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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 has several disadvantages such as low fracture toughness and high
brittleness [13], which frequently limit its clinical application for
Titanium alloys are widely used for orthopedic and dental im- weight-bearing bones. Moreover, hydroxyapatite coatings usually
plants under load-bearing conditions because of their low density, cannot bond strongly with titanium alloy substrates, which may
good corrosion resistance and excellent mechanical property [1,2]. peel off due to different thermal expansion coefficients and non-
However, titanium alloys are bioinert materials. When implanted, metallurgical bonding between coatings and substrates [14]. Fortu-
they always tend to create a stress shielding effect at bone–implant nately, these defects could be resolved effectively by adding organic
interfaces because their mechanical properties are different from polymer to hydroxyapatite coatings. Bioactive organic polymers not
those of host bones [3]. The mismatch of Young's modulus (Ti implant: only act as agglomerants between coatings and substrates, but
110 GPa, bone: 10–30 GPa) may easily cause detrimental resorptive also improve the biocompatibility [15–21]. As a natural polymer,
bone remodeling, wearing and loosening, and even result in implant chitosan is widely used in biomedical fields because of its good bio-
failure [4,5]. compatibility, biodegradability, bioresorbability and bactericidal
To overcome the above drawbacks, several synthetic strategies have property [22–24]. Therefore, chitosan/hydroxyapatite coatings on
been developed, including chemical surface treatments, thermal surface Ti6Al4V substrates may possess excellent mechanic properties and
treatments and surface coatings by using different bioactive ceramics biological properties.
such as hydroxyapatite, bioglass and hydrotalcite [6–11]. As we Herein, we develop a new method to fabricate chitosan/carbonated
know, hydroxyapatite possesses excellent biocompatibility, bioactivity, hydroxyapatite composite coatings (CCHCs) with porous structures ac-
osteoconductivity, nontoxicity and nonimmunogenicity because of its cording to the following steps: (i) deposition of CaCO3 particles on
compositional similarity to bone minerals [12]. However, hydroxyapatite 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
⁎ Corresponding authors. Tel./fax: +86 21 64321951.
E-mail addresses: zhuzhenan2006@126.com (Z.-A. Zhu), ypguo@shnu.edu.cn
this work are to fabricate CCHCs, to investigate their formation mecha-
(Y.-P. Guo). nism, and to study their biocompatibility by using human bone mesen-
1
S. Tang and B. Tian contributed equally to this work. 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) 210–216 211

2. Materials and methods This study was approved by the Ethic Committee of the Ninth People's
Hospital of Shanghai Jiao Tong University. The donor was healthy with-
2.1. Materials out metabolic disease, inherited illnesses or other diseases. hBMSCs
were cultured in a-MEM culture medium supplemented with 10%
Hydrochloric acid, phosphoric acid, hydrofluoric acid, acetic acid, so- fetal bovine serum (FBS), 1% penicillin (100 U/mL), and streptomycin
dium dihydrogen phosphate dehydrate, disodium hydrogen phosphate sulfate (100 mg/mL) (Gibico BRL, Grand Island, NY). Cells were incubat-
dodecahydrate, calcium chloride and sodium carbonate anhydrous ed at 37 °C in a humidified atmosphere of 5% CO2 and 95% air, with the
were purchased from National Medicine Group Chemical Reagent Co., growth medium changed every 48 h. hBMSCs at a passage from P3 to P4
Ltd. Chitosan (85% deacetylated form), TRITC phalloidin and 4′,6 were used for these experiments.
diamidino-2-phenylindole (DAPI) were obtained from Sigma Aldrich, The cytoskeletons of hBMSCs on samples were observed by double
USA. Alpha modification of Eagles medium (a-MEM) and fetal bovine fluorescence staining. Briefly, hBMSCs were seeded on CHACs and
serum (FBS) were purchased from Gibco-BRL (Sydney, Australia). CCHCs at a density of 3 × 104 cells per sample in a 12-well plate in trip-
CCK-8 was obtained from Dojindo Molecular Technology, Japan. All licate, respectively. After incubation for 24 h, the samples were gently
chemicals used in this study were of analytical grade. washed with PBS and maintained in 4% paraformaldehyde for 15 min,
followed by soaking in 0.1% Triton X solution for 15 min. TRITC
2.2. Preparation of CCCs phalloidin was used to stain the actin filaments of cells, and 4′,6-
diamidino-2-phenylindole (DAPI) was used to stain the nucleus of
Ti6Al4V substrates were abraded with 1000-grit silicon carbide cells. Cell morphology and spreading of hBMSCs were investigated by
paper, and washed with pure acetone and deionized water in an confocal laser scanning microscopy (CLSM, Leica TCS SP2; Leica
ultrasonic cleaner. Acid treatment was performed by soaking these sub- Microsystems, Heidelberg, Germany).
strates in a 1.0 mol/L H3PO4–1.5 wt.% HF solution for 20 min at room Proliferation of hBMSCs on CHACs and CCHCs was examined with
temperature. After the acid treatment, the substrates were washed a cell counting Kit-8 (CCK-8, Dojindo Molecular Technology, Japan)
with deionized water, and dried at room temperature. 1.25 g of CaCO3 assay. Briefly, hBMSCs were seeded on the coating surfaces at a density
powders was dissolved in 250 ml of ethanol, and dispersed ultrasonical- of 7 × 103 cells per sample in a 12-well plate in triplicate. At each time
ly for 30 min to get a suspension solution. 0.5 ml of an HCl solution point, the medium was removed, and 1 mL medium was added into
(1.0 mol/L) was added into the above suspension solution, and kept each well with 100 μL CCK-8 according to the manufacturer's instruc-
pH value at about 6.5. To deposit calcium carbonate powders on the tions. After incubation for 3 h, optical density was measured at
substrates, an electrophoretic cell using a Ti6Al4V substrate as cathode 450 nm by using an automated plate reader (PerkinElmer).
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 2.6. Statistical analysis
min. After electrophoretic deposition, the products (CCCs) were dried
at room temperature. Data are presented as mean ± standard deviation (SD) from at least
three independent experiments. Statistical analysis was performed by
2.3. Preparation of CHACs and CCHCs 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.
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 3. Results and discussion
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 3.1. Morphologies of CCHCs
water, and dried in a convection oven at 37 °C for 48 h.
With magnetic stirring, 1.0 g of chitosan powders was dissolved The SEM images of CHACs and CCHCs are shown in Fig. 1. CHACs are
into an acetic acid solution (100 ml, 2 vol.%). CHACs were soaked fabricated by deposition of calcium carbonate particles on Ti6Al4V sub-
into the above solution for 20 s, and then moved into deionized water strates via electrophoresis technique followed by treatment with PBS.
for 5 s. Subsequently, the coatings were dipped into a NaOH solution Our previous report has demonstrated that CCCs are composed of
(1.0 mol/L) for 10 s. Finally, the products (CCHCs) were washed by de- many calcium carbonate particles with a particle size of 0.5–10 μm
ionized water, and dried in a convection oven at 37 °C for 24 h. [26]. These calcium carbonate particles in CCCs are stacked loosely
together due to the weak electrostatic bonding during electrophoretic
2.4. Characterization 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-
The microstructures of samples were analyzed by scanning electron magnification image demonstrates that the carbonated hydroxyapatite
microscopy (SEM, XL800, Philips). The crystalline phases of samples particles on CHACs exhibit plate-like structure (Fig. 1b). These plates ag-
were carried out by X-ray powder diffraction (XRD, D8, Bruker) by gregate to form macropores with a pore size of 0.5–2 μm. It is noted that
using Cu Kα radiation within a scanning range of 2θ = 10° to 60° and the gaps among the calcium carbonate particles in CCCs are partly
scanning rate of 4° min−1 at 40 kV/30 mA current. The functional groups remained in CHACs during the transformation of calcium carbonate to
of samples were examined by Fourier transform infrared spectra (FTIR, carbonated hydroxyapatite (Fig. 1a) [26]. In addition, same cracks are
5DX, Nicolet) within a wavenumber range of 4000–400 cm−1 by using observed in the low magnification SEM image (Fig. 1a), which may
KBr pellet technique at room temperature. The thermal behaviors of sam- weaken the adhesion strength between coatings and substrates.
ples were examined by thermo-gravimetric analysis (TG-DTA, Perkin- In order to decrease the coating cracks and improve the biocompat-
Elmer) at a heating rate of 10 °C/min in an alumina crucible in air ibility, CCHCs are fabricated by modification of CHACs with chitosan. In-
atmosphere. The surface wettabilities of samples were determined by terestingly, the uniformly crack-free CCHCs are obtained, as shown in
sessile drops of water on coating surfaces via contact angle measurement. the SEM image (Fig. 1c). Chitosan is distributed homogeneously around
carbonated hydroxyapatite plates, filling the cracks in CHACs (Fig. 1c
2.5. Cell behaviors of CCHCs and d). At the same time, gaps or macropores among carbonated hy-
droxyapatite plates are remained although the pore size decreases
Human bone mesenchymal stem cells (hBMSCs) were isolated and slightly due to the presence of chitosan (Fig. 1d). In terms of CCHCs,
expanded by using the standard method as described previously [25]. 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, is stronger than the standard diffraction pattern (JCPDS card no. 09-
macropores in CCHCs can increase the contacting area for cell attach- 0432) [31], demonstrating the preferential growth of apatite along the
ment and spreading [27,28]. The cross-section SEM image in Fig. 2 c-axis during the conversion process of calcium carbonate to apatite.
shows that the thickness of CCHCs is about 20 μm. Interestingly, an The same orientation is also observed in bone mineralization, as c-axis
amorphous TiOx layer with a thickness of about 2 μm is produced be- of the apatite nanocrystals is parallel to the collagen fibers [32].
tween coatings and substrates after chemical etching of Ti6Al4V sub- CCHCs are obtained after soaking CHACs in an acetic acid chitosan
strates in a 1.0 mol/L H3PO4–1.5 wt.% HF solution for 20 min [29]. The solution followed by treatment with a NaOH solution. As compared
TiOx layer may improve micromechanical interlocking and physico- with CHACs, CCHCs possess the phases of both apatite and chitosan.
chemical bonding of coatings [29]. 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
3.2. Structure and composition of CCHCs (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).
The phase structures of CHACs and CCHCs are detected by XRD pat- Fig. 4 shows the FTIR spectra of chitosan, CHACs and CCHCs. The
terns, as shown in Fig. 3. After soaking CCCs in PBS at 37 °C for 3 days, functional groups of apatite are detected in both CHACs and CCHCs.
CaCO3 particles in the coatings are converted to apatite plates (Figs. 1b The broad band at 3410 cm−1 is attributed to OH group or absorbed
and 3b). The broad peaks in Fig. 3b indicate that the formed apatite water [33]. The absorption bands at 1031, 602 and 562 cm−1 are attrib-
has low crystallinity similar to biological apatite [30]. The poor crystal- uted to PO3−4 group [34]. The absorption band at around 1105 cm−1 is
linity of apatite may be attributed to the following reasons: (i) part of corresponding to HPO24 −, indicating that the obtained products are
Ca2+ and PO3− 4 ions in apatite crystal lattices are subsitituted by impu- calcium-deficient apatite [35]. The bands of B-type CO2− 3 substitution
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. Fig. 3. XRD patterns of different samples: (a) chitosan, (b) CHACs and (c) CCHCs.
S. Tang et al. / Surface & Coatings Technology 251 (2014) 210–216 213

Fig. 4. FTIR spectra of different samples: (a) chitosan, (b) HCACs and (c) CCHCs.
Fig. 6. TG curves of different samples: (a) CHACs and (b) CCHCs.

at 872 and 1418 cm−1 are detected in Fig. 4b and c, indicating that the 3.3. Wettability of CCHCs
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 The wettability of biocoatings plays an important role in adsorbing
of PO3−
4 ions by HPO2−4 and CO2− 3 ions, so the general formula may be proteins and cellular responses [40]. The contact angles of CHACs and
expressed as Ca10 − x − y/2(HPO4)x(PO4)6 − x − y(CO3)y(OH)2 − x. CCHCs are analyzed by sessile drops of water on the coating surfaces,
Chitosan, a random copolymer of N-acetyl-D -glucosamine and as shown in Fig. 7. CHACs possess profound hydrophilicity with a con-
D -glucosamine, is the partially de-acetylated derivative of chitin tact angle of 12.5°. Recently, Webb et al. have reported that moderately
(Fig. 5). The band at 1571 cm−1 is assigned to N\H bending vibration hydrophilic surfaces with a contact angle of 20–40° can promote cell at-
overlapping amide II vibration. C\N stretching vibration occurs at tachment [41]. Interestingly, the contact angle of 29.4° is obtained for
around 1030 cm−1 and overlaps the vibration from carbohydrate ring. CCHCs after modification of CHACs with chitosan. The higher contact
The broad band at around 3415 cm−1 is corresponding to the stretching angle of CCHCs than CHACs is attributed to the presence of chitosan in
vibration of N\H and OH groups. The \CH2 bending vibration occurs at the coatings. As compared with CHACs, the chitosan has lower hydro-
1410 cm−1 [37,38]. After soaking CHACs in an acetic acid chitosan solu- philicity with a contact angle of about 79.7° [42]. Moreover, the chitosan
tion followed by treatment with a NaOH solution, CCHCs including car- layer in CCHCs may reduce surface roughness (Fig. 1), and thus de-
bonated hydroxyapatite and chitosan are obtained (Figs. 1 and 3). The creases the hydrophilicity of CCHCs.
above results can be further confirmed by the FTIR spectra (Fig. 4). As
compared with CHACs, the same characteristic absorption bands 3.4. Formation mechanism of CCHCs
due to carbonated hydroxyapatite are also observed in CCHCs (Fig. 4b
and c). In addition, the characteristic peaks of chitosan are overlapped Recently, many literatures have reported electrophoretic deposi-
by those of carbonated hydroxyapatite (Fig. 4a and c). tion of bioceramic/chitosan composite coatings [16–19]. The electro-
The thermal behaviors of CHACs and CCHCs are determined by using phoretic deposition technique is a simple method for incorporation
TG analysis, as shown in Fig. 6. For both CHACs and CCHCs, the weight of chitosan in bioactive coatings, but chitosan cannot combine tightly
loss between 30 °C and 180 °C is due to the loss of physically adsorbed with bioceramics via electrostatic interactions. In this work, we have
water on coatings [39]. At the temperature range 180–550 °C, the developed a new method to fabricate CCHCs, as shown in Fig. 8. In the
weight loss of 2.5% in CHACs is attributed to the decomposition of car- first stage, CCCs are formed by deposition of calcium carbonate particles
bonated hydroxyapatite (Fig. 6a). For CCHCs, the weight loss of 10% at on Ti6Al4V substrates by electrophoresis technique (Fig. 8b).
the temperature range 180–550 °C belongs to the decomposition of In the second stage, CCCs are converted into CHACs by treatment
both low crystalline apatite and chitosan [30] (Fig. 6b). The content of with PBS via a dissolution–precipitation reaction (Fig. 8c and d), be-
chitosan is about 7.5% of the total in CCHCs, as calculated by using cause calcium carbonate and hydroxyapatite have different solubilities
CHACs as a control. In addition, part of unreacted calcium carbonate is [26]. For example, the logarithmic solubility product (pKs) of calcite at
remained as CCCs are converted to CHACs in PBS at 37 °C. The weight 37 °C is 8.56, while that of hydroxyapatite is 58.63 [43,44]. After soaking
loss of about 1.8% ranging from 600 °C to 800 °C is attributed to the CCCs in PBS at 37 °C, CaCO3 particles begin to dissolve. The released
2−
unreacted calcium carbonate that is not detected by XRD patterns Ca2 + ions react with the PO3− 4 , HPO4 and OH− ions in PBS to form
because of its low percentage in the coatings. 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
Fig. 5. Chemical structure of chitosan. 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 because of the stereoregularity and molecular hydrogen bonding
of environments. When chitosan is dissolved in an acetic acid solution, among chitosan molecules. Moreover, the released Ca2 +, PO34 −,
the free amino groups in chitosan are protonated to form chitosan- HPO2−
4 and CO2−3 ions from CHACs in an acetic acid chitosan solution
NH+ 3 . After soaking CHACs in an acetic acid chitosan solution, the chito- may react again with the OH− ions in a NaOH solution to form carbon-
san is attached to CHACs via chelating and hydrogen bonding, and en- ated hydroxyapatite. Fig. 1c and d shows that crack-free CCHCs are com-
ters into the macropores among carbonated hydroxyapatite plates. posed of both carbonated hydroxyapatite plates and gel-like chitosan.
The immersion time of CHACs in an acetic acid chitosan solution affects
the morphology and chemical composition of CCHCs. The ideal immer- 3.5. Biocompatibility of CCHCs
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 As we know, bone implants must have good biocompatibility with
immersion time may result in the complete dissolution of the carbonate surrounding cells to promote satisfactory osteointegration between im-
hydroxyapatite particles in CHACs according to the following chemical plants and bone tissues. Therefore, the contact and interaction between
equation: cells and biomaterial surfaces are very important physiological process-
es. Cytoskeleton analysis of hBMSCs on CHACs and CCHCs has been
þ
Ca10−x−y=2 ðHPO4 Þx ðPO4 Þ6−x−y ðCO3 Þy ðOHÞ2−x þ ð2−xÞH → performed by using CLSM, as shown in Fig. 9. Cytoskeleton is a highly
2þ 2− 3− 2− ð2Þ
ð10−x−y=2ÞCa þ xHPO4 þ ð6−x−yÞPO4 þ yCO3 þ ð2−xÞH2 O: dynamic network composed of actin polymers and associated proteins.
The function of cytoskeleton is to mediate a variety of essential biologi-
After the chitosan deposits on the surfaces of CHACs, CCHCs are cal activities, including intra-cellular and extra-cellular movements and
obtained by treatment with a NaOH solution (Fig. 8f). Under alkaline structural supports. Orientation distribution of the actin filaments with-
solutions, the chitosan in the coatings is turned into the solid state 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) 210–216 215

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 surroundings [46]. These investigation results are in agreement with
nuclei are stained with DAPI. Fig. 9 shows the long red bundles of stress the previous reports that hBMSCs have good cell adhesion and spread-
fibers composed of actin filaments and good cell–cell contacts with one ing on hydroxyapatite/chitosan scaffolds [22].
another, displaying good cell cytoskeleton morphologies on both CHACs Recently, CCK-8 assay has been widely used to investigate in vitro
and CCHCs. The hBMSCs on CCHCs present a clustering, confluency and biocompatibility of biomaterials. It is a quick and effective method for
multi-layering polygonal morphology at 24 h, while those on HCACs testing mitochondrial impairment and correlates quite well with cell
display a slim, spherical, and fusiform-shaped morphology. Taken to- proliferation. Herein, the cytotoxicity and proliferation of hBMSCs on
gether, the hBMSCs on CCHCs have better cell adhesion, spreading and CHACs and CCHCs have been evaluated by CCK-8 assay. Fig. 10 indicates
cell–cell contact than those on CHACs. Generally, stem cells are regulat- no significant difference between CHACs and CCHCs before culturing
ed by physical and chemical factors from their complex extracellular 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-
Fig. 10. CCK-8 assay results of hBMSCs cultured on CHACs and CCHCs at different days eton organization [41]. Therefore, the cell morphology, cytoskeleton
(mean ± SD,*p b 0.05 and **p b 0.01). organization, cell attachment, spreading and proliferation of hBMSCs
216 S. Tang et al. / Surface & Coatings Technology 251 (2014) 210–216

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