Fluoride Retention in Proximal Plaque and Saliva Using Two NaF Dentifrices Containing 5,000 and 1,450 ppm F with and without Water Rinsing

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1 Original Paper Caries Res 29;43:64 69 DOI: /21592 Received: July 31, 27 Accepted after revision: December 22, 28 Published online: February 1, 29 Fluoride Retention in Proximal Plaque and Saliva Using Two NaF Dentifrices Containing 5, and 1,45 ppm F with and without Water Rinsing A. Nordström D. Birkhed Department of Cariology, Institute of Odontology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Key Words Dental plaque Fluoride dentifrice Interdental sites Tooth brushing Water Abstract A total of 26 healthy volunteers participated in this randomized 4-leg crossover study designed to measure fluoride (F) retention in interdental plaque and saliva. Two NaF dentifrices (5, and 1,45 ppm F) were used, with and without postbrushing water. The 4 tooth brushing methods were carried out twice a day during 2 weeks. Interdental plaque was collected from all proximal sites after each method, using dental floss. Immediately after the plaque sampling, the subjects were asked to brush their teeth with the same toothpaste and use the postbrushing water procedure as previously. Proximal saliva was collected from 4 interdental sites, using small paper points, before and up to 6 min after the brushing. The present study showed that the 5, ppm F toothpaste without postbrushing water resulted in the highest F concentration in both plaque and saliva and the 1,45 ppm F toothpaste with water in the lowest. The difference in the area under the curve of saliva F concentration versus time between the 2 methods was 4.2 times (p!.1). The corresponding difference in F concentration per unit weight of plaque (n = 16) was 2.75 times (p!.5). Water immediately after tooth brush- ing with 5, ppm reduced the F concentration in saliva by 2.4 times (p!.1). The difference in F values in saliva between 5,/ and was minor and not significant. The increase of F in both proximal saliva and plaque, using a dentifrice with 5, ppm F without postbrushing water, may be of clinical importance. Copyright 29 S. Karger AG, Basel The prevalence of caries has declined in many industrialized countries since the late 196s. This is mainly a result of the daily use of fluoride (F) dentifrices [Glass, 1982; Bratthall et al., 1996; Marinho et al., 23]. The Swedish Council on Technology Assessment in Health Care [SBU, 22] reported strong scientific evidence that daily use of F toothpaste is an effective method for preventing dental caries in permanent teeth. Dentifrices with 1,5 ppm F showed a slightly better preventive effect ( ; 1%) compared with those with 1, ppm F [Twetman et al., 23]. Several studies indicate that there is a linear relationship between F concentration in toothpastes ( 1,5 ppm) and caries reduction [Birkeland, 1972; White and Nancollas, 199]. Various factors influence the efficacy of F toothpaste including the concentration of F in the paste, the amount of toothpaste applied on the brush, the frequency of tooth brushing and the postbrushing water [Davies et al., 23]. Fax karger@karger.ch 29 S. Karger AG, Basel /9/431 64$26./ Accessible online at: Dowen Birkhed Department of Cariology, Institute of Odontology Box 45 SE 45 3 Göteborg (Sweden) Tel , Fax , birkhed@odontologi.gu.se

2 In Sweden, the F concentration in dentifrices sold in ordinary stores varies between 4 and 1,5 ppm. Toothpaste with a higher F concentration can only be obtained at a pharmacy as an over-the-counter product. 5, ppm F toothpaste has recently been introduced on the Swedish market for high caries-risk patients. There are few studies evaluating the F concentration in saliva and plaque after using a dentifrice with 5, ppm F [Tavss et al., 23]. In two clinical studies, a 5, ppm fluoride gel was used daily in a similar way as toothpaste with positive results [Englander et al., 1967; Dreizen et al., 1977]. The reversal of root caries after using a dentifrice containing 5, ppm F has been studied by Baysan et al. [21], Lynch et al. [2] and Ekstrand et al. [28]. The intraoral levels of F may differ in subjects with different postbrushing habits [Collins et al., 1991; Duckworth et al., 1991; Richards et al., 1992] and the behavior may be related to the caries experience of the patient [Sjögren and Birkhed, 1993]. However, Machiulskiene et al. [22] showed limited support refraining from water after tooth brushing. The difference between the mean caries increments in children with and without water was only.6 decayed surfaces (DS) and had no statistical significance. The aim of the present study was to investigate the F retention in interdental plaque and saliva using two NaF dentifrices (5, and 1,45 ppm F), with and without postbrushing water, in order to evaluate the potential benefit of a high F dentifrice without water. Materials and Methods Subjects A total of 26 healthy volunteers participated in this randomized 4-leg crossover designed study. Sixteen students at the Dental Hygienist School in Gothenburg aged years (mean 24 years) and 1 subjects from the staff at the Public Dental Service Clinic in the city of Varberg aged years (mean 47 years) were recruited. The subjects were carefully selected in order to be capable of following the instructions given. They were in good general health and had at least 24 teeth. The subjects were instructed not to use any other F products apart from the test toothpaste, not to eat or drink within 2 h after the last tooth brushing, not to use any proximal cleaning and not to use chewing gum or snuff during the test periods. The study was approved by the Ethics Committee of the Sahlgrenska Academy at the University of Gothenburg (Dnr 188-5). S t u dy D e s i g n One week prior to each brushing method, the subjects used F- free toothpaste (Aloe Vera Tandkräm, Forever Living Products, Gothenburg, Sweden) twice daily and a waxed, F-free dental floss (Johnson & Johnson Products, New Brunswick, N.J., USA, or Sunstar Butler, Laholm, Sweden) once daily. Just before starting the experimental period, the teeth were cleaned professionally with a hand piece and a rubber cup, using RDA 17 polish paste (CCS AB, Borlänge, Sweden) and flossed. The subjects were then carefully instructed to use 1 g of toothpaste on their toothbrushes and to brush for 2 min twice a day [Sgan-Cohen, 25]. Each test period lasted for 2 weeks, followed by a washout period of 1 week, using F-free toothpaste. There were some minor differences in procedures between the subjects. The 1 volunteers from Varberg were given stricter instructions regarding the amount of toothpaste applied on their toothbrushes and the amount of water for the procedure. This was considered as an improved standardization. Two dentifrices were used: Duraphat 5, ppm F as NaF (Colgate-Palmolive AB, Danderyd, Sweden) and Pepsodent Superfluor 1,45 ppm F as NaF (Lever Fabergé, Stockholm, Sweden). The subjects used 4 brushing methods and the order was determined using a randomized crossover table. The study was not blinded. Treatments were: (1) Duraphat toothpaste without postbrushing water ; (2) Duraphat toothpaste followed by 1 ml water 3 times, each time for 1 s; (3) Pepsodent toothpaste without postbrushing water, and (4) Pepsodent toothpaste followed by 1 ml water as in treatment 2. The water was carried out with a 1-ml plastic cup. This procedure was also used by Issa and Toumba [24]. During test period 1 and 3, the subjects simply spat out the dentifrice-saliva slurry after brushing and no postbrushing water was allowed. The outcome measures were accumulation of F in proximal plaque and retention of F in proximal saliva based on the area under the curve (AUC). Fluoride Accumulation in Interdental Plaque Interdental plaque was collected 2 h after the last brushing occasion (in order to measure the F accumulated in plaque during the 2 weeks), using F-free, extrawide, waxed dental floss (Johnson & Johnson or Sunstar Butler) according to Sjögren and Birkhed [1993]. Plaque was collected from all proximal sites, except in the lower anterior region. The collected plaque was transferred from the floss to a 2-ml plastic tube (Eppendorf PRC, Sarstedt, Nümbrecht, Germany). This was done by drawing the floss through a slit, which had been cut in the lid of the tube. Plaque adhering to the floss was thereby left on the inside of the tube. The samples were frozen at 2 C until analyzed. For the 16 student hygienists, the Eppendorf tubes were weighed before and after the plaque sampling and the plaque wet weight determined to the nearest.1 mg. For the 1 staff members, no plaque weight data were available and the accumulated F in the extracted plaque suspension was measured and expressed as ppm F. The accumulated F in plaque in the student hygienists was measured and expressed as both ppm F and ng F/mg plaque. Fluoride Retention in Interdental Saliva This experiment was carried out after each 2-week test period and directly followed the collection of plaque. Interdental saliva was sampled from the proximal sites 16/15, 25/26, 35/36, 45/46, using a standardized, triangular-shaped paper point. The paper points were punched from filter paper (Laboratorieläsk, Munktell Filter, Grycksbo, Sweden) to a size of 5! 2 mm, using a special punch instrument. Each paper point absorbs approximately 4 l ( 8 1%) of fluid, when kept in place for 2 s, using a pair of forceps. Immediately after removal from the interdental area, the paper Two NaF Dentifrices Containing 5, and 1,45 ppm F Caries Res 29;43:

3 points were transferred to a.5-ml Eppendorf tube [Sjögren and Birkhed, 1994; Särner et al., 23]. The samples were frozen at 2 C until analyzed. After collection of the baseline samples, the subjects brushed their teeth with the same toothpaste and used the same postbrushing water procedure as during the 2- week test period. Sampling of saliva was then repeated after 1, 3, 5, 1, 3 and 6 min. Fl u o r i d e An a ly s i s The samples were analyzed blinded regarding subjects and methods. A volume of 2 l of liquid, consisting of distilled water and TISAB III (1: 1; Thermo Electron, Waltham, Mass., USA) was added to the plaque and saliva samples. The plaque suspension was homogenized by sonification for 2 s (Branson W185D, Dansbury, Conn., USA) in order to disperse the plaque. The plaque and saliva samples were kept in a refrigerator at +4 C overnight. The tubes were then vibrated in a Minishaker MS1 (IKA, Wilmington, N.C., USA) for 2 s. One hundred microliters of the solution were placed as a drop on a Petri dish. The F concentration was measured by an ion-specific electrode (model 96-9, Orion Research) by carefully lowering the electrode into the fluid. The surface tension of the drop ensured that the liquid enclosed the entire membrane surface of the electrode. In order to calibrate the electrode in saliva, three standard solutions were used (.1, 1. and 1 ppm F). The F concentration was expressed as ppm. The method for analyzing the F concentrations, using 1 l, was evaluated in 5 samples from 1 to 1 ppm F and the r 2 value was calculated as The salivary F concentrations (both as ppm and log ppm) were plotted versus time (in min) and the AUC 6 min was calculated for each individual and each brushing method (No. 1 4) using a computer program (KaleidaGraph 3.1, Synergy Software, Reading, Pa., USA). S t a t i s t i c a l Me t h o d s Two-way ANOVA followed by Scheffé s test was used to compare the four brushing methods, regarding accumulation of F in the proximal plaque (expressed as ppm F or ng F/mg plaque) and the retention of F in proximal saliva, based on the AUC 6 min. p!.5 were considered statistically significant. R e s u l t s Fluoride Accumulation in Interdental Plaque The mean values ( 8 SD) of F concentration in the extracted plaque suspension, expressed both as ppm F and ng F/mg plaque wet weight, are presented in table 1. The highest accumulation of F in proximal plaque was found using toothpaste with 5, ppm F without postbrushing water and the lowest using 1,45 ppm F with 3! 1 ml water. This trend was the same whether F accumulation was expressed as ppm F in the suspension or F/mg plaque wet weight. The increase of F in plaque between and 5,/ was 2.1 times (2.7/1.3) expressed as ppm F and 2.75 (.11/.4) times expressed on a weight basis (both, p!.5). No other differences were statistically significant. Table 1. Mean values 8 SD of F accumulation in proximal plaque expressed both as ng F/mg plaque wet weight and ppm F in the extracted plaque suspension of the student hygienist group (n = 16) and only as ppm F in the plaque suspension of the staff group (n = 1) Student hygienists Staff ng F/mg plaque ppm F ppm F 5,/ ,/ The samples were collected after 2 weeks brushing procedures and 2 h after the last brushing occasion. The difference between 5,/ and was statistically significant (p <.5; n = 16). Table 2. AUC values (mean 8 SD) for proximal saliva expressed as both ppm F! min and log ppm F! min, using two NaF dentifrices (1,45 and 5, ppm F) with and without postbrushing water Student hygienists (n = 16) Staff (n = 1) Total (n = 26) Nonlogarithmic values, ppm F! min 5,/ ,/ Logarithmic values, log ppm F! min 5,/ ,/ AUC data for subgroups and the whole group are presented separately. Statistically significant differences for the whole group (n = 26) are shown in figure 1. Fluoride Retention in Interdental Saliva The mean F concentration at various time points and the AUC values are shown as ppm F! min and as log ppm F! min in figure 1 (n = 26). AUC data for subgroups and the whole group are presented separately in table 2. The difference in logarithmic values between the staff and student groups was minor and the statistical 66 Caries Res 29;43:64 69 Nordström/Birkhed

4 ± Fluoride concentration (ppm) AUC (ppm min) 4 2 5,/ 289 ± ± 25 5,/ 165±79 2 5,/ 5,/ min 4 4. ± 14. Fig. 1. Mean values (n = 26) of F concentration in proximal saliva (expressed as ppm and log ppm) at various time points up to 6 min after using two NaF dentifrices (1,45 and 5, ppm F) with and without postbrushing water. Inserted are also the AUC values ( 6 min) expressed as mean 8 SD. The bars indicate statistically significant differences ( * p!.5; * * p!.1; * * * p!.1). Differences between 5,/ and were not statistically significant. Fluoride concentration (log ppm) AUC (log ppm min ) ,/ 27.2 ± ,/ 25.7 ± ± 1.2 5,/ 5,/ min ** * Two NaF Dentifrices Containing 5, and 1,45 ppm F Caries Res 29;43:

5 significances of differences were just calculated for the whole group (n = 26) in figure 1. The toothpaste with 5, ppm F without postbrushing water gave the highest F concentration in proximal saliva and the 1,45 ppm F toothpaste with water the lowest. The increase of F in saliva (non-log AUC) was 4.2 times (685/165; p!.1). The difference between the 4 methods was most pronounced within the first 3 min. Water immediately after tooth brushing with 5, ppm reduced the F concentration in saliva by 2.4 times (685/289; p!.1). Comparing 5, ppm F toothpaste without postbrushing water with 1,45 ppm F also without water resulted in 2.5 times higher F concentration in saliva (685/274; p!.1). The difference in F values in saliva (AUC) between 5,/ and was minor and not significant. Discussion The main result in the present study was that the concentration of F in the dentifrice had a great influence on the retention of F in saliva. The 5, ppm F toothpaste compared to 1,45 ppm F resulted in higher levels of F in proximal saliva. Regarding the accumulated F in plaque, the increase between the 1,45 and the 5, ppm F toothpaste was up to 2.75 times. This value should be compared with the level of F in the two toothpastes (5, vs. 1,45 ppm = 3.5 times). An explanation for the discrepancy could be that F does not accumulate in plaque over time. This is in agreement with Heijnsbroek et al. [26], who found no F accumulation in plaque after 6 h, using an AmF/SnF 2 dentifrice. The observed differences of F retention in saliva immediately after brushing and the accumulation of F in interdental plaque over time, could be of clinical importance for the prevention of proximal caries. An interesting observation was that postbrushing water had a great influence on the retention of F in the proximal area. The difference between 5,/ and concerning F AUC values in saliva was minor. Previous studies have produced similar results. Duckworth et al. [1991] and Sjögren and Birkhed [1993] observed an increase in F concentrations in saliva when the frequency and the amount of water were reduced. Sjögren and Melin [21] found a tendency towards an increased concentration of F in saliva and plaque when the amount of postbrushing water was reduced. The method of collecting proximal saliva with paper points has been used in several studies [Sjögren and Birkhed, 1994; Kashani et al., 1998; Särner et al., 23]. This sampling method was found to be reproducible according to our experience. Repeated sampling of proximal saliva within 5 min could easily be carried out since the paper points were inserted simultaneously into two quadrants. Oral motor activity influences the production of saliva and thereby the clearance. In order to eliminate this factor, it was considered important to avoid frequent talking during the collection of the samples. The method for sampling and analyzing F in plaque was originally described by Wilson and Ashley [1988]. Using TISAB III and distilled water for extracting the F as shown by Duckworth et al [1994] seems to release as much F from young plaque (24 h old) as perchloric acid. This method was also used by Sjögren et al. [1996] in a similar experiment. Some toxicological concern has been raised using high F toothpaste (5, ppm) [Ekstrand, 26]. Approximately 5 1% of the dentifrice is swallowed during tooth brushing with a toothpaste technique using a minimum amount of water [Sjögren and Birkhed, 1994]. The amount of swallowed fluoride is thereby.25.5 mg F, when using 1 g of a toothpaste containing 5 mg F/g. This quantity corresponds to the amount of F in 1 2 fluoride tablets containing.25 mg F and is considered to have no toxic effect in teenagers and adults. Children under 16 years should not use toothpaste with 5, ppm F according to instructions given by the manufacturer. Perhaps the age of 12 years would be a more appropriate age limit, in order for F to have an effect on the newly erupted premolars and second molars. Adults and teenagers with a high caries risk are a suitable target group for using a dentifrice with 5, ppm F. Adolescents in particular run a certain caries risk when their teeth have just erupted. A high F dentifrice has also been recommended for optimal caries prevention strategies during orthodontic treatment [Derks et al., 24]. Elderly people with hyposalivation due to medication or radiation are another interesting group to keep in mind. The reversal of root caries has already been documented by Baysan et al. [21], Lynch et al. [2] and Ekstrand et al. [28], using a toothpaste with 5, ppm F. To conclude, 5, ppm F toothpaste without postbrushing water resulted in the highest F concentration in both plaque and saliva and 1,45 ppm F toothpaste with 3! 1 ml postbrushing water in the lowest. The increase of F in both proximal saliva and plaque may be of clinical importance. Toothpaste with 5, ppm F, especially without, seems thereby to 68 Caries Res 29;43:64 69 Nordström/Birkhed

6 be a potentially important vehicle for caries prevention and treatment. Further long-term clinical studies are needed to evaluate the effect of 5, ppm F toothpaste on dental caries. A long-term clinical study on caries-active teenagers comparing 5, and 1,45 ppm F dentifrices is in progress at our department. Ac k n ow l e d g m e n t s We gratefully acknowledge the technical assistance of Ms. Ann-Britt Lundberg and the statistical assistance of Dr. Tommy Johnsson. This study was supported by grants from the Counties of Halland and Västra Götaland, the Swedish Patent Revenue Research Fund and the Institute of Odontology, University of Gothenburg. References Baysan A, Lynch E, Ellwood R, Davies R, Petersson L, Borsboom P: Reversal of primary root caries using dentifrices containing 5, and 1,1 ppm fluoride. Caries Res 21; 35: Birkeland JM: Fluoride content of dental plaque after brushing with a fluoride dentifrice. 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Adv Dent Res 1994; 8: Duckworth RM, Knoop DJM, Stephen KW: Effect of mouth- after tooth brushing with a dentifrice on human salivary fluoride levels. Caries Res 1991; 25: Ekstrand K: Tandpasta med 5, ppm fluorid (in Danish). Tandlægebladet 26; 11: Ekstrand K, Martignon S, Holm-Pedersen P: Development and evaluation of two root caries controlling programmes for home-based frail people older than 75 years. Gerodontology 28; 25: Englander HR, Keyes PH, Gestwicki M, Sultz HA: Clinical anticaries effects of repeated topical sodium fluoride applications by mouthpiece. J Am Dent Assoc 1967; 75: Glass RL: The first international conference on declining prevalence of dental caries. J Dent Res 1982; 61: Heijnsbroek M, Gerardu VAM, Buijs MJ, van Loveren C, ten Cate JM, Timmerman MF, van der Weijden GA: Increased salivary fluoride concentrations after post-brush fluoride not reflected in dental plaque. 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