Keywords
octenidine - chlorhexidine - benzydamine - polyhexamethylene biguanide - hexetidine
- staining - black tea - tooth color
Introduction
Antiseptic mouth rinses are used in home oral care to support oral biofilm management
if mechanical plaque control is temporarily impaired, for example, after oral surgical
interventions, during orthodontic therapy with fixed appliances or as an additional
measure for the treatment of gingivitis or periodontitis.[1] In general, antiseptic agents need to have a broad, nonspecific antibacterial efficacy
toward all bacterial species as well as fungi on the one hand and a low systemic cytotoxicity
on the other hand. Furthermore, a long-lasting substantivity is needed to prevent
immediate washout of the active substances from the mouth by the salivary flow.[2]
Currently, chlorhexidine digluconate (CHX), a bisbiguanide, is the most effective
antimicrobial agent used in antiseptic mouth rinses, which has been demonstrated by
a multitude of studies.[3]
[4]
[5] The cationic CHX molecules bind to the negatively charged sites of the bacterial
cell membrane and to the tissue surfaces of the oral cavity, thus providing a sustained
depot effect. The interaction with bacterial cells results in autolysis and cell death.
However, the use of CHX containing antiseptic oral rinses can cause mucosal irritation
and dysgeusia.[6] Studies have shown that CHX application promotes calculus formation.[7] Furthermore, rinsing with CHX in combination with staining dietary components can
lead to accumulation of staining products on the tooth and mucosal surfaces, resulting
in undesired extrinsic tooth discoloration.[8] These possible side effects of CHX led to the development of equivalent alternatives.
Alternatives like octenidine dihydrochloride (OCT) and octenidine dihydrochloride + 2-phenoxyethanol
(OCTP) show better biocompatibility than CHX,[9] possibly attributable to the lack of an amide and ester structure in its molecule.[10] Besides, benzydamine hydrochloride (BNZ), polyhexamethylene biguanide hydrochloride
(PHMB), and hexetidine gluconate (HEX) are further alternative antibacterial agents
in oral rinsing products.[11]
[12] While the staining potential of CHX-containing rinsing solutions has been investigated
extensively in vitro, there is a lack of data from products containing OCT, OCTP, BNZ, PHMB, and HEX.
The staining potential of mouth rinses is assessed either in vivo or in vitro.[13]
[14] In in vivo studies, the discoloration index provides an opportunity to estimate the extent of
discoloration per tooth in the oral cavity and is often used to assess the color,
intensity, and distribution of extrinsic stains among vestibular and lingual tooth
surfaces.[14] Using in vitro models, artificial materials are often treated with saliva and mouth rinses followed
by rinsing with a staining media (e.g., black tea, red wine, coffee). Color changes
are assessed by recording the optical density using a ultraviolet/visible double beam
spectrophotometer[15] or L*a*b values using a spectrophotometer calculating the color difference ΔE.[16]
[17]
[18] Here, the staining behavior of mouth rinses can be evaluated in comprehensive screenings
over few days under standardized conditions. Preceding the in vivo tests, results of such screenings can be used for a preselection of the best suited
rinsing components or products to be included in the investigation. Thus, the efficiency
of the clinical investigations can be significantly increased considering the long
time needed for in vivo tests (e.g., 4 weeks–6 months) for each product.[19]
To provide the most useful results in vitro models should mirror the development of discolorations caused by application of mouth
rinses including cleaning in the oral cavity as realistically as possible. On the
other hand, daily cleaning with toothpaste and toothbrush is currently not considered
in the existing in vitro models. Avoidance of integrating tooth brushing sequences in staining testing models
is probably due to the detrimental effects of cleaning compared to the building up
of staining, in so far, to integrate intermediate cleaning cycles in the testing protocol
may result in a decrease of sensitivity and selectivity when the staining potential
of oral care products is to be evaluated. Possibly, the findings of any clear differences
comparing alternative products are interfered, thus impede the correlation with in vivo testing. At least, it may drastically increase testing cycle numbers and time to
reach significance in the evaluation results, therefore essentially reducing the efficiency
of in vitro testing.
The aim of this study was to develop and test an in vitro test model for investigations of the staining potential of mouth rinses on human
tooth surfaces that also takes tooth brushing into account. In addition to better
mimicking the reality in an oral cavity, the model provides also potential to investigate
the opposite effects of discoloration-built ups by mouth rinse application in combination
with dietary factors on the one hand, and tooth brushing on the other hand. This means
that also the adherence of the staining to the tooth surface and its resistance against
cleaning can be considered. The null hypothesis was that the staining potential of
mouth rinses with different ingredients and concentrations cannot be clearly differentiated
using the developed in vitro model.
Materials and Methods
Sample Preparation
Human teeth were acquired from Indiana University School of Dentistry, Oral Health
Research Institute. Entire human molars with cervical dentine area were used. The
roots were separated, and the crowns were sectioned in halves by a water-cooled diamond
saw (Minitom, Struers). The halved tooth crowns were fixed (UHU plus endfest 300,
two-component epoxy resin adhesive, UHU GmbH & Co. KG, Bühl, Germany) to a microscopic
slide.
Treatment Groups
[Table 1] shows the treatment groups (mouth rinses) as well as the including actives and concentrations,
the brand and manufacturer of each.
Table 1
Treatment products (mouth rinses)
|
Abbreviation of the treatment groups
|
Actives and concentration
|
Brand and manufacturer
|
|
0.15% BNZ
|
0.15% benzydamine hydrochloride
|
Tantum Verde, Angelini Pharma Deutschland GmbH, Munich, Germany
|
|
0.1% PHMB
|
0.1% polyhexamethylene biguanide hydrochloride
|
ProntOral, B. Braun SE, Melsungen, Germany
|
|
0.2% CHX1
|
0.2% chlorhexidine digluconate
|
Meridol med CHX 0.2%, CP GABA GmbH, Hamburg, Germany
|
|
0.2% CHX2
|
0.2% chlorhexidine digluconate
|
Chlorhexamed Forte, 0.2% CHX, GlaxoSmithKline Consumer Healthcare GmbH & Co. KG, Munich,
Germany
|
|
0.1% HEX
|
0.1% hexetidine gluconate
|
Hexoral, Johnson & Johnson GmbH, Neuss, Germany
|
|
0.08% OCTP
|
Octenidine dihydrochloride,
< 1% 2-phenoxyethanol
|
Octenident, Schülke & Mayr GmbH, Norderstedt, Germany
|
|
0.1% OCTP
|
0.1% octenidine dihydrochloride, 2% 2-phenoxyethanol
|
Octenisept, Schülke & Mayr GmbH, Norderstedt, Germany
|
|
0.1% OCT
|
0.1% octenidine dihydrochloride
|
Octenident antiseptic, Schülke & Mayr GmbH, Norderstedt, Germany
|
Experimental Approach
The test procedure is depicted in [Fig. 1]. The experiments were designed to simulate a 15-day consumer application period,
assuming two applications of the rinsing solution per day (30 applications in total)
and tooth brushing twice daily (30 applications in total). Sets of six specimens were
placed in a 50 mL centrifuge tube. The specimens were pretreated with artificial saliva[20] for 30 minutes at 37°C to simulate the pellicle formation. Afterward, the specimens
were shortly rinsed in a beaker with 200 mL of distilled water under standardized
conditions. For the subsequent cyclic procedure, specimens were stored in artificial
saliva (2 min), rinsed with distilled water, exposed to warm black tea (50°C, 1 min)
and rinsed with distilled water again, followed by an optical measurement (only in
cycle 2–30). Then the specimens were brushed for 5 seconds, rinsed with distilled
water, and measured again optically (only in cycle 2–30). Afterward, the specimens
were placed in rinsing solution (30 s) and rinsed with distilled water. The total
number of cycles was 30. Ten cycles were performed per day on 3 consecutive days.
Samples were dried before each optical measurement.
Fig. 1 Developed test procedure.
A standard tea solution was made using Typhoo One cup (Typhoo, United Kingdom). To
receive a defined concentration, the solution was prepared by brewing one bag of tea
in 100 mL of freshly boiled distilled water for 5 minutes. After stirring and filtering
through double layer gauze, the tea solution was stored for distribution at 50°C.
Black tea and rinsing solution were renewed after every second cycle.
Before brushing, the toothbrushes (Oral-B Indicator 35 medium, Procter & Gamble Company,
Cincinnati, Ohio, United States) were wetted with distilled water under standardized
conditions. Brushing was performed in a brushing simulator (ZM 3-8, SD-Mechatronik,
Feldkirchen-Westerham, Germany) with zig-zag movements and an applied load of 2 N
(stroke frequency 1.6 Hz). A time of 5 seconds for brushing a tooth is assumed to
be representative; therefore, 5 seconds per tooth sample per cycle were chosen.[21] The toothpaste was applied as an aqueous slurry. Therefore, the toothpaste (medium
abrasive Colgate Total, Colgate Palmolive Company, New York City, United States) was
used at a dilution of 1:3 with water (w/w). The samples were carefully rinsed with
distilled water after brushing treatment.
Photographs and Color Measurements
Photographic images were taken before the start, after 10, 20, and 30 cycles with
a reflex camera (Canon EOS 600D, Canon GmbH, Munich, Germany) using a photo measuring
point, a black sample background, and with always the same camera settings to demonstrate
possible staining effects. Color measurements were conducted after each staining and
brushing step, that is, 20 times per day using a VITA Easyshade Compact spectrophotometer
(VITA Zahnfabrik, Bad Säckingen, Germany).
All color measurements were performed in the same room and from the same operator.
In accordance with the manufacturer's instructions, the spectrophotometer was calibrated
by measuring a white disc before taking measurements. After each human molar was placed
on a neutral background, the tip of the spectrophotometer was placed in contact with
and perpendicular to the middle of the tooth.
Values were recorded using the Hunter L*a*b* color scale, which is a color space with
coordinates for lightness (white-black, L*), where the maximum for L* is 100 (a perfect
reflecting diffuser) and the minimum would be zero (black). The scale also represents
redness-greenness (a*), where negative a* is green and positive a* is red, and yellowness-blueness
(b*), where negative b* is blue and positive b* is yellow.
ΔE, which is the total color difference between initial situation (before staining)
and treated specimens (after staining or staining and brushing), is calculated according
to the following equation:
The normality of the mean ΔE values was analyzed using the Shapiro–Wilk test. The
normally distributed data was statistical analyzed using one-way analysis of variance
with post-hoc Tukey honestly significant difference-test and Levene's test for analyses
of homogeneity of variance (Origin2019b, OriginLab Corporation Company, United States).
The level of significance α was set to 0.05.
Results
In [Fig. 2], exemplary images are shown for one enamel sample per test group, for the initial
state and the state after 10, 20, and 30 treatment cycles. The respective ∆L and ∆E
values determined by the color measurements are shown in [Figs 3] to [4] and in the [Supplementary Material Figs. 1–2] (available in the online version). The results of the statistical analysis are presented
in the [Supplementary Material Tables 1] to [3] (available in the online version).
Fig. 2 Exemplary photographic images of the enamel specimens before and after 10, 20, 30
treatment cycles with different rinsing solutions in combination with black tea. BNZ,
benzydamine hydrochloride; CHX, chlorhexidine digluconate; HEX, hexetidine gluconate;
OCT, octenidine dihydrochloride; OCTP, octenidine dihydrochloride + 2-phenoxyethanol;
PHMB, polyhexamethylene biguanide hydrochloride.
Fig. 3 Exemplary progression of the ∆E (A) and ∆L (B) mean values (with standard deviation) of the human enamel samples during treatment
with 0.2% chlorhexidine digluconate 2 (CHX2) and 0.08% octenidine dihydrochloride + 2-phenoxyethanol
(OCTP).
Fig. 4 ∆E mean values (with standard deviation) of the human enamel samples after treatment
with different rinsing solutions: after 10 cycles, after 20 cycles, after 30 cycles;
Mean values that do not have a common letter are significantly different (p ≤ 0.05). BNZ, benzydamine hydrochloride; CHX, chlorhexidine digluconate; HEX, hexetidine
gluconate; OCT, octenidine dihydrochloride; OCTP, octenidine dihydrochloride + 2-phenoxyethanol;
PHMB, polyhexamethylene biguanide hydrochloride.
[Fig. 3] shows a representative progression of the ΔE and ΔL values for the products that
caused the highest (0.2% CHX2) and lowest staining intensity (0.08% OCTP). ΔE and
ΔL of the enamel surfaces increased after treatment with all mouth rinses. Due to
the alternating staining and brushing, a zig-zag progression of the ΔE data was obtained
([Fig. 3], [Supplementary Material Fig. 1], available in the online version). For ΔL data, the zig-zag progression is less
pronounced ([Supplementary Material Fig. 2], available in the online version). The results show that black tea staining was
not completely removed by brushing with toothpaste during each cycle. Up to 10 cycles,
ΔL and ΔE values increased for all mouth rinses with each additional cycle number,
that is, the samples became progressively darker, and black tea staining built up
continuously. Between treatment cycle 10 and 30, the ΔL and ΔE values reached a plateau
after treatment with 0.08% OCTP, while ΔL and ΔE values continued to increase for
the treatment with 0.2% CHX2, thus contributing to further discoloration. Similar
progression curves as shown for 0.08% OCTP were also observed for 0.1% OCTP, 0.1 OCT,
and 0.1% HEX ([Supplementary Material Figs. 1B] and [2B], available in the online version). For 0.15% BNZ, 0.1% PHMB, 0.2% CHX1, the curve
progression of ΔL and ΔE was comparable to the one of 0.2% CHX2 ([Supplementary Material Figs. 1A] and [2B], available in the online version).
[Fig. 4] shows the mean ΔE values of the human enamel samples after 10, 20, and 30 cycles.
After cycle 10, lowest staining was measured for treatment with 0.1% HEX and 0.1%
OCT followed by 0.08% OCTP and 0.1% OCTP. Significant differences were found for 0.1%
HEX, 0.08% OCTP, 0.1% OCTP, and 0.1% OCT as compared to 0.2% CHX1 and 0.2% CHX2 ([Fig. 4]). After cycle 20, significantly lower staining was determined for, 0.1% HEX, 0.08%
OCTP, 0.1% OCTP, and 0.1% OCT as compared to all other tested rinsing solutions. After
cycle 30, the lowest staining was recorded for treatment with 0.08% OCTP, 0.1% HEX,
and 0.1% OCT followed by 0.1% OCTP. Significant differences were determined for 0.1%
HEX, 0.08% OCTP, and 0.1% OCT as compared to 0.15% BNZ, 0.1% PHMB, 0.2% CHX1, and
0.2% CHX2. Furthermore, ΔE-values were significantly lower after treatment with 0.1%
OCTP as compared to 0.2% CHX1 and 0.2% CHX2.
Discussion
The experimental approach of this study is new and partially exploratory in nature.
It shows changes in the tooth color after soaking in antiseptic oral rinsing solutions
and black tea as well as after tooth brushing over a simulated application period
of 15 days. To the authors' knowledge, no in vitro data on the development of discoloration on human teeth after cyclic staining treatment
including toothbrushing have been published so far. Most of the published studies
only investigate the staining potential of mouth rinses in combination with coloring
foods[18]
[22] or examine the ability of oral care products (e.g., toothpastes) to remove tooth
staining.[17]
[23]
To investigate the tooth color changes in vitro, samples are usually treated cyclically[16]
[24] or continuously.[25]
[26] To assess the tooth color, different instrumental objective measurements using spectrophotometers,
colorimeters, and image analysis techniques are performed.[8] First, Prayitno and Addy developed an in vitro model to assess dental discolorations caused by rinsing with CHX solutions of different
concentrations in combination with black tea.[15] Therefore, transparent polymethyl methacrylate (PMMA) specimens were alternately
stained by exposing to saliva, a CHX-containing mouth rinse and a black tea solution.
PMMA is used due to a high degree of standardization. A study has shown that these
in vitro tests (without any consideration of daily mechanical cleaning) are in qualitatively
agreement with the clinical situation.[13] Discoloration appears by interaction between the positively charged active (CHX)
and the anionic staining dietary components. The deposited complexes produced in vitro are identical in color to those seen on teeth clinically.[27] CHX alone does not stain but binds selectively to the anionic staining dietary chromogens,
causing discoloration.[24] It is also stated that a salivary pellicle is not essential to perform discoloration
but acts to enhance staining.[27]
The experimental approach[15] which is characterized by cyclic rinsing of PMMA samples with saliva, antiseptic
mouth rinses and black tea, was also used with an integrated brushing step to assess
the stain removal by toothbrushes and toothpastes.[28]
[29] However, since the same study performed in vivo and in vitro showed different results, the author's concluded that the findings of the in vitro approach are unlikely to be of clinical significance and models need to be developed
on dental hard tissues.[28]
[30] Therefore, human tooth crowns were used in this study instead of artificial materials
to develop an in vitro model that mimics the oral situation more accurately and may better predict the progression
of discoloration caused by antiseptic mouth rinses and black tea considering tooth
brushing.
In this in vitro model, treatment times with mouth rinses, staining with black tea, and tooth brushing
were adapted to simulate the situation in the oral cavity in real life as close as
possible. The treatments were highly standardized and controlled. Extremely long treatment
times in the respective medium, as also stated in literature,[26]
[31] were omitted. In addition, the tooth color was measured after each staining cycle
as well as after each brushing step to monitor the progressive stain development,
rather than only once at the end of the experimental procedure.
Brushing with toothpaste was included to mirror oral hygiene practices as per general
recommendations. It means that also the adherence of the staining products to the
tooth surface and their resistance against cleaning is considered in the test results.
It is also reflected in the measurement results that the brushing step is effective
in reducing stain, although complete stain removal was not achieved. The applied discoloration
is thus gradually accumulated with increasing number of cycles, allowing also to assess
the staining kinetics as a function of cycle numbers.
The null hypothesis of the study has to be rejected (see [Fig. 4]) since these findings are in contrast to the assumption that including the detrimental
effects of intermediate tooth cleaning in a staining testing model will impede the
sensitivity and significance of the results or drastically reduce the effectiveness
of the in vitro testing. It was shown that the proposed model allowed to quantify both the discoloration
intensity and kinetics for all products investigated. In particular, the results allowed
a clear differentiation between different mouth rinses, demonstrating the applicability
of the model for a more realistic in vitro evaluation of the staining potential of oral care products.
Regarding a detailed interpretation of the observed differences for the investigated
products, it has to be considered that little background information on the composition
of the formulations was available. Therefore, it is difficult to derive further conclusions
from these findings on the actual active interaction and the dependence on concentration.
The high staining of CHX in combination with black tea was expected (as already mentioned)
and can be explained by binding of positively charged active ingredient on all negatively
charged dental surfaces.[8] Since all other tested mouth rinses include cationic active ingredients (BNZ, PHMB,
HEX, OCT, OCTP), the same or similar staining mechanisms can be assumed. The observed
differences in staining over time may be attributed to different degrees of adhesion
of the active ingredient to the pellicle or enamel surface. It is conceivable that
the accumulated staining caused by treatment with OCT, OCTP and HEX can be more easily
removed by brushing, as demonstrated by only a slight increase in staining over time.
A less pronounced staining of teeth and dentures by HEX has also been described as
compared to CHX[12]
[32] and this study results confirm earlier findings from in vivo studies reported in literature. Furthermore, mouth rinses with active ingredients
BNZ and PHMB caused a higher dental staining as compared to OCT and OCTP and lower
staining than CHX in this study. The results of this study are also in good agreement
with the literature,[11] where BNZ was found to cause lower light and dark shades on a provisional acrylic
resin than after treatment with a CHX-containing mouth rinse. These agreements underline
the applicability of the new model. For PHMB, no data in context of dental discolorations
were found in the literature.
However, the staining potential of antiseptic mouth rinses is an undesirable side
effect compared to the products efficacy—the antibacterial effect. However, some authors
describe that the degree of dental staining caused by CHX-containing mouth rinses
can also be used as an indicator for the antimicrobial activity.[33] Nevertheless, it is currently not known whether this statement also applies to other
antiseptic agents (e.g., for OCT). In comparison to CHX, OCT showed a similar or even
stronger antibacterial effect in clinical studies[34]
[35] and according to a recent review OCT is considered either superior or comparable
to CHX-based mouthwashes in controlling dental plaque.[36] The staining potential can be described as mild[37] or as generally removable by toothbrushing.[38]
[39]
[40] As shown in the literature, relatively little formulations have been studied in
this regard and further studies focusing on antimicrobial activity in relation to
staining potential should be conducted.
The developed model could be a beneficial tool due the consideration of more realistic
treatment conditions and the sensitivity and selectivity regarding different rinsing
components as demonstrated in this study.
Conclusion
A new testing model for the in vivo assessment of the staining potential of oral care mouth rinses has been proposed
and successfully tested. The testing procedure is based on staining treatments of
human teeth but includes also tooth brushing cycles to mimic the reality of dental
hygiene and to consider the adherence of staining products to the tooth surface. It
was shown that the testing model allows a clear differentiation of different mouth
rinses regarding staining intensity and kinetics.
