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Characterization and effect of modified surface on the morphology structure and function of rabbit bone marrow-derived mesenchymal stem cells 1.

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ASIA-PACIFIC JOURNAL OF CHEMICAL ENGINEERING
Asia-Pac. J. Chem. Eng. 2009; 4: 765–770
Published online 28 June 2009 in Wiley InterScience
(www.interscience.wiley.com) DOI:10.1002/apj.334
Special Theme Research Article
Characterization and effect of modified surface on the
morphology, structure and function of rabbit bone
marrow-derived mesenchymal stem cells 1
K. D. Song,1 * K. Liu,2 T. Q. Liu1 * and X. H. Ma1
1
2
Dalian R&D Center for Stem Cell and Tissue Engineering, Dalian University of Technology, Dalian 116024, China
Institute of Near-field Optics and Nanotechnology, Department of Physics, Dalian University of Technology, Dalian 116024, China
Received 31 October 2008; Revised 12 March 2009; Accepted 17 March 2009
ABSTRACT: The surface morphologies modified with chitosan and gelatin respectively, the effects on cell spreading,
adhesion and distribution of rabbit bone marrow-derived mesenchymal stem cells (rMSCs) were observed under
atomic force microscope (AFM). The structures and morphologies of rMSCs on coverslips were qualitatively and
quantitatively assayed under AFM. Thereafter, the growth status and viability of rMSCs on the different coated surfaces
were examined under inverted microscope and by Hoechst/PI double staining. The results showed that the surfaces
modified with different materials presented different morphologies. The rMSCs adhered to the surfaces modified with
2 mg/ml chitosan and 0.5 mg/ml gelatin solution showed better viabilities than those of other groups, and the latter also
presented the optimal cell adhesion, spreading and distribution with clear cellular structure such as cell nucleus, villus
and interaction among various rMSCs. It is indicated that the surface modification played an important role on the
development of cellular structure and function of rMSCs, and thus provided theoretical guidance to the fabrication and
surface modification of embedded biomaterials in further clinical applications.  2009 Curtin University of Technology
and John Wiley & Sons, Ltd.
KEYWORDS: mesenchymal stem cells; surface modification; biomaterials; AFM; morphology
INTRODUCTION
In the last decade, various stem cells have presented
remarkable applications in cell biology, regenerative
and clinical medicine, owing to their ability for selfrenewal in undifferentiated status and multi-potential to
dissimilar committed cells while induced within specific culture environments.[1,2] In particular, of all the
stem cells, mesenchymal stem cells (MSCs) have been
considered the most widely known and characterized
stem cells due to their easy isolation and expansion
as well as multi-differentiation to osteoblasts, chondrocytes, adipocytes, etc. in vitro.[3,4]
The concept of using MSCs by combining them
with biomaterial scaffolds and/or cytokines to repair
tissues has progressively evolved, and the goal of
cell-mediated therapy is to prolong the natural physiological abilities of healing, or substituting them,
when these are lacking, failing or progressing too
slowly.[5] But, because the original surface of some
*Correspondence to: K. D. Song and T. Q. Liu, Dalian R&D Center
for Stem Cell and Tissue Engineering, Dalian University of Technology, Dalian 116024, China. E-mail: kedongsong@yahoo.com.cn
 2009 Curtin University of Technology and John Wiley & Sons, Ltd.
special scaffolds are not suitable for MSCs adhesion,
some proteins or macromolecules, e.g. chitosan and
gelatin, were used as coatings in order to improve
the cell adhesion and spreading in some previous
studies.[6,7] Therefore, it is relevant to investigate the
influence of surface chemistry and topography on cell
behaviour such as cell adhesion, proliferation and
distribution.
Atomic force microscopy (AFM) is a form of scanning probe microscopy developed recently for imaging
the fine surface structures of various types of specimens
at high resolution.[8,9] It provides three-dimensional
views and cross-sections of stem cells at any point
attached to the substrates, with simultaneous measurement of their sizes.
In this study, we therefore set out to investigate the
feasibility of using AFM to detect the effects of surface modification with different concentrations of chitosan and gelatin thin films on the rMSCs viability,
spreading, morphology and function. Simultaneously,
we wanted to find which concentration for each material
was suitable for the better proliferation and distribution
of rMSCs.
766
K. D. SONG ET AL.
Asia-Pacific Journal of Chemical Engineering
Figure 1. The cell morphology, flow cytometry analysis, multi-potential induction and
live/dead assay of rMSCs. A, morphology photograph of rMSCs, primary passage.
B–C, Contents of CD44+ , CD45− , respectively. D–F, Osteogenesis, chondrogenesis and
adipogenesis assays respectively. Live/dead assays on the different surfaces modified
with the control group, and optimal concentration of chitosan and gelatin, G, Control
group; H, C–2%; I, G–0.5%, respectively. Magnification, ×100. This figure is available
in colour online at www.apjChemEng.com.
EXPERIMENTAL
Culture and multilineage differentiation of
rMSCs
The rMSCs were isolated from the femur of a 5-weekold New Zealand rabbit, as previously described in
Ref. [10]. Multilineage differentiation on osteogenic,
chondrogenic and adipogenic induction of rMSCs were
assessed for this study. The rMSCs were prepared by
inoculating cell suspension of 1 × 105 cells/ml onto
the coverslips with different surface modification and
cultured in 37 ◦ C temperature and 5% CO2 incubator
for 3 days for late use. Osteogenic, chondrogenic and
adipogenic inductions were detected as described in
Refs. [11–13], respectively.
Analysis by HO/PI double-staining and flow
cytometry
The rMSCs were cultured in media containing Hoechst
33 258 (HO, Sigma) and propidium iodide (PI, Sigma)
at final concentrations of 5 µg/ml and 2 µg/ml, respectively, as described in Ref. [14]. After an incubation
period of 1 h at 37 ◦ C, the cells were examined under
 2009 Curtin University of Technology and John Wiley & Sons, Ltd.
fluorescence microscope with DAPI filters. Following
this, the cells were photographed and viable, apoptotic
and necrotic cells were counted manually.
Approximately 1.0 × 106 fresh rMSCs were washed
once in PBS buffer with 1% FBS, stained by using antiCD44-FITC and anti-CD45- FITC (BD Pharmingen)
and incubated at 4 ◦ C in the dark for 30 min. Then
the cells were washed with PBS again and analysed
by using a FACSCalibur.
Surface modification with chitosan and gelatin
The 2 × 2 cm coverslips were put into 6-well culture
plates after being autoclaved or sterilized by ethylene
oxide, and radiated under UV for 30 min. The 2%
(W/V) chitosan and gelatin solutions were diluted
into 1, 0.5, 0.25 and 0.125% by using phosphate
buffer saline (PBS), respectively, and each 200 µl
solution with a different concentration was dropped
onto the coverslips uniformly by using the pipette gun.
The treated coverslips with different modification were
marked as C-2, C-1, C-0.5, C-0.25, C-0.125 and G2, G-1, G-0.5, G-0.25 and G-0.125%, respectively.
C represents chitosan and G, gelatin. The coverslips
were then radiated under UV and ventilated to stay
Asia-Pac. J. Chem. Eng. 2009; 4: 765–770
DOI: 10.1002/apj
Asia-Pacific Journal of Chemical Engineering
MESENCHYMAL STEM CELLS: RABBIT BONE MARROW
Modified with C-2%
Modified with G-0.5%
Figure 2. AFM three-dimensional topographic images of the surfaces modified with
3
2.0x10
3
1.5x103
1.5x103
1.0x103
1.0x103
5.0x102
D
0.0
5.0x102
0.0
0.0
0.5
1.0
1.5
2.0
Concentration of Chitosan
solution/(mg/mL)
5x103
5x103
B
C
4x103
4x103
3x103
3x103
2x103
2x103
1x103
1x103
Standard deviation (SD)/nm
2.0x10
2.5x103
Rp-v
SD
Maximum peak to valley (Rp-v) /nm
2.5x103
Standard deviation (SD)/nm
Maximum peak to valley(Rp-v)/nm
C–2 and G–0.5%. This figure is available in colour online at www.apjChemEng.com.
D
0
0.0
0
0.5
1.0
1.5
2.0
Concentration of gelatin solution/(mg/mL)
Figure 3. The comparison of maximum peak-to-valley (Rp-v) and SD on different concentration of
chitosan and gelatin solution. D, the difference between the values of Rp-v and SD. This figure is
available in colour online at www.apjChemEng.com.
overnight for later use. The non-modified coverslips
were regarded as the control group.
AFM detection
The AFM used was for commercial AFM measurement (Agilent, PicoPlusII, USA) equipped with inverted
microscope (Olympus, IX71). Microfabricated silicon
cantilever was applied in the measurement which
performed in contact mode. The images were stable and reproducible during repeated scanning. The
images were analysed by the software of Picoscan
5 provided with the AFM instrument. In this article, the images of surface morphologies of chitosan and gelatin thin films with different concentration on coverslips were acquired by AFM measurement which this operational method has provided spectacular images of a variety of biomacromolecules. At the same time, the configuration of
rMSCs on different surfaces with different treatments
was exhibited.
 2009 Curtin University of Technology and John Wiley & Sons, Ltd.
Statistics analysis
The experiments were repeated three times, and the
data was presented as mean ± standard deviation (SD)
unless otherwise noted, and analysed using t-test, and
the graph was analysed by the origin 7.0 soft.
RESULTS AND DISCUSSION
Biological and live/dead assays of rMSCs
Figure 1A shows the microscope picture of primary passage of rMSCs after 7 days of culture when the cells
presented typical phenotype of MSCs and excellent viability. From B and C, we know that about 60.93%
of the rMSCs expressed CD44, but only 0.91% of
them expressed CD45, which explained that the cells
we acquired were of high purity coefficient rMSCs.
The inductions confirmed multilineage differentiation
of obtained rMSCs (Fig. 1D–F). Von-Kossa staining
Asia-Pac. J. Chem. Eng. 2009; 4: 765–770
DOI: 10.1002/apj
767
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K. D. SONG ET AL.
Asia-Pacific Journal of Chemical Engineering
Figure 4. Analysis of spreading, adhesion and distribution of rMSCs cultured in the control
group. This figure is available in colour online at www.apjChemEng.com.
Figure 5. Analysis of spreading, adhesion and distribution of rMSCs cultured on surface
modified with C–2%. This figure is available in colour online at www.apjChemEng.com.
positively identified calcium depositions (D). In chondrogenic induction, a cartilage-like matrix was stained
positive for toluidine blue (E). The rMSCs grown in adipogenic induction medium started to show fat deposits
after 7 days of culture. These deposits were stained
positive by oil red staining (F). Figure 1G–I showed
the cell live/dead assay with HO/PI double-staining,
the group modified with G–0.5% maintained the better
viability than that of other groups (G–I), and surfaces
modified with chitosan effected negatively on the viability even if C–2% got the best results in chitosan
groups (H).
Three-dimensional topography and analysis of
surfaces
The C–2 and G–0.5% showed the best viability for
each material basing on the above results, so we only
gave the AFM three-dimensional topographic images
of the surfaces in the two groups (Fig. 2). Both the
scan ranges were 50 × 50 µm square. Compared to
 2009 Curtin University of Technology and John Wiley & Sons, Ltd.
the apparent peak-valley surface topography in group
C–2%, the smoothed surface in group G–0.5% was
more suitable for the adhesion of rMSCs. SD and maximum peaks to valley (Rp-v) are important parameters
that affect the smoothness of the film grade. SD is a
measure of the dispersion of a collection of values. To
assist interpretation of the resulting AFM images, the
two parameters from different concentrations on coverslips were compared in the same reference frame,
and the results are shown in Figure 3. We found that
rMSCs could achieve the best viability and spreading
on the surface in which there were the smallest differences between Rp-v and SD. It also confirmed that the
surface topography could significantly affect the cellular attachment, proliferation and differentiation. The
surface morphologies of C–2 and G–0.5% showed suitable and optimal conditions for culturing rMSCs in our
experiment. Moreover, the G–0.5% showed a better
AFM morphology than that of 0.1% treated surface
from Harrison’s research,[15] which probably was the
reason why G–0.5 showed better cellular viability than
that of other groups.
Asia-Pac. J. Chem. Eng. 2009; 4: 765–770
DOI: 10.1002/apj
Asia-Pacific Journal of Chemical Engineering
MESENCHYMAL STEM CELLS: RABBIT BONE MARROW
Figure 6. Analysis of spreading, adhesion and distribution of rMSCs cultured
on surface modified with G–0.5%. This figure is available in colour online at
www.apjChemEng.com.
Cell distribution on different surfaces
Figure 4 showed the spreading, adhesion and distribution of rMSCs cultured in the control group; the cell
presented good shape, extension and adhesion, and the
cellular width was about 25.6 µm with the height about
80 nm (from 0.29 to 0.37 µm in the direction of the
Y axis). From the profile 2, we know that the width
of filopodia was about 1.0 µm. Therefore, these data
could be used to compare with other two different surface modifications. As to the group C–2% (Fig. 5), the
cell could only achieve weak extension and spreading,
and thus caused very thick distribution in the Y axis,
and the height was about 240 nm (from 0.24 to 0.48 µm
in the direction of the Y axis).
Figure 6 showed the analysis of rMSCs cultured on
the surface modified with G–0.5%. The cell presented
very excellent extension, adhesion and spreading. The
cellular width was about 33.9 µm, which was clearly
larger than that in the control and C–2% groups. The
cell nucleus also showed similar structure and shape
as that of the control group. Moreover, the height of
culture-less areas was ranging from 0.20 to 0.30 µm,
but the height of culture-plus areas was from 0.18 to
0.28 µm, so, there were some interactions between cells
and gelatin macromolecules. From the profiles 1 and 4,
we also found that the cell had very excellent extension
and morphology.
CONCLUSIONS
In summary, the surface modified with G–0.5% could
maintain the good cell viability and, presented better
cell adhesion, spreading and distribution than that of
other groups. The chitosan groups, even the best one
C–2%, which still could not show good spreading morphologies, and cell size was quite small in horizontal
direction while cell height was comparatively large in
 2009 Curtin University of Technology and John Wiley & Sons, Ltd.
horizontal direction. Moreover, it confirmed that AFM
could be used for three-dimensional views and crosssections of stem cells at any point attached to the substrates, with simultaneous measurement of their sizes. It
is further indicated that the cytocompatibility and affinity of gelatin to rMSCs are both better than that of
chitosan, and the concentration of 0.5 mg/ml gelatin
could be an ideal choice for surface modification in
terms of fabricating tissue-engineered tissues in vitro.
Acknowledgement
The authors would like to acknowledge the support of
the National Science Foundation of China (30670525,
30700181) and the new teacher Foundation of Ministry
of Education (20070141055).
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Asia-Pacific Journal of Chemical Engineering
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DOI: 10.1002/apj
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