Caries Experience of Fluoride-related and Unrelated Enamel Malformations

Oral Med Pathol 9 (2004) 61 Caries Experience of Fluoride-related and Unrelated Enamel Malformations Yue Nakahara 1, Kaori Sato 2, Hisao Yagishita 2, Yuuichi Soeno 2, Masaaki Ogawa 3 and Takaaki Aoba 2 1 General Dentistry 1, 2 Department of Pathology, and 3 Dental Research Institute, The Nippon Dental University, Tokyo, Japan Nakahara Y, Sato K, Yagishita H, Soeno Y, Ogawa M and Aoba T. Caries experience of fluoriderelated and unrelated enamel malformations. Oral Med Pathol 2004; 9: 61-66, ISSN 1342-0984 The present study aimed at investigating whether part of the enamel opacities found in 20 permanent premolars could be ascribed to uptake of excess fluoride and whether there are appreciable differences in prevalence and progression of dental caries between tooth groups with and without developmental enamel defects. Fluoride content of enamel was assessed by electron probe microanalysis. Based on visual inspection, F/Ca determination, and histopathologic examination, the pooled premolars were divided into three groups: enamel without developmental defects (n=6), enamel with fluoride-relative defects (n=6), and enamel with fluorotic-irrelative developmental defects (n=8). This data shows a high incidence of developmental enamel malformations. The present histopathologic findings indicate that there were no significant differences in caries experience between the tooth groups regarding the entire coronal enamel, but that mild fluorotic enamel is associated with a lower risk of caries susceptibility in these smooth surfaces, while caries development still remains predominant in pits and fissures. Key words: Dental caries, Enamel, Fluoride, Developmental defects, Human premolars Correspondence: Takaaki Aoba, The Nippon Dental University, Department of Pathology, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan Phone: +81-3-3261-8311, Fax: +81-3-3261-8969, E-mail: patho-aoba@tokyo.ndu.ac.jp Introduction For many years, fluoride uptake into the forming and erupted enamel has been the subject of extensive investigations in dentistry because of the well-documented cariostatic effect of this ion (1-3). To date almost all-successful caries-preventive measures have involved the use of fluoride. Surveys in Japan have also documented that the incidence of caries at younger ages continues to decline (4). While the reasons for this are complex, it is most likely that fluorides in various forms and vehicles have been essential in bringing the caries prevalence down to their present levels (5). We previously conducted a clinicopathological examination of permanent teeth that had been extracted from a Japanese population between 1995-97 (6). The results obtained showed a decrease of advanced carious lesions but a high prevalence of incipient caries. An unexpected observation was that teeth displaying enamel hypoplasia and/or white discoloration by hypocalcification were found at relatively high frequencies, mostly in the teeth collected from a younger genera- tion. It has been well documented that, during the forming stage, the incidence of enamel malformation is high and that part of the malformed enamel is related to uptake of excess fluoride while the remainder of the malformation is independent of fluoride (7-9). The issue of whether discrete types of enamel malformations are a predisposing factor for caries development and progression is still controversial. Since the classic work reported by Dean (10), it has been postulated that more severe forms of dental fluorosis may result in a higher risk of dental caries, but that milder forms may be associated with a reduced risk of caries. However, more recent surveys have been unable to detect a difference in caries prevalence between groups with or without dental fluorosis or other types of enamel defects (3,11). Other types of enamel opacity, apart from dental fluorosis, have received less consideration regarding their association with caries susceptibility. In the present study, we address our attention to two specific questions: first, whether part of the enamel

62 Nakahara et al. Caries in malformed enamel opacities found in a pool of permanent premolars could be ascribed to uptake of excess fluoride and, second, whether there are appreciable differences in caries experience between the tooth groups with and without developmental enamel defects, and between enamel surfaces of fluoride-related and -unrelated malformations. In order to ascertain whether the malformed teeth had been exposed to excess fluoride during the developmental period, the fluoride content of the enamel was assessed by electron probe microanalysis (EPMA). Prevalence of carious lesions and their severities in the coronal region of the teeth were validated by whole-sectioning of tooth specimens and stereomicroscopic examination of all serial sections. Materials and methods Tooth collection and preparation The materials under investigation comprised a total of 20 permanent premolars, which had been extracted for orthodontic and/or periodontal reasons from Japanese patients at the Hospital of the Nippon Dental University during the period of 1995 through 1997. The patients ranged in age from 11 through 61 year-old. In the present clinico-pathologic examination, we focused our attention on the occurrence and progression of primary carious lesions in the coronal enamel, so premolars with fillings or large cavities were omitted. Fluoride history of the selected premolars was incomplete or fragmentary, but it was ascertained that no patients had a history of residence in foreign countries with public fluoridated water supply at least during the first 5 years after birth. All tooth specimens were washed with deionized water and then preserved in 10% neutral buffered formalin at 4 until used for analysis. The pooled teeth were examined by visual inspection under a dental light to record existence and location of any discoloration or defects, i.e., mesio-distal proximal regions, bucco-palatal (or lingual) regions, and pits and fissures relative to tooth surfaces regardless of developmental or carious origin. Histopathological validation of developmental anomalies and carious lesions Prior to preparation of ground sections, five tooth surfaces (i.e., occlusal, buccal, lingual or palatal, mesial, and distal) for the individual teeth were photographed using a stereomicroscope. All teeth were resin-embedded and then cut with a hard-tissue slicing machine (EXACT II, Leitz Co.) to prepare serial slices (300-500 m thick) in the bucco-lingual plane. On average, 12 slices were obtained from each of the premolars. Prior to histologic examination, each sliced section was polished on a whetstone and, if necessary after the initial inspection, staining with acidic fuchsin was applied for the purpose of improving discrimination of carious lesions from the surrounding sound areas. Types and progression of enamel lesions due to developmental defects and caries attacks were assessed by scrutinizing consecutive sections under the stereomicroscope. Caries progression toward the tooth substance and pulp was represented at the deepest point among all histologic sections that included the corresponding lesion. To express numerically the status of caries experience for the individual teeth, enamel and dentin on histologic sections were divided into three layers, i.e., outer, middle, and inner thirds (E1, E2, E3, D1, D2, and D3); depending on the deepest progress of the caries lesion, numerical factors 1 through 6 were given for E1 through D3 in order. Caries scores for the entire coronal enamel surface or specific locations (e.g., smooth surfaces vs pits and fissures) of a premolar was expressed by the sum of the lesion numbers multiplied by the corresponding factors: for instance, if a tooth had three caries lesions (two E2 and one D1 lesions), the calculated caries score was 2 2 4 = 8. Elemental analysis of tooth enamel Concentration gradients of Ca, P, and F across the entire enamel from surface to enamel-dentin junction were determined by electron probe microanalysis (Shimazu Ltd, model 8705). Multiple parallel line scans were made on locations covering the incisal through cervical enamel regions in each specimen. In the current analysis, no quantitative measurements were conducted, but experimental values of F/Ca signal ratio (cps/cps in unit) were adopted as an indicator of the elemental profile of teeth. In particular, we paid attention to variations in F/Ca ratio for individual teeth, namely a relatively constant level in the interior enamel (mean) and the highest level at the narrow surface enamel (maximum). Results Among the 20 premolars under examination, 14 teeth exhibited non-carious lesions of developmental origin, which were characterized by white opacity (diffuse or demarcated), pits or grooves, or exaggerated horizontal perikymata. Some teeth showed diffuse opacities, having no clear boundaries from the adjacent normal enamel, which appeared in patchy and/or linear forms. These features are in accord with the clinical criteria for dental fluorosis (12). Neither major anatomical changes nor severe enamel defects, such as large hypoplastic lesions or dark staining, were found in any teeth. Fig. 1 shows representative enamel manifesting fluorotic changes, namely diffuse chalk-like opacity and fine striae of accentuated perikymata over the coronal surface. Histologic examination of serial ground sections prepared from the corresponding tooth revealed that a visually-dark zone developed below a well-mineralized surface layer and was relatively uniform in width from the cuspal to cervical region. Linear scannings by EPMA further proved that the dark enamel zone had accumulated high concentrations of fluoride, while the subsurface hypomineralized layer had relatively lower fluoride

Oral Med Pathol 9 (2004) 63 Fig. 1: A representative case of fluorotic enamel malformation feastured by diffuse, opaque appearance of enamel with grooves along perikymata. Microgram of a slice of the tooth shows the appearance of dark zone along the entire enamel surface under transmission light. A line-scanning of the enamel by EPMA, indicated by a linear line, shows an increase in fluoride in the superficial zone. Scans for calcium and phosphorus were conducted simultaneously. Horizontal bar, 300 m. Fig. 2: Microgram and line-scans of another type of malformed enamel. A typical caries-cone is detected in the proximal enamel, which exhibits a sharp increase in fluoride in the superficial zone (scan a). Sound enamel without visible caries (scan b) yields a relatively flat profile for fluoride. Horizontal bar, 300 m. content. Fig. 2 shows another type of enamel malformation, which was featured by localized hypomineralization and/ or hypoplasia with accentuation of developmental lines on ground sections. Development of carious lesions in those enamel areas was discernible by the characteristic cone-shaped dark areas. Linear scanning for element analysis indicated that there was no marked concentration of fluoride in the surface of non-caries enamel, while a sharp peak of fluoride was detected in the surface enamel covering the caries cone. Table 1 gives the summary of EPMA and pathohistologic findings obtained from the individual tooth specimens. Fluoride concentrations varied widely between individual teeth and in a location-dependent manner within the same teeth. The EPMA data regarding F/Ca ratios were collected from not-yet-caries attacked enamel. The occurrence of hypoplastic (HP)/hypocalcified (HC) developmental lesions was validated by macro-and microscopic diagnosis of the collected photographs and serial sections. Based on the criteria of histopathologic features and F/Ca determinations of the coronal enamel, we divided the pooled premolars into three groups: namely, enamel without developmental defects, i.e., double negative for HP/HC (Group S in the Table, teeth #2, #9, #11, #13, #15, #20), enamel with fluoride-relative lesions featured by higher mean and max. F/Ca levels plus validation of HP/HC lesions (Group F, #5, #6, #8, #10, #12, #19), and enamel with non-fluorotic developmental defects featured by the presence of HP/HC lesions but a lack of F accumulation (Group NF, #1, #3, #4, #7, #14, #16, #17, #18). Histopathologic validation of the caries prevalence in these groups showed that (i) all the teeth under study had caries experience, displaying a wide spectrum of the incipient enamel changes (E1 E3) to advanced dentin caries (D1 D3), (ii) their caries experience, defined as the number of histologically validated lesions per tooth, ranged from 4 to 10 lesions, and (iii) the caries score for the individual teeth in each tooth group varied widely in the range of 9 and 27. Table 2 gives the summary of caries experience among three tooth groups. The results obtained from the entire coronal enamel showed that there were no significant differences in caries experience between the groups. One finding of note was that the F group was the lowest in the number of carious lesions per tooth, due to a fewer caries in the proximal and occlusal enamel, but exhibited high score for pits & fissure caries, due to the occurrence of advanced caries around pits and fissures (see Table 1). The majority of the pits/fissures lesions in the F group remained invisible by clinical inspection, thereby corresponding to the so-called hidden caries (3). Discussion Dental fluorosis is caused by continuous ingestion of excessive amounts of fluoride during tooth development. Particularly, enamel is known to be sensitive to excess fluoride ingestion because of the longer developmental period required for the completion of enamel mineralization (13). Taking into consideration the fluoride accessibility for the past decades in Japan, the most plausible source of fluoride ingestion has been the home-based usage of fluoridated products such as dentifrice and mouthwash. The occurrence of mild and moderate fluorotic enamel opacities have been reported in individuals and populations with fluoride exposure at low concentrations

64 Nakahara et al. Caries in malformed enamel Table 1: Composition, enamel malformations, and caries experience in the coronal enamel of premolars Specimens 1) F/Ca ratios 2) Enamel defects 3) Caries prevalence and progression 4) Caries score 5) (years) Mean / Max. HP/ HC; Group Smooth surfaces Pits & Fissures W / S.S./ P.F. 1) Teeth under examination are numbered in order according to the age of patients when the teeth were extracted due to orthodontic or other reasons. 2) The data are presented by relative F/Ca ratios in terms of signals for F and Ca (cps/cps x10 5 ) obtained from the simultaneous line scans. The values of mean and maximum level correspond to the ratios attained from the interior enamel region and at surface, respectively. 3) Types of developmental enamel defects were diagnosed into two categories, namely hypoplastic (HP) and hypocalcified (HC) types. Based on visual appearance and histopathologic examination, the severity of the developmental defects for the whole coronal enamel was classified into negative (-), questionable ( ), appreciable (+), and prominent (++). Tooth groups correspond to: S, developmentally intact; F, fluoride-dependently malformed; NF, fluoride-independently malformed. 4) Caries prevalence is presented in terms of total carious lesions per tooth. Progression of individual caries lesions is presented by the parameters E1 through D3 corresponding to lesion depth (see text). 5) Score is calculated by a sum of lesion numbers progression indices (1 through 6 for E1 through D3, see text for details). W / S.S./ P.F. correspond to the whole coronal enamel, smooth (proximal, buccal and palatal or lingual) surfaces, and pits & fissures, respectively. Note that the score for W is the sum of scores for S.S. and P.F.. Table 2: Results of histopathological validation regarding caries experience Tooth 1) Entire coronal enamel Smooth surfaces Pits/ Fissures group Prevalence 2) Score 3) Prevalence Score Prevalence Score S (n=6) 6.2 (1.2) 17.6 (5.3) 2.5 (1.5) 4.8 (3.1) 3.7 (1.0) 12.8 (4.0) F (n=6) 5.2 (1.9) 16.3 (6.5) 1.5 (1.0) 3.7 (3.3) 3.7 (1.6) 12.7 (4.5) NF (n=8) 6.9 (2.4) 15.9 (6.9) 4.1 (2.0) 7.3 (4.6) 2.8 (2.1) 8.6 (7.2) 1) Tooth groups: S, developmentally sound (#2, #9, #11, #13, #15, #20, see Table 1); F, fluoride-related defects (#5, #6, #8, #10, #12, #19); NF, fluoride-independent defects (#1, #3, #4, #7, #14, #16, #17, #18). 2) Expressed by the number of carious lesions per tooth. 3) Expressed by a sum of lesion numbers caries progression parameter (1 through 6 for E1 through D3, see Table 1)

Oral Med Pathol 9 (2004) 65 in water (14). It was also appreciated that a variety of factors, e.g., malnutrition, high altitude, chronic illness and genetic factors, may enhance the effect of fluoride or actually cause enamel mottling that might be misdiagnosed as fluorosis (15). Enamel fluorosis is featured as hypomineralization of tooth enamel due to delay of matrix protein removal requisite for enamel maturation (13). In the current report, we diagnosed fluorotic enamel lesions on the basis of their histologic features (i.e., the occurrence of a diffuse relatively hypomineralized region below a well-mineralized surface layer) and their high fluoride content throughout the enamel layer. Although post-developmental changes in fluoride levels occur at the enamel surface, this does not invalidate the value of the interior enamel as a permanent record of exposure to ingested fluoride during the developmental period. Thus we paid attention to the averaged fluoride level of the interior enamel to discriminate fluoride-dependent and independent developmental defects. The mean fluoride levels for both S and NF groups were in a lower and narrower range of 1.13-1.25x10-5 in terms of F/Ca signal count ratios relatively constant, although fluoride levels for surface enamel were variable. The mean fluoride levels for the F group varied widely, ranging from 1.33 to 1.65 x10-5, which were segregated from the F/Ca range for the other groups. Histopathologic validation also substantiated the positive correspondence between the level of ingested fluoride and the severity of structural alterations of formed enamel. The data collected support the high incidence of developmental enamel malformations including fluoriderelative defects as documented in the literature (8). Regarding possible association between developmental enamel defects and caries experience, the data obtained for the coronal enamel in toto indicate that there were no significant differences in caries score among the three tooth groups. When one focuses separately on smooth surfaces and pits/fissures, it is of interest that the integrated values of caries prevalence and score for smooth surfaces are NF > S> F in order, while the corresponding data for pits & fissures are S = F > NF. This supports the possibility that enamel defects of non-fluoridated developmental origin could have acted as a predisposing factor for caries susceptibility of smooth enamel surfaces in combination with poor oral hygiene or other risk factors. The present histopathologic validation also confirmed that mild fluorotic enamel is associated with a lower risk of caries susceptibility in smooth enamel surfaces but caries development still remains predominant in pits and fissures. The results obtained lend support for the concept of occurrence and prevalence of so-called hidden caries around fissures under fluoride regimen, which may mask the spread of caries in underlying tooth substance by encouraging remineralization at the enamel surface (16). In place of conclusive remarks, it should be stressed that all the experimental data and considerations do not rule out the importance of fluoride incorporation into enamel crystals during tooth formation to improve their chemical stability. What we should emphasize is that, while the chemical stability of tooth mineral is essential for the integrity of tooth structure against caries attack or other chemical invasion in the oral environment, a continuous supply of fluoride at low concentration is required for protection of tooth substance and promotion of remineralization. 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66 Nakahara et al. Caries in malformed enamel 14. Rozier RG. The prevalence and severity of enamel fluorosis in North American children. J Public Health Dent 1999; 59: 239-46. 15. Pendrys DG and Katz RV. Risk of enamel fluorosis associated with fluoride supplementation, infant formula and fluoride dentifrice use. Am J Epidemiol 1989; 130: 1199-208. 16. Weerheijm KL, Kidd EAM and Groen HJ. The effect of fluoridation on the occurrence of hidden caries in clinically sound occlusal surfaces. Caries Res 1997; 31: 30-4 (Accepted for publication September 19, 2003)