Journal of Materials Chemistry BMaterials for biology and medicinewww.rsc.org/MaterialsB

ISSN 2050-750X

PAPERChuanbin Mao, Yingjun Wang et al.Directing the fate of human and mouse mesenchymal stem cells by hydroxyl–methyl mixed self-assembled monolayers with varying wettability

Volume 2 Number 30 14 August 2014 Pages 4757–4958

Journal ofMaterials Chemistry B

PAPER

Publ

ishe

d on

27

June

201

4. D

ownl

oade

d by

Uni

vers

ity o

f C

hica

go o

n 16

/09/

2014

04:

45:5

1.

View Article OnlineView Journal | View Issue

aSchool of Materials Science and Engineerin

Guangzhou 510641, China. E-mail: imwangbNational Engineering Research Center for

Guangzhou 510006, ChinacDepartment of Materials Science and En

100084, PR ChinadDepartment of Chemistry and Biochemistry,

University of Oklahoma, Norman, OK 73019

† Electronic supplementary information (quantitative analysis of cell adhesion (Figon day 14 (Fig. S3); the gene expressionDOI: 10.1039/c4tb00597j

Cite this: J. Mater. Chem. B, 2014, 2,4794

Received 14th April 2014Accepted 15th May 2014

DOI: 10.1039/c4tb00597j

www.rsc.org/MaterialsB

4794 | J. Mater. Chem. B, 2014, 2, 479

Directing the fate of human and mousemesenchymal stem cells by hydroxyl–methylmixed self-assembled monolayers with varyingwettability†

Lijing Hao,ab Hui Yang,ab Chang Du,ab Xiaoling Fu,ab Naru Zhao,ab Suju Xu,c

Fuzhai Cui,c Chuanbin Mao*d and Yingjun Wang*ab

Self-assembled monolayers (SAMs) of alkanethiols on gold have been employed as model substrates to

investigate the effects of surface chemistry on cell behavior. However, few studies were dedicated to

substrates with controlled wettability in studying the stem cell fate. Here, mixed hydroxyl (–OH) and

methyl (–CH3) terminated SAMs were prepared to form substrates with varying wettability, which were

used to study the effects of wettability on the adhesion, spreading, proliferation and osteogenic

differentiation of mesenchymal stem cells (MSCs) from human and mouse origins. The numbers of

adhered human fetal MSCs (hMSCs) and mouse bone marrow MSCs (mMSCs) were maximized on

–OH/–CH3 mixed SAMs with water contact angles of 40–70� and 70–90�, respectively. Hydrophilic

mixed SAMs with a water contact angle of 20–70� also promoted the spreading of both hMSCs and

mMSCs. Proliferation of both hMSCs and mMSCs was most favored on hydrophilic SAMs with a water

contact angle around 70�. In addition, a moderate hydrophilic surface (with a contact angle of 40–90�

for hMSCs and 70� for mMSCs) promoted osteogenic differentiation in the presence of biological stimuli.

Hydrophilic mixed SAMs with a moderate wettability tended to promote the expression of avb1 integrin

of MSCs, indicating that the tunable wettability of the mixed SAMs may guide osteogenesis through

mediating the avb1 integrin signaling pathway. Our work can direct the design of biomaterials with

controllable wettability to promote the adhesion, proliferation and differentiation of MSCs from different

sources.

Introduction

The design of biomedical materials and devices will benetfrom an understanding of molecular and cellular interactions atmaterial surfaces. It is widely accepted that biomaterial surfacesaffect protein adsorption and the subsequent activation ofcells.1 Surface wettability plays an important role in regulatingcell behaviors, which has been extensively studied.2,3 However,most research studies have been conducted on specic

g, South China University of Technology,

yj@163.com

Tissue Restoration and Reconstruction,

gineering, Tsinghua University, Beijing

Stephenson Life Science Research Center,

, USA. E-mail: cbmao@ou.edu

ESI) available: Fluorescence images and. S1 and S2); osteogenic differentiationof avb1 integrin at 12 h (Fig. S4). See

4–4801

materials and the conclusions may not be applied to othersystems. Moreover, it is challenging to eliminate the inuenceof other surface parameters and obtain precisely controlledsurface wettability. Self-assembled monolayers (SAMs) of alka-nethiols on gold are chemically well-dened and can serve ascontrollable model surfaces. The use of SAMs is an efficientapproach to tailoring the surface and interfacial properties,enabling their applications in electrochemical, physical, bio-analytical and bioorganic chemistry.4 A host of thiols withdifferent chemical functional groups have been extensivelystudied.5,6 Our previous work also proved that neural stem cells,MCF-7 breast cancer cell line and mesenchymal stem cells(MSCs) on SAMs were greatly affected by the type of chemicalgroups terminated on the surfaces.7–10

Mixed SAMs composed of multiple components are asimportant as uniform ones because they provide a convenientapproach to tuning the surface properties such as wettabilityand charges by adjusting the ratio of different constituents.11

Besides, SAMs with mixed functional groups are closer to thenative matrix found in biological systems. Mixed SAMs cansimplify material surface effects in complex cell–material

This journal is © The Royal Society of Chemistry 2014

Paper Journal of Materials Chemistry B

Publ

ishe

d on

27

June

201

4. D

ownl

oade

d by

Uni

vers

ity o

f C

hica

go o

n 16

/09/

2014

04:

45:5

1.

View Article Online

interactions to clarify how surface properties of biomaterialscontribute to the activity of cells. Y. Arima and H. Iwata exam-ined the adhesion of human umbilical vein endothelial cellsand HeLa cells onto mixed SAMs with different wettability.11

However, few reports have investigated the effect of mixed SAMs(nonionic) with the difference in only wettability on phenotypeexpression of stem cells, especially regarding their osteogenicdifferentiation. MSCs are multi-potent stem cells and candifferentiate into several lineages including adipocyte, osteocyteand chondrocyte.12 In recent years, MSCs have been widelystudied as seeding cells for regenerative medicine applications,especially for bone repair.13 They possess cellular totipotencywith low variability from different adult donors.14 However, theadhesion, spreading, proliferation, and differentiation of MSCswithin a dynamic 3-dimensional (3D) microenvironment areinuenced by many complex factors and interactions. It ischallenging to discriminate the effects from confoundingsurface properties and elucidate the mechanisms of the cellresponse.

In this study, surfaces with a wide range of wettability wereprepared from alkanethiol solutions with mixed functionalgroups of hydrophilic hydroxyl (–OH) and hydrophobic methyl(–CH3). A change in the ratio of –OH and –CH3 terminatedalkanethiols will lead to tuning of the wettability of the mixedSAMs. Moreover, comparison of the response of MSCs fromdifferent species to the same materials with different wettabilityhas not been fully studied. Hence, we explored the response ofhMSCs and mMSCs to –OH/–CH3 mixed SAMs with varyingwettability. This study will provide a fundamental guidance onthe design of biomaterial surfaces for bone regeneration.

Materials and methodsMaterials

The starting materials used in this study included 1-dodeca-nethiol (CH3(CH2)10CH2SH, $98%, Sigma-Aldrich, USA) and11-mercapto-1-undecanol (HSCH2(CH2)9CH2OH, 99%, Sigma-Aldrich, USA). Gold substrates were prepared using an ANELVAL-400EK electron beam evaporator (Canon Anelva Corporation,Kanagawa, Japan). Titanium (10 nm) and gold (40 nm) lmswere sequentially deposited onto silicon wafers (polished/etched, crystal orientation 100). The wafers were diced intopieces (1 cm � 1 cm) using a DS820 automatic dicing saw(Heyan Technology Corporation, Shenyang, China).

Monolayer formation

Pure alkanethiol solutions were prepared in ethanol with a nalconcentration of 1 mM. Surfaces with a wide range of wettabilitywere prepared by mixing the pure solutions (alkanethiolsterminated with –OH or –CH3) with different volume ratios(–OH/–CH3 ¼ 10/0, 9/1, 7/3, 5/5, 3/7, 0/10). Gold slides werecleaned by nitrogen plasma treatment using a HPC plasmacleaner system (Mycro Technologies Corporation, USA) for2 min. Then they were rinsed 3 times with ethanol and highlypuried water, and dried by a stream of nitrogen gas. Thecleaned gold slides were immersed in the alkanethiol solutions

This journal is © The Royal Society of Chemistry 2014

for 24 h at room temperature. Aer incubation, the modiedsubstrates were rinsed and dried again as mentioned.

Surface characterization of SAMs

Static water contact angles were measured by using a contactangle meter (OCA15, Data Physics, Germany) at room temper-ature. The measurement process was repeated 5 times atdifferent spots of each surface. The chemical functional groupswere investigated by Fourier transform infrared attenuated totalinternal reection (FTIR-ATR) (Vector 33, Bruker, Germany)within the wavenumber range of 4000–400 cm�1. Elementalcompositions of the SAM surfaces were characterized by X-rayphotoelectron spectroscopy (XPS) (AXIS-ULTRA DLD, KRATOS,UK) with an Al Ka source. The photoelectrons were analyzed at atake-off angle of 55�. All the spectra were tted using an XPSpeak-tting program (XPSPEAK Version 4.1).

Cell culture

Human fetal MSCs (hMSCs, Cyagen Biosciences Inc., USA)were maintained and expanded in growth medium (GM,Cyagen Biosciences Inc., USA) consisting of hMSC basalmedium, pre-selected fetal bovine serum (FBS), penicillin–streptomycin and glutamine. Early passages (#5) of hMSCswere collected by addition of 0.25% trypsin/1 mM EDTAsolution (Cyagen Biosciences Inc., USA) and used foranalyzing cell adhesion, proliferation and differentiation.Mouse bone marrow MSCs (mMSCs, CRL-12424, ATCC, USA)were propagated in Dulbecco's Modied Eagle's Medium(DMEM, Gibco, USA) with high glucose and supplementedwith 10% FBS (Gibco, USA). Cells in passages from 3 to 4 werecollected and used for the cell assays. In all experiments, themedium was changed every 2 days until end-point assay. AllSAM substrates were placed in a non-treated 24-well plate,sanitized in 75% (v/v) ethanol for 3 h and then equilibrated insterilized phosphate buffered saline (PBS). The cell suspen-sions were then added to the plates and incubated at 37 �C ina humidied incubator with 5% CO2.

Cell adhesion, spreading and proliferation

Cells (5 � 104 cells per cm2) were seeded onto the samples andcultured for 12 h. Then, the cell/substrate constructs weretransferred to a new cell culture plate, washed 3 times with PBSand xed with 4% formaldehyde for 30 min. Aer being rinsedtwice with PBS, cells were immersed rst in 0.1% Triton X-100PBS for 10 min to increase permeability, then in a phalloidin–FITC probe (AAT Bioquest® Inc, USA) for 60 min and nally inDAPI (Beyitime, China) for 10 min. The morphology of cells oneach surface was observed by laser scanning confocal micros-copy (Leica TCS SP5, Germany) (n ¼ 2). The number of adheredcells was evaluated by a colorimetric assay as reported (n ¼ 5).15

In order to characterize the cell spreading, the cells werexed with 4% formaldehyde for 30min, stained with uoresceinisothiocyanate (FITC, Santa Cruz Biotechnology, Inc., USA) for30 min, then rinsed with PBS, and nally imaged using aninverted uorescence microscope (40FLAXZOSKOP, ZEISS,Germany). Eight 10� images from different elds were chosen

J. Mater. Chem. B, 2014, 2, 4794–4801 | 4795

Journal of Materials Chemistry B Paper

Publ

ishe

d on

27

June

201

4. D

ownl

oade

d by

Uni

vers

ity o

f C

hica

go o

n 16

/09/

2014

04:

45:5

1.

View Article Online

for statistical analysis of cell contact areas using image pro-cessing soware (Image J, National Institutes of Health, USA).

Cell proliferation was evaluated by the Cell Counting Kit-8(CCK-8, Dojindo, Japan) assay following the manufacturer'sinstructions (n ¼ 5). A suspension of MSCs (1 � 104 cells percm2) was added onto the samples. Aer 1, 3 and 5 days ofculture, cell/sample constructs were transferred to a new plate.Then 300 mL of CCK-8 working solution was added into eachwell and incubated at 37 �C for 1 h. Then 100 mL of the solutionwas pipetted to a 96-well plate. The absorbance at 450 nm wasquantied by using a microplate reader (Thermo3001, Thermo,USA).

Osteogenic differentiation and quantitative RT-PCR

The cells (5 � 104 cells per cm2) were cultured onto thesamples in the corresponding osteogenic differentiationmedium. The hMSC osteogenic medium was supplementedwith hMSC growth medium, 0.1 mM dexamethasone (Sigma-Aldrich, USA), 10 mM b-glycerophosphate (Calbiochem, USA)and 50 mM ascorbic acid (Sigma-Aldrich, USA). The mMSCosteogenic medium was composed of H-DMEM, 10% FBS, 0.1mM dexamethasone, 10 mM b-glycerophosphate and 50 mMascorbic acid. Cells were cultured in osteogenic medium for 7or 14 days and then transferred to a new culture plate foralkaline phosphatase (ALP) staining and RT-PCR,respectively.

ALP staining was conducted in duplicate following themanufacturer's instructions using the BCIP/NBT phosphatasesubstrate (1-Component) (KPL, USA). The stained surface wasobserved by using a digital three-dimensional video microscope(HIROX KH-7700, Japan). Total RNA was extracted from twoplates and each plate had two parallel samples for each kind ofsurface (n ¼ 4) using HiPure Total RNA Kits (Magentec, China)and reverse transcribed into cDNA using a PrimeScript® RTreagent Kit with gDNA Eraser (TaKaRa Biotechnology, Japan)according to the manufacturer's protocol. The RNA concentra-tion was quantied by using a NanoDrop2000 spectrophotom-eter (Thermo Scientic, USA). RT-PCR reactions were performedusing an SYBR Green System (Invitrogen, USA). Samples wereheld at 95 �C for 10 min, followed by 40 cycles at 95 �C for 15 sand 60 �C for 1 min. The relative quantication of target geneswas carried out using b-actin as a reference. The level ofexpression of target genes was calculated by the 2�DDCt methodusing the following primer and probe sequences (Invitrogen).Human-specic primers: b-actin {50-GCATCCCCCAAAGTTCACAA-30 (forward) and 50-AGGACTGGGCCATTCTCCTT-30

(reverse)}; runx-2 {50-AGAAGGCACAGACAGAAGCTTGA-30

(forward) and 50-AGGAATGCGCCCTAAATCACT-30 (reverse); ALP{50-AGCACTCCCACTTCATCTGGAA-30 (forward) and 50-GAGACCCAATAGGTAGTCCACATTG-30 (reverse)}; osteocalcin {50-CAGCGAGGTAGTGAAGAGA-30 (forward) and 50-GACTGGTGTAGCCGAAAG-30(reverse)}; collagen I {50-CAGCCGCTTCACCTACAGC-30

(forward) and 50-TTTTGTATTCAATCACTGTCTTGCC-30

(reverse)}; av integrin {50-AAACTCGCCAGGTGGTATGTGA-30

(forward) and 50-CTGGTGCACACTGAAACGAAGA-30 (reverse)};b1 integrin {50-AGCTGAAGACTATCCCATTGACCTC-30 (forward)

4796 | J. Mater. Chem. B, 2014, 2, 4794–4801

and 50-TGGTGTTGTGCTAATGTAAGGCATC-30 (reverse)}. Mouse-specic primers: b-actin {50-TGACAGGATGCAGAAG GAGA-30

(forward) and 50-GCTGGAAGGTGGACAGTGAG-30 (reverse)};runx-2 {50-CACTGGCGGTGCAACAAGA-30 (forward) and 50-TTTCATAACAGCGGAGGCATTTC-30 (reverse)}; ALP {50-TGCCTACTTGTGTGGCGTGAA-30 (forward) and 50-TCACCCGAGTGGTAGTCACAATG-30 (reverse)}; osteocalcin {50-AGCAGCTTGGCCCAGACCTA-30 (forward) and 50-TAGCGCCGGAGTCTGTTCACTAC-30 (reverse)}; collagen I {50-TTCTGCTGCTAATGTTC TTGACC-30

(forward) and 50-GGGATGAAGTATT GTGTCTTGGG-30 (reverse)};av integrin {50-TGAACTGCACGGCAGATACAGAG-30 (forward)and 50-ATCCCGCTTGGTGATGAGATG-30 (reverse)}; b1 integrin{50-ATCATGCAGGTTGCGGTTTG-30 (forward) and 50-GGTGA-CATTGTCCATCATTGGGTA-30 (reverse)}.

Statistical analysis

Quantitative experimental results were expressed as mean �standard deviation. Statistical comparison was performed byanalysis of variance (ANOVA) followed by a post-hoc test and p <0.05 was considered to be statistically signicant.

ResultsSurface characterization of SAMs

XPS and FTIR-ATR measurements were performed to prove thatthe –OH and –CH3 functional groups were successfully intro-duced onto the gold substrates. It was observed that the high-resolution spectra of sulphur can be well tted with a doubletstructure.16 In addition, all samples were free of unbound thiolmolecules because no peaks were found in the binding energyregion above 164 eV.17 This demonstrated that the physicallyadsorbed thiols were completely rinsed away. From the FTIR-ATR spectra in the region of interest of 2800–3000 cm�1, thetypical absorption band located at 2965 cm�1 was assigned tothe asymmetric stretching vibration of –CH3.18 The intensitygradually became stronger while the –CH3 composition wasincreased in the mixed SAMs. In addition, the peaks of theasymmetric (na) and symmetric (ns) stretching modes of –CH2–

appeared at 2919 and 2851 cm�1, respectively,18,19 indicatingcrystalline-like packing of the alkyl chains and high degree ofordering of the monolayers.6

The surface wettability can be tuned by mixing variousratios of thiols terminated with –OH and –CH3 constitutingthe SAMs. The result of the water contact angle on eachsurface showed that the –OH group led to a good hydrophilicsurface and –OH/–CH3 (9/1, 7/3, 5/5 v/v) mixed groups createdmoderately wettable surfaces, while –OH/–CH3 (3/7 v/v) and–CH3 groups produced hydrophobic substrates (Table 1 andFig. 1B). It can be seen that a widespread range of wettabilityfrom highly hydrophilic to highly hydrophobic was obtained.The water contact angle increased gradually with the incor-poration of –CH3 on the surface. In Table 1, O/C ratioscalculated by XPS data (Fig. 1A) decreased with the increase of–CH3 in the solution. The compositions of –OH groups in themixed SAMs (X–OH, surface) were calculated from the inten-sity of the O(1s) and Au(4f) peak using the following

This journal is © The Royal Society of Chemistry 2014

Table 1 Water contact angles and the percentage of surface –OH on OH/CH3 mixed SAMs

SAMs–OH/–CH3 (v/v solution) Water contact angle q (�) –OH (surface) (%) O(1s)/C(1s)

–OH 22.8 � 1.1 100 0.1505 � 0.0219/1 46 � 1.6 82.65 � 0.37 0.1405 � 0.0267/3 73.0 � 0.5 39.56 � 0.52 0.1167 � 0.0035/5 89.4 � 1.6 27.97 � 0.73 0.0673 � 0.0293/7 98.1 � 1.8 16.56 � 0.28 0.0401 � 0.017–CH3 107.1 � 0.7 0 0.0448 � 0.010

Fig. 1 (A) XPS S(2p) and FTIR-ATR spectra of –OH/–CH3 mixed SAMs;(B) water contact angles of –OH/–CH3 mixed SAMs. (a) –OH; (b)–OH/–CH3 (9/1 v/v); (c) –OH/–CH3 (7/3 v/v); (d) –OH/–CH3 (5/5 v/v);(e) –OH/–CH3 (3/7 v/v); (f) –CH3.

Paper Journal of Materials Chemistry B

Publ

ishe

d on

27

June

201

4. D

ownl

oade

d by

Uni

vers

ity o

f C

hica

go o

n 16

/09/

2014

04:

45:5

1.

View Article Online

equation.20

where AO1s and AAu4f are the normalized area for O1s and Au4f

�XOH;surface

�XOH;solution

h

�AO1s

�AAu4f

�XOH;solution�

AO1s

�AAu4f

�XOH;solution¼1

� �AO1s

�AAu4f

�XOH;solution¼0

photoelectrons, respectively; XOH,solution denotes a mixed SAMat a specic nominal ratio of –OH and –CH3 terminated thiols;and XOH,solution¼1 and XOH,solution¼0 denote pure –OH and–CH3 terminated thiols, respectively. Table 1 shows that thepercentage of incorporated –OH on the surface was not equalto the nominal ratio of the thiol solution. The –CH3 termi-nated thiols were adsorbed preferentially over –OH termi-nated ones.

Cell adhesion, spreading and proliferation

The morphologies of MSCs on –OH/–CH3 mixed SAMs aer12 h are shown in Fig. 2. The hMSCs spread well on thehydrophilic substrates (Fig. 2a–c) and exhibited a typical

This journal is © The Royal Society of Chemistry 2014

broblast-like morphology, while those on the hydrophobicsurfaces contracted a little into a compressed shape (e and f).Similarly, mMSCs adhered and spread well on –OH/–CH3 (7/3v/v) but exhibited poor spreading on –CH3 SAMs. This resultsuggested that hMSCs and mMSCs responded well tohydrophilic surfaces in terms of overall cell spreading andmorphology. It is consistent with the quantitative analysis ofthe cell spreading area, which showed that hydrophilic mixedSAMs with a contact angle of 20–70� (especially, the –OH/–CH3 (7/3 v/v) SAMs) tended to promote the spreading of bothhMSCs and mMSCs (Fig. S1 and S2†). In order to quantify thecell adhesion, cell cytoplasm was stained with crystal violetand the colorimetric readings, reecting the number ofadhered cells on various SAMs, were collected. As shown inFig. 3A, the number of adhered hMSCs reached maximum on–OH/–CH3 (9/1 v/v and 7/3 v/v). The maximum number wasobserved for the mMSCs on –OH/–CH3 (7/3 v/v). The MSCproliferation on different –OH/–CH3 mixed SAMs wasanalyzed using the CCK-8 assay aer 1, 3 and 5 days ofculture. As the culture time was extended, the cells on allsamples proliferated well (Fig. 3B). We found that bothhMSCs and mMSCs proliferated slower on –OH/–CH3 (3/7)and –CH3 terminated SAMs when compared with othersamples. The hMSCs seemed to grow fastest on –OH/–CH3 (7/3 v/v) SAMs, while the mMSCs proliferated fastest on –OH/–

CH3 (7/3 v/v) and (5/5 v/v) SAMs. Namely, the hydrophilicmixed SAMs with moderate wettability promote the adhe-sion, spreading and proliferation of both hMSCs andmMSCs.

ALP staining and gene expression of osteogenicdifferentiation

ALP staining of MSCs on different –OH/–CH3 mixed SAMs aer7 days are shown in Fig. 4. The formation of dark purple NBT-formazan was a qualitative indicator. The more blue-purpleareas implied higher ALP activity and enhanced osteogenicdifferentiation. It was seen that the ALP activity of hMSCs andmMSCs was increased on –OH/–CH3 (9/1 v/v), (7/3 v/v) and (5/5

J. Mater. Chem. B, 2014, 2, 4794–4801 | 4797

Fig. 4 ALP staining of MSCs on different –OH/–CH3 mixed SAMs after7 days of culture. (a) –OH; (b) –OH/–CH3 (9/1 v/v); (c) –OH/–CH3 (7/3v/v); (d) –OH/–CH3 (5/5 v/v); (e) –OH/–CH3 (3/7 v/v); (f) –CH3.

Fig. 2 The morphology of MSCs on different –OH/–CH3 mixed SAMsafter 12 h of culture was examined by laser scanning confocalmicroscopy. Cells were fixed and stained for F-actin with AlexaFluor488 phalloidin (green). Cell nuclei were counterstained with DAPI(blue). (a) –OH; (b) –OH/–CH3 (9/1 v/v); (c) –OH/–CH3 (7/3 v/v); (d)–OH/–CH3 (5/5 v/v); (e) –OH/–CH3 (3/7 v/v); (f) –CH3.

Journal of Materials Chemistry B Paper

Publ

ishe

d on

27

June

201

4. D

ownl

oade

d by

Uni

vers

ity o

f C

hica

go o

n 16

/09/

2014

04:

45:5

1.

View Article Online

v/v) SAMs, conrming the improvement in osteogenesis by thehydrophilic surfaces.

Runx-2, ALP, osteocalcin and collagen I are well-acceptedmarkers for osteogenic differentiation. Among them, Runx-2,

Fig. 3 (A) The number of adhered cells on different –OH/–CH3 mixedSAMs after 3 h of culture by a colorimetric assay at 595 nm. (B) Cellproliferation of MSCs on different –OH/–CH3 mixed SAMs after 1, 3and 5 days of culture. + indicates that there was significant difference(p < 0.05).

4798 | J. Mater. Chem. B, 2014, 2, 4794–4801

ALP, and osteocalcin are osteo-specic markers whereascollagen I is non-osteo-specic. Runx-2, a member of the runthomology domain transcription factor family, is essential forosteoblastic differentiation and expressed early in the embryoin mesenchyme.21 High expression of ALP as a marker for bonemetabolism indicated active bone formation.22 Osteocalcin andcollagen I were included in the formation of mineralized bone-like nodules and reected in late bone differentiation.23,24 Theexpressions of these osteogenic genes in MSCs aer 7 days ofculture are shown in Fig. 5. The expression of the markers inhMSCs on different –OH/–CH3 mixed SAMs did not show aregular trend. Higher expression of runx-2, ALP and collagen Iwas observed on –OH/–CH3 (9/1 v/v) SAMs. Osteocalcin wasexpressed at a higher level on –OH/–CH3 (7/3 v/v) mixed SAMsthan on other SAMs. As for mMSCs, the gene expression of allfour osteogenic markers, including runx-2, ALP, osteocalcin andcollagen I, was higher on OH/CH3 (7/3 v/v) mixed SAMs than onother SAMs. In addition to the favorite OH/CH3 (7/3 v/v) mixedSAMs, OH/CH3 (9/1 v/v) mixed SAMs also upregulated ALP andcollagen I in comparison to the other SAMs. Runx-2 andosteocalcin were upregulated on OH/CH3 (5/5 v/v) and OHterminated SAMs, respectively. The difference in the expressionof the osteogenic genes in both hMSCs and mMSCs amongdifferent SAMs was similar between day 7 and day 14 (Fig. S3†).These results indicated that the surface wettability inuencedthe overall osteogenic differentiation of MSCs. Overall, theexpression of four osteogenic genes indicated that the differ-entiation of MSCs on the different –OH/–CH3 mixed SAMs canbe affected by the wettability and the cell origins. The surfacewith a water contact angle of about 40� could upregulate thegene expression of the early stage differentiation markers(Runx-2 and ALP) of hMSCs. However, the surface with a contactangle of 70–90� upregulated the expression of the later stageosteogenic markers of osteocalcin and collagen I. As for

This journal is © The Royal Society of Chemistry 2014

Fig. 5 Relative gene expression of runx-2, ALP, osteocalcin andcollagen I using RT-PCR of MSCs on different –OH/–CH3 mixed SAMsagainst –CH3 terminated SAMs after 7 days of culture.+ indicates thatthere was significant difference (p < 0.05).

Paper Journal of Materials Chemistry B

Publ

ishe

d on

27

June

201

4. D

ownl

oade

d by

Uni

vers

ity o

f C

hica

go o

n 16

/09/

2014

04:

45:5

1.

View Article Online

mMSCs, the expression of the four osteogenic genes consis-tently suggested that the surface with a water angle of about 70�

was most favorable for osteogenic expression of mMSCs.

Discussion

As proved above, highly oriented –OH/–CH3 mixed SAMs wereformed on the gold surface and surfaces with a graded range ofwettability could be achieved. Co-adsorption of two or morethiols was not in complete thermodynamic equilibrium butrather competitive adsorption.25 Many factors could mediatethe process of co-adsorption, such as concentration of thiols,head groups, chain lengths and solvents. In our study, theadsorption of –CH3 terminated thiols was favorable on thesurfaces probably because –OH terminated thiols could formhydrogen bonds with the solvent whereas –CH3 terminatedthiols had a higher activity in solution.20 In principle, theincorporation of –OH terminated thiols onto the surface did not

This journal is © The Royal Society of Chemistry 2014

increase linearly with the increase in the proportion of –OHterminated thiols in solution.

We believe that the different wettability of the mixed SAMsmediated the signaling pathway to direct the fate of the MSCs. Itis well known that the integrin receptors mediate cell–matrixinteractions and play a central role in cell adhesion, spreading,migration, proliferation and differentiation.26–30 The avb1integrin enables the cells to bind to extracellular matrix proteinssuch as bronectin and vitronectin, forming focal adhesion andtriggering the intracellular signals with the actin cytoskeleton.26

We found that both hMSCs and mMSCs on the surface withmoderate wettability presented a higher level of gene expressionof avb1 integrin than those on the more hydrophobic surfaces(Fig. S4†), which was consistent with the results of increased celladhesion, spreading, proliferation and osteogenic differentia-tion of both hMSCs and mMSCs on hydrophilic mixed SAMs(Fig. 2–5 and S2†). Therefore, avb1 integrin may be one of thesignaling pathways triggered by the surface hydrophilicity of–OH/–CH3mixed SAMs for directing the fate of both hMSCs andmMSCs including their adhesion, spreading, proliferation andosteogenic differentiation.

Our results show that the morphologies of MSCs adheredonto the different –OH/–CH3 mixed SAMs were quite different(Fig. 2). On the hydrophobic surface, hMSCs and mMSCs bothexhibited a relatively round morphology. The quantication ofthe spreading area revealed that both hMSCs and mMSCsshowed the largest contact areas on –OH/–CH3 (7/3 v/v) SAMsamong all substrates (Fig. S1 and S2†). The cell spreading dataare consistent with the data on the adhesion, proliferation anddifferentiation in that the moderate hydrophilic SAMs tend tofavor cell adhesion, proliferation and differentiation of bothhMSCs and mMSCs. It is also likely that the mixed SAMs withdifferent wettability direct the stem cell fate by modulating theadsorption of the extracellular protein. Cell adhesion and theformation of actin organization were found to be largelymediated by the interaction between the integrin receptor andadsorbed matrix proteins such as bronectin, vitronectin, andbrinogen.31 Keselowsky et al. found that the density andconformation of bronectin may all play a role in the amount ofintegrin receptor.32 It was also found that the –CH3 surfacewould have more adsorbed bronectin than the –OH surfacedue to the hydrophobic interaction.33 However, there was astrong preference for cell adhesion on –OH surfaces.34 There-fore, MSCs were able to organize their cytoskeleton and spreadwell on the hydrophilic –OH/–CH3 mixed SAMs.

A trend was found in the expression of osteocalcin (higheston 7/3) and collagen I (highest on 9/1 and 5/5) as well as runx-2and ALP (highest on 9/1) for hMSCs (Fig. 5). There was also asimilar trend in the expression of osteocalcin, ALP and runx-2(highest on 7/3) as well as collagen I (highest on 9/1 and 7/3) formMSCs (Fig. 5). Namely, different mixed SAMs differentiallyenhanced lineage specication and commitment althoughoverall the moderate hydrophilic surfaces promoted the osteo-genic differentiation of MSCs. This indicates that the modula-tion of these lineage specic markers over time duringdifferentiation is a complex process and thus difficult toquantify. Indeed, we found that –OH/–CH3 mixed SAMs with

J. Mater. Chem. B, 2014, 2, 4794–4801 | 4799

Journal of Materials Chemistry B Paper

Publ

ishe

d on

27

June

201

4. D

ownl

oade

d by

Uni

vers

ity o

f C

hica

go o

n 16

/09/

2014

04:

45:5

1.

View Article Online

moderate wettability promoted the expression of avb1 integrin(Fig. S4†), which might trigger the integrin signaling pathway topromote the osteogenic differentiation. This is in agreementwith the previous nding35 that b1 integrin was critical forosteogenic differentiation of MSCs. A closer look at Fig. S4†showed a similar trend in the expression of av integrin (higheston 7/3) and b1 integrin (highest on 9/1 and 5/5) for hMSCs aswell as in the expression of av integrin (highest on 7/3) and b1integrin (highest on 9/1) for mMSCs, suggesting that differentmixed SAMs differently enhance the expression of integrins aswell. Hence, it is likely that different wettability arising from thedifferent mixed SAMs inuences the expression of integrinsdifferently to direct the osteogenic differentiation of bothhMSCs and mMSCs.

Hence, the adsorption of adhesive ligands of the integrinfamily cell-surface receptors on these SAMs is worth furtherinvestigation in order to understand the stem cell fate directedby the mixed SAMs. In addition, the activities of stem cells fromdifferent sources are regulated by many cues in their respectivelocal tissue microenvironment. Mechanisms of cellular senes-cence in human and mouse cells are distinct.36 Yet so far, thecomparison between hMSCs and mMSCs for the regulation ofdifferentiation by surface chemistry is not clear. To understandthe molecular mechanism and signaling pathway of the differ-entiation in human cells, numerous studies have been doneusing mouse or rat cells.37–39 However, osteogenic gene expres-sion in our studies clearly differs between mouse and humancells, and thus needs to be further elucidated.

Conclusions

The surface hydrophilicity of hydroxyl–methyl mixed SAMs, asmodel substrates with controlled wettability, increased gradu-ally with the incorporation of hydroxyl terminated groups onthe surface of the monolayer. Such control over the wettabilityof the substrate surface has allowed us to systematically studythe effect of wettability on the fate of MSCs from two differentorigins. The adhesion and proliferation of hMSCs and mMSCswere enhanced on hydrophilic surfaces with contact angles of40–70� and 70–90�, respectively. Hydrophilic mixed SAMs with amoderate wettability (water contact angle of 20–70�) alsopromoted the spreading of both hMSCs and mMSCs. Thesurfaces of moderate wettability promoted osteogenic differ-entiation depending on the cell origins (40–90� for hMSCs and70� for mMSCs) in the presence of biological stimuli. Hydro-philic mixed SAMs with a moderate wettability tended topromote the expression of avb1 integrin of both hMSCs andmMSCs, indicating that one of the possible mechanisms fordirecting the stem cell fate by the tunable wettability of themixed SAMs is through mediating the avb1 integrin signalingpathway. The results indicated that not only the cell behaviorwas affected by surface wettability and chemical functionalgroups, but also the cell origins. These ndings can shed lighton the design of biomaterials for stem cell-based regenerativemedicine applications.

4800 | J. Mater. Chem. B, 2014, 2, 4794–4801

Acknowledgements

This work was nancially supported by the National BasicResearch Program of China (Grant no. 2012CB619100), theNational Natural Science Foundation of China (Grant51232002), and the 111 project (B13039). CBMwould also like tothank the nancial support from the National Science Foun-dation (CMMI-1234957, CBET-0854414, CBET-0854465, andDMR-0847758), National Institutes of Health (5R01HL092526,1R21EB015190), Department of Defense Peer Reviewed MedicalResearch Program (W81XWH-12-1-0384), Oklahoma Center forthe Advancement of Science and Technology (070014 and HR11-006) and Oklahoma Center for Adult Stem Cell Research(434003).

Notes and references

1 J. M. Anderson, Annu. Rev. Mater. Res., 2001, 31, 81–110.2 L. Ponsonnet, K. Reybier, N. Jaffrezic, V. Comte, C. Lagneau,M. Lissac and C. Martelet, Mater. Sci. Eng., C, 2003, 23, 551–560.

3 P. Van Wachem, T. Beugeling, J. Feijen, A. Bantjes,J. Detmers and W. Van Aken, Biomaterials, 1985, 6, 403–408.

4 G. A. Hudalla and W. L. Murphy, So Matter, 2011, 7, 9561–9571.

5 J. C. Love, L. A. Estroff, J. K. Kriebel, R. G. Nuzzo andG. M. Whitesides, Chem. Rev., 2005, 105, 1103–1170.

6 A. Ulman, Chem. Rev., 1996, 96, 1533–1554.7 Y.-J. Ren, H. Zhang, H. Huang, X.-M. Wang, Z.-Y. Zhou,F.-Z. Cui and Y.-H. An, Biomaterials, 2009, 30, 1036–1044.

8 B. Bai, J. He, Y.-S. Li, X.-M. Wang, H.-J. Ai and F.-Z. Cui,BioMed Res. Int., 2013, 2013, 361906.

9 H. Yan, S. Zhang, J. He, Y. Yin, X.Wang, X. Chen, F. Cui, Y. Li,Y. Nie and W. Tian, Biomed. Mater., 2013, 8, 035008.

10 X. Liu, J. He, S. Zhang, X. M. Wang, H. Y. Liu and F. Z. Cui, J.Tissue Eng. Regener. Med., 2013, 7, 112–117.

11 Y. Arima and H. Iwata, Biomaterials, 2007, 28, 3074–3082.12 A. I. Caplan, J. Orthop. Res., 1991, 9, 641–650.13 A. I. Caplan, J. Cell. Physiol., 2007, 213, 341–347.14 M. F. Pittenger, A. M. Mackay, S. C. Beck, R. K. Jaiswal,

R. Douglas, J. D. Mosca, M. A. Moorman, D. W. Simonetti,S. Craig and D. R. Marshak, Science, 1999, 284, 143–147.

15 J. Malmstrom, B. Christensen, J. Lovmand, E. S. Sørensen,M. Duch and D. S. Sutherland, J. Biomed. Mater. Res., PartA, 2010, 95, 518–530.

16 D. G. Castner, K. Hinds and D. W. Grainger, Langmuir, 1996,12, 5083–5086.

17 M. C. L. Martins, B. D. Ratner and M. A. Barbosa, J. Biomed.Mater. Res., Part A, 2003, 67, 158–171.

18 M. D. Porter, T. B. Bright, D. L. Allara and C. E. Chidsey,J. Am. Chem. Soc., 1987, 109, 3559–3568.

19 R. G. Nuzzo, L. H. Dubois and D. L. Allara, J. Am. Chem. Soc.,1990, 112, 558–569.

20 C. D. Bain, J. Evall and G. M. Whitesides, J. Am. Chem. Soc.,1989, 111, 7155–7164.

21 T. Gaur, C. J. Lengner, H. Hovhannisyan, R. A. Bhat,P. V. Bodine, B. S. Komm, A. Javed, A. J. Van Wijnen,

This journal is © The Royal Society of Chemistry 2014

Paper Journal of Materials Chemistry B

Publ

ishe

d on

27

June

201

4. D

ownl

oade

d by

Uni

vers

ity o

f C

hica

go o

n 16

/09/

2014

04:

45:5

1.

View Article Online

J. L. Stein and G. S. Stein, J. Biol. Chem., 2005, 280, 33132–33140.

22 R. Aschaffenburg and J. Mullen, J. Dairy Res., 1949, 16, 58–67.23 J. S. Park, H. N. Yang, S. Y. Jeon, D. G. Woo, K. Na and

K.-H. Park, Biomaterials, 2010, 31, 6239–6248.24 O. Frank, M. Heim, M. Jakob, A. Barbero, D. Schafer,

I. Bendik, W. Dick, M. Heberer and I. Martin, J. Cell.Biochem., 2002, 85, 737–746.

25 J. P. Folkers, P. E. Laibinis and G. M. Whitesides, Langmuir,1992, 8, 1330–1341.

26 D. Docheva, C. Popov, W. Mutschler and M. Schieker, J. Cell.Mol. Med., 2007, 11, 21–38.

27 A. Rosa, R. Kato, L. Castro Raucci, L. Teixeira, F. de Oliveira,L. Bellesini, P. de Oliveira, M. Hassan and M. Beloti, J. Cell.Biochem., 2014, 115, 540–548.

28 A. J. Garcia, Biomaterials, 2005, 26, 7525–7529.29 R. O. Hynes, Cell, 1992, 69, 11–25.30 R. O. Hynes, Cell, 1987, 48, 549–554.

This journal is © The Royal Society of Chemistry 2014

31 U. Hersel, C. Dahmen and H. Kessler, Biomaterials, 2003, 24,4385–4415.

32 B. G. Keselowsky, D. M. Collard and A. J. Garcıa, J. Biomed.Mater. Res., Part A, 2003, 66, 247–259.

33 P. Roach, D. Farrar and C. C. Perry, J. Am. Chem. Soc., 2005,127, 8168–8173.

34 C. C. Barrias, M. C. L. Martins, G. Almeida-Porada,M. A. Barbosa and P. L. Granja, Biomaterials, 2009, 30,307–316.

35 S. Gronthos, P. Simmons, S. Graves and P. G. Robey, Bone,2001, 28, 174–181.

36 K. Itahana, J. Campisi and G. P. Dimri, Biogerontology, 2004,5, 1–10.

37 H. Zhu, B. Cao, Z. Zhen, A. Laxmi, D. Li, S. Liu and C. B. Mao,Biomaterials, 2011, 32, 4744–4752.

38 J. Wang, L. Wang, X. Li and C. B. Mao, Sci. Rep., 2013, 3,1242.

39 J. Wang, L. M. Yang, Y. Zhu, L. Wang, A. P. Tomsia andC. B. Mao, Adv. Mater., 2014, DOI: 10.1002/adma.201400154.

J. Mater. Chem. B, 2014, 2, 4794–4801 | 4801