Progress in Organic Coatings 130 (2019) 206–213

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Progress in Organic Coatings

journal homepage: www.elsevier.com/locate/porgcoat

Preparation, characterization, and biocompatibility of different metal oxide/ T

PEG-based hybrid coating synthesized by sol–gel dip coating method for
surface modification of titanium
José Carlos Bernedo Alcázara, Rafaela Moreira Javier Lemosb, Marcus Cristian Muniz Condea,
Luiz Alexandre Chisinia, Mabel Miluska Suca Salasa,c, Bruno S. Norembergb,
Fabiana Villela da Mottad, Flávio Fernando Demarcoa, Sandra Beatriz Chaves Tarquinioa,
⁎
Neftali Lenin Villarreal Carreñoa,b,
a
Post-Graduate Program in Dentistry, Federal University of Pelotas, Pelotas, RS, Brazil
b
Post-Graduate Program in Science and Material Engineering, Federal University of Pelotas, Pelotas, RS, Brazil
c
Department of Dentistry, Federal University of Juiz de Fora, Governador Valadares, MG, Brazil
d
Department of Materials Engineering, Technology Center, Federal University of Rio Grande do Norte, Natal, RN, Brazil

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

Keywords: The aim of the study was to synthesized organic-inorganic experimental nanocomposite materials via sol–gel dip
Sol–gel synthesis coating and to characterized its biological response. The sols consist of an inorganic–organic matrix based on
Dip coating technique ZrO2, TiO2, Li+ and polyethylene glycol (PEG). The obtained hybrid-coated titanium samples have been char-
Organic–inorganic hybrid materials acterized using atomic force microscope (AFM), Scanning Electronic Microscope (SEM), Energy Dispersive X-Ray
Titanium
Analysis (EDX). Biocompatibility of the hybrid coatings was evaluated by cytotoxicity tests. AFM analysis de-
Cell viability
tailed that the obtained materials were rough nanostructured hybrids. SEM results presented covered surfaces
and the presence of particles with different sizes and formats. The EDX data allowed to know the quantitative
percentage of the elements present on the surface of the hybrid-coated titanium samples compatible with the sols
composition. Cytotoxicity showed that all the hybrid-coated titanium samples had non-cytotoxic effect on target
cells, having the groups that included Li+ in the surface higher cellular grown. The sol gel dipcoating method
represents a viable alternative to include inorganic and organic elements on titanium and form stable hybrid
layers. The modified surfaces of titanium with the experimental materials were rough, presented biocompatible
elements and allowed high cellular grown.

1. Introduction biomaterial in the production of all kind of biomedical devices and

The proportion of older population worldwide is increasing, and as
a result, the health and social policy-makers are facing challenges by
the rapid changing burden of general and oral chronic diseases [1].
Edentulism influence the quality of life of people, hence the used of
prosthetic devices and/or dental implants returned successfully the lost
functionally and aesthetics. Although, problems related to implant
commonly occurred in older adults, mainly due to reduced bone for-
mation associated with different health conditions. In this context, the
global biomedical devices industry, especially orthopedic implant, are
constantly looking for advancements in implant designs and materials
that improved biocompatibility, durability, expanding the clinical ap-
plications [2,3]. Titanium (Ti) and its alloys represent the main
symbolize the gold standard in many fields of medicine, in particular,
for orthopedic and dental prosthetic devices into dentistry [4,5]. The
clinical efficacy of these biomedical devices could be greatly enhanced
by the modifying of nanofeatures onto surface that would improve the
bioactivity, in order to induce a bioactive behavior and to enhance
osseointegration between the biomedical devices and the surrounding
bone or where implantable devices are being applied to reconstruct
degraded tissues [6]. Coating and other surface modification are useful
techniques, as they allow us to optimize the surface properties while
retaining favorable bulk properties [7], such as chemical vapor de-
position, plasma spray, ion beam-assisted deposition, radiofrequency
magnetron sputtering [8], notwithstanding, these techniques have
several drawbacks like expensive, complexity, long-term performance,

⁎
Corresponding author at: Post-Graduate Program in Science and Material Engineering, Federal University of Pelotas, Pelotas, RS, Brazil.
E-mail address: neftali@ufpel.edu.br (N.L.V. Carreño).

https://doi.org/10.1016/j.porgcoat.2019.02.007
Received 14 June 2018; Received in revised form 3 February 2019; Accepted 3 February 2019
0300-9440/ © 2019 Elsevier B.V. All rights reserved.
J.C.B. Alcázar et al.
non-homogeneity, non-control of thickness, lack of adherence [3]. A
technique to synthesize a complex hybrid matrix of ceramic and glass
materials, and that allows the doping of various inorganic, organic
materials and biomolecules during the formation of a glassy matrix is
the sol–gel method [5,9].
The sol–gel method uses an alkoxide or aqueous-based chemical
routes, which is based mainly on the hydrolysis and condensation of
metal alkoxides at low temperature [10]. This technique is supported
by different coating techniques: spin, dip, and spray coating. The main
feature is that allow producing organic–inorganic nanocomposite ma-
terials, which makes possible entrapment of notorious thermolabile
substances into the inorganic matrix, for instance, polymers or drugs
[11]. These are considered as biphasic materials, where the organic and
inorganic phases are mixed at nanometer to micrometer scales, and
their properties are derived from a synergy between the individual
contributions of both phases [12]. The sol–gel technique is an en-
vironmental friendliness and makes it an attractive technology for
synthesizing highly bioactive and biocompatible materials [7,9]. The
aim of this study was to develop and characterize the biological re-
sponse of innovating biomimetic coatings on titanium surfaces for
biomedical applications through the synthesis of four experimental
oxide/PEG-based hybrid materials and the sol gel dip-coating method.
2. Material and methods
2.1. Samples preparation
This study used commercially pure grade II Titanium (Ti) F67- UNS
R50400 (Realum®) square-shape samples with standard diameters.
Surfaces were polished under metallographic sander machine (model
Aropol-E, brand ARATEC). Finishing of surfaces was performed with a
colloidal granular deagglomerated alumina suspension (1 μm). Ti
samples were washed by a succession of different ultrasonic baths using
Acetone, alcohol at 75%, and distilled water, then dried and stored in a
gas chamber for nitrogen covering for 4 h. After drying, samples surface
were acid etched in a mixture of hydrochloric and sulphuric acid at
125–130 °C for 6 min. Surfaces roughness were observed and photo-
graphed using an optical microscope (Olympus Model BX41M-LED).
The samples were handled under nitrogen cover gas chamber and
stored in 0.9% NaCl solution for 6 h, and then dried for 4 h. Afterwards,
samples were randomly divided into five groups G0 = Uncoated
Pretreated Titanium; G1 = ZrO2/PEG [2 M]; G2 = TiO2/PEG [2 M];
G3 = TiO2 [2 M]: Li+(15%): Zr(15%)/PEG G4 = TiO2 [2 M]:
Li+(15%)/PEG.
2.2. Sol–gel synthesis
The ZrO2/PEG, TiO2/PEG, TiO2:Li+:ZrO2/PEG, and TiO2:Li+/PEG
organic–inorganic hybrid were synthesized by means of sol–gel process,
using Zirconium (IV) propoxide (Sigma–Aldrich), Titanium (IV) iso-
propoxide (Sigma–Aldrich), and Lithium hydroxide monohydrate
(Sigma–Aldrich), as a precursor of the inorganic matrix, and
Polyethyleneglycol (PEG) (BioUltra, 400) as the organic component.
Previously, solutions with different concentrations of organic and in-
organic components were synthesized in order to achieve adequate
physical properties of sols, including transparency and low viscosity
[13]. Initial concentrations of ZrO2/PEG and TiO2/PEG were based in
previous studies [10,13]. The concentrations of the other hybrids were
experimental and considered saturation and the before mention phy-
sical properties of the sol. Fig. 1 shows the flow chart of the different
hybrids synthesis and coating procedure.
2.3. Coating procedure
The hybrid materials ZrO2/PEG, TiO2/PEG, TiO2:Li+:ZrO2/PEG,
and TiO2:Li+/PEG synthesized by sol–gel process were used in a sol
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Progress in Organic Coatings 130 (2019) 206–213
phase, to coat titanium substrates (Ti F67- UNS R50400, Realum®),
previously polished and acid-etched. As shown in Fig. 1 the coated were
obtained by dip-coating technique (MA765 Marconi® dip-coater).
The Titanium square-shape samples were dip-coated with ZrO2/
PEG, TiO2/PEG, TiO2:Li+:ZrO2/PEG, and TiO2:Li+/PEG hybrid syn-
thesized solutions according to each experimental groups. Withdrawal
speed of the substrate was 35 cm/min. The coated substrate was heat-
treated at 45 °C for 24 h to promote the partial densification of the
coating without any polymer degradation.
2.4. Cytotoxicity assessment
Cytotoxicity assay is considered the most sensitive of the bio-
compatible tests and shall be used to determine adhesion, death,
growth and proliferation of cells in medical devices, materials and/or
their extracts. Cytotoxicity results have correlation with short-term
implant studies and is considered the fists step towards ensuring bio-
compatibility of medical devices [14].
The extracts for investigation were obtained from the treated-coated
titanium surface. The essay was carried out in triplicate for each ran-
domized group. The positive controls were wells seeded at a density of
10 × 104 cells/well (800 μL) without the hybrid-coated titanium sam-
ples, and the negative controls were wells deposited with 800 μL of
media (DMem/FBS 90:10). 3T3/NIH immortalized fibroblasts were
cultured in Dulbecco's modified eagle's medium (DMeM) (Cultilab®)
supplemented with 10% fetal bovine serum (FBS) (Cultilab®), 2% l-
glutamine, penicillin (100 U/mL) and streptomycin (100 mg/mL)
(Gibco-BRL), at a controlled atmosphere of 5% CO2 at 37 °C. At sub-
confluence around 80% adhere cells in cultures were washed using
phosphate buffered saline (PBS) (Gibco®) and afterward detached using
5 mL of 0.25% trypsin/EDTA (Gibco®). The MTT test allows the analysis
of cell viability by determining the levels of activity of mitochondrial
dehydrogenases towards3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide (MTT) (Sigma Aldrich®), through the formation of
insoluble formazan crystals. A 3T3/NIH cell line was seeded on the
hybrid-coated titanium samples into 48 well plates at a density of
10 × 104 cells/well (800 μL), at a controlled atmosphere of 37 °C in 5%
CO2. At 24 h incubation, the hybrid-coated titanium samples were
carefully transferred to a new 48 well plates and were again incubated
with fresh media (800 μL) for 24 h. Afterward, the hybrid-coated tita-
nium samples were washed with PBS. At 48 h, the hybrid-coated tita-
nium samples were treated with800 μL of MTT (0.50 mg/mL) for 4 h at
a controlled atmosphere of 37 °C in 5% CO2. The MTT solution was
then removed and 800 μL of dimethyl sulfoxide (DMSO) was added to
dissolve the formazan produced on the surface of the hybrid-coated
titanium samples. Finally, the absorbance at 540 nm of each well was
determined using a spectrophotometer (Universal ELISA reader). The
cell adhesion viability was expressed by the absorbance values of the
hybrid-coated titanium samples with the extracts compared to a posi-
tive control. Descriptive analysis and comparisons between groups were
performed using the SigmaPlot software version 12.0 (Systat Software
Inc.) The distribution of the samples in each group was obtained by the
Shapiro-Wilk test. Variance test between groups was performed.
Kruskal Wallis test for comparison between groups and Dunn‘s test for
multiple comparisons were used. Analysis was using a 95% confidence
interval and variables with p-value < 0.05 were considered statistically
different.
2.5. Surface characterization
The microstructure of the different hybrid systems-coated titanium
samples was studied by a Digital Instruments Multimode atomic force
microscope AFM (AutoProbe CP, Thermo microscopes), at an inter-
mittent contact mode in a relative humidity 50% and 25 °C. The surface
of the material was also analyzed by scanning electron microscopy –
SEM (Superscan SSX 550, Shimadzu). The determination of the
J.C.B. Alcázar et al.
Fig. 1. Flow chart of different hybrid systems by sol synthesis.
percentages of the different elements present in the hybrid systems-
coated titanium samples was ascertained by Energy Dispersive X-Ray
Analysis (EDX – Ray Ny – EDX 720, Shimadzu), at [quali-quant] easy-
metal mode, at two bands (Ti-U and Na-Sc) with 1 mm X-ray beam.
3. Results
The coatings consisting of polymer polyethylene glycol (PEG) ma-
trix reinforced with sol–gel synthesized PEG-based hybrid ZrO2/PEG,
TiO2/PEG, TiO2:Li+:Zr/PEG, and TiO2:Li+/PEG hybrid fillers, using
different metal oxides zirconium (ZrO2), Titanium (TiO2), Lithium
(Li+). Through simple observation, the four sols solutions were trans-
parent, uniform and homogeneous. Differences between the solutions
were observed in the color being the ZrO2/PEG and the Li+ (15%)/PEG
yellowish and the TiO2 [2 M]: Li+ (15%)/PEG and TiO2/PEG,
TiO2:Li+(15%): Zr (15%)/PEG sols brownish (Fig. 2).
Moreover, after dip coating procedure, titanium surfaces showed a
bright, glassy, transparent, yellowish and uniformed superficial film
(Fig. 3).
Fig. 2. Appearances of sol–gel sols (G1,G2,G3,G4).
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Progress in Organic Coatings 130 (2019) 206–213
3.1. Cytotoxicity test
The description of cell viability due to cytotoxicity is shown in
Fig. 4.
In all groups, sol–gel dip coating test allowed cellular grown on the
treated titanium surfaces. G3 (TiO2/PEG, TiO2: Li+(15%): Zr (15%)/
PEG) showed higher cellular grown followed by G4 and G1. G2 showed
the lowest cellular viability. Multiple comparison results are presented
in Table 2. Experimental groups (G1, G2, G3, G4) showed statistically
lower cellular viability than the control group (p < 0.05). Groups G3
and G4 were statistically different (p < 0.05) from group G2 showing
higher cellular grown.
3.2. Atomic Force Microscopy (AFM) analysis
AFM analyses of surfaces are showed in Fig. 5. Roughness surfaces
are observed in all experimental groups. G1 surfaces presented high
peaks and valleys with a uniform distribution on the surface. Rounded
hexagonal small-sized particles are present combined by middle-sized
and some big hexagonal shape particles (Fig. 5A–D). G2 showed peaked
octagonal big sized particles are present symmetrically positioning
forming small peaks and valleys, with relative uniform distribution but
J.C.B. Alcázar et al.
Fig. 3. Appearances of titanium treated after dip coating methods.
Fig. 4. Cell viability observed in experimental groups of Titanium disks coated by different sol solutions using the sol–gel dip coating method (G1,G2,G3,G4) and
control group (GC) without sol solutions.
with the presence of few clear deep depressions (Fig. 5E–H). Titanium
modified surfaces in G3 presented a uniform distribution, clearly define
small flat peaks and valleys, formed mostly by hexagonal small-sized
particles uniformly distributed forming a continuous compacted de-
fined layer (Fig. 5H–L). In G4, rounded pentagonal small-sized particles
are predominant combined with middle-sized particles distributed
uniformly in a continuous layer forming peaks and valleys are small
with the presence of slightly deeper valleys than G3 (Fig. 5M–P).
3.3. Scanning Electron Microscopy (SEM)
Titanium surfaces are presented in Fig. 6.
In all groups, surfaces are rough, irregular and apparently covered,
which could indicate the existence of bonding with the doped materials,
without cracks. In G1 rounded large and small prominences hetero-
geneously distributed were predominant in the surface. Depressions can
be observed as well as some spherical particles. G2 surfaces exhibited
rounded irregularities, heterogeneously distributed in the surface.
Several depressions and fewer spherical particles than in G1 can be
seen. G3 presented a more uniform, rough surface, without depressions,
prominence or drastic irregularities. Compared to the other groups,
more spherical particles are present, homogeneously distributed. G4
surface showed clearly rounded hexagonal shape prominences with
quite similar sizes, compacted, with a homogeneous and continues
distribution. Almost non spherical particles are present.
3.4. Energy Dispersive X-Ray Analysis (EDX)
EDX analysis results are shown in Table 1. All groups presented
TiO2, SO3, P2O5, CaO. In Group 3 and in Group 1 ZrO2 was also present.
Surfaces in group 3 also had CuO. Modified Titanium disks from groups
2, 3 and 4 additionally had CuO. G3 presented the lowest percentage of
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Progress in Organic Coatings 130 (2019) 206–213
TiO2 (73.11%) on the surface compared to the other groups.
4. Discussion
This study presents titanium surfaces modified with potential new
biocompatible materials based on Titania (TiO2), Zirconia (ZrO2),
polyethylene glycol (PEG) and Lithium oxides by the sol–gel dip coating
method. Sol–gel dip-coating technique can modify metal surfaces due to
the deposition of organic and inorganic elements forming thin films on
them [11]. In fact, the sol–gel method allowed to synthesized materials
containing organic elements preserving their chemical structure and
properties, that can be decomposed when exposed to temperatures
higher than 250 °C surfaces [5,10,15]. The inclusion of organic com-
ponents into inorganic networks, brittle in nature, formed hybrid
sol–gel coating and improved mechanical properties [16]. For instance,
pure inorganic sol–gel materials can produce undesirable cracks in the
surfaces [10]. The final material is considering biocompatible and more
bioactive than others with similar compounds prepared with other
techniques [7], increasing its medical applications [5,9].
In the present study, we synthesized PEG matrix reinforced with
sol–gel based hybrid fillers materials: ZrO2/PEG, TiO2/PEG,
TiO2:Li+:Zr/PEG, and TiO2:Li+/PEG; the last two ones, new experi-
mental sols not reported elsewhere. The sols were transparent, homo-
geneous with low viscosity. Those characteristics were also reported by
other study [7]. Characteristics of the sols can influence the properties
of the final coatings, for instance the viscosity influence the thickness
[7] and homogeneity indicates the existence of connections at mole-
cular level between the phases that can be weak (Class I) or strong
(Class II) consequently determining thermal behavior, mechanical
properties, stability and dispersion of the solution [12]. Better structure
stability influences biological cellular response of the coating [7].
Studies using the sol–gel dip coating technique to modified titanium
J.C.B. Alcázar et al. Progress in Organic Coatings 130 (2019) 206–213

Fig. 5. AFM analysis of experimental modified titanium in groups: G1 = ZrO2/PEG [2 M] (A–D); G2 = TiO2/PEG [2 M] (E–H); G3 = TiO2 [2 M]: Li+ (15%) (I–L): Zr
(15%)/PEG(I-L); G4 = TiO2 [2 M]: Li+(15%)/PEG (M–P); at Distances: 1 μm (A, E, I, M); 3D-images of 1 μm z-scale (B, F, J, N); Distance 2 μm (C,G,K,O) and 3D-
Images of 2 μm z-scale (D, H, L, P).

Fig. 6. SEM micrograph of (a) G1 sample, (b) G2 sample, (c) G3 sample and (d) G4 sample.

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Table 1
EDX analysis of the experimental coated Titanium surfaces.
Experimental groups Percentages (%) of the elements presented on coated titanium material (Ti-U and Na-Sc)
TiO2 ZrO2 SO3 P2O5
G1 79.47 0.045 25.07 1.71
G2 73.93 – 24.00 1.78
G3 73.11 0.012 24.99 1.57
G4 76.24 – 22.39 1.10
demonstrated to enhance cellular response in vitro and in vivo-animal
models [15,17]. Cellular response is measured by several parameters,
being cell adhesion an essential process directly involved in cell growth,
migration and differentiation [18].
In our study, the cytotoxicity results showed that all experimental
groups presented high cellular viability, presenting the TiO2 [0,2 M]:
Li+ (15%): Zr (15%)/PEG group higher cell grown than the other ex-
perimental groups, followed by TiO2 [0,2 M]: Li+ (15%)/PEG group.
Our results can be attributed to the composition and to the superficial
morphologies of the hybrid coatings. AFM and MEV results allowed to
observed more regular surfaces homogeneously distributed particles
and with less depressions in TiO2 [0,2 M]: Li+ (15%): Zr (15%)/PEG
group and in the TiO2 [0,2 M]: Li+ (15%)/PEG group. Cell attachment
depend on the protein that adsorbs onto the surface that determinate
osteoinduction process, which includes the recruitment of un-
differentiated cells and the consequent osteointegration, thus is highly
influenced by the properties of the surface such as chemistry, topo-
graphy and hydrophobicity–hydrophilicity [19]. In sol–gel materials,
the biological response depends on surface properties such as topo-
graphy, hydrophilicity, electronegativity and ionic dissolution in the
body environment [19]. For instance, in titania the contact angle of the
surface depends on the surface roughness and the presence of hydroxyl
groups on the surface. The photocatalytic activity is related to the
chemical state of Titania and the surface area of the coating. Also the
photocatalytic activity of TiO2 is directly related with the crystal
properties, and photoinduced charges are generated in well crystallized
phases, preferably in the anatase allotropic form [10]. Titania can
crystallized in homogeneous mixtured of oxides, however limited to
20Ywt% of titanium. When titanium contents are higher, TiO2 crys-
tallites formed a separate phase [20]. Lower percentages of Titanium
favored the accommodations of Ti atoms in the network, forming more
regular surfaces, more stable, and with regular roughness adequate to
promoted better cellular grown. Undesirable irregular coating layers
modify the surface roughness and produce heterogeneous textures, in-
terfering in osseointegration [21,22]. Roughness should be preserved
even when molecules are incorporated to the surfaces [20,23] since it is
well know that represents an important factor on surfaces topographies
that allowed cell adhesion and enhance osteoblast differentiation [18].
Osteoinduction is increasing in rough surfaces, due to the improvement
of the wetting ability of the cells on the treated surfaces [23,24].
The presence of oxides, as presented in the EDX results compatible
with shapes and morphologies of AFM and MEV results, in our ex-
perimental groups, such as Zirconium and Lithium, appear to be also
responsible for better biological and surface characteristics. The pre-
sence of Lithium can be assumed to influence a better cellular response
Table 2
ANOVA for comparisons between the experimental groups (G1, G2, G3, G4, GC).
Experimental groups G1 G2
Mean (SD) Mean (SD)
Celular viability 2.300 (0.176)bc 2.249 (0.139)c
Different letters and symbols means statistic difference (p < 0.05).
SD: Standard Deviation; G1 = ZrO2/PEG [2 M], G2 = TiO2/PEG [2 M]. G3 = TiO2 [2 M]: Li (15%): Zr (15%)/PEG, G4 = TiO2 [2 M]: Li (15%)/PEG; GC = Control
Group.
Progress in Organic Coatings 130 (2019) 206–213
CaO CuO K2O In2O3 Fe2O3 SiO2 SrO
0.19 – – – – – –
0.28 0.02 – – – – –
0.27 0.02 – – – – –
0.25 0.01 – – – – –
in G3 and G4, since sol–gel derivated Zirconia and Titania are poor
conductors and ionic salts can increase ionic conductivity such as those
based on lithium. Lithium in sol–gel dip coating methods is likely to be
physically incorporated on the coating surface generally as part of it,
that means that lithium ions adsorb or adjoin to crystals and fill in the
spaces between them, increasing conductivity. [25] Studies showed
that Lithium ions used in depressive disorders treatment, reduced the
risk of fractures in patients receiving the treatment and induced bone
formation in animal model [26,27]. Lithium can inhibit the effects of
1,25-dihydroxy vitamin D consequently inhibit bone resorption, due to
the influence in calcium-sensing receptor decreasing the sensitivity to
calcium [27]. Moreover, Lithium ions (Li+) were reported to function
as a activator of the canonical Wnt signaling pathway, factor involved
in mesenchymal stem cells differentiation toward osteoblastic lineage
in vitro [28]. Because of that, studies incorporate Li+ to synthesized
biocompatible materials and have demonstrated proliferation and dif-
ferentiation of osteoblast-like cells in vitro [25,28].
In our study, even though G1 and G2 presented lower cellular
grown, cellular viability was high, and that could be attributed also to
the presence of PEG. In the ZrO2-PEG film deposited on titanium sur-
faces, lower wt% of PEG influenced the superficial morphology, in
which surfaces with low ZrO2-PEG wt% presented high amount of
fractures, different from PEG hybrids with 24–50% that were free from
them [13]. Also, the presence of PEG highly influence crack presence in
TiO2 surfaces, being surfaces without PEG cracked [10]. In our study
titanium modified with TiO2 PEG showed irregular surfaces with the
presence of deep depressions. Cracked surfaced were related to che-
mical instability and problems in adhesion [10]. The cellular viability
improvement in those studies were attributed to the higher con-
centration of PEG polymer in the hybrids, indicating that the sol–gel
process preserved the PEG biocompatibility and transmitted the quality
at hybrids [13]. The presence of PEG, in inorganic networks, reduced
crack structures in the films, which is an important disadvantage of the
sol–gel method that happened during the gelification due to the elim-
ination of water [13]. Also, Ääritalo et al., reported that the presence of
PEG increased hydrophilicity, reducing the contact angles compared to
pure titanium and photocatalytic activity in modified surfaces of TiO2
compared with those without PEG, which was associated to better
crystallized phases [10]. In fact PEG in biological systems has the
ability to increase biocompatibility, intrinsic resistance to protein ad-
sorption and hydrophilicity [29] which increased osseointegration in
vitro and in vivo animal models [30].
After cellular assays, titanium modified samples were submitted to
EDX analysis, showing mostly the presence of Zirconia (ZrO2), titanium
dioxide (TiO2), Phosphoric anhydride (P2O5), Calcium oxide (CaO).
G3 G4 GC
Mean (SD) Mean (SD) Mean (SD)
2.402 (0.096)b 2.385 (0.156)b 2.983 (0.170)a
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Bioactive materials, such as glasses and glass ceramics, promotes strong
cellular bonds and stimulates new bone formation [31]. ZrO2 present
chemical stability, good mechanical properties as well as adequate
biological properties, enable osseointegration [32] and new bone for-
mation observed in animal models [33]. Zinc, present in zirconia, is an
important ion that is necessary for DNA replication and stimulates
protein synthesis. Also promote cellular growth, differentiation, and
development; and directly influence bone calcification and skeletal
growth, since can activate bone formation and inhibit bone resorption
[34]. Titanium is a widely used biomaterial for biomedical purposes
including orthopedic and dental due to the great mechanical properties,
and its high biocompatibility. High biocompatibility of titanium is at-
tributed to a thin ( ± 26 nm) and stable oxide layer of TiO2 mainly,
formed on the surface exposed to air or fluids. Titanium oxide surface
acidic and basic hydroxide types groups, linked to different bonds be-
tween the Ti surface cations and basic hydroxide coordinated to one Ti
cation which increased polarization and electron transfer from the
oxygen atom to the Ti cation [2]. The isoelectric point of TiO2 range
from 5.0 to 6.7, an in basic body, negative charge increase with pH
[24]. In the oxide film, it seems that alloying elements are not present,
however the passive film is continuously dissolved and reconstructed in
aqueous solutions, promoting the incorporation of different elements
from solution onto the titanium oxide, such as calcium and phosphate
forming calcium phosphate and calcium titanium phosphate at the
outermost surface and also releasing elements, such as metal ions from
the titanium alloys generating phagocytosis of particles [35]. Titanium
surfaces coated by Titania were previously performed by sol–gel
method, and studies have shown that titanium coatings can form cal-
cium phosphate [36] - important composite capable to improved
bonding to bone-, precipitate bone-like hydroxyapatite and facilitate
osteogenesis of bone precursor cells in a fluid medium [6,15]. Also,
Calcium oxide (CaO) and Phosphoric anhydride (P2O5) were presented
on the titanium surface. The presence of Ti-OH groups on the surface
combined with the Ca2+ ions present can increase the positive charge
on the surface combine with the negative charge of the phosphate ions
may formed an amorphous phosphate, which spontaneously transforms
into hydroxyl-apatite [Ca10(PO4)6(OH)2] [7,34]. Indeed studies have
reported the spontaneously induced formation of bone-like apatite on
the implant surface [13,16,17]. Calcium and phosphates could regulate
the initiation of cellular signals that enhance osteoblasts differentiation
and influence intracellular signaling molecules in osteoblasts facil-
itating the mineralization process during bone formation. [16]
Limitations should be discussed. Even though the possible influence
of Lithium on cellular responses could be assumed, the measure was not
performed due to limitations of the EDX equipment. Other studies
should be conducted in order to elucidate the influence of Lithium of
the coatings on cellular behavior and on surfaces topography.
As far as we know, the present study is presenting by the first time
the combination of Titanium, Zirconium, Lithium and PEG used as
biocompatible, active materials to modified titanium surfaces for bio-
medical approaches. Our results indicate that all experimental groups
presented adequate biocompatibility. The TiO2 [2 M]: Li+ (15%): Zr
(15%)/PEG group promoted significantly better surfaces characteristics
and higher cellular growth. Further studies must be addressed in-
vestigating the cellular responses of odontoblastic cells types on TiO2
[2 M]: Li+ (15%): Zr (15%)/PEG modified titanium.
Data availability
The raw/processed data required to reproduce these findings cannot
be shared at this time due to technical or time limitations.
Acknowledgments
This study was financed in part by the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior—Brasil
212
Progress in Organic Coatings 130 (2019) 206–213
(CAPES)—Finance Code 001. The authors gratefully acknowledge
CAPES, FAPERGS, and CNPq (Grant Numbers: 458332/2014-3; 2013/
2998/2014/; 17/2551-0001 157-0).
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