Journal of International Dental and Medical Research ISSN 1309-100X Alveolar Bone Remodeling

http://www.ektodermaldisplazi.com/journal.htm Herniyati, and et al

The Role of Rankl and Opg in Alveolar Bone Remodeling and Improvement of Orthodontic
Tooth Movement Post Coffee Brew Administration

Herniyati1*, Ida Bagus Narmada2, Soetjipto3

1. Department of Orthodontic, Faculty of Dentistry, Jember University, Jember, Indonesia.
2. Head, Department of Orthodontic, Faculty of Dentistry, Airlangga University, Surabaya, Indonesia.
3. Department of Biochemistry, Faculty of Medicine, Airlangga University, Surabaya, Indonesia.

Abstract

This study was aimed at elucidating the role of RANKL and OPG in the alveolar bone
remodeling and improvement of tooth movement induced by orthodontic mechanical force (OMF)
post coffee brew administration.
24 rats were divided into 3 groups. Group A: the rats were administered with OMF , group B:
OMF + coffee brew at 20mg / 100 g of BW in 2 ml of distilled water, and group C: OMF + caffeine of
1, 37 mg / 100 g BW in 2 ml of distilled water. OMF was conducted by ligature wires were applied
on permanent maxillary right first molar and both permanent maxillary incisivus. Subsequently, the
permanent maxillary right first molar was moved to mesial with closed coil spring. Observations
were made on day 15 and day 22 to take GCF and measure the tooth movement on the film
resulted from X-ray photograph.
Coffee brew increases the levels of RANKL and OPG , and improves the movement of the
teeth, therefore it may be an alternative to accelerate orthodontic treatment.
Experimental article (J Int Dent Med Res 2017; 10: (1), pp. 84-88)
Keywords: RANKL, OPG, Coffee, alveolar bone remodeling.
Received date: 14 December 2016 Accept date: 17 January 2017

Introduction is a receptor that interacts with RANKL thus

triggering the differentiation of osteoclasts which
Orthodontic tooth movement (OTM) is play a role in bone resorption. RANKL is
induced by mechanical stimuli and facilitated by expressed on fibroblasts and osteoblasts in the
remodeling of the periodontal ligament and periodontal ligament in the compression side as
alveolar bone. A precondition for these well as plays a role in osteoclast differentiation in
remodeling activities, and ultimately for tooth response to the mechanical force. During the
displacement, is the occurrence of an process of alveolar bone resorption, RANKL can
inflammatory process1. also be detected in odontoblasts, osteoclasts,
Bone remodeling is a dynamic interaction and other cells found in the periodontal
between bone-forming osteoblasts and bone- ligament4.
resorbing osteoclasts. The rate of remodeling is Osteoprotegerin (OPG) is a glycoprotein
defined primarily by cells of the osteoblast produced by human periodontal ligament cells5
lineage, which, in addition to bone formation, are and has been found to be a key factor in the
also responsible for the activation and inhibition of osteoclast differentiation and
recruitment of osteoclast precursors2. activation6. OPG also acts as a receptor for
Receptor Activator of Nuclear Factor- RANKL which competes with RANK to bind and
κβ Ligand (RANKL) is a regulator on bone avoid interaction with RANK, thereby occurs
remodeling during orthodontic tooth movement3. inhibition of osteoclastogenesis7. Expression of
Receptor Activator of Nuclear Factor-κβ (RANK) OPG in osteoblasts is regulated by various
cytokines, hormones and growth factors and by
*Corresponding author: the Wnt/β-catenin. Pathway of Wnt/β-catenin
Dr. Herniyati, also regulates bone formation8.
Fakultas Kedokteran Gigi Universitas Jember - Jl. Results of studies show that the tooth
Kalimantan 37, Jember- 68121, Jawa Timur- Indonesia.
E-mail: herniyati@unej.ac.id
applied with force 7N for 7 days using Hyrax,
rapid expansion equipment, greater RANKL

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 Journal of International Dental and Medical Research ISSN 1309-100X Alveolar Bone Remodeling
http://www.ektodermaldisplazi.com/journal.htm Herniyati, and et al

transcripts was found in the compression side United States) of 1.35 mg / 100 g BW dissolved
than that in the tension side, and the OPG in 2 ml of distilled water. OMF in rats was
transcript was found greater in the tension side conducted by anesthetizing rats using ketamine,
than that in the compression side. RANKL and subsequently ligature wire with a diameter of
OPG were increased in the compression and 0.20 mm was set on permanent maxillary right
tension sides compared to the control 9. Besides first molar and both permanent maxillary
administering OPG locally on periodontal tissues incisivus. The permanent maxillary right first
can inhibit relapse after orthodontic treatment molar was moved to the mesial using Tension
through inhibition of osteoclastogenesis10. Gauce to generate power of 10 g / cm2 with a
Examination using PCR also showed RANKL Nickel Titanium Orthodontic closed coil spring of
increased significantly on the compression side 6 mm long19. The observations were carried out
in the case group followed by bone resorption on the days 15 and 22 to take the gingival
caused by orthodontic tooth movement11. crevicular fluid (GCF) by placing the paper points
Various attempts have been made to to gingival sulcus of mesio-palatal permanent
accelerate orthodontic tooth movement, i.e. by maxillary right first molar for 30 seconds and then
using drugs, surgical methods and methods of stored in eppendorf tubes20. Increased tooth
physical and mechanical stimuli12. Coffee today movement in rats was measured on film of X-ray
becomes one of the most popular beverages in photograph by measuring from the distal
the world which is consumed among people. permanent maxillary right second molar to the
Robusta coffee contains caffein13, that increases mesial permanent maxillary right first molar
osteoclastogenesis through enhancement of lessened by mesiodistal width of permanent
RANKL14. The result of the study on the rats maxillary right first molar and permanent
applied with orthodontic mechanical force (OMF) maxillary right second molar using digital
shows that a high dose of caffeine administration calipers. Determination of RANKL and OPG
on the rats increases the amount of osteoclast levels was performed by ELISA method.
and bone resorption on the compression side15. Analysis of the data was conducted using
Robusta coffee also contains Chlorogenic acid Oneway Anova test followed by Tukey HSD
and caffeic acid that possess an effect as test, Kruskal-Wallis test followed by the Mann-
antioxidant16, that may reduce oxidative stress on Whitney test, Wilcoxon Signed Ranks test,
osteoblast17. The results of study show that Paired t-test, and Brown-Forsythe Statistics with
Chlorogenic acid promotes osteogenesis which is 95% level of significance (α = 0,05). This study
indicated by an increase in bone mineralization18. has been approved by the ethical research
This study was aimed at elucidating the committee of the Faculty of Dentistry, Airlangga
role of RANKL and OPG in the alveolar bone University, Number: 18/KKEPK.FKG/II/2015
remodeling and improvement of Orthodontic
tooth movement induced by Orthodontic Results
mechanical force post coffee brew
administration. The results of taking GCF from mice No.
8 in group C were damaged due to poor storage
Materials and methods thus only seven data were resulted from the
group C. The results of the coffee brew effects on
Type of the study was experimental the levels of RANKL and OPG are shown in
laboratory. A number of 24 rats (Spraque Table 1 (see the appendix).
Dauwley), males, aged 3-4 months, weighed The test of RANKL level on the day 15
250-300 gram and stated healthy, were objected among the research groups based Kruskal-Wallis
to the study. The rats were randomly divided into test followed by the Mann Whitney test showed
3 groups. Group A: the rats were applied with that the levels of RANKL in group B was greater
orthodontic mechanical force (OMF), Group B: than in group A and C (p<0.05). RANKL levels in
the rats were applied with OMF and administered group C was greater than in group A (p<0.05).
with dried coffee brew at 20mg / 100 g BW OPG assay on the day 15 among the groups
dissolved in 2 ml of distilled water, and group C using Kruskal-Wallis test subsequenlty followed
as control +: the rats were applied with OMF and by Mann-Whitney test showed that level of OPG
administered with pure caffeine (TCI-American- in group B was greater than that in the groups A

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 Journal of International Dental and Medical Research ISSN 1309-100X Alveolar Bone Remodeling
http://www.ektodermaldisplazi.com/journal.htm Herniyati, and et al

and C (p<0.05). OPG level in group C was permanent maxillary right first molar to mesial on
greater than group A (p>0.05). RANKL level test the day 15 and 22 is showed in table 2. Table 2
on day 22 using Oneway Anova test followed by illustrates the results of measurement of the
the Tukey HSD test results showed that RANKL increase in the movement of permanent maxillary
level of group B was greater than either group A right first molar to mesial in the group A, group B
or group C (p>0.05), and group C was greater and group C on the days 15 and 22 (see the
than the group A (p<0.05). OPG level test on day appendix). Further test results using Mann-
22 using Oneway Anova test followed by Tukey Whitney test on the day 15 showed the increase
HSD test results showed that the level of OPG in of movement of the tooth in the groups B and C
group B was greater than that in groups A and C was greater than that in the group A (p<0.05).
(p<0.05). OPG level in group C was greater than The tooth movement in group B was greater than
that in group A (p<0.05). that in the group C (p>0.05). The test using
Mann-Whitney on the day 22 showed that the
increase in the movement of in group B and C
was greater than that in group A (p<0.05).
Increased movement of the tooth in group B was
greater than that in group C (p>0.05). This
Table 1. Comparison of GCF: RANKL and OPG showed that the coffee brew increased the
Levels of All Groups in the Observation of Day orthodontic tooth movement on the days 15 and
15 and Day 22. 22. Further the test using paired t-test on the
Notes : *based on Oneway Anova, **based on Kruskal-Wallis test, increase of tooth movement between day 15 and
abc
The same superscript in one column shows no difference among day 22 in group A, group C and group B showed
the groups.
no significant differences (p>0.05).

Discussion

Bone resorption and bone formation are

parts of the remodeling process during OTM.
Table 2. Comparison of the Permanent Maxillary Bone is deposited on the alveolar wall on the
Right First Molar Movement of All Groups in the tension side of the tooth with both heavy and light
Observation of Day 15 and Day 22. forces, and newly formed bone spicules follow
Notes: *based on Oneway Anova, **based on Brown-Forsythe
Statistics, ***based on paired t-test, abcthe same superscript in one the orientation of the periodontal fiber bundles.
column indicates no differences among the groups. On the compression side, with light forces,
alveolar bone is directly resorbed by numerous
The test results based on paired t-test in osteoclasts in Howship's lacunae21 .
group A and Wilcoxon Signed Ranks test in Results of the study showed that on the
group B showed a decrease in the levels of day 15 and day 22, the administration of coffee
RANKL on day 22 compared to day 15, however brew and pure caffeine increases the levels of
it was not significant (p> 0.05), while paired t-test either RANKL or OPG greater than that is not
in group C showed a significant decrease (p administered with coffee brew or pure caffeine.
<0.05). This showed that the administration of Besides, the administration of coffee brew and
coffee brew on day 15 increased the levels of pure caffeine also increases OTM. Whereas the
RANKL. The test results based on Wilcoxon increase of OTM in the administration of coffee
Signed Ranks test showed increased levels of brew is greater than pure caffeine.
OPG on day 22 compared to day 15 in group A Increased levels of RANKL in the coffee
but they were not significant (p> 0.05), whereas brew or pure caffeine administration are caused
Wilcoxon Signed Ranks test in group C showed by caffeine that binds adenosine receptors, and
significantly-increased levels of OPG (p <0.05), modulates several other receptors including the
and paired t-test in group B showed glucocorticoid receptor, insulin, estrogen,
insignificantly-decreased levels of OPG (p> androgen, vitamin D, cannabinoid, glutamate and
0.05). adrenergic receptors, which are all expressed in
The results of the study on the effect of osteoblasts or cell osteoprogenitor and possess
coffee brew to the increased movement of important functions during osteoblast

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 Journal of International Dental and Medical Research ISSN 1309-100X Alveolar Bone Remodeling
http://www.ektodermaldisplazi.com/journal.htm Herniyati, and et al

differentiation22,23,24. Furthermore Lacey show of OPG decrease insignificantly. This condition is

that Vitamin D receptors stimulates RANKL because of the decreased strength of OMF, thus
expression in cells such as osteoblasts and bone the activity of osteoblasts to produce RANKL also
marrow-derived stromal cells25. RANKL produced declines. Besides, OPG is a decoy receptor
by osteoblasts subsequently binds to RANK on protein which competes with RANK to bind
the surface of osteoclast precursors and recruits RANKL and inhibits activation of RANKL, thus
the TRAF6 protein adapter, which causes the inhibition osteoklastogenesis occurs1.
activation of NF-kB and translocates to the Increased tooth movement on the
nucleus. NF-kB increases the expression of c- administration of coffee brew or pure caffeine
Fos, and c- Fos interacts with NFATc1 to trigger due to increased levels of RANKL caused by
osteoclastogenic gene transcription. OPG inhibits caffeine, as resulted in this study. Yamaguchi
the initiation process by binding to RANKL26. stated that RANKL plays a role in bone
Further in vivo studies in animals also show that resorption, which binds to RANK on osteoclast
caffeine reduces bone mineral density (BMD) in precursors to activate osteoclasts and leads to
rats with increased osteoclastogenesis27. Active bone resorption1. The strength of orthodontic
Osteoclasts will result in bone resorption28. pressure will be distributed through the teeth to
Caffeine also increases levels of OPG periodontal ligament and alveolar bone, generate
due to increased potential of osteogenic compression side resulting in bone resorption
osteoblasts. The results showed that caffeine and tension side that will form new bone during
increases the potential of osteogenic in the baby tooth movement33. The complex molecular
rat of which mother was administered 50 mg/kg signals cause the cellular response for alveolar
caffeine during pregnancy29, thus the increased bone resorption leading to tooth movement.
levels of OPG is caused by the increased Orthodontic tooth movement will be followed by
potential of osteoblasts that produces OPG. alveolar bone remodeling and periodontal
Results of previous studies have shown that ligament34. Increased OTM on day 22 in coffee
administration of low doses of caffeine ( 0.1 mM ) brew administration occured but insignificant
in vitro on primary - derivated adipose stem cells compared to day 15. It can be concluded that
(ADSCs) and bone marrow stromal cells coffee brew effectively improves OTM on the day
increases osteoblast differentiation through 15.
markers ALP, Osteocalcin, OPG, RUNX 2, and
Sirtuin 130. Conclusions
Coffee brew increases OPG levels
greater than pure caffeine because coffee Coffee brew administration increases the
contains not only caffeine but also caffeic acid levels of RANKL and OPG, and improves OTM
which is a non-phenolic flavonoid acid. Caffeic thus coffee brew can be an alternative to
acid group has an effect as antioxidant that can accelerate orthodontic treatment.
reduce oxidative stress on osteoblasts. Several
studies in vitro and in vivo in animals indicate that Acknoledgements
oxidative stress reduces the rate of bone
formation by decreasing the differentiation and The authors would like to thank the
life of osteoblast. In addition, it has been reported managers of Biomedical Laboratory Faculty of
that Reactive Oxygen Species (ROS) activates Dentistry, University of Jember for services
osteoclasts and thus, increases the bone provided in the process of treatment on animal
resorption31. The antioxidant activity is important and Laboratory Biochemistry, Medical Faculty,
to stimulate osteoblastic activity through specific University of Brawijaya in the process GCF
receptors, thus it supports the growth of bone32. analysis by ELISA.
Decreased levels of RANKL occurs in the
group A, group B and group C on the day 22 if Declaration of Interest
compared to the day 15, despite the decline in
group B is not significant. Moreover, on the day The authors report no conflict of interest
22 group A showed insignificantly-increased and the article is not funded or supported by any
levels of OPG. Whereas in group C levels of research grant.
OPG increase significantly, and in group B levels

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 Journal of International Dental and Medical Research ISSN 1309-100X Alveolar Bone Remodeling
http://www.ektodermaldisplazi.com/journal.htm Herniyati, and et al

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