International Journal of Biological Macromolecules 46 (2010) 199–205

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International Journal of Biological Macromolecules

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

Alginate–chitosan/hydroxyapatite polyelectrolyte complex porous scaffolds:

Preparation and characterization
Jing Han a , Ziyou Zhou a , Ruixue Yin a , Dongzhi Yang a , Jun Nie a,b,∗
a
State Key Laboratory of Chemical Resource Engineering, The Key Laboratory of Beijing City on Preparation and Processing of Novel Polymer Materials,
Beijing University of Chemical Technology, Beijing 100029, PR China
b
College of Chemistry and Molecular Science, Wuhan University, Wuhan 430072, PR China

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

Article history: Porous scaffolds composed of alginate (AG) and chitosan (CS) were fabricated by combining the for-
Received 15 October 2009 mation of polyelectrolyte complex (PEC) with freeze-drying. The AG scaffold was used as a framework
Received in revised form with uniformly distributed and interconnected pore structure. The chitosan or chitosan/hydroxyapatite
15 November 2009
(CS/HA) composite solution was introduced into the pores of AG scaffold to form PEC scaffolds. FT-IR,
Accepted 18 November 2009
XRD and XPS analysis confirmed that CS or CS/HA was coated on the AG scaffold surface. Microstructure,
Available online 24 November 2009
porosity, mechanical strength and thermal stability of PEC scaffolds were also investigated. The AG–CS
PEC scaffold and Ca2+ crosslinked AG scaffold showed smaller average pore diameter and lower porosity
Keywords:
Alginate
than those of uncrosslinked AG scaffold. Moreover, compared with Ca2+ crosslinked AG scaffold, AG–CS
Chitosan PEC scaffold exhibited higher mechanical strength and better thermal stability.
Hydroxyapatite © 2009 Elsevier B.V. All rights reserved.
Polyelectrolyte complex
Scaffold

1. Introduction are appealing as scaffold materials because they can be produced

Tissue engineering as a multidisciplinary science has been con-
sidered as a promising technology to develop biological substitutes
for failing tissues and organs. The most common approach pro-
posed by Langer and Vacanti is based on living cell, signal molecules
and polymer scaffolds [1]. The scaffold serves as an extracellular
matrix to organize cells into a three-dimensional architecture and
guide tissue regeneration by controlled release of signal molecules.
To perform significant function, the scaffolds should have adequate
porosity, suitable pore size and interconnected pore structure for
the transporting cells, metabolites, nutrients and signal molecules.
Besides, the scaffold should be nontoxic, nonimmunogenic, bio-
compatible and biodegradable at ideal rate corresponding to the
rate of new tissue formation [2]. Therefore, the fabrication of vari-
ous functional suitable scaffolds has attracted much attention in
the field of tissue engineering. A variety of synthetic and natu-
rally derived polymers have been explored as scaffolds for tissue
engineering application. Synthetic polymers including polycapro-
lactone [3], poly(ethylene oxide) [4] and poly(vinyl alcohol) [5]
∗ Corresponding author at: State Key Laboratory of Chemical Resource Engineer-
ing, Beijing University of Chemical Technology, Beijing 100029, PR China.
Fax: +86 10 64421310.
E-mail address: niejun@mail.buct.edu.cn (J. Nie).
with specific properties such as molecular weights, block struc-
tures and cross-linking modes. Naturally derived polymers such as
alginate [6], chitosan [7], collagen [8], fibrin [9], gelatin [10] and
hyaluronate [11] are of special interest due to their biological and
chemical similarities to natural tissues. There are many methods to
manufacture porous scaffolds including porogen leaching, emul-
sion freeze-drying, 3D printing, phase separation techniques and
so forth. Madihally et al. prepared porous chitosan scaffolds with
controlled microstructure in several tissue-relevant geometries by
freeze-drying method [12]. Ho et al. fabricated porous PLLA, PLGA,
chitosan and alginate scaffolds by freeze-fixation or freeze-gelation
method [13]. Ang et al. fabricated 3D chitosan–hydroxyapatite scaf-
folds using a robotic desktop rapid prototyping system [14].
Alginate, derived primarily from brown seaweed and bacteria,
is a linear polysaccharide copolymer that consists of two sterically
different repeating units, (1,4)-␤-d-mannuronic acid (M) and ␣-l-
guluronic acid (G) in varying proportions. Alginate has been studied
extensively in tissue engineering as scaffold materials including the
regeneration of skin [15], cartilage [16], bone [17], and cardiac tis-
sue [18] because it can form gel under gentle condition by divalent
cations such as Ca2+ , Ba2+ or Sr2+ cooperatively interact with block
of G monomers to form ionic bridge between different polymer
chains. However, cells do not bind directly to alginates and no pro-
tein absorption usually takes place on the negatively charged gel
[19]. Specific small molecules are incorporated in order to enhance

0141-8130/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijbiomac.2009.11.004
200 J. Han et al. / International Journal of Biological Macromolecules 46 (2010) 199–205

adhesion, proliferation and differentiation of several cell types [20].

Chitosan is the deacetylated derivative of chitin, composed of ␤-
(1,4)-2-amine-2-deoxy-d-glucopyranose units and small amount
of N-acetyl-d-glucosamine residues. Chitosan has been demon-
strated to own unique properties including antimicrobial activity,
biocompatibility, biodegradability and accelerating wound heal-
ing property, and therefore considered as a promising supporting
material in various tissue engineering applications namely skin,
bone, cartilage, liver, nerve and blood vessel [2]. The cationic
nature of chitosan endows electrostatic interactions with anionic
glycosaminoglycans (GAG) and other negatively charged species,
while a lot of cytokines or growth factors are bound and modu-
lated by GAG. That implied chitosan–GAG complex scaffold could
retain and concentrate desirable factors [12].
Fig. 1. Schematic illustration for fabricating various scaffolds.
The combination of alginate and chitosan via ionic interaction
between carboxylate moieties on alginate and protonated amines
on chitosan to form polyelectrolyte complex (PEC) in the form
of membrane [21], capsule [22], fiber [23], scaffold [24] has been 2.3. Preparation of various scaffolds
investigated widely. Previous method usually involved mixing the
components first and then fabricating PEC materials. For exam- The fabrication scheme of various scaffolds was shown in Fig. 1.
ple, Wang et al. prepared chitosan–alginate PEC membrane as a Sodium alginate was dissolved in deionized water to form 4% (w/v)
wound dressing via gradually mixing alginate solution and chitosan solution. The solution was placed into molds and frozen at −40 ◦ C
solution in the presence of acetone [21]. In this research, we fabri- for 12 h. Then the samples were transferred into a freeze-drying
cated alginate scaffold as a framework first and then immerged the vessel for at least 48 h to get porous scaffolds. One group of alginate
scaffold in chitosan solution to obtain AG–CS PEC scaffold based scaffold was crosslinked by 1 wt% CaCl2 solution for 20 min and
on ionic interaction between cationic chitosan and anionic algi- then washed with deionized water for three times and immersed
nate. Hydroxyapatite as inorganic component was also introduced in water overnight to remove unbound CaCl2 . Another group of
into the system via synthesizing chitosan/hydroxyapatite (CS/HA) alginate scaffold was immerged into 2% (w/v) chitosan or CS/HA
composite. Fourier transform infrared spectroscopy (FT-IR), X- composite acetic acid aqueous solution, resulting in the pores of the
ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) alginate scaffold being filled with chitosan solution. The samples
were utilized to confirm the formation of PEC. The properties of were frozen at −40 ◦ C and lyophilized. Finally, the samples were
PEC scaffolds were studied by using scanning electron microscope soaked in graded ethanol to remove acetic acid and then crosslinked
(SEM), mechanical tester and thermogravimetric analysis (TGA). with CaCl2 solution as above procedure.
Uncrosslinked AG scaffold and conventional Ca2+ crosslinked AG
scaffold were used as references. 2.4. FT-IR spectroscopy

FT-IR spectra of sodium alginate, chitosan, CS/HA composite, HA

2. Experimental powder and various scaffolds pelletized with KBr powder were
recorded on Nicolet Spectra 5700 spectrometer (Nicolet Instru-
2.1. Materials ment, Thermo Company, Madison, USA).

Chitosan (Mw = 200 kDa, degree of deacetylation = 86%) and

sodium alginate were purchased from Yuhuan Ocean Biochemical
Co., Ltd. (Zhejiang, China) and Sanpu Chemical Co., Ltd. (Shanghai,
China), respectively. Other reagents and solvents were of analytic
grade and purchased from Beijing Chemical Reagent Company.
2.2. Preparation and characterization of CS/HA composite
CS/HA composite with calculated mass ratio of chitosan/
hydroxyapatite = 5/2 was prepared by using a wet chemical co-
precipitation approach [25]. Briefly, 2.0 g chitosan was dissolved in
2 wt% acetic acid solution under agitation, and then 80 mL 0.1 mol/L
CaCl2 solution and 48 mL 0.1 mol/L KH2 PO4 solution were added
dropwisely. The pH value of mixture was adjusted to 9–10 by using
0.5 M NaOH solution under stirring for 48 h. Finally, the light yel-
low precipitate was obtained from suspension after washed with
deionized water for several times to neutral pH and then dried at
80 ◦ C overnight.
The composite was characterized by FT-IR and XRD. A small
mount of precipitated composite was calcined at 800 ◦ C for 4 h in a
Muffle furnace under air atmosphere to remove chitosan and obtain
white powder. The powder was also characterized by FT-IR and
XRD. According to the weight ratio of composite before and after
calcined, CS/HA composite contained 28 wt% hydroxyapatite.
2.5. XRD
X-ray diffraction (XRD) measurements of chitosan, CS/HA com-
posite, HA powder and AG–CS/HA PEC scaffold were conducted
by a Rigaku D/Max2500VB2+/Pc diffractometer (Rigaku Com-
pany, Tokyo, Japan) with 40 kV and 50 mA using Cu K␣ radiation
( = 0.154 nm). The scanning scope of 2 was 5–50◦ and the scan-
ning rate was 5◦ /min.
2.6. XPS
XPS spectra of scaffold were obtained by using a VG ESCALAB
MKII X-ray photoelectron spectrometer (VG Scientific Ltd., UK) with
Al K␣ radiation. Survey spectra were recorded for 0–1350 eV bind-
ing energy range.
2.7. SEM
The microstructure of various scaffolds was observed by scan-
ning electron microscope (Hitachi S-4700, Hitachi Company,
Japan). The specimens were cut from the scaffolds and fractured
in liquid nitrogen, and then fixed on stubs with sputter coated with
gold before observation.
 J. Han et al. / International Journal of Biological Macromolecules 46 (2010) 199–205 201

2.8. Porosity measurement

The porosity of scaffolds was measured by liquid displacement

method [26]. The procedure of liquid displacement method was
as follow: the volume (V0 ) and weight (W0 ) of the sample were
measured. Then, the sample was immersed into ethanol until it
was saturated by absorbing ethanol. The sample was weighted
again and noted as W1 . The porosity of the scaffold was calculated
according to the formula: Porosity (%) = (W1 − W0 )/(V0 ) × 100 (
represents the density of the ethanol).

2.9. Mechanical testing

The compressive strength was determined by using an Instron

1185 mechanical tester. The specimens were circular discs of 8 mm
in diameter and 12 mm in thickness. The crosshead speed of the
Instron tester was set at 5 mm/min and load was applied until 30%
reduction in specimen height. Five parallel samples were tested for
every scaffold and the final results were calculated averagely.

2.10. TGA

Dynamic thermogravimetric analysis (TGA) was carried out to

examine thermal stability of scaffolds by using a NETZSCH STA 449C
thermogravimetric analyzer (Germany). The experiments were
performed at a heating rate of 20 ◦ C/min from room temperature
to 800 ◦ C under 24 mL/min Ar flow.

3. Results and discussion

3.1. FT-IR analysis

Fig. 2(A) showed the FT-IR spectra of materials including sodium

alginate (a), chitosan (b), CS/HA composite (c) and HA powder
(d). The characteristic peaks of alginate appeared at 3429, 1620
and 1421 cm−1 , corresponding to hydroxyl (–OH), carbonyl (C O)
and carboxyl (COOH), respectively. The chitosan spectrum showed
characteristic bonds of amide (1620 cm−1 ), amino (1157 cm−1 ),
and C–O stretching (1422 cm−1 ). For CS/HA composite, the amide
peak shifted from 1620 to 1631 cm−1 . A small peak at 1035 cm−1
appeared, corresponding to phosphate stretching vibration. For HA Fig. 2. (A) FT-IR spectra of sodium alginate (a), chitosan (b), CS/HA composite (c)
and HA powder (d). (B) FT-IR spectra of various scaffolds: uncrosslinked AG scaffold
powder sample, the characteristic band at 1634 cm−1 corresponded (a), Ca2+ crosslinked AG scaffold (b) and AG–CS PEC scaffold (c).
to hydroxyl vibrations, while those at 1096 and 1037 cm−1 assigned
to phosphate stretching vibrations [27].
In order to confirm the interaction between alginate and calcium
ion or chitosan, FT-IR spectra of uncrosslinked AG scaffold (a), Ca2+
crosslinked AG scaffold (b) and AG–CS PEC scaffold (c) were shown
in Fig. 2(B). For AG–CS PEC scaffold, characteristic peak which is
assigned to amide group appeared at 1608 cm−1 , while no peak at
1157 cm−1 (amino) was observed, which indicated that protonated
amines on chitosan interacted with carboxylate on alginate.

3.2. XRD analysis

Fig. 3 showed the X-ray diffraction patterns of HA powder (a),

chitosan (b), CS/HA composite (c) and AG–CS/HA PEC scaffold (d).
For HA powder, the existence of 2 peaks at approximately 25.8◦ ,
31.8◦ , 32.1◦ , 32.9◦ , 34.0◦ , 39.9◦ , 46.7◦ and 49.4◦ corresponded to
the diffraction planes of (0 0 2), (2 1 1), (1 1 2), (3 0 0), (2 0 2), (1 3 0),
(2 2 2) and (2 1 3) of the HA crystallites [27]. Chitosan as a semi-
crystalline biopolymer presented broad diffraction peaks at 10.2◦ ,
19.8◦ and 21.9◦ [28]. For CS/HA composite, the typical crystalline
peaks of both chitosan and HA still existed (19.9◦ , 25.9◦ and 32.1◦ ),
but the peaks became broader and weaker as compared to HA pow- Fig. 3. XRD patterns of HA powder (a), chitosan (b), CS/HA composite (c) and
der. Besides, not all diffraction planes were clearly identified. The AG–CS/HA PEC scaffold (d).
202 J. Han et al. / International Journal of Biological Macromolecules 46 (2010) 199–205
Fig. 4. (A) XPS survey spectra of AG–CS PEC scaffold. (B) XPS survey spectra of
AG–CS/HA PEC scaffold.
molecular interaction between chitosan and HA led to hybridiza-
tion and therefore influenced the diffraction peaks [25]. AG–CS/HA
PEC scaffold still exhibited similar peaks as the CS/HA composite,
but peaks became weaker.
3.3. XPS analysis
XPS as a complementary technique provides information about
atomic composition of material surface. The AG–CS PEC scaffold
surface showed carbon (binding energy: 285 eV), oxygen (bind-
ing energy: 532 eV) and nitrogen (binding energy: 399 eV) peaks
as illustrated in Fig. 4(A) [29]. There were small peaks appearing
at 347 and 1072 eV which could be assigned to Ca2p and Na1s ,
respectively [30]. Nitrogen peak confirmed the existence of chi-
tosan on the scaffold surface because alginate does not contain
nitrogen. Further, the N1s /O1s ratio for AG–CS PEC scaffold is 0.186,
which is slightly lower than the calculated value (0.245). However,
results reported by other groups revealed that N1s /O1s ratio of chi-
tosan obtained from XPS was also lower than the calculated value
[29,30].
The elemental characteristic peaks of C1s , O1s , N1s and Ca2p
were identified from the surface of AG–CS/HA PEC scaffold, which
was similarly plotted with AG–CS PEC scaffold. A small peak at
133 eV was assigned to the P2p [31]. Because part Ca element
was derived from Ca2+ crosslinked scaffold, therefore the Ca2p /P2p
ratio (1.68) was slightly higher than other reported data (1.54)
[25]. In the design of PEC scaffolds, chitosan was coated on the
alginate scaffold to improve the cell attachment. Cells have neg-
ative charges on their surfaces and therefore they adhere much
more strongly to substrates with basic groups. The XPS results con-
firmed that cationic chitosan was coated on the PEC scaffold as
expected.
3.4. Morphology and porosity
In tissue engineering realm, three-dimensional scaffolds should
have high porosity and interconnected pore structure to enhance
a compatible biological condition for cell attachment, prolifera-
tion and differentiation. The porous structure of our scaffolds was
achieved by freeze-drying method. The SEM images of various scaf-
folds were shown in Fig. 5. All scaffolds exhibited highly porous
structure with good interconnectivity and the pore size ranged
from 80 to 200 ␮m. The size and the shape of pore would affect
the ability of cell attaching and growing, therefore influence the
efficiency of tissue regeneration. The optimal size and geometry of
scaffold pore are dependent on specific cell types. For example, the
optimal porous size for bone in-growth ranged from 75 to 250 ␮m,
while for fibro-cartilaginous, the size should be larger in the range
of 200–300 ␮m [32]. Besides, interconnectivity of pore in scaffold as
a significant structural property affects the migration and prolifer-
ation of cells. Comparing our four scaffolds, it can be seen that the
pore size of scaffold reduced after crosslink by Ca2+ or polyelec-
trolyte complex with chitosan. The average pore diameters were
obtained by analyzing scaffolds SEM images and listed in Table 1.
AG–CS PEC scaffold exhibited porous structure with smaller
pore size, compared with uncrosslinked AG scaffold. In addition,
regular round micro-pores ranging from 20 to 30 ␮m were found
on the wall of scaffold. These micro-pores might facilitate nutrient
and oxygen transportation in the scaffolds. Hydroxyapatite, a major
inorganic component of natural bone, is also an important candi-
date in bone tissue engineering scaffold material because it can
form a direct bond with bone due to its resemblance to bone min-
eral. In our research, HA was also introduced into the PEC scaffold
via preparing CS/HA composite. Even distribution mineral crystals
coated on the scaffold skeleton were observed in AG–CS/HA PEC
scaffold. The scaffold also showed wrinkles structure, different from
other scaffolds.
The porosity of various scaffolds evaluated by a liquid displace-
ment method was listed in Table 1. The porosity of uncrosslinked
AG scaffold was determined to about 90.8%, while the porosities of
Ca2+ crosslinked AG scaffold, AG–CS PEC scaffold and AG–CS/HA
PEC scaffold decreased obviously. But the porosities of all sam-
ples exceeded 70%. The difference between uncrosslinked sample
and crosslinked samples was noticeable. After crosslinked, scaffolds
compacted and contracted, which was consistent with SEM image
results.
3.5. Strength
Scaffolds for tissue engineering must have certain mechanical
strength because the scaffolds should act as temporary physical
support to withstand the stresses until the tissues are regenerated.
Compression tests of scaffolds were carried out, the compressive
strength and Young’s modulus of various scaffolds were shown in
Fig. 6. AG scaffold without and with Ca2+ crosslinked were 0.23,
0.30 and 1.30, 2.35 MPa, respectively. For AG–CS PEC scaffold, the
two parameters were 0.60 and 5.47 MPa.
The reasons for the mechanical strength improvement of
crosslinked scaffold could be strong ionic interactions. For Ca2+
 J. Han et al. / International Journal of Biological Macromolecules 46 (2010) 199–205 203

Fig. 5. SEM micrographs of various scaffolds: uncrosslinked AG scaffold (a), Ca2+ crosslinked AG scaffold (b), AG–CS PEC scaffold (c and d) and AG–CS/HA PEC scaffold (e and
f).

crosslinked AG scaffold, the mechanical strength was obviously interact with carboxylate on alginate, both M units and G units.
enhanced because Ca2+ has strong ionic interaction with COO− The data implied that the compressive strength depended on both
in alginate chain. For AG–CS PEC scaffold, the strong interaction composition and structure, not only on the porosity. The introduc-
existed between NH3 + groups in chitosan and COO− groups in algi- tion of cationic chitosan to alginate scaffold improved mechanical
nate [24]. In addition, the decrease of porosities for crosslinked strength due to the strong electrostatic interactions between the
scaffold would be another factor influencing the mechanical oppositely charged polymers.
strength. The porosities of Ca2+ crosslinked AG scaffold and AG–CS
PEC scaffold were almost the same, however, the compressive
3.6. Thermogravimetric analysis
strength and Young’s modulus of AG–CS PEC were twice as much
as those of Ca2+ crosslinked AG scaffold. In the Ca2+ crosslinked AG
Thermogravimetric analysis, which supplies information on the
scaffold, only the G units were oriented in manner that rendered
relative thermal stability of materials, is widely used to study elec-
the carboxylate moieties accessible for ionic cross-linking. How-
trostatic interactions in PEC materials [33–35]. TGA was conducted
ever, in AG–CS PEC scaffold, protonated amines on chitosan could
on Ca2+ crosslinked AG scaffold and AG–CS PEC scaffold. The intro-
duction of chitosan influenced thermal stability due to the complex
Table 1 interaction as illustrated in Fig. 7. The TGA curves of both scaffolds
Porosities and average pore diameters of various scaffolds. exhibited the same trend: the weight of sample decreased gradu-
Sample name Porosity (%) Average pore diameter ally from room temperature to 200 ◦ C; in the range of 200–300 ◦ C
(␮m) weight declined sharply and then decreased slightly from 300 to
Uncrosslinked AG scaffold 90.8 187 600 ◦ C; after 600 ◦ C, the scaffold lost the weight rapidly. The weight
Ca2+ crosslinked AG scaffold 79.6 149 loss below 200 ◦ C could be ascribed to the water evaporation and
AG–CS PEC scaffold 75.3 138 decomposition of oligosaccharide, while the rapid weight loss upon
AG–CS/HA PEC scaffold 72.7 105
200 ◦ C involved the complex process of degradation of polysaccha-
204 J. Han et al. / International Journal of Biological Macromolecules 46 (2010) 199–205
Fig. 6. (A) The compressive strength of different scaffolds. (B) The Young’s module
of different scaffolds.
rides. The residual mass of AG–CS PEC scaffold was higher than that
of Ca2+ crosslinked AG scaffold over the whole test temperature
range, which could be attributed to the introduction of chitosan
and the formation of polyelectrolyte complex due to the strong
ionic interaction [36]. The electrostatic interactions between the
oppositely charged polymers result in a great thermal stability of
their structure [35].
Fig. 7. TG curves of Ca2+ crosslinked AG scaffold and AG–CS PEC scaffold.
4. Conclusion
In this research, AG–CS PEC porous scaffold were fabricated
and investigated. HA was also introduced into the scaffold via
synthesizing CS/HA composite. XRD and XPS results revealed the
formation of PEC scaffold by ionic interaction between NH3 + on
chitosan and COO− on alginate. SEM results demonstrated that
PEC scaffold showed interconnected porous structure. The AG–CS
PEC scaffold exhibited higher compressive strength compared with
uncrosslinked AG scaffold or Ca2+ crosslinked AG scaffold, although
porosity was slightly lower. Considering the good biocompatibility
of alginate and chitosan, the PEC scaffold would be used for tis-
sue engineering materials. Importantly, the PEC formation process
provides a simple and effective method to fabricate scaffolds with
different surface properties.
Acknowledgements
The authors are grateful to the Program for Changjiang Scholars
and Innovative Research Team in University.
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