J. Biomedical Science and Engineering, 2010, 3, 1146-1155
doi:10.4236/jbise.2010.312149 Published Online December 2010 (http://www.SciRP.org/journal/jbise/ JBiSE
Published Online December 2010 in SciRes. http://www.scirp.org/journal/JBiSE
Silk fibroins modify the atmospheric low
temperature plasma-treated poly
film for the application of cardiovascular tissue engineering
Huaxiao Yang1, Min Sun1, Ping Zhou1, Luanfeng Pan2, Chungen Wu2
1The Key Laboratory of Molecular Engineering of Polymers Ministry of Education, Department of Macromolecular Science, Fudan
University Shanghai, China;
2 Laboratory of Molecular Biology Shanghai Medical College, Fudan University Shanghai, China.
Email: pingzhou@fudan.edu.cn; lfpan@shum.edu.cn
Received 22 Octorber 2010; revised 26 Octorber 2010; accepted 29 Octorber 2010.
Tissue engineered scaffold is one of the hopeful ther-
apies for the patients with organ or tissue damages.
The key element for a tissue engineered scaffold ma-
terial is high biocompatibility. Herein the poly (3-
hydroxybutyrate-co-3-hyd roxyhexanoate) (PHBHHx)
film was irradiated by the low temperature atmos-
pheric plasma and then coated by the silk fibroins
(SF). After plasma treatment, the surface of PHBHHx
film became rougher and more hydrophilic than that
of original film. The experiment of PHBHHx flushed
by phosphate buffer solution (PBS) proves that the
coated SF shows stronger immobilization on the
plasma-treated film than that on the untreated film.
The cell viability assay demonstrates that SF-coated
PHBHHx films treated by the plasma significantly
supports the proliferation and growth of the human
smooth muscle cells (HSMCs). Furthermore, the
scanning electron microscopy and hemotoylin and
eosin (HE) staining show that HSMCs formed a cell
sub-monolayer and secreted a large amount of ex-
tracellular matrix (ECM) on the films after one
week’s culture. The silk fibroins modify the plasma-
treated PHBHHx film, providing a material poten-
tially applicable in the cardiovascular tissue engi-
Keywords: Biocompatible; Cardiovascular Tissue
Engineering; Low Temperature Plasma;
(PHBHHx); Silk Fibroin
Tissue engineering (TE) [1] involves many issues in-
cluding scaffold design [2-4], cell culture and different-
tiation [5], bio-interfacial interaction between cell and
material [6-8], and tissue regenerative induction [9].
Tissue engineering opens a hopeful door for the patients
who need organs or tissues repaired, replaced, or regen-
erated [10]. Apparently, one of the most significant is-
sues of TE is the scaffold design. A sound scaffold not
only supports the cell adhering and proliferating at the
early stage in vitro [8] but also maintains the primary
functions as an artificial organ in vivo [11]. In the past
decade, the bio-synthetic biodegradable polyesters, po-
lyhydroxyalkanoates (PHAs) [2,12,13], have attracted
many attentions and were widely investigated for the
applications in the biomedical engineering, such as the
scaffold of poly(3-hydroxybutyrate-co-3-hydroxyhexa-
noate) (PHBHHx)/hydroxyapatite for bone reconstruct-
tion [14], the biocompatibility of PHBHHx for bone
marrow stromal cells in vitro [15], the microspheres of
PHBHHx for the drug controlled release [16,17], the
nanofibrous matrix of PHA for the better cell grow th [4]
and so on, because the PHAs have adjustable mechanical
properties and biodegrading rates by varying the content
x of hydroxyhexanoate (HH) in PHBHHx [2,12,18,19].
However, like most of polyesters, PHBHHx is also
fair hydrophobic and not so proper for the cell adhesion
and growth, and has no active functional groups for the
attachment of biomolecules [12,20]. As the results, some
methods have been applied to improve the PHBHHx
surface hydrophilicity and biocompatibility, for instance,
the maleic anhydride was grafted onto the PHBHHx to
form the maleated PHBHHx, which showed the im-
proved biocompatibility, suitable mechanical properties
as well as the accelerated biodegradation [21]. In addi-
tion, significant increase in the growth of the fibroblasts
H. X. Yang et al. / J. Biomedical Science and Engineering 3 (2010) 1146-1155 1147
L929 was also observed on the films prepared by the
ultraviolet-radiated PHBHHx powders [22]. Our group
modified the PHBHHx films and porous scaffolds by
coating silk fibroin (SF), demonstrating that the SF-
coating highly improved the hydrophilicity of the scaf-
fold and proliferations of cardiovascular related cells
seeded on them [8,23,24].
Low temperature plasma (LTP) is an effective method
for modifying the material surface properties including
wettability, topography and surface charge states. It can
penetrate the material in depth of 10-100 Å without da-
maging the bulk material [25-28], therefore LTP exhibits
the comprehensive applications in the surface modifica-
tion of polymeric scaffolds [25,29,30]. In addition, LTP
is also used to introduce some specific elements or func-
tional groups onto the surface of polymer, such as pro-
tein [27], peptide [31], and polysaccharide [32]. Among
the plasma techniques, dielectric barrier discharge (DBD)
presents the advantages as compared to the conventional
techniques which use electron, ion and photon beams to
bomb the surface under vacuum condition. Using the
DBD technique, the discharge can be operated at normal
atmospheric pressure with various gases, e.g. corrosive
gases, dry and humid gas [33]. More importantly, many
functional groups can be intro- duced and grafted onto
the surface after plasma treat- ment by altering working
gas [34], for example, -COOH group can be grafted by
CO2 plasma, -NH2 or -NH- group can be grafted by NH3
plasma, -OH and -CO- groups can be grafted by wa-
ter/O2 or air plasma. These grafted functional groups can
improve the hydrophilicity of the material surface [30].
In present work, to have the SF interaction with the
PHBHHx surface stronger and improve the biocompati-
bility of the PHBHHx film, the surface of PHBHHx
films was irradiated by DBD plasma at atmospheric
pressure with argon as working gas, and then coated by
SF. The optimal plasma irradiation time was evaluated
by the water contact angle. The experiment of PHBHHx
flushed by phosphate buffer solution (PBS) under the
rate of physiologic blood flow was designed to test the
strength of SF immobilization on the surface of plasma-
treated PHBHH x films. The p roliferation and morpholo-
gies of the human smooth muscle cells (HSMCs) cul tu r ed
on the films were also investigated.
2.1. Materials
PHBHHx powder (Mw = 100,000, x = 12 mol% for the
HH content) was kindly donated by Prof. Guo-Qiang
Chen in Tsinghua University, China. The PHBHHx was
purified by dissolving it in the dichloromethane (CH2Cl2)
solution, and fu lly refluxed at 40 for half an hour, and
then filtered through a piece of qualitative filter paper,
and re-precipitated in n-hexane solution. The resultant
solid sample was dried at room temperature for more
than 24 hours, and stored in the desiccator for later use.
About 0.6 g purified PHBHHx powder was dissolved in
10 mL CH2Cl2, and then th e solution was cast on a glass
plate in diameter of 60 mm in fume hood at room tem-
perature for 24 hours. A film in thickness of 97 ± 1 μm
was obtained.
The regenerated silk fibroin (SF) solution was pre-
pared following the report of Mei et al [8]. In brief, raw
Bombyx mori silk was degummed twice with 0.5 wt%
NaCO3 solutions at 100 for 1 hour and then washed
with deionized water. The degummed silk was dissolved
in 9.3 mol/L LiBr solution at room temperature. After
dialysis against the deionized water for more than three
days to remove LiBr salt, the solution was filtered to
remove the impurities. The regenerated silk fibroin so lu-
tion with concentration of ~ 2% (w/v) was obtained and
further diluted to the concentration of 1% (w/v).
For easily and quantitatively probing the silk fibroin
content in the dilute so lution, the silk fibroin was labeled
by fluorescein isothiocyanate (FITC) which can be de-
tected by the fluorescent spectrometer. The FITC-labeled
SF solution was prepared as follows: 20 mL 1% (w/v)
SF solution was adjusted to pH = 7.0 by 0.01 mol/L
NaOH solution, then 0.1 mg FITC powder (purity of
90%, J&K Chemical, Sweden) was added into the solu-
tion. The solution was stirred at 20 for 2 hours, and
then stored at 4 for overnight, and then dialyzed
against deionized water for four days. The water was
refreshed every 3 or 4 hours to remove excessive FITC
in the solution. The whole process was operated in a d im
room. The final concentration of FITC-labeled SF solu-
tion was about 0.94% (w/v) and the solution was stored
in the refrigerator at 4 for later use.
2.2. Surface Modification of PHBHHx Film
2.2.1. Atm ospheric Plasma Treatment
The plasma treatment was carried out by a low tempera-
ture atmospheric plasma generator (CTP-2000K, Nan-
jing Shuman Ltd. Company, China) shown in Figure 1.
The quartz DBD generator was operated at a frequency
of 20 MHz. The sample was irradiated by the plasma
generated between two plate electrodes with diameter of
4 cm and distance of 2 cm (shown in Figure 1). The
PHBHHx film in diameter of 1.5 cm was put on the
ground electrode in the quartz chamber. The argon gas
(purity of 99.9%) was conducted into a chamber which
was connected to the air and the plasma was generated
by the power of 50 W at atmospheric pressure. The
plasma irradiating time was changed from 1 to 10 min-
Copyright © 2010 SciRes. JBiSE
H. X. Yang et al. / J. Biomedical Science and Engineering 3 (2010) 1146-1155
Figure 1. Schematic graph of low temperature atmospheric
plasma generating device.©
utes to determine an optimal irradiating time which was
evaluated by the water contact angle of the film. The
smaller water contact angle indicates the better wettabil-
ity of the PHBHHx films.
2.2.2. Morphology measure m e nt by SEM
The morphologies of pristine and plasma treated
PHBHHx films were measured by the scanning electron
microscopy (SEM). Briefly, sample was sputter-coated
by an ultrathin gold layer for 60 seconds with current of
10 mA, and then observed by SEM (TS 5136MM, Tes-
can vega, Czech) with an accelerating voltage of 20 kV.
2.2.3. Measurement of Water Contac t Angle
The water contact angle was measured by OCA 15 plus
equipment (Data Physics, Germany) packaged with
SCA-20 software. A drop of 1.5 μL of distilled water
was dropped onto the sample surface and its contact an-
gle (θ) was measured within 10 seconds. The contact
angle was mean of the ten values.
2.2.4. Silk Fibroins Immobilization
The PHBHHx films were immediately immersed into
1% (w/v) SF solution fo r 30 min after plasma treatment,
and then freeze-dried overnight under vacuum of 0.1 torr
at -50. At the same time, PHBHHx film without plas-
ma treatment was also immersed into 1% (w/v) SF solu-
tion for 30 min and freeze-dried as control.
2.2.5. Test of the Strength of SF Immobilization on
the Plasma Treated PHBHHx Film
The strength of SF immobilization on the plasma treated
PHBHHx film was tested by flushing the sample with
PBS solution. In this experiment, both of the SF-coated
PHBHHx films with and without plasma treatment were
flushed by recycled 90 mL PBS buffer (pH = 7.4) for 30
min at the flow rate of 55 mL/min which mimicked the
physiologic blood fluid in vivo. During flushing, 2 mL
flushed solution was sampled every 10 min for the fluo-
rescent measurement. The content of the flushed SF in
the PBS solution was measured by the FITC fluorescent
at 520 nm (FLS 920, Edinburgh Instrument, Switzerland)
where FITC was labeled on the SF.
2.3. Human Smooth Muscle Cell (HSMCs)
Isolation and Cells Culture in Vitro
HSMCs were isolated from the human umbilical artery
obtained from Shanghai No.1 Hospital for the Protection
of Mother and Baby’s Health and puerperal informed
consent, and the method of isolation was referred to the
literature [24]. In brief, a 25 cm length of human um-
bilical vein was cleaned with the sterile gauze to remove
blood contaminations, and then the vascular tunica me-
dia layer was prudently separated from the vein. The
layer was thoroughly washed with serum free Dul-
becco’s modified eagle medium (DMEM, Gibco, USA)
to wipe off the portion of connective tissue, and then cut
into ten pieces of tissues in 1 mm3. Those pieces of tis-
sues were put on the botto m of culture flask (po lystyrene ,
Corning, USA) in a proper distance between each other
and cultured in an incubator with saturated humid air
and 5% CO2 for 12 hours at 37 to allow the cells ad-
hering and migrating in the flask. After most of tissues
firmly attached on the bottom of flask, the culture me-
dium (DMEM with 10% (v/v) fetal bovine serum (FBS,
Hyclone, USA), 100 U streptomycin and 100 μg/ml pe-
nicillin) was added into the flask, just covering over the
tissue pieces for continually culturing the cells in the
incubator. The medium was refreshed every 2 days until
the cells grew into the confluence by 80%. The cells
were gently digested with 0.25% trypsinase–0.01%
EDTA solution and passed into the next passage of cells
for further culture. The cells within 5 to 8 passages were
used in the study.
2.4. Cells Seeded on the Studied Films
HSMCs (passage of 5-8) were harvested by 0.25% tryp-
sinase—0.01% EDTA and diluted to the concentration of
4.0 × 105 cells/mL. The cells were seeded on four types
of PHBHHx films including PHBHHx film (P), plasma
treated PHBHHx film (PP), SF-coated PHBHHx film
(SP), and plasma treated PHBHHx film coated by SF
(SPP). Those films were carefully put into 24-well cul-
ture plates (CosterTM) and sterilized in 75% (v/v) ethanol
solution overnight, and then rinsed with PBS buffer to
remove the ethanol and then irradiated by ultraviolet for
30 minutes for each side of the film. 100 μL 4 × 105
cells/mL suspended cell solution was diluted in 300 μL
culture medium, and dropped onto each film. After the
cells adhered on the films for 30 min, extra 600 μL c ul-
ture medium was supplemented into each well. The cul-
Copyright © 2010 SciRes. JBiSE
H. X. Yang et al. / J. Biomedical Science and Engineering 3 (2010) 1146-1155 1149
ture medium was refreshed every 2 days.
2.5. Proliferation Analysis of HSMCs on the
Studied Films
2.5.1. Mitoch o ndri al Met ab o l i c Activity of HSMCs on
the Studied Films
Mitochondrial metabolic activities of the HSMCs on the
different types of films were evaluated by the MTT assay.
MTT (3-(4, 5)-dimethylthiah iazo (-z-y1)-3, 5-diphen yte-
trazoliumromide (Merk, USA)) can be reduced by the
succinate dehydrogenase when the mitochondrial of liv-
ing cells interact with the MTT and form the purple for-
mazan deposits. MTT assay can quantitatively assess the
metabolic activity of cells when the HSMCs ar e cultured
on the studied films. Briefly, after the culture medium in
the plate well was removed, the cells on the films were
rinsed by serum free DMEM medium three times and
then incubated continu ally with 400 μL DM E M med i u m
and 40 μL MTT solution (5 mg/ml in PBS) for 4 h under
the humid conditio n at 37, and then th e insoluble pur-
ple formazan crystals were formed. The crystal was dis-
solved by 400 μL dimethylsulfoxide (DMSO, Sigma,
USA) and 200 μL of the solution was transformed into a
96-well plate for the optical density (OD) measurement
by a spectrophotometer (ELx800, BioTek, USA) at 565
nm. The 200 μL pure DMSO was used as blank control.
2.5.2. HE Staining of HSMCs on the Studied Films
Hemotoylin and eosin (HE) staining is a traditional, fast
and reliable histological staining method to observe the
cells. In this study, the HSMCs cultured on the studied
films for 3, 5 and 7 days were rinsed with PBS buffer,
and fixed by 4% polyformaldehyde buffer solution (pH
= 7.4) overnight at 4, and then dehydrated by the etha-
nol with gradually increased concentration of 50, 60, 70,
80, 90, 95 and 100% (v/v). The cells were stained with
hemotoylin and eosin for 1 min respectively, and then
gently washed with distilled water, and sealed with liq-
uid paraffin. The cells were observed under the inverted
light microscope (XDS-1B, Chongqing Optical Co.,
China) and recorded by a digital camera (Nikon D60).
2.6. Morphology Observation of HSMCs
Growing on the Studied Films
Morphologies of the HSMCs cultured on the studied
films for 3 and 7 days were observed by SEM. The
HSMCs were fixed by 4% polyformaldehyde buffer so-
lution (pH = 7.4) overnight at 4, subsequently, dehy-
drated by the ethanol with gradually increased concen-
tration of 50, 60, 70, 80, 90, 95 and 100% (v/v), and then
lyophilized overnight under vacuum of 0.1 torr at -50.
Morphologies of the HSMCs cultured on the studied
films were observed by SEM.
2.7. Statistical Analysis
Data were presented as means ± SD (standard deviation).
Three values were measured for every sample. Statistical
comparisons were performed using ANOVA one-way
method (OriginTM). P < 0.05 was considered statistically
significant differenc e betwee n two data gr oups.
3.1. Influence of Atmospheric Plasma Treatment
on the Films
In this study, the PHBHHx films were irradiated by low
temperature atmospheric argon/air plasma for time of 1,
2, 4, 6, 8 and 10 min. Plasma irradiation can break down
the chemical bonds of the material to form free radicals,
leading to the chemical composition rearranging and the
surface roughening of polymeric matrix [33,35]. After
the plasma treatment, the surface morphology of
PHBHHx film became rougher than that of pristine film
shown in Figure 2. The similar rough ‘zones’ were also
observed when the PHBHHx film was irradiated by the
ion implantation with fluence of 1×1015 ions/cm2 [20].
The hydrophilicity of the treated material surface was
characterized by the water contact angles shown in Fig-
ure 3. The results indicates that the water contact angles
decreased from 90.3° to 70.4° when the films were irra-
diated by plasma from 1 min to 6 min, and reached a
constant valu e around 72° when the irradiation time was
longer. The decreases in the water contact angle are con-
tributed to the plenty of hydrophilic groups, such as hy-
droxyl and carboxyl groups [25] formed on the surface
of PHBHHx film after plasma treatment. The constant
value of water contact angle is due to the hydrophilic
functional groups saturated on the surfaces of films [27].
Considering the efficient treatment and no destruction
for the bulk polymer, 6 min of the plasma irradiation
time was selected as an optimum treating time in our
experiment. The following reports will no longer men-
tion this treating time any more.
3.2. Strength of SF Immobilization on the
Plasma Treated PHBHHx Films
The SF-coated PHBHHx films that with and without
plasma treatment were flushed by PBS buffer for 30 min,
and the FITC-labeled SF in the eluate was detected by the
FITC fluorescence. The results are shown in Figure 4.
The fluorescence increases as the flushing time increases
for the SF-coated PHBHHx film without plasma treat-
ment, indicating that the SF are easily desorbed from the
SF-coated PHBHHx film, whilst very low fluorescence
intensity and nearly no significant fluorescence change are
found for the SF-coated PHBHHx film with plasma
treatment, indicating that the SF were firmly adsorbed
Copyright © 2010 SciRes. JBiSE
H. X. Yang et al. / J. Biomedical Science and Engineering 3 (2010) 1146-1155
Copyright © 2010 SciRes.
(a) (b)
Figure 2. Comparison of the surface morphologies between pristine PHBHHx film (a) and plasma treated PHBHHx film for 6 min
irradiation (b).
Figure 3. The water contact angles versus the plasma irradia-
tion time for the PHBHHx film. Figure 4. The fluorescents of FTIC-labeled SF in the PBS
buffer solutions which were used to flush the SF-coated
PHBHHx films with (in solid line) and without (in dash dot
line) plasma treatment, respectively.
on the plasma-treated PHBHHx film even though under
the physiologic recycle-flow flushing condition, possibly
due to the hydrophilic ch emical groups newly grafted on
the surface. Therefore, the plasma-treated PHBHHx film
is an ideal substrate for SF immobilization, and would be
as a potential candidate material for the artificial blood
vein to bear the high she er force of b lood flow in vivo.
studied PHBHHx films up to 7 days were evaluated by
MTT assay and HE staining method.
The MTT assay results are shown in Figure 5 which
demonstrates the relative optical densities (OD) of four
types of the studied films. The high OD value indicates
the high cell viability. Fro m the Figure 5, HSMCs seeded
on the SF-coated PHBHHx film (SP) have the better cell
viability than that on the PHBHHx film (P) after cul-
tured for 7 days, which is consistent with our previous
3.3. Growth and Proliferation of HSMCs on the
Studied Films
The growth and proliferation of HSMCs cultured on the
H. X. Yang et al. / J. Biomedical Science and Engineering 3 (2010) 1146-1155 1151
PHBHHx film
SF-coated PHBHHx film
Plasma-treated PHBHHx film
SF-coated PHBHHx film treated by plasma
Abersobance at 565 nm
Day 3Day 5Day 7
** **
Figure 5. Cell vitality of HSMCs seeded on different types of
PHBHHx films at day 3, 5 and 7 (*: P < 0.05; **: P < 0.01, n =
results [7,24]. Furthermore, the plasma-treated PHBHHx
films (PP) better support the growth and proliferation of
the cells than the SF-coated film without plasma treat-
ment. Apparently, HSMCs seeded on the SF-coated
PHBHHx film treated by plasma (SPP) show the highest
OD values during culture days, indicating that the film is
suitable for the HSMCs adhering and proliferating. Ar-
mentano et al. reported that the oxygen-based plasma
treated poly (L-lactide) (PLLA) film could improve the
human marrow stromal cells (HMSCs) adhesion and
growth [36]. Similarly, Shen et al found that the 3T3
fibroblasts on the CO2 plasma-treated poly (lactide-co-
glycolide) (PLGA) scaffold immobilized by the basic
fibroblast growth factor (bFGF) showed the highest vi-
ability and the rapidest proliferation among the various
PLGA scaffolds including those with and without plas-
ma treatment and with bEGF immobilization [26]. These
results substantially prove that plasma treatment for the
material is an efficient method to improve the biocom-
patibility of the material.
Moreover, the shuffle-like HSMCs were observed by
HE staining, where the cell nuclei are in dark purple and
the cytoplasm in red shown in Figure 6. The HSMCs
show the lower cell density on the P and SP films (Fig-
ure 6(a) and (b)) than that on the SPP films (Figure 6(d))
in 3 days culture, which show the highest cell den- sity
among those studied films. In 7 days of cell culture, the
proliferation of HSMCs on the pristine (Figure 6(a’))
and plasma-treated PHBHHx films (Figure 6(c’)) show
the higher cell density than that in 3 days culture, and the
HSMCs on the PP film (Figure 6(c’)) not only expanded
into the more cells than that on the P film (Figure 6(a’))
but also started to form the cell network. Moreover, the
HSMCs on the SP film (Figure 6(b’)) and SPP film
(Figure 6(d’)) gradually form the larger cell sub-mono-
layer network than that on the PP film (Figure 6(c’)).
3.4. Morphologies of HSMCs Growing on the
Studied Films
The morphologies of HSMCs growing on the films of P,
SP, PP and SPP in 3 and 7 days culture were observed by
SEM. In Figure 7, the HSMCs on the P film show a low
cell density without typical shuffle-like morphology at
day 3 (Figure 7(a)). Contrastively, the HSMCs growing
on the SP, PP and SPP films show the h igher cell density
with the better cell shapes of shuffle-like morphology
P film SP film PP film SPP film
Day 3
(a) (b) (c) (d)
Day 7
(a’) (b’) (c’) (d’)
Figure 6. Images of HE-stained HSMCs cultured on four types of films at day 3 from (a) to (d) and day 7 from (a’) to (d’). (a) and
(a’): cells cultured on the pristine PHBHHx film (P film); (b) and (b’): cells cultured on the SF-coated PHBHHx film (SP film); (c)
and (c’): cells cultured on the plasma-treated PHBHHx film (PP film); (d) and (d’): cells cultured on the SF-coated PHBHHx film
treated by plasma (SPP film). The cell nuclei were stained in purple with hematoxylin and the cytoplasma was stained in red with
osin (magnification × 100). e
Copyright © 2010 SciRes. JBiSE
H. X. Yang et al. / J. Biomedical Science and Engineering 3 (2010) 1146-1155
(Figure 7 (b), (c) and (d)). At day 7, HSMCs grew into
cell sub-monolayer on the surface of PP and SPP films
and secreted the extracellular matrix (ECM) to fill up the
holes present in the PHBHHx films (Figure 7 (c’) and
(d’)), while the HSMCs on the P and SP films show a
lower cell confluence and less ECM secretion (Figure 7
(a’) and (b’)). Similarly, Kim et al found that the human
bone marrow stromal cells proliferated well to a larger
area when attached to th e hydrophilic surfaces than those
of the hydrophobic surfaces, resulting in the formation
of a more flatten morphology [30]. Also Lampin et al.
[29] reported that the increase in the surface roughness
of poly (methyl methacrylate) film in extent allowed the
expansion of the migration of the chick embryo vascular
and corneal cells and triggered the subconfluent cells to
secrete the extracellular proteins. Accordingly, in our
work, the plasma-treated PHBHHx film improves the
roughness (shown in Figure 2) and hydrophilicity (sho-
wn in Figure 3), leading to the larger amount of ECM
secretion and the better cell migration.
In this work, PHBHHx films were irradiated by the low
temperature atmospheric plasma for 6 min, resulting in
Day 3 Day 7
(a) (a’)
(b) (b’)
Copyright © 2010 SciRes. JBiSE
H. X. Yang et al. / J. Biomedical Science and Engineering 3 (2010) 1146-1155 1153
(c) (c’)
treated by
plasma (SPP)
(d) (d’)
Figure 7. The morphologies of HSMCs cultured on four types of films: (a) and (a’) cells cultured on the pristine PHBHHx film (P) at
day 3 and 7; (b) and (b’) cells cultured on the SF-coated PHBHHx film (SP) at day 3 and 7; (c) and (c’) cells cultured on the plasma
treated PHBHHx film (PP) at day 3 and 7; (d) a nd (d’) cells cultured on the SF-coated PHBHHx film treated by plasma (SPP) at day
3 and 7. Arrows point the positions of HSMCs.
an increased roughness and improved hydrophilic sur-
face with low water contact angle. The SF-coated
PHBHHx films treated by the plasma were flushed by
PBS buffer under the rate of physiological blood flow,
proving that SF on the plasma-treated surface have better
immobilization strength than that on the surface without
plasma treatment. A significant increase in the prolifera-
tions of HSMCs is present on the SF-coated PHBHHx
films with plasma treatment, and the cell sub-monolayer
and the secreted ECM are also formed well on these
We thank Professor Guoqiang Chen in Tsinghua University for kindly
donating the PHBHHx compound. We also thank Mr. Lingxiang Wu in
Fudan University for providing the DBD plasma generator.
Copyright © 2010 SciRes. JBiSE
H. X. Yang et al. / J. Biomedical Science and Engineering 3 (2010) 1146-1155
[1] Armentano, I. (2009) Role of PLLA plasma surface
modification in the interaction with human marrow stro-
mal cells. Journal of Applied Polymer Science, 114,
[2] Chen, G., Zhou, P., Mei, N., Chen, X., Shao, Z.Z., Pan,
L.F. and Wu, C.G. (2004) Silk fibroin modified porous
poly (E-caprolactone) scaffold for human fibroblast cul-
ture in vitro. Journal of Materials Science-Materials in
Medicine, 15, 671-677.
[3] Chen, G.Q. and Wu, Q. (2005) The application of polyhy-
droxyalkanoates as tissue engineering materials. Bioma-
terials, 26, 6565-6578.
[4] Dumitrascu, N., Borcia C. and Borcia G. (2008) Control
of the blood-polymer interface by plasma treatment.
Journal of Biomedical Materials Research Part B-Ap-
plied Biomaterials, 87B, 364-373.
[5] Dumitrascu, N., Borcia, G., Apetroaei, N. and Popa, G.
(2002) Roughness modification of surfaces treated by a
pulsed dielectric barrier discharge. Plasma Sources Sci-
ence & Technology, 11, 127-134.
[6] Hersel, U., Dahmen, C. and Kessler, H. (2003) RGD
modified polymers: biomaterials for stimulated cell ad-
hesion and beyond. Biomaterials, 24, 4385-4415.
[7] Hoerstrup, S.P. (2000) Functional living trileaflet heart
valves grown in vitro. Circulation, 102, 44-49.
[8] Khang, G., Kim, S.W., Cho, J.C., Rhee, J.M., Yoon, S.C.
and Lee, H.B. (2001) Preparation and characterization of
poly (3-hy-droxybutyrate-co-3-hydroxyvalerate) micro-
spheres for the sustained release of 5-fluorouracil. Bio-
Medical Materials and Engineering, 11, 89-103.
[9] Kim, M.S., Shin, Y.N., Cho, M.H., Kim, S.H., Kim, S.K.,
Cho, Y.H., Khang, G., Lee, I.W. and Lee, H.B. (2007)
Adhesion behavior of human bone marrow stromal cells
on differentially wettable polymer surfaces. Tissue Engi-
neering, 13, 2095-2103.
[10] Lampin, M., WarocquierClerout, R., Legris, C, Degrange,
M. and SigotLuizard, M.F. (1997) Correlation between
substratum roughness and wettability, cell adhesion, and
cell migration. Journal of Biomedical Materials Research,
36, 99-108.
[11] Langer, R. and Vacanti, J.P. (1993) Tissue Engineering.
Science, 260, 920-926.
[12] Lee, S.Y. (1996) Bacterial polyhydroxyalkanoates. Bio-
technology and Bioengineering, 49, 1-14.
[13] Li, X.T., Zhang, Y. and Chen, G.Q. (2008a) Nanofibrous
polyhy- droxyalkanoate matrices as cell growth support-
ing materials. Biomaterials, 29, 3720-3728.
[14] Li, X.T., Sun, J., Chen, S. and Chen, G.Q. (2008b) In
vitro investigation of maleated poly (3-hydroxybutyrate-
co-3-hydroxyhexanoate) for its biocompatibility to mou-
se fibroblast L929 and human microvascular endothelial
cells. Journal of Biomedical Materials Research Part A,
87A, 832-842.
[15] Li, Z.G., Lin, H., Ishii, N., Chen, G.Q. and Inoue, Y.
(2007) Study of enzymatic degradation of microbial co-
polyesters consisting of 3-hydroxybutyrate and mdium-
chain-length 3-hydroxyalkanoates. Polymer Degradation
and Stability, 92, 1708-1714.
[16] Mei, N., Chen, G., Zhou, P., Chen, X., Shao, Z.Z., Pan,
L.F. and Wu C.G. (2005) Biocompatibility of poly (epsi-
lon-caprolac-tone) scaffold modified by chitosan - The
fibroblasts proliferation in vitro. Journal of Biomaterials
Applications, 19, 323-339.
[17] Mei, N., Zhou, P., Pan, L.F., Chen, G., Wu, C.G., Chen,
X., Shao, Z.Z. and Chen, G.Q. (2006) Biocompatibility of
poly (3-hy-droxybutyrate-co-3-hydroxyhexanoate) modi-
fied by silk fibroin. Journal of Materials Science- Mate-
rials in Medicine, 17, 749-758.
[18] Niklason, L.E., Gao, J., Abbott, W.M., Hirschi, K.K.,
Houser, S., Marini, R. and Langer, R. (1999) Functional
arteries grown in vitro. Science, 284, 489-493.
[19] Ostrikov, K. and Murphy, A.B. (2007) Plasma-aided
nanofabrication: Where is the cutting edge? Journal of
Physics D-Applied Physics, 40, 2223-2241.
[20] Robinson, D.E., Marson, A., Short, R.D., Buttle, D.J.,
Day, A.J., Parry, K.L., Wiles, M., Highfield, P., Mistry, A.
and Whittle, J.D. (2008) Surface gradient of functional
heparin. Advanced Materials, 20, 1166-1169.
[21] Shangguan, Y.Y., Wang, Y.W., Wu, Q. and Chen, G.Q.
(2006) The mechanical properties and in vitro biodegra-
dation and biocompatibility of UV-treated poly (3-hy-
droxybutyrate-co-3-hydroxyhexanoate). Biomaterials, 27,
[22] Shen, H., Hu, X.X., Bei, J.Z., Wang, S.G. (2008) The
immobilization of basic fibroblast growth factor on
plasma-treated poly (lactide-co-glycolide). Biomaterials,
29, 2388-2399.
[23] Shen, H., Hu, X.X., Yang, F., Bel, J.Z. and Wang, S.G.
(2007) Combining oxygen plasma treatment with an-
chorage of cationized gelatin for enhancing cell affinity
of poly (lactide-c o-glycolide) . Biomaterials, 28, 4219-4230.
[24] Shishatskaya, E.I., Voinova, O.N., Goreva, A.V., Mogil-
naya, O.A. and Volova, T.G. (2008) Biocompatibility of
polyhydroxybutyrate microspheres: in vitro and in vivo
evaluation. Journal of Materials Science-Materials in
Medicine, 19, 2493-2502.
[25] Siow, K.S., Britcher, L., Kumar, S. and Griesser, H.J.
(2006) Plasma methods for the generation of chemically
reactive surfaces for biomolecule immobilization and cell
colonization — A review. Plasma Processes and Poly-
mers, 3, 392-418.
[26] Sodian, R., Hoerstrup, S.P., Sperling, J.S., Daebritz, S.,
Martin, D.P., Moran, A.M., Kim, B.S., Schoen, F.J., Va-
canti, J.P. and Mayer, J.E. (2000) Early in vivo experi-
ence with tissue-engineered trileaflet heart valves. Cir-
culation, 102, 22-29.
[27] Sun, M., Zhou, P., Pan, L.F., Liu, S. and Yang, H.X.
(2009) Enhanced cell affinity of the silk fibroin-modified
PHBHHx material. Journal of Materials Sc ie nc e -M a t er i al s
in Medicine, 20, 1743-1751.
[28] Tamada, Y. and Ikada, Y. (1993) Cell-adhesion to
plasma-treated polymer surfaces. Polymer, 34, 2208-2212.
[29] Wang, Y.W., Wu, Q., Chen, J.C. and Chen, G.Q. (2005)
Evaluation of three-dimensional scaffolds made of blends
of hydroxyapatite and poly (3-hydroxybutyrate-co-3-
hydroxy-hexanoate) for bone reconstruction. Biomate-
rials, 26, 899-904.
[30] Wu, Q., Wang, Y. and Chen, G.Q. (2009) Medical Appli-
cation of Microbial Biopolyesters Polyhydroxyalka-
noates. Artificial Cells Blood Substitutes and Biotech-
Copyright © 2010 SciRes. JBiSE
H. X. Yang et al. / J. Biomedical Science and Engineering 3 (2010) 1146-1155
Copyright © 2010 SciRes.
nology, 37, 1-12.
[31] Xin, X.J., Hussain, M. and Mao, J.J. (2007) Continuing
different- tiation of human mesenchymal stem cells and
induced chondrogenic and osteogenic lineages in elec-
trospun PLGA nanofiber scaffold. Biomaterials, 28,
[32] Yang, M., Zhu, S.S., Chen, Y., Chang, Z.J., Chen, G.Q.,
Gong, Y.D., Zhao, N.M. and Zhang, X.F. (2004) Studies
on bone marrow stromal cells affinity of poly (3- hy-
droxybutyrate-co-3-hydroxyhexanoate). Biomaterials, 25,
[33] Zhang, D.M., Cui, F.Z., Luo, Z.S., Lin, Y.B., Zhao, K.
and Chen, G.Q. (2000) Wettability improvement of bac-
terial polyhy-droxyalkanoates via ion implantation. Sur-
face & Coatings Technology, 131, 350-354.
[34] Zhang, J.C., Wu, L.B., Jin g , D.Y. and Ding, J.D. (2005) A
comparative study of porous scaffolds with cubic and
spherical macropores. Polymer, 46, 4979-4985.
[35] Zhang, Y., Zhou, P., Pa n, L.F., Xie, S.Z. , Sun, M. and Li,
W.T. (2007) Growth of human smooth muscle cells on
the silk fibroin modified-polyhydroxyalkanoate scaffold.
Acta Chimica Sinica, 65, 2935-2940.
[36] Zhao, K., Deng, Y., Chen, J.C. and Chen, G.Q. (2003)
Polyhy-droxyalkanoate (PHA) scaffolds with good me-
chanical properties and biocompatibility. Biomaterials,
24, 1041-1045.