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RESEARCH REPORT |
Dental Caries Research Group, Guy’s, King’s and St Thomas’ Dental Institute, Caldecot Road, Denmark Hill, London, England. SE5 9RW;
* corresponding author, david.beighton{at}kcl.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: cleft palate • plaque • caries-associated micro-organisms • starch • oral clearance
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Oral levels of mutans streptococci and lactobacilli were determined in Dutch children with cleft lip and/or palate (Bokhart et al., 1996), and 50% were colonized with mutans streptococci and 16% with lactobacilli at rates significantly greater than those found in children with no clefting. Teeth adjacent to the cleft were most often affected by caries (Bokhout et al., 1997), while children with cleft palates who had been bottle-fed had higher caries scores than control, bottle-fed children (Lin and Tsai, 1999). These studies suggest that children with clefts retain food in their mouths for longer periods than control children. High-starch-containing foods remain longer on the teeth than high-sucrose- or low-starch-containing foods (Edgar et al., 1975; Linke and Birkenfeld, 1999). The retention of high-starch-containing foods may give rise to prolonged periods of acid production (Kashket et al., 1994, 1996), which may promote tooth decay.
On the basis of these previous reports, we have tested the hypothesis that children with cleft palates have increased oral clearance times, which is reflected in increased salivary levels of starch-derived saccharides, higher levels of caries-associated micro-organisms, and more caries than found in control children.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Clinical Examination
The types of abnormalities present in the children with cleft palates were classified as unilateral cleft lip (n = 6), unilateral cleft and alveolus (n = 9), bilateral cleft and alveolus (n = 5), unilateral cleft lip and palate (n = 29), bilateral cleft lip and palate (n = 16), cleft palate only (n = 11), soft palate (n = 3), and other cleft condition (n = 2). Caries status was determined according to WHO criteria (WHO, 1987) supplemented with radiographs. Oral health was assessed as an oral hygiene index (0 = poor, plaque on all teeth; 1 = fair, plaque on half of all teeth; and 2 = good, plaque on less than half of the teeth). A gingival index (Löe, 1967) and plaque index (0 = no plaque, 1 = plaque 1/3 height of tooth, and 2 = plaque over 1/3 height of tooth) were recorded as the maximum score on the labial surfaces of the upper and low central incisor teeth, the buccal surfaces of the upper first permanent molars or the upper second deciduous molars, and the lingual surfaces of the lower first permanent molars or the lower second deciduous molars. In total, 8 surfaces were examined per subject.
Saliva Collection and Microbial Analysis
The children were asked to sit with their heads inclined forward and to drool into a sterile container. In this way, approximately 2 mL of unstimulated mixed saliva was collected from each child before the clinical examination. The numbers of mutans streptococci, lactobacilli, and yeasts in each saliva sample were determined as log10([cfu+1] per mL) of saliva. Briefly, saliva samples were decimally diluted in Fastidious Anaerobe Broth (FAB; IPT Ltd., Bury, Lancs., UK), and a 100-µL quantity of appropriate dilutions was spread-plated onto selective media for yeasts (Sabouraud Dextrose Agar [SAB], Oxoid, Basingstoke, Hants, UK), lactobacilli (Rogosa Agar [ROG], Oxoid), and Streptococcus mutans and Streptococcus sobrinus (Mitis Salivarius Agar supplemented with sucrose and bacitracin [BMSA]; Becton Dickinson, Cowley, Oxon, UK). The SAB was incubated aerobically for 3 days, whereas the ROG and BMSA plates were incubated for 3 days anaerobically. After incubation, the relevant taxa were enumerated as described previously (Zoitopoulos et al., 1996).
Oral Clearance and Saliva Rinse Analysis
We determined oral clearance for each child by asking each subject to chew half of a commercially available biscuit (Rich Tea, McVitie’s, Middlesex, England). According to the manufacturer’s nutrition information, the ingredients of the biscuit were wheat flour, sugar, vegetable oil and hydrogenated vegetable oil, partially inverted sugar syrup, malt extract, cultured skimmed milk, salt, and raising agents, and each biscuit contained 6.3 g of carbohydrate, of which 1.7 g was sugars. Five minutes after being given the biscuit to eat, each child was asked to rinse his/her mouth with 10 mL of sterile water, which was collected into a sterile container and frozen at -80°C for subsequent chemical analysis, essentially as described previously (Kashket et al., 1996; Luke et al., 1999).
To demonstrate that starch degradation occurred in the mouth within the timeframe of the oral clearance experiments, we crushed a biscuit, suspended it evenly in 2% (w/v) molten agar at a concentration of 10% (w/v) and, while it was still molten, placed it within a short piece of PVC tubing (1.5 cm x 6 mm i.d.). After setting, the agar extending beyond the ends of the tubing was cut flat. Individual pieces of tubing containing the agar were held in the subject’s mouth for pre-determined periods of 1, 5, or 15 min, the agar was extruded from the tubing, and one end approximately 1 mm thick was cut off by means of a sterile scalpel blade for analysis. The samples were weighed and stored frozen until analyzed. Six healthy adult subjects participated in this aspect of the study.
The oral rinse samples were thawed and centrifuged (3000 g) for the removal of debris, including residual biscuit. The supernatant from each sample was split to allow for the separate analysis of the soluble saccharides and the major bacterial acid end-products. We heated the samples for carbohydrate analysis to 95°C for 5 min to inactivate bacterial and host enzymes and to kill any micro-organisms. The samples for organic acid analysis were diluted 1:5 (v/v) in potassium chromate solution in de-ionized water (25 mg/L) and passed through a washed C18 cartridge (Sep-Pak, 50 mg, Waters Ltd., Milford, MA, USA). The agar samples were thawed, 1 mL of de-ionized water was added to extract sugars, and the samples were heated to 95°C for 5 min prior to analysis.
All carbohydrate and acid analyses were performed with the use of a HPLC system (Dionex Corporation, Sunnyvale, CA, USA). A PA1 column and PA1 guard column trap were used to separate the sugars, which were eluted, at a flow rate of 1 mL per min, with the use of 150 mM NaOH in a 0- to 300-mM sodium acetate gradient over 50 min and detected by pulsed amperometric detection (PAD). An ICE-AS6 column was used to separate the organic acids. They were eluted with 0.4 mM heptafluorobutryic acid (0.6 mL/min) and detected by conductivity, after the eluent had passed through a membrane suppresser (Dionex), which was continually regenerated with aqueous 5 mM tetrabutylammonium hydroxide flowing at 5 mL/min. Concentrations in saliva were expressed as µM for the organic acids and mM for the sugars, while in the agar plugs the sugar concentrations were expressed as nmoles per mg of plug.
Analysis of Data
Means, standard errors, and median values were calculated for the continuous variables and compared by appropriate non-parametric statistical tests, while the distributions of categorical variables were compared by 
2 tests (SPSSPC, Ver. 8.0, Chicago, IL, USA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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; p > 0.05). There was no significant difference in the health status, place of residence, or ethnicity of the two groups of children. Fluoride rinses or drops were used by only 10 cleft palate patients but by none of the control subjects. There was no difference (p > 0.05) between the mean number [± SE] of snacks consumed by each group per day, being 2.01 ± 0.11 for the cleft palate children and 2.18 ± 0.16 for the control children.
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). The children with cleft palates had significantly poorer oral hygiene scores (2 = 48.58; p < 0.001), significantly greater plaque scores (2 = 63.44; p < 0.001), and significantly greater GI scores (2 = 57.31; p < 0.001) than the control children (Fig.).
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Salivary Concentrations of Organic Acids and Sugars
The concentrations of sugars originally present in the biscuit or derived from the breakdown of the starch were significantly greater (p < 0.05) in the oral rinse samples from the children with cleft palates than in those from the controls (Table 2). However, the concentrations of acetic, formic, and succininc acids were significantly greater (p < 0.04) in oral rinse samples from the control children than in those from the children with cleft palates. The concentrations of lactic acid in the salivary rinses were not significantly different between the groups of children (Table 2).
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). The sucrose and fructose, derived from the biscuit, were rapidly metabolized so that after only 1 min these were reduced significantly (p < 0.01), by 84% and 73%, respectively, and continued to be reduced over time. The glucose concentration was initially reduced but was present at a higher concentration in the five-minute samples and was reduced to a lower-than-initial concentration in the 15-minute samples. The concentrations of maltose and maltotriose, in particular, increased significantly (p < 0.01) after 1 min, by 7.7- and 72.5-fold, respectively, and remained at these increased levels throughout the exposure period.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, the cleft palate children had higher salivary levels of mutans streptococci, lactobacilli, and yeasts, which may be associated with their poorer oral hygiene or their higher level of caries. Poor oral hygiene is often associated with higher levels of caries-associated micro-organisms in other groups of patients, as was found here for the children with cleft palates. However, there were no significant differences in carriage of mutans streptococci between children with cleft palates and good oral hygiene and control children (Lucas et al., 2000). These observations underline the profound influence of oral health on the oral microflora.
Here we also investigated the oral clearance of a simple biscuit by measuring soluble sugars derived from the biscuit in oral rinse samples taken 10 min after the subject ate the biscuit. We chose to sample the mouth 10 min after the subject ate the biscuit, since, in other studies, it has been shown that the majority of any food, including starch-containing foods, is cleared from the mouth at that time (Luke et al., 1999). We therefore expected that if a difference in oral clearance times of foods were to be evident, then it would be most apparent at that time. Using these parameters, we demonstrated that the oral clearance times of these sugars by the children with cleft palates were apparently greater than those of the control children. The longer clearance time was evident from the higher levels of sucrose, which cannot be generated de novo from the biscuit, in the rinses from the cleft palate children and the generation of saccharides, by the action of salivary amylase, from the starches present in the biscuit. The generation of these sugars confirmed the rapidity of starch degradation observed in vitro (Grenby and Mistry, 2000), and the speed with which sucrose and fructose were utilized by the oral microflora was evident from the analysis of the sugars present in the agar plugs.
pH changes in plaque, which are more relevant to caries, were not measured. Instead, we determined the salivary organic acid concentrations generated by the fermentation of the sugars from the biscuits, mediated by the intra-oral microflora. While the residual sugar concentrations were higher in the saliva of the cleft palate children, the salivary concentrations of organic acids were lower in the children with cleft palates than in the control children. This relationship does not seem to be what one would expect. However, in a recent study, acid production in plaque following sucrose exposure was negatively related to plaque mass, so that acid production, as acid per mg wet weight of plaque, decreased with increasing plaque mass (Borgstrom et al., 2000), and this may reflect changes in biofilm physiology resulting from oral biofilm maturation (Socransky and Haffajee, 2002). Thus, it would be expected that acid production from the children with cleft palates, with poor oral hygiene, and with high plaque score would produce less acid. Although the acid concentrations were less than in the control children, the longer oral clearance times of foods, and increased time for the generation of acids, may contribute to the increased caries levels in the children with cleft palates. The increased oral clearance times of foods have been identified in elderly patients (Lundgren et al., 1997), in patients with myotonic dystrophy (Engvall and Birkhed, 1997), and in rheumatic patients with dry mouths (Risheim et al., 1992) as a factor significantly associated with high caries activity.
Analysis of these data supports our hypothesis and confirms the previous reports of significantly greater levels of caries in children with cleft palates. This was associated with poorer oral hygiene and higher levels of caries-associated microflora. The other critical finding presented here was that children with cleft palates had a significantly longer oral clearance time for a starch-containing biscuit, which would be expected to have deleterious effects on the dentition. Caries prevention in this group of children should therefore underline the need for good oral hygiene and the use of a fluoride-containing toothpaste. Additionally, these patients should be advised on the clinical consequences inherent in the longer oral clearance times of foods and should be advised of simple methods to reduce levels of residual foods following meals, especially snacks.
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ACKNOWLEDGMENTS |
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Received January 6, 2003; Last revision October 16, 2003; Accepted December 3, 2003
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REFERENCES |
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