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Year : 2020  |  Volume : 12  |  Issue : 2  |  Page : 101-106

Effect of Cocoa Administration on Osteoblast Counts and Alkaline Phosphatase Levels During Orthodontic Tooth Movement in Rats

Department of Orthodontics, Faculty of Dentistry, Universitas Gadjah Mada, Yogyakarta, Indonesia

Date of Submission01-Apr-2020
Date of Decision20-Apr-2020
Date of Acceptance18-Jul-2020
Date of Web Publication16-Feb-2021

Correspondence Address:
Ananto Ali Alhasyimi
Department of Orthodontics, Faculty of Dentistry, Universitas Gadjah Mada, Yogyakarta 55281
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jofs.jofs_51_20

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Introduction: The cocoa effect on osteoblast activity during orthodontic tooth movement remains unknown. To analyze the effect of caffeine in cocoa on osteoblast counts and alkaline phosphatase (ALP) levels during orthodontic tooth movement. Materials and Methods: The subjects used in this 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 stabilized on the maxillary incisors. The rats in the treatment group were given 4.8 g of cocoa powder with 2.7 mg of caffeine. All the subjects were euthanized in four consequent time periods (0, 1, 7, and 14 days), and tissue specimens were stained with hematoxylin-eosin. Osteoblasts were observed and counted under a light microscope with an Optilab camera at 400× magnification. ALP levels were examined through ELISA. Data were analyzed through two-way ANOVA followed by LSD post-hoc test. Results: Significant differences were observed in the control and treatment groups and the time of observing osteoblast count and ALP levels (P < 0.05). Osteoblast counts and ALP levels in the treatment 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

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

How to cite this URL:
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 [serial online] 2020 [cited 2021 Jun 13];12:101-6. Available from:

  Introduction Top

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 and improve function and esthetics.[1],[2] Orthodontic tooth movement occurs because of alveolar bone remodeling and periodontal tissue.[3] Bone remodeling needs the coordination of three types of bone cells, namely, osteoblasts, osteocytes, and osteoclasts. When obtaining a mechanical force, osteocytes function as mechanosensors 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 an increase in alkaline phosphatase (ALP) expression.[4] 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 characterized as an indicator of bone remodeling in bone formation throughout orthodontic tooth movement.[5],[6]

Orthodontic treatment results can be achieved within 1–3 years.[7] During a long treatment period, users of orthodontic appliances experience orthodontic treatment complications and risks, such as caries, white spot lesion, periodontitis, gingivitis, and root resorption.[8] 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 movement during orthodontic treatment.[9] 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 acceleration is caffeine.[10]

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 factor (Cbfa1).[11] 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 shorten orthodontic treatment duration.[9] In vitro study proved that caffeine has potential deleterious effect on the osteoblasts activity by significantly decreased alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) levels.[10] Bozchaloei et al.[12] 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 35 mg of caffeine is found in 28 g of low-sugar cocoa,[13] and the amount of caffeine in cocoa is lower than those in coffee, tea, and energy drinks. High caffeine content can have side effects, such as insomnia, tremors, anxiety, and addiction[14]; 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 Top

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 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 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, deep blue-purple nucleus.[15] 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.
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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Statistical analysis

Results were tested using an independent sample t-test to determine significant differences between the two groups. Differences with P < 0.05 were considered significant. All the analysis was done using SPSS Version V.22 (SPSS Inc, Chicago, Illinois).

  Results Top

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).
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

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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).

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  Discussion Top

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 affected osteoblast activities. Tsuang et al.[10] indicated that caffeine can reduce the number of osteoblasts, so it may speed up orthodontic treatments. Caffeine also reduces VDR, which are receptors involved in the biological action of vitamin D.[16] The expression levels of genes involved in calcium and phosphate homeostasis, cell differentiation, proliferation, and immune responses are regulated by VDRs.[17] Osteoblasts express VDRs to bind to 1,25-dihydroxyvitamin D3, modulate cell proliferation, and trigger osteoblast cell differentiation.[16]

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 bone repair.[18] PGE2 production activates Cbfa1 and osterix transcription factors to stimulate the differentiation of MSCs into osteoblasts.[19] Zhou et al.[11] 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 increased. Nanda[20] revealed that the number of osteoblasts in the pressure area decreases on days 1 to 5 and slowly increases thereafter. Choi et al.[21] 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 supporting tissues after orthodontic force application.[22] 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 levels of VDR and ALP in the alveolar bone.[16] Alveolar bone remodeling is also associated with changes in ALP activities in GCF, implying that osteoblast activities improve at high ALP levels.[23] An increase in osteoblast activities and ALP levels also indicates new bone formation.[24] 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 Top

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.


The authors would like to thank the Department of Orthodontics, Faculty of Dentistry, Universitas Gadjah Mada, Yogyakarta, Indonesia.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2]

  [Table 1]


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