Ceramics International 44 (2018) 14940–14946

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Ceramics International
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Short communication

Biological and corrosion behavior of m-ZrO2 and t-ZrO2 coated 316L SS for T
potential biomedical applications
⁎
Gobi Saravanan Kaliaraj, Vinita Vishwakarma , Karthik Alagarsamy, A.M. Kamalan Kirubaharan
Centre for Nanoscience and Nanotechnology, Sathyabama Institute of Science and Technology (Deemed to be University), Chennai 600119, India

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

Keywords: Surface modification is an established technology in biomedical industries to improve the performance of im-
Bioceramics plant materials. In the present study, monoclinic (m-ZrO2) and tetragonal (t-ZrO2) phases of zirconia were de-
m-ZrO2 and t-ZrO2 coatings posited on stainless steel (316L SS) by electron beam physical vapor deposition (EBPVD) to enhance several
Pseudomonas aeruginosa properties of substrate material. m-ZrO2 and t-ZrO2 coatings effectively protected bacterial invasion against
Biocompatibility
pathogenic Pseudomonas aeruginosa (P. aeruginosa) and subsequent biofilm formation. Superior fibroblast cell
Corrosion
viability with improved cells spreading was observed in m-ZrO2 and t-ZrO2 coatings. Furthermore, electro-
chemical impedance spectroscopy (EIS) results exhibited enhanced charge transfer resistance (Rct) and thus,
strongly improved corrosion protection by m-ZrO2 and t-ZrO2 coatings compared to bare 316L SS substrate in
artificial blood plasma (ABP).

1. Introduction adhesion of pathogenic bacteria, the improvement of biocompatibility

Biomaterial implants play a vital role in replacing damaged or de-
fective parts of the human body. The need of implant materials have
been increased multifold due to accidents, trauma, sports injury, age
related problems, osteoporosis and amputation. The implant materials
have been used for several implantation purposes such as hip replace-
ment implants [1,2], ankle fracture [3], cardio vascular stents [4,5],
etc. However, bacterial invasion, corrosion and cytocompatibility are
the challenging factors to be addressed when designing implant mate-
rials. Clinically associated pathogens are frequently found in hospital
surroundings including operation theatres which elevates the risk of
spreading the pathogens to the patients and health care survival
equipments [6]. Among the various implant associated pathogens, P.
aeruginosa is a predominant microorganism which can be found in
burns, cystic fibrosis [7] and clinical surroundings and can survive even
in dry environment and on inanimate surface for several months [8].
Hence, the provision of smooth surface is a prerequisite for avoiding
earlier bacterial invasion as well as biofilm formation. In addition, the
interaction amidst host cells and materials surface is a significant fac-
tors which determines the healing rate as well as longevity of the im-
plant materials. After implantation, the metallic implant materials tend
to corrode and release metal ions from surface to surrounding tissue or
body fluids [9,10], causing inflammatory and cytotoxic response. It is
very complicate to combine the property requirements listed above in
single metallic implant materials. Nevertheless, the prevention of
and the enhancement of corrosion resistance by surface modification is
required. Among the various surface modifying materials such as ni-
trides, oxides, polymers and composites, bioceramic-coated materials
enjoy significant improvement in the implant of materials performance
due to their chemical inertness, corrosion resistance, sufficient me-
chanical strength, non-allergenic and excellent biocompatibility nature
[11,12]. Recently, yttria-stabilized tetragonal zirconia (t-ZrO2) bio-
ceramic has been widely used in the biomedical implants due to its
good aesthetic [13,14], considerable mechanical strength [15], wear
resistance [16] and better biocompatibility nature [15]. Similarly,
monoclinic phase of zirconia (m-ZrO2) also expressed better mechanical
strength, corrosion protection, hemocompatibility, biocompatibility
and inhibited bacterial invasion [17,18]. Mohandoss et al. studied
corrosion behavior of t-YSZ coating in artificial saliva solution and re-
vealed corrosion resistance property of t-YSZ coating [19]. Kohal et al.
observed biomechanical and histological behavior of zirconia (mono-
clinic) in a rat model [20]. However, there is contradictory information
which of the two modifications of ZrO2 (monoclinic and tetragonal)
performs better when coated onto 316L SS substrate. To the best of our
knowledge, no literatures data are available relating to the corrosion
behavior of m-ZrO2 as well as t-YSZ in artificial blood plasma (ABP)
solution. Among the various surface tailoring physical vapor deposition
(PVD) technique such as ion implantation, nitriding and plasma
spraying, electron beam evaporation stand out owing to its con-
venience, environment friendliness and precision of deposition. Hence,

⁎
Corresponding author.
E-mail address: vinitavishwakarma@sathyabama.ac.in (V. Vishwakarma).

https://doi.org/10.1016/j.ceramint.2018.05.103
Received 3 March 2018; Received in revised form 11 May 2018; Accepted 13 May 2018
Available online 15 May 2018
0272-8842/ © 2018 Elsevier Ltd and Techna Group S.r.l. All rights reserved.
G.S. Kaliaraj et al.
the objective of the present work was to deposit two different phases of
ZrO2 (m-ZrO2, t-ZrO2) using electron beam physical vapor deposition
(EBPVD) and to analyze their corrosion behavior in ABP solution.
Bacterial adhesion behavior against P.aeruginosa and cytotoxic studies
using fibroblast cells were also performed.
2. Experimental methods
2.1. Substrate preparation
Medical grade stainless steel (316L SS) (elemental composition: (wt
%) Cr-17.20; Ni-12.60; Mo-2.40; Mn-1.95; Si-1; C-0.03; N-0.02; Fe-
Balance) with dimensions of 2×1×0.2 cm3 was used as substrate
materials. Before deposition, all specimens were polished using dif-
ferent grades of silicon carbide (SiC) sheets (600, 800, 1000, 2000 and
5000 grits) to obtain uniform roughness. After polishing, they were
ultrasonically cleaned with acetone followed by distilled water for
15 min to remove surface residues. Finally, the cleaned samples were
sterilized by autoclave (under steam pressure for 15 min at 121 °C) and
dried.
2.2. Preparation of pellets
m-ZrO2 pellets were prepared from pure ZrO2 powder (99.99%
purity), whereas t-ZrO2 pellets were obtained by mixing 4 mol% of yt-
tria (Y2O3, 99.7% purity) with ZrO2 powder. The pellets were com-
pressed using 5 t load and sintered at 1723 K for 5 h in a muffle furnace
(VB ceramic consultants, India).
2.3. Deposition of m-ZrO2 and t-YSZ by EBPVD method
The sintered pellets were used to deposit m-ZrO2 and t-ZrO2 coatings
onto polished 316L SS substrates by EBPVD (Plassys, France, MEB600)
at a base pressure of 3 × 10−6 mbar. The substrate temperature, vol-
tage and deposition rate of the coating were held constant i.e., 673 K,
8 kV and 1 Å/s for all coatings respectively. During deposition, the
thickness of the coating was monitored by a digital thickness monitor
(DTM) using a quartz crystal (INFICON IC6 controller) and then con-
firmed by a Dektak profilometer (Bruker, USA). A constant coating
thickness of 1 µm was maintained for both types of samples.
2.4. Characterization of the coatings
The deposited films were analyzed by glancing incidence X-Ray
diffraction (GIXRD) analysis. Surface topography and morphology of
coatings were studied by atomic force microscopy (AFM) and scanning
electron microscope (SEM) analysis, respectively.
2.5. Bacterial studies
A pure P.aeruginosa bacterial culture was obtained from Microbial
Type Culture Collection and Gene Bank (MTCC), Institute of Microbial
Technology (IMTECH, Chandigarh). The bacteria were subcultured in
nutrient broth and incubated at 37 °C for 12 h as recommended by
MTCC. The bacterial culture was transferred into 20 ml conical flask
containing nutrient broth and incubated for 3 h in a shaking incubator.
The final bacterial concentration was adjusted to an optical density of
0.1–0.3 (at 660 nm) in which, the approximate bacterial concentration
was expected to be between 1 × 105 and 3 × 105 CFU ml−1 (colony
forming unit per mililitre).
The test samples (m-ZrO2, t-ZrO2 and bare 316L SS) were sterilized
in an autoclave (120 °C, 15 lbs pressure for 15 min) to avoid cross
contamination. The diluted bacterial culture was inoculated into nu-
trient broth containing the test samples and incubated for 24 h at 37 °C
in a shaking incubator. After incubation, the samples were washed with
distilled water and the sample surface was swabbed with sterile swab to
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Ceramics International 44 (2018) 14940–14946
remove adhered bacteria and collected into phosphate buffered saline
(PBS) solution (composition (g/l): NaCl-8.0; Na2HPO4·12H2O-2.08;
KCl-0.20; KH2PO4-0.20) for serial dilution [21]. Finally, 0.1 ml of se-
rially diluted samples were poured onto an agar plate and incubated for
24 h. The bacterial colony was expressed as colony forming unit per
square centimeter (CFU/cm2).
The same set of samples was also used for live/dead cell analysis to
determine the cytotoxic nature of test samples. For this, the incubated
samples were contacted with 20 µl of acridine orange (AO), incubated
for 10 min, and examined by epifluorescence microscopy (Nikon
Eclipse, E600).
2.6. Biocompatibility studies
2.6.1. MTT assay
Cytotoxicity study was performed with mouse fibroblast cells (NIH
3T3 cell line) supplied by National Centre for Cell Sciences (NCCS),
Pune, India. The cells were cultured in standard Dulbecco's Modified
Eagle Medium (DMEM, GIBCO), with 10% fetal bovine serum (FBS) and
1% non-essential amino acids (GIBCO) added. The medium was sub-
cultured at 2 days intervals, and maintained in 95% humidified atmo-
sphere with 5% CO2 at 37 °C. The cells were treated with 0.1% trypsin
and 0.1% ethylene diamine tetraacetic acid (EDTA) for 5 min to detach
the cells from the walls. Cytotoxicity of test samples were evaluated by
a direct method based on ISO-10993-part5 [22]. The test samples were
placed in 12 well plate containing fibroblast cells in DMEM and in-
cubated at 37 °C and 5% CO2 under humidified conditions.
2.6.2. Epifluorescence studies
Biocompatibility of test samples was determined by live/dead cell
analysis by epifluorescence imaging. After 1 of incubation, the samples
were transferred to 24 well culture plates containing fibroblast cells and
stained with fluorescein isothiocyanate (FITC) and propidium iodide
(PI) and analyzed using epifluorescence microscopy.
2.7. Electrochemical studies
The electrochemical behavior of the test samples was determined in
artificial blood plasma solution (ABP) (composition (g/l): NaCl-6.8;
CaCl2-0.200; KCl-0.4; MgSO4-0.1; NaH CO3-2.2; Na2HPO4-0.126;
NaH2PO4-0.026) at room temperature. All electrochemical measure-
ments were performed in a standard three-cell electrode setting with an
exposed area of 1 cm2. Open circuit potential (OCP) was measured for
about 60 min, prior to corrosion experiments to attain a stable potential
for the working electrode. Electrochemical impedance spectroscopy
(EIS) was performed in the frequency range from 100 kHz to 10 mHz
with an AC amplitude of 10 mV. After EIS analysis, potentiodynamic
polarization (PDP) was studied at a potential of ± 500 mV vs SCE with
a scan rate of 10 mV/s. The corrosion potential (Ecorr) and corrosion
current density (Icorr) were determined by Tafel plot.
2.8. Statistical analysis
All experimental data were calculated as the mean ± standard
deviation. Significance tests were performed by one-way ANOVA, and
subsequently confirmed by a student-newman-keuls post hoc test
whereby p < 0.05 was considered significant.
3. Results and discussion
The m-ZrO2 and t-ZrO2 coatings deposited on 316L SS substrates at
400 °C were subjected to XRD analysis as shown in Fig. 1. The m-ZrO2
and t-ZrO2 exhibited strong diffraction peaks as identified by standard
JCPDS No. 88-2390 and 88-1007 respectively.
Fig. 2 shows the surface topography of m-ZrO2, t-ZrO2 and bare
316L SS substrate. The m-ZrO2 and t-ZrO2 coatings surface (Fig. 2a)
G.S. Kaliaraj et al.
Fig. 1. XRD pattern of m-ZrO2 and t-ZrO2 coating.
showed smooth topography and uniform grains distribution on 316L SS
substrate. In contrast, the bare 316L SS substrate (Fig. 2c) showed
surface defects and irregular morphology on its surface. The average
roughness (Ra) and root-mean square (RMS) value of m-ZrO2, t-ZrO2
and bare 316L SS substrates were 3, 4 and 10 and 4, 6, and 12 nm,
respectively.
Fig. 2. Topographical analysis of (a) m-ZrO2, (b) t-ZrO2 coating and (c) bare 316L SS substrate.
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Ceramics International 44 (2018) 14940–14946
Fig. 3. TVC analysis of P. aeruginosa on m-ZrO2, t-ZrO2 coating and bare 316L
SS substrate.
3.1. Bacterial studies
The rate of bacterial invasion was determined as a function of sur-
face charge, roughness and wettability of the materials [23]. Fig. 3
shows the total viable count analysis of bacteria on m-ZrO2, t-ZrO2 and
bare 316L SS substrate after 1, 2, 3 and 4th day of incubation. The
bacterial attachment showed no significant difference between coated
and bare 316L SS substrate (P > 0.05). As shown in the figure, the m-
ZrO2 and t-ZrO2 coated samples exhibited comparatively less bacterial
adhesion behavior during the entire incubation period compared to the
bare 316L SS substrate. The reason for the bacterial reduction in m-ZrO2
and t-ZrO2 coating could be (a) the electrical conductivity of superficial
structure on ZrO2 surface could reduce the bacterial adherence [24], (b)
the t-ZrO2 coated surface donates electrons to bacteria thereby produ-
cing a repulsive force between bacteria and materials surfaces,
G.S. Kaliaraj et al. Ceramics International 44 (2018) 14940–14946

Fig. 4. Epifluorescence images of P. aeruginosa on m-ZrO2, t-ZrO2 coating and bare 316L SS substrate.

counteracting the bacterial attachment [25], (c) the negatively charged

bacterial surface is attracted by to positively charged ions present in the
solution to subsequently form an “electrical double layer” [26]. Since,
positively charged ions are present in the surrounding of bacterial cells,
they create a strong repulsive force on Zr+ ions present in the m-ZrO2
and t-ZrO2 coating surface thereby causing less bacterial adhesion. In
contrast, more bacterial adhesion was observed on the bare 316L SS
substrate, possibly due to higher surface irregularities and roughness
that overcome the repulsive force and thus, enhance bacterial invasion
[17]. Fig. 4 shows epifluorescence microscopy images of P.aeruginosa
bacterial adhesion and cell viability on m-ZrO2 (Fig. 4a) and t-ZrO2
(Fig. 4b) coating and bare 316L SS substrate (Fig. 4c) at 4th day of
incubation. In general, live/dead cell analysis was determined by the
emission of orange and green fluorescence from the cells. When AO
binds with RNA, it exhibits orange fluorescence (live cells) and green
fluorescence is observed when AO intercalates with DNA (dead cell)
[27]. Slight green fluorescence was observed on m-ZrO2 coating
Fig. 5. Cell viability by MTT assay on m-ZrO2, t-ZrO2 coating and bare 316L SS
(Fig. 4a), due to the antimicrobial effect of ZrO2 particles [28,29]. In t-
substrate.
ZrO2 coating, live cells are observed (orange fluorescence), possibly due
to the poor antibacterial performance of YSZ coating [30,31]. In addi-
tion, on the bare 316L SS (Fig. 4c) surface, adhering bacteria (orange 3.3. Corrosion analysis
fluorescence) survived after 4 days of incubation due to the poorer
antimicrobial behavior of 316L SS. However, since the coating mate- 3.3.1. OCP measurement
rials fails to exhibit superior anti-bacterial property, further studies The measurement of OCP is an important test to stabilize the po-
need to be carried out to examine the antibacterial properties by doping tential of the materials before performing the EIS studies. Fig. 7 shows
antimicrobial agents (Ag and Cu) into different phases of ZrO2 (m-ZrO2 the OCP value of m-ZrO2 (−0.180 V), t-ZrO2 (−0.176 V) coatings and
and t-ZrO2). bare 316L SS substrate (−0.253 V) after 60 min incubation. The OCP
result clearly reveals that for m-ZrO2 and t-ZrO2 coatings, the OCP is
shifted in the positive direction compared to the bare 316L SS substrate.
3.2. Biocompatibility studies Dissolution and surface stabilization occurred on the coating surface up
to 30 min of incubation following establishment of a stable potential. In
The viability of fibroblast cells (cell proliferation numbers) were the case of bare 316L SS, a drastic potential drop was observed over a
determined by the absorbance value obtained from MTT assay and the period of time.
results illustrated in Fig. 5. During the entire observation period, the
number of cells is higher on both the coatings than bare 316L SS sub- 3.3.2. Electrochemical impedance spectroscopy (EIS) studies
strates, suggesting that m-ZrO2 and t-ZrO2 coatings have superior bio- EIS is a useful technique to acquire the information on surface re-
compatibility. sistance and capacitance behavior of the test samples. Fig. 8 shows the
Fig. 6 shows epifluorescence microscopy results of m-ZrO2 and t- Nyquist plot for m-ZrO2, t-ZrO2 and bare 316L SS substrate in ABP
ZrO2 coatings and bare 316L SS substrate samples obtained in 24 well electrolyte solution. The results reveal that the magnitude of the im-
culture plates. Distinct fibroblast cells are seen on all sample surfaces. pedance value (1.95 × 105 Ω for m-ZrO2 and 1.795 × 106 Ω for t-ZrO2)
On close observation, the younger fibroblast cells tend towards elon- of coated samples is much greater than that of the bare 316L SS
gation on m-ZrO2 and t-ZrO2 coating by spreading their stress fibers, (1.50 × 104 Ω). It might be due to the stable oxide behavior of m-ZrO2
microfilament network, filopodia and lamellipodia [20,32]. In contrast, and t-ZrO2 coatings [18] which hinder the electrolyte to penetrate
healthy young cells with poor cell elongation were observed on bare through the vulnerable region (coating) thereby elevating higher re-
316L SS substrates. The results indicate that m-ZrO2 and t-ZrO2 coating sistance value. However in close observation, less Rct value was ob-
enhanced cell proliferation and thus will be a platform for bioactivity served in m-ZrO2 rather than t-ZrO2 and it could be due to the less phase
which enhances the wound healing process for better implant dur- stability of t-ZrO2 coating [33].
ability.
3.3.3. Potentiodynamic polarization studies
The corrosion behavior of test samples was examined by DC po-
tentiodynamic polarization measurements. The corrosion current

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G.S. Kaliaraj et al.
Fig. 6. Epifluorescence images of fibroblast cell adhesion on m-ZrO2, t-ZrO2 coating and bare 316L SS substrate.
Fig. 7. OCP measurement of m-ZrO2, t-ZrO2 coating and bare 316L SS substrate.
Fig. 8. Nyquist plot of m-ZrO2, t-ZrO2 coating and bare 316L SS substrate in
ABP solution.
density (Icorr) of each samples was obtained by extrapolation of the
cathodic and anodic branches of the polarization curves. Protection
efficiency of the test samples were determined from polarization curves
by the equation [34],
0
Pi = 100{1 − (icorr / icorr )}
where, icorr and i°corr are the corrosion current densities of coated (m-
ZrO2 and t-ZrO2) and uncoated (bare 316L SS) samples respectively.
The PDP curves are shown in Fig. 9 and their electrochemical para-
meters including Ecorr, Icorr, cathodic Tafel slope (βc), anodic slope (βa),
corrosion rate and protection efficiency (Pi) of the samples were col-
lected in Table 1. It is noted that Icorr values of m-ZrO2 (0.099 µA/cm2)
Ceramics International 44 (2018) 14940–14946
Fig. 9. PDP curves of m-ZrO2, t-ZrO2 coating and bare 316L SS substrate in ABP
solution.
and t-ZrO2 (0.027 µA/cm2) were reduced compared to bare 316L SS
substrate (0.489 µA/cm2). Similarly, the corrosion potential shows
noble direction for coated samples (m-ZrO2 and t-ZrO2) in contrast to
the bare 316L SS substrates. This is due to the ceramic nature of the
ZrO2 surface that protected the substrate surface. However on close
observation, t-ZrO2 improved the corrosion resistance more strongly
than m-ZrO2. This could be related to the higher crystalline content
((111) preferred orientation), fracture toughness and flexural strength
of the t-YSZ coating [35].
The equivalent circuit of m-ZrO2, t-ZrO2 and bare 316L SS substrates
obtained from EIS plots were represented as Rs, R1(Cdl) and R2(Cdl1) in
Fig. 10 which constitute the solution resistance with two resistances
and capacitors in series. The circuit diagram clearly reveals the pre-
sence of R1, and Cdl that express resistance and double-layer capaci-
tance of bare 316L SS substrate. Rs represents the ABP electrolyte re-
sistance which corresponds to the ohmic resistance. R2 and Cdl1
represent the resistance and the double layer capacitance of m-ZrO2 and
t-ZrO2 coatings. Fig. 10b shows the equivalent circuit model belongs to
the EIS spectra of bare 316L SS substrates, which consists of Rs(R1Cdl)
where, R1 and Cdl are the resistance and double-layer capacitance value
of bare 316L SS substrate.
(1) 4. Conclusion
m-ZrO2 and t-ZrO2 coatings were deposited by EBPVD technique.
Monoclinic and tetragonal phase as well as smooth and uniform topo-
graphy were observed by XRD and AFM respectively. m-ZrO2 and t-
ZrO2 coatings protected P.aeruginosa against bacterial invasion and
reduced biofilm formation due to its their high surface smoothness and
electrical double layer effect even after 4th day on incubation.
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G.S. Kaliaraj et al. Ceramics International 44 (2018) 14940–14946

Table 1
Potentiodynamic polarization and EIS data of test samples.
−1 −1
S. No. Materials Rct (Ω) Ecorr (V) βa V dec βc V dec Icorr (A/cm2) Corrosion Rate (µm/y) Protection efficiency (Pi)

6
1 t-ZrO2 1.795 × 10 0.019 0.12 0.08 0.027 0.012 94.5%
2 m-ZrO2 1.95 × 105 −0.047 0.24 0.09 0.099 0.045 80%
3 Bare 316L SS 1.50 × 104 −0.173 0.06 0.15 0.489 0.222 –

Fig. 10. Equivalent circuits for (a) m-ZrO2, t-ZrO2 coating and (b) bare 316L SS
substrate.
Moreover, enhanced cell viability with stress fibers and actin filament
formation was observed on m-ZrO2 and t-ZrO2 coating, suggesting
higher bioactive as well as biocompatibility nature. In addition, the
intact layer of m-ZrO2 and t-ZrO2 coatings enhanced corrosion re-
sistance in ABP electrolyte solution. Hence, it is suggested that ZrO2
modified 316L SS are a good candidates for orthopedic implant appli-
cations.
Acknowledgement
The authors acknowledge Col. Dr. Jeppiaar, Chancellor, Pro-
Chancellor, Vice-President, Pro Vice-Chancellor, Sathyabama Institute
of Science and Technology, Chennai, Tamil Nadu, India for their en-
couragement and financial support.
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