(PDF) Virus activated artificial ECM induces the osteoblastic differentiation of mesenchymal stem cells without osteogenic supplements (2013) | Jianglin Wang | 82 Citations
Virus activated artificial ECM induces the osteoblastic differentiation of mesenchymal stem cells without osteogenic supplements
Jianglin Wang, Lin Wang, Xin Li, Chuanbin Mao +3 moreUniversity of Oklahoma
- 07 Feb2013
- Vol. 3, Iss: 1, pp 1242-1242
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TL;DR: The unique ridge/groove nanotopography and the display of RGD and PHSRN could induce the osteoblastic differentiation of mesenchymal stem cells (MSCs) without any osteogenic supplements.
Abstract: Biochemical and topographical features of an artificial extracellular matrix (aECM) can direct stem cell fate. However, it is difficult to vary only the biochemical cues without changing nanotopography to study their unique role. We took advantage of two unique features of M13 phage, a non-toxic nanofiber-like virus, to generate a virus-activated aECM with constant ordered ridge/groove nanotopography but displaying different fibronectin-derived peptides (RGD, its synergy site PHSRN, and a combination of RGD and PHSRN). One feature is the self-assembly of phage into a ridge/groove structure, another is the ease of genetically surface-displaying a peptide. We found that the unique ridge/groove nanotopography and the display of RGD and PHSRN could induce the osteoblastic differentiation of mesenchymal stem cells (MSCs) without any osteogenic supplements. The aECM formed through self-assembly and genetic engineering of phage can be used to understand the role of peptide cues in directing stem cell behavior while keeping nanotopography constant.
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Virus activated artificial ECM induces the
osteoblastic differentiation of
mesenchymal stem cells without
osteogenic supplements
Jianglin Wang, Lin Wang, Xin Li & Chuanbin Mao
Department of Chemistry and Biochemistry, Stephenson Life Sciences Research Center, University of Oklahoma, Norman,
OK 73019, USA.
Biochemical and topographical features of an artificial extracellular matrix (aECM) can direct stem cell fate.
However, it is difficult to vary only the biochemical cues without changing nanotopography to study their
unique role. We took advantage of two unique features of M13 phage, a non-toxic nanofiber-like virus, to
generate a virus-activated aECM with constant ordered ridge/groove nanotopography but displaying
different fibronectin-derived peptides (RGD, its synergy site PHSRN, and a combination of RGD and
PHSRN). One feature is the self-assembly of phage into a ridge/groove structure, another is the ease of
genetically surface-displaying a peptide. We found that the unique ridge/groove nanotopography and the
display of RGD and PHSRN could induce the osteoblastic differentiation of mesenchymal stem cells (MSCs)
without any osteogenic supplements. The aECM formed through self-assembly and genetic engineering of
phage can be used to understand the role of peptide cues in directing stem cell behavior while keeping
nanotopography constant.
Stem cell niche as a specific extrinsic mircoenvironment integrate a complex array of molecular signals that,
in combination with induced cell-intrinsic regulatory networks, control stem cell function and balance their
numbers in response to physiological demands1,2. In most instances, stem cells in the niche are in contact
with extracellular matrix (ECM), which provides multiple structural and biochemical cues to govern a series of
stem cell behaviors in the temporal and spatial dimension3,4. Thus, more attention is being paid to the design of
artificial ECM (aECM) by integrating some physical, chemical and/or mechanical factors into biomaterials for
directing stem cell functions.
Nanotopograpy as a particular physical factor is now receiving more interest because it has advantageous
features such as a large surface-to-volume ratio and a higher degree of biological plasticity compared with
conventional micro- or macrostructures5. Emerging literature presents many interesting findings on how nano-
topography enhances cell adhesion, alters cell morphology, affects cell expansion, initiates intracellular signaling,
provides contact guidance and mediates stem cell differentiation5–9. Considering nanoscale topography in the
design of biomimetic materials is a fashional idea because the resulting materials resemble thein vivoniche. On
the other hand, biochemical cues as a traditional regulatory factor in the stem cell niche have been widely studied
for a long time10–12. These signals can be classified into three types, including integral membrane proteins,
localized secreted ECM components and soluble proteins like growth factors and cytokines2. The biochemical
cues have been demonstrated to affect stem cell fates by targeting some specific signaling pathways such asb1
integrins activated MAPK signaling, Wnt signaling pathway in the hematopoietic stem cell (HSC) and Notch
signaling in the development of the nervous system13–15. Therefore, it is increasingly interesting to introduce
biochemical factors into artificial materials to directly control cell behaviors.
M13 filamentous phage, a virus that specifically infects bacteria and is harmless to human beings, is a biona-
nofiber (,880 nm long and,6.6 nm wide)8,16. It is made of DNA as a core and protein coat as a sheath that wraps
the core. The coat protein constituting the side wall of phage is termed pVIII and encoded by gene VIII of the
phage DNA. Compared to other nanofibers, M13 phage is unique in that it can not only be used as an organic
building block to build 2-D films and 3-D scaffolds with unique topographical structures through self-assembly,
but also introduce different peptides on the constituent building block to provide biochemical cues by the
SUBJECT AREAS:
BIOMATERIALS-CELLS
TISSUE ENGINEERING AND
REGENERATIVE
MEDICINE
MESENCHYMAL STEM CELLS
BIOMATERIALS-PROTEINS
Received
18 November 2012
Accepted
18 January 2013
Published
7 February 2013
Correspondence and
requests for materials
should be addressed to
C.B.M. (cbmao@ou.
edu)
SCIENTIFICREPORTS| 3 : 1242 | DOI: 10.1038/srep012421
well-established phage display technique7,8. Inserting a foreign gene
into gene VIII leads to the display of a foreign peptide as fusion to
pVIII and the concomitant presentation of foreign peptide on the
side wall of phage. The ease of displaying a peptide on the side wall of
phage nanofibers enables us to use phage to study the peptide cues
(biochemical cues) that can direct the stem cell fate. In addition, the
ease of assembly of phage nanofibers into a nanostructured film
further gives us the capability of studying the stem cell fate on a
nanostructure with specific peptide sequence displayed on the phage
nanofibers that generate a unique nanotopography. These unique
properties of phage allow us to systematically study the effect of
different peptides on the substrates with constant nanotopography
on the stem cell fate (Figure 1).
In this communication, we employed a phage display approach to
generate a virus-activated aECM with well-defined topographical
and biochemical cues to activate the regulation of the morphology,
proliferation and osteoblastic differentiation of rat mesenchymal
stem cells (MSCs). We separately displayed different fibronectin-
derived peptides (RGD, its synergy site PHSRN, and a combination
of RGD and PHSRN) on the side wall of phage nanofibers through
phage display technique, and assembled them into a 2-D film based
on our established layer-by-layer self-assembly method8. We chose
to study the two fibronectin-derived peptides to be displayed on
phage surface based on the following considerations. Fibronectin is
a crucial ECM component of many tissues and regulates a variety of
cell activities predominantly through direct interactions with cell
surface integrin receptors17. The identified adhesive domains of the
fibronectin are comprised of at least two minimal and pivotal peptide
sequences, including an Arg-Gly-Asp (RGD) sequence located in
the10thtypeIIIrepeatingunitandaPro-His-Ser-Arg-Asn
(PHSRN) sequence in the 9th type III repeating unit18. The RGD
and PHSRN sequences as pervasive adhesive peptides can influence
multiple cell behaviors including cell adhesion, proliferation and
differentiation17,19–22.
Results
Self-assembly of phage-based films.Due to the long-rod structure
andmonodispersityofphagenanofibers7,23,theywerefirstly
assembled into bundles, which were further assembled in a parallel
format to form a 2-D phage-based film material on poly-L-lysine
substrate (Figure 1B). The resultant film showed a slightly rough
surfaceandorderedridge/groovetopography(Figure2).The
formation of the phage-based film was driven through liquid
crystalline phase transitions at the air-liquid interface during the
evaporation process6,8,24. In addition, the electrostatic interaction
betweennegativelychargelyphagenanofibersandpositively
chargely polylysine substrate provided another driving force to
promote the assembly of phage bundles into films with a unique
highly ordered topography where phage bundles as ridges were
parallel to each other and separated by grooves.
Morphology and nanotopography of phage-based films.We found
that the specific ordered ridge/groove topography was controlled by
the concentration of phage solution during layer-by-layer assembly.
Figure 1|Schematic diagram of using phage display technique to produce biomaterials with both unique nanostructures derived from a layer-by-layer
method and functional peptides displayed for directing stem cell fate.Foreign peptides ( RGD or PHSRN) derived from fibronectin were separately
inserted and displayed in the N-terminal end of major coat protein (pVIII) constituting the side wall of M13 phage (1A). The phage bundles were
generated based on the unique properties of long-rod structure and monodispersity of phage nanofibers in the desired phage concentration, and the
engineered phage bundles were further used to form phage-based film biomaterials via a layer-by-layer self-assembly method (1B). The polylysine was
introduced as the first positively charged layer on the substrate, and then the engineered phage, which was anionic due to the presence of anionic amino
acid residues in the major coat protein, was deposited as a second negatively charged layer. This process was repeated for three times and a phage-based
film was formed with phage as a terminating layer (1B, a-e). The resultant phage-based films with precisely introduced peptide sequences (surface
chemistry) and well-defined ridge/groove topographical feature were found to direct osteoblastic differentiation of mesenchymal stem cells (MSCs).
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SCIENTIFICREPORTS| 3 : 1242 | DOI: 10.1038/srep012422
The diameter of phage bundles was around 1000 nm at the higher
phage concentration (1014pfu/ml) whereas the diameter was about
500 nm at the lower phage concentration (1012pfu/ml) (Figure 3).
In addition, our current data showed that the roughness of phage-
based films was dependent on the phage concentration and increased
with the rise of the phage concentrations (Figure 4). Therefore, the
surface topography of phage films could be regulated by altering the
size of phage bundles, which could be controlled by varying the phage
concentrations.
Cell adhesion on the phage-based films.The rat MSCs were used
to evaluate the biological functions of the unique biofilm materials.
Our current data confirmed that the ordered ridge/groove structure
represented by the phage films significantly induced the elongation
and parallel alignment of MSCs along phage bundles in phage-based
film materials for all of the peptide sequences displayed on the
constituent phage nanofibers (Figures 5a,5c & 5e). However, cell
elongation and alignment were not detected on the phage film
derived from higher concentration of phage solution of 1014pfu/ml
(Figures 5b,5d & 5f) and the control substrate (i.e., polylysine
substrate without phage material) (Supplementary Figure S1). The
significant cell elongation and alignment was also missing if the
phage concentration was lower than 1012pfu/ml. Therefore, the
optimalconcentrationofphagesolutionwasdefinedbetween
1012pfu/ml and 1014pfu/ml to form the suitable films, which were
used to significantly stimulate cell elongation. As shown inFigure 3d,
Figure 2|Preparation and characterization of phage-based film materials.The M13 phage with nanofiber-like structure and monodispersity was
driven to form a film by liquid crystalline phase transitions at the arc-shaped air-liquid interface and electrostatic interaction between negatively charged
phage nanofibers and positively charged polylysine substrate (a). The morphology and size of individual phage nanofibers before they were used to form a
film were observed by TEM and AFM (b and c). The phage nanofibers were further assembled to form a phage-based film with a unique topography of
ridge/groove nanostructure (d, bright field; e, SEM; f, AFM; the red arrows highlight the highly oriented self-assemblies of phage bundles).
Figure 3|Morphology of phage-based film derived from the different
phage concentrations.The phage bundles derived from the lower
concentration (1012pfu/ml, a and c) of phage solution was smaller than
those from the higher concentration of phage solution (1014pfu/ml, b and
d). The orientation of phage bundles between neighboring domains is
similar and the phage nanofibers showed a longer range parallel alignment
with each other in the lower concentration (a and c). However, the
orientation of phage bundles was similar inside a small domain but different
between neighboring domains in the high concentration (b and d).
Figure 4|The average surface roughness (Ra) of phage-based films
derived from the different phage concentrations.The AFM surface line
scan profile indicated that the surface roughness of phage films was
increased with the rise of phage concentration (a, b and c denoted the
phage concentration of 1012, 1013and 1014pfu/ml, respectively).
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SCIENTIFICREPORTS| 3 : 1242 | DOI: 10.1038/srep012423
on the films assembled from phage at a higher concentration, phage
bundles are nearly aligned inside a domain with a size (20–25mm)
comparable to MSCs, however, the orientation of phage bundles
between neighboring domains is different. Namely, the direction of
elongation of phage bundles in different domains is different and the
parallel alignment of nanofibers is only confined in a domain with
size similar to MSCs. As a result, the MSCs growing on the films
assembled from a higher concentration of phage are not oriented and
aligned. Moreover, in the absence of phage bundles, the cells were
completely randomly oriented due to the lack of contact guidance
by the phage bundles. Therefore, the morphological changes and
parallel alignments of MSCs on the phage-based film materials
were mainly stimulated by the unique ordered ridge/groove surface
topography but not by the peptide sequences displayed on the
surface.
Cell proliferation and differentiation on the phage-based films.
We proceeded to investigate cell proliferation on the films derived
from phage nanofibers with different concentrations and peptides
displayed. MTT results demonstrated that cell proliferation was
influencedbyboththepeptidesequencesdisplayedonthe
constituent phage nanofibers and the concentrations of the phage
solution (Figures 6a & 6b). Since the phage concentration influenced
the size and separation of phage bundles to modulate the nanoto-
pographical cues and the peptide sequences displayed on phage
represented the biochemical cues, this fact implied that the cell
proliferation was controlled by both topographical and bioche-
mical cues.
To investigate the osteoblastic differentiation of MSCs on the
phage-basedfilmmaterials,thecell-materialswereculturedin
both primary and osteogenic differentiation media for 2 weeks.
Immunofluorescence staining as a qualitative analysis at the protein
level was used to verify the differentiation status. We found that
osteocalcin (OCN) and osteopontin (OPN), the two osteogenesis-
specific markers, presented positive staining on all materials in the
primary media (Figure 6d). The OCN and OPN exbihited a higher
expression on all phage-based materials than that on the control
(poly-L-lysine substrate without phage film). Collagen I (COL) as a
positive control of non-osteogenic marker showed high expression
on all materials, and there was no significant difference between
phage-based and control groups. Also, OCN and OPN presented
positive staining in all materials and their expression was significantly
enhanced in the osteogenic differentiation media (Supplementary
Figure S2) as compared to in the primary media. Real-time polymerase
Figure 5|Cell adhesion on the phage-based film derived from both low (a, c, e) and high (b, d, f) phage concentration.The MSCs on the
phage-based film derived from the low phage concentration (1012pfu/ml) were significantly elongated and aligned along phage bundles (a, c, e) whereas
those on the phage-based film derived from the high phage concentration (1014pfu/ml) were randomly oriented and not elongated (b, d, f). Images shown
were taken from bright field optical microscopy (a, b), SEM (c, d) and fluorescence microscopy (e, f). Cell nuclei were stained by DAPI (blue) and F-actin
were stained by FITC-labeled phalloidin (green).
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SCIENTIFICREPORTS| 3 : 1242 | DOI: 10.1038/srep012424
chain reaction (PCR) assay was used to further analyze the relative
gene level of the osteogenic markers associated with MSCs differ-
entiationonthephage-basedmaterialsintheprimarymedia
(Figure 6c). Both OCN and OPN genes were found to show signifi-
cant up regulation on the phage-based film materials compared to
the control group. Among the different engineered phage nanofibers,
RGD/PHSRN-phage presented an extremely high mRNA level of the
two osteogenesis-specific proteins (OCN and OPN) in comparison
to the control group (**, p,0.01). RGD-phage, PHSRN-phage and
WT-phage showed higher expression of OCN and OPN genes than
the control (*, p,0.05). However, COL gene as a non-specific
osteogenic marker did not show significant difference between
phage-based materials and the control. The phage-based groups still
showed significant up-regulation of mRNA level for both OCN and
OPN genes under the condition of osteogenic media (Supplemen-
tary Figure S3). Overall, the phage-based materials enabled to induce
the osteoblastic differentiation of MSCs in the primary media with-
out any osteogenic supplements, and the osteoblastic differentiation
was further enhanced in the presence of both materials and osteo-
genic differentiation media.
ALP as a marker protein specific for the osteoprogenitor activity
was normally used to verify the osteoblastic differentiation of MSCs.
TheALPassaydemonstratedthatbothphage-basedmaterial
and control groups showed positive ALP expression (Figure 6f).
However, the group of RGD/PHSRN-phage presented the highest
ALP activity among all groups. The typical alizarin red staining
was used to confirm the osteogenic mineralization by detecting the
formation of calcium nodule (Figure 6e). The positive staining of
calcium nodule was detected on all materials. Furthermore, the stain-
ing on the phage-based film materials was much stronger than that
in the control. These results suggest the RGD/PHSRN-phage with
unique nanotopography promoted the osteoblastic differentiation of
MSCs, suggesting a synergetic enhancement by both the biochemical
and topographical cues.
Discussion
Compared to other nanofibers, M13 phage is unique because it can
not only be used as a building block to build unique ridge/groove
structures through self-assembly, but also introduce different peptides
on the constituent building block into the resultant ridge/groove
structures by the well-established phage display technique7,8. This
unique property of phage enables us to systematically study the effect
of different peptides on the substrates with constant topography on
the stem cell fate. In this study, we constructed different recombinant
engineered phages to display adhesive signaling peptide of RGD
and PHSRN derived from fibronectin, respectively. Both RGD and
PHSRN motifs have been identified as pervasive adhesive peptides to
mediate multiple cell activities including cell adhesion, proliferation
and differentiation17,19–22, and have been widely used to design the
smart biomaterials. Traditionally, such peptides are physically mixed
into or chemically immobilized onto biomaterials, preventing us from
forming aECM with ordered assembly of peptides and varying only
the peptide sequences without changing topography in studying stem
cell fates. Therefore, phage display is a unique approach to studying
stem cell fate because it allows us to precisely introduce foreign pep-
tide into a nanotopography by genetic means and the nanotopogra-
phy can be generated by its self-assembly behavior. In addition, the
fact that the self-assembly of phage is not affected by the peptide
displayed on its surface makes it possible to form an ECM with
different peptides but a constant nanotopography for us to system-
atically study the effect of peptide cues on the stem cell behavior.
Figure 6|Cell proliferation and differentiation on the phage-based materials in the primary media.Cell proliferation was significantly influenced by
phage concentration and phage types (a, b). Cell differentiation was regulated by different peptides displayed on the engineered phage types (c, d).
The ALP activity assay (f) and alizarin red staining (e) further demonstrated that the osteoblastic differentiation of MSCs was induced by phage-based film
materials. All data represented the mean6standard deviation (n53,*p,0.05,**p,0.01). WT, RGD, PHSRN and RGD/PHSRN denoted films made of
wild type phage, RGD-displayed phage, PHSRN-displayed phage, and a mixture of RGD- and PHSRN-displayed phage, respectively. CON and LYS
denoted poly-L-lysine substrates without phage. BLANK denoted pure glass substrate. OCN, OPN and COL were stained by rhodamine-labeled antibody
(red) and cell nuclei were stained by DAPI (blue) and F-actin were stained by FITC-labeled phalloidin (green).
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SCIENTIFICREPORTS| 3 : 1242 | DOI: 10.1038/srep012425
M13 phage as a natural nanofiber (,880 nm by 6.6 nm) can be
assembled into bundles due to its long-rod structure and monodis-
persity7,23. The phage bundles can be further aligned to form phage-
based 2-D film materials through two driving forces. One is the liquid
crystalline assembly at the air-liquid interface during the evaporation
process6,8,24, another is the electrostatic interaction between nega-
tively chargely phage nanofibers and positively chargely polylysine.
As a result, an ECM was produced with a unique highly ordered
topography where phage bundles as ridges are parallel to each other
and separated by grooves (Figure 2). In addition, tuning the concen-
tration of the phage suspension used for self-assembly on the sub-
strate can control the size and surface roughness of phage bundles
constituting the film (Figures 3 and 4).
It has been reported that the topography of culture substrate influ-
ences the cell behaviors by elongating cell shape5,25–27. Dalby et al
demonstrated that the topographical cue based on the use of dis-
ordered nanopits in the polymethylmethacrylate (PMMA) substrate
can stimulate the osteoblastic differentiation of human MSCs with-
out the osteogenic supplements28. The mechanism might be that the
disordered nanopits resulted in longer adhesion, which impacts
cytoskeleton tension. These changes in adhesion and cytoskeleton
tension will have an effect on cell behavior through an indirect
mechanotransductive pathway. Moreover, Jin et al showed that the
topographical cue generated from the nanotubular-shaped titanium
oxide regulated the osteogenic differentiation of hMSCs5. The pos-
sible mechanism is that topography-induced cell elongation stimu-
lates the stem cell differentiation. Our current data also confirm that
the ordered ridge/groove structure represented by the phage films
significantly induced the elongation and parallel alignment of MSCs
along phage bundles in phage-based film materials for all of the
peptide sequences displayed on the constituent phage nanofibers.
Therefore, the morphological changes of MSCs on phage-based film
materials might further stimulate the mechanical difference of cytos-
keleton, which plays a pivotal role in regulating mechanotransduc-
tive pathways and finally having an impact on multiple stem cell
behaviors26. Although different topographical cues including nano-
pits28, nano-tube5or nano-bundle (our work) are designed on the
different substrates, including polymer (PMMA28), metal (TiO25)
and biomolecule (phage), respectively, to stimulate stem cell beha-
vior, the nature of regulatory mechanism might be similar. That is,
cell shape is changed by modulating cell adhesion on the substrate
materials and finally stem cell fate is directed by biomechanical dif-
ference or mechanotransductive pathway.
The MTT result demonstrates that cell proliferation is influenced
by both the peptide sequences displayed on the constituent phage
nanofibersandtheconcentrationsofthephagenanofibers
(Figures 6a & 6b). Since the phage concentration influences the size
and separation of phage bundles to modulate the nanotopographical
cues and the peptide sequences displayed on phage represents the
biochemical cues, we can conclude that stem cell morphology and
alignment are solely modulated by the topographical cue, whereas
the cell proliferation is directed by both topographical and biochem-
ical cues.
In order to further understand the effect of both topographical and
biochemical cues on cell differentiation, we design two culture sys-
tems including primary and osteogenic media to elucidate which
factors influence stem cell differentiation. It is widely demonstrated
that the osteogenic differentiation media as a chemical stimulation
plays a key role in enhancing osteoblastic differentiation of multiple
stem cells including embryonic stem cells, induced pluripotent stem
cells and adult stem cells29–31. Our current results also support that
conclusion. Moreover, we simultaneously confirm that the successful
induction of osteoblastic differentiation can be performed in the
media without any osteogenic supplements (Figures 6c & 6d). This
fact means that the material itself can direct the osteoblastic differ-
entiationofMSCsthroughitstopographicalandbiochemical
features in the absence of osteogenic supplements. However, the
osteogenic differentiation of MSCs is extremely enhanced when
the MSCs are cultured on the materials in concert with osteogenic
media (Figures S2 & S3).
RGD and PHSRN motifs derived from fibronectin, which is a
major adhesive component in the natural ECM, have been widely
demonstrated to mediate the stem cell behaviors through specific
integrin signal pathway32. The osteoprogenitor cells expressed some
integrins, includinga5b1specifically binding with RGD and RGD-
PHSRN, to regulate osteoblast survival, proliferation, osteogenic
gene expression and matrix mineralization17,21. Our results showed
that a combination of RGD and PHSRN presented on a unique ridge/
groove nanotopography significantly enhanced osteoblastic differ-
entiation (Figures 4b & 4c). Therefore, the osteoblastic differenti-
ation of MSCs on the phage-based film were significantly stimulated
by both topographical and biochemical cues.
In conclusion, designing materials to direct stem cell fate has a
profound impact on stem cell biology and provides insights that will
facilitate the clinical application of stem cells in modern regenerative
medicine. In this study, a virus-activated aECM with controlled bio-
chemical and topographical cues was precisely designed to mediate
stem cell behavior. This specific aECM is characteristic of highly
orderedtopographywithalignedridge/groovenanostructures,
which result from the self-assembly of phage nanofibers, and simul-
taneously presents the biochemical signals made of RGD and
PHSRN peptides by phage display technique. The current data dem-
onstrate that cell alignment and elongation are mainly regulated by
topographical cues. Cell proliferation are greatly influenced via a
combination of topographical and biochemcial cues. Due to the
presence of the unique ridge/groove nanostructure made of phage
nanofibers and the fibronectin derived peptides displayed on the
phage nanofibers, the aECM can stimulate the osteoblastic differ-
entiation of MSCs in the primary media without osteogenic supple-
ments.ThesameaECMcanfurtherenhancetheosteogenic
differentiation of MSCs once in osteogenic media. Our findings
suggest that a proper combination of unique nanotopographical
and biochemical cues can control the stem cell behaviors including
induction of the osteoblastic differentiation. Our phage display
approach represents a novel strategy for generating a virus-activated
aECM, where peptide sequences can be systematically tuned on a
unique, constant nanotopography by genetic means, for probing the
biochemical cues in directing stem cell fate.
Methods
Peptides display and films fabrication.RGD and PHSRN were respectively
displayed on the N-terminus of pVIII, which was the major coat protein constituting
the external side wall of M13 bacteriophage, by following our reported protocols
(Figure 1A)33–35. Filamentous phages were assembled into films following a
layer-by-layer self-assembly method developed by our group (Figure 1B)8. Briefly, the
disc-shaped cover slide was sonicated and washed with DI water, and placed into each
well of 24-well culture plate. The poly-L-lysine solution (0.01%) was added to the well
with cover slide to form the first layer with positive charge on the cover slide. After
drying, the phage solution was added to form the secondary layer with negative
charge. The process was repeated for three times and a film was formed on the cover
slide with phage as a terminating layer. The morphologies of the individual phage
nanofibers were observed by transmission electron microscope (TEM, ZEISS 10A)
and Atomic force microscope (AFM, BioScope Catalyst, Bruker) and the fabricated
films were examined by optical microscope, fluorescence microscope and scanning
electron microscope (SEM, JSM-840A).
Cell culture and seeding.Rat MSCs were purchased from Invitrogen
(No: S1601-100) and expanded in the primary media, which contained Dulbecco’s
Modified Eagle Media (DMEM, Gibco), 15% fetal bovine serum (FBS, Gibco) and 1%
antibiotics (penicillin 100 U/ml, streptomycin 100 U/ml). The MSCs in their third
passage were seeded onto the phage-based films, and then cultured separately in
primary and osteogenic differentiation media (Thermo scientific, Advance STEMO
steogenic Differentiation Kit). The media was replaced twice a week and the culture
was terminated after two weeks.
Cell proliferation.For study of cell viability and proliferation, the MSCs were seeded
onto the phage-based film materials to investigate the biocompatibility of different
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SCIENTIFICREPORTS| 3 : 1242 | DOI: 10.1038/srep012426
materials and the effects of different concentrations of phage used to make the films in
the primary media. The phage concentration was varied from low to high values,
including 1010pfu/ml, 1011pfu/ml, 1012pfu/ml, 1013pfu/ml, 1014pfu/ml,
5.031014pfu/ml and 7.531014pfu/ml. The cell proliferation was then measured by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma)
staining at the designed time points including day 1 and day 3. The cell-film complex
was incubated in the MTT solution (20ml, 5 mg/ml) at 37uC in 5% CO2incubator for
4 h. The intense purple formazan derivative formed via cell metabolism was eluted
and dissolved in 150ml/well dimethylsulfoxide (DMSO, Sigma). The absorbance was
measured at 490 nm on a plate reader (Biotek, USA).
Immunofluorescence staining.All engineered phage films for osteoblastic
differentiation were derived from the constant phage concentration of 1013pfu/ml.
After cultured for 2 weeks in primary and osteogenic differentiation media, the cells
on the films were washed and fixed with 4% paraformaldehyde at 4uC for 30 min.
They were permeablized using 0.3% Triton X-100 for 5 min and then blocked with
5% goat serum solution for 1 h at room temperature. After blocking, the cells were
incubated overnight at 4uC with the primary antibodies targeting the osteo-specific
proteins (Osteocalcin, OCN and osteopontin, OPN ) and non-osteo-specific protein
(collagen I-a1, COL). Secondary antibody labeled by TRITC was used for labeling
OCN, OPN, and COL, respectively, at 1:1000 dilutions in a blocking buffer for 1 h
at room temperature. Alexa Fluor 488 phallodin (1:400 in PBS) and DAPI
(4,6-diamidino-2-phenylindole) were used to stain the actin filaments and nuclei,
respectively. Images of the stained samples were collected with a fluorescence
microscope (Nikon, Ti-S).
Real-time polymerase chain reaction (PCR).Real-time PCR was further assayed by
Ambion Power SYBR Green cells-to-Ct Kit (Invitrogen, US) in both primary and
osteogenic differentiation media. The template cDNA was amplified with real-time
quantitative PCR using gene-specific primers of OCN, OPN and COL. Acidic
ribosomal phosphoprotein (Arbp) was used as a reference gene. Sequences of the
primers in this study were shown inTable S1. The real-time PCR reaction was done
using the following protocol: initial denaturation at 95uC for 5 min and 45 cycles of
PCR (95uC for 30 s, 58uC for 30 s and 72uC for 45 s). The assay was carried out in
triplicate and relative gene expression was calculated with respect to the gene
expression in the control substrate without phage film36.
Assays of alkaline phosphatase and mineralization of the cell-matrix.After culture
for two weeks in the primary media, the MSCs seeded on phage-based film materials
were tested for alkaline phosphatase (ALP) activity and calcium nodule staining. The
ALP activity was performed by p-nitrophenyl phosphate (pNPP) method. Briefly, the
pNPP was used as a substrate for ALP to be hydrolysed to form a soluble yellow
reaction products at pH 10.5 and 37uC. The staining reaction was terminated by the
addition of 3 M NaOH and the final color showed a maximum absorbance at 405 nm.
For calcium nodule staining, the cells were fixed in 4% paraformaldehyde at 4uC for
15 min and then stained with 0.2% alizarin red at pH 5.0 for 15 min. The staining
images were collected with optical microscope.
Statistical analyses.All experimental analysis of cell proliferation, real-time PCR and
ALP assay were performed in triplicate (n53). The data were expressed as
mean6SD (standard deviation) at a significance level ofp,0.05. Differences among
groups were determined by a one-way ANOVA with a Bonferronipost hocanalysis
with SPSS software (version. 17).
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Acknowledgements
We would like to thank the financial support from National Science Foundation
(CBET-0854414, CBET-0854465, CBET-1229309, and DMR-0847758), National Institutes
of Health (5R01DE01563309, 5R01HL092526-02, 1R21EB015190-01A1,
4R03AR056848-03), Department of Defense Peer Reviewed Medical Research Program
(W81XWH-12-1-0384), Oklahoma Center for the Advancement of Science and
Technology (HR11-006) and Oklahoma Center for Adult Stem Cell Research (434003).
CBM would also like to thank Dr. Antoni Tomsia for his kind help during this study.
www.nature.com/scientificreports
SCIENTIFICREPORTS| 3 : 1242 | DOI: 10.1038/srep012427
http://www.nature.com/scientificreports
http://www.nature.com/scientificreports
http://creativecommons.org/licenses/by-nc-nd/3.0
Author contributions
J.W. and L.W. contributed equally to this work. C.M. and J.W. designed the experiments;
J.W. and L.W. performed the experiments; X.L. assisted with AFM characterization; J.W.
and C.M. wrote the manuscript.
Additional information
Supplementary informationaccompanies this paper at http://www.nature.com/
scientificreports
Competing financial interests:The authors declare no competing financial interests.
License:This work is licensed under a Creative Commons
Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this
license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/
How to cite this article:Wang, J., Wang, L., Li, X. & Mao, C. Virus activated artificial ECM
induces the osteoblastic differentiation of mesenchymal stem cells without osteogenic
supplements.Sci. Rep.3, 1242; DOI:10.1038/srep01242 (2013).
www.nature.com/scientificreports
SCIENTIFICREPORTS| 3 : 1242 | DOI: 10.1038/srep012428
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