Surface & Coatings Technology 324 (2017) 6479

Contents lists available at ScienceDirect

Surface & Coatings Technology

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

Inuence of the electrophoretic deposition route on the microstructure

and properties of nano-hydroxyapatite/chitosan coatings on the
Ti-13Nb-13Zr alloy
Dawid Jugowiec a, Alicja ukaszczyk b, ukasz Cieniek a, Kazimierz Kowalski a, ucja Rumian c,
Krzysztof Pietryga c, M. Kot d, Elbieta Pamua c, Tomasz Moskalewicz a,
a
AGH University of Science and Technology, Faculty of Metals Engineering and Industrial Computer Science, Czarnowiejska 66, 30-054 Krakw, Poland
b
AGH University of Science and Technology, Faculty of Foundry Engineering, Reymonta 23, 30-059 Krakw, Poland
c
AGH University of Science and Technology, Faculty of Materials Science and Ceramics, Mickiewicza Av. 30, 30-059 Krakw, Poland
d
AGH University of Science and Technology, Faculty of Mechanical Engineering and Robotics, Mickiewicza Av. 30, 30-059 Krakw, Poland

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

Article history: In this work, nanocomposite HA/chitosan coatings were electrophoretically deposited (EPD) on a near- Ti-
Received 1 March 2017 13Nb-13Zr alloy. The inuence of the state of the HA particles introduced to the colloidal solution of chitosan
Revised 18 May 2017 (nc-HA-p as a nanopowder and nc-HA-s as nanoparticles suspended in ethanol), as well as the chemical compo-
Accepted in revised form 21 May 2017
sition of a multi-component HA-chitosan suspension and EPD parameters, on the homogeneity of coatings has
Available online 22 May 2017
been studied. It was established that the pH value and the chemical composition of the suspension have a sub-
Keywords:
stantial effect on the electrokinetic properties of suspended HA and chitosan particles. These are also inuenced
Electrophoretic deposition (EPD) by the deposition kinetics of EPD and the uniformity of as-deposited coatings. The thickness of the nc-HA-p/chi-
Composite coatings tosan and nc-HA-s/chitosan coatings was up to 750 nm and 1.5 m, respectively. The nc-HA-s/chitosan coating
Chitosan microstructure consisted of HA nanoparticles, homogeneously embedded in an amorphous chitosan matrix.
Hydroxyapatite (HA) The nc-HA-p/chitosan coating microstructure was non-homogeneous, composed of HA agglomerates in a chito-
Ti-13Nb-13Zr alloy san matrix. The presence of thin oxide layer was observed on the coatings/titanium alloy interface. The nc-HA-s/
Corrosion resistance chitosan coating exhibited better adhesion to the titanium alloy substrate than the nc-HA-p/chitosan coating. It
was found that the nc-HA-s/chitosan coating improves the electrochemical corrosion resistance of the Ti-13Nb-
13Zr alloy in Ringer's solution, as well as its bioactivity and other biological properties.
2017 Elsevier B.V. All rights reserved.

1. Introduction
Titanium and its alloys, due to their relatively good mechanical prop-
erties, high electrochemical corrosion resistance and good biocompati-
bility, have been widely used in different medical applications [13]. A
near- Ti-13Nb-13Zr titanium alloy was developed to replace two
phase ( + ) alloys (such as Ti-6Al-4V and Ti-6Al-7Nb) in orthopedic
implants. The alloy exhibits greater biocompatibility and about 30%
lower elasticity modulus than two phase + alloys [4]. Furthermore,
the Ti-13Nb-13Zr alloy contains harmless elements, Nb and Zr, which
do not cause any adverse reaction in the human body [5]. However,
the osteointegration properties of all titanium alloys in clinical condi-
tions are poor, as the bone tissue ingrowth process is too slow and
thus not effective [6,7]. If sufcient bone tissue ingrowth does not
occur in a specic period of time, it will ultimately lead to implant fail-
ure. To improve the adhesion to the living bone tissue, the surface of
Corresponding author.
E-mail address: tmoskale@agh.edu.pl (T. Moskalewicz).
the titanium implant has been modied by sandblasting, acid etching
and bioactive coatings [8]. In particular, hydroxyapatite (HA) is a well-
established material for developing biocompatible and osteoconductive
coatings on metallic surfaces [9].
Hydroxyapatite Ca10(PO4)6(OH)2 is the most abundant form of calci-
um apatite ceramics with a chemical composition similar to the mineral
part of bone tissue [10,11]. The Ca/P molar ratio of stoichiometric HA is
around 1.67 [12]. HA is characterized by high biocompatibility, bioactiv-
ity, biodegradability and osteoconductivity, making it an attractive ma-
terial for bone tissue engineering applications [13]. However, its clinical
application for high load bearing conditions has been limited due to its
slow biodegradation process and poor mechanical properties, such as
low fracture toughness [9,14,15]. On the other hand, it has been report-
ed that HA coatings exhibit poor adhesion to metallic substrates [1]. All
these problems can be solved by combining HA with a suitable polymer-
ic binder [9].
Currently, HA and polymer composite coatings are being investigat-
ed with the aim of enhancing adhesion of the coatings to the substrate,
mechanical stability in clinical conditions as well as biological

http://dx.doi.org/10.1016/j.surfcoat.2017.05.056
0257-8972/ 2017 Elsevier B.V. All rights reserved.
 D. Jugowiec et al. / Surface & Coatings Technology 324 (2017) 6479
properties, such as bioactivity and biocompatibility [9]. Furthermore,
bone can be considered as a natural composite of collagen and HA crys-
tals. To fulll the many requirements of the complex and sensitive sys-
tem that is the human body, combining the advantages of HA and
natural polymer seems to be a promising approach. In this paper, chito-
san, a natural, cationic polysaccharide produced by N-deacetylation of
chitin [11], has been used as a binder. The main advantages of this poly-
mer are its biocompatibility, biodegradability, antimicrobial activity,
mucoadhesiveness and lm forming capability [1618]. Due to its
unique properties, chitosan has been used in the biomedical eld as a
material for drug delivery systems, scaffolds, biosensors and biomedical
implants [17].
Electrophoretic deposition (EPD), among many other coating
methods, is particularly attractive for the deposition of HA and other
biomaterials on metallic surfaces [9]. EPD offers the possibility to pro-
duce relatively homogeneous coatings with controlled microstructure,
thickness and morphology on complex shaped materials [14,15].
Other advantages of EPD are the high purity of deposits, room temper-
ature processing, higher deposition rate compared to other deposition
techniques, low cost of the equipment as well as the option to co-depos-
it various materials [1,15,16]. The EPD process essentially consists of
two steps: (i) electrophoresis, during which charged particles dispersed
in a colloidal solution or suspension are moved under the inuence of an
electric eld, and (ii) deposition, which consists of accumulation and co-
agulation processes of charged particles on the surface of oppositely
charged electrodes [10,14].
The parameters which inuence the EPD process can be grouped
into two categories: (i) the rst group consists of inter-related parame-
ters that are connected to the suspension, such as dielectric constant,
viscosity, temperature, particle size and morphology, content of the dis-
persant in the suspension, electrokinetic properties of particles, such as
zeta potential and electrophoretic mobility, as well as the type of medi-
um and (ii) the second group that includes parameters related to the
electric eld of EPD, namely applied voltage, deposition time, distance
between electrodes, area of electrodes submerged in the suspension,
as well as material of the counter electrode [10,19]. To deposit coatings
with high microstructural uniformity, adequate morphology and thick-
ness as well as good adhesion to the substrate, it is imperative to opti-
mize the chemical composition of the suspension used during EPD, as
well as to adjust the electric conditions during EPD itself. Water-based
suspensions are inferior compared to organic media, such as methanol,
ethanol and propanol, due to their electrolysis and subsequent gas for-
mation at relatively low applied voltages, which deteriorate as-deposit-
ed coating uniformity and adhesion strength [10,14,15]. However,
particles dispersed in an organic medium characterized by low dielec-
tric constant have relatively low zeta potential and electrophoretic mo-
bility, which reduce the stability of the suspension and, consequently,
the nal coating quality. Therefore, a mixture of water and organic sol-
vent, such as ethanol, is preferred.
In the literature, there are several investigation results concerning
the EPD of chitosan [1921], HA [15] and composite HA/chitosan [11,
22] coatings on stainless steel substrates. Furthermore, there is also in-
formation about the EPD of composite HA/chitosan on titanium and
two-phase ( + ) Ti-6Al-7Nb titanium alloy [23,24]. There are also
many published works about the EPD of multi-component coatings con-
taining HA and chitosan, as well as other materials, such as CaSiO3,
bioglass, silica, carbon nanotubes and halloysite on stainless steel, plat-
inum, platinized silicon, graphite, titanium and Nitinol substrates [16,
17,2529]. The referred works were mostly focused on the optimization
of EPD parameters in order to achieve high coating homogeneity. How-
ever, there is little information about the adhesion of composite HA/chi-
tosan coatings to titanium-based substrates [24]. According to our
knowledge, there is no information about the EPD of HA/chitosan on
near- titanium alloys and little information about the biological prop-
erties of HA/chitosan coatings [23]. The novelty of this work is the com-
parative investigation concerning the inuence of introducing HA nano-
65
particles to the EPD colloidal solution of chitosan in two different states,
as nanopowder (nc-HA-p) and as an ethanol-based colloidal solution of
HA (nc-HA-s), on the nal coating microstructural homogeneity and
coating adhesion to the titanium alloy. An important input of our
work to the EPD literature on HA/chitosan coatings is transmission elec-
tron microscopy (TEM) investigation of chitosan particles present in the
colloidal solution during the EPD of coatings and a detailed characteriza-
tion of the coating microstructure on the cross-section. The microstruc-
ture characterization of coatings is very complex and best performed by
TEM on cross-section specimens. Usually, only scanning electron mi-
croscopy (SEM) results of the coating microstructure on a plan-view
specimen are present in the literature, which is insufcient to investi-
gate coating homogeneity correctly.
The aim of the present work was the development of nanocomposite
nc-HA-p/chitosan and nc-HA-s/chitosan coatings by EPD, as well as a
comparative and detailed investigation of their microstructure, adhe-
sion to the underlying Ti-13Nb-13Zr substrate, electrochemical corro-
sion resistance in Ringer's solution and biological properties.
2. Materials and methods
2.1. Coating components
Both nc-HA-p (powder) and nc-HA-s (suspension) were delivered
by the Laboratory of Nanostructures, the Institute of High Pressure
Physics of the Polish Academy of Sciences, IHPP, Poland. The average
crystallite size, evaluated from the X-ray diffraction (XRD) pattern,
was 10 nm. The hydroxyapatite powder was developed by the micro-
wave solvothermal synthesis method (MSS), as described elsewhere
[30,31]. The HA powder obtained by the MSS method was washed
with anhydrous ethanol and then dried in an air ow at 30 C for 16 h
to obtain nc-HA-p, or ltered and washed ten times with ethanol and
suspended in ethanol (to obtain nc-HA-s). The specic surface area
(SSA) of nc-HA-p equaled 209 m2/g and density equaled 2.87 g/cm3.
The SSA of nc-HA-s suspended in ethanol (30 g of HA per 400 ml of eth-
anol, 7.5 wt%) equaled 227 m2/g, while the density of particles equaled
2.85 g/cm3. The required volume of nc-HA-s to be added to the nal sus-
pension used for EPD was calculated from the suspension's density
(0.075 g/cm3) and appropriate mass of HA to reach selected densities
of material in the nal suspension.
The medium molecular weight chitosan, with a degree of
deacetylation of 7585% was delivered by Sigma-Aldrich (product no
448877, USA). The equivalent circle diameter (ECD) of the dry particles,
evaluated from light microscopy (LM) and scanning electron microsco-
py (SEM) images, was in the range of 0.81.2 mm.
2.2. Sample preparation and EPD procedure
A near- Ti-13Nb-13Zr titanium alloy (13.513.7 Nb, 13.513.8 Zr,
0.050.06 Fe, 0.04 C, 0.010.02 N, 0.001 H, 0.11 O; wt%), delivered by
Xi'an Saite Metal Materials Development Co., Ltd., China, was used as a
substrate for the EPD of coatings. The samples were cut from a bar in
the shape of discs of diameter and thickness equal to 27 mm and
2 mm, respectively. Prior to EPD, the samples were ground with succes-
sively ner grit of sandpaper up to 3000-grit, mechanically polished
with the standard colloidal silica suspension (OP-S, 0.04 m) of Struers
and washed with ethanol. A detailed description of the microstructure
of the Ti-13Nb-13Zr alloy has been provided in our earlier works [32,
33]. It was found that the Ti-13Nb-13Zr alloy had a ne acicular mar-
tensitic morphology composed mainly of (hexagonal close-packed;
hcp) and some (orthorhombic, Cmcm space group) laths in
(body-centered cubic; bcc) grains.
Based on our earlier investigation results of the zeta potential and
electrophoretic mobility of chitosan particles [32], chitosan was dis-
solved at a ratio of 2 g/l in 50% ethanol in distilled water containing
66 D. Jugowiec et al. / Surface & Coatings Technology 324 (2017) 6479
0.5% acetic acid. The colloidal solution of chitosan was prepared by mag-
netic stirring at 300 rpm at room temperature for 72 h.
Suspensions of different concentrations of nc-HA-p (1, 2, 3, 4, 5 g/l)
and nc-HA-s (4, 10, 15, 30 g/l), respectively, were prepared by dispers-
ing HA nanoparticles or the HA suspension in the solution prepared as
described above and stirred at 300 rpm for 20 min.
The electrokinetic properties, such as zeta potential and electropho-
retic mobility, of HA in both suspensions were analyzed using a
Zetasizer Nano ZS90 (Malvern Instruments Ltd., UK). The pH values
were measured using a Mettler Toledo EL20 pH-meter (China).
EPD was carried out in a two-electrode cell under constant voltage
conditions in the range of 830 V for a deposition time of 1, 2, 3, 4, 5,
6 min. The counter electrode was an AISI 316L austenitic stainless
steel plate with a size of 100 mm 29 mm 0.5 mm. The distance be-
tween the electrodes in the EPD cells was 10 mm. The inuence of the
applied voltage, deposition time and chemical composition of the sus-
pensions on current density during EPD has been investigated using a
Tektronix DMM 4040 multimeter (USA).
2.3. Characterization of the microstructure and adhesion of the coatings to
the substrate
The microstructure of coatings was characterized by LM, SEM and
transmission electron microscopy (TEM). The SEM investigation was
carried out using an FEI Nova NanoSEM 450 (The Netherlands). The
TEM investigation was carried out using a JEOL JEM-2010 ARP micro-
scope (Japan). Phase identication was performed by means of selected
area electron diffraction (SAED) and by grazing incidence X-ray diffrac-
tometry (GIXRD). The JEMS-SAS software was used to interpret the
SAED patterns. Specimens from the chitosan solution for TEM investiga-
tion were prepared by placing a droplet of the stable solution composed
of chitosan (2 g/l) in 50% ethanol, 0.5% acetic acid and 49.5% distilled
water after magnetic stirring at 300 rpm at room temperature for 72 h
on a carbon lm deposited on 300 mesh grids of copper and nally
dried. Specimens from HA for TEM investigation were prepared by dis-
persing the powder or HA suspension in ethanol and ultrasonic stirring
in order to separate agglomerated particles. Finally, a droplet of the sus-
pension was placed on the copper grid and dried. The lamella from the
cross-section of coated alloy for the TEM investigation was prepared by
a focus ion beam (FIB) using an FEI QUANTA 3D 200i device (the Neth-
erlands). The chemical composition was analyzed by energy dispersive
X-ray spectroscopy (SEM-EDS, TEM-EDS). The GIXRD patterns were re-
corded using a Panalytical Empyrean DY1061 diffractometer (the Neth-
erlands) applying Cu-K radiation on plan-view specimens.
The surface composition of the titanium alloy substrate and the
chemical states of the present elements were studied by X-ray Photo-
electron Spectroscopy (XPS) using an instrument by VSW (Vacuum Sys-
tems Workshop Ltd.) equipped with a concentric hemispherical
electron analyzer with a radius of 150 mm and a two-plate 18-channel
detector (Galileo). The electron analyzer was operated in xed-analyzer
transmission mode (FAT) with a constant pass energy of electrons equal
to 22.5 eV. Samples were exposed to the K Mg (1253.6 eV) X-ray radi-
ation produced by an X-ray lamp working at the power of 208 W (13 kV
- voltage, 16 mA emission current). The background pressure during
the analyses was b5 108 mbar. Calibration of the binding energy
scale was performed by assuming that the position of the C 1s line of
the adventitious carbon (hydrocarbons), corresponding to the bond
C\\H, is equal to 285 eV.
The surface topography of the coatings was characterized by atomic
force microscopy (AFM). The AFM investigation was carried out by
Veeco Dimension Icon SPM (USA) in tapping mode. The probe was
composed of a silicon tip on a nitride lever. The single analyzed surface
area was 40 m 40 m.
The adhesion of coatings to the titanium alloy substrate was investi-
gated by micro-scratch tests using the Micro-Combi-Tester (MCT) de-
vice (CSM Instruments, Switzerland). The micro-scratch tests were
performed using a Rockwell C indenter with a diamond tip radius of
200 m, speed (dx/dt) of 5 mm/min, and load range of 0.0310 N. The
scratch length was 5 mm. The critical loads Lc1 and Lc2 (corresponding
to cohesive and adhesive cracks, respectively) were determined from
LM observations, acoustic emission and friction force signals.
2.4. Electrochemical corrosion resistance analysis
The open circuit potential (OCP), electrochemical polarization and
electrochemical impedance spectroscopy (EIS) studies of the coated
samples were carried out using an Autolab PGSTAT302N potentiostat
(the Netherlands). The corrosive environment was Ringer's solution at
a temperature of 37 C and pH of 7.4. The chemical composition of
Ringer's solutions was (in g/l) 8.6 NaCl, 0.3 KCl, 0.25 CaCl2. The reference
electrode was made of saturated calomel and a platinum plate was used
as the counter electrode. The polarization test was performed at a scan
rate of 1 mV/s from 1.3 V to +1.5 V. For the EIS measurements, the
amplitude was 10 mV and the frequency was from 105 Hz to 103 Hz.
The EIS data were tted by using the ZView software. Minimizing errors
was done using the chi-squared criteria for tting the experimental data
of the EIS.
2.5. Bioactivity in simulated body uid (SBF)
Bioactivity was investigated for the uncoated as well as chitosan and
nc-HA-s/chitosan coated alloy. Potential bioactivity of the samples was
tested by an immersion test in SBF. SBF was prepared by dissolving
NaHCO3, Na2SO4, KCl, K2HPO4, MgCl26H2O, NaCl and CaCl2 (all
chemicals from Avantor, Gliwice, Poland) in UHQ-water (produced in
PureLab, Elga, UK); the pH was adjusted by HCl and TRIS to 7.4 at 37
C according to a method described earlier [34]. The samples (in the
shape of a quarter of a disc of 27 mm diameter and 1 mm thickness)
were immersed in 30 ml SBF and incubated at 37 C for 3 weeks.
Afterwards, the samples were washed, dried, sputter coated with a
thin carbon layer to make them conductive, and examined by SEM at
an acceleration voltage of 15 kV by Nova 200 NanoSEM (FEI, USA).
The chemical composition of the samples was studied by EDS (Link
AN 10000, UK; Noran Vantage EDS system, USA).
2.6. Cytocompatibility in contact with osteoblast-like cells
Before in vitro tests, the samples (10 mm 10 mm 0.3 mm) were
sterilized by UV exposition for 20 min on both sides and placed in 12-
well cell culture plates (EuroClone). MG-63 osteoblast-like cells (Euro-
pean Collection of Cell Cultures, Salisbury, UK) suspended in a minimal
essential medium (MEM, PAN BIOTECH, Germany) supplemented
with 10% fetal bovine serum, 1% penicillin/streptomycin and 2 mM L-
glutamine (PAA, Austria) were seeded on each sample at an initial den-
sity of 8 103 cells/ml at 37 C under 5% CO2 atmosphere and cultured
for 1, 3 and 7 days. Tissue culture polystyrene (TCPS, i.e. bottom of the
well plates) was used as a control reference. Cell viability was evaluated
using the In Vitro Toxicology Assay Kit, based on resazurin (Sigma Al-
drich). 0.1 ml of resazurin reagent was added to each well and the
cells were incubated for 4 h at 37 C. The reduction in resazurin was
measured uorescently (excitation wavelength 530 nm, emission
wavelength 590 nm) (FLUOstar Omega, BMG Labtech, Germany) and
calculated according to the formula:
%Reduction
 of resazurin
  
Sx Scontrol = S100%reduced Scontrol 100% 1
where:
Sx uorescence of samples,
Scontrol uorescence of medium without cells,
 D. Jugowiec et al. / Surface & Coatings Technology 324 (2017) 6479 67

S100%reduced uorescence of reagent reduced by 100% (reagent with
medium was placed in autoclave for 15 min at 121 C).
The results were expressed as average standard deviation (SD)
from three independent samples performed in triplicate. Statistical sig-
nicance was evaluated according to the unpaired t-test; p b 0.05 was
considered as being signicant.
To visualize the cell attachment and distribution on the samples, the
cell cultures were observed using uorescence microscopy (Zeiss
Axiovert, Germany). Live-dead staining was performed to evaluate cell
viability. Samples were rinsed with PBS (PAN BIOTECH, Germany)
and the supernatant was replaced by 1 ml of PBS solution containing 2
l (1 mg/ml) of calcein AM and 2 l (1 mg/ml) of propidium iodide
(Sigma-Aldrich). To visualize the cytoskeleton (actin bers) and nuclei,
the cells were washed twice in PBS and xed with 3.6% formaldehyde
(Sigma-Aldrich) for 30 min at room temperature. They were then
stained in 50 l (6.6 g/ml) of phalloidin (Phalloidyn Alexa Fluor 488
Phalloidin, Thermo Fisher Scientic) for 30 min, rinsed twice with PBS
and stained using 50 l (2.5 g/ml) of DAPI (Sigma Aldrich).
3. Results and discussion
3.1. Characterization of coating components
In the present study, nano-sized particles of HA prepared by the MSS
method in the state of powder (nc-HA-p) and ethanol-based suspen-
sion (nc-HA-s), as well as chitosan, were applied as component mate-
rials for the EPD of composite coatings. Both the nc-HA-p nanopowder
and nc-HA-s nanoparticles suspended in ethanol used for EPD were
characterized by a longitudinal shape (Fig. 1a, b). The average length
and width of the nano-crystals were 45 7 nm and 3.8 1.2 nm,

Fig. 1. Morphology of nc-HA-p (a) and nc-HA-s (b) nanoparticles used for EPD and SAED patterns taken from areas marked A, B in panel a, b with their identication as HA (hp) as well as
bright - (c) and dark eld (d) image of the chitosan nanoparticles occurred in solution composed of chitosan (2 g/l) in 50% ethanol, 0.5% acetic acid and 49.5% distilled water after magnetic
stirring at 300 rpm at room temperature for 72 h, TEM.
68 D. Jugowiec et al. / Surface & Coatings Technology 324 (2017) 6479
respectively. The aspect ratio was about 12:1. Nanocrystalline HA with a
similar shape was also used for the EPD of coatings in other works [22,
24]. Analysis of the SAED patterns revealed the presence of crystalline
HA (hexagonal primitive, hp) phase (SAED patterns marked A, B in
Fig. 1a,b). The continuous rings in the SAED patterns conrm the very
small size of crystals.
It was found that the chitosan particles, after magnetic stirring at
300 rpm at room temperature for a relatively long time equal to 72 h,
did not dissolve completely in the solution composed of 50% ethanol,
0.5% acetic acid and 49.5% distilled water. TEM observations revealed
the presence of numerous chitosan nanoparticles with a spherical-like
shape (Fig. 1c). Their ECD was evaluated from dark-eld TEM images
as up to 10 nm (Fig. 1d). Therefore, both nanoparticles and macromole-
cules of chitosan were present in the colloidal solution used for EPD.
Moreover, nanoparticles of both components of the suspension, HA
and chitosan, exhibited similar size, but different morphology. Wu et
al. [35] studied chitosan in an aqueous solution by dynamic laser light-
scattering and reported that the chitosan chine is slightly extended in
the aqueous solution, and that even in a diluted solution chitosan still
forms aggregates with a respectively large size.
3.2. Electrophoretic deposition of coatings
High microstructural uniformity of coatings, free of cracks and other
discontinuities, can only be achieved by using a stable solution and
proper adjustment of EPD parameters [1,10,11,14]. The level of the sus-
pension stability can be estimated by measuring the electrokinetic
properties of suspended particles in the suspension, such as zeta poten-
tial and electrophoretic mobility [1,14]. Particles of different materials
usually vary greatly in terms of electrophoretic mobility and zeta poten-
tial [36]. Therefore, it is more challenging to deposit coatings from
multi-component suspensions than from single-component suspen-
sions. In our work, the zeta potential and electrophoretic mobility prop-
erties were rstly investigated for particles suspended in multi-
component suspensions. However, the achieved results were not repro-
ducible and were characterized by high standard deviation, therefore
we have not considered them for further research and moved on to sin-
gle-component suspension analysis. In the second attempt, zeta poten-
tial and electrophoretic mobility of particles were investigated for ve
single-component suspensions with different concentrations of nc-
HA-p (1, 2, 3, 4, 5 g/l) and four suspensions containing different concen-
trations of nc-HA-s (4, 10, 15, 30 g/l), all in a mixture of ethanol, acetic
acid and distilled water of the same volume fraction. The highest values
of zeta potential (17.2 0.4 mV and 37.3 4.1 mV), as well as electro-
phoretic mobility (0.58 0.01 cmm/Vs and 1.11 0.12 cmm/Vs),
Fig. 2. Zeta potential of HA for suspensions with different content of nc-HA-p (a) and of nc-HA-s (b).
were achieved for the suspensions of nc-HA-p (4 g/l) and nc-HA-s
(30 g/l), respectively, in a mixture of distilled water and 0.5 vol% of
acetic acid and 50 vol% of ethanol. The zeta potential analysis results
were shown in (Fig. 2a,b). It was established that the impact of nc-
HA-p and nc-HA-s concentration on the electrophoretic mobility of
suspended particles was signicantly lower compared to that of the
concentration of chitosan [32]. However, both states of HA particles
and chitosan molecules were positively charged in the specied suspen-
sion under the inuence of an electric eld, therefore the cathodic polar-
ization approach was expected to be feasible for EPD.
Taking into consideration other works [1,14,37], it was conrmed
that HA concentration and the correlated pH value of suspensions
have a signicant effect on both zeta potential and electrophoretic mo-
bility values. The highest values of zeta potential and electrophoretic
mobility were obtained for suspensions containing nc-HA-p in the pH
range from 4.7 to 4.8 and for suspensions containing nc-HA-s in the
pH range from 4.9 to 5.0 (Fig. 2a,b). Decreases in zeta potential due to
further increases in the pH of the suspension are believed to be caused
by the suppression of surface ionization and decreases in the electrical
double layer thickness [38]. On the other hand, zeta potential also de-
creased at lower pH values than established optimal ranges, which
might be due to low concentrations of suspended particles and, conse-
quently, reduced suspension conductivity [18].
The most adequate EPD parameters were selected by trial-and-error
experiments, differing in potential difference and deposition time
values. According to Mahmoodi et al. [11], chitosan particles can be in-
corporated into HA/chitosan composite coatings by two mechanisms:
(i) chitosan adsorbed on the HA nanoparticles and incorporated in the
deposit with them, (ii) non-adsorbed chitosan present in the suspen-
sion which is incorporated in the deposit by the EPD method. Chitosan
can be adsorbed on the surface of HA particles, especially when its sol-
ubility in the suspension is low [22]. The chitosan dissolution process
depends on the amount of water and acetic acid in the suspension
[18]. Therefore, it is possible to enhance the adsorption rate of chitosan
onto HA particles by using ethanol-water solutions. The adsorption of
chitosan can lead to increases in the zeta potential of HA particles,
which can result in the rise of electrophoretic mobility, stability and, -
nally, deposition rate of HA particles [22]. It is also possible to obtain
higher zeta potential values of HA particles by increasing the HA content
in the suspension [25]. However, high contents of chitosan or HA can
lead to higher viscosity and pH values of the nal suspension, which
has a deteriorating effect on the electrophoretic mobility of the
suspended particles.
In conclusion, according to Pang et al. [22,25], the adsorption of chi-
tosan results in the electrosteric stabilization of HA nanoparticles, which
 D. Jugowiec et al. / Surface & Coatings Technology 324 (2017) 6479
leads to higher charge and higher zeta potential of HA nanoparticles and
thus higher deposition rate. Our investigations of microstructure of both
coatings types, performed by SEM and TEM (results described below in
Section 3.2), indicate that HA nanoparticles from nc-HA-s suspensions
are more stable during EPD than in the case of HA nanoparticles from
nc-HA-p, as they don't form agglomerates. The decisive factor of higher
stability of HA nanoparticles is the presence of chitosan nanoparticles
and macromolecules in both types of suspension, as, due to their signif-
icantly higher electrophoretic mobility compared to HA nanoparticles,
they are the main driving force of EPD. Depending on the electrokinetic
properties of suspended HA nanoparticles, chitosan nanoparticles can
be deposited both together with HA nanoparticles by being adsorbed
(mechanism (i) described above) or separately from HA nanoparticles
(mechanism (ii)). As investigated in our work (Fig. 2a,b), HA nanoparti-
cles from the nc-HA-s suspension have higher zeta potential compared
to HA nanoparticles from the nc-HA-p suspension, therefore, as they are
more stable, they are also more likely to adsorb chitosan nanoparticles,
which will further enhance their stability. We assume that the higher
stability and deposition rate of HA nanoparticles from the nc-HA-s sus-
pension during EPD is due to the adherence of chitosan molecules,
therefore mechanism (i) is preferred. On the other hand, the less stable
HA nanoparticles and chitosan molecules from the nc-HA-p suspension
appear to be deposited separately during EPD on the substrate (mecha-
nism (ii)), as the distribution of both polymeric and ceramic particles in
the coating is highly non-uniform.
The best quality coatings were singled out with visual inspection and
SEM observation. The most homogeneous and continuous composite
nc-HA-p/chitosan and nc-HA-s/chitosan coatings were both deposited
at constant voltages of 10 V for a deposition time of 4 min (Fig. 3). Fur-
ther increases in voltage led to intensive electrolysis of water during
EPD, which has been discussed previously [10,11,20]. This phenomenon
disturbed the stability of deposition rate and, consequently, the
Fig. 3. Ti-13Nb-13Zr alloy samples with nc-HA-p/chitosan (a) and nc-HA-s/chitosan (b) coatings deposited with different voltage values of 10 V and 20 V and a constant deposition time of
4 min and 6 min.
69
homogeneity of the as-deposited coatings. In the case of voltage values
lower than 10 V, the alloy was only partially coated due to the low de-
position rate. Moreover, it was shown that deposition time had a
much lower effect on the deposition rate and nal coating quality com-
pared to the suspension's electrokinetic parameters and potential dif-
ference. Similar observations were published in our previous work
[32]. The nc-HA-p/chitosan coating of the highest quality was deposited
from a suspension which contained 4 g/l of nc-HA-p and 2 g/l of chito-
san, which agrees well with the highest value of HA zeta potential. How-
ever, the most homogeneous and continuous nc-HA-s/chitosan coatings
were deposited from the suspension which contained only 4 g/l of nc-
HA-s and 2 g/l of chitosan at the potential difference of 10 V for 4 min,
which is not consistent with the highest value of HA zeta potential
achieved for the suspension which contained 30 g/l of nc-HA-s. This is
due to the higher HA/chitosan ratio in the nc-HA-s/chitosan coatings,
which led to the formation of microcracks and, as a result, lower
homogeneity.
The mass ingrowth of composite nc-HA-p/chitosan and nc-HA-s/chi-
tosan coatings as a function of deposition time, as well as potential dif-
ference values, is presented in Fig. 4. It was found that the relation
between the deposition weight and deposition time for both types of
coating was almost linear. Moreover, higher voltage values led to the
deposition of coatings characterized by higher mass but also lower ho-
mogeneity, which was also stated in another study [24]. Increased the
velocity of particles, due to higher voltage values, decreases the time
needed for particle arrangement in the coating [1,14]. Furthermore,
Sun et al. [39] found that longer deposition time leads to an increase
in coating thickness, which in turn amplies the repulsion forces of
charged particles in the coatings. The intensied repulsion force of
charged particles reduces the efciency of the ongoing EPD process.
The analysis of current density as a function of deposition time has pre-
sented a typical pattern, i.e. a decrease with deposition time, for both
70 D. Jugowiec et al. / Surface & Coatings Technology 324 (2017) 6479
Fig. 4. Weight gain of nc-HA-p/chitosan and nc-HA-s/chitosan coatings for different deposition time and a constant potential difference value of 10 V (a) as well as for different applied
potential difference values and a constant deposition time of 4 min (b).
types of composite coating (Fig. 5). In addition, current density values
during the EPD of nc-HA-p/chitosan and nc-HA-s/chitosan coatings
were comparable at 5 V and 10 V during EPD. However, in the case of
deposition of the nc-HA-s/chitosan coating at 15 V and 20 V, current
density was lower and more stable compared to the values obtained
during deposition of the nc-HA-p/chitosan coating. Voltage conditions
were the same for both types of coating, therefore we assume that the
only relevant cause of this phenomena is the difference between the
Fig. 5. Effect of deposition time on current density during EPD of nc-HA-p/chitosan and nc-
HA-s/chitosan coatings for different applied potential difference values.
resistance values of both electrodes during EPD. The difference in resis-
tance values is caused by the different uniformity of the deposited coat-
ings and thus their electric insulating properties. It is expected that the
higher stability of HA nanoparticles from the nc-HA-s suspension leads
to higher resistance to the hydrolysis effect and thus better deposition
rate and higher uniformity of the coating, which in turn leads to higher
electric resistance of the substrate and lowers the value of the current
density during EPD.
3.3. Microstructure and surface topography of coatings
Fig. 6a and b show the representative images of nc-HA-p/chitosan
and nc-HA-s/chitosan coating microstructure, both deposited at a po-
tential difference of 10 V for 4 min. The nc-HA-p/chitosan composite
coatings were non-uniform and contained numerous HA agglomerates
of different sizes, up to 28 m, distributed in a chitosan matrix (Fig.
6a). Small microcracks were present in the coating. On the other hand,
nc-HA-s particles were uniformly embedded in an amorphous chitosan
matrix (Fig. 6b). The nc-HA-s in the ethanol suspension was more stable
than the suspension of nc-HA-p in ethanol. Thus, no agglomerates were
present in the nc-HA-s containing the suspension and nally in the nc-
HA-s/chitosan coatings.
SEM observations also revealed that both coatings exhibited open
nanoporosity with the pore size comparable with the size of the HA
nanoparticles. A similar result was presented in our previous work [24].
The GIXRD investigation conrmed the occurrence of HA and chito-
san in both types of coating. A typical GIXRD pattern of the nc-HA-p/chi-
tosan coating is shown in Fig. 7. It reveals the presence of a broad peak of
amorphous chitosan at 2, equal to 1822 and diffraction peaks for
crystalline HA (hexagonal primitive, hp) phase. The diffraction peaks
of the Ti (hcp) phase coming from the substrate material were also
present in the GIXRD pattern. A detailed description of the
 D. Jugowiec et al. / Surface & Coatings Technology 324 (2017) 6479
Fig. 6. Microstructure of nc-HA-p/chitosan (a) and nc-HA-s/chitosan (b) coatings
deposited at a potential difference of 10 V for 4 min on the Ti-13Nb-13Zr alloy, SEM
plan-view specimens. The HA agglomerates are marked in panel a by a circles. The
arrows in panel a (insert in the upper right corner) indicate microcracks that occurred
within the nc-HA-p/chitosan coating.
microstructure of the Ti-13Nb-13Zr alloy used as a substrate was pro-
vided in our previous works [32,33].
TEM investigation performed on cross-section lamellas enables an
unambiguous description of the coating micro/nanostructure and ho-
mogeneity. The thickness of the nc-HA-p/chitosan and nc-HA-s/chito-
san coatings measured on the FIB lamella was up to 750 nm and 1.5
m, respectively. The TEM investigation shows that both coatings
were uniform in thickness (Figs. 8, 9). However, taking into account
the SEM investigation of the coating microstructure in much larger
areas, it should be noted that the nc-HA-p/chitosan coating was not
equal in thickness due to the presence of HA agglomerates (Fig. 6a).
TEM investigation conrmed the presence of particle agglomeration in
the nc-HA-p/chitosan coating. In contrast, the nc-HA-s/chitosan coating
was very homogeneous and consisted of HA nanoparticles homoge-
neously embedded in an amorphous chitosan matrix. SAED pattern
analysis revealed the presence of a nanocrystalline HA phase (SAED pat-
tern marked as A in Figs. 8, 9) in both types of composite coating. More-
over, SAED pattern analysis conrmed the occurrence of an amorphous
71
chitosan phase (SAED pattern marked as B in Fig. 8) in the nc-HA-p/chi-
tosan coating. The SEM-EDS and TEM-EDS microanalysis revealed that
the Ca/P molar ratio of nc-HA-p and nc-HA-s was around 1.8.
During TEM investigation, the presence of a thin layer (thickness up
to 10 nm) was observed on the coating/titanium alloy interface (Fig.
10). The interface between the coatings and the substrate was investi-
gated in detail by XPS (Fig. 11). The XPS depth probe is a few nanome-
ters. Thus, this technique, being surface sensitive, allowed the
investigation of a thin oxide layer naturally formed on the surface of
the Ti-13Nb-13Zr alloy to which the HA/chitosan coatings were at-
tached. Fig. 11 shows the spectra of the strongest lines (O 1s, Ti 2p, Nb
3d and Zr 3d) of elements detected on the surface of the alloy. Each ex-
perimentally determined spectrum (presented by dots) was tted using
theoretical lines which corresponded to the given oxidation state of the
element. The O 1s spectrum was tted using three theoretical singlet
lines. The line of the lowest energy (~530.2 eV) corresponds to the ox-
ygen in metal oxides. The lines of higher binding energy are assumed
to represent the chemical states of the oxygen present in the com-
pounds adsorbed on the surface. Unfortunately, the position of spectral
lines of oxygen bonded to Ti, Nb and Zr are closer to each other than the
spectrometer resolution, and it is impossible to distinguish them. Thus,
they form a single line. Contrary to oxygen, different chemical states of
metal elements are well distinguishable. The Ti 2p spectrum was tted
using three doublet lines corresponding to Ti0 (the lowest binding ener-
gy), Ti2+ and Ti4+ (the highest binding energy) oxidation states. The Nb
spectrum was tted using four doublet lines corresponding to the fol-
lowing oxidation states (from the lowest to the highest binding ener-
gy): Nb0, Nb2+, Nb4+ and Nb5+. And the Zr 3d spectrum was tted by
two doublet lines: Zr0 (lower energy) and Zr4+ (higher energy). Be-
cause the metallic states of Ti, Nb and Zr were found as relatively strong
lines, it means that the oxide layer is very thin, not exceeding a few
nanometers, which is consistent with the TEM observation. The very
strong lines of the highest oxidation states of these elements indicate
that the atoms with the highest oxidation state are placed at the exter-
nal part of the oxide layer, while the atoms with lower oxidation states
are located closer to the substrate. Quantitative analysis of the spectral
line intensities indicated that the ratio of the concentrations of Ti, Nb
and Zr in the oxide layer and in the substrate alloy is the same, suggest-
ing the absence of preferential oxidation of any component of the alloy.
It is well known that the highly developed and porous surface topog-
raphy of a metallic implant improves its osteointegration process with
the surrounding bone tissue [40,41]. The surface topography of investi-
gated composite nc-HA-p/chitosan and nc-HA-s/chitosan coatings is
shown in Fig. 12a and b, respectively. Furthermore, the representative
Fig. 7. GIXRD pattern of the nc-HA-p/chitosan coating deposited at a potential difference of
10 V for 4 min on the Ti-13Nb-13Zr alloy. The pattern was performed at a low incidence
angle of 1.
72 D. Jugowiec et al. / Surface & Coatings Technology 324 (2017) 6479

Fig. 8. Microstructure of the nc-HA-p/chitosan coating on the Ti-13Nb-13Zr alloy and SAED patterns taken from the coating areas marked as A and B, TEM, FIB lamella.

Fig. 9. Microstructure of the nc-HA-s/chitosan coating on the Ti-13Nb-13Zr alloy and SAED pattern taken from the coating area marked as A, as well as its identication as HA (hp), TEM -
FIB lamella.
 D. Jugowiec et al. / Surface & Coatings Technology 324 (2017) 6479
Fig. 10. Microstructure of the nc-HA-s/chitosan coating on the Ti-13Nb-13Zr alloy, TEM -
FIB lamella. The thin oxide layer is present at the coating/substrate interface.
roughness topography parameters, such as Rq, Rmax, Rsk, and Rku, and
image surface area difference (ISAD) for the analyzed surface areas of
both coatings are presented in Table 1. ISAD indicates the difference
Fig. 11. O 1s, Ti 2p, Nb 3d and Zr 3d spectra determined by the XPS technique. Experimental data (dots) are tted using theoretical lines corresponding to the given oxidation state of
elements present at the surface of the Ti-13Nb-13Zr alloy.
73
between the image's three-dimensional surface area and its two-di-
mensional footprint area [42]. A high value of ISAD signies high surface
development of the coating. The ISAD and the rest of the measured to-
pography parameters indicate that the topography of the nc-HA-p/chi-
tosan coating was considerably rougher compared to the nc-HA-s/
chitosan coating. This is due to the non-homogeneous microstructure
of the nc-HA-p/chitosan coating characterized by the occurrence of HA
agglomerates. The rougher surface of the coating is benecial for coating
adhesion to the titanium alloy substrate.
3.4. Coating adhesion to the titanium alloy substrate
Micro-scratch test investigation has revealed that the nc-HA-s/chito-
san coating had better adhesion to the titanium alloy substrate com-
pared to the nc-HA-p/chitosan coating. For nc-HA-p/chitosan coating
rst small area delamination took place at the scratch sides under load
of 0.7 0.3 N (Fig. 13a). The same failure mode was detected at 3
0.9 N load for nc-HA-s/chitosan coating (Fig. 13b). These values were
dened as adhesive critical load Lc2. Below this loads no cohesive cracks
were found, what could indicate the plastic nature of coating deforma-
tion. However, image of the track of nc-HA-p/chitosan at 1.4 0.4 N
load, when total coating delamination from the whole width of the
scratch was observed, looked rather like a failure of stiff, hard coating
on compliant substrate. This could be a result of low coating adhesion
and its low fracture toughness. Some delamination's, but not as cata-
strophic were found at 10.1 2.8 N load for nc-HA-s/chitosan coating.
The whole scratch track is dominated by abrasive wear with visible ma-
terial pile-ups in front and at the sides of the track. However, the larger
amount of piled-up material of the nc-HA-s/chitosan coating than nc-
HA-p/chitosan may be a result of the greater thickness of the rst coat-
ing. It is presumed that the relatively low adhesion of the nc-HA-p/chi-
tosan coating to the substrate is caused by its non-homogeneous
74 D. Jugowiec et al. / Surface & Coatings Technology 324 (2017) 6479

of the nc-HA-s/chitosan coated alloy. Fig. 14a shows the evolution of

OCP for the uncoated Ti-13Nb-13Zr alloy as well as coated ones with
chitosan and nc-HA-s/chitosan coatings. It can be observed that the po-
tential was stable in time for all samples and equal to 0.45 V, 0.45 V,
0.29 V (vs SCE), for the pristine substrate, coated with chitosan, and

Fig. 12. Surface topography of nc-HA-s/chitosan (a) and nc-HA-s/chitosan (b) coatings on
the Ti-13Nb-13Zr alloy, AFM.

microstructure, especially the presence of HA agglomerates. On the

other hand, the uniform distribution of HA particles in the nc-HA-s/chi-
tosan coating contributed to the better coating adhesion to the underly-
ing substrate. Due to its non-homogeneous microstructure, nc-HA-p/
chitosan was not researched further.

3.5. Corrosion resistance

The electrochemical corrosion resistance for the chitosan coated Ti-

13Nb-13Zr alloy in Ringer's solution was investigated and published
in detail in our previous paper [43]. In the current study, we use these
results to compare them with the electrochemical corrosion resistance

Table 1
Selected parameters of surface topography of composite nc-HA-p/chitosan and nc-HA-s/
chitosan coatings.

Roughness parameters nc-HA-p/chitosan coating nc-HA-s/chitosan coating

Ra 360 nm 58 nm 355 nm 91 nm
Rq 468 nm 81 nm 434 nm 106 nm
Rmax 2954 nm 416 nm 2216 nm 220 nm
Rsk 0.84 0.1 0.1 0.3
Fig. 13. Scratch track within the nc-HA-p/chitosan (a) and nc-HA-s/chitosan (b) coatings
Rku 4.1 0.6 2.6 0.6
on the Ti-13Nb-13Zr alloy, SEM secondary electron images. The magnied details of the
ISAD 13.6% 1.3% 4.3% 0.3%
scratch track are shown in insets in panel a, b.
 D. Jugowiec et al. / Surface & Coatings Technology 324 (2017) 6479 75

coated with the nc-HA-s/chitosan composite, respectively. The ob-

served shift in OCP to positive values for nc-HA-s/chitosan samples
compared to the uncoated one may indicate a decrease in the driving
force for the corrosion process and, therefore, improved corrosion resis-
tance [44]. A potentiodynamic polarization test was performed in order
to further investigate the corrosion properties of the uncoated Ti-13Nb-
13Zr alloy, as well as when coated with chitosan and nc-HA/chitosan
composite coatings (Fig. 14b). Due to the lack of a linear pattern, Tafel
extrapolation cannot be applied to evaluate the electrochemical re-
sponse. Therefore, current density which passes through the passivating
lm can be the measure of the corrosion rate [45]. It was observed that
the passive current density for the nc-HA/chitosan coating has been re-
duced from 20 A/cm2 for the uncoated sample to 6 A/cm2 for the
Fig. 15. Equivalent electrical circuit (Randles circuit) for the analysis of the impedance
spectra. CPE is the constant phase element, R1 is the electrolyte resistance and R2 is the
coating resistance.
coated one. As for the sample with a chitosan coating, passive current
density is comparable to that of the uncoated one.
On the other hand, corrosion potential increased from ~ 0.45 (po-
tential of the cathodic-anodic transition of the pristine Ti-13Nb-13Zr
alloy) to 0.54 V for the nc-HA/chitosan coating. This result conrmed

Fig. 14. Electrochemical measurements of uncoated near- Ti-13Nb-13Zr and samples coated with chitosan and nc-HA-s/chitosan composite materials in Ringer's solution at 37 C,
evolution of the stationary potential vs. time (a), polarization curves at 1 mV/s (b) as well as electrochemical Nyquist (c), Bode (d) and Phase Angle Frequency (e) impedance curves.
76 D. Jugowiec et al. / Surface & Coatings Technology 324 (2017) 6479

Table 2
Chi-square (2) values obtained by tting equivalent electrical circuit with Z View software and electrochemical parameters for the uncoated and coated titanium alloy.
1
Samples 2 R1 ( cm2) CPE-T (Fsn cm2) CPE-P R2 ( cm2)

Ti-13Nb-13Zr 0.005 12.30 0.12 4.3137 105 3.0412 107 0.8963 0.002 321,830 6327.6
Chitosan on Ti-13Nb-13Zr 0.003 61.41 0.39 2.8011 105 2.1839 107 0.7651 0.002 99,619 997.01
nc-HA-s/chitosan on Ti-13Nb-13Zr 0.002 38.94 0.21 2.7965 105 1.0359 107 0.9068 0.0009 448,060 12,584

that the composite nc-HA/chitosan coating improves the corrosion re-
sistance of the titanium alloy.
Fig. 14ce show the EIS spectra presented as a Nyquist plot (Fig. 14c)
and Bode plots (Fig. 14e) of the uncoated and coated alloy in Ringer's so-
lution. From Fig. 14d, the higher Z modulus at lower frequency in the
Bode impedance plot indicated a better corrosion resistance of the en-
tire coated alloy. It follows from Fig. 14e that the uncoated and nc-HA-
s/chitosan coated alloys reveal a highly capacitive nature of the imped-
ance from medium to low frequencies, indicating that a stable lm was
formed on the substrates. This result is probably an effect of its thick-
ness, homogeneous microstructure and good adhesion to the substrate
material. On the chitosan coating a signicantly lower capacitive nature
of the impedance was observed, which can be connected with the rela-
tively poor adhesion of the chitosan coating to the titanium alloy sub-
strate and the biodegradable nature of chitosan.
The EIS studies were carried out in order to describe the electro-
chemical interface of the coated substrates in terms of an equivalent cir-
cuit. The equivalent Randles circuit used to t EIS data is presented in
Fig. 15. In order to adjust to a double-layer model for the surface coating,
the equivalent circuit consisted of the constant phase element (CPE),
the electrolyte resistance (R1) and the coating resistance (R2). Experi-
mental and simulated results were well tted and all the parameters
are listed in Table 2.
The chitosan coating had a lower CPE-P, R2 and CPE-T compared to
the Ti-13Nb-13Zr alloy. These results of EIS investigation indicated
that this coating had a worse corrosion resistance than the uncoated
alloy. The nc-HA-s/chitosan composite coating had a higher CPE-P
value, which was near to the capacitance. The coating resistance (R2)
of the nc-HA-s/chitosan coating was also signicantly higher than that
of the Ti-13Nb-13Zr alloy, which suggested that this coating had a better
corrosion resistance than the uncoated alloy.
3.6. Bioactivity in SBF
The pristine near- titanium alloy surface was smooth (Fig. 16a) and
covered with a thin layer of protective oxide containing Ti, Nb and Zr, as
shown above in TEM (Fig. 10) and XPS (Fig. 11) investigation. The chi-
tosan coating surface was also smooth, homogeneous and continuous
(Fig. 16b). The nc-HA-s/chitosan coating was rougher (Fig. 16c) and
consisted of nc-HA particles homogeneously embedded in the chitosan
matrix (Fig. 9). Immersion in SBF resulted in the creation of typical cau-
liower-like hydroxyapatite deposits only on nc-HA-s/chitosan coating
(Fig. 16f). The HA layer showed the presence of: Ca, P and O, i.e. the el-
ements found in calcium phosphates. The Ca/P ratio was 1.5, i.e. differ-
ent to that measured for nc-HA-s (1.8). Although the Ca/P ratio for
stoichiometric hydroxyapatite is 1.67, but it is similar to that of
hydroxycarbonate apatite (Ca/P = 1.5), which is a major component
of the bone tissue [46].
The process of apatite formation on the bioactive materials in the liv-
ing body can be reproduced by incubation in SBF. To a certain degree, it
allows the in vivo bone bioactivity of a material to be predicted [47]. In
our study, we observed that HA nanoparticles in the chitosan layer pro-
moted HA deposition from SBF. This is due to the presence of negatively
charged groups in HA (i.e. OH and PO3 4 ) that attract the positive cal-
cium ions from the surrounding SBF. This process results in changing
the charge of the surface from negative to positive, which further at-
tracts the negatively charged OH and PO3 4 . The adsorbed and nucleat-
ed ions undergo precalcication forming calcium phosphate nuclei with

Fig. 16. SEM/EDX analysis of: Ti-13Nb-13Zr a), Ti-13Nb-13Zr coated by EPD with b) chitosan and c) nc-HA-s/chitosan, Ti-13Nb-13Zr after incubation in SBF d), Ti-13Nb-13Zr with chitosan
after incubation in SBF e), and Ti-13Nb-13Zr with nc-HA-s/chitosan after incubation in SBF f).
 D. Jugowiec et al. / Surface & Coatings Technology 324 (2017) 6479 77

lower surface energy. They facilitate the heterogeneous nucleation of
calcium phosphate and further precipitation and formation of apatite
on the surface [4850]. On Ti-13Nb-13Zr and that alloy coated with chi-
tosan (Fig. 16d, e), HA formation was hindered, most likely due to a lack
of nucleation sites for HA. Recent study of the EPD deposited chitosan on
the AISI 316L stainless steel showed that after 28 days of incubation in
SBF no HA deposition was detected, similarly to our experiments [51].
3.7. Cytocompatibility with bone cells
In vitro tests show that chitosan coated Ti-13Nb-13Zr and nc-HA-s/
chitosan supported cell growth: cells adhering on the surfaces were
alive (no red dead cells were found; Fig. 17a panels 1 and 2), although
the samples affected cell morphology, adhesion and proliferation in a
different way. On day 1, cell morphology on the samples containing chi-
tosan and chitosan with HA was similar: cells were round and less
spread than those grown on the reference controls (Ti-13Nb-13Zr and
TCPS). On nc-HA-s/chitosan cytoskeleton bers were better developed
as compared to those on chitosan, but slightly less developed than on
both controls (phalloidin/DAPI stained cells - Fig. 17a panels 3 and 4).
On day 7, cells cultured on nc-HA-s/chitosan were better spread, had a
polygonal shape and covered almost all the available area in a way sim-
ilar to the control Ti-13Nb-13Zr and TCPS. On Ti-13Nb-13Zr coated with
chitosan after 7 days cell density appeared to be lower as compared to
nc-HA-s/chitosan and controls. Cell metabolic activity on day 1 and
day 3 was signicantly impeded when the cells were cultured on nc-

Fig. 17. In vitro tests on MG-63 osteoblast-like cells cultured on Ti-13Nb-13Zr, and Ti-13Nb-13Zr coated by EPD with chitosan and nc-HA-s/chitosan and on control tissue culture
polystyrene (TCPS): a) live/dead staining (panel 1 and 2) and phalloidin/DAPI (panel 3 and 4) on day 1 (1 d) and day 7 (7 d); b) metabolic activity of cells after 1, 3 and 7 days;
statistical signicance *p b 0.05 according to t-test.
78 D. Jugowiec et al. / Surface & Coatings Technology 324 (2017) 6479
HA-s/chitosan, however, after 7 days no statistically signicant differ-
ence was found between nc-HA-s/chitosan and the controls (Ti-13Nb-
13Zr and TCPS) (Fig. 17b). On the other hand, for chitosan coated Ti-
13Nb-13Zr no difference was found on day 1, but for longer culture
time (3 and 7 days) cell metabolic activity was negatively affected as
compared to non-coated surface and TCPS.
Our study suggests that chitosan and nc-HA-s/chitosan coatings sig-
nicantly inuence the morphology of adhering cells. This may be at-
tributed to the high water uptake of chitosan and the formation of
soft, gel-like structures precluding the adsorption of cell adhesive pro-
teins (bronectin, vitronectin, collagen) from a cell culture medium
supplemented with serum [23,51]. Similar results were obtained by
other groups. It has been reported that surface roughness, topography
at the micro- and nano-scale may inuence cell morphology and prolif-
eration [52]. The addition of hydroxyapatite nanoparticles (nHA), which
enhances surface roughness, may result in the increased adsorption of
bronectin and improved cell adhesion to the surface [53,54]. The pres-
ence of nHA is also known to increase the stiffness of the material which
supports bone cell proliferation and spreading. The compression modu-
lus of hydrated chitosan scaffolds signicantly increased with increasing
nHA content, which can be attributed to the interaction between chito-
san molecules and HA [53]. For example, HA may interact with amino
and hydroxyl groups of chitosan by the formation of hydrogen bonds
[55].
4. Conclusions
1. The nanocomposite nc-HA-p/chitosan and nc-HA-s/chitosan coat-
ings were successfully deposited on the Ti-13Nb-13Zr titanium
alloy by EPD. HA particles were introduced to the EPD suspensions
in two different states, as: (i) powder and (ii) suspension of HA nano-
particles in ethanol. The highest values of electrophoretic mobility
for HA particles were achieved for the suspensions which contained
4 g/l of nc-HA-p and 30 g/l of nc-HA-s in a mixture of distilled water
and 0.5 vol% of acetic acid as well as 50 vol% of ethanol. Accordingly,
the nc-HA-p/chitosan coating of the highest quality was deposited
from the suspension which contained 4 g/l of nc-HA-p and 2 g/l of
chitosan. However, the most homogeneous and continuous nc-HA-
s/chitosan coatings were deposited from the suspension which
contained 2 g/l of chitosan and only 4 g/l of nc-HA-s instead of
30 g/l of nc-HA-s, at the potential difference of 10 V for 4 min This
is due to the higher HA content in the nc-HA-s/chitosan coating,
which contributed to the formation of microcracks.
2. The nc-HA-s/chitosan coating microstructure was composed of
nanocrystalline HA particles, homogeneously embedded in an amor-
phous chitosan matrix. On the other hand, the nc-HA-p/chitosan
coating microstructure was non-uniform, composed of HA agglom-
erates of different sizes. The thickness of the nc-HA-p/chitosan and
nc-HA-s/chitosan coatings evaluated from the FIB lamellas was up
to 750 nm and 1.5 m, respectively.
3. The nc-HA-p/chitosan coatings exhibited more developed surface to-
pography in comparison to the nc-HA-s/chitosan coatings, mainly
due to the presence of HA agglomerates.
4. The nc-HA-s/chitosan coatings exhibited better adhesion to the Ti-
13Nb-13Zr alloy substrate than the nc-HA-p/chitosan ones. The bet-
ter adhesion of the nc-HA-s/chitosan coatings to the substrate is due
to homogeneous microstructure of the coating.
5. The nc-HA-s/chitosan coating improved the corrosion resistance of
the near- Ti-13Nb-13Zr alloy in Ringer's solution at a temperature
of 37 C.
6. On the nc-HA-s/chitosan coating, a thick layer of low-crystalline hy-
droxyapatite was created after incubation in SBF, which might sug-
gest enhanced bioactivity of this surface that may be benecial for
osteointegration in vivo. Both coatings were not cytotoxic; nc-HA-
s/chitosan supported cell adhesion, proliferation and metabolic activ-
ity, especially for longer time points. Thus, nc-HA-s/chitosan seems
to be promising for osteosynthesis, oral implantology and in joint
prostheses xation.
Acknowledgments
This work was supported by the Polish National Science Centre (DEC
2013/09/B/ST8/00145). The authors appreciate the valuable contribu-
tion of Dr. A. Radziszewska (AGH-University of Science and Technology)
to XRD and Dr. M. Gajewska (ACMIN, AGH-UST) for FIB lamella prepara-
tion, as well as Dr. M. Krok-Borkowicz and K. Reczyska (AGH-UST, Fac-
ulty of Materials Science and Ceramics) for their help during in vitro
studies.
References
[1] Araghi, M.J. Hadianfard, Fabrication and characterization of functionally graded hy-
droxyapatite/TiO2 multilayer coating on Ti-6Al-4V titanium alloy for biomedical ap-
plications, Ceram. Int. 41 (2015) 1266812679.
[2] P.S. Nnamchi, C.S. Obayi, I. Todd, M.W. Rainforth, Mechanical and electrochemical
characterisation of new Ti-Mo-Nb-Zr alloys for biomedical applications, J. Mech.
Behav. Biomed. Mater. 60 (2016) 6877.
[3] I.U. Petersson, J.E.L. Lberg, A.S. Fredriksson, E.K. Ahlberg, Semi-conducting proper-
ties of titanium dioxide surfaces on titanium implants, Biomaterials 30 (2009)
44714479.
[4] O. Florncio, P.S. Silva Jr., R. Ribeiro, J.M. Chaves, F.H. S, F.X. Melo, S.G. Schneider, Ef-
fect of different annealing on the anelastic relaxation in Ti13Nb13Zr alloy, Mater.
Sci. Eng. A 521522 (2009) 351353.
[5] M. Geetha, U.K. Mudali, A.K. Goia, R. Asokamani, B. Raj, Inuence of microstructure
and alloying elements on corrosion behavior of Ti13Nb13Zr alloy, Corros. Sci. 46
(2004) 877892.
[6] M. Jarosz, A. Pawlik, M. Szuwarzyski, M. Jaskua, G.D. Sulka, Nanoporous anodic ti-
tanium dioxide layers as potential drug delivery systems: drug release kinetics and
mechanism, Colloids Surf., B 143 (2016) 447454.
[7] J. Raphel, M. Holodniy, S.B. Goodman, S.C. Heilshorn, Multifunctional coatings to si-
multaneously promote osseointegration and prevent infection of orthopedic im-
plants, Biomaterials 84 (2016) 301314.
[8] F. Samanipour, M.R. Bayati, F. Golestani-Fard, H.R. Zargar, A.R. Mirhabibi, V. Shoaei-
Rad, S. Abbasi, Innovative fabrication of ZrO2HApTiO2 nano/micro-structured
composites through MAO/EPD combined method, Mater. Lett. 65 (2011) 926928.
[9] F. Frajkorov, E. Molero, P. Montero, M.C. Gomez-Guillen, A.J. Sanchez-Herencia, B.
Ferrari, Biodegradable bi-layered coatings shaped by dipping of Ti lms followed
by the EPD of gelatin/hydroxyapatite composites, J. Eur. Ceram. Soc. 36 (2016)
343355.
[10] M. Goudarzi, F. Batmanghelich, A. Afshar, A. Dolati, G. Mortazavi, Development of
electrophoretically deposited hydroxyapatite coatings on anodized nanotubular
TiO2 structures: corrosion and sintering temperature, Appl. Surf. Sci. 301 (2014)
250257.
[11] S. Mahmoodi, L. Sorkhi, M. Farrokhi-Rad, T. Shahrabi, Electrophoretic deposition of
hydroxyapatitechitosan nanocomposite coatings in different alcohols, Surf. Coat.
Technol. 216 (2013) 106114.
[12] K. Kulpetchdara, A. Limpichaipanit, G. Rujijanagul, C. Randorn, K. Chokethawai, In-
uence of the nano hydroxyapatite powder on thermally sprayed HA coatings
onto stainless steel, Surf. Coat. Technol. 306 (2016) 181186.
[13] M. Farrokhi-Rad, S.K. Loghmani, T. Shahrabi, S. Khanmohammadi, Electrophoretic
deposition of hydroxyapatite nanostructured coatings with controlled porosity, J.
Eur. Ceram. Soc. 34 (2014) 97106.
[14] A.A. Abdeltawab, M.A. Shoeib, S.G. Mohamed, Electrophoretic deposition of hy-
droxyapatite coatings on titanium from dimethylformamide suspensions, Surf.
Coat. Technol. 206 (2011) 4350.
[15] M. Farrokhi-Rad, T. Shahrabi, Effect of triethanolamine on the electrophoretic depo-
sition of hydroxyapatite nanoparticles in isopropanol, Ceram. Int. 39 (2013)
70077013.
[16] A. Molaei, M. Yari, M.R. Afshar, Investigation of halloysite nanotube content on elec-
trophoretic deposition (EPD) of chitosan-bioglass-hydroxyapatite-halloysite nano-
tube nanocomposites lms in surface engineering, Appl. Clay Sci. 135 (2017) 7581.
[17] D. Zhitomirsky, J.A. Roether, A.R. Boccaccini, I. Zhitomirsky, Electrophoretic deposi-
tion of bioactive glass/polymer composite coatings with and without HA nanoparti-
cle inclusions for biomedical applications, J. Mater. Process. Technol. 209 (2009)
18531860.
[18] S. Clavijo, F. Membrives, G. Quiroga, A.R. Boccaccini, M.J. Santilln, Electrophoretic
deposition of chitosan/Bioglasss and chitosan/Bioglasss/TiO2 composite coatings
for bioimplants, Ceram. Int. 42 (2016) 1420614213.
[19] A. Simchi, F. Pishbin, A.R. Boccaccini, Electrophoretic deposition of chitosan, Mater.
Lett. 63 (2009) 22532256.
[20] F. Gebhardt, S. Seuss, M.C. Turhan, H. Hornberger, S. Virtanen, A.R. Boccaccini, Char-
acterization of electrophoretic chitosan coatings on stainless steel, Mater. Lett. 66
(2012) 302304.
[21] I. Zhitomirsky, A. Hashambhoy, Chitosan-mediated electrosynthesis of organic-inor-
ganic nanocomposites, J. Mater. Process. Technol. 191 (2007) 6872.
[22] X. Pang, I. Zhitomirsky, Electrophoretic deposition of composite hydroxyapatite-chi-
tosan coatings, Mater. Charact. 58 (2007) 339348.
 D. Jugowiec et al. / Surface & Coatings Technology 324 (2017) 6479
[23] Y.Y. Shi, M. Li, Q. Liu, Z.J. Jia, X.C. Xu, Y. Cheng, Y.F. Zheng, Electrophoretic deposition
of graphene oxide reinforced chitosanhydroxyapatite nanocomposite coatings on
Ti substrate, J. Mater. Sci. Mater. Med. 27 (2016) 48.
[24] T. Moskalewicz, M. Kot, S. Seuss, A. Kdzierska, A. Czyrska-Filemonowicz, A.R.
Boccaccini, Electrophoretic deposition and characterization of HA/chitosan nano-
composite coatings on Ti6Al7Nb alloy, Met. Mater. Int. 21 (2015) 96103.
[25] X. Pang, T. Casagrande, I. Zhitomirsky, Electrophoretic deposition of hydroxyapatite
CaSiO3chitosan composite coatings, J. Colloid Interface Sci. 330 (2009) 323329.
[26] I. Deen, X. Pang, I. Zhitomirsky, Electrophoretic deposition of composite chitosan
halloysite nanotubehydroxyapatite lms, Colloids Surf. A Physicochem. Eng. Asp.
410 (2012) 3844.
[27] K. Grandeld, I. Zhitomirsky, Electrophoretic deposition of composite hydroxyapa-
titesilicachitosan coatings, Mater. Charact. 59 (2008) 6167.
[28] K. Grandeld, F. Sun, M. FitzPatrick, M. Cheong, I. Zhitomirsky, Electrophoretic depo-
sition of polymer-carbon nanotubehydroxyapatite composites, Surf. Coat. Technol.
203 (2009) 14811487.
[29] F. Batmanghelich, M. Ghorbani, Effect of pH and carbon nanotube content on the
corrosion behavior of electrophoretically deposited chitosanhydroxyapatitecar-
bon nanotube composite coatings, Ceram. Int. 39 (2013) 53935402.
[30] . Pajchel, V. Kowalska, D. Smole, A. Kdzierska, E. Pietrzykowska, W. ojkowski, W.
Kolodziejski, Comprehensive structural studies of ultra-ne nanocrystalline calcium
hydroxyapatite using MAS NMR and FT-IR spectroscopic methods, Mater. Res. Bull.
48 (2013) 48184825.
[31] D. Smole, T. Chudoba, S. Gierlotka, A. Kedzierska, W. ojkowski, K. Sobczak, W.
wiszkowski, K.J. Kurzydowski, Hydroxyapatite nanopowder synthesis with a pro-
grammed resorption rate, J. Nanomater. 2012 (2012) 19.
[32] D. Jugowiec, M. Kot, T. Moskalewicz, Electrophoretic deposition and characterization
of chitosan coating on near-beta titanium alloy, Arch. Metall. Mater. 61 (2016)
657664.
[33] A. Sak, T. Moskalewicz, S. Zimowski, . Cieniek, B. Dubiel, A. Radziszewska, M. Kot, A.
ukaszczyk, Inuence of polyetheretherketone coatings on the Ti-13Nb-13Zr titani-
um alloy's bio-tribological properties and corrosion resistance, Mater. Sci. Eng. C 63
(2016) 5261.
[34] T. Kokubo, H. Kushitani, S. Sakka, T. Kitsugi, T. Yamamuro, Solutions able to repro-
duce in vivo surface-structure changes in bioactive glass-ceramic A-W, J. Biomed.
Mater. Res. 24 (1990) 721734.
[35] C. Wu, S. Zhou, W. Wang, A dynamic laser light-scattering study of chitosan in aque-
ous-solution, Biopolymers 35 (1995) 385392.
[36] L. Besra, M. Liu, A review on fundamentals and applications of electrophoretic depo-
sition (EPD), Prog. Mater. Sci. 52 (2007) 162.
[37] A.R. Boccaccini, S. Keim, R. Ma, Y. Li, I. Zhitomirsky, Electrophoretic deposition of bio-
materials, J. R. Soc. Interface 7 (2010) S581S613.
[38] F. Pishbin, A. Simchi, M.P. Ryana, A.R. Boccaccini, A study of the electrophoretic de-
position of Bioglass suspensions using the Taguchi experimental design approach,
J. Eur. Ceram. Soc. 30 (2010) 29632970.
[39] F. Sun, X. Pang, I. Zhitomirsky, Electrophoretic deposition of composite hy-
droxyapatite chitosanheparin coatings, J. Mater. Process. Technol. 209
(2009) 15971606.
79
[40] M. Takemoto, S. Fujibayashi, M. Neo, J. Suzuki, T. Kokubo, T. Nakamura, Mechanical
properties and osteoconductivity of porous bioactive titanium, Biomaterials 26
(2005) 60146023.
[41] J.R. Woodard, A.J. Hilldore, S.K. Lan, C.J. Park, A.W. Morgan, J.A.C. Eurell, S.G. Clark,
M.B. Wheeler, R.D. Jamison, A.J. Wagoner Johnson, The mechanical properties and
osteoconductivity of hydroxyapatite bone scaffolds with multi-scale porosity, Bio-
materials 28 (2007) 4554.
[42] S.E. Oraby, A.M. Alaskari, Atomic Force Microscopy (AFM) topographical surface
characterization of multilayer-coated and uncoated carbide inserts, Int. J. Mech.
Aerosp. Ind. Mechatron Manuf. Eng. 4 (2010) 977988.
[43] A. ukaszczyk, T. Moskalewicz, D. Jugowiec, A. Sak, . Cieniek, H. Krawiec, Corrosion
resistance of Ti13Zr13Nb alloy with polymer and composite sol-gel bioactive glass/
polymer coatings in Ringer's solution, Ann. Chim. Sci. Mater. 40 (2016) 3141.
[44] D.J. Blackwood, A.W. Chua, K.H.W. Seah, Corrosion behaviour of porous titanium-
graphite composites designer for surgical implants, Corros. Sci. 42 (3) (2000)
481503.
[45] E. Mc Cafferty, G.K. Hubler, Electrochemical behavior of palladium-implanted titani-
um, J. Electrochem. Soc. 125 (11) (1978) 18921893.
[46] S. Talebian, M. Mehrali, S. Mohan, H.R. Balaji raghavendran, M. Mehrali, H.M.
Khanlou, T. Kamarul, A.M. A, A.A. Abass, Chitosan (PEO)/bioactive glass hybrid
nanobers for bone tissue engineering, RSC Adv. 4 (2014) 4914449152.
[47] A. Zadpoor, Relationship between in vitro apatite-forming ability measured using
simulated body uid and in vivo bioactivity of biomaterials, Mater. Sci. Eng. C 35
(2014) 134143.
[48] G.K. Toworfe, R.J. Composto, I.M. Shapiro, P. Ducheyne, Nucleation and growth of
calcium phosphate on amine-, carboxyl-and hydroxyl-silane self-assembled mono-
layers, Biomaterials 27 (2006) 631642.
[49] B. Feng, Y.J. Chen, S.K. Qi, L. He, J.Z. Zhao, X.D. Zhang, Carbonate apatite coating on
titanium induced rapidly by precalcication, Biomaterials 23 (2002) 173179.
[50] J. Zhang, C. Dai, J. Wei, Z. Wen, S. Zhang, C. Chen, Degradable behavior and bioactiv-
ity of micro-arc oxidized AZ91D Mg alloy with calcium phosphate/chitosan compos-
ite coating in m-SBF, Colloids Surf., B 111 (2013) 179187.
[51] F. Pishbin, V. Mourino, J.B. Gilchrist, D.W. McComb, S. Kreppel, V. Salih, M.P. Ryan,
A.R. Boccaccini, Single-step electrochemical deposition of antimicrobial orthopaedic
coatings based on a bioactive glass/chitosan/nano-silver composite system, Acta
Biomater. 9 (2013) 74697479.
[52] Y.-F. Chou, W. Huang, J.C.Y. Dunn, T.A. Miller, B.M. Wu, The effect of biomimetic ap-
atite structure on osteoblast viability, proliferation, and gene expression, Biomate-
rials 26 (2005) 285295.
[53] W.W. Thein-Han, R.D.K. Misra, Biomimetic chitosan-nanohydroxyapatite composite
scaffolds for bone tissue engineering, Acta Biomater. 5 (2009) 11821197.
[54] L. Kong, Y. Gao, G. Lu, Y. Gong, N. Zhao, X. Zhang, A study on the bioactivity of chi-
tosan/nano-hydroxyapatite composite scaffolds for bone tissue engineering, Eur.
Polym. J. 42 (2006) 31713179.
[55] M. Kikuchi, T. Ikoma, S. Itoh, H.N. Matsumoto, Y. Koyama, K. Takakuda, K. Shinomiya,
J. Tanaka, Biomimetic synthesis of bone-like nanocomposites using the self-organi-
zation mechanism of hydroxyapatite and collagen, Compos. Sci. Technol. 64 (2004)
819825.