# Virus activated artificial ECM induces the osteoblastic differentiation of mesenchymal stem cells without osteogenic supplements

[Jianglin Wang,](/content/authors/jianglin-wang-1hinecyiak/index.html) [Lin Wang,](/content/authors/lin-wang-qi5xt7pvny/index.html) [Xin Li,](/content/authors/xin-li-49eq2ba9pd/index.html) [Chuanbin Mao](/content/authors/chuanbin-mao-5azjggzuez/index.html) +3 more[University of Oklahoma](/content/institutions/university-of-oklahoma-1osft1o6/index.html)

- 07 Feb[2013](/content/journals/scientific-reports-eyrb8707/2013/index.html)

- [Scientific Reports](/content/journals/scientific-reports-eyrb8707/index.html)

- 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).

www.nature.com/scientificreports

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).

www.nature.com/scientificreports

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://www.nature.com/scientificreports")

[http://www.nature.com/scientificreports](http://www.nature.com/scientificreports "http://www.nature.com/scientificreports")

[http://creativecommons.org/licenses/by-nc-nd/3.0](http://creativecommons.org/licenses/by-nc-nd/3.0 "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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•Journal Article• [10.1126/SCIENCE.1171643](https://doi.org/10.1126/SCIENCE.1171643)

[**Growth Factors, Matrices, and Forces Combine and Control Stem Cells**](/content/papers/growth-factors-matrices-and-forces-combine-and-control-stem-4daxw4e632/index.html)

[Dennis E. Discher,](/content/authors/dennis-e-discher-3p1gko9ktp/index.html) [David J. Mooney,](/content/authors/david-j-mooney-4rpr1v8r82/index.html) [Peter W. Zandstra](/content/authors/peter-w-zandstra-20fbhbytrt/index.html) +2 more[University of Pennsylvania,](/content/institutions/university-of-pennsylvania-32r68p8r/index.html) [Harvard University,](/content/institutions/harvard-university-3suqum0d/index.html) [University of Toronto](/content/institutions/university-of-toronto-3dwwuuvf/index.html)

- 26 Jun[2009](/content/journals/science-px8dli2q/2009/index.html)

- [Science](/content/journals/science-px8dli2q/index.html)

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TL;DR: Multifaceted technologies are increasingly required to produce and interrogate cells ex vivo, to build predictive models, and, ultimately, to enhance stem cell integration in vivo for therapeutic benefit.

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