Ceramics International 45 (2019) 1710–1723

Contents lists available at ScienceDirect

Ceramics International
journal homepage: www.elsevier.com/locate/ceramint

Influence of Ti, Zr or Nb carbide adhesion layers on the adhesion, corrosion T

resistance and cell proliferation of titania doped hydroxyapatite to the
Ti6Al4V alloy substrate, utilizable for orthopaedic implants
Alina Vladescua,b, , Vasile Prunac, Sawomir Kuleszad, Viorel Braica, Irina Titorencuc,
⁎

Miroslaw Bramowiczg, Anna Gozdziejewskae, Anca Paraua, Cosmin M. Cotrutb,f, Iulian Panaa,
Mihaela Dinua, Mariana Braica
a
National Institute for Optoelectronics INOE2000, 409 Atomistilor St., 077125 Magurele, Romania
b
National Research Tomsk Polytechnic University, Lenin Avenue 43, Tomsk 634050, Russia
c
Institute of Cellular Biology and Pathology "Nicolae Simionescu" of the Romanian Academy, 8 B.P.HASDEU street, 050568 Bucharest, Romania
d
University of Warmia and Mazury in Olsztyn, Faculty of Mathematics and Computer Science, Sloneczna 54, 10-710 Olsztyn, Poland
e
University of Warmia and Mazury in Olsztyn, Faculty of Environmental Sciences, Oczapowskiego 5, 10-719 Olsztyn, Poland
f
University Politehnica of Bucharest, 313 Spl. Independentei, 060042 Bucharest, Romania
g
Faculty of Technical Sciences, Oczapowskiego 11, 10-719 Olsztyn, Poland

ARTICLE INFO ABSTRACT

Keywords: In the present paper, we study the enhancement of the adhesion between the titania doped hydroxyapatite
B: Electron microscopy, interfaces, X-ray coating and the Ti6Al4V alloy substrate by the addition of three different biocompatible carbides. The coatings
method were prepared by the magnetron sputtering technique. The addition of interlayer preserved the crystalline
C: Corrosion structure of hydroxyapatite. The morphology of the coating is influenced by the interlayer composition. The
D: apatite
adhesion of the coatings was significantly improved by the interlayer, independent of it's composition. The best
E: Biomedical applications
adhesion was found for the coatings with NbC interlayer, which also showed the best corrosion resistance in
simulated body fluid at 37 °C, due to its flat surface grown without any distinct features. As expected, the results
of the in vitro biological tests were almost similar for all coatings comprising an adhesion layer, as the same
titania doped hydroxyapatite layer was on top. The experimental results revealed that the adhesion between
Ti6Al4V and titania doped hydroxyapatite was improved by a hard metal carbide interlayer, without affecting
the required biomedical properties such as good corrosion resistance, high proliferation level of osteoblast cells,
and good gene expression levels.

1. Introduction
As reported in the literature, the adhesion between the metallic
implant and the surrounding tissue is weak [1–6], such as this re-
presents one of the main reason for rejection of the implant by human
body. For improving this bond, many researchers proposed to cover the
metallic implants with coatings which can confer a rapid biological
fixation and prosthetic stability in the early stage of implantation. The
most used are the bioactive coatings, which contain mainly calcium
phosphates, compounds with significant chemical similarity with the
bone matrix [1,2,7–14], being already demonstrated by in vitro and in
vivo studies their capability for rapid cell attachment, growth and in-
growth of the cell bone [1,2,8–12,15]. After insertion of metallic im-
plants into human body, the implant is encapsulated by a thin layer
consisted in tissue cells, with weak adhesion to implant, and prevents
the ingrowth of the cell bone [3–7,16]. If the metallic implants are
coated with calcium phosphates based coatings, the bone cells will be in
direct contact with coatings and the adhesion between them will be
performed without any intervening layer [6]. In this way, the apatite
matrix of the bone creates a strong bond with the apatite matrix of the
coating, generating an excellent acceptance of the coated implant [6].
Stoichiometric hydroxyapatite is one of the most used calcium phos-
phates based coating [9,10,12–14,17], because it has a low dissolution
rate in contact with the corrosive environment of human body [17,18].
A thorough review of the literature showed that the hydroxyapatite
(HAP) coatings can be prepared using various techniques such as
plasma spraying, sol-gel, RF magnetron sputtering and electrophoretic
deposition, laser ablation, hydrothermal and biomimetic methods

⁎
Corresponding author at: National Institute for Optoelectronics INOE2000, 409 Atomistilor St., 077125 Magurele, Romania.
E-mail address: alinava@inoe.ro (A. Vladescu).

https://doi.org/10.1016/j.ceramint.2018.10.053
Received 8 August 2018; Received in revised form 19 September 2018; Accepted 8 October 2018
Available online 11 October 2018
0272-8842/ © 2018 Elsevier Ltd and Techna Group S.r.l. All rights reserved.
A. Vladescu et al.
[17,19–25]. At present, plasma spraying is the most commonly used
method for calcium phosphates synthesis. Nevertheless, the coatings
prepared by plasma-spraying exhibit a lack of crystallinity and poor
adhesion to the metallic substrate [17,20,21]. By RF magnetron sput-
tering, the deposited HAP coatings present high crystallinity and better
adhesion to the metallic substrate, but due to the low sputtering rate,
the deposition time might be quite long [17,20,21,26–31].
In the literature, it was reported that the mechanical, anticorrosive,
antibacterial and antifungal properties of HAP coatings could be im-
proved by doping it with various elements such as Ti, Si and Ag, or
combination of these [15,32–38]. However, all of these characteristics
are dependent on the adhesion between the coating and the metallic
surfaces. It was found the Si doped HAP coatings obtained by RF
magnetron sputtering exhibited a slightly increased adhesion on
Ti6Al4V alloy substrate [32], such as in this study we added a thin
interlayer of transition metal carbide (MeC), namely TiC, ZrC or NbC
between the titania doped HAP (d-HAP) and Ti6Al4V alloy substrate,
expecting a significant augmentation of the adhesion. The carbides
were selected due to the following reasons. TiC is a biocompatible
[39,40], hard coating with low friction and superior wear performance
in dry or wet atmosphere [41] and good adhesion to different metallic
substrates [39,41]. NbC was selected due to its ability to form strong
bonds with metallic substrates, superior biocompatibility and antic-
orrosive properties [42–46]. ZrC is also known as a hard, wear-corro-
sion resistant and biocompatible coating [47,48]. In this study, the d-
HAP was selected based on the previous results which demonstrated
that the titania addition into HAP structure resulted in better bio-
compatibility, as well as in enhanced mechanical properties and re-
duced degradation rate in body fluids [15,38].
The aim of the present work is to obtain an enhanced adhesion of
the d-HAP coated Ti based alloy substrate by adding an adhesion layer
comprising a MeC coating. A special attention was devoted to the in-
vestigation of the adhesion between the d-HAP and Ti alloy substrate,
the effect of the adhesion layer to the formation of the crystalline HAP
structure and its morphology. These films are intended to be used as
coating for metallic orthopaedic implants, such as their resistance to the
corrosive attack in simulated body fluid (SBF) solution and the response
to cytotoxicity assay are of particular interests.
2. Materials and methods
2.1. Coating deposition
The coatings were deposited by RF magnetron sputtering method
using a magnetron deposition system equipped with 3 cathodes in
confocal configuration (Aja International, USA). For the coatings de-
position, HAP, TiO2 and NbC, ZrC or TiC targets (99.9% purity, Kurt J.
Lesker Company) were used. All cathodes were RF biased at constant
power and Ar was used as sputtering gas. Prior the deposition, the
substrates were sputtered using the Ar+ bombardment (1 keV; 15 min).
A schematic of the deposition chamber and position of the each cathode
used for preparing of NbC/d-HAP coating are presented in Fig. 1. The
distance between the cathodes and substrate holder is 25 mm. For the
preparation of d-HAP coating, both HAP and TiO2 cathodes were si-
multaneous worked. For the NbC/d-HAP coating, firstly the NbC layer
was prepared using the NbC cathode, then both HAP and TiO2 cathodes
for obtained d-HAP coating. The same procedure was repeated for the
TiC/d-HAP or ZrC/d-HAP coatings, using TiC or ZrC cathode, respec-
tively. Table 1 presents the deposition parameters for all of the coatings.
As largely acknowledge, the deposition or annealing temperature is an
important parameter for hydroxyapatite based films crystallisation,
despite the grown techniques [49–53]. The deposition temperature was
set at 700 °C, as it proved to produce crystalline HAP coatings [54]. The
deposition time was adjusted to obtain an adhesion layer with thickness
of 50 nm and d-HAP coatings with 500 nm thickness.
The coatings were deposited on Ti-6Al-4V discs (Φ = 10 mm, 3 mm
1711
Ceramics International 45 (2019) 1710–1723
thickness) for all the investigations, except for the EDS and XRD mea-
surements, carried on Si wafers, in order to differentiate the coatings'
elements and compounds, respectively, from those of the substrate, and
AFM measurements.
In the present study, the investigated coatings are labelled as: d-HAP
– titania doped hydroxyapatite; NbC/d-HAP – titania doped hydro-
xyapatite with NbC adhesion layer; TiC/d-HAP – titania doped hydro-
xyapatite with TiC adhesion layer; ZrC/d-HAP – Ti doped hydro-
xyapatite with ZrC adhesion layer. It is important to mention that d-
HAP was used as reference coating.
2.2. Microstructural and mechanical analysis
The elemental analysis was performed by energy-dispersive X-ray
spectrometry (EDS) using a QUANTAX 70 (Brucker) system equipped
with a XFlash 430H detector, specially designed for the Hitachi
TM3000 Tabletop, with a detection limit of 0.1 wt% [55]. The phase
composition was characterized by X-ray diffraction (SmartLab, Rigaku)
at grazing incidence (0.5°) from 30° to 50°.
A special attention was devoted to the evaluation of the adhesion
properties. The progressive scratch test was carried out for each coating
using an indenter speed of 10 mm/min and a scratch length of 10 mm,
test conditions recommended by the BS EN 1071–3:2005 standard [56].
The adhesion was evaluated by an examination of the load at which the
film flaking starts using the UMT-TriboLab platform (Bruker). The tests
were performed according to the EN 1071–3/2005 standard: diamond
indenter with 200 µm tip radius, progressive load from 0 to 50 N,
scratching length – 5 mm, scratching speed −10 mm/min. The normal
load at which failure of the coating occurs, known as critical load (Lc),
was detected by direct microscopic observation and by an acoustic
sensor (EN 1071–3/2005 standard).
2.3. Morphology and analysis of spatial features
The films surface topography was evaluated by atomic force mi-
croscopy using an AFM system (Bruker Innova) working in tapping
mode, using a 8 nm radius Si tip. At least two (10 × 10) μm2 scans were
acquired on each coating to ensure surface representation. AFM images
contain height samples zxy of the surface under study taken at equidi-
stant scan steps.
Statistical analysis usually starts from computation of standard de-
scriptors of the data distribution: root-mean-square roughness Sq,
skewness Ssk and kurtosis Sku [57], while in order to reveal spatial
patterns, the autocorrelation function R turns out to be useful [58].
With increasing shift, the areal autocorrelation function (AACF)
asymptotically decays to zero, and the rate of this decay can be used to
express the degree of directional differences in small, local deviations of
a surface from the perfectly flat plane, referred to as the anisotropy
ratio Str. Dong et al, proposed the anisotropy ratio to be the proportion
of two extreme decay lengths, i.e. the slowest (τa1) and the fastest (τa2)
is referred to as the anisotropy ratio Str [59]:
0 < Str = a1
1
a2 R = 1 0.2 (1)
In general, the higher Str value indicates the more isotropic surface.
Apart from that, however, AACF also serves as a tool for estimating the
size of predominant surface features such as: grains, nanoparticles,
domains etc. Here, the average grain diameter dAACF is defined as an
arithmetic mean of the half-widths at the half maxima of the AACF
curves taken along directions of extreme decays: Da1 and Da2 [31,60]:
Da1 + Da2
dAACF =
2 (2)
Although statistical parameters mentioned above have been widely
applied to surface description, they suffer from ambiguous relation to
the distribution of the data series (height samples in AFM images) in the
A. Vladescu et al. Ceramics International 45 (2019) 1710–1723

Fig. 1. A schematic of the deposition chamber and position of the each cathode used for the deposition of the NbC/d-HAP coating.

length domain. Fortunately, the entire surface geometry is also re- (pH = 7.4), at 37 ± 0.4 °C, using a PARSTAT 4000 Potentiostat/
presented by the power spectrum in the reciprocal domain [58], which Galvanostat. The tests were conducted using a typical three-electrode
can be used to establish alternative characteristics referred to as fractal cell, with a Pt and saturated calomel (0.242 V vs. NHE) counter and
parameters and associated with the way, the surface changes upon reference electrode (sample). The open-circuit potential (EOC) was
scaling. Thomas and Thomas demonstrated that fractality leaves its monitored for 2 h. The Tafel curves were recorded at ± 250 mV vs. EOC
fingerprint in the structure function (SF) defined as [61] : at a scanning rate of 0.167 mV/s. The potentiodynamic polarization
N m N n curves were ranged from − 1 V to + 1 V at a scanning rate of
1 0.167 mV/s. The exposure area of each sample was of 1 cm2.
Smn = (z kl z k + m, l + n)2
(N m)(N n) k=1 l=1 (3) Experiments were performed according to ASTM G 59–97 (2014)
standard. For repeatability, two tests were carried out on two different
Fractal parameters can be derived from angle-averaged SF profiles,
samples prepared at the same conditions. By graphical extrapolation of
which are found to follow allometric dependence upon the spatial shift
the cathodic and anodic branches of the polarization curves in the range
τ defined as the separation of pairs of points in the plane of the surface
of ± 50 mV [69], the corrosion potential (Ei=0), and corrosion current
(AFM image) and its delayed copy [61]:
density (icorr) were determined. The polarization resistance (Rp) was
S( ) = K 2(2 Dp)
(4) calculated using the Stern-Geary equation [70]:

where: Dp – is the fractal dimension, and K – pseudo-topothesy. Final 1 ba | bc | 1

fractal dimension D of a rough surface is one higher than that for the
profile. Beyond the threshold τc referred to as the corner frequency, the
allometric scaling breaks down and the SF asymptotically approaches
2Sq2 value. Our previous studies demonstrated that the corner fre-
quency is found similar to average decay length, and hence to average
grain diameter [31,58]. As a quick characterization tool, fractal ana-
lysis proved to be useful in studies of morphology and mechanical
properties of various surface structures, for example subjected to heat
treatment [60,62,63], nanomechanical processing [32,64], in contact
with body tissues and fluids [65–68], etc.
2.4. In vitro electrochemical analysis
The corrosion behavior of the coatings was tested in SBF solution
Rp =
2.3 ba + bc icor (5)
where the icorr is corrosion current density, ba - anodic slope and bc -
cathodic slope of the polarization curves in the range of ± 50 mV.
2.5. In vitro biological assay
2.5.1. Cells seeding and culture
For biological tests were used human osteosarcoma cells line MG63
(American Type Culture Collection). The samples were sterilized for
24 h in ethanol 70% v/v, then washed with sterile distilled water and
kept for 24 h in the culture medium. After the cells thawing, they were
cultured for maximum 7 days in 1‰ glucose DMEM medium supple-
mented with 10% fetal bovine serum and antibiotics containing of 50U/
L neomycin, 100U/L streptomycin and 100U/L neomycin. The culture

Table 1
Deposition parameters of the coatings.
Coatings pbase (×10-4 Pa) pdeposition (×10-1 Pa) PRF (W) Ubias (V) Tdeposition (°C)

HAP TiO2 NbC TiC ZrC

d-HAP 1.3 6.67 50 25 – – – −60 700

NbC/d-HAP 30 – –
TiC/d-HAP – 30 –
ZrC/d-HAP – – 30

1712
A. Vladescu et al.
Fig. 2. Explanation of the parameters used for volume of cell suspension cal-
culus: V – the volume of cell suspension added in each well (μL), H – the cy-
linder sample height (mm), D – diameter of the well (mm), d – diameter of the
sample (mm), h – thickness of suspension liquid layer above the sample (mm), θ
– cell density (cells/cm2), ρ – suspension density (cells/mL).
conditions were as follows: temperature − 37 °C, relative humidity
− > 95%, CO2 concentration ─ 5% v/v in air. The density of seeded
cells was of about 8500/cm2. The culture medium was renewed every
third day. In order to have the same number of cells on each sample, the
volume of each sample was accurately determined by measuring its
diameter and height using an electronic caliper. The calculated volume
of the cell suspension needed for cell seeding allowed to obtain the
same thickness of liquid layer over the cultured cells (maximum 4 mm).
This represents an important parameter because the thickness of liquid
layer controls the oxygen diffusion level. The cell suspension volume V
was calculated by the formula:
H 2
V= (D d2) + D 2h
4 4 (6)
In Fig. 2 is presented a scheme for each parameter taken into ac-
count for calculating the volume V. The density (ρ) of the cell suspen-
sion (cells number/mL) was calculated using the formula:
10
=
(7)
h
2.5.2. Proliferation evaluation
To estimate the proliferation level (the cell population excess), the
DNA (deoxyribonucleic) amount was measured. After 3 and 7 days, the
culture medium was removed and the cells were washed with phos-
phate buffer saline (PBS). Subsequently, the cell membranes (including
the nucleus membranes) were broken to release the DNA from nucleus.
In order to disrupt the cell membranes, the cell culture was subjected to
extended temperature variation using the liquid nitrogen as the low
temperature source. More specifically, the liquid nitrogen was added
over the cell layer and then kept at room temperature for 4–6 min to
evaporate and reach room temperature. This step was repeated four
times, after which Hoechst 33258 solution (10 mg/mL) was added in
each well, and the plates were placed at 37 °C for 60 min. A standard
curve was performed using salmon DNA (Sigma) – Hoechst complex
fluorescence with DNA concentrations ranging from 0.125 to 25 ng/mL.
The fluorescence measurements of both standard and samples curves
Table 2
The primers used for gene expression levels.
Osteocalcin Forward: 5′-ACC-TGT-ATC-AAT-GGC-TGG-GAG-3′ Reverse: 5′-GGG-AAG-AGG-AAA-GAA-GGG-TG-3′
Osteonectin Forward: 5′-TGC-CTG-TCT-CTA-ACC-CCT-C-3′ Reverse: 5′-CCT-CCT-CTT-CGG-TTT-CCT-CTG-C-3′
BSP II Forward: 5′-CAA-CAC-TGG-GCT-ATG-GAG-AGG-ACG -3′ Reverse: 5′-ACT-GGT-GCC-GTT-TAT-GCC-TTG-TTC -3′
Type I collagen Forward: 5′-GGC-GAC-AGA-GGC-ATA-AAG-GGT-3′ Reverse: 5′-GTC-ACG-GTC-ACG-AAC-CAC-ATT-G-3′
Osteoprotegerin Forward: 5′- CCC-TTG-CCC-TGA-CCA-CTA-CTA-CAC-AG-3′ Reverse: 5′-CAA-ACC-TGA-AGA-ATG-CCT-CCT-CAXC-AC-3′
Beta-actin Forward: 5’-AGC-ATT-TGC-GGT-GGA-CGA-TGG-AG-3′ Reverse: 5′-GAC-CTG-ACT-GAC-TAC-CTC-ATG-A-3′
1713
Ceramics International 45 (2019) 1710–1723
were carried out in the same black plate using a TECAN spectro-
fluorometer. The fluorochrome excitation wavelength was λ = 350 nm
and the wavelength of recorded emission was λ = 460 nm. Using the
standard curve, the recorded fluorescence for the samples was con-
verted into DNA quantities and graphs were drawn.
2.5.3. Immunostaining
After 3 days of culture, the medium was removed and the cells were
washed with warm PBS. Then, they were fixed during 10 min with a 4%
PFA solution in PBS, and then washed again. A permeabilization step
was performed using the Triton X100 non-ionic surfactant for 15 min.
Further, the cells cultured on each sample were washed and incubated,
with anti- type I collagen and anti-osteopontin antibody (Abcam). In the
case of type I collagen, the primary antibody was directly coupled with
Alexa 488 fluorochrome, while for osteopontin immunostaining, a
second antibody was used, being coupled with Alexa 568 fluorochrome
(Thermo Scientific). The images were taken using a fluorescence mi-
croscope (Axio Observer D1, Zeiss).
2.5.4. Quantification of gene expression
After 7 days, the culture medium was removed and cells were wa-
shed with PBS. The total ribonucleic acid (RNA) was isolated by a
specific protocol, after using GENEzol Lysing Reagent (Geneaid) for
cells' lysis. Starting with 1 μg of total RNA, the messenger RNA (mRNA)
was reverse-transcribed to complementary DNA (cDNA), the reaction
being performed by the reverse transcriptase MMLV (Moloney Murine
Leukemia Virus) (Amresco). The obtained cDNA was used as template
for polymerase chain reaction (PCR) reaction, performed for measuring
the transcription level of osteocalcin, osteonectin, human cell-binding
sialoprotein (BSP II), and type I collagen genes. The normalization was
carried out to the gene encoding beta-actin. Table 2 presents the nu-
cleotide sequences of the primers we made use of.
3. Results
3.1. Elemental and phase composition
The EDS experiments were carried out in three different areas on
each sample. Fig. 3 presents the averaged elemental composition for
each element. Note that the presence of the adhesion layer does not
affect the formation of the HAP compound. Compared to the Ca/P ratio
(Ca/P = 1.67) specific for the stoichiometric HAP compound, only
small differences of this ratio were observed in the MeC/d-HAP coat-
ings, these values varying from 1.6 to 1.8, which are quite close to the
standard value. The concentration errors derived from EDS measure-
ments are small, pointing to the homogenous composition of the coat-
ings, evenly distributed on the coatings surfaces.
Fig. 4 presents the XRD diffractograms of the studied coatings, the
identified diffraction planes indicating the presence of the HAP crys-
talline structure and the presence of the specific MeC interlayer. The
main peaks are well matched to the (300), (310), (311) and (400)
planes of the stoichiometric hydroxyapatite (JCPDS no.09-0432), evi-
denced at 32.90°, 39.82°, 42.03° and 44.37°, respectively. The adhesion
interlayer signatures is also visible, according to the JCPDS files: 04-
007-1462 (TiC), 04-007-1462 (Ti2C), 10-01810 (NbC), 19-0870 (Nb2C),
032-1489 (ZrC) and 032-489 (ZrC0.7). The signature of Ti oxides, most
A. Vladescu et al.
Fig. 3. Elemental composition and Ca/P ratio obtained for the coatings de-
posited on Si wafers; the mean values displayed are related to the measure-
ments performed in three different areas on each sample.
probably as TiO2 and Ti3O5, is also visible, according to JCPDS files no.
84-1750 and 82-1137, respectively. Consequently, the large bump ob-
served in the interval 32.6–32.9° may be ascribed to a mixture of dif-
ferent Ti oxides and two hexagonal HAP structures, as detailed in
Table 3. One may notice the presence of two maxima ascribed to the
(300) plane of two hexagonal stoichiometric HAP compounds. The peak
situated at 32.78° is specific to the HAP depicted by the JCPDS file no.
25-0166, with larger lattice constants than the compound with the
maximum situated at 32.90°, according to JCPDS file no. 09-0432.
Table 4 contains lattice parameters values of the d- HAP and MeC/d-
HAP coatings, obtained by the Rietveld refinement algorithm.
3.2. Topography and roughness
Fig. 5 presents AFM images of the coatings deposited on silicon
substrates. Depending on the specific carbide type, the coated surfaces
presented different spatial features. Fig. 5A shows almost a flat surface
of NbC/d-HAP coating, without any distinct features. In contrast,
Figs. 5B and 5C, showing the TiC/d-HAP and ZrC/d-HAP coatings, re-
spectively, reveal a considerable number of small particles, nearly
identical in size and shape, homogeneously spread over the observed
areas. Perfect circular particles, with quite small diameters (~ 117 nm),
were evidenced on the TiC/d-HAP surface, with a density of about
6.7 × 10-8 cm2, although for ZrC/d-HAP film, the particles are less
regular in shape and around one-fourth larger (145 nm), while their
density is almost half of the one observed on TiC/d-HAP coating,
merely approaching ~ 3.7 × 10-8 cm2.
Table 5 summarizes the statistical and fractal characteristics of the
samples under investigation. To start with, NbC/d-HAP sample is the
flattest one, with a roughness parameter value Sq = 0.105 nm, while
ZrC/d-HAP coating is the roughest one (Sq = 4.14 nm). Considering the
1714
Ceramics International 45 (2019) 1710–1723
height distribution, NbC/d-HAP sample presents an almost perfectly
symmetrical one, considering its skewness (Ssk = 0.29), despite it is
slightly leptokurtic (Sku = 2.49). However, the skewness value rises up
to around 3 in the other two samples, due to the presence of particles,
which also affects their kurtosis values. As a result, the height dis-
tribution sharpens, that is, becomes more leptokurtic according to Sku
values, located somewhere between 11 and 13.
Another statistical measure, anisotropy ratio Str, defines the degree
to which the arrangement of spatial features varies with the axis of
observation. The flat NbC/d-HAP sample is deprived of any distinct
surface structures, hence its Str value comes close to 0.93, which is
specific for the nearly isotropic samples. On the other hand, the TiC/d-
HAP and ZrC/d-HAP samples are less isotropic, as their Str values are
approaching 0.85. The obtained anisotropy ratio is high enough, such
as these structures can be considered isotropic, indirectly implying that
the particles are homogeneously distributed over the entire surface.
Table 5 also presents the results of the fractal analysis, which makes
use of multi-scale allometric similarities expressed in terms of the
fractal dimension D, pseudo-topothesy K and corner frequency τ. In
general, each coating appeared to be monofractal, hence it does not
exhibit any long-range particle arrangement, but apart from that,
fractal characteristics reveal qualitative difference between the sample
containing different interlayers. As long as the fractal dimension is
considered, NbC/d-HAP sample exhibits a D value close to 2.79,
whereas in the two other samples it retains a relatively low value of
about 2.20. The fractal dimension controls the relative amplitude of
roughness at different length scales, which means that the height map is
much more stretched along the normal direction in case of NbC/d-HAP
coating, as compared to TiC/d-HAP and ZrC/d-HAP coatings, due to the
increased contribution of short-wave spatial frequencies. On the other
hand, the corner frequency τ sets up the threshold beyond which fractal
characteristics do not obey the scaling law. As it turns out from the data
in Table 5, the corner frequency τ approaches 115 and 134 nm for TiC/
d-HAP and ZrC/d-HAP samples, a value adjacent to the particle dia-
meter estimated using Otsu’s method ─ dOtsu: 117 and 145 nm, re-
spectively. This fact implies that the low fractal dimensions in these
samples actually correspond to a poorly developed surface geometry.
However, NbC/d-HAP sample without spatial features is different from
this point of view, since both corner frequency τ and grain dimension
dAACF are very low, but still non-zero, unlike the particle diameter dOtsu,
which cannot be estimated. However, one should bear in mind that
both the autocorrelation and allometric functions depend continuously
upon the lag, even for nearly flat surfaces, whereas Otsu’s algorithm is
sensitive to the regularities on the surface.
3.3. Adhesion
The adhesion of the coatings was evaluated by the scratch-tests.
According to EN 1071–3:2005 standard, the two main failures are
cracking (Lc1) and spallation (Lc2). Table 6 presents the Lc values at-
tributed to the specific failure modes for all of the investigated coatings.
One may see that the both Lc values of coatings with adhesion layer are
significantly higher than those of reference d-HAP coatings, indicating
the beneficial effect of the adhesion layer to the adhesion between d-
HAP and Ti6Al4V alloy.
Apart from the reference d-HAP coating, in all the MeC/d-HAP
coatings, the first sign of delamination was observed in the interval 7
(TiC/d-HAP) to 9.7 N (ZrC/d-HAP). For mentioned coatings, the con-
tinuous ductile perforation was found for loads higher than 16.2 N,
being close to the value measured for the reference d-HAP coating. The
NbC/d-HAP coating showed excellent adhesion strength on Ti6Al4V
alloy, with visible spallation for load indent values around 20.8 N. In all
coatings the cohesive spallation was evidenced along the scratch track
borders (Fig. 6).
A. Vladescu et al. Ceramics International 45 (2019) 1710–1723

Fig. 4. XRD patterns of the deposited coatings (left) and expanded image of the rectangle area (right).

Table 3 The reference coating exhibited a lower EOC potential than that un-
Possible peak assignment in the angular domain 2θ = 32.6°–32.9° of the coat- coated alloy and other coatings. The EOC potential of the coatings with
ings diffractograms presented in Fig. 3. adhesion layer is relatively stable in time with more electropositive
2θ exp (°) 2θ th (°) Compound (plane) JCPDS file values, indicating the formation of passive films on the coatings surface.
At the end of the test, the EOC values for all coatings with adhesion layer
32.6–32.9 32.601 TiO2 (002) 84–1750 present quite close values, of max. − 0.107 mV for TiC/d-HAP coating,
32.780 HAP (300) 25–0166
demonstrating that the adhesion layer has no significant influence on
32.849 Ti3O5 (203) 82–1135
32.900 HAP (300) 09–0432 the formation of the passive layer at the coating surface.
Based on the Tafel curves, the main electrochemical parameters
were extracted by performing a fit to the Butler-Volmer equation. The
Table 4 values of these parameters are presented in Fig. 8. The coating porosity
The in plane lattice constants for d-HAP and MeC/d-HAP coatings, resulted influences the rate of its dissolution and hereafter the bioactivity of the
from the Rietveld refinement algorithm. HAP based coatings [71]. Therefore, the porosity of the coatings was
Sample In plane lattice constants calculated by Elsener’s equation [72]:

a (Å) c (Å) Rp, substrate Ei = 0

P= 10 ba
Rp, coating (8)
d-HAP 9.416 6.879
NbC/d-HAP 9.404 6.862
TiC/d-HAP 9.452 6.789 where Rp,substrate – the substrate polarization resistance; Rp,coating – the
ZrC/d-HAP 9.472 6.939 coating polarization resistance; ΔEi=0 – the difference between the
corrosion potentials of the coatings and of the substrate, and ba – the
substrate anodic slope.
3.4. In vitro corrosion resistance When discussing the corrosion resistance of a material, the protec-
tive efficiency (Pe) of the surface is another important factor in the
Open circuit potential (EOC) was monitoring for 2 h, starting after evaluation of the coating resistance at the corrosion attack. Nozawa and
samples immersion in the corrosive environment. Fig. 7 shows the time Aramaki proposed the following equation for calculating the protective
evolution of EOC and Fig. 8 presents the values of the electrochemical efficiency of the coating [73]:
parameters obtained at the end of the tests and Fig. 9 illustrates the
potentiodynamic polarization curves of the uncoated, d-HAP and MeC/ icorr , coating
Pe = 1 100
d-HAP coated substrates. The EOC values measured for all of in- icorr , substrate (9)
vestigated coatings tend to move towards more electropositive values.
where icorr,coating and icorr,substrate are the corrosion current densities of the

1715
A. Vladescu et al. Ceramics International 45 (2019) 1710–1723

Fig. 5. AFM images of the investigated samples: (A) NbC/d-HAP, (B) TiC/d-HAP, (C) ZrC/d-HAP.

coating and of the substrate, respectively. Table 6

Comparing the data presented in Fig. 8, one can summarize the Critical load values (Lc) values of the investigated coatings, attributed to spe-
results, as follows: cific failure modes.

• The corrosion potential of the coatings with adhesion layer is shifted

Lc1 (N) Lc2 (N)

towards more electropositive values compared to the uncoated
substrates and coatings without adhesion layer.
•
The coatings with adhesion layers exhibited lower icorr values com-
pared to the reference d-HAP coating, as well as the uncoated sub-
strate.
• All coatings have higher polarization resistances than the uncoated
substrate. The coatings with adhesion layer showed similar icorr
values.
• The reference coating (d-HAP) exhibited the lowest porosity. In the
case of the coatings with adhesion layer, the lowest porosity was
determined for the coating with NbC adhesion layer, followed by
those with TiC and ZrC adhesion layers.
• The protective efficiency (Pe) increased with the addition of adhe-
sion layers. The highest Pe value was obtained for the coating with
NbC adhesion layer.
Comparing all the date of the Fig. 8, it can be concluded that the
addition of NbC adhesion layer significantly improved the corrosion
resistance of the d-HAP coatings. Fig. 9 shows also the breakdown
potential (Ebreak) values for all the deposited coatings and the bare
substrate. All MeC/d-HAP coatings exhibited higher breakdown po-
tential values than the reference coating (226 mV) and the uncoated
substrate (162 mV), indicating that the addition of a metallic carbide
interlayer is benefic, improving the coatings' corrosion performance.
3.5. In vitro biological performance
The interactions between the cells and the material surface can
modulate the cells functions like attachment, migration, division, dif-
ferentiation, survival and cell-cell communication. After the implant
insertion into a biological environment, the interactions between the
cells and the implant involve various and complex processes.
Immediately after implantation, the first interaction takes place be-
tween the implant and the biological macromolecules (i.e. proteins of
extracellular matrix and of cell membrane, secreted and serum
d-HAP 4.4 17.7
NbC/d-HAP 9.3 20.8
TiC/d-HAP 7.0 17.0
ZrC/d-HAP 9.7 16.2
Lc1 – cracking mode; Lc2 – spallation mode.
proteins). Some of these proteins are adsorbed on the implant surface
and modulate different processes, such as adhesion, attachment and
signaling, through large macromolecular assemblies forming sub-cel-
lular structures (focal adhesions). In the process of cell adhesion, the
signals released by the focal adhesions determine the specific proteins
to be adsorbed, as well as their orientation after adsorption [74].
However, all these details dependent also on the specific characteristics
of the sample's surface.
Fig. 10 presents the outcome of osteosarcoma cells proliferation
after 3 and 7 days of culture. Independent on the number of days of
culture, the cells proliferation, quantified as DNA quantity on MeC/d-
HAP coatings was superior to the one obtained on the bare substrate.
Moreover, all MeC/d-HAP coatings provided higher proliferation level
after both 3 and 7 days of culture, compared to the d-HAP reference
coatings. The best proliferation results, after 3 and also 7 days, were
recorded for ZrC/d-HAP coatings, followed by TiC/d-HAP and NbC/d-
HAP. Comparatively with the d-HAP coating, in all MeC/d-HAP coating
the DNA concentration after 3 and 7 days of culture increase, as com-
pared to the bare substrate and to the reference coating. The more
pronounced increase was observed for ZrC/d-HAP coatings, followed by
TiC/d-HAP and NbC/d-HAP coatings. It is to note that after 7 days of
culture, all MeC/d-HAP coatings presented superior cell proliferation
with respect to the bare substrate, while the reference d-HAP coating
exhibited almost the same value with the bare substrate, considering
the measurement errors. Summarizing, the presence of the adhesion
layers appeared to positively influence the cells proliferation after 3 and
7 days of culture of osteosarcoma cells.
The osteopontin is an osteoblastic secreted protein, deposited into
mineralized bone matrix. In addition to other functions, it ensures the

Table 5
Statistical and fractal parameters of the films determined from AFM images: Sq – roughness, Ssk – skewness, Sku – kurtosis, Str – anisotropy ratio, D – fractal dimension,
K – pseudo-topothesy, τ – corner frequency, dAACF – grain diameter from the AACF decay lengths, dOtsu – grain diameter estimated using Otsu’s algorithm.
Sample Sq (nm) Ssk Sku Str D K τ (nm) dAACF (nm) dOtsu (nm)

NbC/d-HAP 0.105 0.23 2.49 0.93 2.79 3.18 × 10-3 52.4 34 –

TiC/d-HAP 1.280 2.94 12.90 0.85 2.22 1.91 × 10-3 115.0 166 117
ZrC/d-HAP 4.140 2.75 10.80 0.86 2.20 1.31 × 10-2 134.0 211 145

1716
A. Vladescu et al.
Fig. 6. Morphologies of scratch tracks and critical loads for the investigated coatings.
Fig. 7. The evolution of open circuit potential as a function of immersion time
(2 h) in corrosive SBF environment at 37 ± 0.4 °C.
attachment of osteoclasts to the mineralized matrix [75], because the
osteoclasts express the integrin αVβ3, one of the osteopontin receptor
[76]. Besides this role, osteopontin is an important mediator in bone
remodeling. Thus, the increase of osteopontin expression decreases the
osteoclacin production and mineralization, while its decreased expres-
sion leads to an opposite effect [77]. The osteopontin promotes bone
resorption, not only by promoting osteoclasts attachment to bone ma-
trix, but also through a mechanism facilitating the differentiation of
osteoclasts from precursors [78].
Fig. 11 presents the fluorescence microscopy images of MG 63 cells
cultured 3 days on tested samples, after immunostaining for type I
collagen (green) and osteopontin (red). The blue appearing nuclei are
stained with DAPI. The immunostaining revealed that all the samples
allowed the synthesis of osteopontin and type I collagen. Both proteins
are important for implant integration and sustainability. Thus, type I
collagen is important for the bone matrix synthesis, and osteopontin for
the bone remodeling cycles. The remodeling process is taking place by
the removal of the old bone matrix by the osteoclasts, while a new
matrix is formed by the osteoblasts. These two processes are crucial for
1717
Ceramics International 45 (2019) 1710–1723
the implant long term maintaining, such as an imbalance of these
processes, especially the increase of the bone resorption by the osteo-
clast, leads to the implant loosening.
In this study, the messenger RNA transcription level of five genes
was analyzed: four genes involved in the bone matrix synthesis and
mineralization (osteocalcin, osteonectin, type I collagen and BSP II) and
a fifth gene involved in the control of the osteoclast differentiation
(osteoprotegerin).
Osteocalcin is a protein produced by the osteoblasts, being the
most abundant non-collagenous protein in the bone matrix. The os-
teocalcin mainly acts as a nucleation center for the HAP crystals. It also
has other effects on incompletely differentiated osteoclasts, inducing
their migration to certain areas of the matrix (chemotactic effect), their
adherence to the matrix, modulation of intracellular signaling processes
(by changing the concentration of Ca2+), and modulation of synthesis
and secretion of osteopontin and bone sialoprotein [79]. The cells
cultured on the coated samples had significantly increased osteocalcin
gene expression, compared to the control samples (uncoated Ti6Al4V) –
Fig. 12. The cells cultured on TiC/d-HAP coatings exhibited higher
osteocalcin expression than those cultured on and NbC/d-HAP, ZrC/d-
HAP and d-HAP coatings, which presented quite similar values, con-
sidering the associated errors.
Osteonectin is a 32 kDa acidic glycoprotein links both HAP and
collagen, having thus an important role in the mineralization process.
This protein is found more frequently in the mineralized matrix than in
the bone cells. When it is linked with collagen, it can also bind calcium,
but not the phosphate ions [80]. PCR results indicated that all tested
sample allowed the osteonectin mRNA transcription (Fig. 12). The cells
cultured on NbC/d-HAP coated samples presented a slightly higher
osteonectin transcription level, compared to the other coatings, in-
dicating their ability to initiate the active mineralization of bone.
Osteoprotegerin is a glycoprotein produced by osteoblasts, such as
it is indispensable in osteoclasts differentiation process [81]. It is in-
directly involved in the bone resorption by osteoclasts (cells that can
resorb bone mineralized matrix) by controlling the number of mature
osteoclasts [82,83]. It acts as a soluble decoy for RANKL factor (re-
ceptor activator of nuclear factor kappa B ligand) [84] secreted by both
mesenchymal stromal cells and osteoblasts; RANKL attach to RANK
A. Vladescu et al.
Fig. 8. Electrochemical parameters of the investigated samples.
(receptor activator of nuclear factor kappa B) on osteoclast precursors
membrane and differentiate them into osteoclasts. By osteoprotegerin-
RANKL coupling, the precursor’s stimulation through RANKL-RANK
mechanism is blocked, thus osteoprotegerin reduces the number of new
osteoclasts. The osteoclast number may be important for the implant
stability in bone matrix, because these cells are responsible for the bone
matrix resorption, which can lead to implant failure in the event of its
increase. But a small osteoclast number can't provide an optimum bone
remodeling process, needed for the old matrix replacement. The gene
expression results point out that all the tested samples allow osteo-
protegerin mRNA transcription (Fig. 12). The slightly increased level
for cells cultured on Ti6Al4V and ZrC/d-HAP samples, indicates a lower
risk for bone degradation around implants due to osteoclasts action.
1718
Ceramics International 45 (2019) 1710–1723
Fig. 9. Potentiodynamic polarization curves of the uncoated and MeC/d-HAP
coated substrates; the marked values are the breakdown potential.
Fig. 10. Proliferation of osteosarcoma cells on the investigated samples after 3
and 7 days of culture. Data show also the mean and SD values.
Type I collagen is the most abundant type of collagen in the bone
matrix (over 95%). The main role of type I collagen is to confer tensile
strength and to support HAP crystals which are found between or
within the collagen fibers. The expression of collagen-I display the same
trend as the osteonectin and osteoprotegerin genes, and a reversed
trend compared to the osteocalcin expression (Fig. 12). It is unclear
why the expression of collagen-I showed the different trend as osteo-
calcin. None of the sample suppress type I collagen gene expression,
although there are some level differences. The highest expression level
was recorded for the TiC/d-HAP sample followed by d-HAP sample.
Once more, the ZrC/d-HAP and NbC/d-HAP coatings presented similar
values.
Bone sialoprotein (or BSPII) is a bone specific glycoprotein that
may be involved in the formation and crystal growth of HAP [85].
Comparing the results, it can be observed that all the coatings allowed
the BSP II gene transcription, as well as the bare substrate. The highest
value was measured for the TiC/d-HAP coatings, followed by NbC/d-
HAP, d-HAP, ZrC/d-HAP, and the uncoated substrate (Fig. 12).
To summarize the gene expression experiments, all tested samples
maintained the osteoblast specific transcriptome, showing small varia-
tions. The best gene expression was observed in the presence of TiC/d-
HAP sample. No differences were found between NbC/d-HAP and ZrC/
d-HAP coatings. For all genes, the highest transcription level was re-
corded for the cells cultured on the coated samples.
4. Discussions
The lattice parameter “a” of the reference, d-HAP coating (9.416), is
almost the same with the one of the stoichiometric HAP (9.418 Å –
A. Vladescu et al.
Fig. 11. Fluorescence microscopy images of MG 63 cells cultured 3 days on tested samples, after immunostaining for type I collagen (green) and osteopontin (red).
The nuclei are blue stained with DAPI. The scale bar represents 20 µm.
JCPDS#09-0432), indicating that the HAP structure was not sig-
nificantly affected by TiOx doping.
The in-plane lattice parameters of TiC/d-HAP and ZrC/d-HAP
coatings increased, more visible for ZrC/d-HAP, which might be the
results of the d-HAP lattice deformation due to the presence of ZrC0.7
interlayer, which promoted the growth of the HAP (400) plane. The
evidenced signature of an understoichiometric Zr carbide might be
ascribed to ZrC oxidation in contact with the Ti alloy surface covered
with a thin native oxide layer, and by the evolution of this interlayer in
the oxidative environment of d-HAP matrix, as also reported by Woo
[86]. We should note that both ZrC0.7 and Ti2C phases have a cubic
structure.
The presence of different diffraction planes in different MeC/d-HAP
coatings might be ascribed to the growth of d-HAP on different tem-
plates produced by the oriented growth of the particular metal carbide
interlayer. As presented in the left part of Fig. 4, except for HAP (300)
plane, the maxima corresponding to the planes HAP (310) and NbC
(200) are superposed, as well as the maxima corresponding to HAP
(311) and Ti2C (400). Considering the HAP (400) plan in ZrC/d-HAP
coating, one may observe that this maximum is more pronounced in
this coating than in the d-HAP one, such as we ascribed the increase of
the signal to the superposition of two maxima: HAP (400) and ZrC0.7
(332). As observed, all MeC/d-HAP coatings evidenced the presence of
the stoichiometric and understoichiometric carbide compounds, most
probably due to the total or partial oxidation of the transition metal
carbides in the oxidative deposition plasma and in the titania doped
HAP environment.
Due to the large difference in atomic radii, Ca atom being larger
(1.97 Å) than Ti (1.47 Å) or Zr (1.60 Å), the significant lattice distortion
in TiC/d-HAP and ZrC/d-HAP might be ascribed to the incorporation of
Ti and Zr ions at the interface into the d-HAP structure. One should note
that the more distorted TiC/d-HAP and ZrC/d-HAP samples present
surfaces with a distinct granular structure, according to the data de-
rived from fractal analysis: grain diameter from the AACF decay
lengths, dAACFTiC/d-HAP = 166 nm and dAACFZrC/d-HAP = 211 nm.
Considering the NbC/d-HAP coating, the observed small value of
the “a” parameter (9.404 Å) compared to d-HAP one, might be de-
termined by the presence of the orthorhombic Nb2C compound (JCPDS
file 19-0870: a = 12.360 Å, b = 10.895 Å and c = 4.968 Å). Similar to
TiC or ZrC interlayer, also in Nb/d-HAP coating the Nb ion with a
smaller radius (1.46 Å) than Ca was incorporated at the interface in d-
HAP structure. However, the growth of d-HAP on the orthorhombic
template of Nb2C produced a flat surface, with grain diameter from the
AACF decay lengths, dAACFNbC/d-HAP = 34 nm, probably due to the in-
creased strain energy which can lead to amorphisation [87].
Summarizing, we suppose that structural stresses within the thin
1719
Ceramics International 45 (2019) 1710–1723
carbide interfacial layers might have induced crystalline discontinuities
which then served as nucleation centers for overgrown d-HAP layers,
with distorted lattices.
The adhesion of the coatings to the metallic substrate represents an
important asset, as they are intended for use in demanding conditions,
specific for the biomaterials used in arthroplasty. In this context, it
should be underlined that the mechanical properties of the substrate
play an important role on the development of cracks in the coated
substrate, as such it is important to consider the coating/substrate
system as a unit, in order to correctly assess the toughness of the coating
[88]. Thus, it is important to know the coating/substrate properties, in
order to estimate the overall mechanical response of the system [89].
For example, a high coating adhesion to the substrate implies a high
resistance to deformation under load, without detachment of the
coatings from substrate, resulting superior tribological and antic-
orrosive characteristics [88–90]. High toughness and adhesion of the
coatings to the substrate can effectively increase the load bearing
ability of the system, this property being required for the materials
aiming for the use in biomedical applications.
As reported in the literature, physical and chemical bonding, as well
as the mechanical interlocking are responsible for the coating adhesion
on the substrate. While the first two are mainly determined by the
materials composition, the third one might be related to the surface
roughness. There are numerous reports on the correlation between the
surface roughness of a coating and its adhesion to the substrate. Miletic
et al. found that the adhesion strength of the coatings decreased con-
comitantly with the coatings roughness [91], for the Sq roughness
parameter spanning from 59 to 6 nm. The authors found that the for-
mation of the first adhesion failure (Lc1) is hindered if a great amount of
plastic deformation is needed, and the rough surfaces with asperities
require more energy for getting plastically deformed. Therefore, higher
normal load is required to develop the failure on rougher samples [91],
because the energy needed for the plastic deformation of surface as-
perities increases as well [63]. Consequently, for rougher surfaces,
greater normal load is needed for developing the same type of failure
[63].
By the start of the 1990s, there are reports about an opposite results,
demonstrating the increase of the critical load with the decrease of the
surface roughness [92]. Our experiments revealed the increase of the
critical load and consequently of the adhesion for the smoother sur-
faces, indicating that the circumference of the asperities influences the
adhesion, in good agreement with the reported results by other re-
searchers [93]. This finding is probably related to the asperities found
on the rougher surface which act as a stress increment factor and in-
crease the probability of failure at a low applied normal load [94]. On
A. Vladescu et al.
Fig. 12. Messenger RNA (mRNA) transcription level of osteocalcin, osteonectin, type I collagen, BSP II (genes involved in bone matrix synthesis and mineralization)
and of osteoprotegerin (inhibitory factor for osteoclast differentiation).
smoother surfaces, the above mentioned stress increment factor is
missing, leading to an increase in the critical load. The disagreement
between different reports on the increased adhesion related to the
roughness value might be ascribed to the absolute values of roughness
considered by different authors, as also observed in the experiments on
cell adhesion, where eventually was established that a certain range of
roughness values promote the successful adhesion and further growth
of a specific type of cells [94].
As known, when embedded in liquids, the metallic surfaces are
coated with a thin oxide layer (several nanometers), which offers a
limited protection against further corrosive attack [95,96]. Eventually,
1720
Ceramics International 45 (2019) 1710–1723
the thin oxide layer is penetrated by the corrosive solutions, such as the
surface is corroded [96]. The most aggressive environments are those
comprising Cl-, due to its small diameter, permitting the swift breach of
the oxide lattice [96]. During the corrosion tests, the breakdown of
oxide layer in the presence of aggressive ions occurs beyond a certain
potential, known as the breakdown potential, used to assess the point
when the damage of the oxide layer takes place [97,98].
Sato reported that the breakdown potential of the coatings in the
presence of aggressive Cl- ions depends on several factors such as
thickness of the coating, defects developed during the deposition pro-
cess, intensity of the electric field in the coating (depending on the
A. Vladescu et al.
coating's electrical conductivity), and pH of the corrosive solution [99].
In this work, the breakdown of the oxide layer during the electro-
chemical tests in SBF took place as a result of the partial dissolution of
the coatings due chlorine adsorption. All MeC/d-HAP coatings ex-
hibited a higher breakdown potential than the reference coating and
the uncoated substrate, indicating that the addition of an interlayer is
benefic for the corrosion performance. This is not a surprising result, as
it was already reported that the corrosion properties of the coatings
deposited on metallic substrates by PVD methods are affected by the
porosity, interface chemistry between the coating and substrate,
structure, surface defect density, coating adhesion and stress levels
[100,101]. Usually, dense, low porosity films present a high corrosion
resistance in aggressive environments [101]. Keeping this in mind, it
can be seen that the NbC/d-HAP coating exhibited the highest break-
down potential, probably due to its low porosity. The poor protective
ability of the reference coatings is due to the relative low adhesion of d-
HAP on the metallic substrate, as already underlined above.
Considering the biological tests, it is well accepted that the pro-
liferation potential decreases as cell differentiation increases, being
influenced by the surface roughness. The presented experiments in-
dicated that the coating with the roughest surface (ZrC/d-HAP) ex-
hibited the highest proliferation level. This behavior was more evident
for the proliferation tests performed after 7 days of culture. However,
the gene expression level was almost similar for all MeC/d-HAP coat-
ings, an expected outcome, as the top d-HAP layer is the same in all
coatings. The observed differences are probably due to the roughness or
porosity of the surface.
Correlating all experimental results, it can be concluded that the
NbC/d-HAP coating represents a valuable biocompatible coatings,
aiming the improvement of the HAP based coatings on Ti6Al4V alloy
substrates.
4.1. Future possibilities
By comparing the mechanical properties of the d-HAP reference
coating with those of MeC/d-HAP coatings, one may conclude that the
metal carbide interlayers improved to the coatings adhesion to the
metal substrate. Considering the in vitro electrochemical and biological
results, the MeC/d-HAP coated alloy exhibited a higher proliferation
rate of the osteosarcoma cells and a superior corrosion resistance in SBF
at 37 °C. Thus, we should consider a compromise between the adhesion
and the corrosion resistance, biocompatibility and cytocompatibility.
For biomedical applications, the corrosion behavior and biocompat-
ibility are features of more significance than the slight differences on
the adhesion characteristics. Unfortunately, it is difficult to conclude
which of the investigated coating might represent the best solution for
the clinical use based solely on the adhesion tests, in vitro electro-
chemical and biological tests. As already demonstrated in literature, the
in vitro behavior of a biomaterial might be quite opposites to the one
exhibited during the in vivo investigations. Thus, the theme of our fu-
ture research is to perform the in vivo assessment of the studied coat-
ings in laboratory animals, in order to evaluate their osseointegration
abilities.
5. Conclusions
In this paper, it was demonstrated that the adhesion between the
titania doped HAP coating and Ti6Al4V alloy substrate was sig-
nificantly improved by the addition of a biocompatible metal carbide
interlayer. The coatings were prepared by the magnetron sputtering
technique. In all coatings, the formation of the HAP crystalline structure
was observed, indicating that the adhesion layer does not affect the
formation of the stoichiometric HAP structure. The NbC/d-HAP coat-
ings exhibited flat surfaces, while those with TiC and ZrC interlayers
were more distorted, corresponding to the granular structure of their
surfaces. All coatings with adhesion layer exhibited superior resistance
1721
Ceramics International 45 (2019) 1710–1723
to plastic and elastic deformation compared to the d-HAP coatings. The
NbC/d-HAP coatings exhibited the best ability to resist permanent da-
mage, compared with others coating, pointing to excellent adhesion
strength of Ti6Al4V alloy, such spallation was observed at about 20.8 N
indentation force. Coated surfaces with interfacial adhesion layers re-
veal spatial features associated with specific carbide. Niobium carbide
leaves almost flat surface with no distinct deposits, but the surface is
characterized with the largest fractal dimension and the smallest corner
frequency specific of surfaces with large component of short wave-
lengths. On the other hand, both titanium and zirconium carbides leave
circular particles of diameters between 120 and 150 nm, being of the
order of the corner frequency. Together with low fractal dimension
such a result defines both surfaces as those with high-order spatial
alignment.
All MeC/d-HAP coatings exhibited high protective efficiency to the
corrosive attack in SBF solution, with similar values of the corrosion
current density. The best corrosion protection was obtained from NbC/
d-HAP coating which presented low porosity and high protective effi-
ciency. The cell proliferation data showed that all of the coatings with
adhesion layer provide better conditions for cell proliferation, whatever
the period of culture, and the results depended significantly on the
roughness of the d-HAP top layer. Independent on the number of days
of culture, the cells proliferation, quantified as DNA quantity, on MeC/
d-HAP coatings was superior to the one obtained on the bare substrate.
All tested samples maintained the osteoblast specific transcriptome,
showing small variations. The best gene expression was observed for
TiC/d-HAP coating. No differences were found between NbC/d-HAP
and ZrC/d-HAP coatings.
To summarize the experimental results, it can be concluded that the
all the MeC adhesion layer might represent a suitable solution for im-
proving the adhesion between the d-HAP coatings and Ti6Al4V alloy
substrate, due to superior corrosion resistance and positive gene ex-
pression, leading to an prolonged service life of such orthopaedic im-
plants.
Acknowledgements
This work was supported by two grants of the Romanian National
Authority for Scientific Research and Innovation, CNCS/CCCDI -
UEFISCDI, project numbers PN-III-P2-2.1-PED-2016-1580 (BioArt) and
PN-II-PT-PCCA-2014-212 (OSSEOPROMOTE), within PNCDI III. SEM&
EDS&XRD analysis were carried out using the equipment acquired by
the Romanian Sectorial Operational Programme “Increase of economic
Competitiveness”, project INOVA-OPTIMA SMIS code 49164, contract
no. 658/2014.
References
[1] B. Carter, G. Norton, Ceramic Materials Science and Engineering, Springer-Verlag
New York, New York, 2007, https://doi.org/10.1007/978-0-387-46271-4.
[2] C.B. Carter, M.G. Norton, Ceramic Materials, Springer, New York, NY, 2013,
https://doi.org/10.1007/978-1-4614-3523-5.
[3] B.D. Ratner, S.J. Bryant, Biomaterials: where we have been and where we are
going, Annu. Rev. Biomed. Eng. 6 (2004) 41–75, https://doi.org/10.1146/
annurev.bioeng.6.040803.140027.
[4] S. Bose, A. Bandyopadhyay, Introduction to biomaterials, Charact. Biomater.
(2013) 1–9, https://doi.org/10.1016/B978-0-12-415800-9.00001-2.
[5] F. Mahyudin, L. Widhiyanto, H. Hermawan, Biomaterials in orthopaedics, in: Adv.
Struct. Mater., 2016, pp. 161–181. 〈http://dx.doi.org/10.1007/978-3-319-14845-
8_7〉.
[6] A.W. Batchelor, M. Chandrasekaran, Service characteristics of biomedical mate-
rials and implants, in: A.W. Batchelor, J.R. Batchelor, M. Chandrasekaran (Eds.),
Ser. Biomater. Bioeng. 3 Imperial College Press, UK, 2004, pp. 1–260.
[7] X. Liu, P.K. Chu, C. Ding, Surface nano-functionalization of biomaterials, in:
Mater. Sci. Eng. R Rep., 2010, pp. 275–302. 〈http://dx.doi.org/10.1016/j.mser.
2010.06.013〉.
[8] W. Cao, L.L. Hench, Bioactive materials, Ceram. Int. 22 (1996) 493–507, https://
doi.org/10.1016/0272-8842(95)00126-3.
[9] R. LeGeros, G. Daculsi, J. LeGeros, Bioactive bioceramics, Orthop. Biol. Med.
Musculoskelet. Tissue Regen. (2008) 153–181, https://doi.org/10.1007/978-1-
59745-239-7.
A. Vladescu et al.
[10] A. Sola, D. Bellucci, V. Cannillo, A. Cattini, Bioactive glass coatings: a review, Surf.
Eng. 27 (2011) 560–572, https://doi.org/10.1179/1743294410Y.0000000008.
[11] S. Lopez-Esteban, E. Saiz, S. Fujino, T. Oku, K. Suganuma, A.P. Tomsia, Bioactive
glass coatings for orthopedic metallic implants, J. Eur. Ceram. Soc. 23 (2003)
2921–2930, https://doi.org/10.1016/S0955-2219(03)00303-0.
[12] B.G.X. Zhang, D.E. Myers, G.G. Wallace, M. Brandt, P.F.M. Choong, Bioactive
coatings for orthopaedic implants-recent trends in development of implant coat-
ings, Int. J. Mol. Sci. 15 (2014) 11878–11921, https://doi.org/10.3390/
ijms150711878.
[13] S.V. Dorozhkin, Calcium orthophosphate deposits: preparation, properties and
biomedical applications, Mater. Sci. Eng. C 55 (2015) 272–326, https://doi.org/
10.1016/j.msec.2015.05.033.
[14] S.V. Dorozhkin, Calcium orthophosphates in dentistry, J. Mater. Sci. Mater. Med.
24 (2013) 1335–1363, https://doi.org/10.1007/s10856-013-4898-1.
[15] A. Vladescu, S.C. Padmanabhan, F. Ak Azem, M. Braic, I. Titorencu, I. Birlik,
M.A. Morris, V. Braic, Mechanical properties and biocompatibility of the sputtered
Ti doped hydroxyapatite, J. Mech. Behav. Biomed. Mater. 63 (2016) 314–325,
https://doi.org/10.1016/j.jmbbm.2016.06.025.
[16] Q. Chen, G.A. Thouas, Metallic implant biomaterials, Mater. Sci. Eng. R. Rep. 87
(2015) 1–57, https://doi.org/10.1016/j.mser.2014.10.001.
[17] P. Layrolle, Calcium phosphate coatings, in: Paul Ducheyne (Ed.), Compr.
Biomater. Elsevier Ltd, 2014, pp. 223–229.
[18] L. Clèries, J.M. Fernández-Pradas, G. Sardin, J.L. Morenza, Dissolution behaviour
of calcium phosphate coatings obtained by laser ablation, in: Biomaterials, 1998,
pp. 1483–1487. 〈http://dx.doi.org/10.1016/S0142-9612(98)00063-5〉.
[19] K. de Groot, Clinical applications of calcium phosphate biomaterials: a review,
Ceram. Int. 19 (1993) 363–366, https://doi.org/10.1016/0272-8842(93)90050-2.
[20] R.A. Surmenev, A review of plasma-assisted methods for calcium phosphate-based
coatings fabrication, Surf. Coat. Technol. 206 (2012) 2035–2056, https://doi.org/
10.1016/j.surfcoat.2011.11.002.
[21] R.A. Surmenev, M.A. Surmeneva, A.A. Ivanova, Significance of calcium phosphate
coatings for the enhancement of new bone osteogenesis - a review, Acta Biomater.
10 (2014) 557–579, https://doi.org/10.1016/j.actbio.2013.10.036.
[22] Y.K. Pan, C.Z. Chen, D.G. Wang, Z.Q. Lin, Preparation and bioactivity of micro-arc
oxidized calcium phosphate coatings, Mater. Chem. Phys. 141 (2013) 842–849,
https://doi.org/10.1016/j.matchemphys.2013.06.013.
[23] J.H. Park, D.Y. Lee, K.T. Oh, Y.K. Lee, K.N.K.M. Kim, K.N.K.M. Kim, Bioactivity of
calcium phosphate coatings prepared by electrodeposition in a modified simulated
body fluid, Mater. Lett. 60 (2006) 2573–2577, https://doi.org/10.1016/j.matlet.
2005.07.091.
[24] J. Wang, C. Sun, Y. Wang, Y. Wang, Early bone apposition and 1-year performance
of the electrodeposited calcium phosphate coatings: an experimental study in
rabbit femora, Clin. Oral. Implants Res. 21 (2010) 951–960, https://doi.org/10.
1111/j.1600-0501.2010.01935.x.
[25] I.-S. Lee, H.-E. Kim, S.-Y. Kim, Studies on calcium phosphate coatings, Surf. Coat.
Technol. 131 (2000) 181–186, https://doi.org/10.1016/S0257-8972(00)00811-2.
[26] E.A. Dos Santos, S. Moldovan, M. Mateescu, J. Faerber, M. Acosta, H. Pelletier,
K. Anselme, J. Werckmann, Physical-chemical and biological behavior of an
amorphous calcium phosphate thin film produced by RF-magnetron sputtering,
Mater. Sci. Eng. C 32 (2012) 2086–2095, https://doi.org/10.1016/j.msec.2012.
05.041.
[27] Y. Yonggang, J.G.C. Wolke, L. Yubao, J.A. Jansen, The influence of discharge
power and heat treatment on calcium phosphate coatings prepared by RF mag-
netron sputtering deposition, J. Mater. Sci. Mater. Med. 18 (2007) 1061–1069,
https://doi.org/10.1007/s10856-007-0119-0.
[28] S. Yokota, N. Nishiwaki, K. Ueda, T. Narushima, H. Kawamura, T. Takahashi,
Evaluation of thin amorphous calcium phosphate coatings on titanium dental
implants deposited using magnetron sputtering, Implant Dent. 23 (2014) 343–350,
https://doi.org/10.1097/ID.0000000000000098.
[29] E.R. Urquia Edreira, J.G.C. Wolke, A.A. Aldosari, S.S. Al-Johany, S. Anil,
J.A. Jansen, J.J.J.P. Van Den, Beucken, Effects of calcium phosphate composition
in sputter coatings on in vitro and in vivo performance, J. Biomed. Mater. Res. -
Part A 103 (2015) 300–310, https://doi.org/10.1002/jbm.a.35173.
[30] A.R. Boyd, C. O’Kane, B.J. Meenan, Control of calcium phosphate thin film stoi-
chiometry using multi-target sputter deposition, Surf. Coat. Technol. 233 (2013)
131–139, https://doi.org/10.1016/j.surfcoat.2013.04.017.
[31] M. Bramowicz, L. Braic, F.A. Azem, S. Kulesza, I. Birlik, A. Vladescu, Mechanical
properties and fractal analysis of the surface texture of sputtered hydroxyapatite
coatings, Appl. Surf. Sci. 379 (2016) 338–346, https://doi.org/10.1016/j.apsusc.
2016.04.077.
[32] D.M. Vranceanu, C.M. Cotrut, M. Bramowicz, I. Titorencu, S. Kulesza, A. Kiss,
A. Berbecaru, V. Pruna, M. Branzei, A. Vladescu, A.V.D.M. Vranceanu,
C.M. Cotrut, M. Bramowicz, I. Titorencu, S. Kulesza4, A. Kiss, A. Berbecaru,
V. Pruna, M. Branzei, Osseointegration of sputtered SiC-added hydroxyapatite for
orthopaedic applications, Ceram. Int. 42 (2016) 10085, https://doi.org/10.1016/
j.ceramint.2016.03.114.
[33] F.A. Azem, A. Kiss, I. Birlik, V. Braic, C. Luculescu, A. Vladescu, The corrosion and
bioactivity behavior of SiC doped hydroxyapatite for dental applications, Ceram.
Int. 40 (2014) 15881–15887, https://doi.org/10.1016/j.ceramint.2014.07.116.
[34] A. Vladescu, I. Birlik, V. Braic, M. Toparli, E. Celik, F. Ak Azem, Enhancement of
the mechanical properties of hydroxyapatite by SiC addition, J. Mech. Behav.
Biomed. Mater. 40 (2014) 362–368, https://doi.org/10.1016/j.jmbbm.2014.08.
025.
[35] A. Vladescu, M.A. Surmeneva, C.M. Cotrut, R.A. Surmenev, I.V. Antoniac,
Bioceramic coatings for metallic implants, in: Iulian Vasile Antoniac (Ed.), Handb.
Bioceram. Biocomposites, Springer International Publishing, 2015, pp. 1–31, ,
1722
Ceramics International 45 (2019) 1710–1723
https://doi.org/10.1007/978-3-319-09230-0_31-1.
[36] F.A. Azem, I. Birlik, V. Braic, M. Toparli, E. Celik, A. Parau, A. Kiss, I. Titorencu,
A. Vladescu, Effect of SiC interlayer between Ti6Al4V alloy and hydroxyapatite
films, Proc. Inst. Mech. Eng. Part H J. Eng. Med. 229 (2015), https://doi.org/10.
1177/0954411915578886.
[37] M. Badea, M. Braic, A. Kiss, M. Moga, E. Pozna, I. Pana, A. Vladescu, Influence of
Ag content on the antibacterial properties of SiC doped hydroxyapatite coatings,
Ceram. Int. 42 (2016) 1801–1811, https://doi.org/10.1016/j.ceramint.2015.09.
143.
[38] M.A. Surmeneva, A. Vladescu, R.A. Surmenev, C.M. Pantilimon, M. Braic,
C.M. Cotrut, Study on a hydrophobic Ti-doped hydroxyapatite coating for corro-
sion protection of a titanium based alloy, RSC Adv. 6 (2016) 87665–87674,
https://doi.org/10.1039/c6ra03397k.
[39] M. Brama, N. Rhodes, J. Hunt, A. Ricci, R. Teghil, S. Migliaccio, C. Della Rocca,
S. Leccisotti, A. Lioi, M. Scandurra, G. De Maria, D. Ferro, F. Pu, G. Panzini,
L. Politi, R. Scandurra, Effect of titanium carbide coating on the osseointegration
response in vitro and in vivo, Biomaterials 28 (2007) 595–608.
[40] Н Gleiter, Biocompatibility of wear-resistant coatings in orthopaedic surgery in
vitro testing with human fibroblast cell cultures, Prog. Mater. Sci. 33 (1989) 233,
https://doi.org/10.1007/BF00120412.
[41] V. Braic, A. Vladescu, M. Balaceanu, C.R. Luculescu, M. Braic, Nanostructured
multi-element (TiZrNbHfTa)N and (TiZrNbHfTa)C hard coatings, Surf. Coat.
Technol. 211 (2012) 117–121, https://doi.org/10.1016/j.surfcoat.2011.09.033.
[42] C.N. Zoita, L. Braic, A. Kiss, M. Braic, Characterization of NbC coatings deposited
by magnetron sputtering method, Surf. Coat. Technol. 204 (2010) 2002–2005,
https://doi.org/10.1016/j.surfcoat.2009.08.050.
[43] M. Braic, V. Braic, M. Balaceanu, A. Vladescu, C.N. Zoita, I. Titorencu, V. Jinga,
F. Miculescu, Preparation and characterization of biocompatible Nb-C coatings,
Thin Solid Films 519 (2011) 4064–4068, https://doi.org/10.1016/j.tsf.2011.01.
193.
[44] G. Ramírez, S.E. Rodil, H. Arzate, S. Muhl, J.J. Olaya, Niobium based coatings for
dental implants, Appl. Surf. Sci. 257 (2011) 2555–2559, https://doi.org/10.1016/
j.apsusc.2010.10.021.
[45] R. Olivares-Navarrete, J.J. Olaya, C. Ramírez, S.E. Rodil, Biocompatibility of
niobium coatings, Coatings 1 (2011) 72–87, https://doi.org/10.3390/
coatings1010072.
[46] A. OrjuelaG, R. Rincón, J.J. Olaya, Corrosion resistance of niobium carbide
coatings produced on AISI 1045 steel via thermo-reactive diffusion deposition,
Surf. Coat. Technol. 259 (2014) 667–675, https://doi.org/10.1016/j.surfcoat.
2014.10.012.
[47] M.H. Ding, H. Sen Zhang, C. Zhang, X. Jin, Characterization of ZrC coatings de-
posited on biomedical 316L stainless steel by magnetron sputtering method, Surf.
Coat. Technol. 224 (2013) 34–41, https://doi.org/10.1016/j.surfcoat.2013.02.
047.
[48] C.L. Chu, H.L. Ji, L.H. Yin, Y.P. Pu, P.H. Lin, P.K. Chu, Fabrication, properties, and
cytocompatibility of ZrC film on electropolished NiTi shape memory alloy, Mater.
Sci. Eng. C 31 (2011) 423–427, https://doi.org/10.1016/j.msec.2010.10.023.
[49] I. Tudor, A. Popescu, R.G. Ripeanu, A.C. Drumeanu, V. Braic, M. Balaceanu,
A. Vladescu, M. Braic, Tribological behaviour of TiSiN/Ti and TiSiN/Cu multilayer
coatings, J. Balk. Tribol. Assoc. 15 (2009) 156–162 〈http://www.scopus.com/
inward/record.url?eid=2-s2.0-68149119164&partnerID=40&md5=
97e63f7c21fc5e24359ad64242a682d9〉.
[50] V. Braic, A.C.C. Parau, I. Pana, M. Braic, M. Balaceanu, M.B.V. Braic, A.C. Parau,
I. Pana, M. Braic, V. Braic, A.C.C. Parau, I. Pana, M. Braic, M. Balaceanu, Effects of
substrate temperature and carbon content on the structure and properties of
(CrCuNbTiY)C multicomponent coatings, Surf. Coat. Technol. 258 (2014)
996–1005.
[51] L. Braic, N.C. Zoita, Influence of the deposition time and temperature on the
texture of InN thin films grown by RF-magnetron sputtering, Optoelectron. Adv.
Mater. Rapid Commun. 4 (2010) 2013.
[52] L. Braic, N. Vasilantonakis, B. Zou, S.A. Maier, N.M. Alford, A.V. Zayats,
P.K. Petrov, Optimizing strontium ruthenate thin films for near-infrared plasmonic
applications, Sci. Rep. 5 (2015) 9118, https://doi.org/10.1038/srep09118.
[53] C.M. Cotrut, L. Braic, D.M. Vranceanu, A. Kiss, M. Dinu, M. Balaceanu, V. Braic,
A. Vladescu, Influence of the annealing treatment on the structure, morphology,
and corrosion resistance of sputtered Zr-Ti-Si-O coatings used for biomedical ap-
plications, Mater. Corros. 68 (2017), https://doi.org/10.1002/maco.201609200.
[54] A. Vladescu, M. Braic, F.A. Azem, I. Titorencu, V. Braic, V. Pruna, A. Kiss, A.C.
Parau, I. Birlik, Effect of the deposition temperature on corrosion resistance and
biocompatibility of the hydroxyapatite coatings, in: Appl. Surf. Sci., 2015, pp.
373–379. 〈http://dx.doi.org/10.1016/j.apsusc.2015.05.059〉.
[55] J. Kim, D. Schildt, S. Chen, D. Patel, Investigation of Foreign- Particle
Contamination, 8, 2016, pp. 34–57.
[56] B.E. 1071-3, Part 3: Determination of adhesion and other mechanical failure
modes by a scratch test, in: Adv. Tech. Ceram. – Methods Test Ceram. Coat., 2005,
pp. 1–50.
[57] E. Mainsah, J. Greenwood (Eds.), Metrology and Properties of Engineering
Surfaces, Plenum Press, Springer US, 2001.
[58] P.R. Nayak, Random process model of rough surfaces, J. Lubr. Technol. 93 (1971)
398–407, https://doi.org/10.1115/1.3451608.
[59] W.P. Dong, P.J. Sullivan, K.J. Stout, Comprehensive study of parameters for
characterizing 3-dimensional surface-topography .4. parameters for characterizing
spatial and hybrid properties, Wear 178 (1994) 45–60, https://doi.org/10.1016/
0043-1648(94)90128-7.
[60] Ş. Ţălu, M. Bramowicz, S. Kulesza, A. Shafiekhani, A. Ghaderi, F. Mashayekhi,
S. Solaymani, Microstructure and tribological properties of FeNPs/a-C:H films by
A. Vladescu et al.
micromorphology analysis and fractal geometry, Ind. Eng. Chem. Res. 54 (2015)
8212–8218, https://doi.org/10.1021/acs.iecr.5b02449.
[61] A. Thomas, T. Thomas, Digital analysis of very small scale surface roughness, J.
Wave Mater. Interact. 3 (1988) 341–350.
[62] M. Szkodo, Bień, M. Antoszkiewicz, Effect of plasma sprayed and laser re-melted
Al2O3 coatings on hardness and wear properties of stainless steel, Ceram. Int. 42
(2016) 11275–11284, https://doi.org/10.1016/j.ceramint.2016.04.044.
[63] M. Szkodo, A. Bien, Influence of laser processing of the low alloy medium carbon
structural steel on the development of the fatigue crack, Surf. Coat. Technol. 296
(2016) 117–123.
[64] Ş. Ţălu, M. Bramowicz, S. Kulesza, S. Solaymani, A. Ghaderi, L. Dejam, S.M. Elahi,
A. Boochani, Microstructure and micromorphology of ZnO thin films: case study
on Al doping and annealing effects, Superlattices Microstruct. 93 (2016) 109–121,
https://doi.org/10.1016/j.spmi.2016.03.003.
[65] Ş. Ţălu, M. Bramowicz, S. Kulesza, T. Lainovi, M. Viloti, L. Blažić, Influence of the
artificial saliva storage on 3-D surface texture characteristics of contemporary
dental nanocomposites, J. Microsc. 264 (2016) 198–206, https://doi.org/10.
1111/jmi.12432.
[66] S. Kulesza, M. Bramowicz, P. Czaja, R. Jabłoński, J. Kropiwnicki, M. Charkiewicz,
Application of atomic force microscopy for studies of fractal and functionals
properties of biomaterials, Acta Phys. Pol. A 130 (2016) 1013–1015, https://doi.
org/10.12693/APhysPolA.130.
[67] Ş. Ţălu, M. Bramowicz, S. Kulesza, A. Ghaderi, V. Dalouji, S. Solaymani, Z. Khalaj,
Microstructure and micromorphology of Cu/Co nanoparticles: surface texture
analysis, Electron. Mater. Lett. 12 (2016) 580–588, https://doi.org/10.1007/
s13391-016-6036-y.
[68] Ş. Ţălu, M. Bramowicz, S. Kulesza, A. Shafiekhani, M. Rahmati, A. Ghaderi,
M. Ahmadirad, S. Solaymani, Microstructure of nickel nanoparticles embedded in
carbon films: case study on annealing effect by micromorphology analysis, Surf.
Interface Anal. 49 (2017) 153–160, https://doi.org/10.1002/sia.6074.
[69] P.F. Gostin, A. Gebert, L. Schultz, Comparison of the corrosion of bulk amorphous
steel with conventional steel, Corros. Sci. 52 (2010) 273–281, https://doi.org/10.
1016/j.corsci.2009.09.016.
[70] M. Stern, L. Geary, Electrochemical polarization I. A theoritical analysis of the
shape of polarization curves, J. Electrochem. Soc. 104 (1957) 56–63.
[71] G. Kaur, G. Pickrell, G. Kimsawatde, D. Homa, H. a. Allbee, N. Sriranganathan,
Synthesis, cytotoxicity, and hydroxyapatite formation in 27-Tris-SBF for sol-gel
based CaO-P2O5-SiO2-B2O3-ZnO bioactive glasses, Sci. Rep. 4 (2014) 4392,
https://doi.org/10.1038/srep04392.
[72] B. Elsener, A. Rota, H. Bohni, Impedance study on the corrosion of PVD and CVD
titanium nitride coatings, Mater. Sci. Forum 29 (1989) 44–45.
[73] K. Nozawa, K. Aramaki, One- and two-dimensional polymer films of modified
alkanethiol monolayers for preventing iron from corrosion, Corros. Sci. 41 (1999)
57–73, https://doi.org/10.1016/S0010-938X(98)00048-1.
[74] B.D. Boyan, T.W. Hummert, D.D. Dean, Z. Schwartz, Role of material surfaces in
regulating bone and cartilage cell response, Biomaterials 17 (1996) 137–146,
https://doi.org/10.1016/0142-9612(96)85758-9.
[75] F.P. Reinholt, K. Hultenby, A. Oldberg, D. Heinegård, Osteopontin–a possible
anchor of osteoclasts to bone, Proc. Natl. Acad. Sci. USA 87 (1990) 4473–4475,
https://doi.org/10.1073/pnas.87.12.4473.
[76] I. Nakamura, L.T. Duong, S.B. Rodan, G.A. Rodan, Involvement of alpha(v)beta3
integrins in osteoclast function, J. Bone Miner. Metab. 25 (2007) 337–344,
https://doi.org/10.1007/s00774-007-0773-9.
[77] W. Huang, B. Carlsen, G. Rudkin, M. Berry, K. Ishida, D.T. Yamaguchi, T.A. Miller,
Osteopontin is a negative regulator of proliferation and differentiation in MC3T3-
E1 pre-osteoblastic cells, Bone 34 (2004) 799–808, https://doi.org/10.1016/j.
bone.2003.11.027.
[78] N. Tani-Ishii, A. Tsunoda, T. Umemoto, Osteopontin antisense deoxyoligonucleo-
tides inhibit bone resorption by mouse osteoclasts in vitro, J. Periodontal. Res. 32
(1997) (480–406).
[79] C. Chenu, S. Colucci, M. Grano, P. Zigrino, R. Barattolo, G. Zambonin, N. Baldini,
P. Vergnaud, P.D. Delmas, A.Z. Zallone, Osteocalcin induces chemotaxis, secretion
of matrix proteins, and calcium-mediated intracellular signaling in human osteo-
clast-like cells, J. Cell Biol. 127 (1994) 1149–1158, https://doi.org/10.1083/jcb.
127.4.1149.
[80] J.D. Termine, H.K. Kleinman, S.W. Whitson, K.M. Conn, M.L. McGarvey,
G.R. Martin, Osteonectin, a bone-specific protein linking mineral to collagen, Cell
26 (1981) 99–105, https://doi.org/10.1016/0092-8674(81)90037-4.
[81] W.C. Dougall, M. Glaccum, K. Charrier, K. Rohrbach, K. Brasel, T. De Smedt,
1723
Ceramics International 45 (2019) 1710–1723
E. Daro, J. Smith, M.E. Tometsko, C.R. Maliszewski, A. Armstrong, V. Shen,
S. Bain, D. Cosman, D. Anderson, P.J. Morrissey, J.J. Peschon, J. Schuh, RANK is
essential for osteoclast and lymph node development, Genes Dev. 13 (1999)
2412–2424, https://doi.org/10.1101/gad.13.18.2412.
[82] D.J. Hadjidakis, I.I. Androulakis, Bone remodeling, in: Ann. N.Y. Acad. Sci., 2006:
pp. 385–396. 〈http://dx.doi.org/10.1196/annals.1365.035〉.
[83] W.S. Simonet, D.L. Lacey, C.R. Dunstan, M. Kelley, M.S. Chang, R. Lüthy,
H.Q. Nguyen, S. Wooden, L. Bennett, T. Boone, G. Shimamoto, M. DeRose,
R. Elliott, A. Colombero, H.L. Tan, G. Trail, J. Sullivan, E. Davy, N. Bucay,
L. Renshaw-Gegg, T.M. Hughes, D. Hill, W. Pattison, P. Campbell, S. Sander,
G. Van, J. Tarpley, P. Derby, R. Lee, W.J. Boyle, Osteoprotegerin: a novel secreted
protein involved in the regulation of bone density, Cell 89 (1997) 309–319
(doi:S0092-8674)(00)(80209-3)(pii).
[84] H. Yasuda, N. Shima, N. Nakagawa, K. Yamaguchi, M. Kinosaki, S. Mochizuki,
A. Tomoyasu, K. Yano, M. Goto, A. Murakami, E. Tsuda, T. Morinaga, K. Higashio,
N. Udagawa, N. Takahashi, T. Suda, Osteoclast differentiation factor is a ligand for
osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/
RANKL, Proc. Natl. Acad. Sci. USA 95 (1998) 3597–3602, https://doi.org/10.
1073/pnas.95.7.3597.
[85] S. Kasugai, T. Nagata, J. Sodek, Temporal studies on the tissue compartmentali-
zation of bone sialoprotein (BSP), osteopontin (OPN), and SPARC protein during
bone formation In Vitro, J. Cell. Physiol. 152 (1992) 467–477, https://doi.org/10.
1002/jcp.1041520305.
[86] J. Woo, Growth of Epitaxial Zirconium Carbide Layers Using Pulsed Laser
Deposition, 2005, pp. 1–111. 〈http://etd.fcla.edu/UF/UFE0013064/woo_j.pdf〉.
[87] M. Braic, M. Balaceanu, A. Vladescu, C.N. Zoita, V. Braic, Deposition and char-
acterization of multi-principal-element (CuSiTiYZr)C coatings, Appl. Surf. Sci. 284
(2013) 671–678, https://doi.org/10.1016/j.apsusc.2013.07.152.
[88] J. Musil, M. Jirout, Toughness of hard nanostructured ceramic thin films, Surf.
Coat. Technol. 201 (2007) 5148–5152, https://doi.org/10.1016/j.surfcoat.2006.
07.020.
[89] S.V. Hainsworth, W.C. Soh, The effect of the substrate on the mechanical prop-
erties of TiN coatings, Surf. Coat. Technol. 163–164 (2003) 515–520.
[90] Y.X. Ou, J. Lin, H.L. Che, W.D. Sproul, J.J. Moore, M.K. Lei, Mechanical and tri-
bological properties of CrN/TiN multilayer coatings deposited by pulsed dc
magnetron sputtering, Surf. Coat. Technol. 276 (2015) 152–159, https://doi.org/
10.1016/j.surfcoat.2015.06.064.
[91] A. Miletić, P. Terek, L. Kovačević, M. Vilotić, D. Kakaš, B. Skorić, D. Kukuruzović,
Influence of substrate roughness on adhesion of TiN coatings, J. Braz. Soc. Mech.
Sci. Eng. 36 (2014) 293–299, https://doi.org/10.1007/s40430-013-0102-2.
[92] C. Subramanian, K.N. Strafford, T.P. Wilks, L.P. Ward, W. McMillan, Influence of
substrate roughness on the scratch adhesion of titanium nitride coatings, Surf.
Coat. Technol. 62 (1993) 529–535, https://doi.org/10.1016/0257-8972(93)
90295-Y.
[93] K. Meine, K. Kloß, T. Schneider, D. Spaltmann, The influence of surface roughness
on the adhesion force, Surf. Interface Anal. 36 (2004) 694–697, https://doi.org/
10.1002/sia.1738.
[94] R.L. Price, K. Ellison, K.M. Haberstroh, T.J. Webster, Nanometer surface roughness
increases select osteoblast adhesion on carbon nanofiber compacts, J. Biomed.
Mater. Res. - Part A 70 (2004) 129–138, https://doi.org/10.1002/jbm.a.30073.
[95] S. Virtanen, Corrosion and passivity of metals and coatings, in: Tribocorrosion
Passiv. Met. Coat., 2011, pp. 3–28. 〈http://dx.doi.org/10.1016/B978-1-84569-
966-6.50001-4〉.
[96] J. Soltis, Passivity breakdown, pit initiation and propagation of pits in metallic
materials - review, Corros. Sci. 90 (2015) 5–22, https://doi.org/10.1016/j.corsci.
2014.10.006.
[97] T.P. Hoar, The production and breakdown of the passivity of metals, Corros. Sci. 7
(1967) 341–355, https://doi.org/10.1016/S0010-938X(67)80023-4.
[98] N. Sato, A theory for breakdown of anodic oxide films on metals, Electrochim.
Acta 16 (1971) 1683–1692, https://doi.org/10.1016/0013-4686(71)85079-X.
[99] N. Sato, Basics of corrosion chemistry, in: Green Corros. Chem. Eng. Oppor.
Challenges, 2011, pp. 1–32. 〈http://dx.doi.org/10.1002/9783527641789.ch1〉.
[100] M. Fenker, M. Balzer, H. Kappl, Corrosion protection with hard coatings on steel:
past approaches and current research efforts, Surf. Coat. Technol. 257 (2014)
182–205, https://doi.org/10.1016/j.surfcoat.2014.08.069.
[101] F.X. Qin, G.Q. Xie, T. Wada, S.L. Zhu, Z.H. Dan, Electrochemical properties of
porous Pd-based bulk metallic glasses, Mater. Trans. 54 (2013) 1347–1350,
https://doi.org/10.2320/matertrans.MF201314.