Materials Science and Engineering C 70 (2017) 1089–1094

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Materials Science and Engineering C

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

Electrospinning of poly(glycerol sebacate)-based nanofibers for nerve

tissue engineering
Jue Hu a, Dan Kai b,⁎⁎, Hongye Ye b, Lingling Tian c, Xin Ding a,⁎, Seeram Ramakrishna c,f, Xian Jun Loh b,d,e,⁎⁎
a
College of Textiles, Donghua University, Shanghai 201620, China
b
Institute of Materials Research and Engineering (IMRE), A*STAR, 2 Fusionopolis Way, Innovis, #08-03, 138634, Singapore
c
Centre for Nanofibers and Nanotechnology, Department of Mechanical Engineering, National University of Singapore, 2 Engineering Drive 3, Singapore
d
Department of Materials Science and Engineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117576, Singapore
e
Singapore Eye Research Institute, 11 Third Hospital Avenue, Singapore 168751, Singapore
f
Guangdong-Hongkong-Macau Institute of CNS Regeneration (GHMICR), Jinan University, Guangzhou 510632, China

a r t i c l e i n f o a b s t r a c t

Article history: Nerve tissue engineering (TE) requires biomimetic scaffolds providing essential chemical and topographical cues
Received 23 January 2016 for nerve regeneration. Poly(glycerol sebacate) (PGS) is a biodegradable and elastic polymer that has gained
Received in revised form 16 February 2016 great interest as a TE scaffolding biomaterial. However, uncured PGS is difficult to be electrospun into nanofibers.
Accepted 13 March 2016
PGS would, therefore, require the addition of electrospinning agents. In this study, we modified PGS by using
Available online 18 March 2016
atom transfer radical polymerization (ATRP) to synthesize PGS-based copolymers with methyl methacrylate
Keywords:
(MMA). The synthesized PGS-PMMA copolymer showed a molecular weight of 82 kDa and a glass transition tem-
PGS perature of 115 °C. More importantly, the PGS-PMMA could be easily electrospun into nanofiber with a fiber di-
Atom transfer radical polymerization ameter of 167 ± 33 nm. Blending gelatin into PGS-PMMA nanofibers was found to increase its hydrophilicity and
Spinnability biocompatibility. Rat PC12 cells were seeded onto the PGS-PMMA/gelatin nanofibers to investigate their poten-
Methyl methacrylate tial for nerve regeneration. It was found that gelatin-containing PGS-based nanofibers promoted cell prolifera-
Scaffolds tion. The elongated cell morphology observed on such nanofibers indicated that the scaffolds could induce the
neurite outgrowth of the nerve stem cells. Overall, our study suggested that the synthesis of PGS-based copoly-
mers might be a promising approach to enhance their processability, and therefore advancing bioscaffold engi-
neering for various TE applications.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction have been employed as various TE scaffolds, including bone, cartilage

and myocardium [8–10]. Such nanofibrous scaffolds have also been
Peripheral nerve injuries commonly caused by traffic or construction applied for nerve regeneration. The fibrous architecture from
accidents or other traumas may lead to life-long disabilities [1]. Periph- electrospinning is similar to that of neutral ECM and is capable of
eral nerve tissue engineering (TE) is an emerging technology to design directing regeneration of nerves across lesions. The porosity of this fi-
advanced nerve guidance biomaterials for regeneration of nerves across brous structure further allows for cell migration, nutrition (and oxygen)
lesions. Suitable bioengineered scaffolds are able to reduce the require- diffusion and axon incorporation [11–13]. Such nanofibers would func-
ments of autologous nerves and physically guide the sprouting axons tion as a temporary ECM to provide essential signalling cues to the cells
from the proximal nerve end. As the interaction between biomaterial in direct contact.
and cells plays an important role in TE, the key for nerve regeneration To date, a wide range of biodegradable natural or synthetic poly-
is to design a bioactive scaffold mimicking both the biological and phys- mers, such as poly(ε-caprolactone), poly(L-lactic acid), poly(DL-lactic-
ical environment of native neutral ECM with optimized biochemical co-glycolic acid), collagen and gelatin, have been engineered into
properties [2–5]. electrospun nanofibers for nerve TE grafts [14]. Polyglycerol sebacate
Electrospinning is a simple technique to produce nanofibrous sub- (PGS) is a relative new biodegradable polymer that is synthesized via
strates with controllable fiber diameters [6,7]. Electrospun nanofibers a simple polycondensation reaction between glycerol and sebacic acid
[15–17]. Its excellent biocompatibility and mechanical properties (stiff-
ness ~0.056 to 1.5 MPa after curing) made it one of the most attractive
⁎ Corresponding author. biomaterials for biomedical applications, such as drug delivery, implant-
⁎⁎ Corresponding authors at: Institute of Materials Research and Engineering (IMRE),
A*STAR, 2 Fusionopolis Way, Innovis, #08-03, 138634, Singapore.
able devices and TE scaffolds [15,18–20]. PGS has been engineered into
E-mail addresses: [email protected] (D. Kai), [email protected] (X. Ding), microfabricated anisotropic accordion-like honeycomb microstructure,
[email protected] (X.J. Loh). microfibers, and core-shell fibrous membranes for cardiac tissue

http://dx.doi.org/10.1016/j.msec.2016.03.035
0928-4931/© 2016 Elsevier B.V. All rights reserved.
1090 J. Hu et al. / Materials Science and Engineering C 70 (2017) 1089–1094

engineering applications [20–23]. Electrospinning seems to be a suit- was characterized by NMR, gel permeation chromatography (GPC),
able approach to process PGS. However, uncured PGS is a highly viscous thermogravimetric analysis (TGA) and differential scanning calorimeter
and sticky liquid, which cannot form 3D structure. After curing, it (DSC).
crosslinks into an elastomer which cannot be dissolved in organic sol-
vents very easily. Therefore, PGS itself is not able to be electrospun,
and other rigid polymers need to be blended to provide mechanical 2.3. Electrospinning of nanofibers
support.
In this study, we synthesized PGS-based copolymers via atom trans- The respective polymer solutions, either PGS-PMMA alone (10 wt%)
fer radical polymerization (ATRP). ATRP is a highly efficient “living” rad- or a blend of PGS-PMMA/gelatin (10 wt%) at two different ratios (75:25,
ical polymerization method to synthesize polymers with predesigned PGS-PMMA/Gel25 and 50:50 PGS-PMMA/Gel50), were prepared by dis-
compositions, topologies and functionalities [24–28]. In our previous solving the polymers in 1,1,1,3,3,3-hexa-fluoro-2-propanol (HFP). Each
work, we have reported the successful synthesis of PGS-poly(ethylene solution was loaded into 3 ml plastic syringes fitted with a blunt 25-
glycol) methyl ether methacrylate (PEGMA) copolymers for the devel- gauge needle. The feed rate of the polymer solution was 1 ml/h using
opment of thixotropic supramolecular hydrogel with self-healing prop- a syringe pump. A high voltage (10 kV) was applied onto the needle
erties [29]. Here, methyl methacrylate (MMA) monomers were grafted to draw the nanofibers onto collector plate covered with aluminum
onto PGS macroinitiators to synthesize PGS-PMMA copolymer. Its spin- foil (~ 15 cm to the needle). After spinning, the obtained fibers were
ning ability of the copolymer was evaluated. PMMA is a FDA-approved dried overnight under vacuum and used for characterization and cell
polymer and has been used for various TE applications. PMMA-based culture study.
copolymers have also been used as nerve guidance channels and it
was demonstrated that PMMA could provide suitable stiffness for
2.4. Characterization of nanofibers
nerve regeneration [30]. A natural polymer, gelatin, was also blended
into the PGS-PMMA nanofibers to enhance the bioactive of the
The structure and morphology of the PGS-PMMA/gelatin nanofibers
electrospun scaffolds. PC12 cells were seeded onto the nanofibrous scaf-
were evaluated by scanning electron microscopy (SEM, 6360LA, JEOL,
folds to investigate their potential for nerve TE.
Japan). The nanofibers were firstly coated with gold by using sputter
coater (JEOL JFC-1200 fine coater, Japan). Fiber diameter was deter-
2. Materials and methods
mined from 50 random measurements per image by using Image J (Na-
tional Institutes of Health, Bethesda, USA).
2.1. Materials and cells
The surface hydrophilic properties of the electrospun nanofibers
were evaluated by sessile drop water contact angle measurement
All chemicals were purchased from Sigma-Aldrich Chemicals and
using sing a VCA Optima Surface Analysis system (AST products, Biller-
used as received except where noted. MMA was run through a basic alu-
ica, USA). Droplets of 0.5 μl deionized water were dropped onto the drop
mina column before use to remove the inhibitor. PGS (Mn = 4080 and
fibers five samples were used for each test.
polydispersity index, PDI = 4.34) was synthesized using previously
published polycondensation methods [29]. Briefly, an equimolar of
sebacic acid and glycerol was weighed and mixed under N2 for 1 day. 2.5. In vitro cell culture
The mixture was reacted at 120 °C under 30 mTorr vacuum for 2 days
to obtain PGS (Supporting Information Fig. S1). Rat PC12 cells were pur- PC12 cells were cultured in DMEM supplemented with 10% horse
chased from ATCC, USA. Fetal bovine serum (FBS), horse serum (HS) serum, 5% fetal bovine serum and 1% antibiotic/antimycotic solution.
and trypsin/EDTA were obtained from GIBCO Invitrogen, USA. After sterilized under UV light for 2 h, the electrospun nanofibers were
placed in 24-well plates and were seeded with PC12 cells at a density
2.2. Synthesis of PGS-PMMA copolymers of 5,000 cells/well. The cells were incubated in a humidified incubator
at 37 °C with 5% CO2 and the medium was changed every 2 days.
PGS (2 g, containing 8 mmol of\\OH groups) was weighted into a To assess cell proliferation, the alamarBlue assay was carried out
reaction flask, and dried at 50 °C in vacuum oven for overnight to re- after 2, 4, 6 and 8 days of culture. At each time point, the seeded scaf-
move water. The dried PGS was dissolved in 30 ml of anhydrous tetra- folds were incubated for 4 h at 37 °C with pure DMEM supplemented
hydrofuran (THF) at room temperature under nitrogen atmosphere. with 10 vol% alamarBlue. Tissue culture plate (TCP) was used as positive
Then triethylamine (48 mmol, 6.69 ml) was added into PGS solution. control. After incubation, 100 μl medium from each well was transferred
After that, 10 ml of anhydrous THF containing 2-bromoisobutyryl bro- to a 96-well black polystyrene microplates and replicated 3 times. Then
mide (BIBB, 40 mmol, 5 ml) was added dropwise into the PGS solution the fluorescence was measured using a microplate reader (Varioskan
under rapid stirring over 1 h in ice-water bath. The reaction mixture was Flash Multimode Reader, Thermo Scientific) with excitation wavelength
left to stir for 1 day at room temperature. After that, the reaction mix- at 560 nm and emission wavelength at 590 nm.
ture was centrifuged and the supernatant was precipitated with Morphological study of PC12 cells on different nanofibers was car-
1000 ml of ether. The PGS macroinitiator (PGS-Br) was obtained and ried out after 8 days of cell culture by SEM. The cell-fiber constructs
dried under vacuum at 50 °C. The number of initiator sites on PGS was were rinsed with PBS and then fixed in 3% glutaraldehyde. The nanofi-
determined by 1H nuclear magnetic resonance (NMR). bers were further rinsed in deionized water and dehydrated with in-
PGS-Br (0.4 g), MMA (30 g), 1,1,4,7,10,10-hexa-methyltriethy- creasing concentrations of ethanol (50% to 100%). Finally, the cell-fiber
lenetetramine (1.76 g) and 100 ml of degassed isopropanol were constructs were treated with hexa-methyldisilazane and air dried in a
added into a dry flask. The mixture was stirred at room temperature fume hood overnight.
and purged with dry nitrogen for 45 min. After that, a trace amount of
copper(I) bromide was added and the mixture was purged with dry ni-
trogen for another 3 min at room temperature before leaving it to react 2.6. Statistical analysis
overnight at room temperature. The final reaction mixture was diluted
with THF and passed through a short basic Al2O3 column to remove All the data presented are expressed as mean ± standard deviation
the copper catalyst. The resulting solution was precipitated with of the mean. Student's t-test and one-way ANOVA were used, and differ-
1000 ml hexane twice. The PGS-PMMA copolymer was collected by cen- ences between the groups are considered statistically significant at
trifugation and dried under vacuum at 40 °C. The obtained PGS-PMMA p b 0.05.
 J. Hu et al. / Materials Science and Engineering C 70 (2017) 1089–1094 1091

Fig. 1. Synthetic route for PGS-PMMA copolymer.

3. Results and discussion of BIBB-to-PGS, partial conversion could be achieved and the
unreacted hydroxyl groups could be used for other modifications
3.1. Synthesis of PGS-PMMA or polymerizations.
Next, PGS-Br was reacted with monomer MMA via ATRP to yield
The PGS-PMMA copolymer was synthesized via ATRP reaction, as PMMA-grafted PGS copolymer. The 1H NMR spectra of PGS-PMMA co-
shown in Fig. 1. PGS contains plenty of hydroxyl groups which reacted polymer was shown in Fig. 2. The PGS peaks were difficult to be distin-
with BIBB to form the PGS-Br ATRP macroinitiator. After the successful guished as PGS was present in a very small mass fraction (b5%) in the
brominization, NMR showed an addition of the distinctive peak at copolymer. On the other hand, characteristic peaks of PMMA were
about 1.9 ppm. This peak corresponded to the methyl protons of the ini- shown at 0.8, 1.0 and 3.6 ppm corresponding to methyl protons. As
tiating sites added to PGS (Supporting Information Fig. S2). After calcu- shown in Table 1, the PGS prepolymer showed a molecular weight of
lation, 94% conversion was achieved consistently with an excess of 5 M 4.1 kDa and a high PDI of 4.34, while the copolymer showed a molecular
equivalents of BIBB added to the PGS prepolymer. In our previous work, weight of 82.2 kDa and a PDI of 1.55. Based on the molecular weight of
we also demonstrated that the degree of brominization was adjustable PGS, the mass fraction of PGS in the copolymer was 4.8%. ATRP is a well-
by varying the BIBB content in the reaction [29]. With lower ratio known polymerization technique that could control the molecular
weight of the grafted polymer chains. In the previous work, PGS-
polyethylene glycol methyl ether methacrylate copolymers with vari-
ous molecular weights were synthesized by varying the feeding ratios.
Another study demonstrated that lignin-PMMA copolymers with differ-
ent molecular weight (ranging from 11 to 107 kDa) could be synthe-
sized by ATRP [26]. Moreover, the DSC result indicated that the
glass transition temperature (Tg) of the copolymer increased to 115 °C
from − 17 °C for the PGS prepolymer. Moreover, tensile test results
showed that the PGS-PMMA exhibited significant higher elongation at

Table 1
Material Characterization of PGS and PGS-PMMA.

Samples Feed ratio Tm (°C) Tg (°C) Mn (g/mol) PDI

[Br]:[MMA]

PGS N.A. 8.3 −17 4,100 4.34

Fig. 2. 1H of NMR spectra of PGS-PMMA. Peaks a, b and c corresponded to the various
PGS-PMMA 1:75 N.A. 115 82,200 1.55
different types of protons in the PMMA segment of the PGS-PMMA.
1092 J. Hu et al. / Materials Science and Engineering C 70 (2017) 1089–1094

break (5.74%) compared to pure PMMA (1.92%) (Supporting Informa- added. This marked increase in viscosity led to the higher fiber diameter
tion Fig. S3), indicating the positive effect of PGS core in PMMA. The of the PGS-PMMA/gelatin nanofibers. This hypothesis was supported by
PGS-PMMA exhibits a solid phase in room temperature, which allows Khademhosseini's group who fabricated PGS/gelatin blending nanofi-
it to be built up to 3D architectures by electrospinning. bers for cardiac TE. They also reported that higher gelatin content result-
ed in thicker nanofibers [41]. In their study, PGS/gelatin nanofibers
3.2. Electrospinning of PGS-PMMA/gelatin nanofibers required chemical-crosslinking treatment to stabilize the scaffolds for
in vitro/vivo applications, as PGS or gelatin alone would degrade too rap-
Electrospinning of fine nanofibers requires polymers with suitable idly in vivo. On the other hand, PMMA grafted PGS copolymer showed
molecular weights. As shown in Fig. 3, PGS-PMMA with Mn of good stability in body fluids, maintaining the nanofibrous architecture
82.2 kDa was able to generate thin and uniform nanofibers with a and mechanical properties of the scaffolds in vitro and probably
fiber diameter of 167 ± 33 nm. It is the first report on the in vivo. Without any toxic chemical-crosslinking treatment, both PGS-
electrospinning of PGS-based copolymers. In previous studies, PGS- PMMA/Gel25 and PGS-PMMA/Gel50 nanofibers retained their fibrous
based electrospun fibers had to be obtained by blending or coaxial spin- structure and integrity even after incubating in vitro for N1 week.
ning with other carrier polymers [31–33]. Khademhosseini's group Water contact angles and hydrophilicity of a TE culture substrate is
engineered electrospun PGS-based fibers by blending PGS with relative to its protein deposition and cell adhesion. The incorporation
poly(ε-caprolactone) (PCL) [17,22,34–36]. They reported that higher of gelatin had proven to improve the hydrophilicity of PGS-PMMA
PGS concentrations in the system would generate fibers with higher di- nanofibers. Fig. 3 demonstrated that with increasing gelatin concentra-
ameters. The fiber diameters increased from 2.7 μm for 2:1 PGS:PCL tion, the water contact angle significantly decreased from 140 ± 2° for
ratio to 8.7 μm for 5:1 PGS:PCL ratio [22]. Electrospun fibers containing PGS-PMMA down to 21 ± 1° for PGS-PMMA/Gel50. It was widely re-
a PGS core and poly(L-lactic acid) (PLLA) shell were fabricated with a ported that blending natural polymers, such as collagen and gelatin,
fiber diameter of ~ 2 μm by using coaxial electrospinning technique could increase the hydrophilicity of electrospun nanofibers [9,13,42].
[20,37]. Core/shell PGS/poly(vinyl alcohol) (PVA) nanofibers ranging The hydrophilic interface together with nanofibrous structure might
from 200 to 500 nm were reported by the same group, and thermal provide essential topographic cues that favor cell attachment, prolifera-
crosslinking processing was required for the fibrous substrates as TE tion and even differentiation.
scaffolds [38]. In comparison, our PGS-PMMA nanofibers were remark-
ably thinner than the reported PGS-based fibers. It was demonstrated 3.3. In vitro culture of PC12 cells on PGS-PMMA/gelatin nanofibers
that fiber diameter would influence the growth of nerve cells. He et al.
reported that the surface morphologies of fibrous scaffolds, including PGS is a well-known biomaterial for cardiac TE as the elastomer's
fiber pattern and size, play important roles in regulating the viability, stiffness is compatible with that of the heart tissue. In this study, we
proliferation and neurite outgrowth of neural cells [39]. Their results demonstrated the potential of PGS-based scaffolds for nerve TE. Fig. 4
showed that cells on the fibrous scaffolds with lower fiber diameters showed the cell proliferation results carried out on electrospun scaffolds
(b500 nm) exhibited longer neurites and higher neural markers com- by using PC12 cells. Higher cell proliferation was observed on PGS-
pared those grown on thicker fibers. PMMA/Gel nanofibers compared to those on PGS-PMMA only nanofi-
Adding gelatin into PGS-PMMA significantly (p b 0.05) increased the bers. Increased gelatin concentration also resulted in higher cell prolif-
fiber diameters from 225 ± 42 nm for PGS-PMMA/Gel25 to 631 ± eration on the nanofibers. At all time points, PGS-PMMA/Gel50
156 nm for PGS-PMMA/Gel50 with 50% gelatin. It was interesting to ob- showed significantly higher cell proliferation values than those on
serve the fiber diameter increasing, as blending gelatin into synthesized PGS-PMMA only scaffolds. After 8 days of cell culture, there was 38.6%
polymer commonly leads to a drop in fiber diameter [40]. Our observa- higher cell proliferation on PGS-PMMA/Gel50 nanofibers compared to
tion might be attributed to the increase in viscosity of the that on PGS-PMMA fibers. This could be attributed to the preferential
electrospinning solution (PGS-PMMA in HFP) when gelatin was proliferation of neuronal cells on nanofibers containing natural

Fig. 3. SEM images showing the morphology of electrospun nanofibers (A) PGS-PMMA, (B) PGS-PMMA/Gel25, (C) PGS-PMMA/Gel50, and water contact angles of the nanofibers (D) PGS-
PMMA, (E) PGS-PMMA/Gel25, (F) PGS-PMMA/Gel50.
 J. Hu et al. / Materials Science and Engineering C 70 (2017) 1089–1094 1093

SEM. There were very few cells attached on TCP and PGS-PMMA nano-
fibers. These cells showed spherical morphology, indicating that they
were still undifferentiated. On the other hand, there were a larger num-
ber of cells observed on both of the PGS-PMMA/gelatin nanofibers com-
pared to that on TCP and PGS-PMMA only, which was in agreement
with the cell proliferation results. Moreover, some of these cells spread
out and elongated to form long processes of neurite varicosities, indicat-
ing their differentiation into a neuron-like phenotype [51,52]. Such cells
also tried to interact with the nanofibrous scaffolds and communicate
with the neighbor cells through nanofibers. It has been known that
PC12 cells require either special chemical treatments or the presence
of growth factors to drive neural differentiation. Binan et al. developed
PLLA/gelatin electrospun non-woven materials with a degradation
rate and mechanical properties similar to that of the peripheral nerve
tissue. They demonstrated that, with the treatment of retinoic acid
and purmorphamine, neural stem-like cells cultured on the bioactive
nanofibers could differentiate into motor neuronal lineages by express-
ing beta-III-tubulin, HB-9, Islet-1, and choactase markers [53]. It was
Fig. 4. Proliferation of rat PC12 cells on PGS-PMMA/gelatin nanofibers, as determined by also reported that the controlled-release of nerve growth factor from
alamarBlue. #p b 0.05 compared to bare tissue culture place (TCP) at each time point; PLA/silk fibroin nanofibers promoted neurite outgrowth and enhanced
⁎p b 0.05 compared to PGS-PMMA nanofibers at each time point.
the differentiation of PC12 cells by expressing neurofilament 200 pro-
tein [54]. However, in this paper, the PGS-PMMA/gelatin nanofibrous
polymers. Various natural polymers, including collagen [43], gelatin scaffold showed the potential to induce the differentiation of PC12
[44], chitosan [45], fibronectin [46] and laminin [47], have been incorpo- cells into neuron-like cells even in the absence of any nerve growth fac-
rated into electrospun nanofibers for nerve TE. Gelatin, a derivative from tor or chemical treatment. Further work will be carried out to optimize
collagen, presented amide functional groups on the surface of the the mechanical properties of the PGS-based nanofibers and investigate
nanofibrous scaffolds to provide chemical and biological cues for cell ad- the mechanism of the scaffold-triggered neural differentiation.
hesion and growth. Ghasemi-Mobarakeh et al. demonstrated that
blending gelatin into PCL nanofibers enhanced the proliferation and dif- 4. Conclusion
ferentiation of nerve stem cells (C17.2 cells) compared to pure PCL
nanofibers [48]. It was also reported that coating gelatin onto the sur- PGS is a recently developed biodegradable polymer that is promising
face of PCL nanofibers favored the spreading and proliferation of endo- for various TE applications. To explore new applications of PGS, PGS-
thelial cells compared to the uncoated fibers [49]. Similarly, rabbit based copolymers were synthesized via ATRP. The new PGS-PMMA co-
cardiomyocytes cultured on PCL/gelatin blending nanofibers showed polymers showed tunable molecular weights, thermal properties and
higher proliferation rate compared to those on PCL fibers [50]. excellent electrospinnability. Uniform electrospun nanofibers were
Fig. 5 shows the morphology of PC12 cells on tissue culture plate engineered from pure PGS-PMMA and PGS-PMMA/gelatin blends. The
(TCP), PGS-PMMA and PGS-PMMA/gelatin nanofibers observed by PGS-PMMA/gelatin nanofibers exhibited good biocompatibility with

Fig. 5. SEM images showing the morphology of PC12 cells on (A) TCP, (B) PGS-PMMA, (C) PGS-PMMA/Gel25 and (D) PGS-PMMA/Gel50 nanofibers. The cell remained spherical on the TCP
and PGS-PMMA surfaces, implying a lack of differentiation. In the presence of gelatin on the nanofibers, the PC12 cells started to elongate and differentiate into a morphology more similar
to that of neural cells.
1094 J. Hu et al. / Materials Science and Engineering C 70 (2017) 1089–1094

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