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Relationship between fluoride release rate and anti-cariogenic biofilm activity of glass ionomer cements Ngoc Phuong Thanh Chau a,b , Santosh Pandit a , Jian-Na Cai a , Min-Ho Lee c,∗∗ , Jae-Gyu Jeon a,∗ a
Department of Preventive Dentistry, School of Dentistry, Institute of Oral Bioscience and BK 21 Plus Program, Chonbuk National University, Jeonju 561-756, Republic of Korea b Department of Odonto-Stomatology, Hue University of Medicine and Pharmacy, Hue University, 06 Ngo Quyen street, Hue City, Viet Nam c Department of Dental Biomaterials, School of Dentistry, Chonbuk National University, Jeonju, Republic of Korea
a r t i c l e
i n f o
a b s t r a c t
Article history:
Objectives. The aim of this study was to evaluate acidogenicity and composition of Streptococ-
Received 6 August 2014
cus mutans biofilms on glass ionomer cements (GICs) and then to determine the relationship
Received in revised form
between the anti-S. mutans biofilm activity and fluoride release rate of the GICs.
16 October 2014
Methods. S. mutans biofilms were formed on discs prepared using five commercial GICs. Acid
Accepted 19 December 2014
production and fluoride release rates of the biofilms on the GIC discs during biofilm forma-
Available online xxx
tion (0–94 h) were determined. Next, 94-h-old S. mutans biofilms on GIC discs were analyzed to evaluate the biofilm composition (dry weight, bacterial cell number, and extra-cellular
Keywords:
polysaccharide (EPS) amount) using microbiological, biochemical, and confocal laser scan-
Glass ionomer cements
ning microscopy (CLSM) methods. Lastly, relationships between the fluoride release rate
Fluoride release
and changes in acidogenicity and composition of the biofilms were determined using a
Cariogenic biofilms
linear-fitting procedure.
Acidogenicity
Results. All of the tested GICs released fluoride ions. Of the GICs, the two that showed the
Biofilm composition
highest fluoride release rates strongly affected acidogenicity, dry weight, and EPS forma-
Relationship
tion of the biofilms. Furthermore, they reduced the bacterial and EPS bio-volumes and EPS thickness. However, the number of colony forming units (CFUs) of the biofilms was higher than that of the control. Generally, changes in the acidogenicity and composition (except for CFU count) of the biofilms on the GICs followed a negative linear-pattern of fluoride release rate-dependence (R = −0.850 to −0.995, R2 = 0.723–0.990). Significance. These results suggest that the anti-cariogenic biofilm activity of GICs is closely correlated with their fluoride release rate during biofilm formation. © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
∗ ∗∗
Corresponding author. Tel.: +82 63 270 4036. Corresponding author. E-mail addresses: [email protected] (M.-H. Lee), [email protected] (J.-G. Jeon).
http://dx.doi.org/10.1016/j.dental.2014.12.016 0109-5641/© 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
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1.
Introduction
It has been well documented that biofilm formation occurs on the surfaces of different restorative materials within a short time after placement in the oral cavity [1,2]. The biofilms on the microgaps between restorative materials and tooth tissues can lead to secondary caries, which is responsible for half of all restorations fail within 10 years [3]. Similar to the dental caries process, the biofilms on these microgaps cause dissolution of the adjacent tooth surfaces and reduce the longevity of the restorative materials [4]. Although several studies have shown that the level of mutans streptococci is not necessarily related to the development of dental caries [5,6], many other studies have revealed Streptococcus mutans as one of the main causative pathogens for dental caries [7,8]. S. mutans can adhere to and accumulate on the tooth surface using extra-cellular polysaccharides (EPSs) produced by glucosyltransferases (GTFs) from sucrose [9]. Furthermore, this bacterium can metabolize dietary sugars to organic acids and withstand rapid and substantial fluctuations in the environmental pH [10]. The combination of these virulence properties allows S. mutans to effectively colonize the tooth surface and be sustained in the oral cavity. To prevent secondary caries, fluoride-releasing restorative materials such as glass ionomer cements (GICs) and resin composites have been developed. Recently, several researchers have shown that nanocomposites containing calcium phosphate nanoparticles may reduce secondary caries [11]. Of the fluoride-releasing restorative materials, GICs have been used in various areas of restorative dentistry for several decades because of their biocompatibility and cariostatic properties [12,13]. Furthermore, GICs can reduce demineralization in adjacent hard tooth tissues [14,15]. Several studies have shown that the effectiveness of GICs may be related to their ability to release fluoride [13,16,17]. Biofilms on GICs can degrade material properties and roughen their surfaces, which in turn promote further biofilm formation, as well as material surface deterioration [18,19]. Recently, several studies have investigated bacterial adherence to and biofilm formation on GICs and have shown that these materials can affect bacterial adherence, acidigenicity, and biofilm formation [13,20]. However, although it is important to obtain information about whether GICs can affect composition of biofilms on GICs, little has been reported. Furthermore, there has been no study on the precise relationship between fluoride release level from GICs and changes in virulence and composition of biofilms on GICs. Considering the advantages and widespread use of GICs in restorative dentistry, it would be worthwhile to test the hypotheses that fluoride level released from GICs can change virulence and composition of cariogenic biofilms. Therefore, the aim of this study was to investigate the changes in virulence, especially acidogenicity, and composition of cariogenic biofilms formed on GICs and then to determine the relationships between fluoride release level from GICs and changes in acidogenicity and composition of cariogenic biofilms using an S. mutans biofilm model.
2.
Materials and methods
2.1.
GIC disk preparation
Table 1 lists the materials used in this study. Five GICs were selected: Glaslonomer FX-II (Gla), Ketac Fil Plus Aplicap (Keta), Riva self-cure HV (Riva), GC Fuji Filling LC (GC), and GC Fuji II LC (GC2). The GICs used in this study were all commercially available, and the shade of all materials was A2 or A3. These GICs were used to prepare disk-shaped specimens according to the manufacturer’s recommendations. Disk-shaped specimens (12 mm in diameter and 1.2 mm in thickness) were prepared using polytetrafluoroethylene (Teflon) molds with a metal holder and glass slides to cover each face. Gla, Keta, and Riva specimens were self-cured. GC and GC2 specimens were light-cured for 20 s on each face using a light curing unit (G-Light, GC Corp., Japan). After curing, all specimens were polished sequentially with # 800 to # 1200 sand papers. Then the specimens were placed in a desiccator at room temperature. Hydroxyapatite discs (12 mm in diameter and 1.2 mm in thickness; Clarkson Chromatography Products, Inc., South Williamsport, PA, USA) were included as a control in this study.
2.2.
Biofilm formation on GIC discs
The microorganism used for this study was S. mutans UA159 (serotype c). S. mutans biofilms were formed on saliva-coated GIC or hydroxyapatite (HA) discs placed in a vertical position in 24-well plates, as detailed elsewhere [21]. Briefly, the saliva-coated discs were generated by incubation with filtersterilized (0.22 m low protein-binding filter) human whole saliva for 1 h at 37 ◦ C. For biofilm formation, the saliva-coated discs were transferred to a 24-well plate containing 1% sucrose (v/v) ultrafiltered (10 kDa molecular-weight cut-off) tryptone yeast-extract (UTE) broth with S. mutans UA159 (2–5 × 106 colony forming units (CFUs)/ml). The biofilms were grown undisturbed for 22 h to allow initial biofilm growth. From this time point (22 h), the culture medium was changed twice daily (9 AM, 6 PM) until it was 94 h old. The culture medium was changed a total of six times. The pH value and fluoride concentration in the old culture medium were determined during the experimental period (until biofilms reached 94 h of age). The 94-h-old biofilms were used to analyze the biofilm composition. Each assay was performed in duplicate in at least six different experiments (n = 12).
2.3. Determination of fluoride concentration and pH value during biofilm formation To evaluate fluoride release from GICs, the concentration of fluoride in the old culture medium was determined during the experimental period (22, 31, 46, 55, 70, 79, and 94 h). For the determination of fluoride concentration, a total of 2.8 ml of each medium was mixed with 280 l of total ionic strength adjustment buffer (TISAB III). The fluorometer (Thermo Fisher Scientific, Orion, MA, USA) was calibrated using four standard solutions (0.1, 1, 10, and 100 ppm F− ). To evaluate the acidogenicity of S. mutans biofilms on GICs, the pH values of the old
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Table 1 – Characteristics of glass ionomer cements used in this study. Product name
Curing
Composition (wt.%)
Manufacturer
Glaslonomer FX-II
Self-cured
- Glass polyalkenoate. - Powder (15 g), liquid (8 ml, 10 g).
Shofu, Inc., Japan
KetacTM Fil Plus AplicapTM
Self-cured
- Glass powder (>99%).
3 M ESPE, Dental Products, Germany
- Liquid: copolymer of acrylic acid-maleic acid (35–55%), water (40–55%), tartaric acid (5–10%). Riva self-cure HV
Self-cured
- Polyacrylic acid (20–30%). - Tartaric acid (10–15%). - Fluoroaluminosilicate glass (90–95%). - Polyacrylic acid (5–10%).
SDI Limited, Australia
GC Fuji Filling LC
Light-cured
- Paste A: Alumino-fluoro-silicate glass (amorphous) (75–85%); 2-hydroxyethyl methacrylate (10–12%); Urethanedimethacrylate (2–5%). - Paste B: Distilled water (20–30%); polyacrylic acid (20–30%); urethanedimethacrylate (12–15%); silicone dioxide (fumed/amorphous) (10–15%).
GC corporation, Japan
GC Fuji II LC (improved)
Light-cured
- Powder: Alumino-fluoro-silicate glass (95–100%). - Liquid: Polyacrylic acid (20–25%); 2-hydroxyethyl methacrylate (30–35%); proprietary ingredient (5–15%); 2, 2, 4, trimethylhexamethylenedicarbonate (1–5%).
GC corporation, Japan
This is based on information provided by safety data sheet of the products.
culture medium were also determined during the experimental period (22, 31, 46, 55, 70, 79, and 94 h) using a pH electrode. To calculate acid production or fluoride release rates at 0–22, 22–31, 31–46, 46–55, 55–70, 70–79, and 79–94 h, the concentrations of H+ or fluoride in the old culture medium was divided by the respective incubation time. In addition, the mean rates of acid production and fluoride release during biofilm formation (0–94 h) were also calculated using the respective acid production or fluoride release rates (0–22, 22–31, 31–46, 46–55, 55–70, 70–79, and 79–94 h).
2.4. Analysis of 94-h-old S. mutans biofilm composition 2.4.1.
Microbiological and biochemical analyses
The 94-h-old biofilms on the test materials were transferred into 2 ml of 0.89% NaCl and sonicated in an ultrasonic bath for 10 min to disperse the biofilms. The dispersed solution was re-sonicated at 7 W for 30 s after adding 3 ml of 0.89% NaCl (VCX 130PB; Sonics and Materials, Inc., Newtown, CT, USA). An aliquot (0.1 ml) of the dispersed solution was serially diluted and plated onto brain heart infusion (BHI) agar plates to count the CFUs. For the determination of the dry weight and amount of water-insoluble extracellular polysaccharides (water-insoluble EPSs), the remaining solution (4.9 ml) was centrifuged (3000 × g) for 20 min at 4 ◦ C. The biofilm pellet was resuspended and washed twice in the same volume of water. The washed pellet was lyophilized and weighed to determine the dry weight. After weighing, the water-insoluble EPSs were extracted from the dry pellet using 1 N sodium hydroxide before determination of polysaccharide amount, as detailed elsewhere [22].
2.4.2.
Confocal laser scanning microscopy study
In addition to the microbiological and biochemical analyses, a confocal laser scanning microscopy (CLSM) study was performed to evaluate bacterial cells and EPSs of the 94-h-old biofilms on GICs, as described by Chau et al. [23]. Briefly, Alexa Fluor® 647-labeled dextran conjugate (10,000 MW; absorbance/fluorescence emission maxima 647/668 nm; Molecular Probes, Inc., Eugene, OR, USA) was added to the culture medium during biofilm formation at a concentration of 1 M. After 94 h, the bacterial cells in the biofilms were labeled by means of 25 M SYTO® 9 green-fluorescent nucleic acid stain (480/500 nm; Molecular Probes, Inc., Eugene, OR, USA) for 30 min [24]. CLSM imaging of the biofilms was performed using the LSM 510 META microscope (Carl Zeiss, Jena, Germany) equipped with argon-ion and helium–neon lasers. Two independent experiments were performed, and seven image stacks (512 × 512 pixel tagged image file format) per experiment were collected. The biofilms were quantified from the confocal stacks using the image-processing software COMSTAT [25]. This software was written as a script in Matlab 5.1 (The MathWorks, Natick, MA, USA) equipped with the Image Processing Toolbox. In this study, the bio-volume and thickness of bacteria and polysaccharides were analyzed. Bio-volume is defined as the volume of the biomass (m3 ) divided by the surface area of the substratum (GIC or hydroxyapatite discs) (m2 ).
2.5.
Statistical analysis
The data are presented as mean ± standard deviation. The intergroup differences were estimated using one-way
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Fig. 1 – (A) Fluoride release rate, (B) acid production rate, and (C) linear-fitting of mean rate of fluoride release versus mean rate of acid production during Streptococcus mutans biofilm formation on glass ionomer discs (0–94 h). Control, hydroxyapatite; Gla, Glaslonomer FX-II; Keta, Ketac Fil PlusAplicap; Riva, Riva self-cure HV; GC, GC Fuji Filling LC; GC2, GC Fuji II LC.
analysis of variance (ANOVA). Values were considered statistically significant when the p value was 0.05). As shown in Fig. 2D, the dry weight of the 94-h-old biofilms on the GICs was negatively correlated with the mean rate of fluoride release (0–94 h) from the GICs (p < 0.05). The R and R2 of the fit line were −0.922 and 0.849, respectively. Interestingly, although all of the dry weights of the 94h-old biofilms on the GICs, except for GC2, were lower than that of the control, and the numbers of bacterial CFUs of the biofilms were higher than that of the control (p < 0.05) (Fig. 2B). As shown in Fig. 2E, the number of bacterial CFUs of the 94h-old biofilms was positively correlated with the mean rate of fluoride release (0–94 h) from the GICs (R = 0.869, R2 = 0.755) (p < 0.05). In this study, we also evaluated the amount of waterinsoluble EPSs of the 94-h-old biofilms on GICs. As shown in Fig. 2C, all of the amounts of water-insoluble EPSs of the GICs were lower than that of the control (p < 0.05). Especially, the amount of water-insoluble EPSs on Keta, GC, and Gla were reduced by up to 46, 40, and 30%, respectively, compared to the vehicle control (p < 0.05). As shown in Fig. 2F, the amount of water-insoluble EPSs of the 94-h-old biofilms was negatively correlated with the mean rate of fluoride release (0–94 h) from
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Fig. 2 – (A) Dry weight, (B) colony forming unit (CFU), and (C) water-insoluble polysaccharide (WIP) of 94-h-old Streptococcus mutans biofilms on glass ionomer (GI) discs. Linear-fitting of (D) dry weight, (E) CFU, (F) and WIP of the 94-h-old biofilms versus mean rate of fluoride release of GI discs during biofilm formation (0–94 h). Control, hydroxyapatite; Gla, Glaslonomer FX-II; Keta, Ketac Fil PlusAplicap; Riva, Riva self-cure HV; GC, GC Fuji Filling LC; GC2, GC Fuji II LC. *p < 0.05: significantly different from the control.
the GICs (p < 0.05). The R and R2 of the fit line were −0.853 and 0.728, respectively.
3.3. Composition changes of 94-h-old S. mutans biofilms in CLSM study and their relationship with fluoride release rate Differences in compositions of 94-h-old S. mutans biofilms on GICs were confirmed by CLSM image analysis. As shown in Fig. 3A and B, although all of the GICs tested in this study were fluoride-releasing materials (Fig. 1A), only Keta and GC affected the bacterial bio-volume and thickness of the 94-hold biofilms. Of the GICs, Keta had the most noticeable effects, with bacterial bio-volume and thickness being reduced up to 41 and 46%, respectively, compared to the vehicle control (p < 0.05). As shown in Fig. 3C, the bacterial bio-volume of the 94-h-old biofilms on the GICs was negatively correlated with the mean rate of fluoride release (0–94 h) (p < 0.05). The R and R2 of the fit line were −0.850 and 0.723, respectively. Fig. 3D shows representative bacterial images of the 94-h-old biofilms on the control (Fig. 3D-1), Keta (Fig. 3D-2), and GC (Fig. 3D-3). As shown in Fig. 3D-2 and D-3, the total volumes of the biofilm cells on Keta and GC were reduced as compared to that of the control (Fig. 3D-1). In addition to bacterial bio-volume, EPS bio-volumes and thicknesses on GICs were analyzed. As shown in Fig. 4A and
B, all of the GICs tested in this study, except for GC2, reduced the EPS bio-volume and thickness of the 94-h-old biofilms. Of the GICs tested, Keta had the most strongly reduced EPS bio-volume and thickness, with reductions of up to 69 and 38%, respectively, compared to the vehicle control (p < 0.05). As shown in Fig. 4C, the EPS bio-volume and thickness of the 94-h-old biofilms on the GICs were negatively correlated with the mean rate of fluoride release (0–94 h) (p < 0.05). The R (R2 ) of the fit line of the EPS bio-volume and thickness versus the mean rate of fluoride release was −0.995 (0.990) and −0.916 (0.839), respectively. Fig. 4D shows representative EPS images of the 94-h-old biofilms on the control (Fig. 4D-1), Keta (Fig. 4D2), and GC (Fig. 4D-3). As shown in Fig. 4D-2 and D-3, the total volumes of EPS on Keta and GC were reduced as compared to that of the control (Fig. 4D-1).
4.
Discussion
Secondary caries, like dental caries, is a biofilm-related disease associated with the increased consumption of dietary sugar [26,27]. Thus, it is possible for any site on a restored tooth that is prone to cariogenic biofilm formation to develop secondary caries. In this study, we investigated acidogenicity and composition of a cariogenic biofilm on GICs. Furthermore, we determined the relationship between anti-cariogenic biofilm
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Fig. 3 – (A) Bacterial bio-volume and (B) thickness of 94-h-old Streptococcus mutans biofilms on glass ionomer (GI) discs in a confocal laser scanning microscopy (CLSM) study. (C) Linear-fitting of mean rates of fluoride release of GI discs during biofilm formation (0–94 h) versus bacterial bio-volume of the 94-h-old biofilms. Representative CLSM images of bacteria from the 94-h-old biofilms on (D-1) Control (hydroxyapatite), (D-2)Keta (Ketac Fil PlusAplicap), and (D-3) GC (GC Fuji Filling LC). Gla, Glaslonomer FX-II; Riva, Riva self-cure HV; GC2, GC Fuji II LC. *p < 0.05: significantly different from the control.
activity of GICs and their fluoride release rate since (i) it has been well demonstrated that fluoride ions, even in low concentrations, can affect the virulence of cariogenic biofilms [21], and (ii) many GICs release fluoride ions into dental biofilms and teeth [17,28,29]. In this study, we used an S. mutans biofilm model to study acidogenicity and composition of cariogenic biofilms. Although the S. mutans biofilm model does not precisely mimic the complex microbial community of cariogenic biofilms, the monospecies biofilm model is advantageous in examining virulence (acidogenicity and EPS formation) of S. mutans, an important pathogen of dental caries, in biofilms. In the study, we performed a linear-fitting procedure (linear regression analysis) to investigate the relationship between anti-cariogenic biofilm activity of GICs and their fluoride release rate. It is well accepted that linear regression analysis is useful for evaluating the linear relationship between two variables and to determine how much of the variation in one variable can be explained by the linear relationship with another variable [30]. The R of the fit line represents the strength of the relationship between two variables, and the R2 demonstrates the percentage of the variation in one variable that can be explained by the linear relationship with another variable [30]. The most relevant virulence factor of cariogenic biofilms in dissolving the hard tooth surfaces is acidogenicity. Recently,
several studies have examined the effects of fluoride-releasing materials on acidogenicity of cariogenic bacteria [13,21,29]. Some investigations demonstrated that fluoride-releasing materials can affect the acidogenicity of cariogenic biofilms. However, studies that show a relationship between fluoride concentration released from restorative materials and acidogenicity of cariogenic biofilms are rare. In this study, our data clearly showed that the acid production rate of S. mutans biofilms on GICs followed a negative linear-pattern of fluoride release rate-dependence during biofilm formation (Fig. 1C), suggesting that acid production of biofilms decreases as fluoride release rate increases during biofilm formation. As shown in Fig. 1C, the R of the fit line was −0.962, suggesting that acid production rate is strongly and negatively correlated with fluoride release rate. However, because R only shows the strength of the relationship between two variables, R2 was also calculated. For the fit line, R2 was 0.926, suggesting that the line may appropriately describe the change in acid production rate of biofilms according to the fluoride release rate, and that at least 92.6% of the variation in acid production rate may be explained by the fluoride release rate. It has been well reported that the release of fluoride from restorative materials is a very complex process, and can be affected by intrinsic variables, i.e. fillers and formulations, and experimental factors, i.e. storage media, frequency of storage
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Fig. 4 – (A) Extracellular polysaccharides (EPSs) bio-volume and (B) thickness of 94-h-old Streptococcus mutans biofilms on glass ionomer (GI) discs in a confocal laser scanning microscopy (CLSM) study. (C) Linear-fitting of mean rates of fluoride release of GI discs during biofilm formation (0–94 h) versus the EPS bio-volume and thickness of the 94-h-old biofilms. Representative CLSM images of EPSs from the 94-h-old biofilms on (D-1) Control (hydroxyapatite), (D-2) Keta (Ketac Fil PlusAplicap), and (D-3) GC (GC Fuji Filling LC). Gla, Glaslonomer FX-II; Riva, Riva self-cure HV; GC2, GC Fuji II LC. *p < 0.05: significantly different from the control.
medium change [17]. Furthermore, several studies have shown that lower pH conditions increase fluoride release for both conventional and resin-modified glass ionomer cements, and glass ionomers can buffer acids [31,32]. Therefore, a more sophisticated and well-designed study will be needed to reveal whether pH change in biofilms can increase fluoride release and the buffer activity of glass ionomers can influence pH change in biofilms. In this study, relationships between fluoride release rate and dry weight and water-insoluble EPS amount of S. mutans biofilms were also investigated. Our results showed that the dry weight and water-insoluble EPS amount of the 94-h-old biofilms are negatively correlated with the fluoride release rate of the GICs during biofilm formation (Fig. 2D and F). As shown in Fig. 2D and F, the Rs of the fit lines were −0.922 and −0.853, suggesting that dry weight and amount of water-insoluble EPSs of S. mutans biofilms are strongly and negatively correlated with fluoride release rate of GICs. The R2 s of the fit lines were 0.849 and 0.728, suggesting that at least 84.9 and 72.8% of the variation in the dry weight and amount of water-insoluble EPSs, respectively, may be explained by the fluoride release rate during biofilm formation. Generally, our data confirmed a previous finding that the dry weights of dental biofilms on
dental restorative materials are reduced as the rate of fluoride release from the materials is increased [20]. In addition, our CLSM data confirmed the relationship between fluoride release rate and EPS amount of the 94h-old biofilms. As shown in Fig. 4C, the EPS bio-volume and thickness of the biofilms followed linear-pattern of fluoride release rate-dependence with R = −0.916; R2 = 0.839 and R = −0.995; R2 = 0.990, respectively. These findings suggest that EPS bio-volume and thickness are strongly and negatively correlated with fluoride release rate, and that at least 83.9 and 99.0% of the variation in the EPS bio-volume and thickness, respectively, may be explained by the fluoride release rate. Furthermore, this result suggests that the fluoride release level of GICs may affect the structure of cariogenic biofilms since EPSs contribute to the bulk, physical integrity, and stability of the biofilm matrix [24]. To our knowledge, this is the first report showing that fluoride release from GICs can affect the formation of EPSs of cariogenic biofilms. Although the dry weights of 94-h-old S. mutans biofilms on the tested GICs, except for GC2, were lower than that of the control (Fig. 2A), the numbers of bacterial CFUs on the GICs were significantly higher than that of the control (p < 0.05) (Fig. 2B). As shown in Fig. 2E, the change in the number of
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bacterial CFUs followed a positive linear-pattern of fluoride release rate-dependence (R = 0.869, R2 = 0.755), suggesting that fluoride release from the GICs may not efficiently affect the attachment and viability of S. mutans in biofilms. However, the bacterial bio-volumes and thickness of the 94-h-old biofilms formed on the GICs were similar to that of the control (p > 0.05) or lower than that of the control (p < 0.05) (Fig. 3A and B). Furthermore, the change in bacterial bio-volume followed a negative linear-pattern of fluoride release rate-dependence (R = −0.850, R2 = 0.723) (Fig. 3C). In this study, the bacterial biovolume in CLSM analysis reflected the total volume of bacterial cells (live + dead cell volume) in biofilms; however, CFU count reflected the total number of live bacterial cells in the biofilms. Thus, the difference between the results of CFU count and CLSM analysis may be due to differences in the number and volume of dead cells on the test materials, suggesting that our results need to be confirmed using other methods such as a live and dead fluorescence assay. In this study, our results clearly showed that fluoride release rate is negatively correlated with acidogenicity, dry weight, bacterial bio-volume, or amount of EPSs of S. mutans biofilms. These results suggest that fluoride release from GICs may play an important role in inhibiting cariogenic bacterial metabolism in biofilms. Of the GICs tested in this study, Keta and GC showed strong anti-biofilm activity against S. mutans biofilms (Figs. 1–4), and the fluoride release rates of these GICs were higher than those of the other test materials (Fig. 1). These results suggest that, if restorative materials that release higher concentrations of fluoride ions are developed, they may have even stronger anti-cariogenic biofilm activity and may more efficiently inhibit subsequent secondary caries. It is well known that inhibition of secondary caries associated with fluoride-releasing materials is usually attributed to a continuous, sustained release of fluoride ions around the margins of a restoration. Although fluoride release from the GICs tested in this study showed anti-S. mutans biofilm activity, it is unclear whether they can maintain their fluoride release rates over a long period of time since it has been reported that the fluoride release from fluoride-releasing materials occurs in a biphasic pattern, with initial rapid decrease and a slower second phase [33]. Therefore, further investigations are needed to determine the anti-cariogenic biofilm activity of fluoride-releasing materials over a longer period of time and to examine the change in the relationship between fluoride release rate and cariogenic composition of in vivo dental biofilms thereafter.
5.
Conclusions
In conclusion, the results of this study revealed that the activity of fluoride against acidogenicity, bacterial bio-volume, and EPS formation of S. mutans biofilms followed a negative linearpattern of fluoride release rate-dependence (R2 = 0.723–0.990). These findings suggest that, if an appropriate fluoride release rate is sustained from restorative materials during biofilm formation, GICs may play an important role in decreasing the virulence of cariogenic biofilms and subsequent secondary caries formation.
Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (2014R1A4A1005309).
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Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.intl.elsevierhealth.com/journals/dema
Relationship between fluoride release rate and anti-cariogenic biofilm activity of glass ionomer cements Ngoc Phuong Thanh Chau a,b , Santosh Pandit a , Jian-Na Cai a , Min-Ho Lee c,∗∗ , Jae-Gyu Jeon a,∗ a
Department of Preventive Dentistry, School of Dentistry, Institute of Oral Bioscience and BK 21 Plus Program, Chonbuk National University, Jeonju 561-756, Republic of Korea b Department of Odonto-Stomatology, Hue University of Medicine and Pharmacy, Hue University, 06 Ngo Quyen street, Hue City, Viet Nam c Department of Dental Biomaterials, School of Dentistry, Chonbuk National University, Jeonju, Republic of Korea
a r t i c l e
i n f o
a b s t r a c t
Article history:
Objectives. The aim of this study was to evaluate acidogenicity and composition of Streptococ-
Received 6 August 2014
cus mutans biofilms on glass ionomer cements (GICs) and then to determine the relationship
Received in revised form
between the anti-S. mutans biofilm activity and fluoride release rate of the GICs.
16 October 2014
Methods. S. mutans biofilms were formed on discs prepared using five commercial GICs. Acid
Accepted 19 December 2014
production and fluoride release rates of the biofilms on the GIC discs during biofilm forma-
Available online xxx
tion (0–94 h) were determined. Next, 94-h-old S. mutans biofilms on GIC discs were analyzed to evaluate the biofilm composition (dry weight, bacterial cell number, and extra-cellular
Keywords:
polysaccharide (EPS) amount) using microbiological, biochemical, and confocal laser scan-
Glass ionomer cements
ning microscopy (CLSM) methods. Lastly, relationships between the fluoride release rate
Fluoride release
and changes in acidogenicity and composition of the biofilms were determined using a
Cariogenic biofilms
linear-fitting procedure.
Acidogenicity
Results. All of the tested GICs released fluoride ions. Of the GICs, the two that showed the
Biofilm composition
highest fluoride release rates strongly affected acidogenicity, dry weight, and EPS forma-
Relationship
tion of the biofilms. Furthermore, they reduced the bacterial and EPS bio-volumes and EPS thickness. However, the number of colony forming units (CFUs) of the biofilms was higher than that of the control. Generally, changes in the acidogenicity and composition (except for CFU count) of the biofilms on the GICs followed a negative linear-pattern of fluoride release rate-dependence (R = −0.850 to −0.995, R2 = 0.723–0.990). Significance. These results suggest that the anti-cariogenic biofilm activity of GICs is closely correlated with their fluoride release rate during biofilm formation. © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
∗ ∗∗
Corresponding author. Tel.: +82 63 270 4036. Corresponding author. E-mail addresses: [email protected] (M.-H. Lee), [email protected] (J.-G. Jeon).
http://dx.doi.org/10.1016/j.dental.2014.12.016 0109-5641/© 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
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1.
Introduction
It has been well documented that biofilm formation occurs on the surfaces of different restorative materials within a short time after placement in the oral cavity [1,2]. The biofilms on the microgaps between restorative materials and tooth tissues can lead to secondary caries, which is responsible for half of all restorations fail within 10 years [3]. Similar to the dental caries process, the biofilms on these microgaps cause dissolution of the adjacent tooth surfaces and reduce the longevity of the restorative materials [4]. Although several studies have shown that the level of mutans streptococci is not necessarily related to the development of dental caries [5,6], many other studies have revealed Streptococcus mutans as one of the main causative pathogens for dental caries [7,8]. S. mutans can adhere to and accumulate on the tooth surface using extra-cellular polysaccharides (EPSs) produced by glucosyltransferases (GTFs) from sucrose [9]. Furthermore, this bacterium can metabolize dietary sugars to organic acids and withstand rapid and substantial fluctuations in the environmental pH [10]. The combination of these virulence properties allows S. mutans to effectively colonize the tooth surface and be sustained in the oral cavity. To prevent secondary caries, fluoride-releasing restorative materials such as glass ionomer cements (GICs) and resin composites have been developed. Recently, several researchers have shown that nanocomposites containing calcium phosphate nanoparticles may reduce secondary caries [11]. Of the fluoride-releasing restorative materials, GICs have been used in various areas of restorative dentistry for several decades because of their biocompatibility and cariostatic properties [12,13]. Furthermore, GICs can reduce demineralization in adjacent hard tooth tissues [14,15]. Several studies have shown that the effectiveness of GICs may be related to their ability to release fluoride [13,16,17]. Biofilms on GICs can degrade material properties and roughen their surfaces, which in turn promote further biofilm formation, as well as material surface deterioration [18,19]. Recently, several studies have investigated bacterial adherence to and biofilm formation on GICs and have shown that these materials can affect bacterial adherence, acidigenicity, and biofilm formation [13,20]. However, although it is important to obtain information about whether GICs can affect composition of biofilms on GICs, little has been reported. Furthermore, there has been no study on the precise relationship between fluoride release level from GICs and changes in virulence and composition of biofilms on GICs. Considering the advantages and widespread use of GICs in restorative dentistry, it would be worthwhile to test the hypotheses that fluoride level released from GICs can change virulence and composition of cariogenic biofilms. Therefore, the aim of this study was to investigate the changes in virulence, especially acidogenicity, and composition of cariogenic biofilms formed on GICs and then to determine the relationships between fluoride release level from GICs and changes in acidogenicity and composition of cariogenic biofilms using an S. mutans biofilm model.
2.
Materials and methods
2.1.
GIC disk preparation
Table 1 lists the materials used in this study. Five GICs were selected: Glaslonomer FX-II (Gla), Ketac Fil Plus Aplicap (Keta), Riva self-cure HV (Riva), GC Fuji Filling LC (GC), and GC Fuji II LC (GC2). The GICs used in this study were all commercially available, and the shade of all materials was A2 or A3. These GICs were used to prepare disk-shaped specimens according to the manufacturer’s recommendations. Disk-shaped specimens (12 mm in diameter and 1.2 mm in thickness) were prepared using polytetrafluoroethylene (Teflon) molds with a metal holder and glass slides to cover each face. Gla, Keta, and Riva specimens were self-cured. GC and GC2 specimens were light-cured for 20 s on each face using a light curing unit (G-Light, GC Corp., Japan). After curing, all specimens were polished sequentially with # 800 to # 1200 sand papers. Then the specimens were placed in a desiccator at room temperature. Hydroxyapatite discs (12 mm in diameter and 1.2 mm in thickness; Clarkson Chromatography Products, Inc., South Williamsport, PA, USA) were included as a control in this study.
2.2.
Biofilm formation on GIC discs
The microorganism used for this study was S. mutans UA159 (serotype c). S. mutans biofilms were formed on saliva-coated GIC or hydroxyapatite (HA) discs placed in a vertical position in 24-well plates, as detailed elsewhere [21]. Briefly, the saliva-coated discs were generated by incubation with filtersterilized (0.22 m low protein-binding filter) human whole saliva for 1 h at 37 ◦ C. For biofilm formation, the saliva-coated discs were transferred to a 24-well plate containing 1% sucrose (v/v) ultrafiltered (10 kDa molecular-weight cut-off) tryptone yeast-extract (UTE) broth with S. mutans UA159 (2–5 × 106 colony forming units (CFUs)/ml). The biofilms were grown undisturbed for 22 h to allow initial biofilm growth. From this time point (22 h), the culture medium was changed twice daily (9 AM, 6 PM) until it was 94 h old. The culture medium was changed a total of six times. The pH value and fluoride concentration in the old culture medium were determined during the experimental period (until biofilms reached 94 h of age). The 94-h-old biofilms were used to analyze the biofilm composition. Each assay was performed in duplicate in at least six different experiments (n = 12).
2.3. Determination of fluoride concentration and pH value during biofilm formation To evaluate fluoride release from GICs, the concentration of fluoride in the old culture medium was determined during the experimental period (22, 31, 46, 55, 70, 79, and 94 h). For the determination of fluoride concentration, a total of 2.8 ml of each medium was mixed with 280 l of total ionic strength adjustment buffer (TISAB III). The fluorometer (Thermo Fisher Scientific, Orion, MA, USA) was calibrated using four standard solutions (0.1, 1, 10, and 100 ppm F− ). To evaluate the acidogenicity of S. mutans biofilms on GICs, the pH values of the old
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Table 1 – Characteristics of glass ionomer cements used in this study. Product name
Curing
Composition (wt.%)
Manufacturer
Glaslonomer FX-II
Self-cured
- Glass polyalkenoate. - Powder (15 g), liquid (8 ml, 10 g).
Shofu, Inc., Japan
KetacTM Fil Plus AplicapTM
Self-cured
- Glass powder (>99%).
3 M ESPE, Dental Products, Germany
- Liquid: copolymer of acrylic acid-maleic acid (35–55%), water (40–55%), tartaric acid (5–10%). Riva self-cure HV
Self-cured
- Polyacrylic acid (20–30%). - Tartaric acid (10–15%). - Fluoroaluminosilicate glass (90–95%). - Polyacrylic acid (5–10%).
SDI Limited, Australia
GC Fuji Filling LC
Light-cured
- Paste A: Alumino-fluoro-silicate glass (amorphous) (75–85%); 2-hydroxyethyl methacrylate (10–12%); Urethanedimethacrylate (2–5%). - Paste B: Distilled water (20–30%); polyacrylic acid (20–30%); urethanedimethacrylate (12–15%); silicone dioxide (fumed/amorphous) (10–15%).
GC corporation, Japan
GC Fuji II LC (improved)
Light-cured
- Powder: Alumino-fluoro-silicate glass (95–100%). - Liquid: Polyacrylic acid (20–25%); 2-hydroxyethyl methacrylate (30–35%); proprietary ingredient (5–15%); 2, 2, 4, trimethylhexamethylenedicarbonate (1–5%).
GC corporation, Japan
This is based on information provided by safety data sheet of the products.
culture medium were also determined during the experimental period (22, 31, 46, 55, 70, 79, and 94 h) using a pH electrode. To calculate acid production or fluoride release rates at 0–22, 22–31, 31–46, 46–55, 55–70, 70–79, and 79–94 h, the concentrations of H+ or fluoride in the old culture medium was divided by the respective incubation time. In addition, the mean rates of acid production and fluoride release during biofilm formation (0–94 h) were also calculated using the respective acid production or fluoride release rates (0–22, 22–31, 31–46, 46–55, 55–70, 70–79, and 79–94 h).
2.4. Analysis of 94-h-old S. mutans biofilm composition 2.4.1.
Microbiological and biochemical analyses
The 94-h-old biofilms on the test materials were transferred into 2 ml of 0.89% NaCl and sonicated in an ultrasonic bath for 10 min to disperse the biofilms. The dispersed solution was re-sonicated at 7 W for 30 s after adding 3 ml of 0.89% NaCl (VCX 130PB; Sonics and Materials, Inc., Newtown, CT, USA). An aliquot (0.1 ml) of the dispersed solution was serially diluted and plated onto brain heart infusion (BHI) agar plates to count the CFUs. For the determination of the dry weight and amount of water-insoluble extracellular polysaccharides (water-insoluble EPSs), the remaining solution (4.9 ml) was centrifuged (3000 × g) for 20 min at 4 ◦ C. The biofilm pellet was resuspended and washed twice in the same volume of water. The washed pellet was lyophilized and weighed to determine the dry weight. After weighing, the water-insoluble EPSs were extracted from the dry pellet using 1 N sodium hydroxide before determination of polysaccharide amount, as detailed elsewhere [22].
2.4.2.
Confocal laser scanning microscopy study
In addition to the microbiological and biochemical analyses, a confocal laser scanning microscopy (CLSM) study was performed to evaluate bacterial cells and EPSs of the 94-h-old biofilms on GICs, as described by Chau et al. [23]. Briefly, Alexa Fluor® 647-labeled dextran conjugate (10,000 MW; absorbance/fluorescence emission maxima 647/668 nm; Molecular Probes, Inc., Eugene, OR, USA) was added to the culture medium during biofilm formation at a concentration of 1 M. After 94 h, the bacterial cells in the biofilms were labeled by means of 25 M SYTO® 9 green-fluorescent nucleic acid stain (480/500 nm; Molecular Probes, Inc., Eugene, OR, USA) for 30 min [24]. CLSM imaging of the biofilms was performed using the LSM 510 META microscope (Carl Zeiss, Jena, Germany) equipped with argon-ion and helium–neon lasers. Two independent experiments were performed, and seven image stacks (512 × 512 pixel tagged image file format) per experiment were collected. The biofilms were quantified from the confocal stacks using the image-processing software COMSTAT [25]. This software was written as a script in Matlab 5.1 (The MathWorks, Natick, MA, USA) equipped with the Image Processing Toolbox. In this study, the bio-volume and thickness of bacteria and polysaccharides were analyzed. Bio-volume is defined as the volume of the biomass (m3 ) divided by the surface area of the substratum (GIC or hydroxyapatite discs) (m2 ).
2.5.
Statistical analysis
The data are presented as mean ± standard deviation. The intergroup differences were estimated using one-way
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Fig. 1 – (A) Fluoride release rate, (B) acid production rate, and (C) linear-fitting of mean rate of fluoride release versus mean rate of acid production during Streptococcus mutans biofilm formation on glass ionomer discs (0–94 h). Control, hydroxyapatite; Gla, Glaslonomer FX-II; Keta, Ketac Fil PlusAplicap; Riva, Riva self-cure HV; GC, GC Fuji Filling LC; GC2, GC Fuji II LC.
analysis of variance (ANOVA). Values were considered statistically significant when the p value was 0.05). As shown in Fig. 2D, the dry weight of the 94-h-old biofilms on the GICs was negatively correlated with the mean rate of fluoride release (0–94 h) from the GICs (p < 0.05). The R and R2 of the fit line were −0.922 and 0.849, respectively. Interestingly, although all of the dry weights of the 94h-old biofilms on the GICs, except for GC2, were lower than that of the control, and the numbers of bacterial CFUs of the biofilms were higher than that of the control (p < 0.05) (Fig. 2B). As shown in Fig. 2E, the number of bacterial CFUs of the 94h-old biofilms was positively correlated with the mean rate of fluoride release (0–94 h) from the GICs (R = 0.869, R2 = 0.755) (p < 0.05). In this study, we also evaluated the amount of waterinsoluble EPSs of the 94-h-old biofilms on GICs. As shown in Fig. 2C, all of the amounts of water-insoluble EPSs of the GICs were lower than that of the control (p < 0.05). Especially, the amount of water-insoluble EPSs on Keta, GC, and Gla were reduced by up to 46, 40, and 30%, respectively, compared to the vehicle control (p < 0.05). As shown in Fig. 2F, the amount of water-insoluble EPSs of the 94-h-old biofilms was negatively correlated with the mean rate of fluoride release (0–94 h) from
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Fig. 2 – (A) Dry weight, (B) colony forming unit (CFU), and (C) water-insoluble polysaccharide (WIP) of 94-h-old Streptococcus mutans biofilms on glass ionomer (GI) discs. Linear-fitting of (D) dry weight, (E) CFU, (F) and WIP of the 94-h-old biofilms versus mean rate of fluoride release of GI discs during biofilm formation (0–94 h). Control, hydroxyapatite; Gla, Glaslonomer FX-II; Keta, Ketac Fil PlusAplicap; Riva, Riva self-cure HV; GC, GC Fuji Filling LC; GC2, GC Fuji II LC. *p < 0.05: significantly different from the control.
the GICs (p < 0.05). The R and R2 of the fit line were −0.853 and 0.728, respectively.
3.3. Composition changes of 94-h-old S. mutans biofilms in CLSM study and their relationship with fluoride release rate Differences in compositions of 94-h-old S. mutans biofilms on GICs were confirmed by CLSM image analysis. As shown in Fig. 3A and B, although all of the GICs tested in this study were fluoride-releasing materials (Fig. 1A), only Keta and GC affected the bacterial bio-volume and thickness of the 94-hold biofilms. Of the GICs, Keta had the most noticeable effects, with bacterial bio-volume and thickness being reduced up to 41 and 46%, respectively, compared to the vehicle control (p < 0.05). As shown in Fig. 3C, the bacterial bio-volume of the 94-h-old biofilms on the GICs was negatively correlated with the mean rate of fluoride release (0–94 h) (p < 0.05). The R and R2 of the fit line were −0.850 and 0.723, respectively. Fig. 3D shows representative bacterial images of the 94-h-old biofilms on the control (Fig. 3D-1), Keta (Fig. 3D-2), and GC (Fig. 3D-3). As shown in Fig. 3D-2 and D-3, the total volumes of the biofilm cells on Keta and GC were reduced as compared to that of the control (Fig. 3D-1). In addition to bacterial bio-volume, EPS bio-volumes and thicknesses on GICs were analyzed. As shown in Fig. 4A and
B, all of the GICs tested in this study, except for GC2, reduced the EPS bio-volume and thickness of the 94-h-old biofilms. Of the GICs tested, Keta had the most strongly reduced EPS bio-volume and thickness, with reductions of up to 69 and 38%, respectively, compared to the vehicle control (p < 0.05). As shown in Fig. 4C, the EPS bio-volume and thickness of the 94-h-old biofilms on the GICs were negatively correlated with the mean rate of fluoride release (0–94 h) (p < 0.05). The R (R2 ) of the fit line of the EPS bio-volume and thickness versus the mean rate of fluoride release was −0.995 (0.990) and −0.916 (0.839), respectively. Fig. 4D shows representative EPS images of the 94-h-old biofilms on the control (Fig. 4D-1), Keta (Fig. 4D2), and GC (Fig. 4D-3). As shown in Fig. 4D-2 and D-3, the total volumes of EPS on Keta and GC were reduced as compared to that of the control (Fig. 4D-1).
4.
Discussion
Secondary caries, like dental caries, is a biofilm-related disease associated with the increased consumption of dietary sugar [26,27]. Thus, it is possible for any site on a restored tooth that is prone to cariogenic biofilm formation to develop secondary caries. In this study, we investigated acidogenicity and composition of a cariogenic biofilm on GICs. Furthermore, we determined the relationship between anti-cariogenic biofilm
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Fig. 3 – (A) Bacterial bio-volume and (B) thickness of 94-h-old Streptococcus mutans biofilms on glass ionomer (GI) discs in a confocal laser scanning microscopy (CLSM) study. (C) Linear-fitting of mean rates of fluoride release of GI discs during biofilm formation (0–94 h) versus bacterial bio-volume of the 94-h-old biofilms. Representative CLSM images of bacteria from the 94-h-old biofilms on (D-1) Control (hydroxyapatite), (D-2)Keta (Ketac Fil PlusAplicap), and (D-3) GC (GC Fuji Filling LC). Gla, Glaslonomer FX-II; Riva, Riva self-cure HV; GC2, GC Fuji II LC. *p < 0.05: significantly different from the control.
activity of GICs and their fluoride release rate since (i) it has been well demonstrated that fluoride ions, even in low concentrations, can affect the virulence of cariogenic biofilms [21], and (ii) many GICs release fluoride ions into dental biofilms and teeth [17,28,29]. In this study, we used an S. mutans biofilm model to study acidogenicity and composition of cariogenic biofilms. Although the S. mutans biofilm model does not precisely mimic the complex microbial community of cariogenic biofilms, the monospecies biofilm model is advantageous in examining virulence (acidogenicity and EPS formation) of S. mutans, an important pathogen of dental caries, in biofilms. In the study, we performed a linear-fitting procedure (linear regression analysis) to investigate the relationship between anti-cariogenic biofilm activity of GICs and their fluoride release rate. It is well accepted that linear regression analysis is useful for evaluating the linear relationship between two variables and to determine how much of the variation in one variable can be explained by the linear relationship with another variable [30]. The R of the fit line represents the strength of the relationship between two variables, and the R2 demonstrates the percentage of the variation in one variable that can be explained by the linear relationship with another variable [30]. The most relevant virulence factor of cariogenic biofilms in dissolving the hard tooth surfaces is acidogenicity. Recently,
several studies have examined the effects of fluoride-releasing materials on acidogenicity of cariogenic bacteria [13,21,29]. Some investigations demonstrated that fluoride-releasing materials can affect the acidogenicity of cariogenic biofilms. However, studies that show a relationship between fluoride concentration released from restorative materials and acidogenicity of cariogenic biofilms are rare. In this study, our data clearly showed that the acid production rate of S. mutans biofilms on GICs followed a negative linear-pattern of fluoride release rate-dependence during biofilm formation (Fig. 1C), suggesting that acid production of biofilms decreases as fluoride release rate increases during biofilm formation. As shown in Fig. 1C, the R of the fit line was −0.962, suggesting that acid production rate is strongly and negatively correlated with fluoride release rate. However, because R only shows the strength of the relationship between two variables, R2 was also calculated. For the fit line, R2 was 0.926, suggesting that the line may appropriately describe the change in acid production rate of biofilms according to the fluoride release rate, and that at least 92.6% of the variation in acid production rate may be explained by the fluoride release rate. It has been well reported that the release of fluoride from restorative materials is a very complex process, and can be affected by intrinsic variables, i.e. fillers and formulations, and experimental factors, i.e. storage media, frequency of storage
Please cite this article in press as: Chau NPT, et al. Relationship between fluoride release rate and anti-cariogenic biofilm activity of glass ionomer cements. Dent Mater (2015), http://dx.doi.org/10.1016/j.dental.2014.12.016
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Fig. 4 – (A) Extracellular polysaccharides (EPSs) bio-volume and (B) thickness of 94-h-old Streptococcus mutans biofilms on glass ionomer (GI) discs in a confocal laser scanning microscopy (CLSM) study. (C) Linear-fitting of mean rates of fluoride release of GI discs during biofilm formation (0–94 h) versus the EPS bio-volume and thickness of the 94-h-old biofilms. Representative CLSM images of EPSs from the 94-h-old biofilms on (D-1) Control (hydroxyapatite), (D-2) Keta (Ketac Fil PlusAplicap), and (D-3) GC (GC Fuji Filling LC). Gla, Glaslonomer FX-II; Riva, Riva self-cure HV; GC2, GC Fuji II LC. *p < 0.05: significantly different from the control.
medium change [17]. Furthermore, several studies have shown that lower pH conditions increase fluoride release for both conventional and resin-modified glass ionomer cements, and glass ionomers can buffer acids [31,32]. Therefore, a more sophisticated and well-designed study will be needed to reveal whether pH change in biofilms can increase fluoride release and the buffer activity of glass ionomers can influence pH change in biofilms. In this study, relationships between fluoride release rate and dry weight and water-insoluble EPS amount of S. mutans biofilms were also investigated. Our results showed that the dry weight and water-insoluble EPS amount of the 94-h-old biofilms are negatively correlated with the fluoride release rate of the GICs during biofilm formation (Fig. 2D and F). As shown in Fig. 2D and F, the Rs of the fit lines were −0.922 and −0.853, suggesting that dry weight and amount of water-insoluble EPSs of S. mutans biofilms are strongly and negatively correlated with fluoride release rate of GICs. The R2 s of the fit lines were 0.849 and 0.728, suggesting that at least 84.9 and 72.8% of the variation in the dry weight and amount of water-insoluble EPSs, respectively, may be explained by the fluoride release rate during biofilm formation. Generally, our data confirmed a previous finding that the dry weights of dental biofilms on
dental restorative materials are reduced as the rate of fluoride release from the materials is increased [20]. In addition, our CLSM data confirmed the relationship between fluoride release rate and EPS amount of the 94h-old biofilms. As shown in Fig. 4C, the EPS bio-volume and thickness of the biofilms followed linear-pattern of fluoride release rate-dependence with R = −0.916; R2 = 0.839 and R = −0.995; R2 = 0.990, respectively. These findings suggest that EPS bio-volume and thickness are strongly and negatively correlated with fluoride release rate, and that at least 83.9 and 99.0% of the variation in the EPS bio-volume and thickness, respectively, may be explained by the fluoride release rate. Furthermore, this result suggests that the fluoride release level of GICs may affect the structure of cariogenic biofilms since EPSs contribute to the bulk, physical integrity, and stability of the biofilm matrix [24]. To our knowledge, this is the first report showing that fluoride release from GICs can affect the formation of EPSs of cariogenic biofilms. Although the dry weights of 94-h-old S. mutans biofilms on the tested GICs, except for GC2, were lower than that of the control (Fig. 2A), the numbers of bacterial CFUs on the GICs were significantly higher than that of the control (p < 0.05) (Fig. 2B). As shown in Fig. 2E, the change in the number of
Please cite this article in press as: Chau NPT, et al. Relationship between fluoride release rate and anti-cariogenic biofilm activity of glass ionomer cements. Dent Mater (2015), http://dx.doi.org/10.1016/j.dental.2014.12.016
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bacterial CFUs followed a positive linear-pattern of fluoride release rate-dependence (R = 0.869, R2 = 0.755), suggesting that fluoride release from the GICs may not efficiently affect the attachment and viability of S. mutans in biofilms. However, the bacterial bio-volumes and thickness of the 94-h-old biofilms formed on the GICs were similar to that of the control (p > 0.05) or lower than that of the control (p < 0.05) (Fig. 3A and B). Furthermore, the change in bacterial bio-volume followed a negative linear-pattern of fluoride release rate-dependence (R = −0.850, R2 = 0.723) (Fig. 3C). In this study, the bacterial biovolume in CLSM analysis reflected the total volume of bacterial cells (live + dead cell volume) in biofilms; however, CFU count reflected the total number of live bacterial cells in the biofilms. Thus, the difference between the results of CFU count and CLSM analysis may be due to differences in the number and volume of dead cells on the test materials, suggesting that our results need to be confirmed using other methods such as a live and dead fluorescence assay. In this study, our results clearly showed that fluoride release rate is negatively correlated with acidogenicity, dry weight, bacterial bio-volume, or amount of EPSs of S. mutans biofilms. These results suggest that fluoride release from GICs may play an important role in inhibiting cariogenic bacterial metabolism in biofilms. Of the GICs tested in this study, Keta and GC showed strong anti-biofilm activity against S. mutans biofilms (Figs. 1–4), and the fluoride release rates of these GICs were higher than those of the other test materials (Fig. 1). These results suggest that, if restorative materials that release higher concentrations of fluoride ions are developed, they may have even stronger anti-cariogenic biofilm activity and may more efficiently inhibit subsequent secondary caries. It is well known that inhibition of secondary caries associated with fluoride-releasing materials is usually attributed to a continuous, sustained release of fluoride ions around the margins of a restoration. Although fluoride release from the GICs tested in this study showed anti-S. mutans biofilm activity, it is unclear whether they can maintain their fluoride release rates over a long period of time since it has been reported that the fluoride release from fluoride-releasing materials occurs in a biphasic pattern, with initial rapid decrease and a slower second phase [33]. Therefore, further investigations are needed to determine the anti-cariogenic biofilm activity of fluoride-releasing materials over a longer period of time and to examine the change in the relationship between fluoride release rate and cariogenic composition of in vivo dental biofilms thereafter.
5.
Conclusions
In conclusion, the results of this study revealed that the activity of fluoride against acidogenicity, bacterial bio-volume, and EPS formation of S. mutans biofilms followed a negative linearpattern of fluoride release rate-dependence (R2 = 0.723–0.990). These findings suggest that, if an appropriate fluoride release rate is sustained from restorative materials during biofilm formation, GICs may play an important role in decreasing the virulence of cariogenic biofilms and subsequent secondary caries formation.
Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (2014R1A4A1005309).
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