A method to measure clinical erosion: the effect of orange juice consumption on erosion of enamel

PII:

Journal

s0300-5712(97)00025-0

of Dentistry, Vol. 26, No. 4, pp. 329-335, 1998 0 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0300-5712/98 $19.00+0.00

ELSEVIER

A method to measure clinical erosion: the effect of orange juice consumption on erosion of enamel N. X. West*, A. Maxwell*, J. A. Hughes *, D. M. Parker’, R. G. Newcombe’ and M. Addy* *Department of Restorative Dentistry (Periodontology), Bristol, UK +SmifhKline Beecham Consumer Healthcare, Coleford, Gloucestershire, UK pDepartment of Medical Computing and Statistics, University of Wales College

of Medicine,

Cardiff, UK

ABSTRACT Objective: Acidic soft drinks are frequently implicatedin dental erosion,but there are limited supporting

data. Researchis problematicdue to the insidiousnature of erosionand accuracyin assessing tissueloss. The aim of this study was to develop and validate, using a negative control, a model to accurately measureerosion irzsitu due to a singleaetiologicalagent over a relatively short time period. Methods:An intra-oral appliancecapableof retaining an enamelsamplewasdesignedin order to assess the effect of orangejuice consumptionon enamel.The study wasa singlecentre, randomized,placebo controlled, blind, crossoverdesign. Results: Ten subjects,eachconsuming1 1 of orangejuice per day for 15days, showedsignificantlymore erosionon the enamelspecimens than the samesubjectsconsuming1 1 of water per day over the same time period, measurements undertakenwith surfometry. The sameinvestigationwasperformedin vitro. Again, orangejuice wassignificantly more erosive;indeed,it wasin the order of 10timesthat produced in situ. Surfacemicrohardnesstestingin situ and in vitro demonstratedstatistically significantdifferences betweenexposedand unexposedareasafter orangejuice treatment. Conclusions: Changesproducedby water either in situ or in vitro were always well within the baseline measurementparameters(f 0.3pm) setdown for the method and hencevalidated the clinical model in termsof reproducibility and accuracyin measurement. It is concludedthat this methodhasconfirmedthe erosivepotential of orangejuice in situ. The methodcould havemany applicationsto study dentalerosion under highly controlled conditionsand over realistictime periods.0 1998ElsevierScienceLtd. All rights reserved KEY WORDS: J. Dent

1998;

Enamel

erosion,

26: 329-335

Clinical

(Received

study,

Citric

14 November

acid 1996;

accepted

INTRODUCTION The potential erosive effect of food and drink on teeth has been documented over many years. Early reports of erosion were made by Darby’ in 1892 and Miller2 in 1907. Recently there has been an increase in the number of articles discussing tooth wear; however, it is unknown whether this is due to increased awarenessor an Correspondence Restorative Hospital, 9284504;

should be addressed to: M. Addy,

Dentistry (Periodontology), Lower Maudlin Street, Bristol Fax: 0117 9284100.

Department of Bristol Dental School and BSl 2LY, UK. Tel: 0117

1 April

1997)

increase in prevalence of the condition. Much of the literature is based on case report data and as yet there are no longitudinal studies to answer this question3. Further, due to the multifactorial process of tooth wear, it can be extremely difficult to define the contribution of erosion from that of abrasion and attrition. Tooth erosion is thought to be predominantly due to either intrinsic acid from regurgitation processes or extrinsic acid such as acidic food and drink4. Anecdotal data suggest that acid reflux is prevalent in our population, but statistics are few. Indeed, it has been indicated that up to 7% of healthy individuals can

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be expected to experience reflux symptoms daily5. Also, sales figures from the soft drinks industry indicate wider availability and increased consumption of acidic soft drink&j. Much of the literature has concentrated on the erosion from acidic soft drinks, as this is a very accessible area in which to work. However, it is not definitely proven that this is a major aetiological agent responsible for tooth erosion. The majority of the research has been based on in vitro models which are extremely useful for demonstrating propensity characteristics of an erosive substance but cannot replicate the oral environment with all its biological variations. Similar methods are difficult to apply in viva, primarily because of controlling for numerous variables and difficulties in making accurate measurements of tooth substance loss. The aim of this study was to develop and validate a methodology whereby accurate measurements of relatively small amounts of enamel loss, due to a single erosive agent, could be made within a reasonably short period of time. To these ends a model iI2 situ was developed for application in the classical crossover design study to demonstrate the erosive characteristics of orange juice on enamel compared to water. Similar investigations in vitro were conducted concurrently to determine the variation in erosion between the two environmental conditions. Two hypotheses were proposed for the study based on the highly controlled conditions of the crossover design of the studies. Firstly, orange juice would produce appreciably more erosion of enamel than water. Secondly, in validating the methodology, orange juice would cause progressive erosion of enamel over time, whereas water would have no detectable effect on baseline measurements of specimens.

MATERIALS

AND METHODS

Pilot investigation The clinical study to be described was based on a pilot investigation designed to show that polished enamel samples could be measured repeatedly on a surfometer and readings remain within a f0.3pm deflection profile. The study involved five specimens profiled, using a surfometer across two zones and then placed into acrylic intra-oral appliances in vitro. Specimens were returned after 30 min for remeasuring. This procedure was repeated four times. The same experiment was performed in situ with specimens in the appliances worn by the respective five volunteers for 30 min on four occasions. These 40 procedures produced 80 profiles, all within the 5 0.3 pm baseline tolerance set down for the method.

Clinical study The study was a single centre, randomized, single blind two-phase crossover design involving healthy

Fig.

1. Ma-oral

appliance

with enamel

sample

in situ.

volunteers derived from the staff at the Bristol Dental School and Hospital. Ethical approval was granted by the University of Bristol Healthcare Trust Ethics Committee. The study was designed and conducted to conform with the Guidelines for Good Clinical Practice. Subjects were given verbal and written information concerning the l-week study and signed consent to participate. Human unerupted wisdom teeth were collected, tissue remnants removed and sterilized with sodium hypochlorite for 2 weeks. The teeth were sectioned and the buccal/lingual area of enamel embedded in epoxy resin, producing samples of 8 x 5 x 2 mm in dimension. A stainless steel die was constructed, to the exact measurements of the specimens, in order to facilitate profiles to be determined by surfometry. The outer surface of the enamel samples was lightly ground on an automatic lapping and polishing unit (Kermet International Ltd., Maidstone, UK), using 600-grit abrasive discs (Kermet International Ltd.) to produce a flat surface, with no more than an average of f0.3pm deflection on a baseline profile using a surfometer (Planer Products Ltd., Sunbury-on-Thames, UK). The diamond measuring stylus had a tip radius of 20pm with a head velocity of 10 mm min - ‘. The force of the stylus varies linearly with deflection at a rate of 8 rngprn-’ up to a maximum of 1 g at 100 pm. An upper, acrylic, intra-oral appliance was constructed on a dental impression with clasps on the first molars for retention (Fig. I). This appliance permitted fixation of the enamel sample palatally during a treatment period, whilst still allowing withdrawal and replacement in order to procure frequent measurements. Two baseline profiles of each enamel sample were made using the surfometer and then the samples were taped with PVC tape (RF Components Ltd., Corby, UK) to allow a 2 mm zone to be exposed in the oral environment.

West et a/.: Enamel

Ten subjects were enrolled and randomly assigned to the two treatment order groups. Subjects had to be aged between 18 and 60 years, healthy, with no relevant medical or pharmacotherapy histories which could interfere with the conduct of the study. Also, subjects had to be dentally fit, with a high standard of oral hygiene and gingival health and without evidence of excessive tooth wear or dentine hypersensitivity. A regimen of wearing an intra-oral appliance fitted with an enamel sample from 9 a.m. to 5 p.m. on each working day for 15 days was followed. Either 250 ml of orange juice (SmithKline Beecham, Coleford, UK) or 250 ml of water was sipped over 10 min at 0900, 1100, 1300 and 1500 h. The drinks were consumed at room temperature and under supervision. The appliance and enamel sample were dipped in chlorhexidine gluconate mouthrinse 0.2% for 1 min at the beginning and end of the day to prevent plaque accumulation. The appliance was removed for 1 h over lunch (12-1 p.m.) and stored in saline. Over night the appliance and sample were stored in isotonic saline at room temperature. Only tea, coffee and water could be consumed while the appliance was in situ and no toothbrushing was allowed throughout the day. Volunteers were allocated a toothbrush and a conventional fluoride toothpaste to use morning and night throughout the trial. At the end of each study day the samples were taken from the appliances and the tape removed. Two surface profiles were again taken of each specimen. The profile was taken from just within the taped zone on one side, across the exposed area and just into the opposite taped zone. The instrument then calculates the average gain (+) or loss (-) in height of the exposed area compared to two chosen fixed points in the previously taped zones, i.e. corresponding to the original baseline height. Enamel samples were then retaped exactly as before for placement in situ on the next study day. A compliance questionnaire was completed on each day of treatment. All adverse events were reported immediately. The first period was followed by 1 week’s washout, when volunteers returned to normal daily routines and without wearing appliances. The study was then repeated for another 3 weeks with the alternative beverage and fresh enamel samples in the appliances. A safety margin of 20 pm loss of enamel was set and, if achieved, subjects were to be exited from the study. After study completion, specimens were assessed five times on the exposed and unexposed areas for surface microhardness hardness (SMH) using a Wallace Micro-Indentation Tester (H.W. Wallace & Co. Ltd., Croydon, UK). The indentation load was 300 g through a Vickers stylus, with measurements recorded in Wallace Hardness Units (WHU). Laboratory

investigation

The same investigation was performed with enamel samples in the laboratory with no saliva present. Ten

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331

specimens were agitated in 250 ml of each beverage for 10 min four times a day for 15 days. Between exposures and overnight, specimens were stored in isotonic saline at room temperature. The enamel samples were assessed in an identical manner to the in situ specimens, both for enamel loss and SMH. Statistical

methods

The results from the clinical study were analysed using the paired nonparametric Wilcoxon test. This analysis disregarded the possibility of carryover effects, as in this study, like period effects, it is reasonable to assume they are implausible. The results from the investigation performed in vitro were analysed by unpaired t-tests. The results of the SMH testing from the exposed and unexposed areas of each sample were compared using paired t-tests.

RESULTS Clinical

study

The subject group consisted of 4 males and 6 females with a mean age of 24 years (range 20-30 years). All of the subjects completed the study and no adverse events were observed or reported. The 20pm loss safety margin incorporated in the protocol was not approached by any subject. The mean daily losses of enamel, expressed in microns, after exposure to orange juice or water in situ are shown in Table I. The results show progressive loss of enamel with time during orange juice consumption. During water consumption there was no evidence of specimen changes outside the set baseline parameters (& 0.3 pm); indeed, readings remained within a mean of +O.O7pm of baseline. Orange juice consumption at 15 days was significantly (P
study

The mean daily losses, expressed in microns of enamel, after exposure to orange juice or water in vitro are shown in Table ZZ. There was progressive loss of enamel with orange juice over the 15-day period. Specimens exposed to water showed no deviation in measurements outside baseline parameters; indeed, mean daily readings did not vary more than & 0.04 pm from the baseline mean. Compared to the clinical trial, a greater degree of erosion by orange juice was apparent. There was a significant difference (P
332

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West et al.: Enamel

Table 111. SMH of enamel specimens beverage in situ or in vitro (Wallace

In situ study Orange juice Unexposed Exposed Water Unexposed Exposed In vitro study Orange juice Unexposed Exposed Water Unexposed Exposed d.f.=degrees

after exposure to 15 days Hardness Units)

Mean

s.d. for variation between 10 specimens

s.d. for repeatability of reading (40 d. f.)

48.3 50.5

3.4 3.5

2.2 1.9

49.9 50.1

4.1 4.3

1.9 1.9

49.5 60.4

5.5 7.1

2.0 2.4

44.5 44.6

4.9 5.0

1.9 1.6

of

of freedom.

loss of enamel due to orange juice exposure compared to water; 24.20pm loss compared to 0.01 pm gain, respectively. The microhardness data, expressed in WHUs, for exposed and unexposed areas on enamel specimens treated with orange juice or water for the in situ and in vitro studies, are shown in Table III. Statistical analyses showed that there were significant differences between the exposed and unexposed areas of the enamel samples subjected to orange juice under the in situ and in vitro conditions (P=O.O02, P
DISCUSSION There appears to be a general consensus among those with an interest in the subject, that tooth wear has increased in prevalence and severity over recent decades. This could, of course, be artefactual and result from increased research in the area7. Case report data on erosion due to acidic food stuffs are plentiful; however, it is only recently that well constructed surveys*-” have been conducted highlighting the problem of dental erosion. The increased incidence of excessive tooth wear may be explained by a number of reasons. Firstly, in many countries, a considerably greater number of teeth are retained into old age”. Secondly, the aetiological

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clinical trial

333

factors themselves may have increased in prevalence12, notably dietary acid intake13, particularly over the last 5 yearQ. To date, the measurement of tooth wear has been based largely on either case report data or crosssectional epidemiological research. Such data only provide information on the total outcome measure of tooth wear, but cannot directly separate out specific aetiological agents. Certainly, case report data may provide information indicating the role of one particular factor in a single individual, for example erosion associated with behavioural eating disorders”. On the other hand, epidemiological data can only provide associations between possible variables and a particular condition. Such studies measure tooth wear using clinical indices similar to those used in many disciplines of dentistry and as such are relatively crude and unable to detect minor changes in tooth morphology. This has meant that the evaluation of potential aetiological agents in tooth wear has been difficult if not impossible to perform to date. Accurate measurement of tooth loss in vivo, over short periods of time, is problematic for a variety of reasons. Firstly, it is difficult to obtain fixed points from which to take measurements and, secondly, instruments which can measure minute changes in tooth morphology in vivo are as yet not available. Replica techniques are limited by the accuracy of the impression material and often give information on morphological changes rather than measurements of hard tissue 10s~‘~. Even if accurate measurements in vivo could be made, there are numerous variables which could affect erosive and abrasive aetiological factors. Modelling to study a single variable therefore would be difficult. To overcome the clinical problems, studies on erosion and abrasion have concentrated on methods in vitro. Tissue loss has been determined by a variety of methods, including gravimetric changes”, radioactive calcium release”, scanning electron microscopy2’, surface profilometry2i, replication techniques2’, and digital imaging analysis23. These methods of analysis vary considerably from those used in clinical procedures24, however, do allow a number of variables to be studied. Erosion is perhaps a more complex process than abrasion, with factors such as pH, titratable acidity, pK, temperature and type of acid influencing erosion characteristics, as will saliva and the presence of pellicle and fluoride25. Thus, once a reproducible in vitro methodology has been developed, not only can single variables be evaluated, but possible combinations of variables, including abrasion and erosion, can be assessed for their additive or synergistic effects. The limitation of methods in vitro to study tooth substance loss lies in extrapolating the findings to possible effects in vivo, particularly for a single agent. It was this problem which formed the basis of the present investigation. The enamel sample placed intraorally allowed surface profiles to be undertaken with an

334

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instrument with 0.01 pm resolution of accuracy. One limitation of this type of surface profiling is that only measurements from flat surfaces can be obtained, requiring the aprismatic layer to be removed during specimen preparation. It is likely that the prismatic enamel exposed to the acid is more vulnerable than the aprismatic layer26. Also ideally, specimen placement would have been on the labial or lingual aspects of the upper incisor teeth, as this is clinically the most frequent site of erosion from excessive acidic soft drink consumption3. Whilst preliminary studies showed that this was technically possible, volunteer co-operation was compromised due to comfort, aesthetics and function. The palatal site was therefore chosen for volunteer acceptibility and with the expectation that specimens would be contacted by the test fluids. However, provided that the above factors are fulfilled, the siting of specimens is secondary to the aims and design of the study. Thus, the blind, controlled, randomized crossover clinical trial design was chosen to compare the effects of two agents in an environment where most, if not all, other variables were standardized during both periods. The study used a negative control, water, and provided information in two areas. Firstly, the intake of water would not be expected to have any effect whatsoever upon the enamel, and therefore the changes seen, given the method, would be due entirely to the consumption of orange juice. There is the possibility that frictional action of the tongue could have aggravated the effects of the orange juice. Given the hardness values for the eroded enamel and the results from the control period this seems highly unlikely. Secondly, the negative control validated the reproducibility of the measurement technique. Had there been measurement error, this should have shown during the numerous measurements taken from specimens during the placebo period. Thus when specimens were in a non-erosive environment, 80 profiles from the pilot study, 300 profiles from the clinical study and 300 profiles from the laboratory study all remained well within the & 0.3 pm baseline variability established for the method. Comparison of the results in vitro and in situ leads to the predictable conclusion that investigating erosion in this particular laboratory setting grossly overestimated the amount of surface tissue loss that might be expected in situ. Thus, there was a dramatic

was designed to provide extended acid exposure by requesting volunteers to sip the fluids over considerable periods of time, one can only estimate that the actual contact time would be dramatically less than the 100% contact time of the in vitro method. Results from the in situ and in vitro SMH investigation indicated statistically significant changes between the exposed and unexposed (tape masked) enamel surfaces soaked in orange juice. One explanation for the differences between sites on the same specimen could be explained by surface demineralization. This same methodology may provide some evidence for the rate of remineralization of enamel in situ, which is a matter of some interest. Accurate evaluation of remineralization in vivo after acid erosion has been the subject of little research. In summary, a controlled method was devised to study erosion in situ due to a single agent, orange juice, over a relatively short period of time, and to compare this with erosion produced by the samemethod applied in vitro. From the results it must be concluded that the

method was validated and that studies in vitro considerably exaggerate effects in vivo. Accepting the limitations of specimen siting, the study is one of the first of its kind to model erosion in vivo by a single potential aetiological agent under highly controlled conditions. Clearly, the method could be refined, but as it stands, it has many apparent applications in the study of dental

erosion.

Acknowledgements The trial was sponsored by a grant from SmithKline Beecham Consumer Healthcare, Coleford, Gloucestershire, UK. References 1. 2.

Cosmos; 1907, 49, l-23, 3.

difference between

the loss of enamel due to orange juice exposure in the two environments, indeed of the order of IO times. This can be explained in part since there was no protection for the enamel in vitro, whereas pellicle formation occurring in vivo would afford some benefit26. The specimensin vitro had no opportunity for remineralization, nor was the effect of the orange juice limited by the buffering capacity of saliva. Moreover, the orange juice had total contact with the specimensin vitro, whereas in situ the specimenswere exposed to a passing acid fluid mixed with saliva. Even though the methodology

Darby, E. T., Dental erosionand the gouty diathesis.Are they associated? Dental Cosmos, 1892, 34, 629. Miller, W. D., Experiments and observations on the washing of tooth tissuevariously designatedas erosion, abrasion, chemical abrasion, denudation etc. Dental

4.

Journal of Dentistry, 5. 6. I. 8.

109-24, 22547.

Nunn, J. H., Prevalenceof dental erosionand the implications for oral health. European Journal of Oral Sciences, 1996,104, 156-161. Eccles,J. D. and Jenkins,W. G., Dental erosionand diet. 1974, 2, 153-159.

Scheutzel,P., Etiology of dentalerosion-intrinsic factors. European Journal of Oval Sciences, 1996,104, 178-190. British Soft Drinks Association, Report of Seminar in Heidleberg.Factsheetnumber9-7, 1991,p. 91. Shaw,L. and Smith, A. Erosionin children: an increasing clinical problem. Dental Update, 1994, 4, 103-106. Milosevic, A., Young, P. and Lennon, M. A., The prevalence of tooth wear in 14year old school children in Liverpool. Community and Dental Health, 1993, 11, 83-86.

West et al.: Enamel

9. Millward, A., Shaw, L. and Smith, A.J., Continuous intra-oral pH monitoring after consumption of acidic beverages.Journal of Dental Research, 1994, 73(Abstr. 397), 836. 10.

The Office Of Population Censuses And Surveys(OPCS), Dental

Caries among Childraen in the United Kingdom

in

1993. Publication No. SS9411.Office of Population Censuses and Surveys,London 1994. 11. Jarvinen, V. K., Rytmaa, I. I. and Meinonen, 0. P., Risk factors in dental erosion. Journal of Dental Research, 1991,7os, 942-941. 12. Imfeld, T., Dental erosion.Definition and links. European Journal of Oral Sciences, 1996,104, 151-155. 13. National Food and Drink Survey Committee,Household Food Consumption and Expenditure. HMSO, London, 1956-1972. 14. Johnson,G. K. and Sivers,J. E., Attrition, abrasionand erosion: diagnosis and therapy. Clinical Preventative Dentistry, 1987,9, 12-16. 15. Bargen,J. A. and Austin, L. T., Decalcificationof teeth as a result of obstipation with long continued vomiting. Report of a case.Journal of the American Dental Association, 1937,24, 1271-1273. 16. Smith, B. N. F. G. and Knight, J. K., An index for measuringthe wear of teeth. British Dental Journal, 1984, 156, 435438. 17.

Absi, E. G., Addy, M. and Adams, D., Dentine hypersensitivity: the developmentand evaluation of a replica technique to study sensitiveand nonsensitivecervical dentine.Journal of Clinical Peviodontology, 1989, 16, 190.

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18. Sexson,J. C. and Phillips, R. W., Studieson the effectsof abrasiveson acrylic resins.Journal of Prosthetic Dentistry, 1951,1,454471. 19. Wright, K. H. R. and Stevenson,J. I., The measurement and interpretation of dentifrice abrasiveness.Journal of the Society of Cosmetic Chemistry, 1967,18, 3877411. 20. Wictorin, L., Effect of toothbrushing on acrylic resin veneering material. II Abrasive effect of selecteddentifrices and toothbrushes.Acta Odontologica Scandinavica, 1972,30, 338-395. 21. Manly, R. S., Factors influencingtestson the abrasionof dentin by brushing with dentifrice. Journal of Dental Research, 1944,23, 59-72. 22. Mannerberg, F., Appearanceof tooth surfaceasobserved in shadow replicasin various age groups, in long-term studiesafter toothbrushing, in casesof erosionand after exposureto citrus fruit juice. Odontologica Revy Thesis, 1960,llS, 166-168. 23. Mistry, M. and Grenby, T. H., Erosion by soft drinks of rat molar teeth assessed by digital imageanalysis.Caries Research, 1993,27, 21-25. 24. Grenby, T. H., Methods of assessing erosion and erosive potential. European Journal of Oral Sciences, 1996,104, 207-214. 25. Zero, D. T., Etiology of dentalerosion- extrinsic factors. European Journal of Oral Sciences, 1996,104, 162-171. 26. Meuman, J. H. and Frank, R. M., Scanningelectron microscopic study of the effect of salivary pellicle on erosion.Caries Research, 1991,25, l-6