Materials Science and Engineering C 69 (2016) 675–684

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Materials Science and Engineering C

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

Synthesis and characterization of silver doped hydroxyapatite

nanocomposite coatings and evaluation of their antibacterial and
corrosion resistance properties in simulated body fluid
Majid Mirzaee a,⁎, Mohammadreza Vaezi b, Yahya Palizdar b
a
School of Metallurgy and Materials Engineering, College of Engineering, University of Tehran, P.O. Box 11155-4563, Tehran, Iran
b
Research Department of Nano-Technology and Advanced Materials, Materials & Energy Research Center, Iran

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

Article history: Silver-doped hydroxyapatite (Ca10 − xAgx(PO4)6(OH)2 − x) films were synthesized and deposited on anodized
Received 29 March 2016 titanium (Ti) using electrophoretic. The influence of different silver-dopant contents (X = 0, 0.02, 0.05, 0.08
Received in revised form 21 June 2016 and 0.1) on the phase formation and microstructure of the powders were characterized by means of X-ray dif-
Accepted 20 July 2016
fraction (XRD), transmission electron microscopy (TEM), X-ray photoelectron spectroscope (XPS), and Fourier
Available online 22 July 2016
transform infrared spectrum analysis (FT-IR). XRD analysis confirmed the formation of Hexagonal structure of
Keywords:
hydroxyapatite (HAp) annealed at 600 °C with a small shift in the major peak position toward lower angles
Sliver doped hydroxyapatite with adding silver. FT-IR spectroscopy disclosed the presence of the different vibrational modes matching to
TiO2 nanotubes phosphates and hydroxyl groups and the absence of any band characteristics to silver. XPS analysis showed
Electrophoretic that 75% and 23% of silver was in the chemical states of Ag2+ and Ag+, respectively. However, only about 2% of
Corrosion stability silver was in the Ag0 state, resulting in the high quality of nanocomposite films. The anodization treatment im-
proves the bond strength between the Ag doped HAp deposited layers on TiO2. HAp and silver doped HAp
(X = 0.05) are regarded to be hydrophilic due to a large number of –OH groups on the surface. The sample
with content of silver (x = 0.05) also showed excellent antimicrobial efficacy (N99% reduction in viable cells).
Electrochemical reveals the passive current densities of the HAp coated anodized Ti are lower than those of silver
doped HAp coated anodized Ti, leading to a slightly lower corrosion resistance.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction are their mechanical properties, especially for macroporous scaffolds

Hydroxyapatite (HAp) materials have drawn great interest from re-
searchers because they are widely applied as biomedical materials, in-
cluding such uses as bone fillers [1–3], bone tissue engineering
scaffolds [4], bioactive coatings [1], soft tissue repairs [5–8], drug/pro-
tein/gene loading, delivery systems [9–15], and column chromatogra-
phy for the rapid fractionation of biomolecules [16,17] because of their
excellent biocompatibility, osteoconductive properties, and similarity
to the inorganic component of human bones [18]. Pure HAp is a stoi-
chiometric apatite phase with a Ca/P molar ratio of 1.67, the most stable
calcium phosphate salt at normal temperatures and pH between 4 and
12[19]. The structure consists of an array of PO4 tetrahedra held togeth-
er by Ca ions interspersed among them. The Ca ions occur in two mark-
edly different sites, in accurately aligned columns (Ca (I)) and in axes,
and the adjacent OHs point in opposite directions [20]. HAp bioceramics
are usually implanted in the form of granules and porous scaffolds.
However, the major limitation to use HAp as load-bearing biomaterials
⁎ Corresponding author.
E-mail address: majidmirzaee7@gmail.com (M. Mirzaee).
[21]. So far, it is still a great challenge to improve the mechanical prop-
erties of HAp materials. The application of nano-sized Al2O3, ZrO2, TiO2,
metals, and carbon nanotubes, etc., as reinforcement components is the
most common method to solve this problem [22,23]. One of the rare but
critical problems in orthopedics and dentistry is implant associated in-
fection. Removing the implant is usually needed due to biofilm forma-
tion and the consequent resistance of the organisms to antibiotic
therapy even if the bacteria are inherently sensitive. Biofilm formation
starts with adherence to a surface with the help of flagella and pili in
Gram-negative bacteria or surface proteins in Gram-positive bacteria.
The biofilm grows quickly and produces extracellular matrix, comprised
of expolysaccharides, and small amounts of protein, DNA, bacterial lytic
products, and compounds of the host. Finally, a dispersal stage in which
some bacteria are released from the biofilm matrix for colonizing new
surfaces to restart the cycle happens. Biofilms are found in N60% of
chronic wound infections. The wounds are sometimes colonized by
one type of microorganisms or, more often, by several species, the
most common bacteria being Staphylococcus aureus and Pseudomonas
aeruginosa. One of the salient features of biofilms is their increases tol-
erance to antimicrobial agents. Biofilms can resist up to 100–1000 times

http://dx.doi.org/10.1016/j.msec.2016.07.057
0928-4931/© 2016 Elsevier B.V. All rights reserved.
676 M. Mirzaee et al. / Materials Science and Engineering C 69 (2016) 675–684

higher concentrations of antibiotics and disinfectant s than planktonic
cells. In the following, some of the proposed mechanisms for contribut-
ing to biofilm phenotypic resistance are explained. The
expolysaccharide matrix and biofilm structure decrease diffusion and
operate as a primary barrier, preventing the entrance of polar and
charged antibiotics. Alginate and extracellular DNA, components of
the extracellular matrix, have shown antibiotic-chelating activity. Such
a resistance relies on the existence of antibiotic and can be specific or
non-specific for a particular antimicrobial. The upregulation of efflux
pumps is one of the most universal non-specific mechanisms. Many
studies revealed differential expression of several conventional and
biofilm-specific antibiotic resistance genes in biofilms as compared
with planktonic growth. Furthermore, the phenotypes produced by spe-
cific resistance mechanisms are able to mainly differ between biofilm
and planktonic growth. The biofilm structure leads effective horizontal
gene transfer between bacteria, having a significant role in the
development of resistance to antibiotics. [24–26]. Because of this and
the problem of increasing global antibiotic resistance, alternative strat-
egies are required [27]. Titanium stands for the most stable implant
component with minimal effects on the surrounding body tissue involv-
ing the inflammation. Titanium biomaterials in general rely upon the
formation of an extremely thin (about 5–20 nm thick), hard adherent,
protective titanium oxide film that is principally TiO2.·The enhance-
ment or interference with this adherent TiO2 film caused by alloying el-
ements defines whether alloys lead to the promotion or limitation of
general corrosion resistance and superior biocompatibility comparing
with pure Ti grades. A feasible solution is adding silver to calcium phos-
phate ceramics, which has already turned out to have acceptable results
[28,29]. Silver doped HAp shows promising antimicrobial properties,
but the concentration of released silver ions must be kept low to avoid
cytotoxic effects [30]. Effective suppression of accelerated grain growth
is critical for the fabrication of nano-ceramics, in which lower sintering

Fig. 1. A flow chart for silver doped HAp deposited on anodized Ti.
 M. Mirzaee et al. / Materials Science and Engineering C 69 (2016) 675–684
temperature, faster calcination rate, and shorter sintering time are al-
ways required. There are assorted methods to deposit silver doped
HAp coatings on metal surfaces such as pulsed laser deposition,
sputtering, sol-gel, electrophoresis, and plasma spraying. Also, electro-
phoretic deposition (EPD) appears as a way of choice as a result of its
simple assemble and formation of uniform coatings on substrates with
complex shapes. Many papers reported high concentrations for doping,
which are dangerous for body due to the poisonous essence of this ele-
ment, and the coating strength is low. In this paper, with small amount
of Ag and exact control of the Ag amount using XPS and releasing tests
and high adherence due to the existence of anodized titanium oxide, the
highest assurance level was achieved for placing the sample in the sim-
ulated environment for body. The aim of this research is to investigate
the corrosion stability and bioactivity of silver doped hydroxyapatite
coatings on anodized titanium in simulated body fluid (SBF). The syn-
thesized samples were characterized by XRD, FT-IR, AAS, TEM, Contact
Angle, XPS and EIS. Bioactivity test through SBF and the antibacterial ef-
ficacy against Escherichia coli and S. aureus by agar diffusion test was
studied.
2. Material and methods
2.1. Materials
Calcium nitrate tetrahydrate (Ca(NO3)2·4H2O), diammonium hy-
drogen phosphate ((NH4)2HPO4), ethylene glycol and ammonium fluo-
ride were obtained from Sigma-Aldrich, Canada. All chemicals were of
analytical grade or higher and used as received. Ammonium hydroxide
(NH4OH, 30%), anhydrous ethanol (EtOH), and silver nitrate (AgNO3)
were purchased from Sigma-Aldrich, Canada and used as received.
2.2. Film preparation
Titanium dioxide nanotubes were grown on foils of Ti (99.7% purity,
Sigma-Aldrich) with a geometric area of 1 cm2 and 0.25 mm thick. Very
briefly, Ti stripes were polished with emery papers no 400–3000 to ob-
tain a mirror finish and subsequently, cleaned in acetone and deionized
water in an ultrasonic bath. The cleaned samples were anodized at 40 V
for 2 h in ethylene glycol containing 0.5 wt% ammonium fluoride and
2.5 wt% of water at room temperature in a two-electrode cell using plat-
inum foil as a counter electrode. The anodized samples were further
washed with deionized water and ultrasonically cleaned for 60 s in de-
ionized water to remove surface debris and subsequently, dried in air.
The anodized samples were annealed at 450 °C for 2 h to form the ana-
tase phase.
2.3. Preparation of silver doped HAp-TiO2 coatings
The general procedure is as follows: first, (NH4)2HPO4 was dissolved
deionized water. The solution was stirred for 30 min with a magnetic
stirrer, and then the pH value was adjusted to 10 by adding ammonium
hydroxide. After this, Ca(NO3)2·4H2O and AgNO3 were dissolved in
46.67 mL of DI water. Afterward, the Ca(NO3)2·4H2O + AgNO3 solution
was added to the (NH4)2HPO4 solution dropwise while stirring.
Dropwise addition of ammonia was used to adjust the pH of the solution
to 10. The resulting white suspension was then stirred at room temper-
ature for 4 h and then filtered, washed thoroughly with hot distilled
water to remove the remaining NH− 3 and NH3 ions in the filtrate. Finally,
the product centrifuged to separate the precipitates from the suspen-
sion. This procedure gave the silver doped HAp precursor. Silver
doped HAp suspension was prepared by dissolving 1.0 g of nanosized
silver doped HAp powder in 100 mL absolute ethanol. Then, HCl was
added until the pH value of 2.00 was reached to obtain stable suspen-
sions. Subsequently, the silver doped HAp suspensions were ultrasoni-
cally treated for 15 min to obtain a homogeneous suspension. A three-
electrode cell arrangement was used for cathodic electrodeposition.
677
The working electrode was an anodized titanium foil, and the counter
electrodes were two titanium foils, placed parallel to the working elec-
trode at a distance of 1.5 cm. The coatings were deposited from ethanol
suspensions, using the constant voltage method. The experiments were
performed at 40 V for a deposition time of 300 s, at room temperature.
Electrodeposited coatings were dried for 12 h at room temperature. Af-
terward, the coatings were sintered at 600 °C for 30 min in an air atmo-
sphere with the initial heating rate of 5 °C/min. Fig. 1 shows the overall
flowchart for the silver doped HAp deposited on anodized Ti using the
electrophoretic technique.
2.4. Characterization of coatings
A transmission electron microscope (TEM; JEOL 2010) was used to
investigate the microstructures of the HAp and silver doped HAp. The
surface morphology and elemental composition of the composite depo-
sition were examined using SEM (SEM-JEOL JSM-6400, Japan). The stat-
ic water contact angles were measured at 25 °C by a contact angle meter
(Rame-Hart-100) employing drops of SBF. The readings were stabilized
and taken within 120 s after adding. An X-ray diffractometer (XRD;
X'pert PRO, Philips, The Netherlands) was used to identify the crystal-
line phases of the coatings. Fourier transform infrared spectroscopy
(FTIR; Nicolet 5700, Thermo) was used to determine the chemical com-
position of the deposited coatings. The adhesive strengths of the coat-
ings were measured using a universal testing machine (Instron 5567).
The adhesion strength was calculated as the load at failure divided by
the area of samples. The data were the average of three samples. X-ray
photoelectron spectroscopy (XPS) as a surface chemical analysis of
sintered Ag/HAp coatings was performed with an Axis-Ultra spectrom-
eter (Kratos) using non- and monochromatized Al Kα radiation
(1486.6 eV) and a hemispherical analyzer. For electrochemical imped-
ance spectroscopy (EIS) measurements, electrodeposited Ag/HAp/TiO2
coating and bare titanium, were exposed to the SBF solution at a
temperature of 37 °C for 24 h. The SBF solution was prepared by dis-
solving the reagent-grade salts NaCl, NaHCO3, KCl, K2HPO4·3H2O,
MgCl2·2H2O, CaCl2, and Na2SO4 in deionized water and buffered in
trishydroxymethyl aminomethane, in pH adjusted to 7.40 with 1 M hy-
drochloric acid, at 37 °C. The working electrode was coated titanium or
thermally treated bare titanium (tested surface area of 1 cm2), the coun-
ter electrode was a platinum mesh, and the reference electrode was a
saturated calomel electrode (SCE). The impedance data were obtained
at the open-circuit potential using a Reference 600 Potentiostat/
Galvanostat/ZRA (Gamry Instruments, Inc., Warminister, PA), over a fre-
quency range of 300 kHz to 10 mHz using a 5 mV amplitude of sinusoi-
dal voltage.
Fig. 2. XRD patterns of HAp and Ag-HAp layers on the anodized surface with different Ag-
doping contents.
678 M. Mirzaee et al. / Materials Science and Engineering C 69 (2016) 675–684

Table 1 2.5. Ag release and antibacterial tests

XRD parameters silver doped HAp with different Ag-doping contents calculated for major
(211) peak.
To examine Ag release, silver doped HAp coatings were soaked in
Sample Crystallite d-Spacing Unit cell parameter Unit cell parameter 5 mL of PBS at various time periods (1 to 14 days). The Ag concentration
size (nm) (nm) (a-axis (A0)) (c-axis (A0)) of the collected solution was evaluated by using an atomic absorption
HAP 58 ± 0.36 0.29123 9.4180 ± 0.005 6.8750 ± 0.006 spectrophotometer (AAS; Z-2300, Hitachi, Japan). Staphylococcus aureus
HAP-0.02 Ag 49 ± 0.092 0.29197 9.4220 ± 0.006 6.8770 ± 0.001 (ATCC 25923) and Escherichia coli (ATCC 25923) were used to appraise
HAP-0.05 Ag 43 ± 0.86 0.29238 9.4230 ± 0.002 6.8780 ± 0.002
the antibacterial activity of the coatings. The culture was maintained
HAP-0.1 Ag 37 ± 0.9 0.29238 9.4270 ± 0.006 6.8790 ± 0.004
through subculturing every two months on solid nutrient medium at

Fig. 3. Typical XPS spectra for silver doped HAp powders annealed at 600 °C with Ag-doping contents of 0.05: (a) wide scan spectra; (b) Ca 2p; (c) P 2p (d) O 1s and (e) Ag 3d.
 M. Mirzaee et al. / Materials Science and Engineering C 69 (2016) 675–684 679

37 °C and kept at 4 °C between experiments. Test bacteria was activated where λ, β, and θ are the wavelength of the X-rays, the full width at half
by two successive precultures in LB broth (composition yeast extract maximum (FWHM) of (211) diffraction peak, and the diffraction angle
5 g L−1, tryptone 10 g L−1, NaCl 5 g L−1) and incubated at 37 °C during of the XRD spectra, respectively . The resulting d-spacing (nm) and crys-
the night. Before antibacterial test, overnight culture, not older than tallite size (nm) are shown in Table 1 as a function of different silver
18 h, was diluted in physiological solution (10−1) and 2% (v/v) of cul- doping contents. According to this table, the crystallite size of the
ture was used to inoculate test tube with 7 mL of sterile modified films decreased with the increase of the Ag-doping level. The crystal lat-
phosphate-buffered (PB) solution (pH = 7.4) with 15 mg titanium coat- tice parameters, a and c, were calculated using the Eq. (3),
ed samples. The initial number of bacterium in each suspension was be-
tween 106–107 CFU mL− 1, and the concentration of coating material    2 

was approximately 2 mg mL−1. Thus, the prepared samples were incu- 1
¼
4 2 2
h þ k þ hk þ l
2 a 1
ð3Þ
2 3 c2 a2
bated for 24 h at 37 °C, without shaking. Titanium coated samples with- dhkl
out silver (HAp coatings) were also used as control [31]. The number of
bacterium in a sample was monitored at the beginning of the experi-
ment and after 1.5 h, 3 h, and 4.5 h of the incubation. The percentage
of microorganism reduction (R) was calculated by Eq. (1):
3.2. Surface elemental property (XPS)
C0 −C
R¼  100% ð1Þ The X-ray photoelectron spectroscopy of silver doped HAp powders
C0
is shown in Fig. 3a. In the XPS spectrum of the Ag doped HAp, the bind-
ing energies of Ca (2p, 347.3 eV), P (2p, 133.09 eV), and O (1s, 532.1 eV)
where C0 and C are the number of microorganism colonies on the con-
are distinct (Fig. 3a). In the XPS spectrum of the HAp powders with sil-
trol tube and microorganism colonies on the samples, respectively.
ver nanoparticles was observed that the Ag 3d-line was formed by the
contribution of two states where the peaks of Ag (Ag (3d5/2) 367.6 eV)
3. Result and discussion
and Ag ((3d3/2) 373.5 eV) corresponded well with the values in litera-
ture [34]. Silver is a metal that has anomalous properties in BE shifts
3.1. X-ray diffraction studies (XRD)
while being oxidized, i.e. the Ag 3d peaks shift to lower BE values [35].
Usually, positive BE shifts in the metal core-level peaks are observed
The XRD patterns of the samples after depositing the HAp and Ag-
when metal is oxidized, which is explained by considering the electro-
HAp layers on the anodized surface are shown in Fig. 2. The typical
negativity differences between metal atom and cation. Factors such as
HAp (JCPDS no. 09-4302), anatase phase (JCPDS 21-1272) and Ti
lattice potential, work function changes, and extra-atomic relaxation
(JCPDS 44-1294) diffraction peaks can be seen in both HAp and Ag-
energy lead to negative BE shift in the case of Ag [36]. When silver nano-
HAp coatings. No intense diffraction peaks corresponding to the Ag
particles containing nanocomposites were prepared by thermal treat-
were observed, which is possibly cause by the stable silver-ammine
ment, different states of silver, contributing to the Ag3d-line in the
complex ion and the fast reaction between phosphate ion and silver
XPS spectra, have been observed (Fig. 2e). The binding energies of the
ion in the solution. However, the changes in the crystal structure
Ag (3d5/2) core level for Ag, Ag2O, and AgO are 368.6, 368.2, and
show the substitution effect, where a slight shift to the lower degree po-
367.8 eV, respectively. Based on the deconvolution analysis, about 75%
sition in Fig. 2 was observed, which suggests the significant incorpora-
and 23% of silver was in the chemical state of Ag2+ and Ag+, respective-
tion of the dopants into the HAp lattice and an increment in the unit
ly; however, only about 2% of silver is in the Ag0 state. The spectra of O
cell of the HAp in the a- and c-axes as well as the unit cell volume.
1s (Fig. 2d) can be split into two peaks at 529.57 eV and 531.18 eV. The
When a part of the Ca2 + (0.099 nm) sites is replaced by Ag+
first transition belongs to HAp system. The shifting of O1s spectra in the
(0.128 nm), the packing of the larger size atoms tends to distort the lat-
second transition could be related to the change of the surrounding ox-
tice parameters. Another possibility for this deviation may be due to a
ygen atoms.
strain effect due to the thermal expansion coefficient mismatch be-
tween the film and the substrate. The shift of the diffraction peaks to-
ward lower angles observed at 600 °C may be a result of a distortion
in the hexagonal structure due to the increase of the thermal
microstrains in the lattice, particularly observed for high index surfaces
[32]. The observed new diffraction peaks suggested the formation of
crystalline phases, such as CaO (JCPDS No. 74-1226), confirming the
slight decomposition of HAP during the sintering process.
The peak broadening and intensity reduction with Ag doping may be
due to the decrease in crystallites size of HAp powders and crystallinity
with adding Ag dopants. The observed peak shift with increasing Ag-
doping content can be described as follows. From a crystal-chemical
point of view, the substitution of Ag+ (1.28 A °) ions takes place for
Ca2+ (0.99 A°) preferentially in the Ca(1) site of HA, and this leads to
an increase in the lattice parameters linearly with the amount of silver
added in the range of atomic ratio Ag/(Ag + Ca) between 0 and 0.055.
The proposed general formula is Ca10-xAgx(PO4)6(OH)2-x, with vacan-
cies at the hydroxyl site due to charge imbalance caused by Ag+ for
Ca2+ ions, although PO3− 2−
4 for HPO4 is also a likely substitution to com-
pensate the charge imbalance [33]. The crystallite size (D) of the films
was calculated using the Scherrer's Eq. (2),

0:89λ
D¼ ð2Þ Fig. 4. FTIR spectra of the HAp and silver doped HAp powders at 150 °C with different Ag-
ðβ cosθÞ doping contents.
680 M. Mirzaee et al. / Materials Science and Engineering C 69 (2016) 675–684

3.3. FTIR spectra of silver doped HAp coatings 3.4. Microstructural studies and static contact angle measurement

0.09pt?>Fig. 4 shows the FTIR spectra of HAp prepared from differ-
ent silver contents at 150 °C. The spectra display typical phosphate
−1
(PO3−
4 ) characteristic bands. The 963 cm and 470 cm−1 are assigned
to the γ1 and γ2 vibration modes, 1034 and 1092 cm−1 derive from the
γ3 mode, and 565 and 601 cm−1 correspond to the γ4 mode. Moreover,
the bands at 3569 and 631 cm−1 represent stretching and librational vi-
bration of OH−, respectively. Additional weak bands at 870, 1410, and
1460 cm−1 are assigned to carbonate species (CO2−3 ) in the apatite lat-
tice [37,38]. Although the carbonates were not detected by XRD results,
these peaks in the FTIR spectrum can be attributed to the reaction be-
tween CaO, confirmed in the XRD pattern (Fig. 2), and CO2 from the at-
mosphere. The band at ca. 1640 cm−1 is from water (bending modes).
Although the attendance of silver does not affect the structure of the
HAp, no difference between the HAp and silver doped HAp powders
in the shapes of the bands was discerned.
The surface morphology and size of the synthesized HAp and silver
doped HAp nanoparticles with Ag = 0.05 at.% are presented in Fig. 5a,
b, respectively, which were estimated by TEM. As shown in these im-
ages, HAp and silver doped HAp nanoparticles are particles with ap-
proximately 50–70 and 30–45 nm in size, respectively. The addition of
impurity (silver) increases interfacial energy, and they act as nucleation
centers. Therefore, the nucleation rate increases; as a result, further
crystal growth is inhibited [39]. The surface coverage for both thin
films is similar, without any remarkable cracks. The microstructure of
silver doped HAp was more homogenous than the microstructure of
HAp. EDAX examination clearly exhibited the proximity of Ca, P, O and
Ag in the HAP. Afterwards, EDAX investigation confirmed that no unde-
sirable components were discovered in the structure.
Fig. 5c displays well-developed and arrayed TiO2 nanotubes, which
are desirable. It was found that the average diameter of the nanotubes

Fig. 5. TEM micrographs and EDAX spectra of sample (a) undoped HAp (b) silver doped HAp annealed at 600 °C with Ag-doping contents of X = 0.05. The inset shows contact angle
measurements of SBF solution at the surface of (a) undoped HAp and (b) silver doped HAp (c) SEM images of orderly aligned TiO2 nanotubes (d) cross-section of Ag doped HAp
coating on TiO2.
 M. Mirzaee et al. / Materials Science and Engineering C 69 (2016) 675–684 681

was 100 nm. Fig. 5d shows the cross-sections of Ag doped HAp coating
display a thickness of roughly 24.2 μm on a Ti substrate. The hydrophi-
licity of HAp and silver doped hydroxyapatite could be measured with
the contact angle to describe their biocompatibility. A low contact
angle is usually favorable in order to achieve high surface coverage. As
inset shows, the contact angles of HAp/anodized Ti and silver doped
HAp/anodized Ti were 34.3 ± 1.1° and 19.0 ± 0.3°, respectively. HAp
and silver doped HAp are regarded to be hydrophilic due to a large num-
ber of –OH groups on the surface [40,41].

3.5. Adhesion strength

Fig. 6 shows the adhesive strength of the pure HAp and silver doped
HAp deposited on Ti foil ware 16.9 and 15.2 MPa respectively. However,
there is an improvement in the adhesion strength of Ag doped HAp de-
posited on TiO2 at room temperature (16.2 MPa), which is even higher
than the adhesion strength of HAP. Moreover, the anodization treat-
ment improves the bond strength between the Ag doped HAP deposited
layers on TiO2 because of the anchoring effect, nucleation, and growth of Fig. 7. Silver ion release from silver doped HAp annealed at 600 °C with Ag-doping
the HAp inside the nanotubes [42]. contents of X = 0.05 coating into PBS during 14 days of immersion.

3.6. In-vitro and antibacterial activity properties of the silver doped HAp inactivation of proteins and critical enzymes for the respiratory chain.
nanocomposite Similar results have been shown for a wide range of bacterial species
and strains, including MRSA, making silver a broad-spectrum antibacte-
In Fig. 7, the Ag+ ions are released quickly from the sample in the rial agent. These bactericidal effects are enhanced by the use of AgNPs,
first several days. The Ag release and antibacterial activity values were which by means of their small size, they can release more Ag+ due to
measured at three different samples, and the average values were re- an increased surface to the volume ratio and can also disturb or
ported. The Ag+ ion release rate is directly correlated with the antibac-
terial activity of the coating. After 7 days, the release speed diminishes
and reaches a near steady-state, which indicates a long-term sustain-
able release. The Ag+ ion of 0.2 ppm is released during the first day,
which then reached 1.52 ppm. Both values are higher than the mini-
mum required value of 0.1 ppm to have a bactericidal effect in the cul-
ture, but the values are lower than the cytotoxic concentration of
1.6 ppm for somatic. The good bactericidal effect without cytotoxicity
implies excellent behavior of the long-term Ag+ ion release that form
the Ag HAp coating [43].
Almost all antimicrobial and immunological tests are routinely de-
veloped using planktonic inoculums. Bacterial biofilms protect the mi-
crobial community from external damage and promote the
persistence of chronic infections. Interactions between human macro-
phages and bacterial inoculums of planktonic and biofilm modes of
growth have been explored using Escherichia coli (E. coli) and
S. aureus. Antimicrobial mechanism of action of silver ions to microor-
ganisms' cells is very complex. It was shown that Ag+ interferes with
bacterial DNA molecules of E. coli and S. aureus, causing DNA condensa-
tion that result in lost replication abilities [44]. The same study also sug-
gested that Ag+ interact with essential thiol groups, inducing the

Fig. 8. Counting of viable cell numbers (CFU/mL) for (a) E. coli and (b) S. aureus treated
Fig. 6. Adhesion strength of coated samples. with HAp or silver doped HAp samples.
682 M. Mirzaee et al. / Materials Science and Engineering C 69 (2016) 675–684

penetrate the bacterial cell membrane to induce further damage [45].

Comparing the AgNO3 treated E. coli and S. aureus with the control set-
ting, they were found that: the free state of DNA changed to a con-
densed form in the cells; many electron-dense granules appeared
surrounding the cell wall, and the existence of silver in the cytoplasm
and DNA molecules. The above results lead to the following suggestions
about the bactericidal mechanism of silver ions against E. coli and
S. aureus: as a reaction against the denaturation effects of silver ions,
DNA molecules become condensed and lose their replication abilities,
and silver ions interact with thiol groups in protein, which induce the
inactivation of the bacterial proteins [45]. In a study, TEM analysis of un-
stained bacteria showed the external morphological features of the two
bacterial strains. The untreated S. aureus cells retained their coccal mor-
phology (ca. 600 nm in diameter) and seemed to be normal. In contrast,
S. aureus cells treated with the silver ion solution for 2 h appeared to un-
dergo lysis, resulting in the release of their cellular contents into the sur-
rounding environment, and finally became disrupted. It was common to
find electron-dense particles or precipitates around damaged bacterial
cells that were electron translucent in comparison to undamaged cells. Fig. 10. Polarization curves of the samples in SBF solution at 37 °C.

However, significant morphological changes were observed in

S. aureus cells treated with the silver ion solution. They showed lysed
cells with broken walls and membranes and decreases and heterogene-
ity in electron density in the cytoplasm. The localized separation of the
cell membrane from the cell wall could be discerned. E. coli cells diluted
in PBS showed normal morphology having many filaments, such as fla-
gella and fimbriae. The cells showed aberrant morphology; they were
cracked and ruptured. Electron-dense particles or precipitates were
also observed around damaged bacterial cells. The internal showing a
multilayered cell surface consisting of an outer membrane, a peptido-
glycan layer in the periplasmic space, and a cytoplasmic membrane.
Damaged cells showed either localized or complete separation of the
cell membrane from the cell wall. The efficacy of the silver ion solution
showed better activity against the gram-negative E. coli than against the
gram-positive S. aureus. This was possibly due to the thickness of the
peptidoglycan layer, which may prevent the action of the silver ions
through the bacterial cell wall, and this result was consonant with the
results of other studies [46]. The results of pertaining to quantity antimi-
crobial tests in the liquid medium exhibit that all silver doped HAp sam-
ples show a viable cell reduction of all microorganism species (Fig. 8).
The reduction is most pronounced with the HAp-0.1Ag sample, which
suggests that the antimicrobial efficacy of the materials is a function of
the silver content. In Fig. 9a and b, the viable cell reduction percentages
of the specimens are illustrated. Studies showed that E. coli and S. aureus
are the most vital pathogens in biomaterial associated with infections.
Silver which contains antimicrobial biomaterials is comprised of silver
salts/silver complexes in organic or inorganic matrices (polymers or
bioglasses/HAp respectively). This is more remarkable with E. coli,
where a complete reduction of viable cell number is attained within
3 h of contact with the HAp-0.1Ag sample (Fig. 9(a)). Sample HAp-
0.05Ag demonstrated N99% reduction in viable cells after first hour.
The sample with smallest content of silver also showed excellent

Fig. 11. Nyquist plots of the samples in SBF solution after 24 h of exposure to SBF solution
at 37 °C. The inset shows equivalent electrical circuits and physical models for silver doped
Fig. 9. Antimicrobial activity of undoped HAp and silver doped HAp with different Ag- HAp annealed at 600 °C with Ag-doping contents of X = 0.05 coating to SBF solution at
doping contents coatings against (a) E. coli and (b) S. aureus. 37 °C.
 M. Mirzaee et al. / Materials Science and Engineering C 69 (2016) 675–684
Table 2
Fitting values of electrical circuit parameters.
Sample Rs (Ω·cm2) Qb (μF·cm−2) Rb (kΩ·cm2)
Ti 22.5 38.8 52.6
TiO2 31.5 – –
Ag-HAp 51.4 – –
antimicrobial efficacy. As a result, Ag can inhibit the growth of gram-
positive bacteria, and bacteria attachment on Ag-doped HAp will be re-
duced when compared to HAp coatings and Ti surfaces. According to the
results illustrated in Fig. 9(b), bacterial adhesion was significantly re-
duced for S. aureus on silver doped HAp surfaces compared to HAp
and Ti surfaces, but the S. aureus strain is not considerably less capable
to all silver doped HAp samples than E. coli. S. aureus were inhibited at
the mild concentration of Ag nanoparticles, whereas the growth-
inhibitory on E. coli effects were at the low concentration of Ag nanopar-
ticles. HAp-0.1 Ag showed the highest antimicrobial activity, reducing
the number viable cells of S. aureus for N99%. One of the possible expla-
nations of that difference in the capability between E. coli and S. aureus
bacterium is the differences in the cell wrapper structure of them, which
is described in other works [47–49]. HAp without silver ions showed
some cell reduction as well. This suggests that there is some adhesion
of microorganism cells to the particles of HAP, which may contribute
to the overall reduction of cell number by tested samples.
3.7. Corrosion behavior testing & electrochemical impedance spectroscopy
of silver doped HAp coatings
According to potentiodynamic polarization plots presented in
Fig. 10, the nanotubular structure decreases corrosion current density
of the Ti substrate in SBF. Coatings with uncompressed structures such
as HA on Ti (coatings with open defects); allow easy access to oxygen
in the solution and free flow of species, which enhances the regenera-
tion of protective oxide films and better corrosion protection, but this
assertion is not true about substrate's own oxide film [50]. HA coating
deposited on TiO2 nanotubes, hinders electron transport and ion at the
coating/electrolyte interface and also promotes the regeneration of the
TiO2 protective layer through its uncompressed structures, which re-
sults in corrosion protection of the substrate and reduced icorr values.
Because the surface morphology of the silver doped HAp coating is loos-
er than the HAp coating the passive current densities of the HAp coated
Ti are lower than those of silver doped HAp coated Ti, leading to a slight-
ly lower corrosion resistance.
The electrochemical impedance results (Nyquist plot) of bare Ti and
TiO2/Ag doped HAP composite coated Ti immersed in SBF solution are
demonstrated in Fig. 11. Low frequency extent is ascribed to the charac-
teristics of the passive titanium oxide film, whereas the high frequency
extent illustrates the characteristics of silver doped HAp coating. The
equivalent circuit used for the fitting of impedance data for TiO2/silver
doped HAp coating during initial time of disclosure comprises of, Ra im-
pedance at hydroxyapatite/SBF solution interface; Rc impedance at TiO2
coating/SBF solution interface, and Rb impedance at Ti metal/SBF solu-
tion interface, where Qb and Qc are the double layer capacitance of bar-
rier and coated layers, respectively. The formation of a new apatite layer
on the coating surface in SBF solution could also be indicated. After 24 h,
the coating pores filled with the SBF solution were growing into a very
resistant passive film. The calculated values are presented in Table 2.
4. Conclusion
In this study, the characteristics of different silver-dopant contents
doped HAp were studied. The analysis of FTIR, XRD and TEM showed
that particles of silver doped HAp samples are of nano size and homog-
enous in the composition, while a lower value of contact angle in SBF,
determined by the drop test, proved its better wettability compared to
683
Qc (μF·cm−2) Rc (kΩ·cm2) Qa (μF·cm−2) Ra (kΩ·cm2)
– – – –
149.3 246.9
– – 88.5 11.9
the HAp coating. XRD analysis confirmed the formation of hexagonal
structure of hydroxyapatite (HAp) annealed at 600 °C with a small
shift in the major peak position toward lower angles with adding silver.
XPS spectra confirmed the formation of the chemical states of Ag2 +,
Ag+ and Ag0. The results showed that due to the anchoring effect, the
TiO2 nanotubes layer improves the bond strength by N16 MPa. In accord
with the results of antimicrobial tests, antimicrobial efficiency of the sil-
ver doped HAp is a function of quantities of silver ions, which silver
doped HAp samples with low silver content have excellent activity
against E. coli and S. aureus. The corrosion tests also indicated the silver
doped HAp coatings have effective corrosion resistance. Therefore, sil-
ver doped HAp with X = 0.05 could be a better choice especially to pre-
vent device associated infections.
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