materials

Article

Cytotoxicity Evaluation of Anatase and Rutile TiO2

Thin Films on CHO-K1 Cells in Vitro
Blanca Cervantes 1,2 , Francisco Lpez-Huerta 3 , Rosario Vega 1 , Julin Hernndez-Torres 4 ,
Leandro Garca-Gonzlez 4 , Emilio Salceda 1 , Agustn L. Herrera-May 4 and Enrique Soto 1, *
1

2
3
4

Instituto de Fisiologa, Benemrita Universidad Autnoma de Puebla, 14 sur 6301, Col. San Manuel,
72570 Puebla, Mexico; [email protected] (B.C.); [email protected] (R.V.);
[email protected] (E.S.)
Instituto de Investigaciones Biomdicas Alberto Sols, Consejo Superior de Investigaciones
Cientficas-Universidad Autnoma de Madrid, Arturo Duperier, 4, 28029 Madrid, Spain
Facultad de Ingeniera, Universidad Veracruzana, Calzada Ruiz Cortines 455, Boca del Ro,
94294 Veracruz, Mexico; [email protected]
Centro de Investigacin en Micro y Nanotecnologa, Calzada Ruiz Cortines 455, Boca del Ro,
94294 Veracruz, Mexico; [email protected] (J.H.-T.); [email protected] (L.G.-G.);
[email protected] (A.L.H.-M.)
Correspondence: [email protected]; Tel.: +52-222-244-4053

Academic Editor: Jordi Sort

Received: 10 June 2016; Accepted: 12 July 2016; Published: 26 July 2016

Abstract: Cytotoxicity of titanium dioxide (TiO2 ) thin films on Chinese hamster ovary (CHO-K1) cells
was evaluated after 24, 48 and 72 h of culture. The TiO2 thin films were deposited using direct current
magnetron sputtering. These films were post-deposition annealed at different temperatures (300, 500
and 800 C) toward the anatase to rutile phase transformation. The root-mean-square (RMS) surface
roughness of TiO2 films went from 2.8 to 8.08 nm when the annealing temperature was increased
from 300 to 800 C. Field emission scanning electron microscopy (FESEM) results showed that the
TiO2 films thickness values fell within the nanometer range (290310 nm). Based on the results of the
tetrazolium dye and trypan blue assays, we found that TiO2 thin films showed no cytotoxicity after
the aforementioned culture times at which cell viability was greater than 98%. Independently of the
annealing temperature of the TiO2 thin films, the number of CHO-K1 cells on the control substrate
and on all TiO2 thin films was greater after 48 or 72 h than it was after 24 h; the highest cell survival
rate was observed in TiO2 films annealed at 800 C. These results indicate that TiO2 thin films do not
affect mitochondrial function and proliferation of CHO-K1 cells, and back up the use of TiO2 thin
films in biomedical science.
Keywords: biocompatibility; sensors; cytotoxicity; titanium; titanium dioxide; MTT

1. Introduction
The applications of titanium dioxide (TiO2 ) films include photocatalysis, photoelectrolysis, and the
manufacture of sensors and solar cells. These applications depend on the following characteristics
of the TiO2 films: specific surface area, crystal and grain size, phase, concentration and dopant.
TiO2 films can be synthesized through different methods which include sol-gel, hydrothermal, spray
pyrolysis, and physical vapor deposition (PVD) [16]. In health sciences, TiO2 is used as a matrix to
produce biosensors because of its high conductivity, chemical stability, and good biocompatibility [7].
These sensors can be used in the detection of tumor markers such as the carcinoembryonic antigen
and alpha-fetoprotein [810] as well as in photodynamic therapy for cancer, and in drug delivery
systems [11]. Previous studies have shown that the surface of TiO2 thin films deposited by direct
current magnetron sputtering had good quality, homogeneity, roughness, and biocompatibility.
Materials 2016, 9, 619; doi:10.3390/ma9080619

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These films were suitable for the culture of functional living neurons that display normal electrical
behavior [12]. On account of these findings, we proposed these TiO2 thin films to be deposited on
the microelectrode surface and the readout circuit of complementary metal oxide semiconductor
and micro-electromechanical systems (CMOS-MEMS) for biomedical applications [12,13], for which
evaluation of their potential cytotoxicity is required. In order to avoid experimental animal exposure
to unjustified risk, studying in vitro cytotoxicity is an essential step prior to the use of TiO2 thin
films on living animals [14,15]. The cytotoxicity testing of materials is addressed by the International
Organization for Standardization 10993 (ISO 10993-5) which presents guidelines to choose suitable
tests and define the important principles underlying them [1618].
Cytotoxic effects in vitro are evaluated by morphological changes, by analysis of the cell growth
rate, or by the study of specific aspects of cellular metabolism [14]. Cells respond rapidly to toxic stress
by altering their metabolic and cell growth rates [19]. Therefore, the study of these parameters in cell
lines provides valuable information to determine the possible toxic effect of diverse materials.
The CHO-K1 is a well-established cell line derived from Chinese hamster ovary and considered
one of the most sensitive cell lines for cytotoxicity studies [1921]. The tests commonly used to evaluate
cytotoxicity are the colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT), and the trypan blue exclusion assay [22,23]. The MTT test determines the viability and
proliferation of cells [15]. MTT is a water-soluble yellow dye which can be reduced to water-insoluble
purple formazan crystals through cleavage of the tetrazolium ring by living cells mitochondrial
succinic dehydrogenase [24]. Formazan is retained in the cells and can be released by solubilization;
thus, the concentration of dissolved formazan crystals can be quantified by spectrophotometry, giving
a direct measurement of metabolically active living cells. The results are compared to appropriate
control samples [2,22,2427]. The trypan blue exclusion test is a rapid method to assess cell viability
and cell proliferation in response to environmental insults [23]. This test is based on the principle
that live (viable) cells do not take up certain dyes, whereas dead (non-viable) cells do because their
membrane becomes permeable to the colorant, so analyzing the number of stained as opposed to
non-stained cells provides a direct evaluation of the percentage of dead cells in a population and,
in addition, the staining aids visualization of the cell morphology [2830].
The objectives of this study were to determine the surface morphology, thickness and roughness
of the TiO2 thin films, and to evaluate the potential in vitro cytotoxicity of the films in crystalline forms
(anatase and rutile) on CHO-K1 cells using the MTT and trypan blue assays after 24, 48 and 72 h
of culture.
2. Results
2.1. Cytotoxicity Analysis (MTT and Trypan Blue Assays)
To assess the cytotoxicity of TiO2 thin films, cell viability and cell proliferation on control substrate
and on TiO2 were determined using the MTT assay and the trypan blue exclusion test. CHO-K1 cells
cultured on the control and on the TiO2 thin film surfaces (annealed at 300, 500 and 800 C) showed
no ostensive morphological differences (Figure 1). The MTT assay showed that the optical density
in TiO2 thin film surfaces (annealed at 300, 500 and 800 C) was not significantly different from that
of the control after 24, 48 or 72 h of culture (p > 0.05; Figure 2A). As expected, optical density in the
Triton-X control was lower than that of the control and the TiO2 thin films (p < 0.01; Figure 2A), which
shows that cells cultured in the Triton-X control did not survive to the application of the detergent
Triton-X 1%. The percentage of cell viability on TiO2 thin films was similar to that observed on the
control substrate after 24, 48 or 72 h of culture (p > 0.05). The optical density in the control substrate
and in all TiO2 films after 48 or 72 h was greater than that after 24 h (p < 0.01; Figure 2A), revealing that
cell proliferation activity was not influenced by the presence of TiO2 thin films. The optical density in
the control substrate and in TiO2 films after 48 h of culture was not significantly different from the one
measured after 72 h (p > 0.05).

Materials 2016, 9, 619

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3 of 11
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33 of

Figure1.
1. Representative
RepresentativeCHO-K1
CHO-K1cells
cellsin
inculture
culturewith
withthe
thenegative
negativecontrol
controlcondition
conditionand
andin
inTiO
TiO222thin
thin
Figure
Figure
CHO-K1
cells
in
culture
with
the
negative
control
condition
and
in
TiO
thin
C.
filmsannealed
annealedatat
at300,
300,500
500and
and
800
C.
CHO-K1
cells
grew
with
an elongated-ovoid
elongated-ovoid
morphology
in
films
800
CHO-K1
cells
grew
with
an an
elongated-ovoid
morphology
in the
films
annealed
300,
500
and
800
C.
CHO-K1
cells
grew
with
morphology
in
the
control
substrate
and
in
all
TiO
2
thin
films.
The
round
cells
seen
in
all
the
images
are
cells
detached
control
substrate
and
in
all
TiO
thin
films.
The
round
cells
seen
in
all
the
images
are
cells
detached
the control substrate and in all TiO
2 2 thin films. The round cells seen in all the images are cells detached
from
fromthe
thesubstrate.
substrate. Scale
Scalebar
bar=== 20
20 m
m for
for all
all the
the images.
images.
from
the
substrate.
bar
m

Figure 2.
2. TiO
TiO222 thin
thin films
films did
did not
not affect
affect cell
cell proliferation
proliferation of
of CHO-K1
CHO-K1cells
cellsafter
after48
48 h.
h. (A)
(A) Optical
Optical density
density
Figure
CHO-K1
cells
after
48
Figure
thin
films
affect
corresponding
to
Triton-X
control
(borosilicate
glass
plus
Triton-X),
control
(borosilicate
glass)
and
control (borosilicate
(borosilicate glass
glass plus
plus Triton-X),
Triton-X), control
control (borosilicate
(borosilicate glass)
glass) and
and
corresponding to Triton-X control
TiO222 films
films after
after 24,
24, 48
48and
and72
72hhhin
inculture.
culture.The
Theoptical
opticaldensity
densityin
incontrol
controlsubstrate
substrateand
andin
inTiO
TiO222films
films
TiO
48
and
72
in
culture.
The
optical
density
in
control
substrate
and
in
TiO
films
TiO
wasgreater
greaterafter
after48
48and
and72
72hhhthan
thanafter
after24
24hhh(p
(p<<<0.05)
0.05)which
whichindicates
indicatescell
cellproliferation;
proliferation;(B)
(B)Number
Number
was
greater
after
48
and
72
than
after
24
(p
0.05)
which
indicates
cell
proliferation;
(B)
Number
was
of unstained
unstained CHO-K1
CHO-K1 cells
cells (in
(in millions)
millions)
on
control
substrate
and
on
TiO
films
after
24,24,
4848
and
72
of
22 thin
films
after
24,
48
and
72
millions)on
oncontrol
controlsubstrate
substrateand
andon
onTiO
TiO
thin
films
after
and
2thin
h.
The
number
of
CHO-K1
cells
on
the
control
substrate
and
TiO
2
thin
films
was
greater
after
48
and
h. The
number
of of
CHO-K1
cells
ononthe
and
72
h. The
number
CHO-K1
cells
thecontrol
controlsubstrate
substrateand
andTiO
TiO22thin
thinfilms
filmswas greater after 48 and
72hhhthan
thanafter
after24
24hhh(p
(p<<<0.05)
0.05)except
exceptfor
forTiO
TiO222 films
films annealed
annealed at
at 300
300C.
C.
All results
results are
are reported
reported as
asthe
the
72
C. All
than
after
24
(p
0.05)
except
for
TiO
as
the
mean
standard error
error and
andall
allexperiments
experimentswere
wereperformed
performedatat
at
least
three
times.
Triton
X-100
24vs.
mean
least
three
times.
* Triton
X-100
24 h24
mean
standard
and
all
experiments
were
performed
least
three
times.
** Triton
X-100
hh
# 24
vs. Triton
Triton
X-100
4872or
orh;72
72
h; ##h24
24
vs.h;
48and
h; and
and
&h24
24
vs.h.
72 h.
h.
Triton
X-100
48 or48
vs.hh 48
& 24&
vs.hh 72
vs.
X-100
h;
vs.
48
h;
vs.
72

In the
the trypan
trypan blue
blue exclusion
exclusion test,
test, cell
cellviability
viabilityon
onthe
thecontrol
control(borosilicate
(borosilicateglass)
glass)and
andon
onall
allTiO
TiO22
In
the
trypan
blue
exclusion
test,
cell
viability
on
the
control
(borosilicate
glass)
and
on
all
TiO
In
2
thin
films
was
greater
than
98%
after
24,
48
and
72
h
(Figure
3
and
Table
1).
The
number
of
viable
thin
films
was
greater
than
98%
after
24,
48
and
72
h
(Figure
3
and
Table
1).
The
number
of
viable
thin films was greater than 98% after 24, 48 and 72 h (Figure 3 and Table 1). The number of viable
CHO-K1 cells
cells on
on TiO
TiO22 thin
thin films
films after
after 24,
24, 48
48 and
and 72
72 h
was not
not significantly
significantly different
different from
from that
that on
on the
the
CHO-K1
cells
on
TiO
thin
films
after
24,
48
and
72
hh was
was
not
significantly
different
from
that
on
the
CHO-K1
2
controlsubstrate
substrate(p
(p>>> 0.05;
0.05; Figure
Figure 2B).
2B). However,
However,except
exceptfor
forTiO
TiO22films
filmsannealed
annealedat
at300
300C
C
which were
were
control
substrate
(p
0.05;
Figure
2B).
However,
except
for
TiO
films
annealed
at
300
which
were
control
C which
2
not
significantly
different
(p
>
0.05),
the
number
of
viable
CHO-K1
cells
on
the
control
substrate
and
not
significantly
different
(p
>
0.05),
the
number
of
viable
CHO-K1
cells
on
the
control
substrate
not significantly different (p > 0.05), the number of viable CHO-K1 cells on the control substrate andand
on
on
all
TiO
2
films
after
48
or
72
h
was
greater
than
that
found
after
24
h
(p
<
0.01;
Figure
2B).
The
on
all
TiO
2 films after 48 or 72 h was greater than that found after 24 h (p < 0.01; Figure 2B). The
all TiO2 films after 48 or 72 h was greater than that found after 24 h (p < 0.01; Figure 2B). The number of
number
of viable
viable
CHO-K1
cells substrate
on the
the control
control
substrate
and
on
TiO
films
after 48
48 hh different
was not
not
number
of
cells
on
22 films
after
was
viable
CHO-K1
cellsCHO-K1
on the control
and onsubstrate
TiO2 filmsand
afteron
48 TiO
h
was
not significantly
significantly
different
from
the
number
of
cells
found
after
72
h
(p
>
0.05),
indicating
that
cells
did
not
significantly
different
from
the
number
of
cells
found
after
72
h
(p
>
0.05),
indicating
that
cells
did
not
from the number of cells found after 72 h (p > 0.05), indicating that cells did not further proliferate
further
after
48in
of culture,
culture,
either
in
control
or in
in TiO
TiO
films indicating
indicating
that normal
normal
cell
further
48
hh control
of
control
or
films
that
cell
after
48 proliferate
hproliferate
of culture,after
either
or ineither
TiO2 in
films
indicating
that22 normal
cell proliferation
was not
proliferation
was
not
affected
by
TiO
films.
proliferation
was
not
affected
by
TiO
22 films.
affected by TiO films.
2

Materials 2016, 9, 619

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Figure 3. Typical microscope images of cells detached from substrate (control and thin films annealed
at 300, 500 and 800 C after 48 h culture) and stained with trypan blue for analysis of the ratio of living
(the
cells
with anmicroscope
halo and excluding
blue)
and
dead cells
(the and
cellsthin
stained
in a
Figure
3. Typical
images of the
cellstrypan
detached
from
substrate
(control
filmsblue)
annealed
Figure 3. Typical
microscope
images
of cells
detached
fromorsubstrate
(control
and
thin films annealed
Neubauer
chamber.
The
arrows
show
blue
stained
cells
lack
of
blue
halo
surrounding
the
cell,
at 300, 500 and 800 C after 48 h culture) and stained with trypan blue for analysis of the ratio of living
at 300, 500its
and
800 ability
C after 48
h culture)
andblue.
stained
with trypan
blue the
for four
analysis of the ratio of
indicating
lack
exclude
trypan
Calibration
applies
(the cells with
an of
halo andtoexcluding
the trypan
blue)
and dead
cellsto(the
cells images.
stained blue) in a
living (the cells with an halo and excluding the trypan blue) and dead cells (the cells stained blue) in
Neubauer chamber. The arrows show blue stained cells or lack of blue halo surrounding the cell,
a Neubauer
The
blue2 thin
stained
cells
or lackprepared
of blue halo
surroundingbetween
the cell,
Table
1. Cellchamber.
viability in
thearrows
controlshow
and TiO
films
surfaces
at temperatures
indicating its lack of ability to exclude trypan blue. Calibration applies to the four images.
indicating
of 24,
ability
exclude
trypan blue. Calibration applies to the four images.
300
and 800itsClack
after
48 orto72
h.
Table 1. Cellofviability
in the control and TiOTiO
2 thin films surfaces prepared at temperatures between
Percentage
Cell in
300
TiO2 Films
500
TiObetween
2 Films 800
the control
and TiO2 thin2 Films
films surfaces
prepared
at temperatures
Table 1. Cell viability
Control
300 and
800
C
after
24,
48
or
72
h.
Viability
C
C
C
300 and
800 C after 24, 48 or 72 h.
24
h
(n
=
3)
99.0

0.6
99.2

0.1
99.0

0.5
98.5
0.8800
Percentage of Cell
TiO2 Films 300
TiO2 Films 500
TiO2 Films
Control
C
C
C
Percentage
of
Cell
Viability
Control
TiO
Films
300
TiO
Films
500
TiO
Films
800
48
h
(n
=
3)
99.6

0.06
99.5

0.08
99.3

0.06
99.4

2
2
2
Viability
C
C
C0.02
72
99.1
99.0
0.13
99.2
0.12
= 3)
99.0
0.6
99.2
99.0
0.5
98.599.0
0.80.19
24 h
h24(n
(nh ==(n3)
3)
99.0
0.09
0.6
99.20.1
0.1
99.0
0.5
98.5
0.8
48 h (n = 3)

99.6 0.06

99.5 0.08

Mean 99.5
standard
48 h72(nh =(n3)
99.6
0.08error.
= 3)
99.1
0.06
0.09
99.0 0.13
72 h (n = 3)
99.1 0.09Mean standard
99.0 0.13
error.
2.2. Surface Roughness of TiO2 Thin Films Mean standard error.

99.3 0.06
99.3
0.06
99.2
0.12

99.4 0.02
0.02
99.099.4
0.19

99.2 0.12

99.0 0.19

To characterize
topography
of the TiO2 films, the samples were analyzed by atomic force
2.2. Surface
Roughnessthe
of TiO
2 Thin Films
2.2. Surface Roughness of TiO2 Thin Films
microscopy
(AFM) (JSPM-5200,
JEOL,
in a non-contact mode and processed using
To characterize
the topography
of Tokyo,
the TiOJapan)
2 films, the samples were analyzed by atomic force
To
characterize
the
topography
of
the
TiO
2
films,
the
samples4AC
were
analyzed
by atomic
force
Gwyddion
(Gwyddion,JEOL,
Brno,Tokyo,
Czech Japan)
Republic).
Figure
show
typical
topography
microscopysoftware
(AFM) (JSPM-5200,
in
a non-contact
mode
and
processed
using
microscopy
(AFM)(3D)
(JSPM-5200,
JEOL,
in aarea)
non-contact
mode
and
processed
using
Gwyddion software
(Gwyddion,
Brno,
Czech
Figure
4AC
show
typical
topography
three-dimensional
images (5.5
mTokyo,
5.5 Japan)
Republic).
m scan
of TiO
2 films
annealed
at different
three-dimensional
(3D)
images (5.5
m Czech
without
5.5 m
scan area)
of
TiO
atannealing
different
Gwyddion
software
(Gwyddion,
Republic).
Figure
4AC
show
typical
topography
2 films
temperatures.
The TiO
2 films were Brno,
uniform
voids
when
crystallized
atannealed
the
500 C
C annealing
temperatures.
The
TiO
films
were
uniform
without
voids
when
crystallized
at
the
500
three-dimensional
(3D)2 images
(5.5 m
5.5for m
area)annealing
of TiO2 films
annealedareatpresented
different
temperature.
The calculated
roughness
values
thescan
different
temperatures
temperature. The calculated roughness values for the different annealing temperatures are presented
temperatures.
The TiO2 films were uniform without voids when crystallized at the 500 C annealing
in
2.
in Table
Table 2.
temperature. The calculated roughness values for the different annealing temperatures are presented
A
B
C
in Table 2.
A

Figure
4. AFM
Figure 4.
AFM images
images of
of the
the surface
surface of
of TiO
TiO22 films
films deposited
deposited by
by DC
DC magnetron
magnetron sputtering
sputtering and
and

annealed at different temperatures: (AC:

(AC: 300, 500 and 800 C,
C, respectively).
respectively). The
The images
images show
show that
that
annealed
incrementing
the
annealing
temperature
produces
an
increase
in
the
average
roughness
as
a
result
of
Figure
4.
AFM
images
of
the
surface
of
TiO
2 films
deposited
by
DC
magnetron
sputtering
and
incrementing the annealing temperature produces an increase in the average roughness as a result of
the transformation from the anatase to rutile phase.
annealed
at different
temperatures:
300,
500 and 800 C, respectively). The images show that
the
transformation
from
the anatase (AC:
to rutile
phase.
incrementing the annealing temperature produces an increase in the average roughness as a result of
the transformation from the anatase to rutile phase.

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Table
RoughnessofofTiO
TiO2thin
thin film
film obtained
obtained from
from AFM
AFM measurements.
measurements. Columns
thethe
Table
2. 2.Roughness
Columnsshow
show
2
annealing temperature, root-mean square surface roughness (RMS), roughness average (Ra) and area
annealing temperature, root-mean square surface roughness (RMS), roughness average (Ra) and
of each sample.
area of each sample.

TiO2 films (Temperature)

TiO2 Films
(Temperature)
800
500
800
500
300
300

RMS (nm)
RMS
(nm)
8.08

3.66
8.08
3.66
2.80
2.80

Ra (nm)
Ra (nm)
6.67

2.31
6.67
2.31
2.30
2.30

Area (m2)
2
Area (m
5 5)

5 5 5 5
5 5 5 5
55

The increase in the annealing temperature increased the average roughness up to 8.08 nm (Table

in the annealing
the average
roughness
to 8.08 nm
(Table 2)
2) The
due increase
to a transformation
from temperature
the anatase toincreased
rutile phase
[12,31] as
the X-rayup
diffraction
patterns
due
to
a
transformation
from
the
anatase
to
rutile
phase
[12,31]
as
the
X-ray
diffraction
patterns
for TiO2 thin films annealed at different temperatures show (Figure 5). The X-ray diffraction patternfor
TiOof2 the
thinTiO
films
annealed at different temperatures show (Figure 5). The X-ray diffraction pattern
2 thin film, post-deposition-annealed at 800 C, revealed the coexistence of anatase and
of the TiO2 thin film, post-deposition-annealed at 800 C, revealed the coexistence of anatase and
rutile phases; the intensity of the rutile phase compared to the anatase phase increased as a result of
rutile phases; the intensity of the rutile phase compared to the anatase phase increased as a result of
increment
thethermal
thermalannealing
annealingtreatment.
treatment. A
2 thin film has a
thethe
increment
ofof
the
A low
low RMS
RMSvalue
valuemeans
meansthe
theTiO
TiO
2 thin film has a
dense
and
homogenousstructure.
structure.The
TheTiO
TiO22-anatase
-anatase phase
more
dense
and
homogenous
phasehas
hasaastructure
structurethat
thatisisconsiderably
considerably
more
homogeneous
than
thatofofthe
theTiO
TiO
2
-rutile
phase
Small
clusters
of
increasing
size
were
produced
homogeneous
than
that
-rutile
phase
[32].
Small
clusters
of
increasing
size
were
produced
2
C.
by by
heat
treatment
ofof
temperatures
heat
treatment
temperaturesranging
rangingfrom
from300
300 to
to 800 C.

Figure 5. X-ray diffraction patterns for TiO2 thin films annealed at different temperatures (300, 500
Figure 5. X-ray diffraction patterns for TiO2 thin films annealed at different temperatures (300, 500
and 800 C). X-ray diffraction showed the coexistence of anatase-rutile at 800 C. The increment in
and 800 C). X-ray diffraction showed the coexistence of anatase-rutile at 800 C. The increment in
intensity of the rutile phase over the anatase phase was produced by the increase of the thermal
intensity of the rutile phase over the anatase phase was produced by the increase of the thermal
annealing treatment.
annealing treatment.

The FESEM images were recorded with an acceleration voltage of 2 kV at high vacuum (HV)

The FESEM images were recorded with an acceleration voltage of 2 kV at high vacuum (HV)
using a JEOL SEM model JSM-5610LV (Hitachi, Tokyo, Japan). The films were placed in a specimen
using a JEOL SEM model JSM-5610LV (Hitachi, Tokyo, Japan). The films were placed in a specimen
stub with double-sided adhesive carbon tape, and magnified 40,000 times. Figure 6AC show typical
stub with double-sided adhesive carbon tape, and magnified 40,000 times. Figure 6AC show typical
FESEM
micrographsofofTiO
TiO2thin
thinfilm
filmsurfaces
surfaces obtained
obtained at
from
300
to to
800800
C. C.
FESEM
micrographs
attemperatures
temperaturesranging
ranging
from
300
2
FESEM
measurements
of
the
TiO
2
thin
films
were
performed
both
on
the
surface
and
on
FESEM measurements of the TiO2 thin films were performed both on the surface and on cross-sections.
cross-sections.
All TiO2 films were uniform, smooth, and composed of small and compact grains on the surface
(Figure 6A,B). However, the increase of the temperature during heat treatment caused the formation of

Materials 2016,
Materials
2016, 9,
9, 619
619

6 of
11 11
6 of

All TiO2 films were

surface
were uniform,
uniform,smooth,
smooth,and
andcomposed
composedofofsmall
smalland
andcompact
compactgrains
grainsononthe
the
surface

All2016,
TiO9,
2 films
Materials
619

6 of 11

(Figure 6A,B).
(Figure
6A,B). However,
However, the
the increase
increaseof
ofthe
thetemperature
temperatureduring
duringheat
heattreatment
treatmentcaused
causedthe
theformation
formation
of
clusters
approaching
a
few
hundred
nanometers
in
size
(Figure
6C),
which
coincides
with
the
of
clusters
approaching
a few
hundred
nanometers
in (Figure
size (Figure
6C), which
coincides
with
the
clusters
approaching
a few
hundred
nanometers
in size
6C), which
coincides
with the
results
results
of
AFM
(Figure
7A).
FESEM
imaging
of
a
cross-section
of
the
TiO
2 films shows that their
results
AFM 7A).
(Figure
7A).imaging
FESEMofimaging
of a cross-section
of theshows
TiO2 that
filmstheir
shows
that their
of AFMof
(Figure
FESEM
a cross-section
of the TiO2 films
thickness
had
thickness
had
values
around
300
nm
(Figure
7B).
values around
300 nmaround
(Figure300
7B).
thickness
had values
nm (Figure 7B).
A
A

BB

CC

Figure 6. FESEM images (top view) of TiO2 thin films annealed at different temperatures: (AC: 300,
FESEM images
images (top
(top view)
view) of
of TiO
TiO2 thin
thin films
films annealed
annealed at
at different
different temperatures:
temperatures: (AC:
(AC: 300,
300,
Figure 6. FESEM
500 and 800 C, respectively). Grain size of2 the TiO2 films increased with the rise of the annealing
500 and
and800
800C,C,
respectively).
size
the
TiO2increased
films increased
of the
500
respectively).
GrainGrain
size of
theof
TiO
2 films
with the with
rise ofthe
therise
annealing
temperature.
annealing temperature.
temperature.

A
A

B
B

Figure 7. Analysis of TiO2 thin films annealed at 300 C. (A) AFM 3D image (top view) of TiO2 films
Figure
7. Analysis
TiO2area).
thin films
annealed
C. (A)
3D image
(top view)
of TiO
films
(1.5 1.5
0.084 mof3 scan
The gray
scale at
to 300
the right
of AFM
the image
represents
the values
on2 the
Figure 7. Analysis of3 TiO2 thin films annealed at 300 C. (A) AFM 3D image (top view) of TiO2 films
(1.5
1.5
0.084
mthe
scan
area).
The
gray
scale
to
the
right
of
the
image
represents
the
values
on
the
Z axis:
white
being
maximum
value
(84
nm)
and
black
the
minimum
(0
nm);
(B)
FESEM
cross(1.5 1.5 0.084 m3 scan area). The gray scale to the right of the image represents the values on
Z
axis: of
white
the maximum
(84 nm)ofand
nm);times.
(B) FESEM
crosssection
TiO2being
film recorded
with anvalue
acceleration
5 kVblack
at HVthe
andminimum
amplified(0
25,000
The image
the Z axis: white being the maximum value (84 nm) and black the minimum (0 nm); (B) FESEM
shows that
TiO
2 films
had homogenous
thickness of about
section
of TiO
2 film
recorded
with an acceleration
5 kV at300
HVnm.
and amplified 25,000 times. The image
cross-section of TiO2 film recorded with an acceleration of 5 kV at HV and amplified 25,000 times.
shows that TiO2 films had homogenous thickness of about 300 nm.
The image shows that TiO2 films had homogenous thickness of about 300 nm.

3. Discussion
3. Discussion
Titanium dioxide is widely used in medical applications due to its excellent biocompatibility
3. Discussion
and Titanium
good mechanical
Crystalline
TiO
2 occurs in due
three
anatase,
rutile, and
dioxide strength
is widely[6].
used
in medical
applications
to phases:
its excellent
biocompatibility
Titanium dioxide is widely used in medical applications due to its excellent biocompatibility and
brookite.
anatase and
rutile have
the capability
to2form
bioactive
hydroxyl
apatite
layers
in vitro
and
goodBoth
mechanical
strength
[6]. Crystalline
TiO
occurs
in three
phases:
anatase,
rutile,
and
good mechanical strength [6]. Crystalline TiO2 occurs in three phases: anatase, rutile, and brookite.
and
have
good
biocompatibility
[6].
As
a
result
of
its
compatibility,
the
rutile
and
anatase
TiO
2
brookite. Both anatase and rutile have the capability to form bioactive hydroxyl apatite layers in vitro
Both anatase and rutile have the capability to form bioactive hydroxyl apatite layers in vitro and have
surfaces
serve
as substrates [6].
for As
growing
different
cell types [2,3336].
Neurons
from the
and
havecan
good
biocompatibility
a result
of its compatibility,
the rutile
and anatase
TiO2
good biocompatibility
[6]. As
a result
of its
compatibility,
the rutile
and
anatase
TiO2 surfaces
can
mammalian
central
nervous
system
(CNS)
have
a
good
survival
rate
on
TiO
2
film
surfaces
for
up
surfaces can serve as substrates for growing different cell types [2,3336]. Neurons from to
the
serve
as
substrates
for
growing
different
cell
types
[2,3336].
Neurons
from
the
mammalian
central
10 days in culture;
surfaces
offer
good
adherence
axonal
cultured
rat cortical
mammalian
centralrutile
nervous
system
(CNS)
have
a good and
survival
rategrowth
on TiOof
2 film
surfaces
for up to
nervous [34].
system
(CNS) have
a good
survival
rate on
TiO
for up to 10maintain
days in culture;
2 film surfaces
neurons
Moreover,
has
alsooffer
been
reported
that
hepatocytes
their
10
days in culture;
rutileitsurfaces
good
adherence
and axonalproliferate
growth of and
cultured rat cortical
rutile
surfaces
offer
good
adherence
and
axonal
growth
of
cultured
rat
cortical
neurons
[34]. Moreover,
metabolic
activity
in
long-term
culture
on
rutile
and
anatase
TiO
2
[2,36,37].
neurons [34]. Moreover, it has also been reported that hepatocytes proliferate and maintain their
it hasWe
alsoassessed
been reported
that hepatocytes
proliferate
and maintain
their
metabolic
in long-term
physical
properties
theand
possible
cytotoxic
effect
of TiO2activity
thin films
in their
metabolic
activitythe
in long-term
culture onand
rutile
anatase
TiO2 [2,36,37].
culture
on
rutile
and
anatase
TiO
[2,36,37].
2
crystalline
forms, anatase
and anatase/rutile,
using
CHO-K1 cytotoxic
cells that were
culture
We assessed
the physical
properties and
the possible
effectmaintained
of TiO2 thininfilms
in for
their
We assessed the physical properties and the possible cytotoxic effect of TiO2 thin films in their
crystalline forms, anatase and anatase/rutile, using CHO-K1 cells that were maintained in culture for
crystalline forms, anatase and anatase/rutile, using CHO-K1 cells that were maintained in culture for
24, 48 or 72 h on TiO2 thin film surfaces. The MTT and trypan blue assays indicated that CHO-K1

Materials 2016, 9, 619

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cells grew equally well on TiO2 thin films as on the control substrate, pointing out that the TiO2 thin
films did not affect cell viability or proliferation. In addition, these cells were viable and functionally
similar to those grown on the control substrate. The MTT assay demonstrated they had normal
mitochondrial function. These results are consistent with previous data where dorsal root ganglion
neurons from the rat were maintained in culture for 18 and 24 h on TiO2 thin films retaining their
normal electrophysiological properties, proving they were viable and functionally similar to those
grown on the control substrate [12]. In contrast to neuronal cultures where cells do not reproduce,
the use of CHO-K1 cells added information about the proliferation and metabolic capabilities of living
cells on TiO2 films.
Cell viability on TiO2 thin films was similar to that on the control substrate after 24, 48 or
72 h. The cell count and optical density on the control substrate and on all TiO2 films after 24 h
were significantly lower than those after 48 and 72 h, which shows cell proliferation in these
cultures. However, the cell number and optical density after 48 h were similar to those found at
72 h. This indicates that cells grow steadily until they occupy all the available growth surface; after
48 h in culture, they stop their proliferation both on the control substrate and on TiO2 thin films.
After cells are seeded it takes them around 1224 h to recover from trypsinization (i.e., reconstruct
their cytoskeleton, secrete matrix to aid attachment, and spread out on the substrate) which enables
them to reenter the cell cycle. Later on, cells enter their proliferative phase which ends when all the
growth surface is occupied or the culture medium exhausted [38]. This explains the lack of increase in
cell number after 48 and 72 h in culture. However, our results have shown that TiO2 thin films were
not cytotoxic in culture even after 72 h.
Increased cellular proliferation, adhesion and greater efficiency in promoting apatite formation
were observed in osteoblasts cultured on TiO2 nanotubes annealed at 600 C in contrast to those grown
on nanotubes annealed at other temperatures. The results indicated that tubes annealed to a mixture of
anatase and rutile were clearly more efficient than those in their amorphous or plain anatase state [39].
It has been suggested that under this condition, TiO2 nanotubes promoted greater cell adhesion and
cell proliferation due to their crystalline structure and its morphology, and this would have a common
influence on the apatite growth, thereby improving the bioactivity of TiO2 nanotubes annealed at
600 C [39]. In our results cell cultures grown on TiO2 thin films annealed at 800 C produced higher
optical density and a larger number of living cells after 72 h, which suggests that at an annealing
temperature of 800 C, the changes in surface morphology and the ratio of anatase to rutile on the TiO2
thin films are optimal, among the conditions tested, for the viability and proliferation of CHO-K1 cells.
4. Materials and Methods
4.1. TiO2 Thin Films
TiO2 thin films were deposited on a quartz substrate at room temperature by direct current (DC)
magnetron sputtering using a titanium target with a diameter of 50.8 mm. A TiO2 ceramic material
was located on 20% of the titanium target surface; both materials with a purity of 99.99%. The quartz
substrate was cleaned in an ultrasonic bath of acetone (C3 H6 O), ethanol (C2 H6 O), and distilled water
during 5 min at room temperature; this procedure was repeated four times. The TiO2 thin film
deposition was made under an Argon (Ar) atmosphere and a chamber pressure of 7.46 106 mBar.
Argon flow was kept to 15 standard cubic centimeters per minute (sccm) during the TiO2 deposition by
DC magnetron sputtering. The power supply and substrate temperature were controlled to 100 W and
25 C, respectively. The TiO2 films were then subjected to thermal-annealing treatment to achieve their
phase transformation. For this, a thermo scientific thermolyne muffle furnace (model F48025-60-80,
Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to keep the temperature constant for
one h in each heat treatment. The duration of each heat treatment was lower than that reported
elsewhere [40], yet it was enough to reach the required recrystallization and transformation phases.

Materials 2016, 9, 619

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Lastly the TiO2 thin films were post-deposition annealed at different temperatures (300, 500 and 800 C)
to the anatase to rutile phase transformation.
The physical properties of the thin films, including film thickness and phase structure, strongly
depend on the deposition technique and growth parameters. Therefore, the dependence of the surface
morphology and cross-section formation of the TiO2 thin films, prepared with different annealing
temperatures, were analyzed by Field Emission Scanning Electron Microscopy (FESEM) and Atomic
Force Microscopy (AFM).
4.2. Cell Culture
The substrates for cell culture (TiO2 films and control glass) were rinsed with deionized water
and dried on flat paper towels in a laminar flow hood for 30 minutes. Once dry, the substrates were
sterilized by UV light irradiation during 20 minutes. CHO-K1 cells were seeded on the substrates and
cultured in Dulbeccos Modified Eagle's medium (DMEM) supplemented with 10% Fetal Bovine Serum
(FBS), 1% L-glutamine, 1% Pyruvate, and 1% penicillin/streptomycin (all of these substances were
purchased from Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA). The cells plated on these
substrates were incubated in 55 cm2 culture dishes (Sigma-Aldrich, St. Louis, MO, USA) in a humidified
incubator at 37 C with 95% air and 5% CO2 . The cells were grown to 80% confluence and dissociated
with 2.5% trypsin (Gibco) at 37 C for 2 min to obtain complete cell detachment. Then, 4 mL of culture
medium, supplemented with FBS to inactivate the trypsin, was added. The cell suspension was
centrifuged at 1500 revolutions per minute (rpm) for 5 min; after this, the supernatant culture medium
was removed and the cell pellet was suspended with 4 mL of fresh culture medium [4143]. Finally,
about 1 106 cell/mL of cell suspension were plated on control (standard borosilicate coverslip),
Triton-X control (borosilicate coverslip plus 1% Triton X) and various TiO2 thin films. These cells were
incubated for 24, 48 or 72 h in an atmosphere of 95% air and 5% CO2 at 37 C before assay.
4.3. MTT Cytotoxicity Assay
Following incubation, the culture medium was renewed and the cells were incubated with
0.5 mg/mL MTT (SigmaAldrich, St. Louis, MO, USA) for 4 h in an atmosphere of 95% air and
5% CO2 at 37 C. After this time, 85% of the culture media (1.7 mL) was replaced with dimethyl
sulfoxide (DMSO) (Sigma-Aldrich) 1.7 mL in each well; this procedure destroys the cells and
releases the formazan derived from MTT. The concentration of dissolved formazan crystals was
spectrophotometrically quantified in a microplate reader at a wavelength of 570 nm (Epoch Microplate
Spectrophotometer, BioTek, and Winooski, VT, USA). All experiments were done for at least three
times and results expressed as the mean optical density standard error. The surviving fraction of
cells was calculated for each assay as the percentage of cell viability = (optical density test sample) /
(optical density control sample) 100 [23,43,44]. One-way ANOVA and Duncan's multiple range
post-test were used for all comparisons between the control, Triton-X control and TiO2 thin films,
considering as significant a p < 0.05 (although P-values lower than 0.01 were included since it means a
larger statistical significance level).
4.4. Trypan Blue Exclusion Assay
After cell incubation, the cell pellet was suspended in 200 L of PBS from which 100 L were
obtained and placed in 100 L trypan blue 0.4% (Sigma-Aldrich) solution for staining. To determine
the cell number on the control substrate and on the thin films, both sides of an hemocytometer were
loaded with 10 L of the suspension and four corners and the middle squares of each side counted.
The number of stained and unstained cells was determined. The unstained cell count was taken as
a measure of viable cells. Given that each square of the hemacytometer has a surface area of 1 mm2
and a depth of 0.1 mm, its volume is 0.1 mm3 . Since 1 cm3 is approximately equal to 1 mL, the cell
concentration/mL is the average count per square 104 . The number of living and dead cells was
counted on a Leica DM1000 light microscope (Leica Microsystems Inc., Wetzlar, Germany) using digital

Materials 2016, 9, 619

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images obtained with a digital camera ProgRes C10 plus (Jenoptik, Thuringia, Germany) and the
associated software ProgRes Capture Pro 2.1 (Jenoptik, Jena, Germany).
All the experiments were performed in three independent series, and each figure represents data
from 10 independent counts from different samples. The percentage of cell viability was estimated as
unstained cells/total cells (stained and unstained) 100 [27]. To compare the results of the control,
Triton-X control, and TiO2 thin films, one-way ANOVA and Duncan's multiple range post-test were
used, considering as significant a p < 0.05 (although P-values lower than 0.01 were included since it
means a larger statistical significance level).
5. Conclusions
These results confirm the feasibility to use TiO2 thin films in the crystalline form of anatase and
rutile phase as substrates for cell culture. These films allowed the survival and proliferation of CHO-K1
cells. Our results confirmed the in vitro biocompatibility of TiO2 thin films, proving that the survival
of CHO-K1 cells and dorsal root ganglion neurons was similar; there is also the fact that proliferative
and metabolic cell activity were maintained for at least 72 h. Further work will include the study
of the biocompatibility of TiO2 thin films in vivo, and the study of the mechanical properties and
nanoindentation that will explore the possibility of utilizing TiO2 thin films on microelectrode surfaces
and to include the readout circuit to construct a CMOS-MEMS device that might allow the recording
of relevant biological parameters such as micro-potentials caused by pH changes.
Acknowledgments: This work was financed by Secretara de Educacin Pblica (SEP) Programa de
Mejoramiento del Posgrado (PROMEP) posdoctoral grant DSA/103.5/14/5782 to Blanca Cervantes, grant
PROMEP-RED Estudio de dispositivos electrnicos y electromecnicos con aplicacin en fisiologa y
optoelectrnica and Programa Institucional de Fortalecimiento al Posgrado (PIFI) 2013-2014, by Centro
Universitario de Vinculacin y Transferencia de Tecnologa de la Benemrita Universidad Autnoma de Puebla
(BUAP) grants DITCo32 and DITCo 2016-9 to Enrique Soto, and by National Council of Science and Technology of
Mxico (CONACyT) grant CA Neurociencias 229866. Authors thank Jesua Roberto Bueno Gasca for proofreading
the English text.
Author Contributions: Francisco Lpez-Huerta, Agustn L. Herrera-May, Julin Hernndez-Torres and Leandro
Garca-Gonzlez prepared and characterized the TiO2 films; Blanca Cervantes and Octavio Gonzlez realized
the MTT and trypan blue assays; Blanca Cervantes, Rosario Vega and Enrique Soto prepared the figures;
Blanca Cervantes, Francisco Lpez-Huerta, Rosario Vega, Agustn L. Herrera-May, Emilio Salceda and
Enrique Soto wrote and corrected the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations
The following abbreviations are used in this manuscript:
AFM
CMOS-MEMS
CHO-K1
DC
FESEM
MMT
RPM
RMS
SCCM
TiO2

Atomic Force Microscopy

Complementary Metal Oxide Semiconductor and Micro-Electromechanical Systems
Chinese hamster ovary
direct current
Field Emission Scanning Electron Microscopy
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
revolution per minute
root-mean square surface roughness
standard cubic centimeters per minute
Titanium Dioxide

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