IJMS

Vol 44, No 5, September 2019 Original Article

Combined Hydroxyapatite Scaffold and

Stem Cell from Human Exfoliated Deciduous
Teeth Modulating Alveolar Bone
Regeneration via Regulating Receptor
Activator of Nuclear Factor-Κb and
Osteoprotegerin System
Abstract
Chiquita Prahasanti1, DMD, PhD; Background: Tissue engineering using Stem cell from Human Exfoliated Decidu
Lieke Halim Subrata1, DMD; Tania
Saskianti2, DMD, PhD; Ketut Suardita3, Results: The independent t test showed that the differences were statistically sign
DMD, PhD; Diah Savitri Ernawati4, DMD, Conclusion: Hydroxyapatite scaffold and SHED increase Osteoprotegerin and de
PhD
Please cite this article as: Prahasanti Ch, Subrata LH, Saskianti T, Suardita K, Ernawati DS. Combin
1
Departement of Periodontology,
Faculty of Dental Medicine, Universitas
Airlangga, Surabaya, Indonesia; Keywords ● Mesenchymal stromal cells ● Tissue engineering ●
2
Departement of Pediatric Dentistry, Hydroxyapatites ● Osteoprotegerin ● RANK ligand
Faculty of Dental Medicine, Universitas
Airlangga, Surabaya, Indonesia;
3
Departement of Conservative
Dentistry, Faculty of Dental Medicine,
Universitas Airlangga, Surabaya,
Indonesia; 4Departement of Oral
Medicine,
Faculty of Dental Medicine, Universitas
Airlangga, Surabaya, Indonesia

Correspondence:
Chiquita Prahasanti, DMD, PhD;
Department of Periodontology. Faculty
of Dental Medicine, Universitas
Airlangga Jl. Prof. Dr. Moestopo 47
Surabaya Indonesia
Tel\Fax: +62 31 5030255
Email: chiquita-p-s@fkg.unair.ac.id
Received: 17 September 2017
Revised: 27 January 2018
Accepted: 04 March 2018

What’s Known
• Stem Cells from Human
Exfoliated Deciduous Teeth (SHED)
are also an ideal source of stem cells
employed in the repair of damaged
bone.
• SHED demonstrate adult stem
cell characteristics with high
proliferation potential, self-renewal
capacity, and potential differentiation
into various cells with good viability
potential and proliferation.

What’s New

• SHED can be attached to and

proliferate within a Hydroxyapatite (HA)
scaffold.
• HA with SHED increases
Osteoprotegerin (OPG) expression and Introduction
decreases Receptor Activator of NF-Κb
ligand (RANKL) expression.
• SHED have good potential as
alveolar
Iran J Medbone defect regeneration.
Sci September 2019; Vol 44 No 5 1
 Therapy to repair bone damage can be either resective or
reconstructive (regenerative) in nature. Regeneration sometimes

2 Iran J Med Sci September 2019; Vol 44

 HA scaffold and SHED modulating alveolar bone

involves the application of a graft as supporting the cells playing a role include progenitor cells,
material. Various types of graft have begun to ostoblast, osteocytes, and osteoclasts.12 Under
be widely studied and developed rapidly to normal circumstances, the remodeling process
achieve periodontal tissue regeneration.1 will demonstrate a balance between resorption
A graft constitutes a synthetic or natural and bone formation while, under pathological
material acting as a filler which also serves as a conditions, bone resorption is more dominant
scaffold and is able to initiate the process of leading to pathological bone destruction.
bone formation.2 Osteoblast cells require a Walsh and Choi (2104) and Gibertoni and
scaffold and induction mediator to migrate into others, highlighted the main lines defining
defective areas for bone remodeling. Scaffolds the remodeling process of bonding as being
play a role in supporting cell attachment and Osteoprotegerin (OPG), Receptor Activator
proliferation in the defective area and stabilizing of NF-κB (RANK), and Receptor Activator of
blood clots to prevent tissue damage during the NF-κB ligand (RANKL) initiated by osteoblasts
primary phase of regeneration. Ideally, missing and osteoclasts.13, 14 OPG and RANKL will
or damaged tissues are replaced by induce processes referred to as osteogenesis
biomaterials possessing a high level of and osteoclastogenesis.14, 15 OPG and RANKL
biocompatibility. This encourages many expression biomarkers can be used at the initial
clinicians and researchers to apply the concept stage in the bone formation process marking to
of tissue engineering using stem cells from assess the effectiveness of treatment outcome.
human organs and scaffolds derived from OPG protects the skeleton from excessive bone
natural biomaterials.3 resorption by binding to RANKL and preventing
BioHydrox® products made from bovine it from binding to its receptor, RANK. Thus,
bone are biomaterials that have the same RANKL/OPG ratio is an important determinant
microarchitecture and mineral composition as of bone mass and skeletal integrity.16 The aim of
human bone does.4 Hydroxyapatite (HA) is one this study was to analyze the OPG and RANKL
of the materials classified as bioactive in nature expression combination HA with SHED.
and has osteointegrative, osteoconductive,
osteoinductive, and osteogenetic properties
Material and Methods
when used as a bone graft.5
Stem Cells from Human Exfoliated
This study received approval through an ethical
Deciduous Teeth (SHED) are also an ideal
clearance letter from the Faculty of Veterinary
source of stem cells employed in the repair of
Medicine, Universitas Airlangga 042/HRECC.
damaged tooth structure and bone regeneration
FODM/IV/2017. This study was conducted
while also demonstrating the ability to
according to the guideline of animal ethics and
differentiate into osteoblasts. SHED
welfare.17 The rats were housed at 21–23 °C
demonstrate adult stem cell characteristics with
and under controlled humidity (50±5%) for less
high proliferation potential, self-renewal
than a 12-hour artificial light cycle (7 am to 7
capacity, and potential differentiation into
pm). All the animals had access to water and
various cells.6, 7 A previous study conducted by
the AIN93M diet 1 week before the
Puspitasari and others confirmed that SHED
commencement of the study for adaptation.
possessed good viability potential and
This research was experimental in nature,
proliferation by observing the development of
incorporating post-test-only control group
living cell counts. Similarly, the potential for
design. 14 male Wistar rats weighing from 260
stem cell differentiation of SHED was proved to
to 280 g were used as the animal study model
be largely positive.8
in this research. The samples were divided
A previous study conducted by Iohara
randomly into two groups. The animals were
described an innovative dental treatment
randomly assigned to an experimental group
employing tissue engineering technology. It
HA+SHED (Group II) and control group HA
showed that dentin regeneration using Dental
(Group I). Sample stem cells were isolated from
Pulp Stem Cells (DPSCs) combined with
SHED according to certain inclusion criteria
Bone Morphogenic Protein-2 could stimulate
such as the presence of vital conditions, the
odontoblastic cell differentiation as a means
absence of caries, and root resorption of
of forming new dentin.9 Studies conducted by
primary teeth being more than 1/3 of the tooth
Sloan and Smith confirmed that DPSCs played
root or intact. Pulp tissue was placed in the
a role in the regeneration and repair of
medium within a 15 ml conical tube within a
damaged dentin.10
cool box before immediately being sent to the
Bone quality can be established based Tissue Bank at the Diagnostic Center, Dr.
on its size and shape, remodeling process, Soetomo Surabaya General Hospital. Pulp
mineral composition, and collagen content.11 tissue was subsequently
In the constantly ongoing remodeling process,
Iran J Med Sci September 2019; Vol 44 No 5 3
 HA scaffold and SHED modulating alveolar bone

cultured in Dulbeccos Modified Eagle Medium 5% at 37 °C). Media culture was replaced every
(DMEM®, Life Technologies, Gibco BRL™, 3 days after which the culture in the 26
USA) with the addition of 20% fetal bovine HA+SHED was transplanted to the alveolar
serum (FBS, Biochrom AG®, Germany), 5mm bone defect. Immunostaining was performed
L-glutamine (Gibco Invitrogen®, 25, USA), 100 after 8 weeks to examine OPG and RANKL
U/ ml penicillin-G, 100 ug/ml streptomycin, and expression using a marker kit (Sigma Aldrich™,
100 ug/ml kanamycin (Gibco Invitrogen®, 25, Germany).8
USA). After 3 days, the medium was disposed Data were analyzed by independent t test
of in order to remove that part of the cell not to compare the treatment and control group.
attached to the dish and transfer it to a new The correlation between OPG and RANKL
medium. At this stage, Fibroblast Growth expression were analyzed using Pearson’s
Factor-2 was added. The confluent passage correlation test. The P value<0.05 was
was performed using 0.05% trypsin-EDTA considered to be significant. Statistical analysis
before the cell was washed and cultured a was performed using R statistical software
second time in either a 60 mm or 100 mm version 3.4.0.
tissue culture dish. After the cells had reached
the point of confluence, they were ready to be
Results
used for further research. Any cells not used
immediately must be stored in liquid N .8
SHED can be attached to and proliferate within
The alveolar bone defect model was induced
2 an HA scaffold. An Immunohistochemistry
in Wistar rats as the animal study model by
(IHC) examination using anti-OPG monoclonal
extracting the mandible anterior teeth using
antibodies was observed. The IHC result of
sterile needle holder clamps. 20 mL suspension
OPG expression in the control and treatment
of SHED at passage 3-5 with a density of 106
groups on Day 7 can be seen in figure 1. In the
cells was added to HA before being placed in a
present study, the result of OPG expression in
24-well tissue culture plate. After 2 hours, 980
the treatment group was higher than that of the
μl Dulbecco’s Modified Eagle Medium (DMEM)
control group. The mean±SD of OPG
(Gibco™, Thermo Fisher Scientific, Waltham,
expression was 6.0±1.00 and 11.6±1.14 for the
MA USA) was added to each well. In addition,
control and treatment groups, respectively. The
the cells were incubated in the incubator (CO2
mean of OPG expression between group
samples showed a significant difference
between control

Figure 1: Immunohistochemical staining shows OPG Expression (1000× magnification). A positive reaction produced a brown
color in the cytoplasm due to antigen (OPG) (yellow arrow) with monoclonal antibodies (anti OPG); (A) Expression of OPG in
osteoblast on Group I day 7, (B) Expression of OPG in osteoblast on Group II day 7; (C) Expression of OPG in osteoblast on
group I day 14, (D) Expression of OPG in osteoblast on group II day 14.
Iran J Med Sci September 2019; Vol 44 4
 Prahasanti Ch, Subrata LH, Saskianti T, Suardita K, Ernawati

and intervention groups. Independent t test Table 1: OPG expression in the control (group I) and treatment group (group I
showed that the differences between the mean
of OPG expression between group samples Group Mean±SD P value
were statistically significant (table 1). I (Treatment) 11.6±1.14 0.0004*
The IHC result of RANKL expression in the II (Control) 6.0±1.00
control and treatment groups on day 7 and 14 Statistical tests used independent t test, *Significant
can be seen in figure 2. Moreover, RANKL P<0.05
expression in the treatment group was lower
than that of the control group. The mean of
RANKL expression in control group was Table 2: RANKL expression in the control (group I) and treatment group (group I
12.67±2.08 while it was 4.80±1.30 for the
treatment group. The mean RANKL expression Group Mean±SD P value
between the groups showed a significant I (Treatment) 4.80±1.14 0.0005
II (Control) 12.67±1.00
difference (table 2). The OPG expression had a
Statistical tests used an independent t test, *Significant
strong reverse significantly significant
P<0.05
correlation with RANKL expression (r=-0.912,
P=0.0016).
increase in OPG and RANKL expression.11. 19, 20
Discussion RANKL and OPG play an important role in
bone remodeling, modulation, and osteoclasts
In this study, based on the statistical test, it differentiation. Osteoclast maturation will
was found that there was a strong relationship produce a mature osteoblast so that the
in OPG/RANKL ratio. The increased OPG ratio bone remodeling can work well. The balance
compared to RANKL shows SHED’s ability to between OPG-RANKL ratios plays an important
stimulate OPG to bind to RANKL, thus inhibiting role in maintaining bthe one homeostasis of
osteoclastogenesis. The increased expression phosphorus and calcium. The remodeling
of OPG is supported by the research process is maintained by the formation of bone
undertaken by Walsh and Choi. Belibasakis matrix through osteoblast and bone resorption
asserted that OPG expression increase due to by osteoclast.21
an elevated level of TGF-β1 and RUNX2.13, 18 OPG and RANKL can be detected in the
TGF-β1 induced OPG expression in OPG cells. gingival tissue as well as biological fluids such
The osteoblasts produce OPG and RANKL. as saliva, serum, and gingival cervical fluid. In
The higher the number of osteoblasts, the
greater the resulting

Figure 2: Immunohistochemical staining shows RANKL Expression (1000× magnification). A positive reaction produced a
brown color in the cytoplasm due to antigen (RANKL) (yellow arrow) with monoclonal antibodies (anti RANKL); (A) Expression
of RANKL in osteoblast on Group I day 7, (B) Expression of RANKL in osteoblast on Group II day 7; (C) Expression of
RANKL in osteoblast on group I day 14, (D) Expression of RANKL in osteoblast on group II day 14.

4 Iran J Med Sci September 2019; Vol 44

 HA scaffold and SHED modulating alveolar bone

their study, Belibasakis and Bostanci stated that biocompatible surface provides ideal conditions
the ratio of OPG/RANKL in periodontitis for cell growth and tissue differentiation.4, 5, 7, 21
patients showed an increased ratio of
OPG/RANKL. The balance of OPG/RANKL
ratio showed success in the treatment of
periodontitis. Increased RANKL or decreased
OPG expression will lead to bone resorption.18
OPG expression was higher than the RANKL
expression treatment group that showed no
bone resorption occurred in our study. In a
previous study conducted by Lapin on a
periodontitis patient, OPG expression
decreased that showed the severity of the
disease. OPG released by osteoblasts and
inhibits bone resorption inhibits osteoclast
differentiation and activity, by binding to
RANKL receptors. A balanced ratio between
OPG/RANKL is a protective property against
bone loss.13, 19, 20
Bone graft is a material used for
augmentation and stimulation of new bone
formations in certain cases of bone defect.22
Based on the graft materials ability in bone
regeneration, the use of autograft is golden
standards because it comes from the patient’s
bone. Technological advancements allow for the
development and combination of bone graft
materials and SHED. The combination of bone
graft and SHED was used in this study. The
most famous bone graft biomaterial was HA
scaffold because of its osseointegration,
osteoconduction, osteoinduction, and
osteogenesis properties. Progenitor cells
infiltrate porosity in HA which then proliferates
and forms new bone on the surface of the graft
material with subsequent replacement or
combination with HA material. The properties of
osteoinduction HA are derived from the
microporous and macropore surface. The
presence of a microporous surface provides an
interconnection between the macropores that
support the occurrence of interstitial fluid
circulation along the matrix. After the graft is
applied to the bone defect, the HA granules will
dissolve to form a biological apatite layer of HA.
This coating will produce a chemically
hospitable environment and possesses a high
affinity that can induce mesenchymal
differentiation of cells into osteogenic cells.23
HA scaffold has a porous matrix whose size
may vary and depend on the volume of scaffold
produced. The advantages of HA scaffold
include its allowing the cells to move through
existing pores as well as creating pore
conditions for nutrient transport, tissue
infiltration, and vascularization.6 Pores in HA
can bind strongly to bone tissue due to the
structure of HA with regular porosity being
similar to that of natural bone tissue. The pores
have an open structure with the result that the

Iran J Med Sci September 2019; Vol 44 4

 Prahasanti Ch, Subrata LH, Saskianti T, Suardita K, Ernawati
HA contains calcium and phosphate,
potentially increasing the affinity of bone
tissue, resulting in an ion exchange in calcium
phosphate and the interaction between
calcium and cell surfaces. The ion exchange
takes place on the surface of the adjacent
phosphate compound of the surrounding water
(liquid phase) through the surfaces of Ca2+,
PO 3-, and existing impurities such as CO 2-,
4
Cl- or F-. In addition, the ion exchange is also
3
bound to fibronectin protein which is
fundamental in the SHED attachment process.
When SHED interact with HA, it will stick to it
and obtain nutrients from the suspension
medium, resulting in SHED growth and
proliferation.3, 12, 24
Ions can enter on membranes that are
hydrophobic in nature. The cell membrane is
coated by a skeleton in the cytoplasm
rendering the organelle crescent-like cilia or
filipodia which expands the surface area in
order to increase the nutrient absorption by
the cell. The surface area in hydroxyapatite is
cuboid leading to greater nutrient absorption
and promotion of cellular activity which, in
turn, result in more attachment and SHED
proliferation.4, 5, 11, 22
In addition to the HA contained in human
hard tissues, ions such as carbonate, sodium,
and magnesium are also present in them.4, 5
The results of this study proved that HA
scaffolding could lead to attachment, with
SHED proliferation occurring even though
variation in the number of SHED existed within
the scaffold. HA Scaffold can be used as a
carrier for application of SHED and also
increasing cell growth and regenerating the
damaged tissues such as bone. SHED
proliferation in scaffolds can be used as an
alternative to osteogenic tissue engineering.
SHED can induce bone formation with nerve
marker expression. It demonstrates an
encouraging potential for clinical application.7,
23
While SHED cannot directly differentiate
into osteoblasts, they possess the capacity
to stimulate new bone formation by means of
an osteoinductive template in order to obtain
osteogenic cells. This suggests that primary
teeth not only provide a guide for permanent
tooth eruption but also involve bone formation
during this process.8, 25

Conclusion

Hydroxyapatite scaffold and Stem cells from

Human Exfoliated Deciduous Teeth increase
OPG and decrease RANKL expression via
regulating OPG-RANKL system that has a
high potential to be used as an effective
alternative tissue engineering biomaterial for
alveolar bone defect regeneration.

4 Iran J Med Sci September 2019; Vol 44

 HA scaffold and SHED modulating alveolar bone

Acknowledgement acterization of stem cells from human exfoli-

ated deciduous teeth using trypsin enzym).
The authors would like to thank the Universitas Dental Journal (Majalah Kedokteran Gigi).
Airlangga (UNAIR), Faculty of Dental Medicine
and Dr. Soetomo Surabaya General Hospital.
This research was supported by Airlangga
University (Hibah Mandat Research Grants,
2016).

Conflict of Interest: None declared.

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