Journal of Biomaterials and Nanobiotechnology, 2010, 1, 42-49
doi:10.4236/jbnb.2010.11006 Published Online October 2010 (
Copyright © 2010 SciRes. JBNB
Preparation and Characterization of Homogeneous
Hydroxyapatite/Chitosan Composite Scaffolds via
In-Situ Hydration
Hong Li1,2*, Chang-Ren Zh ou1,2, Min-Ying Zhu1, Jin-Huan Tian1,2, Jian-Hua Rong1,2
1Department of Materials Science and Engineering, Jinan University, Guangzhou, China; 2Education Ministry Research Centre of
Artificial Organs & Materials, Jinan University, Guangzhou, China.
Email: *,
Received May 14th, 2010; revised June 5th, 2010; accepted June 30th, 2010.
Hydroxyapatite(HAP)/Chitosan (CS) composite is a biocompatible and bioactive material for tissue engineering. A
novel homogeneous HAP/CS composite scaffold was developed via lyophilization and in situ hydration. A model CS
solution with a Ca/P atom ratio of 1.67 was prepared through titration and stirring so as to attain a homogeneous dis-
persion of HAP particles. After lyophilization and in situ hydration, rod-shaped HAP particles (5 μm in diameter)
within the CS matrix homogeneously scattered at the pore wall of the CS scaffold. X-ray diffraction (XRD) and Fou-
rier-Transformed Infrared spectroscopy (FTIR) confirmed the formation of HAP crystals. The compressive strength in
the composite scaffold indicated a significant increment over a CS-only scaffold. Bioactivity in vitro was completed by
immersing the scaffold in simulated body fluid (SBF), and the result suggested that there was an increase in apatite
formation on the HAP/CS scaffolds. Biological in vivo cell culture with MC 3T3-E1 cells for up to 7 days demonstrated
that a homogeneous incorporation of HAP particles into CS scaffold led to higher cell viability compared to that of the
pure CS scaffold or the HAP/CS scaffold blended. The results suggest that the homogeneous composite scaffold with
better strength, bioactivity and biocompatibility can be prepared via in vitro hydration, which may serve as a good
scaffold for bone tissue engineering.
Keywords: Hydroxyapatite, Chitosan, Scaffold, Composite, Hydration
1. Introduction
Tissue engineering, which applies methods from engi-
neering and life sciences to create artificial constructs to
direct tissue regeneration, has attracted many scientists
and surgeons with a hope to treat patients in a minimally
invasive and less painful way. The important process of a
tissue engineering paradigm is to isolate specific cells to
grow them on a scaffold. A scaffold should be in combi-
nation with support for tissue architecture, biomolecules
and selective transportation of ions in body fluids. Chi-
tosan (CS) is the partially deacetylated form of chitin that
can be extracted from crustacean. Apart from being bio-
resorbable, it is biocompatible, non-harmful, non-toxic
compounds and biofunctional. In addition, CS is easy to
mould a 3-dimensional scaffold which can support tissue
ingrowth, aid in the formation of tissue structure, and
promote growth and mineral rich matrix deposition by
osteoblast in culture for bone tissue engineering [1]. It is
important to note that CS in combination with hy-
droxyapatite (HAP, Ca10(PO4)6(OH)2), further enhance
tissue regenerative efficacy and osteoconductivity [2-4].
HAP can accelerate the formation of bone-like apatite on
the surface of implant and can induce bone formation [5].
By the way, incorporation of HAP into a CS polymer
matrix has also been shown a significant enhancement of
mechanical strength [6].
Several studies have focused on the composite scaffold
for bone tissue engineering [7-9], such as CS/calcium
phosphate [7], CS/HAP bilayer scaffold [8]. The com-
posite had been prepared by different processing, such as
mechanical mixing of HAP powder in a solution [10,11],
co-precipitation [12], and biomimetic process [13,14].
Generally, HAP powder was mixed with CS dissolved in
2% acetic acid solution, poured into a mould, and freez-
ing-dried to make sponge composites. The final material
was heterogeneous, which was shown in the macro-
scopically less homogeneous. However, to ensure a more
Preparation and Characterization of Homogeneous Hydroxyapatite/Chitosan
Composite Scaffolds Via In-Situ Hydration
effective contact between scaffold and tissue, a homoge-
neous composite scaffold should be prepared. In addition,
a uniform distribution of inorganic particles in polymer
matrix theoretically and experimentally improves me-
chanical property [15,16].
The present work aims to design and develop a homo-
geneous composite scaffold fabricated from biopolymer
CS and bioceramic HAP as a candidate for bone tissue
engineering applications. It is hypothesized that a homo-
geneous HAP dispersion could lead to an enhancement
on mechanically competent, bioactivity and biocompati-
bility. Generally, a homogeneous dispersion can be ob-
tained if the materials mixed and formed in an aqueous
environment. However, CS is acid-soluble while HAP
usually forms in a solution with pH > 10. Therefore, in
order to achieve a homogeneous HAP/CS composite
scaffold, the combination of the lyophilization method
and in situ hydration in alkine aqueous was applied in
this work. The composition, morphology, mechanical
property, bioacitivity and biocompatibility of the ho-
mogenous composite scaffold were studied.
2. Experimental Procedures
2.1. Preparation of the Composite Scaffold
CS powder was supplied commercially with the degree
of deacetylation over 97% (Shanghai Boao Biotechnol-
ogy Co., Shanghai, China; the viscosity-average relative
molecular weight was 1.8 × 106 Da.). A CS aqueous so-
lution of 2 wt% was prepared by dissolving CS powder
into deionized water containing 2 wt% acetic acid. Then,
under agitation, a stoichiometric 2 mol/L CaCl2 solution
was slowly added into the CS solution. Subsequently, a
1.2 mol/L K2HPO4 solution, with a Ca/P atom ratio of
1.67, was added dropwise. The ratio of HAP to CS solu-
tion was 60/100 by weight. After stirring, the suspension
was put into dishes (diameter of 30 mm, and depth of 5
mm) and 24-well plates (diameter of 14 mm, and depth
of 14 mm), and then rapidly transferred into a freezer at
presented temperature –40oC to solidify the water and
induced phase separation. The solidifying route main-
tained at that temperature over night. In the next stage,
frozen samples were lyophilized using a freeze-dryer
(Uniequip, Germany) for 24 hrs. The obtained scaffolds
were hydrated with a mixture of 0.1 N sodium hydroxide
solution and pure ethanol with a 2:1 volumetric ratio for
different time periods. After in situ hydration, the sam-
ples washed with deionized water till the pH of washout
water was about 7. Finally, the samples treated were
freeze-dried again to obtain the CS/HAP porous scaffolds.
The samples were denoted by D.
As a control, HAP/CS composite scaffold was pre-
pared via blending method. Briefly, HAP powder (Boao
Bio. Tech. Com., Shanghai, China) was added into a CS
aqueous solution of 2 wt% acetic acid (HAP/CS solution
is 60/100, wt/wt) with magnetic stirring and ultrasonic
treatment. After stirring, the mixture was moulded, fro-
zen and lyophilized as described above. The samples
were denoted by E, while the pure CS scaffold was de-
noted by A.
2.2. SEM Examination
The lyophilized scaffolds were cut with a razor blade to
expose the inner surfaces. After being coated with gold
in a sputtering device, the samples were examined with a
scanning electron microscope (XL-30 ESEM, Philips Co.,
Netherland) with an accelerating voltage of 20 kV.
2.3. XRD
The composite scaffold samples were ground to fine
powder after frozen in liquid N2 for 30 minutes, and then
characterized by X-rays diffraction (XRD; MASL, Bei-
jing, China, 40 kV, 20 mA, 3°/min) .
2.4. FTIR
The HAP/CS scaffolds prepared via lyophilization and in
situ hydration were analyzed by Fourier-transform infra-
red attenuated total reflective spectroscopy (FT-IR;
EQUINOX 55, Bruker, Germany). The scaffolds were
frozen in liquid N2 for 30 minutes and were ground into a
fine powder. The powder samples were mixed with KBr
powder and compressed into pellets for FT-IR examina-
tion. The spectra were collected over the range of
4000-400 cm-1.
2.5. Compressive Strength Measurement
The samples of each of the three type scaffolds (A, CS-
only; D, HAP/CS composite in situ hydrated; E: HAP/CS
composite blended) were cut into rectangle (5.0 mm ×
5.0 mm × 5.0 mm). The compressive strength was meas-
ured using a computer-controlled Universal Testing Ma-
chine (AG-1, Shimadzu Co., Tokyo, Japan) with a guide-
line set in ASTM D5024-95a. The strength was calcu-
lated by using the yield point load divided by the speci-
men’s cross sectional area. Five parallel samples were
evaluated for each of the scaffolds.
2.6. Incubation in Simulated Body Fluid (SBF)
To evaluate the bioactivity of the composite scaffolds in
vitro, the samples were incubated in SBF (the prescrip-
tion of SBF referring to Ref. [17]). The 1 g samples were
placed into SBF (50 ml) and incubated at 37°C. The
concentrations of Ca and P ions in SBF were measured at
1, 3 and 7 days incubation by inductively coupled plasma
atomic emission spectrometry (Optima 2000 DV, Perkin
Copyright © 2010 SciRes. JBNB
Preparation and Characterization of Homogeneous Hydroxyapatite/Chitosan
Composite Scaffolds Via In-Situ Hydration
Elmer Co., USA). Morphologies of the incubated scaf-
folds were observed by SEM as described before.
2.7. Cell Culture
Osteoblast Cells line MC 3T3-E1 (a clonal preosteoblas-
tic cell line derived from newborn mouse calvaria, which
is often used in bone tissue engineering research) were
cultured in DMEM supplemented with 10% fetal bovine
serum (GIBCO Co., U.S.A.), 100 U/mL penicillin
(Sigma, St. Louis, MO), and 100 μg/mL streptomycin
(Sigma). Cells were incubated at 37°C in a 5% CO2 in-
cubator, and the medium was changed every 2 days.
When the cells reached the stage of confluence, they
were harvested by trypsinization followed by the addition
of fresh culture medium to create a cell suspension. A
cell suspension with a concentration of 2 × 106 cells/mL
was loaded into the 3-D porous scaffolds (14 mm in di-
ameter and 2 mm in thickness), with 200 μL of suspen-
sion for each scaffold. The scaffolds were put in a poly-
styrene 24-well flat-bottom culture plate and incubated at
37°C in a 5% CO2 incubator. After cells attached at about
6 hrs, fresh culture medium was added until the total me-
dium volume was 500 μL. Culture medium was changed
every 2 days.
2.8. Cell Viability Assessment
A MTT assay was applied in this study to quantitatively
assess the number of viable cells attached and grown on
the tested scaffolds. Briefly, all the tested scaffolds with
cultured cells at pretermined time points were fetched to
a new 24-well flat-bottom culture plate. 1 mL of se-
rum-free medium and 100 μL of MTT (Sigma) solution
(5 mg/mL in PBS) were added to each sample, followed
by incubation at 37°C for 4 h for MTT formazan forma-
tion. The upper solvent was removed and 1 mL of 10%
sodium dodecyl sulfate (Sigma) in 0.01N HCl was added
to dissolve the formazan crystals for 6 h at 37°C. During
the dissolving period, the spongy scaffolds were squee-
zed every 30 min to ensure the complete extraction of the
formazan crystals. The optical density (OD) at 490 nm
was determined against the sodium dodecyl sulfate solu-
tion blank. Five parallel replicates were analyzed for
each sample.
3. Results and Discussion
3.1. Phase Analysis
The XRD patterns of the D sample before and after hy-
dration for different hydration periods are summarized in
Figure 1. The XRD patterns were verified by the Power
Diffraction File (HAP: Card No. 090432; CS: Card No.
391894; DCPD: No. 720713; KCl: No. 730713). It indi-
cated that, the DCPD and KCl crystalline phases mainly
occurred in the scaffolds D before hydration. The longer
the hydration ripening time, the smoother the peaks be-
long to DCPD and KCl. After 24 h of ripening, DCPD
and KCl crystalline phases disappeared in the composite
scaffold D. The broad peak that appeared around 20° was
assigned to CS (20.305°, 21.290°), and the sharp diffrac-
tion characteristic peaks that appeared at around 31.8°
and 25.9° correspond to the peaks of HAP (31.773°,
For pH of HAP formation more than 10, it was ob-
served that DCPD (brushite), and (or) amorphous CaP
occurred when Ca2+ and HPO4
2- were directly dropped
into a CS solution with pH < 7 (Figure 1(b)). During the
process of in situ hydration in the mixture solution of
sodium hydroxide solution and pure ethanol, the unstable
brushite, as well as the other amorphous CaP phases
transformed into a more stable HAP phase, according to
the following Equations (1) and (2) [18].
10CaHPO4+12OH Ca10(PO4)6OH2+10H2O+4PO3
4++ACP+OH Ca10(PO4)6OH2 (2)
As Pang’s report [19] and our study, after 24 h of rip-
ening, the transformation of brushite and amorphous CaP
to HAP was found nearly completely.
XRD patterns show the presence of KCl in the CS
composite before hydration due to the precipitation of
KCl during the lyophilization. After the composites were
hydrated and washed, KCl solved and disappeared as
indicated in Figure 1.
Figure 1. XRD patterns of (a) KCl, HAP/CS composite
scaffold D before hydration (b), after 1 hr (c), after 3 hrs (d),
after 6 hrs (e), 12 hrs (f) and 24 hrs (g) hydration.
Copyright © 2010 SciRes. JBNB
Preparation and Characterization of Homogeneous Hydroxyapatite/Chitosan
Composite Scaffolds Via In-Situ Hydration
3.2. FTIR
An infrared absorption spectra of the scaffold is summa-
rized in Figure 2. The absorption bands at 3540 cm-1,
3487 cm-1 and 633 cm-1 respectively correspond to the
stretching and vibration of the lattice OH- ions, while the
bands of absorbed water are shown at 3287 cm-1, 3163
cm-1, 1648 cm-1. The characteristic bands for HPO4
2- were
assigned at 1133 cm-1, 1064 cm-1, 989 cm-1, 875 cm-1,
577 cm-1, 527 cm-1 [20]. The magnitude of these bands
became weaker with the development of in situ hydration
and finally disappeared. The characteristic bands for
3- appeared at 963 cm-1 for the ν1 mode [21-22]. The
signal became clearly as the hydration processing. The
observation of the ν3 symmetric P-O stretching vibration
at 1032/1042 cm-1 as a distinguishable peak, together
with the bands 566/602 cm-1 corresponding to ν4 bending
vibration indicates the presence of HAP in the samples as
summarized in Figure 2(c, d, e, f, g, h). Theses peaks
show obviously stronger after 24 hours ripening, in ac-
cord with the XRD results.
3.3. Morphology Analysis
The morphologies of the scaffolds were examined with
SEM. The CS-only scaffold A, composite scaffold D
after hydration showed a similar spongy appearance
(Figure 3) in macroscopic morphology, which indicated
that both adding the HAP in the system and hydrating the
scaffolds did not influence the porous structure. Due to
the artifact of the sample preparation for SEM, the pores
Figure 2. IR spectra of DCPD (a), CS (b), composite scaf-
fold D before hydration (c), after 1 hr (d), after 3 hrs (e),
after 6 hrs (f), 12 hrs (g) and 24 hr s (h) hydration.
in the A scaffold were collapsed as illustrated in Figure
3(a). The D scaffolds depict more regular porous struc-
ture (Figure 3(e)), for its relative high strength can resist
the distortion during the sample preparation. However,
the microscopic morphology on pore-wall surfaces was
quite different. The surface of the scaffold A is smooth as
shown in Figure 3(b). Before hydration processing was
applied, the walls of D are embedded with flower-shaped
large particles, as indicated in Figures 3(c) and (d). After
hydration, the rod like HAP particles with about 5 µm in
diameter were homogeneously scattered in the pore-wall
surfaces of the composite scaffold D as shown in Figure
3(f). The SEM results suggest that HAP particles can be
homogeneously incorporated with CS matrix via lyophi-
lization and in situ hydration process.
3.4. Mechanical Property
The compressive strength of A, D and E are illustrated in
the Figure 4. Sample D has the highest compressive
strength when compared to the control. Li reported that
the incorporation HAP into CS matrix via blending me-
thod would result in the decrease of mechanical proper-
ties of HAP/CS material due to the weaker interfacial
bonding between HAP filler and CS matrix [23]. How-
ever, in our study, no obvious decrease appeared in as-
pect of mechanical property blended HAP/CS sponge E.
Figure 3. SEM morphologies of CS-only scaffold (a), the
pore wall of the CS-only scaffold (b), composite scaffold D
before hydration(c)(d) and after hydration (e)(f).
Copyright © 2010 SciRes. JBNB
Preparation and Characterization of Homogeneous Hydroxyapatite/Chitosan
Composite Scaffolds Via In-Situ Hydration
Figure 4. Compressive strength of the scaffolds A, D after
24 hrs hydration and composite via blending E. Data rep-
resented the mean ± SD for five samples. p < 0.01 compared
with pure CS.
The composite scaffold D prepared by in situ hydration,
with HAP particles homogeneously dispersion, has a
little increment in compressive strength and less deriva-
tion, as compared to the control E. The compressive
strength indicated that the observed homogeneous parti-
cle dispersion would be helpful to enhance the scaffold
mechanically competent.
3.5. Bioactivity
According to Kokubo et al., the in vitro immersion of
bioactive materials in SBF was thought to reproduce in
vivo surface structures [13,24,25]. The grown layer is
sometimes called a bone-like apatite [25]. A bone-like
apatite layer plays an important role in establishing the
bone-bonding interface between biomaterials and living
tissue [4]. As shown in Figure 5, the surface of the
soaked scaffolds in SBF showed spherical particles con-
taining tiny crystals which correspond to apatite [26-28].
The size and number of the apatite particles formed on
the D scaffold was obviously larger than those of the
particles on the scaffolds A and E. The apatite crystals on
the sample D also depict a relatively uniformly size ac-
cording Figure 5, unlike those on the sample E that lar-
ger particles occurred. With the immersion periods going,
the quantities of apatite particles increased in macro-
scopic morphology, but the difference still existed. The
scaffolds A and E were covered with tiny apatite crystals
while some larger particles dotted on the E scaffold, but a
layer of particle crystals fully covered the wall of the D
scaffold. This result is also supported by the results of the
Ca and P concentration decrease in SBF.
Figure 6 displays the concentrates of Ca and P ions in
SBF, which soaked the samples. An abrupt decrease in
the concentrates of Ca and P ions during the first three
days followed by a continuous slow decrease in the next
days. The result is in agreement with the SEM observa-
tion for changes in macroscopic morphologies after the
first three days. This appreciable decrease in Ca and P
concentrations can be attributed to the formation of apa-
tite crystals on the specimen surfaces. However, the de-
creases of Ca and P concentrations in the SBF, which the
composite scaffolds D and E were soaked, were larger
than that of scaffold A. It is just confirmed that the
amount of apatite formed on the scaffold A was less than
that of apatite on the composite scaffolds D and E as
shown in Figure 5. The large apatite crystals dotted on
the scaffold E also led to the decrease in the concentra-
tions of Ca and P ions more rapidly than that of scaffold
D in the first three day.
According to Figure 6, as a result of the difference of
crystallinity of HAP, a little increase of Ca and P con-
centrates at the first day in Figure 6 D sample is shown.
The HAP particles synthesized at low temperatures have
been shown to have low crystallinity and high solubility
[29]. Therefore, the poorly crystallized HAP in the D
scaffolds formed via in situ hydration within the solution
has high solubility, which led to ions release in the SBF
media at early time. There was an increment in the con-
centration of both Ca and P ions after 12 hrs immersion
of the scaffold D in SBF. In the scaffold A, the increment
of Ca and P concentrates might be the de-chelate release
of CS-Ca chelate at neural environment.
Despite the difference of HAP particles, the scaffolds
with HAP (D and E) still show better bioactivity as
compared to the CS scaffold A when the scaffolds were
soaked in SBF. HAP would be favor to the nucleation of
bone-like apatite for HAP particles could act as nuclea
tion sites in a metastable calcium phosphate solution
Figure 5. SEM morphologies of the pore walls of the sam-
ples A, composite D after 24 hrs hydration and composite
via blending E in SBF after 1, 3 and 7 days immersion.
Copyright © 2010 SciRes. JBNB
Preparation and Characterization of Homogeneous Hydroxyapatite/Chitosan
Composite Scaffolds Via In-Situ Hydration
Copyright © 2010 SciRes. JBNB
Figure 6. Concentrations of Ca ions (a) and P ions (b) in SBF in which the samples were immersed (A. CS-only; D. composite
D after 24 hrs hydration; E. composite via blending).
such as SBF [30]. However, a homogeneous dispersion
of HAP in composite can obviously induced a homoge-
neous precipitation of bone-like apatite in SBF, and
would be improve the bioactivity more effectively.
from newborn mouse calvaria, which usually is used to
evaluate the biocompatibility of the materials for bone
tissue engineering. At early 3 day time, a MTT value of
sample A has the highest one. After 7 days’ culture, the
value of the D scaffold was the higher than the others
scaffolds, which indicated that the MC 3T3-E1 cells
showed much better viability property on D. It is an in-
dication of both the process and the component of HAP
might have significant difference in some degree on the
biocompatibility of these scaffold materials.
3.6. Cell Test
The biocompatibility of the scaffolds A, D and E was
assessed on cells' proliferation. Cell proliferation was
examined with MTT assay (Figure 7). The same amount
of MC 3T3-E1 cells were seeded on the scaffolds A, D
and E. MC 3T3-E1 is a preosteoblast cell line derived 4. Conclusions
In this paper, a homogeneous HAP/CS composite scaf-
fold was prepared and investigated. HAP particles were
combined homogeneously with CS matrix through ly-
ophilization and in situ hydration in alkaline solution. As
compared to the controls, the composite scaffold indi-
cated an increment in mechanical strength, altogether
with a homogeneous bone-like apatite precipitation in
SBF. The difference processing for fabricating the
CS/HAP composite scaffold also showed significant dif-
ference in cell’s biocompatibility according to this study.
The results on the homogeneous composite indicate that
this novel process is a new approach to fabricating bone
tissue engineering scaffolds especially for composite
scaffold. Further reports about in vivo study will be re-
ported in the near future.
5. Acknowledgements
Figure 7. MTT assay of cells grown on CS scaffold and
composite porous scaffolds. Data represent the mean ± SD
for three samples. p < 0.01 compared with pure CS. (A:
CS-only; D: composite D after 24 hrs hydration; E: com-
posite via blending).
The authors wish to thank the National High Technology
Development Program of China (2007AA091603) and
the National Science Foundation of China (30870612,
Preparation and Characterization of Homogeneous Hydroxyapatite/Chitosan
Composite Scaffolds Via In-Situ Hydration
20604010) for supporting this research.
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