Index 332 Subject Index

Cariology? van Nieuw Amerongen, A.; Bolscher, J.G.M.; Veerman, E.C.I. (Amsterdam)

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Caries Res 2004;38:167 DOI: 10.1159/000077750

Introduction

In July 2003, the European Organization of Caries Research (ORCA) celebrated its 50th anniversary. This event was marked by a special symposium in connection with the 50th annual congress, held at ORCA’s birthplace, in Konstanz, Germany. The title of the symposium was ‘Cariology in the 21st century – state of the art and future perspectives’. The current issue of Caries Research is devoted to the proceedings of this symposium. The symposium aimed to provide an update on the core issues of cariology and included aspects of diagnosis, prevention and management of the disease. In accordance with the objectives of ORCA, a session was specifically devoted to the problem of how to translate research findings into clinical practice. The format of the symposium was based on short presentations, in which each presenter was asked to review the literature within a certain field and to generate suggestions for future research. Presenters were specifically asked to address the potential impact of the research on clinical practice. Each session was followed by a structured discussion around designated themes. A short summary of the discussions is placed at the end of this issue. The purpose of publishing this special issue of Caries Research is to make the knowledge generated at the symposium available to as broad an audience as possible. Bente Nyvad, Århus J.M. (Bob) ten Cate, Amsterdam Colin Robinson, Leeds Symposium Organizers and Guest Editors

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Caries Res 2004;38:168–172 DOI: 10.1159/000077751

Clinical Manifestations and Treatment of Caries from 1953 to Global Changes in the 20th Century K.G. König Department of Preventive and Community Dentistry and Pedodontology, University Medical Center St. Radboud, Nijmegen, The Netherlands

Key Words Caries manifestations W Caries treatment W Caries risk W Caries prevention

Abstract Manifestations and treatment of caries are strongly dependent on caries risk and the severity of the attacking factors which determine the degree of caries activity. Caries activity in turn will be modified and can be minimized by effective preventive measures. Exemplary cases and events will be discussed to illustrate what has happened since the establishment of ORCA 50 years ago. Copyright © 2004 S. Karger AG, Basel

Relation of Caries Manifestations and Treatment to Caries Risk and Prevention

The clinical manifestations of caries and the principles of caries treatment are closely interrelated, and both depend on caries risk and caries activity. In 1953, at the University of Würzburg, caries treatment was still being taught according to the principles laid down by G.V. Black [1914]. Hand instruments were used

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especially for opening a cavity, and for ‘extension for prevention’ according to the risk of future (secondary) manifestations of caries. The less clean a mouth was, the larger the box of a cavity had to be both buccally and lingually [Black, 1899; Hofheinz, 1902]. Cavity preparation according to the local microenvironment was one of Black’s important treatment principles. It illustrates the fact that treatment must be guided not only by the manifestations of caries, but also by the assessment of the individual’s caries risk. As we well know, this risk depends on the preventive agents and services which are available to the individual and the extent to which these are used effectively by the patient. After World War II, in both West and East, the reindustrialization of a starving Europe and the boom which followed led to an overconsumption of sweets and many other types of luxury food. Tito’s Yugoslavia spent nearly all its Western hard currencies to buy wheat from the West, simply because Tito’s people associated white bread with progress, and it was necessary to demonstrate that in a modern socialist country progress was actually taking place. In particular, sugar consumption in many countries almost reached a level of 1 kg per person per week, and this combined with the lack of good oral hygiene resulted in a tremendous rise in the incidence of caries. Adequate

Prof. Dr. K.G. König Weezenhof 2906 NL–6536 HM Nijmegen (The Netherlands) Tel. +31 24 3449624, Fax +31 24 3442019 E-Mail [email protected]

individual dental care became nearly impossible, not least, for instance, in the juvenile populations of Switzerland. In its mountainous areas dentitions were so bad that a most welcome wedding gift was not the usual dowry, but payment for the extraction of all teeth, with the subsequent provision of full dentures. This radical approach solved two problems once and for all: firstly, there would be no recurrent problem of toothache occurring in midwinter in a high mountain valley, and, secondly, the elimination of a potential financial burden on young husbands, in the form of recurring high dentist’s bills. The same sort of ‘caries treatment’, that is full clearance, was also practiced and well documented in Scotland between 1967 and 1972.The numbers of teeth sacrificed especially in people with no previous denture experience are shocking. In 52% of the subjects 21 or more teeth were extracted and full dentures fitted. Many patients were less than 30 years old when a full clearance was performed [Todd and Whitworth, 1974; Marthaler, 2002]. Obviously in post-war Europe there was not only the problem of individual dental care, but a public health problem plaguing entire populations from birth to death. That was the desperate state of dental conditions when the ORCA was founded in 1953. While the dental professions found exodontism unacceptable, the shortage of dentists could not be alleviated instantly – so disease prevention needed to be practiced on the largest scale possible.

Influence of Prevention versus Non-Prevention on Caries Manifestations and Treatment

The topic of this paper is the manifestations and treatment of caries, and not prevention and its history over the past 50 years. It is, nonetheless, essential to direct attention to prevention because effective preventive measures have clearly influenced manifestations of caries and, secondarily, treatment principles. The preventive effect of fluoride on caries had been unequivocally established by Trendley Dean and his co-pioneers [Dean et al., 1942]. By 1953, even in Europe there was no doubt that this was the key to preventing caries. The original name of ORCA chosen in 1953 was ‘Organisme Européen de Coordination des Recherches sur le Fluor et la Prophylaxie de la Carie Dentaire’ or ‘European Association for the Coordination of Research on Fluoride and Prevention of Dental Caries’. This was logical both as a name and as a programme. North American investigators had shown that fluoride (in drinking waters) would

Manifestions, Treatment and Prevention of Caries

reduce the number of new caries lesions, but there was more: a prominent ORCA member, Dr. Gutherz, a school dentist in Basel, had a graph composed which he used widely in the early 1960s when he was fighting for the introduction of water fluoridation in his Kanton. It showed that not only was the number of lesions halved by fluoride, but that lesions were much smaller. This would permit minimally invasive cavity preparation, as opposed to the more difficult, if not impossible, larger cavity preparations needed in the absence of fluoride. In Zürich the school dentists applied – as a treatment principle – the extraction of all four first permanent molars soon after their eruption; this was done for three reasons: (1) The molars became carious with rapid progression which had already begun during eruption, so that when the child saw the dentist it was often too late for successful treatment. (2) Usually, due to premature loss of deciduous molars, before the age of 6 the space for the erupting premolars and canines had already been lost. (3) Rather than keep the ruins of four heavily filled teeth in a child’s mouth, the first molars were extracted early giving the second and third molars a much better chance to survive in proper function. It was questionable whether these arguments were valid but, that apart, the very fact that systematic extraction of all first molars was practiced on a large scale as a treatment strategy in Switzerland illustrates how serious the caries problem was in the 1950s and 60s. So extreme an approach to treatment of caries and its sequelae would otherwise never have been considered. In the Netherlands (Nijmegen) in 1969 during the baseline examination of approximately 1,000 children aged 7 years, it was very obvious that all the dental practitioners had adopted the principle not to fill any deciduous teeth and the amalgam fillings in the permanent molars were unpolished [Plasschaert and König, 1973]. The explanation was that The Netherlands (like Belgium) had the lowest dentist:population ratio in Europe, 1:4,000, and the need and unmet demand for dental treatment was tremendous. As a matter of fact, in the Nijmegen sample of nearly 1,000 children only 3 were found to be cariesfree. This may not have been representative, but a health education slide series issued by the ‘Ivory Cross’, also in 1969, described a situation which was not very much better than the one in Nijmegen [Van zoet naar zuur, 1969]. Especially sad was the fact that exodontism rather than repair resulted in too many young adults being edentulous.

Caries Res 2004;38:168–172

169

Water Fluoridation Effects versus Caries Decline in Non-Fluoridated Areas

In the Netherlands public health dentists under the leadership of ORCA Honorary Member Otto Backer Dirks had been fighting for water fluoridation since 1953. In 1953 fluoridation of drinking water was started in the city of Tiel on an experimental basis; in 1970 the gradual increase in the number of fluoridated areas resulted in nearly half the population of the Netherlands receiving fluoridated water. However, due to the untiring efforts of antifluoridationists, fluoridation stopped in the whole country after a supreme court decision in 1973 [Kalsbeek and Verrips, 1990]. This generated great concern amongst the dental professionals because everybody was afraid that the deplorable situation of dental ill health experienced in the 1950s and 60s would recur. But this miraculously did not happen; on the contrary, the changes in the average DMFT of 12-year-old children over the whole country show that the decrease of caries prevalence had only just started when water fluoridation ceased in 1973; the average DMFT of 8 at that time in 12-year-old children consistently decreased to a DMFT of 1 by the mid 1990s [Truin et al., 1994, 1998; Marthaler, 2003]. The most plausible reason for this success is that dental health educators and enlightened mothers and fathers were alarmed when they realized that after water fluoridation stopped good dental health could no longer be obtained out of their water taps but needed their individual effort. It was therefore obvious that everybody had to become active and apply preventive measures on an individual basis. The results of this in the Netherlands were quite unexpected. Although health educators had persistently and clearly said ‘eat less sugar’, this did not happen. Sugar consumption did not decrease substantially: being 38.5 kg per person per year in 1985, and the same quantity in 1992, it was still more than 90% of what it had been in 1965. Therefore the improvement in dental health could not be attributed to improvement in our eating habits. It also could not be attributed to administration of fluoride supplements, because the sales of fluoride tablets had always been low and were decreasing continuously. Topical application of fluoride gels and growing popularity of sugarless chewing gum may have contributed, but the most important reason for the improvement was a rapid spread of good oral hygiene habits and the use of fluoride toothpastes. What certainly played a role in this success story was the fact that from the early 1950s preventive dentistry was embraced by dental professionals,

170

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the community health authorities, and academic dentistry. By 1969 all five dental schools in the Netherlands had a chair in this discipline. Moreover, individual communities had started large-scale health education campaigns, covering not only the importance of healthy nutrition, but also stressing the great importance of oral hygiene. At the same time, hygiene in general became socially desirable. Since the mid-1980s most children have had very little or no plaque and are using a fluoride-containing toothpaste. The analysis of the Delphi investigation into the reasons for the decline in caries by Bratthall et al. [1996] showed that an overwhelming majority, 96% of the 55 experts, thought that the use of fluoride toothpastes was ‘very important’ or ‘important’. Regarding sugar consumption, 87% of the experts thought this was of little or no importance.

Sugar Intake as a Caries Risk Factor

Sreebny in 1982 had published an analysis of the sugarcaries relationship. He had based the analysis on the caries data in deciduous dentitions from 23 countries and on data in permanent dentitions from 47 countries. He found that every 20-gram increase in the sugar consumed more per person per day (or 7.3 kg per year) resulted in 0.5 dmft increase in 5- to 6-year-old children, and 1 DMFT increase in 12-year-olds. Soon after it had been published this observation was proved not to be valid. There were some countries where between 1982 and 1985 the sugar consumption had increased, but where, nevertheless, regular epidemiological monitoring of caries data had shown that the caries prevalence in children continued to decrease: these were Sweden [Birkhed et al., 1989], Norway [Rølla and Øgaard, 1987], and New Zealand [König, 1990]. Shortly after the publication by Walker and CleatonJones [1989], Marthaler [1990] published his analysis of the secular trends in caries prevalence and came to the confirmatory conclusion that in many highly developed industrialized countries there was a ‘lack of correlation between the decline of caries prevalence and average sugar consumption’. This is a comforting statement. However, there are still high-risk populations who demand our attention. These are found in developing countries, or in subpopulations (mostly ethnic minority groups) in the highly developed ‘low-caries countries’, such as the refugees from former Yugoslavia in Switzerland [Menghini et al., 2003]. These at-risk subpopulations should be the target of appropriate,

König

effective prevention and treatment strategies. A specific analysis of risk factor(s) per risk group is necessary, and a specific package of preventive measures should be composed to limit the manifestations of caries and reduce treatment need.

Root Caries and the Secular Change of Caries Prevalence in the 1980s

The urgent problems caused by the high incidence and rapid progression of caries in young people made us neglect a typical problem inflicting the elderly with gingival recession, root caries. In the 3 decades between 1954 and 1983 only 14 studies on root caries were carried out [Wagg, 1984]. One of them was the first representative population study on root caries by the Finnish group of Vehkalati et al. [1983], the results of which they started to publish in 1983. It showed that the prevalence was less in women than in men (1.19 vs. 2.23% of teeth affected). At that time the authors already assumed that the prevalence of root caries differed widely in populations of different ethnic, cultural and socio-economic background. Whether this manifestation of caries fully deserves the increased attention it received in the late 1980s, or whether the problem of root caries has, in the meantime, been replaced by the problem of toothbrush abrasion of the exposed roots will depend on one’s perspective. However, root caries is another interesting example on how the manifestations of caries and its treatment can be drastically modified by preventive measures; Nyvad and Fejerskov [1986] persuaded patients with root caries to practice meticulous toothbrushing with a toothpaste containing 1,000 ppm F. Within 2–6 months the lesions changed from a clinically active stage into inactive stages of caries. Subsequently, this success which tended to make traditional drilling and filling superfluous, has been repeated using various modes of fluoride applications [Lynch and Baysan, 2001].

time became available for the aesthetic aspects of treatment, tooth-coloured adhesive materials being applied in anterior and even in posterior teeth. Some older and somewhat reactionary dentists adhered mentally to the previous desolate scene of rampant caries, and rapid progression of small caries lesions. They tended to overestimate the caries risk and did not trust modern recommendations to wait and give remineralization a chance. They practiced overtreatment, excavating and filling small lesions instead of sealing fissures or applying remineralizing agents to sites of incipient attack. The reference in the title to ‘global changes’ should not be taken literally. The situation worldwide was and remains today extremely variable and changes are occurring in different directions. In industrialized countries, there has been great improvement, but in some developing countries deterioration has been observed. There are other instances where the manifestations of caries are pretty stable. However, some countries, in particular on the African Continent, are developing very slowly and due to lack of financial resources cannot provide modern dental services; in these cases the method of atraumatic restorative treatment (ART) is an adequate alternative [Frencken and Holmgren, 1999; Massara et al., 2002]. The filling materials are modern glass-ionomers, and the preparation is minimally invasive, but the preceding excavation is carried out with hand instruments, and so – again – we return to G.V. Black. It is obvious that this atavistic treatment method was born out of the necessity to help patients in poor developing countries, while progress with new, advanced techniques is available in the highly industrialized rich countries. The conclusion therefore must be that the highest priority should not be the development of even more advanced treatment techniques, but the global fight against poverty which threatens the health of large parts of the world population.

Caries Manifestations, Treatment, Overtreatment and Undertreatment

Some manifestations of caries which accompanied the generally improved dental health in highly developed countries are very characteristic: smooth surface caries has become very rare; approximal caries declined drastically, and fissures were sealed or treated by minimally invasive methods without extension for prevention. More

Manifestions, Treatment and Prevention of Caries

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References Birkhed D, Sundin B, Westin SI: Per capita consumption of sugar-containing products and dental caries in Sweden from 1960 to 1985. Community Dent Oral Epidemiol 1989;17:41– 43. Black GV: Susceptibility and immunity to dental caries. Dental Cosmos 1899;41:826–836. Black GV: Konservierende Zahnheilkunde. Berlin, Meuser, 1914, vol 2: Die Technik des Zahnfüllens. Bratthall D, Hänsel-Petersson G, Sundberg H: Reasons for the caries decline: What do the experts believe? Eur J Oral Sci 1996;104:416–422. Dean HT, Arnold FA, Elvove E: Domestic water and dental caries. V. Additional studies of the relation of fluoride domestic waters to dental caries experience in 4,425 white children aged 12–14 years of 13 cities in 4 states. Public Health Rep 1942;57:1155–1179. Frencken J, Holmgren CJ: Atraumatic Restaurative Treatment. Nijmegen, STI Book bv, 1999. Hofheinz RH: Extension for prevention. Dental Cosmos 1902;44:914–919. Kalsbeek H, Verrips GHW: Dental caries prevalence and the use of fluorides in different European countries. J Dent Res1990;69(special issue):728–732. König KG: Changes in the prevalence of dental caries: How much can be attributed to changes in diet? Caries Res 1990;24(suppl 1):16–18. Lynch E, Baysan A: Reversal of primary root caries using a dentifrice with a high fluoride content. Caries Res 2001;35(suppl 1):60–64.

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Marthaler TM: Changes in the prevalence of dental caries: How much can be attributed to changes in diet? Caries Res 1990;24(suppl 1):3–15. Marthaler TM: Dentistry between pathology and cosmetics. Community Dent Oral Epidemiol 2002;30:3–15. Marthaler TM: Dental caries – past and present: Changes in dental caries 1953–2003. Caries Res 2004;38:173–181. Massara MLA, Alves JB, Branda˜o PRG: Atraumatic restorative treatment: Clinical, ultrastructural and chemical analysis. Caries Res 2002;36: 430–436. Menghini G, Steiner M, Marthaler TM, Helfenstein U, Brodowski D, Imfeld C, Weber R, Imfeld T: Kariesprävalenz von Schülern in 16 Zürcher Landgemeinden in den Jahren 1992 bis 2000. Schweiz Monatsschr Zahnmed 2003; 113:267–277. Nyvad B, Fejerskov Ø: Active root surface caries converted into inactive caries as a response to oral hygiene. Scand J Dent Res 1986;94:281– 284. Plasschaert AJM, König KG: Die Wirkung von Zahngesundheitsinformatiion und von Fluoridtabletten auf den Karieszuwachs bei Schulkindern. Schweiz Monatsschr Zahnheilkd 1973;83;421–445. Rølla G, Øgaard B: Reduction in caries incidence in Norway from 1970 to 1984 and some considerations concerning the reasons for this phenomenon; in Frank RM, O’Hickey S (eds): Strategy for Dental Caries Prevention in European Countries According to Their Laws and Regulations. Oxford, Information Retrieval, 1987, pp 223–229.

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Sreebny LM: Sugar and human dental caries. World Rev Nutr Diet 1982;40:19–65. Todd JE, Whitworth A: Adult Dental Health in Scotland 1972. London, Office of Population Censuses and Surveys, Social Survey Division, HMSO, 1974. Truin GJ, König KG, Bronkhorst EM, Frankenmolen F, Mulder J, van’t Hof MA: Time trends in caries experience of 6- and 12-year-old children of different socioeconomic status in The Hague. Caries Res 1998;32:1–4. Truin GJ, König KG, Bronkhorst EM, Mulder J: Caries prevalence amongst schoolchildren in The Hague between 1969 and 1993. Caries Res 1994;28:176–180. Van zoet naar zuur (From Sweet to Acid): Dental Health Education slide series. Rotterdam, Het Ivoren Kruis, 1969. Vehkalahti M, Rajala M, Tuominen R, Paunio I: Prevalence of root caries in the adult Finnish population. Community Dent Oral Epidemiol 1983;11:188–190. Wagg BJ: Root surface caries: A review. Community Dent Health 1984;1:11–20. Walker ARP, Cleaton-Jones PE: Sugar intake and dental caries: Where do we stand? J Dent Child 1989;56:30–35.

König

Caries Res 2004;38:173–181 DOI: 10.1159/000077752

Changes in Dental Caries 1953–2003 T.M. Marthaler Center for Dentistry, University of Zurich, Zurich, Switzerland

Key Words Dental caries W Caries epidemiology W Time trends

Abstract In the first half of the 20th century, indices and methods of conducting surveys of the level of dental diseases were developed. Modern epidemiological studies began in the fifties and many reliable studies have been conducted after 1960. In the following decades, a substantial decline of caries prevalence was documented in the majority of the highly industrialized countries, with reductions of lifetime caries experience exceeding 75%. The decline comes to an end when low or very low levels of prevalence are reached. Children of low socioeconomic status and immigrants from outside Western Europe, however, generally have higher disease levels and may cause increases in caries prevalence. For this and other reasons, caries epidemiology will remain an indispensable part of dental public health. Copyright © 2004 S. Karger AG, Basel

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Early Phases and Progress in Sampling Theory

It was around 1900 that the first statistics on dental decay were published, which was approximately the time when the first university dental institutes were training students in dentistry. The number of these early statistics was very low and they are difficult to interpret. Half a century later, a special commission of the International Dental Association prepared a survey covering the period 1950–1963, which showed that numerous epidemiological studies were carried out at that time. From the 14 most active countries in Europe, a total of 420 publications were compiled [FDI, 1964]. Increasingly, DMFT counts were used. There were two main purposes: (1) purely epidemiological, i.e. to assess the dental status, and (2) identification of the caries-inhibiting effect of fluoride in the drinking water with levels either below 0.3 or above 0.8 ppm. Up to the sixties, the tendency was to draw samples in towns or cities close to dental schools or in areas where special circumstances were expected. Usually it was not specified how the samples were drawn; nowadays they would be regarded as ‘convenience’ samples. In fact, papers on concepts and theory of random sampling began to appear in the 1930s only in specialized statistical journals. It was only in 1949 that the first textbook on this new topic appeared [Yates, 1949]. Its focus was on agricultural research in England. The second textbook, by Cochran

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Table 1. Percentages of children examined from the total of the children selected at random in each of the 16 communities

1964 1968 1972 1976

No examination of teeth at all

Exam yes, but no radiographs

Total nonresponse

0 0 0 0

!1 !1 !1 !2

!1 !1 !1 !2

New law on protection from radiation 1980 !2 no records 1984 !3 14

no records !17

Accident at the Chernobyl atomic reactor in 1986 1988 6* 32 1992 8* 35

36 40

Radiation exposure reduced from 0.34 to 0.12 s 1996 10* 25 2000 11* 19

32 28

* Based on detailed records obtained in 1988; approximately one third were in fact outright rejections, others not examined were sick at the examination day(s), wore extensive orthodontic appliances or had moved. The percentages of children not examined because of rejection were accordingly 4–6 % points lower than the figures presented for 1988 through 2000.

[1953], was relatively easy to understand and was widely used. The earliest caries studies based on random sampling were those carried out in the USA: 1960–62 in adults, 1963–65 in children and 1966–70 in 12- to 17-year-old youths [National Center for Health Statistics, 1967, 1971, 1974]. Similar studies based on random selection procedures were conducted in 1968 with adults in England and Wales [Gray et al., 1970] and in 1973 with children [Todd, 1975]. It took another 20 years until other highly industrialized countries had carried out comparable surveys. This was in part due to three circumstances: (1) The theory and practice of drawing samples were relatively new. (2) In general, it was – and may be even today – difficult to obtain true random samples, i. e. samples in which each individual of a nation or a province etc. envisaged has the same probability of being included in the sample. (3) Once the sample has been selected at random from available lists, each individual chosen should be examined. In the last decades, this prerequisite has become increasingly difficult to fulfill as illustrated in the following paragraph.

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The surveys repeated every 4 years in the Canton of Zurich were carried out within the school dental services. The services have for decades included one mandatory clinical examination of every schoolchild per year, and the examinations for statistical/epidemiological purposes were declared as part of the school dental service (specifically serving quality control of treatments and prevention). Up to 1976, rejection rates were below 2%, but as shown in table 1, rejection rates increased after a new law on radiation protection and even more so after the accident in the Chernobyl atomic reactor. The highest rejection rate was 40% in 1992. As of 2000, the overall rejection rate was down to 28%, with 19% rejecting exclusively the radiographic examination. In very recent times, some adolescents just object to sit down on the examination chair, with teacher and parents often commenting that ‘this is their personal freedom’. In such situations, attempts to obtain random samples may in fact be futile due to the fact that often more than half of the selected subjects cannot be examined. In a recent study in the USA, parents had at first to be asked whether they were interested in a survey including the examination of the teeth and an assessment of urinary fluoride excretion. Those who did hand in the signed one-page document were then given a two-page detailed information sheet in which at the end they were asked to sign a text like ‘with my signature I decide that my child is allowed to take part in...’.

The Early Years of the Decline in Western Europe

The surveys published up to the sixties suggested that dental caries prevalence was high in children of Western European countries. Children 12 years of age often had on average more than 5 DMFT, and at the age of 15 the DMFT averages were often above 10. In the countries with comprehensive school dental services, high caries prevalence was of course known from the excessive burden of restorative treatment and the frequent destruction of teeth beyond repair. This deplorable situation was often the starting point for the search for preventive measures. The discovery of the cariostatic effects of fluoride rapidly inspired many activities in both research and practical dentistry. A considerable number of projects were begun around 1960. Local uses of fluorides were preferred in the Scandinavian studies while in other Western European countries the majority of projects attempted to assess the caries-pre-

Marthaler

On the occasion of the 25th Anniversary of ORCA in 1978, this organization published a supplement to Caries Research with the title Progress in Caries Prevention [Ericsson, 1978]. In the preface, Yngve Ericsson ventured to state that ‘In no other period of history, outside the times of enforced rationing and shortages in war and famine, have these countries enjoyed so great an improvement of dental health’; these countries were those ‘where preventive methods have been systematically implemented on a large scale’ [Ericsson, 1978]. In 1985, a Commission of the FDI compiled data demonstrating a caries decline in 9 countries [Renson et al., 1985]. Four of them were the Northern European countries Denmark, Finland, Norway and Sweden. The remaining 5 were Australia, the Netherlands, New Zealand, the United Kingdom and the USA. After publication of this report, it became widely acknowledged that a secular decline was going on

in many industrialized countries. The greatly improved dental health up to 1993 was further documented at the ‘Second International Conference of Declining Caries’, held in London in April 1994 [Naylor, 1994]. The decline took various courses in Western Europe. This may be exemplified by using data from the Netherlands and Switzerland. The decrease in the Netherlands, as studied in 12-year-old children, was summarized in a very simple manner: ‘The average DMFT decreased steadily from 8 in 1965 to one in 1993’ [König, 2002]. Figure 1 illustrates that in fact the decrease tended to be linear (the customary regression line was not calculated as the beginning and the end of the decline cannot be determined; in addition, the data came from different towns and cities and were based on variable numbers [Truin et al., 1994]; the line drawn is sufficient for the illustration intended here). A decrease of 7 DMFT in 28 years is equivalent to a decrease of 0.25 DMFT per year. The decrease of the DMFT averages in the Canton of Zurich took a different course. As is evident from figure 2, the reduction was most rapid in the mid-sixties but became gradually smaller in numerical terms. Accordingly, the logarithms of the DMFT averages closely followed a straight line for all of the four age groups studied (8-, 10-, 12- and 14-year-olds) [Marthaler et al., 1994]. The course of the decline of the DMFT averages was obviously different from the one in the Netherlands. Most of the European data on the decline up to 1993 were presented at the ‘Second International Conference on Declining Caries’ [Naylor, 1994]. At the ORCA Symposium of 1995, the decline of the DMFT in several Western European countries was found to be still continuing until 1994 [Marthaler et al., 1996]. In the case of Eastern Germany, a large dataset was available over a period of up to 30 years. The data available until 1995, extensively reviewed by Künzel [1997], documented that a decline occurred between 1985 and 1995, obviously connected with the ‘Westernization’ of former Eastern Germany (the former German Democratic Republic). The earlier statistics, dating back to 1959, showed that the fluoride level in the drinking water had been the main determinant of dental caries prevalence. In recent years, an increasing number of papers has shown that caries prevalence was highest in the lower socioeconomic strata. Bratthall’s [2000] significant caries index (SiC) is a reliable tool for focusing on children with high caries experience. The SiC is the average DMFT in the one third of children with the highest caries experience; accordingly, the SiC does not depend on assessments of socioeconomic status (SES), the definition of

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Caries Res 2004;38:173–181

ventive potential of daily tablet intake. Many of these projects were done by school dental services in cooperation with dental schools, which provided professional advice and often carried out the examination of the children’s teeth and the statistical evaluations. Many of the early Scandinavian reports of a caries decline were cited by von der Fehr [1994] and von der Fehr and Haugejorden [1997]. The latter paper showed that in 5 of the 14 Norwegian counties, the decline began around 1967. The authors concluded that in Norway the decline started when fluoride brushing or rinsing programs were introduced. Widespread use of fluoride toothpastes could become a factor at the earliest in 1971/72, that is 4 years later. According to a Danish report typical of that period, a reduction of caries increments of slightly above 50% was obtained from 1962 to 1966 through a comprehensive school-based program comprising multiple topical fluorides [Kann, 1968]. In Switzerland, decline of caries became obvious in the early sixties [Wegelin, 1964; Marthaler and König, 1967; Marthaler, 1969]. Rieder [1967] reported a rapidly decreasing number of fillings necessary in the school dental service. In the Canton of Zurich, the onset of a caries decline was documented already for the period 1964–1968 [Marthaler, 1972]. Early reports on a decline appeared also in Germany [e.g. Sigrist and Marthaler, 1975] and Austria, but there were no conferences or review papers summarizing them.

The Declines of Caries Prevalence in Selected Western European Countries

175

Fig. 1. Average DMFT of 12-year-old children in various towns and cities of the Netherlands [data points from Truin, 1997]. The line symbolizes the steady fall of the averages, equal to approximately 0.25 less DMFT per year from 1965 (8 DMFT) to 1993 (1 DMFT).

which varies from one country to another. In the Swiss Canton of Zurich, the average DMFT of all 12-year-old children examined in 1964 was 7.9 while their SiC was 13.1. In 1996, presumably the end of the period of decline regarding the age group 12, the averages were 0.84 and 2.38, respectively [unpublished data drawn from the existing datasets of the Canton of Zurich, which is the database for the paper by Menghini et al., 2003b]. The reduction of the SiC by 82%, from 13.1 to 2.38, was a dramatic improvement for the one third of children with the highest caries risk. The children of the lowest tercile had an average DMFT = 0, since 62% of the examined children were caries-free.

Reasons for the Decline

Different or sometimes widely diverging opinions exist regarding the reasons of the decline. An inquiry including 52 selected experts, carried out in the mid-nineties, revealed that the daily use of fluoridated toothpastes, preferably twice a day, was considered to be the most important single factor by most experts [Bratthall et al., 1996]. In controlled randomized studies comparing dentifrices with and without fluoride, the reductions ascribable to fluoride were often between 20 and 40% and rarely exceeded 50%. If we assume that 6.0 DMFT were the approximate DMFT in 12-year-old children prior to the decline, fluoride in dentifrices would have lowered the

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Fig. 2. Average DMFT in children (permanent residents) in 16 communities of the Canton of Zurich in which surveys were conducted every 4 years since 1964.

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DMFT to 3.0. In several countries, however, the DMFT averages have fallen to 1.0 or even below. What are the reasons for the reduction from 3 to 1.0 DMFT, equivalent to 67%? In part, it may be due to improved toothbrushing habits: more frequent and more thorough toothbrushing would strengthen the fluoride effect, lower the ‘aggressivity’ of dental plaque and remove fermentable food remnants more thoroughly. However, other important factors are likely to be involved in the dramatic decline of dental caries prevalence at school age. Unfortunately, analytical epidemiological studies often do not provide useful or reliable data to support or disprove specific hypotheses. Therefore, the role of other favorable factors is still a matter of discussion [Bratthall et al., 1996]. Among the factors considered unimportant by Bratthall et al. [1996], placement of sealants needs to be reconsidered. In case of DMFT averages above 3, there will be much caries apart from fissures and pits and the role of sealants will be limited (except perhaps in projects in which they were applied on all fissures and pits of first molars). There is also the irrefutable fact that declines above 70% were obtained in several Western European countries before fissure sealants were commonly used, that is before 1985–1990. However, in the countries in which DMFT averages are now below 2.0, most of the caries occurs in fissures and pits of the first molars until the age of 12 years. Consequently, there are reasons to assume that sealants can be a very important, or even the main factor in lowering the DMFT from say 1.5 to 1.0. Finally, there is agreement that the various and continued uses of fluorides, often applied in combination, are by far the most important factors of the decline. Some confusion has arisen from the fact that in a few specific situations, water fluoridated to around 1 ppm has lost part of its effectiveness of reducing DMFT experience by 50– 60%, as documented up to 1980. In modern societies using fluorides in toothpastes and other topical applications, water fluoridation cannot be clearly demonstrated.

Table 2. Average dmfs and DMFS counts in the Hague: Dutch

children with high or low SES and immigrants from Turkey and Morocco Dutch nationals

Turkey

Morocco

high SES

low SES

Age 6, dmfs 1996 1998 2002

0.8 0.5 0.7

4.7 4.3 4.1

5.3 6.8 7.4

5.1 4.1 4.0

Age 12, DMFS 1996 1998 2002

0.3 0.1 0.4

1.6 2.0 0.6

3.4 2.1 1.0

2.8 1.5 0.9

From Truin et al. [unpublished data].

Regarding the primary dentition, Downer [1994] assumed that the decline ended in England and Wales in 1983. Likewise, in Swiss children of the Canton of Zurich, an end of the decline in the primary teeth became evident in 1988 [Steiner et al., 1991]. In the first survey of 1964, the average dmft was 7.6. In 1988, 1992, 1996 and 2000, Swiss children had dmft averages between 1.5 and 1.8.

This corresponds to reductions of 76–80% from 1964 to the ‘stable’ period of 1988–2000. Recent caries statistics from children having high and low SES in The Hague are presented in table 2. In the high-SES children aged 6, the average dmfs varied between 0.5 and 0.8 from 1996 to 2002. By contrast, in the low-SES children, the average dmfs remained at 4.7 (1996) and 4.1 (2002), thus showing little if any improvement in the 6 years. Children immigrated from Turkey and Morocco also had on average between 4.0 and 7.4 dmfs in the 6-year period. At the age of 12 years, the average DMFS in the Dutch high-SES children was very low, between 0.1 and 0.4 DMFS. In the low-SES children, 1.6 and 2.0 DMFS were counted in 1996 and 1998, respectively, but the latest average, of 2002, was as low as 0.6 DMFS. In the Turkish and Moroccan children, the averages were still at 3.4 and 2.8 in 1996, but had fallen to 1.0 and 0.9, respectively, by 2002. There is no doubt that numbers of legal as well as illegal immigrants will increase in the near future. For proper interpretation of epidemiological data, both the immigration status, country of origin as well as the length of stay in the guest country need to be recorded and reported. These data illustrate that once the average dmf or DMF counts are low or very low, there will be instability or oscillation. In the high-SES Dutch children (the averages of whom are presented in table 2), 79–93% had dmfs or DMFS equal to zero. Accordingly, the few children with counts of 2 or higher can make the averages unstable. Another factor may even be more important: fillings

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Observations regarding the End of the Decline

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Table 3. Average dmft counts in Swiss

Immigrated in last 2 years from

children and immigrants

Ex-Yugoslavia other

all

City of Zurich immigrants 1994, age 7–8

7.2

3.8

5.9

City of Zurich, age 7 children

Swiss

Immigrants

All

1993 1998

1.7 1.7

4.9 3.8

2.9 2.7

Canton of Zurich1, age 7 children

Swiss1

Immigrants from Ex-Yugoslavia

All

1988 1992 1996 2000

1.8 1.6 1.5 1.8

– – 5.9 6.9

– – 1.9 2.4

City of Winterthur, Canton of Zurich 2001, age 5

1.7

7.8

2.4

1

16 communities, not comprising the cities Zurich and Winterthur. Data compiled from Menghini et al. [2003a, b] and Steiner et al. [1988, 1994].

placed by dentists who continue to have diverging ideas about when to place a filling and when this should not be done. ‘White’ fillings have also become a problem. It is inevitable that part of them are not identified by the examiner with the effect that the F component, which in Western Europe has long become much larger than the D and M component, is underestimated to some extent. At the very low caries levels in highly industrialized countries, it is therefore difficult to identify minor changes in caries prevalence, which might be very interesting for scientific research. However, the conventional examination procedures are sufficient to detect substantial increases in caries prevalence should they in fact occur. New diagnostic methods, particularly for fissure caries where the majority of caries lesions and fillings occur, may provide more reliable bases for determining whether decay levels decrease even further, remain constant or whether they increase [Lussi and Francescut, 2003]. The intensity of research regarding the identification of carious predilection sites is evident from ORCA abstracts No. 50–68 [Caries Res 2003;37:284–290]. The effect of immigrated children on overall caries experience is more obvious in Switzerland than in the Netherlands. Swiss children 7 years of age in the City of Zurich on the one hand and 16 other towns and villages on the other all had dmft averages between 1.5 and 1.8

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(table 3). A special investigation in 1994 showed that immigrant children from the former Yugoslavia had 7.2 dmft on average while other immigrants had 3.8, still twice as high as that of Swiss children in the nineties (first line in table 3). When the Swiss children were pooled with those of foreign origin, who constituted as much as 42% of all schoolchildren in the City of Zurich in 2000, the dmft average increased from 1.7 to 2.9 (1993) and from 1.7 to 2.7 (1998). In the 16 communities outside the City, pooling Swiss with foreign children led to an increase of the average dmft from 1.5 to 1.9 (1996) and from 1.8 to 2.4 (2000); in the entire Canton, the percentage of non-Swiss schoolchildren was 27% but has been increasing since. Figure 3 shows dmft averages of 7-year-old children, comprising Swiss and immigrants, in 5 locations in various parts of Switzerland. The increases of caries prevalence after 1988 were at least partially due to increasing immigration. The fact that in 2001 children of Swiss origin in Winterthur (the second largest city in the Canton of Zurich) already had 1.70 dmft at the age of 5 years [Menghini et al., 2003a] suggests that levels of primary tooth caries might increase in Swiss children, too. Extensive surveys carried out in Great Britain compared dmft data from the period 1989/90 with most of those of 2001/2002. Pitts et al. [2003] concluded ‘that the marked geographic variation seen previously is still evi-

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Fig. 3. Average dmft in 7-year-old children (permanent residents and immigrants pooled) in various towns or regions of Switzerland.

Table 4. Average dmft/DMFT counts in

various parts of the United Kingdom (% with counts equal to zero, or caries-free) Age 5, dmft1 Age 12, DMFT Age 14, DMFT2

Year of survey

England Southwest

England Northwest

Wales

Scotland

2001/02 2000/01 1998/99

1.11 (67%) 0.78 (65%) 1.35 (52%)

2.06 (51%) 1.25 (51%) 2.16 (37%)

2.26 (47%) 1.31 (49%) 2.25 (37%)

2.75 (32%)

Age 5: from Pitts et al. [2003]; age 12: from Pitts et al. [2002]; age 14: from Pitts et al. [2000]. 1 South and North instead of South-West and North-West. 2 For Northern Ireland, the respective figures were 3.65 (22%).

dent’. Similarly, from 1996 to 2000, the average DMFT at age 12 was reduced by approximately 0.1 but the typical disparities between the regions persisted [Pitts et al., 2002], the DMFT averages being lowest in the Southern parts of England. The results confirm Downer’s [1994] statement that the data available up to 1993 (from England and Wales) ‘suggest that caries levels have now levelled out...’ while ‘the national data conceal widespread disparities between different regions of the country...’. In fact, for 12-year-old children, the overall average was 0.89 DMFT in 2000/01 as compared to 1.1 and 1.2 in 1992/93 (averages from two different surveys), but in 2000/2001 Wales and the North West still had DMFT averages of 1.31 and 1.25 [Pitts et al., 2002]. It is evident from table 4, showing averages for ages 5, 12 and 14, that caries counts were consistently lowest in South-West England. These averages, based on examinations of large numbers of chil-

dren (between 3,750 and 61,324), suggest that the ‘bottom’ prevalence eventually reached may be at different levels for counties, regions or countries. It is important to note that the decline is carried on into adult age. This was most clearly assessed in military recruits. In Switzerland, their DMFT at age 20 was 16.0 in 1970 but by 1996 had decreased to 4.8 (including extracted premolars) or 4.4 (counting only extracted first molars, which were very rare, in the DMFT) [Menghini et al., 2001]. These authors summarized that in recruits of Australia, Denmark, England and Wales, Germany, Norway, Sweden and the USA, reductions ranging from 18 to 66% have occurred in recent years. Secular improvements of dental health in adults is a vast field, and many more papers on this topic may appear in the near future.

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179

Central and Eastern Europe

This part of Europe comprises countries with different economic situations, all being substantially less strong economically than Western Europe. The three Baltic countries, the Czech Republic, Hungary, Poland, Slovakia, Slovenia with a total population of approximately 70 million will become members of the European Union in May 2004. Apparently only one country among them, Slovenia, has experienced/obtained a substantial and continued decline of dental caries prevalence. In 1987, the average DMFT in children 12 years of age was 5.1, but had decreased to 1.8 in 1998. The corresponding averages for 18-year-olds were 12.7 and 7.0, a reduction by 46%. The advantage in this country was that the school dental service was not thrown overboard when the one-party system was abandoned. The preventive measures and the treatment level offered by the school dental service were maintained or improved [Vrbic, 2000]. Until 1989, Eastern Germany was ruled under the communist one-party system and in essence isolated from Western Europe. For decades, large numbers of children were examined in many cities. The findings were analyzed in detail [Künzel, 1997]. In the present context, the last 15 years are of special interest. In 1986/87, just before the borders between the two Germanies fell, 12-year-old children had 3.8 DMFT on average. By 1994, the average had fallen to 2.5, a reduction of 34%. Recent reports indicate that the average in this part of Germany continues to decrease. The most recent statistics from Eastern Germany indicate average DMFT counts between 1.2 and 1.4 (unpublished latest reports from Chemnitz, the former Karl Marx Stadt, where water fluoridation was abandoned in 1989, and from Dresden and Erfurt). The encouraging developments in Slovenia and Eastern Germany may illustrate how rapidly caries prevalence can be reduced when conditions are favorable and/or proper action is taken. In the countries joining the European Union in 2004, caries is higher than in Western Europe. Available data suggest that in some countries, minor declines may have occurred since 1989. The introduction of organized prevention such as school-based dental health education has been difficult since the previously existing systems of school dental care were mostly abolished in the early nineties. Additional stumbling blocks are the high prices of ‘state-of-the-art’ fluoride toothpastes and toothbrushes, prices which are 2–4 times higher than in Western Europe when related to the typical family incomes in these countries. Under these circumstances it is difficult to promote dental hygiene and the use of topical fluorides such as in

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dentifrices, gels and rinses. Salt fluoridation, which would be by far the cheapest measure, is often authorized; however, initiatives to induce people to use such salt are minimal in most countries. This unfavorable situation is coupled with the fact that dental treatment will remain unaffordable for the lower or even middle SES for many years to come. It seems to be difficult to convince the ministries of health and the majority of the population that the cost of prevention is very low when compared to treatment cost, and that the improvements in dental health would be substantial and cost of treatment reduced in the long run. Implementation of prevention was a relatively slow process in Western Europe. There is a new situation in the sense that many dentists in Central Europe – and sometimes health officers as well – are aware of the dramatic decline of caries prevalence which has taken place in Western Europe. This may hopefully stimulate preventive efforts in these countries so that positive developments may be expected in the near future, particularly in the countries joining the European Union in May 2004.

Outlook

The decline in caries prevalence in Western Europe has been very substantial. It has received much attention until recently but is now often taken for granted. However, caries prevalence, still very different when looking at various parts of Europe, may undergo unexpected changes due to various factors. Increasing immigration has been identified as a new factor, leading to increases of the overall dental caries prevalence in Switzerland (20% non-Swiss residents), the Netherlands and Germany. Caries epidemiology continues to be an important issue in both oral health surveillance and research into refined methods for caries diagnosis. Some pertinent observations may be summarized as follows: (1) Politicians in some countries tend to think that dental caries is no longer an urgent topic; they may even reassure themselves with the illusion that the problem has been solved ‘for ever’. (2) Dental schools have been reluctant in their reaction to reform and adapt the dental curriculum to the new situation. (3) Antifluoridationists persist in opposing various uses of fluorides but their impact is fading: the predicted adverse effects have not occurred and the success of preventive dentistry is obvious; their ideas, however, continue to surface when steps are taken to intensify measures based on fluorides.

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(4) In Central and Eastern Europe, caries prevalence is still high and there are no signs of substantial improvements; in addition, misleading contentions of untoward effects of fluorides (as used for prevention of dental caries) are still widespread. (5) On the global scale, only a minority of children benefit from caries prevention and fluorides.

Acknowledgment The help of Dr. G. Menghini and Dr. M. Steiner in setting up the figures and reading the text, tables and references is gratefully acknowledged.

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Pitts NB, Evans DJ, Nugent ZJ, Pine CM: The dental caries experience of 12-year-old children in England and Wales. Surveys coordinated by the British Association for the Study of Community Dentistry in 2000/2001. Community Dent Health 2002;19:46–53. Renson CE, Crielaers PJA, Ibikunle SA, Pinto VG, Ross CB, Sardo Infirri J, Takazoe I, Tala H: Changing patterns of oral health and implications for oral health manpower: Part I. Int Dent J 1985;35:235–251. Rieder F: Sechsjahrresultate mit kombinierter Kariesprophylaxe nach den Empfehlungen des SSO-Seminars für Jugendzahnpflege 1961. Schweiz Monatsschr Zahnheilkd 1967;77: 1058–1059. Sigrist H, Marthaler TM: Abfall der DMF-Zahnzahl bei 8- und 9jährigen Kindern nach 4 Jahren überwachten Zähnebürstens. Dtsch Zahnärztl Z 1975;30:294–299. Steiner M, Marthaler TM, Bandi A, Menghini G: Prävalenz der Milchzahnkaries in 16 Gemeinden des Kantons Zürich in den Jahren 1964 bis 1988. Schweiz Monatsschr Zahnmed 1991;101:738–742. Steiner M, Menghini GD, Curilovic Z, Marthaler TM: Kariesbefall der Schüler der Stadt Zürich im Zeitraum 1970–1993. Schweiz Monatsschr Zahnmed 1994;104:1210–1218. Todd JE: Children’s Dental Health in England and Wales 1973. Office of Population Censuses and Surveys. London, Her Majesty’s Stationery Office, 1975. Truin GJ: Kariesprävention und die Erfolge der kinderzahnärztlichen Betreuung in den Niederlanden. Zahnärztl Gesundheitsdienst 1997; 27:8–13. Truin GJ, König KG, Bronkhorst EM: Caries prevalence in Belgium and the Netherlands. Int Dent J 1994;44:379–385. Vrbic V: Reasons for the caries decline in Slovenia. Community Dent Oral Epidemiol 2000;28: 126–132. Wegelin H: Erfahrungsbericht und statistische Auswertung der Kariesprophylaxe bei Schulkindern im Kanton St. Gallen. Schweiz Monatsschr Zahnheilkd 1964;74:1043–1059. Yates F: Sampling Methods for Censuses and Surveys. London, Griffin, 1949.

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Caries Res 2004;38:182–191 DOI: 10.1159/000077753

Changing Paradigms in Concepts on Dental Caries: Consequences for Oral Health Care O. Fejerskov Royal Dental College, Faculty of Health Sciences, University of Aarhus, Aarhus, Denmark

Key Words Biofilms W Caries W Dental plaque W Fluoride W Preventive dentistry

Abstract Kuhn proposed in his Structure of Scientific Revolutions (1962) that the theoretical framework of a science (paradigm) determines how each generation of researchers construes a causal sequence. Paradigm change is infrequent and revolutionary; thereafter previous knowledge and ideas become partially redundant. This paper discusses two paradigms central to cariology. The first concerns the most successful caries-preventive agent: fluoride. When it was thought that fluoride had to be present during tooth mineralisation to ‘improve’ the biological apatite and the ‘caries resistance’ of the teeth, systemic fluoride administration was necessary for maximum benefit. Caries reduction therefore had to be balanced against increasing dental fluorosis. The ‘caries resistance’ concept was shown to be erroneous 25 years ago, but the new paradigm is not yet fully adopted in public health dentistry, so we still await real breakthroughs in more effective use of fluorides for caries prevention. The second paradigm is that caries is a transmittable, infectious disease: even one caused by specific microorganisms. This paradigm would require caries prevention by

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vaccination, but there is evidence that caries is not a classical infectious disease. Rather it results from an ecological shift in the tooth-surface biofilm, leading to a mineral imbalance between plaque fluid and tooth and hence net loss of tooth mineral. Therefore, caries belongs to common ‘complex’ or ‘multifactorial’ diseases, such as cancer, cardiovascular diseases, diabetes, in which many genetic, environmental and behavioural risk factors interact. The paper emphasises how these paradigm changes raise new research questions which need to be addressed to make caries prevention and treatment more cost-effective. Copyright © 2004 S. Karger AG, Basel

One of the buzzwords in the health sciences literature is ‘evidence-based medicine’. The word ‘evidence’ is often used uncritically, implying that any written communication which has found its way into the scientific literature is considered as having a value of its own which adds to ‘scientific knowledge’. Even when dealing with scientific contributions published in the most prestigious international journals it is important to appreciate that the evidence normally drawn on by scientists is dictated by an overriding contemporary paradigm. This concept was forcefully argued by Kuhn [1962/1970], who emphasised how a paradigm will shape the way in which any given

Ole Fejerskov Royal Dental College, Faculty of Health Sciences University of Aarhus, Vennelyst Boulevard DK–8000 Aarhus C (Denmark) Tel. +45 33181950, Fax +45 33150626, E-Mail [email protected]

History shows that the development of science has always been highly influenced by paradigms, and revolutionary shifts in paradigms have not easily been brought about [for famous examples see Kuhn, 1962/1970]. When a scientific theory or concept has become a paradigm, this is only dismissed once an alternative paradigm can replace it, in a scientific revolution. The interesting point is that a paradigm is rarely challenged in such a way that attempts are made to falsify it by direct comparison with the particular events or diseases that are studied. Of course this does not mean that scientists do not reject scientific theory. All it shows is that when this happens, such rejections are always in need of finding a new paradigm in order to facilitate the shift. A transition from one paradigm to another is not a cumulative process or a process in which one just expands

on the old paradigm in order to have an impact on research or health activities. Rather a transition necessitates a reconstruction of the field which significantly changes some of the discipline’s most basic theoretical generalisations as well as rejecting several of the methods and interpretations performed in the past. It should be appreciated that very often it is the same set of data which constituted the basis for the old paradigm that is reconsidered, and a new interpretation or paradigm is built on more or less the same set of data. Therefore, it is common to see within the same field the old and the new paradigms living side by side for a substantial period of time. This is because two or more groups of researchers apparently work in ‘different worlds’ where they study different phenomena although they are standing in the same place and are looking in the same direction. However, it is obvious that they do not see the same ‘world’. In certain areas they are definitely seeing different things (things they are looking for simply as a result of the paradigm), but they may equally well see the same phenomena although in a different relative context. This is one of the good reasons why some cannot even consider a causal relationship, which appears intuitively obvious to others. Moreover, this obvious discrepancy between different paradigms is often further enhanced by the fact that even within disciplines communication across revolutionary gaps is very incomplete. Researchers may use similar words, but in reality define them rather differently. More than half a century passed before Newton’s Principia was generally accepted. Also Darwin [1889] wrote in the Origin of Species that he by no means expected to be able to convince other scientists of his views, but that only in the future, when a new generation of researchers grew up, would they be able to appreciate his observations. Max Planck [1940] emphasised that a new scientific truth does not convince its opponents, but only gains strength because its opponents gradually die out. Thus a characteristic aspect of science is the maintenance of a resistance towards changes in paradigms, in particular from those whose careers have been based on existing paradigms. Prestigious institutions or international bodies will often have made strong stands in terms of ‘scientific documentation and subsequent recommendations’. For such bodies a new paradigm will be seen as a threat and resistance will be based on the concept that the old established paradigm would be able to explain the phenomena studied so that ‘nature can be put into the box built by the paradigm’. In the following these thoughts should be kept in mind because – when dealing with diseases – paradigms will

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generation of researchers construes a causal sequence. The term ‘paradigm’ is in this case used to describe the guiding theoretical concepts of a science. In other words, a paradigm will form an umbrella under which a given discipline establishes a common view which often sets the rules and norms for how theories, methods etc. are handled in relation to the discipline. Moreover, results or data analysis and interpretations are conducted within the framework of the paradigm. Most established textbooks within a given discipline bring the paradigms in a ‘digested form’ to the next generations of students and young researchers and they often falsely give a younger generation the impression that scientific growth within a given discipline has the character of a progressive march towards a complete understanding of ‘reality’. Science is an exquisite blend of data and theory, fact and hypotheses, observations and views, which in most disciplines at any given time are embedded in a particular paradigm. The aim of this paper is to demonstrate how such paradigms play an important role in cariology. The paper will first focus on how paradigms in general influence science. Then two paradigms dominating cariology during the past half century will be presented and it will be argued why scientific revolutions – shifts in paradigms – are needed in order to advance new knowledge and improve health in populations. Finally, the impact of such revolutions on cariology and oral health will be presented and the consequences for immediate future research activities will be discussed.

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inevitably have a decisive influence on how we diagnose, prevent and treat a disease. When considering dental caries this is certainly the case.

Fluoride and Dental Caries

Fluorides play a key role in the prevention and control of dental caries. There is no doubt that the discovery of the anti-cariogenic properties of fluoride was one of the most important landmarks in the history of dentistry. The history of the old and the new paradigms explaining the possible causal effects of fluoride on teeth (and bone) represents an excellent example of the need for changing concepts in medicine in order to improve health. In the 1930s experimental animal studies and human epidemiological studies established both the association and the cause-and-effect relationship between fluoride in drinking water and mottled enamel (dental fluorosis) [for reviews see Fejerskov et al., 1977, 1994]. In addition, the presence of fluoride in water supplies was associated with a lower prevalence of dental caries [Dean, 1946]. It was thus reasonable to conclude that ingestion of fluoride was important to reduce the number of cavities in teeth while at the same time affecting enamel formation, so that Dean [1946] could conclude that ‘... amounts not exceeding 1 part per million expressed in terms of fluorine (F) are of no public health significance’. The focus from then on was to explain how children born and reared in communities with about 1 ppm fluoride in water supplies could have about 50% less cavities than expected [Arnold et al., 1956; Backer Dirks, 1974]. The toxic effects of fluoride on amelogenesis were thought to be a result of the secretory ameloblasts being particularly susceptible to fluoride [for review see Fejerskov et al., 1977]. This concept prevailed until experimental studies in pigs showed that dental fluorosis can develop by only affecting the maturation stage of amelogenesis [Richards et al., 1986]. This new understanding resulted in extensive research on chemical, biochemical, and cellular events during enamel maturation [Smith, 1998], but we are still far from understanding how fluoride affects mineralising dental tissues [Aoba and Fejerskov, 2002]. The paradigm on how fluoride ‘prevents dental caries’ resulted from the following line of thinking. Teeth formed in areas with a fluoride content of about 1 ppm in water supplies have an increased fluoride content in surface enamel [Brudevold et al., 1956; Isaac et al., 1958]. Fluorine is the most electronegative of the elements and has a strong affinity for exchange with hydroxyl ion in hydroxy-

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apatite [Kay et al., 1964; Elliott, 1969]. The electrostatic attraction between Ca2+ and the F – will be greater than between Ca2+ and OH –, making the fluoridated apatite lattice more crystalline [Zipkin et al., 1962; Newsely et al., 1963; Frazier et al., 1967] and more stable. As a consequence it is less soluble in acid [Volker, 1939; Kutnerian and Kuyper, 1957; Young, 1975; Brown et al., 1977]. By combining these data it is understandable how the paradigm of fluoride ‘making teeth more resistant to caries attack’ became established, and then for more than half a century entirely influenced caries prevention and research. The hypothesis was that increased intake of fluoride during tooth formation raises the fluoride concentration in enamel and hence increases acid resistance. As a consequence fluoride had to be taken systemically and artificial fluoridation of drinking waters became the ‘optimal’ solution. If this could not be achieved fluoride should be ingested in the form of tablets, vitamin drops, lozenges, salt, added to milk, etc. As ingestion of increasing amounts of fluoride during tooth mineralisation results in a gradual hypomineralisation of the final outer enamel [Fejerskov et al., 1974, 1975, 1994], not least the public health-oriented sections of the dental profession downplayed the toxicological properties of fluorides. Thus diagnosis of early stages of dental fluorosis was questioned (or even ignored). Dean’s [1946] original data interpretation was incorrectly referred to. Early signs of dental fluorosis were described as ‘pearl like’ teeth to be considered normal, whereas fully matured enamel with its yellowish colour was characterised as ‘fluoride-deficient teeth’. In some parts of the world fluoride was added to school drinking water in excessive amounts based on the philosophy that ‘if little is good (to reduce decay) more is better’. In other words, it is apparent that this predominant paradigm fully involved all the general features of scientific paradigms described in the scientific literature [Kuhn, 1962/1970]. Likewise, the scientific revolution leading to the new paradigm also followed the abovedescribed general path of development. Dean’s [1946] data are unique and often confirmed. The effect of fluoride on appetite crystallinity and stability is unquestionable. Artificial water fluoridation in populations with a substantial caries prevalence results in marked reductions in dental cavities [Arnold et al., 1953, 1962; Backer Dirks et al., 1961; Brunelle and Carlos, 1990]. But could it be that fluoride affects the caries process in a way entirely different from what the paradigm claimed? If so, may we then develop new and more effective ways of using fluorides? Reinterpretion of the avail-

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able data combined with new studies led to the scientific revolution as follows: Deciduous teeth from a fluoride area contain much less fluoride in enamel than permanent teeth both from a 1-ppm fluoride area and a very low ( ! 0.2 ppm F) area [Mellberg, 1977], but the systemic effect on caries prevalence is the same in both dentitions irrespective of fluoride incorporated during tooth formation [Thylstrup, 1979]. The difference in fluoride concentration in surface enamel between permanent teeth from low-fluoride and fluoridated areas is very limited [Brudevold et al., 1956; Isaac et al., 1958; Mellberg, 1977; Kidd et al., 1980; Larsen et al., 1986; Richards et al., 1989]. It is hardly likely that such a small difference should explain a 50% reduction in cavities. Complete substitution of OH – in human enamel corresponds to a fluoride content of 3.7% (about 39,000 ppm). Even in the most severe cases of human dental fluorosis ever analysed, less than one quarter of the OH – (about 10,000 ppm F) have been replaced by F [Richards et al., 1992]. It has not been possible to demonstrate a clear-cut inverse relationship between fluoride content of surface enamel and dental caries [DePaola et al., 1975; Poulsen and Larsen, 1975; Richards et al., 1977; Schamschula et al., 1979; Spector and Curzon, 1979]. In vitro experimental caries using gel techniques has shown exactly similar degrees of lesion development in teeth from low- and ‘optimal’-fluoride areas [Kidd et al., 1980] and shark enamel containing fluorapatite develops caries lesions in an in situ model [Ögaard et al., 1988]. Such a thing as relative resistance of enamel to caries attack does not exist [Weatherell et al., 1984], and Brudevold et al. [1965] and Brown et al. [1977] have stressed that the rate of acid solubility is of little importance in protecting the tooth against caries. Clinical observations show that children having their teeth formed and mineralised before moving into a fluoridated region experience a significant reduction in dental caries prevalence [Arnold, 1957]. This author was probably the first to mention that water-borne fluoride has a posteruptive cariostatic effect. This so-called ‘topical’ effect was for very long claimed to be inferior to the postulated ‘systemic effect’ and subsequent clinical trials appeared to show that the capacity of topical fluoride to reduce caries was disappointingly lower (20–30% reduction) than that of the about 50% reduction by water fluoridation. The apparently lower efficacy of topical fluorides, however, was most likely a result of unfair comparison between results of several short-term (1–3 years) clinical trials with a result of water fluoridation programmes lasting for more than 10 years.

The fact that a cariostatic effect of fluoride could be obtained without a concomitant increase of fluoride in sound enamel [Brudevold et al., 1967; Aasenden et al., 1972; Shern et al., 1977] whereas enamel fluoride concentration increased in enamel undergoing a carious challenge [Hallsworth et al., 1971; Richards et al., 1977] became very important. By combining the above data with the results from theoretical and laboratory experiments on enamel solubility [Larsen, 1975; Larsen et al., 1976] it was tempting to suggest ‘a possible explanation of the predominant cariostatic effect of fluoride’ in 1981 [Fejerskov et al., 1981]. This new paradigm was a result of a concomitant reconsideration of the pathogenesis of dental caries lesions. Hitherto, dental caries was always recorded in epidemiological studies as cavities. As such, all clinical measurements (the DMFT/S) only comprised very late stages in the caries process (fig. 2). Thereby it was ignored that a clinically detectable lesion (even the non-cavitated white spot) is a result of innumerable pH fluctuations in the microbiota covering the enamel. The enamel surface is a sponge, which by no means is chemically inert. The constantly ongoing pH fluctuations taking place even in socalled ‘resting plaque’ [Küseler et al., 1993; Baelum et al., 1994], and dramatically enhanced during exposure to fermentable carbohydrates [Stephan, 1940; Fejerskov et al., 1992], will be associated with chemical exchange reactions between the tooth mineral and the surrounding plaque fluid [Margolis et al., 1988]. We therefore proposed that ‘the major reason for the cariostatic effect of fluoride can be ascribed to its ability to influence these processes, even at very low (0.2–1 ppm) F concentrations by facilitating calcium phosphate precipitation’ [Fejerskov et al., 1981]. This was in accord with Brudevold et al. [1965], who suggested that the role of fluoride in caries prevention could be ascribed to its unique ability to induce apatite formation from solutions of calcium and phosphate. Because of saliva’s inorganic composition [Andresen, 1921; Koulourides et al., 1965] it is an excellent fluid for ‘remineralisation’, and fluoride in slightly elevated concentrations enhances this potential as pH is lowered [Larsen, 1975]. The simplistic ‘story’ about ‘remineralisation’ was a reality. Moreover, it was remarkable that only traces of fluoride are required in a solution with calcium present at pH 4.2 to reduce markedly the rate of dissolution of enamel [Manley and Harrington, 1959; Larsen et al., 1976], as theoretically convincingly illustrated by ten Cate and Duijsters [1983]. So, in 1981, we concluded that ‘what appears to be important in reducing the solubility of the

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enamel is the fluoride ion activity in the oral fluid rather than a high content of fluoride in enamel’ [Fejerskov et al., 1981]. When these ways of interpreting available data were first presented in a comprehensive way (at an IADR symposium in Osaka, Japan, in 1981) the reaction was extremely vigorous from parts of the predominantly public health-oriented research community. This was – seen in retrospect – in full accord with the way in which paradigm shifts have been received in the history of science [Kuhn, 1962/1970]. By 1981 it was still a question if the new paradigm could be verified by clinical/epidemiological data. Therefore Groeneveld [1985] re-examined the meticulously collected caries data obtained from the well-controlled Dutch water fluoridation study in Tiel-Culemborg in the 1950s [Backer Dirks et al., 1961]. He could demonstrate that, when both enamel and dentine lesions where diagnosed, there was virtually no difference in the total number of lesions between fluoridated and non-fluoridated areas. However, when only dental lesions (cavities) were recorded, the ‘expected’ reduction of about 50% was found. Thus, the data could be explained as a result of fluoride in the oral environment affecting the dissolution/ reprecipitation processes in the tooth to retard the rate of lesion progression as predicted by the new paradigm. Groeneveld and Backer Dirks [1988] concluded that ‘on the initiation of a caries lesion no pre- nor posteruptive effect of fluoride can be observed. In fact apart from a small retardation of new lesions at younger ages there is almost no effect at all. However, retardation of the progression into dentinal lesions is far more pronounced’. Moreover, despite the fact that in fluoridated areas there is a higher concentration in the fluoride in the outer enamel layers, the difference in prevalence of lesions is so small between fluoride and non-fluoride areas that the conclusion that ‘fluoride concentration in the enamel plays a role of minor importance in caries reduction [Fejerskov et al., 1981]’ is justified [Groeneveld and Backer Dirks, 1988]. A very impressive and open-minded conclusion drawn by one of the most prominent fluoride researchers in the history of ORCA during half a century.

Dental Caries – an Infectious Disease?

The above scientific revolution in fluoride research was partly brought about because the natural history of caries lesion development was reconsidered. Let us, therefore, in the following consider if a paradigm shift also is

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going on in our understanding of dental caries as a disease. The use of the concept ‘an infectious disease’ immediately signals that a disease is caused by a particular microorganism or agent, which has ‘infected’ an individual. Or, as defined by Last [1995], who uses infectious disease synonymously with communicable disease: ‘an illness due to a specific infectious agent or its toxic products that arises though transmission of that agent or its products from an infected person, animal or reservoir to a susceptible host...’. For about half a century caries was defined as an ‘infectious and transmittable’ disease on the following premises. Not all types of microorganisms in the oral cavity are equally able to ferment carbohydrates, so it has been logical to look for major caries pathogens. While research in the first half of the previous century focused on the role of Lactobacillus [Rodriques, 1931], the latter half of that century focussed on the role of mutans streptococci [van Houte, 1980; Loesche, 1986; Bowden, 1991]. The concept of dental caries being ‘infectious and transmittable’ grew out of the elegant rodent studies performed in the 1950s [Keyes, 1960]. Caries only developed in rodents when they were caged with or ate the faecal pellets of groups of caries-active rodents. Further proof emerged when certain streptococci isolated from caries lesions in hamsters, unlike other types of streptococci, caused rampant decay in previously caries-inactive animals [Fitzgerald and Keyes, 1960]. These bacteria, later identified as Streptococcus mutans (SM), gave rise to the concept of caries being due to a specific infection with mutans streptococci, a concept that has gained wide support within the field of caries microbiology over the past four decades [Loesche, 1986; Tanzer, 1989]. It is occasionally claimed that because SM cannot be detected in some patients with no caries increment this is a ‘proof’ that you need to be ‘infected’ with SM to get a lesion. It is ignored that in patients with excellent oral hygiene there is virtually no ‘plaque’ on the tooth surfaces and therefore SM may be below detection level. This should be combined with the fact that mutans streptococci are not primary colonisers of tooth surfaces [Nyvad and Kilian, 1990]. Mutans streptococci belong to the resident microflora and are ubiquitous in populations worldwide. The relationship between SM and dental caries is not absolute. Relatively high proportions of SM may persist on tooth surfaces without caries progression while caries may develop also in the absence of these species [Marsh and Martin, 1992]. The presence of SM can explain only about

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It is not merely semantics, but a paradigm shift, to consider dental caries as a ‘complex disease caused by an imbalance in physiologic equilibrium between tooth mineral and biofilm fluid’ [Fejerskov and Nyvad, 2003]. However, it is acknowledged, as stressed by Davies [2003], that biofilms according to an NIH announcement account for over 80% of microbial infections in the body. Therefore dental caries is of course microbially induced, but the important point is that it is by endogenous bacteria – not exogenous, specific bacteria which infect the individual. The resident flora in the oral cavity will inevitably form biofilms on teeth, e.g. a biofilm defined as a population or community of bacteria living in organised structures at an interface between a solid and liquid. Biofilm bacteria live in microcolonies encapsulated in a matrix of extracellular polymeric substances. Although fixation and dehydration techniques during processing of biofilms for transmission electron microscopy result in shrinkage of the tissue to a varying extent, it has for many years been appreciated that oral biofilms vary extensively in structural arrangement with the colonies deep within the biomass being densely packed, and often being separated by

loosely packed bacteria and larger ‘empty’ pathways. Confocal laser scanning microscopy of biofilms in general has revealed that the biomass comprises ‘open’ water channels through which nutrients and metabolic waste products sieve [Laurence and Neu, 1999]. From dealing with ‘oral debris’ and later ‘dental plaque’ we now appreciate that within an oral biofilm, each group of bacteria occupies microenvironments, which are determined by surrounding cells, proximity to the larger diffusion pathways, the distance from the outer surface, etc., all of which are likely to determine pH, availability of nutrient, relative degrees of saturation of calcium phosphates, etc. Most studies in caries microbiology have dealt with cells in the planktonic phase. It has only recently been appreciated that the same bacteria attached to surfaces and forming biofilms may respond significantly differently to a variety of stimuli such as antimicrobial agents [for review see Davies, 2003]. Many organisms can respond with considerable flexibility to a changing environment; a single set of genes can generate a range of characteristics or phenotypes depending on the environment. With this new general knowledge on biofilm physiology and the appreciation of the fact that the rate of oral biofilm formation and its structural composition vary substantially between individuals [Nyvad and Fejerskov, 1989], not to mention that biofilm ‘age’ may play a decisive role in the response to stimuli, it will be obvious that we need renewed research on oral biofilm physiology in relation to stages of progression of oral diseases (caries, gingivitis and periodontal loss of attachment). Dental plaque is an established designation for any clinically detected microbial mass in the dentition, but it is probably not immediately synonymous with oral biofilm. The paradigm claiming the biofilm as the cause of dental caries has several implications. Lesions develop where biofilms are allowed to mature and remain for prolonged periods of time. Therefore, dental caries occurs at occlusal surfaces (being particularly at risk during the long-lasting eruption into functional occlusion), in interproximal areas below contact points/facets, and along the marginal gingiva. Once exposed, the enamel-cementum junction of course represents an obvious ‘retention site’. Depending on the environmental conditions in the oral cavity, of the individual in general, or at specific sites within an individual, the physiological equilibrium between tooth and biofilm may be disturbed, resulting in a net loss of mineral. If a frank cavity is allowed to form, such a site represents an ecological niche where the biofilm composition gradually adapts to a declining pH environment. Within cavities the biofilm metabolism and diffusion characteris-

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6–10% of the caries experience in a given population [Sullivan et al., 1989; Granath et al.,1993]. The level of mutans streptococci in saliva of children cannot predict future carious increments in children [Matee et al., 1993] and high salivary mutans-streptococcal counts do not add to the prediction when combined with conventional caries experience parameters [van Palenstein Heldermann et al., 2001]. The number of mutans streptococci or lactobacilli in plaque does not explain the variation in caries experience [Sullivan et al., 1996]. A dramatic decline in caries experience has been documented over a few years in populations without apparent changes in salivary mutans levels in the population [Bjarnason et al., 1994]. Collectively, these observations imply that mutans streptococci do not play a specific role in dental caries. Rather, the outgrowth of mutans streptococci should be explained by a disturbance of the homeostasis in the dental biofilm. If the homeostasis of the oral microflora is lost, then an opportunistic infection can occur, i.e., these infections are derived from microorganisms that are endogenous to the host [Marsh and Martin, 1992] and an ecological plaque hypothesis seems more attractive [Marsh, 1994].

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Fig. 1. Schematic illustration of the relationship between the aetiological factor – the microbial deposit and the tooth and biological determinants (inner circle) which influence lesion development at the single tooth surface. In the outer circle are listed various behavioural and socio-economic factors (or confounders) which influence the likelihood for lesion development at an individual and population level. Modified from Fejerskov and Manji [1990].

tics are significantly different from those of biofilm covering sound or inactive caries surfaces [Fejerskov et al., 1992]. Once this is appreciated, the complex character of the disease is highly relevant as numerous biological factors may influence the likely outcome at the single site and in the individual as a whole. In the schematic illustration (fig. 1) the complex interplay between saliva, dietary habits, and the many biological determinants determine biofilm composition and metabolism. In concert with innumerable other factors (several of which we do not even know about yet), the oral and biofilm fluids will determine the likelihood for a net loss of mineral and the rate at which this occurs at any given site. At the individual as well as the population level many of these variables (oral hygiene, diet, etc.) will be highly influenced by the behavioural and socio-economic conditions prevailing. Once this concept of the complexity of the disease and its manifestations is appreciated it will be fully understood why it is so difficult to interpret data about associa-

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tions concerning dental caries, and why no good predictor models are available [Hausen, 1997].

Conclusion and Implications for Oral Research, Prevention and Clinical Management of Dental Caries

The two scientific revolutions (paradigm shifts) in cariology which are described in this paper necessitate a substantial rethinking of the design of future research projects in terms of data analysis and interpretation, and in advancement of new prevention and treatment strategies for dental caries. By appreciating that dental caries belongs to the group of common diseases considered as ‘complex’ or ‘mulifactorial’ such as cancer, heart diseases, diabetes, and certain psychiatric illnesses, we have to realise that there is no simple causation pathway. It is not a simplistic problem such as ‘elimination of one type of microorganism’, or a

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Fig. 2. Schematic illustration of the concept of dental caries as conceived in this paper. Because of continuous exposure to the metabolically active biofilm disease control must be maintained lifelong [Fejerskov and Nyvad, 2003].

matter of improving ‘tooth resistance’. Complex diseases cannot be ascribed to mutations in a single gene or to a single environmental factor. Rather they arise from the concerted action of many genes, environmental factors, and risk-conferring behaviours. As stressed recently by Kiberstis and Roberts [2002], one of the greatest challenges facing biomedical researchers today is to sort out how these contributing factors interact in a way that translates into effective strategies for disease diagnosis, prevention and therapy. Let us keep in mind that dental caries is ubiquitous in all populations [Fejerskov and Baelum, 1998], but the incidence rate varies greatly within and between populations. It is important to appreciate that the caries incidence rate in a group of individuals appears fairly constant throughout life if no special efforts to control lesion progression are made [Hand et al., 1988; Luan et al., 2000]. These new paradigms help to explain the nature of lesion initiation and progression and accordingly why dental caries cannot truly be ‘prevented’, but rather ‘controlled’ by a multitude of interventions. Figure 2 schematically illustrates the concept of dental caries as presented in this paper and explains why dental caries has to be controlled lifelong if a functional dentition is to be maintained. From the figure it will also be appreciated that diagnosis should be performed at non-cavitated stages because the dynamic nature of lesion progression allows for arrest of further mineral loss by restoring

physiological equilibrium between tooth mineral and oral fluids. The whole concept of non-operative treatment has its rationale in these new paradigms, and it will be apparent why any restorative treatment must be accompanied by simultaneous disease control at the individual level. A consequence of dental caries being a complex disease will be that on a population basis we may have success with a particular ‘preventive programme’ in one population in one country, but not necessarily in another population in another country with different cultural and behavioural habits. Moreover, we may organise our dental health care very differently in neighbouring countries, and apply fluorides in very different ways (mouth rinsing, toothpaste, water fluoridation and supervised brushing etc.) and obtain rather similar caries reductions as exemplified by comparing the Scandinavian countries. There is no one single ‘programme’ to be superimposed uncritically on all populations – the important question remains how to control caries lesion progression as cost-effectively as possible. These new concepts explain why we have experienced that several of the ‘old’ recommended preventive programmes are no longer effective. It is of course not because the agents we used in prevention are no longer efficacious. They just become ineffective because the caries incidence rate has changed as the environment has changed. At the individual patient level we have successfully ‘controlled’ the physiologic balance of the intra-oral envi-

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ronment with topical fluorides, dietary monitoring, ‘plaque control’, etc., but the well-trained clinician knows that some patients require much more and ‘closer’ monitoring than others to avoid new lesions. The consequence of the paradigms is to appreciate that the risk of developing new lesions is never zero. Therefore dental caries can never be 100% preventable at the individual and much less at the societal level because of its complex nature. Dental caries is as old as mankind. Whatever directions caries research should take from here it will require a multidisciplinary approach to solving complex problems. More than ever, well-educated clinical dentists should set the stage and in collaboration with colleagues trained in the multitude of new fields in the basic sciences (biophysics, functional genomics, proteomics, chemical biology, nano-technology, etc.) address clinically relevant questions. Let ORCA remain the forum – marketplace, if you like – where clinical dentistry meets basic

sciences in fruitful and challenging exchange of new knowledge to the benefit of the health of populations. History has shown that European caries research has an intellectual flexibility and scientific creativity which allows for ongoing stimulating debates. Let ORCA benefit from this by encouraging more in-depth scientific debates in conjunction with its meetings in the future. Let us bear Charles Darwin’s dictum in mind: ‘All observations must be for or against some view to be of any service’. Let the discussion in ORCA be fearless in conceptual daring, but humble in its respect for observation and facts.

Acknowledgements The present paper would not have been possible without the many discussions and close collaboration over many years with: Vibeke Baelum, G. Dahlén, M.J. Larsen, F. Manji, Bente Nyvad, A. Richards and A. Thylstrup to all of whom I am very grateful.

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Fejerskov O, Silverstone LM, Melsen B, Møller IJ: Histological features of fluorosed human dental enamel. Caries Res 1975;9:190–210. Fejerskov O, Thylstrup A, Larsen MJ: Clinical and structural features and possible pathogenic mechanisms of dental fluorosis. Scand J Dent Res 1977;85:510–534. Fejerskov O, Thylstrup A, Larsen MJ: Rational use of fluoride in caries prevention: A concept based on possible cariostatic mechanisms. Acta Odontol Scand 1981;39:241–249. Fitzgerald RJ, Keyes PH: Demonstration of the etiological role of streptococci in experimental caries in the hamster. J Am Dent Assoc 1960; 61:9–19. Frazier PD, Little MF, Casciani FS: X-ray diffraction analysis of human enamel containing different amounts of fluoride. Arch Oral Biol 1967;12:35–42. Granath L, Cleaton-Jones P, Fatti LP, Grossman ES: Prevalence of dental caries in 4- to 5-yearold children partly explained by presence of salivary mutans streptococci. J Clin Microbiol 1993;31:66–70. Groeneveld A: Longitudinal study of prevalence of enamel lesions in a fluoridated and non-fluoridated area. Community Dent Oral Epidemiol 1985;13:159–163. Groeneveld A, Backer Dirks O: Fluoridation of drinking water, past, present and future; in Ekstrand J, Fejerskov O, Silverstone LM: Fluoride in Dentistry. Copenhagen, Munksgaard, 1988, pp 229–251. Hallsworth AS, Robinson C, Weatherell JA: The chemical pattern of carious attack. Caries Res 1971;5:16–17. Hand JS, Hunt RJ, Beck JD: Incidence of coronal and root caries in an older adult population. J Public Health Dent 1988;48:14–19. Hausen H: Caries prediction – state of the art. Community Dent Oral Epidemiol 1997;25:87– 96. van Houte J: Bacterial specificity in the etiology of dental caries. Int Dent J 1980;30:305–326. Isaac S, Brudevold F, Smith FA, Gardner DE: The relation of fluoride in the drinking water to the distribution of fluoride in enamel. J Dent Res 1958;37:318–325. Kay MI, Young RA, Posner AS: Crystal structure of hydroxy-apatite. Nature 1964;204:1050–1052. Keyes PH: The infectious and transmissible nature of experimental dental caries: Findings and implications. Arch Oral Biol 1960;1:304–320. Kiberstis P, Roberts L: It’s not just the genes. Science 2002;296:685. Kidd EAM, Thylstrup A, Fejerskov O, Bruun C: The influence of fluoride in surface enamel and degree of dental fluorosis on caries development in vitro. Caries Res 1980;14:196–202. Koulourides T, Feagin FF, Pigman W: Remineralisation of dental enamel by saliva in vitro. Ann NY Acad Sci 1965;131:751–757. Kuhn TS: The Structure of Scientific Revolution (1962), ed 2. Chicago, University of Chicago Press, 1970. Kutnerian H, Kuyper AC: The influence of fluoride on the solubility of bone salt. J Biol Chem 1957;233:760–763.

Changing Paradigms in Cariology

Küseler A, Baelum V, Fejerskov O, Heidmann J: Accuracy and precision in vitro of Beetrode® microelectrodes used for intraoral pH measurements. Caries Res 1993;27:183–190. Larsen MJ: Enamel Solubility Caries and Erosions; thesis Royal Dental College, Aarhus, 1975. Larsen MJ, von der Fehr FR, Birkeland JM: Effect of fluoride on the saturation of an acetate buffer with respect to hydroxyapatite. Arch Oral Biol 1976;21:723–728. Larsen MJ, Kirkegaard E, Poulsen S, Fejerskov O: Enamel fluoride, dental fluorosis and dental caries among immigrants to and permanent residents of five Danish fluoride areas. Caries Res 1986;20:349–355. Last JM: A Dictionary of Epidemiology, ed 3. Oxford, Oxford University Press, 1995. Laurence JR, Neu TR: Confocal laser scanning microscopy for analysis of microbial biofilms. Methods Enzymol 1999;310:131–144. Loesche WJ: Role of Streptococcus mutans in human dental decay. Microbiol Rev 1986;50: 2118–2135. Luan W-M, Baelum V, Fejerskov O, Chen X: Tenyear incidence of dental caries in adult and elderly Chinese. Caries Res 2000;34:205–213. Manley RS, Harrington DP: Solution rate of tooth enamel in acetate buffer. J Dent Res 1959;38: 910–919. Margolis HC, Duchworth JH, Moreno EC: Composition of pooled resting plaque fluid from caries-free and caries susceptible individuals. J Dent Res 1988;67:1468–1475. Marsh PD: Microbial ecology of dental plaque and its significance in health and disease. Adv Dent Res 1994;8:263–271. Marsh PD, Martin M: Oral Microbiology, ed 3. London, Chapman & Hall, 1992. Matee MIN, Mikx FHM, De Soet JS, Maselle SY, De Graaff J, Van Palenstein Helderman WH: Mutans streptococci in caries-active and cariesfree infants in Tanzania. Oral Microbiol Immunol 1993;8:322–324. Mellberg JR: Enamel fluoride and its anticaries effects. J Prevent Dent 1977;4:8–20. Newsely JW, McConnell D, Armstrong WD: The nature of carbonate contents in tooth mineral. Experientia 1963;19:620–621. Nyvad B, Fejerskov O: Structure of dental plaque and the plaque-enamel interface in human experimental caries. Caries Res 1989;23:151– 158. Nyvad B, Kilian M: Comparison of the initial streptococcal microflora on dental enamel in caries-active and in caries-inactive individuals. Caries Res 1990;24:267–272. Øgaard B, Rølla, Ruben J, Dijkman T, Arends J: Microradiographic study of deminerlization of shark enamel in a human caries model. Scand J Dent Res 1988;96:209–211. van Palenstein Heldermann WH, Mikx FHM, van’t Hof MA, Truin GJ, Kalsbeek H: The value of salivary bacterial counts as a supplement to past caries experience as caries predictor in children. Eur J Oral Sci 2001;109:312–315. Planck M: Scientific Autobiography and Other Papers (translated by Gaynor F). New York, 1940.

Poulsen S, Larsen MJ: Dental caries in relation to fluoride content of enamel in the primary dentition. Caries Res 1975;9:59–65. Richards A, Fejerskov O, Baelum V: Enamel fluoride in relation to severity of human dental fluorosis. Adv Dent Res 1989;3:147–153. Richards A, Kragstrup J, Josephsen K, Fejerskov O: Dental fluorosis developed in post-secretory enamel. J Dent Res 1986;65:1406–1409. Richards A, Larsen MJ, Fejerskov O, Thylstrup A: Fluoride content of buccal surface enamel and its relation to dental caries in children. Arch Oral Biol 1977;22:425–428. Richards A, Likimani S, Baelum V, Fejerskov O: Fluoride concentrations in unerupted fluorotic human enamel. Caries Res 1992;26:328–332. Rodriques FE: Quantitative incidence of Lactobacillus acidophilus in the oral cavity as a presumptive index of susceptibility to dental caries. J Am Dent Assoc 1931;18:2118–2135. Schamschula RG, Agus H, Charlton G, Duppenthaler JL, Un P: Associations between fluoride concentration in successive layers of human enamel and individual dental caries experience. Arch Oral Biol 1979;24:847–852. Shern RJ, Driscoll WS, Korts DC: Enamel biopsy results of children receiving fluoride tablets. J Am Dent Assoc 1977;95:310–314. Smith CE: Cellular and chemical events during enamel maturation. Crit Rev Oral Biol Med 1998;9:128–161. Spector PC, Curzon MEJ: Surface enamel fluoride and strontium in relation to caries prevalence in man. Caries Res 1979;113:227–230. Stephan RM: Changes in hydrogen-ion concentration on tooth surfaces and in carious lesions. J Am Dent Assoc 1940;27:718–723. Sullivan Å, Borgström MK, Granath L, Nilsson G: Number of mutans streptococci or lactobacilli in a total dental plaque sample does not explain the variation in caries better than the numbers in stimulated whole saliva. Community Dent Oral Epidemiol 1996;24:1559–163. Sullivan Å, Granath L, Widenheim J: Correlation between child caries incidence and S. mutans/ lactobacilli in saliva after correction for confounding factors. Community Dent Oral Epidemiol 1989;17:240–244. Tanzer JM: On changing the cariogenic chemistry of coronal plaque. J Dent Res 1989;68(special issue):1576–1587. Thylstrup A: Clinical Evaluation of Fluoride Derived Enamel Changes: A Critical Review; thesis Royal Dental College, Aarhus, 1979. Volker JF: Effect of fluoride in solubility of enamel and dentin. Proc Soc Exp Biol Med 1939;42: 725–727. Weatherell JA, Robinson C, Hallsworth AS: The concept of enamel resistance: A critical review; in Guggenheim B (ed): Cariology Today. Basel, Karger, 1984, pp 223–230. Young RA: Biological apatite vs. hydroxyapatite at the atomic level. Clin Orthop 1975;113:249– 262. Zipkin I, Posner AS, Eanes ED: The effect of F on x-ray diffraction pattern of apatite of human bone. Biochim Biophys Acta 1962;59:255– 258.

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Caries Res 2004;38:192–198 DOI: 10.1159/000077754

Diagnosis versus Detection of Caries B. Nyvad School of Dentistry, Faculty of Health Sciences, University of Aarhus, Aarhus, Denmark

Key Words Dental caries, detection W Dental caries, diagnosis W Diagnostic threshold W ‘Gold standard’ W Lesion activity W Randomized controlled trial W Reliability W Treatment decision W Validity

Abstract Caries diagnosis is the art or act of identifying a disease from its signs and symptoms. This is distinct from the detection of the signs and symptoms themselves. The diagnosis forms the basis for making informed treatment decisions. Hence, if there is no diagnostic step expressed in terms of the probability of present and future occurrence of disease, practitioners may resort to treatments guided by previous experiences with similar clinical manifestations. This paper reviews various methodological aspects of caries diagnostic testing. It is concluded that rather than continuing to search for the truth of the diagnosis, it may be more informative to consider the consequences of the diagnosis. This view is supported by results from caries-preventive trials in which the activity of carious lesions has been monitored longitudinally over years. Copyright © 2004 S. Karger AG, Basel

ORCA was among the first scientific fora to focus on the challenges of diagnosing caries in populations with low rates of lesion progression. In the concluding remarks

ABC

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Accessible online at: www.karger.com/cre

of the Symposium of the ORCA Caries Diagnosis Working Group in Helsinki in 1992 it was stated: ‘The development of methods for determining whether a carious lesion is stable or progressing is a priority in caries research and a mechanism in terms of estimated progression will be invaluable. Detection of small carious lesions is only part of the problem, the appropriate method for therapeutic and preventive care that can be applied to the lesion is the aspect facing the clinician daily’ [Pine and ten Bosch, 1996]. Regrettably, in spite of these promising visions, most research on caries diagnosis since then has concentrated on the development and testing of new methods for the detection of small lesions. There has been almost no focus on whether the use of such new methods may facilitate treatment decisions or lead to better health outcomes for the patients. The purpose of this paper is first to revisit some issues pertaining to the evaluation of caries diagnostic tests. Secondly, to highlight some new developments within the field of clinical caries diagnosis that may have important implications for caries diagnostic decision making and the delivery of non-operative preventive care.

To Diagnose or to Detect?

The art of diagnosis rests on the assumption that diseases can be distinguished from their signs and symptoms. Diagnostic reasoning is an extremely complex process that involves elements of simple pattern recognition,

Bente Nyvad School of Dentistry, Faculty of Health Sciences University of Aarhus, Vennelyst Boulevard DK–8000 Aarhus C (Denmark) Tel. +45 89424074, Fax +45 86202202, E-Mail [email protected]

Signs: Clinical examination Supplementary tests Symptoms: Anamnestic information

Diagnosis

Treatment decision

Fig. 1. The classical diagnostic decision process.

probabilistic considerations and hypothetico-deductive thinking [Wulff and Gøtzsche, 2000]. Diagnostic decision making is a balancing act. The clinician must not overlook diseases in need of treatment, and, at the same time, he must not make a diagnosis when it is not warranted. The choice of diagnosis is further complicated by the fact that the clinician must take into account the consequences for the patient. The inherent complexity of the diagnostic process explains why nobody has ever been able to unveil how clinicians think when they examine their patients and seek the right diagnosis. It also explains why diagnostic skills cannot be learnt from textbooks alone, but require clinical training. Due to the deductive nature of the diagnostic process, the term diagnosis should not be used synonymously with the term detection. To diagnose is ‘the art or act of identifying a disease from its signs and symptoms’ [MerriamWebster, 2003]. This is distinct from the detection of the signs and symptoms themselves. During the diagnostic process the clinician attempts to assign a label to a set of signs and symptoms brought together from various sources (e.g. interview, clinical examination and supplementary tests). This information is used to assess the probability that the patient has a certain condition. In medicine the diagnosis is a pivotal step for making treatment decisions. Therefore, the diagnostic step has sometimes been referred to as ‘a mental resting place on the way to intervention’ [Baelum and Fejerskov, 2003]. Figure 1 illustrates the classical diagnostic decision process as outlined above. It has been proposed that the diagnostic problem in caries differs from that of other medical diseases. Hence,

Diagnosis of Caries

because there are virtually no symptoms of caries (at least in the early stages of the disease process!) it has been claimed that there is also no diagnostic ‘step’ and consequently no diagnosis in caries [Bader and Shugars, 1996]. Caries examination becomes primarily a question of detection (caries yes or no). This concept may have led to the unfortunate situation that dentists have merged their diagnostic activities with treatment planning decisions [Beck, 1995]. Moreover, it may explain why we have never had a true evidenced-based approach to non-operative preventive caries treatment. Thus, if there is no diagnosis, expressed in terms of the probability of present or future occurrence of caries, practitioners may easily resort to a treatment pattern where previous experiences with similar clinical manifestations will guide the choice of treatment.

How to Evaluate a Caries Diagnostic Test?

In view of the above considerations it is not sufficient to have a good diagnostic test; the test must also work well [Bader and Shugars, 1996]! Traditionally, the performance of a diagnostic test is evaluated in terms of its validity (the degree to which a measurement measures what it purports to measure with reference to an independent ‘gold standard’) and reliability (the degree to which the results obtained by a measurement procedure can be replicated) [Last, 1995]. However, it is also important to assess the consequences for the patient in response to the diagnosis and/or treatment provided. Last but not least, the evaluation should include considerations about the potential negative aspects for the patient as well as considerations about the cost-effectiveness of using the test. Only when all the above issues have been adequately addressed, should a new diagnostic test or tool be implemented for routine use in clinical practice [Abramson, 1990].

The ‘Gold Standard’

Validity may be appraised in different ways although in caries criterion validity has been the most commonly applied. Assessment of criterion validity requires the existence of external criteria – so-called ‘gold standards’ – for the phenomenon. So far most evaluations of caries diagnostic criteria have focused on depth of lesion penetration as assessed either histologically, clinically or radiographically [Hintze and Wenzel, 2003]. There is no strict rule as

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to what the ‘gold standard’ should be; it is a matter of choice [ten Bosch and Angmar-Månsson, 2000]. The investigator should always choose a ‘gold standard’ that represents the highest level of truthfulness for the phenomenon under study. Depth of lesion penetration may not necessarily be the most appropriate ‘gold standard’ for caries. Furthermore, for practical or ethical reasons it may not always be possible to use the most exact ‘gold standard’. Researchers and clinicians should be aware of this when evaluating the performance of caries diagnostic tests. A new diagnostic method that compares favourably with an accepted ‘gold standard’ can be no better than the ‘gold standard’ being used. Likewise, if a new method fails to compare favourably with a ‘gold standard’, the test may not necessarily be useless. It may in fact be superior to the ‘gold standard’ used [Beck, 1995]. In the absence of an external ‘gold standard’, validity can sometimes be appraised by seeing whether a followup study shows an association between the measurement and a subsequent event (e.g. cavity formation) that is believed to be an outcome of what was measured (predictive validity). Another way of appraising validity is to see whether there are associations with other variables that there is reason to believe should be linked with the characteristic under study (construct validity) [Abramson, 1990]. Recently, the latter methods were successfully used to determine the validity of caries activity assessments in a clinical trial of the preventive effect of fluoride toothpaste [Nyvad et al., 2003].

Qualitative versus Quantitative Methods

When summarizing the data from studies that have assessed criterion validity of current clinical caries diagnostic methods (e.g. conventional clinical examination and radiography) most of the studies have shown a rather poor validity with low sensitivity and moderate specificity [for reviews, see Pine and ten Bosch, 1996; Ismail, 1997; Bader et al., 2002]. This implies that caries diagnosis, as normally performed in daily clinical practice, is an inexact procedure that results in both over- and underdiagnosis. In reality, the diagnostic performance may be much poorer than assumed because the majority of the validation studies have been performed under laboratory conditions, excluding the difficulties introduced by the presence of plaque and saliva. The rather poor diagnostic performance of the conventional caries diagnostic methods has prompted some researchers to search for quantitative detection methods,

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such as electrical conductance measurements, light scattering methods, laser fluorescence methods. There were at least three motives for this development: (1) that quantitative methods may detect lesions at an earlier stage than conventional methods; (2) that quantitative measurements are more reliable than qualitative measurements, and (3) that quantitative assessments may provide the means for non-destructive monitoring of the course of disease [ten Bosch and Angmar-Månsson, 2000]. Indeed, some quantitative detection methods have shown a high degree of reliability [Lussi et al., 1999; Tranaeus et al., 2002]. Likewise, it has been demonstrated that quantitative light-induced fluorescence methods may be suitable for the longitudinal quantification of non-cavitated carious lesions on smooth tooth surfaces, especially for scientific purposes [Angmar-Månsson et al., 1996; Tranaeus et al., 2001]. However, laser fluorescence suffers from a poor correlation with mineral loss [Shi et al., 2001]. Furthermore, the cut-off points used to classify the numerical output of the latter devices into sound, enamel and dentin caries have not been finally determined in vivo [Lussi et al., 2001]. Therefore, until such methods have been properly tested and have proved superior to conventional caries diagnostic methods, such as clinical examination [Attrill and Ashley, 2001], they should not be recommended for routine clinical use.

How Early Should a Lesion Be Detected?

Along with the development of quantitative methods for caries lesion detection there has been a parallel effort to detect lesions as early as possible, even prior to the non-cavitated stage of lesion formation [Stookey, 2000; Pitts, 2001]. This development has been driven by the belief that early identification provides better estimates of disease and improves the possibility for successful preventive intervention. However, from a clinical point of view this can be questioned. A low diagnostic threshold not only leads to detection of more small lesions but also to more false positive diagnoses. Furthermore, because a significant number of the early lesions are likely to arrest or regress without professional intervention [Backer Dirks, 1966; Baelum et al., 2003], such a strategy may not be cost-effective. Setting the diagnostic threshold is therefore a question of balance. At present, for clinical purposes it would seem essential to diagnose lesions at a non-cavitated stage but not at such an early stage that they are invisible to the naked eye. This position is supported by the observation that non-cavitated carious

Nyvad

Fig. 2. The relative risk (RR) that a fluorideexposed surface versus a control surface undergoes lesion transition, as observed in a 3year caries-preventive trial of daily supervised brushing with fluoride toothpaste in young teenagers. RR ! 1 indicates inhibition of lesion progression; RR 1 1 indicates promotion of lesion regression. Data modified after Nyvad et al. [2003].

Sound

Active non-cavitated

Inactive non-cavitated

Cavity, filling or extracted

Cavity, filling or extracted

Cavity, filling or extracted

: :

RR < 1 RR > 1

lesions can be controlled sufficiently by non-operative preventive interventions such as topical fluorides [Marinho et al., 2003].

Because of low rates of lesion progression in many Western countries there has over the past decade been an interest in developing clinical diagnostic criteria for assessing the activity state of non-cavitated carious lesions. The theoretical foundation of one such system [Nyvad et al., 1999] is the assumption that lesion activity, defined as net progression or net regression [Fejerskov and Manji, 1990], will be reflected in the surface features of the lesion: matt, ‘chalky’ and rough enamel lesions being ‘active’, and shiny, smooth enamel lesions being ‘inactive’ or ‘arrested’ [for review, see Thylstrup et al., 1994]. By definition, these categories refer to the activity state of the lesion as inferred at the time of examination. Such distinctions have no bearing on what may occur to the lesion over time. Hence, if an ‘active’ lesion is subjected to efficacious preventive intervention, such as oral hygiene or topical fluorides, the activity state of the lesion is likely to change [Baelum et al., 2003]. Only if the local environmental conditions of a lesion remain unchanged can the activity state be expected to stay the same. Clinical studies using this new diagnostic method in contemporary North-European child populations have

shown that caries activity assessments may be both reliable and valid, regardless of the diagnostic threshold chosen. In well-trained examiners the kappa values ranged between 0.74 and 0.85 for intra-examiner agreement and between 0.78 and 0.80 for inter-examiner agreement [Nyvad et al., 1999]. The pattern of distribution of the diagnoses showed that the diagnostic criteria were effective in separating ‘active’ from ‘inactive’ lesions. However, as with other studies assessing non-cavitated carious lesions [Ismail et al., 1992], it was often difficult to differentiate between a diseased surface and a sound surface, possibly because of insufficient plaque removal prior to the examination. Due to the lack of an independent ‘gold standard’ for caries activity, construct validity of the criteria was evaluated by means of the ability of the criteria to reflect known effects of fluoride on caries [Nyvad et al., 2003]. When reanalysing the results from a 3-year trial of daily supervised brushing with fluoride toothpaste [Machiulskiene et al., 2002] it was found that the relative risks for lesion transitions in the experimental group versus the control group imitated the anticipated effect of fluoride on caries lesion dynamics [ten Cate and Featherstone, 1996]; hence, at all stages of lesion formation brushing with fluoride toothpaste inhibited the development or progression of caries and at the same time enhanced arrest or regression of caries (fig. 2). These effects were most pronounced for ‘active’ non-cavitated lesions supporting the notion that fluoride exerts its predominant effect on the active caries process [Fejerskov et al., 1981]. It was concluded,

Diagnosis of Caries

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Recent Developments in Clinical Caries Diagnosis

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The status of each tooth surface

Level 1:

Sound

Inactive (non-progressing)

Level 2:

Level 3: (Treatment decision)

Lesion

NOTAL (NO Treatment At alL)

Filling

Active (progressing)

NOT (Non-Operative Treatment)

No defect

OT (Operative Treatment)

Defect

NOREP (NOREPlacement)

REP (REPlacement)

Fig. 3. Decision-making tree for dental caries according to Nyvad and Fejerskov [1997]. Note that the flow chart does not take into account factors operating at the level of the individual.

therefore, that the diagnostic criteria had construct validity for the assessment of caries lesion activity. A further finding of the study was that caries activity assessments had predictive validity [Nyvad et al., 2003]. Thus, ‘active’ non-cavitated lesions had a considerably greater risk of progressing to a cavity than ‘inactive’ noncavitated lesions, an effect that was more pronounced in subjects not regularly exposed to fluoride. The latter observation implies that caries activity assessments may be used to advise the subsequent course of treatment. In fact, we have previously proposed a decision-making tree for dental caries that has included activity assessment as a key factor in the decision process (fig. 3) [Nyvad and Fejerskov, 1997]. According to this model the first level of the decision process is to determine the state of the tooth surface, whether sound, having a lesion or being filled. Suppose it has been decided that there is a lesion, one may proceed to level 2, which is to assess the activity state of the lesion: ‘inactive’ (non-progressing) or ‘active’ (progressing). The third step is to generate a proper treatment decision. ‘Inactive’ lesions, because of their slowly or nonprogressing nature, do not need professional treatment. ‘Active’ lesions, on the other hand, because of their progressive nature, demand professional treatment. The professional treatment of ‘active’ lesions will depend on the

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accessibility to plaque removal. If plaque control is likely to be insufficient, such as in cavitated lesions, the lesion should be filled. However, for non-cavitated ‘active’ lesions, non-operative preventive interventions are to be preferred. This example illustrates how activity assessment could serve as an important diagnostic means for making evidence-based treatment decisions.

Concluding Remarks

When dealing with diagnostic questions one should bear in mind that the diagnosis is not a goal by itself. The ultimate goal of making a diagnosis is always to select the best possible treatment. Except for the above systematic approach, researchers have shown very little interest in the long-term outcomes of caries diagnostic procedures. This is surprising as for both the clinician and the patient the outcome in terms of the appropriateness of the treatment provided in response to a given diagnosis is of principal interest [Bader et al., 2002; Baelum and Fejerskov, 2003]. Future research on caries diagnosis should be much more concerned about evaluating the long-term consequences of diagnostic methods (e.g. in response to refined diagnostic methods or different modes of lesion

Nyvad

Therapeutic effect Hazard ratio increased by 50–85%

INACTIVE

ACTIVE

Preventive effect Hazard ratio decreased by 25–50 %

Fig. 4. The effect of fluoride on hazard ratios for caries transitions

from inactive to active and active to inactive state, respectively, as observed in a 3-year caries-preventive trial of daily supervised brushing with fluoride toothpaste in young teenagers. Percentages refer to different types of tooth surfaces. Data modified after Baelum et al. [2003].

classification) than by studying the immediate outcomes in terms of accuracy and reliability. This is not to say that the latter issues are not important. However, if we appreciate that the performance of most caries diagnostic tools is poor, it may be more pertinent to consider the conse-

quences of the diagnosis than to dwell on the truth of the diagnosis [Wulff, 1979]. The views presented here imply that in addition to assessing validity and reliability, the performance of caries diagnostic methods should also be evaluated in randomized controlled trials. Such a methodology may, in fact, have additional benefits. We were recently fascinated to learn how survival time analysis of lesion transitions in a caries-preventive trial [Machiulskiene et al., 2002] provided novel information about the preventive and therapeutic actions of fluoride. Thus, when lesions were dichotomized into ‘inactive’ and ‘active’ stages the hazard ratios for transitions from ‘inactive’ to ‘active’ were decreased by 25–50% in the fluoride group compared to the control group while the corresponding ratios for transitions from ‘active’ to ‘inactive’ were increased by 50–85%, depending on the type of surface examined (fig. 4) [Baelum et al., 2003]. Collectively, these observations suggest that the therapeutic effect of fluoride may by far exceed the preventive effect, an observation that is in concert with the marked retardation of caries progression beyond the stage of enamel caries in individuals living in water-fluoridated areas [Groeneveld, 1985].

References Abramson JH: Survey Methods in Community Medicine: Epidemiological Studies, Programme Evaluation, Clinical Trials, ed 4. New York, Churchill Livingstone, 1990, pp 47–56, 151–164. Angmar-Månsson B, Al-Khateeb S, Tranaeus S: Monitoring the caries process: Optical methods for clinical diagnosis and quantification of enamel caries. Eur J Oral Sci 1996;104:480– 485. Attrill DC, Ashley PF: Occlusal caries detection in primary teeth: A comparison of DIAGNOdent with conventional methods. Br Dent J 2001; 190:436–443. Backer Dirks O: Posteruptive changes in dental enamel. J Dent Res 1966;45:503–511. Bader JD, Shugars DA: Issues in the adoption of new methods of caries diagnosis; in Stookey GK (eds): Early Detection of Dental Caries. Indianapolis, Indiana University School of Dentistry, 1996, pp 11–26. Bader JD, Shugars DA, Bonito AJ: A systematic review of the performance of methods for identifying carious lesions. J Public Health Dent 2002;62:201–213. Baelum V, Fejerskov O: Caries diagnosis: ‘A mental resting place on the way to intervention’?; in Fejerskov O, Kidd E (eds): Dental Caries: The Disease and Its Clinical Management. Oxford, Blackwell Munksgaard, 2003, pp 101–110.

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Baelum V, Machiulskiene V, Nyvad B, Richards A, Væth M: Application of survival analysis to carious lesion transition in intervention trials. Community Dent Oral Epidemiol 2003;31: 252–260. Beck JD: Issues in assessment of diagnostic tests and risk for periodontal diseases. Periodontol 2000 1995;7:100–108. ten Bosch JJ, Angmar-Månsson B: Characterization and validation of diagnostic methods; in Faller RV (ed): Assessment of Oral Health. Monogr Oral Sci Basel, Karger 2000, vol 17, pp 174–189. ten Cate JM, Featherstone JDB: Physicochemical aspects of fluoride-enamel interactions; in Fejerskov O, Ekstrand J, Burt BA (eds): Fluoride in Dentistry. Copenhagen, Munksgaard, 1996, pp 252–272. Fejerskov O, Manji F: Risk assessment in dental caries; in Bader JD (ed): Risk Assessment in Dentistry. Chapel Hill, University of North Carolina Dental Ecology, 1990, pp 215–217. Fejerskov O, Thylstrup A, Larsen MJ: Rational use of fluorides in caries prevention: A concept based on possible cariostatic mechanisms. Acta Odontol Scand 1981;39:241–249. Groeneveld A: Longitudinal study of prevalence of enamel lesions in a fluoridated and non-fluoridated area. Community Dent Oral Epidemiol 1985;13:159–163.

Hintze H, Wenzel A: Diagnostic outcome methods frequently used for caries validation. Caries Res 2003;37:115–124. Ismail AI: Clinical diagnosis of precavitated carious lesions. Community Dent Oral Epidemiol 1997;25:13–23. Ismail AI, Brodeur J-M, Gagnon P, Payette M, Picard D, Hamalian T, Oliver M, Eastwood BJ: Prevalence of non-cavitated and cavitated carious lesions in a random sample of 7–9-yearold schoolchildren in Montreal, Quebec. Community Dent Oral Epidemiol 1992;20:250– 255. Last JM: A Dictionary of Epidemiology. ed 3. Oxford, Oxford University Press, 1995. Lussi A, Imwinkelried S, Pitts N, Longbottom C, Reich E: Performance and reproducibility of a laser fluorescence system for detection of occlusal caries in vitro. Caries Res 1999;33:261– 266. Lussi A, Megert B, Longbottom C, Reich E, Francescut P: Clinical performance of a laser fluorescence device for detection of occlusal caries lesions. Eur J Oral Sci 2001;109:14–19. Machiulskiene V, Richards A, Nyvad B, Baelum V: Prospective study of the effect of post-brushing rinsing behaviour on dental caries. Caries Res 2002;36:301–307.

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Marinho VC, Higgins JP, Sheiham A, Logan S: Fluoride toothpastes for preventing dental caries in children and adolescents. Cochrane Database Syst Rev 2003;1:CD002278. Merriam-Webster 2003:www2.Merriam-webster. com/cgi-bin/mwmednlm. Nyvad B, Fejerskov O: Assessing the stage of caries lesion activity on the basis of clinical and microbiological examination. Community Dent Oral Epidemiol 1997;25:69–75. Nyvad B, Machiulskiene V, Baelum V: Reliability of a new caries diagnostic system differentiating between active and inactive caries lesions. Caries Res 1999;33:252–260. Nyvad B, Machiulskiene V, Baelum V: Construct and predictive validity of clinical caries diagnostic criteria assessing lesion activity. J Dent Res 2003;82:117–122.

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Pine CM, ten Bosch JJ: Dynamics of and diagnostic methods for detecting small carious lesions. Caries Res 1996;30:381–388. Pitts NB: Clinical diagnosis of dental caries: A European perspective. J Dent Educ 2001;65: 973–980. Shi XQ, Tranaeus S, Angmar-Månsson B: Comparison of QLF and DIAGNOdent for quantification of smooth surface caries. Caries Res 2001; 35:21–26. Stookey GK: Practical applications of early caries detection methods; in Stookey GK (ed): Early Detection of Dental Caries. II. Indianapolis, Indiana University School of Dentistry, 2000, pp 357–363. Thylstrup A, Bruun C, Holmen L: In vivo caries models – mechanisms for caries initiation and arrestment. Adv Dent Res 1994;8:144–157.

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Tranaeus S, Al-Khateeb S, Bjorkman S, Twetman S, Angmar-Mansson B: Application of quantitative light-induced fluorescence to monitor incipient lesions in caries-active children: A comparative study of remineralization by fluoride varnish and professional cleaning. Eur J Oral Sci 2001;109:71–75. Tranaeus S, Shi XQ, Lindgren LE, Trollsas K, Angmar-Mansson B: In vivo repeatability and reproducibility of the quantitative light-induced fluorescence method. Caries Res 2002;36:3–9. Wulff HR: What is understood by a disease entity? J R Coll Physicians Lond 1979;13:219–220. Wulff HR, Gøtzsche PC: Rational Diagnosis and Treatment: Evidence-Based Clinical Decision Making, ed 3. Oxford, Blackwell Science, 2000.

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Caries Res 2004;38:199–203 DOI: 10.1159/000077755

Diagnostic Levels in Dental Public Health Planning Amid Ismail School of Dentistry, University of Michigan, Ann Arbor, Mich., USA

Key Words Dental caries, diagnosis W Public health W Epidemiology of caries stages W Dental caries, prevention

Abstract This concept paper discusses the rationale for using different diagnostic criteria for dental caries in public health programs. The author contends that epidemiological data or needs assessment surveys should collect data to provide information on the epidemiology of different stages of the caries process in order to enable planners to design tailored programs to prevent dental caries. In a rapidly progressing caries environment, conventional approaches to delivering preventive measures may not work. The author also contends that dental public health programs should expand their vision to influence how dentists are detecting, diagnosing and managing dental caries. Dentists’ restorative decisions have significant impact on the oral health status of a nation. Henceforth, detecting the early or noncavitated caries level and preventing these lesions from progressing to the cavitated stage (or being restored) could have a significant impact on the oral health status around the world. There is a need for more research on the best methods to detect, prevent and treat early carious lesions.

Dental public health planning and practice around the world are based on different concepts, goals and approaches. All dental public health programs share common principles. The bedrock of successful planning in dental public health is information on the epidemiology of a disease and the modifiable risk factors that could be targeted to prevent it. The first step in planning of public health programs is to collect and analyze information on the prevalence or incidence and risk factors of a disease. Measurement of these key markers is an important first step in public health planning. Unfortunately, in most countries, the dental public health community has paid little or even no attention to the complex issues associated with measuring, detecting or diagnosing dental caries.

What Is the Case Definition of Dental Caries?

In spite of the progress in understanding the caries process, there is still some significant level of confusion among members of the dental community on what is dental caries. During most of the 20th century, dental caries was detected and managed as if the caries process is synonymous with ‘cavities’. The practice of dentistry has focused on developing the best ‘drill and fill’ interventions. Even today some European and American re-

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Prof. A. Ismail School of Dentistry, University of Michigan 1011 N. University Ave., D2361 Ann Arbor, MI 48109-1078 (USA) Tel. +1 734 647 9190, Fax +1 734 936 1597, E-Mail [email protected]

searchers and public health specialists still focus on the end stage of the caries process. In my opinion, planning public health programs that focus only on the prevention of cavitated carious lesions could, sometimes, be detrimental to oral health. In this paper, and for reasons to be presented in the next sections, I propose that we should consider and define the stages of dental caries in prevention, health promotion or treatment programs. A stage-based prevention and management of dental caries represents a new approach in planning dental public health programs in the 21st century. Failure to consider the stages of the caries process when designing prevention and treatment programs could lead to inappropriately removing healthy tooth structure in a tooth with a carious lesion that could remineralize. There is evidence that dentists have different levels of knowledge on caries etiology and prevention as well as on when to restore a tooth [Moon et al., 1998; Lewis et al., 1997; Mejàre et al., 1999]. Though these findings have been known since the early 1980s [Elderton, 1985], they have not yet been the subject of debate among dental public health experts. The misclassification of sound tooth surfaces due to relatively less than perfect specificity of detection tools and criteria of dental caries has a significant impact on public health. Even a small percentage of false-positive classifications of teeth can result in a significant contribution to the restorative burden in individuals who predominately have sound teeth. Filling sound teeth, due to misdetection or misclassification, leads to a spurious increase in dental caries indices [Ismail et al., 1997]. This misclassification problem should present an ethical dilemma for promoters of health. In a longitudinal study of restorative decisions made by a sample of private practitioners in Montreal, Canada, it was found that 50% of new class I restorations placed in maxillary first permanent molars of 6- to 9-year-old children within a period of 3 months after the baseline examination were placed in clinically sound teeth (with no signs of staining, active or arrested early noncavitated or cavitated carious lesions) [Ismail et al., 1997]. For mandibular permanent molars, 33% of class I restorations were placed in sound teeth within 3 months after the baseline examination. In the first 3 months of the second year of followup, it was found that the majority of class I restorations were placed in noncavitated pits and fissures (68.8% for maxillary molars and 31.8% for mandibular molars). In this study, the trained and calibrated dental examiners were instructed to score high when in doubt to give the benefit of any doubt in classification of the caries status of a tooth surface to the dentists who provided the dental

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care for each child enrolled in the study. If this degree of error due to misclassification of caries detection in clinical practice is found in other countries, then we have an ethical problem that we so far have refrained from discussing. In my opinion, it is unfortunate that the current standard for detection and assessment of dental caries in planning for dental public health programs in most countries is based on the WHO [1997] or NIDCR [Radike, 1968; NIDCR, 1987] criteria which measure dental caries at the cavitation or ‘softness’ level. As a result, all countries have data on the prevalence of cavitated carious lesions but not the prevalence of noncavitated or early carious lesions. In my opinion, having the latter information may help dental public health programs to design targeted secondary prevention programs to prevent the progression of carious lesions. Such programs are particularly important to promote the dental health of young children and patients who experience rapid progression of dental caries. The issue of what detection level should be used in dental public health planning has not been widely discussed maybe because it has been assumed that ‘caries is caries’ and public health programs should be focused on ‘frank’ cavitated lesions. Since the goal is to prevent ‘cavities’, assessment should focus on measuring their presence. I contend that focusing only on cavitation misses the most important stage of the caries process: early enamel carious lesions.

What Is a Public Health Problem?

In deciding what detection levels of dental caries should be used in planning dental public health programs, it is important to define what a public health problem is. Public health, as Burt and Eklund [1999] contend, cannot easily be defined. There is a general consensus that public health is ‘...fulfilling society’s interest in assuring conditions in which people can be healthy’ [Institute of Medicine, 1988]. This definition implies that public health planning should encompass all programs or services that can promote the health status of a targeted population. Public health programs or services should include activities that focus on health promotion and primary, secondary and tertiary health care. Defining a public health problem is also difficult because there are various cultural, political, personal, community and organizational factors that should be considered for labeling any health condition as a ‘public health problem’. Burt and Eklund [1999] propose that a

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public health problem should meet the following two conditions: (1) there is a condition or situation that is widespread and has an actual or potential cause of morbidity or mortality; (2) there is a perception on the part of the public, government or public health authorities that the condition is a public health problem. Dental caries is a public health problem because it is a widespread condition that is costly to treat and it impacts on the quality of life at all ages. In most of the developing world, dental caries remains untreated [Carino et al., 2003; Yee and Sheiham, 2002]. Fortunately, unlike other infectious diseases, dental caries does not cause significant public alarm because of its low mortality and impact on daily living. Nevertheless, the social and economic costs of dental caries are significant [Yee and Sheiham, 2002]. It is time to rethink the scope of dental public health planning. Traditionally, public health programs have been organized to prevent deadly infectious diseases or promote health by programs designed to reduce the exposure to risk factors in populations. Assessment, policy development and assurance are the three major functions of public health agencies [Institute of Medicine, 1988]. This basic view of public health should be modified to incorporate all activities that the public or health professionals undertake to promote health, prevent disease or manage the damage caused by a disease. Dental public health programs should encompass a wide range of activities throughout life. Dental public health plans should address the following questions: (1) What programs are needed to promote oral health and healthy behaviors? (2) What programs are needed to prevent the development of early carious lesions in enamel? (3) What programs are needed to prevent the progression or reverse early carious lesions in enamel? (4) What programs are needed to minimally treat cavitation limited to enamel? (5) What programs are needed to minimally treat or prevent the progression of noncavitated lesions in enamel and in dentin? (6) What programs are needed to minimally treat cavitated carious lesions? And most importantly: (7) What tools should be used to eliminate the misclassification of dental caries and reduce errors in decision making?

These questions present a model for dental public health planning that is different from the current understanding of the scope of dental public health in some countries. However, we are in the 21st century and on the verge of a significant revolution in diagnostics, regeneration of tissues and genetic testing and therapy. We need to change the current paradigm used in dental public health programs. Such programs should be tailored to the disease status of a targeted population. When the prevalence of noncavitated carious lesions is high, more rapid and progressive prevention programs should be designed than when the prevalence of noncavitated lesions is low. Of course, the prevalence of noncavitated in relation to cavitated carious lesions should be taken into consideration. In developed countries, such as Canada [Ismail et al., 1992], epidemiological data indicate that the ratio of noncavitated to cavitated lesions is higher than in economically developing countries such as Lithuania [Machiulskiene et al., 1998]. Hence in the former, the emphasis should be on conservative methods of management as well as on tailored individualized prevention. In countries where the rate of progression from the noncavitated stage to cavitation is high, the emphasis should also be on tailored but more extensive prevention as well as general preventive measures (such as water or salt fluoridation). While I admit that these decisions are made based upon my expert opinion, I strongly advocate that we start to consider tailoring of dental public health programs based upon disease level and distribution of the different stages of dental caries.

Diagnostic Levels in Public Health Planning

Caries Res 2004;38:199–203

Detection Levels in Dental Public Health Programs

The mission of dental public health planning should be to promote health, prevent the development of disease, prevent the progression of early disease, treat the sequelae of a disease and restore functional health. Dental caries is a process, and its assessment should be determined based on population characteristics and the burden of disease in areas where a public health program is to be implemented. The stages of dental caries that should be considered in public health programs should include enamel and dentinal carious lesions at the noncavitated and cavitated levels. It would also be important in the future to include measures of caries activity. A decision on what stage to target and measure should be made for each program based upon the goals and logistics of the area where the program is to be implemented. Enamel carious lesions can

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be included in detection protocols used in programs targeting early childhood caries and primary prevention programs; dentinal noncavitated lesions can be the focus of public health programs that aim to reduce the surgical management of dental caries, and cavitated lesions can be the focus of programs that aim to prevent the end stage of the caries process. While the prevention of carious cavitation is the ultimate goal of all public health programs, using earlier stages of the caries process may help shift the focus from universal prevention towards targeted prevention and from the ‘drill and fill’ approach to the ‘conservative’ approach of managing dental caries. The focus on high-caries risk populations in public health programs will not result in significant improvement in dental health until we understand that preventive programs that focus only on preventing carious cavitation cannot always succeed in changing the burden of dental caries. The failure to consider the stage of progression of dental caries may be one of the reasons why preventive programs targeting high-caries individuals have not been successful (an example of such programs can be found in Watson et al. [1999]).

Moving Forward

It is sometimes tempting to suggest new models and how to do things. The challenge is how to change and sustain the change. The dental community, especially its academic corps, is slow to change and is extremely conservative. While frequent experimentation with patients using new gadgets and untested concepts is common, dental professionals have been slow to adopt new scientific findings, especially those that are not related to the surgical model of care or do not fit within the context of a dental practice environment. While it is beyond the scope of this paper to analyze the causes and solutions to the current dilemmas facing dental education, dental public health,

dental practice and dental research, it is important that we take actions to improve the status quo. As discussed in this paper, there is a need for research and programs to promote the early detection of dental caries in order to prevent its rapid progression in some populations, to decrease the false-positive detection rates associated with current caries detection systems, to promote nonsurgical approaches for the management of early carious lesions, to use microsurgical approaches to treat small cavitated lesions and to increase access to primary, secondary and tertiary care. Dental public health should shift its sole focus on highrisk groups and populations to all risk groups and all individuals within populations. I am not at all advocating that dental public health programs should change their emphasis on preventing ‘cavities’; rather, I suggest that for some population groups, such as young children and medically compromised patients, there is a need to focus as well on preventing early carious lesions. While there is scarcity of evidence on how to prevent these lesions from progressing to cavitation, secondary preventive programs may be designed with a different frequency and mode of delivery than the design of a similar program targeting only cavitated carious lesions. In the face of a rapidly progressing disease scenario, dental professionals should not rely on ‘simple preventive measures’ [Machiulskiene et al., 1998]. Rather there would be a need to augment a preventive program with dietary counseling, use of alternative sweeteners, frequent professional tooth cleaning and even the use of antimicrobials. Such measures may not be needed if the overall caries activity is low. Finally, while enamel caries was recognized by G.V. Black in 1909 [Black, 1910] as the most important stage in the caries process, the dental public health community, dental educators, researchers and practitioners have not paid much attention to how to detect and manage these lesions. This area should receive significantly more research emphasis.

References Black G: A plea for greater earnestness in the study of caries of the enamel in its relation to the practice of dentistry. Dent Brief 1910;15:161– 178. Burt BA, Eklund SA: Dentistry, Dental Practice, and the Community. Philadelphia, Saunders, 1999, pp 34–36.

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Carino KMG, Shinada K, Kawaguchi Y: Early childhood caries in northern Philippines. Community Dent Oral Epidemiol 2003;31:81–89. Elderton RJ: Implications of recent dental health services research on the future of operative dentistry. J Public Health Dent 1985;45:101– 105. Institute of Medicine: The Future of Public Health. Washington, National Academy Press, 1988.

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Ismail AI, Brodeur JM, Gagnon P, Payette M, Picard D, Hamalian T, Olivier M, Eastwood BJ: Prevalence of non-cavitated and cavitated carious lesions in a random sample of 7–9-yearold schoolchildren in Montreal, Quebec. Community Dent Oral Epidemiol 1992;20:250– 255.

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Ismail AI, Brodeur JM, Gagnon P, Payette M, Picard D, Hamalian T, Olivier M: Restorative treatments received by children covered by a universal, publicly financed, dental insurance plan. J Public Health Dent 1997;57:11–18. Lewis DW, Pharoah MJ, El-Mowafy O, Ross DG: Restorative certainty and varying perceptions of dental caries depth among dentists. J Public Health Dent 1997;57:243–245. Machiulskiene V, Nyvad B, Baelum V: Prevalence and severity of dental caries in 12-year-old children in Kaunas, Lithuania 1995. Caries Res 1998;32:175–180.

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Mejàre I, Sundberg H, Espelid I, Tveit AB: Caries assessment and restorative treatment thresholds reported by Swedish dentists. Acta Odontol Scand 1999;57:149–154. Moon H, Paik D, Horowitz AM, Kim J: National survey of Korean dentists’ knowledge and opinions: Dental caries etiology and prevention. J Public Health Dent 1998;58:51–56. National Institute for Dental and Craniofacial Research (NIDCR): Oral Health of United States Adults. Bethesda, NIDCR, 1987, pp 161–165. Radike AW: Criteria for diagnosing dental caries; in American Dental Association (ed): Proceedings of the Conference on the Clinical Testing of Cariostatic Agents. Chicago, American Dental Association, 1968, pp 87–88.

Watson MR, Howoritz AM, Garcia I, Canto MT: Caries conditions among 2–5-year-old immigrant Latino children related to parents’ oral health knowledge, opinions and practices. Community Dent Oral Epidemiol 1999;27:8– 15. World Health Organization: Oral Health Surveys: Basic Methods. Geneva, WHO, 1997, pp 41– 42. Yee R, Sheiham A: The burden of restorative dental treatment for children in third world countries. Int Dent J 2002;52:1–9.

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Caries Res 2004;38:204–211 DOI: 10.1159/000077756

Dental Plaque as a Microbial Biofilm P.D. Marsh Leeds Dental Institute and Health Protection Agency, Porton Down, Salisbury, UK

Key Words Biofilm W Antimicrobial agents W Antimicrobial resistance W Gene transfer W Gene expression W Cell signalling W Dental plaque

species. A greater understanding of the significance of dental plaque as a mixed culture biofilm will lead to novel control strategies.

Abstract New technologies have provided novel insights into how dental plaque functions as a biofilm. Confocal microscopy has confirmed that plaque has an open architecture similar to other biofilms, with channels and voids. Gradients develop in areas of dense biomass over short distances in key parameters that influence microbial growth and distribution. Bacteria exhibit an altered pattern of gene expression either as a direct result of being on a surface or indirectly as a response to the local environmental heterogeneity within the biofilm. Bacteria communicate via small diffusible signalling molecules (e.g. competence-stimulating peptide, CSP; autoinducer 2); CSP induces both genetic competence and acid tolerance in recipient sessile cells. Thus, rates of gene transfer increase in biofilm communities, and this is one of several mechanisms (others include: diffusion-reaction, neutralization/inactivation, slow growth rates, novel phenotype) that contribute to the increased antimicrobial resistance exhibited by bacteria in biofilms. Oral bacteria in plaque do not exist as independent entities but function as a co-ordinated, spatially organized and fully metabolically integrated microbial community, the properties of which are greater than the sum of the component

Dental plaque can be defined as the diverse community of micro-organisms found on the tooth surface as a biofilm, embedded in an extracellular matrix of polymers of host and microbial origin. There is a high level of interest in the properties of biofilms and microbial communities across all sectors of industrial, environmental and medical microbiology [Allison et al., 2000]. This is because biofilms express properties not exhibited by the same organisms growing in liquid (planktonic) culture, while bacteria are invariably found in nature as part of a consortium, the properties of which are more than the sum of the component species. This review will focus on the significance to oral bacteria of their adoption of a biofilm and microbial community lifestyle, and highlight areas of current controversy and research activity.

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Dental Plaque – Existing Perspective

Research over several decades has provided a solid foundation for current studies of oral biofilms. Numerous cultural studies have reported the diversity of the resident oral microflora, both at the genus and species level in health and disease [Newman and Wilson, 1999]. The

Prof. P.D. Marsh Health Protection Agency Porton Down Centre for Applied Microbiology & Research Salisbury, SP4 0JG (UK) Tel. +44 1980 612287, Fax +44 1980 612731, E-Mail [email protected]

development of dental plaque has been described in detail (a) on a clean surface over time, (b) in people of different ages, from different countries and diets, and with precise deficiencies in their host defences (acquired and innate), and (c) following various therapies [Percival et al., 1991; Nyvad, 1993; Marsh, 2000a]. The composition of dental plaque also varies on distinct anatomical surfaces (e.g. fissures, approximal and smooth surfaces, gingival crevice, dentures) due to the prevailing physical and biological properties of each site [Bowden et al., 1975; Slots, 1977; Theilade et al., 1982]. Recognition of these environmental influences on plaque composition has led to concepts on disease prevention that have embraced ecological principles [Marsh, 2003]. Dental plaque accumulates preferentially at stagnant sites that afford protection from the vigorous removal forces that apply in the mouth. Distinct phases of development can be recognized, including: (a) Adsorption of host and bacterial molecules to the tooth surface. This conditioning film (the acquired pellicle) forms immediately following eruption or cleaning [AlHashimi and Levine, 1989] and directly influences the pattern of initial microbial colonization. Modern techniques offer the opportunity to more fully explore the distribution and composition of pellicle components [Li et al., 2003]. The conformational changes that may occur following adsorption of molecules, and the impact of this on their properties, are now amenable for study: for example, the molecular structure of glucans changes when glucosyltransferases are adsorbed to a surface [Vacca-Smith et al., 1996; Kopec et al., 2001]. (b) Passive transport of oral bacteria to the tooth surface. Weak, long-range physicochemical interactions between the microbial cell surface and the pellicle-coated tooth create a weak area of net attraction that facilitates reversible adhesion [Busscher and van der Mei, 1997]. Subsequently, strong, short-range interactions between specific molecules on the bacterial cell surface (adhesins) and complementary receptors in the pellicle can result in irreversible attachment [Jenkinson and Lamont, 1997; Lamont and Jenkinson, 2000] and can explain microbial tropisms for surfaces. Oral bacteria generally possess more than one type of adhesin on their cell surface and can participate in multiple interactions both with host molecules and similar receptors on other bacteria (coadhesion). (c) Co-adhesion of later colonizers to already attached early colonizers. This stage also involves specific interbacterial adhesin-receptor interactions (often involving lectins) and leads to an increase in the diversity of the bio-

film and to the formation of unusual morphological structures, such as corn-cobs and rosettes [Kolenbrander et al., 2000]. Co-adhesion may also facilitate the functional organization of dental plaque. Bacteria engage in a range of antagonistic and synergistic biochemical interactions [Marsh and Bradshaw, 1999]. The efficiency of metabolic interactions among bacteria in food chains may be enhanced if they are brought into close physical contact. Likewise, the co-adhesion of obligately anaerobic bacteria to oxygen-consuming species can ensure their survival in overtly aerobic oral environments [Bradshaw et al., 1998]. (d) Multiplication of the attached micro-organisms. Cell division leads to confluent growth and, eventually, a three-dimensional spatially and functionally organized, mixed-culture biofilm. Polymer production results in the formation of a complex extracellular matrix made up of soluble and insoluble glucans, fructans and heteropolymers. Such a matrix is a common feature of biofilms and makes a significant contribution to the known structural integrity and general resistance of biofilms; the matrix can be biologically active and retain nutrients, water and key enzymes within the biofilm [Allison, 2003]. Further studies are required to fully understand the influence of the matrix on the architecture and properties of dental plaque. When viewed by conventional light or electron microscopy, mature dental plaque appears as a densely packed structure [Listgarten, 1999; Marsh and Nyvad, 2003]; however, the recent application of novel microscopic techniques has demonstrated a more open architecture (see later). Endogenous substrates (derived from saliva or gingival crevicular fluid) are the main source of nutrients for oral bacteria [Beighton et al., 1986], but their catabolism requires the concerted and sequential action of groups of microbes with complementary enzyme profiles [Bradshaw et al., 1994; Marsh and Bowden, 2000], i.e. plaque functions as a true microbial community. (e) Active detachment. Bacteria can respond to environmental cues and detach from surfaces, enabling cells to colonize elsewhere. For example, enzymes produced by sessile bacteria can hydrolyse the specific adhesins that anchor cells to the surface [Cavedon and London, 1993; Lee et al., 1996]. Once established, the resident plaque microflora remains relatively stable over time and is of benefit to the host [Marsh, 2000b]. The resident microflora of all sites plays a critical role in the normal development of the physiology of the host and also reduces the chance of infection by acting as a barrier to colonization by exogenous (and often pathogenic) species (‘colonization resis-

Plaque as a Biofilm

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Table 1. General properties of biofilms and microbial communities

General property

Dental plaque example

Open architecture Protection from host defences, desiccation etc. Enhanced resistance to antimicrobials Neutralization of inhibitors Novel gene expression Co-ordinated gene responses Spatial and environmental heterogeneity Broader habitat range More efficient metabolism

Presence of channels and voids Production of extracellular polymers to form a functional matrix Increased resistance to chlorhexidine and antibiotics ß-Lactamase production by neighbouring cells to protect sensitive organisms Synthesis of novel proteins; up-regulation of gtfBC Production of cell-cell signalling molecules (e.g. CSP, autoinducer 2) pH and O2 gradients; co-adhesion Obligate anaerobes in an overtly aerobic environment Complete catabolism of complex host macromolecules (e.g. mucins) by consortia

tance’) [McFarland, 2000]. Mechanisms contributing to colonization resistance include more effective competition for nutrients and attachment sites, the production of inhibitory factors and creation of unfavourable growth conditions by the resident microflora. Thus, treatment should attempt to control rather than eliminate the plaque microflora.

Dental Plaque – Paradigm Shifts

Novel non-invasive and non-destructive microscopic techniques, the publication of annotated microbial genomes (which has facilitated new fields such as functional and comparative genomics, transcriptomics and proteomics), the development of molecular tools (e.g. reporter systems to determine gene activity, oligonucleotide probes to identify and locate specific bacteria via PCR or fluorescent in situ hybridization) combined with laboratory and in vivo biofilm models are changing our understanding of the biology of dental plaque (table 1). Selected topics of current research activity are highlighted below.

Structure of Dental Plaque Confocal laser scanning microscopy has confirmed that supragingival plaque has a more open architecture (similar to that of biofilms from other habitats) than was suggested by the earlier electron microscopy reports, with channels traversing from the outside of the biofilm to the enamel surface [Wood et al., 2000; Auschill et al., 2001; Zaura-Arite et al., 2001]. Live/dead stains have suggested that bacterial vitality may vary throughout the biofilm, with the most viable bacteria present in the central part of plaque, and lining the voids and channels [Auschill et al.,

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2001], although the fidelity of such stains for viable/nonviable cells is not 100% [Gelle et al., 2003; Hope and Wilson, 2003]. This more open architecture, combined with the synthesis of a matrix comprised of a diverse range of exopolymers, creates a complex environment for predicting the penetration and distribution of molecules within plaque. Uneven patterns of penetration of radiolabelled fluoride, sucrose and phosphate were found in plaque generated naturally on an in situ biofilm model in volunteers [Robinson et al., 1997], while the diffusion of glucans of increasing molecular size was retarded in laboratory mixed culture biofilms [Thurnheer et al., 2003]. Bacterial metabolism ensures that gradients develop in parameters that are critical to microbial growth (nutrients, pH, oxygen); the gradients in pH are also responsible for enamel demineralization. These gradients are not necessarily linear; the use of two-photon excitation microscopy coupled with fluorescent life-time imaging demonstrated considerable heterogeneity in pH over relatively short distances [Vroom et al., 1999]. Such environmental heterogeneity enables micro-organisms to co-exist in plaque biofilms that would be incompatible with one another in a homogeneous environment; this explains how organisms with contradictory metabolic requirements (e.g. in terms of atmosphere, nutrition) persist at the same site. Bacterial Composition of Dental Plaque Approximately 50% of cells in plaque (especially from subgingival sites) cannot as yet be cultured in the laboratory. Molecular approaches based on nucleotide sequence analysis have characterized the full diversity of dental plaque [Kroes et al., 1999; Wade, 1999; Paster et al., 2001] and identified a large number of novel taxa [Dewhirst et al., 2000]. Improvements in the taxonomy of plaque isolates based on, for example, the unique DNA sequences of the 16S subunit rRNA (16S rDNA) gene

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have resulted in the more valid subdivision of existing species. This can also lead to the finer resolution of species into biovars, genotypes or other subgroups, which facilitates better epidemiological studies [Redmo-Emanuelsson et al., 2003] and the possibility of the closer correlation of particular clonal types with disease, as has occurred with Actinobacillus actinomycetemcomitans and early-onset periodontitis [Haubek et al., 2001]. These unique sequences can be used as templates for nucleotide probes which, when coupled with a reporter system and used in conjunction with confocal microscopy, can visualize and quantify individual species in natural biofilms, e.g. by fluorescent in situ hybridization [Thurnheer et al., 2001; Kolenbrander et al., 2002]. Pilot studies of plaque developing on removable materials in deep periodontal pockets showed that the deepest zones were colonized mainly by spirochaetes and gram-negative bacteria, whereas shallow regions comprised predominantly grampositive cocci [Wecke et al., 2000]. These and similar approaches will enable a more complete description of the plaque microflora in health and disease, and also provide data on the location of, and structural interrelationships among, target species in the biofilm [Thurnheer et al., 2001; Kolenbrander et al., 2002]. The recent application of molecular methods to infected dentine has resulted in the detection and identification of previously uncultured bacteria [Munson et al., 2003].

lowing attachment have been identified in Streptococcus mutans using a whole-cell proteomic approach [Svensater et al., 2001]. Proteins involved in a range of biochemical functions including protein folding and secretion, amino acid and fatty acid biosynthesis, and cell division were up-regulated. Of particular significance, novel proteins of as yet unknown function were expressed by biofilm but not planktonic cells. Similarly, genes associated with glucan (gtfBC) and fructan synthesis (ftf) in S. mutans were differentially regulated in biofilms [Li and Burne, 2001]. There was little influence of surface growth in early biofilm formation (!48 h), but gtf expression was markedly up-regulated in older (7-day) biofilms, whereas ftf activity was repressed. This was interpreted as an indirect effect of biofilm growth on gene expression, i.e. the altered phenotype was probably due to changes in local environmental conditions within the biofilm (e.g. sugar concentration, pH) rather than due to attachment per se [Li and Burne, 2001]. Thus, biofilm growth can have both direct and indirect influences on gene expression by oral bacteria.

Biofilm Regulation of Gene Expression Bacteria in biofilms display a phenotype that is distinct from that exhibited by the same cells growing planktonically. The binding of bacteria to specific receptors can trigger significant changes in both bacterial and host cell patterns of gene expression, e.g. following the initial attachment of Escherichia coli to uro-epithelial cells [Abraham et al., 1998]. Similarly, there is up-regulation of genes involved with alginate synthesis when Pseudomonas aeruginosa colonizes a surface [Boyd and Chakrabarty, 1995]. Similar surface-associated responses are now being identified in plaque bacteria, although the magnitude of this shift in gene expression may be less than that observed in free-living species because of the absolute dependence of oral bacteria on a biofilm lifestyle [Burne, 1998]. The exposure of Streptococcus gordonii to saliva resulted in the induction of genes (sspA/B) encoding adhesins that can bind to salivary glycoproteins and engage in co-aggregation with Actinomyces spp. [Du and Kolenbrander, 2000], implying similar changes may occur during colonization. Changes in protein profile fol-

Cell-Cell Communication In addition to the conventional biochemical and metabolic interactions that have been well catalogued, cells have also been shown to communicate with one another in biofilm communities via small diffusible molecules. Many bacterial species have evolved cell-cell signalling systems (quorum sensing) that help them to adapt and survive various environmental stresses in a cell-densitydependent manner and regulate the expression of genes that also influence their ability to cause disease. In S. mutans, quorum sensing is mediated by a competencestimulating peptide (CSP) [Li et al., 2002b]. This peptide also induced genetic competence in S. mutans so that the transformation frequency of biofilm-grown S. mutans was 10- to 600-fold greater than for planktonic cells [Li et al., 2001]. Lysed cells in biofilms could act as donors of chromosomal DNA, thereby increasing the opportunity for horizontal gene transfer in dental plaque. CSP is also directly involved in biofilm formation; mutants in some of the genes involved in the CSP signalling system (comC, comD, comE and comX) produce defective biofilms [Li et al., 2002b]. This quorum sensing system also functions to regulate acid tolerance in S. mutans biofilms [Li et al., 2002a]. It has been proposed that S. mutans, upon exposure to low pH, could release CSP and initiate a co-ordinated ‘protective’ response among neighbouring cells to such a potentially lethal stress. CSPs are specific for cells of the same species, but other communication systems may function between different taxa [Kolenbrander et al.,

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2002]. Genes encoding autoinducer 2 have been detected in several genera of gram-positive and gram-negative bacteria so that autoinducer 2 may have a broader species range, although its role in plaque remains to be determined. However, mutants of the luxS gene that encodes for the autoinducer 2 synthase in S. mutans and S. gordonii had an impaired ability to produce monospecies biofilms in vitro [Blehert et al., 2003; Merritt et al., 2003]. Future research will identify more of these sophisticated communication networks, and it has been suggested that analogues of the signalling molecules could be used as novel therapeutic agents to manipulate the properties of biofilms. Gene Transfer Cells also communicate with one another in biofilms via horizontal gene transfer. As discussed above, signalling molecules such as CSP markedly increase the ability of recipient cells in biofilms to take up DNA [Li et al., 2002b]. The transfer of conjugative transposons encoding tetracycline resistance between streptococci in model biofilms has been demonstrated [Roberts et al., 2001]. The recovery of resident (S. mitis, S. oralis) and pathogenic (S. pneumoniae) bacteria from the nasopharynx with penicillin resistance genes showing a common mosaic structure confirms that gene transfer can occur in vivo [Dowson et al., 1990; Hakenbeck et al., 1998]. Similar evidence suggests sharing of genes responsible for penicillin-binding proteins among commensal and pathogenic Neisseria [Bowler et al., 1994]. These findings suggest that plaque can function as a ‘genotypic reservoir’ by harbouring transferable mobile elements and genes. Such genetic exchange could have a wider significance given the number of overtly pathogenic bacteria that appear transiently in the mouth [Loo, 2003]. Antimicrobial Resistance A major finding of clinical relevance with respect to micro-organisms growing on a surface is their increased resistance to antimicrobial agents [Gilbert et al., 1997, 2002; Ceri et al., 1999]. For example, P. aeruginosa growing on urinary catheter material can be 500–1,000 times more resistant to antibiotics than the same cells growing in liquid culture. Conventionally, the sensitivity of bacteria to antimicrobial agents is determined on cells grown in liquid culture by the measurement of the minimum inhibitory concentration or minimum bactericidal concentration. Given the decreased sensitivity of an organism on a surface, it has been argued that it would be more appro-

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priate to determine the ‘biofilm inhibitory concentration’ (also described as the ‘biofilm eradicating concentration’ or biofilm killing concentration) [Anwar et al., 1990; Nichols, 1994; Shani et al., 2000; Johnson et al., 2002]. As yet, however, these proposals have not been widely accepted, and there are no generally agreed standardized methods by which these concentrations could be determined. Bacteria growing in dental plaque also display increased resistance to antimicrobial agents, including those used in dentifrices and mouth rinses [Marsh and Bradshaw, 1993; Kinniment et al., 1996; Wilson, 1996; Pratten and Wilson, 1999]. For example, the biofilm inhibitory concentration for chlorhexidine and amine fluoride was 300 and 75 times greater, respectively, when S. sobrinus was grown as a biofilm compared with the minimum bactericidal concentration of planktonic cells [Shani et al., 2000]. Similarly, it was necessary to administer 10– 50 times the minimum inhibitory concentration of chlorhexidine to eliminate S. sanguinis (previously S. sanguis) biofilms within 24 h [Larsen and Fiehn, 1996]. The age of the biofilm can also be a significant factor; older biofilms (72 h) of S. sanguinis were more resistant to chlorhexidine than younger (24 h) biofilms [Millward and Wilson, 1989]. Confocal microscopy of in situ established natural biofilms showed that chlorhexidine only affected the outer layers of cells in 24- and 48-hour plaque biofilms [Zaura-Arite et al., 2001]. Biofilms of oral bacteria are also more resistant to antibiotics (e.g. amoxycillin, doxycycline, metronidazole) [Larsen, 2002; Larsen and Fiehn, 1996]. The mechanisms behind the increased resistance of biofilms to antimicrobial agents are the subject of much research and debate [Gilbert et al., 2002]. Cells can become resistant due to mutations affecting the drug target, the presence of efflux pumps or to the production of modifying enzymes etc., but even innately sensitive bacteria become resistant when growing on a surface. The structure of a biofilm may restrict the penetration of the antimicrobial agent; some charged inhibitors can bind to oppositely charged polymers that make up the biofilm matrix (diffusion-reaction theory). The agent may also bind to and inhibit the organisms at the surface of the biofilm, leaving cells in the depths of the biofilm relatively unaffected. As stated earlier, bacteria growing on a surface display a novel phenotype, and this can result in a reduced sensitivity to inhibitors, while the transfer of resistance genes can occur more readily in biofilm communities such as dental plaque. Growth on a surface may also result in the drug target being modified or not expressed in a

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biofilm, or the organism may use alternative metabolic strategies. Bacteria grow only slowly under nutrientdepleted conditions in an established biofilm and, as a consequence, are much less susceptible than faster-dividing cells. In addition, it has also been proposed that the environment in the depths of a biofilm may be unfavourable for the optimal action of some drugs [Gilbert et al., 2002]. The matrix in biofilms can also bind and retain neutralizing enzymes (ß-lactamase, IgA protease; see above) [Allison, 2003]. At present, it is not clear whether some or all of these effects account for the observed resistance of cells in plaque biofilms. Plaque as a Community The evidence outlined above on the ability of plaque bacteria to interact with neighbouring cells in biofilms provides compelling support for the concept that oral bacteria do not exist as independent entities but rather function as a co-ordinated, spatially organized and metabolically integrated microbial community [Marsh and Bradshaw, 1999]. Benefits of a community lifestyle to micro-organisms include: (a) a broader habitat range for growth, e.g. oxygen-consuming species create environmental conditions suitable for obligate anaerobes; (b) a more efficient metabolism, e.g. complex host macromolecules can only be degraded by consortia of oral bacteria; (c) increased resistance to stress and antimicrobial agents, and (d) enhanced virulence (‘pathogenic synergism’) [Caldwell et al., 1997; Shapiro, 1998; Marsh and Bowden, 2000] (table 1). Microbial community effects can render a sensitive organism as apparently ‘resistant’ to an antibiotic if neighbouring, non-pathogenic cells produce a neutralizing or drug-degrading enzyme (‘indirect pathogenicity’). This has been demonstrated in animal models where a penicillin-sensitive pathogen (S. pyogenes) is protected by a ß-lactamase-producing commensal strain (Moraxella catarrhalis) and, as a result, is still capable of causing a lethal infection [Hol et al., 1994]. In the mouth, gingival crevicular fluid can contain sufficient ß-lactamase to inactivate the concentrations of antibiotic delivered to the site [Walker et al., 1987; Herrera et al., 2000].

(a) the development of inhibitors and antiplaque agents that are more effective against surface-associated micro-organisms, coupled with more effective delivery systems for targeting specific bacteria and for improving the retention of agents in the mouth; this will require the development and use of high throughput biofilm models to screen novel compounds not only for their ability to kill or inhibit sessile cells, but also to promote biofilm detachment; (b) interference with communication networks that coordinate or regulate microbial activities within biofilms; in other areas of microbiology (e.g. P. aeruginosa and cystic fibrosis), attempts are being made to block signalling molecules that induce a shift in the host to a more virulent phenotype; (c) preventing colonization of selected organisms (e.g. by interfering with attachment by modifying the conditioning film or by ‘replacement therapy’, whereby organisms are deliberately implanted to prevent subsequent colonization by more pathogenic species) [Hillman, 1999; Tagg and Dierksen, 2003]; (d) affecting biofilm architecture, for example, by the use of enzymes that can degrade the exopolymers that comprise the plaque matrix; (e) the neutralization of parameters that select for the species that are implicated in disease [Marsh, 2003]; thus, strategies that reduce the pH response to dietary carbohydrates will help prevent the enrichment of acidogenic and aciduric bacteria; (f) the identification of pathogenic clones could also improve diagnosis and might predict sites that are more susceptible to disease.

Future Developments

A greater understanding of the significance of dental plaque as a mixed species biofilm will have the potential to impact significantly on clinical practice. Novel areas for future research include:

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Caries Res 2004;38:212–222 DOI: 10.1159/000077757

Application of the Zürich Biofilm Model to Problems of Cariology Bernhard Guggenheim a Merlin Guggenheim a Rudolf Gmür a Elin Giertsen b Thomas Thurnheer a a Institute

for Oral Biology, Section for Oral Microbiology and General Immunology, University of Zürich, Zürich, Switzerland; b Department of Odontology-Cariology, University of Bergen, Bergen, Norway

Key Words Biofilms W Caries W Antimicrobials W Mouth rinses W Demineralization W Remineralization W Three-dimensional morphology W Tortuosity

Abstract The term biofilm is increasingly replacing ‘plaque’ in the literature, but concepts and existing paradigms are changing much more slowly. There is little doubt that biofilm research will lead to more realistic perception and interpretation of the physiology and pathogenicity of microorganisms colonizing plaques in the oral cavity. There is clear evidence that the genotypic and phenotypic expression profiles of biofilm and planktonic bacteria are different. Several techniques are available today to study multispecies biofilms of oral bacteria, each having its particular advantages and weaknesses. We describe a biofilm model developed in Zürich and demonstrate a number of applications with direct or indirect impact on prophylactic dentistry: spatial arrangement and associative behavior of various species in biofilms; multiplex fluorescent in situ hybridization analysis of oral bacteria in biofilms; use of the biofilm model to predict in vivo efficacy of antimicrobials reliably; mass transport in bio-

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films; de- and remineralization of enamel exposed to biofilms in vitro. The potential of biofilm experimentation in oral biology has certainly not yet been fully exploited and dozens of possible interesting applications could be investigated. The overall physiological parameters of multispecies biofilms can be measured quite accurately, but it is still impossible to assess in toto the multitude of interactions taking place in such complex systems. What can and should be done is to test hypotheses stemming from experiments with planktonic cells in monospecies cultures. In particular, it will be interesting to investigate the relevance to biofilm composition and metabolism of specific gene products by using appropriate bacterial mutants. Copyright © 2004 S. Karger AG, Basel

Microorganisms colonizing the oral cavity had been a major research topic even before the epoch-making contributions of W.D. Miller who coined the prevailing view of the etiology of dental caries for decades. Acid formation from dietary starch by salivary bacteria, in particular by lactobacilli, emerged as a sustained paradigm to explain caries [Miller, 1973]. Early observations by Black and many others, who had pointed out the significance of

B. Guggenheim Institute for Oral Biology, Section for Oral Microbiology and General Immunology University of Zürich, Plattenstrasse 11 CH–8028 Zürich (Switzerland) Tel. +41 44 634 3277, Fax +41 44 634 4310, E-Mail [email protected]

species and numerical composition of biofilms are dependent on the prevailing growth conditions. These seem ultimately decisive for the interaction with the host, resulting in health or disease. P.D. Marsh, a pioneer in oral biofilm experimentation, has described this relation as the ‘ecological plaque hypothesis’ [Marsh, 1994]. Various multispecies models of dental plaque have been described and applied to problems of clinical relevance, most notably biofilm permeability and chemical control of plaque. These systems usually consist either of flow cells [Christersson et al., 1987; Larsen and Fiehn, 1995; Sjollema et al., 1989] or of chemostats modified to allow for insertion and removal of colonizable surfaces [Bowden, 1999; Bradshaw et al., 1996; Herles et al., 1994; Kinniment et al., 1996]. While these devices have contributed to our understanding of microbial adhesion and biofilm formation, their use has certain drawbacks. They can be cumbersome to construct and/or difficult to maintain over long periods of time. Since clearance of pulsed substances is a function of flow rate and volume, chemostats operating with low flow rates and relatively large volumes can have quite long mean residence times, rendering them impractical for studies of selected compounds with shortterm exposures, as is common in oral hygiene procedures. Moreover, systems with working volumes of more than a few milliliters preclude the use of media constituted from natural substrates such as saliva. In this short paper, we will focus on biofilm studies, carried out in Zürich, that illustrate a few applications of one particular model with direct or indirect impact on prophylactic dentistry. The following aspects will be covered: description of the model; spatial arrangement and associative behavior of various species in biofilms; mass transport in biofilms; the biofilm model as a reliable tool to predict the in vivo efficacy of antimicrobials, and de- and remineralization of enamel exposed to biofilms in vitro.

the gelatinous nature of dental plaques and also the role of sucrose in the formation of these bacterial biofilms, were rapidly forgotten [Guggenheim, 1970]. The original version of the chemoparasitic caries theory of Miller received its ‘coup de grâce’ by Ron Gibbons in the early 1960s when it was shown that bacteria associated with dental caries must colonize supragingival plaque in high numbers [Gibbons, 1964]. This shifted mainstream research to plaque streptococci and in particular to mutans streptococci [Carlsson, 1967; Guggenheim, 1968]. Paul Keyes introduced the multifactorial caries theory [Keyes, 1960]. Animal experiments and both human cross-sectional and longitudinal studies left no doubt that mutans streptococci were strongly associated with enamel caries [Guggenheim et al., 1965; Hardie et al., 1977]. As a result, a most simple concept evolved: the specific plaque hypothesis. Dental caries was conceived as a mono-infection by mutans streptococci [Kristoffersson et al., 1985; Loesche, 1986]. The hypothesis induced a flood of in vitro studies with planktonically grown Streptococcus mutans and S. sobrinus cells, with the aim to pinpoint and elucidate virulence mechanisms of these streptococci [van Houte, 1994]. From these experiments, in part carried out with most modern molecular biological methods, a number of new paradigms emerged. Here, just a few will be mentioned. Primary bacterial adhesion to pellicle-coated enamel was described as a highly specific process, with the expression and nature of adhesins being the major determinants for the colonization of specific oral microhabitats [Liljemark and Bloomquist, 1996]. By analogy, secondary colonization was envisioned as entirely dependent on specific coadherence. It was considered predictable from in vitro tests assessing the coaggregation patterns of planktonic bacterial cells that had been mixed in suspensions under no-growth conditions [Kolenbrander and London, 1993]. Glucosyltransferases (GtfB, GtfC, GtfD) were claimed to play a crucial role in bacterial attachment, and their products were thought to act as diffusion-limiting macromolecules in plaque [Ooshima et al., 2001]. Only more recently has the picture started to shift. Studies with bacteria growing in biofilms, in particular in multispecies biofilms, clearly indicated that genotypic and phenotypic expression profiles of biofilm bacteria are different from those of planktonic bacteria [Costerton et al., 1994]. Such biofilm ecologies have been compared to higher multicellular organisms [Costerton et al., 1995]. They show complex intercellular interactions including communication by specific signaling molecules [Davies et al., 1998]. The resistance of biofilm bacteria against antimicrobials is increased [Gilbert et al., 1997]. Both the

In contrast to most other biofilm models, our multispecies model is based on a batch culture approach and not on a continuous flow culture system. The following microorganisms representative for supragingival plaque are used to generate biofilms: Streptococcus oralis, Streptococcus sobrinus, Actinomyces naeslundii, Veillonella dispar, Fusobacterium nucleatum and Candida albicans. Biofilms are formed in 24-well cell culture dishes incubated anaerobically at 37 ° C. A detailed description of the experimental procedures as well as data validating the

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The Zürich Biofilm Model

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Fig. 1. Schematic presentation of experimental procedures used for biofilm formation. The hydroxyapatite disks were incubated in a mixture of saliva and medium either containing 50% saliva and 50% medium or 70% saliva and 30% medium according to the aim of the experiment.

model have been published previously [Guggenheim et al., 2001a; Shapiro et al., 2002]. Therefore, only the main features are recapitulated. Biofilms are formed either on hydroxyapatite or bovine enamel disks that have been preconditioned in pooled, unstimulated saliva. An experiment, including the preparatory phase, lasts from Monday to Friday; the most important steps comprising the timing are summarized in figure 1. In flow models or constant depth film fermenters as well as in vivo, biofilms are subjected to shear forces that are absent in a batch culture system. The disks are, therefore, dipped in saline 3 times daily (fig. 1). At each time point, the biofilms are dipped 3 times in saline, thereby being subjected to passages through an air-liquid interface. The shear forces exerted by this procedure are high and have been estimated to be 0.1 ÌN/cell and passage [Bos et al., 1999]. When the effects of antimicrobials were investigated, the biofilms were exposed beforehand to test solutions for 1 min, involving even a fourth passage.

Spatial Arrangement and Associative Behavior of Species in Biofilms

Polyspecies microbial consortia typically consist of cells in microcolonies embedded in exopolymer matrices. These were hitherto thought to be interwoven by a chan-

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nel system [Davey and O’Toole, 2000; Donlan and Costerton, 2002]. Dental plaque is a clinically relevant example of such a consortium that may mediate oral diseases. The resistance or resilience of biofilms to antimicrobials [Reid, 1999], their diffusion properties [Dibdin and Shellis, 1988; Hojo et al., 1976] and metabolic interactions between members of the consortium [Møller et al., 1998] may be linked to their distinctive architectures. In addition, roles of specific adherence mechanisms and of coadherence [Kolenbrander et al., 2000] in primary and in secondary colonization of bacteria on surfaces are widely accepted. Since these paradigms have evolved mainly from in vitro experiments with resting cell suspensions, we have subjected them to the scrutiny of experiments with growing cells in polyspecies biofilms [Guggenheim et al., 2001b]. Species-specific fluorescence-labeled antibodies in conjunction with confocal laser scanning microscopy (CLSM) allowed characterization of the spatial arrangement and interspecies associations of all members of the consortium during biofilm formation in the 50:50 model (fig. 1). In parallel, after 15 min, 16.5, 40.5 and 64.5 h, the adherent biofilms were quantitatively analyzed using culture techniques. All species pairings were visualized in biofilms after labeling the bacteria with monoclonal antibodies coupled with 1 of 3 different fluorescent dyes in 10 nonredundant pairwise combinations. Cell number esti-

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mates by image analysis were close to culture data. Interspecies coaggregations of all strains using planktonic cells were tested in buffer and in the biofilm medium in nonredundant pairwise combinations. With the exception of F. nucleatum and S. sobrinus that coaggregated in medium as well as in buffer, the coaggregation patterns were different in the two fluids. When bacteria scraped from hydroxyapatite disks after an adherence phase of 15 min were analyzed by culture and image analysis, it was evident that early (S. oralis, A. naeslundii, V. dispar) as well as late colonizers (S. sobrinus, F. nucleatum) adhered in high numbers on the pellicle-covered surface (fig. 2). Image analyses revealed further that interbacterial coadherence was not a dominant mode of (indirect) adhesion to the salivary pellicle during this initial phase, since the proportion of interspecies coadhering cells was for all nonredundant pairwise combinations !4%. These findings allowed us to question the widely accepted ‘Kolenbrander paradigm’ proposing that specific adherence mechanisms and in vitro ‘coaggregations’ reflect crucial mechanisms explaining the order of colonization of bacteria in plaque [Kolenbrander and London, 1993]. We gained insight into the structural features of all species during biofilm development, and into the associative behavior of the strains within the biofilm, that were classified into 5 spatial types; some examples are shown in figure 3. More information is provided by Guggenheim et al. [2001b].

Fig. 2. Number of bacteria as estimated by culture (CFU) or image

analysis on salivary-coated hydroxyapatite disks after an initial adherence phase of 15 min: S. sobrinus (Ss), S. oralis (So), V. dispar (Vd), F. nucleatum (Fn), A. naeslundii (An).

The ability to generate biofilms with high repeatability and the technical skills to visualize these in high quality in native form by CLSM allowed us to tackle experimentally their diffusion properties. Knowledge of the kinetics of mass transport within oral biofilms is essential for understanding how they achieve their characteristic architecture, how they manifest their pathogenic potential and for optimizing strategies to control or eradicate biofilms. 64.5-hour biofilms of the 70:30 model (fig. 1), formed on hydroxyapatite disks preconditioned with saliva, were incubated for defined periods at room temperature with fluorescent markers with molecular weights ranging from 3 to 900 kD. Dextrans (3, 10, 40, 70 kD), IgG (150 kD), F(ab))2 fragments of IgG (100 kD), R-phycoerythrin (240 kD) and IgM (900 kD) amongst others were used in recently published experiments [Thurnheer et al., 2003]. Biofilm-carrying disks were incubated with the fluores-

cent probes for 2, 60, 120, 300 or 600 s, washed by two 60-second dips in saline and then embedded upside down in Mowiol [Guggenheim et al., 2001b] to block further diffusion. They were examined by CLSM at 5 randomly selected positions. A few examples of the images collected are shown in figure 4. It is evident that 240-kD phycoerythrin penetrated poorly into biofilms, whereas 900-kD IgM accumulated on the surface and penetrated hardly at all. From reported hydrodynamic radii for these molecules, the limiting diameter of the biofilm pores can be estimated as slightly greater than 11 nm. A control experiment showed that microspheres (M = 20 nm) did not penetrate the biofilm either. Subsequent analyses revealed that the mean square penetration depth for all tested macromolecules except IgM and 3-kD dextran increased linearly with time, diffusion coefficients being linearly proportional to the cube root of the molecular weight of the probes. Diffusion in the biofilms was markedly slower than in water. Analysis of diffusion phenomena through oral biofilms suggested tortuosity as the most probable explanation. The retardation of molecules with hydrodynamic radii ! approx. 10 nm and the generally proposed existence of a channel network in biofilms [Hall-Stoodley and Stoodley, 2002; Wood et al., 2000] with diameters in the micrometer range are conflicting notions. Such ‘black holes’ are also present in Syto13 or triple fluorescent in

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Mass Transport of Macromolecules within an in vitro Grown Biofilm

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Fig. 3. CLSM images of 64.5-hour biofilms (50:50 model) stained with species-specific monoclonal antibodies. A S. sobrinus (green) plus F. nucleatum (red). B F. nucleatum (green) plus A. naeslundii (red). C S. oralis (green) plus F. nucleatum (red). D V. dispar (green) plus S. sobrinus (red).

situ hybridization [Thurnheer et al., 2004] stained biofilms (fig. 5A, B). However, when such biofilms are in addition stained with the exopolysaccharide stain Calcofluor [Thurnheer et al., 2003], it becomes evident that multispecies biofilms formed in the presence of oral strep-

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tococci and sucrose consist of microbial microcolonies embedded in a compact polysaccharide hydrogel without channels (fig. 5C). Diffusion experiments with such double-stained biofilms revealed that dextrans 610 kD cannot diffuse through the extracellular polysaccharide moi-

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The Zürich Biofilm Model as Reliable Predictor for the Clinical Efficacy of Antimicrobials

Fig. 4. CLSM images of cross-sections of 64.5-hour biofilms (70:30

model) showing the diffusion of macromolecules with different molecular weights after 120 s (3-kD dextran) or after 600 s (10- to 240kD probes).

ety and must find their way on winding pathways through microcolonies, thus providing a more direct picture of the term tortuosity (fig. 5D). In contrast, 3-kD dextrans are able to diffuse through the exopolysaccharide moiety having a pore diameter of 2.8–4.6 nm (data not shown), thus explaining the higher diffusion rate and the lower tortuosity. These findings may explain the lower cariogenic potential of starch in comparison to low-molecular-weight saccharides.

Impact of Biofilm Models on Dentistry

For decades the potential of antimicrobials for oral use was tested in classical MIC and MBC tests utilizing planktonic monocultures and prolonged contact times. In comparison to clinical tests the resulting inhibitory concentrations were 100–1,000 times too low [Shapiro and Guggenheim, 1998]. Thus, they allowed only relative comparisons and were poorly predictive for the clinical efficacy of antiseptic mouth rinses. The obvious reasons are the brief exposure times to e.g. mouth rinses (!3 min/day) and proliferation of surviving microorganisms in plaque during the rest of the day and night between rinsings. It is furthermore widely accepted that bacteria in biofilms express a more resistant phenotype than planktonic bacteria [Marsh, 2003]. Using the 50:50 biofilm model (fig. 1), we devised a simple in vitro model of supragingival plaque whose response towards triclosan and chlorhexidine digluconate (CHX) mimicked closely clinical results reported for these antimicrobial agents [Guggenheim et al., 2001a]. However, when biofilms were exposed 3 times daily during 1 min to commercially available mouth rinses, in particular in low concentrations, the effect did not perfectly match results of clinical studies. Therefore, efforts have been directed towards fine-tuning the model in order to further improve the correspondence between the biofilm response to antimicrobial agents and the effect of these agents on plaque in vivo. It appeared that the effect of antimicrobials on the biofilm was dependent on its growth rate, which is in turn related to the ratio of saliva and medium in the incubation fluid. Using 0.1, 0.12 and 0.2% CHX and a saliva:medium ratio of 70:30, a dose-dependent response was observed, and this ratio was adopted for subsequent experiments (‘70:30 model’). The discriminative power of the model is illustrated in figure 6. The efficacies of 12 different mouth rinses – proprietary products containing CHX, hexetidine, octenidine, triclosan, plant extracts or amine fluoride/stannous fluoride – vis-à-vis biofilm inhibition were compared by Shapiro et al. [2002]. An excerpt from these data is shown in figure 7. In general, there is a good overall agreement between results obtained using our in vitro model and those reported in relevant clinical trials. However, there is a discrepancy in antimicrobial efficiency between pure CHX-containing solutions and some commercial CHX mouth rinses due to the vitiating effect of product formulation [Shapiro et al., 2002]. On the other hand, auxiliary ingredients may also increase the efficacy of a pure com-

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Fig. 5. CLSM images of 6-species biofilms stained for all bacteria with Syto13 (A), for F. nucleatum (red), S. oralis (green) and V. dispar (blue) by triple fluorescent in situ hybridization (B), for bacteria (Syto13) and exopolysaccharides (Calcofluor, blue) (C) and for bacteria (Syto13, green), 10-kD dextran (red) and exopolysaccharides (Calcofluor, blue) (D). The images A and C show the same spot of the biofilm.

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Fig. 6. Box plots showing the effect of 2 concentrations (0.1 and 0.2%, i.e. 1.1 and 2.2 mM ) of CHX and 0.2% (6.9 mM ) triclosan (TC) on 5-species biofilm formation compared to a water control (n = 9). Differences between control and all treatments were highly significant at the 99.9% level. Differences between treatments were not significant.

Fig. 7. Box plots depicting viable cell recov-

ery from 6-species biofilms (n = 9) treated with different commercially available mouth rinses. Significant differences between treatments at the 99 or 95% level are indicated by ** and *, respectively; n.s. = not significant.

pound [Shapiro et al., 2002]. When comparing the effect of antimicrobials, the time point of analysis after the last exposure is crucial. An ideal product should prevent plaque regrowth over extended periods and we chose 16 h. If an antimicrobial has a low microbicidal effect, even slight differences in the numbers of survivors can lead to large variation due to biofilm growth in the posttreatment period. Although our in vitro model cannot mirror oral distribution of a mouth rinse, it has the great advantage

that a mouth rinse can be applied to and removed from the system virtually instantaneously, simulating the brief exposure of supragingival plaque to mouth rinses. In addition, the model can be set up in any microbiology laboratory with standard equipment. It is at present the only model allowing in vitro a reliable prognosis of the in vivo performance of antimicrobials for oral use and is thus a valuable tool for preclinical evaluation of antiplaque formulations.

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Fig. 8. QLF images of bovine enamel disks before (A1, A3) and after demineralization (A2, A4). B1 Image of a disk demineralized with an acidic gel. B2 The same disk after pretreatment with Elmex gel diluted 1:5 and remineraliza-

tion under a biofilm for 64.5 h in the presence of 5 mM Ca2+.

Demineralization and Remineralization of Enamel in the Zürich Biofilm Model

More recently, we explored whether our biofilm model could be used to achieve demineralization and remineralization of bovine enamel in vitro [Guggenheim et al., 2003]. The necessary prerequisite was to find a method allowing a reproducible and rapid assessment of the degree of mineralization of enamel. Bovine enamel disks were prepared from incisors of cows. Demineralization was measured by quantitative light-induced fluorescence (QLF) [Al-Khateeb et al., 1997; van der Veen and de Josselin de Jong, 2000] using a modification of the ‘in vivo’ technique, with prototype hardware and software. We found buffer and carbohydrate concentrations in the medium to be the main parameters controlling demineralization. These variables were tested in a checkerboard arrangement in order to find conditions resulting in substantial demineralization in the 70:30 biofilm model. Six buffer concentrations from 0 to 100% 0.66 M Sørensen phosphate buffer, pH 7.2, were applied in the medium. In the vertical direction, 4 carbohydrate concentrations (glucose + sucrose 1:1) between 0.5 and 5% were

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tested. At a concentration of 0.5% carbohydrate, no demineralizations were observed at any buffer concentration. At a concentration of 1% carbohydrate, demineralizations were observed that decreased with increasing buffer concentration. At a concentration 11% carbohydrate bovine enamel was strongly demineralized independently of the buffer strength. The results led to the choice of 0% buffer and 1% carbohydrate, which gave a mean demineralization of –30.5 B 3.3% (n = 6) with a very narrow range (–27.3 to –35.4%). In figure 8A, representative QLF images of disks before and after demineralization are shown. Differences in fluorescence before and after biofilm exposure are clearly visible. For remineralization experiments, enamel disks demineralized in vitro by acid gels [Schmidlin et al., 2002] were used. The degree of demineralization was assessed by QLF prior to using them as substrate for biofilm formation in the 70:30 model. Ideally, disks with a ¢F score of –25 to –30% were chosen. We found fluoride and calcium to cause heavy precipitation in the 70:30 biofilm medium. Therefore, fluoride was applied prior to pellicle formation. Enamel disks were brushed for 2 min with fluoride preparations, stored in a wet chamber for 2 h and

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different microbiological ecologies, the pathogenicity of single strains and the effect of different fluoride pretreatments on enamel remineralization may be studied.

Concluding Remarks

Fig. 9. Remineralization of bovine enamel disks under biofilms is expressed as increase in ¢F. A water control is compared with disks incubated with an addition of 5 mM Ca2+ to the medium. Disks pretreated with a 1:5 diluted Elmex gel only were compared with disks after fluoride pretreatment and additional incubation with 5 mM Ca2+ in the medium.

subsequently rinsed for 15 s with tap water. Biofilms were formed on fluoride or water-pretreated enamel disks exposed to 5 mM calcium in the medium for 64.5 h. ¢F was calculated before and after exposure to biofilms. Results shown in figure 9 reveal that the addition of 5 mM calcium chloride resulted in a remineralization of approximately 7.5%. Fluoride-pretreated disks exposed in addition to 5 mM calcium remineralized by 12.5%. Remineralization was comparatively slow but could still be seen by eye (fig. 8B). Thus, our model may be used for studying demineralization and remineralization under biofilms. In particular, inhibition of demineralization, the effect of

The Zürich in vitro biofilm model is reproducible and reliable. It may be used for the study of basic, but also for very application-oriented questions that could not be addressed before. Only very few applications could be shown because of space limits; there would have been many others, and there is even much more to explore. The use of biofilm models allows us to address a multitude of questions that could hitherto not be studied with planktonic monocultures. A new area of research in oral biology is now open with promising prospects for preventive dentistry. We have questioned paradigms that have evolved by extrapolating characteristics from mainly Pseudomonas aeruginosa biofilms to oral biofilms or that have been derived from results of in vitro studies with planktonic bacteria under nonphysiological conditions. It is now more and more accepted that in different environments biofilms with widely different properties are formed and that the prevailing growth conditions are the overall dominating factor [Klausen et al., 2003].

Acknowledgments The authors are grateful for excellent technical assistance by André Meier, Martin Gander and Verena Osterwalder. The work was supported by the Universities of Zürich and Bergen, Patentmedelsfonden för Odontologisk Profylaxforskning (Sweden), Colgates Forskningsfond (Norway) and A/S Norsk Dental Depots Fond for Odontologisk Forskning (Bergen).

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Caries Res 2004;38:223–229 DOI: 10.1159/000077758

Antimicrobials in Future Caries Control? A Review with Special Reference to Chlorhexidine Treatment

Svante Twetman Department of Odontology, Pediatric Dentistry, Umeå University, Umeå, Sweden

Key Words Antimicrobials W Chlorhexidine W Clinical trials W Caries prevention

Abstract The aim of this paper was to examine recent evidence for the effect of the antibacterial approach to prevent and control caries with special reference to the use of chlorhexidine (CHX). Existing information from the mid 1990s provided limited evidence for the effectiveness of CHX gels, rinses and toothpaste in preventing caries in permanent teeth of children and adolescents. An updated literature search on CHX intervention in controlled clinical trials from 1995 to May 2003 unveiled 22 studies covering over 4,500 patients with clinical caries as end point. The vast majority (n = 21) were dealing with CHX-containing varnishes. Since the studies exhibited disparities in design, diagnosis and intervention, the findings were subgrouped with respect to caries type and localization. According to the ranking system of the Swedish Council on Technology Assessment in Health Care, the evidence for an anticaries effect of CHX varnishes was rated as inconclusive for caries-active schoolchildren and adolescents with regular fluoride exposure. Regarding fissure Paper presented at the ORCA Anniversary Symposium in Konstanz, Germany, 2003.

ABC

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caries, a preventive effect of CHX varnishes was demonstrated in 4 studies out of 5 when compared to no treatment in children with low fluoride exposure. The evidence for arresting root caries in dry-mouth patients and frail elderly subjects was inconclusive. In conclusion, the evidence from the recent literature was inconclusive for the use of CHX varnishes for caries prevention in risk groups. Copyright © 2004 S. Karger AG, Basel

It is generally understood that dental caries is an infectious disease of bacterial origin [van Houte, 1994] and, therefore, it must be considered relevant to utilize an antimicrobial approach to prevent and control the disease. The ultimate goal of antimicrobial therapy is to achieve a shift from an ecologically unfavourable to an ecologically stable biofilm [Marsh, 1994, 2003]. By suppressing the proportion of acidogenic and aciduric bacteria that have a growth advantage in low pH conditions, less acid is formed in the aqueous interphase between plaque and enamel, which enables and enhances remineralization by fluoride [ten Cate, 1999]. A wide range of antibacterial agents and products, including fluoride and sugar substitutes, are commonly used in preventive dentistry, and numerous in vitro and in vivo reports are available on their influence on bacterial growth and metabolism. This paper is focused on the ‘traditional’ antiseptic

Dr. S. Twetman Department of Odontology, Pediatric Dentistry Medical and Odontological Faculty, Umeå University SE–901 87 Umeå (Sweden) Tel. +46 90 785 6230, Fax +46 90 770 330, E-Mail [email protected]

agents and restricted to studies measuring clinical caries as end point. This standpoint may be justified by two main reasons. Firstly, recent findings indicate that surrogate end points, such as the effect of an antimicrobial agent on levels of mutans streptococci (MS) or plaque reduction may not always correlate with eventual caries reduction [Caufield and Dasanayake, 2001; Dasanayake et al., 2002; Anderson, 2003]. Secondly, the really important outcome from the patient’s and dentist’s perspective is proven reductions in caries. There seems to be consensus over the current indications for a chemotherapeutic approach for caries prevention, limiting its use to cariesactive individuals and to subjects with an increased caries risk and ongoing caries activity [Kidd, 1991; Emilson, 1994; Rozier, 2001]. The aim of this paper was to review and discuss evidence for the efficacy of the antimicrobial approach to prevent dental caries and to identify questions of interest for future research.

Existing Information

It is generally accepted that chlorhexidine digluconate (CHX) remains the gold standard as antiplaque and antigingivitis agent [Matthijs and Adriaens, 2002]. Although CHX has substantial antimicrobial properties against caries-causing bacteria, its use as anticaries agent remains more controversial. The efficacy in caries prevention has been established in several clinical trials as thoroughly reviewed by Emilson [1994]. It was concluded that CHX gel administered in trays was the most effective regimen and that the outcome of the treatment should be monitored by follow-up bacterial samplings. A meta-analysis by van Rijkom et al. [1996] including 8 clinical trials performed between 1975 and 1994 with gels, rinses and toothpaste in schoolchildren and adolescents at risk (n = 612) revealed a prevented fraction of 46% (95% confidence interval 35–57%). This meta-analysis was however mostly based on studies conducted two decades ago in study populations with a higher caries prevalence than today, some were of short duration and some lacked a true control group. It was therefore justified to update the literature search for recent randomized (RCT) or controlled clinical trials (CCT) with CHX rinse, gel or varnish as main intervention with and without additional use of fluoride. The search terms were ‘caries’, ‘chlorhexidine’, ‘chlorhexidine rinsing’, ‘gel’, ‘varnishes’ and ‘antibacterial treatment’, and only papers published in English were considered. The outcome measure was limited to the incidence or progression/regression of manifest and incipient

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caries lesions on crowns and roots as diagnosed by visual inspection, probing and/or radiographs. Furthermore, interim reports or double publications were excluded. The PubMed (US National Library of Medicine) database disclosed 22 papers published from 1995 to May 2003 in which the efficacy of CHX-containing varnishes and rinses in over 4,500 subjects of various ages was investigated (tables 1 and 2). An immediate reflection was that no studies employing CHX gel with caries as end point seemed to have been performed in recent years. The selected papers were reviewed and subgrouped with respect to caries localization and type. The level of evidence was judged in 4 grades according to the protocol of the Swedish Council on Technology Assessment in Health Care [Britton, 2000; www.sbu.se]: 1 = strong evidence, requiring at least 2 studies with a high level of evidence (A) or a good systematic review; 2 = moderate evidence, requiring 1 study with level A and at least 2 studies with a moderate level of evidence (B); 3 = limited evidence, requiring at least 2 studies with level B; 4 = inconclusive evidence, less than 2 studies with level B. All Tooth Surfaces Three recent studies regarding CHX-containing varnish and caries increment in the young permanent dentition of risk and caries-active subjects were found [Forgie et al., 2000; Splieth et al., 2000; de Soet et al., 2002]. In an RCT with more than 1,200 Scottish schoolchildren selected at risk based on past caries experience and high salivary MS levels, Forgie et al. [2000] failed to demonstrate any reduction in caries increment over a 3-year period when a 10% CHX varnish or a placebo varnish was applied 6–12 times. The applications were initially frequent but reduced by time, which to some extent could explain the lack of efficacy. In the second study, Splieth et al. [2000] selected caries-active schoolchildren that had developed more than 1 new lesion per year for the past years for a treatment combination of CHX varnish and fluoride gel versus fluoride gel controls. Although the experimental group developed less caries over 12 months, the difference was not statistically significant. The study was however small, and larger experimental groups and prolonged study duration would have increased the power of this setting. The third study was performed in a high caries community (Surinam) with semi-annual CHX varnish applications [de Soet et al., 2002], and, again, no effect on caries increment in schoolchildren was demonstrated. On the contrary, it was speculated that a high carbohydrate intake in combination with the CHX treatments could even be detrimental for caries development.

Twetman

Table 1. Summary of studies identified between 1995 and May 2003 with CHX varnish intervention and clinical caries outcome Authors

Material

Young permanent dentition Forgie et al. [2000] 1,240 De Soet et al. [2002] 238 Splieth et al. [2000]

Age years

Risk

Design

InterControl vention

Duration years

Drop- Diagnosis out, %

11–13 13–14

6105 CFU high-caries population 1 1 DMFS/year

RCT, DB RCT, DB

1 3

placebo neutral gel

3 2.5

16 19

CCT, SB

2 + Fg

Fg

1

4

clin.

1.2/2.1 DMFS n.s.

split, DB

2

placebo

2

14

BW

CCT, SB

2 + Fv

Fv

3

0

BW

difference –0.21 3.8/3.0 DFSa

n.s.

56

8–10

85

8

219

12

Petersson et al. [2000]

180

13–14

Twetman and Petersson [1999]

174

Fissures Araujo et al. [2002] Baca et al. [2002] Bratthall et al. [1995] Fennis-Ie et al. [1998] Joharji et al. [2001]

Proximal sites Haukali and Poulsen [2003] Petersson et al. [1998]

Outcome

Statistics

clin. + BW 6.8/6.4 DMFS n.s. clin. 2.1/1.7 DMFS n.s.

RCT, SB

2

Fv

3

8

BW

3.1/2.7 DFSa

n.s.

8–10

61 proximal lesion 61 proximal lesion 62 proximal lesions 6105 CFU

CCT, SB

2

untreated

2

2

BW

22/20% DFSa

n.s.

16 229 502 332 200

6–8 6–7 5–12 5–12 7–14

no no no no no

split RCT SB split, SB RCT, DB split, SB

2 2 2 3 2

untreated untreated untreated placebo cleaning

2 2 2 3 0.75

0 21 16 5 9

clin. + BW clin. clin. clin. clin.

0/50% 0.9/1.8 DFSo 7/16% 0.6/0.6 DFSo 18/49%

p ! 0.01 p ! 0.05 p ! 0.001 n.s. p ! 0.001

White spot lesions Jenatschke et al. [2001] Øgaard et al. [2001] Madlena et al. [2000] Twetman et al. [1995]

33 220 24 18

11–18 12–15 13–23 11–18

6105 CFU orthod. orthod. orthod.

RCT RCT split split

3 2 + Fv 2 2

placebo Fv placebo placebo

debond. debond. debond. debond.

0 0 0 0

clin. + BW clin. clin. clin.

31/32% 58/61% 0.7/2.1 DS 6/6%

n.s. n.s. p ! 0.05 n.s.

Root caries Banting et al. [2000] Brailsford et al. [2002]

240 134

45–75 70–80

dry mouth frail elderly

RCT; DB RCT, DB

1 2 + Fv

placebo 1 placebo + Fv 1

24 19

clin. clin.

Powell et al. [1999]

297

660

low income

RCT, SB

R

program

32

clin.

0.8/1.3 p ! 0.05 42/30% n.s. improved 23% reduction n.s.

3

n.s.

Intervention: 1 = Chlorzoin (10% CHX, Oralife, Canada); 2 = Cervitec (1% CHX, Vivadent, Schaan, Liechtenstein); 3 = EC-40 (40% CHX, Explore Biodent BV, Arnheim, the Netherlands); R = 0.12% CHX rinse; Fg = fluoride gel; Fv = fluoride varnish; outcome = caries increment in test/control (mean values of surface or percentage of surfaces); CFU = colony-forming units of mutans streptococci per millilitre saliva; RCT = randomized clinical trial; CCT = controlled clinical trial; split = split-mouth; DB = double-blind; SB = single-blind; clin. = clinical examination; BW = bitewing radiographs; n.s. = not significant.

Table 2. Summary of recent studies with maternal antibacterial intervention with clinical caries as outcome measure in the mother’s

children Authors

Dasanayake et al. [2002] Günay et al. [1998] Isokangas et al. [2000] a b c

Mothers

75 86 195

Risk

Design

Intervention

Time

Control

Child age years

Dropout, %

Outcome

Statistics

selected selected

RCT CCT

CHX varnish CHX rinse + varnishb CHX varnish

6–36 monthsa 2nd trimester to 4 years 3–24 monthsc

placebo untreated

4 4

? 45

dft 2.5/2.1 dfs 1.5/7.0

n.s. p ! 0.001

F varnish

5

27

dmft 3.2/2.9

n.s.

1 105 CFU RCT

9 applications (10% CHX varnish). CHX treatments as part of a comprehensive preventive programme. 3 applications (40% CHX varnish).

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The reason could be that in frequent low-pH situations, the CHX-induced reduction of the sensitive bacteria may lead to an overgrowth of highly aciduric species such as MS and lactobacilli. It was therefore concluded that antibacterial treatments should always be accompanied by other preventive measures in caries-active children. Approximal Caries Four studies were identified in which only the approximal caries incidence in posterior teeth of caries-active schoolchildren was taken into account. The subjects were selected on the basis of having either proximal enamel lesions or elevated salivary bacterial counts. Two reports were 3-year CCTs with parallel arms in which CHX varnish or a mix of CHX and fluoride varnishes were tested against fluoride varnish applications in semi-annual or quarterly regimes [Petersson et al., 1998, 2000]. Caries incidence was determined from bitewing radiographs exposed with a film holder. Both studies were unable to unveil an additional caries-preventive effect of the CHX varnishes over the fluoride varnish alone. The other two papers were 2-year trials in schoolchildren, one with a split-mouth design [Haukali and Poulsen, 2003] and the other evaluated proximal caries incidence progression in relation to the degree of MS suppression [Twetman et al., 1999]. Both studies concluded that CHX varnish applications did not affect the overall proximal progression rate. In the latter study however, children who exhibited significantly suppressed MS counts after the treatments exhibited a lower incidence and progression rate compared with those with a less marked suppression. The results support previous findings that the outcome of the employed topical intervention must be monitored in order to decide whether or not to continue with further antibacterial treatments [Emilson, 1994]. Fissure Caries Five studies were identified with fissure caries as end point [Bratthall et al., 1995; Fennis-Ie et al., 1998; Joharji and Adenubi, 2001; Araujo et al., 2002; Baca et al., 2002]. Three of the reports were split-mouth studies of first and second permanent molars in which a 1% CHX/thymol varnish was applied 3 times per year versus untreated controls. The findings were all in favour of the antibacterial varnish, but it must be underlined that the investigations were carried out in subjects where the regular use of fluoride toothpaste or exposure to fluoride supplements was low or uncertain. Moreover, with one exception, the diagnosis of fissure caries was based on clinical examination only and without the aid of bitewing radiographs.

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Two studies were RCTs in newly erupted first [Baca et al., 2002] and in first and second permanent molars [FennisIe et al., 1998]. Radiographs were not used. In the latter study, a 40% CHX varnish was applied semi-annually for 3 years with a placebo varnish as control. The results disclosed no significant differences between the treatments when the entire study groups were taken into account. However, 15% of the children harboured high counts of salivary MS (106 CFU/ml mixed saliva) at baseline. In that subgroup, a post hoc statistical analysis indicated that the number of carious permanent molars was significantly reduced (p ! 0.05) at the termination of the study. In the report by Baca et al. [2002], a CHX/thymol varnish was applied every third month for 2 years and the incidence of caries in the first molars was compared with that of untreated controls. A small but statistically significant reduction was disclosed, and the authors conclude that the antibacterial agent was a useful alternative to prevent fissure caries when appropriate dental facilities and resources were lacking. All papers explain the effectiveness of the varnish treatments in preventing fissure caries by the retentive nature of the occlusal surfaces, enabling a slow release of the antibacterial agent. White Spot Lesions Four studies dealing with early enamel lesion development were identified, all performed in patients undergoing treatment with fixed orthodontic appliances [Twetman et al., 1995; Madlena et al., 2000; Jenatschke et al., 2001; Øgaard et al., 2001]. Insertion of appliances may interfere with oral hygiene procedures, resulting in plaque accumulation and an increased risk for ‘white spot lesions’ adjacent to the luted bands or bonded bracket bases. Two studies were very small and utilized a splitmouth design with CHX varnish versus a placebo varnish applied during the time of active treatment. Conflicting results were reported. Madlena et al. [2000] found a significant reduction of white spot lesions following CHX varnish treatments among children with a higher caries increment while no effect could be found in a Swedish low-caries population [Twetman et al., 1995]. The two most recent investigations were RCTs comparing CHX varnish with placebo or fluoride varnish as controls [Jenatschke et al., 2001; Øgaard et al., 2001]. In the former study, the orthodontic patients were screened and recruited with high counts of salivary MS while the other included 220 non-selected cases with varying levels of MS. Both studies were unable to disclose any benefit from frequent CHX varnish applications on white spot lesion development during treatment with fixed orthodontic ap-

Twetman

pliances, in spite of significant reductions in MS colonization. Root Caries Three papers dealt with CHX treatments and root caries development in elderly and low-income older adults [Powell et al., 1999], dry-mouth risk patients [Banting et al., 2000] and in frail institutionalized people [Brailsford et al., 2002]. Powell et al. [1999] demonstrated a nonsignificant reduction of root caries events following weekly 0.12% CHX rinses and fluoride varnish compared with a group receiving ‘usual’ care from private practitioners. The other studies were 1-year placebo-controlled randomized trials, and one of them demonstrated a significant reduction and control of root caries lesions, suggesting regular antibacterial applications to be beneficial for these patient groups [Banting et al., 2000]. Notably, neither Powell et al. [1999] nor Banting et al. [2000] found any significant impact on coronal caries increment, but to be fully conclusive on this matter, increased size of study groups and prolonged duration would have been desirable. The results of the root caries studies may however indicate that the antimicrobial therapy may act differently for lesions and cavities located in dentine and enamel, respectively.

children were 6, 12 and 18 months of age, but no differences in caries increment up to 5 years of age were noticed between the CHX and fluoride varnish groups. Recently, Dasanayake et al. [2002] have published a placebo-controlled RCT in which they evaluated the efficacy of CHX varnish treatments of mothers during the eruption of their children’s first teeth and during the second year of life. Although a significant reduction of MS levels in both mothers and children could be seen, no effect on caries development was found among the children. Thus, topical applications of antibacterial agents may reduce the transmissions of oral MS from host to host, but this does not necessarily result in less caries. This approach merits to be further elucidated in terms of effectiveness and efficiency.

Other Antibacterial Agents of Clinical Interest

Mother-Child Transmission The primary preventive concept to interfere with the mother-child transmission route of MS has gained continuous interest during the recent decades. The ‘classic’ studies by Köhler et al. [1984] and Tenovuo et al. [1992] clearly showed that CHX gel treatments of highly infected mothers could reduce MS colonization and caries development in their children. Since then, a number of additional papers on this issue have been presented [Brambilla et al., 1998; Söderling et al., 2000; Gripp and Schlagenhauf, 2002; Thorild et al., 2003], but only three have reported caries as end point. These studies are compiled in table 2. In a German study, Günay et al. [1998] offered a comprehensive preventive programme for mothers and children that started during pregnancy and continued for 4 years. The programme, which included CHX-containing rinses and varnishes, resulted in significantly improved oral health for both the mothers and their children, but the role of the antibacterial agents could not be distinguished from the other measures within the programme. Isokangas et al. [2000] focused mainly on xylitol but included treatments with either CHX- or fluoridecontaining varnishes of highly colonized mothers as controls. The mothers were treated on 3 occasions, when their

Triclosan is a broad-spectrum biocide that may affect many types of oral bacteria. The agent has been incorporated into dentifrices together with a copolymer, and reductions in supragingival plaque and gingivitis have been claimed [Gaffar et al., 1997]. A number of cariesfocused RCTs with triclosan/copolymer-containing fluoride toothpastes have been carried out in schoolchildren and adults [Hawley et al., 1995; Feller et al, 1996; Mann et al., 1996]. The results clearly showed that the addition of the antibacterial agent neither compromised nor enhanced the anticaries effect of the toothpaste. However, in a recent study from Israel, the effect of unsupervised tooth brushing with two 0.243% sodium fluoride dentifrices with and without 0.3% triclosan and 2% copolymer on coronal caries was evaluated in adults [Mann et al., 2001]. The 2-year findings were significantly in favour of the dentifrice with triclosan, indicating an additional anticaries effect that should be further investigated. The antibacterial properties of povidone-iodine as a mucosal antiseptic in medicine are well established, but the agent is rarely utilized in dentistry. Povidone-iodine is water soluble and non-irritating and exhibits no adverse effects such as discoloration and taste alterations, but iodine hypersensitivity, thyroid pathosis and pregnancy are contra-indications. The efficacy of a 10% povidoneiodine solution to prevent early childhood caries has recently been evaluated in a randomized double-blind placebo-controlled trial [Lopez et al., 2002]. The results showed that this topical antimicrobial therapy increased the time of ‘disease-free survival’ in toddlers with high risk of early caries development.

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Future Perspectives for Research

Despite the recent decline, caries is still the most prevalent dental infectious disease with a giant unmet treatment need in many countries. A general concern is that the dental practitioner is still treating caries with a surgical approach rather than with prevention or pharmacotherapeutics. An urgent task for the scientific community is therefore to initiate clinical studies to confirm the efficacy and safety of non-surgical treatment of caries with focus both on the population and selected patient groups. It is not only a question of performing more investigations but also better ones. Well-designed multi-centre trials with antibacterial intervention according to a standard protocol would be desirable, and special efforts should be made to select representative study groups with enough power to ensure firm conclusions. Future research must also be extended to incorporate preschoolers, adults and elderly subjects. No studies can be designed without regular exposure of fluoride from toothpastes or any other commonly used alternative non-surgical treatment for ethical reasons. Since the existing antibacterial agents seem to be less effective for the caries-active patients with the highest need, more potent and long-lasting drugs with as few side-effects as possible need to be developed. In order to improve efficiency and compliance, it seems reasonable in the future to move from topical administrations by professionals to consumer products and homecare procedures, such as antibacterial constituents incorporated into dentifrices and chewing gums. The early intervention concept is interesting as it may be easier to affect the caries-associated bacteria before their perma-

nent colonization compared to later in life when the resident oral flora is firmly established [Könönen, 2000]. The screening of mothers and potentially long treatment duration are however drawbacks in terms of cost and compliance. A full-scale investigation at the population level, including both parents, would be a project of high priority. It may very well be that antibacterial agents are underutilized and factors such as dentist’s knowledge and attitudes, poor patient compliance and low willingness to pay may likely play a role. In today’s evidence-based care, also the patient’s wishes and demands must be taken into account. Therefore, qualitative studies should be planned and undertaken parallel with, and linked to the intervention protocol in order to unveil the patient’s thinking. Further, studies on how to communicate the preventive health message and increase motivation in caries risk patients and vulnerable groups should be encouraged.

Conclusions

The evidence for an anticaries effect of CHX-containing varnishes was rated as inconclusive for caries-active schoolchildren and adolescents with daily exposure to fluoride as well as for root caries arrest in elderly subjects. It must however be underlined that ‘inconclusive evidence of effect’ is not the same as ‘evidence of no effect’ in the sense that antibacterial methods are of no value and should be abandoned. It is however definitely a call for further research and development of well-designed studies.

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Future of Antimicrobials?

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Petersson LG, Magnusson K, Andersson H, Almquist B, Twetman S: Effect of quarterly treatments with a chlorhexidine and a fluoride varnish on approximal caries in caries-susceptible teenagers: A 3-year clinical study. Caries Res 2000;34:140–144. Petersson LG, Magnusson K, Andersson H, Deierborg G, Twetman S: Effect of semi-annual applications of a chlorhexidine/fluoride varnish mixture on approximal caries incidence in schoolchildren: A three-year radiographic study. Eur J Oral Sci 1998;106:623–627. Powell LV, Persson RE, Kiyak HA, Hujoel PP: Caries prevention in a community-dwelling older population. Caries Res 1999;33:333– 339. van Rijkom HM, Truin GJ, van’t Hof MA: A metaanalysis of clinical studies on the caries-inhibiting effect of chlorhexidine treatment. J Dent Res 1996;75:790–795. Rozier RG: Effectiveness of methods used by dental professionals for the primary prevention of dental caries. J Dent Educ 2001;65:1063– 1072. Söderling E, Isokangas P, Pienihäkkinien K, Tenovuo J: Influence of maternal xylitol consumption on acquisition of mutans streptococci by infants. J Dent Res 2000;79:882–887. de Soet JJ, Gruythuysen RJM, Bosch JA, van Amerongen WE: The effect of 6-monthly application of 40% chlorhexidine varnish on the microflora and dental caries incidence in a population of children in Surinam. Caries Res 2002; 36:449–455. Splieth C, Steffen H, Rosin M, Welk A: Caries prevention with chlorhexidine-thymol varnish in high risk schoolchildren. Community Dent Oral Epidemiol 2000;28:419–423. Tenovuo J, Häkkinen P, Paunio P, Emilson CG: Effects of chlorhexidine-fluoride gel treatments in mothers on the establishment of mutans streptococci in primary teeth and the development of dental caries in children. Caries Res 1992;26:275–280. Thorild I, Lindau B, Twetman S: Effect of maternal use of chewing gums containing xylitol, chlorhexidine or fluoride on mutans streptococci colonization in the mother’s infant. Oral Health Prev Dent 2003;1:53–57. Twetman S, Hallgren A, Petersson LG: Effect of an antimicrobial varnish on mutans streptococci in plaque from enamel adjacent to orthodontic appliances. Caries Res 1995;29:188–191. Twetman S, Petersson LG: Interdental caries incidence and progression in relation to mutans streptococci suppression after chlorhexidinethymol varnish treatments in schoolchildren. Acta Odontol Scand 1999;57:144–148.

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Caries Res 2004;38:230–235 DOI: 10.1159/000077759

A Caries Vaccine? The State of the Science of Immunization against Dental Caries

Michael W. Russell a Noel K. Childers b Suzanne M. Michalek c Daniel J. Smith d Martin A. Taubman d a Departments of Oral Biology and Microbiology and Immunology, University at Buffalo, Buffalo, N.Y., Departments of b Oral Biology and c Microbiology, University of Alabama at Birmingham, Birmingham, Ala., and d Department of Immunology, Forsyth Institute, Boston, Mass., USA

Key Words Mutans streptococci W Salivary IgA antibodies W Vaccine antigen W Mucosal immunization

Abstract Studies performed in numerous laboratories over several decades have demonstrated the feasibility of immunizing experimental rodents or primates with protein antigens derived from Streptococcus mutans or Streptococcus sobrinus against oral colonization by mutans streptococci and the development of dental caries. Protection has been attributed to salivary IgA antibodies which can inhibit sucrose-independent or sucrose-dependent mechanisms of streptococcal accumulation on tooth surfaces according to the choice of vaccine antigen. Strategies of mucosal immunization have been developed to induce high levels of salivary antibodies that can persist for prolonged periods and to establish immune memory. Studies in humans show that salivary antibodies to mutans streptococci can be induced by similar approaches, and that passively applied antibodies can also suppress oral re-colonization by mutans streptococci. Progress towards practical vaccine development requires evaluation of candidate vaccines in clinical trials. Promising strategies of passive immunization also require further clinical evaluation. Copyright © 2004 S. Karger AG, Basel

ABC Fax + 41 61 306 12 34 E-Mail [email protected] www.karger.com

© 2004 S. Karger AG, Basel

Accessible online at: www.karger.com/cre

The concept of vaccination against dental caries has existed almost from the time that this disease was recognized to result from colonization of the teeth by acidogenic bacteria, even though the etiological agents were originally thought to be lactobacilli. Since then, Streptococcus mutans and Streptococcus sobrinus and their relatives, collectively known as mutans streptococci, have become recognized as the principal organisms responsible for initiating caries in humans [Loesche, 1986], and considerable progress has been made in elucidating the factors involved in their pathogenic activity, culminating recently in the sequencing of the entire S. mutans genome [Ajdic et al., 2002]. Likewise, enormous strides have been made in comprehending the workings of the mucosal immune system by which secretory IgA (S-IgA) antibodies are generated in saliva and other secretions [Ogra et al., 1999]. This system is functional in newborn infants, and although at birth salivary IgA levels are almost zero, infants promptly develop salivary IgA antibodies concomitantly with oral microbial colonization [Smith and Taubman, 1992; Smith et al., 1998]. The mechanisms of action of salivary IgA antibodies against mutans streptococci include interference with their sucrose-independent and sucrose-dependent attachment to, and accumulation on, tooth surfaces, as well as possible inhibition of their metabolic activities [Russell et al., 1999]. The goal of immunizing infants and young children against colonization by mutans streptococci and hence diminishing the develop-

Michael W. Russell, PhD Department of Microbiology, Farber 138 University at Buffalo, 3435 Main Street Buffalo, NY 14214 (USA) Tel. +1 716 829 2790, Fax +1 716 829 2169, E-Mail [email protected]

Although over the years numerous surface or secreted products of mutans streptococci have been proposed as vaccine antigen candidates, attention has become focused on three protein antigens: the surface fibrillar adhesins known as AgI/II (synonyms: antigen B, P1, SpaP, PAc, SpaA, PAg), the glucosyltransferases (GTF) and the glucan-binding proteins, all of which have demonstrable associations with virulence and the process of tooth surface colonization [Jenkinson and Lamont, 1997]. While some early efforts utilized parenteral injection which was successful in rodent and primate models [Lehner et al., 1976; Russell et al., 1982] probably because of gingival transudation of circulating antibodies [Challacombe et al., 1978], most authorities have long recognized that mucosal routes of immunization, designed to stimulate the common mucosal immune system and induce potent salivary S-IgA antibodies, will not only be more efficacious but also be more acceptable and circumvent some concerns over safety. This and other vaccine goals have driven the development of novel strategies for effectively stimulating mucosal immune responses [Russell, 2003]. Several of these have been applied to mutans streptococcal antigens, including the delivery of immunogens in liposomes and other microparticles, co-administration of mucosal adjuvants such as enterobacterial enterotoxins and their detoxified mutants, coupling of immunogens to the nontoxic B subunits of enterotoxins and the expression of mutans streptococcal antigens in attenuated Salmonella strains [Eastcott et al., 2002; Hajishengallis et al., 1995; Harokopakis et al., 1997; Huang et al., 2001; Martin et al., 2000; Michalek et al., 1992; Russell and Wu, 1991; Smith et al., 2000]. In addition, molecular engineer-

ing of protein antigens by recombinant DNA technology as well as the construction of synthetic peptides representing identified antigenic epitopes have been pursued [Jespersgaard et al., 1999; Smith et al., 2003; Takahashi et al., 1991; Taubman et al., 1995; Zhang et al., 2002]. Numerous experiments in a variety of animal models comprising rodents and primates have demonstrated the induction of salivary S-IgA and circulating IgG antibodies to mutans streptococcal antigens by oral or intranasal immunization with AgI/II, GTF or glucan-binding proteins [reviewed in Childers et al., 2002; Koga et al., 2002; Russell et al., 1999; Russell, 2001; Smith, 2002]. Upon subsequent oral challenge with virulent mutans streptococci and the institution of a high-sucrose diet, these models have further demonstrated reductions in colonization and diminished development of dental caries lesions. Despite these successes, rodent models in particular have limitations in predicting applicability of findings to the human situation for a variety of reasons, including the short duration of the experiments compared with the time scale of caries development in humans. Thus, it is important that the generation of salivary IgA antibodies by immunization procedures developed in rodents has been achieved in primates [Russell et al., 1996] and in human experiments (see below). An important aspect of mucosal immunity centers around the question of immunological memory and the recall of responses upon subsequent exposure to antigens. Most studies of memory have focused on systemic antibody and cellular responses, and indeed earlier concepts, especially those founded upon experiments using simple methods of oral immunization with killed microorganisms or purified protein antigens, held that memory was poorly developed in the mucosal immune system. More effective strategies of mucosal immunization, especially those exploiting the extraordinary immunogenicity and adjuvanticity of cholera and related enterotoxins, however, have shown that memory can be induced and recalled by mucosal immunization [Harrod et al., 2001; Vajdy and Lycke, 1993]. While many details of the cellular and regulatory mechanisms underlying this remain to be elucidated, this finding has important implications for the development of vaccines against many mucosal infections including caries. Particularly in this case, it may be desirable that a salivary antibody response should be induced and sustained throughout the ‘window of infectivity’, the period from approximately 18 to 32 months of age when infants are most likely to become infected with mutans streptococci [Caufield et al., 1993]. It may also be desirable that responses should be recallable either by

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ment of caries might be accomplished by applying new strategies of mucosal vaccination that would induce salivary IgA antibodies without the complications of parenteral injection. A large body of experimental work over several decades has demonstrated the feasibility of inducing protective immunity against mutans streptococci and the subsequent development of dental caries in animal models. Information has also accrued from several smallscale trials in adult volunteers attesting to the applicability of these approaches to humans. For other recent reviews of this subject, see Childers et al. [2002], Koga et al. [2002], Russell et al. [1999], Russell [2001] and Smith [2002].

Current Approaches and Findings in Active Immunization

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booster immunization or by natural exposure to mutans streptococci, if further opportunities for infection arise at later times, such as when children enter school or their permanent teeth erupt. Thus, we have found that salivary IgA responses to AgI/II induced by mucosal immunization with AgI/II coupled to cholera toxin B subunit or expressed in recombinant Salmonella can persist for up to 1 year in mice (i.e. for half their normal life-span; table 1) and are amenable to prompt recall by booster immunization even after 2 years [Hajishengallis et al., 1996; Harokopakis et al., 1997; Harrod et al., 2001; Russell and Wu, 1991; Wu et al., 2000].

Human Trials

Several small-scale human trials in adults have shown that it is feasible to increase levels of salivary S-IgA antibodies to mutans streptococci, and in some cases to inter-

Table 1. Persistence of serum and salivary antibodies to AgI/II in mice after intranasal immunization with AgI/II conjugated to cholera toxin B subunit

Time after immunization Before 4 months 8 months 12 months

Serum

Saliva

IgG, Ìg/ml

IgA, Ìg/ml

0.25 !/&2.31 571 !/&1.84 175 !/&1.58 136 !/&1.83

1.42 !/&1.38 43.1 !/&1.50 16.1 !/&1.69 24.2 !/&1.40

fere with mutans streptococcal colonization (table 2). Human volunteers immunized orally with S. sobrinus GTF packaged in enteric capsules (14 young adults, compared with 11 placebo controls) developed increased levels of parotid salivary IgA antibodies to GTF and showed delayed reaccumulation of mutans streptococci in their oral microbiota [Smith and Taubman, 1987]. In a further study on 23 young adults, topical application of GTF to the lower lip intended to stimulate local antibody production in the minor salivary glands also delayed oral recolonization with mutans streptococci although antibody levels were not significantly increased [Smith and Taubman, 1990]. Oral immunization with preparations of S. mutans GTF that also contained a truncated form of AgI/ II in enteric capsules was also successful in elevating salivary IgA antibodies to the antigen preparation [Childers et al., 1994]. When similar antigen preparations were administered intranasally or by topical application to the tonsils, either in soluble form or incorporated in liposomes, salivary IgA antibodies were likewise increased [Childers et al., 1997, 1999, 2002, 2003; Li et al., 2003]. These studies now need to be extended into progressively younger age groups in controlled trials aimed at establishing whether equivalent responses can be induced in children and whether the responses obtained can suppress oral colonization by mutans streptococci.

IgA (Ab/Ig), % 0 60.5 !/&1.41 13.6 !/&1.76 21.6 !/&1.54

Geometric mean !/& SD, n = 5; from Wu et al. [2000].

Passive Immunization – An Alternative Approach

An alternative approach lies in the development of antibodies suitable for passive oral application against dental caries. This has considerable potential advantage in that it completely avoids any risks that might arise from active immunization. Conversely, in the absence of any active response on the part of the recipient, there is no

Table 2. Trials in adult humans: active immunization with S. mutans protein antigens Antigen

Route

n

Predominant antibody response (protective effect)

Reference

GTF

oral

25

Smith and Taubman [1987]

topical (MSG) oral nasal nasal or tonsillar (topical) nasal nasal

23 7 5, 21 21 12 26

increased salivary IgA antibody (delayed reaccumulation of indigenous S. mutans) (delayed reaccumulation of indigenous S. mutans) increased salivary IgA2 antibody (n.t.) increased nasal IgA1, salivary IgA1 and IgA2 antibodies (n.t.) IgA1 nasal and salivary antibodies in nasal group (n.t.) salivary IgA1 antibodies (n.t.) IgA1 nasal and salivary antibodies (n.t.)

GTF (+ AgI/II)

Smith and Taubman [1990] Childers et al. [1994] Childers et al. [1997, 1999] Childers et al. [2002] Li et al. [2003] Childers et al. [2003]

MSG = Minor salivary glands ; n.t. = not tested.

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induction of immunological memory, and the administered antibodies can persist in the mouth for only a few hours at most or up to 3 days in plaque [Ma et al., 1990]. Strategies include the development of antibodies to mutans streptococcal antigens in cow’s milk and hen’s eggs and the genetic engineering of human-like S-IgA antibodies in plants [Hamada et al., 1991; Hatta et al., 1997; Loimaranta et al., 1998; Ma et al., 1995; Mitoma et al., 2002]. Animal experiments have been encouraging: for example, the administration of chicken egg IgY antibodies to glucan-binding proteins diminished the development of caries lesions in a rat model [Smith et al., 2001]. Mouse monoclonal antibodies to AgI/II applied topically inhibited oral colonization by mutans streptococci and development of caries in monkeys for at least 1 year [Lehner et al., 1985]. Similar treatment, after extensive oral prophylaxis, of a small number of human adult volunteers with this IgG, or with engineered ‘human’ SIgA antibodies derived from the same monoclonal antibody, also suppressed the re-emergence of mutans streptococci for up to 2 years or 4 months, respectively [Ma et al., 1990, 1998]. The plausible though unproven explanation offered for these findings was that once mutans streptococci had been displaced by prophylaxis, passive application of antibody prevented their immediate re-colonization so that their oral ‘niche’ became occupied by other species with the result that their re-emergence was suppressed for far longer than the antibody persisted in the mouth. Unfortunately, further experiments on larger numbers of adults have not consistently demonstrated equivalent long-term reductions in colonization [Weintraub et al., 2001]. Whether a similar application of antibodies to young infants might inhibit subsequent oral colonization by mutans streptococci remains to be determined. However, in spite of these disappointments, collectively these studies clearly demonstrate the potential of antibodies to interfere with the ability of mutans streptococci to colonize teeth and to inhibit caries development. The key question then becomes: how can such antibodies be effectively delivered orally in caries-susceptible individuals and maintained at a protective level for the required length of time? Active vaccination has the advantage of inducing the endogenous production of salivary antibodies and the establishment of immune memory but requires a commitment to performing the human trials necessary to establish safety and efficacy. Passive administration of preformed exogenous antibodies offers the advantage of evading risks, however small, that are inherent in any active immunization procedure, but the need to provide a continuous source of antibodies to

maintain protection over a prolonged time remains a major challenge. Although new technologies for antibody engineering and production in animals or especially in plants (‘plantibodies’) offer the prospect of reducing the costs sufficiently to enable these materials to be incorporated into products for daily use, such as mouthwashes and dentifrices, long-term efficacy has yet to be reliably demonstrated.

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Future Prospects and Potential Impact

Given that dental caries usually develops slowly and can occur throughout life, it may be anticipated that immune protection would need to be similarly long-lasting. Thus, the duration and anamnestic recall of salivary antibody responses are important factors. While it is now clear that mucosal immune responses can persist and that memory is established if the priming stimulus is sufficient, relatively little is known about the parameters that govern memory in the mucosal immune system. The characteristics of specific mucosal memory cells, their location, and how they can be recalled and directed to particular effector sites such as the salivary glands to produce IgA antibodies for transport into the secretion are important subjects for investigation. Although current understanding holds that oral colonization with mutans streptococci mainly occurs during a ‘window of infectivity’ at around 2 years of age after primary teeth begin to erupt, it is unclear whether further opportunities for colonization exist, for example when children enter school and mix socially with a much larger group of their peers, or when the permanent teeth erupt. Two corollaries arise from such considerations: (i) that it would be necessary to immunize infants or young children in order to provide immune protection prior to initial colonization with mutans streptococci; (ii) that booster immunization to recall responses might be desirable to forestall colonization at later time points. As the transmission of mutans streptococci appears to be primarily from mother to infant [Li and Caufield, 1995], a third possibility is that young mothers might be immunized actively or passively with the objective of reducing their oral load of mutans streptococci (possibly in combination with conventional prophylaxis or other interventions), thereby diminishing the probability and extent of transmission to their infants. If the transferred bacteria are coated with maternal salivary antibodies, this would likely reduce their capacity to colonize the infant’s mouth. It has been suggested that immunization of young mothers to induce the generation

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of antibodies to mutans streptococcci in breast-milk could be exploited to provide passive immunity against caries to their infants. However, it seems unlikely that this strategy would have significant impact at least in Western societies, where breast-feeding, if given, usually terminates well before the ‘window of infectivity’ for mutans streptococci opens. Regardless of the mechanism by which immune protection against dental caries is achieved, further advances to make immunization against caries practicable will depend upon clinical trials aimed at establishing whether the findings from animal experiments can be transferred to humans. Particular goals for such studies include determining whether appropriate immune responses can be safely generated in humans, especially in the susceptible age groups, and whether such responses will afford desirable levels of protection. The goals for vaccination against most other, mainly acute, infectious diseases are usually to provide near-complete protection of the individual against infection, and to

achieve a sufficiently high prevalence of immunity in a population that the chain of transmission is broken and the pathogen cannot sustain itself in the community. However, the biology of caries is different from that of acute infections, and as with other modalities of intervention, it is conceivable that immunization will not attain complete effectiveness. Nevertheless, efficacy as low as 50% could have significant impact on the burden of disease, and the social and economic costs associated with it. Given that the bulk of dental caries occurs among a highrisk sector of the population (at least in the USA), targeting an effective vaccine to such individuals would increase its impact.

Acknowledgements The authors’ studies have been supported by USPHS grants DE06746, DE09846, DE07026, DE08182, DE09081, DE04733 and DE06153 from the National Institute of Dental and Craniofacial Research.

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A Caries Vaccine?

Michalek SM, Childers NK, Katz J, Dertzbaugh M, Zhang S, Russell MW, Macrina FL, Jackson S, Mestecky J: Liposomes and conjugate vaccines for antigen delivery and induction of mucosal immune responses. Adv Exp Med Biol 1992; 327:191–198. Mitoma M, Oho T, Michibata N, Okano K, Nakano Y, Fukuyama M, Koga T: Passive immunization with bovine milk containing antibodies to a cell surface protein antigen-glucosyltransferase fusion protein protects rats against dental caries. Infect Immun 2002;70:2721–2724. Ogra PL, Mestecky J, Lamm ME, Strober W, Bienenstock J, McGhee JR (eds): Mucosal Immunology. San Diego, Academic Press, 1999. Russell MW: Potential for vaccines in the prevention of caries lesions. Oper Dent 2001;suppl 6:51–60. Russell MW: Mucosal immunity; in Ellis RW, Brodeur BR (eds): New Bacterial Vaccines. Georgetown, Landes Bioscience, 2003. Russell MW, Hajishengallis G, Childers NK, Michalek SM: Secretory immunity in defense against cariogenic mutans streptococci. Caries Res 1999;33:4–15. Russell MW, Moldoveanu Z, White PL, Sibert GJ, Mestecky J, Michalek SM: Salivary, nasal, genital, and systemic antibody responses in monkeys immunized intranasally with a bacterial protein antigen and the cholera toxin B subunit. Infect Immun 1996;64:1272–1283. Russell MW, Wu H-Y: Distribution, persistence, and recall of serum and salivary antibody responses to peroral immunization with protein antigen I/II of Streptococcus mutans coupled to the cholera toxin B subunit. Infect Immun 1991;59:4061–4070. Russell RRB, Beighton D, Cohen B: Immunization of monkeys (Macaca fascicularis) with antigens purified from Streptococcus mutans. Br Dent J 1982;152:81–84. Smith DJ: Dental caries vaccines: Prospects and concerns. Crit Rev Oral Biol Med 2002;13: 335–349. Smith DJ, King WF, Akita H, Taubman MA: Association of salivary immunoglobulin A antibody and initial mutans streptococcal infection. Oral Microbiol Immunol 1998;13:278–285. Smith DJ, King WF, Barnes LA, Peacock Z, Taubman MA: Immunogenicity and protective immunity induced by synthetic peptides associated with putative immunodominant regions of Streptococcus mutans glucan-binding protein B. Infect Immun 2003;71:1179–1184. Smith DJ, King WF, Godiska R: Passive transfer of immunoglobulin Y antibody to Streptococcus mutans glucan binding protein B can confer protection against experimental dental caries. Infect Immun 2001;69:3135–3142.

Smith DJ, Taubman MA: Oral immunization of humans with Streptococcus sobrinus glucosyltransferase. Infect Immun 1987;55:2562– 2569. Smith DJ, Taubman MA: Effect of local deposition of antigen on salivary immune responses and reaccumulation of mutans streptococci. J Clin Immunol 1990;10:273–281. Smith DJ, Taubman MA: Ontogeny of immunity to oral microbiota in humans. Crit Rev Oral Biol Med 1992;3:109–133. Smith DJ, Trantolo DJ, King WF, Gusek EJ, Fackler PH, Gresser JD, De Souza VL, Wise DL: Induction of secretory immunity with bioadhesive poly(D,L-lactide-co-glycolide) microparticles containing Streptococcus sobrinus glucosyltransferase. Oral Microbiol Immunol 2000;15: 124–130. Takahashi I, Okahashi N, Matsushita K, Tokuda M, Kanamoto T, Munekata E, Russell MW, Koga T: Immunogenicity and protective effect against oral colonization by Streptococcus mutans of synthetic peptides of a streptococcal surface protein antigen. J Immunol 1991;146: 332–336. Taubman MA, Holmberg CJ, Smith DJ: Immunization of rats with synthetic peptide constructs from the glucan-binding or catalytic region of mutans streptococcal glucosyltransferase protects against dental caries. Infect Immun 1995; 63:3088–3093. Vajdy M, Lycke N: Stimulation of antigen-specific T- and B-cell memory in local as well as systemic lymphoid tissues following oral immunization with cholera toxin adjuvant. Immunology 1993;80:197–203. Weintraub JA, Hilton JF, White JM, Hoover C, Pelino JE, Tran K, Wycoff K, Larrick JW, Yu L, Featherstone JDB: Results of a plant-derived mutans streptococci antibody clinical trial (abstract 201). J Dent Res 2001;80(special issue). Wu HY, Abdu S, Stinson D, Russell MW: Generation of female genital tract antibody responses by local or central (common) mucosal immunization. Infect Immun 2000;68:5539–5545. Zhang P, Jespersgaard C, Lamberty-Mallory L, Katz J, Huang Y, Hajishengallis G, Michalek SM: Enhanced immunogenicity of a genetic chimeric protein consisting of two virulence antigens of Streptococcus mutans and protection against infection. Infect Immun 2002;70: 6779–6787.

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Caries Res 2004;38:236–240 DOI: 10.1159/000077760

How Much Saliva Is Enough for Avoidance of Xerostomia? C. Dawes Department of Oral Biology, Faculty of Dentistry, University of Manitoba, Winnipeg, Man., Canada

Key Words Xerostomia W Hyposalivation W Dry mouth W Residual volume W Salivary film W Evaporation W Mucosal fluid absorption W Flow rate W Palatal dryness

Abstract Xerostomia, the subjective sensation of dry mouth, occurs when the salivary flow rate is less than the rate of fluid loss from the mouth by evaporation and by absorption of water through the oral mucosa. Evaporation can only occur during mouth-breathing but could reach a maximum rate of about 0.21 ml/min at rest, although normally it would be much less. Water absorption through the mucosa can occur because saliva has one sixth the osmotic pressure of extracellular fluid, thus creating a water gradient across the mucosa. The maximum absorption rate is calculated to be about 0.19 ml/ min, declining to zero as the saliva reaches isotonicity. A recent study found the residual saliva volume, the volume of saliva left in the mouth after swallowing, to be 71% of normal in patients with severe hyposalivation and whose mouths felt very dry. Saliva in the residual volume is present as a thin film which varies in thickness with site. The hard palate has the thinnest film and when this is ! 10 Ìm thick, evaporation during mouth-breathing and/or fluid absorption may rapidly decrease it to zero, resulting in xerostomia. This is also generally asso-

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ciated with reduced secretion from the soft palate minor glands, which may contribute to the film on the hard palate. Thus, xerostomia appears to be due, not to a complete absence of oral fluid, but to localized areas of mucosal dryness, notably in the palate. Unstimulated salivary flow rates 1 0.1–0.3 ml/min may be necessary for this condition to be avoided. Copyright © 2004 S. Karger AG, Basel

Xerostomia and Hyposalivation

In deciding how much saliva is enough for avoidance of the sensation of dry mouth, it is important to distinguish between xerostomia and hyposalivation [Nederfors, 2000]. Xerostomia is the subjective sensation of dry mouth, while hyposalivation is the objective finding of a reduced salivary flow rate. Patients with low salivary flow may experience many problems which include: xerostomia; an increase in caries, often at sites not normally prone to caries, such as the incisal edges [Odlum, 1991]; reduced clearance of bacteria and food, leading to mucosal soreness, gingivitis, cheilitis, fissuring of the tongue and infection of the salivary ducts; recurrent yeast infections; difficulty in chewing, speaking and swallowing; increased frequency of calculus deposition in the salivary ducts; burning mouth, and difficulty in retention of dentures [Sreebny et al., 1992].

Dr. C. Dawes Department of Oral Biology, Faculty of Dentistry University of Manitoba, 780 Bannatyne Avenue Winnipeg, Man. R3E 0W2 (Canada) Tel. +1 204 789 3512, Fax +1 204 789 3943, E-Mail [email protected]

Several large studies [Dawes, 1987] have shown that the mean flow rate of unstimulated whole saliva is about 0.3 ml/min but with a remarkably large range, which was 0.008–1.85 ml/min in one study of 661 apparently healthy individuals who did not complain of xerostomia [Becks and Wainwright, 1943]. Clearly, the flow rate of saliva which is ‘enough’ varies considerably among individuals. However, Sreebny et al. [1992] and others regard an unstimulated salivary flow rate of !0.1 ml/min as evidence of hyposalivation. Food consumption normally stimulates salivary flow, and an inadequate flow during meals may make the swallowing of dry foods difficult. Sreebny et al. [1992] regard a stimulated flow rate of !0.5 ml/min to be evidence of hyposalivation in response to the chewing of paraffin wax.

Fluid Balance in the Mouth

Fluid enters the mouth from the ducts of the various salivary glands, while fluid loss may occur by swallowing, by evaporation or by absorption through the oral mucosa. Usually, the rate of fluid input exceeds the rate of fluid loss by evaporation or absorption through the oral mucosa, and the excess is periodically swallowed. The volume of saliva in the mouth varies from a mean of 1.07 ml (range 0.52–2.14 ml) prior to swallowing (Vmax) to a mean of 0.77 ml (range 0.38–1.73 ml) after swallowing [Lagerlöf and Dawes, 1984], which is the residual volume. When flow is unstimulated, the volume of saliva swallowed is about 0.3 ml with each swallow and if the unstimulated flow rate is 0.3 ml/min, the swallowing frequency will be about once per minute. The swallowing frequency is less during sleep [Lear et al., 1965], when the salivary flow rate is reduced, but whether the residual volume changes during sleep is unknown. If evaporation of saliva and absorption of fluid through the oral mucosa did not occur, it would be expected that anyone with even a low flow rate of saliva would not experience xerostomia, since there would be no reason why the residual volume would not remain constant. The individual could simply reduce the swallowing frequency to compensate for the reduced rate of input of saliva. However, if the rates of fluid loss by evaporation of saliva and absorption of fluid through the oral mucosa are greater than the unstimulated salivary flow rate, the residual volume would be expected to decrease, although it would be temporarily increased if flow were stimulated or if a drink were consumed.

How Much Saliva Is Enough for Avoidance of Xerostomia?

Rate of Water Evaporation from the Mouth

If the mouth remains closed during breathing so that no air passes through it, no evaporation of water from saliva will occur. However, with mouth-breathing, the rate of water evaporation from saliva will be influenced by the respiratory minute volume, the percentage of the inspired air which passes through the mouth, and the temperature and relative humidity of the inspired air. Proctor [1977] reported that air inspired through the nose becomes almost saturated with water vapour and reaches a temperature of 33 ° C during the few seconds that it takes to reach the nasopharynx. Even when the ambient temperature is –20 ° C, the inspired air is heated to about 27 ° C by the time it reaches the nasopharynx. Air which passes through the mouth would presumably also be heated similarly and be brought to a high relative humidity from the fluid in saliva by the time the air reaches the pharynx. According to Niinimaa et al. [1981], the proportion of the population who are mouth-breathers is 10–15%, and in such persons about 50% of the inspired air passes through the mouth. This is not unexpected, since the combined cross-sectional area of the nasal passages just posterior to the nostrils is only about 60 mm2 [Proctor, 1986] and this area would be equalled by a quite small opening of the mouth. During normal conversation, by nonmouth-breathers, more than half the inspired air passes through the mouth [Camner and Bakke, 1980]. Given that the respiratory minute volume at rest is about 6 l/min [Ganong, 1999] and that air at 33 ° C and 100% relative humidity contains 35.68 g of water/m3 [Weast and Astle, 1978–1979], the maximum rate of water evaporation from saliva into inspired air that was initially dry would be as high as 0.21 ml/min in complete mouth-breathers. Inspired air which already contained some moisture would, of course, pick up fluid at less than that rate. The involvement of saliva evaporation in xerostomia has received little attention, although it is well known that certain animals, such as dogs, use water evaporation from the tongue, in place of sweat, as a means of body temperature control. In winter, it is a common experience in cold climates, such as that of Winnipeg, that water drips from the nose as a result of condensation of water from expired air (which has 100% relative humidity at 37 ° C) as it is suddenly cooled at the nostrils. It seems possible that fluid may be supplied to the mouth by condensation of water on the lips from air which is expired orally when the ambient temperature is low. However, this does not seem

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to have been tested. Theoretically, if it were possible to cool a region of a denture or an appliance, condensation of water from air expired through the mouth would provide an inexhaustible source of fluid for the mouth. Unfortunately, the power requirements for such a cooling system would appear to make it impractical for clinical use.

Rate of Water Absorption through the Oral Mucosa

The permeability of the oral mucosa can be characterized by a permeability coefficient, Kp (cm/min), derived from the relationship: Kp = Q/[A(Co – Ci)t] [Siegel et al., 1981], where Q (mol) is the quantity of permeant crossing the epithelium, A (cm2) is the area of the tissue, Co and Ci (mol/l) are the concentrations of the permeant on the outside and inside of the mucosa and t is time (min). There are relatively few studies of the permeability to water of human oral mucosa, and autopsy specimens have been employed, with tritiated water as the permeant. The advantage of tritiated water is that its concentration on one side of the mucosa (Ci) can be maintained very low in comparison with that on the other side (Co). However, the Kp values for water are identical for transport in both directions across the oral mucosa, and there is no active transport. Thus, water can only diffuse across the oral mucosa if there is a concentration gradient. Lesch et al. [1989] reported that the Kp for water movement across the oral mucosa was about 0.97 ! 10 –4 cm/ min, but more recent values average about 4.8 ! 10 –4 cm/min [Healy et al., 2000; Howie et al., 2001] for different regions of the oral mucosa at 20 ° C. Unstimulated saliva has an osmotic pressure which is about one sixth of that of extracellular fluid (ECF). Thus, there is normally a concentration gradient for water to pass from saliva through the oral mucosa. However, if the saliva were not continuously replenished, the concentration of salivary electrolytes would increase with time, and the net rate of diffusion of water would fall exponentially and cease when the saliva was isotonic with ECF. For calculation of the maximum rate of water transfer across the oral mucosa, it is necessary to calculate the difference in the molar concentrations of water in saliva and in ECF. ECF is isotonic with 0.9% NaCl, whereas unstimulated whole saliva is isotonic with about 0.15% NaCl. Thus, the molarities of water in ECF and saliva must be about the same as those in the two salt solutions.

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The molecular weight of water is 18.0153 and the densities of 0.9 and 0.15% NaCl at 20 ° C are 1.0046 and 0.9993, respectively [Weast and Astle, 1980–1981]. A 0.9% NaCl solution contains 991 g of water in 1,000 g of solution, or 55.0088 mol of water/1,000 g. To convert the 1,000 g to millilitres, division by the density gives 995.421 ml. Thus, the molarity of the water in ECF = 55.0088 ! 1,000/995.421 = 55.2618 mol/l. A similar calculation for 0.15% NaCl yields a water molarity of 55.3863. Thus, the difference in water molarity between saliva and ECF is 0.1245 mol/l. Since the surface area of the oral mucosa averages 178 cm2 [Collins and Dawes, 1987], the maximum rate of water transfer from saliva across the oral mucosa will be: 4.8 ! 10 –4 cm/min ! 178 cm2 ! 0.124 mol/l = 0.0106 mol/min or 0.19 ml/min. In this calculation there are uncertainties in that the Kp value: (1) was derived at room temperature rather than mouth temperature, (2) is for tritiated water rather than H2O, (3) was measured on autopsy mucosa rather than on fresh mucosa and (4) is for ventral tongue mucosa rather than being a weighted average of all sites (Lesch et al. [1989] found site specificity in Kp values). A computer simulation, iterative at 1-second intervals, shows that with the normal residual volume, it would take !6 min to reach 95% of isotonicity if no further saliva entered the mouth. Although water would be expected to pass across the mucosa into the mouth when the mucosal epithelium is dry, this does not seem to occur, possibly because distortion of the epithelial cells by the drying obliterates the water channels normally present.

Saliva as a Thin Film

Collins and Dawes [1987] measured the surface area of the mouth and found it to be 214.7 B 12.9 cm2 (mean B SD, n = 20). From the values for the residual volume and Vmax determined by Lagerlöf and Dawes [1984], they calculated that if the saliva were evenly distributed throughout the mouth, it would be present as a thin film, varying from 72 to 100 Ìm in thickness after and before swallowing, respectively. This calculation assumes that opposing surfaces in the mouth, such as the palate and the dorsum of the tongue, are in contact with each other, except for the interposition of the fluid film. Subsequent investigators have usually measured the fluid thickness on individual surfaces not in contact with other surfaces, and thus the values which they obtain would be expected to aver-

Dawes

age, over the mouth as a whole, 36–50 Ìm, only half of those calculated by Collins and Dawes [1987]. Kleinberg and his colleagues have recently provided some very important data on the thickness of the salivary film at many different sites in the mouth. DiSabato-Mordarski and Kleinberg [1996a] found marked site-specific variation in the thickness of the surface fluid layer on the oral mucosa, with mean values ranging from 70 Ìm on the posterior dorsum of the tongue to 10 Ìm on the hard palate. The maximum film thicknesses in embrasures and occlusal fissures are larger than these values [DiSabatoMordarski and Kleinberg, 1996b], but the rate of fluid turnover in these sites is uncertain. Wolff and Kleinberg [1998] reported that in patients with or without a dry mouth and an unstimulated flow rate of 10.1 ml/min, the mean mucosal fluid thicknesses were 27.8 and 41.8 Ìm, respectively. In those with a dry mouth, the hard palate and the lips had the lowest fluid thicknesses, and values of at least 10 Ìm on the palate appeared necessary to avoid complaints of dry mouth. Wolff and Kleinberg [1999] induced dry mouth in healthy volunteers by pharmacological means and found that the mean onset of oral dryness occurred when the total salivary flow rate had been reduced to just less than 50% of normal, a value in accordance with the findings of Dawes [1987]. When the volunteers experienced dryness, the pattern of variation in fluid film thickness at different sites was the same as that at baseline, with all sites showing a reduced thickness. Again, the surface of the hard palate had the lowest fluid thickness with values !10 Ìm. Wolff and Kleinberg [1998, 1999] have emphasized the importance of dryness of the palatal mucosa for the sensation of oral dryness. The anterior part of the hard palate contains very few minor salivary glands, and a fluid coating is dependent on tongue movements for transfer of fluid from other regions of the mouth. Niedermeier et al. [2000] have summarized in English a great deal of their excellent work, originally published in German, which demonstrates the importance of the palatal mucous gland secretions for retention of the upper denture and that dry mouth and burning mouth syndrome are likely to occur when the flow rates from these glands are !6 Ìl/cm2 of palatal mucosa/min. In persons not wearing dentures, these secretions probably also contribute to the film of saliva covering the mucosa of the hard palate.

How Much Saliva Is Enough for Avoidance of Xerostomia?

Discussion

An important conclusion from the above analysis of fluid balance in the mouth is that commonly used ways of measuring the flow rate of whole saliva are not assessing the total fluid output by the different salivary glands. Rather they assess the net output of saliva after loss of fluid by evaporation and/or by mucosal absorption. That these latter two processes are clinically significant is suggested by the fact that patients with a low salivary flow rate usually experience a dry mouth, rather than maintaining a normal residual volume and swallowing less frequently. In a recent study by Dawes and Odlum [2004], the mean residual volume was found to be reduced by 29% in patients with severe hyposalivation and who stated that they had a very dry mouth. This, along with the studies of Kleinberg and colleagues, shows that such patients do certainly not have a complete lack of oral fluid. It emphasizes the probable importance of localized areas of dryness in the mouth, and especially on the hard palate, for the condition of xerostomia. Saliva production by the palatal minor salivary glands appears to be particularly important for providing an adherent fluid film on the hard palate. The latter site and anterior dorsum of the tongue are where xerostomia symptoms are most pronounced. If there the film thickness is !10 Ìm [Wolff and Kleinberg, 1999], the fluid will be very susceptible to removal by absorption and by evaporation during mouth-breathing as the palate and anterior dorsum of the tongue will be the main areas to receive initial contact with the inspired air. Non-pharmacological ways for the dry-mouth patient to reduce the severity of the condition include: keeping well hydrated to maintain maximum unstimulated salivary flow; avoiding mouth-breathing as a far as possible to reduce evaporation of saliva; using a humidifier to increase the relative humidity during the winter months, and especially in the bedroom, since mouth-breathing commonly occurs during sleep; avoiding tobacco, caffeine and alcohol to reduce their drying or diuretic effects; avoiding mouthwashes containing alcohol; using sugarfree chewing gum or candy to stimulate salivary flow, and using water or saliva substitutes. While several clinical trials suggest that pilocarpine, which stimulates salivary flow, is effective in relieving dry mouth in some patients [Brennan et al., 2002], this drug also has some undesirable side-effects. A recently developed preventive measure for patients about to receive radiation treatment for cancer of the pharynx or larynx is the transfer of one submandibular gland to the submental

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region, to shield it from the radiation beam [Jha et al., 2003]. So far, over 60 patients in Canada have successfully received this treatment, which maintains flow from at least one major salivary gland. Interestingly, the transferred gland appears to undergo hypertrophy, in a similar way to residual rat salivary glands after selective desalivation [Schwartz and Shaw, 1955]. In conclusion, saliva is probably ‘enough’ for avoidance of xerostomia if its flow rate exceeds the rate of fluid loss by mucosal absorption and evaporation. In practice, the unstimulated flow rate may need to be at least 0.1– 0.3 ml/min. Because of an increase in the survival rate for

patients with head and neck cancer and an increase in the elderly population, there is an increased need for further research on the alleviation of xerostomia and for treatment of the deleterious effects of this condition.

Acknowledgements I thank Dr. H.D. Gesser, University of Manitoba, and Dr. C.A. Squier, University of Iowa, for useful discussions on mucosal water absorption, and Dr. O. Odlum, University of Manitoba, for her clinical insights.

References Becks H, Wainwright WW: Human saliva. XIII. Rate of flow of resting saliva of healthy individuals. J Dent Res 1943;22:391–396. Brennan MT, Shariff G, Lockhart PB, Fox PC: Treatment of xerostomia: A systematic review of therapeutic trials. Dent Clin North Am 2002;46:847–856. Camner P, Bakke B: Nose or mouth breathing? Environ Res 1980;21:394–398. Collins LMC, Dawes C: The surface area of the adult human mouth and thickness of the salivary film covering the teeth and oral mucosa. J Dent Res 1987;66:1300–1302. Dawes C: Physiological factors affecting salivary flow rate, oral sugar clearance, and the sensation of dry mouth in man. J Dent Res 1987; 66(special issue):648–653. Dawes C, Odlum O: Salivary status in a treated head and neck cancer patient group. J Can Dent Assoc 2004, in press. DiSabato-Mordarski T, Kleinberg I: Measurement and comparison of the residual saliva on various oral mucosal and dentition surfaces in humans. Arch Oral Biol 1996a;41:655–665. DiSabato-Mordarski T, Kleinberg I: Use of a paper-strip absorption method to measure the depth and volume of saliva retained in embrasures and occlusal fossae of the human dentition. Arch Oral Biol 1996b;41:809–820. Ganong WF: Review of Medical Physiology, ed 19. Stamford, Appleton & Lange, 1999, p 620. Healy CM, Cruchley AT, Thornhill MH, Williams DM: The effect of sodium lauryl sulphate, triclosan and zinc on the permeability of normal oral mucosa. Oral Dis 2000;6:118–123.

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Howie NM, Trigkas TK, Cruchley AT, Wertz PW, Squier CA, Williams DM: Short-term exposure to alcohol increases the permeability of human oral mucosa. Oral Dis 2001;7:349–354. Jha N, Seikaly H, Harris J, Williams D, Liu R, McGaw T, Hofmann H, Robinson D, Hanson J, Barnaby P: Prevention of radiation induced xerostomia by surgical transfer of submandibular salivary gland into the submental space. Radiother Oncol 2003;66:283–289. Lagerlöf F, Dawes C: The volume of saliva in the mouth before and after swallowing. J Dent Res 1984;63:618–621. Lear CSC, Flanagan JB Jr, Moorrees CFA: The frequency of deglutition in man. Arch Oral Biol 1965;10:83–99. Lesch CA, Squier CA, Cruchley A, Williams DM, Speight P: The permeability of human oral mucosa and skin to water. J Dent Res 1989;68: 1345–1349. Nederfors T: Xerostomia and hyposalivation. Adv Dent Res 2000;14:48–56. Niedermeier W, Huber M, Fischer D, Beier K, Müller N, Schuler R, Brinninger A, Fartasch M, Diepgen T, Matthaeus C, Meyer C, Hector MP: Significance of saliva for the denturewearing population. Gerodontology 2000;17: 104–118. Niinimaa V, Cole P, Mintz S, Shephard RJ: Oronasal distribution of respiratory airflow. Respir Physiol 1981;43:69–75. Odlum O: Preventive resins in the management of radiation-induced xerostomia complications. J Esthet Dent 1991;3:227–229.

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Proctor DF: The upper airways. I. Nasal physiology and defense of the lungs. Am Rev Respir Dis 1977;115:97–129. Proctor DF: Form and function of the upper airways and larynx; in Fishman AP, Macklem PT, Mead J, Geiger SR (eds): Handbook of Physiology. Bethesda, American Physiological Society, 1986, section 3, vol III, pp 63–73. Schwartz A, Shaw JH: Studies on the effect of selective desalivation on the dental caries incidence of albino rats. J Dent Res 1955;34:239– 247. Siegel IA, Izutsu KT, Watson E: Mechanisms of non-electrolyte penetration across dog and rabbit oral mucosa in vitro. Arch Oral Biol 1981; 26:357–361. Sreebny LM, Banoczy J, Baum BJ, Edgar WM, Epstein JB, Fox PC, Larmas M: Saliva: Its role in health and disease. Int Dent J 1992;42:291– 304. Weast RC, Astle MJ: CRC Handbook of Chemistry and Physics, ed 59. West Palm Beach, CRC Press, 1978–1979, p E-41. Weast RC, Astle MJ: CRC Handbook of Chemistry and Physics, ed 61. Boca Raton, CRC Press, 1980–1981, p D-261. Wolff M, Kleinberg I: Oral mucosal wetness in hypo- and normosalivators. Arch Oral Biol 1998;43:455–462. Wolff M, Kleinberg I: The effect of ammonium glycopyrrolate (Robinul®)-induced xerostomia on oral mucosal wetness and flow of gingival crevicular fluid in humans. Arch Oral Biol 1999; 44:97–102.

Dawes

Caries Res 2004;38:241–246 DOI: 10.1159/000077761

Salivary Enhancement Therapies Philip C. Fox Department of Oral Medicine, Carolinas Medical Center, Charlotte, N.C., USA

Key Words Saliva W Secretagogues W Xerostomia W Caries W Sjögren’s syndrome W Radiotherapy

Abstract When salivary output is reduced chronically to a significant extent, there is a marked increase in dental caries. As the role of saliva in protection of the oral hard tissue is well recognized, there have long been efforts to enhance salivary function in conditions with associated secretory hypofunction. The rationale is that by stimulating salivary output, caries and other oral complications will be reduced or eliminated. The most widely used method for increasing salivary function is a combination of masticatory and gustatory stimulation. A large number of systemic agents have also been proposed as secretagogues, but only a few have shown consistent salivary enhancing properties in well-designed, controlled trials. Pilocarpine has been shown to improve symptoms of oral dryness and to increase salivary output in patients with Sjögren’s syndrome and postradiation xerostomia. Recently, cevimeline has shown significant salivary enhancement in Sjögren’s syndrome. Pilocarpine and cevimeline have a similar mechanism of action, side effect profile and duration of activity. No secretagogues have been linked directly in clinical trials to either caries prevention or a reduction in the existing caries rate of salivary dysfunction patients. Improved secretagogues are needed, with fewer side effects, increased duration of

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activity and greater potency. Future research directions include gene therapeutic approaches to direct salivary growth and differentiation or modify remaining tissues to promote secretion, creation of a biocompatible artificial salivary gland and salivary transplantation. With improved secretagogues, the effects of conditions that result in reduced salivary function and increased caries will be ameliorated. Copyright © 2004 S. Karger AG, Basel

Saliva is a major protective factor in the oral cavity, providing protection for oral hard and soft tissues and support for critical oral functions [Mandel, 1989]. Salivary gland performance may be affected by a number of conditions. Reductions in saliva output and symptoms of oral dryness are a result of many systemic diseases, medical interventions and hundreds of pharmaceuticals [Fox et al., 1985]. When salivary function is compromised, there is a significant increase in oral complications. Prominent among these is a marked increase in dental caries. The loss of the antimicrobial, buffering, remineralizing and cleansing properties of saliva when secretory output is reduced may lead to rapid and severe caries. Other complications of salivary hypofunction include an increase in oral infections (particularly fungal species), mucosal pain and friability, difficulties with chewing, swallowing and speaking as well as prominent complaints of oral dryness (xerostomia).

Philip C. Fox P.C. Fox Consulting LLC, 6509 Seven Locks Road Cabin John, MD 20818 (USA) Tel. +1 301 320 8200, Fax +1 301 320 3884 E-Mail [email protected]

Table 1. Salivary enhancement therapies

Local/topical secretagogues Gustatory stimulation Masticatory stimulation Oral rinses, gels, mouthwashes, artificial saliva Anhydrous crystalline maltose Acupuncture Systemic secretagogues Pilocarpine HCl Cevimeline HCl Interferon · Bromhexine Anethole trithione Traditional Asian mixtures Essential fatty acids LongoVital® Yohimbine Infliximab

In an effort to address these complications in individuals who have reduced secretory function and complaints of dryness, many approaches have been proposed to enhance salivary output [Fox, 1997]. The rationale is that by providing greater quantities of saliva and its natural protective factors, caries and other oral complications will be reduced or eliminated. While this is a logical assumption, there is actually little proof from clinical research. Salivary function can be enhanced significantly, in a transient manner, but there is an absence of research in humans demonstrating a concomitant reduction in dental caries or other oral complications associated with salivary hypofunction. The problem is not one of negative studies, but of a lack of any studies of secretagogues which have used caries as an outcome variable. In general, clinical trials of salivary enhancement therapies have utilized improvement in oral dryness complaints as the primary outcome measure. Secondary outcome variables are usually measures of symptomatic changes in oral functions, such as speaking and swallowing, or other oral symptoms. Clinical trials of secretagogues have not measured parameters such as caries. This is likely due to the relative ease with which xerostomia and other subjective criteria can be monitored, compared to the lengthy and intensive processes necessary to quantify caries. Ideally, clinical trials should monitor subjective (symptomatic) and objective improvement from treatments designed to enhance salivary output. Salivary enhancement therapies may be divided into local or topical approaches and systemic therapies (ta-

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ble 1). While there have been many agents and techniques proposed for this purpose [Grisius, 2001], the following discussion will be restricted to those interventions that have controlled clinical trials for review. Although even this group is large, there are few interventions that have been studied in an adequate number of subjects in welldesigned and appropriately controlled trials [Brennan et al., 2002].

Topical and Local Therapies

It is recognized that saliva output can be stimulated by oral activity. Chewing will result in a robust increase in saliva output. Salivation is also responsive to taste, particularly sour and bitter. The use of flavored gums and lozenges will increase secretory output and remains a mainstay of palliative therapy of xerostomia. The combination of gustatory and masticatory stimulation can transiently increase salivation and relieve symptoms of oral dryness. Patients with diminished salivation may be instructed to use sugar-free gums, lozenges, candies or mints for symptomatic relief of xerostomia. The use of sugarfree products must be stressed, as otherwise the addition of sugar bathing the dentition will only increase the caries risk and negate the benefit of increased salivary output. Although not strictly a ‘local’ therapy, acupuncture relies on application of the needles to specific locations, often in close proximity to the oral cavity. There have been a number of clinical studies of acupuncture to treat xerostomia associated with Sjögren’s syndrome, radiotherapy or nonspecific causes [Blom et al., 1992, 1996; List et al., 1998; Blom and Lundeberg, 2000]. Results have been generally favorable, with the authors reporting some benefit for relief of symptoms and improvement in salivary output. It is mainly stimulated salivary function which has been positively affected by the therapy. One problem with these studies is the difficulty in providing for appropriate placebo controls in clinical trials. An attempt at using superficial, non-site-specific acupuncture as a control found that the control group had similar improvements as the active acupuncture group. Other difficulties include a small sample size in the studies, a lack of double-blinding and the subjective nature of the reporting. Recent research has attempted to define a mechanism by which acupuncture might affect salivary function. One group has identified at least two neuropeptides (vasoactive intestinal peptide and calcitonin gene-related peptide) which are increased in saliva following acupuncture

Fox

treatments [Dawidson et al., 1998, 1999]. Since these can stimulate salivary function, it is possible that generation of increased amounts of neuropeptides could be responsible for any increase in salivation found. At present, acupuncture remains a possible approach to enhancing salivary function that requires further study. As one of the studies notes, acupuncture may serve as a ‘useful adjunct’ to management of the dry-mouth patient. A very large number of agents – e.g. artificial salivas, oral rinses and gels, flavored mouthwashes – have been proposed to treat dry mouth. All these topical therapies likely provide some degree of transient salivary stimulation. There are few well-designed and controlled clinical trials that have tested these in a formal manner. It appears that different palliative treatments are favored by patients primarily based on personal preference. There is the suggestion that mucin-containing products may meet with better patient acceptance [Gravenmade and Vissink, 1993]. Recent clinical trials have reported on the use of a lozenge composed of anhydrous crystalline maltose as a treatment for dry mouth in Sjögren’s syndrome. In uncontrolled studies, salivary function increased and dry-mouth complaints decreased over 24 weeks of treatment. Due to the design of the trial, it was shown that this benefit was not the result of direct gustatory or masticatory stimulation [Fox et al., 2001, 2002].

Systemic Therapies

There are many systemic agents that are capable of stimulating salivary output (table 1). The drug with the most extensive clinical evidence is pilocarpine HCl. Pilocarpine Pilocarpine is a parasympathomimetic agent with mild ß-adrenergic stimulating properties. It has been proposed as a treatment for dry mouth for over 100 years. A number of well-designed and well-controlled studies of substantial size have examined the affects of pilocarpine on dry mouth and salivary function in patients with Sjögren’s syndrome and postradiation salivary gland hypofunction [Fox et al., 1991; Johnson et al., 1993; LeVeque et al., 1993; Vivino et al., 1999; Horiot et al., 2000]. These clinical trials have consistently demonstrated that at doses of 5–10 mg 3 or 4 times daily, pilocarpine can significantly improve symptoms of dry mouth and increase salivary output.

Saliva Enhancement Therapies

Serious adverse events are rare with pilocarpine. While side effects such as sweating, flushing and urinary frequency are common, they are typically of mild or moderate intensity and of relatively short duration [Wiseman and Faulds, 1995]. Use of pilocarpine is contraindicated in patients with uncontrolled asthma, narrow-angle glaucoma or acute iritis. Caution is advised with use in patients with cardiovascular disease. Clinical trials have utilized symptoms of dry mouth as the primary outcome variable. Secondary variables included salivary output, other oral dryness symptoms and the patient’s perceptions of oral functioning. Salivary secretion is maximally stimulated approximately 1 h after dosing with pilocarpine, and increases over baseline salivary output are found for 3–4 h [Wiseman and Faulds, 1995]. No tolerance to the secretagogue effects of pilocarpine has been reported, nor has long-term improvement in baseline salivary function been found. Increased salivary output is transient, dose-related and consistent [Bell et al., 1999]. Caries or changes in oral flora have not been studied in pilocarpine trials. There is a need for longer-term human trials with pilocarpine which measure caries or cariogenic bacteria, in order to demonstrate that improvement in xerostomia and an increase in saliva lead to a reduction in caries. There are some animal data supporting this, but further studies are needed [Bowen et al., 1988]. Cevimeline Another parasympathomimetic agent, cevimeline HCl, has also been studied in large, well-controlled trials. At doses of 30 mg 3 times daily, cevimeline was shown to significantly improve symptoms of dry mouth and increase salivary output in patients with Sjögren’s syndrome [Petrone et al., 2002]. Cevimeline has a similar pharmacological profile to pilocarpine, although the onset of increased salivation may be somewhat later and the duration of action longer. The safety and adverse event profiles are very similar to pilocarpine as well, with sweating and nausea common complaints among patients. Cevimeline has been reported to have a high selective affinity for M3 subtype muscarinic receptors, the predominant receptor subtype in the salivary glands. This drug is currently being evaluated in clinical trials for use in postradiation xerostomia. Bromhexine Bromhexine has been proposed as a saliva enhancement therapy. However, there are no well-controlled studies which demonstrate that this agent will increase sali-

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vary output or improve dry-mouth symptoms [FrostLarsen et al., 1978; Manthorpe et al., 1981; Prause et al., 1984]. There may be some benefit for dry-eye symptoms in Sjögren’s syndrome [Tapper-Jones et al., 1980], but this has not been shown for the oral cavity. Anethole Trithione This agent has been demonstrated to increase salivation in individuals with mild dysfunction. Doses studied were 25 mg 3 times daily [Hamada et al., 1999]. In more severe cases of secretory hypofunction in Sjögren’s syndrome patients, anethole trithione was ineffective [Schiodt et al., 1986]. There was an interesting report suggesting a synergistic effect between anethole trithione and pilocarpine [Epstein and Schubert, 1987]. The mechanisms responsible for salivary stimulation may relate to upregulation of substance P and ·-calcitonin gene-related peptide by the drug [Nagano and Takeyama, 2001]. There are inadequate clinical trials of this agent. Yohimbine There are limited studies for dry-mouth therapy with this ·2-receptor antagonist. In a trial of dry mouth induced by antidepressants, significant secretagogue properties were found [Bagheri et al., 1997]. Further controlled clinical trials of sufficient size need to be conducted. Interferon · A number of large clinical trials have been reported using interferon · (IFN-·), as a high-dose injectable or a low-dose lozenge, for treatment of dry mouth and decreased salivation in Sjögren’s syndrome [Ferraccioli et al., 1996]. The injectable IFN-· is a recombinant protein, while the lozenge is a natural (cell line-derived) IFN-·. The low-dose lozenge formulation, at 150 IU 3 times a day, has been shown to reduce xerostomia and increase salivary output [Ship et al., 1999]. In one study in Sjögren’s syndrome patients, this preparation also improved minor salivary gland histopathology, reducing inflammatory infiltration and increasing normal-appearing acini, after 6 months of treatment [Shiozawa et al., 1998]. In a recent, large, well-controlled trial, unstimulated salivary function was reported to be significantly increased after 24 weeks of therapy with 150 IU lozenges 3 times daily, although complaints of oral dryness were not significantly improved [Cummins et al., 2003]. Side effects and adverse events were minimal. Further clinical trials will be necessary to define appropriate doses and to demonstrate fully the efficacy of this agent.

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Essential Fatty Acids Evening primrose oil and Á-linolenic acid have been investigated as a treatment for the dry mouth and eyes of Sjögren’s syndrome [Manthorpe et al., 1984]. While some improvements have been noted in ocular parameters, in a controlled trial, there was no significant benefit found versus a placebo control for oral or ocular signs and symptoms [Oxholm et al., 1986]. LongoVital ® LongoVital® is a herbal-based preparation with added vitamins. In a randomized, crossover trial of 40 Sjögren’s syndrome patients lasting 8 months, improvement was reported in salivary function during and following active treatment [Pedersen et al., 1999]. Certain inflammatory markers were also affected. A difficulty with this study is that it is unknown which of the multiple constituents of the preparation may be having an effect. As this is a single study, further clinical trials in larger numbers of subjects will be necessary before the efficacy of this agent is proven. Infliximab This biological agent is a tumor necrosis factor · blocker used in the treatment of rheumatoid arthritis. In preliminary studies in Sjögren’s syndrome, infliximab has shown significant benefit in a number of clinical and functional parameters, including increased salivary flow rate and improvement in symptoms of oral dryness [Steinfeld et al., 2001, 2002]. These results need to be replicated in larger studies. There is also concern about the risk of lymphoma with use of these agents, particularly in a condition such as Sjögren’s syndrome, where there is an underlying increased risk of this complication.

Future Directions for Saliva-Enhancing Therapies

There is a need for improved secretagogues that will have fewer side effects, an increased duration of activity and greater potency. Current therapies are restricted to agents which act primarily via the muscarinic receptor. Future drugs may be directed to other receptors on salivary cells. It is also possible that small-molecule drugs may be developed which target salivary receptors with greater specificity and consequently fewer adverse effects. Research should be directed towards targeting specific salivary glands and altering salivary composition to increase oral defenses. With better understanding of the

Fox

mechanisms of cell damage in systemic diseases such as Sjögren’s syndrome or following radiation therapy, new therapies will be devised directed at correction of underlying pathologies. These will likely have a beneficial effect on the salivary dysfunction accompanying these conditions. Novel approaches will have to be found for individuals with too little remaining salivary function to be helped by saliva-enhancing therapies. In these individuals, directed

salivary cell growth and repair may be possible, perhaps using gene therapeutic techniques. This will be feasible with improved knowledge of cell growth control. The goal would be natural repair of the salivary gland. There is also the possibility of salivary transplantation or use of a biocompatible artificial salivary gland. It is likely that a combination of these approaches will result in many more therapeutic options in the near future.

References Bagheri H, Schmitt L, Berlan M, Montastruc JL: A comparative study of the effects of yohimbine and anetholtrithione on salivary secretion in depressed patients treated with psychotropic drugs. Eur J Clin Pharmacol 1997;52:339– 342. Bell M, Askari A, Bookman A, Frydrych S, Lamont J, McComb J, Muscoplat C, Slomovic A: Sjögren’s syndrome: A critical review of clinical management. J Rheumatol 1999;26:2051– 2061. Blom M, Dawidson I, Angmar-Månsson B: The effect of acupuncture on salivary flow rates in patients with xerostomia. Oral Surg Oral Med Oral Pathol 1992;73:293–298. Blom M, Dawidson I, Fernberg JO, Johnson G, Angmar-Mansson B: Acupuncture treatment of patients with radiation-induced xerostomia. Eur J Cancer B Oral Oncol 1996;32B:182– 190. Blom M, Lundeberg T: Long-term follow-up of patients treated with acupuncture for xerostomia and the influence of additional treatment. Oral Dis 2000;6:15–24. Bowen WH, Pearson SK, Young DA: The effect of desalivation on coronal and root surface caries in rats. J Dent Res 1988;67:21–23. Brennan MT, Shariff G, Lockhart PB, Fox PC: Treatment of xerostomia: A systematic review of therapeutic trials. Dent Clin North Am 2002;46:847–856. Cummins MJ, Papas A, Kammer GM, Fox PC: Treatment of primary Sjögren’s syndrome with low-dose natural human interferon alpha administered by the oromucosal route: Combined phase III results. Arthritis Rheum 2003;49: 585–593. Dawidson I, Angmar-Månsson B, Blom M, Theodorsson E, Lundeberg T: Sensory stimulation (acupuncture) increases the release of vasoactive intestinal polypeptide in the saliva of xerostomia sufferers. Neuropeptides 1998;32: 543–548. Dawidson I, Angmar-Månsson B, Blom M, Theodorsson E, Lundeberg T: Sensory stimulation (acupuncture) increases the release of calcitonin gene-related peptide in the saliva of xerostomia sufferers. Neuropeptides 1999;33:244– 250.

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Epstein JB, Schubert MM: Synergistic effect of sialagogues in management of xerostomia after radiation therapy. Oral Surg Oral Med Oral Pathol 1987;64:179–182. Ferraccioli GF, Salaffi F, De Vita S, Casatta L, Avellini C, Carotti M, Beltrami CA, Cervini C, Bartoli E: Interferon alpha-2 (IFN alpha 2) increases lacrimal and salivary function in Sjögren’s syndrome patients: Preliminary results of an open pilot trial versus OH-chloroquine. Clin Exp Rheumatol 1996;14:367–371. Fox PC: Management of dry mouth. Dent Clin North Am 1997:41:863–875. Fox PC, Atkinson JC, Macynski AA, Wolff A, Kung DS, Valdez IH, Jackson W, Delapenha RA, Shiroky J, Baum BJ: Pilocarpine treatment of salivary gland hypofunction and dry mouth (xerostomia). Arch Intern Med 1991;151: 1149–1152. Fox PC, Cummins MJ, Cummins JM: Use of orally administered anhydrous crystalline maltose for relief of dry mouth. J Altern Complement Med 2001;7:33–43. Fox PC, Cummins MJ, Cummins JJ: A third study on the use of orally administered anhydrous crystalline maltose for relief of dry mouth in primary Sjögren’s syndrome. J Altern Complement Med 2002;8:651–659. Fox PC, van der Ven PF, Sonies BC, Weiffenbach JM, Baum BJ: Xerostomia: Evaluation of a symptom with increasing significance. J Am Dent Assoc 1985;110:519–525. Frost-Larsen K, Isager H, Manthorpe R: Sjögren’s syndrome treated with bromhexine: A randomised clinical study. Br Med J 1978;i:1579– 1581. Gravenmade EJ, Vissink A: Mucin-containing lozenges in the treatment of intraoral problems associated with Sjögren’s syndrome: A doubleblinded crossover study in 42 patients. Oral Surg Oral Med Oral Pathol 1993;75:466–471. Grisius MM: Salivary gland dysfunction: A review of systemic therapies. Oral Surg Oral Med Oral Pathol 2001;92:156–162. Hamada T, Nakane T, Kimura T, Arisawa K, Yoneda K, Yamamoto T, Osaki T: Treatment of xerostomia with the bile secretion-stimulating drug anethole trithione: A clinical trial. Am J Med Sci 1999;318:146–151.

Horiot JC, Lipinski F, Schraub S, Maulard-Durdux C, Bensadoun RJ, Ardiet JM, Bolla M, Coscas Y, Baillet F, Coche-Dequeant B, Urbajtel M, Montbarbon X, Bourdin S, Wibault M, Alfonsi M, Calais G, Desprez P, Pene F, Lapeyre M, Vinke J, Maral J: Post-radiation severe xerostomia relieved by pilocarpine: A prospective French cooperative study. Radiother Oncol 2000;55:233–239. Johnson JT, Ferretti GA, Nethery WJ, Valdez IH, Fox PC, Ng D, Muscoplat CC, Gallagher SC: Oral pilocarpine for post-irradiation xerostomia in patients with head and neck cancer. N Engl J Med 1993;329:390–395. LeVeque FG, Montgomery M, Potter D, Zimmer MB, Rieke JW, Steiger BW, Gallagher SC, Muscoplat CC: A multicenter, randomized, double-blind, placebo-controlled, dose-titration study of oral pilocarpine for treatment of radiation-induced xerostomia in head and neck cancer patients. J Clin Oncol 1993;11:1124– 1131. List T, Lundeberg T, Lundstrom I, Lindstrom F, Ravald N: The effect of acupuncture in the treatment of patients with primary Sjögren’s syndrome: A controlled study. Acta Odontol Scand 1998;56:95–99. Mandel ID: The role of saliva in maintaining oral homeostasis. J Am Dent Assoc 1989;119:298– 304. Manthorpe R, Frost-Larsen K, Hoj L, Isager H, Prause JU: Bromhexine treatment of Sjögren’s syndrome: Effect on lacrimal and salivary secretion, and on proteins in tear fluid and saliva. Scand J Rheumatol 1981;10:177–180. Manthorpe R, Hagen-Petersen S, Prause JU: Primary Sjögren’s syndrome treated with Efamol/ Efavit: A double-blinded cross-over investigation. Rheumatol Int 1984;4:165–167. Nagano T, Takeyama M: Enhancement of salivary secretion and neuropeptide (substance P, alpha-calcitonin gene-related peptide) levels in saliva by chronic anethole trithione treatment. J Pharm Pharmacol 2001;53:1697–1702. Oxholm P, Manthorpe R, Prause JU, Horrobin D: Patients with primary Sjögren’s syndrome treated for two months with evening primrose oil. Scand J Rheumatol 1986;15:103–108.

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Pedersen A, Gerner N, Palmvang I, Hoier-Madsen M: LongoVital in the treatment of Sjögren’s syndrome. Clin Exp Rheumatol 1999;17:533– 538. Petrone D, Condemi JJ, Fife R, Gluck O, Cohen S, Dalgin P: A double-blind, randomized, placebo-controlled study of cevimeline in Sjögren’s syndrome patients with xerostomia and keratoconjunctivitis sicca. Arthritis Rheum 2002;46: 748–754. Prause JU, Frost-Larsen K, Hoj L, Isager H, Manthorpe R: Lacrimal and salivary secretion in Sjögren’s syndrome: The effect of systemic treatment with bromhexine. Acta Ophthalmol 1984;62:489–497. Schiodt M, Oxholm P, Jacobsen A: Treatment of xerostomia in patients with primary Sjögren’s syndrome with sulfarlem. Scand J Rheumatol Suppl 1986;61:250–252.

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Shiozawa S, Tanaka Y, Shiozawa K: Single-blinded controlled trial of low-dose oral IFN-alpha for the treatment of xerostomia in patients with Sjögren’s syndrome. J Interferon Cytokine Res 1998;18:255–262. Ship JA, Fox PC, Michalek JE, Cummins MJ, Richards AB: Treatment of primary Sjögren’s syndrome with low-dose natural human interferon-alpha administered by the oral mucosal route: A phase II clinical trial J Interferon Cytokine Res 1999;19:943–951. Steinfeld SD, Demols P, Salmon I, Kiss R, Appelboom T: Infliximab in patients with primary Sjögren’s syndrome: A pilot study. Arthritis Rheum 2001;44:2371–2375. Steinfeld SD, Demols P, Appelboom T: Infliximab in primary Sjögren’s syndrome: One-year follow-up. Arthritis Rheum 2002;46:3301–3303.

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Tapper-Jones LM, Aldred MJ, Cadogan SJ, Walker DM, Dolby AE, Hopkins R, Nuki G, Beck L: Sjögren’s syndrome treated with bromhexine: A reassessment. Br Med J 1980;280:1356. Vivino FB, Al-Hashimi I, Khan Z, LeVeque FG, Salisbury PL 3rd, Tran-Johnson TK, Muscoplat CC, Trivedi M, Goldlust B, Gallagher SC: Pilocarpine tablets for the treatment of dry mouth and dry eye symptoms in patients with Sjögren syndrome: A randomized placebo-controlled fixed-dose multicenter trial. Arch Intern Med 1999;159:174–181. Wiseman LR, Faulds D: Oral pilocarpine: A review of its pharmacological properties and clinical potential in xerostomia. Drugs 1995;49:143– 155.

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Caries Res 2004;38:247–253 DOI: 10.1159/000077762

Salivary Proteins: Protective and Diagnostic Value in Cariology? A. van Nieuw Amerongen J.G.M. Bolscher E.C.I. Veerman Department of Oral Biochemistry, Academic Centre for Dentistry Amsterdam, Amsterdam, The Netherlands

Key Words Agglutinin W Antimicrobial peptides W Cathelicidin Histatins W Lactoferrin W Mucins W Saliva W Salivary proteins

W

Abstract Saliva is essential for a lifelong conservation of the dentition. Various functions of saliva are implicated in the maintenance of oral health and the protection of our teeth: (i) The tooth surface is continuously protected against wear by a film of salivary mucins and proline-rich glycoprotein. (ii) The early pellicle proteins, proline-rich proteins and statherin, promote remineralization of the enamel by attracting calcium ions. (iii) Demineralization is retarded by the pellicle proteins, in concert with calcium and phosphate ions in saliva and in the plaque fluid. (iv) Several salivary (glyco)proteins prevent the adherence of oral microorganisms to the enamel pellicle and inhibit their growth. (v) The salivary bicarbonate/carbonate buffer system is responsible for rapid neutralization of acids. An overview is presented on the major antimicrobial systems in human saliva. Not only the well-known major salivary glycoproteins, including mucins, proline-rich glycoprotein and immunoglobulins, but also a number of minor salivary (glyco)proteins, including agglutinin, lactoferrin, cystatins and lysozyme,

ABC

© 2004 S. Karger AG, Basel 0008–6568/04/0383–0247$21.00/0

Fax + 41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/cre

are involved in the first line of defense in the oral cavity. Besides, small cationic antimicrobial peptides, e.g. defensins, cathelicidin and the histatins, have come into focus. These are potentially suited as templates for the design of a new generation of antibiotics, since they kill a broad spectrum of microorganisms, while hardly evoking resistance, in contrast to the classical antibiotics. Copyright © 2004 S. Karger AG, Basel

Saliva contains a large number of proteins that participate in the protection of the oral tissues, for instance lysozyme, lactoferrin, lactoperoxidase, immunoglobulins, agglutinin and mucins [e.g. Nieuw Amerongen and Veerman, 2002]. In addition, several peptides with bacteriakilling activity have been identified. These include histatins, defensins and the only human cathelicidin, LL-37 (table 1). Because all these proteins and peptides have a broad spectrum of antimicrobial activity there seems to be a considerable overlap in functionality. This may account for the observation that susceptibility to oral diseases can apparently not be related to the concentration of a single component [Rudney et al., 1999]. The exact reason for this ‘redundancy’ is not really understood but different features may play a role. The oral cavity is the home of numerous different microorganisms, of which many still await identification

Dr. A. van Nieuw Amerongen Vrije Universiteit, Department of Oral Biochemistry Van der Boechorststraat 7, NL–1081 BT Amsterdam (The Netherlands) Tel. +31 20 4448675, Fax +31 20 4448685 E-Mail [email protected]

Table 1. Antimicrobial proteins in

glandular salivas

Salivary (glyco)protein

Tissue of origin

Relative %

MUC5B (mucin MG1) MUC7 (mucin MG2) Immunoglobulins Proline-rich glycoprotein Cystatins Histatins EP-GP (= GCDFP15, SABP, PIP) Agglutinin (= DMBT1, gp340) Lysozyme Lactoferrin Lactoperoxidase Cathelicidin (hCAP18, LL37) Defensins

all mucous salivary glands all mucous salivary glands B lymphocytes: in all salivary glands parotid submandibular 1 sublingual parotid and submandibular submandibular, sublingual parotid 1 submandibular 1 sublingual sublingual 1 submandibular, parotid all salivary glands: mucous 1 serous parotid 1 submandibular salivary glands, neutrophils salivary glands, epithelial cells, neutrophils

5–20 5–20 5–15 1–10 10 5 1–2 1–2 1–2 1–2 !1 !1 !1

and characterization. In addition, an unknown number of microorganisms are temporary guests that are transiently present. To cope with such a wide variety of potential invaders, the oral defense should be equipped with a diverse armament to prevent uncontrolled colonization by microorganisms. In this context it has to be noted that the conditions in the oral cavity for some defense systems are suboptimal. For instance, the microbicidal activity of cationic antimicrobial peptides like defensins, histatins and LL37 is known to be sensitive to the ionic environment as evidenced by a reduction in the presence of elevated salt concentrations or low concentrations of divalent cations. Each type of salivary gland secretes a characteristic spectrum of proteins. The complete arsenal of antimicrobial proteins present in whole saliva is thus the sum of contributions from different glands. As a consequence, the concentration of a single antimicrobial protein will vary over the day in accordance with the activity of its glandular source. Functional overlap in defensive systems means that no single component is necessary for the overall antimicrobial capacity of the salivary defense system. The salivary armory contains defensive components/ systems protecting specifically the dentition. Examples of such ‘tooth-specific’ systems are the carbonate/bicarbonate buffer system (for rapid neutralization of acids) and specific proteins that form a protective coating on the enamel surface, which serves as a barrier to prevent free diffusion of acid. In addition, generic protective systems are present, comprising antimicrobial proteins and peptides, that afford protection against microbial infections and are found in other protective secretions as well. With the exception of the immunoglobulins, antimicrobial

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components in saliva are not focused on elimination of specific (cariogenic) species, such as Streptococcus mutans (table 2). Rather they prevent massive overgrowth of microorganisms, and govern the establishment and maintenance of a stable ecosystem in which harmless species outnumber potentially dangerous species, thus forming a protection in its own right. In this paper different aspects of the antimicrobial action of a number of salivary protective systems will be discussed.

Protective Properties of the Major Salivary Proteins

The most important antimicrobial proteins in saliva, and their glandular source, are summarized in table 1. The salivary immunoglobulins belong primarily (185%) to the IgA subclass and to a lesser extent to the IgG subclass. Together they make up about 5–15% of total salivary proteins. Salivary IgA is synthesized by B lymphocytes located in the vicinity of secretory epithelia. After secretion in the interstitial fluid, it is taken up by acinar and ductal cells of the salivary gland and subsequently secreted into saliva. IgG in saliva mainly derives from crevicular fluid leaked into the oral cavity. Because of its highly specific binding characteristics, a single immunoglobulin idiotype binds and agglutinates just one or at best a few cross-reactive microbial species. However, the entire population of salivary immunoglobulins binds the majority of microorganisms present in saliva, thus presenting a broad-spectrum defense system. In contrast to immunoglobulins in serum, IgA in saliva does not function as an opsonizing agent, since under normal condi-

van Nieuw Amerongen/Bolscher/Veerman

Table 2. Salivary proteins: protective

properties

Salivary protein

Properties

Agglutinin Cathelicidin (LL37) Cystatins/VEGh Defensins EP-GP Histatins Immunoglobulins Lactoferrin Lactoperoxidase Lysozyme MUC5B (mucin MG1) MUC7 (mucin MG2) Proline-rich glycoprotein Proline-rich proteins (aPRPs) Proline-rich proteins (bPRPs) Statherin

aggregation of bacteria broad-spectrum killing of bacteria protease inhibitor broad-spectrum killing of bacteria unknown broad-spectrum killing of bacteria inactivation and aggregation of bacteria growth inhibition growth inhibition killing proton-diffusion barrier in pellicle aggregation unknown: aggregation? adherence unknown: membrane disturbing? adherence

tions no cytotoxic T cells are present in saliva. Also components of the complement system, which in serum cause direct killing of bacteria, are absent in saliva. Thus, the main functions of salivary immunoglobulins tentatively will be inhibition of bacterial adherence and colonization, e.g. by blocking surface structures involved in binding. Mucins constitute another important class of salivary glycoproteins. In unstimulated whole saliva they are the major components, making up 20–30% of the total protein. Two types of genetically different salivary mucins can be distinguished [Levine et al., 1987; Loomis et al., 1987]: MG1, high-molecular-weight mucin (Mr 10–30 MDa), encoded by the MUC5B gene, now designated MUC5B [Thornton et al., 1999], and the low-molecularweight MG2 (Mr F130 kDa), the translation product of the MUC7 gene, now designated MUC7 [Bobek et al., 1993]. Characteristic of mucins is the abundance of carbohydrate side chains which are covalently attached to the polypeptide backbones, forcing the molecule into an extended conformation. On a weight basis, the carbohydrates comprise 60% (for MUC7) to 80% (for MUC5B) of the molecule. The large dimensions and elongated form of MUC5B, in combination with the presence of a hydrophilic sugar coat, are responsible for the characteristic viscoelastic character of MUC5B-containing solutions [van der Reijden et al., 1993]. MUC5B is synthesized exclusively in mucous acinar cells of all (sero)mucous salivary glands [Nieuw Amerongen et al., 1995; Veerman et al., 2003]. MUC5B is a constituent of the protein layers that form on dental enamel after prolonged incubation with saliva, and is indispensable for the proton-barrier func-

tion of these so-called pellicles [Nieuw Amerongen et al., 1987]. Because of its hydrophilic properties, MUC5Bcontaining pellicles lubricate the dental surfaces, protecting them against mechanical wear. Despite its highly diverse population of oligosaccharides, which are potential receptors for bacterial adhesins, MUC5B binds to relatively few oral microorganisms, including Haemophilus parainfluenzae [Veerman et al., 1995] and Helicobacter pylori [Veerman et al., 1997a]. The low-molecular-weight mucin MUC7 differs from MUC5B in structure, localization and function. MUC7 is a single monomeric protein, decorated with short oligosaccharide side chains, which are two or three residues long. MUC7 is synthesized in serous acinar and demilune cells of the (sero)mucous glands [Veerman et al., 1997b, 2003] and is detectable in all (sero)mucous glandular salivas [Bolscher et al., 1999]. In contrast to MUC5B, MUC7 binds a wide variety of bacterial species, including S. mutans [Liu et al., 2000]. Both mucins have been implicated in the protection against viruses [Bergey et al., 1993a, b; Bolscher et al., 2002]. The proline-rich glycoprotein, only present in parotid saliva, makes up about 15–20% of all parotid proteins. In unstimulated saliva it is a minor component that increases with increasing stimulation of the parotid glands to about 10% in stimulated whole saliva. This small cationic glycoprotein (Mr 36 kDa) interacts particularly with Fusobacterium nucleatum and is involved in plaque formation [e.g. Kolenbrander and London, 1993].

Salivary Proteins in Cariology

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Fig. 1. Main functions of saliva in relation to its constituents.

Protective Properties of Minor Salivary Proteins

Besides the major proteins described above, which account for approximately 50% of its total protein, saliva contains a number of antimicrobial proteins that are present in lower concentrations (table 1). A number of these are enzymes which even in low concentration can exert significant biological activity. Examples of antimicrobial proteins with enzymic activity are lactoperoxidase and lysozyme. Lactoperoxidase catalyzes the oxidation of SCN – by hydrogen peroxide, resulting in the formation of OSCN – . Lysozyme (muramidase) is another example of an antimicrobial enzyme. By hydrolyzing cell wall polysaccharides, it makes bacteria more vulnerable to lysis due to e.g. hypo-osmotic conditions in saliva, or other antimicrobial components. Strikingly, after heat inactivation, lysozyme still exhibits bactericidal activity, probably through its cationic character. This suggests a two-step working mechanism involving initial enzymatic cleavage of the cell wall, followed by killing of the bacterium due to physical-chemical perturbation of the cell membrane by lysozyme itself, or by other antibacterial systems. Studies on the cooperative action of salivary defense systems under physiological conditions are scarce, but it is conceivable that the concerted action of proteins having different mechanisms of action enhances the power of the oral defense.

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Lactoferrin is an example of a nonenzymic antimicrobial protein. Its antimicrobial action is generally attributed to its iron-chelating property, which deprives microorganisms of this essential element. In addition, lactoferrin exhibits in vitro anti-inflammatory activities. Moreover, several domains are present within its polypeptide chain that exhibit antimicrobial activities. One of these is lactoferricin, an N-terminal peptide of 40 amino acid residues that is liberated upon combined pepsin and trypsin digestion. Lactoferricin is a cationic peptide which has a broadspectrum bactericidal activity [Groenink et al., 1999]. Another domain of lactoferrin has been implicated in the binding to salivary agglutinin, suggesting that both salivary proteins can act together [van der Kraan et al., 2004]. Salivary agglutinin was originally characterized as an S. mutans-agglutinating glycoprotein isolated from parotid saliva [Ericson and Rundegren, 1983; Lamont et al., 1991; Carlén and Olsson, 1995], but it is also present in submandibular and sublingual saliva [Ligtenberg et al., 2000; Bikker et al., 2002b]. It has now become clear that, besides S. mutans, a variety of other microbes are bound by agglutinin. The binding appears to be mediated by a relatively short peptide stretch, in the Scavenger Receptor Domains, which occur as tandemly repeating domains in agglutinin [Bikker et al., 2002a]. Besides being in saliva, agglutinin or closely related proteins have been detected in lung fluid, designated gp-340, and in brain, designated DMBT1 [Prakobphol et al., 2000; Ligtenberg et al., 2001].

van Nieuw Amerongen/Bolscher/Veerman

Protective Properties of Salivary Peptides

In saliva at least three types of antimicrobial peptides can be distinguished: histatins, defensins and hCAP18/ LL37, a human cathelicidin. Of these antimicrobial salivary peptides the histatins have attracted the most attention over the last decades. These antimicrobial peptides have a broad antimicrobial activity not only against bacteria, but also against yeasts. Such peptides can be used as templates to develop a new generation of antibiotics, because they work very rapidly and efficiently, while they are negligibly cytotoxic [Helmerhorst et al., 1999; van ‘t Hof et al., 2001] and do not evoke resistance. Years before the discovery of the magainins, it was reported that histidine-rich proteins in human saliva had killing activity against Candida albicans and S. mutans [MacKay et al., 1984; Pollock et al., 1984]. Since then most of the research on histatins has focussed on their fungicidal activity [Helmerhorst et al., 1997, 1999, 2001; Edgerton et al., 2000; Gyurko et al., 2001; Ruissen et al., 2001, 2003; Faber et al., 2003]. The histatins are synthesized in the parotid and submandibular glands, meaning that under both stimulated and nonstimulated saliva flow conditions, histatins will be secreted into saliva. The fungicidal, and to a lesser extent the bactericidal activity, of histatins is sensitive to ionic strength, diminishing with increasing salt concentrations [Helmerhorst et al., 1997]. The salivary glands contribute relatively little to the defensin population in saliva, which mostly derives from epithelial cells and neutrophils [Mathews et al., 1999]. Particularly during oral inflammations the expression of e.g. ß-defensin-2 is up-regulated [Abiko et al., 2002; Sawaki et al., 2002]. The same holds true for hCAP18/LL-37, derived from both neutrophils and the salivary glands [Murakami et al., 2002; Woo et al., 2003]. For hCAP18, the precursor of LL-37, no biological activity has been demonstrated thus far. Activation of hCAP18 results in the release of LL-37, consisting of the C-terminal 37 amino acids, which has broad-spectrum antimicrobial activity [Sörensen et al., 2001; den Hertog et al., 2004].

whole polypeptide chain of biologically active proteins but instead only peptides encompassing the functional domain. This opens new perspectives for the application of peptides as instruments to fight multiresistant microorganisms, or as additives in mouthrinses, to restore functionality in patients in whom the natural protection is compromised.

Potential Impact on Clinical Practice

New formulations containing antimicrobial peptides derived from natural salivary components have been tested for their applicability in the treatment of oral inflammatory processes such as gingivitis and periodontitis. An example of a potential clinically applicable antimicrobial peptide is IB-367, a protegrin-derived synthetic peptide with in vitro and in vivo antimicrobial activity against the microflora associated with oral mucositis [Mosca et al., 2000]. Protegrin is the porcine analogue of human cathelicidin. Another example is P113, an 11-amino acid fragment of histatin-3 [Rothstein et al., 2001]. Topical oral application of P113, or use of a mouthrinse containing P113, leads to significant reduction in experimental gingivitis [Paquette et al., 2002] without causing side effects. In addition, the nonhydrolyzable derivative P113D retains its killing activity on Pseudomonas aeruginosa in the presence of sputum having increased electrolyte concentrations from cystic fibrosis patients [Sajjan et al., 2001].

Future Perspectives for Research

Insight into the mechanism of action, in addition to knowledge of the structure-function relationship of antimicrobial proteins and peptides, makes it possible to design small, biologically active peptides that can be used as natural antimicrobials. In many cases it is not necessary to biosynthesize by recombinant techniques the

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Groenink J, Walgreen-Weterings E, van ’t Hof W, Veerman ECI, Nieuw Amerongen AV: Cationic amphipathic peptides, derived from bovine and human lactoferrins, with antimicrobial activity against oral pathogens. FEMS Microbiol Lett 1999;179:217–222. Gyurko C, Lendenmann U, Helmerhorst EJ, Troxler RF, Oppenheim FG: Killing of Candida albicans by histatin 5: Cellular uptake and energy requirement. Antonie Van Leeuwenhoek 2001;79:297–309. Helmerhorst EJ, van ’t Hof W, Breeuwer P, Veerman ECI, Abee T, Troxler RF, Nieuw Amerongen AV, Oppenheim FG: Characterization of histatin 5 with respect to amphipathicity, hydrophobicity, and effects on cell and mitochondrial membrane integrity excludes a candidacidal mechanism of pore formation. J Biol Chem 2001;276:5643–5649. Helmerhorst EJ, van ’t Hof W, Veerman ECI, Simoons-Smit AM, Nieuw Amerongen AV: Synthetic histatin analogs with broad-spectrum antimicrobial activity. Biochem J 1997;326: 39–45. Helmerhorst EJ, Reijnders IM, van ’t Hof W, Veerman ECI, Nieuw Amerongen AV: A critical comparison of the hemolytic and fungicidal activities of cationic antimicrobial peptides against Candida albicans cells and human erythrocytes. FEBS Lett 1999;449:105–110. den Hertog AL, Wong Fong Sang HW, Kraayenhof R, Bolscher JGM, van’t Hof W, Veerman ECI, Nieuw Amerongen AV: Interactions of histatin 5 and histatin 5-derived peptides with liposome membranes: surface effects, translocation and permeabilization. Biochem J 2004, in press. van ’t Hof W, Veerman ECI, Helmerhorst EJ, Nieuw Amerongen AV: Antimicrobial peptides: Properties and applicability. Biol Chem 2001;382:597–619. Kolenbrander PE, London J: Adhere today, here tomorrow: Oral bacterial adherence. J Bacteriol 1993;175:3247–3252. van der Kraan MIA, Groenink J, Nazmi K, Veerman ECI, Bolscher JGM, Nieuw Amerongen AV: Lactoferrampin: a novel antimicrobial peptide in the N1-domain of bovine lactoferrin. Peptides 2004, in press. Lamont RJ, Demuth DR, Davis CA, Malamud D, Rosan B: Salivary-agglutinin-mediated adherence of Streptococcus mutans to early plaque bacteria. Infect Immun 1991;59:3446–3450. Levine MJ, Reddy MS, Tabak LA, Loomis RE, Bergey EJ, Jones PC, Cohen RE, Stinson MW, Al-Hashimi I: Structural aspects of salivary glycoproteins. J Dent Res 1987;66:436–441. Ligtenberg TJM, Bikker FJ, Groenink J, Tornoe I, Leth-Larsen R, Veerman ECI, Nieuw Amerongen AV, Holmskov U: Human salivary agglutinin binds to lung surfactant protein-D and is identical with scavenger receptor protein gp340. Biochem J 2001;359:243–248. Ligtenberg AJM, Veerman ECI, Nieuw Amerongen AV: A role for Lewis a antigens on salivary agglutinin in binding to Streptococcus mutans. Antonie Van Leeuwenhoek 2000;77:21–30.

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Liu B, Rayment SA, Gyurko C, Oppenheim FG, Offner GD, Troxler RF: The recombinant Nterminal region of human salivary mucin MG2 (MUC7) contains a binding domain for oral Streptococci and exhibits candidacidal activity. Biochem J 2000;345:557–564. Loomis RE, Prakobphol A, Levine MJ, Reddy MS, Jones PC: Biochemical and biophysical comparison of two mucins from human submandibular-sublingual saliva. Arch Biochem Biophys 1987;258:452–464. MacKay BJ, Denepitiya L, Iacono VJ, Krost SB, Pollock JJ: Growth-inhibitory and bactericidal effects of human parotid salivary histidine-rich polypeptides on Streptococcus mutans. Infect Immun 1984;44:695–701. Mathews M, Jia HP, Guthmiller JM, Losh G, Graham S, Johnson GK, Tack BF, McCray PB: Production of ß-defensin antimicrobial peptides by the oral mucosa and salivary glands. Infect Immun 1999;67:2740–2745. Mosca DA, Hurst MA, So W, Viajar BSC, Fujii CA, Falla TJ: IB-367, a protegrin peptide with in vitro and in vivo activities against the microflora associated with oral mucositis. Antimicrob Agents Chemother 2000;44:1803–1808. Murakami M, Ohtake T, Dorschner RA, Gallo RL: Cathelicidin antimicrobial peptides are expressed in salivary glands and saliva. J Dent Res 2002;81:845–850. Nieuw Amerongen AV, Bolscher JGM, Veerman ECI: Salivary mucins: Protective functions in relation to their diversity. Glycobiology 1995; 5:733–740. Nieuw Amerongen AV, Oderkerk CH, Driessen AA: Role of mucins from human whole saliva in the protection of tooth enamel against demineralizatiuon in vitro. Caries Res 1987;21:297– 309. Nieuw Amerongen AV, Veerman ECI: Saliva – the defender of the oral cavity. Oral Dis 2002;8: 12–22. Paquette DW, Simpson DM, Friden P, Braman V, Williams RC: Safety and clinical effects of topical histatin gels in humans with experimental gingivitis. J Clin Periodontol 2002;29:1051– 1058. Pollock JJ, Denepitiya L, MacKay BJ, Iacono V: Fungistatic and fungicidal activity of human parotid salivary histidine-rich polypeptides on Candida albicans. Infect Immun 1984:44:702– 707. Prakobphol A, Xu F, Hoang VM, Larsson T, Bergstrom J, Johansson I, Frangsmyr L, Holmskov U, Leffler H, Nilsson C, Boren T, Wright JR, Stromberg N, Fisher SJ: Salivary agglutinin, which binds Streptococcus mutans and Helicobacter pylori, is the lung scavenger receptor cysteine-rich protein gp-340. J Biol Chem 2000; 275:39860–39866. van der Reijden WA, Veerman ECI, Nieuw Amerongen AV: Shear rate dependent viscoelastic behavior of human glandular salivas. Biorheology 1993;30:141–152.

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Rothstein DM, Spacciapoli P, Tran LT, Xu T, Roberts FD, Dalla Serra M, Buxton DK, Oppenheim FG, Friden P: Anticandida activity is retained in P-113, a 12-amino acid fragment of histatin 5. Antimicrob Agents Chemother 2001;45:1367–1373. Rudney JD, Hickey KL, Ji Z: Cumulative correlations of lysozyme, lactoferrin, peroxidase, SIgA, amylase, and total protein concentrations with adherence of oral viridans streptococci to microplates coated with human saliva. J Dent Res 1999;78:759–768. Ruissen ALA, Groenink J, Helmerhorst EJ, Walgreen-Weterings E, van ’t Hof W, Veerman ECI, Nieuw Amerongen AV: Effects of histatin 5 and derived peptides on Candida albicans. Biochem J 2001;356:361–368. Ruissen ALA, Groenink J, Krijtenberg P, Walgreen-Weterings E, van ’t Hof W, Veerman ECI, Nieuw Amerongen AV: Internalisation and degradation of histatin 5 by Candida albicans. Biol Chem 2003;384:183–190.

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Sajjan US, Tran LT, Sole N, Rovaldi C, Akiyama A, Friden PM, Forstner JF, Rothstein DM: P113D, an antimicrobial peptide active against Pseudomonas aeruginosa, retains activity in the presence of sputum from cystic fibrosis patients. Antimicrob Agents Chemother 2001; 45:3437–3444. Sawaki K, Mizukawa N, Yamaai T, Fukunaga J, Sugahara T: Immunohistochemical study on expression of ·-defensin and ß-defensin-2 in human buccal epithelia with candidiasis. Oral Dis 2002;8:37–41. Sörensen OE, Follin P, Johnson AH, Calafat J, Tjabringa GS, Hiemstra PS, Borregaard N: Human cathelicidin, hCAP18, is processed to the antimicrobial peptide LL37 by extracellular cleavage with proteinase 3. Blood 2001;97: 3951–3959. Thornton DJ, Khan N, Mehrotra R, Howard M, Veerman E, Packer NH, Sheehan JK: Salivary mucin MG1 is comprised almost entirely of different glycosylated forms of the MUC5B gene product. Glycobiology 1999;9:293–302. Veerman ECI, Bank CMC, Namavar F, Appelmelk BJ, Bolscher JGM, Nieuw Amerongen AV: Sulfated glycans on oral mucin as receptors for Helicobacter pylori. Glycobiology 1997a;7: 737–743.

Veerman ECI, Bolscher JGM, Appelmelk BJ, Bloemena E, van den Berg TK, Nieuw Amerongen AV: A monoclonal antibody directed against high Mr salivary mucins recognizes the SO33Galß1-3GlcNAc moiety of sulfo-Lewisa: A histochemical survey of human and rat tissue. Glycobiology 1997b;7:37–43. Veerman ECI, van den Keijbus PAM, Nazmi K, Vos W, van der Wal JE, Bloemena E, Bolscher JGM, Nieuw Amerongen AV: Distinct localization of MUC5B glycoforms in the human salivary glands. Glycobiology 2003;13:363– 366. Veerman ECI, Ligtenberg AJM, Schenkels LCPM, Walgreen-Weterings E, Nieuw Amerongen AV: Binding of human high-molecular-weight salivary mucins (MG1) to Hemophilus parainfluenzae. J Dent Res 1995;74:351–357. Woo JS, Jeong JY, Hwang YJ, Chae SW, Hwang SJ, Lee HM: Expression of cathelicidin in human salivary glands. Arch Otolaryngol Head Neck Surg 2003;129:211–214.

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Caries Res 2004;38:254–257 DOI: 10.1159/000077763

Fluorides in Caries Prevention and Control: Empiricism or Science J.M. ten Cate Department of Cariology/Endodontology/Pedodontology, Academic Centre for Dentistry Amsterdam (ACTA), Amsterdam, The Netherlands

Key Words Evidence-based dentistry W Fluoride W History of caries research W Remineralization W Review

tinue to be directed at improving our understanding of fluoride, in particular on topics where success so far has failed. Copyright © 2004 S. Karger AG, Basel

Abstract The caries-preventive effects of fluoride are beyond any reasonable doubt! Inclusion of fluoride use in caries prevention protocols has resulted in significant reduction in caries prevalence in the majority of the population. Nevertheless, even in low-caries prevalence populations up to 20% of individuals may suffer to an unacceptable degree from caries. In the history of caries research various phases can be discerned. Starting with the initial – laboratory – studies to reveal the mode of action of fluoride, attention later shifted to intra-oral studies and in situ product testing. Currently much emphasis is given to evidence-based dentistry and guidelines for clinical practice, which trend has also focussed the research on fluoride and caries. While on some topics, such as the efficacy of fluoride toothpastes, evidence is convincing, additional research is indicated to resolve remaining questions. One such question is that of high-prevalence individuals for which a comprehensive research programme focussing both on caries aetiological and behavioural aspects should be further developed. Efforts should con-

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Accessible online at: www.karger.com/cre

In recent years, more emphasis is given to evidencebased dentistry, both in the dental curricula and in guidelines for clinical practice. This implies that data generated by the research community are re-evaluated by expert committees, subjected to systematic reviews or become part of large-scale meta-analysis. The outcomes have been consensus reports, clinical guidelines and other official documents. The aim of these activities is to improve dissemination of (reliable) information from research to dental practice, to encourage focussing the research questions and improving the quality of research. In reports prepared by expert groups data are often ranked by the level of evidence. This ranges from strong scientific support for a preventive strategy or product to limited scientific support. A more specific approach is to rank recommendations from evidence from meta-analysis of randomized controlled trials (‘hard’ data) to evidence obtained from expert opinions (considered ‘softer’ data). The need for consensus information is currently widely expressed [Newbrun, 2001; Reich, 2001]. Although meta-analyses

Prof. J.M. ten Cate Department of Cariology/Endodontology/Pedodontology Academic Centre for Dentistry Amsterdam (ACTA), Louwesweg 1 NL–1066 EA Amsterdam (The Netherlands) Tel. +31 20 5188 440, Fax +31 20 669 2881, E-Mail [email protected]

and Cochrane type reviews are needed and provide useful information, they also pose new problems. Scrutinizing previously published studies is not a process free from value judgement [Volpe et al., 1993]. The choice of selection criteria is often subjective, not to mention the political impact of a statement that 90% of the published work in a field does not stand up to current criteria for good research. In addition, the methodologies of (clinical) research develop and the world in which these studies are performed changes continuously [e.g. Schuller and Kalsbeek, 2003]. As an example: what is the value of a clinical study on a fluoride product performed in a period with a higher caries prevalence, or even when performed today in a population with a different caries prevalence or distribution? Can we extrapolate findings from studies on 12to 15-year-olds to other age groups? What does a metaanalysis on, for instance, fluoride applications mean in terms of fluoride efficacy for adults, elderly or special groups? Without question there is convincing evidence for a general efficacy of fluorides in caries prevention. Fluoride as a caries-preventive agent was discovered as the side effect of fluorosis in teeth in areas with elevated levels of fluoride in the drinking water [Beltran-Aguilar et al., 2002]. At the time it was difficult to determine small (subppm) concentrations of fluoride in drinking water. Nevertheless, the early studies on fluoridation of the drinking water were convincing and initiatives were taken to add various types of fluorides to other oral hygiene products. Generally, fluoridation of the drinking water and of toothpastes is now considered the preferred mode of fluoride administration, appraised by the highest level of evidence [e.g. SBU, 2002]. Likewise, there is overall consensus on the primary mode of action of fluoride. Fluoride can be incorporated into the tissue mineral hydroxyapatite (HAP). Due to the lower solubility of F-HAP, dissolution is reduced in solutions containing its common ions (Ca, phosphate, F), while precipitation is enhanced. These thermodynamically driven reactions are – for dental researchers – translated as ‘fluoride inhibits demineralization and enhances remineralization’, a sentence found in all modern textbooks of cariology [e.g. ten Cate et al., 2003]. Science more than empiricism has led to the evidence generated on this topic. Other modes of action that are often quoted, like the effects of fluoride on bacteria growth and metabolism, have been demonstrated in carefully designed studies, but their role in and contribution to caries prevention in the real-life ‘experiment’ are questionable. The literature on the above-mentioned processes is extensive. In the Pubmed database of the Nation-

al Library of Medicine, a search of the combination of fluoride and enamel/dentine solubility or de-/remineralization gives over 3,000 ‘hits’. Is there equally convincing evidence for the methods of fluoride supplementation? On this topic and that of fluoride formulation, numerous issues have been discussed in the past decades. Sodium fluoride is often quoted as being more effective than other types of fluoride, e.g. monofluorophosphate [Stookey et al., 1993]. Even though this conclusion was challenged [Volpe et al., 1993], most modern toothpastes contain sodium fluoride, unless other fluorides are used for economic reasons (sodium monofluorophosphate with chalk abrasive) or to provide additional antimicrobial benefits (e.g. amine and stannous fluorides). The small differences in efficacy of the fluoride actives are probably outweighed by variables like frequency of use and intra-oral retention of the ‘active’ component [Chesters et al., 1992; O’Mullane et al., 1997]. Various types of vehicle for fluoride have been proposed. First, there are products used by dental professionals, e.g. high fluoride varnishes, lacquers, rinses, foams. For some of these, Cochrane reviews or meta-analyses have been published [Marinho et al., 2002a, b; van Rijkom et al., 1998]. Such reviews have also been published for fluoride toothpastes [Marinho et al., 2003]. Second, fluoride is added to frequently consumed consumables like salt, sugar or milk [Banoczy et al., 1997; Gyurkovics et al., 1992; Marthaler, 2002]. Many of these initiatives are taken to broaden fluoride use and increase compliance to using fluoride products. Oral hygiene products, notably fluoride toothpastes, rinses, fluoridated toothpicks and floss, have, in total, the largest market, but are only adequately used by dentally motivated individuals. Expert opinion has qualified various treatments (for instance fluoride tablets) as being supported by limited scientific evidence and raised doubts on the additional benefits when they are used in conjunction with fluoridated drinking water or toothpaste [SBU, 2002; SIGN, 2000]. Often, new products are put on the market without a scientific rationale or clinical data. While in modern prevention we wish to formulate our advice on solid clinical evidence, obtaining such evidence has become increasingly difficult. The turnover time for oral care and restorative products no longer seems to allow for the typical 3-year clinical study, which in turn has been so costly that producers have sought alternative methods of evaluation. These range from more sensitive systems of caries detection with the desired shortened time of clinical experimentation, to advanced laboratory or in situ trials [e.g. Zero, 1995]. However, careful scruti-

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nizing of the latter has increased the number of conditions to be fulfilled (e.g. periodic validation of the model, inclusion of various (gold) standard, number of panellists), such that the advantages have partly evaporated [Zero, 1995]. Studies on fluoride efficacy have gone through various phases in the past decades (fig. 1). After the first discovery of fluoride as a caries-preventive agent, efforts were aimed at unravelling its mode of action. From this period date many publications on in vitro findings and their validation in in situ models. In addition, models were developed to test fluoride-containing products prior to fullscale clinical testing. The next wave of reports deals with drawing conclusions from the previous work in terms of clinical guidelines, evidence-based dentistry, etc. Obviously the ultimate aim of all this work was to improve the oral health of the population. Although the use of fluoride has caused a substantial decrease in caries prevalence, many patients still suffer from dental caries and current attention has therefore focussed on the group of high-risk individuals. It is not clear whether this group would benefit from new products with increased efficacy, as lack of compliance to proven methods of caries prevention, often as simple as twicedaily tooth brushing, makes this group not likely users of new products. Even products that do not require daily attention to hygiene measures (e.g. slow release fluoride devices) depend on periodic consultation with dental professionals. An intensive prevention programme to highrisk individuals failed as caries incidence in the special attention group and in a corresponding control group was not significantly different [Hausen et al., 2000]. If we confront our knowledge on the aetiology and pathogenesis of dental caries with the current caries prevalence data, several questions emerge. In my view the most prominent one is how much additional success in reducing the overall caries prevalence could be attained by increasing our knowledge of the pathophysiological aspects of caries. Alternatively, should we turn our attention more to behavioural aspects? A detailed analysis of the characteristics of high-risk groups could provide the basis for answering this dilemma. Translating observations on high-risk groups into mode of action studies and designing appropriate models could be a next research aim. In recent years our research group has tried to better understand caries at plaque retention sites with limited access to saliva and fluoride. It was shown that fluoride efficacy at such sites was lower than at freely accessible open enamel or dentin surfaces that are commonly used in laboratory and in situ studies [Lagerweij et al., 1996].

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1930–1960

Evolution of fluoride research

Epidemiology artificial fluoridation of tap water fluoride addition to toothpastes

Improved oral health (but also health inequalities)

1960–1980 mode of action

Changed patient behaviour

1980–1990 In situ studies clinical studies

1990–

Meta-analysis

Guidelines for clinical practice 2000– Evidence-based dentistry

Acceptance issues political, commercial

Curriculum

Fig. 1. Developments in fluoride research showing how the focus in research has shifted in time. The current dichotomous nature of caries prevalence in society has resulted in new research questions aimed at understanding specific caries susceptibility issues in the high-prevalence (risk) group.

More specifically on fluoride-related caries-preventive products and protocols many topics should be addressed, such as: The dose-response question of fluoride in toothpastes regarding clinical efficacy is still far from being answered unequivocally. Few and rather old studies have addressed the concentration range 0–1,000 ppm F, which is now under focus for products to be used by small children [Ammari et al., 2003]. Similarly, more studies are indicated in the concentration range above 1,500 ppm, where again scarce and conflicting data are available. In conclusion, caries research has gone a long way and major successes have been made. However, caries is still far from having become extinct and the remaining tasks are difficult but challenging.

ten Cate

References Ammari AB, Bloch-Zupan A, Ashley PF: Systematic review of studies comparing the anti-caries efficacy of children’s toothpaste containing 600 ppm of fluoride or less with high fluoride toothpastes of 1,000 ppm or above. Caries Res 2003; 37:85–92. Banoczy J, Fazekas A, Mari A, Pinter A, Szabo J, Szoke J: Recommendation of the introduction of salt fluoridation for caries prevention in Hungary. Fogorv Sz 1997;90:351–358. Beltran-Aguilar ED, Griffin SO, Lockwood SA: Prevalence and trends in enamel fluorosis in the United States from the 1930s to the 1980s. J Am Dent Assoc 2002;133:157–165. ten Cate JM, Larsen MJ, Pearc EIF, Fejerskov O: Chemical interactions between the tooth and oral fluids; in Fejerskov O, Kidd EAM (eds): Dental Caries: The Disease and Its Clinical Management. Munksgaard, Blackwell, 2003, pp 49–69. Chesters RK, Huntington E, Burchell CK, Stephen KW: Effect of oral care habits on caries in adolescents. Caries Res 1992;26:299–304. Gyurkovics C, Zimmermann P, Hadas E, Banoczy J: Effect of fluoridated milk on caries: 10-year results. J Clin Dent 1992;3:121–124. Hausen H, Karkkainen S, Seppä L: Application of the high-risk strategy to control dental caries. Community Dent Oral Epidemiol 2000;28:26– 34.

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Lagerweij MD, Damen JJ, ten Cate JM: The effect of a fluoridated toothpaste on dentinal lesions in plaque-filled grooves: An intra-oral crossover study J Dent Res 1996;75:1687–1691. Marinho VC, Higgins JP, Logan S, Sheiham A: Fluoride varnishes for preventing dental caries in children and adolescents. Cochrane Database Syst Rev 2002a;CD002279. Marinho VC, Higgins JP, Logan S, Sheiham A: Fluoride gels for preventing dental caries in children and adolescents. Cochrane Database Syst Rev 2002b;CD002280. Marinho VC, Higgins JP, Sheiham A, Logan S: Fluoride toothpastes for preventing dental caries in children and adolescents. Cochrane Database Syst Rev 2003;CD002278. Marthaler TM: Dentistry between pathology and cosmetics. Community Dent Oral Epidemiol 2002;30:3–15. Newbrun E: Topical fluorides in caries prevention and management: A North American perspective. J Dent Educ 2001;65:1078–1083. O’Mullane DM, Kavanagh D, Ellwood RP, Chesters RK, Schafer F, Huntington E, Jones PR: A three-year clinical trial of a combination of trimetaphosphate and sodium fluoride in silica toothpastes. J Dent Res 1997;76:1776–1781. Reich E: How to measure the effects of fluoride treatments in clinical trials? The role of caries prevalence and caries assessment. Caries Res 2001;35(suppl 1):34–39.

van Rijkom HM, Truin GJ, van’t Hof MA: A metaanalysis of clinical studies on the caries-inhibiting effect of fluoride gel treatment. Caries Res 1998;32:83–92. SBU: Prevention of Dental Caries: A Systematic Review. Rep No 161. Swedish Council on Technology Assessment in Health Care, 2002, pp 1–26. Schuller AA, Kalsbeek H: Effect of the routine professional application of topical fluoride on caries and treatment experience in adolescents of low socio-economic status in the Netherlands. Caries Res 2003;37:172–177. SIGN, Scottish Intercollegiate Guidelines Network: Preventing dental caries in children at high caries risk. SIGN Publication 47, 2000. Stookey GK, DePaola PF, Featherstone JD, Fejerskov O, Moller IJ, Rotberg S, Stephen KW, Wefel JS: A critical review of the relative anticaries efficacy of sodium fluoride and sodium monofluorophosphate dentifrices. Caries Res 1993;27:337–360. Volpe AR, Petrone ME, Davies RM: A critical review of the 10 pivotal caries clinical studies used in a recent meta-analysis comparing the anticaries efficacy of sodium fluoride and sodium monofluorophosphate dentifrices. Am J Dent 1993;6:S13–S42. Zero DT: In situ caries models. Adv Dent Res 1995;9:214–230.

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Caries Res 2004;38:258–262 DOI: 10.1159/000077764

Systemic versus Topical Fluoride ´ .M. Lennon E. Hellwig A a Department of Operative Dentistry and Periodontology, Dental Clinic and Dental School, Albert Ludwigs University Freiburg, Freiburg, and b Department of Operative Dentistry, Preventive Dentistry and Periodontology, University of Göttingen, Göttingen, Germany

Key Words Caries prevention W Posteruptive fluoride W Pre-eruptive fluoride W Systemic fluoride W Topical fluoride

Abstract The actual mechanism of fluoride action is still a subject of debate. A dogma has existed for many decades, that fluoride has to be ingested and acts mainly pre-eruptively. However, recent studies concerning the systemic effect of fluoride supplementation concluded that the caries-preventive effect of fluoride is almost exclusively posteruptive. Moreover, epidemiologists have cast doubt on the validity of the ‘old’ studies dealing with fluoride use. The concept of the posteruptive fluoride effect is supported by in vitro and in situ investigations demonstrating that the mode of action of fluoride can be attributed mainly to its influence on de- and remineralization kinetics of dental hard tissues. Therefore, topical fluoride application (e.g. in the form of fluoridated dentifrices) should be encouraged. There are still important questions open that need to be answered despite existing knowledge about the caries-preventive effect of fluoride. Copyright © 2004 S. Karger AG, Basel

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Existing Information

Fluoride is still the cornerstone of modern non-invasive dental caries management. However, the actual mechanism of fluoride action remains the subject of debate. The belief that fluoride has to be ingested and acts preventively by becoming incorporated into tooth mineral during its development originated from the early studies of Dean et al. [1942] and McKay [1952]. At this time many clinical trials were designed to prove the pre-eruptive (systemic) mode of action of fluoride. It could be demonstrated that the prevalence of overt carious lesions in the permanent as well as in the primary dentition was lower in residents from areas with fluoridated drinking water compared to those living in non-fluoridated areas [Backer Dirks et al., 1978; Thylstrup et al., 1982; Newbrun, 1989; Ripa, 1993]. Additionally, laboratory analyses revealed that fluoride concentration in surface enamel was higher in teeth that developed under the influence of water fluoridation [Chan et al., 1989; Takeuchi et al., 1996]. It was also found that the prenatal administration of fluoride supplements could reduce caries prevalence in deciduous teeth [Glenn et al., 1982]. As early as 1955, Bibby et al. compared the caries-preventive efficacy of fluoride lozenges with fluoride pills in a group of 5- to 14year-old children. While the lozenges were sucked, the coated pills were swallowed before any of the contained fluoride could come into contact with the teeth. They

E. Hellwig Department of Operative Dentistry and Periodontology University Clinic of Dentistry, Albert Ludwigs University Freiburg Hugstetter Strasse 55, DE–79106 Freiburg (Germany) Tel. +49 761 270 4950, Fax +49 761 270 4762, E-Mail [email protected]

were able to demonstrate that in the group using the lozenges fewer carious lesions developed compared to the group using the pills. They concluded that the caries reduction produced by such lozenges was the result of fluoride acting on the external surfaces of the teeth. Lemke et al. [1970] investigated the dental effects of discontinuation of controlled water fluoridation in Antigo (Wisconsin). They came to the conclusion that the (caries) inhibiting effect tends to persist as long as fluoride exposure is continued, but tends to be gradually lost after fluoride exposure is discontinued. They suggested that periodic or continuous renewal of the fluoride content of tooth enamel is required to maintain the maximum caries-inhibiting effect. However, these early indications of the posteruptive effect of fluoride were neglected and the dogma of the pre-eruptive mode of action of fluoride remained the basis for fluoride research. In this context, LeGeros et al. [1985] performed physicochemical investigations of enamel from deciduous teeth of a small number of children with and without prenatal fluoride supplementation. They found that enamel from children who were subjected to prenatal fluoridation exhibited more homogeneous and less extensive patterns of acid etching, denser crystal populations in intraprismatic regions, larger prism dimensions, greater total mineral density, a higher degree of crystallinity, smaller a-axis dimensions, more fluoride and less carbonate contents. These findings are always cited as evidence for the importance of systemic fluoridation, although they have not been verified since, particularly not for permanent teeth.

By the 1970s and 1980s, some doubts had emerged regarding the exclusively pre-eruptive effect of fluoride. Primary teeth were protected against caries even though prenatal incorporation of fluoride into unerupted teeth was insignificant. Additionally, a randomized, doubleblind, longitudinal study testing the caries-preventing efficacy of prenatal fluoride supplementation in children followed until age 5 failed to support the hypothesis that prenatal fluoride has a strong caries-preventive effect [Leverett et al., 1997]. Hellwig and Klimek [1985] found that children 12.5–16 years old who had been exposed all their life to naturally fluoridated water exhibited significantly fewer carious lesions compared to a control group. However, they also found that even children who consumed fluoridated water for only for 2 years showed a distinctly decreased DMFT score compared to the control children (fig. 1). Künzel and Fischer [1997] analyzed the rise and fall of caries prevalence in two German towns and its relationship to changing drinking water F concentrations. During the first three decades of the study the caries prevalence correlated strictly with the F concentration in the drinking water. Water fluoridation was followed by a caries decline, while interruptions in fluoridation were followed by increasing caries levels. However, since 1987 a significant caries decline occurred despite the fact that only poor water fluoridation was available. They concluded that one of the reasons might be the broader availability of other fluoride-containing products compensating for water fluoridation, e.g. F dentifrices. A similar result was reported by König [2001] for the Netherlands. From 1953 to 1973, drinking water in Tiel was fluoridated and consequently children aged 12 years had significantly lower caries prevalence if compared to children from a control town, namely Culemborg. However, caries prevalence decreased gradually in both towns during the subsequent years and by 1980 was in quite the same order for both towns. He concluded that there is no need for ‘systemic fluoridation’ when topical fluoride application is available, e.g. as fluoridated dentifrices. About 10 years later, Groeneveld et al. [1990] recalculated the Tiel-Culemborg data and came to the conclusion that there was some pre-eruptive fluoride effect especially in pits and fissures. However, Limeback [1999] questioned their estimates since they did not offer any error analyses. Reich et al. [1992] investigated the caries prevalence of 5-year-old children, who had been subjected to different regimens of fluoride supplementation. One group received fluoride supplements from birth, the other group starting from 7 months. There was no statistically significant difference in dmfs scores in the primary teeth

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Fig. 1. Mean DMFS scores of children (12.5–16.0 years) from three different areas: F area (naturally fluoridated drinking water since birth), F area since 2 years, and control (no fluoridated drinking water). Data from Hellwig and Klimek [1985].

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at age 5, indicating that fluoride exerts a posteruptive effect and that fluoride ‘supplementation’ starting from birth is unnecessary. Stephen and Campbell [1978] were able to demonstrate a considerable caries-reducing effect for fluoride tablets when they are sucked and fluoride is allowed to act topically. All the above-mentioned clinical studies suggest that fluoride action is predominantly posteruptive. When reviewing the pre- and posteruptive effects of fluoride, Burt [1999] came to the conclusion that the cariostatic benefit of continuous fluoride exposure in a community is cumulative, i.e. fluoride has its effect by other means than pre-eruptive incorporation into the hydroxyapatite crystal. Otherwise caries-preventive benefits should be maximized in a group of children born when water fluoridation began and caries prevalence would not drop further as a result of water fluoridation. But epidemiological studies demonstrated a further decline in caries prevalence in subsequent cohorts, although no additional fluoridation measure was available [Arnold, 1957; Johnston et al., 1986]. At the same time laboratory studies came to conflicting results. While some could demonstrate that the solubility of enamel originating from residents of a fluoridated region was low, the others could not confirm these results and no direct correlation between fluorapatite in enamel and caries levels in populations could be demonstrated [Armstrong and Brekhus, 1938; Mellberg and Ripa, 1983]. Moreover, it was reported that even shortly after eruption the surface enamel is partly abraded physiologically and fluoride-rich enamel is lost [Aasenden, 1975]. Consequently, it seemed inconceivable that a rather low increase in surface enamel fluoride content due to fluoride ingestion could explain the caries-preventing efficacy of fluoride supplementation. In this context, Ögaard [1990] demonstrated that even shark enamel consisting mainly of fluorapatite demineralizes in an intra-oral caries model. He could also show that topical application of fluoride inhibits the development of caries lesions in human enamel, while it did not interfere with demineralization of shark enamel. The results of more recent epidemiological and laboratory studies can be summarized by stating that posteruptive (topical) application of fluoride plays the dominant role in caries prevention. It may be argued that fluoride might be recycled via the salivary glands after systemic administration, thereby affecting the rate of progression of caries lesions. Oliveby et al. [1989] investigated fluoride excretion in human saliva and its relationship to plasma fluoride levels after ingestion of 1 mg fluoride as NaF. The fluoride concentration in saliva is 2/3 that in simultaneously collected plasma

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and this relationship is maintained when fluoride is ingested [Ekstrand and Oliveby, 1999]. About 40 min after fluoride intake, the peak fluoride concentration in saliva is reached. After 120 min, salivary fluoride concentration decreases distinctly and it is unlikely that the small amount of fluoride recycled into the oral cavity per se can exert a significant caries-preventive effect. However, since plaque can accumulate fluoride [Dawes et al., 1965; Grobler et al., 1982; Ekstrand and Oliveby, 1999], it is conceivable that increasing the salivary fluoride concentration might be of some importance. But even if this is the case, it would be more advisable to increase plaque fluoride concentration directly by topical application. More recently, epidemiologists have questioned the validity of the ‘old’ studies dealing with systemic fluoride use. Since epidemiology was less advanced as a science, many cross-sectional studies were biased. Different grades of oral cleanliness, use of additional fluorides, selected or self-selecting groups, lack of examiner blindness, no concurrent controls, high dropout rates, retrospective analysis, differences in caries activity, no randomization, and different levels of dental awareness were some of the inherent interfering factors [Burt, 1999; Riordan, 1999]. Today it is well accepted that long-term exposure to topical fluorides mediates a reduction in caries prevalence similar to that obtained through ‘fluoride supplementation’. Clinical findings are supported by in vitro and in vivo studies demonstrating that the mode of action of fluoride can be mainly attributed to its influence on the de- and remineralization kinetics of dental hard tissues [Fejerskov et al.,1981; ten Cate and Featherstone, 1991; ten Cate, 1999]. Thus, fluoride should be present in the oral cavity throughout life, particularly during the period when the teeth are erupting [Thylstrup, 1990]. However, in the clinical situation the optimum fluoride level to prevent caries development is not known. In conclusion, one must state that to date there is no placebo-controlled, randomized, prospective study available determining how much of the anticaries effect can be attributed to pre-eruptive or posteruptive fluoride. However, carefully considering the present evidence from clinical and laboratory studies, it can be concluded that the caries-preventive mode of action of fluoride is mainly posteruptive. An entirely different problem with fluoride supplementation has been pointed out by Clark [1993]. He came to the conclusion that fluoride supplements are not particularly effective because of compliance problems. It should also be taken into account that fluoride supplementation increases the risk of fluorosis [Thylstrup et al., 1979; Riordan, 1993, 1999].

Hellwig/Lennon

State of the Art

Future Perspectives for Research

The daily use of an optimally formulated fluoride-containing dentifrice offers the chance for optimum caries prevention on a community and individual level, since it combines oral hygiene with fluoride supplementation. Moreover, this advice follows the idea that small amounts of fluoride should be present during a caries attack. Fluoride-containing mouthrinses, varnishes and gels offer an additional opportunity for caries prevention among people with moderate and high caries activity. Fluoride tablets or lozenges can be used as an aid for topical fluoride application, when children or adults are not able or willing to brush their teeth with a fluoridated dentifrice. People using them should be advised to suck them slowly and not to swallow them immediately after application. In areas with fluoridated drinking water the application of fluoride tablets is not advisable for toxicological reasons. The use of fluoride dentifrices by children living in these areas should be limited to those who are able to spit out adequately after toothbrushing.

In 1999 an international panel of scientists considered 10 priorities concerning fluoride research at large [Clarkson, 2000]. Focusing on the topic of the present paper the following, additional questions are still unanswered: Is there a difference with respect to caries development and caries progression between a group of children who used fluoridated dentifrice since the eruption of the first deciduous tooth and a group of children who used coated tablets since birth and brushed their teeth with an non-fluoridated dentifrice? Is there a measurable effect of fluoride ‘supplementation’ on tooth morphology resulting in a measurable caries-preventive effect? Does fluoride supplementation really promote body growth and/or formation of more stable bone architecture? Is caries development or lesion progression influenced by topical application of fluoride? What is the optimum fluoride concentration for topical treatment under clinical conditions?

References Aasenden R: Post-eruptive changes in the fluoride concentrations of human tooth surface enamel. Arch Oral Biol 1975;20:359–363. Armstrong WD, Brekhus PJ: Possible relationship between the fluoride content of enamel and resistance to dental caries. J Dent Res 1938;17: 393–399. Arnold F: Grand Rapids fluoridation study: Results pertaining to the eleventh year of fluoridation. Am J Public Health 1957;47:539–545. Backer Dirks O, Künzel W, Carlos JP: Caries preventive water fluoridation. Caries Res 1978; 12(suppl 1):7–14. Bibby BG, Wilkins E, Witol E: A preliminary study of the effects of fluoride lozenges and pills on dental caries. Oral Surg Oral Med Oral Pathol 1955;8:213–216. Burt BA: The case for eliminating the use of dietary fluoride supplements for young children. J Public Health Dent 1999;59:269–274. ten Cate JM: Current concepts on the theories of the mechanism of action of fluoride. Acta Odontol Scand 1999;57:325–329. ten Cate JM, Featherstone JDB: Mechanistic aspects of the interactions between fluoride and dental enamel. Crit Rev Oral Biol Med 1991;2: 283–296. Chan JT, Qiu CC, Whitford GM, Weatherred JG, Clardy RK: The distribution of fluoride of prenatal origin in the rat: A pilot study. Arch Oral Biol 1989;34:885–888.

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Clark DC: Appropriate uses of fluorides for children: Guidelines from the Canadian Workshop on the Evaluation of Current Recommendations Concerning Fluorides. Can Med Assoc J 1993;149:1787–1793. Clarkson JJ (ed): International collaborative research on fluoride. J Dent Res 2000;79:893– 904. Dawes C, Jenkins GN, Hardwick JL, Leach SA: The relationship between the fluoride concentrations in the dental plaque and in drinking water. Br Dent J 1965;119:164–167. Dean HT, Arnold FA, Elvove E: Domestic water and dental caries. Public Health Rep 1942;57: 1155–1179. Ekstrand J, Oliveby A: Fluoride in the oral environment. Acta Odontol Scand 1999;57:330–333. Fejerskov O, Thylstrup A, Larsen MJ: Rational use of fluorides in caries prevention: A concept based on possible cariostatic mechanisms. Acta Odontol Scand 1981;39:241–249. Glenn FB, Glenn WD, Duncan RC: Fluoride tablet supplementation during pregnancy for caries immunity: A study of the offspring produced. Am J Obstet Gynecol 1982;143:560–564. Grobler SR, Reddy J, van Wyk CW: Calcium, phosphorus, fluoride, and pH levels of human dental plaque from areas of varying fluoride levels. J Dent Res 1982;61:986–988. Groeneveld A, van Eck AAMJ, Backer Dirks O: Fluoride in caries prevention: Is the effect preor post-eruptive? J Dent Res 1990;69(special issue):751–755.

Hellwig E, Klimek J: Caries prevalence and dental fluorosis in German children in areas with different concentrations of fluoride in drinking water supplies. Caries Res 1985;19:278–283. Johnston DW, Grainger RM, Ryan RK: The decline of dental caries in Ontario school children. J Can Dent Assoc 1986;52:411–417. König KG: Reasons for increasing the fluoride content of children’s toothpastes. Oralprophylaxe 2001;23:27–31. Künzel W, Fischer T: Rise and fall of caries prevalence in German towns with different F concentrations in drinking water. Caries Res 1997; 31:166–173. LeGeros RZ, Glenn FB, Lee DD, Glenn WD: Some physico-chemical properties of deciduous enamel of children with and without pre-natal fluoride supplementation. J Dent Res 1985;64: 465–469. Lemke CW, Doherty JM, Arra MC: Controlled fluoridation: The dental effects of discontinuation in Antigo, Wisconsin. J Am Dent Assoc 1970;80:782–786. Leverett DH, Adair SM, Vaughan BW, Proskin HM, Moss ME: Randomized clinical trial of the effect of prenatal fluoride supplements in preventing dental caries. Caries Res 1997;31: 174–179. Limeback H: A re-examination of the pre-eruptive and post-eruptive mechanism of the anti-caries effects of fluoride: Is there any anticaries benefit form swallowing fluoride? Community Dent Oral Epidemiol 1999;27:62–71.

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McKay FS: The study of mottled enamel (dental fluorosis). J Am Dent Assoc 1952;44:133–137. Mellberg JR, Ripa LW: Fluoride in Preventive Dentistry: Theory and Clinical Applications. Chicago, Quintessence, 1983. Newbrun E: Effectiveness of water fluoridation. J Public Health Dent 1989;49:279–289. Ögaard B: Effects of fluoride on caries development and progression in vivo. J Dent Res 1990; 69(special issue):813–819. Oliveby A, Lagerlöf F, Ekstrand J, Dawes C: Studies on fluoride excretion in human whole saliva and its relation to flow rate and plasma fluoride levels. Caries Res 1989;23:243–246. Reich E, Schmalz G, Bergmann RL, Bergler H, Bergmann KE: Kariesbefall von Kindern nach unterschiedlich langer Applikation von Fluoridtabletten. Dtsch Zahnärztl Z 1992;47:232– 234.

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Riordan PJ: Fluoride supplements in caries prevention: A literature review and proposal for a new dosage schedule. J Public Health Dent 1993;53:174–189. Riordan PJ: Fluoride supplements for young children: An analysis of the literature focusing on benefits and risks. Community Dent Oral Epidemiol 1999;27:72–83. Ripa L: A half-century of community water fluoridation in the United States: A review and commentary. J Public Health Dent 1993;53:17– 44. Stephen KW, Campbell D: Caries reduction and cost benefit after 3 years of sucking fluoride tablets daily at school: A double-blind trial. Br Dent J 1978;144:202–206.

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Takeuchi K, Nakagaki H, Toyama Y, Kimata N, Ito F, Robinson C, Weatherell JA, Stösser L, Künzel W: Fluoride concentrations and distribution in premolars of children from low and optimal fluoride areas. Caries Res 1996;30:76– 82. Thylstrup A: Clinical evidence of the role of preeruptive fluoride in caries prevention. J Dent Res 1990;69(special issue):742–750. Thylstrup A, Bille J, Bruun C: Caries prevalence in Danish children living in areas with low and optimal levels of natural water fluoride. Caries Res 1982;16:413–420. Thylstrup A, Fejerskov O, Bruun C, Kann J: Enamel changes and dental caries in 7-year-old children given fluoride tablets from shortly after birth. Caries Res 1979;13:265–276.

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Caries Res 2004;38:263–267 DOI: 10.1159/000077765

How to Improve the Effectiveness of Caries-Preventive Programs Based on Fluoride H. Hausen Institute of Dentistry, University of Oulu, Oulu, Finland

Key Words Dental caries W Effectiveness, program W Fluoride W Prevention

Abstract During recent years, an increasing number of reports have been published in which the observed caries-preventive effect of fluoride has been lower than could have been expected on the basis of the earlier literature. The current low levels of caries occurrence and the widespread use of fluoridated toothpastes as well as other fluoride products and methods have been suggested as reasons for the reduced relative effect of fluoride from any single source. Theoretically, one can improve the effectiveness of fluoride in caries-preventive programs by using measures that are more effective than the previous ones and still safe and feasible in everyday conditions. Another possibility is to direct fluoride-based prevention to high caries risk susceptible individuals who are most likely to benefit from it. Thirdly, one can enhance the intensity of existing fluoride prevention by increasing the frequency of applications, but this is of course worthwhile only if the recipients are lacking sufficient exposure to fluoride. In theory, people themselves could easily take care of their adequate fluoride supply by using fluoride toothpastes and/or other home use

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products frequently enough to maintain a sufficient salivary fluoride concentration at all times. Many people are unwilling to adopt healthy lifestyles, however, and therefore caries-preventive programs will remain necessary for the foreseeable future. Fluoride is the backbone of all such programs. Since conditions strongly determine the usefulness of caries prevention including different fluoride regimes, more research is still needed to monitor the effectiveness of caries-preventive programs and their components in variable conditions of today and tomorrow. Copyright © 2004 S. Karger AG, Basel

During recent years, an increasing number of reports have been published in which the observed caries-preventive effect of fluoride has been lower than could have been expected on the basis of the earlier literature. This is true for both systemic and topical methods such as water fluoridation [Parviainen et al., 1985], fluoridated school milk [Ketley et al., 2003], fluoride mouthrinses [Stamm et al., 1984] and professional applications of topical fluoride [Schuller and Kalsbeek, 2003] including fluoride varnish applications [Hausen et al., 2000]. The current low levels of caries occurrence and the widespread use of fluoridated toothpastes as well as other fluoride products and methods have been suggested as reasons for the reduced rela-

Hannu Hausen Institute of Dentistry, University of Oulu PO Box 5281 FI–90014 Oulu (Finland) Tel. +358 8 537 5582, Fax +358 8 537 5560, E-Mail [email protected]

tive effect of water fluoridation [Horowitz, 1996]. In the same way, the fact that people are today commonly exposed to fluoride from multiple sources is likely to dilute the effect of fluoride from any single source. The moderate usefulness of added fluoride exposure at the population level today may also be due to the fact that individually applicable fluoride regimes are most likely to reach people who least need them. The individuals whose dental health-related lifestyles are most unfavorable and who are not visiting a dentist regularly are likely to be least exposed to fluoride and it is not easy to provide them with any individual protection against caries. The advantage of community water fluoridation is that it reaches even the least advantaged segments of the population. If the risk for caries is high, however, water fluoridation alone cannot provide full protection against the onset of cavities. Despite the generally low average DMF figures in the Western industrial countries today, dental decay is still far from being a rare disease. Roughly half of the 12-year-olds in two Finnish cities had at least one DMF surface in the 1990s [Seppä et al., 2000] and, at the same time, almost half of the participants of a caries study in Vantaa, Finland, who had no DMF surfaces at the age of 12, developed at least one carious lesion by the age of 15 years with the maximum DMF increment being twelve surfaces within a period of 3 years [Hausen et al., 2000]. It seems to be possible to introduce caries activity into a previously healthy dentition at any age. For a minority of the contemporary Western people, caries still is a true problem [Kaste et al., 1996; Seppä et al., 2000]. Recent reports reveal that the long-time declining trend in the occurrence of dental caries has leveled off at least in some countries [Poulsen and Scheutz, 1999; Haugejorden and Birkeland, 2002]. Consequently, there is still ample need for more effective measures of controlling caries. Efficacy, effectiveness and efficiency reveal different aspects of the effect of an intervention. This nomenclature was originally developed by Cochrane [1972]. Efficacy is the extent to which a measure produces a beneficial effect under ideal conditions, while effectiveness deals with the corresponding extent under everyday circumstances in the field. Efficiency considers the effect achieved in relation to the resources expended [Last, 2001]. These concepts constitute a hierarchy. If efficacy is lacking, there cannot be any effectiveness, which in turn is a basic requirement for efficiency. However easily and cheaply a measure can be applied, the application does not pay off if there is no beneficial effect.

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In the 1960s, Schwartz and Lelouch [1967] introduced the concepts of explanatory and pragmatic trials. The evidence for the effect of fluoride preventives usually stems from explanatory clinical trials that aim at assessing the efficacy of the measure under study. The idea of an explanatory clinical trial is to give the measure a chance to show its effect under the most favorable conditions that are conceivable. Whenever possible, efficacy is assessed among carefully selected, motivated and compliant randomly assigned subjects who are not exposed to any factors that could mask or dilute the beneficial effect of the studied intervention. Especially in the early explanatory clinical trials, participants who dropped out of the study or changed groups were often excluded from the statistical analyses. Thereby the internal validity of the trial was maximized, but at the expense of reduced external validity, i.e. the generalizability of the results. To be useful for the practitioner, the results also need to be applicable to real-world situations. Unfortunately, there tends to be an inverse association between the internal validity of clinical trials and the generalizability of their results to the everyday practice of health care. Many reasons limit the value of the results from purely explanatory clinical trials for designing caries-preventive programs to be implemented in the field under everyday conditions. First, no strict entry criteria are applicable in everyday life where the whole range of manifestations of both dental caries and its determinants are represented and where the compliance of people may be highly variable. Second, the beneficial effect of caries-preventive efforts is known to be strongly dependent on the characteristics of the target population, such as the level of caries occurrence and factors determining the rate of onset and progression of carious lesions. Third, caries-preventive programs often comprise multiple preventive measures for which it is important to know whether each of them retains its effect when applied together with the others. Pragmatic trials aim at finding out the effectiveness of an individual measure or a program including multiple approaches, which means that they focus on the usefulness of the intervention in everyday conditions [Schwartz and Lelouch, 1967; Roland and Torgerson, 1998]. Theoretically, pragmatic trials that are well designed and conducted provide the practitioners with the best possible evidence that is needed for planning and implementing caries prevention in the field. It is somewhat surprising that even though the idea of a pragmatic clinical trial was introduced to the dental research community already in the middle 1970s [O’Mullane, 1976], the number of such trials has been limited. Moreover, even the results of prag-

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matic clinical trials cannot be extrapolated to circumstances that are different from those in which the intervention was studied. In designing contemporary clinical trials, procedures that improve the generalizability of the results are fortunately often preferred even in cases where the planned trial is not explicitly pragmatic. To summarize the effects of various fluoride-based interventions, several well-conducted systematic reviews have been published recently [McDonagh et al., 2000; Marinho et al., 2003a, b, e], and there are quite a few under preparation [Marinho et al., 2003c, d, f, g; Yeung and Tickle, 2003]. A considerable part of the original research included in these reviews was conducted a long time ago in conditions that are different from those of today. Therefore, the value of their results for reducing uncertainties related to upcoming decisions on the policies of today and tomorrow should be considered carefully in each particular case. The conditions that determine the occurrence of caries in a population can change rapidly, resulting in both steep increases and decreases of caries frequency in different parts of the world. Consequently, it is not easy to find from the literature timely and accurate information that would be applicable as such to the everyday conditions of a particular setting. This is especially true for the effectiveness of individual fluoride measures in programs that use multiple approaches to control caries. Extrapolation of information related to efficiency is even more problematic since costs may vary between settings. Consequently, preventive programs are often run without an exact idea of their expected usefulness. Theoretically, one can improve the effectiveness of preventive programs by including in them components that are more effective than the previous ones and still safe and feasible in everyday conditions. If the costs do not rise, even the efficiency will improve. As for fluoride, improvement could possibly be achieved by using novel fluoride formulations or using the existing ones in more efficacious forms like stronger concentrations. Another possibility is to direct fluoride-based prevention to high caries risk susceptible individuals who are most likely to benefit from it. Thirdly, one can enhance the intensity of existing fluoride prevention by increasing the frequency of applications, but this is only worthwhile if the recipients are lacking sufficient exposure to fluoride. At present, none of the above lines of action is likely to bring about a dramatic breakthrough. Much effort has been and is currently made to find out whether novel formulations could make it possible to increase the effect of fluoride in caries prevention. Titanium tetrafluoride makes an example of a formulation regarding which the

results of several studies look promising [Reed and Bibby, 1976; Derand et al., 1989; Skartveit et al., 1991; de Oliveira and Cordeiro, 1995; Buyukyilmaz et al., 1997; Tezel et al., 2002]. Until now, however, there is no evidence stemming from large-scale randomized clinical trials on its applicability and superiority in everyday life. For fluoride toothpastes, there is evidence that the caries-preventive effect increases along with an increasing fluoride concentration [Marks et al., 1992]. So far, this observation has not been fully exploited. Overall, however, it can be concluded that there are no new methods of using the fluoride at hand, whose effect would be substantially higher than that of the preventives in general use today. Targeting fluoride prevention to high-risk individuals is problematic since it is difficult to distinguish between individuals who will and who will not develop cavities in the future. In spite of the poor accuracy of the currently available methods for assessing caries risk, the costs of screening the supposed high-risk individuals can be substantial. In addition, there is no good evidence of the successfulness of the high-risk approach in controlling caries [Hausen et al., 2000]. By using the directed population approach in which prevention is targeted to high-risk groups rather than individuals, one can avoid the costs of screening individuals, but even for this approach the rate of success still remains unclear. Increasing the frequency of fluoride applications may lead to somewhat better effectiveness, but in many populations today this will most likely happen at the cost of reduced efficiency, at least when the applications are performed at dental clinics. If the increased frequency of fluoride exposure can be achieved with little additional cost, however, this possibility is worth considering. Theoretically, people themselves could easily take care of their adequate fluoride supply by using fluoride toothpastes and other home use products frequently enough to maintain a sufficient salivary fluoride concentration at all times. A considerable part of European children brush their teeth only once a day or less often [Kuusela et al., 1997]. The concept of the reversed prevention paradox [Rose, 1992] includes the idea that when many people each receive a little benefit, the total benefit may be large. Accordingly, a considerable reduction in the occurrence of caries could possibly be achieved with little cost if everybody started using fluoridated toothpaste and/or other fluoride products suited for daily use twice a day or, if specially needed, even more often. There is recent evidence from the UK that caries among 5- to 6-year-olds could be reduced simply by providing free toothpaste with an adequate fluoride concentration [Davies et al., 2002].

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It is important to realize that people themselves can do a lot for maintaining good oral health. Many oral health care professionals, however, do not believe in the possibility of introducing changes in the lifestyles that affect the onset and progression of cavities among their patients. This is understandable since patient compliance does vary, and favorable changes seldom appear immediately. Therefore, it is not uncommon that patients are not given advice on proper self-care when they visit dental clinics. At least in Finland, the fact that caries was declining rapidly in the 1970s and 1980s at the same time as the numbers of professional preventive measures at dental clinics were increasing sharply may also have contributed to the overestimation of the potential of the oral health care system and to the underestimation of the resources of people themselves in promoting and maintaining oral health. Everyone should know, however, that for most individuals whose general health and oral conditions are not impaired, observing moderate snacking habits and using fluoridated toothpaste twice a day is probably sufficient to prevent cavities from occurring. If a caries-preventive program is run among such people, its effectiveness and efficiency are likely to be very low. Many people are

unwilling to adopt healthy lifestyles, however, and therefore caries-preventive programs will remain necessary for the foreseeable future. Fluoride is the backbone of all such programs. If the oral conditions are impaired and/or the patient’s lifestyle strongly favors the onset and progression of carious lesions, however, even an ideal fluoride regime may not be sufficient to prevent cavities from developing. Consequently, when designing programs to control caries it is essential to appreciate the multifactorial etiology of dental caries and not to rely merely on measures increasing the resistance of teeth. Recent literature has revealed instances where a considerable reduction of the level of preventive efforts has not been followed by an increase in caries frequency [Seppä et al., 2000] and vice versa [Hausen et al., 2000]. This must have been due to the fact that the studied preventive methods, that had proved to be effective elsewhere, were not effective and efficient in those particular settings. Since conditions strongly determine the usefulness of caries prevention including different fluoride regimes, more research is still needed to monitor the effectiveness of caries-preventive programs and their components in variable conditions of today and tomorrow.

References Buyukyilmaz T, Øgaard B, Duschner H, Ruben J, Arends J: The caries-preventive effect of titanium tetrafluoride on root surfaces in situ as evaluated by microradiography and confocal laser scanning microscopy. Adv Dent Res 1997;11:448–452. Cochrane AL: Effectiveness and Efficiency: Random Reflections on Health Services. London, The Nuffield Provincial Hospitals Trust, 1972. Davies GM, Worthington HV, Ellwood RP, Bentley EM, Blinkhorn AS, Taylor GO, Davies RM: A randomised controlled trial of the effectiveness of providing free fluoride toothpaste from the age of 12 months on reducing caries in 5–6 year old children. Community Dent Health 2002;19:131–136. Derand T, Lodding A, Petersson LG: Effect of topical F – solutions on caries-like lesions in root surfaces. Caries Res 1989;23:135–140. Haugejorden O, Birkeland JM: Evidence for reversal of the caries decline among Norwegian children. Int J Paediatr Dent 2002;12:306–315. Hausen H, Kärkkäinen S, Seppä L: Application of the high-risk strategy to control dental caries. Community Dent Oral Epidemiol 2000;28:26– 34. Horowitz HS: The effectiveness of community water fluoridation in the United States. J Public Health Dent 1996;56:253–258.

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Kaste LM, Selwitz RH, Oldakowski RJ, Burnelle JA, Winn DM, Brown LJ: Coronal caries in the primary and permanent dentition of children and adolescents 1–17 years of age: United States, 1988–1991. J Dent Res 1996;75(special issue):631–641. Ketley CE, West JL, Lennon MA: The use of school milk as a vehicle for fluoride in Knowsley, UK: An evaluation of effectiveness. Community Dent Health 2003;20:83–88. Kuusela S, Honkala E, Kannas L, Tynjälä J, Wold B: Oral hygiene habits of 11-year-old schoolchildren in 22 European countries and Canada in 1993/1994. J Dent Res 1997;76:1602–1609. Last JM (ed): A Dictionary of Epidemiology, ed 4. Oxford, Oxford University Press, 2001. McDonagh M, Whiting P, Bradley M, Cooper J, Sutton A, Chestnutt I, Misso K, Wilson P, Treasure E, Kleijnen J: A Systematic Review of Public Water Fluoridation. York, NHS Centre for Reviews and Dissemination, 2000. Marinho VCC, Higgins JPT, Logan S, Sheiham A: Fluoride gels for preventing dental caries in children and adolescents. Cochrane Database Syst Rev 2003a. Marinho VCC, Higgins JPT, Logan S, Sheiham A: Fluoride varnishes for preventing dental caries in children and adolescents. Cochrane Database Syst Rev 2003b.

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Marinho VCC, Higgins JPT, Sheiham A, Logan S: Combinations of topical fluorides (varnishes, gels, rinses, toothpastes) versus one topical fluoride for preventing dental caries in children and adolescents. Cochrane Database Syst Rev 2003c. Marinho VCC, Higgins JPT, Sheiham A, Logan S: Fluoride rinses for preventing dental caries in children and adolescents. Cochrane Database Syst Rev 2003d. Marinho VCC, Higgins JPT, Sheiham A, Logan S: Fluoride toothpastes for preventing dental caries in children and adolescents. Cochrane Database Syst Rev 2003e;1:CD002278. Marinho VCC, Higgins JPT, Sheiham A, Logan S: One topical fluoride (varnishes, or gels, or rinses, or toothpastes) versus another for preventing dental caries in children and adolescents. Cochrane Database Syst Rev 2003f. Marinho VCC, Sheiham A, Logan S, Higgins JPT: Topical fluoride (toothpastes, mouthrinses, gels or varnishes) for preventing dental caries in children and adolescents. Cochrane Database Syst Rev 2003g. Marks RG, D’Agostino R, Moorhead JE, Conti AJ, Cancro L: A fluoride dose-response evaluation in an anticaries clinical trial. J Dent Res 1992; 71:1286–1291.

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de Oliveira, Cordeiro JG: The effect of various fluoride compounds on the development of experimental root surface caries in hamsters. Bull Tokyo Med Dent Univ 1995;42:105–116. O’Mullane DM: Efficiency in clinical trials of caries preventive agents and methods. Community Dent Oral Epidemiol 1976;4:190–194. Parviainen K, Ainamo J, Nordling H: Changes in oral health from 1973 to 1982 of 13–15-yearold schoolchildren residing in three different fluoride areas in Finland. J Dent Res 1985;64: 1253–1256. Poulsen S, Scheutz F: Dental caries in Danish children and adolescents 1988–1997. Community Dent Health 1999;16:166–170.

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Reed AJ, Bibby BG: Preliminary report on effect of topical applications of titanium tetrafluoride on dental caries. J Dent Res 1976;55:357–358. Roland M, Torgerson DJ: What are pragmatic trials? BMJ 1998;316:285. Rose G: The Strategy of Preventive Medicine. Oxford, Oxford University Press, 1992. Schuller AA, Kalsbeek H: Effect of the routine professional application of topical fluoride on caries and treatment experience in adolescents of low socio-economic status in the Netherlands. Caries Res 2003;37:172–177. Schwartz D, Lelouch J: Explanatory and pragmatic attitudes in therapeutic trials. J Chronic Dis 1967;20:637–648. Seppä L, Kärkkäinen S, Hausen H: Caries trends 1992–1998 in two low-fluoride Finnish towns formerly with and without fluoridation. Caries Res 2000;34:462–468.

Skartveit L, Spak CJ, Tveit AB, Selvig KA: Cariesinhibitory effect of titanium tetrafluoride in rats. Acta Odontol Scand 1991;49:85–88. Stamm JW, Bohannan HM, Graves RC, Disney JA: The efficiency of caries prevention with weekly fluoride mouthrinses. J Dent Educ 1984;48:617–626. Tezel H, Ergucu Z, Onal B: Effects of topical fluoride agents on artificial enamel lesion formation in vitro. Quintessence Int 2002;33:347– 352. Yeung CA, Tickle M: Fluoridated milk for preventing dental caries in children and adolescents. Cochrane Database Syst Rev 2003.

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Caries Res 2004;38:268–276 DOI: 10.1159/000077766

The Effect of Fluoride on the Developing Tooth C. Robinson a S. Connell a J. Kirkham a S.J. Brookes a R.C. Shore a A.M. Smith b a Leeds

Dental Institute, and b Department of Physics and Astronomy, University of Leeds, Leeds, UK

Key Words Apatite W Cell structure W Enamel W Fluoride W Fluorosis W Proteases W Protein-matrix

Abstract This review aims to outline the effects of fluoride on the biological processes involved in the formation of tooth tissues, particularly dental enamel. Attention has been focused on mechanisms which, if compromised, could give rise to dental fluorosis. The literature is extensive and often confusing but a much clearer picture is emerging based on recent more detailed knowledge of odontogenesis. Opacity, characteristic of fluorotic enamel, results from incomplete apatite crystal growth. How this occurs is suggested by other changes brought about by fluoride. Matrix proteins, associated with the mineral phase, normally degraded and removed to permit final crystal growth, are to some extent retained in fluorotic tissue. Fluoride and magnesium concentrations increase while carbonate is reduced. Crystal surface morphology at the nano-scale is altered and functional ameloblast morphology at the maturation stage also changes. Fluoride incorporation into enamel apatite produces more stable crystals. Local supersaturation levels with regard to the fluoridated mineral will also be elevated facilitating crystal growth. Such changes in crystal chemistry and morphology, involving stronger ionic and hydrogen bonds, also lead to greater binding of modulating matrix proteins and proteolytic enzymes. This results in reduced

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degradation and enhanced retention of protein components in mature tissue. This is most likely responsible for porous fluorotic tissue, since matrix protein removal is necessary for unimpaired crystal growth. To resolve the outstanding problems of the role of cell changes and the precise reasons for protein retention more detailed studies will be required of alterations to cell function, effect on specific protein species and the nano-chemistry of the apatite crystal surfaces. Copyright © 2004 S. Karger AG, Basel

Fluoride ion has played a major role in dramatically reducing dental caries over the past 40 years. The discovery was made by comparing caries incidence in individuals exposed to so-called high-fluoride water supplies with that in individuals exposed to lower levels [Dean et al., 1942]. It was deduced from these data that fluoride exposure during tooth development was a prime cause of caries reduction. Since teeth from high-fluoride areas had accumulated higher concentrations of fluoride compared with those from low-fluoride areas, fluoride content of the dental tissues was cited as a major factor in reduced caries incidence. The effect of fluoride on the dentition is dose-dependent and is not confined to increased caries resistance. Above certain levels in the water supply, visible changes to the teeth, particularly the enamel, become evident. This is the condition known as dental fluorosis.

Prof. C. Robinson Division of Oral Biology, Leeds Dental Institute Clarendon Way Leeds LS2 9LU (UK) Tel./Fax +44 113 233 6158, E-Mail [email protected]

Visible Effects of Fluoride on Dental Enamel

The effects of fluoride on dental enamel are well documented [Dean and Elvolve, 1937; Fejerskov et al., 1977; Thylstrup and Fejerskov, 1978]. At about 1 ppm fluoride (53 ÌM) in the water supply, visible signs of fluorosis begin to become obvious on the enamel surface as opacities, implying some porosity in the tissue. As dose increases, these become more obvious until at 10 ppm (530 ÌM) or so, the porosity is such that the enamel is physically compromised and large pieces may be fractured from the tooth especially after eruption. The porosity appears to derive from incomplete crystal growth such that the normal close juxtaposition and interlocking of crystals does not occur.

Selective Effect of Fluoride on the Mineralised Tissues The reasons for the apparent selective effect of fluoride on the skeletal and dental tissues and enamel in particular have been related to the interaction between fluoride ions and the skeletal mineral, calcium hydroxyapatite, dealt with in detail below. Fluoride is the most electronegative of the elements and is of small ionic diameter. Its resulting high charge density endows it with a great capacity to form strong ionic and hydrogen bonds. This provides the fluoride ion with a potential for interacting both with mineral phases and organic macromolecules. Because of these properties, particularly its small size, it can also act as a ‘structure former’ in water. This can decrease the mobility of water molecules in solution and in hydration layers of proteins and apatite surfaces with concomitant effects on ligand binding and exchange. Interactions with the mineral phase have two kinds of effect. First a direct effect on the properties of the mineral itself and its relationship with the enveloping and modulating extracellular organic matrix. Second, the selective concentration of fluoride at the surfaces of mineralised tissues [Robinson et al., 1996] may give rise to elevated fluoride in the immediate vicinity of mineralised tissue cells such that local concentrations may be much higher than those of the tissue fluids in general. Information on local fluoride concentrations in tissue fluids is limited, however, especially in the immediate neighbourhood of fluoridated apatite. In the enormous amount of work which has been carried out the actual concentrations responsible for any given effect are perhaps the most difficult of areas to clarify. Plasma fluoride

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concentrations seem to be of particular relevance, relating closely to dietary supply and being around 1 ÌM at 53 ÌM in the water supply rising to about 4 ÌM at 265 ÌM [Guy et al., 1976; Speirs, 1986]. Wherever possible, therefore, plasma concentrations or those in the immediate tissue environment such as enamel fluid or culture medium have been quoted.

Site(s) of Action of the Fluoride Ion during Odontogenesis While the effects of fluoride on odontogenesis are well established, the precise site(s), stage(s) of development, timing, and mechanism of action are still unclear. The most likely sites are: (a) cells of the tooth-forming tissues: proliferation, differentiation, functional morphology; (b) extracellular matrix of tooth tissues: matrix protein synthesis secretion, processing and loss; (c) mineral phase: initiation, crystal growth, chemical properties, and (d) extracellular matrix-mineral interactions in tooth tissues.

Effect of Fluoride on Odontogenic Cells Stage of Fluoride Uptake In enamel, long considered to be the most susceptible of the dental tissues, fluoride accumulates throughout the developing tissue but especially at its surface. This occurs selectively both very early in amelogenesis and later, across the transition/maturation stage border [Weatherell et al., 1975, 1977]. At this late stage, full tissue thickness has been achieved; the supporting extracellular matrix largely replaced by fluid and considerable growth in crystal thickness begins. Selective uptake may thus be due to the highly porous, hydrated nature of this developmental stage [Hiller et al., 1975; Robinson et al., 1981, 1988]. Substantial amounts of fluoride are then lost during subsequent maturation. This implies that much of this fluoride may be labile and together with the reported lowering of pH at this stage, which would dissolve mineral surfaces [Sasaki et al., 1991], locally elevated fluoride concentrations are likely. Cells associated with matrix withdrawal and crystal growth during maturation could thus be exposed to locally high fluoride concentrations. Suggestions that fluorosis can be induced by elevating concentrations only at this latter stage are consistent with these data [Richards et al., 1986]. While efforts to determine fluoride concentrations in enamel fluid have been made [1 ÌM, Aoba and Moreno, 1987], this did not distinguish

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between developmental stages and must be regarded as an average. Cell Proliferation Work with tissue culture using ‘pre-ameloblasts’ has so far revealed no alterations in DNA synthesis at fluoride concentrations up to 1.31 ÌM [Bronckers and Wöltgens, 1985] or in frequency of mitotic figures at concentrations up to 1.06 mM [Lyaruu et al., 1986]. While the effect of fluoride on proliferating odontogenic cells is equivocal, it is worth noting that bone cells in culture have shown sustained mitosis in response to fluoride [Wergedal et al., 1988 (20 ÌM); Khokher and Dandona, 1990 (1250 ÌM)]. This was attributed to intracellular signalling pathways associated with mitotic activity. Inhibition by fluoride, of tyrosine phosphorylase phosphatase, part of the mitogen-activating protein kinase (MAPK) system, has received particular attention [Lau and Baylink,1990] together with activation of G proteins which stimulate protein kinase C. Inhibition of this phosphatase would tend to sustain mitotic activity by maintaining levels of active tyrosine phosphorylase, a mediator of mitotic activity. Why ameloblasts have not shown increased mitosis is not clear. The effect indicated may be specific to bone cells. However, dividing ameloblasts may already be near maximum ‘mitotic activity’ during tooth formation and any increase over such high activity may not be discernible. Such modest enhancement of mitosis might, however, be in part responsible for alterations to tooth size and morphology attributed to fluoride [Cooper and Ludwig, 1965]. Cell Differentiation and Functional Morphology Almost no effects of fluoride on odontogenic cell differentiation were detected at F50 ÌM peak plasma fluoride concentrations [Walton and Eisenmann, 1974] and up to 265 ÌM in culture medium [Bronckers et al., 1984a], although at higher concentrations (3 mM) a delay in differentiation was reported [Kerley and Kollar, 1977]. While not strictly an effect on differentiation, effects on ameloblast cytoskeletal components have been reported recently in abstract form [Gibson et al., IADR Meeting, Gothenburg, 2003]. The amelogenin gene is nested within a RhoGAP gene, which regulates intracellular signalling by activation of Rho G protein and elevation of F actin. Fluoride at 4 mM for 30 min was shown to inactivate RhoGAP, activating Rho and elevating F actin. In ameloblasts this was localised to actin-rich ameloblast cell junctions and Tomes processes.

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While this concentration is relatively high, lesser concentrations could affect the dramatic alterations to functional cell morphology and cell-cell interactions which accompany the transformation from secretory to maturation phases and presumably reflect changes in cell function from secretion to maturation. This may explain changes in the periodicity of ameloblast cell membrane modulation which occurs during fluorosis (10 ÌM F in plasma) [Denbesten et al., 1985]. The modulation, between smooth and ruffle-ended ameloblasts, is thought to be involved in final crystal growth. This is again especially pertinent since labile fluoride accumulates in enamel precisely at this developmental stage [Weatherell et al., 1975, 1977].

Effect of Fluoride on Matrix Protein Synthesis and Secretion The effects of fluoride on cell activity, for example, rate of protein secretion, has been examined but with equivocal results [Denbesten, 1986; Aoba et al., 1990; Robinson and Kirkham, 1990; Aoba and Fejerskov, 2002]. However, a direct effect on matrix composition per se is difficult to discern from data published so far. Only aminoacid compositions have been looked at in detail and no substantial changes due to fluoride have been reported. Since these investigations, a number of distinct protein species have emerged as components of the enamel matrix, e.g. amelogenin, enamelin and ameloblastin together with a number of specific degradative enzymes and other proteins such as albumin and ·HS2 glycoprotein and small amounts of sulphated proteins [for reviews see Robinson et al., 1998a; Fincham et al., 1999]. The effect of fluoride on the relative concentrations of these species, their alternatively spliced variants and/or their individual functions remains to be investigated in detail. Interpretation of existing data is also complicated by post-synthetic protein processing including post-translational modification and the controlled degradation prior to maturation, which produces a highly consistent pattern of breakdown products [Robinson et al., 1998a; Fincham et al., 1999]. It is therefore difficult to separate effects on protein production per se from effects on post-synthetic or post-secretory activity. A particular case in point is the level of matrix phosphorylation. Judging from the similarity of 32P uptake and of two-dimensional protein gel patterns between fluoridated and control enamel organ cultures, fluoride up to 1.325 mM in culture medium had little effect on matrix

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phosphorylation levels [Denbesten, 1986]. This implies little effect of fluoride on either post-translational phosphorylation or any post-secretory dephosphorylation. The investigation, however, predated the identification of specific phosphorylated proteins of the matrix, in particular enamelin (2%) [Fukae et al., 1996], which might benefit from further investigation. Extracellular Matrix Processing and Loss A major feature of normal enamel development is the almost complete and selective degradation and loss of enamel matrix proteins, particularly the amelogenins. What remains comprises small peptides, amino acids and insoluble tuft protein [for review see Robinson et al., 1998a]. In mature fluorotic enamel this situation was altered with retention of proline-rich components [Eastoe and Fejerskov, 1984; Wright et al., 1989, 1996]. The precise identity of retained molecular species is unknown but, from their amino acid composition, they did not appear to be intact amelogenin and may be a mixture of degradation products [Wright et al., 1989]. Reports concerning developing fluoridated enamel [Drinkard et al., 1983 (370 ÌM F peak plasma); Denbesten and Crenshaw, 1984; Robinson and Kirkham, 1984b; Denbesten, 1986] revealed a relative increase in 25-kD components in developing enamel containing nascent amelogenin, much of which was mineral-bound [Robinson et al., 2003a]. Smaller components were also retained during maturation, but these data were less clear. The most likely explanation for these changes is fluoride-induced retention of intact and degraded protein species together with reduced extracellular proteolysis [for review see Robinson and Kirkham, 1990; Aoba and Fejerskov, 2002; Robinson et al., 2003a]. Lowered calcium activity, due to a less soluble mineral phase, has been suggested to slow down proteolysis by Ca++-dependent, secretory stage proteases [Aoba and Fejerskov, 2002]. While this is possible, it is unlikely as a major factor. First, given the relatively high calcium levels and small amount of enzyme present, extremely severe reductions in calcium would be necessary. Second, major protein destruction occurs at transition via a serine protease (kallikrein 4) which is not Ca++-dependent [Simmer and Hu, 2002]. Third, the timescale for protein removal and maturation is hugely variable between species. In the rat incisor this is about 2 weeks, in the cow and pig about 2 months, and in human teeth this may take years. These latter periods would appear to be quite sufficient for complete processing and removal of matrix in the order of only hundreds of micrograms [Robinson and Kirkham,

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1984a, 1990]. It should also be noted that porcine enamel did not appear to mineralise to the same extent as other species, attaining only 55% mineral by weight as opposed to 80–90%. This should be borne in mind when using the pig as a model for fluorosis [Kirkham et al., 1988; Robinson and Kirkham, 1990]. Since direct inhibition of enzyme activity has not been demonstrated convincingly [Drinkard et al., 1983; Gerlach et al., 2000], it is likely that enhanced protein interaction with the mineral, described below, is responsible for both protein retention and reduced proteolysis in fluorosed tissue. Increased binding of both undegraded amelogenin [Robinson et al., 2003a] and enamel proteases to the mineral phase [Brookes et al., 1998; Aoba and Fejerskov, 2002] have been reported. This may be especially important at the transition/maturation stage, where final degradation occurs via a specific serine protease (kallikrein 4) and where fluoride accumulates selectively. Effect of Retained Protein The result of fluoride-induced protein retention may also explain the incomplete crystal growth which characterises fluorosis, since it has been demonstrated that matrix removal is a necessary prerequisite for unimpaired crystal growth in enamel [Robinson et al., 1989] and synthetic apatites [Aoba et al., 1987]. In this context, one area worthy of further exploration is the role of ameloblastin. Fluorosis involves incomplete crystal growth at prism peripheries and it is at this site that degradation products of ameloblastin accumulate during development [Uchida et al., 1997; Robinson et al., 1998b]. Impaired removal of ameloblastin due to fluoride could be responsible for incomplete crystal growth in this region.

Effect of Fluoride on the Mineral Phase Initiation of Precipitation during Secretion While it is established that mature enamel crystals comprise a substituted calcium hydroxyapatite, the precise nature of initial mineral phases, whether in enamel or dentine, is still a matter of some controversy. These range from amorphous short range order calcium phosphates [Posner, 1985] through brushite-like phases to octacalcium phosphate [Brown et al., 1987; Iijima et al., 1992; Johnsson and Nancollas, 1992]. These are often said to be stabilised by carbonate or magnesium. Whatever the nature of this phase, there seems to be general agreement that the presence of fluoride ion during initial deposition can delay formation of an initial apatite precursor, proba-

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bly by stabilising very early precursor entities [Bachra and Fischer, 1969]. Work by Bronckers et al. [1984b] using hamster tooth germs is consistent with this view and showed that protein matrix formed under an elevated fluoride regime did not mineralise at all unless fluoride was removed, indicating a reversible effect on the matrixassociated initiation process. Precisely how and where initiation occurs is still a matter of discussion. Recently, Robinson et al. [2003c] suggested that crystal formation in enamel may involve fusion of precursor protein/mineral-ion subunits, the established degradative processing of the matrix facilitating initial mineral precipitation. Stabilisation of these protein mineral subunits by fluoride per se would also prevent or delay initiation. Crystal Growth Once the initial mineral phase has formed, fluoride facilitates more rapid deposition [Bachra and Fischer, 1969; Varughese and Moreno, 1981]. This may be due to fluoride-induced conversion of acidic precursors such as amorphous calcium phosphate or octacalcium phosphate to apatite [Iijima et al., 1992]. Perhaps a more likely explanation would be the higher relative supersaturation of tissue fluids with respect to a precipitating fluoridated mineral phase. This would be facilitated by the effect of fluoride in reducing the incorporation of destabilising extraneous ions such as carbonate [Nikiforuk and Grainger, 1965]. Stimulation of crystal growth during early secretion is supported by the work of Bronckers et al. [1984b], who showed, in culture, that with access to fluoride (up to 26.5 ÌM), partially mineralised matrix became hypermineralised. This would be consistent with a more rapid mineral deposition due to a higher relative supersaturation for fluoride-containing mineral. While much in vitro data suggests that fluoride can increase apatite crystal growth in the a and b axes [Eanes and Hailer, 1998], there is little evidence that, at least in enamel, this results in significant alteration to apatite crystal morphology or size [Yanagisawa et al., 1989]. Such changes which have been reported were restricted to the outer enamel and attributed to post-eruptive alterations [Yanagisawa et al., 1989]. Effect of Fluoride on Mineral Properties During mineral deposition, fluoride is incorporated into the growing hydroxyapatite crystals either by accretion or by heteroionic substitution. Fluoride is known to occupy the hydroxyl site in the long c axis of the crystal. The charge symmetry and high

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negative charge density allow a better fit in the lattice compared with the larger asymmetric hydroxyl ion. The effects are profound. The sense of the hydroxyl columns is altered such that adjacent hydroxyls will hydrogen bond to the fluoride ion. In addition, protons associated with acid phosphate groups might be more tightly orientated towards the fluoride ion [Posner et al., 1963; Kay et al., 1964; Van der Lugt et al., 1971]. In terms of overall crystal behaviour, energy levels are much reduced. This explains the lower solubility product for fluoridated compared with non-fluoridated mineral and the fact that the crystal is less reactive. With regard to resorption in dentine and dissolution in caries, the fluoridated crystal is much more acid-resistant. In addition, the larger asymmetric substituent, carbonate tends to be excluded from the crystal, further increasing stability [McCann and Bullock, 1957]. Magnesium might be expected to exert a similar effect to carbonate since it does not fit well in the lattice (about 0.2% maximum). However, an increase in fluoride is usually accompanied by an increase in magnesium content [McCann and Bullock, 1957; Robinson et al., 1983]. This may relate to the fact that magnesium is at highest concentrations during secretion [Hiller et al., 1975] and like fluoride shows some selective uptake during transition [Robinson et al., 1984; Kirkham et al., 1988]. This has been attributed to close affinity of magnesium for fluoride during incorporation into the crystals [Okazaki, 1987]. It may also be surface-located [Neuman and Mulryan, 1971], its higher concentrations being due to specific surface complexes and/or by a greater surface area of the rougher crystal surfaces [Kirkham et al., 2001] (see below).

Matrix-Mineral Interactions During enamel maturation, crystal growth, especially in the final stages, is clearly compromised since fluorosis is characterised by greater intercrystalline space, particularly at the prism peripheries [Fejerskov et al., 1977]. Since enamel matrix removal appears to be a prerequisite for normal crystal growth [Aoba et al., 1987; Robinson et al., 1989], impairment of crystal development in vivo has been associated with the demonstrated retention of mineral-bound protein matrix [Drinkard et al., 1983; Denbesten, 1986; Robinson et al., 2003a]. The mechanism of enhanced retention and the molecular species involved (see above) are not yet clear. Fluoridated mineral may bind proteins more effectively [Tanabe et al., 1988] due to

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greater hydrogen bonding or a less polar surface [Wu and Nancollas, 1999]. Increased magnesium, if located in the Helmholz double layer, could provide cationic bridging for proteins. This does not exclude the possibility that increased magnesium per se could impair growth [Bachra and Fischer, 1969]. More recent work using the atomic force microscope (AFM) has suggested that fluoride could also influence crystal surface morphology and perhaps the mode of crystal growth. This might contribute towards a unifying view of fluoride action. AFM studies of crystal surfaces at the molecular level have indicated that during enamel development, in the rat, the surface roughness of crystals normally decreased in moving from secretion to maturation phases [Kirkham et al., 1998]. This may have resulted from changes in matrix binding but may also reflect a decrease in kink and step site density due to a growth/ healing process perhaps involving a shift from polynuclear towards spiral growth. Enamel produced under fluorotic conditions, however, did not show such a reduction in roughness. Not only was roughness greater than in non-fluorotic teeth but it was also maintained throughout development [Kirkham et al., 2001]. Since fluoride is taken up selectively during transition and maturation stages [Weatherell et al., 1977], local supersaturation levels would be relatively high in terms of the fluoridated depositing phase. This high supersaturation would favour polynuclear growth and thus increased surface roughening. Such increased roughening could, together with changes in crystal surface chemistry, account for the increased magnesium typical of fluorosed enamel, a view supported by the fact that magnesium, like fluoride, is selectively taken up at the transition/maturation stage [Hiller et al., 1975; Robinson et al., 1984; Kirkham et al., 1988]. The increased surface area due to roughening could also facilitate protein binding/retention [Gathercole et al., 1996]. Use of the AFM in chemical force mode has also revealed novel information concerning enamel crystal surface properties. Using carboxyl- or hydroxyl-functionalised tips their binding strength to apatite surfaces was measured as a function of pH. This revealed pK values for apatite surfaces an order of magnitude lower than solution phosphate, implying greater electronegativity. When fluoride was present, binding values were higher and pK values were even lower, indicating further increased, presumably hydrogen, bonding with phosphate groups or fluoride itself [Robinson et al., 2003b] somewhat similar shifts to those seen in bulk synthetic systems [Wu et

al.,1991]. Such studies offer important possibilities for future studies of fluoride-mediated changes to crystal surface properties, not only of crystals, but of the modulating organic matrix. While much of the consideration of the effect of fluoride resides with its effect on the lattice proper, the roughness findings described above also suggest that the crystal surface/fluid interface should be considered. Given the great propensity for fluoride to form hydrogen bonds it is likely that it could affect ligand binding and exchange with the Helmholz/Gouy-Chapman layers and thus with the lattice itself.

Fluoride on the Developing Tooth

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Dentine

The effect of fluoride on dentine is only detectable at concentrations much higher than those required for enamel fluorosis. Overall effects, however, are similar in that hypomineralisation results [Fejerskov et al., 1979].

Matrix Synthesis and Composition While no fluoride-mediated alterations to the main extracellular component, type 1 collagen, have been reported, specific changes to non-collagenous components do occur. Perhaps of greatest current interest is a reported effect on the dentine phosphoproteins [Milan et al., 1999]. Rats rendered fluorotic by dietary fluoride revealed lower molecular sizes for dentine phosphoprotein (phosphophoryn) which, together with lowered phosphate content, was attributed to a lower degree of phosphorylation. Investigations into casein kinase II and alkaline phosphatase – both enzyme types present in developing dentine – also revealed fluoride-mediated inhibition [Milan et al., 2001]. Clearly fluoride is capable of affecting the metabolism of dentine phosphoproteins. The reduction of phosphorylation, in particular, might well decrease mineral ion binding and probably their capacity for crystal initiation. Analysis of proteoglycans from fluorosed rat dentine in vivo revealed no alterations to the protein core. However, glycosylaminoglycans (GAGs) appeared to be smaller and more anionic, possibly due to the additional presence of dermatan and heparan sulphate [Hall et al., 1996]. Interaction of these GAGs with mineralising collagen, the main extracellular matrix component, may be affected, possibly restricting mineral initiation, while binding to the mineral phase could result in less mineral deposition.

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Mineral Phase The mineral phase of dentine takes the form of very small apatite crystals 50 ! 70 ! 5 nm, embedded within a highly cross-linked type 1 collagen matrix. There is little direct information on fluorosed or fluorotic dentine mineral. Early reports did, however, suggest that it might contain reduced levels of carbonate and elevated magnesium similar to fluorotic enamel [McCann and Bullock, 1957]. The implications would be related as in enamel to a reduction in the supersaturation levels required for mineral precipitation and crystal growth with a concomitant reduction in acid solubility.

Concluding Remarks

Despite a very large and often confusing literature concerning the mechanisms which lead to dental fluorosis a relatively clear and well-supported concept is emerging, at least for exposure to concentrations which are not overtly toxic. It seems likely that, at least in enamel, the effect on the developing mineral phase per se coupled with associated effects on the surrounding and modulating protein matrix could account for most of the observed effects of the fluoride ion on tooth development. The most obvious feature of fluorosis – impaired growth of apatite crystals – seems attributable to retention of modulating matrix proteins through enhanced binding of mineral to matrix proteins and/or enhanced binding of the proteases responsible for processing prior to matrix removal.

There are still many unanswered questions, however. Information with regard to effects on specific molecular species is still sparse and more detailed studies of the effect of fluoride on the recent unsuspected substructure of apatite crystal surfaces is unclear. With regard to the effect of fluoride on odontogenic cells, the latest information suggests that functional cell structure might be altered at a stage affecting final crystal growth and matrix withdrawal. The following lists include those areas where information is still lacking or unclear.

Avenues for Future Research Effect of Fluoride on: E Effect of fluoride on cytoskeletal components through the RhoGAP system. E MAPK kinase phosphatase and similar mitosis-sustaining pathways in odontogenesis cells. E Relative secretion/amounts of specific protein species with established sequences. E Alternative splicing of matrix proteins. E Post-translational processing of specific proteins (phosphorylation, glycosylation, sulphation). E Post-secretory processing of specific proteins (proteolysis, dephosphorylation, de-glycosylation). E Surface/molecular morphology of crystals. E Surface chemistry of crystals (double layer and water structure). E Specific protein-mineral interactions (motif changes?). E Regrowth of fluorosed crystals (re-activation of crystal surfaces). E Effect of increased magnesium on crystal growth.

References Aoba T, Fejerskov O: Dental fluorosis: Chemistry and biology. Crit Rev Oral Biol Med 2002;13: 155–170. Aoba T, Fukae M, Tanabe T, Shimizu M, Moreno EC: Selective adsorption of porcine-amelogenins onto hydroxyapatite and their inhibitory activity on hydroxyapatite growth in supersaturated solutions. Calcif Tissue Int 1987;41:281– 289. Aoba T, Moreno EC: The enamel fluid in the early secretory stage of porcine amelogenesis: Chemical composition and saturation with respect to enamel mineral. Calcif Tissue Int 1987;41:86– 94.

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Aoba T, Moreno EC, Tanabe T, Fukae M: Effects of fluoride on matrix proteins and their properties in rat secretory enamel. J Dent Res 1990; 69:1248–1250. Bachra BE, Fischer HRA: The effect of some inhibitors on the nucleation and crystal growth of apatite. Calcif Tissue Res 1969;3:348–357. Bronckers ALJ, Jansen LL, Wöltgens JHM: Longterm effects of exposure to low concentrations of fluoride on enamel formation in hamster tooth-germs in organ culture. Arch Oral Biol 1984a;29:811–819. Bronckers ALJ, Jansen LL, Wöltgens JHM: A histological study of the short-term effects of fluoride on enamel and dentine formation in hamster tooth-germs in organ culture in vitro. Arch Oral Biol 1984b;29:803–810.

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Bronckers ALJ, Wöltgens JHM: Short-term effects of fluoride on biosynthesis of enamel-matrix proteins and dentine collagens and on mineralization during hamster tooth-germ development in organ culture. Arch Oral Biol 1985;30: 181–191. Brookes SJ, Shore RC, Kirkham J, Robinson C: Enzyme compartmentalization during biphasic enamel matrix processing. Connect Tissue Res 1998;39:393–403. Brown WE, Eidelman N, Tomazic BB: Octacalcium phosphate as a precursor in biomineral formation. Adv Dent Res 1987;1:306–313.

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Cooper VK, Ludwig TG: Changes in tooth morphology as affected by fluoridated drinking water. N Z Dent J 1965;61:399–408. Dean HT, Arnold FA, Elvolve E: Domestic water and dental caries: Additional studies of the relation of fluoride in domestic waters to dental caries in 4425 white children, age 12–14 years of 13 cities in 4 states. Public Health Rep 1942; 57:1155–1179. Dean HT, Elvolve E: Further studies on the minimal threshold of chronic endemic dental fluorosis. Public Health Rep 1937;52:1249–1264. Denbesten PK: Effects of fluoride on protein secretion and removal during enamel development in the rat. J Dent Res 1986;65:1272–1277. Denbesten PK, Crenshaw MA: The effects of chronic high fluoride levels on forming enamel in the rat. Arch Oral Biol 1984;29:675–679. Denbesten PK, Crenshaw MA, Wilson MH: Changes in the fluoride-induced modulation of maturation stage ameloblasts of rats. J Dent Res 1985;64:1365–1370. Drinkard CR, Crenshaw MA, Bawden JW: The effect of fluoride on the electrophoretic patterns of developing rat molar enamel. Arch Oral Biol 1983;28:1131–1134. Eanes ED, Hailer AW: The effect of fluoride on the size and morphology of apatite crystals from physiologic solutions. Calcif Tissue Int 1998; 63:250–257. Eastoe JE, Fejerskov Ø: Composition of mature enamel proteins from fluorosed teeth; in Fearnhead RW, Suga S (eds): Tooth Enamel IV. Amsterdam, Elsevier, 1984, pp 326–330. Fejerskov Ø, Thylstrup A, Larsen MJ: Clinical and structural features and possible pathogenic mechanism of dental fluorosis. Scand J Dent Res 1977;85:510–534. Fejerskov Ø, Yaeger JA, Thylstrup A: Microradiography of acute and chronic administration of fluoride on human and rat dentine and enamel. Arch Oral Biol 1979;24:123–130. Fincham AG, Moradian-Oldak J, Simmer JP: The structural biology of the developing enamel matrix. J Struct Biol 1999;126:270–299. Fukae M, Tanabe T, Murakami C, Dohi N, Uchida T, Shimizu M: Primary structure of the porcine 89-kDA enamelin. Adv Dent Res 1996;10: 111–118. Gathercole LJ, Swan AJ, Price G, Dieppe P: Nanometre-scale surface features of arthropathic microcrystals and their relation to protein adsorption: A study by scanning probe microscopy and wide angle X-ray diffraction. J Mater Sci Mater Med 1996;7:511–516. Gerlach RF, Souza AP, Cury JA, Line SRP: Fluoride effect on the activity of enamel matrix proteinases in vitro. Eur J Oral Sci 2000;108:48– 53. Gibson CW, Li Y, Yuan ZA, MacDougall M, Sciutto Kuell J: The Rho signalling pathway in enamel organ cells. J Dent Res 2003;82:144. Guy WS, Taves DR, Brey WS: Organic fluorocompounds in human plasma: Prevalence and characterization. Biochemistry Involving Carbon Bonds Am Chem Soc Symp 1976, pp 117– 134.

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Hall RC, Embery G, Waddington RJ: Modification of the proteoglycans of rat incisor dentinepredentine during in vivo fluorosis. Eur J Oral Sci 2003;104:286–291. Hiller CR, Robinson C, Weatherell JA: Variations in the composition of developing rat incisor enamel. Calcif Tissue Res 1975;18:1–12. Iijima M, Tohda H, Suzuki H, Yanagisawa T, Moriwaki Y: Effects of fluoride on apatite-octacalcium phosphate intergrowth and crystal morphology in a model system of tooth enamel formation. Calcif Tissue Int 1992;50:357–361. Johnsson MSA, Nancollas GH: The role of brushite and octacalcium phosphate in apatite formation. Crit Rev Oral Biol Med 1992;3:61–82. Kay MI, Young RA, Posner AS: Crystal structure of hydroxyapatite. Nature 1964;12:1050–1052. Kerley MA, Kollar EJ: Regeneration of tooth development in vitro following fluoride treatment. Am J Anat 1977;149:181–196. Khokher MA, Dandona P: Fluoride stimulates [3H] thymidine incorporation and alkaline phosphatase production by human osteoblasts. Metabolism 1990;39:1118–1121. Kirkham J, Brookes SJ, Shore RC, Bonass WA, Smith DAM, Wallwork ML, Robinson C: Atomic force microscopy studies of crystal surface topology during enamel development. Connect Tissue Res 1998;38:89–100. Kirkham J, Brookes SJ, Zhang J, Wood SR, Shore RC, Smith DAM, Wallwork ML, Robinson C: Effect of experimental fluorosis on the surface topography of developing enamel crystals. Caries Res 2001;35:50–56. Kirkham J, Robinson C, Weatherell JA, Richards A, Fejerskov O, Josephsen K: Maturation in developing porcine enamel. J Dent Res 1988; 67:1156–1160. Lau WKH, Baylink DJ: Molecular mechanism of fluoride on bone cells. J Bone Miner Res 1990; 13:1660–1667. Lyaruu DM, De Jong M, Bronckers ALJ, Wöltgens JHM: Ultrastructural study of fluoride-induced in vitro hypermineralisation of enamel in hamster tooth germs explanted during the secretory phase of amelogenesis. Arch Oral Biol 1986;31:109–117. McCann HG, Bullock FA: The effect of fluoride ingestion on the composition and solubility of mineralized tissues in the rat. J Dent Res 1957; 36:391–398. Milan AM, Waddington RJ, Embery G: Altered phosphorylation of rat dentine phosphoproteins by fluoride in vivo. Calcif Tissue Int 1999;64:234–238. Milan AM, Waddington RJ, Embery G: Fluoride alters casein kinase II and alkaline phosphatase activity in vitro with potential implications for dentine mineralization. Arch Oral Biol 2001; 46:343–351. Neuman MF, Mulryan BJ: Synthetic hydroxyapatite crystals. IV. Magnesium incorporation. Calcif Tissue Res 1971;7:133–138. Nikiforuk G, Grainger RM: Fluoride carbonatecitrate inter-relations in enamel; in Stack MV, Fearnhead RW (eds): Tooth Enamel 1. Bristol, John Wright and Sons, 1965, pp 26–31.

Okazaki M: Mg2+-F – interaction during hydroxyapatite formation. Magnesium 1987;6:296– 301. Posner AS: Short range order-amorphous precursor in bone: The mineral of bone. Clin Orthop 1985;200:87–99. Posner AS, Eanes ED, Harper RA, Zipkin I: X-ray diffraction analysis of the effect of fluoride on human bone apatite. Arch Oral Biol 1963;8: 549–570. Richards A, Kragstrup J, Josephsen K, Fejerskov O: Dental fluorosis developed in post-secretory enamel. J Dent Res 1986;65:1406–1409. Robinson C, Briggs HD, Atkinson PJ, Weatherell JA: Chemical changes during development of human deciduous enamel. Arch Oral Biol 1981;26:1027–1033. Robinson C, Brookes SJ, Shore RC, Kirkham J: The developing enamel matrix: Nature and function. Eur J Oral Sci 1998a;106(suppl 1): 282–291. Robinson C, Brookes SJ, Wood SR, Kirkham J, Shore RC: The effect of fluoride on the developing mineralised tissues: A brief review. Oralprophylaxe 2003a;25:33–38. Robinson C, Connell S, Brookes SJ, Shore RC, Kirkham J, Smith DAM: pH behaviour of biological hydroxyapatite surfaces, apparent pK values and the effect of fluoride. J Dent Res 2003b:82–83. Robinson C, Hallsworth AS, Kirkham J: Distribution and uptake of magnesium by developing deciduous bovine incisor enamel. Arch Oral Biol 1984;29:479–481. Robinson C, Kirkham J: Is the rat incisor typical? INSERM 1984a;125:377–386. Robinson C, Kirkham J: Enamel matrix components, alterations during development and possible interactions with the mineral phase; in Fearnhead RW, Suga S (eds): Tooth Enamel IV. Amsterdam, Elsevier, 1984b, pp 261–265. Robinson C, Kirkham J: The effect of fluoride on the developing mineralised tissues. J Dent Res 1990;69:685–691. Robinson C, Kirkham J, Hallsworth AS: Volume distribution and concentration of protein, mineral and water in developing dental enamel. Arch Oral Biol 1988;33:159–162. Robinson C, Kirkham J, Shore RC, Brookes SJ, Wood SR: Enamel matrix function and the tuft enigma: A role in directing tissue architecture – a partial sequence of human ameloblastin. Chemistry and Biology of Mineralized Tissues, Proc 6th Int Conf 1998b, vol 34, pp 209–213. Robinson C, Kirkham J, Stonehouse NJ, Shore RC: Control of crystal growth during enamel maturation. Connect Tissue Res 1989;22:139–145. Robinson C, Kirkham J, Weatherell JA: Fluoride in teeth and bone; in Fejerskov Ø, Ekstrand J, Burt BA (eds): Fluoride in Dentistry. Copenhagen, Munksgaard, 1996, p 70. Robinson C, Shore RC, Wood SR, Brookes SJ, Smith DAM, Wright JT, Connell S, Kirkham J: Subunit structures in hydroxyapatite crystal development in enamel: Implications for amelogenesis imperfecta. Connect Tissue Res 2003c;44:1–7.

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Uchida T, Murakami C, Dohi N, Wakida K, Satoda T, Takahashi O: Synthesis, secretion, degradation and fate of ameloblastic during the matrix formation stage of the rat incisor as shown by immunocytochemistry using region-specific antibodies. J Histochem Cytochem 1997;45: 1329–1346. Van der Lugt W, Knottnerus DIM, Young RA: Nuclear magnetic resonance determination of the fluorine position in hydroxyapatite; in Fearnhead RW, Stack MV (eds): Tooth Enamel II. Bristol, John Wright and Sons, 1971, pp 24– 30. Varughese K, Moreno EC: Crystal growth of calcium apatites in dilute solutions containing fluoride. Calcif Tissue Int 1981;33:431–439. Walton RE, Eisenmann DR: Ultrastructural examination of various stages of amelogenesis in the rat following parenteral fluoride administration. Arch Oral Biol 1974;19:171–182. Weatherell JA, Deutsch D, Robinson C, Hallsworth AS: Fluoride concentrations in developing enamel. Nature 1975;256:230–232. Weatherell J, Deutsch D, Robinson C, Hallsworth AS: Assimilation of fluoride by enamel throughout the life of the tooth. Caries Res 1977;11:85–115.

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Wergedal JE, Lau KHW, Baylink DJ: Fluoride and bovine bone extract influence cell proliferation and phosphatase activities in human bone cell cultures. Clin Orthop 1988;233:274–282. Wright JT, Chen SC, Hall KI, Yamauchi M, Bawden JW: Protein characterization of fluorosed human enamel. J Dent Res 1996;75:1936– 1941. Wright JT, Chen SC, Heffernan LN: Enamel proteases in secretory and maturation enamel of rats ingesting 0 and 100 ppm fluoride in drinking water. Adv Dent Res 1989;3:199–202. Wu L, Forsling W, Schindler PW: Surface complexation of calcium minerals in aqueous solutions. I. Surface protonation at fluorapatite interfaces. J Colloid Interface Sci 1991;147:178– 185. Wu W, Nancollas GH: Kinetics and surface energy approaches to the crystallization of synthetic and biological calcium phosphates. Phosphorus Sulfur Silicon 1999;144–146:125–128. Yanagisawa T, Takuma S, Tohda H, Fejerskov Ø, Fearnhead RW: High resolution electron microscopy of enamel crystals in cases of human dental fluorosis. J Electron Microsc 1989;38: 441–448.

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Caries Res 2004;38:277–285 DOI: 10.1159/000077767

Sugars – The Arch Criminal? D.T. Zero Department of Preventive and Community Dentistry, Oral Health Research Institute, Indiana University School of Dentistry, Indianapolis, Ind., USA

Key Words Dental caries W Diet W Fluoride W Sugars W Sucrose

Abstract Numerous lines of evidence have conclusively established the role of sugars in caries etiology and the importance of sugars as the principal dietary substrate that drives the caries process has not been scientifically challenged. While sugars appear to differ little in acidogenic potential, sucrose has been given special importance, as the sole substrate for synthesis of extracellular glucans. Water-insoluble glucans might enhance accumulation of mutans streptococci on smooth tooth surfaces and appear to enhance virulence by increasing plaque porosity, resulting in greater acid production immediately adjacent to the tooth surface. Data indicating that the sugar consumption/caries relationship is now weaker have led to suggestions that recommendations to restrict sugar consumption are no longer necessary. Clearly, fluoride has raised the threshold of sugar intake at which caries will progress to cavitation, but fluoride has its limits, and caries remains a serious problem for disadvantaged individuals in many industrialized countries and is a rising problem in many developing countries. A weakening of the sugar/caries relationship may also be explained by many technical, biological, behavioral and genetic factors. Future research should aim to determine the biologic and behavioral factors that influence caries risk. Measures to educate the public on the dangers of fre-

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quent sugar consumption, combined with recommendations for proper oral hygiene and fluoride use, are still warranted. Individual dietary counseling is highly recommended for patients at high caries risk. As dental caries is a preventable disease, each country must decide: what level of disease is society willing and able to tolerate? Copyright © 2004 S. Karger AG, Basel

Existing Evidence

The role of the consumption of sugars in dental caries is a very broad topic, which is somewhat controversial and eminently important. The title of this article has been carefully crafted by the Symposium Planning Committee and refers to ‘sugars’ and not sugar, which is often used synonymously for sucrose. The term sugars includes all the monosaccharides and disaccharides, the most common of which are glucose, fructose, sucrose, maltose and lactose (table 1) [Moynihan, 1998]. Sucrose, which has been considered to play a special role in dental caries [Newbrun, 1969], will be discussed separately later in the paper. The understanding that sugars are an important etiologic factor in dental caries has been with us since the dawn of civilized man, but the controversy surrounding this subject is a more recent phenomenon. Consistent with comments made by Prof. Fejerskov [2004, this issue], we must keep in mind that science is not conducted

D.T. Zero, DDS, MS Department of Preventive and Community Dentistry, Oral Health Research Institute Indiana University School of Dentistry, 415 Lansing Street Indianapolis, IN, 46202-2876 (USA) Tel. +1 317 274 8822, Fax +1 317 274 5425, E-Mail [email protected]

Table 1. Carbohydrate nomenclature

Monosaccharides Glucose (dextrose) Fructose (fruit sugar) Galactose Invert sugar (1:1 glucose and fructose) Disaccharides Sucrose (table sugar) Maltose Lactose (milk sugar) Trehalose (mushroom sugar) Natural and manufactured oligosaccharides (3–10 units) Polysaccharides (1 10 units) Starch Adapted from Moynihan [1998].

in a vacuum, and many social, political and economic forces come into play, both in the execution of research as well as in its interpretation. Table 2 provides a partial list of review articles that have looked at the role of sugars in dental caries, with most authors supporting the relationship up until fairly recently. This subject has been the focus of many recent review articles and considerable debate, with some authors reaching different conclusions from basically the same studies. There is an extensive literature on this topic. A PubMed search conducted in April 2003, covering papers back to 1965, identified 2,784 articles using the terms ‘dental caries’ AND ‘sugar’. If only articles written in English are included, the number is lowered to 2,027. This short review cannot be all-inclusive and will mainly focus on the clinical data, while recognizing that other model systems, especially animal models, have added considerably to our understanding on this subject. Classic Evidence The classic evidence supporting the role of sugar in dental caries in man is summarized in table 3, and includes studies that are readily recognizable by name – The Vipeholm Study, Turku Sugar Study, World War II Food Rationing, Hopewood House Study, Tristan da Cunha, Hereditary Fructose Intolerance, Experimental Caries in Man, and Stephan Plaque pH Response. No doubt many other studies could be included on this list. This literature collectively still forms in many respects the basis of our understanding of the etiology of dental caries. In particular, the Vipeholm Study has been praised, criti-

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cized and even condemned. In the author’s opinion, the Vipeholm Study remains one of the most important contributions in the entire dental literature and definitively established that the more frequently sugar is consumed, the greater the risk, and that sugar consumed between meals has a much greater caries potential than when consumed during a meal. National Surveys Additional evidence is provided from the analysis of national surveys comparing data on caries experience and sugar supply data (table 4). These reports have established a relationship between sugar consumption and dental caries at the population level. However, it is also evident from the later surveys that the nature of this relationship has changed in most industrialized countries where fluoride use in all forms has dramatically decreased the prevalence of caries at the dentinal caries level in young children [Marthaler et al., 1996]. The weakening of this relationship in industrialized countries may also be explained by the high level of sugar consumption by a majority of the population and the well-known problem of obtaining accurate data on sugar intake [Sreebny, 1982b; Honkala and Tala, 1987; Marthaler, 1990]. The low caries prevalence and high sugar consumption in industrialized countries leaves little room to establish a clear relationship. This relationship is further complicated by the wide variation in sugar consumption patterns among individuals as well as many other factors that will be discussed in the next section of the article. Comparison of the relationship between sugar consumption and caries among different countries is also limited by the reliability of sugar consumption data as well as the reliability of the caries data. There is a lack of consistency in how sugar consumption is reported among countries and in many cases estimates are based on ‘disappearance’ data (and not actual consumption) that are provided by industry or government sources. A wide range of terms are used, including sugar intake, sucrose intake, added sugar, and nonmilk extrinsic sugar, calculated as grams/ person/day, kilograms/person/year, or sugar intake as percentage of total energy intake. Caries data are limited by the many well-known problems, including examiner calibration across different countries and the impact of treatment effects on the F component of DMF scores among countries. It is remarkable that, given these limitations and the complex nature of dental caries, a relationship between sugar intake and caries has been consistently demonstrated.

Zero

Table 2. Review articles on the relationship between sugar (diet) and dental caries

Author(s)

Main conclusions

Marthaler [1967]

foodstuffs containing simple sugars are far more cariogenic than common starchy foods

Newbrun [1969]

called for the specific elimination of sucrose or sucrose-containing foods rather than restricting total carbohydrate consumption

Bibby [1975]

snack foods share importance with sucrose in caries causation

Sreebny [1982a]

total consumption and frequency of intake contribute to dental caries; lacking evidence about the precise definition of the relationship

Newbrun [1982a]

compelling evidence that the proportion of sucrose in a food is one important determinant of its cariogenicity

Sheiham [1983]

sugar is the principal cause of caries in industrialized countries; recommended that sugar consumption be reduced to 15 kg/person/year or below

Shaw [1983]

studies in animals consistent with the clinical evidence on the relationship between sugar and caries

Rugg-Gunn [1986]

cariogenicity of staple starchy foods is low; the addition of sucrose to cooked starch is comparable to similar quantities of sucrose; fresh fruits appear to have low cariogenicity

Bowen and Birkhed [1986]

frequency of eating sugars is of greater importance than total sugar consumption

Walker and Cleaton-Jones [1989] degree of incrimination of sugar as a cause of caries is grossly exaggerated; questioned predictions of reductions in caries from decreases in sugar and snack intakes Marthaler [1990]

in spite of dramatic reductions in caries due primarily to widespread use of fluoride, sugars continue to be the main threat to dental health

Rugg-Gunn [1990]

dietary modification involving restriction on the frequency and amount of extrinsic sugars can be more effective than other control measures

König and Navia [1995]

acknowledged the relationship between frequency and sugar intake and caries; recommended removing the focus away from elimination of sugar and towards improved oral hygiene and use of fluoride toothpaste

Ruxton et al. [1999]

evidence strongly supports formulation of advice on frequency of consumption, not amount

König [2000]

dental health problems do not require any dietary recommendations other than those required for maintenance of general health

van Loveren [2000]

if good oral hygiene is maintained and fluoride is supplied frequently, teeth will remain intact even if carbohydrate-containing food is frequently eaten

Sheiham [2001]

sugars, particularly sucrose, are the most important dietary cause of caries; the intake of extrinsic sugars greater than 4 times a day increases caries risk; sugar consumption should not exceed 60 g/day for teenagers and adults and proportionally less for younger children

Systematic Review There have been many additional epidemiologic (cohort, case-control and cross-sectional) studies evaluating the relationship between sugar consumption and caries risk. The topic has recently been the subject of a systematic review by Burt and Pai [2001], which was conducted as

part of the NIH/NIDCR Consensus Development Conference on Diagnosis and Management of Dental Caries throughout Life. This review specifically addressed the question: ‘in the modern age of extensive fluoride exposure, do individuals with a high level of sugar intake experience greater caries severity relative to those with a lower

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Table 3. Classic evidence from humans supporting the role of sugar in dental caries

Study

Reference(s)

Main conclusions

Vipeholm Study

Gustafsson et al. [1954]

the more frequently sugar is consumed the greater the risk; sugar consumed between meals has much greater caries potential than when consumed during a meal

Turku Sugar Study

Scheinin et al. [1976]

when sugars are almost completely replaced by non-fermentable sugar substitutes (xylitol), caries increment is dramatically reduced; fructose is less cariogenic than sucrose

World War II

Toverud [1957a, b] Takeuchi [1961]

caries decreased and increased with sugar consumption during and after the war, respectively

Hopewood House

Harris [1963]

modern diet more cariogenic than vegetarian low sugar diet

Tristan da Cunha

Holloway et al. [1963] Fisher [1968]

introduction of a modern diet including sugar and refined carbohydrates to this remote island greatly increased caries prevalence

Hereditary Fructose Intolerance

Marthaler [1967] Newbrun et al. [1980]

less caries in individuals that must avoid sucrose and fructose, but not other sugars and complex carbohydrate

Experimental Caries in Man

von der Fehr et al. [1970] Geddes et al. [1978]

incipient caries can be rapidly induced by frequent rinsing with highconcentration sucrose solutions in the absence of oral hygiene

Stephan Plaque pH Response

Stephan [1940, 1944]

demonstrated the relationship between sugar exposure resulting in the acidification of dental plaque and caries experience

Table 4. Data from national surveys

Reference

Parameters

Main findings/conclusions

Sreebny [1982b]

dmft in 6-year-olds from 23 nations and DMFT in 12-year-olds from 47 nations; sugar supply (g/person/day)

significant positive correlation (r = 0.72; p ! 0.005) between caries prevalence and national sugar supplies for 12-year-olds only; ingestion of 50 g of sugar/day may be the upper limit of ‘safe’ or ‘acceptable’ sugar consumption

Woodward and Walker [1994]

DMFT in 12-year-olds from 90 nations; sugar supply (kg/person/year)

linear relationship between DMFT and sugar consumption when all 90 nations were included; no evidence of a relationship with a separate analysis of 29 industrialized nations

Miyazaki and Morimoto [1996]

DMFT in 12-year-olds (kg sugar/year); 1957–1987; low fluoride exposure

excellent correlation (r = 0.91; p ! 0.01) between DMFT and per capita sugar consumption in Japan

van Palenstein Helderman et al. [1996]

caries experience in Africa, Europe and North America; salivary mutans streptococci

caries experience on three continents is attributable to dietary differences and not prevailing mutans streptococci species

Downer [1999]

dmft in 5-year-olds and DMFT in 12-year-olds; available sugar (kg/person/year)

strong positive correlation over time (50 years) between caries experience and national sucrose availability in the UK

level of intake?’ A total of 809 papers were identified in their initial MEDLINE and EMBASE search. Of these, 69 papers met their inclusion-exclusion criteria and were scored and recorded in evidence tables. Thirty-six papers with a quality score of 55 or higher were rated for the

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strength of the relationship between sugar and caries and were used as the basis for their conclusions. They reported that only 2 papers found a strong relationship, 16 found a moderate relationship and 18 found the relationship to be weak-to-none (table 5). Based on this systematic review,

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Table 5. Systematic review evaluating the relationship between sugar consumption and caries risk

Other Evidence Other indirect evidence in support of the role of sugars in caries comes from animal studies, in situ studies, plaque pH studies, and laboratory studies. In particular, animal studies have been highly supportive of the clinical

data [Shaw, 1983], and have added key elements to our current understanding. The main use of these model systems has been to evaluate the cariogenic potential of individual food items with the aim of ranking them, which is something that cannot be done in human clinical trials due to the impact of a highly variable background diet. Based on two consensus conferences, one in San Antonio, Tex., USA, sponsored by the American Dental Association in 1985, and a more recent one in Hertfordshire, UK, sponsored by the British Dental Association in 1999, the animal caries and human plaque pH models were considered acceptable methods [Stamm et al., 1986; Curzon and Hefferren, 2001]. The more recent UK conference also supported the use of in situ models for this purpose. A working group consensus report from the San Antonio conference stated that ‘the true cariogenicity of a food can only be established by experimentally determining in humans the extent of tooth decay associated with a given food’, while cariogenic potential was defined as ‘a food’s ability to foster caries in humans under conditions conducive to caries formation’ [Stamm et al., 1986]. The cariogenic potential of a particular food or beverage is influenced by its properties, most importantly the sugar content and the presence of protective factors, and the consumption pattern, most importantly the frequency of consumption [Bowen et al., 1980]. Edgar [1985] further divided the possible factors that can influence the cariogenicity of foods into food factors (amount and type of carbohydrate; food pH and buffering power; food consistency and retention in the mouth; eating pattern; factors influencing the oral flora; factors modifying enamel solubility; sialogogue properties, and other substrates for bacterial metabolism) and cultural and economic factors (availability and distribution; selection, and marketing). Several approaches have been recommended for ranking foods. Bowen et al. [1980] developed the cariogenic potential index that uses the rat caries model. The cariogenic potential index is calculated by dividing the rat caries score for the test food by the rat caries score for pure sucrose. Several authors [Krasse, 1985; Burt and Ismail, 1986] have supported the contention that a combination of the tests would be a more valid way of ranking the relative cariogenic potential of foods as has been proposed by Matsukubo et al. [1985]. Using the various models, a wide array of foods with varying types and concentrations of sugar have been shown to have cariogenic potential. Some methods in particular, such as the indwelling plaque pH method, seem to be very sensitive to even low sugar foods that are not normally implicated in dental caries, and may accentuate

Sugars – The Arch Criminal?

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Study design

Relation between sugar intake and caries strong

moderate

weak

totals

Cohort Case-control Cross-sectional

1 0 1

6 1 9

5 0 13

12 1 23

Totals

2

16

18

36

Strong = Risk ratio (odds ratio or relative risk) 62.5; moderate = risk ratio between 1.5 and 2.4; weak = risk ratio ^1.4 (adapted from Burt and Pai [2001]).

the authors concluded that while the relationship between sugar consumption and caries is not as strong as it was in the prefluoride era, restriction of sugar consumption still has an important role in caries prevention. There are limitations to these types of epidemiological studies as well. Most studies use different kinds of dietary surveys including 24-hour recall interviews, 2-, 3- and 7day diet diaries, 7-day weighed inventories, and food frequency questionnaires. However, these tools have only rarely been validated. Furthermore, the role of sugar in caries etiology is quite complex because sugar is rarely eaten in a pure form. The cariogenicity of sugar-containing foods can be modified by many factors including the amount and type of carbohydrates (sucrose vs. other sugars, sugar/starch combinations), protective components (proteins, fats, calcium, phosphate, fluoride), and physical and chemical properties (liquid vs. solid, retentiveness, solubility, pH, buffering capacity, sialagogue properties). While some studies have measured frequency of ingestion, most studies do not account for other behaviors associated with food consumption, such as eating sequence in relationship to other foods, eating before bedtime, late night snacks, and behaviors after food consumption, such as oral hygiene and fluoride use, and gum chewing. In addition, environmental, genetic, social, economic, political and educational factors may confound the relationship between sugar consumption and caries, if not controlled for.

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food retention, creating a worst case scenario [Edgar and Geddes, 1986]. Starches have also been shown to have cariogenic potential in many model systems, including in vitro models [Renz and Bibby, 1989], plaque acidity models [Lingström et al., 1993], animal caries models [Firestone et al., 1982; Mundroff et al., 1990] and in situ caries models [Brudevold et al., 1988; Kashket et al., 1994; Lingström et al., 1994; Pollard, 1995]. However, definitive data in humans are lacking [Lingström et al., 2000], suggesting that some degree of caution should be used when interpreting results from these models. While the ability to rank foods based on their relative cariogenic potential seems desirable, there are several problems with this approach. Both the human plaque acidity models and animal caries models do not account for how foods are actually consumed, in regard to the frequency of ingestion, patterns of ingestion, or relationship of the dietary intake of other foods, which can greatly modify the actual cariogenicity of a given food. Furthermore, the actual susceptibility of a given individual to caries will vary mainly based on the composition of their oral microflora, their salivary flow rate and composition, and fluoride exposure. In situ demin/remin models have several advantages in this regard. The question has also been raised as to how can the relative ranking of foods with different cariogenic potential be employed for dietary counseling and caries control [Burt and Ismail, 1986]. Once a food is determined to have cariogenic potential, is it ethical to recommend that food item to a patient over another food that is ranked slightly more cariogenic based on cariogenicity testing? Special Role of Sucrose While the original claim that ‘Sucrose is the Arch Criminal of Dental Caries’ [Newbrun, 1969] has been softened over the years, it continues to be the most common form of added sugar in the diet, even with continuation of the trend towards increased used of high fructose corn syrup in many industrialized countries. There does not appear to be any difference in the acidogenic potential [Imfeld, 1977] or the ability to directly induce in situ enamel demineralization [Koulourides et al., 1976] among the common sugars, sucrose, maltose, glucose and fructose. Lactose has less acidogenic potential than the other sugars and, as a constituent of milk is not considered to be cariogenic mainly due to the protective factors in milk. Sucrose has been given special importance due to its involvement as the sole substrate in the synthesis of extracellular (water-soluble and water-insoluble) glucans medi-

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tated by microbial glycosyltransferases, which have been the subject of intense study for many years. Glucans can form a major component of the structural intermicrobial matrix of dental plaque [Guggenheim, 1970]. It has been proposed that water-insoluble glucans enhance the ability of mutans streptococci to accumulate on the smooth surfaces of teeth [Gibbons, 1984]. When compared to other dietary sugars (glucose, fructose, and lactose) in the rat caries model, sucrose has been shown to be more cariogenic in some studies. However, the effect appears to be strain specific and is also influenced by the type of animal model and the effect is generally associated with smooth surfaces [Frostell et al., 1967; Tanzer, 1979; Van Houte and Russo, 1986]. However, the specific cariogenicity of sucrose compared to equimolar mixtures of glucose + fructose was not supported by a study conducted in monkeys [Colman et al., 1977]. Based on differences in sucrose consumption between the USA and Britain, Burt [1993] suggested that replacement of sucrose by monosaccharides may reduce proximal- and smooth-surface caries. Several studies have indicated that the caries-associated virulence of glucan may have more to do with an alteration in plaque ecology than effects on the accumulation of specific bacteria in plaque, whereby sucrose-mediated synthesis of glucans increases the porosity of plaque, permitting deeper penetration of dietary sugar into the biofilm and greater acid production immediately adjacent to the tooth surface [Dibdin and Shellis, 1988; van Houte et al., 1989; Zero, 1993]. In studies using an intraoral caries model, Streptococcus mutans plaque prepared from sucrose-containing cultures was found to have markedly enhanced demineralization potential compared with glucose grown plaque [Zero et al. 1986; Cury et al. 2000]. The effect has been attributed to an alteration of the diffusion properties of plaque due to the presence of water-insoluble glucan synthesized from sucrose. Recent studies have found a relationship between water-insoluble glucan synthesis by mutans streptococcal strains and caries incidence in young children and suggested that the capacity of mutans streptococci to synthesize insoluble glucans may be more important than their levels in plaque [Mattos-Graner et al., 2000; Nobre dos Santos et al., 2002].

Where Are We Now?

While the classical literature continues to inform us, more recent data indicate that the relationship between sugar consumption and dental caries is not as strong as it

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was in the prefluoride era. In industrialized countries where fluoride exposure has become the norm through the use of fluoride dentifrice and/or water/salt fluoridation and other vehicles of fluoride delivery, the relationship between sugar and caries has been more difficult to demonstrate. This has led some authors to conclude that recommendations to restrict sugar consumption may no longer be necessary [König, 2000; van Loveren, 2000]. Clearly, fluoride has raised the threshold at which the caries process will progress to a frank cavitation, in that a higher cariogenic diet can be tolerated before caries occurs in many individuals. However, fluoride has its limits, and caries remains a serious problem for economically disadvantaged individuals and new immigrants in many highly industrialized countries. It is a rising problem in many developing countries, where sugar consumption is increasing, fluoride use has not been widely adopted and the provision of dental care is not available. The expected results from these population-based human experiments have unfortunately been shown time and again. There continues to be a discussion about the nature of the relationship between sugar intake and caries and whether there is a safe level of sugar intake. Newbrun [1982b] proposed that the relationship can be best described by an S-shaped curve based on animal studies, and speculated that the S-shaped curve may have moved to the right in the postfluoride era (fig. 1a and b, respectively). Woodward and Walker [1994] reported that the relationship is linear based on their analysis of sugar consumption in 90 countries (fig. 1c). In individuals with good oral hygiene and regular fluoride exposure, higher levels of sugar consumption may be tolerated before caries occurs (author’s conjecture) (fig. 1d).

Future Perspectives for Research

Given that not everyone on a high sugar diet will get caries, research should be directed at determining the biologic and behavioral factors that influence diet-related caries risk. The importance of sugar concentration in relationship to plaque accumulation at caries-susceptible sites requires further investigation, especially in regard to fluoride regimes necessary to counteract the caries challenge and the limitations thereof. There is a need for clinical studies that specifically address the role of sucrose compared to other sugars, including the clinical testing of existing intervention strategies and the development of new strategies directed at blocking the glucan-mediated cariogenic effects of sucrose. A related question for study

Sugars – The Arch Criminal?

Fig. 1. Proposed relationships between sugar intake and caries. a S-shaped relationship in the prefluoride era [Newbrun, 1982b]. b S-shaped relationship shifted to the right in postfluoride era [Newbrun, 1982b]. c Linear relationship calculated from data in 90 countries [Woodward and Walker, 1994]. d Individuals with good oral

hygiene and regular fluoride exposure (author’s conjecture).

is – how much dietary sucrose/sugar exposure is necessary to change the ecological conditions in plaque biofilm in favor of caries progression? Another conjecture worthy of investigation is that if glucan also enhances the diffusion of fluoride as well as sugar substrates through plaque, this may explain in part why fluoride is effective in counteracting the cariogenicity of sucrose. Research should be directed at the problem of increased soft drink consumption, especially high sugar/high caffeine products marketed at populations (adolescents). With an aging dentate population, the relationship between diet and root caries needs more attention. The advances in food science and the interest in ‘functional foods’ create many opportunities to decrease the cariogenic potential of high sugar foods by including protective additives.

Summary and Implications

While some questions have been raised by recent epidemiological data, the importance of sugars as the principal dietary substrate that drives the caries process has not

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been scientifically challenged. The fact that sugars are readily metabolized by oral bacteria, leading to the production of organic acids in sufficient concentration to lower the pH of dental plaque was first shown in clinical studies by Stephan in the forties [1940, 1944]. The direct linkage of the frequent exposure to sugar with dental caries was firmly and irrefutably established by the Vipeholm Study. Experimental caries models in man, animals, and in vitro have all confirmed this linkage. The overall weight of this evidence is exceptionally strong, and based on this review, as well as many others, a causal relationship between sugars and dental caries has been established. This does not mean that other carbohydrates such as starches or different combinations of sugars and starch are not cariogenic. Given the availability of food at every turn and our human propensity to graze, it is very likely that many starch-containing processed foods can contribute to caries formation. All research data are subject to criticism and it is relatively easy to create a climate of doubt in the minds of the public as is evidenced by the effectiveness of antifluoridationists in some parts of the world. To put it in another context, the clinical evidence implicating frequent consumption of sugar in the etiology of caries is much stronger than the evidence supporting the widely held belief that caries is an infectious disease caused by mutans streptococci. This contention is

mainly based on studies in coprophagous rodents, and attempts to control caries using this approach have been unsuccessful thus far. However, there are not any multibillion-dollar industries with a vested interest in discounting the role of a specific microorganism. Although caries has declined in many industrialized countries (even in the presence of increased sugar consumption), we should not be complacent. Dental caries still remains a very costly and widespread disease that in many industrialized countries affects mainly disadvantaged individuals and is of serious concern in many developing countries. At the population level, in industrialized countries, measures to educate the public on the dangers of frequent sugar consumption (especially foods with high sugar concentration) in conjunction with recommendations for proper oral hygiene and fluoride use are still warranted. In developing countries, public health strategies need to be developed to ensure that adequate educational resources and dental public health manpower are available before dental health problems manifest. On an individual basis, dietary counseling is highly recommended for patients that show signs of caries activity and/or are at high caries risk (hyposalivation, iatrogenic factors such as orthodontic brackets). Given that dental caries is a preventable disease, each country must decide: what level of disease is society willing and able to tolerate?

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Caries Res 2004;38:286–293 DOI: 10.1159/000077768

Sugar Alcohols: What Is the Evidence for Caries-Preventive and Caries-Therapeutic Effects? C. van Loveren Department of Cariology/Endodontology/Pedodontology, Academic Centre for Dentistry Amsterdam, Amsterdam, The Netherlands

Key Words Sugar alcohols W Xylitol W Sorbitol W Dental caries W Preventive effect W Therapeutic effect

Abstract The most widely used sugar alcohols are: xylitol, sorbitol, mannitol, maltitol, lactitol and the products Lycasin® and Palatinit®. It is often claimed that xylitol is superior to the other sugar alcohols for caries control. This paper examines clinical studies on the caries-preventive and therapeutic effects of sugar alcohols with emphasis on sorbitol and xylitol. It is concluded that chewing sugarfree gum 3 or more times daily for prolonged periods of time may reduce caries incidence irrespective of the type of sugar alcohol used. It may be sufficient to do this only on school days. Sucking xylitol-containing candies or tablets may have a similar effect as chewing xylitol chewing gum. Clinical trials suggest greater caries reductions from chewing gums sweetened with xylitol than from gums sweetened with sorbitol. However, the superiority of xylitol was not confirmed in 2 out of 4 clinical trials comparing the caries-preventive effect of xylitol- with sorbitol-sweetened gums. The caries-preventive effects of polyol-containing gums and candies seem to be based on stimulation of the salivary flow,

ABC

© 2004 S. Karger AG, Basel 0008–6568/04/0383–0286$21.00/0

Fax + 41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/cre

although an antimicrobial effect cannot be excluded. There is no evidence for a caries-therapeutic effect of xylitol. These conclusions are in line with those of recent reviews and with the conclusions of the Scientific Committee on Medicinal Products and Medical Devices of the EU Commission. Copyright © 2004 S. Karger AG, Basel

The most widely used sugar alcohols are: xylitol (a pentitol), sorbitol, mannitol (both hexitols), maltitol, lactitol (both 12-carbon polyols) and the products Lycasin® (6– 8% sorbitol, 50–55% maltitol, 20–25% maltotriitol and 10–20% polysaccharide alcohols) and Palatinit® (1:1 mixture of two 12-carbon polyols). Other sugar alcohols such as erythritol are emerging [Kawanabe et al., 1992; Mäkinen et al., 2001]. All sugar alcohols have been tested in vitro for fermentation by oral micro-organisms and can be classified as hypo- or non-acidogenic. There is a reduced or virtually no extracellular polysaccharide production from sugar alcohols. Hypo- and non-acidogenicity of the sugar alcohols are confirmed by plaque pH measurements. From animal experiments and intra-oral cariogenicity tests, it is concluded that sugar alcohols are (extremely) low or non-cariogenic. In vitro, adaptation of mutans streptococci by frequent subculturing in sorbitol,

C. van Loveren Department of Cariology/Endodontology/Pedodontology Academic Centre for Dentistry Amsterdam, Louwesweg 1 NL–1066 EA Amsterdam (The Netherlands) Tel. +31 20 5188662, Fax +31 20 6692881, E-Mail [email protected]

Xylitol in chewing gum may reduce the amount of dental plaque [Topitsoglou et al., 1983; Söderling et al., 1989; Mäkinen et al., 1989]. Some studies, however, suggest that sorbitol might be as effective [Wennerholm et al., 1994; Söderling et al., 1989; Birkhed et al., 1979]. Moreover the quantitative effect – a reduction of 10–20% in plaque index – should not be overemphasized from a cariological point of view [Birkhed, 1994]. An interesting effect of xylitol is its ability to reduce the population of mutans streptococci in plaque [Isokangas et

al., 1991; Mäkinen et al., 1989] and to loosen the plaque and mutans streptococci binding to the tooth surfaces [Söderling et al., 1991]. This effect was found to depend on the frequency of chewing and the initial level of mutans streptococci [Mäkinen et al., 1989] and seemed to persist after the habitual use of xylitol had stopped [Isokangas et al., 1991]. However, a 3-month experiment in which 2 lozenges were sucked (in total 4 g of xylitol) 4 times daily showed no reduction of the numbers of mutans streptococci in dental plaque [Birkhed et al., 1979]. Tenovuo et al. [1997] did not find a reduction of the numbers of salivary mutans streptococci after 1 month’s regular use of 3 xylitol lozenges a day. After a 2-week experiment, the numbers of mutans streptococci were not significantly different in plaque or saliva whether either a xylitol (65% w/w), xylitol-sorbitol (37.5% xylitol and 37.5% sorbitol w/w) or unsweetened chewing gum base was chewed 3–5 times a day for approximately 3 min each time [Söderling et al., 1997]. After a period of approximately 4 weeks, no differences were observed in numbers of mutans streptococci in saliva when chewing gum sweetened with 70% xylitol, 35% xylitol and 35% sorbitol or 17.5% xylitol and 52.5% sorbitol was chewed for 5 min 12 times daily [Wennerholm et al., 1994]. In contrast, in patients wearing fixed orthodontic appliances 6 times daily use of gums sweetened with xylitol or a xylitol-sorbitol mixture (4:1 or 3:2) reduced the numbers of mutans streptococci in plaque and saliva [Isotupa et al., 1995]. Reduced salivary numbers of mutans streptococci were also observed after a 3-week period of chewing xylitol gum 3 times a day [Autio, 2002]. Hildebrandt and Sparks [2000] used xylitol gum (3 times daily) to maintain a chlorhexidine-borne reduction of salivary mutans streptococci for 3 months. Wennerholm et al. [1994] showed that when gums were sweetened with 70% sorbitol, there was a small but statistically significant increase in numbers of mutans streptococci from 5.9 B 1.0 to 6.6 B 0.6 log CFU/ml saliva. Also Söderling et al. [1989] found an increase in the numbers of mutans streptococci in saliva after the shortterm use of a sorbitol gum. Concomitantly, they found a lower resting pH in interproximal plaque [Söderling et al., 1989]. Mäkinen et al. [2002] confirmed an increase in the numbers of mutans streptococci in dental plaque after 64 days of use of sorbitol gum 5 times a day. The mutansincreasing effect of sorbitol seems to be counteracted when xylitol is added to the gum [Wennerholm et al., 1994]. In conclusion, the data show that regular use of xylitol is more likely to reduce numbers of mutans streptococci in saliva and plaque than the regular use of sorbitol. How-

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maltitol, lactitol and Lycasin occurs, but this is not likely to be important in vivo when the sugar alcohols are given in combination with a diet rich in sucrose. In all these experiments, xylitol stands out and is widely believed to possess anticaries properties, which may render it superior to the other sugar alcohols for potential caries control [for reviews, see Tanzer, 1995; Trahan, 1995]. With rare exceptions, xylitol is not fermented by oral micro-organisms. Xylitol inhibits the growth of mutans streptococci [Vadeboncoeur et al., 1983] even selectively in mixed chemostat cultures [Bradshaw and Marsh, 1994; Rogers et al., 1991]. It interferes with glycolysis when glucose is used as energy source [Wåler and Rölla, 1983] although this may not be a stable phenomenon in vivo [Scheie et al., 1998]. In vivo there was also no reduction of the acidogenic response of dental plaque to sucrose after periods of using xylitol chewing gums [Wennerholm, et al., 1994] or xylitol mouth rinses [Lingström et al., 1997]. It has been proposed that xylitol weakens the caries-inductive properties of dental plaque colonizing newly erupting tooth surfaces [Isokangas et al., 1993] and that such a caries-protective effect might persist several years after the cessation of use of xylitol products [Hujoel et al., 1999]. From animal experiments, it has been concluded that xylitol is anticariogenic [Havenaar et al., 1984]. The interesting biological properties of sugar alcohols, xylitol in particular, have prompted researchers to explore the clinical effects of these compounds. There is no doubt that substitution of sucrose with polyols in chewing gums and lozenges has a caries-preventive effect. However, a number of claims may not be so well supported. In this paper, the caries-preventive and caries-therapeutic effects of xylitol will be discussed and, where appropriate, compared to those of sorbitol. The present paper does not intend to review exhaustively all aspects of xylitol or any other sugar alcohol.

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ever, the studies on the effect of xylitol did not all confirm a mutans-reducing effect. Moreover the clinical relevance of these reductions of mutans streptococci remains to be shown.

the control group to the test products cannot always be controlled. Most of these potential confounders cannot be ruled out and unless identified and controlled for, it is impossible to estimate their effects on the outcome of the study.

Adaptation to Xylitol Clinical Trials with Sorbitol

Frequent exposure to xylitol may increase the proportions of xylitol-resistant mutans streptococci in the oral cavity [Trahan et al., 1992]. Xylitol-resistant mutans streptococci are lacking the fructose phosphotransferase system responsible for xylitol uptake. So, this adaptation does not imply a risk of acid formation from xylitol but merely minimizes the antimicrobial effects of xylitol. Cariogenic traits of xylitol-resistant mutans streptococci have not been shown to be different from those of xylitol-sensitive strains [Assev et al., 2002; Beckers, 1988]. Moreover, in rat experiments the caries reduction due to the consumption of xylitol was independent of whether the animals were superinfected with the xylitol-resistant or the wild-type strain [Beckers, 1988]. Trahan et al. [1992] observed that the proportion of xylitol-resistant mutans streptococci did not increase in dental plaque but did so in saliva. The explanation frequently offered for this observation is that xylitol-resistant or xylitol-adapted strains shed more easily from the tooth surface than xylitol-sensitive strains. Subsequently, this explanation is often used to explain reduced plaque formation and reduced colonization of mutans streptococci following transmission after a period of frequent use of xylitol. However, evidence is lacking to justify this cascade of explanations. There is no direct evidence for a stable decrease in the adhesive properties of mutans streptococci after the use of xylitol.

Clinical Trials with Sugar Alcohols

Conclusions from clinical trials with chewing gums have in general to be interpreted with caution. Both within and between studies there may be differences that affect the results. There may be differences in socio-economic status of the populations, caries risks, dietary and oral hygiene habits, motivation for oral health and in compliance to the programme. Also the diagnostic criteria may be different or differently interpreted. When study groups are randomized into school classes, the study may not be truly blind. Other confounders such as drop-out rates might be different and have a great effect. Access of

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In the first clinical study with sorbitol, children receiving sorbitol tablets developed 48% less caries than control children that did not get tablets [Slack et al., 1964]. However, frequency of intake and quantity of consumption were not reported. In an early chewing gum study, children that used sorbitol gum 3 times daily had 10% less caries over a 2-year period compared to control children that got no chewing gum [Møller and Poulsen, 1973; Møller, 1977]. A 3-year study in Hungary revealed a 48% caries reduction in children using chocolate sweetened with sorbitol compared to children eating sucrose-sweetened chocolate [Banoczy et al., 1981]. A test group of 7- to 11year-old children consuming 2 sorbitol gums per day did not develop less caries in a 2-year period than control children not receiving gums [Glass, 1983]. The effect of chewing sorbitol gums 3 times daily after meals at school was only small compared to no gum chewing [Beiswanger et al., 1998]. The effect was 8% in the total group and 11% for the children at high risk for caries. No significant caries reductions were observed of 3 nor of 5 times daily chewing at school of a gum sweetened with 55.5% sorbitol and 4.3% xylitol and containing 2% carbamide compared to no gum chewing at school [Petersen and Razanamihaja, 1999]. A recent 3-year-study performed at schools in Lithuania showed no statistically significant reduction in the increment of cavitated lesions after the use of two types of sorbitol gums compared to when no gums were delivered at schools [Machiulskiene et al., 2001]. When non-cavitated lesions were included in the results, a significant 27% reduction was found for one of the sorbitol gums tested. The other sorbitol gum was not effective. In another study, an approximately 38% caries reduction (33% when white spot lesions were included in the analysis) was observed in children that were instructed to use sorbitol gum (65% sorbitol and mannitol) after breakfast (supervised at school), lunch (supervised at school) and evening meal (unsupervised at home) compared to a group that chewed no gums after meals [Szöke et al., 2001]. A 40-month study designed to measure the relative effects of chewing gums sweetened with sorbitol alone, sorbitol/xylitol mixtures and xylitol alone (Belize study)

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In the first clinical xylitol study, the Turku sugar study, dietary sucrose was almost completely replaced by xylitol. After 2 years, the sucrose group had developed 7.2 new DMFS and the xylitol group 0 [Scheinin et al., 1975b]. In a parallel 1-year chewing gum study, the group receiving a sucrose-sweetened gum developed approximately 3 new DMFS, while in the xylitol group (chewing gums sweetened with 50% xylitol and 6% sorbitol) the number of DMFS decreased by 1 DMFS [Scheinin et al., 1975a]. A collaborative WHO field study in Hungary showed a caries-preventive effect in a group of children using xylitol in gums, chocolate and other confectionery daily [Scheinin et al., 1985]. This xylitol group developed 35% less caries than a fluoride control group and 45% less than a nonfluoride, non-xylitol control group. Because of the different daily exposures to fluoride in the 3 groups, it was not clear to what extent the effect could be attributed to the xylitol consumption. Another series of collaborative WHO studies in Thailand and French Polynesia compared fluoride chewing gums sweetened with a mixture of xylitol and sorbitol or sweetened with sucrose with fortnightly fluoride rinsing (0.2 % NaF) [Khambanonda et al., 1983; Barmes et al., 1985]. The results from these studies are conflicting. While one found the fluoride rinsing to be effective, the other did not. Since the experimental designs were not very well controlled, the scientific value of these studies can be questioned.

The effects of chewing gums sweetened with 65% xylitol or with a mixture of 15% xylitol and 50% sorbitol, respectively, were compared with a non-gum group in children of low socio-economic status with a high caries rate [Kandelman and Gagnon, 1990]. The two experimental groups had a DMFS increment of 2.24 surfaces, compared with 6.06 surfaces for the control group. In Finland, the Ylievska studies evaluated the effect of chewing frequency: ^1.5 pieces of xylitol gum a day, 1.5–2.5 pieces or 3 pieces [Isokangas et al., 1988]. All gums were sweetened with xylitol. After 2 years, the DMFS increment in the ^1.5 gum group was not different from the no-gum control group, in the 1.5- to 2.5-pieces group there was a non-significant difference of approximately 30% while in the 3-pieces group the reduction was significant at approximately 55–60%. The Belize study [Mäkinen et al., 1995a] studied the effect of mixtures of sweeteners compared to a no-gum group and a sucrose gum group. Experimental gums were pellet gums sweetened with xylitol or mixtures of xylitol and sorbitol in the ratios 3:2 and 1:3 and gum sticks sweetened with xylitol as the only sweetener. The xylitol gums were chewed either 3 or 5 times a day and the mixtures 5 times a day. The children chewing xylitol pellet gums 5 or 3 times a day developed 73 and 59% fewer caries lesions, respectively, than the nogum chewers. For the xylitol stick gum chewers, these values were 56 and 52%, respectively. The caries reduction was lower when the mixtures were chewed (51% for the 1:3 mixture and 44% for the 3:2 mixture), but the differences were only statistically significant compared with the group using 5 xylitol gum pellets. In a field study in Estonia, the effect of the use of a chewing gum with 65% xylitol 3 times a day for 10 min supervised at school days was compared to the consumption of candies sweetened with 49% xylitol supplemented with maltitol or polydextrose with the same intensity and a non-gum control group [Alanen et al., 2000]. After 3 years, both xylitol groups showed a significant 35–60% reduction in caries increment. There were no statistically significant differences between the experimental groups. A recent 3-year-study in Lithuania showed a 36% reduction in the increment of cavitated lesions (30%, when non-cavitated lesions were included) when children were instructed to use a xylitol gum 5 times a day [Machiulskiene et al., 2001]. In conclusion, there is a considerable amount of evidence that the use of gum or sweets sweetened with xylitol or a mixture of xylitol and sorbitol prevents dental caries when used several times daily. Compared to controls without gum use the effectiveness varied between 30 and 60%.

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showed that originally 10-year-old children chewing sorbitol pellet gums 5 times daily developed 26% fewer caries lesions compared to no-gum chewers and 37% compared to children using sugared chewing gum [Mäkinen et al., 1995a]. In 6-year-old children, chewing sorbitol pellet gum resulted in a 55% decrease in the caries increment in the primary dentition when the children were compared to no-gum users after the 2-year evaluation period [Mäkinen et al., 1996a]. When sorbitol stick gums were chewed, the reduction was 30%. In conclusion, most clinical trials with sorbitol-sweetened gums indicate that between- or after-meal consumption of sorbitol-sweetened chewing gum has a cariespreventive effect in comparison with controls without gum use. The effectiveness may vary between 0 and 30% for the permanent dentition. Chewing sorbitol gum may also be effective in the prevention of caries in the primary dentition. But because of the limited number of studies, it is impossible to quantify this effect.

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Clinical Trials Comparing the Effects of Chewing Xylitol or Sorbitol Gum

Up till now only 4 clinical trials have attempted to compare the caries-preventive effect of xylitol and sorbitol in the same setting. The results of these studies are equivocal. In the Belize study [Mäkinen et al., 1995a], the lowest caries onsets varying from 4.6 to 7.8 lesions per 1,000 surfaces per year were observed among initially 10year-old children that chewed xylitol pellet gums 5 times daily or xylitol sticks 3 times daily. Those children who chewed 5 sorbitol pellets a day developed 12.6 caries lesions per 1,000 surfaces per year. These results indicate that xylitol is superior to sorbitol in reducing the risk of caries onset. The difference should, however, be interpreted with caution. An important confounder may be the drop-out rate, which was as low as 7% in the sorbitol group, but 24 and 49% in the xylitol groups, respectively. For 6-year-olds in the Belize study [Mäkinen et al., 1996a], the caries increments in the primary dentition were not statistically significantly different between the xylitol and sorbitol gum chewers. The caries risks for a primary tooth surface in the xylitol pellet and sorbitol pellet gum groups were 1.8 and 2.2% per year, respectively, while in the no-gum group 5% of the primary tooth surfaces developed caries. When the children used gum sticks sweetened with either xylitol or sorbitol, 2.4 and 3.7%, respectively, of the primary tooth surface developed caries within 1 year. Hence, the pellet gums tended to perform better than the stick gums. The study groups were not completely homogeneous with regard to socio-economic factors; furthermore, the xylitol pellet gum group comprised less than half the number of children as did the other groups. In the Lithuanian chewing gum study with 9- to14year-old children, the xylitol gum group (mean 3-year caries increment 8.1 DMFS) did not perform statistically significantly better than the sorbitol gum group (mean 3-year caries increment 9.0 DMFS) [Machiulskiene et al., 2001]. In this study, the children in the xylitol group were slightly younger and had fewer surfaces present than children in the sorbitol group at the start of the experiment. However, the statistical analyses were adjusted for the effects of age, gender, number of erupted surfaces and baseline DMFS values. In elderly individuals the effect of polyol sweetened gum and dragées on root surface caries was studied in two small groups of approximately 40 individuals each. Approximately 15% of the subjects used gum, the others used the dragées. In the xylitol group there were 2.6 lesion

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onsets per 1,000 surfaces a year contrasting with 13.7 onsets in the sorbitol group [Mäkinen et al., 1996b]. Potential confounders of this study were related to the fact that the individuals in the xylitol group were younger than the individuals in the sorbitol group, and the prebaseline root surface caries increment rates were lower in the sorbitol than in the xylitol group. In conclusion, superiority of xylitol is not supported in 2 out of the 4 studies comparing the caries-preventive effect of xylitol- and sorbitol-sweetened gum directly.

Remineralization

Remineralization as a result of using sugar alcohols has been suggested, but the evidence from clinical trials is less clear. In the Turku sugar studies, there were 3 groups consuming either a sucrose-, fructose- or xylitol-sweetened diet. In the xylitol group, the mean caries increment over a 2-year period dropped during the last 3-month period from 0.8 to 0 [Scheinin et al., 1975b]. These results were interpreted as evidence of remineralization. However, careful clinical and radiographic examination revealed the same number of reversals from ‘caries without a defect’ to ‘sound’ in each diet group. Reversals of more advanced lesions were not observed. In the parallel Turku chewing gum study, a negative increment of 1 surface was observed during 12 months’ use of xylitol gum. However, the report of this study did not differentiate between onsets and reversals [Scheinin et al., 1975a]. So it is not clear whether the negative increment was due to an increased number of reversals, a decreased number of onsets or both. In the Belize chewing gum study with the younger children, ‘rehardening’ was observed in 10–27% of the dentinal lesions [Mäkinen et al., 1995b]. The use of xylitol chewing gum 5 times daily tended to be more effective than when the gum was chewed less frequently or when it was sweetened with sorbitol. However, the types of lesions shown in the publication were mostly large open occlusal caries cavities, which are easily subjected to functional attrition and plaque removal. It is likely therefore that the mechanical effect of chewing, and not the sugar alcohol itself, was responsible for these clinical changes. In an in situ model, Manning et al. [1992] compared the remineralizing effect on preformed caries-like lesions in enamel specimens of chewing 5 times per day gum sweetened with sorbitol or a mixture of sorbitol/xylitol (3:1) after 3 daily meals and 2 sugary snacks. No significant differences were observed between the remineraliz-

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In many of the studies referred to above, the total amount of xylitol was reported, suggesting that a certain amount of xylitol should be consumed to be effective in caries prevention. However, the studies with gums containing different amounts of xylitol do not support this suggestion. For instance, the study of Kandelman and

Gagnon [1990] suggests that as little as 0.9 g of xylitol in chewing gum was sufficient for caries prevention, while the Ylivieska study suggests that at least 7 g of xylitol would be necessary [Isokangas et al., 1988]. Also when comparing the mixed xylitol/sorbitol groups in the Belize study those consuming the lowest daily amount of xylitol (2 versus 6 g a day) had the lowest caries onset risk, although the difference was not statistically significant [Mäkinen et al., 1995a]. In most studies on the effects of sugar substitutes, the participants were asked to chew 3–5 pieces of gum a day. In fact, in a study in which the participants were asked to chew 2 sorbitol-containing gums a day, no caries-inhibitory effect was found [Glass, 1983]. In the Ylivieska study, only 3 gums a day gave protection, while ^2.5 gums did not [Isokangas et al., 1988]. In the Belize study there was no statistically significant difference when the stick gums were chewed 3 or 5 times daily [Mäkinen et al., 1995a]. When chewing xylitol pellet gums, the 5-times chewers demonstrated a lower caries onset risk than the 3-times chewers [Mäkinen et al., 1995a]. So the data suggest that for caries control, gums should at least be used 3 times a day for prolonged periods of time. In the Belize study, children were re-examined 5 years after the original 2-year clinical trial [Hujoel et al., 1999]. It was very unlikely that the children had used any of the experimental products over that 5-year period, since they were not available within a distance of 180 km. Five years after the experiment, the children still had less caries than the original control children. Closer examination revealed that teeth erupted after the experimental period were better protected in children that had chewed gums sweetened with xylitol or a xylitol/sorbitol mixture compared to children that had chewed sorbitol-sweetened gum. Compared to the no-gum group, the protection was most prominent in the group of children that used the gum with 100% xylitol. A continued caries-preventive effect after cessation of using xylitol was also observed in the study of Alanen et al. [2000]. These data suggest a prolonged change in the homoeostasis and cariogenicity of dental plaque. It has been speculated that the adhesive properties of mutans streptococci decrease due to the exposure and adaptation to xylitol [Söderling et al., 1987]. This latter mechanism would also explain why children from mothers that use xylitol frequently are less colonized with mutans streptococci than control children [Isokangas et al., 2000; Söderling et al., 2000; Thorild et al., 2003]. However, there is no direct evidence that there is a stable decrease in the adhesive properties of mutans streptococci after the use of xylitol. The results may at least partly be related to an

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ing effects of the gums, when the specimens were evaluated by transversal microradiography [Manning et al., 1992]. In conclusion, the present state of the art does not support an anticariogenic or caries-therapeutic effect of xylitol.

Chew or Polyol Effect

To measure the caries-preventive effect of the polyols per se, polyol-sweetened gums should be compared to a control gum that does not contain polyols but is sweetened by a non-acidogenic/non-cariogenic sweetener. Recently it has been shown that such a control gum was as effective as a sorbitol- or xylitol-sweetened gum, indicating that the caries-preventive effect of chewing sugar-free gum is related to the chewing process rather than being an effect of the polyols [Machiulskiene et al., 2001]. The importance of chewing would also explain why gum pellets with a harder texture were more effective in caries prevention than were softer gum sticks as demonstrated in the Belize study [Mäkinen et al., 1995a, 1996a]. Chewing stimulates salivary flow [Rosenhek et al., 1993] as does sucking of lozenges [Tenovuo et al., 1997]. It is therefore not surprising that the caries-preventive effect of candies sweetened with 49% xylitol with maltitol or polydextrose has been reported to be similar to that of chewing xylitol (65%) chewing gum [Alanen et al., 2000]. If there were an effect of polyols per se it might become visible when subjects rinsed with a polyol solution. Giertsen et al. [1999] showed, however, no effect of 4 weeks 3 times daily rinsing for 1 min with a 40% xylitol solution on salivary flow rate, on the total number of colony-forming units, streptococci or mutans streptococci in saliva, on dental plaque accumulation, gingivitis development or the acidogenic potential of plaque. This study does not exclude a xylitol effect per se, since the results may partly be explained by the fact that the rinsing protocol did not give sufficient substantivity to a xylitol effect.

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increased awareness for dental health as a result of a longterm, intensive, compliance-demanding preventive regimen.

Conclusions

Regular use of xylitol is more likely to reduce the numbers of mutans streptococci in saliva and plaque than regular use of sorbitol; however, not all studies on the effect of xylitol confirm a mutans-reducing effect. Moreover, the clinical relevance of a reduction of the intra-oral load of mutans streptococci is not clear. Chewing of sugar-free chewing gum 3 or more times daily for prolonged periods of time may reduce caries incidence irrespective of the type of sugar alcohol added. It may be sufficient to do this only on school days. Sucking of xylitol-containing candies

or tablets may have a similar effect as chewing xylitol chewing gum. Clinical trials suggest a higher caries-preventive effect of chewing gum sweetened with xylitol than with sorbitol. However, superiority of xylitol was not confirmed in 2 out of 4 clinical trials comparing the cariespreventive effect of xylitol- and sorbitol-sweetened gums. The caries-preventive effects of polyol-containing gums and candies seem to be based on stimulation of the salivary flow, although an antimicrobial effect cannot be excluded. There is no evidence for a minimal therapeutic dose or a caries-therapeutic effect of xylitol. These conclusions are in line with those of previous recent reviews [Imfeld, 1994; Scheie and Fejerskov, 1998] and with the conclusions of the Scientific Committee on Medicinal Products and Medical Devices of the EU Commission [EU Commission, 2003].

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Mäkinen KK, Benett CA, Hujoel PP, Isokangas PJ, Isotupa KP, Rape HR, Mäkinen P-L: Xylitol chewing gums and caries rates: A 40-month cohort study. J Dent Res 1995a;74:1904– 1913. Mäkinen KK, Hujoel PP, Benett CA, Isotupa KP, Mäkinen P-L, Allen P: Polyol chewing gums and caries rates in primary dentition: A 24month cohort study. Caries Res 1996a;30:408– 417. Mäkinen KK, Isotupa KP, Kivilompolo T, Mäkinen P-L, Murtomaa S, Petaja J, Toivanen J, Söderling E: The effect of polyol-combinant saliva stimulants on S. mutans levels in plaque and saliva of patients with mental retardation. Spec Care Dentist 2002;22:187–193. Mäkinen KK, Isotupa KP, Kivilompolo T, Mäkinen P-L, Toivanen J, Söderling E: Comparison of erythritol and xylitol saliva stimulants in the control of dental plaque and mutans streptococci. Caries Res 2001;35:129–135. Mäkinen KK, Mäkinen P-L, Pape HR Jr, Allen P, Benett CA, Isokangas PJ, Isotupa KP: Stabilisation of rampant caries: Polyol gums and arrest of dentine caries in two long-term cohort studies in young subjects. Int Dent J 1995b;45: 93–107. Mäkinen KK, Pemberton D, Mäkinen P-L, Chen C-Y, Cole J, Hujoel PP, Lopatin D, Lambert P: Polyol-combinant saliva stimulants and oral health in Veterans Affairs patients – An exploratory study. Spec Care Dentist 1996b:16:104– 115. Mäkinen KK, Söderling E, Isokangas P, Tenovuo J, Tiekso J: Oral biochemical status and depression of Streptococcus mutans in children during 24- to 36-month use of xylitol chewing gum. Caries Res 1989;23:261–267. Manning RH, Edgar WM, Agalamanyi EA: Effects of chewing gums sweetened with sorbitol or a sorbitol/xylitol mixture on the remineralisation of human enamel lesions in situ. Caries Res 1992;26:104–109. Møller IJ: Sorbitol containing chewing gum and its significance for caries prevention. Dtsch Zahnärztl Z 1977;32(suppl 1):66–70. Møller IJ, Poulsen S: The effect of sorbitol-containing chewing gum on the incidence of dental caries, plaque and gingivitis in Danish schoolchildren. Community Dent Oral Epidemiol 1973; 1:58–67.

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Petersen PE, Razanamihaja N: Carbamide-containing polyol chewing gum and prevention of dental caries in Madagascar. Int Dent J 1999; 49:226–230. Rogers AH, Pilowsky KA, Zilm PS, Gully NJ: Effects of pulsing with xylitol on mixed continuous cultures of oral streptococci. Aust Dent J 1991;36:231–235. Rosenhek M, MacPherson LMD, Dawes C: The effects of chewing-gum stick size and duration of chewing on salivary flow rate and sucrose and bicarbonate concentrations. Arch Oral Biol 1993;38:885–891. Scheie AA, Fejerskov OB: Critical review – Xylitol in caries prevention: What is the evidence for clinical efficacy? Oral Dis 1998;4:268–278. Scheie AA, Fejerskov OB, Danielsen B: The effect of xylitol-containing chewing gums on dental plaque and acidogenic potential. J Dent Res 1998;77:1547–1552. Scheinin A, Mäkinen KK, Tammisalo E, Rekola M: Turku sugar studies. XVIII. Incidence of dental caries in relation to 1-year consumption of xylitol chewing gum. Acta Odontol Scand 1975a;33(suppl 70):307–316. Scheinin A, Mäkinen KK, Ylitalo K: Turku sugar studies. V. Final report on the effect of sucrose, fructose and xylitol diets on caries incidence in man. Acta Odontol Scand 1975b;33(suppl 70):67–104. Scheinin A, Pienihäkkinen K, Tiekso J, Banoczy J, Szoke J, Esztari I, Zimmermann P, Hadas E: Collaborative WHO xylitol field studies in Hungary. VII. Two-year caries incidence in 976 institutionalized children. Acta Odontol Scand 1985;43:381–387. Slack GL, Millward E, Martin WJ: The effect of tablets stimulating salivary flow on the incidence of dental caries: A two-year clinical trial. Br Dent J 1964;116:105–108. Söderling E, Alaräisänen L, Scheinin A, Mäkinen KK: Effect of xylitol and sorbitol on polysaccharide production by and adhesive properties of Streptococcus mutans. Caries Res 1987;21: 109–116. Söderling E, Isokangas P, Pienihäkkinen K, Tenovuo J: Influence of maternal xylitol consumption on acquisition of mutans streptococci by infants. J Dent Res 2000;79:882–887. Söderling E, Isokangas P, Tenovuo J, Mustakallio S, Mäkinen KK: Long-term xylitol consumption and mutans streptococci in plaque and saliva. Caries Res 1991;25:153–157.

Söderling E, Mäkinen KK, Chen CY, Pape HR Jr, Loesche W, Mäkinen PL: Effect of sorbitol, xylitol, and xylitol/sorbitol chewing gums on dental plaque. Caries Res 1989;23:378–384. Söderling E, Trahan L, Tammialai-Salonen T, Häkkinen L: Effects of xylitol, xylitol-sorbitol, and placebo chewing gums on the plaque of habitual xylitol consumers. Eur J Oral Sci 1997;105: 170–177. Szöke J, Ba´no´czy J, Proskin HM: Effect of aftermeal sucrose-free gum chewing on clinical caries. J Dent Res 2001;80:1725–1729. Tanzer JM: Xylitol chewing gum and dental caries. Int Dent J 1995;45:65–76. Tenovuo J, Hurme T, Ahola A, Svedberg C, Ostela I, Lenander-Lumikari M, Neva M: Release of cariostatic agents from a new buffering fluoride- and xylitol-containing lozenge to human whole saliva in vivo. J Oral Rehabil 1997;24: 325–331. Thorild I, Lindau B, Twetman S: Effect of maternal use of chewing gums containing xylitol, chlorhexidine or fluoride on mutans streptococci colonization in the mothers’ infant children. Oral Health Prev Dent 2003;1:53–57. Topitsoglou V, Birkhed D, Larsson L-Å, Frostell G: Effect of chewing gums containing xylitol, sorbitol or a mixture of xylitol and sorbitol on plaque formation, pH changes and acid production in human dental plaque. Caries Res 1983;17:369–378. Trahan L: Xylitol: A review of its action on mutans streptococci and dental plaque – Its clinical significance. Int Dent J 1995;45:77–92. Trahan L, Derling ES, Drean MF, Chevrier MC, Isokangas P: Effect of xylitol consumption on the plaque saliva distribution of mutans streptococci and the occurrence and long-term survival of xylitol resistant strains. J Dent Res 1992;71:1785–1791. Vadeboncoeur C, Trahan L, Mouton C, Mayrand D: Effect of xylitol on the growth and glycolysis of acidogenic oral bacteria. J Dent Res 1983; 62:882–884. Wåler SM, Rölla G: Effect of xylitol on dental plaque in vivo during carbohydrate challenge. Scand J Dent Res 1983;91:256–259. Wennerholm K, Arends J, Birkhed D, Ruben J, Emilson CG, Dijkman AG: Effect of xylitol and sorbitol in chewing gums on mutans streptococci, plaque pH and mineral loss of enamel. Caries Res 1994;28:48–54.

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Caries Res 2004;38:294–304 DOI: 10.1159/000077769

Are We Ready to Move from Operative to Non-Operative/Preventive Treatment of Dental Caries in Clinical Practice? N.B. Pitts Dental Health Services Research Unit and Centre for Clinical Innovations, University of Dundee, Dundee, UK

Key Words Clinical management W Dental caries W Treatment planning W Treatment strategies

Abstract This review focuses on the clinical interactions between patients and the dental team, not on caries prevention at a public health level. Many dentists no longer take a narrow surgical view seeking to apply interventive treatment as a one-off event at a certain trigger point of disease severity and the evidence that caries is an initially reversible, chronic disease with a known multi-factorial aetiology is being appreciated more widely. The caries process should be managed over time in an individualized way for each patient. Very few individuals can be considered to be truly ‘caries free’ when initial lesions as well as more advanced dentine lesions are considered. It is now very clear that, by itself, restorative treatment of the disease does not ‘cure’ caries. The caries process needs to be managed, in partnership with patients, over the changing challenges of a lifetime. The answer to the question posed in the title should be, in many cases, that we are ready to move to non-operative/preventive care (if we have not done so already). However, this should be for appropriate stages of lesion extent and in patients who respond to advice on recall frequency and preventive behaviours.

The aims of this paper were to bring together the very broad areas of evidence relevant to the important clinical question posed by the organizers and to present this in a framework accessible to an audience including, but not limited to, the caries research community and those undertaking clinical caries management. Although dental public health aspects are mentioned, their full consideration is outside the remit of this review. In northern Europe, an overtly preventive philosophy has existed for many years. For example, in the Nordic public dental services, recommendations to be circumspect with operative intervention have long been in place. Although the overall decline in caries can be attributed to a range of factors outside the impact of direct dental care, not least the widespread use of fluoride toothpastes, a more restrained approach to placing the first restoration has also been seen to have a beneficial impact on oral health [Heidmann et al., 1987]. In the USA, however, Ismail et al. [2001] found that much of general dentistry is still within the ‘restorative era’, although there is a growing interest in preventive management. Since 1998, pilot studies of new forms of general dental practice service – the personal dental services – have been made in England [MacLeod et al., 2003]. Some of the studies provided an opportunity for the personal dental services dentists to promote a less interventionist approach to dental care. One study reduced the rate of filled teeth (from 81 to 61 per 100 adult registrations) and then

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Fig. 1. The evidence-based dentistry (EBD) matrix and ORCA’s objectives.

sustained the low rate compared with local general dental practitioners. The capitation-based studies illustrated the potential for dentists to change their prescribing behaviour and provide care which was commonly viewed as being in the long-term benefit of their patients [MacLeod et al., 2003]. If we are to ascertain how to manage caries, we must, in this era of evidence-based health care (EBH) and evidence-based dentistry (EBD), reflect both systematic reviews (SRs) of the literature (the objective state of the science) as well as expert and consensus views on clinical practice (the state of the art). The science and the art can then be integrated into a rational framework for clinical practice, which will determine the pace of the move from operative to non-operative/preventive treatment. The move to EBH is a global phenomenon, but is taking place at a variety of speeds in different countries. The EBH philosophy requires a more open approach from health professionals. Higher value is now given to robust research findings. Lower quality research and expert opinion alone are given limited credence but become important guides in areas where there is no high quality research

relating directly to the clinical question. In a number of countries, patients and their views are also becoming increasingly empowered, for example in the Options for Change developments in England [Department of Health, 2002]. Research in a particular field is now objectively assessed and synthesised by formal SRs, ideally of randomised clinical trials. EBD is designed to help the clinician and patient when addressing specific clinical dental questions. It has been usefully defined by the American Dental Association [American Dental Association, 2002] as ‘an approach to oral health care that requires the judicious integration of (1) systematic assessments of clinically relevant scientific evidence, relating to the patient’s oral and medical condition and history, with (and this is the part that is often missed) (2) the dentist’s clinical expertise and (3) the patient’s treatment needs and preferences.’ To fulfil these requirements, a body of knowledge must first be identified, then objectively synthesised, before being communicated to clinicians and their patients in ways that encourage effective implementation of evidence in practice. Figure 1 is a flow chart illustrating the various

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processes which together make up EBD. It also shows how the resulting clinical practice should then feed back to new primary research evaluating the new practice once again. This figure also demonstrates that the modern EBD approach to oral health care is in fact very well served by the formal objectives of ORCA.

Scientific Background – Summary

In order to review, discuss and advance clinical caries management, it is imperative to agree on and employ modern definitions of the disease process and diagnosis of the disease which are both scientifically and clinically viable. This area was considered at length at the International Consensus Workshop on Caries Clinical Trials [Pitts and Stamm, in press]. The agreed consensus statements relating to disease definition express that: (1) the caries process occurs as an interaction between the biofilm and the tooth surface and subsurface; the caries lesion is the manifestation of the stage of the process at one point in time; (2) caries progression occurs when the demineralisation and remineralisation equilibrium is out of balance, leading to net mineral loss, and (3) remineralisation can arrest or reverse progression of disease and can lead to changes in mineral quality; the understanding of the caries process has progressed far beyond the point of restricting the evidence for dental caries to the D2 (caries in enamel only) or D3 (caries in enamel and dentine) levels of cavitation. The agreed consensus statements relating to the detection and diagnosis of caries express that there is considerable confusion with the terminology employed in the literature. The three agreed key terms of direct relevance to preventive caries care were: (1) lesion detection: implies an objective method of determining whether or not disease is present; (2) lesion assessment: aims to characterise or monitor a lesion, once it has been detected, and (3) caries diagnosis: should imply a human, professional, summation of all available data. It is now appreciated that caries is an initially reversible, chronic, disease process with a known multi-factorial aetiology [Fejerskov and Manji, 1990; Kidd, 1996; Baelum and Fejerskov, 2003]. The stages of the caries process, including clinical lesions in enamel, are well documented over many years [Backer-Dirks et al., 1951; Marthaler, 1965; Møller, 1966; Silverstone, 1973; World Health Organization, 1979; Manji et al., 1991; Kidd, 1996; Fejerskov et al., 2003].

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The state of the art in understanding lesion detection recognises that very few individuals can be considered to be truly ‘caries free’. This is not a new observation; Magitot [1886] some 118 years ago suggested that we switch our focus away from defining dental caries only as ‘cavities’ and should recognise the value of detecting the stages of the caries process. The stages of caries have been represented graphically as an iceberg of dental caries experience [Pitts, 1997a], which is summarised in a simplified form as figure 2. The earliest changes to the dental enamel (at the base of the iceberg) are subclinical, subsurface demineralisations [Fejerskov et al., 2003], often at inaccessible sites. These lesions are extremely common and can often be found when apparently sound surfaces on extracted teeth are subsequently examined histologically. The next level (or diagnostic threshold) up comprises lesions which can be visualised as caries in enamel, usually with apparently ‘intact’ surfaces. Because of the intrinsic insensitivity of unaided visual detection, a proportion of these lesions will be unseen by even a diligent examiner, and thus ‘missed’. There is clear evidence that these non-cavitated enamel lesions are a ‘stage’ of dental caries and not some predisease state [Kidd, 1996]. A smaller number of surfaces in the same individual may have discernible lesions in dentine; again a proportion of these will be unseen and therefore missed during clinical examination. Finally, at the tip of the iceberg are the comparatively extensive and more clinically obvious lesions extending into the dental pulp. Although many values for caries prevalence and incidence are expressed using the DMF index, the values reported will not be directly comparable if different diagnostic thresholds have been used. Diagnostic thresholds vary significantly between different types of research, as well as between traditional epidemiological surveys and clinical practice [Murray and Pitts, 1997]. Although this trap has been recognized within caries research circles for many years [Backer-Dirks et al., 1951; Moller, 1966; Pitts and Fyffe, 1988], it is only recently that clinicians, dental public health workers, service planners and patients are starting to appreciate these complexities with debate around the outcomes of preventive caries control strategies, which seek to prevent the need for fillings wherever possible. Thus, figure 2 demonstrates how the ‘dentineonly D3MF’, used traditionally as an epidemiological measure of caries prevalence, will inevitably produce a lower value than ‘enamel + dentine D1MF’. The latter in turn will produce lower values than ‘enamel + dentine D1MF’ used together with diagnostic aids, such as bite-

Pitts

Reporting terms used in the literature and tools in practice Pulpal caries Unseen dentine caries

Visible dentine caries Visible enamel caries

Unseen enamel caries

Subclinical caries

Dentine only D3MF

Enamel and Dentine D1MF Regular use of diagnostic aids in clinical research and in practice

Diagnostic levels of clinically detected tooth decay –

Fig. 2. Diagnostic levels of clinically detected tooth decay – use in the literature and in clinical practice using visual methods of caries detection.

wing radiographs [Murray and Pitts, 1997; Machiulskiene et al., 1999]. The preventive methods available to the dental team range from topically applied fluorides in a variety of forms (with toothpastes and varnishes being found to be particularly effective, while rinses and tablets are becoming less favoured); a focus on helping the patient achieve optimal plaque control (particularly on occlusal surfaces during eruption); strategies to modify diet with respect to the frequency of sugar consumption, and methods of increasing the resistance of the tooth, such as pit and fissure sealants [National Institutes of Health, 2001].

Existing Reviews and Consensuses

SRs of randomised clinical trials (RCTs) judged to be of high quality and comparing operative with non-operative intervention for caries directly are unfortunately very sparse. However, there are a number of more general SRs and a range of differing types of evidence evaluating the individual elements which comprise operative and non-

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operative care. As with many SRs in medicine, the quality of studies reported is very variable and there are more studies which are of poor quality by modern standards than is desirable. There are comparatively few data on the patient or quality-of-life aspects of these two approaches to clinical caries management. The Cochrane Oral Health Group, part of the International Cochrane Collaboration, undertakes and maintains an ever-widening series of robust systematic reviews on RCTs of interventions in oral health [The Cochrane Library, 2003]. The attention to methodological detail and the international refereeing process ensures that for RCTs these reviews are seen as the gold standard. A step forward in documenting and assessing the evidence in the area of the question posed here was the National Institutes of Health (NIH) Consensus Development Conference on Diagnosis and Management of Dental Caries throughout Life [National Institutes of Health, 2001; http://www.consensus.nih.gov], which went to great lengths to control bias and adopted rigorous processes for SRs. The Journal of Dental Education published a whole issue devoted to the Consensus Development Conference

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[National Institutes of Health, 2001], which included a full SR commissioned by NIH from the Research Triangle Institute [Bader et al., 2001], as well as 31 other SRs and semi-systematic reviews together with invited external expert comments on the Research Triangle Institute review. Although some aspects considered by the NIH were broader than the review topic here and new findings have been reported since March 2001, the Consensus Development Conference report still gives an excellent overview of clinical caries research. Other relevant SRs are those from the International Consensus Workshop on Caries Clinical Trials [Pitts and Stamm, in press], the Scottish Intercollegiate Guidelines Network [Scottish Intercollegiate Guidelines Network, 2000], the National Institute for Clinical Excellence [National Institute for Clinical Excellence, in press], and the Swedish Council on Technology Assessment in Health Care [http://www.sbu.se].

State of the Art

The question posed about how best to manage caries refers to selecting an appropriate treatment (or care) philosophy. In order to plan appropriate clinical caries management, a firm foundation of knowledge about caries detection, assessment and then diagnosis is required. As the following sections will discuss, clinicians should ideally unite: (a) a thorough and up-to-date understanding of evidence about the pathogenesis of caries [Fejerskov et al., 2003]; (b) an understanding of when to use appropriate diagnostic levels [Kidd et al., 2003], and (c) an ability to interpret evidence and clinical information derived from all sources [Pitts, 1997; Kidd and Nyvad, 2003; National Institute for Clinical Excellence, in press]. While this synthesis of related information and evidence seems straightforward and uncontentious to some, it is viewed as radical and somewhat alien by others. This is not surprising when the variations in the teaching content in this area of undergraduate and continuing education across countries and health systems are considered. A further threat to understanding in this clinical field is a failure to be clear about the focus of a clinical or research question. These can be posed from a surface or tooth perspective, from the individual patient perspective or from the population, community or society perspective [Baelum and Fejerskov, 2003]. All are appropriate in certain situations. In the restorative era, the decision process for managing caries centred on an almost unconscious planning of

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which surface to fill with what material [Bader and Shugars, 1995; Ismail et al., 2001], and this approach remains in use with many in general dental practice today. However, many dentists in many geographic locations are no longer taking only a narrow surgical view, seeking to apply interventive treatment as a one-off event at a certain trigger point of disease severity. Classical restorative treatment of the disease only surgically removes the carious tissue and does not, by itself, result in a ‘cure’ for caries. There is established evidence that, although the time frames vary, once a restoration is placed [NHS Centre for Reviews and Dissemination, 1999], the tooth is likely to be subjected to a series of replacement restorations, tending to increase in size, complexity and cost. The limitations of ‘repeat restorative alone’ care [Elderton, 1990; Mjör and Toffeneti, 2000] are increasingly acknowledged and replicated worldwide. Restorations have been shown to fail by a number of factors, with inadequate preparation, marginal failure of the restoration or tooth and secondary caries at approximal sites being the principal problems. It is clear that, at the individual patient level, the disease process of dental caries has to be managed over an extended time by combating each of the multiple known aetiological factors [Kidd and Nyvad, 2003]. It should be appreciated that this discussion about how best to manage and control the disease of dental caries at the encounter between patient and dental health professional in no way undercuts the importance of broader health and community interventions for primary prevention at the population level ‘upstream’ from the dentist. These interventions, combined with oral health promotion linked to other non-dental health promotion activities should (if these are delivered effectively) ensure that the burden of disease for the population (and thus those attending dental clinics and surgeries) is reduced. The public health aspects are beyond the scope of this review but need not be at odds with clinically effective preventive dental care delivered in a primary care setting. From the key SRs of caries prevention [The Cochrane Library, 2003] and management (such as those presented at the NIH Consensus Development Conference [National Institutes of Health, 2001]), we know that in addition to the improvements in caries associated with water fluoridation, fluoride toothpaste and public health initiatives, there have been successes at the practice level in reducing caries prevalence and that ‘effective professional practices such as the use of fluoride, sugarless products and dental sealants were reconfirmed’ [National Institutes of Health, 2001]. However, the Consensus Development Confer-

Pitts

ence also warned of inadequacies in current diagnostic practices: limitations which have to be borne in mind when making clinical decisions. The consensus statement’s section on clinical decision making was that ‘current information indicated that the opportunity now exists to extend prevention and treatment of caries to nonsurgical methods. These include prevention, remineralisation, and arrest of early non-cavitated lesions’ [National Institutes of Health, 2001]. The panel went on to point up the need for reimbursement methods to reward non-surgical treatment and for educational institutions and accreditation bodies to support the growing evidence for prevention and non-surgical treatment, where indicated. The state of the art in modern clinical disease management is to use evidence-based clinical guidelines to inform the dentists and patients when they need to have an up-to-date synthesis of recent research findings [National Institutes of Health, 2001]. The Scottish Intercollegiate Guidelines Network [Scottish Intercollegiate Guidelines Network, 2001; www.sign.ac.uk] has been producing such guidelines to aid medical practice since 1993 and is an example of this approach to facilitating evidence-based care. In 2000, they published a guideline on providing appropriate care for 6- to 16-year-old children attending dental practices in Scotland [Scottish Intercollegiate Guidelines Network Guideline, 2000]. This recommends a preventive approach to clinical management. One of its key recommendations is that, because of the ‘polarisation’ of the population into a low-caries majority and a cariesactive minority, ‘an explicit caries risk assessment should be made for each child presenting for dental care’. The polarisation phenomenon [Poulsen et al., 2001] as caries declines is important. Changes in Danish 15-yearold children between 1980 and 1995 show that: ‘with increasing divergence from a Poisson distribution, caries risk is itself skewed. This means that high risk groups would continue to have high risk to future caries attack and progression’ [Poulsen et al., 2001]. This does not mean that those at the lower-caries end of the distribution are not at risk of new disease or caries progression. This is why it is important to have both a public health strategy – to continue to pull the distribution even further to the low-caries side for everyone [Sheiham and Fejerskov, 2003] – and an element of high-risk strategy to provide appropriate care for those with a high burden of disease. When patients present at dentists for clinical care, it is important that risk is considered so that care is tailored to the needs of the individual [Kidd and Nyvad, 2003]. The following factors should be considered when assessing

caries risk: medical history; social history, especially socioeconomic status; clinical evidence of previous disease; dietary habits, especially frequency of sugary food and drink consumption; use of fluoride; plaque control, and salivary factors [Scottish Intercollegiate Guidelines Network Guideline, 2000; Kidd and Nyvad, 2003]. Although there has been a focus on children in defining this type of preventive care, and the bulk of the available evidence comes from studies of children and adolescents, it should be appreciated that both adults and children are important and that at all ages the disease is driven predominantly by the intensity of the cariogenic challenge for the individual at a single time rather than by chronological age alone. The philosophy of ‘wellness management’ is already the state of the art in some communities and is increasingly prominent in US health care. This reflects both an attempt to ensure that preventive care is optimised by early intervention and an understanding of many patients’ increasing desire to optimise and maintain health, rather than to suffer from a disease and its consequences. This philosophy is gaining support amongst consumer groups and some of those seeking to respond to spiralling general health care costs. Under this model of care, waiting until you have a disease is too late; one needs to pro-actively modify risk factors to avoid the transitions to the diseased state, or to catch the disease at an earlier stage in its development. This approach may have dental applications, but clinical outcome studies are needed. The model of care underlying the clinical management of dental caries is changing from the traditional, but increasingly outmoded one in which the dentist restoring caries is seen as a surgeon, to the model which sees the dentist as a physician. The physician and team are concerned with the prevention and control of the disease wherever possible, as opposed to the surgeon, who manages caries by the restoration and re-restoration of teeth. Traditionally the dentist as a surgeon provided treatment based upon excision of diseased tissue, detection/ intervention, treating signs and symptoms, rehabilitation of lost tissue with early intervention and extension. By contrast, the dentist as a physician now provides care based on: health maintenance; early detection and monitoring; controlling causal agents; use of appropriate pharmacological agents, and minimal intervention. These two roles are, however, not mutually exclusive as, depending on the needs of the patient and the outcome of professional and self-care, the dentist may have to fulfil both roles for the same patient. Although the term ‘medical model of caries care’ has been used widely in this more preventive

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The ‘iceberg of dental caries’

(1995 >) by contemporary patient advice and treatment need D

4

P&OCA Preventive & Operative Care Advised

lesions in to pulp clinically detectable lesions in dentine clinically detectable ‘cavities’ limited to enamel

D

3

D

2

clinically detectable enamel lesions with ‘intact’ surfaces

D

PCA Preventive Care Advised

1

lesions detectable only with traditional diagnostic aids

Fig. 3. The iceberg of dental caries linked to

subclinical initial lesions in a dynamic state of progression/regression

contemporary patient advice and treatment need [Pitts and Longbottom, 1995].

clinical caries management context [National Institutes of Health, 2001], this has been taken to imply an almost exclusive focus upon caries as an infectious disease and, in turn, an antimicrobial approach. As the caries process has a multi-factorial aetiology [Fejerskov and Manji, 1990], it is preferable to use a broader approach (the dentist/team as physician model), which allows a clearer role for oral hygiene and diet-related preventive measures, as well as antibacterial and pharmacological interventions in caries control. A care philosophy which integrates all the scientific information underpinning modern clinical caries management is summarised in figure 3, which, like figure 2, illustrates the iceberg phenomenon, but this time linked to contemporary patient advice and treatment need [Pitts and Longbottom, 1995]. The two more extreme care treatment options are the most straightforward. Very small subclinical initial lesions require no active care in many lower-caries risk patients who need only surveillance at appropriate, individualised time intervals. For more caries-active individuals, the appearance and progression of small lesions are a marker of disease activity and an indication that more aggressive preventive care is required urgently. At the other end of the spectrum, the presence of large D4 lesions extending into the pulp indicates that some form of operative intervention or care is required urgently and this should be advised in addition to the preventive care advice.

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NAC No Active Care Advised

DHSRU/2003

Determining the cut-offs at both ends of the severity scale for the intermediate group of lesions, which require only preventive care advised, is more complex and contentious. These cut-offs have to be decided for individual lesions and patients by dentists utilising all the information available from the diagnostic assessments, the patient and the medical, dental and social histories. Clinical preventive care is focussed on ensuring, at an early stage, that new disease can be avoided if possible, and limited or controlled if it cannot be. The potential long-term wellbeing, health and economic benefits for individual patients are immense. One aspect highlighted by this overview is the urgent need in many countries for improving education about cariology. There are concerns over the lack of basic science-led theory and preventive practice in clinical cariology when compared to other disciplines taught in undergraduate schools, as well as gaps in postgraduate and continuing dental education [Ismail et al., 2001]. This often produces widespread and alarming ignorance among graduates about both the basic science of the disease process and the clinical strategies to best manage the disease. Education of the wider dental profession and the public, patients and planners on the potential for caries prevention, is also poor in many countries. Examples include the continuing misuse and misunderstanding of terms such as ‘caries-free’, a lack of appreciation of the continuing risk of caries throughout life and a failure to

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understand that the minority of those who still have dental disease carry very significant quality-of-life burdens. The key point for clinical caries management is that the caries process needs to be managed for each individual by the dental team, in partnership with the patient, over the changing and continuing cariogenic challenges of a lifetime. The frequency of visits at which assessments should be made should reflect the particular needs of the individual patient taking into account current caries status and an individually determined prognosis [Kidd and Nyvad, 2003; National Institute for Clinical Excellence, in press].

Future Research

We need to increase our scientific understanding of the caries process so that it can be prevented and controlled more effectively. For this reason, we need rigorous research to better understand caries aetiology, pathogenesis, activity, detection (informing diagnosis), diagnosis, monitoring (informing prognosis with minimal harm) and risk assessment [National Institutes of Health, 2001]. This research will then inform the development of new adjuncts to preventive caries control, which will need to be tested in realistic clinical settings. Key recommendations of the 2001 NIH Consensus Development Conference for future research were: (1) epidemiology of primary and secondary caries to collect information on natural history, treatment and outcomes across ages; (2) clinical trials, to modern standards, of established and new treatment methods; (3) studies of clinical practice with respect to effectiveness, appropriateness, quality of care, outcomes and health-related quality of life, and (4) studies to identify genes and genetic markers of diagnostic, prognostic and therapeutic value. There have been enormous changes in design and statistical analysis of medical clinical trials, as well as in the ethical, economic and regulatory environments over the last 20 years. Despite the dramatic changes in our knowledge about research, epidemiology, detection systems and the effectiveness of clinical care for dental caries over the same period, there have been no coordinated formal initiatives to review the methodological requirements for RCTs of caries-preventive interventions [Pitts and Stamm, in press]. One consensus statement expressed the view that recording only cavitated lesions as an outcome measure is becoming outmoded, and that for future caries clinical trials, recording of non-cavitated lesions is essential [Pitts and Stamm, in press].

Moving from Operative to Preventive Clinical Caries Management?

To specifically advance non-operative clinical caries management, we need research into the effectiveness of preventive interventions and caries control for individuals [National Institutes of Health, 2001]. This should bring together basic science and applied technology. A key area of clinical need where more research is required is on lesion assessment, which ascertains the ‘activity’ of caries in individuals and at specific sites [Pitts and Renson, 1987]. At present, most research in this area is concerned with assessing changes in lesion characteristics over specific time periods, but this could and should be complemented with valid assessments of whether or not a lesion is active, made on a single occasion. This has recently been attempted clinically by Nyvad et al. [1999, 2003]. What is needed is a way of differentiating active from inactive lesions and it is important to highlight the different types of behaviour that may be exhibited. The clinical decisions that would be made would clearly be different between the active and inactive lesions if this information were known. Similarly, the optimal timing of care and the planning of individualized recall intervals would be enhanced by this type of information. It would also be very useful for both clinical practice and research studies to be able to reliably identify each type of active and inactive lesion. One of the successes of ORCA is the range of international collaborations that have been initiated and developed between members from a range of different countries. This tradition will remain important in order to achieve generalisable and valid research findings. A recent initiative is the formation of a committee which has started the development of an international caries detection and assessment system [Pitts, in press]. The idea is to work, to develop and test an open framework, which will allow sufficient standardisation of caries criteria to facilitate future SRs. The system will provide a range of methodological tools based on a synthesis of the published literature and suitable for a variety of applications. The aim is to bring together methodology from epidemiological surveys, clinical research and clinical practice. These unifying, predominantly visual criteria would code the characteristics of clean, dry teeth. They will be able to record both enamel and dentine caries and, in future research, should help to explore the measurement of caries activity. It is hoped that the development and use of the international caries detection and assessment system might lead to better quality information to inform decisions about appropriate diagnosis, prognosis and clinical management at both the individual and ultimately public health levels. It should also provide a common framework to val-

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idate and explore the impact of a number of detection aids such as explorers, magnification, tooth separation, fibre optic transillumination, radiography and other diagnostic aids. New studies in these areas are still needed as the synthesis of the results of many existing studies is compromised by the conflicting criteria and methodologies employed [Bader et al., 2001]. A further problem to be overcome is the validation of caries registration methods [Sjogren et al., 2003]. In the future, there should also be more research into treatment choices for clinical caries management. These should include studies of the dynamics of communications and behaviour change, in both patients and health professionals. Such studies should be linked with outcome evaluations to see if the potential of health informatics and clinical decision support [Department of Health, 2002] can be realised. Assessing cost effectiveness as well as clinical effectiveness will be important in determining how well preventive caries interventions can compete for scarce health resources. In routine dental care, choices are frequently made between alternative approaches. The choices between preventive and restorative alternatives are not always easy or straightforward and can be important [National Institutes of Health, 2001; Department of Health, 2002]. The financial arrangements for the dentist, dental team and patients in caries management have often evolved from historical tradition in each country. Most systems have not explicitly asked what is the evidence supporting each option and how it matches the financial incentives for the dentists, the dental team and the patients receiving care. Research is needed to try to inform dental public planning, which should seek to ensure that dental remuneration systems and incentives are linked to optimal and effective caries prevention and control. Recently, the English Department of Health [2002] published a radical document and subsequent legislation is bringing about changes in the organisation of dental services, dentist remuneration and the way in which oral health is assessed. A key aim of this modernisation process is to move towards a more preventively oriented and clinically effective way of meeting patients’ needs [Pitts, 2003]. Under the new arrangements, a comprehensive oral health assessment will comprise three elements: diagnosis, prevention and initial treatment planning. The changes to the remuneration system are designed to ‘remove existing perverse incentives’ which favour restorative care. Appropriate strategies for use in developing countries are also required so that the understandable desire to have equity with and copy the ‘developed’ countries does not

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result in the introduction of inappropriate and inefficient types of caries prevention and control. The restoratively focused era of the 1960s to the 1980s in the developed world is not a desirable model to copy, as caries prevalence and incidence have now changed, as has our knowledge about dental caries and its control [National Institutes of Health, 2001]. Cost-effective and locally appropriate preventive approaches should be adopted from the outset and should meet local objectives.

Potential Impact on Clinical Practice

Non-operative caries management supported by meticulous caries detection, lesion assessment and diagnosis, when combined with modern, proven, caries prevention technologies, should change the face of routine clinical practice. Dentists can and should help their patients control caries preventively and manage their oral health [Kidd and Nyvad, 2003]. Primary caries prevention (preventing new disease) will be an important activity at the population, group and individual levels. For patients attending a dentist, secondary caries prevention – the early detection and prompt, efficacious treatment of disease – is likely to be improved in the coming years by the introduction of new technologies. In more and more patients, this type of clinical care should prevent many lesions from ever reaching the stage when operative intervention is needed [Fejerskov and Nyvad, 2003]. From the patients’ perspective, the realisation that the need for many fillings can be avoided entirely will be very attractive. When tertiary prevention and operative intervention are clearly needed, careful instrumentation maintaining more sound tooth structure, combined with the introduction of ‘smart’ materials with better physical properties, should improve our very limited ability at durable repair [Ismail et al., 2001]. The incorporation of cariostatic materials (such as slow-release fluorides) within restorations has the potential to aid continuing caries control in the future, provided more sustained low-dose fluoride delivery can be achieved in vivo. The use of slow-release fluoride reservoirs bonded to tooth structure is also an attractive prospect for changing clinical practice. EBD should combine with new knowledge services and information technology to ensure that quality-assured objective information about prevention, disease control and treatment choices are readily available and accessible to both patients and the dental team. The gap between research and practice should be reduced in time.

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Non-operative caries care should be more clinically effective, appropriate to patients’ needs and more costeffective than traditional operative care. This new type of care should be provided within a number of new delivery systems in several countries as the design and economics of health care delivery systems and insurance contracts evolve. Funding systems will change, and are already doing so, in response to changes in disease patterns, the evidence base and professional and patient expectations [Department of Health, 2002]. The recommendations made in this non-systematic review must be graded at the consensus or expert opinion level. This is below that which would be accorded to specific meta-analyses of high-quality SRs focusing only on this question, but we do not yet have such reviews. Taking into account the evidence reviewed above, we are, in many cases, ready to move to non-operative/preventive caries treatment in clinical practice if we have not done so already. This style of care should be optimal for appropriate stages of lesion extent when surfaces are macroscopically intact, and with co-operative patients. Where

significant cavity formation and disease progression are seen, operative intervention is still indicated. This should, however, be minimally invasive and executed to a high technical standard. In the future, it should be technically possible, through partnerships between patients, the dental profession, society and industry, for a patient of any age to benefit from high-quality, on-going, preventive caries care in clinical practice. The clinical team should be up to date with the developing evidence. Treatment should be based on a thorough diagnostic work-up, which would help dentists and patients agree to individualised, preventive, caries control plans to manage and control the disease process throughout life.

Acknowledgements and Disclaimer Thanks are due to Dr. Chris Longbottom, with whom the PCA/ OCA caries management approach has been developed. The views expressed are those of the presenter and do not necessarily reflect those of any UK Health Department, the Medical Research Council or any partner or sponsor of the Centre for Clinical Innovations.

References American Dental Association Policy on EvidenceBased Dentistry: American Dental Association Position Statements, 2002:www.ada.org/prof/ resources/positions/statements/evidencebased.asp. Backer-Dirks O, van Amerongen J, Winkler KE: A reproducible method for caries evaluation. J Dent Res 1951;30:346–359. Bader JD, Shugars DA: Variation in dentists’ clinical decisions. J Public Health Dent 1995;55: 181–188. Bader JD, Shugars DA, Bonito AJ: Systematic reviews of selected dental caries diagnosis and management methods. J Dent Educ 2001;65: 960–968. Baelum V, Fejerskov O: Caries diagnosis: A mental resting place on the way to intervention?; in Fejerskov O, Kidd EAM (eds): Dental Caries – The Disease and Its Clinical Management. London, Blackwell Munksgaard, 2003, pp 101– 110. Department of Health: NHS Dentistry: Options for Change. London, Department of Health, 2002. www.doh.gov.uk/cdo/optionsforchange. Elderton RJ: Clinical studies concerning re-restoration of teeth. Adv Dent Res 1990;4:4–9. Fejerskov O, Manji F: Risk assessment in dental caries; in Bader JD (ed): Risk Assessment in Dentistry. Chapel Hill, University of North Carolina Dental Ecology, 1990, pp 215–217.

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Fejerskov O, Nyvad B: Is Dental Caries an Infectious Disease? Diagnostic and Treatment Consequences for the Practitioner. Nordic Dentistry 2003. Copenhagen, Quintessence Copenhagen, 2003, pp 141–152. Fejerskov O, Nyvad B, Kidd EAM: Clinical and histological manifestations of dental caries; in Fejerskov O and Kidd EAM (eds): Dental Caries – The Disease and Its Clinical Management. London, Blackwell Munksgaard, 2003, pp 71– 97. Heidman J, Holund U, Poulsen S: Changing criteria for restorative treatment of approximal caries over a 10-year period. Caries Res 1987;21: 460–463. Ismail AI, Hasson H, Sohn W: Dental caries in the second millenium. J Dent Educ 2001;65:953– 959. Kidd EAM: The carious lesion in enamel; in Murray JJ (ed): Prevention of Oral Disease, ed 3.. Oxford, Oxford University Press, 1996, pp 95– 106. Kidd EAM, Nyvad B: Caries control for the individual patient; in Fejerskov O, Kidd EAM (eds): Dental Caries – The Disease and Its Clinical Management. London, Blackwell Munksgaard, 2003, pp 303–312. Machiulskiene V, Nyvad B, Baelum V: A comparison of clinical and radiographic caries diagnoses in posterior teeth of 12-year-old Lithuanian children. Caries Res 1999;33:340–348.

MacLeod HST, Morris AJ, Hill KB: Evaluation of personal dental services (PDS) first wave pilots: The alternative to general dental services (GDS) offered by the capitation based pilots. Br Dent J 2003;195:644–650. Magitot E: Therapeutic indications in dental caries. Part 1. Br J Dent Sci 1886;29:405–410. Manji F, Fejerskov O, Baelum V, Luan W-M, Chen X: The epidemiological features of dental caries in African and Chinese populations: Implications for risk assessment; in Johnson NW (ed): Risk Markers for Oral Diseases. Dental Caries Markers of High and Low Risk Groups and Individuals. Cambridge, Cambridge University Press, 1991, vol 1, pp 62–100. Marthaler TM: The caries-inhibiting effect of amine fluoride dentifrices in children during three years of unsupervised use. Br Dent J 1965;119:153–163. Mjör I, Toffenetti F: Secondary caries: A literature review with case reports. Quintessence Int 2000;31:169–71. Møller IJ: Clinical criteria for the diagnosis of the incipient carious lesion. Adv Fluorine Res 1966;4:67–72. Murray JJ, Pitts NB: Trends in oral health; in Pine CM (ed): Community Oral Health. Dundee, University of Dundee, Reed Educational and Professional, 1997, pp 126–146.

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National Institutes of Health: The diagnosis and management of dental caries throughout life. National Institutes of Health Consensus Development Conference, Washington March 26th– 28th 2001. J Dent Educ 2001;65:1162–1168. National Institute for Clinical Excellence: Dental Recall: Recall Interval between Routine Dental Examinations – Guideline. London, National Institute for Clinical Excellence (NICE), Department of Health, in press. NHS Centre for Reviews and Dissemination: Restoration Longevity: Effectiveness Matters Bulletin. York, 1999. Nyvad B, Machiulskiene V, Baelum V: Reliability of a new caries diagnostic system differentiating between active and inactive caries lesions. Caries Res 1999;33:252–260. Nyvad B, Machiulskiene V, Baelum V: Construct and predictive validity of clinical caries diagnostic criteria assessing lesion activity. J Dent Res 2003;82:117–122. Pitts NB: Review of ICW-CCT meeting and philosophy and approach of ICDAS. Proc Third Indiana Conf Early Detect Dent Caries, Indiana, in press.

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Pitts NB, Fejerskov O, von der Fehr FR: Caries epidemiology, with special emphasis on diagnostic standards; in Fejerskov O, Kidd EAM (eds): Dental Caries – The Disease and Its Clinical Management. London, Blackwell Munksgaard, 2003, pp 140–163. Pitts NB, Fyffe HE: The effect of varying diagnostic thresholds upon clinical caries data for a low prevalence group. J Dent Res 1988;67:592– 596. Pitts NB, Longbottom C: Preventive Care Advised (PCA)/Operative Care Advised (OCA) – Categorising caries by the management option. Community Dent Oral Epidemiol 1995;23:55– 59. Pitts NB, Renson CE: Monitoring the behaviour of posterior approximal carious lesions by image analysis of serial standardised bitewing radiographs. Br Dent J 1987;162:15–21. Pitts NB, Stamm JW: Proceedings of the International Consensus Workshop on Caries Clinical Trials ICW-CCT. J Dent Res, in press. Poulsen S, Heidman J, Vaeth M: Lorenz curves and their use in describing the distribution of ‘the total burden’ of dental caries in a population. Community Dent Health 2001;18:68–71. Scottish Intercollegiate Guidelines Network: SIGN guidelines: A Guideline Developer’s handbook. Edinburgh, Scottish Intercollegiate Guidelines Network, 2001, SIGN publication No 50.

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Scottish Intercollegiate Guidelines Network Guideline: Targeted Caries Prevention in 6- to 16Year-Olds Attending for Dental Care. Edinburgh, Scottish Intercollegiate Guideline Network, 2000. Sheiham A, Fejerskov O: Caries control for populations; in Fejerskov O, Kidd EAM (eds): Dental Caries – The Disease and Its Clinical Management. London, Blackwell Munksgaard, 2003, pp 313–325. Silverstone LM: Structure of carious enamel including the early lesion. Oral Sci Rev 1973;3: 100–160. Sjogren P, Ordell S, Halling A: Validation methodology in publication describing epidemiological registration methods of dental caries: A systematic review. Community Dent Health 2003;20: 251–259. Swedish Council on Technology Assessment in Health Care: http://www.sbu.se. The Cochrane Library: CD ROM Update Software Ltd, Oxford, 2003. http://www.cochrane. co.uk. World Health Organization: A Guide to Oral Health Epidemiological Investigations. Geneva, World Health Organization, 1979.

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Caries Res 2004;38:305–313 DOI: 10.1159/000077770

How ‘Clean’ Must a Cavity Be before Restoration? E.A.M. Kidd Guy’s, King’s and St. Thomas’ Dental Institute, London, UK

Key Words Caries removal W Cavity preparation W Stepwise excavation

Abstract The metabolic activity in dental plaque, the biofilm at the tooth surface, is the driving force behind any loss of mineral from the tooth or cavity surface. The symptoms of the process (the lesion) reflect this activity and can be modified by altering the biofilm, most conveniently by disturbing it by brushing with a fluoride-containing toothpaste. The role of operative dentistry in caries management is to restore the integrity of the tooth surface so that the patient can clean. Thus, the question, ‘how clean must a cavity be before restoration?’ may be irrelevant. There is little evidence that infected dentine must be removed prior to sealing the tooth. Leaving infected dentine does not seem to result in caries progression, pulpitis or pulp death. However, some of the bacteria survive. What is their fate and if they are not damaging, why is this? Copyright © 2004 S. Karger AG, Basel

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Fax + 41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/cre

Caries the Process, Caries the Lesion

The disease dental caries is a dynamic process taking place in dental plaque, the microbial deposit (biofilm) on the tooth surface, which results in a disturbance of the equilibrium between tooth substance and the overlying biofilm. Over time, there may be a net loss of mineral, leading to dissolution of the dental hard tissues and possibly a carious lesion that can be seen [Baelum and Fejerskov, 2003]. In this definition of the caries process, it is the metabolic activity in the biofilm that is the all-important driving force. The demineralization of the enamel and dentine beneath may be seen as a reflection of the dynamic events taking place in the biofilm. The implication of this definition is that the symptoms of the process (the lesion) can be modified by alteration of the biofilm; for instance, the lesion can be modified by regular disturbance of the plaque with a brush and a fluoride-containing toothpaste, the fluoride controlling the rate of lesion progression [ten Cate and Featherstone, 1996]. However, at an advanced stage of caries, sometimes a hole (cavity) in a tooth retains the biofilm and careful brushing cannot remove it. Now operative dentistry has a role to play in caries management to restore the integrity of the tooth so that the patient can clean effectively; but once the enamel is cavitated, the dentine becomes demineralized and infected and now the essential question is: what is driving the caries process? Is it the biofilm at

Prof. E.A.M. Kidd Floor 25, Guy’s Tower London Bridge, SE1 9RT (UK) Tel. +44 207 188 1573, Fax +44 207 188 1583

the cavity surface or the infected dentine within the cavity, or both? If it is only the biofilm that drives the caries process, the question, ‘how ‘‘clean’’ must a cavity be before restoration?’ becomes an irrelevance because what matters is sealing the hole in the tooth so that the biofilm can be removed. And yet, the concept of removing infected, demineralized tissue and its replacement by a filling material has spawned a profession, a public and political paymasters who consider that the removal of infected tissue and filling teeth is an essential management of dental caries. The discussion as to how much tissue must be removed in order to arrest the caries process is not new. In 1859, John Tomes [1859] wrote, ‘it is better that a layer of discoloured dentine should be allowed to remain for the protection of the pulp rather than run the risk of sacrificing the tooth’, but in 1908, G.V. Black [1908] disagreed claiming ‘... it will often be a question of whether or not the pulp will be exposed when all decayed dentine overlaying it is removed ... it is better to expose the pulp of a tooth than to leave it covered only with softened dentine’. The following discussion will look for evidence to confirm or refute the current practice of cleaning infected tissue out of the cavity prior to placing a restoration. This review of evidence must be preceded by a brief description of caries pathology.

Pulpo-Dentinal Reactions in Response to Caries

The shape of the enamel lesion is governed by the activity of the bacteria in the overlying biofilm and the orientation of the enamel crystals. The corresponding pulpo-dentinal reactions are similarly influenced by the biofilm with transmission of the stimulus through the enamel being in the direction of the prisms [Thylstrup and Qvist, 1986]. The implication of this is that when acid production ceases at the surface, due to regular disturbance or removal of the biofilm, lesion progression arrests [Bjørndal, 2002]. The demineralized enamel and dentine remain as scars in the tissue. In non-cavitated enamel lesions, the level of bacterial invasion is very low, if present at all. But once the demineralized enamel crumbles and a cavity forms, the biofilm will form in the hole and the dentine becomes infected. Once the biofilm is directly on the dentine, the lesion spreads laterally along the enamel-dentine junction at the edges of the cavity, as well as back through the sound, undermined enamel. In these deep lesions, there may be large variations and changes within the lesion environment. The central part

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of the lesion may be cavitated, open and accessible to plaque removal, by chewing and cleaning. In this area, the lesion may progress slowly. However, peripheral parts of the same cavity may still be protected by undermined enamel with heavy plaque accumulation. In these areas, the lesion may progress more rapidly. Thus, it is possible to have slowly and rapidly progressing parts within the same lesion. It is also possible that an entire lesion is rapidly or slowly progressing and the responses of the pulpdentine complex to these two speeds of progression vary. As Massler [1967] pointed out a long time ago, it is essential to differentiate active from arrested lesions if one is to make any sense of the biological reactions. In slowly progressing lesions, increased mineralization of the dentine beneath the enamel lesion is normal. Formation of highly mineralized peritubular dentine corresponding to the affected dentinal tubules reduces the diameter of the tubules. Furthermore, tertiary dentine forms at the pulpal end of the affected tubules (reactionary dentine). The more active the lesion, the more irregular the structure of this dentine. Together, these processes serve to protect the pulp against exogenous destructive stimuli [Bjørndal and Mjör, 2001]. In rapidly progressing lesions, there may be destruction of the odontoblasts and a lack of formation of tertiary dentine. Now the pulpal tissue will react to the transmission of microbial products through a permeable dentine. There will be inflammatory changes in the pulp leading to either reversible or irreversible pulpitis, sometimes associated with sensitivity or pain. Even though the odontoblasts have been destroyed, new odontoblast-like cells may differentiate from the pulp to form tertiary dentine (reparative dentine) if the cariogenic environment is removed or altered [Bjørndal and Mjör, 2001]. It can be seen from the preceding discussion that the lesion entirely reflects the activity in the biofilm, so it is hardly surprising that modifying the biofilm will modify the lesion. How should this understanding of caries pathology influence the operative dentist?

Root Caries

Root caries lesions, accessible to cleaning, are of particular relevance to this discussion. The dentine in such lesions is infected at a relatively early stage in lesion progression [Nyvad and Fejerskov, 1990]. Despite this, active lesions can be converted to inactive lesions over a period of months by regular cleaning and fluoride application [Nyvad and Fejerskov, 1986]. Thus, in these lesions,

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Table 1. Chronological overview of studies placing sealants over carious dentine Study

Treatment

Period

Jeronimus et al. [1975]

occlusal lesions of varying depths on bitewing etching, sealant (n = 33) etching, sealant (n = 33) etching, sealant (n = 30) etching, sealant (n = 25)

10 min 2 weeks 3 weeks 4 weeks

Handelman et al. [1976]

etching, sealant (n = 60)

0–2 years

Going et al. [1978]

etching, sealant (n = 46)

Mertz-Fairhurst et al. [1979a]

Control

Indication of caries activity

Result and conclusion

gross observation of carious dentine micro-organisms (% positive cultures)

many sealants lost; where sealant was intact, dentine became dry, dark, leathery; decrease in micro-organisms in shallow lesions, but persist in deeper lesions

untreated (n = 29)

clinical observation, radiography, micro-organisms

no increase in radiographic lesion depth; large reduction of micro-organisms by comparison to controls, increased with time

5 years

untreated (n = 21)

clinical observation, micro-organisms

sealed teeth caries arrested; on re-entry either sterile or large reduction in micro-organisms in comparison to controls, but Streptoccocus mutans and lactobacilli survived

occlusal lesions at DEJ on X-ray; etching, sealant (n = 4)

6–12 months

untreated (n = 4)

lesion depth measurements, micro-organisms

no increase in lesion depth in test; control lesions increased in depth; absence of microorganisms in test sealed teeth

Mertz-Fairhurst et al. [1979b]

occlusal lesions at DEJ on X-ray; etching, sealant (n = 4)

6–12 months

untreated (n = 6)

clinical observations, radiographs

under sealant dentine powdery, dry, white with hard, glassy, smooth dentine beneath, control dentine spongy, soft, yellow; sealed teeth – no or small increase in depth; control – increase in depth

Jensen and Handelman [1980]

etching, sealant (n = 106)

0–12 months

unsealed, unsealed and etched

micro-organisms

etching alone reduced micro-organisms by 75%; in sealed teeth, bacterial counts reduced with time

Handelman et al. [1981]

etching, sealant (n = 108)

2–5 years

contralateral routine amalgam

radiographic lesion depth

decrease in lesion depth provided sealant intact

Mertz-Fairhurst et al. [1986]

etching, sealant (n = 14)

1–17 months

unsealed (n = 14)

direct lesion depth measurements and radiographs; micro-organisms

unsealed lesions got deeper but sealed lesions did not; all but I sealed lesion, no microorganisms

Weerheijm et al. [1992]

teeth already etched and sealed but occlusal radiolucency in dentine (n = 30)

micro-organisms: total colony forming units lactobacilli, mutans streptococci, non-mutans streptococci; clinical observation of dentine

cariogenic micro-organisms found in 50% of teeth despite sealant; dentine soft, moist, dark (not leathery, dry)

the operative dentist has no need to cut away the infected dentine in order to arrest the lesion. Subsequently, the arrested root caries lesion is only superficially colonized [Beighton et al., 1993] presumably because the soft, infected dentine has been brushed away. Does this mean that it is not necessary to remove infected dentine when preparing coronal cavities to receive fillings? Once the restoration is in place, there is no chance for the patient to brush the infected material away. What is the fate of these micro-organisms, entombed by the restorative dentist? Do these lesions remain active or are they arrested?

Caries Removal

Fissure Sealant Studies

Table 1 gives a chronological overview of studies investigating the consequences of placing sealants over carious dentine. All studies, with the exception of Weerheijm et al. [1992], were prospective and in many there were unsealed, control, lesions. Caries activity was assessed in a number of ways including clinical observation, lesion depth measurement, radiographic lesion depth measurement and microbiological sampling. Observation periods varied from 2 weeks to 5 years.

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The disparity of methodologies militates against a systematic review of the studies, but some uniform themes emerge. Sealed lesions appeared to arrest both clinically and radiographically. Investigations of the fate of the sealed bacteria showed a decrease in micro-organisms with time or their complete elimination. There was no pulpitis reported in sealed teeth. On the other hand, lesions progressed where sealants were lost and in unsealed, control teeth. The study of Weerheijm et al. [1992] is an interesting outlier. This work was a retrospective examination of sealed teeth where radiographs showed radiolucency in dentine beneath a sealant that was clinically intact. This methodology precluded microbiological sampling before the sealant was placed, which is unfortunate because there can be no comparison of microbial counts before and after sealing. Nevertheless, it is worrying that cariogenic microorganisms were found in 50% of the teeth and the dentine was often soft and moist, rather than leathery or dry. This would seem to indicate active lesions. The microbiological examination in this work was more detailed than in many other studies examining for lactobacilli, mutans streptococci and non-mutans streptococci. Since there was no preoperative sample, it is impossible to know whether sealing had changed the numbers or the distribution of the microflora.

Classical Caries Excavation

The operative tradition is to remove softened dentine in order to eliminate infected tissue. This approach assumes that both the biofilm and the micro-organisms within the carious dentine drive the caries process. In fact, it is not possible to eliminate all the micro-organisms because a few will remain even if all soft dentine is removed [Lager et al., 2003]. At the enamel-dentine junction, some schools teach that the area should be stain-free as well as hard, but a few bacteria remain whatever approach is adopted and thus it seems logical to leave stain in this area as a more conservative approach [Kidd et al., 1996]. Over the pulpal surface, contemporary teaching recommends that carious dentine that is ‘firm and leathery’ should be left where its removal might expose the pulp [Hilton and Summitt, 2000]. A calcium hydroxide liner is placed over the demineralized area of dentine and this medicament has been shown to significantly reduce the number of remaining bacteria [Leung et al., 1980]. This procedure is called indirect pulp capping. Vigorous exca-

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vation is positively contraindicated, but the student will find that one teacher’s definition of ‘firm and leathery’ is another’s ‘rather soft’ interpretation. The subjective clinical assessment of carious dentine led Fusayama [1988] to develop a caries dye (acid red in propylene glycol) to differentiate clinically ‘infected’ from ‘affected’ dentine. He reported that the more superficial zone of infected dentine was an irreversibly damaged, bacterially infected layer that would never remineralize. The deepest affected dentine was shown to harden as a result of remineralization [Eidelman et al., 1965]. Fusayama’s group suggested the dye staining front coincided with the bacterial invasion of the dentine. However, several studies have reported that the dye does not discretely discriminate the bacterially infected from softened affected tissues [Anderson et al., 1985; Boston and Graver, 1989; Kidd et al., 1993]. Consequently, its injudicious use may lead to over-preparation of the tissues, encouraging excess removal at the enamel-dentine junction [Kidd et al., 1993] as well as unnecessary removal of dentine over the pulpal surface [Yip et al., 1994]. Soft dentine is usually wet but sometimes, particularly when an old restoration has been removed, the dentine may appear crumbly and dry. This dry dentine has been shown to be minimally infected [Kidd et al., 1995] and it may represent residual caries that a previous dentist left during cavity preparation. This may indicate that there is no need to remove soft, wet dentine. The process may be arrestable by simply sealing it in place.

Stepwise Excavation

Stepwise excavation, described by Bodecker [1939], differs from the classical excavation of carious lesions described above. Only part of the soft, dentine caries is removed at the first visit during the acute phase of caries progression. The cavity is restored and re-opened after a period of weeks. Further excavation is now carried out prior to a definitive restoration. The objective of the exercise is to arrest lesion progression and allow the formation of tertiary dentine before final excavation, making pulpal exposure less likely. This procedure has been investigated scientifically for more than 30 years. These studies have involved baseline investigations of carious dentine and then a re-analysis after a period of sealing it in the tooth. This work is important evidence of the consequences of sealing infected dentine into teeth.

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Table 2. Chronological overview of stepwise excavation studies Study

Toothtype, lesion depth

Treatment

Law and Lewis [1961]

deciduous and permanent, deep lesions

Schouboe and Macdonald [1962]

Control

Time to re-entry

Indication of carious activity

Result and conclusion

access to caries then Ca(OH)2 + H2O on dentine; amalgam (n = 66); re-entry at 6 months; excavation completed (n = 57)

6–24 months

clinical observation; observation dentine on re-entry, radiographs

76% clinically (no exposure) and radiographically (no pathology) successful

molars with occlusal caries

access, carious dentine sampled; gold plate over dentine, then amalgam (n = 17)

69–139 days

micro-organisms

positive cultures in 14 cases, on re-entry a different flora

King et al. [1965]

? deciduous, deep lesions, no pulpitis

only deepest layer decayed, dentine left; capped with Ca(OH)2 or ZnO/Eug or amalgam; restored amalgam (n = 51)

25–206 days

observation dentine on re-entry, microorganisms

initial samples of deep, soft dentine, infected dentine harder on re-entry with Ca(OH)2 and ZnO/Eug but not with amalgam; 3/8 teeth exposed after further caries removal with amalgam; micro-organisms on re-entry; Ca(OH)2 teeth 61.4% sterile; ZnO/Eug teeth 81.8% sterile; amalgam teeth 0% sterile but numbers of organisms reduced

Kerkhove et al. [1967]

deciduous and permanent, deep lesions

only deepest layer decayed, dentine left; 41 teeth Ca(OH)2 and amalgam, 35 teeth ZnO/ Eug and amalgam

3–12 months

observation of dentine on re-entry, radioopacity relative to control area assessed visually and densitometrically

92% clinical success; on reentry dentine dry, hard, brownish yellow; increased radio-opacity; very slight time but not material dependant

Magnusson and deciduous, Sundell [1977] deep lesions, no pulpitis

partial excavation; calcium full excavation hydroxide, zinc oxide and (n = 55) eugenol cement; at re-entry all soft carious dentine excavated (n = 55)

4–6 weeks

clinical observation, observation dentine on re-entry

15% treatment group pulp exposed, 53% control group pulp exposed

Weerheijm et al. permanent [1993] molars, small visible occlusal lesions

part of lesion opened to denas treatment but tine; this filled glass ionomer Delton sealant cement (GIC); remainder used (n = 4) sealed GIC; at re-entry all caries removed and composite placed (n = 20)

7 months

clinical observation of dentine on re-entry micro-organisms

poor retention GIC sealant, micro-organisms 100 times less in re-entry sample but still found in 90% of second samples

Leskell et al. [1996]

permanent, bulk carious dentine excadeep, no pulpitis vated; calcium hydroxide, zinc oxide and eugenol cement; at re-entry all soft dentine removed, excavators or burs (n = 57)

8–24 weeks all soft caries removed, Ca(OH)2, ZnO/Eug cement, then GIC composite or amalgam (n = 57)

clinical observation

17.5% treatment group exposed, 40% control group exposed

Kreulen et al. [1997]

permanent molars, occlusal caries on radiograph

lesions opened to dentine, filled resin modified glass ionomer (n = 40)

as treatment but filled amalgam (n = 40)

6 months

clinical observation of dentine on re-entry micro-organisms

dentine darker and harder on re-entry; substantial decrease in total viable count, mutans streptococci and lactobacilli; more reduction with resin modified glass ionomer than amalgam

Weerheijm et al. permanent [1999] molars, occlusal caries on radiograph

lesions opened to dentine, filled resin modified glass ionomer (n = 33)

as treatment but filled amalgam (n = 33)

2 years

micro-organisms

25 patients reviewed; substantial decrease in total viable count, mutans streptococci and lactobacilli; more decrease in glass ionomer than amalgam; micro-organisms not cultured in 11 out of 50 cases +

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Table 2 (continued) Study

Toothtype, lesion depth

Bjørndal et al. [1997]

Treatment

Control

Time to re-entry

Indication of carious activity

Result and conclusion

permanent teeth, peripheral excavation and no pulpitis, deep excavation ‘cariogenic lesions biomass’ and superficial demineralized dentine; calcium hydroxide and temporary filling; at re-entry, complete excavation (n = 31)

6–12 months

clinical observation of dentine on re-entry; micro-organisms

no pulpal exposures at final excavation; at re-entry dentine darker, harder, dryer; substantial reduction in colony-forming units – not time-dependent

Bjørndal and Thylstrup [1998]

permanent teeth, peripheral excavation and no pulpitis, deep excavation ‘cariogenic lesions biomass’ and superficial demineralized dentine; calcium hydroxide and temporary restoration (n = 94)

2–9 months clinical observation of dentine on re-entry; follow up clinical and radiographic examination 1 year after final restoration

dentine harder and darker on reentry; 5 exposures on final excavation (2 sensitive to pressure, 2 inadequate seal): 88 cases symptomless at 1 year; 1 case lost temporarily and needed root treatment

Bjørndal and Larsen [2000]

permanent teeth, as above + microbiological no pulpitis, deep sampling (n = 9) lesions

4–6 months clinical observation of dentine on re-entry; micro-organisms

dentine harder and darker on reentry; colony-forming units and proportion lactobacilli substantially reduced; gram-negative rods declined; flora dominated by Actinomyces naeslundii and various streptococci

Maltz et al. [2002]

permanent teeth, cavity walls made hard; no pulpitis, deep incomplete caries removal lesions pulpally; calcium hydroxide and zinc oxide and eugenol cement

6–7 months clinical observation of dentine before and after re-entry; radiographic examination; microorganisms

dentine dryer, harder, darker on re-entry; increase in radio-opacity during study period; bacterial counts decreased significantly

Table 2 gives a chronological overview of stepwise excavation studies. The majority of these studies have no control. Most have been done on permanent teeth with deep lesions. The amount of carious dentine removed at the initial excavation varies from access to caries only, to removing the bulk of the carious dentine. The restorative materials are also very variable. They include calcium hydroxide, zinc oxide and eugenol, amalgam, glass ionomer cement and composite resin. Times to re-entry are also very variable, the shortest being 3 weeks, the longest 2 years. Caries activity has been assessed clinically, radiographically and often by microbiological examination at initial entry and on re-entry. With such differing methodologies, a systematic review is not possible but some themes emerge. (1) The clinical success rate appears high. Exposure is usually avoided using the stepwise technique and symptoms rarely arise between excavations. Control lesions are often exposed by conventional excavation. (2) Some studies report the dentine is altered on re-entry, being dryer, harder and darker. (3) Microbiological monitoring indicates substantial reductions in cultivable flora.

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Some teeth appear sterile, but in most some micro-organisms survive. Two studies [Bjørndal and Larsen, 2000; Maltz et al., 2002] suggest that the cultivable flora is altered on re-entry to a less cariogenic flora. (4) There is a possibility that there may be an effect from the dental material on the outcome, but very few studies have addressed this in a controlled manner.

Why Re-Enter?

The studies in table 2 seem to show that the depth of the first excavation is not relevant to the level of infection of the soft, dry dentine that is found on re-entry. The final excavation allows the dentist to be sure there is no exposure and removes the remaining infected dentine. The logic here is that the carious process may continue, albeit slowly, in this infected tissue. However, perhaps there is no need to re-enter and indeed this is the basis of the indirect pulp capping technique [Hilton and Summitt, 2000], although most of the demineralized tissues is removed in this procedure. In

Kidd

Table 3. Randomized controlled clinical trials of ‘complete’ versus ‘incomplete’ caries removal Study

Tooth type, lesion depth

Treatment

Control

Observation period

Results and conclusions

Magnusson and Sundell [1977]

deciduous, deep but no pulpitis

cavity washed microbiocidal solution; partial excavation, calcium hydroxide, zinc oxide and eugenol cement; at re-entry, all soft carious dentine excavated (n = 55)

all softened dentine excavated regardless of risk of exposure (n = 55)

re-entry: 4–6 weeks in treatment group

treatment: 2 cases pulpitis between visits, dentine ‘altered’ on re-entry; 15% pulps exposed; control: 53% pulps exposed

Leskell et al. [1996]

permanent, deep but no pulpitis

bulk carious tissue excavated, calcium hydroxide, zinc oxide and eugenol cement; at re-entry all soft dentine removed with excavator or burs (n = 57)

all softened dentine removed; if no exposure, calcium hydroxide, zinc oxide and eugenol cement, glass ionomer cement; in some teeth composite or amalgam on top of this (n = 70)

re-entry: 8–24 weeks in treatment group

treatment: 17.5% pulps exposed; easy to distinguish ‘soft’ and ‘hard’ dentine on re-entry; control: 40% pulps exposed

Mertz-Fairhurst et al. [1998]

permanent; occlusal lesions no deeper than halfway into dentine on radiograph

DEJ not made caries free; moist, soft, infected dentine left at DEJ and over pulp; restored bonded, sealed, composite (n = 156)

complete caries removal; amalgam + sealant group (n = 77); conventional amalgam group (n = 79)

no re-entry; 10-year follow-up

no exposure during caries removal; treatment: 85 teeth reviewed at 10 years, caries apparently arrested, 1 lesion ‘caved in’; control: some conventional amalgam rest failed with new caries at margin

Ribeiro et al. [1999]

deciduous, no pulpitis, no exposure expected

DEJ made caries free with round bur but moist, soft, infected dentine left over pulp; restored dentine bonding agent and composite (n = 24)

caries removal with slow round bur guided by caries dye; all dye stained dentine removed; restored dentine bonding agent and composite (n = 24)

no re-entry: followed for 1 year; assessed on radiograph and histology

treatment: all restorations retained; excellent marginal integrity after 1 year; on radiograph: 46% regressed, 25% progressed, 29% unchanged; adhesive system formed altered hybrid layer histologically; control: pulpal necrosis in 1 tooth, all other restorations retained; excellent marginal integrity, adhesive system formed hybrid layer

stepwise excavation, on the other hand, soft, wet dentine is left in place. Is it now necessary to re-enter? After all, if the caries process is driven by the activity in the biofilm, the process should be arrested simply by sealing the cavity. The persistence of a few micro-organisms may be irrelevant. Perhaps they are just opportunistic squatters adapted to the new environment in which they find themselves.

Are there deleterious consequences after incomplete caries removal? Only randomized controlled clinical trials will answer this question and table 3 documents 4 such studies.

Two of these selected deep lesions in deciduous [Magnusson and Sundell, 1977] or permanent [Leskell et al., 1996] teeth where exposure seemed likely following conventional caries removal. Both studies strongly support a stepwise approach (using calcium hydroxide after initial excavation) if pulp exposure is to be avoided. In these cases, conventional caries removal was deleterious; both studies re-entered. The other two studies in table 3 selected less advanced lesions and did not re-enter to remove the remaining soft dentine in the treatment groups. Both studies sealed incompletely excavated cavities with dentine bonding agents and composite resins. The work of Ribeiro et al. [1999] on deciduous teeth concluded that the clinical performance of the restorations was not adversely affected by

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Randomized Controlled Clinical Trails

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the incomplete caries removal after 1 year. The study by Mertz-Fairhurst et al. [1998] was remarkable for a 10-year follow-up of occlusal restorations placed over moist, soft, infected dentine left both at the enamel-dentine junction and over the pulp. Lesion progression was arrested and there were no more clinical failures in this group than in control groups with conventional caries removal.

What Does the Evidence Tell Us about Our Current Operative Approach?

This review makes uncomfortable reading for those of us teaching operative dentistry. There is no clear evidence that it is deleterious to leave infected dentine, even if it is soft and wet, prior to sealing the cavity. Indeed, this cautious approach may be preferable to vigorous excavation because fewer pulps will be exposed and sealing the dentine from the oral environment encourages arrest of lesion progression. The reparative processes of tubular sclerosis and tertiary dentine are encouraged, thus reducing the permeability of the remaining dentine. The residual micro-organisms are now in a very different environment. They are entombed by the seal of the restoration on one side and the reduced permeability of the remaining dentine on the other. The apparent irrelevance of the infected dentine is biologically logical if it is accepted that the caries process is driven by the biofilm and its reflection is the lesion in the dental hard tissues.

Further Research

One of the most intriguing aspects of this review is the fate of the residual micro-organisms. How do they survive? Is their survival time dependant? Do they change, either phenotypically or genotypically? Do they continue to demineralize the dentine, albeit very slowly? How does

the pulp react to their presence in the short and long terms? In view of the numerous studies that show the pulp can be compromised by leakage of bacteria around restorations [Bergenholtz et al., 1982], it is remarkable that their presence does not result in pulpitis and pulp death. It is probably highly relevant that the studies relating bacterial leakage around restorations to pulp pathology are done on caries-free teeth. Thus, cavity preparation will open up millions of tubules, each one a pathway to the pulp. There is a dearth of research that relates the activity of a carious lesion to the histological changes in the underlying pulp. Is it really necessary to extract a tooth to examine pulpal pathology? There seems a need to find a way of monitoring what is going on in vivo. The stepwise excavation studies in table 2 show many disparate methodologies. Randomized, controlled clinical trials should be designed to compare: the results of the stepwise technique in shallow and deep lesions; superficial caries removal with a deeper excavation; the relevance of the medicament (e.g. calcium hydroxide, zinc oxide and eugenol) and the filling material (amalgam, composite, glass ionomer cement) to the outcome, and the relevance of the time before re-entry to the clinical and microbiological outcome. In addition, this methodology might examine techniques designed to kill bacteria in infected dentine such as ozone treatment [Baysan et al., 2000] photo-activated disinfection [Burns et al., 1995; Williams et al., 2003]. Would these techniques help, hinder or be irrelevant to the clinical outcome? Further long-term, randomized, controlled clinical trails will be important, but those who have attempted such work must look at the 10-year results of Mertz-Fairhurst et al. [1998] with admiration. The problem in clinical trials is usually an unacceptable loss of patients, but they seemed able to recall many of their patients. Stable populations will be required for these essential long-term studies.

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Jeronimus DJ, Till MJ, Sveen OB: Reduced viability of microorganisms under dental sealants. J Dent Child 1975;42:275–280. Kerkove BC, Herman SC, Klein AI, McDonald RE: A clinical and television densitometric evaluation of the indirect pulp capping technique. J Dent Child 1967;34:192–201. Kidd EAM, Joyston-Bechal S, Beighton D: The use of a caries detector dye during cavity preparation: A microbiological assessment. Br Dent J 1993;174:245–248. Kidd EAM, Joyston-Bechal S, Beighton D: Marginal ditching and staining as a predictor of secondary caries around amalgam restorations: A clinical and microbiological study. J Dent Res 1995;74:1206–1211. Kidd EAM, Ricketts D, Beighton D: Criteria for caries removal at the enamel-dentine junction: A clinical and microbiological study. Br Dent J 1996;180:287–291. King JB, Crawford JJ, Lindahl RL: Indirect pulp capping: A bacteriologic study of deep carious dentine in human teeth. Oral Surg Oral Med Oral Pathol 1965;20:663–671. Kreulen CM, de Soet JJ, Weerheijm KL, Van Amerongen WE: In vivo cariostatic effect of resin modified glass ionomer cement and amalgam on dentine. Caries Res 1997;31:384–389. Lager A, Thornqvist E, Ericson D: Cultivable bacteria in dentine after caries excavation using rose-bur or carisolv. Caries Res 2003;37:206– 211. Law DB, Lewis TM: The effect of calcium hydroxide on deep carious lesions. Oral Surg Oral Med Oral Pathol 1961;14:1130–1137. Leskell E, Ridell K, Cvek M, Mejare I: Pulp exposure after stepwise versus direct complete excavation of deep carious lesions in young posterior permanent teeth. Endod Dent Traumatol 1996;12:192–196. Leung RL, Loesche WJ, Charbeneau GT: Effect of Dycal on bacteria in deep carious lesions. J Am Dent Assoc 1980;100:193–197. Magnusson BO, Sundell SO: Stepwise excavation of deep carious lesions in primary molars. J Int Assoc Dent Child 1977;8:36–40. Maltz M, de Oliveira EF, Fontanella V, Bianchi R: A clinical, microbiologic, and radiographic study of deep caries lesions after incomplete caries removal. Quintessence Int 2002;33:151– 159. Massler M: Pulpal reactions to dental caries. Int Dent J 1967;17:441–460. Mertz-Fairhurst EJ, Curtis JW, Ergle JW, Rueggeberg FA, Adair SM: Ultraconservative and cariostatic sealed restorations. J Am Dent Assoc 1998;129:55–66.

Mertz-Fairhurst EJ, Schuster GS, Fairhurst CW: Arresting caries by sealants: Results of a clinical study. J Am Dent Assoc 1986;112:194– 198. Mertz-Fairhurst EJ, Schuster GS, Williams JE, Fairhurst CW: Clinical progress of sealed and unsealed caries. 1. Depth changes and bacterial counts. J Prosthet Dent 1979a;42:521–526. Mertz-Fairhurst EJ, Schuster GS, Williams JE, Fairhurst CW: Clinical progress of sealed and unsealed caries. 11. Standardized radiographs and clinical observations. J Prosthet Dent 1979b;42:633–637. Nyvad B, Fejerskov O: Active root surface caries converted into inactive caries as a response to oral hygiene. Scand J Dent Res 1986;94:281– 284. Nyvad B, Fejerskov O: An ultrastructural study of bacterial invasion and tissue breakdown in human experimental root surface caries. J Dent Res 1990;69:2218–2225. Ribeiro CCC, Baratieri LN, Perdigao J, Baratieri NMM, Ritter AV: A clinical, radiographic, and scanning electron microscope evaluation of adhesive restorations on carious dentin in primary teeth. Quintessence Int 1999;30:591–599. Schouboe T, Macdonald JB: Prolonged viability of organisms sealed in dentinal caries. Arch oral Biol 1962;7:525–526. Thylstrup A, Qvist V: Principal enamel and dentine reactions during caries progression; in Thylstrup A, Leach SA, Quist V (eds): Dentine and Dentine Reactions in the Oral Cavity. Oxford, IRL Press, 1986, pp 3–16. Tomes J: A System of Dental Surgery. London, John Churchill, 1859, p 336. Weerheijm KL, de Soet JJ, van Amerongen WE, de Graaff J: Sealing of occlusal caries lesions: An alternative for curative treatment. J Dent Child 1992;59:263–268. Weerheijm KL, de Soet JJ, van Amerongen WE, de Graaff J: The effect of glass ionomer cement on carious dentine. Caries Res 1993;27:417–423. Weerheijm KL, Kreulen CM, de Soet JJ, Groen HJ, van Amerongen WE: Bacterial counts in carious dentine under restorations: 2-year in vivo effects. Caries Res 1999;33:130–134. Williams JA, Pearson GJ, Colles MJ, Wilson M: The effect of variable energy input from a novel light source on the photo-activated bactericidal action of toludine blue O on streptococcus mutans. Caries Res 2003;37:190–193. Yip HK, Stevenson AG, Beely JA: The specificity of caries detector dyes in cavity preparation. Br Dent J 1994;176:417–421.

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Caries Res 2004;38:314–320 DOI: 10.1159/000077771

The Future Role of a Molecular Approach to Pulp-Dentinal Regeneration D. Tziafas Department of Endodontology, School of Dentistry, Aristotle University of Thessaloniki, Thessaloniki, Greece

Key Words Dentin W Growth factors W Odontoblasts W Odontoblast-like cells W Pulp W Reactionary dentin W Reparative dentin W TGF-ß W Vital pulp therapy

Abstract The ultimate goal of a regenerative pulp treatment strategy is to reconstitute normal tissue continuum at the pulp-dentin border, regulating tissue-specific processes of tertiary dentinogenesis. Experimental investigations in mature teeth have shown that a network of extracellular matrix molecules and growth factors signal tertiary dentinogenesis. Application of dentin matrix components or growth factors in deep dentinal cavities stimulated up-regulation of biosynthetic activity of primary odontoblasts (reactionary dentin formation). Pulp-capping studies with a broad spectrum of biological agents, including growth factors and extracellular matrix molecules, showed formation of osteodentin and/or tertiary dentinogenesis (reparative dentin formation). Promising biologically active substances should be subjected to careful evaluation in well-designed preclinical investigations as well as in long-term clinical trials before their introduction in clinical practice. Copyright © 2004 S. Karger AG, Basel

ABC

© 2004 S. Karger AG, Basel 0008–6568/04/0383–0314$21.00/0

Fax + 41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/cre

Vital pulp therapy aims to treat reversible pulpal injury and maintain pulp vitality and function. It includes two therapeutic approaches: indirect pulp capping in cases of deep dentinal cavities and direct pulp capping/pulpotomy in cases of pulp exposures. Successful outcome for vital pulp therapy is very dependent on the type and location of injury, age of the tooth, treatment modality (capping material) and integrity of the cavity restoration [for reviews, see Mjör, 2002; Horsted-Bindslev and Bergenholtz, 2003]. This paper focuses on the potential therapeutic role of biologically active molecules as treatment modalities in vital therapy. Whilst the biological processes directed by the treatment strategy have received much attention during the last four decades, controversy still exists regarding the biological basis of the mechanism by which the capping material regulates healing and repair of the pulp in vital pulp therapy [Nyborg, 1955; Fitzgerald, 1979; Cox et al., 1985; Horsted et al., 1985; Schroder, 1985; Cvek et al., 1987; Stanley, 1989; Mjör et al., 1991]. Advances in biomedical research open directions to design new methods of dental treatment, aiming at regeneration of the dentinpulp complex. Numerous agents, delivering biologically active molecules at pulp exposure, were under investigation during the last decade. New approaches have been based on the understanding of the molecular and cellular mechanisms regulating dentinogenesis during dental tissue repair and their potential for clinical exploitation.

Dr. D. Tziafas Department of Endodontology, School of Dentistry Aristotle University of Thessaloniki GR–54124 Thessaloniki (Greece) Tel./Fax +30 2310 999 626, E-Mail [email protected]

Tertiary Dentinogenesis in Vital Pulp Therapy

The dental pulp possesses the ability to form a dentinlike matrix (tertiary dentin) as a part of repair in the dentin-pulp organ [Baume, 1980]. Vital pulp therapy aims to treat reversible pulpal injury, whenever dentin and pulp are affected by caries, restorative procedures or trauma. The injury may or may not involve pulpal exposure and is followed by a classical wound healing process of the connective tissue. Wound healing and new hard tissue formation beneath the injury are prerequisites for long-term control of post-operative infection and pulp survival in vital pulp therapy. It is well recognized that the nature and specificity by which a traumatized tissue area is healed determine the biological properties of the newly formed tissues. The end result of the healing process in vital pulp therapy would be a reconstitution of normal tissue architecture and tertiary dentin formation at the wound area, or formation of scarlike soft tissue and fibrodentin formation. The healing pattern may be dependent, partly at least, on the type and extent of tissue injury and the effect of the associated defence reaction on the structural and functional integrity at the dentin-pulp border [for a review, see Smith, 2002]. Principally, whenever the dentin-pulp complex is affected by injury, three different physio-pathological conditions could be observed at the dentin-pulp border. (a) In the case of mild injuries, odontoblasts may survive, e.g. non-cavitated stages of enamel caries, slowly progressing dentinal caries, mild abrasion, erosion, mechanico-chemical irritation or fracture involving enameldentin. The odontoblast layer is stimulated to form tertiary dentin matrix beneath the injury (reactionary dentin), while peritubular dentin formation is seen in the dentinal tubules [Frank, 1968; Stanley et al., 1983; Bjørndal and Mjör, 2001; Murray and Smith, 2002]. Reactionary dentin shows many anatomical, biochemical and functional similarities to the primary and secondary dentin and can effectively oppose exogenous destructive stimuli to protect the pulp [Smith et al., 2002]. Reactionary dentinogenesis represents up-regulation of the biosynthetic activity of primary odontoblasts, restricted to those cells affected by the injury. Peritubular dentin formation should be distinguished from atypical intratubular calcification, which has been suggested to represent a non-vital process [Frank, 1968]. (b) With severe dentinal injuries without pulp exposure, odontoblasts are destroyed subjacent to the affected dentin, e.g. rapidly progressive carious lesions, severe tissue damage due to cavity preparation or cytotoxic injury

on pulpal cells due to restoration [Stanley et al., 1983; Kitamura et al., 2001; Bjørndal and Mjör, 2001]. A cascade of inflammatory and healing events rapidly occurs in the area of degenerating odontoblasts [Kim, 1990; Chiego, 1992]. As a part of the connective tissue healing, pulpal cells proliferate and migrate toward the circumpulpal dentin. Initially, fibroblast-like cells align themselves against the dentin and atubular fibrodentin is laid down at the dentin-pulp border. In an appropriate metabolic state of the dentin-pulp complex, a new generation of odontoblast-like cells may differentiate and form tubular tertiary dentin (reparative dentinogenesis) in a polar predentin-like pattern [Baume, 1980; Bjørndal and Darvann, 1999]. It must be emphasized that under clinical conditions matrix formed at the pulp-dentin interface often comprises reactionary dentin, reparative dentin or fibrodentin formation. It is impossible to distinguish these processes at the in vivo level and the processes may also from a biochemical and molecular point of view be indistinguishable. (c) In the case of pulpal exposure, the amputated pulp can be repaired by itself or after application of capping materials [Nyborg, 1955; Kakehashi et al., 1965; Yamamura, 1985]. Pulpal exposure due to caries shows very limited potential for pulp recovery due to bacterial infection of the pulp for a substantial period of time, which compromises the defence reaction [Bergenholtz, 2001]. Favourable conditions for pulp repair after oral exposure require an environment free of bacteria, absence of severe haemodynamic changes and absence of severe inflammatory cell infiltration. Whether subsequent reactions lead to pulp healing and repair or to generalized pulp inflammation and necrosis will depend on the extent of defence reactions [Trowbridge, 1981]. As a part of the wound healing process in the repairing pulp, the dentinogenic potential of pulpal cells can be expressed [for a review, see Tziafas, 1997]. Proliferation, migration and differentiation of progenitor cells can give rise to a new generation of reparative dentin-forming cells (odontoblast-like cells) reconstituting the lost continuum at the pulp-dentin border [Fitzgerald, 1979; Fitzgerald et al., 1990; Mjör et al., 1991]. The cellular processes taking place after pulp exposure have been elucidated by using calcium hydroxide-based materials or other materials producing a low-grade irritation to the pulp. Initially, the pulpal cells under the capping material proliferate, migrate and elaborate new collagen in contact with a firm necrotic zone of the treated pulpal area. Then, mineral salts precipitate on the necrotic zone and in the associated new collagen matrix. Finally,

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Fig. 1. Transdentinal stimulation of reac-

tionary dentinogenesis in the case of mild dentinal injury. The ultimate goal of a regenerative treatment strategy is to up-regulate the biosynthetic activity of survived primary odontoblasts corresponding to the involved area.

reparative dentinogenesis is initiated; a layer of odontoblast-like cells is formed in association with the superficial calcification and a tubular mineralized matrix is secreted in a polar predentin-like pattern [Schroder, 1985; Cvek, 1987]. However, many studies have shown that the wound healing mechanism often results in early formation of fibrodentin with osteotypic appearance at the traumatized area [Baume, 1980; Cox et al., 1996; Higashi and Okamoto, 1996]. Osteotypic hard tissue cannot provide the necessary barrier effect to protect the pulp from exogenous destructive stimuli.

Therapeutic Regulation of Tertiary Dentinogenesis: Existing Knowledge and Future Perspectives for Research

The ability of the pulp-dentin complex to respond to therapeutic applications by specific cellular processes and hard tissue formation has long been recognized. Current research has provided insights into the basic molecular events underlying dental tissue repair, induction of tertiary dentin formation, competence of the responsive cells and how these phenomena could be integrated into the clinical approach to the problem of vital pulp therapy [for reviews, see Lesot et al., 1994; Smith et al., 1995; Rutherford, 1999; Tziafas et al., 2000]. Transdentinal Stimulation of Reactionary Dentinogenesis The aim of a regenerative treatment strategy in the case of mild dentinal injuries (fig. 1) is to stimulate localized peritubular dentin formation and to provide a regional and time-limited effect on surviving odontoblasts, in order to up-regulate their biosynthetic activity. The optimal end result is sclerosis of primary dentin and an

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underlying zone of reactionary dentin at the pulp-dentin border corresponding to the involved area. The molecular processes underlying reactionary dentinogenesis have recently been approached in experimental models in vitro and in vivo and in carious human teeth [Magloire et al., 1996; Sloan and Smith, 1999]. Our understanding in this field has significantly advanced with appreciation that dentin extracellular matrix does not represent an inert material, but it contains bioactive molecules potentially available for release during pulp healing and repair [Finkelman et al., 1991; Magloire et al., 2001; Smith et al., 2001]. Carious demineralization of the dentin has been suggested to release bioactive molecules, which signal the associated dentinogenic events [Smith, 2002]. Dental materials may also solubilize and release growth factors (TGF-ßs) from the soluble fraction of dentin components [Zhao et al., 2000]. Application of EDTAsoluble dentin components in unexposed cavities of ferret teeth demonstrated stimulation of odontoblasts forming a zone of reactionary dentin beneath the injury [Smith et al., 1994, 2001]. Transdentinal stimulation of tertiary dentinogenesis was also seen after application of osteogenic protein-1 (bone morphogenetic protein-7) in unexposed cavities of monkey teeth [Rutherford et al., 1995]. In unexposed deep cavities of dog teeth, human recombinant TGF-ß1 was placed on acid-treated dentin. A specific effect of TGF-ß1 producing intratubular mineralization in a superficial zone of the treated dentin was seen [unpubl. data]. Further studies are still required to fully understand the kinetics of growth factors and the associated stimulation of dentinogenic events. Transdentinal Stimulation of Reparative Dentinogenesis A regenerative therapeutic approach, in the case of severe localized injury without pulp exposure (fig. 2), may

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Fig. 2. Transdentinal stimulation of reparative dentinogenesis in the case of severe dentinal injury. The ultimate goal of a regenerative treatment strategy is to favour the biological activity of dentin matrix, which in an appropriate pulpal environment is able to trigger differentiation of new odontoblastlike cells replacing lost primary odontoblasts.

result in differentiation of odontoblast-like cells for replacement of the lost odontoblasts and a time-limited formation of reparative dentin corresponding to the involved area. The debate on the origin of odontoblast-like cells and the associated signaling mechanisms controlling migration, orientation, attachment and cytodifferentiation of pulpal cells remains to be resolved. It has been strongly suggested that the exposed dentinal surface and the accumulated bioactive molecules might provide the necessary signals that determine the underlying cell function [Heritier et al., 1990]. Implantation of autogenous demineralized, or native, or unmineralized dentin matrices into the pulp at a distance from the site of mechanical pulp exposure allowed us to study potential interactions between dentinal matrix and the pulpal cells [Tziafas et al., 1992]. It is evident that the normal sequence of reparative events do not take place in the intrapulpal test model, but the biological effects of exogenous matrices or molecules on pulpal cells with minimal tissue trauma can be evaluated. In close proximity to demineralized dentin, we found stimulated spindle-shaped or polygonal cells after 3 days, groups of cells undergoing differentiation in relation to a newly formed matrix after 7 days and mineralized reparative dentin with a new layer of odontoblast-like cells after 2 weeks. The response of the pulpal cells to demineralized dentin was also characterized by deposition of fibrodentinal matrix before initiation of reparative dentinogenesis. Direct odontoblast-like cell differentiation in close proximity to the implanted unmineralized dentin matrix (predentin) was seen 3 days after implantation. These data indicate two mechanisms for reparative dentinogenesis onto the dentin surface: direct induction of odontoblast-like cells by the dentin matrix or indirect differentiation of odontoblast-like cells on an intermediate fibrodentinal matrix. Hence, predentin surface seems to represent

an appropriate substratum for direct induction of reparative dentin. Already in 1985, Mjör [1985] reported early formation of atubular fibrodentin (or interface dentin) before the onset of tertiary dentin formation after localized odontoblast destruction in deep dentinal cavities. The dentinogenic activity of dentin matrix might be attributed to the soluble fraction of dentin components. Morphological and functional differentiation of odontoblast-like cells was seen in close proximity to Millipore filters containing EDTA-soluble dentin components after 8 days [Tziafas et al., 1995]. The dentinogenic activity of this dentin fraction had previously been demonstrated in vitro [Begue-Kirn et al., 1992] and it was shown that the dentinogenic activity could be abolished by preincubation of the components with an antibody blocking the biological activity of TGF-ß molecules. Similarly, any effect of antibody-treated dentin on pulpal cells was completely or partially lost in vivo, indicating that the dentinogenic activity of dentin matrix can at least partly be ascribed to the TGF-ß molecules [Tziafas, 1995]. It seems that the endogenous pools of TGF-ßs and other growth factors in the dentin matrix may provide a natural delivery system, regulating reparative dentinogenesis after destruction of primary odontoblasts in deep dentinal cavities. Further studies are required to clarify whether the biological effect of dentin matrix on initiation of reparative dentinogenesis could be triggered therapeutically.

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Direct Induction of Reparative Dentinogenesis The ultimate goal of a regenerative treatment strategy in direct pulp capping or pulpotomy situation is to induce differentiation of odontoblast-like cells forming reparative dentin at the pulp-capping material interface (fig. 3) and to stimulate the biosynthetic activity of surrounding primary odontoblasts [Mjör, 2002]. The optimal end result is the reconstitution of dentinal defect with a bridge

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Fig. 3. Direct induction of reparative dentinogenesis in pulp exposure. The ultimate goal of a regenerative treatment strategy is to induce differentiation of odontoblast-like cells at the pulp-capping material interface and to up-regulate the biosynthetic activity of primary odontoblasts around the pulpal exposure to reconstitute the lost continuum of the pulp-dentin border.

of reparative dentin in direct continuum with reactionary dentin formed around the pulp exposure. The signaling mechanisms regulating reparative dentinogenesis after pulp capping have not been fully understood. The nature of the pulp wound healing mechanism depends on the defence reaction, possible contamination with oral bacteria, bleeding during surgery or cramming of dentinal chips into the pulp space [Heys et al., 1981, 1990; Cvek et al., 1987; Cox et al., 1987; Stanley, 1989]. It has been postulated that a network of interactions between extracellular matrix molecules, including fibronectin [Yoshiba et al., 1996; Tziafas et al., 1995], and growth factors regulates odontoblast-like cell differentiation and reparative dentinogenesis in the repairing pulp environment [Lesot et al., 1994]. The presence of a mechanical support seems to be of critical importance. Intermediate fibrodentinal matrix may act as the basement membrane does for odontoblast differentiation during tooth formation [Ruch, 1985]. In numerous animal studies, application of biologically active growth and morphogenetic factors and extracellular matrix molecules as capping materials resulted in hard tissue formation. Bone morphogenetic proteins (BMP), such as BMP-2, BMP-4 and BMP-7 (osteogenic protein1), induced formation of osteodentin in large amounts followed by tubular reparative dentin [Nakashima, 1994a, b; Rutherford et al., 1993; Jepsen et al., 1997]. Capping experiments with insulin-like growth factor-I have demonstrated complete dentinal bridging and occasionally tubular reparative dentin formation [Lovschall et al., 2001]. Osteodentin followed by homogeneous and wellmineralized atubular reparative dentin was seen after capping treatment with bone sialoprotein [Decup et al., 2000]. Hard tissue formation at a distance from the capping was found after placement of enamel matrix derivatives in the exposed pulp [Nakamura et al., 2001].

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Intrapulpal implantation of Millipore filters containing either EDTA-soluble dentin constituents [Tziafas et al., 1995] or human TGF-ß1 [Tziafas et al., 1998] induced specific dentinogenic events in close proximity to the implants. Cytological differentiation of odontoblast-like cells and reparative dentin formation were seen around the filters. Implantation of Millipore filters containing other growth factors, such as basic fibroblast growth factor or insulin-like growth factor-II, showed increased dentinogenic effect at a distance from the implant [Tziafas et al., 1998]. It seems that while TGF-ß1 appears to be an effective signal for odontoblast-like cell differentiation within the pulp, its ability to do so at the wound surface is very limited. Nakashima [1994b] showed inhibition of reparative dentin formation after pulp capping with collagen containing TGF-ß1. Experimental applications of several artificial substrates, such as Millipore filters, hydroxyapatite granules, pure titanium, as carriers for recombinant human TGF-ß1 used for pulp capping in dog teeth failed to induce any dentinogenic effect. Only preset calcium hydroxide soaked in recombinant human TGF-ß1 stimulated differentiation of odontoblast-like cells and reparative dentin, while the control teeth, which were capped with pre-set calcium hydroxide only, did not show any particular response [Tziafas et al., 2001]. It is clear that a broad spectrum of biological substances stimulate reparative dentin formation in the exposed pulp. However, it is important to recognize that in most of these cases the reparative dentinogenesis proceeded via formation of fibrodentin matrix. As has been previously stated [Tziafas et al., 2000], formation of fibrodentin perhaps implies an indirect effect, e.g. stimulation of the biosynthetic activity of pulpal cells, which is later superseded by the tissue-specific dentinogenic response. The clinical problem with the indirect effects of biologically active molecules on pulpal cells is that fibro-

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dentin formation does not represent a guided natural regeneration at the dentin-pulp border. Development of new capping materials for delivery of exogenous signaling molecules offers exciting opportunities for the future. However, a number of critical considerations, such as the dose-response effects, the nature of the delivery system, half-life of the molecules and their possible side-effects need to be addressed before any introduction of new treatment modalities into clinical practice.

Modern materials able to exploit endogenous biologically active molecules could also be used in the shorter-term at least. In any case, promising new treatment strategies should be exposed to careful evaluation in properly designed preclinical investigations with a large number of capping experiments and in well-designed clinical trials to account all possible variables that may exist clinically [Bergenholtz, 2001].

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Kim S: Neurovascular interactions in the dental pulp in health and inflammation. J Endod 1990;16:48–57. Kitamura C, Kimura K, Nakayama T, Toyoshima K, Terashita M: Primary and secondary induction of apoptosis in odontoblasts after cavity preparation of rat molars. J Dent Res 2001;80: 1530–1534. Jepsen S, Hans-Karl A, Fleiner B, Tucker M, Rueger D: Recombinant human osteogenic protein-1 induces dentin formation: An experimental study in miniature swine. J Endod 1997;23:378–382. Lesot H, Smith AJ, Tziafas D, Begue-Kirn C, Cassidy N, Ruch JV: Biologically active molecules and dental tissue repair: A comparative review of reactionary and reparative dentinogenesis with the induction of odontoblast differentiation in vitro. Cells Mater 1994;4:199–218. Lovschall H, Fejerskov O, Flyvbjerg A: Pulp capping with recombinant human insulin growth factor-I (rhIGF-I) in rat molars. Adv Dent Res 2001;15:108–112. Magloire H, Joffre A Bleicher F: An in vitro model of human dental pulp repair. J Dent Res 1996; 75:1971–1978. Magloire H, Romeas A, Melin M, Couble M-L, Bleicher F, Farges J-C: Molecular regulation of odontoblast activity under dentin injury. Adv Dent Res 2001;15:46–50. Mjör IA: Dentin-predentin complex and its permeability: Pathology and treatment overview. J Dent Res 1985;64:621–627. Mjör IA: Pulp-dentin biology in restorative dentistry. 7. The exposed pulp. Quintessence Int 2002;33:113–135. Mjör IA, Dahl E, Cox CF: Healing of pulp exposures: An ultrastructural study. J Oral Pathol Med 1991;20:496–501. Murray PE, Smith AJ: Saving pulps – A biological basis. An overview. Prim Dent Care 2002;9: 21–26. Nakamura Y, Hammarström L, Lundberg E, Ekdahl H, Matsumoto K, Gestrelius S, Lyngstadaas SP: Enamel matrix derivative promotes reparative processes in the dental pulp. Adv Dent Res 2001;15:105–107.

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Nakashima M: Induction of dentin formation on canine amputated pulp by recombinant human bone morphogenetic proteins (BMP)-2 and -4. J Dent Res 1994a;73:1515–1522. Nakashima M: Induction of dentine in amputated pulp of dogs by recombinant human bone morphogenetic proteins -2 and -4 with collagen matrix. Arch Oral Biol 1994b;39:1085–1089. Nyborg H: Healing processes in the dental pulp on capping. A morphologic study. Experiments on surgical lesions of the pulp in dog and man. Acta Odontol Scand 1955;13(suppl 16):1–130. Ruch JV: Odontoblast differentiation and the formation of odontoblast layer. J Dent Res 1985; 64:489–498. Rutherford BR: Regeneration of the pulp-dentin complex; in Lynch SE, Genco RJ, Marx RE (eds): Tissue Engineering. Applications in Maxillofacial Surgery and Periodontics. Chicago, Quintessence Publishing Co, Inc, 1999, pp 185–199. Rutherford B, Spangberg L, Tucker M, Charette M: Transdentinal stimulation of reparative dentine formation by osteogenic protein-1 in monkeys. Arch Oral Biol 1995;40:681–683. Rutherford RB, Wahle J, Tucker M, Rueger D, Charette M: Induction of reparative dentine formation in monkeys by recombinant human osteogenic protein-1. Arch Oral Biol 1993;38: 571–576. Schroder U: Effects of calcium hydroxide-containing agent on pulp cell migration, proliferation and differentiation. J Dent Res 1985;64:541– 548. Sloan AJ, Smith AJ: Stimulation of the dentinepulp complex of rat incisor teeth by TGFbeta isoforms 1–3 in vitro. Arch Oral Biol 1999;44: 149–156.

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Caries Res 2004;38:321–324 DOI: 10.1159/000077772

Getting Research into Clinical Practice – Barriers and Solutions J.E. Clarkson University of Dundee, Dental Hospital and School, Dundee, UK

Key Words Implementation W Practice W Primary care

Abstract The success of current efforts towards evidence-based health services in many countries depends on efficient transfer of research findings to health practitioners. However, there is a lag in research being adopted. In part this is due to difficulties in interpreting or generalising research findings, in part to inertia, organisational structures and information. Clinical guidelines are usually cited as being the most effective product of evidence assessment and means of getting research into practice. The processes by which they are prepared and disseminated are discussed. Current clinical practice requires that health professionals adapt to changing systems and adopt new techniques. Therefore, in future, practice research to evaluate (a) clinical interventions and (b) dissemination and implementation strategies will become increasingly important. Recognised barriers to such research include lack of interest, lack of involvement, lack of time and lack of remuneration. High-quality research in dental primary care requires academics and dental service providers working in partnership on topics that are relevant both to clinicians and policy makers. Good project management, education and training are essential.

Understanding factors that influence the implementation of research findings in clinical practice is perhaps one of the greatest challenges members of ORCA face. In this presentation, I will discuss issues concerned with getting research into clinical practice, both barriers and solutions. I will consider the existing information, state of the art, future perspectives and potential impact on practice. I should first like to remind you of the motivation to establish this organisation. The five founding members of ORCA were general dental practitioners who recognised the need to promote the dissemination of caries research in dental practice. The recommendation to ORCA in the 1970s for there to be inclusion of social and behavioural scientists recognised a need to understand behaviour change and that probably still holds true for today. With regard to existing information, it is well recognised that the bulk of research evidence has not been generated in settings where most clinical care is provided. Academic or hospital-based research is more common than research in primary care and there is resistance to generalising research findings because of possible differences in the population groups and clinical environment. So there is a lag time in research being adopted; additional reasons for this include inertia, organisational structures, information overload and interpretation difficulties where there are incomplete or inconsistent results and/or conflict [NHS Centre for Reviews and Dissemination, 1999; McGlone et al., 2001]. Professional behaviour has

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to change. The linear model that providing knowledge will change attitudes and therefore behaviour I believe to be too simplistic. However, access to appropriate knowledge is essential and clinical guidelines are usually cited as being the most effective product of evidence assessment and means of getting research into practice. This brings me to the state of the art. I would like to discuss the most recent systematic reviews evaluating guideline dissemination and implementation and give an overview of the Cochrane Oral Health Group, as this is the resource most appropriate for dentistry. Guidelines are systematically developed statements to assist practitioner decisions and patient decisions about appropriate health care for specific clinical circumstances [Field and Lohr, 1990]. EPOC (Effective Practice and Organisation of Care) is one of the 50 review groups in the Cochrane Collaboration that undertake the development and maintenance of systematic reviews concerned with the effectiveness of practice and organisation of care. This group has recently completed an update of a previous systematic review evaluating the effectiveness of dissemination and implementation strategies for clinical guidelines. This update found education to be effective, which is in contrast to the original review and contains 235 studies, of which 110 (47%) were cluster randomised controlled trials [Grimshaw et al., 2001, 2004]. Cluster trials represent best practice because they are considered to be the most appropriate methodology for implementation research: where the practice or practitioner is the unit of analysis and not the patient. The cluster trials contained 309 comparisons of different implementation strategies, the most common being reminders, education, audit and feedback. Audit and feedback involve practitioners taking part in an audit relevant to a guideline and reflecting on feedback; education includes dissemination of guidelines and related events; reminder systems are mechanisms that identify (flag up) patients with certain characteristics. The synthesised results of a change in practice, that is, implementation of the guideline, indicate a similar median size effect for audit and feedback (7%, range 1–16%) and education (8%, range 4–17%). The overall effect for reminders was not significant (13%, range –1 to 34%). The difference between the original and updated reviews indicates the importance of including contemporary evidence and maintaining systematic reviews. Access to best evidence is important for patients, practitioners, guideline implementation groups and policy makers. Systematic reviews are considered the highest level of research evidence. The Cochrane Oral Health Group

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[http://www.cochrane-oral.man.ac.uk] was founded in 1994 and is considered to have the most comprehensive database of trial evidence in dentistry. In the most recent version of the Cochrane Library [Update Software, Issue 2, 2003] using the research terms ‘caries’ or ‘decay’ retrieves 14 completed systematic reviews of randomised clinical trials and 26 protocols registered for ongoing systematic reviews. Also the Database of Abstracts of Reviews of Effectiveness (DARE) contains 24 items which are critically appraised – not any of which are Cochrane systematic reviews related to caries. In the trial register, there are over 16,000 entries concerned with oral health and 2,000 of these involve trials of interventions for caries management. The series of 4 systematic reviews evaluating the effectiveness of topical fluorides contains data from over 125 randomised clinical trials and 60,000 participants [Marinho et al., 2003a–d]. These reviews compared the effectiveness of fluoride toothpaste, gel, varnish and mouth rinses in relation to placebo and the summary statistics include preventive fractions and numbers needed to treat. The number needed to treat is the reciprocal of the risk or rate difference and can be interpreted as the number of patients needed to be treated for one to be cured. In many countries, policy makers and service providers are exploring ways to make health services evidence based. The Cochrane Oral Health Group has recently collaborated with the Dental Health Services Research Unit and the Italian Cochrane Centre to supply information about evidence in dentistry to the Italian Government for future service planning. From this mapping exercise of trials in dentistry, comparing the characteristics of trials for caries with those of other trials in dentistry demonstrates that these are more likely to be with children and involve more participants. This finding reflects issues concerned with the conduct and cost of caries research. There is a lack of clinical research evidence from general practice. I think that in the future the two important areas for practice research will be clinical interventions and the evaluation of dissemination and implementation strategies. Current clinical practice requires that health professionals adapt to changing systems and adopt new techniques. In terms of complexity of science, it would be akin to a complex adaptive system [Plsek and Greenhalgh, 2001]. This is defined as a collection of individual agents with freedom to act in ways that are not always totally predictable, and whose actions are interconnected so that one agent’s actions change the context for other agents. In the past, we have trained dentists with a focus on competence, training them to repeat similar tasks in familiar

Clarkson

environments. Perhaps that would have been sufficient 50 years ago, when amalgam was the main restorative material. However, today we should be training individuals to adapt to information which is becoming available and to deal with the increasingly broad ranging and large body of research evidence [Fraser and Greenhalgh, 2001]. An example of initiatives designed to support research in dental primary care is the Scottish consortium for development and education in dental primary care [Clarkson et al., 2000]. Its initiatives include higher training fellowships and the Scottish Dental Practice-Based Research Network which is housed in the Scottish School of Primary Care. Currently, the network has over 300 members. Support is given to take projects from initial ideas to grant proposals, while an online journal [www.tuith.co.uk] provides updates on research activity and access to clinical guidelines. We have found that general practitioners are keen to access research evidence and Tuith receives over 5,000 hits per month, with the most frequently used link being to guidelines. An example of how this has worked is in the development of what is referred to as the Hall technique. The sequence of events for this particular project was that a regional audit identified a dentist who was placing stainless steel crowns on deciduous teeth without prior removal of decay or local anaesthetic. In order to evaluate this technique further, a regional network was funded to conduct a pilot project [Evans et al., 2000]. One of the participating dentists is now lead investigator for a randomised controlled trial with joint government and industry funding which supports both her salary and her study for a PhD. Research into the evaluation of implementation strategies in dentistry is sparse. We have experience of evaluating the effectiveness of audit and feedback compared to computer-aided learning strategies, for the dissemination of SIGN (Scottish Intercollegiate Guideline Network) Third Molar Guidelines [Bahrami et al., 2002]. The background to the development of these guidelines was the high cost of treatment and the high level of inappropriate treatment. The trial demonstrated the challenge of recruiting practitioners to participate in research: out of 565 approached, only 63 were recruited and only 51 took part in the final analysis. It raised the issue of whether a representative sample (of practitioners) is important for this kind of research, because the pre-intervention agreement with the guideline was high at 74%. However, this might not be a problem of selection bias so much as a reflection of current practice, because national data of third molar extractions in Scotland peaked in the 1990s, with a sudden downturn in 1998.

Table 1. Summary of barriers and solutions

Getting Research into Practice

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Barriers

Solutions

Lack of interest Lack of involvement

Research topics relevant to general practice Invite practitioners to participate in taking forward a research idea Reduce practice time by Coordinating research with efficient management systems Utilising routine data Recruiting multiple practices/ practitioners Payment for time taken and reward of continuing professional development

Lack of time

Lack of remuneration

Recognised barriers to research in primary care include lack of interest, lack of involvement, lack of time and lack of remuneration. All of these issues present a challenge to the current demand for an increase in capability and capacity for research in primary care. In Scotland, with its high caries incidence, we have an ideal environment for trials for caries prevention. An example is a current study evaluating the effectiveness of implementation strategies based on remuneration and training in evidence-based health care. The outcome is clinical practice in accordance with SIGN guidelines on targeted caries prevention for 6- to 16-year-olds [Royal College of Physicians of Edinburgh, 2000]. Within this cluster randomised controlled trial, we will be evaluating 6 theories of behaviour change and measuring how closely theoretical constructs can predict professional behaviour. The reason for this is to find out not only whether our interventions work, but why. The result will inform future implementation strategies in dentistry. A particular challenge in conducting cluster trials in dentistry is our observation that the intra-class correlation coefficient of clinical treatment appears much higher for dental practice than for medical practice. The implication of this is that larger trials involving more practices and patients need to be conducted. There is an increasing amount of empirical research available to assist the design of such research, for example, a systematic review on questionnaire design [Edwards et al., 2002]. I believe that in order to increase the conduct of research in dental primary care, it must be of high quality; there need to be partnerships between academic units and service providers; the research should be relevant not only to clinicians but also to policy makers. Project management will determine the success or failure of a study, so

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investment is required, and the role of education and training in promoting research needs to be considered. We are beginning to see the benefits of addressing barriers and the solutions require partnerships and resources (table 1). I hope I have provided an overview of existing information, state of the art and future perspectives. So, what is the potential impact on clinical practice? The use of best evidence will enable high quality care to be provided and that will improve oral health. However, if this is to come about, there has to be a change in research practice, too.

Research needs to be relevant and researchers require a more sophisticated understanding of policy process. There needs to be collaboration to enable quality research and it is important that the results are generalisable. This presents a particular challenge when interventions are skill based as opposed to therapeutic or drug based. If ORCA is to take forward the vision of its founding members, then I think heed should be paid to the recommendation in the 1970s to include social and behavioural scientists. Understanding professional behaviour change will make a difference to getting research into practice.

References Bahrami M, Bonetti D, Clarkson JE, et al: Effectiveness of different dissemination and implementation strategies for evidence-based guidelines for third molar problems in primary dental care. 7th Eur Forum Qual Improv Health Care, Edinburgh, 2002. Clarkson JE, Murray M, Pitts NB, MacFarlane TW, Newton JP, Burke FJT, Bain CA, Ibbetson R, Rennie JS: Scottish consortium for development and education in dental primary care. Br Dent J 2000;189:222–223. Edwards P, Roberts I, Clarke M, DiGuiseppi C, Pratap S, Wentz R, Kwan I: Increasing response rates to postal questionnaires: Systematic review. BMJ 2002;324:1–9. Evans DJP, Southwick CAP, Foley JI, Innes NP, Pavitt SH, Hall N: The Hall technique: A pilot trial of a novel use of preformed metal crowns for managing carious primary teeth. 2000. Tuith Online: http://www.dundee.ac.uk/tuith/ Articles/rt03.htm Field MJ, Lohr KN (eds): Clinical Practice Guidelines: Directions for a New Program. Washington, National Academy Press, 1990.

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Fraser SW, Greenhalgh T: Coping with complexity: Education for capability. BMJ 2001;323:799– 803. Grimshaw JM, Shirran L, Thoase R, et al: Changing provider behaviour: An overview of systematic reviews of interventions. Med Care 2001;39:II2–II45. Grimshaw JM, Thomas RE, Maclennan G, et al: Effectiveness and efficiency of guideline dissemination and implementation strategies. Health Technol Assess 2004;8:1–84. Marinho VCC, Higgins JPT, Logan S, Sheiham A: Fluoride toothpastes for preventing dental caries in children and adolescents (Cochrane Review); in: Cochrane Library, Issue 3, 2003a. Oxford, Update Software. Marinho VCC, Higgins JPT, Logan S, Sheiham A: Fluoride gels for preventing dental caries in children and adolescents (Cochrane Review); in: Cochrane Library, Issue 3, 2003b. Oxford, Update Software. Marinho VCC, Higgins JPT, Logan S, Sheiham A: Fluoride varnishes for preventing dental caries in children and adolescents (Cochrane Review); in: Cochrane Library, Issue 3, 2003c. Oxford, Update Software.

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Marinho VCC, Higgins JPT, Logan S, Shieham A: Fluoride mouth rinses for preventing dental caries in children and adolescents (Cochrane Review); in: Cochrane Library, Issue 3, 2003d. Oxford, Update Software. McGlone P, Watt R, Sheiham A: Evidence-based dentistry: An overview of the challenges in changing professional practice. Br Dent J 2001; 190:636–639. NHS Centre for Reviews and Dissemination: Getting evidence into practice. Eff Health Care Bull 1999;5:1–16. www.york.ac.uk/inst/crd/ ehc51.pdf Plsek PE, Greenhalgh T: The challenge of complexity in health care. BMJ 2001;323:625– 628. Royal College of Physicians of Edinburgh: Preventing Dental Caries in Children at High Risk: Targeted Prevention of Dental Caries in the Permanent Teeth of 6- to 16-Year-Olds Presenting for Care. Edinburgh, Scottish Intercollegiate Guideline Network, 2000, No 47.

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Summaries of Discussions Caries Res 2004;38:325–329 DOI: 10.1159/000077773

Summaries of Discussions at 50th Anniversary ORCA Symposium

Defining and Diagnosing Caries

How to Prevent Caries? Role of the Biofilm

Chairs: B. Angmar-Månsson, A.S. Lussi

Chairs: D. Beighton, C.H. Sissons

Several presenters pointed out the importance of early detection of caries lesions, which would improve the possibility of successful preventive intervention. However, early detection of the signs of caries should never trigger early operative intervention. In the future, early detection should take into account molecular and chemical aspects of the process. Meaningful research should not aim at repetition of known facts but should implement knowledge in new designs of studies. These studies will be more complex in the future, in line with the multifactorial nature of caries and the skewed distribution of the disease in the population, with a relatively small number of individuals having a large caries experience. A preventive programme has to be adapted to the society where it is supposed to work; no programmes can be used in all societies. Further, it is very difficult, if not impossible, to implement preventive programmes as long as politicians and patients consider caries not to be a problem. As long as these groups hold this belief, they will ignore preventive programmes. Preventive programmes can only be successful if behavioural scientists are involved.

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Three main topics were discussed. The responses from the speakers and from the floor underlined a lack of commonality of views. Caries is not an infectious disease but a dysfunction of the normal microflora – there are no caries pathogens. Sissons agreed that oral bacteria were functioning ‘normally’, although perhaps in a different environment, but their proportions may have changed. Marsh stated that plaque is variable but all sites in all people, even disease-free individuals, harbour most bacteria, including those described as ‘pathogenic’. Changes in the oral environment, e.g. sugar, inflammation, smoking, drugs affecting saliva, lead to an increase in ‘pathogens’ caused by ecological imbalances. Guggenheim agreed. Since 1967, caries microbiology had been dominated by Streptococcus mutans research, but S. mutans infection was opportunistic, as explained by the ecological plaque hypothesis. While S. mutans had an important role in caries, it was not the only cariogenic organism. Twetman thought that this implied that we should not throw away chlorhexidine, to which people respond differently with respect to S. mutans numbers. There was no direct association of mutans streptococci with caries. Russell thought more work was needed on host/bacteria interactions, but it was a sound principle to keep approaches simple.

Summary: There was a degree of unanimity amongst the panel members, but the audience was not as united, especially as to the role of S. mutans. S. mutans has a minor role in caries. W.H. Bowen emphasised the importance of pellicle as a conditioning film that determines initial adherence and contains many enzymes, in particular glucosyl transferases (GTF). Because the major polymer in plaque is ·-1,3-glucan, which also binds GTF, an important role of mutans streptococci is to provide the glucan responsible for this stickiness. A decrease in GTF leads to a caries reduction in rats, suggesting both the importance of S. mutans and a need to focus on experiments to decrease virulence factors. He also pointed out that antimicrobial agents are not effective against caries. Marsh agreed that pellicle and plaque matrix need more research. Guggenheim thought that there was a question as to whether glucan-induced adhesion is specific or non-specific. Russell said results in rats on immunization against AgI/II showed that GTF has a role in caries. When asked why, if vaccines are successful, the normal immune system cannot control mutans streptococci, he replied that there is evidence for the production of antibody to plaque S. mutans, but that we do not know the level of mucosal immunity required to give protection. Organisms also adversely direct immune responses for their own benefit. Summary: It has been clearly demonstrated that immunization against ‘S. mutans caries’ in rats works, but these experiments have yet to be replicated in humans. Only the study of plaque biofilms is relevant to in vivo processes. C. Robinson said in his group’s in vivo system F does not completely penetrate into plaque and commented that in the analysis of biofilm structure there can be problems with limited stain uptake and masking by EPS. Guggenheim replied that staining was not a problem in his group’s model. S. Petti pointed out that there is a need to focus on the floras associated with different types of caries – root, enamel and dentine. C. Splieth asked why studies seem to be confined to only five species: why not use pooled plaque? Guggenheim stated that standardization would be lost, and a reproducible procedure is needed for testing antimicrobials (referring to his in vitro model). Summary: Methods need to be developed to study human plaque in situ, to fully understand the complex microbial-host interactions required for the development of oral diseases.

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How to Prevent Caries? Role of Saliva Chairs: J. Tenovuo, L. Tabak

It was agreed that caries is a ‘complex disease’, influenced by host and microbial genes, infectious agents, diet/nutrition, environment, behaviour and societal factors. The oral milieu is dominated by a complex fluid – saliva – which contains many molecules that interact with each other, with micro-organisms and with host cells. All these interactions differ somewhat between individuals and to get a better picture of their role in the caries process the scale of analysis needs to be increased, using genomic, proteomic and other new molecular approaches to interrogate all salivary proteins simultaneously. This should be done in real time to identify all relevant components at various stages of the disease, perhaps exploiting advances in biosensors. It was agreed that no single salivary protein or component was decisive in initiation and progression of caries. Mucins and water were believed to be important, but interactions with other protective proteins and agents must also be considered. These events were very difficult to study in vivo because no patients (except those with selective IgA deficiency) were known to lack only one protein. Clearly salivary antimicrobials were present at insufficient concentrations to prevent dental caries totally and might be more important in keeping non-oral bacteria away than in regulating the oral microflora. One reason for the many problems associated with dry mouth was likely to be the reduced output of all protective salivary factors in severe hyposalivation. Reduced clearance of carbohydrates and acids likely contributed as well. Minor salivary glands, with their rich content of protective proteins, were very important in creating healthy local environments.

How to Prevent Caries? Role of Fluoride Chairs: J.D.B. Featherstone, L.C. Chow

The key points made by the speakers during the discussion period are summarized here. ten Cate Availability of more effective caries-preventive products. This often depends more on corporate marketing decisions than on scientific or technological advances. A major manufacturer once stated that it is less costly and

Summaries of Discussions

more profitable to launch a new toothpaste that claims improved whitening than one that claims superior anticaries effects. Mechanisms of fluoride action. A usually neglected fact is that dental mineral is a defective carbonated hydroxyapatite that is much more acid soluble than hydroxyapatite, which in turn is more soluble than fluorapatite. During remineralization, the crystal surfaces become closer to fluorapatite, and are hence much less soluble than the original mineral. The true importance of any antibacterial action of fluoride needs to be tested clinically in a cariogenic environment. Why a fluoride product may be less effective on certain individuals. The ability of a product/treatment to deliver sufficient fluoride to sites that are most vulnerable to caries, especially where there is a high bacterial challenge, should be given more attention in future research. Dose response to fluoride in toothpastes and other products. The concept of dose response, as it applies to fluoride products, may need a better definition. The efficacy of delivery rather than concentration of fluoride in the product may be the key to success. The concept of slow-release fluoride has not been put into practice, but should be.

Robinson On the effects of fluoride and magnesium on tooth mineral formation. A study on enamel mineral in growing rat incisors showed that a higher serum fluoride level led to a lower acid phosphate content, while a higher serum magnesium level led to a higher acid phosphate content. On the effects of fluoride on cells and tooth development. Studies on tooth-forming cells in contact with fluoride at realistic micromolar concentrations are needed.

How to Prevent Caries? Role of Sugars and Diet Chairs: W.H. Bowen, P. Lingström

Hausen There are no novel fluoride measures in sight with an efficacy substantially higher than that of the preventives in general use today. Recent studies showed that oral fluoride retention from mouth rinse can be increased significantly without increasing the fluoride concentration in the rinse. A recent clinical study showed that simply controlling increased daily use of fluoride toothpaste reduced caries. These examples suggest that significantly more effective and practical fluoride products can be formulated. Discussants considered there is considerable room for improved fluoride delivery methods.

Although sucrose is recognized as an important factor in the aetiology and pathogenesis of dental caries, it was pointed out that sucrose is rarely consumed alone and that attention should be paid to the diverse effects of food combinations. For example, a sucrose/maize mixture causes plaque pH to fall, whereas a milk/maize/sugar mixture raises it to alkaline levels. Inefficient swallowing might delay carbohydrate clearance from the mouth, and thus enhance the cariogenic potential of foods. The suggestion was made that we no longer need to recommend restriction of sugar consumption as a means of preventing caries because of the advent of fluoride in various forms. In response, it was pointed out that inappropriate use of sugar was a public health problem. Caries was now strongly associated with poverty and even a small reduction in sugar use showed benefits. The question was also raised whether, from a public health perspective, it would be beneficial to re-consider adding cariostatic agents to sugar. Several questions concerned the use of non-sucrose sweeteners such as polyols. Whether xylitol was simply non-cariogenic or actually caries-preventive remained unresolved. It was stated that chewing gum containing polyols when used by the mentally handicapped had a beneficial cleansing effect. Infants whose mothers used xybitol harboured much fewer S. mutans than controls. Users of xylitol also had plaque that was not very adhesive and harboured fewer S. mutans. Populations of S. mutans increased when sucrose was used. It was questioned whether the preventive effect of xylitol might be greater in relation to root surface caries compared with enamel caries. Although topical application of sorbitol usually results in a small fall in plaque pH, one speaker mentioned a patient who developed rampant caries from persistently

Summaries of Discussions

Caries Res 2004;38:325–329

Hellwig The primary caries-preventive mode of action of fluoride is post-eruptive. The evidence strongly supports this conclusion, but there is also evidence for some pre-eruptive effect and discussants insisted that we do not lose sight of a systematic effect. What is the optimum fluoride concentration for topical treatments? There is ample evidence that topical fluoride affects lesion progress. However, fluoride concentration and retention in the oral environment rather than fluoride concentration in the product should be given more attention. The optimum for individuals will depend on the demineralization challenge.

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using sorbitol-containing lozenges, and in whom plaque pH fell to 3.5 following topical sorbitol application. All sweeteners currently on sale are generally regarded as safe, but it was questioned whether there are sufficient data to consider them safe for use by children. In relation to early childhood caries, it was stated that most caregivers are well aware of what constitutes poor feeding behaviours, but persist in these poor habits nevertheless. Obtaining compliance with good dietary advice was regarded as a universal problem. The complexity of the diet/caries relationship was addressed. The exact role of diet was thought difficult to assess and it was stressed that not only what is consumed, but also individual patterns of consumption should be taken into account.

How to Manage Caries Chairs: J.P. van Amerongen, B.H. Clarkson

Are we ready to move from operative to non-operative/preventive treatment? J. Hamilton asked whether treatment strategy should be modified when delivering preventive non-operative care to patients who, like 50% of the US public, do not attend the dentist regularly. Pitts replied that it was important to adapt the preventive message for irregular attenders. In England, for example, the National Health Service is developing clinical care pathways for evidencebased preventive care and specific pathways for irregular attenders will present recommendations to patients and discuss the limitations of restoring teeth without addressing the causes of the disease. S. Petti pointed out that in Italy, which has no dental public health and a high caries incidence, there was no time for prevention and all caries has to be treated by operative procedures. Pitts replied that it was vital to address the causes of caries, as the evidence shows the futility, cost and limited outcomes associated with repeat restorative care. Zero said that efforts had been made for a number of years to get the ‘medical model’ of practice (in contrast to ‘surgical care’) accepted in the USA. This designation suggests that caries is an infectious disease. Pitts replied that it was vital to recognise that caries is a multifactorial disease; in most patients, it is not caused by bacteria alone, diet alone, poor oral hygiene alone or lack of fluoride alone. It was therefore important to give a valid, balanced message to both public and colleagues and not emphasise only one factor. He suggested that, rather than just talk of the ‘medical’

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model, perhaps the dentist should be called an oral health physician to provide a better contrast to the traditional dental ‘surgeon’ who ‘treated’ caries rather actively and irreversibly. C.M. de Almeida commented that the best way to interrupt the caries process (and allow salivary remineralization) might be to teach patients to remove plaque completely and/or remove it professionally. M. Maltz agreed that the only available option for health professionals to promote oral health was to move towards non-operative/preventive strategies. For at least 20 years, sufficient data have been available to show that operative procedures can only prevent further development of a cavitated lesion. They do not prevent occurrence of new lesions that are likely to develop when the disease is not under control. Effective control required interference with aetiological and determinant factors. Pitts agreed but emphasised the need to convince dentists who have yet to make the conversion. In some countries, most dentists have already changed and practice in this way. In others, despite the evidence, many teachers and dentists choose to manage caries in the traditional way. ORCA and others have a continuing job to provide best evidence and best practice. How ‘clean’ must a cavity be before restoration? M. Maltz commented that asking whether or not to leave bacteria beneath a restoration overlooked the wellestablished consensus that conventional caries removal normally leaves bacteria beneath restorations. However, after incomplete removal of carious dentine and tooth sealing, the number of residual bacteria was significantly reduced, and the dentine underneath the provisional restoration acquired the characteristics of an inactive caries lesion. An important question was whether there were any differences in the residual bacteria after conventional carious dentine removal and after incomplete carious dentine removal and tooth sealing. Kidd agreed. M.J. Noack thought it was not a problem that teaching with respect to caries excavation during the last decades had been in error, because dentists had not followed this teaching in any case. There was evidence that in most cases some carious dentine is left, especially at the dentine-enamel junction. Kidd agreed. Noack thought two questions might influence strategy: how should non-compliant patients, who do not control plaque properly, be dealt with? As dental restorations in general leak, is the proposal for a less invasive excavation approach applicable only to adhesively sealed restorations or can it also be used for conventionally cemented restorations? Kidd thought more research was needed. C.M. de Almeida thought that the

Summaries of Discussions

main problem in pulp survival was not bacteria in dentine, given a perfect seal at the interface with the cavity wall, but bacteria that had already penetrated to the pulp, producing reversible or irreversible pulpitis. Often the production of sclerotic and tertiary dentine was defined as a pulpal defence mechanism, but should perhaps be regarded more as a sign of a degenerative process: the consequence of inflammation, resulting in fibrosis and calcification. J.P. van Amerongen asked about the teaching of the new approaches and especially the criteria for determining the extent of excavation at the lesion periphery. Kidd enumerated the following: discussion of the literature, particularly the work of Mertz-Fairhurst (which she considered should be repeated by another group); when removing demineralised dentine, the enamel-dentine junction should be made hard (but not stain-free); demineralised tissue over the pulp should be excavated to the level of firm dentine provided there was no likelihood of pulpal exposure; deep lesions in symptomless, vital teeth should be gently excavated; soft, demineralised dentine can remain where its removal might expose the pulp; a permanent restoration is placed; there was no re-entry; caries dyes were not used. The future role of a molecular approach to pulp-dentinal regeneration A. Sotirovska-Ivkovska asked how, when the pulp is exposed because of caries, reversible and irreversible pulpal changes can be distinguished. When should the pulp be stimulated for dentine bridge formation? Tziafas answered that in the presence of irreversible pulpal changes, tissue preservation was not possible. Under active caries, irritating factors from bacteria and tissue breakdown affect the dentinogenic unit at the dentinepulp interface for long periods and progressively destroy it, so therapeutic regulation of tissue healing, regardless of treatment modality, is not possible. C.M. de Almeida suggested that pulp-dentinal regeneration might depend more on levels of bacterial penetration and of pulpal inflammation than on a molecular approach. Tziafas disagreed; the most important determinant of vitality and function of the dentine-pulp complex was restoration of the dentinogenic unit at the pulp-dentine interface. Bacteria, other exogenous stimuli, including traditional thera-

Summaries of Discussions

peutic modalities, and inflammatory changes adversely affect the healing capacity of the dentinogenic unit. As a result, irregular hard tissue of unpredictable defensive nature is deposited, but this cannot block exogenous destructive stimuli effectively. New molecular approaches were aimed at specifically controlling the restoration of the dentinogenic unit. Tertiary dentine was the most appropriate matrix to protect the pulp from secondary infection. O. Fejerskov, following up de Almeida’s comment, pointed out that pulpal inflammation had been shown to attenuate the ability of the pulp to respond to BMP application, which in the non-inflamed pulp had resulted in new dentine formation. O. Fejerskov then asked about the role of the superficial necrotic zone subjacent to the Ca(OH)2 in the formation of new matrix, given that Ca(OH)2 containing TGF-ß1 induces dentine bridge formation. Tziafas answered that odontoblast-like cells differentiated and reparative dentine was formed in relation to this zone. Preliminary reports had shown that a network of bioactive molecules, including fibronectin and growth factors, was created at the surface of vital tissue. A necrotic zone was not formed under experimental conditions in relation to pre-set (inert) Ca(OH)2 and it was hypothesised that addition of TGF-ß1 substituted for the zone of tissue necrosis and the associated molecular network.

Getting Research into Clinical Practice Chair: N.B. Pitts

It was agreed necessary to acquire data on all age groups, particularly adults. J. Brown felt that research by general practitioners was necessary to acquire generalisable data and to ensure that training on prevention was delivered to general practitioners. W.H. Bowen asked about procedures for liability and ethics approval of research conducted in general practice. Clarkson replied that all projects went through the usual Research Ethics Committee procedures and that liability was covered through the National Health Service Trusts in which the dentists worked.

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ORCA Announcement Caries Res 2004;38:330

ORCA Summer School, July 3–4, 2004, Marburg, Germany

Theme: The conduct, interpretation and publication of clinical research. Good clinical practice and statistics

The ORCA summer school will be held at the end of the ORCA congress in Marburg, Germany. The registration fees for the summer school is EURO 200 per participant and will be limited to 30 participants. Registration includes overnight accommodation (incl breakfast) on Saturday July 3rd, all meals including lunch on Sunday.

The cost of additional overnight stay on Sunday night is Euro 35. Places will be allocated on first come first serve basis. This summer school will be of particular interest to young researchers. To book your places please initially contact Professor Monty Duggal at the following: [email protected] or mail to: Professor Monty Duggal, Child Dental Health, Leeds Dental Institute, Clarendon Way, Leeds LS2 9LU, U.K. Fax: +44 113 233 6140

Programme for the ORCA Summer School 2004, Marburg, Germany The conduct, interpretation and publication of clinical research. Good clinical practice and statistics

Saturday 2.00–2.40

Introduction – Types of studies

Prof. Monty Duggal, UK

2.40–3.20

The conduct of clinical research

Dr. Helen Whelton, IRE

3.20–3.45

COFFEE

3.45–4.30

Ethical issues in clinical research

4.35–5.30

DISCUSSION OF ALL PRESENTATIONS MODERATED BY PROF. DUGGAL AND CLOSE FOR THE DAY

Prof. Dom Zero, USA

Sunday 9.00–9.40

Day to data running of clinical studies

Melissa Mau, USA

9.40–10.20

Regulatory issues within Europe

Glaxo SmithKline

10.20–10.40

COFFEE

10.40–11.20

Modern statistical methods

Dr. Michael Cronin, IRE

11.20–12.00

Writing up studies for publication

Dr. Peter Shellis, UK

12.00–12.30

Evidence-based Research

Dr. Cor van Loveren, NL

12.30–1.30

DISCUSSION OF ALL PRESENTATIONS MODERATED BY PROF. DUGGAL AND CLOSE

Supported by Glaxo SmithKline and GABA

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Author Index Vol. 38, No. 3, 2004

Bolscher, J.G.M. 247 Brookes, S.J. 268

Ismail, A. 199 Kidd, E.A.M. 305 Kirkham, J. 268 König, K.G. 168

Cate, J.M. ten 167, 254 Childers, N.K. 230 Clarkson, J.E. 321 Connell, S. 268

Lennon, A´.M. 258 Loveren, C. van 286

Shore, R.C. 268 Smith, A.M. 268 Smith, D.J. 230 Taubman, M.A. 230 Thurnheer, T. 212 Twetman, S. 223 Tziafas, D. 314

Dawes, C. 236 Marsh, P.D. 204 Marthaler, T.M. 173 Michalek, S.M. 230

Fejerskov, O. 182 Fox, P.C. 241 Giertsen, E. 212 Gmür, R. 212 Guggenheim, B. 212 Guggenheim, M. 212

Nieuw Amerongen, A. van 247 Nyvad, B. 167, 192

Hausen, H. 263 Hellwig, E. 258

Robinson, C. 167, 268 Russell, M.W. 230

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Veerman, E.C.I. 247 Zero, D.T. 277

Pitts, N.B. 294

© 2004 S. Karger AG, Basel

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331

Subject Index Vol. 38, No. 3, 2004

Agglutinin 247 Antimicrobial agents 204, 212, 223 – –, clinical trials 223 – peptides 247 – resistance 204 Apatite 268

Epidemiology, caries stages 199 Evaporation, fluid loss 236 Evidence-based dentistry 254

Proteases 268 Public health 199 Pulp 314

Flow rate, saliva 236 Fluoride 182, 254, 263, 268 Fluorosis 268

Radiotherapy, xerostomia 241 Randomized controlled trial, caries detection/diagnosis 192 Reactionary dentin 314 Remineralization 212, 254 Reparative dentin 314 Residual volume, saliva 236

Biofilms 182, 204, 212 Caries, see Dental caries Cathelicidin 247 Cavity preparation, restoration 305 Cell signalling 204 – structure 268 Chlorhexidine 223 Demineralization 212 Dental caries 173, 182, 212, 241, 263, 277, 286, 294 – –, clinical practice 294 – –, detection 192 – –, diagnosis 192, 199 – –, epidemiology 173 – –, manifestations 168 – –, prevention 168, 182, 199, 223, 263, 286 – –, removal 305 – –, risk 168 – –, treatment 168, 192, 286, 294 – plaque 182, 204 Dentin 314 Diagnostic threshold, caries 192 Diet 277 Dry mouth 236 Effectiveness, caries-preventive programs 263 Enamel 268

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Gene expression/transfer, bacteria 204 ’Gold standard’, caries detection/diagnosis 192 Growth factors 314 Histatins 247 History, caries research 254 Hyposalivation 236 Implementation strategies, dental primary care 321 Lactoferrin 247 Lesion activity 192 Matrix proteins 268 Mouthrinses 212 Mucins 247 Mucosal fluid absorption 236 – immunization 230 Mutans streptococci 230 Odontoblast-like cells 314 Odontoblasts 314 Palatal dryness 236 Posteruptive fluoride 258 Practice research 321 Pre-eruptive fluoride 258 Primary care 321

Saliva 236, 241, 247 Salivary film 236 – IgA antibodies 230 – proteins 247 Secretagogues 241 Sjögren’s syndrome 241 Sorbitol 286 Stepwise excavation 305 Sucrose 277 Sugar alcohols 286 Sugars 277 Systemic fluoride 258 Therapeutic effects, sugar alcohols 286 Three-dimensional morphology 212 Time trends, dental caries 173 Topical fluoride 258 Tortuosity 212 Transforming growth factor beta 314 Vaccine antigen 230 Vital pulp therapy 314 Xerostomia 236, 241 Xylitol 286