Keywords
caries - energy-dispersive X-ray spectroscopy - hydroxyapatite - oral care - dental
plaque - calcium and phosphorus levels
Introduction
The presence of cariogenic dental biofilms (dental plaque) is a major factor for the
development of dental caries which is a disease globally affecting millions of individuals.[1]
[2] Preventive strategies for oral care are mainly based on (1) mechanical dental plaque
removal (e.g., tooth brushing and flossing), (2) antibacterial strategies based on
chemical agents (e.g., chlorhexidine, metal salts), and (3) remineralizing agents
(e.g., fluorides and calcium phosphates).[2]
[3] Interestingly, even by dental staff (i.e., dentists, dental students, and dental
assistants) it is not possible to achieve a complete plaque removal.[4] Dental plaque itself has a complex composition and contains more than 700 different
bacterial species.[5] While most of the oral microorganisms are commensals, some of the bacteria and fungi
(e.g., Candida albicans) are promoting the dental caries process. These bacteria are mainly acidic (acid
producer) and aciduric (acid resistant) species from the genera Streptococcus, Lactobacillus, Prevotella, Veillonella, and others.[2]
[6]
[7]
[8] Consequently, it is important to control the oral biofilm using mechanical and chemical
approaches. However, dental biofilms can also persist interdental or in dental fissures.[9] Therefore, agents are needed that can interact with dental biofilms and decrease
its ability to demineralize the tooth mineral and increase the remineralization.
Due to its high similarity to human enamel crystallites, particulate hydroxyapatite
(HAP, Ca5(PO4)3(OH)) is a promising biomimetic agent in oral care formulations that is used in different
products such as toothpastes, mouthwashes, and gels.[10]
[11] HAP has an excellent biocompatibility and, in contrast to fluorides, does not lead
to dental fluorosis, that is, it is ideally suited for all age groups including infants
and young children.[12]
Its application, in any reasonable dose, does not pose any risks for a patient’s health.[13] HAP particles interact with enamel surfaces as shown in vitro,[14] in situ,[15] and in vivo.[16]
[17] Clinical studies have shown effects of HAP-based toothpastes, for example, in prevention
of enamel caries[17] and reducing dentin hypersensitivity,[18] as well as in improving periodontal health.[19] Additionally, HAP particles reduce bacterial colonization to enamel surfaces in
situ similar to chlorhexidine without having antibacterial effects, but showing antiadherent
properties.[15]
HAP for oral care applications may differ in composition (e.g., substitutions of foreign
ions for calcium like zinc[16]), as well as in crystallite morphology, and crystallite size.[10] A recently published review article describes the multifunctionality of HAP and
its modes of action in preventive oral health care.[20] One of the described modes of action of HAP is the release of calcium and phosphate
ions (i.e., a partial dissolution of HAP) after an acidic attack (pH < 5.5), that
is, HAP may act as a calcium and phosphate reservoir when present at the tooth surface
and/or in the plaque. The simplified corresponding equation is given as follows:
Ca5(PO4)3(OH) (s) + 4 H+ (aq) → 5 Ca2+ (aq) + 3 HPO4
2- (aq) + H2O (l)
(s = solid; aq = aqueous; l = liquid)
Acidic pH values are easily reached in the oral cavity both due to erosion (e.g.,
by soft drinks: pH, approximately 2.5–3.5)[21] and by cariogenic biofilms (pH < 5.5).[2]
Shaw et al showed that plaque samples of caries-free children contain more calcium
and phosphorus than plaque of caries-positive children.[22] Bayrak et al also found higher calcium and phosphorus levels in plaque of caries-free
individuals compared with caries-positive individuals; however, the differences were
not statistically significant.[23] In the field of erosion protection, the presence of calcium and phosphate ions reduces
the erosive potential of acidic foods (e.g., milk or yogurt).[24] Likewise, adding HAP to acidic sport drinks decreases their erosive effect.[25]
Thus, increasing calcium and phosphate levels at the tooth surface and/or in plaque
by using calcium phosphate-based oral care products, is a promising preventive strategy
to influence the demineralization/remineralization process positively to remineralization.[20]
[26]
[27]
[28]
Therefore, the aim of this in vivo study was to analyze the 3-day effects of a newly
developed hydroxyapatite-based oral care gel on the calcium and phosphorus levels
within the dental plaque of children. To the best of the authors’ knowledge, this
is the first study analyzing the effects of HAP in dental plaque under in vivo conditions.
Materials and Methods
General Information
The study was registered at ClinicalTrials.gov (NCT03956992). It was conducted in
Kebon Padangan, Bali, Indonesia (August 08–10, 2019) and was approved by the local
health department. Parents and children gave their informed consent prior to their
inclusion into the study. We chose this region because the children there have high
amounts of dental plaque because daily tooth brushing is not common. Children were
recruited with the help of the local school. The following inclusion and exclusion
criteria were applied:
Inclusion criteria included the following:
Exclusion criteria included the following:
-
Allergy to one or more ingredients of the gel.
-
Use of medications (e.g., antibiotics).
-
Other significant reasons as decided by the principal investigator.
Case report forms were used which contained patient no., sex, date of birth (month/year),
DFM-T (permanent teeth) and dmf-t (primary teeth) indices (DMF-T/dmf-t: decayed, missing,
and filled teeth), inclusion and exclusion criteria, and an application diary.
In total, 34 patients (22 boys, 12 girls, mean age: 8.9 years; mean DMF-T: 0.6; mean
dmf-t: 4.5) were included in the study. All patients completed the study from start
to end. For each patient, baseline values (calcium and phosphorus) were used as control
(no treatment) and compared with the values measured in the dental plaque samples
collected at the end of the study.
Composition and Application of the Hydroxyapatite Gel
The formulation of the oral care gel was based on a newly developed gel (Karex gelée;
Dr. Kurt Wolff GmbH & Co. KG, Bielefeld, Germany), but slightly modified with the
aim to exclude all calcium-based ingredients, except for the main active ingredient,
that is, HAP microclusters.[14] Thus, possible changes before/after the study in plaque calcium and phosphorus levels
could be assigned to only one ingredient, that is, to HAP. The gel was filled in optically
neutral tubes and contained the following ingredients (International Nomenclature
of Cosmetic Ingredients [INCI]):
Ingredients: AQUA, HYDROXYAPATITE, GLYCERIN, HYDROGENATED STARCH HYDROLYSATE, HYDROXYETHYLCELLULOSE,
PEG-40 HYDROGENATED CASTOR OIL, XYLITOL, HYDROXYACETOPHENONE, 1,2-HEXANEDIOL, CAPRYLYL
GLYCOL, AROMA, STEVIA REBAUDIANA LEAF/STEM POWDER, PROPYLENE GLYCOL, SODIUM HYDROXIDE,
LIMONENE, CITRAL.
The safety of the HAP gel was confirmed by a cosmetic product safety report according
to the Regulation (EC) 1223/2009 on cosmetic products before starting the study.
The gel was manually applied (finger) for 3 days and thrice daily by an experienced
dentist (H.S.) using approximately 2 cm of the oral gel for the upper jaw and lower
jaw, respectively. The gel remained in the mouth, no immediate rinsing with water
was performed after the application. After application, the patients were sent home
without further instructions regarding eating, drinking, and others. In general, all
patients did not brush their teeth, as oral hygiene is not commonly used in this region.
Dental plaque was collected with a dental excavator (sterile, for single-use only).
To exclude any bias which might occur during the plaque collection, sampling altered
from subject to patient (i.e., A, B, A, B, …):
-
Plaque collection (baseline): quadrant one-fourth; plaque collection (after 3 days):
quadrant two-thirds.
-
Plaque collection (baseline): quadrant two-thirds; plaque collection (after 3 days):
quadrant one-fourth.
Plaque samples were stored in 2-mL Eppendorf tubes (DNA LoBind tubes PCR clean).
Course of the study
Day 1 (August 08, 2019):
Plaque collection (buccal and labial surfaces; no surfaces with calculus):
-
Application of the gel in the morning (after collecting of plaque).
-
Application of the gel at noon (after lunch).
-
Application of the gel in the evening (after dinner).
Day 2 (August 09, 2019):
-
Application of the gel in the morning (after breakfast).
-
Application of the gel at noon (after lunch).
-
Application of the gel in the evening (after dinner).
Day 3 (August 10, 2019):
-
Application of the gel in the morning (after breakfast).
-
Application of the gel at noon (after lunch).
-
Application of the gel in the evening (after dinner).
One hour after application of the gel: plaque collection (buccal and labial surfaces;
no surfaces with calculus).
Note: 29 patients received nine applications of the gel; 1 patient received eight
applications of the gel, 1 patient received seven applications of the gel, 3 patients
received six applications of the gel, 5 patients missed some applications for unknown
reasons; however, they received a minimum of six oral care gel applications (i.e.,
an average of twice daily application was ensured). Consequently, they were included
in the further analysis.
Analysis of Dental Plaque Samples
Calcium and phosphorus in dental plaque samples of 29 patients were quantitatively
analyzed by energy-dispersive X-ray spectroscopy[29] (EDX; Thermo Scientific UltraDry EDS X-ray detector; in combination with a scanning
electron microscope, Apreo S LoVac Thermo Scientific) at the University of Duisburg-Essen
(inorganic chemistry). Note that 34 patients were included into the study. We first
tried to analyze plaque samples of four randomly selected patients by atomic absorption
spectroscopy (AAS) because it is a very accurate analytical technique for the quantitative
analysis of calcium. However, due to insufficient quantity of plaque, we could not
perform AAS analyses. Additionally, one patient was excluded from statistical analysis
because of extremely high calcium values in the baseline samples (mean calcium for
this patient was 20.6 ± 2.71 wt% and mean calcium of all other patients was 0.74 ±
1.82 wt%). For the remaining 29 patients, we conducted three EDX analyses of plaque
samples at baseline and three EDX analyses of plaque samples after the study. Afterward,
the mean values were calculated for each patient (weight %), the plaque samples were
applied onto silicon single crystal substrates and dried at 60°C for 1 hour in air.
The EDX analyses were performed at 10 kV at a probe current of 0.9 nA. Quantitative
analysis was performed by calculating the relative concentrations of chemical elements
in the sample from the relative X-ray counts and applying matrix corrections. Here,
a standard less iterative Phi-Rho-Z matrix correction process was used for the depth distribution function (Phi), the
mass density (Rho), and the mean atomic number (Z), as well as for fluorescence correction. Element concentrations were determined
by multiplying a measured K-ratio by the matrix correction. The data are normalized
to 100% (C, N, O, Na, Mg, K, Ca, P, S, and Cl). Overall medians for both calcium and
phosphorus were calculated using the means of the 29 patients.
Statistical Tests
All means and standard deviations, as well as medians, were calculated using Microsoft
Excel. To test any statistical significance of the data (baseline means versus end
of study means), a two-sided t-test was performed using Microsoft Excel.
Results and Discussion
This study was conducted under in vivo conditions and showed that both median calcium
and phosphorus levels in dental plaque samples of children increased after using the
HAP-gel for 3 days ([Table 1]). The application of HAP led to an increase of calcium and phosphorus in the dental
plaque. However, the increase was not statistically significant (p > 0.05) but showed a tendency. Following this, the study indicates a potential interaction
of HAP and the oral biofilm and/or the tooth surface (i.e., new plaque grown on HAP-coated
tooth surfaces). This is in line with studies showing the interaction of HAP particles
with enamel surfaces.[14]
[15]
[16] For example, Kensche et al found an accumulation of HAP particles into the pellicle
after using a HAP-based mouthwash in a transmission electron microscopic (TEM) study.[30] The interaction may be based on mineral bridges between HAP particles and enamel
surfaces as shown by Fabritius-Vilpoux et al in a scanning electron microscopy (SEM)
study.[14] The interaction of HAP particles with oral bacteria was also shown in different
studies, for example, by Venegas et al[31] and Kensche et al,[15] Venegas et al analyzed the adhesion of HAP to S. mutans and different Lactobacillus
spp.,[31] which are prominent bacteria of dental plaque.[2] Some bacteria showed adhesion to enamel surfaces, dependent on the calcium-concentration
of the aqueous medium, used in this in vitro study.[31] Kensche et al analyzed spit-out samples after using a HAP-mouthwash (i.e., a HAP-water-dispersion,
without additives) and found an accumulation of HAP particles around bacteria by TEM.
This could explain the reduction of initial bacterial colonization to enamel surfaces
by using HAP-mouthwashes in situ.[15]
[32]
Table 1
Calcium and phosphorus levels of dental plaque at baseline and after 3 days application
of the HAP-gel in vivo (29 children)
|
EDX analyses
|
Baseline
|
After the study
|
|
Abbreviations: EDX, energy-dispersive X-ray; HAP, particulate hydroxyapatite; SD,
standard deviation.
|
|
Calcium
|
Median: 0.25 wt%
Mean with SD: 0.74 ± 1.82 wt%
|
Median: 0.40 wt%
Mean with SD: 0.54 ± 0.49 wt%
|
|
Phosphorus
|
Median: 1.17 wt%
Mean with SD: 1.25 ± 0.75 wt%
|
Median: 1.41 wt%
Mean with SD: 1.43 ± 0.58 wt%
|
Our study shows that HAP may be incorporated into the oral biofilm and/or may adhere
to dental plaque. The strength of the study is the in vivo setting within a highly
cariogenic study population (mean DMF-T 0.6; mean dmft-t 4.5) under real live conditions.
Each patient served as its own control: biologically occurring differences, that is,
in saliva composition, salivary flow, and others can be excluded. All patients used
only the HAP gel for oral hygiene instructions. Additionally, the diet was nearly
the same with all patients during the study period. The application of the gel was
standardized as it was performed by only one experienced dentist. Thus, variations
of the applications can be excluded. Sampling was also performed by the same dentist
at the beginning and at the end of the study. To exclude weakly attached HAP, collection
was performed 1 hour after the last gel application.
It is well known that atomic absorption spectroscopy (AAS) is the analytical method
to measure calcium at high accuracy. However, this method requires a minimum amount
of dry mass from a biologic sample. This amount could not be reached within this study.
Therefore, EDX-analyses were used to measure the quantity of calcium and phosphorus
in the samples. EDX only needs small amounts as it is coupled to SEM. However, it
only gives semiquantitative data unless the samples are polished and calibration standards
are used.[29] Other studies quantified calcium and phosphorus in plaque samples by AAS[22]
[33] (Ca), ion chromatography[23] (Ca and phosphate), or by microcolorimeter[34] (Ca and phosphate). Further suitable methods are inductively coupled plasma mass
spectrometry and X-ray fluorescence spectroscopy.
Nevertheless, the calcium and phosphorus content is comparable to results reported
by Shaw et al,[22] they analyzed dental plaque samples of 23 caries-free and 32 caries-active children
and found calcium contents for caries-active children (posterior plaque: 1.63 µg/mg
≙ 0.163 wt% and anterior plaque: 2.57 µg/mg ≙ 0.257 wt%). Although we did not differentiate
between anterior and posterior plaque, these values are comparable to our EDX-results
([Table 1]).
Furthermore, Shaw et al reported calcium levels of caries-free children (posterior
plaque: 3.57 µg/mg ≙ 0.357 wt% and anterior plaque: 11.55 µg/mg ≙ 1.155 wt%). These
results clearly show that a naturally (i.e., by saliva) increased calcium content
of the dental plaque is correlated with a decreased rate of caries.[22] It appears reasonable that by regularly using a HAP-based gel, the calcium content
of the dental plaque may be artificially increased.
The variation in our data was high as shown in [Table 1] which was also reported by Shaw et al (e.g., calcium [anterior plaque]: 1.8–36.5
µg/mg). These big variations can be explained by a high heterogeneity between the
individual patients.
HAP was the only calcium source of the gel. Therefore, it is likely that the increased
calcium content is due to an incorporation of HAP into the oral biofilm.
Zhang et al showed in an in vitro pH-cycling study that the calcium level in S. mutans biofilms treated with HAP was approximately eight-times higher than in a group treated
with sodium fluoride (NaF) This resulted in an enhanced enamel demineralization protection
as shown by transversal microradiography. However, no enamel remineralization was
observed.[33] In contrast, other in vitro and in situ studies have shown a remineralizing effect
of HAP-containing toothpastes on enamel and/or dentin surfaces. Recent studies were
published by Tschoppe et al[27] (in vitro study), Najibfard et al,[26] and Amaechi et al[28] (the latter are in situ studies).
Besides the release of calcium and phosphate ions (i.e., remineralization and minimizing
demineralization), HAP may also act as a buffer of organic acids in cariogenic biofilms.[17]
[35] Nedeljkovic et al analyzed the acid-buffering properties of different tooth restoration
materials in vitro. HAP and amalgam showed superior acid-buffering properties, compared
with a glass-ionomer cement and giomer (composite with surface prereacted glass ionomer
fillers; lower buffering capacity) and a conventional composite (no buffering capacity).
The authors concluded that restorative materials without buffering properties increase
the risk of shifting plaque composition to more cariogenic bacteria.[35] The buffering effect of HAP can be explained by the release of (hydrogen/dihydrogen)
phosphates after an acidic attack which may act similar to the phosphate buffer system
present in saliva.[2]
Due to the explorative character of this pilot study, limitations are that only gel
with HAP was used but no placebo gel or control gel. Additionally, patients were not
under observation after gel applications. Thus, it was not possible to monitor their
food intake. Finally, future studies should focus on a longer study period and a larger
number of patients.
This first study clearly emphasizes the need for further studies to investigate the
protective properties of biomimetic HAP acting as calcium and phosphorus reservoir
for remineralization of initial carious lesions and its possible buffering potential.
This is relevant, for example, for individuals with a high caries risk, such as orthodontic
patients (high plaque values),[17] and for children, as well as for individuals, with a reduced salivary flow (hyposalivation)
because these individuals have a lack of calcium and phosphate ions in the oral cavity.[36]
Besides hydroxyapatite, the administration of other calcium phosphate minerals has
led to increased calcium and phosphate levels in dental plaque. For example, Vogel
et al reported on a chewing gum which contained α-tricalcium phosphate, α-Ca3(PO4)2 (which is also a calcium phosphate mineral) and its effects on the composition of
plaque and saliva after sucrose ingestion. They found higher calcium and phosphate
concentrations in plaque fluid and saliva compared with a control chewing gum.[34] Additionally, a paste containing CPP-ACP (casein phosphopeptides and amorphous calcium
phosphate; Cax(PO4)y . n H2O) increased the calcium and phosphate levels in both saliva and plaque.[37] In general, the various structures of calcium phosphates, used in oral care products,
may have different efficacies.[38] A systematic review and meta-analysis show that HAP reduces dentin hypersensitivity
while ACP does not seem to be effective.[18]
Conclusion
Calcium phosphates (e.g., hydroxyapatite, α-tricalcium phosphate, and amorphous calcium
phosphate) are promising agents to increase the calcium and phosphate levels in dental
plaque.[33]
[34]
[37]
[38]
Due to the high biocompatibility of HAP and the absence of any fluorosis risk for
children,[12]
[17] increasing HAP dosing by the combination of different HAP-based oral care products
(such as HAP-toothpaste, HAP-mouthwash, or HAP-gel) may further increase calcium and
phosphorus levels in dental plaque and consequently HAP’s efficacy in preventive oral
care.[14]
[17]
To conclude, within the limitations of this study, it was shown for the first time
that a 3-day administration of an oral HAP-gel in a group of children (mean age, 8.9
years; mean DMF-T, 0.6; mean dmft-t, 4.5) increased medians of both calcium and phosphorus
levels in plaque. Thus, a positive influence on the remineralization/demineralization
process is very likely.[20]
[26]
[27]
[28]
