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Colloids and Surfaces B: Biointerfaces 100 (2012) 169–176

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Colloids and Surfaces B: Biointerfaces

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Physicochemical characterization and biocompatibility in vitro of biphasic

calcium phosphate/polyvinyl alcohol scaffolds prepared by freeze-drying method
for bone tissue engineering applications
Lei Nie a , Dong Chen b , Jinping Suo a,∗ , Peng Zou a , Shuibin Feng a , Qi Yang a , Shuhua Yang b , Shunan Ye b
State Key Laboratory of Mould Technology, College of Materials Science and Engineering, Huazhong University of Science and Technology, Wuhan 430074, PR China
Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, PR China

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

Article history: In this study, a well developed porous biphasic calcium phosphate (BCP)/polyvinyl alcohol (PVA) scaffold
Received 23 February 2012 was prepared by emulsion foam freeze-drying method possessed moderate inter-connected pores and
Received in revised form 11 April 2012 porosity. The SEM analysis showed that BCP nano-particles could disperse uniformly in the scaffolds, and
Accepted 29 April 2012
the pore size, porosity, and compressive strength could be controlled by the weight ratio of BCP/PVA.
Available online 31 May 2012
The in vitro degradation and cytocompatibility of scaffolds were examined in this study. The degradation
analysis showed the prepared scaffolds have a low variation of pH values (approximately 7.18–7.36)
in SBF solution, and have the biodegradation rate of BCP/PVA scaffolds decreased with the increase of
Porous scaffold
Biphasic calcium phosphate
PVA concentration. Moreover, MTT assay indicated that the BCP/PVA porous scaffold has no negative
PVA effects on cells growth and proliferation, and the hBMSCs possessed a favorable spreading morphology
Cell culture on the BCP/PVA scaffold surface. The inter-connected pore structure, mechanical strength, biodegradation
rate and cytocompatibility of the prepared BCP/PVA scaffold can meet essential requirements for blame
bearing bone tissue engineering and regeneration.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction defect clinical requirements on the bone substitutes. Currently, the

critical issue of concern for a newly developed scaffold is how to
Every year, millions of people are suffering from bone defects simultaneously achieve its moderate mechanical strength, degra-
arising from trauma, tumor or bone related diseases and of course dation rate, porosity, and reasonable pore size distribution.
several are dying due to insufficiency of ideal bone tissue [1]. Due to the compositional similarity with bone, Calcium
Biologically produced bone structures are known for self heal- phosphate-based biomaterials, such as hydroxyapatite (HA,
ing. However, large bone defects do not heal spontaneously and Ca10 (PO4 )6 (OH)2 ), ␤-tricalcium phosphate (␤-TCP, Ca3 (PO4 )2 ), and
require surgical intervention for restoration, and the current ther- biphasic calcium phosphate (BCP), a mixture of HA and ␤-TCP,
apies include autografts, allografts. Nonetheless, autografts maybe have received most attention for bone repair applications in the
associated with donor shortage and donor site morbidity whereas literature [10–13]. Among the various calcium phosphate bioma-
allografts may have the risk of disease transmission and immune terials used in orthopedic surgery, BCP is an important candidate
response [2–4]. The aforesaid limitations and the expected shortage of the bone substitutes that combines high mechanical strength,
of bone grafts for surgical procedures, motivated materials scien- excellent bioactivity of HA, and faster degradation rate of ␤-TCP.
tists to find suitable bioactive materials, such as three-dimensional Especially, the surface of BCP allows osteoblasts/osteoclasts to act
(3D) porous scaffold designed with the required porosity, mechan- in a more natural way than that of pure HA or pure ␤-TCP. Such
ical strength and a favorable environment for bone cell attachment, BCP can be assumed to co-exist with living bone tissue, receiving
to provide mechanical stability in the defect region and launch tis- the natural action of bone cells during the bone remodeling process
sue regeneration with specific living cells [5–9]. With the rapid [14]. Having the ceramic origin, any bioceramics made of calcium
development of biomaterial-based bone substitutes, great progress phosphates possess poor mechanical properties that do not allow
has been made in the last decade. However, as advancements are them to be used in load-bearing areas, such as artificial teeth or
made, new challenges also emerge, which comes from the bone bones [15]. A polymeric component was introduced into inorganic
materials in order to form an organic–inorganic composites used
to overcome the mechanical weakness of inorganic-based scaffolds.
∗ Corresponding author. Among biodegradable and biocompatible polymers, Polyvinyl alco-
E-mail address: (J. Suo). hol (PVA) is one of the most commonly used polymer in biomedical

0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
170 L. Nie et al. / Colloids and Surfaces B: Biointerfaces 100 (2012) 169–176

applications [16–22]. Zhang et al. [29] showed that the tensile (0.2 M) solutions were added in a three necked flask by simultane-
strength and modulus of PVA/HA composite both increased com- ous drops at room temperature while the pH was adjusted at 11
pared to pure PVA hydrogel, the tensile strength first increased using buffers, the resultant white color product was washed sev-
and then decreased with the increase of HA concentration. The eral times with distilled water to completely remove the buffer.
tensile strength reached to the maximum value when HA concen- The powder obtained was thermally treated at 1125 ◦ C for 1 h, to
tration was 7.5%. As HA concentration further increased the tensile produce biphasic calcium phosphate. The BCP powder was crushed
strength decreased. While previous research have focused on PVA in a mortar and pestle and classified using stainless steel sieves to
film blended with inorganic particles, to the authors knowledge, provide particles of size <75 ␮m.
little reports are available on developing moderate porous scaf-
folds based on hydrophilic PVA polymer and BCP nano-particles. 2.3. Fabrication of BCP/PVA porous scaffold
The porous nano-HA/PVA hydrogel scaffolds were fabricated with
emulsion foam method by Mu et al. [23] showed that nano- The porous BCP/PVA scaffold was prepared by emulsion foam
hydroxyapatite existed in the composite in the shape of short-rod freeze-drying method. Briefly, BCP powder was dispersed in dis-
and the HA nano-particles in the composite could dispersed tilled water under stirring to prepare BCP slurry (40 wt%). Aqueous
uniformly. Because of narrow pore size in the scaffolds, the cyto- 8 wt% PVA solution was prepared by soaking pre-weighed quan-
compatibility and biodegradation were not researched. For bone tities of dry PVA in distilled water for 6 h and heating them at
tissue engineering, the most important morphological properties 90 ◦ C for 3 h. PVA solution and BCP slurry were added in a three
of a biomaterial scaffold are porosity and pore size, however the necked flask, polyethylene glycol octyl phenyl ether (OP) as the
required porosity and pore size cannot be set as a general guideline emulsifier (50 wt% compared to the final weight of scaffold) was
for optimal bone tissue scaffolds due to the wide range of bone fea- added in mixed solution, and the mixed solution was mechani-
tures in vitro and in vivo [24,25]. The development of nanomaterials cally stirred for 48 h at 60 ◦ C to disperse the BCP particles in the
or nanostructures in bone tissue engineering may be preferable as polymer matrix in homogeneous manner. To change the weight
they can be fabricated with controlled size, shape, composition, ratio of PVA concentration, the volume of PVA solution added in
surface chemistry and other physicochemical properties. mixed solution was adjusted while the volume of BCP slurry was
Although BCP and PVA exhibit high biocompatibility, lit- not changed. The milky white resultant solution was then trans-
tle researchers paid close attention to the BCP/PVA composites ferred to appropriate 12 well plates and quenched at −20 ◦ C, five
because this kind of composites do not show the complex hier- freeze thawing cycles were carried out (20 h at −20 ◦ C and 4 h at
archical nanostructures and high mechanical property to meet the 25 ◦ C). The samples were further freezed at −80 ◦ C for 48 h and
demand of bone substitute. Our approach to solve these problems lyophilized with freeze dryer to form porous scaffolds. Finally, the
was to develop BCP nano-particles mixed with PVA polymer uni- solid samples were removed from the freeze-dryer and immersed
formly by emulsion foam method, furthermore, the freeze-thawing in distilled water for 24 h to remove the emulsifier by leaching.
method was applied to prepare the scaffold. PVA can strongly inter- The distilled water was refreshed at 4 h interval. The samples were
act with the surface of BCP, because BCP has hydroxyl groups on the then placed in a vacuum-dryer for 48 h at −80 ◦ C to remove any
surface and a high concentration of hydroxyl pendant groups in of the remaining solvent. In this way, BCP/PVA scaffolds with a
PVA makes it uniquely capable of being cross-linked physically and well developed porous structure were prepared. The designation
without the incorporation of any chemical additives [26–29]. The of BP20, BP30, BP40 and BP50 represents the final weight ratios of
physical cross-linking process of PVA by freeze–thawing method PVA concentration (wt%) in scaffolds were 20%, 30%, 40% and 50%.
was to establish the hydrogen bonds among hydrogen and oxy-
gen atoms in two parallel PVA polymeric chains. The physical 2.4. Physicochemical characterization of the scaffolds
cross-linking mechanism avoids the necessity to use chemical
cross-linkers in PVA for bone tissue applications. 2.4.1. Morphology
In this study, by controlling the concentration of PVA, the porous The BCP nanoparticles were characterized by FE-scanning
BCP/PVA bone scaffolds with highly inter-connected macropores electron microscopy (FE-SEM) (Sirion 200, Holland FE). The cross-
were fabricated by emulsion foam freeze-drying method. These section of the porous scaffolds were coated with gold to prevent
scaffolds were characterized in terms of morphology, porosity and imaging artifacts from electrical charging. And their morpholo-
compressive strength, moreover, the pH values and biodegradation gies were examined by FEI-Scanning Electron Microscopy (SEM)
rate in simulated body fluid (SBF) solution and the cytocompatibil- (Quanta 200, Holland FEI). The pore size was calculated from SEM
ity of BCP/PVA scaffolds in vitro was evaluated. pictures by selecting five arbitrary areas for measurement.

2.4.2. Fourier-transform infrared spectroscopy (FTIR)

2. Materials and methods
FTIR (VERTEX, Bruker) was used to characterize the presence of
specific chemical groups in BCP/PVA scaffolds. FTIR spectra were
2.1. Materials
obtained within the range between 4000 and 500 cm−1 on VERTEX
70 FTIR spectrometer with a resolution of 1 cm−1 , using attenuated
PVA was obtained from Shanxi Sanwei Group Co. Ltd. (polymer-
total reflectance (ATR) technique.
ization degree ∼1799 and hydrolysis degree ∼99%). Diammonium
hydrogen phoshate ((NH4 )2 HPO4 ) and Calcium nitrate tetrahy-
2.4.3. X-ray diffraction (XRD)
drate (Ca(NO3 )2 ·4H2 O) were obtained from Sinopharm Chemical
The samples were analyzed by X-ray diffraction (X-Pert PRO,
Reagent Co. Ltd. Ammonia was obtained from Wuhan Chemical
PANalytical B.V.). This instrument works with voltage and current
Reagent Co. Ltd. All the chemical reagents used in this work were
settings of 40 kV and 40 mA respectively and uses Cu-K␣ radiation
in analytical reagent level.
(1.540600 Å). For qualitative analysis, XRD diagrams were recorded
in the interval 10◦ ≤ 2 ≤ 90◦ at scan speed of 2◦ /min.
2.2. Fabrication of BCP particles
2.4.4. Porosity
Calcium-deficient apatite powder was prepared by aqueous pre- The open porosity can be calculated by the liquid displacement
cipitation reaction. Briefly, Ca(NO3 )2 ·4H2 O (0.4 M) and (NH4 )2 HPO4 method. The scaffold is submerged in a known volume (V1 ) of
L. Nie et al. / Colloids and Surfaces B: Biointerfaces 100 (2012) 169–176 171

ethanol that is not a solvent for the scaffold and a series of brief 2.7. Culture of human bone mesenchymal stem cells (hBMSCs)
evacuation repressurization cycles is conducted to force the liq- and adhesion on scaffolds
uid into the pores of the scaffold. After these cycles the volume of
the liquid and liquid-impregnated scaffold is V2 . When the liquid- Human bone mesenchymal stem cells (hBMSCs) were cultured
impregnated scaffold is removed, the remaining liquid volume is according to supplier’s recommendations in Dulbecco’s modified
V3 and open porosity is given as: Eagle’s medium (DMEM), containing 10% fetal bovine serum and
1% antibiotic/antimycotic mixture at 37 ◦ C in a 5% CO2 humidi-
V1 − V3
p= fied environment. Cells were harvested by gentle digestion with
V2 − V3
0.05% trypsin/EDTA, and suspended in fresh media. To investigate
the cell adhesion on the BCP/PVA scaffolds, the BCP/PVA scaffolds
2.4.5. Mechanical properties were placed in the bottom of 6-well cell-culture plate and ster-
The porous BCP/PVA scaffolds were subjected to a compres- ilized with ethylene oxide at room temperature for 24 h. Human
sion strength test using an electromechanical universal testing bone mesenchymal stem cells were then seeded and cultured with
machine (SANSCMT4503, China) at a crosshead speed of 2 mm/min. DMEM at 37 ◦ C in a 5% CO2 humidified atmosphere. After incu-
The compressive offset yield stress was determined from the bated for 2 day, the membranes were removed from the wells and
stress–strain curve at a 2% strain. Five replicate determinations washed twice in phosphate-buffered saline (PBS), and then fixed
were undertaken for each formulation. with 2.5% glutaraldehyde at pH 7.4 overnight. After rinsed in 0.1
PBS, specimens were dehydrated in ethanol solutions of varying
2.5. Biodegradation in SBF solution concentrations (i.e. 30%, 50%, 70%, 90%, and 100%, respectively).
After being dried in 100% hexamethyldisilazane (HMDS; Kelong
The biodegradation of the prepared scaffolds in vitro was inves- Chemicals, Chengdu, China) for 15 min and then placed in a des-
tigated by soaking them in SBF solution at 37 ◦ C. The SBF solution iccator for removal of HMDS, the scaffolds were observed by an
was prepared as previously reported [30]. Cylinder-shaped scaf- electron scanning microscopy after sputtering a thin layer of gold.
folds were immersed in SBF solution at 37 ◦ C for 1, 5, 10, 15, 20 and
25 days at a solid/liquid ratio of 50 mg/ml. All samples were held in 2.8. Statistical analysis
plastic flasks which were sealed to minimize the change in pH and
microorganism contamination. The SBF solution was not refreshed All the statistical data are expressed as the mean ± SD and were
during the experiment. After soaking, the samples were filtrated, obtained from five independent experiments. For statistical analy-
rinsed with distilled water and dried at 40 ◦ C for 4 days and the sis, first Levene’s test was performed to determine the homogeneity
final weight of samples carefully measured. The weight loss was of variance for all the data, and then Tamhane Post Hoc tests was
calculated according to percentage of initial weight. Five scaffolds performed for the comparison between different groups. PASW
were used to measure the weight loss and pH change, and results statistics program was employed for all statistical analysis and dif-
were expressed by as mean ± SD. ferences were the data was accepted at a p value of <0.05 for 95%
2.6. Cytotoxicity testing-MTT assay

The immortalized rat osteoblastic ROS 17/2.8 cell line (obtained 3. Results and discussion
from Orthopedics department, Union Hospital, Tongji Medical
College of Huazhong University of Science and Technology) was 3.1. Physical characterization of BCP powders
utilized in this study. These cells were incubated in a humidified
atmosphere of 5% CO2 at 37 ◦ C. Cells were cultured in Dulbecco’s To illustrate the BCP powders properties, XRD, FE-SEM and FTIR
modified Eagle’s medium (DMEM, Invitrogen, Paisley, UK) and sup- were investigated. Fig. 1 shows the distinct high resolution FE-SEM
plemented with 10% fetal bovine serum (FBS, Invitrogen, Paisley, morphologies of the BCP nano-particles prepared in this study.
UK), 100 mg/ml streptomycin and 100 U/ml penicillin. The culture Fig. 1B shows the BCP powders were oblong nano-particles, and
media was changed every alternate day. particles had an average width of 50 nm and length of 100 nm.
The cytotoxicity of the BCP/PVA scaffolds with different The HA and ␤-TCP phases cannot be differentiated [31]. Further-
PVA concentration were investigated by the MTT (3-[4,5- more, the particles were agglomerated in micron scale according
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay to Fig. 1A. Fig. 2 shows the XRD pattern of BCP powders treated
which was used to assess cell proliferation by measuring mito- at 1125 ◦ C. By quantitative analysis for the XRD patterns, it was
chondrial succinate dehydrogenase activity. The samples were confirmed that biphasic calcium phosphate that consisted of about
fixed in each bottom of a 24-well cell culture plate and were 40% HA and 60% ␤-TCP was acquirable. The HA and ␤-TCP peaks
sterilized with ethylene oxide (ETO) steam for 24 h at room were present in prepared BCP samples, the peaks at 2 = 25.9◦
temperature; then 1 ml cell suspension was seeded evenly onto (0 0 2), 28.2◦ (1 0 2), 29.7◦ (2 1 0), 31.8◦ (2 1 1), 32.3◦ (1 1 2) and
each sample. The culture medium was changed by fresh medium 34.6◦ (2 0 2) matching closely with the diffraction peaks of stoi-
every 2 days. After seeding for 1, 3, and 5 days, 100 ␮l of MTT chiometric HA (JCPDS No. 9-0432). The peaks of ␤-TCP appears at
(5 mg/ml) solution was added to each well and incubated at 37 ◦ C 2 = 22.9◦ (3 3 −2), 24.2◦ (3 6 −1), 30.8◦ (4 3 −4), 34.2◦ (3 3 −5) and
for 4 h to allow the formation of formazan crystals, respectively; 34.6◦ (4 2 −1) (JCPDS No. 9-0169). XRD pattern showed no other
removed supernatants, 650 ␮l of dimethyl sulfoxide (DMSO) was impurity was found in the BCP nano-powders.
added to each well for dissolving the blue formazan crystal, then
the solution was transferred to 96-well plates. The absorbance 3.2. BCP/PVA scaffold characteristics
of each well was measured at 570 nm using an ELISA microplate
reader (Bio-Rad). The cells without scaffold were served as the 3.2.1. Fourier transform infrared spectroscopy (FTIR) analysis
negative control in this study. A mean value was obtained from FTIR spectroscopy was carried out to analyze any complex
the measurement of five test runs. The results were statistically structural changes as well as interactions between BCP and PVA.
analyzed to express the mean 5 the standard deviation (SD) of the Typical FTIR spectra of the pure PVA, BCP and BCP/PVA scaffold
mean. are shown in Fig. 3. As shown in Fig. 3, line B, the phosphate
172 L. Nie et al. / Colloids and Surfaces B: Biointerfaces 100 (2012) 169–176

Fig. 1. FE-SEM micrographs of (A) the BCP powders agglomerate state at 1500× magnification and higher magnification (80,000×) images of oblong BCP nanoparticles (B).

ions, PO4 3− , were the principal molecular components of BCP con- the absorption bands associated with H2 O molecules appeared at
tributing to the IR absorbance in the 1200–550 cm−1 region. The around 3315 and 1639 cm−1 in the spectrum of BCP. In the BCP/PVA
characteristic peaks at 1033 and 1088 cm−1 correspond to the scaffold, the higher peak at 3315 cm−1 is only narrowed without
stretching vibration of PO4 3− and at 599 cm−1 to the deforma- reduction in its intensity, while the peak at 1639 cm−1 showed a
tion vibrations of PO4 3− . The characteristic absorption bands of large reduction of its intensity.
PVA occurred at 3278 cm−1 (stretching of OH), 2935 cm−1 (asym-
metric stretching of CH2 ), 2906 cm−1 (symmetric stretching of 3.2.2. Scaffold morphology and microstructure analysis
CH2 ), 1417 cm−1 (wagging of CH2 and bending of OH), 1143 cm−1 It is well known that interconnected porous scaffolds play a crit-
(stretching of CO from crystalline sequence of PVA), 1088 cm−1 ical role in bone tissue engineering. They provide a template for cell
(stretching of CO and bending of OH from amorphous sequence attachment and bone extracellular matrix formation, and the struc-
of PVA), 919 cm−1 (bending of CH2 ) and 838 cm−1 (rocking of CH) tural support for the newly formed tissue and provide sufficient
[32]. Fig. 3, line A exhibited the characteristic absorption bands space for blood circulation. Fig. 4 showed the macroporous network
of BCP/PVA scaffold, after incorporating BCP nano-particles into morphologies and strut microstructure of the porous BCP/PVA scaf-
the PVA the predominant broad absorption band associated with fold fabricated by emulsion foam freeze-drying method. From the
the OH stretching of PVA centered at 3278 cm−1 shifts to a higher SEM pictures, the expected 3D inter-connected porous structure
wave number region (OH = 3296 cm−1 ). Due to the high surface with open macropores was observed. For BP20 samples (Fig. 4A,
area of BCP nano-particles, the surface OH groups strongly inter- B), the pore size of open macropores were about 400–700 ␮m. In
acted with PVA macromolecules forming interfacial layers on their addition, the small pores of 5–10 ␮m formed in the phase sepa-
surface. In addition, the major absorption band of PO3 −4 stretch- ration process and smaller pores induced by solvent vaporization
ing, which appeared at 1019 cm−1 in the BCP spectrum, moved to in the pore walls could be observed. Compared to BP20, the small
1033 cm−1 . This red shift might be attributed to the interactions pores with a pore size of 5–10 ␮m could not be observed on the
between the PVA molecules and BCP particles. Also the spectra other samples. At a concentration of 30% PVA (Fig. 4C, D), the
of PVA adsorbed on BCP clearly influenced the recovering of the PVA fibers were observed in the scaffold and BCP particles were
carbonyl-stretching region [33]. It is of interest to note here that

Fig. 2. XRD pattern of biphasic calcium phosphate. Fig. 3. The FTIR spectra of pure BCP/PVA scaffold (A), BCP (B) and PVA (C).
L. Nie et al. / Colloids and Surfaces B: Biointerfaces 100 (2012) 169–176 173

Fig. 4. ESEM images of the morphologies and microstructures of the prepared porous scaffolds at different magnification: (A, B) BP20, (C, D) BP30, (E, F) BP40 and (G, H) BP50
(arrows: the pores in the scaffolds).
174 L. Nie et al. / Colloids and Surfaces B: Biointerfaces 100 (2012) 169–176

At the PVA concentration of 20 wt%, the BCP nano-particles were

incorporated onto porous scaffold, and the scaffold presented
the ceramic property because of the low concentration of PVA
component. As the PVA concentration increased to 30 wt%, the BCP
nano-particles mixed with PVA uniformly and the biochemical
binding affinity between BCP and hydrophilic PVA led to the
increase of compressive strength. While the PVA concentration
increased, the mechanical property of PVA polymer led to the
decrease of compressive strength. It could be deduced from Fig. 5
that PVA concentration has stronger influence on the strength of
BCP/PVA scaffolds. The results illustrated that 30 wt% addition of
PVA was appropriate in the BCP matrix when comprehensively
considering the requirement of pore size, porosity, and mechanical
strength for tissue engineering scaffold.
In the case of the porous scaffold there has always been a
tradeoff between sufficient mechanical strength and porosity. An
inhomogeneous distribution and agglomeration of the particles
in the polymer matrix might result in poor mechanical proper-
Fig. 5. The porosity and compressive strengths of the BCP/PVA scaffolds with differ- ties, while homogeneous disturbed nanopaticles would lead to a
ent PVA concentrations. Error bars represent the mean ± standard deviation, n = 5. significant improvement in mechanical properties [34–39]. Our
p < 0.05.
approach to solve these problems is to adjust the PVA concentration
in porous scaffolds with freeze-thawing method. The compressive
observed in PVA phase homogeneously. An increase of PVA concen- strength of porous polymer/inorganic composites was in the range
tration led to the pore size of scaffold decreased from 400–700 ␮m of 0.2–0.4 MPa, and for the spongy bone (not the strut), the com-
to 150–350 ␮m approximately while the porous network struc- pressive strength was in the range of 0.2–4 MPa [40,41]. While the
ture of scaffold remained unchanged, and surface morphology was scaffolds used for the blame bearing bone application, the com-
altered, where some difference in microstructure of nanocompos- pressive strength value was lower than used for load bearing bone
ite scaffolds was seen at the higher magnification. At a much higher application. Furthermore, the compressive strength property can-
concentration of PVA, the pore size became much smaller (approxi- not be set as a guideline for the blame bearing bone application. For
mately 50–150 ␮m), some pores were fully clogged and the surface BCP/PVA scaffold, the pore size was similar to natural spongy bone,
morphologies of scaffolds have changed significantly (Fig. 4E, F, and compressive strength could be adjusted by the PVA concen-
G, H). This is further supported by porosity analysis of the pre- tration with changing the porosity of prepared scaffolds through
pared scaffolds showing that the porosity decreased from 87% to freeze-drying method. Typically, for the BP20 samples, the com-
73% while the PVA concentration increased from 20 to 50 wt%. pressive strength value was at the spectrum of the acceptable range,
suggesting the potential use for blame bearing bone regeneration in
tissue engineering. In addition, for porous bone scaffold, an incom-
3.2.3. Scaffold porosity and compressive strengths analysis plete pore interconnection could constrain the overall biological
Fig. 5 showed the porosity and compressive strengths of the system and limit the blood vessel invasion, which is essential for
BCP/PVA scaffolds with different PVA concentrations. As the PVA tissue in-growth into the scaffolds, for this reason, interconnect
concentration increased from 20 to 50 wt% in the BCP/PVA scaf- structure is a key factor for porous scaffold.
folds, the porosity of the BCP/PVA scaffolds gradually decreased,
however, the compressive strength increased to 0.26 MPa at the 3.3. Scaffolds in vitro biodegradation in SBF solution
PVA concentration of 30 wt% and decreased to 0.19 MPa slowly
at the PVA concentration of 50 wt%. Generally, the compressive Fig. 6A represented the mass loss of the composites soaked
strength of a scaffold was related to its composition and porosity. in SBF solution different soaking times. For BP20 scaffold, after

Fig. 6. The weight loss (A) and pH value (B) of the prepared scaffolds in SBF solution after different soaking times. Error bars represent the mean ± standard deviation, n = 5.
p < 0.05.
L. Nie et al. / Colloids and Surfaces B: Biointerfaces 100 (2012) 169–176 175

25 days of soaking, the weight loss was 38% relative to initial

weight and showed a greater weight loss than BP30, BP40 and
BP50 scaffolds. The high concentration of BCP nano-particles led to
some part of nano-particles dissolved in SBF solution because the
portion of nano-particles was not incorporated into PVA matrix.
The result indicated that the degradation of the scaffold could be
controlled by the concentration of BCP nanoparticles. Dispersion of
BCP nano-particles in PVA polymer matrix is not an easy process,
especially when the particle size decreases to the nanometer scale,
as nano-particles have a strong tendency to self-agglomerate
(Fig. 1A). The gaps between inorganic particles and polymer matrix
could provide a channel for the penetration of water into polymer
led to weight loss of scaffolds and the pH change in SBF solution.
As can be seen in Fig. 4C, D, for BP30 scaffolds, BCP nano-particles
distributed uniformly in PVA polymer matrix, and significant dif-
ference in the surface morphology was found compared to BP20,
BP40, and BP50 scaffolds. In addition, the biodegradation rate of
nanocomposite porous scaffolds in SBF solution might be con-
trolled by BCP nanoparticles concentration in BCP/PVA scaffolds
and the porosity of scaffolds [42]. An increase porosity of scaffolds
led to the increase of biodegradation rate in SBF solution. The BP30
Fig. 7. Formazan absorption at 570 nm in MTT assays rat osteoblasts seeded on the
scaffold showed slower dissolution than BP20 scaffold and quicker BCP/PVA scaffolds after 1, 3 and 5 days and the negative control group, Data were
dissolution than BP40, BP50 scaffolds as measured by weight loss expressed as means ± standard deviation, n = 5. * p < 0.05.
of scaffolds in SBF solution. The graphs of the pH value changed in
terms of soaking time also illustrated the dissolution behavior of
BP50 scaffolds. The reason for which might be owing to their surface
prepared scaffolds as shown in Fig. 6B. The pH values of SBF with-
morphologies and BCP concentration, nanocomposites containing
out scaffolds were stable throughout the experimental period. The
BCP had good cell attachment and proliferation owing to BCP had
pH values of prepared scaffolds exhibited approximately 7.18–7.36
excellent bioactivity [44–46]. Therefore, the BCP/PVA scaffold pre-
and a low variation was observed for all BCP/PVA scaffolds.
pared in this study can be a promising biomaterial for bone tissue
regeneration application due to their good cytocompatibility.
3.4. Scaffolds cytotoxicity testing-MTT assay
3.5. hBMSCs grown on the porous prepared scaffolds
Indispensably, all materials implanted into the body for bone
regeneration must be of good biocompatibility, which does not Mesenchymal stem cells are well-known to exhibit prolifera-
elicit an immunological or clinically detectable foreign body reac- tion, self-renewal and multipotent differentiation capacities [47].
tion. In this study, rat osteoblast cells were cultured on four groups MSCs are able to differentiate into cell types of mesodermal ori-
of porous scaffolds for 1, 3, 5 days, respectively, after treatment of gin, such as osteoblasts, chondrocytes, adipocytes and muscle
the cell-scaffold constructs with the MTT solution, dark blue crys- cells [48]. For bone tissue engineering, porous composites scaf-
tals of formazan were seen, indicating the presence of metabolically folds, when combined with human bone mesenchymal stem cells
active cells. The MTT absorption was measured at 570 nm with a (hBMSC), demonstrate superior properties of osteoconduction and
background subtraction at 630 nm, a higher absorbance indicates osteoinduction [49–51]. Fig. 8 displayed the distinct morphologies
that there are either more cells or that they are metabolizing [43], of hBMSCs observed on scaffold after 4 days culture. The hBM-
the results were presented in Fig. 7. It showed that the MTT absorp- SCs were mostly polygonal and unevenly dispersed on the surface
tion of all samples increased with time indicating cell proliferation (Fig. 8A) and numerous filopodia-like structures anchored the cell
on the scaffold, in particular, the osteoblast cells exhibited an out- bodies and entered the pores (Fig. 8B). These results suggested
standing proliferation rate on BP20 scaffold than BP30, BP40 and that the hBMSCs possessed a favorable spreading morphology on

Fig. 8. hBMSC morphologies observed by SEM after 4 days on BCP/PVA scaffolds which the concentration of PVA was 30 wt%.
176 L. Nie et al. / Colloids and Surfaces B: Biointerfaces 100 (2012) 169–176

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