Materials Science and Engineering C 68 (2016) 397405

Contents lists available at ScienceDirect

Materials Science and Engineering C

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

Silane crosslinking of electrospun poly (lactic acid)/nanocrystalline

cellulose bionanocomposite
M. Rahmat a, M. Karrabi b,, I. Ghasemi a, M. Zandi c, H. Azizi a
a
b
c

Department of Plastics, Iran Polymer and Petrochemical Institute, P.O. Box: 14965/115, Tehran, Iran
Department of Rubber, Iran Polymer and Petrochemical Institute, Nano and Smart Polymers Center of Excellence, P.O. Box: 14965/115, Tehran, Iran
Department of Biomaterial, Iran Polymer and Petrochemical Institute, P.O. Box: 14965/115, Tehran, Iran

a r t i c l e

i n f o

Article history:
Received 18 March 2016
Received in revised form 18 May 2016
Accepted 24 May 2016
Available online 26 May 2016
Keywords:
Biocomposites
Electrospinning
Cellulose nanocrystals
Poly (lactic acid)
Silane crosslinking
Tissue engineering

a b s t r a c t
Biodegradable nanobrous mats fabricated by electrospinning are commonly used in tissue engineering, however, lack of essential mechanical properties of such nanobers is a challenging issue. In this work,
vinyltrimethoxysilane (VTMS) was grafted onto poly (lactic acid) (PLA) and the silane grafted PLA was subsequently applied in electrospinning process. Electrospun nanobrous mats based on PLA/nanocrystalline cellulose
(NCC) and PLA-g-silane/NCC nanocomposites were fabricated and immersed in hot water (70 C) for crosslinking
of silane grafted PLA. It was found that introducing NCC to the samples cause to reduction in ber diameter and
the other hand the silane crosslinking of PLA increase the mean ber diameter. DSC thermograms also revealed
that silane grafting caused a reduction in mobility of polymer segments, and consequently reduction of crystallinity. On the contrary, the NCC in the PLA-g-silane samples effectively inuenced the crystal nucleation, while
in the PLA nanobers the nucleation was lower. The impact of NCC on tensile strength enhancement of samples
was notable. The results suggested that the chemical crosslinking remarkably improves the mechanical properties of PLA nanobers. Furthermore, biocompatibility of such modied nanobers was also evaluated through cytotoxicity results, therefore the modied PLA nanocomposite can be considered as a practical candidate for hard
tissue engineering applications.
2016 Elsevier B.V. All rights reserved.

1. Introduction
One of the processing methods to produce non-woven nanobrous
mats is the electrospinning process, which has attracted a great interest
in recent years. Submicron diameters of electrospun bers lead to a
large area, making them an ideal candidate for medical applications
such as scaffold and high performance lter media [1,2].
Biodegradable polymers have found a fast-growing area of polymer
science [3]. Among the variety of biomaterials, poly (lactic acid) (PLA) is
an interesting polymer for preparing electrospun bers due to its biocompatibility and biodegradability in medical applications, especially
in tissue engineering [4,5]. However, it has been pointed out that, due
to its weak mechanical properties, PLA is not proper for the preparation
of hard tissues such as bone regeneration [6,7]. Many efforts, such as
changing polymer molecular structures [8,9], forming polymer blend bers [10,11] and adding nanoparticles [2,1214], have been used to improve the mechanical properties of PLA nanobers.
The mechanical properties of the nanobers can be enhanced using
different methods [6]. For example, introducing nano-structures results
in a high performance nanocomposite development. A wide variety of
Corresponding author.
E-mail address: M.karabi@ippi.ac.ir (M. Karrabi).

http://dx.doi.org/10.1016/j.msec.2016.05.111
0928-4931/ 2016 Elsevier B.V. All rights reserved.

nano particles including hydroxyapatite [2,15], clay [12,16], carbon

nanotube (CNT) [13,17,18] and nanocrystalline cellulose (NCC) [7,14,
19] have been incorporated in PLA nanobers to adapt the bers for particular applications. Nano clay particles have been added in electrospun
PLA bers to control their mechanical and biodegradation properties for
potential biodegradable packaging applications [16]. Electrospun PLA
nanobers for potential use as bone graft materials have been reinforced with incorporating carbon nanotubes [18].
NCC extracted from many renewable sources including wood, cotton, bacteria and tunicates have drawn increasing attention as reinforcing agents in nanocomposite bers [20,21]. High aspect ratio (length to
thickness ratio), high stiffness and low density, are some of NCC advantages beside biodegradability and relatively low in cost, that make it an
appropriate ller as a reinforcing agent in polymer nanocomposites
[2224].
A major drawback of NCC is its inherent hydrophilicity which results
in poor miscibility with common hydrophobe polymeric matrices. Surface modication of NCC by grafting various hydrophobic moieties to
improve its compatibility with polymers, is carried out to overcome
this problem [2527].
Xiang et al. [19] prepared nanocomposite bers of PLA/NCC
electrospun at elevated temperatures from PLA/DMF solutions. After
optimization of electrospinning conditions, they studied the effects of

398

M. Rahmat et al. / Materials Science and Engineering C 68 (2016) 397405

the nanocrystalline cellulose content on physical properties of the nano

bers. Presence of NCC, as a nucleating agent, increases crystallinity of
electrospun PLA nanocomposite bers. Because of increase in crystallinity and reduction of ber diameter, the tensile modulus of non-woven
fabrics was improved by incorporation of NCC into PLA. Shi et al. [7]
have found that the average ber diameter of PLA/NCC nanobers decreased in the higher NCC content. Their tensile results revealed that
NCC loading can improve strength and modulus of PLA nanobers.
Ramirez et al. [14] produced highly porous PLA/NCC nanobers using
electrospinning. The mechanical properties showed an increase in tensile strength value up to 200% for the nanocomposite scaffolds including
5% NCC. Cacciotti et al. [5] studied PLA based electrospun bers loaded
with NCC and silver nanoparticles. They prepared bers with average
diameter of about 500 nm and found that llers did not remarkably affect the PLA ber morphology and size. Thermal analysis data revealed
that the cellulose nanoparticles acted as crystallization nuclei, thus promoting a different rearrangement of the PLA chains. Mechanical analysis
showed a slight improvement in the nano ber composites properties.
Chemical crosslinking is a method to introduce covalent linkage between polymer molecules. Crosslinking of nanobers is carried out by
radiation or adding a chemical agent [9,28]. Silane agents are one of
the chemical materials used in crosslinking of some polymers [29,30].
The silane crosslinking method is a well-known two-stage process. In
the rst stage, vinyl silane is grafted to polymer chains through a peroxide free radical. In the second stage, the alkoxy groups of grafted silane
molecules are hydrolyzed in contact with hot water. Afterwards, these
hydroxyl groups are condensed to form stable Si-O-Si linkages as
crosslinks. It is worth noting that a few studies have been reported on
silane crosslinking of PLA nanobers [29,31]. Fig. 1 schematically illustrates the silane crosslinking of PLA.
Well dispersion of NCC and load transfer in the polymer/NCC interfaces are challenging issues facing improvement of mechanical properties in nanocomposites. It is clear that the stronger interfaces between
the NCC and polymer matrix improve the mechanical properties of the
nanocomposite nanobers [22]. Surface modication of NCC is one of
the methods used to improve PLA/NCC interaction. Further, it is expected that introducing covalent bonds improves interfacial interactions
leading to a greater reinforcing effect due to NCC incorporation in
electrospun PLA nanobers. Thus the motivation for this work is fabrication of covalently bonded NCC to PLA chains using silane modied PLA
in the electrospinning process. The effects of silane crosslinking and

NCC content on morphology, thermal and mechanical properties, and

cytotoxicity of the nanober were evaluated.

2. Experiment section
2.1. Materials
Poly (lactic acid), PLA, (IngeoBiopolymer 3251D) was purchased
from Nature works LLC, with a melt ow index 30 g/10 min (190 C,
2.16 kg) and a density of 1.24 g/cm3. Vinyltrimethoxysilane, VTMO
(YAC-V171, 98% purity) was supplied from Lanyachem and Dicumyl
peroxide, DCP, (99% purity), obtained from Aldrich, Germany. Microcrystalline cellulose powder (MCC) was purchased from Merk, Germany. Sulfuric acid (96.1%, density, 1.84 g/cm3) for hydrolysis of
cellulose, N,N-dimethylformamide (DMF) and chloroform were purchased from Merck, Germany. All reagents were used without further
purication.
L929 mouse broblast cell lines were obtained from the Cell Bank
Department of Pasteur Institute of Iran, (4,5-dimethylazol-2-yl)-2,5MTT was purchased from Sigma-Aldrich, Germany. Roswell Park Memorial Institute-1640 Medium (RPMI-1640), fetal bovine serum (FBS),
and Trypsin-EDTA 0.25% solution were obtained from Gibco, Canada.
Phosphate buffered saline (PBS) were prepared using sodium chloride
(NaCl), potassium chloride (KCl), disodium hydrogen phosphate
(Na2HPO4), and monopotassium phosphate (KH2PO4).

2.2. Preparation of silane grafted PLA (PLA-g-silane)

According to our pervious study [29], silane grafting of PLA was
accomplished via melt mixing using an internal mixer (Brabender,
Germany). The mixing temperature was held at 190 C and rotor
speed was set at 75 rpm. At rst, PLA was dried in an oven at 80 C
for 8 h, afterwards, it was added to mixing chamber and 30 s after
its complete melting, solution of DCP (0.5%) in VTMO (3%) was
poured gradually by a syringe (during the period of 30 s). Mixing
was continued for 5 min and the obtained compound was immediately compressed at 200 C in a hot press (Toyoseiki, Japan). To
avoid premature crosslinking, PLA sheets were stored in desiccators
after pressing.

Fig. 1. Mechanism of silane crosslinking of PLA (VTMO is a vinyl silane and DCP is a peroxide).

M. Rahmat et al. / Materials Science and Engineering C 68 (2016) 397405

2.3. Preparation of NCC

The preparation method of NCC followed the procedure mentioned
by Bondeson et al. [32]. According to this method, NCC suspension
was prepared from hydrolyzing MCC by sulphuric acid. The MCC was
treated by 63.5 wt% sulfuric acid in water solution. The ratio of MCC to
acid solution was 10.2 g to 100 ml. The MCC in the suspension was
then hydrolyzed at 44 C for 130 min. The obtained suspension was
washed by decanting and centrifugation with distilled water until PH
reached the value of 3. Subsequently, NCC suspension dialyzed against
distilled water with dialysis tubing until reaching a pH value of 7. The
nal neutral suspension was freeze dried and then used in further
processes.
2.4. Electrospinning of nanobers
The freeze dried NCC were redispersed in DMF/chloroform mixture
(1:3 v/v). This solution was sonicated for 2 min using a probe at 100%
amplitude (Sonopuls, BANDELIN electronic, Germany). PLA was then
added to the NCC/solvent suspension and the mixture was stirred at
40 C for 30 min until all of PLA pellets dissolved in DMF/chloroform
mixture. This solution was then sonicated for 10 min in the ultrasonic
bath (Elmasonic P, Elma Schmidbauer GmbH, Germany) until the well
dispersion of NCCs. Afterwards, the suspension was ltered by stainless
steel screen to remove any large particle and undesirable gels before
electrospinning. Immediately, the solution was poured into a 10 ml syringe and subjected to the electrospinning apparatus (ANSTCO, Iran)
using a horizontal system with a rotating aluminum drum (Diameter,
50 mm) covered by an aluminum foil at room temperature.
Electrospinning was started at 15 kV and at 1.5 ml/h feed rate. In all experiments the distance between the needle tip and the collector was set
to 170 mm and all samples were collected during 45 min. After deposition, nanobers were separated from the aluminum foil by immersing
in ethanol, then dried under vacuum and stored in a desiccator. The
sample codes and the related compositions are summarized in Table
1. The polymer concentration in all solutions was constant at 10%.

399

2.5.2. Morphology of electrospun mats

The morphology of the nanobers mats was observed by means of
scanning electron microscopy (SEM) (VEGA, TESCAN, Czech). The surface mats were coated with gold prior to SEM observation. The average
diameter of the nanobers was evaluated from geometrical measurements (using ImageJ software) on the SEM Images. About 100 bers diameter measurements were assessed from four different areas of the
same sample.
2.5.3. Differential scanning calorimetry (DSC)
The crystallization and melting behavior of the electrospun nanobers were considered using differential scanning calorimetric (DSC)
method (Mettler-Toledo, Switzerland). For intended purpose, a
heating-cooling-heating regime was employed. Approximately 5 mg
of each sample, in a nitrogen atmosphere, was heated to 200 C and
then kept for 5 min at this temperature, after that the specimen was
cooled to room temperature with a rate of 10 C/min and reheated up
to 200 C, with a rate of 10 C/min.
The crystallinity of the nanobers based on the DSC heating curves
was calculated through Eq. (1) [33].
xc

Hm Hcc


 100%
H0m

where xc is the crystallinity of the nanobers, Hm (J/g) is the heat of fusion , Hcc (J/g) is the heat of cold crystallization, and H0m is the heat of

2.5. Test methods

2.5.1. Atomic force microscopy (AFM) of NCC
After NCCs extraction, a drop of dilute NCC suspensions was deposited onto freshly cleaved mica. After 3 min the excess liquid was removed from mica surface and the remaining lm was allowed to be
dried. AFM tests were carried out using a DUALSCOP (95-200E, DEM,
Denmark) operating in air atmosphere and intermittent non-contact
mode with a 15 nm tip.

Table 1
Sample codes and composition.
Immersion in hot water

NCC content (%)

Polymer type

Sample code

No
No
No
No
No
No
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes

0
0.5
1
2
0
0.5
1
2
0
0.5
1
2
0
0.5
1
2

PLA
PLA
PLA
PLA
PLA-g-silane
PLA-g-silane
PLA-g-silane
PLA-g-silane
PLA
PLA
PLA
PLA
PLA-g-silane
PLA-g-silane
PLA-g-silane
PLA-g-silane

P0
P0.5
P1
P2
Ps0
Ps0.5
Ps1
Ps2
P0C
P0.5C
P1C
P2C
Ps0C
Ps0.5C
Ps1C
Ps2C

Fig. 2. (a) AFM image (topography mode, 10 10 m2) of NCC, (b) histogram of
nanoparticle length. In corner of AFM image, height distribution showed as a graph.

400

M. Rahmat et al. / Materials Science and Engineering C 68 (2016) 397405

fusion for 100% crystalline PLA which was taken as 93 J/g [7]. The crystallinity of the polymer in the nanocomposites was calculated as xp from
xc divided by weight fraction of PLA (w) in the nanocomposites, according to Eq. (2):

xp

xc
w

2.5.4. Tensile properties of electrospun mats

Tensile properties of the PLA nanobers mats were measured with a
universal testing machine (SMT-20, Santam, Iran) equipped with a
10 kN load cell under a crosshead speed of 5 mm/min at room temperature. The sample mats, after carefully peeling off from the surface of
aluminum foil by immersing in ethanol, were placed between two
pieces of paper frame to avoid any direct touch damage on the mat surfaces during tensile test. At least ve rectangular specimens with dimension of 30 mm in length, 10 mm in width, and 50100 m in
thickness were used for each sample. The stress and strain values

were measured through the force and displacement respectively,

which are related to the initial cross-section area and gauge length.
2.5.5. Cytotoxicity test
The biocompatibility and cytotoxicity of the nanobrous mats were
evaluated according to ISO 10993-5, L929 mouse broblast cells were
cultured in the vicinity of the selected samples (P0, P1 and Ps1C, 50
100 m thickness). The UV radiation was used in this study to sterilize
the substrate. Both sides of the prepared substrates were exposed to
UV radiation for 20 min. Cell culture was started from a frozen stock.
After thawing rapidly in a 37 C water bath, the cells were cultured (at
density of 5 104 cells per well) in a standard medium consisting of
0.5 ml RPMI-1640 with 10% (v/v) fetal bovine serum (FBS) (RPMI1640-10% FBS) and 1% (v/v) antibiotic-antimycotic in a 50-cm3 ask
and incubated under a CO2 (5%) atmosphere at 37 C for 48 h. The medium was then replaced with fresh RPMI-1640-10% FBS. Cell viability of
each sample was assayed by MTT test and the absorbance of culture
media was measured at 540 nm using a microplate reader on an Elisa
Reader ELX 808. The cell viability was calculated by the normalization
of optical density against the negative control.

Fig. 3. SEM images of PLA nanobers with various concentrations of NCC before and after hot water immersion. (A cluster of NCC is shown beside hc).

M. Rahmat et al. / Materials Science and Engineering C 68 (2016) 397405

3. Results and discussion

3.1. Characterization of NCC
Cellulose nanocrystals were achieved from the sulfuric acid hydrolysis of MCC and the simultaneous breakdown of cellulose microcrystals.
The distribution of cellulose nanocrystals dimensions are shown in
Fig. 2. According to AFM microscopy, the average length of cellulose
nanocrystals is 237 72 nm. Longitudinal line proles on images of isolated cellulose nanocrystals also showed that the height of nano particle
is 4.79 nm (the prole is shown in the corner of Fig. 2a), and therefore
the aspect ratio (l/d = 237/4.79) of these NCC is 50.

3.2. Morphology of nanobers

SEM micrographs of electrospun brous mats of PLA and PLA-g-silane, before and after immersion in hot water, including different NCC
contents are shown in Fig. 3. The corresponding ber diameter histograms are given in the corner of each SEM gure. All nanobers are homogeneous, and a reduction in their diameter with increasing NCC
content in PLA and PLA-g-silane is observed (Fig. 4) [5,7]. This phenomenon could be attributed to the higher conductivity of the nanocomposite suspensions leading to a more elastic behavior of the electrospinning
jet and therefore a reduction in the electrospun ber diameter. Such behavior has also been reported in the literature [5,34,35]. In comparison

401

with neat PLA solution, electrical conductivity of the PLA/NCC suspensions signicantly increases due to presence of SO
3 groups on the surface of NCC [36]. In addition, with increasing NCC content, there is a
considerable enhancing in electrical conductivity for nanocomposite
suspensions. Higher charge density on the surface of Taylor cone during
electrospinning makes more elongation forces to drive the jet under the
electrical eld. Therefore, the diameter of nanobers becomes substantially smaller with rising of NCC contents [7].
All the nanober mats show a porous morphology with randomly
aligned bers, but the mats that were immersed in hot water (Fig.
3(achc)) exhibit more dense morphology and relatively thicker bers
compared to counterparts unimmersed in water [Fig. 3(ah)]. As illustrated in Fig. 4, the average ber diameters of all sample increases
after immersion in hot water. But in the case of PLA based nanobers,
with no capability of crosslinking, this trend is only minor. In PLA samples (P0, P0.5, P1 and P2), the increase in diameter could be related to
shrinkage of oriented bers above their glass transition temperature
(Tg). It means that the PLA chains oriented in the electrospinning process, relax the imposed stresses and back to random coil structure as a
result of rising temperature above their Tg (about 60 C). It is noteworthy to mention that possible hydrolytic degradation which leads to
chain breaking, consequently, favors reversing molecules to their relaxed position [37,38]. However, in the case of PLA-g-silane samples,
in addition to relaxation of oriented molecules, a partially crosslinked
structure is also formed during immersion in hot water. Therefore,
such a partial crosslinking which forms a few Si-O-Si linkages causes
more shrinking effects, hence, bers diameter becomes thicker. A similar result in the chemical crosslinking of PLA has been reported by Qiao
et al. [9], however, they related the thickening of nanober to formation
of a crosslinked layer on the ber surface.
It is expected that with the existence of NCC, nanocrystals also
affect the recoiling of PLA molecules. Cellulose whiskers in the ber
may cause a restriction on PLA molecules movement above Tg .
Thus, ber thickening should be reduced after immersing them in
hot water. Fig. 5 shows a schematic view of different shrinkage
modes of nanobers after immersion in hot water. However, it is to
be noted that the tight network in the crosslinked bers leads to a
different behavior, because the bers with a more crosslink density
show more shrinkage in transverse direction of the bers, and therefore
it causes a reduction in diameter [39].
3.3. Thermal analysis

Fig. 4. Fiber diameter in different NCC content (a) before and (b) after hot water
immersion.

In order to investigate the effects of NCC on the thermal properties of

the electrospun nanobers, DSC analyses were carried out on neat PLA
and PLA-g-silane based samples. Fig. 6 shows the DSC thermograms of
the electrospun nanocomposite bers with varying NCC loadings in
the rst and second heating scans. Table 2 lists the glass transition
(Tg), melting (Tm), cold crystallization (Tcc) temperatures, the melting
(Hm) and cold crystallization (Hcc) enthalpies, and the crystallinity
(Xp), for the samples.
DSC results show that silane grafting reaction leads to reduction of
crystallinity of PLA (Ps0 vs. P0), due to generating new structures with
lower mobility and therefore lower crystallinity [29]. Nevertheless, addition of NCC in the PLA-g-silane causes a sharp rising in crystallinity
(a 136% rise for Ps2 in comparison with Ps0), while in the similar PLA
nanocomposites there is a slight increase in crystallinity (a 16.5% rise
for P2 in comparison with P0). This observation could be interpreted
as effectiveness of nucleation effect of NCC in the PLA-g-silane samples,
whereas in the PLA based samples, less nucleation effect of NCC is obtained. It can be explained according to hydroxyl groups of grafted silane and similar groups on the surface of cellulose crystals. This
similarity causes more compatibility between polymer and NCC, and
therefore a better crystal nucleation effect is observed. As seen in
Table 2, crosslinking reaction cause to reduction of Xc in Ps0 sample
from 9.8% to zero in Ps0C. The signicant reduction in crystallinity

402

M. Rahmat et al. / Materials Science and Engineering C 68 (2016) 397405

Fig. 5. Schematic of shrinkage modes of bers after hot water immersion.

Fig. 6. DSC thermograms of samples, (a) rst heating and (b) second heating scans.

after crosslinking has also been observed in another report [9]. However,
describing the simultaneous effects of crosslinking and NCC presence on
crystallinity is a nontrivial task. Because there is a competition between
nucleation effect of NCC which leads to increase the crystallinity and
hindering effect of crosslinking on crystal growth.
Returning to the Fig. 6, concerning the second heating scans, the Tcc
values of all samples are higher than those of the rst scans, revealing
the process history effect of electrospinning which inuences the organization of the molecular chains in nanobers [5]. DSC data (Table
2) show that in the second heating scans the Tcc of PLA/NCC nanocomposites increases with growing NCC contents. In the contrary,
in PLA-g-silane in the second heating scan, NCC promoted the cold
crystallization at lower temperatures. It is likely attributed to the
low compatibility of NCC with PLA, and therefore NCC does not act
as nucleating agent in comparison with the PLA-g-silane, due to the
decreased contact area between NCC and PLA matrix. [7,19]. In all samples with increasing NCC content, cold crystallization peak becomes
broader. This broadening is related to an increment in the heterogeneity
of the segmental mobilities and, thus, of the relaxation times distribution [5,40].
According to the rst heating scans (Fig. 6a), in respect of the samples with high nanocrystal contents a shoulder is appeared in the melting peak. The shoulder observed in high loaded NCC nanocomposites is
associated with the reorganization of molecular segments and the development of heterogeneous crystal phases in PLA during
electrospinning [7]. Moreover, this occurrence is not visible in the case
of PLA-g-silane, which conrms more compatibility of NCC with PLAg-silane rather than PLA.
Silane grafting of PLA causes a slight increase in Tg of the resulting
nanocomposite bers. However, incorporation of NCC shows negligible
inuence on the Tg [19]. Some other studies showed a different effect of
NCC on glass transition temperature, so that incorporation of NCC led to
Tg rising [7,41].

M. Rahmat et al. / Materials Science and Engineering C 68 (2016) 397405

403

Table 2
Thermal properties and crystallinity of samples (2nd heating data are listed).
Sample

P0
P0.5
P1
P2
Ps0
Ps0.5
Ps1
Ps2
Ps0C
Ps0.5C
Ps1C
Ps2C

Tg(C)

Tcc(C)

Hm

Hcc

Hc

Peak

Onset

Midpoint

Peak

Onset

Peak

Onset

(J/g)

(J/g)

(J/g)

(%)

168.04
168.1
168.08
168.83
165.35
164.53
165.81
166.15
163.95
164.19
165.04
165.17

161.37
161.83
161.88
162.18
153.27
152.21
154.95
155.11
154.35
153.04
152.31
152.79

60.5
60.91
60.34
60.98
61.76
61.78
61.43
61.2
61.19
61.89
61.94
61.96

96.68
100.58
102.19
104.05
107.71
108.17
105.18
104.99
107.69
103.83
106.68
106.68

88.34
93.85
92.78
92.09
100.25
98.35
96.05
92.39
98.68
92.21
97.21
97.79

99.72
99.48
99.89
101.21
103.88
104.71
104.74
106.4
99.17
106.55
105.71
105.4

116.94
112.99
115.1
119.37
120.63
121.7
122.24
123.01
117.88
122.43
121.96
121.42

47.63
38.97
42.3
41.55
32.72
30.52
38.4
35.4
29.42
35
31.93
40.04

23.28
15.57
19.21
13.73
23.56
18.83
19.72
14.21
29.35
16.36
17.02
13.77

10.37
10.03
12.19
16.37
5.12
9.34
11.67
16.45
4.12
14.72
9.87
17.12

26.1828
25.28773
25.07874
30.52447
9.849462
12.63306
20.28891
23.24995
0.075269
20.14373
16.1942
28.82379

Tm(C)

Tc(C)

3.4. Mechanical properties of nanober mats

The behavior of nanobers during tensile deformation is strongly
dependent on the level of the nanobers alignment within the mat,
ber lay-ups and interface adhesion of berber contact [42,43]. Usually, the tensile strength and modulus of the non-woven ber mat are
lower than that of the mats with uniaxially oriented bers [44].
As it has been already remarked [5,45], the mechanical properties of
nanober mats are affected by combination of several factors such as:
(a) intrinsic properties of the polymer which inuence the bers deformation mechanisms (that has a direct relationship with ultimate tensile
stress); (b) packing density of ber mats (interaction and mechanical
interlocking that increase the modulus); (c) bers average diameter
size (thicker bers correspond to a lower packing density in a constant
mat thickness); (d) presence of defects in the bers (such as broken bers) and (e) degree of ber orientation in the mat.

Fig. 7. Stress-strain curves of nanober mats.

Xp

The average stress-strain curves of the samples were calculated and

the results are compared in Fig. 7. Since the cohesion force between bers plays an important role in the beginning of the stretching, therefore, bers in the mats hardly move. In the second stage, most bers
reach the tightened form leading to yielding in the stress-strain curve.
Afterwards, with increasing tensile strain, the bers are drawn out to
highly orientation lengthways. At the ultimate stress, most bers are
necked and destroyed, and gradually leads to rupture of mats. Regarding these explanations, ultimate tensile stress is the important parameter to compare intrinsic properties of samples [7]. The ultimate stress
values of the uniaxial tensile tests of PLA and PLA-g-silane mats are
compared in Fig. 8.
According to Fig. 7a tensile strength of electrospun PLA/NCC nanocomposite mats improves with increasing NCC content levels, making
mats stiffer and stronger, compared with the neat electrospun PLA
mats. Nevertheless, PLA nanober containing NCC shows a slight improvement in tensile strength, while this effect is insignicant in silane
grafted PLA. The latter may be related to chain scission occurred in the
grafting reaction [29]. A similar result has been reported on maleic anhydride modication of PLA, indicating a suffering from substantially
low grafting yields and complex side-reactions leading to chain scission
[46]. The results reveal that ultimate stress increases after immersion in
hot water in the P0C (about 28%, comparing with P0). The improvement
in tensile strength could be caused by higher ber packing density, because of the shrinkage of nanobers. On the other hand, PLA-g-silane
shows a different trend, and after immersion in hot water the tensile
strength values reach 4.1, 5, 5.1 and 4.2 MPa for Ps0C, Ps0.5C, Ps1C
and Ps2C, respectively. This signicant improvement in tensile strength
is related to partial crosslinking of PLA as well as the high ber packing
density of nanober mats. It is worth noting that crystallinity of the

Fig. 8. Ultimate stress of nanober mats.

404

M. Rahmat et al. / Materials Science and Engineering C 68 (2016) 397405

electrospun nanocomposite bers is also expected to affect the mechanical properties [19], nevertheless according to DSC results, crystallinity
of PLA and silane crosslinked PLA nanocomposites are practically in
the same range.
The improvement in tensile strength implies that crosslinking of
PLA-g-silane/NCC results in the effective load transfer from polymer to
NCC via interactions between PLA and nanollers. In addition, the
formed networks between modied polymer chains and NCC also contribute in tensile strength enhancement. However, at the 2 wt% NCC
content, the tensile strength of Ps2C decreases. It is likely interpreted
by the presence of NCC agglomerates at high loading levels in the
electrospun bers (Fig. 3 Ps2C sample).
3.5. Cytotoxicity
In tissue-engineered substrate, the role of matrix characteristics such
as morphology, functional groups, hydrophilicity on cell viability is important. Fibroblast cell behavior on tissue culture plate (TCP) in vicinity
of fabricated substrates was evaluated under optical microscopy (OP).
Fig. 9 shows the broblast cells close to the nanobrous substrate exhibited typical spindle like morphology. The lopodia were also observed
around the cells which indicated the cellular growth and expansion.
The nanobers based on PLA and NCC has shown good biocompatibility [47,48] in comparison with control, and non-grafted VTMS molecules may lead to the cell death [49]. Fig. 9 illustrates that the samples
have shown no cytotoxicity effect on the survival rate of cells. The culture system covered with proliferated and spread cells.
The viability of seeded L929 was followed for 24 h, MTT assay was
performed to quantify the cell viability. Fig. 10 shows the absorption
value in 540 nm for mouse broblast cells seeded in vicinity of the
nanobrous mats during the culture period. The MTT results showed
that P0 has a good biocompatibility and cell growth results. Moreover,
the nanobrous mats containing NCC (P1 and Ps1C) showed better biocompatibility than the pristine PLA (P0) which is attributed to cellulose
nanocrystals incorporation due to possess a lot of OH groups on the surfaces. But in the Ps1C slight reduction in viability was observed. It could
be related to ungrafted silane molecules with less biocompatibility [49].
Another suggestion is that some hydroxyl groups of NCC may be involved in the silane crosslinking network.

Fig. 9. L929 cell line was cultured for 24 h in the vicinity of nanobrous mats.

Fig. 10. Cytotoxicity studies of PLA/NCC nanoparticles after MTT assay. Formazan
absorbance expressed for three samples (P0, P1, and Ps1C versus TCP as control).

In general, these results showed that all samples have good biocompatibility and no cytotoxicity is observed, thus silane crosslinking in
presence of NCC does not induce any cytotoxic activity on the seeded broblast cells.
The evaluation of the morphology of cultured L929 broblast cells on
the surface of Ps1C substrates was carried out by SEM after 48 h (Fig.
11). As it is obvious, cells have proper connection with the substrates,
which proves the results of the MTT assay. In tissue engineering, designing and fabrication of the substrates are considered as an important role
because substrates act as ECM in the body. The results show the substrates with nano-topographical feature have signicant impact on the
cell behavior and eventually on tissue regeneration. Finally, the SEM micrograph conrms that the substrate have suitable stability in culture
media after 48 h and keep the brous structure.
4. Conclusion
This work is motivated by the current researches in development of
PLA nanocomposites as a biocompatible material for tissue engineering.
Effects of NCC and silane crosslinking on morphology, thermal and

Fig. 11. SEM micrograph of cultured L929 broblast cells on the Ps1C surface after 48 h.

M. Rahmat et al. / Materials Science and Engineering C 68 (2016) 397405

mechanical properties and biocompatibility were monitored using SEM,

DSC, tensile and cytotoxicity tests. The following results can be drawn
from this study:
Because of higher conductivity of nanocomposite suspensions, a reduction of nanobers diameter with increasing NCC content was noticed. Also immersion of nanobers in hot water generally increased
their diameter because of nanobers longitudinal shrinkage. It was
shown that partial crosslinking favors recoiling of polymer molecules
while NCC nano particles restrict this effect.
The DSC thermograms conrmed that NCC has a well nucleation effect
in PLA-g-silane rather than PLA due to good compatibility between silane grafted PLA and NCC.
Tensile strength of nanober mats showed a signicant effect of
crosslinking on the mechanical properties in the presence of NCC.
The cytotoxicity test suggested that all samples have good biocompatibility and no more cytotoxicity was observed.

References
[1] L.-T. Lim, R. Auras, M. Rubino, Processing technologies for poly (lactic acid), Prog.
Polym. Sci. 33 (8) (2008) 820852.
[2] H.W. Kim, H.H. Lee, J. Knowles, Electrospinning biomedical nanocomposite bers of
hydroxyapatite/poly (lactic acid) for bone regeneration, J. Biomed. Mater. Res. Part A
79 (3) (2006) 643649.
[3] I. Armentano, M. Dottori, E. Fortunati, S. Mattioli, J. Kenny, Biodegradable polymer
matrix nanocomposites for tissue engineering: a review, Polym. Degrad. Stab. 95
(11) (2010) 21262146.
[4] F. Yang, R. Murugan, S. Wang, S. Ramakrishna, Electrospinning of nano/micro scale
poly (L-lactic acid) aligned bers and their potential in neural tissue engineering,
Biomaterials 26 (15) (2005) 26032610.
[5] I. Cacciotti, E. Fortunati, D. Puglia, J.M. Kenny, F. Nanni, Effect of silver nanoparticles
and cellulose nanocrystals on electrospun poly (lactic) acid mats: morphology, thermal properties and mechanical behavior, Carbohydr. Polym. 103 (2014) 2231.
[6] J.H. Lee, T.G. Park, H.S. Park, D.S. Lee, Y.K. Lee, S.C. Yoon, J.-D. Nam, Thermal and mechanical characteristics of poly (L-lactic acid) nanocomposite scaffold, Biomaterials
24 (16) (2003) 27732778.
[7] Q. Shi, C. Zhou, Y. Yue, W. Guo, Y. Wu, Q. Wu, Mechanical properties and in vitro
degradation of electrospun bio-nanocomposite mats from PLA and cellulose
nanocrystals, Carbohydr. Polym. 90 (1) (2012) 301308.
[8] H. Li, T. Qiao, P. Song, H. Guo, X. Song, B. Zhang, X. Chen, Star-shaped PCL/PLLA
blended ber membrane via electrospinning, J. Biomater. Sci. Polym. Ed. 26 (7)
(2015) 420432.
[9] T. Qiao, P. Song, H. Guo, X. Song, B. Zhang, X. Chen, Reinforced electrospun PLLA ber
membrane via chemical crosslinking, Eur. Polym. J. 74 (2016) 101108.
[10] A.K. Jaiswal, S.S. Kadam, V.P. Soni, J.R. Bellare, Improved functionalization of
electrospun PLLA/gelatin scaffold by alternate soaking method for bone tissue engineering, Appl. Surf. Sci. 268 (2013) 477488.
[11] B. Liu, F. Xu, M.-Y. Guo, S.-F. Chen, J. Wang, B. Zhang, Electrospun PLLA bers coated
with chitosan/heparin for scaffold of vascular tissue engineering, Surf. Coat. Technol.
228 (2013) S568S573.
[12] Y.H. Lee, J.H. Lee, I.-G. An, C. Kim, D.S. Lee, Y.K. Lee, J.-D. Nam, Electrospun dual-porosity structure and biodegradation morphology of montmorillonite reinforced
PLLA nanocomposite scaffolds, Biomaterials 26 (16) (2005) 31653172.
[13] S.D. McCullen, K.L. Stano, D.R. Stevens, W.A. Roberts, N.A. Monteiro-Riviere, L.I.
Clarke, R.E. Gorga, Development, optimization, and characterization of electrospun
poly (lactic acid) nanobers containing multi-walled carbon nanotubes, J. Appl.
Polym. Sci. 105 (3) (2007) 16681678.
[14] M.A. Ramirez, 2010.
[15] A. Sonseca, L. Peponi, O. Sahuquillo, J.M. Kenny, E. Gimnez, Electrospinning of biodegradable polylactide/hydroxyapatite nanobers: study on the morphology, crystallinity structure and thermal stability, Polym. Degrad. Stab. 97 (10) (2012)
20522059.
[16] Z. Huajun, K. Kyoung-Woo, G. Emmanuel, J. Yong Lak, Nanobers from Polylactic
Acid Nanocomposites: Effect of Nanoclays on Molecular Structures, American
Chemical Society, Polymeric Nanobers, 2006 217230.
[17] F. Mei, J. Zhong, X. Yang, X. Ouyang, S. Zhang, X. Hu, Q. Ma, J. Lu, S. Ryu, X. Deng, Improved biological characteristics of poly (L-lactic acid) electrospun membrane by incorporation of multiwalled carbon nanotubes/hydroxyapatite nanoparticles,
Biomacromolecules 8 (12) (2007) 37293735.
[18] S.S. Khan, (Drexel) 200209
[19] C. Xiang, Y.L. Joo, M.W. Frey, Nanocomposite bers electrospun from poly (lactic
acid)/cellulose nanocrystals, J. Biobased Mater. Bioenergy 3 (2) (2009) 147155.
[20] M.S. Peresin, Y. Habibi, A.-H. Vesterinen, O.J. Rojas, J.J. Pawlak, J.V. Seppl, Effect of
moisture on electrospun nanober composites of poly (vinyl alcohol) and cellulose
nanocrystals, Biomacromolecules 11 (9) (2010) 24712477.

405

[21] J.O. Zoppe, M.S. Peresin, Y. Habibi, R.A. Venditti, O.J. Rojas, Reinforcing poly (caprolactone) nanobers with cellulose nanocrystals, ACS Appl. Mater. Interfaces
1 (9) (2009) 19962004.
[22] P. Lu, Y.-L. Hsieh, Cellulose nanocrystal-lled poly (acrylic acid) nanocomposite brous membranes, Nanotechnology 20 (41) (2009) 415604.
[23] H. Dong, K.E. Strawhecker, J.F. Snyder, J.A. Orlicki, R.S. Reiner, A.W. Rudie, Cellulose
nanocrystals as a reinforcing material for electrospun poly (methyl methacrylate) bers: formation, properties and nanomechanical characterization, Carbohydr.
Polym. 87 (4) (2012) 24882495.
[24] M.A. Hubbe, O.J. Rojas, L.A. Lucia, M. Sain, Cellulosic Nanocomposites: A Review,
2008.
[25] K.-Y. Lee, J.J. Blaker, A. Bismarck, Surface functionalisation of bacterial cellulose as
the route to produce green polylactide nanocomposites with improved properties,
Compos. Sci. Technol. 69 (15) (2009) 27242733.
[26] P. Tingaut, T. Zimmermann, F. Lopez-Suevos, Synthesis and characterization of
bionanocomposites with tunable properties from poly (lactic acid) and acetylated
microbrillated cellulose, Biomacromolecules 11 (2) (2009) 454464.
[27] A.-L. Gofn, J.-M. Raquez, E. Duquesne, G. Siqueira, Y. Habibi, A. Dufresne, P. Dubois,
From interfacial ring-opening polymerization to melt processing of cellulose
nanowhisker-lled polylactide-based nanocomposites, Biomacromolecules 12 (7)
(2011) 24562465.
[28] X. Zhang, M. Kotaki, S. Okubayashi, S. Sukigara, Effect of electron beam irradiation on
the structure and properties of electrospun PLLA and PLLA/PDLA blend nanobers,
Acta Biomater. 6 (1) (2010) 123129.
[29] M. Rahmat, I. Ghasemi, M. Karrabi, H. Azizi, M. Zandi, M. Riahinezhad, Silane
crosslinking of poly (lactic acid): the effect of simultaneous hydrolytic degradation,
Express Polym Lett 9 (12) (2015) 11331141.
[30] H. Azizi, J. Morshedian, M. Barikani, Silane grafting and moisture crosslinking of
polyethylene: the effect of molecular structure, J. Vinyl Add. Tech. 15 (3) (2009)
184190.
[31] C. Han, J. Bian, H. Liu, L. Han, S. Wang, L. Dong, S. Chen, An investigation of the effect
of silane water-crosslinking on the properties of poly (L-lactide), Polym. Int. 59 (5)
(2010) 695703.
[32] D. Bondeson, A. Mathew, K. Oksman, Optimization of the isolation of nanocrystals
from microcrystalline cellulose by acid hydrolysis, Cellulose 13 (2) (2006) 171180.
[33] K.-Y. Lee, M. Tang, C.K. Williams, A. Bismarck, Carbohydrate derived copoly (lactide)
as the compatibilizer for bacterial cellulose reinforced polylactide nanocomposites,
Compos. Sci. Technol. 72 (14) (2012) 16461650.
[34] S. Ramakrishna, K. Fujihara, W.-E. Teo, T.-C. Lim, Z. Ma, An Introduction to
Electrospinning and Nanobers, World Scientic, 2005.
[35] A. Greiner, J.H. Wendorff, Electrospinning: a fascinating method for the preparation
of ultrathin bers, Angew. Chem. Int. Ed. 46 (30) (2007) 56705703.
[36] C. Zhou, Q. Wang, Q. Wu, UV-initiated crosslinking of electrospun poly (ethylene
oxide) nanobers with pentaerythritol triacrylate: effect of irradiation time and incorporated cellulose nanocrystals, Carbohydr. Polym. 87 (2) (2012) 17791786.
[37] M. enkiewicz, R. Malinowski, P. Rytlewski, A. Richert, W. Sikorska, K. Krasowska,
Some Composting and Biodegradation Effects of Physically or Chemically
Crosslinked Poly (Lactic Acid), Polymer Testing, 2011.
[38] N. Nagasawa, A. Kaneda, S. Kanazawa, T. Yagi, H. Mitomo, F. Yoshii, M. Tamada, Application of poly (lactic acid) modied by radiation crosslinking, Nucl. Instrum.
Methods Phys. Res., Sect. B 236 (1) (2005) 611616.
[39] X. Zhang, M. Kotaki, S. Okubayashi, S. Sukigara, Effect of electron beam irradiation on
the structure and properties of electrospun PLLA and PLLA/PDLA blend nanobers,
Acta Biomater. 6 (1) (2010) 123129.
[40] R. Picciochi, Y. Wang, N.M. Alves, J.F. Mano, Glass transition of semi-crystalline PLLA
with different morphologies as studied by dynamic mechanical analysis, Colloid
Polym. Sci. 285 (5) (2007) 575580.
[41] C. Zhou, R. Chu, R. Wu, Q. Wu, Electrospun polyethylene oxide/cellulose nanocrystal
composite nanobrous mats with homogeneous and heterogeneous microstructures, Biomacromolecules 12 (7) (2011) 26172625.
[42] C.Y. Xu, R. Inai, M. Kotaki, S. Ramakrishna, Aligned biodegradable nanobrous structure: a potential scaffold for blood vessel engineering, Biomaterials 25 (5) (2004)
877886.
[43] X.-F. Wu, Y.A. Dzenis, Size effect in polymer nanobers under tension, J. Appl. Phys.
102 (4) (2007) 044306.
[44] A. Baji, Y.-W. Mai, S.-C. Wong, M. Abtahi, P. Chen, Electrospinning of polymer nanobers: effects on oriented morphology, structures and tensile properties, Compos.
Sci. Technol. 70 (5) (2010) 703718.
[45] W.-J. Li, J.A. Cooper Jr., R.L. Mauck, R.S. Tuan, Fabrication and characterization of six
electrospun poly(-hydroxy ester)-based brous scaffolds for tissue engineering
applications, Acta Biomater. 2 (4) (2006) 377385.
[46] C. Nyambo, A.K. Mohanty, M. Misra, Effect of maleated compatibilizer on performance of PLA/wheat straw-based green composites, Macromol. Mater. Eng. 296
(8) (2011) 710718.
[47] K.M.Z. Hossain, M.S. Hasan, D. Boyd, C.D. Rudd, I. Ahmed, W. Thielemans, Effect of
cellulose nanowhiskers on surface morphology, mechanical properties, and cell adhesion of melt-drawn polylactic acid bers, Biomacromolecules 15 (4) (2014)
14981506.
[48] R.M. Domingues, M.E. Gomes, R.L. Reis, The potential of cellulose nanocrystals in tissue engineering strategies, Biomacromolecules 15 (7) (2014) 23272346.
[49] A. Dupraz, S.v. Meer, J. De Wijn, J. Goedemoed, Biocompatibility screening of silanetreated hydroxyapatite powders, for use as ller in resorbable composites, J. Mater.
Sci. Mater. Med. 7 (12) (1996) 731738.