DSpace at VNU: Biomimetic scaffolds based on hydroxyapatite nanorod poly(D,L) lactic acid with their corresponding apatite-forming capability and biocompatibility for bone-tissue engineering

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Colloids and Surfaces B: Biointerfaces
journal homepage: www.elsevier.com/locate/colsurfb

Biomimetic scaffolds based on hydroxyapatite nanorod/poly(d,l)
lactic acid with their corresponding apatite-forming capability and
biocompatibility for bone-tissue engineering
Nguyen Kim Nga a,∗ , Tran Thanh Hoai a , Pham Hung Viet b
a
b

School of Chemical Engineering, Hanoi University of Science and Technology, 1 Dai Co Viet Road, Hanoi, Viet Nam
Research Center for Environmental Technology and Sustainable Development, Hanoi University of Science, 334 Nguyen Trai Street, Hanoi, Viet Nam

a r t i c l e

i n f o

Article history:
Received 14 October 2014
Received in revised form 27 February 2015
Accepted 1 March 2015

Available online xxx
Keywords:
Biomimetic scaffolds
Poly(d,l) lactic acid
Hydroxyapatite nanorods
Apatite
Biocompatibility
Bone tissue engineering

a b s t r a c t
This study presents a facile synthesis of biomimetic hydroxyapatite nanorod/poly(d,l) lactic acid
(HAp/PDLLA) scaffolds with the use of solvent casting combined with a salt-leaching technique for
bone-tissue engineering. Field emission scanning electron microscopy, Fourier transform infrared spectroscopy, and energy-dispersive X-ray spectroscopy were used to observe the morphologies, pore
structures of synthesized scaffolds, interactions between hydroxyapatite nanorods and poly(d,l) lactic
acid, as well as the compositions of the scaffolds, respectively. Porosity of the scaffolds was determined
using the liquid substitution method. Moreover, the apatite-forming capability of the scaffolds was
evaluated through simulated body fluid (SBF) incubation tests, whereas the viability, attachment, and
distribution of human osteoblast cells (MG 63 cell line) on the scaffolds were determined through alamarBlue assay and confocal laser microscopy after nuclear staining with 4 ,6-diamidino-2-phenylindole and
actin filaments of a cytoskeleton with Oregon Green 488 phalloidin. Results showed that hydroxyapatite
nanorod/poly(d,l) lactic acid scaffolds that mimic the structure of natural bone were successfully produced. These scaffolds possessed macropore networks with high porosity (80–84%) and mean pore sizes
ranging 117–183 ␮m. These scaffolds demonstrated excellent apatite-forming capabilities. The rapid formation of bone-like apatites with flower-like morphology was observed after 7 days of incubation in SBFs.
The scaffolds that had a high percentage (30 wt.%) of hydroxyapatite demonstrated better cell adhesion,
proliferation, and distribution than those with low percentages of hydroxyapatite as the days of culture
increased. This work presented an efficient route for developing biomimetic composite scaffolds, which
have potential applications in bone-tissue engineering.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction
Bone repair and regeneration have become a serious challenge in orthopedic surgery because of the increase in clinical
bone diseases (e.g., bone infections, bone tumors, and bone loss

through trauma) [1]. Current therapies for bone defects include
autografts, allografts, and other bone substitutes [2]. Autografts
(bones obtained from another anatomical site in the same subject) comprise the gold standard for the treatment of bone defects,
but this surgical method still has major disadvantages, such as
the possibility of donor site morbidity, shortage of donor bone
supply, anatomical and structural complications, as well as graft

∗ Corresponding author. Tel.: +84 4 38680 110; fax: +84 4 38680 070.
E-mail address: (N.K. Nga).

sorption [1]. Allografts (bones from donors or another species) can
be used as an alternative; however, this method causes inherent
problems (e.g., disease transmission and immunogenic responses)
[3]. Synthetic bone substitutes, which are mostly made of metals or
bioceramics and glasses, have osteoconductive properties instead
of osteoinductive properties, thus limiting their use [4]. Bone-tissue
engineering (BTE) has attracted scientific attention for being more
effective than conventional methods in terms of bone repair and
reconstruction. The core of BTE is to combine a biodegradable
matrix (scaffold) and living cells to grow tissue in vitro prior to
implantation of the subject [5]. Scaffolds for BTE should possess
several properties, such as high porosity, a macroporous network
for in vitro cell migration, adhesion, proliferation, and further tissue
growth, biocompatibility and biodegradability to non-toxic products, as well as sufficient mechanical strength [1]. To achieve these
properties, BTE scaffolds are often designed to mimic the structural

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and biological functions of a naturally occurring extracellular
matrix (ECM) in terms of both chemical composition and physical
structure [6,7].
In this regard, biodegradable polymers, such as poly(l-lacticco-glycolic acid) [8], poly(␧-caprolactone) [9], poly(l-lactic acid)
[10], and poly(d,l-lactic acid) (PDLLA) [11], have been processed
into three-dimensional (3D) scaffolds. These polymers show good
mechanical properties, with shapes and degradation rates that
are easily adjustable. The main disadvantage of these polymeric
materials is their pure biocompatibility, given that they do not
provide a favorable surface for cell attachment and proliferation because of the lack of specific cell-recognizable signals [1].
Bioactive inorganic materials, such as hydroxyapatite (HAp) [12],
␤-tricalcium phosphate [13,14], and bioactive glasses [15,16], have
been designed as 3D porous scaffolds for bone regeneration. The
advantages of bioactive ceramics are their excellent osteoconductivity and biocompatibility; however, their inherent brittleness and
low mechanical strength (for porous specimens) are major disadvantages in developing 3D scaffolds, thus limiting their applications
[13,15,17].
Polymer/bioceramic composite scaffolds are attracting increasing attention in the field of bone regeneration because of their
mechanical stability and biocompatibility [18,19]. Among such
scaffolds, HAp/polymer composites have received considerable
interest because of their composition and structural similarity to
natural bone [20,21]. Bone is a complex composite that comprises

an organic phase (90% type I collagen, other non-collagenous proteins (e.g., proteoglycans)), minor amounts of lipids and osteogenic
factors (e.g., bone morphogenetic proteins), and a mineral phase
[22]. The mineral phase consists of one or more types of calcium phosphates comprising 65–70% of bone and embedded in
a protein matrix [22–24]. Among the CaP salts, hydroxyapatite
(Ca10 (PO4 )6 (OH)2 , HAp) is the most stable calcium phosphate in
body fluids and is the most similar to the mineral part of bone [23].
The biological HAps found in physiological hard tissues are irregularly rod-like or plate-like crystals of variable lengths and widths
(30–45 nm) with a thickness of approximately 5 nm [22]. These
HAps are oriented to the c-axis, which is parallel to the collagen
fibrils [22,25] in which different cell types, including osteoblasts,
osteoclasts, and osteocytes, reside. A survey of the literature shows
that bioceramics that mimic bone mineral in terms of composition, structure, and morphology can promote osteointegration and
subsequent bone tissue formation [26–28].
PDLLA is a nontoxic, biocompatible, and biodegradable material that has been used as sutures and tissue-engineering scaffolds
[29]. However, PDLLA presents strong hydrophobicity owing to
the absence of hydrophilic groups and suitable functional groups
(e.g., NH2 , OH), which results in the absence of osteoconductivity. A recent study focused on fabricating bioactive nanocomposite
PDLLA/nano-HAp membranes using an electrospinning method to
improve the osteoconductivity of the polymer [30]. However, the
products are characterized by small pore sizes with a mean diameter of 4.8 ␮m and are thus unsuitable for use as BTE scaffolds,
given that the minimum pore size requirement for bone scaffolds
is 100 ␮m [31].
Several methods have been developed to fabricate 3D scaffolds.
These methods include fiber bonding, freeze drying, phase separation, super-critical fluid technology, solvent casting combined with
particulate leaching, and melt molding [1]. Among these methods,
solvent casting combined with particulate leaching has been widely
used for fabricating 3D scaffolds because of its simplicity and efficiency. This method allows to produce highly porous scaffolds with
porosity up to 93% and mean pore diameter up to 500 ␮m by varying porogen particles size, and weight ratio of polymer to porogen
without the need of the specialized equipment [1,32]. As a result,
in this study, solvent casting combined with particulate-leaching

method has been used as an efficient control to synthesize 3D
HAp nanorod/poly(d,l) lactic acid (HAp/PDLLA) scaffolds. The rodshaped HAp nanoparticles with the same size as bone minerals
were successfully prepared in past studies [33,34] and have since
been used as an inorganic phase to incorporate into the PDLLA
matrix and to prepare biomimetic HAp/PDLLA scaffolds for BTE.
The apatite-forming capability of the scaffolds was determined by
assessing the formation of bone-like apatite on the surface of the
scaffolds after immersing in simulated body fluids (SBFs). Meanwhile, biocompatibility was investigated in direct contact with
human osteoblast cell line MG 63 through in vitro tests.
2. Experimental
2.1. Chemicals
All reagents were of analytical grade and used as received without further purification. Calcium chloride dihydrate (CaCl2 ·2H2 O),
sodium monophosphate dihydrate (Na2 HPO4 ·2H2 O), NaOH,
C2 H5 OH, 1,4-dioxane, hydrochloric acid (HCl), pluronic co-polymer
PEO20 –PPO70 –PEO20 (P123), NaCl, NaHCO3 , KCl, K2 HPO4 ·3H2 O,
MgCl2 ·6H2 O,
Na2 SO4 ,
Tris-hydroxymethyl
aminomethane
((HOCH2 )3 CNH2 ), phosphate buffered saline (PBS) were obtained
from Sigma–Aldrich. PDLLA was purchased from Boehringer
Ingelheim (Ingelheim, Germany). Deionized water was used to
prepare all solutions and reagents.
2.2. Scaffold production
The HAp nanorods used in this study were prepared using
the hydrothermal method assisted by a pluronic co-polymer
PEO20 –PPO70 –PEO20 . The preparation and characterizations of
these HAp nanorods were conducted in the same manner as in our
previous work [33]. HAp nanorod/PDLLA scaffolds were then prepared using solvent casting combined with salt-leaching method

with NaCl as the porogen. A 7% polymer solution was produced by
dissolving 0.455 g of PDLLA in 6.5 mL of 1,4-dioxane for 3 h at 32 ◦ C.
A suspension of C2 H5 OH with various amounts of HAp nanorods
(0–30 wt.% HAp to PDLLA) was added dropwise into the PDLLA
solution. The resulting mixture was vigorously stirred on a magnetic stirrer at a speed of 500 rpm for 2 h to achieve homogeneity.
The homogeneous mixture was then cast into a 55 mm glass Petri
dish containing 9 g of NaCl with particle sizes of 300–450 ␮m. The
samples were then air-dried under a chemical hood for 24 h and
vacuum-dried for another 24 h to remove all solvents. The resulting scaffolds were then immersed in distilled water at 35 ◦ C for 3 d
(water was changed 3–4 times each day) to leach out the salt. The
produced HAp/PDLLA scaffolds were further air-dried and vacuumdried and then stored in a desiccator until use. Finally, four scaffolds
were obtained according to the percentage of HAp nanorods. These
scaffolds were labeled as S1, S2, S3, and S4 for 0 wt.% HAp, 10 wt.%
HAp, 20 wt.% HAp, and 30 wt.% HAp, respectively.
2.3. Scaffold characterization
The morphologies and pore structures of the produced scaffolds
were examined using field emission scanning electron microscopy
(FE-SEM) (Supra 40, Zeiss, Germany) at low (200×) and high (500×,
1000× and 5000×) magnifications, as well as optical microscopy
(SMZ800, Nikon, Japan). Prior to FE-SEM observation, the dry samples of the scaffolds were cut into small disks, which were mounted
on an SEM stub and then sputter-coated with a thin chromium layer
(Quorumtech, Q150T ES Turbo Chromium Sputter-Evaporator). The
mean pore diameter and pore wall thickness of the scaffolds were
measured by using scanning electron microscopic (SEM) images
through ImageJ software. The Fourier transform infrared spectra

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(FTIR) of the scaffolds were recorded on a Nicolet 6700 spectrometer using KBr pellet technique in the range of 4000–400 cm−1
with a resolution of 4 cm−1 . All measurements were performed
at 25 ◦ C. The composition of HAp nanoparticles on the surface of
HAp/PDLLA scaffolds was examined through energy-dispersive Xray spectroscopy (EDXS) (Nova NanoSEM 450, FEI).
The porosity of the HAp/PDLLA scaffolds was measured using
the liquid substitution method [35]. Distilled water was used as
the displacement liquid. In brief, a dry sample of each scaffold
was weighed and then immersed in a graduated cylinder containing a known volume V1 of water and left to stand to enable
the water to penetrate into the pores of the scaffold sample
until no air bubbles emerged from the scaffold. The total volume of water and water-impregnated scaffold was recorded as
V2 . The volume difference (V2 − V1 ) represents the volume of the
HAp/PDLLA scaffold skeleton. The water-impregnated scaffold was
then removed from the graduated cylinder, and the residual water
volume was recorded as V3 . The total volume of the scaffold is given
by V = (V2 − V1 ) + (V1 − V3 ) = (V2 − V3 ). By measuring the initial and
final weights Wi and Wf of the scaffolds before and after immersing
in water, we can calculate the pore volume of the scaffold as
Wf − Wi
H2 O

.

(1)

The porosity of the scaffold can be calculated using the following
equation:
Porosity =

(Wf − Wi )/

H2 O

V2 − V3

(2)

At least five measurements were conducted for each scaffold,
and the results were averaged from these five measurements.
2.4. In vitro mineralization tests
In vitro mineralization tests were conducted through the incubation of the HAp/PDLLA scaffolds in SBFs. SBF was prepared as
described previously [36], filtered using a 0.22 ␮m Millipore filter
system to eliminate bacterial contamination, and then stored in
a plastic bottle in a refrigerator at 4 ◦ C. After disinfection in 70%
ethanol at 4 ◦ C, three samples of each scaffold were immersed in
30 mL of SBF solution placed in closed polyethylene containers
at 37 ◦ C. After being immersed in SBF for the designed days, the
samples were removed, gently rinsed with deionized water, and
dried under warm flowing air. The rinsing process has been used to
remove the residual ions of the SBF solutions that remained on the
scaffold samples and could affect the scaffold structure. All operations were conducted in a laminar airflow hood to avoid bacterial
infection. The apatite-forming capability of HAp/PDLLA scaffolds
was assessed through FE-SEM and EDXS.
2.5. In vitro cell culture experiments
Human osteoblast cells (MG 63 cell line, IZSLER Biobanking of

Veterinary Resources, Brescia, Italy) were used to evaluate biocompatibility of the prepared HAp/PDLLA scaffolds. Cells were grown
in a minimum essential medium (Gibco) supplemented with 10%
(v/v) fetal bovine serum (Gibco), 2% (v/v) l-glutamine (Gibco),
1% (v/v) antibiotic, 1% (v/v) sodium pyruvate, and 1% (v/v) nonessential amino acids (Gibco) at 37 ◦ C and 5% CO2 . The medium
was changed every 2 d. The adherent cells were allowed to reach
confluence, then detached with 0.1% trypsin ethylenediaminetetraacetic acid, and suspended in a fresh culture medium for the
experiments.
Prior to cell seeding, the prepared HAp/PDLLA scaffold samples
with a diameter of 8 mm were sterilized in 70% ethanol at 4 ◦ C for
24 h, washed with sterile distilled water, and immersed in a culture

3

medium for 1 h before seeding. Thereafter, the samples were placed
on 48-well plates. 20 ␮L of cell suspension at a density of 7.5 × 104
cells per sample was seeded on scaffold samples. The samples were
then incubated at 37 ◦ C for 2 h to allow cell attachment to the scaffold surfaces, followed by adding 250 ␮L of culture medium into
each well. Culture plates were then transferred to an incubator at
37 ◦ C and 5% CO2 . Culture times were 3, 5, and 7 d, and the fresh
culture medium was changed every 2 d.
Cell viability and proliferation were determined after each incubation time using alamarBlue assay. The assays were performed
after 3, 5, and 7 d of cell seeding, according to the manufacturer’s
protocol. Briefly, at the end of each incubation time, the culture
medium was removed from the wells and fresh culture medium
with 10% (v/v) alamarBlue was added to each well. After 4 h of
incubation at 37 ◦ C, aliquots of 100 ␮L were pipetted into 96-well
plates, and fluorescence was recorded on a microplate reader at an
excitation wavelength of 565 nm and an emission wavelength of
595 nm.
Cell morphology, growth, and distribution were visualized

after staining with Oregon Green 488 phalloidin (Life Technologies; Carlsbad, CA, USA) and 4 ,6-diamidino-2-phenylindole (DAPI,
Sigma–Aldrich). Cells were fixed with 4% (w/v) formaldehyde in
PBS, permeabilized with 0.2% Triton X in PBS, and stained with
Oregon Green 488 and DAPI, according to the manufacturer’s protocol. After three rinses with PBS, the samples were examined using
confocal laser microscopy.
2.6. Statistical analysis
Results were averaged and expressed as mean ± standard deviation. Statistical analysis was performed using ANOVA. A value of
P < 0.05 was considered statistically significant.
3. Results and discussion
3.1. Characterization of the synthesized scaffolds
Four HAp/PDLLA scaffolds were synthesized with different
weight percentages of HAp (one scaffold without HAp filling and the
other three scaffolds with HAp filling). The compositions and other
characteristics of the produced scaffolds are summarized in Table 1.
Fig. I1 (supporting material) show digital camera images of the scaffold samples (S3 and S4 scaffolds) and the optical images of a typical
S3 scaffold sample (Fig. I2). These scaffolds have stable shapes,
thicknesses of 2 mm (Fig. I1), and porous structures (Fig. I2). Surface
morphologies and pore structures were then examined through
FE-SEM imaging. The SEM images of all synthesized scaffolds are
presented in Fig. 1 and Fig. I3. Observations show that the surface
morphologies of the scaffolds become coarser as the HAp content increases. A smooth surface morphology was produced for S1
PDLLA scaffolds (Fig. 1a and I3A), whereas coarse surface morphologies were obtained for HAp/PDLLA scaffolds. HAp nanoparticles
were homogeneously deposited within the pore walls of the scaffolds (Fig. 1b–d). FE-SEM images with high magnifications (Fig.
I3B–D) indicated that no aggregates of nanoparticles appeared on
the pore walls for all composite scaffolds. Moreover, thin pore walls
of 1.48 ± 0.28 ␮m were observed (Fig. I3B) for S2 scaffold with a
low HAp percentage of 10 wt.%. A further increase in HAp percentage produced scaffolds with thicker pore walls of 1.81 ± 0.3 and
3.94 ± 0.63 ␮m (Fig. I3C and D) for S3 and S4 scaffolds, respectively.
Fig. I3E (FE-SEM image of a higher magnification for S4 scaffold)
showed that tiny rod-like HAp particles were embedded on the

polymer phase. According to Table 1, increasing HAp percentage
resulted in a decrease in pore sizes of the produced scaffolds. Furthermore, statistical analysis and pore size distribution of S1 PDLLA

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Table 1
Composition and some characteristics of HAp/PDLLA composite scaffolds.
Sample codes

HAp massa (g)

PDLLA mass (g)

Weight percent (%) (HAp/PDLLA)

Pore sizesb (␮m)

S1
S2
S3

S4

0
0.0455
0.091
0.136

0.455
0.455
0.455
0.455

0
10
20
30

274
183
122
117

a
b
c

±
±
±
±

76
60
58
60

Porosityc (%)
89.39
84.46
83.37
80.18

±
±
±
±

2.86
2.17
1.81
0.78

Actual amount of HAp in total PDLLA.
Mean value ± standard deviation (SD); n = 30.
Mean value ± standard deviation; n = 5.

scaffolds (Fig. I4A) demonstrate that these scaffolds have pore sizes,
which are significantly larger than those of the HAp/PDLLA scaffolds (I P < 0.001) and are mainly in the range of 100–450 ␮m with a
mean pore size of 274 ␮m. S2 and S3 HAp/PDLLA scaffolds have
mean pore sizes of 183 and 122 ␮m (Table 1) within the range

of 100–350 ␮m and 50–300 ␮m, respectively (Fig. I4B and C); differences in their pore sizes are statistically significant (I P < 0.001).
S4 HAp/PDLLA scaffolds have the smallest pore sizes with a mean
pore size of 117 ␮m (Table 1). However, statistical analysis showed
that pore sizes of S4 HAp/PDLLA scaffolds are significantly smaller
than those of S1 PDLLA and S2 HAp/PDLLA scaffolds (I P < 0.001),
but not significantly smaller than those of S3 HAp/PDLLA scaffolds
(P > 0.05). The obtained results confirm that HAp content greatly
affects the pore structure and overall morphology of the composite
scaffolds. Higher HAp content resulted in the production of scaffolds with small pore sizes and thick pore walls. The past studies
demonstrated that pores in scaffolds play an important role in bone
tissue formation. Large pores (100–150 and 150–200 ␮m) showed
substantial bone ingrowth, while pores (75–100 ␮m) resulted in
ingrowth of unmineralized osteoid tissue, and smaller pores (10–44
and 44–75 ␮m) were penetrated only by fibrous tissues [37,38]. Our
results indicated that the produced composite scaffolds have mean
pore sizes of 117–183 ␮m, which is believed to be suitable for BTE
scaffolds.
To evaluate the interaction between PDLLA matrix and the
inorganic phase, FTIR spectra of nano-HAp (a), typical HAp/PDLLA

scaffolds (b and c), and PDLLA scaffolds (d) are presented in Fig. 2A.
The spectra of the HAp/PDLLA scaffolds do not exhibit significant
differences, which reveal the presence of HAp on the composite scaffolds. In fact, the stretching and bending vibrations of the
PO4 3− groups for HAp are visible at 1090, 1030, 953, 602, 561, and
469 cm−1 in the spectra of the HAp/PDLLA scaffolds. However, these
vibrations are detected at 1095, 1032, 955, 604, 563, and 471 cm−1
in the spectrum of the HAp sample. A strong band at 1749 cm−1
can be observed in the spectrum of S1 PDLLA scaffold. This band
is assigned to the vibration of the carbonyl group (C O) of PDLLA.
However, this band shifts to 1754 and 1752 cm−1 for S3 and S4

HAp/PDLLA scaffolds, respectively. In addition, the characteristic
peaks for C H vibrations were detected at 2999 and 2951 cm−1
for the S1 PDLLA scaffold, but observed at 2996 and 2946 cm−1
and at 2998 and 2948 cm−1 for S3 and S4 HAp/PDLLA scaffolds.
These results reveal that some molecular interactions between
HAp nanoparticles and PDLLA in the composite scaffolds may have
occurred. Moreover, the weak peak at 3569 cm−1 is characteristic
of the vibration of the OH group of HAp nanoparticles. However,
this peak appeared at a lower region at 3501 and 3496 cm−1 for the
S3 and S4 scaffolds, respectively. This result suggests that hydrogen
bond was formed between the OH group of HAp and the C O group
of PDLLA, thus making the HAp nanoparticles stable in the polymer
matrix.
The presence of HAp on the surface of the HAp/PDLLA scaffolds was verified by EDXS analysis. The EDXS spectra and

Fig. 1. FE-SEM images of scaffolds, synthesized at different percentages of HAp to PDLLA: (a) S1 PDLLA scaffold, (b) S2 HAp/PDLLA scaffold, (c) S3 HAp/PDLLA scaffold, and
(d) S4 HAp/PDLLA scaffold at low magnification of (200×). The insets represent FE-SEM images at higher magnification of (500×).

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3.2. Apatite-forming capability

Fig. 2. (A) FTIR spectra of (a) nano-HAp powder, (b) S3 HAp/PDLLA scaffold, (c)
S4 HAp/PDLLA scaffold, and (d) S1 PDLLA scaffold and (B) EDXS spectrum of S4
HAp/PDLLA scaffold.

components of a typical S4 HAp/PDLLA scaffold are illustrated
in Fig. 2B. Three elements, namely, O, P, and Ca, are the
major constituents of the synthesized HAp/PDLLA scaffold with
45.03 ± 1.63 at.%, 5.64 ± 0.47 at.%, and 9.58 ± 0.52 at.%, respectively.
A trace of Cl (0.71 ± 0.06 at.%) was detected, which could be
attributed to a residue of the synthesis reaction, and was incompletely eliminated from the scaffold sample through washing.
Moreover, the presence of C in the scaffold samples can possibly be
attributed to the use of carbon adhesive tape to mount the samples
or the presence of C in the polymer matrix. Since delicate particles
could easily be affected, therefore carbon adhesive tape could affect
the scaffold morphology. However, the Ca/P ratio was 1.69, which
was close to the stoichiometric value of 1.67. This result confirms
that the HAp nanoparticles were successfully incorporated into the
scaffolds.
The porosity data of the synthesized scaffolds are shown in
Table 1. The experimental results indicate that the porosity changes
linearly with the increase in HAp content and exhibits a downward
trend with HAp loading. Within the HAp content range studied, the
porosity values varied from 89.39 ± 2.86% to 80.18 ± 0.78%, which
show relatively high porosity for all produced scaffolds. The high
porosity levels of the synthesized scaffolds suggested that these
scaffolds would be beneficial for in vitro cell adhesion, ingrowth,
and survival.

An important characteristic of the scaffolds is their capability to
form an apatite layer on their surfaces. Fig. 3 presents the surface
morphologies of typical HAp/PDLLA scaffolds after being immersed
in SBF for 5 and 7 d. The FE-SEM image of S3 HAp/PDLLA scaffold
with low magnification (Fig. 3a) shows that a mineral layer was
already formed. This layer covered the scaffold surface after 5 d
of soaking in SBF. The image with higher magnification (Fig. 3b)
confirms that the mineral layer consisted of aggregated flower-like
particles with a mean diameter of 1.08 ␮m. These particles were
formed on the surface and inside the scaffold pores. The growth of
the flower-like particles was observed when the soaking time was
prolonged to 7 d. As shown in the low-resolution FE-SEM image in
Fig. 3c, the particles grew larger (their mean diameter increased to
1.81 ␮m). The FE-SEM image with a high resolution (Fig. 3d) indicates the formation of the flower-like mineral layer consisting of
tiny needle-like crystals on the scaffold surface after 7 d of soaking
in SBF. The surface morphologies of S4 HAp/PDLLA scaffold after 7 d
of soaking in SBF are similar to those of the S3 HAp/PDLLA scaffold.
However, the intense formation of such particles was observed on
the entire surface of the S4 HAp/PDLLA scaffold (Fig. 3e). The highresolution FE-SEM image (Fig. 3f) showed that a rose-like mineral
layer was exclusively produced for the S4 HAp/PDLLA scaffold. This
mineral layer morphology is typical for bone-like apatite [36,39].
The obtained results confirm the complete formation of the mineral layer on the surfaces of both S3 and S4 HAp/PDLLA scaffolds
after 7 d of soaking in SBF. To explore the effect of HAp nanoparticles on the apatite-forming capability of the composite scaffolds,
in vitro mineralization experiments of pure PDLLA and HAp/PDLLA
scaffolds with 10 wt.% of HAp were examined. Fig. I5 shows the
FE-SEM images of the S1 PDLLA scaffold after being immersed in
SBF for 5 and 7 d and that of S2 HAp/PDLLA scaffold after 7 d. Only
several spherical-like mineral particles formed on the surface of
the S1 PDLLA scaffold after 5 d of soaking in SBF (Fig. I5A). The
number of the mineral particles increased after 7 d in SBF, and

some bundles of aggregated mineral-like crystals (Fig. I5B) were
found on the scaffold surface because of the conjunction of such
particles, indicating poor mineralization-forming capability. The
addition of HAp at 10 wt.% to the PDLLA scaffold almost did not
improve the mineralization-forming capability of the composite
scaffolds. The FE-SEM image in Fig. I5C showed that the severe
aggregation of mineral-like crystals was observed on the surface
of the S2 HAp/PDLLA scaffold after being immersed in SBF for 7 d.
Meanwhile, the HAp/PDLLA scaffolds with high HAp amounts (20
and 30 wt.%) already showed full coverage by the mineral layer with
flower-like morphology on their surfaces after 7 d, which revealed
a significantly higher in vitro mineralization response than those of
pure PDLLA scaffolds and scaffolds with 10 wt.% of HAp. Mineralization involves the nucleation and growth of bone-like apatite onto
biomaterials, which is associated with uptake of calcium and phosphate ions from the physiological environment. The HAp/PDLLA
scaffolds with high HAp percentages provide more nucleation sites
(Ca2+ ) for apatite formation than the same composite scaffolds with
low HAp percentages (e.g., 10 wt.% HAp) and accordingly demonstrate better in vitro mineralization. Our results indicate that the
HAp content in the scaffolds significantly affects the induction of
the mineral layer on their surfaces. HAp content of 20–30 wt.%,
which is relative to PDLLA is optimal for producing HAp/PDLLA
scaffolds with high in vitro mineralization.
The chemical composition of the released mineral layer was
further analyzed using EDXS method. EDXS analyses were conducted for the typical composite scaffolds after being soaked in
SBF. Table I1 presents the elemental compositions of the mineral
layers released on S3 HAp/PDLLA scaffold after being soaked in SBF
for 5 and 7 d. Ca, P, and O are three main elements found in the

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Fig. 3. FE-SEM images of representative HAp/PDLLA composite scaffolds after immersion in SBF: (a and b) at 5 d with magnifications of (5000×) and (10,000×), and (c and
d) at 7 d with magnifications of (5000×) and (50,000×) for S3 HAp/PDLLA scaffold; (e and f) at 7 d with magnifications of (5000×) and (50,000×) for S4 HAp/PDLLA scaffold.

released mineral layers after soaking in SBF for 5 and 7 d. However, Na and Cl were detected as trace elements (for the mineral
layer, released after 5 d in SBF), which was attributed to the residue
from the reaction synthesis of the scaffolds. According to Table
I1, the Ca/P ratios were 1.24 and 1.55 for the mineral layers produced on the scaffold after 5 and 7 d of soaking in SBF, respectively.
Apart from apatite mineral, a number of other calcium phosphate
minerals (e.g., amorphous calcium phosphate, dicalcium phosphate
dihydrate, octacalcium phosphate, tricalcium phosphate, as well as
␣- and ␤-Ca3 (PO4 )2 ) can be produced under the same in vitro conditions that form apatite. The results suggest that after 7 d in SBF a
new bone-like apatite phase was already produced on the S3 scaffold, but after 5 d in SBF dicalcium phosphate and/or octacalcium
phosphate as intermediate phases were probably released on the
scaffold. Moreover, the Ca/P ratio of the produced mineral layer was
1.55, which was lower than the stoichiometric value of 1.67, and
was attributed to the calcium-deficient carbonated hydroxyapatite,
which suggests that the formed apatite is carbonated.
Table I2 compares the apatite-forming capability of the
HAp/PDLLA scaffolds as synthesized in this study with other
composite materials reported in previous studies. The complete
formation of the bone-like apatite layer was observed after 7 d of
incubation in SBF for the HAp/PDLLA scaffolds in this study. However, the same layer was obtained after a longer time (21 and 14

d) for the nano-HAp/PDLLA films [40] and Akermanite/PDLLA scaffolds [41], respectively. Moreover, the bone-like apatite layer with
flower-like morphology was obtained for the HAp/PDLLA scaffolds
synthesized in this study (Fig. 3e and f). Meanwhile, the bone-like
apatite layer formed on nano-HAp/PDLLA films consisted of numerous uniform-orbicular aggregates, and a worm-like morphology
was produced on Akermanite/PDLLA scaffolds, which significantly
differ from those released on the HAp/PDLLA scaffolds in this study.
The comparison revealed that the HAp/PDLLA scaffolds demonstrated better in vitro mineralization than the other composite
materials, which could be attributed to the similarity (e.g., in
morphology and composition) of the inorganic component of the
scaffolds to that of natural bone. As shown in Table I2, the inorganic
component of the HAp/PDLLA scaffolds in this study is composed
of rod-shaped HAp particles with diameter ranging from 25 to
30 nm and length ranging from 100 to 130 nm, which is similar
to the values for bone minerals. The inorganic components of the
other materials exhibit morphological and compositional differences from those of the HAp/PDLLA scaffolds and bone minerals
(see Table I2). Based on the comparison of the in vitro mineralization in the present and previous studies, the morphology and
composition of the inorganic part of the scaffolds have a crucial
function in their bioactivity and in promoting bone-like apatite
formation.

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Fig. 4. Cell viability and proliferation on the scaffolds after 3, 5, and 7 d of culture
through alamarBlue assay. All data are expressed as means ± SD; n = 6. *P < 0.001
(data compared with those at longer culture time, and with S1 PDLLA scaffold),
**P < 0.01 and ***P < 0.05 (data compared with S1 PDLLA).

3.3. In vitro biocompatibility
Cell–scaffold interactions are the basis of initial cell attachment
and influence cell phenotypes and functions. In this study, in vitro
cell culture tests were conducted to evaluate biocompatibility of
the synthesized scaffolds in terms of cell viability, proliferation, and
attachment.
Fig. 4 shows results of the alamarBlue assay, which was performed after MG 63 cells cultured on the scaffolds and the cell
culture plates (served as the control group) for 3, 5, and 7 d. The
alamarBlue reduction indicated the viability and proliferation of
MG 63 cells. Fig. 4 shows no significant difference in the cell viability between all scaffold groups and the control group after 3 d of
culture. Statistical analysis also showed that differences in the cell
viability among all groups were not significant after 3 d (P > 0.05).
As shown in Fig. 4, the fluorescence values for all scaffold groups
(at higher levels than those of the control groups) increased as
the culture time increased to 5 and 7 d, which revealed that the
cell viability of the scaffolds significantly increased within this culture duration (*P < 0.001). At 5 d, the cell viability on S2 HAp/PDLLA
scaffold was significantly higher than that on pure PDLLA scaffold
(**P < 0.01), but the cell viability for S3 and S4 HAp/PDLLA scaffolds
showed no significant difference with that of pure PDLLA scaffold.
At 7 d, the cell viability for all HAp/PDLLA scaffolds became significantly higher than that of pure PDLLA scaffold (*P < 0.001). The S2
HAp/PDLLA scaffolds (with 10 wt.% HAp) demonstrated better cell
viability than the other scaffolds for different culture times (5 and
7 d) (***P < 0.05), whereas the cell viability for S3 and S4 scaffolds
was comparable for all the culture times (Fig. 4).

Previous studies proved that small pores might prevent cellular
penetration and migration within scaffolds [31]. Among the scaffolds studied, S1 PDLLA scaffold was characterized by the largest
pore sizes, but the cell viability of S1 PDLLA scaffold was lower
than that of the HAp/PDLLA scaffolds. This result can be attributed
to the absence of the HAp component in the composition of S1
PDLLA scaffold. As a result, the surface of S1 PDLLA scaffold is
lacking in functional groups as cell-recognition signals (e.g., OH
groups) and does not support that attachment of many cells to the
PDLLA scaffold, even if many MG 63 cells can migrate to the scaffold. Among the three composite scaffolds, S2 HAp/PDLLA scaffolds
present higher cell viability than the other two scaffolds, which
may be attributed to the fact that they possess larger pore sizes

7

(mean pore size of 183 ␮m) than S3 and S4 scaffolds (mean pore
sizes of 122 and 117 ␮m, respectively). This result probably caused
more beneficial cell migration and higher cell viability on the S2
scaffolds than on the S3 and S4 scaffolds. The results of the alamarBlue assay indicate that all the scaffolds exhibit good cell viability,
and the addition of HAp nanorods to the PDLLA scaffold significantly enhanced the viability of MG 63 cells. However, the high
content of HAp in the scaffolds may inhibit the increase in cell
viability.
Confocal laser microscopy was used to examine cell attachment
and distribution. Fig. I6 and Fig. 5 show the confocal laser scanning
microscopy images of MG 63 cells cultured on the scaffolds after 3,
5, and 7 d. The staining methods for nuclei by DAPI and for actin filaments by Oregon Green 488 phalloidin indicate the cell attachment
and cytoskeleton distribution on the surfaces of scaffolds. In fact,
few cells could be observed on the S1 PDLLA and S2 HAp/PDLLA
scaffolds at 3 d (Fig. I6A and B). Meanwhile, qualitatively higher
MG 63 cells were observed on the S3 HAp/PDLLA and S4 HAp/PDLLA
scaffolds (Fig. I6C and D). Additionally, the anchorage of the cellular

cytoskeleton to the scaffolds was observed at 3 d, revealing that cell
attachment occurred on these scaffolds early in the culture time. A
further increase in culture time to 5 and 7 d resulted in a significant
increase in the number of cells for all scaffolds (Fig. 5). At 5 d, all scaffolds showed a cytoskeleton arrangement of MG 63 cells, which was
attached to the scaffold surfaces (Fig. 5a, c, e and g). Among them,
the S2 HAp/PDLLA scaffold demonstrated a uniform cytoskeleton
arrangement with a high density of MG 63 cells interconnecting
the entire scaffold at 5 d, but the corresponding cytoskeleton distribution was gradually lost at 7 d (Fig. 5d). The S1 PDLLA scaffold
exhibited a relatively uniform cytoskeleton distribution at 5 d, however, such a cytoskeleton arrangement in the S1 scaffold was lost
at 7 d (Fig. 5b). The results suggested that HAp nanorods may have
an effect on the cell attachment in these scaffolds. The S1 and S2
scaffolds with small HAp percentages (0–10 wt.% HAp) in their composition probably did not support the formation of high amount of
stress fibers in these scaffolds, which maintain actin bundles and
focal contacts between MG 63 cells and the scaffolds for a longer
culture time. This resulted in weaker cell attachment and gradual
loss of cytoskeleton organization in S1 and S2 scaffolds at 7 d. By
contrast, a more extended cytoskeleton network was observed in
the S3 HAp/PDLLA (Fig. 5f) and S4 HAp/PDLLA scaffolds (Fig. 5h)
at 7 d than at 5 d. These observations confirm that high HAp percentages (20–30 wt.% HAp) enabled S3 and S4 scaffolds to produce
higher amount of stress fibers, which led to stronger cell attachment and the extended cytoskeleton organization in these scaffolds
with increasing culture time to 7 d. Moreover, a well-organized
cytoskeleton network with a large number of adhered cells was
exclusively produced in the S4 HAp/PDLLA scaffold at 7 d, which
was attributed to the highest HAp percentage in the S4 scaffold
composition among the scaffolds studied. The results revealed that
all the scaffolds possess good cell adhesion, proliferation, and distribution. A high percentage of HAp in the composite scaffolds should
be helpful in supporting cell adhesion, proliferation, and distribution for longer culture periods.
The alamarBlue and confocal laser microscopy results demonstrated that all the scaffolds showed good MG 63 cell affinity
in terms of cell viability, proliferation, and adhesion. The S4
HAp/PDLLA scaffolds have the inorganic component consisting of

HAp nanorods with a mean diameter of 28 nm and a mean length
of 120 nm [33] and comprise 30 wt.% of the inorganic component
to the polymer, which are the closest similarity to natural bone
among the scaffolds studied. As a result, the S4 scaffolds showed
higher biocompatibility (better cell attachment and well-organized
cytoskeleton arrangement), compared to the other scaffolds with
lower HAp content with increasing number of culture days. The
S4 HAp/PDLLA scaffolds thus are promising candidates that will

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Fig. 5. Confocal laser scanning microscopy images of MG 63 cells stained nuclei with DAPI (blue) and actin filaments of cytoskeleton with Oregon Green 488 phalloidin
(green) after 5 and 7 d of seeding on (a and b) S1 PDLLA scaffold, (c and d) S2 HAp/PDLLA scaffold, (e and f) S3 HAp/PDLLA scaffold, and (g and h) S4 HAp/PDLLA scaffold. Scale
bars represent 100 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

promote production and organization of ECM with mineralization
and expression of osteopontin, collagen type I, bone sialoprotein,
which are typically expressed in native bone and will be further
investigated in the next phase of our work.

4. Conclusion

This study demonstrated that biomimetic 3D hydroxyapatite nanorod/poly(d,l) lactic acid scaffolds were successfully

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synthesized using solvent casting combined with salt-leaching
technique. The synthesized scaffolds are porous, have thicknesses of 2 mm, macropore networks with mean pore sizes of
117–183 ␮m, and high porosity. Both in vitro mineralization and
in vitro cell culture tests showed that the composite scaffolds with
high HAp contents, which have a structure that is the closest to that
of natural bone, induced the rapid formation of bone-like apatite
after a quick soaking time in SBF and demonstrated better cell
adhesion, proliferation, and distribution with increasing culture
days. Within this study, it is concluded that HAp/PDLLA scaffolds
with high HAp percentages are potential biomaterials to be used as
BTE scaffolds for further studies that will be aimed at performing
long-term in vitro cell culture experiments, degradation studies, and deeper characterizations (mechanical strength, surface
roughness).
Acknowledgments
This study was funded by the Vietnam National Foundation for
Science and Technology Development (NAFOSTED) under Grant
number 104.02-2012.42.
The authors would like to thank Prof. C. Migliaresi and A. Motta,

Department of Industrial Engineering and BIOtech Research Centre,
University of Trento, Italy, for supporting cell culture experiments.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at />2015.03.001.
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