Toughening of electrospun poly(L-lactic acid) nanofiber scaffoldswith unidirectionally aligned halloysite nanotubes

Ning Cai • Qin Dai • Zelong Wang •

Xiaogang Luo • Yanan Xue • Faquan Yu

Received: 27 June 2014 / Accepted: 4 November 2014 / Published online: 13 November 2014

� Springer Science+Business Media New York 2014

Abstract The mechanical properties of the tissue engi-

neering scaffold are important as they are tightly related

the regeneration of structural tissue. The application of

poly(L-lactic acid) (PLLA) nanofiber scaffolds in tissue

engineering has been hindered by their insufficient

mechanical properties. In the study, halloysite nanotubes

(HNTs) were used to reinforce the mechanical properties of

PLLA-based nanofibers. 4 wt% HNT/PLLA nanofiber

membranes possess the best mechanical performance,

which represents 61 % increase in tensile strength, 100 %

improvement of Young’s modulus, 49 % augment of

elongation to break, as well as 181 % elevation in energy

to break compared with neat PLLA samples. The satis-

factory enhancement effect of HNTs can be attributed to

the effective dispersion and incorporation of HNTs in

PLLA matrix, which have been confirmed by the analysis

of SEM, TEM, and FTIR. The addition of HNTs also

improves the degree of crystallization and thermal stability

of PLLA-based nanofibers. HNT-incorporated PLLA

nanofiber membranes possess higher protein adsorption

from fetal bovine serum than the neat PLLA specimen.

Therefore, the introduction of HNTs can effectively

enhance the mechanical properties of PLLA nanofiber

scaffolds. HNT/PLLA nanofiber scaffolds possess potential

application in skin tissue engineering.

Introduction

One of the key objectives in tissue engineering is to

develop a scaffold for supporting three-dimensional tissue

regeneration [1]. To fulfill this goal, some specific criteri-

ons should be observed in designing tissue engineering

scaffolds [2]. A high porosity with adequate pore size is

necessary to facilitate cell seeding and nutrients’ diffusion

[3]. Biodegradability is also an essential factor, since

scaffolds should be absorbed by the surrounding tissues

during the new tissue regeneration [4]. In addition, the

sufficient mechanical strength and the structural integrity

of the scaffolds are very important for handling an implant

and maintaining the desired structure prior to the formation

of new tissue [5]. Indeed, it has been found that the strength

and deformability of scaffold influence in vitro cell

migration, proliferation and differentiation, along with cell

morphology [6].

Electrospinning biopolymer to generate nanometer to

micrometer-scale fibers has emerged as a prominent

method for fabricating 3D scaffolds with tissue-like

microstructures. Numerous natural and synthetic biopoly-

mers have been successfully electrospun to produce micro-

or nanofibers for tissue engineering and other related

applications [7]. Among them, poly(L-lactic acid) (PLLA)

is one of the most extensively used biopolymers, as it is

one of the few bioresorbable polymers that have been

approved by the U.S. Food and Drug Administration for

in vivo applications. However, electrospun PLLA nanofi-

ber scaffolds normally have weak mechanical strength,

partially resulted from high porosity and random alignment

of fibers, which limit their biomedical applications, espe-

cially as tissue engineering scaffolds [8, 9].

To address this issue, different types of fillers, including

inorganic particles, such as hydroxyapatite, carbon

N. Cai � Q. Dai � Z. Wang � X. Luo � Y. Xue � F. Yu (&)

Key Laboratory for Green Chemical Process of Ministry of

Education, School of Chemical Engineering and Pharmacy,

Wuhan Institute of Technology, Wuhan 430073, China

e-mail: fyuwucn@gmail.com; fyu@mail.wit.edu.cn

Z. Wang

Department of Research, Hunan Xiangjiang Kansai Paint Co.,

Ltd., Changsha 410003, China

123

J Mater Sci (2015) 50:1435–1445

DOI 10.1007/s10853-014-8703-4

nanotubes and graphene oxide, have been co-electrospun

into the polymer nanofibers for improving mechanical

properties [10]. For instance, the improved mechanical

stiffness has been shown on carbon nanotube-incorporated

polymeric nanofibers [11]. However, these extensively

used nanofillers do not possess satisfactory biocompati-

bility, which becomes the major hurdle in extending their

potential applications to biomedical engineering [12].

The environmental friendliness and biocompatible nat-

ure make halloysite clay nanotubes [Al2Si2O5(OH)4�nH2O]

an attractive nanoreinformcent candidate. Halloysite clay

nanotubes (HNTs) are aluminosilicate tubes with length of

100–1000 nm, diameter of ca 50 nm, and internal diameter

of 15 nm [13]. Because of its good biocompatibility and

low cost, HNTs have attracted increasing interests of

researchers due to their unique advantages. HNTs have

been demonstrated to be an ideal reinforcement agent for

fabricating polymeric composites with improved mechan-

ical performance [14]. Recently, several types of HNT-

filled composite nanofibers have been successfully pro-

duced. However, there were no reported studies on the

application of HNTs for improving mechanical perfor-

mance of electrospun PLLA nanofibers. The properties of

HNT/PLLA composite nanofibers were also not studied

systematically.

The aim of this work is to evaluate the effect of addition

of biocompatible halloysite nanotubes (HNTs) on the

mechanical properties of electrospun PLLA nanofiber

membranes. The structure and morphology of the electro-

spun nanofiber membranes were examined by scanning

electron microscopy (SEM), transmission electron

microscopy (TEM), Fourier transform infrared spectros-

copy (FTIR), and X-ray diffraction (XRD). Crystallization

behavior and thermal stability were studied by differential

scanning calorimetry (DSC) and thermogravimetric ana-

lysis (TGA) methods, respectively. The effect of addition

of HNTs on mechanical properties was examined by the

tensile tests and the reinforcement mechanism was dis-

cussed. Protein adsorption of HNT/PLLA from fetal bovine

serum (FBS) was also evaluated.

Experimental

Materials

PLLA with molecular weight of 7,3000 kD was obtained

from Sigma-Aldrich, and its density was 1.25 g/cm3. HNTs

were mined from the deposit of Hubei province of China,

which had an average diameter of 60 nm, a length of about

1.2 microns, and a density of 2.5 g/cm3. Before usage,

HNTs were purified according to a reported protocol [15].

In brief, dry halloysite was added into water to prepare

10 wt% water solution of halloysite followed by the

addition of 0.05 wt% sodium hexametaphosphate. The

obtained solution was stirred for 30 min and left to stand

for 20 min at room temperature. By filtration, the clay

aggregate and impurities precipitated in the bottom were

removed. The supernatant was collected and centrifuged to

obtain HNTs. The purified HNTs were dried and stored for

further use. Chloroform, dimethylformamide (DMF) and

other reagents were purchased from Sinopharm Chemical

Reagent Co. Ltd., China.

Preparation of electrospun HNT/PLLA nanofiber

membranes

Solution intercalation technique was employed for the

fabrication of composites. Initially, HNTs were suspended

in chloroform and DMF mixture solution (The volume

ratio of chloroform to DMF was 6:1) by stirring for 12 h,

followed by sonication of 1 h at room temperature to

achieve good dispersion. PLLA pellets were gradually

added into the HNTs suspension with slowly raising the

temperature up to 60 �C. The suspension was continuously

stirred at 60 �C for 2 h to dissolve PLLA. The electrical

conductivity and viscosity of the HNT/PLLA solution were

measured by an electrical conductivity meter (DDS 307A,

Shanghai Rex Instrument, China) and an AR2000 rheom-

eter (TA Instruments, United States) at 22 �C, respectively.

The HNT/PLLA nanofibers were prepared by electros-

pinning solution using an electrospinning system (Beijing

Kangsente Co., China), which comprised a syringe pump

and a high voltage power supply generating positive DC

voltage. A 10 mL syringe containing electrospinning

solution was connected to a stainless steel needle with an

inner diameter of 0.6 mm. The needle tip was set up hor-

izontally. A vertical metal plate wrapped with aluminum

foil was used to collect the electrospun nanofibers. The

electrospinning parameters were fixed as follows: feeding

rate, 2.0 mL/h; voltage, 20 kV; distance between needle tip

and collector, 10 cm; humidity, 40–50 %; temperature,

25 �C. The nanofiber membranes were then peeled off

from the aluminum foil for further characterization.

Structural and thermal characterization

The morphology of HNTs and the electrospun HNT/PLLA

nanofiber membranes was investigated using a SEM (JEOL

JSM-5510LV) at an accelerating voltage of 10 kV. The

nanofiber diameter distribution was obtained by analyzing

at least three distinct images using ImageJ software. The

dispersion of the HNTs within the composite nanofibers

was examined by a TEM (FEI TecnaiG2 20 S-Twin). FTIR

was recorded with a Nicolet 6700 FTIR spectrometer in the

range of 4000–600 cm-1 to determine the chemical

1436 J Mater Sci (2015) 50:1435–1445

123

signatures. XRD analysis was performed with a Bruker D8

ADVANCE X-ray diffractometer at a voltage of 40 kV

with Ni-filtered Cu Ka radiation. The 2h scan data were

collected from 10.0� to 50.0� at a scanning speed of 1.0�/

min. The thermal stability was studied with thermo gravi-

metric analysis (TGA) using a thermogravimeter (Netzsch,

Germany). A 5 mg sample was placed in an aluminum pan

and a heating rate of 10 �C/min was employed under

nitrogen flow. DSC measurements were carried out in the

20–200 �C range using a SII DSC 6220 equipment appa-

ratus (Seiko Instruments, Japan), at a heating rate of 10 �C/

min in nitrogen atmosphere. The degree of crystallinity was

obtained by using the equation [16].

Xc ¼DHm � DHcc

DH0mð1�Wf Þ

� 100 %;

where Xc (%) is crystallinity, DHm is the melting enthalpy,

DHcc is the cold crystallization enthalpy, DHm0 is the

melting enthalpy of completely crystallized PLLA (93.7 J/

g according to Ref. [17]), and Wf is the weight fraction of

HNTs in the composites.

Mechanical properties testing

The mechanical properties of electrospun fiber membranes

were measured with an MTS (CMT 800, USA) tensile

testing machine using a 200 N load cell. Rectangular

specimens were cut to 60 mm 9 5 mm and extended at a

constant speed of 10 mm/min with a 40 mm gauge length.

Each specimen was tested for five times to acquire the

mean value. The thickness of each specimen was the

average of three measurements taken along the gauge

length with a digital micrometer. The force displacement

data were taken from the tensile machine and converted to

engineering stress-engineering strain results. Engineering

stress was defined as the ratio of force to the initial cross-

sectional area, and engineering strain was defined as the

ratio of the change in length to the original gauge length.

Protein adsorption onto HNT/PLLA nanofiber

membranes

Electrospun PLLA and HNT/PLLA fibrous membranes

were cut into round pieces in a diameter of 15 mm. The

samples were placed in a 24-well tissue culture plate and

immersed in 0.01 M PBS. After being equilibrated with

PBS overnight, the samples were incubated in 0.5 mL of

FBS (10 %) for 24 h at 37 �C. The concentration of FBS

solution before or after adsorption was determined by the

absorbance read at 280 nm in a SpectraMax M2e spec-

trophotometer. Independent measurements were performed

in four samples and the amount of the adsorbed protein was

calculated based on the difference of FBS solution con-

centration before and after adsorption.

Statistical analysis

All the data were shown as a mean ± standard deviation. A

one-way analysis of variance (ANOVA) was performed to

compare the mean values among different groups. Statis-

tical significance was tested at p \ 0.05.

Results and discussion

Morphological and structural analysis

HNTs consist of gibbsite octahedral sheet (Al–OH) groups

on the internal surface and siloxane groups (Si–O–Si) on

the external surface [18]. Figure 1 shows the TEM image

of HNTs. As exhibited in Fig. 1, HNTs possess the tubular

structures. Following the analysis by ImageJ, it is deter-

mined that the HNTs used in this study range in length

from 0.5 to 1.2 lm and in diameter from 50 to 100 nm.

The morphology of electrospun HNT/PLLA nanofiber

membranes was examined through SEM inspection. It is

shown in Fig. 2a that the neat PLLA nanofibers are smooth,

and no broken ends as well as beads were found. The

smooth surface of PLLA-based nanofibers is still main-

tained after the incorporation of low content of HNTs.

However, C2 wt% addition of HNTs resulted in nanofibers

with uneven surface. It is clear that HNT/PLLA nanofiber

membranes containing 6 wt% loading of HNTs possess

rather rough surfaces. In addition, the incorporation of

Fig. 1 TEM image of halloysite nanotubes

J Mater Sci (2015) 50:1435–1445 1437

123

Fig. 2 SEM micrographs and

the corresponding diameter

distribution of electrospun

HNT/PLLA nanofiber

membranes containing 0 (a), 1

(b), 2 (c), 4 (d), and 6 wt%

(e) loading of HNTs

1438 J Mater Sci (2015) 50:1435–1445

123

HNT particles into PLLA also induces the change of the

nanofiber diameter. As shown in Fig. 2a–e, the composite

nanofiber diameter gradually increases from 307 ± 82 to

917 ± 121 nm when the HNT content is elevated gradu-

ally up to 6 wt%. The influence of HNT particles on the

morphology of PLLA nanofibers presumably results from

the impaired stretching of the fiber during the electros-

pinning process. It is known that the viscosity and the

charged density of electrospinning solution contribute to

the change of diameter of electrospun nanofibers [19].

According to our measurement, the incorporation of HNTs

induces the gradual increase of viscosities of electrospin-

ning solution from 0.09 Pa s (neat PLLA) to 0.22 Pa s

(6 wt% HNT/PLLA). High viscosity restricts the stretching

of the liquid jet, beneficial for the formation of larger

electrospun fibers [20]. With addition of HNTs, the elec-

trical conductivity of electrospinning solution demonstrates

a ascending trend with the augment of HNT contents

(0.27 lS cm-1 for PLLA and 3.78 lS cm-1 for 6 wt %

HNT/PLLA). Thus, the introduction of negatively charged

HNTs into the electrospinning solution results in the

increased charge density of electrospinning solution, pro-

moting the formation of smaller nanofibers. As shown in

Fig. 2, the diameter of HNT/PLLA nanofibers demon-

strates an increasing trend with HNT content. Therefore, it

is the elevation in viscosity following the addition of HNTs

that induces the increase of the diameter with HNT content

for electrospun HNT/PLLA nanofibers [21].

FTIR spectroscopy was utilized to study the chemical

structures of the neat PLLA and HNT/PLLA composite

nanofiber membranes. As illustrated in Fig. 3, FTIR spec-

tra of HNT exhibits two characteristic bands at 3623 and

3695 cm-1 which are assigned to the stretching vibration

of inner O–H and the O–H located at the inner-surface of

the nanotubes, respectively [22]. A strong absorption peak

centers at 1028 and 1008 cm-1, which is attributed to the

stretching vibration band of in-plane Si–O–Si of HNT [23].

For HNT/PLLA nanocomposites, the peak at 912 cm-1

corresponding to deformation vibration of inner O–H of

HNT [24] can be identified when HNT content is raised

over 2 wt%. Under the influence of the incorporated HNTs,

the strong peak attributed to the stretching vibration of

C=O of PLLA gradually shifts to lower wavenumber, from

1750 cm-1 (neat PLLA) to 1746 cm-1 (6 wt% HNT/

PLLA). The shift may be mainly stemmed from hydrogen-

bonding interactions between the carbonyl groups (C=O) of

PLLA and the hydroxyl groups of HNTs. In fact, the

hydroxyl groups of PLLA can also interact with the Si–O–

Si groups of HNT via hydrogen bonding interactions [25].

Liu et al. have reported that the peak of the C=O stretching

vibration at 1756 cm-1 for neat PLLA is shifted to

1750 cm-1 for the 40 wt% HNT/PLLA nanocomposites

[25]. Therefore, there is not conspicuous difference in the

shift of the peak assigned to C=O stretching vibration

between HNT/PLLA composites and electrospun HNT/

PLLA composite nanofibers. The influence of nanofillers

on characteristic FTIR band shift of PLLA was also

reported in other composite systems [26, 27]. Obviously,

these interactions result in effective adhesion between

HNTs and PLLA matrix, which probably affect the

mechanical and thermal properties of PLLA-based com-

posite nanofibers.

To illustrate the interactions of HNTs with PLLA, XRD

experiment was conducted. For neat PLLA, only a broad

scattering reflection, locating at around 2h = 16�, is found

in the XRD spectrum, indicating that it does not crystallize

during the electrospinning in the sample preparation pro-

cess [28]. The XRD pattern of the original halloysite

sample, shown in Fig. 4, is in good agreement with a

Fig. 3 FTIR spectra of electrospun HNT/PLLA composite nanofiber

membranes containing 0 (a), 1 (b), 2 (c), 4 (d), and 6 wt% (e) loading

of HNTs and pristine HNTs (f)

Fig. 4 XRD spectra of HNTs (a) and electrospun HNT/PLLA

composite nanofiber membranes containing 0 (b), 1 (c), 2 (d), 4

(e) and 6 wt% (f) loading of HNTs

J Mater Sci (2015) 50:1435–1445 1439

123

previously published pattern for halloysite [29]. Three

distinct XRD peaks at 2h = 12.1�, 20.0� and 24.9� in

relation to reflection planes (0 0 1), (0 2 0), (1 1 0) and (0 0

2) are observed, which correspond to d = 0.732, 0.446 and

0.358 nm, respectively [30]. It should be pointed out that

the reflection of HNTs at around 20.0� seems to disappear

in all the HNT/PLLA nanocomposites, which may be

attributed to the low intensity of XRD peak of HNT at

2h = 20.0�.

Thermal analysis

PLLA is semicrystalline polymer and its mechanical and

physical properties are governed by the crystal micro-

structure. The introduction of nanosized HNTs may indu-

ces the formation of the interfacial interactions between

PLLA and HNTs, affecting the crystallization behavior of

composites. As shown in Fig. 5, there is a step-like change

for all samples in the temperature range of 50–70 �C,

which is assigned to the glass transition region of PLLA

[31]. It can be seen in Fig. 5 and Table 1 that the glass

transition temperature (Tg) of the HNT/PLLA nanocom-

posites decreases slightly with the increment of HNT

content within 2 wt%.

Incorporating isotropic nanoparticles including nanodi-

amonds [32] and nano-TiO2 [33] into the PLLA matrix

usually induces the increase of Tg. Following the addition

of nanofillers, polymer chain may interact with the fillers

through intermolecular attractions, such as van der Waals

force and hydrogen bonding [34]. Thus, the mobility of the

polymer chain segment in the vicinity of the nanofiller

surface may be inhibited by the inorganics [35].

For the anisotropic nanofillers, the influence of nanof-

illers on Tg of polymer matrix could be different, which

were due to oversize in at least one dimension for the

nanofillers [36, 37]. For instance, the diameter of HNTs

ranges from 50 to 100 nm. Although the diameter of HNTs

is on the nanoscale, their length is usually in the microscale

([1 lm), which far exceeds the typical gyration radii of

polymer chains. Consequently, the packing of the polymer

chains is prevented and the free volume near HNT surface

increases [38]. With the increase of free volume, the

increase mobility of the polymer chains is allowed [39]. Of

course, the incorporated HNTs can still inhibit the motion

of PLLA chains due to HNT-PLLA intermolecular inter-

actions. However, the inhibition of motion of polymer

segments is offset by the promotion of free movement

resulted from the increased free volume [38]. Furthermore,

the latter has become the major factor in determining the

Tg. As a result, the incorporation of HNTs tends to lower

the Tg of the PLLA-based nanocomposites. The decrease of

Tg with the increase nanofiller content has also been

reported in HNT/EPDM (Ethylene Propylene Diene

Monomer) [37] and MWCNTs/PMMA [40] systems. It

should be pointed out that the Tg maintains almost constant

value in the range of HNT content from 2 to 6 wt%, which

suggests the enhancing effect of newly generated free

volume is just canceled out by the restraining effect of

additional HNTs incorporated in PLLA matrix on the free

motion of PLLA chain segments.

The introduction of HNTs also induces the change of

cold crystallization peak temperature (Tcc). It is shown in

Table 1 that Tcc maintains a descending trend within the

4 wt% HNT content and then goes up with the increase of

HNT content from 4 wt% upwards. This lowering of Tcc

could be attributed to the nucleating effect of HNTs on

polymer crystallization. Generally, small amount of HNT

can serve as an effective nucleating agent for PLLA [41].

When HNT content is too high ([4 wt%), nucleating effect

is on long pronounced and Tcc starts to go down with the

increase of HNT content, which could be explained by the

reduction of nucleation surface resulted from the aggra-

vation of HNT agglomeration [42]. This explanation is

consistent with TEM results (Fig. 8).

Due to the nucleating effect of HNTs, the degree of

crystallinity (Xc) of HNT/PLLA nanocomposites also

makes change, which increases from 20.1 to 40.8 % within

Fig. 5 DSC curves of electrospun HNT/PLLA composite nanofiber

membranes

Table 1 Thermal properties of electrospun HNT/PLLA nanofiber

membranes

HNT

content

(wt%)

Tg

(�C)

Tcc

(�C)

Tm

(�C)

DHm

(J/g)

Xc

(%)

T10wt%

(�C)

T50wt%

(�C)

0 63.9 82.5 165.5 30.2 20.1 314.8 353.6

1 62.4 77.6 166.9 39.2 25.3 328.1 357.3

2 61.6 77.5 166.9 36.8 32.3 334.9 361.4

4 61.8 77.1 166.5 41.1 40.8 343.9 366.1

6 61.8 80.5 165.8 37.6 36.7 342.6 367.0

1440 J Mater Sci (2015) 50:1435–1445

123

4 wt% of HNT content. The ascending trend of Xc is

reversed when HNT content exceeds 4 wt%. Based on the

analysis of Tg and Xc, 4 wt% as the critical concentration,

can be supposed to an indicator which reflects the distri-

bution status of HNTs in PLLA matrix. For melting tem-

perature (Tm) of HNT/PLLA nanocomposite, no obvious

change is observed, as halloysites are mineral fillers [43].

The thermal stability of the neat PLLA and HNT/PLLA

nanocomposites were characterized by TGA. As shown in

Fig. 6, neat PLLA begins to decompose at *254 �C.

Comparatively, higher temperature is required to initiate

the decomposition of HNT/PLLA nanocomposite. Fol-

lowing the decomposition of neat PLLA, there was almost

no residue. In contrast, HNT/PLLA cannot decompose

completely even under 600 �C, the residue of which is

related to the HNTs. Comparing the thermal decomposi-

tion temperatures (Td) of neat PLLA and HNT/PLLA

nanocomposites for 10 and 50 % weight loss, it is found

that T10 and T50wt% of HNT/PLLA are higher than those of

neat PLLA. Therefore, HNT/PLLA nanocomposites pos-

sess better thermal stability than neat PLLA. It was

reported the improvement of thermal stability through

introducing fillers was largely dependent on the dispersion

of fillers in matrix. In our study, serious agglomeration of

HNTs is not found in TEM observation. Therefore, rela-

tively uniformly dispersed HNTs in the matrix act like a

barrier to the passage of the volatile pyrolized products of

PLLA, eventually retarding thermal decomposition of the

HNT/PLLA [44]. The present TGA results are consistent

with the previous reported thermal stability results of silica/

PLLA [45], and graphene/epoxy nanocomposites [46].

Mechanical properties

The mechanical properties of the electrospun nanocom-

posite membranes were investigated by the tensile stress–

strain testing method. Representative stress–strain curves

of the HNT/PLLA nanofiber membranes are presented in

Fig. 7a, and their tensile strength, Young’s modulus,

elongation at break and toughness are summarized in

Table 2. For all the samples, it is found that the improve-

ment of tensile strength and Young’s modulus is achieved

on HNT/PLLA samples. With the addition of 4 wt% HNT,

the tensile strength and Young’s modulus demonstrate an

elevation of 61 and 100 %, respectively, which indicates

that the nanofiber membranes become stiffer and stronger.

However, extra addition of HNT nanotubes no longer

produces enhancing effects. In fact, both tensile strength

and Young’s modulus start falling down with the increase

of HNT content from 4 to 6 wt%, as demonstrated in

Table 2. To quantitatively characterize the toughness of

nanocomposite membranes, the energy to break was

determined by integrating the stress–strain curves in

Fig. 7a. It is shown in Table 2 that neat PLLA possesses

the energy to break of 0.32 MJ/m3. Accompanied by the

addition of HNTs, the energy to break for the nanocom-

posite is elevated up to 0.90 MJ/m3 (at 4 wt%). Therefore,

the incorporation of HNTs induces conspicuous increase in

toughness.

According to the reported studies, the Young’s modulus

of human skin ranges from 15 to 150 MPa, dependent to

different age [47]. As shown in our study, Young’s mod-

ulus of PLLA-based nanofiber membranes can be elevated

from 7.1 MPa (neat PLLA) to 14.2 MPa (4 wt% HNT/

PLLA), which is just lying in the aforementioned range. Of

course, if higher Young’s modulus is required, the scaf-

folds fabricated in our study still need further processing

including thermal annealing to achieve the desired

mechanical properties.

The mechanical reinforcement performance of fillers on

composites is relied on the effective load transfer from the

matrix to the fillers [48], which can be achieved when there

are strong interactions at the nanofiller-matrix interface and

the nanofillers are dispersed uniformly in the matrix [49].

There exist three main mechanisms for interactions

between matrix and fillers, which are micromechanical

interlocking, chemical bonding, and van der Waals force.

There are effective interactions between HNT walls and

PLLA chains due to the existence of hydrogen bonding.

As-received HNTs are held together in bundles van der

Waals force. Thus, it is crucial to disperse nanotubes well

in polymer matrix to acquire satisfactory mechanical per-

formance of the composites. Ultrasonication was employed

in the fabrication of HNT/PLLA composite nanofibers. To

monitor the distribution of HNT within the polymer matrix,

TEM observation was performed. As shown in Fig. 8a, the

incorporated nanotubes are straight and aligned along the

fiber axis. In fact, agglomeration of HNTs in the 2 wt%

HNT/PLLA nanofibers is seldom observed, indicating good

dispersion of HNTs. Thus, applied external load can be

Fig. 6 TGA curves of electrospun HNT/PLLA composite nanofiber

membranes

J Mater Sci (2015) 50:1435–1445 1441

123

effectively transferred to HNTs, inducing the improvement

of mechanical properties for PLLA-based composite

nanofibers. However, if HNT content is too high (e.g.,

6 wt%), agglomeration becomes evident. As shown in

Fig. 8b, the two HNTs in the fibers are assembled

‘‘shoulder by shoulder’’. Good particle–matrix interfacial

adhesion cannot form in aggregative nanotubes, which

impairs the effective load transfer from the polymer matrix

to the fillers. Thus, these aggregates act as defects, result-

ing in degraded mechanical performance [35]. In the results

of DSC, 4 wt% is deduced to be the critical concentration

to judge whether the dispersion of HNTs is aggravated.

HNT/PLLA nanofiber membranes exhibit their best

mechanical performance at 4 wt% of HNT content, which

is consistent of DSC results.

It should be emphasized that nanotubes are preferen-

tially oriented along the longitude of fiber, which is

induced by the shear force during electrospinning pro-

cessing. Longitudinal alignment rather than random ori-

entation of nanofillers in nanofibers is beneficial for the

improvement of the mechanical properties of the HNT/

PLLA nanocomposite [25].

To gain better estimation of the enhancing effect of

HNT particles, Reuss and Voigt models equation [46, 50]

was used to estimate the reinforcement effect of the HNT

particles on the Young’s modulus of the composite. The

Fig. 7 Stress-strain and Young’s modulus of electrospun HNT/PLLA nanofiber membranes

Table 2 Mechanical properties of electrospun HNT/PLLA nanofiber

membranes

HNT

content

(wt%)

Tensile

strength

(MPa)

Young’s

modulus

(MPa)

Elongation

at break (%)

Toughness

(MJ/m3)

0 0.75 ± 0.02 7.1 ± 0.6 59.6 ± 6.1 0.32 ± 0.03

1 0.92 ± 0.04 12.0 ± 1.3 74.9 ± 7.9 0.52 ± 0.04

2 1.05 ± 0.02 12.9 ± 1.2 79.3 ± 8.2 0.63 ± 0.05

4 1.21 ± 0.02 14.2 ± 1.3 88.7 ± 9.0 0.90 ± 0.07

6 1.13 ± 0.03 13.7 ± 1.3 71.6 ± 7.2 0.62 ± 0.05

Fig. 8 TEM images of

electrospun 2 wt% (a) and

6 wt% (b) HNT/PLLA

nanofibers

1442 J Mater Sci (2015) 50:1435–1445

123

lower and upper bounds of Young’s modulus deduced by

Reuss and Voigt models are given by:

Elowerc ¼ Ef Em

�Ef 1� Vf

� �þ EmVf

� �ð1Þ

Eupperc ¼ Ef Vf þ Em 1� Vf

� �; ð2Þ

where Eclowerand Ec

upperare lower- and upper-bound of

composite’s modulus, Ef and Em are modulus of filler and

matrix, and Vf is volume fraction of filler. Ef of HNT was

assumed to be 300 GPa [51]. Here, 1200 and 60 nm are

used as lf and df, respectively, from the SEM image (data

not shown). The volume fraction of the HNT can be cal-

culated according to

Vf ¼Wf

Wf þqf

qm

� �� qf

qm

� �Wf

; ð3Þ

where Wf is the weight fraction of the HNT, and qf and qm

are the densities of the HNT and the polymer matrix,

respectively. HNT density and PLLA density are taken as

2.5 and 1.25 g/cm3, respectively. The experimental data,

theoretical lower- and upper-bound of Young’s modulus

are showed in Fig. 7b. It is found that the experimental data

lie between these two bounds, confirming the reasonability

of our results.

Protein adsorption onto HNT-doped PLLA fibrous

scaffolds

An ideal scaffolding material should allow good protein

adsorption onto the material surface, to provide sufficient

nutrition to promote cell growth and migration. As dem-

onstrated in Fig. 9, neat PLLA and HNT-incorporated

PLLA nanofiber scaffolds have greater absorption of FBS

than cover slips which are lack of porous fibrous structure.

In addition, the HNT/PLLA nanofiber membranes can

absorb more protein than PLLA samples without HNTs.

The protein adsorption onto PLLA scaffolds with 1, 2 and

4 wt% HNT loading is 1.1–1.8 times higher than that of

neat PLLA scaffolds. Therefore, the incorporation of HNT

favors the protein adsorption, which could be related to the

difference in surface composition of nanofibers due to the

incorporation of HNTs [52]. In fact, rough surface of

nanofiber with high HNT content may also make partial

contribution to the higher protein absorption [53].

Conclusions

HNTs were employed for enhancing the mechanical proper-

ties of PLLA-based nanofibers scaffolds. Substantial

improvement of mechanical properties of HNT/PLLA com-

posite nanofibers is achieved with the addition of HNTs. At

4 wt% of HNT content, HNT/PLLA nanofiber membranes

possess the optimum mechanical performance, which repre-

sents 61 % increase in tensile strength, 100 % improvement

of Young’s modulus, 49 % augment of elongation to break, as

well as 181 % elevation in energy to break. The results of

SEM and TEM demonstrate the effective dispersion of HNTs

in PLLA matrix. Strong interactions between nanofillers and

PLLA are confirmed by FTIR, XRD, and DSC. Therefore,

efficient transfer of applied load from the matrix to HNTs is

enabled, which explains the reinforcement effect of incorpo-

rated HNTs on PLLA nanofibers. The introduction of HNTs

also improves the degree of crystallization and thermal sta-

bility of PLLA-based nanofibers. Furthermore, HNT-rein-

forced PLLA nanofiber membranes possessed higher protein

adsorption from FBS than neat PLLA specimen, which pos-

sesses potential application in tissue engineering.

Acknowledgements This research was supported by the National

Natural Science Foundation of China (Grant No. 21071114), the

Excellent Program of Activity of Science and Technology for Over-

seas-Returned Scientists founded by the Ministry of Human Resources

and Social Security of the People’s Republic of China, the Program for

Innovative Research Teams of Hubei Provincial Department of Edu-

cation, the Scientific Research Foundation for Returned Overseas

Chinese Scholars of State Education Ministry, Key Natural Science

Foundation of Hubei Province (Grant No. 2012FFA100), the Inno-

vative Team Incubation Program in High-Tech Industry of Wuhan

City (Grant No. 2014070504020244) and Graduate Innovative Fund of

Wuhan Institute of Technology (Grant No. CX2013010).

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