Thin Solid Films 660 (2018) 477–483

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Thin Solid Films

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Bactericidal activity and cytotoxicity of a zinc doped PEO titanium coating T

a,b b a,⁎
L. Sopchenski , K. Popat , P. Soares
a
Department of Mechanical Engineering, Polytechnic School, Pontifícia Universidade Católica do Paraná, Curitiba, PR 80215-901. Brazil
b
Department of Mechanical Engineering, School of Biomedical Engineering, Colorado State University, Fort Collins, CO 80523, USA

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

Keywords: Metallic implants are susceptible to bacterial colonization even years after the implantation impairing the os-
Bactericidal seointegration process. The treatment of a colonized implant is highly demanding, and in most cases implant
Zinc replacement is the only effective solution. To avoid the bacterial attachment and proliferation, bactericidal
Titanium coatings are proposed as a long-term prevention tool. Those coatings must assure a bactericidal activity for a
TiO2
long period and cannot induce cytotoxic responses in eukaryotic cells. Among all the bactericidal agents, Zinc is
Plasma electrolytic oxidation
one of the most investigated due to its broad bactericidal activity spectrum and its stimulatory effect on bone
formation. The aim of this study is to obtain a titanium oxide coating containing Zinc and evaluate its bacter-
icidal activity, cytotoxicity and ion release profile. The coating was obtained by Plasma Electrolytic Oxidation
(PEO) on commercially pure titanium grade 4 at 350 V for 60 s. Samples were divided in two groups; the re-
ference group was obtained in a base electrolyte containing calcium acetate and calcium glycerophosphate
(called CaP group). The experimental group had Zinc acetate added as a Zinc source to the base electrolyte
(called Zn-CaP group). The surface was characterized by Scanning Electron Microscopy (SEM) and X-ray
Photoelectron Spectroscopy (XPS), while the ion dissolution was evaluated by Inductively Coupled Plasma -
Atomic Emission Spectroscopy (ICP-AES). The bactericidal activity was determined against Staphylococcus aureus
by fluorescence microscopy using a live/dead viability kit. The cytotoxicity against eukaryotic cells was eval-
uated using adipose derived stem cells (ADSC) using the lactate dehydrogenase (LDH) assay. Zinc, Calcium and
Phosphorous were incorporated to the titanium oxide coating and no changes on the coating structure and
morphology were observed by the addition of Zn to the electrolyte. ICP-AES results show the coatings released
Ca, P and Z ions after 28 days of immersion in DI water. The ICP-AES profile suggests the ion release reach an
equilibrium state after 7 days of immersion. The Zn-CaP coating presented bactericidal activity against S. aureus,
showing a higher number of dead bacteria after 6 h of incubation and a lower number of living bacteria after
24 h compared to the CaP group. No cytotoxic effect was observed against ADSC by the presence of Zn on the
coating, indicating the Zn-CaP coating has a potential to prevent bacterial colonization in metallic implants.

1. Introduction infection in implants still have an expressive mortality rate, Lora-Ta-

Endosseous implants are susceptible to be colonized by bacteria
even years after the implantation, when endogenous bacteria may mi-
grate to the prosthesis surface [1]. This bacterial contamination impairs
the osseointegration process leading to the implant failure, where the
implant replacement is the only effective solution [2–4]. Staphylococcus
aureus and Pseudomonas aeruginosa are one of the most common colo-
nizers strains on orthopedic and dental implants. Those bacteria form
biofilms, an organized bacteria community involved by an extracellular
matrix who protects the microorganisms from the host immune system
and the antibiotic action, being the prosthesis removal the only effec-
tive treatment [5, 6]. Despite all the prophylactic measures bacterial
mayo et al. showed 7% of deaths among patients infected just with S.
aureus in total knee arthroplasty [7]. To avoid the bacterial attachment
and proliferation, bactericidal coatings that do not induce cytotoxic
responses in eukaryotic cells are proposed as a long-term prevention
tool.
Multifunctional coatings that prevent the bacterial adhesion and
improve the bone growth by the presence of osteoinductor elements can
be obtained by plasma electrolytic oxidation (PEO). This electro-
chemical technique allows the incorporation of bactericidal elements
along with Ca and P on titanium surface. Ishizawa et al. showed for the
first time the possibility to incorporate Ca and P on the TiO2 structure
by PEO; those coatings reduce the Ti4+ ion release, increase osteoblast

⁎
Corresponding author at: Pontifícia Universidade Católica do Paraná, Department of Mechanical Engineering, Rua Imaculada Conceição, 1155 – Prado Velho, Curitiba, PR 80710-230,
Brazil.
E-mail address: pa.soares@pucpr.br (P. Soares).

https://doi.org/10.1016/j.tsf.2018.05.055
Received 21 March 2018; Received in revised form 18 May 2018; Accepted 18 May 2018
Available online 28 June 2018
0040-6090/ © 2018 Elsevier B.V. All rights reserved.
L. Sopchenski et al. Thin Solid Films 660 (2018) 477–483

Fig. 1. Top view SEM images of the CaP and Zn-CaP coatings and cross-sectional image of the Zn-CaP coating. Number 1 outlines the outmost porous layer, while
number 2 indicates the inner dense layer, both characteristic from coatings obtained by PEO.

Table 1
Elemental composition of the coatings determined by XPS.
Element (% at)

Ti O Ca P Zn

CaP 6.3 67.4 12.7 13.6 –

Zn-CaP 3.9 66.7 8.4 11.3 9.7

adhesion and proliferation in vitro, and improve the osseointegration in

Fig. 2. XRD pattern of the CaP and Zn-CaP coatings. The XRD peaks were in-
dexed by the following PDFs: Anatase [01-078-2486], Rutile [00-021-1276]
and Titanium [01-089-3073].
vivo [8–11].
Zinc is an oligoelement present in abundance on the bone tissue
[12]. Zinc influences the bone growth and mineralization and its de-
privation impairs the bone metabolism [13]. Moonga et al. showed zinc
is also involved in the control of bone resorption in vitro, by inhibiting
the osteoclasts action [14]. Besides playing an important role on bone
tissue, zinc is also essential for the function of > 300 enzymes, proteins,
it regulates DNA synthesis, influences hormonal regulation and cell
division [15–17].
Zinc has been extensively investigated as a broad spectrum bacter-
icidal agent specially under the zinc oxide form [18]. Zinc oxide may
act by the dissolution of zinc ions or the generation of reactive oxygen
species (ROS), capable of disrupting the bacteria membrane [19]. Ad-
ditionally, zinc oxide nanoparticles and structures may interact and
attach to the cell wall, impairing the membrane function and possibly
disrupting it [20]. Zinc ions are believed to compete with di-valent

Fig. 3. Coatings high-resolution XPS spectra of a) Ca 2p, b) P 2p and c) Zn 2p.

478
L. Sopchenski et al.
Fig. 4. Elemental release of the CaP and Zn-CaP coatings assessed by ICP after
the immersion in deionized water.
binding sites on proteins, impairing the bacteria's metabolism, as in the
case of PsaA protein, where the binding zinc ion prevent the manganese
absorption, resulting in the bacteria death [21]. In biomaterials zinc has
been widely studied as an antibacterial agent in hydroxyapatites, dental
composites, bioglasses, and dopant on metallic materials due to its
bactericidal activity against different pathogens [22–26].
The aim of this study was to evaluate the effect of zinc incorporation
into PEO - TiO2 coatings containing Ca and P on cytotoxicity and
bactericidal activity against S. aureus and P. aeruginosa. The coating's
Ca, P and Zn ion release profile was determined up to 28 days of testing.
2. Materials and methods
2.1. Samples preparation
TiO2 coatings were obtained on commercially pure titanium grade 4
on disc samples (12 mm Ø). Samples were previously grinded with 600
SiC abrasive paper and cleaned in a sequence of acetone, ethanol and
deionized water. Plasma electrolytic oxidation was based on a previous
Fig. 5. Live and dead coverage of S. aureus and P. aeruginosa after 6 h and 24 h of culture on the coatings.
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Thin Solid Films 660 (2018) 477–483
work, and conducted at 350 V for 60 s at room temperature, using a
conventional cell where the titanium discs are the anode and a platinum
plate the cathode [27]. To produce samples doped with calcium and
phosphorous (CaP samples), an electrolyte containing calcium acetate
(0.15 M) and calcium glycerophosphate (0.02 M) were used. To pro-
duce samples doped with calcium, phosphorous and zinc (Zn-CaP
samples), zinc acetate (0.02 M) was added to the previous electrolyte.
After that, the anodization samples were rinsed in deionized water,
dried and stored at 37 °C.
2.2. Coating characterization
Coating morphology was evaluated by scanning electron micro-
scopy (SEM, Vega3, Tescan), by top-view and cross-sectional images.
The chemical composition of the coatings was assessed by X-ray pho-
toelectron spectroscopy (XPS, Physical Electronics PHI-5800 spectro-
meter). For XPS, the C 1 s peak was set to 284.6 eV to compensate any
residual charging effects. Data for percent elemental composition, ele-
mental ratios and peak fit analysis were calculated using Multipack and
XPS Peak 4.1 software. To determine the crystalline phases of the oxide
layers, samples were analyzed by X-ray diffraction (XRD, XRD-7000
Shimadzu). XRD analysis was performed using a thin-film geometry,
CuKα radiation, with an incidence angle of 5° at 40 kV and 30 mA, and
scanning speed of 1°/min.
To measure the Ca, P and Zn ion release from the coatings, samples
were immersed in 5 mL of deionized water in a sealed bottle at 37 °C for
1, 7, 14 and 28 days. After immersion the ion concentration was mea-
sured by inductively coupled plasma-mass spectroscopy (ICP-MS)
(ELAN DRC II, Perkin Elmer, Norwalk, CT).
2.3. Bactericidal activity
The bactericidal activity of the coatings was assessed using a live/
dead staining kit (LIVE/DEAD® BacLight, Thermo Fisher) against
Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aer-
uginosa). Stocks of S. aureus and P. aeruginosa, previously prepared in a
glycerol solution and stocked at −80 °C, were thaw at room tempera-
ture and centrifuged at 4700 rpm for 10 min. The glycerol supernatant
was discarded, and the pellet was resuspended in 50 mL of Lysogeny
broth media (LBM) and allowed to grow for 24 h at 37 °C. After the
growth, the bacteria solution was adjusted to an OD600 nm ∼0.35 and
an aliquot of 500 μL of this solution was added on the coatings in a 48
well plate. The bacteria were cultured for 6 h and 24 h, after this period
the samples were gently rinsed with the phosphate buffer solution
L. Sopchenski et al.
Fig. 6. Live/dead fluorescence images of S. aureus and P. aeruginosa cultured for 24 h on the coatings. Both cultures were stained with propidium iodine (PI) to dye
the dead bacteria red and SYTO® 9 to dye the living bacteria green.
(PBS).
To evaluate the coverage of dead and live attached bacteria, a live/
dead kit was used (LIVE/DEAD™ BacLight™, ThermoFisher). After the
culture, the bacteria were fixed with formaldehyde for 15 min, rinsed
with PBS and incubated for 25 min at 37 °C in the staining solution
prepared following the manufacturer's instructions. The staining solu-
tion contains Propidium iodide (PI) to stain the dead bacteria and
SYTO® 9 to stains the living bacteria, which together allow to determine
the relative proportion of dead and live bacteria. Fluorescence images
of the live and dead bacteria were recorded in a fluorescence micro-
scope (Zeiss Imager-A2) and analyzed by ImageJ software.
The bacterial morphology and attachment on the coatings were
evaluated by SEM. The bacteria were cultured for 6 h and 24 h, rinsed
and fixed for 45 min in a solution containing glutaraldehyde 3%, so-
dium cacodylate 0.1 M and sucrose 0.1 M, followed by immersion in a
buffer solution. Samples were dehydrated in a sequence of ethanol
(from 30% to 100%) followed by 10 min in hexamethyldisilazane
(HDMS). Prior to the SEM analysis the samples were gold coated.
2.4. Cytotoxicity analysis
2.4.1. Cell culture
Adipose derived stem cells (ADSCs) were used to evaluate the bio-
compatibility of the coatings. The ADSCs were cultured at 37 °C in a
humidified atmosphere of 5% CO2 in 75 cm2 flasks containing α-MEM
medium, supplemented with 1% penicillin-streptomycin and 10% of
Fetal Bovine Serum (FBS). Prior to cell seeding on coatings, the samples
were cleaned, and sterilized in a sequence of acetone, alcohol and
phosphate buffered saline (PBS), followed by UV radiation exposure for
30 min. ADSCs were seeded onto the coatings in a 48-well plate at a
density of 104 cells per well.
2.4.2. Cytotoxicity
The cytotoxicity of the coatings was evaluated by Lactate
Dehydrogenase (LDH) assay kit (Cayman Chemical) after 1 day of cul-
ture. Besides the cells cultured on the coatings, a set of 10 wells re-
ceived just the cells in α-MEM. Five wells were used as positive control,
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Thin Solid Films 660 (2018) 477–483
and other 5 received 10% of Triton-X to be used as negative control.
After 24 h of culture 100 μL of the cell culture media was transferred
from each well to a 96-well plate together with 100 μL of LDH solution.
The 96-well was incubated for 30 min on an orbital shaker and then the
absorbance of each well was measured at a wavelength of 490 nm using
a plate reader (BMG Labtech).
2.5. Statistical analysis
The quantitative data were presented as the means ± standard de-
viations. The statistical differences were compared at p < .001 using
one-way ANOVA and Tukey's HSD test.
3. Results and discussion
3.1. Characterization of the CaP and Zn-CaP coatings
The PEO process generates an homogeneous porous coating on the
titanium surface, with volcano shaped structures, typical from coatings
obtained in a calcium‑phosphorus based electrolyte [28]. No difference
on the coating morphology was observed by the addition of 0.02 M of
zinc acetate on the electrolyte, as seen on the SEM images in Fig. 1. The
porous distribution and dimensions were not affected by the zinc in-
corporation, as observed on the top-view SEM images. The cross-section
SEM image shows a 1.0 ± 0.3 μm dense inner layer, followed by a
4.1 ± 2.2 porous out layer, characteristic from coatings obtained by
PEO.
It is well established that the coating crystallinity is directly de-
pendent on the applied potential and the electrolyte composition [29].
The Zn-CaP coating presented the same crystalline structure as the CaP
one, showing that the introduction of zinc acetate on the electrolyte did
not disarrange or originate new crystalline phases, as observed on the
XRD patters on Fig. 2. Peaks from anatase, rutile and titanium were
observed. The coating crystallinity is higher on the external portion of
the coating, and improves the bone apposition in vivo, as the atomic
arrangement of anatase and rutile are suitable for the epitaxy of apatite
crystals [30–32].
L. Sopchenski et al.
Fig. 7. SEM adhesion of S. aureus and P. aeruginosa after 24 h of cultivation on the coatings.
Fig. 8. Cytotoxicity of the surfaces after 24 h of ADSC culture assessed by LDH
absorbance. P < .001.
The zinc incorporation was confirmed by XPS analysis, where the
presence of metallic zinc was confirmed by the observation of the
double peak at 1021.6 and 1044.7 eV in the XPS spectra in Fig. 3.
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Thin Solid Films 660 (2018) 477–483
Calcium and phosphorus were incorporated as shown on Ca 2p and P 2p
spectra. Ca 2p peaks at 347.1 eV and 350.6 eV are ascribed to calcium
in Ca3(PO4)2, in accordance with the P 2p peak at 133.1 eV, related to
the PeO bond in phosphate ions [33]. The elemental composition of the
samples is presented in Table 1. During the oxidation the anions
(PO43−) are moved to the anode surface by electromigration, dictated
by the external potential applied. Previous studies showed the Ca2+
anions are driven to the anode surface by diffusion, the same me-
chanism should be expected for the Zn2+ anion [34]. It was observed a
decrease on the incorporation of calcium on the Zn-CaP coating, pos-
sibly due to the presence of zinc on the electrolyte.
The release of calcium, phosphorus and zinc ions from the coatings
were measured after 1, 7, 14 and 28 days of immersion in deionized
water. Values under 10 ppm were found after 28 days of immersion,
indicating the release of ions is minimal, as seen in the ion release
profile in Fig. 4. Within the immersion time, the ions release increase
gradually and tend to a stabilization state after 7 days of immersion.
The slight difference of Ca ion released on both coatings, and the re-
duced amount of Zn ion released, is in accordance with the Zn-CaP
coating elemental composition determined by XPS.
3.2. Bactericidal activity
It is well established that the implants are more susceptible to
bacterial infections during the first hours of implantation, so the bac-
tericidal activity of the coatings was evaluated after 6 and 24 h of
culture. After just 6 h of culture the amount of dead S. aureus and P.
L. Sopchenski et al.
aeruginosa had significantly increased on Zn-CaP coating compared to
the CaP coating, indicating the bactericidal action of zinc. The same
pattern was observed after 24 h of culture, as observed by the bacterial
coverage in Fig. 5 and in the fluorescence images in Fig. 6. Subbiahdoss
et al. showed S. aureus and P. aeruginosa cause the death of adhering
human osteoblasts after 24 h of coculture [35]. This aggressive infec-
tion may be controlled by the Zn-CaP action with the release of < 1
ppm within this 24 h period.
The number of live bacteria increased from 6 h to 24 h of culture on
the CaP coatings, showing bacterial proliferation in the absence of a
bactericidal agent. Significant reduction in the amount of the live P.
aeruginosa was observed on the Zn-CaP coating after 24 h of culture in
comparison to the CaP coating. No significant increase in the live S.
aureus was observed on the Zn-CaP coating from 6 h to 24 h of culture,
showing no spread of the bacterial colonization.
The SEM images confirm the results from the live/dead assays,
showing a significant reduction in the number of adherent bacteria on
the Zn-CaP both for S. aureus and P. aeruginosa after 24 h of culture
(Fig. 6). These results show the Zn-CaP coating present a great ability to
inhibit the bacterial proliferation in the first 24 h of contact. Previous
works showed the bactericidal properties of doped PEO coatings con-
taining Ca, P, Zn and Ag-nanoparticles, where the bactericidal ability
against S. aureus was credited to the combination of Zn and Ag-nano-
particles [36]. Silver nanoparticles have been extensively researched as
an antibacterial agent on implant coatings, although its indiscriminate
use may originate adverse side effects as dermal argyria and toxic ef-
fects at molecular level [37–39]. Our results show the incorporation of
Ag-nanoparticle is not mandatory since Zn itself present bactericidal
ability against S. aureus and P. aeruginosa (Fig. 7).
3.3. Cytotoxicity
In order to determine whether the Zn present on the Zn-CaP coating
impair the cell adhesion and proliferation, the cytotoxicity was eval-
uated by the LDH assay. After 24 h of ADSCs cultivation on the coatings,
no obvious cytotoxicity was observed, the absorbance of the cells cul-
tured on the Zn-CaP coating was comparable to the ones cultured on the
CaP coating and the control group, corresponding to the cells on the
culture media. This result shows the release of 0.30 ± 0.11 ppm of Zn
ions does not induce cytotoxic effects on the ADSC cells. Early reports
demonstrate the release of up to 2.8 ppm of Zn ion does not induce
cytotoxic effects but increase the stem cell growth and proliferation
[40] (Fig. 8).
4. Conclusions
In this study, we showed the viability of incorporating Zn on a TiO2
coating and profiting from its minimal dissolution to assure the bac-
tericidal ability without inducing cytotoxic effects. A Zn, Ca, P doped
coating was obtained on titanium by plasma electrolytic oxidation and
the elements incorporation was confirmed by XPS. The presence of
osteoinductors (Ca and P) and bactericidal (Zn) elements on the coating
did not change the coating morphology and crystallinity, factors that
are already proved to improve the osseointegration. The Zn in-
corporation grant the coating bactericidal ability against S. aureus and
P. aeruginosa after 6 h of culture. The coating did not induce cytotoxic
effects on eukaryotic cells. Those results indicate the Zn doped coating
can be employed as a tool to prevent the bacterial colonization on en-
dosseous implants, however, its performance needs to be validated by in
vivo studies.
Acknowledgments
Research reported in this publication was supported by CNPq
(Grants 427025/2016-8 and 305715/2016-0), and Capes (Grant PDSE
88881.132290/2016-01) for scholarship to L.S. Sopchenski to conduct
482
Thin Solid Films 660 (2018) 477–483
part of this research at Colorado State University.
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