Keywords
teeth whitening - hydroxyapatite - oral care - spectrophotometry
Introduction
Oral care products and formulations for teeth whitening have gained an increased attention
worldwide. Many people prefer white teeth since its discoloration may affect their
quality of life.[1] Besides, professional applications (e.g., bleaching with peroxides and professional
dental cleaning), various whitening products for home use are commercially available.
This includes, for example, whitening toothpastes, mouthwashes, and gels.[1]
[2]
Whitening formulations often contain abrasive agents (e.g. hydrated silica, alumina,
perlite), chemical agents (e.g. phosphates), or optical agents (e.g. blue covarine).[1]
[2] However, highly abrasive agents and peroxides may induce unwanted side effects.[3]
[4] Toothpastes with high radioactive dentin abrasion (RDA) values may be harmful to
exposed dentin and gingiva. Additionally, the use of peroxides could lead to tooth
sensitivity and unwanted damage of the organic matrix of both enamel and dentin.[1] Therefore, research has focused on alternative nonoxidative, and less-abrasive tooth
whitening agents.[5] One of these agents belong to the group of calcium phosphates like particulate hydroxyapatite
(HAP; Ca5[PO4]3[OH]).[1]
[5]
[6]
[7]
[8]
[9] In particular, biomimetic HAP is inspired by structure and composition of natural
enamel crystallites[10] and shows a broad range of applications in preventive oral health care,[11]
[12]
[13]
[14]
[15]
[16]
[17] such as antibiofilm properties.[18] A recently published in vivo study has shown its positive properties to release
calcium in dental biofilms.[19] This can be helpful to buffer cariogenic biofilms, and with increased calcium-levels
leading to a shift from tooth demineralization to remineralization.[11]
[17]
The aim of this in vitro study was to test the whitening effect of a newly developed
gel formulation based on microcrystalline HAP. To exclude any abrasive influence by
toothpaste abrasives harder than natural enamel,[20] and more importantly by the toothbrush that is used in daily oral care, this HAP-gel
formulation does not contain commonly used abrasives (e.g. hydrated silica) and was
applied by finger. This fact is of importance since the whitening effect of the toothpaste
is mainly determined by abrasive ingredients and the mechanical cleaning efficacy
of the toothbrush.[1] The assumed mode of action of the newly developed whitening formulation with HAP
is based on its adhesion of particles on the enamel surface.
Consequently, the focus of this in vitro study was the analysis of the specific whitening
effect of a HAP-containing containing gel. This would be helpful for individuals who
prefer a brighter appearance of their teeth but are suffering from dentin hypersensitivity
or patients suffering from gingivitis or periodontitis. Our hypothesis was that HAP
would change the appearance of the natural tooth color to a brighter (whiter) color
without having abrasive and/or oxidizing properties. A nonabrasive, commercially available
whitening mouth rinse based on ethanol and phosphates served as positive control,
and distilled water as negative control. The whitening effects of the different formulations
were tested on bovine enamel samples using a pre–post design.
Materials and Methods
Treatment Products and Control Group
The whitening properties of three different groups were tested as follows:
-
Test product: HAP-based (15% w/w) oral care gel (Karex gelée; Dr. Kurt Wolff GmbH
& Co. KG, Bielefeld, Germany).
Aqua, hydroxyapatite, glycerin, hydrogenated starch hydrolysate, calcium lactate,
hydroxyethylcellulose PEG 40, hydrogenated castor oil, xylitol, calcium carbonate,
hydroxyacetophenone, 1,2-hexanediol, caprylyl glycol, aroma, stevia rebaudiana leaf/stem
powder, propylene glycol, sodium hydroxide, limonene, citral.
-
Positive control: whitening mouth rinse (Listerine Advance White, Johnson & Johnson
GmbH, Neuss, Germany).
Aqua, alcohol, sorbitol, tetrapotassium pyrophosphate, pentasodium triphosphate, citric
acid, poloxamer 407, sodium benzoate, eucalyptol, thymol, menthol, sodium sacharin,
sodium fluoride (220 ppm F), tetrasodium pyrophosphate, propylene glycol, sucralose,
aroma, disodium phosphate.
-
Negative control: distilled water (pH = 6.8).
Sample preparation
Bovine incisors were cleaned and embedded in epoxy resin (EpoFix; Struers, Cleveland,
Ohio, United States). The buccal enamel surface was grinded to 1,200 grit by SiC abrasive
paper (Struers, Cleveland, Ohio, United States). All enamel samples were stored in
distilled water for 24 hours before starting the experiments.
Treatment with Test Product and Control Group
To ensure a comparison of the experimental data, all enamel samples were treated for
1 minute with the test products. The whole test procedure is depicted in [Fig. 1].
Fig. 1 Overview of the test procedure for the analysis of (a) HAP-gel, (b) mouth rinse (positive control), and (c) distilled water (negative control). HAP, hydroxyapatite.
-
(a) HAP-gel: The HAP gel was applied by finger on the enamel surface (2 seconds, n = 8). After 1 minute, the samples were rinsed under agitation with distilled water
and air dried at room temperature. The treatment procedure was repeated twice (three
applications in total). Color measurements were carried out after first and third
cycles.
-
(b) Mouth rinse and (c) control group: Enamel samples... were stored in group b (mouth
rinse [n = 8]) or c (distilled water [n = 4]) for 1 minute by using a laboratory shaker. Afterwards the samples were rinsed
under agitation with distilled water and air dried at room temperature. The treatment
procedure was repeated eight times (nine applications in total). Color measurements
were carried out after first, third, and ninth cycles. Nine treatment cycles were
chosen for the mouth rinse testing in order to simulate a longer treatment period
which is according to the manufacturers’ instructions.
Color Measurements
Tooth color and color changes (ΔE) were analyzed by a spectrophotometer (CM-3600A,
Konica Minolta Sensing Europe B.V., Bremen, Germany) using a (L*a*b*) color space
with coordinates: white–black (±L*), redness–greenness (±a*), and yellow–blueness
(±b*). Color changes between the different measurement and the baseline measurement
in each group were calculated by using the following equation:[21]
[22]
ΔL*= L* after treatment–L* initial
Δa*= a* after treatment–a* initial
Δb*= b* after treatment–b* initial
Statistical Analysis
Statistical analyses were conducted by one-way analysis of variance (ANOVA) with post
hoc Bonferroni’s test and Levene’s test for analyses of homogeneity of variance (Origin
2019b; OriginLab Corporation Company, Northampton, Massachusetts, United States).
The level of significance of α was set at ≤0.05.
Scanning Electron Microscopy
Surface analyses of the enamel samples at baseline and after one treatment cycle were
performed by scanning electron microscopy (SEM; Quanta 3D FEG scanning electron microscope,
FEI Company, Hillsboro, Oregon, United States). The samples were coated with an ultra-thin
carbon film by evaporation before the SEM analyses.
Results
Color Measurement
Color measurement shows a significant increase in ∆E* after one cycle (5.14 [±2.66],
p ≤ 0.006) and after three cycles (11.2 [±3.11], p < 0.0001) in group (a) (HAP-group) compared to group (b) (whitening mouth rinse),
and group (c) (water), respectively ([Fig. 2]). No significant increase in ∆E was measured in group (b) after one cycle. Group
(b) showed an increase in ∆E after three cycles (2.77 [±1.01], p = 0.02) and nine cycles (ΔE = 3.27 [±1.61], p = 0.006) compared to group (c). A similar trend as for ∆E* could be revealed for
ΔL* and Δa* ([Table 1]). These values increased after treatment with HAP-gel (group a) and whitening mouth
rinse (group b) compared to water (group c), representing an increase in brightness
and reddish (minor color shift). (minor color shift). For groups (a) and (b), a correlation
on ΔL* and Δa* was observed with an increasing cycle number.
Fig. 2 ΔE* values of the HAP-gel (group a), mouth rinse (group b), and water (group c).
In (a) color measurements were performed after 1 cycle and 3 cycles, while in (b) and (c) color measurements were also performed after 9 cycles. HAP, hydroxyapatite.
Table 1
Mean ∆L*, ∆a*, and ∆b* values after cycle 1, 3, or 9
|
∆L*
|
After cycle 1
|
After cycle 3
|
After cycle 9
|
|
Abbreviation: HAP, hydroxyapatite.
|
|
HAP-gel
|
4.51 [±2.48]
|
10.66 [±2.97]
|
Not performed
|
|
Mouth rinse
|
0.93 [±0.81]
|
2.31 [±1.02]
|
3.08 [±1.43]
|
|
Water
|
0.15 [±0.33]
|
0.32 [±0.43]
|
0.07 [±0.22]
|
|
∆a*
|
|
HAP-gel
|
0.59 [±0.17]
|
1.10 [±0.19]
|
Not performed
|
|
Mouth rinse
|
0.08 [±0.13]
|
0.15 [±0.16]
|
0.29 [±0.35]
|
|
Water
|
0.15 [±0.33]
|
0.06 [±0.06]
|
0.01 [±0.03]
|
|
∆b*
|
|
HAP-gel
|
-1.77 [±1.60]
|
-2.87 [±1.39]
|
Not performed
|
|
Mouth rinse
|
-0.62 [±1.00]
|
-0.88 [±1.19]
|
-0.07 [±1.12]
|
|
Water
|
0.16 [±0.35]
|
-0.29 [±0.35]
|
-0.23 [±0.33]
|
∆b* decreased significantly in group a, representing a decrease in yellowness of the
enamel surface in the HAP-group. For groups (b) and (c), this effect could not be
detected.
Scanning Electron Microscopy
SEM images show that the initial enamel surface is characterized by grinding marks
resulting from the sample preparation ([Fig. 3A]). After the treatment with the group (a), deposited particles were visible on the
enamel surfaces ([Fig. 3B]). It can be assumed that the particles consist of HAP. High-resolution SEM images
of the deposited particles revealed rod and flat shaped crystallites ([Fig. 4]), that could be assigned to HAP since its microstructure is known and described.[10]
Fig. 3 SEM images of enamel surfaces before and after treatment: (A) baseline, (B) after HAP-gel treatment, (C) after mouth rinse treatment, (D) after water treatment. HAP, hydroxyapatite; SEM, scanning electron microscope.
Fig. 4 High-resolution SEM image of the deposited particles after HAP-gel treatment. HAP,
hydroxyapatite; SEM, scanning electron microscope.
After treatment with group (b), the enamel surface seems to be slightly smoothed by
a thin layer formation compared to the initial sample surface ([Fig. 3C]). Furthermore, single particles were visible on the surface. The origin of these
particles is not known. No changes could be detected after treatment with group (c)
([Fig. 3D]).
Discussions
This in vitro study shows that the tested HAP oral gel led to an increase in ΔE, thus
to a whiter tooth color already after one-time application. This effect could be enhanced
by regu- larly performed applications of the gel ([Fig. 2]). The tooth whitening properties of the HAP-gel can be explained by HAP’s adhesion
to the tooth surface. Fabritius-Vilpoux et al showed in an in vitro study that HAP
particles of a mouth rinse adhere to enamel surfaces;[10] this is confirmed by the SEM investigation performed in the present study. The
quantity of adhesion can be increased by higher concentrations of HAP, i.e. 10% HAP
showed a higher enamel coverage compared to 1 and 5% HAP.[10] Additionally, Niwa et al found a whitening optimum by using 15% HAP in a toothpaste
formulation.[9] Based on these findings, the concentration of HAP was chosen to be of 15% in the
tested oral care gel. Kensche et al and Lelli et al confirmed the adhesion of HAP
to tooth surfaces also under in situ conditions and with an ex–in vivo study design,
respectively.[23]
[24] Our results are in good agreement with other studies that analyzed the teeth whitening
effects of HAP.[5]
[6]
[7]
[8]
[9]
[25] Niwa et al, for example, analyzed the in vivo whitening effect of toothpastes with
0, 3, and 15% HAP. They found that the whitening effect could be increased by higher
HAP concentrations. Interestingly, in an additional in vitro experiment it was shown
that the polishing properties were not altered when higher HAP concentrations were
used.[9] This clearly underlines that HAP, in contrast to abrasives with a high-relative
hardness (e.g. perlite, a mineral of silicate; alumina, Al2O3),[20] is a suitable whitening agent which does not lead to a damage of tooth or gingiva.
Moreover, HAP reduces the roughness of the teeth.[25] Dabanoglu et al and Jin et al showed that different calcium phosphates including
HAP contribute to tooth whitening in vitro.[5]
[7] Besides the adhesion of HAP particles to the tooth surface as described above, remineralization
effect of HAP may also contribute to tooth whitening (i.e. due to a smoother surface
on which stains cannot attach).[9]
[11]
[26]
[27] An advantage of the use of the HAP in a gel formulation is the good adhesion to
the tooth surface. It can be easily applied by using the finger after tooth brushing
and also showed both remineralization effects[26] and erosion protective properties.[17]
[28] Bommer et al reported that a self-assembling peptide matrix can act as an adhesive
for HAP particles improving the whitening effect of HAP alone.[6] A similar effect could be observed in HAP group of our study. The matrix of the
HAP-gel may further increase the HAP-adhesion to the tooth surface compared to HAP-particles
alone (which already show a good adhesion to tooth surfaces[5]
[10]
[23]). Future studies should be carried out to analyze the whitening effect of the HAP-gel
also under in vivo conditions. To date, only a few in vivo studies on the whitening effects of HAP been published.[6]
[9]
[29]
In situ studies show an efficient reduction of bacterial colonization to enamel surfaces
by using HAP-based mouth rinses.[18]
[23]
[30] This might be also an important factor for tooth whitening since stains are often
incorporated into dental plaque.[1]
In contrast to the HAP-gel, the tested mouth rinse showed only minor whitening effects
in our in vitro setting after one, three, and nine cycles. This may be explained by
the different mode of action compared to HAP. The SEM images showed that HAP adheres
to the tooth surface; however, only a thin layer of unknown deposits was detectable
in the mouth rinse group ([Figs. 3B]
[3C]). Thus, the mouth rinse may support (e.g. by its ingredients ethanol and phosphates,
as well as by an acidic pH) the stain removal action of toothbrush and toothpaste,[31] but does not lead to the formation a white (protective) layer on the tooth surface.
Other test approaches are necessary to understand completely the whiting mechanisms
of such mouth rinses. Further testing should also involve model refinement, i.e. it
would be of interest to include experimental steps in the protocol in order to examine
the stability of the whitening effect against chemical (mimicking acid challenges
of daily food intake) and/or mechanical (mimicking tongue and mucosa movement or saliva
flow or tooth brushing) stress.
Additionally, oral-care formulations based on HAP show not only whitening properties,
but was also tested to be effective in preventing cavities, improvement of gingival
health, and reduction of dentin hypersensitivity.[11] Biomimetic ingredients based on HAP used in oral care show a high compatibility,
since the mineral phase of the teeth and bones mainly consists of calcium and phosphate.[32]
[33]
Conclusion
To conclude, the in vitro model tested in this study can be used as basic-approach for further testing of nonabrasive
whitening agents.
In this study, the nonabrasive HAP-gel showed the highest values on tooth-whitening
when compared to a mouth rinse with whitening-agents. Therefore, HAP particles may
be suited to be a gentle, fast, and biomimetic approach for cosmetic tooth whitening.
