Surface & Coatings Technology 251 (2014) 210–216

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Surface & Coatings Technology

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

Chitosan/carbonated hydroxyapatite composite coatings: Fabrication,

structure and biocompatibility
Sha Tang a,1, Bo Tian b,1, Ya-Jun Guo a, Zhen-An Zhu b,⁎, Ya-Ping Guo a,⁎
a
The Education Ministry Key Lab of Resource Chemistry, Shanghai Key Laboratory of Rare Earth Functional Materials, Shanghai Normal University, Shanghai 200234, China
b
Shanghai Key Laboratory of Orthopedic Implant, Department of Orthopedic Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China

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

Article history: Chitosan/carbonated hydroxyapatite composite coatings (CCHCs) were fabricated according to the following
Received 12 November 2013 steps: (i) preparation of calcium carbonate coatings (CCCs) on Ti6Al4V substrates by electrophoretic deposition;
Accepted in revised form 11 April 2014 (ii) transformation of CCCs into carbonated hydroxyapatite coatings (CHACs) in a phosphate buffer solution
Available online 24 April 2014
(PBS); and (iii) formation of CCHCs by modification of CHACs with chitosan. Inorganic constituents in CCHCs
are plate-like carbonated hydroxyapatite particles with a low crystallinity. Interestingly, macropores with a
Keywords:
Carbonated hydroxyapatite
pore size of 0.5–2 μm still remain among the carbonated hydroxyapatite plates even after the chitosan deposits
Chitosan homogeneously on the coatings. Moreover, CCHCs have moderately hydrophilic surfaces with a contact angle of
Coating 29.4° due to the presence of the chitosan. Biocompatibility tests have been carried out by using human bone
Macropore mesenchymal stem cells (hBMSCs) as cell models. The hBMSCs show better cell morphology, adhesion, spreading
Biocompatibility and proliferation on CCHCs than on CHACs. The excellent biocompatibility of CCHCs is mainly attributed to the
Electrophoretic deposition organic/inorganic compositions, macroporous structure and moderately hydrophilic surfaces. Hence, CCHCs
have great potentials for bone implants.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction
Titanium alloys are widely used for orthopedic and dental im-
plants under load-bearing conditions because of their low density,
good corrosion resistance and excellent mechanical property [1,2].
However, titanium alloys are bioinert materials. When implanted,
they always tend to create a stress shielding effect at bone–implant
interfaces because their mechanical properties are different from
those of host bones [3]. The mismatch of Young's modulus (Ti implant:
110 GPa, bone: 10–30 GPa) may easily cause detrimental resorptive
bone remodeling, wearing and loosening, and even result in implant
failure [4,5].
To overcome the above drawbacks, several synthetic strategies have
been developed, including chemical surface treatments, thermal surface
treatments and surface coatings by using different bioactive ceramics
such as hydroxyapatite, bioglass and hydrotalcite [6–11]. As we
know, hydroxyapatite possesses excellent biocompatibility, bioactivity,
osteoconductivity, nontoxicity and nonimmunogenicity because of its
compositional similarity to bone minerals [12]. However, hydroxyapatite
⁎ Corresponding authors. Tel./fax: +86 21 64321951.
E-mail addresses: zhuzhenan2006@126.com (Z.-A. Zhu), ypguo@shnu.edu.cn
(Y.-P. Guo).
1
S. Tang and B. Tian contributed equally to this work.
has several disadvantages such as low fracture toughness and high
brittleness [13], which frequently limit its clinical application for
weight-bearing bones. Moreover, hydroxyapatite coatings usually
cannot bond strongly with titanium alloy substrates, which may
peel off due to different thermal expansion coefficients and non-
metallurgical bonding between coatings and substrates [14]. Fortu-
nately, these defects could be resolved effectively by adding organic
polymer to hydroxyapatite coatings. Bioactive organic polymers not
only act as agglomerants between coatings and substrates, but
also improve the biocompatibility [15–21]. As a natural polymer,
chitosan is widely used in biomedical fields because of its good bio-
compatibility, biodegradability, bioresorbability and bactericidal
property [22–24]. Therefore, chitosan/hydroxyapatite coatings on
Ti6Al4V substrates may possess excellent mechanic properties and
biological properties.
Herein, we develop a new method to fabricate chitosan/carbonated
hydroxyapatite composite coatings (CCHCs) with porous structures ac-
cording to the following steps: (i) deposition of CaCO3 particles on
Ti6Al4V substrates by electrophoresis technology; (ii) transformation
of calcium carbonate coatings (CCCs) into carbonated hydroxyapatite
coatings (CHACs) in a phosphate buffer solution (PBS); and (iii) forma-
tion of CCHCs by modification of CHACs with chitosan. The main aims of
this work are to fabricate CCHCs, to investigate their formation mecha-
nism, and to study their biocompatibility by using human bone mesen-
chymal stem cells (hBMSCs) as cell models.

http://dx.doi.org/10.1016/j.surfcoat.2014.04.028
0257-8972/© 2014 Elsevier B.V. All rights reserved.
 S. Tang et al. / Surface & Coatings Technology 251 (2014) 210–216
2. Materials and methods
2.1. Materials
Hydrochloric acid, phosphoric acid, hydrofluoric acid, acetic acid, so-
dium dihydrogen phosphate dehydrate, disodium hydrogen phosphate
dodecahydrate, calcium chloride and sodium carbonate anhydrous
were purchased from National Medicine Group Chemical Reagent Co.,
Ltd. Chitosan (85% deacetylated form), TRITC phalloidin and 4′,6
diamidino-2-phenylindole (DAPI) were obtained from Sigma Aldrich,
USA. Alpha modification of Eagles medium (a-MEM) and fetal bovine
serum (FBS) were purchased from Gibco-BRL (Sydney, Australia).
CCK-8 was obtained from Dojindo Molecular Technology, Japan. All
chemicals used in this study were of analytical grade.
2.2. Preparation of CCCs
Ti6Al4V substrates were abraded with 1000-grit silicon carbide
paper, and washed with pure acetone and deionized water in an
ultrasonic cleaner. Acid treatment was performed by soaking these sub-
strates in a 1.0 mol/L H3PO4–1.5 wt.% HF solution for 20 min at room
temperature. After the acid treatment, the substrates were washed
with deionized water, and dried at room temperature. 1.25 g of CaCO3
powders was dissolved in 250 ml of ethanol, and dispersed ultrasonical-
ly for 30 min to get a suspension solution. 0.5 ml of an HCl solution
(1.0 mol/L) was added into the above suspension solution, and kept
pH value at about 6.5. To deposit calcium carbonate powders on the
substrates, an electrophoretic cell using a Ti6Al4V substrate as cathode
and a graphite plate as anode was mounted with two electrodes about
10 mm apart. The electrophoretic process was carried out at 90 V for 1
min. After electrophoretic deposition, the products (CCCs) were dried
at room temperature.
2.3. Preparation of CHACs and CCHCs
CCCs were immersed into a beaker with PBS (NaH2PO4 + Na2HPO4)
of pH = 7.4. The beaker was then placed in a bio-cultivating box, kept at
37 °C for 3 days. To maintain pH value at 7.4, the PBS was replaced
every day. Finally, the products (CHACs) were washed with deionized
water, and dried in a convection oven at 37 °C for 48 h.
With magnetic stirring, 1.0 g of chitosan powders was dissolved
into an acetic acid solution (100 ml, 2 vol.%). CHACs were soaked
into the above solution for 20 s, and then moved into deionized water
for 5 s. Subsequently, the coatings were dipped into a NaOH solution
(1.0 mol/L) for 10 s. Finally, the products (CCHCs) were washed by de-
ionized water, and dried in a convection oven at 37 °C for 24 h.
2.4. Characterization
The microstructures of samples were analyzed by scanning electron
microscopy (SEM, XL800, Philips). The crystalline phases of samples
were carried out by X-ray powder diffraction (XRD, D8, Bruker) by
using Cu Kα radiation within a scanning range of 2θ = 10° to 60° and
scanning rate of 4° min−1 at 40 kV/30 mA current. The functional groups
of samples were examined by Fourier transform infrared spectra (FTIR,
5DX, Nicolet) within a wavenumber range of 4000–400 cm−1 by using
KBr pellet technique at room temperature. The thermal behaviors of sam-
ples were examined by thermo-gravimetric analysis (TG-DTA, Perkin-
Elmer) at a heating rate of 10 °C/min in an alumina crucible in air
atmosphere. The surface wettabilities of samples were determined by
sessile drops of water on coating surfaces via contact angle measurement.
2.5. Cell behaviors of CCHCs
Human bone mesenchymal stem cells (hBMSCs) were isolated and
expanded by using the standard method as described previously [25].
211
This study was approved by the Ethic Committee of the Ninth People's
Hospital of Shanghai Jiao Tong University. The donor was healthy with-
out metabolic disease, inherited illnesses or other diseases. hBMSCs
were cultured in a-MEM culture medium supplemented with 10%
fetal bovine serum (FBS), 1% penicillin (100 U/mL), and streptomycin
sulfate (100 mg/mL) (Gibico BRL, Grand Island, NY). Cells were incubat-
ed at 37 °C in a humidified atmosphere of 5% CO2 and 95% air, with the
growth medium changed every 48 h. hBMSCs at a passage from P3 to P4
were used for these experiments.
The cytoskeletons of hBMSCs on samples were observed by double
fluorescence staining. Briefly, hBMSCs were seeded on CHACs and
CCHCs at a density of 3 × 104 cells per sample in a 12-well plate in trip-
licate, respectively. After incubation for 24 h, the samples were gently
washed with PBS and maintained in 4% paraformaldehyde for 15 min,
followed by soaking in 0.1% Triton X solution for 15 min. TRITC
phalloidin was used to stain the actin filaments of cells, and 4′,6-
diamidino-2-phenylindole (DAPI) was used to stain the nucleus of
cells. Cell morphology and spreading of hBMSCs were investigated by
confocal laser scanning microscopy (CLSM, Leica TCS SP2; Leica
Microsystems, Heidelberg, Germany).
Proliferation of hBMSCs on CHACs and CCHCs was examined with
a cell counting Kit-8 (CCK-8, Dojindo Molecular Technology, Japan)
assay. Briefly, hBMSCs were seeded on the coating surfaces at a density
of 7 × 103 cells per sample in a 12-well plate in triplicate. At each time
point, the medium was removed, and 1 mL medium was added into
each well with 100 μL CCK-8 according to the manufacturer's instruc-
tions. After incubation for 3 h, optical density was measured at
450 nm by using an automated plate reader (PerkinElmer).
2.6. Statistical analysis
Data are presented as mean ± standard deviation (SD) from at least
three independent experiments. Statistical analysis was performed by
Student's t-test using SPSS 13.0 software (SPSS Inc., USA). Difference
was considered significant at *p b 0.05 or **p b 0.01.
3. Results and discussion
3.1. Morphologies of CCHCs
The SEM images of CHACs and CCHCs are shown in Fig. 1. CHACs are
fabricated by deposition of calcium carbonate particles on Ti6Al4V sub-
strates via electrophoresis technique followed by treatment with PBS.
Our previous report has demonstrated that CCCs are composed of
many calcium carbonate particles with a particle size of 0.5–10 μm
[26]. These calcium carbonate particles in CCCs are stacked loosely
together due to the weak electrostatic bonding during electrophoretic
deposition. After soaking CCCs in PBS at 37 °C for 3 days, CCCs are
converted to CHACs with a uniform surface (Fig. 1a). The high-
magnification image demonstrates that the carbonated hydroxyapatite
particles on CHACs exhibit plate-like structure (Fig. 1b). These plates ag-
gregate to form macropores with a pore size of 0.5–2 μm. It is noted that
the gaps among the calcium carbonate particles in CCCs are partly
remained in CHACs during the transformation of calcium carbonate to
carbonated hydroxyapatite (Fig. 1a) [26]. In addition, same cracks are
observed in the low magnification SEM image (Fig. 1a), which may
weaken the adhesion strength between coatings and substrates.
In order to decrease the coating cracks and improve the biocompat-
ibility, CCHCs are fabricated by modification of CHACs with chitosan. In-
terestingly, the uniformly crack-free CCHCs are obtained, as shown in
the SEM image (Fig. 1c). Chitosan is distributed homogeneously around
carbonated hydroxyapatite plates, filling the cracks in CHACs (Fig. 1c
and d). At the same time, gaps or macropores among carbonated hy-
droxyapatite plates are remained although the pore size decreases
slightly due to the presence of chitosan (Fig. 1d). In terms of CCHCs,
the chitosan acts as a “bridge” among the carbonated hydroxyapatite
212 S. Tang et al. / Surface & Coatings Technology 251 (2014) 210–216
Fig. 1. SEM images of CHACs: (a) low magnification; (b) high magnification. SEM images of CHACs: (c) low magnification; (d) high magnification.
plates, making a good interconnection among them. Moreover,
macropores in CCHCs can increase the contacting area for cell attach-
ment and spreading [27,28]. The cross-section SEM image in Fig. 2
shows that the thickness of CCHCs is about 20 μm. Interestingly, an
amorphous TiOx layer with a thickness of about 2 μm is produced be-
tween coatings and substrates after chemical etching of Ti6Al4V sub-
strates in a 1.0 mol/L H3PO4–1.5 wt.% HF solution for 20 min [29]. The
TiOx layer may improve micromechanical interlocking and physico-
chemical bonding of coatings [29].
3.2. Structure and composition of CCHCs
The phase structures of CHACs and CCHCs are detected by XRD pat-
terns, as shown in Fig. 3. After soaking CCCs in PBS at 37 °C for 3 days,
CaCO3 particles in the coatings are converted to apatite plates (Figs. 1b
and 3b). The broad peaks in Fig. 3b indicate that the formed apatite
has low crystallinity similar to biological apatite [30]. The poor crystal-
linity of apatite may be attributed to the following reasons: (i) part of
Ca2+ and PO3− 4 ions in apatite crystal lattices are subsitituted by impu-
rities such as Na+, CO2−3 and HPO2− 4 ions; (ii) the low reaction temper-
ature at 37 °C tends to decrease the crystallinity of apatite crystals. It is
noted that the intensity ratio between the (002) and (211) diffraction
Fig. 2. Cross-section SEM image of CCHCs.
is stronger than the standard diffraction pattern (JCPDS card no. 09-
0432) [31], demonstrating the preferential growth of apatite along the
c-axis during the conversion process of calcium carbonate to apatite.
The same orientation is also observed in bone mineralization, as c-axis
of the apatite nanocrystals is parallel to the collagen fibers [32].
CCHCs are obtained after soaking CHACs in an acetic acid chitosan
solution followed by treatment with a NaOH solution. As compared
with CHACs, CCHCs possess the phases of both apatite and chitosan.
The crystallinity and crystal orientation of apatite in CHACs are similar
to those in CCHCs (Fig. 1b and c). In addition, chitosan exhibits a
broad peak at around 20° because it is a semi-crystalline material
(Fig. 3a). The characteristic peaks due to chitosan are also observed in
CCHCs (Fig. 3c), as confirmed by the SEM images (Fig. 1c and d).
Fig. 4 shows the FTIR spectra of chitosan, CHACs and CCHCs. The
functional groups of apatite are detected in both CHACs and CCHCs.
The broad band at 3410 cm−1 is attributed to OH group or absorbed
water [33]. The absorption bands at 1031, 602 and 562 cm−1 are attrib-
uted to PO3−4 group [34]. The absorption band at around 1105 cm−1 is
corresponding to HPO24 −, indicating that the obtained products are
calcium-deficient apatite [35]. The bands of B-type CO2− 3 substitution
Fig. 3. XRD patterns of different samples: (a) chitosan, (b) CHACs and (c) CCHCs.
 S. Tang et al. / Surface & Coatings Technology 251 (2014) 210–216
Fig. 4. FTIR spectra of different samples: (a) chitosan, (b) HCACs and (c) CCHCs.
at 872 and 1418 cm−1 are detected in Fig. 4b and c, indicating that the
PO3−
4 ions in apatite crystals are replaced partly by CO2−
3 ions [36]. The
obtained coatings are calcium-deficient apatite with the part substitution
of PO3−
4 ions by HPO2−4 and CO2− 3 ions, so the general formula may be
expressed as Ca10 − x − y/2(HPO4)x(PO4)6 − x − y(CO3)y(OH)2 − x.
Chitosan, a random copolymer of N-acetyl-D -glucosamine and
D -glucosamine, is the partially de-acetylated derivative of chitin
(Fig. 5). The band at 1571 cm−1 is assigned to N\H bending vibration
overlapping amide II vibration. C\N stretching vibration occurs at
around 1030 cm−1 and overlaps the vibration from carbohydrate ring.
The broad band at around 3415 cm−1 is corresponding to the stretching
vibration of N\H and OH groups. The \CH2 bending vibration occurs at
1410 cm−1 [37,38]. After soaking CHACs in an acetic acid chitosan solu-
tion followed by treatment with a NaOH solution, CCHCs including car-
bonated hydroxyapatite and chitosan are obtained (Figs. 1 and 3). The
above results can be further confirmed by the FTIR spectra (Fig. 4). As
compared with CHACs, the same characteristic absorption bands
due to carbonated hydroxyapatite are also observed in CCHCs (Fig. 4b
and c). In addition, the characteristic peaks of chitosan are overlapped
by those of carbonated hydroxyapatite (Fig. 4a and c).
The thermal behaviors of CHACs and CCHCs are determined by using
TG analysis, as shown in Fig. 6. For both CHACs and CCHCs, the weight
loss between 30 °C and 180 °C is due to the loss of physically adsorbed
water on coatings [39]. At the temperature range 180–550 °C, the
weight loss of 2.5% in CHACs is attributed to the decomposition of car-
bonated hydroxyapatite (Fig. 6a). For CCHCs, the weight loss of 10% at
the temperature range 180–550 °C belongs to the decomposition of
both low crystalline apatite and chitosan [30] (Fig. 6b). The content of
chitosan is about 7.5% of the total in CCHCs, as calculated by using
CHACs as a control. In addition, part of unreacted calcium carbonate is
remained as CCCs are converted to CHACs in PBS at 37 °C. The weight
loss of about 1.8% ranging from 600 °C to 800 °C is attributed to the
unreacted calcium carbonate that is not detected by XRD patterns
because of its low percentage in the coatings.
Fig. 5. Chemical structure of chitosan.
213
Fig. 6. TG curves of different samples: (a) CHACs and (b) CCHCs.
3.3. Wettability of CCHCs
The wettability of biocoatings plays an important role in adsorbing
proteins and cellular responses [40]. The contact angles of CHACs and
CCHCs are analyzed by sessile drops of water on the coating surfaces,
as shown in Fig. 7. CHACs possess profound hydrophilicity with a con-
tact angle of 12.5°. Recently, Webb et al. have reported that moderately
hydrophilic surfaces with a contact angle of 20–40° can promote cell at-
tachment [41]. Interestingly, the contact angle of 29.4° is obtained for
CCHCs after modification of CHACs with chitosan. The higher contact
angle of CCHCs than CHACs is attributed to the presence of chitosan in
the coatings. As compared with CHACs, the chitosan has lower hydro-
philicity with a contact angle of about 79.7° [42]. Moreover, the chitosan
layer in CCHCs may reduce surface roughness (Fig. 1), and thus de-
creases the hydrophilicity of CCHCs.
3.4. Formation mechanism of CCHCs
Recently, many literatures have reported electrophoretic deposi-
tion of bioceramic/chitosan composite coatings [16–19]. The electro-
phoretic deposition technique is a simple method for incorporation
of chitosan in bioactive coatings, but chitosan cannot combine tightly
with bioceramics via electrostatic interactions. In this work, we have
developed a new method to fabricate CCHCs, as shown in Fig. 8. In the
first stage, CCCs are formed by deposition of calcium carbonate particles
on Ti6Al4V substrates by electrophoresis technique (Fig. 8b).
In the second stage, CCCs are converted into CHACs by treatment
with PBS via a dissolution–precipitation reaction (Fig. 8c and d), be-
cause calcium carbonate and hydroxyapatite have different solubilities
[26]. For example, the logarithmic solubility product (pKs) of calcite at
37 °C is 8.56, while that of hydroxyapatite is 58.63 [43,44]. After soaking
CCCs in PBS at 37 °C, CaCO3 particles begin to dissolve. The released
2−
Ca2 + ions react with the PO3− 4 , HPO4 and OH− ions in PBS to form
carbonate hydroxyapatite as the ionic activity product exceeds the
thermodynamic solubility product. Carbonated hydroxyapatite plates
deposit in situ on the coatings to form CHACs (Fig. 1a and b). The general
scheme of this transformation reaction can be expressed as the follow-
ing chemical equation:
2þ 2− 3− 2− −
ð10−x−y=2ÞCa þ xHPO4 þ ð6−x−yÞPO4 þ yCO3 þ ð2−xÞOH →
Ca10−x−y=2 ðHPO4 Þx ðPO4 Þ6−x−y ðCO3 Þy ðOHÞ2−x :
ð1Þ
In the third stage, uniform CCHCs with a good combination between
carbonated hydroxyapatite and chitosan have been fabricated by
immersion of CHACs in an acetic acid chitosan solution followed by
treatment with a NaOH solution. The presence of amino groups makes
214 S. Tang et al. / Surface & Coatings Technology 251 (2014) 210–216

Fig. 7. Contact angle measurement of water on the surface of different samples: (a) CHACs and (b) CCHCs.

chitosan exist in a soluble or solid state, which depends on the pH value
of environments. When chitosan is dissolved in an acetic acid solution,
the free amino groups in chitosan are protonated to form chitosan-
NH+ 3 . After soaking CHACs in an acetic acid chitosan solution, the chito-
san is attached to CHACs via chelating and hydrogen bonding, and en-
ters into the macropores among carbonated hydroxyapatite plates.
The immersion time of CHACs in an acetic acid chitosan solution affects
the morphology and chemical composition of CCHCs. The ideal immer-
sion time should be controlled at 20 s. If the immersion time is too
short, few chitosan deposits on CHACs. On the contrary, the long
immersion time may result in the complete dissolution of the carbonate
hydroxyapatite particles in CHACs according to the following chemical
equation:
þ
Ca10−x−y=2 ðHPO4 Þx ðPO4 Þ6−x−y ðCO3 Þy ðOHÞ2−x þ ð2−xÞH →
2þ 2− 3− 2−
ð10−x−y=2ÞCa þ xHPO4 þ ð6−x−yÞPO4 þ yCO3 þ ð2−xÞH2 O:
After the chitosan deposits on the surfaces of CHACs, CCHCs are
obtained by treatment with a NaOH solution (Fig. 8f). Under alkaline
solutions, the chitosan in the coatings is turned into the solid state
because of the stereoregularity and molecular hydrogen bonding
among chitosan molecules. Moreover, the released Ca2 +, PO34 −,
HPO2−
4 and CO2−3 ions from CHACs in an acetic acid chitosan solution
may react again with the OH− ions in a NaOH solution to form carbon-
ated hydroxyapatite. Fig. 1c and d shows that crack-free CCHCs are com-
posed of both carbonated hydroxyapatite plates and gel-like chitosan.
3.5. Biocompatibility of CCHCs
As we know, bone implants must have good biocompatibility with
surrounding cells to promote satisfactory osteointegration between im-
plants and bone tissues. Therefore, the contact and interaction between
cells and biomaterial surfaces are very important physiological process-
es. Cytoskeleton analysis of hBMSCs on CHACs and CCHCs has been
performed by using CLSM, as shown in Fig. 9. Cytoskeleton is a highly
ð2Þ
dynamic network composed of actin polymers and associated proteins.
The function of cytoskeleton is to mediate a variety of essential biologi-
cal activities, including intra-cellular and extra-cellular movements and
structural supports. Orientation distribution of the actin filaments with-
in cells is, therefore, an important determinant of cellular shape and

Fig. 8. Schematic representation of the fabrication strategy of CCHCs: (a) Ti6Al4V substrate pretreated by physical and chemical methods; (b) deposition of CaCO3 particles on Ti6Al4V by
electrophoresis to obtain CCCs; (c) immersion of CCCs in PBS at 37 °C; (d) formation of CHACs; (e) modification of CHACs in an acetic acid chitosan solution; (f) formation of CCHCs after
treatment in a NaOH solution.
 S. Tang et al. / Surface & Coatings Technology 251 (2014) 210–216
Fig. 9. CLSM image of cytoskeletal morphology of hBMSCs cultured on different samples for 24 h: (a–c) CHACs and (d–f) CCHCs. The nuclei were stained with DAPI (blue), and the actin
filaments were stained with TRITC phalloidin (red).
functionality [45]. Actin filaments are stained with TRITC phalloidin, and
nuclei are stained with DAPI. Fig. 9 shows the long red bundles of stress
fibers composed of actin filaments and good cell–cell contacts with one
another, displaying good cell cytoskeleton morphologies on both CHACs
and CCHCs. The hBMSCs on CCHCs present a clustering, confluency and
multi-layering polygonal morphology at 24 h, while those on HCACs
display a slim, spherical, and fusiform-shaped morphology. Taken to-
gether, the hBMSCs on CCHCs have better cell adhesion, spreading and
cell–cell contact than those on CHACs. Generally, stem cells are regulat-
ed by physical and chemical factors from their complex extracellular
Fig. 10. CCK-8 assay results of hBMSCs cultured on CHACs and CCHCs at different days
(mean ± SD,*p b 0.05 and **p b 0.01).
215
surroundings [46]. These investigation results are in agreement with
the previous reports that hBMSCs have good cell adhesion and spread-
ing on hydroxyapatite/chitosan scaffolds [22].
Recently, CCK-8 assay has been widely used to investigate in vitro
biocompatibility of biomaterials. It is a quick and effective method for
testing mitochondrial impairment and correlates quite well with cell
proliferation. Herein, the cytotoxicity and proliferation of hBMSCs on
CHACs and CCHCs have been evaluated by CCK-8 assay. Fig. 10 indicates
no significant difference between CHACs and CCHCs before culturing
for 3 days. Interestingly, after culturing for 5 days, the number of the
hBMSCs on CCHCs is 15.6% higher than that of CHAC. With prolonging
the culture time to 7 days, the superiority of CCHCs appears special
evident to 23.4%. These results suggest clearly that CCHCs are nontoxic
to hBMSCs, so they can serve as good candidates for bone implants.
Although the initial cell adhesions on CCHCs are similar to those on
CHACs, the hBMSCs cultured on CCHCs have better cell proliferation
than those on CHACs. The excellent adhesion, spreading and prolifera-
tion of the hBMSCs on CCHCs may be attributed to the following
reasons. Firstly, the chitosan originated from the hard shell of insects
and crustaceans has good biocompatibility, biodegradability and bioac-
tivity. The presence of chitosan in CCHCs may improve the adhesion,
proliferation of hBMSCs [22,47]. Secondly, crack-free CCHCs with the
macroporous structure possess the large contacting areas, which are
preferable for the cell attachment, spreading and proliferation [27,28].
Thirdly, the wettability of biocoatings has a great effect on cell attach-
ment, spreading and cytoskeleton organization. Among different sur-
faces, moderately hydrophilic surfaces with a contact angle of 20–40°
can promote the high level of cell attachment, spreading, and cytoskel-
eton organization [41]. Therefore, the cell morphology, cytoskeleton
organization, cell attachment, spreading and proliferation of hBMSCs
216 S. Tang et al. / Surface & Coatings Technology 251 (2014) 210–216
are better on CCHCs with a contact angle of 29.4° than those on CHACs
with a contact angle of 12.5°.
4. Conclusion
CCHCs have been fabricated according to the following steps: (i) de-
posit of CaCO3 particles on Ti6Al4V substrates by electrophoretic deposi-
tion; (ii) transformation of CCCs into CHACs in PBS; and (iii) modification
of CHACs with chitosan. The inorganic constituent in CCHCs is carbonat-
ed hydroxyapatite plates with a low crystallinity. Macropores with a
pore size of 0.2–5 μm exist among the apatite plates. The organic con-
stituent in CCHCs is the chitosan, which is distributed homogeneously
on the coating surfaces. CCHCs possess moderately hydrophilic surfaces
with a contact angle of 29.4°, while that of CHACs is only 12.5°. Cell tests
demonstrate that hBMSCs have better cell morphology, attachment,
spreading and proliferation on CCHCs than on CHACs. The excellent
biocompatibility of CCHCs suggests that they possess great potentials
for bone implants.
Acknowledgments
This research was supported by Key Disciplines of Shanghai Munic-
ipal Education Commission (no. J50206), Natural Science Foundation of
China (nos. 51002095, 51372152 and 30973038), Science and Technol-
ogy Commission of Shanghai Municipality (no. 12JC1405600), Program
of Shanghai Normal University (nos. DZL124 and DCL201303), Innova-
tion Foundation of Shanghai Education Committee (no. 14ZZ124), and
State Key Laboratory for Modification of Chemical Fibers and Polymer
Materials, Donghua University (no. LK1206).
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