journal of the mechanical behavior of biomedical materials 65 (2017) 428 438

Available online at www.sciencedirect.com

www.elsevier.com/locate/jmbbm

Preparation and characterization of electrospun

alginate/PLA nanobers as tissue engineering
material by emulsion eletrospinning

Weihong Xua,1, Renzhe Shenc,1, Yurong Yana,n, Jie Gaob,**

a
Department of Polymer Materials and Engineering, South China University of Technology, Guangzhou 510640,
People's Republic of China
b
The Afliated Hospital of Guangzhou Medical University, Guangzhou, People's Republic of China
c
The Afliated Zhongshan Hospital of Xiamen University, People's Republic of China

ar t ic l e in f o abs tra ct

Article history: Scaffolds made by biomaterials offer favorite environment for cell grow and show a wide
Received 4 July 2016 potential application in tissue engineering. Novel biocompatibility materials polylatic acid
Received in revised form (PLA) nanober membranes with favorable biocompatibility and good mechanical strength
6 September 2016 could serve as an innovative tissue engineering scaffold. Sodium alginate (SA) could be
Accepted 7 September 2016 used in biomedical areas because of its anti-bacterial property, hydrophilicity and
Available online 13 September 2016 biocompatibility. In this article, we chose PLA as continuous phase and SA as dispersion

Keywords: phase to prepare a W/O emulsion and then electrospun it to get a SA/PLA composite

Polylatic acid nanober membranes. The CLSM images illustrated that the existence of SA was located

Alginate on the surface of composite bers and the FTIR results conrmed the result. A calcium ion

Emulsions electrospinning replacement step was used as an after-treatment for SA/PLA nanober membranes in

Tissue engineering order to anchor the alginic ion in a form of gelated calcium alginate (CA). The single ber
tensile test shows a good mechanical property of CA/PLA nanober membranes, and the
nanober membranes are benecial for cell proliferation and differentiation owing to MTT
array as well as Alizarin red S (ARS) staining test.
& 2016 Elsevier Ltd. All rights reserved.

1. Introduction tissue formed will become versatile (Langer and Vacanti,

Tissue engineering is an interdisciplinary eld that combines
the engineering with the life sciences to biological substi-
tutes. Biomaterials play an important role in the particular
elds by providing matrices for cellular growth, proliferation,
and new tissue formation. With more precisely controlling
scaffold material, the types of scaffold and the quality of
2016). In the past ten years, some obvious development in
electrospun nanobrous scaffolds have been used for tissue
engineering. Biodegradable polymer like poly(lactic-co-glyco-
lic) acid (PLGA) (Mehrasa et al., 2015), poly(-caprolactone)
(PCL) (Hu et al., 2016), polylactic acid (PLA) (Santoro et al.,
2016) have been used as tissue engineering scaffold materials
in many potential application elds.

n
Corresponding author.
nn
Corresponding author.
E-mail addresses: yryan@scut.edu.cn (Y. Yan), 48014713@qq.com (J. Gao).
1
These authors contributed equally to this study and should be considered as co-rst authors.

http://dx.doi.org/10.1016/j.jmbbm.2016.09.012
1751-6161/& 2016 Elsevier Ltd. All rights reserved.
 journal of the mechanical behavior of biomedical materials 65 (2017) 428 438
Polylactic acid (PLA) has been widely used in variety of
biomedical application owing to its biocompatibility, biodegrad-
ability and good solubility in some organic solvents like
dichoromethane, chloroform, acetone, N,N-Dimethylforma-
mide and Dimethylacetamide (Casasola et al., 2014). When
designing to be a cell scaffold, Polylactic acid, alone or combin-
ing with other biodegradable material, provides an excellent
environment for cell growth because of its physical properties
(Armentano et al., 2013; Santoro et al., 2016). However, the
hydrophobic surface of PLA is application limitation in a
hydrophilic bio-environment. For example, it will occur large
dimensional changes in the scaffold which would be harmful to
the anchored cells. The hydrophobicity of PLA also results in
low cell afnity and it can elicit an inammatory response from
the living host upon direct contact with biological uids (Guo
et al., 2015; Murariu and Dubois, 2016; Stankevich et al., 2015). In
order to expand the use of PLA, hydrophilic polymer like
polyethylene oxide (PEO) (Zhao et al., 2012), poly(vinyl alcohol)
(PVA) (Abdal-hay et al., 2016) have been used in the modica-
tions of PLA to improve its hydrophilicity.
Sodium alginate (SA) has various applications such as
environmental, clinical, biomedical and other areas because
of its anti-bacterial, hydrophilicity and biocompatibility. It's
one of the widely used dressing materials because it tends to
dissolve or gel in the wound bed and form a kind of swelled
calcium alginates after the ion-change reaction between
sodium alginate and Ca2 after it contact with blood or tissue
uid, and then block the further bleeding of wound. Also
because of its good hydrophilicity and biocompatibility, it
have been used in dental impression material (Won et al.,
2014; Wang et al., 2003; Yan et al., 2013). Zhu et al. (Zhu and
Shen, 2002) successfully use sodium alginate to modify PLA
lms via an entrapment method, which enhanced the hydro-
philicity of the PLA lms. However, this simple surface
treatment method is just suitable for lms, limiting the
application of PLA in other elds.
Electrospinning was rst patented by Du Pont and got
further improvement over the next decades to produce com-
posite, shaped or matrix bers composed of a mixture of two
or more components (Arthur and Edwin, 1956; Hener, 1942).
The emulsions applied for electrospinning contain an water
phase which could dissolve bioactive protein, hydrophilic drug
and biomaterial, and the oil phase which is a polymer
(biocompatible and biodegradable) dissolved in organic solvent
(Tian et al., 2012). During the process of electrospinning,
emulsion containing oil phase and aqueous phase can be
elongated and form bers with coreshell structure (Li et al.,
2010) or aqueous phase will be randomly distributed within
the ber membrane ix (Chew et al., 2005). Li et al. (2013)
electrospun CNC-PLA water-in-oil(W/O) emulsion to get core
shell or hollow structure bers, and founding a correlative
relationship between emulsion droplet size and ber structure.
In our research, nanobers with sodium alginate and
polylactic acid were fabricated by using w/o emulsion elec-
trospinning. Here, sodium alginate can gel and block the pore
of the dressing then provide an environment for the wound
bed, and PLA can provide the tensile strength for the whole
dressing. In this paper we will discuss whether this kind of
special composite ber have the potential application as a
kind of tissue engineering material.
429
2. Experiment
2.1. Material
Sodium alginate A2158 was purchased from Sigma-Aldrich,
surfactant Span80 (chemical grade) was purchased from
Tianjin Fuchen Chemical Reagent Factory. Polylactic acid
(PLA) was supplied by Natureworks LLC, with the product
code of PLA 403 2D. Chloroform (analysis grade) was pur-
chased from Tianjin Fuchen Chemical reagent factory (Tian-
jin, China), DMSO and Cholamine were purchased from
Tianjin Fuyu Fine-Chemicals Co., Ltd. (Tianjin, China), and
Rhodamine B was purchased from Sigma-Aldrich. All of the
solvents were used without further purication.
2.2. Preparation and characterization of SA/PLA emulsion
2.2.1. Preparation of SA/PLA emulsion
SA/PLA emulsions were prepared as follows (Yan et al., 2013):
SA was dissolved in distilled water to become 40 mg/ml
aqueous solution. Span80 (0.3 g) was rst dissolved in 10 ml
Chloroform; subsequently, different amount of SA solutions
were added under high stirring speed of 7000 r/min for 10 min.
Finally, PLA was added into the solution, and the mixture was
stirred at 240 rpm for 2 h to obtain uniform emulsions.
2.2.2. Characterization of SA/PLA emulsion
The viscosities of emulsions were measured on a NDJ-79
rotational viscometer (Shanghai Changji Instruments Co.,
Ltd) at 25 1C. The surface tensions of emulsions were tested
on OCA-20-LHT surface tensiometer (Dataphysics, Germany)
at the temperature of 25 1C.The conductivities of the emul-
sion were measured on a CON510 conductivity meter (Eutech
Instrument, Singapore) at the test temperature of 25 1C.
2.3. SA/PLA emulsion electrospinning
Before the electrospinning, emulsions were stirred at the speed
of 7000 r/min for ten minutes to get a stable emulsion systems
and then emulsions were placed in a horizontally positioned
plastic syringe (1 ml) with a at end metal needle (0.6 mm in
inner diameter) respectively. The syringe was operated by a
syringe pump to feed solution at a rate of 0.5 ml/h, and the
needle was subjected to a high-voltage DC power of 15 kV. The
collector was positioned 15 cm away from the needle tip, which
was covered by a sheet of aluminum foil to collect bers.
2.4. Characterization of SA/PLA ber membrane
2.4.1. Scanning electron microscope
The Fiber morphology and its diameter distribution were
observed on a Nano430 scanning electron microscope. Each
sample was coated with gold with 5-nm Au/Pd in a Vacuum
sputtering before SEM observation. The diameter of bers in
SEM images was analyzed by using image analysis program.
2.4.2. Confocal laser scanning microscope analysis
In order to identify the sodium alginate domain in the SA/PLA
ber, we dyed ber with Rhodamine B, and tested it on Leica
430 journal of the mechanical behavior of biomedical materials 65 (2017) 428 438
TCS SPE Confocal Laser Scanning Microscope (Germany).
Rhodamine B was rst dissolved in Dimethyl sulfoxide
(DMSO) to prepare 1 mg/ml solution and 50 L of this solution
were added into SA solution. After stirring at 40 1C for 1 h,
25 L ethanol amine were added into the solution to termi-
nate the reaction. In the Confocal laser scanning microscope
analysis, laser wavelength of 532 nm was used to excite
uorescence to track the Rhodamine B in the bers (Olivier
and Moine, 2013).
2.4.3. Fourier transform infrared analysis
Infrared spectra for PLA, SA powder and SA/PLA ber mem-
brane (before and after immerged distilled water) were taken
on Fourier transform infrared spectroscopy (Vector 33, Bru-
ker, Germany). All the spectra were used attenuated total
reectance module in the spectral range of 4000500 cm  1 by
accumulation of 30 scans. The SA/PLA ber membrane
immerged distilled water for 24 h and then dried in a vacuum
oven at 50 1C for 12 h before the FTIR test.
2.5. Preparation and mechanical tests of CA/PLA
membrane
2.5.1. Preparations of CA/PLA membrane
Ion exchange is a good method to change the water-soluble
SA into a water-insoluble CA because the CaCl2 solution was
used to replace the Na to get calcium alginate (CA) which
will not dissolve in the hydrophilic environment. In this
research, we added 1 ml of 0.2 g/ml CaCl2 solution on the
SA/PLA membrane by electrostatic spraying to obtain a
CA/PLA membrane (a). To prepare a comparison sample,
SA/PLA membrane was immersed in 100 ml of CaCl2 solution
with the concentration of 0.01 g/ml (b) (Chan et al., 2002; Fu
et al., 2011). All CA/PLA membranes were dried in a vacuum
oven at 40 1C for 24 h.
2.5.2. Mechanical test
Mechanical properties of membranes were determined by
YGOO4D single Fiber Strength Tester (Changzhou Second Textile
Machines Co., Ltd, China) at ambient condition. All samples,
including PLA, SA/PLA, CA/PLA(a), CA/PLA(b) membranes respec-
tively, were cut into 30  1 mm for the nal test, and each
sample were tested at least 15 times and an average value was
as a nal result.
2.5.3. Hydrophilic test
Hydrophilic/hydrophobic nature of the PLA, CA/PLA mem-
brane were evaluated by a CAST2.0 contact angle analysis
system (OCA20LHT-TEC700-HTFC1500, Dataphysics, Ger-
many) at room temperature and the tested ber membranes
were 1  1 cm. Images of the droplet on membranes were
taken after 15 s of contact time, and the hydrophilicity of the
ber membranes were measured by the acquired images
according to GB/T 30447-2013.
2.5.4. Cell proliferation assay
The ber membrane were precut into 10 mm in diameter and
washed by phosphate buffered saline (PBS) three times before
placed in a 24-well plate. Cells were detached by adding 1 ml of
0.25% trypsin containing 0.1% EDTA and then centrifuged,
counted as well as seeded on the membrane. They were
cultured in media for 1, 3, 5 days in 37 1C in a 5% CO2 incubator.
Cell proliferation was evaluated using MTT test, in which 50 L
of water-soluble MTT (3-(4,5-dimethyl-2-thiazolyl)2,5-diphe-
nyl-2-H-tetrazolium bromide) solution in each well and incu-
bated for 4 h at 37 1C. After removal of the medium, the
converted dye was dissolved in 500 L/well DMSO (dimethyl
sulfoxide). Solution(150 L) of each sample was transferred to a
96-well plate. Absorbance of converted dye is measured at a
wavelength of 490 nm using a micro-plate spectrophotometer
(model 680; Bio-Rad Laboratories, HercLes, CA).
2.5.5. Cell differentiation assay
The ber membrane were precut into 10 mm in diameter and
washed by a phosphate buffered saline (PBS) three times
before placed in a 24-well plate. Cells were detached by
adding 1 ml of 0.25% trypsin containing 0.1% EDTA and then
centrifuged, counted as well as seeded on the membrane.
They were cultured in media for 14 days in 37 1C in a 5% CO2
incubator. The level of cell mineralization was evaluated by
Alizarin red S (ARS) staining. The cell was washed with PBS
3 times and xed in 4% (v/v) formaldehyde at room tempera-
ture for 10 min. The xed cells were stained with 5% Alizarin
red S (pH 7.0) for 15 min at 25 1C. Then, the cell was washed
with deionized water and observed by Stereoscopic micro-
scopes (Leica EZ4).
3. Results and discussion
3.1. Physical properties of SA/PLA emulsions
According to Table 1, SA/PLA emulsions composing oil phase,
surfactant and different ratios of water phase were prepared.
The viscosity and surface tension are two factor inuen-
cing the eletrospinning process of polymer solution, and all of
them will be inuence by the second phase of SA in SA/PLA
emulsion solution. In order to study how SA distributed in the
PLA solution and its inuence on solution properties, we
studied the viscosity and surface tension of solutions, and
variation trend were shown in Fig. 1.
Emulsions were composed by two kinds of immiscible
liquids like water and oil. An emulsifying agent was added as
the third component to reduce the interaction between water
and oil and stabilize the dispersed-phase micelles. The viscosity
of the dispersed-phase (40 mg/ml SA solution) was 740 mPa/S
which was much higher than the viscosity of emulsions,
showing that the SA was packed perfectly in micelles.
Table 1 SA/PLA emulsions with different compositions.
Samples Volume of SA Span80 (g) CHCl3 (ml) PLA (g) SA/
solution PLA
(40 mg/ml) (w.t)
(ml)
A 0.0 0.3 18 1.92 0.0
B 1.2 2.5
C 2.4 5.0
D 3.6 7.5
E 4.8 10.0
 journal of the mechanical behavior of biomedical materials 65 (2017) 428 438
Fig. 1 The inuence of the percentage of SA on the viscosities and surface tensions of SA/PLA emulsions.
Fig. 2 The status of SA/PLA emulsion (Yan et al., 2013).
According to Figure, we can nd that viscosities of SA emul-
sions with the SA content of 2.5%, 5%, 7.5% and 10% were apart
from that of the pure PLA solution(37.2 mPa s) with the increas-
ing of SA content at the tested values of 45.8 mPa/S, 53.9 mPa/S,
59.6 mPa/S and 64.9 mPa s for the emulsions respectively.
According to Taylor's analysis (Taylor, 1932), the size of
dispersed-phase micelles increased with the percentage of SA
adding up, leading to the increasing of the viscosity of
emulsions.
The surface tension between water and oil phase was
reduced by the surfactants, thus facilitating the emulsica-
tion of the immiscible phases. Sorbitan monooleate (Span80)
is an nonionic, amphiphilic surfactant with a HLB value of
4.3, making it suitable for the preparation of water-in-oil
emulsion. Surrounded by span80 molecular, the SA solution
phase formed micelles, isolating SA solution from the PLA
solution, as shown in Fig. 2.
The surface tension of the 40 mg/ml SA solution were
64.2 mN/m, and the surface tension of PLA solution is
26.5 mN/m. Surface tensions of the emulsions increased
from 26.5 mN/m to 28.9 mN/m, 30.2 mN/m, 30.9 mN/m, and
32.8 mN/m as the SA concentration added from 0% to 10%
which were all below the 64.2 mN/m (surface tension of pure
SA solution), proving the contribution of the emulsion shown
in the Fig. 2. The polymer (like PLA) which was not amphi-
philic makes no obvious contribution to the surface tension
when dissolving (Zhu and Rosen, 1984), so the increase in the
surface tension of emulsions may be attributed to the change
431
of micells in the interface. The higher content of SA solution
added, the more micells formed in SA/PLA emulsion systems,
which reduce the Span80 molecule distributing in the emul-
sion surface. Surfactant can lower the surface tension, so the
surface tension of the emulsions increased with the decrease
of Span80 on the interface (Eriksson and Ljunggren, 1989).
The electrical conductivity of PLA solution and emulsions
were all too low to be measured, while the electrical con-
ductivity of 40 mg/ml SA solution was 8.76 mS/cm which was
much higher than the formers. This could be interpreted by
the following reasons: Firstly, The chloroform is a kind of
non-conducting solvent, thus making the solution shows
evident non-conductivity. When the PLA was added in the
chloroform, there were few ions in the PLA solution, which
will not contribute to the improvement of the electrical
conductivity of solution (Hou and Papadopoulos, 1997;
Nampoothiri et al., 2010). Secondly, Span80 is a kind of
nonionic surfactant which make no effects to the conductiv-
ity when dissolved in SA/PLA emulsions (Son et al., 2004). The
formation of micelles made the SA solution surrounded by
Span80, therefore, the conductivity of the emulsions were not
obvious.
3.2. Morphology of SA/PLA emulsion electrospun ber
membrane
During electrospinning, a polymer jet would be elongated
many times in tens of milliseconds, which eventually lead to
the formation of nanobers. Meanwhile, emulsion droplets
could be drawn with the polymer jet elongating, accompa-
nied by the fast jet solidication which was caused by the
evaporation of solvent in 0.1 s after bending instability (Fong,
2007).
The diameter of electrospun nanobers is controllable
owing to the mature electrospun technology. A wide range
of parameters can be manipulated to produce nanobers with
different diameter, and the parameters (Mitchell, 2015) can be
divided into three categories including feedstock properties
(like concentration, viscosity, surface tension, conductivity,
solvent, etc.) (Wang et al., 2009; Lu et al., 2010). Experimental
setup (like electrostatic eld strength, feed rate, etc.) (Li et al.,
432 journal of the mechanical behavior of biomedical materials 65 (2017) 428 438

2007; Lu et al., 2011) and environmental conditions (like
temperature, humidity, etc.) (Huang et al., 2011).
However, in our experiment, we use chloroform as a single
solvent to dissolve the polylactic acid (PLA) in order to
compare it with the alginate/PLA emulsion whose oil phase
is PLA/chloroform solution, which makes it difcult to control
the diameter of the PLA nanobers at experimental
established concentration conditions. The morphology and
diameter of electrospun nanobers strongly depends on the
type of polymer/solvent system and its concentration
(Casasola et al., 2014) and the solvent system we used to
prepare the PLA solution is not a most optimal state for
electrospinning at the established condition, so the diameter
of PLA nanobers is uncontrollable.

Fig. 3 Impact of the SA concentration on the morphology and diameter of electrospun SA/PLA ber membrane: (A) 0%
(B) 2.5% (C) 5% (D) 7.5% (E) 10% and (F) mean diameter of SA/PLA bers.
 journal of the mechanical behavior of biomedical materials 65 (2017) 428 438
Fig. 3 shows the SEM micrographs of the morphology and
diameter distribution of the electrospun SA/PLA bers. The
ber diameters of the brous scaffold without SA solution
were 7807200 nm in average (Fig. 3A), which was obviously
higher than SA/PLA emulsion electrospun bers. Affected by
the polymer jet elongation as well as the induction of the
electric eld, the behavior of demulsifying happened,
destroying the status of SA/PLA emulsion, which contributed
to the release of alginate and Na (Li et al., 2009, 2010). The
appearance of ions in the polymer jet results in a high charge
density on the surface of electrospinning jet, leading to a
higher elongation forces under the electrical eld. It's well
known that the overall tension in the bers depends on the
self-repulsion of the excess charges on the jet. Thus, as the
charge density increases, the nal ber diameter becomes
thinner (Stanger et al., 2009; Zong et al., 2002). In addition, the
morphology of SA/PLA bers with different concentration of
SA solution did not evident difference, and their diameters
were about 250790 nm.
3.3. The distribution of SA in ber membrane
In confocal laser scanning microscopy (CLSM) the light from
out-of-structures is fade out and this technique enables a
Fig. 4 CLSM images of nanobers with different concentration of dispersed content: (A) 2.5% SA (B) 5% SA (C) 7.5% SA (D) 10% SA.
433
non-destructive view through the ber, making the material
become transparent. By using different kinds of uorescence
labels, it is possible to make the unambiguous identication
of several compounds (Lamprecht et al., 2000). Fig. 4 listed the
CLSM images of nanobers with different concentration of SA
solution in SA/PLA emulsions and they showed no evident
distinction according to the changing concentration of dis-
persed content. It is obvious that the SA labeled by Rhoda-
mine B could be clearly observed along the ber direction, but
we could not distinguish whether the SA distributed on the
ber surface or inside the ber matrix from the CLSM images.
Since Span80 shows high-viscosity at the room temperature,
it is possible to have relatively high-mobility in the electro-
spinning polymer jets and Span80 would migrate from
emulsion spheres to the outer region and even the surfaces
of nanobers owing to the charge repulsion, bringing part of
the sodium alginate at the same time (Li et al., 2010). On the
other hand, during the processing of electrospinning, phase
separation took place and a few SA and Span80 molecules
transferred to the surface of PLA matrix owing to the
evaporation of solvents (Hu et al., 2013).
In order to verify this hypothesis, we used FTIR to prove
that part of the SA distributed on the surface of nanobers and
the results were shown in Fig. 5. For pure PLA, the strong
434 journal of the mechanical behavior of biomedical materials 65 (2017) 428 438
characteristic absorption bands at 1755 cm  1 attributed to
stretching vibration of CO bond. As for SA, the sharp peak
obtained at 1612 cm  1 was due to COC stretching of Pyrone
compounds. After comparing the characteristic peaks between
PLA and SA, we tried to nd out the change of FTIR spectra of
SA/PLA ber membrane before and after immersed in the
water. As shown on Fig. 6, there were no evident distinction
between the two FTIR spectra except the peak obtained at
1612 cm  1. Before immersed in the water, there existed a peak
at about 1612 cm  1 which belongs to the COC stretching of
SA, and it almost entirely disappeared after water treatment.
This phenomenon corresponded to the assumption that part
of the SA migrated outside during electrospinning, leading to
its distribution on the surface of nanobers.
3.4. Mechanical properties of CA/PLA membrane
Sodium alginate is widely used in biomedical materials owing
to its ability to form gels with divalent cations such as
Fig. 5 The FTIR spectra of pure PLA and SA powders.
Fig. 6 FTIR spectra of SA/PLA ber membrane before and
after water treatment.
calcium under mild condition. Calcium alginate have been
used in controlled release drug delivery system, wound
dressing and dental impression materials (King et al., 2008).
The sodium alginate with calcium ions is described by the
egg-box model, where calcium cations interact with guluro-
nic acid monomers in the cavities formed in the alginate
molecular chains (Fu et al., 2011; Gombotz and Wee, 2012)
(Fig. 7).
The rule of mixtures (ROM) is a model used to predict the
elastic modulus of composites. Which allows elastic modulus
calculation for composites by assuming equal amount of
strain on both bers and matrix (Facca et al., 2006). The
equation (Jones, 1975) of the model is expressed as below:
Ec Vf Ef Vm Em 1
where Vf and Vm are the volume fraction of the bers and
matrix, Ef and Em represent the elastic modulus of bers and
matrix respectively, and Ec is the modulus of composite.
Krenchel's factor (Krenchel, 1964) is added into the rule of
mixtures when considering the effect of ber length and
orientation, and it is expressed as below:
Ec l 0 Vf Ef Vm Em 2
Where l is the ber length distribution factor and 0 is the
Krenchel orientation factor. This rule of mixture provides a
simple way to estimate the elastic properties of the ber-
reinforced composites (Puska et al., 2004).
For the ber-reinforced composites, we could use those
rules to calculate the theoretical values of elastic modulus of
composites. However, in our experiment, the volume fraction
of the calcium alginate (Vm) is not a deterministic value,
which makes the calculation for elastic modulus inaccurate.
The reasons are as following: rstly, during the process of
emulsion electrospinning, the sodium alginate is formed into
two parts. One part is located into Span80 micelles in the
bers, and the other part is distributed randomly on the
surface of bers because some Span80 transfer to the outer
region of ber matrix (Li et al., 2010). Only the latter part
would transform from sodium alginate into calcium alginate
after Ca2 ions replacement reaction, which can contribute to
the elastic modulus. Secondly, during the process of replace-
ment reaction, some alginate will loss in the hydrophilic
environment, which reduce the volume fraction of the cal-
cium alginate in ber matrix. As a result, it is difcult to
Fig. 7 Sketch map of egg-box model (Fu et al., 2011).
 journal of the mechanical behavior of biomedical materials 65 (2017) 428 438 435

calculate a precise theoretical result of elastic modulus with- alginate gels (Clark and Ross-Murphy, 1987; Cuadros et al.,
out accurate volume fraction of the calcium alginate (Vm). 2012), which evidently promoted the mechanical properties
Fig. 8 showed the typical stressstrain curves of PLA, of the nanober membrane. Also, comparing to the electro-
SA/PLA and two kinds of CA/PLA nanober membranes. The static spraying method, the immersing sample showed
values of some mechanical properties including Young's higher degree of cross-linking ratio, which contributed the
modulus, tensile strength and elongation at break were better mechanical properties.
summarized in Table 2.
Compared with the pure PLA nanober membrane, the
3.5. Biological properties of CA/PLA membrane
elongation at break of the SA/PLA nanober membrane was
obviously higher, with the value sharply increasing thresh-
When material applied in the tissue engineering scaffold, the
old. After the replacement of Ca2, both the tensile strength
characteristic of hydrophilicity become very important,
and modulus of nanober membrane increased, especially
because this property could enhance the cell viability and
for the CA/PLA nanober membrane produced by immersing
proliferation (Maretschek et al., 2008). Fig. 9 showed the
method whose tensile strength and modulus rose from
hydrophilicity of pure PLA nanober membrane and CA/PLA
0.6470.05 MPa to 3.1370.24 MPa and 0.5170.07 MPa to
nanober membrane. Polylactic acid is a hydrophobic poly-
1.7870.11 MPa respectively. This could be result of the den-
mer, which could be easily observed from the picture (contact
sication of the cross-linked interaction in the calcium
angle higher than 901), while the CA/PLA nanober mem-
brane showed great hydrophilicity. Owing to the distribution
of alginate on the surface of ber during electrospinning, the
hydrophilic alginic acid ions existed on the surface. And after
the replacement of Na into Ca2, the alginic acid ions were
stayed while the surfactant Span80 and Na was removed,
which lead to the hydrophilic performance of nanober
scaffold.
Fig. 10 showed the cell proliferation of several kinds of
nanober membranes. Compared to the control group, the
pure PLA nanober membrane showed higher cell prolifera-
tion. This could be explained that in the natural tissues, cells
are surrounded by extracellular matrices (ECMs), whose
physical structural features ranging from nanometer scale
to micrometer scale. Owing to the previous study, the scales
of electrospinning nanober which were as closely as ECMs
were suitable for the cell's native in vivo environment (Mo
Fig. 8 Stressstrain curve of different nanober membrane.
et al., 2004), which were benecial for the cell growth and
(A) PLA (B) SA/PLA (C) CA/PLA electrostatic spraying sample
proliferation. In addition, the CA/PLA nanober scaffold play
(D) CA/PLA immersed sample.
a role as synthetic extracellular matrices for cell immobiliza-
tion and cell transplantation. The scaffold replace many
Table 2 Mechanical properties of nanober membrane. functions of the native ECMs, providing mechanical integrity
to the new tissue and a hydrated space for the diffusion of
Sample of Elastic Tensile stress Elongation at
nanober modulus (MPa) break (%) nutrients and metabolites, organizing cells to adhere and
membrane (MPa) grow (Jen et al., 1996; Putnam and Mooney, 1996). And
comparing to the smooth surface of PLA nanober, the sur-
PLA 0.2770.02 0.2570.03 31.274.7
face of composite nanober is coarser. And this irregularity
SA/PLA 0.6470.05 0.5170.07 118.5714.1
surface caused by disorder distribution of alginic provide
CA/PLA(a) 1.3470.08 1.3670.10 79.377.6
CA/PLA(b) 1.7870.11 3.1370.24 110.8710.9 enough sites for adequate cells to attach and grow, which
we can named coarsed adhesion and growth.

Fig. 9 Hydrophilic test photos of (A) PLA membrane and (B) CA/PLA membrane.
436 journal of the mechanical behavior of biomedical materials 65 (2017) 428 438
Fig. 11 showed the cell differentiation of the electrospin-
ning nanober membranes. The mineralized nodules, which
are the symbols of cell differentiation, would be stained by
Alizarin red S (Gregory et al., 2004). Two group of the CA/PLA
nanober membranes showed obvious color change, while
the control group and PLA nanober membranes had no
evident color change. Alginate gels are benecial for the cell
transplantation in tissue engineering, and the calcium algi-
nate hydrogels which were on the surface of the nanober
are used to deliver cells to the desired site, providing a space
for new tissue formation (Lee and Mooney, 2012). With the
help of the alginates, the CA/PLA nanober membranes were
good for the cell differentiation. As biomaterials, alginates
can easily be formulated into soft, elastic gels, which provide
an environment in which appropriate regulation of cell
behavior (e.g., adhesion, proliferation, migration and differ-
entiation) can occur then some kind of functional tissue can
form. And it would become a cell-delivery vehicle and a cell
immobilization matrix in the presence of calcium ions by
forming the egg-box model (Atala and Koh, 2005).
Animal tissue is composed of cells which embedded in an
extracellular matrix (ECM), and from a structural perspective,
natural ECM is a three-dimensional network consisted of
various protein and polysaccharide bers with diameters
ranging from tens to hundreds of nanometers (Wang et al.,
Fig. 10 The cell proliferation of different nanober
membrane: (A) control group, (B) PLA, (C) CA/PLA
electrostatic spraying, (D) CA/PLA immersed.
Fig. 11 The cell differentiation of different nanober membrane: (A) control group, (B) PLA, (C) CA/PLA electrostatic spraying,
(D) CA/PLA immersed. (For interpretation of the references to color in this gure legend, the reader is referred to the web
version of this article.)
2013). The diameter of electrospun alginate/PLA nanobers is
between 100 and 300 nm, which is in the size range of ECM,
making it suitable for cell proliferation and differentiation. In
our experiment, we focus on the comparison between PLA
nanobers with and without alginate, the effect of different
ion exchange method, which help us to conrm a compara-
tively good method to fabricate the scaffold. Previous results
show that, as the diameter of nanober decreased (from
7497153 to 283745), higher degree of proliferation was
observed (Christopherson et al., 2009). In our paper, the
diameter of aligate/PLA nanobers (4 groups: B, C, D, E) show
no evident difference, and the size of them is about
250790 nm. So in the following experiment, we will try to
fabricate aligate/PLA nanobers with evidently different dia-
meter and compare their results of cell proliferation and
differentiation.
Adhesive interactions between cells and the extracellular
matrices are governed primarily by integrins, which act as
cell surface adhesion receptors. Upon binding to extracellu-
lar ligands, integrins can associate with a diverse set of
cytoplasmic proteins, initiate signaling cascades and form
associations with cytoskeleton to provide physical links
between the cytoskeleton and the ECMs. And the nature of
adhesive interactions governs the survival, growth, motility
and differentiation of cells (Boudreau and Jones, 1999; Davey
et al., 1999; Dike et al., 1999). The alginate including the
sequence arginineglycineaspartic acid (RGD) have been
extensively used as model adhesion ligands, due to the
wide-spread presence of integrin receptor for this ligand
on various cell types (Koo et al., 2002; Lee and Mooney, 2012).
Appropriate ligands in alginate are crucial to promote and
regulate cellular interactions, especially for cell culture and
tissue engineering applications.
4. Conclusion
A method for producing electrospun SA/PLA membrane from
a single setup had been achieved by emulsion electrospin-
ning, which alleviates the need for two sets of separate
electrospinning systems or a coaxial electrospinning setup.
The possibility of having SA/PLA nanobers had been con-
rmed by SEM images and the distribution of SA on the ber
surface had been conrmed by CLSM images and FT-IR
spectra. Meanwhile, the nanober membranes showed good
 journal of the mechanical behavior of biomedical materials 65 (2017) 428 438
hydrophilic and preferable mechanical property after Ca2
ions replacement. The coarsed adhesion and growth of cells
on nanober scaffold materials and the integrins between
alginate and cells were benecial for cell proliferation and cell
differentiation. Due to these results, the prepared alginate/
PLA emulsion electrospun nanober membranes can serve as
an innovative type of tissue engineering material.
Acknowledgments
Weihong Xu and Renzhe Shen contributed equally to this
work and should be considered co-rst authors. The research
Grants supported by National Natural Science Foundation of
China Youth Foundation (No. 5110620) and National Natural
Science Foundation of China the state Key Program of (No.
21234003) are appreciated.
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