Colloids and Surfaces B: Biointerfaces 112 (2013) 146–154

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Colloids and Surfaces B: Biointerfaces

journal homepage: www.elsevier.com/locate/colsurfb

Surface functionalization of styrenic block copolymer elastomeric

biomaterials with hyaluronic acid via a “grafting to” strategy
Xiaomeng Li a,b , Shifang Luan a,∗ , Shuaishuai Yuan a,b , Lingjie Song a,b , Jie Zhao a,b ,
Jiao Ma a,b , Hengchong Shi a , Huawei Yang a , Jing Jin a , Jinghua Yin a,∗
a
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 a b s t r a c t

Article history: As a biostable elastomer, the hydrophobicity of styrenic block copolymer (SBC) intensely limits its
Received 13 May 2013 biomedical applications. In order to overcome such shortcoming, the SBC films were grafted with
Received in revised form 2 July 2013 hyaluronic acid (HA) using a coupling agent. The surface chemistry of the modified films was exam-
Accepted 26 July 2013
ined by ATR-FTIR and XPS techniques, and the surface morphology was visually described by AFM. The
Available online 3 August 2013
biological performances of the HA-modified films were evaluated by a series of experiments, such as
protein adsorption, platelet adhesion, and in vitro cytocompatibility. It was found that the HA-modified
Keywords:
samples showed a low adhesiveness to fibroblast at the initial stage; however, it stimulated the growth
Styrenic block copolymer (SBC)
Hyaluronic acid (HA)
of fibroblast. The L929 fibroblast growth presented a strong dependence on the molecular weight (MW)
Surface functionalization of HA. The samples modified with 17 kDa HA exhibited the worst wettability and platelet adhesion, while
Hemocompatibility providing the best results of supporting fibroblast proliferation.
Cytocompatibility © 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 poly-
olefine soft segment. The high glass transition temperature (Tg )
styrenic regions as physical crosslinking points provide strength for
this elastomer, while the low Tg olefinic regions contribute flexibil-
ity 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 vul-
canized 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 [1–4],
such as disposable medical devices, microchips [5], urinary tract [6],
glaucoma shunt [7], artificial heart valve [8] and stent drug delivery
coating [9,10].
For some specific biomedical applications, SBC bioelastomer
should be further subjected to surface modification. A desired
surface can be tailored by immobilizing various biocompati-
ble substances [11,12], such as poly(ethylene glycol) (PEG) and
its derivatives [13–15], poly(vinylpyrrolidone) (PNVP) [16–19],
heparin [20–22], zwitterionic materials [23–25], peptides [26,27],
∗ Corresponding authors. Tel.: +86 431 85262109; fax: +86 431 85262109.
E-mail addresses: sfluan@ciac.jl.cn, luanshifang@163.com (S. Luan),
yinjh@ciac.jl.cn (J. Yin).
proteins [28], chitosan [29,30] and hyaluronic acid (HA) [31–35].
As reviewed by Burdick and Prestwich [36], HA (Fig. S1(b))
is an attractive starting material in the construction of hydro-
gels for various tissue engineering applications. A huge number
of HA-based hydrogels with desired morphology, stiffness and
bioactivity have been tailored by two main strategies: addition
or condensation reactions [37], and free radical polymerization
reactions [38]. Jia et al. [39,40] have pioneered techniques for
the production of HA-based hydrogel particles (HGPs) of micron
to submicron dimensions via inverse emulsion polymerization
processes. The presence of nanoscale pores within the HGPs
makes them ideal candidates as release depots for protein-based,
biomacromolecular drugs. Up to now, just a few articles have
reported that HA could be used as biocompatible surface modi-
fier for a polymeric substrate, but they have aroused great interest
[32–35]. For instance, Lee’s group [35] reported a novel mussel-
inspired method for the homogeneous and robust coating of
HA onto various substrates via Michael addition or Schiff base
reactions between HA and the surface-adherent polydopamine
layer.
In this work, HA was used to modify a SBC film using a coupling
agent. The surface densities of bioactive HA were quantified, and
the surface morphology was visually described by AFM. The biolog-
ical interactions of the substrates modified with different molecular
weight HAs were evaluated by a series of experiments, such as pro-
tein adsorption, platelet adhesion, and in vitro response of L929
fibroblast.

0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.colsurfb.2013.07.048
 X. Li et al. / Colloids and Surfaces B: Biointerfaces 112 (2013) 146–154
2. Experimental
2.1. Materials and reagents
Poly(styrene-b-(ethylene-co-butylene)-b-styrene) copolymer
(Kraton G 1652) was purchased from Shell Chemicals. Diethyle-
netriamine (DETA) was provided by Shanghai Aladdin Chemicals.
HA (Molecular weight (MW) = 17, 47 and 310 kDa, MW was
tested by light scattering) was purchased from Liuzhou Chem-
icals. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)
hydrochloride and N-hydroxysuccinimide (NHS) were provided
by Alfa Aesar. Phosphate-buffered saline (PBS, pH 7.4), bovine
serum albumin (BSA), bovine serum fibrinogen (BFg), MES buffer
(2-(N-morpholino) ethanesulfonic acid, pH 5.4) and sodium
dodecyl sulfate (SDS) were provided by Dingguo Biotechnology.
Micro BCATM protein assay reagent kit (AR1110) was provided by
Boster Biotechnology. Ninhydrin was provided by Shanghai Huishi
Biochemical Reagent. Human hyaluronic acid binding protein
(HABP) ELISA Kit was supplied by Shanghai kanu biotechnology.
Dulbecco’s modified Eagle’s medium (DMEM) and 0.25 wt.% trypsin
were purchased from Beijing Solarbio Science & Technology. Sterile
filtered fetal bovine serum (FBS) was supplied by Beijing Yuan-
hengjinma Biotechnology. Fluorescein Isothiocyanate Labeled
Phalloidin (FITC-Phalloidin) and 4 ,6-diamidino-2-phenylindole
(DAPI) dihydrochloride were provided by Sigma. Other reagents
were AR grade and used without further purification.
2.2. Surface functionalization of SBC elastomer
The HA-functionalized substrate was prepared as follows
(Fig. 1). The SBC films (1.5 cm × 1.5 cm for each film) were washed
with deionized water and ethanol, and dried for use. Then they
were subjected to an approximate 10 Pa oxygen atmosphere in a
DT-03 plasma apparatus (Suzhou Omega Technology, China) with
a plasma power of 30 W for 30 s. The pre-treated SBC films were
soaked in an acetonitrile solution containing DETA (0.04, 0.08, 0.12,
0.16 and 0.20 mol/L) at 75 ◦ C for 16 h. The films were respectively
washed with deionized water and ethanol, and finally dried under
vacuum for 24 h to obtain the DETA-modified film (denoted as SBC-
DETA). 1 mL solution of HA in 0.05 mol/L MES buffer (0.5, 1.0, 5.0 and
1.0 mg/mL) was prepared, and followed by adding 1 mL 0.1 mol/L
EDC and 1 mL 0.05 mol/L NHS solution for each film. The DETA-
modified films were soaked in the as-prepared HA solution at room
temperature for 16 h for the HA immobilization, then rinsed with
deionized water, and dried in a vacuum oven. The samples modi-
fied by HA with a MW of 17 kDa, 47 kDa and 310 kDa were denoted
as HA17, HA47, and HA310.
2.3. Surface chemistry characterization of the SBC films
ATR-FTIR spectra were obtained from a Fourier transform
infrared spectrometer (FTIR, BRUKER Vertex 70). A total of 32
scans were accumulated with a resolution of 4 cm−1 for each spec-
trum. Surface elemental compositions were examined by an X-ray
photoelectron spectroscopy (XPS, VG Scientific ESCA MK II Thermo
Avantage V 3.20 analyzer) with Al/K (h = 1486.6 eV) anode mono-
X-ray source at the detection angle of 90◦ . Scans between 1200 and
0 eV were performed.
2.4. Surface morphology of the SBC films
Surface morphology was examined in an atomic force
microscopy (AFM) with contact mode (SPA300HV with a
SPI 3800 controller, Seiko Instruments Industry, Japan). The
root-mean-square (RMS) roughness was evaluated directly from
147
AFM images. The reported data were the mean values of triplicate
specimens.
2.5. Surface amine density of the SBC substrate
Surface amine density was tested via a ninhydrin method.
Firstly, the films (1.5 cm × 1.5 cm) were placed in 3 mL test tubes
with 0.5 mL 0.1 mol/L sodium citrate (pH 5) and 1 mL ninhydrin
reagent, and heated to 100 ◦ C for 15 min. Finally, the solution
was cooled to room temperature and the colorimetric response
was measured at 490 nm by a microplate reader (TECAN SUN-
RISE, Swiss). A standard curve was prepared from a series of DETA
standard solutions. The reported data were the mean values of
triplicate specimens.
2.6. Surface HA density of the SBC substrate
Surface HA density was measured by human hyaluronic acid
binding protein (HABP) ELISA kit. Briefly, the HA-grafted films
reacted with 50 ␮L hyaluronic acid binding protein conjugated with
horseradish peroxidase (HRP) at 37 ◦ C for 30 min to create HA com-
plexes on the substrate. After completely rinsing with PBS solution,
50 ␮L HRP-conjugate reagent was added, and incubated at 37 ◦ C for
30 min, and followed by adding 50 ␮L chromogen solution A and
50 ␮L chromogen solution B, finally the reaction was terminated
by adding 50 ␮L stop solution. The absorbance values of the solu-
tions were tested by a microplate reader (TECAN SUNRISE, Swiss)
at a wavelength of 450 nm. A standard curve was calculated from a
series of HA standard solutions. Each result is an average of at least
five parallel experiments.
2.7. Wettability
Water contact angle (WCA) of the SBC films was measured with
a drop shape analysis instrument (DSA, KRÜSS GMBH, Germany) at
room temperature. Each result is an average of at least five parallel
experiments.
2.8. Protein adsorption
After soaking in PBS solution at room temperature for 12 h, the
SBC films were dipped into PBS solution containing BSA (0.1, 0.5
and 1.0 mg/mL) or BFg (0.1, 0.5 and 1.0 mg/mL) at 37 ◦ C for 2 h.
Each film was sequentially rinsed five times with fresh PBS, soaked
in an aqueous solution of 1.0 wt.% SDS, and oscillated at 37 ◦ C for
1.0 h to remove the adsorbed proteins from the substrate. Based on
bicinchoninic acid (BCA) protein assay kit, the absorbance values
of the SDS solution containing proteins was measured at 570 nm
with a microplate reader (TECAN SUNRISE, Swiss), and amount of
the adsorbed proteins was calculated. Each result is an average of
at least three parallel experiments.
2.9. Platelet adhesion
The SBC films were soaked in PBS solution at room tempera-
ture for 2 h. Fresh platelet-rich plasma (RRP) was obtained from a
healthy rabbit by centrifugation at 800 rpm for 10 min. Then 20 ␮L
RRP was dropped on the films and incubated at 37 ◦ C for 1 h. After
washing three times with PBS solution, the platelets adhered on the
films were fixed by 2.5 wt.% glutaraldehyde at 4 ◦ C for at least 10 h.
Finally, the films were washed three times with PBS, and sequen-
tially dehydrated with a series of ethanol aqueous solution (10, 30,
50, 70, 90, 100 vol%). The samples were observed with field emitted
scanning electron microscopy (SEM, XL 30 ESEM FEG, FEI Company,
USA). The number of platelet adhesion on the SBC substrates was
148 X. Li et al. / Colloids and Surfaces B: Biointerfaces 112 (2013) 146–154
Fig. 1. The preparation procedure of the HA-functionalized surface.
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 fibroblast 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 N-
2-Hydroxyethylpiperazine-N -2-ethanesulfonic acid (HEPES) and
100 ␮g/mL streptomycin and maintained in a humidified 5%
CO2 /95% air incubator at 37 ◦ C.
The SBC films 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 films were then soaked in cell culture medium
and kept in cell culture incubator. After reaching 95% confluency,
cells were detached by 0.25 wt.% trypsin and then suspended in cul-
ture 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 viabil-
ity and cell numbers were measured after cultured for 1, 2 and 4
days. The cell culture medium was removed and followed by exten-
sive rinsing in PBS, and the cell adhered on the films were fixed
by 4.0 wt.% paraformaldehyde at 4 ◦ C for 30 min to obtained the
cell-immobilized samples. For observing the fluorescence images of
cells in different stages, the cell-immobilized samples were stained
via adding 0.2 mL fluorescence-staining solution at room temper-
ature 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 fluorescence 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 spread-
ing area and cell density by using Image J software. Cell density and
cell surface area on the SBC substrates were analyzed using a one-
way 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 modifiers are based
on PEG and its derivatives [41–45]. We have found that the intro-
duction of PEG could impart good hemocompatibity to a SBC
elastomeric substrate [46,47]; however, these modifiers simulta-
neously suppressed cell adhesion and proliferation. The covalent
attachment of acrylated HA to this substrate via a photograft-
ing polymerization could obtain both hemocompatiblity and
cytocompatiblity [48]. In view of the typical characteristics of
photo-initiated free radical polymerization and the inhomoge-
neous distribution of photoinitiator physically adsorbed on the
surface, a large polydispersity index and inhomogeneous distri-
bution of the grafted chains were inevitable. The morphology
of the modified substrate was certainly heterogeneous on the
micro- and macro-scales. This rendered the dependence of bio-
logical response on the MW of the immobilized HA ambiguous.
In this study, HA with different MW were respectively immobi-
lized 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-modified
systems.
3.1. Surface chemistry
3.1.1. ATR-FTIR
As ATR-FTIR spectra shown in Fig. 2a, two new characteris-
tic peaks at 3387 cm−1 (N H stretching) and 1265 cm−1 (C N
stretching) appeared after surface modification of the SBC sub-
strate with diethylenetriamine. As for the HA-modified substrates,
the ATR-FTIR spectra further changed. Specifically, characteristic
peaks at 3387 cm−1 (N H stretching) and 1265 cm−1 (C N stretch-
ing) strengthened, and new peaks at 1665 cm−1 (C O stretching),
1099 cm−1 (C OH stretching) and 812 cm−1 (C O C stretching)
appeared.
3.1.2. XPS
Surface composition of the SBC films was detected by XPS
(Fig. 2b). After sequential surface modification 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 spec-
tra of the virgin SBC film were divided into two peaks using a
Gaussian peak fitting algorithm (Fig. S2(a)). After covalent immo-
bilization with DETA, a new peak at 285.6 eV attributing to C N
group appeared (Fig. S2(b)). As for the HA-modified film, the high-
resolution C1s spectra were decomposed into five 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,
 X. Li et al. / Colloids and Surfaces B: Biointerfaces 112 (2013) 146–154
Fig. 2. ATR-FTIR (a), and XPS (b) spectra of the SBC films. (I) Virgin SBC, (II) SBC-DETA, (III) HA17, (IV) HA47, and (V) HA310.
respectively (Fig. S2(c)–(e)). The composition ratios of N C O were
larger than that of O C O, suggesting that activated by EDC/NHS
partner, carboxyl groups in HA chains reacted with amine groups
on the substrate (Table S2).
3.1.3. Surface amine density of the SBC films
As quantified using a ninhydrin assay, the surface amine den-
sity gradually rose with the increment of DETA concentration, and
reached a maximum of ∼60 ng/cm2 at a DETA concentration of
0.20 mol/L (Fig. 3a). After the HA immobilization (10 mg/mL HA
(17 kDa) in the loading solution), the exposed amines on the sub-
strates decreased, confirming that DETA participated in the HA
grafting reactions. It should be noted that the secondary amines,
which already existed in HA chains and newly created by the link-
ing reaction between HA and the grafted DETA, also reacted with
the ninhydrin reagent, so amines were still detected for the HA-
modified films.
149
3.1.4. Surface HA density of the SBC substrate
Surface HA density was determined by a quantitative assay
based on the binding of labeled HA-binding protein (HABP) to the
grafted HA [49]. As shown in Fig. 3b, the amount of the grafted
HA generally increased as both HA concentration and MW rose.
A maximum density of the grafted HA reached ∼53 ng/cm2 at a
concentration of 10.0 mg/mL HA with a 310 kDa MW. Using the
solutions of 10.0 mg/mL HA with a 17 kDa MW, 5.0 mg/mL HA with
a 47 kDa MW, and 1.0 mg/mL HA with a 310 kDa MW, a similar HA
density of ∼40 ng/cm2 could be tailored and used in the subsequent
experiments.
For the HA binding and recognition, HABP required HA having a
continuous segments of ∼1200–2000 Da, that is, a 3–5 disaccharide
repeating units, so the grafted amount of HA was underestimated
by this assay kit [33]. This underestimation was supported by the
ATR-FTIR and XPS results. Similarly, the biological functions of HA
to other proteins also required a minimum HA repeating sequence,
and the biological interaction between HA and cells was expressed
150 X. Li et al. / Colloids and Surfaces B: Biointerfaces 112 (2013) 146–154
Fig. 3. Surface density of amine groups (a) (n = 3) and HA (b) (n = 5) on the SBC films.
through cell surface proteins [50]. This HA measurement appeared
inappropriate, but accurately presented the amount of bioactive HA
which had biological response to protein and cell.
3.2. Surface morphology
Chemical and physical signals from a substrate, such as sur-
face energy, topography, electrostatic charge and wettability, play
a key role in stimulating cell adhesion and influencing cell growth
behavior. As indicated by AFM three-dimensional images, there
were several big holes and a few large granules on the SBC con-
trols (Fig. S3(a)), and the RMS value was ∼27 nm (Fig. S3(e)). Once
HA was grafted to the substrate, the RMS value could increase up
to ∼65 nm. The constrained mobility stemming from the solid sub-
strate strongly prevented the short chains of the low MW HA from
spreading. The high MW HA chains had a larger steric hindrance,
but the long HA chains had good mobility, and their grafting yields
was much larger, so fully spread out on the substrates as a homo-
geneous and continuous HA layer. Thus, the RMS value had an
inverse correlation to the MW of the grafted HA, i.e., ∼65 nm for
HA17, ∼59 nm for HA47 and ∼39 nm for HA310, respectively (Fig.
S3(b)–(e)).
Fig. 4. Amount of protein adsorption on the SBC surfaces. (a) BSA, and (b) BFg. (The
error bars: standard deviations, n = 3.)
3.3. Biocompatibility of the SBC films
Protein adsorption is the first event in blood–material inter-
actions 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 prop-
erties, 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). Corre-
spondingly, 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 throm-
bosis would be initiated by platelets adhesion and activation and
breakage of platelets. In contrast, as for the HA-modified sam-
ples (a WCA of ∼62–69◦ ) (Table S3), HA hydration layer prevented
the hydrophobic interaction between protein and substrate, so
the amount of BSA and BFg adsorbed on the HA-modified sub-
strates 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-modified substrates had a
slight variation, the adsorbed amount of BSA was generally reduced
by ∼20–50%, 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
 X. Li et al. / Colloids and Surfaces B: Biointerfaces 112 (2013) 146–154
Fig. 5. Adhesion of platelets on the SBC films. (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.)
receptor, and formed bridges between platelets, resulting in
platelet activation and aggregation, while BSA was generally non-
adhesive for platelet. In addition, the ratio of fibrinogen to albumin
adsorbed on the substrate decreased, suggesting that the HA-
immobilized surfaces has good hemocompatibility [51].In the case
of platelet adhesion, the hydrophilic HA chains quickly hydrated
when contacting with platelet-rich plasma, which weakened the
interaction force between platelets and the substrate because
of the steric hindrance effect, bringing about the less adhered
platelets (Fig. 5c–e). The values of the adhered platelets were
∼1.5 × 104 cells/mm2 (HA17 and HA47) and 0.9 × 104 cells/mm2
(HA310) relative to ∼2.5 × 104 cells/mm2 of SBC controls (Fig. 5f).
The difference between the SBC controls and the HA-modified
groups was statistically significant, i.e., p < 0.05 for HA17 and HA47,
p < 0.01 for HA310 (Fig. 5f). In addition, HA17 and HA47 groups
had statistically significant differences in comparison with HA310
group. As for the SBC controls, the ratio of activated platelets was
151
∼71% (Table S4), while that of the HA-modified surfaces was respec-
tively reduced to 54%, 52% and 38%, confirming 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-modified films 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-modified films
significantly promoted cell proliferation, especially for HA17, as
compared with the SBC controls. As for the HA17 films, 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 fibro-
blast proliferation. With respect to the ∼10–20 ␮m size of cells, the
152 X. Li et al. / Colloids and Surfaces B: Biointerfaces 112 (2013) 146–154

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.)
 X. Li et al. / Colloids and Surfaces B: Biointerfaces 112 (2013) 146–154
Fig. 8. Cell density (a) and cell surface area (b) on the SBC substrates for culturing
1, 2 and 4 days. (The error bars: standard deviations, n = 3; data analyzed using a
one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001.)
HA layer on the SBC surface could be thought as homogeneous and
continuous. The HA17 and HA47 samples had the similar rough-
ness, while the fibroblast growth of HA17 samples was statistically
significant better than that of HA47 samples. Therefore, it could be
concluded that the L929 fibroblast growth had a strong dependence
on the MW of the HA immobilized on the SBC substrate, similar to
that of the HA in other forms [53].
Fine topographies of L929 cells were obtained by SEM (Fig. 7),
and the cell spreading area as well as morphology were summa-
rized in Fig. 8b and Table S5, respectively. After 1 day culturing, the
SBC controls were mostly covered with round-shaped cells, while
the HA-modified substrates were mainly adhered by the spreading
cells, i.e., spindle-shaped, triangle-shaped and stellate-shaped cells
(Fig. 7A, Table S5). The cell-spreading area of HA17 was larger than
those of the SBC controls, HA47 and HA310 groups, and it had sta-
tistically significant differences as compared with the SBC controls
and HA310 group (Fig. 8b). As culture time increased, cells rapidly
proliferated and fully spread out on the HA-modified substrates
(Fig. 7). Comparing with the SBC controls, the HA-modified films
had more spreading cells (Table S5), as well as larger cell-spreading
area (Fig. 8b), especially for HA17. It should be noticed that flu-
orescent photographs (Fig. 6) and SEM images (Fig. 7) presented
obvious differences as for cell morphology, particularly for the HA-
modified samples at initial stage of cell culturing. It was probably
due to that the cells were not tightly bound to the HA-modified
substrates, so the treatment for SEM observation resulted in the
153
de-adhesion and recovery of these cells. Thus, the cell-spreading
area of the HA-modified samples was actually much larger than
that analyzed from SEM images. The testing results of cell adhe-
sion, proliferation, morphology and spreading area suggested that
HA imparted good cytocompatibility to the SBC substrates.
As we know, a single molecule chain of HA could bind to multiple
cell surface receptors, the HA binding domains to different cell sur-
face proteins were different, and new cell surface recognizing HA
proteins was continuously discovered, so the biological response
of HA to cell was still incompletely elucidated even for the HA in
aqueous solution. The constrained mobility of the grafted HA made
the interaction between HA and cell more complex.
4. Conclusions
The SBC elastomeric biomaterials modified with hyaluronic acid
(HA) significantly inhibited protein adsorption and platelet adhe-
sion relative to the SBC controls. The HA-modified samples had
a low adhesiveness to fibroblast at the initial stage; however, it
stimulated the growth of fibroblast. The L929 fibroblast growth
showed a strong dependence on the MW of HA. The samples
modified with the 17 kDa HA presented the worst wettability and
platelet adhesion, while producing the best results of supporting
fibroblast proliferation. Although the complex interaction between
the immobilized HA and cell remained to be incompletely eluci-
dated, the HA modification imparted excellent hemocompatiblity
and cytocompatibility to the SBC biomaterials.
Acknowledgements
The authors acknowledge the financial support of National
Science Foundation of China (Project number: 21274150 and
51273200), Chinese Academy of Sciences-Wego Group High-tech
Research & Development Program (2011–2013), and Scien-
tific Development Program of Jilin Province (Project number:
20130102064JC).
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2013.
07.048.
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