Hydroxyapatite nanorods/poly(vinyl pyrolidone) composite nanofibers, arrays and three-dimensional fabrics: electrospun preparation and transformation to hydroxyapatite nanostructures.

Abstract

Electrospinning has been recognized as an efficient technique for fabricating polymer nanofibrous biomaterials. However, the study of electrospun inorganic biomaterials with well-designed three-dimensional (3-D) structures is still limited and little reported. In this study hydroxyapatite (HAp) nanorods with an average diameter of approximately 7 nm and length of approximately 27 nm were synthesized through a simple precipitation method and used for the fabrication of inorganic/organic [poly(vinyl pyrolidone) (PVP)] composite nanofibers by electrospinning in ethanol solution. 3-D fabrics and aligned nanofiber arrays of the HAp nanorods/PVP composite were obtained as precursors. Thereafter, 3-D single phase HAp fabrics, tubular structures and aligned nanofiber arrays were obtained after thermal treatment of the corresponding composite precursors. Cytotoxicity experiments indicated that the HAp fabric scaffold had good biocompatibility. In vitro experiments showed that mesenchymal stem cells could attach to the HAp fabric scaffold after culture for 24h.

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Hydroxyapatite nanorods/poly(vinyl pyrolidone) composite nanofibers, arrays
and three-dimensional fabrics: Electrospun preparation and transformation
to hydroxyapatite nanostructures
Feng Chen, Qi-Li Tang, Ying-Jie Zhu*, Ke-Wei Wang, Mei-Li Zhang, Wan-Yin Zhai, Jiang Chang
State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050,
People’s Republic of China
a r t i c l ei n f o
Article history:
Received 16 October 2009
Received in revised form 12 January 2010
Accepted 10 February 2010
Available online 16 February 2010
Keywords:
Hydroxyapatite
Electrospinning
Nanofiber
Nanorod
Porous scaffold
a b s t r a c t
Electrospinning has been recognized as an efficient technique for fabricating polymer nanofibrous bioma-
terials. However, the study of electrospun inorganic biomaterials with well-designed three-dimensional
(3-D) structures is still limited and little reported. In this study hydroxyapatite (HAp) nanorods with an
average diameter of ?7 nm and length of ?27 nm were synthesized through a simple precipitation
method and used for the fabrication of inorganic/organic [poly(vinyl pyrolidone) (PVP)] composite nanof-
ibers by electrospinning in ethanol solution. 3-D fabrics and aligned nanofiber arrays of the HAp nano-
rods/PVP composite were obtained as precursors. Thereafter, 3-D single phase HAp fabrics, tubular
structures and aligned nanofiber arrays were obtained after thermal treatment of the corresponding com-
posite precursors. Cytotoxicity experiments indicated that the HAp fabric scaffold had good biocompat-
ibility. In vitro experiments showed that mesenchymal stem cells could attach to the HAp fabric scaffold
after culture for 24 h.
? 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction
Bone is a multiphase composite, with the main constituents of
bone being collagen matrix and assembled hydroxyapatite (HAp)
crystals [Ca10(PO4)6(OH)2]. HAp is the major inorganic constituent
in human bone [1]. Synthetic HAp particles, films, coatings and
porous skeletons with high biocompatibility are widely used in
various biomedical applications [2]. Numbers of methods have
been used for the synthesis of HAp, such as the wet chemical, mec-
hano-chemical [3,4] and sol–gel methods [5,6]. HAp materials with
different morphologies, including nanorods [7], plate-like nano-
crystals [8], nanoparticles [9] and three-dimensional (3-D) struc-
tures [10–13] have been synthesized. They can be used as a kind
of ideal biomaterial for application in tissue engineering, drug/gene
delivery systems and other fields [14–16].
Electrospinning has been recognized as an efficient technique
for fabricating polymer nanofibers which can be widely used in
biomedical areas [17,18]. A great number of polymer and compos-
ite nanofibers have been prepared by electrospinning [19]. Recent
developments in fiber electrospinning have shown that it is a
promising way to produce future advanced composite systems.
For instance, nanofibers are ideally suited to form a scaffold on
which multi-functional components can be hierarchically orga-
nized [20]. Electrospun fibers can also form well-designed patterns
with complex ordered architectures using patterned conductive
collectors [21,22]. Cell adhesion, growth and biomineralization
on hybrid membranes are usually better than on pure polymer
membranes [23–25]. However, investigations on electrospun inor-
ganic biomaterials with well-designed 3-D structures are still lim-
ited and little reported. However, the production of non-woven
HAp fibers by electrospinning using a solution containing polyvinyl
alcohol has been reported [26,27].
In this study HAp nanorods were synthesized through a simple
precipitation method and then incorporated into PVP nanofibers to
form HAp nanorods/PVP composite nanofibers through electros-
pinning. 3-D fabrics with different shapes and aligned nanofiber
arrays of the HAp nanorods/PVP composite were obtained by
changing the collectors used during electrospinning. 3-D fabrics,
tubular structures and aligned nanofiber arrays of single phase
HAp were obtained by thermal treatment of corresponding com-
posite precursors. Cytotoxicity experiments indicated that a HAp
fabric scaffold had good biocompatibility.
2. Experimental section
All chemicals were analytical grade reagents and were used as
received, without further purification. For the preparation of HAp
1742-7061/$ - see front matter ? 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.actbio.2010.02.015
* Corresponding author. Tel.: +86 21 52412616; fax: +86 21 52413122.
E-mail address: y.j.zhu@mail.sic.ac.cn (Y.-J. Zhu).
Acta Biomaterialia 6 (2010) 3013–3020
Contents lists available at ScienceDirect
Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat

Page 2

nanorods 0.916 g of CaCl2?2H2O and 5.334 g of acrylamide were
dissolved in 30 ml of deionized water under continuous stirring.
An aliquot of 0.508 g of (NH4)2HPO4 was dissolved in 15 ml of
deionized water and was then slowly added to the above solution.
The pH value of the solution was kept at 10 using NH3?H2O. The
reaction system was aged for about 1 h. The product was separated
by centrifugation and washed with deionized water and absolute
ethanol several times.
Thereafter, as prepared HAp nanorods and 0.8 g of PVP (K-90)
were dispersed in 10 ml of absolute ethanol and used for electros-
pinning. As a control experiment a solution of PVP in ethanol at a
concentration of 8 wt.% was used for electrospinning under the
same conditions. The solution for electrospinning was fed into a
plastic syringe with a needle (inner diameter ?210 lm). A syringe
pump (WZS-50F6, Smiths Medical Instruments, China) was used to
feed the solution to the needle at a rate of 1.0 ml h?1. The voltage
applied was 12 kV using a high voltage power supply (BGG6-358,
BMEI, China). A grounded foil and stainless steel tube were used
to collect nanofibers at a fixed distance (15 mm from the needle
tip). Aligned nanofibers were obtained with two parallel grounded
aluminum slats. To prepare HAp nanofibers and fabrics the HAp/
PVP composites were used as precursors. They were heated to
600 ?C and held at this temperature for 6 h in air in a muffle fur-
nace (Shanghai YI-Feng Electrical Furnace Co.). The strategies for
fabrication of HAp/PVP composite nanofibers and fabrics and single
phase HAp nanofibers and fabrics are presented in Fig. 1.
Transmission electron microscopy (TEM) was performed with a
JEOL JEM-2100F field-emission transmission electron microscope
with an accelerating voltage of 200 keV. Scanning electron micros-
copy (SEM) was performed with a JEOL JSM-6700 field-emission
Fig. 1. Scheme of the strategy for fabrication of HAp/PVP composite nanofibers and fabrics (A) and the process of formation of HAp nanofibers (B).
Fig. 2. Transmission electron micrographs of HAp nanorods (A) and HAp nanorods/PVP composite fibers (B).
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scanning electron microscope. X-ray powder diffraction (XRD) was
performed in a Rigaku D/max 2550 V X-ray diffractometer with
high intensity CuKaradiation (k = 1.54178 Å) and a graphite mono-
chromator. Fourier transform infrared (FTIR) spectroscopy was
performed with a Nicolet 380 spectrophotometer (Thermo Scien-
tific, Waltham, MA) by the KBr pellet technique. Differential scan-
ning calorimetric (DSC) and thermogravimetric (TG) analyses were
carried out with a STA-409PC/4/H Luxx simultaneous TG-DTA/DSC
apparatus (Netzsch, Germany) at a heating rate of 10 ?C min?1un-
der flowing air. Alignment of nanofibers was analyzed using the
two-dimensional (2-D) fast Fourier transform approach [28].
Mesenchymal stem cells (MSCs) were isolated from the bone
marrow of New Zealand rabbits and maintained in Dulbecco’s
modifiedEagle’smedium(DMEM)(4.5 g l?1glucose) supplemented
with 10% fetal bovine serum (FBS), penicillin (100 U ml?1) and
streptomycin (100 lg ml?1) at 37 ?C in a 5% CO2humidified atmo-
sphere. For cytotoxicity analysis cells were seeded on sterilized
HAp fabrics in 96-well plates at a density of 1500 cells per well.
The cells were washed with phosphate-buffered saline (PBS) on
days 1 and 3. Aliquots of 50 ll of MTT solution in DMEM
(2 mg ml?1) were added to each well. Plates were incubated for
an additional 2 h at 37 ?C. The MTT-containing medium was re-
moved and 100 ll of DMSO was added to dissolve the formazan
crystals formed by living cells. The optical density (OD) was mea-
sured at a wavelength of 490 nm using an enzyme-linked immuno-
adsorbent assay plate reader (ELx 800, Biotek, Winooski, VT). To
0200 400600800
0
5
10
15
20
25
30
35
289 nm
Number of nanofibers
Diameter (nm)
0200400600 800
0
10
20
30
40
50
Number of nanofibers
277 nm
Diameter (nm)
C C
F F
Fig. 3. Scanning electron micrographs of as prepared HAp nanorods/PVP composite nanofibers (A and B), pure PVP nanofibers (D and E) and the corresponding diameter
distribution (C and F).
Fig. 4. XRD patterns (A and C) and FTIR (B and D) spectra of as prepared samples. (C and D) XRD pattern in the range of low 2h angle and FTIR spectrum in the range of low
wavenumbers of 450–700 cm?1.
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investigate the morphology of cells on the HAp fabric cells cultured
for 24 h on the fabric and on coverslips were fixed with 2.5% glutar-
aldehyde and dehydrated in 50, 75, 90 and 100% alcohol before
observation by SEM.
3. Results and discussion
Fig. 2A shows a transmission electron micrograph of the HAp
sample prepared by precipitation using (NH4)2HPO4 and CaCl2.
The as prepared HAp sample consisted of nanorods with an aver-
age diameter of ?7 nm and length of ?27 nm. As shown in
Fig. 2B, the HAp nanorods were incorporated into PVP nanofibers
by electrospinning using ethanol solution to form HAp nanorods/
PVP composite nanofibers. The HAp nanorods were well dispersed
in the PVP nanofibers.
The morphology and diameter of HAp/PVP composite nanofi-
bers prepared by electrospinning were investigated by SEM
(Fig. 3). Pure PVP nanofibers were also prepared for comparison.
Compared with pure PVP nanofibers one can see that the blending
of HAp nanorods with PVP had little influence on the morphology
and diameter of the composite nanofibers. The average diameter of
HAp/PVP composite nanofibers was ?289 nm, similar to that of
pure PVP nanofibers (277 nm). However, the HAp/PVP composite
nanofibers displayed protuberances in places, while, in contrast,
the pure PVP nanofibers were more uniform and smooth.
Fig. 4A shows the XRD patterns of single phase HAp nanorods
and HAp/PVP composite nanofibers. The peaks in the XRD pattern
can be related to HAp with a hexagonal structure (JCPDS No. 09-
0432). The diffraction peaks of single phase HAp nanorods at
2h ? 25.8?, 28.9?, 31.7?, 34.0?, 40.5?, 46.7?, 49.5?, 53.1? and 64.1?
correspond to the (0 0 2), (2 1 0), (2 1 1), (2 0 2), (2 2 1), (2 2 2),
(2 1 3), (0 0 4) and (3 0 4) crystal faces of HAp. During the prepara-
tion of HAp by the precipitation method it is likely to form inter-
mediate phases of calcium phosphate [for example octacalcium
phosphate (OCP)]. We performed additional XRD characterization
of the product in the low 2h range – this data is shown in
Fig. 4C. Fig. 4C shows that there was no obvious peak at around
2h = 4.7?, where the main diffraction peak of OCP is located. The
main characteristic peaks of HAp were also observed for HAp/
PVP composite nanofibers, although the peak intensities were low-
er as a result of the dispersion of HAp nanorods in the PVP polymer
matrix.
As shown in the FTIR spectra in Fig. 4B, there is an obvious
absorption peak at 1658.5 cm?1for PVP, which can be assigned
to the amide I group, consisting of >C@O and C@N. In the FTIR
spectrum of HAp nanorods bands corresponding to PO43?are ob-
served at 1031.7, 603.6 and 563.1 cm?1, while the peak at
?3571.5 cm?1can be assigned to the hydroxyl group of HAp. The
detailed FTIR spectrum of HAp nanorods corresponding to PO43?
in the range 450–700 cm?1is presented in Fig. 4D. These character-
istic peaks also appear in the FTIR spectrum of composite nanofi-
bers, consistent with the above discussed XRD, TEM and SEM
results, indicating that a HAp/PVP composite had been successfully
fabricated by the electrospinning method.
The thermal properties, such as the thermal stability, of the
polymer and composite are very important for their application,
and are usually studied by TG and DSC [29]. TG and DSC curves
for the as prepared single phase HAp nanorods, HAp/PVP compos-
ite nanofibers and PVP nanofibers are shown in Fig. 5. The weight
loss of single phase HAp nanorods was ?13.4% at about 195 ?C in
the TG curve, accompanied by an endothermic peak at near
195 ?C in the DSC curve. This was due to the loss of water mole-
cules from the product. For the PVP nanofibers without HAp the
weight loss of ?90% from 250 ?C to 650 ?C is due to decomposition
and oxidation of the PVP polymer nanofibers, accompanied by exo-
thermic peaks at about 375 ?C and 500 ?C in the DSC curve. For the
HAp/PVP composite nanofibers the weight loss of ?57.05% from
230 ?C to 660 ?C was due to oxidation of the PVP polymer in the
nanofibers, and exothermic peaks at about 375 ?C and 500 ?C ap-
peared in the DSC curve.
A thermal treatment route has been designed to prepare single
phase HAp fabrics with different shapes using HAp/PVP composite
nanofibers as the precursor. Characterization of the as prepared
single phase HAp fabrics is shown in Fig. 6. From Fig. 6C one can
see that the as prepared single phase HAp fabric was composed
of HAp nanofibers. The shapes of the precursor HAp/PVP composite
nanofibers were well preserved during the calcination process.
Thus, single phase HAp fabrics with a well-defined shape can be
obtained by using HAp/PVP composite nanofibers with a similar
200400600800
Temperature (°C)
Temperature (°C)
Temperature (°C)
B
DSC Exothermal
DSC
TG
0
20
40
60
80
100
90.0 %
Weight (%)
200400600800
30
40
50
60
70
80
90
100
DSC Exothermal
DSC
TG
Weight (%)
57.05 %
C
200400600800
DSC
TG
DSC Exothermal
13.4 %
76
80
84
88
92
96
100
Weight (%)
A
Fig. 5. TG and DSC curves of single phase HAp nanorods (A), PVP nanofibers (B) and
HAp/PVP composite nanofibers (C).
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shape as the precursor. SEM image of the single phase HAp fabric
also shows a porous structure (Fig. 6C). The peaks in the XRD pat-
tern in Fig. 6D can be related to single phase HAp with a hexagonal
structure.
A fabric composed of HAp/PVP composite nanofiber arrays has
been obtained with two parallel grounded aluminum slats. As
shown in Fig. 7A and B, the HAp/PVP composite nanofibers were
parallel, forming HAp/PVP composite nanofiber arrays. This can
be reasonably explained by the change in electric force during
the electrospinning process. There are two kinds of electric force
in the electrospinning process which can influence the arrange-
ment of the electrospun nanofibers: the electrostatic force result-
ing from the electric field and Coulomb interactions between
positive charges on the nanofibers and negative charges on the col-
lectors [30,31]. When using parallel substrates as collectors the
electric field is changed and the nanofiber arrays are collected be-
tween two substrates.
After calcining the fabric composed of HAp/PVP composite
nanofiber arrays as precursor a fabric composed of single phase
HAp nanofiber arrays was obtained. As shown in Fig. 7D, each sin-
gle HAp nanofiber was an assembly of HAp nanoparticles with
diameters of several tens of nanometers, different from the precur-
sor HAp/PVP composite nanofibers. Upon calcination neighboring
HAp nanorods in the PVP matrix can grow to form HAp nanoparti-
cles and then form single phase HAp nanofiber arrays. This novel
fabric composed of single phase HAp nanofiber arrays has potential
application as a porous scaffold which can be used in tissue engi-
neering, surface modification and medical implants. Fig. 7E shows
the raw output of the 2-D fast Fourier transform (FFT) alignment
analysis of the fabric composed of single phase HAp nanofiber ar-
rays corresponding to Fig. 7C. Pixel intensities and the distribution
of the intensities in this output image correlate with the directional
content of the original image. The slender profiles in the raw out-
put of the 2-D FFT indicate the arrays of nanofibers. Fig. 7F shows a
radial plot of the summation of relative pixel intensity at a radius
versus the angle (?) caused by the inherent symmetry of the raw 2-
D FFT output. Curve (a) shows two peaks at 90? and 270?, indicat-
ing the general direction in which the nanofibers were oriented. A
fabric composed of randomly distributed nanofibers without align-
ment used as a control [curve (b) in Fig. 7F] displayed four peaks at
0?, 90? 180? and 270?.
MSCs are multipotent stem cells that can differentiate into a
variety of cell types and are widely used as seed cells in tissue engi-
neering. In this study as prepared HAp fabric scaffolds were com-
pared with reference tissue culture plates (TCP) in terms of
in vitro cytotoxicity (Fig. 8) using MSCs. As the culture time in-
creased from 24 to 72 h the OD values, which indicate the viability
of cells cultured on the HAp fabric scaffold and TCP, increased from
0.17 and 0.16 to 0.22 and 0.21, respectively. The OD values for the
HAp fabric scaffold were close to those of TCP at both 24 and 72 h.
These results indicate that the as prepared HAp fabric scaffold has
essentially no in vitro cytotoxicity and is comparable with the tis-
sue culture plates.
The HAp fabric was tested as a substrate for MSC culture. Rep-
resentative SEM images of MSCs cultured on the HAp fabric are
shown in Fig. 9. MSCs cultured on coverslips were also investigated
for comparison. The MSCs attached well to the HAp fabric substrate
after culture on the fabric substrate for 24 h. A magnified image
(Fig. 9C) presents details of interconnections between the cells
and HAp fabric substrate.
PVP polymer is virtually bioinert and has vast potential for bio-
medical application [32,33]. During the electrospinning process no
additives were used. In previous studies hybrid nanofibers of
poly(L-lactic acid) (PLLA)/HAp were prepared by electrospinning.
Cell adhesion and growth on a PLLA/HAp hybrid membrane were
far better than on a pure PLLA membrane [23,24]. Having a similar
crystalline phase to the inorganic component of natural bone, HAp
ceramics have been extensively used as substitutes in bone grafts
[34,35]. However, it is still difficult to develop porous HAp ceram-
ics with interpore connections, which would be expected to be
Fig. 6. The single phase HAp fabrics prepared by calcination of HAp/PVP composite nanofibers as the precursor. (A) Film, (B) tubular fabric, (C) scanning electron micrograph
and (D) XRD pattern.
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ideal bone substitutes [36–38]. This study has proved that electros-
pinning is a promising way to obtain HAp fabrics composed of
nanofibers with a porous structure. Electrospinning has also been
used as a promising technique for shaping biomaterials [21,22].
Herein, inorganic/organic composite nanofibers consisting of HAp
nanorods and PVP were prepared by electrospinning using an eth-
anol solution containing PVP. 3-D fabrics with different shapes and
aligned nanofiber arrays of HAp nanorods/PVP composite were ob-
tained. 3-D single phase HAp fabrics, tubular structures and
aligned nanofiber arrays were obtained through thermal treatment
of the composite precursors. These results show that HAp nanorods
can be used for the fabrication of 3-D fabrics and aligned nanofi-
bers as novel bio-inorganic fibrous scaffolds by electrospinning. It
is expected that a variety of structures consisting of HAp nano-
rods/PVP composite or single phase HAp prepared by the present
method have great potential application in the biomedical field.
4. Conclusions
A simple method has been developed for the preparation of
HAp/PVP composite nanofibers, 3-D fabrics with different shapes
and aligned nanofiber arrays by electrospinning. Single phase
HAp fabrics, tubular structures and aligned nanofiber arrays can
be obtained by thermal treatment of the corresponding composite
Fig. 7. Characterization of the fabrics composed of HAp/PVP composite nanofiber arrays and of single phase HAp nanofiber arrays. (A and B) Optical micrographs of the fabric
composed of HAp/PVP composite nanofiber arrays. (C and D) SEM images of the fabric composed of single phase HAp nanofiber arrays. (E) Raw output of the 2-D FFT
alignment analysis of the fabric composed of single phase HAp nanofiber arrays corresponding to (C). (F) Radial plot of the summation of relative pixel intensity at a radius
versus the angle (?) caused by the inherent symmetry of the raw 2-D FFT output. Pattern (a) corresponds to the SEM image in (C), pattern (b) corresponds to the SEM image of
Fig. 6C for comparison.
0.00
0.05
0.10
0.15
0.20
0.25
0.30
2472
MTT OD (490 nm)
TCP
HAp fabrics
Time (hour)
Fig. 8. In vitro cytotoxicity assays of MSCs cultured on tissue culture plates (TCP)
and HAp fabric for 24 and 72 h.
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precursors. One advantage of this method is that it is easy to obtain
HAp fabric scaffolds with well-designed structures. The as pre-
pared HAp fabric was used as the substrate for MSC culture. The re-
sults show that the HAp fabric scaffold was biocompatible and cells
could attach well to it. These results show that the HAp nanorods
could be used to fabricate 3-D fabrics and aligned nanofiber arrays
as novel bio-inorganic fibrous scaffolds by electrospinning. It is ex-
pected that a variety of structures consisting of HAp nanorods/PVP
composite or single phase HAp prepared by the present method
have great potential application in the biomedical field.
Acknowledgements
Financial support from the National Natural Science Foundation
of China (50772124 and 50821004), the Fund for Nano-Science and
Technology of the Science and Technology Commission of Shang-
hai (0852nm05800), the Program of Shanghai Subject Chief Scien-
tist (07XD14031), Shanghai-Unilever Research and Development
Fund (09520715200) and the Key Project for Innovative Research
(SCX 0606) of the Shanghai Institute of Ceramics is gratefully
acknowledged.
Appendix A. Figures with essential colour discrimination
Certain figures in this article, particularly Figures 1 and 4–8, are
difficult to interpret in black and white. The full colour images can
be found in the on-line version, at doi:10.1016/j.actbio.2010.
02.015).
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