Original Paper Caries Res 2015;49:394–400 DOI: 10.1159/000381960
Received: June 30, 2014 Acepted after revision: March 27, 2015 Published online: June 24, 2015
Effectiveness of a Toothpaste with Low Fluoride Content Combined with Trimetaphosphate on Dental Biofilm and Enamel Demineralization in situ Eliana M. Takeshita Marcelle Danelon Luciene P. Castro Kikue T. Sassaki Alberto C.B. Delbem Araçatuba Dental School, UNESP – Universidade Estadual Paulista, Araçatuba, Brazil
Abstract Objective: The aim of the present study was to evaluate in situ whether a toothpaste with low fluoride associated with sodium trimetaphosphate (TMP) would provide similar effect to that of a 1,100 ppm F toothpaste. Design: This crossover double-blind study consisted of 4 phases (14 days each), during which 10 volunteers wore oral appliances containing 4 enamel bovine blocks. The cariogenic challenge was performed by the application of a 20% sucrose solution (6×/day). The toothpaste treatments (2×/day) were: placebo, 500 ppm F, 500 ppm F plus 1% TMP, and 1,100 ppm F. At the end, enamel mineral loss and biofilm composition were analyzed. Results: The toothpaste with 500 ppm F plus 1% TMP showed the lowest mineral loss (p < 0.05). Regarding the fluoride and calcium concentrations in the enamel and in the biofilm, there were no significant differences between 500 ppm F plus 1% TMP, and 1,100 ppm F toothpastes (p > 0.569), but they were significantly different when compared to toothpaste with 500 ppm F (p < 0.050). Conclusion: The addition of 1% TMP to a low-fluoride toothpaste reduces enamel demineralization in situ similar to a 1,100 ppm F toothpaste. © 2015 S. Karger AG, Basel
© 2015 S. Karger AG, Basel 0008–6568/15/0494–0394$39.50/0 E-Mail [email protected] www.karger.com/cre
In the past decades, the use of fluoridated products has led to a decline in the prevalence of dental caries, mainly owing to the fluoridation of public water supplies and the use of toothpastes [Browne et al., 2005]. However, the use of fluoride toothpastes has led to an increase of dental fluorosis prevalence in some areas [Mascarenhas, 2000]. In this sense, the reduction of the fluoride concentration in toothpastes can play an important role in minimizing fluoride ingestion by children. However, a comprehensive review has confirmed the benefits of using a toothpaste only with fluoride concentrations of ≥1,000 ppm [Walsh et al., 2010]. In order to increase the anticaries effect of fluoride toothpastes, researchers have increased the concentration of fluoride or aimed at identifying other sources of fluoride that have superior efficacy to conventional sources [Biesbrock et al., 2001; Mensinkai et al., 2012]. Since the risk of fluorosis would increase in children using toothpastes with high fluoride concentration, fluoride in toothpaste could be partly replaced by alternative agents to reduce F exposure in a fluorosis-prone child population, while keeping the overall caries-preventive efficacy. One way to enhance the anticaries efficacy of toothpaste is to combine it with polyphosphates [Takeshita et al., 2009; Takeshita et al., 2011; Delbem et al., 2012; Amaral et al., 2013; Zaze et al., 2014]. A longitudinal study showed that fluoride intake by children using toothpaste with 500 ppm F associated Alberto C.B. Delbem Department of Pediatric Dentistry, Faculdade de Odontologia de Araçatuba, Universidade Estadual Paulista, Rua José Bonifácio 1193 Araçatuba, SP 16015-050 (Brazil) E-Mail adelbem @ pq.cnpq.br
Downloaded by: National Taiwan University 198.143.44.1 - 6/28/2015 9:03:50 PM
Key Words Biofilm · Fluoride · Ionic strength · Polyphosphates · Tooth demineralization
n = 160
a
Enamel block preparation
20% sucrose solution
Enamel block selections through surface hardness test
Dentifrice slurry (1:3, w/w)
Acrylic palatal appliance
b
c
Intraoral phase
h
After each phase
Fluoride, Ca and P content in enamel analysis
Surface hardness test after each phase
i
Cross-sectional hardness test
f
d
e
g
Biofilm collection and analysis
Fig. 1. a Enamel block selections through surface hardness test. b Acrylic palatal appliance preparation. c Intraoral phase. d After each phase, enamel blocks and biofilm were collected from each slot of palatal appliance. e Fluoride, Ca, P, and EPS content in bio-
film analysis. f Surface hardness test after each phase. g Enamel blocks were sectioned longitudinally. h Fluoride, Ca, and P content in enamel analysis in 1 of the halves of the enamel block. i Crosssectional hardness test in the other half of the enamel block.
to organic or inorganic phosphate is significantly reduced when compared to 1,100 ppm F toothpaste [Amaral et al., 2014]. Sodium trimetaphosphate (TMP) is a cyclic polyphosphate that adsorbs to enamel surfaces, thereby reducing enamel demineralization [Takeshita et al., 2009; Takeshita et al., 2011; Danelon et al., 2014] and changing the affinity between the enamel surface and salivary proteins [Nordbö and Rölla, 1972]. Takeshita et al. [2009] showed that the addition of TMP to low-fluoride toothpastes resulted in a similar effectiveness to that of a standard toothpaste (1,100 ppm F) in an in vitro study. They also showed that at concentrations >1%, TMP reduced the mineral loss and increased the fluoride content in enamel treated with low-fluoride toothpastes. In addition, this combination of fluoride and TMP promotes less demineralization in the underlying caries lesion in originally sound enamel [Takeshita et al., 2011]. Since the abovementioned studies were carried out using a pH cycling model, which has some limitations in predicting clinical effectiveness [Roberts, 1995], the results obtained should
not be considered definitive. Considering the lack of information about the effect of toothpastes with a low fluoride concentration supplemented with TMP on the biofilm composition as well as their influence on the enamel underneath the biofilm formed in situ, the aim of this study was to evaluate the effectiveness of such a toothpaste in an in situ model, comparing it to a standard toothpaste. The null hypothesis was that low-fluoride toothpaste associated to TMP would lead to a similar anticaries effect when compared to low-fluoride toothpaste without TMP.
Low-Fluoride Toothpaste with Trimetaphosphate
Caries Res 2015;49:394–400 DOI: 10.1159/000381960
Material and Methods Experimental Design This study was previously approved by the human ethical committee (protocol No. 2007/01367), and all participants read and signed informed consent statements prior to the study initiation. The experimental design is presented in figure 1. This crossover double-blinded study was performed in 4 phases of 14 days each. Ten volunteers, with ages ranging from 20 to 30 years, were selected
395
Downloaded by: National Taiwan University 198.143.44.1 - 6/28/2015 9:03:50 PM
Enamel blocks sectioned longitudinally
Enamel block collections
Palatal Appliance Preparation and Treatments The palatal appliance was prepared in acrylic resin (Jet, Articles Classic Odontológico, Sao Paulo, Brazil), and 4 enamel blocks were fixed in the region of the premolars and first molar, with a different device used in each phase of the experiment. In order to allow biofilm accumulation on the enamel blocks, a piece of plastic mesh was fixed to the acrylic appliance leaving a space of 1 mm from the block surface [Amaral et al., 2013]. To provide a cariogenic challenge, the volunteers were instructed to remove the device and drip 20% sucrose solution (Sucrose, Synth, Diadema, Brazil) [Paes Leme et al., 2004] on each enamel block 6×/day. Five minutes later, the device was reinserted into the mouth. Treatments with the toothpastes were performed 2×/day, during the volunteers’ habitual oral hygiene routine. The device was removed, and toothpaste slurries (toothpaste/deionized suspension, 1: 3 w/w) were dripped onto the block, which was kept outside the mouth for 1 min (ex vivo). A fresh suspension was prepared in the early morning of each day of the experiment for use throughout the day. The volunteers brushed their natural teeth 3×/day (after the main meals) with the same toothpaste that had been dripped onto the block during the experimental period [Paes Leme et al., 2004]. After this, the device was washed in deionized water and reinserted into the
396
Caries Res 2015;49:394–400 DOI: 10.1159/000381960
300 240 180 Sound enamel Placebo 500 ppm F 1,100 ppm F 500 TMP
120 60 0
0
55
110
165
220
275
330
Depth (μm)
Fig. 2. Cross-sectional hardness profiles (mean KHN, n = 10) at different depths (micrometers) in enamel blocks according to the toothpaste treatments.
mouth. The volunteers were instructed to remove the palatal appliance before drinking or eating. During a 7-day preexperimental period and washout periods (7 days between each phase), the volunteers brushed their teeth with a toothpaste without fluoride and TMP. The volunteers received all instructions before the initiation of the experiment. Hardness Analysis The enamel surface hardness was determined before and after each phase in each specimen by using a Shimadzu HMV-2000 microhardness tester (Shimadzu Corp., Kyoto, Japan) under a 25-g load for 10 s. At each enamel block, 5 indentations (baseline), spaced 100 μm from each other, were made in the center of the enamel block. After each phase, 5 indentations (surface hardness) were made, spaced 100 μm from the baseline indentations. For the cross-sectional hardness measurements, the enamel blocks were longitudinally sectioned through their center, embedded in acrylic resin and gradually polished. Three sequences of 8 indentations at different distances (10, 30, 50, 70, 90, 110, 220, and 330 μm) were made from the surface of the enamel in the central region spaced 100 μm from each other under a 25-g load for 10 s [Delbem et al., 2012]. Using the hardness values (KHN) obtained, the ΔKHN (KHN × μm) was calculated [Spiguel et al., 2009] (fig. 2). Fluoride, Ca and P in Enamel The enamel biopsy was performed according to the study by Weatherell et al. [1985], as modified by Alves et al. [2007]. The blocks were fixed to a mandrel and attached to a handpiece (N 270, Dabi-Atlante, Ribeirão Preto, Brazil) which was fixed to the top of a modified microscope. Self-adhesive polishing discs (13 mm of diameter) and 600 grit silicon carbide (Buehler, Lake Bluff, Ill., USA) were fixed to the bottom of polystyrene crystal tubes (J-10, Injeplast). A 50-μm deep layer was removed from each enamel block, and the fluoride was determined using a specific electrode
Takeshita/Danelon/Castro/Sassaki/ Delbem
Downloaded by: National Taiwan University 198.143.44.1 - 6/28/2015 9:03:50 PM
Toothpaste Formulation The experimental toothpastes were prepared in a laboratory and had the following ingredients: carboxymethylcellulose, sodium methyl-p-hydroxybenzoate, sodium saccharin, peppermint oil, glycerol, hydrated silica, sodium lauryl sulfate, and water [Takeshita et al., 2009]. The fluoride (NaF, Merck, Darmstadt, Germany) concentration in the experimental toothpaste was 500 ppm F, to which 1% TMP (Sigma-Aldrich Co., St. Louis, Mo., USA) was added. To compare and validate the results, the following toothpastes were also manufactured: placebo (without F and TMP), 500 ppm F, and 1,100 ppm F with the same formulation as described previously.
360
Hardness (KHN)
considering the criteria described previously by Delbem et al. [2010] and Vanichvatana and Auychai [2013]. The number of subjects was based on previous studies [Delbem et al., 2005; Amaral et al., 2013] considering the primary outcome from surface hardness and integrated subsurface hardness recovery analysis, the mean difference between the groups (45 and 1,700, respectively), standard deviation (28 and 1,000, respectively), an α error of 5%, and a β error of 20%. The subjects wore an acrylic palatal appliance with sound bovine enamel blocks (4 mm × 4 mm, n = 160) previously polished and selected using a surface hardness (baseline) test [330 up to 370 Knoop hardness number (KHN); p = 0.991]. Then, the specimens were allocated to 4 treatments: placebo (without F and TMP), toothpaste with 500 ppm F, toothpaste with 500 ppm F combined with 1% TMP (500 TMP), and toothpaste with 1,100 ppm F (positive control). After each phase of 14 days of cariogenic challenge, the biofilm was collected for the analysis of fluoride, calcium (Ca), phosphorus (P), and insoluble extracellular polysaccharides (EPS). In the enamel blocks, the surface hardness was assessed again. The blocks were sectioned longitudinally, and 1 of the halves was subjected to a cross-sectional hardness test to calculate the integrated loss of subsurface area (ΔKHN) of enamel. In the other half, fluoride, Ca, and P content in the enamel were determined.
Table 1. Means of the variables analyzed according to the toothpastes treatments (n = 10)
Variables
SH, KHN ΔKHN, KHN × μm Fluoride/enamel, μg/mm3 Ca/enamel, μg/mm3 P/enamel, μg/mm3 Fluoride/biofilm, mmol/kg Ca/biofilm, mmol/kg P/biofilm, mmol/kg EPS/biofilm, μg/g
Toothpastes placebo
500 ppm F
1,100 ppm F
500 TMP
117a (21) 7,167a (679) 0.58a (0.15) 787a (119) 230a (112) 1.04a (0.26) 56a (11) 169a (63) 53.6a (11.6)
244b (47) 4,717b (597) 0.84b (0.30) 794a (272) 235a (86) 3.66b (2.62) 127b (41) 186a (59) 38.2b (11.2)
301c (27) 3,207c (721) 1.31c (0.63) 1,160b (165) 338b (57) 7.96c (4.79) 236c (104) 273a (124) 20.6c (12.2)
324d (20) 3,027c (791) 1.26c (0.47) 1,039b (96) 323b (48) 6.20c (4.20) 183c (54) 219a, b (104) 23.1c (9.1)
Distinct superscript letters indicate statistical significance among the dentifrices in each analysis (Student-Newman-Keuls’s test; p
Received: June 30, 2014 Acepted after revision: March 27, 2015 Published online: June 24, 2015
Effectiveness of a Toothpaste with Low Fluoride Content Combined with Trimetaphosphate on Dental Biofilm and Enamel Demineralization in situ Eliana M. Takeshita Marcelle Danelon Luciene P. Castro Kikue T. Sassaki Alberto C.B. Delbem Araçatuba Dental School, UNESP – Universidade Estadual Paulista, Araçatuba, Brazil
Abstract Objective: The aim of the present study was to evaluate in situ whether a toothpaste with low fluoride associated with sodium trimetaphosphate (TMP) would provide similar effect to that of a 1,100 ppm F toothpaste. Design: This crossover double-blind study consisted of 4 phases (14 days each), during which 10 volunteers wore oral appliances containing 4 enamel bovine blocks. The cariogenic challenge was performed by the application of a 20% sucrose solution (6×/day). The toothpaste treatments (2×/day) were: placebo, 500 ppm F, 500 ppm F plus 1% TMP, and 1,100 ppm F. At the end, enamel mineral loss and biofilm composition were analyzed. Results: The toothpaste with 500 ppm F plus 1% TMP showed the lowest mineral loss (p < 0.05). Regarding the fluoride and calcium concentrations in the enamel and in the biofilm, there were no significant differences between 500 ppm F plus 1% TMP, and 1,100 ppm F toothpastes (p > 0.569), but they were significantly different when compared to toothpaste with 500 ppm F (p < 0.050). Conclusion: The addition of 1% TMP to a low-fluoride toothpaste reduces enamel demineralization in situ similar to a 1,100 ppm F toothpaste. © 2015 S. Karger AG, Basel
© 2015 S. Karger AG, Basel 0008–6568/15/0494–0394$39.50/0 E-Mail [email protected] www.karger.com/cre
In the past decades, the use of fluoridated products has led to a decline in the prevalence of dental caries, mainly owing to the fluoridation of public water supplies and the use of toothpastes [Browne et al., 2005]. However, the use of fluoride toothpastes has led to an increase of dental fluorosis prevalence in some areas [Mascarenhas, 2000]. In this sense, the reduction of the fluoride concentration in toothpastes can play an important role in minimizing fluoride ingestion by children. However, a comprehensive review has confirmed the benefits of using a toothpaste only with fluoride concentrations of ≥1,000 ppm [Walsh et al., 2010]. In order to increase the anticaries effect of fluoride toothpastes, researchers have increased the concentration of fluoride or aimed at identifying other sources of fluoride that have superior efficacy to conventional sources [Biesbrock et al., 2001; Mensinkai et al., 2012]. Since the risk of fluorosis would increase in children using toothpastes with high fluoride concentration, fluoride in toothpaste could be partly replaced by alternative agents to reduce F exposure in a fluorosis-prone child population, while keeping the overall caries-preventive efficacy. One way to enhance the anticaries efficacy of toothpaste is to combine it with polyphosphates [Takeshita et al., 2009; Takeshita et al., 2011; Delbem et al., 2012; Amaral et al., 2013; Zaze et al., 2014]. A longitudinal study showed that fluoride intake by children using toothpaste with 500 ppm F associated Alberto C.B. Delbem Department of Pediatric Dentistry, Faculdade de Odontologia de Araçatuba, Universidade Estadual Paulista, Rua José Bonifácio 1193 Araçatuba, SP 16015-050 (Brazil) E-Mail adelbem @ pq.cnpq.br
Downloaded by: National Taiwan University 198.143.44.1 - 6/28/2015 9:03:50 PM
Key Words Biofilm · Fluoride · Ionic strength · Polyphosphates · Tooth demineralization
n = 160
a
Enamel block preparation
20% sucrose solution
Enamel block selections through surface hardness test
Dentifrice slurry (1:3, w/w)
Acrylic palatal appliance
b
c
Intraoral phase
h
After each phase
Fluoride, Ca and P content in enamel analysis
Surface hardness test after each phase
i
Cross-sectional hardness test
f
d
e
g
Biofilm collection and analysis
Fig. 1. a Enamel block selections through surface hardness test. b Acrylic palatal appliance preparation. c Intraoral phase. d After each phase, enamel blocks and biofilm were collected from each slot of palatal appliance. e Fluoride, Ca, P, and EPS content in bio-
film analysis. f Surface hardness test after each phase. g Enamel blocks were sectioned longitudinally. h Fluoride, Ca, and P content in enamel analysis in 1 of the halves of the enamel block. i Crosssectional hardness test in the other half of the enamel block.
to organic or inorganic phosphate is significantly reduced when compared to 1,100 ppm F toothpaste [Amaral et al., 2014]. Sodium trimetaphosphate (TMP) is a cyclic polyphosphate that adsorbs to enamel surfaces, thereby reducing enamel demineralization [Takeshita et al., 2009; Takeshita et al., 2011; Danelon et al., 2014] and changing the affinity between the enamel surface and salivary proteins [Nordbö and Rölla, 1972]. Takeshita et al. [2009] showed that the addition of TMP to low-fluoride toothpastes resulted in a similar effectiveness to that of a standard toothpaste (1,100 ppm F) in an in vitro study. They also showed that at concentrations >1%, TMP reduced the mineral loss and increased the fluoride content in enamel treated with low-fluoride toothpastes. In addition, this combination of fluoride and TMP promotes less demineralization in the underlying caries lesion in originally sound enamel [Takeshita et al., 2011]. Since the abovementioned studies were carried out using a pH cycling model, which has some limitations in predicting clinical effectiveness [Roberts, 1995], the results obtained should
not be considered definitive. Considering the lack of information about the effect of toothpastes with a low fluoride concentration supplemented with TMP on the biofilm composition as well as their influence on the enamel underneath the biofilm formed in situ, the aim of this study was to evaluate the effectiveness of such a toothpaste in an in situ model, comparing it to a standard toothpaste. The null hypothesis was that low-fluoride toothpaste associated to TMP would lead to a similar anticaries effect when compared to low-fluoride toothpaste without TMP.
Low-Fluoride Toothpaste with Trimetaphosphate
Caries Res 2015;49:394–400 DOI: 10.1159/000381960
Material and Methods Experimental Design This study was previously approved by the human ethical committee (protocol No. 2007/01367), and all participants read and signed informed consent statements prior to the study initiation. The experimental design is presented in figure 1. This crossover double-blinded study was performed in 4 phases of 14 days each. Ten volunteers, with ages ranging from 20 to 30 years, were selected
395
Downloaded by: National Taiwan University 198.143.44.1 - 6/28/2015 9:03:50 PM
Enamel blocks sectioned longitudinally
Enamel block collections
Palatal Appliance Preparation and Treatments The palatal appliance was prepared in acrylic resin (Jet, Articles Classic Odontológico, Sao Paulo, Brazil), and 4 enamel blocks were fixed in the region of the premolars and first molar, with a different device used in each phase of the experiment. In order to allow biofilm accumulation on the enamel blocks, a piece of plastic mesh was fixed to the acrylic appliance leaving a space of 1 mm from the block surface [Amaral et al., 2013]. To provide a cariogenic challenge, the volunteers were instructed to remove the device and drip 20% sucrose solution (Sucrose, Synth, Diadema, Brazil) [Paes Leme et al., 2004] on each enamel block 6×/day. Five minutes later, the device was reinserted into the mouth. Treatments with the toothpastes were performed 2×/day, during the volunteers’ habitual oral hygiene routine. The device was removed, and toothpaste slurries (toothpaste/deionized suspension, 1: 3 w/w) were dripped onto the block, which was kept outside the mouth for 1 min (ex vivo). A fresh suspension was prepared in the early morning of each day of the experiment for use throughout the day. The volunteers brushed their natural teeth 3×/day (after the main meals) with the same toothpaste that had been dripped onto the block during the experimental period [Paes Leme et al., 2004]. After this, the device was washed in deionized water and reinserted into the
396
Caries Res 2015;49:394–400 DOI: 10.1159/000381960
300 240 180 Sound enamel Placebo 500 ppm F 1,100 ppm F 500 TMP
120 60 0
0
55
110
165
220
275
330
Depth (μm)
Fig. 2. Cross-sectional hardness profiles (mean KHN, n = 10) at different depths (micrometers) in enamel blocks according to the toothpaste treatments.
mouth. The volunteers were instructed to remove the palatal appliance before drinking or eating. During a 7-day preexperimental period and washout periods (7 days between each phase), the volunteers brushed their teeth with a toothpaste without fluoride and TMP. The volunteers received all instructions before the initiation of the experiment. Hardness Analysis The enamel surface hardness was determined before and after each phase in each specimen by using a Shimadzu HMV-2000 microhardness tester (Shimadzu Corp., Kyoto, Japan) under a 25-g load for 10 s. At each enamel block, 5 indentations (baseline), spaced 100 μm from each other, were made in the center of the enamel block. After each phase, 5 indentations (surface hardness) were made, spaced 100 μm from the baseline indentations. For the cross-sectional hardness measurements, the enamel blocks were longitudinally sectioned through their center, embedded in acrylic resin and gradually polished. Three sequences of 8 indentations at different distances (10, 30, 50, 70, 90, 110, 220, and 330 μm) were made from the surface of the enamel in the central region spaced 100 μm from each other under a 25-g load for 10 s [Delbem et al., 2012]. Using the hardness values (KHN) obtained, the ΔKHN (KHN × μm) was calculated [Spiguel et al., 2009] (fig. 2). Fluoride, Ca and P in Enamel The enamel biopsy was performed according to the study by Weatherell et al. [1985], as modified by Alves et al. [2007]. The blocks were fixed to a mandrel and attached to a handpiece (N 270, Dabi-Atlante, Ribeirão Preto, Brazil) which was fixed to the top of a modified microscope. Self-adhesive polishing discs (13 mm of diameter) and 600 grit silicon carbide (Buehler, Lake Bluff, Ill., USA) were fixed to the bottom of polystyrene crystal tubes (J-10, Injeplast). A 50-μm deep layer was removed from each enamel block, and the fluoride was determined using a specific electrode
Takeshita/Danelon/Castro/Sassaki/ Delbem
Downloaded by: National Taiwan University 198.143.44.1 - 6/28/2015 9:03:50 PM
Toothpaste Formulation The experimental toothpastes were prepared in a laboratory and had the following ingredients: carboxymethylcellulose, sodium methyl-p-hydroxybenzoate, sodium saccharin, peppermint oil, glycerol, hydrated silica, sodium lauryl sulfate, and water [Takeshita et al., 2009]. The fluoride (NaF, Merck, Darmstadt, Germany) concentration in the experimental toothpaste was 500 ppm F, to which 1% TMP (Sigma-Aldrich Co., St. Louis, Mo., USA) was added. To compare and validate the results, the following toothpastes were also manufactured: placebo (without F and TMP), 500 ppm F, and 1,100 ppm F with the same formulation as described previously.
360
Hardness (KHN)
considering the criteria described previously by Delbem et al. [2010] and Vanichvatana and Auychai [2013]. The number of subjects was based on previous studies [Delbem et al., 2005; Amaral et al., 2013] considering the primary outcome from surface hardness and integrated subsurface hardness recovery analysis, the mean difference between the groups (45 and 1,700, respectively), standard deviation (28 and 1,000, respectively), an α error of 5%, and a β error of 20%. The subjects wore an acrylic palatal appliance with sound bovine enamel blocks (4 mm × 4 mm, n = 160) previously polished and selected using a surface hardness (baseline) test [330 up to 370 Knoop hardness number (KHN); p = 0.991]. Then, the specimens were allocated to 4 treatments: placebo (without F and TMP), toothpaste with 500 ppm F, toothpaste with 500 ppm F combined with 1% TMP (500 TMP), and toothpaste with 1,100 ppm F (positive control). After each phase of 14 days of cariogenic challenge, the biofilm was collected for the analysis of fluoride, calcium (Ca), phosphorus (P), and insoluble extracellular polysaccharides (EPS). In the enamel blocks, the surface hardness was assessed again. The blocks were sectioned longitudinally, and 1 of the halves was subjected to a cross-sectional hardness test to calculate the integrated loss of subsurface area (ΔKHN) of enamel. In the other half, fluoride, Ca, and P content in the enamel were determined.
Table 1. Means of the variables analyzed according to the toothpastes treatments (n = 10)
Variables
SH, KHN ΔKHN, KHN × μm Fluoride/enamel, μg/mm3 Ca/enamel, μg/mm3 P/enamel, μg/mm3 Fluoride/biofilm, mmol/kg Ca/biofilm, mmol/kg P/biofilm, mmol/kg EPS/biofilm, μg/g
Toothpastes placebo
500 ppm F
1,100 ppm F
500 TMP
117a (21) 7,167a (679) 0.58a (0.15) 787a (119) 230a (112) 1.04a (0.26) 56a (11) 169a (63) 53.6a (11.6)
244b (47) 4,717b (597) 0.84b (0.30) 794a (272) 235a (86) 3.66b (2.62) 127b (41) 186a (59) 38.2b (11.2)
301c (27) 3,207c (721) 1.31c (0.63) 1,160b (165) 338b (57) 7.96c (4.79) 236c (104) 273a (124) 20.6c (12.2)
324d (20) 3,027c (791) 1.26c (0.47) 1,039b (96) 323b (48) 6.20c (4.20) 183c (54) 219a, b (104) 23.1c (9.1)
Distinct superscript letters indicate statistical significance among the dentifrices in each analysis (Student-Newman-Keuls’s test; p
