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Colloids and Surfaces A: Physicochem. Eng. Aspects 434 (2013) 110– 117

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces A: Physicochemical andEngineering Aspects

jo ur nal ho me p ag e: www.elsev ier .com/ locate /co lsur fa

dsorbed BMP-2 in polyelectrolyte multilayer films for enhancedarly osteogenic differentiation of mesenchymal stem cells

eng Cai, Zhongyuan Xue, Wei Qi ∗, Hua Wang ∗∗

ey Laboratory of Life-Organic Analysis, Key Laboratory of Pharmaceutical Intermediates and Analysis of Natural Medicine, School of Chemistry andhemical Engineering, Qufu Normal University, 57 Jingxuan West Road, Qufu, Shandong 273165, China

i g h l i g h t s

The multilayer film was constructedto adsorb and stabilize growth factorBMP-2.The activity of the adsorbed BMP-2and the response of MSCs to it wereevaluated.The enhanced early osteogenic differ-entiation of MSCs was observed.The system may be used in localdelivery of growth factors for medicalimplants.

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

rticle history:eceived 10 April 2013eceived in revised form 10 May 2013ccepted 13 May 2013vailable online 20 May 2013

eywords:MP-2

a b s t r a c t

The present work was focused on the development of multilayer films to adsorb and stabilize growthfactors initially for delivery to mesenchymal stem cells (MSCs). The multilayer was constructed bypoly(allylamine hydrochloride) (PAH) and poly(sodium 4-styrenesulfonate) (PSS) via layer-by-layerassembly. The adsorption and bioactivity of bone morphogenetic protein 2 (BMP-2) in the films wereexplored. Then, the response of MSCs to the adsorbed BMP-2 was evaluated. It was demonstrated thatthe adsorbed BMP-2 could retain its activity toward enhanced early osteogenic differentiation of MSCs.Alkaline phosphatase (ALP) activity of MSCs seeded on the BMP-2 loaded film was even higher than that

olyelectrolyte multilayer filmayer-by-layer assemblysteogenic differentiationesenchymal stem cells

on the non-loaded film with BMP-2 added to the culture media. The results confirmed that a localizedand sustained delivery of BMP-2 was more effective in vivo. It is noteworthy that these films can bedeposited on a wide range of substrates with different shapes, sizes and composition, and can load othergrowth factors for facilitating stem cell differentiation toward specific lineages. Thus, such a system mayfind diverse applications in local delivery of immobilized growth factors for medical implants and fortissue-engineered constructs.

∗ Corresponding author at: School of Chemistry and Chemical Engineering, Qufuormal University, 57 Jingxuan West Road, Qufu Shandong 273165 China.el.: +86 537 4458208.∗∗ Corresponding author at: School of Chemistry and Chemical Engineering, Qufuormal University, Qufu, Shandong 273165, China. Tel.: +86 537 4456306.

E-mail addresses: qiwei@iccas.ac.cn, qf qw@163.com (W. Qi),uawangqfnu@126.com (H. Wang).

927-7757/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.colsurfa.2013.05.041

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Mesenchymal stem cells (MSCs) isolated from bone marroware attractive tools for tissue engineering and cell-based therapies[1–3]. They are capable of differentiation into multiple lineages,such as ostoblasts, chondrocytes and adipocytes. When exposedto specific inducers or growth factors they can be induced to dif-

ferentiate into desired cell types [4–7]. For instance, in the fieldof bone tissue engineering, bone morphogenetic protein 2 (BMP-2) has been used commonly to stimulate differentiation of MSCstoward osteoblastic lineage [8,9]. However, bolus injections of

P. Cai et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 434 (2013) 110– 117 111

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Scheme 1. Scheme representing the osteogenic differentiation of MSCs derived

rowth factors are rapidly cleared from the blood stream and theirctivities are lost on exposure to blood. Moreover, supraphysiologicose of growth factor are needed for injections due to growth factorilution in a large body volume [10–12]. Therefore, there is a needor a controlled and localized delivery of growth factors from a bio-

aterial surface, which could offer the potential to concentrate therowth factor and to protect it from degradation by enzymes in tis-ue fluids. An efficient attempt is to design a coating that can retainnd locally concentrate growth factors in an active form [13–17].his calls for versatile, surface-based methodologies that allow foroating of a current biomedical device.

Layer-by-layer (LbL) method is an ideal candidate for suchn application due to the ease of production and the control oflm architecture, thickness, mechanical properties, etc. [18–25].urthermore, as the deposition process is achieved in aqueousedium, incorporation of biomolecules is possible. As reported

26–31], the embedded proteins in the LbL films had been shown toetain their activities. Hence, the multilayer films fabricated withbL technique represent a promising surface coating for the deliv-ry of growth factors in a localized, stabilized and controlled releaseay.

In this work, we reported the adsorption of BMP-2 inhe polyelectrolyte multilayer film composed of poly(allylamineydrochloride) (PAH) and poly(sodium 4-styrenesulfonate) (PSS)

abricated via LbL assembly technique. PAH/PSS multilayer film washe most widely used polyelectrolyte system and has been useds biologically compatible substrate [32,33]. The purpose of the

resent work is to evaluate performance of the adsorbed BMP-2 inhe multilayer film on inducing osteogenic differentiation of MSCs,s shown in Scheme 1. For that, we access the cell adhesion, prolif-ration and osteogenic differentiation of MSCs from bone marrow

bone marrow on (PAH/PSS)22 film loaded with BMP-2. Scale bars are 200 �m.

seeded onto the multilayer film. The results confirmed the abilityof adsorbed BMP-2 in PAH/PSS multilayer film to stimulate MSCssurvival, growth and osteogenic differentiation. It can be expectedthat such a system may be used as a biocompatible, versatile coat-ing for implant engineering, tissue engineering and other medicalapplications.

2. Materials and methods

2.1. Materials

Polyethyleneimine (PEI, Mw: 25,000), poly(sodium 4-styrenesulfonate) (PSS, Mw: 70,000), poly(allylamine hydrochlo-ride) (PAH, Mw: 58,000), tetramethylrhodamine isothiocyanate(TRITC), 5(6)-carboxyfluorescein N-hydroxysuccinimide ester (CF),dexamethasone, �-glycerol phosphate and ascorbic acid werepurchased from Sigma–Aldrich (USA). Recombinant human bonemorphogenetic protein 2 (BMP-2) was obtained from Invitrogen(USA). Water used in this work was purified by a Milli-Q watersystem (Millipore, USA).

2.2. Film preparation

To ensure the successful adsorption, a precursory layer of PEIwas first deposited on glass coverslips. PSS and PAH were thenalternately assembled onto the substrate. All the polyelectrolyteswere prepared at a final concentration of 2 mg/mL in aqueous solu-

tions, containing 0.15 M NaCl. Glass coverslips and silicon waferswere used as the substrates for cell culture and AFM characteriza-tion, respectively. They were cleaned in piranha solution (7:3, v/vH2SO4/H2O2) before use, followed by rinsing with water and dried

112 P. Cai et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 434 (2013) 110– 117

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the control and the test well plates. After 24 h, osteogenesiswas induced by replacing the original medium with osteogenicmedium, which was changed every 3 days up to confluence (15

ig. 1. AFM images (5 �m × 5 �m) of the surface morphology of (PAH/PSS)22 films w.037 nm and 7.461 nm, respectively.

nder a smooth stream of N2. The multilayer film with 22 bilayersas assembled and expressed as (PAH/PSS)22.

.3. Loading and release of BMP-2

BMP-2 was incorporated into the films pre-equilibrated for0 min in the medium in which BMP-2 was suspended (20 mMcetic acid) [34,35]. A volume of 50 �L for 96-well plates or 0.3 mLor 24-well plates of BMP-2 was deposited on to the films and lefto adsorb overnight at 4 ◦C. 150 �L of PBS, pH 7.4 were then addedo each well and left at room temperature for 15 min after that,he coated slides were then dipped into PBS for the release studiesnd the measurements were performed at different time points.ubsequently, their release behaviors were also studied in normalell culture medium after being thoroughly washed for 12 h in PBS.he amounts of BMP-2 released were determined with the Quan-ikine BMP-2 immunoassay (R&D systems, France) using Wallac420 VICTOR2 multilabel counter (Perkin-Elmer, USA). The experi-ents were carried out at least in triplicate, with three independent

amples per condition in each experiment.

.4. Cell culture

Rat bone marrow derived MSCs were obtained from commercialources (Tianjin Weikai Bioeng Ltd., China). Cells were cultured in

humidity-controlled environment under 5% CO2 and fed every 3ays with Dulbecco’s modified Eagle’s medium (Gibco, USA), sup-lemented with 10% fetal bovine serum (Invitrogen, USA) and 1%enicillin/streptomycin (Invitrogen, USA). Cells were received atassage 1, expanded at 5000 cells/cm2, and subcultured at 70–80%onfluence. Passage 4 cells were used in the experiments. Cell via-ility was studied with cells cultured in normal stem cell medium.tem cell differentiation was examined in cells cultured on conven-ional osteogenic media containing 10 mM �-glycerol phosphate,0−8 M dexamethasone, and 0.2 mM ascorbic acid.

For cell viability, the MSCs (20,000 cells/well, 24-well plate)ere seeded on different surfaces, namely, coverslips (control)

PAH/PSS)22 film-coated coverslips, BMP-2-loaded (PAH/PSS)22lm-coated coverslips and non-loaded (PAH/PSS)22 film in BMP-2

edium. Cell counting kit-8 (CCK-8) assays (Dojindo, Japan) were

arried out for all the above cells. The morphology of the MSCs onifferent surfaces was obtained with inverted phase microscopeOlympus CKX31, Japan) at day 1, 4, 7, and 10.

t (A) and with (B) adsorbed BMP-2. The root mean square roughnesses (Rms) were

2.5. Alkaline phosphatase (ALP) staining and quantification

The MSCs (20,000 cells/well, 24-well plate) were seeded into

Fig. 2. (A) Kinetics of BMP-2 adsorption on (PAH/PSS)22 film and coverslip. The initialBMP-2 loading concentration was 20 �g/mL in 20 mM acetic acid. (B) Time-courseof BMP-2 cumulative release from (PAH/PSS)22 film in PBS and subsequently in cellculture medium.

P. Cai et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 434 (2013) 110– 117 113

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ig. 3. (A) CLSM observation of (PAH/PSS)22 film with adsorbed BMP-2. TRITC-PAHcid) and rinsed for 12 h in PBS before observation. (B) The corresponding intensity

ays). ALP staining was performed using BCIP/NBT stock solutionBeyotime, China) and the ALP expressed by MSCs seeded on dif-erent surfaces was quantified using the protocol from Alkalinehosphatase Assay Kit (abcam, Hong Kong).

.6. Optical characterization

Atomic force microscopy (AFM) images were performedn a Digital Instruments multimode microscope controlled byanoscope IIIa apparatus (Digital Instruments, Santa Barbara, CA,SA). The film vertical structure was imaged using FV-1000 con-

ocal system (Olympus, Japan) for films on 14 mm coverslips. Foronfocal microscopy (CLSM) observations, BMP-2 was labeled withF according to the report [34], and PAH labeled with TRITC wasrepared with the reported methods [36]. The amount of chemi-al inducers adsorbed was determined from change in adsorptionefore and after addition of (PAH/PSS)22 films using a UV-vis spec-rophotometer UV-1601 (Shimadzu, Japan).

.7. Statistical analysis

All experiments were repeated at least three times. Error bars

epresent standard errors, and statistical analysis was performedsing SPSS 13.0 to evaluate the statistical differences (*p < 0.05,*p < 0.01) among all samples or between samples and controls,espectively.

sed to bulidup the film and CF-BMP-2 was adsorbed at 20 �g/mL (in 20 mM acetices were given along with z-direction.

3. Results and discussion

3.1. Morphology of (PAH/PSS)22 film without and with adsorbedBMP-2

Firstly, AFM technique was used to analyze morphology of the(PAH/PSS)22 film. AFM is one of the surface imaging techniques atthe micro- and nanoscale. It has been widely used to monitor sur-face modifications, especially for biological molecules immobilizedon solid surfaces [37,38]. Tapping mode images of the (PAH/PSS)22film without and with BMP-2 on silicon wafer revealed the typ-ical topography of multilayer film fabricated with LbL technique(Fig. 1). It was observed that BMP-2 loading did not significantlyaffect the film’s overall morphology. According to AFM analy-sis, the root mean square roughness (Rms) was 9.037 nm for the(PAH/PSS)22 film without BMP-2 loading (Fig. 1A), and that of the(PAH/PSS)22 film loaded with BMP-2 was 7.461 nm, as shown inFig. 1B. A decrease in surface roughness was observed, which maybe attributed to the coating of BMP-2.

3.2. Kinetics of BMP-2 adsorption and release

Then, kinetics of BMP-2 adsorption in the (PAH/PSS)22 film wasstudied (Fig. 2A). The adsorbed BMP-2 amounts in the film areexpressed in ng/cm2 for ease of comparison. It could be seen thatthe maximum loading was reached in about 1.5 h with 697 ng/cm2

114 P. Cai et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 434 (2013) 110– 117

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ig. 4. (A) MSCs cell viabilities cultured for 1, 4, 7, 10, 15 days on (PAH/PSS)22 filmith the coverslip as the control detected by CCk-8 assay. (B) Morphology of MSCs

edium. Scale bars are 200 �m.

f BMP-2. The initial BMP-2 loading concentration was 20 �g/mLn 20 mM acetic acid. Releases of film-adsorbed BMP-2 in phos-hate buffer solution (PBS, pH 7.4) and normal stem cell cultureedium were also investigated, as shown in Fig. 2B. The release

n PBS reached an equilibrium value in 10–12 h, releasing 23.1%.s the films were always thoroughly washed in PBS prior toeing introduced to the culture medium, the release in the cul-ure medium was performed after 12 h of release in PBS. Actually,t was found that the release amount in the culture medium wasow and less than 5% of the effective adsorbed amount was observedn 4 days. Accordingly, the quantity of BMP-2 remaining in the filmfter 12 h of release in PBS was taken as the effective incorporatedmount (537 ng/cm2 of BMP-2).

A visual and qualitative analysis of BMP-2 adsorption and diffu-ion in the film was obtained from confocal microscopy images, ashown in Fig. 3. For these experiments, BMP-2 was labeled with CFnd PAH labeled with TRITC was used to construct film with a thick-ess of 151 nm determined with AFM at dried state. The overlay ofreen and red channel (Fig. 3A) for the (PAH/PSS)22 film loadedith BMP-2 as well as the intensity profiles along the z-direction

Fig. 3B) indicated that BMP-2 had been adsorbed and loaded in thelm. The results also showed that BMP-2 appeared to accumulate

n the upper part of the film with a limited diffusion within the film34].

P-2-loaded (PAH/PSS)22 film and non-loaded (PAH/PSS)22 film in BMP-2 mediumed for 1, 4, 7, 10, 15 days on BMP-2-loaded (PAH/PSS)22 film in normal cell culture

3.3. MSCs response to the adsorbed BMP-2

In order to determine how the adsorbed BMP-2 would play arole in the cellular response and differentiation, we seeded MSCs onthe (PAH/PSS)22 multilayer films with and without BMP-2 loaded,respectively. A glass coverslip was used as a control surface. Also,the experiments were conducted where the cells were cultured onthe (PAH/PSS)22 film without BMP-2 in media supplemented withBMP-2 (600 ng/mL). According to the previous report [35], a highconcentration of BMP-2 (600 ng/mL) was used when delivered insolution to saturate endogenous BMP-2 receptors on cells.

Cell viabilities were evaluated on all four surfaces by CCK-8 assaywhen the cells were cultured in normal stem cell medium (Fig. 4A).After 3 days of culture, the cell viabilities on BMP-2-loaded filmsin normal cell culture medium and on non-loaded films in BMP-2 medium were significantly greater than that on the coverslips.Phase contrast images of MSCs cultured for 1, 4, 7, 10, 15 dayson the (PAH/PSS)22 films with adsorbed BMP-2 were presented inFig. 4B. MSCs were found to spread on the film surfaces and grew ina whirlpool manner, lined up in order and exhibited a shuttle-shape

appearance. These results suggested that the polyelectrolyte mul-tilayer film did not hamper the normal growth of stem cells and theincorporation of growth factors could enhance the proliferation ofthe stem cells. Importantly, it demonstrated that the (PAH/PSS)22

P. Cai et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 434 (2013) 110– 117 115

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have the ability to preconcentrate typical chemical inducers, whichmade it possible to induce the osteogenic differentiation of MSCs[39].

ig. 5. ALP staining results of MSCs for 15 d following osteogenic induction on covend non-loaded (PAH/PSS)22 film (bottom right) in BMP-2 medium. Scale bars are 2

ultilayer film and the adsorbed BMP-2 would not affect the phys-ological conditions of the microenvironment of stem cells, whichhereby inform future bone and tissue regenerative models for thexploration of clinical relevance of LbL growth factor delivery films.

Next, we measured the bioactivity of film-adsorbed BMP-2 byuantifying the activity of ALP of MSCs seeded on it. The expres-ion of ALP is typically used as an early marker of the osteogenichenotype. Firstly, we compared the extent of ALP production onach surface of the above four, with the results manifested in Fig. 5.ith the 15 days time frame of the experiments, the control rep-

esented by the coverslips in osteogenic medium did not show anyLP production. In contrast, ALP staining for the adsorbed BMP-

in (PAH/PSS)22 film was strong positive. The cytoplasm of theolygon was stained blue-black. Spectrophotometric quantifica-ion confirmed these above observations, as shown in Fig. 6. The ALPctivity increased from 2 days to 15 days indicating the progressivesteogenic differentiation of MSCs induced by the adsorbed BMP-. Interestingly, the activity of ALP for the adsorbed BMP-2 wasreater than that in solution. This verified that a localized deliveryas more appropriate for obtaining an optimal efficacy of BMP-

, which was consistent with the previous reports [13,34]. As therevious reports suggested [34], the adsorbed BMP-2 may favorirect contact with the receptor chains on cells by restricting theiriffusion and/or internalization. There was still a lot work to beone to get exact explanations for such an improved activity of thedsorbed growth factors.

Additionally, we observed an obvious ALP production with theare (PAH/PSS)22 film in the osteogenic medium. To derive theechanism of its ability to induce osteogenic differentiation ofSCs, the adsorption of the film for typical osteogenic chemi-

al inducers, namely, dexamethasone, �-glycerol phosphate andscorbic acid was further examined using optical spectroscopyechniques. The loading capabilities of the film for the three chem-cals were determined and the results were summarized in Table 1.

top left) (PAH/PSS)22 film (top right), BMP-2-loaded (PAH/PSS)22 film (bottom left).

From Table 1, one can note that the film could adsorb pretty highamount of �-glycerol phosphate (236 �g/cm2) and ascorbic acid(6.85 �g/cm2). While in the case of dexamethasone the resultscould not be obtained due to too low concentration to be deter-mined. The results preliminarily suggested that the film might

Fig. 6. Activity of ALP of MSCs cultured for 2, 6, 10, 15 days on coverslip (PAH/PSS)22

film, BMP-2-loaded (PAH/PSS)22 film and non-loaded (PAH/PSS)22 film in BMP-2medium.

116 P. Cai et al. / Colloids and Surfaces A: Physicoch

Table 1Adsorption ability of (PAH/PSS)22 multilayer film for dexamethasone, �-glycerolphosphate and ascorbic acid.

Chemical inducer in PBS Mass adsorbed (�g/cm2 of PEM film)

Dexamethasone (10−8 M) N.A.

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�-Glycerol phosphate (10 mM) 472 ± 39.1Ascorbic acid (0.2 mM) 13.7 ± 1.92

. Conclusions

In summary, we have demonstrated the MSCs adhesion, pro-iferation and osteogenic differentiation on the LbL-constructedolyelectrolyte multilayer films. The obtained evidences indicatedhat such a simple multilayer film could be employed for theelivery of BMP-2 to achieve the enhanced MSCs osteogenic differ-ntiation. The presence of multilayer films and the adsorbed BMP-2howed no influence on the shape and the growth of the cells inormal stem cell media. Also, the (PAH/PSS)22 films with adsorbedMP-2 were initially demonstrated to be more effective systems

or enhancing MSCs early osteogenic differentiation, as comparedith non-loaded (PAH/PSS)22 film in BMP-2 medium. Furthermore,

n the presence of an osteogenic medium, the (PAH/PSS)22 filmnduced the differentiation of MSCs obviously, in contrast to theoverslip control, presumably due to the effective preconcentra-ion of osteogenic inducers on the multilayer film. The present workepresented a promising system for the development of biomedicalurface modification by the LbL technique with its easy perfor-ance and cost efficiency. It is noteworthy that the as-developedultilayer film holds great potential for loading other kinds of

rowth factors to facilitate the stem cell differentiation towardpecific lineages. Additionally, the proposed protocol can allow forhe deposition of the stem cell-growing films onto a wide range ofiomedical substrates with different shapes, sizes and composition.herefore, such a system should find diverse applications in tissuengineering and regenerative medicine fields.

cknowledgments

This work was financially supported by the National Naturecience Foundation of China (No. 21003084), Shandong Provinceromotive Research Foundation for Excellent Young and Middle-ged Scientists (No. BS2010CL023), Student Research Trainingrogram of Qufu Normal University (Nos. 201210446022 and012A033) and the Taishan Scholar Foundation of Shandongrovince, China.

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