In Vitro Cell.Dev.Biol.
Animal
DOI 10.1007/s11626-015-9925-8
Regulation of astrocyte activity via control over stiffness
of cellulose acetate electrospun nanofiber
Seul Ki Min & Sang Myung Jung & Jung Hyeon Ju &
Yeo Seon Kwon & Gwang Heum Yoon & Hwa Sung Shin
Received: 26 January 2015 / Accepted: 20 May 2015 / Editor: T. Okamoto
# The Society for In Vitro Biology 2015
Abstract Astrocytes are involved in neuron protection fol-
lowing central nervous system (CNS) injury; accordingly,
engineered astrocytes have been investigated for their useful-
ness in cell therapy for CNS injury. Nanofibers have attracted
a great deal of attention in neural tissue engineering, but their
mechanical properties greatly influence physiology. Cellulose
acetate (CA) has been studied for use in scaffolds owing to its
biocompatibility, biodegradability, and good thermal stability.
In this study, stiffness of CA nanofibers controlled by heat
treatment was shown to regulate astrocyte activity. Adhesion
and viability increased in culture as substrate became stiffer
but showed saturation at greater than 2 MPa of tensile
strength. Astrocytes became more active in terms of increas-
ing intermediate filament glial fibrillary acidic protein
(GFAP). The results of this study demonstrate the effects of
stiffness alone on cellular behaviors in a three-dimensional
culture and highlight the efficacy of heat-treated CA for astro-
cyte culture in that the simple treatment enables control of
astrocyte activity.
S. K. Min : S. M. Jung : J. H. Ju : Y. S. Kwon : G. H. Yoon :
H. S. Shin (*)
Department of Biological Engineering, Inha University,
Incheon 402-751, Korea
e-mail: hsshin@inha.ac.kr
S. K. Min
e-mail: skmin1206@inha.edu
S. M. Jung
e-mail: sangmyungjung11@gmail.com
J. H. Ju
e-mail: jjh6750@nate.com
Y. S. Kwon
e-mail: kyslove@naver.com
G. H. Yoon
e-mail: myyoon21c@naver.com
Keywords Cellulose acetate . Electrospun nanofiber . Heat
treatment . Stiffness . Astrocyte tissue engineering
Introduction
The central nervous system (CNS) contains a large number of
nerve cells supported by glial cells. When the CNS is injured,
it is difficult for the neural cells to regenerate. Accordingly,
many studies have investigated treatment of injured CNS with
drugs and transplants. Astrocytes, the most abundant cell in
the brain, are a type of glial cell that occupy approximately
3050% of the brain and play important roles in supporting
neural cells and the brain-blood barrier (BBB), regulating the
concentration of ions and maintaining homeostasis for neuro-
nal function.
Implantable materials for brain disease have purpose to
regenerate the damaged tissue by filling the cavity and
supporting structure (Wang et al. 2014). For example, ische-
mic stroke obstructs cerebral blood supply, and it causes loss
of brain tissue resulting in the formation of infarct cavity. The
infarct area leads to a larger cavity and worsens by inflamma-
tion or immune reaction as time goes by. Therefore,
bioengineered astrocytic tissue has been actively investigated
for their potential as implantable tissue constructs for treat-
ment of injured neural tissue (Yucel et al. 2010; Meng et al.
2012). Astrocytes are of particular interest because lost and
healthy tissue consists primarily of astrocytes that do not di-
vide well in the normal state and astrocytes have various func-
tions that help regenerate injured neurons, such as the ability
to release neurotrophic factors (Sofroniew and Vinters 2010).
Nanofiber scaffold produced by electrospinning is now widely
used for astrocyte tissue engineering because its structure and
topography facilitate tissue constructs similar to the actual
 MIN ET AL.
tissues (Ma et al. 2005; Ladd et al. 2011). Nanofibers can be
produced from synthetic polymers, poly (-caprolactone)
(PCL), poly (lactic-co-glycolic acid) (PLGA), and poly (eth-
ylene oxide) (PEO), as well as from naturally derived poly-
mers such as collagen, gelatin, cellulose acetate, alginate, and
dextran (Huang et al. 2003; Kweon et al. 2003; Pham et al.
2006).
Mechanical properties influence cell function, differentia-
tion, morphology, and composition (Blacklock et al. 2010;
Monici and Cialdai 2008; Wells 2008). Nanofiber is suitable
for being used as a scaffold because it has same morphological
features with extracellular matrix (ECM). Therefore, stiffness
is appropriate to control not inducing morphological changes
for using nanofiber. Physical and chemical reactions are re-
quired to change the stiffness of a scaffold. Nanofiber stiffness
is easily modified by chemical reactions or variations in fab-
rication, but these methods induce differences in surface
chemistry, topography, or mechanical properties among nano-
fibers, making analysis of the stiffness effects difficult due to
contamination with other causes (Liu et al. 2011). Physical
modifications that do not induce any chemical, topological,
or mechanical variations other than stiffness are preferable.
Stiffness of extracellular matrix generally plays a pivotal role
in the formation and function of a tissue, and several studies
have shown that tissues such as the bone, cartilage, brain, and
stem cells detect changes in substrate stiffness via mechanical
stress-sensing cascades (Mason et al. 2012). However, stiff-
ness is known to have different effects among cell types (Tee
et al. 2009). Several studies have recently investigated the
effects of scaffold stiffness on astrocyte activities based on
hydrogel culture systems (Jiang et al. 2007; Moshayedi et al.
2010). However, hydrogel stiffness is controlled by monomer
and cross-linker densities; therefore, structural or electro-
chemical variations could differ. In addition, nanofiber-based
studies of the effects of stiffness on cellular behaviors have
rarely been performed.
Cellulose is one of the most abundant natural polysaccha-
ride polymers on earth, and cellulose acetate (CA) is a cellu-
lose derivative that can be obtained by acetylation of the hy-
droxyl group of cellulose. CA has been applied to biological
applications such as filters, scaffolds for artificial tissues, and
dressings due to its good thermal stability, chemical resistance,
and biodegradability. For example, cellulose acetate is active-
ly used for bone and cartilage tissue engineering because of its
biocompatibility and mechanical strength (Katoh and Urist
1993; MayerWagner et al. 2011). In addition, CA has been
investigated as a material for vascular tissue and peripheral
nervous system recovery (Han and Cheung 2011; Pooyan et al.
2012). However, few studies have employed 3D cultures of
astrocytes on CA nanofiber. In this study, we fabricated an
array of electrospun nanofibrous mats with cellulose ace-
tate and manipulated their stiffness by a simple heat treat-
ment. Structural and chemical changes were investigated
after thermal treatment to ensure that there were no chem-
ical or physical variations among nanofibers except for
stiffness, and primary astrocytes were then assessed for
their physiology after cultivation on nanofibrous mats with
different levels of stiffness. Therefore, it is believed that
this material could be considered to implant for the regen-
eration of lost brain tissue.
Materials and Methods
Fabrication and thermal modification of electrospun CA
nanofibrous mat. CA (Mw =30,000, Sigma Aldrich) was dis-
solved by mixing with a 1:1 mixture of acetone and acetic acid
at 15% (w/v) for 24 h. The solution was then loaded into a
plastic syringe connected to an 18 gauge needle mounted on a
syringe pump (KD Scientific) and electrospun onto a drum
collector 17 cm from the needle tip under 17 kV at 1 ml h1.
Thermal treatment was then conducted as previously de-
scribed (Wells 2008; Pooyan et al. 2012). Briefly, the CA
nanofibrous mat was thermally treated in a drying oven
at 207C for 0, 1, and 2 h so that cellular behavior
could be observed according to changes in the stiffness
of the mat.
Characterization of the electrospun CA nanofibrous mat. -
Contact angle was examined to compare wettability and hy-
drophilicity among scaffolds subjected to thermal treatment
for different lengths of time. Briefly, 4-l water droplets were
placed onto the samples through a 22G syringe needle, after
which the contact angle was measured using a contact angle
meter (SEO, South Korea). The morphologies of the CA
nanofibers were then observed via scanning electron micros-
copy (SEM) (Hitachi). Next, the fabricated scaffold was cut to
11 cm2 and sputtered with platinum for 180 s, and the sam-
ples were then observed at 15 kV under 1000 magnification.
Additionally, the thickness of nanofiber strands was manually
measured in the SEM images using image J (NIH, v1.47). The
data were analyzed after measuring 200 nanofibers. The ten-
sile strength and viscoelasticity of the CA nanofibrous mat
was measured using a Universal Testing Machine (Instron).
All tensile testing samples were in the form of a rectangle
30 mm long, 16 mm wide, and 140 m thick, and tests were
carried out at a constant drawing speed of 10 mm min1 in the
length direction at room temperature. To confirm the structural
changes in the nanofiber component after heat treatment, the
infrared from the FT-IR (Bruker) was reflected off the samples
and analyzed. Each graph was plotted using data obtained
from at least 30 scans and peaks and was compared with
reference peaks.
Primary astrocyte cell culture. Primary astrocytes were ob-
tained from the cerebrum of neonatal Sprague-Dawley rats
 HEAT-TREATED CA NANOFIBER FOR THE ASTROCYTE CULTURE
(Samtako, South Korea) using a previously described protocol
(Fedoroff and Richardson 2001). The brain was then isolated
from the skull, and its meninges were gently removed from
the cerebrum. The cerebrum was then minced with a Pasteur
pipette and passed through a 0.7-mm pore cell strainer. For
astrocyte isolation, various cells with culture media were
poured into a T-75 polystyrene-coated tissue culture flask
(5106 cells) and then incubated under 5% CO2 at 37C for
1 wk. The culture medium consisted of Dulbecco-modified
eagles medium (DMEM-high glucose, GIBCO) with 10%
FBS and 1% antibiotic-antimycotic solution. After 1 wk, sam-
ples were cultured on a shaker at 200 rpm in a CO2 incubator
for 48 h to remove the other glial cells. Astrocytes were
strongly adhered and therefore remained on the T-75 cell cul-
ture flask.
Adhesion and viability of astrocytes onto the CA nanofiber. -
To measure the adhesion and viability, astrocytes were seeded
onto a CA nanofiber mat in a 24-well cell culture plate at a
concentration of 2104 cells/well and 1104 cells/well, re-
spectively. The adhesion and viability of astrocytes were
then measured at 2, 8, and 24 h and 1, 4, and 7 d,
respectively. To investigate the viability and adhesion, a
WST-1 assay was conducted according to the manufac-
turers instructions.
Western blotting analysis of GFAP. Western blotting was con-
ducted to measure the level of glial fibrillary acidic protein
(GFAP). First, astrocytes were seeded on a CA nanofiber mat
that had been thermally treated. Proteins were then extracted
from the astrocytes using RIPA buffer, after which they were
quantified with a bicinchoninic acid (BCA) protein assay kit
(Thermo Scientific). The same amount of proteins from each
sample was separated by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE), after which
separated proteins were transferred to a poly (vinylidene fluo-
ride) (PVDF) membrane (Sigma Aldrich). The membrane was
then blocked by incubation in blocking buffer (5% skim milk
in triethanolamine-buffered saline (TBS)) at 4C overnight.
Next, the PVDF membrane was treated with rabbit anti-
GFAP polyclonal IgG antibody as primary antibody in TBS
containing 5% skim milk overnight at 4C followed by goat
anti-rabbit IgG-HRP as the secondary antibody for 1 h. The
PVDF membrane was washed with TBST three times for
5 min each between antibody treatment steps. And then, anti-
bodies attached on the PVDF membrane reacted with ECL
solution (RPN2232, GE healthcare, UK) and the membrane
developed on the film in a dark room.
Statistical analysis. All experiments were carried out three
times for statistical analyses and expressed as the meansstan-
dard deviation (S.D.). A Students t test was then used to iden-
tify significant differences among groups with a p value <0.05.
Results
Mechanical properties of thermally treated CA nanofibers. -
To measure the thickness of the nanofiber and condition of the
CA nanofibrous mat after heat treatment for various times,
mats were observed by SEM and analyzed using the Image J
software after each sample was collected (Fig. 1). The mean
thickness of the CA nanofiber before heat treatment was
436.8154.1 nm, while it was 44 2153.0 nm and 438.3
128.1 nm after heat treatment for 1 and 2 h, respectively. The
numerical values represent the average and standard deviation
from about 200 strands of nanofiber. The results indicate that
there was no significant change in the thickness of the CA
nanofiber mat in response to heat treatment and that structural
changes were too small to detect or did not occur. Differences
in heat treatment time did not lead to different contact angles
between the surface of the CA nanofiber and the water drops,
with treatment for 0, 1, and 2 h resulting in contact angles of
135.4182.762, 136.4691.474, and 134.0971.506, re-
spectively. These findings indicate that the wettability of the
CA nanofibrous mat was unaffected by heat treatment.
The chemical composition of the CA nanofibrous mat accord-
ing to heat treatment time was analyzed by FT-IR to identify
changes in chemical composition. The position of peaks
shown in Fig. 2 at 1050/cm (-C-O-), 1250/cm (-R-O-R- ether
bonding), 1750/cm (C=O), 3000/cm (-CH), and 3400/cm
(OH) was consistent with FT-IR spectra of cellulose acetate.
Other peaks were not observed in all samples. Overall, these
findings indicate that the chemical components of the CA
nanofibrous mat were maintained during heat treatment.
The tensile strength of the CA nanofibrous mats was mea-
sured after heat treatment for up to 2 h to observe changes in
stiffness. The tensile strength of CA nanofiber was 0.423
0.013, but those of heat-treated CA nanofibers were around
2 MPa (1.9080.055 and 2.0310.030, respectively), indicat-
ing that the stiffness increased in response to heating, regard-
less of the heat treatment time (Fig. 3). Viscoelasticity was
also measured as tensile modulus by the universal testing ma-
chine (UTM) using the following equation:
E 1
where E indicates the tensile modulus and and indicate the
tensile strength and tensile strain, respectively. The tensile
modulus, indicating viscoelasticity, was 24.016.44 MPa be-
fore heat treatment, while it was 79.8910.39 and 72.62
10.53 MPa after 1 and 2 h of heat treatment, respectively.
The viscoelasticity of the CA nanofiber increased during the
first 1 h of heat treatment, after which it was saturated. These
results indicate that each CA nanofiber showed different vis-
coelasticity depending on the heat treatment and saturated
 MIN ET AL.
Figure 1 Characterization of CA nanofiber with and without heat
treatment. SEM images of CA nanofiber at 1000 (A, D, G) and 4000
(B, E, H). Histogram of CA nanofiber diameter of each sample (C, F, I).
value after 1 h of heat treatment. Overall, the simple heat
treatment generated stiffer CA nanofibrous mats, while not
influencing topography, surface chemistry, or wettability of
the CA nanofibrous mat. Therefore, the subsequent cellular
responses to CA nanofibers were caused by stiffness alone.
Behavior of astrocytes on CA nanofibers. MTT assay to eval-
uate the cytotoxicity of the CA nanofibrous mats revealed that
astrocyte viability did not differ significantly among tissue
culture polystyrene (TCPS) and 0, 1, and 2 h heat-treated
CA nanofibers (Fig. 4). These findings confirm that CA
nanofibrous mats are nontoxic to astrocytes, which is similar
to the results of previous studies4. The ability of astrocytes to
adhere to each sample was confirmed by a WST-1 assay. As-
trocytes cultured on TCPS as a positive control showed
(A, B, C) nonheat-treated CA nanofiber, (D, E, F) 1-h heat-treated CA
nanofiber, (G, H, I) 2-h heat-treated CA nanofiber (scale bars; 10 m for
A, D, and G, 4 m for B, E, and H)
normal adhesion behavior, ensuring that the prepared cells
were active. There was no difference in adhesion among
heat-treated samples, while adhesion to nontreated CA grad-
ually decreased with time (Fig. 5a). The WST-1 assay indicat-
ed that the viability depended on stiffness of the CA nanofiber.
Astrocytes cultured on TCPS were used to identify the normal
viability of cells as a positive control. The viability decreased
from days 1 to 4 on CA nanofiber that was not subjected to
heat treatment (P<0.001). However, viability was maintained
on heat-treated samples from days 1 to 4, after which an in-
crease in cell growth occurred until day 7 (Fig. 5b). The ex-
pression of GFAP was investigated by Western blot analysis to
investigate the activity of astrocytes on TCPS and CA mats
that were thermally treated for 0, 1, and 2 h (Fig. 5c). The
sample on TCPS showed the highest GFAP expression, while
 HEAT-TREATED CA NANOFIBER FOR THE ASTROCYTE CULTURE
Figure 2 FT-IR data of CA nanofiber with and without heat treatment
samples treated for 1 and 2 h showed greater expression than
those that were not heat-treated. To confirm that CA could be
used as a scaffold on which astrocytes maintain their viability
and reorganize extracellular matrix (ECM) in long-term cul-
ture, astrocytes were cultured in CA scaffolds for 3 wk, and
their viability and expression of collagen and glycosaminogly-
can (GAG) were evaluated. The long-term culture confirmed
that astrocytes on CA maintain their viability, and heat-treated
Figure 3 Graphs of (a) tensile
strength and (b) modulus of no
heat- and heat-treated CA
nanofiber with time
Figure 4 Cytotoxicity graph of no heat- and heat-treated CA nanofiber
via MTT assay. *P < 5.0 10 2, **P < 1.0 102, ***P < 1.0 103 ,
compared to the control group
CA increased astrocyte density (Fig. 6) relative to nontreated
CA. Even though they were not quantified, the collagen and
GAG of astrocytes were well organized in CA nanofibrous
mats. As shown in Fig. 6, cellular behaviors such as cell ex-
istence and secreted metabolites for extracellular matrix
(ECM) were observed. The first row of Fig. 6 shows images
of cell nuclei on nanofiber mats. Comparison of Fig. 6a, b, and
c reveals that fewer astrocytes were attached to nontreated CA
nanofibers than heat-treated nanofibers. However, there was
no significant difference between the two heat-treated CA
nanofibers. Similarly, pink-stained collagen was observed
sparsely on nontreated CA nanofiber (Fig. 6d). In contrast,
CA nanofibers heat-treated for 1 and 2 h were completely
covered by collagen (Fig. 6e, f). Moreover, the GAG-stained
blue and nuclei showed similar tendency with collagen stain-
ing. As shown in Fig. 6gi, less expression was observed on
nontreated CA nanofiber than on heat-treated CA nanofiber.
Discussion
Electrospun CA nanofibrous mats are known to have less
molecular conjugation between fiber strands after fabrication,
resulting in poor mechanical strength (Tang et al. 2008).
Therefore, several studies have been conducted to increase
 MIN ET AL.
Figure 5 Cellular behavior of astrocytes on no heat- and heat-treated CA
nanofiber. (a) Adhesion rate of astrocytes. (b) Viability rate of astrocytes
after 1 wk of cultivation. (c) Western blot data of GFAP expression at day
the mechanical strength of CA nanofibers (Tang et al. 2008;
Zhou et al. 2011). The results of these studies have shown that
heat treatment is a simple method to manipulate the stiffness
of CA nanofibers. However, CA will be destroyed by heat
treatment at temperatures above 210C, while its mechanical
strength will not be affected by treatment at temperatures un-
der 205C. In the present study, no visual differences in the
form and fiber diameter were observed in response to heat
treatment at 207C (Fig. 1). Moreover, as shown in Figs. 1
Figure 6 Stained images of astrocytes at day 21. (a, b, c) Astrocyte
nuclei stained with Hoechst 33342. (d, e, f) Collagen synthesized by
astrocytes stained with Sirius red. (g, h, i) Glycosaminoglycan
7. *P<5.0102, **P<1.0102, ***P<1.0103, compared to the
control group
and 2, the surface characteristics and molecular structure were
unchanged after heat treatment. However, as shown in Fig. 3,
heat treatment effectively increased the mechanical strength of
the CA nanofiber. The glass transition point of CA is 198
205C, and the melting point is 224230C; therefore, when
fabricated CA nanofiber was treated at the glass transition
point, a glass transition occurred while maintaining the nano-
fiber morphology and cross-links formed between the CA
molecules and the fiber strands. For this reason, the stiffness
synthesized by astrocytes stained with alcian blue. (a, d, g) No heat-
treated CA nanofiber, (b, e, h) 1-h heat-treated CA nanofiber (c, f, i) 2-h
heat-treated CA nanofiber (scale bar 1 m)
 HEAT-TREATED CA NANOFIBER FOR THE ASTROCYTE CULTURE
of the entire CA nanofibrous mat increased in response to heat
treatment. Additionally, the results confirmed that heat treat-
ment at the glass transition temperature modified stiffness of
the CA nanofiber saturated after 1 h of heat treatment by the
tensile strength and tensile modulus. Figure 4 shows the bio-
compatibility of each nanofiber with astrocyte culture. The ad-
hesion to heat-treated CA samples was higher than that to
nontreated samples, which was similar to the cell viability dur-
ing short- and long-term culture. Adhesive cells show a close
relationship between adhesion and viability because complete
adhesion of the cells is required for increased cellular viability.
Therefore, adhesion and viability changed in a similar trend in
response to changes in the stiffness of CA nanofiber.
GFAP is a marker for evaluation of astrocyte activity that
was observed to increase in response to increased surface
stiffness. The upregulation of GFAP indicates that astrocytes
are activated with many alterations such as secretion of neu-
rotrophic factors, especially in the case of CNS injury (Bren-
ner 2014). Therefore, GFAP have been analyzed in many
studies for treatment of CNS injury using astrocytes. When
cells adhere to a surface, stiffer surfaces might allow attached
cells to become wider, resulting in increased GFAP expres-
sion. It has been reported that adhered cells sense mechanical
strength via a cascade, leading to expression of integrin and
subsequent changes in the cytoskeleton such as upregulation
of intermediate filament GFAP (Fuchs and Weber 1994).
Therefore, it is likely that astrocytes sensed stiffness of the
CA nanofiber, which led to changes in the expression of
GFAP. In addition, the stimuli exerted on the plasma mem-
brane are transferred to regulation of DNA transcription for
the cell cycle and activity via the cytoskeleton-matrix interac-
tion. In the case of astrocytes, a cytoskeleton GFAP transfers
surface stimuli to intracellular signaling, leading to enhanced
gene expression of mitosis via several kinases, microtubule-
associated binding protein, and actin-binding proteins such as
paxillin, pectin, and -actin (Rutka et al. 1997; Breuls et al.
2008; Ulrich et al. 2009). Many studies have noticed about the
positive role of increased activity of astrocytes, and we also
focused on that. As previous studies, it has been confirmed
that when astrocytes are activated, they have clinical effect on
the brain disease such as Alzheimers disease as well as in-
crease the expression of neurotrophic factors such as BDNF,
NGF, and CNTF for neuroprotection through a variety of pre-
vious studies (Sofroniew and Vinters 2010). We could make
combination version of materials which inhibit the negative
effect of activated astrocytes as further study.
There are several requirements for using as artificial mate-
rials in tissue engineering. One of these is that materials must
have ability to stimulate proliferation of cells in order to fill up
tissue cavity as well as adhere to surface of scaffold. Also, it
should activate expression of ECM molecules such as GAG
and collagen for making cells stable and having interaction
with real tissue when it is implanted.
This is the first study to demonstrate that stiffness alone
influences astrocytes without any other causes due to chemis-
try, topography, and density of the scaffold. In addition, nano-
fiber has attracted a great deal of attention owing to its poten-
tial for use as a scaffold for tissue engineering, and it is well
known that scaffold stiffness regulates intracellular signal
transduction; however, the effects of nanofiber stiffness on
cell behaviors have rarely been investigated. One previous
study did indicate that increased stiffness of CA/chitosan
nanofibers improved the adhesion of endothelial cells
(Georges et al. 2006). Therefore, this research is meaningful
in that the controlled stiffness of heat-treated CA nanofiber
mats can be used to regulate astrocyte activity, which is one
of the targets for neural regeneration and astrocyte tissue
engineering.
Conclusion
Scaffold stiffness has recently been used to finely regulate cell
physiology so that the engineered tissue can mimic structure,
morphology, and characteristics of real tissue well. In this
study, the chemical and topological variations in heat-treated
CA nanofibrous mats were minimized to evaluate the effects
of stiffness alone. Specifically, cells were cultured on
stiffness-controlled nanofibers and assayed for cellular behav-
iors by several assays, which revealed increased cellular ad-
hesion, viability, GFAP expression, and ECM expression in
response to increased stiffness of scaffolds. The information
provided in this study improves our understanding of the ef-
fects of nanofiber stiffness on astrocytes and will facilitate the
use of stiffness-controllable CA nanofiber to engineer astro-
cytes suitable for transplant or culture in special environments.
Acknowledgment This work was supported by the National Research
Foundation of Korea (NRF) grant funded by the Korea government
(MSIP) (NRF-2014R1A2A1A11052143).
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