International Journal of Biological Macromolecules 51 (2012) 1079–1085

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International Journal of Biological Macromolecules

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

In vivo evaluation of porous hydroxyapatite/chitosan–alginate composite

scaffolds for bone tissue engineering
Hyeong-Ho Jin a , Dong-Hyun Kim a , Tae-Wan Kim a , Keun-Koo Shin b , Jin Sup Jung b ,
Hong-Chae Park a , Seog-Young Yoon a,∗
a
School of Materials Science and Engineering, Pusan National University, San 30, Jangjeon-dong, Gumjeong-gu, Busan 609-735, Republic of Korea
b
Department of Physiology, School of Medicine, Pusan National University, Yangsan 626-770, Republic of Korea

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

Article history: Porous hydroxyapatite (HAp)/chitosan–alginate composite scaffolds were prepared through in situ co-
Received 22 May 2012 precipitation and freeze-drying for bone tissue engineering. The composite scaffolds were highly porous
Received in revised form 3 August 2012 and interconnected with a pore size of around 50–220 ␮m at low concentrations of HAp. As the HAp con-
Accepted 23 August 2012
tent increased, the porosity of the scaffolds decreased from 84.98 to 74.54%. An MTT assay indicates that
Available online 30 August 2012
the obtained scaffolds have no cytotoxic effects on MG-63 cells, and that they have good biocompatibil-
ity. An implantation experiment in mouse skulls revealed that the composite scaffold provides a strong
Keywords:
positive effect on bone formation in vivo in mice. Furthermore, that HAp/chitosan–alginate composite
Hydroxyapatite
Chitosan
scaffold has been shown to be more effective for new bone generation than chitosan–alginate scaffold.
Alginate © 2012 Elsevier B.V. All rights reserved.
Scaffold
In vivo

1. Introduction non-toxic, and biofunctional. It has been studied as a useful bio-

Recently, tissue engineering has emerged as one such promising
approach for bone repair and reconstruction [1]. Tissue engineer-
ing involves the expansion of cells from a small biopsy, followed
by the culturing of the cells in temporary porous scaffolds to
form the new organ or tissue [2–4]. With this approach, the
porous scaffold serves an important role in the manipulation of
the functions of osteoblasts and a central role in the guidance of
new bone formation into desired shapes. Therefore, the scaffold
materials must be biocompatible, osteoconductive, and osteointe-
grative, and have enough mechanical strength to provide structural
support.
Hydroxyapatite (Ca10 (PO4 )6 (OH)2 , HAp) has been widely used
in medicine and dentistry because it is biocompatible, osteocon-
ductive, and has excellent chemical and biological affinity with
bony tissue [5]. As a result, HAp is accepted as a bioactive scaf-
fold material for guided bone regeneration. In addition to the
requirements for chemical composition of the scaffold material, an
interconnected porous structure is necessary to allow cell attach-
ment, proliferation, and differentiation, and to provide pathways
for biofluids. Chitosan is a natural cationic polymer that is bio-
logically renewable, biodegradable, biocompatible, non-antigenic,
∗ Corresponding author. Tel.: +82 51 510 2487; fax: +82 51 512 0528.
E-mail address: syy3@pusan.ac.kr (S.-Y. Yoon).
material in diverse tissue engineering applications because of its
hydrophilic surface promoting cell adhesion, proliferation and
differentiation, good biocompatibility and good host response,
high biochemical significance in hemostasis, angiogenesis and
macrophage activation, biodegradability by lysozyme and other
enzymes, bactericidal/bacteriostatic activity, and capacity to main-
tain a predefined shape after cross-linking [3,6–8]. Alginate is a
biocompatible, hydrophilic, and biodegradable anionic polymer
under normal physiological conditions and is widely used as an
instant gel for bone tissue engineering [9–11].
Chitosan–alginate scaffolds have been tested in the regeneration
of various tissues and bone with promising results [12,13]. How-
ever, many biopolymers are fragile and do not exhibit undoubtedly
biocompatible behavior. Some polymer and bioactive ceramics
composites have been developed for bone tissue engineering in
order to increase the bioactivity and mechanical properties of
the materials [14–17]. Hydroxyapatite (HAp)/polymer composites
have attracted a great deal of attention because they exhibit osteo-
conductivity because of the presence of HAp [18,19], which has a
similar chemical composition and structure as the mineral phase
of human bones and hard tissues.
In this study, porous HAp/chitosan–alginate composite scaffolds
were prepared through in situ co-precipitation and freeze-
drying for bone tissue engineering. With the aim of fabricating
HAp/chitosan–alginate composite scaffolds and in vivo culture of
bone tissue engineered constructs.

0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijbiomac.2012.08.027
1080 H.-H. Jin et al. / International Journal of Biological Macromolecules 51 (2012) 1079–1085
Fig. 1. (a) Photograph of HAp/chitosan–alginate composite scaffolds, and SEM morphology of the composite scaffolds with different HAp contents; (b) 0, (c) 10, (d) 30, (e)
50 and (f) 70 wt%.
2. Experimental
2.1. Preparation of HAp/chitosan–alginate composite scaffolds
The HAp/chitosan–alginate composite scaffolds were synthe-
sized through in situ co-precipitation. A chitosan aqueous solution
was prepared by dissolving 3.84 g of the chitosan powder (vis-
cosity > 200 cP:1 wt% solution in the 1 wt% acetic acid solution in
Brookfield, Sigma–Aldrich) in 64 ml 1 M acetic acid. To prepare
the alginate solution, 3.84 g of the sodium alginate powder (vis-
cosity 200–400 cP for 1 wt% solution at 20 ◦ C, Sigma–Aldrich) was
dissolved in 96 ml of 1 M NaOH. H3 PO4 and Ca(OH)2 were added
to the chitosan aqueous and alginate solutions, respectively, to
form the HAp/chitosan–alginate composite scaffolds. The ratios
of chitosan to H3 PO4 and alginate to Ca(OH)2 were adjusted so
that the final HAp/chitosan–alginate weight ratios were 10/90 and
70/30, respectively. The resulting suspension was mixed under con-
stant stirring in a blender for 1 h. Acetic acid was gradually added
Fig. 2. Pore diameter of HAp/chitosan–alginate composite scaffolds with different
HAp contents.
drop-wise to the suspension until a pH of 7.4 was obtained. The
slurry was placed into 24-well cell culture plates and stored in
a freezer at −15 ◦ C until frozen. Subsequently, the samples were
lyophilized in a freeze dryer at −80 ◦ C for 24 h. The dried samples
were cross-linked with a 1% (w/v) CaCl2 solution for 15 min and
then immersed in distilled water for 24 h to remove any residual
sodium acetate and unbound CaCl2 [12]. After immersion, the sam-
ples were washed three times and then freeze-dried at −80 ◦ C for
24 h to obtain the HAp/chitosan–alginate composite scaffolds.
The morphologies, pore configuration, and pore size of the
HAp/chitosan–alginate composite scaffolds were investigated
using scanning electron microscopy (SEM, S-4300, Hitachi, Japan).
The porosity and density were measured by the liquid displacement
method [18].
2.2. Cell culture
The cytotoxicity of the HAp/chitosan–alginate composite
scaffolds was assessed using the MTT (3-[4,5-dimethylthiazol-
2-yl]-2,5-diphenyltetrazolium bromide) assay [20]. MG-63 cells
Fig. 3. Density and porosity of HAp/chitosan–alginate composite scaffolds with dif-
ferent HAp contents.
 H.-H. Jin et al. / International Journal of Biological Macromolecules 51 (2012) 1079–1085
Fig. 4. (a) Cell viability (MTT assay) of HAp/chitosan–alginate composite scaffolds with different HAp contents. (b) SEM micrographs of the surface and cross-section of the
30 wt% HAp/chitosan–alginate composite scaffold after MTT test.
seeded in 24-well plates with the composite scaffolds at a density of
1 × 105 cells/well, and the plates was incubated at 37 in 5% CO2 . The
MG-63 cells were treated with chitosan–alginate scaffold as control
[12] and the composite scaffolds for 1, 3, 5, and 7 days. MTT solution
was added to each well (final concentration ∼50 mg/ml). The cells
were incubated at 37 ◦ C for 3 h and dimethyl sulfoxide (DMSO) was
added to dissolve the formazan crystals. The absorbance was read
at 570 nm in an ELISA reader (E-MAX Molecular Devices) against a
reference wavelength of 490 nm.
2.3. In vivo test of the composite scaffolds
The HAp/chitosan–alginate composite scaffolds were evaluated
qualitatively in vivo using critical-sized calvarial bone defects in
adult (6-week-old) severe combined immunodeficient (SCID) mice.
The surgical procedures were performed in aseptic conditions
under general anesthesia. Briefly, a linear incision (1 cm long) was
made on the left side of the skull and the scalp was dissected to
expose the calvaria. The periosteum was carefully peeled off and 2
lateral 4-mm-wide calvarial bone defects were performed in each
animal using a 3-mm-diameter trephine bur using a slow-speed
dental drill. To avoid tissue damage due to overheating, 0.9% saline
was dripped onto the contact point between the bur and bone
1081
and great care was taken to avoid dura mater injury. One defect
was then used for implanting with HAp/chitosan–alginate com-
posite scaffolds while the contralateral site was implanted with
chitosan–alginate scaffolds as a control [12].
The animals were euthanized after 4 and 8 weeks by expo-
sure to hyperbaric carbon dioxide. At each time point, the skulls
were harvested and fixed in 4% paraformaldehyde for 12 h. Rep-
resentative skulls stained with 1 mg/ml alizarin red for 12 h
to view bone formation. Calvaria were X-rayed using a volu-
metric computed tomography (CT) scanner (NFR-MXSCAN-G90:
NanoFocusRay, Iksan, Korea) at 50 kVp, 65 ␮A, and 470-ms per
frame and then decalcified overnight with decalcifying solution
(10% EDTA). Samples were then trimmed, processed, and embed-
ded in paraffin wax. A micro-CT image of mouse calvaria was
taken using the CT scanner without having to move the ani-
mal’s head position. Paraffin-embedded samples were sectioned
at 10-mm thickness with a microtome. Sections were floated in
a water bath at 40 ◦ C, placed on poly-l-lysine-coated polysine
microscope slides and baked at 37 ◦ C overnight. For hematoxylin
and eosin (H&E) staining, sections were dewaxed in xylene and
rehydrated in ethanol baths. The sections slides were stained
with H&E viewed under an optical microscope for histological
observation.
1082 H.-H. Jin et al. / International Journal of Biological Macromolecules 51 (2012) 1079–1085
Fig. 5. Micro-CT images of the mouse skull after transplantation for (a) 4 and (c) 8 weeks. Photographs of mice skulls stained with alizarin red after transplantation for (b) 4
and (d) 8 weeks. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
3. Results and discussion
3.1. Structure of the composite scaffolds
The SEM micrographs and the pore size of the
HAp/chitosan–alginate composite scaffolds with different HAp
contents were shown in Figs. 1 and 2. The chitosan–alginate
polyelectrolyte complex was highly porous and interconnected
with a pore size of around 50–220 ␮m. At low HAp contents,
the pore structure of the composite scaffolds (Fig. 1(b) and (c))
was similar to the chitosan–alginate polyelectrolyte complex
(Fig. 1(a)). However, the pore size decreased with increasing HAp
content above 30 wt%, and eventually the pore structure locally
collapsed and appeared to be agglomerated (Fig. 1(d) and (e)).
Characterization of the composite scaffolds was described in our
previous report [21]. Chitosan and alginate, the polymer matrix of
the composite scaffolds, were cross-linked, and the synthesized
HAp was low crystallized.
The density and porosity of the composite scaffolds with differ-
ent HAp contents were shown in Fig. 3. Porosity is based on the
presence of open pores which are related to properties such as per-
meability and surface area of the porous structure. High porosity
usually means a high surface area/volume ratio, and thus favors
cell adhesion to the scaffold and promotes bone tissue regeneration
[22]. As the HAp content increased, the porosity of the composite
scaffolds decreased, and the density increased. These phenomena
would be attributed that the pores became interconnected with
more dense and thicker pore walls with increasing of the HAp
content. It is worthwhile mentioning that the density of a scaf-
fold decreases with the level of porosity at constant HAp content.
The porosity of the composite scaffolds ranged between 84.98 and
74.54%.
3.2. The cytotoxicity of the scaffolds
Fig. 4 shows the results of the MTT test and the SEM micrographs
of the HAp/chitosan–alginate composite scaffolds. In Fig. 4(a), the
density on control and the HAp/chitosan–alginate composite scaf-
folds increased with increasing time. The density on the composite
scaffolds was higher than that on control. These results show that
the composite scaffolds exhibited no cytotoxic effects on the MG-
63 cells, and have good biocompatibility. After the MTT test, the
surface and cross-section of the composite scaffolds with 30 wt%
HAp are shown in Fig. 4(b). The cells were observed at both sur-
face and cross-section of the composite scaffolds, indicating that
the structure of the HAp/chitosan–alginate composite scaffold was
favorable for cell attachment and new bone tissue growth. There-
fore, 30 wt% HAp/chitosan–alginate composite scaffold could have
good biocompatibility and osteoconductivity for bone tissue engi-
neering as the results in Figs. 1–4.
3.3. In vivo tissue compatibility
Bone defects of the skull were implanted with two types of scaf-
folds. One site was implanted with chitosan–alginate scaffold as a
control. 30 wt% HAp/chitosan–alginate composite scaffold show-
ing the good cell affinity as the result in Fig. 4, was inserted on the
opposite site. During the in vivo experiment, mice remained in good
health and did not show any wound complications. At explana-
tion, no inflammatory signs or adverse tissue reactions were seen.
It indicates that the composite scaffold could be suitable for cell
attachment and proliferation as bone substitute materials. Fig. 5
shows micro-CT images and alizarin red analysis of a mouse skull
after 4, 8 weeks. After 4 weeks in vivo, low-density mineralization in
the area of the bone defect was produced in the site implanted with
 H.-H. Jin et al. / International Journal of Biological Macromolecules 51 (2012) 1079–1085 1083

Fig. 6. Histological analysis of (a) the mouse skull with H&E staining after 4 weeks, (b) the site implanted with control sample, (d) high magnification of (b), (c) the site
implanted with HAp/chitosan–alginate composite scaffold, (e) high magnification of (c) (HBT: host bone tissue, NBT: new bone tissue, Ob: osteoblast).

the composite scaffold, whereas mineralization was not detected in
the control site (Fig. 5(a)). It indicates that the site implanted with
the composite scaffold had started to heal with new generation
bone. These features were more obvious after 8 weeks of implan-
tation as shown in Fig. 5(c). After 8 weeks of implantation, the site
implanted with the composite scaffold had formed sufficient bone
to span most of the bone defect, even though the control site had
just observed low-density mineralization.
Alizarin red analysis of the mouse skull after in vivo for 4 and
8 weeks was shown in Fig. 5(b) and (d). Alizarin red is used in
study new bone generation because it stains free calcium and cer-
tain calcium compounds a red or light purple color [23]. After 4
weeks in vivo, the light red color can be seen in both implanted
sites in Fig. 5(b), however the site implanted with the composite
scaffold was observed dark red color after 8 weeks in vivo as shown
in Fig. 5(d). Alizarin red staining localized bone formation to areas of
mineralization onto the site implanted with the composite scaffold.
Figs. 6 and 7 show histological analysis of a mouse skull
with H&E staining after 4 and 8 weeks in vivo. H&E staining
showed characteristic bone morphology on the implanted sites.
After 4 weeks, the appearance and shape of both implanted sites
showed no obvious change, although slight connection between
the material and surrounding tissue with osteoblast was detected
as shown in Fig. 6. However, in Fig. 7, newly formed bone tis-
sue which filled the area of the bone defect was observed in the
site implanted with the composite scaffold after 8 weeks in vivo,
whereas bone tissue was not observed in the control site. The
new bone tissue in the site implanted with the composite scaf-
fold had not only osteoblast but also osteoclast and Howship’s
lacuna. In general, bone regeneration is accompanied by bone reab-
sorption and bone formation with osteoblast and osteoclast [24].
Therefore, the new bone tissue in the site implanted with the
composite scaffold was actively generated, suggesting that degra-
dation of HAp promoted osteoid production [25,26]. In summary,
implantation experiments in mouse skulls in the present study
have revealed that HAp/chitosan–alginate composite scaffold is
more effective for new bone generation than chitosan–alginate
scaffold.
1084 H.-H. Jin et al. / International Journal of Biological Macromolecules 51 (2012) 1079–1085

Fig. 7. Histological analysis of (a) the mouse skull with H&E staining after 8 weeks, (b) the site implanted with control sample, (d) high magnification of (b), (c) the site
implanted with HAp/chitosan–alginate composite scaffold, (e) high magnification of (c) (HBT: host bone tissue, NBT: new bone tissue, Ob: osteoblast, Oc: osteoclast, B: bone,
Hl: Howship’s lacuna).

4. Conclusions
Porous HAp/chitosan–alginate composite scaffolds were pre-
pared through in situ co-precipitation and freeze-drying for
bone tissue engineering. At low HAp concentrations, the
HAp/chitosan–alginate composite scaffolds were highly porous and
interconnected with a pore size of around 50–220 ␮m. As the
HAp content increased, the porosity of the scaffolds decreased
from 84.98 to 74.54%, whereas the density increased from 0.12
to 0.24 g/cm3 . An MTT assay indicates that the obtained scaffolds
have no cytotoxic effects on MG-63 cells, and that they have good
biocompatibility. 30 wt% HAp/chitosan–alginate composite scaf-
fold could have good biocompatibility and osteoconductivity for
bone tissue engineering. The cells were observed at both the sur-
face and cross-section of the composite scaffolds after MTT test.
After 8 weeks of implantation, the site implanted with the com-
posite scaffold had formed sufficient bone to span most of the bone
defect, even though the control site had just observed low-density
mineralization. Alizarin red staining localized bone formation to
areas of mineralization onto the site implanted with the composite
scaffold. Newly formed bone tissue which filled the area of the bone
defect was observed in the site implanted with the composite scaf-
fold after 8 weeks in vivo, whereas bone tissue was not observed in
the control site.
Acknowledgment
This work was supported by National Research Foundation of
Korea Grant funded by the Korean Government Ministry of Educa-
tion, Science and Technology (NRF-2011-355-D00021).
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