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ssBioMed CentHead & Face Medicine
Open AcceResearch
Induction of osteogenic markers in differentially treated cultures of
embryonic stem cells
Jörg Handschel*1, Karin Berr1, Rita A Depprich1, Norbert R Kübler1,
Christian Naujoks1, Hans-Peter Wiesmann2, Michelle A Ommerborn3 and
Ulrich Meyer1
Address: 1Department for Cranio- and Maxillofacial Surgery, Heinrich-Heine-University Düsseldorf, Moorenstr. 5, 40225 Düsseldorf, Germany,
2Department for Cranio- and Maxillofacial Surgery, Westfälische-Wilhelms-Universität Münster, Waldeyerstr. 30, 48149 Münster, Germany and
3Department for Operative and Preventive Dentistry and Endodontics, Heinrich-Heine-University Düsseldorf, Moorenstr. 5, 40225 Düsseldorf,
Germany
Email: Jörg Handschel* - handschel@t-online.de; Karin Berr - handschel@med.uni-duesseldorf.de; Rita A Depprich - depprich@med.uni-
duesseldorf.de; Norbert R Kübler - kubler@med.uni-duesseldorf.de; Christian Naujoks - christian.naujoks@med.uni-duesseldorf.de; Hans-
Peter Wiesmann - HansPeter.Wiesmann@ukmuenster.de; Michelle A Ommerborn - ommerborn@med.uni-duesseldorf.de;
Ulrich Meyer - ulrich.meyer@med.uni-duesseldorf.de
* Corresponding author
Abstract
Background: Facial trauma or tumor surgery in the head and face area often lead to massive
destruction of the facial skeleton. Cell-based bone reconstruction therapies promise to offer new
therapeutic opportunities for the repair of bone damaged by disease or injury. Currently,
embryonic stem cells (ESCs) are discussed to be a potential cell source for bone tissue engineering.
The purpose of this study was to investigate various supplements in culture media with respect to
the induction of osteogenic differentiation.
Methods: Murine ESCs were cultured in the presence of LIF (leukemia inhibitory factor), DAG
(dexamethasone, ascorbic acid and β-glycerophosphate) or bone morphogenetic protein-2 (BMP-
2). Microscopical analyses were performed using von Kossa staining, and expression of osteogenic
marker genes was determined by real time PCR.
Results: ESCs cultured with DAG showed by far the largest deposition of calcium phosphate-
containing minerals. Starting at day 9 of culture, a strong increase in collagen I mRNA expression
was detected in the DAG-treated cells. In BMP-2-treated ESCs the collagen I mRNA induction was
less increased. Expression of osteocalcin, a highly specific marker for osteogentic differentiation,
showed a double-peaked curve in DAG-treated cells. ESCs cultured in the presence of DAG
showed a strong increase in osteocalcin mRNA at day 9 followed by a second peak starting at day
17.
Conclusion: Supplementation of ESC cell cultures with DAG is effective in inducing osteogenic
differentiation and appears to be more potent than stimulation with BMP-2 alone. Thus, DAG
treatment can be recommended for generating ESC populations with osteogenic differentiation
Published: 10 June 2008
Head & Face Medicine 2008, 4:10 doi:10.1186/1746-160X-4-10
Received: 30 July 2007
Accepted: 10 June 2008
This article is available from: http://www.head-face-med.com/content/4/1/10
© 2008 Handschel et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 7
(page number not for citation purposes)
that are intended for use in bone tissue engineering.
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Background
Facial trauma or tumor surgery in the head and face area
often lead to massive destruction of the facial skeleton [1].
The reconstruction of damaged or lost bone is a clinical
challenge in modern reconstructive surgery. The repair of
bone defects still poses a significant problem for many cli-
nicians. In the early decades of bone reconstruction sur-
geons used artificial tissue substitutes containing metals,
ceramics, and polymers to maintain skeletal function [2].
These artificial materials have facilitated surgeons to
restore the form and – to some extent – the function of
defective bones. Nevertheless, these artificial materials
have specific disadvantages, and thus encouraged sur-
geons to develop alternative approaches including cell-
based devices. Transplantation of autografts is a fre-
quently used treatment strategy in routine clinical practice
and has gained the "gold standard" in bone reconstructive
surgery, despite donor site morbidity and donor shortage
[3].
Modern cell-based bone reconstruction techniques may
offer new therapeutic opportunities for the repair of bone
damaged by disease or injury. Generally, the combination
of scaffolds, bioactive factors, and living cells provides a
surgically implantable product for use in tissue regenera-
tion and functional restoration [4,5]. Numerous attempts
were undertaken with various success to restore bone
defects by various biomaterials alone [6-10] or in combi-
nation with bioactive cytokines such as bone morphoge-
netic protein (BMP)-7, BMP-2 or BMP-2-mutants [11,12].
Cell-based strategies in bone tissue engineering use differ-
ent cell sources including autologous cells as well as allo-
genic and xenogenic cells [13-16]. There are some reports
that use totipotential embryonic stem cells in tissue engi-
neering of bone [17,18].
Embryonic stem cells (ESCs) are routinely derived from
the inner cell mass of blastocysts and represent pluripo-
tential embryonic precursor cells that give rise to all cell
types in the developing organism. ESCs have historically
been maintained in co-culture with mitotically inactive
fibroblasts [19-21]. This co-culture system is unnecessary
if the medium is supplemented with leukemia inhibitory
factor (LIF) [22,23]. In the absence of LIF embryonic stem
cells will differentiate into a morphologically mixed cell
population expressing features of endoderm and meso-
derm lineages [24]. By definition ESCs have the potential
to differentiate into osteogenic cells under selective cul-
ture conditions. Specifically, it has been shown by various
investigators that ESCs can differentiate into osteogenic
cells under selective culture conditions [17,18,25]. How-
ever, it is unclear which medium is most suitable to initi-
ate osteogenic differentiation. BMP-2 and a mixture of
the time-dependent expression of the osteoblastic mark-
ers osteopontin [26], collagen I [27], alkaline phos-
phatase [28], and osteocalcin [29] in ESC cells.
Methods
Culture of ESCs with biomaterials
Feeder-independent murine ESCs were derived from the
inner cell mass of blastocysts extracted from C57BL/6
mice. The ESCs were kindly provided by K. Pfeffer (Insti-
tute for Microbiology, Heinrich-Heine-University, Ger-
many). The cells were tested to be positive for the stem cell
marker Pouf1 (alias Oct4) and Foxd3 [30] (data not
shown). A total number of 1.5 × 106 cells per petri dish
(10 cm in diameter) were cultured in Dulbecco's Eagle
medium (DMEM). The medium was supplemented with
5 mM glutamine, 100 units/ml penicillin, 100 μg/ml
streptomycin, 50 μM 2-mercaptoethanol and 15% fetal
calf serum (FCS). The ESCs were divided into four groups
and cultured for 25 days as follows: group I; control, sup-
plemented with LIF to prevent differentiation, group II;
no additional supplement, group III; supplemented with
BMP-2 (10 ng/ml), and group IV; supplemented with
DAG (dexamethasone (0.1 μM), ascorbic acid (50 μM)
and β-glycerophosphate (10 mM).
Microscopical analyses
To detect mineralization in the differently treated cell cul-
tures, the cells were washed two times with PBS (phos-
phate-buffered saline) before fixation with 3%
glutardialdehyde in PBS for 30 minutes. The cells were
washed with distilled water and incubated in 5% silver
nitrate (Sigma Aldrich) for 1 hour. The cells were washed
again with distilled water. A solution of 5% sodium car-
bonate and 10% formaldehyde was added for 2 minutes
before the cells were washed again and fixed with 1%
sodium thiosulfate. Calcium-phosphate deposits stained
black [31,32].
Quantitative real time PCR
Quantitative real time PCR was employed to assess the
influence of the biomaterials on gene expression. Total
RNA was isolated from specimens using the RNeasy Mini
Kit (Qiagen, Hilden, Germany) according to the manufac-
turer's instructions. For cDNA synthesis 800 ng total RNA
was used as a template for Superscript II (Invitrogen, Pais-
ley, UK) and OligodT-Primers (Peqlab, Erlangen, Ger-
many) in a total volume of 20 μl. Amplification was
performed with 1 μl of cDNA and the following specific
primer pairs (MWG-Biotech AG, Ebersberg, Germany):
CD34; 5'-CACAGAACTTCCCAGCAAACTC-3' and 5'-
CATGTTGTCTTGCTGAATGGCC-3', osteopontin; 5'-
CCCGGTGAAAGTGACTGATT-3' and 5'-TTCTTCAGAG-
GACACAGCATTC-3', osteocalcin; 5'-GCCCTGAGTCT-Page 2 of 7
(page number not for citation purposes)
dexamethasone, ascorbic acid and β-glycerophosphate
(DAG) are good candidates [19,25]. Thus, we examined
GACAAAGGTA-3' and 5'-GGTGATGGCCAAGACTAAGG-
3', collagen type I; 5'-AAGGGGTCTTCCTGGTGAAT-3'
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and 5'-GGGGTACCACGTTCTCCTC-3', alkaline phos-
phatases; 5'-AAGGCTTCTTCTTGCTGGTG-3' and 5'-
GCCTTACCCTCATGATGTCC-3', and GAPDH; 5'-CAAT-
GAATACGGCTACAGCAAC-3' and 5'-AGGGAGATGCT-
CAGTGTTGG-3'. For quantitative real time PCR the
iCycler Thermal Cycler Base (Bio-Rad Laboratories
GmbH, München, Germany) and qPCR MasterMix, No
Rox, #RT-QP2X-03NR (Eurogentec, Köln, Germany) was
used. The increase in reaction products during PCR was
monitored by measuring the increase in fluorescence
intensity caused by the binding of SYBR green to double-
stranded DNA that accumulated during PCR cycles. Reac-
tion mixtures were set up as suggested by the manufac-
turer. Threshold cycle values of target genes were
standardized against GAPDH expression and normalized
to the expression in the control culture (group I). All real
time experiments in this study have been performed with
regard to the publication of Pfaffl [33]. We have applied
the mathematical model given there to eliminate devia-
tions due to sample preparation. In order to apply this
model it is necessary to choose a reference gene (e.g.
GAPDH) for calculating relative expression levels. The
quantitative real time PCR was performed in samples
obtained at day 5, 9, 11, 13, 15, 17, 19, 21, 23, and 25 of
culture, respectively. Following PCR agarose-gel electro-
phoresis was performed using β-actin as a reference.
Results
In ESC cultures supplemented with DAG we found the
largest deposition of calcium phosphate-containing min-
erals, as judged by von Kossa staining (Fig. 1). ESCs cul-
tured in the presence of BMP-2 exhibited less
mineralization, and there were no signs of mineral depo-
sition in unstimulated control cells or cells stimulated
with LIF.
In order to assess the differentiation of ESCs cultured
under different conditions, we used the hematopoetic
stem cell marker CD34. Only in ESC cultures without any
additional stimulus (ESCs without LIF) the expected
amplicon appeared in agarose-gel electrophoresis. ESCs
which were differentiated with BMP-2 or DAG have
downregulated this marker (Fig. 2).
Next the kinetics of gene expression in ESCs during differ-
entiation and matrix formation were evaluated. The val-
ues were plotted as a multiple of the expression in the
control group (ESCs with LIF). Expression of osteopontin
was reduced in ESC treated with LIF as compared to all
other samples (without LIF, with BMP-2 or with DAG).
The low level of osteopontin mRNA synthesis persisted in
the presence of DAG, and in BMP-2-treated cells showed
a steep increase after 2.5 weeks of culture. ESCs without
LIF showed similar expression rates as the DAG group
(Fig. 3).
Starting at day 9 of culture, a strong increase in collagen I
expression was recorded in the DAG culture, which was
Results from qualitative PCR showing amplification of the h ma opoetic stem cell marker CD34 in ESC cells trea ed (a) without LIF, (b) with BM -2, or (c) DAGFig re 2
Results from qualitative PCR showing amplification
of the hematopoetic stem cell marker CD34 in ESC
cells treated (a) without LIF, (b) with BMP-2, or (c)
Mineral deposition at day 14 in differently treated embryonic stem cells (ESCs)Figu e 1
Mineral deposition at day 14 in differently treated embryonic
stem cells (ESCs). Cells were exposed to (a) LIF (leukemia
inhibitory factor), (b) without LIF, (c) DAG (dexamethasone,
ascorbic acid and β-glycerophosphate) or (d) BMP-2. Shown
are von Kossa stainings with arrows pointing to the deposi-
tion of calcium phosphate-containing minerals that stained in Page 3 of 7
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DAG. Beta-actin was used as control.black.
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paralleled to a lesser extent by the collagen expression in
the BMP-2-treated cells. After three weeks of culture the
expression level of collagen I mRNA was similar in all
groups of the differentially treated cells (Fig. 4). Only the
DAG culture showed a second but smaller increase at day
23.
The transcription of mRNA coding for alkaline phos-
phatase was slightly increased in cells stimulated with
BMP-2. ESCs exposed to DAG did not significantly differ
from the control culture (Fig. 5). Expression of osteocal-
cin, which is regarded as a highly specific marker for oste-
oblasts, demonstrated showed a double-peaked curve in
the DAG-treated cells. ESCs cultured in DAG-supple-
mented medium showed a prominent peak after 9 days
and a second peak beginning at day 17. The first increase
was also seen in ESCs cultured in the presence of BMP-2
or the absence of LIF. Interestingly, in all the differentially
treated cells a second peak of osteocalcin transcription
was observed 7 days later (Fig. 6). All three ESC cultures
showed similar expression pattern of the hematopoetic
stem cell marker CD34 (Fig. 7).
Discussion
Currently, there are many efforts to establish cell-based
strategies in bone tissue engineering. ESCs are one of
many different cell populations, which are being tested
for their feasibility for these treatment options. The pur-
osteogenic differentiation in ESC cultures. In addition, we
investigated the kinetics of gene expression during in vitro
differentiation.
The results of our microscopical analysis revealed that
ESCs cultured in the presence of DAG show by far the
highest extent of mineralisation as determined by the
occurrence of calcium-phosphate-containing crystals.
With respect to extracellular matrix maturation and min-
eral deposition as crucial steps in the osteogenic cascade
[34], DAG seems to be the most promising supplement
for inducing osteogenic differentiation in ESCs. In accord-
ance with our microscopical results, a strong increase of
collagen I expression was observed at day 11 in the DAG-
treated cells. Stimulation with BMP-2 also increased colla-
gen synthesis. Expression of osteocalcin mRNA followed a
different pattern and appeared as a double-peaked curve,
when ESCs were supplemented with osteogenic agents
(DAG or BMP-2). However, the peak induction of osteo-
calcin mRNA in the BMP-2-treated cells was lower and
delayed as compared to DAG-exposed cells. Taken
together, these results support the use of DAG as a potent
agent for inducing in vitro differentiation of ESCs into
osteoblast-like cells.
There are only few reports addressing osteogenic differen-
tiation of ESCs published in the literature so far
[18,25,34,35]. In agreement with these results we describe
here that mineralisation is microscopically evident as
Expression of collagen I transcripts in differentially treated SCs cultures: DAG , BMP-2 and without additional supple-ments (ESC without LIF) Figure 4
Expression of collagen I transcripts in differentially treated
ESCs cultures: DAG , BMP-2 and without
additional supplements (ESC without LIF) . Values
are calculated as multiples of the transcription level of the
control culture (ESC with LIF) and shown as mean values and
standard deviations after normalisation against GAPDH.
X
mRNA levels for osteopontin in ESCs cultured with DAG , BMP-2 or without additi al supplements (ESC without LIF) Figure 3
mRNA levels for osteopontin in ESCs cultured with DAG
, BMP-2 or without additional supplements
(ESC without LIF) . Values are calculated as multiples
of the transcription level of the control culture (ESC with
LIF). Shown as mean values and standard deviations normal-
ized to the expression of GAPDH.
XPage 4 of 7
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pose of this investigation was to determine which supple-
ments in culture medium are most suitable to initiate
early as two weeks of culture. Buttery and co-workers also
used DAG as a culture supplement and found that miner-
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alisation was detectable when dexamethasone was added
only at day 14 or later [35]. By following this protocol the
differentiation process was delayed as compared to the
findings in our ESC cultures. While Buttery used only
microscopical methods for studying osteogenic differenti-
ation, zur Nieden and colleagues performed also gene
expression analyses for osteogenic markers [34]. With
respect to the time-course of gene expression with an early
increase of collagen I and a later increase of osteocalcin
transcripts, their data are comparable to our findings as
shown above. Unlike to the findings of zur Nieden and
colleagues, an early peak of osteocalcin expression and a
minor increase of osteopontin were found in the pre-
sented study. The differences could be explained by differ-
ent concentrations of supplements used for cell
differentiation. Zur Nieden et al. used 1,25-OH vitamin
D3 instead of dexamethasone. According to Zhang et al.
vitamin D3 increases osteopontin expression in osteob-
lasts and inhibits expression of osteocalcin [36]. Chaud-
hry and co-workers replaced dexamethasone with retinoid
acid, which was found to be an inductor of mineralization
in three-dimensional scaffolds [25]. Notably, alkaline
phosphatase was constitutively expressed at high levels in
undifferentiated cells [37]. In this experimental setting the
mineralisation process was delayed and was detectable
only after day 21. Treatment with DAG appeared to be
equal or even superior to BMP-2 stimulation regarding the
induction of osteogenic differentiation in ESCs. Other
authors have used BMP-2 in combination with osteogenic
supplements for this purpose [18,38].
An advantage of using ESCs instead of tissue-derived pro-
genitor cells is that ESCs are immortal and could poten-
tially provide an unlimited supply of differentiated
osteoblast and osteoprogenitor cells for transplantation.
In contrast to embryonic cells, the proliferative, self-
renewal and differentiation capacity of cells derived from
adult tissues generally decreases with age [39,40]. One
major challenge pointing to the use of ESCs lies in over-
coming immunological rejection from the transplant
recipient. Interestingly, Burt and colleagues performed
mRNA levels of CD34 expression in ESCs cultured with DAG , BMP-2 and without additional supplements (ESC without LIF) Figure 7
mRNA levels of CD34 expression in ESCs cultured with
DAG , BMP-2 and without additional supple-
ments (ESC without LIF) . Data are presented as in
X
mRNA levels of alkaline phosphatase in ESCs cultured with DAG , BMP-2 and without additional supplements (ESC without LIF) Figure 5
mRNA levels of alkaline phosphatase in ESCs cultured with
DAG , BMP-2 and without additional supple-
ments (ESC without LIF) . Data are presented as in
Fig. 3.
X
mRNA levels of osteocalcin in ESCs cultured with DAG , BMP-2 and with ut additional supplements (ESC without LIF) Figure 6
mRNA levels of osteocalcin in ESCs cultured with DAG
, BMP-2 and without additional supplements
XPage 5 of 7
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Fig. 3.(ESC without LIF) . Data are presented as in Fig. 3.
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