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149]

Original Article

Effect of Cocoa Administration on Osteoblast Counts and Alkaline

Phosphatase Levels During Orthodontic Tooth Movement in Rats

Abstract Thalia Ayu Arianda,

Introduction: The cocoa effect on osteoblast activity during orthodontic tooth movement remains Putri Rezqita,
unknown. To analyze the effect of caffeine in cocoa on osteoblast counts and alkaline phosphatase Pinandi Sri
(ALP) levels during orthodontic tooth movement. Materials and Methods: The subjects used in this
Pudyani,
study were 24 male Sprague–Dawley rats aged 2.5–3 months. They were divided into treatment and
control groups (n =1 2). A three-spin stainless steel coil spring with a 35 cN orthodontic force was Niswati Fathmah
stabilized on the maxillary incisors. The rats in the treatment group were given 4.8 g of cocoa powder Rosyida,
with 2.7 mg of caffeine. All the subjects were euthanized in four consequent time periods (0, 1, 7, and 14 Ananto Ali
days), and tissue specimens were stained with hematoxylin-eosin. Osteoblasts were observed and Alhasyimi
counted under a light microscope with an Optilab camera at 400× magnification. ALP levels were Department of Orthodontics,
examined through ELISA. Data were analyzed through two-way ANOVA followed by LSD post-hoc Faculty of Dentistry, Universitas
test. Results: Significant differences were observed in the control and treatment groups and the time of Gadjah Mada, Yogyakarta,
observing osteoblast count and ALP levels (P < 0.05). Osteoblast counts and ALP levels in the treatment Indonesia
group were significantly lower than those in the control group. Conclusion: Caffeine in cocoa might
inhibit osteoblast activities by decreasing ALP levels and osteoblast count.

Keywords: Alkaline phosphatase, cocoa, caffeine, osteoblast, orthodontic tooth movement

Introduction an increase in alkaline phosphatase (ALP)

Orthodontic treatment has been widely
explored because of a high level of public
awareness about malocclusion and the need
to improve esthetics. It aims to obtain good
esthetics, regular tooth position, harmonious
occlusion, and a good relationship between
teeth and their supporting tissues. This
treatment also aims to correct malocclusion
[1,2]
and improve function and esthetics.
Orthodontic tooth movement occurs because
of alveolar bone remodeling and periodontal
[3]
tissue. Bone remodeling needs the
coordination of three types of bone cells,
namely, osteoblasts, osteocytes, and
osteoclasts. When obtaining a mechanical
force, osteocytes function as mechano-
sensors to identify alterations in the bone
fluid flow in bone canaliculi and react by
transmitting signals to osteoblasts through a
syncytial process. Osteoblasts act as bone-
forming cells, while osteoclasts resorb the
alveolar bone. Osteoblast proliferation
throughout bone formation is identified by
[4]
expression. ALP is a basic phosphatase
hydrolase that creates an alkaline pH
through the hydrolysis of monophosphate
ester bonds, thereby increasing the local
concentration of phosphate ions. ALP
expression is an initial marker of osteogenic
cell differentiation. The analysis of ALP
components is a noninvasive method that
can be used to detect cellular tissue
responses under periodontal ligaments
during orthodontic tooth movement.
Previous studies also confirmed that ALP
detected in gingival crevicular fluid (GCF) is Address for correspondence:
characterized as an indicator of bone Ananto Ali Alhasyimi, DDS,
remodeling in bone formation throughout MDSc, Ph.D., Department of
orthodontic tooth movement.
[5,6] Orthodontics, Faculty of
Dentistry, Universitas Gadjah
Orthodontic treatment results can be achieved Mada, Yogyakarta 55281,
[7] Indonesia.
within 1–3 years. During a long treatment E-mail: anantoali@ugm.ac.id
period, users of orthodontic appliances
experience orthodontic treatment compli-
cations and risks, such as caries, white spot Access this article online
Website: www.jofs.in
Received: 1-4-2020 Revised: 20-4-2020 DOI: 10.4103/jofs.jofs_51_20
Accepted: 18-7-2020 Published: 16-02-2021 Quick Response Code:

This is an open access journal, and articles are distributed under

the terms of the Creative Commons Attribution-
NonCommercial-ShareAlike 4.0 License, which allows others
to remix, tweak, and build upon the work non-commercially, as
long as appropriate credit is given and the new creations are
licensed under the identical terms.
For reprints contact: reprints@medknow.com
How to cite this article: Arianda TA, Rezqita P,
Pudyani PS, Rosyida NF, Alhasyimi AA. Effect of
Cocoa Administration on Osteoblast Counts and
Alkaline Phosphatase Levels During Orthodontic
Tooth Movement in Rats. J Orofac Sci
2020;12:101-6.

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Arianda, et al.: Cocoa alleviate osteoblast activity during OTM
[8]
lesion, periodontitis, gingivitis, and root resorption. Efforts to
accelerating orthodontic treatment should be performed to
overcome the side effects of a relatively long-term
orthodontic treatment. Various therapeutic types, including
local application of the parathyroid hormone, the receptor
activator of nuclear factor kappa β ligand, osteocalcine, and
prostaglandins, have been carried out to accelerate tooth
[9]
movement during orthodontic treatment. However, the use
of natural ingredients to accelerate orthodontic treatment has
not been extensively studied. One of the natural ingredients
with potential for application in orthodontic treatment
[10]
acceleration is caffeine.
Caffeine is an active pharmacological substance that is often
used and naturally found in coffee plants, cocoa, and tea. This
substance reduces bone mineral density (BMD) by decreasing
the expression of vitamin D receptors (VDRs). Caffeine can
also reduce the differentiation of mesenchymal stem cells
(MSCs) into osteoblasts by decreasing alpha-1 core binding
[11]
factor (Cbfa1). Exposure to the right dose of caffeine can
inhibit osteoblast cell formation and cause a decrease in BMD.
A decreased BMD can trigger accelerated bone remodeling to
[9]
shorten orthodontic treatment duration. In vitro study proved
that caffeine has potential deleterious effect on the osteoblasts
activity by significantly decreased alkaline phosphatase (ALP)
[10] [12]
and lactate dehydrogenase (LDH) levels. Bozchaloei et al.
showed caffeine alters alkaline phosphatase activity of human
gingival fibroblasts in vitro.
Today, much attention has been given to natural products
with health-promoting advantages. An example of a popular
yet natural caffeine-containing food is cocoa. Approximately
[13]
35 mg of caffeine is found in 28 g of low-sugar cocoa, and
the amount of caffeine in cocoa is lower than those in coffee,
tea, and energy drinks. High caffeine content can have side
[14]
effects, such as insomnia, tremors, anxiety, and addiction ;
thus, cocoa is a safe choice of caffeine source for
consumption. This study was proposed to examine the
effect of cocoa administration on osteoblast counts and
ALP levels during orthodontic tooth movement in rat models.
Materials and Methods
Ethical approval
Ethical approval for this study (protocol No. 001348/KKEP/
FKG-UGM/EC/2018) was provided by the Ethics Committee
of the Research of Dentistry Faculty, Universitas Gadjah
Mada, Indonesia, on 5 March 2018.
Animal experiments
In this laboratory experiment, 24 10-week-old male
Sprague–Dawley rats (weighing 250–300 g) were
randomly divided into two groups that were further
divided into four subgroups with three animals
representing four observation time points: 0 (3 h), 1, 7, and
14 days after orthodontic appliance installation. The rats were
maintained in individual polycarbonate cages under normal
102
laboratory conditions and adapted to a 12 h/12 h light/dark
cycle at a constant temperature of 25°C and a humidity of
50%. During the experiments, the rats were fed with a pellet
diet (expanded pellets; Stepfield, UK) and provided with tap
water ad libitum. They were regularly investigated in terms of
food consumption and fecal characteristics.
The rats were intramuscularly anesthetized with a mixture of
ketamine (35 mg/kg BW) and xylazine (5 mg/kgBW) during
orthodontic appliance installation. A noninvasive technique
was applied to move the teeth distally by utilizing 35 g force,
which was adequate for a rat model. The force was delivered
by using a three-spin loop spring (diameter = 2 mm; wire arm
length = 5 mm) made of 0.012ʹʹ stainless steel archwire
(DiynaFlex, Missouri, USA). The ends of the wire arms were
connected to the orthodontic fixed incisor band attached to
the two maxillary incisors of the rats by using flowable
composites (3M Orthodontics, USA) to move the teeth
distally. The appliance was not reactivated during the
experiment. Shortly after orthodontic appliance
installation, 4.8 of unsweetened cocoa (Hershey’s, USA)
with approximately 2.7 mg of caffeine was given to the
rats in the treatment groups. The cocoa powder was
dissolved in 5 mL of distilled water and orally delivered to
the treatment group once a day by utilizing oral sonde at 9 am.
Isolation of GCF
GCF was collected on four subsequent times (0, 1, 7, and 14
days after orthodontic appliance installation; day 0 represents
baseline, day 1 represents initial phase, day 7 represents lag
phase, and day 14 represents post-lag phase) by cleaning the
incisors with cotton swabs to remove supragingival plaque,
isolated with cotton wool, and dried. A paper point of size 15
(Sendoline, UK) was entered about 1 mm into the gingival
sulcus of the maxillary incisors for 30 s at 90 s intervals to
increase the volume of GCF taken from each side. Three
dipped paper points were initially inserted into 350 μL of
physiological saline solution and subsequently removed. The
supernatant solution was stored at −80°C until ALP activity
tests were carried out. ALP activities were examined at the
Laboratory of Molecular Biology, Faculty of Medicine,
Universitas Gadjah Mada.
Afterward, 50 μL of 40 mM carbonate buffer at pH 9.8 was
mixed with 3 mM MgCl2 and placed in a microplate by using
a pipette. The same well was added with 50 μL of GCF
sample and 50 μL of p-nitrophenylphosphate. The microplate
was incubated at 37 °C for 30 min. The enzyme reaction was
stopped by adding 50 μL of 0.6 M sodium hydroxide.
Absorbance was measured immediately at a wavelength of
405 nm by using a spectrophotometer. ALP activity was
expressed in the form of enzyme units (U), defined as the
amount of p-nitrophenol (mol) released per minute at 37°C.
Histological preparation
Following GCF isolation, the alveolar bone tissues of the
maxillary incisors were obtained through the cervical
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Arianda, et al.: Cocoa alleviate osteoblast activity during OTM

dislocation of euthanized rats in the control and treatment
groups on four subsequent times (0, 1, 7, and 14 days after
orthodontic appliance installation). The tissue sections were
cleaned with 0.9% NaCl and soaked in 10% formalin for 24 h.
Decalcification was performed using 10% EDTA (Sigma-
Aldrich, USA), and this procedure was repeated twice for 2
months until the specimens softened and could be cut. The
specimens were impregnated with liquid paraffin at 480 °C.
The obtained paraffin block was cut mesiodistally (parallel
to the long axis of the incisor) to a thickness of 4–6 μm in
a rotary microtome. Hematoxylin–eosin staining was
conducted to histologically examine the number of
osteoblasts on the distal surface of the incisors root
(pressure side).
Histological analysis
Histological examination was conducted on six fields
randomly selected as regions of interest (ROIs), extending
from the incisal to the apical of the maxillary bone on the
incisors on the pressure side [Figure 1]. The number of
osteoblasts was accomplished by examining the samples
under a light microscope connected to a digital camera
(OptiLAB LLC Phoenix, USA) at 400× magnification. The
number of osteoblasts per field was calculated using ImageJ®
(NIH, Maryland, USA). The total number of osteoblasts was
obtained by calculating the mean values across six ROIs from
both incisors. The hematoxylin–eosin-stained osteoblasts
found on the edge surfaces of the alveolar bone and
appearing as cuboidal cells were characterized by a single,
[15]
deep blue-purple nucleus. All measurements were performed
by two-trained observers who were blinded to the applied
sample and repeated twice. The examiners revealed a good
level agreement in their analysis (κ = 0.87), designating
satisfactory intra-examiner and inter-examiner reliability.
The mean of these measurements was used as the
representative value.
Statistical analysis
Results were tested using an independent sample t-test to
determine significant differences between the two groups.

Figure 1: Experimental design of the region of interest (a) divided into three regions: (b) 1/3 incisal, (c) 1/3 medial, and (d) 1/3 apical of alveolar bones. AB,
alveolar bones; PDL, periodontal ligament; d: dentin.

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Arianda, et al.: Cocoa alleviate osteoblast activity during OTM
Differences with P < 0.05 were considered significant. All
the analysis was done using SPSS Version V.22 (SPSS Inc,
Chicago, Illinois).
Results
In general, giving cocoa at the selected dose did not cause any
general toxicity, edema or deaths (by clinical observation),
and all the animals were well-tolerated to all the experimental
procedures. The mean and standard deviations of the number
of osteoblasts (cells/field) and ALP levels (U/mg) of the two
groups are presented in Table 1. Histological examination
revealed that osteoblast number in both groups decreased
from days 1 to 7 but increased from days 7 to 14 (Figure 2).
The mean number of osteoblasts in the group receiving cocoa
was significantly lower than that in the control group on days
0, 7, and 14 (P < 0.05). Meanwhile, as shown in Table 1, the
ALP levels showed a decreased on days 0 to 1, both in the
control and treatment groups. On days 7 to 14, the ALP levels
in both groups increased. The mean number of ALP levels in
the group receiving cocoa was significantly lower than that in
the control group on days 7 and 14 (P < 0.05).
Discussion
This investigation validated the hypothesis that cocoa
containing caffeine could inhibit osteoblast activities by
decreasing ALP levels and osteoblast counts, thus
potentially accelerating orthodontic tooth movement. The
independent t-test revealed that the control group and the
treatment group had significantly different osteoblast counts
and ALP levels. This result confirmed that caffeine in cocoa
[10]
affected osteoblast activities. Tsuang et al. indicated that
caffeine can reduce the number of osteoblasts, so it may speed
up orthodontic treatments. Caffeine also reduces VDR, which
Table 1: Means and standard deviation of osteoblast
count (cells/field) and ALP levels (U/mg) between the two
groups tested on days 0, 1, 7, and 14
Observation time Control Treatment P-
(Day) group group valuea
Osteoblast count(cells/
field)
0 37.42 ± 2.95 33.25 ± 3.30 0.021*
1 30.08 ± 3.38 28.42 ± 4.82 0.061
7 41.01 ± 3.66 36.42 ± 4.01 0.009*
14 48.83 ± 3.63 41.58 ± 3.76 0.001*
ALP levels(U/mg)
0 26.28 ± 1.43 23.02 ± 3.03 0.048
1 15.75 ± 2.03 14.73 ± 2.05 0.091
7 44.24 ± 3.02 25.12 ± 3.12 0.001*
14 55.06 ± 2.03 40.12 ± 3.38 0.001*
Values are presented as mean ± standard deviation. aTested by
independent t-test of variance. * = significant differences between
groups (P < 0.05)
104
[16]
are receptors involved in the biological action of vitamin D.
The expression levels of genes involved in calcium and
phosphate homeostasis, cell differentiation, proliferation,
[17]
and immune responses are regulated by VDRs.
Osteoblasts express VDRs to bind to 1,25-dihydroxyvitamin
D3, modulate cell proliferation, and trigger osteoblast cell
[16]
differentiation.
The mechanical force produced by an orthodontic device
increases prostaglandin E2 (PGE2) concentrations because of
the activation of COX-2, which is an enzyme that can regulate
[18]
bone repair. PGE2 production activates Cbfa1 and osterix
transcription factors to stimulate the differentiation of MSCs
[19] [11]
into osteoblasts. Zhou et al. also demonstrated that
caffeine exposure can reduce Cbfa1, thereby decreasing
the differentiation of MSCs into osteoblasts. This
phenomenon agrees with our results and reveals the
connection between cocoa administration and osteoblast
count reduction.
Each group experiences a change in the number of osteoblasts
on each day of observation. The number of osteoblasts in the
control and treatment groups on days 0 to 1 decreased, and the
number of osteoblasts on days 1 to 7 and on days 7 to 14
[20]
increased. Nanda revealed that the number of osteoblasts in
the pressure area decreases on days 1 to 5 and slowly
[21]
increases thereafter. Choi et al. showed that the number
of osteoblasts continues to increase because of the appearance
of byglican, which is useful in the formation of osteoblasts,
on day 5. Byglican is a component that plays a role in bone
repair and functions as a modulator of bone morphogenetic
protein-2 in osteoblast cell formation. The results showed
significant differences in osteoblast counts in almost all
groups, but no significant differences were observed in the
control group on day 1 possibly because the dynamics of cells
and the inhibitory effect of caffeine with less than the optimal
dose in cocoa on the formation of osteoblast cells can cause
nonsignificant differences.
The results also showed that the ALP levels between the
two groups significantly differed, whereas the ALP activity
of the groups that received cocoa was significantly lower
than that of the control group. ALPs are biological markers
of osteoblast activities during new bone formation. ALP
levels indicate biochemical changes that occur in
[22]
supporting tissues after orthodontic force application.
Detected ALP levels in GCF can be used as an indicator
of alveolar bone apposition in orthodontic tooth movement.
The use of 2.7 mg of caffeine can reduce ALP activities in
the alveolar bone. The results of this study indicated that the
caffeine content in cocoa could reduce ALP levels. The
ALP levels of the treatment group that received cocoa
drinks were lower than those of the control group. This
result was consistent with a previous finding, which showed
that caffeine content in cocoa can reduce the expression
[16]
levels of VDR and ALP in the alveolar bone. Alveolar
bone remodeling is also associated with changes in ALP
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Arianda, et al.: Cocoa alleviate osteoblast activity during OTM

Figure 2: Osteoblasts stained with hematoxylin–eosin on days 0, 1, 7, and 14 in the control and treatment groups. Difference could be found in both groups
by observing the number of osteoblasts shown in the pictures (indicated by black arrows; 400× magnification).

activities in GCF, implying that osteoblast activities
[23]
improve at high ALP levels. An increase in osteoblast
activities and ALP levels also indicates new bone
[24]
formation. With regard to tooth movement
acceleration, osteoblast activities likely decreased.This
result indicates that using cocoa is an effective
pharmacological choice to locally control osteoblasts
activity for purposes such as orthodontic tooth movement
acceleration. It is a limitation of our study that we have done
HE staining for labeling of osteoblasts, but did not use any
specific marker such as von-Kossa or alkaline phosphatase.
This method of counting osteoblasts may have been
somewhat subjective. Therefore, further investigations
using biomarkers of osteoblasts are required to
strengthen this result conclusion.
Conclusion
Within the limitations of the study it can be concluded that
cocoa containing caffeine can inhibit osteoblast activities by
decreasing ALP levels and reducing osteoblast count during
orthodontic tooth movement in rats. However, further studies
are needed to confirm the efficacy and potency of cocoa in
clinically accelerating tooth movement.

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Arianda, et al.: Cocoa alleviate osteoblast activity during OTM
Acknowledgment
The authors would like to thank the Department of
Orthodontics, Faculty of Dentistry, Universitas Gadjah
Mada, Yogyakarta, Indonesia.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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