Vol 49 No 1 Jan-Mrt 2016.indd


22

Research Report

Dental Journal
(Majalah Kedokteran Gigi)

2016 March; 49(1): 22–27

The potential of chitosan combined with chicken shank collagen 
as scaffold on bone defect regeneration process in Rattus 
norvegicus

Fitria Rahmitasari,1 Retno Pudji Rahayu,2 and Elly Munadziroh3
1Department of Dental Materials, Faculty of Dentistry, Universitas Hang Tuah
2Department of Oral Pathology and Maxillofacial, Faculty of Dental Medicine, Universitas Airlangga
3Department of Dental Materials, Faculty of Dental Medicine, Universitas Airlangga
Surabaya - Indonesia

ABSTRACT

Background: In the field of dentistry, alveolar bone damage can be caused by periodontal disease, traumatic injury due to tooth 
extraction, cyst enucleation, and tumor surgery. One of the ways to regenerate the bone defect is using graft scaffold. Thus, combination 
of chitosan and collagen can stimulate osteogenesis. Purpose: The aim of this study was to examine the potential of chitosan combined 
with chicken shank collagen on bone defect regeneration process. Method: Twelve Rattus norvegicus were prepared as animal models 
in this research. A bone defect was intentionally created at both of the right and left femoral bones of the models. Next, 24 samples 
were divided into four groups, namely Group 1 using chitosan – collagen scaffold (50:50), Group 2 using chitosan collagen-scaffold 
(80:20), Group 3 using chitosan scaffold only, and Control Group using 3% CMC-Na. On 14th day, those animals were sacrificed, and 
histopathological anatomy examination was conducted to observe osteoclast cells. In addition, immunohistochemistry examination was 
also performed to observe RANKL expressions. Result: There was a significant difference in RANKL expressions among the groups, 
except between Group 3 using chitosan scaffold only and control group (p value > 0.05). The highest expression of RANKL was found 
in Group 1 with chitosan – collagen scaffold (50:50), followed by Group 2 with chitosan-collagen scaffold (80:20). Moreover, there 
was also a significant difference in osteoclast generation, except between Group 1 using chitosan – collagen scaffold (50:50) and 
Group 2 using chitosan-collagen scaffold (80:20), p value < 0.05; and between Group 3 using chitosan scaffold only and control 
group, p value > 0.05. Less osteoclast was found in the groups using chitosan – collagen scaffold (Group 1 and Group 2). Conclusion: 
Combination of chitosan and chicken shank collagen scaffold can improve regeneration process of bone defect in Rattus novergicus 
animals through increasing of RANKL expressions, and decreasing of osteoclast. 

Keywords: chitosan scaffold; chicken shank collagen; bone regeneration

Correspondence: Fitria Rahmitasari, Department of Dental Materials, Faculty of Dentistry, Universitas Hang Tuah. Jln. Arief Rahman 
Hakim No. 150 Surabaya 60111, Indonesia. E-mail: fitri.rahmitasari@gmail.com

INTRODUCTION

In the field of dentistry, alveolar bone damage can be 
caused by periodontal disease, traumatic injury due to 
tooth extraction, post-cyst enucleation, and post-tumor 
surgery. Biotechnology development actually has already 
introduced tissue engineering concept by using xenografts 
to help bone regeneration process since it does not require 
additional surgery, cause low morbidity, and reduce the risk 

of disease transmission.2 However, in the last few years, 
innovation of bone tissue engineering has been developed, 
as a result, biomaterials are focused on a physicochemically 
suitable scaffold design for cell attachment, proliferation, 
differentiation, and specific organ tissue formation.3 

Scaffold is the term for the synthesis of the extracellular 
matrix. Scaffold also becomes a place of attachment and 
growth of new cells. Thus, scaffold must be made of 
biodegradable materials that can be metabolized in the 

Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 56/DIKTI/Kep./2012.
Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG
DOI: 10.20473/j.djmkg.v49.i1.p22-26

mailto:fitri.rahmitasari@gmail.com
http://e-journal.unair.ac.id/index.php/MKG


2323Rahmitasari, et al./Dent. J. (Majalah Kedokteran Gigi) 2016 March; 49(1): 22–26

body and eventually disappear when the new cells have 
started to grow a lot, be healthy, and survive. Some of the 
polymeric materials, which have been developed in tissue 
engineering, are chitosan and collagen.4

Chitosan is an amino polysacharide (poly-1,4-
D-glucosamine), widely used as polymers in tissue 
engineering.5 These polymers have been regarded as a 
material which has many functional advantages because it 
has high biocompatibility, biodegradable properties, and 
low toxicity.6,7 Chitosan is also known to stimulate the 
growth and differentiation of osteoblasts in cell culture. 
The existence of chitosan alone is not osteoconductive 
enough, so the ability in new bone formation is still less than 
optimal. Approaches to overcome the weaknesses has been 
conducted, such as designing a composite by combining the 
strengths of different materials to minimize the weaknesses 
of two different materials.8

Another organic material playing an important role in 
tissue engineering is collagen.9 Collagen can be found in 
cartilage, bone, intervertebral disc, blood vessels, tendons, 
ligaments, skin, and a major component of the extracellular 
matrix. Collagen can also be found in chicken shank. 
Collagen contains RGD (Arg-Gly-Asp) and non-RGD 
peptides that can bind to cell surface related to integrin, 
consequently, collagen can facilitate migration, adhesion, 
proliferation, and differentiation of cells.10 

Collagen, furthermore, has been used in numerous 
applications in tissue engineering since it has good 
biocompatibility and biodegradable properties, as well as 
low antigenicity.11,12 This material, thus, is considered as a 
material suitable for repairing damaged tissue and organ.9 
Collagen actually has many disadvantages, such as rapid 
degradation time and weak mechanical strength. Therefore, 
a combination of the two polymer materials is needed to 
produce better material. Chitosan and collagen can be 
combined into a new material to form a unique structure 
that can improve the mechanical strength and decrease the 
biodegradation rate of collagenase.13,14 The concentrations 
used to make a good combination of chitosan and collagen 
scaffold with good mechanical strength are 50: 5015,16 and 
80: 20.17 

In addition, bone healing is characterized by a series 
of cellular and molecular processes, as well as tissue 
transformation consisted of resorption and formation of hard 
and soft tissues. Bone formation by osteoblasts and bone 
resorption by osteoclasts regulate skeletal remodeling.18 
These processes are fundamental in maintaining bone mass 
and architecture.19 Osteoclasts play an important role in 
bone resorption both physiologically and pathologically. 

Osteoclasts require the presence of cytokine receptor 
activator of nuclear factor-κB ligand (RANKL) as a key 
cytokine inducing osteoclastogenesis.20 In this research, 
two organic natural ingredients were used, namely 
chitosan combined with chicken shank collagen as a 
scaffold to determine the potential of those material on 
the healing process of bone defects in Rattus norvegicus 
rats on day 14 with by observing RANKL expressions and 

osteoclast count. Similarly, a previous research also show 
that bone callus would increase on the 14th day after the 
administration of bone defect.21

MATERIALS AND METHODS

This research was an experimental laboratory research. 
Extraction process of chicken shank collagen and 
manufacture of chitosan and collagen scaffold were 
performed in Unit Research Services (ULP) of Pharmacy 
Faculty, Universitas Airlangga and the Laboratory of 
Human Genetics-Tropical Disease Center of Universitas 
Airlangga. Treatment in experimental animals was 
conducted at the Laboratory of Biochemistry - Faculty 
of Medicine, Universitas Airlangga. Histological and 
immunohistochemical preparations was carried out in 
Diagnostic Center - Dr. Soetomo Hospital in Surabaya.

Gel base for the control group was made of carboxy 
methyl celulose sodium (CMC Na) at a concentration of 
3%. Chitosan used in this research was chitosan with the 
degree of deacetylation of >75-85%. Chitosan gel was made 
by 200 mg of chitosan powder mixed with 5 ml of 0.1M 
acetic acid, and then added with 15 ml of 0.1 M NaOH. 
After that, centrifuge at 9000 rpm was carried out to obtain 
pure chitosan gel.

Moreover, collagen was obtained from the extraction 
of chicken shank collagen. Chicken shank was cut into 
small pieces and mashed. Smoothed chicken shank was 
mixed with 250 U/ mg of trypsin enzyme powder, and then 
incubated at 37° C for 24 hours. Glacial acetic acid was 
added and stored at 4° C for 48 hours. The preparations were 
mixed using a mixer to form a fiber, and then centrifuged 
to obtain a supernatant. They were centrifuged twice 
to obtain a pure supernatant. Supernatant obtained was 
mixed with 0.5M acetic acid to dissolve, and then added 
with 5% NaCl to form bands of collagen. The process was 
repeated three times. Those collagen bands were filtered 
with filter paper. After that, the collagen was dialysed and 
centrifuged again. 

Furthermore, manufacture of chitosan and collagen 
scaffold was conducted by mixing chitosan gel and chicken 
shank collagen gel homogeneously with ratios of 50:50 and 
80:20. The gel was inserted into scaffold mold made of 
teflon, then frozen at -20° C for 2 hours, and was followed 
by freeze dry for 24 hours. Scaffold then was sterilized 
using a clean bench UV.

In addition, samples used were 12 male Wistar rats 
(Rattus norvegicus) weighed 250-300 mg. Their left 
and right femur bone then was defected. Thus, the total 
of samples used in this research was 24 samples. Those 
samples were classified into four groups each of which 
consisted of six samples. The research groups were control 
group (3% CMC Na), Group 1 using chitosan – collagen 
scaffold (50:50), Group 2 using chitosan collagen-scaffold 
(80:20), and Group 3 using chitosan scaffold only. Femur 
bone defect then was made with a diameter and height of 

Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 56/DIKTI/Kep./2012.
Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG
DOI: 10.20473/j.djmkg.v49.i1.p22-26

http://e-journal.unair.ac.id/index.php/MKG


24 Rahmitasari, et al./Dent. J. (Majalah Kedokteran Gigi) 2016 March; 49(1): 22–26

3 mm using a low-speed round bur under anesthesia 
condition using ketamine and xylazine. The scaffold 
material then was applied to the defected bone in accordance 
with the treatment of each group. 

On day 14, the defected bones in the control and 
treatment groups were taken under the effects of 
inhalation anesthetics. Those bones were processed for the 
manufacture of histological preparations with hematoxylin 
eosin staining (HE) to examine the number of osteoclasts. 
Immunohistochemical examination then was carried out 
using RANKL antibody to observe RANKL expressions. 
Finally, osteoclast count and RANKL expressions were 
measured manually on five of the visual field examination 
using a light microscope with a magnification of 400x.

RESULTS

The results of this research showed the average values 
of osteoclast count and RANKL expressions in the control 
group, the treatment group using chitosan scaffold only, 
the treatment group using chitosan-collagen scaffold with 
the ratio of 80:20, and the treatment group using chitosan-
collagen scaffold with the ratio of 50:50 during the healing 
process of those femur bone defect as shown in Table 1.

The most RANKL expressed by osteoblasts was found 
in the group using chitosan-collagen scaffold with the 
ratio of 50:50. Meanwhile, the least RANKL expressed by 
osteoblasts was in the control group. The highest number 
of osteoclasts was widely obtained in the control group, 
while the least number of osteoclasts was in the group 
using chitosan-collagen scaffold with the ratio of 50:50. 
The results of histopathological examination then were 
obtained by observing the number of osteoclasts in the 
control and treatment groups using a light microscope with 
a magnification of 400x as seen in Figure 1.

The fewest osteoclasts were found in the group using 
of chitosan-collagen scaffold with the ratio of 50:50 (d), 
following the group using of chitosan-collagen scaffold 
with the ratio of 80:20 (c), the group using chitosan (b), 
and the control group (a) (Figure 1). RANKL expressions 
in each treatment group and the control can be seen in 
Figure 2.

The illustration photo above show the results of 
immunohistochemical examination that the combination 
of chitosan and chicken shank collagen scaffold can 
increase RANKL expressions during the healing process 
of the fermur bone defect. However, the combination of 
chitosan-collagen scaffold with the ratio of 50:50 was more 
effective in increasing RANKL expression than with the 
ratio of 80:20.

Table 1. The average values of osteoclast count and RANKL expressions 

Group Number of samples
Average values (Mean)

Osteoclast count RANKL expressions

Control 6 4.8333 5.8333

Chitosan scaffold 6 4.6667 8.1667

Chitosan scaffold with the ratio of 80:20 6 2.1667 17.5000

Chitosan scaffold with the ratio of 50:50 6 1.3333 28.0000

Figure 1. Osteoclast cells in each group. a) the control group; b) 
the group using chitosan scaffold only; c) the group 
using chitosan collagen-scaffold (80:20); d) the group 
using chitosan–collagen scaffold (50:50).

Figure 2. RANKL expressions in each group. a) the control 
group; b) the group using chitosan scaffold only; c) the 
group using chitosan collagen-scaffold (80:20); d) the 
group using chitosan – collagen scaffold (50:50).

Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 56/DIKTI/Kep./2012.
Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG
DOI: 10.20473/j.djmkg.v49.i1.p22-26

http://e-journal.unair.ac.id/index.php/MKG


2525Rahmitasari, et al./Dent. J. (Majalah Kedokteran Gigi) 2016 March; 49(1): 22–26

The research data of osteoclast count and RANKL 
expressions in each group were then tested using one way 
Anova test. Before the one way Anova test conducted, 
the results of Kolmogorov Smirnov test showed that the 
distribution of the data in this research was normal (p> 
0.05). Based on one way Anova test, the significance value 
obtained was 0.000 (p<0.05). It means that there was a 
significant difference in osteoclast count and RANKL 
expressions between the control group, the group using 
chitosan scaffold only, the group using chitosan collagen-
scaffold (80:20), and the group using chitosan–collagen 
scaffold (50:50). Thus, Post Hoc test and Tukey HSD test 
were performed to know differences between one group 
and another group.

There was a significant difference in the number of 
osteoclast cells between one group and another group. 
Nevertheless, there was no significant difference in the 
number of osteoclast cells (p>0.05) between the group 
using chitosan scaffold only and the control group, as well 
as between the group using chitosan-collagen scaffold with 
the ratio of 50:50 and the group using chitosan-collagen 
scaffold with the ratio of 80:20. In addition, there were 
significant differences in RANKL expressions between 
one group and another group. However, there was no 
significant difference in RANKL expressions (p>0.05) 
between the group using chitosan scaffold and the control 
group (p> 0.05). 

DISCUSSION

On the 14th day, histopathological anatomy observation 
was conducted to examine the number of osteoclasts. 
Osteoclasts play an important role in bone resorption, both 
in physiological and pathological conditions. Osteoclast are 
derived from myeloid cells and macrophages, producing 
many cytokines and regulating macrophages and dendritic 
function. Osteoclast, moreover, are located on the surface 
of endosteal cells in the Havers channels along cortical 
and trabecular bones.22 Osteoclasts are the largest, 
multinucleated, irregularly shaped cells with pale cytoplasm 
color.23 The least average number of osteoclasts was found 
in the group using chitosan – collagen scaffold (50:50), 
following the group using chitosan–collagen scaffold 
(80:20), and the group using chitosan scaffold only. The 
highest average number of osteoclasts was found in the 
control group. 

Osteoclasts, furthermore, are known to contribute to 
bone resorption. As a result, the least number of osteoclasts 
was found in the groups using chitosan–collagen scaffold. It 
may indicate that bone formation is stimulated dominantly 
by osteoblasts during the healing process of the femur bone 
defect in those experimental animals. RGD (Arg-Gly-
Asp) contained in the collagen can inhibit the expression 
of RANK by blocking the integrin, αvβ3, so possibility 
of RANKL to bind to RANK is lower and inhibits the 
formation of mature osteoclasts.24

The statistical results, however, showed that there were 
significant differences between each groups, except between 
the group using chitosan-collagen scaffold with the ratio 
of 50:50 and the group using chitosan-collagen scaffold 
with the ratio of 80:20, as well as between the group using 
chitosan scaffold only and the control group (p>0.05). This 
is due to the fact that there was not much different in the 
average pore size of the scaffold between in the the group 
using chitosan-collagen scaffold with the ratio of 50:50 
and in the group using chitosan-collagen scaffold with the 
ratio of 80:20 about 183 m and 123 m. Thus, there was no 
significant effect in reducing the number of osteoclasts 
useful in the process of bone healing. The greater porosity 
size actually can trigger a better vascularization so that the 
healing process can be more optimal.17 Both the treatment 
group using chitosan scaffold only and the control group, 
consequently, did not produce statistically significant 
differences in the number of osteoclasts because the 
chitosan alone is not enough osteoconductive. Therefore, 
the bone healing process was less than optimal. In this case, 
the ability to produce osteoclasts was not much different 
between the control group and the treatment group using 
3% CMC-Na on the 14th day. 

The expressions of RANKL by osteoblasts, moreover, 
were more visible in the treatment groups using chitosan-
collagen scaffold combination than the group using 
chitosan scaffold only and the control group due to a 
possible increase in the number of osteoblasts and the 
expression of osteoprotegerin (OPG), playing important 
roles in bone formation. In other words, the combination 
of chitosan-collagen scaffold can stimulate the occurrence 
of osteogenesis by facilitating adhesion, proliferation, 
and differentiation of cells. Collagen can also enhance 
osteoblast differentiation and increase bone formation by 
activating genes RUNX-2 to stimulate pre-osteoblasts into 
osteoblas.25 Therefore, it can be said that the greater level 
OPG expressions than the level of RANKL expression 
may trigger bone formation. RANKL is a key cytokine in 
stimulating osteoclastogenesis.19 RANKL can stimulate 
differentiation, maintain viability, and activate mature 
osteoclasts. 

All those functions can be run because of the interaction 
between RANKL and receptor activator of nuclear factor-
κB (RANK). RANK is a transmembrane protein expressed 
by the pre-osteoclasts. In the bone healing process, there is 
a protein, OPG, which can inhibit osteoclast development. 
OPG functions as a decoy receptor by binding to RANKL, 
resulting in inhibiting RANK signaling.26 Besides, since 
RANKL is expressed by osteoblasts, the high number of 
osteoblasts may also contribute to the high expressions of 
RANKL. According to a research conducted by Wang, 
RANKL and OPG levels can significantly increase 
immediately after a fracture in a bone fracture until the 
4th week compared to the control group of healthy bone.27 
Although both increase, the presence of RANKL is less 
than OPG, thus showing that the number of osteoblasts 
will increase over that period and bone formation is more 

Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 56/DIKTI/Kep./2012.
Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG
DOI: 10.20473/j.djmkg.v49.i1.p22-26

http://e-journal.unair.ac.id/index.php/MKG


26 Rahmitasari, et al./Dent. J. (Majalah Kedokteran Gigi) 2016 March; 49(1): 22–26

dominant in playing the role during the bone healing 
process. 

Finally, the results of this research need to be analyzed 
further. Further researches should reveal the location of 
RANK and OPG expressions since RANK/ RANKL/ 
OPG is a signaling system that is responsive to control 
osteoclastogenesis. Properties of chitosan itself have 
good mechanical strength less than in combination with 
other polymers so that chitosan scaffold is less optimal in 
facilitating osteogenesis. In this case, the ability to express 
RANKL in bone defects was not much different from the 
control group on the 14th day. It can be concluded that the 
combination of chitosan and collagen scaffold can improve 
the healing process of bone defect in Wistar rats through 
an increase in RANKL expressions and a decrease in the 
number of osteoclasts. 

REFERENCES

 1. Fokkema SJ. The central role of monocytes in systemic immune 
effects induced by the chronic periodontal infection. Wageningen: 
Ponsen & Looijen BV; 2002. p. 104-16. 

 2. Mehta M, Schmidt-Bleek K, Duda GN, Mooney DJ. Biomaterial 
delivery of morphogens to mimic the natural healing cascade in 
bone. Adv Drug Deliv Rev 2012; 64(12): 1257-76.

 3. Niu X, Fan Y, Liu X, Li X, Li P, Wang J, Sha Z, Feng Q. Repair of 
bone defect in femoral condyle using microencapsulated chitosan, 
nanohydroxyapatite/collagen and poly (L-lactide)-based microshere-
scaffold delivery system. Artificial Organs 2011; 35(7):119. 

 4. Chen G, Ushida T, Tateishi T. Scaffold design for tissue engineering. 
Macromol Biosci 2002; 2(2): 67-77.

 5. Amaral IF, Cordeiro AL, Sampaio P, Barbosa MA. Attachment, 
spreading, and short-term proliferation of human osteoblastic cells 
cultured on chitosan films with different degrees of acetylation. J 
Biomaterial Science Polymer 2007; 18(4): 469-85. 

 6. Ven katesan J, K im S. Chitosan composites for bone tissue 
engineering–an overview. Mar Drugs 2010; 8(8): 2252-66. 

 7. Sonia TA, Sharma CP. Chitosan and its derivatives for drug delivery 
perspective. Adv Polym Sci 2011; 243: 23-54. 

 8. Ariani MD, Matsuura A, Hirata I, Kubo T, Kato K, Akagawa Y. 
New development of carbonate apatite-chitosan scaffold based 
on lyophilization technique for bone tissue engineering. Dental 
Materials Journal 2013; 32(2): 317-8. 

 9. Cui K, Zhu Y, Wang XH, Feng QL, Cui FZ. A porous scaffold from 
bone-like powder loaded in a collagen-chitosan matrix. Journal of 
Bioactive and Compatible Polymers 2004; 19(1): 17-31.

10. K ruger TE, Miller AH, Wang J. Collagen scaffolds in bone 
sialoprotein-mediated bone regeneration. ScientificWorld Journal 
2013; 2013: 812718.

11. Ma L, Gao C, Mao Z, Zhou J, Shen J, Hu X, Han C. Collagen/
chitosan porous scaffold s with improved biostability for skin tissue 
engineering. Biomaterials 2003; 24(26): 4833-41.

12. Osidak EO, Osidak MS, Ak hmanova MA, Domogatskii SP. 
Collagen-A biomaterial for delivery of growth factors and tissue 
regeneration. Russian Journal of General Chemistry 2014; 84(2): 
368-9. 

13. Arpornmaeklong P, Suwatwirote N, Pripatnanont P, Oungbho 
K. Growth and differentiation of mouse osteoblasts on chitosan-
collagen sponges. Int.J.Oral Maxillofac 2007; 36(4): 328-9. 

14. Yoo JH, Lee MC, Lee JE, Jeon KC, Kim YM, Jung MY, Ahn 
HJ, Seong SC, Choi SJ, Lee JH. Evaluation of chondrogenesis in 
collagen/chitosan/ glycosaminoglican scaffolds for cartilage tissue 
engineering. J Korean Orthop Res Soc 2005; 8(1): 28-40.

15. Tangsadthakun C, Kanokpanont S, Sanchavanakit N, Banaprasert 
T, Damrongsakkul S. Properties of collagen/chitosan scaffold s for 
skin tissue engineering. Journal of Metals, Materials, and Minerals 
2006; (1): 38. 

16. Sionkowska A, Kaczmarek B, Stalinska J, Osyczka AM. Biological 
properties of chitosan/collagen composites. Key Engineering 
Materials 2014; 587: 205. 

17. Suryati, Agusnar H, Gea S, Ilyas S. Nonporous chitosan/collagen 
scaffold for skin tissue engineering. Proceeding of the 2nd Annual 
International Conference Syiah Kuala University 2012; 2(2): 376.

18. Bab IA, Sela JJ. Cellular and molecular aspects of bone repair. 
Principles of Bone Regeneration Jerusalem: Springer Science + 
Business Media; 2012. p. 11-28. 

19. Nakashima T, Hayashi M, Takayanagi H. New insights into 
osteoclastogenic signaling mechanisms. Trends in Endocrinology 
and Metabolism 2012; 23(11): 582. 

20. Clarke B. Normal bone anatomy and physiology. Clin J Am Soc 
Nephrol 2008; 3 Suppl 3: S131-9.

21. Kon T, Cho TJ, Aizawa T, Yamazaki M, Nooh N, Graves D, 
Gerstenfeld LC, Einhorn TA. Expression of osteoprotegerin, receptor 
activator of NF-kappaB ligand (osteoprotegerin ligand) and related 
proinflammatory cytokines during fracture healing. J Bone Miner 
Res 2001; 16(6): 1004-14.

22. Lorenzo J, Horowitz M, Choi Y, Takayagi H. Osteoimmunology. 1st 
edition. UK: Academic Press; 2011. p. 10-235. 

23. Baron R. Anatomy and ultrastructure of bone histogenesis, growth, 
and remodelling. In: Arnold A, editor. Disease of bone and mineral 
metabolism. MDText.com, South Dartmouth MA. Web 2010.

24. Mochizuki A, Takami M, Miyamoto Y, Nakamaki T, Tomoyasu S, 
Kadono Y, Tanaka S, Inoue T, Kamijo R. Cell adhesion signaling 
regulates RANK expression in osteoclast precusors. PLoS One 2012; 
7(11): e48795.

25. Uchihashi K, Aoki S, Matsunobu A, Toda S. Osteoblast migration 
into type I collagen gel and differentiation to osteocyte-like cells 
within a self-produced mineralized matrix: A novel system for 
analyzing differentiation from osteoblast to osteocyte. Bone 2013; 
52(1): 102-10.

26. Pérez-Sayáns M, Somoza-Martín JM, Barros-Angueira F, Rey 
JM, García-García A. RANK/RANKL/OPG role in distraction 
osteogenesis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 
2010; 109(5): 679-86. 

27. Wang XF, Zhang YK, Yu ZS, Zhou JL. The Role of the serum 
RANKL/OPG ratio in the healing of intertrochanteric fractures in 
elderly patients. Mol Med Rep 2013; 7(4): 1169-72.

Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 56/DIKTI/Kep./2012.
Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG
DOI: 10.20473/j.djmkg.v49.i1.p22-26

http://e-journal.unair.ac.id/index.php/MKG