1

Effect of Decellularized Cartilage Bovine Scaffold and Hypoxic

Condition on Stem Cell Differentiation to Chondrocyte : An In
Vitro Study
Tri Wahyu Martanto, MD, Aries Rakhmat Hidayat, MD

Background: Cartilage healing is poor because of its specific characteristic. In literature,

about 60% cartilage injury accidentaly found in routine diagnostic arthroscopy procedures.
Various treatment had been evaluated and Autologous Cartilage Implantation (ACI) has become
a gold standard. Development of tissue engineering has potential to cover the disadvantage of
ACI with a single stage surgery without donor site morbidity. This study is engaged in cartilage
tissue engineering biomaterials.

Methods: This in vitro experimental laboratory study used decellularized bovine cartilage
scaffold and hypoxic stem cell differentiation. This study conducted comparative test between 3
groups with chondrogenic medium, scaffold, and a combination of chondrogenic medium and
scaffold. Each group was performed normoxic and hypoxic conditions within phase of stem cell
differentiation. The evaluation was done using immunohistochemical SOX9, RUNX2, and
collagen type II staining.

Results: After 5 weeks treatment period, we obtained the best SOX9 and collagen type II
evaluation result in group with combination of chondrogenic medium and scaffold. Expression
of RUNX2 is lowered in hypoxic medium chondrogenic group but it did not happen on scaffold
or combination group. There’s no significant difference of collagen type II between hypoxic
scaffold group and combination group.

Conclusions: Decellularized cartilage bovine matrix is a raw material that acts as a scaffold
and contains growth factor by providing good microenvirontment to induce stem cell
differentiation into chondrocytes. Hypoxic conditions increase the production of type II collagen
by adult chondrocytes with a hypoxic positive effect mechanism depicted by the increased
amount of SOX9 expression and decreased expression of RUNX2.

Keywords: cartilage defect, decellularized cartilage bovine scaffold, hypoxic stem cell.

C
artilage of knee joints has its own
characteristics compared to other
tissues. Unlike other tissues,
cartilage of the knee joint has no
vascularization (avascular), has no neural
network (aneural), and has no lymphatic
system (alymphatic) (1). The presence of
damage to cartilage tissue is complicated by
its location, being intra-synovial and intra-
articular. This causes the process of
haematoma formation to be very difficult
and aggravates the complexity of any
regeneration process. (2)
Various methods of cartilage
restoration have been well-known, including
microfracture, osteochondral autograft
 2
transplantation, osteochondral allograft
transplantation, and autologous chondrocyte
implantation. Although autograft remains
the gold standard method for repairing
musculoskeletal tissue damage, this method
causes donor site morbidity and has limited
availability. Allograft and xenograft are
potential sources of tissue, although it has
some degree of risk in enhancing immune
responses and rejections associated with the
presence of cellular components and the
expression of major histocompatibility
complexes (MHC). (3)
Autologous Chondrocyte
Implantation (ACI) is a method aimed at
regenerating the articular cartilage.
Although some research has been done and
ACI itself shows promising results, this
method is not without any weakness. One
study mentioned cultured chondrocyte cells
were eliminated before starting to produce
any extracellular matrix. (4)
With its ability to regenerate and
differentiate, stem cells provide new hope in
the treatment of cartilage defects. This
method has the main advantage, that is being
a minimally-invasive option. Nevertheless, it
turns out after further examination that this
concept does not provide optimal results in
the handling of cartilage defects. With the
concept of tissue engineering in the last
decade, the attention and research in the
field of stem cell is progressing very rapidly.
However, in vitro research and the use of
mesenchymal stem cells which have been
widely practiced in the orthopedic field have
been largely using conventional culture with
free air (normoxia), which in turn resulted in
shock state and reduced viability of stem
cells prior to transplantation. (5)
Mesenchymal stem cells
physiologically require optimal precondition
in the form of low O2 tension of 1-3% in
bone marrow (6). The condition of hypoxia
increases the differentiation of stem cells
into chondrocytes, but it also inhibits the
occurrence of terminal differentiation into
hypertrophic chondrocytes (7). The terminal
differentiation of chondrocytes into
hypertrophic state is correlated with the
pathophysiology of osteoarthritis (8).
Therefore, keeping the chondrocytes
phenotype within normal and preventing
them from becoming into hypertrophic ones
are essential in the treatment of cartilage
defects.
With the new technique of
decellulerization, cellular components that
can induce an immune response can be
eliminated from allograft and xenograft, as
well as being able to prove its potential as an
alternative source of microenvironment stem
cells.
Materials and Methods
T his study was conducted for 6 months
at the Institute of Tropical Disease
(ITD) Airlangga University and The
Tissue Bank of Dr. Soetomo General
Hospital, Surabaya. This research took place
from July 2016 until December 2016.
This research is an experimental in
vitro laboratory study of Bone Marrow
Mesenchymal Stem Cells (BMSCs) culture
on decellularized bovine cartilage scaffold
under hypoxic condition. The experimental
unit was divided into three different
treatment groups, each divided by the
treatment of normoxic and hypoxic
conditions, then evaluated at the same
incubation period. The experimental unit in
this study was mesenchymal stem cell taken
from the bone marrow of male New Zealand
white rabbit femoral bone, weighing at least
3 kg, aged between 6-9 months.
Implementation of this research is
divided into 4 stages of research. First, the
isolation of MSCs from the bone marrow of
healthy male New Zealand rabbit strain.
Second, MSCs culture on three different
mediums of decellularized bovine cartilage
scaffold, chondrogenic medium, and the
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combination of scaffold and chondrogenic

medium that already contained growth plate.
Third, providing a hypoxic culture of stem
cells in differentiation phase with two doses
of concentration of O2 (21 and 1%). Fourth,
analyzing the stem cell differentiation into
chondrocytes, which includes: (1) Collagen
type II production on extracellular matrix
formed (immunocytochemically and
immunohistochemically); (2) Increased
mature cholesterol (based on
immunocytochemical and
immunohistochemical SOX9 expression); Figure 1. Expression of Collagen Type II
And (3) Decreased formation of Chondrogenic Medium with 100x
hypertrophic chondrocytes (based on Magnification. A-B. Condition of Hypoxia,
immunocytochemical and C-D. Conditions of Normoxia
immunohistochemical RUNX2 expression).
From Figure 1 and Graphic 1 it is
known that the amount of type II collagen
Results expressed on chondrogenic medium exposed

D ifferent expressions of type II

collagen, SOX9, and RUNX2 were
compared between decellularized
cartilage scaffolds with a combination of
decellularized cartilage scaffold and
to hypoxic conditions under constant
surveillance showed much greater value
than those exposed to normoxic condition
and this difference was statistically
significant (p <0.05).
chondrogenic medium. Moreover, this
research also conducted a study using
monolayer culture plate for measuring the
number of type II collagen, SOX9 and
RUNX2 in chondrogenic medium exposed
under hypoxic and normoxic conditions.
After that we performed an
immunocytochemistry (ICC) assay to clarify
the expression of type II collagen and also
we perform an immunofluorescence
examination to calculate the number of cells
expressing SOX9 and RUNX2. Graphic 1. Comparison of type II collagen
With imunocytochemistry/ICC expression on chondrogenic medium
imaging, it is clear that the difference in type between normoxic and hypoxic conditions
II collagen expression with surrounding
cells is as shown in Figure 1. The expression Furthermore, through Figure 2 and
of type II collagen formed is then calculated Graphic 2 it is known that the number of
on 5 planes with a 100x magnification light SOX 9 expressed and fluorescensed by
microscope. chondrogenic medium exposed to hypoxic
conditions is more than those exposed to
normoxic condition and this difference again
was statistically significant (P <0.05).
 4
Figure 2. SOX 9 Expression on
chondrogenic medium. A-B. Condition of
Hypoxia, C-D. Condition of Normoxia
Graphic 2. The comparison of SOX9
expression on chondrogenic medium
between normoxic and hypoxic conditions
Different results are shown in Figure
3 and Graphic 3 below where RUNX2
counts are expressed more in chondrogenic
medium under normoxic condition than
fluorescent cells found in preparations
exposed to hypoxic conditions. The result is
statistically significant (p <0.05).
Figure 3. RUNX2 Expression on
Chondrogenic Medium. A-B. Condition of
Hypoxia, C-D. Conditions of Normoxia
Graphic 3. The comparison of RUNX2
expression on chondrogenic medium
between normoxic and hypoxic conditions
From overall comparation, Graphic 4
below showed that the largest expression of
type II collagen is shown by scaffold
combined with chondrogenic medium
exposed to hypoxic conditions and these
results are significant (p <0.05).
 5
Graphic 4. Comparison of type II collagen
expression between Scaffold and a
combination of scaffold and chondrogenic
medium under normoxic and hypoxic
conditions
The interesting point is shown in
Graphic 5 in which scaffolds combined with
chondrogenic medium exposed to both
hypoxic and normoxic conditions showed
significant differences in SOX9 expression
when compared with the scaffold only group
(p <0.05).
Graphic 5. Comparison of SOX9 expression
between Scaffold and a combination of
Scaffold and chondrogenic medium under
normoxic and hypoxic conditions
Graphic 6. Comparison of RUNX2
expression between Scaffold and a
combination of Scaffold and chondrogenic
medium under normoxic and hypoxic
conditions
From a series of
immunohistochemistry assay results of
decellularized cartilage scaffold above, it
was found that scaffolds exposed to hypoxic
conditions will express a higher amount of
collagen, with SOX 9 and RUNX 2 being no
different from the scaffold exposed to
normoxic conditions. It was also found that
in the decellularized cartilage scaffold
combined with chondrogenic medium
group, exposure to hypoxic conditions
expressed the higher number of collagen
type II, SOX9, and RUNX2 levels compared
with the combination of scaffold and
chondrogenic medium exposed to normoxic
conditions.
Discussion
T he biomaterials used in this study
have been tested in previous studies
under an electron microscope (15).
The decellulerization technique was done
physically, chemically, and enzymatically.
With decellulerization technique, it is
expected that the biomaterial used in this
research can be classified as raw material
which did not serve only as a scaffold but
also contain natural growth factor.
 6
Raw material also contains growth
factor proven by the emergence of SOX9
and RUNX2 expressions in the study group
of decellularized cartilage bovine by seeding
stem cells (Figure 4) without the provision
of growth factor from outside. This proves
the theory that there is interaction between
cells and biomaterials; The more
physiological the biomaterials inhabited by
stem cells, the better the growth and
development of those cells (15,16). Even cell
differentiation into chondrocytes in
decellularized cartilage bovine has been able
to produce collagen type II which is
essential in the formation of hyaline
cartilage.
Figure 4. Decellularized cartilage bovine
after stem cell seeding was observed under
an electron microscope
Stem cells underwent the
differentiation phase in hypoxic condition
showed better chondrogenic potency than in
normoxic condition. This is supported by the
fact that more SOX9 and collagen type II
were expressed in stem cell cultures under
hypoxic conditions. The hypothesis that
hypoxic conditions inhibit the chondrocytes
becoming hypertrophic was demonstrated by
lower RUNX2 expression in hypoxic
conditions compared with normoxic ones.
In the decellularized bovine cartilage
group without the addition of growth factor
(scaffold), we obtained that the collagen
type II was produced more under hypoxic
conditions. This supports previous research
groups on chondrogenic mediums that
hypoxic conditions stimulate
chondrogenesis better than normoxic ones.
However, the exact mechanism in which the
hypoxic conditions in the scaffold yields
more type II collagen is still inexplicable.
This is because SOX9 and RUNX2
expressions are not significantly different
between hypoxic and normoxic conditions.
The absence of growth factor in this sample
group may also be the cause of SOX9 and
RUNX2 expressions that do not fit the
theory.
Samples with a combination of
scaffold and chondrogenic medium under
hypoxic conditions have significantly higher
type II collagen production than others. This
condition represents ideal conditions that
can be achieved in tissue engineering:
optimally processed cells (hypoxic), well
processed scaffold (physiological scaffold),
and availability of growth factor. The results
of this study support the theory of tissue
engineering triad (cells, scaffold, and growth
factors) with the most superior results
obtained in the combination group, both
with the expression parameters of SOX9 and
type II collagen.
The inconsistent phenomenon of
RUNX2 expression on scaffold in this study
is an interesting one. In the scaffold group
without the chondrogenic medium under
hypoxic conditions, RUNX2 expression
decreased but was not statistically
significant when compared with the
normoxic ones in the same group.
Meanwhile, in the combination group of
scaffold under hypoxic conditions, RUNX2
expression increased significantly compared
to the normoxic ones in the same group.
Meretoja et al stated that the medium
change of the 2D monolayer chondrogenic
medium on the culture plate into a 3D
scaffold one provides an oxygen gradient
change (12). Changes in the oxygen gradient
within the scaffold porosity lead to some
 7
changes in the mechanism of hypoxic
influence on differentiation of chondrocytes.
A study from Foldager et al
compared the culture process on a 2D
monolayer medium with a 3D medium
under hypoxic condition. The study stated
that there was a combination of positive
effects between 3D culture medium with
hypoxic condition. This confirmed the
theory that hypoxic condition is not the sole
factor that affects the differentiation of
chondrocytes in the tridimensional medium
(13)
. Changing the culture medium from 2D
to 3D itself is an important factor in
inducing chondrogenesis (14). This could
explain the inconsistency of RUNX2
expression in this study and other studies (12-
14)
. for reaching great effectiveness and
Although it can not be compared
between groups of chondrogenic mediums
with scaffold, researchers can still draw the
conclusion that hypoxic conditions produce
better quality and quantity than the
normoxic ones because the expression of
SOX9 and type II collagen is consistently
produced more in hypoxic conditions within
all sample groups. In a semi-quantitative
comparison between the scaffold groups and
the combination of scaffold and
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Aries Rakhmat Hidayat, MD1
1
Department of Orthopaedics and
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