Surface & Coatings Technology 403 (2020) 126381

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Surface & Coatings Technology

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

Development of novel dual-action coatings with osteoinductive and T

antibacterial properties for 3D-printed titanium implants
Alejandra Rodríguez-Contrerasa, Diego Torresa,c, Jordi Guillem-Martia, Patricia Serenoa,
Maria Pau Ginebraa,b, Jose A. Caleroc, José María Maneroa, Elisa Rupéreza,
⁎

a
Biomaterials, Biomechanics and Tissue Engineering group (BBT), Dept. Materials Science and Engineering, Universitat Politècnica de Catalunya (UPC), Escola
d'Enginyeria de Barcelona Est (EEBE), Eduard Maristany 16, 08019 Barcelona, Spain
b
Institute for Bioengineering of Catalonia (IBEC), Baldiri Reixac 10-12, 08028 Barcelona, Spain
c
AMES GROUP, Carrer de Laureà Miró, 388, 08980 Sant Feliu de Llobregat, Barcelona, Spain

ARTICLE INFO ABSTRACT

Keywords: Gallium (Ga) has been recently proposed as a novel therapeutic agent, since it promotes bone formation and
Porous structures exhibits antibacterial properties. This work focuses on the optimization of a thermochemical treatment that
Gallium incorporates Ga ions by the addition of the body-friendly Ga nitrate approved by the Food and Drug
Biomaterials Administration. The objective was to simultaneously provide the inner and the outer surfaces of porous‑titanium
Antibacterial activity
surfaces obtained by 3D-printing with bioactivity and antibacterial properties. The apatite-forming ability of the
Titanium implants
coating, as well as the antibacterial activity and SaOS-2 cell adhesion, proliferation, differentiation and mi-
3D-printing
neralization were evaluated and compared with untreated Ti surfaces. The characterization of the surfaces re-
vealed the presence of a Ga-containing calcium titanate layer, which was non cytotoxic and in simulated body
fluid produced a homogeneous apatite coating well adhered to the substrate. The formation of this apatite layer
was accelerated with increasing Ga amounts present on the surface, resulting also in an increase in thickness. An
initial quick release of Ga ion promoted the antibacterial effect against gram positive strains, especially for
Pseudomonas aeruginosa, one of the most frequent resistant pathogens in nosocomial infections. SaOS-2 cells
adhered and proliferated on the Ga-doped Ti surfaces, its presence contributed to cell differentiation and to
considerably increase the mineralization levels. Thus, the developed multifunctional coatings could provide
bioactivity to the porous Ti implants while protecting them from the most frequent gram-negative pathogens.

1. Introduction a bonelike apatite layer on their surfaces when immersed in simulated

The need for body well-accepted biomaterials demands a constant
improvement of the implant materials. Thus, biomaterials with special
properties such as antibacterial or bioactive are continuously developed
to overcome the principal difficulties related to implant surgery and
post-operative complications.
Ti and its alloys are widely used as biomaterials for medical devices
due to their good mechanical properties, excellent corrosion resistance
and favorable biocompatibility. Moreover, different strategies have
been explored to improve the interaction of Ti-based materials with
bone, focused specifically on enhancing the osseointegration, by mod-
ifying their surface properties such as topography, chemical composi-
tion, and surface energy [1–3]. In 1996, Kokubo et al. [4,5] established
a simple thermochemical methodology for inducing bioactivity in Ti
and its alloys. A thin sodium titanate layer was formed, which produced
body fluid (SBF), with a strong bonding to the substrate, which resulted
in a uniform gradient of stress transfer from bone to the implants [4,5].
It has also been reported that various types of metal ions such as Ca [6],
Sr [7], Mg [8], and Ag [9] can be incorporated onto the coating by
modifying the conventional treatment [7,8].
The trivalent semimetal Ga has shown therapeutic efficacy, being
effective against cancer-induced hypercalcemia, inhibiting the pro-
liferation of tumor cells, and has shown activity against non-Hodgkin's
lymphoma and bladder cancer in clinical trials [10–13]. Ga has also
shown some relevant properties concerning the osseointegration pro-
cess. More specifically, Ga showed to directly inhibit bone osteolysis,
prevent bone calcium release and augment bone mass [11,13]. Besides,
recent studies have shown that Ga exerts a significant inhibitory ac-
tivity against numerous bacteria including Staphylococcus aureus, Rho-
dococcus equi, Pseudomonas aeruginosa, Acinetobacter baumannii and

⁎
Corresponding author at: Escola d'Enginyeria de Barcelona Est (EEBE), Eduard Maristany 16, 08019 Barcelona, Spain.
E-mail address: elisa.ruperez@upc.edu (E. Rupérez).

https://doi.org/10.1016/j.surfcoat.2020.126381
Received 16 May 2020; Received in revised form 30 August 2020; Accepted 1 September 2020
Available online 02 September 2020
0257-8972/ © 2020 Published by Elsevier B.V.
A. Rodríguez-Contreras, et al.
Escherichia coli [14,15]. Yamaguchi et at. [16] has recently reported a
variant of the traditional Kokubo's process where Ga was added to the
Ti surface in the form of gallium chloride (GaCl3). However, this
strategy shows a major inconvenience that is its dangerousness. Gallium
chloride is considered hazardous by the 2012 OSHA Hazard Commu-
nication Standard (29 CFR 1910.1200) [17], since, among other char-
acteristics, it reacts hardly and violently with water and decomposes in
hydrogen chloride gas which is a hazard product.
Thus, this study was focused on the development of gallium-doped
Ca titanate coatings on porous Ti structures by a modification of the
traditional thermochemical treatment (TT) developed by Kokubo. The
main objective was to endow the inner and outer surfaces of porous Ti
structures obtained by 3D printing with a double feature: on one side to
increase the bioactivity and on another to confer antibacterial proper-
ties. The possibility of replacing the GaCl3 used in Yamaguchi's work
with the more biocompatible gallium nitrate (Ga(NO3)3), which was
already approved by the FDA (Food and Drug Administration agency)
for the treatment of cancer-associated hypercalcaemia, was assessed.
The determination of the optimal gallium nitrate concentration limited
by the highest non-toxic concentration and the one that permits Ga
releasing for longer time, the evaluation of the antibacterial properties
of the coatings and the assessment of the osteoblastic-like cell adhesion,
proliferation, differentiation and mineralization on the coatings devel-
oped were also objectives of this work.
2. Materials and methods
2.1. Materials
Porous cylindrical samples (7 mm in diameter and 4 mm in height),
produced by direct ink writing with an orthogonal pattern were pro-
vided by AMES (Catalonia, Spain) (Fig. 1). For the ion release, cyto-
toxicity and cell response studies, commercially pure, grade 2 Ti discs of
10 mm diameter and 2 mm thickness were used. They were ground with
silicon carbide papers of grit 320, 800, 1200, and 2500 (Struers, Spain),
and mirror polished with colloidal silica (0.05 μm particle size). Pre-
vious to their use, the porous Ti samples and the Ti discs were ultra-
sonically cleaned with acetone, 2-propanol and distilled water for 5 min
each, and dried with nitrogen gas.
2.2. Specimen preparation
Both the solid discs and the porous cylinders were soaked in 5 M
NaOH solution (10 mL/sample) for 24 h at 60 °C [4]. After gently
Fig. 1. Image of the surface of a porous Ti cylindrical sample fabricated by 3D printing, and FESEM images of its outer (above) and inner (below) surfaces. Cylindrical
samples had a macroporosity of 347 ± 1 μm and a microporosity 8.6 ± 0.2 μm.
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Surface & Coatings Technology 403 (2020) 126381
washing the samples with distilled water, samples were immersed in
calcium chloride (100 mM), with 5, 10, 50 or 100 mM gallium nitrate at
40 °C for 24 h [16]. The samples were then heated at 5 °C/min until
600 °C and maintained 1 h at this temperature. In a final phase (water
step), they were dipped in water at 80 °C for 24 h to facilitate the ionic
release [6]. The treated Ti samples were dried with nitrogen and stored
in a vacuum. Samples subjected to the thermochemical treatment
without gallium nitrate were used as controls (TT).
2.3. Surface characterization
2.3.1. Field emission scanning electron microscopy (FESEM)
The inner and outer surface morphology and topography of porous
treated-Ti samples were examined using a field emission scanning
electron microscope JEOL JSM-7001F (Toyo, Japan) operating at
20 kV. The chemical composition of the surfaces was analyzed using an
energy dispersive X-ray analyzer (EDS, JEOL jsm-6400) at 10 keV.
High-resolution images allowed titanate and apatite layer thickness to
be determined using a Java-based image processing ImageJ.
2.3.2. Fourier transform confocal laser Raman spectrometry (Raman)
A inVia™ Qontor® confocal Raman microscope (Renishaw Centrus
2957 T2, Gloucestershire, UK) with a regular mode laser with a wave-
length of 532 nm with a grating of 2400 l/mm was used as the laser
source. Spectra were acquired with an objective of 50 magnifications,
1 s of exposure time and 40 accumulations.
2.3.3. X-ray diffraction (XRD)
The phases present on the treated-Ti surfaces were identified by a
low angle X-ray diffraction (Bruker D8 advance, MA, USA) and a data
base from EVA software (Bruker). The measured angle range was from
20 to 60° with a step size of 0.02° and a step time of 1 s, with a sample
inclination of 1°.
2.4. Ga ion release assay
The release of Ga from the porous Ti surfaces treated with and
without the water step was evaluated according to the ISO-10993-12
standard. The structures were immersed in 3.5 mL of previously filtered
Hanks solution (1 mL per sample gram) and incubated at 37.5 °C for 2 h,
8 h, 1 day and 3 days under mild stirring. At these intervals, Hanks
solution samples were taken and analyzed by mass spectrometry with
inductively coupled plasma (Agilent 5100 SVDV ICP-OES, CA, USA) for
Ga ion release assessments. The same volume taken from the samples
A. Rodríguez-Contreras, et al.
was replaced with filtered Hanks solution. This evaluation was per-
formed three times per sample.
2.5. Evaluation of the apatite-forming ability
Simulated body fluid (SBF) prepared according the standard ISO
23317:2014 was used to study the formation of the apatite layer on Ti
surfaces in vitro, as a method to predict its bioactivity in living bone
[18,19]. Briefly, SBF was prepared by dissolving reagent-grade NaCl,
NaHCO3, KCl, K2HPO4.3H2O, MgCl2.6H2O, CaCl2 and Na2SO4 in ultra-
pure water, and buffered at pH = 7.40 [19,20]. Then, treated-Ti sam-
ples were soaked in filtered SBF (25 ml) and incubated at 37 °C for 3, 5 y
7 days. The fluid was renewed every 48 h. Then, the samples were
gently rinsed with distilled water and dried in the oven at 40 °C. The
hydroxyapatite formed on treated-Ti surfaces was evaluated by FESEM.
2.6. Antibacterial properties assay
Porous Ti structures doped with Ga were sterilized by washing them
three times in ethanol (70%, v/v) for 5 min, and they were then kept
under UV overnight. The antibacterial properties were assessed using
the agar diffusion test, commonly used procedure for studying the an-
tibacterial action of the coated surfaces was followed [21,22]. The assay
was performed with both Staphylococcus epidermidis (S. epidermidis,
CECT 231) and Staphylococcus aureus (S. aureus, CECT 59) as gram-
positive and Pseudomonas aeruginosa (P. aeruginosa, CECT 110) and
Escherichia coli (E. coli, CECT 101) as gram-negative organisms. The
overnight-incubated bacteria solutions were diluted with sterile PBS
solution to reach an absorbance value of 0.2 at 600 nm (bacterial
concentration about 108 CFU/ml). The solutions were used to inoculate
(100 μL) the Petri plates with triptone soy agar media. The samples
were placed in the middle of the inoculated surface. The plates were
incubated at 37 °C for 24 and 48 h, and the diameter of the growth
inhibition halo was then measured. Untreated Ti and Ti surfaces treated
with the conventional TT were used as controls. Tests were carried out
in triplicate.
2.7. Cell response
2.7.1. Cell culture
SaOS-2 osteoblast-like cells (ATTC, USA) were cultured in McCoy's
5A medium (Sigma-Aldrich, USA) supplemented with 10% fetal bovine
serum (FBS), 20 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic
acid buffer solution, penicillin/streptomycin antibiotics (50 U/mL and
50 μg/mL, respectively), and 2 mM L-glutamine (all from Thermo Fisher
Scientific) at 37 °C in a humidified atmosphere and 5% CO2.
2.7.2. Cytotoxicity
The methodology used to analyze the cytotoxicity of treated-Ti
surfaces was an indirect in vitro test carried out with human osteoblast-
like SaOS-2 cells (ATCC, Manassas, VA) according to ISO 10993-5.
Although SaOS-2 cells are an osteosarcoma cell line, their suitability as
osteoblast cell model has been widely demonstrated [23–26]. Each
specimen was immersed (1 mL/g) in Dulbecco's modified Eagle's
medium (DMEM) for 72 h at 37 °C. Afterwards, the extraction medium
was removed and diluted with DMEM (dilution 1/0; 1/1; 1/10; 1/100;
1/1000) and added to previously-seeded SaOS-2 cells. After 24 h of
incubation, cells were lysed with M-PER (Pierce, USA) and the activity
of the lactate dehydrogenase (LDH) enzyme was analyzed using the
Cytotoxicity Detection Kit LDH (Cytotoxicity Detection Kit-LDH, Roche
Diagnostics, Mannheim, Germany). Absorbance's were recorded at
492 nm using a Synergy HTX Multi-Mode Reader (Bio-Tek, USA). As a
reference for 100% maximum survival, cells were placed in TCPS
(Tissue culture polystyrene).
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Surface & Coatings Technology 403 (2020) 126381
2.7.3. Cell adhesion and proliferation
Cells were seeded at a concentration of 50 000 cells/sample on
treated Ti porous cylindrical samples and allowed to adhere for 6 h. For
the proliferation studies, cells were cultured for 4 h, 3 days, 7 days,
14 days, 21 days, and 28 days changing the medium thrice a week. After
each incubation period the cells were rinsed thrice in PBS and lysed
with 300 μL of mammalian protein extraction reagent (M-PER, Thermo
Fisher Scientific). The number of living adhered cells was measured by
quantifying the released lactate dehydrogenase (LDH) activity using the
Cytotoxicity Detection KitPLUS (LDH) (Roche, USA). The obtained ab-
sorbances at 492 nm in a Synergy HTX multimode reader (Bio-Tek,
USA) were expressed as cell number using a calibration curve with
increasing number of cells. Untreated Ti and Ti surfaces treated with
the conventional TT were used as controls.
2.7.4. Cell differentiation
The same cell lysates from the cell proliferation assay were used to
quantify the activity of alkaline phosphatase (ALP). Samples were in-
cubated at 37 °C using the SensoLyte pNPP Alkaline Phosphatase Assay
Kit (AnaSpec Inc., USA) and absorbance was registered at 405 nm using
a PowerWave HT Microplate reader (Bio-Tek). Results were extra-
polated from a calibration curve using purified ALP from the kit. The
resulting ALP quantity was normalized versus the time of incubation
and their corresponding cell numbers obtained in the cell proliferation
assay.
2.7.5. Cell mineralization
Cells were seeded under the same conditions described for the cell
proliferation assays for 28 days. Then, the cells were fixed with 4% PFA
for 15 min and washed twice with Milli-Q water. Calcium deposits were
stained with 500 μL/sample of 40 mM Alizarin Red S (Sigma-Aldrich)
for 20 min with gentle shaking. Afterwards, 10% cetylpyridinium
chloride in 10 mM NaH2PO4 was added to extract the staining. The
supernatants were then collected and spectrophotometrically measured
at 570 nm in a Synergy HTX multimode reader (Bio-Tek). Untreated Ti
and Ti surfaces treated with the conventional TT were used as controls.
Results were normalized versus their corresponding cell numbers ob-
tained in the cell proliferation assay.
2.8. Statistical analyses
Biological results were expressed as a mean value of standard de-
viation (SD) for each sample. The t-test was used with a 95% confidence
interval to evaluate statistical differences of the means between two
groups.
3. Results
3.1. Surface physicochemical characterization
Regardless of the concentration of gallium nitrate, a porous acicular
feather-like structure was obtained with the thermochemical treatment
in all treated Ti surfaces (Fig. 2a). For all concentrations, the thickness
of the layer was similar (1.67 ± 0.38 μm) (Fig. 2b). This value is si-
milar to those obtained by Yamaguchi after similar treatment with
gallium chloride [16]. EDS analyses revealed a tendency to increase the
presence of Ga and decrease Ca when the concentration of gallium ni-
trate increased (Fig. 2c). The EDS analyses of the inner and outer sur-
faces of the porous structures showed that both Ga and Ca ions are
present on the entire surface of the sample, confirming that the treat-
ment penetrates the porous Ti structures.
Raman analyses of porous Ti surfaces subjected only to the initial
stages of the treatment (NaOH and Ca chloride with gallium nitrate
processes) showed main bands at 270, 440 and 690 cm−1, corre-
sponding to both gallium hydrogen titanate (Ga-HT) or gallium-con-
taining calcium hydrogen titanate (Ga-Ca-HT) (Fig. 2d) [6,16]. With
A. Rodríguez-Contreras, et al.
the alkaline treatment, Ti oxides practically disappear because they
dissolved in alkali hydroxides to produce water-insoluble titanates
(Fig. 2d). When Ti surfaces are subjected to the thermal treatment,
anatase and rutile phases were formed on the Ti surfaces, since the
thermal treatment enhances the oxidation process. Consequently, the
oxides band appeared stronger (Fig. 2e). Simultaneously, hydrogenated
titanates (with Ga atoms) oxidized giving rise to oxides with the pre-
sence of Ga in their crystal lattice and water (evaporates). The DRX
analyses demonstrated the presence of Ca titanate with Ga (Ga-Ca-T) on
the Ti surfaces subjected to the complete thermochemical treatment
(Fig. 2f). The diffraction peaks at 25ᴼ and 48ᴼ can be attributed to
gallium-containing calcium titanate with the following possible stoi-
chiometry: GaxCa1-1.5xTi4O9, GaxCa1-1.5xTi2O4, GaxCa1-1.5xTi4O5. While
the rutile phase is shown with the diffraction peak at 37.5ᴼ, anatase
diffraction peak may overlap with the first Ga-Ca-T peak at 25ᴼ [16].
3.2. Bioactivity analyses
The bone-bonding activity and the bioactivity degree of a surface
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Surface & Coatings Technology 403 (2020) 126381
Fig. 2. Surfaces characterization: (a) FESEM images
of the feather-like structure of Ti surface treated with
the conventional TT, 5 mM and 100 mM Ga(NO3)3.
(b) FESEM images of Ti surface cross section after
thermochemical treatment with a concentration of
100 mM of gallium nitrate. (c) Elemental composi-
tion (atomic%) of Ti surfaces treated with 5, 10, 50
and 100 mM gallium nitrate by EDS analyses of the
inner and outer surfaces of the porous structures.
Raman spectra of Ti samples (d) without and (e)
with the thermal treatment for the different gallium
nitrate concentrations. X-HT bands correspond to
gallium hydrogen titanate (Ga-HT) or to gallium-
containing calcium hydrogen titanate (Ga-Ca-HT),
where X = Ga or GaeCa. A and R correspond to
anatase and rutile, respectively. (f) X-ray diffraction
pattern of a treated Ti surface with 5 mM of gallium
nitrate. Ga-Ca-T correspond to Ca titanate containing
Ga, R correspond to rutile and T to α-Ti.
can be assessed by examining the apatite formation on its surface in SBF
[20]. Thus, the bioactivity of the internal and external surfaces of Ti
samples treated with the proposed gallium nitrate concentrations (5,
10, 50 and 100 mM) was evaluated by SBF immersion for 3, 5 and
7 days for the assessment of apatite formation (Fig. 3). The EDS results
indicated that the samples after being immersed in SBF generated a
layer on the surface composed of calcium and phosphorus, most cer-
tainly in the form of calcium phosphates, with a Ca/P ratio of 1.7.
According to the literature, this ratio agrees with that of the apatite
[27,28] (Fig. 3c). Regardless of the gallium nitrate concentration,
homogeneous coatings of apatite on the treated-Ti inner and outer
surfaces were formed after 5 days of incubation in SBF. At longer in-
cubation time, the apatite coating thickness increased. Moreover, the
highest concentration of gallium nitrate (100 mM) resulted in a faster
formation of the apatite layer, already visible and noticeably more
compact at 3 days (Fig. 3a). Regarding the interface, homogeneous
apatite coatings, with a good and continuous contact with the substrate
were observed for the gallium-containing surfaces (Fig. 3b and c). In
contrast, the interface between the apatite layer and the gallium-free
A. Rodríguez-Contreras, et al.
treated-Ti samples showed some discontinuities. After 7 days in SBF,
the control and the treated surfaces with 5, 10 mM gallium nitrate
showed apatite coatings of similar thickness with no significant dere-
ferences (Fig. 3d). The thickness of the apatite layer enlarged when
gallium nitrate concentration increased, especially for the higher gal-
lium nitrate concentration (100 mM). Therefore, the presence of Ga on
the surface of Ti triggered and accelerated the nucleation of an apatite
layer and resulted in an increased thickness.
3.3. Ion release assays
Ga-based compounds have been used for treating bacterial infec-
tions due to the antibacterial activity shown by this ion both in vitro
and in vivo against several important bacterial pathogens [29]. More-
over, Ga has shown clinical efficacy in suppressing osteolysis, bone pain
and has been suggested as a treatment for osteoporosis [11]. It is
through the release of the ion that the metallic agent is effective.
Therefore, the cumulative concentration of Ga ion released was de-
termined for treated Ti surfaces with and without the final phase of the
thermochemical process (water step) to evaluate if this benefits the ion
release. Results from the ion release assay confirmed that Ti surfaces
subjected to the whole thermochemical treatment showed a greater Ga
release than the samples treated without the water step (Fig. 4a). This is
in agreement with the results reported in the literature with Ca [6].
Therefore, the water step of the thermochemical treatment contributes
to Ga ion release, mainly attributed to an increase of ion mobility [6].
The ion-release cumulative curves showed an initial quick release of
Ca and Ga ions within the first 10 h of incubation in Hanks solution,
followed by a stabilization phase of around 24 and 48 h, suggesting no
more ion release (Fig. 4a). These results coincide with the reported
results in the literature, where Ga ions are greatly released in an initial
5
Surface & Coatings Technology 403 (2020) 126381
Fig. 3. (a) FESEM micrographs (x10k) of Ti surfaces
treated with 5 mM and 100 mM gallium nitrate and
immersed in FBS for 3 days and 7 days. (b) FESEM
micrograph (x200) of the cross-section of the porous
Ti structures. Arrows indicate the examined cross
section areas. (c) FESEM micrographs (x10k) of the
cross-section after immersion in SBF for 7 days:
Treated Ti by TT without Ga (control), surface
treated with 5 mM gallium nitrate, surface treated
with 100 mM gallium nitrate. Arrows in the control
sample indicate the discontinuity of the apatite-sur-
face interface (d). EDS analyses (atomic%) of the
coating formed on a Ti surface treated with 5 mM
gallium nitrate by SBF immersion during 7 days. (e)
Apatite layer thickness after 7 days in SBF with re-
spect to the gallium nitrate concentration applied in
the thermochemical treatment. Values showing sig-
nificant differences (p < 0.05) are indicated with an
asterisk.
phase and short afterward it reaches a stable phase where it stops re-
leasing [16]. Furthermore, the Ga ion release increased up to around
15 ppm when the metal was subjected to the water step, this being four-
fold the concentration obtained for gallium chloride (3.75 ppm) [16].
Although the Ga ion release results showed no further release after
24–48 h, Ga is still present in the surface of the structure as showed by
the EDS analyses after the 7-day immersion test in SBF (Fig. 3d) in the
form of gallium-containing calcium titanate as the surface character-
ization showed.
3.4. Antibacterial properties assay
Indwelling device infections are associated with considerable mor-
bidity and extremely high cost. Ga is a novel attractive inorganic an-
timicrobial agent, hypothetically due to its ability to replace Fe3+ in
bacterial metabolism. Both ions, Ga3+ and Fe3+, share some simila-
rities in terms of charge, ionic radius, mass, electronic configuration,
and coordination number. However, Ga3+ cannot be reduced to lower
oxidation states by the vital redox processes, provoking the bacterial
biological functions to cease [15,29,30].
In this study, the response of bacterial strains to porous Ti surfaces
treated with Ga was uneven (Fig. 4c, d, e). While surfaces used as
controls (untreated porous Ti surfaces and treated with the conven-
tional TT) did not induce an inhibition halo for any of the strains tested,
Ga-treated Ti surfaces showed an inhibition halo for P. aeruginosa, ir-
respective of the Ga concentration used (Fig. 4c). Furthermore, the
diameter of the inhibitory halo was larger after longer times (48 h) of
bacterial incubation, suggesting an antibacterial effect for this strain
which depends on the Ga diffusion through the agar. For E. coli a
growth inhibition was found only when the concentration of Ga used in
the treatment was the highest (100 mM). The relevance of these finding
A. Rodríguez-Contreras, et al. Surface & Coatings Technology 403 (2020) 126381

Fig. 4. (a) Cumulative release of Ga ions from Ti surfaces

treated with 5 mM gallium nitrate. (b) Table with the
diameters of the growth inhibition halo (∅ in mm) of the
four bacterial strains tested Staphylococcus epidermidis (S.
epi), Staphylococcus aureus (S. aureus) as gram-positive
and Pseudomonas aeruginosa (P. aeru), Escherichia coli (E.
coli) as gram-negative organisms for the different Ga
concentrations used in the thermochemical treatment.
Samples that did not show any growth-inhibition halo are
marked with N. Photographs of the growth inhibition
halo of P. aeruginosa and S. epidermidis on non-treated Ti
(Control), treated-Ti without Ga (control TT).
Photographs of the growth inhibition halo of P. aerugi-
nosa on treated-Ti with the minimum concentration
proposed (5 mM gallium nitrate). Here, blue and red
marks correspond to the halo of growth inhibition gen-
erated after 24 and 48 h of incubation, respectively.
Photographs of the growth inhibition halo test for S.
epidermidis on treated-Ti with the maximum concentra-
tion proposed (100 mM gallium nitrate). (c) Cytotoxicity
study for treated-Ti samples with 5 mM and100 mM
gallium nitrate concentrations compared to non-treated
Ti sample (Control). The black line shows the limit at
70% of cytotoxicity according to ISO 10993-5. (For in-
terpretation of the references to colour in this figure le-
gend, the reader is referred to the web version of this
article.)

6
A. Rodríguez-Contreras, et al.
is linked to the fact that Escherichia coli is one of the first causes of gram-
negative related orthopedic implant infections [31] and P. aeruginosa is
the most frequent gram-negative etiologic agent associated with in-
fections of indwelling catheters and body implants. Indeed, P. aerugi-
nosa represents 10% of all microorganisms involved in hip prosthesis
infection [32]. In both cases, the samples did not show any growth
inhibition halo after 48 h, showing no more ion diffusion in the agar
and coinciding with the ion release results. Thus, the antibacterial effect
of the surfaces treated with Ga occurs in the first hours, coinciding with
the initial rapid release of ions. Furthermore, it is important to consider
that the presence of antibacterial Ga could protect the Ti surface from
the problematic bacterial strains. The generation of resistant bacterial
strains is most unlikely since some literature demonstrated that only
long and massive release of Ga can promote the appearance of bacteria
resistance [33].
In contrast to the results obtained for the gram-negative strains, no
inhibitory effect of the Ga-containing Ti surfaces was acquired for the
gram-positive bacteria S. epidermidis and S. aureus, even when the
maximum Ga concentration was used (100 mM) (Fig. 4d). Baldoni et al.
[34] reported that the minimal inhibitory concentration (MIC) of a Ga
salt for S. aureus is found between 375 and 2000 μg/ml and for S. epi-
dermidis is established between 94 and 200 μg/ml. Therefore, for bac-
terial inhibition, the Ga release should be greater than these values
[29], and, as the ion release assay showed, the maximum Ga ion lib-
erated was about 15 ppm. A strategy to overcome this lack of protection
against gram-positive bacteria would be to rely on the synergy with
other antibacterial ions more effective against this type of bacteria, or
on increasing the presence of Ga on the surface but simultaneously
controlling the cytotoxicity of the surfaces.
3.5. Cytotoxicity
The cytotoxicity of Ti samples treated with 5 and 100 mM gallium
nitrate was analyzed and compared with untreated Ti samples.
Regardless of the dilution degree, the level of cell viability was higher
than 70%, the minimum required by ISO 10993-5: 2009, for all surfaces
evaluated (Fig. 4b). Therefore, Ti samples treated with the proposed
concentration of gallium nitrate were not cytotoxic.
3.6. Cell adhesion, proliferation and mineralization
In vitro assays with SaOS-2 cells with the minimum and maximum
gallium nitrate concentrations used in the thermochemical process (5
and 100 mM) were performed to assess cell adhesion, proliferation and
mineralization (Fig. 5). Cell count showed no significant differences in
cell adhesion after 4 h of incubation between the controls (Ti and TT)
and the treated Ti samples with the different gallium nitrate con-
centrations used (Fig. 5a inset).
Cells proliferated on all the substrates, but the rate of proliferation
was different. Comparing both controls, proliferation on Ti treated with
no Ga (TT) was lower than on the untreated Ti sample (Ti). Also
compared to this last control, cell proliferation was lower on surfaces
treated with Ga, especially with the highest concentration (100 mM).
Previous studies have shown that microroughness in Ti surfaces can
inhibit proliferation and promote differentiation of osteoblasts. This
effect of the surface seems to be associated with activation of the in-
tegrin signaling in osteoblasts [35–37]. Thus, the effect of the micro-
topography created by the thermochemical treatment explains cell be-
havior on the TT control subjected to treatment with no Ga. This
microstructure is also present on samples doped with Ga (Fig. 2a) and
could influence cell proliferation.
Theoretically, the process of osteoblast differentiation consists of 3
steps: Cell proliferation, extracellular matrix formation and maturation,
and bone matrix mineralization. Each differentiation period involves
the expression and regulation of distinct genes. During bone matrix
maturation and mineralization, osteoblast proliferation is reduced [38].
7
Surface & Coatings Technology 403 (2020) 126381
Thus, while lower proliferation rates were obtained with the highest Ga
doping (100 mM gallium nitrate), differentiation and mineralization
were strongly promoted in these samples (Fig. 5b and c), as higher le-
vels of ALP activity and calcium deposition were quantified, respec-
tively, at 28 days of cell incubation (Fig. 5b). In the case of Ti treated
with 5 mM gallium nitrate, the values obtained for calcium deposition
by means of Alizarin Red staining were similar to those found for the TT
control at 28 days. At the same time, ALP activity of both samples
showed no significant differences. Therefore, the mineralization effect
should be associated with the microroughness rather than to the pre-
sence of Ga ions on the surface. Taken together these results suggest
that above a given threshold, the presence of Ga on the Ti surfaces
promotes the differentiation and mineralization on Saos-2 cells.
4. Conclusion
In this work, not only the suitability of applying the thermochemical
treatment to porous structures produced by 3D-printing was confirmed,
but also demonstrated how a simple, inexpensive and biocompatible
change in the treatment improved the bioactivity of the surface and
provided it with some antibacterial activity. The incorporation of gal-
lium nitrate in the process provoked the formation of a homogeneous
layer of gallium-containing calcium titanate, anatase, and rutile on the
inner and outer surfaces of microporous Ti scaffolds. This new chemical
configuration on Ti surface promoted apatite precipitation, accelerating
its formation and producing thicker and noticeably more compact layer
with the highest concentration of gallium nitrate (100 mM) tested. The
presence of Ga on the Ti surfaces showed an antibacterial effect against
gram-negative strains in the first hours, coinciding with the initial rapid
release of Ga ions. The presence of Ga on the surfaces after that release,
enhance the osteoinductive effect, promoting the differentiation and
mineralization on Saos-2 cells. Thus, the strategy proposed creates a
coating with a dual-action effect appropriate and beneficial to apply to
the new generation of structural implants.
CRediT authorship contribution statement
Alejandra Rodríguez-Contreras, Diego Torres, Jordi Guillem-Marti,
Patricia Sereno, Maria Pau Ginebra, Jose A. Calero, José María Manero,
Elisa Rupérez
All authors made substantial contributions to conception and de-
sign, acquisition of data, or analysis and interpretation of data; took
part in drafting the article or revising it critically for important in-
tellectual content; gave final approval of the version to be published;
and agree to be accountable for all aspects of the work.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
The authors acknowledge the Ministry of Science and Innovation of
Spain for financial support through the RTI2018-098075-B-C21 project,
cofounded by the EU through the European Regional Development
Funds (MINECO-FEDER, EU). Authors also acknowledge Generalitat de
Catalunya for the funding through 2017SGR-1165 project and the
ICREA Academia award of MPG. Dr. Rodríguez-Contreras acknowl-
edges the KTT Excellence Program, funded by the European Union
through the European Regional Development Fund (EDF), the
Government of Catalonia and the UPC.
A. Rodríguez-Contreras, et al.
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