Professional Documents
Culture Documents
State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR
China
b
University of Chinese Academy of Sciences, Beijing 100049, PR China
a r t i c l e
i n f o
Article history:
Received 13 May 2013
Received in revised form 2 July 2013
Accepted 26 July 2013
Available online 3 August 2013
Keywords:
Styrenic block copolymer (SBC)
Hyaluronic acid (HA)
Surface functionalization
Hemocompatibility
Cytocompatibility
a b s t r a c t
As a biostable elastomer, the hydrophobicity of styrenic block copolymer (SBC) intensely limits its
biomedical applications. In order to overcome such shortcoming, the SBC lms were grafted with
hyaluronic acid (HA) using a coupling agent. The surface chemistry of the modied lms was examined by ATR-FTIR and XPS techniques, and the surface morphology was visually described by AFM. The
biological performances of the HA-modied lms were evaluated by a series of experiments, such as
protein adsorption, platelet adhesion, and in vitro cytocompatibility. It was found that the HA-modied
samples showed a low adhesiveness to broblast at the initial stage; however, it stimulated the growth
of broblast. The L929 broblast growth presented a strong dependence on the molecular weight (MW)
of HA. The samples modied with 17 kDa HA exhibited the worst wettability and platelet adhesion, while
providing the best results of supporting broblast proliferation.
2013 Elsevier B.V. All rights reserved.
1. Introduction
Styrenic block copolymer (SBC) (Fig. S1(a)) typically consists of
two polystyrene hard segments on each end and a central polyolene soft segment. The high glass transition temperature (Tg )
styrenic regions as physical crosslinking points provide strength for
this elastomer, while the low Tg olenic regions contribute exibility and elasticity. The physical crosslinking points are reversible
upon heating, therefore imparting the multiple melt processing
properties to this bioelastomer, which makes it superior to a vulcanized rubber. As a result of its physiological inertness, good
thermal and oxidative stability, as well as low toxicity, the biostable
SBC elastomer have a wide range of biomedical applications [14],
such as disposable medical devices, microchips [5], urinary tract [6],
glaucoma shunt [7], articial heart valve [8] and stent drug delivery
coating [9,10].
For some specic biomedical applications, SBC bioelastomer
should be further subjected to surface modication. A desired
surface can be tailored by immobilizing various biocompatible substances [11,12], such as poly(ethylene glycol) (PEG) and
its derivatives [1315], poly(vinylpyrrolidone) (PNVP) [1619],
heparin [2022], zwitterionic materials [2325], peptides [26,27],
Corresponding authors. Tel.: +86 431 85262109; fax: +86 431 85262109.
E-mail addresses: suan@ciac.jl.cn, luanshifang@163.com (S. Luan),
yinjh@ciac.jl.cn (J. Yin).
0927-7765/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.colsurfb.2013.07.048
2. Experimental
147
AFM images. The reported data were the mean values of triplicate
specimens.
148
analyzed using a one-way ANOVA method (*p < 0.05, **p < 0.01).
Each result is an average of at least three parallel experiments.
2.10. Cytocompatibility
Cytocompatibility was studied using murine broblast cell
line L929. Cells were grown in DMEM supplemented with
10 vol% FBS, 4.5 g/L glucose, 100 units/mL penicillin, 5958 mL/L N2-Hydroxyethylpiperazine-N -2-ethanesulfonic acid (HEPES) and
100 g/mL streptomycin and maintained in a humidied 5%
CO2 /95% air incubator at 37 C.
The SBC lms with a diameter of 16 mm were pretreated with
75 vol% ethanol aqueous solution for 1 h, and followed by extensive
rinsing in PBS. Those lms were then soaked in cell culture medium
and kept in cell culture incubator. After reaching 95% conuency,
cells were detached by 0.25 wt.% trypsin and then suspended in culture medium at a density of 1.0 105 cells/mL. Then 1 mL medium
and 0.5 mL cell suspension were added to each sample. Cell viability and cell numbers were measured after cultured for 1, 2 and 4
days. The cell culture medium was removed and followed by extensive rinsing in PBS, and the cell adhered on the lms were xed
by 4.0 wt.% paraformaldehyde at 4 C for 30 min to obtained the
cell-immobilized samples. For observing the uorescence images of
cells in different stages, the cell-immobilized samples were stained
via adding 0.2 mL uorescence-staining solution at room temperature for 30 min. Finally, they were washed three times with PBS
and once with deionized water, and vacuum freeze dehydration.
Fluorescence images of FITC and DAPI-stained cells were obtained
by an inverted TE-2000-U digital uorescence microscope (Nikon)
attached with a digital camera (DXM1200F). For observing the SEM
images of cells, the cell-immobilized samples were washed three
times with PBS and once with deionized water, and vacuum freeze
dehydration. Images from scanning electron microscopy (SEM, XL
30 ESEM FEG, FEI Company, USA) were used to calculate cell spreading area and cell density by using Image J software. Cell density and
cell surface area on the SBC substrates were analyzed using a oneway ANOVA method (*p < 0.05, **p < 0.01, ***p < 0.001). Each result
is an average of at least three parallel experiments.
3. Results and discussion
The most widely used hydrophilic surface modiers are based
on PEG and its derivatives [4145]. We have found that the introduction of PEG could impart good hemocompatibity to a SBC
elastomeric substrate [46,47]; however, these modiers simultaneously suppressed cell adhesion and proliferation. The covalent
attachment of acrylated HA to this substrate via a photografting polymerization could obtain both hemocompatiblity and
cytocompatiblity [48]. In view of the typical characteristics of
photo-initiated free radical polymerization and the inhomogeneous distribution of photoinitiator physically adsorbed on the
surface, a large polydispersity index and inhomogeneous distribution of the grafted chains were inevitable. The morphology
of the modied substrate was certainly heterogeneous on the
micro- and macro-scales. This rendered the dependence of biological response on the MW of the immobilized HA ambiguous.
In this study, HA with different MW were respectively immobilized onto a SBC substrate under mild condition via a grafting
to strategy (Fig. 1). Both the biocompatibility and the MW
effect of HA were systematically investigated for the HA-modied
systems.
3.1. Surface chemistry
3.1.1. ATR-FTIR
As ATR-FTIR spectra shown in Fig. 2a, two new characteristic peaks at 3387 cm1 (N H stretching) and 1265 cm1 (C N
stretching) appeared after surface modication of the SBC substrate with diethylenetriamine. As for the HA-modied substrates,
the ATR-FTIR spectra further changed. Specically, characteristic
peaks at 3387 cm1 (N H stretching) and 1265 cm1 (C N stretching) strengthened, and new peaks at 1665 cm1 (C O stretching),
1099 cm1 (C OH stretching) and 812 cm1 (C O C stretching)
appeared.
3.1.2. XPS
Surface composition of the SBC lms was detected by XPS
(Fig. 2b). After sequential surface modication with DETA and HA,
the intensity of N1s peak nearby 399 eV immensely strengthened,
and the ratios of N/C increased from 0.85% up to 2.85% (Table
S1). Due to oxygen contamination, the high-resolution C1s spectra of the virgin SBC lm were divided into two peaks using a
Gaussian peak tting algorithm (Fig. S2(a)). After covalent immobilization with DETA, a new peak at 285.6 eV attributing to C N
group appeared (Fig. S2(b)). As for the HA-modied lm, the highresolution C1s spectra were decomposed into ve peaks: a C H
(C C) peak at 284.6 eV, a C N peak at 285.6 eV, a C O C peak at
286.2 eV, a N C O peak at 288.2 eV and a O C O peaks at 289.0 eV,
149
Fig. 2. ATR-FTIR (a), and XPS (b) spectra of the SBC lms. (I) Virgin SBC, (II) SBC-DETA, (III) HA17, (IV) HA47, and (V) HA310.
150
Fig. 3. Surface density of amine groups (a) (n = 3) and HA (b) (n = 5) on the SBC lms.
Fig. 4. Amount of protein adsorption on the SBC surfaces. (a) BSA, and (b) BFg. (The
error bars: standard deviations, n = 3.)
Protein adsorption is the rst event in bloodmaterial interactions and some proteins in blood, which plays an important
role in material-associated clotting. It is widely recognized that a
hydrophilic surface possesses good anti-protein adsorption properties, a moderate hydrophilicity is generally optimal for a polymer.
The high amounts of protein adsorption were observed on the
hydrophobic SBC samples (a WCA of 103 ) (Table S3). Correspondingly, a large number of disk-shaped (a typical stage of early
activation), and spreading (a totally activated status) platelets were
observed on the virgin SBC surface (Fig. 5a). The serious thrombosis would be initiated by platelets adhesion and activation and
breakage of platelets. In contrast, as for the HA-modied samples (a WCA of 6269 ) (Table S3), HA hydration layer prevented
the hydrophobic interaction between protein and substrate, so
the amount of BSA and BFg adsorbed on the HA-modied substrates was smaller than that of the hydrophobic SBC controls
(Fig. 4). Depending on protein concentration, although the amounts
of the proteins adsorbed on the HA-modied substrates had a
slight variation, the adsorbed amount of BSA was generally reduced
by 2050%, while that of BFg was even up to 91% relative to
the SBC controls. The amount of protein adsorption was mainly
dependent on the concentration and species of protein, rather than
the MW of HA (Fig. 4). BFg could bind to the platelet GP IIb/IIIa
151
Fig. 5. Adhesion of platelets on the SBC lms. (a) virgin SBC, (b) SBC-DETA, (c) HA17, (d) HA47, (e) HA310, and (f) the statistical number of adherent platelets. (The error bars:
standard deviations, n = 3; data analyzed using a one-way ANOVA, *p < 0.05, **p < 0.01.)
71% (Table S4), while that of the HA-modied surfaces was respectively reduced to 54%, 52% and 38%, conrming that the hydrophilic
HA graft chains suppressed platelet spread and activation.
Usually, the interaction force between the vinculin on the Cell
Membrane and the substrate was also weakened because of the
hydrated layer effect, bringing about the less adhered cells. For
example, anti-fouling PEG and its derivatives inevitably inhibited
cell adhesion and proliferation. As shown in Figs. 6, 8 and S4, the
HA-modied lms suppressed L929 cell adhesion (Fig. 8a), which
was consistent with the previous reports that the low adhesiveness
of the HA surfaces to cells [52]. However, the HA-modied lms
signicantly promoted cell proliferation, especially for HA17, as
compared with the SBC controls. As for the HA17 lms, the number
of L929 cell cultured on their surfaces increased in time dependent
manner, their increasing rates are much larger than that of HA47
and HA310 groups, presenting the best results of supporting broblast proliferation. With respect to the 1020 m size of cells, the
152
Fig. 6. Fluorescent photomicrographs showing L929 cell numbers at different time points on various surfaces. (I) virgin SBC, (II) HA17, (III) HA47, and (IV) HA310, after
incubation for 1 day (A), 2 days (B) and 4 days (C). (Size of the scale bars: 100 m.)
Fig. 7. SEM images of L929 cell at different time points on various surfaces: (I) virgin SBC, (II) HA17, (III) HA47, and (IV) HA310, after incubation for 1 day (A), 2 days (B) and
4 days (C). (Size of the scale bars: 200 m.)
153
154
[16] U. Edlund, M. Kllrot, A.-C. Albertsson, J. Am. Chem. Soc. 127 (2005) 8865.
[17] S.F. Luan, J. Zhao, H.W. Yang, H.C. Shi, J. Jin, X.M. Li, J.C. Liu, J.W. Wang, J.H. Yin,
P. Stagnaro, Colloids Surf. B Biointerfaces 93 (2012) 127.
[18] Z.M. Liu, Z.K. Xu, L.S. Wan, J. Wu, M. Ulbricht, J. Membr. Sci. 249 (2005) 21.
[19] H.Y. Yu, Z.K. Xu, Y.J. Xie, Z.M. Liu, S.Y. Wang, J. Membr. Sci. 279 (2006) 148.
[20] U. Edlund, S. Danmark, A.C. Albertsson, Biomacromolecules 9 (2008) 901.
[21] F.J. Xu, Y.L. Li, E.T. Kang, K.G. Neoh, Biomacromolecules 6 (2005) 1759.
[22] Y.-C. Kuo, Y.-T. Tsai, Colloids Surf. B Biointerfaces 82 (2011) 616.
[23] J. Yuan, L. Chen, X.F. Jiang, J. Shen, S.C. Lin, Colloids Surf. B Biointerfaces 39
(2004) 87.
[24] J. Zhao, Q. Shi, L.G. Yin, S.F. Luan, H.C. Shi, L.J. Song, J.H. Yin, P. Stagnaro, Appl.
Surf. Sci. 256 (2010) 7071.
[25] Y.-F. Yang, Y. Li, Q.-L. Li, L.-S. Wan, Z.-K. Xu, J. Membr. Sci. 362 (2010) 255.
[26] D. Li, H. Chen, W.G. McClung, J.L. Brash, Acta Biomater. 5 (2009) 1864.
[27] D. Li, H. Chen, S.S. Wang, Z.Q. Wu, J.L. Brash, Acta Biomater. 7 (2011) 954.
[28] L.P. Zhu, J.H. Jiang, B.K. Zhu, Y.Y. Xu, Colloids Surf. B Biointerfaces 86 (2011)
111.
[29] O. Wiarachai, N. Thongchul, S. Kiatkamjornwong, V.P. Hoven, Colloids Surf. B
Biointerfaces 92 (2012) 121.
[30] Y.F. Wang, Q.F. Hong, Y.J. Chen, X.X. Lian, Y.F. Xiong, Colloids Surf. B Biointerfaces 100 (2012) 77.
[31] M. Kallrot, U. Edlund, A.C. Albertsson, Macromol. Biosci. 8 (2008) 645.
[32] J.G. Alauzun, S. Young, R. DSouza, L. Liu, M.A. Brook, H.D. Sheardown, Biomaterials 31 (2010) 3471.
[33] M. Morra, Biomacromolecules 6 (2005) 1205.
[34] T.W. Chuang, K.S. Masters, Biomaterials 30 (2009) 5341.
[35] H. Lee, S.M. Dellatore, W.M. Miller, P.B. Messersmith, Science 318 (2007) 426.
[36] J.A. Burdick, G.D. Prestwich, Adv. Mater. 23 (2011) H41.