Biocompatibility of osteogenic predifferentiated human cord blood stem cells with biomaterials and the influence of the biomaterial on the process of differentiation.

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Biocompatibility of Osteogenic
Predifferentiated Human
Cord Blood Stem Cells with
Biomaterials and the Influence
of the Biomaterial on the
Process of Differentiation
CHRISTIAN NAUJOKS, FABIAN LANGENBACH*, KARIN BERR,
RITA DEPPRICH, NORBERT KU¨BLER, ULRICH MEYER
AND JO¨RG HANDSCHEL
Department for Cranio- and Maxillofacial Surgery, Heinrich-Heine-University
Du¨sseldorf, Moorenstr. 5, 40225 Du¨sseldorf, Germany
GESINE KO¨GLER
Institute for Transplantation Diagnostics and Cell
Therapeutics Heinrich-Heine-University Du¨sseldorf, Moorenstr. 5
40225 Du¨sseldorf, Germany
ABSTRACT: Modern cell-based bone reconstruction therapies offer new
therapeutic opportunities and tissue engineering represents a more biological-
oriented approach to heal bone defects of the skeleton. Human unrestricted
somatic stem cells (USSCs) derived form umbilical cord blood offer new
promising aspects e.g., can differentiate into osteogenetic cells. Furthermore
these cells have fewer ethical and legal restrictions compared to embryonic stem
cells (ESCs). The purpose of this study was to evaluate the compatibility of
osteogenic pre-differentiated USSCs with various biomaterials and to address
the question, whether biomaterials influence the process of differentiation of the
USSCs. After osteogenic differentiation with DAG USSCs were cultivated with
various biomaterials. To asses the biocompatibility of USSCs the attachment
*Author to whom correspondence should be addressed.
E-mail: fabian.langenbach@med.uni-duesseldorf.de
JOURNAL OF BIOMATERIALS APPLICATIONS Volume 00 — Month 2009 1
0885-3282/09/00 0001–16 $10.00/0 DOI: 10.1177/0885328209358631
� The Author(s), 2009. Reprints and permissions:
http://www.sagepub.co.uk/journalsPermissions.nav
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and the proliferation of the cells on the biomaterial were measured by a
CyQUANT� assay, the morphology was analyzed by scanning electron
microscopy and the influence of the gene expression was analyzed by real time
PCR. Our results provide evidence that insoluble collagenous bone matrix
followed by b-tricalciumphosphate is highly suitable for bone tissue engineering
regarding cell attachment and proliferation. The gene expression analysis
indicates that biomaterials influence the gene expression of USSCs. These
results are in concordance with our previous study with ESCs.
KEY WORDS: biocompatibility, bone tissue engineering, gene expression,
scaffold, scanning electron microscopy, stem cell.
INTRODUCTION
Bone defects of the cranio- and maxillofacial skeletal system canoccur congenital or can be originated by manifold causes like
trauma, tooth loss, infection, age depending atrophy of the jaw, and
tumor resection. To achieve a sufficient quality of life it is necessary to
regenerate or reconstruct these bony defects in order to recover aesthetic
and functional aspects of the stomathognatic system. Despite the
substantial progress in field of tissue engineering the use of autologous
bone grafts, especially in patients with alterations of the tissue caused by
radiotherapy[1], is still the gold standard [2,3] for tissue repair even
though there is significant morbidity from donor site procedures and
quantitative limitations [4–6]. Modern cell-based bone reconstruction
therapies may offer new therapeutic opportunities and tissue engineering
represents a more biological-oriented approach to heal tissue defects [7].
By using cells, scaffolds, and growth factors, the three-foldmodel of tissue
engineering, a specialized tri-dimensional tissue is grown in vitro and can
be implanted into the bone defect [8]. Different types of scaffolds, growth
factors and cell sources – alone [9–14] or in various combinations with
bioactive cytokines like bonemorpheogenetic protein (BMP)-7, BMP-2, or
BMP-2-mutants [15–17] – have been applied for development of
bioartificial bone tissues [18–20].
Whereas transfer of the patient’s own tissue as an in situ stimulation
relies on autologous cells, extracorporal tissue engineering and genetic
engineering can be done with a wide variety of cells in different stages of
cell differentiation and maturation including autologous cells as well as
allogenic and xenogenic cells. [21–23].
It has been shown by various investigators that embryonic stem cells
(ESCs), representing pluripotent embryonic precursor cells, can differ-
entiate under selective culture conditions into osteogenic cells [24–26].
The most common way to initiate osteogenetic differentiation in stem
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cells is to supplement the medium with dexamethasone, ascorbic acid,
and b-glycerolphosphate [26–28].For this reason in the presented study
we performed osteogenic predifferentiation of the USSCs by the
supplementation of DAG to the medium. An advantage of using ESCs
instead of tissue-derived progenitor cells is that ESCs are immortal and
could potentially provide an unlimited supply of differentiated osteoblasts
and osteoprogenitor cells for transplantation. In contrast the prolifera-
tive, self-renewal and differentiation capacity of cells derived from the
adult tissue decreases with age [29,30]. In a previous animal study in rats,
the osteoinductive potential of ESCs was demonstrated. ESCs were able
to promote ectopic bone formation in vivo when they were used with
demineralized bone (insoluble collagenous bone matrix, ICBM) [31].
Recently, a new promising stem cell source was established by Ko¨gler
and co-workers [32]. Human unrestricted somatic stem cells (USSCs), a
multipotent cell line derived from cord blood, also have the ability to
differentiate into osteogenetic cells. Moreover, compared to the ESCs the
USSCs do not show any immunological rejection. Hematopoetic differe-
ntiated cells from cord blood are already used in the therapy of
hematopoetic and genetic disorders [33]. In addition, we could show
that USSCs can perform an osteogenic differentiation and format
hydroxyl apatite in micromass culture technology [34] in a previous
study (data not shown, paper in progress). Due to these facts and the
above-mentioned disadvantages of ESCs we analyzed the biocompat-
ibility of USSCs with biomaterials in the presented study.
Beside the cells the scaffold is another important fold of tissue
engineering. There are high demands on an ideal scaffold like cytocom-
patibility with the cells and biodegradability. Furthermore, the scaffold
should support the attachment and proliferation of the cells and may be
remodeled by the cells themselves. To date, several different bone
substitutes have been studied as scaffold material for applications in
tissue engineering [20]. Deproteinized bovine bone (Bio Oss�), b-tricalci-
umphosphate (b-TCP) (Cerasorb�), multiporose b-TCP (Cerasorb M�),
collagen (Resorba�), and ICBM are commonly used scaffold materials. In
a previous study, we analyzed the cytocompatibility of ESCs and the
influence of the biomaterial on the differentiation of the cells on these
biomaterials. We were able to show that ICBM followed by b-TCP is most
suitable for bone tissue engineering regarding cell attachment and
proliferation as well as the influence on the phenotype of the cells [35].
Despite the fact that scaffolds as well as the extracellular matrix
have both direct and indirect influence on cells and their behavior, e.g.,
the rate of proliferation and gene expression profile, common
approaches to engineer bone ex vivo are based on a combination of
Biocompatibility of Human Cord Blood Stem Cells 3
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cells and scaffolds [36]. There is still controversy concerning the use of
artificial scaffolds compared to natural matrix because the physico-
chemical properties of the biomaterials influence the proliferation and
the gene expression of the cells [12,18,19]. On this account, we analyzed
the influence of the biomaterial on the process of differentiation of
USSCs by the gene expression profile.
Summarized our previous results regarding ESCs and USSCs as well
as the findings of the literature conducted us to evaluate the
compatibility of pre-differentiated USSCs with various biomaterials
and to address the question whether biomaterials influence the process
of differentiation of the USSCs.
MATERIALS AND METHODS
Culture of USSCs with Biomaterials
USSC were kindly provided by the Jose´ Carrereas Stammzellbank,
Heinrich-Heine-University Du¨sseldorf, Germany. The cells were isolated
from cord blood with informed consent of the mother, isolated, and
cultivated according to a standardized protocol published by Ko¨gler et al.
[32]. Briefly, Ficoll (Biochrom) gradient centrifugation was used to
isolate the mononuclear cell fraction. Cells were plated out at 5 to
7� 106 cells/mL on T25 culture flaks (costar) in low glucose DMEM
(Cambrex), supplemented with 30% FCS, dexamethasone (10�7M;
Sigma-Aldrich), penicillin (100U/mL; Gru¨nenthal), streptomycin
(100mg/mL; Hefa-pharm), and ultraglutamine (2mM; Cambrex).
Later on in the expansion of the cells, the dexamtehasone was left out
of the medium. Cells were incubated in a humidified atmosphere at 378C
in 5% CO2. The cells were split when confluency reached 80%, by
detaching the cells with 0.25% trypsin (Lonza) and re-plating at ratio
1 : 3. After proliferation of the cells the osteogenic pre-differentiation
was performed by addition of 0.1 mM dexamethasone, 50 mM ascorbic
acid, and 10mM b-glycerolphosphate (all from Sigma) to the normal
growth medium according to previous published works [26,27,37,38].
After 3 days of osteogenic predifferentiation 200,000 cells suspended in
300 mL medium were seeded on each biomaterial-disk and cultured
under the above-mentioned standardized conditions without DAG for
24 h in a 48-well-plate. The biomaterial disks were put into medium for
24 h before seeding them with cells. To standardize the surface of the
specimen for each biomaterial a specimen-disk of 0.5 cm thickness and
1 cm diameter was used. After 24 h the disks were conveyed to a 6-well-
plate to minimize the influence of cells that were not bound to
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the biomaterial. Half of the specimen was analyzed at this time (day 1) of
cultivation by CyQUANT�-assay reflecting the attachment of the cells
on the different biomaterials. The second half of the specimen was
cultured under the above mentioned conditions without DAG for 7 days.
Medium change was performed every second day. On day 7 the second
half of the specimen was analyzed by CyQUANT�-assay representing
the proliferation of the cells on the biomaterial. Furthermore scanning
electron microscopy was performed for morphological analysis of the
specimen. In order to analyze the influence of the biomaterial on the
osteogenic gene expression profile of the cells, real time PCR was carried
out analyzing the gene expression profile. The following biomaterials
were used: deproteinized bovine bone (Bio Oss� Fa. Geistlich Pharma
AG, Wolhusen, Switzerland), b-TCP (Cerasorb�, Cerasorb M�, Fa.
Curasan AG, Kleinostheim, Germany), collagen (RESORBA�, Fa.
Resorba Wundversorgung GmbH þ Co. KG, Nu¨rnberg, Germany), and
ICBM produced in our laboratory.
Preparation of ICBM
One centimetre thick slices of bovine femur spongiosa were defatted
by three washing steps (24 h each) with chloroform–methanol solution
(3 : 1; Merck). After a washing step in aqua destillata for 30min the
slices were bleached with H2O2 (Merck) for 15min and washed again in
aqua destillata. Washing the slices three times 90min in 0.5M HCl
(Merck) was used for demineralization. Disks with a diameter of 10mm
and a height of 5mm were cut and incubated in 4M Guanidin-HCl/
50mM Tris-HCl (pH 7.0) (Merck) for 16 h at 48C. The slices were
incubated with 50mM Tris-HCl (pH 7.0)/0.15M NaCl (Merck) for 4 h at
48C and washed once again with aqua destillata for 30min.
Attachment-/Proliferation Assay
To assess the attachment and proliferation of the cells on the various
biomaterials the CyQUANT� assay was used (CyQUANT Cell
Proliferation Assay Kit�, Fa. Invitrogen, Karlsruhe, Germany). The
CyQUANT� cell proliferation assay is a highly sensitive, fluorescence-
based microplate assay for determining numbers of cultured cells. The
assay employs CyQuant GR dye, which produces a large fluorescence
enhancement upon binding to cellular nucleic acids that can be
measured using standard fluorescein excitation (485nm) and emission
(535nm) wavelengths. The fluorescence emission of the dye-nucleic acid
complexes correlates linearly with the cell number over a large range
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using a wide variety of cell types. Under the recommended assay
conditions, the readouts of the experiments lay well within the detection
limits. The assay was performed as previously described[39]. Between
8000 and 63,000 cells were analyzed per biomaterial disc.
PCR
To determine the influence of the biomaterials on the gene expression
pattern of the USSCs, quantitative real time PCR was used. Because
osteogenic differentiation was the main focus, genes were selected, which
are known to play a key role in osteogenic cells. Total RNA was isolated
from specimens using the RNeasy Mini Kit (Quiagen, Hilden, Germany)
according to themanufacturer’s instructions. For cDNA synthesis 800 ng
total RNA was used as a template with Superscript II (Invitrogen,
Paisley, UK) and OligodT-Primers (Peqlab, Erlangen, Germany). A total
of 1mL cDNA (dilution 1 : 10) was used for amplification performed with
specific primers (Fa. MWG-Biotech AG, Ebersberg, Germany) for
collagen type I (forward primer: 50-AAGGGGTCTTCCTGGTGAAT-30
and reverse primer: 50-GGGGTACCACGTTCTCCTC-30), alkaline phos-
phatase (forward primer: 50-AAGGCTTCTTCTTGCTGGTG-30 and
reverse primer: 50-GCCTAACCCTCATGATGTCC-30) GAPDH (forward
primer: 50-CAATGAATACGGCTACAGCAAC-30 and reverse primer:
50-AGGGAGATGCTCAGTGTTGG-30) and osteonectin (forward primer:
50-GTGCAGAGGAAACCGAAGAG-30 and reverse primer: 50-TGTTTGC
AGTGGTGGTTCTG-30). For quantitative real time PCR the iCycler
Thermal Cycler Base (Fa. Bio-Rad Labortatories GmbH, Munich,
Germany) and qPCR MasterMix, No Rox, #RT-QP2X-03NR
Eurogentec, Cologne, Germany, were used. The increase in reaction
products during PCR was monitored by measuring the increase in
fluorescence caused by the binding SYBR� Green to double-stranded
DNA accumulating during PCR cycles. Reaction mixtures were set up as
suggested by the manufacturer. Threshold cycle values of target genes
were standardized against GAPDH expression and normalized to
expression in cultures of USSCs which were cultured under the same
conditions but had no contact to any biomaterial. This control group was
differentiated for 3 days with osteogenic medium similar to the cells with
contact to a biomaterial. All real time experiments in this study have been
performed in accordance with to the publication of Pfaffl [40]. We have
applied the mathematical model given there to eliminate deviations 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. Differences were statistically analyzed by the 2���Ct method, a
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convenient way to analyze the relative changes in gene expression from
real-time quantitative PCR experiments. In this method, the expression
of a target gene in a specific sample is normalized to the expression of the
house keeping gene GAPDH (�Cttarget gene¼Cttarget gene�Cthouse keeping
gene) and is then compared to the normalized expression of a control
group. The complete equation to calculate the relative change in gene
expression is 2���Ct¼ (Cttarget gene�Cthouse keeping gene)Sample� (Cttarget
gene�Cthouse keeping gene)Control.
SCANNING ELECTRON MICROSCOPY
For Scanning electron microscopy (SEM) the biomaterial discs were
fixed for 3 h with 2.5% glutaraldehyde in 0.1M PBS (pH¼ 7.3) and
subsequently washed in 0.1M PBS for 30min three times. The samples
were dehydrated in increasing concentrations of acetone (from 50% to
100%, 10% steps). After critical point drying using CO2 as transitional
fluid (Bal-Tec Dryer CPD-030,) the specimens were sputter-coated with
gold and observed in SEM (REM S 3000, Hitachi).
RESULTS
The biocompatibility of human USSCs on biomaterials was assessed
by the attachment and the proliferation of the cells on the different
materials. Furthermore, the influence of the biomaterial on the process
of differentiation of the cells was monitored by an analysis of osteogenic
gene expression profile. Morphological analysis of the USSCs was
performed with SEM. USSCs are multipotent cell line that would be
expected to be able to form bone cells and facilitate extracorporal tissue
engineering of bone.
The number of cells on the biomaterial assessed by CyQUANT� assay
on day one represents the attachment of USSCs on the biomaterial
whether the cell count on day 7 reflects the proliferation of the cells on
the biomaterial. The attachment and the proliferation of cells on
biomaterials can be equalized with the biocompatibility of the biomater-
ial. ICBM and Cerasorb M� showed the highest cell count on day one
revealing that the USSCs show better attachment on these two
biomaterials compared to the other. The Influence of the biomaterial on
the attachment was tested by ANOVA and showed a high statistical
significance (Figure 1 (a) and (b)). Cerasorb M� and ICBM showed no
significant difference regarding the attachment. Interestingly, the
proliferation of the cells from day 1 to day 7 only took place on ICBM
and Cerasorb� and not on Cerasorb M�. The Influence of the biomaterial
Biocompatibility of Human Cord Blood Stem Cells 7
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p<0.05
p<0.05
p<0.05
p<0.05
p<0.005
p<0.005
p<0.05 p<0.05 p<0.05
p<0.005
60,000
(a)
(b)
50,000
40,000
30,000
Ce
lls
o
n
bi
om
at
er
ia
l
Ce
lls
o
n
bi
om
at
er
ia
l
20,000
10,000
0
60,000
50,000
40,000
30,000
20,000
10,000
0
Tag 1
Cerasorb M® Cerasorb®Collagen ICBM Bio Oss®
Tag 7 Tag 1 Tag 7 Tag 1 Tag 7 Tag 1 Tag 7 Tag 1 Tag 7
Tag 1
Cerasorb M® Cerasorb®Collagen ICBM Bio Oss®
Tag 7 Tag 1 Tag 7 Tag 1 Tag 7 Tag 1 Tag 7 Tag 1 Tag 7
p<0.005
p<0.005
p<0.005
Figure 1. Relative attachment and proliferation values. Shown are the means of the
CyQUANT� assay on day 1 and 7 which are a relative measure for attachment or the
proliferation of the cells on the biomaterial. ANOVA analysis determined the biomaterial
specific differences as statistically significant. (a) ICBM and Cerasorb M� showed a
significant better attachment of USSCs compared to Collagen, Cerasorb� and Bio Oss�. (b)
Cerasorb� and ICBM show a significant increase of the cell count reflecting a good cell
proliferation. Bio Oss� showed a significant decrease of the cell count. Note that on
Cerasorb M� the cell count decreases even if this reached no statistical significance. ICBM
showed the highest total cell count after 7 days of proliferation.
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on the proliferation was tested by ANOVA and showed a high statistical
significance regarding the increase on ICBM and Cerasorb. All other
biomaterials including Cersorb M showed a decrease of the cell count to
day seven, but these findings reached statistical significance only on Bio
Oss� (Figure 1(c)). On day 7 ICBM showed the highest cell number
compared to all other biomaterials. This difference is highly statistically
significant compared to all other biomaterials (Figure 1(d)).
The CyQUANT� results were confirmed with SEM. On Bio Oss no
cells were observable, whereas on all other biomaterials a layer of cells
was visible. The USSCs had direct contact to the biomaterial. An
invasion of the cells in the pores of the biomaterial could only be detected
on ICBM. It seems that the pore size of both tricalciumphosphate
materials and the collagen is too small to be invaded. Especially on
Cerasorb M�, ICBM and Collagen the cells did not show the
morphological characteristics of undifferentiated USSCs but rather of
mesenchymal cells (Figure 2). The cells showed a cubic, flattened
appearance and seemed to be in close attachment to each other like an
epithelial layer. Compared to the other biomaterials especially on ICBM
a particularly dense cell layer with a close attachment to each other has
been formed. In addition the cells on ICBM resemble most closely to the
phenotype of osteoblasts.
The gene expression analysis showed varying results. The gene
expression levels of the analyzed osteogenic markers varied on every
biomaterial so that every biomaterial generated a specific gene
expression pattern. Interestingly, also the gene expression pattern
varies between the two b-TCP (Cerasorb�, Cerasorb M�) scaffolds. The
highest levels of collagen I and osteonectin were seen on Cerasorb M�
and Collagen (Figure 3). In conformance with the previous results with
ESCs and the CyQUANT� analysis, the RNA content on Bio Oss� was
not sufficient for generating cDNA and real time PCR analysis.
DISCUSSION
Besides growth factors, the two main folds of bone tissue engineering
are the scaffolds with their material specific properties and the chosen
cell line. The USSCs are a promising cell source for tissue engineering of
bone. In this study, it has been shown that there are remarkable
differences in the compatibility of USSCs with biomaterials. In addition,
these biomaterials apparently influence the gene expression profile of
the USSCs.
The biocompatibility of different biomaterials and USSCs was
evaluated by vital cells on day 1 and day 7 after cell-application analyzed
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with a fluorescence based microplate assay (CyQUANT� assay).
Compared to other assays which detect ATP, the CyQUANT� assay
had a lower suspectibility of the intracellular DNA content compared to
the ATP concentration measured by other assays. Thus this assay is
more sensitive leading to a high correlation between the measured
concentration and the cell number in the CyQUANT� assay [39].
The results of the CyQUANT� assay show impressively that after 7
days the highest number of living cells appeared on the demineralized
bone (ICBM). The difference to the other biomaterials reached high
statistical significance. Even if there were no differences between ICBM
and Cerasorb M� regarding the attachment of the cells, the proliferation
Ce
ra
so
rb
M
®
Bi
o
O
ss
®
Ce
ra
so
rb
®
IC
BM
Co
lla
ge
n
Figure 2. SEM of biomaterials with USSCs. On Bio Oss� there are hardly any cells
observable. The other biomaterials show a layer of cells. Morphologically, the cells do not
show the characteristics of undifferentiated USSCs but rather of mesenchymal cells and
they seem to be in close attachment to each other. An invasion of the cells into the pores is
only observable on ICBM.
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of the cells only took place on ICBM and not on Cerasorb M�. These
findings underline that the USSCs show the best attachment on ICBM;
furthermore, they proliferate best on ICBM compared to the other
materials. In addition, the SEM analysis supports theses findings. A
close correlation between proliferation assay and SEM in compatibility
testing was also detected by other authors [41]. On the biomaterials, the
cells have lost their spheroidal shape, which is characteristic for USSCs,
and are attached to the scaffolds by their pseudopods. Thus these cells
resemble osteoblast-like cells. However, regarding the gene expression,
which does not reflect osteoblast-like expression patterns, this cell shape
could also resemble mesenchymal (stem) cells. These results correlate
with previously published results from our work regarding the
biocompatibility of ESCs on different biomaterials [35].
Biocompatibility of biomaterials are influenced by the tridimensional
topography and the physico-chemical properties of the material surface
[19]. Many studies that have demonstrated that osteoblasts are very
sensible to the gross topography of the surface of a biomaterial [42–44].
According to the structure of the surface, cells adapt with their
orientation, migration and attachment kinetics [45–47]. The porosity
of the material is one of the major properties that affects the structure of
Cerasorb® Cerasorb M® Collagen
ALP
5
4
3
2
1
0
ICBM
Collagen I Osteonectin
Figure 3. Gene expression analysis. Shown are the multiple of gene expression levels
compared to the culture of USSCs without contact to any biomaterial. expression of
alkaline phosphatase; expression of collagen I; expression of osteonectin. Note:
On Bio Oss� no values are detectable.
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the surface and should be about 100 mm [48]. Regarding the macropores
ICBM has the highest porosity and the largest pores of the materials
tested in this study. Theses pores are interconnected to each other and
thus may improve bone formation. These geometric properties may have
a positive effect on the formation of bone [49]. This is, however, obvious
since ICBM is a natural product made of bovine bone, whereas the
products of Cerasorb and collagen are synthetic constructs that try to
meet the biological properties. The size of the pores may also be the
reason for the differences regarding the proliferation of the cells between
Cerasorb� and Cerasorb M�. These materials are both made of b-TCP
and only differ in the size of the pores.
Furthermore, the physico-chemical properties of the materials may
influence the behavior of the cells. Interestingly, ICBM, which consists
primarily of collagen type I, provides the best conditions for the cell
attachment and proliferation. Various studies support these findings and
could prove that the attachment of osteoblasts in the first hours mainly
depends whether the surface of the material is similar to proteins [43,50].
These authors have described that the highest number of mesenchymal
stem cells was found on biomaterials with a high content of collagen. The
fact that ICBM has by far the highest portion of collagen may be an
adequate explanation for the best biocompatibility of ICBM.
Regarding the attachment of cells Turhani et al. showed recently that
the attachment on Bio Oss� is worse compared to peptid P15 coated
hydroxylapatite. Furthermore, the presented results of this study are in
coexistence with the results of Petrovic and co-workers [51], which could
show significant higher proliferation and attachment of cells on
biomaterials with large fraction of collagen compared to materials with
less or without collagen. In the presented study ICBM showed the highest
fraction of collagen apart from the collagen sponge (RESORBA�). The
fact that RESORBA� has significantly smaller pores compared to the
ICBM, may be a possible explanation for the reduced proliferation of
the cells compared to the ICBM. Furthermore, it may be possible that
ICBM contains relicts of growth factors which could extend the
osteogenic stimulation of the cells [15].
Regarding the gene expression we observed very different expression
patterns depending on the biomaterial. This means that the biomaterial
does affect the gene expression. However, the underlying reasons are not
known. These findings are in concordance with our previous results
regarding the ESCs [35].
In conclusion, ICBM is highly suitable for bone tissue engineering
with USSCs regarding cell proliferation and phenotype. However, this
does not infer that this biomaterial induces ostogenic differentiation.
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That might provide new options in the therapy of periodontal bone
defects or bone loss due to trauma, tooth loss, and atrophie.
Furthermore, we could demonstrate that the biomaterial influences
the osteogenic differentiation of the cells.
ACKNOWLEDGMENT
Fabian Langenbach is supported by a grant of Deutsche
Forschungsgemeinschaft (DFG) (HA 3228/2-1). The work of Prof.
Ko¨gler is supported by the DFG grant KO2119/6-1.
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