You are on page 1of 6
Journal of Dentistry Journal of Dentistry 28 (2000) 265–270 www.elsevier.com/locate/jdent Relative susceptibility of deciduous and permanent dental hard tissues to erosion by a low pH fruit drink in vitro M.L. Hunter a,*, N.X. West b, J.A. Hughes b, R.G. Newcombe c, M Addy b a Dental Health and Development, University of Wales College of Medicine Dental School, Heath Park, Cardiff CF14 4XN, UK b Restorative Dentistry, University of Bristol Dental School, Lower Maudlin Street, Bristol BS1 2LY, UK c Medical Computing and Statistics, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK Received 26 April 1999; received in revised form 1 October 1999; accepted 23 November 1999 Abstract Objectives: The objectives of this study were two-fold: (1) to determine (by surfometry) loss of deciduous and permanent enamel and dentine following 15 days’ exposure to a single low pH orange drink; and (2) to determine (by surfometry) loss of deciduous and permanent enamel and dentine following exposure to the product 2 versus 4 times per day for 15 days. Methods: This in vitro study employed the validated methodology described by West and co-workers [Journal of Dentistry, 1998;26:329– 335.] Results: In all four tissues, erosion was progressive over time, though this pattern was more linear in enamel than in dentine. In general, erosion of enamel was greater in the deciduous tissue, while erosion of dentine was greater in the permanent tissue. However, these differences were rarely of statistical significance. Increasing frequency of exposure resulted in a non-proportional increase in tissue loss. Conclusions: Differences in susceptibility of deciduous and permanent tissues to erosion by a low pH drink in vitro appear to exist, though these may not be of statistical significance. Care may be indicated in the delivery of dietary advice, since reduced frequency of exposure to a low pH drink does not appear to result in a proportional reduction in tissue loss. q 2000 Elsevier Science Ltd. All rights reserved. Keywords: Erosion; Enamel; Dentine 1. Introduction In recent years, the dental profession has noted an increase in the number of children and adolescents presenting with localised loss of anterior tooth tissue to which an erosive aetiology has been ascribed [2,3]. A number of epidemiological studies published during the last 5 years have examined the prevalence of erosion in these younger age groups [4–9], but it is perhaps in those focusing on preschool and younger school-age children [4,6–8] that the most disturbing statistics lie. The early supervention of sensitivity, or even frank exposure, in the deciduous dentition has usually been attributed to the reduced amount of tooth tissue present [10], though this may be an over-simplification. While the enamel and dentine layers of the primary dentition are certainly known to be much thinner than those of its permanent successor * Corressponding author. Tel. 144-2920-742458; fax: 144-2920742421. E-mail address: hunterml@cf.ac.uk (M.L. Hunter BDS, MScD, FDS (Paed) RCS (Edinburgh)). [11,12], other differences between deciduous and permanent tissues may also be of importance. For example, deciduous teeth demonstrate a higher degree of enamel porosity [13] and a lower degree of mineralisation [14–16] than permanent teeth. In addition, deciduous enamel has repeatedly been noted to have a higher content of carbon dioxide and carbonate, as well as a lower content of phosphorous than the permanent tissue [14,17–21]. Featherstone and Melberg [22] and Tyler and co-workers [23] have demonstrated that deciduous teeth are more susceptible to caries-like acid attack than permanent teeth in vitro. Lending support to this observation, Shellis [24] found that, on average, artificial caries-like lesions in deciduous teeth were 75% deeper than in permanent tissue. It is therefore logical to question whether the susceptibility of deciduous and permanent tissues to erosion, for example by low pH drinks, is also inherently different. If this were to be so, care would be indicated in the extrapolation to the deciduous dentition of data derived from experiments using specimens derived from permanent teeth. No published study has specifically examined the relative susceptibility of deciduous and permanent hard tissues to 0300-5712/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved. PII: S0300-571 2(99)00074-3 266 M.L. Hunter et al. / Journal of Dentistry 28 (2000) 265–270 erosion by low pH drinks, though Johansson and co-workers [25] have recently investigated the susceptibility of deciduous and permanent enamel to erosion by 2% citric acid. In this latter study, the microhardness of the two tissues was shown to be statistically significantly different both before and after exposure. The authors concluded that deciduous enamel seemed to be more susceptible to dental erosion than its permanent analogue. The primary aim of this study was therefore to examine the existence of a difference in susceptibility of deciduous and permanent enamel and dentine to erosion by a single low pH drink. The primary objective was to determine the amount of deciduous and permanent enamel and dentine lost by erosion following 15 days’ exposure to an orange drink (Asda Farm Stores No Added Sugar Orange Squash. Asda Stores Limited, Leeds, UK) in vitro. Determination of tissue loss at 5 and 10 days was considered to be a secondary objective. As with caries, frequency, rather than total consumption, of low pH drinks is thought to be critical to the erosive process. However, until now, there has been a dearth of evidence to support this assumption [26]. Therefore, as a secondary aim, this study also sought to determine the role of frequency in the erosion of deciduous and permanent enamel and dentine. This was accomplished by comparing the amount of tissue loss resulting from two versus four exposures per day. 2. Methods Specimens of permanent enamel and dentine were derived from recently extracted, caries free, unerupted third permanent molars. These were collected from patients aged between 18 and 30 years of either gender. Specimens of deciduous enamel and dentine were derived from recently extracted, caries free, deciduous canines. These were collected from children of either gender who were undergoing their extraction for the relief of crowding. At the time of extraction, donors were resident in areas where the water supplies contained less than 0.3 ppm fluoride. However, details of previous residence were not available and it is also likely that fluoride-containing toothpaste were being used. Following extraction, each tooth was soaked for at least 24 h in 50% sodium hypochlorite before being scraped of any remaining tissue with a scalpel. It was then rinsed in copious amounts of distilled water. Finally, the crown was sectioned from the root and both portions cut vertically to produce approximately equal sections of enamel and dentine, respectively. In the case of the third permanent molars, four or six equal sections were cut from the crown of each tooth, depending on its size. However, due to differences in both size and morphology, it was only possible to cut two sections from the crown of each deciduous canine. Each section was embedded in a vacuum-formed poly- urethane mould filled with epoxy resin. Twenty-four hours later, when the epoxy resin had cured, the specimen was removed and, using an automatic lapping and polishing unit (Kermet International, Maidstone, UK), ground to fit a stainless steel jig. This had been specifically constructed to hold the specimen precisely during surfometry and ensure that a stable horizontal platform was maintained. Using abrasive discs of decreasing coarseness (Kermet International Ltd., Maidstone, UK), a smooth, flat area of buccal or lingual/palatal enamel or dentine (as required) was exposed, care being taken to remove the minimum amount of tissue. This process was monitored by surfometry, employing an SF200 surfometer (Planer Products Ltd., Sunbury-on-Thames, UK). Each specimen was only accepted for use when two consecutive readings across the exposed area fell within the range 20.3 to 10.3 mm. Thirtytwo specimens of each tissue were prepared in this way. Following acceptance, each specimen was given a unique reference number which was recorded on its reverse side in indelible ink. It was then stored in isotonic saline at room temperature in an eppendorf tube marked with the same reference number, and its two baseline surfometry measurements recorded for future reference. Immediately before use, an area of enamel or dentine was delineated by placing PVC insulating tape (RF Components Ltd., Corby, UK) over the specimen, leaving a 2 mm wide zone of hard tissue exposed. On each of 15 consecutive working days, the sugar free concentrate was diluted 1:4 with bottled mineral water (Volvic, Premier Waters Ltd, London, UK). Prior to each exposure, at least 1 l of the prepared drink was warmed to 378C in a water bath. Specimens of deciduous and permanent enamel and dentine were placed in large glass beakers. The four tissues were segregated, the beakers being clearly marked with the nature of the tissue which they contained. Two hundred millilitres of the prepared, previously warmed drink were added to the specimens, and the beakers placed in a water bath at 378C. The mixture was then stirred with an overhead paddle stirrer for 10 min, a stopwatch being used for monitoring. Following each exposure, the specimens were removed from the drink, rinsed in a total of 1 l of distilled water, and finally stored in isotonic saline at room temperature. The ‘used’ drink was discarded. Following the first exposure, the ‘treated’ area was marked using indelible ink. This aided the repositioning of the tapes following measurement of tissue loss. Sixteen specimens of each tissue were exposed to the drink at 0900 and 1100 h. The remaining sixteen specimens of each tissue were exposed to the drink at 0900, 1100, 1300 and 1500 h. Overnight, and at weekends, the specimens were stored in isotonic saline at room temperature. This was contained in sealed beakers clearly marked with the nature of the tissue therein. At completion of days 5, 10 and 15, the PVC tape was removed from each specimen and tissue loss determined by surfometry as described by West and co-workers [1]. Two M.L. Hunter et al. / Journal of Dentistry 28 (2000) 265–270 267 Table 1 Progression of erosion (in mm) over time by frequency of exposure Tissue Exposures/day Day 5 Mean (SD) Day 10 Mean (SD) Day 15 Mean (SD) Deciduous enamel 2 4 2 4 2 4 2 4 212.35 (5.82) 218.90 (7.40) 210.84 (3.80) 214.71 (5.79) 212.23 (2.20) 219.00 (4.27) 212.07 (3.94) 219.44 (3.32) 221.29 (7.26) 239.80 (12.30) 217.99 (4.32) 234.14 (5.93) 217.45 (5.04) 223.43 (6.65) 217.20 (5.38) 228.94 (8.88) 231.70 (10.30) 254.50 (11.70) 227.58 (5.99) 256.15 (7.35) 216.83 (3.30) 224.76 (7.85) 220.04 (3.64) 229.48 (7.04) Permanent enamel Deciduous dentine Permanent dentine surface profiles were taken from each specimen. Each profile was taken from just within the taped zone on one side, across the exposed area and just into the opposite taped zone. This enabled the instrument to calculate the average gain (1) or loss (2) of the exposed area compared to two chosen fixed points in the previously taped zones, i.e. corresponding to the original baseline height. Following measurement on days 5 and 10, the area to be exposed was once more delineated with PVC tape. It was possible to evaluate differences between deciduous and permanent teeth and the effect of frequency of exposure. Enamel and dentine were considered separately. The primary outcome measure was mean tissue loss on each measurement day. This was calculated by taking the mean of the two replicate readings on days 5, 10 and 15 and subtracting the mean of the two replicate readings at baseline. Unpaired t tests were employed to examine differences between erosion in deciduous and permanent tissues and between the two frequencies of exposure. 3. Results Two specimens each of deciduous enamel, deciduous dentine and permanent dentine were not measured at day 15. In the case of the deciduous enamel specimens, it was evident that erosion had progressed into dentine, while in that of the dentine specimens, it was considered that the PVC tapes had moved, thereby significantly altering the area to be measured. There were no other missing data. Means for erosion at days 5, 10 and 15 are presented in Table 1. This illustrates progression of erosion over time for all four tissues and for both exposure frequencies. In general, erosion in all four tissues was progressive over Table 2 Statistical significance of differences between erosion of specimens subjected to two versus four exposures per day Tissue Day 5 P value Day 10 P value Day 15 P value Deciduous enamel Permanent enamel Deciduous dentine Permanent dentine 0.0096 0.035 0.0000 0.0000 0.0000 0.0000 0.0079 0.0001 0.0000 0.0000 0.0029 0.0002 time, though this pattern was more linear in enamel than in dentine. In deciduous dentine, at least 70% of the total erosion had occurred by day 5; in permanent dentine, at least 60% of the total erosion had occurred by the same time point. It is apparent, though not tested formally, that, at day 15, in vitro erosion was much more severe in enamel than in dentine. Generally, deciduous enamel was seen to erode more severely than permanent enamel, whereas deciduous dentine showed less erosion than permanent dentine. The statistical significance of these differences was determined using unpaired t tests. Differences between deciduous and permanent enamel did not reach conventional levels of statistical significance. Differences between deciduous and permanent dentine were statistically significant only at day 15 and at the lower exposure frequency …p ˆ 0:016†: Tissue loss was consistently greater in specimens exposed four times daily than in those exposed twice daily. The difference between the two regimes was always statistically significant, often highly so (Table 2). However, it should be noted that in only one case (day 15 for permanent enamel) did doubling the frequency of exposure actually result in doubling the amount of erosion recorded. Further, no consistent ratio for the degree of erosion produced by 4 versus 2 exposures per day emerged. 4. Discussion From the results of Johansson and co-workers [25] one might logically have expected the degree of erosion seen in deciduous enamel to be greater than that in permanent enamel. However, although deciduous enamel was generally seen to erode more severely than permanent enamel, while deciduous dentine showed less erosion than permanent dentine, only one contrast in each case reached a conventional level of statistical significance. The whole pattern could therefore simply be regarded as a chance finding. Despite the absence of statistical significance, one inevitably starts looking for explanations for these observations. With regard to deciduous and permanent enamel, the most obvious assumption is that differences in the rate of erosion arise from the differences in structure which are known to exist between the two tissues. Unfortunately, structural differences between deciduous and permanent dentine do 268 M.L. Hunter et al. / Journal of Dentistry 28 (2000) 265–270 not appear to have been investigated previously. This is clearly an area which may repay attention. Though already noted, it should be reiterated here that deciduous enamel exhibits a higher degree of enamel porosity [13]. Shellis [24] postulated that this difference might arise from variations in the relative volume contributions of (a) the prism junctions and (b) the interprismatic enamel. In his study, he showed that the interprismatic fraction and the prism-junction density were indeed significantly greater in deciduous enamel than its permanent analogue …p , 0:001†: These results supported, and provided a structural basis for, the hypothesis that differences in caries lesion formation between deciduous and permanent teeth can be accounted for in terms of enamel porosity [27]. We would venture to suggest that differences in porosity might also contribute, at least in part, to the observed variation in rate of erosive lesion formation. The process of diffusion plays a central role in the progression of erosion. Of likely importance in explaining the results of the studies reported here, Linden and co-workers [28] have produced data suggesting that the apparent diffusion coefficient (Da) is greater for deciduous enamel than for the permanent tissue, an observation consistent with the relative mineral contents [16]. An index, determined by magnetic spin resonance and related to the degree of dispersion of crystal orientation within the enamel, has also been found to be greater in deciduous than in permanent enamel [29,30]. In addition, carbonate substitutions in the hydroxyapatite crystals are also known to change both the charge and solubility of the surface [31–33]. Although increased frequency of exposure resulted in increased erosion of all tissues, the effect was clearly not proportional. Patients receiving dietary advice aimed at controlling their erosion not infrequently (subjectively at least) initially respond by suggesting that they ‘cut down’ rather than ‘cut out’ their consumption of erosive drinks. From this study, doubling the frequency of exposure did not result in double the amount of erosion. However, it is important to emphasise that this observation can also be interpreted as ‘reducing the frequency of exposure by half does not result in half the amount of erosion’. If this observation were to be replicated in an in situ or in vivo study, there would clearly be important implications for the delivery of dietary advice. The somewhat surprising observation that erosion in dentine appeared to progress in a less linear manner than in enamel is of particular interest, since this phenomenon does not appear to have been reported previously. For reasons of availability, both deciduous and permanent dentine specimens employed in this study were prepared from sectioned roots. Fogel and co-workers [34] have shown the hydraulic conductance of radicular dentine to be much lower than that of coronal dentine, this difference being largely attributable to variations in tubule diameter and density. Since convection or hydraulic conductance varies with the fourth power of the tubule radius, while diffusion only varies with its square [35], hydraulic conductance results tend to be more pronounced than would be expected for diffusion data. However, the results of Fogel and co-workers [34] would predict that diffusive permeation across radicular dentine would also be lower than that measured in coronal dentine in a manner analogous to their relative hydraulic conductance. It is not possible to explain our observations on the basis of variations in the relative hydraulic conductance of inner and outer radicular surfaces. Indeed, Fogel and co-workers [34] showed the hydraulic conductance of inner radicular dentine to be approximately 20% that of coronal dentine, while outer radicular dentine has a hydraulic conductance approximately 2% that of coronal dentine. Since it was the outer surface of the root dentine which was exposed, one might therefore have expected tissue loss to increase over time, rather than decrease as observed. It is obviously tempting to suggest that the marked dentine loss observed in the first five days was due to removal of the smear layer, since this is known to be extremely acid labile [36,37], being readily removable by exposure to dietary fluids known to contain organic acids [38]. The depth of the smear layer varies widely depending on whether the dentine is cut wet or dry, the amount and composition of the irrigant, and the instrumentation employed. The thickest smear layers reported (,10– 15 mm) are those produced in vitro with a coarse diamond blade mounted in a metallurgical saw [39]. The specimens used in these experiments were prepared using silicon carbide discs irrigated with copious amounts of water. Although no data are available relating to the depth of smear layer thus created, it seems more likely that this would fall at the lower end of the range (1–5 mm) noted for clinically-produced smear layers [40]. In our opinion, removal of the smear layer cannot be the sole explanation for the loss of at least 60% of the tissue in the first five days. Clearly, further research is required to establish the aetiology of such non-linear tissue loss. The aim of this study was to determine whether deciduous and permanent tissues differed in their susceptibility to erosion. Consequently, differences between enamel and dentine per se were not tested formally. It was anticipated that enamel would be far more resistant to acid attack than dentine, though it was noted that Davis and Winter [41] had found that the rate of erosion in enamel was often as fast as that for dentine. In the light of this expectation, the results of this in vitro study were somewhat surprising. At day 15, erosion was much more severe in enamel than in dentine, though this relationship was not the same at all measurement points. At the present time, it is not possible to explain why this should have occurred. However, it may be postulated that the reduced hydraulic conductance (and by extrapolation, the reduced diffusion) associated with radicular dentine [34], might have, in some way, limited the amount of erosion which could take place in vitro. It is M.L. Hunter et al. / Journal of Dentistry 28 (2000) 265–270 our suggestion that this in vitro study be repeated using coronal dentine, albeit accepting the logistic problems which this poses. In relation to the erosion of enamel, it has been recognised that the particular in vitro methodology used here dramatically over-estimates the amount of tissue loss which can be expected in the oral environment [1]. During ‘treatment’, the drink has total contact with the specimens, whereas in situ or in vivo, the drink is diluted with saliva both before and after it reaches the latter [42–44], thereby altering its thermodynamic properties [45]. Further, the effect of the drink is not limited by the buffering capacity of saliva. During storage, the specimens have no access to CaPO4 to facilitate remineralisation. These shortcomings may be addressed by the use of artificial (or natural) saliva in the ‘treatment’ and storage of specimens, thus simulating the oral environment. However, it is difficult to see how one can replicate the dynamics of the latter. Pellicle (and plaque) formation undoubtedly confers some benefit in with respect to erosion of enamel [46,47]. However, it should be emphasised that little information is available on the characteristics of pellicle formation on dentine and its possible protective effects. Maupome´ and co-workers [48] have emphasised that the introduction of carefully controlled saliva environments and salivary pellicles is necessary if we are to achieve a more realistic simulation of how an erosive challenge posed by frequent soft drink consumption is met in situ or in vivo. In retrospect, it may have been preferable to incorporate the development of salivary pellicles in this study. 5. Conclusions From the results of this in vitro study, it can be concluded that differences in susceptibility of deciduous and permanent tissues to erosion by a low pH drink appear to exist. Though not of consistent statistical significance, the increased susceptibility of deciduous enamel to erosion is of clinical significance. Deciduous teeth have a maximum covering of 1–1.3 mm of enamel [11]. Indeed, a recent study by Harding and co-workers [12], seeking to examine the thickness of human deciduous incisor enamel, found that the mean thickness of enamel (in millimetres) of the buccal incisal and cervical thirds was 0:39 ^ 0:08 and 0:15 ^ 0:05; respectively, with the mean palatal thickness being 0:22 ^ 0:04: Such a thin covering of enamel coupled with an increased rate of wear would appear to render deciduous teeth more likely to exhibit exposure of dentine (or pulp) during their lifetime. From this point of view, our findings perhaps go a long way towards explaining the high percentage of 5- and 6-year-olds in whom the palatal surfaces of the maxillary deciduous incisors are seen to be eroded into dentine or pulp [6]. Increasing frequency of exposure to a low pH drink 269 appears to result in a non-proportional increase in erosion. Likewise, halving the frequency of exposure does not result in a similar reduction in tissue loss. This observation may prove to be of relevance in the delivery of dietary advice to those in our care. It is recognised that in vitro methodology is incapable of replicating the biological variations of the oral environment and that actual erosion in vivo largely depends on consumption practices. The results of this study are of limited value if they cannot be correlated with those arising from an in situ or in vivo study. Therefore, it is our recommendation that this study be replicated in the oral environment. References [1] West NX, Maxwell A, Hughes JA, et al. A method to measure clinical erosion: the effect of orange juice consumption on erosion of enamel. Journal of Dentistry 1994;26:329–35. [2] Bishop K, Briggs P, Kelleher M. The aetiology and management of localized anterior tooth wear in the young adult. Dental Update 1994;21:153–60. [3] Shaw L, Smith A. Erosion in children: an increasing clinical problem. Dental Update 1994;21:103–6. [4] Millward A, Shaw L, Smith A. Dental erosion in four-year-old children from differing socioeconomic backgrounds. ASDC Journal of Dentistry for Children 1994;61:263–6. [5] Milosevic A, Young PJ, Lennon MA. The prevalence of tooth wear in 14-year-old school children in Liverpool. Community Dental Health 1994;11:83–6. [6] O’Brien M. Children’s dental health in the United Kingdom, 1993. London: HMSO, 1994. [7] Hinds K, Gregory JR. National diet and nutrition survey; children aged 1 1/2 to 4 1/2 years. Report of the dental survey, vol. 2. London: HMSO, 1995. [8] Jones SG, Nunn JH. The dental health of 3-year-old children in East Cumbria. Community Dental Health 1995;12:161–6. [9] Bartlett DW, Coward PY, Nikkah C, et al. The prevalence of tooth wear in a cluster sample of adolescent schoolchildren and its relationship with potential explanatory factors. British Dental Journal 1998;184:125–9. [10] Nunn J, Shaw L, Smith A. Tooth wear—dental erosion. British Dental Journal 1996;180:349–52. [11] Huszar G. Observations sur l’epaisseur de l’email (Studies on the thickness of dental enamel). Bulletin Du Group International Pour La Recherche Scientifique En Stomatologie Et Odontologie 1971;14:155–67. [12] Harding AM, Satanovskiy Y, Simmelink JW, et al. Thickness of human primary incisor enamel. Journal of Dental Research 1996;75:196. [13] Fejerskov O, Stephen KW, Richards A, et al. Combined effect of systemic and topical fluoride treatments on human deciduous teeth—case studies. Caries Research 1987;21:452–9. [14] Naujoks R, Schade H, Zelinka F. Chemical composition of different areas of the enamel of deciduous and permanent teeth. (The content of Ca, P, CO2, Na and N2.) Caries Research 1967;1:137–43. [15] Thylstrup A, Fejerskov O, Joost Larsen M. Polarized light microscopy of enamel structure in incisors from newborn infants. Scandinavian Journal of Dental Research 1976;84:243–54. [16] Wilson PR, Beynon AD. Mineralization differences between human deciduous and permanent enamel measured by quantitative microradiography. Archives of Oral Biology 1989;34:85–8. [17] LeGeros Z, Trautz OR, LeGeros JP, et al. Apatite crystallites: effects of carbonate on morphology. Science 1967;155:1409–11. 270 M.L. Hunter et al. / Journal of Dentistry 28 (2000) 265–270 [18] Cutress TW. The inorganic composition and solubility of dental enamel from several specified population groups. Archives of Oral Biology 1972;17:93–109. [19] Ingram GS. The role of carbonate in dental mineral. Caries Research 1973;6:217–30. [20] Feagin F, Thiradilok S, Aponte-Merced L. The carbonate and fluoride in surfaces of remineralised enamel. Journal of Oral Pathology 1977;6:338–42. [21] Nelson DGA. The influence of carbonate on the atomic structure and reactivity of hydroxyapatite. Journal of Dental Research 1981;60:1621–9. [22] Featherstone JDB, Mellberg JR. Relative rate of progress of artificial caries lesions in bovine, ovine and human enamel. Caries Research 1981;15:109–14. [23] Tyler JE, Poole DFG, Shellis RP. Artificial carious lesion formation in human enamel: deciduous/permanent and high/low fluoride comparisons. Journal of Dental Research 1982;61:562. [24] Shellis RP. Relationship between human enamel structure and the formation of caries-like lesions in vitro. Archives of Oral Biology 1984;29:975–81. [25] Johansson A-K, Sorvari R, Meurman JH, et al. In vitro effect of citric acid on deciduous and permanent enamel. Caries Research 1998;32:310. [26] Milosevic A. Toothwear: aetiology and presentation. Dental Update 1998;25:6–11. [27] Poole DFG, Shellis RP, Tyler JE. Rates of formation in vitro of dental caries-like enamel lesions in man and some non-human primates. Archives of Oral Biology 1981;26:413–7. [28] Linden L-A, Bjo¨rkman W, Hattab F. The diffusion in vitro of fluoride and chlorhexidine in the enamel of human deciduous and permanent teeth. Archives of Oral Biology 1986;31:3–37. [29] Cevc G, Cevc P, Schara M, Skaleric U. The caries resistance of human teeth is determined by the spatial arrangement of hydroxyapatite microcrystals in the enamel. Nature 1980;286:425–6. [30] Skaleric U, Ravnik C, Cevc P, et al. Microcrystal arrangement in human deciduous dental enamel studied by electron paramagnetic resonance. Caries Research 1982;16:47–50. [31] Kibby CL, Hall WK. Surface properties of calcium phosphate. In: Hair ML, editor. The chemistry of biosurfaces, 2. New York: Marcel Dekker, 1972. p. 663–729. [32] Besic FC, Bayard M, Wiemann MR, et al. Composition and structure [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] of dental enamel: elemental composition and crystalline structure of dental enamel as they relate to its solubility. Journal of the American Dental Association 1975;91:594–600. Arends J, Jongebloed WL. Dislocations and dissolutions in apatites: theoretical considerations. Caries Research 1977;11:186–7. Fogel HM, Marshall FJ, Pashley DH. Effects of distance from the pulp and thickness on the hydraulic conductance of human radicular dentine. Journal of Dental Research 1988;67:1381–5. Merchant MJ, Livingston MJ, Pashley DH. Dentin permeation: comparison of diffusion with filtration. Journal of Dental Research 1977;56:1161–4. Pashley DH. The influence of dentin permeability and pulpal blood flow on pulpal solute concentrations. Journal of Endodontics 1979;5:355–61. Addy M, Absi EG, Adams D. Dentine hypersensitivity. The effects in vitro of acids and dietary substances on root planed and burred dentine. Journal of Clinical Periodontology 1987;14:274–9. Absi EG. Studies on the aetiology, appearance, and treatment of hypersensitive dentine. (Thesis), University of Wales, 1989. Pashley DH, Michelich V, Kehl T. Dentin permeability: effects of smear layer removal. Journal of Prosthetic Dentistry 1981;46:531–7. Bra¨nnstro¨m M. Dentin and pulp in restorative dentistry. London: Wolfe Medical Publications, 1982. Davis WB, Winter PJ. The effect of abrasion on enamel and dentine and exposure to dietary acid. British Dental Journal 1980;148:253–6. Speirs RL. Saliva and dental health (1). Dental Update 1984;11:541– 2. Speirs RL. Saliva and dental health (1). Dental Update 1984;11:544. Speirs RL. Saliva and dental health (1). Dental Update 1984;11:546. Ireland AJ, McGuiness N, Sherriff M. An investigation into the ability of soft drinks to adhere to enamel. Caries Research 1995;29:470–6. Meurman JH, Frank RM. Scanning electron microscopic study of the effect of salivary pellicle on enamel erosion. Caries Research 1991;25:1–6. West NX. Dentine hypersensitivity; clinical and laboratory studies of toothpastes, their ingredients and acids, Thesis. University of Wales College of Medicine, 1995. Maupome´ G, Diez-de-Bonilla J, Torres-Villasenor G, et al. In vitro quantitative assessment of enamel microhardness after exposure to eroding immersion in a cola drink. Caries Research 1998;32:148–53.