A. KOCIJAN et al.: COMPARATIVE STUDY OF SUPERHYDROPHILIC AND SUPERHYDROPHOBIC TiO2/EPOXY ...
355–361
COMPARATIVE STUDY OF SUPERHYDROPHILIC AND
SUPERHYDROPHOBIC TiO
2
/EPOXY COATINGS ON AISI 316L
STAINLESS STEEL: SURFACE PROPERTIES AND
BIOCOMPATIBILITY
PRIMERJAVA SUPERHIDROFILNIH IN SUPERHIDROFOBNIH
TiO
2
/EPOKSI PREVLEK NA NERJAVNEM JEKLU AISI 316L:
POVR[INSKE LASTNOSTI IN BIOKOMPATIBILNOST
Aleksandra Kocijan
1
, Marjetka Conradi
1
, Matej Ho~evar
1
, Damjana Drobne
2
1
Institute of Metals and Technology, Lepi pot 11, SI-1000 Ljubljana, Slovenia
2
Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia
aleksandra.kocijan@imt.si
Prejem rokopisa – received: 2017-11-15; sprejem za objavo – accepted for publication: 2018-01-09
doi:10.17222/mit.2017.190
TiO2/epoxy coatings were successfully applied to the surface of AISI 316L stainless steel to change the wetting properties, with
the aim being to improve the biocompatibility of the superhydrophobic/superhydrophilic surfaces. Contact-angle measurements
were used to evaluate the wetting properties of the non-coated, epoxy-coated, as-received TiO2/epoxy-coated and fluoroalkyl-
silane (FAS)-TiO2/epoxy-coated substrates. The as-received TiO2/epoxy coating and FAS-TiO2/epoxy coating showed super-
hydrophilic and superhydrophobic characteristics, respectively. The average surface roughness (Sa) of the superhydrophobic
surface was higher compared to the superhydrophilic surface due to the formation of agglomerates. The biocompatibility
evaluated by cell attachment showed that AISI 316L stainless steel with a hydrophilic nature and low Sa is the most favourable
surface for bone osteosarcoma cells (MG-63) growth. On the other hand, the two limiting cases of surfaces, superhydrophilic
and superhydrophobic coatings with increased roughness compared to AISI 316L, showed lower biocompatibility.
Keywords: stainless steel, epoxy coating, TiO
2, wetting properties, biocompatibility
Na povr{ino nerjavnega jekla tipa AISI 316L smo uspe{no nanesli TiO2/epoksi prevleke z namenom regulacije omo~itvenih
lastnosti, ki vplivajo na izbolj{anje biokompatibilnosti superhidrofobnih/superhidrofilnih povr{in. Z meritvami kontaktnih kotov
smo dolo~ili omo~itvene lastnosti ~iste jeklene povr{ine ter jeklene povr{ine prevle~ene z epoksidno smolo, s TiO2/epoksi
prevleko in fluoroalkilsilan (FAS)-TiO2/epoksi prevleko. TiO2/epoksi prevleka je pokazala superhidrofilno, FAS-TiO2/epoksi
prevleka superhidrofobno naravo. Povpre~na hrapavost povr{ine (Sa) superhidrofobne povr{ine je bila vi{ja kot na
superhidrofilni povr{ini zaradi opa`ene tvorbe aglomeratov. Analiza biokompatibilnosti je pokazala, da je nerjavno jeklo AISI
316L s hidrofilno naravo in nizko Sa najprimernej{a povr{ina za rast kostnega osteosarkoma (MG-63). Po drugi strani pa sta oba
mejna primera prevlek s superhidrofilno in superhidrofobno naravo z ve~jo hrapavostjo v primerjavi z AISI 316L, pokazala
manj{o stopnjo biokompatibilnosti.
Klju~ne besede: nerjavno jeklo, epoksi prevleka, TiO2, omo~itvene lastnosti, biokompatibilnost
1 INTRODUCTION
The major issues in the biomedical applications of
stainless steels are related to understanding the relation-
ship between the surface properties of the material and
the cellular responses, accompanied by the risk of micro-
bial infections.
1
The interaction of nanoscale surface
topographies with cells was proven to play a crucial role
in the biocompatibility of implants.
2
Various nanoscale
surface modifications have been proposed in order to
enhance the biocompatibility and antibacterial activity of
medical implants.
3
Epoxy resins are widely used as
long-term protective coatings for stainless steels in
various applications.
4,5
However, their susceptibility to
abrasion
6
and poor resistance to crack propagation, due
to a highly cross-linked structure
7
, have inspired major
research efforts to develop improved nanostructured
composite coatings by adding various inorganic nano-
fillers such as silica
8–10
or TiO
2
.
11,12
The unique surface
properties and high surface energy of the nanomaterials
provide additional available sites for protein adsorption,
which enhances the interaction between the cells and the
implant.
13
K. Webb et al.
1
reported that hydrophilic
surfaces promote significantly greater cell attachment,
cell spreading, and cytoskeletal organization compared
to hydrophobic surfaces. D. S. Kommireddy et al.
14
studied super-hydrophilic TiO
2
nanoparticle thin films
prepared by using a layer-by-layer nano-assembly
method. The biocompatibility of the coating was demon-
strated by the successful attachment of human dermal
fibroblast cell culture. K. Mehrabi et al.
15
reported that
the presence of nanoscale features on the surface of
implants enhanced the growth and attachment of
osteoblast bone-forming cells. Such enhancement is due
to an increase in the surface area that provides more area
for the cell–substrate interaction, more surface energy,
Materiali in tehnologije / Materials and technology 52 (2018) 3, 355–361 355
UDK 620.1:667.648.2:62-4 ISSN 1580-2949
Original scientific article/Izvirni znanstveni ~lanek MTAEC9, 52(3)355(2018)

more protein adsorption, integrin clustering, and as a
result, higher cell adhesion. K. Anselme et al.
16
reported
on the influence of topography, chemistry and surface
energy on osteoblast/material interaction. These surface
characteristics determine how the biological molecules
will adsorb to the surface and more particularly deter-
mine the orientation of the adsorbed molecules. K. Das
et al.
17
showed that osteoblast proliferation was signi-
ficantly higher on an anodized nanotube surface.The
surface properties changed with the emergence of
nanoscale morphology. The higher nanometer-scale
roughness, low contact angle and high surface energy in
the nanoporous surface enhanced the osteoblast-material
interactions. A growing number of studies report that a
topographical modification of the cell-substrate interface
is a significant regulator of cellular adhesion and
function.
18
The future surface modifications will go in
the direction of controlling cellular interactions, with the
aim being to enhance tissue regeneration, to regulate
cellular adhesion and their differentiation.
19
In the present study we focus on the biocompatibility
of epoxy coatings on AISI 316L stainless steel by adding
as-received and surface-modified TiO
2
nanoparticles
with an emphasis on tuning the wetting properties bet-
ween the two limiting cases, i.e., superhydrophobic/
superhydrophilic coatings. The surface morphology,
surface roughness, contact-angle and surface energy, as
well as the biocompatibility of as-received and sur-
face-modified TiO
2
/epoxy coatings on the AISI 316L
stainless steel, were evaluated and compared with the
characteristics of bare and epoxy-coated AISI 316L
stainless steel.
2 MATERIALS AND METHODS
Materials. • Austenitic stainless steel AISI 316L
(17 % Cr, 10 % Ni, 2.1 % Mo, 1.4 % Mn, 0.38 % Si,
0.041 % P, 0.021 % C, < 0.005%Si nmass fraction)
was used as the substrate.
A two-component USP Class VI biocompatible
epoxy EPO-TEK 302-3M (EPOXY TECHNOLOGY,
Inc.) was mixed in the % of mass fractions ratio 100:45.
TiO
2
nanoparticles with mean diameters of 30 nm were
provided by Cinkarna Celje, whereas the 300-nm
particles were supplied by US Research Nanomaterials,
Inc.
Surface modification of TiO
2
. • The TiO
2
particles
were functionalized in1%o fv olume fractions of
fluoroalkylsilane or FAS17 (C
16
H
19
F
17
O
3
Si) ethanol
solution. The solution was shaken for a few minutes and
left overnight prior to use in the experiments.
Steel substrate preparation. • The steel sheet with a
thickness of 1.5 mm was cut into discs of 25-mm
diameter. The steel discs were grinded mechanically with
SiC emery paper (up to 4000 grit), diamond polished (up
to 1 μm), ultrasonically cleaned with ethanol and dried in
warm air.
Coating preparation. • Prior to the TiO
2
nanoparticle
adsorption, the diamond-polished AISI 316L substrate
was spin-coated with a 300-nm layer of epoxy (as
determined by ellipsometry) and then cured for1ha t
70 °C and post-cured at 150 °C for another hour.
20
We
decided on a thin base epoxy layer to improve the TiO
2
nanoparticle adhesion as the oxide layer growing on the
surface of the clean AISI 316L substrate prevents adhe-
sion and is very difficult to remove. The nanoparticles
were further applied to the epoxy-coated AISI 316L sur-
face by spin-coating 20 μL of3%ofmass fractions of
TiO
2
nanoparticle ethanol solution. We consecutively
applied three deposits of dual-size, dual-layer coating
consisting of 30-nm and 300-nm FAS-TiO
2
nano-
particles. The coating was then dried in an oven for
approximately 30 min at 100 °C. The same procedure
was repeated with the as-received, non-functionalized
TiO
2
nanoparticles.
Surface roughness. • An optical 3D metrology sys-
tem, Alicona Infinite Focus (Alicona Imaging GmbH),
was employed for the surface-roughness analysis. At
least three measurements per sample were performed at a
magnification of 20× with a lateral resolution of 0.9 μm
and a vertical resolution of about 50 nm. IF-Measure-
Suite (Version 5.1) software was used for the roughness
analysis. The software offers the possibility to calculate
the average surface roughness, S
a
, for each sample, based
on the general surface-roughness Equation (1):
Sa
LL
zxy xy
xy
L L y x
=
∫ ∫
11
0 0
(,)dd (1)
where L
x
and L
y
are the acquisition lengths of the sur-
face in the x and y directions and z(x,y) is the height.
The size of the analyzed area was 714×542 μm
2
.T o
level the profile, corrections were made to exclude the
general geometrical shape and possible measurement-
induced misfits.
Contact-angle measurements. • The static contact-
angle measurements of water (W) on a clean AISI 316L
diamond-polished sample, on the epoxy-coated AISI
316L substrate, on the TiO
2
/epoxy-coated AISI 316L
substrate and on the FAS-TiO
2
/epoxy-coated AISI 316L
substrate were performed using a surface-energy evalu-
ation system (Advex Instruments s.r.o.). Liquid drops of
5 μL were deposited on different spots of the substrates
to avoid the influence of roughness and gravity on the
shape of the drop. The drop contour was analysed from
the image of the deposited liquid drop on the surface and
the contact angle was determined using Young-Laplace
fitting. To minimize the errors due to roughness and
heterogeneity, the average values of the contact angles of
the drop were calculated approximately 30 s after the
deposition from at least five measurements on the
studied coated steel. All the contact-angle measurements
were carried out at 20 °C and ambient humidity. As the
contact angles were only available for water, an
A. KOCIJAN et al.: COMPARATIVE STUDY OF SUPERHYDROPHILIC AND SUPERHYDROPHOBIC TiO2/EPOXY ...
356 Materiali in tehnologije / Materials and technology 52 (2018) 3, 355–361

equation-of-state approach
21
was used to calculate the
corresponding surface energies.
Scanning Electron Microscopy (SEM). • SEM anal-
ysis using a FE-SEM JEOL JSM-6500F was employed
to investigate the surface morphology of the coatings as
well as the morphology and distribution of the attached
cells after a biological evaluation. The samples were
sputtered with gold prior to the imaging. The thickness
of the coatings was determined by cross-section SEM
images. The specimen cross-sections for SEM were
prepared using a JEOL Cross Section Polisher
SM-09010.
Biocompatibility evaluation. • Fast-growing bone
osteosarcoma cells (MG-63; (ATCC® CRL-1427™)
were used for the assessment of cytocompatibility/bio-
compatibility on sterilized epoxy, epoxy-TiO
2
and
stainless-steel samples (given/obtained by the Krasteva
Institute of Biophysics and Biomedical Engineering,
Bulgarian Academy of Sciences, Sofia, Bulgaria). MG63
cells were cultured under controlled conditions (37 °C,
5%CO
2
, high humidity) in Dulbecco’s modified Eagle’s
medium (DMEM), supplemented with etal bovine serum
(FBS; 10 %, v/v) and 4-mM L-glutamine (all the
reagents were purchased from Sigma-Aldrich, Stein-
heim, Germany). Cells were routinely subcultured once a
week or when they reached 65–70 % confluence. Before
harvesting with Trypsin-0.25 % EDTA (Sigma-Aldrich,
Steinheim, Germany) for approximately 10 minutes at
37 °C, the cells were washed three times with Phosphate
Buffered Saline without Ca
2+
and Mg
2+
(PBS; Sigma-
Aldrich, Steinheim, Germany). The cells were then
re-suspended in the growth medium, centrifuged at 200 g
for 5 min, and plated at a seeding density of3×1 0
4
cells/cm
2
in 6-well plates (Sigma-Aldrich, TPP ®,
Steinheim, Germany) containing epoxy, epoxy-TiO
2
and
stainless-steel samples, all in 3 mL of growth medium
( 2 7×1 0
4
cells/well) and incubated for 24 h under
controlled laboratory conditions (5 % CO
2
/95 % air at
37 °C). Then, the cells were further processed for SEM
fixation. After 24 h of incubation, the cell-culture
medium was removed, cells were washed three times
with PBS and fixed for 24 h at 4 °C using a modified
Karnovsky fixative, composed of 2.5 % glutaraldehyde
(SPI Supplies, West Chester, PA, USA) and 0.4 %
paraformaldehyde (Merck KGaA, Darmstadt, Germany)
in a 0.1-M Na-phosphate buffer (NaH
2
PO
4
2·H
2
O and
Na
2
HPO
4
2·H
2
O; all the chemicals from Merck KGaA,
Darmstadt, Germany). Then the samples were washed in
t h eb u f f e rf o r3×1 0m i na n dpost-fixated with 1 %
osmium tetroxide (OsO
4
) (SPI Supplies, West Chester,
P A,USA;1×60min), followed by washing in the buffer
for 3 × 10 min. The samples were dehydrated through an
alcohol gradient (30–100 %) (EtOH; Merck KGaA,
Darmstadt, Germany) with each step lasting 10 min.
Further dehydration steps were conducted with a mixture
of Hexamethyldisiloxane (HMDS; SPI Supplies, West
Chester, PA, USA) and absolute EtOH (1:1; v/v; 10
min), 3:1 (HMDS: absolute EtOH, v/v; 10 min) and
absolute HMDS (10 min) and HMDS (2 h), which was
finally left to evaporate. The SEM was used to visualize
the distribution, the number and the shape of the attached
M63 cells.
Optical Microscopy. • Optical microscope Microphot
FXA (Nikon, Japan) with an Olympus DP73 camera was
used to visualise the number and the distribution of the
attached MG63 cells. Twenty images from different
fields of view were randomly taken per sample, and each
of the four different surfaces was investigated in tripli-
cate. The software program Stream Motion was used to
quantify (the percentage of coverage) the number and
distribution of adhered cells and the results are shown as
the arithmetic mean ± the standard deviation. The diffe-
rence in the median-measured parameters in the exposed
and unexposed groups was tested with the non-para-
metric Mann-Whitney U test. All the calculations were
performed with Statgraphics Plus 4.0. All the data were
A. KOCIJAN et al.: COMPARATIVE STUDY OF SUPERHYDROPHILIC AND SUPERHYDROPHOBIC TiO2/EPOXY ...
Materiali in tehnologije / Materials and technology 52 (2018) 3, 355–361 357
Figure 1: SEM images of surface morphology of: a) as-received TiO
2
/epoxy coatings on AISI 316L stainless steel and b) FAS-TiO
2
/ epoxy
coatings on AISI 316L stainless steel; the zoomed-out insets show the significant morphology difference between the two coatings

submitted for an analysis of variance (ANOVA) and
Duncan’s multiple range test, where appropriate.
3 RESULTS AND DISCUSSION
3.1 Surface properties
The morphology of the as-received TiO
2
/epoxy and
FAS-TiO
2
/epoxy coatings was compared by SEM
imaging (Figure 1). The SEM images reveal a difference
in the morphology between the two coatings, which is
mostly reflected in the different length scales of the
average size of the nanoparticle agglomerates. FAS
functionalization apparently does not homogenize the
particle distribution as the formation of large agglome-
rates up to a few tens of microns is observed during the
deposition of the FAS-TiO
2
nanoparticles (Figure 1b). In
contrast, in the as-received TiO
2
nanoparticle coatings,
the nanoparticles are more finely dispersed and agglome-
rates of the order of only a few microns are observed
(Figure 1a). This is confirmed by the average surface-
roughness measurements, which are reported in Table 1.
To analyse the surface wettability, we performed five
static contact-angle measurements with water (W) on
different spots all over the sample and to calculate the
corresponding surface energy. The average surface-
roughness parameter was used to evaluate the morphol-
ogy difference between the two coatings under investiga-
tion.
Table 1: Surface properties of diamond-polished AISI 316L, epoxy
coated AISI 316L and TiO
2
coatings prepared from FAS-TiO
2
and
as-received TiO
2
nanoparticles: static water contact angles ( W
), the
corresponding surface energies ( ) and the average surface roughness
(S
a
). All data are reported as mean ± standard deviation (n=5)
Substrate q
W
(°) g (mN/m) S
a
(nm)
0065 AISI 316L 57±1 49±5 31±4
epoxy coated AISI 316L 71±1 41±3 50±5
TiO 2/epoxy coated AISI
316L
< 5 72±7 423±20
FAS-TiO 2/epoxy coated
AISI 316L
160±3 0.24±0.02 618±30
With the contact-angle measurements it was con-
firmed that we were able to synthesise two limiting cases
of coatings concerning their wetting characteristics. The
FAS-TiO
2
/epoxy coating was superhydrophobic and the
as-received TiO
2
/epoxy coating was superhydrophilic.
As the contact angles were only available for water,
an equation-of-state approach
21,22
was used to calculate
the surface energies with the Equation (2):
cos
()
   
=− +
−−
12
2
s
l
sl
e
b
(2)
For a given value of the surface tension of a probe
liquid  l
(i.e., for water  l
= 72.8 mN/m)
23
and  W
measured on the same solid surface, the constant  and
the solid surface-tension s
values were determined using
the least-squares analysis technique. For the fitting with
equation (2), a literature value of  = 0.0001234
(mJ/m
2
)
–2
was used, as weighted for a variety of solid
surfaces.
21,22
The calculated values of the solid surface
energy are listed in Table 1 and confirm the two limits of
the superhydrophobic and the superhydrophilic surface.
The measured average surface roughness indicates a
slight difference in the average surface-roughness
parameter S
a
(Table 1) between the FAS-TiO
2
/epoxy and
the as-received TiO
2
/epoxy coatings, which is in agree-
ment with the surface morphology evaluated with the
SEM. The rougher FAS-TiO
2
/epoxy surface is covered
with large agglomerates, whereas in the as-received
TiO
2
/epoxy coatings the nanoparticles are more finely
dispersed, resulting in a reduced roughness (Table 1 and
Figure 1).
As a reference we also measured the contact angles,
surface energies and average surface roughness on
diamond-polished AISI 316L and epoxy-coated AISI
316L.
3.2 Biocompatibility
Biomaterials are never truly inert, but they are bio-
tolerable to different degrees. The cyto-compatibility of a
material can be assessed in vitro by observing the growth
and differentiation that is manifested by the cellular
density and morphological characteristics of cells grown
on specific surfaces. After the initial cell attachment, the
cells are transformed with time: a decrease in the cell
widths and an increase in the cell lengths. The time
dependence of the cell morphological transition could be
explained in terms of the re-organization of focal
contacts and the intracellular cytoskeleton.
24
The cytocompatibility/biocompatibility of the AISI
316L stainless steel, the epoxy-coated AISI 316L, the
as-received TiO
2
/epoxy coating on AISI 316L and the
A. KOCIJAN et al.: COMPARATIVE STUDY OF SUPERHYDROPHILIC AND SUPERHYDROPHOBIC TiO2/EPOXY ...
358 Materiali in tehnologije / Materials and technology 52 (2018) 3, 355–361
Figure 2: SEM images of MG63 cells attached to AISI 316L
stainless-steel surface: a) distribution, attachment pattern and shape of
the cells, b) flatten cells with one or two longer protrusions, c) round-
shaped cell has many outgrowths in various shapes, mostly tubular or
vesicular structures of up to 2 μm in size

FAS-TiO
2
/epoxy coating on AISI 316L were evaluated
using the attachment of bone osteosarcoma cells.
In the case of bare AISI 316L stainless steel (Fig-
ure 2), the cells were randomly distributed over the sur-
face of the specimens and half of the surface was
covered with attached cells (Table 2). The majority of
the cells were 50–100 μm. The morphology of the cells
was 95 % polygonal, with less than 5 % being round
shaped. They differed in terms of size and the degree of
flatness. The flatter the cell, the larger the area they were
occupying. Most of the cells had one or two longer pro-
trusions. Some cells had one very long protrusion, which
was even longer than the entire cell and could reach up
to 100 μm or even more. The surface morphology of the
flat part was less structured with rare tubular protrusions.
The round cells or rounded parts of the flat cells had
many outgrowths in various shapes, but they were mostly
tubular or vesicular structures of up to 2 μm in size. In
some round-shaped cells the vesicular outgrowths pre-
vailed. The round-shaped cells were, in most cases, not
attached to the surface by filamentous expansions, but
there were some exceptions, which appeared to be a
transitional phase.
Table 2: Cell attachment after 24 h of exposure expressed as a
percentage of the covered surface; all data are reported as mean ±
standard deviation (n=5)
Material
Cell attachment
(% of covered surface)
AISI 316L 55±9
Epoxy coated AISI 316L 20±6
TiO 2
/epoxy coated AISI 316L 12±2
FAS-TiO 2
/epoxy coated AISI 316L 7±2
In the case of the epoxy-coated AISI 316L samples
(Figure 3), the adhesion pattern was similar to the bare
AISI 316L stainless steel. However, the cells were more
randomly attached over the surface and the percentage of
the covered surface was two times lower (Table 2). The
size of the cells was up to 100 μm along the longest axis.
Most of the cells (up to 90 %) had two morphological
features. One part of the cell was flat, cuboidal-to-elon-
gated in one axis, with numerous delicate cellular
filamentous extensions attached to the material surface.
Some cells had one very long filamentous protrusion.
The other part of the same cell was round shaped and
10–30 μm in diameter. This region was higher in the z
axis, rounded, with edges detached from the surface. The
surface morphology of the round and flat parts of the cell
was also significantly different. The surface of the flat
part was less structured with rare tubular protrusions.
The upper surface of the round part of a cell had many
outgrowths in various shapes, ranging from tubular,
lamellar to vesicular structures up to 2 μm in size.
In the case of the superhydrophilic as-received
TiO
2
/epoxy coating on AISI 316L (Figure 4), the cells
were distributed more randomly and formed cluster
patterns across the surface, compared to the AISI 316L
and epoxy-coated AISI 316L samples. The cells covered
8 % less surface than observed on the epoxy-coated AISI
316L samples (Table 2). The majority of the cells
measured approximately 50 μm in diameter. Most of the
cells (95 %) were elongated, spindle-shaped. Some (less
than 5 %) were round shaped, but not completely
rounded. No cells or parts of them were flat and tightly
attached to the surface by delicate filamentous protru-
sions. Most of the cells had one very long filamentous
protrusion, sometimes exceeding the size of the cells.
Occasionally, the protrusions of different cells were
tightly connected. The round cells or parts of the cells
were more structured than the flatter parts of the cell.
The cell surface was covered with mostly tubular protru-
A. KOCIJAN et al.: COMPARATIVE STUDY OF SUPERHYDROPHILIC AND SUPERHYDROPHOBIC TiO2/EPOXY ...
Materiali in tehnologije / Materials and technology 52 (2018) 3, 355–361 359
Figure 4: SEM images of MG63 cells attached to hydrophilic
as-received TiO
2
/epoxy-coated AISI 316L surface: a) distribution,
attachment pattern and shape of the cells, b) cells are predominantly
rounded and tightly attached to the surface with only one very long
filamentous protrusion, c) magnified part of the cell
Figure 3: SEM images of MG63 cells attached to epoxy-coated AISI
316L surface: a) distribution, attachment pattern and shape of the
cells, b) cells with flat and cuboidal to elongated parts, with numerous
delicate cellular filamentous extensions, c) round-shaped part of the
cell

sions of different densities. Vesicles apparently adsorbed
to the surface were also observed.
In the case of the superhydrophobic FAS-TiO
2
/epoxy
coating on AISI 316L (Figure 5), the cells attached to
the surface exhibited a similar pattern of attachment as in
the case of the as-received TiO
2
/epoxy-coated AISI 316L
samples. The percentage of covered surface with cells is
the lowest of all the compared samples: only7%ofthe
surface was covered by cells (Table 2). The cells pre-
ferred to grow on flat surfaces with fewer TiO
2
particles.
However, they were also found to overgrow the TiO
2
particles deposited on the flat surface. The majority of
the cells measured approximately 30–50 μm in diameter.
Most of the cells were spindle-shaped with a rounded
central part; they exhibited different degrees of round-
ness. Approximately 10 % of the cells were irregularly
round shaped. Some spindle-like cells had a stocky
protrusion that was strongly attached to the surface by
delicate filamentous protrusions. Some cells had one or
two very long filamentous protrusions up to 200 μm. The
protrusions of the different cells were occasionally
connected. The round cells or parts of the cells were
more structured than the flatter parts of the cell. The cell
surface was covered with mostly tubular protrusions of
different densities. Vesicles apparently adsorbed to the
surface were also observed.
Several studies have indicated superior cell attach-
ment and cell spreading on (super)hydrophilic surfaces
compared to hydrophobic surfaces.
1,14
We evaluated two
limiting cases of surface wettability, i.e., superhydro-
phobic vs. superhydrophilic coatings, and the results
were comparable with the literature data.
1,14
This study
shows that both surface topographical and chemical cues
modulate the cell morphology and spreading. We ob-
served a variability in cell abundance, size, shape,
number, shape and size of the protrusions as well as the
surface structures of the cells. We found that different
surface characteristics significantly modulated the mor-
phological features of cells and their abundance. The
results indicated improved cell adhesion on the bare
AISI 316L stainless steel compared to the superhydro-
phobic/superhydrophilic coatings. This suggests that
surfaces with moderate hydrophilic nature (i.e., contact
angles around 50–60°) are more appropriate for pro-
posed applications. In addition to the appropriate wetting
properties, we have shown that the low surface rough-
ness also plays a crucial role in improving the cell
adhesion. On AISI 316L surface, the cells appeared
flattened and were able to maintain their osteoblastic
morphology. The cells were attached to the material
surface by numerous fine filamentous extensions. This is
in line with the literature data, which report that cell
adhesion is generally better on hydrophilic surfaces.
25
The cell growth indicated by a portion of covered mate-
rial was less pronounced on the other three materials,
especially on the superhydrophobic surface. However, all
three materials exhibited some unique characteristics that
were manifested in the different morphological
responses of the cells. Optimal tissue regeneration can be
induced only by the selective cell adhesion/activation for
cells of interest, but not for others.
4 CONCLUSIONS
In our work we were able to significantly influence
the wetting properties and biocompatibility of coated
AISI 316L stainless steel by altering the surface
topographical and chemical features. Superhydrophobic
FAS-TiO
2
/epoxy and superhydrophilic as-received
TiO
2
/epoxy coatings were successfully fabricated, result-
ing not only in opposite wetting characteristics but also
in different average surface-roughness properties. This
was reflected in the formation of larger agglomerates in
the FAS-TiO
2
/epoxy coating, making the superhydro-
phobic surface more rough compared to the super-
hydrophilic surface. The biological evaluation showed a
significant variation in the cell abundance, size and
shape as well as the surface structure of the cells. The
results showed that AISI 316L stainless steel is the most
favourable surface for bone osteosarcoma cells (MG-63),
followed by the epoxy, superhydrophilic and superhydro-
phobic coatings, respectively.
The information provided in our work shows that the
four applied surfaces interact very differently with bone
osteosarcoma cells in terms of attachment pattern and
could thus be used for different purposes where low or
high attachment intensity is required. The results pre-
sented in this paper can inspire new strategies for the
preparation of titanium dioxide nanoparticle surfaces for
biomedical applications.
A. KOCIJAN et al.: COMPARATIVE STUDY OF SUPERHYDROPHILIC AND SUPERHYDROPHOBIC TiO2/EPOXY ...
360 Materiali in tehnologije / Materials and technology 52 (2018) 3, 355–361
Figure 5: SEM images of MG63 cells attached to hydrophobic
FAS-TiO
2
/epoxy-coated AISI 316L samples: a) distribution, attach-
ment pattern and shape of the cells at lower magnification, b) cells are
spindle-shaped with rounded central part and they exhibit different
degrees of roundness, c) magnified part of the cell

Acknowledgments
The authors acknowledge the project (Antibacterial
Nanostructured Surfaces for Biological Applications, ID
J2-7196) that was financially supported by the Slovenian
Research Agency.
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