International Journal of Pharmaceutics 577 (2020) 118950

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International Journal of Pharmaceutics

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

Gold as a dopant in selenium-containing carbonated hydroxyapatite fillers of T

nanofibrous ε-polycaprolactone scaffolds for tissue engineering
⁎
M.K. Ahmeda, , S.F. Mansourb, Reem Al-Wafic, M. Afifid, Vuk Uskokoviće
a
Department of Physics, Faculty of Science, Suez University, Suez, Egypt
b
Physics Department, Faculty of Science, Zagazig University, Egypt
c
Physics Department, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia
d
Ultrasonic Laboratory, National Institute of Standards, Giza, Egypt
e
Department of Mechanical and Aerospace Engineering, University of California, Irvine, CA, USA

A R T I C LE I N FO A B S T R A C T

Keywords: The necessity for finding a compromise between mechanical and biological properties of biomaterials spurs the
Carbonated hydroxyapatite investigation of the new methods to control and optimize scaffold processing for tissue engineering applications.
Electrospinning A scaffold composed of ε-polycaprolactone fibers reinforced with carbonated hydroxyapatite (CHAP) dually
Gold doped with selenite oxyanions (Se) and cationic gold (Au) was synthesized using the electrospinning technique
HFB4
and studied at different contents of Au. Despite the fact that the amount of the Au dopant was relatively low,
Nano
variations to it induced significant microstructural changes, affecting the cell response and mechanical prop-
Toughness
erties in return. Au nanoparticles segregated as a separate, ternary phase at the highest Au content, corre-
sponding to x = 0.8 in the AuxCa10−1.5x(PO4)5.8(SeO2)0.2−x(CO3)x(OH)2 stoichiometric formula of Au/Se-CHAP.
Their appearance coincided with a rapid degeneration in the density and adhesion of osteoblastic cells grown on
the scaffolds. In spite of this adverse effect, the cell spreading and proliferation improved with increasing the
amount of the Au dopant in the Au/Se-CHAP particles of the scaffold in the x = 0.0–0.6 range, suggesting that
the biological effects of Au in the ionic and in the nanoparticulate form on the implant integration process may
be diametrically opposite. The addition of Au had a dramatic effect on some mechanical properties, such as
toughness and strain at break, which were both reduced twice upon the introduction of Au into Se-CHAP at the
lowest amount (x = 0.2) compared to the Au-free composite. The significant variation of physical and biological
properties of these composite scaffolds with trace changes in the content of the Au dopant inside the ceramic
filler particles is promising, as it provides a new, relatively subtle avenue for tailoring the properties of tissue
engineering scaffolds for their intended biomedical applications.

1. Introduction
Bone tissue engineering has grown in recent years into an indis-
pensable avenue for the regeneration of bone functions impaired due to
age, disease or trauma (Ahmed et al., 2018). The ideal structure of a
scaffold for bone replacement has to meet versatile properties, such as
biocompatibility, osteoconductivity, a sufficient specific surface area,
adequate porosity ratio, and an optimal biodegradation rate to en-
courage cell growth, differentiation, and proliferation (Ahmed et al.,
2019a,b; Mansour et al., 2017c). The electrospinning technique is one
of the most common and easily applicable methods for the fabrication
of tissue engineering scaffolds with adjustable properties, such as in-
terconnected microporosity, high tensile strength, and resistance to
wear. It is also a method often used to synthesize core@shell structures
and thus obtain multifunctional biomaterials that enhance the biolo-
gical response, including cell attachment, proliferation and migration
(Chen et al., 2019; Wang et al., 2019b). Although electrospinning is
developing fast and can be currently used to produce coaxial (Zhou
et al., 2019), tri-axial (Yu et al., 2018), modified coaxial (Wang et al.,
2019a) and modified tri-axial (Yang et al., 2019a) core-shell structures,
as well as side-by-side (Wang et al., 2018a) structures such as Janus
ones, the single-fluid electrospinning is still the mainstream method in
this field (Yang et al., 2019b) and its products could be explored as
templates for new functional fibrous nanostructures. The present study
falls along this line of effort in its attempting to create an elementally
complex bio-functional inorganic-polymeric core-shell structure.
However, achieving an optimal compromise between mechanical
and biological properties of such biomaterials is not always a

⁎
Corresponding author.
E-mail address: mkaa@zu.edu.eg (M.K. Ahmed).

https://doi.org/10.1016/j.ijpharm.2019.118950
Received 10 October 2019; Received in revised form 30 November 2019; Accepted 7 December 2019
Available online 16 December 2019
0378-5173/ © 2019 Elsevier B.V. All rights reserved.
M.K. Ahmed, et al.
straightforward approach, even though it is a prerequisite for their use
as scaffolds in tissue engineering. For example, if mechanical properties
are improved by compaction of the scaffold structure, this will be done
at the expanse of the optimal porosity and will adversely affect the cell
proliferation. Hybridizations between polymeric and bioceramic phases
via a core@shell nanofiber design is one way of enhancing the me-
chanical properties of electrospun composites without compromising
their porosity on which the favorable cell response depends. This effect
is owed to an increase in toughness by incorporation of bioceramic
particles inside the polymeric fibers (Ni et al., 2019) and presents only
one of the many potential synergistic effects resulting from the pre-
paration of electrospun composite nanofibers.
One of the most interesting bioceramics that can be proposed for use
in these composites is hydroxyapatite [HAP: (Ca10 (PO4 )6 (OH )2 )]. The
unit cell of pure synthetic HAP consists of 44 atoms and crystallizes in
hexagonal symmetry and the space group of P63/m. Owing to its high
level of chemical and crystallographic resemblance to the mineral part
of bone tissues, HAP is a material characterized by an exceptional
biocompatibility, low cytotoxicity and osteoconductivity, all of which
enhances cell adhesion and proliferation. However, its brittleness and
poor mechanical properties represent obstacles that hinder its utiliza-
tion in load-bearing applications. These restrictions of HAP in the ap-
plicative domain can be compensated for partially by taking advantage
of its structure. Namely, HAP lattice is susceptible to ionic substitutions,
offering a simple avenue for overcoming these drawbacks. Ca ions in
the crystal lattice of HAP could be classified into two categories de-
pending on their sites, namely Ca(1) and Ca(2), while tetrahedral
(PO43 −) is found in six different positions and two (OH )− ions are con-
fined to c-axis channels. Both Ca sites could be replaced with a plethora
of cations, ranging from the monovalent to the pentavalent, while
(PO43 −) positions could be occupied by similarly monovalent and poly-
valent anions and either monovalent or divalent anions may substitute
the (OH )− sites (Basirun et al., 2017; El-dek et al., 2017; Luo et al.,
2018; Szurkowska et al., 2018).
In contrast to pure synthetic HAP, biogenic HAP undergoes a
spontaneous substitution with different ions, the most prominent of
which is (CO32 −) at 3–8 wt% (Uskokovic and Uskokovic, 2011). Car-
bonated hydroxyapatite (CHAP) gets resorbed faster because of its
higher solubility (Germaini et al., 2017), in spite of its lower affinity for
the osteoblastic cell attachment compared to the pure synthetic one
(Redey et al., 2000). Carbonation also occurs spontaneously upon the
precipitation of HAP under atmospheric conditions, typically yielding
B-type CHAP, where carbonates occupy the place of phosphate groups
in the crystal structure of HAP. Most ionic substitutions, including
carbonate, distort the HAP lattice and lower its crystallinity. In the case
of natural HAP, the multiple dopants often compensate for each other’s
effects on contractions and expansions in specific lattice directions, as
the result of which the crystals maintain their tendency to grow pre-
ferentially along the c-axis and in conformity with their hexagonal
symmetry, in which form they are found scattered through the col-
lagenous matrix of bone. Therefore, to mimic the extracellular matrix
(ECM) of bone, an ideal stimulation requires both approaches; that is,
both composition and structure should be adjusted to emulate the
conditions present in natural bone via the design of appropriate or-
ganic-inorganic composites (Zhao et al., 2018). One way of achieving
this is through the synthesis of multi-doped CHAP dispersed in a bio-
polymer.
One of the polymers that has been frequently used for clinical bone
regeneration, particularly in the form of fibrous scaffolds, is ε-poly-
caprolactone (PCL). PCL is known for its biocompatibility and adequate
tensile strength. In addition, it is a compound approved by the United
States Food and Drug Administration (FDA) and can be easily processed
in the form of fibers using electrospinning. Chen et al. investigated the
core-shell nanostructure of gelatin-chitosan/HAP and found out that
HAP deposited on the fiber surface encouraged the osteoblast cell
proliferation (Chen et al., 2019). Moreover, Sarkar et al. studied the
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International Journal of Pharmaceutics 577 (2020) 118950
ternary composition of carboxymethyl cellulose fibers reinforced with
carbon and HAP and demonstrated an increase in the flexural strength
and the compressive strength of the matrix up to circa 140 MPa and
118 MPa, respectively, while the flexural modulus was in the 9–22 GPa
range (Sarkara et al., 2019). Furthermore, Zhu et al. studied the me-
chanical properties of a ternary composition containing different con-
tents of HAP nanoparticles and poly(glycolide) (PGA) fibers inter-
spersed within a HAP/poly(lactide-co-glycolide (PLGA) matrix, and the
results showed that the bending strength reached 17 MPa at 70% of
PGA (Zhu et al., 2017). In addition, Aragon et al. fabricated PCL fibers
with the diameters of 150–300 nm, containing needle-shaped HAP
particles with the diameter of 20 nm and the length of 150 nm inter-
spersed within them (Aragon et al., 2017).
Selenium (Se) is one of the crucial trace elements in the human body
due to its roles as an antioxidant, an antimicrobial and an element fa-
cilitating the resistance to bone fracture (Wei et al., 2017). Owing to its
anticancer activity, Se could be used as a dually or even multiply
functional element that inhibits the formation of cancerous and other
abnormal cells, while enhancing the growth of bone tissues (Wei et al.,
2017). For instance, Wei et al. studied the crystal structure of HAP
doped with different amounts of selenite and concluded that the lattice
distortion increased and the particle size decreased with an increase in
the Se content (Wei et al., 2017). Sun et al. found out that selenite ions
are incorporable successfully into HAP lattice, where they have a sig-
nificant influence on the morphology and particle size of the as-syn-
thesized Se-HAP nanoparticles (Sun et al., 2017). Wang et al. in-
vestigated the anticancer activity of HAP doped with selenite in both in-
vitro and in-vivo conditions and found out that Se-HAP containing 10 wt
% Se induced cancer cell apoptosis (Wang et al., 2016b).
In the medical field, gold nanoparticles (AuNPs) have been in-
vestigated for drug delivery purposes, for use in biosensors and other
imaging applications, and as a material for photodynamic thermo-
therapies (Chai et al., 2018; Ferreira dos Santos et al., 2015; Shoueir
et al., 2019). Besides, a hybrid material combining HAP with AuNPs has
been examined for bone regeneration and the improvement of hemo-
compatibility (Chai et al., 2018). Chai et al. incorporated Au3+ ions into
HAP extracted from fish scales and investigated the interaction with
albumin, demonstrating enhanced protein adsorption in both cell cul-
ture and the human blood (Chai et al., 2018). Santos et al. synthesized
AuNPs on the surface of HAP and showed that the interaction between
mesenchymal stem cells and the AuNPs-HAP hybrid leads to improved
osteoblastic differentiation (Ferreira dos Santos et al., 2015).
Such fabrication of AuNPs on the surface of HAP crystals, however,
results in effective phase separation, which may suppress the elicitation
of an ideal behavior in the tissue. In addition to this, AuNPs in this case
shield the more bioactive and osteoconductive surface of HAP, which
may hinder the bone tissue integration process. Finally, the biode-
gradation of AuNPs may not necessarily proceed with as little of side
effects as the clearance of Au ions. To avoid these obstacles, Au ions are
here incorporated into HAP lattice co-doped with Se and carbonate
anions and dispersed within electrospun PCL fibers. Despite the limit in
the dopant concentration, doping HAP with Au ions and its subsequent
dispersion within the polymeric nanofibers may be the basis for the
formation of a novel composition that could act as a promising bio-
material for bone-related applications. No prior work has been done on
HAP doped simultaneously with Au and Se, let alone on its combination
with electrospun polymers. Therefore, in this work we introduce these
novel composite comprising Au/Se-CHAP@PCL core-shell nanofibers.
We also study its crystal structure, morphological behavior and biolo-
gical interaction, including human fibroblast cell viability and attach-
ment.
M.K. Ahmed, et al.
2. Materials and methods
2.1. Synthesis of Au/Se-CHAP/PCL microfibrous
Calcium chloride dihydrate [CaCl2·2H2O], diammonium hydrogen
phosphate [(NH4)2HPO4], gold chloride [HAuCl4], selenium oxide
[SeO2], chloroform (99.5%) and methanol (99%) were purchased from
LOBA, India, and they were utilized without further purifications. PCL
(Mw = 80,000 g/mol) was purchased from Sigma-Aldrich.
As the first step in the synthesis, viscous PCL gel was prepared by
dissolving 8 g of PCL pellets in 100 ml of the solvent composed of
66.7 ml of chloroform and 33.3 ml of methanol. CHAP was synthesized
by precipitation method. Meanwhile, to obtain Au-free Se-HAP, solu-
tions comprising equal volumes of 0.5 M calcium chloride dihydrate,
0.29 M diammonium hydrogen phosphate, and 0.01 M selenium oxide
were prepared separately. Then (P + Se) were combined and added
slowly, at the rate of 1 ml/s, into the Ca solution while the pH value was
kept at 11.00 ± 0.05 and the stirring was steady at around 1200 rpm.
Au-containing Se-HAP samples were synthesized by following the same
procedure and adding 0.01, 0.02, 0.03 or 0.04 M of HAuCl4 to replace
Ca concentration in the 0.5 M calcium chloride solution, per the fol-
lowing equation:
xHAuCl4 ·5H2 O + 0.2SeO2·6H2 O + 5(NH4 )2 HPO4 + (10 − 1.5x ) CaCl2
·2H2 O → Au x Ca(10 − 1.5x ) (PO4 )5.8 (SeO2)0.2 (OH )2
↓ + 3(NH4 )(NO3) + 6(NH4 ) Cl (1)
where 0.0 ≤ x ≤ 0.8, with a step of 0.2.
After one hour of continuous stirring, the solution containing the
precipitate was aged for one day, after which it was washed several
times using double distilled water. The obtained gel was dried at
50–60 °C.
The nanofibers were synthesized as follows: 110 mg of each ob-
tained powder were added to 10 ml of PCL (8%) in a sealed glass bottle
to be suspended via stirring overnight. The suspensions were then so-
nicated using an ultrasonic probe (Branson Digital Sonifier) for 15 min
to increase the homogeneity and dispersibility of the composition and
make it ready for electrospinning. The composite samples were then
introduced to a syringe pump to be processed by electrospinning, using
a custom-made electrospinning setup and fixed parameters for all
samples. The operating parameters were as follows: the high voltage
was kept at 18 ± 0.1 kV, the injection rate was 1 ml/h, the gap be-
tween the electrode and the target was 16 cm, and the syringe needle
was 22φ. The flow chart of the synthesis procedure is displayed in
Fig. 1.
2.2. XRD measurements
X-ray diffraction (XRD) analysis was carried out on an X-ray dif-
fractometer (analytical-x' pertpro, Cu kα1 radiation, λ = 1.5404 Å,
45 kV, 40 mA, Netherlands). The data were collected in the range of
4° ≤ 2θ ≤ 60° with a step size of 0.02° and the irradiation time of 0.5 s
per step.
The crystallite size in the polymeric phase was calculated using
Scherrer’s formula (El-Shabasy et al., 2019; Mansour et al., 2017c;
Salama et al., 2017):
kλ
D=
βhkl cosθ (2)
where D refers to the crystallite size in nm, k represents the shape factor
(0.9), λ is the wavelength of the X-rays (1.54056 Å for CuKα radiation),
while βhkl is the full width at half maximum expressed in radians, and θ
is the Bragg diffraction angle.
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International Journal of Pharmaceutics 577 (2020) 118950
2.3. FTIR measurements
Fourier transformed infrared (FTIR) spectra were scanned using an
FTIR spectrometer (Perkin-Elmer 2000) in the wavenumber range of
4000–400 cm−1.
2.4. Particle microstructure and morphology
The high-resolution transmission electron microscopy (HRTEM,
JEOL/JME 2100) was used to investigate the morphology and micro-
structure of the composite fibers. The operating voltage was 200 kV.
The sample preparation process involved the collection of fibers
through a water-filled tube, after which the copper grid was submerged
into the tube for 10 s. Field emission scanning electron microscope
(FESEM) coupled to an energy-dispersive X-ray (EDX) spectroscopic
probe was used to scan the fibrous surfaces and assess the sample mi-
crostructures and morphologies under an operating voltage of 20–30 kV
(model: QUANTA-FEG250, Netherlands).
2.5. In vitro cell viability tests
The human osteoblast cell line, HFB4, was cultured in Dulbecco's
modified Eagle's medium (DMEM, Gibpco) at 37 °C and 5% CO2 to
investigate the viability of cells seeded onto the microfibers. Cells
seeded at the density of 5 × 103 cells/cm2 were cultured on the fibers
in 12-well plates. After three days of incubation at 37 °C, the medium
was removed and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide) injected into each well, after which the cell viabi-
lity was detected via an optical analyzer.
Cell viability was defined as the percentage of the viable cells
compared to the total cell number (Mansour et al., 2018; Shaban et al.,
2019):
Mean optical density of test samples
Viability (%) = × 100
Mean optical density of the control (3)
2.6. Cells growth on the scaffold
FESEM was used to observe the growth behavior of human osteo-
blastic HFB4 cells seeded on the microfibers. For this purpose, each
sample was cropped into two pieces of 0.5 × 0.5 cm, which were then
exposed to a UV lamp in 12 well plates for 30 min for sterilization. Then
1.5 ml of HFB4 cells (5 × 105 cells) were added into each well. Lastly,
the plate was covered and incubated at 37 °C for three days. After this
time, the fibers were washed with phosphate buffer saline (PBS). To
keep the cells fixed on the nanofiber surface, the scaffolds were sub-
merged in a glutaraldehyde solution (4% concentration) for 1.0 h. They
were then dehydrated in the air for 15 min. Finally, they were sputtered
with gold for 2 min to be ready for FESEM imaging.
2.7. Roughness measurements
Micrographs of microfiber samples obtained using FESEM before
treating with cells were processed with Gwyddion 2.45 software to
investigate for their roughness (Mansour et al., 2017a). 3D micrographs
were obtained for each sample and the resolution of the micrographs
was fixed at 1450 × 900 pixels. The graphed edges were eliminated to
avoid unwanted boundaries. Finally, the roughness parameters were
computed using the same software in nm.
2.8. Density and porosity
Porosity and real density were measured using a Quantachrome
Instruments analyzer (UltraPyc 1200e). The analyses were done by
processing a rectangular, (2 × 3) cm area for each sample. Then the
helium gas flowed through the cell and the averages of porosity and real
M.K. Ahmed, et al. International Journal of Pharmaceutics 577 (2020) 118950

Fig. 1. Flow chart describing the preparation of the working fluid for the electrospinning of Au/Se-CHAP/PCL.

density were derived from three independent measurements on each density is expressed in mass/volume units (Xu et al., 2006), while the
sample. total calculated porosity is given as a ratio between the theoretical and
The theoretical density (Dx) of Au/Se-CHAP was computed as measured density (ρ):
(Mansour et al., 2017b):
TDx ⎞
ZM P = ⎜⎛1 − ⎟ × 100

Dx = ⎝ ρ ⎠ (6)
NV (4)

where M is the molecular weight of the Au/Se-CHAP/PCL fibers, N is

Avogadro's number, Z is the number of molecules per unit cell, and V is
the unit cell volume. However, the contribution of PCL must be taken
into consideration and the total theoretical density can be represented
as (Mansour et al., 2017a):
TDx = ADx1 + BDx 2 (5)
where A and B represent the relative fractions of the two phases,
namely Au-Se-CHAP and PCL, respectively, equaling A = 12.09% and
B = 87.91%. With the Dx of PCL being 1.145 g/cm3, the measured
2.9. Mechanical testing
The samples were processed into rectangular strips with the di-
mensions of 100 × 20 × 0.1 mm. Strips were used instead of the
standard, dog-bone shapes in order to avoid the creation of stress
concentration points along the specimen geometry, which might dete-
riorate the tensile strength. The stress/strain test was then carried out
by pulling the nanofibers at the rate of 5 mm/minute up to the fracture
point, as in accordance with the standard code ASTM D882.

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M.K. Ahmed, et al. International Journal of Pharmaceutics 577 (2020) 118950

at the higher, x = 0.8 content of Au.

The substitution of calcium ions in the HAP lattice by Au ions has
not been studied before, so analogies with previous studies may be
found in studies of other trivalent metals, given that the trivalent gold is
the more stable of the two possible Au valences: +1 and +3. In case of
the trivalent cation substitution such as Al3+, Wang et al. studied the
geometric optimization of HAP unit cell upon its doping with Al3+
using density functional theory (DFT) and found out that both a- and c-
axes contract with an increase in the foreign ion content (Wang et al.,
2016a). In addition to this, this theoretical study showed that Al3+
preferably accommodates itself on Ca(II) sites, with a vacancy forming
on the same lattice plane. Fahami et al. concordantly showed that both
a- and c-axes contract with an increase in the Al3+ content up to
x = 0.6, after which the a-axis begins to expand significantly (Fahami
et al., 2017).
The following explanation can be provided to describe the observed
changes in crystal structure parameters. First, it should be invoked that
Ca2+ ions can occupy two sites in the HAP crystal lattice: Ca(I) and Ca
Fig. 2. Normalized XRD patterns of Au/Se-CHAP@PCL fibers at different Au (II), with the coordination states (C.S.) 9 and 7, respectively, and the
contents (♦: CHAP and *: Au), along with the standard pattern of HAP (ICDD radii of around 1.18 and 1.06 Å, respectively. In contrast, Au3+ has two
01-073-0293). C.S., namely 4 and 6, with the corresponding radii of 0.68 Å and 0.85 Å,
respectively (Shannon, 1976). Herein, there are four potential ways by
3. Results and discussion which Ca sites can be replaced by Au3+ and they could be explained by
considering first that the selenite anion is trivalent, with the radius of
3.1. Structural analysis around 2.28 Å, which is very close to the radius of the phosphate anion
(2.39 Å) (Sun et al., 2017). The two oxyanions, however, possess dif-
ferent structural symmetries. Specifically, the phosphate is tetrahedral,
Fig. 2 displays the XRD patterns of the fibrous Au/Se-CHAP-PCL
while the selenite is trigonal (Sun et al., 2017). The substitution of
scaffolds at different contents of Au. The peaks that could be assigned to
selenite for phosphate causes a significant variation in lattice para-
PCL were detected at 2θ = 21.452 and 2θ = 23.814°. The half width of
meters as well as in the morphology. Previous studies did not show a
these peaks visibly decreased as the content of Au in Au/Se-CHAP in-
consensus over the effects of this substitution on the lattice parameter a,
creased, but only up to × = 0.4, after which the opposite effect was
but they all unequivocally demonstrated that the lattice parameter c
noticed and the peak half widths increased together with the Au content
decreases due to this substitution. Thus, Wei et al. studied the structural
in Au/Se-CHAP. Simultaneously, the diffraction peaks became more
behavior of HAP crystals due to the ionic substitution of selenite ions
diffuse, suggesting the reduction of crystallinity of the polymeric phase
and found out that the lattice parameter a increases and the lattice
as the Au content in the nanoparticle filler increased from × = 0.4 to
parameter c decreases with an increase in the selenite content (Wei
× = 0.8. Moreover, a closer peak analysis demonstrates that a binary
et al., 2017). Uskoković et al. demonstrated that both a- and c-axes
composition including PCL and HAP forms at the low contents of Au, all
contract with the addition of selenite (Uskoković et al., 2017), while
the way up to x = 0.6. However, at the highest content of Au, i.e.
Sun et al. indicated that the c-axis contracts, while the lattice parameter
x = 0.8, a ternary composition is detected. Pure HAP crystallized in the
a fluctuates with an increase in the selenite content (Sun et al., 2017).
hexagonal symmetry conforming to the ICDD card no. 01-073-0293, but
In addition to this, there is a competition between selenite and carbo-
was unnoticeable with the naked eye because of its filling mostly the
nate for the phosphate sites in the HAP lattice, for which reason the
interior of the fibers. As it is reported in Table 1, the calculated
parameter a is larger and the parameter c lower than the standard va-
Scherrer’s crystallite size starts from 25.2 nm at x = 0.0 and then it
lues reported in the ICCD. However, an increase in the Au content up to
follows the crystallinity trend by increasing to 38.8 nm at x = 0.4 and
x = 0.4 causes an expansion of the lattice in the a-axis direction and a
then decreasing steadily down to 11.3 nm at x = 0.8.
contraction along the c-axis, as seen in Fig. 3, which is due to the
The calculated lattice parameters demonstrate a similar trend of
supposedly most energetically favorable replacement of Ca(II)
attaining maxima/minima at Au contents between x = 0.4 and x = 0.6.
Specifically, the lattice parameter a starts from 9.47 Å for the Au-free (O.S. = 7) by Au3+ (O.S. = 6). This demonstrates that the Ca → Au
substitution may compensate for the effects of the phosphate → selenite
composition, after which it increases steadily with the Au content up to
9.53 Å at x = 0.4 and then decreases down to 9.47 Å at x = 0.8. The substitution. Moreover, the Au contents higher than x = 0.4 are ac-
lattice parameter c, in contrast, follows the opposite trend by starting companied by a decrease in the lattice parameter a, which could be
from 6.83 Å at x = 0.0, after which it enters a plateau and then drops to attributed to the saturation of the HAP lattice with Au ions, which
6.52 Å at x = 0.6, before rising back to 6.86 Å at x = 0.8. Interestingly, precipitate as a minor ternary phase with the zero oxidation state at
both lattice parameters undergo changes with the Au content, but get x = 0.8.
restored to the close vicinity of values typifying the Au-free composition
3.2. FTIR spectra
Table 1
Crystallite size, lattice parameters a and c, and the unit cell volume (V) of Au/ Fig. 4 displays the characteristic vibrational bands of the composite
Se-CHAP-PCL at different contents of Au. fibers, while the band wavenumber values and the corresponding as-
x Crystallite size (nm) a (Å) c (Å) V (Å3) signments are reported in Table 2. In the case of x = 0.0 composition,
the bands appearing at 453.2, 568.9 and 599.8 cm−1 could be attrib-
0.0 25.2 9.47 6.83 530.05 uted to the (ν4) bending mode of PO34− (Surmenev et al., 2019). In ad-
0.2 26.5 9.49 6.88 536.26
dition, the band at 1043.3 cm−1 refers to the asymmetric stretch of
0.4 38.8 9.53 6.84 537.23
0.6 19.1 9.49 6.53 508.71
PO34− (ν3), while the bands at 3149.2–3533.9 cm−1 are assigned to the
0.8 11.3 9.47 6.86 532.44 stretching oscillation of the OH group (Mansour et al., 2017a; Mansour
et al., 2018). The stretching vibrational mode of CO33 − (ν3) appears at

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M.K. Ahmed, et al. International Journal of Pharmaceutics 577 (2020) 118950

1463.7 cm−1 (Youness et al., 2018). The asymmetric stretching bands

of the CH2 group are detected at 2936.1 and 2861.8 cm−1, while the
band of the (C = O) group appears at 1726.9 cm−1. The band at
1292.1 cm−1 refers to the CeO and CeC vibrations, whilst the bands at
1240 and 1180.2 cm−1 are due to the asymmetric oscillations of the
CeOeC group (Gorodzha et al., 2018). The bands around 1160 cm−1
that would be originating from the amorphous phase were not detected,
proving the crystalline nature of the PCL fibers, providing a good match
with the XRD patterns (Fig. 2).

3.3. Morphological and microstructural analyses

Fig. 5(a, b) displays the HR-TEM micrographs of Au/Se-CHAP@PCL

Fig. 3. Lattice parameters a and c of the Au/Se-CHAP phase in Au/Se-CHAP@
PCL at different contents of Au.
Fig. 4. Normalized FTIR spectra of Au/Se-CHAP@PCL fibers at different con-
tents of Au.
fibers at x = 0.6. The low magnification image (Fig. 5a) shows that the
interconnected nanofibers form a network that may support its me-
chanical properties. The diameters of fibers ranged from 115 to 235 nm.
In addition, the fibers appeared loose, suggesting their ability to absorb
a relatively high energy before fracture in comparison with their more
rigid counterparts. In general, the design of non-taut, interconnected,
nanofibrous structures is considered a way to obtain a higher fracture
toughness in materials. The high magnification micrograph (Fig. 5b)
shows that Au/Se-CHAP particles are embedded in the shell of PCL fi-
bers. The multi-doped HAP particles appear ellipsoidal in shape, with
dimensions ranging from 10 × 20 nm to 20 × 40 nm. This embedment
within the bulk of the fibers and no tendency for surface segregation of
the particle fillers indicates a solid loading capacity of the PCL fibers
and the possibility for their tailoring to be adequate for advanced drug
delivery applications.
Fig. 6(a)–(h) shows the surface morphology of Au/Se-CHAP@PCL at
different contents of Au. It could be noticed from Fig. 6(a, b) that at
x = 0.0, the fibrous scaffold is randomly oriented, with a broad dis-
persity of fiber diameters, in the range of 0.15–0.7 µm. In addition, the
intergranular porosity with a high porosity is observed. Increasing the
content of Au up to x = 0.4 (Fig. 6(c, d)) lowers the porosity of the
fibers, however the diameters distribution becomes bimodal, as the fi-
bers tend to be divided to two subgroups: the thin fibers with diameters
from 0.15 to 0.3 µm and larger ones with diameters from 0.6 to
0.85 µm. Fig. 6(e, f) shows the composition Au/Se-CHAP@PCL at
x = 0.6, where the porosity gets restored to its high initial value, while
the fibers also adopt a curvier appearance than at lower Au contents. In
addition, there is a significant gap between the diameters of the thin
fibers and the large fibers: while the large ones have diameters of
1.15–1.30 µm, the diameters of the thin fibers do not exceed 0.04 µm.
The thin fibers appear to reinforce the large fibers by forming trans-
verse connections. Fig. 6(g, h) displays the sample with the highest Au
content, x = 0.8, where fibers are similarly classified to two classes

Table 2
Characteristic FTIR bands of Au/Se-CHAP/PCL fibers at different contents of Au.
x = 0.0 x = 0.2 x = 0.4 x = 0.6 x = 0.8 Assignment Ref.

453.2 452.2 452.2 452.2 452.2 (ν4) of OePeO Mansour et al. (2017a)
568.9 568.9 566.0 567.9 576.6 (ν4) ofPO34− Duta et al. (2017), Tang et al. (2014), Wang et al. (2017)
599.8 599.8 597.8 599.8 — (ν4) ofPO34− Mansour et al. (2018), Tang et al. (2014)
730.9 731.9 730.9 730.9 731.9 (ν3) of SeeO Youness et al. (2018)
960.4 960.4 960.4 959.4 960.4 (ν1) ofPO34− Gorodzha et al. (2018), Murugan et al. (2018)
1043.3 1044.3 1044.3 1042.3 1045.2 (ν3) ofPO34− Fadeeva et al. (2012)
1101.2 1103.1 1102.1 1101.2 1104.1 PeO stretch Zhang et al. (2016)
1180.2 1179.3 1179.3 1176.4 1179.3 CeOeC Sattary et al. (2018)
1240.0 1240 1239.0 1238.1 1240 CeOeC Murugan et al. (2018), Sattary et al. (2018)
1292.1 1293.1 1292.1 1291.1 1293.0 CeO and CeC Gorodzha et al. (2018), Sattary et al. (2018)
1463.7 1463.7 1463.7 1461.8 1463.7 (ν3) ofCO33 − Wang et al. (2018b), Zhang et al. (2016)
1726.9 1728.9 1727.9 1725.0 1728.9 C=O Gorodzha et al. (2018), Sattary et al. (2018)
2861.8 2862.8 2861.8 2859.9 2862.8 CeH stretch Gorodzha et al. (2018), Sattary et al. (2018)
2936.1 2938.0 2936.1 2932.2 2939.0 CeH stretch Sattary et al. (2018)
3433.6 3437.5 3433.6 — 3435.6 OeH stretch Ahmed et al. (2018), Murugan et al. (2018), Wang et al. (2017)
— 3526.2 — — 3533.9

6
M.K. Ahmed, et al.
Fig. 5. HRTEM micrographs of Au/Se-CHAP@PCL at x = 0.6 and at different resolutions: low (a) and high (b).
based on their diameters: 0.4–1.15 µm and 0.1–0.25 µm. The most
notable detail detected in this sample is the appearance of bright, star-
like points distributed across the fiber surface. These points could be
attributed to the AuNPs, thus matching the XRD patterns (Fig. 2).
AuNPs are in the 15–27 nm diameter range and are well dispersed
throughout the fibers, showing no signs of agglomeration. The quanti-
tative EDX analysis of this representative sample led to the detection of
all the constitutive atomic species in the composite fibers, namely C at
77.0 at.%, O at 20.1 at.%, Ca at 1.2 at.%, Se at 0.8 at.%, Au at 0.5 at.%,
and P at 0.4 at.% (Fig. 6i). Finally, there is a consistent increase in the
diameters of the larger fibers in the composite samples with an increase
in the Au content, which may be an effect related to the lowering of
crystallinity that accompanies this increase in the Au content. Namely,
the lowering of crystallinity leads to the lowering of surface energy
because of the entropic reasons, which favors the sticking of the fiber
precursors during the extrusion process and the thickening of the fibers.
As it is reported in Table 3, the roughness average (Ra) starts from
28.8 nm at x = 0.0, then it increases with the Au content up to 33 nm at
x = 0.8, albeit marking a drop at x = 0.6. The increase of Ra in the
lower range of the parameter x(0–0.4) may be due to an increase in the
density of surface defects. Then, the Ra drop in the medium range of the
parameter x(0.4–0.6) may be due to the loss of crystallinity in this re-
gion, given that higher crystallinity usually corresponds to sharper
edges and a higher roughness. Finally, the jump recorded in the Ra
value in the highest range of the parameter x(0.6–0.8) could be as-
signed to the formation of Au nanoparticles on the surface of the na-
nofibers. The other two roughness values exhibit a similar trend to the
Ra. Thus, the root means square roughness (Rq) starts from 40.3 nm for
the Au-free nanofibers (x = 0.0), increases to 42.4 nm at x = 0.4, drops
down to 38 nm at x = 0.6, but then it soars to 49 nm at x = 0.8. The
maximum height of the roughness (Rt) likewise begins at 482.6 nm for
x = 0.0, drops down to 401 nm at x = 0.6, but then jumps to 519 nm at
x = 0.8.
In view of these results, it is obvious that Au substitution has a
significant and complex influence on the surface roughness owing to its
ability to induce crystal defects, as it can be seen in Fig. 7(a)–(d).
Crystal defects on the surface are attractive sites to form new chemical
bonds with host tissues, which may enhance the cell adhesion and fa-
cilitate the implant/tissue interlocking process. If this premise is cor-
rect, then this critical interaction could be controlled with composi-
tional parameters such as the type and concentration of the dopant
inside the particle filler of the electrospun nanofibers.
3.4. Cell viability
The viability of cells is an essential indicator of biocompatibility and
7
International Journal of Pharmaceutics 577 (2020) 118950
a determinant of the potential of the material to be used as an implant
in bone tissue replacement procedures. Therefore, the viability of HFB4
cells grown on Au/Se-CHAP/PCL fibers was examined at different
contents of Au after 3 days of culturing. As seen in Fig. 8, the cell
viability was 97.3% at x = 0.0 and 96.0% at x = 0.8, whereas the
lowest value recorded was 94.7% at x = 0.2. There was no statistically
significant difference between the viability of cells depending on the Au
content in the material and viability values for all the samples speak in
favor of excellent biocompatibility.
3.5. Cell attachment onto the fibrous scaffolds
These high viability values allowed the cells to spread on the surface
of the fibers, as it is shown in Fig. 9(a)–(j). As for the fibers of the
x = 0.0 composition, which are shown in Fig. 9(a, b), the cells do not
only spread on their surface, but also tend to cover the insides of their
network. The density of cells spread on the surface of the material is so
high that they appear to form a membranous sheath over the whole
surface. Fig. 9(c, d) shows the x = 0.2 composition, with an even more
copious cell growth compared to x = 0.0. In this case, however, the
growth of cells is even more in-depth, occurring through the pores, and
is not limited to the surface. Also, the filopodia of the cells tend to
follow the path of the fibers, which gives them more adhesion strength
that is supportive of the growth process. Fig. 9(e, f) illustrates that the
surface covered by the cells further increases at x = 0.4 compared to
the lower Au contents. In addition to this, cells displayed a solid form,
with thicknesses larger than at the lower Au contents. The x = 0.6
composition, comprising rougher surface fibers as in agreement with
the earlier presented data, is shown in Fig. 9(g, h). Herein, the increased
curvature of the fibers shows its benefits, as the cells proliferate through
the curves, which support the cell growth both mechanically and bio-
logically. The cells adhere to the fibers, which allow the metabolically
active ions to be released from the fibers, while the curves and the
resulting pores facilitate the transfer of nutrients across the volume of
the scaffold. The highest Au content (x = 0.8) is seen in Fig. 9(i, j), and
in this case the cell spreading activity is lower than at lower Au contents
despite the comparatively high cell viability measured for this compo-
sition. One factor causing this is the presence of AuNPs on the fiber
surface detected in this composition. For example, it was reported that
AuNPs with sizes lower than 15 nm could cause cytotoxicity in tissues
(Coradeghini et al., 2013). Another factor of influence is the relatively
low porosity in comparison with the compositions with lower Au con-
tents. Despite this, the cells still do grow both on and under the surface
of the material, which is indicative of their ability to achieve sufficient
adhesion strength necessary to promote a proper tissue integration
process.
M.K. Ahmed, et al. International Journal of Pharmaceutics 577 (2020) 118950

Fig. 6. FESEM micrographs of Au/Se-CHAP@PCL at different Au contents: x = 0.0 (a, b), x = 0.4 (c, d), x = 0.6 (e, f), and x = 0.8 (g, h), along with the EDX pattern
for the x = 0.8 composition (i).

8
M.K. Ahmed, et al. International Journal of Pharmaceutics 577 (2020) 118950

Table 3
Roughness parameters of Au/Se-CHAP, including roughness average (Ra), root
mean square roughness (Rq), and the maximum height of the roughness (Rt) at
different contents of Au.
x Ra (nm) Rq (nm) Rt (nm)

0.0 28.8 40.3 482.6

0.4 30.7 42.4 404.9
0.6 27 38 401
0.8 33 49 519

Therefore, the fibroblast cells display positive and promising pro-

liferation on the Au/Se-CHAP/PCL fibers and their growth is somewhat
affected by the Au content in the material. The cell growth increases up
to x = 0.6, but then it deteriorates in parallel with the formation of the
AuNP phase and the segregation of the AuNPs on the surface of the
material. In general, there are positive effects that come not only from
the addition of the bioactive multi-doped Au/Se-CHAP phase into the
PCL fibers and the phase that rectifies the hydrophobicity of PCL.
Another important effect that favors the cell growth comes from the Fig. 8. Viability of the HFB4 cell line in interaction with Au/Se-CHAP@PCL
presence of Au, but strictly in the ionic form. As soon as the ions pre- fibers at different Au contents.
cipitate in the form of metallic NPs, these positive effects are lost. This
dependency of the proliferation and spread of the bone cells on the concentrations of the heavy element that Au is. The total density,
content of Au in the composition supports the view that the properties however, does not show a large variation because of the comparatively
of biomaterials for clinical investigation could be tailored by controlling low contribution of the ceramic phase to the total composition
a relatively minor amount of an ionic dopant in a relatively minor (13.75 wt%). The measured density varies within a relatively wide
component of the composite material. range, from the lowest value of 0.115 g/cm3 at x = 0.6 to the highest
one of 0.201 g/cm3 at x = 0.8. High porosity is an essential structural
property of materials in tissue engineering, given the key role of pores
3.6. Mechanical properties
in the transfer of the nutrients across the overall geometry of the
scaffold and in increasing its surface area available for interaction with
From the theoretical densities calculated from the XRD data for the
the cells. Like density, porosity varied too, achieving the lowest value of
CHAP co-doped with Au/Se and reported in Table 4, it is obvious that
85.58% at x = 0.8 and the highest one of 91.72% at x = 0.4. Although
the values increase gradually with x. This effect is due to the higher

Fig. 7. Surface roughness of Au/Se-CHAP@PCL at different Au contents: x = 0.0 (a), x = 0.4 (b), x = 0.6 (c), and x = 0.8 (d).

9
M.K. Ahmed, et al.
Fig. 9. HFB4 cell adhesion and proliferation on Au/Se-CHAP@PCL at different Au contents: x = 0.0 (a, b), x = 0.2 (c, d), x = 0.4 (e, f), x = 0.6 (g, h), and x = 0.8 (i,
j).
high porosities are required for biological applications, their drawbacks
come in the form of deteriorating the mechanical properties of the
material, the reason for which a compromise must be made between the
two properties by a precise compositional and microstructural control.
Fig. 10(a)–(e) illustrates a change in the mechanical properties of
the fibrous composites with the Au content. Fig. 10a shows the stress-
strain curves, which could be used to compute other mechanical
parameters, such as toughness presented in Fig. 10b. Toughness
10
International Journal of Pharmaceutics 577 (2020) 118950
undergoes a dramatic drop with the introduction of Au to the Se-CHAP
component, from 6.68 MJ/m3 at x = 0.0 up to 2.89 MJ/m3 at x = 0.2.
The lowest value of 1.72 MJ/m3 was achieved at x = 0.6 and the rather
slight decrease in toughness in the x= 0.4–0.8 range is accompanied by
the reduction in crystallinity of the polymer. Crystallinity can affect
toughness in more than one way, including the increased propensity for
particle agglomeration, which may amplify the overall brittleness,
adding to the increased rigidity coming from the incorporation of CHAP
M.K. Ahmed, et al. International Journal of Pharmaceutics 577 (2020) 118950

Table 4 into the fibrous scaffold in the first place. A change in the maximum
Total theoretical density (TDx), measured density (ρ), and porosity of Au/Se- tensile strength as a function of the Au content is less dramatic than that
CHAP/PCL fibers at different Au contents. detected for toughness and is illustrated in Fig. 10c. It starts from the
x Dx (Au/Se-CHAP) TDx (g/cm3) ρ (g/cm3) Porosity (%) highest value of 5.85 MPa in the material free of the Au dopant and
deteriorates slightly with an increase in the amount of Au in the com-
0.0 3.11 1.38 0.130 90.61 posite, up to 5.34 MPa at x = 0.8, with the lowest value once again
0.2 3.10 1.38 0.134 90.32
being detected at x = 0.6. The significant difference between the de-
0.4 3.12 1.38 0.115 91.72
0.6 3.32 1.41 0.126 91.05 terioration of the tensile strength and of toughness with an increase in
0.8 3.20 1.39 0.201 85.58 the Au content could be assigned to the timing of their measurements.
Specifically, while the tensile strength is measured for fibers im-
mediately before the fracture, toughness is the total absorbed energy
measured at the endpoint. Changes to the elongation ratio shown in
Fig. 10e are directly proportional to the crystallinity of the polymer;

Fig. 10. Dependency of the mechanical properties of Au/Se-CHAP@PCL fibers on the Au content: (a) stress-strain, (b) toughness; (c) tensile strength, (d) Young’s
modulus, and (e) maximum strain before fracture.

11
M.K. Ahmed, et al.
namely, while they both increase in the x = 0.0–0.4 range of the Au
content, they begin to deteriorate and rapidly decrease at higher Au
contents, specifically x = 0.4–0.8. Finally, the strain at break displays a
similarly intense reduction with the addition of the Au dopant inside
the Se-CHAP particles. Thus, the strain at break of the composition at
x = 0.0 was 206.48%, but it decreased to 84.82% at x = 0.4, which
was the lowest measured value. Here we have come upon yet another
important compromise, namely that between the flexibility of the
polymeric fibers and the rigidity of the bioactive ceramic phase. This
compromise must be precisely optimized by taking into consideration
the ionic dopant contents in order to design an adequate structure
suitable for clinical investigations.
4. Conclusion
A scaffold composed of PCL fibers reinforced with CHAP dually
doped with Se and Au was synthesized using the electrospinning
technique and studied at different contents of Au. Despite the fact that
the amount of the Au dopant was relatively low, variations to it induced
significant microstructural changes in the material, affecting its cell
response and mechanical properties in return. Crystallinity of the
scaffolds improved with the addition of Au, but then deteriorated at
higher Au contents. AuNPs segregated as a separate, ternary phase at
the highest Au content, corresponding to x = 0.8 in the
AuxCa10−1.5x(PO4)5.8(SeO2)0.2−x(CO3)x(OH)2 stoichiometric formula of
Au/Se-CHAP. HR-TEM imaging showed that Au/Se-CHAP particles got
incorporated inside the PCL fibers, while FE-SEM imaging indicated
that the fiber diameters increased with the Au content in Au/Se-CHAP,
with the highest value of 0.4–1.15 µm being reached at the highest
content of Au, i.e. x = 0.8. The ternary phase of AuNPs at x = 0.8
formed spherical nanoparticles with sizes in the 15–27 nm range and
their appearance coincided with a rapid degeneration in the density and
adhesion of osteoblastic cells grown on the scaffolds. In spite of this
adverse effect, the cell spreading and proliferation improved in direct
proportion with the increasing amount of Au dopant in Au/Se-CHAP
particles of the scaffold, showing gradual improvements throughout the
entire x = 0.0–0.6 range, suggesting that the biological effects of Au in
the ionic and the nano particulate form on the implant integration
process may be diametrically opposite. The porosity and the surface
roughness all underwent changes with the amount of Au in the Au/Se-
CHAP particles and their highest values were 91.72% at x = 0.4 and
33 nm at x = 0.8. The addition of Au had a dramatic effect on some, but
not all mechanical properties. The most intense effect was detected on
toughness and strain at break, which both recorded twice lower values
upon the introduction of Au into Se-CHAP at the lowest amount
(x = 0.2). On the other hand, changes imposed on the tensile strength
and elongation rate were relatively minor, paralleling for the most part
the aforementioned changes to the crystallinity of the polymeric phase
caused by the variations in the amount of Au in the Au/Se-CHAP par-
ticles. The significant variation of physical and biological properties of
these composite scaffolds with trace changes in the content of the Au
dopant inside the HAP particles used as a filler is promising, as it
provides a new, relatively subtle avenue for tailoring the properties of
tissue engineering scaffolds for their intended biomedical applications.
CRediT authorship contribution statement
M.K. Ahmed: Investigation, Methodology, Funding Acquisition,
Writing – Review & Editing. S.F. Mansour: Supervision, Funding
Acquisition, Writing – Original Draft. Reem Al-Wafi: Data Curation,
Visualization, Writing – Original Draft. M. Afifi: Investigation,
Visualization, Methodology, Writing – Original Draft. Vuk Uskokovic:
Supervision, Writing – Review & Editing.
12
International Journal of Pharmaceutics 577 (2020) 118950
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
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