Keywords
toothpaste - biomaterial - biosilicate - white spot lesion - dental remineralization
Introduction
The surface of dental enamel is often exposed to changes in pH that occur in the oral
cavity. These changes are related to bacterial[1] and nonbacterial acid exposure (intrinsic and extrinsic),[2] which progress toward the dissolution of hydroxyapatite, resulting in caries and
dental erosion, respectively.[1]
[2]
Dental caries happen when the remineralization phase cannot control demineralization.[3]
[4]
Streptococcus mutans and other species involved in the etiology of caries produce large amounts of lactic
acid in the presence of fermentable sugars, causing loss and impairment of tooth structure.[5]
[6] The initial stage of demineralization results in white opaque areas, known as active
white spot lesions (WSLs).[7]
WSLs are seen as white spots without cavity formation and are characterized by subsurface
demineralization with an intact surface layer of enamel. These lesions are frequent
in children and are associated with bad oral health habits and high sugar intake.[8] Sometimes oral hygiene is not performed after nighttime feeding, increasing the
risk of caries.[8]
The use of fluorides is the most studied strategy to strengthen dental enamel and
is related to lower white spot prevalence.[9] They prevent caries, inhibit demineralization, reinforce remineralization, and reduce
the metabolic activity of bacteria and the enamel permeability.[10]
[11] However, one of the limitations in their use is that fluorides have limited effectiveness
in preventing mineral loss due to caries disease. The calcium fluoride layer formed
during topical application tends to be gradually dissolved by acidic activity from
the diet.[12]
So, there is a need to look for at-home alternatives that can arrest the progression
of caries and induce remineralization of WSLs. Bioactive glasses have been investigated
as remineralizing agents.[13] These materials are capable of chemical bonding to dental hard tissues and are composed
of calcium, sodium, phosphorus, and silica oxides that promote their bioactivity.[14] These biomaterials can be applied directly to dental tissues and form a hydroxyapatite
layer on mineralized tissues to promote remineralization.[13]
[14]
[15]
[16] They have also been incorporated into toothpaste to determine their remineralization
potential, showing promising results.[14]
Biosilicate (Bio), a bioactive glass ceramic with fully crystallized particles, has
been introduced to combine the high mechanical bioactivity of glass with the strength
of glass ceramics.[12]
[13]
[14] It has also shown promising results on sound, and caries-affected dental tissues
applied as suspension or air-abrasion.[16] Considering that one of the first chemical reactions that occur when using Bio in
the presence of fluids is the increase in pH, its use as a therapeutic agent to control
oral pH and the presence of biofilm could help maintain oral health, in addition to
reduce the incidence of caries, especially in cases where sucrose intake is frequent.[12]
However, little is known about the effect of this additive on dental enamel remineralization
when incorporated into toothpaste, one of the most efficient ways to deliver therapeutic
agents to tooth surfaces. Thus, the aim of this in vitro study was to evaluate the
effect of incorporating Bio particles into experimental toothpaste (ET) on their abrasiveness
and remineralization capacity for WSL. The null hypotheses tested were that there
would be no significant differences among the treatments in terms of (1) their abrasiveness
or (2) their remineralization capacity.
Materials and Methods
Sample Preparation
The sample size calculation was performed after a pilot study comparing the mean values
(www.openepi.com), with a confidence interval of 95% and sample power of 80%.
Sound bovine teeth were selected and cut into 32 fragments of 6 mm × 6 mm × 2 mm (metallographic
precision saw Isomet 100 Buehler, Illinois, United States). Buccal faces were planned
and polished with 600- and 1,200-grit silicon carbide sandpaper strips to remove grooves,
and the surface roughness was standardized (≤0.2 μm).
Cariogenic Challenge
The samples were submitted to cariogenic challenge to simulate enamel demineralization
in WSLs. The protocol was performed with 8% methylcellulose gel and lactic acid. The
dentin of the fragments was protected with cosmetic nail polish (Colorama, Loreal
Brasil, Rio de Janeiro, RJ, Brazil), and 1.5 mL of gel was added over it (pH = 4.6),
which remained in contact with the surface of the fragment for 12 hours in a cold
chamber (4°C). After this period, 1.5 mL of 0.1 M lactic acid (pH = 4.6) was added.[17]
After the cariogenic challenge, the samples were separated into four groups (n = 8) according to the treatment performed ([Table 1]).
Table 1
Experimental groups
|
Groups
|
Products
|
Composition
|
|
G1
(Control)
|
Colgate Smiles toothpaste (Batman and Wonder Woman) 100 mg
|
Sodium fluoride 0.242%, Sorbitol, Water, Hydrated silica, PEG-12, Cellulose gum, Sodium
lauryl sulfate, Flavor
Sarcarina sodica, Sodium fluoride, Mica (CI77019), Titanium dioxide (CI77891), FD&C
Rojo No. 40 9CI1603
|
|
G2
|
Experimental toothpaste with fluoride
|
Carboxymethylcellulose (2.0 g), Glycerin (74.68 g), Tixosil 73 (10.0 g), Tixosil 43B
(13.9 g), Sodium fluoride (1,450 ppm)
|
|
G3
|
Experimental toothpaste with biosilicate
|
Carboxymethylcellulose (2.0 g), Glycerin (74.68 g), Tixosil 73 (10.0 g), Tixosil 43B
(13.9 g), Biosilicate 10% (5.0 g)
|
|
G4
|
Biosilicate suspension (10%) in distilled and deionized water
|
Distilled and deionized water (20 mL) and biosilicate (10% by weight)
|
Obtaining the Toothpaste
Two similar formulations of ET based on carboxymethylcellulose (CMC) were obtained
as described in [Table 1]. CMC was dissolved in 5 wt% of glycerol at 95°C, and the other components were added
in the necessary proportion to obtain the proper consistency. One of the toothpastes
had 1,450 ppm of sodium fluoride, and the different formulation was obtained by adding
10 wt% of Bio powder (average size of 4 µm) immediately before use, manually mixed
with a spatula, until homogeneous distribution was achieved without separation of
phases between the thickener and Bio.
Both ETs were compared with a commercial toothpaste (Colgate Smiles Batman and Mulher
Maravilha, Colgate-Palmolive Company, São Bernardo do Campo, SP, Brazil) and the same
concentration of Bio in suspension form after the addition of 10 wt% of Bio microparticles
in distilled and deionized water, prepared immediately before application.
pH Test
Before the treatments, the pH of the toothpaste and the BS was measured using a digital
pH meter (Kasvi model K39-2014B, Paraná, Brazil) calibrated with standard solutions.[18]
Simulated Brushing
For the treatment with the toothpaste, the samples were submitted to brushing in a
toothbrushing simulator (Pepsodent, MAVTEC Comércio De Peças Acessorios E Serviços
Ltda Me, Ribeirão Preto, SP, Brazil). A soft children's toothbrush (Tek, Johnson &
Johnson Ind. Com. Ltd., São José dos Campos, SP, Brazil) was used for each sample.
The brush heads were fitted and fixed in the machine. The fragments were submitted
to 14,600 brushing cycles under a constant load of 200 g and a traveled course of
3.8 cm at a speed of 356 rpm, simulating 1 year of brushing by a healthy individual.[19] The samples were fixed in acrylic resin plates (Acrilpress Artefatos de acrylic
Ltda, Ribeirão Preto, SP, Brazil) with hot glue, allowing immobilization of the samples
at the time of brushing.
The toothpastes were diluted in a proportion of 1:1 in distilled water immediately
before use to obtain a slurry with uniform consistency. For each sample, 10 mL of
the slurry was poured, and the fragments were brushed.
Suspension Immersion
After the addition of the Bio powder in distilled and deionized water, the suspension
was vigorously shaken for 3 minutes, and then, 1.5 mL of the BS was pipetted into
Eppendorf. The samples were immersed in the suspension for 8 hours, and after that,
they remained in artificial saliva for 16 hours at 37°C. This treatment was performed
for 60 days, making up 1,440 cycles.
Surface Roughness
Surface roughness analysis (Rugosimeter SE 1700 Surfcorder, Kosakalab, Tokyo, Japan)
was performed before and after treatments, with three cutoff lengths of 0.8 mm, totalizing
a reading length of 2.4 mm, at a speed of 0.25 mm/s.
Three readings were taken at different locations on the surface of the samples, perpendicular
to the brushing direction: one central, one 1 mm to the right, and one 1 mm to the
left. The average of these values was used as surface roughness measurement. The surface
roughness variation was calculated by the difference between the final and initial
values.
Microhardness Analysis
Knoop microhardness number (KHN) analysis (Microhardness Tester HMV-2, Shimadzu, Tokyo,
Japan) was performed with a vertical static load of 25 g applied for 5 seconds at
40× objective. When activated, the penetrator tip compresses the surface of the pattern,
generating a geometric figure in the form of an inverted pyramid. The diamond makes
it possible to determine the surface KHN of the material from the measurement of its
largest diagonal, whose value is applied in the formula:
where
KHN, Knoop hardness number;
F = 25 g;
d = length of the longest diagonal in the indentation.
Five different readings were performed on the samples, each one equidistant at 1 mm
from each other. The average of the five readings was considered as the KHN value.
The KHN change (ΔKHN) was calculated considering the relative differences in relation
to the initial values by the formula:
Where ΔKHN is the relative KHN calculated, KHNi is the initial value, and KHNf is
the final value obtained. The remineralizing potential of the treatments applied was
also calculated using the final values and the values obtained after the cariogenic
challenge by the formula:
Where KHNc corresponds to the KHN value after the cariogenic challenge.
Scanning Electron Microscopy
Qualitative analyses of the enamel surface were performed. Two samples were randomly
selected from each group, which were observed under scanning electron microcopy (SEM;
Jeol JSM-6610LV Microscope, Sony, Tokyo, Japan) to compare the sound and treated enamel.
The fragments were fixed on aluminum stubs and sputter-coated with gold–palladium
alloy (Bal-Tec, model SCD 050 sputter coater, Balzers, Liechtenstein). The surfaces
were analyzed at 500 × , 1,000 × , and 2,000× magnifications (20 kV, 30 mm working
distance [WD], and spot size 28 mm).
Statistical Analysis
Quantitative data were analyzed according to their distribution using the Shapiro–Wilk
test, with a significance level of 95% (α = 0.05). Data distribution was considered
normal for all the parameters analyzed and, therefore, they were analyzed by one-way
analysis of variance (ANOVA) followed by Tukey's test, with a significance level of
95%.
Results
pH
pH values of the treatments were 7.6 for Control, 7.34 for experimental toothpaste
with fluoride, 8.4 for experimental toothpaste with biosilicate, and 9.5 for 10% BS.
Surface Roughness
The surface roughness means and their comparisons (one-way ANOVA, Tukey's test, p < 0.05) can be seen in [Table 2]. Control group showed more remarkable surface roughness alteration (p < 0.05) than the groups treated with the fluoride toothpaste and the BS presented
the slightest alteration, different from all the other groups (p < 0.05). The toothpaste containing Bio resulted in surface roughness alteration similar
to the Control and the group treated with the fluoride toothpaste (p > 0.05).
Table 2
Comparison of means (standard deviation) of surface roughness change, relative microhardness,
and remineralizing potential between groups
|
Control
|
Fluoride toothpaste
|
Biosilicate toothpaste
|
Biosilicate suspension
|
|
Surface roughness
|
0.70 (0.23)a
|
0.34 (0.25)b
|
0.45 (0.15)ab
|
0.06 (0.04)c
|
|
Relative microhardness (Percentage)
|
75.3 (5.1)ab
|
66.3 (12.8)b
|
78.9 (8.3)a
|
75.2 (4.9)ab
|
|
Remineralizing potential
|
33.1 (15.7)a
|
55.0 (22.5)a
|
54.4 (20.9)a
|
55.3 (17.8)a
|
Different superscript letters, between the columns, indicate a statistically significant
difference.
Microhardness Analysis
The comparison of the relative KHN means (percentage) is shown in [Table 2]. The treatments were not able to fully recover the initial KHN of the enamel after
demineralization. There was no difference between the groups (p > 0.05), except when comparing the fluoride toothpaste and the one with Bio (p < 0.05). The fluoride toothpaste had the lowest relative KHN values but similar (p > 0.05) to the Control and the group treated with the BS.
The mean comparison of the remineralizing potential (Kruskal–Wallis, Dunn's test,
p < 0.05) is presented in [Table 2]. There was no difference between the groups (p > 0.05).
Scanning Electron Microscopy
Representative SEM images are shown in [Fig. 1]. Sound enamel surfaces showed a scratched appearance after grinding with sandpaper.
The Control group (brushed with conventional toothpaste) demonstrated a more polished
enamel surface than the sound enamel. The samples brushed with the fluoride toothpaste
had an enamel surface with a reticular structure. At higher magnification, an abraded
and irregular enamel surface was observed. The samples brushed with the toothpaste
containing the Bio revealed a more polished enamel surface than the sound enamel and
Bio particles on the surface. The samples treated with the BS also presented grooves
and scratches on the surface resulting from the polishing process. In addition, Bio
particles were also seen on the surface, and at higher magnification, it is possible
to verify regions of remineralization.
Fig. 1 Representative scanning electron microscopy images of each group. Images were obtained
at three magnifications: 500 × , 1,000 × , and 2,000 × . BS, Biosuspension; ETB, experimental
toothpaste with biosilicate; ETF, experimental toothpaste with fluoride.
Discussion
This study aimed to evaluate the impact of incorporating 10% Bio into an ET on their
abrasiveness and ability to remineralize artificially created WSL. The ET containing
Bio particles was compared with a conventional children's toothpaste (Control) and
regularly marketed,[8]
[9] and an ET with 1,450 ppm of fluoride, that was also included to isolate the influence
of other ingredients on dental enamel. The effectiveness of applying a 10% BS was
also tested, since previous studies have proven its remineralizing capacity.[17]
[20]
The first null hypothesis, which posited similar abrasiveness among treatments, was
rejected, based on the study results (p < 0.05). The conventional toothpaste was significantly more abrasive than the other
treatments, causing greater surface roughness compared with the ET with fluoride and
BS, which had little impact on surface roughness of the enamel.[21]
Commercial toothpastes formulations typically contain abrasives for stains and biofilm
removal.[22]
[23] However, these abrasives may also cause enamel wear, gradually increasing the surface
roughness of the enamel over time.[22] SEM analysis showed linear scratches on untreated enamel surfaces due to sandpaper
polishing. Interestingly, after toothbrushing with the conventional toothpaste, the
enamel surface appeared more polished, demonstrating that the abrasives can indeed
contribute to enamel wear.[21]
The nature of abrasives, including type, quantity, and particle size, significantly
influences a toothpaste's abrasiveness.[23] The conventional toothpaste contained hydrated silica as its abrasive component,[24] known for its potential to induce dental wear.[25] According to Ali et al 2020,[25] hydrated silica's higher Mohs hardness scale value compared with enamel (ranging
from 2.5 to 5 vs. 3.5) can lead to enamel surface scratches and increased roughness.
Furthermore, in the present study, we simulated incipient caries lesions (WSLs) by
inducing superficial and subsurface demineralization of the enamel surface. Due to
the mineral loss, the affected lesion surfaces become softer and more susceptible
to abrasion from toothbrushing.[23]
In contrast, the ET with fluoride functioned solely as a vehicle for delivering the
therapeutic agent and did not include abrasive particles, which could explain its
low abrasiveness. Moreover, the fluoride in this toothpaste may have prevented the
dissolution of calcium hydroxyapatite,[26] helping to maintain the surface roughness. SEM observations showed sediments on
the enamel surface, suggesting the formation of a filamentous network due to the deposition
of fluoride, which is insoluble in glycerin. Additionally, despite dilution in water
to create the slurry, sodium fluoride is poorly soluble in water.[27]
The ET with Bio showed intermediate surface roughness results, attributed to the presence
of Bio particles with an average size of 4 µm. Bio particles are highly bioactive
and undergo a rapid multistage surface reaction when in contact with an aqueous media,
ultimately resulting in the formation of hydroxycarbonate apatite.[15] Therefore, ET with Bio was not formulated with water or simulated body fluid; instead,
it was formulated with CMC and glycerin as a vehicle[28] and was exposed to water only immediately before the simulated brushing.
Furthermore, despite their high bioactivity, Bio particles take some time to completely
dissolve, as observed in a previous study[29] and confirmed in our study through SEM analysis. The simulated brushing lasted for
41 minutes, which appeared to be insufficient time for their complete dissolution.
Consequently, the partially dissolved Bio particles may have acted as abrasives. However,
this abrasive effect might have been mitigated by the remineralization process, resulting
in intermediate results.
In contrast, the treatment with BS involved immersion, which explains the low surface
roughness alteration and the presence of some grooves due to the polishing process,
as no simulated toothbrushing occurred.
The second null hypothesis, regarding remineralizing potential, was accepted as there
were no significant differences among the treatments. The most common and efficient
strategy for reducing enamel demineralization is toothbrushing with a fluoride toothpaste.[11]
[30]
[31] Fluoride replaces the hydroxyl ions in apatite structure, forming fluorapatite.
This process enhances the enamel hardness, as demonstrated in the present study, and
makes it more acid resistant.[31]
[32]
[33]
[34]
[35] According to Toti et al (2022), daily fluoride toothpaste use has been associated
with reduced WSL prevalence in children.[8]
While fluoride prevents dental demineralization, Bio acts as a remineralizing agent
by inducing the formation of a silica gel-rich layer on the dental surfaces. This
layer facilitates the deposition of calcium and phosphate ion, creating an amorphous
calcium phosphate (ACP) layer. Over time, this ACP layer undergoes crystallization
and transforms into hydroxy carbonate apatite (HCA).[15] Thus, Bio promotes mineral replacement, while fluoride protects existing minerals.
A previous study by Ferreira et al (2022), revealed that the treatment of caries-affected
dentin with 10% BS and silver diamine fluoride yielded comparable microhardness values.[36] This suggests that both treatments have a similar remineralizing capacity. Other
studies have also reported similar efficiency in remineralizing initial lesions when
comparing a fluoride toothpaste to a fluoride-free toothpaste containing microcrystalline
hydroxyapatite.[24]
[37]
[38]
[39] However, Chinelatti et al 2017, found that Bio has demonstrated superior and continuous
remineralization compared with topical fluoride application.[12] Notably, our study assessed the incorporation of Bio into toothpaste, a novel approach.
Despite similar remineralizing capacity among treatments, enamel treated with Bio-containing
ET exhibited higher relative KHN values compared with fluoride-containing ET (p < 0.05). Toothpaste pH influenced the efficiency of these remineralizing agents.
A lower pH in fluoride-based agents promotes calcium fluoride formation,[34]
[35] as observed in a previous study where reducing the pH of the toothpaste enhanced
the fluoride uptake in the dental biofilm.[35]
[36]
[37] In this study, both commercial and experimental fluoride toothpastes had a neutral
pH (7.6 and 7.3, respectively), potentially limiting fluoride's effectiveness.
Conversely, the ET with Bio had a basic pH (8.4) due to the initial reaction between
the glass–ceramic and aqueous media. This reaction leaches alkaline ions from the
glass[15] resulting in a basic pH that favors the formation of the silica gel-rich layer.
Thus, in this case, the pH was favorable for remineralization.
Toothbrushing with fluoride toothpastes leads to immediate high fluoride levels on
enamel surfaces,[37] but its effectiveness is limited since salivary flow rapidly washes away the deposited
fluoride.[38] In contrast, Bio particles, with their high bioactivity, rapidly form an HCA layer
similar to natural enamel mineral, explaining their superior KHN values.
The BS had a more basic pH (= 9.5) due to direct dissolution in water, accelerating
the reaction rate. However, excessively high reaction rates can hinder the formation
of the silica gel-rich layer, reducing ion uptake, and consequently, remineralization.[39] This likely explains why BS resulted in a similar relative KHN to fluoride toothpastes,
rather than a higher relative KHN as observed with ET containing Bio.
In conclusion, all evaluated treatments showed similar remineralizing capacity. However,
both Bio and fluoride have limitations. Bio requires time to initiate the remineralization
process, while fluoride, although readily available, is swiftly washed away from the
enamel surface.
One limitation of our study is that the ET with fluoride lacked water, potentially
limiting its efficiency. Further studies should explore the effectiveness of these
toothpaste formulations, including variations in concentrations and particle sizes
of abrasives and Bio. Additionally, investigating the synergy between Bio and fluoride
could yield valuable insights, as prior studies suggest potential benefits, such as
fluoride release in the oral cavity and potential anticarcinogenic properties.[40]
[41]
Conclusion
Within the limitations of the study, we can conclude that the toothpaste incorporating
10% Bio particles exhibited comparable abrasiveness to the commercial toothpaste.
While there was no disparity in remineralization potential between the treatments,
it is worth noting that the KHN of dental enamel following treatment with the experimental
Bio-containing toothpaste exceeded that of the fluoride-based toothpaste. Consequently,
the Bio-infused toothpaste may be a viable option for addressing WSL in children.
