Original
Caries Research
Paper
Caries Res 2002;36:347-352 DOI: 10.1159/000065959
Received: December 24, 2001 Accepted afler revision: June 28, 2002
Relationship among Dental Plaque Composition, Daily Sugar Exposure and Caries in the Primary Dentition M. Nobre dos Santos L.Meio dos Santos S.B. Francisco J.A. Cury Faculty of Dentistry of Piracicaba, State University of Campinas, Piracicaba, SP, Brazil
Key Words Dental plaque Sucrose
. Nursing caries . Primary
dentition
.
suggest that the dietary sugar experience changes the biochemical and microbiological composition of dental plaque, which could partly explain the different caries patterns observed in primary dentition. Copyright@2oo2
S. Karger AG, Basel
Abstract The relationship among daily sugar exposure, dental plaque composition and caries patterns in the primary dentition is not known. Three groups of 20 children (1848 months old) were studied: (1) caries-free (CF), (2) presenting pit and fissure caries (PFC), and (3) with nursing caries (NC). Dental caries index (dmfs) was assessed and the frequency of daily sugar exposure (TS) and its forms were estimated by questionnaires. Fluoride (F), calei um (Ca), inorganic phosphorus (Pj),insoluble polysaccharide (IP) and mutans streptococci were quantified in dental plaque. The three groups of children were statistically different regarding daily sugar exposure. Concentrations of F, Ca and Pjwere lower and the IP and mutans streptococei statistically higher in dental plaque from NC children than those from CF children. PFC children also showed statistically lower F and P levels in their plaque than CF children. Statistically significant correlations were found between IP, TS and dmfs from NC and PFC children. A statistically significant correlation between mutans streptococci in plaque from anterior teeth and dmfs for NC children was also observed. These results
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Dental caries is a diet-dependent bacterial disease and the properties of the biofilm formed on tooth surfaces may explain the different caries pattems found. Among the dietary components, sucrose (sugar) is considered the most critical, regarding the biochemical and microbiological change that it induces in dental plaque composition. The changes in dental plaque due to sugar exposure are not limited to the organic composition, because the inorganic composition of dental plaque is also influenced by sugar exposure [Cury et aI., 1997]. Although the data on low concentrations of calcium (Ca) and inorganic phosphorus (Pi) in human dental plaque exposed to sugar are not consistent [Ashley and Wilson, 1977a; Geddes et aI., 1978], Cury et aI. [1997] showed, in a well-controlled experimental in situ study, that the concentration ofthese ions decreased with increasing frequency of sucrose exposure. Ca and Pi are important ions involved in the equilibrium between the demineralization and remineralization of enamel as a 'saturation buffer' [Pearce, 1998], and recently Pearce et aI. [2002] showed that the concentration
Prof. Jaime A. Cury Av. Limeira 901 13414-903 Piracicaba, SP (Brazil) Tel. +551934125302/5303, Fax +551934125218 E-Mail
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of Ca in artificial mouth plaque microcosm biofilms decreased as sucroseapplication frequency increased. Additionally, some authors have suggested that there is an inverse relationship between the concentrations of these ions in dental plaque and caries experience or increment [Ashley,1975;Ashleyand Wilson, 1977b; Schamschulaet aI., 1978;Grobler et aI., 1982;Shawet aI., 1993]. Fluoride (F) is another important ion in relation to dental caries and recent publications have shown that its concentration in dental plaque is low when the biofilm is formed in the presence of sucrose exposure [Cury et aI., 1997, 2000]. It has a very important physicochemical effect on enamel demineralization and remineralization and its constant presence in dental plaque is important to control dental caries. Although the concentrations of Ca, Pi and F in dental plaque are important, the cariogenicityofthis biofilm has been explored more extensivelywith respect to its microbiologicalcomposition and its glucan-richmatrix. Mutans streptococci have been the bacteria most c1earlyassociated with dental caries [Emilsonand Krasse, 1985;Loesche, 1986] and it has been suggested that the levels of these bacteria increase in the presence of sucrose [Hamada and Slade, 1980].Moreover, according to de Stoppelaar et aI. [1970] and Scheie et aI. [1984],there is a positive correlation between the sucrose content of the diet and the leveI of mutans streptococci in dental plaque, but this trend was not confirmed in a controlled in situ study [Cury et aI., 2001]. Nevertheless, mutans streptococci are specialized in producing extracellular polysaccharides from sucrose, of which the insoluble glucan is mainly implicated in dental caries. A correlation between dental plaque cariogenicity and extracellular insoluble polysaccharide (IP) was suggestedby some authors [Zero et aI., 1986;Dibdin and Shellis, 1988; van Houte, 1994] and has been confirmed experimentally [Cury et aI., 2000]. Furthermore, Mattos-Graner et aI. [2000] showed that caries incidence in children was more dependent on the capacity of mutans streptococci to synthesize insoluble glucans than on the number ofthese bacteria in dental plaque. Among the different patterns of caries, nursing caries (NC) is a worldwide public health problem which has been attributed to sweetened baby bottles [Ripa, 1988]. In dental plaque of children with NC, high levelsof mutans streptococci have been found [van Houte et aI., 1982;Berkowitzet aI., 1984; Milnes and Bowden, 1985; Alaluusua et aI., 1996; Kreulen et aI., 1997]. Thus, the data in the literature suggest that the biochemical and microbiological composition of dental plaque may be involved with dental caries. Considering 348
Caries Res 2002;36:347-352
that there are no published data evaluating sugar exposure, dental plaque composition and the different patterns of caries in the primary dentition, we conducted the present study. Materiais and Methods Subjects The study group comprised 60 preschool children of both sexes, ali of low socioeconomic levei, aged 18-48 months. The children attended public nursery schools in Piracicaba town, state ofSão Paulo, Brazil. Three groups of 20 preschool children were formed: presenting NC, pit and fissure caries (PFC), or caries-free (CF). Two hundred and six children were examined and 20 were found to present NC. This group was formed on the basis of a questionnaire answered by their parents containing questions about infant feeding practices, with emphasis on the use of nursing bottles, and from the clinical examination. The inclusion criteria for presenting NC was that the children had at least two carious maxillary anterior teeth and a history of prolonged exposure to the nursing bottle [WHO, 1987; Ripa, 1988]. The two other groups of 20 children, one having caries in pits and fissures and the other caries-free, were randomly formed from the remaining 186 children. The pit and fissure pattem was defined as including ali occlusal fissures, buccal pits of mandibular second molars and lingual grooves of maxillary second molars [Douglass et aI., 1994]. The three groups presented the following characteristics: NC (9 girls and II boys, mean age 39.7 :t 10.8 months); PFC (13 girls and 7 boys, mean age41.1 :t 9.0 months); CF (10 girls and 10 boys, mean age 36.3 :t lU months). The Ethical Committee of the University of Campinas, School of Dentistry of Piracicaba, approved this research and the parents of the children signed a written informed consent. Ali the children were reported to use fluoridated water (0.7 ppm F) and F dentifrice. Their teeth were brushed at the daycare centers twice a day using fluoridated dentifrice. The children stayed at the daycare center from 7 a.m. to 5 p.m. and ali of them were fed with the same meals. CariesAssessment Dental examinations, using a mouth mirror and a dental probe and under focusable flashlights, were carried out by one of the authors. Replicate examinations were performed prior to the beginning of the study, on a random sample of 10 children ofthe subjects studied. The number of decayed tooth surfaces was determined in each child using Radike's [1972] criteria and recorded in a way that each primary tooth received a designation of sound (s), decayed (d), missing due to caries (m) and filled (O. No radiographs were performed and only primary teeth were included in the scoring. DietarySugarConsumptionEvaluation To determine the children's daily frequency of meals containing sugar, the mothers and the health agents ofthe school nurseries participating in the study were asked to fill a diet chart for 3 consecutive days, including children's feeding practices and snack food frequency. They were asked to specify the time of the day that the children eat or drink anything and, additionally, the content of ali meals and snacks. Based on the analysis of the dietary charts, the mean daily frequencies of meals and snacks containing sugar in both solid and liquid forms, and also the total frequency of sugar exposure, were
Nobre dos Santos/Meio dos Santos/ Francisco/Cury
estimated. Inc1uded in the frequency were not only foods already sweetened with sugar but also juice, milk and dessert prepared with sugar. Collection of Plaque Samples Two plaque collections were performed, one for biochemical analysis and another for microbiological analysis, with a 2-week interval between the collections. The sampling was carried out at least I h after previous food intake in the beginning ofthe moming or aftemoon lesson period, after children had refrained from having their teeth brushed for 24 h. For F, Ca, Pi and IP determinations, plaque was collected from all buccal and lingual smooth surfaces, except for lower incisors and canines, using a sterile excavator and placed in a capped microcentrifuge tube. To assess the number of colony-forming units (CFU) ofmutans streptococci, two plaque sampies from buccal and lingual surfaces were collected from each child, one being from upper incisors and canines (anterior) and the other from upper and lower molars (posterior). Dental plaque was collected with sterilized plastic curettes, placed in preweighed microcentrifuge tubes, and the wet weight of each plaque sample was determined.
(ANOVA) after transformation, respectively, by inverse function and inverse of root square. The solid and total sugar exposure frequencies were not submitted to any transformation. The results for Ca, F, IP and mutans streptococci in posterior plaque were transformed by 10glOwhile mutans streptococci in anterior plaque and Pi data were transformed by the power 0.2 and square root, respectively. These variables were also analyzed by ANOVA. For all analyses an alpha levei of 0.05 and the software SAS/Lab were used. The differences between the mean values for each of the tested parameters were evaluated by Tukey test with an alpha levei of 0.05. Additionally, Pearson correlation coefficients were ca1culatedbetween dmfs and F, Ca, Pi, mutans streptococci in anterior and posterior plaque, sugar exposure frequency and IP.
Results
Table I shows that the three groups of children presented statistically significant differences (p < 0.05) in daily sugar exposurefrequency, whether total, solid or liqBiochemical Analysis uid. The differencesin dmfs were also significant. Dental plaque was dried for 16 h in vacuum at 40°C over P20s [Pearce, 1984] and the dry weight was obtained using an analytical Table 2 summarizes the biochemical and microbiologbalance (:t 10 Ilg). For carbohydrate and inorganic analysis, 0.5 M ical composition of dental plaque samples. The plaque HCI was added to the tube in the proportion of 0.25 mllmg plaque concentrations of F, Ca and Pi were significantly lower in dry weight. After extraction for 3 h at room temperature under conNC children than in CF children. In addition, except for stant agitation, the same volume ofTISAB 11,pH 5.0 (containing 20 g NaOH/I), was added to the tube as a buffer [Benelli et aI., 1993;Cury Pi, these concentrations were also statistically lower (p < et aI., 1997,2000]. The samples were centrifuged (11,000 g) for I min 0.05) than in PFC children. It can also be noted (table 2) and the supematant retained for determination of acid-soluble F, Pi that PFC children had significantlylower(p < 0.05) F and and Ca. To the precipitate 1.0 N NaOH (0.1 ml/l O mg plaque dry Pi in their plaque than CF children. However, plaque Ca weight) was added. The samples were homogenized for I min, agitated for 3 h at room temperature and the concentration of alkali- concentration in PFC children was not significantly difsoluble carbohydrate (IP) determined in the supematant. F was ana- ferent from that in CF children. Table 2 also shows that Iyzed in the acid extract of plaque, using an ion-selective electrode plaque from NC children had significantly more (p < (Orion 96-09) and an ion analyzer (Orion EA-940), Pi was deter- 0.05) IP than did plaque from CF subjects. The NC group mined colorimetrically [Fiske and Subbarow, 1925] and Ca was anaalso had a higher plaque IP concentration than the PFC Iyzed by atomic absorption spectrophotometry using lanthanum to group but the difference was not significant. The levels of suppress phosphate interference. Alkali-soluble carbohydrate was determined colorimetrically [Dubois et aI., 1956]. The results were mutans streptococciwere significantlyhigher (p < 0.05) in expressed as micrograms per milligram of plaque dry weight. anterior plaque from NC children than in plaque from either PFC or CF children (table 2). Additionally, in posMicrobiologicalAnalysis terior plaque from NC children, the levels of mutans The plaque samples were transferred to tubes containing 10 glass streptococci were higher than in plaque from the other beads and sterile 0.1 Mphosphate bUffer,pH 7.4 (1.0 mllmg). The two groups but only the difference between the NC and tubes were vortexed for 30 s and the suspension was serially diluted with phosphate buffer. For each dilution, 50 III of the samples was CF groups reached statistical significance.From table 2 it plated in duplicate on the selective SB 20 agar [Davey and Rogers, can also be noted that PFC children exhibited significant1984]containing 20% sucrose and 0.2 units bacitracin/ml. The plates ly higher (p < 0.05) levels of mutans streptococci in both were incubated for 48 h at 37 °C in anaerobic jars containing a H21 anterior and posterior plaque than CF children. CO2 mixture (Gas PaK BBL System) according to van Palenstein Table 3 shows the correlation coefficients between Helderman et aI. [1983]. Representative colonies with typical mutans streptococci morphology were counted using a stereomicrodmfs from PFC and NC groups and Ca, Pi, F, IP, mutans scope. The number of CFU was transformed to naturallogarithm streptococci in dental plaque and total sugar exposure freand the results expressed as In(CFU/mg) of plaque wet weight. quency. Statistical analysis revealed significant correlations between dmfs from NC children and mutans strepStatistical Analysis For statistical analysis the data on dmfs and liquid sugar expo- tococci in anterior plaque (p = 0.0001),IP (p = 0.0008), sure frequency were analyzed by a one-way analysis of variance solid sucrose exposure frequency (p = 0.0004) and total Plaque Composition and Caries in the Primary Dentition
Caries Res 2002;36:347-352
349
Table 1. Means :t SD (n
=20) of dental
caries experience (dmfs) and
the dailyfrequencyof sugar-basedmealsin the three groupsof children Groups
dmfs
total
liquid
solid
2.88:t 0.22a 3.90:t0.38b 5.32:t0.75c
0.40:t0.28a 1.00:t0.24b 1.40:t0.32c
2.48:t 0.30a 2.90:t0.38b 3.92:t 0.59c
Groups whose means are followed by distinct letters differ statistically (p < 0.05).
Table 2. Plaque analyses in the three groups of children Analysis
Groups CF
NC
PFC
~
F,l1g/.!ftg -5&.1.:t*='30a Ca,l1g/mg 1O.6:t5.44a Pj,l1g/mg 6.0:t 2.92a IP, I1g/mg 39.2:t 7.42a Mutans streptococci, In(CFU/mg) Anterior teeth 8.6:t 2.00a Posteriorteeth, 9.8:t2.18a
~
~:tMt3b 7.9:t 4.33a 4.0:t 1.87b 47.4:t 8.92b
~:t1;93c 3.3:t 2.56b 2.6:t 1.25b 55.6:t 17.56b
11.3:t 1.40b 12.0:t 1.92b
14.3:t0.63c 12.6:t 1.71b
Means :t SD (n = 20). Groups whose means are followed by distinct letters differ statistically (p < 0.05).
Table 3. Pearson correlation coefficients (r) and probabilities of statistical significance (p) between caries (dmfs) and the variables anaIyzed among NC and PFC children Variables
Groups of children NC p
PFC r
p
0.25 0.16 -0.18
0.3179 0.4778 0.4314
-0.14 -0.04 -0.22
0.5843 0.8707 0.3520
0.85 -0.28 0.94 0.62 0.12 0.72
0.0001 0.2666 0.0001 0.0035 0.6072 0.0004
-0.23 0.03 0.79 0.72 0.15 0.63
0.3681 0.9073 0.0001 0.0003 0.5178 0.0031
r F Ca Pj MS in plaque Anterior teeth Posterior teeth IP Total sugar/day Liquid sugar/day Solid sugar/day MS
350
=Mutans
solid sugar exposure frequency (p = 0.0031) and total sug-
ar exposure (p =0.0003) in PFC children.
Sugar/day
2.40:t0.90a 5.60:t 3.40b
CF PFC NC
sugar exposure frequency (p = 0.0035). Strong correlations were also found between dmfs and IP (p = 0.0001),
streptococci.
Caries Res 2002;36:347-352
Discussion
This study shows for the first time that there is a relationship between the biochemical and microbiological composition of dental plaque, sugar exposure frequency and caries pattern in primary dentition. A trend for lowconcentrations ofF, Ca and Pj in dental plaque with increase of caries severity was shown (table 2). Thus, statistically higher concentrations of F, Ca and Pj were found in dental plaque of CF children than in NC children. These results are in agreement with those observed in dental plaque fluid from CF and caries-positive permanent teeth [Margoliset aI., 1988, 1993;Margolis and Moreno, 1992;Hartshorne et aI., 1994].However, it should be emphasized that most of the studies published have focused on the difference between dental plaque composition from individuaIs who are either caries-free or with high caries experience. The present data showed that even dental plaque composition from PFC children is different from that ofCF children. In addition, F was the only inorganic component that differed significantly between CF, PFC and NC children. The trend of a low concentration of F, Ca and Pj in dental plaque from the children evaluated may be a consequenceof their diet. Thus, table 1 showsthat these children present statlstical~',\ ly different sucrose exposure frequencies and recent ex- . perimental studies have shown that the inorganic composition of dental plaque depends on the presence and frequency of sucrose exposure [Cury et aI., 1997,2000]. Of the inorganic components, only F seems to be sensitive to diet changes.Thus, the plaque F concentration in CF children was 9.4 times higher than in NC children. The plaque Ca and Pj concentrations in CF children were also higher than in NC children, but the difference was lower (2.6-3.5 times). Regarding Ca and Pj, our results are not in agreement with Ashleyand Wilson [1977a],who found an inverse relationship between dietary sugar experience and Ca and Pj concentrations in dental plaque from permanent teeth. We were not able to show a significant correlation between the inorganic plaque composition and the caries experience of PF and NC children (table 3), although the highest concentrations of inorganic components were found in CF children (table 2). The present data for caries in primary dentition are not in agreement Nobre dos Santos/Meio Francisco/Cury
dos Santos/
with data for permanent teeth, which showed a relationship between the inorganic composition of dental plaque and caries experience [Ashley, 1975; Hartshorne et aI., 1994]. In summary, a1thoughour data showed that there is a trend towards low plaque F, Ca and Pi concentrations and the pattern of caries in the primary dentition, the correlation with dmfs was not statistically significant. One explanation could be that dental plaque was collected in a nonfasting condition, so that plaque composition might have been affected by the last meal. Furthermore, considering the limitations of the cross-sectional design used in the present study and the important role of these ions in maintaining the equilibrium between enamel and its environment, a longitudinal evaluation should be carried out. With respect to the concentration of IP, our data showed a significant1y higher concentration in dental plaque of PFC and NC children than in CF children (table 2). IP is formed from sucroseand the difference found can be explained by the children's diet. In fact, table 1 shows that NC children ingested sugar more frequent1y than PFC or CF children. These data are in accordance with our experimental study [Cury et aI., 1997] showing that the concentration of IP in dental plaque depends on sucrose exposure. IP changes dental plaque matrix and is responsible for the high cariogenicity of sucrose [Cury et aI.,2000]. In the present study wefound a high correlation between plaque IP concentration and dental caries in the primary dentition (table 3). Regarding the levels of mutans streptococci, our data showed a higher concentration in dental plaque from PFC and NC children than in CF children. The very high concentration of these bacteria in dental plaque from NC children is in agreement with severalstudies showingthat these children are heavily infected with mutans streptococci [van Houte et aI., 1982; Berkowitz et aI., 1984; Milnes and Bowden, 1985;Alaluusua et aI., 1996]. Sugar increases the levels of mutans streptococei in dental plaque [Hamada and Slade, 1980;Loesche, 1986]and our data showed that NC children are more frequent1y exposed to sugar (table 1)and present high levels ofmutans streptococci in their plaque (table 2). However, it is known that a heavy infection by mutans is not a sine qua non forcaries manifestation. Recent data have shownthat the ability of these bacteria to synthesize IP might be more important than their levels [Mattos-Graner et aI., 2000]. Furthermore, according to Alaluusua et aI. [1996], children with NC are colonized with more than one donal type of mutans streptococci and the acquisition of these bacteria may be influenced by frequent sugar consumpPlaque Composition and Caries in the Primary Dentition
tion. Our data give support to these inferences, because we showed that children with NC ingested sugar more frequent1y (table 1) and their dental plaque had mutans streptococci that produce IP (table 2). Thus, the present study suggests that the determination of IP in dental plaque could be a predictor of dental caries and a longitudinal study should be designed to evaluate this possibility. In summary, our data showed that children with NC and PFC had lower concentrations of inorganic components, higher concentrations of IP and of mutans streptococei in their plaque, and also reported higher frequencies of daily sugar exposure. Thus, the results presented give support to the concept that a sugar-rich diet changes the biochemical and microbiological composition of dental plaque, which could partly explain the different caries patterns noticed in the primary dentition.
Acknowledgments This research was partly supported by FAEP, State University of Campinas, SP, Brazil, grant No. 0315/97. The authors would like to specially acknowledge Mariza de Jesus Carlos Soares and Waldomiro Vieira Filho for their valuable laboratory assistance. We also wish to thank the Secretary of Education of Piracicaba town and the health agents who participated in this study. The authors thank DI. Cínthia P.M. Tabchoury, FOP-UNICAMP, for the linguistic review of the manuscript.
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