Materials Science and Engineering C 35 (2014) 36–42

Contents lists available at ScienceDirect

Materials Science and Engineering C

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

Hydroxyapatite-silver nanoparticles coatings on porous

polyurethane scaffold
Gabriela Ciobanu ⁎, Simona Ilisei, Constantin Luca
“Gheorghe Asachi” Technical University of Iasi, Faculty of Chemical Engineering and Environmental Protection, Prof. dr. docent Dimitrie Mangeron Rd., no. 63, 700050, Iasi, Romania

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

Article history: The present paper is focused on a study regarding the possibility of obtaining hydroxyapatite-silver nanoparticle
Received 3 July 2013 coatings on porous polyurethane scaffold. The method applied is based on a combined strategy involving hydroxy-
Received in revised form 28 September 2013 apatite biomimetic deposition on polyurethane surface using a Supersaturated Calcification Solution (SCS), com-
Accepted 19 October 2013
bined with silver ions reduction and in-situ crystallization processes on hydroxyapatite-polyurethane surface by
Available online 31 October 2013
sample immersing in AgNO3 solution. The morphology, composition and phase structure of the prepared samples
Keywords:
were characterized by scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (SEM-
Polyurethane EDX), X-ray diffraction (XRD), UV-Vis spectroscopy and X-ray photoelectron spectroscopy (XPS) measurements.
Hydroxyapatite The data obtained show that a layer of hydroxyapatite was deposited on porous polyurethane support and the
Silver nanoparticles silver nanoparticles (average size 34.71 nm) were dispersed among and even on the hydroxyapatite crystals.
Coating Hydroxyapatite/polyurethane surface acts as a reducer and a stabilizing agent for silver ions. The surface plasmon
resonance peak in UV-Vis absorption spectra showed an absorption maximum at 415 nm, indicating formation of
silver nanoparticles. The hydroxyapatite-silver polyurethane scaffolds were tested against Staphylococcus aureus
and Escherichia coli and the obtained data were indicative of good antibacterial properties of the materials.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction plasma spraying, sol–gel, electrophoretic deposition, hydrothermal

and biomimetic methods are usually applied to produce bioactive
One of the most important applications of a biomaterial (polymeric hydroxyapatite coatings over the solid implants [6–8].
or metallic) is the replacement of a damaged hard tissue. Implant Recent research efforts are focused on combining the best character-
surface properties are of key importance for initial tissue interactions, istics of both hydroxyapatite and polymers to create a material to
the acceleration of bone healing and osseointegration [1]. be used for hard tissue replacement applications [9]. The polymer/
In recent times, the coating of implants has produced much interest hydroxyapatite composites have attractive features as candidates for
for improving osseointegration and preventing adverse tissue reactions. novel bone substitutes because they may show bone-bonding capacity
The concept of multiple functionalities for surface coating of implants is and mechanical performances derived from the organic substrate
still in the initial stage of development. These multifunctional coatings [10,11]. Using the biomimetic method, the hydroxyapatite can be
should be nontoxic, to mitigate possible adverse tissue responses added to synthetic polymers to develop biomimetic composites for
including the foreign body reaction and implant infection, to be easily bone regeneration. Consequently, these polymers/hydroxyapatite hy-
applied, efficient and cost-effective [2]. brids are expected to act as an artificial bone exhibiting bioactivity and
Much research has been directed to the coating of orthopedic and mechanical performance similar to those of natural bone. The polyure-
dental implants with porous layers to increase hard tissue integration thane polymer is widely engaged in numerous biomedical applications
in vivo [3]. The hydroxyapatite, Ca10(PO4)6(OH)2, is one of the materials due to the excellent mechanical properties and high flexural endurance
able to form a direct and strong binding between the implant and bone [12]. In addition to that, the hydroxyapatite/polyurethane composites
tissue [4]. The synthetic form of hydroxyapatite has been widely inves- are developed to enhance the mechanical and bioactive properties for
tigated due to the similar chemical composition to the mineral matrix of bone repair or substitute [13,14].
bone, which is generally referred to as hydroxyapatite. The coatings A very important problem of using implants is the occurrence of bac-
with hydroxyapatite can be considered as bioactive because they pro- terial infections when placed within the human body. This shortcoming
mote a direct bone–implant contact without an intervening connective can be overcome by modifying the implant surfaces by means of
tissue layer leading to a proper biomechanical fixation of implants [5,6]. antibacterial coatings while maintaining the good biocompatibility.
A diversity of coating methods including ion sputtering, laser ablation, Among the different antimicrobial agents, silver has been the most ex-
tensively studied, being used since ancient times against infections
⁎ Corresponding author. Tel.: +40 741025163. and for preventing the spoilage. The antibacterial, antifungal and antivi-
E-mail address: [email protected] (G. Ciobanu). ral properties of silver ions, silver compounds and silver nanoparticles

0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.msec.2013.10.024
 G. Ciobanu et al. / Materials Science and Engineering C 35 (2014) 36–42 37

have been extensively studied [15]. The silver was found to be non-toxic where D is the crystallite size (nm), λ is the wavelength of Cu Kα radi-
to human organism in low concentrations. It has also strong inhibitory ation (λ = 0.15418 nm), B1/2 is the full width at half maximum intensi-
and bactericidal effects as well as a broad spectrum of antimicrobial ac- ty value for the diffraction peak under consideration (rad), θ is the
tivities. The microorganisms do not develop as much resistance against diffraction angle of the corresponding reflection (°), and k is the broad-
silver as the antibiotics do since the silver attacks a broad range of ening constant varying with crystal habit and chosen as 0.89 for the Ag
targets in the microbes [16]. particles. For quantitative determinations, the peak at 2θ = 38.4°
The silver incorporation into hydroxyapatite coatings is an alterna- for (111) reflection was used to evaluate the crystallite size of Ag parti-
tive that can provide good antibacterial properties of these coatings. cles. This peak was chosen since it is well resolved and shows no
There are several methods to introduce silver into hydroxyapatite interferences.
coatings, such as electrochemical deposition, laser deposition, plasma The surface analysis of samples was performed by X-ray photoelec-
spraying, ion beam-assisted deposition, magnetron sputtering, micro- tron spectroscopy (XPS) using a PHI-5000 VersaProbe photoelectron
arc oxidation and sol-gel technology [17–20]. spectrometer (Φ ULVAC-PHI, INC.) with a hemispherical energy
Currently there are no studies on biocomposites consisting of analyser (0.85 eV binding energy resolution). A monochromatic Al Kα
hydroxyapatite-silver coating on polyurethane polymer. Consequently, X-ray radiation (hν = 1486.7 eV) was used as an excitation source.
in this paper a combined strategy involving biomimetic approach and sil- The optical properties of the coatings were characterized by UV-Vis
ver reduction process has been advanced to deposit a hydroxyapatite/ spectroscopy with a Shimadzu UV-2450 UV-Vis spectrophotometer
silver layer on polyurethane scaffolds able to regenerate the natural (Shimadzu Co., Japan) with an integrating sphere attachment. The
bone and mitigate implant infection. The bactericidal activity of the absorbance was recorded at a resolution of 0.5 nm at 300–800 nm.
hydroxyapatite/silver/polyurethane scaffolds against Escherichia
coli and Staphylococcus aureus bacteria was investigated and the in- 2.3. Antibacterial activity test
hibition ratio evaluated as the antimicrobial efficiency.
The antibacterial test was performed by the LB broth method [19].
2. Experimental Two types of microorganisms, Escherichia coli as Gram-negative bacteria
and Staphylococcus aureus as Gram-positive bacteria, were taken in an-
2.1. Materials and methods tibacterial experiments. These bacteria were supplied by the collection
of microorganisms and cell cultures of the microbiology laboratory of
The porous polyurethane scaffolds were prepared by phase inver- our institution. The bacteria were cultivated at 37 °C in a sterilized
sion method described in previous works [21,22]. The used materials broth (peptone 10 g, glucose 20 g, agar 15 g, distilled water 1000 mL)
were: polyurethane polymer (Institute of Macromolecular Chemistry in a rotary shaker at 100 rpm for 24 h. The concentration of all the bac-
“P. Poni” of Iasi, Romania), N,N-dimethylformamide (DMF, Sigma terial cell suspensions was of about 104 colony-forming units (CFU)/mL.
Aldrich, Germany) as a solvent, and deionized water as a nonsolvent. With the LB broth method, the hydroxyapatite/silver/polyurethane
The polyurethane films obtained were about 100–500 μm thickness. samples (0.5 g) were put into the flasks containing 10 mL aqueous me-
The supersaturated calcification solution (SCS) was prepared by dissolv- dium at 37 °C for 24 h. Then, 10 mL of the bacteria medium was added
ing the following chemicals in deionized water: CaCl2·2H2O, NaH2PO4·H2O and the incubation was continued for another 24 h. The culture with
and NaHCO3 (Sigma-Aldrich, Germany). The ion concentrations of SCS pure broth served as control sample.
solution were of: 4.0 mmol/L Na+, 5.0 mmol/L Ca2+, 10.0 mmol/L Cl−, The bacterial concentrations in liquid cultures were determined by
2.5 mmol/L (H2PO4)−, and 1.5 mmol/L (HCO3)−. measuring the optical density as absorbance at 600 nm (OD600) using a
The biomimetic method applied to coating polyurethane surface Shimadzu UV-2450 UV-Vis spectrophotometer (Shimadzu Co., Japan).
with hydroxyapatite layer consists in immersion of polymeric samples The inhibition ratio based on OD600 data was evaluated as the antimicro-
in SCS solution at 37 °C, for certain period of time (1–7 days), as bial efficiency, calculated according to the equation:
presented elsewhere [23]. Then, in order to obtain a silver coating, the
polymeric samples coated with hydroxyapatite layer were rapidly im- At −Ao
IR%¼100−  100 ð2Þ
mersed in 50 mL of freshly prepared 0.5 M AgNO3 (Sigma-Aldrich, Acon −Ao
Germany) solution with pH = 6.5 at 22 °C, for certain period of time
(1–2 days). Finally, the samples were taken off and carefully rinsed where Ao is the OD600 value of the bacterial broth medium before incuba-
with deionized water followed by drying in air at 60 °C for 24 h. tion; At and Acon are OD600 values of the bacterial broth medium for tested
All chemicals used in this study were of reagent grade and used with sample (containing the Ag nanoparticles) and the control sample respec-
no other purifications. tively, after incubation for a certain period of time (24 h).
All the experiments were run in triplicate. The average and standard
2.2. Samples characterization deviation were calculated. Statistical analysis was performed by using
the ANOVA test of Microsoft Excel.
The morphology and chemical composition of sample surface were
elucidated by scanning electron microscopy (SEM) coupled with energy 3. Results and discussion
dispersive X-ray spectroscopy (EDX), with QUANTA 200 3D Dual Beam
scanning electron microscope (FEI Co., USA). For the SEM-EDX investi- The biomimetic method has been shown to be a good way to obtain
gations, gold sputtering was used to create a conductive coating surface. hydroxyapatite coatings which are homogeneous and would be more
The coatings formed on polyurethane support were characterized favorable to biological interaction. Generally, a surface on which hy-
by X-ray diffraction (XRD) with X'PERT PRO MRD X-ray diffractometer droxyapatite is precipitated in the biomimetic solution shows well
(PANalytical, Netherlands), using monochromatic CuKα radiation biomaterial-bone in a living body [24]. By the biomimetic method,
(λ = 0.15418 nm), operating at 40 kV and 50 mA, over a 2θ range the hydroxyapatite coatings can be produced in aqueous solutions
from 2° to 70°. The average crystallite size (D) was calculated from at physiological temperatures using artificial solutions with ionic con-
XRD data using the Scherrer equation: centrations similar to those of human blood plasma. In practice, the
most widely used artificial biomimetic solutions are: Simulated Body
Fluid (SBF) and Supersaturated Calcification Solution (SCS) [24,25].
kλ The hydroxyapatite coatings can provide an osteoconductive and an
D¼ ð1Þ
B1=2  cos θ osteoinductive approach for enhancement of the bone formation on
38 G. Ciobanu et al. / Materials Science and Engineering C 35 (2014) 36–42

implants. These biological properties may be augmented further by sample was found to exhibit many areas of noncoverage while after
adding other atoms/molecules to produce a truly osteoinductive platform, 48 h it was completely covered with a silver layer on the polymeric sur-
which would improve the physiochemical properties of these coatings face. As shown in SEM images in Fig. 2, after 48 h of immersion the Ag
being also able to increase their safety and efficacy. The silver impregnat- aggregates are distributed uniformly on the polyurethane surface
ed coatings represent very attractive alternatives for mitigate infection of forming a layer with intergranular micropores. All the prepared samples
implants. In particular, silver-containing hydroxyapatite coatings on exhibit the same porous morphology. From the high-magnification SEM
implants can effectively inhibit bacterial adhesion and growth without image (Fig. 2b), the size of Ag aggregates can be estimated to be about
affecting the activity of osteoblasts and epithelial cells [2]. 70–150 nm. The bigger Ag particles can be formed by coalescence of
In the present study the biomimetic method combined with reduction smaller ones during the reduction process. Also, a large number of the
process and in-situ crystallization was used to co-deposit the hydroxyap- Ag nanoparticles were formed on the surface of hydroxyapatite crystals,
atite crystals and silver nanoparticles on the porous polyurethane scaffold as seen in Fig. 2c. The size of these Ag nanoparticles can be estimated to
surface by means of polymeric sample treatment in SCS and AgNO3 be less 50 nm, consistent with the XRD results.
solutions.

3.1. SEM analysis

In this study, the polyurethane scaffolds with an asymmetric and

interconnected porous structure were obtained by applying a phase
inversion process, as mentioned elsewhere [21,22]. These asymmetric
scaffolds have a very thin skin layer supported by a more open porous
substructure. The formation of such a skin layer was attributed to the
solvent evaporation during the microphase separation process. The
size of the pores in the skin layer is very small compared to that in
the substructure. The polyurethane scaffolds have internal micropores
(2–5 μm) and macropores (20–50 μm). At external surface of the
scaffold, on the skin layer, many nanopores (10–100 nm) were formed
during phase inversion process. The porosity of the scaffold is high, of
about 60%. This porous structure provides a good microenvironment
for cell adherence, growth and proliferation.
We have also demonstrated that biomimetic treatment in SCS solu-
tion promoted the nucleation and growing rate of the hydroxyapatite
coating on porous polyurethane surface [23]. Coatings of the polyure-
thane substrates by immersion in SCS solutions were obtained using
rather short exposure times (120–144 h), when the SCS solution was
refreshed every two days. The SEM micrograph of a sample stored in
SCS for 120 h presented in Fig. 1 shows that polyurethane surface is
good enough covered with a hydroxyapatite layer. It can be observed
that the crystalline apatite layer exhibits a petal rose-like morphology.
After hydroxyapatite coating formation the samples were immersed
in AgNO3 solution for a certain time. During this stage, silver ions
are transferred from the solution to the scaffold surface where their
reduction takes place.
Along the immersion time in AgNO3 solution, the spheroidal
clusters/aggregates containing Ag nanoparticles were formed on the
polyurethane surface among hydroxyapatite crystals. The Ag concentra-
tion in the scaffold increased with increasing immersion time in the
AgNO3 solution. Based on SEM studies, after 24 h of immersion the

Fig. 2. Low-magnification (a) and high-magnification (b,c) SEM images of the

Fig. 1. SEM image of the surface of porous polyurethane sample after 120 h soaking in SCS hydroxyapatite-silver layer deposited on polyurethane surface after 48 h soaking in
solution. AgNO 3 solution.
 G. Ciobanu et al. / Materials Science and Engineering C 35 (2014) 36–42 39

3.2. EDX analysis

The energy dispersive X-ray spectroscopy (EDX) results are indica-

tive of the Ag particles formation. Fig. 3 shows the EDX spectrum
(given along with SEM image in inset) of hydroxyapatite-silver nano-
particle layer deposited on porous polyurethane scaffold, confirming
that the coating contains Ag (existing in nanoparticles and spherical
aggregates), and Ca, P and O (in apatite crystals) atoms. The peaks ob-
served at 2.9, 3.1 and 3.3 keV correspond to the binding energies of Ag
Lα, Ag Lβ and Ag Lβ2 respectively, as presented in literature [26].
These optical absorption peaks are typical for the absorption of metallic
silver nanocrystalline due to surface plasmon resonance. Based on the
above mentioned results, the presence of nanocrystalline elemental sil-
ver on the scaffold was confirmed. In EDX spectrum the other small
peaks correspond to Ca, P and O in hydroxyapatite crystals found in
the proximity of the silver particles. According to SEM-EDX results, the
Fig. 4. XRD patterns of the hydroxyapatite/polyurethane (a) and hydroxyapatite/silver/
apatite crystallites deposited onto polyurethane surface are composed polyurethane (b) samples.
mainly of hydroxyapatite that exhibits a molar Ca/P ratio of ∼1.67, in
good agreement with literature data [27]. Also, the carbon peak in
EDX spectrum can be attributed to the presence of polyurethane in with literature data [27]. The diffraction peaks of Ag were distinct
the scaffold. It should be mentioned that the signal attributed to the which indicated that Ag was well crystallized. The average crystallite
Au comes from the gold layer that covers the outer layer of the sample size of the Ag particles, calculated by Scherrer's formula, was found to
in order to prepare it for the SEM images. EDX analysis indicated that be 34.71 nm, confirming SEM studies.
the Ag content in the hydroxyapatite-silver layer deposited on porous Also, the XRD patterns of both samples show a diffraction peak near
polyurethane scaffold was about 8 wt.%. 21° which correspond to the characteristic diffraction pattern for the
polyurethane polymer. This diffraction peak appears to be very broad
3.3. XRD analysis due to the amorphous nature of polyurethane.
The XRD analysis demonstrated that the coatings were composed of
The XRD patterns of the coated samples are illustrated in Fig. 4. The hydroxyapatite and/or Ag nanoparticles.
hydroxyapatite/polyurethane sample shows the most intense peaks at
2θ values of about 25.9°, 31.67°, 32.8°, 34.1°, 39.7° and 46.6° representing 3.4. UV-Vis spectroscopy
the reflexion planes (002), (211), (300), (202), (310) and (222), which
are similar to the standard hydroxyapatite having hexagonal crystal The formation and growth of the Ag nanostructures on hydroxyapatite/
system and 63/m space group (JCPDS card no. 09-432). polyurethane surface was supported by UV-Vis spectroscopy. The surface
In the case of the hydroxyapatite/silver/polyurethane sample, after plasmon resonance plays a major role in the determination of optical
deposition of the Ag nanoparticle onto scaffold surface, new peaks absorption spectra of Ag nanoparticles. The surface plasmon band of
were detected in addition to the hydroxyapatite peaks, as seen in nanoparticles is known to be sensitive to the size, shape, and spatial dis-
Fig. 4. The diffraction peaks were observed at about 38.4°, 44.5°, 64.6°, tribution of particles, which shifts to a longer wavelength with increasing
and 77.5° representing the (111), (200), (220) and (311) Bragg's reflec- particle size [28]. In Fig. 5 the UV-Vis spectra of the samples in the range
tions in the face centered cubic (fcc) phase structure of silver nanopar- 300–800 nm are depicted. It can be seen that the polyurethane and
ticles (JCPDS card no. 04.0784). These results are in a good agreement hydroxyapatite/polyurethane samples do not show absorption peaks

Fig. 3. EDX spectrum and SEM image (inset) of the hydroxyapatite-silver layer deposited on polyurethane surface after 48 h soaking in AgNO3 solution.
40 G. Ciobanu et al. / Materials Science and Engineering C 35 (2014) 36–42

Fig. 5. UV-Vis spectra of the polyurethane (a), hydroxyapatite/polyurethane (b) and hydroxyapatite/silver/polyurethane (c) samples. Inset shows the optical image of hydroxyapatite/
silver/polyurethane sample.

in the 400–500 nm region, while a broad absorption peak with a maximum 3.5. XPS analysis
at 415 nm can be noticed with the hydroxyapatite/silver/polyurethane
sample. The symmetrical shape of this plasmon band suggests that the The oxidation state of silver on the scaffold surface was examined by
reduced silver particles are well dispersed and sphere-shaped. It is gen- XPS. Fig. 6 shows the XPS spectrum of the hydroxyapatite/polyurethane
erally accepted that the absorption peak with the maximum within the sample after 48 h soaking in AgNO3 solution, indicating the presence of
410 to 520 nm domain is related to the formation of silver metal nano- the Ag, Ca, P, O and C elements, which correspond with the SEM-EDX
particles and its height gives information on the concentration of silver analysis. The full XPS spectrum shows the major peaks for Ag 3d5/2
metal nanoparticles [28]. Our data support the silver nanoparticles oc- (368 eV), Ag 3d3/2 (374 eV), Ca 2p (345 eV), P 2p (133 eV), O 1s
curring by means of polyurethane polymeric chains and hydroxyapatite (532 eV) and C 1s (286 eV).
crystals to be successfully formed on the scaffold surface acting as a Inset of Fig. 6 shows the binding energies for Ag electron configura-
nanoreactor for the silver nanoparticle formation. tions of 3d5/2 and 3d3/2 of 368 eV and 374 eV, respectively, with a differ-
The Ag nanoparticles are well known to exhibit yellow to dark brown ence of 6.0 eV. This is comparable to the standard Ag 3d binding
colors with increasing particle size. The colors arise due to the excitation energies (3d5/2 = 368.3 eV, 3d3/2 = 374.3 eV, Δ = 6.0 eV) for metallic
of surface plasmon resonance of the Ag nanoparticles. The formation of silver, as mentioned in literature [29]. In addition, the shift of Ag peaks
Ag nanoparticles on hydroxyapatite/polyurethane surface is accompanied was found to be 0.3 eV for both 3d5/2 and 3d3/2, respectively, compared
by color changes in time. Instead, when the hydroxyapatite/polyurethane to standard metallic silver. The shift is caused by the chemical environ-
samples (colorless) were immersed in AgNO3 solution, a gradual ment and fine particle size effect of Ag nanoparticles. The complex elec-
change in color from colorless through yellow to reddish brown (inset tronic environment on the surface of the scaffold heterostructure reveals
of Fig. 5) was noticed with increasing incubation time (from 24 h to strong electronic interactions between silver and hydroxyapatite/
48 h), confirming the reduction of the Ag+ ions and the subsequent for- polyurethane in the case of the hydroxyapatite/silver/polyurethane
mation of Ag nanoparticles on the scaffold surface. sample. Therefore, it can be said that this shift indicates that the Ag+

Fig. 6. The full XPS spectrum of the hydroxyapatite/silver/polyurethane sample after 48 h soaking in AgNO3 solution. Inset show the XPS narrow scan spectrum of Ag 3d region.
 G. Ciobanu et al. / Materials Science and Engineering C 35 (2014) 36–42 41

ions have been transformed into Ag nanoparticles, confirming the with hydroxyapatite as reducing agent and polyurethane as protecting
reduction of the Ag+ ions into the metallic Ag°. Also, the XPS analysis in- and stabilizing agent.
dicated that the Ag content in the hydroxyapatite-silver layer deposited The in-situ formation of the Ag nanoparticles on the hydroxyapatite/
on the porous polyurethane scaffold was about 7.1 wt.%, consistent polyurethane scaffolds could be explained by a mechanism involving
with the SEM-EDX results. following processes: reduction of the Ag ions to Ag atoms by hydroxy-
In Fig. 6 the C 1s peak at 286 eV can be attributed to the presence of apatite, coordination of the Ag atoms by the polar groups of the
polyurethane in the scaffold. Also, the presence of the Ca and P peaks at hydroxyapatite/polyurethane matrix, nucleation of metallic silver and
345 eV and 133 eV, respectively, explains the existence of the hydroxy- the growth of the Ag nanoparticles on the solid scaffold surface.
apatite deposited on the polyurethane surface. The reduction of silver ions occurs by the electron transfer from the
hydroxyl groups on the surface of hydroxyapatite acting as both reduc-
3.6. Ag nanoparticles formation ing and binding agent as mentioned in the literature [42]. Then the Ag
atoms are coordinated by the polar groups (i.e., hydroxyl, amide and
The use of silver nanoparticles was explored as a constituent of coat- urethane) of the hydroxyapatite/polyurethane matrix that act as a stabi-
ings on medical devices and implants. Many different strategies have lizer for the Ag nanoparticles as well as a matrix for their homogeneous
been developed to obtain silver nanoparticles coatings on solid (poly- distribution and immobilization [37–41]. After a short incubation time
meric, ceramic or metallic) supports, including silver ions reduction when the supersaturation concentration of the Ag atoms on solid
(chemical, photochemical or electrochemical reductions) with and with- surface is reached, the nucleation process of the Ag atoms begins leading
out stabilizers (surfactants, polymers, copolymers, solid matrix, etc.) to aggregation of Ag atoms into small Ag nanoparticles. These nanopar-
[30,31]. Widespread synthesis protocols used for silver nanoparticle ticles can coalesce with each other giving larger aggregates. That could
production often require the use of harsh organic solvents/surfactants explain the existence of some small and other large spherical Ag
(dimethyl formamide, ethylene glycol, cetyltrimethylammonium bro- nanoparticles on hydroxyapatite and polyurethane surfaces, respectively,
mide, sodium dodecyl sulfate, etc.) and strong reducing agents (e.g., bo- as made evident by the SEM images (Fig. 2).
rohydride, hydrazine hydrate, etc.) [32,33]. These methods typically Thus, the hydroxyapatite/polyurethane scaffold of a porous struc-
generate large quantities of hazardous waste and often involve absorp- ture could offer a platform for nucleation and growth of the Ag nanopar-
tion of harsh chemicals on the surfaces of silver nanoparticles raising ticles. The petal rose-like morphology of hydroxyapatite coating on
the toxicity issues. Hence, silver nanoparticle synthesis procedures that the polyurethane surface improves the diffusion of the silver solution
eliminate the use of hazardous reagents and afford greener, more cost- promoting then the growth of Ag particles on the material as made
effective alternatives are becoming more desirable as the number of evident by SEM (Fig. 2). The reduced Ag nanoparticles attached on
nanoparticle applications increases [34,35]. Therefore, in the present the hydroxyapatite surface have no influence on the structure and
study, a simple but “green” synthesis of silver nanoparticles using morphology of the hydroxyapatite crystals. Therefore, Ag nanoparticles
non-toxic hydroxyapatite/polyurethane matrices as both reducing and and Ag aggregates are formed and deposited on the porous structure of
stabilizing agents was studied. the material.
As mentioned in literature in the field, the current trend in nano-
technology research is the use of biocompatible or biological friendly 3.7. Antibacterial activity
polymers (starch, gelatin, cellulose, chitosan, polyvinyl alcohol, etc.)
which can provide reduction as well as stabilization functions in the The bactericidal activity of the hydroxyapatite/silver/polyurethane
preparation of silver nanoparticles [35]. The polymers are known to be scaffold (sample denoted as HA-Ag-PU) against Escherichia coli and
often used for obtaining silver nanoparticles where the polar groups of Staphylococcus aureus bacteria was investigated and the inhibition
the polymer interact directly with the metal particles strongly influenc- ratio evaluated as the antimicrobial efficiency. The antibacterial proper-
ing the particle shape [32,36]. In recent years, several investigations ties of the HA-Ag-PU scaffold were evaluated by the comparison with
have been carried out concerning the possibility of Ag nanoparticle de- the pure porous polyurethane scaffold (sample denoted as PU) and
position on polyurethane films/foams [37–41]. These studies revealed with the porous polyurethane scaffold coated with hydroxyapatite
that the adsorption of metallic silver on the organic polymeric substrate (sample denoted as HA-PU).
could be attributed to silver nanoparticles interaction with the nitrogen As shown in Table 1 the HA-Ag-PU sample exhibited a very good an-
in the –NH– group of polyurethane. tibacterial activity against both bacteria. Thus, the antimicrobial proper-
Incorporation of silver into ceramic materials has been studied for ties of the hydroxyapatite coatings were improved after Ag treatments.
several years. There are some examples in the literature supporting The inhibitory activity of the Ag on the hydroxyapatite/polyurethane
the silver nanoparticle deposition on hydroxyapatite particles [42–45]. surface is higher in case of Gram negative bacteria, as mentioned in the
Recently Arumugam and co-workers [42] reported for the first time literature [46]. It is known that the antibacterial activity of the Ag can be
the development of a novel method for the synthesis of anisotropic attributed to the adsorption of the Ag nanoparticles on the cell wall and
silver nanocrystals by the spontaneous reduction of aqueous silver to the reaction of the Ag particles with the protein present there. Then,
ions (Ag+) on the hydroxyapatite surface. They explain the reduction the denaturation of the cell wall proteins occurs resulting in the forma-
of silver ions to occur by the electron transfer from the hydroxyl groups tion of porous structures which causes the permeability increase and
on the surface of hydroxyapatite. The hydroxyl groups on the surface of accumulation of external fluids [47]. Therefore, the conclusion can be
hydroxyapatite act as both reducing and binding agents. This process drawn that the hydroxyapatite-silver polyurethane scaffolds show a
results in the formation of silver nanoparticles that are bound on the good antibacterial activity. Incorporating silver into hydroxyapatite
surface of hydroxyapatite and the observed silver nanocrystals show
anisotropic structure. Thus, hydroxyapatite crystals can be used as a
Table 1
new class of inorganic scaffolds for the synthesis of nanomaterials The results of the antibacterial activity tests after 24 h incubation of the samples on the
with implications in designing inorganic–inorganic hybrid nanocom- bacteria medium.
posites for medical and other various applications.
Sample Antimicrobial efficiency (%)
In the present paper we suggest the obtaining of the Ag nanoparti-
E. coli S. aureus
cles by reducing Ag ions on porous hydroxyapatite/polyurethane scaf-
fold surface when the scaffold is placed into a concentrated aqueous PU 36.1 35.7
solution of silver nitrate. Silver nanoparticles have been deposited on HA-PU 3.9 1.4
HA-Ag-PU 94.3 92.5
scaffold surface at ambient temperature and under natural sunlight
42 G. Ciobanu et al. / Materials Science and Engineering C 35 (2014) 36–42

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