Keywords
toothpaste - nanoparticles - bioactive glass - zinc oxide - fluoride release - antibacterial
Introduction
Brushing teeth with fluoridated toothpaste has been widely practiced worldwide for
dental caries prevention, as it removes biofilm mechanically, which adds to the therapeutic
effects of fluoride. Other ingredients for functions such as whitening, antiplaque,
antibacterial, antitartar, and erosive prevention can be added.[1] Availability and stability of fluoride in dentifrices are basic requirements for
effectiveness against tooth decay.[2]
[3] Fluoride ions can inhibit the production of bacterial acids in dental plaque due
to the influx of hydrogen fluoride into bacterial cells, and the dissociation to the
H+ and F– ions in the cytoplasm.[4] For caries control, dentifrice formulation should contain a minimum of 1000 parts
per million (ppm) fluoride, which must be in a soluble form to impart the anticaries
effect.[5]
[6] The factors which control the soluble fluoride content in dentifrice are its composition
(i.e., fluoride source) and storage conditions (i.e., temperature and aging). It is
reported that low-pH toothpastes containing 500 to 550 ppm fluoride showed similar
results on enamel demineralization as the one containing 1100 ppm fluoride.[7]
Dentifrice composition plays a crucial role in disabling free fluoride ions, which
may lead to creation of a low-solubility product with a diminished anticaries outcome.[2]
[3] Currently, there are no regulations to specify how much of total fluoride should
be maintained in a dentifrice formulation.[5] There has been a growing interest in the use of “smart” bioactive materials (i.e.,
amorphous calcium phosphate, hydroxyapatite, bioactive glass, etc.) for tooth remineralization.
It is reported that the remineralization effect of nano-hydroxyapatite crystals is
generally limited to the surface of the initial carious lesion.[8] Bioactive glass is one such material being considered as a novel dental material
with remineralization potential. Some in vitro studies have shown that bioactive glass-containing
dentifrices minimized dentin hypersensitivity by dentinal tubule occlusion and apatite
formation.[9]
[10]
Recently, fluoride-doped bioactive glass nanoparticles (F-nBG) have been synthesized
by our group, and the long-term fluoride release behavior of F-nBG and its effect
on pH was evaluated.[11] Zinc oxide (ZnO) powder has been added as a preservative in dentifrices, which,
in aqueous suspension, not only inhibits dentin demineralization but also induces
antimicrobial action by yielding zinc ions (Zn2+ ) and reactive oxygen species.[12]
[13]
[14] The purpose of this study was to assess and compare the antimicrobial efficacy and
fluoride release behavior from the suspensions and elutes of experimental dentifrices
and a commercial dentifrice.
Materials and Methods
In this study, all chemicals were analytical grade. Initially, basic ingredients were
mixed in optimized ratios and allowed to mix homogeneously. Then 1.5 wt% titanium
dioxide (Sigma Aldrich; St. Louis, MO, United States) and 3 wt% zinc oxide nanoparticles
were added in increments to avoid agglomeration. The ZnO nanoparticles were synthesized
by our group as described previously.[15] The scanning electron microscopy (SEM) image and X-ray diffraction (XRD) pattern
of prepared ZnO nanoparticles are shown in [Fig. 1]. The morphological pattern showing the nanostructure of prepared ZnO and the diffractogram
is in accordance with JCPDS N 00–036–1451 (standard X-ray peaks of zinc oxide). F-nBG (5% mol. concentration of F- in F-BG) was used in this study and incorporated in experimental dentifrices in two
ratios: 1.5% and 4% wt/wt. Colgate Cavity Protection (Colgate-Palmolive, Guildford, UK) was used as the control. The composition of both the control
and the experimental dentifrices is given in [Table 1].
Table 1
Grouping and composition of dentifrices used in the study
|
Dentifrice
|
Abrasive
|
Other ingredients (% wt)
|
Fluoride agent % age
|
Group name
|
|
Abbreviation: SMFP, sodium monofluorophosphate.
|
|
Colgate cavity protection (Colgate-Palmolive, Guildford, UK) Lot-number: 10141222
|
Calcium carbonate
(CaCO3)
|
Water
Glycerin
Sodium lauryl sulfate
Sodium saccharin
Cellulose gum
Flavoring agent
Tetrasodium pyrophosphate
|
SMFP
0.76 w/w
(0.15 w/v)
|
CD
|
|
Experimental dentifrices
|
Calcium carbonate
(CaCO3) 27% wt
|
Water ~ 36%
Glycerol ~ 32%
Sodium benzoate ~1%
Methyelcellulose ~1%
Flavoring agent ~ 1%
Sodium lauryl sulfate ~ 2%
Water ~ 36%
Titanium dioxide (TiO2) 1.5%
Zinc oxide (ZnO) nanoparticles 3%
|
F-nBG with 5% mol fluoride
1.5% F-nBG (w/w)
|
ED1
|
|
F-nBG with 5% mol fluoride
4% F-nBG (w/w)
|
ED2
|
Fig. 1 SEM image and X-ray diffractogram of synthesized zinc oxide. SEM, scanning electron
microscope.
Fluoride Release Analysis
The fluoride research protocol used in this study was modified from the one suggested
by Pearce.[16] The study was performed in quadruplicates. From each group, a 100 mg dentifrice
sample was homogenized in 10 mL deionized water to form dentifrice suspensions, from
which half of the suspension was split in two centrifuge tubes, each containing 2.5
mL suspension. The remaining half dentifrice suspension was centrifuged (Hettich EBA
20; Hettich, Tuttlingen, Germany) at 6000 rpm for 10 min (2461 × g) to remove the
insoluble fluoride bound to filler particles to extract elute into two centrifuge
tubes, each containing 2.5 mL of elute. The same procedure was repeated for all dentifrice
groups. Then 2.5 mL of 2M HCl (BDH Chemicals; Hull, East Yorkshire, UK) was added
to all the centrifuge tubes containing the dentifrice elute and suspension samples,
which were then conditioned at 45°C for 1 hour followed by an addition of 5 mL of
1M NaOH (Sigma Aldrich) and 1 mL TISAB III reagent (Hanna Instruments; Woonsocket,
Rhode Island, United States). Finally, the samples were analyzed using precalibrated
ion selective electrode (ISE) potentiometry (Hanna HI3222 pH/ISE meter and fluoride
electrode; Hanna Instruments, Woonsocket, Rhode Island, United States).
Antimicrobial Susceptibility Test
For the antimicrobial susceptibility test, freeze-dried microbial strains Streptococcus mutans (ATCC-25175) and Lactobacillus casei (ATCC-393) were obtained from American Type Culture Collection (ATCC; Manassas, VA,
United States). Dehydrated culture media was obtained from Oxoid (Basingstoke, Hampshire,
England) and Merck Millipore (Darmstadt, Germany). Microorganisms were revived from
freeze-dried vials in their respective culture media—MRS (de Man Rogosa) broth for
L. casei and BHI (Brain Heart Infusion) broth for S. mutans—as per American type culture collection (ATCC) guidelines. For the test procedure,
2 g of selected dentifrice ([Table 1]) was mixed in 2mL of sterile deionized water, and this yielded a 1:1 dilution. The
same procedure was applied to prepare three dilutions of each dentifrice in the ratios
of 1:1, 1:2, and 1:4, respectively. Dentifrice slurries were vortexed in an electric
shaker (Heidolph REAX 2000; Gemini BV Laboratory, DG Apeldoorn, the Netherlands).
For inoculum preparation, three to five well-isolated, similar colonies were selected
and transferred to tubes containing 5 mL of 0.9% saline. The suspensions were then
adjusted to a density equivalent to 0.5 standard of the McFarland Turbidity Scale
(DensiCHEK plus; bioMérieux, NC, United States). Within 15 min of the preparation
of the adjusted inoculum suspensions, it was swabbed evenly over the entire surface
of the respective agar plates of prepared media to create a bacterial lawn.
Agar Well-Diffusion Assay
A sterile cork borer was used to create four wells in each of the agar plates. The
punched agar circles were picked with a sterile needle and removed from the plates.
The wells were 8 mm in diameter and 4 mm in depth and were placed at distances of
20 mm in each of the plates. Then, 0.2 mL of the three dilutions (1:1, 1:2, and 1:4)
of each dentifrice and a control containing deionized water were poured into separate
wells in each agar plate using a micropipette (Genex Beta; Guangdong China). The pH
of prepared, solid agar mediums used for this study was similar to the physiological
pH of plaque.[17]
All the plates were made in triplicate, and S. mutans was incubated in both aerobic and anaerobic conditions and tested on Muellar–Hinton
agar plates. In the case of L. casei, the sets of triplicate were tested in microaerophilic as well as predominant anaerobic
conditions on chocolate blood agar plates (Gas-Pak sachet-Oxoid AnaeroGen; Thermo
Fisher Scientific, Bartlesville, OK, United States).
Determination of Minimum Bactericidal Concentration
Susceptibility break points are expressed as minimum inhibitory concentration (MIC)
and/or minimum bactericidal concentration (MBC) using appropriate dilution methods,
whereby two-fold dilutions of the experimental and commercial groups were prepared
as 1:1, 1:2, 1:4, 1:8, and 1:16 (2 g/2 mL yielded a 1:1 dilution). MBC was determined
using modified broth dilution method for dentifrices.[18] Then, 1 mL of each dilution of dentifrice was mixed with 1 mL of respective broth
to yield a defined volume of 2 mL in each of the five test tubes. A test tube without
dentifrice, containing 2 mL of deionized water for each bacterial strain, was used
as control.
All the test tubes, including controls, were inoculated using a sterile wire loop.
The test tubes inoculated with S. mutans were incubated aerobically for 24 hours at 37°C, whereas the test tubes inoculated
with L. casei were incubated for 48 hours at 37°C. To ensure reproducibility, this step was repeated
twice. Following incubation, all test tubes were sub-cultured to Mueller–Hinton agar
(S. mutans) and chocolate blood agar plates (L. casei) in duplicate. The plate which showed no visible growth was taken as MBC and reported
in g/mL of the corresponding dilution.
Statistical Analysis
SPSS version 22 (IBM; Armonk, New York, United States) was used to calculate the
means and standard deviations of the groups along with differences within and between
the groups by one-way ANOVA, and a posthoc Tukey’s test was used for pairwise comparison
between groups.
Results
Fluoride Release Analysis
The fluoride release in ppm for both the elutes and the suspensions of all the study
groups are presented in [Fig. 2]. Among elutes of the dentifrices, maximum mean fluoride release (± standard deviation)
was detected in ED2 (13.22 ± 0.09 ppm), followed by ED1 (9.74 ± 0.05 ppm), and the
control dentifrice (6.28 ± 0.05 ppm). Among suspensions of the study dentifrices,
the maximum mean fluoride release was exhibited by ED2 (14.20 ± 0.09 ppm), followed
by ED1 (10.07 ± 0.09 ppm), and the control group (6.63 ± 0.05 ppm). In both elutes
and suspensions of experimental dentifrices, a rise in mean fluoride release was observed
with a rise in F-nBG filler content. The posthoc Tukey’s test showed a statistically
significant difference between the mean fluoride release of elutes of all the study
dentifrice groups and between the mean fluoride release of suspensions of all study
dentifrice groups (p = 0.000).
Fig. 2 Mean fluoride elusion values with standard deviations (error bars) in the elutes
and suspensions of the experimental dentifrices.
Antimicrobial Analysis
Antimicrobial Susceptibility Test: Agar Well Diffusion Assay
Following incubation, resultant zones of inhibition were observed on the agar plates.
The clear and circular halos surrounding the wells were observed. The zone of inhibition
against S. mutans appeared after 24 hours, and the inhibition zone against L. casei appeared after 48 hours. The inhibition zones increased in a dose-dependent manner
for the dilutions 1:1, 1:2, and 1:4 of dentifrice slurries. The negative control wells
containing deionized water produced no observable inhibitory effect for any of the
bacteria in each of the triplicate sets of agar plates. The assessment of antibacterial
potential against caries-causing odonto-pathogens was done by recording the diameters
of zones of inhibition (ZOIs) (mm) using a Vernier caliper ([Table 2]). One-way AVOVA (analysis of variance) revealed that only ED1 and CD indicated statistical
significance (p < 0.05). Pair-wise comparisons using a posthoc Tukey’s test revealed differences between
the concentration groups. ED1 and ED2 were statistically insignificant (p > 0.05) for both bacterial strains under all conditions.
Table 2
Mean diameter and SDs of ZOI against microbial strains
|
ZOIs against S. mutans
|
|
Experimental
|
Mean ± SD
Dilution 1:1
|
Mean ± SD
Dilution 1:2
|
Mean ± SD
Dilution 1:4
|
|
Aerobic
|
Anaerobic
|
Aerobic
|
Anaerobic
|
Aerobic
|
Anaerobic
|
|
Abbreviation: ZOI, zone of inhibition.
|
|
ED1
|
F-BG 1.5%
|
22.43 ± 0.61
|
21.8 ± 1.29
|
20.07 ± 1.46
|
17.83 ± 1.43
|
17.27 ± 0.90
|
15.26 ± 1.15
|
|
ED2
|
F-BG 4%
|
25.10 ± 0.69
|
23.23 ± 1.45
|
21.90 ± 1.51
|
20.16 ± 0.84
|
17.84 ± 1.03
|
16.76 ± 0.88
|
|
CD
|
SMFP 0.76%
|
29.83 ± 0.23
|
29.83 ± 1.31
|
25.83 ± 1.31
|
26.5 ± 1.47
|
21 ± 0.81
|
20.73 ± 0.90
|
|
ZOIs against
L. casei
|
|
Experimental
|
Microaerophilia
|
Anaerobic
|
Microaerophilia
|
Anaerobic
|
Microaerophilia
|
Anaerobic
|
|
ED1
|
F-BG 1.5%
|
10.5 ± 0.41
|
11.17 ± 1.02
|
8.17 ± 0.62
|
6.5 ± 0.41
|
7.0 ± 0.82
|
5.83 ± 0.24
|
|
ED2
|
F-BG 4%
|
13.1 ± 0.54
|
15.33 ± 0.85
|
10.17 ± 1.03
|
11.0 ± 0.816
|
8.67 ± 0.47
|
8.83 ± 0.62
|
|
CD
|
SMFP 0.76%
|
16.33 ± 1.25
|
17.67 ± 0.47
|
11.33 ± 0.62
|
14.33 ± 0.47
|
10.4 ± 0.43
|
11.33 ± 0.47
|
Determination of Minimum Bactericidal Concentration
ED2 showed increased effectiveness and was bactericidal (S. mutans) even at the dilution of 1:16, similar to CD. In the case of L. casei, ED1 was effective up to a dilution 1:4 only and growth was observed on plates at
a dilution of 1:8. This finding also corresponded with the smallest mean ZOIs observed
for ED1 against L. casei using agar well-diffusion assay ([Table 2]). The remaining dentifrices were bactericidal (L. casei) up to a dilution of 1:8. For both bacterial strains, marked growth was observed
on the plated controls.
Discussion
In the current study, evaluation of the proportions of F-nBG fillers in dentifrices
that yield maximum fluoride elusion among novel dentifrices was performed. Upon increasing
the F-nBG filler content in experimental dentifrice groups, an increase in fluoride
release rates in both elutes and suspensions of dentifrices was observed, whereas
a direct proportionality of fluoride release rate with the F-nBG filler loading in
novel dentifrices was observed.
Dentifrices are used in suspension form during tooth brushing procedures; therefore,
the fluoride elusion in dentifrice suspensions is of greater clinical significance
than the fluoride elusion in the dentifrice elutes. However, no remarkable difference
was observed in fluoride release values of elutes and suspensions of the same group.
This may possibly be due to instant release of fluoride ions upon dissolution of fluoride
compounds used in experimental dentifrices in aqueous media. Regarding the bactericidal
effect of fluoride, it is reported that fluoride release of 20 ppm exhibits direct
bactericidal action.[19] However, due to the buffering action of saliva, this amount drops and may not be
as relevant clinically. The elutes and suspension of ED2 showed 13.72 ppm and 14.20
ppm and close to the required amount.
In a previous study conducted by our group, F-nBG particles with 5 wt% fluoride showed
burst fluoride release for the initial 9 days.[11] As dentifrices are used to brush teeth for only a few minutes, therefore, the burst
fluoride release by F-nBG filler particles with 5 wt% fluoride is of clinical significance
in dentifrices. Addition of fluoride to bioactive glass formulation can enhance its
potential for converting tooth surface hydroxyapatite to more acid-resistant fluorohydroxyapatite
and its ability to inhibit bacterial enzymes.[20]
[21] For apatite formation in teeth, the F-nBG particles must stay on the teeth and their
surroundings for sufficient time to induce remineralization without undergoing dilution
by saliva and beverage intake. As salivary flow is lowest at night during sleep, the
desired time for apatite formation should be less than 8 hours[22] following the use of dentifrices enriched with F-nBG fillers.
For efficacy of fluoride dentifrice, free available fluoride in an appropriate concentration
is required. This means that the fluoride agent in dentifrice should not chemically
react with any other dentifrice ingredients.[23] Fluoride compounds generating fluoride ions like sodium fluoride, stannous fluoride,
and ammonium fluoride are not compatible with chalk-based abrasives as ionic fluoride
reacts with ionic calcium forming insoluble calcium fluoride.[23]
[24] By contrast, sodium monofluorophosphate [SMFP (Na2PO3F)] displays greater compatibility with calcium-based abrasives, as the monofluorophosphate
anion is stable and therefore cannot bind to ionic calcium.[23] In the mouth, protective action in SMFP dentifrices results either by the direct
effect of the monofluorophosphate anions or by the fluoride ions released due to hydrolytic
degradation of SMFP in the mouth.[25]
[26]
Studies have shown that SMFP dentifrices with calcium-based abrasives lose an average
of 25% to 35% ionic fluoride upon aging at 22°C and 29°C, respectively, within 1 year.[27]
[28]
[29]Although the commercial dentifrices used in this study were within 3 years of expiration,
high-temperature storage conditions (before coming to laboratory) could be blamed
for the presence of smaller amounts of free fluoride available in the elutes and
suspensions of the control group.
The literature mostly focuses on the total fluoride content of dentifrices and does
not deliver sufficient information about the effective concentrations of free available
fluoride in toothpaste to yield best anticaries effectiveness. The guidelines for
cosmetic dentifrices mostly focus on total fluoride content only and state that this
should not surpass 1500 ppm fluoride. The US Food and Drug Administration states that
NaF/SMFP dentifrices comprising total fluoride of 850 to 1150 ppm should hold ≥ 65
ppm and ≥ 800 ppm free fluoride, respectively. The justification for these values
remains unclear. ISO Standard 11609[30] at present mentions requirements for the physical and chemical properties of dentifrices,
provides guidelines for suitable test methods including total fluoride content of
dentifrice, and does not report the necessity to specify the amount of free fluoride
in dentifrice, although this is a vital condition for anticaries efficiency. ISO7405[1][31] specifies and quantitative requirements for freedom from biological and toxicological
hazards, however, no specific ISO guidelines for microbiological studies available.
In addition, not a single study was found covering fluoride release of dentifrice
containing F-nBG fillers. As all the novel dentifrices contain calcium carbonate as
an abrasive, therefore, the effect of ionic calcium on free available fluoride in
elutes and suspensions of novel dentifrices cannot be established. This study not
only highlights the fluoride release behavior of the novel dentifrices enriched with
F-nBGs but also covers the antimicrobial analysis of such dentifrices.
There was a need to incorporate a bactericidal agent in the dentifrice. The partial
dissolution of ZnO particles releases Zn2+ ions in an aqueous suspension.[14] Zinc ions (Zn2+ ) display antimicrobial action against many bacterial strains through direct contact
with cell walls.[14] For this purpose, ZnO nano powder was incorporated. In this study, the methods include
the content and intent of the recommended methodology of the American Microbiological
Society (ASM). For antimicrobial analysis, slurries of all the included dentifrices
were made in various dilutions, as the level at which antimicrobial properties are
buffered or lost in dilution by saliva is important. Agar well diffusion tests revealed
that inhibition zones decreased in a dose-dependent manner for the dilutions 1:1,
1:2, and 1:4 of dentifrice slurries, further supporting the direct proportionality
of filler loading of F-nBG. No increase in zone size was observed after 48 hours.
All tested dentifrices exhibited greater antibacterial activity against the major
cariogenic pathogen, that is S. mutans, than L. casei. With regard to the evaluation of the facultative nature of S. mutans, the recorded diameters of inhibition zones in both aerobic and anaerobic conditions
were similar, whereas the inhibition zones recorded for L. casei were larger in the case of anaerobic conditions when compared with the CO2 jar.
The inhibition zones observed for ED2 were greater than ED1 because of greater fluoride
content along with ZnO and consequent fluoride release. The ZOIs for CD were slightly
elevated when compared with the inhibition zone for ED2, most likely due to incorporation
of anticalculus agent—tetrasodium pyrophosphate in the commercial dentifrice—for enhanced
cavity protection. According to previous studies, some dentifrices containing such
agents may provide reduction in biofilm formation by approximately 15%.[32]
[33]
MBC values of tested dentifrices revealed that all the test dentifrices had greater
effectiveness against S. mutans compared with L. casei. It also revealed that 3 wt% ZnO and an increased concentration of F-nBG directly
corresponded to increased bactericidal potential of experimental dentifrices. MBC
values for ED2 (4 wt% F-nBG) were same as that for CD (Colgate® Cavity protection). It is important to consider that MIC values are bacteriostatic,[34] whereas MBC values are bactericidal as they corresponded to subcultured plates which
showed absence of bacterial growth.[35] Further, in vitro studies need to be performed to assess the tooth remineralization
potentials of these dentifrices to gauge their efficacy in relieving enamel white
spot lesions and dentin hypersensitivity. Finally, cytotoxicity testing and in vivo
usage tests should be performed to introduce the most suitable composition commercially.
Conclusion
It is concluded that in dentifrices, mainly the composition of the fluoride source,
governs fluoride release behavior. Ionic fluoride in elutes and suspensions of both
novel dentifrices was directly proportional to F-nBG filler loading. Among novel dentifrices,
maximum mean fluoride release was detected in elutes and suspensions of group ED2,
which contained the highest filler loading, that is, 4 wt% F-nBG. Filler loading matched
the fluoride release of the commercial dentifrice.
Statistical analysis indicated that ED2 (F-nBG 4 wt.%) demonstrated similar antimicrobial
potential to that of an ordinary, non-tetra sodium pyrophosphate-containing fluoride
dentifrice in terms of ZOIs for both bacterial strains under all growth conditions.
The MBC obtained for ED2 was also similar to obtained MBC values for CD. Incorporation
of F-nBG and ZnO provide a multi-benefit approach to simultaneously treating early
white spot lesions, reducing bacterial growth, and providing core plaque control.
